Plants for Immunity and Conservation Strategies [1st ed. 2023] 9819928230, 9789819928231

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Manoj Kumar Mishra Nishi Kumari   Editors

Plants for Immunity and Conservation Strategies

Plants for Immunity and Conservation Strategies

Manoj Kumar Mishra • Nishi Kumari Editors

Plants for Immunity and Conservation Strategies

Editors Manoj Kumar Mishra Faculty of Engineering and Technology Department of Biotechnology Rama University Kanpur, India

Nishi Kumari Department of Botany, Mahila Mahavidyalaya Banaras Hindu University Varanasi, India

ISBN 978-981-99-2823-1 ISBN 978-981-99-2824-8 https://doi.org/10.1007/978-981-99-2824-8

(eBook)

# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

This is the time of pandemic year 2021. The people around us could not think of any idea to deal with it. But they believed in one thing that the naturally present flora can give them immunity against this disease. That is why many people took decoctions of natural medicines on the advice of Vaidya, which increased their immunity and helped them combat the epidemic. However, due to all these reasons, unrestricted collection by local people and pharma companies has destroyed many habitats of wild flora. Understanding all these things, I thought of making a book which tells about such medicinal plants which provide immunity and how to preserve them. So, we have given the title of this book Plants for Immunity and Conservation Strategy. The present edited book is primarily designed for students and research scholars as well as general audience. This book provides detailed information about medicinal plants which are local, less explored as well as endangered. Some of the chapters cover the origin, classification, important bioactive compounds of vital medicinal plants, plant metabolite biosynthesis, therapeutic importance, and description of conservation strategies. In addition, some chapters cover economically and medicinally important plant resources in different ranges of the Himalayas and challenges or limitation of in vitro culture. This edited book explains the importance of selected medicinal plants for students and researchers and describes the importance and synthesis of various biomolecules present in them and their role in our daily life. Also, it is offering significant advantages in terms of the development of plantderived pharmaceutical compounds and inadequate tissue culture information for germplasm protection. I express my heartfelt gratitude to my teachers and corresponding authors for their kind co-operation and valuable suggestions for the successful compilation of this book, which have been a constant source of encouragement and inspiration to me. Without the understanding and encouragement of my family, I would never been able to undertake the task of editing and writing of this book. Kanpur, India

Manoj Kumar Mishra

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Contents

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A Literature Update on Strategies for Harnessing and Conserving the Bioactive Phytochemicals from Tinospora cordifolia: Current Status, Challenges, and Future Prospects . . . . . . . . . . . . . Archana Prasad, Preeti Patel, Mamta Kumari, Gauri Saxena, Debasis Chakrabarty, and Satya Shila Singh

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Medicinally Important Phytoconstituents and Conservation Strategies of Neem: A Critical Overview . . . . . . . . . . . . . . . . . . . . . Kavita Arora and Sangeeta Sen

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An Insight into Coptis Teeta Wall., an Endangered Medicinal Plant and Its Conservation Strategies . . . . . . . . . . . . . . . . . . . . . . . Manoj Kumar Mishra

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Strategies for Conservation and Production of Bioactive Phytoconstituents in Commercially Important Ocimum Species: A Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mamta Kumari, Archana Prasad, Laiq-Ur-Rahman, Ajay Kumar Mathur, and Archana Mathur

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Studies of Natural Product Synthesis of Withania somnifera and Their Conservation Strategy Through In Vitro Method . . . . . . Gaurav Singh

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In Vitro Studies in Andrographis paniculata Pertaining to Andrographolides Accumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . M Joe Virgin Largia

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Identification of Bioactive Compounds in Berberis Species and In Vitro Propagation for Conservation and Quality . . . . . . . . . 113 Shalini Tiwari and Charu Lata

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Bioactive Compounds in Solanum viarum: Medicinal Properties, In Vitro Propagation, and Conservation . . . . . . . . . . . . 123 Shatrujeet Pandey and Samir V. Sawant

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Biosynthesis of Essential Oils in Artemisia Species and Conservation through In Vitro Propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Pankaj Kumar Verma and Shikha Verma

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Immunostimulatory Properties of Echinacea purpurea and Conservation Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Syed Saema, Laiq-Ur-Rahman, Nafisa Shaheen, and Vibha Pandey

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An Insight of Phytochemicals of Shatavari (Asparagus racemosus) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Vibha Pandey, Manju Shri, Sonali Dubey, Syed Saema, and Shivani Tiwari

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Ex Situ Conservation of Shatavari (Asparagus racemosus) . . . . . . . 207 Vibha Pandey, Sonali Dubey, Ravi Kant Swami, Manju Shri, Shivani Tiwari, and Akanksha Bhardwaj

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Recent Developments in Natural Compounds of Guggul and Production of Plant Material for Conservation and Pharmaceutical Demand Commiphora wightii (Arn.) Bhandari . . . . 239 Gulwaiz Akhter and Ghazala Javed

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Assessment of Economically and Medicinally Important Plant Resources in Sangla Valley Region of Indian Himalaya . . . . . . . . . 259 Usha Devi, Pankaj Sharma, J. C. Rana, R. Murugeswaran, Anees Ahmad, and Asma Sattar Khan

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Ethnomedicinal Pertinence and Antibacterial Prospective of Himalayan Medicinal Plants of Uttarakhand in India . . . . . . . . . 311 Shobha Mehra, Varun Kumar Sharma, Charu Tygai, and Lomas Kumar Tomar

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An Immune Modulator Constituent in Mucuna Pruriens L. (DC) and Biotechnological Approach for Conservation . . . . . . . . . . 349 Naushad Alam and Gul Naaz

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In Vitro Cultures: Challenges and Limitations . . . . . . . . . . . . . . . . 371 Nishi Kumari, Ashish Gupta, Brajesh Chandra Pandey, Renu Kushwaha, and Mohd Yaseen

Editors and Contributors

About the Editors Manoj Kumar Mishra is currently working as assistant professor in Department of Biotechnology, at Rama University, Kanpur. He completed his Ph.D. from Banaras Hindu University, Varanasi. After Ph.D., he joined as postdoctoral researcher at CISH, Rahman Khera, Lucknow. He was awarded the prestigious Dr. D.C. Kothari Postdoc Fellowship in 2015. He also served as Senior Project Associate and Senior Research Associate in CSIR-NBRI, Lucknow and ICAR-NBFGR, Lucknow, respectively. Nishi Kumari is professor and section in-charge in Department of Botany, MMV. She completed her graduation, postgraduation, and Ph.D. from BHU, Varanasi. After Ph.D., she joined postdoc fellowship in Delhi University for three years. She joined BHU in 2004 as lecturer in Botany.

Contributors Anees Ahmed CCRUM-Drug Standardization Research Institute (DSRI), PCIM&H Campus, Ghaziabad, India Gulwaiz Akhter Central Council for Research in Unani Medicine, Ministry of AYUSH, Government of India, New Delhi, India Naushad Alam Botany Department, Aligarh Muslim University, Aligarh, India Kavita Arora Department of Botany, National P.G. College, Lucknow, Uttar Pradesh, India Akanksha Bhardwaj ICAR-Indian Agricultural Research Institute, Delhi, India Debasis Chakrabarty Molecular Biology and Biotechnology Division, Council of Scientific and Industrial Research-National Botanical Research Institute, Lucknow, Uttar Pradesh, India

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Usha Devi CCRUM-Drug Standardization Research Institute (DSRI), PCIM&H Campus, Ghaziabad, India ICAR-National Bureau of Plant Genetic Resources, Shimla, India Sonali Dubey School of Biosciences, IMS Ghaziabad University Courses Campus, Ghaziabad, India Ashish Gupta Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi, India Ghazala Javed Central Council for Research in Unani Medicine, Ministry of AYUSH, Government of India, New Delhi, India Asma Sattar Khan CCRUM-Drug Standardization Research Institute (DSRI), PCIM&H Campus, Ghaziabad, India Mamta Kumari Division of Plant Biotechnology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Council of Scientific and Industrial Research, Lucknow, India Nishi Kumari Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi, India Renu Kushwaha Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi, India Laiq-Ur-Rahman Division of Plant Biotechnology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Council of Scientific and Industrial Research, Lucknow, India M Joe Virgin Largia Department of Botany, St. Xavier’s College, Palayamkottai, Tamil Nadu, India Charu Lata CSIR-National Institute of Science Communication and Policy Research, New Delhi, India Ajay Kumar Mathur Division of Plant Biotechnology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Council of Scientific and Industrial Research, Lucknow, India Archana Mathur Division of Plant Biotechnology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Council of Scientific and Industrial Research, Lucknow, India Shobha Mehra Department of Biotechnology and Microbiology, School of Sciences, Noida International University, Gautam Budh Nagar, Uttar Pradesh, India Manoj Kumar Mishra Faculty of Engineering and Technology, Department of Biotechnology, Rama University, Kanpur, India R. Murugeswaran National Medicinal Plant Board, Ministry of AYUSH, Government of India, New Delhi, India

Editors and Contributors

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Gul Naaz Botany Department, Aligarh Muslim University, Aligarh, India Brajesh Chandra Pandey Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi, India Shatrujeet Pandey CSIR-National Botanical Research Institute, Lucknow, India Vibha Pandey CSIR-National Botanical Research Institute, Lucknow, India Preeti Patel Molecular Biology and Biotechnology Division, Council of Scientific and Industrial Research-National Botanical Research Institute, Lucknow, Uttar Pradesh, India Department of Botany, Banaras Hindu University, Varanasi, Uttar Pradesh, India Archana Prasad Department of Botany, University of Lucknow, Lucknow, Uttar Pradesh, India Laiq Ur Rahman CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India J. C. Rana ICAR-National Bureau of Plant Genetic Resources, Shimla, India Bioversity International, New Delhi, India Syed Saema Department of Environmental Science, Integral University, Lucknow, India Samir V. Sawant CSIR-National Botanical Research Institute, Lucknow, India Gauri Saxena Department of Botany, University of Lucknow, Lucknow, Uttar Pradesh, India Sangeeta Sen Plant Biotechnology, Bangalore, India Nafisa Shaheen Department of Environmental Science, Integral University, Lucknow, India Pankaj Sharma ICAR-National Bureau of Plant Genetic Resources, Shimla, India Varun Kumar Sharma Department of Biotechnology and Microbiology, School of Sciences, Noida International University, Gautam Budh Nagar, Uttar Pradesh, India Manju Shri School of Applied Sciences and Technology, Gujarat Technological University, Ahmedabad, India Gaurav Singh Aix Marseille Univ, CEA, BIAM, Saint Paul-lez-Durance, France CEA-The Aix-Marseille Institute of Biosciences and Biotechnologies, Saint-Paullez-Durance, France Satya Shila Singh Department of Botany, Banaras Hindu University, Varanasi, Uttar Pradesh, India Ravi Kant Swami Jamia Hamdard, Delhi, India

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Editors and Contributors

Shalini Tiwari School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Shivani Tiwari Azad Institute of Pharmacy and Research, Lucknow, India Lomas Kumar Tomar Department of Biotechnology and Microbiology, School of Sciences, Noida International University, Gautam Budh Nagar, Uttar Pradesh, India Charu Tygai Department of Biotechnology, VSPG (PG) College, CCS University, Meerut, Uttar Pradesh, India Pankaj Kumar Verma French Associates Institute for Agriculture and Biotechnology of Dryland, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boqer Campus, Midreshet Ben-Gurion, Israel Shikha Verma French Associates Institute for Agriculture and Biotechnology of Dryland, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boqer Campus, Midreshet Ben-Gurion, Israel Mohd Yaseen Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi, India

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A Literature Update on Strategies for Harnessing and Conserving the Bioactive Phytochemicals from Tinospora cordifolia: Current Status, Challenges, and Future Prospects Archana Prasad, Preeti Patel, Mamta Kumari, Gauri Saxena, Debasis Chakrabarty, and Satya Shila Singh Abstract

Tinospora cordifolia (Family Menispermaceae), commonly known as “Guduchi,” is an extensively used medicinal plant in modern as well as traditional Ayurvedic systems of medicine. The constitutive occurrence of various bioactive constituents such as terpenes, alkaloids, glycosides, aliphatic compounds, and flavonoids attributes to its inexplicable efficacy towards various chronic ailments due to its antidiabetic, anti-inflammatory, hepatoprotective, immunomodulatory, antiperiodic, antileprotic, antispasmodic, antiarthritic, antioxidant, antistress, antimalarial, and antineoplastic activities. The whole plant parts viz. roots, stem, and leaves act as a repository of these important bioactive constituents and are utilized in the preparation of various pharmaceutical, nutraceutical, and

Archana Prasad and Preeti Patel have equal contribution to this work. A. Prasad (✉) · G. Saxena Department of Botany, University of Lucknow, Lucknow, Uttar Pradesh, India P. Patel Molecular Biology and Biotechnology Division, Council of Scientific and Industrial ResearchNational Botanical Research Institute, Lucknow, Uttar Pradesh, India Department of Botany, Banaras Hindu University, Varanasi, Uttar Pradesh, India M. Kumari Division of Plant Biotechnology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIRCIMAP), Council of Scientific and Industrial Research, Lucknow, Uttar Pradesh, India D. Chakrabarty Molecular Biology and Biotechnology Division, Council of Scientific and Industrial ResearchNational Botanical Research Institute, Lucknow, Uttar Pradesh, India S. S. Singh Department of Botany, Banaras Hindu University, Varanasi, Uttar Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_1

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cosmeceutical formulations. A scan of the published literature survey on T. cordifolia clearly indicates that an array of reviews is available on the phytochemical and pharmacological activities of this plant, but systematic compilation for its conventional and nonconventional mode of propagation, conservation, and strategies for the sustainable production of its metabolites is still lacking. Hence, the present review is an attempt in this direction focused on understanding the different means of propagation that act as an alternative platform for strategizing the production as well as the conservation of the bioactive constituents of this important medicinal plant. A critical evaluation of the future implications as well as breakthrough for the commercialization of T. cordifolia is also discussed. Keywords

Tinospora cordifolia · Natural products · Conservation · Propagation

1.1

Introduction

Since the ancient period, medicinal plants have piqued man’s interest. The use of medicinal plants has been practiced by almost all civilizations. For their primary medical care, around 80% of people in developing nations around the world use traditional medicine, and about 85% of traditional medicine utilizes plant extracts (Sharma and Thokchom 2014). Plant extracts have been known to have extraordinary biological effects since the dawn of time (Preeti 2011; Mishra et al. 2013). T. cordifolia, often known as Amrita and Guduchi, is a member of the Menispermaceae family and a potential medicinal plant. It is an herbaceous vine with tropical adaptations that is indigenous to Asia and found in China, India, and Sri Lanka (Spandana et al. 2013; Sharma et al. 2019; Akash et al. 2022). Indigenous medical systems generally utilize this plant, and it is an important drug for Indian Systems of Medicine (ISM) (Sivarajan and Balachandran 1994; Sinha et al. 2004; Maurya et al. 2012). This plant is frequently employed as a drug in India’s 5000year-old Ayurvedic system of traditional medicine. According to Ayurveda, T. cordifolia is a Rasayana plant, which can either purify the blood or treat a wide range of clinical symptoms (Shirolkar et al. 2020). T. cordifolia has enormous economic value as a potential therapeutic plant. The leaf and the stem parts are enriched with various bioactive metabolites, some of them are phenolics, saponins, terpenes, glycosides, steroids, alkaloids, and flavonoids, and have therapeutic potential (Upadhyay et al. 2010; Reddy and Reddy 2015). According to reports, stem extracts can help to treat diabetes, fever, flatulence, hypertension, and leucorrhea (Rajalakshmi et al. 2009; Upadhyay et al. 2010). This plant is well known for its hepatoprotective, antipyretic, antispasmodic, antiallergic, antineoplastic, hypoglycemic, and hypolipidemic effects. Additionally, it is utilized for general weakness, digestive issues, children’s fever, appetite loss, diarrhea, gonorrhea, urinary tract

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infections, viral hepatitis, rheumatoid arthritis, leprosy, and anemia (Singh et al. 2003; Sinha et al. 2004; Jagetia and Rao 2006; Adhvaryu et al. 2007; Sharma et al. 2019). Guduchi-satva is a starch made from a stem that is packed with nutrients, easily digested, and used to treat a variety of illnesses. It is frequently used in Ayurveda because of its ability to strengthen the immune system and the body’s ability to withstand illnesses (Singh et al. 2016). The substance has undergone intensive phytochemical, pharmacological, and clinical investigations over the past decades, and numerous intriguing discoveries in the fields of immunomodulation, anticancer, and hypoglycemia have been recorded (Sinha et al. 2004; Sharma et al. 2019).

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Botanical and Pharmacogenetic Descriptions

1.2.1

Botanical Description and Distribution

T. cordifolia is a large, deciduous, glabrous, succulent, and extensively spreading climbing shrub with several elongated twining branches (Reddy and Reddy 2015). Guduchi is a perennial plant with a flimsy and mushy stem that can be found all throughout India. In India, the T. cordifolia plant is known by a number of different names, including Tippa-teega (Telugu), Amruthu, Chittamruthu (Malayalam), Shindil, Amrutha balli (Kannada), Seendal kodi (Tamil), Rasakinda (Sinhala), Garo (Gujarati), Amrita, Amritavalli, Madhparni, Kundalini, Chakralakshanika, Vatsadani, Tantrika (Sanskrit), Gurcha, Giloy (Hindi), Gilo (Punjabi), Amrita, Gilo (Kashmiri), Guduchi (Marathi), Guluchi (Oriya), and Tinospora (English) (Chaudhari and Shaikh 2013; Gaur et al. 2014; Akash et al. 2022). The name “Guduchi,” which derives from Sanskrit, means “one who defends the whole body.” The name “amrita” refers to a substance that is said to promote youthfulness, energy, and longevity (Chaudhari and Shaikh 2013). T. cordifolia stem is either gray or creamy white, strongly split longitudinally and spirally, and the gaps between the lenticels are dotted with large lenticels that resemble rosette-like structures. The wood is white, porous, and soft. When exposed to air, the freshly cut surface immediately develops a yellow hue. The thread-like aerial roots emerge from the stem. Generally, the wood is porous, white, and soft. The leaf is smooth, heartshaped, simple, alternate, exstipulate, long petiolate, and displays multicoated reticulate venation. The yellowish flowers grow in bunches during the rainy season. This plant bears pea-like fruits that are noticeable in winter in India (Reddy and Reddy 2015; Sharma and Thokchom 2014; Akash et al. 2022). Flowers bloom in the summer and fruits in the winter (Chaudhari and Shaikh 2013). The plant is grown up to 1200 meters above sea level in the tropical region of India, which stretches from Kumaun to Assam in the north and through West Bengal, Bihar, the Deccan, Konkarn, Karnataka, and Kerala in the south. It grows well in tropical climates. It is a typical plant that grows over hedges and small trees in deciduous and dry forests (Sinha et al. 2004; Reddy and Reddy 2015).

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Classification (by Chaudhari and Shaikh 2013) Kingdom Division Class Order Family Genus Species

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Plantae Magnoliophyta Magnoliopsida Ranunculales Menispermaceae Tinospora Cordifolia

T. cordifolia: Chemical Constituents

Numerous chemical compounds have been identified from different plant parts as a result of the detailed scientific study that has been conducted on this plant. Some of the categories include terpenoids, alkaloids, steroids, flavonoids, sesquiterpenoids, phenolics, lignans, lactones, tannins, vitamins, polysaccharides, and aliphatic compounds (Sinha et al. 2004; Mittal et al. 2014; Reddy and Reddy 2015). Several plant components have been identified such as tinosporic acid, tinosporonesyringen, berberine, cordifolisides A to E, giloin, crude giloininand, gilenin, arabinogalactan polysaccharide, bergenin, picrotene, gilosterol, tinosporidine, tinosporol, sitosterol, heptacosanol, cordifol, octacosonal, columbin, tinosporide, chasmanthin, palmatosides C and F, palmarin, amritosides, tinosponone, ecdysterone, cordioside, makisterone A, magnoflorine, hydroxyecdysone, tembetarine, glucan polysaccharide, syringine, syringine apiosylglycoside, palmatine, isocolumbin, jatrorrhizine, and tetrahydropalmaitine (Singh et al. 2003; Sharma et al. 2010) (Table 1.1 and Fig. 1.1).

1.2.3

Phytochemical Constituents of T. cordifolia and Their Bioactivities

Several biologically active substances have been extracted from different plant body parts. According to reports, these substances are involved in a number of biological roles in the cure of various ailments (Reddy and Reddy 2015). The plant’s chemical components such as diterpenoid lactones, glycosides, steroids, sesquiterpenoids, phenolic compounds, essential oils, a combination of fatty acids, and polysaccharides are what really potentialwly lead to the pharmacological bioactivities of the plant (Kidwai et al. 1949; Khan et al. 2016). According to reports, these substances play a variety of biological roles in several ailments. The list of natural products (active compounds) that have been isolated from various plant parts is given here, along with details on how they work biologically (Mittal et al. 2014) (Table 1.2).

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Table 1.1 Some of the essential chemical constituents of T. cordifolia Bioactive component Alkaloids

Terpenoids

Lignans Others

1.2.4

Constituents Tinosporine, berberine, magnoflorine, choline, 1,2-substituted pyrrolidine, jatrorrhizine, palmatine Tinosporide, furanolactone clerodane diterpene, furanolactone diterpene, furanoid diterpene, ecdysterone makisterone and several glucosides isolated as poly acetate, tinosporaside, phenylpropene disaccharides cordifolioside A, B, and C, cordifoliside D and E, cordioside, tinocordioside, palmatosides C and F, sesquiterpene, tinocordifolin, sesquiterpene glucoside tinocordifolioside 3 (a, 4-dihydroxy-3-methoxybenzyl)4-(4-hydroxy-3-methoxybenzyl) Giloin, tinosporal acetate, tinosporan acetate, tinosporidine, octacosanol, heptacosanol, sinapic acid, tinosponone, two phytoecdysones, an immunologically active arabinogalactan

Reference Qudrat-I-Khuda et al. (1966); Bisset and Nwaiwu (1983), Mahajan et al. (1985), Pathak et al. (1995a, b), Sarma et al. (1995), Choudhary et al. (2013) Khuda et al. (1964), Bhatt et al. (1988), Hanuman et al. (1988), Bhatt and Sabata (1989), Khan et al. (1989), Gangan et al. (1994, 1995, 1996), Wazir et al. (1995), Maurya and Handa (1998)

Hanuman et al. (1986a, b) (Khaleque et al. (1970), Maurya et al. (1995), Khuda et al. (1964)

Medicinal Applications

1. Several helpful drugs could be made from this plant. T. cordifolia functions as an immunomodulator in disorders including sepsis, hepatic fibrosis, peritonitis, and obstructive jaundice. It lowers body heat, so it is used to treat jaundice (Sangeetha et al. 2013). It has also been demonstrated that the arabinogalactan in the Guduchi stem aqueous extract induces immunological activity (Mishra et al. 2013). 2. The plant stem is used to treat urinary disorders, dyspepsia, fever, and general debility (Singla 2010). In skin problems, stem extracts are also beneficial (Shefali and Nilofer 2013). 3. T. cordifolia increases the ability of PMN cells and macrophages to scavenge and deslough. These phagocytic cells support pro-healing activities that are typically reduced and hindered in chronic wounds, such as growth factor activation, angiogenesis, and the formation of granulation tissue (Purandare and Supe 2007). 4. To treat cancer, powder of root and stem along with milk is used (Raghunathan 1969). The MCF-7 cell line is very sensitive to the phytochemicals present in T. cordifolia, which have significant anticancer properties (Mishra et al. 2013).

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Fig. 1.1 Structure of some chemical constituents of T. cordifolia

5. T. cordifolia may be used in addition to other treatments for HIV/AIDS (Bhatt and Sabnis 1987). A polyherbal preparation of T. cordifolia has been found to have positive effects on HIV patients (Srivastava 2011). T. cordifolia extract is an immunostimulant that significantly affects HIV symptoms as observed. Extract from the leaves has demonstrated anti-HIV 1 action. As a result, it can be inferred that a biological extract from this plant will unquestionably be helpful for both the prevention and treatment of a range of viral diseases in people (Estari et al. 2012). 6. Asthma can also be treated with juice from the stem (Sinha et al. 2004). 7. This plant is used along with L-DOPA while treating Parkinson’s disease since it is a potent antioxidant. When dopamine is created, free radicals are produced by L-DOPA. T. cordifolia, therefore, counteracts a drug’s harmful effect (Srivastava 2011). 8. Diabetes mellitus, a chronic disease affecting people worldwide and caused by low insulin levels, is characterized by increased blood sugar levels. Type 1 and Type 2 diabetes are the two main subtypes of the disease. In both healthy and Alloxan-induced diabetic mice, T. cordifolia significantly lowered blood sugar

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Table 1.2 The biological processes occurring in various plant parts of T. cordifolia Plant parts Alkaloids Stem, Choline, Berberine, root Tembetarine, Tinosporin, Magnoflorine, Palmetine, Aporphine alkaloids, Isocolumbin, Jatrorrhizine, Tetrahydropalmatine Steroids Shoot δ-sitosterol, β-sitosterol, ecdysterone, 20 β-hydroxyecdysone, giloinstero, makisterone A

Glycosides Stem Furanoid diterpene glucoside, 18-norclerodane glucoside, Tinocordifolioside, Tinocordiside, Cordifolioside Syringin, Cordioside, Syringinapiosyl glycoside, Palmatosides, Pregnane glycoside, Cordifolioside A, B, C, D, and E Diterpenoid lactones Whole Clerodane derivatives plant [(5R,10R)-4R8Rdihydroxy-2S3R:15,16-diepoxycleroda-13 (16),14-dieno17,12S:18,1Sdilactone], Furanolactone, Tinosporon, Jateorine, Tinosporides, Columbin

Biological activity

Reference

Anticancer, antiviral infections, antidiabetes, antioxidant activity, neurological, inflammation, immunomodulatory, psychiatric conditions

Rout (2006), Jagetia and Rao (2006), Patel et al. (2009), Upadhya et al. (2010), Gupta and Sharma (2011), Sharma et al. 2019)

Glucocorticoid-induced osteoporosis in early inflammatory arthritis, IgA neuropathy, induce cell cycle arrest in G2/M phase and apoptosis through c-Myc suppression, antistress activity, inhibits TNFα, IL-1 β, IL-6, and COX-2

Sarma et al. (1996), Lv et al. (2012), Mckeown et al. (2012), Sundarraj et al. (2012)

Treats neurological disorders like ALS, dementia, Parkinson’s, motor and cognitive deficits and neuron loss in hypothalamus and spine, immunomodulation, inhibits NF-kB and acts as nitric oxide scavenger to show anticancer activities

Karpova et al. (1991), Kapil and Sharma (1997), Baldwin (2001), Chen et al. (2000), Ly et al. (2007), Kim et al. (2008)

Inhibits Ca++ influx, Vasorelaxant: Relaxes norepinephrine induced contractions, antiinflammatory, antihypertensive, antimicrobial, antiviral, inhibits bcl-2, induces apoptosis in leukemia by activating caspase-3 and bax

(Kohno et al. (2002), Zhao et al. (2008), Dhanasekaran et al. (2009), Sriramaneni et al. (2010), Sriramaneni et al. (2010)

(continued)

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Table 1.2 (continued) Plant parts Sesquiterpenoid Stem Tinocordifolin Whole Octacosanol, Nonacosanplant 15-one dichloromethane, Heptacosanol

Others Root, whole plant

9.

10.

11.

12.

13.

Tinosporidine, Cordifol, 3,(a,4-di hydroxy-3methoxy-benzyl)-4-(4compounds hydroxy-3methoxy-benzyl)tetrahydrofuran, Jatrorrhizine, Giloinin, Cordifelone, Giloin, Tinosporic acid, N-transferuloyltyramine as diacetate

Biological activity

Reference

Antiseptic Antinociceptive and antiinflammatory, downregulate VEGF and inhibit TFN-α from binding to the DNA, protection against 6-hydroxydopamineinduced parkinsonisms in rats

Maurya and Handa (1998) (Thippeswamy et al. (2008), Wang et al. (2010)

Protease inhibitors for HIV and drug-resistant HIV

Ghosh et al. (2008), Mukherjee et al. (2010)

levels. Blood sugar levels are lowered by plant extracts (Chattopadhyay 1999; Shefali and Nilofer 2013). Monkey malaria is frequently treated with giloy. Giloy juice, a combination of Tulsi leaves and Giloy herb, has been demonstrated in studies to enhance body resistance by up to three times and act as a potent defense against Plasmodium viral assaults (Vashist et al. 2011). Guduchi is also used to treat sore and sensitive digestive tract mucous membranes. By boosting mucin production, it protects the duodenum and stomach. It is recognized as one of the best psychoactive substances in India. The juice from the roots is extremely good for urinary disorders (Meshram et al. 2013). Tumor suppression is another area where T. cordifolia is useful. According to studies, the metastatic lung colonies were inhibited when T. cordifolia’s polysaccharide fraction was administered intraperitoneally to mice (Pingale 2010). The giloy root is used to treat intestinal blockage and acts as a strong emetic. Additionally, T. cordifolia’s root and stem are recommended for snake bite and scorpion sting remedies (Meshram et al. 2013). T. cordifolia’s dry bark contains antispasmodic, antipyretic, allergic, antiinflammatory, and antileprotic effects. There are numerous ways in which

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T. cordifolia is said to strengthen the immune system. The immunomodulatory activity of T. cordifolia’s alcoholic and aqueous extracts has been satisfactorily tested. It was discovered through clinical testing that the plant’s antistress and tonic properties had positive effects on kids with mild behavioral problems and intellectual deficits. Additionally, the I.Q. levels have greatly increased (Shefali and Nilofer 2013). 14. The medicinal herb T. cordifolia has antimycobacterial properties that are used to cure leprosy and tuberculosis (Mittal et al. 2014).

1.3

Production of High-Value T. cordifolia Secondary Metabolites Through Viable In Vitro Culture-Based Platform

The agri-based production of secondary metabolites in plants encounters the limitations of low yield of the desired metabolites that leads to prompt the interest of research workers to search for alternate production platforms. Plant cell and tissue culture always opt as the first choice for developing viable renewable resources in the latter stage of the twentieth century. These in vitro cultures offer various advantages such as rapid growth rate, easy to handle, free from seasonal and geographical influences, implementation of bioreactor-based upscaling, feasibility for elicitors/precursor feeding/biotransformation, and low-cost downstream processing for the desired compounds. These advantages enhance the interest of various researchers to exploit plant cells/tissue systems for the production and upregulation of commercially important compounds. The utility of in vitro-based culture approach for the production of bioactive compounds and chemical fingerprinting of in vitro cultures of T. cordifolia has been attempted by limited workers summarized below. Kumari (2012) found that the methanolic extract of in vitro grown micropropagated plants exhibits antimicrobial activity against E. coli (MTCC1), Bacillus subtilis (MTCC8), Salmonella typhi (MTCC737), and Staphylococcus aureus (MTCC98). The maximum bioactivity was found against Staphylococcus aureus with the largest inhibition zone of 18 mm. Other preliminary screening of callus, leafy shoots, and roots showed the presence of total saponin, alkaloids, carbohydrates, steroids, and flavonoids. In 2015, Sinha and Sharma successfully standardize a rapid clonal propagation protocol for T. cordifolia through in vitro micropropagation. They have done the phytochemical screening of in vitro propagated shoots and showed the presence of tannins, alkaloids, phlobatannins, saponins, terpenoids, and other bioactive compounds in this potentially important medicinal plant. Mittal and Sharma (2017) establish an efficient regeneration protocol from nodal explants of T. cordifolia. They performed clonal fidelity using intersimple sequence repeat (ISSR) marker and found homogeneity among the regenerants and mother plants. The chemical quantification of berberine content was done by LCMS QToF and its presence was further confirmed using mass spectrometric analysis. The maximum berberine content was found in in vitro callus

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cultures followed by stem and the leaves of field-grown plants. Srivastava and Chaturvedi (2018) successfully established an efficient large-scale clonal propagation using nodal explants. The leaves of elite clones were used for the generation of high-yielding protoberberine alkaloid callus cell lines. The purification and identification of alkaloids from the high-yielding cell culture of T. cordifolia were done using mass spectrometry, thin layer chromatography, and high-performance liquid chromatography. Priti and Rani (2019) compared the presence of the total alkaloid content in wild, in vitro propagated, and callus tissue of T. cordifolia. For this, they used different solvents viz. ethyl acetate, methanol, water, chloroform, and diethyl ether for maximum recovery of alkaloids. They found the different accumulation of alkaloid content based on the explant used and the extraction solvents. Trailing of this study, in another study they have reported that the extracts prepared from wild and in vitro regenerated plants in different solvents viz. diethyl ether, methanol, aqueous, ethyl acetate, and chloroform showed the presence of only phenol, alkaloids, terpenoids, flavonoids, and steroids while saponins, tannins, and glycosides were absent in the screened extracts. These extracts also showed the differential potential of antioxidative activity in diverse solvents. In 2020, two groups (Sudan et al. 2020; Sahu et al. 2020) reported the in vitro propagation protocol establishment using nodal explant. The first group, Sudan et al. (2020), found that the in vitro raised cultures and shoots were enriched with berberine content analyzed by thin layer chromatographic techniques. The other group Sahu et al. (2020) performed the chemo-profiling of crude extracts of different plant parts, i.e., leaf, stem, and root of in vitro grown plants of T. cordifolia. They have also analyzed the extracts for the antimicrobial activity against different bacterial species. They found that T. cordifolia showed most antibacterial activity against Bacillus subtilis over other tested bacterial species. In most of these studies, the optimization of culture conditions/medium was done for their utilization of the production of bioactive constituents of T. cordifolia. Efforts for the upscaling of bioactive compounds using different biotechnological strategies such as elicitation, precursor feeding, transformation in complementation with genomics and metabolomics tools need to be implemented to provide better insight into the investigation of the biosynthetic pathway of T. cordifolia bioactive compounds.

1.4

Conservation Strategies for Selection of Elite Germplasm of T. cordifolia

The increasing popularity of plant-based medicines led to an increase in the trade of their medicinal products throughout the world. The rising demands for green medicines lead to severe constraints on the wild resources resulting in the depletion or extinction of some economically valuable germplasms (Fay 1992; Sarwat et al. 2011). The collection from random and wild resources results in the high adulteration of the raw materials used in different phytopharmaceutical industries. As a result of which the quality of the phyto-formulations may face rejection from quality

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assurance department or sometimes they may cause certain side effects. Hence, it is an essential step to identify and characterize the elite, high-yielding promising germplasm to support the quality of the phyto-formulation. Farming and pharming are the two approaches that can be used for further production of genotypes with high content of bioactive phytomolecules. Characterization and evaluation of elite high-yielding germplasm and knowledge of their genetic diversity are one of the prerequisite steps for maintaining them under in situ/ex situ conditions for conservation and further utilize them by the pharmaceutical industries (Singh et al. 2006). These are strategies useful for the selection and conservation of elite genotypes for sustainable development and further cultivation and uses.

1.4.1

In Situ Conservation Using Molecular Markers

Recent advancement in the field of molecular markers plays an imperative role in this direction as they compile the information ranging from diversity/similarity at the nucleotide level (SNPs) to gene and frequencies of allele (genotype information), distribution, and extent of genetic diversity, and population structure (Pourmohammad 2013; El-Bakatoushi and Ahmed 2018). The utilization of molecular markers to evaluate the genetic diversity in T. cordifolia has been studied by several workers (Ishnava and Mohan 2010; Paliwal et al. 2016; Lade et al. 2020). During the last decades, molecular markers such as ISSR and RAPD (Shinde and Dhalwal 2010; Rana et al. 2012; Lade et al. 2018; Malik et al. 2019), gene-specific SCoT markers (Paliwal et al. 2013), cpDNA and ITS-based markers (Ahmed et al. 2006), SSR and genomic SSR (Paliwal et al. 2016; Gargi et al. 2017; Lade et al. 2020), and isozyme (Kalpesh and Mohan 2009) have been studied for the genetic diversity/polymorphism and monomorphism among different germplasms of T. cordifolia collected from different geographical regions. Recently, Singh et al. (2022) generated a meta-transcriptome and created the first TinoTranscriptDB (Tinospora cordifolia Transcriptome Database) for the utility of SSR and other transcription factors. The above studies will be helpful for the selection and identification of the improved accessions of T. cordifolia both at the genetic level, and further these accessions can be recommended for their utilization in developing good agricultural practices (GAP) for T. cordifolia.

1.4.2

Ex Situ Conservation Platform

Plant micropropagation technology possesses three major roles in the herbal trade industries: (a) generation of good quality plants of elite genotype for their commercial cultivation and/or generating the stocks for the phyto-industries; (b) production of chemotypes with desired traits via transgenic plant approach, and (c) synthesis of in vitro culture-based desired bioactive metabolites at commercial scale. All these values raise the plant tissue culture as an alternate production platform for the mass multiplication of reliable and sustainable sources of the bioactive targeted

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compounds. The in vitro micropropagation technique serves as an important tool for mass production and conservation of various endangered and vulnerable medicinal plants. It offers incredible opportunities for the production of a stock culture of required genotypes for their possible commercial utilization. These techniques constitute various genetic improvement programs for crop designing and also provide viable upscaling options for the production of valued pharmaceutically important bioactive molecules at the cellular level under in vitro conditions. It will also ease the pressure of overexploitation of wild species and hence can contribute to the conservation of various rare and endangered species (Mulabagal and Tsay 2004; Karuppusamy 2009). According to a task force on sustainable and conservation use of medicinal plants, T. cordifolia is one of the most extensively valuable plants used in various pharmaceutical industries. Approximately, 10,000 tonnes are needed for each year to fulfill the demand for this particular species in the Indian System of Medicine (Singh and Warrier 2004). The rising market demands always search for an alternate production platform to meet the need for quality plant material. Plant tissue culture serves as a powerful technology that has always made a significant contribution to conservation research for a series of medicinal plants. Various workers have extensively explored tissue culture methodologies for achieving different levels of the organization viz. cell/tissue/organ cultures in T. cordifolia. Some of these studies performed during the last decades have been presented in Table 1.3. Influences of auxins and cytokinins in the medium have been documented for callus induction, somatic embryogenesis, and whole plantlet regeneration in T. cordifolia. In most of the studies, benzyl aminopurine (BAP) and indole-3-butyric acid (IBA) were found to be efficient for the shoot and root induction. In one study performed by Sivakumar et al. (2014) silver nitrate was also found to be effective for shoot induction but in this case leaching of phenolics in the medium was found. In one recent report by Singh et al. (2021) alginate encapsulated shoots were used for the formation of synthetic seeds, as an efficient short-term conservation technique. These encapsulated seeds showed 94% germination frequency. The established protocol can be exploited for the exchange of elite planting material irrespective of any seasonal and geographical limitations. The enhancement of bioactive metabolites using different biotechnological strategies has also been attempted by some researchers. Elicitation is one of the efficient technologies used to enhance the metabolites in different plant cell/organ cultures. These elicitors are classified into biotic and abiotic, physical and chemical, exogenous and endogenous depending on their origins. They trigger a variety of defence responses in plants that protect the plants against diseases and various environmental stresses. The use of elicitor in cell suspension cultures of T. cordifolia for the enhancement of protoberberine and berberine alkaloids was studied by some researchers (Kumar et al. 2017, Pillai and Siril 2022). These studies showed that both biotic and abiotic elicitors at specific dose and growth stage showed upregulation in the production of metabolites in cell suspension cultures of T. cordifolia. Kumar et al. (2017) found that culture filtrate of P. indica (5% v/v) showed 4.2- and 4.0-fold enhanced production of jatrorrhizine and palmatine, respectively, over the control. The cell suspension cultures elicited with P. indica

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Table 1.3 The summary of tissue culture studies carried out during the last decade in T. cordifolia Explant used Nodal

Nodal

Nodal

Nodal

Nodal

Stem

Nodal, shoot tip Shoot tip

Nodal

Nodal

Basal medium/PGR/ additives MS + 5 μM BA + 150 μM glutamine; ½ MS + 0.5 μM IBA MS + 6.97 μM kin; MS + 4.4 μM BAP + 13.42 μM NAA MS + 8.88 μM BAP + 18.6 μM Kn + 0.91 μM TDZ; ½ MS + 8.28 μM IBA MS + 4.36 μM Kn; MS + 4.36 μM Kn + 20% silver nitrate; ½ MS + 06.43 μM IBA MS + 6.98 μM Kn + 79.4 μM Phloroglucinol; ½ MS 7.4 μM IBA + 793.7 μM Phloroglucinol MS + 17.76 μM BAP MS + 32.22 μM NAA

Morphogenetic response 100% shoot emergence with 4 shoots/ explants; 100% rooting with 6.8 cm root length

Reference Mishra et al. (2010)

Shoot induction and proliferation; rooting response

Singh et al. (2009)

Shoot emergence with 10.29 shoots/ explants; 80% rooting response with 10–12 roots/ explant 70% shoot emergence with 1.8 shoots/ explants; due phenolic exudation shifted into another medium gives 100% induction frequency; 85% rooting response with 5.2 roots/ explant 84% shoot emergence with 7.5 shoots/ explants; 81.1% rooting response with 3.2 roots/ explant

Sultana and Handique (2013)

90% shoot emergence with 4–5 shoots/ explants; 90% rooting response

MS + 8.88 μM BAP + 1.14 μM IAA

65% and 60% shooting response with 4.5 and 4.2 shoots/explants

MS + 22.2 μM BAP; MS + 4.44 μM BAP + 1.734 μM GA3; MS + 4.9 μM IBA; MS + 2% sorbitol +2% mannitol MS + 4.44 μM BAP + 2.46 μM 2iP; ½ MS + 2.45 μM IBA

95% shoot emergence with 16.5 shoots/ explants; highest shoot length in 12.5 cm 82% rooting response with roots/shoot; 95% survival response at 10 °C stored up to 8 month

MS + 8.88 μM BAP; MS + 2.22 μM BAP + 2.32 μM Kn + 0.571 μM IAA; Shoot base +treated with 980 μM mg/l IBA for 3 min

86% shooting response with 7.9 shoots/ explant and shoot length 9.3 cm; 89% rooting response with the 8.3 cm root length 87% shooting response with 3.8 shoots/ explants; modified MS medium showed 15 shoots/explants; 87% concurrent ex vitro rooting response

Sivakumar et al. (2014)

Jani et al. (2015)

Sinha and Sharma (2015) Tupe and Pandhure (2015) Chatterjee and Ghosh (2016)

Mittal and Sharma (2017) Panwar et al. (2018)

(continued)

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Table 1.3 (continued) Explant used Nodal

Basal medium/PGR/ additives MS + 2 μM BAP; Micro-shoots treated with 30 μM IBA

Nodal

MS + 13.95 μM Kn; MS + 5.71 μM IAA

Nodal

Nodal

MS + 8.0 μM kin + 2.0 BA μM; ½ MS + 6.5 μM IBA MS + 8.87 μM BAP; MS + 8.87 μM BAP + 5.71 μM IAA; ½ MS + 2.22 μM BAP + 4.92 μM IBA 2.5% sodium alginate + 100 mM calcium chloride; MS + 8.88 μM BAP + 1.07 μM NAA B5 + BA

Nodal

MS + BAP + 2-iP

Nodal

Nodal

Morphogenetic response 75% shooting response, 2.95 shoots/ culture with 4.89 cm shoot length; 87.77% ex vitro rooting response with 7.29 roots/shoots 100% shooting response with 2.7 shoots/ explants; 90% rooting response with 5 roots/shoot Showed 4.5 shoots/explants; produced 4.5 roots/shoot

Reference Pillai and Siril (2019)

Priti et al. (2019) Shankar et al. (2020)

Showed 6.3 shoots/explants; maximum shoot elongation; maximum rooting response

Sahu et al. (2020)

Optimum concentration for encapsulation of shoots to form synthetic seed formation and short-term conservation; showed 94% synthetic seed germination percentage

Singh et al. (2021)

Showed the maximum shoot bud induction frequency

Mishra et. al. (2020) Patel and Pandya (2022)

86% showed the shoot bud induction frequency

resulted in achieving maximum jatrorrhizine and palmatine content than other abiotic elicitors (methyl jasmonate) and biotic elicitors (chitin and chitosan). In another study performed by Pillai and Siril (2022) the cell suspension cultures of T. cordifolia were elicited by adding silver nitrate (AgNO3), methyl jasmonate (MJ), and salicylic acid (SA). The cell treated with 100 μM MJ produced maximum berberine content (3077.76μgg-1dw, 5.57-fold higher than the control). These studies clearly support that the efficacy of elicitors for enhancing growth and metabolites highly depends on different factors such as elicitors specificity, their concentration, exposure time, age of culture, and nutrient composition.

1.5

Future Prospects and Conclusion

The published literature on pharmacological values of chemical compounds and in vitro-based production of these bioactive compounds leads to an emerging area of research interest in T. cordifolia presented in this report. The present compilation

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suggests that this medicinal herb is a very totipotent and amenable system for in vitro biomass production and studies for modulation of the biosynthetic pathways of bioactive compounds. Protocols for cell/tissue culture are now available in T. cordifolia. But further refinements of these technologies need to be strengthened for upscaling the end products to ensure the commercial supply of elite genotypes, which is an alarming issue in this important medicinal plant. The nutritional, environmental, and hormonal parameters play a very important role in the biosynthesis and accretion of bioactive compounds of T. cordifolia, which is also one of the important areas that can be explored for a better understanding of the genetic and biochemical regulation of their synthesis. The implementations of various biotechnological tools such as precursor feeding, elicitation, transformation, induction of novel transgene, and genome editing can serve as major breakthroughs in the -omics studies of T. cordifolia in the upcoming future. In addition, reinforcement towards different breeding programs may also lead to the cultivation of high-yielding chemically and genetically characterized elite genotypes developed through tissue culture interventions to meet the high industrial demand for this herb. Acknowledgments All the authors are grateful to the University of Lucknow as well as the Director of the CSIR-National Botanical Research Institute, Lucknow, for the research facilities. AP and PP are thankful to the University Grant Commission (UGC), New Delhi, India, or the award of Dr. D. S. Kothari Post-Doctoral Fellowship (BSR/BL/18-19/0078) and Junior research fellowship (No. 2121530609, Ref No: 20/12/2015(ii) EU-V), respectively. PP is also grateful to Banaras Hindu University (BHU), Varanasi, for her Ph.D. (Sept.-2016/317) registration.

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Chen S, Wu K, Knox R (2000) Structure-function studies of DT-diaphorase (NQO1) and NRH: quinone oxidoreductase (NQO2). Free Radic Biol Med 29:276–284 Choudhary N, Siddiqui MB, Azmat S et al (2013) Tinospora cordifolia: ethnobotany, phytopharmacology and phytochemistry aspects. Int J Pharm Sci 4:891 Dhanasekaran M, Baskar AA, Ignacimuthu S et al (2009) Chemopreventive potential of epoxy clerodane diterpene from Tinospora cordifolia against diethylnitrosamine-induced hepatocellular carcinoma. Investig New Drugs 27:347–355 El-Bakatoushi R, Ahmed DGA (2018) Evaluation of genetic diversity in wild populations of Peganum harmala L., a medicinal plant. J Genetic Eng Biotechnol 16:143–151 Estari M, Venkanna L, Reddy AS (2012) In vitro anti-HIV activity of crude extracts from Tinospora cordifolia. BMC Infect Dis 12:1–1 Fay MF (1992) Conservation of rare and endangered plants using in vitro methods. In Vitro Cell Dev Biol 28:1–4 Gangan VD, Pradhan P, Sipahimalan AT et al (1996) Palmatosides C, F: diterpene furan glucosides from Tinospora cordifolia. Structural elucidation by 2D NMR spectroscopy. Chem Inform 27 Gangan VD, Pradhan P, Sipahimalani AT (1995) Norditerpene furan glycosides from Tinospora cordifolia. Phytochemistry 39:1139–1142 Gangan VD, Pradhan P, Sipahimalani AT et al (1994) Cordifolisides a, B, C: norditerpene furan glycosides from Tinospora cordifolia. Phytochemistry 37:781–786 Gargi M, Thakur S, Anand SS et al (2017) Development and characterization of genomic microsatellite markers in Tinospora cordifolia. J Genet 8:e25–e30 Ghosh AK, Chapsal BD, Weber IT et al (2008) Design of HIV protease inhibitors targeting protein backbone: an effective strategy for combating drug resistance. Acc Chem Res 41:78–86 Gupta R, Sharma V (2011) Ameliorative effects of Tinospora cordifolia root extract on histopathological and biochemical changes induced by aflatoxin-B1 in mice kidney. Toxicol Int 18:94 Hanuman JB, Bhatt RK, Sabata B (1988) A clerodane furano-diterpene from Tinospora cordifolia. J Nat Prod 51:197–201 Hanuman JB, Bhatt RK, Sabata BK (1986a) A diterpenoid furanolactone from Tinospora cordifolia. Phytochemistry 25:1677–1680 Hanuman JB, Mishra AK, Sabat B (1986b) A natural phenolic lignan from Tinospora cordifolia Miers. J Chem Soc Perkin Trans 1:1181–1185 Ishnava K, Mohan JSS (2010) Assessment of genetic diversity in medicinal climber of Tinospora cordifolia (Willd.) Miers (Menispermaceae) from Gujarat. India Asian J Biotechnol 1:93–103 Jagetia GC, Rao SK (2006) Evaluation of the antineoplastic activity of guduchi (Tinospora cordifolia) in Ehrlich ascites carcinoma bearing mice. Biol Pharm Bull 29:460–466 Jani JN, Jha SK, Nagar DS et al (2015) Phloroglucinol plays role in shoot bud induction and in vitro tuberization in Tinospora cordifolia- a medicinal plant with multi-therapeutic application. Adv Tech Biol Med 3:2 Kalpesh I, Mohan JSS (2009) Assessment of genetic diversity in the medicinal climber Tinospora cordifolia (Willd.) Miers (Menispermaceae) from Gujarat, India. Afr J Biotechnol 8:6499–6505 Kapil A, Sharma S (1997) Immunopotentiating compounds from Tinospora cordifolia. J Ethnopharmacol 58:89–95 Karpova EA, YaV V, Dudukina TV et al (1991) 4-Trifluoromethylumbelliferyl glycosides as new substrates for revealing diseases connected with hereditary deficiency of lysosome glycosidases. Biochem Int 24:1135–1144 Karuppusamy S (2009) A review on trends in production of secondary metabolites from higher plants by in vitro tissue, organ and cell cultures. J Med Plant Res 3:1222–1239 Khaleque A, Maith MAW, Huq MS (1970) Tinospora cordifolia IV. Isolation of heptacosanol, ß sitosterol and three other compounds tinosporine, cordifol and cordifolone. Pakistan J Sci Industry Res 14:481–483 Khan MA, Gray AI, Waterman PG (1989) Tinosporaside, an 18-norclerodane glucoside from Tinospora cordifolia. Phytochemistry 28:273–275

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Mittal J, Sharma MM (2017) Enhanced production of berberine in In vitro regenerated cell of Tinospora cordifolia and its analysis through LCMS QToF. 3 Biotech 7:25 Mittal J, Sharma MM, Batra A (2014) Tinospora cordifolia: a multipurpose medicinal plant-A. J Med Plant Res 2:32–47 Mukherjee R, De UK, Ram GC (2010) Evaluation of mammary gland immunity and therapeutic potential of Tinospora cordifolia against bovine subclinical mastitis. Trop Anim Health Prod 42:645–651 Mulabagal V, Tsay HS (2004) Plant cell cultures-an alternative and efficient source for the production of biologically important secondary metabolites. Int J Appl Sci Eng 2:29–48 Paliwal R, Kumar R, Choudhury DR et al (2016) Development of genomic simple sequence repeats (g-SSR) markers in Tinospora cordifolia and their application in diversity analyses plant. Gene 5:118–125 Paliwal R, Singh R, Singh AK et al (2013) Molecular characterization of Giloe (Tinospora cordifolia (Willd.) Miers ex Hook F & Thoms.) accessions using Start Codon Targeted (SCoT) markers. Int J Med Arom Plants 3:413–422 Panwar D, Patel AK, Shekhawat NS (2018) An improvised shoot amplification and ex vitro rooting method for offsite propagation of Tinospora cordifolia (Willd.) Miers: a multi-valued medicinal climber Ind. J Plant Physiol 23:169–178 Patel DS, Pandya A (2022) Combination of PGRS for rapid and enhanced micropropagation of Tinospora cordifolia. Inter J Innovat Sci Res Technol 7:233–240 Patel SS, Shah RS, Goyal RK (2009) Antihyperglycemic, antihyperlipidemic and antioxidant effects of Dihar, a polyherbal ayurvedic formulation in streptozotocin induced diabetic rats. Indian J Exp Biol 47:564–570 Pathak AK, Agarwal PK, Jain DC (1995a) NMR studies of 20p-hydroxyecdysone, a steroid; isolated from Tinospora cordifolia. Indian J Chem 34:674–676 Pathak AK, Jain DC, Sharma RP (1995b) Chemistry and biological activities of the genera Tinospora. Int J Pharmacogn 33:277–287 Pillai SK, Siril EA (2019) Elite screening and in vitro propagation of Tinospora cordifolia (Willd.) Miers ex Hook F. & Thoms. Proc Natl Acad Sci 89:551–557 Pillai SK, Siril EA (2022) Exogenous elicitors enhanced berberine production in the cell suspension cultures of Tinospora cordifolia (Willd.) Miers ex Gook F. & Thoms. Proc Natl Acad Sci 92: 209–218 Pingale SS (2010) Hepato suppression study by Tinospora cordifolia. Der Pharma Chemica 2:83– 89 Pourmohammad A (2013) Application of molecular markers in medicinal plant studies. Agric Env 5:80–90 Preeti S (2011) Tinospora cordifolia (Amrita)-a miracle herb and lifeline to many diseases. Int J med Aromat Plants 1:57–61 Priti RS (2019) Estimation of total alkaloids in wild and in-vitro regenerated Tinospora cordifolia. Int J Pharm Sci Res 10:2777–2784 Priti, Rani S (2019) Estimation of total alkaloids in wild and in-vitro regenerated Tinospora cordifolia. Int J Pharm Sci Res 10:2777-2784 Purandare H, Supe A (2007) Immunomodulatory role of Tinospora cordifolia as an adjuvant in surgical treatment of diabetic foot ulcers: a prospective randomized controlled study. Indian J Med Sci 61:347–355 Qudrat-I-Khuda M, Khaleque A, Bashir A et al (1966) Studies on Tinospora cordifolia-isolation of tinosporon, tinosporic acid and tinosporol from fresh creeper. Sci Res 3:9–12 Raghunathan K (1969) The aqueous extract of T. cordifolia caused reduction of blood sugar in alloxan induced hyperglycemic rats and rabbits. J Res Ind Med 3:203–211 Rajalakshmi M, Eliza J, Priya CE, Nirmala A, Daisy P (2009) Antidiabetic properties of Tinospora cordifolia stem extracts on streptozotocin-induced diabetic rats. African J Pharm and Pharmaco 3:171–180

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Rana V, Thakur K, Sood R et al (2012) Genetic diversity analysis of Tinospora cordifolia germplasm collected from north-western Himalayan region of India. J Genet 91:99–103 Reddy NM, Reddy RN (2015) Tinospora cordifolia chemical constituents and medicinal properties: a review. Sch Acad J Pharm 4(8):364–369 Rout GR (2006) Identification of Tinospora cordifolia (Willd.) Miers ex Hook F. & Thomas using RAPD markers. Z Naturforsch 61:118–122 Sahu B, Behera L, Priyadarshini A et al (2020) Vitro plantlet regeneration from Tinospora cordifolia (Willd.) Miers - a highly valuable medicinal plant and its chemo-profiling. Int J Chem Stud 8:1424–1429 Sangeetha MK, Priya CM, Vasanthi HR (2013) Anti-diabetic property of Tinospora cordifolia and its active compound is mediated through the expression of Glut-4 in L6 myotubes. Phytomedicine 20:246–248 Sarma DK, Khosa RL, Chansauria JN (1995) Effect of Tinospora cordifolia on brain neurotransmitters in stressed rats. Fitoterapia (Milano) 66:421–422 Sarma DNK, Khosa RL, Chansauria JPN (1996) Antistress activity of Tinospora cordifolia and Centella asiatica extracts. Phytother Res 10:181–183 Sarwat M, Nabi G, Das S et al (2011) Molecular markers in medicinal plant biotechnology: past and present. Crit Rev Biotechnol 32:74–92 Shankar G, Upadhyay N, Soni R et al (2020) Optimization of quick in vitro regeneration protocol using nodal segments of Tinospora cordifolia: a therapeutic reservoir. Indian Res J Genet Biotech 12:200–208 Sharma A, Gupta A, Singh S et al (2010) Tinospora cordifolia (Willd.) Hook. F. & Thomson-A plant with immense economic potential. J Chem Pharm Res 2:327–333 Sharma P, Diwivedee BP, Bisht D et al (2019) The chemical constituents and diverse pharmacological importance of Tinospora cordifolia Heliyon 5(9):e02437. https://doi.org/10.1016/j. heliyon.2019.e02437 Sharma S, Thokchom R (2014) A review on endangered medicinal plants of India and their conservation. J Crop Weed 10:205–218 Shefali C, Nilofer S (2013) Gaduchi-the best ayurvedic herb. J Pharm Innov 2:97–102 Shinde VM, Dhalwal K (2010) DNA fingerprinting of Tinospora cordifolia using RAPD analysis. J Glob Pharm Technol 2:38–42 Shirolkar A, Yadav A, Mandal TK et al (2020) Intervention of Ayurvedic drug Tinospora cordifolia attenuates the metabolic alterations in hypertriglyceridemia: a pilot clinical trial. Diabetes Metab J 19:1367–1379 Singh A, Sah SK, Pradhan A et al (2009) In vitro study of Tinospora cordifolia (Willd.) Miers (Menispermaceae). J Plant Sci 6:103–105 Singh BG, Warrier RR (2004) Tinospora cordifolia. Indian For 130:1806 Singh JS, Singh SP, Gupta SR (2006) Ecology, environment and resource conservation. Anamaya Publishers, New Delhi Singh R, Kumar R, Mahato AK et al (2016) De novo transcriptome sequencing facilitates genomic resource generation in Tinospora cordifolia. Funct Integr Genomics 16:581–591 Singh R, Mahato AK, Singh A et al (2022) TinoTranscriptDB: a database of transcripts and microsatellite markers of Tinospora cordifolia, an important medicinal plant. Genes 13:1433 Singh S, Tripathi MK, Tiwari S et al (2021) Encapsulation of nodal segments for propagation and short-term storage of Giloe (Tinospora cordifolia Willd.): a medicinally important plant species. Curr J App Sci Technol 40:15–24 Singh SS, Pandey SC, Srivastava S et al (2003) Chemistry and medicinal properties of Tinospora cordifolia (Guduchi). Indian J Pharmacol 35:83 Singla A (2010) Review of biological activities of “Tinospora cordifolia”. WebmedCentral. Pharmaceut Sci 1(9):WMC0060 Sinha A, Sharma HP (2015) Micropropagation and phytochemical screening of Tinospora cordifolia (Willd.) Miers Ex. Hook. F. & Thoms.: a medicinal plant. Int J Adv Pharm Biol chem 4:114–121

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Sinha K, Mishra NP, Singh J et al (2004) Tinospora cordifolia (Guduchi), a reservoir plant for therapeutic applications. a review 3:257–270 Sivakumar V, Rajan MSD, Sadiq AM et al (2014) In vitro micropropagation of Tinospora cordifolia (Willd.) Miers ex Hook. F. & Thoms - an important medicinal plant. J Pharmacog Phytochem 3:5–10 Sivarajan VV, Balachandran I (1994) Ayurvedic drugs and their plant sources. Oxford and IBH publishing Spandana U, Ali SL, Nirmala T (2013) A review on Tinospora cordifolia. Int J Current Pharma Rev Res 4:61–68 Sriramaneni RN, Omar AZ, Ibrahim SM et al (2010) Vasorelaxant effect of diterpenoid lactones from Andrographis paniculata chloroform extract on rat aortic rings. Pharm Res 2:242 Srivastava (2011) Tinospora cardifolia (Amrita). A miracle herb and lifeline too many diseases. Int J Med Aromat Plant 1(2):57–61 Srivastava V, Chaturvedi R (2018) Optimized micropropagation protocol to establish high-yielding true-to-type plantations of elite genotypes of Tinospora cordifolia for consistent production of therapeutic compounds. Acta Hortic 1212:259–260 Sudan SS, Batra B, Gusain T et al (2020) In vitro propagation of Tinospora cordifolia and estimation of berberine content by Chromatographic analysis. Eco Env Cons 26:S58–S62 Sultana CS, Handique PJ (2013) TDZ enhances multiple shoot production from nodal explants of Tinospora cordifolia-a commercially important medicinal plant species of NE India. Res J Biotech 8:31–36 Sundarraj S, Thangam R, Sreevani V (2012) γ-Sitosterol from Acacia nilotica L. induces G2/M cell cycle arrest and apoptosis through c-Myc suppression in MCF-7 and A549 cells. J Ethnopharmacol 141:803–809 Thippeswamy G, Sheela ML, Salimath BP (2008) Octacosanol isolated from Tinospora cordifolia downregulates VEGF gene expression by inhibiting nuclear translocation of NF- B and its DNA binding activity. Eur J Pharmacol 588:141–150 Tupe M, Pandhure N (2015) In vitro propagation of ayurvedic important plant Tinospora cordifolia (willd.). Miers Int J Recent Trend Sci Technol 15:62–64 Upadhya K, Kumar K, Kumar A et al (2010) Tinospora cordifolia (Willd.) Hook. f. and Thoms. (Guduchi)–validation of the Ayurvedic pharmacology through experimental and clinical studies. Int J Ayurveda Res 1:112 Upadhyay AK, Kumar K, Kumar A, Mishra HS (2010) Tinospora cordifolia (Willd.) Hook.f. and Thoms. (Guduchi)—validation of the Ayurvedic pharmacology through experimental and clinical studies. Int J Ayurveda Res 1:112–121 Vashist N, Drabu S, Nand P et al (2011) Treatment strategies for monkey malaria. Res J Pharm Biol Chem Sci 2:478–487 Wang T, Liu YY, Wang X et al (2010) Protective effects of octacosanol on 6-hydroxydopamineinduced Parkinsonism in rats via regulation of ProNGF and NGF signaling. Acta Pharmacol Sin 31:765–774 Wazir V, Maurya R, Kapil RS (1995) Cordioside, a clerodane furano diterpene glucoside from Tinospora cordifolia. Phytochemistry 38:447–449 Zhao F, He EQ, Wang L et al (2008) Anti-tumor activities of andrographolide, a diterpene from Andrographis paniculata, by inducing apoptosis and inhibiting VEGF level. J Asian Nat Prod Res 10:467–473

2

Medicinally Important Phytoconstituents and Conservation Strategies of Neem: A Critical Overview Kavita Arora and Sangeeta Sen

Abstract

Neem or the Margosa tree, botanically known as Azadirachta indica, Family: Meliaceae, has been one of the most commonly valued traditional medicinal plants, found mostly in India, Nigeria, and the USA. The analysis of its bioactive components reveals its antioxidant, antiallergenic, anti-itch, anti-inflammatory, antihyperlipidemic, antimicrobial, antipyretic, antiglycemic, immunity boosting, hepatoprotective, and many other beneficial characteristics. These medicinal properties have been attributed to the fact that this plant is a rich source of various phytochemicals, which has been validated even by modern medicine. Due to these reasons, neem is found to be highly significant in the global context. Therefore, it becomes extremely urgent to investigate the current updates on neem phytochemistry and its conservation status. This review intends to explore those organic compounds obtained from different plant parts such as leaves, roots, seeds, stem, fruits, flowers and bark, their chemistry, level of abundance, and its usefulness in many disorders including SARS-COV-2. Moreover, this review highlights the multiple conservation strategies employed towards such a potentially rich plant in order to ensure a steady supply of these active chemicals for the near future. Keywords

Azadirachta indica · Bioactive compounds · Conservation · Neem · Phytochemistry

K. Arora (✉) Department of Botany, National P.G. College, Lucknow, Uttar Pradesh, India S. Sen Phd in Plant Biotechnology, Bangalore, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_2

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2.1

K. Arora and S. Sen

Introduction

Neem, botanically known as Azadirachta indica A. Juss, Family: Meliaceae, has been one of the most commonly valued traditional medicinal plants, found mostly in the Indian subcontinent (Wylie and Merrell 2022). It belongs to the Rutales order, Rutinae suborder, subfamily Melioideae, and tribe Melieae (Maji and Modak 2021). Morphologically, it is an arid tropical and subtropical “majestic” evergreen perennial tree, native to India and Myanmar (Singh and Chaturvedi 2013; Singh et al. 2019; Wylie and Merrell 2022). It is also known as the Margosa tree or the Indian Lilac (Maji and Modak 2021). Geographically, its distribution has spread to other countries such as Pakistan, Nepal, Nigeria, Fiji, and the USA along due to the gradual migration of Indians (Singh and Chaturvedi 2013). Conventionally, neem propagation occurs through seeds, which usually starts flowering in 3 to 5 years of birth and can live for multiple centuries. However, the seeds of neem are shortly viable when stored (Singh et al. 2019) and mostly recalcitrant (Rohini et al. 2021). Neem is drought resistant and flourishes well in temperatures ranging from zero degrees to about forty-nine degrees and “sandy stony shallow soils” along with soils having hard clay with a pH flexibility extending from 4 to 10 (Maji and Modak 2021; Khanal 2021). Typically, a neem tree tends to grow up to 30 m in height with a girth of 2.5 m (Gowda et al. 2019). Economically, neem is highly important with multipurpose uses obtained from each and every plant part, starting from timber to ayurvedic properties (Singh and Chaturvedi 2013). Due to its vast medicinal uses, it also called as the “bitter gem,” “noble tree,” “nature’s drug store,” and considered as a “village dispensary” in the Indian subcontinent (Maji and Modak 2021) and “tree of the 21st century” in the USA (UNEP 2012) as well as “tree of forty” in the African belt (Bello et al. 2022). Its medicinal properties have been attributed to the fact that neem is considered to be a potentially excellent source of secondary metabolites with a capability of producing more than 300 such unique phytochemical compounds (Wylie and Merrell 2022). Phytochemicals have been defined as chemicals that are produced by the plants to counter “against fungi, bacteria and virus inflammation, protect from exhaustion of insects and other animals” (Khanal 2021) and “contribute to the colour, aroma and flavor of the plant” (Uwague 2019). The phytochemical screening of the neem extracts from various sources reveals the nature of the bioactive compounds. Table 2.1 summarizes some of the recent studies conducting the qualitative analysis obtained from various neem extracts. The most preferred solvent was observed to be ethanol as it yielded maximum extract when compared with diethyl ether, chloroform, ethyl acetate, and water (Gebremedhin et al. 2020). In a comparative study between leaves, seeds and stem bark, polyphenols, steroids, and tannins were present only in the leaves and stem bark and not in the seeds (Khanal 2021). The quantitative analysis showed that there was hardly much variation in the levels of alkaloids, flavonoids, saponins, and terpenoids within the plant parts. However, it was noted that the neem phytoconstituents varied from place to place (Seriana et al. 2021). This makes commercialization of these products extremely difficult, where consistency and

Dried leaves

Fresh leaves

Leaves, seeds and stem bark Leaves

Fresh leaves

Bark

Leaves

Fresh leaves

3.

4.

5.

7.

8.

9.

10.

6.

Fresh leaves

2.

Nigeria

Nashik, India

Tigray, Ethiopia

Kajhu, Limpok, Indonesia Pakistan

Gaidakot, Nepal

Saliyamangalam, Tamil Nadu, India Khyber Pakhtunkhwa, Pakistan Agbani farm, Nigeria

Ethanol

Methanol

Ethanol

Ethanol

Ethanol

Methanol

Ethanol

Methanol

Methanol

Alkaloids, flavonoids, tannins, phenols, terpenoids, saponins, steroids Terpenoids, flavonoids, phenolic compounds, saponins, tannins Flavonoids, glycosides, alkaloids, steroids

Alkaloids, flavonoids, tannins, saponins, steroids Tannins, glycosides, alkaloids, saponins

Phytochemicals present Tannin, saponin, flavonoids, steroids, terpenoids, triterpenoids, alkaloids, carbohydrates, protein, polyphenol, glycoside Tannin, phlobatannins, Saponin, flavonoids, terpenoids, triterpenoids, alkaloids, polyphenol Alkaloids, carbohydrates, reducing sugars, phenolic compounds, flavonoids, terpenoids, steroids, tannins Alkaloids, saponins, tannins, steroid, terpenoids, glycosides, flavonoids, phenol, oxalic acid Alkaloids, flavonoids, saponins, terpenoids, polyphenols and tannins, steroids

Saponin

Alkaloids and glycosides

Cardiac glycosides, volatile oils

Flavonoids and triterpenes

Terpenoids

Glycosides and coumarins

–

Azhagu (2021)

Steroids, carbohydrates, protein, anthroquinone, glycoside Saponin, glycosides, proteins, and amino acids

(continued)

Seriana et al. (2021) Aziz et al. (2020) Gebremedhin et al. (2020) Kale et al. (2020) Uwague (2019)

Khanal (2021)

Baig and Yousaf (2021) Ujah et al. (2021)

Reference Azhagu (2021)

Phytochemicals absent Phlobatannins, anthroquinone

Solvent for extraction Ethanol

Plant part Fresh leaves

Table 2.1 Phytochemical screening of neem extracts

Sl. 1.

Medicinally Important Phytoconstituents and Conservation Strategies. . .

Location of the plant Saliyamangalam, Tamil Nadu, India

2 23

Plant part Leaves

Leaves

Sl. 11.

12.

Table 2.1 (continued)

Udgir, India

Location of the plant Nigeria

Water

Solvent for extraction Ethanol

Alkaloids, tannins, phytosterols, saponins

Phytochemicals present Flavonoid, anthocyanin, quinones, terpenoids, acids

Phytochemicals absent Alkaloids, saponins, glycosides, cardiac glycosides, phenols, tannins Glycosides, phenolic compounds

Virshette et al. (2019)

Reference Babatunde et al. (2019)

24 K. Arora and S. Sen

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Medicinally Important Phytoconstituents and Conservation Strategies. . .

25

reproducibility become an issue (Kumar et al. 2014). Therefore, phytochemical screening of neem plants should be mandatorily done before extracting these bioactive compounds for commercial purposes. Due to the vast potential of the phytoconstituents of neem, this review intends to focus on the pathways of phytochemical synthesis of important bioactive compounds obtained from neem, their overall therapeutic uses, bioactivities of its extracts, and finally the strategies towards conservation of neem.

2.2

Phytochemical Synthesis of Important Bioactive Compounds and Related Pathways in Neem

Among these 300 or more identified secondary metabolites obtained from neem, the prominently identified bioactive compounds include azadirachtin, nimbolinin, nimbanene, gedunin, nimbin, nimbolide, nimbandiol, salanin, tignic acid, sitosterol, valassin, and ascorbic acid (Maji and Modak 2021; Saravanan 2022; Srivastava et al. 2020), most of which are terpenoids (Kumar et al. 2014). Besides these, other bioactive isolates obtained from neem include catechin, 6-desacetylnimbinene β-sitosterol, epicatechin, n-hexacosanol, non-acosane, and quercetin (Paul et al. 2021). The phytochemical synthesis of the most important bioactive compound and their related pathways (if known) in medicinal plants has been elaborated below. In case of azadirachtin, the chemical synthesis has been found to be extremely challenging and has taken 22 years to understand a total of 71 steps that yield 1.5 × 10-5 percent of the compound only (Jauch 2008; Rangiah and Gowda 2019). Squalene, the precursors of azadirachtin come from the mevalonate pathway (Rani and Akhila 1994), which gets cyclized by terpene synthase enzyme to form Euphol or Tirucallol. In the next step, these are further allylic isomerized to gain a furan ring to form butrospermol. This further oxidizes and undergoes rearrangement through a Wagner-Meerwein 1,2-methyl shift to form Apoeuphol and apotirucallol. Thereafter, four carbons are cleaved from the side change to form a limonoid, which further gets cleaved of ring C to eventually form azadirachtin. Figure 2.1a summarizes the probable pathway for the biosynthesis of azadirachtin along with limonoids as described by Kumar et al. (2014). However, recent studies by Wang et al. (2020) still claim that there is uncertainty in the pathway related to biosynthesis of azadirachtin, therefore, forms the basis for enhancing focus on these processes. Alternatively, it was construed that squalene undergoes epoxidation to form 2,3-oxisqualene, which undergoes transformation by the 2,3-oxidosqualene cyclase to form a compound, which further undergoes demethylation once, dual oxidations, and three hydroxylations, that finally changes from azadirone to form azadirachtin A (Fig. 2.1b). Coming to the synthesis of other bioactive molecules, almost all the bio-constituents such as nimbin, a tetranortriterpenoid, and many more are formed as intermediates of the azadirachtin pathway. Further, gedunins are formed due to the oxidation of the D-ring in the azadirones such as nimbinin, nimbicinol, etc. (Kumar et al. 2014). Another study shows that the biosynthesis pathways of gedunin starts with tetranortriterpenoid; the details of which are explained in Fig. 2.2.

26

K. Arora and S. Sen

Fig. 2.1 Biosynthesis of azadirachtin. (Source: a: Based on Kumar et al. 2014; b: Wang et al. 2020)

Fig. 2.2 Biosynthesis of gedunin. (Source: Braga et al. 2020)

2.3

Bioactivity of Neem Extracts

The use of neem has been reported a long time ago back in 2500 BC (Srivastava et al. 2020). Since its discovery, neem trees are known for their restorative bioactivities that have reached common households as a source of medicines as well as a nontoxic biopesticide (Adhikari et al. 2020; Azhagu 2021; Wylie and Merrell 2022). The mode of administration of neem extract has primarily been oral (Aggarwal and Shishu 2011). According to the traditional Ayurvedic medicinal system, the medicinal usefulness of neem extends to its multiple benefits exhibiting antacid, antidiarrheal, antioxidant, antiallergenic, anticancer, antidermatitic, anti-itch, anti-inflammatory, antihyperlipidemic, antimicrobial, antipyretic, antiparasitic, antiglycemic properties, antiulcer as well as immunity boosting, contraceptive, hepatoprotective, and many

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Medicinally Important Phytoconstituents and Conservation Strategies. . .

27

Table 2.2 Bioactivity of neem based on the type of plant part (Source: Maji and Modak 2021; Azhagu 2021; Srivastava et al. 2020) Sl. 1. 2. 3.

Plant part Twigs Barks Leaves

4. 5.

Flowers Seeds

Bioactivities Oral deodorant, teeth cleaner, relieving tooth aches, reduce swollen gums Antiallergenic, antidermatitic, antifungal, antiprotozoal, antitumor, antimalarial Anticlotting, anticholinergic, antiseptic, antiemetic, antifungal, antihelminthic, antituberculosis, antitumor, antiviral, insecticidal, nematocidal, epidermal dysfunctions, wound healer, antidandruff, anti-irritation, anti-itching, antieczema, immunity boosting Analgesic, stimulant, treat anorexia, nausea, contraceptive Analgesic, anticholinergic, antifungal, antihelminthic, antihistaminic, antiprotozoal, antipyretic, antiviral, insecticidal, bactericidal

other beneficial characteristics (Jahan et al. 2021; Seriana et al. 2021; Wylie and Merrell 2022). The antibacterial activity has been found to be strong even for antibiotic resistant strains including Enterococcus faecalis, Escherichia coli, Helicobacter pylori, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, Shigella flexneri, Staphylococcus aureus, Streptococcus mutans, and Vibrio cholerae (Wylie and Merrell 2022). Additionally, neem extracts act against viruses such as dengue, herpes, influence, encephalitis virus, human immunodeficiency virus (HIV), hepatitis, and severe acute respiratory syndrome coronavirus disease (SARS-COV-2), against fungus such as Aspergillus flavus, A. niger, Candida albicans, and C. tropicalis and against other parasites such as Plasmodium falciparum, Leishmania donovani, and Schistosoma mansoni. Along with this, it has also been found to display antiarthritic, diuretic, antimalarial, and spermicidal effects (Paul et al. 2021). In a preliminary study, a prophylactic role towards COVID was observed in individuals who were given capsules of neem (Nesari et al. 2021). Moreover, extracts from neem leaves also have protective properties specific to the liver, kidney, heart, and nervous system along with treating any inflammation in the skin, psoriasis, or break out of the epidermis (Azhagu 2021). It has been found to be involved in immunomodulation and reduce the formation of malignant growths. Additionally, neem extracts have been found to repel dandruffs (Gebremedhin et al. 2020) and reduce serum cholesterol and hypertension (Srivastava et al. 2020). Hardly, any adverse effects due to neem have been observed in humans (Wylie and Merrell 2022). Besides these direct benefits to humans, neem has been successfully employed as broad-spectrum herbal pesticides, that is extremely effective against plant pests such as whitefly and aphids affecting the okra and potato cultivation (Baig and Yousaf 2021) and also with worms and vectors of jungle fever (Saravanan 2022). The bioactivity has been observed from almost all the body parts of the matured tree, including twigs, barks, leaves, roots, gum, flowers, fruits, and seeds (Maji and Modak 2021; Khanal 2021). The medicinal properties vary depending upon the type of body parts of neem plant as explained in Table 2.2. Highest bioactivity was obtained from the seeds, and thus have been further employed as a feed for animals,

28

K. Arora and S. Sen

or used as a soil fertilizer and a neutralizer because of its antimicrobial properties (Wylie and Merrell 2022). Table 2.3 elaborates on the bioactivity of neem extracts depending upon the phytochemicals and their mode of action. Overall neem extracts act by restricting the life processes crucial for the viruses by obstruction the entry of cells and hindering their replication (Wylie and Merrell 2022). In case of HIV, neem extracts tend to provide protection to the T cell population, decrease any activation of immunity and to neutralize any toxicity of the antiretroviral drugs that are being taken by the patients. The administration of neem orally also reduces the peripheral sugar, which leads to reduced need for insulin (Paul et al. 2021). The insecticidal properties are primarily due to the active chemical compound called “azadirachtin” present in various plant part extracts (Saravanan 2022). It acts as a wide spectrum insecticide by inducing sterility in male insects by meddling with the sperm formation. It also targets the mouthparts, other chemoreceptors, guts, cuticle, muscles, dividing cells, and the machinery for cell synthesis (Adhikari et al. 2020). Along with this, limonoids are known to cause severe irritation in the insects, thereby forcing neem to become a bio-repellant (Saravanan 2022). Other products such as chlorogenic acid, kaempferol and its derivative, querticin, and myricetin contribute to antioxidation and anti-inflammation properties, while Rutin causes antihyperglycemia (Maji and Modak 2021). Similarly, compounds extracted from bark such as gallic acid, epicatechin, catechin, and peptidoglycan induce immunomodulatory and anti-inflammatory properties, whereas flavonoids and phenols cause oxidation and polysaccharides have antitumor effect. Similarly, gedunin, mahmoodin, margalone, margolonone, isomargolonone, flavonoids, saponins, and tannins fight off microbes.

2.4

Conservation Strategies of Neem

As discussed before, neem is a perennial tree that bears fruits within 3 to 5 years of plantation and lives for more than few decades (Gowda et al. 2019). Traditionally, it is cultivated either from germination of seeds, sprouts, root suckers, and other approaches in the Indian subcontinent. However, the seeds are known to lose their viability quite early, thus have short life (Singh et al. 2019). Moreover, heterozygosity leads to variation in the azadirachtin content (Bello et al. 2022). Due to its medicinal omnipotence, versatile nature, and pharmaceutical industrial over use, neem requires conservation (Agasimundin et al. 2019). Conservation of bio/phytodiversity is a “holistic approach” which “involves both in situ and ex situ methods of conservation” and typically depends upon the type of plant in question (Sharma et al. 2012). Each conservation strategy has its own merits and constraints. Figure 2.3 illustrates the various conservation methods specifically employed for neem. Each strategy in the context of neem has been elaborated below.

Nimbin (Meliacin)

Nimbolide (Terpenoid)

Meliacinolin (Trinortriterpenoids)

Gedunin (tetranorterpenoids)

Nimbandiol (Pentanortriterpenoid) Nimbolide

Limonoids Glycolipid sulfonoquinovosyldiacylglyceride (SQDG) Nimbidin (mixture of tetranortriterpenes)

2.

3.

4.

5.

6. 7.

8. 9.

10

Name and class of the compound Azadirachtin (Meliacin)

Sl. 1.

Seeds

Seeds Leaves

Leaves Seeds

Seeds

Leaves

Oil of seed kernels, leaves Seeds

Source Leaf

Table 2.3 Bioactivity of compounds from neem extracts

Hypo-glycemic, antiarthritic, antigastric, antifungal, antibacterial, diuretic, antipyretic, spermicidal, anti-inflammatory

Antiplasmodial Antiviral against herpes virus, antihelminthic

Antituberculosis, antimalaria Antibacterial

Vasodilator, antitumor, antimalarial, antidiabetic activity, antifungal, anticancer, neurosupport

Antimalarial, antioxidant, antibacterial Antidiabetic

Bioactivity Anticerebral malaria, antioxidant, anti-inflammatory Antihistamine, antiulcer, antifungal, antidengue, antioxidant, spermicidal

Antifeedant Binds covalently with E3 ligase Block early sporogenesis Inhibit the synthesis of nucleic acids, restrict binding of glycoproteins Function suppression of macrophages and neutrophils

Inhibits α-amylase and α-glucosidase Inhibition of salivary and pancreatic amylase

Antiradical hunting

Blocks the protease and envelope protein development

Mode of action Binds to Gephyrin E

Maji and Modak (2021)

Tapanelli et al. (2016) Wylie and Merrell (2022)

Aarthy et al. (2018), Herrera-Calderon et al. (2019), Mazumdar et al. (2020) Lin et al. (2021) Blum et al. (2019)

Kumar et al. (2018), Maji and Modak (2021) Paul et al. (2021)

Paul et al. (2021), Shanmugam et al. (2020)

Reference Okoh et al. (2018)

2 Medicinally Important Phytoconstituents and Conservation Strategies. . . 29

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K. Arora and S. Sen

Biosphere Reserves

National Parks

In situ

Wildlife Sanctuaries

Home gardens

Sacred Grooves Conservation strategies Botanical Gardens Cryopreservation

Herbal gardens Ex situ

Shoot culture Seed Banks Somatic embryogenesis In vitro approaches Synthetic seeds

Excised root cultures

Fig. 2.3 Conservation strategies common to neem

2.4.1

In Situ Conservation of Neem

In situ conservation strategies commonly include biosphere reserves, national parks, wildlife sanctuaries, sacred groves, and other protected areas where plants are grown in their natural habitats (Sharma et al. 2012). Since this plant is endemic to Indian subcontinent, therefore, neem trees are commonly found all over India and neighboring areas (Gowda et al. 2019). However, proper cataloging of any of these trees has not been done officially; therefore, the actual numbers are not known. There are only some sporadic studies reporting any in situ conservation of neem across time. Review of these literature construes about the collection of neem trees harboring endophytic fungi in leaves from Pachamarhi Biosphere Reserve located in Madhya Pradesh, India (Tenguria and Khan 2011). Outside India, Lawachara National Park, located in Bangladesh, has grown neem plants along with other plants and used them in traditional practice of medicine (Uddin et al. 2012). Apart from medicinal purposes, Singra National Park also from Bangladesh has cultivated neem trees for afforestation and reforestation purposes (Ali et al. 2020). Coming to wildlife sanctuaries, Hazarikhil Wildlife Sanctuary of Chittagong in Bangladesh has reported the cultivation of neem plants along with other rare and endangered species (Rahman 2017). Similarly, a network of 13 protected areas in

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different districts of Punjab, India, has been established that gives shelter to neem (Dhami 2018). Even though, there are still sparing reports on neem plants growing in biosphere reserves, wildlife sanctuaries, and national parks located either in India or abroad, cataloging of each neem tree can be helpful in its germplasm protection. However, one of the less documented methods of in situ conservation applied for neem is through maintenance of locally available plants in home gardens (Tiwari et al. 2017; Shrestha et al. 2002). Apart from these, there are sacred groves, which are relic forest patches that have been preserved in the name of religion and culture. These are considered as “Sacred natural sites” or sacred groves by International Union for Conservation of Nature (IUCN) (Oviedo and Jeanrenaud 2007). In India, several such sacred groves have been developed since ancient times especially with the Gujjar community of Rajasthan, where neem trees are worshipped as “God Devnarayan” (Singh 2016). The Kota district in Rajasthan witnesses such practice that has led to the existence of about 70,000 neem trees from different age groups in the area (Agarwal 2016). In a recent note, Dey et al. (2022) documented about a single tree from localities such as Madhurpur and Bankura, rural areas of West Bengal being conserved as culturally protected site. From all this, it can be implied that in situ conservation requires development of protected areas with appropriate maintenance measures. Absence of this causes many practical problems to arise in maintaining these areas. Some of the common ones are risk of pathogen attack, occurrence of natural calamities, anthropogenic disturbances, increased costs, and issues pertaining to maintenance (Possiel et al. 1995; Sharma et al. 2012).

2.4.2

Ex Situ Conservation

Ex situ conservation involves “off-site conservation” and includes botanical gardens, arboreta, herbal gardens, seed banks, pollen banks, DNA banks, and other biotechnological approaches such as in vitro banks (Sharma et al. 2012). Neem, because of its medicinal properties and other utilizations, is considered as an appropriate candidate to be grown in botanical and herbal gardens. It is a common practice for various institutes to maintain in-campus botanical as well as herbal gardens. The Forest Botanic Garden in State Forest Research Institute (FRI), Jabalpur, Madhya Pradesh, India, has a collection of neem plantations raised through tissue culture (Chaubey et al. 2015). Along with this, a recent report shows neem being cultivated in herbal gardens of Dayal Bagh Educational Institute (DEI), Agra, Uttar Pradesh, India (Prasad et al. 2019). Since the neem seeds lose viability within days of collection (Maithani et al. 1989; Sacandé et al. 1998), therefore, several attempts have been made for the storage of viable neem seeds. Nayal et al. (2000) showed that seeds observed a 20-fold increase in longevity, when dried to 4.6% moisture content over seed: silica gel (1:1) at 15 ° C. Under these conditions, maximum half viability period of 663 days was achieved. Moreover, moisture content and temperature also played a key part in the germination process; germination percentage was observed to be 86% after desiccation. In a separate study, Neya et al. (2004) mentioned in their report that

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the aging of neem seeds is related to imbibitional stress and damages. Seeds were stored for 10 weeks and were compared with seeds stored for 10 months for imbibitional damage at 4% as well as 7% moisture content. Seeds stored for 10 months at 7% moisture content were sensitive to imbibitional stress and got affected more with 4% moisture content. More studies were oriented towards the extension of period of storage as well as to enhance germination percentage after storage (Sacandé and Hoekstra 1999; Kumar et al. 2014). Singh et al. (2019) reported increased effectiveness of seed germination by providing pre-sowing treatments through seed priming. It was observed that the best germination percentage was obtained when the seeds were stored at 10 ° C temperature (43.3%), followed by seeds which were coated with gum (41.7%). Storing the seeds between 10 and 20 ° C temperature was the best treatment to keep the seeds viable even after 40 days compared to other methods, such as coating the seed with wax, clay, or wet storing, or sun drying. Among the in vitro approaches of conservation, most widely used methods that have been successful for neem include cryopreservation, shoot cultures, synthetic seeds, and use of excised root cultures. Since heterozygosity leads to variation in azadirachtin content, therefore, clonal propagation is recommended for neem (Padilla et al. 2021). Cryopreservation is well known in case of neem as a means of long-term conservation, since the first successful report of Chaudhury and Chandel (1991), where the desiccated seeds were stored at 196 °C. Similarly, seeds (Varghese and Naithani 2008) and embryonic axes (Malik and Chaudhury 2019) have been used for cryogenically storing neem. Moreover, National Bureau of Plant Genetic Resources (NBPGR), New Delhi, India, presently has 2612 neem accessions with them stored in cryobanks obtained successfully using zygotic and embryonic axes (Okoh et al. 2018). Uniform uninfected seeds were desiccated by mixing seeds and silica gel (1:20 w/w) in a glass desiccator. Highest survival of 94–96% was achieved during the first month of cryostorage and thereafter germination decreased. Some of the limitations that were observed include a reduction in seed germination percentage when seeds were cryopreserved up to 12 months (Varghese and Naithani 2008). Post-thawing and pre-heat treatment showed little enhancement in the seed recovery percentage. Coming to a latest 2022 report, synthetic seeds of neem have been developed by encapsulation of shoot tips taken from in vitro grown plantlets (Kader et al. 2022). Such seeds were stored at 4 and 24 °C with results showing that the seeds at 24 ° C had more germination frequency and regeneration potentiality in all storage periods that varied from 15 to 120 days. In another report, in vitro derived nodal segments were encapsulated and stored at 4, 8, and 12 °C for 4 weeks (Padilla et al. 2021). The sprouting of encapsulated nodal segment stored at 12 °C increased from 20% to 75% when these segments were incubated with acetylsalicylic acid for 4 weeks prior to encapsulation. The best hydrogel complexation was observed when 3% sodium alginate and 75 mM calcium chloride were used for a period of 20 min (Kader et al. 2022). These results were compared with the findings of Padilla et al. (2021). In their study, Kader et al. (2022) obtained germination (ca. 77%) after 120 days

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storage as against obtained by Padilla et al. (2021) where germination remained completely inhibited even after 4 weeks. Field survival has not been reported by Padilla et al. (2021); however, Kader et al. (2022) have mentioned field survival to be 85.9% which is quite high and desirable for the success of establishing a conservation protocol. Additionally, successful cyclic somatic embryogenesis has been achieved by Srivastava and Chaturvedi (2018), and these somatic embryos have been utilized for production of synthetic seeds. For the establishment of shoot cultures of neem, almost every vegetative part has been used as an explant (Win and Ko 2020). A summary of the best media practices used for shoot regeneration from different explants is presented in Table 2.4. This includes nodal stem segments (Chaturvedi et al. 2004; Bello et al. 2022; Dhandapani et al. 2021; Arora et al. 2010), leaf/leaflet segments (Ramesh and Padhya 1990; Arora et al. 2009; Phukan et al. 2017), root segments (Arora et al. 2011), shoot tips (Bello et al. 2022), unpollinated ovary (Srivastava et al. 2009), etc. Along with these, seeds and seedling explants, like cotyledons and immature zygotic embryo have been utilized for raising in vitro shoot cultures (Dhandapani et al. 2021). Within the studies, Arora et al. (2009) reported the best medium for leaflet was found to be a modified Murashige and Skoog (Murashige and Skoog 1962) medium supplemented with cytokinin, Benzyl amino purine (BA). To control the regeneration pathway, a pulse treatment for 5 days was given in a medium containing BA (8.88 μM) and adenine hemi-sulfate (ADS, 81.43 μM). This was followed by transferring the explants in one tenth concentration of 0.88 μM BA and 81.43 μM of ADS. The results demonstrated an average number of 17.4 shoot buds per leaflet formed due to the shoot proliferation medium which comprised of modified MS supplemented with BA (1.11 μM), IAA (Indole-3-acetic acid; 1.43 μM), and ADS (135.72 μM). A hundred percent rooting of these explants could be attained in modified MS medium supplemented with indole butyric acid (IBA 2.46 μM). After rooting, survival of plants in pots was reported to be 100%. The maximum regeneration occurred from the basal part, while the segment of leaflet was least in the upper part. Such reporting is not there in case of neem except Ramesh and Padhya (1990) where leaf discs were suggested promoting regenerant differentiation. In a separate study, the comparative regenerant differentiation capacity of different vegetative organs was assessed (Arora unpublished data; Fig. 2.4). Modified MS medium was used for regenerant differentiation which was supplemented with different cytokinins. Among the used cytokinins, BA along with ADS was found to be most conducive for leaf, while BA along with IAA and ADS was employed for stem. Along with this, a combination of BA, 2-isopentenyladenine (2iP), IAA, and ADS was used for root explants. Through these treatments, minimum intervening callus formation with a moderate number of differentiated regenerants was observed. Results showed that the leaflet had maximum regenerant capacity (with an average of 7.82 regenerants per explant) followed by stem (average 5.75 regenerants per explant), and root (average 3.5 regenerants per explant). Shoot proliferation and rooting was done following the protocols given in Arora et al. (2010). It can be implied that the use of more regenerative explants manifolds the rate of multiplication. Similar results showing variation in regeneration capacity of different explants have been reported by Bello et al. (2022). When

Explant source Lateral and terminal buds

De-embryonated, cotyledon and nodal segments (matured tree)

Leaf

Leaf and seed

Nodal segments (matured tree)

Root segments (in vitro grown plantlets)

Sl. 1.

2.

3.

4.

5.

6.

Mod. MS + BA (8.88 μM) + 2iP (9.84 μM) + IAA (5.71 μM) + ADS (81.43 μM) + Pu (2.27 μM) for 2 days followed by transfer to 1/10th of BA+2iP + IAA + ADS (81.43 μM) + Pu (2.27 μM)

Callus induction For Leaf MS medium+2, 4-D (0.3 mg/ l) + Kn (0.3 mg/l) + NAA (0.3 mg/l) For seed MS medium + 2, 4-D (0.5 mg/ l) + BA (0.5 mg/l) MS + BA (8.88 μM)

CB + NAA + BA (in different concentrations)

Establishment medium MS+ BA (0.1 mg/l) + IBA (0.01 mg/l) 1/2 MS + BA (3 mg/l) + NAA (0.5 mg/l) + CH (1 g/l)

Table 2.4 In vitro raised shoot cultures employed for conservation of neem

Mod. MS + BA (1.11 μM), IAA (1.43 μM) + ADS (135.72 μM)

MS + BA (4.44 μM)

MS medium + BA (1.0 mg/l) + NAA (0.5 mg/l)

1/2 MS + BA (1.5 mg/l) + NAA (0.5 mg/l) + CH (400 g/l) CB+ NAA (105 M) + BAP (10-6 M)

Shoot proliferation medium MS + BA (0.7 mg/l)

MS + Tryp (146.89 μM) Mod. MS + IBA (2.46 μM)

CB + NAA + BA (in different concentrations) 1/2 MS medium + NAA (3 mg/l)

Rooting medium LS+ IBA (4.0 mg/ l) 1/2 MS+ IBA (2 mg/l)

Transplanted but data not given

Gehlot et al. (2014) Arora et al. (2011)

Prithviraj et al. (2019)

87.5%

N.M.

Win and Ko (2020)

Reference Bello et al. (2022) Dhandapani et al. (2021)

N.M.

N.M

Field success (% survival) 80%

34 K. Arora and S. Sen

Leaflet segments (in vitro grown plantlets)

Unfertilized ovaries

Nodal segments from mature (15-year-old) trees and greenhouse-grown juvenile (1.5-yearold) seedlings

8.

9.

10.

Mod. MS + BA (8.88 μM) + ADS (81.43 μM) for 5 days followed by transfer to BAP (0.88 μM) + ADS (81.43 μM) Callus induction MS (9% sucrose) + 2, 4-D (1 μM) + BA (5 μM) Mod. MS + BA (2.0 mg/l), IBA (0.3 mg/l)

Mod. MS + BA (1.11 μM) + IAA (1.43 μM) + ADS (81.43 μM)

MS + BA (1 μM) + CH (250 mg/dm3) MS + BA (1 mg/l) + NAA (0.5 mg/l)

Same as establishment medium + ADS (135.72 μM) Mod. MS + BA (1.11 μM), IAA (1.43 μM) + ADS (135.72 μM) N.M.

81.1%

72%

¼ MS + IBA (0.5 μM) ½ MS + IBA (2 mg/l)

100%

-do-

Mod. MS + IBA (2.46 μM)

Srinidhi et al. (2008)

Srivastava et al. (2009)

Arora et al. (2009)

Arora et al. (2010)

2iP 2-isopentenyl, 2,4-D 2,4-dichlorophenoxy acetic acid, ADS adenine hemi-sulfate, BA benzyl adenine, CH casein hydrolysate, IAA indole-3-acetic acid, IBA: indole-3-butyric acid, Kn Kinetin, LS Linsmaier and Skoog (1965) medium, Mod. Modified, MS Murashige and Skoog, NAA naphthalene acetic acid, N.M. not mentioned, Pu putrescine, tryp tryptophan

Nodal stem segments (mature tree)

7.

2 Medicinally Important Phytoconstituents and Conservation Strategies. . . 35

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K. Arora and S. Sen

Fig. 2.4 Relative regenerant differentiation potentiality of different vegetative organs taken from in vitro grown plantlets. (a) Leaf segment; (b) Stem segment; (c) Root segment (Source: Arora, unpublished)

the regeneration potentiality of de-embryonated cotyledon, immature zygotic embryo, and nodal segments from a 30-year-old neem plus tree was compared, it was found that the de-embryonated cotyledon was more responsive compared to others. Moreover, the success rate in this method also depended on the choice of medium at proliferation stages and at the rooting stage. The medium used by most of the workers for initiation of shoots for bud break at the time of establishment was the Murashige and Skoog (1962) medium (Srivastava et al. 2009; Bello et al. 2022), whereas modified MS medium or its half strength was found to be best for shoot initiation (Arora et al. 2009, 2010, 2011; Dhandapani et al. 2021). As reported by Arora et al. (2010), nodal stem segments (obtained from 40 years old tree) were found to be good explants for establishment of neem cultures. Moreover, the effect of position of node on bud break was observed where the third and fourth node from tip (middle order nodes) were found to be more responsive. Such details were not provided in the earlier reports. Goodness of nodal segments as explants has been assured by other workers also (Chaturvedi et al. 2004; Srinidhi et al. 2008). The best month for explant collection was observed to be between March to May (Dhandapani et al. 2021). During hardening, the plantlets were exposed to half strength Knop’s Solution (Knop 1865) fortified with trace elements and iron of MS medium for the first time. These plants were then transplanted to soil: manure (3: 1) medium in pots and hardened in glasshouse. Srinidhi et al. (2008) suggested

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vermiculite as the best potting mix and reported a 72% survival in pots while 80% survival was observed by Bello et al. (2022). A far better field survival success of 100% was achieved by Arora et al. (2010). Further, performance in the field was observed for two years and then samples were subjected to molecular analysis through. RAPD, which ascertained the genetic fidelity of plants. Dhandapani et al. (2021) reported sand:soil:vermiculite (1:1:1) as the best potting mix for transfer till greenhouse only; however, there was no mention of the transplantation percentage. Along with this, neither field transplantation nor field performance after transplantation was mentioned in the earlier reports (Chaturvedi et al. 2004; Bello et al. 2022). Previous reports did not follow-up their protocol with any further molecular analysis (Srinidhi et al. 2008; Bello et al. 2022). In a separate study, Padilla et al. (2015) tested the effect of several growth retardants on the sprouting and development from nodal buds of neem and conducted histological analysis for meristem development. The recovery was found to be good only from acetylsalicylic acid (ASA) at low concentrations (25 μM). This study proved that these growth retardants can be suitable for the purpose of slow growth culture in neem. One of different regeneration pathways include the induction of somatic embryos (SE), which can also potentially be used as a method for ex situ conservation as these are useful for the production of synthetic seeds or can be maintained as cultures. Due to the disadvantages faced in synthetic seeds, these are not worth storing for longterm purposes, thereby defeating the purpose of germplasm conservation through seed banks. Somatic embryogenesis as an alternative provides a way for long-term preservation. It may occur as an embryogenic calli or through somatic embryos (Singh and Chaturvedi 2013). Table 2.5 summarizes the studies reporting somatic embryogenesis in neem. Phukan et al. (2017) used leaf and stem explants collected from a young plant of 2–3 months old. Leaf was found to be more responsive for somatic embryo formation. About 180 somatic embryos were formed in 35 days when incubated in medium supplemented with 1.0 mg/l BA. However, somatic embryos on germination were found to be monopolar. Thus, only shoots could be regenerated and thereafter rooting was obtained in MS medium supplemented with 1.0 mg/l IBA along with 0.1 mg/l IAA. With the aging of callus, the number of somatic embryos increased till third subculture in which maximum numbers (180) were obtained after which the number of somatic embryo formation reduced. There was no mention about field transplantation or survival for the plants by this study. Ramirez and Fernández Da Silva (2018) also reported induction of somatic embryos in leaf and cotyledon explants. Non-embryogenic calli developed in leaf explants as against cotyledon where calli were embryogenic. Best somatic embryo induction was observed in higher concentration of BA (2.5 mg/l) with production of 77% of primary embryos. In consonance with results of Phukan et al. (2017), it was observed that as incubation period increases, the embryogenic response also increases till the cultures are 8 weeks old. Further, rooting was done on ½ MS without any auxin; these findings were contrary to the findings of Phukan et al. (2017). Here also, no field data was provided by the researchers. However, Shekhawat et al. (2009) observed a high level of field success.

Leaf, nodes, and roots

6.

For leaf: Callus induction Mod. MS + TDZ (2.3– 4.5 μM) + 2,4-D (0.5 μM)

MS + KN (1.5 mg dm3) + IAA (1.5 mg dm-3) MS + BA (1.11 μM) + 2,4D (4.52 μM)

Callus induction: MS + BA (2.5 mg/l) MS + BA (0.5 mg/l)

Same as Proembryogenic calli + 2,4-D (0.45 μM) For leaf, nodes, and roots: Same as Proembryogenic calli

N.M

N.M

–

For leaf, nodes, and roots: Mod. MS + BA (2.2 μM) + GA3 (0.3 μM) [Bipolar]

80– 83.5%

N.M

N.M

–

–

1/2 MS + one time vermiculite + Mod. nutrient medium MS + BA (1.0 mg/ l) + IAA (0.1 mg/l)

Medium for rooting –

½ MS (with 2% sucrose) + 0.94 μM ABA [Bipolar]

MS + BA (2 mg/l) + IAA (0.1 mg/l) [Monopolar-shoot] ½ MS [Bipolar]

–

–

For leaf: MS + BA (0.5 mg/l) for stem: MS+ BA (1 mg/l) Same as Proembryogenic calli

Medium for germination 1/2 MS + TDZ (0.2 mg/l) [Bipolar]

Medium for somatic embryo induction Same as Proembryogenic calli

Field success (% survival) N.M

ABA abscisic acid, TDZ: thidiazuron, GA3 gibberellic acid, 2,4-D 2,4-dichlorophenoxyacetic acid, Mod. Modified, MS Murashige and Skoog

Immature zygotic embryos

Leaf and stem (2–3-monthold seedling) In vivo leaflets

Explant source Immature zygotic embryos Leaves and cotyledons

5.

4.

3.

2.

Sl. 1.

Medium for proembryogenic calli/mass 1/2 MS + TDZ (0.2 mg/l)

Table 2.5 Somatic embryogenesis: an in vitro approach employed for conservation of neem

Akula et al. (2003)

Rout (2005)

Shekhawat et al. (2009)

Ramirez and Fernández Da Silva (2018) Phukan et al. (2017)

Reference Dhandapani et al. (2021)

38 K. Arora and S. Sen

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An innovative approach of germplasm preservation was developed in the form of regenerative excised root cultures for neem (Sharma et al. 2012). In this method the excised root is cultured as a system for germplasm preservation. Some of the merits of this method include the use of simple incubation conditions in moderate temperature (25°-35 °C) which is unaffected by temperature fluctuations, no requirement for light, low maintenance cost, long intervals between subcultures which can be extended to even 4 to 6 months, economy of space as several meters long roots can be accommodated in small containers, and potential for producing enormous propagules (clonal plants) per culture of roots. All these aspects make this method superior to other approaches that have been discussed so far. This system allows safe exchange of germplasm over long distances across the international boundaries unaffected by lack of light and temperature fluctuations during transit, which has otherwise been found to be extremely damaging for shoot cultures that are used for exchange of germplasm. In case of neem, it was established that by using root segments taken from in vitro grown plantlets, 2 years old root cultures were established by growing them in modified MS medium (pH 5.2) supplemented with IBA (0.05 μM). Like leaflets, here also pulse treatment of 2 days was effective for direct regenerant differentiation. A higher concentration of BA, 2iP, IAA along with adenine sulfate (81.43 μM) and Putrescine (2.27 μM) was proven to be effective for the formation of shoot buds. The regenerant differentiation capacity of root segments was found to be low as compared to leaflets (Arora et al. 2011). Since roots preserved for 2 years were employed as excised root cultures that were shown to be regenerative, therefore, this method holds future scope for long-term preservation in neem.

2.5

Summary

Over the years, neem has become one the most enterprising instruments in pharmacology showing nontoxic therapeutic impacts recognized not only in India but also in the entire globe. Due to the numerous positive bioactive properties of neem, it has been isolated, measured, and recognized by many researchers. Increasingly, it has become an indispensable part of the agricultural industry, pharmaceutical industry, and pesticide industry. Alongside, neem also finds its forthcoming applications in the treatment of cosmetics (Padilla et al. 2021), gastrointestinal problems, dentistry, and food industry (Wylie and Merrell 2022). In dentistry, neem extracts form ingredients for mouthwashes, toothpastes, and irrigants required post root canal treatment. Extracts from neem leaves are being incorporated into “alginated fibers” which are applied for dressing the wounds (Hussain et al. 2017). Along with this, it has also been used as a “natural cleanser” that restores any skin lesions (Azhagu 2021). It offers an answer to existing diseases, ecological disturbances but also possess potential for future problems may it be related to health or environment arising due to modern lifestyles. Therefore, neem being the green gold of India, need special attention in terms of phytoconstituent availability and conservation. The future of neem lies in nanoparticles as these are “untapped source of novel

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K. Arora and S. Sen

therapeutics” (Wylie and Merrell 2022) and should be used as bioactive compounds in synergy with the traditional antibiotics. Efforts to develop effective conservation strategies for neem are lacking as none of the methods ensures long-term conservation. The excised root culture holds some promise in this regard. Moreover, cryopreservation and seed storage longevity of storage and post storage germination need to be worked upon. Along with this, special focus needs to be given to enhance the success rate of the tissue-cultured plants to the field.

References Aarthy T, Mulani FA, Pandreka A, Kumar A, Nandikol SS, Haldar S, Thulasiram HV (2018) Tracing the biosynthetic origin of limonoids and their functional groups through stable isotope labeling and inhibition in neem tree (Azadirachta indica) cell suspension. BMC Plant Biol 18(1):1–21 Adhikari K, Niraula D, Shrestha J (2020) Use of neem (Azadirachta indica A. Juss) as a biopesticide in agriculture: a review. J Agric Appl Biol 1(2):100–117 Agarwal M (2016) Conserving water & biodiversity: traditions of sacred groves in India. Eur J Sustain Develop 5(4):129. https://doi.org/10.14207/ejsd.2016.v5n4p129 Agasimundin VB, Rangiah K, Sheetal A, Gowda M (2019) Neem microbiome. In: Gowda M, Sheetal A, Koole C (eds) The neem genome. Springer, Cham, pp 111–123 Aggarwal N, Shishu (2011) A review of recent investigations on medicinal herbs possessing antidiabetic properties. J Nutrition Disorder Ther 1(102):2 Akula C, Akula A, Drew R (2003) Somatic embryogenesis in clonal neem, Azadirachta indica A. Juss. and analysis for in vitro azadirachtin production. In Vitro Cell Develop Biol Plant 39: 304–310. https://doi.org/10.1079/IVP2003415 Ali MM, Akter N, Kabir MR, Hasan MM, Rahman MM, Bari MS (2020) The biodiversity status and conservation activities of Singra National Park (SNP) in the link of co-management strategy. Int J Environ Clim Change 10(10):136–146. https://doi.org/10.9734/ijecc/2020/ v10i1030256 Arora K, Sharma M, Sharma AK (2009) Control of pattern of regenerant differentiation and plantlet production from leaflet segments of Azadirachta indica A. Juss.(neem). Acta Physiol Plant 31(2):371–378. https://doi.org/10.1007/s11738-008-0244-5 Arora K, Sharma M, Srivastava J et al (2010) Rapid in vitro cloning of a 40-year-old tree of Azadirachta indica A. Juss. (Neem) employing nodal stem segments. Agrofor Syst 78:53–63. https://doi.org/10.1007/s10457-009-9230-1 Arora K, Sharma M, Srivastava J, Ranade SA, Sharma AK (2011) In vitro cloning of Azadirachta indica from root explants. Biol Plant 55(1):164–168. https://doi.org/10.1007/s10535-0110023-9 Azhagu MS (2021) Phytochemical analysis and anticancer activity of Azadirachta indica ethanolic extract against A549 human lung cancer cell line. J Biomed Res Environ Sci 2(4):280–285 Aziz F, Taqdees M, Ifrah I, Sayyada GN (2020) Phytochemical screening and antibacterial activity of neem extracts on uropathogens. Pure Appl Biol 9(1):148–153 Babatunde ED, Otusemade GO, Elizabeth Ojewumi M, Agboola O, Oyeniyi E, Deborah Akinlabu K (2019) Antimicrobial activity and phytochemical screening of neem leaves and lemon grass essential oil extracts. Int J Mechan Engineer Technol 10(3):882–889 Baig B, Yousaf S (2021) Phytochemical screening of neem and black pepper for bioefficacy against insect pests of okra and potato. Sarhad J Agric 37:697–705 Bello A, Kamali Aliabad K, Saravi A, Sodaei Zade H (2022) Determination of the best culture medium and plant growth regulators for micropropagation of neem tree (Azadirachta indica A. Juss). Int J Hort Sci Technol 9(2):237–245

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Srivastava SK, Agrawal B, Kumar A, Pandey A (2020) Phytochemicals of Azadirachta indica source of active medicinal constituent used for cure of various diseases: a review. J Sci Res 64(1):385–390 Srivastava V, Chaturvedi R (2018) Somatic embryogenesis in neem. In: Jain SM, Gupta P (eds) Step wise protocols for somatic embryogenesis of important Woody plants. Springer, Cham, pp 369–386 Tapanelli S, Chianese G, Lucantoni L, Yerbanga RS, Habluetzel A, Taglialatela-Scafati O (2016) Transmission blocking effects of neem (Azadirachta indica) seed kernel Limonoids on plasmodium Berghei early Sporogonic development. Fitoterapia 114:122–126. https://doi.org/10.1016/ j.fitote.2016.09.008 Tenguria R, Khan F (2011) Distribution of endophytic fungi in leaves of Azadirachta indica A. JUSS. (neem) of Panchmarhi biosphere reserve. Cur Bot 2:27–29 Tiwari V, Negi KS, Rawat R, Mehta PS (2017) In-situ conservation and traditional uses of medicinal plants: a case study of home gardens in Nainital, Uttarakhand. Asian Agri-Hist 21(1):47–61 Uddin MZ, Hassan MA, Rahman M, Arefin K (2012) Ethno-medico-botanical study in Lawachara National Park, Bangladesh. Bangladesh J Bot 41(1):97–104. https://doi.org/10.3329/bjb.v41i1. 11087 Ujah II, Nsude CA, Ani ON, Alozieuwa UB, Okpako IO, Okwor AE (2021) Phytochemicals of neem plant (Azadirachta indica) explains its use in traditional medicine and pest control. GSC Biol Pharmaceut Sci 14(2):165–171 United Nations Environment Programme (2012) Neem: the UN’s tree of the 21st century. United Nations Environment Programme, Nairobi; http://www.unep.org/wed/tree-a-day/neem.asp Uwague A (2019) Comparative potential qualitative and quantitative phytochemical evaluation of neem and Moringa oleifera leaf plants in Ozoro. Delta State, Nigeria, pp 120–340 Varghese B, Naithani SC (2008) Oxidative metabolism-related changes in cryogenically stored neem (Azadirachta indica A. Juss) seeds. J Plant Physiol 165(7):755–765 Virshette SJ, Patil MK, Deshmukh AA, Shaikh JR (2019) Phytochemical analysis of different extract of Azadirachta indica leaves. Int J Pharm Sci Rev Res 59(1):161–165 Wang H, Wang N, Huo Y (2020) Multi-tissue transcriptome analysis using hybrid-sequencing reveals potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (Azadirachta indica). BMC Genomics 21(1):1–17 Win T, Ko SNN (2020) In vitro organogenesis and antibacterial activity in leaves extracts of Neem (Azadirachta indica AJuss). 3rd Myanmar Korea Conf Res J 3(3):990–997 Wylie MR, Merrell DS (2022) The antimicrobial potential of the neem tree Azadirachta indica. Front Pharmacol 13:1–16

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An Insight into Coptis Teeta Wall., an Endangered Medicinal Plant and Its Conservation Strategies Manoj Kumar Mishra

Abstract

Coptis teeta wall. is an endemic plant belonging to the Ranunculaceae family listed as an endangered species growing in the North-East region of India and Yunnan state of China. A biochemical analysis reported that the plant rhizome and roots contained a wide range of pharmacologically important bioactive molecules. These identified compounds include benzylisoquinoline alkaloids, lignans, flavonoids, terpenoids, organic acids, sterol, and sterol glycosides. Because of the medicinal value of the whole plant, it was collected unrestrictedly by indigenous peoples as well as by the pharma industry resulting in population decline in their habitats. Over the years, good agricultural practices, in situ conservation and ex situ conservation methods have been adopted for the conservation and sustainable use of these elite plant generations. However, among all conservation methods, ex situ conservation is preferable in C. teeta because it has a narrow area of habitats and is not easy to grow. Keywords

Benzylisoquinoline alkaloids · Phytochemistry · Pharmacology · Conservation

3.1

Introduction

Coptis, a member of the Ranunculaceae (buttercups) family, is one of the most significant plant genera for pharmaceutical purposes, endemic in Eastern Asia region (China province, North-East India) (Bajpay et al. 2019; Wang et al. 2019). Rhizoma M. K. Mishra (✉) Faculty of Engineering and Technology, Department of Biotechnology, Rama University, Kanpur, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_3

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Fig. 3.1 Coptis teeta Wall: A complete plant shows pinnatifid leaves

Coptidis (RC), an eminent traditional Chinese medicine known for bringing down fever, banishing dampness, and expelling fire toxins, is made from dried rhizomes of Coptis plants (Wang et al. 2019). Coptis plants have been used in numerous Chinese herbal medicines for over 2000 years. They were first mentioned in the earliest monograph on Chinese materia medica, Sheng Nong’s Herbal Classic, which was written during the eastern Han dynasty (25–220 AD) (Mukherjee and Chakraborty 2019; Wang et al. 2019). Coptis chinensis a well-known species of this genera has been extensively grown in China, and the majority of its rhizomes are exported to other nations. The rhizomes of C. japonica are occasionally substituted for those of C. chinensis in Korea and Japan (Yang et al. 2017; Wang et al. 2019; Liu et al. 2021). Similar species, also known as Coptis teeta Wall or Mishmi teeta, are abundant in temperate areas of India, including the Mishmi Hills and Arunachal Pradesh, Sikkim, and Bhutan (Mukherjee and Chakraborty 2019). It is also known by golden thread variety in India and also called “Mamira” by local peoples. This plant was discovered in Mishmi Hills of Indian flora in 1825 by R. Wilcox and Captain Bedford. C. teeta is a small (30–50 cm), perennial herbaceous with reduced stem having 2n = 18 chromosome number (Pandit and Babu 1993; Bajpay et al. 2019). Pinnatifid (pinnately divided into lobes that extend more than halfway to the midrib) leaves with a 10–20 cm long glabrous petiole around, 3-lobed lamina that is glossy and slimy with ovate-lanceolate leaves (Fig. 3.1). Inflorescence panicled; flowers are pedicelled, small, regular, whitish in colour with shadowed hair follicles (Bajpay et al. 2019; Mukherjee and Chakraborty 2019). Rhizomes are horizontal to oblique shape (5–15 cm long) with a bitter taste, brownish yellow colour, and covered with numerous fibrous roots, and are widely used as a medicine in the Indian medical systems of Ayurveda, Siddha, and Unani. The interior of the transverse section of rhizome is yellow-orange, but the central pith is darker. Fruits are follicles and multi-seeded with black coloured seed (Bajpay et al. 2019). Reported flowering period of C. teeta is between February and April and fruiting occurs from May to July in summer. According to researchers, the people of

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North-East India also already have knowledge of cultivation of C. teeta and their uses (Pandit and Babu 1998). Local people of Mishmi and other tribes of Arunachal Pradesh used it for the treatment of various health problems like malaria, dysentery, cold allergy, diarrhoea, typhoid, hypertension, tuberculosis, febrifuge, and epidemic cerebrospinal meningitis. It is also used to prevent inflammation, relieve the pain of eyes conjunctives, clear away heat, dry dampness, purging fire and detoxification, as well as to cure skin races or other related problems, abdominal pain and bowel movement, liver and urine disorders, and inflammation (Pandit and Babu 1998; Bajpay et al. 2019; Mukherjee and Chakraborty 2019).

3.2

Cultivation

C. teeta is an endemic plant that is primarily restricted to the state of Arunachal Pradesh, Dibang and Lohit valleys (Fig. 3.2). It is classified as a RET (Rare, Endangered, and Threatened) species in Indian flora (Pandit and Babu 1998; Bajpay et al. 2019).

Fig. 3.2 Cultivation of Coptis teeta: The plant (green colour icon shows Coptis teeta) is cultivated in small areas of the Lohit and Dibang valley districts of Arunachal Pradesh in India and China province

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This plant can only be grown in certain climates and geographic areas. It does thrive in temperate regions that are covered in snow in the winter (Mukherjee and Chakraborty 2019). Usually, the natural population of this plant occurs in temperate forest regions. This plant is cultivated at the lower altitude of 1700 m and high altitude of 2800 m (Pandit and Babu 1998; Huang and Long 2007). The forest floor was covered with several layers of leaf litter and humus, as well as a high moisture content. Due to the physical characteristics of the habitat and its edaphic characteristics, it has been demonstrated that this species engages in small-scale agriculture in its natural habitats in India (Arunachal Pradesh) and China (Yunnan) (Huang and Long 2007). For instance, C. teeta likely grows in acidic, shady places with high levels of humus and moisture in the soil. It prefers peaty sandy loam soil that is well drained. In some areas of Arunachal Pradesh, the local tribal people began growing this plant as a result of awareness campaigns. Additionally, the forest department of Arunachal Pradesh has taken on the duty of cultivating this plant in small areas of the Lohit and Dibang valley districts (Fig. 3.2). This plant is not easy to grow and cultivate in other habitats. Therefore, due to low production in habitats and high demand in the pharma industry, it is very costly and is sold for about Rs. 2000/kg in the local market (Pandit and Babu 1998; Huang and Long 2007; Mukherjee and Chakraborty 2019).

3.3

Phytochemistry

In C. teeta, roots and rhizomes are valuable parts of plants, which have a bitter taste due to the occurrence of a wide range of natural bioactive alkaloids, i.e. berberine, jattrozhine, coptisine, and palmatine (Bajpay et al. 2019; Wang et al. 2019). These alkaloids were classified as benzylisoquinoline alkaloids (BIA) and played an effective role in the treatment of numerous ailments as well as in the prevention of various microbes (Mishra et al. 2020a, b). The benzylisoquinoline alkaloids are a diverse class of metabolites that exhibit a wide range of pharmacological activities and are synthesised through plant biosynthetic (benzylisoquinoline alkaloid (BIA)) pathways made up of complex enzyme activities and regular strategies (Fig. 3.3) (Mishra et al. 2020a; Hagel and Facchini 2013; Liu et al. 2021). They are frequently found in the Papaveraceae, Ranunculaceae, and Berberidaceae families (Beaudoin and Facchini 2014). These alkaloids are primarily derived from an important amino acid phenylalanine/tyrosine that occurs in a variety of diverse but related monomeric and dimeric structural forms (Zenk et al. 1985; Hagel and Facchini 2013). The first success in the chemical analysis of C. teeta plants achieved from the extraction of berberine as an alkaloid. This report was published for the first time in 1862 (Perrins 1862). Previous chemical reports stated that the Coptis genera have had more than 100 chemical components isolated and identified to date. Among all chemical components, berberine and coptisine are important constituents of this plant (Fig. 3.4). Various alkaloids, including palmatine, epiberberine, columbamine, tetradehydroscoulerine, jatrorrhizine, groenlandicine, berberastine, worenine, 8-oxyberberine, 8-oxycoptisine, 3-hydroxy-2-methoxy-9,10-methylenedioxy-8-

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L -tyrosine NCS (S)-Norcoclaurine (S)-3ʹ-Hydroxy-Nmethylcoclaurine 4’-OMT

Reculine

(S)-tetrahydrocolumbamine CAS CjCYP719A (S)-canadine STOX Berberine

SOMT1

BBE

OMT (S)-Scoulerine CFS EC CYP719A5 (S)-Cheilanthifoline SPS EC CYP719A2 ECCYP719A3 (S)-Stylopine STOX

(S)-tetrahydropalmatrubine CYP719 dihydroberberine STOX Epiberberine

Copsine Fig. 3.3 Benzylisoquinoline alkaloids pathway: Pathway shows the biosynthesis of reticuline is a more important intermediate product, which synthesises most valuable berberine, coptisine, and epiberberine in roots or rhizome parts through different enzymatic pathways in Coptis genera. The dotted line shows Pictet–Spengler condensation reaction by NCS. Enzymes involved in BIA pathway, i.e. norcoclaurine synthase (NCS), 3′-hydroxy-N-methylcoclaurine 4’-Omethyltransferase (4’-OMT), berberine bridge enzyme (BBE), scoulerine 9-O-methyltransferase (SOMT1), (S)-tetrahydroproto-berberine oxidase (STOX), canadine synthase (CYP719A1), and stylopine synthase (CYP719A2)

oxyprotoberberine, 8-oxyepiberberine, 8-oxyberberrubine, (-)-5-hydroxyl-8oxyberberine, (þ)-5-hydroxyl-8-oxyberberine, tetrahydroscoulerine, and 8,13dioxocoptisine hydroxide, are also present (Table 3.1, Fig. 3.4) (Yoshikawa et al. 1995; Wang et al. 2014, 2019). Berberine is the most prevalent chemical component among the BIA alkaloids, which have been proposed to be the primary active components of C. teeta (Wang et al. 2015). Along with alkaloids, C. teeta has also been found to contain quinones, organic acids, coumarins, phenylpropanoids, and coumarin derivatives.

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Alkaloids berberine

coptisine

epiberberine

jatrorrhizine

palmatine

Lignans

Woorenoside I

Woorenogenin

Phenylpropanoid

protocatechuic acid

Wogonin

Ferulic acid

Fig. 3.4 Chemical analysis: Chemical structures of identified bioactive constituents extracted from root and rhizome parts of Coptis teeta plants Table 3.1 Phytochemical constituents of C. teeta identified in different plant parts S. No. 1.

Bioactive compound Alkaloids

2.

Organic acid

3.

Lignans and flavonoids

4.

Other compounds

Constituent Magnoflorine, jatrorrhizine hydrochloride, columbamine, epiberberine, coptisine, palmatine chloride, berberine hydrochloride Quinic acid, acetic acid, malic acid, succinic acid, tartaric acid, oxalic acid, citric acid 3, 5, 7-trihydroxy-6, 8-dimethylflavone, ferulic acid, Z-octadecyl caffeate, protocatechuic acid, methyl-3, 4-dihydroxyphenyl lactate, 3, 4-dihydroxyphenethyl alcohol, 3, 5-dihydroxyphenethyl alcohol-3-Oβ-D-glucopyranoside, (+)-Lariciresinol, Woorenoside I, Woorenoside II Longifolroside A (+)-Syringaresinol-4-O-β-Dglucopyranoside Limonin, β-sitosterol, fixed oil, lignin, and sugar

Leaf, roots, and rhizomes

Reference Yang et al. (2017)

Whole plants

Li et al. (2018)

Roots and rhizomes

Meng et al. (2013)

Roots and rhizomes

Bajpay et al. (2019), Wang et al. (2019)

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Additionally, lignin, fixed oil, albumin, carbohydrates, sterol, and other glycoside compounds are present (Li et al. 2018; Bajpay et al. 2019).

3.4

Pharmacology

According to several pharmacognosy reports, C. teeta has a variety of biological activities because it contains a variety of bioactive molecules in its whole plant parts, including alkaloids, glycosides, triterpenoids, and other bioactive compounds (Wang et al. 2019). All identified bioactive molecules showed strong therapeutics properties and serve as a blending component in various ailments for the treatment of various diseases (Fig. 3.5) (Mishra et al. 2021). Berberine is the most important active monomer of C. teeta and has demonstrated potential antimicrobial and antibacterial activity (Yu et al. 2005). Gram-positive bacteria like Streptococcus agalactiae and Staphylococcus aureus as well as Gram-negative bacteria like Escherichia coli and Actinobacillus pleuropneumoniae can both exhibit significant inhibition of progression (Yu et al. 2005; Kang et al. 2015). Another isomer of berberine known as epiberberine may act as a urease inhibitor to treat Helicobacter pylori infection. Berberine also showed the inhibitory effect against the respiratory syncytial virus, herpes simplex virus, coronavirus, influenza virus, enterovirus, and cytomegalovirus. Palmatine was found to be the most effective alkaloid against H1 N1 infection with an IC50 of 50.5 mM. It also showed significant inhibition against yellow fever virus and dengue virus (Wang et al. 2019). In addition to berberine, coptisine, palmatine, epiberberine, and jatrorrhizine, plants containing alkaloids also appeared

Anmicrobial Anhyperlipidemic

An-diarrheal

Cops teeta

Anoxidant acvity

Cholesterol reducon

Aninflammatory

Anhypertensive

An - diabec

Fig. 3.5 Pharmacological importance of C. teeta

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to inhibit body weight gain, lower serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-c), and raise levels of high-density lipoprotein cholesterol (HDL-c), which aid in the prevention of cardiovascular diseases (He et al. 2016; Yang et al. 2016; Wang et al. 2019). Moreover, a common chronic disease such as diabetes mellitus (DM) which shows disorders of the blood sugar level that seriously threaten human health (Ni et al. 2015). C. teeta extracts from the roots and rhizomes, or their decoction, have antidiabetic effects by enhancing insulin resistance (IR), pancreatic beta cells, and gut microbiota regulation (Han et al. 2011; Bajpay et al. 2019).

3.5

Conservation Strategy

Due to the growing demand for herbal products, pressure is rising on Himalayan medicinal plants. These important plants, which are native to high altitude areas, are not seriously attempted to be cultivated commercially in this area. It was pointed out that some valuable herbal plants have been rapidly going extinct over the past few decades as a result of irresponsible, excessive, and unregulated extraction of medicinal plants from their natural habitat and haphazard development in their niche. Due to its valuable therapeutic properties, C. teeta is more in demand in the pharmaceutical industry and among the local population. As a result of over-exploitation for these purposes, deforestation and the plant’s slow regenerative success have caused its population in the Eastern Himalayan regions of India to decline at an alarming rate (Pandit and Babu 1998). Because of all of these factors, this plant is recorded as an endangered species in the Indian Red Data Book (Pandit and Babu 1998; Bajpay et al. 2019). However, the threat of extinction can be reduced by adopting various scientific approaches in the collection, in situ, ex situ conservation, adulteration, and agro-technology to ensure sustainable economic benefits as well as increase yield and use value (Mishra et al. 2020b). In this context, we offer some helpful details about the methods for the selection and preservation of elite C. teeta genotypes for continued cultivation and beneficial uses.

3.5.1

Traditional Harvesting Practices

According to several reports, the species’ decline was largely brought on by habitat invasion and indiscriminate harvesting. It has been observed that habitat loss is a threat to a wide range of taxa, so that conservation of a single species cannot be focused on. However, over-exploitation creates problems for a few taxa including ornamental plants, medicinal plants, and timber trees. Such over-exploitation has targeted C. teeta, gravely jeopardising the species’ chances of surviving. The traditional harvesting practice used by the local farmers is good in many ways as the rhizomes were harvested by the local people only on promising or auspicious days of the month and after the plant has completed its sexual cycle. This method of collection allowed for the emergence of some variation in a population with a

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consistent genetic make-up and ensured that sexual reproduction could occur (Pandit and Babu 1998; Mukherjee and Chakraborty 2019). Therefore, the species could be used sustainably only through traditional cultivation practices and cultural norms.

3.5.2

In Situ Conservation

It is argued that in situ conservation is one of the prominent methods for on-site conservation of genetic resources in natural habitats as well as the maintenance and recovery of healthy populations of species in their natural environments. Various studies in ecological biodiversity suggested the edaphic factors have played a significant role in ecological preference, natural distribution, and evolutionary divergence of the species (Pandit and Babu 1998; Huang and Long 2007). The distribution range, demography, ecology, cytology, reproductive biology, and population genetic structure of C. teeta’s have been found to be endemic to a small zone, occupying a very narrow habitat, and being highly dispersed with very small population sizes, according to an investigation report (Pandit and Babu 1998). This implies that not all types of soil and climate are suitable for growing this plant but it has highly specific microsite requirements such as it is suitable to grow well in temperate areas covered with snow during winter and prefers well-drained peaty sandy loam soil. Other characteristics of the species include a “K” strategy, high male sterility, poor reproductive success and efficiency, insufficient seed dispersal, and little genetic variation (Pandit and Babu 1998; Mukherjee and Chakraborty 2019). Therefore, the interaction of these genetic barriers with external threats like habitat destruction and over-exploitation for economic gain may lead to its extinction. For conservation management policy of the species, it is recommended that the habitat of the species be designated as a protected area with active participation from the local population, including the sharing of conservation benefits. A cooperative conservation management programme might be started to ensure that the interests of conservationists and local people do not conflict at the same time that the traditional rights of local people must be taken into consideration. The local population would be required to regulate and control activities like domestic cattle grazing and good agricultural practises, and the conservation authorities would have to share any benefits that might result, like from the controlled sale of plant products and eco-tourism, with the local population.

3.5.3

Ex Situ Conservation

Ex situ conservation deals with the “off-site conservation” of the wild genetic resources in natural habitat. It includes the collection, preservation, and maintenance of certain genetic resources from wild. Ex situ method of conservation is a complementary action to conserve the genetic diversity, thereby reducing pressure on wild environments and enhancing raw material availability. For many species of medicinal plants their wild population is on a life-threatening level, making it unsuitable

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for in situ conservation action. It can be served as field gene banks and also helps to engage the number of stakeholders in production and regeneration of medicinal plant. Ex situ conservation is concerned with “off-site conservation” of certain genetic resources from wild. It includes the collection, preservation, and maintenance of wild genetic resources in their natural habitat. According to forest department officials of India, many medicinal plant species and their wild populations are in danger of extinction and are unsuitable for in-situ conservation action. A recent report from Uttarakhand has claimed that the state has covered 1145 flora species through in situ and ex situ conservation techniques and that almost 90% of it has been done by ex situ conservation method (Uttarakhand News 2021). Ex situ conservation methods, such as field gene banks or conservation labs, may play a more prominent role in involving a larger number of stakeholders in the production and regeneration of various vital genotypes of traditional medicinal plants (Mishra et al. 2020a, b). Nowadays a variety of biotechnological tools are used for crop improvement such as regeneration, micropropagation, and genetically modify medicinal plants in order to increase the production of secondary metabolites in vitro. As a result of all these factors, plant tissue culture is now being considered as a potential alternative production platform for the large-scale synthesis of sustainable and reliable sources of bioactive targeted compounds. These contemporary methods are crucial for the selection, reproduction, and preservation of the essential genotypes of conventional medicinal plants. The C. teeta in vitro germplasm conservation report is extremely concerning because there hasn’t been much done to ensure its survival. However, there aren’t many published studies on C. teeta in vitro propagation. An eminent plant scientist at the North-Eastern Hill University in Shillong Dr. P.K. Tandon and his team worked on this plant and established the in vitro micropropagation technique for the first time (Tandon et al. 2007). This method suggested that explants collected between March and April were better for micropropagation, which could be attributed to the favourable weather conditions for vigorous and juvenile plant growth. For micropropagation, initially they used a portion of surface sterilised rhizome containing approximately 8–10 per axillary bud for in vitro culture in different treatments of nutrient medium. They achieved the maximum of 55% culture initiated in ½ strength MS medium containing 4.42 μM BAP and 0.56 μM 1AA within 5–6 weeks. However, a hypocotyl segment was used for callus production in nutrient medium containing 4.5/μM 2,4-D and 0.5/μM kinetin at 25 °C for in vitro regeneration (Tandon and Rathore 1992). In 6 weeks, the hypocotyl segments were completely covered by a mass of yellowish-white friable callus, which was then subcultured in the same medium for nearly 2 years. Within 6–7 weeks, 7–8 microshoots were produced per culture.

3.6

Future Prospects

C. teeta is a promising source of therapeutically important bioactive alkaloids that can be used for the treatment of various health disorder. Hence, the synthesis of these bioactive compounds and in vitro production of these plants leads to an emerging

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area of research interest in C. teeta. However, few reports for in vitro culture are now available in C. teeta but further refinements of these technologies need to be strengthened for upscaling the end products to ensure the commercial supply of elite genotypes, which is an alarming issue in this important medicinal plant. While the development of biotechnology has led to the feasibility of large-scale biosynthesis of natural products, tissue culture, genetic engineering, and genome editing can serve as major breakthroughs in the -omics studies of C. teeta. In addition, reinforcement towards various breeding programs and good agriculture practices may also lead to the cultivation of high yielding chemically and genetically characterised elite genotypes developed through tissue culture interventions to meet the high industrial demand for this herb.

References Bajpay A, Nainwal RC, Singh D (2019) Coptis teeta: a potential endemic and endangered medicinal plant of eastern Himalayas. J Pharmacogn Phytochem 8(4):245–248 Beaudoin GA, Facchini PJ (2014) Benzylisoquinoline alkaloid biosynthesis in opium poppy. Planta 240(1):19–32 Hagel JM, Facchini PJ (2013) Benzylisoquinoline alkaloid metabolism: a century of discovery and a brave new world. Plant Cell Physiol 54(5):647–672 Han J, Lin H, Huang W (2011) Modulating gut microbiota as an anti-diabetic mechanism of berberine. Med Sci Monit 17(7):RA164 He K, Kou S, Zou Z, Hu Y, Feng M, Han B, Ye X (2016) Hypolipidemic effects of alkaloids from Rhizoma Coptidis in diet-induced hyperlipidemic hamsters. Planta Med 82(08):690–697 Huang J, Long C (2007) Coptis teeta-based agroforestry system and its conservation potential: a case study from Northwest Yunnan. AMBIO J Hum Environ 36(4):343–349 Kang S, Li Z, Yin Z, Jia R, Song X, Li L, Yin L (2015) The antibacterial mechanism of berberine against Actinobacillus pleuropneumoniae. Nat Prod Res 29(23):2203–2206 Li D, Zhou L, Wang Q, He Y (2018) Determination of organic acids for quality evaluation in Coptis herbs by ion chromatography. 3 Biotech 8(6):1–6 Liu Y, Wang B, Shu S, Li Z, Song C, Liu D, Nie J (2021) Analysis of the Coptis chinensis genome reveals the diversification of protoberberine-type alkaloids. Nat Commun 12(1):1–13 Meng F, Wang L, Zhang J, Yin Z, Zhang Q, Ye W (2013) Non-alkaloid chemical constituents from the rhizome of Coptis teeta. J China Pharmaceut Univ 44(4):307–310 Mishra MK, Pandey S, Misra P, Niranjan A, Srivastava A (2020a) An efficient protocol for clonal regeneration and excised root culture with enhanced alkaloid content in Thalictrum foliolosum DC.—an endemic and important medicinal plant of temperate Himalayan region. Ind Crop Prod 152:112504 Mishra MK, Pandey S, Misra P, Niranjan A (2020b) In vitro propagation, genetic stability and alkaloids analysis of acclimatized plantlets of Thalictrum foliolosum. Plant Cell Tissue Organ Cult (PCTOC) 142(2):441–446 Mishra MK, Pandey S, Niranjan A, Misra P (2021) Comparative analysis of phenolic compounds from wild and in vitro propagated plant Thalictrum foliolosum and antioxidant activity of various crude extracts. Chem Pap 75(9):4873–4885 Mukherjee D, Chakraborty S (2019) Coptis Teeta: conservation and cultivation practice-a rare medicinal plant on Earth. Curr Invest Agric Curr Res 6:845–851 Ni WJ, Ding HH, Tang LQ (2015) Berberine as a promising anti-diabetic nephropathy drug: an analysis of its effects and mechanisms. Eur J Pharmacol 760:103–112 Pandit MK, Babu CR (1993) Cytology and taxonomy of Coptis teeta Wall. (Ranunculaceae). Botan J Linn Soc 111(3):371–378

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Pandit MK, Babu CR (1998) Biology and conservation of Coptis teeta wall.–an endemic and endangered medicinal herb of eastern Himalaya. Environ Conserv 25(3):262–272 Perrins JD (1862) XLIII.—on berberine—contributions to its history and revision of its formula. J Chem Soc 15:339–356 Tandon P, Rathore TS (1992) Regeneration of plantlets from hypocotyl-derived callus of Coptis teeta. Plant Cell Tissue Organ Cult 28(1):115–117 Tandon P, Rathore TS, Kumaria S (2007) Micropropagation of Coptis teeta Wall.–threatened medicinal plant of Arunachal Pradesh, India. IJBT 6(2):280–282 Uttarakhand News (2021) https://static.pib.gov.in/WriteReadData/specificdocs/documents/2022/ jan/doc20221207001.pdf. Wang L, Zhang SY, Chen L, Huang XJ, Zhang QW, Jiang RW, Ye WC (2014) New enantiomeric isoquinoline alkaloids from Coptis chinensis. Phytochem Lett 7:89–92 Wang N, Tan HY, Li L, Yuen MF, Feng Y (2015) Berberine and Coptidis Rhizoma as potential anticancer agents: recent updates and future perspectives. J Ethnopharmacol 176:35–48 Wang J, Wang L, Lou GH, Zeng HR, Hu J, Huang QW, Yang XB (2019) Coptidis Rhizoma: a comprehensive review of its traditional uses, botany, phytochemistry, pharmacology and toxicology. Pharm Biol 57(1):193–225 Yang W, She L, Yu K, Yan S, Zhang X, Tian X, Zhang X (2016) Jatrorrhizine hydrochloride attenuates hyperlipidemia in a high-fat diet-induced obesity mouse model. Mol Med Rep 14(4): 3277–3284 Yang Y, Peng J, Li F, Liu X, Deng M, Wu H (2017) Determination of alkaloid contents in various tissues of Coptis chinensis Franch. By reversed phase-high performance liquid chromatography and ultraviolet spectrophotometry. J Chromatogr Sci 55(5):556–563 Yoshikawa K, Kinoshita H, Kan Y, Arihara S (1995) Neolignans and phenylpropanoids from the rhizomes of Coptis japonica var. dissecta. Chem Pharm Bull 43(4):578–581 Yu HH, Kim KJ, Cha JD, Kim HK, Lee YE, Choi NY, You YO (2005) Antimicrobial activity of berberine alone and in combination with ampicillin or oxacillin against methicillin-resistant Staphylococcus aureus. J Med Food 8(4):454–461 Zenk MH, Rueffer M, Amann M, Deus-Neumann B, Nagakura N (1985) Benzylisoquinoline biosynthesis by cultivated plant cells and isolated enzymes. J Nat Prod 48(5):725–738

4

Strategies for Conservation and Production of Bioactive Phytoconstituents in Commercially Important Ocimum Species: A Review Mamta Kumari, Archana Prasad, Laiq-Ur-Rahman, Ajay Kumar Mathur, and Archana Mathur

Abstract

The genus Ocimum belonging to the Lamiaceae family comprises a number of species that are used to treat many diseases since ancient times. Ocimum plants have been a globally valuable source of many herbal phyto-formulations as well as in cosmetic products, thereby increasing the demand and supply at the commercial level. Due to the high demand and supply of this herb, there is a need to develop efficient strategies for proper cultivation and conservation of uniform plant material. Ocimums have a great variation in batch-to-batch phytoconstituents production because this is a cross-pollinated plant. Ocimum species biosynthesize various phytoconstituents like phenolics, flavonoids, alkaloids, tannins and saponins having a specific value in pharma industries. These phytoconstituents possess many bioactivities like antibacterial, immunomodulatory, antidiabetic, anti-inflammatory, antifungal and anticancer. In this chapter, the production of these bioactive phytoconstituents and various strategies for conservation of different Ocimum species based on plant tissue culture (PTC) and breeding conservation technology have been emphasized. PTC and breedingbased approaches like micropropagation, meristem culture, synthetic seed technology and molecular marker-based selection are effective for the conservation of the Ocimum species and development of uniform planting material. The production and enrichment of secondary metabolites to fulfil the rising commercial demands are also achieved by focusing on biotic and abiotic elicitors in callus

M. Kumari (✉) · Laiq-Ur-Rahman · A. K. Mathur · A. Mathur Division of Plant Biotechnology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIRCIMAP), Council of Scientific and Industrial Research, Lucknow, India A. Prasad Department of Botany, University of Lucknow, Lucknow, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_4

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and cell suspension cultures of Ocimum species have also been highlighted in the present compilation. Keywords

Ocimum · Phytoconstituents · Conservation · Micropropagation

4.1

Introduction

Medicinal plants and their various parts are being used as medicines to combat numerous diseases since ancient times. These plants possess an enormous wealth of chemical compounds with high drug potential. Significant therapeutic uses of medicinal plants have been claimed for many diseases, including their safety, economic potential, efficacy and ease of accessibility (Board 2002). Due to these benefits, traditional practitioners have used medicinal plants for treating the population around 90% in Bangladesh, 85% in Burma and 80% in India (Rahman and Parvin 2014). Traditionally various parts (leaves, flower, stem, root, seed and even entire plant) of medicinal plants are being utilized for curing of various ailments. Plants are being used as drugs in the form of powders, capsules, tonics, ointments, dietary supplements, natural health products and phyto-cosmetics. Herbal medicines often claim assurance of quality and efficacy of drugs by rendering details of their component plant species. Exact identification and quality (their constituents) of plant material are, therefore, a need and critical prerequisite to ensure reproducible quality of herbal products. The quality of herbal products depends on the raw materials that vary according to the environment, collection, manufacturing and storage techniques. Hence, a standard system is required that ensures that every dosage should have a predefined amount of quantity/quality of constituents that can induce intended therapeutic effect (Ekor 2014). The herbal product market is around USD 85 billion globally that is increasing in a steady manner and it is expected to reach USD 7 trillion by 2050 (Pandey et al. 2013; Singh and Kumar 2021). To meet this growing demand, certain expensive and rare medicinal plant species are often substituted or adulterated by morphologically similar, less expensive or easily available species (Prakash et al. 2013). Most regulatory guidelines and pharmacopoeias suggest microscopic analysis and chemical fingerprinting of the plant materials for quality control and standardization (WHO 1998). Thus, in order to ensure product quality, identification of the correct plant species that constitute these herbal products is mandatory. Basils are important aromatic herb of the genus Ocimum which is one of the most versatile genera of medicinal and aromatic crops and is also known as Tulsi in Sanskrit, which means “matchless one”. It has been cultivated extensively since ancient times both as an ornamental and medicinal plant. Geographically this genus is spread over South and Central America, tropical and subtropical regions of Asia and the main centre of diversity belonging to Africa (Sahoo et al. 1997). Traditionally the whole plant parts like stem, flowers, leaves, seeds and roots of Ocimum

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species have been used as medicines as they harbour volatile constituents with high therapeutic efficacy (Khare 2007; Shahrajabian et al. 2020). Epidermal glands present on the hairy leaves also secrete volatile oils which give representative fragrances to numerous species. Ocimum sanctum L. (Tulsi), O. basilicum (Bubui Tulsi), O. americanum, O. kilimandscharicum (Kapoor Tulsi), O. canum (Dulal Tulsi) and O. gratissimum (Ram Tulsi) are some important species to have tremendous medicinal potential. Major secondary metabolites of Ocimums are phenols, flavonoids, terpenoids, alkaloids and essential oils (methyl chavicol, eugenol, linalool, methyl eugenol and camphor) (Gurav et al. 2022). This genus has been used as a pharmaceutical agent since ages because of its antimicrobial, antidiabetic, antiasthmatic, antistress, insecticidal, diuretic, expectorant, analgesic and hepatoprotective properties (Wei and Shibamoto 2010; Gurav et al. 2022). Inter/intra-specific hybridization and polyploidy that occur within the Ocimum genus create lots of taxonomic confusion in understanding the genetic relationship between the species (Grayer et al. 1996). The variability in morphology amongst various Ocimum spp. is further enhanced by their cultivation over the years which are complemented with the occurrence of chemotypes that differ in leaf, shape, size and pigmentation (Simon et al. 1990). The variability in genetic and chemical background limits its utility at commercial scale (Dode et al. 2003). Standardization of quantity and quality of the required constituents from the plant of Ocimum is an essential prerequisite in different pharmaceutical/cosmeceutical industries. In this context, micropropagation can play a significant role to overcome these boundaries and conserve the high-yielding and true-to-type elite planting material (Saha et al. 2014a, c; Prasad et al. 2015; Kumari et al. 2017; Rodrigues et al. 2020). Utilization of molecular and chemical maker-based strategies helps in the identification and characterization of highyielding genotypes. The application of biotechnological tools such as genetic engineering, elicitation, biotransformation, precursor feeding etc. enables the modification of the regulatory biosynthetic pathway that leads to up/down stream processing of the synthesis of various bioactive metabolites in different Ocimum species. The present review is the compilation of the recent research advancement of in vitro-based propagation and production/enhancement of bioactive metabolites in some industrially important Ocimum species. In addition, reports on various in vitro and in vivo conservation strategies of the high-yielding genotype are also highlighted.

4.2

Plant Description, Taxonomy and Geographical Distribution of Ocimum

The genus Ocimum is one of the most valuable aromatic herbs used in Ayurveda (Charak Samhita) since 5000 BC. Ocimum is widely known as Basil (English) and Tulsi (Hindi). In Sanskrit Tulsi means “matchless one”. Geographically it occurs in tropical and subtropical regions of Asia, South and Central America, and Africa

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(Paton et al. 1999). In many Asian and African countries, different species of Ocimum are traditionally used by various medicinal systems such as Ayurveda, Greek, Roman, Siddha and Unani for curing various ailments. The genus Ocimum is a well-known medicinal herb, and it is the largest genus of this family and includes 50–150 species and sub-species that comprise annual and perennial herbs/shrubs (Labra et al. 2004). The taxonomic classification of this species is very broad and complex.

4.2.1

Classification

Kingdom Subkingdom Super division Division Class Order Family Genus

Plantae Tracheobionta Spermatophyta Magnoliophyta Magnoliopsida Lamiales Lamiaceae Ocimum

There are a lot of confusion regarding its taxonomic classification due to the highly cross-pollinating nature of these plants that results in the occurrence of various varieties, cultivars and chemotypes within the species. Paton et al. (1999) classified Ocimum into two major groups Basilicum and Sanctum based on cytology and morphological characters. The updated information about the classification by Kumar et al. (2018) showed a total of seven species from Indian origin. Earlier to this report, many studies were performed by different researchers who classified the Ocimum species based on morphology (stem, leaf, flower and fruit), chromosome number and chemical constituents. The Basilicum group comprises annuals, perennials herbaceous plants (chromosome number x = 12) with black, ellipsoid, mucilaginous seeds. The perennial shrubs comprising the Sanctum group have the chromosome number x = 8, and their seeds are brown globose and non-mucilaginous. Epidermal glands are present on hairy leaves of Ocimum species which accumulate the volatile oils that are highly important for fragrances and perfumery industries. All the vegetative plant parts such as leaves, stem, seeds, flowers and roots contain bioactive constituents that are used in different cosmetic and pharmaceutical companies (Jamshidi and Cohen 2017). The chemical composition varies in different Ocimum species and possesses various classes of phytoconstituents, i.e. monoterpene derivatives (limonene, camphor, citral, linalool, 1,8-cineole and geraniol), phenylpropanoid derivatives (methyl cinnamate, methyl eugenol, eugenol, methyl chavicol and chavicol) and sesquiterpene derivatives (bergamotene, bisabolene and caryophyllene) (Martins et al. 1999). O. sanctum L. (Holy Basil), O. basilicum (Sweet basil), O. kilimandscharicum (Camphor basil), O. gratissimum (Clove basil) and O. americanum (Hoary basil) are very important

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Fig. 4.1 Plants of Ocimum species: O. gratissimum (a), O. sanctum (CIM-Angana) (b), O. Kilimandscharicum (c), O. basilicum (CIM-Saumya) (d)

due to their economical and medicinal properties that will be discussed in succeeding sections (Fig. 4.1 and Table 4.1).

4.3

Some Commercially Important Ocimum Species

4.3.1

Ocimum basilicum L

O. basilicum is the most popular species of this genus, and more than 30 chemotypes are found in tropical and subtropical countries (Vieira and Simon 2006). This herb is commonly known as Babui tulsi (Bengali), Sabza (Gujrati), Marwa dana (Hindi), Vishva tulasi (Sanskrit), Tirunitrupachai (Tamil) and Rudrajeda (Telegu). This aromatic herb is native to North-West India and Central Asia. It is widely cultivated in Indonesia, France, Egypt, United States of America, Greece, Hungary and Morocco. In India, it is cultivated in West Bengal, Madhya Pradesh, Maharashtra, Jammu, Uttar Pradesh, Bihar, Assam, etc. (Lal et al. 2004). The genus Ocimum commonly known as basil is derived from the Greek word “ozo” which means smell and Basileus, meaning “King”, or basilikon meaning “royal”. This herb is grown throughout the world but the content of the volatile oil varies based on variety and environmental conditions. The height of the herb is 30–60 cm, semi-erect, branching stems, opposite and simple leaves with lanceolate shape, verticillaster inflorescence,

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Table 4.1 Phytochemical constituents present in different Ocimum species and their bioactivities No. 1

Species O. basilicum Herb (x = 12)

Phytochemical constituents Methyl chavicol, α-humulene, β-ocimene, β-caryophyllene, linalool, 1,8-cineole, germacrene-D

2

O. gratissimum Shrub (x = 10)

Linalool, eugenol, β-caryophyllene, limonene, β-ocimene, camphor, borneol, 1,8-cineole

3

O. tenuiflorum (syn. O. sanctum) Herb (x = 9)

Eugenol, methyl eugenol, β-elemene, β-caryophyllene, bisabolene, 1,8-cineole, methyl chavicol, α-pinene, circimaritin, isothymusin, apigenin, rosmarinic acid, orientin, vicenin

4

O. kilimandscharicum Guerke shrub (2x = 76)

Camphor, trans-sabinene hydrate, cis-sabinene hydrate, germacrene-D, p-cymene, trans-caryophyllene, linalool, γ-terpinene, terpinen-4-ol α-terpinolene, α-terpinene, 1,8-cineole, myrtenol, β-ocimene, limonene

Pharmacological activity Antimicrobial, antifungal, antibacterial, insecticidal, analgesic, antioxidant, antispasmodic, antiinflammatory Antimicrobial, antifungal, anticancer, anti-inflammatory, antihypertensive, antibacterial, analgesic, antidiarrheal, leishmanicidal, ovicidal, antioxidants, hepatoprotective, immunostimulatory, antimutagenic, antidiabetic, wound healing Antimicrobial, anticancer, larvicidal, antioxidant, antidiabetic, antifertility, radioprotective, immunomodulatory, cardioprotective, hepatoprotective, mosquito repellent, anticarcinogenic, anti-inflammatory, antistress Antioxidant, antimicrobial, wound healing, antibacterial, antifungal

Source: Gill et al. 2012; Purushothaman et al. 2018; Singh and Chaudhuri 2018; Bhavani et al. 2019

corolla and calyx bilabiate and black or brown seeds (also called nutlets) (Suddee et al. 2005). In India and other countries, O. basilicum is extensively grown for its volatile oil used in different food, pharma and cosmetic industries. Traditionally, the herb is remarkably known for curing various ailments such as cancer, deafness, whooping cough, convulsion, hiccup, impotency, insanity, diarrhoea, nausea, sore throat, gout, toothaches and epilepsy (Ahmed et al. 2019). The herb has been classified into four chemotypes (eugenol rich, methyl chavicol/linalool rich, methyl cinnamate rich and camphor rich) based on major components present in its essential oil (Paton et al. 1999). The quantity and composition of phytochemical constituents depend on their geographical distribution and seasonal variation. Plants possess many pharmacological activities such as anti-hyperlipidaemic, antioxidant, anticonvulsant, antimicrobial, antifungal, antibacterial, insecticidal, anti-inflammatory, antispasmodic and

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analgesic (Purushothaman et al. 2018). The leaves contain approximately 0.9% of volatile oil that is bright yellow in colour. The seeds also contain oil which is known as “fixed oil” that comprises alkaloids, glycosides and saponins. Some other phytoconstituents, i.e. citric acid, maleic acid and tartaric acid, are also present in trace amounts in the fixed oil of seeds.

4.3.2

Ocimum sanctum L. (Syn. Ocimum tenuiflorum L.)

O. sanctum plant is an aromatic herb or undershrub, mainly spread over tropical and subtropical provinces of West Africa, Australia, Asia (India, Malaysia, Sri Lanka, China and Thailand) and some Arab countries (Kumar et al. 2013). This plant is native to India and widely found up to a height of 5900 ft (1800 m) in the Himalayas (Shah et al. 2018). It is also distributed in the geographical region of Northern and Eastern Africa, Hainan Island and Taiwan and found in the dry and sandy areas of Sichuan and Hainan (China), as well as in Laos, Vietnam, the Philippines, Myanmar, Cambodia and Indonesia. O. sanctum is the most sacred plant in India, commonly grown in homes and temples and it is a well-integrated part of ancient Hindu tradition. The stem and roots of tulsi are used as small beads to make a string (Mala), which is used in meditation, chanting, spiritual practices and consequently ceremonially connect mind, body and spirit. The earliest history of this plant is found in “Rigveda”, supposed to be the oldest repository of human knowledge (Bhateja and Arora 2012). There are two kinds of holy basils—one with green leaves recognized as Shree/Ram Tulsi and other bearing purple leaves identified as Krishna Tulsi. The plant is collectively known as Holy basil (English), Tulsi (Hindi), Manjary (Sanskrit), Trittava (Malayalam), Maduruthala (Telugu), Tulshi (Marathi) and Thulasi (Tamil). The plant is erect, branched attaining the height of about 30–60 cm. It bears opposite, simple, oblong, elliptic leaves with dentate margins and hairs on adaxial and abaxial surfaces. O. sanctum flowers are small, purplish and arranged in elongate racemes in close whorls. The plant is bitter and acrid with small fruits having reddish-yellow seeds (Gupta et al. 2002). Tulsi has been well documented for therapeutic potentials in the form of Kaphaghna (anti-cough) and Dashemani Shwasaharni (antiasthmatic) (Singh et al. 2011). Basil is famous as “the elixir of life or queen of herb”. Traditional uses of holy basil for curing various diseases are to cure oral infections, cough, colds, asthma, ulcers, tumours, cancer, headache, flu, malaria, colic pain, insomnia, hepatic diseases, skin diseases, respiratory inflammation, arthritis, hypoglycaemia, bronchitis, night blindness, cardiovascular problems and as an antidote of scorpion sting and many more. Leaves are also used as memory enhancers. O. sanctum is another Ocimum species with a large amount of volatile oil (approx. 0.7%) primarily found in its leaves than other plant parts. The volatile oil of O. sanctum mainly includes eugenol (70%) and methyl eugenol (20%). Besides these constituents, the oil also contains sesquiterpene (β-caryophyllene), phenylpropanoids (circimaritin, rosmarinic acid, isothymusin and apigenin) and flavonoids (vicenin and orientin) and minerals (vitamin C and A, calcium,

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phosphorous, chromium, copper, zinc and iron) (Singh and Chaudhuri 2018). The plant possesses many pharmacological activities such as antidiabetic, radioprotective, cardioprotective, hepatoprotective, antimicrobial (antibacterial, antifungal and antiviral), antifertility, immunomodulatory, antistress, anti-inflammatory, mosquito repellent/larvicidal and insecticidal properties (Singh and Chaudhuri 2018). Fixed oils are obtained from the seeds which contain five fatty acids, namely stearic, linolenic acids, palmitic, linoleic and oleic along with alkaloids, glycosides, saponins and some other phytoconstituents (citric acid, tartaric acid and maleic acid) in trace amounts.

4.3.3

Ocimum gratissimum (L.)

O. gratissimum L. is a perennial, woody shrub, commonly known as Vriddhi tulsi (Sanskrit), Ram tulsi (Hindi and Bengali), Avachibavachi (Gujarati), Elumicham tulsi (Tamil), Rama tulsi (Marathi) and Nimmatulsi (Kannada). It is extensively cultivated in the tropical regions of Africa, South America, Asia, Ceylon and Nigeria (Shah et al. 2018). In India, it is geographically distributed in Haryana, Uttar Pradesh, Jammu, Maharashtra, Madhya Pradesh, Punjab, Bihar and Kerala (Gupta et al. 2011). The plant of O. gratissimum is erect, highly branched and attains the height of approx. 50–300 cm. Leaves are elliptical to lanceolate in shape, acute, coarsely crenate, serrate, pubescent on both sides with dotted glands, 6.3–12.5 cm long, and the petioles are slender and 2.5–6.3 cm long. Flowers are simple, in close whorls, bracts longer than calyx and sessile, corolla 4 mm long and pale greenish yellow. This plant is traditionally used in South America and Africa for curing diabetes, bacterial infections and diarrhoea (Aguiyi et al. 2000). In Nigeria, O. gratissimum is used for curing of skin diseases (eczema, dermatitis and scabies), asthma and bronchitis, cough, wounds, gastrointestinal infections (diarrhoea, dysentery), stroke, insect bites, nose-bleeding and anaemia. O. gratissimum is monoterpene and sesquiterpene rich species that contains mainly eugenol, α-thujene, limonene, citral, thymol, germacrene-D, sabinene, α-pinene, phellandrene, β-ocimene, 1,8-cineole, α-copane, β-caryophyllene, α-bergamotene and camphene. This species also contains phenolic, flavonoids and alkaloids compounds (Bhavani et al. 2019). On the basis of the presence of major phytoconstituents, it is categorized into six chemotypes, i.e. ethyl cinnamate type, geraniol type, citral type, linalool type, eugenol type and thymol type (Pino et al. 1996). O. gratissimum possesses many pharmacological activities such as antimutagenic, antifungal, leishmanicidal, antidiarrhoeal, anti-inflammatory and analgesic, wound healing, antimicrobial, antihypertensive, immunostimulatory, antidiabetic, antioxidants and cardiovascular activity (Bhavani et al. 2019).

4

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4.3.4

65

Ocimum kilimandscharicum Guerke

O. kilimandscharicum is an evergreen aromatic shrub which is commonly known as Kapur tulsi (Hindi), Kapura tulasi (Sanskrit), African basil, Camphor basil, Hoary basil, Kilimanjaro basil, and fever plant (English). O. kilimandscharicum is native to Kenya (East Africa). It is distributed in tropical and subtropical regions of Rwanda, Athens, Nigeria, Sudan, Ghana and India. In India, it is cultivated in Dehradun, Jammu, Darjeeling, West Bengal, Uttar Pradesh, Maharashtra, Mysore and Kerala. It is an erect, highly branched pubescent shrub attaining maximum height up to 100–200 cm. Leaves are simple, opposite decussate, acute, elliptic-ovate to oblong, deeply serrated, narrow at the base and pubescent on both sides. Flowers are small in 4–6 whorls with long villose racemes, bract sessile, ovate to lanceolate. The calyx is bi-lipped and hirsute with long white hairs. The plants can survive in high temperatures, provided it gets sufficient moisture; it cannot resist low temperature below 0 °C. It is used for curing various illnesses including malaria, cold, cough, abdominal pain and diarrhoea (Gill et al. 2012). In Burundi and Africa, the shrub is traditionally used as veterinary medicine. O. kilimandscharicum is camphor rich (45.9–78.3%) among the various Ocimum species (Verma et al. 2013). The highly valuable essential oil yield depends on the season, location and stage of the plant. Leaves contain maximum oil followed by flower and stem. The plant contains various classes of compounds like monoterpenes—ocimene, camphene and limonene; oxygenated monoterpenes— 1,8-cineole, camphor and linalool; sesquiterpenes—caryophyllene, oxygenated sesquiterpenes and alcohols. Terpenes and sesquiterpenes are the most abundant compounds of O. kilimandscharicum. This plant exhibits several pharmacological activities like fungicidal, antimicrobial, insecticidal, nematicidal, herbicidal, antioxidant and wound healing (Gill et al. 2012).

4.4

Conservation of Elite Ocimum Species Using Different Strategies

4.4.1

Tissue Culture-Based Strategies for Conservation of Elite Species

The Ocimum species is usually cultivated through the seeds which are not very economical due to low seed viability and germination percentage (≤10%). The seed germination also depends on the season. Furthermore, a seed-derived progeny does not provide the uniform populations due to cross-pollination that may change in ploidy within the genus (Heywood 1978). This genus has taxonomic confusion that makes it difficult to distinguish the relationship between the species at the genetic level (Grayer et al. 1996). The complicated taxonomy and similar morphological characteristics create confusion in the identification and distinguishment of various cultivars and varieties in the Ocimum genus (Simon et al. 1990). Hence, there is an urgent need to use in vitro approaches to conserve and generate a clonally identical

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elite population with high essential oil yield. In this context in vitro studies have been conducted in some industrially important Ocimum species like O. sanctum L. (Holy Basil), O. kilimandscharicum Guerke (Camphor Basil), O. gratissimum (Clove Basil), O. americanum (Hoary Basil) and O. basilicum (Sweet Basil). Tissue culture is a valuable tool that utilizes the totipotent nature of plant cells and offers many strategies for genetic improvement and genetic engineering in plant systems. The success of plant tissue culture (PTC) protocol depends upon the selection of plant growth regulators (PGRs), nutrient medium, the sources of explant, type of explants and controlled physical conditions. The use of tissue culture technique in Ocimum species has been mainly confined to micropropagation for the large-scale production of elite plants that are free from the disease.

4.4.1.1 Micropropagation Micropropagation offers a rapid multiplication and conservation of industrially and economically valuable medicinal plant species. This technique is used significantly for micropropagation of elite genotypes, haploid production, and elimination of breeding barriers, producing disease-free plants, germplasm conservation and selection of desirable traits. Many researchers have tried various medium compositions/ concentrations/combinations of auxins/cytokinins for achieving different morphogenetic responses like callus development, somatic embryogenesis and regeneration of the whole plantlet in different Ocimum species as summarized in Table 4.2. Shoot regeneration was achieved by the direct and indirect induction using different explants. Shoot induction was observed from the axillary buds of nodal explants, which were further used for generating and multiplying various plants. Direct/indirect shoot regeneration of Ocimum species has been reported from nodal, shoot tip, cotyledon, cotyledonary node and hypocotyl explants of O. kilimandscharicum, O. basilicum, O. sanctum, O. viride and O. gratissimum (Dode et al. 2003; Siddique and Anis 2007, 2008; Daniel et al. 2010; Saha et al. 2010a; Asghari et al. 2012; Shahzad et al. 2012; Khan et al. 2015; Kumari et al. 2017; Shukla et al. 2021). The emergence of shoots from the nodal segments excised from young plants of Ocimum species was observed under the influence of cytokinins (TDZ, 2iP, BA and Kn) alone and in combinations with auxins (IAA and NAA). Different basal medium formulations such as Murashige and Skoog’s (1962) (MS) and White’s medium (White 1943) (WM) were used, out of which only MS media was found to be suitable for the growth and development of shoot culture of Ocimum species (Singh and Sehgal 1999). Various types of cytokinins (BA, Kn, 2iP, TDZ and Zeatin) have been used, whereas BA was the most efficient PGR for shoot bud induction and propagation in the Ocimum species (Begum et al. 2002; Gopi et al. 2006; Banu and Bari 2007; Shukla et al. 2021). Most of the researchers used nodal explants for establishing the shoot cultures of Ocimum species. In vitro, axillary shoot proliferation on MS medium fortified with different concentration BA from nodal explants in O. basilicum was attempted by various workers. Begum et al. (2002) and Shahzad et al. (2012) found the highest bud break (100%) using the nodal explants with BA alone, respectively. While Daniel et al. (2010) used MS media fortified with BA (4.44 μM) in combination with of IAA (0.85 μM) and observed

O. basilicum

O. basilicum

Cotyledonary leaves Leaf

Shoot tip

MS + 0.88 μM BA MS + 0.537 μM NAA

Nodal

MS + 22.2 μM BA+1.07 μM NAA MS + 4.53 μM 2,4D MS + 2.26 μM 2,4D + 4.44 μM BA MS + 4.44 μM BA+5.37 μM NAA + 2.32 μM Kn

Formation of shoot from the embryos

100% shoot emergence and rooting, 7.5 shoots/ explant with 5.5 cm length 75% shoot formation and 100% rooting, 4.1 shoots/ explant with 3.0 cm length Emergence of shoot from the callus, 66% shoot and 90% root formation, 5 shoots/explants Callus induction; formation of somatic embryos Enlarged embryos

MS + 16.8 μM TDZ

Leaf

O.basilicum (3 varieties) 1 Sweet Dhani 2 Methyl cinnamate 3 Green purple ruffles O. basilicum

MS + BA (1.1, 2.2, 4.4 μM)

Axillary buds

O. basilicum, O. gratissimum, O. americanum O. sanctum

Morphogenetic responsea 95% shoot emergence, 93% rooting, 10.5 shoots/ explant with 4.8 cm length, GA3 along with BA enhanced the bud break Produced non-morphogenetic callus Direct multiple shoot emergence with 96% induction frequency, 11.2 shoots/explant with 2.8 cm length 4% sodium alginate and 75 mM calcium chloride used for encapsulation of axillary buds; various substrates used (TW, AG, VC, SM, GS, MS, MS + BA) for shoot induction from encapsulated axillary buds. MS + BA media found to be the most suitable for shoot induction from axillary buds Shoot induction achieved from the non-morphogenetic callus derived from leaf explant

Basal medium/PGR/additives MS + 4.4 μM BA MS + 4.4 μM BA+1.2 μMGA3 ½ MS + 4.9 μM IBA MS+ 2.26 μM 2,4-D MS + 4.44 μM BA+ 0.285 μM IAA

Young inflorescence

Source of explant Nodal

O. sanctum

Species O. basilicum

Table 4.2 In vitro morphogenetic studies in Ocimum species

(continued)

Dode et al. (2003) Gopi and Ponmurugan (2006)

Begum et al. (2002)

Phippen and Simon (2000)

Mandal et al. (2000)

Singh and Sehgal (1999)

Reference Sahoo et al. (1997)

4 Strategies for Conservation and Production of Bioactive. . . 67

Shoot tips

Nodal

Shoot tips

Nodal

Nodal

Nodal

Nodal, cotyledon, hypocotyls Nodal

Nodal

Shoot tips

O. basilicum

O. basilicum

O. sanctum

O. basilicum (sweet basil)

O. basilicum

O. kilimandscharicum

O. basilicum (sweet basil)

O. gratissimum

O. kilimandscharicum

O. basilicum

Source of explant Nodal

Species O. gratissimum

Table 4.2 (continued)

MS + 5 μM BA MS + 2.5 μM BA+0.5 μM IAA MS + 1 μM IBA MS + 0.88 μM BA MS + 0.54 μM NAA MS +4.44 μM BA+ 0.85 μM IAA ½ MS + 7.35 μM IBA MS + 2.22 μM BA ½ MS+ 5.37 μM NAA MS + 4.44 μM BA ½ MS +7.35 μM IBA MS + 11 μM BA ½ MS + 11 μM BA+2.85 μM IAA MS + 10 μM BA MS + 10 μM BA+205.28 μM glutamine ½ MS + 5 μM IBA MS + 4.44 μM BA ½ MS + 7.35 μM IBA MS + 4.44 μM BA ½ MS + 7.35 μM IBA

Basal medium/PGR/additives MS + 2.22 μM BA+1.42 μM IAA ½ MS + 2.85 μM IAA MS + 50 μM TDZ

Saha et al. (2012) Saha et al. (2014c)

Shahzad et al. (2012)

100% shoot formation and rooting, 13.4 shoots/ explant, glutamine essential for shoot sprouting 91% shoot formation and rooting, 5.17 shoots/ explant with 2.5 cm length Encapsulated shoot tips (using 3% sodium alginate and 75 mM calcium chloride) showed (83%) shoot regeneration

Saha et al. (2010b) Saha et al. (2010a) Asghari et al. (2012)

Banu and Bari (2007) Daniel et al. (2010)

Siddique and Anis (2007) Siddique and Anis (2008)

Reference Gopi et al. (2006)

95% shoot formation and 82% rooting, 6.2 shoots/ explant with 3.7 cm length 93% shoot formation and 81% rooting, 6 shoots/ explant with 3.8 cm length 96.67% shoot formation and 83% rooting, 5 shoots/ explants

78% regeneration frequency, 11 shoots/explant with 4.8 cm length 80% shoot induction, 12 shoots/explant 16 shoots/explant 87% rooting 90% shoot formation/rooting, 5.8 shoots/explant with 3.39 cm length 82% shoot formation and 89% rooting

Morphogenetic responsea 100% shoot formation and rooting, 14 shoots/ explant with 6.8 cm length

68 M. Kumari et al.

Nodal

Shoot tips

Apical bud

Nodal

Cotyledonary node

Nodal

Shoot tip

Nodal

O. canum Sims

O. basilicum var. pilosum (Willd.)

O. basilicum

O. sanctum (CIM-Ayu)

O. gratissimum

O. sanctum

O. sanctum

O. sanctum Purple leaves O. sanctum Green leaves O. basilicum

Hypocotyl, epicotyls, shoot tip

Nodal

Shoot tip

O. gratissimum

MS + 4.44 μM BA+2.85 μM IAA ½ MS + 7.35 μM IBA MS + 8.88 μM BA+2.68 μM NAA MS + 4.76 μM NAA MS + 2.22 μM BA ½ MS + 4.9 μM IBA MS + 4.44 μM BA ½ MS + 4.9 μM IBA MS+ 43.82 μM ZnSO4
7H2O + 5.71 μM IAA

MS + 2.22 μM BA ½ MS + 4.9 μM IBA MS + 4.65 μM Kn + 2.68 μM NAA MS+ 4.9 μM IBA MS + 8.88 μM BA MS + 9.3 μM Kn MS + 4.44 μM BA+ 2.68 μM NAA MS + 4.9 μM IBA MS + 6.66 μM BA+2.34 μM Kn ½ MS

MS + 4.44 μM BA ½ MS + 7.35 μM IBA

57% shoot induction and 100% rooting, 15 shoots/ explant

92% shooting and 90% rooting, 7 shoots/explants

89% shooting and 87% rooting, 5 shoots/explants

90% shoot induction and rooting, 9 shoots/explant

Khan et al. (2015)

98% shoot regeneration frequency with 9 shoots/ explant, 70% rooting, A. tumefaciens LBA4404 containing binary vector pBI121, maximum transformation frequency (20% ± 0.7) 82% shoot induction and 89% rooting

Strategies for Conservation and Production of Bioactive. . . (continued)

Verma et al. (2016)

Saha et al. (2016)

Jamal et al. (2016)

Mishra (2015)

Leelavathi et al. (2014) Gaddaguti et al. (2015)

Saha et al. (2014b) Gopal et al. (2014)

Saha et al. (2014a)

86% shoot induction with 15 shoots/explants; 70% shoot regeneration 88% shoot induction and 80% rooting, 9 shoots/ explant with 4.0 cm length

95% shoot formation and 86% rooting, 7 shoots/ explant with 4.0 cm length

Encapsulated shoot tips (using 3% sodium alginate and 75 mM calcium chloride) showed (98%) shoot regeneration 91% shooting and 80% rooting, 5.32 shoots/explant

4 69

Nodal

O. sanctum

Basal medium/PGR/additives MS+ 2.67 μM BA ½ MS+ 0.27 μM NAA MS + 1.1 μM BA+ 0.3 μM GA3 + 0.6% AC MS + 0.5 μM IBA + 0.6% AC

Morphogenetic responsea 80.67% shooting response with 9.5 shoots/explants and 75% rooting response 95% shooting and 89% rooting

MS-Murashige and Skoog (1962) AC activated charcoal, TW tap water, AG Agar, VC vermicompost, SM soilrite mix, GS garden soil, SMM shoot multiplication media

a

Source of explant Nodal

Species O. basilicum (CIM-Saumya)

Table 4.2 (continued) Reference Kumari et al. (2017) Shukla et al. (2021)

70 M. Kumari et al.

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71

only 82% bud break and shoot induction in O. basilicum. However, Sahoo et al. (1997) observed BA (4.44 μM) with gibberellic acid (1.2 μM) to be the best for increasing the bud induction frequency (95%). In O. basilicum (CIM-Saumya) nodal explants showed 80.67% induction frequency with 9.5 shoots per explants (Kumari et al. 2017). Siddique and Anis (2007) used two different explants, i.e. shoot tip and nodal part, on MS media fortified with TDZ and BA with IAA and registered 78% (11 shoots/explant) and 80% (16 shoots/explant) shoot bud induction, respectively. Verma and his co-workers (Verma et al. 2016) studied on micropropagation of O. basilicum using different concentrations of zinc sulphate (ZnSO4.7H2O) with IAA on various types of explants viz. hypocotyls, epicotyls and shoot tip. They found the maximum number of shoot development from the hypocotyl explants (15 shoots/explant) and 100% rooting. Direct multiple shoots differentiated from shoot tip and nodal segments of O. sanctum within 1–2 weeks when inoculated on MS medium containing BA alone or in combination with NAA or GA3 and AC (Banu and Bari 2007; Gaddaguti et al. 2015; Mishra 2015; Jamal et al. 2016; Saha et al. 2016; Shukla et al. 2021). In one study performed by Singh and Sehgal (1999), direct multiple shoot induction was observed using young inflorescence explant on MS medium supplemented with 4.4 μM BA and 0.285 μM IAA. An efficient micropropagation protocol of O. gratissimum was also established using nodal and cotyledonary explants on MS medium with varying concentrations of different cytokinins (BA, Kn and 2-iP) alone or in combination with auxins (IAA and NAA). Saha et al. (2014a) established a successful micropropagation protocol with 91% bud induction in O. canum using nodal explants (Table 4.2). The micropropagation of O. kilimandscharicum nodal explants was inoculated on MS medium containing various concentrations and combinations of 2-iP, BA and Kn (Saha et al. 2010a). Khan et al. (2015) established the Agrobacterium-mediated genetic transformation protocol using cotyledonary node explants in O. gratissimum. The finding of scanned literature has been observed that BA was the most suitable PGR for shoot induction and multiplication of Ocimum species. Most of the studies are summarized in Table 4.2. The rhizogenic response was observed in MS or ½ MS medium fortified with various concentrations of IBA/NAA/IAA or without PGR. Finally, developed plantlets were acclimatized and established successfully in vermicompost and soil before transferring to the field. The mature plants were successfully hardened in the glasshouse and further transferred to the experimental field (Banu and Bari 2007; Gaddaguti et al. 2015; Mishra 2015; Jamal et al. 2016: Shukla et al. 2021).

4.4.1.2 Somaclones Somatic embryogenesis (SE) in plants is generally present by two modes, i.e. direct SE and indirect SE (Williams and Maheswaran 1986). The somatic embryos are produced on the surface of the explants (without callus phase) by direct SE, while indirect SE is produced by multistep regeneration process, as follows: (1) callus induction, (2) formation of embryogenic callus, (3) maturation of somatic embryos and (4) plantlet regeneration (Von Arnold et al. 2002). The plants regenerated from SE, calli, and organ cultures may exhibit some genetic and phenotypic variations

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(Orbović et al. 2008). SE is a most effective tool for the genetic manipulation of in vitro plant regeneration producing transgenic plant, germplasm conservation and artificial seeds (Ebrahimie et al. 2006). Some researchers explored callus culture for indirect regeneration of shoots via SE in O. basilicum (Table 4.2). Young leaves and cotyledonary explants were used for induction of shoot buds and formation of regenerative callus (Phippen and Simon 2000; Dode et al. 2003; Gopi and Ponmurugan 2006). SE was observed on MS medium containing various concentrations/combinations of BA, Kn and NAA. Phippen and Simon (2000) observed shoot formation from non-morphogenetic callus on MS media containing TDZ whereas morphogenetic callus was observed on 2,4-dichlorophenoxyacetic acid (2,4-D) containing MS medium. This callus was further transferred on to MS medium enriched with cytokinins and auxins for shoot induction (Gopi and Ponmurugan 2006). Ibrahim et al. (2019) observed SE formation and plant regeneration in O. basilicum on MS medium containing BA (4.4 μM) in combination with 2,4-D (2.26 μM). Some researchers have reported embryogenic callus (globular embryo) formation in basil on MS medium containing only 2,4-D (Hakkim et al. 2011c: Mathew and Sankar 2011).

4.4.1.3 Synthetic Seed Technology Most of the plants can be propagated vegetatively; however, the traditional methods are expensive, time consuming and cannot produce plants at larger scale. Many researchers explored synthetic seed technology for producing high quality and elite genotype production at large-scale level. In this context, Saha et al. (2014b, c) used the shoot tip as explants for alginate encapsulation and stored at 4 °C and 25 °C. Maximum shoot emergence (93%) from encapsulated shoot tips was observed on MS medium fortified with 4.44 μM BA stored at 25 °C (Saha et al. 2014c). Shoots transferred onto ½ strength MS media with 7.35 μM IBA showed rhizogenesis and further these well-developed plantlets were hardened in plastic pots filled with soil: vermiculite (1:1) mixture and finally shifted to the field. Mandal et al. (2000) also used synthetic seed technology using axillary buds in four Ocimum species (O. sanctum, O. basilicum, O. americanum, O. gratissimum) as presented in Table 4.2.

4.5

In Vitro Production of Secondary Metabolites

Plant cell cultures can serve as an efficient platform for the production of valuable compounds in large scale. In vitro raised plants, cell and organ cultures have a shorter culture cycle and faster proliferation rates that have led to higher production of desirable compounds when compared to field grown plants (Rao and Ravishankar 2002). In addition to this unlike field grown plants, it is also not affected by various physiological factors likes climate variations, ecological and pathogens infection. In recent years, various types of biotechnological approaches such as medium optimization, selection of cell line, culture environments, precursor feeding, elicitation, immobilization, permeabilization, metabolic engineering and biotransformation

4

Strategies for Conservation and Production of Bioactive. . .

73

have been utilized for improving biomass accumulation and secondary metabolites production under in vitro conditions (Ochoa-Villarreal et al. 2016). Among them, the use of elicitors was observed to be an effective tool for enhancement of metabolites in different Ocimum species. Phenolic compounds are present in all parts of the plant of Ocimum species. These compounds exhibit strong antioxidant bioactivity that is of immense interest for human health. The in vitro production of phenolic compounds in callus, cell suspension culture, shoots and adventitious/genetically transformed root cultures of Ocimum species was enhanced by incorporation of elicitors (biological/chemical) in the living system (Tada et al. 1996; Kintzios et al. 2003, 2004; Rady and Nazif 2005; Hakkim et al. 2011a, b). Kintzios et al. (2003, 2004) studied the accumulation of rosmarinic acid in cell suspension, shoots and immobilized cells cultures of Ocimum basilicum. Out of these in immobilized cells culture showed minimum accumulation of rosmarinic acid (0.015 mg/g DW). Hakkim et al. (2011a) studied the accumulation of rosmarinic acid in cell suspension culture of O. sanctum using elicitors (methyl jasmonate and yeast extract) and precursors (phenylalanine and sucrose). Enhanced production of RA (2.7, 4.1, 7.0 and 8.6 fold) was observed in cell suspension cultures of O. sanctum through sucrose (5%), phenylalanine (0.25 g/ L), yeast extract (0.5 g/L) and methyl jasmonate (100 M), respectively (Table 4.3). Hakkim and co-workers (2011) in another study showed that the embryogenic cell suspension and callus culture of O. sanctum also accumulate RA. Tada et al. (1996) reported the presence of RA in hairy root cultures of O. basilicum. Five different clones of hairy roots viz. A1, A2, J1, J2 and J3 were developed on MS media without plant growth regulators. Maximum production of RA (6.8 mg/g DW) was observed by J1 clone. O. basilicum hairy root cultures have also been subjected to SA, JA, chitosan and fungal cell wall (Phytophthora cinnamoni) treatments but only fungal cell wall showed a positive effect on biomass and RA accumulation (Bais et al. 2002). Rady and Nazif (2005) reported the presence of RA (3.01 mg/g DW) in multiple shoots of O. americanum. Kiferle et al. (2011) showed the presence of RA along with caffeic, trans-cinnamic and FA in in vitro and hydroponically grown plants of O. basilicum (sweet basil). On the other hand, Nazir et al. (2019) studied the production of RA 52.22 mg/g DW, cyanidin 16.39 mg/g DW, CA 44.67 mg/g DW, peonidin 10.77 mg/g DW and chicoric acid 43.89 mg/g DW in callus culture of O. basilicum (purple basil). In another study, callus culture of O. basilicum (Var. purpurascens) treated with YE produced peonidin 2.7 mg/g DW, RA 15.19 mg/g DW, chicoric acid 2.13 mg/g DW, cyanidin 1.57 mg/g DW, and eugenol 0.25 mg/g DW (Zaman et al. 2022). However, most of the studies on Ocimum species were concentrated on cell suspension culture rather than callus and hairy root cultures for elicitor treatment (Table 4.3). The lower content of RA has been detected in shoot culture of Ocimum species compared to cell suspension culture (Rady and Nazif 2005; Hakkim et al. 2011a, b). Similarly, higher content of RA (2.7 mg/g DW) was in leaf-derived callus culture of O. sanctum as compared to the field grown plants leaves (0.25 mg/g DW) (Hakkim et al. 2007). Most of the studies were focused on production of bioactive constituents in Ocimum species using biotic and abiotic elicitation strategies and optimization of

Shoots

Leaf, stem, inflorescence callus

O. americanum var. pilosum

O. sanctum

MS + 4.53 μM 2,4-D+ 0.456 μM Kn

None

None

MS +4.44 μM BA+4.9 μM IBA

Shoots (bioreactor) CS (bioreactor)

O. basilicum (sweet basil)

MS + 4.65 μM Kn +10.74 μM NAA MS + 4.44 μM BA+ 4.42 μM IAA

0.5% sucrose, 0.567 mM ascorbic acid, 3.02 mM phenylalanine Immobilized cells in calcium alginate beads [1.5, 2 or 3% (w/v) alginate] None

MS + 9.06 μM 2,4-D + 10.74 μM NAA

CS

O. basilicum (sweet basil)

Additivesb None

Fungal cell wall elicitors (CWE), SA, JA, chitosan

Hairy roots

O. basilicum

Basal medium/PGRs MS without PGR

MS without PGR

Type of culturea Hairy roots

Ocimum species O. basilicum (sweet basil)

Table 4.3 A brief account of reports on in vitro production of phenolics in Ocimum species

RA 2.7, 2.2, 1.4 mg/g DW; leaf, stem and inflorescence, respectively

RA 3.01 mg/g DW

RA 0.178 mg/g DW RA 0.029 mg/g DW

RA 0.015 mg/g in immobilized cells

Amount of the phenolicsc RA 6.84 mg/g DW and Lithospermic acid A 17 mg/g DW, Lithospermic B 1.7 mg/g DW Nonelicited hairy roots—RA 29.8 mg/g FW; elicited with Phytophthora cinnamoni 81 mg/g FW, elicited with SA, JA, and chitosan negative effect on growth as well as RA production RA 10 mg/g DW

Rady and Nazif (2005) Hakkim et al. (2007)

Kintzios et al. (2004)

Kintzios et al. (2003)

Bais et al. (2002)

References Tada et al. (1996)

74 M. Kumari et al.

CS

CS

Shoot/ hydroponics

Coculture (hairy roots and Rhizophagus irregularis)

C

CS

O. sanctum

O. sanctum

O. basilicum

O. basilicum

O. basilicum (purple basil)

O. basilicum

None

½ MS + 16.11 μM NAA + 1.16 μM Kn MeJ

None

MS + BAP + NAA

None

5% sucrose, 1.51 mM phenylalanine, 1.82 mM YE, MeJ 100 M None

MS + 4.53 μM 2,4-D+ 0.456 μM Kn

MS + 976.78 μM glutathione +MES 2.56 μM; shoot forming media—MS + 1.1 μM BA; rooting media—½ MS without PGR MS without PGR

None

MS + 4.53 μM 2,4-D+ 0.456 μM Kn

RA 52.22 mg/g DW, CA 44.67 mg/g DW, chicoric acid 43.89 mg/g DW, cyanidin 16.39 mg/g DW, peonidin 10.77 mg/g DW RA 15.73 mg/g DW, BA 14.63 mg/g DW, UA 4.71 mg/g DW and OA 0.91 mg/g DW Enhanced content of above metabolites

RA 76.41 mg/g DW in HR5 line after 60 days inoculation but CA 1.74 in HR4 line after 50 days of inoculation RA 140.53 mg/g DW in HR4 line, CA 2.04 mg/g DW in HR2 lines

RA 63 mg/g DW but in trace amounts caffeic, FA, transcinnamic acid also found

RA 10.9 mg/g, 12.22 mg/g, 13.6 mg/g, 14.96 mg/g DW

5.84 mg/g DW

(continued)

Pandey et al. (2019)

Srivastava et al. (2016b) Nazir et al. (2019)

Srivastava et al. (2016a)

Hakkim et al. (2011b) Hakkim et al. (2011a) Kiferle et al. (2011)

4 Strategies for Conservation and Production of Bioactive. . . 75

Type of culturea C

Basal medium/PGRs MS + BAP + NAA

Additivesb YE

Amount of the phenolicsc YE 100 mg/L conc. was effective for maximum production of RA 15.19 mg/g DW, chicoric acid 2.13 mg/g DW, peonidin 2.7 mg/ g DW, cyanidin 1.57 mg/g DW, eugenol 0.25 mg/g DW, 0.037 mg/g DW

b

C callus, CS cell suspension, HR hairy roots YE yeast extract, MeJ methyl jasmonate, SA salicylic acid c RA rosmarinic acid, BA betulinic acid, FA ferulic acid, CA caffeic acid, DW dry weight, MES 2-(N-morpholino) ethanesulfonic acid

a

Ocimum species O. basilicum (var purpurascens)

Table 4.3 (continued) References Zaman et al. (2022)

76 M. Kumari et al.

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culture conditions/medium. There is a need to explore various biotechnological strategies such as precursor feeding, metabolomics tools and transformation for the upscaling of these important bioactive compounds of Ocimum species.

4.6

Genetic Fidelity and Marker-Based Selection and Conservation of Ocimum Species

Molecular markers offer various advantages such as easy to handle, stability, costeffectiveness and serve as an important tool for a wide range of applications viz. genetic diversity, genome mapping, phylogenetic analysis, gene tagging, and forensic investigations (Agarwal et al. 2008; Gupta et al. 2021). These molecular markers have been extensively used in establishing the genetic fidelity/diversity in microcloned progeny. In vitro micropropagated plants may face the problem of somaclonal variation due to prolonged culture period, and use of high concentrations of plant growth regulators may affect their genetic uniformity (Largia et al. 2015). Therefore, to test genetic uniformity/diversity to confirm the quality of elite clones for their commercial application is very much needed. At present, different types of molecular markers viz. ISSR (inter-simple sequence repeats), SSR (simple sequence repeats), RAPD (random amplified polymorphic DNA) and SCoT (start codon targeted) are used to evaluate the genetic uniformity in micropropagated plants (Gupta et al. 2014, 2021). The RAPD, ISSR and SCoT techniques are based on differential PCR amplification of genomic DNA. These molecular markers offer various advantages such as easy to handle, quick and cost-effective methods. These techniques require a small amount of DNA without any previous information of the genome sequence of the test species (Lakshmanan et al. 2007). The RAPD technique deduces the DNA polymorphisms generated by “deletions or rearrangements at or between primer binding sites of oligonucleotide in the genome” which used short random mostly 10 bases long oligonucleotide sequences (Williams et al. 1990) while the simple sequence repeats (microsatellites) are targeted by the ISSR technique that is scattered and abundant throughout the genome. ISSR markers have higher reproducibility than RAPD primers due to the longer primer length. SCoT markers are based on short conserved region in plants genes, i.e. ATG sequence known as initiation codon. It showed high polymorphism and reproducibility in plants. These techniques are a powerful tool for the evaluation of genetic fidelity/diversity, species and cultivar identification, DNA finger printing in different species and for quantitative trait loci (QTL) mapping (Agarwal et al. 2008; Largia et al. 2015; Gupta et al. 2021). Some researchers have reported on the evaluation of genetic fidelity/similarity in in vitro established Ocimum species viz. O. kilimandscharicum, O. sanctum, O. canum and O. basilicum using PCR-based RAPD and ISSR molecular markers (Saha et al. 2010a, 2014a, b, 2016; Kumari et al. 2017). Patel et al. (2015) studied genetic diversity using RAPD and ISSR molecular markers between 17 genotypes from 5 different Ocimum species. In another report, only RAPD markers have been used for the detection of genetic divers ity in various accession and species of

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Ocimum (Chowdhury et al. 2017). Gupta et al. (2021) evaluated the genetic diversity in 36 accessions of Ocimum species collected from different geographical regions of the world by using two PCR-based molecular marker systems, i.e. ISSR and SCoT. Apart from the molecular markers, various authors evaluated the genetic fidelity using flow cytometry (Faisal et al. 2014; Alatar et al. 2017; Kumari et al. 2017). Flow cytometry (FCM) is one of the predominant methods for the determination of nuclear DNA content and estimation of the ploidy status in plants (Shapiro 2003). In FCM, DNA is stained using specific fluorochromes such as propidium iodide (PI), ethidium bromide, DAPI (4, 6-diamidino-2-phenylindole) and SYBR green that are used for quantification on the basis of the amount of light scattered by each nucleus in liquid suspension (Doležel and Bartoš 2005). Recently, this technique has been used for the determination of genetic variation in cell/tissue caused by the prolonged culture duration in the presence of plant growth regulators (Mallón et al. 2010). These variations may occur at the biochemical and genetic level, thereby leading to polyploidy, aneuploidy, chromosomal breakage, deletion, translocation, gene amplification and mutations. Flow cytometry has been earlier used to evaluate the genetic uniformity in many in vitro grown plants like Solanum aculeatissimum, Prunus cerasus, Mentha arvensis and Solanum lycopersicum (Ghimire et al. 2012; Vujovic et al. 2012; Faisal et al. 2014; Alatar et al. 2017). One of the reports has been found on O. basilicum (CIM-Saumya) in which genetic fidelity was evaluated by flow cytometry and the obtained histogram of the mother plant was compared with the in vitro propagated plants (Kumari et al. 2017). These techniques would be frequently used to study for establishing the genetic uniformity/diversity in the in vitro multiplied and field grown plants of various Ocimum species.

4.7

Breeding Approaches for Conservation of Elite Ocimum Species

Agronomic characteristics of the elite cultivars/species are developed and conserved through conventional breeding. Thus, maintaining the genetic purity is a prerequisite for the elite cultivars/species by the breeder. In this context, CSIR-Central Institute of Medicinal and Aromatic Plants, India, developed many genetically improved Ocimum varieties through various intensive breeding approaches which have traditional significance. The genetically improved varieties/cultivars/species have a quality plant type with high herb, consistently producing high yield of phenylpropanoid eugenol and other industrially important active constituents to make value-added products. O. sanctum (Var. CIM-Ayu) was developed through conventional breeding which is high eugenol-rich oil-yielding variety (Lal et al. 2003). An early maturing, short-duration methyl chavicol and linalool-rich variety of O. basilicum CIM-Saumya (Indian basil) was developed by Lal et al. (2004). Another variety O. sanctum CIM-Angana was also developed by Lal et al. (2008), which has dark purple pigmentation on the leaves. Lal et al. (2018) have developed a new variety of O. basilicum, which has a unique fragrance due to the presence of chavibetol. Some

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breeders are also focusing towards the development of new varieties/cultivars of Ocimum species with enriched disease resistance efficiency against downy mildew caused by Peronospora species (Römer 2010) and Fusarium wilt by Fusarium oxysporum (Dudai et al. 2002). Römer (2010) also released one improved variety of Ocimum species with tolerance efficiency towards cold stress. Chemical characterization and selection of Ocimum genotype and their traditional uses in medicine were described by various authors (Singh and Sehgal 1999; Chowdhury et al. 2017; Kumar et al. 2018). Yield and their related traits correlation were studied in O. basilicum and characterization of germplasm (Srivastava et al. 2018). The present compilation of research work will be helpful for the identification and selection of the improved varieties/cultivars of Ocimum species at genetic and chemical level, and further these varieties/cultivars can be recommended for their utilization in developing good agricultural practices (GAP) for Ocimum species.

4.8

Conclusion and Future Perspectives

Ocimum comprises a wide range of utility due to the occurrence of broad class of bioactive compounds and their pharmacological activities. There is a library of reports available on bioactivity studies of chemical compounds from different Ocimum species but still identification of novel compounds and/or their derivatives always has promising future and utility in different formulations of pharmaceutical and cosmeceutical industries. Plant cell and tissue cultures always offer a sustainable platform for the production of plant-based bioactive metabolites. Ocimum species are also extensively explored for the rapid production of their bioactive compounds in cell as well as organized organ culture under in vitro conditions. The present review provides an overview of the feasibility of in vitro-based cell, tissue and organ cultures for the production of bioactive secondary metabolites and further the execution of various biotechnological tools for the conservation of elite genotypes and modulation of secondary metabolites in commercially important Ocimum species. Environmental factors either in vitro (elicitors, stresses, media composition, etc.) or in vivo (genotype, seasons, soil, humidity, etc.) significantly affect the biosynthesis of Ocimum bioactive compounds. The targeted studies for the understanding and regulation of Ocimum metabolite via proteomics, genetics and metabolomics will prospect the upscaled production of these compounds in heterologous plant and microbial-based system. In addition, the identification of key regulators using genome editing tools will not only be helpful to elicit the production of desired metabolites but also unveil the role of various regulators involved in the biosynthesis of different classes of metabolic end products in this commercially important plant. Acknowledgements All the authors are grateful to the Director of the CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, for providing the research facilities. MK is thankful to Indian Council of Medical Research (ICMR) for the award of Senior Research Fellowship (No. 3/1/ 3/JRF-2012/HRD) and AP is thankful to University Grant Commission (UGC), New Delhi (No. F.4-2/2006 (BSR)/BL/18-19/0078), for the financial support carried out during this investigation.

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Shahrajabian MH, Sun W, Cheng Q (2020) Chemical components and pharmacological benefits of basil (Ocimum basilicum): a review. Int J Food Prop 23(1):1961–1970 Shahzad A, Faisal M, Ahmad N (2012) An efficient system for in vitro multiplication of Ocimum basilicum through node culture. Afr J Biotecnol 11:6055–6059 Shapiro HM (2003) Practical flow cytometry, 4th edn. Wiley-Liss, New York, NY Shukla MR, Kibler A, Turi CE et al (2021) Selection and micropropagation of an elite melatonin rich Tulsi (Ocimum sanctum L.) germplasm line. Agronomy 11(2):207 Siddique I, Anis M (2007) Rapid micropropagation of Ocimum basilicum using shoot tip explants pre-cultured in thidiazuron supplemented liquid medium. Biol Plant 51:787–790 Siddique I, Anis M (2008) An improved plant regeneration system and ex vitro acclimatization of Ocimum basilicum L. Acta Physiol Plant 30:493–499 Simon JE, Quinn J, Murray RG (1990) Basil: a source of essential oils In: Janick, J., Simon, J. E; Advances in new crops. Timber, Portland, OR, pp 484–989 Singh D, Chaudhuri PK (2018) A review on phytochemical and pharmacological properties of holy basil (Ocimum sanctum L.). Ind Crop Prod 118:367–382 Singh S, Kumar S (2021) Medicinal plant sector in India: status and sustainability. Int J Econ Plants 8(2):081–085 Singh NK, Sehgal CB (1999) Micropropagation of “holy basil” (Ocimum sanctum L.) from young inflorescences of mature plants. Plant Growth Regul 29:161–166 Singh V, Vimal B, Suvagia V (2011) A review on ethnomedical uses of Ocimum sanctum (tulsi). Int Res J Pharm 2:1–3 Srivastava S, Conlan XA, Adholeya A et al (2016a) Elite hairy roots of Ocimum basilicum as a new source of rosmarinic acid and antioxidants. Plant Cell Tissue Organ Cult 126:19–32 Srivastava S, Conlan XA, Cahill DM et al (2016b) Rhizophagus irregularis as an elicitor of rosmarinic acid and antioxidant production by transformed roots of Ocimum basilicum in an in vitro co-culture system. Mycorrhiza 26:919–930 Srivastava A, Gupta AK, Sarkara S et al (2018) Genetic and chemotypic variability in basil (Ocimum basilicum L.) germplasm towards future exploitation. Ind Crop Prod 112:815–820 Suddee S, Paton AJ, Parnell JAN (2005) Taxonomic revision of tribe Ocimeae Dumort. (Lamiaceae) in continental South East Asia III. Ociminae. Kew Bull 60:3–75 Tada H, Murakami Y, Omoto T et al (1996) Rosmarinic acid and related phenolics in hairy root cultures of Ocimum basilicum. Phytochemistry 42:431–434 Verma RS, Padalia RC, Chauhan A (2013) Exploring compositional diversity in the essential oils of 34 Ocimum taxa from Indian flora. Ind Crop Prod 45:7–19 Verma SK, Sahin G, Das AK et al (2016) In vitro plant regeneration of Ocimum basilicum L. is accelerated by zinc sulfate. In Vitro Cell Dev Biol-Plant 52:20–27 Vieira RF, Simon JE (2006) Chemical characterization of basil (Ocimum spp.) based on volatile oils. Flavour Fragr J 21:214–221 Von Arnold S, Sabala I, Bozhkov P et al (2002) Developmental pathways of somatic embryogenesis. Plant Cell Tissue Organ Cult 69:233–249 Vujovic T, Cerovic R, Ruzic D (2012) Ploidy level stability of adventitious shoots of sour cherry and Gisela 5 cherry rootstock. Plant Cell Tissue Organ Cult 111:323–333 Wei A, Shibamoto T (2010) Antioxidant/lipoxygenase inhibitory activities and chemical compositions of selected essential oils. J Agric Food Chem 58:7218–7225 White PR (1943) Nutrient deficiency studies and an improved inorganic nutrient for cultivation of excised tomato roots. Growth 7:53–65 WHO (1998) Quality control methods for medicinal plant materials. World Health Organization, Geneva Williams EG, Maheswaran G (1986) Somatic embryogenesis: factors influencing coordinated behaviour of cells as an embryogenic group. Ann Bot 57(4):443–462 Williams JGK, Kubelik AR, Livak KJ et al (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:6531–6535

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Zaman G, Farooq U, Bajwa MN et al (2022) Effects of yeast extract on the production of phenylpropanoid metabolites in callus culture of purple basil (Ocimum Basilicum L. var purpurascens) and their in-vitro evaluation for antioxidant potential. Plant Cell Tissue Organ Cult 150:543–545

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Studies of Natural Product Synthesis of Withania somnifera and Their Conservation Strategy Through In Vitro Method Gaurav Singh

Abstract

Plant natural products are small molecules produced as primary and secondary metabolites in a cell. Withania somnifera, an important Indian medicinal herb, is well known as a reservoir of pharmaceutically active primary and secondary metabolites such as withanolides. Withanolides are a group of triterpenoids based C28-steroidal lactone compounds which synthesized via rearrangement and secondary modification of the isoprene chain. Indeed, withanolides have been well characterized in terms of clinical profiling, but the knowledge about their biosynthesis route is still limited. Despite this, recent emerging trends to explore advanced transcriptomics, genomics and metabolomics studies have opened a glimpse of hope about the withanolide pathway engineering. Apart from withanolides, W. somnifera may have several unexplored economically important bioactive molecules such as iridoids, terpenoids, flavonoids, phenolic acids and their glycosides which can be characterized in future. However, due to deforestation, global warming and overexploitation, conservation strategies need to be adopted to improve the population and genetic pools of W. somnifera. Moreover, with some standard biotechnological approaches, strict measures should be taken for the conservation of this medicinal herb in future. Keywords

Withania somnifera · In vitro conservation · Secondary metabolite · Micropropagation

G. Singh (*) Aix Marseille Univ, CEA, BIAM, Saint Paul-Lez-Durance, France # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_5

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Introduction

Withania somnifera (Ashwagandha, Family: Solanaceae) is an important medicinal herb, also known as winter cherry, Indian ginseng and poison gooseberry. Due to its numerous health benefits, different plant parts have been used in the pharmaceutical field differently for more than 3000 years ago (Dutta et al. 2019). Recently, several researchers have explored the importance of plant extract in improving lower blood pressure and depression (Mirjalili et al. 2009; Bonilla et al. 2021). In addition to the adaptogenic effect, plant extracts have been studied for their anti-inflammatory, antitumor, antioxidant and hypoglycaemic property (Bonilla et al. 2021). These medicinal properties have sparked a great deal of scientific interest in studying the biochemical composition of W. somnifera at different levels. After deep biochemical analysis of plant parts, it has been noted that W. somnifera has several important bioactive molecules such as flavonoids, alkaloids and steroidal lactones. Withanolide and its glycosides are important steroidal lactones which have been well-described for their medicinal importance. Using molecular docking, an in silico method, we recently demonstrated the biomedical application of withanoside V (a type of glyco-withanolides) in the treatment of cancer, Parkinson’s and Alzheimer’s disease (Singh et al. 2018). Another potential in silico approach has been studied against SARS-CoV2 as a protease inhibiter to describe the antiviral role of withanolide (Tripathi et al. 2021). Some of the important constituents such as Withaferin A and Sitoindosides VII–X have been studied to have anti-stress activity against acute models of experimental stress (Singh et al. 2011). The above-described properties emphasize the medicinal and economic values of this herbal plant. Withanolides, also known as triterpenoid saponins, are highly complex and structurally diverse bioactive molecules. These triterpenoids are synthesized via mevalonate (MVA) and non-mevalonate (MEP) pathways with a series of modifications, such as oxidation, cyclization and glycosylation. Other than different classes of withanolides, W. somnifera contains over 35 bioactive molecules in the root with multifunctional properties such as isopelletierine, anaferine, cuseohygrine and anahygrine (Afewerky et al. 2021). Under different environmental conditions, bioactive molecules and their responsive biosynthetic genes have been widely characterized in the different plant parts (Mishra et al. 2013; Singh et al. 2016a, 2017b), but their regulation and conservation still need more emphasis. Due to the extensive use of metabolites in various herbal products, there is a rise in demand and production of plant extract in the global market. The major companies that have been involved in the W. somnifera extract market are Life Extension, Taos Herb Company, General Nutrition Centers, Jarrow Formulas, Hugh Mountains, Organic India and Vitamin Shoppe (data used from Afewerky et al. (2021)). An increase in consumer consciousness and trust in herbal supplements has unquestionably covered the growth of W. somnifera market. Due to its very little or no risk with high medicinal value, USP (US Pharmacopeia) has gained attention for brain tonic production showing its importance in the global market. The popularity of W. somnifera can be seen in the “Amul Memory Milk”, a product by Gujarat Cooperative Milk Marketing Federation (GCMMF, an Indian Dairy manufacturing

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company) where Ashwagandha is used in the sterilized homogenized flavoured toned milk to enhance the memory. These properties enhance the economic value of W. somnifera globally. Therefore, large-field cultivation and in vitro conservation are highly important for this plant. In India, the annual production of W. somnifera is 1500 tonnes whereas the anticipated consumption is over 4-times more than the production value (Kaur et al. 2022). So, various approaches of germplasm conservation and in vitro propagation methods can enhance the plant production and demand.

5.2

Withanolide Biosynthesis of Withania somnifera

Withanolides are an important class of secondary metabolite chemically named as 22-hydroxy ergostane-26-oic acid. Some of the withanolide candidates, like withaferin A, withanolide A and withanone (Fig. 5.1), have been demonstrated to have significant medicinal values. On the contrary, the plant itself uses these bioactive molecules for the defence mechanism against different biotic and abiotic stresses (Mishra et al. 2013; Singh et al. 2016a, 2018). W. somnifera is also known as a factory for natural product biosynthesis in which leaf, root and stem produce different amounts of withanolides. Due to the characteristics and difference in the amount (less in root and more in leaf), root withanolides are possibly transported from the leaf. Withanolide biosynthesis starts via a dual complex regulatory network and reaction intermediates of cytosolic mevalonate (MVA) and plastid-specific 2-Cmethyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose-5-phosphate (MEP/DOXP) pathway. MAV pathway involves seven different precursors for the IPP and DMAPP biosynthesis, important connecting intermediate products between MVA and MEP (Dhar et al. 2015). In the plastid-specific MEP pathway, the first step is catalysed by the 1-deoxy-D-xylulose 5-phosphate synthase (DXS) enzyme which converts pyruvate and glyceraldehyde 3-phosphate (products from glycolysis) into 1-deoxy-Dxylulose 5-phosphate (DXP) (Cordoba et al. 2009). DXP further modifies at several steps to produce DMAPP in the plastid. DMAPP and IPP (from MVA) are the isomers, catalyses by isopentenyl diphosphate isomerase and interchange their concentration in the cytosol and plastid. DMAPP acts as a precursor of geranyl diphosphate (GPP) which is the synthetic precursor of zeatin, monoterpene, diterpenoid and carotenoid. In the cytosolic MVA pathway, two IPP units from the MVA pathway condense with one DMAPP unit of MEP and form a 15-carbon farnesyl pyrophosphate (FPP), the precursor of saponins. This crucial step may also involve maintaining and exchanging the pool of sterols and terpenes in the cell. Finally, squalene synthase (SQS) condenses two FPP units and generate a linear 30-carbon precursor squalene which is further cyclized by oxidosqualene cyclases (OSCs) and cycloartenol synthase (CAS) to generate cyclic backbone of sterols (Singh et al. 2018). 24-methylenecholesterol is the product of the MVA pathway which goes for the hydroxylation and δ-lactonization to produce withanolide (Fig. 5.2). There are enormous possibilities to enhance withanolide production by

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Fig. 5.1 Chemical structure of some withanolide derivatives isolated from W. somnifera. (a) Withanolide A, (b) Withanolide B, (c) Withanone, (d) Withanolide D, (e) Withaferin A. Chemical structures are acquired from PubChem (https://pubchem.ncbi.nlm.nih.gov)

targeting at the intermediate steps of biosynthetic pathway. In 2015 Dhar et al. published a review article in the Frontiers in Plant Science journal explaining the importance of some intermediate genes in the sitosterol, stigmasterol and withanolide production (Dhar et al. 2015). Now there is a piece of growing evidence that MVA and MEP pathways might be interconnected and involved to fulfil different intermediate precursor pools in the cell (Rodríguez-Concepción et al. 2004).

5.3

In Vitro Conservation Strategies of W. somnifera

Due to the high resource of important natural products, W. somnifera has always played a high impact on human health since ancient times. A list of different biomedical functions and cellular targets of different withanolides is given in Table 5.1.

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MVA pathway Acetyl Co-A

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MEP pathway

Cytosol

DXP

Plasd IPP GPPS Geranyl pyrophosphate FPPS Farnesyl pyrophosphate SS

⍺-Amyrin

SE

AAS β-Amyrin

Squalene

BAS

LAS

LS

CAS

Lanosterol

2-3, oxidosqualene

Lupeol

Cycloartenol

24-Methylenecholesterol Phytosterol

Withanolides

Fig. 5.2 Schematic diagram of withanolide biosynthetic pathway (pathway and structural skeleton are taken from KEGG, https://www.genome.jp/pathway/map00900). Key enzymes mentioned in the pathway are as follows—GPPS geranyl diphosphate synthase, FPPS farnesyl diphosphate synthase, SS squalene synthase, SE squalene epoxidase, CAS cycloartenol synthase, AAS α-amyrin synthase, BAS β-amyrin synthase, LS lupeol synthase. Enzymes denoted in red colour can be used as important targets to enhance withanolide production in plant

However, the enormous use of this species has been severely affected by overpopulation, human activities and climate change. Therefore, sustainable utilization and conservation of W. somnifera are necessary to involve a long-term, coordinated and scientifically oriented action programme. The in vitro technique is one of

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the feasible options that can be an important alternative for the conservation of the W. somnifera plant. The basic procedure of this technique is to conserve a part of the plant in a flask or tube containing artificial media, under controlled environmental conditions. The main aim of in vitro conservation is to generate the whole organism from tissue and cells, producing genetically similar individuals or clones. This technique is delivering a constant renewable plant source for large-scale plant culture and withanolide production. Several works have been done applying different in vitro strategies such as cell, organ and root cultures to produce withanolides accumulation (Sangwan et al. 2007; Sivanandhan et al. 2012; Singh et al. 2017a). In vitro, conservation of W. somnifera can mainly be referred to as median or long-term conservation. Mainly the such type of conservation starts with the tissue culture technique to eliminate the pests and diseases and for the exchange and dissemination of germplasm. Here, we are discussing a few methods of the plant in vitro conservation.

Table 5.1 Natural withanolides and their biomedical functions No. 1

Targets COX-2

2

Active compounds 2,3Dihydrowithaferin A Withanoside IV

3

Isodeoxyelephantopin

4

Withaferin A

5

Withanolide D

6

Withanolide sulfoxide

7

Withanone

TNF-induced NF-kB COX-2 Nrf2, and IL-6 COX-2 STAT3 COX-2, PGE2, STAT1, and STAT3 LPS-mediated HMGB1 release, NF-kB and AP-1 Ubiquitin proteasome pathway and NF-kB network COX-2 and TNFα-induced NF-B BIR5 domain of survivin

Induces neurite outgrowth

Diseases involve Inflammation, cancer Dementia and Alzheimer’s disease Inflammation, cancer Inflammation, cancer Microglial activation Vascular HMGB1 release, inflammatory diseases such as atherosclerosis

Ref Ichikawa et al. (2006) Kuboyama et al. (2006) and Joyashiki et al. (2011) Ichikawa et al. (2006) Suttana et al. (2010) and Ichikawa et al. (2006), Lee et al. (2012) and Ahmed (2014)

Age-related macular degeneration

Bargagna-Mohan et al. (2006)

Inflammation, cancer

Mulabagal et al. (2009)

Cancer

Wadegaonkar and Wadegaonkar (2013)

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In Vitro Multiplication

Micropropagation is an important tissue culture method for the rapid multiplication of the plant in a very short time duration by optimizing external environmental conditions such as nutrient media, explant type and growth hormones. Due to rapid multiplication and mass production, this is one of the simple methods of in vitro plant conservation. Micropropagation of W. somnifera was initially done on the seed and shoot tip using different concentrations of 6-benzyladenine (BA) (2.2, 4.4 and 8.9 μM) (Sen and Sharma 1991). Then several in vitro multiplication strategies such as somatic embryogenesis and organogenesis have been developed to improve plant regeneration (Rani et al. 2004; Supe et al. 2006; Saritha and Naidu 2007; Shukla et al. 2010; Kanungo and Sahoo 2011; Fatima and Anis 2012; Singh et al. 2016b, 2017a). Further, the in vitro multiplication conservation method of W. somnifera has been extensively used for research purposes. For instance, using the Agrobacteriummediated transformation method on the areal part of the plants, in vitro multiplication and regeneration were used to develop transgenic W. somnifera with a high amount of withanolides (Pandey et al. 2010; Saema et al. 2016; Udayakumar et al. 2014). In 2013, an in vitro encapsulation technology was developed where a non-embryonic, synthetic seed was encapsulated in calcium alginate (3% sodium alginate+100 mM calcium chloride) containing ½ MS medium with hormonal supplement (Fatima et al. 2013). The described in vitro technique can be highly useful for the long-term conservation of plants as well.

5.3.2

Slow Growth Conservation

In this method, fewer nutrients and other chemicals are used to reduce plant growth. For the slow growth conservation of W. somnifera, 2% sorbitol and 2% mannitol can be used to reduce the plant growth rate (Tuhin and Biswajit 2012). Although until now, it is not validated yet by other researchers for W. somnifera however, for the slow growth conservation of Poincianella pyramidalis and Tinospora cordifolia mentioned method works very well (Chatterjee and Ghosh 2016; dos Silva et al. 2019). Classically, some other methods is applied to conserve strawberry, apple and plum plants which now can be used to conserve W. somnifera for certain periods (Druart 1985). For example, strawberry plantlets have been conserved in the dark at 4  C for 6 years with the regular addition of liquid medium drops (Mullin and Schlegel 1976). The slow-growth conservation method has several advantages over other methods. For instance, depending upon the species, it allows clonal plant conservation for several months to a year under aseptic conditions with limited space and low cost.

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Long-Term Conservation

Cryopreservation (storage at an ultra-low temperature in liquid nitrogen at 196  C or liquid nitrogen vapour at 160 temperature) is the only method for long-term plant conservation. At this temperature, all the cell metabolic activity stops, and thoracically, plant samples can be stored for a limitless time. Since the whole plant can regenerate from frozen culture, cryopreservation provides a better opportunity to conserve W. somnifera. Cryopreservation of the cell culture has been effectively reported for other endangered medicinal plant species such as D. lanalta, Rauvolfia serpentina, Hyoscyamus and A. belladonna (Bajaj 1988). In modern days, cryopreservation is based on the “vitrification” process which avoids intracellular ice crystallization which makes this technique more advantageous.

5.4

Conclusion

W. somnifera has been used as an immense source of medicinally important metabolites for thousands of years. The demand for W. somnifera extracts is growing continuously in the medicinal industries. To produce herbal products plant materials are harvested without sufficient knowledge of plant growth, propagation and origin. As a result of over-harvesting and climate change, the plant is endangered and has been listed by IUCN in the “Red Data Book” (Kumari and Mishra 2020). In vitro conservation through micropropagation and tissue encapsulation methods not only improve storage and transportation but also promotes a high rate of regeneration (Chen et al. 2016). Altogether, the conservation of W. somnifera is necessary to maintain the supply and demand in the pharmaceutical market and protect the plant from extinction.

References Afewerky HK, Ayodeji AE, Tiamiyu BB et al (2021) Critical review of the Withania somnifera (L.) Dunal: ethnobotany, pharmacological efficacy, and commercialization significance in Africa. Bull Natl Res Cent 45:176 Ahmed LA (2014) Renoprotective effect of Egyptian cape gooseberry fruit (Physalis peruviana L.) against acute renal injury in rats. Sci World J 273870:1–7 Bajaj YPS (1988) Cryopreservation and the retention of biosynthetic potential in cell cultures of medicinal and alkaloid-producing plants. In: Bajaj YPS (ed) Medicinal and aromatic plants I. Springer, Berlin, pp 169–187 Bargagna-Mohan P, Ravindranath PP, Mohan R (2006) Small molecule anti-angiogenic probes of the ubiquitin proteasome pathway: potential application to choroidal neovascularization. Invest Ophthalmol Vis Sci 47:4138–4145 Bonilla DA, Moreno Y, Gho C et al (2021) Effects of Ashwagandha (Withania somnifera) on physical performance: systematic review and Bayesian meta-analysis. J Funct Morphol Kinesiol 6:20. https://doi.org/10.3390/jfmk6010020

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Chatterjee T, Ghosh B (2016) Efficient stable in vitro micropropagation and conservation of Tinospora cordifolia (Willd.) Miers: an anti-diabetic indigenous medicinal plant. Int J Bio Resour Stress Manag 7:814–822 Chen S-L, Yu H, Luo H-M et al (2016) Conservation and sustainable use of medicinal plants: problems, progress, and prospects. Chin Med 11:37. https://doi.org/10.1186/s13020-0160108-7 Cordoba E, Salmi M, León P (2009) Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants. J Exp Bot 60:2933–2943 Dhar N, Razdan S, Rana S et al (2015) A decade of molecular understanding of Withanolide biosynthesis and in vitro studies in Withania somnifera (L.) Dunal: prospects and perspectives for pathway engineering. Front. Plant Sci 6:1031 dos Silva TS, Nepomuceno CF, Soares TL, de Santana JRF (2019) In vitro conservation of Poincianella pyramidalis (Tul.) LP Queiroz under minimal growth conditions. Ciência e Agrotecnologia 43:1–11 Druart P (1985) In vitro germplasm preservation technique for fruit trees. Adv Agric Biotechnol 14: 167–171 Dutta R, Khalil R, Green R et al (2019) Withania somnifera (Ashwagandha) and withaferin a: potential in integrative oncology, vol 20. Int J Mol, Sci, p 20 Fatima N, Anis M (2012) Role of growth regulators on in vitro regeneration and histological analysis in Indian ginseng (Withania somnifera L.) Dunal. Physiol Mol Biol Plants 18:59–67 Fatima N, Ahmad N, Anis M, Ahmad I (2013) An improved in vitro encapsulation protocol, biochemical analysis and genetic integrity using DNA based molecular markers in regenerated plants of Withania somnifera L. Ind Crop Prod 50:468–477 Ichikawa H, Nair MS, Takada Y, Sheeja DB et al (2006) Isodeoxyelephantopin, a novel sesquiterpene lactone, potentiates apoptosis, inhibits invasion, and abolishes osteoclastogenesis through suppression of nuclear factor-kappaB (nf-kappaB) activation and nf-kappaB-regulated gene expression. Clin Cancer Res 12(19):5910–5918 Joyashiki E, Matsuya Y, Tohda C (2011) Sominone improves memory impairments and increases axonal density in Alzheimer's disease model mice, 5XFAD. Int J Neurosci 121:181–190 Kanungo S, Sahoo SL (2011) Direct organogenesis of Withania somnifera L. from apical bud. Int Res J Biotechnol 2:58–61 Kaur K, Dolker D, Behera S, Pati PK (2022) Critical factors influencing in vitro propagation and modulation of important secondary metabolites in Withania somnifera (L.) dunal. Plant Cell Tissue Organ Cult 149:41–60 Kuboyama T, Tohda C, Komatsu K (2006) Withanoside IV and its active metabolite, sominone, attenuate Abeta(25-35)-induced neurodegeneration. Eur J Neurosci 23:1417–1426 Kumari V, Mishra PK (2020) Withania somnifera- an endangered medicinal plant from Lohardaga district of Jharkhand. J Emerg Technol Innov Res 7:203–207 Lee W, Kim TH, Ku SK, Min KJ, Lee HS, Kwon T, Bae JS (2012) Barrier protective effects of withaferin a in HMGB1-induced inflammatory responses in both cellular and animal models. Toxicol Appl Pharmacol 262:91–98 Mirjalili MH, Moyano E, Bonfill M et al (2009) Steroidal lactones from Withania somnifera, an ancient plant for novel medicine. Molecules 14:2373–2393 Mishra MK, Chaturvedi P, Singh R et al (2013) Overexpression of WsSGTL1 gene of Withania somnifera enhances salt tolerance, heat tolerance and cold acclimation ability in transgenic Arabidopsis plants. PLoS One 8(e63064):1–16 Mulabagal V, Subbaraju GV, Rao CV, Sivaramakrishna C, Dewitt DL, Holmes D, Sung B et al (2009) Withanolide sulfoxide from Aswagandha roots inhibits nuclear transcription factorkappa-B, cyclooxygenase and tumor cell proliferation. Phytother Res 23:987–992 Mullin RH, Schlegel DE (1976) Cold storage maintenance of strawberry meristem plantlets 1. Hort Sci 11:100–101 Pandey V, Misra P, Chaturvedi P et al (2010) Agrobacterium tumefaciens-mediated transformation of Withania somnifera (L.) Dunal: an important medicinal plant. Plant Cell Rep 29:133–141

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Rani G, Virk GS, Nagpal A (2004) Somatic embryogenesis in Withania somnifera (L.) Dunal. J Plant Biotechnol 6:113–118 Rodríguez-Concepción M, Forés O, Martinez-García JF et al (2004) Distinct light-mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development. Plant Cell 16:144–156 Saema S, Rahman LU, Singh R, Niranjan A, Ahmad IZ, Misra P (2016) Ectopic overexpression of WsSGTL1, a sterol glucosyltransferase gene in Withania somnifera, promotes growth, enhances glycowithanolide and provides tolerance to abiotic and biotic stresses. Plant Cell Rep 35(1): 195–211 Sangwan RS, Chaurasiya ND, Lal P et al (2007) Withanolide a biogeneration in in vitro shoot cultures of ashwagandha (Withania somnifera DUNAL), a main medicinal plant in Ayurveda. Chem Pharm Bull (Tokyo) 55:1371–1375 Saritha KV, Naidu CV (2007) In vitro flowering of Withania somnifera Dunal—an important antitumor medicinal plant. Plant Sci 172:847–851 Sen J, Sharma AK (1991) Micropropagation of Withania somnifera from germinating seeds and shoot tips. Plant Cell Tissue Organ Cult 26:71–73 Shukla DD, Bhattarai N, Pant B (2010) In-vitro mass propagation of Withania somnifera (L.) Dunal. Nepal J Sci Technol 11:101–106 Singh N, Bhalla M, de Jager P, Gilca M (2011) An overview on Ashwagandha: a rasayana (rejuvenator) of ayurveda. Afr J Tradit Complement Altern Med 8:208–213 Singh G, Tiwari M, Singh SP et al (2016a) Silencing of sterol glycosyltransferases modulates the withanolide biosynthesis and leads to compromised basal immunity of Withania somnifera. Sci Rep 6:25562 Singh P, Guleri R, Pati PK (2016b) In vitro propagation of Withania somnifera (L.) Dunal. Methods Mol Biol 1391:201–213 Singh G, Saema S, Singh S, Misra P (2017a) Effect of antioxidant protection system on regeneration potential of different chemotypes of Withania somnifera (L.) Dunal—a comparative analysis. Ind J Exp Biol 55:242–250 Singh G, Tiwari M, Singh SP et al (2017b) Sterol glycosyltransferases required for adaptation of Withania somnifera at high temperature. Physiol Plant 160:297–311 Singh G, Dhar YV, Asif MH, Misra P (2018) Exploring the functional significance of sterol glycosyltransferase enzymes. Prog Lipid Res 69:1–10 Sivanandhan G, Arun M, Mayavan S et al (2012) Optimization of elicitation conditions with methyl jasmonate and salicylic acid to improve the productivity of withanolides in the adventitious root culture of Withania somnifera (L.) Dunal. Appl Biochem Biotechnol 168:681–696 Supe U, Dhote F, Roymon MG (2006) In vitro plant regeneration of Withania somnifera. Plant tissue Cult Biotechnol 16:111–115 Suttana W, Mankhetkorn S, Poompimon W, Palagani A, Zhokhov S, Gerlo S, Haegeman G, Berghe WV (2010) Differential chemosensitization of P-glycoprotein overexpressing K562/Adr cells by withaferin a and Siamois polyphenols. Mol Cancer 9:99 Tripathi MK, Singh P, Sharma S et al (2021) Identification of bioactive molecule from Withania somnifera (Ashwagandha) as SARS-CoV-2 main protease inhibitor. J Biomol Struct Dyn 39: 5668–5681 Tuhin C, Biswajit G (2012) Mass propagation and in vitro conservation of Indian ginseng-Withania somnifera (L.) Dunal. Glob J Res Med plants Indig Med 1:529–538 Udayakumar R, Kasthurirengan S, Mariashibu TS et al (2014) Agrobacterium-mediated genetic transformation of Withania somnifera using nodal explants. Acta Physiol Plant 36:1969–1980 Wadegaonkar VP, Wadegaonkar PA (2013) Withanone as an inhibitor of survivin: a potential drug candidate for cancer therapy. J Biotechnol 168:229–233

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In Vitro Studies in Andrographis paniculata Pertaining to Andrographolides Accumulation M Joe Virgin Largia

Abstract

Andrographis paniculata, belonging to the family Acanthaceae, is the most valuable Indian medicinal plant. It is widely distributed throughout tropical Southeast Asian countries and popularly called as Kalmegh because of its bitter taste. It is widely utilized in various Indian and Chinese medicines for the treatment of respiratory tract infection, common cold, fever, and liver disorders. Diverse pharmacological benefits of the plant include antiviral, anti-HIV, antimalarial, anti-helmintic, antioxidant, anti-inflammatory, antibacterial, and anticancer activity. This herb produces more than 80 pharmaceutically important molecules belonging to diterpenoids, flavonoids, quinic acids, xanthones, etc. Increasing demand for plant-based drugs leads to large-scale commercial production of plant materials, their extracts and metabolites. Biotechnological approaches elicitation, permeabilization, immobilization, alteration in media composition, transformation result in enhanced production of phytochemicals, thereby conserve the treasured plant germplasm. This chapter discusses the actions of significant phytomolecules of A. paniculata and highlights the various plant, cell, and tissue culture-based in vitro strategies pertaining to its conservation. Keywords

Andrographis · In vitro culture · Andrographolide · Elicitation · Gene expression · Transcription factors

M. J. V. Largia (✉) Department of Botany, St. Xavier’s College, Palayamkottai, Tamil Nadu, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_6

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Abbreviations AD AP BAP DDAD DW GA IAA IBA Kn mM MS NAA NAD

6.1

Andrographolide Andrographis paniculata Benzyl amino purine 14-deoxy 11,12-didehydroandrographolide Dry weight Gibberellic acid Indole-3-acetic acid Indole-3-butyric acid Kinetin Millimolar Murashige & Skoog Naphthalene acetic acid Neoandrographolide

Introduction

Andrographis paniculata (Burm. f.) Wall. ex Nees (AP), commonly called as the “king of bitters” is a notable medicinal plant of Acanthaceae family. It is an annual, branched, erect, and herbaceous plant which grows in hedgerows throughout the plane lands, hill slopes, waste ground, farms, moist habitat, seashores, and roadsides. The stem is dark green, quadrangular with longitudinal furrows and wings on the angles of the young parts and slightly enlarged at the nodes. Leaves are glabrous, lanceolate, pinnate, acute, and entire. Flowers are white with rose-purple spots on the petals, small, in axillary and terminal racemes or panicles. Seeds are linear-oblong capsules and acute at both ends. It is widely cultivated and used in South Asian countries including Bangladesh, China, Hong Kong, India, Pakistan, Sri Lanka, Philippines, Malaysia, Indonesia, and Thailand (Kandanur et al. 2019) for treating several maladies such as snake bite, bug bite, diabetes, dysentery, malaria, fever, common cold, bronchitis, gastrointestinal disorders, hypertension, cancer, and urinary infection. The plant has been proved to possess a broad range of pharmacological effects such as anticancer, anti-diarrheal, anti-HIV, anti-hyperglycemic, antiinflammatory, antimicrobial, antimalarial, antioxidant, cytotoxic, hepatoprotective, immunostimulatory, etc. (Murthy et al. 2021). Extracts of the aerial part of AP contain diterpenoids, diterpene glycosides, lactones, flavonoids, and flavonoid glycosides. Among these, three diterpene lactones such as andrographolide (AD), 14-deoxy 11,12-didehydroandrographolide (DDAD), and neoandrographolide (NAD) are valued much for their pharmaceutical importance (Chao and Lin 2010; Sareer et al. 2014). The chemical structures of these phytomolecules are illustrated in Fig. 6.1. They are derived from C5 unit isopentenyl diphosphate, which can be synthesized via the mevalonate pathway in the cytoplasm and the methylerythritol

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Fig. 6.1 Some popular market products based on Andrographis paniculata

phosphate pathway in the chloroplasts of plants (Nagegowda 2010). These three phytocompounds were reported to possess specific pharmacological effects and are most important to treat many important diseases. Several pharma products based on AP extracts are available in the market currently (Fig. 6.1) and Table 6.1 provides the details of some of those products. Some notable studies have been carried out to assess the quantity of three significant metabolites in wild plants of AP. In 2007, Prathanturarug et al. reported that the average AD content varied from 12.44 to +33.52 mg/g and DDAD content varied from 0.23 to 2.08 mg/g in dried leaves of AP. Furthermore, they isolated the elite individual plants containing high amounts of AD and DDAD (up to 52.57 and 3.46 mg/g dried leaves, respectively). In 2019, Tajidin et al. stated that AP young leaves at pre-flowering harvest age were found to be richer in AD, DDAD, and NAD compared to mature leaves. In 2021 Dalawai et al. quantified the amount of diterpene lactones in leaves and stem of different species of Andrographis and reported that the most abundant diterpenoid was AG and highest amount of 68.35 mg/g DW was recorded in a population of AP. NAG was optimum in the leaves of A. macrobotrys (98.43–102.03 mg/g DW). DDAD was higher in the leaves of AP (16.01 mg/g DW). From these studies it is very clear that the biosynthesis and accumulation of AD, NAD, and DDAD depends upon the species, genotypes/cultivars, phenological stage, and plant part used and geography of the region of cultivation. In such a case, large-scale commercial production of these metabolites by using wild plants is cumbersome and can be achieved via in vitro plants through plant cell, tissue, and organ culture techniques and advancements. Various strategies such as strain improvement, optimization of culture medium/environment, elicitation, nutrient/ precursor feeding, hairy root culturing, Agrobacterium mediated transformation, and other bioprocess technologies could be applied with in vitro cell and organ

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Table 6.1 Details of some popular products based on Andrographis paniculata in the market S. No. 1.

2. 3.

Name of the product Kalmegh— capsules Andrographis— capsules Andrographis capsules

4.

Kalmegh capsules

5.

Andrographis paniculata dilution Andrographis paniculata— tablets Andrographis paniculata— capsules Nilavembu Kudineer Chooranam Andrographis tablets

6.

7.

8.

9.

Name of the company Bixa botanical, India Nature’s way, USA Terry naturally, USA Biotic nature products, India SBL Pvt. ltd., India Sri Sri TATTVA, India Herba diet, India Granniez green herbs, India Planetary herbals, USA

Details of AP extract 20% AD

Purpose Liver tonic

10% AD

Immune support

80 mg of AD

Liver support, immune function, and joint health

AP extract

Strength and vigour, liver tonic, antibacterial, and antioxidant

AP extract

A homeopathic medicine for treating fever, skin problems and to boost immunity Healthy liver function, immune health, boosts digestion, and skin health For supporting body’s defense

AP whole plant— 500 mg AP leaf extract— 400 mg AD

10% AD

Siddha medicine for fever, cold, cough, and immune booster Not mentioned

cultures for higher accumulation of secondary metabolites (Murthy et al. 2014). Therefore, this chapter is focused on exploring the different methodologies and approaches followed so far to produce efficient in vitro cultures and mass production of three noteworthy chemicals.

6.1.1

In Vitro Regeneration

Protocols for in vitro regeneration of A. paniculata have been devised by several workers. The very first report was published by Martin et al. in 2004 for regeneration through somatic embryogenesis. They initiated friable callus from leaf and internode explants on MS medium supplemented with different concentrations of 2,4-D and callus subcultured on the medium with reduced concentration of 2,4-D (2.26 mM) became embryogenic which gave rise to the highest number of embryos (mean of 312 embryos) after being transferred to half-strength MS basal liquid medium. The addition of 11.7 mM silver nitrate to half-strength MS liquid medium resulted in

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71% of embryos undergoing maturation, while 83% of embryos developed into plantlets after being transferred to agar medium with 0.44 mM BAP and 1.44 mM GA. Most plantlets (88%) survived under field conditions and were morphologically identical to the parent plant. In 2008, Purkayastha et al. established a rapid and high frequency regeneration protocol through nodal explants on MS medium with 10 μM BAP, which induced an average of 34 shoots in 94% of the cultures within 4 weeks. Elongation of the induced shoots was achieved on MS basal medium supplemented with 1.0 μM GA3 within 2 weeks. Rooting was best induced in 94% of shoots cultured on MS medium supplemented with 2.5 μM IBA, within a week. The plantlets were successfully transferred to soil after hardening with a 92% survival rate. An efficient protocol for direct regeneration of A. paniculata from stem base explants was given by Roy et al. (2009). Maximum number of adventitious shoots (62.0 ± 4.2/explant) was obtained on MS media supplemented with 2 mg/L Zeatin. Shoot elongation from proliferated shoots occurred on MS media supplemented with BAP (2 mg/L). Shoots rooted after they were dipped in IBA (1 g/L) and upon its further transfer to MS basal media. In 2010, Kataky and Handique established high frequency shoot proliferation of A. paniculata via nodal explants in half-strength MS medium supplemented with BAP (1 mg/L) and adenine sulfate (1.5 mg/L). This hormonal combination has produced an average of 18.36 numbers of shoots after 45 days of culture and rooting was achieved using IBA (0.5 mg/L) after 7 days of culture. The regenerated plants were successfully acclimatized in the greenhouse and transferred to the field with 98% survival rate. Further this study has evaluated the antioxidant potential of the 8-months old field established micropropagated plants using 2, 2- diphenyl-1, picrylhydrazyl (DPPH) radical scavenging activity with various organic and aqueous solvents. Methanol extract gives a maximum percentage inhibition of 80.94% at 50 g/mL concentration. In 2011, Kataky and Handique tested the efficiency of three different media formulations such as MS, Gamborg’s B5, and Nitsch selection medium for the optimum growth response of the explants of A. paniculata. In MS medium initial bud break was observed within 5 days of culture with a maximum average of 2.2 shoots after 30 days of culture. In Gamborg’s medium slight greening was observed but multiple shoot formation was entirely absent after 30 days of culture whereas, no shoot induction was found in the Nitsch medium. The rate of shoot proliferation was found higher in ½ MS as compared to full MS medium. Shoots tips were found to produce lesser number of multiple shoots than the nodal explants. Rooting was achieved in 0.5 mg/L IAA or IBA. The regenerated plants were successfully acclimatized in the greenhouse and transferred to field with 98% survival rate. Dandin and Murthy (2012) reported a micropropagation protocol by using nodal explants in which they recorded a maximum of 39 shoots per explant on MS medium supplemented with 1.0 μM BAP and 5.0 μM Kn. Rooting was induced on transferring the developed shoots to half-strength MS medium supplemented with IBA (2.0 μM) and the rooted plantlets were successfully acclimatized. In addition, they analyzed the genetic fidelity using RAPD markers which did not exhibit any type of

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polymorphism. They have also determined the considerable amount of andrographolide in in vitro regenerated plants through HPLC technique. In 2013, Bansi and Rout achieved micropropagation from leaf and stem explants on MS medium supplemented 3.0 mg/L BAP, 1 mg/L NAA, and 50 mg/L adenine sulfate after 6 weeks of culture. The elongated shoots were rooted within 9–11 days on ½ strength MS medium supplemented with 0.5 mg/L IBA with 2% sucrose. The rooted plantlets were survived in the greenhouse. Roy in 2014 developed an in vitro regeneration protocol from shoot tip and nodal segment explants on MS medium supplemented with 11.10 μM BAP, 10% coconut water. Addition of 100 mg/L urea and 2.0 g/L activated charcoal to the medium showed proper shoot elongation. The isolated shoots were rooted well (90%) on half-strength MS medium fortified with 9.80 μM IBA. The plantlets were acclimatized successfully in polybags containing a mixture of soil, sand, and compost in 2:1:1 ratio. High frequency of regeneration, axillary flower induction, and fruit formation was achieved on MS medium containing BAP (3 mg/L) along with IAA (0.2 mg/L). Elongated shoots rooted well on half-strength MS basal medium and were successfully acclimatized to the garden conditions (Mohammed et al. 2016). A rapid method for the large-scale propagation of AP through in vitro culture of embryonic explants has been developed. Adventitious shoot regeneration from embryonic explants was possible in case of cotyledons and root decapitated embryonic axis among all the tested embryonic explants. High frequency of adventitious shoot formation was observed within 30 days from embryonic explants and shoot induction followed either direct regeneration on MS medium supplemented with BAP 3 mg/L or indirect adventitious shoot regeneration in 1.0 mg/L of TDZ. Shoots elongated in MS media with GA3 0.5 mg/L are effectively rooted in half-strength MS media with IBA 0.5 mg/L and a high percentage of (82.5%) plants were successfully hardened in soil, sand, and vermiculite (1:1:1) (Paritala et al. 2017). Yadav et al. (2022) reported in vitro multiplication and rooting of A. paniculata using BAP and IAA. Rapid multiplication protocol from nodal segments was described by Jagadibabu et al. in 2022. Among the various studies, the best media composition for maximum multiple shoot production was MS + 2 mg/L zeatin, which produced 62 shoots per explant (Roy et al. 2009).

6.2

Andrographolide Production Through In Vitro Cultures

6.2.1

Adventitious Culture

Adventitious roots were induced directly from leaf segments on MS medium with 5.3 μM NAA and were cultured in MS liquid medium with 2.7 μM NAA for higher accumulation of biomass and andrographolide within 4 weeks. Seven-fold increment of fresh biomass was evident in suspension cultures along with 3.5-fold higher andrographolide compared to natural plants (Praveen et al. 2009). Among the different auxin treatments in adventitious root culture, only NAA was able to induce adventitious roots. Adventitious roots grown in modified strength MS medium

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showed the highest root growth (26.7 +/- 1.52), as well as the highest amount of andrographolide (133.3 +/- 1.5 mg/g DW) as compared with roots grown in halfand full-strength MS medium. Growth kinetics showed maximum biomass production after 5 weeks of culture in different strength MS liquid medium. The produced andrographolide content was 3.5–5.5 folds higher than that of the natural plant, depending on the medium strength. (Sharma et al. 2013). Supplementation of IBA to the NAA + Kn-containing MS medium boosted the overall growth and AD/NAD synthesis in the adventitious roots. Compared to control leaves, the adventitious root exhibited about 2.61-and 8.8-fold higher contents of AD and NAD, respectively (Singh et al. 2018).

6.2.2

Callus and Cell Suspension Culture

Callus was induced by culturing leaf discs on MS medium with different concentrations and combinations of plant growth regulators. Best callus induction was obtained at lower concentration of 2,4-D (0.5 and 1.0 mg/L), combination of 2,4D + NAA (1 + 1 mg/L) 2,4 D + KN (1.0 + 0.5 mg/L) and BAP + NAA (1.0 + 1.0 mg/L). Callus from the 2,4-D + NAA (1 + 1 mg/L) was friable and best to release cells in suspension culture. The highest amount of andrographolide (32.40 ± 2.22 mg/g FW) is found in 2, 4-D + NAA (1 + 1 mg/L) cell suspension culture followed by 2,4-D + KN (1.0 + 0.5 mg/L) (31.95 ± 2.21 mg/g FW) (Sharmila et al. 2013).

6.2.3

Shoot Cultures

Worakan et al. (2017) reported a significant increase in leaf biomass and andrographolide contents after treating with the synthetic cytokinin, cytokinin-1(2-chloro-4-pyridyl)-3-phenylurea (CPPU). It was found that CPPU could significantly enhance new axillary bud formation, branching, FW and DW than the control. Application of CPPU at 5 mg L-1 significantly promoted the highest contents of total reducing sugar at 2.5-fold in leaves and at 1.5-fold in roots. Interestingly, 5 mg L-1 CPPU could enhance andrographolide content (2.2-fold higher than the control) after 24 h treatment.

6.2.4

In Vivo Plants

Growth regulators such as IAA, NAA, and GA3 were used at different concentrations (25, 50, 75, 100 mg/L) to find their effect on andrographolide content in leaves of A. paniculata. Treatment of detached leaves with growth regulators showed increase in andrographolide content and maximum enhancement was observed with NAA at 100 mg/L (Vidyalakshmi and Ananthi 2013).

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Talei et al. (2015) reported that salinity decreased the photosynthetic parameters, protein content, total dry weight, and total crude extract but increased the AD content. In addition, the leaf protein analysis revealed that the two polymorphic protein bands as low- and medium-sized of 17 and 45 kDa acted as the activator agents for the photosynthetic parameters and AD content. A study carried out by Zhong et al. (2021) reported that organic N (NO3-, NH4+, urea, glycine) sources decreased carbohydrate depletion by reducing N metabolism and promoted plant growth and andrographolide biosynthesis synergistically. It is also reported that increasing S (nutrient solution containing sulfur) application rate enhanced the accumulation of andrographolide compounds (AGCs) in A. paniculata. Simultaneously, salicylic acid (SA) and gibberellic acid (GA4) concentrations were increased by high S, suggesting that they were involved in the S-mediated accumulation of phytochemicals. Taken together, the results indicated that increasing the S application rate is an effective strategy to improve AGC accumulation in A. paniculata (Jian et al. 2021).

6.3

Improved Production of Andrographolide Through Different Approaches

6.3.1

Biotic Elicitation

On testing the effect of different biotic elicitors such as yeast, Escherichia coli, Bacillus subtilis, Agrobacterium rhizogenes 532, and Agrobacterium tumefaciens C 58, yeast was found out to accumulate maximum amount of andrographolide (13.5 mg/g DCW), which was found to be 8.82-fold higher than the untreated cultures (Gandi et al. 2012). The treatment with fungal elicitors (Aspergillus niger and Penicillium expansum) for eliciting andrographolide production in the cell suspension culture of A. paniculata was studied by Vakil and Mendhulkar (2013b). A. niger extract (1.5 mL with10 days treatment duration) revealed 6.94-fold increase in andrographolide content (132 μg) and P. expansum elicitor (0.6% with 8 days treatment duration) could reveal 6.23-fold enhancement in andrographolide content (81.0 μg).

6.3.2

Chemical Elicitation

Salicylic acid (0.05 mM) application for 24 h duration resulted in about 18.5-fold increment in andrographolide content (37.0 μg/g). The chitosan treatment with 20 mg for 48 h duration explored highest elicitation of andrographolide (119.0 μg/ g, 59.5-fold) compared to the respective control and rest of the treatments. Chitosan mediated elicitation treatment was superior over the treatments of salicylic acid (Vakil and Mendhulkar 2013a). Among the various concentrations of MJ tested at different time periods, 5 μM MJ yielded 5.25 times more andrographolide content

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after 24 h of treatment. The accumulation of andrographolide was correlated with the expression level of known regulatory genes (hmgs, hmgr, dxs, dxr, isph, and ggps) of mevalonic acid (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. These results established the involvement of MJ in andrographolide biosynthesis by inducing the transcription of its biosynthetic pathways genes. The coordination of isph, ggps, and hmgs expression highly influenced the andrographolide biosynthesis (Sharma et al. 2015). Treatment of JA at 1.0 μM concentration resulted in 1.322% DW with 2.6-fold increase in andrographolide production after fifth week in shoot cultures of A. paniculata. However, JA treatment at 25 and 50 μM promoted 3.3, 3.0-fold enhancement in andrographolide production (1.624 and 1.481% DW), respectively, after eighth week compared to control. Treatment of 10, 20, and 50 μM SA resulted in 3.0, 3.4, and 3.1-fold andrographolide content (1.479, 1.654, and 1.483% DW), increase after eighth week, respectively, compared to control (0.478% DW) (Zaheer and Giri 2016). The influence of different chemical elicitors was evaluated for increased andrographolide production using adventitious root cultures of Andrographis paniculata. Maximum andrographolide content of 10.8-fold (2.548% DW) was obtained after the first week using adventitious root cultures elicited with 25 μM JA compared to control (0.234% DW). Among the concentrations of SA and its derivatives, elicitation with MeSA at 100 μM for 7 days promoted 2.6-fold increase in andrographolide production. JA was found superior than SA, and JA stimulated increased andrographolide content in adventitious root cultures of A. paniculata (Zaheer and Giri 2017). The production of andrographolide in the cell suspension cultures of Andrographis paniculata by eliciting with arachidonic acid was investigated by Ganeshkumar et al. in 2019. The maximum andrographolide production was found to 109.62% w/w over the control cell cultures (26.79% w/w) with the addition of arachidonic acid (100 μM) on day 7. Optimum callus induction was obtained with cotyledon and hypocotyls of the plant on Skoog and Hilderbrandt (SH) medium containing 2.0 μg/mL 2,4-D and 0.1 μg/mL BAP. Half MS medium containing 20 g/L sucrose and 20 h photoperiod showed highest cells fresh weight (CFW) (17.96 ± 0.06 g/50 mL), growth index (10.95 ± 0.96), and andrographolide yield (4.61 ± 0.688 mg/g DCW). The addition of copper sulfate (500 μM/L), methyl jasmonate (25 mg/L), chitin (500 mg/L), or fungal mycelium (500 mg/L) in separate experiments showed significant increase in bioproduction of andrographolide to the extent of 29.42 ± 0.31 mg/g DCW, 13.13 ± 0.11 mg/g DCW, 19.45 ± 0.68 mg/g DCW, and 13.629 ± 1.12 mg/g DCW, respectively, copper sulfate thus proved to be the most effective one (Dawande and Sahay 2020). A study by Das and Bandyopadhyay in 2021 revealed silver nitrate as a potent elicitor of andrographolide production in in vitro callus culture, when added in combination with the pathway inhibitors. The highest andrographolide production was obtained in callus treated with a combination of silver nitrate and lovastatin,

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indicating a predominant role of the plastidial DXP (deoxy-xylulose phosphate) pathway in andrographolide biosynthesis.

6.3.3

Hairy Root Culturing

Development of the hairy root culture of A. paniculata was conducted for growing the hairy roots and production of andrographollide. Different strains of Agrobacterium rhizogenes (R-1000, A4, ATCC 15834), different types of explants (cotyledons, hypocotyls, and leaves), and different infection time of A. rhizogenes (1, 2, 3 days) were tested to induce hairy roots of A. paniculata. The results indicated that the best strain, type of explants, and infection time for hairy roots induction were found in strain ATCC 15834, the explants of cotyledon, and the 2 days of infection, respectively. The best medium for growing the hairy roots was liquid half-strength MS medium with the addition of 5.0 μM IBA. The highest amount of andrographolide was observed in the medium with the addition of 5.0 μM IBA on week 2, as much as 0.54%. Integration of T-DNA of A. rhizogenes in hairy roots was confirmed by polymerase chain reaction (PCR) analysis with specific primer for rolA and rolC genes of the plasmid. Visualization of the PCR products on agarose gel electrophoresis showed two fragments with lengths of 248 bp and 490 bp which correspond to rolA and rolC genes from Ri plasmids of ATCC 15834 (Marwani et al. 2015).

6.3.4

Studies Revealing the Biosynthetic Pathway

In plants, plastidial 2C-methyl-D-erythritol-4-phosphate (MEP) and cytosolic mevalonic acid (MEV) pathways provide two 5C isoprenoid building blocks, dimethylallyl diphosphate (DMAPP), and isopentenyl diphosphate (IPP), for the biosynthesis of diverse terpene metabolites. IPP and DMAPP derived from the MEP pathway are converted to monoterpenes, diterpenes, and tetraterpenes, whereas those derived from the MEV pathway are converted to sesquiterpenes and triterpenes. Important metabolites of A. paniculata such as AD, NAD, DDAD belong to the group diterpene lactones and are synthesized through ent-LRD pathway. This involves the head-to-tail condensation of three IPP and one DMAPP to a C20 compound geranylgeranyl diphosphate (GGPP). This prenyl transfer reaction is catalyzed by the plastidial geranylgeranyl diphosphate synthase (GGPS). It is further cyclized into ent-diterpenyl diphosphate, e.g., ent-copalyl diphosphate (ent-CPP) following protonation-initiated cyclization mechanism catalyzed by the class II diterpene synthase (diTPS). Ent-diterpenyl diphosphate then acts as substrate for the class I diTPS that catalyzes further cyclization and/or rearrangement reactions to form AD, NAD, and DDAD (Garg et al. 2015). Misra et al. (2015) carried out a comparative transcriptional analysis using leaf and root tissues and identified 389 differentially expressed transcripts, including 223 transcripts that were preferentially expressed in leaf tissue. Analysis of the

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transcripts revealed various specialized metabolic pathways, including transcripts of the ent-LRD biosynthetic pathway. Two class II diterpene synthases (ApCPS1 and ApCPS2) along with one (ApCPS1′) and two (ApCPS2′ and ApCPS2″) transcriptional variants that were the outcomes of alternative splicing of the precursor mRNA and alternative transcriptional termination, respectively, were identified. ApCPS1 and ApCPS2 encode for 832- and 817-amino acids proteins, respectively, and are phylogenetically related to the dicotyledons ent-copalyl diphosphate synthases (ent-CPSs). The spatio-temporal patterns of ent-LRD metabolites accumulation and gene expression suggested a likely role for ApCPS1 in general (i.e., primary) metabolism, perhaps by providing precursor for the biosynthesis of phytohormone gibberellin (GA). However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Bacterially expressed recombinant ApCPS2 catalyzed the conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP), the general precursor of diterpenes to ent-copalyl diphosphate (ent-CPP), the precursor of ent-LRDs. Taken together, these results advance our understanding of the tissue-specific accumulation of specialized ent-LRDs of medicinal importance (Garg et al. 2015).

6.4

Molecular Advancements Related to AD Accumulation

6.4.1

Gene Transcripts

Shen et al. (2016a) demonstrated the expression of ApCPS in all tissues of A. paniculata at all growth stages, which is consistent with andrographolides accumulating in these organs. They also have revealed the induced expression of ApCPS gene and enhanced accumulation of andrographolides in upon MJ elicitation. Gene silencing of ApCPS resulted in decreased accumulation of andrographolides significantly with HPLC analysis. It was also shown that HMGR, DXS, and GGPS genes exhibited inducible gene expression with MeJA treatment as well as ApCPS, indicating pleiotropic regulation of MJ on andrographolides biosynthesis (Shen et al. 2016b). The qRT-PCR involving nine key pathway genes was studied, which revealed up regulation of GGPS1 and HMGR1/2 genes and downregulation of DXS1/2 and HDR1/2 genes in the adventitious root as compared to that in the control leaves. Such observations highlight that in vitro cultures can serve as efficient production alternatives for AD/NAD as the cytosolic genes (HMGR1/2 of MVA pathway) are competent enough to take over from the plastidial genes (DXS1/2 and HDR1/2 of MEP pathway), provided the accredited first branch-point regulatory gene (GGPS) expression and the culture requirements are optimally fulfilled (Singh et al. 2018). A quantitative RT-PCR study on expression of tissue-specific ApDXS (A. paniculata 1-deoxy-D-xylulose-5-phosphate synthase) and ApHMGR (A. paniculata 3-hydroxy-3-methylglutaryl-coenzyme) and genes revealed maximum fold expression in the leaves compared to other parts in JA treated in vitro adventitious root cultures. The highest expression of both genes was found at 25 μm

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JA elicitation which were correlated with increased AD accumulation (9.38-fold) (Srinath et al. 2021). Out of 6853 transcripts, 1370 of transcripts were represented by terpenoid biosynthetic pathway, which involved in secondary metabolite andrographolide biosynthesis. in which nine important differentially expressed transcripts related to MEP (2C methyl-d-erythritol 4-phosphate) and MVA (mevalonic acid) andrographolide biosynthesis pathways. The highest expression of gene 13-hydroxy-3-methylglutaryl-coenzyme a reductase (HMGR) was reported, which is responsible for accumulation of andrographolide in leaf. These upregulated genes could be overexpressed to enhance the andrographolide content using genetic engineering of these metabolic pathways. It will also give an idea to the breeder for development of molecular markers for direct screening of the genotypes (Patel et al. 2020).

6.4.2

Transcription Factors

Various transcription factors such as DREB’s, MYC’s, WRKY, and MED which uniquely influenced terpenoid biosynthesis were identified through differential expression study following JA elicitation in A. paniculata plantlets (Bindu et al. 2020). WRKY transcription factors related to andrographolide biosynthesis were systematically identified, including sequences alignment, phylogenetic analysis, chromosomal distribution, gene structure, conserved motifs, synteny, alternative splicing event, and gene ontology (GO) annotation by Zhang et al. (2021). A total of 58 WRKYs were identified and the combination of binding site prediction, genespecific expression patterns, and phylogenetic analysis suggested that 7 WRKYs (ApWRKY01, ApWRKY08, ApWRKY12, ApWRKY14, ApWRKY19, ApWRKY20, and ApWRKY50) might regulate andrographolide biosynthesis.

6.4.3

Proteome Analysis

Maximum increase in AG content was noticed during salinity stress. Interestingly, the leaf protein analysis revealed that the two polymorphic protein bands as low- and medium-sized of 17 and 45 kDa acted as the activator agents for the photosynthetic parameters and AG content (Talei et al. 2015). Protein samples from both JA treated and untreated control plantlets analyzed by Q-TOF–LC–MS/MS showed the enhanced expression of 22 proteins involved in the isoprenoid pathway in treated and three with untreated plantlets. About 40 metabolic processes identified by functional annotation of proteins and highly elevated (5.7%) post-translational modifications were observed in JA treated. It also unveiled the appearance of additional secondary metabolism related proteins; primarily phenylpropanoids, isoprenoids, and flavonoids from elicited cultures (Bindu et al. 2020). The strategies followed in the research of in vitro production of AP and its metabolites have been summarized in Fig. 6.2.

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Fig. 6.2 Overview of different approaches followed so far to improve the production of important metabolites of A. paniculata

6.4.4

Concluding Remarks

Andrographis paniculata is a most potent medicinal plant with noteworthy effects on many pharmacological conditions. Andrographolide, the significant triterpene lactone of the plant is potent to treat upper respiratory tract infections. In addition, the whole plant extract of AP is used as liver tonic, immunity booster, etc. As per the report published by Marketandresearch.biz, the global AP extract market is expected to grow from USD 125.20 million in 2021 to USD 305.01 million by 2030. Plant tissue culture-based strategies offer an effective platform to meet this burgeoning market demand by providing genetically stable in vitro cultures and methods to accumulate the vital chemical compounds. Reproducible protocols for in vitro regeneration of AP were framed by a number of researches. Fungal cells and chemicals such as methyl jasmonate, salicylic acid, arachidonic acid, and copper sulfate have been reported to enhance the production of AD. Agrobacterium rhizogenes mediated hairy root culturing has also been reported by one researcher. is available. Molecular studies determine the increased expression of gene transcripts, transcription factors, and proteome associated with the biosynthesis of triterpenoid lactones. But still, studies related to approaches such as precursor feeding, nano-elicitation, Agrobacterium tumefaciens transformation, bioreactorbased culture production should be fully investigated. Hence this review would invite young researches to explore the unexplored areas of AP biotechnology.

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Murthy HN, Dalawai D, Bhat MA, Dandin VS, Paek KY, Park SY (2021) Biotechnological production of useful phytochemicals from adventitious root cultures. In: Ramawath KG, Ekiert HM, Goyal S (eds) Plant cell and tissue differentiation and secondary metabolites: fundamentals and applications. Springer Nature, Switzerland, pp 469–486 Nagegowda DA (2010) Plant volatile terpenoid metabolism: biosynthetic genes, transcriptional regulation, and subcellular compartmentation. FEBS Lett 584:2965–2973 Paritala V, Mohammed A, Kayat F (2017) In vitro plant regeneration of Andrographis paniculata Nees using mature zygotic embryonic explants. Agri Res Tech 5(5):121–128 Patel AA, Shukla YM, Kumar S, Sakure AA, Parekh MJ, Zala HN (2020) Transcriptome analysis for molecular landscaping of genes controlling diterpene andrographolide biosynthesis in Andrographis paniculata ( Burm f.) Nees. 3 Biotech 10(12):512 Prathanturarug S, Soonthornchareonnon N, Chuakul W, Saralamp P (2007) Variation in growth and diterpene lactones among field cultivated Andrographis paniculata. J Nat Med 61:159–163 Praveen N, Manohar SH, Naik PM, Nayeem A, Jeong JH, Murthy HN (2009) Production of andrographolide from adventitious root cultures of Andrographis paniculata. Curr Sci 96: 694–697 Purkayastha J, Sugla T, Paul A, Solleti A, Sahoo L (2008) Rapid in vitro multiplication and plant regeneration from nodal explants of Andrographis paniculata: a valuable medicinal plant. In Vitro Cell Dev Biol Plant 44:442–447 Roy PK (2014) In vitro propagation of Andrographis paniculata Nees—a threatened medicinal plant of Bangladesh. Jahandgirnagar Univ J Biol Sci 3:67–73 Roy S, Giri A, Bhubaneswari C, Narasu LM, Giri CC (2009) High frequency plantlet regeneration Vis direct organogenesis in Andrographis paniculata. Med Aromat Plant Sci Biotechnol 3(1): 94–96 Sareer O, Ahmad S, Umar S (2014) Andrographis paniculata: a critical appraisal of extraction, isolation appraisal of extraction, isolation, and quantification of and other active constituents. Nat Prod Res 28:2061–2101 Sharma SN, Jha Z, Sinha RK (2013) Establishment of in vitro adventitious root cultures and analysis of andrographolide in Andrographis paniculata. Nat Prod Commun 8:1045–1047 Sharma SN, Jha Z, Sinha RK, Geda AK (2015) Jasmonate-induced biosynthesis of andrographolide in Andrographis paniculata. Physiol Plant 153(2):221–229 Sharmila R, Subburathinam KM, Sugumar P (2013) Effect of growth regulators on andrographolide production in callus cultures of Andrographis paniculata. Adv BioTech 12(9):17–19 Shen Q, Li L, Jiang Y, Wang Q (2016a) Functional characterization of ent-copalyl diphosphate synthase from Andrographis paniculata with putative involvement in andrographolides biosynthesis. Biotechnol Lett 38:131–137 Shen Q, Liu Q, Li L, Fu Y, Wnag Q (2016b) Functional characterization of ApCPS involved in andrographolides biosynthesis by virus-induced gene silencing. Acta Bot Boreal Occid Sin 1: 17–22 Singh S, Pandey P, Ghosh S, Banerjee S (2018) Anti-cancer labdane diterpenoids from adventitious roots of Andrographis paniculata: augmentation of production prospect endowed with pathway gene expression. Protoplasma 255:1387–1400 Srinath M, Shailaja A, Bindu BBV, Giri CC (2021) Molecular cloning and differential gene expression analysis of 1-Deoxy-D-xylulose 5-Phosphate Synthase (DXS) in Andrographis paniculata (Burmf) Nees. Mol Biotechnol 63:109–124 Tajidin NEA, Shaari K, Maulidiani M, Salleh NS, Ketaren BR, Mohamad M (2019) Metabolite profiling of Andrographis paniculata (Burm. F.) Nees. Young and mature leaves at different harvest ages using 1H NMR-based metabolomics approach. Sci Rep 14(9):16766 Talei D, Valdiani A, Maziah M, Sagineedu SR, Abiri R (2015) Salt stress-induced protein pattern associated with photosynthetic parameters and andrographolide content in Andrographis paniculata Nees. Biosci Biotechnol Biochem 79(1):51–58

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Identification of Bioactive Compounds in Berberis Species and In Vitro Propagation for Conservation and Quality Shalini Tiwari and Charu Lata

Abstract

The Berberis (Berberidaceae) genus consist large deciduous shrub that is found in Western Himalaya, which includes approximately 500 species worldwide. Berberis plants have been shown to have a wide spectrum of medicinally and nutritionally significant phytochemical components. Chemical profiling of plant tissues, including fruit, leaf, root, and stem showed the presence of various bioactive compounds, mainly including magnoflorine, berbamine, berberine, etc. in it. These compounds are alkaloids, tannins, phenolic, sterols, and triterpenes in nature and have medicinal properties as antimicrobial, antipyretic, anti-inflammatory, anti-arrhythmic, anti-cholinergic, anti-leishmaniasis, antimalaria, and as sedative. Being a traditional medicine since many centuries, there is an increasing trend to enhance the production of bioactive compounds from it. Therefore, to tackle the problem of overexploitation, the present chapter mainly focuses on the identification of bioactive compounds and in vitro propagation for conservation and assessment of quality of micropropagated plants for the sustainable use of Berberis species plants. Keywords

Bioactive compounds · Benzylisoquinoline · Protoberberine · Ex situ conservation

S. Tiwari (*) School of Life Sciences, Jawaharlal Nehru University, New Delhi, India C. Lata CSIR-National Institute of Science Communication and Policy Research, New Delhi, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_7

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7.1

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Introduction

Extracts of plants that can be used for the treatment of different ailments either directly or indirectly are categorized as medicinal plants. In most developing countries, traditional medicine and medicinal plants play a major role in the maintenance of good health. Due to the presence of bioactive compounds in the different parts of plants, various plants have medicinal values, i.e. they can be used in drug development either pharmacopoeial, non-pharmacopoeial, or synthetic drugs and have the capability to produce physiological actions in humans (Fitzgerald et al. 2020). Common bioactive compounds that are found in medicinal plants are alkaloids, glycosides, resins, gums, mucilages, etc. (Rabizadeh et al. 2022). Among various plants of medicinal value, Berberis L. is one of the largest genera under the family Berberidaceae which is known for its vast medicinal properties. This genus includes approximately 500 species distributed in tropical and subtropical regions of the northern hemisphere (Mokhber-Dezfuli et al. 2014). South America and Asia have the greatest species diversification, although native species also exist in Europe, Africa, and North America. In India, the genus Berberis has ~55 species with the majority distribution in the Himalayan region. Apart from the Himalayan region, five species, namely B. asiatica, B. hainesii, B. nilegrica, B. tinctoria, and B. wightiana are found in Nilgiri Hills, Chhota Nagpur Plateau, and Pachmarhi Hills of Madhya Pradesh (Tiwari et al. 2012). Berberis plants are mainly valued due to the presence of the chief alkaloid berberine. This alkaloid is effective against various infections and diseases. It also possesses antipyretic, diaphoretic, astringent, and stomachic properties. Due to these diverse medicinal characteristics, plants of this genus are overexploited for the production of various drugs. The utilization of medicinal plants for the development of new drugs is becoming more and more prevalent in industrialized cultures which leads to biodiversity loss of them. Consequently, scientists and pharmaceutical companies nowadays are inclined toward the in vitro propagation of plants throughout the world for the conservation of plants as well as to explore the various bioactive components of medicinal plants to help humanity. Therefore, in the present chapter, we discussed the identification of bioactive components of Berberis genus plants, their medicinal properties as well as the in vitro methods for their conservation.

7.2

Medicinal Significance of Berberis

Berberis genus plants are known for their medicinal properties. Several studies around the globe reported its pharmaceutical usage. Berberis aristate commonly known as Tree turmeric or Daruharidra possesses antibacterial, antiseptic, antiinflammatory, and antipyretic properties. This herb is used as a cholagogue, stomachic, laxative, and diaphoretic (Choudhary et al. 2021). In Indian traditional knowledge, Sushruta Samhita, Agnivesh Charak Samhita, and Glossary of Indian medicinal Plants mentioned the use of Daruharidra (B. aristate) for the treatment of kidney stones and other kidney disorders. Recently in the year 2021, CSIR-NBRI

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developed a medicine named URO-5 for urolithiasis (kidney stone) in which B. aristate is one of the key components. Being a traditional medicine and vast medicinal properties of URO-5, the Government of India has included it under SVASTIK—Scientifically Validated Societal Traditional Knowledge for effective communication of traditional knowledge to the public. Previous studies suggested that B. vulgaris can also be used as therapeutics for different forms of urolithiasis (Alok et al. 2013; Akram and Idrees 2019). A review documented the ethnopharmacological uses of Berberis species plants in the diabetes, hypertension, and obesity treatment (Belwal et al. 2020). This group also reported that B. lycium is used for the treatment of fever, intestinal colic, diarrhoea, jaundice, ophthalmic disorders, bone fractures, rheumatism, menorrhagia, and diabetes mellitus. In Indian traditional medicine B. aristata stem is widely used for diabetes treatment (Upwar et al. 2011). A bisbenzylisoquinoline alkaloid named Berbamine extracted from B. thunbergii shows a number of potential pharmacological traits and have capability to prevent cardiac ischemia (Zheng et al. 2017). In addition, berbamine can also treat infection of influenza virus (Wang and Yang 2021). Berbamine can also prevent Japanese encephalitis virus infection by interfering endolysosomal trafficking of the low-density lipoprotein receptor’s (Huang et al. 2021a). Same group of researchers also investigated that berbamine inhibits the infection of flaviviruses and SARS-CoV-2 similarly by compromising endolysosomal trafficking (Huang et al. 2021b). Marahatha et al. (2022) showed that berbamine along with Neferine has the potential to inhibit the SARS-CoV-2 RNA-dependent RNA polymerase. A study using in silico approach suggested that berbamine, rutin, and oxyacanthine from B. asiatica act as anti-SARS-CoV-2 compounds (Joshi et al. 2021). A recent study by Wang and Yang (2021) mentioned Chinese herbal medicine that fights SARS-CoV-2 infection on all fronts contains a component names Berbamine that was isolated from the plant Berberis thunbergii DC. However, for detailed medicinal significance a list of Berberis genus plants found in India and their medicinal usage are listed in Table 7.1.

7.3

Phytochemical Components and Their Identification from Berberis Family Plants

Phytochemical analyses of Berberis genus plants have revealed the presence of approximately 105 bioactive compounds with varied structural confirmations. This phytochemical screening involves determining the presence of several secondary metabolites, including flavonoids, alkaloids, steroids, triterpenoids, sugars, tannins, and others, and estimating their amounts (Belwal et al. 2020). The majority of bioactive components in the Berberis genus is alkaloids. There are several different alkaloids that have been identified from Berberis species; however the most prevalent ones include berberine, berbamine, jatrorrhizine, palmitine, and isotetrandrine. Berberine is one of the significant and potential compounds of the Berberis genus, which is benzylisoquinoline alkaloids of the protoberberine group. The protoberberine is a family of organic cations with a distinctively yellow colour. It

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Table 7.1 List of Berberis genus plants and their medicinal usage S. No. 1.

Name Berberis affinis

Medicinal properties Jaundice, skin, and eye problem

2.

Berberis aristata

3.

Berberis asiatica Berberis atrocarpa Berberis baluchistanica Berberis brandisiana Berberis calliobotrys Berberis chitria

Diarrhoea, haemorrhoids, gynaecological disorders, HIV-AIDS, osteoporosis, diabetes, eye and ear infections, wound healing, jaundice, skin diseases, and malarial fever Blood purifier, conjunctivitis, fever, cough

4. 5. 6. 7. 8.

9.

Berberis crataegina

10.

Berberis glaucocarpa Berberis heteropoda Berberis holstii

11. 12. 13.

Berberis integerrima

14.

Berberis jaeschkeana Berberis kongboensis Berberis kumaonensis Berberis libanotica Berberis lycium

15. 16. 17. 18.

19.

Berberis microphylla

Weight control, reduce blood glucose and cholesterol Jaundice, digestive problems, gynaecological problems, snakebite Arthritis, dysentery, sore throat, wound healing Internal wound Jaundice, enlargement of spleen, leprosy, rheumatism, fever, morning/evening sickness, and snakebite Antifungal activity, anti-inflammatory, antipruritic, and diuretic effects Antileishmanial, anti-inflammatory, antipyretic Dysentery, enteritis, pharyngitis, stomatitis, eczema, and hypertension Cough, malaria, stomachache, pneumonia, sexually transmitted disease Hypoglycemic, anti-hypertensive, antipyretic, anti-gout, jaundice, blood purifier Diuretic, blood purifier, eye disorder, jaundice, menorrhea, skin disease Diarrhoea, gastrointestinal diseases Diuretic, blood purifier, eye disorders, jaundice, skin disease Rheumatism, neuralgic disease, diarrhoea Jaundice, diabetes, diarrhoea, eye disorder, toothache Cardiovascular disease, reduce cholesterol level

References Sharma and Devi (2013) Potdar et al. (2012)

Shrestha and Dhillion (2003) Abudureheman et al. (2022) Bibi et al. (2014, 2015) Bibi et al. (2015) Jan et al. (2011) Srivastava and Rawat (2014) Demirci et al. (2021) and Eroğlu et al. (2020) Alamzeb et al. (2021a, b) Sun et al. (2022) Maliwichi-Nyirenda et al. (2011) Amiri et al. (2014)

Gaur et al. (1983) and Singh et al. (2009) Chen et al. (2021) Gaur et al. (1983) Jan et al. (2011) and Diab et al. (2015) Kapoor et al. (2013) and Sharma and Devi (2013) Olivares-Caro et al. (2020) (continued)

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Table 7.1 (continued) S. No. 20. 21. 22. 23. 24. 25. 26.

27. 28. 29. 30.

31.

Name Berberis nummularia Berberis orthobotrys Berberis pachyacantha Berberis petiolaris Berberis pruinosa Berberis pseudumbellata Berberis tinctoria Berberis ulicina Berberis umbellata Berberis vulgaris Berberis wallichiana

Berberis xanthophloea

Medicinal properties Weight control, reduce blood glucose and cholesterol Diabetes, eye disorder, dysentery, bone fracture, wound Fever, stomach disorder

References Abudureheman et al. (2022) Khan et al. (2015)

Treat malarial fever, diarrhoea, conjunctivitis, and jaundice Diarrhoea

Neag et al. (2018) and Karimov (1993) Li et al. (2015)

Jaundice, cold, cough, fever, eye irritation

Singh (2012)

Rheumatism, jaundice, diabetes, fever, stomach disorder, skin disease, malaria fever, eye and ear disease Ring worm

Rawat and Srivastava (2007)

Skin problem, fever Hepatic problems, indigestion, antipyretic, anti-gout, diarrhoea, dysentery Heal wounds, diarrhoea, fever, eye diseases, jaundice, pregnancy vomiting, rheumatism, kidney stones, and gallstones, cytotoxicity against several cancer cell lines Diarrhoea, gastrointestinal diseases

Singh et al. (2009)

Buth and Navchoo (1988) Sharma and Devi (2013) 49, 72 Eroğlu et al. (2020) Bui et al. (2022)

Chen et al. (2021)

has four fused benzene rings in which nitrogen links two ring pairs, and one is modified by two oxygen atoms at each end. The berberine content of the plants gradually rises with age of plants. The cortical tissues present in the roots and stems of Berberis contain the majority of the plant’s alkaloids. The largest concentration of alkaloids is found in the bark of old roots. Very low concentrations of alkaloids are found in the top portions of the stem; however, no alkaloids are detected in young leaves (Srivastava et al. 2015). Apart from it, alkaloid content also differs between Berberis plants of different geographical locations. A vast number of bioactive components are present in plants at various developmental stages and in different plant tissues such as roots, leaves, fruit samples, etc. Various techniques have been used till date to investigate the components present in plants. Phytochemicals were extracted from leaves of Berberis thunbergii using HPLC-/ESI-MSn and from Berberis hispanica using HPLC–DAD–MS/MS (Fernández-Poyatos et al. 2019, 2021). Moldovan et al. (2021) developed an optimized drying process for the recovery of bioactive compounds from the autumn fruits of Berberis vulgaris L. Villinski et al. (2003) also investigated alkaloid content

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from B. thunbergii using HPLC-DAD-ESI/MSn. Profile of alkaloids was also studied in B. thunbergii using UPLC-MS/MS (Hussain et al. 2017; Och et al. 2017). Gulfraz et al. (2004) used column chromatography and thin layer chromatography to separate alkaloids from the root and fruit of Berberis lyceum.

7.4

Conservation by Means of In Vitro Propagation

Excessive exploitation of medicinal plants for the benefits of humankind causes biodiversity loss at mass level. As in the case, the COVID-19 pandemic sickness is having a detrimental influence on human society in most of the world’s nations, while biodiversity is benefiting from decreased human activity as a result of the lockdown in most of the nations. This predicament also emphasizes the current requirement for the protection and conservation of plants. Apart from the ecological conservation and restoration, in vitro conservation also plays essential role in maintenance of plant genetic diversity and endurance of plant species. The main purpose of in vitro techniques is for rapid multiplication, disease-free plant, and gene bank conservation. Establishing an effective in vitro micropropagation technology is a great strategy for both rapid plant growth and ex situ conservation of the plant germplasm specifically medicinal plants germplasm (Mishra et al. 2020). Apart from this in vitro propagation also helps quality improvement, i.e. augmenting bioactive compounds. Mishra et al. (2021) reported the augmentation in phenolic content in the in vitro propagated Thalictrum foliolosum plant. In a recent study researchers micropropagated B. asiatica for harnessing its potential as a source of berberine and natural antioxidants (Bisht et al. 2022). In a phytochemical assessment of B. chitria Pandey et al. (2013) observed that total phenol component is comparatively higher in in vitro propagated plants than wild types. Thus, in vitro propagation of medicinal plants for rapid growth and quality improvement is the need of the hour for the sustainable use of the limited bioresources and emerging pharmaceutical industries.

7.5

Conclusion and Future Perspectives

The in-depth study of medicinal plants and their traditional use across the world has increased during the past several decades. Folk medicine reports that were then subjected to serious scientific review have offered the world more sources for remedial, preventative, and even somewhat curative actions in a variety of ailments. Among the most significant traditional herbs, Berberis species have a wide range of pharmacological actions. A greater potential for cardiovascular, hepatoprotective, antibacterial, and anticancer properties was found in Berberis and its separated alkaloids. This chapter also attempts to assess the current status of the application of various in vitro approaches for the conservation of threatened medicinal plants.

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Bioactive Compounds in Solanum viarum: Medicinal Properties, In Vitro Propagation, and Conservation Shatrujeet Pandey and Samir V. Sawant

Abstract

Solanum is one of the largest genera of the family Solanaceae that contains about 1500 species mainly dispersed around the tropical and subtropical regions of the globe. A great number of economically important plant species used for food, ornamental, and medicinal purposes belong to Solanum. Fruits of S. viarum are known as rich sources of steroidal alkaloids and glycoalkaloids, which exhibit a variety of biological activities such as anti-inflammatory, anticancerous, antifungal, antimicrobial, antiviral, insecticidal, etc. Various reports also show the presence of significant amounts of other important phytochemicals such as phenolics, flavonoids, saponins, and terpenoids. These plants provide the raw material for the commercial production of several pharmaceutically important steroidal drugs, which are used for the treatment of several chronic diseases. Due to the paramount medicinal properties of these plants, considerable effort has been made to unravel the phytoconstituents for in vitro establishment and for the enrichment of essential bioactive compounds. This chapter provides detailed information about phytochemistry and pharmaceutical uses, in vitro mechanisms for large-scale micropropagation and enrichment of vital phytochemicals, and conservation approaches for these natural resources. Keywords

Alkaloid · Solanum viarum · In vitro conservation · Phenolics and flavonoids

S. Pandey · S. V. Sawant (✉) CSIR-National Botanical Research Institute, Lucknow, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_8

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Introduction

In recent years, the world has seen increasing interest in medicinal plants. They are traditional medicines or natural remedies in their effective and pure form and characterized by effective high therapeutic activities and chemical preparations without side effects. Medicinal plants occupy a special place in agricultural production. It is currently considered one of the most important strategic materials in the pharmaceutical industry, that is, in the middle of the prefix in the chemical composition of drugs (Djaafar and Ridha 2014). Solanum, one of the largest and most complex genera among the Angiosperms, is considered as the largest and most representative genus in the Solanaceae family. About 1500 species constitute this genera, which are dispersed across subtropical and tropical regions of Asia, non-arid Africa, tropical Africa, Americas, Australia, and India. Many of these species are commercially very important. The S. lycopersicum, S. melongena, and S. tuberosum are widely used as food crops. S. aviculare, S. capsicastrum, S. crispum, S. laciniatum, S. laxum, S. pseudocapsicum are important ornamental species. Several pharmacological studies have been conducted to validate the traditional medicinal uses of many plants of the genus Solanum. This genus has drawn enormous attention in chemical and biological investigations during the last 30 years. It is a rich source of steroidal alkaloids, saponins, phenolics, and terpenoids present in various parts of the plant that exhibit various pharmacological activities. Many species of nightshade are often used in folk remedies. The presence of the steroid alkaloid solasodine, an important raw material for the synthesis of steroid hormones, is a characteristic active ingredient of solanum, which greatly influences the economic and medicinal use of this genus worldwide. The lack of immediately known uses for some members of this group has led to neglect and subsequent genetic erosion. This potential depletion of useful plant resources must be addressed with an emphasis on germplasm studies and conservation. This study provides details of the bioactive compounds of Solanum viarum used in traditional and folk remedies worldwide for the treatment of various diseases like leprosy, diabetes mellitus, and toothache (Tag et al. 2012; Udayan et al. 2006; Deka and Deka 2007). In vitro propagation and conservation strategies will also be discussed. Solanum viarum Dunal (Solanum khasianum var. chatterjeeanum Sengupta & Sengupta) is known as the tropical soda apple, and in India commonly known as “kanthakari.” It is widely spread in north-east, north-west, south, and central India and extends to Burma and China (Sreeramu 2004). The aerial part of S. viarum like stem, leaf, and petioles including calyx is packed with a sharp and large prickle, which makes harvesting of the fruit very laborious, tedious, and time-consuming (Singh and Kaushal 2007). The mechanized harvesting of ripe berries is not possible due to their non-synchronous maturity. Considering economic value significant effort to develop prickleless verity has been made and CSIR-National Botanical Research Institute, Lucknow, has obtained a prickleless variety during the breeding program of S. viarum (Singh et al. 1998; Khanna and Singh 1985). The prickleless variety (INGR19030) having a high content of alkaloids was later named as

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“Nishkantak” and submitted to the National Bureau of Plant Genetic Resources (Misra et al. 2020). Nishkantak has highly rich in Solasodine content in the field (berries, leaves, roots, and stems) and under in vitro (berries, leaves, roots, and stems) conditions (Patel et al. 2021; Misra et al. 2020). Other medicinally important phytochemicals like phenolics and flavonoid content also improved (Patel et al. 2021; Pandey et al. 2018, 2020). Due to improved biosynthesis of bioactive phytomolecules, prickleless variety is a better alternative for the pharmaceutical production of steroidal alkaloids.

8.2

Bioactive Compounds of Solanum Viarum

S. viarum is a medicinal plant having valuable status in the drug and pharmaceutical industry (Eltayeb et al. 1997) due to the presence of steroidal alkaloids solasodine. Solasodine is a nitrogenous analogue of diosgenin and easily convertible to 16-dehydro pregnenolone acetate (16-DPA), a key intermediate in the synthesis of several steroidal drugs, such as progesterone and cortisone (Shilpha et al. 2015; Bhatnagar et al. 2004). This plant is also rich in several other steroidal alkaloids and glycoalkaloids such as α-solanine (glycoalkaloid), solamargine (glycoalkaloid), solanidine (aglycone), solasonine (glycoalkaloid), etc. (Table 8.1, Figs. 8.1 and 8.2). These metabolites possess a wide variety of therapeutic activities such as anti-obesity (Khasero and Somani 2016), anticancerous (Daunter and Cham 1990), antifungal (Chataing et al. 1998), antimicrobial, antiviral (Ripperger 1998; Tadeusz 2007), antibiotic, insecticidal, anti-inflammatory, anthelmintic (Patel et al. 2013; Jarald et al. 2008), and anti-herpes activities (Ikeda et al. 2000). Biosynthesis of steroidal alkaloid and glycoalkaloid is initiated with the acetylCoA (C5) via cytosolic mevalonate or isoprenoid pathway. The enzyme 3-hydroxy3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the first step in the biosynthesis of isoprenoids and plays a key role in their biosynthesis (Stermer et al. 1994). A schematic biosynthesis pathway is represented in Fig. 8.1. The accumulation of α-solanine, solanidine, and solasodine in the aerial part of S. viarum under in vitro conditions is in an age-dependent manner and influenced by the expression of mevalonate kinase (MVA), HMGR, and UDP-galactose/ solanidine galactosyltransferase (SGT1), UDP-glucose/solanidine glucosyltransferase (SGT2), and farnesyl diphosphate synthase (FPS) the key enzymes of the biosynthetic pathways (Prasad et al. 2020). S. viarum the richest source of steroidal alkaloid serves as an alternate source for the steroid drug industry to ensure the sustainable supply of the raw material for the commercial production of the pharmaceutically important contraceptive steroid (Eltayeb et al. 1997). Although steroidal alkaloids are also found in other members of Solanaceae but the S. viarum is a promising source for the commercial production of solasodine in India (Singh and Kaushal 2007). Traditionally, the ripe berries of S. viarum are considered as the richest source of solasodine and production of quality fruits that need 6–8 months. Several studies on plants grown under in vitro conditions have now emerged as alternate strategies for the production of solasodine

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Table 8.1 Chemical constituents in different tissues of Solanum viarum Dunal Group Steroidal alkaloids and glycosides

Chemical constituents Solasodine

Solanidine

α-Solanine

Phenolics and flavonoids

Solaviaside A Solaviaside B Solaviaside C Indioside C Solamargine Anguivioside XV Aculeatiside A Protodioscin Gallic acid

Caffeic acid

Sinapic acid Ferulic acid

Benzoic acid Catechol Rutin

Quercetin

Kaempferol Viarumacid A Viarumacid B

Origins Leaf, stem, root, fruits, and stem epidermis Leaf, stem, root, fruits, and stem epidermis Leaf, stem, root, fruits, and stem epidermis Fruits Fruits Fruits Fruits Fruits Fruits

References Ono et al. (2009), Pandey et al. (2018) and Patel et al. (2021)

Fruits

Ono et al. (2009)

Fruits Leaf, stem, root, fruits, and stem epidermis Leaf, stem, root, fruits, and stem epidermis Leaf, stem, root, and fruits Leaf, stem, root, fruits, and stem epidermis Leaf, stem, root, and fruits Leaf, stem, root, and fruits Leaf, stem, root, fruits, and stem epidermis Leaf, stem, root, fruits, and stem epidermis Leaf, root, and stem epidermis Fruits Fruits

Ono et al. (2009) Pandey et al. (2018) and Patel et al. (2021)

Pandey et al. (2018) and Patel et al. (2021) Pandey et al. (2018) and Patel et al. (2021) Ono et al. (2009) Ono et al. (2009) Ono et al. (2009) Ono et al. (2009) Ono et al. (2009) Ono et al. (2009)

Pandey et al. (2018) and Patel et al. (2021) Patel et al. (2021) Pandey et al. (2018) and Patel et al. (2021) Patel et al. (2021) Patel et al. (2021) Pandey et al. (2018) and Patel et al. (2021) Pandey et al. (2018) and Patel et al. (2021) Pandey et al. (2018), Patel et al. (2021) Wu et al. (2012) Wu et al. (2012)

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Fig. 8.1 A schematic biosynthesis pathway for steroidal alkaloids/glycosides Solanaceous plant. Broken arrows indicate the involvement of multiple steps. HMGR 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, GAME glycoalkaloid metabolism, SGT1 UDPgalactose/solanidine galactosyltransferase, SGT2 UDPglucose/solanidine glucosyltransferase, SGT3 rhamnosyltransferase

in a comparatively shorter time frame. Pandey et al. (2020) have established an efficient procedure for in vitro cultivation using different aerial parts and estimated solasodine yields. In addition to berries, these alkaloids and glycoalkaloids were also analyzed in a noteworthy amount in the aerial part of in vitro-grown plants (Prasad et al. 2020). These alkaloids and glycoalkaloids are also produced via hairy root

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Fig. 8.2 Chemical structures of important Solanum alkaloids (https://pubchem.ncbi.nlm.nih.gov/)

culture (Srivastava et al. 2016). Thus, in vitro cultivation of various parts of S. viarum could use an alternate strategy for large-scale commercial production of solasodine. Thermal stress (cold/heat) enhances the accumulation of steroidal alkaloids, glycoalkaloids, phenolics, and flavonoids in S. viarum (Patel et al. 2022). Besides the bioactive alkaloids, other pharmaceutically important phytochemicals like phenolics and flavonoids also accumulate in several parts of the plant such as berries (Braguini et al. 2018; Patel et al. 2021), leaves, stems, and roots of field-grown plants (Patel et al. 2021) and the stem, root (Patel et al. 2021), leaves (Patel et al. 2021; Pandey et al. 2020), and epidermis (Pandey et al. 2018) of in vitro-grown plants (Table 8.1). These phenolics and flavonoids contribute to various antioxidant activities (Braguini et al. 2018; Prasad et al. 2022).

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In Vitro Propagation and Conservation

According to the International Union for Conservation of Nature (IUCN), of the more than 12,000 plant species, about 70% are under threatened and 19% are in the critically endangered category (Trejgell et al. 2015). The development of a conservation strategy for every economically important plant is vital to preserving their pure genetic resources. Traditionally, plant germplasm is conserved in the field as a whole plant, which has the risk of contamination by various infections of microbial agents (bacteria, viruses, fungi) and losses due to abiotic disasters, and it is expensive too. In vitro conservation of plant germplasm is an ex situ biotechnological approach to preserve plant biodiversity free from vulnerable depletion by harsh environmental (biotic/abiotic) conditions (Paunescu 2009; Dhillon and Saxena 2003). In vitro conservation is used for the vegetatively propagated plant species, threatened/endangered species, and genetically modified and elite genotypes of plants. In vitro preservation could be categorized as short, mid, and long term based on the period of storage. Usually short- and mid-term storage is used for plants because long-term storage (-196 °C) has technical complexity and is too expensive. It is applied shortly to medium-term storage of vegetative culture under slow growth under optimized in vitro conditions (Chauhan et al. 2019). Various parts of S. viarum such as the stem, leaf, root, and petiole have efficient regeneration capability. TDZ induces shoot induction up to 100% without influencing genetic stability and thus could use for ex situ conservation in the future (Pandey et al. 2020). Nodal segment and excised root culture are being used for in vitro multiplication and maintenance from ~35 to 40 years to conserve genetic stability of the elite genotype (prickleless genotype) of S. viarum at National Botanical Research Institute (NBRI), Lucknow (Misra et al. 2020). Furthermore, the hairy root culture has been established for in vitro multiplication and conservation of S. viarum (Srivastava et al. 2016). To the best of my knowledge, long-term storage is still not reported for this plant.

8.4

Conclusion

S. viarum is a promising source of solasodine used for the production of contraceptive steroids by the pharmaceutical industry. Its extract is used by local people for various remedial activities like the cure of leprosy, diabetes mellitus, and toothaches for a long time (Tag et al. 2012; Udayan et al. 2006; Deka and Deka 2007). In vitro conservation of plants is a potential tool being used to preserve pure genetic stock for a long time with the least input (Chauhan et al. 2019). Considering the global need and medicinal importance, immense efforts for the improvement of alkaloid and glycoalkaloid levels, for development of efficient metabolite extraction procedure, validation of therapeutic activities under various medical conditions, identification and quantification of novel metabolic compounds and their therapeutic uses, efficient strategies for large-scale in vitro multiplication and establishment have been done.

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Acknowledgments The authors are grateful to Director CSIR-NBRI, Lucknow, for providing essential facilities. First author SP acknowledge CSIR for the award of fellowship. This manuscript bears NBRI manuscript number CSIR-NBRI_MS/2023/01/06.

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Patel P, Prasad A, Srivastava D, Niranjan A, Saxena G, Singh SS, Misra P, Chakrabarty D (2022) Genotype-dependent and temperature-induced modulation of secondary metabolites, antioxidative defense and gene expression profile in Solanum viarum Dunal. Environ Exp Bot 194:104686 Paunescu A (2009) Biotechnology for endangered plant conservation: a critical overview. Rom Biotechnol Lett 14(1):4095–4103 Prasad A, Patel P, Pandey S, Niranjan A, Misra P (2020) Growth and alkaloid production along with expression profiles of biosynthetic pathway genes in two contrasting morphotypes of prickly and prickleless Solanum viarum Dunal. Protoplasma 257(2):561–572 Prasad A, Patel P, Niranjan A, Mishra A, Saxena G, Singh SS, Chakrabarty D (2022) Biotic elicitor–induced changes in growth, antioxidative defense, and metabolites in an improved prickleless Solanum viarum. Appl Microbiol Biotechnol 106(19):6455–6469 Ripperger H (1998) Solanum steroid alkaloids–an update. Alkaloids Chem Biol Pers 12:103–185 Shilpha J, Satish L, Kavikkuil M, Largia MJV, Ramesh M (2015) Methyl jasmonate elicits the solasodine production and anti-oxidant activity in hairy root cultures of Solanum trilobatum L. Ind Crop Prod 71:54–64 Singh K, Kaushal R (2007) Comprehensive notes on commercial utilization, characteristics and status of steroid yielding plants in India. Ethnobot Leafl 2007(1):9 Singh S, Khanna K, Sudhir S (1998) Breeding of Solanum viarum: current status as steroid bearing plant. J Med Aromat Plant Sci 20(2):423–431 Sreeramu B (2004) Cultivation of medicinal and aromatic crops. Universities Press, Hyderabad Srivastava M, Sharma S, Misra P (2016) Elicitation based enhancement of secondary metabolites in Rauwolfia serpentina and Solanum khasianum hairy root cultures. Pharmacogn Mag 12(Suppl 3):S315 Stermer BA, Bianchini GM, Korth KL (1994) Regulation of Hmg-Coa reductase-activity in plants. J Lipid Res 35(7):1133–1140 Tadeusz A (2007) Alkaloids–secrets of life. Alkaloid chemistry, biological significance, applications and ecological role, 1st edn. Elsevier, Amsterdam Tag H, Kalita P, Dwivedi P, Das A, Namsa ND (2012) Herbal medicines used in the treatment of diabetes mellitus in Arunachal Himalaya, Northeast, India. J Ethnopharmacol 141(3):786–795 Trejgell A, Kamińska M, Tretyn A (2015) In vitro slow growth storage of Senecio macrophyllus shoots. Acta Physiol Plant 37(11):1–9 Udayan P, George S, Tushar K, Balachandran I (2006) Medicinal plants used by the Malayali tribe of Servarayan Hills Yercaud Salem District Tamil Nadu India. Zoos Print J 21:2223–2224 Wu S-B, Meyer RS, Whitaker BD, Litt A, Kennelly EJ (2012) Antioxidant glucosylated caffeoylquinic acid derivatives in the invasive tropical soda apple, Solanum viarum. J Nat Prod 75(12):2246–2250

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Biosynthesis of Essential Oils in Artemisia Species and Conservation through In Vitro Propagation Pankaj Kumar Verma and Shikha Verma

Abstract

The Artemisia genus is the widely distributed genera of the angiosperm family Asteraceae. It consists of around 500 different species spread across Asia, North America, and Europe’s temperate zones. The Artemisia species contain biologically active compounds and secondary metabolites having broad-spectrum activity so extensively used in traditional medicine. The stem of the Artemisia species contains natural phytochemicals like sesquiterpene lactones, terpenoids, flavonoids, alkaloids, lignans, phenolic acids, and steroids. The major compounds are essential oil containing various types of mono- and sesquiterpenoid constituents. The genus Artemisia has come to attention because of the sesquiterpene, artemisinin having antimalarial properties. Due to the less quantity and limited production of these important metabolites, in vitro prorogation is highly desirable. For several Artemisia species, in vitro propagation has also been recognized as a crucial tool for conservation and reintroduction studies. This chapter summarizes the recent developments in biosynthetic processes of the major classes of essential oils and several factors affecting the regulation and biosynthesis of essential content in Artemisia species. Keywords

Artemisia · Biosynthesis · Essential oil · Propagation

P. K. Verma (✉) · S. Verma French Associates Institute for Agriculture and Biotechnology of Dryland, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boqer Campus, Midreshet Ben-Gurion, Israel # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_9

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9.1

P. K. Verma and S. Verma

Introduction

Artemisia belongs to the Asteraceae family, among the largest and most diverse genera containing more than 500 species (Zhu et al. 2011). Various species of this genus grow wildly in North America Asia and Europe (Bora and Sharma 2011). Artemisia species are widely used for ornamental, aromatic, and medicinal functions since ancient times due to their specific aroma and taste. Many species are used worldwide to cure several diseases like malaria, inflammation, hepatitis, cancer, and bacterial, fungal, and viral infections (Adewumi et al. 2020). The Artemisia species produces essential oils that can be recovered from leaves and flowers. Due to the presence of several active components or specific metabolites, essential oils have broad-spectrum bioactivity. Based on the technique of extraction from the plants, mostly distillation, essential oils contain volatile molecules like aliphatic compounds, terpenes, and phenolic derivatives. The Artemisia genus comprises important medicinal plants, nowadays gaining a lot of attention in the pharmaceutical sector due to essential oil production and their vast biological and chemical diversity (Trendafilova et al. 2021). For example, A. annua L. which contains sesquiterpene endoperoxide lactone artemisinin, mainly present in leaves and inflorescence used as a malarial drug (Soni et al. 2022). The extensive research work leads to the development of artemisinin-based drugs, artesunate, arteether, and artemether which are effective against malaria-causing Plasmodium falciparum and cerebral malaria (Das and Prabhu 2022). Due to the vast importance and pharmaceutical usage, Artemisia species are used as an important biological system to explore variation in plant secondary metabolites, particularly terpenoids of essential oils. For instance, Artemisia annua has been widely used as a model to understand the terpenoid biosynthetic process. Several studies were focused on a wide range of intra- and inter-species, organ-wise composition, and distribution of essential oils from Artemisia species. This chapter presents a comprehensive review focusing on essential oil biosynthesis and progress to understand the genetic control of essential oil biosynthesis in Artemisia species as well as the conservation of Artemisia species through in vitro propagation.

9.2

Essential Oils from the Genus Artemisia

Essential oils are naturally occurring, volatile compounds with a strong odor. These are usually extracted by diverse methods of which steam or hydro-distillation are the common extraction methods. Hydro-distillation mainly involves low- or highpressure distillation using hot steam or boiling water. The plant produces essential oils to protect itself against numerous biotic stressors such as bacterial, fungal, and viral diseases as well as decreased herbivory. These essential oils possess a diverse range of pharmaceutical properties such as antipyretic, antimicrobial, anticancer, anthelminthic, etc. and therefore can be used to treat human diseases. Numerous Artemisia species generate essential oils that are utilized in traditional and modern medicine, as well as cosmetics, and the pharmaceutical industry (Nigam et al. 2019).

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Due to the presence of numerous active metabolites, essential oils typically have a wide range of bioactivity. Terpenoids, as well as aromatic and aliphatic components produced from phenols, are among the volatile compounds found in Artemisia essential oils (Taleghani et al. 2020). Chemically, essential oils are natural mixtures that contain between 20 and 60 components in varying concentrations. They are distinguished by the presence of a few major components in high concentrations ranging from 20 to 70%, with other components present in trace amounts. The biological activities of the essential oil are generally defined by these key major components. The components are divided into two categories with distinct biosynthetic origins: the major group is made up of terpenes, while the other is made up of aromatic and aliphatic constituents, both groups are known for having low molecular weights. The aromatic odor of Artemisia species is caused by high concentrations of volatile terpenes, which are constituents of their essential oils, particularly in leaves and flowers (Ivănescu et al. 2021). In several Artemisia species from all over the world, the chemical composition of essential oils has been thoroughly investigated. Numerous studies have shown that the terpene components of essential oils vary significantly within the Artemisia species. The harvesting season, fertilizer input, soil type, and pH, as well as dry conditions, extraction method, geographic location, subspecies or genotype, and choice of a plant part, all have an impact on the quality and yield of essential oils from Artemisia species (Bilia et al. 2014). Table 9.1 summarizes the list of bioactive compounds, their metabolite class and their chemical structure.

9.3

Essential Oil Biosynthesis

Essential oils have several usages ranging from the pharmaceutical, cosmetic, and aromatherapy to food industries. The essential oil contains some specific active volatile organic compounds such as terpenes, and phenylpropanoids, which are fatty acid and amino acid derivatives. These are synthesized by coordinated and tightly regulated pathways.

9.3.1

Synthesis of Terpenes

Terpenes are the major constituents of the essential oils of the Artemisia species. Different forms of terpenes such as monoterpenes, sesquiterpenes, and diterpenes and their modified forms constitute the bulk of Artemisia essential oils. The biosynthesis pathway of terpenes is accomplished in three phases: first, the production of C5 building blocks; second, the condensation of C5 units to produce C10, C15, C20, and C25 prenyl diphosphates; and third, the use of prenyl diphosphates to produce terpenes (Dudareva et al. 2013).

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Table 9.1 Bioactive compounds discovered in the essential oil of Artemisia species Metabolite class Monoterpene

Compound Borneol

Formula C10H18O

Monoterpene

Camphene

C10H16

Monoterpene

Camphor

C10H16O

Monoterpene

Chrysanthenone

C10H14O

Monoterpene

p-Cymene

C10H14

Monoterpene

Eucalyptol (1,8-Cineole)

C10H18O

Monoterpene

Geranyl acetate

C12H20O2

Monoterpene

Limonene

C10H16

Monoterpene

Linalool

C10H18O

Monoterpene

β-Myrcene

C10H16

Monoterpene

trans-Ocimene

C10H16

Monoterpene

α-Pinene

C10H16

Structure

(continued)

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Table 9.1 (continued) Metabolite class Monoterpene

Compound β-Pinene

Formula C10H16

Monoterpene ketone

Piperitone

C10H16O

Monoterpene

Sabinene

C10H16

Monoterpene

γ-Terpinene

C10H16

Monoterpene ketone

α-Thujone

C10H16O

Monoterpene ketone

β-Thujone

C10H16O

Sesquiterpene lactone

Artemisinin

C15H22O5

Sesquiterpene

γ-Cadinene

C15H24

Sesquiterpene alcohol

α-Cadinol

C15H26O

Sesquiterpene

Caryophyllene

C15H24

Structure

(continued)

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Table 9.1 (continued) Metabolite class Sesquiterpene oxide

Compound Caryophyllene oxide

Formula C15H24O

Sesquiterpene

Chamazulene

C14H16

Sesquiterpene

Farnesene

C15H24

Sesquiterpene

Germacrene D

C15H24

Sesquiterpene

Spathulenol

C15H24O

Sesquiterpene alcohol

β-bisabolol

C15H26O

Diterpene alcohol

Phytol

C20H40O

Phenylpropene

trans-Anethole

C10H12O

Enones

Artemisia ketone

C10H16O

Monoterpene

Bornyl acetate

C12H20O2

Tetrahydrofuran

Davanone

C15H24O2

Sesquiterpene

γ-Elemene (Elixene)

C15H24

Structure

(continued)

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Table 9.1 (continued) Metabolite class Sesquiterpene

Compound Epiglobulol

Formula C15H26O

Sesquiterpene

cis-Lanceol

C15H24O

Phenylpropene

Methyl chavicol (Estragole)

C10H12O

Fatty aldehyde

9,12,15Octadecatrienal (Linolenyl aldehyde) trans-Sabinyl acetate

C18H30O

Monoterpene

Structure

C12H18O2

9.3.1.1 Phase 1: Synthesis of 5-Carbon (C5) Building Blocks The mevalonic acid (MVA) pathway produces 5-carbon (C5) isomeric molecules such as isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the cytoplasm (cytosol, endoplasmic reticulum, peroxisomes) and plastids by methylerythritol phosphate (MEP) pathway (Vranová et al. 2013). The MVA pathway consists of six enzymatic reactions. Condensation of three molecules of acetylCoA results in 3-hydroxy-3- methyl glutaryl-CoA (HMG-CoA). Mevalonate is formed by NADPH-reduction of HMG-CoA in two steps. Mevalonate is transformed into IPP in three steps reactions, two phosphorylation steps, and a decarboxylation/elimination step, all three are ATP-dependent (Fig. 9.1). There are seven enzymatic steps in the MEP pathway. In the first stage, D-glyceraldehyde 3-phosphate (CoAP) derived from the pentose phosphate pathway, glycolysis, and plastidic pyruvate is converted into 1-deoxy-D-xylulose 5-phosphate (DXP). MEP is formed from DXP by NADPH-dependent reduction and its isomerization. In five more steps, MEP is converted into IPP and DMAPP. In both MVA and MEP pathways, IPP is converted into DMAPP upon isomerization by isopentenyl diphosphate isomerase (Berthelot et al. 2012; Smoak et al. 2022). The MEP and MVA pathways are interconnected such that MEP pathways supply IPP and DMAPP to the cytoplasm (Hemmerlin et al. 2012).

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Fig. 9.1 Terpenoid biosynthesis pathway. The steps of MEP, MVA, and phenylpropanoid pathways accomplished in the chloroplast (plastids) are shown broadly in green color and those performed in the cytoplasm (cytosol) are shown in black color. The crosstalk between MEP and MVA pathways is depicted in red color. The abbreviation used are DXP 1-deoxy-D-xylulose-5phosphate, DMAPP dimethylallyl pyrophosphate, FPP farnesyl pyrophosphate, GA3P glyceraldehyde-3-phosphate, GPP geranyl pyrophosphate, IPP isopentenyl pyrophosphate, HMG-CoA 3-hydroxy-3methyl-glutaryl coenzyme A, MEP 2-C-methyl-D-erythritol-4-phosphate, MVA mevalonate, PEP phosphoenolpyruvate

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9.3.1.2 Phase 2: Condensation of C5 Units to Produce C10, C15, C20, and C25 Prenyl Diphosphates The C5 building blocks, IPP and DMAPP are condensed to produce a series of prenyl diphosphates, including the following: GPP (C10, geranyl diphosphate), NPP (C10, neryl diphosphate), FPP (C15, farnesyl diphosphate), GGPP (C20, geranyl diphosphate), and GFPP (C25, geranyl farnesyl diphosphate) in cytoplasm and plastids. The higher-order prenyl phosphates, such as C30 and C40, are formed by condensation of lower-order (FPP and GGPP) prenyl phosphates. A range of shortchain prenyltransferases catalyzes the condensation reactions. Different prenyl diphosphates serve as the building blocks for various terpene classes (Nagegowda and Gupta 2020), (Fig. 9.2).

Fig. 9.2 Mevalonic acid (MVA) pathway in Artemisia annua produces building blocks for terpene biosynthesis, including sesquiterpenes and artemisinin, in the cytoplasm of glandular trichome cells. The abbreviation used are AACT acetyl-CoA acetyltransferase, FDS farnesyl diphosphate synthase, HMGS 3-hydroxy3methyl-glutaryl coenzyme synthase, HMGR HMG-CoA reductase, IDI isopentenyl diphosphate isomerase, MDPC mevalonate diphosphate decarboxylase, MVK mevalonate kinase, PMK phosphomevalonate kinase, TS terpene synthase

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9.3.1.3 Phase 3: Use of Prenyl Diphosphates to Produce Terpenes This phase comprises enzymatic reactions that synthesize terpenes and further modify them. Terpene synthases (TPSs) transform prenyl diphosphates into terpenes in both plastids and cytoplasm. Monoterpenes, sesquiterpenes, and diterpenes are synthesized from GPP and NPP, FPP and GFPP, and GGPP, respectively. Some terpene synthases produce multiple terpene kinds from the same prenyl diphosphate (Pazouki and Niinemets 2016). The enzymatic reactions mediated structural modifications such as cyclization, hydroxylation, dehydroxylation, oxidation, reduction, or glycosylation on specific terpenes produce their variants (Shang and Huang 2019), (Fig. 9.3).

Fig. 9.3 Methylerythritol phosphate (MEP) pathway in Artemisia annua produces building blocks for the terpene (monoterpenes) biosynthesis in chloroplasts of glandular trichome cells. The abbreviations used are CMK 4-diphosphocytidyl-2-Cmethyl-D-erytritol kinase, DXS 1-deoxy-D-xylulose-5phosphate synthase, DXR 1-deoxy-D-xylulose-5phosphate reductoisomerase, GS geranyl diphosphate synthases, HDR 4-hydroxy-3methylbut-2-eryldiphosphate reductase, HDS 4-hydroxy-3methylbut-2-eryldiphosphate synthase, MCT 2-C-methyl-Derythritol 4-phosphate cytidyltransferase, MDS 2-Cmethyld-erythritol 2, 4-cyclodiphosphate synthase, MS monoterpene synthases

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9.3.2

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Synthesis of Phenylpropanoid Volatiles

Phenylpropene class of volatile molecules are the second most pronounced compounds found in essential oils of several Artemisia species. The major Phenylpropene class of volatiles such as chavicol, methyl chavicol, eugenol, and methyleugenol is present in high concentrations. Phenylpropenes are comprised of a benzene ring (C6) with a propyl (C3) side chain. In some compounds such as in eugenol and chavicol, a modified benzene ring is present which is modified by the para-hydroxyl group. The prominent precursor of phenylpropenes is L-phenylalanine (Phe) an aromatic amino acid (Yadav et al. 2020). Phenylpropanoids biosynthesis starts with the deamination of phenylalanine to trans-cinnamic acid by the action of L-phenylalanine ammonia-lyase (Mohammed et al. 2021). The biosynthesis is carried out by the cinnamate 4-hydroxylase enzyme, which reacts with cinnamic acid to produce p-coumaric acid. A class II 4CL (4-coumarate CoA ligase) specific to phenylpropanoid metabolism then converts p-coumaric acid to p-coumaroyl-CoA. From here onward the pathway branches to produce coniferyl alcohol on the one hand and coumaryl alcohol on the other hand. At this stage, an acetyltransferase acetylates coniferyl alcohol to coniferyl acetate and coumaryl alcohol to coumaryl acetate. Subsequently, the eugenol/chavicol synthase (EGS), the NADPH-dependent reductase, derives eugenol from coniferyl acetate and chavicol from p-coumaryl acetate (Fig. 9.4). The enzymes eugenol-omethyl transferase (EOMT) and chavicol-o-methyl transferase (COMT) react with eugenol and chavicol, respectively, to produce methyleugenol and methyl chavicol (estragole). Whereas phenylalanine is biosynthesized via shikimate and aromatic amino acid pathways in chloroplasts, the phenylpropenes are synthesized in the cytoplasm of glandular trichomes in Artemisia annua.

9.4

Traditional Applications and Biological Activities of Artemisia Species Essential Oils

The Artemisia genus contains significant medicinal plant species that have been utilized for pharmacological and culinary purposes since ancient times. As a result, there are several biopharmaceutical products on the market today for treating various diseases that contain Artemisia extracts.

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Fig. 9.4 Pathway of phenylpropene (C6-C3) biosynthesis that produces phenylpropanoid volatiles in the cytoplasm of glandular trichome cells of Artemisia annua. The abbreviations used are 4CL 4-Coumarate-CoA ligase, C4H cinnamate-4-hydroxylase, CAAT coniferyl alcohol acetyltransferase, CAD cinnamyl alcohol dehydrogenase, CC3H p-Coumaroyl-CoA-3-hydroxylase, CCOMT caffeoyl-Co-A-O-methyl transferase, CCR cinnamoyl CoA-reductase, EGS eugenol (and chavicol) synthase, PAL phenylalanine ammonia-lyase

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Artemisia species

Common name

Chemical compound

Uses

References

Artemisia abrotanum L.

Southern wormwood

Cineole, borneol, p-cymene

Ekiert et al. (2021a, b)

Artemisia herbaalba

White wormwood

Cineole, thujone, borneol

Artemisia absinthium L.

Romanian wormwood

Absinthin, thujone, chamazulene

Artemisia afra Jacq. ex Willd.

African wormwood

Thujone, cineole, camphor, germacrene, cadinene, terpineol

Artemisia annua L.

Sweet wormwood

Artemisinin

Astringent, stimulant, spasmolytic, anti-septic, and febrifuge Anti-diabetes, antihypertensive, antioxidant, and antimicrobial, analgesic, and anti-spasmodic Treatment of fevers, stomach-ache, act as a diuretic, anti-helminthic, antimicrobial, antioxidant, and hepatoprotective Treatment of coughs, colds, headaches, diabetes, dyspepsia, malaria, and disorders of the kidney and bladder Treatment of fevers (including malaria) and chills

Artemisia arborescens L.

Tree wormwood

Thujone, camphor, terpinen-4-ol, pinene

Anti-inflammatory, antibacterial, and anti-viral

Artemisia vulgaris L.

Wild wormwood

Analgesic, allelopathic, antioxidant, larvicidal, cytotoxic, antimalarial, and anti-hyperlipidemic

Artemisia capillaris Thunb.

Fragrant wormwood

Artemisia dracunculus L.

Tarragon

Artemisinin, camphene, camphor, sabinene, scopoletin, vulgarin, derivatives of quercetin and kaempferol Capillarisin, apigenin, hesperidin, coumaric acid, β-caryophyllene, α-pinene β-pinene, and capillene Estragole and methyleugenol

Artemisia japonica Thunb.

Oriental wormwood

Borneol, bornyl acetate, camphor, cineole, thujone, artemisia ketone

Wound healing, depurative

Bertella et al. (2018), Kadri et al. (2022) and Mohammed et al. (2021) Boudjelal et al. (2020), Moacă et al. (2019) and Szopa et al. (2020) Adeogun et al. (2018) and du Toit and van der Kooy (2019) Abate et al. (2021), Brown (2010) and Mirbehbahani et al. (2020) Janaćković et al. (2019), Jaradat et al. (2022) and Plescia et al. (2022) Chaudhary et al. (2021) and Siwan et al. (2022)

Neuroprotective, antiepileptic, allelopathic, anticancer, and antimicrobial

Jung et al. (2008) and Sailike et al. (2022)

Hyperglycemic, anticoagulant, antihyperlipidemic, antiepileptic, anti-spasmodic agent and laxative, carminative

Basiri and Nadjafi (2019), Ekiert et al. (2021a, b), Liu et al. (2018) and Smoak et al. (2022) Akhil et al. (2022) and Kwon and Lee (2001)

(continued)

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(continued) Artemisia species

Common name

Artemisia indica Willd.

Indian wormwood

Artemisia songarica Schrenk.

Artemisia argyi H. Lév. & Vaniot

Silvery wormwood

Artemisia selengensis Turcz.

Riverside wormwood

Artemisia montana (Nakai) Pamp. Artemisia keiskeana Miq. Artemisia nilagirica (Clarke) Pamp.

Large mugwort

9.5

False mugwort Indian wormwood

Chemical compound

Uses

References

β-Thujone, herniarin, sabinyl acetate, 1, 8-cineol, estragole, cis-chrysanthenyl acetate, terpineol, davanone, trans-ethyl cinnamate, piperitone, artemisolide, eupatilin Bisabolol oxide II, nerolidol, bisabolol, bisabol oxide A, spathulenol Eucalyptol, cyclohexanol, artemisia alcohol, borneol, spathulenol, eugenol, camphor

Treatment of dyspepsia, chronic fever, and other hepatic ailments as well as having anti-fungal, anti-feedent, antioxidant, and anticancer properties

Dahal et al. (2021)

Insecticide and repellent activities

Zhang et al. (2022)

Anti-inflammatory, antioxidant, anticancer, neuroprotective, immunomodulatory, anti-osteoporotic and anticoagulant, antimicrobial, and insecticidal activities Antioxidant and immunostimulatory, phytotoxic Anti-inflammatory, antimicrobial

Song et al. (2019) and Xiao et al. (2019)

Anticancer activity

Choi and Kim (2013) Joshi (2020)

α-Pinene, β-sabinene, eucalyptol, β-thujone, cis-sabinol Borneol, 3-cyclohexen-1-o1, camphor Sabinene, β-pinene, α-curcumene α-Pinene, sabinene, artemisia ketone, perillene, carvone, germacrene D

Antimicrobial, antioxidant

Shi et al. (2021)

Lee et al. (2020)

Conservation of Artemisia

The Artemisia genus contains numerous bioactive compounds that are used in the pharmaceutical, cosmetic, and food industries. Because of the immense importance of artemisia essential oil, the species is being ruined due to rising demand, resulting in the genus’s biodiversity being lost. Climate change also affects the biodiversity of the artemisia genus. As a result, the germplasm of the Artemisia genus is in danger of extinction (Wang et al. 2022). Therefore, substantial and immediate action must be made to ensure its conservation. The creation of efficient propagation procedures can help to conserve vulnerable indigenous species with therapeutic value. Early reports suggested that conventional methods of propagating Artemisia species are ineffective due to the small seed size, which requires symbiotic association with microflora

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for germination. The current work on micropropagation shows that it is a worthwhile method for quick multiplication (Turi et al. 2014).

9.5.1

In Situ Conservation of Artemisia Genus

Artemisia annua had the best in situ protection status in Europe, both without and with human modification and was considered as a medium urgency for further action. It is also protected in Asia, North America, and South America. As a result, the taxa were not adequately conserved in situ. Although A. annua is widely distributed throughout Asia, it's in situ conservation status is substantially poor than other continents. The conservation status of Artemisia in Asia is even worse than Australia and Oceania (Wang et al. 2022).

9.5.2

Ex Situ Conservation of Artemisia Genus

The ex situ conservation of the extremely threatened population of Artemisia chamaemelifolia was successfully achieved by in vitro cultivation techniques (Hristova et al. 2012). In an effort to conserve and improve the yield of volatile compounds, numerous studies were conducted for in vitro cultivation. These studies focused on the impact of plant growth regulator ratios and concentrations and their effect on different explants such as leaf, root, hypocotyl seed, etc. For instance, treating Artemisia annua seeds using 0.5 μm or 2.0 μm GA3 and 0.5 μm BAP in MS media increases seed germination by suppressing the phenolics secretions (Tahir et al. 2013). Therefore, it may provide a way for in vitro regeneration of Artemisia plants in other species also. Another study suggests the impact of different explants and plant growth regulators on micropropagation of in vitro grown seedlings of A. absinthium (Mannan et al. 2012). The axenic culture was used for A. tridentata in vitro conservation, which served as a tool for comprehending secondary metabolite production in aseptic conditions, however, they were unable to start rooting from individual shoot tips (Turi et al. 2014). Another extensive study employing varied auxin concentrations, indole-3butyric acid, which promotes root development, focused on vegetative proliferation from stem cuttings (Abate et al. 2021; Alvarez-Cordero and McKell 1979). Thus, in order to induce adventitious roots in the regenerated branch tips of various Artemisia species, auxins such as indole-3-butyric acid, indole-3-acetic acid (IAA), and naphthalene acetic acid (NAA) have been widely utilized (Abate et al. 2021). For instance, A. vulgaris developed 98.2% of its roots at a concentration of 1.5 mg L1 IAA (Sujatha and Kumari 2007). Another study found that incorporating 1 mg L-1 IBA into the culture medium boosts root formation in regenerated A. pallens shoots (Alok et al. 2016). The maximum root development (85.8%) was observed in regenerated shoots employing 0.5 mg L-1 NAA in an in vitro regeneration investigation of A. annua (Dangash et al. 2015). According to another study, in a media devoid of growth regulators, diploid A. tridentata subsp. tridentata shoot tip cuttings

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may be able to generate adventitious roots. Nonetheless, the adventitious root generation is highly dependent on the seedling sources from which the shoot tips were collected. Overall, the internal auxin concentration (either accumulated during seed germination or synthesized in plant tissues) plays an important role in adventitious root formation along with the eternally supplied auxin (Yu et al. 2017). Shekhawat and Manokari (2015) created an effective micropropagation strategy using ex vitro rooting to boost the survival rate of plantlets using juvenile nodal explants for the regeneration of shoots of A. absinthium. Another protocol has been developed for Artemisia nilagirica var. nilagirica (Indian wormwood) in vitro propagation using MS medium with 2.5 μM IAA and then subculture on MS medium containing 2.5 μM BAP with 7.5 μM 2-isopentenyl adenine (2-iP) (Shinde et al. 2016). Many other different efficient in vitro micropropagation protocols were established by different researchers applying the combination of phytohormones. For example, MS medium with 1.78 μM benzyl adenine and 0.27 μM naphthalene acetic acid is best suited for the micropropagation of Artemisia absinthium L (Nin et al. 1994). While Artemisia annua L. cv: Anamed was best performed on MS medium containing 1 mg L-1 BAP with 0.1 mg L-1 IBA for regeneration and 1/2 MS with 0.5 mg L-1 IBA for root generation (Hailu et al. 2013). For Artemisia petrosa, an endemic to the central Apennines that is currently endangered due to indiscriminate harvest for commercial uses, a micropropagation method was created (Pace et al. 2004). The therapeutic herb Artemisia vulgaris L., also known as mugwort, is in danger of extinction. The mass propagation of its plantlets was achieved by in vitro liquid culture using MS medium with 4.44 μM 6-benzyl adenine (BA). To initiate root, individual shoots were placed on MS medium with 8.56 M IAA (Govindaraj et al. 2008). Different auxin and cytokinin concentrations were also tested on the critically endangered and endemic plant Artemisia amygdalina to produce maximum shoot tip. These shoot tips were transferred to MS basal media for rooting. The protocol is best suited for A. amygdalina with a focus on the conservation of its unique germplasm (Rafia et al. 2013). Although several studies were focused on micropropagation, still a consistent and productive method for Artemisia species micropropagation and secondary metabolites production is required in order to conserve natural vegetation.

9.6

Conclusion

The phytochemical analysis of natural flora has gained interest from researchers and pharmaceutical industries to identify innovative herbal components and their therapeutic uses with no side effects. Artemisia genus may prove as a prominent source for the discovery of novel approaches for therapeutic purposes. The Artemisia genus possesses a wide range of volatiles that can be used for their medicinal properties. Because of the extensive demands of pharmaceutical industries several Artemisia species were overexploited and tend to extinct. We must assure widespread cultivation of Artemisia using conventional and micropropagation procedures in order to maintain output and availability.

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Immunostimulatory Properties of Echinacea purpurea and Conservation Strategy

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Syed Saema, Laiq-Ur-Rahman, Nafisa Shaheen, and Vibha Pandey

Abstract

Echinacea purpurea (L.) Moench, a member of the Asteraceae (Compositae) family is an important and well-known medicinal plant. The plant is used in chemoprevention and chemotherapy for infectious disorders of the upper and lower respiratory tracts. Toothaches, gut pain, snake bites, skin problems, epilepsy, chronic arthritis, and cancer have all been treated with this species in the past. For instance, research has demonstrated the plant’s ability to cause antianxiety, anti-depression, cytotoxicity, and anti-mutagenic effects. Echinacea has immense decorative potential in addition to its potential medical benefits. Alkamides, caffeic acid derivatives, polysaccharides, and glycoproteins are some of the plant’s secondary metabolites that have immunostimulatory properties. Echinacea’s anti-inflammatory properties are largely due to its polysaccharides. In vitro culture offers the ability to overcome a variety of problems associated with Echinacea propagation, such as bottlenecks in growth and poor seed germination, and also to meet up the increased demand. Alkamides are thought to be responsible for the immunomodulatory actions of Echinacea extracts both in vitro and in vivo.

S. Saema (✉) · N. Shaheen Department of Environmental Science, Integral University, Lucknow, India Laiq-Ur-Rahman CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India V. Pandey CSIR-National Botanical Research Institute, Lucknow, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_10

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Keywords

Phytochemicals · Echinacea purpurea · Alkamides · Caffeic acid · Polysaccharides

10.1

Introduction

Echinacea purpurea (L.) Moench (EP) is a perennial herbaceous blooming plant of the Asteraceae family also known as purple coneflower. In comparison to other plant families, a large number of species in the Asteraceae family have been used for therapeutic purposes due to the availability of chemicals with a wide range of therapeutic properties as well as the fact that the Asteraceae family of plants is one of the most notable and well-known (Kołodziejczyk-Czepas et al. 2015; Nadaf et al. 2019). The 60–180 cm tall stems have short, silky hairs and may branch at the top. The bottom leaves are widely lanceolate to oval in shape and have coarse, uneven teeth (the most useful trait for identifying this species). The middle cone frequently has vivid orange tips (probably the second-best distinguishing characteristic). Pale tips are straight and flexible. The body length of the central cone’s bristles is halved. The ray flowers range from rose to deep purple, and white is an unusual color. It flourishes in wide-open forests, plains, and thickets. In this species, the plant is the one that is most frequently grown for medicinal purposes (McKeown 1999). It has primarily been utilized in chemotherapy and chemoprevention for infectious disorders of the upper and lower respiratory systems (Grimm and Müller 1999; Patel et al. 2008). Additionally, the anti-inflammatory effects of this plant are the main reason it is used traditionally, independent of the location or kind of inflammation, such as skin inflammation or inflammation brought on by an immune response. Herbal tea made from blossoms is supposed to strengthen the immune system. Echinacea has tremendous promise as a decorative plant in addition to potential medical applications. As a field-grown specialty cut flower, E. purpurea, the only species for which ornamental cultivars have been developed, is both productive and profitable (Starman et al. 1995). Alkamides have been linked to the immunomodulatory effects of Echinacea extracts in vitro and in vivo, according to several studies (Gertsch et al. 2004). Additionally, several species of Echinacea contain caffeic acid, which may be used to authenticate and oversee the quality of plant extracts. Polysaccharides are crucial to the preparation of Echinacea’s anti-inflammatory effects (Laasonen et al. 2002). Due to the numerous conventional and contemporary medical applications of Echinacea purpurea, numerous plant components are utilized to varying degrees. All parts of the plant—flowers, leaves, stems, and roots—are employed in medical practices. Recent years have seen an unprecedented increase in the consumption of echinacea. The top-selling medicinal health products are made with echinacea. Echinacea has become well-known in the medical community because of its wide range of health advantages. Furthermore, there is no recent proof of any negative effects brought on by this herb. Numerous licensed herbalists prescribe it for the treatment

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of skin conditions, UTI infections, and ear infections in addition to other immunological advantages due to its healing properties. There are several different segments of echinacea. The Echinacea market is divided into the following product categories: tablets, capsules, ointments, extracts, and creams. The Echinacea market is divided into two categories based on the form: liquid and powder. The Echinacea market is split into seven regions: Japan, North America, Latin America, Western and Eastern Europe, and Asia Pacific excluding Japan. Asia Pacific is also the fastest-growing region in this market due to the region’s customers’ increased preference for natural and herbal products.

10.2

Bioactive Metabolites of Echinacea purpurea

Several biological activities that were connected to the main chemical components of E. purpura have been identified (Bauer 1999). For instance, the polysaccharide fraction was discovered to promote macrophage activity and several other processes connected to the synthesis of cytokines, and specific groups of phenolic compounds and alkamides were discovered to have antiviral and antifungal activities (Bauer 1999; Binns et al. 2002; Goel et al. 2002; Merali et al. 2003; Randolph et al. 2003; Rininger et al. 2000). Alkamides, caffeic acid esters (cichoric acid), polysaccharides, and polyacetylenes are all present in Echinacea purpura, Table 10.1 (Chen et al. Table 10.1 The chemical constituents present along with their concentrations in the root of E. purpurea Class Alkamides

Concentration (%) 0.01–0.70

Caffeic acid

2.0–2.8

Polysaccharides

Volatile oil

Others

0.1

Chemical compounds Isobutyl amides of straight-chain fatty-acids with olefinic and/or acetylenic bonds, e.g. isomeric dodeca2E,4E,8Z,10E/Z-tetraenoic isobutyl amide. Undeca-2Z,4Ediene-8,10- dioic acid isobutyl amide Cichoric acid (2,3-O-di-caffeoyl tartaric acid, 1.7–2.4%) derivatives and (2-O-caffeoyl tartaric acid, ca. 0.2–0.8%) also echinacoside, verbascoside, caffeoylechinacoside, chlorogenic and is chlorogenic acids Arabinogalactans and an arabinogalactan and glycoproteins containing glycoprotein with a sugar component consisting of arabinose (64–84%), galactose (2–5%), and galactosamine (6%) Caryophyllene, caryophyllene oxide, humulene, limonene, camphene, aldehydes, and dimethyl sulfide. As per WHO monograph, Penta deca-(1,8-Z)- diene (44%), 1-pentacene, ketoalkynes, and ketoalkenes are also present Small amounts of polyacetylene compounds polyynes (0.01 mg/%including Tribeca-1-en3,5,7,9,11-pentane, Tribeca-1,11-dien 3,5,7,9,- tetraine,Tribeca-8,10,12-triene2,4,6-triine). Effective alkaloids: Tussilagine, isotussilagine (0.006%, as per WHO)

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Table 10.2 Biological and pharmacological effects of the bioactive compounds of Echinacea purpurea Bioactive compounds Alkylamides

Polysaccharides

Biological and pharmacological effects Immunomodulators, antiinflammatories, macrophage modulation, NO reduction and tumor necrosis factor-α, antiviral immunity mediators, and type 2 cannabinoid receptor Antitumor, antioxidant, antimicrobial, antifungal, antiviral, immunomodulator, hypoglycemic, hepatoprotective, gastrointestinal protector, and antidiabetic

Glycoproteins

Immunomodulatory

Flavonoids

Antioxidant, antiinflammatory, anti-ulcer, anti-allergic, and antiviral activity Anti-inflammatory, antioxidant activity, antiosteoporotic activity, antimicrobial, antitumor, and neuroprotective action

Caffeic acid derivatives

References Rios and Olivo (2014), Woelkart and Bauer (2007), Mudge et al. (2011), Cech et al. (2010) and Schumacher and Friedberg (1991)

Shariatinia (2019), Balciunaite et al. (2015), Cai et al. (2014), Stimpel et al. (1984), Sharma et al. (2010), Mazzio and Soliman (2009), Vickers (2002), Voaden and Jacobson (1972), Yao et al. (2019), Tsai et al. (2012), Abreu et al. (2011), Luettig et al. (1989), Sharif et al. (2021), Jiang et al. (2016) Guiotto et al. (2008), Kim et al. (2014), and Bodinet and Beuscher (1991) Kurkin et al. (2011), Agrawal (2011), Lee et al. (2010) and Speroni et al. (2002)

Ekeuku et al. (2021), Zhang et al. (2014), Senica et al. (2019), Tsai et al. (2012), Jiang et al. (2016), Xing et al. (2011), Liu et al. (2018), Jia et al. (2009), and Pires et al. (2016)

2005; Dalby-Brown et al. 2005). These crucial substances provide Echinacea with its antitumor, antioxidant, antimicrobial, antifungal, antiviral, immunomodulatory properties, etc., Table 10.2.

10.3

Caffeic Acid Derivatives (Phenolic Compounds)

A phenolic acid called caffeine is found naturally in many plant-based foods like carrots, cabbage, tomatoes, and several berries. Additionally, it can be found in a variety of drinks like wine, fruit juices, and coffee. One of the main active ingredients in many traditional Chinese remedies is caffeic acid (Collins 2017). The primary hydroxycinnamic acid present in the diets of humans is caffeic acid (CA), a polyphenol formed by the secondary metabolism of vegetables, such as olives, coffee beans, fruits, potatoes, carrots, and propolis (Genaro-Mattos et al. 2015; Silva et al. 2014; Verma and Hansch 2004). The primary hydroxycinnamic acid present in human diets is found in propolis and carrots These phenolic

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compounds can be found in the simple form (monomers) as amides, glycosides, organic acid esters, sugar esters, dimers, and trimers, as well as in more complex forms like dimers, trimers, and derivatives of flavonoids. They can also be bound to proteins and other polymers in the vegetable’s cell wall (Chen and Ho 1997; Collins 2017; Manayi et al. 2015; Scalbert and Williamson 2000). Since CA inhibits the growth of bacteria, fungi, and insects, it helps protect plant leaves from ultraviolet B (UV-B) radiation and contributes to the defensive mechanism of plants against predators, pests, and illnesses (Gould et al. 2000; Tošović 2017). Caffeic acid is a relatively prevalent phenolic acid that can exist in both free and esterified forms that accounts for between 75 and 100% of the total amount of hydroxycinnamic acid in fruits (Manach et al. 2004). However, Caffeic acid is prevalent in meals in its esterified form, which makes it challenging for the body to absorb (Kołodziejczyk-Czepas et al. 2015; Manach et al. 2004; Scalbert and Williamson 2000). One of the hydroxycinnamate and phenylpropanoid metabolites that are more broadly distributed in plant tissues is caffeic acid (3,4-hydroxycinnamic). Numerous foods contain this polyphenol, including coffee, blueberries, apples, and cider (Clifford 2000). In addition to food, caffeic acid can be found in several commonly used drugs, many of which are propolis-based (Lustosa et al. 2008). It is known to have antioxidant and antibacterial activity in vitro in addition to serving as a carcinogenic inhibitor (Greenwald 2004; Huang and Ferraro 1992), and it may help prevent atherosclerosis and other cardiovascular problems (Sánchez-Moreno et al. 2000; Spagnol et al. 2016; Vinson et al. 2001). Echinacea purpurea contains caffeic acid derivatives such as caftaric acid, chlorogenic acid, caffeic acid, cynarin, echinacoside, and cichoric acid as active components. In the recent past, efforts have been made to produce caffeic acid compounds from adventitious root cultures (Murthy et al. 2014). Cichoric acid, the most predominant phenolic component in the root and petiole, is thought to be the most significant derivative of caffeic acid in Echinacea purpurea (L.) Moench species (Erkoyuncu and Yorgancilar 2021; Liu et al. 2006; Pellati et al. 2004; Thygesen et al. 2007). The most prevalent phenolic substance in the root and petiole of Echinacea purpurea (L.) Moench is cichoric acid. Cichoric acid, which has a concentration range of 1.2–3.1% and 0.6–2.1% of dry weight, is the main active component present in Echinacea purpurea roots and flowers, respectively (Mistrikova and Vaverkova 2006). A bioactive substance called caffeine can be found in a wide range of plants, including fruits, vegetables, herbs, and beverages. It is a significant representative of the polyphenol subgroup of hydroxycinnamic acids and a member of the enormous group of compounds known as polyphenols. Caffeic acid primarily manifests itself in food as the quinic acid ester chlorogenic acid. Similar to other polyphenols, caffeine is thought to provide several health advantages brought on by its antioxidant qualities, including the reduction of diabetes, cancer, and neurological illnesses (Birková et al. 2020). The beneficial chemicals known as caffeine acid derivatives (CADs) are mostly generated by the Echinacea species, Echinacea purpura, Echinacea Angustifolia, and Echinacea pallida. Echinacea is a well-known dietary supplement that is used all over the world as well as a well-known herbal remedy

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Fig. 10.1 Caffeic acid

for the treatment of the common cold (Murthy et al. 2014). The 50% ethanolic Echinacea purpurea flower extract’s extraction yield, total phenols, caffeic acid derivatives (CAD), and antioxidant qualities were assessed, also looked at type 2 diabetes-related angiotensin-converting enzyme (ACE) and amylase, a-glucosidase, and 50% ethanolic extract’s in vitro inhibitory effects on these enzymes (Chiou et al. 2017). All plant species routinely produce hydroxycinnamate and phenylpropanoid, which are the precursors to caffeine. It can be found in many foods that are renowned for being high in antioxidants. Caffeic acid, an antioxidant, reduces reactive oxygen species, which have been linked to bone loss. Studies have emphasized the benefits of caffeic acid in preventing bone resorption (Ekeuku et al. 2021). Caffeic acid (CA) is widely present in a variety of foods, including fruits, vegetables, tea, coffee, and oils. Due to its inherent therapeutic and medical characteristics, CA and its derivatives have been used for many millennia. Antioxidant, anti-inflammatory, anticancer, and neuroprotective benefits are only a few of the biological and pharmaceutical properties of CA. Through the regulation and inhibition of transcription and growth factors, CA may have therapeutic benefits. In human cell cultures and animal models, CA has demonstrated potential neuroprotective and anticancer properties (Alam et al. 2022). Similar to other polyphenols, caffeine is thought to provide several health advantages brought on by its antioxidant qualities, including the reduction of diabetes, cancer, and neurological illnesses. The usage of naturally occurring bioactive compounds, such as caffeic acid, is becoming increasingly popular in modern society. Therefore, knowledge of their characteristics and roles is crucial (Birková et al. 2020). Caffeic acid has shown antibacterial activity and may hold promise in the treatment of skin illnesses in addition to its potent antioxidant activity, boosting collagen formation, and prevention of premature aging. The usage of caffeic acid, which has become more widespread in humans, is the basis for the significance of this study (Magnani et al. 2014), (Fig. 10.1).

10.4

Polysaccharide Derivatives

A type of naturally occurring macromolecular polymer known as a polysaccharide is often made up of more than 10 monosaccharides linked together by glycosidic bonds in either linear or branching chains. Its molecular weight can reach tens of thousands or even millions (Xie et al. 2016). As renewable and sustainable sources of bioactive

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compounds, derivatives of polysaccharides have drawn interest. Recent studies on the biological characteristics of polysaccharide derivatives and the derivatization methods, including sulfated-, acetylated-, phosphorylated-, carboxymethylated-, aminated-, benzoylated-, C-glycosylated-, hydroxypropylated-, and selenizedpolysaccharides. Native polysaccharides changed their physical, chemical, and, most significantly, biological properties after the addition of a new functional group (s). The molecular weight, method of modification, native polysaccharide type, circumstances of the modification process, solubility, and conformation of polysaccharide derivatives all affect their biological qualities. However, some polysaccharide derivatives and hybrid derivatives (with numerous functional groups) have gotten less focus (Simsek et al. 2021). Sulfated, acetylated, phosphorylated, carboxymethylated, aminated, benzoylated, C-glycosylated, hydroxypropylated, and selenized polysaccharides are just a few of the newly modified native polysaccharides that have undergone these modifications. These alterations are possible because polysaccharides have certain chemical properties. Since hydroxyl groups act as nucleophiles and contain saccharide oxygen, polysaccharides undergo etherification and esterification processes. Polysaccharides containing uronic acid are capable of nucleophilic and electrophilic processes including esterification and amide production due to the presence of carboxyl groups (Yalpani 1985). Echinacea purpurea is one of the most popular immunostimulant plants. It has immunostimulatory and anti-inflammatory properties (Manayi et al. 2015). Polysaccharides, glycoproteins, caffeic acid derivatives, alkamides, and melanins are some of its key active ingredients. The extraction method can be used broadly because it is effective for figuring out the polysaccharide content of flowers and leaves, summer and fall plants, plants with green and red stems, and plants from two distinct plantations. It was found that plants with green stems contained much more polysaccharides than plants with red stems and that flowers had a higher polysaccharide content than leaves. The amount of polysaccharides in Echinacea purpurea was 159.8 ± 12.4 mg/g dry weight (DW), whereas extracts made by using 55% ethanol at 55 °C had 11.0 ± 1.0 mg gallic acid equivalent/g DW of the total phenolic component. When compared to ascorbic acid at the same concentration, the 0.1 mg/ mL of EP extracts’ Trolox comparable antioxidant capacity showed just a 30% activity (Lee et al. 2009). Because they are non-toxic, biodegradable, biocompatible, and less expensive than their synthetic counterparts, polysaccharides and their derivatives have benefits over synthetic polymers. These benefits give polysaccharides and their derivatives a wide range of uses in a variety of industries, including the biomedical or pharmaceutical, food, and cosmetic industries. Today, polysaccharides play significant roles in conventional disease prevention and healthcare (such as those derived from herbs and dietary fibers as discussed above), while numerous new application areas are also being investigated. These include tissue engineering, drug delivery, the treatment of internal and external wounds, cancer prevention, diagnosis, and therapy, and the management of bacterial and viral diseases (Khan and Ahmad 2013; Klein 2009; Lindblad et al. 2007).

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Fig. 10.2 Echinacoside (polysaccharide)

In general, there are two categories of applications for polysaccharides: The first is the use of pharmaceutical materials, drug release agents, and plasma substitutes using polysaccharides that are simple to gel, have high osmotic pressure, high viscosity, and water absorption. The second is the biological activities of polysaccharides, their antigenicity, antitumor, and other biological function to prepare vaccines or new drugs. The objective of this article is to examine recent developments in the study of bioactive polysaccharides that have been extracted from natural sources and their application to pharmacology and biological medicine. Additionally, it covers the numerous polysaccharide bioactivities, such as effects on immune regulation, tumor prevention, virus defense, and anti-inflammation (Yu et al. 2018), (Fig. 10.2).

10.5

Alkamides Derivatives

It has been shown that the alkamides found in the genus Echinacea (Asteraceae) have high bioavailability as well as immunomodulatory effects. Alkamides are organic compounds created naturally when different amines and straight-chain, typically unsaturated, aliphatic acids are linked together by an amide bond. From eight plant families made up of diverse combinations of 200 acids and 23 amines, more than 300 compounds are recognized. Alkamides with unsaturated acid portions

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are classified as compounds with entirely olefinic patterns and those with olefinic and acetylenic connections, with a few saturated derivatives being the exception. While acetylenic acid portions are typical for Asteraceae, alkamides with extended olefinic acid parts are primarily found in Piperaceae and Brassicaceae (Greger 2016). The roots of E. purpura, as well as the aerial sections, are principally where alkamides are found, some experts believe that alkamides are still the essential and biologically active components of Echinacea (Bauer 1999; Miller 2000). Any specific species of Echinacea has a different distribution of alkamides in different regions of the plant. E. purpurea root and aerial parts have the lowest alkamide contents in Echinacea collections (Bauer et al. 1988). The quantities of alkamides and cichoric acid discovered in the root material utilized in this investigation are in good agreement with data for Danish-grown E. purpurea that have previously been published (Mølgaard et al. 2003). The resulting alkamide combination was representative of the alkamides in the 80% ethanol extract of Echinacea purpurea root because the HPLC fingerprint of the alkamide mixture separated from the 80% ethanol extract was identical with the alkamide section of the HPLC chromatogram of the complete 80% ethanol extract. A significant variety of biological activity, including immunomodulatory, antibacterial, antiviral, larvicidal, insecticidal, diuretic, pungent, analgesic, cannabimimetic, and antioxidant activities, are exhibited by the group of bioactive natural chemicals known as alkamides. Along with inhibiting prostaglandin biosynthesis, RNA synthesis, and arachidonic acid metabolism, these natural chemicals also potentiate several antibiotics. Alkamide-containing plant species have been employed in conventional medicine by numerous cultures all over the world (Rios and Olivo 2014). The common cold and various upper respiratory tract illnesses are the principal conditions for which Echinacea purpurea alkamides are sold. They might have both immunostimulatory and anti-inflammatory properties (Woelkart and Bauer 2007). Purified cichoric acid and alkamides, two of Echinacea purpurea’s ingredients, were compared to the antioxidant activity of extracts of the plant’s stems, leaves, and roots (Thygesen et al. 2007), (Fig. 10.3).

10.6

Conservation of Echinacea purpurea

The botanical and conservation communities have emphasized that echinacea’s commercial exploitation is unsustainable as its popularity has increased. There was concern about the plant’s potential to regenerate within a few years after it was introduced to the health sector because of the quantity obtained (Sayre 1903). Echinacea is often propagated by crown divisions, seeds, and root cuttings. Typically, seeds are sown in the direct ground or grown in a greenhouse before being transplanted into the ground (Miller 2000). However, due to the physiological makeup of the seed and its surroundings during growth, soil moisture, and pH, Echinacea seed germination and transplant production efficiency varies widely, from no germination to varying frequency (Macchia et al. 2001). Different techniques and

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Fig. 10.3 Alkamides

approaches, such as the rapid introduction of new cultivars with desirable features, as well as the rapid multiplication of axenic, healthy plants, have been created to meet the growing need for this significant medicinal plant. Techniques for in vitro tissue cultivation have shown to be highly beneficial in this area.

10.7

Echinacea purpurea Mass Propagation Using Plant Tissue Culture

Plant regeneration and in vitro culture offer advantages over traditional vegetative propagation due to a plant’s capacity for rapid growth. Additionally, these techniques might be helpful for maintaining species that are less susceptible to traditional cloning (Harbage 2001), Table 10.3. Different explants, such as in vitro seedlings and fully grown plants in the wild, have been used to produce different Echinacea species. For commercially viable Echinacea species, several regeneration techniques have been documented (Coker and Camper 2000; Choffe et al. 2000; Harbage 2001; Lakshmanan et al. 2002; Zobayed and Saxena 2003; Koroch et al. 2003; Pan et al. 2004; Sauve et al. 2004; Zhao et al. 2006; Jones et al. 2007), and almost all techniques have used explants from embryonic or in vitro-grown seedlings. Anthers, mesophyll protoplasts, petioles, stems, seeds, flower stalks, leaf sections, hypocotyls, and cotyledons were employed as explants in early trials to induce callus, which later developed into shoots and roots. Over the past 5 years, substantial advancement has been achieved in the creation of Echinacea in vitro systems for regeneration. The establishment of axenic cultures and the regeneration of tissues, organs, and entire plants are thus possible using several protocols. The plants can be grown until they are fully formed under regulated conditions. The in vitro Echinacea production methods yield numerous plants, as well as regenerants that develop swiftly in the greenhouse and can provide physiologically stable vegetation for the year of pharmaceutical manufacture (Murch et al. 2006; Zheng et al. 2006, Jones et al. 2007). The market tendency toward control production of plants as opposed to wild plants collecting is projected to accelerate as a result of the overall advancements made in Echinacea biotechnology. The need for higher standards of the quality of raw and processed plant material is becoming more widely acknowledged in the natural product sector. The growing herbal market demands and anticipates better

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Table 10.3 Plant tissue culture studies in Echinacea purpurea Explant Petiole

Hypocotyl, cotyledon

Plant growth regulators Indole-3-acetic acid, Thidiazuron, naphthaleneacetic acid, benzyl amino purine, 2,4dichlorophenoxyacetic acid Indole-3-butyric

Hypocotyl

Naphthaleneacetic acid, kinetin

Seed, shoot tip Leaf

Benzyl amino purine Benzyl amino purine, kinetin, indole-3-butyric acid Indole-3-acetic acid, benzyl amino purine

Leaf, petiole, hypocotyl, cotyledon Leaf Leaf

Naphthaleneacetic acid, benzyl amino purine, isopentenyl adenine Benzyl amino purine, naphthaleneacetic acid

Leaf

Benzyl amino purine, naphthaleneacetic acid

Leaf, cotyledon, root

Benzyl amino purine, indole-3-acetic acid

Leaf

Benzyl amino purine, naphthaleneacetic acid

Flower, leaf section, hypocotyl, cotyledon Mesophyll

Naphthaleneacetic acid, Thidiazuron

Leaf, petiole, root

Benzyl amino purine, naphthaleneacetic acid

Leaf

D 2,4- dichlorophenoxyacetic acid, dicamba, Thidiazuron

Benzyl amino purine, indole-3-butyric acid

Reference Choffe et al. (2000) Choffe et al. (2000) Coker and Camper (2000) Harbage (2001) Lakshmanan et al. (2002) Bhatti et al. (2002) Zhao et al. (2006) Koroch et al. (2002) Mechanda et al. (2003) Zobayed and Saxena (2003) Koroch et al. (2003) Sauve et al. (2004) Pan et al. (2004) Wang and To (2004) Jones et al. (2007)

quality control methods, including standardization techniques. Here, in vitro technology can immediately and significantly contribute to the production of vast amounts of high-quality, chemically consistent raw material for the Echinacea market.

References Abreu RMV, Ferreira ICFR, Calhelha RC, Lima RT, Vasconcelos MH, Adega F, Chaves R, Queiroz M-JRP (2011) Anti-hepatocellular carcinoma activity using human HepG2 cells and hepatotoxicity of 6-substituted methyl 3-aminothieno [3, 2-b] pyridine-2-carboxylate derivatives: in vitro evaluation, cell cycle analysis and QSAR studies. Eur J Med Chem 46(12):5800–5806 Agrawal AD (2011) Pharmacological activities of flavonoids: a review. Int J Pharm Sci Nanotechnol 4(2):1394–1398

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Alam M, Ahmed S, Elasbali AM, Adnan M, Alam S, Hassan MI, Pasupuleti VR (2022) Therapeutic implications of caffeic acid in cancer and neurological diseases. Front Oncol 12:860508 Balciunaite G, Juodsnukyte J, Savickas A, Ragazinskiene O, Siatkute L, Zvirblyte G, Mistiniene E, Savickiene N (2015) Fractionation and evaluation of proteins in roots of Echinacea purpurea (L.) Moench. Acta Pharma 65(4):473–479 Bauer R (1999) Chemistry, analysis and immunological investigations of Echinacea phytopharmaceuticals. In: Immunomodulatory agents from plants. Springer, Cham, pp 41–88 Bauer R, Remiger P, Wagner H (1988) Echinacea vergleichende DC und HPLC: analyse der Herba–Drogen von Echinacea purpurea, E. pallida und E. angustifolia. Dtsch Apoth Ztg 128: 174–180 Bhatti SM, Myles EL, Long DE, Sauve R (2002) In vitro regeneration of St. Johns wort and coneflowers. SNA Research Conference 47:340–342 Binns SE, Hudson J, Merali S, Arnason JT (2002) Antiviral activity of characterized extracts from Echinacea spp. (Heliantheae: Asteraceae) against herpes simplex virus (HSV-I). Planta Med 68(09):780–783 Birková A, Hubková B, Bolerázska B, Mareková M, Čižmárová B (2020) Caffeic acid: a brief overview of its presence, metabolism, and bioactivity. Bioact Compd Health and Dis 3(4):74–81 Bodinet C, Beuscher N (1991) Antiviral and immunological activity of glycoproteins from Echinacea purpurea radix. Planta Med 57(S2):A33–A34 Cai C, Chen Y, Zhong S, Ji B, Wang J, Bai X, Shi G (2014) Anti-inflammatory activity of N-butanol extract from Ipomoea stolonifera in vivo and in vitro. PLoS One 9(4):e95931 Cech NB, Kandhi V, Davis JM, Hamilton A, Eads D, Laster SM (2010) Echinacea and its alkylamides: effects on the influenza A-induced secretion of cytokines, chemokines, and PGE2 from RAW 264.7 macrophage-like cells. Int Immunopharmacol 10(10):1268–1278 Chen JH, Ho C-T (1997) Antioxidant activities of caffeic acid and its related hydroxycinnamic acid compounds. J Agric Food Chem 45(7):2374–2378 Chen Y, Fu T, Tao T, Yang J, Chang Y, Wang M, Kim L, Qu L, Cassady J, Scalzo R (2005) Macrophage activating effects of new alkamides from the roots of Echinacea species. J Nat Prod 68(5):773–776 Chiou S-Y, Sung J-M, Huang P-W, Lin S-D (2017) Antioxidant, antidiabetic, and antihypertensive properties of Echinacea purpurea flower extract and caffeic acid derivatives using in vitro models. J Med Food 20(2):171–179 Choffe KL, Victor JMR, Murch SJ, Saxena PK (2000) In vitro regeneration of Echinacea purpurea L.: direct somatic embryogenesis and indirect shoot organogenesis in petiole culture. In Vitro Cell Dev Biol-Plant 36(1):30–36 Clifford MN (2000) Chlorogenic acids and other cinnamates–nature, occurrence, dietary burden, absorption and metabolism. J Sci Food Agric 80(7):1033–1043 Coker PS, Camper ND (2000) In vitro culture of Echinacea purpurea L. J Herbs Spices Med Plants 7(4):1–7 Collins HR (2017) Caffeic acid: sources, potential uses and health benefits. Nova Science Publishers Incorporated, Hauppauge, NY Dalby-Brown L, Barsett H, Landbo A-KR, Meyer AS, Mølgaard P (2005) Synergistic antioxidative effects of alkamides, caffeic acid derivatives, and polysaccharide fractions from Echinacea purpurea on in vitro oxidation of human low-density lipoproteins. J Agric Food Chem 53(24): 9413–9423 Ekeuku SO, Pang K-L, Chin K-Y (2021) Effects of caffeic acid and its derivatives on bone: a systematic review. Drug Des Devel Ther 15:259 Erkoyuncu MT, Yorgancilar M (2021) Optimization of callus cultures at Echinacea purpurea L. for the amount of caffeic acid derivatives. Electron J Biotechnol 51:17–27 Genaro-Mattos TC, Maurício ÂQ, Rettori D, Alonso A, Hermes-Lima M (2015) Antioxidant activity of caffeic acid against iron-induced free radical generation—a chemical approach. PLoS One 10(6):e0129963

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An Insight of Phytochemicals of Shatavari (Asparagus racemosus)

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Vibha Pandey, Manju Shri, Sonali Dubey, Syed Saema, and Shivani Tiwari

Abstract

Asparagus racemosus is known to be a very important species due to its vital application in various diseases. Shatavari is the popular name of A. racemosus. In Ayurveda, shatavari has been documented for its curative and preventive use in aging, with improved mental function and increased longevity, along with supplementing vigor and vitality to the body. A. racemosus has also been used in dyspepsia, nervous disorders, inflammation, tumors, hepatopathy, and neuropathy. There are reports regarding the pharmacological activities of extracts of A. racemosus that include antioxidant, anti-cancerous, anti-diarrheal, immunomodulatory, anti-ulcer, and anti-diabetic activities. Steroidal saponins (shatavarinI–X) are the main active components of shatavari root extract with pharmacological activity. In the same category, shatavarinIV has been classified as glycosides of sarsasapogenin. Along with shatavarin, there are a few other active components that have been identified and characterized, such as quercetin, rutin, and immunoside. Not only the roots, flowers, fruits, as well as leaves also

Vibha Pandey and Manju Shri contributed equally with all other contributors. V. Pandey (✉) CSIR-National Botanical Research Institute, Lucknow, India M. Shri (✉) School of Applied Sciences and Technology, Gujrat Technological University, Ahmedabad, India S. Dubey School of Biosciences, IMS Ghaziabad University Courses Campus, Ghaziabad, India S. Saema Department of Environmental Science, Integral University, Lucknow, India S. Tiwari Azad Institute of Pharmacy and Research, Lucknow, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_11

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possess many of these pharmacologically active compounds such as shataverins, diosgenin, and quercetin-3 glucuronide. In this chapter, we will try to collect the state of the art of phytochemicals discovered from shatavari with their biological application that will be beneficial for utilization in the development of specialty/ functional foods. Keywords

Phytochemicals · Steroidal saponins · Shatavari · Secondary metabolites

Abbreviations ABTS●+ CAE CHE DPPH DW ESI FW GAE HPTLC LC MS NMR PDA QRE QTOF RUE TLC TOF

11.1

3-ethyl benzothiazolin-6-sulfonic acid radical cation (blue chromophore) Catechin equivalents Cholesterol equivalent 2,2-diphenylpicrylhydrazyl Dry weight Electrospray ionization Fresh weight Gallic acid equivalent High-performance thin-layer chromatography Liquid chromatography Mass spectroscopy Nuclear magnetic resonance Photodiode array Quercetin equivalent Quadrupole time of flight Rutin equivalent Thin-layer chromatographic Time of flight

Introduction

Consumption of medicinal plants for several diseases is as old as the human heritage. Awareness of medicinal plant usage and sufficiency is usually suggested for treating various human disorders even though their components are not completely identified (Sofowora et al. 2013). The use of therapeutic plants and their supplements has been effectively encouraged worldwide, especially in nations with enriched biodiversity and traditional medicine system (India, South Africa, Brazil, China, etc.). The existence of 500,000 plants has been reported globally and presently approximately

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10% are consumed in the form of food by humans and different organisms (Tuszynska 2010; Singh et al. 2018a, b). The Planning Commission has divided India into 15 agro-climatic regions. Out of 17,000–18,000 varieties of blossoming plants, about 6000–7000 have been identified to possess therapeutic implementation in the community and acknowledged in the Indian medicine systems, like Homeopathy, Ayurveda, Siddha, and Unani (Samal 2016; Singh et al. 2018a, b). Asparagus is a large genus consisting of about 300 species of the family Asparagaceae, including the most cultivated and essential Asparagus racemosus, commonly called “shatavari” to represent the “curer of a hundred diseases.” Shatavari is broadly cultivated in tropical, subtropical, and Himalayan regions that range up to 1500 m altitude in India, Australia, Africa, China, Sri Lanka, and Indonesia (Tuszynska 2010; Janani and Singaravadivel 2014; Ali et al. 2018; Singh et al. 2018a, b; Thakur et al. 2021). Due to the valuable implementation, A. racemosus has been depicted as an established Ayurvedic Rasayana in Rig-Veda and Atharvaveda to impede illnesses and enhance immunity as well as mental activity. Different extracts as well as marketed formulations of A. racemosus have been proven with anti-cancer, anti-microbial, antioxidant, anti-diabetic, anti-ulcer, anti-diarrheal, anti-inflammatory, immunomodulatory, neuroprotective as well as hepatoprotective activities (Tuszynska 2010; Mishra and Verma 2017; Sharma and Sharma 2017; Selvaraj et al. 2019; Ratdiya and Aher 2020; Dhanusha et al. 2021). All plant parts of A. racemosus have shown therapeutic potential based on their metabolic profile containing imperative bioactive constituents, leading with steroidal saponins, alkaloids, flavonoids, carbohydrates, terpenoids, etc. (Tuszynska 2010; Jayashree et al. 2013; Singh et al. 2018c; Kashyap et al. 2020; Ratdiya and Aher 2020; Borse et al. 2021). Steroidal saponins are dominant metabolites of A. racemosus (up to 85%), including shatavarin I–X, as the principal active components. Besides, other active components like ferulic acid, caffeic acid, gallic acid, cinnamic acid quercetin, rutin, diosgenin, lupeol, and β-glucogallin have been also isolated from the plant to establish their therapeutic property (Shameem and Majeedi 2020; Yu and Fan 2021; Thakur et al. 2021). This chapter is focused on the abundance and variety of phytoconstituents of Shatavari in different samples like field material, in vitro culture, and marketed products/formulations. Based on solvent selection and analytical method, the metabolic composition of samples as well as highlighted significance of A. racemosus varies considerably. Qualitative as well as quantitative analysis of metabolites (with substantial medicinal value) has been discussed in detail (Fig. 11.1), to conclude the effective part, solvent, and method required to achieve maximum yield of valuable product (extract/pure compound).

11.2

Qualitative Metabolic Profile

A. racemosus is very well known for its medicinal properties. Phytochemicals present in the plant (either in roots, shoots, or leaves) alone or in combination had been shown to have various therapeutic properties. The crude, semi-purified, or

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Fig. 11.1 Layout of phytochemicals identified from different samples of Asparagus racemosus, extracted using diverse solvent systems for different analyses

purified extract of roots/leaves/tubers is beneficial to health. There are various qualitative and quantitative studies available for the identification, quantification, and purification of phytochemicals from all the parts of A. racemosus. Shatavari is a huge source of various phytochemicals such as carbohydrates, alkaloids, terpenoids, saponins, shataverins, etc. Various studies have been performed with various plant parts, discussing different methods, and extracts to utilize for qualitative metabolic profiling of A. racemosus. Various chemicals have been taken for the extraction methods and variations in results have been observed (Table 11.1). Methanol has been the first choice of authors followed by methanolic water for the extraction of phytochemicals from A. racemosus. Methanolic extracts are detected with alkaloids, terpenoids, diterpenes, triterpenes, tannins, quinines, steroids, fatty acids carbohydrates, flavonoids, reducing compounds, phenolics, polyphenols, saponins, and/or glycosides in different studies (Pise et al. 2011; Kafle et al. 2012; Sivakumar and Gajalakshmi 2014; Haghi et al. 2012; Jayashree et al. 2013, 2015b; Shastry et al. 2015; Saraswathi et al. 2020; Suja and Sivakala 2021; Ashraf et al. 2021; Dhanusha et al. 2021). Behera (2018) obtained a 29.2%

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Table 11.1 Qualitative analyses Compounds From root tissue Alkaloids; flavonoids; sterols; terpenes Fatty acids; flavone; glycosides; glycosides; polyoses; quinines; reducing compounds; saponin sterols; triterpenes Fatty acids; flavonoids; glycosides; lactones; phenolics; phytosterols; saponins; tannins; triterpenoids Carbohydrates; cardiac glycosides; flavonoids; phenol; saponins; steroids; tannins Alkaloids; carbohydrates; flavonoids; glycosides; Phenolics; saponins; Total sugar; Reducing sugar Alkaloids; flavonoids; glycosides; Saponins; steroids; tannins; Terpenoids Flavonoid Phenolics

Flavonoids Polyphenols

Quantity (mg/g)

Extraction solvent

Medicinal activity/property

–

Ethanol

Anti-diarrheal

0.58% (saponins)

Hexane; methanol; aqueous

Anti-microbial

–

Methanol

–

Jayashree et al. (2013)

–

Ethanol

–

Janani and Singaravadivel (2014)

– – 6.42% 5.13%

Ethanol

–

Selvarajan et al. (2014)

–

Methanol

–

Sivakumar and Gajalakshmi (2014)

0.68–0.71% w/w QRE 2.92–3.67% w/w GAE 113 ± 6.8 154 ± 7.4 μg/mg CAE

Methanol

–

Haghi et al. (2012)

Water; methanol

In vitro scavenging (DPPH, ABTS●+);

Jayashree et al. (2015b)

References Gomase and Sherkhane (2010) Kafle et al. (2012)

(continued)

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Table 11.1 (continued) Compounds

Quantity (mg/g)

Extraction solvent

146 ± 8.2 246 ± 11.8 μg/ mgGAE

Amino acids; amides; cellulose; flavonoids; hemicelluloses; lignin; polysaccharides; tannins Alkaloids; carbohydrates; flavonoids, glycosides; steroids; tannins; triterpenoids Flavonoids, glycosides; saponins; steroids; tannins; terpenoids Flavonoid Phenolics

Alkaloids; carbohydrates; flavonoids; glycosides; phenolics; proteins; resins; saponins; steroids; tannins Flavonoids Polyphenols

Flavonoids Phenols Steroids

–

Tissue moisture only, using FT-IRS

Medicinal activity/property protective against H2O2 induced plasmid DNA, colon and muscle cell damage –

References

Mishra et al. (2015)

Ethanol

Anti-microbial

Shrestha et al. (2015)

–

Ethanol

–

Agarwal et al. (2018)

0.80 ± 0.001 mg/ g RUE; 12.90 ± 0.002 mg/g GAE –

Methanol

DPPH radical scavenging

Behera (2018)

Hydroalcoholic

H2O2 scavenging

Thakur et al. (2018)

6.88–12.86 μg/g QRE for SE 1.37–11.63 μg/g QRE for ME 3.17–9.48 mg/g GAE for SE 0.12–7.40 mg/g GAE for ME 56.77 ± 0.19 μg/ mg QRE 412.6 ± 0.42 μg/ mg GAE

Soxhlet; maceration; CO2; ethanol; water

DPPH radical scavenging; anti-diabetic

Hasan and Panda (2020)

Water

Radicals scavenging (DPPH, superoxide,

Saraswathi et al. (2020)

(continued)

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Table 11.1 (continued) Compounds

Quantity (mg/g)

Extraction solvent

Medicinal activity/property

Methanol

hydroxyl, ABTS●+); antioxidant (Mo6+, Fe3+ reducing) –

27.5 ± 0.33 μg/ mg CHE

Alkaloids; – carbohydrates; flavonoids; steroidal saponins – Steroids, alkaloids, flavonoids, glycosides, saponins; diterpenes Carbohydrates; – gums; mucilage; 30.43 ± 0.97 mg/ proteins; g Flavonoids; 69.1 ± 0.42 mg/g Phenols; 55.6 ± 0.89 mg/g Saponins; 63.61 ± 1.17 mg/ Steroids g From other plant parts From root calli: 10.38 ± 0.14 mg/ Saponins g From nodal calli: 7.69 ± 0.136 mg/ Saponins g Steroids (from – leaves) From aerial parts: 34–497 mg/g Flavonoid QRE Phenolics 75–154 mg/g GAE

References

Suja and Sivakala (2021)

Methanol

Cytotoxic and antiproliferative for MDAB-231 cells

Dhanusha et al. (2021)

Hydroalcoholic

In vitro antioxidant

Shalini and Ilango (2021)

Butanol fraction of methanol extract Methanol

–

Pise et al. (2011)

–

Methanol

DPPH radical scavenging

Verma et al. (2013) Ashraf et al. (2021)

a

SE Soxhlet extract, ME maceration extract, FT-IRS Fourier-transform infrared spectroscopic, GAE gallic acid equivalent, QRE quercetin equivalent, RUE rutin equivalent, CAE catechin equivalents, CHE cholesterol equivalent, DPPH 2,2-diphenylpicrylhydrazyl, ABTS●+ 3ethyl benzothiazolin-6sulfonic acid radical cation (blue chromophore)

yield, with 12.90 mg/g DW of total phenolic and 0.80 mg/g DW of total flavonoid contents. However, Jayashree et al. (2015b) demonstrated 113 ± 6.8 and 154 ± 7.4 μg/mg CAE of flavonoids along with 146 ± 8.2 and 246 ± 11.8 μg/mg GAE of polyphenols in aqueous and methanolic root extracts, respectively. The phytochemical screening of Shatavari root ethanolic extract showed the presence of steroids, saponins, carbohydrates, flavonoids, alkaloids, terpenes, tannins, phenolics, proteins and/or amino acids (Gomase and Sherkhane 2010; Janani and Singaravadivel 2014; Shastry et al. 2015; Shrestha et al. 2015; Jadhav 2018; Thakur et al. 2018; Shalini and Ilango 2021). Hydroalcoholic extract of

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A. racemosus roots contains carbohydrates, gums, mucilage, proteins, amino acids, phenols, saponins, steroids, essential oils, vitamins, flavonoids, diterpenes, along with resins, tannins, and glycosides, which were absent in ethanolic root extract (Thakur et al. 2018; Shalini and Ilango 2021). Mishra et al. (2015) used dry powder and fresh juice of tuber roots of A. racemosus and confirmed the presence of various active groups such as phenol, alkyl group, methyl groups, alcohols, ethers, and carboxylic acid, using performed Fourier-transform infrared spectroscopic (FT-IRS). These active groups represent the presence of several biomolecules, like tannins/ flavonoids, amino acids, amides, lignin, cellulose, hemicelluloses, and polysaccharides. The methanolic extract of A. racemosus revealed anti-microbial activity (against Staphylococcus aureus, Salmonella typhi, S. paratyphi, and Shigella dysenteriae (Kafle et al. 2012), free radicals (DPPH, Superoxide, Hydroxyl, ABTS●+) scavenging (Jayashree et al. 2015b; Shastry et al. 2015; Behera 2018; Saraswathi et al. 2020), antioxidant (reduction of phophomolybdenum and ferric; Saraswathi et al. 2020), anticonvulsant (Shastry et al. 2015) along with cytotoxic and antiproliferative activity in MDAB-231 cells (Dhanusha et al. 2021). Methanolic extract of A. racemosus has been also proven to inhibit H2O2 induced plasmid DNA, colon, and muscle cell damage (Jayashree et al. 2015b). Ethanolic root extract was found with anti-diarrheal (Gomase and Sherkhane 2010), free radical scavenging (DPPH, H2O2) anticonvulsant (Shastry et al. 2015; Thakur et al. 2018), and anti-microbial activity against various pathogenic bacteria, such as Bacillus subtilis, Candida albicans, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Saccharomyces cerevisiae, Salmonella typhi, Streptococcus pyogenes, S. pneumonia, Staphylococcus epidermidis, S. aureus, and/or Pseudomonas aeruginosa, (Shrestha et al. 2015; Jadhav 2018). The hydroalcoholic root extract can also be used as an antioxidating agent (Thakur et al. 2018; Shalini and Ilango 2021). High free radical scavenging activity and phytochemical constituents reported from the plant material are likely to be valuable for advanced studies to combat oxidative stress.

11.3

Steroidal Saponins

Saponins belong to the glycoside family and it is widely distributed not only in plants but also in lower marine organisms. Saponins form a large group with steroids, with one or more sugar moiety or triterpenoid aglycone groups attached (Hostettmann and Marston 1995). Steroidal saponins are exclusively present in angiosperms. Steroidal saponins are known to have various pharmacological applications such as inhibitory action on platelet aggregation, insecticidal, along with antiparasitic, antihyperlipidemic, anti-oxidative, antitumor, and anti-diabetic properties (Sparg et al. 2004). Here we briefly discuss the studies (Table 11.2) involved in the identification and purification of steroidal saponins from A. racemosus, which are present not only in roots but also in other plant organs such as fruits, leaves, and stems.

25S-5β-spirostan-3β-yl-O-[O-α-Lrhamnopyranosyl-(1 → 4)]-β-D-glucopyranoside; 25S-5β-spirostan-3β-yl-O-β-D-glucopyranosyl (1 → 2)-O-[O-α-L-rhamnopyranosyl-(1 → 4)]-β-Dglucopyranoside; 25S-5β-spirostan-3 β-yl-O-β-Dglucopyranosyl (1 → 2)-O-{[O-β-Dglucopyranosyl-(1 → 4)]-O-{α-D-arabinopyranosyl (1 → 6]}-β-D-glucopyranoside; immunoside; shatavarin I; shatavarin IV Asparinin A; Asparinin B; Asparoside A; Asparoside B; Curillin H; Curilloside G; Curilloside H; Shavatarin I; Shavatarin II; Shavatarin III; Shavatarin IV; Shatavarin V 3-O-{[β-D-glucopyranosyl(1 → 2)][α-Lrhamnopyranosyl (1 → 4)]-β-D-glucopyranosyl}26-O-(β-D-glucopyranosyl)-(25S)-5β-furostan3β,22α,26-triol; 3-O-{[β-D-glucopyranosyl(1 → 2)] [α-L-rhamnopyranosyl (1 → 4)]-β-Dglucopyranosyl}-(25S)-5β-spirostan-3β-ol Shatavarin I; Shatavarin IV; Shatavarin V; Shatavarin VI; Shatavarin VII; Shatavarin IX; Shatavarin X; Immunoside; SchidigerasaponinD5

Compounds Froom roots 8-methoxy-5,6,4′-trihydroxyisoflavone -7-O-β-Dglucopyranoside

Table 11.2 Saponins in Asparagus racemosus Analytical method NMR; MS

SGCC; TLC; HPLC; NMR

RP-HPLC; NMR; LCMS RP-HPLC; NMR; LCMS

RP-HPLC; NMR; LCMS

Extraction solvents –

BuOH-F of AqE

Acetonitrile: Water (9:1)

Acetonitrile: Water (9:1)

Acetonitrile: Water (9:1)

–

Anti-HIV activity Sabde et al. (2011); Anti-cancerous Bhutani et al. (2010)

–

–

–

–

–

–

–

Medicinal property

0.082% DW

Quantity

An Insight of Phytochemicals of Shatavari (Asparagus racemosus) (continued)

Hayes et al. (2008)

Hayes et al. (2006b)

Hayes et al. (2006a)

Saxena and Chourasia (2001) Jadhav and Bhutani (2006)

References

11 177

BuOH-F of AqE Ethyl acetate

Immunoside

Anti-hepatitis B Immuno-stimulant

– –

HPTLC NMR; XRC;

TLC; HPLC; NMR

HPLC-QTOF-MS/ MS

Methanol

–

–

–

0.0–12 mg/g DW; 0.0–2.5 mg/g DW; 0.0–3.1 mg/g DW; 0.0–0.3 mg/g DW; 0.0–0.4 mg/g DW

–

Immunomodulatory Sharma et al. (2013)

–

SGCC; NMR; MS

Medicinal property Immunomodulatory

Quantity 8.53 ± 0.38% 0.038 ± 0.003%

Analytical method HPLC

BuOH-F of MeOH

Methanol

Methanol

Shatavaroside A; Shatavaroside B; Filiasparoside C

(1S,2R,3S,8S,9S,10S,13S,14S,16S,17R,22R,25R)21-nor-18β,27α-dimethyl-1 β,2 β,3 β -trihydroxy25-spirost-4-en-19 β-oic acid Shatavaroside C; Shatavarol; Shatavarin IV; Racemoside A; β-sitosterol; Stigmasterol; Ursolic acid 3-O-{[β-D-glucopyranosyl(1 → 2)] [α-Lrhamnopyranosyl-(1 → 4)]-β-Dglucopyranosyl}-(25S)-5β- spirostan-3β-ol4; 3-O{[β-D-glucopyranosyl (1 → 2)] [α-Lrhamnopyranosyl-(1 → 4)]-β-Dglucopyranosyl}-26-O-(β-d-glucopyranosyl) (25S)-5β-furostan-3β, 22α, 26triol 3 Asparacoside; Shatavarin IX; Shatavarin IV; Asparanin A; Shatavari V

Extraction solvents BuOH-F of AqE

Compounds Shatavarin IV; Immunoside

Table 11.2 (continued)

Onlom et al. (2017a)

References Gautam et al. (2009) Sharma et al. (2009) Sidiq et al. (2011) Sharma et al. (2011) Sharma et al. (2012b) Pasha et al. (2016)

178 V. Pandey et al.

–

BuOH-F of MeOH

Racemoside A (fruits)

–

0.53 μg/g EE

Anti-leishmanial

–

Immunomodulatory

Neuroprotective effect on PC12 cells; antioxidant

–

–

Water; butanol BuOH-F of MeOH

ShatavarinIV; Immunoside RacemosidesA; RacemosidesB; RacemosidesC (fruits)

Anti-cancer

–

0.15–1.45% 0.0053–0.054% –

RIA, HPLC

Methanol

AspoligoninA; AsparacosinA; AsparagosideA; Dioscin; Protodioscin; Sarsasapogenin; ShatavarinIV; ShatavarinIX; ShatavarinX; Schidigera saponinD5; Ferulic acid; Nyasol From other plant pats Phytoecdysteroids (from seeds)

Inhibition of lipid peroxidation

–

HPTLC, NMR TLC, NMR, ESI-TOF

HPCPC; NMR; ESI/MS UPLCQTOF; LC/MS; RP-HPLC;

Acetonitrile: Water (9:1) Water

ELISA; ICC; LC–MS/MS

Acetonitrile: Water (9:1)

Asparacoside; Shatavarin IX; Asparanin A; Shatavarin V; Shatavarin X; 3-O-{[β-D-glucopyranosyl(1 → 2)][α-Larabinopyranosyl(1 → 4)]-β-D-glucopyranosyl}(25S)-5β-spirostan-3β-yl; 3-O-{[α-Larabinopyranosyl (1 → 6)][β-D-glucopyranosyl (1 → 2)]-β-D-glucopyranosyl}-(25S)-5β-spirostan3β-yl; 3-O-{[β-D-arabinopyranosyl(1 → 4)]-β-Dglucopyranosyl}-(25S)-5β-spirostan-3β-yl; 3-O{[α-l-arabinopyranosyl(1 → 4)]- β-Dglucopyranosyl}-(25S)-5β-spirostan-3β-yl Asparacoside; ShatavarinIX; AsparaninA; ShatavarinV

An Insight of Phytochemicals of Shatavari (Asparagus racemosus) (continued)

Dinan et al. (2001) Satti et al. (2006) Mandal et al. (2006) Dutta et al. (2007)

Onlom et al. (2017c) Kashyap et al. (2020)

Onlom et al. (2017b)

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HPLC

LC-TOFMS; LC-QMS; LC-MS/MS; HPLC SGCC; TLC; NMR; MS

BuOH-F of MeOH Methanol

Methanol

Analytical method HPLC

Extraction solvents BuOH-F of MeOH Medicinal property – – –

–

Quantity 1.1% (20-fold enhanced)

11.48 ± 0.61 mg/g; 4. 02 ± 0.09 mg/g –

–

Verma et al. (2013)

Kumeta et al. (2013)

Pise et al. (2012)

References Pise et al. (2011)

EtAc-F ethyl acetate fraction, BuOH-F butanol fraction, AqE aqueous extract, MeOH methanol, CC cell culture, SM spent media, CSP commercial shatavari products, ELISA enzyme-linked immunosorbent assay, HPCPC high-performance centrifugal partition chromatography, HPTLC high-performance thin-layer chromatography, ICC immunoaffinity column chromatography, MS mass spectroscopy, RIA radioimmunoassay, TLC thin-layer chromatographic, SGCC silica gel column chromatography, NMR nuclear magnetic resonance, LC liquid chromatography, QTOF quadrupole time of flight, ESI electrospray ionization, TOF time of flight, UPLC ultra performing liquid chromatography

a

Compounds (from root calli; nodal calli) ShatavarinIV; Sarsapogenin; ShatavarinIV; Sarsapogenin (from roots; CC; SM) ShatavarinI; ShatavarinIII; ShatavarinIV; ShatavarinV; ShatavarinVI; ShatavarinVII; ShatavarinIX; ShatavarinX; Sarsasapogenin; Dehydrosarsasapogenin (from CSP) Spirostan-5-en-3β -ol3-O-[α-L-rhamnopyranosy(1 → 6)-β-D-glucopyranoside (from leaves)

Table 11.2 (continued)

180 V. Pandey et al.

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11.3.1 Steroidal Saponins in Roots Roots of A. racemosus are highly explored organs for the study of various phytochemicals including steroidal saponins. Shatavarin I–X are examples of steroidal saponins that are present in A. racemosus. A large number of extraction methods and chemicals have been used by authors for the identification and purification of steroidal saponins (Table 11.2). After examination of the root aqueous extract, Gautam et al. (2009) acknowledged the presence of shatavarinIV (8.53 ± 0.38%) and immunoside (0.038 ± 0.003%) through HPLC. Later, aspoligonin A, asparacosin A, asparagoside A, sarsasapogenin, shatavarin IX, shatavarin X, schidigera saponin D5, ferulic acid, dioscin, nyasol, and protodioscin were added by Kashyap et al. (2020) in the list of contents using UPLC-QTOF. Butanolic fraction of root ethanolic extract has opted for co-TLC, HPLC (Jadhav and Bhutani 2006), HPTLC (Sidiq et al. 2011), TLC, HPLC, and NMR (Pasha et al. 2016) analyses. Two new sarsasapogenin glycosides, 25S-5β-spirostan-3β-yl-O-βD-glucopyranosyl (1 → 2)-O-[O-α-L-rhamnopyranosyl-(1 → 4)]-β-Dglucopyranoside and 25S-5β-spirostan-3β-yl-O-[O-α-L-rhamnopyranosyl-(1 → 4)]β-D-glucopyranoside were identified by NMR along with 25S-5β-spirostan-3β-yl-Oα-L-rhamnopyranosyl (1 → 2)-O-[O-α-L-rhamnopyranosyl-(1 → 4)]-β-Dglucopyranoside (known as immunoside), 25S-5β-spirostan-3 β-yl-O-β-Dglucopyranosyl (1 → 2)-O-{[O-β-D-glucopyranosyl-(1 → 4)]-O-{α-Darabinopyranosyl (1 → 6]}-β-D-glucopyranoside, shatavarinI and shatavarinIV (Jadhav and Bhutani 2006). Sidiq et al. (2011) demarcated only immunoside, while Pasha et al. (2016) identified 3-O-{[β-D-glucopyranosyl (1 → 2)][α-Lrhamnopyranosyl- (1 → 4)] -β-D-glucopyranosyl}-(25S)-5β -spirostan-3β-ol4 and 3-O-{[β-D-glucopyranosyl(1 → 2)][α-Lrhamnopyranosyl-(1 → 4)]-β-Dglucopyranosyl}-26-O-(β-d-glucopyranosyl)(25S)-5β-furostan-3 β, 22α,26-triol-3, from the respective extract. Roots of A. racemosus were extracted using 90% acetonitrile/water and analyzed through RP-HPLC, NMR (Hayes et al. 2006a, b), immunoaffinity column, LC-MS/ MS (Onlom et al. 2017b) as well as high-performance centrifugal partition chromatography (HPCPC; Onlom et al. 2017c). Shatavarin V, which is (3-O→ 2)][β-D-glucopyranosyl(1 → 4)]-β-D{[α-L-rhamnopyranosyl(1 glucopyranosyl}-(25S)-5β-spirostan-3β-ol), was identified along with asparanin A, asparanin B, asparoside A, asparoside B, curillin H, curilloside G, curillosie H, shavatarin I and shavatarin IV (Hayes et al. 2006a). Two phytochemicals known as shatavarin I and IV, were elucidated as 3-O-{[β-D-glucopyranosyl(1 → 2)][α-Lrhamnopyranosyl(1 → 4)]-β-D-glucopyranosyl}-26-O-(β-D-glucopyranosyl)-(25S)5β-furostan-3β,22α,26-triol and 3-O-{[β-D-glucopyranosyl(1 → 2)][α-Lrhamnopyranosyl(1 → 4)]-β-D-glucopyranosyl}-(25S)-5β-spirostan-3β-ol (Hayes et al. 2006b). Later, Hayes et al. (2008) also acknowledged the presence of shatavarin IV–X; along with shatavarin I (or asparoside B), immunoside, and schidigerasaponin D5 (or asparanin A) in the root extract of A. racemosus, through RP-HPLC, NMR, and LC-MS. Along with saponin glycosides (asparacoside,

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asparaninA, shatavarinV, shatavarinIX) 4-minor peaks of 3-O-{[β-Dglucopyranosyl(1 → 2)][α-L-arabinopyranosyl (1 → 4)]-β-D-glucopyranosyl}(25S)-5β-spirostan-3β-yl, 3-O-{[α-L-arabinopyranosyl (1 → 6)][β-D-glucopyranosyl (1 → 2)]-β-D-glucopyranosyl}-(25S)-5β-spirostan-3β-yl, 3-O-{[β-Darabinopyranosyl (1 → 4)]-β-D-glucopyranosyl}-(25S)-5β-spirostan-3β-yl and 3-O-{[α-L-arabinopyranosyl (1 → 4)]- β-D-glucopyranosyl}-(25S)-5β-spirostan3β-yl were also detected during LC-MS/MS (Onlom et al. 2017b). Sharma et al. (2009) studied the methanolic root extract of A. racemosus and elucidated structures of shatavaroside A, shatavaroside B and filiasparoside C after chromatographic detection. In continuation, Sharma et al. (2012b) isolated shatavaroside C, shatavarol, shatavarin IV, racemoside A, β-sitosterol, stigmasterol and ursolic acid from the same extract, using column chromatography. NMR was used to determine the structure of shatavaroside C as 3-O-{[α-L-arabinopyranosyl(1 → 2)][α-L-rhamnopyranosyl-(1 → 4)]-β-D-glucopyranosyl}-26-O-β-Dglucopyranosyl-22α-methoxyl-(25S)-5β-furostan-3β,26-diol together with the structure of shatavarol as 5,5-di-(4-hydroxybenzene)-pent-3E-en-1,2-diol. In a different study, Sharma et al. (2011) isolated unique polyhydroxylated steroidal sapogenin acid, from the ethyl acetate root extract of A. racemosus. The compound was determined as (1S,2R,3S,8S,9S,10S,13S,14S,16S,17R,22R,25R)-21-nor-18β,27α-dimethyl-1 β,2 β,3 β -trihydroxy-25-spirost-4-en-19 β-oic acid. Following the extraction protocol of Jadhav and Bhutani (2006), Bhutani et al. (2010) targeted the anti-cancer activity of phytochemicals, while Sabde et al. (2011) confirmed the anti-HIV activity of the plant. Shatavarin IV observed a maximum of 10%, reduction in cell viability of colon cancer cells at 6 μM, while only immunoside induced apoptosis (Bhutani et al. 2010). The immunomodulatory activity of Shatavari root aqueous extract, along with isolated polyhydroxylated steroidal sapogenin acid as well as purified shatavaroside A and shatavaroside B was accurately acknowledged by Gautam et al. (2009), Sharma et al. (2011, 2013), respectively. The protective effect of root aqueous extract of A. racemosus was confirmed against hepatitis B surface antigen (Sidiq et al. 2011), and Aβ induced neurotoxicity, H2O2 induced toxicity and anti-cholinesterase activity (Kashyap et al. 2020). The aqueous extract also has DPPH free radical scavenging activity and inhibited β-APP cleaving enzyme 1 (BACE1), monoaminoxidase-B (MAO-B), in PC12 cells (Kashyap et al. 2020). Onlom et al. (2017b, c) demonstrated the effect of root extracts (90% acetonitrile/water) against lipid peroxidation as well as human prostate- (LNCaP) and hepato- (HepG2) carcinoma cell lines, respectively. Individual saponin glycosides were devoid of anti-lipid peroxidation (IC50 > 200 μg/mL). However, the recombination of components enhanced inhibiting potential (IC50 24 μg/mL). The saponin-enriched extract exhibited more lipid peroxidation (IC50 11.3 μg/mL) and cytotoxic activity (IC50 87.3–207 μg/mL) as compared to defatted ethanolic extract (lipid peroxidation with IC50 41.1 μg/mL and cytotoxic with IC50 469–798 μg/mL). LNCaP and HepG2 cell lines were observed to be more sensitive for shatavarinV (IC50 16.5 μg/mL) as well as asparacoside (IC50 13.6 μg/mL) and asparanin A (IC50 14.1 μg/mL), respectively.

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11.3.2 Steroidal Saponins in Other Plant Parts Seeds, fruits, leaves, in vitro cultures, and Shatavari-containing marketed products have also been found rich in saponin contents, as listed in Table 11.2. Only, Satti et al. (2006) used aqueous and butanol extract of A. racemosus with HPTLC and NMR to conclude 0.15–1.45% of shatavarinIV and 0.0053–0.054% of immunoside. The first detection technique used was radioimmunoassay (Dinan et al. 2001) for methanolic seed extract to detect very low levels of (0.53 μg/g ecdysone equivalent) phytoecdysteroids. Following that, butanol fraction of methanolic extracts from defatted fruits (Mandal et al. 2006; Dutta et al. 2007), in vitro callus cultures (from the root and nodal segments; Pise et al. 2011), along with roots, cell culture, and spent media (Pise et al. 2012) were examined. Defatted fruits revealed racemosideAC as (25S)-5β-spirostan-3β-ol-3-O-{β-D-glucopyranosyl (1 → 6)-[α-Lrhamnopyranosyl (1 → 6)-β-D-glucopyranosyl(1 → 4)]-β-D-glucopyranoside}, (25S)-5β-spirostan-3β-ol-3-O-α-L-rhamnopyranosyl (1 → 6)-β-D-glucopyranosyl and (25S)-5β-spirostan-3β-ol-3-O-{α-L(1 → 6)-β-D-glucopyranoside, rhamnopyranosyl-(1 → 6)-[α-L-rhamnopyranosyl (1 → 4)]-β-D-glucopyranoside} through TLC, NMR and ESI-TOF (Mandal et al. 2006) along with racemoside A (Dutta et al. 2007). Between callus cultures, root calli accounted for a maximum of 10.38 ± 0.14 mg/ g saponins (shatavarin IV and sarsapogenin) as compared to 7.69 ± 0.136 mg/g from calli of nodal explants. Also, HPLC proved that root calli contained 1% (20-fold more) of shatavarin IV as compared to 0.05–0.08% of wild-type roots. Examination of cell culture determined maximum biomass (28.30 ± 0.29 g/L), and saponins (20-fold) after 25 days of cultures with pH in the range of 3.4–5.6. Media containing NAA (1 mg/L), 2,4-D (1 mg/L), BAP (0.5 mg/L), casein hydrolysate (2 g/L), and pectinase (0.005%) exposed to the maximum level of sarsapogenin (4.02 ± 0.09 mg/ g) as intracellular as well as secreted in media. However, media supplemented with 2,4-D (2 mg/L), casein hydrolysate (2 g/L), and pectinase (0.005%) exhibited a maximum amount of shatavarinIV (11.48 ± 0.61 mg/g), as compared to natural plant roots. Accumulation of sarsapogenin varied intracellular as well as in secreted form based on media composition, while shatavarinIV was detected mostly in secreted form. Methanol has been the primary choice of extraction and used for the extraction of 11 commercial shatavari products (Kumeta et al. 2013), leaves (Verma et al. 2013) in addition to samples (root, stem, leaves) from 5-different locations (Bangkok, Phetchabun, Phitsanulok, Rayong, Aurangabad) of Thailand and India (Onlom et al. 2017a). Commercial shatavari products revealed the presence of shatavarin I, shatavarin III–VII, shatavarin IX–X, dehydrosarsasapogenin and sarsasapogenin through LC–TOFMS, LC–QMS, LC-MS/MS or HPLC. The structure of steroidal saponin called spirostan-5-en-3β -ol3-O-[α-L-rhamnopyranosy-(1 → 6)-β-Dglucopyranoside was established from leaf extract using TLC, NMR and MS (Kumeta et al. 2013). Comparative study of samples from different locations demonstrates that root samples were rich in 5-saponin glycosides, (asparacoside, asparanin A, shatavarin V, shatavarin IV and shatavarin IX), while only traces of

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asparacoside were found in sample (Bangkok) of leaves and stem. Maximum and minimum asparacoside were quantified from Phetchabun (12 mg/g DW) and Indian (absent) samples, respectively. ShatavarinIV (3.1 mg/g DW), followed by shatavarinV (0.4 mg/g DW) was the chief component in Indian samples. ShatavarinIV was absent in all the Thai samples, while shatavarinV was present in only the Rayong Thai sample (0.3 mg/g DW). Asparanin A was present (0.3 mg/g DW) in only Rayong samples (Verma et al. 2013). Shatavari extracts presented agonistic and antagonistic properties, which were absent in seed extracts (Dinan et al. 2001). Use of A. racemosus in the form of aqueous extract, shatavarin IV and immunoside (Satti et al. 2006) as well as fruit extract (Dutta et al. (2007) unveiled immunomodulatory activity and antileishmanial activity.

11.3.3 Shatavarin IV Shatavarin IV also known as “Asparanin B” or “Sarsasapogenin-3-O-4Grhamnosylsophoroside” with chemical nomenclature as “3-O-{[β-D-glucopyranosyl (1 → 2)][α-L-rhamnopyranosyl-(1 → 4)]-β-D-glucopyranosyl}-(25S)-5β-spirostan3β-ol,” is the prime steroidal saponins of A. racemosus. Table 11.3 enlisted all the studies involving isolation and identification of shatavarin IV only. Roots of A. racemosus have been the preferred choice to identify shatavarin IV using methanol as the prime solvent of extraction. Jadhav and Bhutani (2006) and Satti et al. (2006) identified shatavarin IV (along with other metabolites) for the first time from aqueous extracts. Using methanolic extracts, Sharma et al. (2012a) invented a convenient, inexpensive TLC protocol for the detection of saponins (shatavarinIV). Subsequently, HPLC-UV (Haghi et al. 2012, using two kinds of extracts), ultra-high-performance liquid chromatography (UHPLC) with photodiode array (PDA) detector (Kishor et al. 2019) and TLC with HPTLC (Gohel et al. 2015) were utilized to quantify shatavarin IV 0.36–3.42% w/w, 0.08 mg/g, and 401.1 mg/ 250 g (66% pure), respectively. The physical properties of the compounds were confirmed by FT-IR-MS and ESI-MS. Mitra et al. (2012) quantified shatavarin IV from different fractions (hexane, ethyl acetate, n-butanol) of roots extracts (chloroform, methanol, and chloroform: methanol) of A. racemosus. Ethyl acetate insoluble fraction of chloroform:methanol (2:1) extract observed with maximum shatavarin IV (5.05%), which was further crystallized and purified up to 84.69% using HPTLC and MS. HPTLC was also used by Smita et al. (2021) to identify shatavarin IV from methanol and chloroform/ methanol root extract of the plant. HPLC-ESI-MS/MS method was used to evaluate root aqueous extract (Patil et al. 2014a) as well as commercially available dried roots, aqueous extract along with marketed formulations in 90% acetonitrile:water (Patil et al. 2014b) with 1.301 ± 0.07 μg/mg as well as 0.2941–0.4745%, 0.1301% and 0.0085–0.2983% (w/w) of shatavarin IV, respectively. While examining ethanolic root extract, Thakur et al. (2012) confirmed 0.56% (w/w) of shatavarin IV (saponin) through HPTLC

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Table 11.3 ShatavarinIV in Asparagus racemosus Extraction solvents Roots Ethyl acetate insoluble fraction of chloroform: Methanol (2:1) Methanol Ethanol

Methanol

Aqueous extract

Methanol

Methanol

Methanol Methanol Hydroalcoholic; water Methanol; Chloroform + methanol Hydroethanolic extract Other plant parts Acetonitrile-water (9:1): Roots; Acetonitrile-water (9:1): AE; Acetonitrile-water (9:1): MF

Analytical method#

Quantitya

Medicinal property

References

HPTLC; MS

4–5%

Anti-cancerous against MCF-7, HT-29, A-498

Mitra et al. (2012)

HPLC-UV

0.36–3.42% w/w

–

TLC; HPTLC; SEC; GC-FID; HPAEC TLC

0.56% w/w

Natural killer cell

Haghi et al. (2012) Thakur et al. (2012)

–

–

HPLCESI-MS/ MS Patil et al. (2009) SGCC; HPTLC; FT-IR-MS; ESI-MS UHPLC

1.301 ± 0.07 μg/ mg

401.1 mg/ 250 g

Insignificant CYP3A4 inhibitory potential as chemotherapeutic agent –

0.08 mg/g

–

LC-MS/ MS LC-MS/ MS UHPLCPDA; MS HPTLC

8.53 μg/g

–

152.06 μg/g

–

–

In silico antiviral

0.13 mg/g FW 0.12 mg/g FW

Neuro-modulatory potential

TLC, HPTLC

4.63 ± 0.4 mg/kg

Anticonvulsant effect

Pahwa et al. (2022)

HPLC; ESI-MS/ MS

0.2941–0.4745% (w/w); 0.1301% (w/w); 0.0085–0.2983% (w/w)

–

Patil et al. (2014b)

Sharma et al. (2012a) Patil et al. (2014a)

Gohel et al. (2015)

Kishor et al. (2019) Saran et al. (2019) Saran et al. (2020) Borse et al. (2021) Smita et al. (2021)

(continued)

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V. Pandey et al.

Table 11.3 (continued) Extraction solvents Methanol: MF1 Methanol: MF2

(PFb) chloroform: Acetic acid: Water: Methanol (5:3.5: 1.5:1 v/v)

Analytical method# HPLC

HPTLC

Quantitya 5.79–6.45% (w/w); 3.87–4.43% (w/w) 0.048 ng/g

Medicinal property –

References Deshmukh et al. (2020)

–

Shalini and Ilango (2022)

a

FW fresh weight, DW dry weight, AE aqueous extract, MF marketed formulations, HPTLC highperformance thin-layer chromatography, TLC thin-layer chromatographic, SEC size exclusion chromatography, GC-FID gas chromatography with flame ionization detector, HPAEC highpressure anion exchange chromatography, ESI electrospray ionization, MS mass spectroscopy, Q-TOF quadrupole time of flight, LC-MS liquid chromatography-mass spectrometry, UHPLC ultra-high-performance liquid chromatography, PDA photodiode array, FT-IR-MS Fouriertransform infrared mass spectroscopy b PF: Polyherbal formulation containing Asparagus racemosus; Bauhinia variegate; Caesalpinia bonducella; Saraca asoca; Symplocos racemose

analysis. Borse et al. (2021) as well as Pahwa et al. (2022) examined hydroalcoholic and water (UHPLC-PDA and MS) as well as hydroethanolic extracts (TLC and HPTLC) for the presence and quantification (4.63 ± 0.4 mg/kg) of shatavarin IV, respectively. Different accessions of A. racemosus were compared for morphological and yield-attributing variables, before to methanol extraction and quantification, through LC-MS/MS. Roots were harvested from the plants grown under different green shade net intensity (SNI: 0, 25, 50, 75 and 90%; Saran et al. 2019) as well as from 12-accessions (Saran et al. 2020) and harvested after 6–18 months as well as 4 years (2015–16 to 2019–20), respectively. Maximum shatavarin IV (8.53 μg/g and 152.06 μg/g) was obtained with mild shade intensity of 25% (Saran et al. 2019) as well as in DAR-14 accession with maximum root length (39.50 cm; Saran et al. 2020), while other accessions have variability in considered 15-traits. Such a brief examination might be very beneficial to determine morphological and biochemical markers to lead to the proper harvesting of shatavari. Besides, harvested plant parts, marketed formulations (root powder, MF1 and tablet, MF2) were also studied by Deshmukh et al. (2020) to reduce microbial contamination (0.87–1.42 log-fold), after subjecting to gamma-irradiation (5–10 kGy). Formulations dissolved in methanol were observed with negligible change in shatavarin IV content (6.45–5.79% and 4.43–3.87% w/w), with or without irradiation. Therefore, irradiation treatment can help increase the shelf life (up to 12 months) of herbal formulations. Shatavarin IV enriched extract (250 and 500 mg/kg b.wt.) displayed in vitro cytotoxic effect toward MCF-7 (human breast cancer), HT-29 (human colon adenocarcinoma), and A-498 (human kidney carcinoma) cell lines as well as in vivo anti-

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cancer activity in cancerous mice. A. racemosus extract enhanced the average life span in normal test animals (Swiss albino mice) by 6 days, while reduced body weight (4 g), viable tumor cell count (4 × 107cells/mL), tumor volume (1.40 mL) and packed cell volume (0.60 mL) in cancerous mice (Mitra et al. 2012). However, aqueous root extract was found insignificant to inhibiting human CYP3A4 isoenzyme, which regulates the metabolism of some chemotherapy agents (Patil et al. 2014a). Shatavarin IV containing hydroethanolic (Pahwa et al. 2022), as well as methanol and/or chloroform root extract (Smita et al. 2021) of A. racemosus, was confirmed for its neuro-modulatory potential and anticonvulsant effect in a mouse model of Catamenial Epilepsy, respectively.

11.4

Other Metabolites

Along with saponins (shataverins) described in the above sections, there are many more phytochemicals present in A. racemosus that will be described in this section. A racemosus is a huge reservoir of various pharmaceutically active chemicals such as alkaloids, carbohydrates, saponins, phenolic acids, terpenes, etc., mentioned in Table 11.4. All of these phytochemicals alone or even in combination with one another make the extracts of A. racemosus useful in various health-related conditions. Apart from these discussed metabolites, several vitamins (A, B1, B2, C, E) and minerals (Ca, Co, Cu, Fe, K, Mg, Mn, P, Se, Zn) have been reported from different parts of A. racemosus (Singh et al. 2018b; Thakur et al. 2021).

11.4.1 Carbohydrates Madan (1972) harvested tubers, leaves, and shoots of A. racemosus with high fructosans and extracted them in boiling water. Only, tuber extract was recognized with the presence of 4-fructosans with different mobility. After 40 min of boiling in water, the complete dissolution of fructosans was observed in polymerized form, not as reducing sugar. Chromatographic studies revealed the presence of sucrose and 3-other low-molecular polyfructosans, however, high-molecular polyfructosans (like insulin) were absent. Sucrose and fructose were also detected as faint spots. Thakur et al. (2012) confirmed the presence of mono-, di- 8.8% (w/w), and polysaccharides 28% (w/w) in root ethanolic (60%) extract form A. racemosus, using enzymatic evaluation. The nature of polymerization was confirmed by size exclusion chromatography (SEC), gas chromatography with flame ionization detector (GC-FID), and high-pressure anion exchange chromatographic (HPAEC) analyses. The activity of Natural Killer cells was also improved by 51% at 25 μg/ mL fructo-oligosaccharides rich extract of A. racemosus. Selvarajan et al. (2014) used ethyl alcohol for the extraction of alkaloids, carbohydrates, flavonoids, glycosides, phenolics, saponins, total sugar (6.42%), and reducing sugar (5.13%) from roots of A. racemosus and created HPTLC chromatogram.

Leaves

Roots

Quercetin 3-O-β-D-glucuronide

Gallic acid; Glycetin; Diazin; Genestin; Ferulic acid; quercetin; Apegenin; Naringenin; Catechin; trans-chalcone

Petroleum ether; EtAc; methanol Soxhlet; maceration; CO2; ethanol

Methanol

CH2Cl2

Roots

CSP

–

Roots

8-methoxy5,6,4′-trihydroxyisoflavone 7-O-β-D-glucopyranoside Racemofuran; Asparagamine A; Racemosol

Asparagamine A

EtAc-F of EtOH

Roots

Racemosol

Ethanol

Root

Ethanol

Hot water

Root

Roots

Extraction solvents

Plant part

Alkaloid, Phenolics and flavonoids Asparagamine A

Compounds Carbohydrates Total carbohydrate; Fructose Mono-; Di-saccharides; Polysaccharides

Table 11.4 Other metabolites in Asparagus racemosus

HPLC

SGCC; HR-EIMS; UV; MS; NMR LC-TOFMS; LC-QMS; LC-MS/MS; HPLC TLC

SGCC; HR-EIMS; NMR; XRC SGCC; HR-EIMS; NMR NMR; MS

HPTLC; SEC; GC-FID; HPAEC

PC; AC

Analytical method

DPPH radical scavenging

–

– DPPH radical scavenging; antidiabetic

– –

Absent

–

Antiabortifacient; Antitumor –

Stimulates natural killer cell activity

–

Medicinal property

0.082% DW

–

–

1048 μg 700 μg 8.8% (w/w); 28% (w/w)

Quantity

Verma et al. (2014) Hasan and Panda, (2020)

Kumeta et al. (2013)

Sekine et al. (1994, 1995) Sekine et al. (1997) Saxena and Chourasia (2001) Wiboonpun et al. (2004)

Madan (1972) Thakur et al. (2012)

References

188 V. Pandey et al.

Methanol; hexane; acetone

Ethanol

Methanol

Roots

Roots

BuOH-F of EtOH

Methanol; EtAc; chloroform;

Roots

Roots

β-Glucogallin

Sterols, triterpenes and other 9-hexadecanoic acid-9Octadecenylester (Z,Z); β-Sitosterol; Stigmasterol-3-O-β-Dglucopyranoside; Diosgenin Urs-1,12,19- trien-3β-ol-28-oic acid3β-D-glucopyranosyl (4′-1″)-β-Dglucopyranoside; n-octadecanyl hexadecanoate; nhexacosanyl-n-octadecanoate; β-sitosterol; Stigmasterol; 1-lauryl 3-arachidyl glycerol-3-phosphate Protodioscin; Pseudoprotodioscin; ASP-VII; ASP-V; AS-1A; vanillin; AsparasideA; AsparaninA; AsparaninB5; 3-O-[β-Dglucopyranosyl(1 → 2)-β-Dglucopyranosyl]-(25S)-5β-spirostan3β-ol; AspafiliosideC; AsparaninB8; AsparaninB9; 26-O-β-Dglucopyranosylfurostane-3-β,26-diol3-O-β-D-xylopyranosyl (1 → 4)-β-Dglucopyranoside; Chelidonic acid;

Arial parts

Myricetin; Quercetin; p-coumaric acid; Caffeic acid

LC-MS; MS/MS

TLC; NMR; ESI-MS; IRS

SGCC

SGCC; NMR

HPTLC

–

–

An Insight of Phytochemicals of Shatavari (Asparagus racemosus) (continued)

Jaiswal et al. (2014)

Siddiqui et al. (2013)

–

137.7 mg (0.0077% yield)

Ahmad et al. (2022)

Ashraf et al. (2021)

Kafle et al. (2012)

Antiglycation

DPPH radical scavenging

Anti-microbial

0.58% (saponins)

436–745 μg/ g DW; 183–411 μg/ g DW; 228–362 μg/ g DW; 377–632 μg/ g DW –

11 189

Asparacemosone A; Asparacemosone B; Asparacemosone C; Asparacemosone D;

glycerol ester PMV70P691–117; 1,2-dithiolan-4-carboxylic acid 6-Dα/ß-glucopyranose ester; β-sitosterylD-glucoside-6′-palmitate Isoagatharesinol; Gobicusin A; AsparacosinA; Muzanzagenin 22-O-methylprotodioscin; 3″-Methoxynyasol; Asparacosin A; AsparagosideA; AsparosideB; Asparenydiol; AspoligoninA; Nyasol; Chalconaringenin 2′-rhamnosyl(1 → 4)-glucoside; Ferulic acid; Immunoside; OfficinalisninI; Protodioscin; ShatavarinI; ShatavarinV; ShatavarinVII; ShatavarinVIII; ShatavarinIX; ShatavarinX; Sarsasapogenin; Schidigera saponin D5; transConiferyl alcohol Acemosin; Asparacosin A; Stigmasterol

Compounds

Table 11.4 (continued)

CH2Cl2-F of MeOH

Roots

EtAc; MeOH

Methanol

Roots

Roots

Methanol

Extraction solvents

–

Plant part

SGCC; NMR; IRS; MS

HPLC; NMR, HR-MS; XRC

LC-ESI-MS/MS

SGCC; NMR

Analytical method

Quang et al. (2018)

Tantapakul et al. (2020)

α-Glucosidase inhibitory

Shah et al. (2014) Jayashree et al. (2015a)

References

Anti-cancer

In vivo antioxidant activity; prevented t-BHP induced hepatic and DNA damage in rats

–

8.7 mg/ 945.9 g; 276 mg/ 945.9 g; 23.6 mg/ 945.9 g 21.7 mg/ 2 kg DW; 51.1 mg/ 2 kg DW;

Antibacterial

Medicinal property

–

Quantity

190 V. Pandey et al.

Hydroalcoholic; water

Hexane:EtAc (6:4); EtAc: Acetic acid: Formic acid; MeOH (10:1.1:1.1:2.6)

Roots

PFa HPTLC

UHPLC-PDA; MS

0.067 ng/g; 0.053 ng/g

81.2 mg/ 2 kg DW; 12.9 mg/ 2 kg DW; 49.9 mg/ 2 kg DW; 19.7 mg/ 2 kg DW; 66.6 mg/ 2 kg DW; 4.4 mg/2 kg DW; 3.0 mg/2 kg DW; 4.5 mg/2 kg DW; – – Borse et al. (2021)

Shalini and Ilango (2022)

In silico antiviral

–

PF: polyherbal formulation containing Asparagus racemosus; Bauhinia variegate; Caesalpinia bonducella; Saraca asoca; Symplocos racemose; bF fraction, EtAc ethyl acetate, BuOH-F butanol fraction, EtOH ethanol, MeOH Methanol, CH2Cl2 Dichloromethane, CSP commercial shatavari products, PC paper chromatography, AC adsorption chromatography, XRC X-ray crystallography, SGCC silica gel column chromatography, HPTLC high-performance thinlayer chromatography, SEC size exclusion chromatography, UHPLC-PDA ultra-high-performance liquid chromatography-photo- diode array, IRS infrared spectroscopy, RIA radioimmunoassay, HR-EI-MS high-resolution electron ionization mass spectrometry, GC-FID gas chromatography with flame ionization detector, HPAEC high-pressure anion exchange chromatography, TLC thin-layer chromatographic, t-BHP tert-Butylhydroperoxide, NMR nuclear magnetic resonance, LC liquid chromatography, QMS quadrupole mass spectrometry, ESI electrospray ionization, TOF time of flight, HR-MS high-resolution mass spectrometry

a

Asparagamine A; Asparanin A; Rutin; Muzanzagenin; Isoagatharesinol; ShatavarinI; ShatavarinV; ShatavarinIX; ShatavarinVI; ShatavarinVII; ShatavarinX Lupeol Rutin

Asparacosin A; (25R)-12β-acetyl-17α-hydroxyspirost4-en-3-one; Nyasol; Asparenydiol; 4-[5-(4-methoxyphenoxy)-3-penten1-ynyl] phenol; 3″-methoxyasparenydiol; Stigmasterol

11 An Insight of Phytochemicals of Shatavari (Asparagus racemosus) 191

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11.4.2 Alkaloids, Phenolics, and Flavonoids Asparagamine A is a polycyclic pyrrolizidine alkaloid and a very well-known antioxidant, which was extracted from the roots of A. racemosus. Sekine et al. (1994, 1995) isolated and elucidated the stereo-structure of asparagamine A, the first alkaloid from the ethanolic root extract of A. racemosus. The molecular formula of asparagamine A was detected by high-resolution electron impact mass spectrometry (HR-EI-MS) as C22H27NO5. However, structure and stereochemistry were established with the help of NMR and crystal X-ray diffraction analysis, respectively. This study confirmed the complicated polycyclic structure of asparagamine A, fused with a pyrrolizidine ring. The significance of asparagamine A was established with oxytocin-induced anti-abortifacient (38–68% inhibition by 10-6 to 10-5 mg/cm3) and dose-dependent in vitro antitumor activity (10–100 μg). Conversely, Kumeta et al. (2013) mentioned the non-existence of asparagamine A in 11 commercial shatavari products, extracted using methanol claiming asparagamine A is not found in any identified A. racemosus plant. However, Wiboonpun et al. (2004) and Borse et al. (2021) identified asparagamine A from dichloromethane (CH2Cl2) hydroalcoholic/water root extracts using HR-EI-MS and UHPLC-PDAMS, respectively. Ethyl acetate (Sekine et al. 1997) and n-butanol (Ahmad et al. 2022) fraction of ethanolic root extract of A. racemosus was used to isolate and identify racemosol as and 9,10-dihydro-1,5-dimethoxy-8-methyl-2,7-phenanthrenediol (C17H18O4) β-glucogallin (phenolic acid), respectively. New isoflavone, confirmed as 8-methoxy-5,6,4′-trihydroxyisoflavone7-O-β-D-glucopyranoside (0.082% DW) was detected from root extract (Saxena and Chourasia 2001), of A. racemosus. Different from above, Wiboonpun et al. (2004) tried dichloromethane extract and separated racemofuran, asparagamine A, and racemosol using silica gel chromatography. HR-EI-MS/IR and NMR/MS revealed the stereochemistry of the compounds. Successive extraction from leaves (Verma et al. 2014) and aerial parts of A. racemosus (Ashraf et al. 2021) was performed using petroleum ether, ethyl acetate, and methanol as well as hexane, chloroform, ethyl acetate, and methanol, respectively. Methanolic extract elucidated quercetin-3-O-β-D-glucuronide using TLC (Verma et al. 2014), myricetin (745.17 μg/g DW), quercetin (411.21745.17 μg/g DW), p-coumaric acid (362.38745.17 μg/g DW), and caffeic acid (632.55745.17 μg/g DW) through HPTLC (Ashraf et al. 2021). Hexane extract presented minimum phenolic (75.13 mg GAE/g) and flavonoid (34 mg QRE/g) with very poor myricetin, quercetin, p-coumaric acid, and caffeic acid. A much different method of extraction of Soxhlet, maceration, and supercritical fluid (CO2) with water and/or ethanol as co-solvent utilized treated roots (by enzyme treatment and/or sonicated) of A. racemosus (Hasan and Panda 2020). Soxhlet extraction (SE) provided a better yield (7.62–76.27% w/w) as compared to maceration extraction (ME; 0.59–65.91% w/w) with all the methods of extractions. Also, total flavonoid 6.88–12.86 μg/g QRE for SE and 1.37–11.63 μg/g QRE for ME as well as phenolic components 3.17–9.48 mg/g GAE for SE and 0.12–7.40 mg/g GAE for ME, respectively, were quantified. Gallic acid, glycetin, diazin, genestin, ferulic

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acid, quercetin, apigenin, naringenin, catechin, and trans-chalcone were also examined through HPLC in all the extracts. Racemofuran of dichloromethane root extract was confirmed with maximum DPPH radical scavenging activity (IC50 130 μM), followed by racemosol (IC50 300 μM) and asparagamine A (IC50 500 μM), as mentioned by Wiboonpun et al. (2004). Also, consistent with metabolic content, soxhlet extract (53.69–98.02 μM/ mg-Trolox equivalent; Hasan and Panda 2020) as well as methanolic extract (Ashraf et al. 2021) observed with better DPPH scavenging activity as compared to maceration extract (3.97–95.86 μM/mg-Trolox equivalent) or ethyl acetate and chloroform extract. Also, Soxhlet and maceration extract revealed good viability (80.14 and 94.37%, respectively) and improved insulin released (0.65 and 0.82 ng/mL, respectively) in β-pancreatic RINm-5f cells (Hasan and Panda, 2020). While β-glucogallin from A. racemosus confirmed antiglycation activity (Ahmad et al. 2022).

11.4.3 Sterols, Triterpenes, Lignan, Non-lignan, and Others Shatavari samples were further explored to understand the chemo-diversity in A. racemosus based on sample or solvent selection. Root being the preferred tissue with methanol (Jayashree et al. 2015a) as the favorite solvent of extraction was utilized during most of the studies, including velamen, cortex, vascular bundles, and pith regions of roots (Jaiswal et al. 2014) along with different fractions of extract like dichloromethane (Quang et al. 2018) or n-hexane, dichloromethane, ethyl acetate, and water (Shah et al. 2014). As mentioned in Table 11.4, more than 18- (Jaiswal et al. 2014) and 22-compounds including flavonoids, saponins, and shatavarins (Jayashree et al. 2015a) were detected through LC-MS. While, isoagatharesinol, gobicusin A, asparacosin A, muzanzagenin (Shah et al. 2014), 8.7 mg acemosin, 276 mg asparacosin, and 23.6 mg stigmasterol (Quang et al. 2018), purified by column chromatography and HPLC (from 945.9 g roots), respectively. Few of identified compounds can be found in all tested parts, while few are specific for particular parts of roots. Some studies either used hexane and water (Kafle et al. 2012), ethyl acetate with methanol (chromatographic fractionation; Tantapakul et al. 2020), ethanol (Siddiqui et al. 2013), or alcohol: water and water (Borse et al. 2021) to extract roots of A. racemosus. Hexane root extract was detected with fatty acids, sterols, and triterpenes, while methanol and aqueous extract were distinguished with glycosides, quinines, flavone glycosides, reducing compounds, and polyoses along with 0.58% of saponins. Further, column chromatography revealed 9-hexadecanoic acid-9octadecenylester (Z,Z) and β-Sitosterol from hexane extract, while stigmasterol-3O-β-D-glucopyranoside and diosgenin, from acetone and methanol extracts, respectively (Kafle et al. 2012). The ursane glycoside called urs-1,12,19-trien-3β-ol-28-oic acid-3β-D-glucopyranosyl (4′-1″)-β-D-glucopyranoside was revealed from ethanolic extract and quantified up to137.7 mg with 0.0077% yield after column elution, along with few other metabolites (Table 11.4; Siddiqui et al. 2013). Six spirosteroids, nyasol, stigmasterol, and three acetylenic norlignans were quantified from different

194

V. Pandey et al.

chromatographic fractions of 2Kg dried roots (Table 11.4; Tantapakul et al. 2020). Borse et al. (2021) quantified asparagamine A, asparanin A, isoagatharesinol, muzanzagenin, rutin, shatavarin I, shatavarin V, shatavarin IX, shatavarin VI, shatavarin VII, and shatavarin X through UHPLC-PDA and MS. Shalini and Ilango (2022) selected polyherbal formulations (Asparagus racemosus; Bauhinia variegate; Caesalpinia bonducella; Saracaasoca; Symplocosracemose) for quantification of metabolites by HPTLC. Extractions using n-hexane: ethyl acetate (6:4 v/v), ethyl acetate: acetic acid: formic acid; methanol (10:1.1:1.1:2.6 v/v) and chloroform: acetic acid: water: methanol (5:3.5: 1.5:1 v/v) resulted in lupeol (0.067 ng/g), rutin (0.053 ng/g), and shatavarin IV (0.048 ng/g), respectively. Identified compounds from A. racemosus showed maximum and minimum (undetected) antibacterial activity against S. aureus and S. typhi, respectively, while moderate activity was observed for E. coli. (Shah et al. 2014). A. racemosus extract was observed to prevent tert-butylhydroperoxide (t-BHP) induced damage in rats, with a dose-dependent reduction in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), lipid peroxidation (malondialdehye content), ROS generation, hence hepatic and DNA damage due to enhanced antioxidant activity of antioxidant enzymes (Jayashree et al. 2015a). Acemosin exhibited better cytotoxic activity against HepG2 cancer cells (IC50 value of 87.3 μg/mL) as compared to asparacosin A with IC50 > 128 μg/mL (Quang et al. 2018). In vitro, αglucosidase inhibition study of recognized compounds revealed that four nor-lignans presented strong inhibition (IC50 values 0.003–0.004 μM) as compared to spiorosteroids (IC50 values 12–48 μM) and 5 × 104 fold higher than standard acarbose (Tantapakul et al. 2020).

11.5

GC-MS Profiling of A. racemosus

GC/MS analysis is a perfect tool for the identification of any unknown compound in the sample derived from diverse origins. With the help of GC-MS not only the unknown compounds can be identified but the impurities present in traces can also be recognized. GC-MS has been applied widely for the identification of various phytochemicals, present in the A. racemosus using root/root tubers as the main source for extract. A detailed list of compounds identified from the roots of A. racemosus through GC-MS have been mentioned in Table 11.5. For the GC-MS, methanol has been chosen by Sivakumar and Gajalakshmi (2014), Leema and Prakash (2019), Suja and Sivakala (2021) and Dhanusha et al. (2021). Sivakumar and Gajalakshmi (2014) confirmed presence of 2-propanone-1,3dihydroxy, 2-fruancarboxyaldehyde-5-(hydroxymethyl), hexadecanoic acid-, n-hexadecanoic acid- and ethanol,2(Octyloxy)-, 1,9-nonanediol. Leema and Prakash (2019) revealed the presence of 36 metabolites, varied from 0.01 to 0.02% to 24.4 to 22.76%, in rhizomes of two (Pili and safed) shatavar varieties, respectively. Eighteen- and twenty-one compounds were also detected by Suja and Sivakala (2021) and Dhanusha et al. (2021), respectively.

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Table 11.5 GC-MS profiling of root/root tubers of Asparagus racemosus Compounds 2-Propanone-1,3-dihydroxy; 2-Fruancarboxyaldehyde5-(hydroxymethyl); Hexadecanoic acid; n-Hexadecanoic acid; Ethanol,2(Octyloxy)-; 1,9-NonanediolPropane, 1,1,3- triethoxy; 2-Fruancarboxyaldehyde, 5-(hydroxymethyl)-; 1,2-Dithiolane-3-carboxylic acid; 1,6-Anhydro-β-d-talopyranase; Dodecanoic acid; Tetradecanic acid; n-Hexadecanic acid; oleic acid; Octadecanoic acid; 4H-Pyran- 4-one,2,3 dihydro -3,5 dihydroxy-6 methyl; 9,12-Octadecadienoic acid Disulfide,bis(1-methylpropyl); Benzene,1,3-bis(1,1 dimethylethyl)-; hexadecane; Phenol,2,4-bis (1,1-dimethylethyl)-; Heptadecane; Octadecane; (E)Hex-3-enyl (E)-2-methylbut-2-enoate; Nonadecane; Nonadecane,4-methyl-; phthalic acid,butyl tetradecyl ester; Jatamansone; Eicosane,2-methyl-; Cyclohexane, nonadecyl-; benzene,(1-methyltridecyl)-; Heneicosane; n-Pentadecylcyclohexane; Cyclohexane,undecyl-; Tetracosane; Heptacosane; squalene; Octadecane,2methyl2,6,10-Trimethyltetradecane; Tert-Hexadecanethiol; 2-Hexyl-1-decanol; Dibutyl phthalate; Eicosane; Androst-5,7-dien-3-ol-17-one; Pentacosane; N-Phenyl-2naphthylamine; Tetracosane; Hetriacontan; Bis (2-ethylhexyl) phthalate Neopentyl glycol; chlorobenzene; ethylbenzene; benzyl chloride; 2-(Formyloxy)-1-phenylethanone; Ethoxybenzene; N,N-Dimethylbenzylamine 2-Fruancarboxyaldehyde -5-(hydroxymethyl); 1,9-Nonanediol; Hexadecanoic acid; 2-Propanone-1,3dihydroxy; 2-Furaldehyde; Hydrazine-1,1-dimethyl; Ethanimidic acid Di-n-octyl phthalate; Decane,1-chloro-; 1-tridecyne; 9-Octadecenoic acid (Z)ijmap; 1-undecanol-; 2-tridecene,2-chloro-1,1,1-trifluoro-,(Z); (MESO)-3,4dihydroxymethyl-3,4-dimethylhexane; 2-tridecene,2chloro-1,1,1-trifluoro-,(Z)-; Naphthalene,1,6-dimethyl-4(1-methylethyl); 3-methyl-1-cyclooctene; N, N-dimethyldecanamide; decanal; Oxirane, [(dodecyloxy)methyl]-; Phenol,2,4-bis(1-phenylethyl); Methanone,[1,4-dimethyl-7-(1-methylethyl)-2-azulenyl] phenyl; Methanone,[1,4-dimethyl-7-(1-methylethyl)-2azulenyl]phenyl; 4,5-2H-oxazole-5-one,4- [3,5-di-Tbutyl-4-methoxyphenyl]methylene-2-phenyl; 2,4,6-tris(1-phenylethyl)-phenol Methyl ester: Cyclopentaneundecanoic acid; Pentadecanoic acid,14methyl-; Heptacosanoic acid; 9-Octadecenoic acid (Z)-;

Extraction solvents Methanol

References Sivakumar and Gajalakshmi (2014)

Ethanol

Janani and Singaravadivel (2014)

Hexane

Jayashree et al. (2015a)

Chloroform

Gravel et al. (2017)

Acetonitrile

Gravel et al. (2017)

Ethanol

Agarwal et al. (2018)

Hydroalcoholic

Thakur et al. (2018)

(continued)

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Table 11.5 (continued) Compounds Heptadecanoic acid,16-methyl-; Dodecanoic acid,8-nitro11-oxo-; Dodecanoic acid Other ester and ether: Diethylene glycol monododecyl ether; phthalic acid,4cyanophenyl nonyl ester; 1,2-Benzenedicarboxylic acid (butyl octyl ester); phthalic acid (butyl undecyl ester); 1,2-Benzenedicarboxylic acid (dinonyl ester) Ethyl propionate; methyl sorbate; Di-isopropyl 2-oxomalonate; dimethyl fumarate; 5-(hydroxymethyl)2-furaldehyde; dimethyl maleate; 1,3-dichlorocyclopentane; 3-deoxy-d-mannoic lactone; oleic acid; 2-Hexadecanoyl glycerol; 1-Heptanol; 2-Etradecanol octanoate; Hexanoic acid, 5-hydroxy-, methyl ester; Cyclopentane, 1-acetyl-1,2-epoxy-; 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-; 2-butanone, 4-hydroxy-3-methyl; 1,2,3-Propanetriol; ethyl 3-(acetyloxy)-2-(hydroxymethyl)propanoate; Tetradecanoic acid; Tridecane; Oleoyl chloride; 1,2,3,5tetraisopropylcyclohexane; sucrose; Neotigogenin acetate; 1-allyl-2-methylenecycloheptano; 2-propanone, 1,3-dihydroxy; 2-Fruancarboxyaldehyde, 5(hydroxymethyl); Hexadecanoic acid; n-Hexadecanoic acid; Ethanol,2(octyloxy)-; 1,9-Nonanediol; Tetranorlipoic acid; 1,6-anhydro-β-d-talopyranase; propane, 1,1,3- triethoxy; Dodecanoic acid 2-Phenyl-1,3-Cyclohexadiene; Benzidine; Pentadecane; flavone; Phenol,2,6-bis(1,1-dimethylethyl)-4[(4-hydroxy-3,5-dimethylphenyl) methyl]-; But-2endiamide-N,N′-bis (4-methoxyphenyl)-; formic acid,1dimethylamino-,[2-methoxy-4-(3-oxobutyl)]phenyl ester; Cyclopentanone,3-acetyl-4(methoxycarbonylmethyl)-; Piperazine-2,5-dione, 1,4-(4-methylphenyl)1H-indole, 1,2-dimethyl-; Indolizine, 2,6-dimethyl; pyrimidine, 2,4,5-triami; propane phosphonic acid; Xanthone; methyl-p-2-phenyl-1ben; 1,2 benzenedicarboxylic; Retinal,9-cis-; 2,3-Dihydroxypropyl elaidate; Heptadecanoic acid,-15methyl-methyl ester; 1-Hentetracontanol; Tetrapentacontane,1,54-; Spirost-5-en-3-ol, acetate; 9,12Octadecadienoic acid (Z,Z)-2,3-dipropyl ether; glycine, N-[(3.α.,5.β.,12.α.); 4,4-dimethyl-5a-cholesta-8,24-dien3-b-ol; Diosgenin; γ-Sitosterol 5-Hydroxymethylfurfural; 1,2,3-propane triol; (S)-(-)1,2,4-Butanetriol, 2-acetate; Stigmasta-5,22-Dien-3-ol; 2-Nonene; guanosine; Cycloheptasiloxane, tetradecamethyl-; 3-Furanacetic acid, 4-hexyl-2,5dihydro-2,5-dioxo-; Cyclooctasiloxane, hexadecamethyl; α.-methyl-l-sorboside; guanosine; δ.1,.α.-

Extraction solvents

References

Methanol

Leema and Prakash (2019)

Aqueous

Saraswathi et al. (2020)

Methanol

Suja and Sivakala (2021)

Methanol

Dhanusha et al. (2021)

(continued)

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Table 11.5 (continued) Compounds Cyclohexaneacec acid; Heptasiloxane, Hexadecamethyl; 3,3-dimethyl-5-oxocyclohexane -carbaldehyde; 2-Monopalmin; Pent-3-ene-2-one, 3-phenyl-, oxime; Stigmasterol; γ-Sitosterol; Methyl ester: Hexadecanoic acid; linoleic acid; 9-Octadecenoic acid (z)-; Octadecanoic acid 2,2′-Bioxirane; 2-Furanmethanol; 6-Oxabicyclo (3.1.0) Hexan-3-one; 4-H-Pyran-4-one, 2,3 dihydro3,5dihydroxy-6; isosorbide; 5-Hyroxymethyl furfural; D-Glucitol, 1,4-anhydro; 9,12-Octadecadienoic acid

Extraction solvents

References

Hydroalcoholic

Shalini and Ilango (2021)

Propane,1,1,3-triethoxy-, 1,2-dithiolane-3-carboxylic acid [tetranorlipoic acid], 1,6-Anhydro-β-d-talopyranase-, dodecanoic acid, tetradecanic acid, oleic acid, 4H-Pyran-4-one,2,3 dihydro -3,5 dihydroxy-6 methyl-, octadecanoic acid, and 9,12-octadecadienoic acid (Janani and Singaravadivel 2014) as well as 1,9-nonanediol, 2-propanone-1,3-dihydroxy, 2-furaldehyde, hydrazine-1,1-dimethyl and ethanimidic acid (Agarwal et al. 2018), along with common hexadecanic acid and 2-fruancarboxyaldehyde5-(hydroxymethyl) were identified from ethanolic root extract of the plant. Aqueous root extract of Shatavari reported 2-phenyl-1,3-cyclohexadiene, benzidine, pentadecane, flavone, phenol,2,6-bis(1,1-dimethylethyl)-4-[(4-hydroxy-3,5dimethylphenyl) methyl]-, but-2-endiamide, N,N′-bis (4-methoxyphenyl)-, pentadecane-2,4-dione, formic acid,1-dimethylamino-,[2-methoxy-4-(3-oxobutyl)] phenyl ester, cyclopentanone,3-acetyl-4-(methoxycarbonylmethyl)-, and piperazine-2,5-dione,1,4-(4-methylphenyl)- (Saraswathi et al. 2020). Whereas hydroxymethyl root extract (Thakur et al. 2018) demonstrated the presence of 30-active phytochemicals including 7-methyl esters and di-n-octyl phthalate (89.58%), pentadecanoic acid, 14-methyl-, (methyl ester; 1.45%) and 1,2-benzenedicarboxylic acid (butyl octyl ester; 1.56%). Differences in polarity of hexane as a solvent resulted in the documentation of completely different 21-compounds (Jayashree et al. 2015c), named disulfide, bis (1-methylpropyl), benzene,1,3-bis(1,1-dimethylethyl)-, hexadecane, phenol,2,4-bis (1,1-dimethylethyl)-, heptadecane, octadecane, (E)-Hex-3-enyl (E)-2-methylbut-2enoate, nonadecane, nonadecane,4-methyl-, phthalic acid (butyl tetradecyl ester), jatamansone, eicosane,2-methyl-, cyclohexane,nonadecyl-, benzene, (1-methyltridecyl)-, heneicosane, n-pentadecylcyclohexane, cyclohexane,undecyl-, tetracosane, heptacosane, squalene, and octadecane,2-methyl-. Gravel et al. (2017) revealed 11- and 7-phytochemicals from chloroform and acetonitrile extracts, respectively. Androst-5,7-dien-3-ol-17-one (a phytosteroidal; 1.16%) of chloroform extract, can be considered a marker substance. N-phenyl-2naphthylamine (20.66%), as well as ethoxybenzene (24.39%) and chlorobenzene

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(20.32%), predominates the volatile fraction of the chloroform as well as acetonitrile extract, respectively. The most recent study by Shalini and Ilango (2021) explored 2,2′-bioxirane, 2-furanmethanol, 6-oxabicyclo (3.1.0) hexan-3-one, 4-H-pyran-4one, 2,3 dihydro-3,5 dihydroxy-6, isosorbide, 5-hyroxymethyl furfural, D-glucitol, 1,4-anhydro, and 9,12-octadecadienoic acid from the hydroalcoholic root extract of A. racemosus. These investigations conclude that more than 150 metabolites can be recognized through methanolic, ethanolic and/or aqueous extracts, which are extra affluent for extraction. Hexadecanoic acid and 2-fruancarboxyaldehyde-5-(hydroxymethyl) were reported in four GC-MS analyses. Three evaluations mentioned the identification of dodecanoic acid, n-hexadecanoic acid, 4-H-pyran-4-one,-2,3dihydro3,5dihydroxy-6-methyl, 1,9-nonanediol, and 2-propanone-1,3-dihydroxy. At least two studies stated the presence of 1,2,3-propanetriol, 1,6-anhydro-β-d-talopyranase, 2-tridecene,2-chloro-1,1,1-trifluoro-,(Z), 5-hydroxymethylfurfural, 9,12octadecadienoic acid, 9-octadecenoic acid, ethanol,2(octyloxy)-, guanosine, heneicosane, octadecanoic acid, oleic acid, pentacosane, propane 1,1,3- triethoxy, tetracosane, tetradecanic acid and γ- sitosterol. Several derivatives of cyclohexane, cycloheptane, octadecenoic acid, phthalic acid, etc. were also mentioned among identified components of A. racemosus extract. Methanol (Leema and Prakash 2019), aqueous (Saraswathi et al. 2020) and hydroalcoholic (Shalini and Ilango 2021) extracts might be responsible for the radical scavenging, antioxidant and other curative activities of the plant. Metal chelating potential of two (Pili and safed) shatavar varieties were 536.66–612 μg/ mL (IC50), 79.06–177.12 μg/mL (GAE; IC50) and 271.21–599.78 μg/mL (IC50), respectively (Leema and Prakash 2019). Also, the methanolic root extract of A. racemosus is considered cytotoxic with antiproliferative activity in MDAB-231 cells (Dhanusha et al. 2021).

11.6

In Silico Studies

In the urge of finding potent antiviral and/or anti-cancerous components from A. racemosus, few studies utilized in silico platforms to distinguish significant phytochemicals responsible for the individual therapeutic property of the plant. Association of A. racemosus was discovered with 18 immune pathways and 19immune targets, including Bcl-2-like protein 1 (BCL2L1), glycogen synthase kinase-3 beta (GSK3B), Interleukin-2 (IL2), prostaglandin G/H synthase 1 and 2 (PTGS1 and PTGS2), prothrombin (F2), and signal transducer and activator of transcription 3 (STAT3), of Th17 cell differentiation, IL-17 signaling, etc. (Borse et al. (2021). Therefore, Borse et al. (2021) selected 11, while Chikhale et al. (2021) selected 32 compounds from the plant. These metabolites were targeted against the ReceptorBinding Domain of Spike Protein (RBD), the Main Protease (Mpro), and the RNA-dependent RNA polymerase (RdRp) as well as NSP15 Endoribonuclease and 2019-nCoV-spike receptor-binding domain (SRBD), respectively, using

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docking simulation. Asparagamine A, asparanin A, muzanzagenin, shatavarin IV, shatavarin VI, shatavarin VII and shatavarin IX as well as asparoside C, asparoside D and asparoside F were observed with maximum efficiency (docking score) against SARS-CoV-domains, respectively. These ligands interact with Mpro through hydrogen bonds with His163, Gln89, Arg188, Thr190, and/or Glu166, electrostatics interaction with Met49, hydrophobic interactions with Phe140, Asn142, His164, Met165 and/or Pro186 along with few Van der Walls (vdW) interactions. Interaction with RdRp involved H-bond using Asp623, Ser795 and/or Cys622 as well as Phe140, Leu141, Leu167 and/or Ala191. However, interaction with RBD involves H-bond with Arg403, Ser494, Tyr495, Thr500 and/or Asn501 along with other non-ploar interactions involving Arg403, Asn501, Gln493, Gln498, Gly496, Leu455, Phe456, Phe497, Ser494, Tyr495, Tyr505, and/or Tyr453. The binding capacity for RBD was maximum with asparoside C and asparoside D using 7-H-bond (and other interactions) with 7.542 and 7.069 Kcal/mol, of docking scores, along with -62.61 and - 66.49 Kcal/mol binding energy, respectively. However, asparoside C, asparoside F, and asparoside D were potent for NSP15 with docking scores of -7.165, -6.615, and 6.445, along with binding energy of -51.42, -55.30, and - 72.46 Kcal/mol, respectively. Qazi and Raza (2021) mentioned that quercetin from A. racemosus displayed a high binding affinity of -8.0 kcal/mol, inhibitory effects against prostate ovary testis embryo expression (POTEE) protein receptor, which is a potential ovarian cancer target. The study was conducted after active site prediction in POTEE followed by molecular docking. The large structure of quercetin provided conformational flexibility with the POTEE complex and utilized large conformational space (1224) and good replica-exchange molecular dynamics (REMD) score of 50 ns. Seven H-bond and 1-hydrophobic bonds are involved in the structure formation and have less steric interferences. The immunomodulatory activity of these phytochemicals is the foundation for these remedial activities. Therefore, these can be proposed as antiviral (pre- and post- COVID-19) and anti-cancerous based on the bioavailability of the Rasayana. These studies confirmed the therapeutic nature of phytoceuticals (muzanzagenin, asparagamine A), which have the competence to penetrate the blood-brain barrier (BBB) and are orally bioavailable.

11.7

Conclusion

More than 120 metabolites including 10 shataverins have been isolated from mostly roots of A. racemosus, which was enhanced with the identification of more than 150 metabolites through GC-MS. It is evident from the above details that, aqueous extract and its butanol fraction along with acetonitrile: water (9:1) extract has been a rich source of steroidal saponins from roots of A. racemosus. Shatavarin I-X are dominant metabolites of A. racemosus, which were found in multiple studies. The most significant shatavarin IV alone was isolated in more than 25 analyses.

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Therapeutic plants like A. racemosus is considered storage house of phytoceuticals. Also, the root aqueous extract followed by methanolic/ethanolic extract is full of medicinal properties with dominating antioxidant and anti-cancerous actions. Diversity in chemical components and their quantity varies with tissue, extraction solvent, and analytical method, as well as due to geographic and horticulture circumstances. Therefore, there are many possibilities to harvest opportunities for chemical characterization of different parts of the plants, under consideration of environmental, geographical as well as in vitro conditions.

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Ex Situ Conservation of Shatavari (Asparagus racemosus)

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Vibha Pandey, Sonali Dubey, Ravi Kant Swami, Manju Shri, Shivani Tiwari, and Akanksha Bhardwaj

Abstract

Globally, medicinal plant conservation is among the most pressing issues. To satisfy the demand for medicinal plants, natural resources are being depleted at an exponential rate. A rich legacy of spirituality along with culture underpins the nation’s strategies, plans for conservation, sustainable use, and equitable resource stewardship. Asparagus racemosus (Shatavari) has been described as the “queen of herbs” for its medicinal properties. The multitude of uses of A. racemosus in conjunction with its constant, high demand, resulted in fluctuated and insufficient supply, which makes it a critical resource for conservation. The conservation of Shatavari can be achieved by using ex situ techniques such as the establishment of gene banks, field banks, seed banks, in vitro plant tissue banks, cryopreservation, vitrification, artificial propagation of plants for reintroduction into the wild, as well as the implementation of nurseries and home gardens. This chapter will provide detailed information about the advantages, disadvantages, preparations for ex situ tools (conventional and biotechnological), as well as the role of key V. Pandey (✉) CSIR-National Botanical Research Institute, Lucknow, India S. Dubey (✉) School of Biosciences, IMS Ghaziabad University Courses Campus, Ghaziabad, India R. K. Swami Jamia Hamdard, Delhi, India M. Shri School of Applied Sciences and Technology, Gujrat Technological University, Ahmedabad, India S. Tiwari Azad Institute of Pharmacy and Research, Lucknow, India A. Bhardwaj ICAR-Indian Agricultural Research Institute, Delhi, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_12

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institutions involved in ex situ conservation. A series of themed issues are intended to provide a window into the current research concerning the conservation of plants by ex situ means. Keywords

Conservation · Ex situ · In vitro culture · Shoot multiplication · Propagation · Encapsulation

Abbreviation 2ip 8-HQC ADS DKW EtOH GA3 H2SO4 LS MS NaOCl NN WM

12.1

2-Isopentyl adenine 8-Hydroxyquinoline citrate Adenine sulfate Driver and Kuniyuki Walnut media Ethyl alcohol Gibberellic acid Sulfuric acid Linsmaier and Skoog (1965) media Murashige and Skoog’s (1962) media Sodium hypochlorite Nitsch and Nitsch (1969) media White’s (1963) media

Introduction

India’s long, ancient history has been inextricably linked with herbal medicine, especially with the oldest system of health care known as Ayurveda. Many people have been turning to herbal products and medicines in the last few years in an attempt to improve their health (Bopana and Saxena 2007; Ravishankar and Shukla 2007; Semwal et al. 2019). One of the well-known Ayurvedic herbs is Asparagus racemosus (family Asparagaceae) also called Shatavari and often found at lower altitudes in India. There are approximately 300 species in the genus Asparagus worldwide. Of those 300 species, 22 are native to India. The herb is very effective for treating nervous disorders, dyspepsia, diarrhea, tumors, inflammation, hyperdipsia, hepatopathy, cough, bronchitis, hyperacidity, liver disease, and certain infectious conditions. A major factor underlying their medicinal properties is the presence of secondary metabolites that are stimulated by natural environments and may not manifest under controlled conditions (Alok et al. 2013; Thakur et al. 2015a; Kumar et al. 2020; Saran et al. 2020; Encina and Regalado 2022). A. racemosus was among 13.19% of ethnopharmacological climbers in Bangladesh (Kadir et al. 2012) and 11.7% of climbers in Zambia (Chinsembu

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2016) with more than 8 (1.86 RI value) and 4 (pneumonia, cough, diarrhea, syphilis) pharmacological properties, respectively. In India, A. racemosus has been listed among 73 ethnobotanical species of South Surguja district, Chhattisgarh (Kala 2009), 10% climbers of 71 plant species from 4-forest ranges of Odisha (Mohapatra et al. 2013; Kumar et al. 2021), 57-ethnomedicinal plants, 14% climbers of West Bengal (Biswas et al. 2017) as well as 107-plant taxa and 287-voucher specimens of the Kashmir Himalaya (Bhat et al. 2021). The use of A. racemosus for a variety of purposes has continued to increase its demand, but the supply of it has been erratic and insufficient due to destructive harvesting, habitat destruction along with deforestation. As a result, the plant is now listed as vulnerable in its natural habitat (Bopana and Saxena 2007; Thakur et al. 2015a). The purpose of medicinal plant conservation refers to the stewardship of their use by humans to ensure that present and future generations will benefit (medicinally, economically, socially, and culturally) from them in a sustainable way. Conserving these plants implies protecting, maintaining, utilizing, restoring, and enhancing them (Dulloo et al. 2010; Thakur et al. 2015a, b). Therefore A. racemosus is among 117 medicinal and aromatic plants, selected by National Medicinal Plant Board (NMPB) for conservation practices (Gowthami et al. 2021; Patel et al. 2022). We present an analysis of ex situ contributions to Shatavari conservation in this chapter that emphasizes a myriad of considerations pertinent to well-established interventions. This chapter will address the elements of a long-term conservation system that might directly or indirectly impact the efficacy, efficiency, and effectiveness of conservation and the facilitation of the use of Shatavari in all aspects. A major objective of the ex situ conservation of Shatavari is to retain and restore wild plant diversity. The goal can be reached, however, by improving working practices and facilities. Due to the lack of a general understanding of ex situ tools, they are undervalued and subsequently underused. Our purpose in this chapter is to examine the value, advantages, disadvantages, and range of possible ex situ tools for the conservation of Shatavari.

12.2

Preparation and Steps for Conservation of Shatavari

India is known as the “Medicinal or Botanical Garden” of the world due to its 8% of biodiversity and 4-global biodiversity hotspots (GBH), with 1748 endemic medicinal plant species only in the Indian Himalayan region. Globally about 7500 species are being utilized for indigenous health practices (including 1200 species in Ayurveda) as well as in modern medicines. The market for herbal medicines has increased about 100 times (more than 2000 tons per annum), which led to the uncontrolled harvesting of therapeutic plants as well as deforestation. India is second after China in the global trade of medicinal plants, including 75% of modern medicines manufactured from herbal sources (Bopana and Saxena 2007; Semwal et al. 2019; Gowthami et al. 2021; Gupta et al. 2022).

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The threat status of medicinal plants in India has been assessed from time to time by several institutes. Several Conservation Assessment and Management Prioritization (CAMP) workshops have been conducted all over India, to assess and prioritize the threat status of medicinal plants. Also, NMPB has assigned the Indian Council of Forestry Research and Education (ICFRE) along with the Foundation for Revitalization of Local Health Traditions (FRLHT) to evaluate time-to-time production and utilization of Indian medicinal plants (Gowthami et al. 2021). The demand for Asparagus racemosus has also increased, which influenced National Medicinal Plants Board (NMPB) to include the plant in the list of plants required for conservation and provide a 30% subsidy for the cultivation of the plant. NMPB also started a consortium related to A. racemosus cultivation (https://www.nmpb.nic.in). A. racemosus has been selected for several conservational approaches, following the steps mentioned by Maxted (2013): 1. 2. 3. 4. 5. 6.

Selection of target taxa Project Commission Ecogeographic survey/preliminary survey mission Conservation objectives Field exploration Conservation strategies

A. racemosus can only be harvested after 3 years of propagation, which creates the necessity to create effective propagation/conservation techniques to compete with the harvesting demand of the plant. Therefore, A. racemosus becomes a choice of taxa, based on logical, scientific, and economic considerations for an effective conservation approach. Due to escalating demand for Shatavari, its cultivation tactic provides a better target area with relevant environmental conditions for conservation rather than assortment from the wild. As suggested by Bopana and Saxena (2007) as well as outlined by Maxted (2013) in situ and ex situ are the two prior methods of conservation that include seed or in vitro storage, botanical garden, genetic reserve, or conservation at natural habitat.

12.3

Ex Situ Conservation

The term “ex situ conservation” simply demarcated the conservation “outside their natural habitats.” Ex situ collections limit the need for repeated sampling of wild populations by acting as a source of supply. Ex situ conservation of plant species includes germplasm banks, common garden archives, seed banks, DNA banks, and various techniques, including tissue culture, cryopreservation as well as the incorporation of disease, pest, and stress tolerance traits through genetic transformation (Gupta et al. 2022; https://bsi.gov.in/page/en/ex-situ-conservation).

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211

Seed Germination

Several cultivation trials have been initiated for field propagation of A. racemosus using seeds, although Shatavari can also be propagated using the rhizomatous disc. Germination of seeds for Shatavari and other plants varied significantly among different batches due to several environmental factors. Table 12.1 represents all the treatments studied for surface sterilization and breaking seed dormancy to get better seed germination. Gupta et al. (2002) tried physical (using a needle) and chemical scarring (5–30% H2SO4 and 144, 288, 577 μM GA3), while Laxmi et al. (2014) analyzed the effect of temperature 15, 20, 25, 30, and 35 °C, along with access of light and air using different sowing postures. Scarring was performed 3–4 h before sowing, to break the seed dormancy, while the top of the moist paper (15–35 °C), as well as in soil, sand, between the paper and between rolled towel paper (20 °C) was used to access the effect of light and air. Best germination was observed with 20% of H2SO4 (61–86%), as compared to control (35–60%), in two varieties. Germination occurred only in June–August, during higher temperatures and humidity. Also, better germination was observed (90.5%) when put in between the paper and the top of the paper at 20 ° C, due to proper accessibility of light and air. Sowing conditions (in vitro and in vivo) for fresh and 1-year-old seeds were tested following 13- different types of treatments alone or in combination, by Tiwari and Dubey (2017), to break seed dormancy. These treatments included physical scarification on seed coat using sandpaper (1 min), chemical scarification using 95% H2SO4, 63.1% HNO3, or 35% HCl (for 1 h), 10 mg/L GA3 with 0.2% KNO3 (24 h), hot water treatment (70 °C for 1 h), and cow dung treatment (12 h). Fresh seeds of A. racemosus did not germinate with or without physical or chemical scarring followed by water soaking. However, hot water and cow dung treatment with or without associated GA3 treatment followed by water soaking resulted in 3.5–46% as well as 4.5–47% seed germination in fresh as well as 1-year-old seeds. Fresh as well as 1-year-old seeds were germinated in vitro after physical scarring and treatment with GA3, KNO3 as well as cow dung. Treatment of fresh and old seeds with hot water followed by water soaking resulted in 46–47% seed germination. Qadir and Khan (2018) also tried so many pre-treatments including stratification, scarring, electric current, mechanical injury, alternately high and low temperature, KNO3, thiourea, kinetin, IAA, GA3, H2SO4, coumarin as well as brassinolide, to break the seed dormancy. Physical scarring using sand, pre-treatment with IAA, hot water, and pre-soaking in water resulted in recognized 80%, 74%, 74%, and 68% germination, respectively, on the 17th day of sowing. Sequential treatment includes pre-treatment as temperature stratification (5 °C and 35 °C) for 30 days (dark) followed by different treatments including water soaking, 1% KNO3, 50 ppm GA3, and 100 ppm NAA (for 20 days in the dark at 25 ° C) have been also accessed for breaking seed dormancy (Prabha et al. 2018). Control condition supported only 20% of seed germination which enhanced up to 66.66% using KNO3, GA3 as well as using NAA after warm stratification. Germination was

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Table 12.1 Seed germination of Asparagus racemosus

Treatment No treatment H2SO4 (5–30%) GA3 (144, 288, 577 μM) PSR PSR + GA3 –

Soil Sand Top of the paper Between the paper Between rolled towel paper –

PSR: Sand paper (1 min) CSR: H2SO4/HNO3/HCl/ (1 h)/ 0.2% KNO3 (24 h) 10 mg/L GA3 (24 h) Hot water treatment (70 ° C; 1 h) Cow dung treatment (12 h) –

Control

Water soaking 1% KNO3 50 ppm GA3 100 ppm NAA

Media/ special conditiona Soil; sand; farmyard manure

Sterilization/culture conditionb,c 30 ± 5 °C; 80% RH

MS + 3% sucrose; 0.8% agar; 0.2, 0.4, 0.8, 1.0 mg/L TDZ Water

SD-H2O: 2–3 times; 5% Labolene; 0.01% HgCl2: 5 min; SD-H2O: 5–6 times; 70% EtOH: 1 min; SD-H2O: 5–6 times 0.15% HgCl2: 10 min; SD-H2O: 3 times/15, 20, 25, 30, 35 °C

½MS + 3% sucrose; activated charcoal Water soaking Fresh/1-yearold seeds In vivo/ in vitro sowing

Results 35–60% 61–86% 50–80% 45–70% 50–72% Callus

Reference Gupta et al. (2002)

Trivedi et al. (2010)

30.5% 00.0% 90.5% 63% 90.5%

Laxmi et al. (2014)

Tween-80; SD-H2O

–

Jat et al. (2014)

–

0.67–17% 0–6% 0–34%

Tiwari and Dubey (2017)

0–20% 46–47% 0.67–35.5%

MS

Before stratification: Dark; 30 days

Liquid detergent: 15 min; R-H2O: 45 min; 90% EtOH: 5 min; SD-H2O; 0.2% HgCl2: 5 min; SD-H2O: 4 times –

–

Pant and Joshi (2017, 2018a, b)

55%

Prabha et al. (2018)

61.66% 61.66% 33.33%

(continued)

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Table 12.1 (continued) Media/ special conditiona

Treatment

Cold (5 °C) stratification

Warm (35 ° C) stratification Control

No treatment Water soaking 1% KNO3 50 ppm GA3 100 ppm NAA No treatment Water soaking 1% KNO3 50 ppm GA3 100 ppm NAA No treatment

Scarification Hot water IAA Pre-soaking Brassinolide Mechanical injury Stratification Thiourea KNO3 H2SO4 Electric current Coumarin Alt. high and low temp. GA3 Kn Control Dark light

Sterilization/culture conditionb,c

Results 20%

Reference

40%

After stratification: 25 °C; 20 days; dark; moist Whatman no. 1 filter paper

50% 45% 26.66% 20% 75% 96.66% 90% 61.66% 20%

Moist filter paper pads

R-H2O: 3–4 times; 0.1% HgCl2: 5 min; SD-H2O

Water/MS/½ MS/¼ MS/wet cotton beds

Savlon: 5-6 min; R-H2O: 10 min; 70% EtOH: Quick rinse; 0.1% HgCl2: 5 min; SD-H2O: 4 times

80% 74% 74% 68% 65% 57% 53% 53% 49% 48% 41% 40% 38% 36% 27% 7% –

Qadir and Khan (2018)

Lomror et al. (2018)

(continued)

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Table 12.1 (continued)

Treatment --

Media/ special conditiona MS+ 3% sucrose; 0.7% agar;

Sterilization/culture conditionb,c R-H2O; liquid detergent: 15 min; D-H2O: 3–4 times; 0.2% Bavistin: 5 min;

Results –

Reference Kumari et al. (2020)

a MS was used as basal media, with 3% sucrose, 0.8% agar as solidifying agent and pH 5.6–5.8, unless mentioned otherwise. Condition for autoclave has been: 121 °C; 15 lbs./1.06 kg/cm2; 15–20 min b Culture conditions have been set with 1000–3500 Lux; 16/8 h light/dark; 25 ± 2 °C, unless mentioned otherwise c EtOH ethyl alcohol, R-H2O running water, D-H2O distilled water, SD-H2O sterile distilled water, NaOCl sodium hypochlorite, SC subculturing, MS Murashige and Skoog’s (1962) media, NN Nitsch and Nitsch (1969) media, WM White (1963) media, LS Linsmaier and Skoog (1965) media, RH relative humidity, ME malt extract, Kn kinetin, ADS adenine sulfate, 2ip 2-isopentyl adenine, PG phloroglucinol, CH casein hydrolysate, AC activated charcoal, 8-HQC 8-hydroxyquinoline citrate, H2SO4 sulfuric acid, GA3 gibberellic acid, PSR physical scarification, CSR chemical scarification, RH relative humidity

reduced with cold stratification, while warm stratification was better with 75%, 90%, and 96.66% after water soaking, GA3, and KNO3 treatment, respectively. MS media was utilized by Trivedi et al. (2010) with 60% seed germination within 20–25 days as well as by Pant and Joshi (2017, 2018a, b) and Kumari et al. (2020). MS media of half strength with activated charcoal was also effective for in vitro seed germination (Jat et al. 2014).

12.5

In Vitro Conservation

In vitro, culture techniques are the best way to multiply those species that cannot easily regenerate by conventional means and easily produce disease-free plant material. Tissue culture-derived conserved in vitro plants can serve as a backup for Field Gene Banks, or use cryogenic technology to cryopreserve seeds, pollen, and/or plant tissues over long periods. In vitro grown propagules or explants can be collected for the propagation of clonal material at any time to maintain elite genotypes, regardless of the flowering period for each species also virus-free genetic stocks can be produced by meristem culture from contaminated tissues. Additionally, in vitro techniques have other advantages like germination of difficult seeds or embryos, reliable distribution of healthy germplasm across borders, and reduced storage space (Jain et al. 2012b; Verma et al. 2012; Jalli et al. 2015; Rajasekharan and Sahijram 2015; Encina and Regalado 2022). Various biotechnological approaches are used to conserve plant genetic resources; however, plant tissue culture/micropropagation and cryopreservation have been deemed to be the most

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critical. This technology can effectively be used to meet the growing demand for clonally uniform elite plants of Asparagus racemosus. Encina and Regalado (2022) discussed in vitro conservation strategies being developed for A. officinalis and its wild relatives. Table 12.2 described all the methods utilized for surface sterilization, media composition, growth regulators, or any other supplement, to establish in vitro conservation of A. racemosus.

12.5.1 Normal Growth Culture Tissue culture is the repeated practice of propagating plants from plant parts or single cells or groups of cells in a test tube under extremely controlled and hygienic conditions. Various methods such as clonal propagation, somatic embryogenesis, organogenesis, and callus differentiation could achieve in vitro propagation of endangered, threatened, and rare plant species. In vitro material can either be protoplasts, cell suspension, meristem cultures, or an organized plantlet. Starting from the explant. Callus production is preferred followed by shoots and root regeneration or direct organogenesis provides auxiliary explants, which develop into whole plants. After acclimatization, developed plants can be relocated to the field (Jain et al. 2012b; Niazian 2019; Deepa and Thomas 2020; Encina and Regalado 2022).

12.5.1.1 In Vitro Shoot Regeneration and Multiplication Most studies involving in vitro shoot regeneration utilized MS basal media with 3% sucrose and 0.8% agar (solidifying agent) at pH 5.6–5.8 and autoclaved at 121 °C and 15 lbs. or 1.06 kg/cm2 for 15–20 min. Inoculated explants were cultured at 25 ± 2 °C, under lights with 1500–3500 Lux intensity for a 16/8 h light/dark period. 12.5.1.1.1 Indirect Shoot Proliferation The first report of in vitro indirect shoot multiplication was established by Kar and Sen (1985a) after developing callus culture from shoot segments. Calli were inoculated on MS media supplemented with BAP (1 mg/L) and IAA (0.1 mg/L) for rapid regeneration of shoots (3–12 shoots/explant). Later, multiplied shoots were subjected to IBA (0.5 mg/L) for best root induction. For rooting in regenerated shoots, NAA (1 mg/L) alone displayed 62–69% rooting in MS media of full and half strength, as compared to IAA and IBA or NAA in MS of quarter strength. Reddy et al. (2007) selected shoot tips and nodal buds and tried 1–4 mg/L BAP with 0.1 mg/L NAA in MS media. High BAP (4 mg/L) supported callusing, differently lower BAP resulted in shoot induction (2 mg/L BAP) and multiplication (1 mg/L BAP) with 11.9 shoots/crown. Rooting was observed in MS of half strength with 7 mg/L IBA, among tried 1–8 mg/L NAA and IBA. 12.5.1.1.2 Direct Shoot Proliferation Kulkarni et al. (1994) inoculated surface sterilized shoot tips in media containing BAP (0.25–2.0 mg/L) and kinetin (0.5–1.0 mg/L). A maximum of 18 shoots/explant

5% lab wash; 70% Spirit: 3 min; 0.2% HgCl2: 1 min; D-H2O: 5 times

R-H2O: 30 min; 0.2% Bavistin +0.2% Cetrimide +5–6 drops Savlon: 30 min; 2%(v/v) Tween20: 15 min; D-H2O: 1 time; 0.1% HgCl2: 3 min; SD-H2O: 3–4 times

70% EtOH: Quick rinse; Teepol: 10 min; R-H2O: 15 min; 0.1% HgCl2: 10 min; SD-H2O: 3 times

ST; NS (1 cm)

NS

R-H2O: 60 min; 5% Teepol: 20 min; 95% EtOH: 1 min; 0.05% HgCl2: 10 min; SD-H2O

–

washing 5% Teepol; 0.1% HgCl2: 8–10 min; SD-H2O: 3 times

ST; NS cladodes

Spear sections: IVC ST (2 cm)

Explant (size) SS (5 cm)

Table 12.2 In vitro cultures of A. racemosus

½ MS + 7 mg/L IBA 2.22–6.66 μM BAP ± 2.32–6.97 μM Kn 2.46–7.38 μM 2ip ± 2.22–3.33 μM BAP

1 mg/L BAP + 0.1 mg/L NAA

0.1 mg/L NAA + 0.1–0.5 mg/L IBA 0, 1, 2 mg/L 2,4-D ± 0–1 mg/L NAA + 1 mg/L Kn ± 15% v/v coconut water 0.25–2.0 mg/L BAP ± 0.5–1.0 mg/L Kn ¼ MS/½ MS/MS + (0.5–2.0 mg/ L) IAA/IBA/NAA ± Kn (0.5 mg/ L) 3 mg/L BAP ± 3 mg/L Kn ± 1 mg/L NAA 3 mg/L BAP ± 2 mg/L Kn ± 0.1 mg/L NAA 4 mg/L BAP + 0.1 mg/L NAA 2 mg/L BAP + 0.1 mg/L NAA

Growth regulatorsa 1 mg/L 2,4-D + 1 mg/L Kn 1 mg/L BAP + 0.1–0.5 mg/L IAA

250–500 mg/L ME; 79.30–198.25 μM PG; 98.91 ADS; 100 mg/L CH; 100 mg/

–

–

7/17 h light/dark;

Frequency of polyploidy (55% RH; SC 45 days)

Especial condition/treatment/ observationb,c (50–60% RH; SC 30–35 days)

Callus Shoot induction Shoot elongation Roots Shoot multiplication Shoot proliferation

Shoots

Callus

Roots

Shoots

Callus

Results Callus (80%) Shoots (3–12/ explant) Roots (70%)

Bopana and Saxena (2008)

Reddy et al. (2007)

Vijay and Kumar (2004)

Kar and Sen (1985b) Kulkarni et al. (1994)

References Kar and Sen (1985a)

216 V. Pandey et al.

70% EtOH: 2–25 sec; Teepolwater: 10 min; R-H2O: 15 min; 0.1% HgCl2: 10 min; SD-H2O

R-H2O: 10–15 min; 0.1% HgCl2: 10 min SD-H2O: 5–6 times

SD-H2O: 2–3 times; 5% Labolene; 0.01% HgCl2: 5 min; SD-H2O: 5–6 times; 70% EtOH: 1 min; SD-H2O: 5–6 times Detergent + D-H2O: 30 min; 0.1% HgCl2: 3 min; 70% EtOH: 5 min; SD-H2O R-H2O: 10–15 min; Bavistin; D-H2O; EtOH: 2–3 min; 20%

NS

NS (2–4 mm)

Seeds (for NS: 1 cm)

ST; NS

NS; roots

Liquid detergent: 15 min; R-H2O: 45 min; 90% EtOH: 5 min; 0.2% HgCl2: 5 min; SD-H2O: 4-times

ST; NS (0.5–1 cm)

0–3 mg/L NAA ± 0–4 mg/L 2,4-D ± 0–2 mg/L BAP

½ MS ± 2.68–8.05 μM NAA ± 0.23–0.46 μM Kn ± 2.85–8.56 μM IAA ± 2.46–7.38 μM IBA 0.1, 0.5, 1.0, 2.0 mg/L NAA ± BAP/Kn; 0.5, 1.0, 2.0 mg/L Kn 0.1–0.5 mg/L NAA + 1–2 mg/L BAP 0.1–2.0 mg/L BAP ± 0.1–1.0 mg/L NAA 1.0 mg/L NAA 3.69 μM 2ip ½ MS + 1.61 μM NAA + 0.46 μM Kn + 98.91 μM ADS +500 mg/L ME +198.25 μM PG 0.1–0.5 mg/L BAP ± 0.05–0.25 mg/L NAA or IBA 0.05–0.1 mg/L BAP ± 0.1, 0.3, 0.5, 1.0, 2.0 mg/L IBA 0.2, 0.4, 0.8, 1.0 mg/L TDZ

Callus

Shoot induction

–

Callus

Roots

Shoots

Roots Shoots Roots

Shoots

Buds

Callus

85% roots

170.0–500.0 mg/L KH2PO4 Saponin production

–

0.7% agar

SC 30 days

100 mg myo-inositol

L AC (12/12 h light/dark; SC 30 days)

Ex Situ Conservation of Shatavari (Asparagus racemosus) (continued)

Jain et al. (2012b)

Pise et al. (2011)

Trivedi et al. (2010)

Afroz et al. (2010)

Saxena and Bopana (2009)

Pant and Joshi (2009)

12 217

–

Liquid detergent: 5 min; R-H2O: 60 min Bavistin +8HQC + Indophyl: 7 min; 0.1% HgCl2: 5–7 min; SD-H2O: Many-times –

IVC

NS (1–1.5 cm)

BAP or Kn ± IAA or NAA 2,4-D; IAA; NAA 0.25–0.5 mg/L BAP

0.5–1.5 mg/L BAP ± 0.5–1.5 mg/L Kn ± 100 mg/L ADS 0.5–2 mg/L BAP ± 1–1.5 mg/L Kn ± 0.5–1.0 mg/L NAA 0.5–2 mg/L 2,4-D ± 1–3 mg/L NAA ± 0.5 mg/L BAP ½MS/¼MS/MS (with 10, 20 g/L mannitol or 40, 80 mg sorbitol or 30, 100, 150 g/L sucrose) 1–2.5 mg/L BAP ± 0.2–0.5 mg/L IAA ½MS/MS/± 0.1 mg/L NAA/IBA; dark/light

HgCl2: 5 min; NaOCl: 5 min; SD-H2O

ST; NS (1–1.5 cm)

EN (26.5 mm)

1–3 mg/L NAA ± 0.5–2 mg/L 2,4-D ± 0.5 mg/L BAP ± 2 g/L CH

Growth regulatorsa 0.5–1.5 mg/L BAP ± 0.5–1.5 mg/L Kn ± 100 mg/L ADS 1.5 mg/L BAP ± 100 mg/L ADS

30% tween 20: 30 min; 0.1% HgCl2: 3 min; 70% EtOH: 5 min; SD-H2O

NaOCl: 5–6 min; SD-H2O: 1-time; 0.1%HgCl2: 5 min; SD-H2O: 5–6 times

washing

NS (15 mm)

Explant (size)

Table 12.2 (continued)

50–60% RH; 28 ± 2 °C; SC 4–5 weeks

Shoots

–

Shoots Callus Shoots

Roots

Slow growing culture

Callus

Roots

Shoots

Shoot proliferation Cell suspension culture

Results

25 °C and 4 °C

170.0–500.0 mg/L KH2PO4; Saponin production; (0.6% Phytagel/80 rpm; SC 3 weeks) –

Especial condition/treatment/ observationb,c

Jat et al. (2014)

Pandey and Sinha (2013) Singh et al. (2013)

Jain and Kumar (2013)

Pise et al. (2012)

References

218 V. Pandey et al.

1.5–3% sucrose; 2–4% mannitol; 2–4% sorbitol 3% sodium alginate; 100 mM CaCl2 (15–25 min) 2, 4-D ± NAA ± BAP ± Kn

–

0.1% HgCl2: 10 min; SD-H2O

0.1% Bavistin: 5–10 min; 70% EtOH: 2 min; 20% NaOCl: 5 min; 0.1% HgCl2: 3 min; SD-H2O: 6 times 0.1% Bavistin: 15 min; D-H2O: 2–3 times; 70% EtOH: 1 min; 0.1% HgCl2: 3 min; SD-H2O: 5 times

NS

NS (0.4–1.0 cm)

Different explants

50–60% RH; SC 21 days

–

25 ± 2 °C and 8 ± 2 °C

25 ± 2 °C and 15 ± 2 °C

Roots

Shoots (85%)

Callus

Shoots

Callus culture; cell suspension Slow growing culture Encapsulation

Roots

Shoots

Callus

Cell suspension

(continued)

Thakur et al. (2016a)

Pandey et al. (2016)

Thakur et al. (2015b)

Pise et al. (2015)

Thakur et al. (2014) Sherathiy et al. (2014) Patel and Patel (2015)

Ex Situ Conservation of Shatavari (Asparagus racemosus)

NS

0.1 mg/L 2,4-D + 0.2 mg/L BAP, 0.2 mg/L IAA + 0.1 mg/L NAA 0.1–1.5 mg/L BAP + 0.15 mg/L IAA 0.1–3.0 mg/L IAA + 0.1 mg/L Kn 0–4 mg/L BAP + 0–3 mg/L Kn

50–60% RH

(0.1–2.0 mg/L) NAA ± BAP ± Kn ± 2,4D; 0.1–2.0 mg/L BAP ± 0.1, 0.5, 1.0 mg/L IBA/NAA 0.1–2.0 mg/L IBA ± 0.1–2.0 mg/ L BAP 1.0 mg/L NAA + 1.0 mg/L 2,4-D + 0.5 mg/L BAP

R-H2O: 30 min; D-H2O; 1% Bavistin: 30 min; NaOCl: 5 min; 0.1% HgCl2: 2–5 min; SD-H2O

NS

ST; NS (1–2 cm)

Saponin production

NAA ± 2, 4 D ± BAP ± Zeatin ± kinetin

–

LS, RS, NS

SC 3 weeks

Coconut milk/De-oiled rice bran/ dextrose/lactose/maltose

0.1% HgCl2: 10 min; SD-H2O: 5–6 times

NS (1 cm)

12 219

–

–

Liquid detergent: 5 min; R-H2O: 60 min; Bavistin +8HQC + Indophyl: 7 min; 0.1% HgCl2: 5–7 min; SD-H2O (Savlon: 5 min; R-H2O: 10 min; 70% EtOH: Quick rinse; 0.1% HgCl2: 8 min; SD-H2O) R-H2O: 30 min; 5% Teepol: 5 min; SD-H2O; 0.1% HgCl2: 3 min; SD-H2O

Liquid detergent + R-H2O: 60 min; D-H2O: 4–5 min; 0.1%

NS (IVC) (0.5–1.0 cm)

NS: PLB

AB; RS; ST; (NS); ZE

ST (0.5–2.0 cm)

NS

–

washing 0.1% HgCl2: 10 min; SD-H2O: 3 times

Fresh pollen

Explant (size)

Table 12.2 (continued)

0.01, 0.1, 0.5, 1.0 mg/L NAA ± 0.5, 1.0 mg/L BAP ± 0.1, 0.5, 1.0, 1.5, 2.5 mg/L Kn

0.5–2.0 mg/L BAP + 1.5 mg/L Kn

IBA + BAP (1.0 mg/l) or NAA (0.1 mg/L) ½MS + 2% sucrose +1.5, 2.5, 3.5% sodium alginate ±50, 75, 100 mM CaCl2 (15–25 min) 0, 1.1, 1.54, 2.2 mg/L 2,4-D ± 0.43 mg/L Kn

0.1 mg/l NAA Sucrose (1–50%) ± boric acid (25-500 ppm) NAA ± BAP ± Kn (0.1–2.0 mg/ L)

Growth regulatorsa

SC 4–6 weeks

2% sucrose

Dark; SC 4 weeks

SC 6–12 weeks

–

Especial condition/treatment/ observationb,c MS, NN, WM, LS; (0.75% Agar; 50–60% RH; SC 4 weeks)

Shoots (11–24 shoots/ explant) Shoots (3–13 shoots/ explant)

Callus/ somatic embryo induction

Results Shoots (15 shoots/ cluster) Roots In vitro pollen germination Callus/ somatic embryo Germination of SE Encapsulation

Paudel et al. (2018)

Mani and Yadav (2018)

Soni and Sharma (2017) Lomror et al. (2018)

Pal et al. (2017) Pant and Joshi (2017)

References Thakur et al. (2016b)

220 V. Pandey et al.

–

5% Teepol; 0.1% HgCl2: 8–10 min; SD-H2O

R-H2O; 0.3% Bavistin +0.03% streptomycin: 10 min; D-H2O: 2 times; Savlon: 10 minutes;

ZE

NS (4–6 cm)

NS, IS, LS

NS (IVC) (0.5–1 cm)

NaOCl: 10 min; SD-H2O: 3–4 times –

Radical scavenging activities; secondary metabolites

DKW; MS liquid media; SC 2–3 weeks

–

Shoots

Roots 40%

Shoots

Somatic embryo germination

Callus/ somatic embryo induction

Shoot bud

–

1–2 mg/L BAP/Kn + 0.1–1.0 mg/ L IAA/IBA/NAA 0, 1.1, 1.54, 2.2 mg/L 2,4-D ± 0.43 mg/L Kn 0.05, 0.1 mg/L NAA ± 0.5, 1.0 mg Kn ± 0.5, 0.75, 1.0 gm/L Ancymidol 0.05–0.1 mg/L NAA + 0.5–1.0 mg/L Kn ± 0.75 mg/L Ancymidol ±600–800 mg/L glutamine ±400–500 mg/L CH + 3 or 5% sucrose 0, 0.125, 0.25, 0.5 μM: NAA + Kn + 3 or 6%: Glucose/ fructose/sucrose/maltose ±0.5–1 μM Ancymidol/ Paclobutrazol 0, 10, 15 or 20 μM IBA/IAA/ NAA + 3% or 6% sucrose ±0.5–1 μM Ancymidol MS

Shoots

–

1–2 mg/L Kn ± 0.1–2.0 mg/L IBA/IAA

Ex Situ Conservation of Shatavari (Asparagus racemosus) (continued)

Kumar et al. (2020)

Haque et al. (2020)

Pant and Joshi (2018a) Pant and Joshi (2018b) Chaudhary and Dantu (2019)

12 221

NS (IVC)

0.1–3.5 mg/L BAP ± 0.25 mg/L NAA or 0.25–2.5 mg/L ± 0.25 mg/L IAA 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0 mg/L NAA

0.2, 0.5, 1.0, 1.5, 2.0 mg/L NAA ± 0.2, 0.5, 1.0, 1.5, 2.0 mg/L 2,4-D ± 0.2, 0.5, 1.0, 1.5, 2.0 mg/ L BAP ± 0.2, 0.5, 1.0, 1.5, 2.0 mg/L Kn 1–2 mg/L 2,4-D ± 1–2 mg/L NAA + 0.5 mg/L BAP 1–4 mg/L BAP + 0.5 mg/L NAA

Growth regulatorsa

–

–

Especial condition/treatment/ observationb,c

Roots

Shoots

Axillary buds

Callus

Callus

Results

Sulava et al. (2020)

Nabi et al. (2020)

Kumari et al. (2020)

References

a MS was used as basal media, with 3% sucrose, 0.8% agar as solidifying agent and pH 5.6–5.8, unless mentioned otherwise. Condition for autoclave has been: 121 °C; 15 lbs./1.06 kg/cm2; 15–20 min b Culture conditions have been set with 1500–3500 Lux; 16/8 h light/dark; 25 ± 2 °C, unless mentioned otherwise c SS shoot segments, ST shoot tips, NS nodal segments, INS internodal segments, LS leaf segments, IVC in vitro culture, EN epicotyledonary node, AB auxiliary buds, ZE zygotic embryos, EtOH ethyl alcohol, R-H2O running water, D-H2O distilled water, SD-H2O sterile distilled water, NaOCl sodium hypochlorite, SC subculturing, MS Murashige and Skoog’s (1962) media, NN Nitsch and Nitsch (1969) media, WM White’s (1963) media, LS Linsmaier and Skoog (1965) media, DKW Driver and Kuniyuki Walnut media, RH relative humidity, ME malt extract, Kn Kinetin, ADS adenine sulfate, 2ip 2-Isopentyl adenine, PG phloroglucinol, CH casein hydrolysate, AC activated charcoal, 8-HQC 8-hydroxyquinoline citrate, PLB protocorm-like bodies

Twigs; ST; INS; LS

R-H2O: 30 min; Cetrimide; D-H2O; 70% EtOH: 30 min; SD-H2O; 0.15% HgCl2: 1 min; SD-H2O: 3 times R-H2O: 30 min; 5% Labolene: 3–5 min; D-H2O; 0.1% HgCl2: 3–4 min; SD-H2O: 3 times

SD-H2O: 2 times; 0.01% HgCl2: 5 min; SD-H2O –

NS; INS

washing

Explant (size)

Table 12.2 (continued)

222 V. Pandey et al.

12

Ex Situ Conservation of Shatavari (Asparagus racemosus)

223

were obtained using 0.5 mg/L of BAP, after 3–4 sub-culture on the same medium. Kinetin stimulated callusing along with shoot proliferation. Following this, Thakur et al. (2014) used in vitro multiplied shoots grown in presence of 0.25–0.5 mg/L BAP and tested several carbon sources for shoot multiplication. Coconut milk and de-oiled rice bran reinforced good shoot production similar to dextrose, lactose, and maltose, however, sucrose created the best shoot proliferation and shoot length as compared to any other carbon sources. Based on the response of BAP alone, axillary bud breaking is ensured by using 1.0 mg/L BAP in 3–4 days with 28 shoots/explant. The bud breaking was delayed till 10–11 days using 0.1 mg/L or 3.25 mg/L BAP (4–11 shoots/explant). Opposite to these observations, surface sterilized nodal explants were observed with observed 1.68-fold increased shoot multiplication using 5.81 μM of kinetin only, as compared to BAP alone or in combination with kinetin (Bopana and Saxena 2008). Changing the growth regulator to 3.69 μM of 2ip, after culturing in MS basal media, resulted in 3.5-fold higher (6–8 shoots/ cluster) shoot proliferation. Therefore, the use of both cytokinins BAP and kinetin was considered during several studies. Shoot tips and nodal segments of field grown (Jain et al. (2012a) as well as in vitro plants (Jain and Kumar 2013) were inoculated on media supplemented with 0.5–1.5 mg/L BAP and/or kinetin along with or without 100 mg/L ADS. Shoot induction of 80% achieved with 1 mg/L each of BAP and kinetin together, followed by 1.5 mg/L BAP or kinetin as well as 1.5 mg/L BAP with 100 mg/L ADS. Further shoot as well as root proliferation was completed by using 1.5 mg/L BAP with 100 mg/L ADS as well as BAP (1 mg/L), kinetin (l mg/L), and NAA (0.5 mg/L). Based on the observation, Thakur et al. (2016b) observed the effect of different media compositions, after initiating a culture of nodal explant using different concentrations of BAP (0–4 mg/L) and kinetin (0–3 mg/L) in MS media. Initiated shoots were transferred in MS media with different BAP and kinetin as well as in MS, LS, NN, and WM containing 0.25 mg/L BAP. Best shoot multiplication was obtained with 0.5 mg/L BAP and 0.5 mg/L kinetin in MS media, with more number (15 shoots per cluster), height (3.35 cm) and health of shoots, followed by NN medium. WS medium was observed with the lowest growth rate. Mani and Yadav (2018) noticed that a combination of BAP 1.5 mg/L and kinetin 1.5 mg/L was most effective for regeneration with 24 shoots/explant. However, Pandey et al. (2016) observed best shoot emergence with sequential application of 2 mg/L kinetin followed by 4 mg/L BAP. The regenerative capacity of shoot segments (nodes, internodes, shoot tips), cladodes and root explants of A. racemosus were compared using different combinations of BAP, kinetin, NAA, 2,4-D, IAA and IBA (Vijay and Kumar 2004; Pant and Joshi 2009). Nodal explant (Vijay and Kumar 2004) and shoot segments (nodes, internodes, shoot tips) were more regenerative (Pant and Joshi 2009) using 0.1 mg/L NAA and 2 mg/L kinetin (15–25 shoots) as well as 1–2 mg/L BAP 1–2 mg/L alone and in combination with 0.1 mg/L NAA (6–8 shoots/explant), respectively. Increasing concentration of NAA (1 mg/L) caused callusing, and BAP was less effective for shoot regeneration (5–10 explants) at higher concentrations

224

V. Pandey et al.

(3.0 mg/L). Explants were non-responsive in the presence of 2,4-D (all concentrations). Among cytokinins, BAP was most effective for shoot regeneration, therefore, a significant number of studies tried the combination of BAP with different auxins in MS media for in vitro culture establishment using shoot tips or nodal segments of A. racemosus. Supplementation of 0.1 mg/L BAP with 0.05 mg/L NAA (Afroz et al. 2010), 2 mg/L BAP with 0.1 mg/L NAA or 1.0 mg/L IBA (Pant and Joshi 2018b), 0.5 mg/L BAP with 0.1 mg/L NAA (Paudel et al. 2018), 1–2 mg/L BAP with 0.5–1.0 mg/L NAA (Patel and Patel 2015), as well as 2 mg/L BAP with 0.5 mg/L NAA (Nabi et al. 2020), produced 20 shoots/nodal explant (90%), 10.33–11.6 shootbuds/explants, 6–13 shoots/shoot tips explant (5–6 weeks), 8.4–9 shoots/nodal explant (80–83%), 3 shoots/nodal explant (40%), respectively. A combination of 2 mg/L BAP with 0.2 mg/L IAA (Singh et al. 2013), as well as 1.5 mg/L BAP with 0.15 mg/L IAA (Thakur et al. 2016a), resulted in 7 shoots/nodal explants in 20 days and 85% shoot proliferation, respectively. Epicotyledonary nodes (Jat et al. 2014) and nodal segments of in vitro germinated seedlings (Pant and Joshi 2018a) have presented good shoot induction in the combination of kinetin with IAA, IBA or NAA. Most suitable combinations having 13.93 μM kinetin with 5.70 μM IAA (Jat et al. 2014), 2.0 mg/L kinetin combined with 0.1 mg/L IAA and 0.5 mg/L IBA (Pant and Joshi 2018a), presented 12 shoots/ explant as well as 5.8 and 8.3 shoots/explants respectively. Haque et al. (2020) tested Driver and Kuniyuki Walnut (DKW) and MS media supplemented with 0, 0.125, 0.25, 0.5 μM of growth regulators (NAA and Kn) and 3 or 6% carbohydrate (glucose, fructose, sucrose or maltose) for shoot regeneration of A. racemosus. DKW media having 0.5 μM kinetin with 0.125 μM of NAA as well as 87.6 mM and 175.3 mM glucose or fructose corresponded to a greater number of nodes and shoots. A low concentration of NAA supported less development of callus. DKW or MS media in liquid and solid conditions reinforced shoot thickness and percentage shoot induction, respectively. Pant and Joshi (2018b) mentioned that individual use of 0.5 mg/L IAA and 0.1 mg/L IBA (auxins) resulted in 4.8 and 2.5 shoots/explants. Kumar et al. (2020) proliferated shoots on MS basal media with optimal conditions to analyze radical scavenging activity and the metabolic composition of in vitro plant extracts. BAP has been used more frequently for shoot induction and NAA for root induction as compared to other cytokinins and auxins, respectively. 12.5.1.1.3 Rooting in In Vitro Cultures NAA (1 mg/L) alone displayed 62–69% rooting in MS media of full and half strength, as compared to IAA and IBA or NAA in MS of quarter strength (Kar and Sen 1985a). A higher dose of malt extract (500 mg/L) and lower dose of phloroglucinol (198.25 μM) was more favorable for rooting (85%) along with NAA (1.61 μM), kinetin (0.46 μM) and adenine sulfate (98.91 μM) supplementation in MS media of half strength (Bopana and Saxena 2008; Saxena and Bopana 2009). NAA at any concentration (0.1–2 mg/L) alone, and 1 mg/L along with 0.1 mg/L of BAP effectively produced a higher number of roots (Pant and Joshi 2009). Proving

12

Ex Situ Conservation of Shatavari (Asparagus racemosus)

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this, longer and 6% more roots were observed with 2.0 mg/L NAA as compared to other concentrations of NAA (Sulava et al. 2020). Besides NAA, rooting resulted in 5 roots/shoots and 3.6 roots/explant from MS media in dark (Singh et al. 2013) and adding 1.5 mg/L IBA alone or 1.0 mg/L IBA in combination with 0.1 mg/L BAP (Patel and Patel 2015) as well as 3 mg/L IAA with 0.1 mg/L kinetin (Thakur et al. (2016a). Also, 85% of regenerated shoots were obtained in MS media of half strength with 0.05 mg/L BAP and 0.1 mg/L IBA (Afroz et al. 2010). There was no rooting in DKW media, while MS media with 10 μM IBA and 0.5 μM ancymidol produced roots within 6 weeks, as compared to NAA (14 weeks) and IAA (12 weeks) Haque et al. (2020).

12.5.1.2 Callus Culture Kar and Sen (1985a) introduced in vitro culture of A. racemosus for the first time, through callus culture using shoot segments inoculated in MS media having 1 mg/L of both 2,4-D and kinetin. Callusing was also observed with 4 mg/L BAP supplemented along with 0.1 mg/L NAA, in MS media using shoot tips and nodal buds as explants (Reddy et al. 2007). Pant and Joshi (2009) established better regenerative capacity of shoot segments (nodes, internodes, shoot tips) and proved that maximum callusing was observed with 0.1 mg/L NAA (with or without 0.5 mg/ L BAP) and 1–2 mg/L kinetin. Compared to other auxins, higher concentrations of NAA supported callus and root growth, 2,4-D also resulted in only some callusing. Trivedi et al. (2010) used nodal segments of in vitro germinated seedlings to obtain callus cultures using different concentrations (0.2, 0.4, 0.8, and 1.0 mg/L) of TDZ in MS media. Developed callus with 40% callusing in 0.4 mg/L TDZ containing media was shifted to TDZ free MS media for further shoot formation in a minimum of 12 days. Utmost callusing was observed with the use of 1.0 mg/L NAA, 1.0 mg/L 2,4-D and 0.5 mg/L BAP, in 25–30 days from nodal segments (Pise et al. (2011), within 2–3 weeks from shoot tips and nodal explants (Jain and Kumar 2013) or in 4-weeks in 40% nodal/internodal explants (Nabi et al. 2020). Increasing (2 mg/L) or decreasing (0 mg/L) the concentration of 2,4-D or NAA, resulted in less callus formation. Calli were maintained in the same media with a slight increase in the concentration of 2,4-D. The use of additional KH2PO4 (170–500 mg/L) reduced induction time by half. Callus culture was used to extract Shatavari (Pise et al. (2011). Inoculation of nodal explants in 0.1 mg/L NAA or 1.0 mg/L NAA with 0.5 mg/L BAP produced 93% (0.82 gm DW) and 91% (0.80 gm DW) of callus, respectively (Patel and Patel 2015). Similarly, 0.1 mg/L NAA, 0.1 mg/L 2,4-D, 0.2 mg/L IAA and 0.2 mg/L BAP induced callusing in explants (Thakur et al. 2016a). Increasing NAA alone to 0.2 mg/L NAA developed 90.36% compact greenish yellow callus, followed by friable 88.78%, 88.62% as well as compact 82.48%, callus due to 2 mg/ L kinetin, 1 mg/L BAP with 1 mg/L NAA as well as 0.5 mg/L NAA, respectively (Kumari et al. 2020).

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12.5.1.3 Somatic Embryo Callus developed from hypocotyl and zygotic embryos (Chaudhary and Dantu 2019) along with nodal explants (Lomror et al. 2018) of in vitro germinated seedlings (Pant and Joshi 2017) have been used to produce somatic embryos. Nodal segments (from seedlings) were inoculated on media containing different combinations of NAA, BAP, and kinetin (0.1–2.0 mg/L) and sub-cultured every 6–12 weeks. Media containing 0.1 mg/L NAA with 1.0 or 2.0 mg/L of BAP proved to be most effective to generate somatic embryoids. Other explants were subjected to media having 0, 1.1, 1.54, 2.2 mg/L 2,4-D with or without 0.43 mg/L kinetin, for callus induction to conclude callusing in 38.4% of hypocotyl explants at 1.5 mg/L 2,4-D which enhanced up to 62.7 or 74% with the addition of 0.43 mg/L kinetin (Lomror et al. 2018; Chaudhary and Dantu 2019). Callus induction was also obtained using kinetin (0.5–1.0 mg/L) and replacing 2,4-D with NAA (0.05–0.1 mg/L). Calculated 4.83 embryos/culture using 0.1 mg/L NAA and 0.5 mg/L kinetin were enhanced up to 14 embryos/culture by the addition of 0.75 mg/L Ancymidol. Further addition of 600–800 mg/L glutamine or 400–500 mg/L casein hydrolysate with 3 or 5% sucrose promoted up to 65% of somatic embryos germination. Microscopic examination confirmed the development of somatic embryos showing various stages of somatic embryoids. Germinating embryos were transferred to MS media containing different concentrations of BAP (0, 0.002, 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 mg/L), resulting in the highest 15.87 shoots/cluster using 0.08 mg/L BAP (Chaudhary and Dantu 2019). 12.5.1.4 Cell Culture Pise et al. (2012, 2015) established cell culture from nodal segments via callusing in MS media containing NAA (1–3 mg/L), 2,4-D (0.5–2 mg/L) and BAP (0.5 mg/L), within 10–21 days. Pise et al. (2012) also included KH2PO4 (170–500 mg/L) in supplemented media. The same medium without solidifying agent was used to develop a cell suspension culture, to extract Shatavari from the callus as well as spent media (Pise et al. 2015). Besides, nodal segments, leaf and root segments were also included by Sherathiy et al. (2014) to generate callus followed by cell suspension culture, using 1 mg/L zeatin and 1 mg/L BAP and analyzed for saponin production. 12.5.1.5 Other Aspects of Normal In Vitro Cultures Kar and Sen (1985b) studied ploidy levels in callus culture originating in MS media containing 2,4 D (0–2 mg/L) or NAA (0–1 mg/L) with kinetin (0–1 mg/L) and/or coconut water (0–15% v/v), from in vitro spear sections of the plant. Up to 90 days, about 90% of callus cells were diploid (2n = 20). However, polyploidy increase with the age of culture from 135–450 days, under influence of 2,4-D and coconut water, as compared to NAA and kinetin. Pal et al. (2017) collected during anthesis and sown on sucrose (1–50%) and boric acid (25-500 ppm) solutions for in vitro pollen germination to understand the first momentous morphogenetic event of plant production (pollen germination). Germination (4–6%) can begin at 2% sucrose or 25 ppm boric acid (91 and 26 μm pollen

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tube, respectively) and reach up to 86% and 24% germination with 715 and 182 μm long pollen tubes, in 20% sucrose and 50 ppm boric acid, respectively. A combination of 20% sucrose and 50 ppm boric acid resulted in 98% of pollen germination with a tube length of 780 μm. Boron of female gametophyte is required in low concentration during pectin synthesis for pollen tube growth and enhances uptake of important respiratory substrate and osmogene, sucrose. Therefore, the combined treatment was more effective due to the formation of the sugar-borate complex which supports better translocation.

12.5.2 Slow-Growth Culture and Synthetic Seeds The most practiced technique of in vitro conservation is “slow-growth cultures,” which provide storage of clonal plant material for 1–15 years and require subculturing from time to time to avoid contamination. Slow-growth methods for active collection are designed to reduce cell division and growth so that longevity can be increased without causing genetic changes. As a result of this, the time between transfers is extended, which prolongs storage and reduces maintenance costs. It is accomplished by the use of osmotica, growth restriction retardants or low temperature and light intensities (Rajasekharan and Sahijram 2015; Niazian 2019; Encina and Regalado 2022). The use of synthetic (artificial) seeds, which refer to the alginate encapsulation of plant explants, has been among strategies for the conservation and micropropagation of medicinal plants, because of their advantages, including genetic stability, ease in handling and transportation, effectiveness in terms of space, labor, time, and cost (Rajasekharan and Sahijram 2015; Niazian 2019; Encina and Regalado 2022). Pandey and Sinha (2013) studied the effect of sorbitol, mannitol, and excess sucrose supplementation of MS media, along with ½MS and ¼MS media, for in vitro conservation of A. racemosus at 4 °C or 25 °C. The best suitable temperature, as well as media for conservation, was noted as 4 °C as well as MS media supplemented with sorbitol (80 mg), sucrose (100 or 150 g) or sucrose (30 g) together with mannitol (10 g) suited best for the conservation drive. Another slowgrowth culture was maintained using ½ MS with 1.5% of sucrose, 2% mannitol or sorbitol for up to 6 months at 15 or 25 °C as a conservation strategy by Thakur et al. (2015b). Although cultures at 15 °C exhibited reduced shoot growth, cultures at 25 ° C were better in more percentage of survival. These shoots were also encapsulated in calcium alginate beads (3% sodium alginate and 100 mM CaCl2) for 75 days of storage under culture conditions. Re-growth of cultures can be initiated by using 1.11 μM BAP in MS media, followed by rooting in 1.61 μM NAA, 0.46 μM kinetin, 98.91 μM ADS, 500 mg/L malt extract, and 198.25 μM phloroglucinol (PG) and acclimatization. Later, Soni and Sharma (2017) understood the different natures of synthetic seeds prepared using different concentrations of sodium agitate (1.5%, 2.5%, 3.5%) and CaCl2 (50, 75,100 mM CaCl2) on protocorm-like bodies (PLB) obtained from nodal segments of A. racemosus. Seeds generated from 3.5% sodium agitate were rigid,

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firm, clear, and isodiametric when combined with 100 mM CaCl2, as well as with a short tail when combined with 50 or 75 mM CaCl2. Ideal uniform, solid and clear seeds were created using 2.5% sodium agitate and 100 mM CaCl2. Further, a decrease in the concentration of sodium agitates and CaCl2 produced seeds that were not suitable as they had solid clusters or were very soft. These synthesized seeds gave 94.9% germination on MS media in 3 weeks.

12.6

Conventional Methods

Ex situ seed/embryo collection and storage is the most convenient and widely used method of genetic conservation. Conservation ex situ, however, interrupts evolutionary and ecological processes while also limiting the genetic variation and adaptability of species. Moreover, ex situ conservation has significantly higher costs, risks, and research requirements than in situ conservation. There are many ex situ conservation methods, such as botanic gardens and field gene banks.

12.6.1 Germplasm Collection The germplasm of plants is a collection of propagules: seeds, pollen, cuttings, buds, rhizomes, or cell cultures for preserving specific genetic blends. It is mandatory to conserve all the germplasm with appropriate data in National Gene Bank (seed gene bank/in vitro/field gene banks) along with a backup at a regional level to ensure accessibility and preservation of threatened medicinal plants for future practices (Gowthami et al. 2021). Ethiopia holds the title of one of the world’s richest sources of genetic resources. The following institutes of India are participating in the conservation of medicinal plants like A. racemosus: 1. 2. 3. 4. 5. 6.

ICAR-National Bureau of Plant Genetic Resources (NBPGR) ICAR-Indian Agricultural Research Institute (IARI) CSIR- Central Institute of Medicinal and Aromatic Plants (CIMAP) CSIR-National Botanic Research Institute (NBRI) CSIR-Indian Institute of Integrative Medicine (IIIM) KSCSTE-Jawaharlal Nehru Tropical Botanic Garden and Research Institute (KSCSTE - JNTBGRI) 7. Department of Biotechnology (DBT) 8. Botanical Survey of India (BSI) Users can obtain information and plant material from gene banks by registering, studying, describing, and documenting them. Gene banks ensure the long-term viability of numerous genetically intact accessions (or samples) for easy access and progression of plant breeding and basic biological research. Gene banks include:

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Seed bank Field Gene bank Pollen gene bank. Spores gene bank Gene bank of vegetative parts of plants

A seed bank is sometimes referred to as a cryogenic facility, in which seeds can be preserved without losing their fertility for up to a century or more. The germplasm in Field Gene Banks is kept as plants, which are held permanently as living collections.

12.6.1.1 Seed Collecting Parveen et al. (2011) collected seeds of A. racemosus from 20 different geographical locations in 10-state of India and planted 6-month-old seedlings in a field 1 m × 1 m apart. All growing plants were briefly examined on morphological parameters for the evaluation of variance among them. Root production displayed the best critical difference (CD) of 0.91, followed by root diameter (0.68), shoot weight (0.49), number of shoots (0.42), shoot height (0.39), number of roots (0.39), and root length (0.15). Shoot height was observed with the highest genotypic coefficient of variance (GCV), phenotypic coefficient of variance (PCV), genetic advance (308.29), and genetic gain (162.96). Root weight and root length had a genetic gain of 11.28% and 62.41%, respectively, along with 0.36–0.57 heritability values. A maximum and minimum significant correlation were calculated between shoot height and the number of roots (0.64) as well as root diameter and the number of roots (-0.47), respectively. Based on these analyses, seed sources with superior genetic gain for commercially imperative traits can easily be selected. Pantnagar (Uttarakhand) was found most promising seed source for the production of roots. Currently, the seed collection of A. racemosus along with other 96 plant species has been going on in the Model Nursery of Medicinal and Aromatic Plants at NAU, Navsari, India. (Patel et al. 2022). These seeds can be easily accessed by anyone interested in the cultivation/conservation of the plant. The most suitable time for Asparagus racemosus propagation is March–April, using seeds collected earlier (Bopana and Saxena 2007, 2008; Encina and Regalado 2022). 12.6.1.2 Botanical Garden Ex situ conservation takes place in botanical gardens, where whole, protected specimens are housed for reproduction and possible reintroduction into the wild. Aside from providing housing and care for endangered species, this facility provides educational opportunities as well (Maxted 2013; Jalli et al. 2015; O’Donnell and Sharrock, 2017). Only in India, more than 140 botanical gardens including universities, parks, nurseries, and agri-horticultural gardens conserve more than 250 living plants, including more than 32 species from Indian Red Data Book (Jalli et al. 2015). Conservation of medicinal and aromatic plants (MAPs) through nurseries is one of the major promotional schemes nowadays promoted by the National Medicinal Plant Board (NMPB) and various state medicinal plant boards. The core of the

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nursery is to raise the MAPs plants from the seed and through vegetative propagation to deliver excellent planting material. Model Nursery on MAP’s at Navsari Agricultural University (4 hectares) has been introduced under National Horticulture Mission, in the AES zone-III (Heavy rainfall zone). Among 117-MAPs species that have been conserved and multiplied by various vegetative as well as non-vegetative methods include 49 herbs, 21 shrubs, 11 climbers, 4 grass, and 32 tree species. Navsari MAP’s nursery also accommodated 3 species of the genus Asparagus, A. racemosus, A. sprengeri, and A. gonocladus (Patel et al. 2022).

12.6.2 Field Cultivation Filed cultivation of any plant/crop requires good knowledge related to the properties of localized soil along with annual weather structure. Proper knowledge of filed requirements and propagation specifics benefits and high yield of Shatavari. Therefore, Pathak et al. (1987) and Sharma et al. (1993) briefly studied the red, black, and mixed soil of Bundelkhand, India as well as the soil of Pune, India, respectively. High yield can be achieved by planting germinated saplings of A. racemosus (100 × 100 cm apart) on the sandy red soil of Bundelkhand with sufficient application of NPK urea. An average of 31% of seed germination and faster-growing sprouts was observed in Pune, 8–10 days after the first monsoon shower or plantation (60 × 60 cm apart). Treatment Farm Yard Manure provided better yield as compared to NPK urea, in Pune soil. Sharma et al. (1993) also considered harvesting ages of 15, 27, and 40 months, and observed a 7-times increase in root yield from 27 to 40 months of age. Economically, growing A. racemosus (in Pune) is estimated at Rs 12/kg which by calculation is profitable as compared to cash crops like sugarcane and potato. Based on the profit calculation of Sharma et al. (1993), Saran et al. (2020) proceeded with a high-density plantation (45 × 60 cm) in a 2.5 ha area of Bhavnagar (Gujarat), India, during 2016–2017, with drip irrigation system. Front-line demonstrations (FLD) of A. racemosus produced 40.8 t/ha fresh and 4.13 t/ha dry root yield after 24 months, replacing Rs. 1.6 Lakhs/ha/year outcome from cotton, groundnut, jeera, and gram cultivation. Personal interviews with pre-tested questionnaires, a simple cost accounting method, and a costs-returns comparison were used to collect primary data and examine economics and financial feasibility, respectively. Therefore, A. racemosus could be planted as a crop in sandy or stony soil for high returns per investment as compared to long-standing crops.

12.7

Genetic Conservation

Conservation of a specimen in the form of the genetic reserve comprises location, description, management, and intensive care of genetic diversity in a precise native place. Inspecting and confirming genetic stability has been an imperative facet of in vitro conservation. Molecular markers and DNA polymorphism have been listed

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as more sophisticated technology to determine the genetic stability of specimen (Maxted 2013; Rajasekharan and Sahijram 2015). These include: 1. Restriction fragment length polymorphisms (RFLP) 2. Randomly amplified polymorphic DNA (RAPD) markers 3. Single sequence repeats (SSR) markers Vijay et al. (2009) collected 7-accessions of A. racemosus from different regions of Madhya Pradesh to isolate genomic DNA and performed RAPD PCR amplification, using 6-random primers, to check genetic diversity. Amplification with 71 bands, including 39 (54.92%) polymorphic, 25 monomorphic, and 7 unique (9.85%) bands, were observed with 4 primers. Accession Ar-9 from Indiranikunj nursery and RP-3 primer presented maximum polymorphism, while the least polymorphism was observed in Ar-5 and Ar-3. Therefore, morphologically similar accession might have a difference at the DNA level. Saxena and Bopana (2009) were the first to determine clonal fidelity from randomly selected leaves from in vitro grown and acclimatized A. racemosus plants as well as from related species. The fingerprint pattern of clonal propagates was determined using Inter Simple Sequence Repeats (ISSR) PCR assay. Later, Idrees et al. (2018) developed designed SSR markers using 72,953 Asparagus nucleotide (NCBI) to determine genetic relationships among 14 species of Asparagus including A. racemosus of Pakistani regions. Di-nucleotide SSR was most abundant (13.6%), followed by tri- (2.3%), tetra- (0.84%), and penta- (0.27%) nucleotide SSR. The most and least frequent SSR were AA/TT (73.9%) and TA/TA (1.18%), respectively. A total of 143 nucleotide sequences were recognized with SSR, and 40 of them were designated to design 14 SSR primers. Amplification with 10 primers produced 156 bands including 144 (88.36%) polymorphic bands with approximately 14.4 alleles per primer. Cluster analysis based on UPGMA grouped the Asparagus species and its cultivars into two main clusters. Results of genetic similarity coefficients (0.52 to 0.94) discovered a high level of genetic distinctions among Asparagus species from Pakistan. Geetha and Siril (2022) collected 20 genotypes from 10-diverse agro-economical regions of Kerela, India to reveal genetic diversity using SSR markers. Previously developed 22 microsatellite primers for A. officinalis SSR loci showing 100% transferability in 7- and 20-accessions with 18 and 15 primers of the same, respectively. After amplification 46 alleles were recognized with 37 polymorphic loci and 16-primers showed polymorphism with 123 bp to 506 bp variation in band size. An average of 82.81% polymorphism was discovered from comprehensive and systematic inspections among these 20 accessions, with 100% polymorphic 9-primers, moderately polymorphic 7-primers, and monomorphic 2-primers. Among selected microsatellite loci 5-loci have one allele each and 4-loci revealed 6 unique alleles (using 5 primers). A comparison of genetic variation among 20 accessions proves Ar5 and Ar6 as well as Ar11 and Ar12 have the lowest (0.43) as well as highest (0.88) similarity index from the Onattukara region and Palakkad plains of Kerela,

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respectively. Based on the similarity index, A. racemosus accessions have been placed into three clusters. These results signify SSR analysis’s usefulness in demonstrating the transferability and practicality of SSR markers to characterize and analyze the genetic diversity of medicinal plants like A. racemosus.

12.8

Conclusion and Prospects

Medicinal plant conservation is one of the most pressing issues in the world today. The daily demand for medicinal plants is rising exponentially, depleting naturally occurring resources. Therefore, we must focus on conserving medicinal plants to compensate for the loss of wild plant sources, as well as meet the demand for medicinal plants. A long and rich tradition of spirituality and culture anchors the nation’s strategies and plans for the conservation, sustainable, and equitable use of biological diversity. Indian Vedic scripture describes Shatavari (Asparagus racemosus) as the “queen of herbs,” with its medicinal benefits. A supplementation of Shatavari can be used as an appetizer, a reproductive tonic, and a rejuvenating tonic, which can also improve hormonal balances and also balance weight in women. Hence, various ex situ conservation techniques are discussed above in this chapter owing to conserving Shatavari. In this chapter, we outline two very important conservation techniques: conventional and biotechnological. The conservation and sustainable utilization of medicinal and aromatic plants must necessarily involve a long-term, integrated, and scientifically oriented action program. The conservation of Shatavari can be accomplished by the utilization of ex situ methods, i.e., outside of its natural habitats, such as the establishment of gene banks, field banks, in vitro plant tissue banks, artificial propagation of plants for reintroduction into the wild, as well as the development of nurseries and home gardens. A field Gene Bank is readily accessible and useable for characterization, evaluation and crop improvement. The main limitation is that Field Gene Banks is that it takes up a great deal of space and is sometimes difficult to maintain and protect from natural disasters. As an integral part of a broader strategy for conserving and sustaining plants, seed banks are vitally important. The cost-effectiveness of seed banking technology is highly dependent on the seed biology of the target species, and the role that the collection will play in long-term conservation and ecological restoration. Seed storage remains the most cost-effective and efficient method for their conservation and sustainable use in the immediate future. Despite the ability to withstand desiccation, seeds from many species may be considered “minimally orthodox” even though their lifespan in conventional gene bank storage may not exceed many decades. Future cryobanks will likely hold a vastly greater diversity of germplasm than they do now. Though it has been found that the two latest cryopreserve methods, droplet-vitrification, and cryo-plate, are preferred for cryopreserving complex organs (e.g., shoot tips) in tropical species, the cryopreservation of explants from recalcitrant seeds remains challenging for the vast majority of species. In addition to being centers of botanical expertise, botanic gardens and arboreta are crucial links between people and plants

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and play a vital role in plant conservation. A revolution in the allocation of resources, funding, and attention will be necessary for gardens to achieve the results they need to avert the plant extinction crisis. Biotechnological methods of plant improvement have benefited greatly from plant tissue culture techniques. A multitude of applications has been made possible by these tools, both for propagating genetically engineered superior clones and for conserving valuable germplasms ex situ. Today, micropropagation and in vitro conservation have been standardized for various plant species. It is no longer an empirical science, and now it is used to unravel intricate pathways of plant metabolites and to understand molecular genomics in plants. This series of themed issues aims to provide a glimpse into current research activities concerning the conservation of plants by ex situ means. In the future, these techniques will lead to the emergence of the ex-conservation of medicinal plants and also open the doors for ignited awareness.

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Recent Developments in Natural Compounds of Guggul and Production of Plant Material for Conservation and Pharmaceutical Demand Commiphora wightii (Arn.) Bhandari

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Gulwaiz Akhter and Ghazala Javed

Abstract

Commiphora wightii (Arnott.) is a dioecious, slow-growing, balsamic tree of family Burseraceae that is primarily found in desert regions of India, Pakistan, and Bangladesh. The states of Rajasthan, Gujarat, Assam, Madhya Pradesh, and Karnataka have the suitable condition for growing C. wightii. Since ancient times, C. wightii, sometimes known as “Guggul,” has been used to treat a variety of illnesses and problems due to the presence of the steroidal component guggulsterone in the oleo-gum resin. Guggulsterone’s bioactive isomers E and Z are mainly responsible for its effects on lipid and cholesterol levels. Recent research has also revealed anticancerous properties. Guggul is a poor choice for social forestry since it takes several years to grow and blossoms slowly. This shrub is used to produce firewood and gum-resin, among other forest products. The relentless exploitation of this species and insufficient conservation efforts have led to its placement in the list of endangered plant species maintained by the International Union for Conservation of Nature. Field surveys over the past several decades have revealed a decline in its wild population. Due to improper harvesting for their oleo-gum resin, the plant dies after two to three years. This chapter summarizes the natural compound, pharmaceutical demand, and conservation and future directions of research for this important endangered medicinal plant.

G. Akhter (✉) · G. Javed Central Council for Research in Unani Medicine, Ministry of AYUSH, Government of India, New Delhi, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_13

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Keywords

Ayurveda · Commiphora wightii · Guggul · Guggulsterone · Pharmaceutical Unani

13.1

Introduction

The name Commiphora derived from the Greek word “KOMMI” (which means “gum”) and phero (means “to bear”). The oleo-gum resin of Commiphora wightii has been used as a natural substance that is effective, multi-targeted, and exceedingly safe because it is the foundation of many conventional and natural medical systems, including Ayurveda, Siddha, Unani, and Chinese medicine (Barve and Mehta 1993; Shishodia et al. 2008; Harsha et al. 2017). In Ayurveda, the oleo-gum known as “Guggul” is used to address a variety of illnesses, including obesity, lipid metabolic abnormalities, bone fractures, arthritis, and inflammation. Guggal (oleo-gum resin) was reported to have therapeutic and regenerative qualities in the Sushruta Samhita, an ancient Ayurvedic text from 3000 years ago. The anticancer and antioxidative properties have also been explored to fight cancer (Kumar et al. 2004; Hanuš et al. 2005; Harsha et al. 2017).

13.2

Distribution

Commiphora wightii (Arnott.) Bhandari (Syn. C. mukul, Balsamadendron mukul) belongs to the family Burseraceae, is primarily grown in arid regions of Pakistan, Bangladesh, and India. Rajasthan, Gujarat, Assam, Madhya Pradesh, and Karnataka are among the Indian states that have it (Barve and Mehta 1993; Deng 2007; Kant et al. 2010).

13.3

Current Status

The plant is in danger due to resin exploitation, weak plant growth, and inadequate seed germination. Guggul is a poor choice for social forestry since it takes several years to grow and blossoms slowly. This shrub is used to produce firewood and gum-resin, among other forest products. The relentless exploitation of this species and insufficient conservation efforts have led to its placement in the list of endangered plant species maintained by the International Union for Conservation of Nature. Field surveys over the past several decades have revealed a decline in its wild population. Due to improper harvesting for their oleo-gum resin, the plant dies after two to three years.

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13.4

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Biology

It grows as a short, bushy tree with prickly branches that secrete a yellow-colored gum resin known as guggul through tiny channels spread throughout its bark. Incisions in the bark are made to access the trees. Before being collected, the resin, which oozes out, is given time to solidify. From November to January, the tree is tapped, and from May to June, the resin is gathered. Each harvesting season, the guggul tree produces between 700 and 900 g of dried resin (Deng 2007; Yamada and Sugimoto 2016). Oleo-gum resin can be found in the balsam canals at the stem base and the large leaf veins of the plant.

13.4.1 Chemistry of Gum-Resin The yellowish exudates from the trunk of C. wightii contain a large number of compound like aliphatic esters, gum (carbohydrates) steroids, diterpenoids, and minerals (Table 13.1) (Ramawat et al. 2008). However, the presence of guggulsterones in resin of guggul makes significant differences from other 184 species of genera, Commiphora (Schauss and Munson 1999. In addition, Boswellia serrata, a different tree in the Burseraceae family, produces a gum resin known as “Salai guggul” or “white guggul” that lacks guggulsterones. Oleo-gum resin of C. wightii is a composite mixture of sterols (guggulsterol- I, II, III, IV, V), sterone (Z, E, M-guggulsterone and dehydroguggulsterone-M), essential oils, flavanones, ferrulates, gum, and lignans. Various bioactive compounds like diterpenoids, fatty tetrol esters, triterpenoids, steroids, and lignans are also found in ethyl acetate soluble fraction (Kant et al. 2010). Oleo gum-resin contains 1.45% volatile oil, 3.2% organic foreign matter, 19.5% minerals, 38.5% resin, 32.3%gum, and other impurities. Further separation results in 95% neutral, 4% acidic, and 1% basic fractions based on the pH gradient. During the separation, a ketonic compound was said to have been present in the neutral fraction up to 5.13%. Z-guggulsterol (0.01%), guggulsterol-VI (0.02%), Z-guggulsterone (1.6%), E-guggulsterone (0.4%), guggulsterol III (0.03%), guggulsterol-I (0.8%), guggulsterol-IV, guggulsterol-V, and some defense-related secretory ketones are among the biologically significant active principles of C21 or C27 found in the neutral fraction (Patil et al. 1973; Purushothaman and Chandrasekharan 1976; Prasad and Dev 1976). Purification of oleo-gum resin includes 45% soluble and 55% insoluble components with the help of ethyl acetate, alcohol, or petroleum-ether. The toxic effects are reported due to insoluble fraction; moreover, the soluble fraction contains the guggulsterones and other chemical constituents that are of biological importance, viz. anti-inflammatory. A method of high performance liquid chromatography (HPLC) was developed for quantification of E- and Z guggulsterones in C. wightti resin (Mesrob et al. 1998). Details of the separation of various components from the resin are discussed In review by (Dev 1987). Using a different method, Meselhy (2003) separated guggulsterone and extracted guggulsterone-M using column chromatography. The most significant bioactive fraction is the ketonic component, which

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Table 13.1 Shoot Bud induction Class Sterols

Structure

Reference Bajaj and Dev (1982)

Purushothaman and Chandrasekharan (1976)

Benn and Dodson (1964)

Dev (1987)

(continued)

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Table 13.1 (continued) Class

Structure

Reference Dev (1987, 1999)

Dev (1987, 1999)

Dev (1987, 1999)

Guggulsterol Y

Kimura et al. (2001)

Steroids

Dev (1987); Meselhy (2003),

Guggulsterone M

(continued)

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Table 13.1 (continued) Class

Terpenoids and alcohols

Structure

Reference Dev (1987); Meselhy (2003),

Kimura et al. 2001

Patil et al. (1973) Kimura et al. (2001)

Prasad and Dev (1976)

Asres et al. (1998)

(continued)

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Table 13.1 (continued) Class

Structure

Reference Asres et al. (1998)

Shah et al. (2012)

Bajaj and Dev (1982)

Zhu et al. (2001a)

Zhu et al. (2001a)

(continued)

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Table 13.1 (continued) Class

Structure

Reference Shah et al. (2012)

Sarup et al. (2015)

Lignans

Dev (1987); Chadha et al. (1970); Hanuš et al. (2005)

(continued)

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Table 13.1 (continued) Class

Structure

Reference Dev (1987); Chadha et al. (1970); Hanuš et al. (2005)

accounts for 12% of the ethyl acetate-soluble fraction and is composed of roughly two dozen different substances, including sterols, guggulsterone-E, and guggulsterone-Z. Other plants and lower animals are known to contain guggulsterone, guggulsterol, and its derivatives (Table 13.2). These substances aid in insect molting and aid in protecting the insects from predators (Dev 1987) (Patil et al. 1973). Two isomers, guggulsterone-E and guggulsterone-Z, in callus and cell cultures of the plant are grown in our laboratory under various conditions. Similarly, guggulsterone-E was transformed into guggulsterone-Z and various other derivatives by fungus (Aspergillus niger, Cephalosporium aphidicola) in in vitro (Dev 1999). Table 13.2 Guggulsterone and related compound found in other medicinal plants and animal species Molecule Guggulsterol III

Source Mediterranean gorgonian Leptogorgia Sarmentosa

Guggulsterone

Ailanthus grandis

Z-guggulsterol and derivatives Z-guggulsterol and derivatives Z-guggulsterol and derivatives Z-guggulsterol and derivatives

Defensive secretion of Dytiscus Marginalis Acitus Sulcatus Prothoracic defensive gland secretion of Cybister tripunctatus, Ilybius fenestratus Bark of Grewia tiliifolia Vahl

Reference Chadha et al. (1970) Benvegnu et al. (1982) Hung et al. (1995) Chadha et al. (1970) Schildknacht (1970) Kuruvilla and Anilkumar (2020)

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Essenr al oil Foreign , 1% Resin , 38%

Miner als , 20%

Maer, 5%

Gum, 32% other, 4%

Gsterol IV, 1%

GS-Z, 56%

b

GS-E , 14% Gsterol I, 28%

Gsterol III, 1%

a

Gsterol , 0%

Ketonic , 12%

Non-ketonic , 88%

c

Fig. 13.1 (a–c) Chemical composition of oleo-gum resin and ketonic fraction containing guggulsterones. G guggul, GS-E guggulsterone, EGS-Z guggulsterone-Z

When the neutral fraction is further fractionated, it yields fractions that are 88% non-ketonic and 12% ketonic. The ketonic fraction was used to produce several steroids, including the two isomers E-(cis-) and Z-(trans-) GS [4, 17 (20)pregnadiene-3, 16-dione]. The GS contains 2% gum guggul and nearly 5% guggulipid by weight (Deng 2007; Shishodia et al. 2008; Harsha et al. 2017; Kunnumakkara et al. 2018) (Fig. 13.1).

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13.4.2 Gum-Resin Production The resin canals develop schizogenously in the young stem and the stem is tapped from February to June for gum Ramawat et al. (2008). Plants with a basal diameter of at least 7.5 cm and at least 5 years old are suitable for tapping. Circular incision up to 1.5 cm deep is made on the main stem and branches at uniform distance of 30 cm apart and at an angle of 60° to the stem. Through the cuts, the golden, fragrant latex leaks out and gradually solidifies into vermicular or stalactitic bits that are carefully removed. Gum resin is thereafter collected at intervals of 10–15 days. Typically, a plant yields 200–500 g of dried guggul in a single growing season. However, the production of guggul is increased 22-fold when ethephon is applied to the incisions compared to the control condition. Conversely, prolonged use of ethephon exhausts the plant and eventually causes its death due to excessive production.

13.4.3 Economic Importance In addition to its use in medicine, guggul also plays important role in the incense and perfume industries (Barve and Mehta 1993; Patil and Belge 2019). Many multinational companies like Aie Pharmaceuticals Inc., Lorience, Gamble, Proctor, and Unilever have been cherished by preparing detergent, lotions, cream, and perfumes (Patil and Belge 2019). In food industries, oleo-gum resins are extensively used for making beverage, chewing gums, candies, gelatine, nut product, and confectioneries (Siddiqui et al. 2013; Al-Bishri and Al-Attas 2013). Swami et al. (2022) revealed immense biological properties of guggul due to secondary metabolites known as phytochemicals and identified to use in different therapeutic actions. The United States Food and Drug Administration has authorized the usage of myrrh (Myrrh is a gum-resin extracted) for food products (21 Code of Federal Registration-CRF 172.510); however, the European council included myrrh in the list of plants and is acceptable as food (Lemenith and Teketay 2003. Tadesse et al. (2007) found the repellent properties for flies, mosquito, and termite as incense stick.

13.4.4 Pharmaceutical Importance The therapeutic and medical benefits of guggul have been used for human since long back. Its intriguing past includes the Indian Vedas, an old classic text, Shishodia et al. (2008) and Ramawat et al. (2008). The well-known Ayurvedic literature Sushruta Samhita discusses the therapeutic applications of oleo-gum resin, produced from the tree C. wightii, for conditions like obesity and problems with lipid metabolism (Urizar and Moore 2003). C. wightii was valued as much as gold and mentioned in numerous verse in The Old Testament in Biblical time. In Judaism prophet Moses was commanded to make incense and anointing oil has reported. The Greek soldiers do not go to the battle without a poultice of myrrh to apply as a dressing on their wounds, Hanuš et al. (2005). Nowadays all human civilization be it Indian, Greek,

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Chinese, Egyptians, Arabs, and Romans are benefited from the C. wightii as natural remedy (Lemenith and Teketay 2003). In 1966, Indian researcher, Satyavati figured out valuable medicinal properties of guggul based on ancient Ayurvedic text. She found the hypolipidemic activities on rabbit. Thereafter, successful experiments were conducted and ayurvedic formulations were approved for marketing purpose as hypolipidemic drug (Deng 2007; Siddiqui 2011). Many guggul-based ayurvedic products are currently available for purchase in India, including Yograj Guggul, Laksha Guggul, Kaishore Guggul, Gokshuradi Guggul, and Kanchanara Guggul, Panchamritalouha, Panchatikta, Punarnava, Simghanada, Trayodasanga, and Triphala. Among these, crucial herbal remedies include “Laksha guggul” alleviate joint and bone fracture pain and is also used to treat heart problems, malnutrition, and vitiligo; “Kaishore Guggul,” another herbal remedy, is taken as a dietary supplement for blood purification and anti-allergic effects, and additionally helpful for arthritis and diabetes (Lather et al. 2011). The removal of toxins from the body for the maintenance of body physiology and metabolism is one of the common treatments for rheumatoid arthritis known for Yograj Guggul. In old age, Triphala Guggul is used for piles and Vatari guggul is used backache (Pradhan and Pradhan 2011).

13.4.5 Anti-inflammatory and Antioxidant Guggul from the C. wightii plant has been shown to have anti-inflammatory activities in osteoarthritis by Arora et al. (1971), Sharma (1977), and Singh et al. (2001). According to Kar and Panda (2003), guggulsterone components have antioxidative and triiodothyronine-promoting properties as well as anti-cancer properties. In rats, guggulsterone’s Z-isomer has stronger cardioprotective and antioxidant effects than its E-isomer. Z-guggulsterone markedly increased thyroid hormone production and free radical scavenging capacity, Chander et al. (2003). The anti-inflammatory and antioxidant activities of oleo-gum resin were first demonstrated over 50 years ago, Shishodia et al. (2008). Deng (2007) guggulsterone prevents hypercholesterolemia due to anti-inflammatory and antioxidative properties. Shishodia et al. (2008) reported that guggulsterone inhibited the tumor cell proliferation, arrest S-phase and promotes apoptosis by activation of c-Junterminal kinase, suppression of Akt pathway and downregulated of anti-apoptotic gene expression. Goyal et al. (2011) found that the gugulipid in proniosomal gel increased entrapment efficiency which allows the faster release of the drugs long term anti-inflammatory activities in rate.

13.4.6 Cardioprotective Effects Many authors have emphasized the cardioprotective benefits of guggul resin (Kimura et al. 2001; Deng 2007). The steroid guggulsterone found in guggul has cardioprotective properties. In the study, it was found that pre-inducing myocardial

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necrosis in rats with isoproternol led to a considerable increase in the blood serum levels of SGPT and creatine phosphokinase (glutamate pyruvate transaminase). Following a sharp decrease in the levels of cholesterol, phospholipids, and glycogen, additional parameters such as phospholipase, lipid peroxides, and xanthine oxidase were simultaneously raised in the pathological manifestation of ischemic heart state. In rats with pre-induced ischemia, oral administration of guggulsterone components (50 mg/kg) considerably reduced damage to the cardiovascular system as indicated by the process of blood flow reversal and biochemical cardiac parameters Chander et al. (2003), Zhu et al. (2001b). The heart-protective properties of guggul resin have been noted by numerous publications (Kimura et al. 2001; Deng 2007). The human dietary system must include phenolic chemicals, which are also significant because of their cardioprotective characteristics (Manach et al. 2004; Balasundram et al. 2006; Randhir et al. 2004). Guggulsterone’s Z-isomer exhibits stronger cardioprotective and antioxidant action in rats than the E-isomer, according to Al-Howiriny et al. (2005). Additionally, it was found that Z-guggulsterone enhanced both the activity of free radical scavengers and the synthesis of thyroid hormone. Hypotensive and cardiovascular effects studies conducted in the lab revealed that the lipopolysaccharide (LPS)-activated murine macrophage J774 cells fraction of the methanolic/ethanolic extract acts as a powerful inhibitor of nitric oxide (related with rheumatoid arthritis and cardiovascular disorders) generation. Shah et al. (2012) studied the Z- and E-guggulsterones, which are biologically active components and discovered that nuclear receptors modulate the expression of proteins with carcinogenic properties. They discovered that guggulsterones are also known to modulate other molecular targets, such as steroid receptors, nuclear factor (NF)-B, and signal transducer and activator of transcription (STAT) factors, which are transcription factors. Inflammation, neurological disorders, hyperlipidemia, and related cardiac disorders like hypertension and ischemia, skin disorders, cancer, and urological problems can all be treated with gum guggul, according to a large body of scientific research. Gupta et al. (2022), who conducted a more recent research, suggest that guggulsterone may be used to treat malignancies, urinary diseases, and skin conditions. The expression of proteins involved in carcinogenic activity is modulated by guggulsterone. It exhibits its multi-level and multi-targeted pleiotropic effects by acting on a variety of molecular targets, including transcription factors like nuclear factor, signal transducer, and activator of transcription and steroid receptors.

13.4.7 Inflammatory Bowel Disease Chronic inflammatory diseases of the gastrointestinal system are grouped together as inflammatory bowel disease (IBD). Both Crohn’s disease (CD) and ulcerative colitis (UC) are symptoms of this persistent, progressive disease of the colon and intestines (Mencarelli et al. 2009). Guggulsterone has anti-inflammatory properties, so Cheon et al. (2006) used experimental models of murine colitis and intestinal epithelial cells to study the molecular mechanism underlying its efficacy in the treatment of IBD.

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On production of lipopolysaccharides or IL-1b, guggulsterone inhibited the expression of the intercellular adhesion molecule (ICAM)-I gene. IKK activity was prevented by repressing IkB phosphorylation activation. The transcriptional activity of NF-kB was also reduced by its binding to the DNA elements in the nucleus. The severity of the mice’s dextran sulfate sodium (DSS)-induced murine colitis significantly decreased after guggulsterone administration, according to a clinical evaluation. These results suggested the use of guggulsterone in targeting the IKK complex to obstruct the NF-kB signaling pathway and attenuation of murine colitis could be looked upon as a therapeutic treatment of IBD (Cheon et al. 2006). In mice models of inflammation induced by oxazolone and trinitrobenzene sulphonic acid (TNBS), guggulsterone’s anti-inflammatory action has recently been studied. The inhibition of interleukin production and T cell proliferation by guggulsterone raises the possibility that it could be used as a treatment for colitis (Mencarelli et al. 2009).

13.4.8 Hypolipidemic Activity A well-known anti-hyperlipidemic medication, according to Vyas et al. (2015), is guggul. Guggul samples that had just freshly collected and those that were 1 year old were processed in gomtra. Patients who satisfied inclusion criteria of hyperlipidemia were divided into two groups at random and given the medication twice daily in a dose of 1 g with lukewarm water for 8 weeks. With treatment, both groups’ symptoms of medoroga and lipid profiles significantly improved. Fresh samples of Guggul had a stronger effect on lowering serum cholesterol, triglyceride, and very low density lipoprotein levels, while older samples of guggul had a less effect on lowering blood triglyceride VLDL and a non-significant increase in serum HDL-cholesterol. Additionally, the body weight and BMI significantly decreased in the older guggul sample.

13.4.9 Antifertility Activity When guggul was given orally to female rats weighing 100 g and 2 and 20 mg, antifertility action was shown. Guggul may be useful as an antifertility agent because the weight of the uterus, ovaries, and cervix dropped, while the amounts of glycogen and sialic acid in these organs increased (Azharhusain et al. 2022; Radheshyam et al. 2022).

13.4.10 Cytotoxic Activity Ferulates, substantial bioactive components contained in the resin, were discovered to play a significant impact in in vitro cytotoxicity (Chaudhary 2012). According to Zhu et al. (2001b), ferulate chemical is utilized in various ways to prevent and treat aberrant cell growth and proliferation associated with inflammation, neoplasia, and

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cardiovascular disease. Ethyl acetate extract displayed notable cytotoxicity. Additionally, they stated that a fraction of the ferulates exhibited cytotoxic and scavenging activity. According to Xiao et al. (2011), administration with gugulipid to the androgendependent LNCaP human prostate cancer cell line and its androgen-dependent variation (C-81) with IC 50 of M (24 h treatment) dramatically decreased the viability of the cell line, confirming its cancer-preventive apoptosis. The findings of this study showed that whereas a normal prostate epithelial cell line is resistant to growth inhibition and apoptosis induction by this hormone ingredient, guggulsterone decreased proliferation of PC-3 cells in culture by inducing apoptosis. These findings justified additional preclinical and clinical testing of guggulsterone for its effectiveness against prostate cancer.

13.5

Conservation Strategies

Unfortunately, due to its slow growth rate, poor seed germination rate, intensive agriculture, lack of cultivation, excessive urbanization, and unprofessional gum resin harvesting by pharmaceutical industries, the plant C. wightii has become threatened. This problem is more acute in arid region of Northwest India wherein plant supports numerous livelihoods and serves as sources of food, fuel, fiber, timber, and medicine and functions as an integral part of local agricultural production systems. With the help of community engagement, Soni (2010) made major efforts to educate the Aravalli hills’ rural and tribal residents about the value of and need to protect C. wightii. Commiphora wightii should be preserved in its natural environment (in situ protection) by creating networks of protected areas. Several themes occur in numerous collections of suggestions that have been brought together about the protection of guggul. A thorough analysis of various natural populations in areas where the plant is endangered must be part of the conservation strategy for C. wightii. This analysis must be followed by physio-morphological and molecular characterization of these populations to identify their active constituents. For ex situ conservation, it is important to record and reproduce the climatic conditions, edaphic components, and ecological associations for the places where the plant thrives. However, the species’ gene pool may also be preserved in its natural environment for the growth of genetic variants and extension of the genetic base that would otherwise be lost due to overexploitation for commercially available pharmaceutical preparations. It is necessary to gather and store the available seeds from various populations for later use (Fig. 13.2).

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Fig. 13.2 Conservation/cultivation strategy for Commiphora wightii

13.6

Conclusion and Future Prospects

The Commiphora wightii can be found in India, Bangladesh, and Pakistan. It is present in the states of Gujarat, Assam, Madhya Pradesh, Rajasthan, and Karnataka. Increasing demand and ruthless exploitation of this plant have created a serious problem. No significant efforts have been made to improve the status of the plant through selection and conventional breeding methods. The wild population of C. wightii has declined by more than 80% over the past 84 years (three generation duration) as a result of habitat loss and degradation along with uncontrolled oleogum resin harvesting and tapping. As a result, this species is considered to be critically endangered. Some of the important issues must need attention, comprise conservation and multiplication more rigorously, improved scientific tapping processes, pest and disease and in vitro production of guggulsterone. The micropropagation and somatic embryogenesis techniques for bulk production of plant material may play a significant role. However, these techniques have many limitations including, explants yellowing and death during culturing, slow growth, low rooting rates, low field establishment rates, asynchronous development and low conservation rates.

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Guggul is susceptible to various types of pathogens and climatic restraints during cultivation like other perennials plants. Therefore, development of genetically engineered plants proficient to counter such abiotic and biotic stresses is important. Conversely, it is necessary to understand the guggulsterone biosynthesis process in greater depth in order to create transgenes with higher guggulsterone levels. The availability of a broad genetic base is essential to initiate the breeding programs in guggul crop. The superior quality of genetic material, with a significant level of guggulsterone content to characterize so that conservation efforts can be linked. The available genetic base may be expanded by using modern biotechnology tools, including in vitro selection of somaclonal variations and mutagenesis and transgenic. Oleo-gum resin from C. wightii has a significant medicinal and therapeutic potential, which has resulted in uncontrolled, ruthless exploitation of the plant resources in areas of its natural distribution. Without a question, this species, C. wightii, has a bright future ahead of it. Therefore, it is necessary that the valuable germplasm be protected and conserved. No conservation efforts will be successful without the involvement of local people, villagers, and forest developers. Save guggul project, started in Rajasthan, is one of the good examples of conservation of guggul plant. As part of this program, several education campaigns regarding the value and preservation of guggul plants are being conducted throughout Rajasthan (Soni 2010). Additional scientific methods are also required to prevent damage to the plant after tapping. If systematic reforestation efforts are implemented in the area, C. wightii has the potential to enhance the ecological and economic growth of the local population. More extensive research are needed to explore the important qualities of C. wightii. The world can gain more from humankind if C. wightii resources are exploited and used wisely. Acknowledgments Central Council for Research in Unani Medicine are gratefully acknowledged by the authors.

References Al-Bishri WM, Al-Attas OS (2013) Guggul resin extract improve hyperglycemia and lipid profile in streptozotocin induced diabetes mellitus in rats. Life Sci J 10(1):2735–2741 Al-Howiriny T, Al-Sohaibani M, Al-Said M, Al-Yahya M, El-Tahir K, Rafatullah S (2005) Effect of Commiphora opobalsamum (L.) Engl. (Balessan) on experimental gastric ulcers and secretion in rats. J Ethnopharmacol 98(3):287–294 Arora RB, Kapoor V, Gupta SK, Sharma RC (1971) Isolation of a crystalline steroidal compound from Commiphora mukul and its anti-inflammatory activity. Indian J Exp Biol 9(3):403–404 Asres K, Tei A, Moges G, Sporer F, Wink M (1998) Terpenoid composition of the wound-induced bark exudate of Commiphora tenuis from Ethiopia. Plantamedica 64(05):473–475 Azharhusain SM, Shrivastava B, Quazi A, Shaikh MAJ, Patwekar M (2022) A review on guggul [Commiphora wightii (Arn.) Bhand.], its phytochemical constitution and mode of action. Int J Ayurveda Pharma Res:74–79 Bajaj AG, Dev S (1982) Chemistry of Ayurvedic crude drugs—V: Guggul (resin from commiphora mukul)—5 some new steroidal components and, stereochemistry of guggulsterol-I at C-20 and C-22. Tetrahedron 38(19):2949–2954

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Assessment of Economically and Medicinally Important Plant Resources in Sangla Valley Region of Indian Himalaya

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Usha Devi, Pankaj Sharma, J. C. Rana, R. Murugeswaran, Anees Ahmad, and Asma Sattar Khan

Abstract

The current study was assessed with the aim to explore the most important plant resources growing in the Sangla Valley region of the Indian Northwest Himalaya. Data were collected during 2011–2017 at 1800–4600 m altitudes to the assess plant resources, their pre-existing traditional information and documentation of ethnomedicinal utilization. In this study, a total of 320 important plant species of 202 genera belonging to 70 families were reported. Of these, angiosperms were found as a dominant group consisting of 302 species, 192 genera, and 63 families. Whereas gymnosperms having 13 species, 7 genera, and 4 families and represented as a sub-dominant group. Conversely, pteridophytes contain a minimum number of 5 species belonging to 3 genera and 3 families. All these reported plants were distributed in different life forms like trees (29 spp.), shrubs (42 spp.), and herbs (249 spp.). Out of 70 families of angiosperm, Asteraceae (49 spp.), Rosaceae (20 spp.), Apiaceae (18 spp.), Ranunculaceae (18 spp.), and Fabaceae U. Devi (✉) CCRUM-Drug Standardization Research Institute (DSRI), PCIM&H Campus, Ghaziabad, India ICAR-National Bureau of Plant Genetic Resources, Shimla, India P. Sharma ICAR-National Bureau of Plant Genetic Resources, Shimla, India J. C. Rana ICAR-National Bureau of Plant Genetic Resources, Shimla, India Bioversity International, New Delhi, India R. Murugeswaran National Medicinal Plant Board, Ministry of AYUSH, Government of India, New Delhi, India A. Ahmad · A. S. Khan CCRUM-Drug Standardization Research Institute (DSRI), PCIM&H Campus, Ghaziabad, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_14

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(14 spp.) families were found as dominant. Of which, 25 families were recorded as a monotypic represented by only a single species. Out of 202 genera, Artemisia (7 spp.), Saussurea (6 spp.), Anaphalis, Berberis, Rosa, Thalictrum (5 spp. each) and Bupleurum, Geranium, Juniperus, Lactuca, Nepeta, Persicaria, Poa, Polygonum, Potentilla, Salix (4 spp. each) were recorded as dominant genera. Other 14 and 34 genera represented only 3 and 2 species, respectively, and remaining 138 genera were represented only a single species. Out of 320 species, 182 were native to the Himalayan region and the remaining were non-natives as they were from different biogeographic domains of the world. Many plant species were recorded as endemic (45 spp.) and near endemic (185 spp.). Whereas 5 critically endangered, 11 endangered, and 10 vulnerable species were recorded. Consequently, the presented data reflects the significance of the Sangla Valley region with great diversity of medicinal plant resources. Keywords

Bioresource · Conservation · Diversity · Endemic · Medicinal Plants · Sangla Valley

14.1

Introduction

World Health Organization (WHO) has evaluated about 80% of the population of developing countries basically depends on herbal drugs. Among them, approximate 25% of raw drugs are obtained from natural plant resources (Ahmad et al. 2018). Recently, many countries, including India are facing serious problems in conserving medicinally or economically valued plant resources because they are continuously exploited. Anthropogenic activities and climate change also affect many plant resources. Besides this, medicinal plants are key resources of traditional medicine system and pharmaceutical industries. This resource would provide a big revenue and health security for poor communities in many developing countries. The Himalayas are a big mountain range in Asia continent. They are separating the plains of the Indian subcontinent from the Tibetan Plateau. The Himalayas are home to a big diversity of medicinal plants. Its stretches over nearly 3000 km, almost from the borders of the North of Burma in the East to Afghanistan in the West (27°–36° N, 72°–91° E) and they connect the mountains of the Near East and Central Asia with those of East Asia. The Himalaya Mountain has many sub-ranges and extensions such as the Karakoram, the Hindu Kush, and the Pamir. This mountain encompasses parts of Afghanistan, Pakistan, India, Burma, and China, as well as all of Bhutan and Nepal (Allison 2012). Due to its distinctive biological, geo-hydrological, esthetic, social, and cultural beliefs, it is regarded as the lifeline of the Indian subcontinent (Johnsing et al. 1998). The geo-dynamically young mountains are vital for climate because they are provider of life and water to a large part of the Indian subcontinent. Also, they are a big harbor of flora, fauna, and cultural diversity (Singh 2006). This may be due to its

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unique topography, diverse habitats, and a large altitudinal range. Uncontrolled harvesting of fuel wood, fodder, grazing, and non-timber forest products (NTFPs) may be the most widespread pressure on forest resources because most of rural communities significantly depend on household and livelihood needs (Misra 2010; Malik et al. 2016; Jiju et al. 2021). Additionally, the old traditional value directly attached to the forest and diverse forest products has attained immense significance, especially in the Himalayan region (Kandari et al. 2014). According to Kumar et al. (2011) the Indian Himalayan Region (IHR) barely occupies 15% of the country’s geographical area; however, it possesses a unique variety of the world’s mountainous habitats. Singh and Hajra (1996) suggested that this ecosystem alone supports about 18,440 plant species, out of which approximately 45% of these have therapeutic value. Because of their excellent multipurpose properties such as wild edible, fuel, fodder, fibers, spices, dyes, timber, agriculture tools, esthetic, religious, etc., these plant resources have been utilized by the several native communities of the region (Samant and Dhar 1997). Numerous studies have suggested that the Indian Himalayan Region is home to a wide range of plant species, including 675 species of wild edibles, 1748 species of medicinal plants,155 species of sacred belief, 118 essential oil yielding medicinal plants, and 279 fodder plants (Samant and Dhar 1997; Samant et al. 1998; Samant and Palni 2000). About 643 medicinal plants were listed in the northwestern Himalayan region, of which 374 non-natives, 269 natives, 17, endemic and 131 are near endemic (Samant et al. 2007). Generally, native plant species play an important role in conserving local ecosystem. However, the rapid explosion of human population, climate change, and alien species are main causes for increase pressure on the survival of the native species (Levine et al. 2003; Serrill 2006; Ahmad et al. 2022). The local elimination of any species from natural habitat may cause biodiversity reduction and alteration of natural ecological processes. In previous studies it has been clearly observed that a large number of scientific studies on ethnobotany and pharmacology have been conducted by many researchers especially in the Northwest Himalayan region or Himachal Pradesh. For instance, many researchers prepared a checklist of vascular plants in Sangla Valley and Kinnaur region of Himachal Pradesh (Chawla et al. 2012; Devi et al. 2014). Devi and Thakur (2011) studied the ethnobotany of wild plants from cold desert of Himachal Pradesh. Sharma and Devi (2013) reported the Ethnobotanical uses of bio-fencing plants in Himachal Pradesh. Another study by Lal and Prasher (2016) discussed conservation practices of plant resources with the help of studying indigenous knowledge in Kinnaur district, whereas Bhardwaj et al. (2020) studied the ethnomedicinal plants diversity of Kinnaur district. Whereas Prakash et al. (2020) described the wild edible resources tribal community (Pangwal) in Pangi valley. Prakash et al. (2022) described the ethnoveterinary medicines used by the tribal migratory shepherds of Northwestern Himalaya. However, Dutt and Negi (2007) and Sharma et al. (2014) gave only phytosociological account and distribution pattern of vegetation in the Sangla Valley of Himachal Pradesh. But all the studies showed lack of distribution patterns, biogeographic affinities of plant resources in the Sangla Valley. Therefore, it becomes imperative to study on a

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database of the economically important species of the Sangla Valley region and to identify the issues related to stakeholder interaction with natural plant resources in view of the loss of biodiversity for its sustainable utilization.

14.2

Materials and Methods

14.2.1 Study Area Sangla Valley (31°10′01.00′′–31°30′17.16′′N Latitudes and 78°10′26.52′′–78°52′ 41.75′′E Longitudes) is commonly known as ‘Baspa Valley’ or ‘Baspa Basin’. This valley is located in the southeast corner of Kinnaur District of Himachal Pradesh (Fig. 14.1). Deota et al. (2011) reported that Baspa River is a major tributary of the Satluj river which starts from near the Indo-Tibetan border and forms a valley from Chitkul (3475 m) to the junction with the Sutlej River at Karchham (1770 m). The Sangla Valley is highly glaciered and located in the higher-altitude range. After winter season this valley is mostly contributed by snow melt rather than rainfall. In this region the climate varies from dry, temperate to alpine. The vegetation of this region is mainly of temperate, alpine, and sub-alpine types. The forests of Sangla Valley are dominated by broadleaved and coniferous species. The economies of the local folks are based on agriculture, cattle rearing, and several less important activities. Most of the population engaged in traditional farming involving few

Fig. 14.1 Study area

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animals. Transhumance of cattle and sheep are particularly important activity in the area. The Sangla Valley region is mainly surrounded by big mountains with an average height of 5480 m and the lowest point (1800 m) lies at the entrance to valley in Karchham. Srikantia and Bhargava (1998) reported that these mountains are primarily composed of different matters such as quartz schist, carbonaceous slates, garnetiferous schists, phyllite, quartzite, and lenticular limestone. The valley inhabited by indigenous tribes “Kinnauri” community. They are Hindu, Buddhist, or mixture of the two. This region is inhabited by people of different villages like Sangla, Kamru, Kuppa, Rakchham, Chanshu, Chhitkul, Boring-Saring, Kupa, Batseri, etc. Local communities of this remote Himalayan region have very close relationship with their adjacent plant resources due to their direct dependency on plant-based natural products for their food, fodder, health, shelter, fuel wood, and many other cultural purposes. In socio-economic terms, the Sangla Valley is important as many hydropower stations are being planned in this basin and a few were already functional (e.g., 300 MW Baspa Stage II Hydroelectric Project). Economically as well as environmentally forests are the most significant natural resource in Sangla Valley. Also, the forest ranges in the catchment are protected forests which cover more than 40.66 km2 area. Due to the heavy reliance on natural resources, the plant resources of the Sangla Valley were under too much stress.

14.2.2 Survey, Sampling, and Data Collection The present study was based on field surveys conducted in the Sangla Valley along an altitudinal gradient from 1800 to 4600 m during year 2011–2017. The information on economical plant species were collected through interviews, semi-structured questionnaire and discuss with local tribal people of the valley. Local inhabitants were inquired about the indigenous uses of the native flora and their occurrence in the area. There are many species of medicinal plants of the study area which are used by the inhabitants of other parts of IHR and their pre-existing traditional information was also included under the category of medicinal species (Jain 1991; Kala 2006; Khare 2007; Samant and Palni 2000; Samant et al. 2001, 2007). Most of the species were identified on the same sites but some non-identified samples were brought to the National Bureau of Plant Genetic Resources (NBPGR), Shimla (Himachal Pradesh) and were identified with the help of various regional floras already developed by many botanists (Aswal and Mehrotra 1994; Dhaliwal and Sharma 1999; Murti 2001) and authenticated with specimens lying with the herbarium (BSD) of Botanical Survey of India (BSI), Dehradun. In the present study, Angiosperm Phylogeny Group (APG) IV classification system was adopted for documentation of economically important plant resources found in the Sangla Valley region. The nomenclatures of these plants were updated with the help of The World Flora Online (WFO). All these identified families were described alphabetically and depicted in Table 14.2. Furthermore, the plant species restricted to the Indian Himalayan Region were considered as endemic and the species having the Himalayan origin has been considered as natives. Whereas those species that have an extended distribution to

264

U. Devi et al.

neighboring Himalayan countries, including China-Tibet Province, Bhutan, Nepal, Pakistan, Afghanistan, were considered as a near-endemic species. The endemic category of plant species for the Indian Himalayan Region was assessed by following Nayar (1996) method. Whereas threatened status of the species was assessed by following IUCN criteria (Ved et al. 2003). All the climber species were considered as a shrub while fern a herb. Data compiled and analyzed for species diversity, distribution pattern, indigenous uses and nativity (Anonymous 1970; Samant et al. 1998).

14.3

Results

14.3.1 Species Diversity and Distribution The complete enumeration of vascular plants in the Sangla Valley revealed 639 plant species belongs to 321 genera and 99 families (Devi et al. 2014). In the present study, we recorded 320 species of plants belonging to 202 genera and 70 families. Of these, Angiosperms were a dominant group having 68 families, 190 genera and 302 species, followed to Gymnosperms (4 families, 7 genera, and 13 species) and Pteridophytes (3 families, 3 genera, and 5 species). All these plant species were spread in different life forms such as 29 tree species, 43 shrub species, and 248 herb species (Tables 14.1 and 14.2). In case of angiosperm, 49 species of Asteraceae, 20 species of Rosaceae, 18 species of each Apiaceae, Ranunculaceae and 14 species of Fabaceae were found as dominant families. However, about 25 families were reported as a monotypic in nature and they have represented by only a single species. Among 202 genera, 07 species of Artemisia and 06 species of Saussurea, 05 species of each Anaphalis, Berberis, Rosa and Thalictrum and 04 species of each Bupleurum, Geranium, Juniperus, Lactuca, Nepeta, Persicaria, Poa, Polygonum, Potentilla and Salix were recorded as dominant genera. Consequently, the occurrence of 320 species of economically important medicinal plants of the Sangla Valley region indicates own suitable natural environmental condition, particularly shady moist and forest habitats are most favorable for the growth and development. The current study was carried out at altitudes ranging from 1800 to 4600 m. Considering species with different altitude ranges, a maximum abundance exhibited in between the altitude ranges of 2800–3800 meters (291 spp.). This may be due to diverse habitats, favorable climatic condition and a large catchment area of the Sangla Valley. The most common economically important species in this area Table 14.1 Taxonomic account of plant resources of Sangla valley Taxonomic group Angiosperms Gymnosperms Pteridophytes Total

Families 63 4 3 70

Genera 192 7 3 202

Species 302 13 5 320

Herbs 244 – 5 249

Shrubs 38 4 – 42

Trees 20 9 – 29

Kanta-Chaulai

Amaranthus retroflexus L.

Amaranthus spinosus L.

Chenopodium album L.

Chenopodium foliosum Asch.

Dysphania botrys (L.) Mosyakin & Clemants syn. Chenopodium botrys L.

3.

4.

5.

6.

7.

Amaryllidaceae 8. Allium humile Kunth@@

Puthkanda

Amaranthaceae 2. Achyranthes aspera L.

Jungli Lassan

Sokana

Parangh

Bathua sag

Cholai

2680– 3445 m

Bhutni, Kimota

3000– 3600 m

2700– 3550 m 2300– 3400 m

2200– 3900 m

2500– 3200 m 2000– 3000 m

2500– 2800 m

Altitudinal range (m)

Local name/ common name

Family/Taxa Adoxaceae 1. Viburnum cotinifolium D. Don@

Reg Himal

Reg Bor

Iran

Reg Temp et Trop

Amer bor

N Amerer

Asia trop

Reg Himal

Phytogeographic affinities

H

H

H

H

H

H

H

S

Life form

Table 14.2 Diversity and distribution of economically and medicinally important plant resources of Sangla Valley

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

M: Indigestion, stomachache [Bl]

M: Skin problem, liver complaint, dry cough, joint pain, toothache, dysentery, stomachache, ear, eye complaint, anthelmintic [Wp] M: Stomachache, menstruation problem [Wp] M: Boils, burns, snakebite, laxative, tonic, gonorrhea, veterinary uses [Wp]; OU: Vegetable [Lf] M: Joint pain, skin problem, indigestion, stomach pains, constipation, urine problem [Wp]; OU: Vegetable [Ap] M: Indigestion [Fr, Lf]; OU: Edible [Fr, Lf] M: Leucorrhea, eye infections, gastric problem, headache, anthelmintic, liver problem [Wp]

M: Liver tonic, digestive problem, menorrhagia [Bk, Fr, St]; OU: Fodder and fuel [Ap], edible [Fr]

Indigenous uses

14 265

Sapal, Chaura

Apiaceae 10. Angelica glauca Edgew @@EN

3117– 3445 m 2750– 3448 m 3000– 3550 m

Kaligewa, Janglijira –

Bupleurum falcatum L. @@

Bupleurum hamiltonii N. P. Balakr

Bupleurum longicaule Wall. ex DC.

Carum carvi L.

13.

14.

15.

16.

Zira, Shingu Jeera

–

3500– 4150 m 2670– 3455 m

Kaligewar

Bupleurum candollei Wall.ex DC.@

12.

2900– 3200 m

Kalagira

Bunium persicum (Boiss.) B. FedtschVU

2800– 3510 m

Altitudinal range (m) 3000– 3150 m

11.

9.

Local name/ common name Pharna

Family/Taxa Allium stracheyi Baker @@VU

Table 14.2 (continued)

EuropOriens; Asia

Reg Himal

China

Reg Himal

Reg Himal

Persia

Reg Himal

Phytogeographic affinities Reg Himal

H

H

H

H

H

H

H

Life form H

M: Liver problems, stomach disorders, tonic, fever, wound, sour, anticoagulant [Wp] M: Digestive problems, depression, liver disorders, and loss of appetite [Wp] M: Abdominal inflammation, fever, indigestion, malaria [Rt] M: Dysentery, piles, body weakness, cold, cough, fever, joint pain, liver disease, back pain, nose pain, veterinary uses [Wp]

M: Constipation, dysentery, vomiting, typhoid, bronchitis, appetizer, liver tonic, veterinary uses [Rt]; OU: Flavoring agent, incense, condiment [Rt], fodder [Ap] M: Liver complaints, diarrhea, fever, gastric complaint, stimulant, cold, cough, fever, loss of appetite, joint pain, fever, worm infestation, dysentery in animal [Wp]; OU: Condiments [Sd] M: Stomachache, liver complaints [Wp]

Indigenous uses M: Indigestion, stomachache [Wp]; OU: Vegetable, condiment [Lf]

266 U. Devi et al.

Nesar

Pleurospermum brunonis (DC.) Benth. ex C.B. Clarke@@

Pleurospermum candollei (DC.) Benth. ex C.B. Clarke @@ Pimpinella tomentosa Engl.

22.

23.

24.

27.

26.

Selinum wallichianum (DC.) Raizada & H.O. Saxena syn. S. tenuifolium Salisb.@ Selinum vaginatum C.B. Clarke

Aschak, Karpo, Agu Nesar, Losar

Heracleum thomsonii C.B. Clarke@@

21.

Butkeshi, Mathosla

Mathosal

–

3460– 3800 m 2000– 3000 m 3100– 3400 m

Poral

Heracleum candicans Wall. ex DC.

20.

25.

2700– 3520 m 3400– 3800 m

Khaidmo

Ferula jaeschkeana VatkeVU

19.

2710– 3500 m

3400– 3950 m 2780– 3516 m

Ampang, Shakrag –

Chaerophyllum villosum Wall.ex DC. syn C. reflexum Lindl.@ Cortia depressa (D. Don) C. Norman@

3100– 3200 m 2770– 36,700 m 3900– 4310 m

18.

–

Chaerophyllum aromaticum L.

17.

Reg Himal

Reg Himal

Reg Himal

H

H

H

H

H

Reg Himal

Reg Himal

H

Reg Himal

H

H

H

Reg Himal

Himalaya Border Occ Turk Reg Himal

H

H

Reg Himal

Europ

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

M: Skin disease, menstrual complaints, nerve tonic, sedative [Rt]; OU: Fodder [Ap]

M: Antibacterial, sedative [Rt]; OU: Incense, insecticidal, fodder [Rt, Ap]

M: Fevers, skin disease, cold, cough [Wp]; OU: Perfumery industry, insect repellent, incense, offered to deities [Ap] M: Renal pain, fever, flatulence, stomachache [Fr, Rt] M: Dysentery [Wp]

M: Indigestion [Wp]; OU: Flavoring agents [Wp] M: Indigestion [Wp]; OU: Flavorings agents [Wp] M: Stomach complaints, rheumatism, sedative, fever, liver complaints, eye diseases, gastric pains, muscular pains, skin diseases, tonic, swelling [Wp, Rt] M: Pain in chest or back, fatigue, rheumatism, wounds, bruises [Rt, St] M: Leucoderma, ringworm infection, piles, giddiness [Rt, Fr]; OU: Fodder [Ap] M: Oxytocic [Wp]; OU: Fodder [Ap]

14 267

Polygonatum verticillatum (L.) All.VU

Aspleniaceae 36. Asplenium trichomanes L.

35.

Asparagaceae 32. Asparagus filicinus Buch.-Ham. ex D. Don 33. Polygonatum cirrhifolium Wall. (Royle)@EN 34. Polygonatum multiflorum (L.) All.VU

–

Salam-misri

Meda, Salammisri –

Sansbai, Elipali

Kira aloo

Jemul

Araceae 30. Arisaema flavum (Forssk.) Scott

Arisaema Jacquemontii Blume@

Tarkuch

Aquifoliaceae 29. Ilex dipyrena Wall.

31.

3117– 3400 m

–

3151– 3410 m

2600– 3263 m 2800– 3520 m 2590– 3610 m 2808– 3400 m

2575– 3523 m

2708– 3154 m

1900– 2800 m

Altitudinal range (m)

Local name/ common name

Family/Taxa Apocynaceae 28. Vincetoxicum hirundinaria Medik.

Table 14.2 (continued)

–

Reg Himal; Burma Reg Himal Asia bor Europ Asia bor Afghan Europ Asia borRhm

Reg Himal

Arab

Reg Himal

Europ; Reg Cauc; Asia

Phytogeographic affinities

H

H

H

H

S

H

H

T

H

Life form

M: Cold [Rz]

M: Kidney disorder, piles, wounds, appetizer, nerve tonic, urinary problems, spermatorrhea [Tb]; OU: Edible [Tb]

M: Diabetes, stomachache, leucorrhea, hair fall [Rt, Sd] M: Appetizer, blood pressure, blood purifier, fever [Tb, Lf] M: Tonic, urogenital disorders [Tb, Ap]

M: Bronchitis, skin disease [Fr, Bl]; OU: Used in local wine making [Fr], Pesticidal [Ap, Bl] M: Ringworms [Bl]; OU: Edible [Lf]

Fodder [Lf], fuel [Wd]

M: Skin problem, scorpion and snakebite [Wp]

Indigenous uses

268 U. Devi et al.

Kyamali

Khapchho, Khashmal

Berberis jaeschkeana C.K.Schneid.@

Berberis lycium Royle @@

Berberis vulgaris L.

Sinopodophyllum hexandrum (Royle) T. S. Ying. syn. Podophyllum hexandrum Royle @EN

42.

43.

44.

45.

Papra, Bankakri

Kashmal

Berberis coriaria Royle ex Lindl.@@

41.

Chutrum, Kashmal

–

Impatiens sulcata Wall. @@

39.

Berberidaceae 40. Berberis aristata DC.@

Tilpara

Impatiens scabrida DC.@@

–

38.

Balsaminaceae 37. Impatiens glandulifera Royle @

2900– 3122 m 3189– 4000 m

2692– 3260 m

2300– 2700 m 2400– 3416 m

2292– 3359 m

3189– 3523 m 2575– 3560 m 2840– 3423 m

Reg Himal

Europ Asia Temp

Reg Himal

Reg Himal

Reg Himal

Ind or

Reg Himal

Reg Himal

Reg Himal

H

S

S

S

S

S

H

H

H

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

M: Cancer, cough, fever, gastric ulcers, skin problem, tuberculosis, leucorrhea, warts, cuts, wounds, liver problems, vermifuge, body weakness, [Fr, Rt]; OU: Edible [Fr]

M: Acidity, fever, jaundice, bronchitis, diabetes, snakebites, boil, piles, malaria, eye infection [Wp]; OU: Dye [Rt], fibers [St], edible [Fr], fencing in crop fields M: Intestinal antiseptic, antiinflammatory [Wp] M: Fever, stomach disorders, eye trouble, skin diseases, blood purifier, diuretic, jaundice [Wp]; OU: Edible [Fr], fencing crop in fields M: Dysentery, tonic, ulcers, leucorrhea, jaundice, fever, cough, throat infection, eye infection [Wp]; OU: Edible [Fr], fencing crop fields Edible [Fr], fencing crop fields

M: Tonic, burn, joints pain, diuretic [Lf, Fl, Sd]; OU: Dye [Lf] M: Abortion [St]; OU: Edible [Fr], fodder [Ap] M: Eczema, pimples [Sd, Fr]

14 269

3000– 3800 m 2000– 3400 m

2850– 3300 m 2876– 3345 m

Thales cress Girahkat

–

Hackelia uncinata (Benth.) C.E.C. Fisch. Brassicaceae 51. Arabidopsis thaliana (L.) Heynh

Capsella bursa-pastoris (L.) Medik.

Descurainia sophia (L.) Webb ex Prantl

Lepidium apetalum Willd.

52.

53.

54.

–

–

–

Eritrichium canum (Benth.) Kitam.@

49.

50.

2000– 3450 m 2700– 3445 m 3260– 3500 m

Kochi-Shuver

Cynoglossum wallichii G. Don.

48.

3400– 4150 m

2800– 3500 m

Bhuj, Bhoj Shakpang

Masari, Ratanjot

Altitudinal range (m)

Local name/ common name

Boraginaceae 47. Arnebia benthamii Wall. ex G.. Don. I. M. Johnst. @CR

Family/Taxa Betulaeae 46. Betula utilis D. Don@EN

Table 14.2 (continued)

Russia Sibir

Reg Temp

Reg Bor Temp

Reg Himal

Reg Himal

Ind or Burma

Reg Himal

Reg Himal Japon

Phytogeographic affinities

H

H

H

H

H

H

H

H

T

Life form

M: Blood pressure, blood purifier, diarrhea, dropsy, gonorrhea, urinary trouble, cuts, fever, wounds, stimulant [Wp, Sd] M: Anti-inflammatory, fever, ulcers [Ap, Sd] M: Skin problem, promotes sweating, stomachache [Wp]

M: Mouth ulcer, stomachache [Wp]

M: Piles [WP]; OU: Fodder [Ap]

M: Assists child birth [Wp]

M: Arthritis, piles, antiseptic, boils, cuts, wounds, fungal hair infection, [Rt]; OU: Food coloring [Rt] M: Wounds, muscular pain [Lf, Rt]

M: Antiseptic, burns, cuts, wounds, cough, ear infection, jaundice, antiseptic, joint pain, tonic [Bk, St, Lf]; OU: Fuel [Wd], religious ceremonies, timber

Indigenous uses

270 U. Devi et al.

Baang Kharmu – Bishkandara

Caprifoliaceae 60. Abelia triflora R.Br. ex Wall.

Lonicera hypoleuca Decne.

Morina coulteriana Royle @@

Morina longifolia Wall.ex DC.@

Valeriana hardwickii Wall.

61.

62.

63.

64.

Nakhniani

Bhang

Sardandi, Khiri

Cannabaceae 59. Cannabis sativa L.

Codonopsis viridis Wall.@

Nepali bikh

Campanulaceae 57. Campanula pallida Wall.

58.

Khubkalan

Sisymbrium irio L.

56.

–

Nasturtium officinale R.Br.

55.

2200– 3000 m 2708– 3502 m 3300– 3625 m 3200– 3527 m 2750– 3210 m

2000– 3170 m

2550– 3090 m 2800– 3300 m

2750– 3146 m

2800– 3300 m

Reg Himal Malaya

Reg Himal

Reg Himal

Reg Himal

Reg Himal

Asia Centr Himal BorOcc

Reg Himal

Ind or Afghan

Europ; Asia et Afrbor

Reg bor Temp

H

H

H

S

S

H

H

H

H

H

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

M: Wounds, boils [Rt]; OU: Incense [Rt] M: Skin problem, antidote to poisonous insects, hysteria [Wp]; OU: Religious ceremonies, insecticidal [Ap, Rt]

M: Eye complaints [Fl, Rt]

Fuel [Ap], fodder [Ap]

Walking stick, fuel, fodder [Ap]

M: Paralysis, joint pain, anthelmintic, bronchitis, cuts, arthritis, insect bite, cough, sedative, piles, skin disorder, cold, cough, cramp, wounds, sores, epilepsy [Wp]; OU: Seeds edible, fiber [Sd, St]

M: Bruises, ulcers, swollen joints, and healing of wounds [Rt, Lf]

M: Wounds [Ap]

M: Carminative, constipation, vermifuge, kidney trouble [Ap] OU: Vegetable [Ap] M: Itching, body pain, bloody stool [Lf, Sd]; OU: Vegetable [Lf]

14 271

Allardia tomentosa Decne. syn. Waldheimia tomentosa (Decne.) Regel Anaphalis busua (Buch.-Ham.) DC.

Anaphalis contorta (D.Don) Hook.f

72.

74.

73.

Ainsliaea aptera DC.@

71.

–

Dhareu

Phillu

Karu- buti

Saijum

Asteraceae 70. Achillea millefolium L.

Stellaria media (L.) Vill. @@

68.

–

Khokhua-bhaji

Silene vulgaris (Moench) Garcke

67.

Celastraceae 69. Parnassia nubicola Wall. ex Royle

Gandoli

Gypsophila cerastoides D. Don @

Local name/ common name

66.

Family/Taxa Caryophylaceae 65. Cerastium cerastoides (L.) Britton.

Table 14.2 (continued)

2700– 3300 m 3600– 4400 m 2000– 2790 m 2700– 3200 m

2800– 3600 m

2700– 3300 m

Reg Himal

2800– 3150 m 2794– 3150 m 3080– 3185 m 2750– 3345 m

Reg Himal

Reg Himal

Tibet Occ

Reg Himal

Europ

Reg Himal

Reg Himal

Reg Himal

Reg Himal

Phytogeographic affinities

Altitudinal range (m)

H

H

H

H

H

H

H

H

H

H

Life form

M: Colds, cough, fevers, blood purifier, rheumatism, piles, toothache, liver complaints, gastric complaint, ulcer, worms, tonic, piles, laxative, leucorrhea [Wp]; OU: Insect repellent [Ap] M: Crushed roots are used for gastric problems, leucorrhea, fever [Wp, Rt] M: Rheumatism, cut, wounds [Wp]; OU: Incense [Wp] M: Cuts, wounds, stop bleeding [Lf, Fl]; OU: Fiber [St] M: Antibacterial, cuts, wound, boils, stop bleeding, cold, cough, [Wp] OU: Insect repellent [WP]

M: Food poisoning, snakebite [Tb]

M: Bronchitis, asthma, stomach pai [Lf, St]; OU: Edible as vegetable [Ap] M: Burns, boils, bone fracture, wounds, skin infections [Wp]; OU: Edible [Ap]

M: Bodyache, renal pain, cough, headache [Wp] M: Boils, wounds [Wp]

Indigenous uses

272 U. Devi et al.

Anaphalis triplinervis (Sims) Sims ex C.B. Clarke Anthemis cotula L.

Arctium lappa L.

Artemisia annua L.

Artemisia biennia Willd.

Artemisia capillarisThunb.

Artemisia japonica Thunb.@@

Artemisia maritima L.

Artemisia roxburghiana Wall. ex Besser

77.

79.

80.

81.

82.

83.

84.

85.

78.

76.

Anaphalis griffithii Hook.f. syn. A. royleana C.B. Clarke Anaphalis nepalensis (Spreng) HandMazz.

75.

2500– 3400 m

2200– 3167 m

–

2876– 3448 m 3000– 3400 m 2600– 3516 m 2575– 3200 m

2900– 3800 m 2400– 2950 m 2750– 3350 m

2750– 3900 m 2700– 3850 m

Seski

Khamtso, Nurcha Nireha, Jonkhar

Kampa

–

Jangli Kuth

–

Yaktso

Monpig

Kirchee

Europ

Europ Reg Cauc; Asia Sibir

Ind or Burma

Amer Bor; Sibir; Reg Himal China

Asia

Europ

Europ; Afr

Reg Himal

Reg Himal

Reg Himal

H

S

H

H

H

H

H

H

H

H

H

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

M: Swelling, spermatorrhea, indigestion [Wp]; OU: Used in ritual ceremonies [Wp], Buds are Edible M: Diuretic [Wp]; OU: Edible [Bd], used in ritual ceremonies [Wp] M: Stomach complaints [WP, Fl]; OU: Ornamental M: Diabetes, burn, gastric, skin and kidney problems, gout, asthma [Wp, Sd]; OU: Insect repellants, manure [Wp] M: Abdominal pain, blood purifier, worm troubles, asthma, nervine [Lf] M: Stomachache, reduces excess body fat [AP] M: Joint pain, earache, intestinal bleeding (animals) [Lf] M: Carminative, vermifuge, throat infection, aromatic [Lf, Fl, Sd]; OU: Insecticide [Lf] M: Blood purifier, toothache, antimalarial, anthelmintic, stomachache, asthma, fever, cough, cuts, gastric complaints, tonic, stimulant, antiseptic, anti-inflammatory, joint pain, [Wp]; OU: Fodder, for making brooms, incense [Ap] M: Eczema, pimples, sores, diabetes, fever, tonic, skin allergy [Lf]; OU: Plant used in religious ceremonies [Ap]

Edible [Bd]

14 273

Bursa

– Bashakar

Cirsium wallichii DC.@@

Cousinia thomsonii C.B.Clarke

Echinops cornigerus DC.

Erigeron alpines L. @@

Erigeron canadensis L.

Erigeron multiradiatus (Lindl. ex DC). Benth.ex Hook.f. Galinsoga parviflora Cav.

Jurinella macrocephala (Royle) Aswal & Goel@

89.

90.

91.

92.

93.

94.

96.

95. Dhoop

Pipulughas

–

Palit

Batasatsuak

Tisu

88.

87.

–

Local name/ common name Jhyang

Askellia flexuosa (Ledeb.) W.A.Weber. syn. Youngia glauca Edgew Carduus edelbergii Reich.f.@

86.

Family/Taxa Artemisia vestita Wall. ex Besser

Table 14.2 (continued)

3250– 3450 m 2700– 3500 m 3150– 3500 m 2700– 2900 m 3112– 3305 m 2575– 3400 m 3050– 3900 m

Altitudinal range (m) 2700– 3445 m 3345– 3600 m 2500– 3200 m 2700– 2900 m

Reg Himal

Mexico

Reg Himal

Amerphig

Ind or Audrey truschke Reg Bor et Arct

Reg Himal

Reg Himal

Reg Temp Asia Bor Afghan

Phytogeographic affinities Reg Himal

H

H

H

H

H

H

H

H

H

H

Life form H

M: Wound bleeding, eczema [Ap]; OU: Fodder [Ap] M: Stomachache, diarrhea, wounds fever, laxative, antiseptic, cardiac tonic, fever, gout, rheumatism [Rt]; OU: Incense, insect repellent [Rt]

M: Stomachache, brain tonic [Ap]

M: Gastric problems, dysentery, cough, swelling, headache [Wp]; OU: Fodder [Ap] M: Swelling, body pain, join pain [Wp]; OU: Edible, incense [St] M: Fever, cold, cough, cuts, wounds, tonic, antibacterial [Wp] M: Cough, cold, rheumatism, cough, cold, fodder [Wp] M: Diarrhea, dysentery, ringworm [Wp]

M: Fever, blood purifier, tonic [Fl, Wp]

Indigenous uses M: Antibacterial inflammatory diseases [Ap] M: Fever, jaundice, indigestion, [Ap]

274 U. Devi et al.

Lactuca lessertiana (Wall. ex DC.) Wall. ex C.B. Clarke. Lactuca macrorhiza (Royle) Hook. f.

99.

Saussurea albescens Hook. f & Thomson@@ Saussurea costus (Falc.) Lipsch.@@

Saussurea gossypiphora D. Don.@CR

Saussurea obvallata (DC.) Edgew.@CR

Saussurea roylei C.B. Clarke@

Saussurea taraxacifolia (Lindl.) Wall. ex DC.

103.

105.

106.

107.

108.

104.

–

Ligularia amplexicaulis DC.@

102.

–

Gugghibadshah Dongar, Barhmkamal, Dodaphoo –

Kuth, Koth

–

Dhoop

Laphangium affine (D. Don) Tzvelev syn. Gnaphalium affine D.Don

–

–

Gringoli

Dudhali

101.

100.

98.

Lactuca brunoniana (DC.) Wall. ex C.B. Clarke syn. Prenanthes brunoniana Wall. ex. DC. Lactuca dolichophylla Kitam. @@

97.

3400– 3900 m 3400– 3900 m

3800– 4000 m 3600– 4000 m

2750– 3100 m 2740– 3510 m 2876– 3509 m 2780–3160 2780– 3160 m 3200– 3400 m 2810– 3320 m 3550– 4000 m

3100– 3600 m

Reg Himal

Reg Himal

Reg Himal

Reg Himal

Reg Himal

Reg Himal

H

H

H

H

H

H

H

H

Reg Himal

Reg Himal

H

H

H

Reg Himal

Reg Himal

Reg Himal

H

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

M: Bronchitis, digestive problem, skin eruption, [Lf] M: Cardiac stimulant, antiseptic, joint pains, asthma, blood purifier, bronchitis, dysentery, skin problem, toothache headache [Rt]; OU: Insect repellent [Rt] M: Burns, cuts, wounds, asthma, cuts, wound healing [Wp] M: Cuts, wounds, bruises [Wp]; OU: Offered for deities in religious ceremonies [Fl] M: Antidote, swelling, wounds, aching joints [Rt] M: Cold, cough, headache, ulcers, [Wp]

M: Astringent, bone fracture [Wp]

M: Fever, throat infection, cough, influenza [Wp]

M: Constipation, jaundice [Wp]

M: Cough, kidney pain [Ap]

Vegetable [Lf]

Fodder [Ap]

14 275

Family/Taxa Scorzonera virgata DC.

Senecio chrysanthemoides DC.

Senecio glaucus L. subsp. coronopifolius (Maire) C. Alexander syn. Senecio desfontainei Druce Solidago virga-aurea L.

Sonchus asper (L.) Hill

Sonchus oleraceus (L.) L.

Tagetes minuta L.

Taraxacum campylodes G.E. Haglund syn. Taraxacum officinale (L.) Weber ex F.H. Wigg

Waldheimia glabra (Decne.) Regel

Youngia japonica (L.) DC.

109.

110.

111.

112.

113.

114.

115.

116.

117.

118.

Table 14.2 (continued)

–

Phillu

Dudhli, Dulal, Aachak

–

Dodak

4050– 4550 m 2708– 3050 m

2550– 3523 m

3150– 3480 m 2600– 3000 m 2700– 3000 m 2200– 2600 m

–

–

Parpal

Altitudinal range (m) 3100– 3600 m 2692– 3200 m 2880– 3240 m

Local name/ common name

Asia Austr

Tibet Occ

Reg Temp Bor et Austr

Amer trop

Cosmop

Cosmop

Reg Bor Temp

Reg Himal

China Mongol

Phytogeographic affinities China Mongol

H

H

H

H

H

H

H

H

H

Life form H

M: Jaundice, fever [Ap]; OU: Vegetable [Lf] M: Skin problem, muscular pain, stomachache, vermifuge, fever, piles, earache [Wp, Fl, Lf] M: Kidney diseases, blood purifier, wounds, boils, dislocation of joints, dysentery, ulcers, headache, fever, digestive problem [Wp]; OU: Edible [Lf] M: Cuts, wounds, burns, food poison [Wp]; OU: Incense [Wp] M: Cancer, urine complaint, headache [Wp]

M: Antiseptic, throat infection, asthma, fever, [Wp, Lf] Cuts wounds, boil [Wp]

M: Respiratory problem, fever, stomachache, sore throat [Wp] Fodder [Ap]

Indigenous uses M: Stomachache, constipation [Ap]

276 U. Devi et al.

Sedum multicaule Wall. ex Lindl.@

Shur

Juniperus polycarpos C. Koch@EN

Juniperus recurva Buch.-Ham. ex D. Don

127.

128.

Mant Thelu

Dhoop

Juniperus indica Bertol.

Juniper, Thaleru

Tindi

Moshughas

126.

Cupressaceae 125. Juniperus communis L@

124.

Crassulaceae 121. Rhodiola heterodonta (Hook. f. & Thomson) Boriss.@VU 122. Rosularia rosulata (Edgew.) H. Ohba@@ 123. Sedum ewersii Ledeb. –

Amar bel

120.

Cuscuta reflexa Roxb.

Haranpadi

Convolvulaceae 119. Convolvulus arvensis L.

3300– 4000 m 3465– 3800 m 3263– 3500 m

3254– 3448 m

3030– 4000 m 2700– 3400 m 2708– 3800 m 2600– 3263 m

2730– 3220 m 2814– 3400 m

Soongar; Reg Himal Persia; Reg Himal Reg Himal

Reg Himal

Reg Himal Sibir Altaic Reg Himal China

Reg Himal

Reg Himal

Ind or

Geront Temp

S

T

S

S

H

H

H

H

H

H

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

M: Tumors, piles, bronchitis, asthma, liver and spleen complaints, joint pains, heart disease, nervous disorder, antibiotic for animals [Ap, Sd]; OU: Insect repellent, religious as incense, fuel [Ap] M: Stomachache, asthma, vermifuge, tumors, bronchitis, piles [Ap] M: Asthma, stomach cramp, [Wp]; OU: Insect repellent [Ap] Incense, fuel, ornamental [Lf, Wd]

M: Gastric problems, burn, headache, toothache, cuts, wounds [Wp] OU: Insecticidal [Lf]

M: Stomachache, cough, lung infection, sexual potency, diarrhea [Ap, Rt] M: Skin problem [Rt]

M: Cuts, wounds, burns, bruises, laxative [Wp] M: Body pain, bone fracture, jaundice, rashes, itching, indigestion, wounds [Wp]; OU: Rituals ceremonies

14 277

Ephedraceae 135. Ephedra intermedia Schrenk & C.A. Meyer

134.

Hippophae salicifolia D. Don syn. Elaeagnus salicifolia (D. Don) A. Nelson@ Hippophae tibetana Schltdl.@

133.

2800– 3200 m

3200– 3590 m

Chharma

Khanna, Chhe

2780– 3450 m

2400– 2700 m 3300– 3500 m

Surcham, Chharma

Chharma, Gartsak

Ghyayeen

Elaeagnaceae 131. Elaeagnus parvifolia Wall. ex Royle

Elaeagnus rhamnoides (L.) A. Nelson syn. Hippophae rhamnoides L. VU

Shinglimingli

Dioscoreaceae 130. Dioscorea deltoidea Wall. ex Griseb.EN

132.

2100– 2400 m

Munji

2775– 3350 m

Altitudinal range (m)

Local name/ common name

Family/Taxa Cyperaceae 129. Eriophorum comosum (Wall.) Nees

Table 14.2 (continued)

Centr Asia Himalaya

Tibet

Reg Himal (Nepal)

Europ; Asia Temp

Japan

Ind or

Ind or

Phytogeographic affinities

S

S

S

S

S

H

H

Life form

M: Asthma, fever, cold, skin pimples, eight sight, sedative, intoxication [Wp]; OU: Religious ceremonies [Ap], edible [Fr]

M: Indigestion, skin wrinkles, lung disease, blood pressure, blood purifier, constipation, wounds, cough, antibacterial [Wp]; OU: Fruits edible, made into sauce and juice M: Wounds, cuts, ulcers, cough, fever, dandruff, skin disease, whooping cough [Fr, Bk, Lf]; OU: Edible [Fr], fuel [Ap] M: Lung disease, leucorrhea [Fr]; OU: Fruits edible, made into sauce and juice

Fuel, fodder [Ap], edible [Fr]

M: Joints pains, oral contraceptive, fever, gout, asthma, piles, cold, hair growth, digestive ailments [Tb]; OU: Used for washing woolen cloths, edible [Tb]

Fiber [Wp], religious ropes for local deities [Wp]

Indigenous uses

278 U. Devi et al.

3500– 3800 m 2630– 2800 m

–

Dudawaj

– Yamcho, Cho

Rhododendron lepidotum Wall. ex G. Don.@@VU Euphorbiaceae 141. Euphorbia helioscopia L.

Fabaceae 142. Astragalus chlorostachys Lindl. @

143.

144.

140.

Astragalus rhizanthus subsp. candolleanus (Benth.) Podlech. (Syn. Astragalus candolleanus Benth.)@ Astragalus rhizanthus Benth.@

@VU

Rhododendron campanulatum D. Don

Cho

3200– 3500 m

Sairmanang

139.

3300– 4050 m

Buransh

@@VU

Rhododendron anthopogon D. Don

138.

2876– 3448 m

3250– 3527 m 3300– 4200 m

2800– 3750 m

Salu

Ericaceae 137. Cassiope fastigiata (Wall.) D. Don@

2690– 2900 m

–

Equisetaceae 136. Equisetum arvense L.

Reg Himal

Reg Himal

Reg Himal

Europ et Asia Bor

Reg Himal

Reg Himal

Reg Himal

Reg Himal

Alaska

S

S

H

H

S

S

S

S

H

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

Fuel, browsed by mule, goat, sheep [Wp]

M: Blood purifier, cough, skin disease, tuberculosis [Wp, Rt]

Fodder [Ap]

M: Febrifuge and vermifuge [Lf, St]

M: Fire burns, wound, bloody dysentery, itching [Wp, Lf]; OU: Incense [Wp] M: Cold, cough, chronic bronchitis, stomach problem, joint pain, skin disease, fever [Wp]; OU: Tea is made from the leaves M: Joint pain, skin disease, boils, cold, cough, tonic, fever, boils, piles, bone fracture of animals [Wp]; OU: Fuel [Ap], eaten raw or juice, religious ceremonies [Fl] M: Cold, cough, boils, bronchitis [Wp]

M: Kidney disease, acidity [Wp, plant ash]

14 279

Kali Kathi

Indigofera heterantha Brandis @

Lotus corniculatus L.

Medicago falcata L.

Medicagolupulina L.

Oxytropis lapponica (Wahlenb.) Gay

Robinia pseudoacacia L.

Trifolium pratense L.

Trifolium repens L.

Trigonella emodi Benth.

146.

147.

148.

149.

150.

151.

152.

153.

154.

155.

Fagaceae 156. Quercus floribunda Lindl. ex A. Camus@@

Sambar

Desmodium elegans DC. @@

145.

Moru

Tuljima

Triptra

Kikar, honeylocust Purple clover

–

Hop clover

Bird’s-foot trefoil –

Local name/ common name –

Family/Taxa Cicer microphyllum Benth.@

Table 14.2 (continued)

2550– 2900 m

2200– 2750 m 2876– 3400 m 2900– 3445 m 2981– 3501 m 2900– 3400 m 2400– 3000 m 2575– 3350 m 2575– 3400 m 2708– 3500 m

Altitudinal range (m) 2708– 3350 m 2200– 2681 m

Reg Himal

Reg Himal

Europ; Asia Temp GerontBor Temp

AmererBor

Europ; Asia Bor

GerontBor Temp

Geront Temp Austr GerontBor Temp

Reg Himal

Reg Himal China

Phytogeographic affinities Reg Himal

T

H

H

H

T

H

H

H

H

S

S

Life form H

Fuel, fodder, agriculture tools [Lf, St, Wd]

M: Cough, bronchitis, dandruff [Wp]; OU: Fodder [Lf] Aromatic, fodder [Ap]

Fodder [Ap]

Fodder [Lf], fuel, implements [Wd]

Fodder [Ap]

M: Fever, cough, tonic, diuretic, chronic, vomiting, asthma [Wp, Rt]; OU: Fodder, fuel, used as toothbrush, fiber for ropes [Lf, St] M: Headache, chest pain [Rt, Lf]; OU: Fodder [Ap] M: Good source of carotene [Ap]; OU: Fodder, edible [Ap] M: Wound healing [Ap]; OU: Fodder [Ap] Fodder [Ap]

Indigenous uses Edible, fodder [Wp, Rt]

280 U. Devi et al.

Pitpapra

Halenia elliptica D. Don@

162.

Porlo Laljari

Likatur

Geranium nepalense Sweet.

Geranium pratense L.

Geranium wallichianum D. Don ex Sweet @

166.

167.

168.

Polo, Laljari

Chirettah, N epaliChirata

Tikta

Lomatogonium carinthiacum (Wulfen) A.Braun 164. Swertia ciliata (D. Don ex G. Don) B.L. Burtt.@ Geraniaceae 165. Geranium himalayense Klotzsch

163.

Chateek

Gentianopsis detonsa (Rottb.) Ma

161.

–

–

Kharshu

Titka

Quercus semecarpifolia Sm.@

Gentianaceae 158. Gentiana argentea (Royle ex D. Don) Royle ex D.Don@ 159. Gentiana coronata (D. Don ex Royle) Griseb.@ 160. Gentiana tianschanica Rupr. ex Kusn.

157.

2690– 3500 m 2575– 3527 m

2700– 3445 m 2575– 3385 m

3100– 3500 m 2980– 3260 m 3000– 4000 m 29813448 m

2780– 3490 m 3400– 3980 m 3250– 3527 m

2500– 3050 m

Reg Himal

Europ; Asia Bor

Ind or China

Europ; Asia Bor

Reg Himal

Reg Himal

Reg Himal

Temp bor

Asia centr

Reg Himal; China Reg Himal

Reg Himal

H

H

H

H

H

H

H

H

H

H

H

T

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

M: Astringent, kidney disease, toothache, stomach problem, cuts, jaundice, ulcer, wounds, [Wp]; OU: Dye [Rt] M: Jaundice, cough, bruises, gastric, headache [Wp]; OU: Black dye [Rt] M: Cuts, wounds, toothache,, ear & eye problem, jaundice [Fl, Rt]; OU: Red purple dye [Rt]

M:Skin problem, stomach pain [Rt, Fl]

M: Blood purifier, cough, cold, fever, joint pain [Fl] M: Malaria, cold, fever, headache, fever [Ap, Lf]

M: Fever, liver inflammations [Wp]

M: Urinary disorder, tonic, stomachic, blood purification, jaundice [Rt]; OU: Edible [Rt] M: Jaundice [WP]

M: Stomach pain, fever [Rt]

M: Sore throat [Fl, Lf]

Fuel, fodder, agriculture tools [Lf, St, Wd]

14 281

–

Iridaceae 172. Iris hookeriana Foster@@

176.

Elsholtzia eriostachya (Benth) Benth.@

Lamiaceae 175. Clinopodium vulgare L.

Juglandaceae 174. Juglans regia L.@@

Betso

–

Dandasa/ Akhrot

–

Basant

Hypericaceae 171. Hypericum perforatum L.VU

Iris kemaonensis Wall. ex D. Don.@

–

Hydrangeaceae 170. Deutzia staminea R. Br. ex Wall.@

173.

2800– 3450 m

Pilikcha

2630– 3500 m 2800– 3450 m

2200– 3100 m

2770– 3445 m 2900– 3927 m

2300– 3340 m

2575– 3400 m

Altitudinal range (m)

Local name/ common name

Family/Taxa Grossulariaceae 169. Ribes alpestre Wall. ex Decne.@

Table 14.2 (continued)

Reg Himal

Europ; Canada

Asia Occ Reg Himal

Reg Himal

Reg Himal

Europ

Reg Himal

Reg Himal

Phytogeographic affinities

H

H

T

H

H

H

S

S

Life form

Edible condiment (as chutney) [Lf]

M: Tonic, skin problem, astringent [Ap]

M: Skin disease, anthelmintic, astringent, diarrhea toothache, fungicide, insecticide, hypertension, cleaning and sparkling teeth [Bk, lf, Fr]; OU: Timber for making furniture

M: Fever, toothache, epilepsy, urine complaints [Rt, Sd, Lf]

Ornamental

M: Cough, cold, cuts, immunity, malaria, anticancer, antiviral [Wp]

Kill the house fleas (Wp)

M: Vomiting, dysentery, gastric problem [Ap, Fr]; OU: Fuel, fodder [Ap], edible [Fr]

Indigenous uses

282 U. Devi et al.

Brun –

Nepeta eriostachya Benth.@

Nepeta discolor Royle ex Benth.@@

Nepeta elliptica Royle ex Benth.

Nepeta podostachys Benth.

Origanum vulgare L.

Phlomoides bracteosa (Royle ex Benth.) Kamelin & Makhm.@ Prunella vulgaris L.

Salvia nubicola Wall. ex Sweet

Thymus linearis Benth

179.

180.

181.

182.

183.

184.

185.

186.

187.

Ban ajwain

–

–

–

Banajwain, Baslughas

Ribuksu

–

Podina, Jungli pudina

Mentha longifolia (L.) L.

178.

–

Leonurus cardiaca L.

177.

2700– 3509 m 2770– 3150 m 2708– 3399 m 2100– 3700 m

3185– 3500 m 2680– 3400 m 2708– 3523 m 2780– 3440 m 2981– 3527 m

2750– 3100 m 2800– 3400 m

Europ; Austr Reg Himal Europ Asia et Afrbor

Reg Himal Temp

Reg Himal

Europ Asia et Afrbor

Afghan

E Afghan; Nepal

Reg Himal

Reg Himal

Reg bor Temp

Reg Bor Temp

H

H

H

H

H

H

H

H

H

H

H

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

M: Whooping cough, skin eruption, antifungal, antibacterial, cold, toothache, hookworms, liver problem, fever [Wp]

M: Cold, cough, fever, gastric problem, headache [Wp] M: Cough, cold, cuts, wounds, [Lf, Rt]

M: Cold, cough, fevers, kidney disorder [Wp, Rt, St] M: Cold, whooping cough, fever, bronchitis, antiseptic, diarrhea, menstrual disorder, boils, wounds, cuts, ulcers [Wp] M: Eye problems [Ap]

M: Dysentery, uterine problem [Sd]

M: Cold, cough, eyes infection [Wp]

M: Fever, headache, vomiting, antiseptic, diarrhea, dysentery, joint pain [Ap, Lf]; OU: For making chutney, flavoring agent, herbal tea [Ap, Lf] M: Diuretic, eye problem [Wp]

M: Stomachache [Lf, Sd]

14 283

Malva verticillatta L.

191.

194.

Jasminum officinale L.

Melanthiaceae 192. Trillium govanianum Wall. ex D. Don@@ Oleaceae 193. Fraxinus xanthoxyloides (G. Don) Wall. ex A.DC.@@

Malva sylvestris L.

190.

Family/Taxa Liliaceae 188. Fritillaria cirrhosa D. Don. syn. Fritillaria roylei Hook.@EN Malvaceae 189. Malva neglecta Wallr.

Table 14.2 (continued)

2700– 3100 m

24003300 m

Thum

White Jasmine

3400– 3509 m

Nag Chhatri, Satwa

25753600 m

–

2800– 3600 m

Khubar, Sonchala

2800– 3700 m

3000– 3250 m

–

Marsh-mallow

Altitudinal range (m)

Local name/ common name

Ind bor; Occ; China

Reg Himal

Reg Himal

Europ Asia et Afrbor

Hungary

Europ

Europ Asia et Afrbor

Phytogeographic affinities

S

S

H

H

H

H

H

Life form

M: Skin problem, fracture, abdominal disorder in veterinary medicine [Ap, Bk, St] M: Stomachache, blood purifier, emollient, aromatic, toothache, cough, fever, milk production in cattle [Fl, Lf]; OU: Ornamental

M: Joint pain, birth control, dysentery [Tb]

M: Scurvy, piles, bronchitis, cough, leucorrhea, malaria, kidney disorder, laxative [Wp, Sd]; OU: Vegetable, fodder [Ap] M: Jaundice, cold, whooping cough, sore throat, stomach cramp, [Wp] OU: Vegetable [Wp], ornamental M: Kidney problem, whooping cough, bladder, piles, ulcer, urinary problem [Ap]; OU: Vegetable, fodder [Wp]

M: Asthma, fever, eye problem, bronchitis, burns [Bl]

Indigenous uses

284 U. Devi et al.

Olea europaea subsp. cuspidata. (Wall. & G.Don) Cif. syn. Olea ferruginea Royle

Epilobium royleanum Hausskn.@

Luak –

Orobanche alba Stephan ex Willd

Pedicularis bicornuta Klotzsch@@

203.

204.

Herminium monorchis (L.) R. Br.

201.

–

Goodyera fusca (Lind.) Hook. f.@

200.

3150– 3400 m 3000– 3400 m 2700– 3500 m

2500– 3600 m 3000– 3709 m 2400– 2900 m

3000–3400

2600– 3250 m

–

Panja Salampanja

3000– 4000 m.

1900– 2300 m

Dharshak

Kohu, Wild olive

Orobanchaceae 202. Euphrasia simplex D. Don@

Epipactis helleborine (L.) Crantz

199.

Orchidaceae 198. Dactylorhiza hatagirea (D. Don.) Soo@CR

197.

Onagraceae 196. Epilobium angustifolium L.

195.

Europ; Oriens; Asia Bor Reg Himal

Nepal

Java

Reg Himal

Europ; Asia Bor

Reg Himal

Reg Himal

Europ Asia Bor Amer Bor

Reg Oriens

(continued)

M: Joint pain, burns, gout, body pain, sedative [Ap]

M: Joint pain [Wp]

M: Jaundice, cold [Wp]

M: Kidney problem, antiseptic [Tb]

M: Diarrhea, dysentery, blood purifier, nervine tonic, bone fracture, antibiotic, cuts, wounds, fractures, cough, cold, joint pain, diabetic, weakness, lose motions [Tb] M: Blood purifier, fever, aphrodisiac [Lf, Rz] M: Blood purifier

M: Stomachache, kidney problem, liver complaints, intestinal infections [Ap]; OU: Ornamental M: Ringworm, astringent, cattle warts, poisonous to livestock [Rt, Lf]

M: Antiseptic, astringent, toothache, stomachache, diuretic, soar throat, toothache [Lf, Bk]; OU: Agricultural tools specially ploughs and handles, timber, fuel, fodder [Lf, Wd]

Assessment of Economically and Medicinally Important Plant Resources. . .

H

H

H

H

H

H

H

H

H

T

14 285

2600– 2900 m

213.

Abies spectabilis (D. Don) Mirb. @

Pinaceae 212. Abies pindrow (Royle ex D. Don) Royle.@ 2800– 3250 m 2800– 3000 m

Krok rai

–

–

–

Meconopsis horridula Hook. f. & Thomson@ Phytolacaceae 211. Phytolacca acinose Roxb. @

–

Meconopsis aculeata Royle @@EN

209.

210.

3200– 3500 m 3100– 3300 m

Bhutkesi

Corydalis govaniana Wall.@@

208.

2500– 4150 m 3117– 3950 m

Bhutkesi

Papaveraceae 207. Corydalis cashmeriana Royle @@

2200– 3263 m

Altitudinal range (m) 3159– 3527 m

–

Local name/ common name Michren

Oxalidaceae 206. Oxalis corniculate L.

205.

Family/Taxa Pedicularis punctata Decne.

Table 14.2 (continued)

Reg Himal

Reg Himal

Reg Himal Asia trop

Reg Himal

Reg Himal

Reg Himal; Asia Trop Reg Himal

Amerphig Temp Trop

Phytogeographic affinities Reg Himal; Persia

T

T

H

H

H

H

H

H

Life form H

M: Swelling, fever, asthma, bronchitis, cough, chest infection [St, Lf, Bk]; OU: Timber M: Asthma, headache, bronchitis cold, cough [Lf, Wd] OU: Fuel, timber, furniture [Lf, Wd]

M: Wounds, skin problem, body pain, dysentery, cattle pneumonia [Lf, Rt]

M: Skin problem, gastric pains, joint pain, liver tonic, eye problem, tonic, leprosy [Wp] M: Fever, backache, colic, tonic, renal pain [Wp] M: Swelling, fever [Wp]

Fodder [Ap]

M: Cut, cough, dysentery, eye problem, swelling, stomachache [Wp]; OU: Edible [Lf]

Indigenous uses M: Body ache, cough, cold [Wp]

286 U. Devi et al.

Lim, Kail

Pinus wallichiana A.B. Jacks. @

218.

221.

Plantago asiatica subsp. erosa (Wall.) Z. Yu Li. syn. P. erosa Wall. Plantago depressa Willd. Isabagol

JangliIsabgol

Chil

Pinus roxburghii Sarg.@

217.

220.

Ree, Chiri, Neoza

Pinus gerardiana Wall. ex D. Don.@

216.

Karu, Kutki

Rou, Royang Tosh, Rai

Picia smithiana (Wall.) Bioss.@

215.

Plantaginaceae 219. Picrorhiza kurrooa Royle@@EN

Devdar

Cedrus deodara (Roxb. ex D. Don) G. Don@

214.

3100– 3263 m 2100– 3000 m

3200– 3900 m

2250– 3527 m

2000– 2600 m

2250– 2890 m

2981– 3350 m

2026– 3285 m

Europ et Amer Bor Sibir

Reg Himal

Reg Himal

Reg Himal

Afghan

Reg Himal

Reg Himal

H

H

H

T

T

T

T

T

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

M: Asthma, appetizer, cold, cough, fever, stomachache, bile trouble, jaundice, leprosy, constipation, leucoderma, blood purifier, blood pressure reducer [Rt] M: Cough, diarrhea, piles, skin troubles, bone fracture, cooling agent [Rt, Lf] M: Pulmonary diseases, laxative, cough, asthma, stomach pain, dysentery, wounds, piles [Wp]

M: Skin disease, worm, joint pain, ulcers, fever, piles [Lf, St, Bk, Oil]; OU: Repel insect in cattle [Lf], timber, religious ceremonies, incense, fuel [Ap] M: Body pain, cuts, sores, [Lf, Wd]; OU: Timber, packing case, litter for cattle, fuel, cones for decoration purpose M: Tonic, carminative, appetizer, aphrodisiac, ulcer, skin diseases [resin, Sd]; OU: Edible [Sd] M: Stomachache, asthma, bronchitis, ulcer, itching, pile, toothache [resin, Wd]; OU: Timber, cones for decoration M: Joint pain, cough, asthma, piles, ulcer, toothache, cuts, wound, fractures [Wp]; OU: For making booms, brush, furniture, house construction, packing cases, bridges and beams, roof thatching materials, fuel

14 287

Festuca rubra L.

Pennisetum glaucum (L.) R. Br. syn. Setaria glauca (L.) P. Beauv. Phleum alpinum L.

Poa alpina L.

Poa annua L.

228.

229.

231.

232.

230.

Dactylis glomerata L.

227.

Chirua

–

–

Bandra

–

–

Dhroov ghas

–

Cynodon dactylon (L.) Pers.

224.

2875– 3527 m 2800– 3500 m 2750– 3700 m 2900– 3500 m 2900– 4000 m 2700– 3445 m

2200– 3550 m 2000– 3100 m

3100– 3400 m 2100– 3400 m

–

226.

Veronica beccabunga L.

223.

Binu Bajha

Veronica anagallis-aquatica L.

222.

Altitudinal range (m) 2675– 3527 m

Local name/ common name Isabgol, Luhuriya

Poaceace 225. Chrysopogon gryllus (L.) Trin.

Family/Taxa Plantago major L.

Table 14.2 (continued)

Reg Bor Temp

Reg Bor et Arct

Europ; Asia Temp Reg Bor et Arct

Reg Bor Temp

Europ; Asia bor

Reg Trop et Subtrop Cosmo

Reg bor temp

Reg bor Temp

Phytogeographic affinities Europ

H

H

H

H

H

H

H

H

H

H

Life form H

Fodder

Fodder

Fodder

Fodder

Fodder

M: Menstrual complaints, headache, eye problem, nasal bleeding, cuts, wounds, piles, diuretic, fever, diarrhea, urinary complaints [Ap]; OU: Fodder, religious ceremonies Fodder

Fodder

M: Wounds, cuts, burns, piles [Wp]; OU: Used as vegetable [Ap]

Indigenous uses M: Gastric pain, dysentery, joint pain, laxative, fever, cough, cuts, astringent, tonic, wounds, fever, weakness, [Sd, Lf, Fr, Wp] M: Skin problem, scurvy [Lf]

288 U. Devi et al.

243.

242.

241.

2750– 3100 m 2700– 3520 m 2560– 4000 m.

–

Khuliya

–

2650– 3100 m

–

Persicaria capitata (Buch. -Ham. ex D. Don) H. Grosssyn. Polygonum capitatum Buch. -Ham. ex Don@ Persicaria hydropiper (L.) Delarbre syn. Polygonum hydropiper L. Persicaria wallichii Greuter & Burdet syn. Polygonum polystachyum Wall.ex Meissn. Polygonum affine D. Don

240.

2700– 3500 m

–

Persicaria alpina (All.) H. Gross. syn. Polygonum alpinum All.

239.

2789– 3500 m

Oxyria digyna (L.) Hill

238.

2560– 4000 m 2100– 3400 m.

2700– 3900 m 3800– 4600 m 2700– 3400 m

Shupchi

Ogal

Fagopyrum esculentum Moench

–

237.

Trisetum spicatum (L.) K. Richt.

235.

–

–

Poa supina Schrad.

234.

–

Polygonaceace 236. Bistorta affini Greene@@

Poa himalayana Nees ex Steud. @

233.

Reg Himal

Reg Temp bor et Austr Ind or Asia trop

Reg Himal

Europ; Austr; Asia bor

Reg Bor Alp et Arct

Europ; Asia Bor

Reg Himal

Austr

Cosmop

Reg Himal

H

H

H

H

H

H

H

H

H

H

H

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

M: Cold, diarrhea, flatulence, dysentery [Wp, Fl, RT, Sd]

M: Skin disease, ringworm [Lf, Fl]; OU: Fodder [AP] M: Acidity, indigestion [Ap]; OU: Fodder [Ap]

M: Cough, cold, vomiting, diarrhea (Wp) M: Joint pain, sunburn, lung disease, typhoid, sunburns, colic [Rt, Sd, Lf]; OU: Flour, vegetable [Sd, St] M: Indigestion, constipation, laxative, edible, skin problem, fever, appetite fever, liver tonic [Wp]; OU: Vegetable [Lf] M: Diarrhea, joint pain, purgative, fever, menstruation disorder [Ap]; OU: Fodder [Ap] M: Snakebite, boils [Wp]; OU: Fodder [Ap]

Fodder

Fodder

Fodder

14 289

Arch, Chukri

–

Rheum australe D. Don. syn. Rheum emodi Wall. ex Meisn.@EN

Rumex hastatus D. Don@

Rumex nepalensis Spreng

247.

248.

249.

Guna

–

Primula denticulate Sm.@

Primula rosea Royle@

252.

–

251.

Primulaceae 250. Androsace rotundifolia Hardw.@

Jangli palak

–

Polygonum recumbens Royle ex Bab.@

246.

3510– 4000 m

2950– 3300 m 3200– 3400 m

2145– 3350 m 2150– 3527 m

3100– 3400 m 3100– 3500 m 2876– 3527 m

–

245.

244.

Altitudinal range (m) 2400– 3200 m

Local name/ common name Sarbguni

Family/Taxa Polygonum amplexicaulis (D. Don) Ronse Decr syn. Bistorta amplexicaule (D. Don) Greene@ Polygonum plebeium R.Br.

Table 14.2 (continued)

Reg Himal

Reg Himal; China Reg Himal

Europ Asia bor

Reg Himal

Reg Himal

Reg Himal

Reg Himal

Phytogeographic affinities Reg Himal

H

H

H

H

H

H

H

H

Life form H

M: Cough, stomachache, gastric problem [Ap] M: Diabetes, headache, urinary ailments, appetizer, cough, liver problem, pulmonary disease, kill lice [Ap, Fl, Rt] Ornamental

Indigenous uses M: Sores, wounds, cough, dysentery, tonic, heart, fever, joints pain [Rt, Ap]; OU: Fodder [Ap] M: Sikn problem, baldness, diarrhea, dysentery [Wp] M: Skin rashes, cuts, wounds, scabies, blood purifier [Wp] M: Bone fracture, muscular injury, cuts, wounds, headache, stomach pains, constipation, dysentery, tonsillitis, asthma, cough, fever, piles, skin diseases, ulcers [St, Rt, Ap] M: Nasal bleeding, skin problem, cuts, wounds [Lf]; OU: Edible [Lf] M: Scurvy, boils, colic, cooling, diuretic, pimple, swelling, stomachache [Lf, Rt, St]; OU: Tender shoots as vegetable, fodder [Ap]

290 U. Devi et al.

Rattanjog

Jakri

Lamo

Actaea spicata L.

Anemone obtusiloba D. Don@@

Anemone rivularis Buch. -Ham. ex DC.@

Aquilegia fragrans Benth.@@

Caltha palustris L.

258.

259.

260.

261.

262.

Munire, Pipling-tasha

Onayalkas, Mitha Patish Mamira

@@VU

Aconitum violaceum Jacq. Ex Stapf

257.

Atis, Patish

2700– 3600 m 2500– 3550 m

2700– 3700 m

3000– 3525 m

3000– 4200 m 3000– 3600 m

2900– 3950 m

Adiantum venustum D. Don

255.

Ranunculaceae 256. Aconitum heterophyllum Wall. Ex Royle.@@CR

2610– 3310 m 2680– 2850 m

–

Adiantum pedatum L.

254. Damtuli, Hansraj

2640– 2930 m

Hansraj

Pteridaceae 253. Adiantum capillus-veneris L.

H H

Reg Bor Temp et Arct

H

H

H

H

H

F

F

F

Reg Himal

Reg Himal

Reg Himal

Reg Bor Temp

Reg Himal

Reg Himal

Afghan India Border Afghan India Border

S Europ

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

M: Cold, cough, fever, diarrhea, diabetes, dysentery, stomachache, vomiting, toothache, piles [Rt] M: Stomachache, renal pain, cough, cold, fever, piles, joint pain, tonic [Rt] M: Joint pain, eye complaint, asthma, body pain, nerve tonic, sedative [Rt]; OU: Dye [Rt] M: Urogenital infections, toothache, headache, joint pain, menorrhea, cooling, emetic and purgative [Wp, Rt, Sd] M: Bone fracture, indigestion, gastric problem, headache, wounds, ear complaints, appetizer, sores, [Wp] M: Rheumatism, kidney stones, headache [Rt, Sd, Fl] M: Joint pain, leprosy, cattle wounds, hand cleaning [Rt, Lf]

M: Stomachache, cold, bronchitis tonic, skin eruptions, diuretic, hypothermic, headache, piles, wound, fever, hair fall [Wp]

M: Sore throat, cold, cough, fever, menstrual problem, hypertension, bronchitis, tonic [Wp]; OU: Fodder [Fd] M: Fever, skin eruption [Wp]

14 291

3000– 3600 m 2770– 3350 m 2708– 3527 m 2575– 3500 m 3150– 3400 m 3080– 3300 m

Raskalch

Losker

–

– – – Mamiri

Delphinium cashmerianum Royle@@

Delphinium denudatum Wall. ex Hook f & Thomson@@

Ranunculus diffuses DC.

Ranunculus laetus Wall. ex Hook. f. & J.W. Thomson Thalictrum alpinum L.

Thalictrum cultratum Wall.@

Thalictrum foetidum L.

Thalictrum foliolosum DC.@

264.

265.

266.

267.

268.

270.

271.

272.

269.

2890– 3450 m

Kasturilata

Delphinium brunonianum Royle@

263.

Jaldhar, Jaldra

Altitudinal range (m) 2770– 3340 m 3200– 3850 m 2700– 4000 m

Local name/ common name –

Family/Taxa Clematis grata Wall.

Table 14.2 (continued)

Reg Himal

EuropSibir

Reg Himal

Reg Bor et Arct

Reg Himal

Ind or; Malaya

Reg Himal

Reg Himal

Phytogeographic affinities Reg Himal China; Afr Reg Himal

H

H

H

H

H

H

H

H

H

Life form C

M: Stomach pains, blood purifier, boils, eczema, earache, eye problem, fever, leucorrhea, piles, joint pain, gout, tonic, toothache, foot and mouth disease of animals [Rt]

M: Fever, eye disease[Ap]

M: Urinary infection [Rt]

M: Fever [Rt, Lf]

M: Skin problem [Lf]

M: Skin diseases, cuts, wounds, dysentery, worm, fever abdominal pain, renal pain, cough, cold, fever, headache, swelling (Wp, lf, St); OU: Ornamental M: Joint pain, stomachache, toothache, fever, ulcer, fever, tonic, anthelminthic, respiratory disorder, hair loss (Rt) M: Boils [Wp]

Indigenous uses M: Baldness, ringworm, skin disease [St] M: Toxic, destroy ticks on animals [Lf]

292 U. Devi et al.

Potentilla argyrophylla Wall. ex Lehm.@ Potentilla atrosanguinea G. Lodd. ex D. Don@@ Potentilla indica (Andrews) The.Wolf syn. Fragaria indica Andrews Prinsepia utilis Royle @

278.

Prunus armeniaca L.

Prunus cornuta (Wall. ex Royle) Stued

282.

283.

281.

280.

279.

277.

Fragaria nubicola (Lindl. ex Hook.f.) Lacaita Potentilla anserina L.

276.

Krun, Jamu

Chuli

Bhenkul

Bhumra

Lamasu

Cinguefoil, silverweed –

Bale Bhasu

Re

Cotoneaster microphyllus Wall.ex Lindl.@

275.

–

Reunsh

Thalictrum minus L@

Rosaceae 274. Cotoneaster bacillaris Wall. ex Lindl.@

273.

2800– 3250 m

2000– 3250 m

2280– 3980 m 2130– 3700 m 3100– 3800 m 3000– 3900 m 2150– 3150 m 1800– 2200 m

2700– 3800 m

2700– 3516 m

2700– 3450 m

T

T

Europ Asia Bor Ind or

S

H

Reg Cauc

Ind; Malaya China Reg Himal

H

H

Reg Himal Reg Himal

H

H

S

S

H

Europ

Reg Temp

Reg Himal

Reg Himal

Reg Himal; Asia

Assessment of Economically and Medicinally Important Plant Resources. . . (continued)

M: Toothache, wound healing, burns [Rt, Fl] M: Diarrhea, earache, leucorrhea [Lf, Fr]; OU: Edible [Fr] M: Rheumatic pains, diarrhea, wounds, burns, cuts, rheumatism [Rt, Sd, Bk, Fr]; OU: Fuel [Wd] edible [Fr] M: Body massage, arthritis, fever, cosmetics, hair growth [Sd, Lf] OU: Cattle feed, fuel, for making boxes, fresh or dehydrated fruits are edible, seed oil for cooking M: Cuts, wounds, joint pain, [Sd, Lf, St, Fr]; OU: Fuel [Wd], fodder [Lf], edible [Fr]

M: Astringent, scabies, arthritis [Lf, St]; OU: Fuel, Fodder, timber, walking sticks [Lf, Wd] M: Cuts, wounds, gastric problem, diarrhea [Fr, Rt, Lf] OU: Fodder, edible, fuel, walking sticks [Lf, Fr, Wd] M: Fever, purgative [Ap]; OU: Edible [Fr] M: Kidney stones, leucorrhea, diarrhea, arthritis [Wp] M: Cuts, wounds healing (Lf)

M: Joint pain, eye disease, fever [Rt, Ap]

14 293

Sea, Pashu

– –

Rosa niveus Thunb.

Rosa paniculatus Sm

Rosa webbiana Wall. ex Royle@

Rubus ellipticus Sm.

Sibbaldianthe bifurca (L.) Kurtto & T. Erikss. syn. Potentilla bifurca L. Sorbaria tomentosa (Lindl.) Rehder@

Spiraea canescens D. Don@@

286.

287.

288.

289.

290.

291.

293.

Rubiaceae 294. Galium aparine L.

292.

Manger

Rosa moschata Herrm. Syn. R. brunonii Lindl.

285.

Kathir, Nilakari

–

Chosho

Kala hinur

Kuja

Jungli-gulab, Kuja

Rosa macrophylla Lindl.@@

284.

Local name/ common name Behmi, Reck

Family/Taxa Prunus mira Koehne

Table 14.2 (continued)

2680– 3516 m

2200– 2676 m 3200– 4000 m 2675– 3200 m 3200– 3700 m

2600– 3000 m 2700– 2900 m 2980– 3527 m

2680– 3500 m

Altitudinal range (m) 2100– 3100 m 3050– 3520 m

Reg bor Temp et Magell

Reg Cauc; Asia Asia Reg Himal Asia bor Reg Himal

Ind or

Reg Himal

Reg Himal

Reg Himal

Oriens

Reg Himal China

Phytogeographic affinities

H

S

S

H

S

S

S

S

S

S

Life form T

M: Jaundice, diuretic, antiscorbutic, skin disease [Wp]

M: Antiseptic, cuts, wounds, asthma [Fr, St] M: Cuts, wounds, sores [Bk]

M: Headache, hepatitis, jaundice, asthma, stomachache stomach pain [Fr, Fl]; OU: Fodder, fuel, fencing M: Stomach pain, dysentery, dysentery, malaria [Fr, Rt]; OU: Edible [Fr] M: Headache, snakebites [Rt, St]

M: Stomach pain, fever, bile disorders [Fl, Fr]; OU: Edible [Fr], fuel, fencing as hedges M: Stomach disorder, diarrhea, wounds, eye disease [Fl, Lf, Fr]; OU: Edible [Fr], fuel, fodder, religious ceremonies M: Menstrual disorder [Rt, Fr]; OU: Edible [Fr] M: Diarrhea, stomachache [Lf, Fr]

Indigenous uses Edible [Sd, Fr]

294 U. Devi et al.

Rubia cordifolia L.@

296.

Salix acmophylla Boiss.

Salix alba L.

Salix daphnoidesVill.

Salix fragilis Forssk.

300.

301.

302.

303.

Acer caesium Wall. ex Brandis@@VU Mndru

Mandru

Chanker, RichangJangli Beli –

2689– 3189 m 3189– 3300 m

3300– 3900 m

2700– 3100 m 2900– 3509 m 2700– 3600 m

2700– 2900 m 2670– 3163 m

Safeda, Jangifrast Poplar

Bada, Bed, Jangli Beli Bis, Bhushan

2650– 3502 m

2708– 3527 m 2680– 3000 m

Pisumarbuti

–

Reg Himal

Reg Himal

Europ; Asia bor

Europ; Asia et Afrbor Europ; Asia bor

Oriens Ind or

Reg Himal

Europ; Asia bor

Reg Himal; Japon

Europ; Asia Temp Asia Trop et Temp

T

T

T

S

T

T

T

T

H

H

H

(continued)

M: Skin problem, abortifacient [Bk, Ap]; OU: Fodder [Lf], fuel [Wd] M: Skin problem [Bk]; fodder [Lf]; fuel [Wd]

M: Dental problem [Lf]; OU: Fodder [Lf]

Fuel, fodder

M: Fever, joint pain, gastric and hepatic disorders [Wp] M: Blood purifier, tonic, bone fracture mouth and foot diseases of livestock, [St, Lf, Bk]; OU: Fuel, fodder [Wd, Lf] M: Fever [Ap]; OU: Fuel, making kitchen utensils Green fodder

M: Wound healing, toothache, [Ap]; OU: Kills fleas, lice, other insects [Ap]

M: Astringent, stomachache, insects & snakebites, leucoderma, inflammation, jaundice, liver complaints, menstrual disorder, paralysis, urine complaints, ulcers [Rt, St]; OU: Dye [Rt]

M: Diuretic, skin problem [Wp]

Assessment of Economically and Medicinally Important Plant Resources. . .

305.

Sapindaceae 304. Acer acuminatumWall.ex D. Don@

Populus ciliata Wall. ex Royle@

299.

Rutaceae 297. Boenninghausenia albiflora (Hook.) Rchb. ex Meisn. Salicaceae 298. Populus alba L.

Galium asperifolium Wall.

295.

14 295

Tambhaku

Dhatura

Solanaceae 312. Datura stramonium L.

2350– 2650 m

2000– 3523 m

3510– 4000 m

–

Saxifraga flagellaris Willd.Ex Sternb.@

310.

Scrophulariaceae 311. Verbascum thapsus L.

2700– 4000 m

Lao, Pashanbed

2689– 3850 m

Bergenia stracheyi (Hook. f. & Thomson) Engle.@VU

KhanorBankhor

309.

307.

Altitudinal range (m) 2600– 3289 m 2800– 3100 m

Lao, Pashanbhed

Aesculus indica (Wall. ex Camb.) Hook.

306.

Local name/ common name Mandru

Saxifragaceae 308. Bergenia pacumbis (Buch.-Ham. ex D. Don) C.Y. Wu& J.T. Pan syn. Bergenia ligulate (Wall.) Engl.@

Family/Taxa Acer cappadocicum Gled.

Table 14.2 (continued)

Cosmo Trop et Temp

Europ; Reg Himal

Reg Himal

Reg Himal

Reg Himal

Reg Himal

Phytogeographic affinities Asia Min

H

H

H

H

H

T

Life form T

M: Toothache, asthma, diarrhea, rheumatism, sleepiness, boils, earache [Lf, Sd]

M: Rheumatism, diarrhea, cough, constipation, stomach pains, asthma, piles, febrifuge, sunburns, pulmonary disease of cattle [Lf, Sd, Rt]; OU: Used in religious ceremonies [Ap]

M: Burns, cuts, wounds, boils, fever, piles, joint pain, kidney and bladder stone, cough, earache, hair tonic, veterinary use; OU: Edible, fencing crop fields M: Indigestion, fever, boils, wounds, antiseptic, kidney stones, joint pain, scurvy, astringent, fever [Rt, Lf] M: Antiseptic, wounds, jaundice, (Ap)

M: Skin cracks, wound, joint pains, vermifuge, diuretic, dislocated joints, leucorrhea, veterinary medicine (Sd); OU: Fuel, furniture, agricultural implements (Wd), fodder, manure (Lf), edible (Sd)

Indigenous uses Fodder [Lf], fuel [Wd]

296 U. Devi et al.

Physochlaena praealta (Decne.) Miers.

Solanum nigrum L.

@VU

Violaceae 319. Viola canescens Wall.@ Banfasa

Nupun

Urticaceae 317. Urtica dioica L.

Verbenaceae 318. Verbena officinalis L.

Himalayan elm

Yamdal, RakhalSangcha

Makoi

–

Ulmaceae 316. Ulmus wallichiana Planch.@@EN

Taxaceae 315. Taxus baccata L.EN

314.

313.

2700– 3250 m

2550– 3115 m

2275– 3154 m

2490–2790

2400– 3550 m

3700– 4000 m 2700– 3200 m

Reg Himal; Ind or Malaya China

China AsiaTemp

Reg bor Temp

Ind or

Reg Bor Temp

Amerphig

Reg Himal

H

H

H

T

T

H

H

(continued)

M: Cold, cough, asthma, bronchitis, cuts, wounds, malaria, fever, jaundice [Wp]

M: Febrifuge, nerve tonic, joint pains, antidote to snakebite [Wp]

M: Skin problem, jaundice, blood purifier, baldness, [Lf, Rt, Sd]; OU: Cooked as vegetable (Lf)

M: Joint dislocation, bone fracture in animal and human beings [Ap, Bk]; OU: Fodder, fuel, timber [Lf, Wd]

M: Bone fracture, cold, asthma, bronchitis, anticancerous, fever, headache, blood purifier, [Bk, Lf]; OU: Used for making black tea, fuel, edible [Lf, St, Bk]

M: Boils, vermifuge, ulcers [Wp, Sd, Lf] M: Urinary complaints, skin problem, jaundice, diarrhea, piles, dysentery, fever, pimples, asthma, whooping cough [Lf, Rt, Fr]; OU: Edible [Fr]

14 Assessment of Economically and Medicinally Important Plant Resources. . . 297

Local name/ common name Bansfasa

Altitudinal range (m) 2800– 3600 m

Phytogeographic affinities Ind or Malaya China

Life form H Indigenous uses M: Fever, cold, sore throat problem, headache [Wp]

Afr Africa, Alp Alpine, Am America, Amphig Amphigaea, Amur N.Mongolia Russia and China, Ap Aerial part, Arab Arabia, Arct Arctic, As Asia, Austr Australia, Baluchist Baluchistan, Bk Bark, Bl Bulb, Bor Boreal (North Temperate Zone), Caucas Caucasus, Centr Central, Cosmop Cosmopolitan, CR Critically Endangered, EN Endangered, et And, Europ Europe, F Fern, Fd Fodder, Fr Fruit, Geront Gerontia (Greece), H Herb, H Herb, Himal Himalayan, Hisp Hispanic (Latinamerica), Ind India, Inf Inflorescence, Lf Leaf, LF Life form, M Medicinal Uses, Mediterr Mediterranean, Min Minor, Mongol Mongolia, N.Zel New Zealand, Occ Occidental (Western hemisphere), or origin, Oriens Oriental, OU Other Uses, Reg Region, Rt Root, Rz Rhizome, S Shrub, Sd Seed, Sh Shrub, Sibir Siberia, Soongar Soongarica, St Stem, SubTrop SubTropical, T Tree, Tb Tuber, Temp Temperate, Trop Tropical, Turkist Turkistan, VU Vulnerable, Wd Wood, Wp Whole plant, @@ Endemic, @ Near Endemic

320.

Family/Taxa Viola pilosa Blume syn. V. serpens Wall. ex Ging.

Table 14.2 (continued)

298 U. Devi et al.

14

Assessment of Economically and Medicinally Important Plant Resources. . .

299

were Actaea spicata, Angelica glauca, Betula utilis, Bunkum persicum, Carum carvi, Ferula jaeschkeana, Fritillaria roylei, Pleurospermum brunonii, Sinopodophyllum hexandrum, etc. It was followed by 1800–2800 m altitude zone having 197 species. The commoners were Achyranthus aspera, Amaranthus spinosus, Rubus ellipticus, Datura stramanium, Desmodium elegans, Prinsepia utilis, etc. Whereas the zone above 3800 m altitude having only 43 species due to extremely harsh and non-conducive climatic conditions (Fig. 14.2). The valuable species at this zone are Aconitum violaceum, Arnebia benthamii, Dactylorhiza hatagirea, Saussurea gossypiphora and Saussurea obvallata, etc. The occurrence of less plant species in this area is due to low moisture retention, poor rainfall, minimum humidity, very cold climate, and high-altitude condition.

14.3.2 Phytogeographic Affinities Everyone knows that the Himalaya is an extremely rich source of plant biodiversity. In this region a large number of native plants are evolved naturally in a particular biogeographic region before human introduction from distant places. Knowing the status of any plant species as to whether it is native or introduced in a particular area is a major interest for conservation program. According to Levine et al. (2003), species invasions beyond their native range constitute a global driver of change as non-native species threaten biodiversity and change ecosystem functioning. Out of 320 species, 182 were reported native as they have the Himalayan origin, whereas, remaining are being non-natives as they belong to different biogeographic domains of the world. As a result, the trend in plant nativity in the Sangla Valley region was reported as follows as the Himalayan region (182 spp.) > European/Oriental region (28 spp.) > Asia (25 spp.) > European region (16 spp.) > Temperate region (13 spp.) > Indian region (10 spp.) > India/Oriental region (8 spp.) > America (7 spp.) > European/Oriental/African, Temperate and Arctic (6 spp. of each) > Cosmopolitan (5 spp.) > Australian (4 spp.) > Amphigaea (3 spp.) > Arctic, European/ Oriental/American and Oriental (2 spp. of each) and European/African (1 spp.). Such trend of nativity is depicted in Table 14.2 and Fig. 14.3. Many plant species of the valley were recorded as endemic (45 spp.) and near endemic (185 spp.), whereas 5 spp. were critically endangered, 11 spp. endangered and 10 spp. vulnerable in Table 14.2. Native and endemic species are part of the ecological heritage of any region, and if those species lost, there will be a global loss of species (Volenzo and Odiyo 2020). The presence of a high proportion of native and endemic species in a particular area is indicative of its high conservation value. Plant species such as Heracleum candicans, Heracleum thomsonii, Pleurospermum brunonis, Allium humile, Berberis lycium, Stellaria media, Artemisia japonica, Saussurea costus, Pedicularis bicornuta, Rosa macrophylla, Delphinium cashmerianum, Delphinium denudatum, etc. are native as well as endemic species having multipurpose uses such as medicinal, fuel, fodder, edible, incense, ritual, fencing insecticide, ornamental, etc. Overexploitation of these species put the area under biotic pressure, which would result in

300

U. Devi et al.

Fig. 14.2 Altitudinal distribution economically important plant resources of Sangla Valley region. (T tree, S shrub, H herb, Fn fern)

deterioration of phytodiversity of the valley. Similarly, species Aconitum heterophyllum, Aconitum violaceum, Angelica glauca, Picrorhiza kurrooa, etc. have been reported endangered as well as endemic to the Himalayan region. These species are under great stress because of their extraction from wild for local uses as well as for sale purpose because of their high market price. Endemic and rare plant species are typically more vulnerable to anthropogenic threats due to their restricted distribution (Isik 2011; Coelho et al. 2020). It leads to their risk of extinction if the main concern for their conservation would not address.

14.3.3 Utilization Pattern A total of 320 species were reported as economically important plants in the Sangla Valley region. All the plant species have been used in different purposes such as medicinal (278 spp.) followed by fodder (69 spp.), fuel wood (36 spp.), edible (46 spp.), vegetable (15 spp.), timber (11 spp.), ornamental (07 spp.), fence/hedge (01 spp.), agricultural tool (04 spp.), flavoring agent (08 spp.), tea substitute (03 spp.), fiber yielding (05 spp.) and poisonous (03 spp.) (Fig. 14.4). In spite of above mentioned uses, many plants were also used in religious ceremonies, preparation of local beverages, spices/condiments, oil extraction, etc. (Table 14.2). In the present study, about 86.9% (278 spp.) species were recorded for medicinal purposes and about 40% (130 spp.) have multiple uses. Several plant species have been used to cure more than two diseases. Medicinal plant species such as Anaphalis busua, Bistorta affinis, Cannabis sativa, Delphinium cashmerianum, Geranium wallichianum, Mentha longifolia, Origanum vulgare, Oxyria digyna, Polygonum amplexicaule, Polygonatum verticillatum, Saussurea costus, Thymus linearis, Geranium nepalense, Rheum australe, Rumex nepalensis, etc. were more commonly used due to their abundance in local forests. Tree species such as Aesculus indica, Abies

14

Assessment of Economically and Medicinally Important Plant Resources. . .

301

Fig. 14.3 Biogeographic affinities pattern of economically important plant resources of Sangla Valley region. (Afr Africa, Amer America, Amphig Amphigaea, Arct Arctic, Aust Australia, Cosmo Cosmopolitan, Euro Europe, Orient oriental, Temp temperate)

spectabilis, Betula utilis, Cedrus deodara, Juglans regia, Pinus gerardiana, Prunus cornuta, etc. and shrub species such as Artemisia maritima, Berberis aristata, Hippophae salicifolia, Juniperus communis, Juniperus indica, Rhododendron campanulatum, Desmodium elegans, Ribes alpestre, Prinsepia utilis, Rosa moschata, etc. were widely distributed and have been used for multiple purpose. Multiple uses of plant resources and their overexploitations have also created some impact on the local ecosystem. Thus, a sustainable approach must be adopted to utilize natural resources in such a way that they can regenerate their productive capacity.

14.3.4 Medicinal Use and Their Applications Different plant parts of medicinally important plant species such as whole plants (131 spp.), aerial part (107 spp.), stem bark (10 spp.), leaves (93 spp.), inflorescences (42 spp.), fruits (65 spp.), seeds (28 spp.), roots (97 spp.) and tubers (9 spp.), etc. have been used for different medicinal purposes (Table 14.2; Fig. 14.5). Most of medicinal plant species of the Sangla Valley were utilized by the many stakeholders traditionally to cure of various ailments such as fever, asthma, cough and cold, blood purification, rheumatism, piles, toothache, liver complaints, gastric complaint, ulcer,

302

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Fig. 14.4 Utilization pattern of economically important plant resources of Sangla Valley region. (M medicinal, Fd fodder, Ed edible, Fl fuel, Ti timber, Flav flavoring agent, Orn ornamental, Fib fiber, AgT agricultural tools, Tea tea substitutes, Poi poisonous)

worms, tonic, piles, etc. by various mode of application such as decoction, powder, extraction, raw edible, paste etc. Traditionally the applications of medicinal plants have been used in different forms such as decoction, paste, massage, extraction, etc. If external use of plant-based drugs, it would convert into a paste or directly rubbed or massaged on the affected area of the human body. Such as leaves paste of Abies pindrow has been applied for general swelling after an injury. Leaf paste of Anaphalis contorta, Anaphalis busua, Polygonum amplexicaule and Origanum vulgare have been applied on cuts, wound, boils and sores. Root paste of Achyranthus aspera applied on joint pains, whereas root past of Saussurea obvallata has been applied in cuts and bruises. As per local tribal community, powder of flowering shoots of Pleurospermum brunonii mixed with fresh butter made up of cow’s milk and massaged over entire body to alleviate fever and pain. The oil extracted from the seeds of Prunus armeniaca used as a massage, a very effective remedy for relieving joint pain. Leaves of Rumex nepalensis and Cannabis sativa have been rubbed on body part for irritation caused by Urtica dioca. If oral administrated, the plant parts have either eaten as a raw drug or made up in powder or decoction form. Such as Sinopodophyllum hexandrum root powder has been orally consumed to cure gastric ulcers and leucorrhea. Whereas Dactylorhiza hatagirea tuber powder given to the person who suffering from general weakness, diabetes, loose motions etc. Powder of Arnebia benthamii rhizome mixed with honey and utilized in cure of asthma. Picrorhiza kurrooa roots powder has been used in cure of cold-cough, jaundice and blood purification. A decoction made up of Rhododendron anthopogon leaves has been used to cure cold. The fruits of

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Fig. 14.5 Radar diagram exhibited different parts of plant resources are commonly used for making traditional drug formulations. (Ap aerial parts, Bk bark, Fr fruits, Inf inflorescence, Lea leaves, Rt roots, Sd seeds, Tb tubers, Wp whole plant)

Hippophae salicifolia were used in cure whooping cough and skin diseases. The flower buds of Achillea millefolium chewed in toothache by the villagers. In case of joint dislocation, the wood strip of Pinus wallichiana, Cedrus deodara, and Pinus roxburghii has been used as a bandage. The bark of Juglans regia (locally known as Dandasa/Akhrot) has been used as miswak for teeth cleaning. Nowadays, most of the highly valued medicinal plants (i.e. Aconitum heterophyllum, A. violaceum, Arnebia benthamii, Artemisia maritima, Bergenia stracheyi, Betula utilis, Bunium persicum, Carum carvi, Cassiope fastigiata, Corydalis govaniana, Dactylorhiza hatagirea, Dioscorea deltoidea, Ferula jaeschkeana, Elaeagnus rhamnoides, Heracleum candicans, Hypericum perforatum, Jurinella macrocephala, Sinopodophyllum hexandrum, Picrorhiza kurrooa, Rheum australe, Saussurea obvallata, etc.) of Sangla Valley are used by many pharmaceutical industries. Also, such species are utilized by local people for their traditional healing purposes. Many of traditional uses described in the present study are in compliance with earlier findings on the Indian Himalaya Region (Boktapa and Sharma 2010; Samant et al. 2011; Rana and Samant 2011; Thaplyal et al. 2012; Devi et al. 2013; Sharma et al. 2015; Pant and Wani 2020; Haq et al. 2021).

14.3.5 Fodder and Fuel Resources The Sangla Valley region is a completely rural area. The livestock was one of the major sources of their livelihood and integral part of their economy. Up to some extent the fodder requirement of the area was met from the agricultural and agroforestry systems. Populus, Salix, Robinia species were the most common species planted along with roadside and they were used as fodder and fuel wood. But

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most of their livestock rely on fodder from wild. Although unsustainable harvesting of fuel wood or fodder have serious concern for the ecology, forest, and vegetation degradation. Therefore, such localized deforestation leads to accelerated soil erosion also (Arnold et al. 2006; San et al. 2012). In the valley, utilization pattern of fodder species varies from season to season. In rainy season, mostly grasses with herbs have been used as fodder. Herbs such as Galinsoga parviflora, Hackelia uncinata, Heracleum candicans, H. thomsonii, Medicago falcata, M. lupulina, Polygonum amplexicaule, Persicaria capitatum, P. polystachyum, P. hydropiper, Selinum wallichiana, S. vaginatum, Trifolium pretense, T. repens, and T. emodi etc., along with grasses such as Poa alpina, P. annua, P. himalayana and Dactylis glomerata, etc. have been used during the rainy season. During summer and winter mostly shrubs e.g., Abelia triflora, Cotoneaster bacillaris, Desmodium elegans, Indigofera heterantha, Lonicera hypoleuca, Prinsepia utilis, Rosa webbiana, R. macrophylla, Ribes alpestre, Viburnum cotinifolium and trees like, Acer acuminatum, A. caesium, Aesculus indica, Quercus floribunda, Olea ferruginea and Prunus cornuta have been used as fodder. Besides this, fuel wood obtained from forest has the major resource of household energy. Many trees i.e., Acer spp., Cedrus deodara, Abies pindrow, Pinus roxburghii, P. wallichiana, Picea smithiana, and Betula utilis etc., and shrubs i.e., Berberis aristata, B. lycium, Cotoneaster bacillaris, Desmodium elegans, Hakea salicifolia, Rosa sp., Viburnum cotinifolium and Rhododendron campanulatum were used as fuel wood along with twigs of the various horticultural crops. Rawat and Vishvakarma (2011) have shown the similar finding during their explorations in the Kullu and tribal communities of Lahaul valley. Samant et al. (2000) explored in Askot Wildlife Sanctuary in Kumaun Himalaya, whereas Sharma and Samant (2014) reported in Nargu Wildlife Sanctuary of Himachal Pradesh.

14.3.6 Wild Edible Resources Many scientific studies reported more than 300 million people all over world are dependent for food or other livelihood options on forest resources (Burlingame 2000; Abbasi et al. 2013). Indigenous plant resources in rural areas form an integral part of their livelihoods. In the study area, edible fruits and seeds from wild habitat have been obtained from a number of species such as Berberis jaeschkeana, B. aristata, B. lycium, Chenopodium foliolosum, Duchesnia indica, Ephedra intermedia, Fragaria nubicola, Sinopodophyllum hexandrum, Prunus mira, Prunus cornuta, Ribes alpestre, Rubus niveus, R. ellipticus, Rosa macrophylla, R. webbiana and Solanum nigrum etc. Different products such as Juice, sauce and tea made up from fruit extracts of Elaeagnus rhamnoides, Hippophae salicifolia, H. tibetiana is considered as high nutritional drinks. Juice made from the flowers of Rhododendron campanulatum is considered good for chronic rheumatism, sciatica and syphilis and good cooling agent. Prunus armeniaca fruits have been eaten fresh or in dehydrated form, to store for winter. Oil obtained from its kernels has been used in cooking and considered good for hair nutrition also. Its seed cake has also a good cattle food. The

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oil obtained from seeds of Prinsepia utilis is used for cooking purpose. Nuts of Pinus gerardiana (Chilgoza) and Juglans regia have high nutritious value. The Chilgoza seeds is their own traditional value as they have been used in every traditional auspicious occasion of the area and people wore it as an ornament also. Besides, the seeds of Chilgoza are very important commodity for local communities from a commercial view point. It carries good sale value in the local market. Leaves of many plants such as Amaranthus spinosus, Capsella bursa-pastoris, Chenopodium album, Lactuca dolichophylla, Malva verticillatta, Nastrutium officinale, Oxyria digyna, Silene vulgaris, Sisymbrium orientale, Sisymbrium irio, Stellaria media, Taraxacum officinale and tender shoots of Rumex nepalensis, Rheum australe have locally used as vegetables. Leaves of Lotus corniculatus, Oxalis corniculatam, Rumex hastatus, whereas, the flower buds of Anaphalis nepalensis, Anaphalis royleana and Anaphalis triplinervis have been eaten as a raw. Leaves of Lactuca dolichophylla, Sonchus oleraceous, Taraxacum officinale have been used as a salad. Shepherds of the area eat leaves of Chenopodium foliosum with boiled milk and consider it as a nutritious diet. Leaves of Elsholtzia eriostachya and Mentha longifolia have been used for making chutney (Indian cuisine that usually contains some mixture of spices) and considered as a table delicacy. Leaves of Allium stracheyi and Mentha longifolia have been used for seasoning. Roots of Angelica glauca and seeds of Bunium persicum, Carum carvi have been used as condiment. Stem bark of Taxus baccata has been used by the local people for making tea and considered to be anticancerous. Though, it is bitter in taste but assumed to be refreshing and energetic. Indigenous people of the Sangla Valley were roast seeds of Cannabis sativa with wheat and store it for use in the winter season when there is snow all around and scarcity of food items. These uncultivated and wild species are known for their high nutritive values and sometimes their content value is higher than several known common vegetables and fruits (Orech et al. 2007). Therefore, there seems an urgent need to improve the utility of wild edibles by making value added products for better economic benefits to the tribal farmers of the Sangla Valley.

14.3.7 Miscellaneous Uses Beside the above mentioned there are many more miscellaneous uses of the plant resources of the Sangla Valley. For example, timber of four species viz., Cedrus deodara, Juglans regia, Pinus wallichiana and Pinus roxburghii have been used in house construction, bridges, beams, etc. Cedrus deodara is the most valuable timber, which fetches a very high price. Walnut wood is one of the most valued timbers for making different type of furniture and other decorative items. Female cones of Pinus used for decoration purpose. Many agricultural tools, especially plows and handles were made from wood of Aesculus indica, Olea ferruginea, Quercus spp., Picias mithiana. The spiny nature of Berberis jaeschkeana, B. aristata, B. lycium, Rosa macrophylla, R. webbiana, and Rubus ellipticus makes them ideal as fence and hedge and prevents stray animals entering the crop fields. Other species like

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Cotoneaster bacillaris and C. microphylla used for making walking sticks. Also, there are a number of species which have ritual values viz., Artemisia roxburghiana, Cedrus deodara, Betula utilis, Cynodon dactylon, Ephedra intermedia, Rosa moschata, Saussurea obvallata, Rhododendron campanulatum etc. Young shoots of Berberis aristata and Principia utilis are kept in all four corners as well as at each door of the house with the belief that evil spirits would not enter the house. Different parts of many plants such as stem barks and young twigs (Cassiope fastigiata, Cedrus deodara, Juniperus indica, Juniperus communis, Juniperus squamata, Juniperus polycarpos), roots (Jurinella macrocephala and Selinum wallichianum) and whole plant (Waldheimia glabra) have aromatic properties and they were utilized in many religious ceremonies for making incense. Many other plants, including Epilobium angustifolium, Epilobium latifolium, Iris hookeriana, Iris kemaonensis, Jasminum officinale, Rosa spp. have been used as ornamental plants by local communities of the Sangla Valley. Cannabis sativa yields a high-quality fiber and also is a high value narcotic species. Here this plant also has commonly used for making ropes. Arnebia benthamii has been extensively used for hair growth and used as hair tonic. Dried plants of Pleurospermum brunonii are kept in the boxes with clothes as a preservative against the attack of moth, silver fish etc. Many species like Caltha palustris, Epilobium royleanum, and Vincetoxicum hirundinaria were considered as poisonous in nature. Due to the hardy and rough nature of Equisetum diffusum, it has been used as scrubber for cleaning the home utensils.

14.4

Discussion

Overexploitation of wild plants can affect growth, reproduction, and survival of the natural populations and their dynamics (Ticktin 2004; Schmidt et al. 2011). Nowadays, rapid increase of human population develops an extra pressure on forest resources and livelihoods as a result of their shortages (Chettri and Sharma 2006). Day-to-day need for forest-based resources, especially for fuel wood, fodder, medicine, food, timber, and house building has increased the pressure on forest trees and shrubs to a great extent. During the present study all these conditions were particularly predominant in the study area. The presence of 320 species of economically important plants indicates richness and high value of socio-economic of the Sangla Valley. These species were mostly used as medicine followed by fodder, wild edible/ food, fuel, timber, flavoring agent, ornamental, tea substitute, fibers, making agricultural tools etc. These economic as well as medicinal plant species found in the area are also comparable to other areas of Himachal Pradesh (Devi et al. 2019; Thakur et al. 2020; Haq et al. 2021). During survey and interaction with local people, it was observed that populations of many plant species like Aconitum heterophyllum, A. violaceum, Arnebia benthamii, Dactylorhiza hatagirea, Jurinea macrocephala and Picrorhiza kurrooa were being greatly reduced during last two decades. Regeneration of these species was highly affected due to migratory livestock, which graze upon the high-altitude pastures (Kanda) of Sangla, Rupin and Chansu etc. and destroy the vegetation

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through physical injuries. Many medicinal plant species Angelica glauca, Betula utilis, Hypericum perforatum, Polygonatum cirrhifolium, Polygonatum verticillatum, Dioscorea deltoidea, Rhododendron anthopogon, Rhododendron campanulatum etc. occurred in the valley are in IUCN listed globally as well for Himachal Pradesh (Ved et al. 2003), however, their occurrence in the valley was widespread. So, due to rapid destruction of natural habitat, such plant species may become locally threatened. The major problem is destructive harvesting of the underground parts of these plants or even the plant as a whole. In most cases, the aerial part of plant is used for medicinal purpose but whole plant is uprooted. Unsustainable uses of these plant resources may result in their permanent depletion from natural habitats. Therefore, successful implementation of alternative harvesting methods of wild plants is likely to involve agencies such as the forest department. Besides, awareness to the villagers or stakeholders is essential. Development of small nurseries at each in situ site is imperative, so as to propagate the species and reintroduce them where populations are low. Furthermore, overexploitation of threatened and native species should be banned. However, medicinal plant species that could be exploited for both commercial and community development purposes should be conserved by ex situ means. This would boost the income of rural people and in turn help in the conservation of the resources. The high preference of these species and continuous extraction from the wild for trade has caused increased pressure, which may cause the extinction of these species from the area in near future.

14.5

Conclusion

The rural communities in the remote area of Sangla Valley have directly dependent on forest resources for their livelihood. An important conclusion to be made from this study is that overexploitation, habitat destruction, deforestation, grazing in the forest, expansion of agriculture, selective removal of species and more importantly, natural habitat degradation would pose a danger to the local abundance of these plant species. Given this, there is vital to develop suitable approach and action plan for the conservation and management of natural habitats, species, and communities. Efforts toward the conservation of plants will ultimately lead to the development policies and their sustainable consumption. However, an increasing potential threat to biodiversity is the negative environmental factor that warrants immediate remedial measures for sustainable management and conservation. Acknowledgment The authors acknowledge Department of Science and Technology, Government of India for funding support (no. SR/DGH/HP-1/2009 and SB/DGH-96/2015). The local inhabitants of the valley are also acknowledged for their willingness to share the valuable knowledge and wholehearted co-operation. Conflict of Interest There is no conflict of interest.

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Ethnomedicinal Pertinence and Antibacterial Prospective of Himalayan Medicinal Plants of Uttarakhand in India

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Shobha Mehra, Varun Kumar Sharma, Charu Tygai, and Lomas Kumar Tomar

Abstract

Uttarakhand is a northern state in India and has a strong traditional medicinal system because of the rich and diverse population of ethnomedicinal plants. Transfer of knowledge to generations on herbal wealth and ancient therapeutic systems has been the tradition among inhabitants of this region. This herbal wealth has been widely utilized as antimicrobial, anticancer, anti-inflammatory, antioxidant, antidiabetic, antifungal, antispasmodic, anthelmintic, antirheumatic, antihepatotoxic, hepatoprotective, analgesic, and immunostimulant for therapeutic treatment strategies. A recent World Health Organization global report on traditional and complementary medicine 2021–22 confirms the 88% member states have acknowledged their use of traditional and complementary medicine which corresponds to 170 member states. Traditional systems of medicine namely Ayurveda, Unani, Homeopathy, and Siddha are almost completely based on plant-based extracts. Plants which have been studied and investigated pharmacologically and chemically have been helpful in providing active principle in modern medicine and in some cases giving leads for partial or total synthesis of new drugs. Accessibility of modern medical facilities has also played a role in keeping local peoples dependent on traditional methods of using plants as a source for treating ailments. The aim of this study is to review salient reports on traditional, pharmacology, ethnomedicinal, and biological activities of some medicinal plants of Uttarakhand region of India. S. Mehra · V. K. Sharma · L. K. Tomar (✉) Department of Biotechnology and Microbiology, School of Sciences, Noida International University, Gautam Budh Nagar, Uttar Pradesh, India C. Tygai Department of Biotechnology, VSPG (PG) College, CCS University, Meerut, Uttar Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_15

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Furthermore, medicinal plants are therapeutic agents and raw material for manufacturing traditional and modern medicines. For a better clinical use of Herba Patriniae, it is urgent to establish systematic pharmacology, quality control, and pharmacokinetics validation of these precious heritage plants. Keywords

Antibacterial activity · Ethnomedicine · Himalayan plants · Phytochemical · Uttarakhand

15.1

Introduction

The traditional medicine system has an efficient procedure for extracting effectual substances; hence it can be an effective and potential source of medical advancement. Socioeconomic and ecological perspectives (Negi et al. 2011) have highlighted the importance of medicinal plants in the Himalayan region (Larsen et al. 2000; Olsen 2005). Comparatively the slow rate of development and less accessibility of mountainous terrain in the Himalayas have kept the larger part of traditional knowledge of the use of various plant species still intact with the indigenous people (Kala 2000; Farooquee et al. 2004). Located in the central Himalayan region of the Indian state, Uttarakhand (20° 26′ and 31° 38′ N latitude and 77° 49′ and 80° 6′ E longitude) covering an area of 53,483 km2 is gifted with a large variety of plant species having medicinal properties. Medicinal plants play a significant role in the lives of the people of Uttarakhand providing basic health care and employment to the farmers (Ghayur and Kop 2005). For future understanding, research, and sustainable management of medicinal plants, existing ethno-botanical knowledge is not only helpful, but this knowledge is also essential for the poor people who are in need of immediate relief and cannot afford expensive medicines. The flora of Garhwal (Uttarakhand) has been already extensively explored and studied by several botanists (Bhatti and Vashishtha 2008). Out of 15,000 species of flowering plants found in India, about 17% have their medicinal value and several species are from the Indian Himalayan region and found in Uttarakhand. The local people of this state are partially or completely dependent on forest resources for medicine, food, and fuel. For thousands of years, traditional plant-derived medicines have been used in most parts of the world and their use in fighting microbial diseases is becoming the focus of study (Bhavnani and Ballow 2000). Intensive studies on extracts and biologically active compounds isolated from medicinal plants have played an essential role in drug discovery in the last few decades. Various parts of such plants like roots, tubers, bark, flowers, leaves, and seeds are used for medicinal purposes. The aim of this study is to review salient reports on traditional, pharmacology, ethnomedicinal, and biological activities of some medicinal plants of Uttarakhand region of India.

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Plants with Ethanomedicinal and Antimicrobial Potential (Described with Family Classification)

15.2.1 Family—Acanthaceae: Adhatoda zeylanica Medic Adhatoda zeylanica Medic is commonly known as Adulsa in Hindi. It is an evergreen perennial shrub, gregarious, 1–2.5 m height, with opposite ascending branches. The plant is distributed throughout India, up to an altitude of 1300 m and mainly found in sub-Himalayan region, also found in Nepal, Pakistan, and Germany. Leaves are ovate or lanceolate with tapered base, acuminate apex, petiolate, and ex-stipulate with 5–20 cm length and 3–10 cm width having a characteristic odor and bitter taste. The plant is used in preparation of herbal cough syrup “Zee Tuss.” Ethanobotanical and medicinal use: Roots, leaves, flowers, and fruits are used for treating common remedies like common cold and cough, whooping cough to chronic bronchitis and asthma, used as antispasmodic, anthelmintic, and sedative expectorant. It is reported to be antimicrobial, anticancerous, and antitussive. The plant

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leaves have insecticidal properties, anti-inflammatory and antifungal activity. It is also used in ringworm, migraine, hair loss, and louse control in hen (Singh et al. 2003). Antimicrobial activity: Different organic solvent extracts of the leaves and root showed antibacterial activity against different clinically important strains at different concentrations of 100, 200, and 400 μg/disc by agar diffusion method against E. faecalis, Actinomycin, S. epidermidis, Vibrio parahaemolysis, S. heamophytenus, Bactere maserins, Serratries masciens, and Vibrio cholerae. The zone of inhibition was measured by the dimension of zone calculated with the number of bacterial growth around the disc. The antibacterial activity of chloroform extract in the concentration of 400 μg/mL and hexane extract (400 μg/mL) has shown better activity than other extracts (Ilango et al. 2009).

15.2.2 Family—Apiaceae 15.2.2.1 Centella asiatica L. Centella asiatica is found in India and other Asian countries in the tropical and subtropical areas with an altitude up to 600 m. Leaves are orbicular or oblongated elliptical with seven veins and long petioles and yellowish green in color. The plant grows with red or green stolon combined horizontally with each other with roots underground. C. asiatica is a perennial creeper herb with faint aroma which attains a height up to 15 cm with hairless stem, striated and rooting at the nodes. Flowers are in fascicled umbels, every inflorescence consisting of 3–4 white to pink or purple flowers, flowering happens within the month of April–June. It is one of the chief herbs for treating skin problems, to heal wounds. Ethanobotanical and medicinal use: Traditional knowledge suggests that C. asiatica has antimicrobial, antibacterial, anti-inflammatory, antioxidant, antidiabetic, and antifungal activity, and it can improve blood circulation, helps to decrease anxiety, stress, and fatigue, strengthens veins, and can be a good remedy for skin conditions such as eczema, chronic ulcers, and urethritis. It is one of the most valuable herbs in Ayurvedic medicine because as per Ayurveda knowledge it is used as Madhya Rasayana, i.e., Brain tonic; it can revitalize nerve and brain cells, increases memory and concentration, and has an overall rejuvenating effect on our body. Antimicrobial activity: Different organic solvent extracts of C. asiatica leaf have been studied for antibacterial activity against opportunistic bacterial pathogens isolated. Inhibition zone around each disc caused by diffusion of antibacterial properties from disc containing plant extract into surrounding medium was observed in vitro. C. asiatica (30 mg/disc) leaf extract with ethyl acetate, ethanol, acetone, chloroform, and petroleum ether showed antibacterial activity to at least two of the tested bacterial pathogens. Among all extract with ethyl acetate showed high activity against Brevibacterium paucivorans (12.33 mm), Staphylococcus haemolyticus (11 mm), and Bacillus cereus (10.33 mm) respectively. The same extract showed moderate inhibition against E. amnigenus and K. pneumoniae (8 mm), S. aureus

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(9 mm). Similarly, ethanol extract showed the second highest inhibition zone against S. marcescens (9 mm), K. oxytoca (9.5 mm), and S. haemolyticus (10 mm) (Okwu and Josiah 2006).

15.2.2.2 Apium graveolens L Apium graveolens is an important aromatic plant locally known as Ajmod (Hindi). The root of the A. graveolens is shallow and thickened in the middle. The stem is branched, succulent, furrowed, and rigid. The leaves are pinnate and ovate in shape. The size of the flower is small, and it is white/greenish white. Fruits are schizocarp with two mericarps, suborbicular to ellipsoid in shape, and slightly bitter in taste. Ethanobotanical and medicinal use: The leaves, stalks, and seeds of the plant are used for anthelmintic, antispasmodic, carminative, diuretic, laxative, arthritis, gout, rheumatism, and urinary tract inflammation (Al-Asmari et al. 2017). Antimicrobial activity: Seed extracts of A. graveolens showed the broad spectrum of antibacterial activity on selected microorganisms. The methanol extract showed 16 mm zone for E.coli, which is the maximum zone of inhibition followed by aqueous (5 mm), and diethyl ether (15 mm) was found as effective for the zone of inhibition, and the methanol extract exhibited 17 mm zone for P. aeruginosa that is the maximum zone of inhibition, subsequently aqueous (8 mm), and diethyl ether (10 mm) extracts (Tyagi et al. 2013; Sisay et al. 2019).

15.2.3 Family—Asteraceae 15.2.3.1 Eclipta alba (L.) Hassk Eclipta alba is an annual herbaceous plant, commonly known as Bhringaraj, which is found as a common weed throughout India ascending up to 6000 ft. It is prostrate or erect, much branched, annual, roughly hairy, and rooting at the nodes; the leaves are sessile, opposite, and lanceolate (Wagner et al. 1986). Ethanobotanical and medicinal use: It is used as a tonic and diuretic in hepatic and spleen enlargement liver cell generation and skin diseases. It is traditionally used for its anticancer, hepatoprotective, anti-inflammatory, analgesic, immunomodulatory, antihypertensive, antihyperlipidemic, antioxidant, and hair growth promoting activities. Antimicrobial activity: The antibacterial activities of extracts of aerial parts of E. alba using different solvents such as methanol, acetone, aqueous, ethanol, and hexane extract were evaluated against selected bacterial species. The hexane extract showed best activity against E. coli and S. aureus (MIC 90.0 μg/mL), and also against, K. pneumoniae, P. mirabilis, and S. typhi (MIC 125.0 μg/mL). Methanol, ethanol and acetone, extracts showed better activity on selected bacterial species. With the MIC of 100–500 μg/mL, aqueous extract showed good activity against bacterial species (Pandey et al. 2011).

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15.2.3.2 Anaphalis contorta (D. Don) Hook.F Anaphalis contorta is an erect herb. The leaves are crowded, linear, and shortly lobed at the base, margins recurved sometimes. Flower and spreading in fruit outer ones are purple. It is widely distributed in the temperate Himalayan region at a height of 1500–4500 m (Wollenweber et al. 1993). Ethanobotanical and medicinal use: The paste of fresh leaves of this plant is applied on cuts, wounds, and boils (Raturi et al. 2012). Antimicrobial activity: The in vitro antimicrobial activity was evaluated by the disc diffusion method. The antimicrobial activity showed A. contorta against the selected microorganisms at three different concentrations (0.25 μg/mL, 0.125 μg/ mL, and 0.062 μg/mL). The oil was found to be more active against the microorganism Pseudomonas aeruginosa (gram-negative) followed by Staphylococcus aureus (gram-positive) bacteria and Microsporum canis (fungi) at a concentration of 0.125 μg/mL (Joshi 2011). 15.2.3.3 Eupatorium odoratum L Eupatorium odoratum grows in tropical areas as well as at the edge of jungles in most parts of India. It is a tall and multi-stemmed shrub to 2.5 m (100 in.) in open areas. It has soft bark and stem, but the base of the shrub is woody. It is a noxious invasive weed in most parts of India invading field crops. It has been reported as the most problematic invasive species of plants within protected rainforests of Africa. Ethanobotanical and medicinal use: The Eupatorium odoratum leaf extract has diverse biological activities like inhibition of the growth of some bacteria to enhancement of anti-inflammatory, hemostasis, blood coagulation, astringent, diuretic and hepatotropic activities. Phytochemical analysis revealed the presence of many secondary metabolites. The young leaves of E. odoratum are used to treat skin wounds (Rani and Abraham 2006). Antimicrobial activity: The antibacterial activity of methanol extract of E. odoratum flower was studied using agar well diffusion method. Antimicrobial activity (zone of inhibition) of different solvent methanol and ethanol extract of the 317 strains, 8 strains were sensitive. Nine strains were inhibited by ethanol-based extraction only while 93 strains were inhibited by methanol-based extraction discs. Among bacterial strains sensitive to both ethanol- and methanol-based extractions 7 were isolated from house gecko and one from Axone. All the 8 bacterial strains inhibited by both ethanol- and methanol-based extraction were members of Enterobacteriaceae (C. freundii 1, S. houtenae 1, C. amalonaticus 2, K. pneumoniae 1, E. coli 1, Pragia fontium 2). The 9 strains which were inhibited by only ethanol-based extraction were isolated from house gecko and included two strains each of Enterococcus dispar and S. aureus and one strain each of E. ananas, L. ghrimontii, K. pneumoniae, P. aeruginosa, and E. coli (Singh et al. 2015).

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15.2.4 Family—Agavaceae 15.2.4.1 Agave americana L Agave americana is commonly known as Rambans (Hindi). The leaf has a yellow or white marginal or central stripe from the base to apex cultivated. It has a powerful leaf rosette with gray-blue or gray-green leaves that can in tropical areas grow up to 1.75 m long and 20 cm wide. It also provides stable economic return to local communities especially through the sale of wild harvested material. The natural fibers produced under different environmental conditions such as soil type and weather can significantly affect the fiber properties. Ethanobotanical and medicinal use: Traditionally, it was used to treat stomach inflammation, tuberculosis, jaundice, ulcers, liver diseases, syphilis, and menstrual problems. Additionally it is used as a treatment for cough and high fever by inducing sweat. A. americana has antiseptic, wound healing, and anti-inflammatory properties, which explains its uses externally as a medicinal herb to treat burns, bruises, minor cuts, injuries, and skin irritation caused by insect bites. Antimicrobial activity: The antibacterial activity showed the zone of inhibition against gram-negative bacterial pathogens namely P. aeruginosa and E. coli and gram-positive bacterial pathogens namely K. pneumoniae and S. aureus by agar well diffusion method. The zone of inhibition test results of Agave americana methanolic leaf extract showed good antibacterial activity against gram-negative pathogens namely P. aeruginosa (17 mm) and E. coli (20 mm) than gram-positive pathogens namely S. aureus (18 mm) and K. pneumoniae (24 mm). The extract proved that it has better control over the positive pathogens than compared to the negative pathogens (Krishnaveni 2017).

15.2.5 Family—Apocynaceae 15.2.5.1 Holarrhena pubescens Buch-Ham Holarrhena pubescens is commonly known as Kuru (Hindi). It is an Indian medicinal tree 30–40 ft. high and up to 4 ft. in girth. The tree is common in the forest of India and Pakistan, indigenous to the tropical Himalaya region. Ethanobotanical and medicinal use: The bark of this plant commercially known as “kurchi” has antidiarrheal, astringent, antidysenteric, and anti-anthelmintic properties. The bark is used for the treatment of chest pain, piles, colic, dyspepsia, and diuresis. It is also used for the treatment of spleen and skin disease. The seeds are effective in diarrhea, fever, jaundice, and bladder stones. The main steroid alkaloid has been used for the treatment of vaginitis and amoebic dysentery. Antimicrobial activity: The extract of H. pubescens was found to be significantly effective against 4 out of 13 g-positive and 1 out of 15 g-negative bacteria tested at 1000 mg per disc concentration. Separation of H. pubescens extract through the classical method yielded five fractions (HP-EA, HP-B, HP-N-E, HP-N-PE, and HP-NEA). They were found to be active against several gram-negative and grampositive bacteria at this concentration. Alkaloidal fraction HP-B showed significant

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activity against 14 g-negative and 14 g-positive bacteria. This fraction, on further separation according to the method reported earlier (Siddiqui et al. 1993), yielded conessine as the main alkaloid (Othman et al. 2019).

15.2.6 Family—Acoraceae 15.2.6.1 Acorus calamus L. Acorus calamus is commonly known as Sweet Flag. It is a semiaquatic, perennial, and smelly plant, found in both temperate and subtemperate zones. It grows up to 6 ft. tall with sword-shaped leaves, small yellow/green bowers, and branched rhizomes. Ethanobotanical and medicinal use: The herb is used both internally and externally. In rheumatism, rheumatic fever, and inflamed joints, the paste applied externally alleviates the pain and swelling. Internally sweet flag is valuable in a vast range of diseases. It is effective for digestive ailments such as flatulence, loss of appetite, abdominal dull pain, and worms. The powder of sweet flag given with lukewarm salt water induces vomiting and relieves phlegm, while easing coughs and asthma. Antimicrobial activity: Antibacterial activities of leaf and rhizome extracts were evaluated by agar well diffusion method. Ethyl acetate-based extracts of leaf and rhizome exhibited strong inhibitory action against the selected microorganisms tested; however extracts in other solvents did not show any antibacterial activities. Antimicrobial potentials of the extracts were evaluated by measuring the diameter inhibition of zone (mm) as well as minimum inhibitory concentration (MIC). Only E. coli (MTCC901 and NCIM) was found to be highly sensitive to both leaves and rhizomes extracts. Rest of the bacteria gram-positive as well as gram-negative were found to be resistant to rhizome and leaves extract. Rhizome and leaves extract showed marked antifungal activity against A. Flavus (MTCC 2799), A. niger (MTCC 1344), Microsporum canis (MTCC 2820) except P. chrysogenum (MTCC 2725), with zone of inhibition ranged 18–25 and 20–28 mm, respectively, along with MIC value 2–4 mg/mL for each rhizome and leaves extract (Priya and Ganjewala 2007).

15.2.7 Family—Betuleae 15.2.7.1 Betula utilis, D. Don Betula utilis is a moderate-sized tree that grows up to 20 m in height. The bark is shining, smooth, reddish white or white, with white horizontal lines. The outer bark consists of narrow thin papery layers, exfoliating in broad horizontal rolls. The inner cortex is moist and red. The leaves are elliptic, ovate-acuminate, and irregularly serrate. The flowers bloom in June–July, in pendulous spikes. Seeds are thin and winged (Mishra et al. 2016). Ethanobotanical and medicinal use: The bark contains oleanolic acid, betulin, acetyloheanolic acid, betulinic acid, lupeol, lupenone, sitosterol, methyl betulate,

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methyle betulonate, karachic acid, and a new triterpenoid. It is aromatic and has antiseptic, therapeutic, carminative, aromatic, and contraceptive properties. Antimicrobial activity: Antibacterial activity of different solvents and aqueous extracts tested at 50 μL concentrations against fourteen important human pathogenic bacteria is presented. A significant antimicrobial activity was observed with ethanoland methanol-based extracts. Results showed that S. boydii was highly susceptible to methanol-based extract while P. mirabilis was found susceptible to both methanoland ethanol-based extract. P. aeruginosa was found to be highly susceptible to ethanol extract. Methanol extract exhibited similar antibacterial activity against S. paratyphi, S. typhimurium, S. aureus and S. faecalis with inhibition zone of around 14–16 mm (Kumaraswamy et al. 2008).

15.2.8 Family—Bignoniaceae 15.2.8.1 Oroxylum indicum (L) Vent Oroxylum indicum is native to the Indian subtropical region, in the Himalayan foothills with a part extending to southern China and Bhutan, in Indo-China and the Malaysia ecozone. It is a medium- to small-sized deciduous tree, of height 12–15 m. The bark of the plant is light grayish brown in color and soft, spongy in texture having corky lenticels. The leaves are 90–180 cm long, ovate, elliptical, bipinnate or tripinnate or broad; leaflets are 2–4 pair ovate or elliptical, 5 in. long and 2–3 in. broad having sharp edges, glabrous, and acuminate. The flowers are numerous; corolla is fleshy and dark purple in color from outside, pinkish-yellow and pale within, 10 cm long (Deka et al. 2013). Ethanobotanical and medicinal use: The root bark is a well-known tonic and astringent useful in fever, diarrhea, dysentery, bronchitis, intestinal worms, leucoderma, asthma, inflammation, anal troubles. It is diaphoretic and used in rheumatism. Antimicrobial activity: The antimicrobial activity of ethanolic extracts of leaves of O. indicum was tested against P. aeruginosa. The inhibition zone measured by the alcoholic extract of leaves of O. indicum against P. aeruginosa was found to be 0.6 cm. The well method for B. subtilis measured the zone of inhibition as 0.5 cm. This indicates that the leaf extracts of O. indicum produce some compounds that act against the growth of microorganisms.

15.2.9 Family—Bombacaceae 15.2.9.1 Bombax ceiba L Bombax ceiba is commonly known as Simbal, Indian bombax, or Red Silk cotton tree. It is widely found in tropical Asia, temperate Asia, Australia, and Africa. In India, it can be found at altitudes up to 1500 m. In peninsular India, the tree is most

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common in the dry as well as moist deciduous near rivers and forests. The bark is silver gray to pale ash in color. Flowers are large in diameter, red in color, and numerous with copious nectar. Ethanobotanical and medicinal use: Stem bark is considered as acrid, diuretic, demulcent, inflammation, slightly astringent, and tonic. It is applied on swelling, boil, and burning sensation and applied on face in facial complaints such as acne vulgaris, freckles, and other cutaneous as well as pigmentation disorders (Rameshwar et al. 2014). Antimicrobial activity: Plant extract showed antibacterial activity ( p < 0.05) against both gram-negative and gram-positive bacteria in a dose-dependent manner as shown. The results showed that S. typhi with a diameter of 19 mm of zone of inhibition was the most resistant microbe against the tested extract among the selected microbial strains while S. aureus was the most susceptible to the extract with 24 mm zone of inhibition. It was observed that the extract was active against gram-negative strains as well as gram-positive strains ( p > 0.05) even at lower concentration (Akhtar and Mustafa 2017).

15.2.10 Family—Combretaceae 15.2.10.1 Terminalia bellirica Roxb Terminalia bellirica Roxb commonly known as myrobalan is a deciduous tree found throughout the Indian forests and plains. It is known as Bahera in India and has been used for Ayurveda, a holistic system of medicine. The tree is about 30–40 m in height and 2–3 m in girth. The leaves are broadly elliptic clustered, and the stem is straight, leaves near the end of the branches. The flowers are simple, solitary in axillary spikes. The fruit is ovoid 1–2 cm in diameter drupe of gray to dark brown in color (Chanda et al. 2013). Ethanobotanical and medicinal use: Seed extract produces yellow colored oil which has medicinal uses and is used as astringent and a laxative tonic. It is a major constituent of Triphala, an Ayurvedic medicine. It is used in coughs and sore throat. Its pulp is used in dysentery, diarrhea, and liver disorders. It is also useful in hair care, leprosy, and fever. This plant exhibits several pharmacological effects including antimalarial, antidiabetic, antifungal, antibacterial, anti-HIV, antioxidant, and antimutagenic effects (Alam 2011). Antimicrobial activity: The crude methanol-based extracts of T. bellerica were strongly inhibitory to S. aureus, forming a large zone of inhibition of 30 mm and 28 mm respectively. The crude extract was less effective against Y. enterocolitica as it formed 15.5 mm zone of inhibition. The methanol extract (14 mm) was less effective against P. aeruginosa than the crude extract (20 mm). The lowest MIC values of methanol-based extract and crude extracts were against S. aureus suggesting that T. bellerica was most effective against S. aureus.

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15.2.10.2 Terminalia arjuna Terminalia arjuna is called arjuna in Hindi. It is an evergreen and deciduous tree distributed throughout India and also found in Sri Lanka, Burma, and Mauritius, growing up to a height of 65 to 95 ft. Leaves of T. arjuna are oblong, simple, or elliptic with pale brown lower surface and dark green upper surface. Flowers are sessile, bisexual, and white arranged in short axillary spikes or in terminal pannicule. The bark is pinkish, smooth outside, and flakes off in large, curved and rather flat pieces (Dwivedi 2007). Ethanobotanical and medicinal use: The bark and leaves of this plant have been used in indigenous system of medicine for curing different diseases, the bark in the treatment for angina, antidysenteric, expectorant, purgative, laxative, anemia, leucoderma, hyperhidrosis, tumors, asthma, and other cardiovascular disorders. Antimicrobial activity: T. arjuna leaf extract showed antibacterial activity followed in acetone, petroleum ether, and aqueous water extract. The maximum inhibition was found against S. pneumoniae (17.3 ± 0.57 mm) followed by H. influenzae (16.6 ± 0.57 mm), P. aeruginosa (14.6 ± 0.76 mm), S. pyogenes (13.6 ± 0.28 mm), and S. aureus (12.6 ± 0.28 mm) respectively. The minimum inhibition zone was measured against C. albicans (11.3 ± 0.28 mm) (Sanjay et al. 2014). The antimicrobial potential of T. arjuna has been found effective against selected fungal and bacterial species. T. arjuna alcoholic leaf extract was found to be most effective against S. aureus (28 mm) followed by Proteus mirabilis (27.6 mm), Acinetobacter species (16.6 mm), and P. aeruginosa (16 mm) (Aneja et al. 2012).

15.2.11 Family—Caesalpiniaceae 15.2.11.1 Cassia fistula L Cassia fistula is one of the most important trees widely spread in the forest of India. It usually occurs in deciduous forests throughout the greater part of India, ascending up to an altitude of 1220 m in the sub-Himalayan areas and the outer Himalayas. It is a deciduous tree with gray greenish bark, compound leaves; leaflets are each 6–14 cm long pairs (Luximon-Ramma et al. 2002). Ethanobotanical and medicinal use: Cassia fistula is one of the most commonly used plants is Unani and Ayurvedic medicine. It has been used against liver troubles, skin diseases, and tuberculous glands, and its use in the treatment of leucoderma, hematemesis, diabetes, and pruritus has been suggested. Medicinally it shows different types of pharmacological activities like antimicrobial, antifungal, antipyretic, analgesic, anti-inflammatory, larvicidal, antioxidant, antitumor, hepatoprotective, antidiabetic, and hypoglycemic activities. Antimicrobial activity: Antimicrobial and antifungal activities of plant extracts against four pathogenic bacteria (2 g-negative and 2 g-positive) and three pathogenic fungi were investigated by the agar disc diffusion method. The antimicrobial activity of both the plant extracts of C. fistula was studied in different concentrations (5 μg/ mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL) against four pathogenic bacterial strains 2 g-negative (E. coli MTCC 443, P. aeruginosa MTCC 424) and

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gram-positive (S. aureus MTCC 96, S. pyogenes MTCC 442), and three fungal strains (A. clavatus MTCC 1323, A. niger MTCC 282, C. albicans MTCC 227). The zone of inhibition was measured by ranged from 10 to 20 mm for all the sensitive bacteria, and ranged from 12 to 21 mm for fungal strains (Bhalodia et al. 2012).

15.2.12 Family—Ephedraceae 15.2.12.1 Taxus baccata L Taxus baccata locally known as “Thuner” in various parts of the Western Himalaya has greater economic and medicinal values than the other gymnosperms in the region. Taxus is an evergreen tree found in temperate forests in the Pacific, East Asia, north-west of North America, North Africa, and Europe. It is widely distributed in the tropical region of Himalaya (India) between 1800 m and 3300 m above mean sea level. Ethanobotanical and medicinal use: The paste of bark is applied as a plaster for fractured bones. It is also used in the treatment of headache. The extract from the leaves and bark is also used for the treatment of various diseases like asthma, bronchitis, poisonous insect bites, and also as an aphrodisiac (Purohit et al. 2001). Antimicrobial activity: The antimicrobial activity of ethanol extract and ethyl acetate extract showed antimicrobial effect against all gram-negative bacteria while only ethyl acetate extract showed antimicrobial effect against all gram-positive bacteria. The MIC values ranged from 39.06 to 156.25 μg/mL of water extract and ethanol extract on all the selected microorganisms. However, at the tested MIC limit of 312.5 μg/mL, the inhibitory activity of n-hexane extract and dichloromethane extract was noted on 10 (80%) of the 13 tested microorganisms while that of ethyl acetate and water extract was observed on 12 (92.3%) and 11 (84.6%) respectively (Patel et al. 2009).

15.2.13 Family—Euphorbiaceae 15.2.13.1 Euphorbia neriifolia L Euphorbia neriifolia is an erect, branched, prickly, small fleshy glabrous shrub cultivated in gardens. The leaves arise from the sides of wings toward the end of the branches and are fleshy, oblong-obviate, and 5–15 cm long. Ethanobotanical and medicinal use: It is used in the treatment of asthma, tumors, bronchitis, leucoderma, inflammation, piles, enlargement of spleen, ulcers, fever, anemia, and in chronic respiratory troubles. It is used as anti-inflammatory, hepatoprotective, analgesic, immunostimulant, mild CNS depressant, and radioprotective agent and for wound healing (Yadav et al. 2012). Antimicrobial activity: Antimicrobial activity of crude saponin of E. neriifolia against bacteria like E. coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 10145), Staphylococcus aureus (ATCC 25923), and Candida albicans (MTCC

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227) was performed using agar well diffusion method. E. neriifolia did not show antimicrobial activity up to 10 mg/mL concentration. The antimicrobial effect of different extracts viz. ethanol, chloroform, ethyl acetate, butanol, and aqueous of leaves of E. neriifolia was studied using E coli, S. aureus, K. pneumonia, P. fluorescens, and P. vulgaris. The highest effect was seen in chloroform-based extract against P. vulgaris with the zone of inhibition of 8 mm followed by ethanolbased extract against K. pneumonia with the zone of inhibition of 5 mm. The water extract and ethyl acetate extract exhibit very little effect. The antimicrobial efficacy of methanol-based extract of the stem of E. neriifolia was assessed against S. aureus (ATC-2245), E. coli (K-88), P. aeruginosa, P. vulgaris (CC-52), S. typhi (12), Aspergillus niger (36), and Candida albicans using disc diffusion and microdilution dilution assays (Sultana et al. 2022).

15.2.13.2 Emblica officinalis Gaertn Emblica officinal is commonly known as Amla or Indian gooseberry and is a small and medium-sized deciduous tree found throughout India, the fruits of which are highly valued in traditional medicine (Asmawi et al. 1993). Ethanobotanical and medicinal use: Emblica officinalis is used as an antioxidant agent and is used to treat anemia, fever, dysentery, gravel, and sores. Leaves have been used for anti-inflammatory and antipyretic treatments. Antimicrobial activity: Different solvent extracts of E. officinalis leaves and fruits were evaluated by agar well diffusion method against different pathogenic bacteria. Highest antibacterial activity was observed against E. coli (ZOI = 17.0 ± 1.0 mm and AI = 0.939) by methanol extract followed by the zone of inhibition of E. coli by aqueous extract (ZOI = 14.5 ± 0.5 and AI = 0.801) of E. officinalis fruits while hexane extract slightly inhibited growth of Serratia marcescens and remaining bacteria showed resistance to the extract. 15.2.13.3 Mallotus philippensis, Linn, Muell Mallotus philippinens commonly called Kamala is a common perennial shrub or small tree found in outer Himalayas ascending to 1500 m. Leaves are mostly acute or acuminate at apex, hairy and reddish glandular beneath, conspicuously 3-nerved, petiole size 1–4 cm long, puberulous, and reddish brown in color. Seeds are subglobose and black in color and 4 mm across (Thakur et al. 2005). Ethanobotanical and medicinal use: All parts of the plant like glands and hairs from the capsules or fruits are used as anthelmintic, heating, purgative, vulnerary, maturant, and carminative and are useful in the treatment of bronchitis and abdominal diseases. The leaves are used externally for different types of skin infections and infected wounds. Antimicrobial activity: The methanolic extracts of M. philippensis showed considerable growth inhibition of test bacteria at different concentrations (30%, 50%, 70%, 100%) as compared to acetone fruit extract of the plant. The methanolic extract of M. philippensis was found to be most effective against S. aureus at 20 mm at 100% followed by 17 mm at 70%, 14 mm at 50%, and 13 mm at 30%, and it offered minimum zone of inhibition in P. aeruginosa (14 mm at 100%, 12 mm at 70%,

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10 mm at 50%, and 9 mm at 30%). The acetone extract of M. philippensis was found to be most effective against S. aureus at 15 mm at 100% followed by 13 mm at 70%, 14 mm at 50%, and 11 mm at 30%, and it showed minimum inhibition toward P. aeruginosa (13 mm at 100%, 10 mm at 70%, 9 mm at 50%, and Nil at 30%). It was concluded from the results that methanolic and acetone fruit extract of M. philippensis were quite effective in inhibiting the growth of S. aureus which is considered as a serious human pathogen causing infections in wounds (Rana et al. 2016).

15.2.14 Family—Ericaceae 15.2.14.1 Rhododendron arboreum Smith Rhododendron arboreum is a small tree or evergreen shrub with a showy display of bright red color flowers. Rhododendron is the national flower of Nepal and known as Laligurans and is the state tree of Uttarakhand. Leaves are 10–20 cm long and 3.6 cm wide, oblong-lanceolate. Ethanobotanical and medicinal use: The flowers are used in the treatment of checking bloody dysentery and diarrhea. R. arboreum is the active constituents of Ayurvedic preparation “Asoka Aristha,” which possesses estrogenic, oxytocic, and prostaglandin synthetase-inhibiting activity. Antimicrobial activity: Antibacterial activities of methanolic extract of flowers, leaves, bark, stem, and roots of R. arboreum against the mentioned human pathogens were observed. Methanolic extract of flower showed potent antibacterial activity against E. coli, B. subtilis S. aureus, and S. typhi in the following order: E. coli (80%) > B. subtilis (60%) > S. typhae (50%) > S. aureus (45%). Flower extract showed good to significant activity against the mentioned bacterial strains. 15.2.14.2 Lyonia ovalifolia Wallich Drude Lyonia ovalifolia locally known as Angyar is a deciduous medium-sized tree about 12–16 m tall. It is commonly distributed in the slopes of mountain Himalaya associated with Oak and Rhododendron Forest between the altitude of 1200 and 3000 m in Uttarakhand Himalaya in India, Pakistan, China, Myanmar, and S.E Asia (Sahu and Arya 2017). Ethanobotanical and medicinal use: Lyonia ovalifolia is used for the treatment of wounds, burns, cuts, scabies, etc. Lipid peroxidation has a major role in the progression of various life-threatening conditions like cancer, cardiovascular diseases, inflammation, and infection. Antimicrobial activity: The antimicrobial activity in two different concentrations of extracts (500 mg/mL and 250 mg/mL) was observed. The methanolic extract of young leaves and apical buds inhibited the growth of P. aeruginosa (n-hexanne frcation of leaves) and gave maximum zone of inhibition 10.50 ± 0.86 (mm) with P. aeruginosa at 500 (mg/mL).

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15.2.15 Family—Fabeacae 15.2.15.1 Butea monosperma (Lam) Kuntz. Syn Butea monosperma is locally called as palas. Generally it grows extrovertly on open grasslands and spread in mixed forest. It is a 10–15 m tall tree with irregular branches and crooked trunk. The bark is rough and ash colored whereas the young parts are downy. Leaves are 3-foliate, stipules linear lanceolate, petioles 10–15 cm long finely silky and conspicuously reticulate veined beneath. Ethanobotanical and medicinal use: The flowers are used in the treatment of hepatic disorders, diarrhea, viral hepatitis, anti-inflammatory and anticonvulsive agent, and as tonic. The roots are useful in the treatment of piles, night blindness, ulcers, tumor and antispermatic activity, liver disorders, antifertility activity, and gout. The gum is a powerful astringent. The stem bark possesses antifungal activity and dermal wound healing activity (Shahavi and Desai 2008). Antimicrobial activity: The ability of the extracts to inhibit the growth of bacteria was determined using the agar disc diffusion method. The strongest antibacterial activity was seen against B. subtilis followed by P. aeruginosa, S. typhimurium, and E. coli. MIC for P. aeruginosa was observed with 31.25 mg/mL concentration and no activity was found in the lower concentrations. MIC for K. pneumonia, P. vulgaris, and S. aureus was observed with 125 mg/mL concentration and no activity was found in the lower concentrations. The inhibition of the growth of E. aerogenes was observed with 250 mg/mL concentration only and no activity was found in the lower concentrations. The flower possesses hepatoprotective activity against CCl4-induced hepatitis in rats. 15.2.15.2 Pueraria tuberosa (Roxb. Ex Willd) It is common in Central India and ascending up to 1300 m mean sea level and also found in the hills of the western Himalayan region. Leaves are long petioles, three foliolate with pinnately compound. Fruits are membranous. Pods are of 5.0–7.5 cm long, flat, and jointed. It contains 2–3 seeds clothed with long, silky bristly brown hairs. Ethanobotanical and medicinal use: The edible tubers are aphrodisiac, cardiotonic, galactogogue, and diuretic. The tubers are used for the treatment of dysuria, emancipation, and spermatorrhea and given for malarial fever. Antimicrobial activity: Among all the extracts only petroleum ether extract showed positive results against Klebsiella pneumoniae. The maximum zone of inhibition of 6 mm was observed in petroleum ether extract against Klebsiella pneumoniae (Theng and Korpenwar 2012).

15.2.16 Family—Geraniaceae 15.2.16.1 Geranium nepalense Sweet Geranium nepalense is one of the most common medium flowered species in the Himalayas. It is a branched annual herb, hairy, with mostly procumbent branches,

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leaves palmately 3–5, rarely 7-lobed, up to 7 cm broad, upper ternate, segments irregularly lobed, stipules lanceolate, 10–12 mm long, flowers pale pink, 8–15 mm across, on 2-flowered axillary up to 12 cm long peduncles, sepals 5–6 mm long, with about 1 mm long mucro, 3-nerved, pubescent, petals slightly longer than sepals, obovate, slightly notched, and mericarps hairy with up to 15 mm long beak (Lu et al. 2012). Ethanobotanical and medicinal use: Used as dysentery, influenza, antiphlogistic and analgesic tonic, stomachic, rheumatism, renal diseases, hemostatic and antidiabetic drugs. The whole plant is antibacterial and astringent (Lu et al. 2012).

15.2.16.2 Geranium wallichianum D. Don Ex Geranium wallichianum is commonly known as Kaphla (Hindi). The length of plant is 50 cm or more. Stem ascending, adolescent and 1–4 ft. length. Leaves palmate partite, reniform, 5.5–7 cm wide, lamina pubescent-villous, 5-angled lobes rhomboid cuneate, size of petiole is 7 cm, at the base of lamina dense villous (Shaheen et al. 2017). Ethanobotanical and medicinal use: Root is used in backache, joint pain, colic, jaundice, and kidney and spleen disorder. The whole plant is used to cure a wide range of disorders from simple toothache to complex illnesses of diabetes and blood pressure (Ahmad et al. 2003).

15.2.17 Family—Hypoxidaceae 15.2.17.1 Curculigo orchioides, Gaerth Curculigo orchioides is commonly known as Kalimusli (Hindi). It is a linear lanceolate, perennial herb with a rosette of sensible membranous leaves and bright yellow color flowers close to ground. The plant is native to India and is distributed from subtropical Himalayas, Assam, West Bengal, Konkan, West peninsula to Kanyakumari. Ethanobotanical and medicinal use: Rhizomes are widely used as a diuretic, aphrodisiac, demulcent, and aromatic tonic in the treatment of nervous disease and leprosy. Rhizomes of C. orchioides have been reported for their medicinal properties like antioxidant activity, platelet regeneration, antipyretic activity, hepatoprotective efficacy, and immune stimulant properties (Susindran and Ramesh 2014). Antimicrobial activity: The antimicrobial activity of the methanol extract of Curculigo orchioides callus was examined, both qualitatively and quantitatively at 10 mg/mL (100 μg) concentration by the presence or absence of microbial growth and zone of inhibition on agar well plates. The callus extract of Curculigo orchioides showed good inhibitory activity against only gram-positive organisms like S. aureus (16.0 ± 1.3) and Bacillus cereus (14.7 ± 2.5) and average antibacterial activity against gram-negative organisms like E. coli (10.1 ± 1.5), S. typhimurium (12.9 ± 1.6), P. vulgaris (10.9 ± 1.2), and P. aeruginosa (8.7 ± 1.5). However the methanol extract showed good zone of inhibition in C. albicans (13.3 ± 0.5). The zone of inhibition of Ciprofloxacin (10 mg/mL) against tested organisms shows the

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range between 25.0 ± 4.52 (C. albicans) and 17.0 ± 0.95 (P. aeruginosa) (Garapati et al. 2016).

15.2.18 Family—Juglandaceae 15.2.18.1 Juglans regia L Juglans regia is a deciduous, large tree attaining heights of 25–35 m, and a trunk up to 2 m diameter, commonly with a broad crown and short trunk, though taller and narrower in dense forest competition. The bark is olive brown, smooth when young, and silvery gray on older branches, and features scattered broad fissures with a rougher texture. Ethanobotanical and medicinal use: Juglans regia leaves have been used mostly in traditional medicines worldwide as antimicrobial, anthelmintic, astringent, keratolytic, antidiarrheal, hypoglycemic, depurative, tonic, carminative, and for the treatment of sinusitis, cold, and stomachache. Antimicrobial activity: The antimicrobial activity of the acetone-based extract and aqueous extracts of J. regia at different concentrations of 150 μg, 200 μg, 250 μg, and 300 μg. Aqueous extract exhibited zones of inhibition against most of the tested samples whereas the acetone-based extract exhibited zones of inhibition against selected samples. A concentration of 300 μg/disc with the average zone of inhibition 16.50 mm in aqueous extract and 14.25 mm in acetone-based extract is found to inhibit the growth of salivary microbial flora of the in vitro test samples of saliva (Nael and Mohammad 2011).

15.2.19 Family—Linaceae 15.2.19.1 Linum usitatissimum L Linum usitatissimum is commonly known as flax and linseed. The leaves are alternate, slender lanceolate, grayish green, 2 to 4 cm long and 3 mm broad. The flowers are white or bright blue, with five petals, 1.5 to 2.0 cm in diameter. Linseed grows practically all over the world. Ethanobotanical and medicinal use: The seed is analgesic, emollient, demulcent, laxative, pectoral, and resolvent. The leaves and bark are used in the treatment of gonorrhea. The flowers are cardiotonic and nervine. The plant has a long history of folk use in the treatment of cancer diseases. Antimicrobial activity: The antibacterial activity of the petroleum ether extract showed antibacterial activity against all tested bacteria, with zone of inhibition between 10.2 and 23.5 mm in diameter, K. pneumoniae showed highest susceptibility toward petroleum ether extract compared with the antibiotics cephalexin and ampicillin, with an zone of inhibition 23.5 mm using the extract concentration 50 mg/cm3; this extract concentration was the optimal concentration inhibiting K. pneumoniae and S. aureus (Jabeen et al. 2014).

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15.2.20 Family—Lythraceae 15.2.20.1 Woodfordia fruticosa L. Kurz W. fruticosa is also known as Dhawala (Hindi). The plant is widely distributed in the Himalaya region, throughout India, ascending up to an altitude of about 1600 m, and also in a majority of the countries of Far East Asia and South East like China and Sri Lanka. The full-grown leafy shrub is about 3.2 m tall, having spread and long branches with fluted stems. The leaves are opposite or sub-opposite in nature. Flowers are innumerable, brilliant red, arranged in dense axillary paniculate-cymose clusters, with short glandular pubescent pedicels (Rani et al. 2015). Ethanobotanical and medicinal use: It is being used as a source of medicinal agents for anthelminthic, antibacterial, astringent, febrifuge, emetic, sedative, and stimulant. The decoction of the flower is used for burns, hemorrhage, leprosy, diabetes, and skin diseases. It is used in leucorrhea, menorrhagia, and antitumor activity. Antimicrobial activity: The methanolic extracts and ethanolic extracts showed maximum antibacterial activity against all the bacterial strain used with a zone of inhibition ranges from 7.4 to 23.0 mm and less activity was observed in the aqueous extract with the zone of inhibition ranges from 6.0 to 15.5 mm. The standard antibiotic Ciprofloxacin (1 mg/mL) shows highest zone of inhibition against S. paratyphi, i.e., 35 mm and the test drug was against S. sonnei, i.e., 23 mm of zone of inhibition. The plant extracts were also screened for qualitative analysis to know the presence of phytochemicals which may be responsible for the potent antibacterial activity (Kumar et al. 2014).

15.2.21 Family—Lamiaceae 15.2.21.1 Nepeta ciliaris Benth The genus Nepeta comprises about 250 species. Locally, this plant is known as Jufa. The plant is perennial, sub-shrub, around 40–70 cm tall. N. ciliaris is used for the preparation of joshandah, extensively used by the masses in India for the treatment of catarrh, common cold, cough, and associated respiratory disease and fever. Ethanobotanical and medicinal use: The decoction of leaves and seeds is taken for treating fever. N. ciliaris is used as an antipyretic and antitussive agent. The pharmaceutical cough syrups and drugs use it as a principal ingredient. The liquid extract (Araq-e-Zuufaa) and squash (Sharbat-e-Zuufaa) prepared from N. ciliaris are prescribed when phlegm is thick and sticky and chest is congested. Antimicrobial activity: Antibacterial activity may be indicative of the presence of some metabolic toxins or broad spectrum. The methanol extract, water extract, acetone extract, and petroleum ether extracts were active against all the selected pathogens. The N. ciliaris extracts were found to be less effective as compared to erythromycin. In the case of S. pneumoniae, S. aureus and S. pyogenes acetone

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extract exhibited the highest degree of antimicrobial activity as compared to methanol extract, aqueous extract, and petroleum ether extracts. The maximum zone of inhibition by acetone extract found against S. aureus and S. pneumoniae was 14 mm and 17 mm respectively. While in case of P. aeruginosa the methanol extract was most active and showed maximum inhibition (15 mm) followed by acetone extract, aqueous extract, and petroleum ether extracts. The minimum inhibitory concentration determined against the four respiratory tract pathogens showed positive inhibition and found that the lowest value was exhibited by S. pyogenes, S. aureus, and S. pneumoniae at 40 mg/mL and P. aeruginosa at 50 mg/mL concentrations.

15.2.22 Family—Mimosaceae 15.2.22.1 Acacia catechu L Acacia catechu is a small or medium-sized, thorny tree up to 15 m long; its bark is grayish brown or dark gray. Flowers are 5–10 cm long, pentamerous, axillary spikes, white to pale yellow. The plant has diverse pharmacological actions and has been widely used in ayurveda for processing various formulations. Ethanobotanical and medicinal use: The plant extract is used to treat diarrhea and sore throats, also useful in high blood pressure, dysentery, gastric problems, colitis, bronchial asthma, leucorrhea, cough, and leprosy. It is used as mouthwash for mouth, sore throat, gum, gingivitis, dental and oral infections. Antimicrobial activity: Different organic solvent extracts of leaves are shown to have the zone of inhibition ranging from 18 to 24 mm. This extract was equally inhibitory for gram-positive bacteria as well as gram-negative bacteria. Among all the selected bacterial strains tested, S. aureus was found most susceptible with the maximum zone of inhibition by methanolic extract producing the zone of inhibition >20 mm. The methanolic extracts were found to be a stronger inhibitor than other extracts. MIC of this extract was 1000 μg/mL against S. aureus and B. subtilis while it was 700, 1500, and ≤ 2000 μg/mL for gram-negative S. typhimurium, E. coli, and P. aeruginosa respectively (Negi et al. 2012).

15.2.23 Family—Myrtaceae 15.2.23.1 Eucalyptus tereticornis Smith Eucalyptus tereticornis is a tree up to 35 m tall. Trunk is erect, 1–1.8 m in diameter, open or fairly dense, variable, whitish, bark smooth, peeling in irregular thin sheets or large flakes. Leaves are alternate, drooping on slender leaf stalks, hairless, with many fine side veins at an angle and a distinct vein along edge. Flower clusters (umbel) as single at leaf base (Jain et al. 2010). Ethanobotanical and medicinal use: Essential oil has traditionally been used to treat bronchitis, sinusitis, and respiratory tract disorders such as pharyngitis. The leaves are also a good source of important triterpenoids such as ursolic acid and

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betulinic acid, which have a wide range of pharmacological activities and therapeutic uses. Antimicrobial activity: The antimicrobial activity of methanolic solvent leaf and bark extracts of eucalyptus tree was evaluated against gram-negative and grampositive bacteria S. aureus, E. coli, S. mutans, and a yeast C. albicans. Methanolic bark extract of eucalyptus was more effective in inhibiting all the test pathogens with the zone of inhibition measuring between 17 mm and 27 mm as compared to methanolic leaf extract (18–24 mm) (Jain et al. 2010).

15.2.24 Family—Moraceae 15.2.24.1 Ficus religiosa L. F. religiose is found throughout the plains of India up to 1700 m altitude in the Himalayas. Leaves are alternate, spirally arranged and broadly ovate, glossy, coriaceous (leathery). Bark is gray with brownish specks, smooth, exfoliating in irregular rounded flakes. Ethanobotanical and medicinal use: Leaves have been used to treat skin diseases whereas bark has been used in the treatment of gonorrhea and ulcers as antidiarrheal, astringent, antibacterial, antiprotozoal, and antiviral agents. Latex is used as a tonic, and fruit powder is used to treat laxatives and asthma (Singh et al. 2003). Antimicrobial activity: Different concentrations of methanol extract and diethyl ether extractions (100, 200, 300, and 400 mg/mL) of both bark and leaves of F. religiosa were used for the assay. Disc diffusion method was used to carry out the assay. The methanol extracts of leaves and bark showed antimicrobial activity against three bacteria. At lower concentrations methanol extracts showed less antimicrobial activity and showed higher activity at 400 mg/mL concentration against the three tested bacteria. Both leaf and bark methanol extracts measured by zone of inhibition 2.8 and 2.2 mm respectively in S. aureus and 2.4 and 1.8 mm respectively in E. coli. P. aeruginosa is measured by a small zone of inhibition (2.2 and 1.1 mm) in methanol extracts of leaves and bark. But at lower concentrations no activity was observed whereas at higher concentrations (40 mg/mL) very less activity was observed against fungi (A. niger) (Ramakrishnaiah and Hariprasad 2013).

15.2.25 Family—Myricaceae 15.2.25.1 Myrica esculenta D. Don M. esculenta is commonly known as Boxberry, Kaiphal (Hindi). The genus M. esculenta constitutes about 97 species of aromatic shrubs and small trees. These are reported to be globally distributed in both temperate and subtropical regions of the world. Leaves are lanceolate with entire or serrate margin, having pale green at lower surface and dark green at upper surface, about 9–12 cm in length and 3–3.5 cm in width, and are mostly crowded toward the ends of branches.

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Ethanobotanical and medicinal use: Fruits and roots are used to prepare ayurvedic formulations such as Chwayanprash and Brahmarasayan to enhance digestion, memory, intelligence, concentration and physical strength. Used in abdominal tumors, asthma, fever, piles, irregular bowel function, anemia, nausea, oral disorders, cough, and dyspnea. Useful to retain placenta and bone fracture. The juice of the unripe fruit is used as an anthelmintic. Antimicrobial activity: Methanol extract of M. esculenta fruits showed antibacterial activity against S. epidermis and S. aureus comparable to that of the standard tetracycline with a maximum zone of inhibition 18 ± 0.5 and 16 ± 0.5 mm, respectively (Rawat et al. 2011).

15.2.26 Family—Oleaceae 15.2.26.1 Olea europaea L Olive (Olea europaea) is one of the oldest known cultivated plant tree species. The wild olive tree is a prolonged, evergreen species, extensive as a native plant in the Mediterranean province. It is a tree bearing silvery green leaves, feathery white and small flowers. It is globally distributed especially in the tropical and subtropical region (Ross 2005). Ethanobotanical and medicinal use: The olive tree has an extensive history of therapeutic and nutritional values. This herb is used for kidney problems, backache, and orally for sore throat infection. Leaf infusions are used elsewhere as a lotion to treat eye disease or a gargle to relieve sore throat (Sanjay et al. 2014; Ross 2005). Antimicrobial activity: O. europaea extracts showed antibacterial activity against tested pathogens. The fruit extract of O. europaea antimicrobial activity showed against S. aureus (16 mm) at concentration 800 μg/mL (Charu et al. 2008). O. europaea aqueous water extracts were screened for their antimicrobial activity against six bacteria such as B. subtilis, B. cereus, S. aureus, P. aeruginosa, E.coli, and K. pneumoniae and two fungi such as C. neoformans and C. albicans (Bensehaila et al. 2022). Methanolic extract of dried fruits was inactive against E. coli. Different research groups (Anesini and Perez 1993; Sanjay et al. 2014) reported antimicrobial potential of crude extracts acetone, methanol, water and petroleum ether of O. europaea against dental pathogens (S. sanguinis, S. aureus, S. mutans, S. sobrinus, S. salivarius, L. acidophilus, and C. albicans). The isolated phenolic components of O. europaea showed inhibitory effect against some foodborne pathogens such as Helicobacter pylori, Campylobacter jejuni, and S. aureus.

15.2.27 Family—Pinacae 15.2.27.1 Pinus roxburghii Sergeant P. roxburghii sergeant is commonly known as “chir pine” and has a long history of medicinal use. Pinus consists of 110–120 species that are distributed throughout temperate regions of the northern Himalayan. It is a large tree, branches are more or

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less whorled, and bark is dark gray, often reddish, rough, deeply fissured and exfoliating in longitudinally elongated plates. Leaves are in clusters of three, 20–30 cm, long, finely toothed, triquetrous, light, wings long, green, membranous. In soldering process, resin is used to get rid of oxide compounds in the surface of metal, synthetic rubber, and chewing gums. Ethanobotanical and medicinal use: P. roxburghii is used in many traditional medicines as diuretic, diaphoretic, antiseptic, ionic, vermifuge, and rubefacient as well as has cultural uses like herbicide, charcoal, pigment, resin, and wood (Siddiqui et al. 2009). In ayurvedic medicine, P. roxburghii is prescribed as an antidyslipidemic, intestinal antiseptic, spasmolytic, and antioxidant. The resin is applied to cure boils and administered orally to combat gastric problems (Puri et al. 2011). Antimicrobial activity: The antibacterial activity of the essential oil of Pinus roxburghii was tested against a panel of six bacterial strains used such as P. aeruginosa (MTCC-1688), Bacillus subtilis (MTCC-441), S. aureus (MTCC 96), E. coli (MTCC-443), K. pneumonia (MTCC-19), and P. vulgaris (MTCC1771). The essential oil of P. roxburghii showed antibacterial effect against the entire selected microorganisms used for screening. This oil was mainly effective against E. coli and P. vulgaris with the highest zone of inhibition 30 and 32 mm respectively. Streptomycin sulfate was used as a positive control which showed the zone of inhibition between 20 and 30 mm against different bacterial species. Therefore the antibacterial activity of Pinus roxburghii essential oil seems closer to reference antibiotic. The MIC value of P. vulgaris was found between 6.4 and 12.8 mg/mL whereas the MIC of other selected bacteria was found within the range of 12.8 mg/mL (Qadir et al. 2014).

15.2.28 Family—Papaveracea 15.2.28.1 Argemone mexicana L The plant is an annual herb, an erect prickly of about 1 m high; leaves are usually 5 to 11 cm long, and more or less blotched with white and green, glaucous broad at the base, half-clasping the stem, spiny, and prominently sinuate-lobed. The flowers are yellow and become 4 to 5 cm in diameter, terminal, and scentless. The capsule is elliptic-oblong, spiny and obovate about 3 cm in length. The seeds are spherical, shining, black, and pitted. Ethanobotanical and medicinal use: The plant possesses analgesic, narcotic, antispasmodic, and sedative properties. The fresh yellow, milky, seed extract contains protein dissolving substances which are effective in the treatment of warts, cold sores, cutaneous infectious, skin diseases, itches, and dropsy jaundice, and the smoke of the seeds is used to relieve toothache. Antimicrobial activity: The in vitro antibacterial activities of A. mexicana extracts against the employed bacteria were assessed by the presence or absence of the growth zone of inhibition. The lowest concentration of ethyl acetate extract, acetone extract, and petroleum ether extract that resulted in complete growth inhibition of the

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tested organisms was found in the range of crude to 512 μg mL-1. The petroleum ether extract showed higher antibacterial activity by having lower minimum inhibitory concentration value than ethyl acetate extract and acetone extract (Rahman et al. 2009).

15.2.29 Family—Rosaceae 15.2.29.1 Pyrus pashia Buch Ham. Ex D.Don Pyrus pashia, the Himalayan Pear, is a small or medium-sized deciduous tree, found in the Himalayas. Leaves are long-pointed, ovate to broadly lance-shaped, toothed, hairless, and shining. The leaf is 5–10 cm long and shoots are often 3–5-lobed. Flowers are 2–2.5 cm across, with darker veins with white obovate petals. The sepal cup is urn-shaped with spreading white-woolly sepals. The fruit is round, dark brown, covered with raised pores and 1.3–2.5 cm long (Janbaz et al. 2015). Ethanobotanical and medicinal use: Traditionally, leaves and fruits crush is used in wounds, cuts, bacterial and fungal infection. Fruit juice and ripe fruits of P. pashia are used for treating eye injury and mouth sours (Negi et al. 2011). 15.2.29.2 Rubus ellipticus Sm Rubus ellipticus, commonly known as Yellow Himalayan Raspberry, is mostly found in forest edges, and numerous forests exist over wide areas of mountains and lowlands of India and Sri Lanka. Branches are purplish brown or brownish, pubescent, with sparse, curved prickles and dense, purplish brown bristles or glandular hairs. It usually grows 100–300 cm tall. Ethanobotanical and medicinal use: Traditionally used to cure diabetes mellitus, ulcers, and inflammatory disorders. The juice extracted from the root has also been used for gastric problems, fevers, diarrhea, and dysentery, and the root paste, applied to wounds, promotes healing and has antimicrobial, antifertility, analgesic, and antiepileptic properties (Saini et al. 2014). Antimicrobial activity: Methanolic extract of R. ellipticus showed significant zone of inhibition whereas hexane was moderate, and ethyl acetate and methanol extract showed less activity against all the selected species. Further, all the bacteria were found to be more susceptible to methanolic leaf extracts. Ethyl acetate and methanolic extract showed the maximum zone of inhibition produced against the bacteria S. aureus (14, 17, 19 mm and 16, 20, 22 mm) followed by M. luteus (12, 15, 17 mm and 12, 15, 16 mm) (Saini et al. 2014).

15.2.30 Family—Rutaceae 15.2.30.1 Murraya koenigii L Murraya koenigii, commonly called Curry leaves in trade, occurs throughout India up to an altitude of 1500 m. It abundantly occurs in outer Himalayas, Assam, Chittagong, upper and lower Burma, and Andaman Islands (Ramsewak et al. 1999).

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Ethanobotanical and medicinal use: The fruits and leaves are also a source of an essential oil which finds use as a fixative for heavy type of perfume soap. The juice of roots provides relief from renal pain, dysentery, and diarrhea and prevents vomiting. Antimicrobial activity: Antibacterial activity of M. koenigii (leaf extract of ethanol with DMSO) was assayed in vitro by agar cup method against clinical isolates of P. aeruginosa and E. coli. The sequence of antibacterial activity of leaf extract against E.coli exhibited no antimicrobial activity in 25 μL but produced 11 mm and 17 mm zones of inhibition in 50 μL and 100 μL concentrations, respectively. With respect to P. aeruginosa the plant extract had shown no antimicrobial activity in 25 μL and 50 μL concentrations but produced a 15 mm zone of inhibition in 100 μL concentration. Higher concentration of the leaf extract shows highest antibacterial activity (Tachibana et al. 2001).

15.2.31 Family—Ranunculaceae 15.2.31.1 Aconitum heterophyllum Wall. Ex Royle Aconitum heterophyllum, commonly known as Atis, is one of the most important medicinal plants. It is native to the northern Himalayas and found in Kashmir, Uttarakhand, Sikkim, and Nepal at altitudes between 2500 and 4000 m. Stem is simple, erect or branched, from 15 to 20 cm tall. Glabrous below, finely crispopubescent in the upper part. Ethanobotanical and medicinal use: The plant is used to treat patients with reproductive disorders and is known to have hepatoprotective, analgesic and antipyretic, antioxidant, alexipharmic, anodyne, anti-flatulent, anti-atrabilious, antiperiodic, anti-phlegmatic, and carminative properties. Antimicrobial activity: The antibacterial activity was compared with the two standard antibacterial antibiotics Cefuroxime and Amoxicillin. The MIC of the methanolic extract was found to be 40 mg/well against S. aureus and for B. subtilis, it was 50 mg/well. Determination of the surviving fractions of strains against the increasing concentrations of methanol revealed that there was a considerable decrease in the surviving fractions with the increasing concentration of the extracts. Less surviving fractions (0.0047) were found in the case of B. subtilis, while maximum were in the case of P. aeruginosa (0.543), when the concentration of extracts was 60 mg/mL (Srivastava et al. 2011).

15.2.32 Family—Sterculiaceae 15.2.32.1 Abroma augusta L. Abroma augusta is distributed from India throughout southern China to South-East Asia, Solomon Islands, and the northern Australia. It is a small tree or erect shrub up to 10 m tall. Leaves are simple, alternate, and highly variable. The dry roots have 0.5–1.0 mm thick, brown barks which are highly fibrous. The thickness of the bark

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varies according to the age and girth of the root. Flowers are 5 cm in diameter, dark red, yellow or purple in color occurring on few flowered cymes (Gupta et al. 2011). Ethanobotanical and medicinal use: The plant are useful in treating diabetes, stomachache, dermatitis, leucorrhea, scabies, gonorrhea, cough, leukoderma, jaundice, nerve stimulant, weakness, hypertension, uterine disorders, rheumatic pain of joints, and headache with sinusitis. It is also used in dermatitis, anti-inflammatory, and analgesics (Al-Mamun et al. 2010). Antimicrobial activity: The antibacterial activity of acetone-based plant extract was studied, and zones of inhibition of 27.0 and 26.0 mm diameter were recorded against S. typhi and B. megaterium, respectively. The lowest antibacterial activity of plant extract was 21.0 mm diameter of zone of inhibition observed against S. dysenteriae at the concentration of 300 μg/disc (Saikot et al. 2012).

15.2.33 Family—Solanaceae 15.2.33.1 Hyoscyamus niger L Hyoscyamus niger, commonly known as Henbane, is widely distributed in Europe and Asia. Within India, it has been recorded in the Western Himalayan region of Jammu & Kashmir, Himachal Pradesh, and Uttarakhand in an altitude range of 2100–3300 m. Leaves are gray-green, alternate, covered with short glandular hairs, oblong to lanceolate, short-stalked (lower) to sessile (upper), 5–20 cm long, coarsely toothed to acutely pinnate-lobed, with conspicuous pale veins covered with long glandular hairs (Cuneyt et al. 2004). Ethanobotanical and medicinal use: It is used in mental disorders, epileptic mania, and chronic dementia with insomnia, paralysis, agitans, convulsions, neuralgia, odontalgia, bleeding gums, dental caries, mammillitis, orchitis, rheumatoid arthritis, cardiac debility, epistaxis, hematemesis, whooping cough, asthma, bronchitis, cephalalgia, fever, meningitis, anxiety, insomnia, scabies, diabetes, spermatorrhea, dysmenorrhea, leucorrhea, amenorrhea, neuralgia, and beneficial in irritable infections in urinary tract. Antimicrobial activity: The antibacterial activity of H. niger extracts against the S. aureus examined in this study was qualitatively and quantitatively assessed by the presence of the zone of inhibition. The methanol extracts of H. niger had strong antibacterial effects against the selected bacterial strains, with the zone of inhibition at 9.0 to 25.0 mm. Notably, S. aureus is most susceptible to the extract of H. niger as compared to standard antibacterial antibiotics ampicillin and tobramycin (inhibition zone is 25.0 mm) (Dulger et al. 2010).

15.2.34 Family—Salvadoraceae 15.2.34.1 Salvodara persica L Salvodara persica is commonly known as Kharjal in Hindi. It is an evergreen, large much-branched shrub or a tree, found in the dry and arid regions of India, and on

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saline lands and in coastal regions just above the high water mark. The bark is deeply cracked, dull gray or gray-white, and leaves are variable in shape, lanceolate ovate, or elliptic ovate and somewhat fleshy. Flowers are smooth, pedicellate, greenish yellow or greenish white in lax panicles, drupes are globose or round, and red when ripe (Kumar et al. 2016). Ethanobotanical and medicinal use: Leaves are bitter and possess corrective, antiscorbutic, deobstruent, liver tonic, diuretic, anthelmintic, analgesic, and astringent properties and used in piles, scabies, strengthen the teeth, leucoderma, and other nose troubles. Antimicrobial activity: Antimicrobial activity against all the selected pathogens at 200 mg/mL. Methanol extract showed the maximum antibacterial activity against S. mutans and L. acidophilus followed by water, ethyl acetate extract, and petroleum ether extract. Methanol extract showed the best activity against L. acidophilus (22.3 ± 0.76 mm) and S. mutans (21.6 ± 0.76 mm) followed by S. sobrinus (19.3 ± 0.76 mm), S. aureus (19.3 ± 0.28 mm), S. salivarius (18.0 ± 0.50 mm), S. sanguinis (18.6 ± 0.76 mm), and C. albicans (14.0 ± 0.50 mm). Water and methanol extracts of S. aureus were investigated for their antibacterial activities against seven isolated oral pathogens including S. mutans, S. faecalis, S. pyogenes, P. aeruginosa, L. acidophilus, and C. albicans. The ethanol and methanol extracts of S. persica extracts showed antibacterial activity against S. aureus, E. faecalis, and K. pneumonia (Al-Bayati and Sulaiman 2008).

15.2.35 Family—Urticaceae 15.2.35.1 Urtica dioica Linn Urtica dioica Linn. is commonly known as Kandali (Hindi). It is broadly disseminated all through the mild and tropical regions around the world. It is found in the Himalayas from Kashmir to Kumaon (Uttarakhand), India, at heights of 2100–3200 m. Ethanobotanical and medicinal use: The leaves and underlying foundations of the plant are utilized inside as a blood purifier (Kataki et al. 2012). It is widely used by the traditional medicinal practitioners to cure various diseases such as hematuria, nephritis, jaundice, arthritis, menorrhagia, and rheumatism. U. dioica has various pharmacological activities like antioxidant, antibacterial, anti-inflammatory, immunomodulatory, analgesic, hepatoprotective, antiviral, anti-colitis, and anticancer effects. Antimicrobial activity: Antibacterial activity of the ethanolic and methanolic extracts of U. dioica was surveyed by Kirby-Bauer disc diffusion method. The zone of inhibition around each disc was measured in mm and recorded. Both extracts were active against B. cereus, S. aureus, S. epidermidis, and E. coli 10, 14, 16, and 18 mm (methanolic extract) and 9, 11, 16, and 17 mm (ethanolic extract) zone of inhibition. The MIC of methanolic extract against S. aureus and S. epidermidis was 40 and 10 mg/mL, respectively (Gülçin et al. 2004).

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15.2.36 Family—Verbenaceae 15.2.36.1 Clerodendrum serratum (L) Clerodendrum serratum L. is commonly known as Bharangi in Hindi. It is a shrub with a height of 0.9–2.4 m, scarcely woody, not much branched with stems bluntly quadrangular. Leaves are often ternate and opposite reaching as much as 28 cm long but usually 12.5–14 by 5.7–6.3 cm. Flowers are numerous, showy, in lax pubescent dichotomous cymes, with a pair of acute bracts at each branching and a flower in the fork (Patel et al. 2014). Ethanobotanical and medicinal use: Leaves of C. serratum have been used as traditional medicine for the treatment of cancer, malaria, stomach upset, labor pain, high fever, cough, etc. The root is used for the treatment of rheumatism, dropsy, typhoid, and asthma (Singh et al. 2012). Antimicrobial activity: The ether and saline extracts of the leaves exhibited antibacterial activity against S. aureus, while they were found to be inactive against E. coli. The sulfuric acid, phosphate buffer, and acetate buffer extracts were inactive against both the bacteria. The 80% ethanolic extract of the leaves at 25 mg/mL showed inhibition of E. coli, P. aeruginosa, S. aureus, and B. subtilis (Charu et al. 2008).

15.2.37 Family—Valerianaceae 15.2.37.1 Valeriana jatamansi Jones Valeriana jatamansi is commonly known as Indian Valerian (Mushkibala in Hindi). V. jatamansi is a perennial dwarf, small, hairy, rhizomatous herb having thick roots covered with fibers. The plant grows at an altitude of 1220–3000 m (Rather et al. 2012). Ethanobotanical and medicinal use: Valeriana jatamansi is regarded as an antispasmodic, tranquilizer, antiseptic, expectorant, febrifuge, ophthalmic, sedative, and tonic useful in hysteria, cholera, snakebite, scorpion sting, asthma, and neurosis. Roots are acrid and bitter which are used as carminative, laxative, and are also used for curing blood diseases, burning sensation, cholera, skin disease, throat troubles, and ulcers (Negi et al. 2012). Antimicrobial activity: The antimicrobial activity of the plant extracts in different solvents in varying concentrations was observed. Ethanolic extract of V. jatamansi showed maximum activity against all selected bacterial strains except B. subtilis, which was found sensitive to methanol extract of the species. H. intermedia and V. jatamansi extracts in the solvents were also found to inhibit the growth of E. coli (Agnihotri et al. 2011).

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15.2.38 Family—Zingiberaceae 15.2.38.1 Kaempferia rotunda, Linn It is also known as Bhumi-Champaka, or Hallakam, and widely distributed throughout India in wet and shaded regions. Leaves are broad-lanceolate, measuring around 17–27 cm long. The upper side of the leaf is dark green, and light green along the mid-rib. Flower arises from the underground rhizome, white two purple lower lobes. Ethanobotanical and medicinal use: The young leaves and rhizome are used as a spice to add flavor to food. The rhizome juice is used to treat throat infection, but the juice can cause vomiting and much salivation. It has antibacterial, anti-inflammatory, antiulcer, antitumor, and wound healing properties. Antimicrobial activity: Antibacterial activity for different solvent extracts was tested. Ethyl acetate extract showed maximum zone of inhibition against L. acidophilus (MTCC 447) (17.3 ± 0.57 mm), S. pyogenes (16.6 ± 0.28 mm), S. pneumonia (16.6 ± 0.28 mm), and P. aeruginosa (15.3 ± 0.28 mm). The methanol extract exhibited moderate activity against P. aeruginosa (15.6 ± 0.28 mm), S. pneumonia (15.3 ± 0.57 mm), and S. pyogenes (15.3 ± 0.28 mm). In addition, methanolic extract of K. rotunda reportedly showed potential antiplatelet aggregation (Rashel Kabir et al. 2011) (Table 15.1).

15.3

Discussion

India has agelong culture associated with the use of medicinal plants based on local and indigenous beliefs, traditional knowledge, and skills. Even today local people primarily depend on several plants for medicinal purposes on a day-to-day basis. These plants are easily available to the locals in their surroundings or nearby wastelands and forests. Plants which have been studied and investigated pharmacologically and chemically have been helpful in providing active principle in modern medicine and in some cases giving leads for partial or total synthesis of new drugs. Accessibility of modern medical facilities has also played a role in keeping local peoples dependent on traditional methods of using plants as a source for treating ailments. The natives have been traditionally using almost all parts like leaves, roots, and fruits and in some cases flowers, bark, stem, seeds as well as whole plants as a source of medicines. Common ailments like stomachache, fever, cold and cough, bleeding and wounds, fungal infections, insect bites, and rheumatic pains are treated by plant extracts which can be found in nearby surroundings. According to the WHO (2005) report, “traditional healers such as herbalists, midwives and spiritual healers constitute the main source of assistance with health problems for at least 80% of rural population in developing countries.” Indigenous knowledge of flora and fauna has led to the production of a number of pharmaceutical products and therapeutic practices. People with knowledge of traditional medicines and practicing the same to treat several diseases are known to the communities with different names as traditional folk healers, Vaidya, Hakeem, Amchi, Dai, etc. On religious grounds a small patch of forest is saved by local communities known as sacred groves. These

Holarrhena pubescens BuchHam Acorus calamus L.

Eupatorium odoratum L. Agave americana L.

Anaphalis contorta (D. Don) Hook. f. Bidens pilosa L.

Kuru

Vacha

Apocynaceae

Acoraceae

Agavaceae

Kharna, bakura Rambans

Kumur

Asteraceae

Asteraceae

Buglya

Asteraceae

Leaves and rhizome

Bark

Leaf

Whole plant

Plant extract

Leaf

Leaves

Fruit, seeds Whole plant

Seeds

Leaves

Antispasmodic, antibacterial, antifungal, antioxidant, antihepatotoxic,

Indigestion, flatulence, constipation, jaundice, and dysentery Dysentery, febrifuge

Burns, skin diseases, and wounds

Caraway Whipcord cobra lily Bhringaraj

Apiaceae Araceae

Asteraceae

Ajmod

Apiaceae

Apium graveolens L. Carum carvi L. Arisaema tortuosum (Wall.) Schott Eclipta alba (L.) Hassk.

Brahmi

(continued)

Priya and Ganjewala (2007)

Siddiqui et al. (1993)

Krishnaveni (2017)

Dagawal and Ghorpade (2011) Singh et al. (2015)

Joshi (2011)

Pandey et al. (2011)

Gupta et al. (2011) Rameshwar et al. (2014)

Tyagi et al. (2013)

Udoh et al. (2012)

Apiaceae

Centella asiatica L.

References Ilango et al. (2009), Sayeed et al. (2009)

Family Acanthaceae

Plant species Adhatoda zeylanica medic

Ethanomedicinal use Treatment of bleeding piles, wound healing, antibacterial, antiulcer, hepatoprotective, cardioprotective, anti-inflammatory Asthma, skin disorders, ulcers and body aches, nervine tonic, improving memory, wound healing, cytotoxic and antitumor, treatment of leprosy, urethritis, and leucorrhea Bronchitis, liver, asthma, spleen disease, arthritic pain, and rheumatism Dysuria, hematuria Constipation, indigestion, abdominal pain, and dysentery Liver cell generation, diuretic in hepatic and spleen enlargement, skin diseases, antiviral activity against Ranikhet disease virus Paste applied on cuts, wound, boils, and insect repellent With honey used in cough and bronchitis

Table 15.1 List of ethnomedicinal plants with antibacterial properties Parts used All parts of the plant

Ethnomedicinal Pertinence and Antibacterial Prospective of. . .

Local name Adulsa

15 339

Semal Bahera

Arjun

Simara

Dhoop

Sal

Thuner

Bombacaceae Combretaceae

Combretaceae

Caesalpiniaceae

Compositae

Dipterocarpaceae

Ephedraceae

Bombax ceiba L. Terminalia bellirica Roxb. Terminalia arjuna

Cassia fistula L.

Jurinea dolomiaea, Boiss. Shorea robusta, Gaertn Taxus baccata L.

Anwala

Rolli

Euphorbiaceae

Euphorbiaceae

Emblica officinalis Gaertn. Mallotus philippensis, Linn, Muell.

Patashij

Euphorbiaceae

Euphorbia neriifolia L.

Bhojpatra Shyonaka

Betuleae Bignoniaceae

Betula utilis, D. Don Oroxylum indicum (L.) Vent

Local name

Family

Plant species

Table 15.1 (continued)

Fruit

Juice, root, stem, and leaves Fruit

Bark

Bark

Root

Fruit, bark

Leaf

Root, stem Seed, nut, fruit

Bark Root, bark, leaves

Parts used

Antiseptic, antidote of snake and scorpion bite, asthma, bronchitis, and skin diseases Extract of root is used in treatment of rheumatism, gout, fever, and skin eruptions Bark extract and gum are used in diarrhea, dysentery, and skin allergies Plaster on fractured bones, headache, taxolanticancer Asthma, syphilis, dropsy, general anasarca, leprosy, ulcers, scabies, antiseptic, bronchitis, piles, diuretic, cough, and cold Vitamin C, several disorder, diarrhea, dysentery, and eye diseases Fruit extract used in wound healing, ulcers, cough, ringworm, hemorrhages, and skin disorders

immunosuppressive, insecticidal, antiulcer, and antispasmodic Bark paste used in the treatment of broken bone Biliousness, fevers, bronchitis, intestinal worms, vomiting, leucoderma, asthma, diarrhea, dysentery, antimicrobial activity Aphrodisiac, leucorrhea, digestive disorder Dry prolonged cough, dropsy, diarrhea, and leprosy Antibacterial

Ethanomedicinal use

Gangwar et al. (2014)

Asmawi et al. (1993)

Sultana et al. (2022)

Purohit et al. (2001)

Vashisht et al. (2016)

Singh et al. (2015)

Sanjay et al. (2014), Aneja et al. (2012) Bhalodia et al. (2012)

Rameshwar et al. (2014) Chanda et al. (2013)

Mishra et al. (2016) Deka et al. (2013)

References

340 S. Mehra et al.

KaliMusli

Hypoxidaceae

Nepeta ciliaris Benth Acacia catechu L.

Linaceae

Linum usitatissimum L. Woodfordia fruticosa L. Kurz.

Jufa

Khair

Mimosaceae

Flax and linseed Dhawa

Lamiaceae

Lythraceae

Juglandaceae

Geraniaceae

Walnut

Phori, syunli Kaphla

Geraniaceae

Fagaceae

Chemical goldmines Baanj

Dhak or palas

Fabaceae

Fabaceae

Anyar

Ericaceae

Juglans regia L.

Pueraria tuberosa (Roxb. ex Willd.) Quercus leucotrichophora A. Camus Geranium nepalense Sweet Geranium wallichianum D. Don ex Sweet Curculigo orchioides, Gaerth.

Lyonia ovalifolia, Wallich Drude Butea monosperma (Lam.) Taub.

Bark

Seeds, oil, and flower Leaves, fruits, flowers, and gum Whole plant

Leaves

Rhizome and leaves

Root

Whole plant

Leaf, bark

Leaves, bark, seed oil, flowers Tuber

Seed

Powder of rhizome used in urinary disorder, diarrhea, jaundice, aphrodisiac tonic, and piles; paste of leaves used in wound healing Antimicrobial, anthelmintic, astringent, keratolytic, antidiarrheal, hypoglycemic, and depurative Anti-inflammatory, analgesic, vesicant, chest cleanser, aphrodisiac, and phlegm expectorant Antitumor activity, astringent, hemostatic, anthelminthic, wound healing, antibacterial, and antidysenteric Cold, catarrh, respiratory distress, fever, antimicrobial Diarrhea, dysentery, bronchitis, menstrual disorder

Used as influenza, dysentery, antiphlogistic and analgesic tonic Paste of root used in joints pain; root extract used in cholera, dysentery, and cold

Gonorrheal, asthma, hemorrhages, diarrhea

Shahavi and Desai (2008)

Anti-inflammatory, antifungal activity, bactericidal and fungicidal, liver disorders, antiestrogenic, and anthelmintic Antioxidant and adaptogenic activity

Ethnomedicinal Pertinence and Antibacterial Prospective of. . . (continued)

Negi and Dave (2010)

Shankar et al. (2012)

Rani et al. (2015)

Jabeen et al. (2014)

Nael and Mohammed (2011)

Susindran and Ramesh (2014)

Ahmad et al. (2003)

Lu et al. (2012)

Theng and Korpenwar (2012) Sati et al. (2011)

Sahu and Arya (2017)

Wounds and boils

15 341

Chir

Satyanashi

Mehal

Padam

Hisalu

Gandela

Atis

Ulatkambal

Henbane

Papaveracea

Rosaceae

Rosaceae

Rosaceae

Rutaceae

Ranunculaceae

Sterculiaceae

Solanaceae

Murraya koengii L.

Aconitum heterophyllum wall. ex Royle Abroma augusta L. f. Hyoscyamus niger L.

Olive

Oleaceae

Pinaceae

Kaphal

Peepal

Moraceae

Myricaceae

Local name Safeda

Family Myrtaceae

Pinus roxburghii Sargent Argemone mexicana L. Pyrus pashia Buch Ham. ex D.Don. Pyracantha crenulata (Don) Roem. Rubus ellipticus Sm.

Myrica esculanta D. Don Olea europaea

Plant species Eucalyptus tereticornis Smith Ficus religiosa L.

Table 15.1 (continued)

Roots, leaves, barks All parts of the plant

Flower, bark, root, leaves Dried tuberous roots

Bark

Fruit

Roots, leaves, seeds Fruit

Antibacterial

Fruit, flowers, stems Wood

Antidiabetic, anti-inflammatory, antifungal, antibacterial, and insecticidal Antispasmodic, anticholinergic, and analgesic

Saini et al. (2014)

Fevers, gastric troubles, diarrhea and dysentery, colic Piles, skin diseases, and bacterial infection; increases digestion; and controls dysentery Antibacterial and enzyme inhibition activities

Cuneyt et al. (2004)

Gupta et al. (2011)

Tachibana et al. (2001), Ramsewak et al. (1999) Srivastava et al. (2011)

Saklani and Chandra (2012)

Janbaz et al. (2015)

Rahman et al. (2009)

Charu et al. (2008), Bensehaila et al. (2022) Qadir et al. (2014)

Bhalerao and Sharma (2014) Rawat et al. (2011)

References Jain et al. (2010)

Dysentery

Antiseptic, hemostatic, foul ulcers, asthma, gonorrhea, epilepsy Leprosy, skin diseases, inflammations, and bilious fevers Digestive disorder, diarrhea, cancer

Digestive disorder

Ethanomedicinal use Chronic cough, asthma, bronchitis, pyorrhea, burns, dyspepsia, skin, insect repellent Bronchitis and skin ailments

Fruit, leaves

Bark

Parts used Leaves, bark

342 S. Mehra et al.

Bharangi

Balchhari, sumaya Bhui champa Gokharu

Violaceae

Verbenaceae

Valerianaceae

Viola odorata L.

Clerodendrum serratum (L.) Moon Valeriana jatamansi Jones Kaempferia rotunda, Linn. Tribulus terrestis, L.

Zygophllacea

Zingiberaceae

Kandali

Urticaceae

Vanfsa

Bhimal

Tiliaceae

Grewia optiva JR.D ex B Urtica dioica Linn.

Kharjal

Salvadoraceae

Salvodara persica L.

Whole plants

Rhizome

Leaves and roots Root

Seeds and leaves Whole plant

Stem, bark

Stem and bark

Respiratory disorders, fever, inflammation, liver disorders, and antiasthmatic potential Aphrodisiac, mental disorder, paste of root used in wound healing and blisters Antibacterial, anti-inflammatory, antitumor, antiulcer, wound healing Decoction of leaf and root used in treatment of kidney stone

Leaf extract believed to stop baldness and roots in various skin ailments Bronchitis, cough, cold

Antibacterial, antifungal activity

Antibacterial activity

Baburao et al. (2009)

Negi et al. (2012), Agnihotri et al. (2011) Rashel Kabir et al. (2011)

Singh and Dhariwal (2018) Patel et al. (2014)

Kataki et al. (2012)

Al-Bayati and Sulaiman (2008), Kumar et al. (2016) Arora (2011)

15 Ethnomedicinal Pertinence and Antibacterial Prospective of. . . 343

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sacred groves have been a traditional way of conservation of biodiversity of medicinal plants and serve as an in situ repository of medicinal plants too.

15.4

Conclusion

In India a major share of the health care system is occupied by medicinal plants and serves as an asset to the nation. This review tries to focus on the importance of medicinal plants having a vital role in the medicinal system of the Himalayan region in the state of Uttarakhand, India. Various tribal and local people use these medicinal plants to cure different ailments like fever, cough and cold, diarrhea, injuries, wounds cuts, ulcers, swelling, bone fractures, skin care, night blindness, dental problems, and antidotes. In modern-day world due to negligence of these traditional practices and lack of people having sound knowledge of these plants and their use, the growth and availability of these plants are getting affected. There is a need for constructive measures to make people aware of the use of these traditional medicines and conserve this knowledge and medicinal biodiversity with better documentation and human involvement programs. Social responsibilities have played a role in the protection of this natural wealth of Himalayas and should play a major role in the future too.

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An Immune Modulator Constituent in Mucuna Pruriens L. (DC) and Biotechnological Approach for Conservation

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Naushad Alam and Gul Naaz

Abstract

Mucuna pruriens is one of the high-value medicinal plants and an immune booster in modern medicines and Ayurveda systems. In its natural habitat, its propagation is limited, so micropropagation practice is one of the best substitute methods to fulfil its requirement on the industrial level. The present study provides an improved protocol for propagation by using axenic cotyledonary node (CN) explants excised from a 10-day-old plant. Murashige and Skoog’s medium augmented with different strengths (0.5–5.5 μM) of BAP (6-benzyl adenine) and Kin (Kinetin) alone or combined by different concentrations of CdCl2 (0.5–50.0 μM) with an optimal concentration of BA and Kin (2.5 μM) were used for studying the comparative morphogenic response of used explant. The results found that CdCl2 (2.5 μM) in the combination of BAP (2.5 μM) gave the highest multiplication of shoot (18.40), while Kin (2.5 μM) + CdCl2 (2.5 μM) induced 15.00 shoots per explants after 56 days of inoculation. Survival rates were also found to be maximum at the optimized medium. The best rooting was documented at 4.40 ± 0.51 mean root number on half strength of MS salt after 28 days of incubation. The in vitro-grown rooted plants were successfully acclimatized in Soilrite, then successful transfer to garden soil, where they grew well. Keywords

Velvet beans · Immune buster · Aphrodisiac · Micropropagation

N. Alam (✉) · G. Naaz Botany Department, Aligarh Muslim University, Aligarh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_16

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Introduction

The presence of enormous numbers of plant species, India has earned the title of “the world herbal garden,” with over 45,000 species of herbal medicinal plants spread over hotspots in the Western Ghats, Eastern Himalayas, and the Andaman and Nicobar Islands (Anonymous 2003). Many of these plant species are employed for medicinal purposes in traditional and modern medicine. From the beginning of civilization, medicinal plants have been used with great interest as they offer a valuable renewable resource for numerous drugs and chemicals. In the Ayurveda and Unani system of medicine, we find an exhaustive account of medicinal plants, their properties, and their practices for supporting and improving different body functions. For thousands of years, medicinal plants have been intimately linked with the human body’s strength and immune system. In the past few decades and recently, during the COVID-19 pandemic, there has been a resurgence of attention towards the use and study of medicinal plants in human civilization and recognition of the importance of plant-based products to human health care. Nowadays, compared to synthetic drugs, the health care system is dominated by herbal Medicare as it is considered safer. Recently, plant-based products such as dietary supplements and drug manufacturing at the industrial level have been significantly improved. The diversity of medicinal plants represents the significant natural wealth of our country. They provide healthcare facilities to ordinary people and are helpful in the economic growth of the country through the export of medicinal plants (Bhat and Lone 2017; Van Wyk and Prinsloo 2018). A prominent member of medicinal plant species is at a high risk of extinction because of continuous collection and over-exploitation from the wild for commercial uses. We are losing about one potentially vital medicinal plant every 2 years. The loss of possible sources of vitamins, protein-rich foods, principal crops, and bioactive substrates also represents the extinction of plant species from their natural habitat. Continuous loss of medicinal plants produces huge fears about income, health care, and living safety in developing and underdeveloped countries. Depletion occurs due to the destructive, unsustainable, and continuous harvesting of such potent medicinal plants resulting in the lack of genetic resources from natural habitats, which may also lead to a severe global shortage of these products (Hamilton 2004; Negi et al. 2018). In India, the medicinal plant sector significantly influences the socio-cultural, therapeutic, and economic situation of tribal and rural populations. Thus, the Indian government has established a “Department of Indian system of Medicine and Homeopathy,” in addition to the “Medicinal Plant Board,” which promotes, regulates, and develops the sector for conservation and sustainable consumption. The government and other private agencies have recognized the protection of pharmaceutical plants as one of their main goals and have started their conservation programs in the threatened zones. The main aim of conservation programs is the appropriate use of natural resources, encouraging sustainable progress without disturbing the wild species. The sustainable use of pharmaceutical plants includes collection, propagation, evaluation, characterization, disease indexing, exclusion, storage, and distribution of medicinal plants by certified organizations (Sharma 2003; Sarkar et al. 2015).

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Mucuna pruriens (velvet beans), belonging to the Fabaceae family, is a highly valuable climbing legume that consists of about 150 annual or perennial species, some being endemic to India or other tropical regions. They are found in bushes and hedges in damp places, ravines, and scrap jungles throughout the plains of India, Africa, and the West Indies (Singh et al. 1996; Anonymous 2003). Almost all the plant’s parts are used to produce medicinal compounds, in which L-dopa (a dopamine precursor) is one of its main active constituents. Its highest percentage is in seeds (7–10%), while its lowest is in leaves (1%). It is used in Parkinson’s disease treatment (Bell and Janzen 1971; Morris and Wang 2018). L-DOPA (3, 4-dihydroxy phenyl-L-alanine) is a non-protein amino acid obtained from its seeds. Seeds also contain glutathione, gallic acid, and beta-sitosterol. Seeds also possess unidentified tryptamine bases such as mucunadine, mucunine, prurienine, and prurieninine (Majumdar and Santra 1953; Kapoor 2017). Chemical compounds such as indole-3-alkylamines-N, and N-dimethyltryptamine, are obtained from the plant parts, while the compound Serotonin is only present in seed pods (Khare 2004; Liu et al. 2016; Upadhyay 2017). The seeds also contain oils, including palmitic acid, oleic acid, linoleic acids, nicotinic acid, lecithine, n-hexadecanoic acid, ascorbic acid, squalene, and octadecanoic acid (Singh et al. 1996; Upadhyay 2017). Apart from these, the seed also contains 5-hydroxytryptophan, bufotenine, 5-hydroxytryptamine, 6-methoxyharman, tryptamine, palmitic acid, stearic acid, 3-methoxy-1,1-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroquinoline,1-dimethyl7,8-dihydroxy-1,2,3.4-tetrahydroquinoline, Indole-3-alkylamines-N, N-dimethyltryptamine (Liu et al. 2016; Upadhyay 2017). Protein extracts from seeds of M. pruriens are active against snake bites (Tan et al. 2009), and seeds are also used as antidiabetic (Shanmugavel and Krishnamoorthy 2018). All plant parts are rich in the phenolic compound, which exhibits significant antioxidant and free radical scavenging capacity (Rajeshwar et al. 2005; Neta et al. 2018). The beans of Mucuna pruriens are a rich source of carbohydrates and protein and are used as an instant energy food to gain muscle mass. Its demand as a protein supplement for muscle gain and supplementary nutrition has increased manifold (Musthafa et al. 2018; Briguglio et al. 2018). M. pruriens is a powerful aphrodisiac to increase sperm count and testosterone levels (Jadhao 2013; Singh et al. 2017). Leaves are harvested as fodder and used as a green cover crop. The species is well known for its nematicidal effects and antimicrobial activity when used in rotation with several commercial crops (Anonymous 2003). Foundation of Revitalization of Local Health Traditions (FRLHT), working under the Ministry of Environment and Forest and the ministry of health, Government of India, identified 178 medicinal plant species with high trade potential. M. pruriens found its place among these plants with an estimated trade of annual 1000 metric tons per year. However, the wild population of the species has been decreasing at an alarming rate due to unsustainable harvesting from the wild, and is likely to become endangered. The pharmaceutical industry primarily meets the need for L-Dopa by extracting the compound from wild populations. The second reason for its low population is its annual habit, short life span, and propagation only through seeds having highly allergic properties causing uncontrolled itching.

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Besides these, Mucuna has a vast global demand for its active constituent. Thus, conventional propagation through seed is not an adequate solution to meet the demand. Therefore, there is an urgent need to use alternative techniques for conservation and sustainable utilization. Nowadays, the micropropagation system is mainly used for the bulk production of planting stock material for further increase in biomass manufacture. Thus, considering the immense opportunities offered by the submission of tissue culture techniques with the current status of M. pruriens, investigations have been conducted to develop a reproducible, cost-effective protocol for mass production from cotyledonary node explants. The metal tolerance potentiality of the legume plant was also analyzed through micropropagation.

16.2

Ursolic Acid (UA) Mediated Immune Modulation

Ursolic acid is a natural pentacyclic triterpenoid carboxylic acid in many plants (Liu 2005). Several biochemical and pharmacological effects of UA have antiinflammatory, antioxidant, anti-proliferative, anti-cancer, anti-mutagenic, anti-atherosclerotic, antihypertensive, anti-leukemic, and antiviral properties been reported in a number of experimental systems (Shanmugam et al. 2013). UA has shown antiinflammatory effects in RAW264.7 cells (Mouse monocyte-macrophage cell line) by attenuating iNOS (inducible nitric oxide synthase) and COX-2 (Cyclooxygenase-2) expression (Ryu et al. 2000). UA’s anti-proliferative, anti-tumor, and anti-leukemic properties are mediated via suppression of NF-kB (nuclear factor κ-light-chainenhancer of activated B cells) activation and inhibiting the expression of NF-kB regulated genes like lipoxygenase, COX-2, MMP-9, and iNOS (Shishodia et al. 2003). It is eminent that activation of NF-kB, MAPKs, AP-1, and NF-AT followed by primary histocompatible complex-T cell receptor (MHC-TCR) interaction is vital for the antigen-induced lymphocyte proliferation, cytokine secretion, and survival (Wan and Lenardo 2010). In resting T cells, NF-kB is isolated into an inactive state by the cytoplasmic inhibitor of NF-kB (IkB-α). T cell activation through TCR leads to the rapid activation of the IkB kinases (IKKs) via protein kinase C. It results in phosphorylation and subsequent degradation of IkB proteins, allowing nuclear translocation of NF-kB (Li and Verma 2002). Since dysregulation of NF-kB function is associated with inflammation, thus, any molecule which interferes with NF-kB activation is a potential candidate for therapeutic strategy in the treatment of inflammatory diseases. In 2012, Checker et al. reported that UA’s potent antiinflammatory activity is mediated by suppressing NF-kB, AP-1, and NF-AT (Checker et al. 2012). In 2001, Baricevic et al. said about the topical antiinflammatory activity of Salvia officinalis L. Leaves, in which they also explored the relevance of UA (Baricevic et al. 2001). In 2006, Vasconcelos et al. have reported the in vivo analgesic and anti-inflammatory activities of UA and oleanoic acid from Miconia albicans (Melastomataceae). Thus, it can be said that in Mp extract, UA might show immunomodulatory activity by suppressing the NF-kβ, AP-1, and NF-AT of astrogliyal cell/microglial cell.

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16.3

353

Immune Modulation Action

Environmental toxicants that cross the blood–brain barrier and enter the central nervous system cause neuro-inflammation directly or indirectly. In basal ganglia, the toxicant enters the glial cell (astrogliyal cell and microglial cell), which may activate the cytosolic NFkB. During PD (Parkinson’s disease) pathology, a wide variety of substances, including reactive oxygen species, reactive nitrogen species, pro-inflammatory prostaglandins, and cytokines, are produced by the activated microglia under the influence of T cells. These lethal chemicals cause neurodegeneration and may initiate various biochemical pathways involved in neurodegeneration, including further localized microglial activation and inflammation. Thus, there is a complex interplay between local inhibitory and stimulatory influences in shaping microglial responses. Proin-flammatory cytokines then accompany microglial activation, functional changes of brain vascular endothelial, and recruitment of immune system cells into the damaged tissue (Raivich et al. 1999). The constant localization of activated microglia in areas of ongoing neurodegeneration in PD supports a prominent role for this cell type in extending the disease process. Numerous animal models like MPTP (1 methyl-4-phenyl-1,2,3,6tetrahydropyridine) and PQ (Paraquat) of the PD reinforce this concept. These microglial cells and astrogliyal cells perform neuro-degeneration with the help of NF-kB. The most common NF-kB complex seems to have of p65, p50, and IκBα in neurons (Simpson and Morris 1999). Depending upon factors such as the neurons’ developmental state and location within the nervous system, other complexes are also found in neurons, and their composition may vary (Levenson et al. 2004). The mechanism of NF-kB activation involves phosphorylation of the inhibitory Ikβ subunit by the Ikβ kinase complex (IKK) (May and Ghosh 1999). The Ikβ phosphorylation targets it for ubiquitination and subsequent proteasomal degradation, releasing the active NF-kB factor dimer. NF-kB then translocates to the nucleus and binds to kβ sites in promoters of target genes. NF-κB is critical in regulating neuroinflammation-associated disease pathogenesis, as it plays a central role in neuroinflammation. Besides neurons, the roles of NF-kB in astroglia/microglia have been studied concerning brain injury (Rai et al. 2017). Microgliosis is, pathologically, a neurodegenerative disorder. Microglial activation of NF-kB plays a central role related to the delivery of reactive oxygen species and pro-inflammatory cytokines (such as IL-1β, interferon-γ, and TNF-α) that can cause secondary neurotoxicity (Rai et al. 2017). NF-kB is inducible and regulates inflammatory processes that exacerbate inflammation-induced neurodegeneration in glial cells. Thus, any herbal plant/extract with anti-inflammatory properties may suppress NF-kB activation in glial cells. Although the normal function of NF-kB is neuro-protective, in a diseased condition, it plays a major role in the advancement of Parkinson’s disease and other neurodegenerative disorders. The signals that can activate NF-kB in neurons include TNF-α (Barger et al. 1995), the excitatory neurotransmitter glutamate (Guerrini et al. 1995), nerve growth factor (NGF), (Maggirwar et al. 1998) activity-dependent neuro-trophic factor (ADNF), (Glazner et al. 2000) a secreted form of amyloid precursor protein and cell adhesion molecules (Barger and Mattson 1996).These molecules can activate NF-kB through kinase

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cascades, including calcium/calmodulin-dependent kinase II (Meffert et al. 2003), Akt (Rojo et al. 2004), and protein kinase (Wooten 1999). One or more of these signaling pathways can describe the high constitutive activity of NF-kB in neurons compared to non-excitable cells (Guerrini et al. 1995). Thus NF-kB activates the pro-inflammatory cytokines and vice versa in diseased conditions. It contains various alkaloids and the polyphenolic compound, which is mainly responsible for its mechanism of action. In 2020, Rai et al. determined the L-DOPA and UA in the seed (Rai et al. 2020). In the last 5 decades, profuse works have been carried on LDOPA, which provides only symptomatic relief, and after 2–3 years, it causes a side effect called drug-induced dyskinesia. In 2015, Rai et al. described that UA attenuates oxidative stress in nigrostriatal tissue and improves the neurobehavioral activity in MPTP—an induced Parkinsonian mouse model. Thus, the anti-oxidative activity of UA was already shown by Rai et al. in 2015, but no report explores UA’s anti-inflammatory activity. It can be said that UA directly or indirectly inhibits neuro-inflammation in Parkinson’s disease by modulating the activity of different pro-inflammatory cytokines and transcription factor NF-kB. Kim et al. in 2015 reported that α-Asarone attenuates microglia-mediated neuro-inflammation by inhibiting NF-kB activation and alleviating MPTP-influenced behavioral shortfall in a mouse model of Parkinson’s disease. Kim et al. in 2015 also showed that α-asarone potentially inhibited microglia-mediated neuro-inflammatory responses in vitro and attenuated the behavioral deficits observed in the MPTP-induced mice model of PD in vivo. The MPTP is used worldwide to induce an animal model of Parkinsonism. MPTP intoxication also results in early microglial activation and increases immune molecules, causing neuro-inflammation (Kim et al. 2015).

16.4

Conservation Strategy Through Micropropagation

16.4.1 Materials and Methods 16.4.1.1 Explant Source, Surface Sterilization, and Establishment of Aseptic Culture Healthy, mature seeds were harvested from the Mucuna pruriens plant propagated at the botanical garden department of Botany, Aligarh Muslim University, Aligarh, India. The seeds were washed with water for 25 min, then with a laboratory detergent (labolene, Qualigens, India) 4% (v/v) for 8 min, followed by 5–6 washing with sterile Milli Q water. Surface sterilization was done with 0.1% (w/v) mercuric chloride for 3 min, followed by repeated washing with autoclave Milli Q water. Ten-days-old germinated seeds have existed for axenic Cotyledonary nodes as explants and cultured on a sterile shoot induction medium. 16.4.1.2 Shoot Bud Induction Multiplication Media The growth and morphogenesis of plant tissue in in vitro are primarily governed by the composition of culture media. Murashige and Skoog’s (1962) medium was used as the basal medium. MS basal media augmented with different concentrations of BAP and Kin (0.0, 0.5, 1.5, 2.5, 3.5, 4.5, and 5.5) for shoot induction. Responsive or

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shoot-inducing explants were transferred on optimized BAP, and Kin medium supplemented with different concentrations (0.0, 0.05, 0.1, 0.5, 1.0, and 1.5) of auxins (IAA, IBA, and NAA) and CdCl2 (0.5, 2.5, 5.0, 10.0, 20.0, 30.0, 40.0 or 50.0 μM).

16.4.1.3 Media and Culture Condition All the experimental media contained sucrose (3%) with pH at 5.8 maintained with HCL (1 N) or NaOH (1 N), and agar (7.5%) was used as a solidifier. All the culture vessels with culture medium and instruments were autoclaved at 121 °C for 20 min. Inoculated cultures and hardened plants were kept at 24 ± 2 °C in a culture room (16/8 h photoperiod) with a photosynthetic photon flux density of 50 μmol-2 m-2 s2 provided by a cool white florescent lamp (2 tubes × 40 W, Philips India) and with maintained humidity (60–65%). 16.4.1.4 Rooting Acclimatization Healthy and well-developed roots and shoots were excised from the clamp or branch of multiple shoots and inoculated on MS medium or ½ MS augmented with different concentrations (0.0, 0.5, 1.0, 1.5, 2.0, and 2.5) of IBA containing 0.25% phytagel. Plantlets with well-developed root and shoot systems were harvested from the culture tubes, washed softly under running tap water, and relocated to cups with sterile soilrite under a 16:8 h photoperiod. Planted cups were enclosed with a glass hood to ensure humidity and irrigated with either ½ MS every alternate day for 21 days. Afterward, the glass hood was removed to harden the plants to field conditions. After 35 days, the acclimatized plants were shifted to pots with garden soil and kept in a polyhause. 16.4.1.5 Data Collection and Analysis All the experiments were conducted with a minimum of 20 replicates per experiment and treatment. Data were recorded on the percent shooting or rooting shoot number or root number per explant after 21 days of incubation. Statistically, data were analyzed using one-way ANOVA by SPSS ver 16 (SPSS, Chicago USA) software. The outcome was a significant difference among means was analyzed through multiple range test (DMRT, Duncan’s at P = 0.05) and denoted as means ± standard error (SE).

16.5

Results

16.5.1 Effect of Cytokinins (BA and Kin) CN explants inoculated on MS basal medium devoid of cytokinins failed to induce shoot buds even after 28 days of inoculation. The type and concentration of cytokinins significantly affect the morphogenic response. Adding different concentrations (0.5–5.5 μM) of cytokinins (BA and Kin) in the MS medium promoted shoot bud induction but at different frequencies. The number of shoots increased on increasing the concentration of cytokinins up to 2.5 μM, and thereafter a gradual decrease in morphogenic response was observed (Table 16.1). The CN

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Table 16.1 Effect of different concentrations of cytokinin (BAP and Kin) on shoot bud induction from CN explants on MS medium

Response % 0

28 days No. of shoots/ explants Mean ± SE 0.00 ± 0.00g

0.5 1.5 2.5 3.5

42 72 85 82 76 60 35 55 80 75

2.00 ± 0.32ef 3.20 ± 0.37bcde 5.40 ± 0.51a 4.20 ± 0.37b 3.60 ± 0.24bcd 2.80 ± 0.58cde 1.40 ± 0.24f 2.60 ± 0.40def 4.00 ± 0.71bc 3.60 ± 0.51bcd

4.5 5.5

65 40

3.00 ± 0.45bcde 2.00 ± 0.32ef

Cytokinin (μM) BAP Kin 0 0.5 1.5 2.5 3.5 4.5 5.5

Shoot length (cm) Mean ± SE 0.00 ± 0.00j 1.40 ± 0.04h 1.72 ± 0.04g 3.30 ± 0.07a 2.86 ± 0.07b 2.03 ± 0.05f 2.24 ± 0.03e 1.17 ± 0.04i 1.97 ± 0.05f 2.64 ± 0.07c 2.34 ± 0.05de 2.45 ± 0.04d 1.70 ± 0.04g

56 days No. of shoots/ explants Mean ± SE 0.00 ± 0.00h 2.80 ± 0.37fg 4.00 ± 0.45cdef 7.00 ± 0.71a 5.80 ± 0.37b 4.40 ± 0.40cde 3.40 ± 0.24deffg 2.20 ± 0.37g 3.20 ± 0.20efg 5.00 ± 0.45bc 4.60 ± 0.51bcd

Shoot length (cm) Mean ± SE 0.00 ± 0.00m 1.53 ± 0.05k 3.45 ± 0.05f 5.50 ± 0.07a 5.05 ± 0.05b 3.73 ± 0.05e 2.65 ± 0.05h 1.29 ± 0.04l 2.30 ± 0.04i 4.66 ± 0.05c 4.20 ± 0.07d

3.60 ± 0.24def 3.00 ± 0.63fg

3.01 ± 0.06g 1.93 ± 0.05j

Values represent means ± SE. Means followed by the same letters within columns are not significantly different (P = 0.05) using Duncan’s multiple range test

explant produced 5.40 ± 0.51 shoots with shoot length (3.30 ± 0.07 cm) in 85% culture when inoculated on MS medium augmented with BA (2.5 μM) after 28 d of inoculation. After the first sub-culturing, an enhancement in shoot number (7.00 ± 0.71) and shoot length (5.50 ± 0.07 cm) was recorded after 56 days (Table 16.1, Fig. 16.2). At equimolar concentration, Kin formed (5.00 ± 0.45) shoots after 56 days of culture. Gradually declines in the shoots were recorded on increasing the concentration beyond the optimal level.

16.5.2 The Combined Effect of Cytokinin and Auxins Application of auxins (IAA, IBA, and NAA) at various concentrations (0.05–1.50 μM) to the optimized cytokinin in MS medium showed an antagonistic effect, hampered shoot bud differentiation, and resulted in the enlargement of explants with irregular basal callusing, which lead to a decrease in the number of shoots per explant. Among the different combinations tested, MS medium augmented with BA (2.5 μM) and IAA (0.05 μM) produced shoots (7.60 ± 0.50) with (1.75 ± 0.09 cm) shoot length in 80% cultures after 56 days of culture. At the same time, IBA and NAA were less effective and resulted in profuse basal callusing (Table 16.2). A combination of Kin and auxins were also tested. MS medium supplemented with 2.5 μM Kin with 0.05 μM IAA resulted in shoot numbers (6.40 ± 0.24) with shoot length of 1.56 ± 0.1 cm in 80% of cultures (Table 16.3).

NAA 0.0 0.05 0.10 0.50 1.00 1.50

Response % 0 80 70 68 55 52 75 65 60 50 48 65 58 48 45 40

28 days No. of shoots/explants Mean ± SE 0.00 ± 0.00j 5.80 ± 0.37a 4.60 ± 0.24bc 4.40 ± 0.24bcd 3.60 ± 0.24ef 3.40 ± 0.24fg 5.00 ± 0.00b 4.20 ± 0.37cde 3.80 ± 0.20def 3.20 ± 0.20fg 2.80 ± 0.20gh 4.20 ± 0.20cde 3.80 ± 0.20def 2.80 ± 0.20gh 2.20 ± 0.20hi 1.60 ± 0.24i 0 0 + + ++ ++ + + ++ +++ ++ + ++ +++ +++ ++

Callus

Shoot length (cm) Mean ± SE 0.00 ± 0.00k 1.66 ± 0.09a 1.28 ± 0.11c 1.10 ± 0.10d 0.64 ± 0.06ghi 0.58 ± 0.05hij 1.48 ± 0.02b 0.88 ± 0.08ef 0.70 ± 0.06gh 0.50 ± 0.00ij 0.46 ± 0.02ij 1.00 ± 0.00de 0.80 ± 0.06fg 0.48 ± 0.02ij 0.44 ± 0.02j 0.42 ± 0.02j

56 days No. of shoots/explants Mean ± SE 0.00 ± 0.00i 7.60 ± 0.50a 5.60 ± 0.24b 5.40 ± 0.24c 3.80 ± 0.20ef 3.40 ± 0.24fg 6.40 ± 0.24b 4.80 ± 0.20cd 4.00 ± 0.31ef 3.20 ± 0.20fg 2.80 ± 0.20g 5.20 ± 0.20c 4.40 ± 0.24de 2.80 ± 0.20g 2.60 ± 0.24g 1.80 ± 0.37h 0 0 + + +++ +++ ++ ++ +++ ++++ +++ ++ +++ ++++ ++++ +++

Callus

Shoot length (cm) Mean ± SE 0.00 ± 0.00k 1.75 ± 0.09a 1.34 ± 0.10bc 1.28 ± 0.09cd 0.80 ± 0.08fgh 0.72 ± 0.07ghi 1.52 ± 0.02b 1.10 ± 0.10de 0.90 ± 0.04efg 0.60 ± 0.07hij 0.50 ± 0.00j 1.24 ± 0.11cd 0.96 ± 0.04ef 0.52 ± 0.02ij 0.48 ± 0.02j 0.46 ± 0.02j

Values represent means ± SE. Means followed by the same letters within columns are not significantly different (P = 0.05) using Duncan’s multiple range test

Auxins (μM) IAA IBA 0.0 0.0 0.05 0.10 0.50 1.00 1.50 0.05 0.10 0.50 1.00 1.50

Table 16.2 Effect of the optimum concentrations of BAP (2.5 μM) with various concentrations of auxins (IAA, IBA, and NAA) on shoot formation from CN explants on MS medium

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NAA 0.0 0.05 0.10 0.50 1.00 1.50

Response % 0 80 70 68 55 52 75 65 60 50 42 65 58 45 40 35

28 days No. of shoots/explants Mean ± SE 0.00 ± 0.00m 4.80 ± 0.20a 4.00 ± 0.31bc 3.60 ± 0.24cd 2.60 ± 0.24fghi 2.40 ± 0.24ghi 4.20 ± 0.20b 3.20 ± 0.20def 2.80 ± 0.20efgh 2.20 ± 0.20hi 1.60 ± 0.24jk 3.40 ± 0.24de 3.00 ± 0.00defg 2.00 ± 0.00ij 1.20 ± 0.20kl 1.00 ± 0.00 l 0 0 + + +++ +++ + ++ ++ +++ +++ + +++ ++++ ++++ ++++

Callus

Shoot length (cm) Mean ± SE 0.00 ± 0.00h 1.44 ± 0.12a 1.14 ± 0.09b 1.10 ± 0.10b 0.56 ± 0.06efg 0.50 ± 0.00fg 1.20 ± 0.12b 0.86 ± 0.09cd 0.66 ± 0.10def 0.44 ± 0.04fg 0.38 ± 0.03g 1.00 ± 0.00bc 0.76 ± 0.11de 0.40 ± 0.04g 0.38 ± 0.03g 0.34 ± 0.02g

56 days No. of shoots/explants Mean ± SE 0.00 ± 0.00l 6.40 ± 0.24a 5.00 ± 0.31bc 4.80 ± 0.37cd 3.20 ± 0.20fgh 3.00 ± 0.00gh 5.60 ± 0.24b 4.20 ± 0.20de 3.60 ± 0.24efg 2.80 ± 0.20hi 2.20 ± 0.20ij 4.60 ± 0.24cd 3.80 ± 0.20ef 2.60 ± 0.24hi 1.80 ± 0.20jk 1.40 ± 0.24k 0 0 ++ ++ +++ +++ ++ +++ +++ ++++ ++++ ++ +++ +++++ +++++ +++++

Callus

Shoot length (cm) Mean ± SE 0.00 ± 0.00j 1.56 ± 0.11a 1.24 ± 0.11bc 1.14 ± 0.09bcd 0.70 ± 0.12fgh 0.60 ± 0.10ghi 1.30 ± 0.12b 1.00 ± 0.00cde 0.80 ± 0.12efg 0.50 ± 0.00hi 0.42 ± 0.04i 1.04 ± 0.04cde 0.90 ± 0.10def 0.46 ± 0.04hi 0.40 ± 0.03i 0.36 ± 0.04i

Values represent means ± SE. Means followed by the same letters within columns are not significantly different (P = 0.05) using Duncan’s multiple range test

Auxins (μM) IAA IBA 0.0 0.0 0.05 0.10 0.50 1.00 1.50 0.05 0.10 0.50 1.00 1.50

Table 16.3 Effect of the optimum concentrations of Kin (2.5 μM) with various concentrations of auxins (IAA, IBA, and NAA) on shoot formation from CN explants on MS medium

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Among the cytokinins tested, Kin was the least influential and induced more basal callus mass than BA, even after 56 days of culture.

16.5.3 The Combined Effect of Cytokinin and CdCl2 A standardized MS medium augmented with BA, or Kin, was considered an optimized medium (OM). Another experiment was conducted to evaluate shoot multiplication responses with different concentrations of CdCl2 (0.5–50.0 μM), documented in Tables 16.4 and 16.5. Among the various combinations tested, MS medium augmented with 2.5 μM of CdCl2 along with BA (2.5 μM) resulted in bud break in 90% of cultures and the highest shoots in number (12.80 ± 0.58) with length (3.28 ± 0.08 cm) was obtained after 28 days of inoculation. Subculturing onto the same fresh medium increased shoot number (18.60 ± 0.50) with 3.48 ± 0.08 cm shoot length after 56 days of culture (Table 16.4, Fig. 16.2b). On the other hand, Kin supplemented with (2.5 μM) CdCl2 gave 10.20 ± 0.58 shoots per explant and length (3.00 ± 0.07 cm) in 85% of cultures after 28 days of inoculation. While data collected after 56 days resulted in 15.00 ± 0.70 shoots with a shoot length (4.28 ± 0.06 cm) (Table 16.5). However, by increasing the strength of CdCl2 above the optimal concentration, the regeneration potential and shoot numbers were hampered. CN explants inoculated on 50.0 μM of CdCl2 became black and eventually died, which is considered lethal. The combined effect of CdCl2 with Kin was found to be less effective than BA. Occasionally, basal callusing was observed at the basal portion of explants, which was removed during sub-culturing as callus hampered shoot proliferation and growth. Table 16.4 Effect of different concentrations of CdCl2 supplied in optimized medium (*OM) on multiple shoot proliferation after 28 and 56 days of culture CdCl2 μM 0.0 0.5 1.0 2.5 5.0 10.0 20.0 30.0 40.0 50.0 60.0

Response % 00 78 80 90 85 75 70 65 55 00 00

No. of shoots/ explants Mean ± SE 0.00 ± 0.00h 8.60 ± 0.24d 9.80 ± 0.73c 12.80 ± 0.58a 11.00 ± 0.54b 7.80 ± 0.20d 6.60 ± 0.24e 3.20 ± 0.20f 1.80 ± 0.20g 0.00 ± 0.00h 0.00 ± 0.00h

Shoot length (cm) Mean ± SE 0.00 ± 0.00g 2.16 ± 0.21d 2.64 ± 0.06c 3.28 ± 0.08a 2.96 ± 0.06b 2.04 ± 0.19d 1.88 ± 0.13de 1.22 ± 0.02f 1.10 ± 0.04f 0.00 ± 0.00g 0.00 ± 0.00g

No. of shoots/ explants Mean ± SE 0.00 ± 0.00j 12.20 ± 0.20d 14.60 ± 0.24c 18.60 ± 0.50a 16.60 ± 0.24b 10.80 ± 0.37e 8.60 ± 0.24f 4.20 ± 0.48h 2.60 ± 0.24i 0.00 ± 0.00j 0.00 ± 0.00j

Shoot length (cm) Mean ± SE 0.00 ± 0.00g 2.32 ± 0.17d 2.70 ± 0.08c 3.48 ± 0.08a 3.10 ± 0.07b 2.22 ± 0.17d 2.04 ± 0.15de 1.34 ± 0.02f 1.18 ± 0.05f 0.00 ± 0.00g 0.00 ± 0.00g

Values represent means ± SE. Means followed by the same letters within columns are not significantly different ( p = 0.05) using DMRT; *OM = MS + BAP (2.5 μM)

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Table 16.5 Effect of different concentrations of CdCl2 supplied in optimized medium (*OM) on multiple shoot proliferation after 28 and 56 days of culture CdCl2 μM 0.0 0.5 1.0 2.5 5.0 10.0 20.0 30.0 40.0 50.0 60.0

Response % 00 76 78 85 83 73 68 63 53 00 00

No. of shoots/ explants Mean ± SE 0.00 ± 0.00i 6.80 ± 0.37d 7.80 ± 0.37c 10.20 ± 0.58a 8.80 ± 0.58b 6.20 ± 0.20de 5.40 ± 0.24ef 2.80 ± 0.37g 1.40 ± 0.24h 0.00 ± 0.00i 0.00 ± 0.00i

Shoot length (cm) Mean ± SE 0.00 ± 0.00i 2.14 ± 0.04d 2.48 ± 0.06c 3.00 ± 0.07a 2.74 ± 0.05b 1.86 ± 0.02e 1.58 ± 0.06f 1.24 ± 0.07g 1.00 ± 0.07h 0.00 ± 0.00i 0.00 ± 0.00i

No. of shoots/ explants Mean ± SE 0.00 ± 0.00i 10.20 ± 0.48d 11.80 ± 0.37c 15.00 ± 0.70a 13.40 ± 0.50b 8.60 ± 0.24e 7.00 ± 0.31f 3.60 ± 0.40g 2.20 ± 0.20h 0.00 ± 0.00i 0.00 ± 0.00i

Shoot length (cm) Mean ± SE 0.00 ± 0.00i 3.18 ± 0.09d 3.54 ± 0.06c 4.28 ± 0.06a 3.90 ± 0.05b 2.54 ± 0.10e 2.04 ± 0.10f 1.52 ± 0.09g 1.24 ± 0.05h 0.00 ± 0.00i 0.00 ± 0.00i

Values represent means ± SE. Means followed by the same letters within columns are not significantly different ( p = 0.05) using DMRT; *OM = MS + Kin (2.5 μM)

16.5.4 In Vitro Rooting and Acclimatization Shoots developed in vitro were cut out from the cultures and put on the rooting medium (MS, 1/2 MS, 1/3 MS, 1/4 MS, IBA). Individual healthy shoots (4 cm) excised from shoot clusters were inoculated on various strengths of MS medium alone or in combination with IBA (Fig. 16.1 and 16.2c). Half strength of MS was found to be better than other strengths, producing 1.80 roots after 28 days of culture. The addition of IBA at different concentrations (0.05–2.5 μM) supplemented with half-strength of MS medium exhibited differential responses for rhizogenesis. Among the various concentrations of IBA (1.00 μM) gave a maximum number (04.40 ± 0.51) of roots. In our results, the frequency of root formation markedly differentiated in all the combinations except phytagel without nutrients or PGRs. The plantlets with fully expanded 4–5 leaves with healthy established roots were effectively hardened off inside the culture room on a selected planting substrate (Soilrite) for 28 days and then transferred to field condition (Fig. 16.2d).

16.6

Discussion

The recent increase in the consumption of herbal medicines due to their lower toxicity and side effects compared to allopathic medicines has led to an unexpected rise in the number of herbal drug manufacturers at both the industrial and non-industrial levels (Tiwari et al. 2018). Most pharmaceutically significant vegetation is harvested from natural populations as a source of raw materials for industrial demands (Jamshidi et al. 2018). Overexploitation and unskillful collection of medicinal plants are reducing the natural vegetation and increasing the risk of extinction of

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Fig. 16.1 In vitro rooting in aseptic micro-shoots of Mucuna pruriens treated with-(T0) Control (without PGRS), (T1) MS, (T2) ½ MS, (T3) 1/3 MS, (T4) ¼ MS, (T5) ½ MS + IBA (0.5 μM), (T6) ½ MS + IBA (1.0 μM), (T7) ½ MS + IBA (1.5 μM), (T8) ½ MS + IBA (2.0 μM), (T9) ½ MS + IBA (2.5 μM). Values represent means ± SE. Means followed by the same letters within columns are not significantly different (P = 0.05) using Duncan’s multiple range test

a species. In the future, they may become endangered, so it is necessary to conserve all the valuable plant species by complacency concerning their conservation and cultivation for sustainable demands of supply (Negi et al. 2018). During the last two decades, dramatic progress has been made in developing and refining various tissue culture techniques to make them competent enough to meet the growing demand in the global market (Swamy et al. 2018; Iannicelli et al. 2018). Nowadays, micropropagation techniques are of great interest for conserving elite germplasm through clonal propagation, multiplication, development of new variants, and production of genetically modified plants through genetic transformation (Salma et al. 2018). The medicinal economic and ecological importance of leguminous plants demands the use of plant tissue culture practices for their mass propagation. During the last few years, many leguminous plant species have been cultivated positively in vitro (Pratap et al. 2018; Ahmad et al. 2018). Therefore, the present study proposed to develop an in vitro regeneration system for a potential medicinal legume, M. pruriens. Shoot bud induction was not found in CN explants cultured on MS basal medium lacking PGRs, even after 21 days of incubation. However, inoculated on MS medium augmented with different cytokinins with different concentrations of BA and Kin, a differential response concerning shoot bud initiation, multiplication, and elongation was observed. Cytokinins effectively remove the meristematic shoot apical dominance, so adding cytokinin at optimum level in

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Fig. 16.2 In vitro propagation of Mucuna pruriens (a) Shoot bud induction on MS medium supplemented with BAP (2.5 M) from cotyledonary node explant after 28 days of culture; (b) Multiple shoot regeneration on MS medium containing BAP (2.5 M) and CdCl2 (2.5 M) after 56 days of culture; (c) In vitro rooting in micro-shoot on ½ MS containing indole-3-butyric acid (1.0 M); (d) Acclimatized plants on Soilrite after 28 days

culture media has a beneficial effect on shoot induction and proliferation. Similar results have been reported in various earlier scientific reports (Alam et al. 2020). Commonly, growth regulators used singly or in combination with auxins for micropropagation gave a positive correlation to direct organogenesis. There are many reports on the beneficial explanation of the combined effect of cytokinins and auxins in plant tissue culture (Chawla 2018; Bridgen et al. 2018). The type of cytokinins also affected the morphogenic response among the tested cytokinins. Among the BAP and Kin, BAP (2.5 μM) was found to be the best for the induction shoots. The stimulatory effect of BAP has been well documented in M. pruriens (Faisal et al. 2006a, b; Sathyanarayana et al. 2008; Lahiri et al. 2011, 2012; Raaman et al. 2013). Also, the efficiency of BAP on shoot bud differentiation has been verified in many other plants like Erythrina variegata (Javed and Anis 2015),

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Curculigo orchioides (Nagesh and Shanthamma 2016), Scadoxus puniceus (Naidoo et al. 2017), and Cunila menthoides (Oliveira et al. 2018). Cell division requires temporal relation between the S phase and cell division, signifying that cytokinin and auxin concentration in culture media is to be carefully matched. Cells are supposed not to pass in the mitosis phase unless cytokinin is present. There are several earlier reports on the beneficial role of cytokinins in promoting axillary bud proliferation and shoot elongation (Aremu et al. 2017; Baskaran et al. 2018). The application of cytokinins in tissue culture media varies according to plant species, types of explants used, and culture practices. MS media, combined with various cytokinins at optimal concentration and optimized auxins, has been most promising and shows a mutual regulatory effect on in vitro shoot multiplication and elongation. Exogenous application of phytohormones in MS media at optimal levels showed positive results in previous studies (Renuka et al. 2017; Faisal et al. 2018). The effectiveness of BAP over other Kin at optimum level has been well documented in Albizia lebbeck (Perveen and Anis 2015). One of the probable descriptions for better response achieved on BAP is that the ribosides and nucleotides are naturally stable in BAP compared to other cytokinins (McGaw et al. 1985; Zalabak et al. 2013). However, an increase in a concentration above the optimal level had a negative impact, and the shoot showed a short with a decreased number of shoots regenerated. These conclusions align with the outcomes obtained in the reports (Martins et al. 2018; He et al. 2019). The present findings recorded a negative response on shoot multiplication and elongation when exogenous auxins are supplied in a cytokininsoptimized medium. The lowest concentration of IAA (0.05 μM) in combination with the optimized concentration of cytokinins was most effective for shoot bud induction in M. pruriens. Adding auxin at a lower concentration in the media nullifies the influence of cytokinins on axillary shoot elongation (Hu and Wang 1983; Masondo et al. 2015). In contrast, adding auxins appears to be non-significant towards the regeneration potential of explants. Data revealed that the shoot number did not increase on increasing the auxin concentration. Our findings showed that auxins harmed shoot regeneration as they stimulated basal callus formation. Thus, the outcome suggests that the MS medium composed of cytokinins singly is sufficient for in vitro propagation of M. pruriens. Workers have documented similar results (Sujatha and Kumari 2007; Alam and Anis 2019). Cadmium has been classified as one of the most toxic elements negatively influencing plant growth and development due to its high toxicity and ubiquitous presence in the environment. Elevated concentration of Cd leads to a decline in photosynthesis and alteration of chlorophyll ratio resulting in reduced net CO2 assimilation (Gill et al. 2012; Perveen et al. 2012; Mohamed et al. 2012; Wiszniewska et al. 2017). However, the sensitivity of plants to Cd fluctuates with species and strengths (Gallego et al. 2012). Some plants can tolerate an elevated level of Cd. These plants, called metallophytes, have more potential to abide and survive in the metal contaminated area (Wiszniewska et al. 2017; Muszynska et al. 2018). Of the various concentrations (0.5–60.0 μM) of CdCl2 tried, 2.5 μM gave positive results on in vitro shoot multiplication, and the effectiveness of cadmium on growth in cultures is following the report in Gypsophila fastigiata (Muszynska et al. 2018). Shoots produced at higher concentrations (above

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2.5 μM) of CdC12 showed abnormalities such as yellowing of leaves, stunting of shoots, and browning of the explants. The influence of CdCl2 in the micropropagation system has been recently observed in many plants such as Vitis vinifera (Cetin et al. 2014), Hypoxis hemerocallidea (Okem et al. 2016), and Arabidopsis thaliana (Sofo et al. 2017). During the present study, the root initiation occurred in the micro-shoots when incubated on all the MS treatments except phytagel. The highest percentage (90%) of root formation was recorded on half MS medium containing IBA. Optimum rooting response using IBA has also been reported in several leguminous plant species, including Bauhinia racemosa (Sharma et al. 2017), Cicer arietinum (Singh et al. 2019). Acclimatization of in vitro regenerated plantlets to natural conditions is a crucial step in developing a successful micropropagation protocol. The most important and significant step in the acclimatization protocol is their transition during hardening from in vitro to ex vitro conditions, along with consequent field performance (Chugh et al. 2018). Successful acclimatization generally depends on several weeks of exposure to a transitional environment to improve the survival percentage of in vitro-raised plants (Coopman and Kane 2018). The potted plants grew well without any detectable phenotypic variation.

16.7

Conclusion

The present study describes the development of a practicable regeneration system for the mass multiplication of genetically stable progenies of Mucuna pruriens which can be reintroduced into the original or favorable habitats for cultivation, conservation, and sustainable development. Furthermore, the plantlets developed through this study will help scavenge the heavy metal contamination in waste and agricultural lands. The results of the investigation can be utilized for the commercialization/ production of secondary metabolites around the year, particularly Dopamine for pharmaceutical preparations. This amenable study will provide new innovative ideas with alternate biotechnological strategies for gaining the desired genetic improvements and pharmaceutical discoveries of more important compounds in Mucuna pruriens.

References Ahmad A, Ahmad N, Anis M (2018) Preconditioning of nodal explants in Thidiazuronsupplemented liquid media improves shoot multiplication in Pterocarpus marsupium (Roxb.). In: Thidiazuron: from urea derivative to plant growth regulator. Springer, Singapore, pp 175–187 Alam N, Anis M (2019) Influence of silver nitrate in enhancing the in vitro shoot regeneration in Mucuna pruriens (L.) dc.-Amultipurpose medicinal legume. Res J Life Sci Bioinform Pharm Chem Sci 5:476–487

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In Vitro Cultures: Challenges and Limitations

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Nishi Kumari , Ashish Gupta, Brajesh Chandra Pandey, Renu Kushwaha, and Mohd Yaseen

Abstract

Plant tissue culture provides an effective system for large scale production of plants. Several rare and threatened plants have been conserved through this technique. It plays pivotal role in the production of hybrids, cybrids, genetically engineered plants, disease-free plants, somaclones, and bioactive compounds, etc. It has several industrial applications. Plant cells are miniature factories of chemicals and in vitro cultures can be successfully used for cost-effective and eco-friendly production of such chemicals. For its commercial application, there is need to identify various issues of tissue culture and scientists should give the proper solution in handling such issues. Keywords

Conservation · Vitrification · Recalcitrance · Synthetic seeds · Somaclonal variation

Growing population and indiscriminate use of land have posed a great threat for the existence of many plants. Plant tissue culture has emersed as major technology and it has wider applications in diverse fields such as large scale production of plants, mass plantation, plant improvement, secondary metabolite production, cosmetics, biofuels, etc. (Lakhera et al. 2018). Growing demands of plants and plant products have enhanced the exploitation of economically important and medicinal plants. Rapid urbanization, infrastructure development, deforestation have compounded the problem more seriously and the existence of several plants are in danger. Plant tissue N. Kumari (*) · A. Gupta · B. C. Pandey · R. Kushwaha · M. Yaseen Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. K. Mishra, N. Kumari (eds.), Plants for Immunity and Conservation Strategies, https://doi.org/10.1007/978-981-99-2824-8_17

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culture technology is the potent tool for their mass propagation and conservation. The technology provides a major platform for biotechnological advancements of plants, plant molecular biology, and advancement in the field of metabolomics. Success of tissue culture depends on several factors and there is a need to minimize the problems in developing in vitro cultures. Some of the major issues, which act as constraints are as follows.

17.1

Challenges (Fig. 17.1)

17.1.1 Contamination Contamination problem is a major constraint of tissue culture in developing axenic culture (Fig. 17.2). Various microbes have been identified as contaminants (Abass 2013). Different ways have been adopted by the researchers to deal with it: by using combination of sterilizants, selection of explants as first flushes, use of meristem tissue as the explant, treatment with antibiotics (Bhojwani and Dantu 2013). Some bacteria remain undetectable on several media and their appearance at later stage may affect cell proliferation, differentiation, cell viability, etc. such bacteria are called as latent, endophytic, or endogenous. The use of antibiotics is generally not suggested as they may cause phytotoxicity at higher concentration. Some antibiotics which are being used to control bacterial infection are kanamycin and streptomycin. Streptomycin and kanamycin act on bacteria by interacting with their ribosomes and affects translation process. Adverse effects of these antibiotics were observed for chloroplasts and mitochondria of cultures, which may develop small and yellow leaves in micropropagated plants. Many disinfection protocols have been developed, which are effective against broader spectrum of microbes. Thermotherapy and

Fig. 17.1 Major problems of plant tissue culture have been summarized in Fig. 17.1

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Fig. 17.2 Main sources of contamination

microbiological quality assurance systems (e.g., Hazard Analysis Critical Control Point- HACCP procedures) are being used by commercial plant tissue culture laboratories. Sterilization with ozone dissolved in water, plant preservative mixture (PPM), nanoparticles, use of antimicrobials, medium acidification, etc. have been found effective in the elimination of microbes (Mitsunaga et al. 2022).

17.1.2 Vitrification In several cases, in vitro cultures have shown hyperhydric conditions, which are responsible for many physiological and morphological abnormalities. Higher accumulations of ethylene and CO2 have been observed in hyperhydric cultures. Such conditions are not favorable for growing and differentiating cultures. Several researchers observed less multiplication of shoots, reduction in culture vigor, and difficulty in field transfer of such micropropagated plants. Plantlets developed in hyperhydric conditions showed many aberrations such as tissue hyperhydricity, hypertrophy, deficiency of chlorophyll a and b, absence or lack of cell wall lignification or large intercellular spaces in the leaves. Many factors are responsible for the process: gelling agent type, medium composition, type of plant growth regulators, growth conditions, etc. High concentration of NH4+ ions and high concentrations of cytokinins favor hyperhydric conditions (Polivanova and Bedarev 2022). High relative humidity develops stress, and it causes increase in ethylene production. Ethylene affects the lignification process by stimulating cell wall degrading enzymes, which causes water uptake by different tissues (Sreelekshmi and Siril 2020). Several steps were taken by the researchers to deal with hyperhydricity problem, such as increase in agar concentration, use of lower concentration of cytokinins, bottom cooling of culture vessels, use of AgNO3, etc. (Saher et al. 2005).

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17.1.3 Somaclonal Variation Somaclonal variation is observed in tissue cultured plants, which may be genotypic or phenotypic (Krishna et al. 2016). These variations may be heritable or non-heriatable. Heritable or genetic variation remains stable in both sexual or asexual type of propagation. Epigenetic or non-heritable variations may be unstable, when propagated sexually. Genetic variations take place due to several factors: change in ploidy number of chromosomes, chromosomal rearrangements, mutations, etc. Epigenetic variations may occur due to gene amplification and gene methylation. Somaclonal variations may be desirable or undesirable. To prevent variations in the culture, various major should be taken, such as avoiding long-term culture, use of axillary shoots as explants, less frequency of subcultures, less use of 2,4-D or use of 2,4-D for short duration, re-initiation of culture by using fresh explants, etc. If the cultures have gametophytic origin, then such variations are called gemetoclonal variation. Somaclonal variation acts as major application for plant improvement, if micropropagated plants show the presence of desirable or improved characters. However, it acts as major drawbacks for the system, where uniformity of micropropagated plants is required (Krishna and Singh 2007).

17.1.4 Phenolic Exudation Plants show the presence of phenolics as major group of secondary metabolites of plants. Secretion of phenolics from explants of woody plants or trees are major hindrance for the culture initiation. Phenolics provide rigidity to the plant cell wall acts as molecular bridges between cell wall components (Ozyigit 2008). Browning of explants occur due to the release of phenolic compounds from their cut ends and oxidation of these compounds takes place by polyphenol oxidases, peroxidases or by air. Oxidation product of polyphenols- quinones are highly reactive and inhibit enzyme activity of the explants leading to their death. Phenolics are found inhibitory to cellular growth (Martini and Papafotiou 2013). Researchers used various methods to overcome browning of explants like choice of juvenile explants, or new growth flushes during (1) the active growth period, (2) culture in darkness, (3) intervals, (4) culture in liquid medium, (5) inclusion of antioxidants in the culture medium, or soaking explants in water or solutions containing antioxidants prior to inoculation, (6) use of adsorbing agents, such as activated charcoal, polyvinylpyrrolidone (Amente and Chimdessa 2021). Sealing the cut ends with paraffin wax was also found effective in controlling browning by preventing exudation. Browning problem of calli occurs due to the formation of quinines. Age of donor plant shows positive correlation with phenolic exudation (Ozyigit 2008). Phenolics are found inhibitory to cellular growth (Amente and Chimdessa, 2021). Browning and subsequent death of the explants is usually depended on the phenolic compounds and the quantity of total phenols. Various problems were observed in in vitro cultures such as media discoloration, rooting deficiencies and explant browning and death due to exudation

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of phenolics. In addition, the amount of phenolics can be more or less in different stages of organogenesis due to metabolic actions.

17.1.5 Recalcitrance Some plant cells or tissues do not show morphogenic response and their such inability is termed as recalcitrance. Establishment of culture of such plants becomes a challenging job for researchers. There is a need to identify the problems causing recalcitrance in plant cells or tissues and then resolving it by proper majors. The problem is common with perennial woody plants. Explants may be taken from seedlings or offshoots. Culture conditions may act as stress and it may be one of the reasons for developing recalcitrance. Modification of culture medium is also one solution. Recalcitrance problem was overcome by the use of Thidiazuron (Lu 1993). To overcome recalcitrance problem, there is a need to study whole plant physiology. Another reason is the formation of ethylene in cultures due to the interaction of exogenous and endogenous hormones (Benson 2000). Shoot organogenesis from cultures of Mentha spp. became possible after the addition of mannitol and TDZ (Faure et al. 1998).

17.1.6 Problems Associated with Somatic Embryogenesis Somatic embryogenesis is a multi-stage process and culture requirements may vary in different stages. Some major problems associated with the protocols are asynchronous development of somatic embryos, lack of desiccation phase in somatic embryos, poor conversion frequency of somatic embryos into plantlets, abnormal embryos (fused embryos, pluricotyledony), secondary embryogenesis, etc. (Ochatt and Revilla 2016). Synchronized somatic embryos and induced desiccation of somatic embryos are efficient solutions for normal maturation of somatic embryos and their conversion of embryos into plantlets. Synchronization of somatic embryos can be achieved by growing homogenous suspension cultures and the absence of plant growth regulators in the medium (Tonon et al. 2001). Desiccation of somatic embryos can be induced by using abscisic acid and higher concentration of sucrose in the medium (Lema Ruminska et al. 2013).

17.1.7 Somatic Hybridization Related Problem In hybridization and cybridization, elimination of chromosomes may take place from hybrid cell. In certain wide crosses, elimination of chromosomes from the hybrid cell is another limitation of somatic hybridization (Shuro 2018). Therefore, desirable hybrids are no longer available. Although some attempts have been made to increase the percentage of fused cells, still it is also a limitation of somatic hybridization.

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Finally, for hybrid identification, selection, and isolation at the culture level, there is no standardized method which is applicable for all material.

17.2

Applications

At present scenario, the loss of habitats of several plants, uncontrolled uses of natural resources, anthropogenic activities have raised a serious concern for the conservation of biodiversity. Plant tissue culture technique has got success in the conservation of several rare, threatened, plants with economic and medicinal importance. Large scale plantation of plants has become possible by using this technique. Dependence on plants and plant products for medicinal purpose has increased considerably, and therefore it is utmost need to conserve such plants (Chen et al. 2016). In vitro cultures of medicinal plants have shown significant medical efficacy and they are being used for the commercial and cost-effective production of pharmaceutically important compounds. Plant tissue culture acts as major platform for the biotechnological advancement of the plants. Plant tissue culture plays a significant role in manufacturing industries of cosmetics and food (Eibl et al. 2018). Plant cultures provide sustainable method for energy crop production (Norouzi et al. 2022). Some of their major applications are as follows (Fig. 17.3).

17.2.1 Mass Propagation of Plants The technology is useful in large scale production of plants. It requires less time, less space, and thus identified as efficient and cost-effective method. Conventional means of propagation has several drawbacks: recalcitrance of seeds in many plants,

Fig. 17.3 Major applications of the plant tissue culture

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loss of seed viability after certain period, poor seed germination in many woody plants, slow seedling growth, limited number of seedlings, low survival of seedlings, absence of sexual mode of reproduction in many plants, etc. (Visscher et al. 2022). Perennial and woody plants produce heterogenous population of progenies due to crossing over during gamete formation and cross fertilization, thus making it difficult to raise uniform population of elite plants. Plant breeding techniques are laborintensive, time consuming and it does not provide means for mass propagation of plants. Regeneration protocols of many plants have been developed by organogenesis and/or somatic embryogenesis (Bhojwani and Dantu 2013). Now commercial production of many plants has become possible such as bamboo, banana, Populus, papaya, etc. Regeneration of many plants has been achieved by organogenesis in different plants. Requirement of plant growth regulators vary in different plants and it depends upon endogenous level of hormones in plant tissues. In general, rooting is favored by higher ratio of auxin and cytokinin and shooting occurs in the lower ratio of cytokinin and auxin. If organogenesis occurs without the formation of callus as intermediate phase, the organogenesis is called as direct, while organogenesis through the differentiation of callus is called as indirect. Most of the scientists observed nodal explants and apical bud explants highly suitable for the induction of organogenesis due to pre-existing buds in the explants. It offers true to plants in less time; however, limited number of shoots is a major constraint. Somatic embryogenesis is an efficient means of propagation as large number of plants can be produced in less time and less labor intake (Guan et al. 2016). In several case of woody and perennial trees, it is difficult to achieve somatic embryo formation from mature explants. In such cases, zygotic embryo or embryo derived tissues may be responsible in inducing somatic embryos, but such explants are considered unproven. Somatic embryogenesis may be direct or indirect (Zhang et al. 2021).

17.2.2 Germplasm Conservation The germplasm is a collective term used to describe seeds, plants or their parts. It ranges from wild plants to genes and it contains all genetic information thus acting as major genetic resources of an organism. There are two major ways of germplasm conservation: in situ and ex situ conservation. In situ methods are being used for the maintenance of plants in the nature and it provides natural selection of plants and the existence of self-sustaining populations of wild plants. In ex situ conservation plants are maintained outside their habitat and different methods used for such conservation are gene banks, botanical gardens, seed banks, in vitro cultures. Tissue culture is most effective ex situ method of germplasm conservation (Ifeanyi et al. 2016). It provides material for short-term, medium-term, and long-term conservation. Shortterm conservation is generally required for transportation of micropropagules, which can easily form the plantlets, for example, synthetic seeds, somatic embryos, in vitro shoots, etc. (Cruz- Cruz et al. 2013). Slow growth culture method is used for the maintenance of in vitro cultures for both the short-term and medium-term conservations. Two kinds of in vitro germplasm preservation were considered:

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slow growth condition culture for mid-term preservation, and cryopreservation using the encapsulation/dehydration technique for long-term preservation. Minimal growth storage can be achieved by manipulating culture media, culture conditions or by adding growth retardants. Cryopreservation is highly useful for long-term storage.

17.2.3 Production of Disease-Free Plants Production of disease-free plants has always remained a big challenge for plant growers. Plant viruses are obligate intracellular parasites that colonize only inside the living cells of the host and can be transmitted by vegetative propagation from generation to generation and insect vectors may infect another healthy plant. The use of chemicals is one way to control the spread of viral diseases. However, tissue culture provides an efficient solution and virus-free plants can be grown through this technique at large scale. For this, various strategies are being used such as meristem culture, micrografting, chemotherapy, thermotherapy, and shoot tip cryotherapy (Galatali et al. 2021). To raise virus-free plants, meristem culture is highly suitable. Transportation of viruses to the meristem region of the plant is prevented due to the lack of transport system in meristem. Tissue culture methods in combination with thermotherapy were reported successful in getting healthy plants of grapevine wood without phytoplasma (Klimenkob et al. 2020). In vitro culture was observed successful in getting disease-free ginger plants (Zhao et al. 2023).

17.2.4 Synthetic Seeds The encapsulation technology provides the protection to somatic embryos or micropropagules (micropropagated shoots, calli, etc.) from any injury and supply of nutrients with during or after encapsulation helps them to rejuvenate growth. Synthetic seeds are easier to handle during transport and it is also ideal for storage (Ravi and Anand 2012). Synthetic seeds can be produced either as coated or non-coated, desiccated somatic embryos or as embryos encapsulated in hydrated gel (Redenbaugh et al. 1987). Successful utilization of synthetic seeds as propagules of choice requires an efficient and reproducible production system and a high percentage of post-planting conversion into vigorous plants. Various gelling agents can be used for encapsulation such as sodium alginate, potassium alginate, sodium pectate, and carrageenan, etc. Sodium alginate is most widely used for this purpose (Rihan et al. 2017). To facilitate growth and survival, various supplements are added to the gel such as carbon sources, plant growth regulators, antimicrobial agents, and others. Desiccated synthetic seeds are suitable only for those somatic embryos, which are desiccation tolerant. The technology is useful for hybrid embryos with unstable genotypes and poor seed viability (Dhabhai & Prakash, 2012).

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17.2.5 Hybrid Plants Through Parasexual Hybridization Somatic hybridization and cybridization have great application in plant improvement programs It helps in overcoming sexual incompatibility among different species or different genera of the plants. The technology facilitates transfer of elite genes showing disease resistant, pest resistant, stress tolerance, etc. Considerable improvement of citrus plants was made by somatic hybridization and cybridization methods (Grosser et al. 2015; Ruiz et al. 2018). Similarly, the improvement of banana was made by somatic hybridization (Matsumoto et al. 2002). Somatic hybridization provides modification and improvement of polygenic traits of the plants (Liu et al. 2015). Improvement of non-flowering and non-tuber bearing plants is possible through this technique. Protoplasts of plants with different ploidy levels can be also fused and thus fertile plants can be obtained. Asymmetric hybridization is another promising approach, where partial genome transfer is being made (Shankar et al. 2013). In cybridization, fusion of nucleated protoplast with enucleated protoplast takes place. Cybridization was used to transfer cytoplasmic male sterility (CMS) in rice. Atrazine resistant Brassica campestris and male sterile Raphanus sativus were developed by this method.

17.2.6 Metabolite Production from Medicinal Plant The natural habitats for a large number of plants are rapidly destroyed leading to extinction of many valuable and even endemic species. In vitro cultures of medicinal plants have become major resources for the production of secondary metabolites (Espinosa- Leal et al. 2018). Fedoreyev et al. (2000) used calli of Maackia amurensis for the production of various isoflavones. In vitro cultures of Taxus sp. were exploited to produce taxol and taxanes (Filova 2014). Hairy root cultures have got great significance in the synthesis of biologically active compounds and their genetic or biosynthetic activity remains unchanged even in successive generations. Commercial production of several pharmaceutically important compounds such as shikonin, alkaloids, dihydroxyphenylalanine, harpagide, etc. Enhanced production of phytochemicals was achieved by elicitation and precursor feeding treatments. Various types of elicitors are used to trigger the synthesis of phytochemicals such as methyl jasmonate, salicylic acid, fungal wall derivatives, etc. (Ochoa-Villarreal et al. 2016).

17.2.7 Production of Genetically Modified Plants Genetically modified plants or transgenic plants are those, whose DNA is modified using genetic engineering techniques (Rani and Usha 2013). By this technique, a new trait is entered in the plant cell and donor plant may be of same species or different species or different genera. This process is now being used for different plant improvement programs. Introduction of genes for biotic and abiotic stress

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resistance, improving shelf time of fruits, higher yield has been achieved in several plants. This technique has been also used for the production of pharmaceutically important compounds or compounds of industrial value. Such plants are also useful in the production of complex vaccines, which are cost-effective and contamination free (Arevalo-Villalobos et al. 2020). By producing pest resistant genetically engineered plants, uses of pesticides and insecticides have been minimized and thus the technique is beneficial for plant growers and environment friendly (Anderson et al. 2019).

17.2.8 Application of Somaclonal Variation in Agriculture Somaclonal variation is considered as one of major drawbacks in the production of true to type plants. However, such variations are advantageous for apomicts and the plants with narrow genetic base (Krishna et al. 2016). It does not require any sophisticated method to obtain variations. Commercially, uniformity of micropropagated plants is desirable, but it gives a scope for the improvement of vegetatively propagated plants, plants with longer juvenile period, difficult to breed, etc. Several cultivars are now preferring plants with desirable variations such as tolerant to biotic and abiotic stresses, disease tolerant, etc. (Zayova et al. 2010).

17.2.9 Industrial Applications Plant tissue culture technique has several industrial applications. Plant based formulations of cosmetics have become popular among the users. Manufacturing company of cosmetics find plant products safer, sustainable and eco-friendly (Zappelli et al. 2016). Plant tissue culture makes the availability of plants throughout the year without depending upon a particular season. By 2050, food requirement of the world will be increased by 60% (Alexandratos and Bruinsma 2012), however, agriculture land is shrinking every year. Plant cell based cellular agriculture will be only solution in future (Nordlund et al. 2018). Many phytochemicals are significant in flavor and aroma industry and their commercial production is feasible from plant cultures. Importance of plant tissue culture in the field of fuel industry is also very significant and it can be the source of fifth generation biofuel (Norouzi et al. 2022). Manipulation of calli can be done to enrich lignin content and such engineered calli will act as major source for the production of high-quality biofuel.

17.3

Conclusion

Plant tissue culture has been identified as effective means for plant propagation, conservation of plants, improvement of plants through genetic engineering, costeffective production of phytochemicals, development of new variants of plants, etc.

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However, the success of work depends upon overcoming different problems, which affect the output of the work.

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