Wild Mushrooms and Health: Diversity, Phytochemistry, Medicinal Benefits, and Cultivation 1032372192, 9781032372198

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Wild Mushrooms and Health Medicinal mushrooms have been used since ancient times. Certain mushrooms can be used to treat numerous conditions, including those related to cardiovascular health, obesity, cholesterol balance, bone health, diabetes, and cancer. Wild Mushrooms and Health: Diversity, Phytochemistry, Medicinal Benefits, and Cultivation presents reports on numerous species of wild medicinal mushrooms with discussion of drug-discovery implications, analysis of bioactive substances, and prospects for cultivation.

FEATURES • Comprehensive review of medicinal mushrooms as sources of promising bioactive molecules and prospective compounds for drug discovery • Information on diversity, distribution, ethnomycology, ecology, cultivation, descriptions of specific species, and folk medicinal uses of mushrooms throughout the world • Emphasis on identification, documentation, bioactive substances, and the nature of mushroom bioactivity • Discussion of the nutraceutical properties of wild mushrooms, including high protein content comparable to that of meat, and low fat content, which make them a complete dietary food source • Exploration of methods used in the collection, identification, documentation, cultivation, analysis, and conservation of mushrooms for drug discovery An installment in the Exploring Medicinal Plants series, this volume is a comprehensive resource for medical researchers, scientists, and pharmaceutical companies. In addition, this resource is appropriate for mycologists and botanists interested in pharmacognosy.

Exploring Medicinal Plants Series Editor Azamal Husen Wolaita Sodo University, Ethiopia Medicinal plants render a rich source of bioactive compounds used in drug formulation and development; they play a key role in traditional or indigenous health systems. As the demand for herbal medicines increases worldwide, supply is declining as most of the harvest is derived from naturally growing vegetation. Considering global interests and covering several important aspects associated with medicinal plants, the Exploring Medicinal Plants series comprises volumes valuable to academia, practitioners, and researchers interested in medicinal plants. Topics provide information on a range of subjects including diversity, conservation, propagation, cultivation, physiology, molecular biology, growth response under extreme environment, handling, storage, bioactive compounds, secondary metabolites, extraction, therapeutics, mode of action, and healthcare practices. Led by Azamal Husen, PhD, this series is directed to a broad range of researchers and professionals consisting of topical books exploring information related to medicinal plants. It includes edited volumes, references, and textbooks available for individual print and electronic purchases. Plants as Medicine and Aromatics: Conservation, Ecology, and Pharmacognosy Mohd Kafeel Ahmad Ansari, Bengu Turkyilmaz Unal, Munir Ozturk and Gary Owens Sustainable Uses of Medicinal Plants Learnmore Kambizi and Callistus Bvenura Medicinal Plant Responses to Stressful Conditions Arafat Abdel Hamed Abdel Latef Aromatic and Medicinal Plants of Drylands and Deserts: Ecology, Ethnobiology and Potential Uses David Ramiro Aguillón Gutiérrez, Cristian Torres León, and Jorge Alejandro Aguirre Joya Secondary Metabolites from Medicinal Plants: Nanoparticles Synthesis and their Applications Rakesh Kumar Bachheti, Archana Bachheti Aquatic Medicinal Plants Archana Bachheti, Rakesh Kumar Bachheti, and Azamal Husen Antidiabetic Medicinal Plants and Herbal Treatments Azamal Husen Ethnobotany and Ethnopharmacology of Medicinal and Aromatic Plants: Steps Towards Drugs Discovery Adnan Mohd, Mitesh Patel and Mejdi Snoussi Wild Mushrooms and Health: Diversity, Phytochemistry, Medicinal Benefits, and Cultivation Kamal Ch. Semwal, Steven L. Stephenson, and Azamal Husen For more information, please visit: www.routledge.com/Exploring-Medicinal-Plants/book-series/ CRCEMP

Wild Mushrooms and Health: Diversity, Phytochemistry, Medicinal Benefits, and Cultivation

Edited by Kamal Ch. Semwal, Steven L. Stephenson, and Azamal Husen

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

First edition published 2024 by CRC Press 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487–2742 and by CRC Press 4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN CRC Press is an imprint of Taylor & Francis Group, LLC © 2024 Taylor & Francis Group, LLC Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978–750–8400. For works that are not available on CCC please contact [email protected] Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. ISBN: 978-1-032-37219-8 (hbk) ISBN: 978-1-032-54248-5 (pbk) ISBN: 978-1-003-33593-1 (ebk) DOI: 10.1201/b23190 Typeset in Times by Apex CoVantage, LLC

Contents About the Editors�����������������������������������������������������������������������������������������������������������������������������vii Preface����������������������������������������������������������������������������������������������������������������������������������������������ix Contributor List���������������������������������������������������������������������������������������������������������������������������������� x Chapter 1 Biological Activity of Six Wild Edible Mushrooms of the Western Ghats, India�������������1 Venugopalan Ravikrishnan, Kandikere Ramaiah Sridhar Chapter 2 Distribution, Ecology, and Taxonomy of Medicinal Mushrooms������������������������������ 11 Steven L. Stephenson, Kamal Ch. Semwal Chapter 3 Wild Medicinal Mushrooms of the Northwest Himalayas of Jammu and Kashmir, India and Their Traditional and Modern Applications����������������������� 23 Komal Verma, Tahir Mehmood, Sanjeev Kumar, Uzma Altaf, Yash Pal Sharma Chapter 4 Ethnomycology, Bioprospection, and Uses of Mushrooms in Costa Rica����������������� 51 Carlos Rojas, Ignacio Arroyo, Robin G. Doss Chapter 5 A Checklist of Medicinal Mushrooms in Kerala State, India������������������������������������ 65 C. K. Pradeep, A. Manoj Kumar Chapter 6 Cultivation of Medicinal Mushrooms for a World in Need��������������������������������������� 95 Steven L. Miller, Adam Sayner Chapter 7 Ethnomycology: Diversity and Utilization of Medicinal Mushrooms in the Philippines������������������������������������������������������������������������������������������������������ 130 Angeles M. De Leon, Jaica Jane C. Balcruz, Justine Reine T. Matillano, Charlyn M. Miguel, John Paul R. Orevillo, Rich Milton R. Dulay Chapter 8 Uses of Mushrooms in Traditional Medicine in China�������������������������������������������� 155 Pu Liu, Yu Li Chapter 9 Diversity and Ecology of Wild Mushrooms in Ethiopia������������������������������������������ 180 Tatek Dejene, Busha Teshome, Wubalem Tadesse, Pablo Martín-Pinto Chapter 10 Wild Edible Mushroom Trade in Europe: Special Focus on Estonia���������������������� 194 Surya Sudheer, Ain Raal, Aivar Jürgenson, Rajeev Bhat

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viContents

Chapter 11 Diversity, Distribution, Ecology, and Utilization of Wild Mushrooms in Bangladesh����������������������������������������������������������������������������������������������������������� 210 F. M. Aminuzzaman, J. F. Tanni, K. Das Chapter 12 Some Medicinal Fungi from the Forests Used by Tribal People in Central India������������������������������������������������������������������������������������������������������������� 232 N. S. K. Harsh Chapter 13 The Current Status of Sri Lankan Mushroom Research������������������������������������������240 Mahesh C. A. Galappaththi, Samantha C. Karunarathna Chapter 14 Bioactive Profile of Xylaria spp.—A Comparative Account����������������������������������� 250 Tahir Syed, Kodandoor Sharthchandra, Gabbalkaje Harish Jithesh, Gopalappa Abhisheka, Shivannegowda Mahadevakumar, Kandikere Ramaiah Sridhar Chapter 15 Some Uncommon Traditionally and Medicinally Important Wild Macrofungi of the Garhwal Himalayas, Uttarakhand, India����������������������������������� 261 Vipin Parkash, Ranjna Kaundal, Supriti Paul, Anuj Chauhan, Ashok Aggarwal Chapter 16 Hidden Treasure of Himalayan Forests: Mushrooms with Curative Potential�������������������������������������������������������������������������������������������������������������������� 274 Priyanka Uniyal, Upendra Singh, Rajendra Prasad Bhatt Index���������������������������������������������������������������������������������������������������������������������������������������������� 289

About the Editors Kamal Ch. Semwal is an Associate Professor in the Department of Biology of the College of Sciences at the Eritrea Institute of Technology in Asmara, where he teaches interdisciplinary subjects in the botanical sciences. He received his Ph.D. from HNB Garhwal University (a Central University), BGR Campus Pauri, Srinagar Garhwal, Uttarakhand, India, in 2004. He began his career as a research associate at the Directorate of Mushrooms in Solan (Himachal Pradesh) and later taught at Gondar University and Mekelle University in Ethiopia (East Africa). His research on mushrooms is well recognized and has been cited numerous times in various research journals. He is the author or coauthor of several dozen papers published in peer-reviewed journals, as well as book chapters and review articles. He also served as a co-editor of a book entitled Ecosystem and Environmental Pollution, published by Discovery Publishing House (P) Ltd., India, and as co-investigator of the study Collection, identification, documentation of wild edible and medicinal mushrooms of Garhwal Himalaya of Uttarakhand funded by UCOST, Dehradun, India. His papers have considered a number of topics within botany, particularly regarding the diversity, taxonomy, and ecology of wild mushrooms in the Northwestern Himalayas of India. He has more than 18 years of experience in fungal taxonomy and ecology. He has described several species of wild mushrooms new to science and is currently serving as a reviewer for many highly reputed international scientific journals throughout the world. Steven L. Stephenson is currently a research professor in the Department of Biological Sciences at the University of Arkansas, USA, where he teaches courses in plant biology, forest ecology, and plant ecology. He received his Ph.D. from Virginia Polytechnic Institute and State University (Virginia Tech) in 1977 and taught at Fairmont State College (now University) for 27 years before moving to the University of Arkansas in 2003. He was a Senior Fulbright Scholar at Himachal Pradesh University, India in 1987, Visiting Scientist at the Australian Antarctic Division in 1995, and William Evens Fellow at the University of Otago in New Zealand in 2003. His studies of myxomycetes (slime molds) and fungi have taken him to all seven continents and every major type of terrestrial ecosystem. He is a Fellow of the Mycological Society of America and the author or coauthor of 18 books and more than 480 book chapters and papers published in peer-reviewed journals. His notable works include The Kingdom Fungi: The Biology of Mushrooms, Molds, and Lichens (2010), Mushrooms of the Southeastern United States (2017), and Secretive Slime Moulds: Myxomycetes of Australia (2021). Azamal Husen served as Professor and Head of the Department of Biology at the University of Gondar, Ethiopia, and is a foreign delegate at Wolaita Sodo University, Wolaita, Ethiopia. Previously, he was a visiting faculty member of the Forest Research Institute and the Doon College of Agriculture and Forest in Dehra Dun, India. His research and teaching experience of 20 years involves studies of biogenic nanomaterial fabrication and application; plant responses to environmental stresses; nanomaterials at the physiological, biochemical, and molecular levels; herbal medicine; and clonal propagation for the improvement of tree species. He has conducted several research projects sponsored by various funding agencies, including the World Bank (FREEP), the National Agricultural Technology Project (NATP), the Indian Council of Agriculture Research (ICAR), the Indian Council of Forest Research Education (ICFRE), and the Japan Bank for International vii

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About the Editors

Cooperation (JBIC). He received four fellowships from India and a recognition award from the University of Gondar, Ethiopia, for excellent teaching, research, and community service. Husen has been on the editorial board and the panel of reviewers of several reputed journals published by Elsevier, Frontiers Media, Taylor & Francis, Springer Nature, RSC, Oxford University Press, Sciendo, The Royal Society, CSIRO, PLOS, MDPI, John Wiley & Sons, and UPM Journals. He is on the advisory board of Cambridge Scholars Publishing, UK. He is a fellow of the Plantae group of the American Society of Plant Biologists and a member of the International Society of Root Research, the Asian Council of Science Editors, and INPST. To his credit are over 200 publications, and he is Editor-in-Chief of the American Journal of Plant Physiology. He is also working as Series Editor for Exploring Medicinal Plants, published by Taylor & Francis Group, USA; Plant Biology, Sustainability, and Climate Change, published by Elsevier, USA; and Smart Nanomaterials Technology, published by Springer Nature Singapore Pte Ltd., Singapore.

Preface We are living in a world with as many as one and a half to six million different species of fungus, only about 144,000 of which have been formally described thus far. Why is there a need to know these yet undescribed fungi, more commonly referred to as mushrooms? First, mushrooms are rich in many bioactive substances, such as alkaloids, flavonoids, polyphenols, saponins, sterols, tannins, triterpenoids, p-coumaric acids, and many more. These bioactive substances are prospective agents for the discovery of new drugs that will benefit mankind. For example, clinical studies have demonstrated that some substances derived from mushrooms have the potential to treat various ailments. Although an unknown number of wild mushrooms have yet to be discovered and examined for the presence of new bioactive substances that could yield new drugs, mushrooms are valuable for other reasons. Many species of wild mushrooms are collected and consumed as food in countries throughout the world. Only a few of these have been domesticated and cultivated to the extent that they have considerable economic value. There is little question that if more of these edible wild mushrooms were systematically identified and cultivated, it would be possible to overcome food security in certain areas of the world. Among the species of wild edible mushrooms, there are many examples that are ectomycorrhizal and thus important components of forest ecosystems. As such, there are some technical and ecological constraints in collecting and cultivating their fruiting bodies. However, accounts of the successful cultivation of wild ectomycorrhizal species provide evidence that there should be a greater effort to increase the number of species that can be cultivated and exploited as a source of food. Although there have been some success stories, there are very large differences in the extent to which mushroom cultivation occurs between different countries. A few countries grow appreciable amounts of edible or medicinal species, but many cultivate only a few species, such as the oyster mushroom (Pleurotus spp.) or the button mushroom (Agaricus spp.), in very low numbers. Our knowledge of the diversity, distribution, and taxonomy of wild mushrooms is incomplete because of the information gap that exists in many countries. For some countries and geographical areas, we do not yet have anything approaching a comprehensive list of the mushroom varieties present. This means that there is no basis for carrying out studies to determine what potentially useful bioactive substances might be discovered, especially with respect to wild mushroom species found only in a particular part of the world. There is little question that additional exploration and documentation of wild mushrooms would lead to new drug discovery opportunities. This book addresses various aspects of the diversity, distribution, ecology, culinary and medicinal uses, ethnomycology, cultivation, and biological activity of wild mushrooms in countries all over the world. In addition, the bioprospection of mushrooms and the economics of commercial mushroom cultivation and marketing are considered. Throughout the book, emphasis is placed on the role of field exploration for wild mushrooms as the first step towards the documentation, identification, and research into their pharmaceutical properties, some of which may have value for treating different ailments, including some currently untreated diseases. The purpose of this book is to disperse the knowledge of wild mushrooms among researchers, scientists, students, and citizen scientists (generally including those interested in wild mushroom consumption or foraging). The editors are grateful to all the individuals who contributed chapters to this book for their time and effort. We also extend our gratitude to Randy Brehm, Tom Connelly, and all associates at Taylor & Francis Group, LLC / CRC Press for their active cooperation. We shall be happy to receive comments and criticism, if any, from subject experts and general readers of this book. Kamal Ch. Semwal Steven L. Stephenson Azamal Husen ix

Contributors Gopalappa Abhisheka Department of Biology Jnanasudha Pre-University College India Ashok Aggarwal Botany Department Kurukshetra University India Uzma Altaf Department of Botany University of Jammu India F. M. Aminuzzaman Department of Plant Pathology, Faculty of Agriculture Sher-e-Bangla Agricultural University Bangladesh Ignacio Arroyo Finca Boryana San Gerardo de Dota, Costa Rica Jaica Jane C. Balcruz Department of Biological Sciences, College of Science Central Luzon State University Philippines Rajeev Bhat Estonian Literary Museum Estonia Rajendra Prasad Bhatt Department of Botany & Microbiology HNB Garhwal University India Anuj Chauhan Forest Pathology Section, Forest Protection Division, Forest Research Institute India

K. Das Department of Plant Pathology, Faculty of Agriculture Sher-e-Bangla Agricultural University Bangladesh Angeles M. De Leon Department of Biological Sciences, College of Science Central Luzon State University Philippines Tatek Dejene Ethiopian Forest Development (EFD) Ethiopia Robin G. Doss Maialu S.A. Santo Domingo de Heredia, Costa Rica Rich Milton R. Dulay Address 1- Department of Biological Sciences, College of Science Central Luzon State University, Science City of Munoz Nueva Ecija, Philippines 3120 Address 2- Center for Tropical Mushroom Research and Development, College of Science Central Luzon State University Science City of Munoz Nueva Ecija, Philippines 3120 Mahesh C. A. Galappaththi Postgraduate Institute of Science (PGIS) University of Peradeniya Peradeniya 20400 Sri Lanka N. S. K. Harsh Former Scientist, Forest Research Institute India Gabbalkaje Harish Jithesh Department of Biosciences Mangalore University India

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Aivar Jürgenson School of Humanities Tallinn University Estonia

Tahir Mehmood Department of Botany University of Jammu India

Ranjna Kaundal Forest Pathology Section, Forest Protection Division Forest Research Institute India

Charlyn M. Miguel Department of Biological Sciences, College of Science Central Luzon State University Philippines

Samantha C. Karunarathna Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering Qujing Normal University China

Steven L. Miller Department of Botany University of Wyoming USA

Yu Li Engineering Research Center of Edible and Medicinal Fungi Ministry of Education China Pu Liu Engineering Research Center of Edible and Medicinal Fungi Ministry of Education China

John Paul R. Orevillo Department of Biological Sciences, College of Science Central Luzon State University, Science City of Munoz Nueva Ecija, Philippines 3120 Vipin Parkash Forest Pathology Section, Forest Protection Division Forest Research Institute India

Shivannegowda Mahadevakumar Forest Pathology Department Division of Forest Protection India

Supriti Paul Forest Pathology Section, Forest Protection Division Forest Research Institute India

A. Manoj Kumar Post Graduate and Research Department of Botany Government College for Women India

C. K. Pradeep Microbiology Division Jawaharlal Nehru Tropical Botanic Garden and Research Institute India

Pablo Martín-Pinto Ethiopian Forest Development (EFD) Ethiopia

Ain Raal Institute of Pharmacy, Faculty of Medicine University of Tartu Estonia

Justine Reine T. Matillano Department of Biological Sciences, College of Science Central Luzon State University Philippines

Venugopalan Ravikrishnan Department of Biosciences Mangalore University Mangalagangotri, Karnataka, India

xiiContributors

Carlos Rojas Department of Biosystems Engineering and Engineering Research Institute University of Costa Rica Costa Rica

Surya Sudheer ERA-Chair for Food (By-) Products Valorisation Technologies (VALORTECH) Estonian University of Life Sciences Estonia

Sanjeev Kumar Department of Botany University of Jammu India

Tahir Syed Department of Biosciences Mangalore University India

Adam Sayner GroCycle Courses UK

Wubalem Tadesse Ethiopian Forest Development (EFD) Ethiopia

Yash Pal Sharma Department of Botany University of Jammu India

J. F. Tanni Department of Plant Pathology, Faculty of Agriculture Sher-e-Bangla Agricultural University Bangladesh

Kodandoor Sharthchandra Department of Biosciences Mangalore University India

Busha Teshome Ethiopian Forest Development (EFD) Ethiopia

Upendra Singh Department of Botany & Microbiology HNB Garhwal University India

Priyanka Uniyal Department of Botany Govt. P. G. College India

Kandikere Ramaiah Sridhar Department of Biosciences Mangalore University India

Komal Verma Department of Botany University of Jammu India

1

Biological Activity of Six Wild Edible Mushrooms of the Western Ghats, India Venugopalan Ravikrishnan, Kandikere Ramaiah Sridhar

1.1 INTRODUCTION Cultivated and wild edible mushrooms serve as attractive functional foods throughout the world owing to their nutritional value, texture, taste, and flavor. In addition, they possess adequate nutritional components (protein, indispensable amino acids, carbohydrates, fiber, and vitamins) and low quantities of fat, calories, sodium, and cholesterol (Mattila et al. 2000). Moreover, cultivated and wild mushrooms serve as a repository of therapeutic bioactive compounds (polyketides, phenolics, steroids, and terpenes [Kues and Liu 2000]). Damage by free radicals is linked to many human diseases (cancer, diabetes, atherosclerosis, and cirrhosis) as well as aging (Halliwell and Gutteridge 1984). Synthetic antioxidants like butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tertiary-butylated hydroxyquinone (TBHQ) exhibit protective activity against reactive oxygen, but they are known for carcinogenesis (BHA and BHT) and may possess tumor-producing/tumor-promoting properties (BHA) (Botterweck et al. 2000). Hence, wild and cultivated mushrooms serve as a potential natural source of antioxidants (Ferreira et al. 2009; Boonsong et al. 2016; Sánchez 2017). Among the antioxidants in mushrooms, phenolics have gained more attention owing to their excellent reactive oxygen quenching potential, anti-inflammatory activities, and anti-tumor potential (Puttaraju et al. 2006; Palacios et al. 2011). Mammalian cells possess intracellular defense molecules such as catalase (CAT), glutathione peroxidase (GP), and superoxide dismutase (SOD) to protect themselves against excessive free radicals. Although many organisms have protective mechanisms against damage caused by reactive oxygen, such support is not enough to prevent such damage, and thus supplementary aids are needed (Simic 1988; Turkoglu et al. 2007). Additional air to protect the oxidative damage comes from several compounds, especially minerals (Se and Zn), vitamins (A, E, and β-carotene), and proteins (albumin, ceruloplasmin, and transferrin) (Ostrovidov et al. 2000). Such aids could be further supplemented by cultivated and wild mushrooms in the human diet. Antioxidant components in mushrooms are found in fruit bodies, mycelia, and culture broth (polysaccharides, tocopherols, phenolics, carotenoids, ergosterol, and ascorbic acid) (Sánchez 2017). Increased knowledge of the harmful reactive oxygen species, synthetic antioxidants, and antibiotic resistance by microorganisms have tremendously promoted an interest in exploring natural bioactive compounds with antioxidant and antimicrobial activities. Mushrooms are a potential source of remediation for several cancers (stomach, esophagus, and lungs) prevalent in Canada, China, Japan, Korea, Russia, and the United States (Ferreira et al. 2007). In addition to antioxidant potential, mushrooms also possess antimicrobial activity (Heleno et al. 2013; Khan et al. 2016). There exists a number of synthetic antimicrobial agents that have been built up to control or kill microorganisms without affecting human metabolism. However, uncontrolled applications of such antibiotics result in the emergence of drug-resistant pathogenic bacteria, which are threatening human life. Many mushrooms serve as natural antibiotics against several

DOI: 10.1201/b23190-11

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bacteria, including multi-drug-resistant bacteria (e.g., Gao et al. 2005; Turkoglu et al. 2007; Barros et al. 2007a, b, 2008a, b; Alves et al. 2012). Extracts of fruit bodies and mycelia from the culture of over 2000 mushroom species have been examined and the successful isolation of diterpene, a pleuromutilin useful in the treatment of mycoplasma infections in animals, has been achieved, leading to the development of the first commercial antibiotic of basidiomycete origin (Kavanagh et al. 1950; Brizuela et al. 1998; Sandven 2000). Domesticated edible mushrooms are available throughout the year due to cultivation, harvest, postharvest, processing, and storage practices (Palacios et al. 2011). Edible wild mushrooms are gaining importance owing to their nutritional, medicinal, and anti-pathogenic potential in the gastrointestinal tract. In view of the utilization of mushrooms as a nutraceutical source, this chapter provides details of the antioxidant and antimicrobial potential of six edible wild mushrooms [Amanita hemibapha (Berk. and Broome) Sacc., Boletus edulis Bull., Hygrocybe alwisii (Berk. and Broome) Pegler., Lentinus polychrous Lév., L. squarrosulus Mont. and Termitomyces schimperi (Pat.) R. Heim] occurring in the foothills of the Western Ghats.

1.2  MUSHROOMS AND PROCESSING Tender edible wild mushrooms (Amanita hemibapha, Boletus edulis, Hygrocybe alwisii, Lentinus polychrous, L. squarrosulus, and Termitomyces schimperi) were collected from three locations in the foothills of the Western Ghats region. Replicate samples were oven-dried (58±2°C) to constant mass. The dried mushrooms were milled and preserved in air-tight containers in the refrigerator.

1.3  ANTIOXIDANT ANALYSES 1.3.1  Total Antioxidant Activity Evaluation of the total antioxidant activity (TAA) of mushrooms was performed according to Prieto et al. (1999). The mushroom flour was extracted with methanol (1 mg/ml) (0.1 ml) mixed with sodium phosphate (28 mM) and ammonium molybdate (4 mM in 0.6 M sulfuric acid). Samples were then incubated (95°C for 90 min) and cooled to room temperature. Absorbance (695 nm) was assessed using a phosphomolybdenum complex with a methanol blank. The TAA was expressed as μM ascorbic acid equivalents per gram of mushroom flour (μM AAEs/g).

1.3.2  Ferrous Ion-Chelating Capacity The Fe2+ chelating capacity of methanolic extracts from mushroom flour was assessed using the method by Hsu et al. (2003). A volume of 2 mM FeCl2 (0.1 ml) and 5 mM ferrozine (0.2 ml) were added to methanol extract (1 ml), and fresh methanol was added to a volume of 5 ml. Samples were incubated at room temperature (10 min) and the absorbance of the Fe2+-ferrozine complex was assessed (562 nm). Samples without methanolic extract served as controls to evaluate the capacity of ferrous ion chelation. ( A Ferrous ion-chelating capacity ) % ( = | 1 s562 ( A c562

( | 100 )

(Where Ac is absorbance by the control and As is absorbance by the sample.)

Biological Activity of Edible Mushrooms of the Western Ghats, India

3

1.3.3 DPPH Free Radical-Scavenging Activity The free radical-scavenging activity of the methanol extract of mushroom flour was determined using the method proposed by Singh et al. (2002). To 1 ml of the extracts, 0.01 mM 2,2-diphenyl1-picrylhydrazyl (DPPH) (4 ml) was mixed and allowed to react for up to 20 min at room temperature. The reagents devoid of methanolic extracts served as controls and their absorbance (517 nm) was assessed to determine free radical–scavenging activity. (A A ) Free radical-scavenging activity (%) = | c517 s517 | 100 A c517 ( ) (Where Ac is absorbance by the control and As is absorbance by the sample.)

1.3.4 Reducing Power The reducing power of mushroom flour was measured using the technique by Oyaizu (1986) with a minor alteration. Varied concentrations (200–1,000 μg) of methanolic mushroom flour extracts were mixed with 2.5 ml phosphate buffer (0.2 M, pH 6.6) followed by 2.5 ml potassium ferricyanide (1%). After mixing, samples were incubated (at 50°C, 20 min). Later, 2.5 ml trichloroacetic acid (10% w/v) was added and samples were mixed and centrifuged (402×g, 10 min). The supernatant (2.5 ml) was blended with distilled water (2.5 ml). To this mixture, 0.5 ml ferric chloride (0.1%) was added, and the absorbance was measured (700 nm). Increased absorbance of the reaction mixture indicated increased reducing power.

1.4  ANTIMICROBIAL ASSAYS 1.4.1  Microbial Strains Strains of Gram-positive bacteria (Bacillus cereus, MTCC430; Bacillus subtilis, MTCC441; Staphylococcus aureus, MTCC96; and Streptococcus pneumoniae, MTCC655), Gram-negative bacteria (Enterobacter aerogenes, MTCC2823; Escherichia coli, MTCC443; Haemophilus influenzae, MTCC3826; Proteus vulgaris, MTCC1771; and Pseudomonas aeruginosa, MTCC424) and yeast (Candida albicans, MTCC183) were obtained from the Microbial Type Culture Collection (MTCC) in Chandigarh, India. Bacteria were maintained on Muller-Hinton agar, whereas the yeast was cultured on Sabouraud dextrose agar. Bacteria were cultured overnight and 200 μl aliquots were transferred into 20 ml of sterile Muller-Hinton broth. Yeast was incubated in Sabouraud dextrose broth (37°C) to stabilize the culture and obtain a concentration of 105 CFU/ml.

1.4.2  Mushroom Extraction Whole tender mushroom powder (5 g) each from triplicate sampling was extracted in 25 ml of methanol using a rotary shaker (120 rpm) for up to 24 h at room temperature. The extract was dried in an oven (50±2°C). The dried methanol extract was dissolved in 20% dimethyl sulfoxide (DMSO) to prepare a stock solution (2 mg/ml). DMSO served as a solvent control and its impact on the growth of microorganisms was examined.

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1.4.3 Cup Diffusion Method The antimicrobial activities of the mushroom extracts were assessed using the cup diffusion method (Bauer et al. 1966). A 24 h bacterial culture (0.1 ml; 105 CFU/ml) was spread on Muller-Hinton agar, while the yeast culture was spread on Sabouraud dextrose agar. Sterile porcelain cups (6 × 6 mm; inner tunnel, 4 × 6 mm) were slightly pressed into the seeded plates on the respective agar medium and then loaded with mushroom extract (200 µg/cup). Each plate comprised four cups (three with mushroom extract as triplicate samples and one with DMSO as a control) with one standard antibiotic disc. The standard antibiotic discs contained vancomycin (30 µg/disc) against bacteria and fluconazole (25 µg/disc) against yeast. The plate cultures were incubated (at 37±1°C, 18–24 h) for the growth of bacteria and for the growth of yeast at 26±1°C (48–72 h). The plates were assessed periodically for growth inhibition. Wherever inhibition was observed, the diameter of the inhibition zone was measured using a dial caliper.

1.5  DATA ANALYSIS The total antioxidant, ferrous ion-chelation, DPPH free radical-scavenging, and reducing activities of the six edible mushrooms were compared by one-way analysis of variance (ANOVA) (SigmaPlot #11; Systat Software Inc., USA).

1.6  ANTIOXIDANT ACTIVITIES All the mushrooms showed considerable total antioxidant activity, with the lowest levels observed in H. alwisii (84 μM AAEs/g) and the highest in L. polychrous (394 μM AAEs/g) (Figure 1.1a). The total antioxidant activity, in increasing order, showed a trend of: H. alwisii < T. schimperi < L. squarrosulus < B. edulis < A. hemibapha < L. polychrous. The ferrous ion-chelating capacity was lowest in L. polychrous (32.6%) and highest in A. hemibapha (62.1%) (Figure 1.1b). Ferrous ion-chelating capacity increased in the following order: L. polychrous < B. edulis < H. alwisii < T. schimperi < L. squarrosulus < A. hemibapha. DPPH radical-scavenging was lowest in L. polychrous (9%) and highest in T. schimperi (28%) (Figure 1.2a). The DPPH radical-scavenging increased in the following order: L. polychrous < L. squarrosulus < A. hemibapha < H. alwisii < B. edulis < T. schimperi (Figure 1.2b). Reducing power was lowest in H. alwisii (1 mg: absorbance, 0.044) and highest in B. edulis (1 mg: absorbance, 0.294). One-way ANOVA revealed that the total antioxidant activity was significant among the wild mushrooms (p