Asian Berries: Health Benefits 9780367251994, 9780429286476, 036725199X

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Asian Berries

Functional Foods and Nutraceuticals Series Series Editor John Shi, PhD Guelph Food Research Center, Canada

Asian Berries: Health Benefits, 2020 Edited by Gengsheng Xiao, Yujuan Xu, and Yuanshan Yu

Phytochemicals in Goji Berries: Applications in Functional Foods, 2019 Edited by Xingqian Ye and Yueming Jiang

Korean Functional Foods: Composition, Processing, 2018 Health Benefits Edited by Kun-Young Park, Dae Young Kwon, Ki Won Lee, and Sunmin Park

Phytochemicals in Citrus: Applications in Functional Foods, 2017 Xingqian Ye

Food as Medicine: Functional Food Plants of Africa, 2016 Maurice M. Iwu

Chinese Dates: A Traditional Functional Food, 2016 Edited by Dongheng Liu, PhD, Xingqian Ye, PhD, and Yueming Jiang, PhD

Functional Food Ingredients and Nutraceuticals, 2015 Processing Technologies, Second Edition Edited by John Shi, PhD

Marine Products for Healthcare: Functional and Bioactive, 2009 Nutraceutical Compounds from the Ocean Vazhiyil Venugopal, PhD

Methods of Analysis for Functional Foods and Nutraceuticals, Second Edition, 2008 Edited by W. Jeffrey Hurst, PhD

Handbook of Fermented Functional Foods, Second Edition, 2008 Edited by Edward R. Farnworth, PhD

Functional Food Carbohydrates, 2007 Costas G. Biliaderis, PhD and Marta S. Izydorczyk, PhD

Dictionary of Nutraceuticals and Functional Foods, 2006 N. A. Michael Eskin, PhD and Snait Tamir, PhD

Handbook of Functional Lipids, 2006 Edited by Casimir C. Akoh, PhD

Asian Berries Health Benefits

Edited by

Gengsheng Xiao, Yujuan Xu, and Yuanshan Yu

First edition published 2021 by CRC Press 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742 and by CRC Press 2 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN © 2021 Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, LLC This book contains information obtained from authentic and highly regarded sources. While all reasonable efforts have been made to publish reliable data and information, neither the author[s] nor the publisher can accept any legal responsibility or liability for any errors or omissions that may be made. The publishers wish to make clear that any views or opinions expressed in this book by individual editors, authors or contributors are personal to them and do not necessarily reflect the views/opinions of the publishers. The information or guidance contained in this book is intended for use by medical, scientific or health-care professionals and is provided strictly as a supplement to the medical or other professional’s own judgement, their knowledge of the patient’s medical history, relevant manufacturer’s instructions and the appropriate best practice guidelines. Because of the rapid advances in medical science, any information or advice on dosages, procedures or diagnoses should be independently verified. The reader is strongly urged to consult the relevant national drug formulary and the drug companies’ and device or material manufacturers’ printed instructions, and their websites, before administering or utilizing any of the drugs, devices or materials mentioned in this book. This book does not indicate whether a particular treatment is appropriate or suitable for a particular individual. Ultimately it is the sole responsibility of the medical professional to make his or her own professional judgements, so as to advise and treat patients appropriately. The authors and publishers have also 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 ISBN: 978-0-367-25199-4 (hbk) ISBN: 978-0-429-28647-6 (ebk) Typeset in Times by codeMantra

Contents Series Preface ...................................................................................................................................vii Preface...............................................................................................................................................ix Editors ...............................................................................................................................................xi Contributors ................................................................................................................................... xiii Chapter 1

Bioactive Components in Mulberry Fruits and Pharmacological Effects ................... 1 Lu Li, Yujuan Xu, Yuanshan Yu, Lina Cheng, Bo Zou, and Jun Li

Chapter 2

Nutritional Value and Processing Technology of Mulberry Fruit Products .............. 21 Lan Ma, Gengsheng Xiao, Jijun Wu, Jing Wen, Zhibin Bu, and Daobang Tang

Chapter 3

Nutritional Composition, Antioxidant Properties, and Health Benefits of Mulberry Fruits .......................................................................................................... 41 Maria R. Bronze, Ana Teresa Serra, Paula R. Augusti, Pilar Legua Murcia, and Francisca Hernández García

Chapter 4

Biological Activities of Mulberry Fruits for Skin and Bone ...................................... 67 Ariya Sarikaphuti, Yousef Rasmi, and Pornanong Aramwit

Chapter 5

Black Mulberry Juice .................................................................................................99 Merve Tomas, Gamze Toydemir, and Esra Capanoglu

Chapter 6

Mulberry Fruits: Characteristic Constituents and Health Benefits .......................... 113 Pallav Sengupta, Sulagna Dutta, Chee Woon Wang, and Zheng Feei Ma

Chapter 7

Longan Syrup and Related Products: Processing Technology and New Product Developments ..................................................................................... 123 Noppol Leksawasdi, Kritsadaporn Porninta, Julaluk Khemacheewakul, Charin Techapun, Yuthana Phimolsiripol, Rojarej Nunta, Ngoc Thao Ngan Trinh, and Alissara Reungsang

Chapter 8

Recent Advances in Longan Polysaccharides and Polyphenols: Extraction, Purification, Physicochemical Properties, and Bioactivities .................................... 149 Yuzhu Mao, Hongshun Yang, Caili Fu, Siyong You, Hui Cao, and Shaojuan Lai

Chapter 9

Quality Changes of Longan Fruits during Storage and Shelf Life Extension ......... 173 Muhammad Rafiullah Khan and Vanee Chonhenchob

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Chapter 10 Litchi Berries: Phytochemical Properties and Cosmetic Benefits ........................... 189 Mayuree Kanlayavattanakul and Nattaya Lourith Chapter 11 Loquat (Chinese Plums): Medicinal and Therapeutic Values and Their Processed Products................................................................................................... 205 M. Selvamuthukumaran, Pramod K. Prabhakar, and Neeraj Ghanghas Chapter 12 Sea Buckthorn Berries: A Richer Source of Nutrition and Potential Health Benefits .......................................................................................... 221 Sampan Attri and Gunjan Goel Chapter 13 Health Perspectives of Anthocyanins from Chinese Bayberry Fruits ..................... 239 Huan Cheng, Xingqian Ye, Jianle Chen, and Haibo Pan Chapter 14 Haskap Berries (Lonicera caerulea L.): Phytochemical Constituents and Health Benefits ......................................................................................................... 249 Hae-Jeung Lee and Anshul Sharma Chapter 15 Health Benefits of Haskap Berries (Lonicera caerulea L.)...................................... 279 Rabie Khattab, H.P. Vasantha Rupasinghe, Marianne Su-Ling Brooks, and Giovana Bonat Celli Chapter 16 Haskap (Lonicera caerulea) Berries ........................................................................ 327 Shusong Wu and De-Xing Hou Chapter 17 Indian Gooseberry (Phyllanthus emblica): – A Wonder Fruit ................................. 343 Jiwan S. Sidhu and Tasleem A. Zafar Chapter 18 Amla (Indian Gooseberry): Characteristics, Therapeutic Potential, and Its Value Addition..................................................................................................... 363 M. Selvamuthukumaran Chapter 19 Pepper Berries (Piper nigrum L.) – Drupes with Therapeutic and Nutraceutical Potential ............................................................................................. 381 Vanshika Adiani and Prasad S. Variyar Index .............................................................................................................................................. 399

Series Preface The general public is interested in nutrition, health, natural compounds, and alternate and complementary approaches to health. Increasingly, consumers prefer to make their purchases based on good scientific information. The book Asian Berries: Heath Benefits describes Asian berries such as mulberries, haskap berries, sea buckthorn fruits, lychee, longan, bayberries, pepper berries, and so on and their by-products for people’s health maintenance and well-being purposes. The book focuses on up-to-date information on chemical properties of Asian berries, processing technologies, functional food products, and health benefits, to fill the gap between Asian berries science, functional food products, and human health. The chapters of this book cover a range of chemical, biochemical, and botanic properties, production in Asian countries, bioactive components and health benefits, postharvest storage technology, processing technology development, ethnic foods, utilization of Asian berries’ by-products, new functional food developments of Asian berries, traditional food technology, as well as food safety issues, which will meet the expectations of the global general public. Based on this new information, the new book Asian Berries: Heath Benefits is published in the series Functional Foods and Nutraceuticals by CRC Press. This book furnishes better understanding of some information regarding traditional ethnic functional foods and their dietary applications. The information from the new book may give an opportunity to meet special common concepts around the world and to promote Asian berry products as ethnic functional foods with advanced processing technology. The book is aimed to the interests of academic and research groups. There is great deal of interest now in the areas of nutraceuticals and functional foods in the traditional ethnic health-promoting ingredients and foods for human health. It can also serve as scientific reading material for college and university students majoring in food science, nutrition, pharmaceutical science, and botanical science. The food, pharmaceutical, and nutraceutical industries will be particularly interested in the content of Asian Berries. These industries are always looking for the most current publications in the area of phytochemicals, functional foods, ethnic foods, and nutraceuticals to keep them well informed and to use the information wherever appropriate to their advantage. Another area that will be of great interest to this group is the current regulatory status relating to use and health claims of berries-based functional foods and nutraceuticals both locally and globally. Manufacturers of nutritional supplements will also be another target group for this book. Additionally, people with health concerns are interested in books that provide sound information on Asian berries and products such as ethnic functional foods. Professionals in food and food ingredient industries, universities, research institutes, and in public health organizations are major audiences. Graduate students and senior undergraduate students may also use this book as a reference book. Based on sound scientific findings, this book is poised to become a best seller. It also will serve as a unique reference for food science professionals pursuing functional foods, marketing expansion, as well as nutritional dietary management. Readers will obtain sound scientific knowledge of the nutritional value and health benefits of the different Asian berry products such as raisins, juice, cakes, soup, snacks, berry wines, berry vinegar, medicated foods, and so on. The Functional Foods and Nutraceuticals series is appropriate for academic use; it will be a good scientific reference for food science and technology, nutrition science, and pharmaceutical science faculty and students. The series can also serve as a reference for food science professionals in either government or industry that are pursuing functional foods, food ingredient developments, and R&D in food companies. Readers will obtain current and sound scientific knowledge and information about functional food products and new developments. It is our hope that the scientific

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community will appreciate our efforts in the promotion of preparing this series and the impact on the advancing frontiers of functional foods and nutraceuticals. Dr. John Shi Series Editor of Functional Foods and Nutraceuticals Series Books of CRC Press/USA Guelph Food Research Center, Agriculture and Agri-Food Canada and University of Guelph Guelph, Ontario, Canada

Preface Asian people have a long history of using ethnic Asian berry fruits and their by-products for health maintenance and well-being purpose. Asian berries such as mulberries, haskap berries, sea buckthorn fruits, lychee, longan, bayberries, pepper berries, and so on are all grown in Asian regions and have been listed in traditional food and herbal medicinal category since the early 16th century. Increasing attention to healthcare and quality of life has sparked interest in natural health products. Consumers are always looking for natural health foods to promote health maintenance and well-being. Much interest has been raised toward the consumption of foods with recognized health properties. Some natural health products with rich antioxidant and high free radical scavenging activity such as Asian berries currently draw high interest for scientific studies, which aim to evaluate their nutritional and health-promoting properties for healthy lifestyle and balanced nutrition. Asian berries grown in China, Thailand, Japan, Indian, Vietnam, Burma, Korea, and so on are well-known traditional nutritional foods and herbal medical material as well as valuable nourishing tonics, which have been used for thousands of years in Asian countries. Asian berries are traditionally employed as the herbal medicinals from ancient times. Recently, Asian berries widely marketed as health foods have become increasingly popular in the Western world because of their health-promoting properties. Because of their antipyretic, anti-inflammation, and antisenile properties, Asian berries have myriad varieties of biological activities and pharmacological functions, which play important roles in preventing and treating various chronic diseases. For example, mulberries, originated from China, are rich in antioxidants, which are actually the main line of defense against free radicals. As there are a diverse range of antioxidants in mulberries, they can neutralize free radicals and reduce health risks. Haskap berries, originated from Japan, have by far the highest levels of anthocyanins. Cyanidin3-O-glucoside (C3G) is the major component of anthocyanin group present in haskap berries, comprising about 79%–92% of total anthocyanin content and more than 60% of the total polyphenols. Evidence shows significant antioxidant, cardioprotective, anti-inflammatory, neuroprotective, anticancer, and antidiabetic properties of C3G-rich haskap berry preparations both in vitro and in vivo. The bioactive components in Asian berries are complicated, and certain investigations have been conducted to confirm and demonstrate its formulating ingredients and pharmacological properties in recent years. The concentrated juice or extracts from these berry fruits are added to beverages or other food formulations as ingredients with the aim of improving people’s health maintenance and well-being purpose, including effects on aging, cancer, cardiovascular issues, stroke, diabetes, and immune system functionality. With the development of analysis methods, various chemical constituents have been identified, including anthocyanins, polysaccharides, carotenoids, and flavonoids, among others. During the past years, anthocyanins, carotenoids, and natural vitamin E isolated from the aqueous extracts of Asian berries have been identified as one of the major active ingredients responsible for biological activities. Many studies on pharmacology and phytochemistry have demonstrated that polyphenolics, anthocyanins, carotenoids, flavonoids, and so on in Asian berries have various bioactivities such as antioxidant, immunomodulation, antitumor, neuroprotection, radioprotection, antidiabetes, hepatoprotection, and so on. Asian Berries: Heath Benefits contains 19 chapters covering broad areas such as chemical and biochemical properties, bioactive components and health benefits, postharvest storage technology, processing technology development, ethnic foods, utilization of Asian berries by-products, and functional foods of Asian berries, as well as food safety issues. We thank all the contributing authors for their cooperation in preparing the book chapters, which we hope will serve as an excellent reference for those interested in the science and technology of bioactive components from Asian berries as health-promoting foods. ix

Editors Professor Gengsheng Xiao is the Vice President, Guangdong Academy of Agricultural Science, China. He earned his master’s degree in Agriculture from South China Agricultural University and master’s degree in Public Administration from Tsinghua University. His work mainly focuses on fruit and vegetable processing. He is also appointed as scientist in mulberry fruit processing by China Ministry of Agriculture. He has led 40 national- and provincial-level scientific and technological projects. He received 18 scientific and technological achievements awards, including three first-place prizes of Guangdong Provincial and Ministry of Agriculture, eight second-place prizes, four third-place prizes, and six city awards. He has 28 invention patents on new mulberry and silkworm varieties approved by the National and Guangdong Provincial Variety Examination Committee. He published 5 books and more than 150 research papers, including more than 30 SCI papers. He has also led/participated in the development of more than 12 new products. He participated in the formation of one agricultural industry standard and two local standards in Guangdong Province. He was awarded as the Guangdong Agricultural Science and Technology Innovation Leader in 2009 and won the Guangdong Ding-Ying Technology Award in 2012. Dr Yujuan Xu is Director in Sericulture and Agri-Food Research Institute of Guangdong Academy of Agricultural Sciences, China. Dr Xu majors in Food Science and Engineering, has received the Scientific and Technological Innovation Leader Award for “Millions of Plans” of China, the State Council Special Allowance Expert of China, the Young and Middle-aged Science and Technology Innovation Leader of the China Ministry of Science and Technology. She has been recognized as an expert in postharvest processing of Lingnan fruits in the Modern Agricultural Industrial Technology System of Guangdong Province. Her fruit and vegetable processing team is known as the “National Agricultural Research Innovation Team” by China Ministry of Agriculture. Working on research and development of fruit and vegetable preservation and processing over 20 years, Dr Xu leads more than 20 national and provincial science and technology projects, including the National “Twelfth Five-Year” Science and Technology Support Program, National Public Welfare Industry, Science and Technology Special Project, and Guangdong Natural Science Foundation Team and Key Projects. She has received 13 awards from Guangdong Province, including four first-place prizes, three second-place prizes of Guangdong Science and Technology, one second-place prize of China Agricultural Science and Technology Awards, the 4th National Women’s Inventor Rookie Award, the 13th Guangdong Ding-Ying Technology Award, and so on. She also has 36 invention patents and published 5 books and 138 research papers. Dr Yuanshan Yu is Director of Fruit and Vegetable Processing Unit, Sericulture and Agri-Food Processing Research Institute, Guangdong Academy of Agricultural Sciences, China. His research interests are in emerging technology for tropical and subtropical fruit processing and fermented fruit juice. Dr Yu was selected as an outstanding young research scientist of the Ministry of Agriculture and Rural Areas (2016), the Top Scientific and Technological Innovative Talents of Guangdong Research Program (2015), and the Outstanding Young Scientific and Technological Talents of agricultural product processing industry (2017) and also named as the Jin-Ying Star of Guangdong Academy of Agricultural Sciences (2016). Over the past 5 years, Dr Yu is the principal investigator of more than 10 research projects, including one project supported by the National Natural Science Foundation for Youths, one project supported by the National Key R&D Program, and eight other projects supported by provincial and municipal funding agencies. Dr Yu has published more than 60 peer-reviewed research papers, from xi

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Editors

which 16 papers as the first author, and registered more than 20 patents in China. Dr Yu has won one first-place prize and one third-place prize of Science and Technology of Guangdong Province, one second-place prize of China Agricultural Science and Technology program, and three first-place prizes of Guangdong Agricultural Science and Technology Extension. Dr Yu’s research unit was selected as the National Agricultural Research Innovation Team by the Ministry of Agriculture of China in 2015.

Contributors Vanshika Adiani Food Technology Division Bhabha Atomic Research Centre Mumbai, India Pornanong Aramwit Center for Cellular and Molecular Research Urmia University of Medical Sciences Urmia, Iran Bioactive Resources for Innovative Clinical Applications Research Unit and Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences Chulalongkorn University Bangkok, Thailand Sampan Attri Department of Biotechnology and Bioinformatics Jaypee University of Information Technology Waknaghat, India Paula R. Augusti Food Science and Technology Institute Federal University of Rio Grande do Sul Porto Alegre, Brazil Maria R. Bronze iBET – Instituto de Biologia Experimental e Tecnológica Oeiras, Portugal Instituto de Tecnologia Química e Biológica António Xavier Universidade Nova de Lisboa (ITQB NOVA) Oeiras, Portugal Faculdade de Farmácia da Universidade de Lisboa Universidade Nova de Lisboa Lisboa, Portugal Marianne Su-Ling Brooks Department of Process Engineering and Applied Science, Faculty of Engineering Dalhousie University Halifax, Nova Scotia, Canada

Zhibin Bu Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Science Key Laboratory of Functional Foods Ministry of Agricultural and Rural Affairs/ Guangdong Key Laboratory of Agricultural Products Processing Guangzhou, China Hui Cao School of Medical Instrument and Food Engineering University of Shanghai for Science and Technology Shanghai, China Esra Capanoglu Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering Istanbul Technical University Maslak, Istanbul, Turkey Giovana Bonat Celli Innovation & Quality The Whole Coffee Company Miami, Florida Jianle Chen College of Biosystems Engineering and Food Science Zhejiang University Hangzhou, China Lina Cheng Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Sciences Key Laboratory of Functional Foods Ministry of Agriculture and Rural Affairs Guangdong Key Laboratory of Agricultural Products Processing Guangzhou, China Huan Cheng College of Biosystems Engineering and Food Science Zhejiang University Hangzhou, China xiii

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Contributors

Vanee Chonhenchob Department of Packaging and Materials Technology Kasetsart University Bangkok, Thailand

Muhammad Rafiullah Khan Department of Packaging and Materials Technology Kasetsart University Bangkok, Thailand

Sulagna Dutta Faculty of Dentistry MAHSA University Kuala Lampur, Malaysia

Rabie Khattab Clinical Nutrition Department Imam Abdulrahman Bin Faisal University Dammam, Saudi Arabia

Caili Fu National University of Singapore (Suzhou) Research Institute Suzhou, China

Julaluk Khemacheewakul Bioprocess Research Cluster Faculty of Agro-Industry Chiang Mai University Chiang Mai, Thailand

Francisca Hernández García Department of Plant Sciences and Microbiology Research Group in Plant Production and Technology Miguel Hernández University Alicante, Spain Neeraj Ghanghas Department of Food Science and Technology National Institute of Food Technology Entrepreneurship and Management Sonepat, India Gunjan Goel Department of Microbiology School of Interdisciplinary and Applied Life Sciences Central University of Haryana Mahendergarh, India

Shaojuan Lai College of Basic Medicine Guizhou University of Traditional Chinese Medicine Guiyang, China Hae-Jeung Lee Department of Food & Nutrition Gachon University Gyeonggi-do, Korea Noppol Leksawasdi Bioprocess Research Cluster Faculty of Agro-Industry Chiang Mai University Chiang Mai, Thailand

De-Xing Hou Department of Food Science and Biotechnology Faculty of Agriculture The United School of Agriculture Science Kagoshima University Kagoshima, Japan

Jun Li Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Sciences Key Laboratory of Functional Foods Ministry of Agriculture and Rural Affairs Guangdong Key Laboratory of Agricultural Products Processing Guangzhou, China

Mayuree Kanlayavattanakul School of Cosmetic Science Mae Fah Luang University Chiang Rai, Thailand Phytocosmetics and Cosmeceutical Research Group Mae Fah Luang University Chiang Rai, Thailand

Lu Li Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Sciences Key Laboratory of Functional Foods Ministry of Agriculture and Rural Affairs Guangdong Key Laboratory of Agricultural Products Processing Guangzhou, China

Contributors

Nattaya Lourith School of Cosmetic Science Mae Fah Luang University Chiang Rai, Thailand Phytocosmetics and Cosmeceutical Research Group Mae Fah Luang University Chiang Rai, Thailand Lan Ma Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Science Key Laboratory of Functional Foods Ministry of Agricultural and Rural Affairs Guangdong Key Laboratory of Agricultural Products Processing Guangzhou, China

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Haibo Pan College of Biosystems Engineering and Food Science Zhejiang University Hangzhou, China Yuthana Phimolsiripol Division of Product Development Technology, Faculty of Agro-Industry Chiang Mai University Chiang Mai, Thailand Kritsadaporn Porninta Bioprocess Research Cluster Faculty of Agro-Industry Chiang Mai University Chiang Mai, Thailand

Zheng Feei Ma Department of Health and Environmental Sciences Emerging and Interdisciplinary Sciences Building Xi’an Jiaotong-Liverpool University Suzhou, China

Pramod K. Prabhakar Department of Food Science and Technology National Institute of Food Technology Entrepreneurship and Management Sonepat, India

Yuzhu Mao Department of Food Science & Technology National University of Singapore Singapore National University of Singapore (Suzhou) Research Institute Suzhou, China

Yousef Rasmi Department of Biochemistry Faculty of Medicine and Center for Cellular and Molecular Research Urmia University of Medical Sciences Urmia, Iran

Pilar Legua Murcia Department of Plant Sciences and Microbiology Research Group in Plant Production and Technology Miguel Hernández University Alicante, Spain Rojarej Nunta Bioprocess Research Cluster Faculty of Agro-Industry Chiang Mai University Chiang Mai, Thailand Division of Food Innovation and Business, Faculty of Agricultural Technology Lampang Rajabhat University Lampang, Thailand

Alissara Reungsang Research Group for Development of Microbial Hydrogen Production Process Khon Kaen University Khon Kaen, Thailand Department of Biotechnology, Faculty of Technology Khon Kaen University Khon Kaen, Thailand H.P. Vasantha Rupasinghe Department of Plant, Food, and Environmental Sciences Faculty of Agriculture Dalhousie University Truro, Nova Scotia, Canada

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Ariya Sarikaphuti Department of Anti-aging and Regenerative Medicine School of Anti-aging and Regenerative Medicine Mae Fah Laung University Bangkok, Thailand M. Selvamuthukumaran Department of Food Technology Hindustan Institute of Technology and Science Chennai, India Pallav Sengupta Faculty of Medicine, Bioscience and Nursing MAHSA University Kuala Lampur, Malaysia Ana Teresa Serra Food and Health Division, Food Functionality and Bioactives Lab iBET – Instituto de Biologia Experimental e Tecnológica Oeiras, Portugal Instituto de Tecnologia Química e Biológica António Xavier Universidade Nova de Lisboa (ITQB NOVA) Oeiras, Portugal Anshul Sharma Department of Food & Nutrition Gachon University Gyeonggi-do, Korea Jiwan S. Sidhu Department of Food Science and Nutrition College of Life Sciences Kuwait University Safat, Kuwait Daobang Tang Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Sciences Key Laboratory of Functional Foods Ministry of Agriculture and Rural Affairs Guangdong Key Laboratory of Agricultural Products Processing Guangzhou, China

Contributors

Charin Techapun Bioprocess Research Cluster Faculty of Agro-Industry Chiang Mai University Chiang Mai, Thailand Merve Tomas Department of Food Engineering, Faculty of Engineering and Natural Sciences Istanbul Sabahattin Zaim University Istanbul, Turkey Gamze Toydemir Department of Food Engineering Alanya Alaaddin Keykubat University Antalya, Turkey Ngoc Thao Ngan Trinh Nong Lam University Ho Chi Minh City, Vietnam Prasad S. Variyar Food Technology Division Bhabha Atomic Research Centre Mumbai, India Chee Woon Wang Faculty of Medicine, Bioscience and Nursing MAHSA University Kuala Lampur, Malaysia Jing Wen Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Sciences Key Laboratory of Functional Foods Ministry of Agriculture and Rural Affairs Guangdong Key Laboratory of Agricultural Products Processing Guangzhou, China

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Contributors

Jijun Wu Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Science Key Laboratory of Functional Foods Ministry of Agricultural and Rural Affairs Guangdong Key Laboratory of Agricultural Products Processing Guangzhou, China Shusong Wu Hunan Collaborative Innovation Center for Utilization of Botanical Functional Ingredients College of Animal Science and Technology Hunan Agricultural University Hunan, China Hongshun Yang Department of Food Science & Technology National University of Singapore Singapore, Singapore National University of Singapore (Suzhou) Research Institute Suzhou, China

Xingqian Ye College of Biosystems Engineering and Food Science Zhejiang University Hangzhou, China Siyong You College of Biological Science and Technology Fuzhou University Fujian, China Tasleem A. Zafar Department of Food Science and Nutrition, College of Life Sciences Kuwait University Kuwait City, Kuwait Bo Zou Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Sciences Key Laboratory of Functional Foods Ministry of Agriculture and Rural Affairs Guangdong Key Laboratory of Agricultural Products Processing Guangzhou, China

1

Bioactive Components in Mulberry Fruits and Pharmacological Effects Lu Li, Yujuan Xu, Yuanshan Yu, Lina Cheng, Bo Zou, and Jun Li Guangdong Academy of Agricultural Sciences

CONTENTS 1.1 1.2

Introduction .............................................................................................................................. 1 Chemical Properties of Bioactive Components in Mulberry Fruits ......................................... 2 1.2.1 Polysaccharides............................................................................................................. 2 1.2.2 Polyphenolics ................................................................................................................ 3 1.2.3 Flavonoids ..................................................................................................................... 4 1.2.4 Anthocyanins ................................................................................................................ 5 1.2.5 Phenolic Acids .............................................................................................................. 7 1.2.6 Melatonin ...................................................................................................................... 9 1.3 Pharmacological Effects ........................................................................................................... 9 1.3.1 Immunomodulatory Activity ...................................................................................... 10 1.3.2 Antioxidant Activity ................................................................................................... 11 1.3.3 Antihyperglycemic Activity ....................................................................................... 11 1.3.4 Hypolipidemic Activity .............................................................................................. 12 1.3.5 Anticancer Activity..................................................................................................... 13 1.3.6 Neuroprotective Activity ............................................................................................ 14 1.3.7 Antiatherosclerosis Activity ....................................................................................... 14 1.4 Summary ................................................................................................................................ 14 Acknowledgments............................................................................................................................ 15 References ........................................................................................................................................ 15

1.1

INTRODUCTION

In most mulberry-growing countries, mulberry fruits are commonly eaten fresh or dried, or processed into wine, fruit juice, and jam because of their delicious taste, pleasing color, low calorie, and high nutrient contents (Pawlowska et al., 2008; Huang et al., 2017; Jelled et al., 2017). In China, Korea, and Japan, mulberry fruits are also used in folk medicine for their pharmacological effects on fever reduction, treatment of sore throat, liver and kidney protection, eyesight improvement, and their ability to lower blood pressure (Chen et  al., 2016; Zhou et  al., 2017). These pharmacological effects have close correlation with the bioactive compounds present in mulberry fruits (Yuan & Zhao, 2017). In recent years, researchers detected many bioactive compounds in mulberry fruits, such as anthocyanins, polysaccharides, alkaloids, resveratrol, oxyresveratrol, and proanthocyanins (Pawlowska et  al., 2008; Jiang  & Nie, 2015). Among them, resveratrol (trans-3,41,5trihydroxystilbene) and oxyresveratrol (trans-2,31,4,51-tetrahydroxy stilbene) are hydroxystilbenes (Bae  & Suh, 2007). Oxyresveratrol has an inhibitory effect on tyrosinase to limit melanin biosynthesis and is used as a cosmetic material and medical agent for hyperpigmentation disorders 1

2

Asian Berries: Health Benefits

(Choi  & Hwang, 2005; Zheng et  al., 2012). Anthocyanins are a group of natural phenolic compounds responsible for coloring of fruits. They serve as the best source of health benefits because of their antioxidant and anti-inflammatory properties (Nuengchamnong et al., 2007). Anthocyanins have high inhibitory ability on lipid oxidation. The mulberry anthocyanin extracts (MAEs) have antimetastatis activity to inhibit migration of B16-F1 cells (Cui et al., 2008; Özgen et al., 2009). Flavonoids are commonly present in mulberry fruit, and there are at least four flavonoids present in the fruit. Flavonoids have been recognized to possess anti-inflammatory, antioxidant, antiallergic, anti-thrombotic, hepatoprotective, antiviral, and anti-carcinogenic activities in human beings (Daimon et al., 2010). Moreover, the polysaccharides purified from mulberry fruits showed antioxidant and hypoglycemic activities (Chen et al., 2015; Chen, You et al., 2016). Therefore, mulberry fruits are often used as functional ingredients in various food and fitness products (Kim, Choi et al., 2010., 2010). To better utilize mulberry fruits, this chapter aims to summarize recent knowledge regarding the bioactive components and pharmacological effects of mulberry fruits.

1.2

CHEMICAL PROPERTIES OF BIOACTIVE COMPONENTS IN MULBERRY FRUITS

In order to investigate the bioactive components in mulberry fruits, numerous studies have been conducted to confirm their chemical constituents, including amino acids, fatty acids, minerals, polysaccharides, flavonoids, and anthocyanins (Jiang & Nie, 2015; Sánchez-Salcedo et al., 2015).

1.2.1

POLYSACCHARIDES

Polysaccharides are the major active components of mulberry resources, and their compositions are listed in Table 1.1 (Liu & Lin, 2012; Wen et al., 2019). Recently, increasing attention has been focused on mulberry fruit polysaccharides (MFPs), and some advances have been made to characterize these polysaccharides and their bioactivities (Yuan & Zhao, 2017; He et al., 2018). To date, several polysaccharides have been identified in mulberry fruits using various extraction methods and purification processes. Physicochemical properties and structural features, such as molecular weight and monosaccharide composition, have been studied using Fourier transform infrared spectra, highperformance liquid chromatography, and methylation analysis. Several polysaccharide fractions with antioxidant and hypoglycemic activities were purified from mulberry fruits using various purification

TABLE 1.1 The Composition of Polysaccharide Isolated from Mulberry Fruits Variety Morus alba M. alba Morus nigra Unknown

Components of Polysaccharide Mannose (1.60 mol%), rhamnose (18.40 mol%), glucose (3.10 mol%), galactose (37.60 mol%), xylose (1.70 mol%), fucose (1.30 mol%), and arabinose (36.30 mol%) Rhamnose (25.98 mol%), glucose (13.06 mol%), galactose (23.10 mol%), galacturonic acid (16.35 mol%), and arabinose (21.51 mol%) Mannose (18.88 mol%), rhamnose (5.94 mol%), glucose (27.62 mol%), galactose (25.00 mol%), xylose (1.75 mol%), galua (7.87 mol%), and arabinose (12.94 mol%) Three components: the first was composed of mannose (8.51 mol%), glucose (28.20 mol%), galactose (18.51 mol%), xylose 6.55 mol%), galacturonic acid (3.97 mol%) and arabinose (34.15 mol%); the second was composed of mannose (2.61 mol%), glucose (38.33 mol%), galactose (29.27 mol%), xylose 4.33 mol%), galacturonic acid (3.18 mol%), and arabinose (22.45 mol%); the third was composed of mannose (5.17 mol%), glucose (12.14 mol%), galactose (30.33 mol%), xylose 11.25 mol%), galacturonic acid (3.19 mol%), and arabinose (37.45 mol%)

References Lee et al. (2013) Chen et al. (2016) Ma et al. (2018) Chen et al. (2017)

Bioactive Components in Mulberry Fruits

3

methods (Chen et al., 2015; Chen et al., 2016). A glycoprotein isolated from the lyophilized powder of mulberry fruit juice (yield 10.6%), with a carbohydrate content of 28.4% and a protein content of 71.6%, showed better antiapoptotic activity than strawberry fruit polysaccharides (Liu  & Lin, 2014). Choi, Synytsya et al. (2016) isolated and investigated the structural properties of a pectic-type polysaccharide from mulberry fruits. The obtained data indicated that the polysaccharides have a rhamnogalacturonan type I (RG I) backbone composed of repeated disaccharide fragments [4-α-DGalpA-1 → 2-α-L-Rhap-1 → ]. The arabinan side chain is composed of (1 → 5)-α-L-Ara attached to the O-4 position of α-L-Rhap. The arabinogalactan type II (AG II) side chain was found to have a (1 → 6)-β-D-galactan core branched at O-3 by α-L-Araf (Figure 1.1). However, the connection patterns between AG II and RG I are still unclear. In the future, structures of polysaccharides will remain to be further determined to provide more information for elucidating the structure–activity relationship.

1.2.2

POLYPHENOLICS

Mulberry fruits are rich sources of polyphenolics. Polyphenolics represent a large family and are classified by their structural characteristics as flavonoids (anthocyanins, flavanols or catechins, flavonols, flavones, flavanones, isoflavonoids), phenolic acids, stilbenes, tannins, and lignans (Liu, 2004; Han et al., 2007). It is well established that intake of foods rich in polyphenols is associated with a reduced risk of cancer, cardiovascular diseases, and neurodegeneration (Han et  al., 2007; Del Rio et  al., 2013). Significant amounts and a wide diversity of phenolic compounds are present in mulberry fruits (Figure 1.2). The total phenolic, flavonoid, and anthocyanin contents in mulberry fruit are

FIGURE 1.1 Structure of polysaccharide isolated from mulberry fruits (Yuan & Zhao, 2017).

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FIGURE  1.2 Chemical structures of the main polyphenol compounds in mulberry fruits (Yuan  & Zhao, 2017).

104.78–215.53 mg gallic acid equivalent (GAE)/100 g dry weight (DW), 64.55–211.01 mg catechin equivalent (CE)/100 g DW, and 45.42–208.74 mg cyanidin-3-O-glucoside (C3G) equivalent/100 g frozen weight, respectively (Butkhup et al., 2013; Natić et al., 2015). The phenolic contents in mulberry fruits vary with different cultivars (Butkhup et  al., 2013; Natić et  al., 2015). Besides the cultivars, maturity stages also have significant influences on the phenolic contents of mulberry fruits. The phenolic contents in mulberry fruits increase as the fruit progresses from unripe stage to fully ripened stages. Lin and Tang (2007) compared the total phenolic and flavonoid contents among four selected fruit species. The results showed that the total phenolic and flavonoid contents were significantly higher in mulberry fruits (1515.9 ± 5.7 mg GAE/100 g fresh matter (FM), 250.1 ± 6.3 mg quercetin equivalent (QE)/100 g FM) than in other deep-colored fruits, such as strawberry (363.7 ± 6.7 mg GAE/ 100 g FM, 14.6 ± 3.0 mg QE/100 g FM), oriental plum (668.0 ± 8.0 mg GAE/100 g FM, 37.6 ± 7.0 mg QE/100 g FM), and loquat (199.4 ± 13.1 mg GAE/100 g FM, 14.2 ± 0.9 mg QE/100 g FM).

1.2.3

FLAVONOIDS

Flavonoids are chemically polyphenolic secondary metabolic compounds universally distributed in green plant kingdom located in cell vacuoles (Ramesh et al., 2014). Flavonoids are found mostly in glycosylated form, and they have complex flavonol glycoside profiles including 13 quercetin derivatives, 5 kaempferol derivatives, and O-methylated flavonol analogs, such as rhamnetin and isorhamnetin (Khalifa et al., 2018). Flavonoids are responsible for the color and aroma of flowers to attract pollinators, spore germination, and growth and development of seedlings (Samanta et al., 2011). Flavonols and flavanols are the main subgroups of flavonoids. Their structures are similar but slightly different in C-2, C-3, and C- 4 positions. Compared with flavanols, flavonols have a double bond between C-2 and C-3 and a carbonyl group at C- 4 of the C ring. Mulberry fruits contain many flavonols, including rutin, quercetin, myricetin, and kaempferol (Table 1.2). Derivatives of quercetin and kaempferol are the major components of mulberry fruit flavonols. Glycosylated forms of

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5

TABLE 1.2 Flavonols and Flavanols Composition in Mulberry Fruits Compound Flavonols

Flavanols

a b

Content

Rutin

0.065–7.728 mg/100 g FWa

Quercetin Quercetin 3-O-rutinoside Quercetin 3-O-glucoside Quercetin 3-O-galactoside Myricetin Kaempferol Kaempferol 3-O-glucoside Kaempferol 3-O-rutinoside Catechin Epigallocatechin gallate Epicatechin Procyanidin B1 Procyanidin B2

31.88–58.42 mg/100 g DWb 2.869 mg/100 g FW 1.069 mg/100 g FW 0.002 mg/100 g FW 0.66–1.18 mg/100 g DW 0.24–1.61 mg/100 g DW 1.623 mg/100 g FW 2.00–14.00 mg/100 g DW 309.26–750.01 mg/100 g DW 0.033–0.086 mg/100 g DW 8.47–17.12 mg/100 g DW 59.64–224.41 mg/100 g DW 1.02–5.66 mg/100 g DW

References Natić et al. (2015) Butkhup et al. (2013) Jin et al. (2015) Jin et al. (2015) Jin et al. (2015) Butkhup et al. (2013) Butkhup et al. (2013) Jin et al. (2015) Sánchez-Salcedo et al. (2015) Butkhup et al. (2013) Natić et al. (2015) Butkhup et al. (2013) Butkhup et al. (2013) Butkhup et al. (2013)

FW, fresh weight. DW, dry weight.

quercetin and kaempferol, such as quercetin 3-O-rutinoside, quercetin 3-O-glucoside, quercetin 3-O-galactoside, kaempferol 3-O-glucoside, and kaempferol 3-O-rutinoside, have been found in some mulberry fruit cultivars (Jin et al., 2015; Sánchez-Salcedo et al., 2015). In general, flavanols do not occur naturally as glycosides. Among them, catechin, epigallocatechin gallate, epicatechin, and procyanidins B1 and B2 have been found in mulberry fruits (Table 1.2). Natic ́ et al. (2015) stated that rutin was the most abundant phenolic compound, contributing 44.66% of the total phenolics in 11 mulberry varieties. These results were consistent with previous findings (Zhang et al., 2008; Gundogdu et al., 2011; Chan et al., 2015).

1.2.4

ANTHOCYANINS

Anthocyanins are water-soluble pigments composed of an anthocyanidin aglycone and a sugar moiety mainly attached at the 3 position on the C ring or, less frequently, at 5 or 7 position on the A ring (Figure  1.3) (Prior  & Wu, 2006; Szajdek  & Borowska, 2008). The biosynthesis pathway of anthocyanidins is shown in Figure  1.4 (Zhang et  al., 2014). Moreover, the anthocyanins are the predominant polyphenols (up to ∼3000 mg/ kg fw) in mulberry fruits (Veberic et al., 2015a). Mulberry fruits have higher anthocyanins content than blueberry, blackberry, blackcurrant, and redcurrant fruits. Anthocyanins are mainly responsible for the color of mulberry fruits (60%) (Gerasopoulos & Stavroulakis, 1997) and also greatly contribute to their health-promoting effects, including antioxidation, anti-inflammatory, blood lipid regulation, insulin resistance improvement, night vision enhancement, antibacterial, and antiaging (Graf et  al., 2013). Various anthocyanins have been qualified and quantified in mulberry fruits (Table 1.3). The qualitative analysis methods used for anthocyanins are paper chromatography, thin-layer chromatography, spectral analysis, chromatography–mass spectrometry, and nuclear magnetic resonance (Harborne, 1958; Ando et al., 1999), while the quantitative methods of anthocyanins mainly include pH difference method and subtraction method. The principal anthocyanin in mulberry fruits is C3G, followed by cyanidin-3-rutinoside (C3R) (Chang et al., 2013; Chen et al., 2016). Small amounts of pelargonidin3-glucoside and pelargonidin-3-rutinoside have also been detected in mulberry fruits in addition

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FIGURE  1.3 Chemical structures of anthocyanidins present in berry with corresponding color (Veberic et al., 2015b).

FIGURE 1.4 Biosynthetic pathway of anthocyanidins. Phenylalanine ammonia lyase (PAL), cinnamate 4hydroxylase (C4H), 4-coumaryol CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavanone 3050-hydroxylase, (F3050H), flavanone 30-hydroxylase (F30H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (A NS), flavonol synthase (F LS), and flavone synthase (FNS).

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TABLE 1.3 Anthocyanins Composition in Mulberry Fruits Compound

Content

References

Cyanidin-3-glucoside

301.75 mg/g MAEa

Cyanidin-3-rutinoside

108.79 mg/g MAE

Pelargonidin-3-glucoside

NAb

Pelargonidin-3-rutinoside

NA

Cyanidin 3-O-(6″-O-α-rhamnopyranosyl-β-D-glucopyranoside) Cyanidin 3-O-(6″-O-a-rhamnopyranosyl-β-D-galactopyranoside) Cyanidin 3-O-β-D-galactopyranoside Cyanidin 3-O-β-D-galactopyranoside Petunidin 3-O-β-glucopyranoside

270 mg/g CMAc 57 mg/g CMA 233 mg/g CMA 33 mg/g CMA 5.1 mg/g CEEd

Chang et al. (2013), Chen et al. (2016) Chang et al. (2013), Chen et al. (2016) Huang et al. (2008), Liu et al. (2009) Huang et al. (2008), Liu et al. (2009) Du et al. (2008) Du et al. (2008) Du et al. (2008) Du et al. (2008) Sheng et al. (2014)

a b c d

MAE, mulberry anthocyanin extract. NA, not available. CMA, crude mulberry anthocyanin. CEE, crude ethanol extract.

to C3G and C3R in some works (Liu et al., 2008; Qin et al., 2010). Using high-speed countercurrent chromatography, Du et  al. (2008) identified five anthocyanins in mulberry fruits: cyanidin 3-O-(6″-O-α-rhamnopyranosyl-β-D-glucopyranoside) (C3RG) (also known as keracyanin), cyanidin 3-O-(6″-O-a-rhamnopyranosyl-β-D-galactopyranoside) (C3RGa), C3G, cyanidin 3-O-β-Dgalactopyranoside (C3Ga), and cyanidin 7-O-β-D-glucopyranoside (C7G) (Figure  1.5). In another analytical study, the main anthocyanin was C3RGa, at 41.3% of MAE, and other isolated pigments were C3RG and petunidin 3-O-β-glucopyranoside (Sheng et al., 2014). In general, fruits of the colored species are somewhat superior to those of non-colored species, and the difference between their contents was reported (Özgen et al., 2009; Sánchez-Salcedo et al., 2015). The utmost anthocyanin content (0.57–1.88 mg C3G g/ DW) was observed in the black mulberry fruits, whereas it lacked (0.01 mg C3G g/DW) in the white mulberry (Özgen et al., 2009; Sánchez-Salcedo et al., 2015). Similarly, Chen et al. (2016) found that the total anthocyanin contents of five Chinese mulberry cultivars, namely, Zhongshen 831, Da 10, Zhongshen 5801, Ding 33, and Taiwanguosang, are less than 900 μg/g fw, and no anthocyanin was observed in the white cultivars such as Zhenzhubai, Jiguihua, and Baiyuwang. Bao et al. (2016) reported that the quantity of C3G ranged from 1.25 to 3.35 g/ kg fw in Morus atropurpurea Roxb. cv Guangdong and J33, respectively, and the quantity of C3R varied from 0.25 to 1.50 g/ kg fw in Morus multicaulis Perr. cv Guangdong and Hongguo, but traces of anthocyanins were detected in white mulberry cultivars. Consistent with visual color change, total anthocyanins content markedly increases as the fruit ripens from white/ light red to black stages (Bae & Suh, 2007). Differences in geographical conditions also resulted in different levels of anthocyanins accumulation (Ercisli & Orhan, 2007; Lee et al., 2004).

1.2.5

PHENOLIC ACIDS

Among the phytochemicals in fruit, phenolic acids are regarded as major functional food components and are thought to contribute to the beneficial health effects of fruit-derived products due to their capacity to prevent various diseases associated with oxidative stress such as cancers,

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FIGURE  1.5 Molecular structures of cyanidin 3-O-(6″-O-a-rhamnopyranosyl-β-D-galactopyranoside), cyanidin 3-O-(6″-O-α-rhamnopyranosyl-β-D-glucopyranoside), cyanidin 3-O-β-D-galactopyranoside, and cyanidin 7-O-β-D-glucopyranoside.

cardiovascular diseases, and inflammation (Scalbert & Williamson, 2000; Lodovici et al., 2001). Phenolic acids constitute about one-third of the dietary phenols and are present in plants in free and bound forms (Robbins, 2003). Mulberry fruits contain a variety of phenolic acids. The composition and contents of phenolic acids in mulberry fruits have been the subject of many studies (Gundogdu et  al., 2011; Mahmood, Anwar, Abbas and Saari 2012; Butkhup et  al., 2013; Natić et  al., 2015). Phenolic acids in mulberry fruits are mainly represented by derivatives of hydroxycinnamic and benzoic acids (Table 1.4). Among hydroxycinnamic acid derivatives, chlorogenic acid, ferulic acid, p-coumaric acid, o-coumaric acid, cinnamic acid, and caffeic acid were found in mulberry fruits. In the group of benzoic acid derivatives, gallic acid, hydroxybenzoic acid, protocatechuic acid, and vanillic acid have been reported (Yuan & Zhao, 2017). These phenolic acids commonly occur in conjugated forms of esters and glycosides, but rarely exist as free acids (Zhao, 2007). Esters make up 53.1% of total phenolic acids, whereas glycosides and free acids account for 43.6% and 3.3%, respectively (Zadernowski et  al., 2005). Glycosidic acid and esters of chlorogenic and protocatechuic acids are the most common forms in mulberry fruits, especially in the black mulberry fruit cultivars (Memon et  al., 2010). Dissimilar results were reported in a previous study (Radojković et al., 2012), probably due to the genetic difference. Another report showed that the protocatechuic acid has anti-inflammatory and antihepatotoxicity effects (Hsu et al., 2012). Meanwhile, levels of chlorogenic acid and its isomers, which have antiobesity properties, decline as mulberries ripen from semimatured to fully matured (Lee, Oh et al., 2016). Mulberry fruits also contain quinic acid, which may help to alleviate urinary tract infections (Bao et al., 2016). Ellagic acid is a hydroxybenzoic acid, but most of it is in the form of ellagitannins, and it is rich in the Spanish species (8.7–15.5 mg 100 g−1) (Calín-Sánchez et al., 2013).

Bioactive Components in Mulberry Fruits

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TABLE 1.4 Phenolic Acids Composition in Mulberry Fruits Compound Chlorogenic acid Ferulic acid p-Coumaric acid o-Coumaric acid Cinnamic acid Caffeic acid Gallic acid p-Hydroxybenzoic acid Syringic acid Protocatechuic acid Vanillic acid

Content

References

5.3–17.3 mg/100 g DW 0.057–2.949 mg/100 g DW 0.024–0.142 mg/100 g DW 0.015 mg/g FW 11.64–15.05 mg/100 g DW 1.06–8.17 mg/100 g DW 7.33–23.34 mg/100 g DW 0.028–0.154 mg/100 g DW 0.049 mg/g FW 0.264–0.794mg/ 100 g FW 0.008 mg/g FW

Mahmood et al. (2012) Natić et al. (2015) Natić et al. (2015) Gundogdu et al. (2011) Butkhup et al. (2013) Butkhup et al. (2013) Butkhup et al. (2013) Natić et al. (2015) Gundogdu et al. (2011) Natić et al. (2015) Gundogdu et al. (2011)

DW, dry weight. FW, fresh weight.

In general, there are discrepancies in the polyphenolic composition and content in mulberry fruit across studies. The differences depend not only on the cultivars used but also on the extraction and analytical methods, genetic differences, and growing conditions, including geographical and environmental conditions such as temperature, humidity, light, and degree of maturity (Mahmood et al., 2012; Butkhup et al., 2013; Natić et al., 2015; Sánchez-Salcedo et al., 2015).

1.2.6

MELATONIN

Melatonin (N-acetyl-5-methoxytryptamine) is a natural compound of almost ubiquitous occurrence (Hardeland & Pandi-Perumal, 2005). It presented in almost all major taxa of organisms, as far as tested, including bacteria, unicellular eukaryotes, macroalgae, plants, fungi, and invertebrate animals. Several studies dealt with melatonin in edible plants, including mulberry fruits (Reiter et al., 2001; Conti et al., 2002; Reiter & Tan, 2002; Wang et al., 2016). Wang et al. (2016) studied dynamic changes in melatonin content during mulberry fruits development (from the fruit setting to ripening) and ethanol fermentation. High levels of melatonin (5.76 ng/g F W) were detected in stage I but then decreased in stages II and III. The melatonin level in mulberry fruit wine was much higher than that in fruits. The melatonin content increased to 31.59 and 28.11 ng/m L during ethanol fermentation at 25˚C and 16˚C, respectively. Melatonin is derived from tryptophan via enzymatic conversion reactions (Figure 1.6). Melatonin plasma concentration varies according to a circadian cycle, and this cyclic production determines the periodic effects at a systemic level (Reiter, 1991). Melatonin is evolutionary conserved and exerts many regulatory functions by modulating cellular behavior via binding to specific receptors and intracellular targets (Carlson et al., 1989; Pandi-Perumal et al., 2008). It is also reported to possess a wide array of physiological activities, such as antioxidant, antiinflammatory effects, and regulation of circadian rhythms and sleep disorders (Cardinali & Pévet, 1998; Bonnefont-Rousselot & Collin, 2010; Radogna et al., 2010).

1.3 PHARMACOLOGICAL EFFECTS Mulberry fruits, rich in nutrients, have always been used for food and medicine. As early as more than 2,000 years ago, it became one of the emperor’s royal drugs. As a traditional Chinese medicine and food resource, mulberry fruits can be used as a raw material for ordinary foods and health foods.

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FIGURE 1.6 Biosynthesis of melatonin.

The mulberry fruits are designated one of the first medicinal and edible plants by the Ministry of Health of China in 1985, and their medical use is recorded in the Chinese pharmacopoeia (Yuan & Zhao, 2017). According to current researches, mulberry fruits have the activities of immunomodulatory, anti-oxidant, anti-hyperglycemic, hypolipidemic, anti-cancer, neuroprotective, and antiatherosclerosis (Chen et al., 2005; Sarikaphuti et al., 2013; Seo et al., 2015; Raman et al., 2016).

1.3.1

IMMUNOMODULATORY ACTIVITY

Studies have shown that mulberry fruit extracts have the function of enhancing immunity (Yuan & Zhao, 2017). Resveratrol is a polyphenol compound and an anti-inflammatory agent found in mulberry fruits; IL-8 (interleukin-8) plays a key role in the initiation and maintenance of inflammatory response. IL-8 production was measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). Resveratrol inhibits IL-8 secretion by blocking mitogen-activated protein kinase (MAPK) phosphorylation and nuclear factor kappa B (NF-κB) activation. Resveratrol modulates THP-1 cell activation under inflammatory conditions (Ramesh et al., 2014). Moreover, several pyrrole alkaloids from mulberry fruits had significantly activated macrophage activity in RAW 264.7 cells by the enhancement of nitric oxide, TNF-α (tumor necrosis factor-alpha), and IL-12 production (K im et al., 2013). A glycoprotein (MP) isolated from mulberry fruits was also examined for its immunomodulatory effects in mouse primary macrophages by Liu and Lin (2012). MP significantly (P < 0.05) decreased proinflammatory cytokines including IL-1β and IL-6, whereas the anti-inflammatory cytokine IL-10 was increased. In addition, MP improved the ratio of Bcl-2/ Bak protein expression, suggesting that MP enhanced cell viability by inhibition of apoptosis. In another recent study from Liu and Lin (2014), similar results were obtained in murine primary splenocytes. Besides, Lee et al. (2013) used ELISA and RT-PCR indicated that MFP can stimulate secretion of chemokines (RANTES and M1P-1α) by RAW in mouse

Bioactive Components in Mulberry Fruits

11

macrophages and promote the secretion of anti-inflammatory factors such as TNF-α. TNF-α and IL-6, which induced the secretion of nitric oxide (NO) and prostaglandin (PGE2), showing certain immunological activity. Dendritic cells (DCs) are the most potent antigen-presenting cells that initiate the majority of immune responses, including tumor-specific T cell responses. Maturation of DCs is usually attenuated in the tumor microenvironment, which is an important immunological problem in DC-based immunotherapy of cancer (He et  al., 2018). Nowadays, plant- and fungalderived polysaccharides have been used as inducers of DC maturation; especially polysaccharides from Grifola frondosa have been assessed in clinical trials of cancer immunotherapy (He et al., 2017). Based on recent experimental findings, MFPs were shown to be capable of inducing phenotypic and functional maturation of DCs, as evidenced by several changes in MFP-treated DCs (Shin et al., 2013). MFPs were confirmed to induce phenotypic and functional maturation of DCs at least in part via Toll-like receptor 4 (TLR4), since DCs having mutated TLR4 are hyporesponsive to MFPs compared with DCs having normal TLR4. Furthermore, Chang et al. (2015) found that mulberry fruit extracts (MFEs) exerted their immunomodulatory activity by TLR4-mediated NF-κB and MAPK signaling pathways. In another research, Qian et al. (2015) found that MFEs were able to attenuate inflammatory responses by the NF-κB/p65 and protein kinase R (PKR)–like endoplasmic reticulum kinase (pERK)/MAPK pathways. In addition, Morus australis anthocyanin extract suppressed inflammation through downregulating the expression of TNFα, IL-6, inducible nitric oxide synthase, and NF-κB genes (Wu et al., 2016). And the prophylactic or therapeutic efficiency in acute inflammation of anthocyanins (extracted from Morus nigra fruit) was investigated by Hassimotto et al. (2013).

1.3.2

ANTIOXIDANT ACTIVITY

According to previous researches, mulberry fruits have good antioxidant and free radical scavenging effects (Shih et al., 2010; Yang & Lee, 2012; Wang, Xiang et al., 2013). Mulberry fruits are rich in anthocyanins, which play an important role in antioxidant effect. For anthocyanins, the 3,4dihydroxy substituents in the B ring were critical for radical scavenging. They react readily with radicals such as hydroxyl (%OH), azide (N 3%), and peroxyl (ROO·) to form stable flavonoid radicals (Du et al., 2008). Indeed, C3G, abundant in mulberries, has antioxidative ability 3.5 times stronger than Trolox, whereas pelargonidin, which was also found in mulberries, was reported to have the equal antioxidant effect to Trolox (Wang et al., 1997). In another study, anthocyanin (extracted from Morus alba fruit) eliminated excessive intracellular free radicals to ameliorate oxidative damage in HepG2 cells, regulated MAPKs, and nuclear factor-erythroid 2–related factor 2 signal pathways to prolong the life span of Caenorhabditis elegans (Yan et al., 2017). Moreover, MFPs also showed good in vitro antioxidant activity. The antioxidant activities of four polysaccharide fractions (MFP1, MFP-2, MFP-3, and MFP-4) from mulberry fruits were determined, and the result suggested that MFP-4, containing more galacturonic acid and a large part of low-molecular-weight fractions, had the greatest ability to scavenge 2,2-diphenyl-l-picrylhydrazyl (DPPH) and hydroxyl radicals (Chen et al., 2016). The carboxyl or carbonyl groups of MFPs may facilitate hydrogen atoms to bind to peroxy radicals and terminate the radical chain reactions. The selenide of the MFPs showed higher peroxy radical scavenging capacity than MFPs in vitro, which may be due to the activation of the hydrogen atom of the anomeric carbon by the selenyl or seleno acid ester groups (Chen et al., 2016).

1.3.3

ANTIHYPERGLYCEMIC ACTIVITY

Diabetes mellitus (DM), a chronic metabolic disorder characterized by hyperglycemia, has become the third most life-threatening disease worldwide (Wang et  al., 2013). Controlling blood sugar (glucose) levels is important. Related studies have shown that mulberry fruits have a good pharmacological effect of reducing blood sugar. Mulberry fruit anthocyanins (125–250 mg/kg) not only prevented the progressive declining of insulin secretion through protecting β cell but also enhanced

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Asian Berries: Health Benefits

hepatic/peripheral tissue glucose uptake, leading to lower the glucose blood levels of Zucker diabetic fatty rats (Sarikaphuti et al., 2013). Wang et al. (2013) studied the antihyperglycemic activity of mulberry fruit ethyl acetate extracts. In vitro, mulberry fruit ethyl acetate extracts showed potent α-glucosidase inhibitory activity, and DPPH free radical and superoxide anion free radical scavenging activity. In vivo, mulberry fruit ethyl acetate extracts significantly reduced fasting streptozotocin (STZ)-induced diabetic mice with blood glucose (FBG), glycated serum protein (GSP), and increased antioxidant enzyme activities (superoxide dismutase, catalase, and glutathione peroxidase). Mahmoud et al. (2014) used a mixture of black mulberry and white mulberry fruit powder to administer diabetic mice. Four weeks later, results showed that the hyperglycemia level of the mice was improved and the activity of α-amylase in the body was reduced. MFEs are rich in phenolics, and flavonoids appeared to be a potent inhibitor of α-glucosidase, which was confirmed recently by Xiao et al (2017). The oral administration of MFEs for 2 weeks significantly reduced fasting blood glucose and glycosylated serum protein in STZ-induced hyperglycemia mice. Polysaccharides obtained from mulberry fruits also have shown inhibitory effects on α-glucosidase, α-amylase, and the diffusion of glucose in vitro and exhibited antihyperglycemic activity in type 2 DM rats (Chen et al., 2015; Chen et al., 2016; Jiao et al., 2017). Using HepG2 cells as a model, the mechanism behind the hypoglycemic activity induced by mulberry fruit anthocyanin extracts was proposed. MAE may accelerate glycogen synthesis, promote gluconeogenesis, and ameliorate insulin resistance via the PI3K/Akt (phosphatidylinositol 3-k inase/protein kinase B) pathway. Furthermore, MAE improved glucose metabolic disorders in db/db mice by activating protein kinase B phosphorylation and its downstream targets in insulinsensitive tissues (Yan, Dai et al., 2016; Yan, Zhang et al., 2016). The hypoglycemic effects of MFEs containing high levels of anthocyanins were studied in diabetic C57BL/KsJ-db/db mice. The results revealed that MFEs can enhance insulin sensitivity, reduce hepatic glucose production, increase glucose transporter 4 (GLUT4) levels in skeletal muscle, and decrease glucose 6-phosphatase and phosphoenolpyruvate carboxykinase levels in the liver. These effects are due to increased phosphorylation of AMP-activated protein kinase and the 160-kDa Akt substrate (Choi et al., 2016).

1.3.4 HYPOLIPIDEMIC ACTIVITY The hypolipidemic activity of mulberry fruit freeze-dried powder was tested in hyperlipidemia Wistar rats that were induced using a high-fat diet. The powder effectively reduced serum triglyceride (TG), total cholesterol (TC), serum low-density lipoprotein (LDL) cholesterol, liver TG, liver TC, and atherogenic index but increased serum high-density lipoprotein cholesterol (Yang et al., 2010). Furthermore, the effect of mulberry fruit consumption on lipid profiles in hypercholesterolemic subjects (aged 30–60 years) was studied. The level of TC and LDL cholesterol significantly decreased compared with the control group, indicating that mulberry fruits could improve lipid profiles in hypercholesterolemic patients (Sirikanchanarod et  al., 2016). Chen et  al. (2005) used mulberry water extracts to administer stomach to reduce atherosclerosis by 42%– 63%, effectively alleviating hyperlipidemia. The hypolipidemic mechanism studies were carried out in hamsters with high-fat/cholesterol diets. Results indicated that mulberry fruit water extract treatment increased LDL receptor expression and uptake of LDL but decreased the expression of HMG-CoA reductase, fatty acid synthase, and glycerol-3-phosphate acyltransferase (Liu et al., 2009). Using HepG2 cells, Chang et al. (2013) investigated the protective effects of mulberry fruit anthocyanin extracts and their underlying mechanisms. Mulberry fruit anthocyanin extracts reduced lipid accumulation induced by oleic acid. Triglyceride synthesis–related proteins and cholesterol biosynthesis–related proteins were suppressed, whereas free fatty acid–related proteins were elevated, indicating that mulberry fruit anthocyanin extracts regulated lipid biosynthesis and lipolysis to exert hypolipidemic effects. There was also evidence to suggest that moracin C in mulberry fruit inhibits the proprotein convertase subtilisin–kexin type 9 (PCSK9) mRNA expression and thereby decreases degradation of LDL

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receptor, which could lower cholesterol levels (Pel et  al., 2017). Thus, in addition to these compounds, other bioactive compounds such as polysaccharides could also be investigated to elucidate the hypolipidemic mechanism of mulberry fruits for many polysaccharides from natural products showing obvious hypolipidemic effects (Liu et al., 2002; Zhao et al., 2012).

1.3.5

ANTICANCER ACTIVITY

Cancer is one of the major causes of death in animals specially felines and canines. Because of high incidence of cancer, many studies are currently being performed with the aim of finding better and safer therapeutic agents (Naderi et al., 2004). Over the years, it has been reported that both crude extracts and compounds obtained from mulberry fruits exhibited antitumor activity through different pathways, which has become one of the most important biological activities of mulberry fruits. Chen et  al. (2006) found that cyanidin 3-rutin and cyanidin 3-glucoside of mulberry fruits can activate c-jun and NF-κB, thereby inhibiting the migration of human lung cancer cells and invading other cells. MFEs induce cell proliferation by inducing apoptosis and causing death of human glioma cells (Jeong et al., 2010). Mulberry fruit anthocyanins inhibit the survival of gastric cancer cells and the formation of tumors by acting on the p38/p53 and c-jun signaling pathways and trigger the apoptosis of established gastric cancer cells, which ultimately leads to the death of gastric cancer cells (Huang et  al., 2011). A recent study investigated the synergistic effect of combined treatments with mulberry fruit water extracts and paclitaxel on human bladder cancer using TSGH 8301 cells and TSGH 8301 xenograft models. The paclitaxel extracted from mulberry fruits can arrest TSGH 8301 cells at the G2/M phase during the cell cycle, inducing mitotic catastrophe and inhibiting the generation of early endosomes, which may be associated with expression of PTEN. Furthermore, animal experiments suggested that the combined treatment groups showed reduced tumor volume through activation of PTEN and caspase 3 expression. The drug combination had a greater effect on cancer in almost all cases, than either drug alone. These results indicate that combinations of paclitaxel and MWE could provide a novel and effective therapeutic option in treating bladder cancer. Chang et al. (2015) also explored the synergistic antitumor effect of MFE and another drug 5-fluorouracil (5-FU) in mice transplanted with CT26 cells. Leukocyte counts, spleen weight, natural killer cells, and cytotoxic T lymphocyte activity in the tumor xenograft mice were significantly increased in the MFE + 5-FU group. The antitumor activity was supposed to be the result of the immunostimulatory effects. Angiogenesis, the formation of new blood vessels from preexisting ones, plays a crucial role in tumor progression (Folkman, 1971; Hanahan & Folkman, 1996), and antiangiogenic agents have been approved for the treatment of cancer through this mechanism (Carmeliet, 2005). Lee et al. (2016) reported that odisolane, a novel oxolane derivative (Figure  1.7) obtained from mulberry fruits, could significantly inhibit tube formation in human umbilical vein vascular endothelial cells (HUVECs). The molecular mechanism of their antiangiogenic effects is associated with inhibition of the expression of the vascular endothelial growth factor, p-Akt, and pERK protein in HUVECs. These results suggest that compounds isolated from mulberry fruits may be beneficial in antiangiogenesis therapy for cancer treatment. In the future, the mechanisms of antitumor activity of crude extracts and pure compounds from mulberry fruits need to be further elucidated.

FIGURE 1.7

Structure of odisolane isolated from mulberry fruits (Lee et al., 2016).

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1.3.6

Asian Berries: Health Benefits

NEUROPROTECTIVE ACTIVITY

Mulberry fruits can calm the nerves, delay aging, attending neurasthenia, insomnia, and forgetfulness. The protective effects of 70% ethanol extracts of mulberry fruits against neurotoxicity in in vitro and in vivo Parkinson’s disease models were examined. Results revealed that mulberry fruits significantly protected the cells from neurotoxicity in a dose- dependent manner (Kim et al., 2010). The beneficial effects of MFE rich in phenolics and anthocyanins were evaluated. Six-month- old senescent-accelerated SAMP8 and SAMR1 mice were fed with a basal diet supplement with 0.18% and 0.9% MFEs for 12 weeks. Mice that were fed with MFE supplement showed significantly less amyloid β-protein and showed improved learning and memory ability. Compared with control mice, MFE-t reated mice showed higher antioxidant enzyme activity and less lipid oxidation in both liver and brain (Zhang et al., 2008). Mulberry fruit anthocyanin-rich extracts neutralized the cytotoxicity and prevented Aβ25–35-induced PC12 cells by downregulating mRNA levels of Alzheimer’s-related genes, such as Apaf 1 and Bace 2 (Song et al., 2014). Neurodegeneration is mostly caused by free radicals production. Neurological disorders such as Parkinson’s and Alzheimer’s diseases have been due to the depletion of γ-a mino butyric acid in brain. The results of these studies suggested that the compounds isolated from mulberry fruits can be used as neuroprotective agents for the treatment of neurodegenerative diseases (Tian et al., 2005).

1.3.7

ANTIATHEROSCLEROSIS ACTIVITY

Atherosclerosis, a chronic inflammatory disease characterized by the accumulation of lipids in the arterial intima, is widely accepted as the main cause of cardiovascular diseases (Williams & Tabas, 2002). Oxidative LDL is an important atherogenic factor (Hertog et al., 1993). Consumption of a diet rich in natural antioxidants is associated with attenuation of the development of atherosclerosis (Kaliora et al., 2006; Gendron et al., 2010). Both mulberry fruit water extracts and mulberry fruit anthocyanin extracts exhibited antioxidation and antherosclerogensis abilities in vitro, could decrease macrophage death induced by oxidative LDL, and also inhibit the formation of foam cells, and anthocyanin components in MFEs could prevent atherosclerosis (Liu et al., 2008). Chen et al. (2005) reported that feeding 0.5% or 1.0% mulberry fruit water extracts (containing 2.5% anthocyanins and 4.6% total phenol) for 10 weeks significantly decreased plasma triglyceride levels in the cholesterol-fed rabbits. Atherosclerotic lesion was significantly reduced by 42%–63% in the aorta from rabbits fed with 0.5% or 1.0% of mulberry water extracts compared with the control. Histological analysis revealed that mulberry fruit water extracts reduced the formation of foam cell and the migration of smooth muscle cells in blood vessel of rabbits. Chan et al. (2015) investigated the antiatherosclerosis activity of mulberry fruit polyphenol extracts and its underlying mechanism of action in vascular smooth muscle cells. They found that mulberry fruit polyphenol extracts could arrest the A7r5 rat thoracic aorta smooth muscle cell cycle at the G0/G1 phase through induction of NO production and AMPK/p53 activation. The major active compounds of mulberry fruit polyphenol extracts were rutin, protocatechuic acid, and other polyphenols, such as caffeic acid and naringenin.

1.4 SUMMARY Mulberry fruits have a variety of functional active ingredients and high nutritional value. As a kind of medicine and food plant, they have high medicinal and edible values. They are a highquality raw material for developing functional foods and medicines, and they are well known across the world. With the continuous advancement of scientific research techniques, researches on the active components of mulberry fruits have also made great progress, identifying the chemical structure, content, and pharmacological activity of various components of mulberry fruits. This chapter

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describes active ingredients contained in mulberry fruits and their functions. Active ingredients such as polysaccharides, polyphenolics, flavonoids, anthocyanin, phenolic acids, and melatonin, which are rich in mulberry fruits, have immunomodulatory, antioxidant, antihyperglycemic, hypolipidemic, anticancer, neuroprotective, and antiatherosclerosis activities. The anticancer and neuroprotective activities indicate that the developments of mulberry fruits’ health products have great potential. However, the lack of quality standards has seriously affected the quality of their products. Moreover, the research on the functional components of mulberry fruits is still in the laboratory stage. In addition, the use of mulberry resources is relatively simple, which also led to a large amount of mulberry resources waste. The quality standards of mulberry products should be formulated as soon as possible to regulate the mulberry product market, increase the research on mulberry active components, integrate them with industrialization, strengthen the utilization of mulberry resources, and make it more reasonable, more standardized, and more effectively develop and utilize Chinese mulberry resources.

ACKNOWLEDGMENTS We acknowledge the China Agriculture Research System (CARS-18-ZJ0506) and the Innovation Team of Modern Agricultural Industry Technology System in Guangdong Province of China (2019KJ116) for financial support.

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2

Nutritional Value and Processing Technology of Mulberry Fruit Products Lan Ma, Gengsheng Xiao, Jijun Wu, Jing Wen, Zhibin Bu, and Daobang Tang Guangdong Academy of Agricultural Science

CONTENTS 2.1 2.2 2.3

Mulberry Fruits....................................................................................................................... 21 Chemical Properties and Nutrient Values .............................................................................. 23 Processing of Mulberry fruits.................................................................................................24 2.3.1 Mulberry Fruit Juice and Mulberry Candy ................................................................24 2.3.1.1 Mulberry Fruit Juice ....................................................................................24 2.3.1.2 Mulberry Candy ........................................................................................... 27 2.3.2 Mulberry Jam and Mulberry Jelly ..............................................................................28 2.3.3 Mulberry Wine ........................................................................................................... 30 2.3.4 Mulberry Fruit Powder and Dried Mulberries ........................................................... 32 2.3.5 Mulberry Yogurt ......................................................................................................... 35 2.3.6 Mulberry Vinegar ....................................................................................................... 36 2.3.7 Utilization and Development Prospect ....................................................................... 36 2.4 Summary ................................................................................................................................ 37 Acknowledgments............................................................................................................................ 37 References ........................................................................................................................................ 37

2.1 MULBERRY FRUITS Mulberries belong to the Moraceae family and are also called Fructus Mori and black scorpion. They are a fast-growing deciduous plant found in a wide range of climatic, topographical, and soil conditions. It is also widely distributed from temperate to subtropical regions of the northern hemisphere to the tropics of the southern hemisphere. As the ancient Chinese Material Medica describes many medicinal benefits of mulberry, the potential nutritional and medicinal values of mulberry have attracted increasing research interest (Wen et al., 2019). Currently, there are more than 3,000 species resources in the world. To date, this genus has 24 species and 100 varieties (Ercisli and Orhan, 2007). Areas cultivated with mulberry are shown in Table 2.1 (Huo, 2002; Singhal et al., 2010). Mulberry fruits are mainly distributed in the Pearl River Basin, the middle and lower regions of the Yangtze River, and the lower stream region of the Yellow River, Sichuan, and Xinjiang Province in China. Mulberry fruits are perennial deciduous arbors, which are cultivated by the monosexual mulberry pollination. Fruits are of an elliptical cylindrical shape with a length of 1–3 cm. The surfaces contain many small achenes. Figure 2.1 shows the fresh mulberry fruits. The flowering period is generally 4–5 months, and the fruiting period is 6–7 months (Baynes, 1991). Usually, the harvesting 21

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Asian Berries: Health Benefits

TABLE 2.1 Areas Cultivated with Mulberries (Huo, 2000; Singhal et al., 2010) Main Regions for Mulberries Xinjiang Uygur Autonomous Region, China Shandong and Hebei Provinces, China Shanxi and Shananxi Provinces, China Zhejiang and Jiangsu Provinces, China Anhui, Hubei, and Hunan Provinces, China Sichuan Province, China Guangdong and Guangxi Provinces, China Yunnan and Guizhou Provinces, China Geneva, Japan North America, USA South America, USA USA, India USA California, Arizona and Texas, USA Kew Korean mulberry Tohoku district From Kyushu to Tohoku district Okinawa islands: Morus kagayamae Koidz and Metrosideros boninensis Koidz India

Himalayas (India) Karnataka Andhra Pradesh Tamil Nadu South India Irrigated KSSRDI, Thalaghattapura South India Rained Eastern and NE India Irrigated Hills of Eastern India CSRTI, Pampore RSRS, Jammu Spain Italy Taiwan Carbacena county, Minas Gerais state Rio de Janeiro state

Varieties He tian Bai Sang Da Ji Guan, Hei Lu Cai Sang, Xuan 792, and Niu Gen Sang Hei Ge Lu Tong Xiang Qing, Hong Cang Sang, Hu Sang 1917, Hu Sang 199, Huo Sang, Nong Sang 8, Yu 2, and Zhong Sang 5801 Hong Pi Wa Sang Hei You Sang, Da Hua Sang, Xiao Guan Sang, and Jia Ling 16 Guangdong Jing Sang, Lun 40, Lun 109, Sha 2, Da 10, and Kang Qing 10 Yun Sang 2 and Dao Zhen Sang Wellington (Morus nigra), Stubbs (Morus rubra) Thorburn and Trowbridge (Morus alba), New American (M. alba) English Black (M. nigra) Downing (M. alba) Beautiful Day (M. alba) Black Persian (M. nigra) Pendulum (M. alba) Morus australis Morus bombycis Koidz Meryta latifolia Morus acidosa Griff Mikania laevigata, MI-0118, MI-0171, MI-0249, MI-0300, MI-0497, MI-0512, MI-0059, MI-0506, MI-0380, MI-0572, ME-0004, ME-0042, Tr-10, MS-9404, S-146, S-36, S-54, Victoria-1.S-7999, S-1635, BC-259 Morus indica, M. laevigata Morus multicaulis M. nigra Miscanthus sinensis Kanva-2 DD S-13, S-34, MR-2 S-1 Tr-10 Goshoerami, China White Chak Majra Branca da Espanha Rosa, Contadini, Calabresa Formosa Siciliana Serra-das-Araras

period is from April to June. As the fruits grow and develop, the colors of the mulberry fruits turn from green to red and then purple (Gerasopoulos and Stavroulakis, 2015). Mature mulberry fruits are fleshy, full of serum, and high in sugar contents. The taste is slightly sour and sweet. The fragrance is pleasant. Among the various types of mulberry fruits, black fruits are considered to have high medicinal value in the folk and some pharmacopoeia, which may be related to their unique active compounds (Calín-Sánchez et al., 2013).

Nutritional Value and Processing Technology

FIGURE 2.1

2.2

23

The picture of fresh mulberry fruits (a) and (b).

CHEMICAL PROPERTIES AND NUTRIENT VALUES

Mulberry fruits are rich in nutrients with complicated chemical compositions. The contents of pulp, peel, seeds, and fruit stalks in mulberry fruits are 71.4%, 23.5%, 2.9%, and 2.2%, respectively (Yang et al., 2006). The fresh mulberry fruits contain 1.42–2.13 g/100 g nitrogen, 0.21–0.31 g/100 g phosphorus, 1.48–2.17 g/100 g kalium, 0.19– 0.43 g/100 g calcium, 0.12– 0.19 g/100 g magnesium, and 0.07–0.11 g/100 g sulfur. The contents of protein are 8.9%–13.33% DW (dry weight) (SánchezSalcedo et al., 2015). The dried mulberry fruits contain 77%–87% organic matter, 76%–84% crude protein, 80%–89% crude fiber, and 52%–63% ash (Kandylis et al., 2009). Amino acid compounds ingredients are currently the hot topic for the study of mulberry nutrients (Eun et al., 2012; SánchezSalcedo et al., 2015). Research on mulberry fruits showed that the essential amino acid contents of fresh mulberries were 64 mg/100 g, the total amino acid contents were 293 mg/100 g, and the essen­ tial amino acids accounted for 22% of the total amino acid content (Wu and Weng, 2005). There are a total of 19 types of free amino acids, and 7 other amino acids except tryptophan are essential amino acids. Xiao et al. (2001) studied two kinds of semiessential amino acids and eight essential amino acids in the fresh mulberry.

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Asian Berries: Health Benefits

Ercilisli et al. (2007) showed that the total fatty acids of mulberry fruits were around 1%. Among them, unsaturated fatty acids accounted for 76%, and the highest linoleic acids were around for 75%. They also contained linolenic acid, palmitic acid, oleic acid, small amounts of tannic acid, caprylic acid, and citric acid, and so on. Phospholipids are mainly bisphosphophenol glycerol, lysophos­ phatidic acid choline, phospholipid choline, phospholipid creatinine, phospholipid oxime ethanol­ amine, and phosphatidic acid (Ercilisli et al., 2007). The properties of leaves and fruits of white mulberries are used more and more com­ monly for production of foods with beneficial effects on human health. Moreover, nutritional effects of mulberry fruits would improve health state and well-being and reduce disease risk after mulberry fruits are incorporated in dietary supplement as functional food productions ( Łochyńska, 2015). There are more dietary products containing extracts or dried mulberry fruits. Polyphenol-rich chocolate, probiotic and prebiotic chocolate, or chocolate with mulberry fruit extracts or anthocyanin extracts was produced (Lin and Tang, 2007). Dried material is also used in Indian cooking for baking bread of wheat flour called “paratha.” In Thailand, pow­ dered white mulberry fruits are added to muesli in order to increase the antioxidant capacity of products too (Joanna et al., 2013). New functional yogurts mixed with mulberry jam were also produced (Lee and Hong, 2010). Mulberry fruits are also used for preparation of jams, ice creams, vinegars, juices, wine, and cosmetic products (Nati et al., 2015). In comparison with grape wine, mulberry wine has higher antioxidant potential and better phenolic profile (Celep et al., 2015). Mulberry fruits in China are matured usually in less than 1 month. Mulberry fruits are difficult to preserve because they have high water content (Yang et al., 2010). According to traditional Chinese medicine, mulberry fruits are used to improve eyesight and protect against liver damage (Yang et al., 2010). Mulberry fruits have been used in traditional Oriental medicine to treat diabetes and premature white hair. The mulberry leaves are used to feed silkworms (Arabshahi-Delouee and Urooj, 2007; Sohn et al., 2009).

2.3

PROCESSING OF MULBERRY FRUITS

China has been a major country for mulberry cultivation since ancient times, and it has the biggest yields of mulberry fruits in the world. The current annual productions from Sichuan Nanchong, Xichong, and other provinces would yield of million mulberry fresh fruits and are used for the food processing industry. Because their maturity is in the April–June period before the rainy season, it is difficult to store mulberry fruits at room temperature with the normal transportation methods due to short harvest period in rainy seasons. Therefore, special attention should be paid to the processing of mulberry production (Tong et al., 2006). In recent years, with the improvement of fruit storage methods and the people’s demand for new natural products, a series of processed products of mulberry fruits have come to the market, such as mulberry juice, mulberry wine, mulberry fruit powder, mulberry fruit yogurt, mulberry fruit vinegar, and mulberry fruit jam (Yan et al., 2017).

2.3.1

MULBERRY FRUIT JUICE AND MULBERRY CANDY

2.3.1.1 Mulberry Fruit Juice Mulberry fruits are traditional Chinese edible fruits that are used effectively in folk medicines to treat fever, protect liver from damage, strengthen the joints, facilitate discharge of urine, and lower blood pressure (Singhal and Khan, 2010). Nowadays, mulberry fruits are used for soft drink market. Studies showed that mulberry fruits had significant effects in antioxidation, reducing low­ density lipoprotein level, delaying aging, and beautifying skin. In some European countries, mul­ berry alba and other mulberries are grown for fruit productions that have certain applications in

Nutritional Value and Processing Technology

FIGURE 2.2

25

The picture of mulberry fruit juice. (https://shop.smartjuice.us/ black-mulberry-cranberry.)

some traditional food stuffs. The mulberry fruits are often harvested by spreading a sheet on the ground and shaking the branches. In some of the genotypes, the fruits are so ripe that just picking them breaks the fragile skin of the fruit and stains the fingers purple with juice. At present, various mulberry fruits are made for fruit juice (Arfan et al., 2012). The picture of mulberry fruit juice is shown in Figure 2.2. Fresh mulberry fruits are more suitable for processing into juice products with high juice yields. The mulberry fruit juice on the market can be divided into two types: mulberry juice and blended juice. The mulberry fruit juice is directly squeezed from fresh mulberry fruits or from rewatered dried fruits. The taste of juice is relatively light, and the price is very high. The mul­ berry blended juice is made by adding other fruit juice, sweeteners, flavors, and other ingredients. It is currently only available in a few areas in China (Yan, 2017). A new UK fruit juice company “Fairjuice” has launched a super fruit drink prepared from pure fresh mulberry fruits, which is full of antioxidants. Mulberry fruits also contain much resveratrol that can be considered to be benefi­ cial for heart health. Mulberry fruit beverages have been reported as a useful beverage against obe­ sity (Duyen et al., 2008). The mulberry cultivars such as Tr-10, Chinese white, Da Shi, MS-9404, Bai Yu, S-146, and Mandalaya can provide lots of fleshy dark purple color sweet fruits from March to April. The highest fruit yield of 11 kg per tree is from the cultivar MS-9404. Fruit characteristics of different mulberry cultivars under subtropical conditions of Asia are listed in Table 2.2 (Singhal et al., 2009). 8.82 L of fruit juice per tree has been produced. Juice taste is very sweet with a little sour. But in the field, the cultivar S-146 is more common for sericulture activities, where one tree provides about 1.847 L of dark purple juice of sweet and sour taste. The juice is found suitable for food industry to prepare fruit jam, pulp, and other horticulture products (Singhal et al., 2009). Process of mulberry juice follows the steps such as selection of the fruits with the variation of the dark purple mature mulberry fruits as the raw material. Fruits are disinfected with the tap water in disinfection equipment such as dual-nuclear ozone sterilization equipment and then soaked in water for a period of time. After the ozone gas is exhausted, the mulberry fruits are washed with the tap water to remove the mud and other debris on the surface of the mulberry fruits. The mulberry fruits contain a certain amount of pectin, which will affect the juice yield when mulberry fruits are processed. At present, enzymatic hydrolysis is commonly used to increase the juice yield of mulberry fruits. Xu et al. (2015) optimized the process conditions for extracting mulberry juice by enzymatic hydrolysis. The results showed that the optimal enzymatic extraction conditions of mulberry juice were shown promising. The amount of pectin added was 0.04%, the enzymatic hydrolysis temperature was 50˚C, the enzymatic hydrolysis time was 90 min, and the

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Asian Berries: Health Benefits

TABLE 2.2 Fruit Characteristics of Different Mulberry Cultivars under Subtropical Conditions of Asia (Singhal et al., 2009) Characteristics of Mulberry fruits Types of Mulberry Fruits

Length of the Fruits (cm)

The Number of Fruits per Tree

The Yield of Fruits per Tree (kg)

The Content of Fruit Juice Per Tree (L)

S-146 Da Shi Tr-10

0.96 4.35 1.02

3,303 1,126 5,736

3.294 5 7.352

1.847 2.5 4.267

Bai Yu MS-9404 Mandalaya

2.5 1.24 0.95

4,500 5,902 2,172

3.33 11.021 1.983

2.331 8.82 1.473

Taste of Mulberry Fruits Highly sweet to a little sour Sweet Moderately sweet to a little sour Sweet with a little sour Highly sweet Moderately sweet to a little sour

The Color of Mulberry Fruits Purple black Purple black Purple black White Pinkish red Purple

juice yield of mulberry fruits reached 73.4%. Xu et al. (2015) found that with 0.04% pectinase treat­ ment at pH 3.5 and 45˚C for 3 h, the juice yield of mulberry increased from 52.8% to 70%. Xu (2015) and other experiments also showed that the effects of pectinase and cellulase alone were effective, and the juice yield could reach 75.21% and 74.55%, respectively. The effects of complex enzymatic hydrolysis can have better results. The mixture of pectinase and cellulase (1.0:1.36) was used for enzymatic hydrolysis at pH 5.0 and 49.5˚C for 2.6 h. The juice yield of mulberry fruits was 86.13%, which was 10.92% higher than single enzyme treatment (Xu, 2015). In addition, the mulberry fruit juice and other juices are mixed to enrich the flavor and nutrition of the mulberry juice. Chen et al. (2014) studied the production process of the new blueberry and mulberry mixture by blending and determined the optimal processing conditions. In enzymatic hydrolysis with pectinase, the enzyme dosage was 0.3%, the enzymatic hydrolysis time was 5 h, and the enzymatic hydrolysis temperature was 40˚C. A kind of blended beverage consists of 140 mL blueberry juice, 8 g sugar, 40 mL mulberry juice, 10 mL rose juice, 2.33% vitamin C, 0.13% citric acid, and 0.02% potassium sorbate. Such blueberry and mulberry combined drink is fresh, elegant, sweet, sour, and nutritious. Fazaeli et al. (2013) studied the processing technology of a mixed and flavored juice by using mulberry leaves and mulberry fruits. The mulberry fruit beverage is sweet and sour, with pure flavor and purple color. It also has the unique aroma of mulberry fruits and mulberry leaves. In addition to the use of processed mulberry fruit alone, the mulberry fruit juice can be mixed with other juices to obtain a new juice product with unique flavor and more compre­ hensive nutrition. Cao (2015) mixed 20% of mulberry fruit juice with 20% of sweet orange juice. The product has a purple red color and a sweet and sour taste. The steps followed in the production of mulberry juice are shown in Figure  2.3. The major steps involved in the processing of black mulberry fruits into juice include selection of black mul­ berry fruits, washing, mechanical milling into pomace, mashing (without enzymes, 45˚C–50˚C, 30 min), cold pressing, pasteurization (107˚C, 3 min), and filling–packing (27˚C). Anthocyanins (cyanidin-3-glucoside and cyanidin-3-rutinoside), phenolic acids (3-caffeoylquinic acid), and fla­ vonols (rutin, quercetin-3-glucoside, and quercetin-malonyl-glucoside) were identified using liquid chromatography– quantitative time-of-flight–mass spectrometry and quantified using highperformance liquid chromatography (HPLC). Approximately, 60%–70% of the fruit anthocyanins were retained in the final juice, which also contained high levels of caffeoylquinic acids from the fruits. The mashing and pressing processes were effective for the recovery of fruit polyphenolics into the juice fraction. Moreover, an in vitro gastrointestinal digestion model, applied to determine

Nutritional Value and Processing Technology

FIGURE 2.3

27

The process of producing mulberry fruit juice.

the effect of processing on the bioavailability of mulberry antioxidants, indicated a higher anthocy­ anin bioavailability for the fruit matrix than for the juice matrix (Tomas et al., 2015). For heat pretreatment of mulberries, superheating phenomenon will happen. During microwave heating and concentration of juice, the changes of color and anthocyanin contents were critical issues. Results showed that the degradation of color and anthocyanins was more pronounced with rotary evaporation compared with microwave heating method (Min et al., 2006). The study on the color change of black mulberry fruit juice during concentration with both microwave and conven­ tional methods showed that all the Hunter color parameters (L, a, and BI) changed significantly. There were obvious changes in the Hunter L and a values, confirming the degradation of visual color components of the juice samples. The values of Hunter L decreased in all cases during con­ centration, which were affected by the operational pressure, especially in the case of conventional heating. The results showed that higher pressure led to a longer process time and lower L values. Concentration of mulberry juices heated by microwave power led to a decrease in the L values. The effects of sugar and the degradation products include the acceleration of anthocyanins breakdown and enhancement of nonenzymatic browning reactions during thermal concentration (Cemeroglu et al., 2006; Suhl et al., 2003). 2.3.1.2 Mulberry Candy Mulberry candy is a kind of traditional ethnic foods in Asian countries. Figure 2.4 shows the pic­ ture of mulberry strips candy. Its unique flavor makes it become a popular snack food today. The processing steps of mulberry fruits include selecting, washing, protecting, hardening, sugaring, drying, finishing fruits, and packaging, as shown in Figure  2.5. Du et  al. (2019) used mulberry fruits as raw materials to process low-sugar mulberry fruit candy and focused on the effects of color-protecting “hardening” sugar process on the quality of low-sugar candied mulberry fruits. The preferred process conditions included vacuum 0.06 MPa, sugar liquid temperature was 70˚C, and the vacuum time was 80 min (Xiao and Li, 2006). Jiang et al. (2013) studied the production of mulberry fruit candy. The results showed that the color was protected by 0.8% citric acid solution for 2 h, 1% calcium chloride solution for 6 h, and then 40% sugar solution for 24 h, and the candy was finally cooked for 2 min. The sugar solution was infiltrated for 6 h and dried at 50˚C–55˚C for 10–12 h (Jiang et al., 2013). Budiman et al. (2019) evaluated the antibacterial activity of black mulberry extracts (Morus nigra L.) in a chewing candy preparation against Streptococcus mutans (S. mutans) and Streptococcus sanguis (S. sanguis). Among them, S. mutans was one of the most common streptococci in the oral flora and was one of the main components of dental plaque. S. sanguis was one of the normal oral bacterial groups. It maintained its stability through hydrophobic interactions and could react with a variety of bacteria. It played an important role in the formation of plaque on the gums. The antibacterial activity of the extracts was determined by using diffusion method. The extract dose was determined from minimum inhibition concentration (MIC) and minimum bactericidal con­ centration (MBC) valued using the microdilution method. The extracts were formulated into three variations of the glucose–sucrose base: F1 (43.5%:8.7%), F2 (34.78%:8.7%), and F3 (26%:26%). The chewing candy of black mulberry extracts was evaluated physically using organoleptic test, prefer­ ence test, and antibacterial activity test. Results show it can resist S. mutans and S. sanguis. The results showed that black mulberry extracts had antibacterial activity with MIC 0.3125% and MBC

28

Asian Berries: Health Benefits

FIGURE 2.4

The picture of mulberry fruit strips. http://www.suning.com/ bigimages/10549919777.html.)

FIGURE 2.5

The process of producing mulberry fruit candy.

0.625% against S. mutans and S. sanguis. The best formulation of chewing candy consisted of 26% w/w of sucrose, 26% w/w of glucose, and 0.625% w/w of black mulberry extracts. The chewing candy from black mulberry fruit extracts had antibacterial activity with an 8.2-mm inhibition zone against S. mutans and one of 10.8 mm against S. sanguis. The chewing candy consisted of 26% w/w of sucrose, 26% w/w of glucose, and 0.625% w/w of black mulberry extracts, which had antibacte­ rial activity against S. mutans and S. sanguis (Budiman et al., 2019).

2.3.2

MULBERRY JAM AND MULBERRY JELLY

Both mulberry jam (Figure 2.6) and mulberry jelly (Figure 2.7) are processed with raw mulberry juice. As a kind of traditional product, jam is a type of fruit processing product with rich nutrition, unique flavor, and convenient consumption. Jam and syrup are often made from fruits in Persian­ derived names, i.e., toot (mulberry) or shahtoot (King’s or “superior” mulberry) (Singhal et al., 2009). It is one of the main processed products in fruit processing industry in Persian area (Tomas et al., 2016). Mulberry jam is a considerable contribution to the Indian economy; about 1,993 kg of fruit jam and 2,794 L of fruit pulp can be produced in subtropical India. The steps involved in the processing

Nutritional Value and Processing Technology

29

FIGURE 2.6 The picture of mulberry fruit jam.

FIGURE 2.7

The picture of mulberry fruit jelly. (https://www.jiankang.com/yinshi/c29118/.)

procedure of mulberry jam include washing fresh mulberry fruits into puree, adding glucose–fructose syrup and water, cooking, adding citric acid and apple pectin, and finally pasteurizing (Tomas et al., 2016). One tree can provide about 1.847 L juice of dark purple color with sweet–sour taste. The juice is suitable for industrial production of preparation of fruit jam (Singhal et al., 2009). Black mulberry jam processing was performed by following standard commercial procedures in a fruit jam factory (Tomas et al., 2017), as shown in Figure 2.8. The steps included selecting frozen black mulberries (12°Brix), adding glucose–fructose syrup (approximately two times the amount of fruits, 68°Brix) and water, cooking (performed at 550 mm Hg and 78˚C until the dry matter content reaches 71°Brix), adjusting the pH through the addition of citric acid and apple pectin, removing seeds, and finally pasteurizing at 95˚C for 25 min. During cooking, the seeds collected are removed at the top of the jam mixture. The sample collections and processing are shown as follows: (1) add­ ing glucose–fructose syrup and water, (2) cooking, (3) adding citric acid and pectin, (4) removing the seeds, and (5) pasteurized jam (final sample).

30

Asian Berries: Health Benefits

FIGURE 2.8 The process of producing mulberry fruit jam.

Jelly has crystal-clear appearance and soft and smooth taste. It is a popular snack food among consumers, especially the youth group. Zhou and Yu (2012) used mulberries, hawthorns, honey­ suckles, and wild chrysanthemums as the main raw materials for a mixed fruit jelly. The processing conditions and formula ratio were selected to develop the child-health mulberry jelly. The product formula was 50% mulberry juice, 10% honeysuckle and wild chrysanthemum extracts, 15% haw­ thorn juice, sugar and acid ratio at 100:1, and 0.5% stabilizer. The child-health mulberry jelly had both edible values and health-promoting effects such as appetizing healthcare (Zhou and Yu, 2012). The jelly products have good sensory properties, with certain nutritional values and healthcare functions (Yue et al., 2009). Kim and Cho (2012) developed jelly using mulberry fruits that were compatible with Korean tastes as health functional food by grafting the method to manufacture jelly consumed as a dessert or a kind of snack in the west. According to the central composited design, mulberry fruit jelly was produced by varying the content of citric acid, sucrose, and gelatin. As the sensory preference of mulberry jelly, except the flavor, the remaining hardness, elasticity, sweet­ ness, color, and the overall quality were also significant. It was also found to be influenced greatly by gelatin content. The optimization point was found to be 6.2 g citric acid, 141.0 g sugar, and 13.5 g gelatin (Kim and Cho, 2012).

2.3.3

MULBERRY WINE

The anthocyanins contained in mulberry wine are five times higher than red grape wine, and the content of resveratrol is 1.38 μg/mL. Regular drinking of mulberry wine can enhance physical fit­ ness and immunity. The picture of mulberry wine is shown in Figure 2.9. Using mulberry fruits as a raw material, it not only can produce mulberry wine with good taste, color, and flavor but also can be mixed with kiwi and wormwood to process various wines. The brewing process of mulberry wine is mainly divided into three parts: raw fruit juice, fermentation process, and posttreatment process. Raw fruit processing includes mulberry fresh fruit sorting, crushing, enzymatic hydro­ lysis, pressing, filtration, and adjustment. The fermentation process shown in Figure 2.10 includes Saccharomyces cerevisiae activation, cultivation, inoculation, fermentation, pouring, and postfer­ mentation. The posttreatment process includes clarification, filtration, aging, and blending steps. Among them, the raw materials, the brewing yeast, the control of the fermentation process, the clarification of the wine, and the analysis of the finished wine are important procedures in the mul­ berry wine brewing process. In the mulberry wine brewing process, the sugar in the mulberry fruits is mainly converted into alcohol and other products by using the brewing yeast. For example, most of Chinese mulberry wine brewing yeasts use active dry brew yeast of Angel Company (Angel Yeast Co., Ltd, China). There are few studies on the special yeast of mulberry wine. Chen et al. (2014) used the mature Wulingshan mulberry fruits as a raw material for fermenting it naturally and then screened four strains of yeast from the fermentation broth, which were identified as winemaking reagents. Yeast (S. cerevisiae) has a fast leaven and a pronounced wine aroma. Cao et al. (2015) used natural mulberry as a raw material

Nutritional Value and Processing Technology

31

FIGURE 2.9 The picture of mulberry fruit wine.

FIGURE 2.10 The process of producing mulberry fruit wine.

to ferment it naturally and then isolated 42 strains of yeast from the fermentation mashes. It was first screened by Du’s small tube fermentation method and then rescreened with alcohol-producing ability and finally with SO2 resistance. The odor activity value (OAV) was subjected to a three-stage screen­ ing to finally obtain the optimal strain JNB-14. It was identified by the 18S rDNA sequence to have a 100% match with a strain of S. cerevisiae (sequence ID: AFD50639.1). In the fermentation process, mulberry wine processing contains several steps as follows, adjustment of sugar, selection of yeast and inoculum, adjustment of acidity, control of fermentation temperature, and termination of fer­ mentation time. When studying the fermentation process of mulberry wine, Huo (2002) chose high­ quality mature Guangdong Yunfu mulberry fruits as a raw material to produce wine. Firstly, 70 mg/ L pectinase was added after mulberry crushing, and enzymatic hydrolysis was carried out at 25˚C for 10 h. Then, 600 mg/ L M06 yeast was added in mulberry juice to be fermented at 20˚C for 48 h. The final alcohol content reached 12% v/v. The final mulberry wine was moderately sour, purple-red, and clarified. In the mulberry wine process, Zeng et al. (2007) evaluated yeast GABA expression ability by HPLC and found that S. cerevisiae JM037 has a strong ability to produce GABA by fermentation, reaching 670 mg/ L. Further analysis of the tolerance and mulberry fermentation performance based on S. cerevisiae JM037. The comprehensive tolerance of alcohol, SO2, glucose, and pH is found to be good. After fermenting mulberry juice, the ethanol output reaches 38.36 g/ L at 168 h, the mass concentration of residual sugar drops to 3.06 g/ L at 216 h, and the mass concentration of GABA increases to 1145 mg/ L at 144 h. Taking Angel yeast as a reference, there is little difference in ethanol

32

Asian Berries: Health Benefits

production and residual sugar concentration, but S. cerevisiae JM037 has a unique ability to produce GABA. Therefore, S. cerevisiae JM037 has the potential to brew GABA-rich mulberry wine. The mulberry wine obtained under those conditions had the unique fruity aroma of mulberry fruits with pure flavor and full mouth feel. Yang (2017) optimized the fermentation process of mulberry wine by designing response surface method to select sucrose addition amount, fermentation temperature, and inoculum. The total acid, total residual sugar, ethanol, total flavonoids, and sensory comprehen­ sive were scored of mulberry wine. The optimal fermentation process parameters obtained by the design expert software were sucrose addition of 12.80 g/100 g, fermentation temperature of 15.3˚C, and active dry yeast inoculum of 8.00 g/ L. Liang and Bao (2018) optimized the fermentation process of mulberry wine, taking mulberry as a raw material and using active dry yeast SY as the fermenta­ tion strain. The optimal fermentation conditions were selected as the active dry yeast inoculum ratio of 0.2%, initial sugar content of 50%, the initial juice fermentation temperature of 26˚C, and the fermentation period of 7 days. Under this condition, the mulberry wine had an alcohol content of 9.3% v/v and pH value of 3.2. The wine was clear and translucent; the color was pure; the taste was mellow, sweet, and sour; and the wine flavor was fruity. Yang et al. (2017) used mulberry fruits as a raw material to optimize the fermentation process parameters of mulberry wine. Mature mulber­ ries from Anhui Yinzhou production area were used as raw materials. The results showed that the inoculation amount of yeast seed solution was 0.6%, the initial pH was 3.6, the initial sugar content was 22.6%, the main fermentation temperature was 15˚C, the wine was clarified and translucent, the taste was mellow and long, and the aroma was appropriate. Ye et al. (2018) used the F15 yeast as the strain and selected the fermentation temperature of 22˚C. The initial sugar content was 21%, the active dry yeast inoculation amount was 0.01%, and the main fermentation time was 4 days. Under these conditions, the prepared mulberry wine had a typical mulberry fruit aroma and wine scent, and the wine was clear, dark red, shiny, soft, and full bodied. After the fermentation, the mulberry wine contained a variety of substances, such as yeast, gum, protein, tannin, cellulose, hemicellulose, and berry tissue fragments. The precipitate would make the wine turbid, which affected the clarity and color of the finished wine. In addition, when the contents of certain metal ions (Fe2+, Cu2+) in the wine were too high, it might cause turbidity. In order to ensure the clarification and transparency of the mulberry wine to stabilize the wine, to improve the quality of the wine, and to prevent the turbidity of the wine, it is necessary to clarify the original wine. Wang et al. (2009) used gelatin (0.05 g/L), egg white protein (0.04 g/L), polysaccharide (0.03 g/L), polyvinylpyrrolidone (0.1 g/L), and bentonite (0.3 g/L). Five single clarifiers mixed with three groups composite clarifying agents for clarification controls experiments. The three groups are polyvinylpyrrolidone (0.05 g/L) and chitosan (0.02 g/L), gelatin (0.03 g/L) and bentonite (0.15 g/L), egg white protein (0.02 g/L) and bentonite (0.15 g/L). The results indicated that among the five single clarifying agents, the combination of polyvinylpyrrol­ idone (0.05 g/L) and chitosan (0.02 g/L) showed the best clarification effect. Zhou et al. (2017) used clarification treatment of mulberry wine with low-temperature freezing and three kinds of clarifying agents of bentonite, gelatin, and pectinase to detect light transmittance and sensory evaluation. The results showed that gelatin (1.25 g/L) had the best effect on the clarification treatment of mulberry wine, and its liquid transmittance was up to 71% and had no effect on the aroma, taste, and color of the liquor. Zhou et al. (2017) selected six kinds of single clarifying agents such as diatomaceous earth, chitosan, tannin, gelatin, and egg white, as well as diatomaceous earth, gelatin, and egg white mixtures clarifying agents for clarification of wine. The results showed that diatomaceous earth (0.6 g/L) and egg white (0.3 g/L) composite clarifying agent had the best effect. The light transmit­ tance was 80.1%. The clarified mulberry wine was light purple red, and the stability was good (Zhou et al., 2017).

2.3.4

MULBERRY FRUIT POWDER AND DRIED MULBERRIES

Mulberry powder is a natural dried product. It completely maintains the unique nutritional value, color, and flavor of mulberry fruits. It is widely used in supplements and health products,

Nutritional Value and Processing Technology

FIGURE 2.11

33

The picture of mulberry fruit powder.

confectionery, cakes, yogurt, and other products. Mulberry fruit powder mainly includes fruit pow­ der and fruit juice powder. Mulberry fruit powder is shown in Figure 2.11. The color of mulberry fruit powder is purple-red, similar to the color of fresh fruits. The mulberry juice powder is of lavender color, which is lighter than that of fresh fruits. Texture is fine and uniform. The mulberry juice powder is used to make fruit powder capsules (Yu, 2014). Mulberry fruits can be dried and stored as a powder. Figure 2.12 shows a kind of dried mul­ berry. About 10 g of dried fruits can provide about 100 mg of anthocyanins. It contains resveratrol; fruit powder can work as an antimutagen which can inhibit the mutation of healthy normal cells into cancerous cells (Hou, 2003). It is believed to prevent heart disease, cancer, and other diseases associated with chronic inflammation. The mulberry fruit powder has an antiaging effect on cells because it combats free radical damage. Fruit powder promotes healthy cholesterol and controls carbohydrate digestion in the human body (Liu et al., 2009). Currently, the drying methods used in fruit powder processing mainly include spray drying, hot air drying, vacuum freeze drying, microwave drying, temperature change, and puffing drying (Bi et al., 2013). The general procedure to produce mulberry powder is shown in Figure 2.13. Fazaeli et  al. (2012) explored the effects of some processing parameters on the moisture content, water activity, drying yield, bulk density, solubility, glass transition temperature, and microstructure of spray-dried black mulberry (M. nigra) as juice powders. A pilot-scale spray dryer was employed for drying process. Maltodextrin with different dextrose equivalent (6, 9, and 20DE) and gum Arabic were used as carrier agents. Independent variables were the inlet air temperatures (110˚C, 130˚C, and 150˚C), the compressed air flow rates (400, 600, and 800 L/ h), the concentrations of drying aids (8%, 12%, and 16%), and percent replacement of maltodextrin (6 and 9DE) by gum Arabic and maltodextrin 20DE (25%, 50%, and 75%). Between the different drying aids, maltodextrin 6DE showed the best effect on the properties of black mulberry juice powders. The drying yield ranges from 45% to 82%. The highest drying yield (82%) and solubility (87%) referred to the blend of maltodextrin 6DE and gum Arabic. The lowest moisture content powders (1.5%) were produced at the compressed air flow rate of 800 L/ h. The inlet air temperatures negatively influenced the bulk density due to the increase of powder’s porosity. The lower the bulk density, the higher the solubility of powder was. With regard to morphology, powders produced with maltodextrin and gum Arabic presented the smallest size (Baráth et al., 2004). However, the spray-dried fruit juice of high tem­ perature would cause the heat-sensitive nutrient losses and the utilization rate of the filter residue to be lower. The hot air drying is relatively cheap and easy to obtain; however, the loss of nutrients is higher. In the vacuum freeze drying, the active substances in the material can be effectively pre­ served, which is a better drying method. Sung (2014) reported the retention of vitamin C in fruit

34

FIGURE 2.12 superfruits.)

Asian Berries: Health Benefits

The picture of dried mulberry fruit. ( https://shop.extremehealthusa.com/collections/organic­

FIGURE 2.13 The process of producing mulberry fruit powder.

powder. Levels of total phenols and anthocyanins by vacuum freeze drying could reach more than 95%. Anthocyanins in mulberry are unstable and can be affected by enzymes, sugars, and other phenolic acids in their own cells, which cause degradation. Freeze drying can solve this problem better than other drying methods (Cao, 2015). Liu (2019) prepared freeze-dried mulberry fruit pow­ der by wet pulverization and freeze drying process for good solubility, high anthocyanin retention rate, and nutrients close to fresh fruits. The experimental results showed that the homogenate time of mulberry fruits was 3 min, then maltodextrin was added, and the liquid-to-feed ratio was 2.5:1 (mL/g). The obtained mulberry fruit powder had good solubility, high anthocyanin retention rate, and low nutrient loss. The flavor is close to the fresh fruits (Liu, 2019). Sun drying is practiced

Nutritional Value and Processing Technology

35

widely in tropical and subtropical countries (Basunia and Abe, 2001). However, there are some problems in sun drying, such as the slowness of the process. The long drying time is undesirable for economic reasons because the product is exposed to danger of contamination during this period (Kostaropoulos and Saravacos, 1995). Therefore, some methods should be used for increasing dry­ ing rate. The drying rate can be increased by means of removing the surface resistance of fruits by chemical pretreatments (hot and cold) such as ethyl oleate, potassium carbonate, and sodium hydroxide (Grncarevic and Hawker, 1971).

2.3.5

MULBERRY YOGURT

Mulberry fruits as an ingredient in yogurt can not only improve the flavor of yogurt products but also have various physiological functions such as regulating body immunity, antioxidation, and preventing cardiovascular and cerebrovascular diseases (Sung and Choi, 2014). The processing of mulberry yogurt is based on mulberry juice, full fat emulsion, and white granulated sugar. Mulberry fruits are uniformly mixed, preheated at 60˚C–70˚C, sterilized at 85˚C for 10 min, and cooled at 43˚C– 45˚C. The steps involved in the production of mulberry yogurt include inoculation, homog­ enization, subpackaging, heat-preservation fermentation at 43˚C for 4–6 h, refrigeration at 4˚C for 12– 4 h, and finally finished products. Cheng (2015) studied mulberry ingredient in yogurt. The results showed that with conditions of the amount of white sugar added 4%, mulberry fruit juice content 6%, fermented inoculum 6%, and fermentation time 6 h, the quality, flavor, and taste of the yogurt were highly accepted. Fu and Tang (2016) used mulberry and milk powder as the major ingredients, with the original flavor of yogurt as a starter to study the production process conditions of mulberry fruit yogurt (Figure 2.14). The results showed that with conditions of 12% of mulberry fruit juice, 8% of white sugar, ratio of milk powder and water 1:10, fermentation at 42˚C°C for 10 h, mulberry fruit yogurt was obtained with pink color, fragrant mulberry scent, fine texture, and sweet and sour taste (Figure 2.15). Wang et al. (2007) used mulberry fruits and skim milk as raw materi­ als, white sugar and stabilizer as auxiliary materials, and domesticated Lactobacillus bulgaricus and Streptococcus thermophilus to ferment and develop yogurt. The mulberry yogurts were rich in the nutrients and delicious with a very high health function (Wang, 2006).

FIGURE 2.14 The picture of mulberry fruit yogurt. (http://www.yantangmilk.com/products_detail/productId= 149.htm.)

36

Asian Berries: Health Benefits

FIGURE 2.15

The process of producing mulberry fruit yogurt.

FIGURE 2.16

The process of producing mulberry fruit vinegar. S. cerevisiae, Saccharomyces cerevisiae.

2.3.6

MULBERRY VINEGAR

Mulberry fruit vinegar has unique health benefits such as promoting digestion, skin care, and beauty; softening blood vessels; and lowering blood fat and blood pressure (Yao et al., 2005). According to Lyu’s (2014) method, mulberries are used as the raw materials. Mulberry fruit juice and mulberry residues both are effectively utilized, and compared with common fruit vinegar, the mulberry fruit vinegar drink is added with a large number of soluble dietary fibers, yield is improved, and cost is lowered. The fermentation process of mulberry fruit vinegar can be carried out in two steps: firstly, mulberry fruit is fermented into mulberry wine by yeast and then fermented further with acetic acid bacteria to obtain mulberry fruit vinegar. There are three methods of fermentation process of mulberry fruit vinegar: solid fermentation method, liquid fermentation method, and solid–liquid mixed fermentation (Figure 2.16). Although the fruit vinegar brewed by the solid-state fermentation method has a good flavor, it has the disadvantages of long fermentation time, low utilization rate of raw materials, and poor hygienic quality of the product. Compared with the solid-state fermenta­ tion method, the liquid fermentation method has a short fermentation cycle, low labor intensity, and greatly improved utilization rate of raw materials (Yao, 2005). Therefore, the liquid fermentation method is currently the most widely used method for fruit vinegar brewing. Yang (2010) used mul­ berry juice as the main raw material, inoculated with 10% of the acetic acid bacteria of Shanghai No. 1.01 and ventilated at a temperature at 31˚C to obtain high-quality and low-cost mulberry vin­ egar. Kim (2012) evaluated the physicochemical properties of various commercially available vin­ egar consumed in the Korean market, including their pH, acidity, sugar, total soluble sugar, total acid, total amino acid content, and antioxidant capacity. The pH values ranged from 2.81 to 3.20, and total acidity ranged from 1.95% to 2.34%. Sugar content ranged from 31.63°Brix to 38.75°Brix. The most commonly occurring acid was acetic acid, which can serve as an indication of the taste quality of the vinegars in quality control. The antioxidant activities of mulberry vinegars had the highest activity and also showed the highest total polyphenol content (Kim et al., 2012).

2.3.7

UTILIZATION AND DEVELOPMENT PROSPECT

Asian countries such as China, India, Thailand, Japan, and Korea have abundant natural mulberry resources. The processing and utilization prospects are optimistic. In recent years, there are mul­ berry juice processing plants for industrial production in China. New products such as mulberry

Nutritional Value and Processing Technology

37

juice, mulberry wine, mulberry jam, mulberry paste, mulberry red pigment, mulberry fruit tea, mulberry fruits, mulberry solid beverages, mulberry lozenges, and many other products are devel­ oped in industrial productions to meet the demand of international markets in the world. Mulberry juice and mulberry wine have excellent color, fragrance, and taste for natural health products. In the production process of mulberry jams and mulberry pastes, the low-temperature vacuum concentra­ tion technology was adopted. The traditional long-time high-temperature process was reformed to maximize the preservation of the nutrient compositions of the mulberry fruits and the vivid color of the mulberry fruits. At the same time, low-sugar mulberry jam was developed to meet the needs of special consumers (Kim and Cho, 2012). Although mulberry fruits have gained an important position in the local soft beverage market, it has biological and pharmacological effects that are still poorly defined (Duyen, 2008).

2.4

SUMMARY

Mulberry fruits belong to “3G fruit.” (3G fruits refer to a type of fruits that are distributed in barren mountains and wild ridges and have not been fully developed, or some newly developed new special fruits. They also have the value of dual uses of medicine and food). Studies have shown that the nutritional benefits of the third generation of fruits, as well as the values of ornamental, medication, and soil and water conservation, were much higher than the previous two generations. Mulberry fruits are rich in nutrients and have high health benefits. Currently, the popular mulberry products are mainly mulberry juice, mulberry wine, mulberry fruit powder, mulberry yogurt, mulberry fruit vinegar, and mulberry jam. Mulberries can be produced into snacks that are easier to carry, light, and easy to eat with nutrients. In addition, with the development of breeding technology, many high-yield and high-quality mulberries have been successfully cultivated. With high yield and large fruit shape, they are suitable for large-scale planting and industrial processing and have a good potential market value. In the future, the processing and utilization of the mulberry fruits, as a new health type of fruit resource, should be a new health food product, in line with modern consumers’ lifestyles and consumption habits.

ACKNOWLEDGMENTS We acknowledge the China Agriculture Research System (CARS-18-ZJ0506) and the Innovation Team of Modern Agricultural Industry Technology System in Guangdong Province of China (2019KJ116) for financial support.

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Cao, S. Q., Liu, L., Zhang, C., Qi, X. Y. (2015). Purification and thermal degradation kinetics of anthocyanins from mulberry. Chinese Journal of Food Science, 15(5), 54– 62. Celep, E., Charehsaz, M., Akyüz, S., Acar, E. T., Yesilada, E. (2015). Effect of in vitro gastrointestinal diges­ tion on the bioavailability of phenolic components and the antioxidant potentials of some Turkish fruit wines. Food Research International, 78, 209–215. Cemeroglu, B., Velioglu, S., Isik, S. (2006). Degradation kinetics of anthocyanins in sour cherry juice and concentrate. Journal of Food Science, 59(6), 1216–1218. Chen, L., Wang, C., Bao, J. J. (2014). Processing and development of blueberry and mulberry compound bever­ age. Agricultural Products Processing, 5, 39– 41. Cheng, S. M., Zeng, Xia., Zhou, G. Y., Yan, X. L. (2015). Development of mulberry compound yoghurt. China Brewing, 34(10), 147–150. Du, M. C., Liu, S. Y., Zhang, Y. X., Zhang, W., Su, E. Z. (2019). Development of ginkgo mulberry soft candy. Agricultural Products Processing, 10, 17–19. Duyen, N. T., Hai, L. T., Minh, N. P., Dao, D. T. A. (2008). Application of natural fermentation to ferment mulberry juice into alcoholic beverage. Journal of Advances in Biology, 17(6), 775–779. Ercisli, S., Orhan, E. (2007). Chemical composition of white (Morus alba), red (Morus rubra), and black (Morus nigra) mulberry fruits. Food Chemistry, 103(4), 1380–1384. Eun, J. K., Ji, Y. L., Choi, I. (2012). Physicochemical properties and antioxidant activities of Korean traditional alcoholic beverage, yakju, enriched with mulberry. Journal of Food Science, 77(7), C752– C758. Fazaeli, M., Hojjatpanah, G., Emam-Djomeh, Z. (2013). Effects of heating method and conditions on the evaporation rate and quality attributes of black mulberry (Morus nigra) juice concentrate. Journal of Food Science and Technology, 50(1), 35– 43. Fu, S. W., Tang, C. Q. (2016). Study on the production process conditions of mulberry yogurt. Contemporary Animal Husbandry, 15(5), 49–51. Gerasopoulos, D., Stavroulakis, G. (2015). Quality characteristics of four mulberry (Morus sp.) cultivars in the area of Chania, Greece. Journal of the Science of Food & Agriculture, 73(2), 261–264. Grncarevic, M., Hawker, J. S. (1971). Browning of Sultana grape berries during drying. Journal of the Science of Food and Agriculture, 22(5), 270–272. Hou, D. X. (2003). Potential mechanisms of cancer chemoprevention by anthocyanins. Current Molecular Medicine, 3(2), 149–159. Huo, Y. K. (2002). Mulberry cultivation and utilization in China, FAO Animal Production and Health Paper. Jiang, R. P., Sun, H. T., Shao, X. R., He, W. B., Ding, J. C. (2013). Researching on processing technology for preserved mulberry. Ginseng Research, 3(7), 24–27. Joanna, K. C., Anna, G. M., Dominik, K., Korczak, J. (2013). Mulberry fruits as an antioxidant component in muesli. Agricultural Sciences, 4, 130–135. Kandylis, K., Hadjigeorgiou, I., Harizanis, P. (2009). The nutritive value of mulberry leaves (Morus alba) as a feed supplement for sheep. Tropical Animal Health & Production, 41(1), 17–24. Kim, S., Cho, H., Shin, H. (2012). Physicochemical properties and antioxidant activities of commercial vin­ egar drinks in Korea. Food Science & Biotechnology, 21(6), 1729–1734. Kostaropoulos, A. E., Saravacos G. D. (1995). Microwave pre-treatment for sun-dried raisins. Journal of Food Science, 60(2), 344–347. Lee, A. C., Hong, Y. H. (2010). Development of functional yogurts prepared with mulberries and mulberry tree leaves. Korean Journal for Food Science of Animal Resources, 30(4), 649– 654. Liang, M., Bao, Y. H. (2018). Optimization of fermentation process of lonicera cider wine and the effect of fermentation process on anthocyanins. Food Science, 10, 151–157. Lin, J. Y., Tang, C. Y. (2007). Determination of total phenolic and flavonoid contents in selected fruits and vegetables, as well as their stimulatory effects on mouse splenocyte proliferation. Food Chemistry, 101(1), 140–147. Liu, L. K., Chou, F. P., Chen, Y. C., Chyau, C. C., Ho, H. H., Wang, H. C. (2009). Effects of mulberry (Morus alba L.) extracts on lipid homeostasis in vitro and in vivo. Journal of Agricultural & Food Chemistry, 57(16), 7605–7611. Liu, Y. (2019). Preparation of mulberry fruit powder and its stability. Food Industry Technology, 40(4), 184–189. Łochyńska, M. (2015). Energy and nutritional properties of the white mulberry (Morus alba L.). Journal of Agricultural Science & Technology A, 5, 709–716. Lyu, Q. M. (2014). Method for making mulberry fruit vinegar drink. Patent CN 104140922 A.[p].2014-12-11. Min, D., Ren, T., Wang, F., Shiow, Y., Bowman, L., Lu, Y. G., Yong, Q., Castranova, V., Jiang, B. H., Shi, X. L. (2006). Cyanidin-3-glucoside, a natural product derived from blackberry, exhibits chemopreventive and chemotherapeutic activity. Journal of Biological Chemistry, 281(25), 17359.

Nutritional Value and Processing Technology

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Sánchez-Salcedo, E. M., Mena, P., García-Viguera, C., Martínez, J. J., Hernández, F. (2015). Phytochemical evaluation of white (Morus alba L.) and black (Morus nigra L.) mulberry fruits, a starting point for the assessment of their beneficial properties. Journal of Functional Foods, 12, 399– 408. Singhal, B. K., Dhar, A., Khan, M. A., Bindroo, B. B., Fotedar, R. K. (2009). Potential economic additions by mulberry fruits in sericulture industry. Plant Horti Tech, 9, 47–51. Singhal, B. K., Khan, M. A., Dhar, A., Baqual, F. M., Bindroo, B. B. (2010). Approaches to industrial exploita­ tion of mulberry (mulberry sp.) fruits. Journal of Fruit and Ornamental Plant Research, 18(1), 83–99. Sohn, B. H., Park, J. H., Lee, D. Y., Cho, J. G., Kim, Y. S., Jung, I. S., Kang, P. D., Baek, N. I. (2009). Isolation and identification of lipids from the silkworm (Bombyx mori) droppings. Journal of the Korean Society for Applied Biological Chemistry, 52(4), 336–341. Suhl, H. J., Noh, D. O., Kang, C. S., Kim, J. M., Lee, S. W. (2003). Thermal kinetics of color degradation of mulberry fruit extract. Food/ Nahrung, 47(2), 132–135. Sung, J. M., Choi, H. Y. (2014). Effect of mulberry powder on antioxidant activities and quality characteristics of yogurt. Journal of the Korean Society of Food Science & Nutrition, 43(5), 690– 697. Tomas, M., Toydemir, G., Boyacioglu, D., Hall, R., Beekwilder, J., Capanoglu, E. (2015). The effects of juice processing on black mulberry antioxidants. Food Chemistry, 186, 277–284. Tomas, M., Toydemir, G., Boyacioglu, D., Hall, R. D., Beekwilder, J., Capanoglu, E. (2016). Processing black mulberry into jam: effects on antioxidant potential and, in vitro, bioaccessibility. Journal of the Science of Food and Agriculture, 97(10), 3106–3113. Tomas, M., Toydemir, G., Boyacioglu, D., Hall, R. D., Beekwilder, J., Capanoglu, E. (2017). Processing black mulberry into jam: effects on antioxidant potential and in vitro bioaccessibility. Journal of the Science of Food and Agriculture, 97(10), 3106–3113. Wang, C., Zhou, Y., Gu, X. Y. (2007). Development of solidified mulberry yogurt. Food Research and Development, 28(5), 64– 67. Wang, T. Y. (2006). The function of fruit vinegar. Chinese Condiments, 6, 10–12. Wang, Y. H., Zeng, Q. W., Chen, S., Lu, H. X., Zhao, H. M. (2009). The influence of different clarifying agents on the clarification effect of mulberry wine. Preservation and Processing, 9(4), 52–54. Wen, P., Hu, T. G., Robert J. L., Liao, S. T., Wu, H., Zou, Y. X. (2019). Mulberry: a review of bioactive com­ pounds and advanced processing technology. Trends in Food Science & Technology, 83, 138–158. Wu, Z. F., Weng, P. F. (2005). Analysis of nutritional components and functional properties of mulberry. Chinese Journal of Food Science, 5(3), 102–107. Xiao, G. S., Xu, Y. J., Liu, X. M., Wu, J. J., Chen, W. D., Yao, X. Z. (2001). Nutrition and health functions of mulberry and its processing and utilization. Chinese Herbal Medicine, 24(1), 70–72. Xiao, C. L., Li, L. H. (2006). Study on the processing technology of low sugar non-sulfur strawberry pre­ served. Food Science, 27(10), 261–265. Xu, L. P., Du, B. L., Li, C. Y. (2015). Optimization of the preparation process of mulberry juice by enzymatic method. Food Industry Technology, 36(18), 165–169. Yan, X. (2017). Research and processing of fruit mulberry. Food Research and Development, 38(16), 209–213. Yang, F., Wang, Z. X., Wang, K. G., Tian, Y. (2017). Research on the fermentation process of mulberry wine. Brewing Technology, 44(5), 42– 47. Yang, X. L., Mao, L. X., Zhang, X. Y. (2006). Studies on the nutritional and antioxidant effects of black mul­ berry. Food Science, 27(2), 248–250. Yang, X. L., Yang, L., Zheng, H. (2010). Hypolipidemic and antioxidant effects of mulberry (Morus alba L.) fruit in hyperlipidaemia rats. Food & Chemical Toxicology, 48 (8), 2374–2379. Yao, Y. Y. (2005). Research on fermentation technology of mulberry fruit vinegar. China Brewing, 6, 58– 61. Ye, X. L., Cheng, S. M., Wen, L. W. (2018). Research on brewing technology of mulberry wine. Journal of Anhui Agricultural University, 45(2), 7–13. Yu, L. (2014). Analysis of Mulberry Composition and Optimization of Process Parameters of Low Sugar Jam. Sichuan Agricultural University: Ya’an. Yue, L., Wu, Z., Li, Z., Fan, J. (2009). Processing technology of the nutrition mulberry jelly. Food  & Fermentation Technology, 3, 70–73. Zeng, L., Tan, X., Zhang, Q., Yang, Y., Tang, J. (2007). Screening of biotransformed γ-aminobutyric acid Saccharomyces cerevisiae and its application in mulberry wine brewing. Food and Fermentation Industries, 43(6), 122–128. Zhou, J. H., Fang, S. L., Cao, J. H. (2017). Clarification and stability study of mulberry wine. Wine, 44(6), 54– 60. Zhou, J. H., Yu, W. W. (2012). The Development of a nutritious and healthy fruit jelly for children. Food Research & Development, 33(5), 83–85.

3

Nutritional Composition,

Antioxidant Properties,

and Health Benefits

of Mulberry Fruits

Maria R. Bronze iBET – Instituto de Biologia Experimental e Tecnológica Universidade Nova de Lisboa (ITQB NOVA) Faculdade de Farmácia da Universidade de Lisboa

Ana Teresa Serra iBET – Instituto de Biologia Experimental e Tecnológica Universidade Nova de Lisboa (ITQB NOVA)

Paula R. Augusti Federal University of Rio Grande do Sul

Pilar Legua Murcia and Francisca Hernández García Miguel Hernández University

CONTENTS 3.1 3.2 3.3

3.4 3.5 3.6 3.7

3.8

Introduction .......................................................................................................................... 42

Nutritional Composition .......................................................................................................44

Bioactive Compounds ...........................................................................................................44

3.3.1 Phenolic Compounds ................................................................................................44

3.3.1.1 Phenolic Profile of Mulberry Fruit ............................................................ 45

3.3.1.2 Phenolic Profile of Mulberry Products ......................................................46

3.3.2 Polysaccharides.........................................................................................................46

Sugar and Organic Acids Composition ................................................................................ 48

Mineral Composition ............................................................................................................ 50

Volatile Compounds ............................................................................................................. 50

Health Benefits...................................................................................................................... 50

3.7.1 Antioxidant Activity ................................................................................................. 50

3.7.2 Antidiabetic Effect.................................................................................................... 53

3.7.3 Antiobesity Effect ..................................................................................................... 53

3.7.4 Effects against Xenobiotic Toxicity .......................................................................... 54

3.7.5 Neuroprotective Effects ............................................................................................ 54

3.7.6 Other Health Effects ................................................................................................. 55

Processed Products ............................................................................................................... 55

41

42

Asian Berries: Health Benefits

3.9

Other Aspects ....................................................................................................................... 56

3.9.1 Bioavailability of Mulberry Bioactive Compounds .................................................. 56

3.9.2 Adverse Effects ......................................................................................................... 56

3.10 Summary .............................................................................................................................. 57

Acknowledgements .......................................................................................................................... 57

References ........................................................................................................................................ 57

3.1 INTRODUCTION Diets that include fruit consumption provide important benefits to the human health, attributed mainly to the presence of secondary metabolites known as phytochemicals (García-Solís & Celis, 2019). Phytochemicals can be defined as plant-derived compounds with therapeutic activities such as anticarcinogenic, antimutagenic, anti-inflammatory, and antioxidant properties (McGuire, 2011). Because of their impact on the human health, the interest in the identification, isolation, and potentization of fruit secondary metabolites in pharmaceuticals and/or supplement forms has increased in the recent years (Skrovankova et  al., 2015; Yuan  & Zhao, 2017). Various types of berry fruits such as blackberries, strawberries, mulberries, and others are important sources of phytochemical compounds with health-promoting effects such as cardiovascular risk reduction (Feresin et al., 2016), glucose metabolism improvement (McDougall et al., 2008; Fotschki et al., 2019), antioxidant properties (Veljkovic et  al., 2019), and anticancer (Navindra et  al., 2006; Li et al., 2019) effects. In general, the Moraceae family, composed of 40 genus and more than 1400 species, is dis­ tributed throughout the world (Rohwer, 1993; Clement & Weiblen, 2009). Broussonetia, Maclura, Ficus, Artocarpus, and Morus are the major genera, and they are economically important because of their use in silkworm feeding (Morus and Maclura), fruit production (Morus, Artocarpus, and Ficus), furniture (Artocarpus and Brossonetia), and paper production (Morus, Broussonetia, and Maclura) (Zerega et al., 2005). Within the family Moraceae, the genus Morus has shown to be the most relevant for its wide geographic distribution and for the nutritional and functional properties of its fruits (Zerega et al., 2005; Donno et al., 2018). The genus Morus consists of diverse species of deciduous trees commonly known as mulberries. The origins of the major mulberry varieties are believed to be in Japan, China, and the Himalayan foothills (Yuan & Zhao, 2017) but are found almost everywhere in the world because of their adapt­ ability (Zhao et al., 2005). They can grow in several types of soil tolerating both wet and dry soils (Zoofishan et al., 2018). The ideal temperature and humidity for growth range from 24˚C to 29˚C and 65% to 80%, respectively, whereas the cold resistance varies depending on the cultivar (Kumar & Chauhan, 2008). However, the plant is able to tolerate great climatic variation, as it develops well in tropical, subtropical, and temperate areas, although the majority of the plant is widespread in Asian countries, such as China, Japan, Korea, and India (Sánchez-Salcedo, 2002). It also tolerates differ­ ent altitudes, with good development from sea level regions to altitudes of 4,000 m (Vijayan et al., 2018). However, the different developmental conditions may influence the chemical composition of the fruit (Ercisli & Orhan, 2008; Imran et al., 2010). The fruits have a cylindrical shape, measure approximately 2 cm in length, and are considered aggregate fruits. In particular, they originate from flowers that have several carpels, in which the individual parts are called fruits (Vijayan et al., 2018). For the formation of the fruit, the corollas become thick by acquiring reserve substances such as sugars and certain organic acids. The seeds have a color ranging from yellow to brown according to species and can measure up to 3 mm. The heartwood is durable, and the leaves are deciduous and free of thorns (Rohwer, 1993; USDA, 2018). Red mulberry, Morus rubra (originated in North and South America); white mulberry, Morus alba (originated in Western Asia); and black mulberry, Morus nigra (originated in Southern Russia) are the most notable species of the genus Morus (Figure 3.1) (Datta, 2002; Ercisli & Orhan, 2007; Özgen et al., 2009). Although its etymology remitted to the fruit color, this parameter should not

Health Benefits of Mulberry Fruits

43

FIGURE 3.1 Mulberry fruits in different maturation stages: (a) Morus alba (white cultivar); (b) Morus rubra (red cultivar); and (c) Morus nigra (black cultivar).

be used as a determinant for species identification, since the same species, namely M. alba, can produce the fruit of several colors (Aramwit et al., 2010). Traditionally, both mulberry leaves and fruits are consumed. While the foliage is normally used to feed the silkworm (Bombyx mori L.) (Naoki et al., 2001; Mishra et al., 2003; Tanaka et al., 2009), the fruits are usually consumed fresh, dried, and/or different processed forms such as juice, ice cream, wine, jam, syrup, fruit leather, yogurt, marmalade, food colorant, and fruit tea and are used as raw materials in the cosmetic, nutritional, and pharmaceutical industries (Kang-Sun et al., 2000; Kim, 2003; Zou et al., 2008; Singhal et al., 2010; Lee & Hong, 2010; Kim et al., 2011; Lee, 2011; Lee et al., 2013; Wang et al., 2015; You et al., 2015; Lee et al., 2016; Tomas et al., 2017; He et al., 2018; Yu et al., 2018).

44

Asian Berries: Health Benefits

Previous studies on mulberry fruits indicated that they are rich sources of phytochemicals with biological effects against degenerative diseases such as Alzheimer’s and Parkinson’s diseases (Kim et  al., 2010; Shin et  al., 2016), glucose metabolism dysfunctions (Wang et  al., 2013; Banu et  al., 2015; Shin et al., 2016), cardiovascular disorders (Chen et al., 2005; Lee et al., 2012), and cancer (Łochyńska, 2015; Zoofishan et al., 2018), among others. This chapter focuses on the nutritional composition, antioxidant properties, and health effects of mulberry fruits through a compilation and synthesis of the available studies. With this, the authors not only intend to contribute to the organization of the state of the art in mulberry, promoting the consolidation and dissemination of fruit properties, but also motivate for new industrial approach and scientific research.

3.2 NUTRITIONAL COMPOSITION The mulberry fruits contain high contents of carbohydrates, lipids, proteins, fiber, minerals, and vitamins but a relatively low energy value (Łochyńska, 2015). Data on nutrient components in mul­ berries are reported in Table 3.1. The values are the average of data found in numerous references and do not report the standard deviation, which reflects the high variation of the different mulberry varieties. Based on Table  3.1, the mean data indicate a total titratable acidity (0.43 g citric acid 100 g−1) and pH (5.11), and high total soluble solid (21.6%) and moisture (80.21%) contents, enabling their use in desserts such as sweets, jams, and juices.

3.3 BIOACTIVE COMPOUNDS 3.3.1

PHENOLIC COMPOUNDS

Phenolics are the products of the secondary metabolism of plants, providing essential functions in their physiology. They are involved in plant morphology (color and mechanical support), growth (some compounds have been related to nutrient uptake, protein synthesis, enzyme activity, and pho­ tosynthesis), and plant reproduction (attracting birds and insects which helps pollination) and are known for being responsible for the protection of plants against attack by pathogens or other stress factors (Gould & Lister, 2006). In human diet, phenolic compounds have been highlighted to be one of the main contributors of the health-promoting effect in humans. In fact, there is substantial evi­ dence from in vitro and in vivo assays with animal models and human intervention studies showing TABLE 3.1 Mulberry Fruit Nutritional Composition

Titratable acidity Total soluble solids pH Moisture Energy Protein Total lipid (fat) Carbohydrate Fiber, total dietary Sugar, total

Unit

Content per 100 g

References

% % – g kcal g g g g g

0.03–1.57 11.20–32.00 3.52–6.70 78.03–88.00 43 0.8–1.44 0.14–0.71 7.8–9.80 0.9–1.70 6.63–17.02

Farahani et al. (2019), Hosseini et al. (2018)

Aljane and Sdiri (2016), Farahani et al. (2019), Hosseini et al. (2018)

Ercisli and Orhan (2007), Farahani et al. (2019), Hosseini et al. (2018)

Imran et al. (2010), Vijayan et al. (2018)

USDA (2018)

Gungor and Sengul (2008), Singhal et al. (2010), USDA (2018)

Imran et al. (2010), USDA (2018)

USDA (2018), Vijayan et al. (2018)

USDA (2018), Vijayan et al. (2018)

Imran et al. (2010), USDA (2018)

Health Benefits of Mulberry Fruits

45

that certain phenolic compounds benefit health status, especially the prevention and management of chronic diseases including obesity, type 2 diabetes, neurodegenerative diseases, cardiovascular diseases, and cancer (Cory et al., 2018). According to their structure, phenolic compounds can be divided into several classes, namely, phenolic acids, flavonoids, stilbenes, coumarins, and tannins (Khalifa et al., 2018b). 3.3.1.1 Phenolic Profile of Mulberry Fruit Mulberry fruits provide an extensive range of phenolic compounds, namely, phenolic acids and flavonoids (mainly flavonol derivatives and anthocyanins). The phenolic composition of mulberry fruits greatly varies with cultivars, climatic conditions, agricultural practices, and process condi­ tions (Khalifa et al., 2018b), and thus, their health-promoting effect can vary accordingly. The total phenolic content of mulberry fruits ranges between 77 and almost 1,500 mg GAE (gallic acid equivalents) 100 g−1 FW (fresh weight) (revised by Khalifa et  al., 2018). In Table  3.2, the contents of the main phenolic compounds identified in different species of mulberry fruits are summarized. Anthocyanins are the major phenolic compounds in mulberry, being responsible for the color and the health benefits of the fruits. Black mulberry cultivars are the ones with the highest anthocyanin content presenting up to 120% more compounds than white fruits (revised by Khalifa et al., 2018). Cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, pelargonidin 3-O-glucoside, and pelargonidin 3-O-rutinoside are the main anthocyanins found in mulberry fruits, especially in black and red cultivars (Isabelle et al., 2008; Khalifa et al., 2018), whereas in some studies, these compounds were not detected in white cultivars (Bao et al., 2016). Moreover, other anthocyanins, namely, cyanidin3-sophoroside, cyanidin-3-glucorutinoside, cyanidin hexoside, cyanidin rhamnosylhexoside, cya­ niding galloylhexoside, cyanidin pentoside, cyanidin hexosylhexoside, pelargonidin hexoside, pelargonidin rhamnosylhexoside, peonidin 3-O-glucoside, petunidin rhamnosylhexoside, delphini­ din rhamnosylhexoside, and delphinidin hexoside were found (Khalifa et al., 2018). It is important to note that some mulberry cultivars are reported to present higher anthocyanin content than other berry fruits such as blueberries, blackberries, blackcurrants, and red currants (Veberic et al., 2015). Like berries, these compounds are indicators of the ripening stage of the colored fruits, which ranged from white/light red to black stage (Bae & Xu, 2017). The other flavonoid compounds that have been highlighted to be present in higher amounts in these fruits include different glycosylated forms of quercetin and kaempferol (revised by (Khalifa et al., 2018)) (Table 3.2). Quercetin, luteolin, isorhamnetin, rhamnetin, kaempferide, and taxifolin have been also identified in different mulberry fruits but at low concentrations when compared with those of glycoside forms (Jin et al., 2015; Sánchez-Salcedo et al., 2015).

TABLE 3.2 Total Phenolic Contents among Different Mulberry Fruits (Morus alba – white, Morus rubra – red, Morus nigra – black) Species TPC (mg GAE 100 g−1 FW) TFC (mg RE 100 g−1 FW) TAC (mg C3GE 100 g−1 FW)

Morus alba

Morus rubra

Morus nigra

77–85 29–37 0.1–0.8

100–1035 21 0.3–20

92–1422 27–260 30–480

C3GE, cyanidin-3-glucoside equivalents; FW, fresh weight; GAE, gallic acid equivalents; RE, rutin equivalents;

TAC, total anthocyanin content; TFC, total flavonoid content; TPC, total phenolic content.

References: Bao et  al. (2016), Calín-Sánchez et  al. (2013), Ercisli and Orhan (2007), Gerasopoulos  &

Stavroulakis (1997), Özgen et al. (2009).

46

Asian Berries: Health Benefits

Concerning no flavonoid compounds, phenolic acids such as hydroxybenzoic acid (protocatechuic, vanillic, and p-hydroxybenzoic acids) and hydroxycinnamic acid (caffeic, p-coumaric, ferulic, chlo­ rogenic, and m-coumaric acids) derivatives are the mostly found components in mulberry fruits (Khalifa et  al., 2018). Among all conjugated forms, esters are dominant followed by glycosides and free acids. Black mulberries are known to mainly present conjugate forms of chlorogenic and protocatechuic acids (Memon et al., 2010). Nevertheless, the environment factors are pointed to be the main contributors to the phenolic acid content of the fruits (Gundogdu et al., 2011; Natić et al., 2015; Sánchez-Salcedo et al., 2015). Other phenolic compounds such as resveratrol analogs (piceid and piceatannol), condensed tan­ nins, and procyanidins are also identified in mulberries but in lower amounts (Bao et  al., 2016; Donno et al., 2015; Jin et al., 2015). 3.3.1.2 Phenolic Profile of Mulberry Products Phenolic compounds are widely recognized to be very sensitive to oxygen, light, pH, heating, and other types of treatment processes, suffering polymerization and oxidative degradation. Therefore, it is expected that some losses in mulberry phenols will occur during the manufacture of some tradi­ tional fruit products such as juice, jam, jelly, and liquors. In Table 3.3, the main findings related with the impact of the different processing treatments on the mulberry phenolic stability are summarized. Among all, centrifugation and thermal treatment applied during cooking and sterilization of products contribute to higher losses in the phenolic content of mulberry (Table 3.3). Despite this, mulberry products are still a source of important phenolic compounds (Table 3.4) that could con­ tribute to enrich the human diet in health-promoting bioactive compounds.

3.3.2

POLYSACCHARIDES

Polysaccharides isolated from mulberry fruits have also attracted increasing attention due to their health-promoting effect. In this field, there are several in vitro and animal studies performed with polysaccharides from M. alba fruits, showing that they present antioxidant activity, hypoglycemic potential, and anti-inflammatory effect, being also associated with the prevention of obesity disor­ ders through the reduction of the viability of preadipocyte cells (revised by Wen et al., 2019). The main polysaccharides identified in mulberry fruits are present in Table 3.5.

TABLE 3.3 Impact of Processing Treatments on the Mulberry Phenolic Content during the Preparation of Juices, Jams, and Alcoholic Beverages Product

Processing Treatment

Mulberry juice

Milling Pasteurization Crushing, clarification, and filtration Centrifugation Thermal processing Alcoholic fermentation

Mulberry alcoholic beverages

Centrifugation and thermal processing Mulberry jam Cooking and Pasteurization

Outcome ↓ TPC (24.9%); ↓ flavonoids (40.7%) ↓ Quercetin (20%) Minor effects on phenolic composition

References Tomas et al. (2015) Yu et al. (2014) Revised by Khalifa et al. (2018)

Lucia et al. (2016) ↓ TAC (67.8%) ↓ TAC (51.2%) ↓ TAC (50%), no changes in flavonols and Pérez-Gregorio, Regueiro et al. (2011) ellagic acid Lucia et al. (2016) ↓ TAC (>50%) ↓ TPC (88%), ↓ TFC (89%), ↓ TAC (97%) (Tomas et al. (2016)

TAC, total anthocyanin content; TFC, total flavonoid content; TPC, total phenolic content.

Health Benefits of Mulberry Fruits

47

TABLE 3.4 Phenolic Content and Composition in Different Mulberry Products Product Mulberry juice

TPC/TFC/TAC TPC: 12.9–18.8 mg GAE g−1 DW

nd

TPC: 19.19 mg GAE g−1 DW

TPC: 2.06-2.25 mg GAE L−1

TAC: 21.83 mg/mL TFC: 8.66–123.67 mg RE L−1 TAC: 22.19–3208.69 mg C3GE L−1 TPC: 2.7 mg GAE g−1 DW TFC: 2.06 mg CE g−1 DW TAC: 0.19 mg C3GE g−1 DW Dried TPC: 9.8 mg GAE g−1 DW mulberry TFC: 3.8 mg CE g−1 DW TAC: 0.61 mg C3GE g−1 DW Mulberry TPC: 1.09 mg GAE g−1 DW jam TFC: 0.92 mg CE g−1 DW TAC: 0.04 mg C3GE g−1 DW TPC: 1.93 mg GAE g−1 DW TFC: 1.58 mg RE g−1 DW TAC: 0.33 mg C3GE g−1 DW Mulberry TPC: 0.25 mg GAE g−1 DW syrup TFC: 0.05 mg CE g−1 DW TAC: 0.001 mg C3GE g−1 DW Mulberry TPC: 4.02 mg GAE g−1 DW molasses TFC: 0.85 mg CE g−1 DW TAC: 0.02 mg C3GE g−1 DW Mulberry ice TPC: 3.78 mg GAE g−1 DW cream TFC: 1.40 mg CE g−1 DW TAC: 1.73 mg C3GE g−1 DW Mulberry wine

TPC: 7.37 mg GAE g−1 DW TAC: 0.27 mg C3GE g−1 DW TFC: 7.51 mg CE g−1 DW TPC: 1937–2493 mg GAE L−1 TAC: 159–719 mg C3GE L−1

Identified Phenolic Compounds F: C3G, cyaniding 3-galactoside, epicatechin, procyanidins A2, 7-hydroxycoumarin, rutin, myricetrin, and kaempferol 3-O-glucoside PA: Protocatechuic, sinapic, chlorogenic, and caffeic acids F: Catechin, rutin, quercetin PA: Chlorogenic, ferulic, O-coumaric, pcoumaric, caffeic, syringic, vanillic, and gallic acids F: C3G, C3R, rutin, quercetin-3-O-glucoside, quercetin-malonyl-glucoside PA: Caffeoylquinic acid F: C3G, C3R, quercetin PA: Gallic, protocatechuic, caffeic, and p-coumaric acids F: C3G, C3R, pelargonidin-3-O-glucoside, pelargonidin-3-O-rutinoside F: C3G, C3R, rutin Other compounds: 1-deoxynojiri-micyn, resveratrol, oxyresveratrol F: C3G, C3R, pelargonidin-3-O-glucoside, rutin, quercetin-3-O-glucoside, quercetin derivatives PA: Gallic, syringic, neochologenic, cafeic acids F: C3G, C3R, pelargonidin-3-O-glucoside rutin, quercetin-3-O-glucoside, quercetin derivatives PA: Gallic, syringic, neochologenic, caffeic acids F: C3G, rutin, quercetin-3-O-glucoside, quercetin derivatives PA: Gallic, syringic, neochologenic, caffeic acids F: C3G, C3R, cyanidin-3,5-diglucoside, pelargonidin-3-O-glucoside, pelargonidin-3-O-rutinoside F: C3G, rutin, quercetin-3-O-glucoside PA: Neochologenic, caffeic acids

References Li et al. (2016)

Akin et al. (2016)

Tomas et al. (2015) Yu et al. (2014)

(Wu et al., 2013) (Song et al., 2009)

Kamiloglu et al. (2013) Kamiloglu et al. (2013) Kamiloglu et al. (2013) Tomas et al. (2016) Kamiloglu et al. (2013)

F: C3G PA: Gallic, syringic, neochologenic, acids

Kamiloglu et al. (2013)

F: C3G, C3R, pelargonidin-3-O-glucoside, pelargonidin-3-O-rutinoside, rutin, quercetin-3O-glucoside, quercetin derivatives PA: Gallic, neochologenic, caffeic acids F: C3G, C3R, rutin, quercetin-3-O-glucoside, quercetin derivatives PA: Gallic, neochologenic, caffeic acids nd

Kamiloglu et al. (2013)

Kamiloglu et al. (2013) Xie et al. (2017)

C3G, cyanidin 3-O-glucoside; C3GE, cyanidin-3-glucoside equivalents; C3R, cyanidin 3-O-rutinoside; CE, catechin equiva­ lents; DW, dry weight; F, flavonoids; GAE, gallic acid equivalents; nd, not determined; PA, phenolic acids; RE, rutin equiva­ lents; TAC, total anthocyanin content; TFC, total flavonoid content; TPC, total phenolic content.

Adapted from Khalifa et al. (2018).

48

Asian Berries: Health Benefits

TABLE 3.5 Composition and Bioactivity of Polysaccharides Isolated from Mulberry Fruits Variety Morus alba

Morus alba

Morus nigra

Unknown

Polysaccharide Components Mannose (1.60 mol%), rhamnose (18.40 mol%), glucose (3.10 mol%), galactose (37.60 mol%), xylose (1.70 mol%), fucose (1.30 mol%), and arabinose (36.30 mol%) Rhamnose (25.98 mol%), glucose (13.06 mol%), galactose (23.10 mol%), galacturonic acid (16.35 mol%), and arabinose (21.51 mol%) Mannose (18.88 mol%), rhamnose (5.94 mol%), glucose (27.62 mol%), galactose (25.00 mol%), xylose (1.75 mol%), galua (7.87 mol%), and arabinose (12.94 mol%) Mannose (8.51 mol%), glucose (28.20 mol%), galactose (18.51 mol%), xylose (6.55 mol%), galacturonic acid (3.97 mol%), and arabinose (34.15 mol%) Mannose (2.61 mol%), glucose (38.33 mol%), galactose (29.27 mol%), xylose 4.33 mol%), galacturonic acid (3.18 mol%), and arabinose (22.45 mol%) Mannose (5.17 mol%), glucose (12.14 mol%), galactose (30.33 mol%), xylose 11.25 mol%), galacturonic acid (3.19 mol%), and arabinose (37.45 mol%)

MW

Bioactivity

References

1,639 kDa

Potential immunomodulator

Lee (2011)

136.6 kDa

Antioxidant and hypoglycemic

Chen et al. (2016)

–

Antioxidant

Ma et al. (2018)

838 kDa 49.5–163 kDa 1.41–15.30 kDa

Ability to reduce the rate and extent of lipid digestion

Chen, Huang et al. (2017)

Adapted from Wen et al. (2019).

3.4 SUGAR AND ORGANIC ACIDS COMPOSITION The average of the total sugar content of fresh mulberries fruits is, generally, 11.91%, but in some cases, it can increase until values maximum of 31%, depending on fruit ripening and mulberry variety (Lin & Lay, 2013; Saracoglu, 2018). Fructose and glucose were the predominant sugars in mulberry fruits with variation between 6.53–8.55 and 4.22–5.37 g 100 g−1, respectively, followed by sucrose (Table 3.6). The fructose, glucose, and sucrose contents of the fruit increased with the progression of fruit ripening (Lee et al., 2016). Because sugars are the initial precursor in the bio­ synthesis of anthocyanins, the high content in mature mulberries may indicate a high content of anthocyanins confirming their important nutritional value (Aramwit et al., 2010). On the other hand, organic acids are considered as ripening index and important flavor contribu­ tors to many fruits and vegetables (Akin et al., 2016). Malic, citric, tartaric, succinic, and quinic acids are the main organic acids responsible for flavor notes for most fruits (Lamikanra, 2002). The most predominant organic acids in mulberry varieties were malic acid and citric acid (Table 3.6). However, in black mulberry, organic acid content was lower than white and red species, as shown in Table 3.6. The difference between species in terms of organic acid content might be caused by genetic factors as well as cultural practices and ecological factors (temperature, light, humidity, etc.) (Aljane & Sdiri, 2016; Gundogdu et al., 2011). In addition, the mulberry fruit also contains vitamins, such as vitamin C (36.4 mg 100 g−1), vitamin A (25 UI), vitamin B6 (0.05 mg 100 g−1), vitamin K (7.8 mg 100 g−1), vitamin E (0.87 mg 100 g−1), thiamin (0.029 mg 100 g−1), riboflavin (0.101 mg 100 g−1), niacin (0.62 mg 100 g−1), and folate (6 μg 100 g−1), with positive impact on human health (Eyduran et al., 2015; USDA, 2018).

Health Benefits of Mulberry Fruits

49

TABLE 3.6 Sugars and Organic Acid Contents in Mulberry Fruits from Principal Varieties of Morus sp. Unit Total organic acids Acetic acid Ascorbic acid Malic acid

Tartaric acid

Citric acid

Lactic acid Succinic acid

Fumaric acid

Sugar, total

Glucose

Fructose

Sucrose

FW, fresh weight

Morus alba

g 100 mL−1 mg/g FW mg/g FW g 100 mL−1 g 100 g−1 g/L g 100 g−1 g 100 g−1 mg/g FW g 100 g−1 FW g 100 g−1 FW g 100 g−1 FW g 100 g−1 FW g 100 g−1 g 100 g−1 g 100 g−1 g 100 g−1 mg/g FW g 100 g−1 FW g 100 g−1 FW g 100 g−1 FW g 100 g−1 FW g 100 g−1 FW g 100 g−1 mg/g FW g 100 g−1 FW g 100 g−1 FW

– – 0.008 – 74.86 5.98 2.13 – – 3.09 1.13–2.10 0.22 0.15–0.43 0.15 2.56 0.63 – – 0.39 0.73–1.03 0.074 0.16 0.26–0.43 0.25 – 0.02 0.10–0.12

g 100 g−1

0.10

g 100 mL−1 g 100 g−1 FW g 100 g−1 FW g 100 g−1 FW g 100 g−1 g 100 mL−1 g 100 g−1 FW g 100 g−1 g 100 g−1 FW g 100 g−1 g 100 g−1 g 100 g−1 FW g 100 g−1 g 100 g−1 g 100 g−1

– – 7.55 2.23 8.31 – 5.33–9.43 4.65–8.13 2.3 7.69 – 4.05–7.70 3.53–6.16 – 0.84–1.21

Morus rubra 3.6–10.4 – 0.015 0.03–0.05 – 6.02 – 3.0–1.3 – 4.46 – 0.33 – – 2.45 – 2.3–9.1 – 0.76 – 0.074 0.13 – – – 0.02

5.7–9.5 – – 4.65 – 2.85–4.96 – 4.64-10.05 5.21 – 2.77–4.66 – – 0.10–0.04 0.74–1.20

Morus nigra

References

3.6–28.8 3.55 0.019 0.03–0.1 – 5.56 3.07 2.3–1.1 2.62 1.32 1.21–3.04 0.12 0.14–0.29 0.29 1.28 0.82 14.5–22.5 0.71 1.08 0.48–0.68 0.049 0.34 0.11–0.36 0.11 6.48 0.01 0.01–0.10

Özgen et al. (2009) Jiang and Nie (2015) Gundogdu et al. (2011) Özgen et al. (2009) Yildiz (2013) Aljane and Sdiri (2016) Gecer et al. (2016) Özgen et al. (2009) Jiang and Nie (2015) Gundogdu et al. (2011) Eyduran et al. (2015) Gundogdu et al. (2011) Eyduran et al. (2015) Gecer et al. (2016) Aljane and Sdiri (2016) Gecer et al. (2016) Özgen et al. (2009) Jiang and Nie (2015) Gundogdu et al. (2011) Eyduran et al. (2015) Gundogdu et al. (2011) Gundogdu et al. (2011) Eyduran et al. (2015) Gecer et al. (2016) Gundogdu et al. (2011) Gundogdu et al. (2011) Eyduran et al. (2015)

0.12

Gecer et al. (2016)

10.4–13.6 5.09–7.26 6.64 5.02 9.55 5.50–7.12 6.17–8.53 – 5.75 8.15 4.86–6.41 4.16–7.16 0.74–0.90 0.01–0.07 –

Özgen et al. (2009) Koyuncu et al. (2014) Imran et al. (2010) Aljane and Sdiri (2016) Gecer et al. (2016) Özgen et al. (2009) Eyduran et al. (2015) Gundogdu et al. (2018) Aljane and Sdiri (2016) Gecer et al. (2016) Özgen et al. (2009) Eyduran et al. (2015) Gundogdu et al. (2018) Özgen et al. (2009) Gundogdu et al. (2018)

50

Asian Berries: Health Benefits

3.5 MINERAL COMPOSITION In the literature, the fresh mulberry fruit also stands out for its mineral contents. Potassium is the major macronutrient (194 mg 100 g−1), followed by calcium (39 mg 100 g−1), phosphorus (38 mg 100 g−1), and sodium (10 mg 100 g−1). Iron and manganese are the major microelements which are important for human health (Akbulut & Özcan, 2009; Singhal et al., 2010; USDA, 2018).

3.6

VOLATILE COMPOUNDS

As mentioned earlier, mulberry fruits have been widely used in the production of mulberry wine, fruit juice, jam, and alcoholic beverages such as the traditional Greek distillate “Mouro” (Soufleros et al., 2004; Singhal et al., 2010), black mulberry liqueurs (Darias-Martín et al., 2003), and a dis­ tillate (Alonso González et al., 2010). Studies based on consumer preferences show that the flavor is the most important attribute of the fruit. In this sense, the volatile composition influences the organoleptic characteristics, particularly the aromatic ones (Baldwin, 2002). Scientific studies on the volatile fraction of mulberry fruit showed significant differences among species and cultivars (Calín-Sánchez et al., 2013). For black mulberry (M. nigra) fresh fruits, around 30 aromatic compounds were detected (Göğüş et al., 2011; Feng et al., 2015), whereas for white mulberry (M. alba) fresh fruits, around 40–50 aromatic compounds were detected (Chen et  al., 2015; Zhang et al., 2011). These differences could be attributed to the climatic conditions, stage of maturation, harvest, postharvest, conservation, and processed treatment (Christophe et al., 2003; Dabbou et al., 2016; Santos Silva et al., 2019; Vendramini & Trugo, 2000). Although studies on mulberry fruit volatile fraction are still limited, compared with those on other fruits, there are several studies focused on processed products such as mulberry wine. These studies identified between 80 and 100 volatile compounds (Butkhup et al., 2011; Feng et al., 2015; Tchabo et al., 2017). Calín-Sánchez et al. (2013) described the alcohols and esters as the major aroma compounds of the Morus genus. C6‐alcohols were the most abundant aromatic alcohols detected in the mulberries. The C6-alcohols were presented in higher concentrations in fresh fruits than dried fruits (Göğüş et al., 2011; Chen et al., 2015). Esters were responsible for 49% of the total amount of aroma com­ pounds (Chen et al., 2015). The major ester component in the free form for mulberry fruit was ethyl acetate, which was also determined as predominant ester in raspberry (de Ancos et al., 2000). The C6- and C9-aldehydes such as hexanal, (Z)-3-hexenal, and (E)-2-hexenal were the predominant free volatile aldehydes identified in the mulberry fruit (Calín-Sánchez et al., 2013; Chen et al., 2015). These aldehydes often play an important role in the construction of a desirable aromatic profile and are described as “green and leafy” and “cucumber-like” aromas, respectively (Meret et al., 2011; Chen et al., 2015). In addition, acids, terpenoids, ketones, and volatile phenols were also present in mulberry fruit but in lower levels, compared with alcohols, aldehydes, and esters (Chen et al., 2015).

3.7 HEALTH BENEFITS Mulberry plants were one of the first to receive the designation of medicinal-and-edible plants by the Ministry of Health of China (Chinese Pharmacopoeia Commission, 2015). Several healthpromoting properties attributed to mulberry fruits were extensively reviewed in recent reports (Zhang et al., 2018), and some of them are displayed in Table 3.7.

3.7.1

ANTIOXIDANT ACTIVITY

The antioxidant activity of plants is often associated with the health benefits they exert. Health benefits of mulberry fruits have been associated with antioxidant activity, and phytochemicals in

Health Benefits of Mulberry Fruits

51

TABLE 3.7 Major Health-Promoting Properties of Mulberry Fruits Mulberry Fruit Presentation

Experimental Model

Gastric and intestinal digested

Human HepG2 cells

Lyophilized ethanolic extract

Human keratinocytes

Ethanolic extract of Human HepG2 black fruits; extract of cells black and white fruits Black fruit juice

Male Wistar rats

Microencapsulated extract

Female Wistar rats

Isolated prenylated arylbenzofuran and flavonoids Juice freeze-dried powder

HT22 mouse hippocampal cell Male BALB/c mice

Methanolic crude extract

Male Sprague– Dawley rats

Ethanolic extract

Male Wistar rats

Ethanolic extract

SHSY5Y cells and male C57bl/6 mice

Powder

Kunming mice

White mulberry polysaccharide solution Cyanidin-3-O-b-Dglucopyranoside fraction

Sprague– Dawley rats Male Sprague– Dawley rats

Health Effect

Mechanism of Action

Ameliorating dietary Improvement of cell resistance to acrylamide-induced oxidative stress cytotoxicity Protection against skin Inhibition of aryl hydrocarbon damage induced by receptor signaling benzo[a]pyrene and its products Protection against ethyl Restoring ROS overproduction, carbamate–induced GSH and lipid peroxidation oxidative damage levels, as well as suppression of mitochondrial dysfunction Anticonvulsivant effect Decrease in malondialdehide levels and in the frequency of epileptiform activity Neuroprotective effects Enhanced cholinergic function, in metabolic syndrome Erk phosphorylation, and during menopause oxidative stress status in hippocampus Neuroprotective effect Antioxidant effect, releasing the antioxidant and phase II enzymes or inhibition of apoptosis Effect against acute Modifying of bacterial content, colitis maintenance of goblet cells, and activation of NLRP6 inflammasomes Inhibition of liver deiodinase Protection against activity along with the increase hypothyroidism in serum T3 and T4 concentrations Neuroprotection in Increase of cholinergic function vascular dementia and decrease of oxidative stress and apoptosis Protection on Antiapoptotic effects and Parkinson’s disease regulation of reactive oxygen species and nitric oxide generation Anti-constipation Modulation of gut microbiota and short-chain fatty acids production, decrease in expression of aquaporins along with the increase in colonic mucus cells Antihypertensive effect Reduction of blood pressure by enhancing endothelial nitric oxide production Protective effects Improvement of maximum against bladder and intravesical pressure, activation erectile dysfunction in of nitric oxide synthase diabetes expression, minimization of apoptosis and oxidative stress

References Zhang et al. (2017) Woo et al. (2017)

Chen et al. (2017), Li et al. (2018) Tubaş et al. (2017) Kawvised et al. (2017)

Seo et al. (2015)

Wang and Hatabu (2019)

He et al. (2019)

Kaewkaen et al. (2012) Kim et al. (2010)

Hu (2019)

Wang et al. (2019) Ha et al. (2012)

(Continued)

52

Asian Berries: Health Benefits

TABLE 3.7(Continued) Major Health-Promoting Properties of Mulberry Fruits Mulberry Fruit Presentation

Experimental Model

White mulberry polysaccharide fraction

Human HepG2 cells

Lyophilized ethanolic extract of mulberry powder

Male Sprague– Dawley rats

Fresh fruits of a new cultivar (J33) submitted to gastrointestinal digestion Ethanolic extract; isolated cyanidin-3-glucoside

Human HepG2 cells

Microencapsulated extract

Mouse MIN6N; pancreatic β-cells Female Wistar rats

Lyophilized aqueous extract

Male C57BL/6 mice

Health Effect

Mechanism of Action

Metabolic dysfunctions Increase in Nrf2 phosphorylation and activation of Nrf2/ARE pathway, as well as enhanced gene expressions in order to protect hepatocytes against palmitic acid–induced toxicity Effect on fatty liver Improvement of serum lipid disease profile, suppression of cholesterol-regulating gene, and amelioration of oxidative stress Effect on fatty liver Prevention of cytotoxicity induced disease by palmitic acid by promoting this fatty acid incorporation into inert triglycerides in response to lipid overload Antidiabetes effect Protection against oxidative damage and apoptosis caused by hydrogen peroxide/oxidative stress Effects in metabolic Improvement in glucose and lipids syndrome during metabolism along with menopause inflammatory and oxidative stress status Antiobesity action Restoring oxidative stress and inflammation markers

References Hu et al. (2019)

Yang and Jo (2018)

Hu et al. (2018a)

Lee et al. (2014, 2015)

Wattanathorn et al. (2019)

Wu et al. (2016)

the fruits, such as anthocyanins, procyanidins, and other phenolic compounds (Arfan et al., 2012), are responsible for those effects. The antioxidant activity varies with cultivar and phenotype, as well as the processing of the fruit (mulberry juice and mulberry marc). In fact, the processing of black mulberries into jam resulted in losses close to 90% of phenolic compounds and antioxidant capacity (Tomas et  al., 2016). Besides, the antioxidant activity depends not just on the phenolic content but also on the identity of these compounds and interactions between them or with other compounds in the fruit (Yuan & Zhao, 2017). The ethyl acetate–soluble extracts of M. alba fruits showed radical scavenging activities against 2,2-diphenyl-l-picrylhydrazyl (DPPH) and superoxide anion radicals in vitro and increased the antioxidant enzymes in streptozotocin (STZ)-induced dia­ betic mice (Wang et al., 2013). In the case of sugar-free extracts of M. nigra and M. alba fruits, both have high antioxidant potential , as determined by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), DPPH, and reducing power assays. Besides, the antioxidant activity of M. nigra was greater than that of M. alba fruits (Arfan et al., 2012). Moreover, the purification of a phenolic-rich extract from mulberry fruit (J33 variety) resulted in increased Fe-chelating ability and protective effects on mouse red blood cell hemolysis and lipid peroxidation ex vivo (Ajay Krishna et al., 2018). Recently, a gel formulation with extracts of black mulberry was developed as a sunscreen, and the formulation presented antioxidant activity in the DPPH assay, with an IC50 value of 104.66 ppm (IC50 value of extracts was 146.73 ppm) (Budiman et al., 2019). A crude polysaccharide fraction from black mulberry exhibited the most effective scavenging activities against DPPH, ABTS, and hydroxyl radicals. The polysaccharide fraction presented high

Health Benefits of Mulberry Fruits

53

reducing power and protected PC12 cells from oxidative damage caused by hydrogen peroxide (Wang & Hatabu, 2019). The antioxidant and anti-inflammatory effects of M. nigra L. pulp extracts were compared with the leaves in C57BL/6 mice with sepsis, being the hydroethanolic extracts from the pulp more effec­ tive in preventing impairment in metalloproteinase type 2 expression (De Pádua Lúcio et al., 2018).

3.7.2

ANTIDIABETIC EFFECT

Morus is known as an allied in diabetes treatment. A recent report described that polysaccha­ rides from the mulberry fruit are able to modulate gut microbiota, enhanced benefic bacteria, and reducing pathogenic ones, which may contribute to their antidiabetic effects (Chen et al., 2018). In Caenorhabditis elegans exposed to glucose, mulberry anthocyanin extracts attenuated malondial­ dehyde and triglyceride accumulation, recovered antioxidant enzymes activities, and reversed the shortening in life span (Yan et al., 2017). However, mulberry wine administration caused no hypo­ glycemic action in diabetic rats, despite the ameliorating effects on markers of oxidative stress in several tissues. This discrepancy may be related to the mulberry treatment after diabetes induction with STZ (Srikanta et al., 2016). Another study showed that the cyanidin-3-O-b-D-glucopyranoside fraction from mulberry fruits improved maximum intravesical pressure in STZ-induced diabetic rat bladder. This effect was associated with apoptosis minimization, reduction in the level of 8­ hydroxy-2-deoxyguanosine, and upregulation in the superoxide dismutase expression (Ha et al., 2013). These mechanisms assigned to cyanidin-3-O-b-D-glucopyranoside were also observed in rats with erectile dysfunction induced by diabetes (Ha et al., 2012). Cyanidin-3-glucoside, another compound isolated from mulberry fruits, has shown to protect MIN6N pancreatic β cells against oxidative stress–induced apoptosis (Lee et  al., 2015). In agreement, mulberry extracts protected pancreatic β cells against features caused by hydrogen peroxide, such as reactive oxygen species (ROS) generation, lipid peroxidation, cell death, and DNA condensation and/or fragmentation (Lee et  al., 2014). In Zucker diabetic fatty rats, mulberry anthocyanin prevented the insulin secretion decline by protecting β cells besides enhanced peripheral tissue glucose uptake, lowering glucose blood (Sarikaphuti et  al., 2013). The anti-α-glucosidase activity is an important mechanism for hypoglycemic effects of mulberry phenolics, since the inhibition of the enzyme delays the postpran­ dial rise in blood glucose (Cheng et al., 2016).

3.7.3 ANTIOBESITY EFFECT The antiobesity mechanism of mulberry fruits was recently detailed by Khalifa et al. (2018). Since obesity correlates with many other health problems, such as diabetes, strategies that ameliorated obesity or obesity-related metabolic stressors can be of great importance. A combined consump­ tion of mulberry leaf and fruit extracts was reported to reduce inflammation and oxidative stress in obese (high-fat diet–induced) mice (Lin et al., 2013). Similarly, combined fruits and leaves of mul­ berry ameliorated cholesterol transfer proteins accompanied by reduction of oxidative stress in the high-fat diet–induced obesity (Valacchi et al., 2014). Mulberry extracts prevented liver damage and serum lipids impairment, along with the suppression of cholesterol-regulating gene and oxidative stress, in rats with fatty liver disease (Yang & Jo, 2018). In another study, it has been observed that an encapsulated mulberry extract also beneficiated female rats with menopause induced by bilateral ovariectomy and with metabolic syndrome induced by high-carbohydrate high-fat diet. The extract improved body weight gain, adiposity index, glucose intolerance, lipid profiles, atherogenic index, and oxidative stress status, besides reversing features in the expressions of TNF-α (tumor necro­ sis factor-alpha), NF-κB (nuclear factor kappa B), and PPAR-γ (peroxisome proliferator–activated receptor-gamma) (Wattanathorn et  al., 2019). It has also been observed that another microen­ capsulated extract of mulberry ameliorated the memory impairment, oxidative stress status, and acetylcholinesterase activity, besides improving neuron density and Erk phosphorylation in the

54

Asian Berries: Health Benefits

hippocampus of rats with menopause and metabolic syndrome (Kawvised et al., 2017). Mulberry anthocyanins could enhance the mitochondrial function and increase the expressions of thermo­ genic genes, PPAR-α, and CPT1, which are transcription factors in initiating the mitochondrial β-oxidation of long-chain fatty acids (You et al., 2015; Peng et al., 2011). In a recent study, the characterization of a polysaccharide fraction from white mulberry and its ability to protect against palmitic acid–induced cytotoxicity in hepatocytes was described (Hu et al., 2019). In particular, this polysaccharide fraction activated Nrf2/ARE signaling pathway, by increas­ ing Nrf2 phosphorylation and its nuclear translocation. In addition, the fraction enhanced HO-1, NQO1, and γ-GCL gene expressions and promoted catalase and glutathione peroxidase activities, which protected hepatocytes against cytotoxicity.

3.7.4 EFFECTS AGAINST XENOBIOTIC TOXICITY Mulberry fruit extracts (before and after gastrointestinal digestion) successfully suppressed ROS overproduction, mitochondrial membrane lipid peroxidation, and glutathione (GSH) depletion caused by acrylamide in human HepG2 cells (Zhang et al., 2017). The authors suggest that mul­ berry fruits improve the cell resistance to acrylamide-induced oxidative stress, rather than react directly with the toxic compound. Cyanidin-3-glucoside was reported as the main component of mulberry extracts able to protect human keratinocytes against pollutant materials. In particular, this compound showed potent inhibitory activity against benzo[α]pyrene-induced cell cycle arrest at the S phase and against the formation of adduct between benzo[α]pyrene metabolite and DNA (Woo et al., 2017). These effects were similar to those presented by mulberry extracts. Mulberry fruit ethanolic extracts also afford protection against ethyl carbamate–oxidative damage in human liver HepG2 cells by scavenging ROS and preventing GSH depletion (Chen et al., 2017). In addition, black mulberry extracts were more effective than white mulberry extracts in ameliorating ethyl carbamate–induced cytotoxicity n HepG2 cells (Li, Bao et al., 2018). Red mulberry fruit aqueous extracts also accelerate ethanol metabolism by increasing expression of alcohol dehydrogenase and acetaldehyde dehydrogenase (Yang & Jo, 2018). Mulberry crude methanolic extracts (30, 60, or 120 mg/kg body weight) also ameliorated the changes in thyroxine and triiodothyronine serum levels and the deiodinase 3 downregula­ tion caused by 28 days’ exposure of rats to nonylphenol (He et al., 2019). In particular, mulberry extracts prevented diphenoxylate-induced constipation by increasing the fecal water content, short­ ening defecation time, promoting gastric evacuation, and increasing the gastrointestinal transit rate in mice (Hu, 2019).

3.7.5

NEUROPROTECTIVE EFFECTS

A prenylated arylbenzofuran and flavonoids isolated from the fruits of M. alba L. presented pro­ tective effects against glutamate cytotoxicity in HT22-immortalized hippocampal cells (Seo et al., 2015). Another study showed that the administration (2, 10, and 50 mg/kg) of a mulberry fruit extract for 7 days before and 21 days after the occlusion of right middle cerebral artery resulted in enhanced memory and increased neuron densities along with decreased oxidative stress in hippo­ campus of rats with vascular dementia. Thus, mulberry fruits may be pointed as a potential natural cognitive enhancer and neuroprotector (Kawvised et al., 2017). Regarding Parkinson’s disease, a 70% ethanol extract of mulberry fruits was reported to have a protective effect (in a dose-dependent way) against neurotoxicity of 6-hydroxydopamine in SH-SY5Y cells and in a Parkinson’s disease animal model (Kim et al., 2010). Authors showed that the protective effect was mediated by their antiapoptotic effects and by regulating ROS and nitric oxide generation. Administration of M. rubra fruit extracts (2.5, 5, 10, 20 mg/kg) to rats resulted in decreased spike frequencies of convulsions along with decreased malondialdehyde blood levels in penicil­ lin-induced epileptiform activity (Tubaş et al., 2017).

Health Benefits of Mulberry Fruits

3.7.6

55

OTHER HEALTH EFFECTS

A high number of health-promoting effects have been attributed to the Morus genus, such as laxa­ tive properties, antibacterial activities, and antihypertensive, antiatherogenic, and anticarcinogenic effects (Rodrigues et al., 2019). The methanolic extracts of dried M. nigra fruits have been described to have laxative activ­ ity, most likely associated with anticholinergic effects (Akhlaq et al., 2016). A recent study indi­ cates that mulberry consumption prevents constipation in mice and may be considered a candidate in a constipation therapy (Hu et al., 2019). In agreement, mulberries increased Lactobacillus and Bifidobacterium numbers and decreased Helicobacter and Prevotellaceae numbers in feces (Hu, 2019). Moreover, mulberry juice freeze-dried powder attenuated colitis severity by ameliorating body weight loss, colon shortening, and colonic inflammation (Wang & Hatabu, 2019). In addition, anthocyanins and flavonoids from M. nigra fruit were reported as antinociceptive and antibacte­ rial, being these activities related to their anti-inflammatory effects (Chen et al., 2018). Regarding cardiovascular diseases, M. nigra fruit ethanolic extracts decreased the serum lipid levels and ath­ erosclerotic lesions and increased the high-density lipoprotein content in Sprague–Dawley rats fed with a hyperlipidic diet (Jiang et al., 2017). White mulberry fruit polysaccharides reduced arterial blood pressure in both normotensive Sprague–Dawley and spontaneously hypertensive rats through enhanced endothelial NO produc­ tion (Wang et al., 2019). Regarding antitumor effects, the mulberry cyanidin-3-glucoside decreased cell viability in MDA-MB-453 human breast cancer cells and inhibited tumor growth when these breast cancer cells were inoculated in mice (Cho et al., 2017). Additionally, gastric carcinoma growth in balb/c nude mice was inhibited after animals had been fed with anthocyanin-rich mulberry fruit extracts for 7 weeks (Huang et al., 2011).

3.8 PROCESSED PRODUCTS The consumer demands black, purple, and white mulberry fruit products not only because they have pleasant taste but also because they can provide abundant natural compounds such as phenolic compounds, organic acids, and sugars, which may promote human health benefits (Kim et al., 2010; Lim et al., 2019; Pham et al., 2017; Sánchez-Salcedo et al., 2014; Shin et al., 2016), increasing the interest in the food industry (Özgen et al., 2009; Aramwit et al., 2010). The principal forms of mul­ berry fruit consumption are fresh, frozen and/or dried, and also processed products such as vinegar, syrup, marmalade, jam, wine, juice, and beverages (Ercisli & Orhan, 2008; Sánchez-Salcedo et al., 2015; Gundogdu et al., 2018). In some cultures, especially in Mediterranean countries, the mulberry fruits are frequently used in traditional dishes, such as “pestil,” “köme,” and “molasses” very popular in Turkey (Yildiz, 2013; Eyduran et al., 2015). Mulberry “pestil” and “köme” are traditionally consumed in winter and are prepared by boiling mulberry juice and sugar with the addition of several types of nuts (Yildiz, 2013). Mulberry “molasses” are used to make “pekmez” that is typically consumed at breakfast (Sengül et al., 2005). Due to the high amounts of sugar, carbohydrates, and protein, these foods are a good source to a rapid supply of energy (Łochyńska, 2015). Mulberry fruits are used also for the production of alcoholic drinks such as “mournoraki,” tradi­ tional fruit-distilled beverage in Greece (Soufleros et al., 2004), and “Tut araghi,” mulberry vodka, consumed in Azerbaijan, Georgia, and Armenia (Farid & Iskandar, 2000). In Asia and China, the mulberry wine, with sweet taste, is a popular alcoholic drink (Tchabo et al., 2017). The traditional culture defends that the consumption of small doses of these drinks offers benefits to human health (Farid & Iskandar, 2000; Soufleros et al., 2004). Finally, mulberry fruits are also used as natural dyes in food and cosmetic industries (Datta, 2002; Ercisli & Orhan, 2008).

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Due to their distinct aroma characteristics and nutritive features, the mulberry-processed prod­ ucts have significant marketing value (Eyduran et al., 2015). In addition, and although it is not the objective of this chapter, the importance of mulberry leaves should be highlighted. Mulberry leaves, traditionally used for feeding silkworms (B. mori L.), are nutritious and can be consumed in different ways as tea, very popular in Thailand (Gryn-Rynko et al., 2016), and as a condiment in soup preparation and curry (Dalmagro et al., 2018). Furthermore, mulberry leaf–carbonated beverage and mulberry leaf chrysanthemum beverage are widely con­ sumed in China (Kumar & Chauhan, 2008; Li et al., 2018). The leaves are also used to feed dairy animals to improve milk yield (Singhal et al., 2010).

3.9

OTHER ASPECTS

3.9.1 BIOAVAILABILITY OF MULBERRY BIOACTIVE COMPOUNDS In vitro studies and animal models have evaluated the bioaccessibility and bioavailability of mul­ berry bioactive compounds. Studies showed that, in general, the bioaccessibility of black mulberry’s anthocyanins (9%) is higher than that of raspberry (5.3%), sour cherry (2.8%), and ruddy mulberry (0.34%) (Liang et al., 2012). Additionally, black mulberry wine exhibited the best bioavailability of phenolic compounds and the highest antioxidant level when compared with cherry and blackberry wines (Celep et al., 2015). However, data on mulberry’s phenolic metabolism and bioavailability may be variable because of the different chemical structures, solubility, and food–matrix interaction. In fact, the anthocya­ nins are more bioaccessible in fruits than in juice (Tomas et al., 2015), whereas the opposite occurs in mulberry jam (Tomas et al., 2016). The absorption of anthocyanins from mulberry in rats occurs very quickly, appearing in plasma 15–60 min after consumption, and the excretion is completed within 6–8 h (Hassimotto, Genovese, & Lajolo, 2008). In another study, black mulberry anthocya­ nins were highly concentrated in stomach, small intestine, and large intestine after 15 min to 3 h of intake by rats. Anthocyanins are metabolized by intestinal microflora to produce easily absorbed phenolic acids, such as protocatechuic, chlorogenic, caffeic, and ferulic acids (Cheng et al., 2016). A recent report drew attention to the fact that, after the digestive process, some compounds of mul­ berry fruits can increase or decrease their bioactivity (Bao et al., 2019). In fact, these authors dem­ onstrated that one mulberry cultivar named Hanguo contained abundant phenolic compounds and exhibited potent antioxidant property even after in vitro digestion and gut microbiota fermentation compared with other mulberry cultivars. After in vitro digestion, the content of anthocyanins in mul­ berry fruits decreased significantly, most likely due to the instability of these compounds in alkaline pH (Zhang et al., 2017). On the other hand, the contents of flavonoid glycosides were increased, and this fact was related to the partial digestion of fiber, which has been associated with phenolic bond­ ing in wine (Saura-Calixto & Díaz-Rubio, 2007). In agreement, the protective effect of digested mulberry fruits against acrylamide-induced oxidative damage enhanced when compared with mul­ berry fruits without digestion (Zhang et al., 2017). In another study, the gastrointestinal digested mulberries (new cultivar J33) reduced palmitic acid–induced cytotoxicity, an important issue in nonalcoholic fatty liver disease, while mulberry extracts without digestion showed no protection. The digested mulberries could act by suppressing ROS accumulation, regulating intracellular GSH, and ameliorating mitochondrial dysfunction, as well as by promoting palmitic acid incorporation into inert triglycerides in order to deal with the lipid overload (Hu et al., 2018b).

3.9.2 ADVERSE EFFECTS Regarding adverse effects of mulberry fruits, there are insufficient studies about the safety of mul­ berry fruits and extracts and their recommended consumption. An extract of mulberry fruits com­ bined with leaf, administrated to obese mice for 12 weeks, did not show liver toxicity (Lim, Yang

Health Benefits of Mulberry Fruits

57

et al., 2013). However, a single dose of mulberry (2 g per 2 mL/kg of body weight) significantly reduced the bioavailability of cyclosporine, a potent immunosuppressant widely used in transplant patients (Hsu et al., 2013). In this way, the consumption of mulberry could diminish the drug effi­ ciency. For the best of our knowledge, until the present moment, no studies describing adverse effects of mulberry fruits were carried out, and their consumption can be considered as safe.

3.10 SUMMARY This chapter presents a concise overview on the nutritional and phytochemical composition of mul­ berry fruits and their health benefits. Recent studies are discussed concerning the evaluation of health function of mulberry fruits in terms of antioxidant activities, antidiabetic and antiobesity effects, protection against xenobiotic toxicity, and neuroprotection, among others. Mulberry fruits are recognized to present a high nutritional value and a high content of bioactive compounds, such as phenolic compounds and polysaccharides. Despite the phenolic content varied within fruit cul­ tivar and with environment conditions and processing treatments, the bioactive effect of different mulberry fruits, their derived products (juices, jams, alcoholic beverages), and bioactive compounds are widely supported by in vitro and in vivo studies using cell and animal models of disease. Up to now, human clinical trials on the bioavailability of mulberry bioactive compounds and on the pharmacological activities of mulberry fruits are still limited. Therefore, future human intervention studies should explore the effect of mulberry fruit consumption on human health and elucidate the mechanisms of action of the main bioactive compounds.

ACKNOWLEDGEMENTS The authors acknowledge iNOVA4Health-UID/Multi/04462/2013, a program financially supported by FCT/Ministério da Educação e Ciência, through national funds, and cofunded by FEDER under the PT2020 Partnership Agreement, and INTERFACE program (Innovation, Technology and Circular Economy Fund [FITEC]). P.R.A acknowledges CNPq (Brazilian National Scientific and Technological Development Council) for the postdoctoral scholarship (process 205295/2018-5).

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Wu, T., Yin, J., Zhang, G., Long, H., & Zheng, X. (2016). Mulberry and cherry anthocyanin consumption prevents oxidative stress and inflammation in diet-induced obese mice. Molecular Nutrition and Food Research, 60(3), 687– 694. doi: 10.1002/mnfr.201500734. Xie, X. H., Zhang, L., & Gao, X. (2017). Phenolic compounds content and antioxidant activity of mulberry wine during fermentation and aging. American Journal of Food Technology, 12, 367–373. Yan, F., Chen, X., & Zheng, X. (2017). Protective effect of mulberry fruit anthocyanin on human hepatocyte cells (LO2) and Caenorhabditis elegans under hyperglycemic conditions. Food Research International, 102, 213–224. doi: 10.1016/j.foodres.2017.10.009. Yang, D. K., & Jo, D. G. (2018). Mulberry fruit extract ameliorates nonalcoholic fatty liver disease (NAFLD) through inhibition of mitochondrial oxidative stress in rats. Evidence-Based Complementary and Alternative Medicine, 2018. doi: 10.1155/2018/8165716. Yildiz, O. (2013). Physicochemical and sensory properties of mulberry products: Gümüşhane pestil and köme. Turkish Journal of Agriculture and Forestry, 37, 762–771. doi: 10.3906/tar-1301- 41. You, Y., Yuan, X., Lee, H. J., Huang, W., Jin, W., & Zhan, J. (2015). Mulberry and mulberry wine extract increase the number of mitochondria during brown adipogenesis. Food & Function, 6(2), 401– 408. doi: 10.1039/C4FO00719K. Yu, Y., Li, H., Zhang, B., Wang, J., Shi, X., Huang, J., … Deng, Z. (2018). Nutritional and functional com­ ponents of mulberry leaves from different varieties: Evaluation of their potential as food materials. International Journal of Food Properties, 21(1), 1495–1507. doi: 10.1080/10942912.2018.1489833. Yu, Y., Xu, Y., Wu, J., Xiao, G., Fu, M., & Zhang, Y. (2014). Effect of ultra-high pressure homogenisation processing on phenolic compounds, antioxidant capacity and antiglucosidase of mulberry juice. Food Chemistry, 153, 114–120. Yuan, Q.,  & Zhao, L. (2017). The mulberry (Morus alba L.) fruit: A review of characteristic components and health benefits. Journal of Agricultural and Food Chemistry, 65, 10383–10394. doi: 10.1021/acs. jafc.7b03614. Zerega, N. J. C., Clement, W. L., Datwyler, S. L., & Weiblen, G. D. (2005). Biogeography and divergence times in the mulberry family (Moraceae). Molecular Phylogenetics and Evolution, 37(2), 402– 416. doi: 10.1016/j.ympev.2005.07.004. Zhang, H., Ma, Z. F., Luo, X., & Li, X. (2018). Effects of mulberry fruit (Morus alba L.) consumption on health outcomes: A mini-review. Antioxidants, 7(5), 1–13. doi: 10.3390/antiox7050069. Zhang, L, Wang, B., & Ecologica, Z. X.-S. X. (2011). Volatile constituents of four moraceous host plants of Apriona germari. Shengtai Xuebao/Acta Ecologica Sinica, 31(34), 7479–7485. Zhang, L., Xu, Y., Li, Y., Bao, T., Gowd, V., & Chen, W. (2017). Protective property of mulberry digest against oxidative stress—A potential approach to ameliorate dietary acrylamide-induced cytotoxicity. Food Chemistry, 230, 306–315. 10.1016/j.foodchem.2017.03.045. Zhao, W., Miao, X., Jia, S., Pan, Y., & Huang, Y. (2005). Isolation and characterization of microsatellite loci

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4

Biological Activities

of Mulberry Fruits for

Skin and Bone

Ariya Sarikaphuti Mae Fah Laung University

Yousef Rasmi Urmia University of Medical Sciences

Pornanong Aramwit Center of Excellence in Bioactive Resources for Innovative Clinical Applications and Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Chulalongkorn University The Academy of Science, The Royal Society of Thailand

CONTENTS 4.1

Background Information on Mulberry Fruit .......................................................................... 68

4.1.1 Nutrients in Mulberry Fruit ...................................................................................... 68

4.1.2 Bioactive Ingredients in Mulberry Fruits ................................................................. 71

4.1.3 P olyphenolics ............................................................................................................ 71

4.1.4 Anthocyanins ............................................................................................................ 72

4.1.5 F lavonols ................................................................................................................... 72

4.1.6 Phenolic Acids .......................................................................................................... 72

4.1.7 Polysaccharides......................................................................................................... 72

4.1.8 Melatonin .................................................................................................................. 72

4.1.9 O ther Bioactive Compounds ..................................................................................... 73

4.1.10 Correlation between the Antioxidant Activity and the Contents of Total

Anthocyanins, Total Polyphenols, Total Flavonoids, and Total Sugars ................... 73

4.2 Health Benefits of Mulberry Fruits and Anthocyanins and Their Mechanisms of Action .... 73

4.2.1 Antioxidant Properties .............................................................................................. 73

4.2.2 Immunomodulatory Activity .................................................................................... 74

4.2.3 Antiatherosclerosis Activity ..................................................................................... 74

4.2.4 Hypolipidemic Activity ............................................................................................ 74

4.2.5 Antihyperglycemic Activity ..................................................................................... 75

4.2.6 Neuroprotective Activity .......................................................................................... 75

4.2.7 Antitumor Activity.................................................................................................... 75

4.3 Properties of Anthocyanins .................................................................................................... 76

4.4 Effects of Anthocyanin on the Skin ....................................................................................... 79

4.5 Effects of Anthocyanin on Bone ............................................................................................ 82

4.6 The Use of Mulberry Fruit for Health Claims and Future Trends ......................................... 86

4.7 Summary ................................................................................................................................ 87

Acknowledgements ..........................................................................................................................87

References........................................................................................................................................ 87

67

68

4.1

Asian Berries: Health Benefits

BACKGROUND INFORMATION ON MULBERRY FRUIT

Mulberry belongs to the genus Morus in the Moraceae family and is globally distributed under var­ ied climatic conditions ranging from tropical to temperate (Yuan et al. 2015, Pel et al. 2017). There are 24 species of Morus and one subspecies with at least 100 known varieties (Ercisli and Orhan 2007), of which Morus alba L. is a dominant species among them because it can be easily culti­ vated and provides healthy foliage. It is an economically important plant that is used for sericulture because it is the sole food plant for the domesticated silkworm, Bombyx mori. Recently, numerous studies have revealed that edible plants are good sources of phytochemicals and play a prominent role in the maintenance of human health (Zafra-Stone et al. 2007). Mulberry has higher a content of polyphenols, including flavonoids, anthocyanins, and carotenoids. However, the contents of total anthocyanins and total flavonoids vary widely across the different species (Liang et al. 2012). The carbohydrate content of mulberry fruits ranges from 3% to 30%. Among them, glucose and fructose are the main sugars (Gundogdu et al. 2011). Mulberry fruits can be simply divided into three types: red mulberry (Morus rubra L.), black mulberry (Morus nigra L.), and white mulberry (M. alba L.). However, mulberry species cannot be identified using the color of their fruits. As mentioned earlier, white mulberry is widely grown, whereas other mulberry fruits are normally discarded due to their short storage life and lack of economy of scale (Butkhup et al. 2013). Several studies have reported the biological activities of mulberry fruits, such as the protective effects of the liver, strengthening of the joints, facilitating the discharge of urine and the lowering of blood pressure, laxative effects, hypoglycemic activities, expectorant, anthelmintic, odontalgic and emetic effects (Zhishen et  al. 1999), and antithrombotic (Yamamoto et  al. 2006), antioxidant (Naderi et  al. 2004), antimicrobial (Takasugi et al. 1979), anti-inflammatory (Kim and Park 2006), and neuroprotective effects (Kang et al. 2006). These activities are generated by phenolics, flavonoids (Ercisli and Orhan 2007), and anthocyanins (Lee et al. 2004). Anthocyanins are the most important constituent of mulberry fruits, which are a group of nat­ urally occurring phenolic compounds that are responsible for the color attributes and biological activities of mulberry fruits. There are also other compounds found in mulberry fruits as shown in Table 4.1. Cyanidin-3-glucoside and cyanidin-3-rutinoside are the major anthocyanins that perform all of the activities (Liu et al. 2004). The antioxidant properties of anthocyanins have drawn sub­ stantial attention since they can be used as a functional food that possesses a potential strategy for preventing acute central nervous system (CNS) injury (Gilgun-Sherki et al. 2002), cardiovascular diseases (Cuzzocrea et al. 2001), and asthma (Kirkham and Rahman 2006). There are several col­ ors of mulberry fruits, such as red, purple-red, or purple, which represent the different amounts of anthocyanins contained within them (Aramwit et al. 2010). Researchers have found that the purplecolored mulberry fruit, the sweetest type, contains more than five times the amount of anthocya­ nins, which is significantly different compared with the red-colored fruit (Aramwit et  al. 2010). Since sugars are the initial precursors in the biosynthesis of anthocyanins (Ruhnau and Forkmann 1988) and the sugar content regulates the intensity of the berry coloration, there is a relationship between anthocyanins and sugars in several berries (González-SanJosé and Diez 1992). In addition to anthocyanins, there are several nutrients in mulberry fruits, which will be mentioned in detail.

4.1.1

NUTRIENTS IN MULBERRY FRUIT

Mulberry fruits from M. alba L. contain abundant proteins, lipids, carbohydrates, fibers, minerals, and vitamins but a low number of calories, which can be a healthy food choice for consumers. It was found that 100 g of fresh mulberry fruits can produce 1.44 g of protein, which is higher than that of strawberries and raspberries (Yuan and Zhao 2017). A total of 18 amino acids, including all nine essential amino acids required by humans, are found in mulberry fruits. The essential amino acid/total amino acid ratio is 42%, which is close to the ratio of some high-quality protein foods such as milk and fish (Jiang and Nie 2015). The essential amino acid score of several essential

Mulberry Fruits for Skin and Bone

69

amino acids is more than 100, which meets the requirements of children or adults according to the Food and Agriculture Organization/World Health Organization/United Nations University (FAO/ WHO/ UNU) reference (Consultation 1991). Fatty acids are also found in mulberry fruits. Essential fatty acids are long-chain polyunsaturated fatty acids derived from linolenic, linoleic, and oleic acids, and they are necessary for the forma­ tion of healthy cell membranes, the proper development and functioning of the brain and nervous systems, and the production of hormone-like substances called eicosanoids (thromboxanes, leu­ kotrienes, and prostaglandins) (Ercisli and Orhan 2007). Polyunsaturated fatty acids are the most abundant fatty acids (76.68%) found in mulberry fruits, followed by monounsaturated fatty acids and saturated fatty acids (Sánchez-Salcedo et al. 2016). The fatty acid profile of mulberry fruits can

TABLE 4.1 Classes and Names of Compounds Isolated from Mulberry Fruits Compound Classes and Names Alkaloids 2α, 3β-Dihydroxynortropane 2β, 3β-Dihydroxynortropane 3β, 6exo-Dihydroxynortropane 2-[2-Formyl-5-(hydroxymethyl)-1-pyrrolyl-]3-methyl pentanoic aid lactone 4-[Formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanoate 4-[Formyl-5-(methoxymethyl)-1H-pyrrol-1-yl] butanoic acid 2-(5ʹ-Hydroxymethyl-2ʹ-formylpyrrol-1ʹ-yl)-3-(4-hydroxyphenyl) propionic lactone 2-(5ʹ-Hydroxymethyl-2ʹ-formylpyrrol-1ʹ-yl)-3-phenylpropionic acid lactone 2-(5-Hydroxymethyl-2ʹ, 5ʹ-dioxo-2ʹ, 3ʹ, 4ʹ, 5ʹ-tetrahydrobipyrrole) carbaldehyde 2-(5-Hydroxymethyl-2-formylpyrrol-1-yl) isovaleric acid lactone 2-(5-Hydroxymethyl-2-formylpyrrol-1-yl) propionic acid lactone 2-(Hydroxymethyl-2-formylpyrrol-1-yl) isocaproic acid lactone Methyl 2-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]-3-(4-hydroxyphenyl) propanoate Methyl 2-[2-formyl-5-(methoxymethyl)-1H-pyrrole-1-yl] propanoate Morroles B-F 2α, 3β, 4α-Trihydroxynortropane 2α, 3β, 6exo-Trihydroxynortropane Flavonoids Epigallocatechin Epigallocatechin gallate Gallocatechin Gallocatechin gallate Isorhamnetin glucuronide Isorhamnetin hexoside Isorhamnetin hexosylhexoside Kaempferol glucuronide Kaempferol hexoside Kaempferol hexosylhexoside Kaempferol rhamnosylhexoside Morin Naringin Quercetin Quercetin glucuronide Quercetin hexoside Quercetin hexosylhexoside Quercetrin Rutin

References Kusano et al. (2002) Kusano et al. (2002) Kusano et al. (2002) Kim et al. (2014) Kim et al. (2014) Kim et al. (2014) Kim et al. (2014) Kim et al. (2014) Kim et al. (2014) Kim et al. (2014) Kim et al. (2014) Kim et al. (2014) Kim et al. (2014) Kim et al. (2014) Kim et al. (2014) Kusano et al. (2002) Kusano et al. (2002)

Natić et al. (2015)

(Continued)

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Asian Berries: Health Benefits

TABLE 4.1 (Continued) Classes and Names of Compounds Isolated from Mulberry Fruits Compound Classes and Names Anthocyanins Cyanidin 3-O-glucoside Cyanidin 3-O-rutinoside Cyanidin 3-O-β-D-galactopyranoside Cyanidin 3-O-β-D-glucopyranoside Cyanidin 7-O-β-D-glucopyranoside Cyanidin galloylhexoside Cyanidin hexoside Cyanidin hexosylhexoside Cyanidin pentoside Cyanidin 3-O-(6ʺ-O-α-rhamnopyranosyl-β-D-galactopyranoside) Cyanidin 3-O-(6ʺ-O-α-rhamnopyranosyl-β-D-glucopyranoside) Cyanidin rhamnosylhexoside Delphinidin acetylhexoside Delphinidin hexoside Delphinidin rhamnosylhexoside Pelargonidin 3-O-glucoside Pelargonidin 3-O-rutinoside Pelargonidin hexoside Pelargonidin rhamnosylhexoside Petunidin rhamnosylhexoside Phenolic acids 3-O-caffeoylquinic acid Ellagic acid Ferulic acid Gallic acid Gentisic acid Hydroxybenzoic acid p-Hydroxybenzoic acid Hydroxyphenylacetic acid methyl ester Jaboticabin Methyl 3-O-caffeoylquinate Methyl 4-O-caffeoylquinate Methyl 5-O-caffeoylquinate Methyl dicaffeoylquinate Protocatechuic acid Protocatechuic acid ethyl ester Protocatechuic acid methyl ester Protocatechuic aldehyde Syringaldehyde Syringic acid Vanillic acid

References Qin et al. (2010)

Qin et al. (2010)

Du et al. (2008)

Du et al. (2008)

Du et al. (2008)

Natić et al. (2015)

Natić et al. (2015)

Natić et al. (2015)

Natić et al. (2015)

Du et al. (2008)

Du et al. (2008)

Natić et al. (2015)

Natić et al. (2015)

Natić et al. (2015)

Natić et al. (2015)

Qin et al. (2010)

Qin et al. (2010)

Natić et al. (2015)

Natić et al. (2015)

Natić et al. (2015)

Hisayoshi et al. (2004)

Hisayoshi et al. (2004)

Hisayoshi et al. (2004)

Hisayoshi et al. (2004)

Hisayoshi et al. (2004)

Memon et al. (2010)

Hisayoshi et al. (2004),

Dat et al. (2010)

Dat et al. (2010)

Dat et al. (2010)

Hisayoshi et al. (2004)

Hisayoshi et al. (2004)

Hisayoshi et al. (2004)

Hisayoshi et al. (2004)

Hisayoshi et al. (2004),

Dat et al. (2010)

Dat et al. (2010)

Dat et al. (2010)

Memon et al. (2010)

Memon et al. (2010)

Memon et al. (2010)

Memon et al. (2010)

be affected by variables including environmental and genetic factors (Liang et al. 2012). In general, linoleic acid (the most abundant and considered essential for disease prevention and human develop­ ment), palmitic acid, and oleic acid are the major fatty acids found in mulberry fruits. Mulberry fruits are an outstanding source of minerals, particularly potassium (521–1,718 mg/ 100 g dried weight) (Liang et  al. 2012), followed by calcium and phosphorus. High amounts of

Mulberry Fruits for Skin and Bone

71

ascorbic acid are present in the fresh fruit (~36.4 mg/100 g weight) (Yuan and Zhao 2017). The sodium content is low, and the potassium/sodium ratio is relatively high, which has been considered to be an advantage from a nutritional point of view (Dini et al. 2008). However, there were signifi­ cant differences in the levels of minerals among the tested mulberry cultivars. Mulberry fruits also provide other vitamins, such as thiamin, riboflavin, niacin, folate, vitamin A, vitamin B6, vitamin E, and vitamin K. Four types of tocopherols have been detected in mulberry fruits, mainly γtocopherol (0.245 mg/g) (Yang et al. 2010).

4.1.2

BIOACTIVE INGREDIENTS IN MULBERRY FRUITS

There are a variety of bioactive ingredients found in mulberry fruits. However, the amount of each bioactive ingredient may vary across studies. The differences depend not only on the cultivars used but also on the extraction and analytical methods, genetic differences, and growing conditions, including geographical and environmental conditions such as temperature, humidity, light, and degree of maturity (Mahmood et al. 2012a, Natić et al. 2015, Sánchez-Salcedo et al. 2015).

4.1.3

POLYPHENOLICS

Polyphenol is considered as a major active compound in several substances. It also possesses strong antioxidant activity, which is related to the prevention of various diseases. Table  4.2 shows main sources of polyphenols with antioxidant activity from natural resources. All berry fruits are rich sources of polyphenolics that can be classified based on their structural characteristics such as flavo­ noids (anthocyanins, flavanols or catechins, flavonols, flavones, flavanones, isoflavonoids), phenolic acids, stilbenes, tannins, or lignans (Liu 2004, Han et al. 2007). The intake of polyphenols is directly associated with a reduced risk of cancer, cardiovascular diseases, and neurodegeneration (Han et al. 2007, Del Rio et al. 2013). The total phenolic, flavonoid, and anthocyanin contents in mulberry fruit are 104.78–215.53 mg gallic acid equivalent (GAE)/100 g DW (dry weight), 64.55–211.01 mg catechin equivalent (CE)/100 g DW, and 45.42–208.74 mg cyanidin-3-O-glucoside (C3G) equivalent/100 g fro­ zen weight, respectively (Butkhup et al. 2013, Natić et al. 2015). The phenolic contents in mulberry fruits vary with different cultivars and maturity stages; normally, the phenolic contents increase as the fruits progress from the unripened to fully ripened stages (Mahmood et al. 2012).

TABLE 4.2 Main Sources of Polyphenols from Natural Resources with Antioxidant Activity (Bosch et al. 2015) Polyphenol Flavonoids Catechins: catechin, epicatechin, galactocatechin, epicatechingallate, epigallocatechin-3-gallate Isoflavones: Genistein Sylimarin Proanthocyanidins (tannins) Anthocyanins Nonflavonoids Phenolic acids Benzoic acids: Gallic acid Cinnamic acids Stilbene Resveratrol

Major Sources

Tea Soy Thistle Grapeseed, mangosteen Mulberry fruit, pomegranate Grape & derivatives Tea Polypodium leucotomos Grape & derivatives Grape, nuts, peanuts

72

Asian Berries: Health Benefits

4.1.4 ANTHOCYANINS Anthocyanins are a subclass of flavonoids and a major contributor to flower and fruit color, rang­ ing from red to blue and purple. Various anthocyanins have been found in mulberry fruits, and the principal anthocyanin is cyanidin-3-O-glucoside, followed by cyanidin-3-rutinoside (Chang et  al. 2013, Chen et al. 2016). Although the amount varies across strains, the amount of anthocyanin in mulberry fruit ranges from 19 to 193 mg/100 g (Liang et al. 2012).

4.1.5

FLAVONOLS

Mulberry fruits contain many flavonols, a subgroup of flavonoids, such as rutin, quercetin, myrice­ tin, and kaempferol. Derivatives of quercetin and kaempferol are the major components of mulberry fruit flavonols (Yuan and Zhao 2017). Normally, flavanols do not occur naturally as glycosides. Catechin, epigallocatechin gallate (EGCG), epicatechin, and procyanidins B1 and B2 have been found in mulberry fruits. It has been reported that rutin was the most abundant phenolic compound, contributing 44.66% of the total phenolics in 11 mulberry samples (Natić et al. 2015).

4.1.6

PHENOLIC ACIDS

Hydroxycinnamic acids and benzoic acid derivatives are the commonly found phenolic acids in mulberry fruits. Among the hydroxycinnamic acid derivatives, chlorogenic acid, ferulic acid, p-coumaric acid, o-coumaric acid, cinnamic acid, and caffeic acid are found in mulberry fruits. It has been reported that chlorogenic acid (5.3–17.3 mg/100 g DW) was the most abundant pheno­ lic acid in mulberry fruits (Mahmood et  al. 2012). Among benzoic acid derivatives, gallic acid, hydroxybenzoic acid, protocatechuic acid, and vanillic acid have been found, whereas gallic acid (7.33–23.34 mg/100 g DW) is considered a major benzoic acid derivative in mulberry fruit cultivars (Butkhup et al. 2013).

4.1.7 POLYSACCHARIDES Several polysaccharides have been found in mulberry fruits, and they are directly related to the biological properties. A glycoprotein with a carbohydrate content of 28.4% and a protein content of 71.6% isolated from the lyophilized powder of mulberry fruit juice (yield 10.6%) has shown better antiapoptotic activity than strawberry fruit polysaccharides (Liu and Lin 2014). Other researchers isolated and investigated the structural properties of a pectic-type mulberry fruit polysaccharides and found that the polysaccharide has a rhamnogalacturonan type I backbone composed of the repeating disaccharide fragments (4-α-D-GalpA-1→2-α-L-Rhap-1→). The arabinan side chain is composed of (1→5)-α-L-Ara attached to the O- 4 position of α-L-Rhap. The rhamnogalacturonan type II side chain was found to have a (1→6)-β-D-galactan core branched at O-3 by α-L-Araf (Choi et al. 2016a). Future research will determine the structure–activity relationship.

4.1.8

MELATONIN

Melatonin can be found in several plants, including mulberry fruits. Researchers have also reported on the dynamic changes in melatonin content during mulberry fruit development (from the fruit setting to ripening) and processing, such as alcohol fermentation (Wang et al. 2016). High levels of melatonin (5.76 ng/g fresh weight) were detected in stage I but then decreased in stage II and stage III. The melatonin level in mulberry wine was much higher than that in fruit – ranging from 28.11 to 31.59 ng/mL after ethanol fermentation at 16˚C and 25˚C, respectively.

Mulberry Fruits for Skin and Bone

73

4.1.9 OTHER BIOACTIVE COMPOUNDS Deep-colored fruits are good sources of carotenoids. It has been found that the β-carotene content in red-colored fruits was significantly higher than that in purple-colored and purple-red-colored fruits (Aramwit et al. 2010). Researchers have also isolated many pyrrole alkaloids, including some new compounds from mulberry fruits, among which some of the pyrrole alkaloids have shown sig­ nificant macrophage activation activity and pancreatic lipase inhibitory activity that are important for the regulation of obesity (Kim et al. 2013, Kim et al. 2014). Recently, 2 new, natural compounds and 13 known compounds were found, including the identified moracin C that has a benzofuran structure and shows cholesterol lowering activity (Yuan and Zhao 2017).

4.1.10 CORRELATION BETWEEN THE ANTIOXIDANT ACTIVITY AND THE CONTENTS OF TOTAL ANTHOCYANINS, TOTAL POLYPHENOLS, TOTAL FLAVONOIDS, AND TOTAL SUGARS Researchers have found a good linear correlation between the antioxidant activity and total poly­ phenols in the extracts of mulberry fruits (Liang et  al. 2012). Previous studies have shown that anthocyanins (especially monoglucosides of cyaniding and delphinidin) as well as nonanthocyanin phenolics (chlorogenic acid, kaempferol, quercetin, etc.) possess high antioxidant activity that is comparable with α-tocopherol and trolox (Kahkonen and Heinonen 2003). The correlation of anti­ oxidants with total polyphenols is better than the correlation with total anthocyanins (Liang et al. 2012). However, there was a negative linear correlation between the antioxidant activity and total sugars. There was a slightly better correlation between antioxidant activity and total polyphenol/ total anthocyanin compared with that of total flavonoids/total sugars, which means that polyphenols and anthocyanins significantly contribute to the antioxidant activities of mulberry fruits (Liang et al. 2012).

4.2 HEALTH BENEFITS OF MULBERRY FRUITS AND ANTHOCYANINS AND THEIR MECHANISMS OF ACTION 4.2.1

ANTIOXIDANT PROPERTIES

Mulberry fruits are well known for their antioxidant properties. The antioxidant properties of fruits, including mulberry fruits, are correlated well with the level of oxygen radical scavengers, such as phenolic compounds (Cai et al. 2004, Isabelle et al. 2008, Giampieri et al. 2012). It has been shown that polyphenol-rich mulberry fruit extracts can strongly inhibit lipid and linoleic acid oxi­ dation and exhibit concentration-dependent free radical scavenging activity against 2,2-diphenyl-l­ picrylhydrazyl (DPPH) , hydroxyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and superoxide anion radicals, as well as reducing power (Bae and Suh 2007, Du et al. 2008, Arfan et al. 2012); however, this activity differed with the cultivar. Nevertheless, the antioxidant activity was found to be dependent not only on the phenolic content but also on the identity of the phenolic compounds, the other phytochemicals present, and the synergistic effects among them (Li et  al. 2005, Cai et al. 2006). Mulberry fruit polysaccharides, especially galacturonic acid, showed good in vitro antioxidant activity (Chen et al. 2016), which may be because the carboxyl or carbonyl groups of mulberry fruit extracts may facilitate hydrogen atoms to bind to peroxy radicals and terminate the radical chain reactions (Chen et al. 2016). There was also a result indicating that 70% ethanol extracts of mulberry fruit could rescue hydrogen peroxide–induced oxidative injury of pancreatic MIN6N β cells via the inhibition of reactive oxygen species (ROS) accumulation and lipid per­ oxidation in a dose-dependent manner (J. S. Lee et  al. 2014). Moreover, mulberry fruit extracts can decrease ROS and O2− accumulation in HepG2 cells under oxidative stress conditions and, at the same time, increase mitochondrial numbers and mitochondrial membrane potential (Yan and Zheng 2017).

74

4.2.2

Asian Berries: Health Benefits

IMMUNOMODULATORY ACTIVITY

Several pyrrole alkaloids from mulberry fruits can significantly activate macrophage activity in RAW 264.7 cells by enhancing nitric oxide, TNF-α (tumor necrosis factor-alpha), and IL-12 (interleukin-12) production (Kim et al. 2013). Moreover, a glycoprotein isolated from mulberry fruits was examined for their immunomodulatory effects, and the results showed that it markedly decreased proinflam­ matory cytokines, including IL-1β and IL-6 and increased the anti-inflammatory cytokine IL-10 (Liu and Lin 2012). In addition, mulberry glycoproteins can increase the ratio of Bcl-2/Bak protein expression, which indicates enhanced cell viability via the inhibition of apoptosis.

4.2.3

ANTIATHEROSCLEROSIS ACTIVITY

Due to the association between antioxidant and inflammatory reactions, consumption of a diet rich in natural antioxidants is also related to attenuation of the development of atherosclerosis – a chronic inflammatory disease characterized by the accumulation of lipids in the arterial intima (Kaliora et al. 2006, Gendron et al. 2010). Researchers found that feeding 0.5% or 1.0% mulberry water extracts (containing 2.5% anthocyanins and 4.6% total phenolics) for 10 weeks significantly decreased plasma triglyceride levels in cholesterol-fed rabbits (Chen et al. 2005). Moreover, athero­ sclerotic lesions were significantly reduced by 42%–63% in the aorta compared with the control. Histological analysis revealed that the mulberry water extracts reduced the formation of foam cells and the migration of smooth muscle cells in the blood vessels of rabbits. In addition, Liu et al. indi­ cated that mulberry water extracts and mulberry anthocyanin extracts scavenged DPPH radicals and inhibited the relative electrophoretic mobility, the formation of thiobarbituric acid reaction substances, and ApoB fragmentation in oxidized low-density lipoprotein (LDL) induced by Cu2+ (Liu et al. 2008). Both extracts can also inhibit macrophage death induced by oxidized LDL and the formation of foam cells. However, the efficacy of mulberry anthocyanin extracts was tenfold higher than that of mulberry water extracts, which indicated that mulberry anthocyanins could decrease atherogenesis. In addition to the water and anthocyanin extracts, mulberry polyphenol extracts have been investigated for antiatherosclerosis activity in vascular smooth muscle cells (Chan et al. 2015). The results indicated that mulberry polyphenol extracts could arrest the A7r5 rat thoracic aorta smooth muscle cell cycle at the G0/G1 phase through induction of nitric oxide production and AMPK/p53 activation. The major active compounds of mulberry polyphenol extracts were further analyzed and found to be rutin and protocatechuic acid together with other polyphenols such as EGCG, caffeic acid, and naringenin.

4.2.4 HYPOLIPIDEMIC ACTIVITY A study indicated that freeze-dried mulberry fruits can reduce serum triglycerides, total choles­ terol, serum LDL cholesterol, liver triglycerides, liver total cholesterol, and the atherogenic index but increase serum high-density lipoprotein cholesterol in rats (Yang et  al. 2010). Furthermore, this effect was further evaluated in hypercholesterolemic subjects and found that the levels of total cholesterol and LDL cholesterol significantly decreased compared with the control group (Anchalee Sirikanchanarod et al. 2016). Regarding its mechanism of action, it was found that mulberry water extract treatment increased LDL receptor expression and the uptake of LDL but decreased the expression of HMG-CoA (3-hydroxy-3-methyl-glutaryl-CoA) reductase fatty acid synthase and glycerol-3-phosphate acyltransferase (Liu et al. 2009). The mulberry anthocyanin extracts suppressed triglyceride synthesis–related proteins and cholesterol biosynthesis–related proteins, whereas free fatty acid–related proteins were elevated, indicating that mulberry anthocyanin extracts regulated lipid biosynthesis and lipolysis to exert hypolipidemic effects (Chang et al. 2013). There was also evidence that the moracin C found in mulberry fruits inhibits proprotein convertase subtilisin-kexin

Mulberry Fruits for Skin and Bone

75

type 9 (PCSK9) mRNA expression and thereby decreases the degradation of LDL receptors, which could lower cholesterol levels (Pel et al. 2017).

4.2.5

ANTIHYPERGLYCEMIC ACTIVITY

A mulberry fruit extract rich in phenolics and flavonoids appeared to be a potent inhibitor of αglucosidase. The study in mice indicated that oral administration of mulberry fruit extracts for 2 weeks significantly reduced fasting blood glucose and glycosylated serum protein levels (Jiao et al. 2017). The polysaccharides obtained from mulberry fruits have also shown inhibitory effects on α-glucosidase, α-amylase, and the diffusion of glucose in vitro; moreover, these polysaccharides exhibited antidiabetic activity against rats with type 2 diabetes mellitus (Chen et al. 2015, Chen et al. 2016). Regarding the mechanism of action, mulberry anthocyanin extracts may accelerate glycogen synthesis, promote gluconeogenesis, and ameliorate insulin resistance via the PI3K/Akt pathway. Furthermore, in vivo studies have indicated that mulberry anthocyanin extracts improved glucose metabolic disorders in mice by activating protein kinase B phosphorylation and its downstream targets in insulin-sensitive tissues (Yan et al. 2016a,b). In diabetic rats, it was shown that mulberry fruit extracts can enhance insulin sensitivity, reduce hepatic glucose production, increase glucose transporter 4 (GLUT4) levels in skeletal muscle, and decrease glucose 6-phosphatase and phospho­ enolpyruvate carboxykinase levels in the liver. These effects are due to the increased phosphoryla­ tion of AMP-activated protein kinase and the 160-kDa Akt substrate (Choi et al. 2016b).

4.2.6

NEUROPROTECTIVE ACTIVITY

ROS-induced damage has been a great concern since it is related to several diseases. A study found that cyanidin-3-O-glucoside significantly increased the cell viability of oxygen–glucose-deprived PC12 cells (Kang et  al. 2006). In middle cerebral artery–occluded animal models, cyanidin-3­ O-glucoside offered more neuroprotective effects against cerebral ischemia than mulberry fruit extracts, which indicated that cyanidin-3-O-glucoside is a major neuroprotective constituent of mul­ berry fruit extracts. A study on Parkinson’s disease models found that mulberry fruit extracts sig­ nificantly protected neurons from neurotoxins through antioxidant and antiapoptotic effects (Kim et  al. 2010). Moreover, mulberry fruit extracts were also able to inhibit olfactory dysfunctions, ameliorate motor deficits and the degeneration of dopaminergic neurons, and inhibit the upregula­ tion of α-synuclein and ubiquitin, as a part of Lewy bodies (Gu et al. 2017). Another in vivo study indicated that dietary mulberry fruit extract supplementation could enhance memory, increase neu­ ron density, and reduce AChE activity in middle cerebral artery occlusion models (Kaewkaen et al. 2012). In addition, mulberry fruit extract–supplemented animals showed positive effects on Bcl-2immunopositive neuron density and antioxidase activity (Kaewkaen et al. 2012). Qiao et al. extracted artoindonesianin-O, a phenolic compound from mulberry, and found that artoindonesianin-O could lower Aβ42- or N-methyl-D-aspartate-induced neurotoxicity and reduced okadaic acid–induced tau hyperphosphorylation in neuronal cells (Qiao et al. 2015). This mechanism is associated with the inhibition of kinase p-ERK1/2 expression. Moreover, the number of dendritic spines increased after artoindonesianin-O treatment. These results strongly suggest that artoindonesianin-O exerts signifi­ cant neuroprotective effects on neurons.

4.2.7

ANTITUMOR ACTIVITY

Several reports have mentioned that not only crude extracts but also compounds obtained from mulberry fruits exhibit antitumor activity through different pathways, which is one of the most important biological activities of mulberry fruits. In 2006, Chen et al. found that mulberry antho­ cyanins inhibited the migration of A549 human lung carcinoma cells (Chen et al. 2006), similar

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Asian Berries: Health Benefits

to the mechanism found by other researchers (Huang et al. 2008). The treatment of human bladder cancer cells using mulberry water extracts together with paclitaxel in TSGH-8301 cells and TSGH8301 xenograft models found that the combination of mulberry water extracts and paclitaxel could arrest TSGH 8301 cells at the G2/M phase during the cell cycle, inducing mitotic catastrophe and inhibiting the generation of early endosomes, which may be associated with expression of PTEN (phosphatase and tensin) (Chen et  al. 2016). Further in vivo studies indicated that the combined treatment groups showed reduced tumor volume through activation of PTEN and caspase 3 expres­ sion. Not only paclitaxel but also mulberry water extracts have been combined with 5-fluorouracil and evaluated in mice transplanted with CT26 cells (Chang et al. 2015). The results indicated that leukocyte counts, spleen weight, NK (natural killer) cells, and CTL (cytotoxicity T lymphocyte) activity were significantly increased in the combination-treated tumor xenograft mice. A derivative from the mulberry fruit, odisolane, has also been investigated for its antitumor activity (Lee et al. 2016). Odisolane significantly inhibited tube formation or angiogenesis, a crucial role in tumor progression, in human umbilical vein vascular endothelial cells. The mechanism of its antiangiogenic effects is associated with inhibition of the expression of the vascular endothelial growth factor, p-Akt, and p-ERK proteins in the cells. Anthocyanins are water-soluble glycosides belonging to the flavonoid family. They are com­ monly referred to as anthocyanidins and comprise a variety of compounds, including pelargonidin, cyanidin, delphinidin, peonidin, petunidin, and malvidin (Krenn et al. 2007).

4.3

PROPERTIES OF ANTHOCYANINS

Anthocyanins are water-soluble pigments that produce bright colors, such as red, purple, and blue. They belong to a class of flavonoids, which are the largest family of polyphenolic compounds. The basic structure of an anthocyanin consists of a flavylium core (2-phenyl-1-benzopyrylium) with various sugar groups attached and two benzoyl rings separated by a heterocyclic ring (Figure 4.1). The sugar-free molecule is called an anthocyanidin. Depending on the number and position of the hydroxyl and methoxyl groups, various anthocyanidins have been described, and of those, six are commonly found in vegetables and fruits, which are pelargonidin, cyanidin, delphinidin, petunidin, peonidin, and malvidin. Anthocyanins always have a positive charge on the molecule, which enables it to absorb light and thus have color. The differences in the number of hydroxyl groups, the number and position of the sugars attached to the molecule, and the nature of the molecule together with the number of aliphatic and aromatic acids attached to the sugars contribute to the variety in chemical struc­ tures of anthocyanins (Yan et al. 2008). Anthocyanins are subdivided into the sugar-free anthocy­ anidin aglycones and the anthocyanin glycosides. Currently, more than 550 different anthocyanins have been reported to be found (Kong et al. 2003). Among the anthocyanin pigments, cyanidin-3glucoside is the major anthocyanin found in most plants.

FIGURE 4.1 Basic anthocyanin structure where R and R’ are functional groups.

Mulberry Fruits for Skin and Bone

77

The stability of anthocyanins depends on the type of anthocyanin pigments, copigments, light, temperature, pH, metal ions, enzymes, oxygen, intramolecular association, and intermolecular asso­ ciation with other compounds and antioxidants (Turturică et al. 2015). Anthocyanins are subject to physiochemical degradation in vivo and in vitro. Anthocyanidin’s stability is also influenced by the B-ring in the anthocyanidin structure and the presence of hydroxyl or methoxyl groups (CastañedaOvando et al. 2009). These groups are known to decrease the anthocyanidin stability in solution. B-ring hydroxylation status and pH have been shown to mediate the degradation of anthocyanins to their phenolic acid and aldehyde constituents (Woodward et al. 2009). Indeed, significant portions of ingested anthocyanins are likely to degrade into phenolic acids and aldehydes in vivo following consumption. This characteristic confounds the scientific isolation of specific anthocyanin mecha­ nisms in vivo. The color of anthocyanins depends on the pH of the solution. This is because of the ionic nature of the molecular structure of anthocyanins (Turturică et al. 2015). In acidic conditions, some antho­ cyanins appear red, a purple color is shown at neutral pH, and the color changes to blue under basic conditions (Khoo et al. 2017). The red-colored pigments of anthocyanins are predominantly in the form of flavylium cations (Bąkowska-Barczak 2005). These anthocyanins are more stable in acidic solution. At lower pH, the flavylium cation formed enables the anthocyanin to be highly soluble in water. The decrease in water concentration increases the rate of deprotonation of the flavylium cat­ ion, thus reducing color stability (Coutinho et al. 2015). In addition to the pH, anthocyanin–tannin polymerization could also increase the color stability at a lower pH (Sims and Morris 1985). At increasing pH conditions, the colorless carbinol pseudobase and chalcone structures are formed, followed by the formation of anionic quinonoidal species. This is due to the kinetic and thermody­ namic competition between the hydration reactions of the flavylium ion (Fossen et al. 1998). This blue quinonoidal species is unstable at lower pH. Under acidic conditions (pH 4–5), an anthocyanin solution has very little hue due to the small amounts of flavylium cation and quinonoidal anion (Khoo et al. 2012). At neutral pH, resonance-stabilized quinonoid anions (purple color of anthocya­ nins) are formed from further deprotonation of the quinonoidal species. The bioavailability of anthocyanins is also of great concern, and the structure of anthocyanins is a key factor that determines their bioavailability and bioactivity. Bioavailability is defined as the rate and extent to which a compound is absorbed and utilized by an organism to perform multiple physiological effects (Yousuf et al. 2016). Due to this content, bioavailability has been considered an essential index in evaluating the efficacy of bioactive compounds. Absorption is the main fac­ tor that influences the bioavailability of anthocyanins. The absorption rate varies depending on the molecular size, sugar moiety, and acylated groups. In addition, interference by other materials within the food matrix is also a considerable factor that affects absorption. An in vitro study indicated that anthocyanins with more free hydroxyl groups and fewer methoxyl groups had lower bioavailability (Yi et al. 2006). Anthocyanidin glycosides exhibited higher bioavailability than anthocyanidin galac­ tosides, while nonacylated anthocyanins have better absorption than acylated anthocyanins (Tsuda et al. 1996, Zhang et al. 2005). Studies also found that anthocyanins can be absorbed mainly in their intact glycosidic forms through the stomach and small intestine (Fang 2014) and can be detected in the plasma within a few minutes after intake (Milbury et al. 2002). An in vivo study showed that the highest absorption of anthocyanins occurred in the jejunum, whereas minor absorption occurred in the duodenum (Matuschek et al. 2006). However, both human (Czank et al. 2013) and mouse studies (Felgines et al. 2010) have demonstrated that most of the cyanidin-3-glucosides that enter the large intestine were excreted in feces. Although anthocyanins display high absorption in the gastrointesti­ nal tract, the bioavailability of anthocyanins is less than 1% (Bub et al. 2001, Matsumoto et al. 2001). Recent studies suggest that anthocyanins, similar to other flavonoids, are metabolized by the colonic microbiota (Aura et al. 2005, Keppler and Humpf 2005) and the metabolic function might be a direct result of metabolomic indicators rather than bioavailability (Vamanu et al. 2019). Most anthocyanin pigments have a high stability under acidic conditions compared with basic conditions, and degradation occurs at higher pH. Cyanidin and delphinidin are examples of

78

Asian Berries: Health Benefits

anthocyanidins, which are stable under acidic conditions. Anthocyanin becomes less stable when exposed to heat, causing a loss in color and browning. Numerous techniques have been used to enhance the stability of anthocyanins, such as microencapsulation using spray drying (Ersus and Yurdagel 2007) and copigmentation of anthocyanins with caffeic, ferulic, gallic, tannic, rosmarinic, ascorbic, and citric acids (Klimaviciute et al. 2015). Compared with the copigmentation method, spray drying is considered a simple technique to produce anthocyanin-encapsulated beads with high stability and a controllable size and degradation profile. Several materials have been used as carriers for encapsulation systems for anthocyanins using the spray drying method, such as natural gums (gum Arabic (Flores et al. 2014), mesquite gum (Jiménez-Aguilar et al. 2011), and gum acacia (Nayak and Rastogi 2010)), proteins (whey (Flores et al. 2014) and soy (Robert et al. 2010)), and polysaccharides (maltodextrin (Oidtmann et al. 2012), inulin (Bakowska-Barczak and Kolodziejczyk 2011), corn starch (Villacrez et al. 2014), yucca starch (Villacrez et al. 2014), alginate (Chatterjee and Bhattacharjee 2015), and alginate-chitosan (Kanokpanont et al. 2018)). The encap­ sulation technique not only stabilizes the anthocyanin content but also reduces the electrostatic charges of the materials, which makes filling them into capsules easier (Figures 4.2 and 4.3).

FIGURE  4.2 Microscopic image of anthocyanins encapsulated in alginate beads (×4, left), and scanning electron microscopic image of anthocyanins encapsulated in alginate beads (×500, right).

FIGURE  4.3 Anthocyanin powder filled into capsules (left), and encapsulated anthocyanins in alginate beads filled into capsules (right).

Mulberry Fruits for Skin and Bone

4.4

79

EFFECTS OF ANTHOCYANIN ON THE SKIN

For skin, the uses of anthocyanins vary from antiaging properties to psoriasis and wound healing. Starting from the most common use, solar ultraviolet (UV) radiation is the most ubiquitously dam­ aging environmental factor. Exposure to UV radiation, particularly UVB, can cause many adverse reactions, including erythema, hyperpigmentation, hyperplasia, immune suppression, photoaging, and skin cancer. Polyphenols, especially anthocyanins, have been reported as potentially effective agents for preventing signs of skin aging and protecting the skin from external injuries caused by UV radiation (Afaq and Katiyar 2011). A clear understanding of the role of UV radiation, ROS, inflammation, and extracellular matrix remodeling in skin pathophysiology has allowed researchers to propose specific molecular targets for anthocyanins, which are mainly involved in all processes, resulting in their effectiveness for skin protection. The well-known antioxidant activity of antho­ cyanins has been a promising strategy for their protection against UV skin damage. An in vitro study showed that a cosmetic formulation containing anthocyanins from purple sweet potato at a concentration of 0.61 mg/100 g cream base could absorb up to 46% of the incident UV radiation (Chan et al. 2010). The results from this study suggested that the topical application of anthocyanins from purple sweet potato at very low doses may prevent UV-induced skin damage by decreasing the amount of UVB radiation reaching the epidermis due to the ability of the anthocyanins to strongly absorb in the visible and UV spectra with maximum absorbances in the ranges of 500–550 nm and 280–320 nm (Harborne 1958). The UV absorption capacity of anthocyanins varies depending on their specific aglycones, sugar conjugation, and acylation patterns. It has been found that acylated anthocyanins containing coumaric acid, caffeic acid, and ferulic acid display enhanced adsorption of UVB radiation (Harborne 1958). Moreover, acidic ethanol–extracted anthocyanins have better radical scavenging ability, higher total phenolic content, and stronger reducing ability than acidic water– extracted anthocyanins (Chan et al. 2010). Skin protection from anthocyanins from other sources, such as berries, has been reported. An extract from blueberries rich in cyanidin-3-glucoside, petunidin-3-glucoside, malvidin-3-glucoside, and delphinidin-3-glucoside prevented UVB-induced overexpression of methane monooxygenases and upregulated the UVB-induced suppression of collagen synthesis in human fibroblasts (Bae et al. 2009). These results suggest that anthocyanins from blueberries may offer protection against photo­ aging. Another report by Cimino et al. (2006) found that cyanidin-3-O-glucoside inhibited the UVinduced translocation of the transcription factors NF-κB and AP-1 (activator protein 1) and other inflammatory responses in keratinocytes. According to their results, cyanidin-3-O-glucoside could provide multifaceted protection against skin damage since NF-κB and AP-1 are the key modulators of several cellular survival programs of skin cells, including the synthesis of inflammatory media­ tors, and are effectors of both innate and adaptive immunities. Cyanidin-3-O-glucoside was also found to prevent the UV-induced overexpression of IL-8, caspase-3 activation, and DNA fragmenta­ tion in human keratinocytes (Cimino et al. 2006). A similar effect was documented using anthocya­ nins from bilberry and human keratinocytes as a model of dermal UV-induced damage (Svobodova et al. 2008). They found that anthocyanins from bilberries reduce UVA-stimulated ROS formation and lipid peroxidation. Moreover, delphinidin, one of the most potent anthocyanins found in fruits and vegetables, could inhibit UVB-mediated oxidative stress and reduce DNA damage, thereby protecting the keratinocyte cells from UVB-induced apoptosis (Afaq et al. 2007). Regarding its mechanism of action, Tsoyi et al. proposed that anthocyanins protect against skin damage in the following ways: (1) by reducing the UVB-induced elevation of cyclooxygenase-2 and prostaglandin E2 through an NF-κB-dependent pathway (Tsoyi et al. 2008a) and (2) by preventing the apoptotic cell death by inhibiting caspase-3 activation and reducing proapoptotic Bax protein levels (Tsoyi et al. 2008b). However, this preclinical evidence is seemingly insufficient to conclude that anthocyanins are solely responsible for the skin-protective properties observed in vitro and in vivo because various polyphenols that are different from anthocyanins may be present in the tested materials. To confirm anthocyanin activity on the skin, a clinical study has been reported using

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Asian Berries: Health Benefits

multicomponent formulation containing anthocyanins and glutathione. The results showed that this formulation significantly reduced skin erythema after radiation therapy in patients with breast can­ cer (Miko Enomoto et al. 2005). Unfortunately, this study provided limited information regarding the specific group of anthocyanins and doses used for topical applications. Further investigation has been performed on the antiwrinkle effects of fruit-extracted antho­ cyanins. A combination of fruit extracts from Punica granatum, Ginkgo biloba, Ficus carica, and M. alba was formulated using a 2% combination extract in a basic formulation, and its efficacy against collagenase inhibition was evaluated together with the clinical antiwrinkle effect (Ghimeray et al. 2015). The results indicated that the fruit extracts could inhibit collagenase effectively in a dose-dependent manner. A concentration of 5 μg/mL extract showed 67.45% enzyme inhibition, whereas lower concentrations of 0.5, 1, and 2.5 μg/mL fruit extract caused 12.03%, 32.90%, and 55.61% enzyme inhibition, respectively. Moreover, the use of combination fruit extract cream on the wrinkles of crow’s feet found that treatment for 56 days significantly reduced the percent of wrinkle depth, length, and area by 11.5%, 10.07%, and 29.55%, respectively, compared with placebo. The dermatological scores of the sides treated with the extract containing the cream decreased signifi­ cantly after 56 days with a 1.5-fold lower score than that of the placebo treatment. Although this study reported the efficacy of wrinkle reduction from a combination fruit extract, it gives funda­ mental data regarding the use of anthocyanins in the cosmeceutical area. Skin pigmentation is a main concern for the skin. Moreover, skin pigmentation can be related to the wellness of the skin. Differences in racial skin pigmentation are due to variations in the amount of melanin produced by the melanocytes and deposited throughout the epidermis (Jimbow et al. 1976). Differences in skin melanin content are likely due to several factors, including (1) the rate of synthesis and decay of tyrosinase, (2) the activity of tyrosinase in melanosomes, (3) the rate of synthesis and melanization of melanosomes, (4) melanosome size, (5) the efficiency of melanosome transfer to keratinocytes, and (6) the rate of degradation of melanosomes in keratinocytes (Seiji et al. 1963, Quevedo 1969). Because tyrosinase is the rate-limiting enzyme for melanin synthesis, it seems likely that the racial differences in human skin color may be primarily due to differences in the tyrosinase activity in the melanocytes from various skin types (Giebel et al. 1990). In addi­ tion, the amount of melanin produced by human melanocytes is strictly dependent on the level of tyrosinase activity (Iozumi et  al. 1993). A previous study indicated that mulberry fruit extracts exhibited antityrosinase activity (Aramwit et al. 2010). However, different colors of mulberry fruits showed different levels of antityrosinase properties (Figure 4.4), which may be due to the various levels of anthocyanins found in mulberry fruits. Anti-inflammatory activity has also been found in an anthocyanin-rich extract (Chao et al. 2013). Topical applications of freeze-dried fruits have been shown to exert some antioxidant and anti-inflammatory activities of anthocyanins (Mallery et al. 2007). Chamcheu et al. also reported that delphinidin possesses antipsoriatic activity in vitro in cell­ free and cell cultures, as well as in vivo in preclinical imiquimod-induced psoriasis-like diseases in mice due to inhibition of the PI3K/Akt and mTOR signal transduction pathways (Chamcheu et al. 2017). This result also indicated the benefit of anthocyanins or their derivatives on skin treatment. In addition to the basic treatment of skin conditions, anthocyanins can also be applied for wound treatment since anthocyanins can promote cell migration and angiogenesis (Table  4.3). Anthocyanins are safe with potential uses as anti-inflammatory agents via cyclooxygenase inhibi­ tion and wound-healing agents due to the scavenging of ROS and stimulation of cell proliferation, migration, and angiogenesis (Castañeda-Ovando et al. 2009). Due to the water-soluble properties and poorly available sites for the local action of anthocyanins, Priprem et  al. formulated antho­ cyanins in niosomal-encapsulated vesicles that were dispersed in a mucoadhesive gel (Priprem et al. 2018). They found that complexation of anthocyanins is possible in the presence of zinc and caffeic acid, resulting in enhanced permeation and improved activity for the promotion of oral wound in rats, which is primarily determined by wound size reduction. The buccal wounds of rats treated with anthocyanin gels demonstrated both anti-inflammatory and wound-healing activities compared with those treated with placebo gel and fluocinolone gel. Moreover, a dose-dependent

Mulberry Fruits for Skin and Bone

FIGURE  4.4 activity.

81

Effect of various colors of mulberry fruit extracts on the inhibition of mushroom tyrosinase

manner for both activities was also observed within the concentration range of the anthocyanin complex of 1%–10%, with no adverse reactions found. The same formulation was further evaluated in healthy volunteers whose oral wounds were small in size and not inflamed to mildly inflamed and rapidly self-healing under normal circumstances (Limsitthichaikoon et al. 2018). Under normal circumstances, the wound-healing process starts within 1 day after being wounded, but the success of this formulation is related to its complex chemotactic and enzymatic activities as well as proper cell migration, adhesion, and specific protein regulations that occur between 2 and 7 days after wounding. The results found that the average size of 10% anthocyanin in mucoadhesive gel–treated wounds was approximately 70% of baseline since day 1 and significantly smaller than that of the placebo gel. There was only 8% anthocyanin in mucoadhesive gel–treated wounds but ≥20%–30% of the placebo gel–treated wounds, which were categorized as worse (larger size) and/or maintained (same size). Mucoadhesive gels containing anthocyanin also promote wound closure, which is one of the essential elements for wound healing. Full-thickness wounds in rats also showed improve­ ment after treatment with anthocyanin solution (5 mg in 0.1 mL) from the black soybean seed coat (Xu et  al. 2013). The wound size decreased dramatically in the group treated with anthocyanin solution compared with the control group (normal saline-treated wounds) from the first week of treatment up to 3 weeks after application. Further evaluation in cell culture found that anthocyanins increased VEGF (vascular endothelial growth factor) expression and decreased TSP1 (a well-known mediator that inhibits angiogenesis and suppresses wound healing) expression in the HaCaT cell line. It can be concluded that anthocyanins enhanced wound healing by increasing angiogenesis. Afaq et al. further evaluated the use of pomegranate fruit extracts containing anthocyanins and hydrolyzable tannins for their chemopreventive (the use of agents to slow the growth, delay the onset, or reverse carcinogenesis) activities (Afaq et al. 2005). They found that topical application of pome­ granate fruit extracts prior to 12-O-tetradecanoylphorbol-13-acetate (TPA, a potent tumor promoter) application to CD-1 mice resulted in a significant decrease in skin edema, hyperplasia, epidermal ornithine decarboxylase (ODC) activity, and protein expressions of ODC and COX-2, which are

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Asian Berries: Health Benefits

TABLE 4.3 Role of Anthocyanins on Cell Migration and Angiogenesis Fraction (s) Delphinidin chloride Delphinidin Cyanidin, delphinidin, pelargonidin, petunidin Delphinidin Cyanidin, delphinidin, malvidin Apigenin, delphinidin, ellagic acid, luteolin Quercetin Quercetin Pelargonidin chloride, pelargonidin-3-glucoside chloride Cyanidin, delphinidin, petunidin, pelargonidin, malvidin, peonidin Cyanidin-3-O-β-glucoside Crude extract Anthocyanins Anthocyanins Anthocyanins Ethanol extract

Anthocyanin extract

Overall Effect

References

10−2 g/L 25 μM 25 μM

Concentration

BAECs HUVECs HUVECs, PASMCs

Model

Antiangiogenesis Antiangiogenesis Antiangiogenesis

Martin et al. (2003) Lamy et al. (2005) Lamy et al. (2008)

10−2 g/L 0.3, 1, 3, 10 μM

HUVECs HUVECs

Antiangiogenesis Antiangiogenesis

5, 10, 15, 20, 25 μM

HUVECs, HMVECs

Antiangiogenesis

Duluc et al. (2014) Matsunaga et al. (2010a) Lamy et al. (2012)

0.1, 1, 10, 25, 50 μMol/L HUVEC HUVEC transgenic 50, 100, 200 μM zebrafish embryos HASMCs, HUVECs 10, 20, 40 μM

Antiangiogenesis Antiangiogenesis

Scoditti et al. (2012) Zhao et al. (2014)

Antiangiogenesis

Son et al. (2014)

2.5, 5, 10, 20, 40 μM

HASMCs Sprague– Dawley rats

Antiangiogenesis

Son et al. (2013)

0.2% (w/w) 0.075% (w/v) 0.3, 1, 3, 10, 30 μM

apoE−/− mouse model HPVAM C57BL/6 mice HUVECs apoE−/− mouse model

Proangiogenesis Antiangiogenesis Antiangiogenesis

Zhang et al. (2013) Liu et al. (2005) Matsunaga et al. (2010b) Mauray et al. (2012)

Diet supplemented with 0.02% of bilberry CAM 30 μL from 180 mL crude extract 31.3, 62.5, 125, 250, HUVEC CAM 500 μg/mL 25, 50, 100, 200 μg 0.002, 8, 15, 60, HUVEC Ibidi 120 μg/mL wound-healing assay

Antiangiogenesis Antiangiogenesis Antiangiogenesis

Antiangiogenesis

Vuthijumnonk et al. (2016) Bae et al. (2016)

Tsakiroglou et al. (2019)

BAECs, bovine aortic endothelial cells; CAM, chick chorioallantoic membrane; HASMCs, human aortic smooth muscle cells; HMVECs, human microvascular endothelial cells; HPVAM, human placental vein angiogenesis model; HUVECs, human umbilical vein endothelial cells; PASMCs, pulmonary aortic smooth muscle cells..

classical markers of inflammation and tumor promotion. Moreover, the inhibition of phosphorylation of mitogen-activated protein kinases (MAPKs) and activation of NF-κB/p65 and IKKα together with the degradation and phosphorylation of IκBα were found in mice treated with pomegranate fruit extracts. These data indicated that pomegranate fruit extracts containing anthocyanins could be a potent antitumor-promoting agent for skin cancer as well as other types of tumors because of their capability to inhibit conventional and novel biomarkers of TPA-induced tumor promotion.

4.5 EFFECTS OF ANTHOCYANIN ON BONE Age-related bone disease has become a public health concern, as average life expectancy has increased. One of the most prevalent forms of age-related bone disease today is osteoporosis, a

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decrease in the bone mineral density including calcium content, which is estimated to affect over 200 million individuals worldwide (Reginster and Burlet 2006). In osteoporosis, the body slows down bone formation, and existing bone is increasingly being resorbed to maintain the calcium bal­ ance (Lewiecki 2011). Complications of bone loss include increased susceptibility to falls, fragile bones, decreased quality of life, and increased risk of mortality (Lewiecki 2011). The causes of osteoporosis are diverse and complex. Major age-related risk factors for osteopo­ rosis include hormonal imbalance, resulting in osteoclast and osteoblast dysfunction, increased oxi­ dative stress, and chronic inflammation (Clarke and Khosla 2010). In women, the most prominent hormonal imbalance associated with age-related bone loss is the decrease in circulating estrogen levels following menopause. Estrogen deficiency has been shown to contribute to increased levels of ROS followed by increased pro-osteoclastogenic cytokine secretion from bone marrow cells (Lean et al. 2003). Estrogen levels are related to the inhibition of osteoclast differentiation (Chen et al. 2014). Moreover, estrogen deficiency is associated with an imbalance in bone metabolism, involving a net increase in bone resorption over formation, leading to excessive and sustained bone loss (Raisz 2005). This increase in bone resorption is the result of increased osteoclastogenesis and decreased osteoclast apoptosis (Raisz 2005, Reid 2013). The generation of oxidative stress and chronic inflammation associated with aging can lead to excess bone resorption, causing osteopo­ rosis (Clarke and Khosla 2010, Weaver et al. 2012). Estrogen deficiency may promote osteoclast resorption directly by stimulating the signaling associated with osteoclast differentiation and recep­ tor activator of NF-κB, or indirectly, by stimulating osteoblast/osteoclast coupling and subsequent osteoclast differentiation through RANK ligand (RANKL) (Ðudarić et al. 2015). Since berries can reduce oxidative stress (Jakesevic et al. 2011, Mane et al. 2011), they can also prevent these reactions and promote bone health. As the body ages, the skeleton becomes more susceptible to oxidative stress damage (Syed and Ng 2010, Almeida 2012). Oxidative stress is the result of an abundance of ROS with an insufficient antioxidant defense system that affects osteoblast and osteoclast regula­ tion (Sanchez-Rodriguez et al. 2007, Almeida 2012). The association between age-related bone loss and the level of oxidative stress (by monitoring advanced oxidation protein products, malondialde­ hyde, and superoxide dismutase) has been found in the femurs of young, adult, and old rats (Zhang et al. 2011). Levels of advanced oxidation protein products and malondialdehyde increase with age, whereas level of superoxide dismutase activity decreases (Hubert et al. 2014). A study indicated that femur bone mineral density was significantly lower in the adult groups compared with that of the young group and was positively correlated with superoxide dismutase activity, suggesting oxidative stress–induced age-related bone loss (Zhang et al. 2011). Normally, the body is able to prevent the excessive production of ROS through the body’s natural antioxidant defense system by produc­ ing enzymes such as superoxide dismutase, catalase, and glutathione (Almeida 2012, Almeida and O’Brien 2013). Mice with superoxide dismutase–deficient conditions exhibited lower bone mass, lower osteoblast and osteoclast numbers, decreased RANKL expression, and higher ROS levels compared with wild-type mice (Nojiri et al. 2011). There are several medications that have been approved by the Food and Drug Administration for the prevention and treatment of osteoporosis, called “antiresorptive medicine,” such as bisphospho­ nates, hormone replacements, and selective estrogen receptor modulators. However, some of these agents have adverse effects that limit their efficacy. For example, hormone replacement therapy is associated with an increased risk of stroke together with other heart conditions (Yang et al. 2013). Bisphosphonates may cause renal impairment, hypocalcemia, hypophosphatemia, influenza-like illnesses, gastrointestinal distress, and musculoskeletal pain (Sanders and Geraci 2013). For these reasons, attention has been paid to complementary and alternative medicines as a natural means of disease prevention. Antioxidant-rich foods, such as dietary polyphenols, may be an option to slow age-related bone loss and improve bone remodeling. Anthocyanins have been shown to reduce oxidative dam­ age and inflammation and increase bone mineral density (Basu et  al. 2010, Welch et  al. 2012). Researchers have found that berries could prevent the deterioration of whole-body bone mineral

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density and prevent bone turnover due to the antioxidative and anti-inflammatory properties of anthocyanins (Shen et al. 2012). Karlson et al. demonstrated that anthocyanins isolated from bil­ berries and blackcurrants efficiently suppressed lipopolysaccharide (LPS)-induced activation of NF-κB in cultured monocytes (Karlsen et al. 2007). Tanabe et al. reported that cranberry extract inhibited RANKL-dependent differentiation of human preosteoclasts and bone resorption activity of osteoclasts (Tanabe et al. 2011). Furthermore, a recent study by Moriwaki et al. demonstrated that anthocyanin compounds extracted from bilberries and blackcurrants inhibited osteoclast for­ mation from osteoclast precursor RAW264.7 cells (Moriwaki et al. 2014). Devareddy et al. demon­ strated that ovariectomized rats fed with blueberry powder supplements (5% w/w) for 4 months had a higher overall whole-body bone mineral density compared with rats in the ovariectomized group (Devareddy et al. 2008). Additionally, they identified that the supplement treatment downregulated ovariectomized-induced elevation of alkaline phosphatase (ALP), collagen, and tartrate-resistant acid phosphatase gene expression, suggesting that the bone-protective effect of blueberries may be due to the suppression of bone turnover. More recently, Zheng et al. identified that ovariectomized mice given a diet containing 1% anthocyanin-rich blackcurrant extracts for 12 weeks had signifi­ cantly greater femur bone mineral density compared with ovariectomized control mice (Zheng et al. 2016). Notably, they demonstrated that the extract reduced the number of tartrate-resistant acid phosphatase-positive osteoclast-like cells and bone resorption activity and concluded that the extract may alleviate bone loss by suppressing osteoclastogenesis and osteoclast function. Another in vitro study investigated the effect of cyanidin-3-glucoside on rat bone marrow–derived mesen­ chymal stem cells and found that growth media mixed with cyanidin-3-glucoside at 50 and 100 μmol could enhance the ALP activity and calcium deposition of bone marrow–derived mesenchy­ mal stem cells; the mulberry extract, which possessed the same amount of cyanidin-3-glucoside, reduced these signs of osteogenic differentiation under the same conditions (Yamdech 2012). These findings indicate that anthocyanins extracted from berry fruits may alleviate bone resorption and bone density loss following menopause in women. However, evidence of the bone-protective effects of anthocyanins from berries outside of an estrogen-deficient model is still limited. In vitro experi­ ments using murine bone marrow macrophages found that anthocyanins suppressed nicotinamide adenine dinucleotide phosphate oxidase, NOX (NOX1 and NOX2), and mRNA expression by over 60% (Lee et al. 2014). This reduction consequently downregulated nuclear factor (erythroid-derived 2)–like 2 (Nrf2) mRNA expression, suggesting that the NOX pathway was the major source of ROS production and that berry anthocyanins effectively inhibited the NOX pathway, thus reduc­ ing ROS production (Melough et  al. 2017). In cultured RAW 264.7 macrophages, anthocyanins significantly inhibited LPS-induced inflammation, as indicated by lower mRNA levels of TNF-α and interleukin-1β and lower nuclear p65 levels, indicating decreased NF-κB activity (Lee et al. 2014). TNF-α plays a central role in inflammation-mediated bone loss by augmenting osteoblastic RANKL-induced osteoclastogenesis and directly stimulates osteoclast formation (Komine et  al. 2001). These results indicated that anthocyanins may be an effective dietary supplement to prevent aging-associated bone deterioration, not only during estrogen deficiency but also directly by inhibit­ ing NOX-mediated formation and indirectly by reducing bone resorption by lowering ROS forma­ tion (Melough et al. 2017). In addition to anthocyanins, some berries are a good source of vitamin C (Del Rio et al. 2010), which also demonstrated positive effects on reduced bone resorption and enhanced bone health (Nieves 2013) and may be a confounding factor in attributing only anthocya­ nins to the preventive effects of osteoporosis. Some studies have indicated that anthocyanin supplementation may not be effective in promot­ ing bone mass for all ages (Sakaki et al. 2018). An in vivo study suggested that blackcurrant, which contains a high amount of anthocyanins, consumption improves trabecular bone mass in young mice that have not already lost a substantial amount of bone mass due to aging. The underlying mechanism appears to be related to an improved antioxidant defense, as evidenced by the increase in glutathione peroxidase activity, although the exact mechanism remains unclear. Blackcurrants were able to moderately reduce inflammation in aged mice but were unable to affect bone mass,

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presumably because there was very little bone to rescue. These data indicated that early consump­ tion of anthocyanin-containing fruits is crucial in preventing age-related bone loss and that the beneficial effects on bone morphology may not be apparent if the dietary intervention is initiated late in age (Sakaki et al. 2018). Phenolic compounds, mainly flavonoids, have a significant role in bone metabolism (Sacco et al. 2013); however, the mechanisms are not fully understood. Polyphenols may work through the reg­ ulation of osteoblasts and osteoclasts (Williams et  al. 2004, Trzeciakiewicz et  al. 2009). It has been proposed that polyphenols affect bones through the upregulation of osteoblastogenesis and downregulation of osteoclastogenesis through a variety of mechanisms (Shen et al. 2012). These include upregulating mechanisms related to transcription factor-2 (Runx2), osteocalcin, the canoni­ cal Wnt signaling pathway, β-catenin, and IGF-1, together with the downregulation of RANKL, TRAP, and several matrix metalloproteinases (MMPs) (Shen et al. 2012). It was reported that the canonical Wnt signaling pathway includes a series of growth factors and cascading phosphoryla­ tion signals regulated by the protein β-catenin to increase the transcription levels of genes respon­ sible for osteoblast proliferation and differentiation (Weaver et al. 2012). β-Catenin synergizes with bone morphogenetic protein-2 to enhance osteoblast differentiation and bone formation during the activation of Runx2 (Mbalaviele et al. 2005). When the oxidative stress is introduced, β-catenin is diverted from the T cell factors to FoxO-mediated transcription, attenuating osteoblastogenesis and bone formation (Mbalaviele et al. 2005). It has been predicted that berries, including mulberry, influence the activation of the Wnt signaling pathway through the phosphorylation of MAPK 38 (Weaver et al. 2012), which is the key regulator in bone loss and inhibition of osteoclastogenesis (Boyle et  al. 2014). In an in vivo experiment in which rats are fed with a blueberry diet, it was found that the phosphorylation of MAPK p38 in femur bone tissue was significantly higher than that in the control-fed rats (Chen et al. 2010). Moreover, the blueberry-fed rats also exhibited an increase in β-catenin and Runx2 expression, and an overall higher bone mass (Chen et al. 2010). To explore this mechanism in vitro, ST2 cells (a bone marrow–derived stroma cell line that is known to undergo osteoblastogenesis in response to Wnt) were treated with serum from male and female blueberry diet and control-fed rats (Chen et al. 2010). The results indicated that ST2 cells treated with the serum from blueberry-fed rats exhibited higher amounts of osteoblast differentiation along with increased ALP levels, osteocalcin gene expression, and mRNA expression of osteoprotegerin (OPG). Phosphorylation of MAPK p38 and activation of β-catechin also increased, indicating the possible role of the Wnt signaling pathway. To confirm this pathway, the β-catenin gene was silenced in some ST2 cells and treated with serum from blueberry-fed rats. The results showed no observed increase in osteoblast differentiation, indicating a connection between the berries and activation of the Wnt/β-catenin pathway (Chen et al. 2010). Since most berries, including mulberry fruits, contain polyphenols, polyphenols have also been investigated, and it was found that polyphenols act through the OPG/RANKL/RANK pathway. OPG, which is produced by osteoblasts, inhibits osteoclastogenesis by interfering with the binding of RANKL to RANK (Trzeciakiewicz et al. 2009). In the rats that are fed with berries, expres­ sion of RANKL mRNA, an osteoclast differentiation marker, decreased in the isolated femur. Osteoclastogenesis was also impaired in these rats. Polyphenol-containing berry-fed rats were asso­ ciated with an increased number of osteoblasts and a decreased number of osteoclasts in vivo (Chen et al. 2010). Additional evidence to support this hypothesis has also been found (Tanabe et al. 2011). Fruits containing proanthocyanidins have been shown to inhibit osteoclast formation, impair cell maturation, and decrease bone resorption in human preosteoclastic cells isolated from human bone marrow (Tanabe et al. 2011). At the transcriptional level, flavonoids affect the expression of NF-κB, which controls the genes involved in the inflammatory process, and normally increase during oxidative stress in the MAPK pathway (Trzeciakiewicz et al. 2009). The polyphenol action on osteoblasts counteracts the effects of osteoclasts due to oxidative stress. Cyanidin, a potent antioxidant, can reduce oxidative stress both in vitro (Choi et al. 2010) and in vivo (Tsuda et al. 2000). Berries rich in cyanidins demonstrated

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anti-inflammatory properties by inhibiting the release of IL-12 in murine bone marrow–derived dendritic cells (Dai et al. 2007), providing evidence of the anti-inflammatory effect of cyanidin, which may account for the effect of anthocyanins on bone. Mulberry fruits together with blueberries have been shown to increase the proliferation of human bone marrow cells, which are also known to be osteoblast progenitor cells (Bickford et al. 2006, Yamdech 2012). Regarding clinical investigations, several studies have evaluated the effects of anthocyanins or anthocyanin-rich foods on metabolic markers or disease risk, especially for cardiovascular diseases and diabetes. However, clinical investigation of the effects of anthocyanins on bone is limited. A clinical study evaluated the effect of the consumption of fermented milk (providing 25 and 20% recommended dietary allowance of calcium and vitamin D, respectively) enriched with a blueberry extract rich in anthocyanins on bone biomarkers in postmenopausal women (Davicco et al. 2013). The results indicated that consumption of the fermented milk enriched with calcium, vitamin D, and blueberry extracts containing high amounts of anthocyanins for 3 months corrected the insuffi­ ciency of vitamin D in postmenopausal women and resulted in improved bone formation. Moreover, its consumption significantly improved serum bone ALP (a marker for osteoblast activity) with­ out any significant change in C-telopeptide cross-linking levels (a marker of bone resorption). Effectively, in a population from the Aberdeen Prospective Osteoporosis Screening Study (APOSS) of Scottish women aged 54.8 years between 1997 and 1999, high intakes of dietary anthocyanins (22 μg/day) were associated with increased bone mineral density and decreased markers of bone resorption (pyridinoline and deoxypyridinoline) (MC Donald et al. 2012). Another clinical trial investigated 3,160 women, a cohort of female twins from the TwinsUK registry, and elucidated the correlation between flavonoid consumption, distinguishing between subclasses (flavanones, anthocyanins, flavan-3-ols, oligomeric proanthocyanidins, flavonols, and flavones) and the women’s bone density (Welch et al. 2012). The results indicated that total flavo­ noid consumption was positively associated with bone mineral density, with anthocyanins resulting in greater bone density of both the hip and spine.

4.6

THE USE OF MULBERRY FRUIT FOR HEALTH CLAIMS AND FUTURE TRENDS

Mulberry fruits are composed of several nutrients and bioactive compounds. They also possess various pharmacological properties. However, to apply the active ingredient of mulberry fruits for health and medical applications, the molecular mechanisms of their biological activities need to be explored in depth. The pharmacokinetics need to be further investigated. Although the chemical composition of mulberry fruits has already been studied extensively, there may still be unknown compounds that contribute to their biological activities. A standardized set of analytical method­ ologies is clearly desirable. The availability of rigorous methods providing more homogeneous results would promote more rapid and productive comparisons between different studies. The current analytical methods have some limitations, such as underestimating anthocyanin levels in plasma and urine. Anthocyanins transformed during metabolism are unable to return to the red flavylium cation following reacidification during sample preparation and thus will not be detected by the current methods of analysis using simple high-performance liquid chromatography (Pojer et al. 2013). Moreover, a consistent phytochemical profile should be developed for consumption and clinical studies. Despite the low bioavailability of anthocyanins, several studies have suggested that the metabolites may actually be responsible for many of their health-promoting activities. This hypothesis should be investigated in new nutritional studies. Sophisticated techniques, such as mass spectrometry–based metabolomics, should be applied for measuring both the native compounds and their main metabo­ lites produced in the organism in the same experiment, together with the perturbations induced by the transitory presence of anthocyanins on endogenous metabolic pathways.

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Numerous studies suggest that anthocyanins may be positively implicated in human health. In addition to their effects on skin and bone, they also exhibit antidiabetic and antiobesity effects and act as neuroprotective agents (Prior and Wu 2006, Tsuda 2012). These compounds exert cardio­ vascular protection (He and Giusti 2010) and inhibit cancer growth (Thomasset et al. 2009). An interesting study found that the intravenous administration of cyanidin-3-glucoside alters certain important cellular metabolites such as bile acids, glutathione, oxidized glutathione, and some lipids in the blood, kidneys and liver of rats (Vanzo et al. 2013). They also reported that anthocyanins affect mammalian metabolism in the brain and plasma, which may be related to several bioactivi­ ties, such as cardiovascular protection and vision improvement. Due to the benefit of anthocyanins on health, it has been traditionally used as folk medicine for the treatment of several conditions such as hypertension, pyrexia, liver disorders, dysentery and diarrhea, urinary problems, and the common cold. Recently, anthocyanin-containing materi­ als are being incorporated into food products such as drink or jam. It has been also widely used as colorants. Anthocyanin applications in food systems are preferably used in acidic food to assure a predominance of the flavylium cation (Shipp and Abdel-Aal 2010). For modern pharmaceuti­ cal area, anthocyanins including mulberry fruit extracts have been developed into a dietary food supplement. Anthocyanins are being sold as supplement called Medox® which incorporates a con­ centrated amount of cyanidin-3-glucoside and delphinidin-3-glucoside extracted from Vaccinium myrtillus and Ribes nigrum (BiolinkGroup 2019). Anthocyanin-containing red rice is also being fermented and sold as a dietary supplement and marketed as Cholestin® to help reduce choles­ terol levels (Pharmanex 2004). Moreover, blue wheat bran can be furthered processed to produce anthocyanin-rich blue wheat powder as a dietary supplement (Pharmanex 2004, Abdel-Aal et al. 2008). Increased development of anthocyanins with enhanced stability and prolonged shelf life will increase food applications and overall consumption and thereby their positive role in human health.

4.7

SUMMARY

The prevention of disease has become increasingly important in modern society, and it is generally understood that certain food sources and compounds found naturally in foods can play an important role in helping organisms to remain healthy. Anthocyanins are natural bioactive agents that clearly facilitate the transfer of information from nutritional and pharmacological research into practical advice toward health-concerned consumers. The benefits of anthocyanins from several sources have been explored intensively. Anthocyanins are safe and exert several health benefits, and the advan­ tages of these compounds have been found in almost all systems. However, to develop these natural substances as food supplements for the prevention or treatment of certain conditions in modern medicine, standardization of the material together with the effective doses for each application needs to be further investigated.

ACKNOWLEDGEMENTS This project is funded by National Research Council of Thailand.

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Sakaki, J., Melough, M., Lee, S. G., Kalinowski, J., Koo, S. I., Lee, S. K. and Chun, O. K. (2018). Blackcurrant supplementation improves trabecular bone mass in young but not aged mice. Nutrients, 10: 1671. Sanchez-Rodriguez, M. A., Ruiz-Ramos, M., Correa-Munoz, E. and Mendoza-Nunez, V. M. (2007). Oxidative stress as a risk factor for osteoporosis in elderly Mexicans as characterized by antioxidant enzymes. BMC Musculoskeletal Disorders, 8: 124. Sánchez-Salcedo, E. M., Mena, P., García-Viguera, C., Martínez, J. J. and Hernández, F. (2015). Phytochemical evaluation of white (Morus alba L.) and black (Morus nigra L.) mulberry fruits, a starting point for the assessment of their beneficial properties. Journal of Functional Foods, 12: 399–408. Sánchez-Salcedo, E. M., Sendra, E., Carbonell-Barrachina, Á. A., Martínez, J. J. and Hernández, F. (2016). Fatty acids composition of Spanish black (Morus nigra L.) and white (Morus alba L.) mulberries. Food Chemistry, 190: 566–571. Sanders, S. and Geraci, S. A. (2013). Osteoporosis in postmenopausal women: considerations in prevention and treatment: (Women’s health series). Southern Medical Journal, 106: 698–706. Scoditti, E., Calabriso, N., Massaro, M., Pellegrino, M., Storelli, C., Martines, G., De Caterina, R. and Carluccio, M. (2012). Mediterranean diet polyphenols reduce inflammatory angiogenesis through MMP-9 and COX-2 inhibition in human vascular endothelial cells: a potentially protective mecha­ nism in atherosclerotic vascular disease and cancer. Archives of Biochemistry and Biophysics, 527: 81– 89. Seiji, M., Shimao, K., Birbeck, M. S. and Fitzpatrick, T. B. (1963). Subcellular localization of melanin biosyn­ thesis. Annals of the New York Academy of Sciences, 100: 497–533. Shen, C. L., von Bergen, V., Chyu, M. C., Jenkins, M. R., Mo, H., Chen, C. H. and Kwun, I. S. (2012). Fruits and dietary phytochemicals in bone protection. Nutrition Research, 32: 897–910. Shipp, J. and Abdel-Aal, E. (2010). Food applications and physiological effects of anthocyanins as functional food ingredients. The Open Food Science Journal, 4: 7–22. Sims, C. A. and Morris, J. R. (1985). A comparison of the color components and color stability of red wine from noble and cabernet sauvignon at various pH levels. American Journal of Enology and Viticulture, 36: 181–184. Sirikanchanarod, A., Bumrungpert, A., Kaewruang, W., Senawong, T. and Pavadhgul, P. (2016). The effect of mulberry fruits consumption on lipid profiles in hypercholesterolemic subjects: a randomized controlled trial. Journal of Pharmacy and Nutrition Sciences, 1: 7–14. Son, J., Jeong, H., Kim, H. B., Kim, Y., Lee, E., Lee, H. and Lee, K. (2014). Pelargonidin attenuates PDGFBB-induced aortic smooth muscle cell proliferation and migration by direct inhibition of focal adhesion kinase. Biochemical Pharmacology, 89: 236–245. Son, J., Lee, E., Jung, S., Kim, J., Oak, M., Lee, K. and Lee, H. (2013). Anthocyanidins, novel FAK inhibi­ tors, attenuate PDGF-BB-induced aortic smooth muscle cell migration and neointima formation. Cardiovascular Research, 101: 503–512. Svobodova, A., Rambouskova, J., Walterova, D. and Vostalova, J. (2008). Bilberry extract reduces UVAinduced oxidative stress in HaCaT keratinocytes: a pilot study. Biofactors, 33: 249–266. Syed, F. A. and Ng, A. C. (2010). The pathophysiology of the aging skeleton. Current Osteoporosis Reports, 8: 235–240. Takasugi, M., Nagao, S., Masamune, T., Shirata, A. and Takahashi, K. (1979). Structures of moracins E, F, G, and H, new phytoalexins from diseased mulberry. Tetrahedron Letters, 20: 4675–4678. Tanabe, S., Santos, J., La, V. D., Howell, A. B. and Grenier, D. (2011). A-type cranberry proanthocyani­ dins inhibit the RANKL-dependent differentiation and function of human osteoclasts. Molecules, 16: 2365–2374. Thomasset, S., Teller, N., Cai, H., Marko, D., Berry, D. P., Steward, W. P. and Gescher, A. J. (2009). Do anthocyanins and anthocyanidins, cancer chemopreventive pigments in the diet, merit development as potential drugs? Cancer Chemotherapy and Pharmacology, 64: 201–211. Trzeciakiewicz, A., Habauzit, V. and Horcajada, M. N. (2009). When nutrition interacts with osteoblast func­ tion: molecular mechanisms of polyphenols. Nutrition Research Reviews, 22: 68–81. Tsakiroglou, P., Weber, J., Ashworth, S., Del Bo, C. and Klimis-Zacas, D. (2019). Phenolic and anthocyanin fractions from wild blueberries (V. angustifolium) differentially modulate endothelial cell migration partially through RHOA and RAC1. Journal of Cellular Biochemistry, 120: 11056–11067. Tsoyi, K., Park, H. B., Kim, Y. M., Chung, J. I., Shin, S. C., Lee, W. S., Seo, H. G., Lee, J. H., Chang, K. C. and Kim, H. J. (2008a). Anthocyanins from black soybean seed coats inhibit UVB-induced inflamma­ tory cylooxygenase-2 gene expression and PGE2 production through regulation of the nuclear factorkappaB and phosphatidylinositol 3-kinase/Akt pathway. Journal of Agricultural and Food Chemistry, 56: 8969– 8974.

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5

Black Mulberry Juice Merve Tomas Istanbul Sabahattin Zaim University

Gamze Toydemir Alanya Alaaddin Keykubat University

Esra Capanoglu Istanbul Technical University

CONTENTS 5.1 5.2 5.3 5.4 5.5

Introduction ............................................................................................................................99

Black Mulberry Fruits, Growing Conditions, and Harvest .................................................. 100

Chemical Composition of Black Mulberry Fruits and Their Juice ...................................... 100

Phytochemicals in Black Mulberry Fruits and Their Health Benefits ................................. 102

Effect of Juice Processing Technology and Process on Antioxidants of Black Mulberry Fruits..................................................................................................................... 106 5.6 Summary .............................................................................................................................. 109

References ...................................................................................................................................... 109

5.1 INTRODUCTION Black mulberry (Morus nigra L.) is a member of Morus genus in the Moraceae family, growing from temperate to subtropical regions of the northern hemisphere and across an extensive range of climatic, geographical, and soil conditions (Figure 5.1) (Ercisli & Orhan, 2007; Tutin, 1996; Vijayan et al., 1997). Black mulberry, originating from Iran, is currently cultivated in Southern Europe and Southwest Asia and is recognized as one of the most important fruits in Mediterranean countries (Ercisli & Orhan, 2007; Hojjatpanah et al., 2011). The fruits of black mulberry are getting more popular every day for their flavor, nutritional quali­ ties, and especially for their health-promoting bioactive substances. As there is increasing interest in black mulberry fruits, it is important to know the variability in chemical characteristics of different black mulberry genotypes cultivated worldwide (Koyuncu et al., 2014). The consumption of black mulberries has been proposed to reduce the risk of several chronic diseases such as cardiovascular diseases and certain types of cancer. These health-protective effects have been widely attributed to the presence of antioxidants (Tomas et al., 2015). Furthermore, black mulberries have gained an important position in the food industry due to the presence of anthocya­ nins. The major compounds identified were cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside (Hojjatpanah et al., 2011). Black mulberry fruits are consumed as fresh or processed into different products including juice, jam, paste, and ice cream. However, processing may trigger several reac­ tions, which may cause undesirable changes in the physicochemical and sensory properties of the processed foods as well as their antioxidant status (Al-juhaimi et al., 2018). On the other hand, both consumers and producers have been searching methods for the maximum level of nutrient retention, due to their increasing demands for healthier products. Therefore, great attention should also be given to avoid or minimize the detrimental effects of technological processing methods (Capanoglu et al., 2010). These requirements lead to alternative methods that involve minimal processing. In 99

100

Asian Berries: Health Benefits

(a)

(b)

(c)

FIGURE  5.1 Mulberry species. (a) Black mulberry (Morus nigra L.). (b) White mulberry (Morus alba L.). (c) Red mulberry (Morus rubra L.).

this sense, nonthermal technologies such as high hydrostatic pressure, pulsed electric field (PEF), and ultrasound have emerged as alternative techniques in food processing to improve nutritional qualities of processed foods (Nayak et al., 2015). The aim of this chapter is to provide a critical evaluation of nutritional compositions and health benefits of black mulberry fruits and juice. Moreover, this chapter also aims to review the effects of juice processing technologies on black mulberry antioxidants.

5.2 BLACK MULBERRY FRUITS, GROWING CONDITIONS, AND HARVEST Black mulberries (M. nigra L.) can grow over a wide range of climatic conditions from warm tem­ perate to subtropical and tropical. While it requires an ideal range of temperature between 24˚C and 28˚C, it can also tolerate temperatures as low as −10˚C. Mulberry is known to grow best in coastal areas having low altitudes, whereas it can also be cultivated at altitudes as high as 4,000 m. Places with an annual rainfall ranging from 600 to 2,500 mm and with an atmospheric humidity between 60% and 80% are ideally suited for black mulberry growth. Black mulberries grow well in soils with good moisture holding capacity and having a pH range of 6.5–6.8 (Ahlawat et al., 2016). Compared with the other mulberry species, including white mulberry (Morus alba L.) and red mulberry (Morus rubra L.), the black mulberry is known to be the least cold-hardy and does not do well in areas with humid summers (Ahlawat et al., 2016; California Rare Fruit Growers, Inc, 1997). Black mulberry plant flowers from April to May, and fruits ripen from July to September (Koyuncu, 2014; Koyuncu et al., 2004). The most commonly used maturity index for harvesting black mulberry fruits is the change in color from reddish to black, accompanied by a glossy surface and typical fruit aroma (Ahlawat et al., 2016). The fruits of black mulberries ripen in summer to late summer depending on the locality and the climate of the growth area, followed by a picking season which is over 3 weeks from August to September. Harvesting is preferably done, in the early morning, by shaking individual secondary branches over a sheet spread on the ground, with an additional orchard ladder usage to reach the fruits at upper branches. The remaining ripe berries are handpicked, and all the harvested fruits are kept in the shade to prevent the accumulation of exces­ sive field heat (Ahlawat et al., 2016).

5.3

CHEMICAL COMPOSITION OF BLACK MULBERRY FRUITS AND THEIR JUICE

Black mulberry fruits are gaining an increasing interest because of their nutritive values, and nowa­ days, they are consumed both in fresh form and in varying processed forms, including juice, jams, syrups, and dried fruits (Ercisli  & Orhan, 2007; Gundogdu et  al., 2011). Recently, a number of studies have been reported on the chemical composition and nutritional constituents of black mul­ berry fruits grown worldwide (Ercisli & Orhan, 2007; 2008; Imran et al., 2010; Jiang & Nie, 2015; Koyuncu et al., 2014; Ozgen et al., 2009; Sánchez-Salsedo et al., 2015, 2016). Ercisli and Orhan

Black Mulberry Juice

101

(2007) analyzed black mulberry fruit samples, harvested in the East Anatolia Region of Turkey, for their moisture, total soluble solids, total fat, and ascorbic acid contents, as well as for their fatty acid and mineral compositions. Average moisture, total soluble solids, total fat, and ascorbic acid contents were found to be 72.6%, 16.7%, 0.95%, and 21.8 mg/100 mL, respectively. The most abundant fatty acids in black mulberry fruits were shown to be linoleic acid (C18:2 ω6) (61.85%), oleic acid (C18:1 ω9) (14.75%), and palmitic acid (C16:0) (12.06%), respectively. The mineral components having the highest levels in black mulberry fruits were reported to be potassium (922 mg/100 g), nitrogen (920 mg/100 g), phosphorus (232 mg/100 g), calcium (132 mg/100 g), magnesium (106 mg/100 g), and sodium (59 mg/100 g), respectively, with an additional presence of iron (4.2 mg/100 g), manganese (4.2 mg/100 g), zinc (3.2 mg/100 g), and copper (0.4 mg/100 g) in trace amounts (Ercisli & Orhan, 2007). In another study of Ercisli and Orhan (2008), five black mulberry genotypes, grown in the Northeast Anatolia Region of Turkey, were evaluated for their total soluble solid, organic acid, fatty acid, and ascorbic acid contents. In accordance with the results obtained in their previous study (Ercisli  & Orhan, 2007), the total soluble solid contents of five black mulberry genotypes were measured to change in between 14.30% and 19.35%, whereas the ascorbic acid contents were in the range of 15.1–18.7 mg/100 mL. The most predominant organic acid was determined to be malic acid with a range of 123–218 mg/g, which was followed by citric acid having a range of 21–41 mg/g in five different black mulberry genotypes. Six fatty acids, including linoleic, myristic, stearic, palmitic, oleic, and nonadecenoic acid, were recorded to be present in black mulberry geno­ types studied, representing linoleic acid as the dominant fatty acid (53.57%–64.41%), followed by palmitic acid (11.36%–16.41%), oleic acid (10.66%–15.98%), stearic acid (3.22%–9.14%), myristic acid (0.87%–3.41%), and nonadecenoic acid (0.22%–1.35%), respectively (Ercisli & Orhan, 2008). The ripe black mulberry fruits, collected from the northern regions of Pakistan, were analyzed for their major nutritional components, including moisture, ash, protein, lipid, total carbohydrate, total sugar, and fiber contents, as well as for their essential mineral and vitamin compositions (Imran et  al., 2010). The contents of moisture, ash, lipids, proteins, fibers, and total carbohydrates were recorded to be 82.40, 0.50, 0.55, 0.96, 11.75, and 13.83 g/100 g DW (dry weight), respectively. Total sugar, reducing sugar, and pectin contents of black mulberry species studied in this work were determined to be 6.64 g/100 g, 4.94 mg/100 g, and 0.76 g/100 g, respectively. The results of vitamin analysis indicated the presence of ascorbic acid, niacin (vitamin B3), and riboflavin (vitamin B2) with the contents of 15.37, 1.60, and 0.040 mg/100 g, respectively. The predominant mineral compo­ nent was potassium (1270 mg/100 g), followed by calcium (470 mg/100 g), sodium (272 mg/100 g), magnesium (240 mg/100 g), iron (77.6 mg/100 g), zinc (59.2 mg/100 g), and nickel (1.60 mg/100 g), respectively (Imran et  al., 2010). Jiang and Nie (2015) described the chemical characteristics of black mulberry fruits cultivated in the Xinjiang Province of China. Their results indicated succinic acid (6.48 mg/g), acetic acid (3.55 mg/g), malic acid (2.62 mg/g), and citric acid (0.71 mg/g) as the most abundant organic acids in black mulberry fruits, which differed from the findings of Ercisli and Orhan (2008). The trend of dominant fatty acids was also distinguished from Ercisli and Orhan (2008), indicating oleic acid (26.0%) as the most abundant fatty acid, followed by palmitic acid (23.8%) and linoleic acid (23.1%), respectively. The protein content of black mulberry fruits ana­ lyzed in this study was measured to be 1.17%, having histidine (0.1%) as the predominant essential amino acid and proline (0.49%) as the predominant nonessential amino acid. Consistent with the results reported by Ercisli and Orhan (2007) and Imran et al. (2010), potassium (297 mg/100 g) was determined to be the most abundant mineral component, followed by calcium (113 mg/100 g), mag­ nesium (36.9 mg/100 g), iron (11.9 mg/100 g), and sodium (5.9 mg/100 g) (Jiang & Nie, 2015). Ripe fruits of wild-grown black mulberry genotypes, grown in Mahmatlar, Turkey, were analyzed for some of their chemical properties (Koyuncu et al., 2014). Proximate composition analysis revealed the moisture, total sugar, crude fat, ash, and crude protein contents to range in between 77.30%– 84.27%, 5.09%–7.26%, 3.15%– 6.79%, 0.12%– 0.36%, and 7.66%–12.93%, respectively. The main mineral component of black mulberry genotypes studied was potassium, followed by sodium, phos­ phorus, and magnesium. The mean values of potassium, sodium, phosphorus, magnesium, calcium,

102

Asian Berries: Health Benefits

iron, manganese, zinc, and copper were 1,041, 277, 170, 128, 32, 7.07, 5.63, 3.02, and 0.34 mg/ 100 g, respectively (Koyuncu et al., 2014). Ozgen et al. (2009) analyzed the individual sugar and organic acid contents of black mulberry fruits, sampled from Turkey. The mean values of glucose, fructose, and sucrose were recorded as 6.39, 5.77, and 0.03 g/100 mL, respectively, whereas the contents of malic acid, ascorbic acid, and citric acid were measured to be 0.16, 0.005, and 1.87 g/100 mL, respectively (Ozgen et al., 2009). In another study, four black mulberry clones, collected in South-Eastern Spain, were analyzed for their mineral and protein contents. Protein contents ranged between 8.90% and 10.85%, and the decreasing order of mineral elements was presented as K > N > Ca > P > Mg > S > Na > Fe > Zn > Cu > Ni (Sánchez-Salcedo et al., 2015). Sánchez-Salcedo et al. (2016) evaluated four black mulberry clones, collected in southeastern Spain, for their fatty acid contents, and reported linoleic acid (4,365–7,789 mg/100 g DW) as the most abundant fatty acid, followed by palmitic acid (972–1,236 mg/100 g DW), oleic acid (325–754 mg/100 g DW), and stearic acid (189–2,751 mg/100 g DW), respectively. Table 5.1 presents the main chemical constitu­ ents reported in black mulberry fruits and juice samples studied worldwide. The variations between the results of different studies may be attributed to the different cultivars, grafting rootstocks, and the geographical conditions (Jiang & Nie, 2015), as well as to the different ecological factors, har­ vesting time, and genetic factors (Koyuncu et al., 2014). Total soluble solids, ascorbic acid, and trace minerals contents were evaluated in black mulberry juice, produced from fresh fruits harvested in Pakistan, by Khalid et  al. (2011). The contents of total soluble solids and ascorbic acid were measured to be 19% and 23 mg/100 mL, respectively. Trace mineral analysis indicated potassium (1300 mg/100 g) as the dominant mineral component in black mulberry juice, followed by sodium (160 mg/100 g), calcium (150 mg/100 g), magnesium (130 mg/100 g), zinc (45 mg/100 g), iron (40 mg/100g), and manganese (7 mg/100 g), respectively, which were in accordance with the results recorded for black mulberry fruit samples (Ercisli  & Orhan, 2007; Imran et al., 2010; Koyuncu et al., 2014; Sánchez-Salcedo et al., 2015). Tomas et al. (2015) reported the moisture content of black mulberry juice, processed from black mulberry fruits collected in Turkey, as 88.4%. Lucia et al. (2016) measured the reducing sugar and ascorbic acid contents of black mulberry juice samples, obtained by centrifugation or by thermal processing of fresh fruit samples grown in Central Slovakia, and reported the contents as 17.24 mg/kg and 0.87 mg/100 g, respectively, for the juice sample obtained by centrifugation, and as 20.04 mg/kg and 0.69 mg/100 g, respectively, for the juice sample obtained by thermal processing.

5.4

PHYTOCHEMICALS IN BLACK MULBERRY FRUITS AND THEIR HEALTH BENEFITS

Phytochemicals are gaining increased attention because of their health benefits. Moreover, epide­ miological studies showed a positive correlation between the consumption of diets rich in fruits and vegetables and a reduced risk of certain chronic diseases, which could partly be attributed to the presence of antioxidants, especially phenolic compounds (Tomas et al., 2015). Black mul­ berries are one of the most commonly consumed berries in the world, and similar to other ber­ ries, they are rich sources of phenolic compounds which are related to their antioxidant activity (Sánchez-Salcedo et  al., 2015). They have been reported to contain phenolic acids and flavonols including p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, neochlorogenic acid, chloro­ genic acid, caffeic acid, 3-caffeoylquinic acid, 4-caffeoylquinic acid, p-coumaric acid, ferulic acid, m-coumaric acid, quercetin 3-O-rutinoside, quercetin 3-O-glucoside, quercetin-malonyl-glucoside, and kaempferol 3-O-rutinoside. On the other hand, the presence of anthocyanins, mainly, cyani­ din 3-O-glucoside, cyanidin 3-O-rutinoside, pelargonidin 3-O-glucoside, and pelargonidin 3-Orutinoside (Figure 5.2) has also been reported (Sánchez-Salcedo et al., 2015; Tomas et al., 2015). All these bioactive compounds have attracted great attention because of their role in the preven­ tion of several chronic diseases (Table  5.2). Cancer is one of the leading causes of death in the world. In 2019, 1,762,450 new cancer cases and 606,880 cancer deaths occur in the United States

–

72.60%

82.40 g/ 100 g

–

82.31%

–

–

Turkey

Turkey

Pakistan

China

Turkey

Turkey

Spain

88.40%

–

Turkey

Slovakia

–

–

Protein

–

–

TC

–

–

Lipid

–

–

–

–

–

0.21%

–

–

–

–

–

–

10.25%

1.17%

–

–

–

–

–

–

–

–

–

–

–

–

5.75%

–

0.50 g/ 0.96 g/ 13.83 g/ 0.55 g/ 100 g dw 100 g dw 100 g dw 100 g dw

–

–

Ash

DW, dry weight; TC, total carbohydrates; “–,” not analyzed/not reported.

–

Pakistan

Juice

Moisture

Fruit

Cultivation Area of Fruit

–

–

26.00%

–

14.75%

10.66– 15.98%

Oleic Acid

–

–

23.80%

–

12.06%

11.36– 16.41%

Palmitic Acid

–

–

–

–

–

–

–

–

–

K

–

1041 mg/100 g

297 mg/100 g

1270 mg/ 100 g

922 mg/ 100 g

–

–

–

1300 mg/ 100 g

4,365– 325–754 mg/ 972–1236 – 7,789 mg/ 100 g dw mg/ 100 g dw 100 g dw

–

–

23.10%

–

61.85%

53.57– 60.65%

Linoleic Acid

Chemical Characteristics 

–

Mg

–

–

128 mg/ 100 g

–

–

–

–

0.16 g/ 100 mL

– –

 

–

–

160 mg/ – 100 g

–

–

Citric Acid

–

–

–

–

–

–

1.87 g/ 100 mL

–

2.62 mg/g 0.71 mg/g 277 mg/ – 100 g

–

150 mg/ 130 mg/ 100 g 100 g

–

–

32 mg/ 100 g

Malic Acid 176 mg/g 31 mg/g

272 mg/ – 100 g

59 mg/ 100 g

–

Na

113 mg/ 36.9 mg/ 5.9 mg/ 100 g 100 g 100 g

470 mg/ 240 mg/ 100 g 100 g

132 mg/ 106 mg/ 100 g 100 g

–

Ca

TABLE 5.1 Chemical Compositions of Black Mulberry Fruits and Juice Samples Studied Worldwide

References

Tomas et al. (2015)

Khalid et al. (2011)

SánchezSalcedo et al. (2016)

Ozgen et al. (2009)

Koyuncu et al. (2014)

Jiang & Nie (2015)

Imran et al. (2010)

Ercisli & Orhan (2007)

0.69–0.87 Lucia et al. mg/ (2016) 100 g

–

23 mg/ 100 mL

–

0.005 g/ 100 mL

–

–

–

21.8 mg/ 100 mL

14.9– Ercisli & 18.7 mg/ Orhan 100 mL (2008)

Ascorbic Acid

Black Mulberry Juice 103

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Asian Berries: Health Benefits

FIGURE  5.2 Chemical structure of (a) cyanidin-3-O-glucoside (National Center for Biotechnology Information, 2019a) and (b) cyanidin-3-O-rutinoside (National Center for Biotechnology Information, 2019b).

(Siegel et al., 2019). In the literature, both in vitro and in vivo studies have shown that berry antioxi­ dants may slow or prevent the development of cancer. In general, berries have been shown to inhibit the NF-κB (nuclear factor kappa B) signaling pathway and downregulate all major inflammatory markers including TNF-α (tumor necrosis factor-alpha), IL-1β (interleukin-1β), IL-6, IL-10, induc­ ible nitric oxide synthase, cyclooxygenase-2, prostaglandin E2, NF-κB, and p-p65 (Kristo et al., 2016). In a study, Turan et al. (2017) evaluated the antiproliferative and apoptotic effects of M. nigra extract on human prostate cancer cells. They reported that M. nigra extract arrested the cell cycle of human prostate adenocarcinoma cells at the G1 phase, induced apoptosis via increased caspase activity, and reduced mitochondrial membrane potential. Another study conducted by Huang et al. (2011) revealed that mulberry anthocyanins inhibited the growth of human gastric carcinoma cells, inducing intrinsic (p38/p53/Bax signaling) and extrinsic (p38/Fas/FasL/caspase 8 signaling) apop­ totic pathway. Furthermore, 0.2% mulberry anthocyanins by both injection and oral gavage revealed a positive effect on tumor inhibition. Similarly, the same group reported that mulberry extracts contain high amounts of anthocyanins, which have been reported to have antitumor effects in vitro and in vivo, triggering Ras/PI3K pathway (Huang et al., 2008). Diabetes mellitus, commonly known as diabetes, is characterized by hyperglycemia result­ ing from defects in insulin secretion, insulin action, or both (American Diabetes Association, 2010). Furthermore, α-amylase inhibitors are important to modulate glucose tolerance in diabetes. Mahmoud et al. (2014) investigated the hypoglycemic effect of black mulberry fruits in diabetic rat. They showed that administration of black mulberry fruits to diabetic rats significantly reduced α-amylase activity. Furthermore, Ştefănuţ et al. (2013) observed that the administration of black mulberry extracts showed a significant decrease in glucose level from 252 mg/dL at the start day to 155 mg/dL at the end of experiment. In another study, black mulberry intake demonstrated thera­ peutic effects in diabetic subjects and improvements in nondiabetic groups with respect to blood glucose, total cholesterol, creatinine, uric acid, and blood pressure levels (Abdalla, 2006). Mahmoud (2013) also carried out studies on the administration of black mulberry fruits to hypercholestrolemic rats. They reported that administrating with different levels (2.5%, 5% and 10%) of black mulberry fruits resulted with a significant decrease in total cholesterol, triglycerides, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) and a significant increase in high-density lipopro­ tein (HDL), when compared with rats that are fed with hypercholestrolemic diet.

Black Mulberry Juice

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TABLE 5.2 Health Benefits of Black Mulberries Chronic Disease Prostate cancer

Gastric cancer

Liver injury Diabetes mellitus

Nonalcoholic fatty liver disease

Hypercholesterolemia

Sepsis Cardiovascular diseases Cognitive impairment Inflammation Oxidative stress

Study Model

Results

Human prostate adenocarcinoma Morus nigra may be a novel candidate (PC-3, ATCC-CRL-1435) cancer against prostate cancer and human normal foreskin fibroblast cells (ATCCCRL-2522) AGS gastric cancer cell and five Mulberry anthocyanins can be a potential Balb/c nude mice therapy agent in preventing the gastric carcinoma Fifty six male Sprague–Dawley M. nigra showed protective effect against rats CCl4 toxicity Thirty male Sprague–Dawley rats Black mulberry fruit significantly reduced α‐amylase activity of diabetic rats Black mulberry showed an antiSprague–Dawley rats hyperglycemic effect 12 type 2 diabetes mellitus and 26 Black mulberry intake demonstrated therapeutic effects in diabetic subjects and nondiabetic subjects improvements in nondiabetic groups in blood glucose, total cholesterol, creatinine, uric acid, and blood pressure Black mulberry ethanol extract Thirty-six male C57BL/6J mice supplementation protected mice from high-fat diet–induced obesity, hepatic steatosis, and insulin resistance Consumption of black mulberry fruits Forty adult male albino Sprague– showed positive effects on Dawley rats hypercholesterolemia M. nigra produced beneficial effects on the Male C57BL/6 modulation of important septis parameters M. nigra extract produced a significant dose Frog’s heart dependent decrease in heart rate. M. nigra has potential in improving Thirty-two Balb-C mice cognitive deficits in mice Total flavonoids of black mulberry possess Kunming male mice anti-inflammatory and analgesic effects Black mulberry extract has a potent ability Human HepG2 cells to cope with EC-induced oxidative stress

Reference Turan et al. (2017)

Huang et al. (2011) Mnaa et al. (2014) Mahmoud et al. (2014) Ştefănuţ et al. (2013) Abdalla (2006)

Song et al. (2016)

Mahmoud (2013) de Pádua Lúcio et al. (2018) Malik et al. (2012) Turgut et al. (2016) Chen et al. (2016) Li et al. (2018)

In a recent study, Deniz et al. (2018) indicated that M. nigra extracts provided significant protec­ tion against CCl4-induced hepatic liver injury. Similarly, de Pádua Lúcio et al. (2018) evaluated the anti-inflammatory and antioxidant properties of M. nigra in a sepsis model induced by lipopoly­ saccharide. Malik et al. (2012) showed that the aqueous methanolic extracts of M. nigra L. fruits produced a significant dose-dependent decrease in the heart rate of frogs. This could be attributed to the presence of polyphenolic compounds. Aging is another main public issue that reduces mental, physical, or social activities in human beings. Turgut et al. (2016) concluded that M. nigra significantly improved learning dysfunctions, increased memory retention, reduced malondialdehyde levels, and elevated superoxide dismutase, glutathione peroxidase, and catalase activities compared with the control group. They also suggested that M. nigra may be beneficial to suppress aging, partially because of its scavenging activity against

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Asian Berries: Health Benefits

free radicals and high antioxidant capacity. Chen et al. (2016) investigated the anti-inflammatory and antinociceptive activities of total flavonoids of black mulberry fruits in mice. They showed that flavonoids in black mulberry fruits had inhibitory activities on xylene-induced ear edema and carrageenan-induced paw edema. Moreover, they had antinociceptive activities in the two nocicep­ tive phases of formalin test. In a more recent study, Li et al. (2018) investigated the effect of black and white mulberry extracts on ethyl carbamate (EC)–induced cytotoxicity and oxidative stress. They observed that black mulberry extracts, which contained more total phenolics, total flavonoids, procyanidins, cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, and pelargonidin-3-O-glucoside, were more effective in ameliorating EC-induced cytotoxicity by inhibiting excessive cellular reac­ tive oxygen species generation, suppressing mitochondrial dysfunction, and increasing cellular glu­ tathione concentration in HepG2 cells compared with white mulberry extracts. Song et al. (2016) evaluated the effects of ethanolic mulberry extracts on high-fat diet–induced hepatic steatosis and insulin resistance in mice. They reported that administration of ethanolic black mulberry extracts decreased diet-induced body weight gain, improved high-fat diet–induced hepatic steatosis and adi­ pose hypertrophy, ameliorated insulin resistance, and improved glucose homeostasis, suggesting that black mulberries might be a potential candidate for the treatment of obesity.

5.5 EFFECT OF JUICE PROCESSING TECHNOLOGY AND PROCESS ON ANTIOXIDANTS OF BLACK MULBERRY FRUITS Black mulberries are commonly consumed fresh but can also be consumed as shelf-stable processed products. Black mulberry juice production includes the steps of thawing (if fruit is frozen), mechani­ cal milling, mashing, pressing, and pasteurizing (Figure 5.3) (Tomas et al., 2015). Moreover, juice processing of black mulberry inevitably includes steps with heat treatments that have potential of affecting the antioxidants of the end product. The aims of these treatments might be to inactivate fruit-borne and added enzymes, to inhibit microbial activity, and to reduce moisture content to concentrate the processed foods (Weber  & Larsen, 2017; Nayak et  al., 2015). These processing steps could be either beneficial or detrimental for the health-promoting phytochemicals. Moreover, the antioxidant capacity of the products may change depending on processing type and conditions (temperature, time, etc.) and the food matrix (Al-juhaimi et al., 2018). Conventional heat treatment is routinely used to prolong the shelf life; to preserve fruits and vegetables and their products; to make them available out of season; to produce practical products especially for home consump­ tion; and to provide better nutritional characteristics (Capanoglu et al., 2010). On the other hand, with increasing awareness of the retention of antioxidants through food processing, researchers and producers go toward nonthermal technologies. In the past decade, novel technologies such as highpressure treatment, PEFs, or ultrasound have been introduced into juice processing (Weber and Larsen, 2017). Table 5.3 gives an overview on the juice processing of black mulberry antioxidants. In a study, Tomas et al. (2015) investigated the effect of industrial-scale juice production on black mulberry antioxidants. They observed that juice processing led to 40%, 16%, 29%–81%, and 3.1fold significantly higher values in the final juice sample compared with the starting fruit, for total phenolics, total flavonoids, total antioxidant capacity, and total monomeric anthocyanins, respec­ tively. Furthermore, they reported that mashing and pressing steps were the two main treatments applied that gave rise to this high recovery and increased representation of black mulberry phenolics in the processed juice fraction. After obtaining the juice, a common method to convert and keep the juice in a solid dry powder form is spray drying, which is a well-established and widely used method. Fazaeli et al. (2012) reported that increasing the maltodextrin concentration, the inlet air temperature and compressed air flow rate had negative effects on total anthocyanin contents of black mulberry juice. Moreover, they suggested that the optimum conditions for total anthocyanin contents (5.85 mg/100 mL) were found to be temperature = 130˚C, maltodextrin concentration = 8%, and compressed air flow rate = 800 L/h.

Black Mulberry Juice

107

Black mulberry

Washing

Milling

Mashing

Press cake

Pressing

Pasteurization

FIGURE 5.3

Juice

Schematic representation of black mulberry juice process.

In some studies, both conventional and nonthermal techniques are used to compare the changes in antioxidants. Similarly, black mulberry juice was concentrated using these methods. Dinçer et al. (2016), for instance, compared the primary quality parameters of black mulberry juice concentrates produced by osmotic distillation and thermal evaporation. They showed that the anthocyanin con­ tent of samples processed with osmotic distillation (604.6 mg/L) did not significantly change as compared to the starting black mulberry juice (646.7 mg/L), whereas thermal evaporation of black mulberry juice caused a significant decrease (541.9 mg/L, p < 0.05). In another black mulberry juice concentrate study, Fazaeli et al. (2013a) observed that applying microwave (7.3 kPa pressure, 300 W) instead of rotary evaporation heating method decreased the degradation of anthocyanins. Similarly, Hojjatpanah et  al. (2011) showed that the degradation of anthocyanins was more pronounced in rotary evaporation compared with microwave heating method. Furthermore, Fazaeli et al. (2013b) indicated that different heating methods caused different results for the degradation of anthocyanins and antioxidant capacity of black mulberry juices. On the other hand, they also observed that during applying both microwave and rotary heating methods, pelargonidin 3-glucoside and pelargonidin 3,5-diglucoside were observed to be more sensitive to temperature and pressure, whereas cyanidin 3-glucoside and delphidin 3-glucoside were more resistant. This could be related to their molecular structure. Thermosonication is another novel and viable technology for fruit juice processing to protect antioxidants compared with conventional thermal treatments. Dinçer and Topuz (2015) reported that sonication and thermosonication treatments led to a decrease in the anthocyanin content of black mulberry juice and the anthocyanin degradation percentage was in the range of 2.4%–4.1%. They suggested that these losses may be due to the cavitation and oxidation reactions. Since the cavitation produces local heat, pressure, and mechanical action, anthocyanins of the juices were degraded (Tiwari et al., 2009). In another study, Engmann et al. (2014) carried out studies on the effect of temperature and high hydrostatic pressure on anthocyanins. They reported that heating at 75˚C for 2 min and pressurizing at 480 MPa for 10 min led to the highest retention of anthocyanins

108

Asian Berries: Health Benefits

TABLE 5.3 Effects of Juice Processing Techniques on the Antioxidants of Black Mulberries Process methods and conditions Mashing (without enzymes, 45˚C–50˚C, 30 min), cold pressing, pasteurization (107˚C, 3 min)

Spray drying: inlet air temperature (110˚C, 130˚C, and 150˚C), compressed air flow rate (400, 600, and 800 L/h), and maltodextrin concentration (8%, 12%, and 16%) Osmotic distillation: PE membrane (0.1 m², ps 0.2 μm) Thermal evaporation: 120 min, 100 rpm, 250 mbar, 80˚C

Sonication treatments: 20 kHz constant frequency: i. Pulsed sonication: 5 s on/off, at three amplitude levels: 60%/80%/100%, 15 min, 25˚C ii. Thermosonication: at only 100% amplitude level, %, 15 min, 30˚C/40˚C/50˚C Temperature: 75˚C, heating time: 2 min, pressure: 480.00 MPa, pressurizing time: 10 min Temperature: 90˚C, heating time: 3.5 min, pressure: 365.00 MPa, pressurizing time: 17.5 min Microwave method: 7.3, 38.5, and 100 kPa, 300 W Rotary evaporation: 38.5 and 7.3 kPa, 120˚C

Microwave method: 7.3 kPa, 300 W Rotary evaporation: 7.3 kPa, 120˚C

Thermal processing: 90˚C for 30 s Microwave processing: 900 W, 30 s Ultrasonic processing: 20 kHz, 30 min, 20˚C

Results

References

TPC: 40% ↑ TFC: 16% ↑ TAC: 29%–81% ↑ TMA: 3.1-fold ↑ TMA: 5.85 mg/mL Optimum conditions: Temperature: 130˚C Maltodextrin concentration: 8% Compressed air flow rate: 800 L/h The osmotic distillation process did not significantly change anthocyanin content, whereas the thermal evaporation of black mulberry juice caused a significant decrease in anthocyanin content (10% loss) TMA content decreased from 2.4% to 4.1%

Tomas et al. (2015)

Highest retention of anthocyanins: 96.09% Lowest retention of anthocyanins: 78.31%

Engmann et al. (2014)

Microwave method decreases the degradation of anthocyanins compared with the rotary evaporation heating

Hojjatpanah et al. (2011) Fazaeli et al. (2013a) Fazaeli et al. (2013b)

Fazaeli et al. (2012)

Dinçer et al. (2016)

Dinçer & Topuz (2015)

AC: 21.55 mg/L TMA: 20.26 mg/L AC:14.71 mg/mL TMA:13.04 mg/mL Anthocyanin degradation and consequent decrease in antioxidant activity were more tended in rotary evaporation compared with microwave heating method TMA Jiang et al. 36–45%↓ (2015) 28–38%↓ 24–34%↓ Ultrasound sterilized juice was found to have higher retention of anthocyanins compared with microwave and thermally processed juice

AC, total anthocyanin content (determined by high-performance liquid chromatography/mass spectrometry); TAC, total antioxidant capacity; TFC, total flavonoid content; TMA, total monomeric anthocyanin content; TPC, total phenolic content; ↓, decrease; ↑, increase.

Black Mulberry Juice

109

(96.09%), whereas treatment at 90˚C for 3.5 min and 365 MPa for 17.5 min resulted in the least anthocyanin retained (78.31%). Furthermore, they indicated that increasing temperature and pres­ sure resulted in significantly diminished amount of anthocyanins. In another study, Jiang et  al. (2015) examined the effect of different sterilization methods (thermal, microwave, and ultrasonic processing) on the anthocyanin of black mulberry juice during selected storage time (8 days) and temperatures (5˚C, 15˚C, and 25˚C). Their results revealed that anthocyanin content of thermal, microwave, and ultrasound sterilized juices was reduced between 36%–45%, 28%–38%, and 24%– 34%, respectively, during storage at 5˚C, 15˚C, and 25˚C for 8 days. They also observed that the higher stability of anthocyanins in black mulberry juice was achieved at the lowest temperature. Therefore, these authors suggested to apply ultrasonic processing as a preservation technique for black mulberry juice during storage where anthocyanin retention is desired.

5.6 SUMMARY Black mulberries (M. nigra L.) are one of the most important berry fruits because of their nutritive values and the presence of health-promoting phytochemicals. Phytochemicals in black mulberries have beneficial effects on chronic diseases including cancer, diabetes, cardiovascular diseases, and inflammation. Processing black mulberries into juice changes the content of antioxidants. The dif­ ferent process conditions applied in juice processing may have different effects on black mulberry bioactives. In general, nonthermal methods were shown to have protective effects on antioxidants. However, consistent with the current trends in juice processing technology, further research should be focused largely on nonthermal methods, such as PEF, and how they affect the fate of black mul­ berry juice antioxidants.

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Mahmoud, M. Y. (2013). Natural antioxidants effect of mulberry fruits (Morus nigra and Morus alba L.) on lipids profile and oxidative stress in hypercholestrolemic rats. Pakistan Journal of Nutrition, 12, 665– 672. Malik, M. N. H., Salma, U., Qayyum, A., & Samreen, S. (2012). Phytochemical analysis and cardiac depres­ sant activity of aqueous methanolic extract of Morus nigra L. Fruit. Journal of Applied Pharmaceutical Science, 2(12), 39. Mnaa, S., Aniess, W., Olwy, Y., & Shaker, E. (2014). Antioxidant activity of white (Morus alba L.) and black (Morus nigra L.) berries against CCl4 hepatotoxic agent. Advanced Techniques in Biology & Medicine, 2014, 1–7. Nayak, B., Liu, R. H., & Tang, J. (2015). Effect of processing on phenolic antioxidants of fruits, vegetables, and grains – a review. Critical Reviews in Food Science and Nutrition, 55(7), 887–918. National Center for Biotechnology Information. (2019a). PubChem Compound Summary for CID 441667, Cyanidin 3-O-glucoside. https://pubchem.ncbi.nlm.nih.gov/compound/Cyanidin-3-O-glucoside (accessed on October 2, 2019). National Center for Biotechnology Information. (2019b). PubChem Compound Summary for CID 441674, Cyanidin 3-O-rutinoside. https://pubchem.ncbi.nlm.nih.gov/compound/Cyanidin-3-O-rutinoside (accessed on October 2, 2019). Ozgen, M., Serce, S., & Kaya, C. (2009). Phytochemical and antioxidant properties of anthocyanin-rich Morus nigra and Morus rubra fruits. Scientia Horticulturae, 119, 275–279. Sánchez-Salcedo, E. M., Mena, P., García-Viguera, C., Martínez, J. J., & Hernández, F. (2015). Phytochemical evaluation of white (Morus alba L.) and black (Morus nigra L.) mulberry fruits, a starting point for the assessment of their beneficial properties. Journal of Functional Foods, 12, 399– 408. Sánchez-Salcedo, E. M., Sendra, E., Carbonell-Barrachina, A. A., Martinez, J. J., & Hernandez, F. (2016). Fatty acids composition of Spanish black (Morus nigra L.) and white (Morus alba L.) mulberries. Food Chemistry, 190, 566–571. Siegel, R. L., Miller, K. D., & Jemal, A. (2019). Cancer statistics, 2019. CA: A Cancer Journal for Clinicians, 69(1), 7–34. Song, H., Lai, J., Tang, Q., & Zheng, X. (2016). Mulberry ethanol extract attenuates hepatic steatosis and insu­ lin resistance in high-fat diet–fed mice. Nutrition Research, 36(7), 710–718. Ştefănuţ, M. N., Căta, A., Pop, R., Tănasie, C., Boc, D., Ienaşcu, I., & Ordodi, V. (2013). Anti-hyperglycemic effect of bilberry, blackberry and mulberry ultrasonic extracts on diabetic rats. Plant Foods for Human Nutrition, 68(4), 378–384. Tiwari, B. K., O’donnell, C. P., & Cullen, P. J. (2009). Effect of non thermal processing technologies on the anthocyanin content of fruit juices. Trends in Food Science & Technology, 20(3– 4), 137–145. Tomas, M., Toydemir, G., Boyacioglu, D., Hall, R., Beekwilder, J., & Capanoglu, E. (2015). The effects of juice processing on black mulberry antioxidants. Food Chemistry, 186, 277–284. Turan, I., Demir, S., Kilinc, K., Burnaz, N. A., Yaman, S. O., Akbulut, K.,  …  & Deger, O. (2017). Antiproliferative and apoptotic effect of Morus nigra extract on human prostate cancer cells. Saudi Pharmaceutical Journal, 25(2), 241–248. Turgut, N. H., Mert, D. G., Kara, H., Egilmez, H. R., Arslanbas, E., Tepe, B., … & Tuncel, N. B. (2016). Effect of black mulberry (Morus nigra) extract treatment on cognitive impairment and oxidative stress status of D-galactose-induced aging mice. Pharmaceutical Biology, 54(6), 1052–1064. Tutin, G.T. (1996). Morus L. In: Tutin, G.T., Burges, N.A., Chater, A.O., Edmondson, J.R., Heywood, V.H., Moore, D.M., Valentine, D.H., Walters, S.M., Webb, D.A. (Eds.). Flora Europa, Psilotaceae to Platanaceae, 2nd ed., vol. 1, Cambridge University Press, Port Melbourne. Vijayan, K., Chauhan, S., Das, N. K., Chakraborti, S.P., & Roy, B. N. (1997). Leaf yield component combining abilities in mulberry (Morus spp.). Euphytica, 98, 47–52. Weber, F., & Larsen, L. R. (2017). Influence of fruit juice processing on anthocyanin stability. Food Research International, 100, 354–365.

6 Characteristic Constituents Mulberry Fruits

and Health Benefits Pallav Sengupta, Sulagna Dutta, and Chee Woon Wang MAHSA University

Zheng Feei Ma Xi’an Jiaotong- Liverpool University

CONTENTS 6.1 6.2 6.3

Introduction........................................................................................................................... 113

Characteristic Components of Mulberry Fruits .................................................................... 114

Health Benefits of Mulberry Fruits ....................................................................................... 115

6.3.1 Antioxidative Potential ............................................................................................. 115

6.3.2 Anti-inflammatory and Antimicrobial Effects .......................................................... 115

6.3.3 Anticancer Properties ................................................................................................ 116

6.3.4 Cardioprotective Effects ........................................................................................... 117

6.3.5 Neuroprotective Effects ............................................................................................ 118

6.3.6 Antidiabetic Effects................................................................................................... 118

6.3.7 Hepatoprotective and Gastroprotective Effects ........................................................ 119

6.3.8 Protection against Skin Diseases .............................................................................. 119

6.4 Conclusion and Future Perspectives ..................................................................................... 119

References...................................................................................................................................... 120

6.1

INTRODUCTION

Medicinal plants have been integral part of therapeutics since 4,000–5,000 BC and have been exten­ sively used in the Asian countries. Their applications reach far and wide including treatment of almost all pathological conditions, including inflammations, cutaneous diseases, gastrointestinal disorders, and even cancer (1). Efficacy and safety of therapeutic use of several medicinal plants have been extensively studied and well accepted (2). These herbs have vital roles mostly in the primary healthcare system because of their accessibility, cost-effectiveness, and modest or no side effects, as compared with modern drugs (2). Since medicinal plants have a vast array of species with a multitude of physiological effects, bulk of research activities have been based on unveiling the exact mechanisms of their action, especially for the herbs used in the treatment of noncommu­ nicable chronic diseases, such as metabolic disorders (1,3). Mulberries are sweet edible fruits from a widespread genus (genus Morus, family Moraceae) of deciduous trees growing in a varied temperate region all over the globe. They are among the mostly used species of plants that find immense applicability in medical realms. The genus Morus covers 15 deciduous plant species including Morus nigra, M. alba, Morus rubra, Morus atropurpurea, Morus australis, Morus notabilis, Morus cathayana, and Morus mesozygia. They are small to medium sized with mono- or dioic plantar, are believed to originate from China, and have spread all over the world owing to their taste, flavor, and nutritive values (4). Mulberry plants are economically 113

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Asian Berries: Health Benefits

essential because almost every part of the plant bears pharmacological value, for example, the leaves of this plant are widely utilized in sericulture. Mulberries contain a great variety of nutrients which are indispensable for the body, such as iron, vitamins C and K, riboflavin, phosphorous, potassium, and calcium. These fruits are also rich in dietary fibers and innumerable organic com­ pounds, which essentially include zeaxanthin, phytonutrients, resveratrol, lutein, anthocyanins, and different polyphenolic compounds. Mulberries are of immense pharmacological interest attributing to the properties that are beneficial to health, such as antioxidant, anticancer, anti-inflammatory, cardioprotective, hepato- and gastroprotective, antidiabetic, and neuroprotective properties (4). This chapter thereby aims to highlight the characteristic components and biological properties of the mulberry fruits to encourage further in-depth research to reveal the exact mechanisms and specific use of the active components of mulberries in healthcare system.

6.2

CHARACTERISTIC COMPONENTS OF MULBERRY FRUITS

Morus species have been estimated to comprise of 24 varieties with minimum of 100 subspecies distributed according to suitable topographical, climatic, and soil conditions. Environmental fac­ tors also determine their characteristic compositions, such that mulberries in different regions vary in terms of nutraceutical contents, which can be used in the treatment of various diseases for their health benefits (5). In this aspect, the commonly used Morus species mainly include M. alba (white mulberry), M. rubra (red berry), and M. nigra (blackberry) (5,6). Focusing on the physicochemical compositions of the mulberries, it is noteworthy that all the commonly used species have weight between a range of 2.0 and 4.0 g, with high water content of approximately 70%. M. alba has the highest pH and highest soluble solid contents as compared with M. rubra and M. nigra. This suggests that M. alba has a sweeter taste than the other two and the most recommended species for processing. The acidity values of the mulberries follow similar pat­ tern with M. nigra having 1.40%, M. rubra having 1.37%, and M. alba with 0.25% (5). Mulberries generally have low lipid content with the highest being in M. alba (1.1%) (5,7). The fatty acid content in the mulberries mainly comprise of linoleic acid, palmitic acid, and smaller amount of oleic acid (5,7). However, mulberries are excellent sources of proteins that can potentially contribute to the daily recommended protein intake. The protein content of blackberries is report­ edly between 8.9% and 10.85%, whereas that of white berries is even greater, between 10.15% and 13.33% (8). Mulberries are rich in important minerals, such as Ca, Mn, Zn, Cu, Mg, Fe, and Se (7). Other than these, the fruits have abundance of sodium, potassium, and phosphate (5). The antioxidant properties of mulberries are among the major qualities of these fruits that account for their wide spectrum of use in healthcare system. These fruits have substantial amount of ascorbic acid (vitamin C), with about 48.4 mg/100 g in blackberries, which prevents and miti­ gates oxidative damage (7). The phytochemicals present in the mulberry fruits also possess essen­ tial antioxidants and provide beneficial health effects, especially in case of inflammatory process, cardiovascular diseases (CVDs), and cancer. Flavonoids are the most prominent phytochemical compounds in mulberry fruits. M. alba and M. nigra contain flavonoids in the form of flavonols, which again has twenty subtypes with major glycolyzed forms being quercetin, soramnetin, and kaempferol. These components are useful in diminishing the risk of type 2 diabetes mellitus and certain types of cancer (9). Moreover, M. nigra contains 1.422 mg, and M. rubra contains 1.035 mg of “gallic acid equivalents (GAE)”/100 g of their fruit weight for total phenols, while having 276 mg and 219 mg “quercetin equivalents (QE)”/100 g of fruit weight for flavonoids (5). The content and value of flavonoids and phenolic compounds vary in different mulberry species as well as within the same species according to their unique characteristics and distribution (5). The constituents of medicinal values in the white mulberries differ with their geographic locations. These constituents essentially include bioactive contents, such as the flavonoids, anthocyanins, and the carotenoids (5,10). The red mulberries are also used extensively for their medicinal properties (11),

Mulberry Fruits

115

particularly for their effects such as the hyporglycemic agent, glibenclamide, and thus, red mulberry extracts help in significant reduction of blood glucose levels. Red mulberry extracts are also used for their efficacy in preventing lipid peroxidation, reducing malonaldehyde (MDA) levels in serum and liver, enhancing antioxidant enzymes activities, and decreasing glutathione (GSH) activities (12). Blackberries have rich antioxidant content (13) and cyanidin-3-O-glucoside and cyanidin-3­ O-rutinoside as the major anthocyanins in these mulberry species (14). In both the white and black mulberries, derivatives of benzoic acid (p-hydroxybenzoic acid, protocatechuic acid, and vanillic acid), cinnamic acid (chlorogenic acid and neochlorogenic acid), p-hydroxybenzoic acid, gallic acid, caffeic acid, ellagic acid, and p-coumaric acid are found (8,15).

6.3 6.3.1

HEALTH BENEFITS OF MULBERRY FRUITS ANTIOXIDATIVE POTENTIAL

Mulberries contain a wide array of phytochemicals, a substantial portion with antioxidative poten­ tial. The fruits of mulberry, M. alba (L.) are considered as nutritious food. As mentioned in the previous section, the nutritious mulberry fruits contain essential flavonoids and polyphenols. These include quercetin, apigenin, luteolin, caffeic acid, morin, gallic acid, chlorogenic acid, rutin, umbel­ liferone, and kaempferol (16). The important fatty acid contents include oleic, palmitic, and linoleic acids which are required for formation of cell membrane, neural development and proper function­ ing, inflammatory responses, and production of eicosanoids (5). Mulberry fruit extracts have been shown to be beneficial when used in vitro on human liver (HepG2) cells after induction of cytotoxicity by ethyl carbamate (17). Another study reported that mulberry fruit consumption could enhance endurance capacity in mice undergoing weight-loaded swimming test, maybe owing to their antifatigue properties (18). It has been suggested that antioxi­ dant properties in mulberries are mainly due to the presence of anthocyanins that effectively act to minimize oxidative stress and physical fatigue caused by excessive exercise or physical activities (Figure 6.1).

6.3.2

ANTI-INFLAMMATORY AND ANTIMICROBIAL EFFECTS

Mulberries have been reported to exhibit immunomodulatory, antiinflammatory, antimicrobial, and antinociceptive properties (Figure 6.1) (17,19). M. alba, Morus mongolica, and M. nigra have been shown to have antinociceptive properties attributing to the presence of cyanoidin-3-O-glycoside, anthocyanins, and flavonoids isoquercetin and rutin. These fruits were shown to significantly decrease the levels of inflammatory cytokine interleukin-6 (IL-6) levels, inhibit nitric oxide synthe­ sis, and stimulate the expression of IL-10, a potent anti-inflammatory cytokine (17). Anti-inflammatory and antinociceptive properties of the fruits of M. nigra have been demon­ strated in mice with xylene-induced ear edema and carrageenan-induced paw edema. This study showed that mulberry fruit flavonoids in a dose of 50 and 100 mg/kg of body weight possess analge­ sic and anti-inflammatory effects. These activities were also suggested to have positive associations with their antioxidant activity (14). The anti-inflammatory effects of M. alba leaves and fruit extracts were also observed in obese animals. In this study, following 12 weeks of treatment, expression of an inflammatory protein (NLRP3) was normalized during the early healing stages and improved healing profile compared with animals that did not receive extract supplementation (20). NLRP3 plays a major role in initia­ tion of inflammatory progression as it activates inflammatory cascades and reduces angiogenesis during the stages of wound healing (20). Research indicates that the flavonoids and anthocyanins found in M. nigra fruits possess anti­ nociceptive and antibacterial activities against certain microorganisms, such as Pseudomonas

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Asian Berries: Health Benefits

FIGURE  6.1 Phytochemical constituents of mulberry fruits, pharmacological properties, and possible mechanisms. LDL, low-density lipoprotein.

aeruginosa, Escherichia coli, and Staphylococcus aureus. Such activities could be linked to the inhibitory impacts on proinflammatory cytokines, nitric oxide synthesis, and NF-κB (17). Thus, from the available reports, it may be inferred that mulberry fruit extracts, especially from M. alba and M. nigra species, demonstrate a reduction in inflammatory markers in different parts of the organism.

6.3.3

ANTICANCER PROPERTIES

Cancer is perhaps the most dreaded public health problem in the world. The natural compounds mostly focus on modulation of inflammatory and immune pathways for cancer prevention and treat­ ment. There are several anticancer compounds in mulberry as well. Morus fructus fruit extracts have been shown to induce cancer cell death both in vitro and in vivo (Figure 6.1). It is suggested that mulberry extracts mediate in vitro cancer cell death via reactive oxygen species–dependent mitochondrial apoptosis (21). M. alba show in vitro anticancer properties on hepatoma cells, owing to the presence of phenolic compounds. The mechanism of action in this case is through cell cycle

Mulberry Fruits

117

arrest at G2-M phase and inhibits activities of topoisomerase II (22). Isolated from M. alba root bark, albanol A induces strong cytotoxicity (IC50 1.7 mM) in HL60 cells alba by inhibiting topoi­ somerase II activity (IC50 22.8 mM). However, it induces early apoptosis through the pathway of cell death receptors and the mitochondrial pathway, observed by exposure to membrane phospho­ lipids, reduced levels of pro caspases 3, 8, and 9, and increased levels of cleaved caspases 3, 8, and 9 (23). The essential oil’s anticancer operation isolated from Morus rotunbiloba koidz has been stud­ ied in human larynx epidermoid carcinoma (Hep2) and human colon adenocarcinoma (SW620) cell lines with African green monkey (Vero) kidney cell line as guide. The 0.1–100 μg/mL oil has had no effect on the viability of Vero cells. The median lethal concentrations (LC50) of the oil on Hep2, SW620, and Vero cytotoxicity were 70, 120, and 280 μg/mL, respectively (24). Resveratrol purified from the methanol extracts of M. alba showed inhibition of heparinase and antimetastatic effects on murine B16 melanoma cells (25). M. alba is a rich source of prenylated cytoatalantoflavones, kaempferols, and so on. Morusin is the most active of them against HeLa cells with an IC50 value of 0.64 μM (26). New 2-arylbenzofuran derivatives (i.e., moracines of different structures from M. alba and wittifurans from Morus wittiorum) with potent cytotoxic activity against various lines of human cancer cells have been recently identified (27,28). Also isolated was a new galactose-binding lectin from M. alba leaves with cytotoxic effect on human breast cancer (IC50–8.5 μg) and colon cancer cells (IC50–16 μg) (29). Anthocyanins are a group of phenolic compounds with beneficial effects because of their anti­ oxidant, anti-inflammatory, and chemopreventive properties in reducing the risk of CVDs and cancer (30). Cyanidin 3-rutinoside and cyanidin 3-glucoside are the two anthocyanins present in mulberry, which have strong anticancer properties. These have been shown to prevent the invasion and migration of human lung cancer A549 cells by downregulating the expression of MMP-2 and plasminogen activator urokinase and enhancing the expression of TIMP-2 and activator plasmino­ gen inhibitor. In this case also, an inhibition of NF-κB and c-jun activation were observed (31). Osajin is a prenylated isoflavone isolated from the fruit of Maclura pomifera, a mulberry family tree. It exerts multiple effects such as loss of mitochondrial transmembrane potential, release of cytochrome C, expression of Fas ligand, suppression of glucose-regulated protein, and activation of various caspases and proapoptotic proteins in human nasopharyngeal carcinoma cells. Thus, osajininduced apoptosis involves extrinsic death receptor pathways and endogenous mitochondrial and endoplasmic reticulum pathways (32). Chalcones, a group of plant aromaticenones, form the central nucleus of a variety of biologically important compounds.

6.3.4

CARDIOPROTECTIVE EFFECTS

CVDs are the leading cause of death, with data showing 17 million worldwide deaths due to CVD (33). This CVD-associated global mortality rate reportedly will sustain in the future as well (34). A major risk factor for CVD is hyperlipidemia (35). Therefore, studies that assess effectiveness of herbal medicine for regulation of lipid profile and cardiac health are steadily increasing (36). The most obvious benefits of herbal treatment over common hypolipidemic medications are fewer side effects and cost-effectiveness (36). Mulberry fruit extracts could potentially reduce levels of serum and liver triglycerides, serum low-density lipoprotein (LDL) levels, and total cholesterol levels and increase serum high-density lipoprotein cholesterol levels, as studied in rats that are fed with high-fat diet (36). Mulberries have high dietary fiber content which helps in inhibiting lipogenesis in liver and stimulating activities of the LDL receptor (37). Furthermore, mulberry fruits may depict hypolipidemic properties owing to the presence of high linoleic acids and dietary fibers (36). A study on white New Zealand rabbits that are fed for 10 weeks with high cholesterol diet together with 0.5%–1.0% mulberry fruit extracts showed lower triglyceride levels, LDL levels, and total cho­ lesterol levels as compared with those only with high cholesterol diet and no mulberry supplemen­ tation (38). Mulberry fruit extracts also could significantly reduce the levels of atherosclerosis in

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Asian Berries: Health Benefits

aorta (38). Treatment with mulberry extracts did not show any adverse alterations in renal or hepatic functions (38). Another study on 58 hypercholesterolemic adults showed significant reduction in the levels of serum LDL and total cholesterol (39) with 6 weeks of mulberry fruit intake (45 g of freeze-dried fruit containing 325 mg of anthocyanins). Thus, mulberry fruits possess substantial therapeutic potential in treating hypercholesterolemia and atherosclerosis owing to their antihyperlipidemic and antioxidant properties (38).

6.3.5

NEUROPROTECTIVE EFFECTS

The amyloid plaques are produced by the proteolysis of the precursor protein by the enzyme a, β, or d secretase. Therapeutic attempts to combat Alzheimer’s disease by blocking certain enzymes have now cantered on stopping this cascade. There are many flavones found in Morus ihrou with inhibi­ tory involvement in β-secretase (40). Cholinesterases are important enzymes that play a major role in cholinergic transmission. Eight flavonoids were isolated from the root bark of M. ihou L., includ­ ing kuwanon U, kuwanon E, kuwanon C morusin, morusinol with cholinesterase inhibitory activity (both acetylcholine and butyrylcholine esterase) (41). The neuroprotective role of oxyresveratrol has been studied in in vitro model of stretch-induced trauma in neuronal and glial cocultures or by exposing cultures to high glutamate levels. Trauma induced pronounced neuronal death, and this death was substantially prevented by oxyresveratrol. Glia microscopic examination indicated signs of toxicity in cultures treated with 100 μM oxyresveratrol, as evidenced by elevated protein release of S-100B and a high proportion of condensed nuclei. Cultures exposed to glutamate for 24 h showed neuronal loss of ~37% that was not blocked by oxyresveratrol (42). Studies have shown that mulber­ ries have beneficial effects on the activation of an antioxidant defense system and improved memory loss in aged animals (43). The neuroprotective role of M. alba fraction cyanidine-3-glucoside (C3G) was examined in oxygen deprivation and glutamate-mediated cell death in primary cortical neurons of rats. C3G did not provide a protective effect against glutamate-induced cell death but preserved the mitochondrial membrane potential (44).

6.3.6

ANTIDIABETIC EFFECTS

Diabetes is characterized by hyperglycemia arising from insulin secretion defects (45). It is linked with several pathological issues, such as CVD and organ failure (45). It has been shown that mul­ berry fruit polysaccharides, namely, MFP50 and MFP90, when given to diabetic rats for 7 weeks, could significantly reduce the levels of fasting serum insulin, fasting blood glucose, insulin resis­ tance, and triglycerides in diabetic rats (10). In a similar study, it was reported that fasting blood glucose in diabetic rats was lowered in 2 weeks with treatment with polysaccharide mulberry fruits (46). Another study by Wang et  al. (47) reported that the levels of fasting blood glucose and glycosylated serum protein have been decreased substantially by diabetic rats that are fed with mulberry extracts with ethyl acetate– soluble for 2 weeks. The authors also found that catalase, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in diabetic rats have significantly increased their antioxidant activity in ethyl acetate–soluble extracts from mulberry fruits (47). Ethylaxillodiphenyl-1-picrylhydrazyl (DPPH) and anion superoxide radicals also possess high α-glucoside and a radically soluble extract of mulberry fruits (47). Xu et al. (48) had reported a lower level of hemoglobin A1c and the reduction of streptozotocin-lesioned pancreatic cells in diabetic mice that are fed with mulberry fruit polysac­ charides. Additionally, B cell 2 levels of insulin and B cell expression were used for the feeding of polysaccharides to diabetic mice (48). Yan et al. (49) reported a significant reduction in cholesterol, fasting blood glucose, leptin, serum insulin, and triglyceride levels and an increase in adiponectin levels in male C57BL6/DB mice that are fed with mulberry extracts at dosages of 50 and 125 mg/kg body weight per day for 8 weeks.

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The authors therefore proposed that mulberry fruit anthocyanin extraction can be used to boost the insulin and leptin resistance (49). Such results together indicate that mulberry fruits may play an important role in diabetes treatment because of their antihyperglycemic and antihyperlipidemic effects (10,46).

6.3.7

HEPATOPROTECTIVE AND GASTROPROTECTIVE EFFECTS

Nonalcoholic liver disease is characterized by excessive lipid accumulation in hepatocytes, and therefore, it is one of the commonest obesity complications. Ethanol extracts from Morus nigra fruits have enhanced the hepatic steatosis of C57BL/6J mice by reducing significantly the existence of lipid droplets in hepatocytes and decreasing serum alanine aminotransferase (ALT) and aspartate aminotransferase levels, liver triglyceride levels, and overall cholesterol levels. Improved glucose and insulin tolerance, as well as induction of fatty acid oxidation as well as reduced fatty acids and cholesterol biosynthesis have been associated with the protective effects of the extract (50). In a study investigating the safety of anthocyanins (the solid portion following juicing) of mul­ berry fruits in male Sprague–Dawley rats on carbon tetrachloride (CC14), Li et al. (51) reported that the levels of ALT, aspartate aminotransferase, collagen type III hyaluronidatic acid, and hydroxy­ proline in rats that are fed with mulberry fruit marc anthocyanins decreased. Another study by Chang et al. (52) reported that the extracts of mulberry fruits reduced synthesis and boosted fatty acid oxidation. The mulberry fruits therefore may prevent the liver diseases, which are not alcoholic. M. alba extract considerably reduced stomach mucosal damage in absolute ethanol-induced experimental rats with substantial leukocyte infiltration in submucosal layer reduction (53).

6.3.8

PROTECTION AGAINST SKIN DISEASES

Depigmentation by mulberroside-A and oxyresveratrol-3-O glucosides has been shown to cause topical impacts, such as decreased melanin indices, inhibition of tyrosinase activity, and reduc­ tion in melanin content in UV-induced hyperpigmentation of guinea pig skin. Melanogenesis was more strongly inhibited in oxyresveratrol and oxyresveratrol-3-O-glucoside than mulberroside-A. The MITF gene, which regulates the transcription of the proteins in melanocyte pigmentation, was reduced by this procedure (54). There are many different components of the carotenoids such as lutein, β-carotene, zeaxanthin, and α-carotene, which include high levels of vitamin A and vitamin E. All these elements act as antioxidants, affecting particularly the skin, tissue, hair, and other parts of the body in which free radicals affect. Mulberries can help in skin care, minimize blemishes and the appearance of old spots, and keep the hair shiny and clean by mitigating actions of free radicals.

6.4

CONCLUSION AND FUTURE PERSPECTIVES

Literature reviews found out that polyphenolic compounds and antioxidants are abundant in mul­ berry fruits (55). This reflects that food and healthcare industries have many opportunities to research on the health benefits of mulberries as the demand for mulberry fruits is rising potentially. Yet bioactive components such as anthocyanins, flavonoids, alkaloids, and polyphenols depend on cultivars. While the bioactive components should function in synergy with health, they do need further research in order to determine the causative link between the use of mulberry fruits and health (56). Published data are insufficient to affirm whether and exactly how mulberry fruits benefit human health, especially as regards chronic diseases such as diabetes, CVDs, and other diseases. Similar to other food products (57–64), most studies demonstrating beneficial health effects of mulberry fruits are animal studies. Such studies also used different varieties of mulberry fruits, solvent forms, and preparation methods, which make it difficult to evaluate the activities of mulberry fruits and require very heterogeneous data. For the effects of mulberry fruit consumption on human health, larger,

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Asian Berries: Health Benefits

well-conceived, randomized controlled trials are necessary. Further research is needed to elucidate structures of some key active components of mulberry fruits and the mechanisms by which their pharmacological characteristics can be enhanced.

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7

Longan Syrup and

Related Products

Processing Technology and

New Product Developments

Noppol Leksawasdi, Kritsadaporn Porninta, Julaluk Khemacheewakul, Charin Techapun, and Yuthana Phimolsiripol Chiang Mai University

Rojarej Nunta Lampang Rajabhat University

Ngoc Thao Ngan Trinh Nong Lam University - Ho Chi Minh City

Alissara Reungsang Khon Kaen University

CONTENTS 7.1

Longan Fruits........................................................................................................................ 123

7.1.1 General Information ................................................................................................. 123

7.1.2 Economic Potential ................................................................................................... 124

7.2 Extraction and Concentrating Processes and Technology.................................................... 126

7.3 Related Products ................................................................................................................... 131

7.3.1 Fructo-oligosaccharides............................................................................................ 131

7.3.2 Extraction of Pericarp and Seed for Bioactive Compounds ..................................... 131

7.3.3 Ethanol and Phenylacetylcarbinol Production .......................................................... 134

7.4 New Product Developments.................................................................................................. 139

7.5 Summary .............................................................................................................................. 140

Acknowledgments.......................................................................................................................... 140

References...................................................................................................................................... 140

7.1 LONGAN FRUITS 7.1.1 GENERAL INFORMATION Longan fruits (Dimocarpus longan Lour.) are subtropical plant species that belong to the soapberry family Sapindaceae(Lithanatudom et al., 2017; Hassler, 2019), which consists of 1,899 plants (1,820 living species) such as longans, lychees, rambutans, akees, pulasans, and khorlaens (Janthasri and Pasuvitayagon, 2005; Hassler, 2019). Longan fruits are nonclimacteric with 1.5–3.0 cm in diameter 123

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consisting of pericarp, aril, and seed (Yang and Chiang, 2019). The quality of longan fruits dete­ riorates rapidly after harvesting at room temperature due to “aril breakdown” process (Lin et al., 2019). The seasonal harvesting period of longan fruits in Thailand is July–August (Phuong, 2018). Hassler (2019) described four intraspecific taxons of longan fruits as follows: (1) D. longan subsp. longan, (2) D. longan subsp. malesianus Leenhouts, (3) D. longan var. longetiolatus Leenhouts, and (4) D. longan var. obtusus (Pierre) Leenhouts. Lithanatudom et  al. (2017) provided genetic relationship evidence based on multilocus molecular markers and morphological characteristics and proposed nomenclatural changes of two species, namely, D. malesianus and D. obtusus. Janthasri and Pasuvitayagon (2005) mentioned the introduction of longan plants from Chinese merchant to Thailand more than 120 years ago during the reign of King Rama V in 1896. Since then, a total of 26 cultivated varieties (cultivars) have been emerged in Thailand in comparison with some 300– 400 cultivars in China (Lithanatudom et al., 2017; Yang and Chiang, 2019). Janthasri and Pasuvitayagon (2005) classified these cultivars based on their adaptability to climate of the cultivation area, namely, subtropical cultivars (such as E-Daw, Biew khiew, Chompoo, Haew) in northern part of Thailand and tropical cultivars (such as Phetsakon) in central Thailand.

7.1.2 ECONOMIC POTENTIAL The FAO (2010) classified tropical fruits based on their economic potential into two subgroups, namely, major and minor tropical fruits. The members of major group are mango, pineapple, papaya, and avocado with global exporting values of USD 10 billion and production volume of 7 million tons (Altendorf, 2017). The minor tropical fruits include lychees, durians, rambutans, guavas, pas­ sion fruits (FAO, 2010), longans, and mangosteens (Altendorf, 2018) with global exporting values of USD 20 billion and production volume of 24 million tons (Altendorf, 2018). Guavas and longans were ranked first and second in terms of economic potential for minor tropical fruits (Altendorf, 2018). The global production volume of longan fruits in 2017 was 3.600 million tons with the aver­ age of 3.445 million tons between 2015 and 2017 (Altendorf, 2018). Three main producers of longan fruits with the total of more than 95% of global production include China (50%), Thailand (30%), and Vietnam (15%) (Chen et al., 2015). The average production volumes between 2015 and 2017 for the three main producers were 1.919, 0.980, and 0.517 million tons, respectively (Altendorf, 2018). The other 5% of longan fruits are cultivated in India, United States (Hawaii and Florida), Australia (joining as a new producer) (Chen et al., 2015), Srilanka, and Myanmar (Altendorf, 2018). Chen et al. (2015) mentioned that China possessed 73.6% of longan global cultivation area (such as Guangdong, Hainan, Guangxi, Sichuan, Yunnan, and Fujian (FreshPlaza, 2019)) and provided up to 59.7% of longan fruits world output in 2014. Kubo and Sakata (2018) stated that domestic longan production in China has been concentrated mainly on the southern part of the country with pro­ duction volume of 1.28 million tons in 2010 with additional imports from Thailand (0.137 million tons) and Vietnam (0.155 million tons). However, the popularity of Thai longan fruits had shifted the importing volume to 57% from Thailand and 43% from Vietnam in 2017 with the total values of USD 438 million. Longans (as shown in Figure 7.1) are an important economic crop of Thailand as evident from the exportation and importation values in 2018 at USD 935 million and USD 0.76 million (OAE, 2019), respectively, or export to import ratio of 1,230 indicating a very strong positive trade balance for this fruit. These could be compared with the total exportation value of agro-industrial products at USD 253 billion in 2018 (MOC, 2019). The total production level of Thai longan fruits in 2018 was 1.031 million tons, a slight increase from the previous year of 1.029 million tons (OAE, 2018). The comparison between longan fruits and some other crops in terms of exporting values and total production levels in 2018 is shown in Table 7.1 (sorting in descending order of exporting value). Fresh longan fruits (1.29 USD/kg) can be processed simply into conventional products such as dried whole longan fruits (6.0–7.5 USD/kg) and longan juice (1.75 USD/kg) (Lithanatudom et al., 2017; Alibaba 2019; NamSamoonPai, 2019; TalaadThai, 2019; Yang and Chiang, 2019). Additional

Longan Syrup and Related Products

125

FIGURE 7.1 Fresh longan fruits.

TABLE 7.1 Total Production Levels and Exporting Values of Longan Fruits and Other Crops in 2018 Type of Crops Cassava Longan Pineapple Durian Rambutan Mangosteen a b

Total Production Levela (million tons)

Exporting Valueb (USD million)

28.46 1.03 2.38 0.74 0.27 0.18

2,411 935 622 193 18 10

OAE (2018). OAE (2019).

processing steps result in products such as canned longan fruits (5.27–7.59 USD/kg), freeze-dried longans (48.6 USD/kg), and longan syrup (176 USD/kg) (Nunta et  al., 2018; MIThai, 2019; Sun Snack FoodTech, 2019; TOPS, 2019), which can valorize longan fruits further. Yang and Chiang (2019) listed a number of bioactive phenolic compounds generally found in pericarp and seed of longan fruits such as gallic acid, corilagin, ellagic acid, epicatechin, as well as glycosides of quer­ cetin and kaempferol. These compounds have been proven to provide positive bioactivities such

126

Asian Berries: Health Benefits

as antityrosinase (mitigate hyperpigmentation relating to aging effect), anticancerous, antiglyca­ tion (prevent covalent bonding between sugars and functioning enzymes that cause several impair­ ments), and memory enhancing effects (Yang and Chiang, 2019). The utilization of longan fruits for production of fructo-oligosaccharide (FOS, 4.19 USD/kg) through enzyme linkage provides additional health benefits for the consumers (Nunta et  al., 2018; IndiaMart 2019a). Ethanol and phenylacetylcarbinol (PAC) production that implemented substandard longan fruits or longan solid waste as alternative substrate through fermentation and biotransformation processes of the same pyruvate decarboxylase (PDC) enzyme is an alternative strategy complementing the previously mentioned conventional longan processes to achieve the goal of zero waste production for this eco­ nomical fruit. Ethanol (0.83 USD/kg) is a powerful and multiversatile solvent and biofuel in many industries (CSF, 2019; GPP, 2019). PAC could be used as a valuable substrate (production cost of 2.85 USD/kg with selling price for up to 146 USD/kg) for production of ephedrine – an antiasth­ matic compound (46.8 USD/kg), as well as pseudoephedrine – a nasal decongestant (61.0 USD/kg) through reductive amination (FDA Thailand, 2011; DOS, 2014; PharmaCompass, 2016a,b; Nunta et al., 2018; IndiaMart 2019b).

7.2 EXTRACTION AND CONCENTRATING PROCESSES AND TECHNOLOGY Longans are nonclimacteric fruits which are sweet, juicy, and aromatic (Li et  al., 2009). There are four volatile compounds of fresh longan fruits, i.e., ethanol, ethyl acetate, and trans- and cis(β)-ocimene, while there are more compounds developed during drying process of longan fruits (Lapsongphol et al., 2007). These volatiles are, for example, 3-methyl butanol, 3-methyl butanal, and phenyl ethyl alcohol (Zhang et al., 2009). With high total soluble solid of fresh longan fruits about 18°Brix to 25°Brix, they are gradually accepted by consumers over the world due to their sweet juicy mouthfeel and health benefits (Rangkadilok et  al., 2007). Further perusal of longan syrup safety for consumption in recent years by Chiranthanut et al. (2020) on acute and chronic oral toxicity assessment of longan sugar extracts derived from whole fruit and from fruit pulp in rats also revealed that whole fruit longan did not cause any toxic effects in rats. Most of the carbohy­ drates in longan fruits are in the form of fructose, glucose, and sucrose, which are easily absorbed by the human body. The main sugar compositions of preconcentrated longan juice are 2.77% glu­ cose, 3.91% fructose, and 14.21% sucrose as reported by Yunchalad et al. (2008). Therefore, longan juice can potentially be used for syrup production. Recently, there have been production of longan syrups in both liquid and powder forms, a new and high commercial product in market (Figure 7.2). Longan syrup is considered to have functional properties such as the ability to act as a sugar replacer and use for the production of FOSs (Surin et al., 2012). Optimization of extraction and concentration

FIGURE 7.2

Longan syrups in both liquid and powder forms.

Longan Syrup and Related Products

127

processes has influences in flavor and aroma of syrup. Main processes for longan syrup production are extraction of longan juice and concentration. Juice extraction is the process in which the liq­ uid part is separated from the solid part. It can be done using a press or an extractor (Oyeleke and Olaniyan, 2007). During this process, juice is exposed to atmosphere, which could cause deteriora­ tion to juice properties and consequently to longan syrup (Li et al., 2009). The pressing is the most commonly used method for juice extraction because it uses the conventional methods such as juicer, screw extraction, hydraulic press, and burr machine (Barwal and Shrera, 2009). Surin et al. (2014) found that extraction using juicer and hydraulic press did not affect physical and sensory proper­ ties, volatile compounds, and sugar contents of longan syrup. The yield percentages of longan syrup produced from the six methods ranged from 10.2% to 14.2%. Acceptance information showed that consumers liked syrup with high floral aroma, but with low caramel aroma. The objectives of implementing concentrating process and technology to fruit and vegetable juice include not only extending the shelf life of the juice by mitigating water activity but also sav­ ing storage space and mitigating transportation cost by removal of undesired water content (An et al., 2019; Darvishi et al., 2019). The preservation of either the content or organoleptic properties of health-beneficial bioactive compounds such as saccharides, vitamins, and polyphenols in the feed juice, which are sometimes thermosensitive, must also be considered at the same time (Sabanci et al., 2018; Borchani et al., 2019; Tamba et al., 2019). Conventionally, fruit and vegetable juice could be concentrated by three main processes (Shoikhedbrod, 2018), namely, evaporation, freezing, and membrane (Ilame and Singh, 2015; Orellana-Palma et al., 2017; Bevilacque et al., 2018). Other con­ centrating technologies under experimental stage include hydrogen electrolysis gas bubbles gener­ ated by applying electric field (Shoikhedbrod, 2018) and by applying alternating current directly to the fruit juice or ohmic heating under vacuum condition (Darvishi et al., 2019; Sabanci et al., 2019), provided rapid and uniform heating (Fadavi et al., 2018) with additional benefits of simultaneous deactivation of contaminant microbes to a certain degree (Tumpanuvatr et al., 2015). The longan fruit extract demonstration pilot plant (as shown in Figure 7.3) for the production of both syrup and powder has been developed by author’s research group starting from (1) raw material selection and screening, (2) grinding, (3) crushed solid removal, (4) crude precipitation, (5) filtration, (6) adjusting

FIGURE  7.3 Longan extract demonstration unit. 1. Raw material reception and washing. 2. Grinder. 3. Crushed solids removal unit. 4. Crude precipitation unit. 5. Filtration plant. 6. Soluble solid ratio adjuster unit. 7. Vacuum evaporator unit. 8. Crystal development unit. 9. Crystal centrifuge separator. 10. Longan extract powder drier. 11. Longan extract powder silo.

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Asian Berries: Health Benefits

soluble solid, (7) vacuum evaporation, (8) crystal development, (9) crystal centrifuge separation, (10) drying longan extract powder, and (11) finally, longan extract powder silo. The most frequently used method of water removal in fruit and vegetable juice industry is mul­ tieffect evaporation with highly efficient countercurrent flow (Ruan et  al., 2015; Orellana-Palma et  al., 2017. The commonly used multieffect evaporators on an industrial scale had the capacity between 3 and 40 m3/h (Cyklis, 2017). This technology has been well recognized for more than 50 years based on the relatively older sugarcane juice technology (Cyklis, 2017). Thermal process­ ing of fruit and vegetable juice generally suffered Maillard reaction and might result in formation of carcinogenic and cytotoxic compounds such as acrylamide and 5-hydroxymethylfurfural (HMF). Further conversion of the latter could lead to a highly undesirable mutagenic 5-sulfoxymethyl­ furfural (Cordova et al., 2019). The extent of formation of these toxic compounds could vary for production of fruit and vegetable juice in comparison with that of sugarcane juice as the maximum limits of evaporating temperature differed in each case, for example, 140˚C for sugarcane juice, 98˚C for apple juice, and 90˚C for blackcurrant juice (Cyklis, 2017). The excessive heat imple­ mentation might result in degradation of nutritious, but rather heat-labile, bioactive compounds in concentrated juice or deterioration in juice appearance as well as aroma and flavors (Ruan et al., 2015; Bozkir and Baysal, 2017). Energy costing is another major concern of thermal processing to produce concentrated juice in which alternative heat transfer from renewable resources such as solar energy is generally proposed (Alizadeh et al., 2019). Additional measures to conserve energy also included steam jet heat pump technology, solution flash, and condensed water flash (Ruan et al., 2015). The former strategy helped elevating the effect of flash evaporation, which in turn decreased the loss of bioactive compounds with low boiling points (Sabanci et al., 2019). Some factories could expend 24%–30% of its total energy on evaporation process or as high as 1% of overall energy expenditure for the whole country such as New Zealand (Cyklis, 2017). Optimization of evapora­ tion process for production of concentrated juice also relied on the accurate determination of heat transfer coefficient and scaling rate of deposits in the system such as shell tube or plate falling film evaporators so that the amount of supplied steam could be minimized (Cyklis, 2017). These crucial parameters could be derived from system information such as juice concentration and temperature, saturated steam temperature, juice level in the evaporator, vapor bleeding level, and juice flow rate (Cyklis, 2017; Chantasiriwan, 2018). Darvishi et al. (2019) mentioned that the conventional heating process leading to evaporation of fruit juice was inefficient as the process relied upon several steps of heat transfer mechanisms involving conduction, convection, or radiation (Fadavi et al., 2018) and could be replaced by other technologies such as ohmic heating for juice with an appropriate electri­ cal conductivity (Tumpanuvatr et al., 2015) or microwave heating to shorten heat and mass transfer with relatively low operating temperature (Bozkir and Baysal, 2017). Cordova et al. (2019) men­ tioned that an industrial evaporation process in production of concentrated apple juice from 15°Brix to 16°Brix original juice involved three-effect evaporator with three conditions, namely, (1) 85˚C for 25 min, (2) 75˚C for 13 min, and (3) 58˚C for 20 min with the highest formation of HMF in stage 3 at 22.3 ± 1.3 mg/kg when the total soluble solid of concentrated apple juice reached 64°Brix– 66°Brix. Alizadeh et al. (2019) designed a novel solar system in combination with liquid desiccant bed to concentrate barberry juice from the original juice of 16°Brix to the concentrated form of 65°Brix resulting in 62% decrease of energy expenditure relative to conventional evaporation system at atmospheric pressure. The installation of an air circulation system with air flow rate of 0.014 kg/s could also decrease the processing time by 25% when compared with the utilization of lower air flow rate at 0.006 kg/s. Borchani et al. (2019) improved the quality of concentrate prickly pear juice (69°Brix) under both conditions of vacuum (60˚C) and atmospheric evaporation (100˚C) by addi­ tion of 5 U pectinase and 20 U cellulase during pulp pretreatment. These enzymes could minimize crystallization or glass transition as well as enhance “cloud stability,” viscosity, and yield of juice. Sabanci et  al. (2019) also concentrated pomegranate juice from 17.5% to 40% based on ohmic heating–assisted vacuum evaporation and pointed out some technology limitations, which must be rectified for future development on an industrial scale, for example, relatively high initial investment

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and operating costs as well as long processing time to achieve the desired level of fruit and vegetable concentrate, especially in the case of low electrical conductivity materials (Fadavi et  al., 2018). HMF could be mitigated from processed juice under ohmic heating, but the relatively high degra­ dation rate of total phenolic content, total monomeric anthocyanin, and antioxidant activity could imply the requirement for usage of electrochemically inert electrodes (Sabanci et al., 2019). Fadavi et al. (2018) also produced concentrate tomato juice from ohmic heating under vacuum condition and pointed out the additional advantages of this combined technology by saving processing time and energy. The minimization of thermal processing time could maximize the quality of concen­ trated juice. Vacuum operation had many benefits over atmospheric counterpart such as minimal changes of properties including pH, turbidity, acidity, and lycopene in the tomato juice. Bozkir and Baysal (2017) applied microwave heating under vacuum condition (MHV) for production of apple juice concentrate from 11.0°Brix in the original juice to 66.5°Brix in the concentrated form with optimal microwave power of 668 W and vacuum level of 500 mbar. In comparison with conven­ tional rotary evaporation and rising film evaporator, MHV was a simpler and faster process with the improved physicochemical, biochemical, and sensory properties of concentrated juice. Freeze concentration or cryo-concentration is the emerging and promising concentrating tech­ nology for fruit juice processing, as organoleptic properties can still be maintained while the losses of vital bioactive compounds such as vitamin C in the original feed juice are relatively minimal (Auleda et al., 2011; Petzold et al., 2016; Orellana-Palma et al., 2017, 2018). Miyawaki et al. (2016) claimed that freeze concentration could concentrate apple juice to the best quality as aroma com­ pounds with low molecular weight, which played a major role in flavor balance, could still be pre­ served after concentration process. Ding et al. (2019) revealed that freeze concentration process is energy conserving as the energy required to evaporate 1 kg of water is 2,440 kJ in comparison with only 334 kJ of heat to be withdrawn from the equivalent mass of water. Although, thermodynami­ cally, the transfer of heat is thus 7.30 times less in the freezing process, the costing for removal of water in multieffect evaporation is still cheaper than freeze concentration by 1.43 times due to gen­ eral availability and the relatively low cost of steam generation. Henao-Ardila et al. (2019) classified freeze concentration into three types based on how the ice crystallization was achieved, namely, suspension, progressive, and block freeze concentration. The suspension freeze concentration sys­ tem was relatively complicate with the requirement of high capital investment cost. The improved design of progressive or falling film freeze concentration technology limited the ice formation to a single site so that ice removal was done efficiently while the concentrated juice could be collected with relative ease (Miyawaki et al., 2016). Orellana-Palma et al. (2017) investigated block freeze concentration process in which three steps of (1) freezing, (2) thawing, and (3) assisted separation such as vacuum, ultrasonication, and centrifugation were involved. In their study, the concentrating effect of blueberry juice could occur under a slight vacuum condition (80 kPa) of stage 3 in both radial and unidirectional flow at −80˚C with the highest total polyphenolic content of 3.64–3.85 times relative to the original blueberry juice. Orellana-Palma et al. (2018) also applied the same system to orange juice resulting in the elevated concentration of total soluble solid and ascorbic acid by 4.4 and 4.1 times, respectively. One-step assisted separation to obtain concentrate juice in freeze concentration process was necessary for feasibility of commercial production and had been tested with several products such as red wine, orange juice, pineapple juice, sea water, and sucrose solution (Petzold et al., 2016, 2017). The integration of block freeze concentration with centrifugation was investigated for blueberry and pineapple juices by Petzold et al. (2015). The flow rate of concen­ trated juice through ice matrix channel was accelerated by centrifugal force (4,600 rpm, 20˚C for 10 min) and repeated for several cycles. The concentration factor after the third cycle was approxi­ mately 2.50 times relative to feed juice. Henao-Ardila et al. (2019) combined both falling film freeze concentration and spray drying technologies to concentrate and produce powder from feijoa juice. Freeze concentration was crucial in this study, as pectin gelation process through evaporation of this juice could be prevented. The feijoa juice was concentrated by 1.62 times after freeze concentration, which could be compared with only 1.12 times when the juice was vacuum evaporated. As feijoa

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juice contained significant pectin content, thermal evaporation – even under vacuum condition – still caused formation of the film through pectin gelation process, which mitigated evaporation rate of water from the juice. The advantages of membrane process in terms of high energy efficiency and relatively low heat utilization based on seven-membrane technologies, such as microfiltration, ultrafiltration, forward osmosis, reverse osmosis (hyperfiltration), nanofiltration, membrane distillation, and osmotic distil­ lation, were clearly pointed out by Ilame and Singh (2015) and Bevilacque et al. (2018). The selec­ tion or sequence combination of these technologies for practical application should be done with care by considering the properties and characteristics of the membranes to be used for clarifying and concentrating the feed juice. For example, ultrafiltration membrane with molecular weight cut­ off (MWCO) between 50 and 100 kDa would retain colloids and suspended matter pigments such as pectin, denatured protein, and insoluble wax while allowing small molecules (MW < 50 kDa) such as some pigments, organic acids, sugars, and inorganic salts to pass or freely pass through (Yang et  al., 2019). Antimicrobial effect was also considered as an additional benefit of adopt­ ing membrane process to concentrate fruit and vegetable juice (Bevilacque et al., 2018). Driving forces of these processes might include difference of applied external pressure, osmotic pressure, and water vapor pressure across two sides of the membrane, namely, feed and stripping solution (Bagci et al., 2019; Rehman et al., 2019; Tamba et al., 2019). The operation under high pressure, susceptibility to membrane fouling, and wetting could result in diminishing concentrating effect of the juice being processed and pose drawbacks of these technologies (An et al., 2019; Rehman et al., 2019; Rouquie et al., 2019). Some techniques to alleviate fouling might include pretreatment with hemicellulase/cellulase/pectinase enzymes mixture (Tamba et al., 2019) and enhance hydrophilic­ ity of membrane surface through low-pressure plasma treatment (Bagci et  al., 2019; Hou et  al., 2019). Tamba et al. (2019) stated that the combination of membrane types, for example, ceramic or organic membranes and pressure levels could be regulated to concentrate, separate, or fraction­ ate solute such as betacyanin in cactus pear juice. In such process, microfiltration was used for the screening of undissolved solutes prior to enzymatic treatment and subsequent separation of the desired solutes through ultra/nanofiltration. The composite or polyethersulfone membranes used in ultra/nanofiltration with nominal MWCO between 0.2 and 1 kDa under operating pressure of 5–25 bar could achieve the concentration factor of cactus pear juice between 9.1 and 9.9. Rouquie et al. (2019) compared two concentration and clarification processes of grapefruit juice and two winery products through immersed microfiltration and cross-flow filtration in 6.3 l scale. Similar quality of both retentate and permeate passing through microfiltration and cross-flow filtration was observed with 99.9% for clarification efficiency. An et  al. (2019) applied the integration process between forward osmosis and membrane distillation to efficiently concentrate apple juice feed and draw solution simultaneously with addition of food preservative and electrolyte – potassium sorbate to act as draw solute. Evidently, this system could be run continuously on the bench scale, resulting in 4.25-fold concentrated juice with the presence of relatively low concentration of potassium sorbate (45% ± 9% of the maximum allowable concentration of 1.00 g/L by CODEX standard) while main­ taining important nutrition parameters such as ascorbic and titratable acids. Such integrated system could improve the concentrating effect by 80% in comparison with reverse osmosis alone. Bagci et al. (2019) combined reverse osmosis and osmotic distillation to concentrate pomegranate juice with augmentation of plasma technology to improve thin-film composite polyamide reverse osmosis membrane. The treated membrane with 90-W plasma power for 15 min enhanced water flux through the membrane allowing for the increase of soluble solid content in the concentrated juice and sub­ sequently shortened the time required for osmotic distillation process by 30%. In this case, reverse osmosis was used as a preconcentration stage so that the preconcentrated juice of up to 18°Brix could be processed further through osmotic distillation resulting in juice syrup with 60°Brix. Rehman et al. (2019) compared the concentrating effect of flat sheet polyvinylidene fluoride (PDVF) and polytetrafluoroethylene (PTFE) membranes in the production of pomegranate juice concentrate based on osmotic distillation. The latter was found to suffer less effect of membrane wetting, which

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eventually corrupted the structure of membrane and quality deterioration of concentrated juice. The decline in hydrophobicity and normalized flux properties of PTFE membrane were also less by 4.83 and 1.51 times relative to PDVF membrane, thereby suggesting the suitability for implementation of this membrane in a large-scale production of pomegranate concentrate juice.

7.3 RELATED PRODUCTS 7.3.1

FRUCTO-OLIGOSACCHARIDES

The advantage of oligosaccharides is that they are natural functional prebiotic ingredients. Prebiotics are being used in the food industry as functional ingredients in several products such as beverages, milk, yogurts, and synbiotic products. FOSs are oligosaccharides that occur naturally in plants such as onion, chicory, garlic, asparagus, banana, and artichoke, among many others (Sridevi et al., 2014). FOSs are a nutritive and low-calorie sweetener. They have been attracted and attributed to the expansion of healthy-sugar market due to prebiotic function. In the food industry, FOSs are used as low-calorie food and nutraceutical ingredients (Diling et al., 2017). Several beneficial aspects of FOSs on human health include the increment of the number of Bifidobacteria in the large intestine, reduction in total cholesterol and lipid in serum, relief of constipation, and general improvement of human health such as immune system activation, resistance to some infections, synthesis of βcomplex vitamins, and calcium absorption (Tomomasu, 1994). Furthermore, FOSs could be used to treat breast cancer, diarrhea, and constipation (Roberfroid et  al., 1998). The compositions of FOSs consist of sucrose molecules to which 1, 2, or 3 additional fructose units are added by a β(2-1)-glycosidic linkage to the fructose unit of sucrose, including 1-kestose (GF2), nystose (GF3), and 1F-fructosylnystose (GF4) (Jung et al., 1989). FOS can be produced from sucrose transforma­ tion by fructosyltransferase (FTase) or β-fructofuranosidase enzymes from bacterial and fungal sources (Ghazi et al., 2007; L’Hocine et al., 2000). FTase possesses a higher transferring activity than β-fructofuranosidase (Koops and Jonker, 1994). The overall stoichiometry of FTase action on sucrose can be characterized by two parallel reaction paths (Jung et al., 1989). A set of disproportion reactions provides FOS and glucose from FTase activity, whereas the enzyme hydrolytic activity results in the formation of glucose and fructose as by-products (Aboudzadeh et al., 2006). A typical composition of the reaction mixture on the mass basis is as follows: 65% FOS, 25% glucose, 5% fructose, and 5% sucrose (Sangeetha et al., 2004). Commercial enzyme preparation called Pectinex Ultra SP-L derived from Aspergillus aculeatus contains several enzymes including pectinase, cel­ lulase, β-galactosidase, and FTase. This enzyme has been used to produce FOS from sucrose due to a high level of FTase activity. This enzyme can convert 450 g/L of sucrose to 272 g/L of FOS, which contained 224 g/ L 1-kestose and 48 g/ L nystose (Del-Val and Otero, 2003; Hang and Woodams, 1996). However, glucose obtained from the reaction can inhibit the FOS production. Efficiency of FOS production can be improved by simultaneous removal of glucose via an enzymatic reaction (Del-Val and Otero, 2003). Surin et al. (2012) found that 60°Brix of longan syrup can be used as a substrate to produce FOS with the mixtures of enzymes. The optimal values for FOS production were Pectinex Ultra SP-L 3.3 U/g sucrose, glucose oxidase 1022 U/g sucrose, and reaction time 8 h 41 min. As a result, the amounts of nystose and 1-kestose were ranged from 28 to 30 g/L and 119 to 123 g/L, respectively. It is suggested that FOS from fruit syrup can be produced and used as a healthy ingredient. Figure 7.4 shows the sample of FOS from longan fruits.

7.3.2

EXTRACTION OF PERICARP AND SEED FOR BIOACTIVE COMPOUNDS

Longan fruits also consist of three important phenolic compounds, namely, gallic acid, corilagin, and ellagic acid, which are generally found in seed, flesh, and peel (Rangkadilok et  al., 2005). The specific content of these compounds in fresh longan fruits could vary depending on the vari­ ety. There are also numerous reports of bioactive compounds being found on processed forms of

132

FIGURE 7.4

Asian Berries: Health Benefits

FOS from longan fruits. FOS, fructo-oligosaccharide.

longan fruits such as dried longan. The specific content of bioactive compounds in dried product is usually higher than in fresh fruits due to evaporating effect (Jaitrong et al., 2006; Rangkadilok et al., 2007; Yahya et al., 2017; Tang et al., 2019). All three compounds have different structures and properties; for example, gallic acid (3,4,5-trihydroxybenzoic acid, C7H6O5) is an organic acid with hydroxyl and carboxylic acid functional groups (Panyathepa et al., 2013). This compound has a myriad of bioactivities such as antifungal, antimutagenic, anti-inflammatory, anticancerous, and antioxidant bioactivities (Soong and Barlow, 2006; Huang et al., 2012; Rangkadilok et al., 2012; Worasuttayangkurn et al., 2012; Rahim et al., 2013; Bai et al., 2019). Corilagin (β-1-O-galloyl-3,6­ (R)-hexahydroxy diphenoyl-D-glucose, C27H22O18) is a phenolic compound in the similar group as tannin, which can be found in several plants. The useful properties include some protective effects against hepatitis, tumor, and sclerosis of blood vessels (Moreira et al., 2013). Ellagic acid (2,3,7,8tetrahydroxy-1-benzopyrano-5,4,3-cde-1-benzopyran-5,10-dione, C14H6O8) is a phenolic compound commonly found in fruits and beans, especially in the form of ellagitannins. The anticancerous property through apoptosis process by ellagic acid is evident after digestion in the stomach. The relatively good antioxidant property was observed at 10 μg/mL, whereas the anticancerous prop­ erty of oral cancerous cells was revealed when testing with ellagic concentration range of 12.5– 100 μg/mL. The induction of apoptosis death was evident for intestine tumor HT-29 and HCT-116 at ellagic concentration level of 100 μg/mL. In addition, drug resistance in cancer chemotherapy and resistance to radiation therapy of cancerous cells could be mitigated upon subjection to the presence of ellagic acid. Certain levels of antimutagenic, antitumor, antihepatitis, antiviral, anti­ inflammatory, antibacterial, and pharmacological properties of this compound were also reported (Soong and Barlow, 2006; Landete, 2011; Huang et al., 2012; Wang et al., 2012; Yang and Chiang, 2019). The antimicrobial activities of gallic acid, ellagic acid, and corilagin were also investigated on a plant pathogen (Erwinia carotovora), human pathogenic bacteria (Staphylococcus aureus and Corynebacterium accolans), and human pathogenic yeast (Candida albicans). The highest level of antimicrobial activity was found to be corilagin followed by ellagic and gallic acids. However, ellagic acid could not inhibit S. aureus (Fogliani et  al., 2005). In contrast with Rangkadilok et  al. (2012) who evaluated antimicrobial activities of longan fruit extracts in comparison to with active compounds. Ellagic acid showed the most potent antimicrobial activity followed by corilagin and gallic acids, respectively. In addition, the results showed that longan seed extracts could inhibit against the opportunistic yeasts (Candida sp. and Cryptococcus neoformans), whereas longan pulp and whole fruit extracts did not demonstrate any inhibitory effects. For antibacterial activity, cori­ lagin and gallic acids had moderate inhibitory effects against S. aureus and Streptococcus mutans, respectively. Moreover, ellagitannin extracts had the ability to inhibit a range of pathogenic organ­ isms including Vibrio cholerae, Shigella dysenteriae, and Campylobacter spp. (Landete, 2011).

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Techapun and Leksawasdi (2011a,b) and Leksawasdi and Techapun (2012) developed a prototype for longan sugar production process, which was in a pilot plant in the Faculty of Agro-Industry in Chiang Mai University. The capacity of this pilot plant was 870 kg/day from assorted grade fresh longan fruits of B and C grades at 7–9 tons/day. The important unit operations included clean­ ing, grinding, membrane filtration, and multieffect vacuum evaporation sets. The brief process for production of longan sugar involved grinding of whole fresh longan fruits which consisted of peel, flesh, and seed. The incorporation of cleaning set was thus necessary to ensure that carbaryl pesti­ cide of carbamate group which was generally implemented for cultivation of longan fruits to remove longan pole borer, flower stem borer, flower eating worm, branch borer, and mealy bug from longan sugar products. The detailed analysis of longan sugar products in both syrup and powder forms did not reveal significant contaminant of this pesticide. In addition, the research by the Faculty of Agriculture also indicated the absence of potassium chlorate (KClO3) – which was usually applied to speed up the flowering process of longan tree – in the whole longan fruits. The safety of longan sugar could thus be rated at the relatively high level. The further step in the processing of longan fruits involved the removal of undesired longan solid waste from the supernatant. The supernatant would then be filtered through membrane and evaporated in multistage vacuum evaporators at the suitable condition with low temperature yielding syrup and powder such that the undesired burning scent, normally resulted from the conventional evaporator, could be prevented. Leksawasdi and Techapun (2013) declared that longan sugar possessed 0.11 ± 0.02% (w/w) gal­ lic acid and 2.03 ± 0.05% (w/w) ellagic acid, respectively, and recommended a daily consumption dosage of 2 g/day in order to achieve the sufficient ellagic acid intake of 40 mg/day. The normal healthy individual should not consume longan sugar products more than 50 g /day, and diabetes mellitus patient should consult a medical doctor prior to consumption. The acute and chronic toxici­ ties of longan fruit extracts were investigated in white mice by research team from the Faculty of Medicines in Chiang Mai University. Longan sugars obtained from whole longan fruit and flesh of longan in both powder and syrup forms did not cause any detrimental effect to blood circulation system as well as other systems in the body, especially pancreas and kidney. Leksawasdi et al. (2019) investigated preliminary single-pass extraction strategy with supercriti­ cal fluid carbon dioxide (SFC) extractor using the mixture of 95% (v/v) commercial grade ethanol and supercritical carbon dioxide as extraction media in 5 L scale for fresh longan seeds within the pressure range of 150–200 bars and 40˚C–60˚C. The experiment was carried out in triplicate. The resulting extract had yellowish color. The extraction condition at 150 bars and 40˚C was optimal with the following extraction yields in the soluble part (mg/kg): gallic acid (5.21 ± 0.19), tannic acid (1.18 ± 0.02), corilagin (0.07 ± 0.01), and ellagic acid (0.14 ± 0.01) with the highest total specific con­ tent of bioactive compounds at 6.85 ± 0.16 mg/kg. These specific contents were statistical, signifi­ cantly much less (p ≤ 0.05) than those of conventional direct extraction in 250 mL Erlenmeyer flask with working volume of 50 mL and stirring speed range of 75–150 rpm in case of preextract whole longan solid waste (Prex-WLSW). Prex-WLSW is the solid by-product obtained from the extraction of longan juice from fresh whole longan fruits for production of longan syrup. In fact, the extrac­ tion of Prex-WLSW using SFC extractor in the same condition as described earlier yields greenish color – an evidence of chlorophyll – extractant without any noticeable peak of bioactive compound. Further optimization in SFC condition which differs from fresh longan seed is thus required in case of Prex-WLSW to attain usable level of bioactive compounds in the extract (Nunta et al., 2019). In addition, Leksawasdi et  al. (2018) revealed the total specific content of bioactive compounds up to 2,280 ± 82 mg/kg with the presence of gallic acid (382 ± 7), corilagin (211 ± 13), and ellagic acid (1,687 ± 63) in the absence of tannic acid when ethanol (60%–70% (v/v)) at 60˚C was used as an extractant for Prex-WLSW. This could thus be used as an evident indicating that there still exists relatively high specific content of bioactive compounds in Prex-WLSW. In other experiments, Leksawasdi et al. (2015) studied extraction of bioactive compounds from dried longan seeds, peel, and solid wastes using varied concentration level of ethanol, extraction time, and extraction tem­ perature in shaking condition of 200 rpm. The optimal extraction condition was 50% (v/v) ethanol

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for 15 h and 60˚C with the specific contents of gallic acid, ellagic acid, and corilagin (in mg/kg) of (2,090 ± 40, 2,860 ± 10, and 3,500 ± 10) from dried longan seeds (170 ± 10, 990 ± 10, and 660 ± 10), from dried longan peel, and (380 ± 10, 2,420 ± 19, and 640 ± 10) from dried longan solid wastes, respectively. These results could also be compared to those of Chindaluang and Sriwattana (2014) and Rangkadilok et al. (2007) who performed multiple passes extraction of fresh longan seeds with­ out prior extraction. Rangkadilok et al. (2007) performed hot water extraction of longan seeds and pulp powder at 70˚C–75˚C for 1 h for three times in a small scale (2 L) before analyses of bioactive compounds which revealed up to 23.0 g gallic acid and 12.65 g ellagic acid per kg of dried longan seeds. Chindaluang and Sriwattana (2014) suggested ultrasonic-assisted water extraction (UAWE) as an alternative method for extraction of bioactive compounds from longan seeds as total polyphe­ nol yield was higher than ethanol extraction method. In fact, UAWE was able to extract ellagic acid at an equivalent level to hot water extraction and at a statistical significantly higher (p ≤ 0.05) level than ethanol extraction method.

7.3.3

ETHANOL AND PHENYLACETYLCARBINOL PRODUCTION

Longan fruit production was affected by not only the common diseases such as witches’ broom and brown spot which decreased its quality (Sittigul et  al., 2005) but also overproduction that dropped the longan fruit prices during certain years (DITP, 2016; Prachachat, 2017). In addition, only AA- and A-grade fresh longan fruits were exported, whereas B-grade fresh longan fruits were processed in the form of canned and dried longans. The C-grade fresh longan fruits were undesir­ able and were produced for more than 50,000 tons (about 200 million Baht) and were destroyed by either burning or burying (OAE, 2005; ARDA, 2010; OAE, 2014). The flesh of longan fruits is sweet and is found to contain high concentration of sugars (sucrose, glucose, and fructose), vita­ mins, minerals, and fibers. Glucose and fructose comprised 70% of total sugars in longan fruits, whereas sugarcane and beetroot contained only sucrose. Upon consumption, monosaccharides, such as glucose or fructose, could be rapidly absorbed into bloodstream, whereas sucrose must be hydrolyzed to monosaccharides prior to absorption (Elliott et  al., 2002). Therefore, longan juice extracted from fresh and dried longan fruits of any grade is thus suitable for utilization as carbon sources in ethanol production. The processing of fresh longan fruits to dried longans for the purpose of added values had encountered a deadstock problem, which prevented 67,000 tons of the processed longan fruits from being transferred aboard during 2003–2004. It took more than 6 years to reach the final resolution to rectify the problem. One alternative strategy is to retrieve dried longan with high level of sugars and utilize it for ethanol production as biofuels. The attained biomass can also be used as biocatalyst for the production of a precursor for medicines, namely, ephedrine and pseudoephedrine with the properties of relieving the allergic and nasal congestion symptoms (Leksawasdi and Pratanaphon, 2010). Processes involved with production of ethanol and/or PAC from various types of longan products/by-products and other biomaterials (Natikarn and Leksawasdi, 2009; Natikarn et al., 2011; Prommajak et al., 2019) had been investigated by our group for a number of years. These included expired dried longan (EDL) (Achavasmith and Leksawasdi, 2009; Agustina et al., 2009; Pudthathep et al., 2009; Leksawasdi and Pratanaphon, 2010; Leksawasdi et al., 2011), fresh longan juice (FLJ) (Nunta et al., 2018, 2019), and WLSW (Wattanapanom et al., 2019). Some recent examples of alco­ hol production for beverage and/or utilization as an alternative energy source from readily ferment­ able agricultural or food waste materials included partially aerobic ethanol production from longan fruits (Chen et al., 2013; Nunta et al., 2018, 2019) or cashew apple juice (Prommajak et al., 2019) and anaerobic butanol production from pineapple juice (Sanguanchaipaiwong and Leksawasdi, 2018) or glycerol (Sanguanchaipaiwong and Leksawasdi, 2017). Further interests on ethanol production from the mixture of pentose/hexose sugars (Leksawasdi et al., 2001; Yuvadetkun et al., 2017) as well as lignocellulosic agricultural and agroindustrial materials (Boonchuay et  al., 2018; Li et  al., 2018) indicated the necessity to achieve zero waste process for eventual implementation in food industries.

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As of 2017, the ethanol production in Thailand (3.4 million L/day) with the installed capacity of 4.4 million L/day (77.3% operational capacity) from 21 factories could not catch up with the ethanol demand (3.9 million L/day), which was equivalent to a deficit of 0.5 million L/day. This was compared with Brazil and United States with corresponding production and demand (million L/day) of (82.7, 60.8) and (159.5, 147.40), respectively (BOT, 2017). Such gap between demand and supply could provide opportunity for ethanol production from a number of readily available raw materials in Thailand (ethanol yield in L/ton of raw materials), for example, corn (375), molasses (260), EDL (209 ± 14), fresh cassava (180), and sugarcane (70) (NESDB, 2006; Leksawasdi et al., 2010). A typical large-scale ethanol production plant from sugarcane (0.2 million L/day) would have a raw material input of 5 tons/day. The optimal initial total sugars concentration was 220–240 g/L (25°Brix–30°Brix) with cultivating temperature of 29˚C–32˚C and pH control between 4.5 and 5.5. The final total sugar concentration after fermentation stage was 9 g/L with the ethanol concentra­ tion of 90 g/L (ethanol mass yield of 0.41 g/g total sugars consumed). This ethanol concentration level was well beyond economic feasibility limit of 40 g/L, thus rendering an economically viable ethanol distillation process. In fact, the ethanol mass yield was also acceptable in comparison with the theoretical mass yield of 0.511 g ethanol/g glucose and 0.538 g ethanol/g sucrose (Natikarn and Leksawasdi, 2009; APEC-ATCWG Biofuels, 2011). Leksawasdi et al. (2013) investigated ethanol production capability of Saccharomyces cerevisiae TISTR 5606 and Candida tropicalis TISTR 5306 using dried longan extracts as carbon source under shaking condition of 200 rpm at 30˚C ± 1˚C. The result showed that S. cerevisiae TISTR 5606 produced the highest ethanol concentration level of 45.1 ± 2.6 g/L with Yp/s of 0.46 ± 0.01 g ethanol/g total sugar. This was compared with C. tropicalis TISTR 5306 which could produce ethanol concentration level of 38.1 ± 3.0 g/L with Yp/s of 0.42 ± 0.01 g ethanol/g total sugar. In addi­ tion, the comparison of ethanol concentration levels obtained by S. cerevisiae TISTR 5606 and C. tropicalis TISTR 5306 using FLJ having sugar concentration of 17°Brix as carbon source was also made. The results indicated that S. cerevisiae TISTR 5606 could produce ethanol concentration level of 38.3 ± 1.3 g/L with Yp/s of 0.42 ± 0.01 g ethanol/g total sugar, which was higher than that of C. tropicalis TISTR 5306 (17.8 ± 0.2 g/L with Yp/s 0.20 ± 0.01 g ethanol/g total sugar) (Leksawasdi et al., 2014). The biotransformation processes were principal in the pharmaceutical sector followed by food and agricultural sectors (Straathof et al., 2002). The transformation of organic compounds using biocatalysts such as enzyme, cell organelles, and whole cells was critical in organic synthesis and had been widely used in the production of steroids, antibiotics, vitamins, and other high-value prod­ ucts. The advantages of such biotransformation were that they were reaction-specific processes and involved high degrees of regio- and stereospecificity (Rogers et al., 1997). Microbial biotransforma­ tion was one of the research fields which had gained popularity for the past years (Joseph and Priya, 2011). This process could be easily carried out under a relatively mild condition of temperature and pH (Schmid et al., 2002; Cherry and Fidantsef, 2003). Microbial biotransformation for the biologi­ cally active chiral compounds was a rapidly growing field. One example of such was the production of PAC which was an organic compound that had two enantiomers of R- and S-configurations or D- and L-configurations. The enantiomeric excess of PAC could be quantified and distinguished between R- and S-enantiomers by high-performance liquid chromatography at 283 nm. The result showed high enantiomeric excess in terms of R-PAC (higher than 90%). PAC had melting point at 172.0˚C, half-life of 240 h, and specific optical rotation at −375.8 (Rosche et al., 2002; Smallridge et al., 2006). PAC was used as an intermediate in the production of ephedrine and pseudoephedrine. These were pharmaceutical compounds used as decongestants and antiasthmatics (Leksawasdi et  al., 2003, 2004; Leksawasdi, 2004). Certain microorganisms could transform aromatic aldehyde sub­ strates to produce acetyl aromatic carbinols in the presence of pyruvate generated during glycolysis. This reaction was catalyzed by an intracellular PDC. Biotransformation could be achieved in the form of either partially purified PDC (Tangtua et al., 2015, 2017) or pretreated yeast whole cells

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(Nunta et al., 2019). PAC production by growing cells of various yeast species had been demon­ strated with various degrees of success (Mandwal et al., 2004). The exact pathway for production of PAC and associated products was shown in detail by Leksawasdi (2004). Pyruvic acid, a prod­ uct of glycolysis, was decarboxylated to “active acetaldehyde” that reacted with benzaldehyde to produce PAC (Leksawasdi et al., 2005a,b). Benzaldehyde could be associated with the formation of benzyl alcohol by-product due to the activity of alcohol dehydrogenase (ADH) and/or other non­ specific oxidoreductases. Some traces of benzoic acid as a by-product had also been reported. The effect of benzaldehyde deactivation on PDC activity had also been elucidated (Leksawasdi et al., 2003; Khemacheewakul et al., 2018). PAC produced from a biotransformation was later converted to ephedrine by a chemical reductive amination with methylamine (Leksawasdi et al., 2006). Current commercial preparation of PAC consists of the process in which the fermentative yeast was cultivated on sugar or alternative carbon source to produce biomass, i.e., pyruvic acid with induction of PDC activity (Zhang et al., 2008). The cells obtained after fermentation process had the potential application for extraction of PDC enzyme, which was able to catalyze the ligation reaction between benzaldehyde and pyruvate to produce PAC. Various yeast strains had been investigated with reference to the production of PAC. Earlier studies had shown PAC production by Brewer’s yeast. PAC was currently produced via microbial transformation process using different species of yeast, fungi, and bacteria such as S. cerevisiae, Saccharomyces carlsbergensis, Hansenula ano­ mala, Rhizopus javanicus, and Zymomonas mobilis (Chandrakant et al., 1997; Rosche et al., 2001; Hauer et al., 2003; Tangtua et al., 2013). Leksawasdi and Pratanaphon (2010) screened six Candida utilis strains which included TISTR 5001, 5032, 5043, 5046, 5198, and 5352 to evaluate the growth and ethanol production kinetics in detail. The database of these yeasts is available online (TISTR Database #147, 2019). C. utilis strain TISTR 5352 was able to produce ethanol at the highest level at 150 mL scale for 192 h cultivation period in a static condition using a carbon source from EDL extract with other supplementary nitrogen sources at 25.6˚C. The second ethanol producer was C. utilis TISTR 5198. The ethanol yield obtained from the cultivation of C. utilis TISTR 5352 in 1,500 mL scale was 0.27 ± 0.01 g/g. The cultivation of C. utilis TISTR 5198 and TISTR 5352 in digested dried longan flesh hydrolysate (DDLFH) medium at total suspended solid levels of 20°Brix and 40°Brix indicated the growth inhi­ bition. The two-phase PAC biotransformation of C. utilis TISTR 5198 using whole cells harvested at 192 h in DDLFH medium with 6.12 g/L dried biomass equivalent resulted in the overall PAC pro­ duction level of 1.76 ± 0.06 mM which was followed by C. utilis TISTR 5352 in EDL medium with PAC production level of 0.75 ± 0.02 mM. Inoculum level at 1% (v/v) was most suitable for a 1,500mL-scale batch cultivation of S. cerevisiae TISTR 5606 using EDL extracts as a carbon source in a static condition for 36 h and 25.6˚C. The consecutive runner-ups were inoculum levels at 5% and 10% (v/v), respectively. The carbon source from EDL medium was most suitable for batch cultiva­ tion in 5,000 mL scale with an initial aeration period of 12 h from the overall 36 h cultivation period at 25.6˚C. Fed-batch system illustrated the toxicity of DDLFH medium in comparison with EDL medium. The two-phase separated PAC biotransformation using whole cells cultivated in 5,000 mL scale with EDL and DDLFH media did not result in PAC production. The attempt to optimize PDC production in other microorganisms was carried out by Leksawasdi and Pratanaphon (2010). The PDC1 gene was amplified from S. cerevisiae 5606, and the amplicon was ligated into pPICZA. The resulting pPICZA-PDC1 was transformed into Pichia pastoris X­ 33. Three clones of recombinant P. pastoris found on selective media containing 500/mL zeocin were cultured and induced with methanol. The activities of PDC1 expressed from P. pastoris were similar to S. cerevisiae TISTR 5606 and C. utilis TISTR 5198. This study allowed possible further studies for optimization of the PDC1 expression. Leksawasdi and Techapun (2019) generated bio­ ethanol from agricultural/agroindustrial biomaterials in Thailand and high value-added compounds using by-products (in the form of whole yeast cells) obtained from bioethanol production process. In the first subproject, bioethanol in 30 and 100 L scales were produced at the concentration levels of 5.9% and 5.7% (v/v), respectively, using separation hydrolysis and fermentation (SHF) from yeast

Longan Syrup and Related Products

137

cultivation of S. cerevisiae (active dry yeast) in pretreated sugarcane bagasse hydrolysate. This bio­ ethanol production process was conducted using a continuous stirred bioreactor of which agitation speed was controlled at 200 rpm and aeration rate of 1 vvm. The economic analysis revealed the operating cost for bioethanol production process from sugarcane bagasse of 0.87 USD/L. The sec­ ond research subproject utilized a mixture of coculture whole yeast cells (S. cerevisiae and C. tropi­ calis), a by-product from ethanol production, as biocatalyst for the production of PAC. The results showed that biphasic biotransformation process consisting of an oil phase and a buffer phase with initial concentration level of benzaldehyde/pyruvate at 100/120 mM could produce the highest con­ centration level of PAC in oil and buffer phases of 14.8 ± 0.4 and 5.0 ± 0.13 mM, respectively. This corresponded to the average PAC concentration level of 9.91 ± 0.24 mM from the reaction period of 360 min. The economic analysis also revealed that the wet biomass of 1 kg whole yeast cells could yield 145 g of PAC with the industrial production cost of 0.42 USD (equivalent to 345 g PAC/USD or 2.90 USD/kg PAC), whereas the possible selling cost of produced PAC could reach 21.31 USD/kg PAC depending on the PAC purity as indicated in Tables 7.2–7.4. Nunta et al. (2018, 2019) pointed out the necessity in examining the utilization of FLJ obtained from C-grade longan fruits (FLJ-C) due to poor marketing demands stemming from the relatively small size of the longan pulp. Nunta et al. (2018) selected and utilized FLJ-C as an inexpensive carbon source for production of ethanol and PAC by comparing production efficiency between C. tropicalis TISTR 5306 and S. cerevisiae TISTR 5606. Five grades of fresh longan fruits, namely, AA (radius > 12.5 mm), A (10.5–12.5), B (9.5–11), C (2%–5% O2 lied in the elastic stress zone where some ethanol was found at the end of storage. Detection of ethanol in longans in this elastic stress zone suggested that induction of fermentation took place as plant tissues needed energy to survive. In this stage, elevated CO2 can affect this relationship by reducing the utilization of sugar and changing the activities of enzymes critical to glycolysis (Beaudry, 1999). Based on the tolerance study (Khan et al., 2017), 5% O2 combined with 5%, 10%, and 15% CO2 was selected to determine the optimum CA and compared with normal air as control at 2˚C. Pericarp browning and decay incidence of longans were significantly (P ≤ 0.05) higher in control than all the

182

Asian Berries: Health Benefits Controlled Atmosphere (CA) Conditions Air

(5 % O2 + 5 % CO2)

(5 % O2 + 10 % CO2)

(5 % O 2 + 15 % CO2)

Day 0

Day 0

Day 0

Day 0

Day 21

Day 21

Day 21

Day 21

Day 35

Day 35

Day 35

Day 35

Day 35

Day 35

FIGURE 9.1 Quality changes and decay incidence of longan fruits during storage under different controlled atmosphere conditions (Khan et al., 2017).

FIGURE  9.2 Ethanol production in longan fruits stored at 2˚C in various O2 (2%, 5%, 10%, 15% O2 and air, balanced with N2) and CO2 (5%, 10%, 15%, and 20% balanced with air) concentrations. (Reprinted with permission from Khan et al., 2017.)

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183

CA treatments. CA storage reduced PPO activity, maintained color (L* value), and slowed down a decrease in TPCs. Pericarp browning was highly correlated with PPO, L*, and TPC. Based on the CA study, the combination of 5% O2 with 5%, 10%, and 15% CO2 showed no significant (P > 0.05) difference in most quality parameters. However, gas composition of 5% O2 + 10% CO2 was found most suitable in terms of quality maintenance and lower ethanol production. Khan et al. (2016) studied the effects of packaging films with various transmission rates on the quality and shelf life of longan fruits (Table  9.3). Sulfur dioxide (SO2)–fumigated longan fruits were stored at 2˚C in PE bags (7 × 11 in.) of different gas transmission rates, namely, PE-3000, PE­ 6000, and PE-10000, compared with commercial polypropylene (PP; PP-1000). Thicknesses of these films were 80, 55, 28, and 80 μm with oxygen transmission rates (OTRs) of 2,861.87 ± 155.02, 6,046.02 ± 380.25, 10,572.22 ± 506.29, and 1,116.23 ± 10.22 cm3/m2/day, respectively, and carbon dioxide transmission rates (CO2TR) of 8,914.56 ± 82.97, 28 581.30 ± 782.58, 57 517.39 ± 488.59, and 2,488.74 ± 239.28 cm3/m2/day1, respectively. PE-3000 and PE-6000 maintained the longest shelf life of 46 days. Fruits in the PE-10000 had the shelf life of 39 days as limited by pericarp browning and decay incidence. The shortest shelf life of 27 days was observed in the PP-1000 due to fermentative metabolism under anaerobic condition (Khan et al., 2016). For detail investigation and commercial applications of the polymeric films/package for longan fruits, the fruit quality in different tolerance levels and optimum CA were compared with the results obtained from the MAP study by Khan et al. (2016), which showed that PE-3000 and PE-6000 pre­ served the longan fruit quality with longest shelf life. The gas composition established in PE-3000 (4% O2 + 9% CO2) was closest to the recommended CA condition (5% O2 + 10% CO2). Nevertheless, the O2 and CO2 levels established in PE-6000 (9% O2 + 5% CO2) corresponded to the results from the tolerance study that 10% O2 was not significantly (P > 0.05) different from 5% O2, whereas 5% CO2 gave similar results to 10% CO2 except for ethanol content. PE-10000 provided shorter shelf life than PE-3000 and PE-6000. The gas composition established in PE-10000 (15% O2 + 2% CO2) was clearly out of the recommended O2 and CO2 ranges as suggested by both CA and tolerance stud­ ies. The results indicated that O2 and CO2 tolerance and CA experiments are important for MAP design and packaging film selection. Therefore, based on the previous experiments on MAP (Khan et  al., 2016) and the tolerance levels and CA storage (Khan et al., 2017) of longans, PE bags (55 ± 4 μm thickness, 5.5 × 15 in.) were developed to provide O2 and CO2 in the range of 4%–9% and 5%–10%, respectively (Khan et al., 2019). The OTR and carbon dioxide transmission rates (CO2TR) of this film were 6,379.35 ± 280 and 28 528.30 ± 782 cm3/m2/day, respectively, at 23˚C. The effect of this packaging film for the inhibition of pericarp browning and decay incidence of longans (cv. Daw) was studied with combination of

TABLE 9.3 Effects of gas transmission rates of packaging films on equilibrium modified atmosphere (EMA) and shelf life of longans at 2°C Films PP-1000 PE-3000 PE-6000 PE-10000

a

OTRa (cm /m2/day)

CO2TRa (cm3/m2/day)

1,116.23 ± 10.22 2,861.87 ± 155.02 6,046.02 ± 380.25 10,572.22 ± 506.29

2,488.74 ± 239.28 8,914.56 ± 82.97 28,581.30 ± 782.58 57,517.39 ± 488.59

3

EMA n/a 4%O2 + 9%CO2 9%O2 + 5%CO2 15%O2 + 2%CO2

Shelf Life (days)

Causes of Deterioration

27 46 46 39

Fungal growth, off-flavor Fungal growth Fungal growth Fungal growth, pericarp browning

OTR (oxygen transmission rate) and CO2TR (carbon dioxide transmission rate) were measured at 23˚C, 0% relative humidity. Adapted from Khan et al. (2016).

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natural antibrowning and antimicrobial compounds (Khan et al., 2019). Fruits were treated with 5% AA, citric acid (CA), OA, TH, and 2% CH for 5 min, sealed under MAP, and stored at 5˚C. Fruits treated with distilled water and sealed under MAP condition (MAPC) and air condition (AIRC) were used for control treatments. Combined effects of chemical treatments and MAP maintained quality of longans for 56 and 49 days in CH and TH, respectively, as compared with 28 days in MAPC and 21 days in AIRC. AA, CA, and OA prevented BI, restrained enzyme activities, and maintained high phenols. AA, CA, and OA were most potent BI inhibitors; however, DI was the limitation of these treatments, whereas CH and TH effectively inhibited DI. CA storage and MAP of longan fruits were previously reported by other researchers. Tian et al. (2002) stored longan fruits (cvs. Chuliang and Shixia) at 2˚C in CA of 4% O2 + 5 or 15% CO2, or 70% O2 + 0% CO2. The authors reported that CA inhibited PPO activity and delayed pericarp browning and fruit decay better than MAP with 15%–19% O2 + 2%– 4% CO2. CA with high O2 concentration effectively decreased ethanol production in the flesh. CA with elevated CO2 (4% O2 + 15% CO2) effectively reduced decay and extended the storage life of longan fruits than other CA treatments. The two cultivars responded differently in terms of PPO enzyme, pericarp browning, and diseases incidence at the same storage condition (2˚C, 95% RH).

9.3.5

CHITOSAN COATING

CH is a natural linear biopolyaminosaccharide obtained by alkaline deacetylation of chitin, which is the principal component of protective cuticles of crustaceans such as crabs, shrimps, prawns, lobsters, and cell walls of some fungi such as aspergillus and mucor (Kumar et al., 2004). Because of economical, biodegradable, and nontoxicity properties, CH is widely used in fields such as bio­ technology, pharmaceutics, cosmetics, textiles, and agriculture and food industries (Ravi, 2000). The antimicrobial activity of CH against a wide range of microorganisms has drawn its attention. Regarding the antimicrobial activity of CH, different researchers have proposed different mecha­ nisms. The positive charges of CH react with the electronegative charges on cell surfaces and thus change the cell permeability, which causes the leakage of intracellular electrolytes and proteinaceous constituents. It was also reported that entrance of CH into fungal cells adsorbs the essential nutrients, which inhibit or slow down the synthesis of mRNA and protein (Avadi et al., 2004; Chen et al., 1998). Due to the versatile properties of CH, its application to fresh produce has widely been studied. CH coating produces a semipermeable film, which can regulate the gas exchange and reduce tran­ spiration losses (water loss) and respiration rate (Bautista-Banos et al., 2006). Jiang and Li (2001) coated longan (cv. Shixia) fruits in 0.5%, 1%, and 2% CH and stored them at 2˚C for 40 days. Fruits dipped in the glacial acetic acid solution were used as control. CH effectively reduced the respiration rate and weight loss, slowed down the PPO activity, delayed the changes in pericarp color, and partially prevented the decay of longan fruits than the control treatment. Vangnai et al. (2006) treated longan fruits (cv. Daw) with 0%, 0.5%, 1.0%, and 1.5% CH and stored them at 4˚C and 90%–95% RH for 20 days. The authors reported that CH at all concentrations prevented decay, reduced the respiration rate and weight loss, and maintained AA contents better than the control treatments. CH at all concentrations reduced increasing activities of PPO but slightly reduced the pericarp browning. Shi et al. (2013) treated the longan fruits (cv. Shijia) with 2% CH, nanosilica, and CH/nanosilica solutions for 4 min. Fruits dipped in glacial acetic acid (0.5%) were used as the control. Fruits were sealed in PE bags at 25˚C for 10 days with 70%–80% RH. All treatments greatly extended the shelf life, delayed weight loss, reduced pericarp browning, inhibited the increase of malondialdehyde amount and PPO and POD activities, and reduced the reduction of total soluble solids, titratable acidity, and AA better than the control treatments. The authors suggested that among all the treatments, CH hybrid film (CH/nanosilica) was the best in maintaining the longan fruit quality. Khan et al. (2019) also reported that longan fruits (cv. Daw) treated with 2% CH and sealed in PE film extended the shelf life of longans to 56 days than 21 days in air (normal air) and 28 days in the MAP control treatments (not treated with CH).

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CH in combination with antibrowning agents showed a synergetic effect in reducing the pericarp browning and decay of longan fruits. Apai et al. (2009) dipped longan fruits (cv. Daw) in 1.2% (w/v) CH coating solution containing 1.0% citric acid, 1.0% citric acid solution, and distilled water for 2 min. All the fruits were packaged in a foam tray, wrapped with polyvinyl chloride film, and stored at 5˚C, 95% RH for 27 days. Combination with citric acid best maintained the quality, and the fruits were acceptable even after 27 days of storage, whereas the fruits dipped only in citric acid or dis­ tilled water were not acceptable after 20 days of storage. Combined treatment of CH and citric acid also maintained low pericarp pH, low PPO activity, high color (L*, Chroma and Hue values), high phenolic content, and consequently low pericarp browning than 1% citric acid or distilled water treatments. Apai et al. (2008) also studied the combined effect of citric acid (1%, 3% and 5%) and CH (1.2%) coating on longan for 20 days at 5˚C and 10˚C. Combination of 1% citric acid with 1.2% CH at 5˚C was the best treatment, which preserved the longan fruits for 20 days with good quality and low decay incidence than other treatments.

9.4 CONCLUSION Because of the high perishable nature and sugar content in longan fruits, pericarp browning, post­ harvest decay, and skin desiccation are the limiting factors for longan postharvest storage life. Color is one of the most important quality attributes for a selection of fruits, and their deterioration causes the fruits unmarketable. Pericarp browning of longan fruits is attributed to the oxidation of phenolic compounds by PPO producing brown pigments. Various attempts have been made to overcome these problems. Most effective chemical treatment is SO2, which has widely been used for longan fruits. However, because of the safety and environmental issues, use of SO2 has been banned and restricted. Several alternatives have been applied in the form of chlorine dioxide fumi­ gation, antibrowning dipping, and CH coating to inhibit pericarp browning and postharvest decay of longan fruits. CA/MAP with low O2 and high CO2 concentrations was shown to prevent pericarp browning and postharvest decay of longan fruit. Low O2 concentration reduces the respiration rate of the fruit, pre­ vents oxidation of phenolic compound, avoids anaerobic fermentation, and delays fruit ripening and onset of ethylene production. Carbon dioxide has shown an inhibitory effect on microbial growth as well as an antagonistic effect on enzymes involved in ethylene biosynthesis. Browning due to dehydration has also been shown to reduce by MAP. Selection of films according to respiration rate, storage temperature, and O2 and CO2 tolerance limits of longan fruit are the important factors for designing MAP system. Any combination of O2 (3%‒10%) and CO2 (5%‒10%) is beneficial for longan fruit storage in both the CA and MAP at low temperatures. Coating of longan fruits with natural antibrowning and antimicrobial solutions in combination with polymeric film providing the aforementioned recom­ mended range of O2 and CO2 levels could potentially be used as the best alternative to SO2 fumiga­ tion for longan fruits at commercial level and export to long-distance markets.

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Sudjaroen, Y., Hull, W.E., Erben, G., Würtele, G., Changbumrung, S., Ulrich, C.M., Owen, R.W. (2012). Isolation and characterization of ellagitannins as the major polyphenolic components of Longan (Dimocarpus longan Lour.) seeds. Phytochemistry, 77: 226–237. Suwanamornlert, P., Sangchote, S., Chinsirikul, W., Sane, A., Chonhenchob, V. (2018). Antifungal activity of plant-derived compounds and their synergism against major postharvest pathogens of longan fruit in vitro. International Journal of Food Microbiology, 271: 8–14. Taylor, S.L., Higley, N.A., Bush, R.K. (1986). Sulfites in foods: uses, analytical methods, residues, fate, expo­ sure assessment, metabolism, toxicity and hypersensitivity. Advanced Food Research, 30: 1–76. Tian, S., Xu, Y., Jiang, A., Gong, Q. (2002). Physiological and quality responses of longan fruit to high O2 or high CO2 atmospheres in storage. Postharvest Biology and Technology, 24: 335–340. Tongchitpakdee, S. (2012). Longan and carambola. In: Siddiq, M. (Ed.). Tropical and Subtropical Fruits: Postharvest Physiology, Processing and Packaging. John Wiley & Sons, Inc., New York, pp. 223–240. Toivonen, P.M.A., David, A.B. (2008). Biochemical bases of appearance and texture changes in fresh-cut fruit and vegetables. Postharvest Biology and Technology, 48: 1–14. Vangnai, T., Wongs-Aree, C., Nimitkeatkai, H., Kanlayanarat, S. (2006). Quality maintaining of ‘Daw’ lon­ gan using chitosan coating. Acta Horticulturae, 712: 599–604. Wall, M.M. (2006). Ascorbic acid and mineral composition of longan (Dimocarpus longan), lychee (Litchi chi­ nensis) and rambutan (Nephelium lappaceum) cultivars grown in Hawaii. Journal of Food Composition and Analysis, 19: 655–663. Whangchai, K., Saengnil, K., Uthaibutra, J. (2006). Effect of ozone in combination with some organic acids on the control of postharvest decay and pericarp browning of longan fruit. Crop Protection, 25: 821–825. Wu, Y.L., Yi, G.J., Zhou, B.R., Zeng, J.W., Huang, Y.H. (2007). The advancement of research on litchi and longan germplasm resources in China. Scientia Horticulturae, 114, 143–150. Yang, B., Zhao, M., Shi, J., Yang, N., Jiang, Y. (2008). Effect of ultrasonic treatment on the recovery and DPPH radical scavenging activity of polysaccharides from longan fruit pericarp. Food Chemistry, 106: 685– 690. Yang, B., Zhao, M., Prasad, K.N. Jiang, G., Jiang, Y. (2010). Effect of methylation on the structure and radical scavenging activity of polysaccharides from longan (Dimocarpus longan Lour.) fruit pericarp. Food Chemistry, 118: 364–368. Yang, B., Jiang, Y., Shi, J., Chen, F., Ashraf, M. (2011). Extraction and pharmacological properties of bioactive compounds from longan (Dimocarpus longan Lour.) fruit- A review. Food Research International, 44: 1837–1842. Yao, F., Huang, Z., Li, D., Wang, H., Xu, X., Jiang, Y., Qu, H. (2014). Phenolic components, antioxidant enzyme activities and anatomicstructure of longan fruit pericarp following treatment with adenylate triphosphate. Scientia Horticulturae, 180: 6–13. Yonemoto, Y., Chowdhury, A. K., Kato, H., Macha, M. M. (2006). Cultivars identification and their genetic relationships in Dimocarpus longan subspecies based on RAPD markers. Scientia Horticulturae, 109: 147–152. Zhang, Y., Gao, B., Zhang, M.W., Shi, J., Xu, Y. J. (2009). Headspace solid-phase microextraction–gas chro­ matography–mass spectrometry analysis of the volatile components of longan (Dimocarpus longan Lour.). European Food Research and Technology, 229: 457–465. Zhang, Z., Zeng, D., Li, G. (2008). Study of the volatile profile characteristics of longan during storage by a combination sampling method coupled with GC/MS. Journal of the Science of Food and Agriculture, 88: 1035–1042. Zheng, J.S., Zhao, Y.F., Lin, H.T., Lin, W.Z., Zhuang, W.D., Tang, H.L. (2009). Effects of bagging time on growth, development and quality of ‘Lidongben’ longan fruit. Chinese Journal of Tropical Crops, 30: 1232–1237.

10 Phytochemical Properties Litchi Berries

and Cosmetic Benefits Mayuree Kanlayavattanakul and Nattaya Lourith Mae Fah Luang University

CONTENTS 10.1 10.2 10.3

Introduction ........................................................................................................................ 190

Skin, Skin Dryness, Skin Wrinkle, and Skin Dullness ...................................................... 190

Naturally Derived Compounds That Are Commonly Used for Cosmetic Benefits ........... 191

10.3.1 Phenolics ............................................................................................................... 191

10.3.1.1 Caffeic Acid .......................................................................................... 191

10.3.1.2 Chlorogenic Acid .................................................................................. 191

10.3.1.3 p-Coumaric Acid .................................................................................. 192

10.3.1.4 Ferulic Acid .......................................................................................... 192

10.3.1.5 Rosmarinic Acid ................................................................................... 193

10.3.1.6 Sinapic Acid .......................................................................................... 193

10.3.1.7 Syringic Acid ........................................................................................ 193

10.3.2 Flavonoids ............................................................................................................. 194

10.3.2.1 Catechins .............................................................................................. 194

10.3.2.2 Quercetin .............................................................................................. 194

10.3.3 Proanthocyanidins................................................................................................. 194

10.3.4 Polysaccharides ..................................................................................................... 195

10.3.4.1 Cellulose ............................................................................................... 195

10.3.4.2 Starch .................................................................................................... 195

10.3.4.3 Pectin .................................................................................................... 195

10.3.4.4 Gum ...................................................................................................... 195

10.3.4.5 Mucilage ............................................................................................... 195

10.4 Phytochemical Actives of Litchi Berries with Cosmetic Benefits...................................... 196

10.4.1 Pericarp ................................................................................................................. 196

10.4.2 Pulp ....................................................................................................................... 196

10.4.3 Seeds ..................................................................................................................... 197

10.5 Conclusions ......................................................................................................................... 198

Abbreviation...................................................................................................................................200

References......................................................................................................................................200

189

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Asian Berries: Health Benefits

10.1 INTRODUCTION Litchi berries (Litchi chinensis Sonn. [Sapindaceae]) are fruits with a characteristic flavor and taste that, based on their biological profile, have many health benefits as evidenced in their pharmaco­ logical records (Ibrahim and Mohamed, 2015). Their phytochemical actives could be relied on fruit, leaf, stem, bark, and root, of which traditionally used fruit-derived compounds are more implied in health and health promotion products including for aesthetic proposes, of which phenolic compounds are those of important pharmacologically actives (Hollman, 2001; Zillich et al., 2015) derived from litchi berries that are recorded. The health benefits are majorly implied by antioxidant activity, and the consequence associated in several diseases and disorders treatment due to oxidative damage not only initiates but also propagates severity of the symptom. Accordingly, antioxidant capability of the natural extract is firstly assessed by several assays designed to replicate those of reactive species during the course of the metabolic pathway in the organism. Thereafter, additional biological activ­ ity assessments are undertaken in harmony with the objective of the therapeutic applications on the basis of the chemical active molecules constituent in the natural extract, of which in vitro antioxidant assessment methods are widely discussed and would be consulted in different chapters of this book. In addition to the in vitro study, ex vivo study or an examination in cell culture is one of the important steps to verify safety and efficiency of the phytochemically active compounds. In the past, animal model assessment is undertaken as the following step from in vitro. However, the advancements of ex vivo would fill in the gap omitting assessment in animal, in accordance with the specific cell, developed from the human organ to yield more accurate and precise result resembling the real scene in human metabolic function. In addition, the consumers’ awareness among animal testing product turns to be unacceptable. Thus, cell culture or ex vivo study is regarded as the efficient substitute studying method instead of animal model prior to in vivo assessment in human volunteers. This chapter will be therefore emphasized on cosmetic benefits of litchi berries, i.e., pulp, peel, and seed. Skin and causes of skin disorders, i.e., skin dryness, skin wrinkle, and skin dullness are briefly addressed in an order to pave an understanding of the litchi berries’ active compounds ben­ eficial for skin. Those of pharmacologically active phenolics are exclusively summarized, in which the commonly used phenolics for cutaneous benefits will be addressed including the associated biological activities appreciable for cosmetics. Preparation of the isolated active fractions or pure compounds derived from pulp, peel, and seed of litchi berries is described together with the relevant cutaneous benefits by means of in vitro, cell culture, and in vivo assessments in animal or human volunteer if available.

10.2

SKIN, SKIN DRYNESS, SKIN WRINKLE, AND SKIN DULLNESS

Skin is composed of epidermis and dermis, of which dermis is the most important skin layer responsi­ ble for skin elasticity contributing in aesthetic property of skin. Dermis is a three-dimensional network of collagen and elastin fibers surrounded by the ground substances. Skin aging process is started once the skin becomes dried and thereafter epidermal and dermal layers become slimmer. The epidermal junction becomes flatter with a higher degree of skin roughness; in the meantime, epidermal sebum is decreasing. Skin chromophores, particularly melanin, are diminishing and becoming irregular. These processes are followed by a deterioration of dermal matrix. Thereafter, the sign of aging is visibly noticed in skin color, translucency, apparent roughness, and skin wrinkles and folds consequently. Skin aging is caused by several factors which damage cell membranes and components. Reactive molecules with unpaired electrons or free radicals initiate cellular damage, disturbing several physi­ ological functions including aging. Natural cellular metabolism generates free radicals in a selfdefense mechanism and efficiently scavenges these species and neutralizes the radicals; however, these are decreased with age. Dermal damage is also induced by UV exposure at the shorter wavelengths (UVB), which are absorbed by the epidermis prior to irradiation of keratinocytes. In the meantime, longer wavelengths

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(UVA) penetrate into the deeper layers of skin and interact with epidermal and dermal cells. Proteolytic enzyme activities are propagated, resulting in degradation of collagen and elastin fibers including glycosaminoglycan (GAG), hyaluronan, chondroitin, keratin, dermatan, and heparin. They are linked to proteins such as collagen (28 types) and elastin and act as lubricants associated with the elasticity and tensile strength of skin. The matrix metalloproteinase (MMP) is the degradation enzyme of the extracellular matrix (ECM), including collagen, elastin, and GAG. The enzyme is therefore classified into MMP-1 to MMP-28. Their activities are exacerbated with age and radicals including inflammatory mediators as well . Therefore, deactivation, inhibition, or suppression of MMP, especially collagenase, elastase, and hyaluronidase, in addition to stimulation of hyaluronan synthase, is regarded as the leading strategy in the management of skin aging (Kanlayavattanakul and Lourith, 2015a; Lourith and Kanlayavattanakul, 2016). Skin hyperpigmentation is caused by several factors, i.e., UV radiation, radicals, inflamma­ tory mediators, and hormones. Briefly, UV radiation causes skin hyperpigmentation by stimulat­ ing keratinocytes to secrete α-melanocyte-stimulating hormone (α-MSH), a small peptide hormone derived from proopiomelanocortin (POMC). Consequently, α-MSH binds to melanocortin 1 receptor (MC1R) expressed on melanocyte surfaces and thereafter induces melanogenesis via multiple sig­ naling pathways resulting from cAMP, protein kinase A (PKA), cAMP response element–binding protein (CREB), and microphthalmia-associated transcription factor (MITF) activity. MITF is a key transcription factor regulating the transcription of melanogenic enzymes, i.e., tyrosinase, tyrosinase­ related protein (TRP)-1, and TRP-2. In addition, UV radiation modulates nuclear factor E2–related factor 2 (Nrf2) and further activates mitogen-activated protein kinases (MAPKs). MAPKs consist of three subtypes: stress-activated protein kinases (SAPKs)/c-Jun NH2-terminal kinases (JNK), p38, and extracellular signal–regulated kinases (ERKs). JNK and p38 kinases are stimulated by proin­ flammatory cytokines and environmentally induced stresses such as exposure to UV irradiation, heat, and hydrogen peroxide, resulting in DNA damage. Melanogenesis is controlled by MAPKs, with MITF being activated by p38 phosphorylation. By contrast, ERK activation inhibits melanin synthesis by downregulating MITF expression (Kanlayavattanakul and Lourith, 2018).

10.3 NATURALLY DERIVED COMPOUNDS THAT ARE COMMONLY USED FOR COSMETIC BENEFITS 10.3.1 PHENOLICS 10.3.1.1 Caffeic Acid Caffeic acid was exhibited to scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS) in a dose-dependent manner. Moreover, at a greater concentration than 5 μM, this antioxi­ dative phenolic turns to function as a prooxidant (Maurya and Devasagayam, 2010). It was shown to suppress hemoglobin-induced oxidation but weak antioxidant against iron-induced oxidative stress. Its properties were by chelating and redox potential (Kristinová et al., 2009). Caffeic acid has a remarkable photoprotective property in addition to its antioxidant capability. This phenolic additionally possesses antimicrobial activities against those of prohibited microbes in cosmetics, i.e., Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans. Caffeic acid cellular activity was shown to be COX-2 inhibitor and cytotoxic to tumor cells but not normal cells and conformed to in vivo assessment in animal models. Migratory capacity of malignant keratinocytes was suppressed by caffeic acid, which is p38-MAPK activation inhibitor (Magnani et al., 2014). 10.3.1.2 Chlorogenic Acid This antioxidative phenolic additionally possesses anti-inflammatory activity. Interleukin-8 (IL-8) activation was suppressed by chlorogenic acid via its ROS activity and alleviating proinflammatory

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cytokines production as well. Furthermore, it was reported to lower blood pressure associated with hypertension treatment as examined in human volunteers administrated with coffee riches in chlo­ rogenic acid. This was in harmony with nitric oxide reduction and relaxing effect on vasodilation in animal models. Antimicrobial activity against those of microbes prohibited in cosmetics was reported as well. Chlorogenic acid was exhibited to inhibit E. coli, S. aureus, and C. albicans including the skin flora Staphylococcus epidermidis (Naveed et al., 2018) that are associated with acne and bad body odor (Kanlayavattanakul and Lourith, 2011a, b). 10.3.1.3 p-Coumaric Acid Antioxidant activity by hydroxyl radical assay of p-coumaric acid was shown to be less potent than the standard vitamin E (IC50 = 256.3 ± 11.9 and 155.7 ± 4.3 μM). Its tyrosinase inhibitory activity by L-tyrosine and L-DOPA inhibitions (463.4 ± 16.4 and 1951.8 ± 157.5 μM) was weaker than the stan­ dard ascorbic and kojic acids (331.5 ± 2.7 and 7.6 ± 0.9 μM) as per its cellular activity against melano­ genesis in B16 melanoma that was weaker than arbutin (5773.2 ± 362.5 and 1110.4 ± 132.8 μM) (Choi et al., 2007). Melanogenesis suppression of p-coumaric acid (1 mM) was shown to be significant as examined in B16F1 cells. The mechanism was revealed by antityrosinase activity. p-Coumaric acid reduced cAMP–CREB phosphorylation downregulating MITF and tyrosinase expressions (Jun et al., 2012). Molecular docking analysis further revealed that p-coumaric acid interacted with five amino acid residues of tyrosinase and its strong binding activity thereafter suppressed the activity of tyrosinase (Kim et al., 2017). UVB exposure not only reduces cell viability but also shortens cel­ lular lifetime. In addition, UVB induces keratinocyte-releasable stratifin (SFN), inducing protein on MMP-1 expression in fibroblast. This cellular communication by SFN is therefore potentially related with premature skin aging. p-Coumaric acid was shown to attenuate the UVB-induced cell death in a dose-dependent manner (3–30 μg/mL) and suppress UVB-stimulated SFN expression. Moreover, its ability to diminish MMP-1 releasing in fibroblast was reported. Thus, p-coumaric acid is confirmed as a promising agent to reduce UV-induced, SFM-centered signaling associated with collagen catabolism during the course of skin photoaging (Seok and Boo, 2015). 10.3.1.4 Ferulic Acid Ferulic acid is one of the emerging important phenolics for health promotions and has been used as a food additive (Kumar and Pruthi, 2014). Similar to caffeic acid, it possesses antioxidant activity both against ROS and RNS (Maurya and Devasagayam, 2010). Ferulic acid was better than caf­ feic acid in anti-ABTS (2,2ʹ-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid) activity, of which its antioxidative function was by chelating ability and redox potential (Kristinová et  al., 2009). Moreover, this antioxidant phenolic can protect UVA- and UVB-induced aging of skin according to its strong UV absorption (Graf, 1992). Its protective effect on proteins degradation from heat shock was accumulated with its cellular antioxidant activity suppressing ROS surplus with its deac­ tivation on MMP-2 and MMP-9 activities, which in vitro and animal model studies were in accor­ dance. Expression of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) were increased by ferulic acid treatment as per HIF-1 induction guide for promotion of cell growth, and it was proved later that ferulic acid was able to accelerate cellular regeneration with wound-healing effect in animal models. Epithelialization during the course of wound healing was increased in a corresponding detection of hydroxyproline and hydroxylysine, which are collagen precursors. In addition to protective effects of ferulic acid on skin structures, its antityrosinase activ­ ity with melanocytic proliferation inhibitory effect opts ferulic acid to be used as a skin-lightening agent (Zduńska et al., 2018) with protecting activity against UVB-induced erythema (Saija et al., 2000). Its function is not only by competitive inhibition with tyrosinase. In addition, IL-8 level in mouse was reduced following oral ferulic acid administered daily. Gram-positive and gram-negative bacteria were inhibited by ferulic acid, which also posed antiviral activity (de Paiva et al., 2013).

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Antityrosinase activity of ferulic acid was shown by L-tyrosine inhibition that is better than that of the standard ascorbic acid (IC50 = 277.6 ± 8.2 and 331.5 ± 2.7 μM), although its cellular antimela­ nogenesis in B16 melanoma was weaker than arbutin (IC50 = 4864.4 ± 237.5 and 1110.4 ± 132.8 μM) (Choi et al., 2007). 10.3.1.5 Rosmarinic Acid Rosmarinic acid is one of the remarkable pharmacological phenolics that has cosmetic benefits. This ester of caffeic acid and 3,4-dihydroxyphenyllactic acid not only exhibits antioxidant activ­ ity but also includes anti-inflammatory capacity with an ability to prevent tissue damage with wound healing as examined in vitro and in vivo studies. Supplementations of rosmarinic acid were evidenced to be safe and efficient for antioxidative and proinflammatory suppression activi­ ties in animal models. Tissue damage protection was revealed by oxidative stress and TNF-α (tumor necrosis factor-α) and NF-κB (nuclear factor kappa B) inhibitions. Furthermore, it was shown to suppress atopic dermatitis following 0.3% rosmarinic cream onto the volunteers’ elbow twice daily for 2 months (Amoah et al., 2016). 10.3.1.6 Sinapic Acid This hydroxycinnamic acid is already used in health products including cosmetics as antioxidants and comparable with caffeic acid. Its IC50 against DPPH radical was shown to be 56 μM, and its superoxide anion radical (O −2 ) scavenging activity was at IC50 of 17.98 μM. Furthermore, its antioxidant activity by hydroxyl radical scavenging assay was shown to be potent with an IC50 of 3.80 mM, whereas that of ascorbic acid was 4.56 mM. Terminating on nitric oxide radical (•NO) of sinapic acid was shown to be more potent than the standard 2-(4-carboxyphenyl)-4,4,5,5­ tetramethylimidazoline-1-oxyl-3-oxide potassium salt (IC50 = 3.99 and 16.73 mM). In addition, its potent activity against peroxynitrite (ONOO −) was also stronger than the standard penicil­ lamine (IC50 = 0.58 and 2.93 mM). Scavenging activity against nonradical oxidant, hypochlorite (ClO −), was examined by chloramine and carbonyl assays, and their IC25 were 1.5 and 1.1 mM, respectively. In addition, lipid peroxidation inhibition of sinapic acid was evidenced by several assays as per its Cu2+ chelating efficacy. In correlation with antioxidant activity, sinapic acid is regarded as one of the anti-inflammatory and anticarcinogenic agents. NF-κB was inactivated by sinapic acid and suppressed proinflammatory mediators expression, i.e., NOS, COX-2, TNF-α, and IL-1β. Antimicrobial activities of sinapic acid against Bacillus subtilis, E. coli, and S. aureus, the prohibited microbes in cosmetics, were reported with MIC of 2, 4, and 1.9 mM, respectively ( Nićiforović and Abramovič, 2014). 10.3.1.7 Syringic Acid Syringic acid significantly inhibited UVB-induced COX-2 and MMP-1 expression as well as PGE2 production as examined in HaCaT cells. Additionally, it significantly inhibited UVB-induced AP-1 transactivity. The mechanism of syringic acid against photoaging was proved by an inhibition of UVB-induced phosphorylation of ERK1/2/JNK1/2/p38, MEK1/2/ MKK4/7/ MKK3/6, B-Raf, Akt, and Src. Phosphorylation of epidermal growth factor receptor (EGFR) suppressing activity of syrin­ gic acid was performed by maintaining protein tyrosine phosphatase-kappa (PTP-κ) activity and preventing an oxidation of PTP-κ that is induced by UVB irradiation. Intracellular ROS following UVB irradiation was suppressed by syringic acid because of its inhibitory effect against NADPH oxidase activity. These cellular activities of syringic acid related to photoaging were confirmed in animal model and evidenced by its inhibitory effect against UVB-induced COX-2 and MMP-13 expressions (Ha et al., 2018).

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10.3.2 FLAVONOIDS 10.3.2.1 Catechins Catechins, especially (–)-epigallocatechin-3-gallate (EGCG), are proved safe to Mel-Ab cells up to a concentration of 10 μM. Cellular melanin production was found to be significantly suppressed at low EGCG concentrations (1 μM), with activity being dose dependent. Moreover, the presence of EGCG also coincided with reduced MITF levels (Kim et al., 2004). 10.3.2.2 Quercetin This dietary flavonoid is accounted as a strong antioxidant due to its two pharmacophore molecules, i.e., catechol and OH moieties, which not only scavenge the radicals but also include metal chelation enhance its antioxidant activity by several mechanism. Its scavenging activities toward ROS were widely examined as per its lipid peroxidation inhibitory effect. Moreover, its ability to increase glu­ tathione level thereafter diminishes additional radical formation. Anti-inflammatory properties of quercetin are one of the widely studied issues. JNK/SAPK and p38 MAP kinase enzymes activities were counteracted by quercetin treatment as revealed in cell cultures. Life span of human fibroblasts was positively prolonged when treated with quercetin, and it was proved to positively affect against senescence of human-derived cell cultures (D’Andrea, 2015). In vitro assessment shows a positive effect of quercetin against collagenase or MMP-1 with an IC50 of approximately 286 μM (Sin and Kim, 2005). An expression of MMP-1 in UVA-irradiated human dermal fibroblast was 21.81% suppressed, at which mRNA expression of MMP-1 was found to be 24.07% inhibited (Sim et al., 2007). At a low concentration, this flavonoid promoted collagen synthesis ( 35°Brix and with final acidity content of 0.1%–0.3%. The product (Figure 11.5, Table 11.4) can be prepared by selecting sound quality fruits followed by washing the fruits, peeling, deseeding, slicing, and crushing the slices to get fine juice; this juice is pasteurized, and then pasteurized juice is blended with strained acidified sugar syrup of 70°Brix, which is prepared by the following steps: the desired amount of sugar and citric acid is

Loquat (Chinese Plums)

213

TABLE 11.4 Ingredients Used for Preparing Loquat Fruit Syrup Name of the Ingredient Loquat fruit juice Sugar Citric acid Potassium metabisulfite Water

FIGURE 11.6

Quantity 1 kg 2 kg 3–4 g 2.9 g 1L

Loquat juice.

boiled in potable water, the boiled solution is strained to remove impurities, and this strained syrup is blended with the desired amount of pasteurized juice and hot-filled in sterilized glass bottles to give loquat-based fruit syrup. The preservatives can also be added, but a high concentration of sugar will act as a preservative.

11.4.3 LOQUAT JUICE The loquat fruit is of sweetish taste; therefore, delicious juice (Figure 11.6) can be prepared from loquat fruits. The juice can be prepared alone or can be blended with other fruit juice such as orange or apricot to have a mixed variety of fruit flavors. For preparing the juice, healthy fruits are selected and spoiled fruits are discarded; then the selected fruits are washed well, peeled, sliced, deseeded, and crushed to get juice. If needed, slight sugar can be added, and the juice is passed through fine sieves, pasteurized at 80˚C for 15–20 min, bottled, capped, and stored airtight at room temperature.

11.4.4 LOQUAT HERBAL TEA The loquat-based herbal tea (Figure  11.7, Table  11.5) can be prepared from loquat leaves. This herbal-based tea has got a lot of therapeutic benefits. The beverage can reduce diabetes by enhanc­ ing insulin level in the body. It detoxifies the liver, cleans kidneys, and boosts the immune system.

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Asian Berries: Health Benefits

FIGURE 11.7

Loquat herbal tea.

TABLE 11.5 Ingredients Used for Preparing Loquat Herbal Tea Name of the Ingredient

Quantity

Loquat leaves Cardamom powder Black pepper powder Dry ginger powder Cloves powder Water Honey Lemon juice

20 g 5g 5g 5g 5g 1L 100 mL 5–10 mL

The tea is prepared by the following steps: the leaves are crushed in a blender, the crushed leaves are transferred to a boiling pan, the desired amount of water is added, various ground spices are incor­ porated, and the contents are allowed to boil for 5–10 min. Then the contents are strained through a strainer, followed by the addition of lemon juice as well as honey, and it can be consumed to gain aforementioned health benefits.

11.4.5

LOQUAT WINE

The wine is a yeast-fermented alcoholic product having an ethanol content of 7%. It has several health benefits; the cardiac people can consume such products to augment antioxidants in their bodies. It can be prepared by choosing the sound fruits such as loquat (Figure 11.8, Table 11.6) fol­ lowed by washing, peeling, deseeding, and pulping (which is known as the must); this must, which is the base for wine production, has to be adjusted with sugar so that the final must has 24°Brix. For the growth of yeast, 0.1% dihydrogen ammonium phosphate can be added, and 0.2% enzymes (pectin esterase) and 200 ppm preservatives (potassium metabisulfite) are also added. The yeast (Saccharomyces cerevisiae) is added at 2%–5% to the must, and the contents are fermented for 10–12 days. During this time, glucose will be converted into simpler units such as maltose and galactose, and at the end of the fermentation, the TSS of the must drastically reduce, thereby pro­ ducing ethanol, CO2, and other fermented products. The fermentation is carried out under anaerobic

Loquat (Chinese Plums)

215

FIGURE 11.8 Loquat wine.

TABLE 11.6 Ingredients Used for Preparing Loquat Wine Name of the Ingredient

Quantity

Loquat pulp Sugar Dihydrogen ammonium phosphate Pectin esterase Dried yeast Potassium metabisulfite Honey

1 kg 100–150 g 1g 2g 30 g 200 ppm 100 mL

conditions at room temperature. The base wine is filtered and matured for 6–12 months; this process is known as aging, which can be carried out in silver oak barrels so that the finished product will have mellow flavor. After 6–12 months, the wine is clarified either by adding chemicals such as bentonite or centrifuged, bottled, pasteurized, cooled, labeled, and stored for further use.

11.4.6 CANNED LOQUAT The stability of the loquat fruits can be increased by the canning process. This process involves the selection of sound and firm healthy fruits, followed by washing, peeling, deseeding, and halving. Once the fruits are halved, they are subjected to mild blanching at 80˚C for 2 min to kill enzymes and microorganisms. The cans are washed, sterilized with steam, and dried for packing the blanched slices. The slices after blanching are filled into cans containing sugar syrup of 40°Brix up to brim, clinched or covered with a lid, and further exhausted at 82˚C for 10 min, followed by seaming the can through can seamer. Then the cans are subjected to processing, i.e., sterilizing the contents at 100˚C for 10 min to kill all microbes; cans are cooled immediately and stored for further use (Figure 11.9).

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Asian Berries: Health Benefits

FIGURE 11.9 Canned loquat.

FIGURE 11.10 Loquat juice concentrates.

11.4.7 LOQUAT FRUIT JUICE CONCENTRATES Loquat fruit juice concentrates are obtained by the evaporation process (Figure 11.10). The fruits are extracted from the sound and well-ripened fruits. The juice is treated with pectinase enzymes at 0.03 g/L and clarified using centrifugal separator or adding chemicals such as bentonite followed by filtration and pasteurization, and finally, clarified juice is evaporated in a vacuum concentrator so that the TSS of the fruit juice will be enhanced to 50°Brix–60°Brix and the product will be packed in sterilized cans, heat-processed at 100˚C, cooled immediately, and stored until further use. The concentration of juice under vacuum conditions may retain several nutrients. The product has got a lot of usages: the concentrates are found to possess nearly five- to sixfold juice content, and therefore, they can be diluted for preparing beverages, jams, wine, and so on. The major advantage of concentrates is that it can be used during the off-season to meet the demand of consumers. The concentrates are the rich source of packed multivitamins and minerals.

Loquat (Chinese Plums)

FIGURE 11.11

217

Loquat fruit juice powder.

11.4.8 LOQUAT FRUIT JUICE POWDER The ready-to-reconstitute spray-dried powder (Figure  11.11) can be prepared from loquat fruits. The sound fruits are selected, washed, peeled, and pulped; the pulp is pasteurized and blended with drying aids like maltodextrin. The fruit pulp contains sugars, if it is dried as such, the product will be sticky instead of free-flowing nature. Therefore, to reduce the stickiness, high-molecular-weight sugar such as maltodextrin at 20%–25% is added to the fruit juice, homogenized well, atomized into fine droplets form under air pressure of 2.1–2.5 kg/cm2, and spray-dried at an inlet air temperature of 165˚C–170˚C and outlet air temperature of 85˚C; the powder is collected in the jar from cyclone separator and stored in aluminum foil pouches or airtight glass jars until its use. The powder can be reconstituted to give RTS (ready-to-serve) drink after incorporating sugar and citric acid.

11.4.9

LOQUAT DRIED SLICES

Loquat is a highly perishable seasonal fruit, and its quality degrades with time. The preservation and availability are challenging as well as crucial. Drying is one of the most common and traditional methods of preservation. Studies have shown that the physicochemical and nutritional attributes of loquat can be preserved effectively by drying. Production of dried loquat slices requires the follow­ ing unit operations: cleaning, washing, seed removing, slicing, immersing in sodium metabisulfite solution (0.5%), and finally drying. The dried fruits are easy to pack, store, transport, and handle. Dried loquat fruits exhibit high shelf life with reduced storage and distribution cost (Farina et al., 2020; El-Safy, 2014).

11.4.10 CHICKEN NUGGETS Chicken nuggets are ready-to-cook-and-eat the product; they are a highly preferred meal for con­ sumers. Nowadays, health-related concerns and demand for sustainable industrial practices have

218

Asian Berries: Health Benefits

pushed the researchers to find novel sources of functional ingredients. Loquat seeds are the waste of loquat fruits, but they are rich in various bioactive compounds. The bioactive phenols, flavonoids, tannins, amygdalin, and so on present in loquat seeds can be used to improve the shelf life of food by adding the loquat seed powder in the formulation of the product. For the preparation of chicken nuggets, loquat seed powder can be used as a replacement (different levels) to wheat flour in the formulation. The formulation consists of minced chicken breast meat (80 g), wheat flour (8 g), skim milk powder (8 g), dried garlic (1.4 g), dried onion (0.8 g), sodium chloride (1 g), pepper powder (0.8 g), and loquat seed powder. All the ingredients are mixed with the minced chicken breast meat to make a homogeneous mixture and keep this mixture in the freezer for 5 min. The chicken nuggets are then shaped and cooled to 5˚C. The cooking of chicken involves deep frying in vegetable oil for 4 minutes at 180˚C (El-Gammal et al., 2018). According to El-Gammal et al. (2018), replacement of 6.6% of wheat flour by loquat seed powder has resulted in significant improvement in the overall acceptability of the product. Also, the addition of loquat seed powder in the formulation reduced microbial load, controlled lipid oxidation, and reduced the cooking loss in chicken nuggets.

11.4.11 COOKIES Loquat leaf powder can be used to increase the nutrition value and overall acceptability of cook­ ies. Cho and Kim (2013) studied the effect of replacement of wheat flour with loquat leaf powder at various extents. The formulation consists of wheat flour (300 g), sugar (150 g), butter (150 g), egg (1), baking powder (8 g), and loquat leaf powder. It was found that replacement of 2% wheat flour with loquat leaf powder gives good results. The overall acceptability of cookies containing 2% loquat leaf flour increased significantly, along with an increase in the DPPH free radical scavenging activity.

11.4.12 PRODUCTION OF α-AMYLASE The α-amylase is widely used in the food industry for starch hydrolysis. Although various meth­ ods are used for α-amylase production, solid-state fermentation is widely used for its production. Waste loquat kernel can act as a good substrate in solid-state fermentation for the production of α-amylase. Erdal and Taskin (2010) optimized the process for the production of α-amylase using fungus Penicillium and loquat kernel as substrates. According to their study, 1012 U enzyme can be produced from per gram of loquat kernel. The optimum condition for enzyme production from loquat includes initial moisture content (70%), particle size (1 mm), pH 6, starch, peptone, methanol (1 mL), and 6 days’ incubation period.

11.5

CONCLUSIONS

The loquat can be commercially processed to give various processed products such as jelly, fruit syrup, juice powder, fruit juice concentrates, wine, and herbal leaf tea. The consumption of such fruit- and leaf-derived products may enhance the antioxidant status of the body of consumers, thereby scavenging the formation of free radicals, which are being generated in the body. To ascer­ tain health benefits, one should keep on continuously consuming various processed products devel­ oped from either loquat fruits or leaves.

REFERENCES Cho, H. S., & Kim, K. H. (2013). Quality characteristics of cookies prepared with loquat (Eriobotrya japonica Lindl.) leaf powder. Journal of the Korean Society of Food Science and Nutrition, 42(11), 1799–1804. DiNicolantonio, J. J., Liu, J., & O’Keefe, J. H. (2018). Magnesium for the prevention and treatment of cardio­ vascular disease. Open Heart, 5(2), e000775.

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El-Gammal, O. E. S., Gaafar, A. M., Salem, R., & El-messiry, D. (2018). Evaluation of chicken nuggets formu­ lated with loquat (Eribotrya japonica) seeds powder. Journal of Food and Dairy Sciences, 9(2), 77–82. El-Safy, F. S. (2014). Drying characteristics of loquat slices using different dehydration methods by compara­ tive evaluation. World Journal of Dairy & Food Sciences, 9(2), 272–284. Erdal, S. E. R. K. A. N., & Taskin, M. E. S. U. T. (2010). Production of α-amylase by Penicillium expansum MT-1 in solid-state fermentation using waste Loquat (Eriobotrya japonica Lindley) kernels as substrate. Romanian Biotechnological Letters, 15(3), 5342–5350. Farina, V., Cinquanta, L., Vella, F., Niro, S., Panfili, G., Metallo, A., & Corona, O. (2020). Evolution of carot­ enoids, sensory profiles and volatile compounds in microwave-dried fruits of three different loquat cultivars (Eriobotrya japonica Lindl.). Plant Foods for Human Nutrition, 75(2), 200-207. Femenia, A., MariaGarcia-Conesa, M., Simal, S., & Rosselló, C. (1998). Characterisation of the cell walls of loquat (Eriobotrya japonica L.) fruit tissues. Carbohydrate Polymers, 35(3–4), 169–177. Ferreres, F., Gomes, D., Valentão, P., Gonçalves, R., Pio, R., Chagas, E. A., & Andrade, P. B. (2009). Improved loquat (Eriobotrya japonica Lindl.) cultivars: Variation of phenolics and antioxidative potential. Food Chemistry, 114(3), 1019–1027. Haddy, F. J., Vanhoutte, P. M., Feletou, M. (2006). Role of potassium in regulating blood flow and blood pres­ sure. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 290(3): R546–R552. He, Q., Li, X. W., Liang, G. L., Ji, K., Guo, Q. G., Yuan, W. M., Zhou, G. Z., Chen, K. S., van de Weg, W. E., & Gao, Z. S. (2011). Genetic diversity and identity of Chinese loquat cultivars/accessions (Eriobotrya japonica) using Apple SSR markers. Plant Molecular Biology Reporter, 29, 197–208. Ito, H., Kobayashi, E., Takamatsu, Y., Li, S. H., Hatano, T., Sakagami, H., Kusama, K., Satoh, K., Sugita, D., Shimura, S., Itoh, Y., & Yoshida, T. (2000). Polyphenols from Eriobotrya japonica and their cytotoxic­ ity against human oral tumor cell lines. Chemical and Pharmaceutical Bulletin (Tokyo), 48(5), 687–693. Jung, H. A., Park, J. C., Chung, H. Y., Kim, J., & Choi, J. S. (1999). Antioxidant flavonoids and chlorogenic acid from the leaves of Eriobotrya japonica. Archives of Pharmacal Research, 22, 213. Kim, M. J., Lee, J., Seong, A. R., Lee, Y. H., Kim, Y. J., Baek, H. Y., … & Yoon, H. G. (2011). Neuroprotective effects of Eriobotrya japonica against β-amyloid-induced oxidative stress and memory impairment. Food and Chemical Toxicology, 49(4), 780–784. Kim, M. S., You, M. K., Rhuy, D. Y., Kim, Y. J., Baek, H. Y., & Kim, H. A. (2009). Loquat (Eriobotrya japon­ ica) extracts suppress the adhesion, migration and invasion of human breast cancer cell line. Nutrition Research and Practice, 3(4), 259–264. Koba, K., Matsuoka, A., Osada, K., & Huang, Y. S. (2007). Effect of loquat (Eriobotrya japonica) extracts on LDL oxidation. Food Chemistry, 104(1), 308–316. Li, X., Xu, C., & Chen, K. (2016). Nutritional and composition of fruit cultivars: Loquat (Eriobotrya japonica Lindl.). In: Simmonds, M., & Preedy, V. (eds.). Nutritional Composition of Fruit Cultivars (pp. 371– 394). Academic Press: San Diego, CA. Liu, Y., Zhang, W., Xu, C., & Li, X. (2016). Biological activities of extracts from loquat (Eriobotrya japonica Lindl.): A review. International Journal of Molecular Sciences, 17(12), 1983. Lutz, M., Fuentes, E., Ávila, F., Alarcón, M., & Palomo, I. (2019). Roles of phenolic compounds in the reduc­ tion of risk factors of cardiovascular diseases. Molecules, 24(2), 366. Noreen, W, Wadood, A., Hidayat, H. K., & Wahid, S. A. (1988). Effect of Eriobotrya japonica on blood glu­ cose levels of normal and alloxan-diabetic rabbits. Planta Medica, 54(3), 196–199. Sultan, M. Z. (2017). Loquat (Eriobotrya japonica Lindl.). In: Yahia, E. M. (ed.). Fruit and Vegetable Phytochemicals: Chemistry and Human Health (2nd Edition, pp. 1107–1126). Wiley: Hoboken, NJ. Tan, H., Sonam, T.,  & Shimizu, K. (2017). The potential of triterpenoids from loquat leaves (Eriobotrya japonica) for prevention and treatment of skin disorder. International Journal of Molecular Sciences, 18(5), 1030. Tan, H., Furuta, S., Nagata, T., Ohnuki, K., Akasaka, T., Shirouchi, B., Sato, M., Kondo, R., & Shimizu, K. (2014). Inhibitory effects of the leaves of loquat (Eriobotrya japonica) on bone mineral density loss in ovariectomized mice and osteoclast differentiation. Journal of Agricultural and Food Chemistry, 62(4), 836–841. Weaver, C. M. (2013). Potassium and health. Advances in Nutrition, 4(3), 368S–377S. Xu, H. X., Li, X. Y., & Chen, J. W. (2014). Comparison of phenolic compound contents and antioxidant capaci­ ties of loquat (Eriobotrya japonica Lindl.) fruits. Food Science and Biotechnology, 23(6), 2013–2020. Zar, P. P. K., Sakao, K., Hashimoto, F., Morishita, A., Fujii, M., Wada, K., & Hou, D. X. (2013). Antioxidant and anti-inflammatory activities of loquat (Eriobotrya japonica) tea. Functional Foods in Health and Disease, 3(11), 447–461.

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Zar, P. P. K., Yano, S., Sakao, K., Hashimoto, F., Nakano, T., Fujii, M., & Hou, D. X. (2014). In vitro anticancer activity of loquat tea by inducing apoptosis in human leukemia cells. Bioscience, Biotechnology, and Biochemistry, 78(10), 1731–1737. Zhang, W., Zhao, X., Sun, C., Li, X., & Chen, K. (2015). Phenolic composition from different loquat (Eriobotrya japonica Lindl.) cultivars grown in China and their antioxidant properties. Molecules, 20(1), 542–555.

12 A Richer Source of Nutrition Sea Buckthorn Berries

and Potential Health Benefits

Sampan Attri Jaypee University of Information Technology

Gunjan Goel Central University of Haryana

CONTENTS 12.1 12.2 12.3 12.4 12.5

Introduction ........................................................................................................................ 222

Sea Buckthorn: Origin and Taxonomy ............................................................................... 222

Distribution of Sea Buckthorn in Asia ............................................................................... 223

Characteristics of Sea Buckthorn Plant and Its Berries ..................................................... 223

Nutritional Attributes of Sea Buckthorn Berries ................................................................ 223

12.5.1 Moisture Content................................................................................................. 227

12.5.2 Ash Content ......................................................................................................... 227

12.5.3 Total Soluble Solids............................................................................................. 227

12.5.4 Carotenoids ......................................................................................................... 228

12.5.5 Vitamins .............................................................................................................. 228

12.5.6 Carbohydrates ..................................................................................................... 228

12.5.7 Organic Acid ....................................................................................................... 228

12.5.8 Proteins and Amino Acids .................................................................................. 229

12.5.9 Volatile Compounds ............................................................................................ 229

12.5.10 Mineral Elements ................................................................................................ 229

12.5.11 Oil........................................................................................................................ 229

12.5.12 Phytosterols ......................................................................................................... 230

12.5.13 Antioxidants ........................................................................................................ 230

12.6 Application of Sea Buckthorn Berries in Food Industry .................................................... 230

12.6.1 Juices and Beverages ........................................................................................... 230

12.6.2 Jams and Jellies ................................................................................................... 230

12.6.3 Dietary Supplements and Food Additives........................................................... 231

12.6.4 Milk Products...................................................................................................... 231

12.6.5 Alcoholic Drinks ................................................................................................. 231

12.7 Therapeutic Use of Sea Buckthorn Berries ........................................................................ 231

12.7.1 Cardiac Diseases ................................................................................................. 231

12.7.2 Antitumor/Anticarcinogenic Effects ................................................................... 232

12.7.3 Gastroenterological Diseases .............................................................................. 232

12.7.4 Hepatoprotective Activity ................................................................................... 232

12.7.5 Anti-inflammatory and Immunomodulatory Activities ...................................... 232

221

222

Asian Berries: Health Benefits

12.7.6 External Applications of Sea Buckthorn Berries ................................................ 232

12.7.7 Prebiotic Activities of Sea Buckthorn Berries .................................................... 233

12.8 Nutraceutical and Cosmeceutical Potential of Sea Buckthorn Berries .............................. 233

12.9 Conclusions ......................................................................................................................... 234

12.10 Summary ............................................................................................................................ 234

References ...................................................................................................................................... 234

12.1 INTRODUCTION Berries are defined as fleshy part of the fruit, which arises from the plant ovary and surrounds the seeds. Generally, the berries are reported for their nutritional components that are formed during the developmental stages. The major compounds involved in the development of ovaries in fruit/ berries can be divided into nutritional and non-nutritional compounds. In case of nutritional compo­ nents, the berries are usually low in calories, fats, and sodium but contain many essential minerals, dietary fibers (including water-soluble fiber such as pectin), antioxidants, and vitamin C (Yang and Kortesniemi, 2015). Most of the berries contain simple sugars such as glucose, sucrose, and fructose that add sweetness to them. All berries contain different kinds of carotenoids, some of which act as precursors to vitamin A. Other than carotenoids, berries are full of nutrient-rich building blocks such as antioxidants, phytochemicals, polyphenols, vitamins, and minerals. The amount of these phytochemicals varies from one plant species to another and is generally affected by plant growth conditions and environmental factors (Skrovankova et al., 2015). Sea buckthorn, also known as sandthorn, sallowthorn, Siberian pineapple, or seaberry, is a berry-bearing, wind-pollinated dioecious shrub. Some nutritionist quotes sea buckthorn berry (SBB) as “Heaven’s Holy Fruit, the wonder berry as nature’s most balanced fruit.” Currently, this extraordinary shrub is currently being cultivated in several regions of the world. Berries of this plant contain more than 190 biologically active compounds, which are highly beneficial for human health (Li, 2003).

12.2 SEA BUCKTHORN: ORIGIN AND TAXONOMY In an undated publication, Xu Mingyu et al. reviewed the historical information on sea buckthorn. Based on the available literature, the authors reported that Chinese people were the first to use sea buckthorn as a drug or medicine. SBBs have been used in ancient Tibetan, Mongolian, and Indian systems of medicine for treatment of digestive, pulmonary, and cardiovascular system dis­ eases (Koskovac et al., 2017). Many centuries ago, this sea buckthorn plant was mentioned in Yue Wang Yao Zhen of the Tang Dynasty during the 8th century in Sibu Yidian literature. Sibu Yidian is a traditional Tibetan medical literature available in four volumes and contains 158 chapters. Out of these 158 chapters, nearly about 30 chapters deal only with therapeutic products derived from sea buckthorn, describing the medicinal effects on sputum induction, enhancing the working abil­ ity of lungs and dispersed dampness. Also in traditional Chinese medicine, sea buckthorn was to tonify the Yin and strengthen the Yang. More than 60 entries refer to the ability of sea buckthorn to strengthen the stomach and spleen, to enhance blood circulation, and to eradicate blood stasis. Also, there are 84 entries related to sea buckthorn, prepared in seven different forms of application. These forms include use of sea buckthorn in the forms of powder, decoction, pill, extract, ash, or tinc­ ture or as a shortbread. During 18th century, Sibu Yidian literature was translated into Mongolian language, and soon after, it was further translated by European countries for detailed study on sea buckthorn. In ancient Greece, the leaves and young branches of sea buckthorn were used in the fod­ der for quick weight gain and a shiny coat of the horses. Therefore, name of this shrub is derived from Latin words “Hippo” that means “horse” and “phaos” that means to “shine.” In 1903, Sibu Yidian literature was further published in Russia in St. Petersburg (Yingcai, 1989). During the 1940s, particularly after World War II, nutritionists and pharmacologists detected the presence of

Sea Buckthorn Berries

223

vitamins in the sea buckthorn, which could be used not only as a foodstuff but also for a potential medicine. In the early 1940s of the 20th century, Hippophaė rhamnoides fruits began to be used on an industrial scale in Russia. In 1943, a Swiss pharmaceutical company launched juices and syrups as a supplement to the daily diet. In 1952, sea buckthorn was rediscovered in Tibet, and a scholarly thesis was written on sea buckthorn by Zhonglu (1956) at Sichuan Medical College, China. The initial research on the fruit juice of sea buckthorn was published in 1956, and sea buckthorn was listed in the Chinese Pharmacopoeia for the first time in the year 1977. Sea buckthorn (genus Hippophaė) belongs to family Elaeagnaceae, naturally distributed in Asia and Europe continents. It includes six species and 12 subspecies, of which H. rhamnoides is most commonly available. Furthermore, H. rhamnoides has many subspecies such as subsp. carpatica, subsp. fluviatilis, subsp. gyantsensis, subsp. mongolica, subsp. rhamnoides, subsp. sinensis, subsp. turkestanica, and subsp. yunnanensis.

12.3

DISTRIBUTION OF SEA BUCKTHORN IN ASIA

The worldwide area under sea buckthorn plantation is unidentified, and country-wise data lack a standardized assessment. In Asia, sea buckthorn is distributed mainly in countries such as China, Russia, India, Mongolia, Pakistan, Turkey, Afghanistan, Kyrgyzstan, Bhutan Kazakhstan, Nepal, Ukraine, and Uzbekistan (Figure 12.1). China is the largest producer of sea buckthorn followed by Mongolia, Russia, and India.

12.4

CHARACTERISTICS OF SEA BUCKTHORN PLANT AND ITS BERRIES

Sea buckthorn is a hardy plant, cold and drought resistant, and useful for land reclamation and farmstead protection by its strong vegetative reproduction and has tough, complex root system with nitrogen-fixing nodules (Rongsen, 1992). It is a deciduous shrub that normally ranges between 0.5 and 6.0 m in height with equivalent spread but may reach up to 18 m in central Asia. This plant can grow at very low temperatures of −40˚C and can bear drought and high saline conditions. For proper growth of sea buckthorn, plant requires full sunlight and cannot bear shady areas near other planta­ tion and trees. The branches of this shrub are firm, thick, and very thorny with both terminal and axillary twig spines. The linear shaped leaves, which are 30–80 mm in length and less than 7 mm wide, are generally dark gray-green on the upper side and a distinct pale, silvery-gray on the lower side (Figure 12.2). Sea buckthorn is dioecian plant, with separate female and male plant. Flowering occurs prior to the leaves, which are confined to the 2-year-old woody area only, in small group on the axils of leaves along the whole length of the branches. Pollination of the flowers on the female plant occurs in mid-May and is completely reliant on environmental factors such as wind to spread pollens from the male flowers. Ripening of sea buckthorn berries occurs approximately 100 days after pollination. Female plant of sea buckthorn produces berries in oval shape. Berries are yellow (Figure 12.3a) to orange-reddish (Figure 12.3b) with 5–8 mm length and 4–7 mm diameter. SBBs generally consist of “pulp (68%), seed (23%), and peel (7.75%)” (Oomah, 2003). The waxy smooth skinned SBBs contain a single wrapped brown seed and a juicy pulp filled with cellular structures of dietary fibers (Beveridge et al., 2002).

12.5 NUTRITIONAL ATTRIBUTES OF SEA BUCKTHORN BERRIES SBBs are one of the most nutritious berries among all the fruits. SBB juice is a rich source of car­ bohydrates, organic acids, fats such as fatty acids, phytochemicals such as phytosterol, flavonoids, vitamins, and mineral elements. One of the major components present in SBBs is vitamin C, which is known for its high importance toward human health. The content of vitamin C ranges from 53 to 3,909 mg/100 g in different species of the plant. It is estimated that there is adequate amount of

FIGURE 12.1 Distribution of the sea buckthorns (genus Hippophaė) in the Asian region. (Adapted from Li and Schroeder, 1996.)

224 Asian Berries: Health Benefits

Sea Buckthorn Berries

FIGURE 12.2

225

Sea buckthorn plant–bearing ripened berries.

(a)

(b)

FIGURE 12.3 Sea buckthorn berries: (a) unripened yellow and (b) ripened orange-colored berries.

vitamin C present in SBBs across the world to meet the dietary demand of the whole human popu­ lation. Other than vitamin C, SBBs also contain other vitamins such as A, E, K, B6, and B2, ribo­ flavin, niacin, and pantothenic acid. The berries also contain considerable amount of carotenoids including “β-carotene, 4-biketone-β-carotene, zeaxanthin, lycopene, polyring-lycopene, progestin, flavoxanthin, cryptoxanthin, violaxanthin, and neoxanthin.” Sea buckthorn is a rich source of poly­ phenols such as quercetin, rutin, gallic acid, catechin, coumaric acid, kaempferol, and ferulic acid. Due to occurrence of these vitamins, carotenoids, and polyphenols in higher amounts, it is impera­ tive that the berries have strong antioxidant potential. SBB juice is also considered a rich source of various free amino acids; 18 kinds of free amino acids have been analyzed in SBB, of which eight kinds of amino acids are essential for human body development. Sea buckthorn seed is a good source of important unsaturated fatty acids such as oleic acid, omega-3, omega-6, omega-7, and omega-9 fatty acids (Table 12.1).

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Asian Berries: Health Benefits

TABLE 12.1 Nutritional Composition of Asian Sea Buckthorn Berries Composition Moisture (%, w/w)

Ash (%) Total suspended solids (°Brix)

Vitamin C (mg/100 g)

Vitamin E (mg/100 g) Vitamin K (mg/100 g) Flavonoids (mg/100 g) Total carotenoids

Iron (Fe; mg/kg)

Magnesium (Mg; mg/kg)

Sodium (Na; mg/kg)

Sea Buckthorn Origin Country China Pakistan China China India India India India India China China India Pakistan Pakistan China China China China China India India India China China China China Turkestan Pakistan China India Soviet Union China India China China Pakistan India China Turkey Pakistan Pakistan China China Turkey Pakistan India Pakistan China Turkey China

Content in Berries

References

74.0 20.0–32. 72.2–75.5 61.5–79.4 52.4 58.7 1.76 1.8 10.7–13.2 10.83–15.55 10.19–22.74 10–12 250–333 150–250 200–2,500 460–1,330 502–1,061 1,348 513–1,676 168.3 509 422–416 502 1,348 513–1,676 780.0 200–1,500 216 162–255 110–230 854 354 6.8-6.9 4.6–12.0 2.0–16.1 4–15 1.6 4.13–10.9 0.46-1.27 64–176 150–240 39.8–103 53.3–165 187–190 50–140 6.9 20–80 17.7–125 172–208 18.0–89.8

Ma and Cui (1987) Sabir et al. (2005) Ma et al. (1989) Zhang et al. (1989) Katiyar et al. (1990) Chauhan et al. (2001) Katiyar et al. (1990) Chauhan et al. (2001) Arimboor et al. (2006) Tong et al. (1989) Zhang et al. (1989) Chauhan et al. (2001) Sabir et al. (2005) Sabir et al. (2003) Zheng and Song (1992) Yao et al. (1992) Ma et al. (1989) Liu and Liu (1989) Zhang et al. (1989) Arimboor et al. (2006) Katiyar et al. (1990) Chauhan et al. (2001) Ma et al. (1989) Liu and Liu (1989) Zhang et al. (1989) Mingyu et al. (2001) Ahmad and Kamal (2002) Zeb (2004) Zhang et al. (1989) Dhyani et al. (2007) Yuzhen and Fuheng (1997) Yuzhen and Fuheng (1997) Chauhan et al. (2001) Ma et al. (1989) Zhang et al. (1989) Sabir et al. (2005) Katiyar et al. (1990) Zhang et al. (1989) Ercisli et al. (2007) Hussain et al. (2014) Sabir et al. (2005) Zhang et al. (1989) Tong et al. (1989) Ercisli et al. (2007) Arif et al. (2010) Katiyar et al. (1990) Sabir et al. (2005) Zhang et al. (1989) Ercisli et al. (2007) Tong et al. (1989) (Continued)

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TABLE 12.1 (Continued) Nutritional Composition of Asian Sea Buckthorn Berries Composition

Sea Buckthorn Origin Country

Potassium (K; mg/kg)

Phosphorous (P; mg/kg)

Calcium (Ca; mg/kg)

Oil content (%)

Pakistan Turkey India China Pakistan Turkey India Pakistan China Pakistan Pakistan India Pakistan China Turkey China Pakistan China

Content in Berries 140–360 636–1,192 62.2 100–806 110–133 610–990 7.4 100–450 82.1–206 70–98 22–68 67.1 35–80 93.9–173 126–547 64–256 1–4.5 0.26–1.46

References Sabir et al. (2005) Ercisli et al. (2007) Katiyar et al. (1990) Tong et al. (1989) Sabir et al. (2005) Ercisli et al. (2007) Katiyar et al. (1990) Arif et al. (2010) Zhang et al. (1989) Sabir et al. (2005) Hussain et al. (2014) Katiyar et al. (1990) Arif et al. (2010) Zhang et al. (1989) Ercisli et al. (2007) Tong et al. (1989) Sabir et al. (2005) Zhang et al. (1989)

Source: Updated from Bal et al. (2011).

12.5.1

MOISTURE CONTENT

Moisture content has been established as an important indicator of shelf life for fruits and berries. The highest moisture content (g/100 g fresh wt) of SBBs in the range of 80%–87% was reported by Lõugas et al. (2006), whereas the lowest moisture content of SBBs with origin in Pakistan in the range of 20.0%–32.0% was reported by Sabir et al. (2005). The variation of moisture content is due to the difference in origin and environmental conditions of plant. But moisture content of SBB pulp of Indian origin was 84.9%–97.6% as reported by Dhyani et al. (2007). Moisture content of seed of SBB was reported to be 5.43%–21.9% for cv. Indian-Summer (Li et al., 1998) and 22.4% for Indian varieties (Chauhan et al., 2001).

12.5.2

ASH CONTENT

The determination of ash content of fruits/berries is conducted by burning away of organic contents, leaving inorganic minerals. This helps determine the amount and type of minerals present in the sample. The ash content of the SBB was reported in the range of 1.76%–1.8% (Chauhan et al., 2001).

12.5.3 TOTAL SOLUBLE SOLIDS Total soluble solid (TSS, °Brix) of SBBs was highest (10.19–22.74) for Chinese varieties as reported by Zhang et al. (1989) and lowest (9.3–17.3) for cv. Indian-Summer as reported by Li et al. (1998). On the other hand, TSSs of the pulp extracted from berries were 26.2–27.9 (Arimboor et al., 2006) and 8.86–9.72 (Dhyani et al., 2007) for Indian varieties. The TSS of the freshly prepared juice from SBB was 10.7–13.2 as reported by Arimboor et al. (2006).

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12.5.4 CAROTENOIDS Oils derived from SBB are an excellent source of carotenoids about 900–1000 mg/100 g of berries, which provides rich orange color to the berries (Mironov, 1989). It is well known that the total amount and the occurrence of different types of carotenoids present in SBB are highly variable depend­ ing on the genetic origin, conditions of cultivation, climate, and harvesting time. Carotenoids vary widely depending on the source of the oil, ranging from 314 to 2,139 mg/100 g for Chinese-grown SBB (Zhang et al., 1989). SBBs are rich in lipoproteins and pigments present in the fleshy pulp and outer membranes of SBB. Caroteno-lipoprotein complexes present especially in SBB membranes may act as bridge component between the polar proteins and nonpolar carotenoids for lipids (Pintea et  al., 2001). The study conducted by Andersson et  al. (2009) has identified lutein, zeaxanthin, β-cryptoxanthin, lycopene, γ-carotene, β-carotene as major carotenoids present in SBBs. Carotenoid content is the main attribute used for commercialization of SBB oils (Beveridge et al., 1999).

12.5.5

VITAMINS

SBBs are well known for their extremely high levels of vitamin C. The amount of vitamin C in berries varies from 0.36 g/100 g of berries for the rhamnoides subspecies (Wahlberg and Jeppsson, 1992) to 2.5 g/100 g of berries for the Chinese subspecies sinensis (Yao and Tigerstedt, 1994). The pulp of the Indian SBB contains 223.2 mg/100 g of vitamin C. Approximately 75% of the vitamin C in the pulp of berries was retained in the juice during processing, resulting in 168.3–184.0 mg/100 g of vitamin C in the final clear juice (Arimboor et al., 2006). SBBs do not possess the enzyme ascor­ bic acid oxidase that catalyzes L-ascorbic acid oxidation to L-dehydroascorbic acid, which leads to its decomposition to nonactive compounds. Therefore, vitamin C is well preserved in berries and their products (Valíček and Havelka, 2008). The Turkestanica SBB is reported to possess vitamin C content in the range of 200–1,500 mg/100 g, which is 5–100 times higher than any other fruits or vegetables (Ahmad and Kamal, 2002). Tocopherols and tocotrienols, commonly known as vitamin E, are also present in SBBs in higher quantities. The oil prepared from whole berries of H. rhamnoi­ des cultivars contained 1.01–1.283 g/kg of total tocopherols, in which α-tocopherol was 62%–68% and δ-tocopherol was 32%–38% of total tocopherols (Zadernowski et al., 2003). Another study by Ma et al. (1989) reported that Chinese variety SBBs seed contains 40.1–103.0 mg/100 g vitamin E. SBBs are also reported to be rich in several other vitamins such as B1, B2, and K and bioflavonoids (Bekker and Glushenkova, 2001).

12.5.6

CARBOHYDRATES

Carbohydrates are a major component of dry matter in SBBs. Total carbohydrates level var­ ies between 400 and 600 g/kg dw (Selvamuthukumaran and Farhath, 2014). Polysaccharides of SBB are especially non–starch-type polysaccharides, which are composed of cellulose, hemicel­ luloses, pectin, and hydrocolloids that are together with lignin the major constituents of dietary fibers (Greenfield and Southgate, 2003). Among monosaccharide and disaccharide sugars, mainly glucose, fructose, and xylose are present in SBBs. Total soluble sugars reported for Chinese-origin SBBs ranged from 5.6% to 22.7% in unprocessed juice. Glucose is the major sugar present in all varieties of SBBs. Both glucose and fructose accounts for around 90% of the total simple sugars content for Russian and Chinese origin berries (Kallio et al., 1999). The presence of sugar alcohols such as mannitol, sorbitol, and xylitol at low levels is also reported (Makinen and Soderling, 1980).

12.5.7 ORGANIC ACID The SBBs contain organic acids, mainly malic and quinic acids which together constitute around 90% of all total organic acids present in different origins. Large variations in concentrations of these acids have been also reported among different varieties of SBBs. Tang, (2002) has reported that SBB contains predominantly malic acid (11–60 mg/L of juice), quinic acid (7–49 mg/L of

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juice), citric acid (up to 0.3% of all acids), tartaric acid (up to 0.013% of all acids), and succinic acid (up to 0.6% of all acids). Russian SBBs showed relatively lower concentration of total organic acids (2.1–3.2 g/100 mL), whereas Chinese SBBs showed the higher concentration of total organic acids (3.5–9.1 g/100 mL) (Kallio et al., 1999). However, no organic acid content has been reported for Indian varieties.

12.5.8

PROTEINS AND AMINO ACIDS

SBBs are also considered a rich source of proteins and amino acids. Total protein contents of various species of SBBs from India were reported to be 11–31 g/kg of fresh berries (Selvamuthukumaran and Farhath, 2014). In Mongolian wild sea buckthorn species, approximately 38% of total protein was found in seeds (Uransanaa et al., 2003) A total of 18 out of 22 known amino acids have been detected in SBBs (Zhang et al., 1989), most of which are essential for human body, since they play a crucial role in various metabolisms such as energy generation, development of tissues and muscles, reduction of unwanted fats, and mood and brain functional modulations. Chen (1988) reported 18 different types of amino acids in the juice of Chinese SBBs. Among these amino acids, eight amino acids (“threonine, valine, methionine, leucine, lysine, trytophan, isoleucine, and phenylalanine”) are essential for the human health.

12.5.9 VOLATILE COMPOUNDS SBBs have a very unique aroma as compared with any other common fruits. The compounds respon­ sible for aroma are mainly short-chain esters, branched or n-fatty acids, and alcohols. The profile of the volatile compounds is evidently dependent upon the time of harvesting of the SBB (Yang, 2001). Kallio et al. (1999) have reported that Chinese SBBs contained higher proportions of “ethyl 3-methylbutanoate, butyl pentanoate, 2-methylpropyl 3 methylbutanoate, pentyl 3-methylbutanoate, ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, and ethyl hexanoate.” Sixty different volatile compounds from SBB oil were detected by the study conducted by Hirvi and Honkanen (1984). Among esters, mainly ethyl and 3-methylbutyl esters were the most commonly found compounds in SBBs. Cakir (2004) identified 30 volatile compounds, mainly alcohols, aliphatic esters, and hydro­ carbons from steam distilled oil of SBB. Other compounds identified in SBB were phenols, ter­ penes, aldehydes, and ketones.

12.5.10

MINERAL ELEMENTS

Many mineral elements that are present in berries and seeds of SBBs such as potassium (K), nitrogen (N), phosphorus (P), iron (Fe), manganese (Mn), boron (B), calcium (Ca), aluminum (Al), silicon (Si), and others are reported (Wolf and Wegert, 1993). Potassium in SBBs ranged between 10.12 and 14.84 ppm in the pulp and between 9.33 and 13.42 ppm in the seeds of the Indian species (Dhyani et al., 2007). In liquors prepared from SBBs, traces of various other mineral elements such as cadmium (Cd), copper (Cu), magnesium (Mg), sodium (Na), lithium (Li), lead (Pb), and zinc (Zn) were also detected by Harju and Ronkainen (1984). The levels of mineral elements were reported to be affected by the maturation period (Bounous and Zanini, 1988). Other macro- and micronutrients, such as Na, Mg, Fe, Cu, and Zn, are also present in low to moderate quantity in pulp and seeds of SBBs.

12.5.11

OIL

The most valuable and commercialized product of the SBBs is their oil. Both berry pulp and seeds have high amounts of lipid content, mostly unsaturated fatty acids of omega-3 and omega-6 fam­ ily along with tocopherols, tocotrienols, and carotenoids (Yang and Kallio, 2002). The oil derived from seeds that are present at a high concentration (140 mg/100 mL) contains over 95% of the recoverable tocopherols, 1% phytosterols, and small amounts of tocotrienols (Parimelazhagan et al.,

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2005). Saturated fatty acids are mainly found in the oil derived from the pulp, mostly palmitic and palmitoleic acids (Kallio et al., 2002). The oils derived from the seeds and pulp of SBB have dif­ ferent fatty acid compositions. Oil extracted from the juice and pulp contains palmitic (16:0) and palmitoleic acids (16:1), whereas the oil extracted from the seeds is rich in unsaturated fatty acids of C18-type oils, linoleic (18:2) and linolenic acids (18:3). The oil extracted from the pulp and seeds is also rich in vitamin E and carotene (Bernath and Foldesi, 1992).

12.5.12

PHYTOSTEROLS

Phytosterols are plant-derived sterols, with structures similar to cholesterol. These phytosterols are reported to lower down the plasma cholesterol in humans. Phytosterols are the major compounds present in unsaponifiable fraction of SBB oil. The major phytosterol present in SBB oil is sitosterol (β-sitosterol) and 5-avenasterol. The sterol content in different varieties of SBBs generally ranges from 1.3% to 2%.

12.5.13

ANTIOXIDANTS

Antioxidants by definition are the compounds that inhibit or delay the oxidation of other biologi­ cal molecules by inhibiting the initiation or propagation of oxidizing chain reactions. The anti­ oxidant activity of SBB has been reported by various researchers. Many phenolic compounds in sea buckthorn, including phenolic acids, flavonoids, and hydrolysable tannins, are responsible for its bioactive and antioxidant properties. Study conducted by Gorbatsova et al. (2007) found high antioxidant content in different varieties of SBBs and major antioxidant compounds responsible for high antioxidant activity such as “trans-resveratrol, catechin, myricetin, quercetin, p-coumaric acid, caffeic acid, L-ascorbic acid, and gallic acid” (linear range of 50–150 mM/L). Velioglu et al. (1998) reported the antioxidant value of 0.036, antioxidant activity of 93.6%, oxidation rate ratio of 0.064, antioxidant activity coefficient of 827.6, and total phenolic content of 1112 mg/100 g in SBBs. They also found that the SBB had the highest antioxidant activity which is 93.6% among all the other medicinal plants studied.

12.6 APPLICATION OF SEA BUCKTHORN BERRIES IN FOOD INDUSTRY Due to the richness of bioactive substances present in SBBs, they are now very often used in pro­ duction of functional and health food on account of constant demand increase among potential cus­ tomers. SBBs belong to the most nutritious and vitamin-rich plants. In the food industry, it may be used as a preservative or food additive, or it can be used to enhance the nutritional and organoleptic values of food (Schroeder and Yao, 1995).

12.6.1

JUICES AND BEVERAGES

One of the most popular and earliest manufactured products made of sea buckthorn are juices and beverages. At the Olympic Games in Seoul in 1992, these were the official drinks of Chinese athletes. Beverages made from this plant were also used in the diet of Indian soldiers – they were receiving herbal multivitamin drinks based on sea buckthorn juice during their work at very low temperatures. Juices are usually obtained by centrifugation of oil fraction from the sea buckthorn fruit pulp (Niesteruk et al., 2013).

12.6.2 JAMS AND JELLIES Despite the sour and exotic taste, SBBs can be used for the production of jams and jellies. Its pun­ gent flavor can be neutralized by mixing the juice or pulp of sea buckthorn and other fruits with

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a much milder taste in different proportions. Research conducted by Marszałek et al. (2014) has shown that SBBs, due to the high content of biologically active compounds, are a valuable material for the production of jams. During the study, a variety of flavors were prepared, by mixing sea buck­ thorn berries with other fruits such as apples, gooseberries, raspberries, and strawberries, which also gave jams different colors, from orange to dark red.

12.6.3 DIETARY SUPPLEMENTS AND FOOD ADDITIVES Food products made of SBBs with the addition of their oil play an important role in enhancement of nutritional properties. Oil from SBBs combined with other vegetable oil can also be used to expand and diversify the offer of currently available cold-pressed oils. SBB extract is also used as a nutritional component in baby food. The remains of the SBB juice are good functional supplements for meat and nonvegetarian diets, which enrich them with plant-derived polyphenols (Bhartee et al., 2014).

12.6.4

MILK PRODUCTS

SBB products can be used as an additive to dairy products such as kefir, yoghurt, or cheese as well. Studies conducted by Liszka et al. (2014) have shown that the addition of SBB pulp resulted in a sig­ nificant increase in antioxidant properties of fermented milk drinks and acidity of tested products. Kefir with supplemented SBBs was characterized by higher number of the mesophilic streptococci and lower level of lactobacilli in the finished product. Addition of SBBs to cheese has resulted in reduction of pathogenic microorganism number and improved the organoleptic properties of cheese (Terpou et al., 2017).

12.6.5

ALCOHOLIC DRINKS

Fruit wines are rich in nutraceutical compounds linked with a reduced risk of cardiovascular and other diseases. Production of alcoholic beverages with the use of SBB has also been reported in lit­ eratures. In Tibet, alcoholic beverage tincture is made from SBB. Negi et al. (2013) have developed sea buckthorn wine, which is reported with higher levels of antioxidants such as rutin, myricetin, and quercetin.

12.7 THERAPEUTIC USE OF SEA BUCKTHORN BERRIES Sea buckthorn has been used for thousands of years as a natural remedy against various ailments. Sea buckthorn has been used in China for over 12 centuries where it was first used for traditional Chinese medicine strengthening stomach, blood circulation, and respiration. Sea buckthorn is cur­ rently being used in juice, sports drinks, jellies, ice cream, cosmetics, and medicines.

12.7.1

CARDIAC DISEASES

Flavonoids from sea buckthorn act by activation of nuclear factor (NF)-kappa by stretching cul­ tured cardiac myocytes, which improve myocardial function for the treatment of hypertension and chronic cardiac insufficiency (Xiao et  al., 2003). The mechanism of action of flavonoids present in SBB may include reduced stress of cardiac muscle tissue by regulation of inflam­ matory mediators and reduce the production of pathogenic thrombosis. Antioxidant of sea buckthorn juice affects the risk factors such as plasma lipid, low-density lipoprotein, platelet aggregation, and plasma soluble cell adhesion protein concentration for coronary heart diseases (Eccleston et al., 2002).

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12.7.2 ANTITUMOR/ANTICARCINOGENIC EFFECTS An alcoholic extract of SBB, which is rich in polyphenols, has been reported to protect the bone marrow from damage due to radiation and provides fast recovery of bone marrow in cancer patients. Fast recovery of hemopoietic system after chemotherapy is also observed after consumption of SBB. The seed oil has been found to enhance nonspecific immunity and to provide antitumor effect in preliminary laboratory studies (Agrawala and Goel, 2002; Let, 1993).

12.7.3 GASTROENTEROLOGICAL DISEASES SBB is traditionally used in the treatment of gastric ulcers, and many laboratory studies have con­ firmed the efficiency of seed oil for ulcer treatment. It can improve the functioning of the stomach and maintain the normal activity of the gastrointestinal tract. Sea buckthorn oil normalizes gastric acid and reduces inflammation by controlling proinflammatory mediators. Hexane extracts of SBB are found in prevention of gastric injury (Suleyman et al., 1997). Many of the preparations of sea buckthorn such as ointment, suppositories, liniments, and liquids are used for oral mucositis, rectum mucositis, and duodenal and gastric ulcers (Li and Schroeder, 1996).

12.7.4

HEPATOPROTECTIVE ACTIVITY

SBB extracts help to normalize liver enzymes, serum bile salts, acids, and immune system markers responsible for liver inflammation and degeneration (Gao et al., 2003). Laboratory studies revealed that SBB oil protects the liver from damaging effects of toxic compounds such as CCl4. Combination of SBB along with antiviral drug proves effective in treatment of hepatitis B as it shortens the dura­ tion of normalizing serum alanine aminotransferase enzyme (Huanng et al., 1911).

12.7.5

ANTI-INFLAMMATORY AND IMMUNOMODULATORY ACTIVITIES

Oxidative damage to cells has been related to the pathogenesis of different types of clinical diseases and their wide range of effects on human body. Alcoholic extracts of SBB show significant cyto­ protection against Na+ nitroprusside-induced oxidative stress in the lymphocytes. The extracts also attenuate the nicotine-induced oxidative stress in rat liver and heart. Total flavonoids content from SBB provides protection against hydrogen peroxide–induced apoptosis on vascular endothelial cells by lowering the caspase-3 expression (Cheng et al., 2011).The antioxidant and immunomodulatory properties of SBB are evaluated in vitro using rat splenocytes, macrophages, and C6 glioma cell line and in vivo using male albino rats. The extract alone stimulates interleukin 2 (IL-2) and interferon (IFN) production in the absence of Con A and also inhibits chromium-induced decline in IL-2 and IFN production. SBBs result in significant immunomodulatory activity and specifically activate the cell-mediated immune response (Geetha et al., 2005). SBBs also show an immune-protective effect against T-2 toxin-induced immunodepression in 15-day-old chicks. Sea buckthorn has been extensively used in oriental traditional medicines for treatment of many inflammatory disorders (Ramasamy et al., 2010). The in vitro and in vivo antioxidant properties of seed oil are also ana­ lyzed, and their observations indicate that SBB oil has significant antioxidant properties. Seed oil also shows strong inhibition of oxidative damage induced by CCl4 in mice model, increases the activities of antioxidant enzymes, and decreases the lipid peroxidation in liver.

12.7.6

EXTERNAL APPLICATIONS OF SEA BUCKTHORN BERRIES

As compared with other vegetable and fruit oils, oil derived from SBB is considered to have a unique composition of fatty acids, carotenoids, and other complex lipids. SBB oil has been used in the treatment of different skin diseases (e.g., eczema, dermatoses, ulceration, psoriasis, and atopic

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dermatitis). Externally applied SBB oil may also reduce bedsores, spots, acne, scars, discoloration, and allergic and inflammatory lesions of the skin (Yang et al., 2000). SBB oil contains a rare palmi­ toleic acid (ω-7 acid), a component of skin lipids, that stimulates skin regeneration processes in the epidermis, minimizes scars, and promotes wound healing (Koskovac et al., 2017). The mechanisms behind the action of SBB oil are the stimulation of epidermis regeneration and collagen synthesis. These mechanisms are also linked to the amount of unsaturated ω-3 and ω-6 fatty acids, carotenoids, and tocopherols present in SBB, which stimulate fibroblast proliferation, collagen biosynthesis, and expression of specific matrix metalloproteinases that induce tissue reparation and angiogenesis. Preparations containing SBB oil have been found to promote wound healing (Ito et al., 2014).

12.7.7 PREBIOTIC ACTIVITIES OF SEA BUCKTHORN BERRIES Prebiotics are defined as “substances such as carbohydrate polymers and sugars that improve host health by stimulating the growth and activity of health-promoting bacteria found in the human digestive system” (Santos et  al., 2006). Roberfroid (2007) has reported that SBBs also contain nondigestible oligosaccharides (NDOs), which act as prebiotics and enhance the activities of health-promoting microbial community present in the human colonic region. Recently, the authors have investigated the effect of releasing bioactive compounds on the beneficial bacterial groups via batch culture as well as establishing in vitro gut systems that mimic the microbial processes with the simulated gut environment of the human gastrointestinal tract. A batch colonic fermentation study using SBB juice has also reported beneficial effect to gut microbial community (Attri et al., 2018) with release of antioxidants in 36 h of fermentation. A study conducted by Gunenc et al. (2016) has reported enhancement of viability of probiotic strains in yogurt by addition of SBBs. Another study by Attri and Goel (2018) has also reported enhancement of beneficial gut microbiota by addition of SBB juice with regular feed using human gut simulation model suggesting the prebiotic activities of the components of SBB. SBB products have also been used in the production of feta cheese, where it acts as biodegradable scaffold on which beneficial probiotic bacteria strain Lactobacillus casei ATCC 393 was able to grow. Thus, SBB may serve as a new prebiotic source for development of functional foods.

12.8 NUTRACEUTICAL AND COSMECEUTICAL POTENTIAL OF SEA BUCKTHORN BERRIES SBB oils and seeds could be considered functional foods due to medicinal and nutritional properties. Because of their functional properties and unique taste and flavor, SBBs can be processed to make juice, candies, jellies, jam, and alcoholic or nonalcoholic beverages and used as flavoring agents for dairy products. The seed and pulp oils of SBB are used as a source of ingredients in food supple­ ments, such as gelatin, vegetable-based capsules, and oral liquids. SBBs have cosmetic values and can be used as liquids, powders, plasters, films, pastes, pills, liniment, suppositories, and aerosols. SBB oil has ultraviolet blocking activity and hence is used in sunblock cream. Besides, this plant is used in skin grafting, cosmetology, and treatment of corneal wound and used in commercially available cosmetic products, such as shampoo. SBB oil is similar to natural skin sebum lipids and provides important healing and antiaging benefits to skin. Sea buckthorn oil is anti-inflammatory, antimicrobial, analgesic, and regenerative. Palmitoleic acid, a fatty acid which is the content of SBB oil, is the main component of skin. It is reported to nourish the skin and useful in treating skin dis­ eases such as atopic dermatitis. This mainly diminishes inflammation, disinfects bacteria, relieves pain, removes blood stasis, increases blood circulation, improves human immunity, and promotes regeneration of tissues. Research on 350 patients treated with sea buckthorn cream revealed the positive therapeutic effect on senile skin wrinkles, melanosis, xanthopsia, and freckles (Patil and Choudhary, 2017).

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12.9 CONCLUSIONS Sea buckthorn products have attracted many people who are interested in the production or utiliza­ tion of nutritional and medicinal components of this exotic plant. The plant produce such as seeds, berries, and oil are used for prophylactic or therapeutic purposes. The berries in particular have their significance as these are rich in nutritional and bioactive substances. The content of each particular group of compounds is different depending on the variety. However, the knowledge of dietary intake of these bioactive components and their bioaccessibility and bioavailability in the human gut are key factors in assessing their significance in human health. The potential application of sea buckthorn parts in a wider range of food and pharmaceutical products should be explored in the future through value addition.

12.10 SUMMARY Sea buckthorn berries (H. rhamnoides L.) are polyphenol-rich berries with interesting bioactivities reported in several in vitro and in vivo studies. The berries are reported for their use for human nutrition due to richer source of antioxidants, fatty acid composition, and other micronutrients. This chapter takes a holistic view of the plant, its geographical distribution, compositional analysis, and potential health benefits of the bioactive compounds. This chapter also highlights the potential application of berries in the food industry.

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13

Health Perspectives of Anthocyanins from Chinese Bayberry Fruits Huan Cheng, Xingqian Ye, Jianle Chen, and Haibo Pan Zhejiang University

CONTENTS 13.1 13.2 13.3

Introduction ........................................................................................................................ 239

Chemical Composition of Anthocyanins in Chinese Bayberry Fruits ............................... 242

Extraction and Purification of Anthocyanins from Bayberry Fruits .................................. 242

13.3.1 Anthocyanin Extraction ........................................................................................ 243

13.3.2 Anthocyanin Purification...................................................................................... 243

13.3.2.1 Solid-Phase Extraction ......................................................................... 243

13.3.2.2 Countercurrent Chromatography .........................................................244

13.4 Stability of Anthocyanins from Bayberry Fruits................................................................244

13.5 Health Benefits and Applications of Anthocyanins from Bayberry Fruits ........................244

13.5.1 Antioxidant Capacity of Anthocyanins from Bayberry Fruits .............................244

13.5.2 Prevention of Diabetic Diseases ........................................................................... 245

13.5.3 Antitumour Activity of Anthocyanins from Bayberry Fruits .............................. 245

13.6 Concluding Remarks ..........................................................................................................246

References......................................................................................................................................246

13.1

INTRODUCTION

The Chinese bayberry (Myrica rubra Sieb. et Zucc.) is one of the berry fruits native to China with high commercial values. Nowadays, Chinese bayberry fruits are widely cultivated in the south of China. Especially in Zhejiang province, the fruits are important agricultural products with production exceeding 300,000 tons per year. The Chinese bayberry has been known for 7,000 years but was started to be cultivated only 2,000 years ago. Ripe berries can be from red to deep red (cv. ‘Biqi,’ ‘Wandao,’ etc.), pink (cv. ‘Fenhongzhong’), or white (cv. ‘Shuijing’), depending on the specific cultivar and fruit maturity (Figures 13.1 and 13.2), and have a pleas­ ant aroma and sweet–sour taste (Cheng, Chen, Chen, Wu, Liu, & Ye, 2015). Consumers seem to be primarily attracted by the red to purple color, special sweet–sour taste, and exquisite flavor of the fruits. Unfortunately, due to ripening in hot and rainy season (normally June and July), Chinese bayberries are highly susceptible to mechanical injury and microbiological decay that limit their postharvest life to 1–2 days. Although they are stored in a cooler (4°C), their bioactive components still decrease rapidly, and shelf life can be only 5 days. Therefore, there is a demand for alternative processes for extending the shelf life or developing other products (Figure 13.3). Bayberry fruits are consumed in various forms, including jam, juice, and wine, or can be canned in syrup (Fang & Bhandari, 2011). It has been reported that Chinese bayberry fruits have strong antioxidant capacity associated with anthocyanins, phenolics, and ascorbic acid. Bayberry fruits have the potential to provide excellent 239

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FIGURE 13.1 Chinese bayberry (Myrica rubra Sieb. et Zucc.) fruits on tree.

FIGURE  13.2 Different cultivars of Chinese bayberry fruits with different colors (Li zhi (a), Bai yangmei (b), Ding ao (c), Zao se (d), Chi se (e), Bi qi (f), Shui mei (g), Dong kui (h), Wan dao (i), Tan mei (j), and Fen hongzhong (k)).

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FIGURE 13.3 Different products made from Chinese bayberries.

FIGURE 13.4 Different cultivars of bayberry juice with different colors.

color in their developed products. The red color of Chinese bayberry juice is mainly due to the pres­ ence of anthocyanins, especially cyaniding-3-glucoside (Figure 13.4). In this chapter, the health benefits of Chinese bayberry fruits by capturing the chemical composi­ tions of anthocyanins from Chinese bayberry fruits are discussed. This is followed by a discussion of studies investigating the extraction and separation, chemical stability, health benefits, and their applications of anthocyanins from bayberry fruits. Recent scientific evidence has presented that anthocyanins from bayberry play a positive and potential role in the treatment of many various human diseases.

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13.2 CHEMICAL COMPOSITION OF ANTHOCYANINS IN CHINESE BAYBERRY FRUITS Anthocyanins are colored water-soluble pigments belonging to the subclasses of phenolic phy­ tochemicals, which are usually in glycosylated forms and generally change color from red through purple, blue, and bluish green as a function of pH. The sugar-free counterparts of antho­ cyanins are termed anthocyanidins. Anthocyanidins are grouped into 3-hydroxyanthocyanidins, 3-deoxyanthocyanidins, and O-methylated anthocyanidins, whereas anthocyanins are in the forms of anthocyanidin glycosides and acylated anthocyanins. The most common types of anthocyani­ dins are cyanidin, delphinidin, pelargonidin, peonidin, petunidin, and malvidin. Acylated antho­ cyanins are also detected in plants besides the typical anthocyanins. Acylated anthocyanins are further divided into acrylated anthocyanins, coumaroylated anthocyanins, caffeoylated anthocya­ nins, and malonylated anthocyanins. Anthocyanins have the basic structure of flavylium cation, lack of a ketone oxygen at the 4-position. The positive charge can move around the molecules. The empirical formula for flavylium cation of anthocyanin is C15H11O+ with a molecular weight of 207.24 g/mol (Khoo, Azlan, Tang, & Lim, 2017). Cyanidin, delphinidin, and pelargonidin are the most common anthocyanidins distributed in bayberry fruits, in which cyanidin-3-glucoside (C3G) is identified as a major anthocyanin in Chinese bayberry, accounting for 95% of total anthocyanins (Zhang et al., 2015). The content of C3G (Figure 13.5) in bayberries is similar to that of blackberries and is much higher than that in cranberries, blueberries, bilberries, lingonberries, and chokeberries (Bao, Cai, Sun, Wang,  & Corke, 2005; Siriwoharn, Wrolstad, Finn,  & Pereira, 2004; Zhang et  al., 2008; Zheng & Wang, 2003). In nature, C3G is a reddish-purple (magenta) pigment, which endows the characteristic red color in bayberry fruits.

13.3 EXTRACTION AND PURIFICATION OF ANTHOCYANINS FROM BAYBERRY FRUITS Bayberry fruits are perishable, which leads to a short shelf life. Large amounts of bayberry fruits are discarded if not consumed in time. Extraction and isolation of anthocyanins is one of the approaches used to process bayberry fruits. The solid–liquid extraction (SLE) is a common approach used to separate anthocyanins from bayberry fruits. Besides, green approaches have been developed to get better extract anthocyanins, including supercritical extraction, pulsed electric field (PEF)–assisted extraction, microwave-assisted extraction, ultrasound-assisted extraction, and pressurized liquid extraction (PLE). However, it is difficult to compare the extraction yields of different methods since compound variation from matrix to matrix hampers comparisons.

FIGURE 13.5 The chemical structure of cyanidin-3-glucoside (C3G).

Chinese Bayberry Fruits

13.3.1

243

ANTHOCYANIN EXTRACTION

SLE as a classic approach can be used to extract not only anthocyanins from bayberry fruits but also other phenolics. Several parameters have been reported to affect SLE yield. Solvent type and temperature are most commonly reported in literature. The common solvents used in SLE are polar, such as water, methanol, ethanol, acetone, and their acidified forms. Combinations of different solvent and acid (HCl, formic acid, acetic, propionic, tartaric, and citric acids) are usually studied to evaluate their effects on the extraction of anthocyanins. Overall, solvent acidification led to the improvement of extraction yield, though the effects were dependent on the types of acids (Silva, Costa, Calhau, Morais, & Pintado, 2017). It has been reported that trifluoroacetic acid increased anthocyanin yield for all solvents except acetone (Barnes, Nguyen, Shen, & Schug, 2009). Methanol combined with citric acid appeared to be the best combination for extraction of anthocyanins from wine pomace. A different behavior was observed that ethanol and acetone proved to be more effec­ tive than methanol, isopropanol, and acetonitrile in the extraction of anthocyanins from blueberry samples. The ratio of water/organic solvent is another interesting variable that significantly influ­ ences the anthocyanin yield. Decreasing acetone/water ratio lowers the yields of anthocyanins from cabernet grapes, whereas the opposite effect was observed with ethanol. The differences of extrac­ tion efficacy among given solvent/acid combinations may result from the differences in anthocyanin compositions in plant. Even so, the most widely used protocol for anthocyanin extraction is metha­ nol acidified with HCl. Besides, the presence of strong acids also gives rise to difficulty for further processing of anthocyanin extracts (Castaneda-Ovando, Pacheco-Hernandez, Paez-Hernandez, Rodriguez, & Galan-Vidal, 2009; Kapasakalidis, Rastall, & Gordon, 2006). When anthocyanins are concentrated via removal of extraction solvent, anthocyanin hydrolysis occurs as the concentra­ tion of the strong acid reaches a certain level.. Thus, sulfured solutions with SO2 were used as the extracts to substitute acids (Cacace & Mazza, 2002). However, SO2 may induce adverse reactions in hypersensitive individuals, which hinders its application in the production of edible extracts. Besides, anthocyanins are sensitive to heat, which should be taken into account when establish­ ing an extraction protocol. The levels of anthocyanins in blackberry and hibiscus pulps reduce to 20% at 90°C for 6 h (Cacace & Mazza, 2003a, b; Vrhovsek, Masuero, Palmieri, & Mattivi, 2012). The best temperature for anthocyanin extraction from blackcurrants seemed to be 30°C–35°C, as higher temperatures lead to lower contents of anthocyanins. Similar results were also observed as aqueous acetone was used to extract anthocyanins from grape residues with higher yield at 20°C than 60°C (Vatai, Škerget, Knez, Kareth, Wehowski, & Weidner, 2008). With the demand for green extraction, several advanced methodologies have been developed to minimize the utilization of solvents and agents, which are unfriendly to environment. Because of their potential advantages, new technologies need to be applied according to the anthocyanin com­ ponents and the inherent characteristics of plant tissues. Microwave can be able to heat both solvent and fruits quickly, efficiently, and evenly. Furthermore, microwave-assisted extraction can shorten the processing time and decrease the use of solvents. However, with the increase of temperature and irradiation time, the anthocyanin yield decreased significantly, which was attributed to the degra­ dation of anthocyanins (Duan, Jin, Zhao, & Sun, 2015). Thus, temperature and irradiation time in microwave-assisted extraction should be considered to reduce anthocyanin degradation.

13.3.2

ANTHOCYANIN PURIFICATION

13.3.2.1 Solid-Phase Extraction Solid-phase extraction (SPE) separates dissolved compounds according to their physicochemi­ cal characteristics. Traditional SPE uses adsorbent sorbents, such as Sephadex, C18, or Amberlite adsorption resins that absorb target components via their hydroxyl or hydrophobic groups. Nonionic acrylic ester resins showed the high adsorption rates and elution capacity of anthocyanins, whereas the silica nonpolar resin did not absorb anthocyanins (Chandrasekhar, Madhusudhan, &

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Raghavarao, 2012). SPE is relatively cheap and easy-to-operate method. However, nonselective absorption leads to low purity of anthocyanins. Other flavonols besides anthocyanins are still pres­ ent in purified anthocyanins from bayberry fruits (Buran, Sandhu, Li, Rock, Yang, & Gu, 2014; Denev, Ciz, Ambrozova, Lojek, Yanakieva,  & Kratchanova, 2010). To improve the selectivity of SPE, the response of anthocyanin charge to pH is used to change the interaction between sorbents and anthocyanins (He & Giusti, 2011). Anthocyanins are introduced into the system at a pH value of 2, where they interact with the negatively charged sorbent and the other compounds can be removed using solvents with various polarities. To remove the anthocyanins, an alkaline eluent is used to shift the anthocyanins into a negatively charged particle that will no longer interact with the resin. The pH-related SPE is superior to the other common SPE in regard to purity and recovery, sorbent capacity, cost, simplicity, and high throughput. 13.3.2.2 Countercurrent Chromatography High-speed countercurrent chromatography (HSCCC) is a form of liquid–liquid chromatography that uses a liquid stationary phase to hold in the place by centrifugal force and is used to separate, identify, and quantify the chemical components of a mixture. HSCCC has been demonstrated to be a feasible method to purify anthocyanins in a large scale. No columns are required, and only cheap solvents are used in HSCCC, which make it a suitable method for industrial production. Tert-butyl methyl ether, n-butanol, acetonitrile, and water acidified with trifluoroacetic acid (2:2:1:5) have been demonstrated to be feasible to purify anthocyanins from a range of sources such as wine, blueber­ ries, and red cabbage (Kostadinovik, Mirhosseini, & Bogeva, 2013).

13.4 STABILITY OF ANTHOCYANINS FROM BAYBERRY FRUITS Anthocyanins are highly susceptible to degradation. The stability of anthocyanins is of great con­ cern for food applications because they are usually less stable and more sensitive to changes in pH compared with synthetic colorants. Consequently, many studies on the stability of anthocyanins from bayberry fruits have been published. Several physicochemical factors are known to affect the stability of anthocyanins, such as pH and temperature. Typically, anthocyanins are more sta­ ble under acidic conditions (Castaneda-Ovando et al., 2009; Fleschhut, Kratzer, Rechkemmer, & Kulling, 2006). This is the reason why anthocyanin extraction is carried out in an acidified environ­ ment although strong acidic media may lead to the hydrolysis of the glycoside bonds. Temperature is another determinant factor for anthocyanin stability as mentioned earlier. To enhance the stability of anthocyanins in solutions, copigments, metallic ions, or antioxidants could be added to protect anthocyanins. The protections with antioxidant compounds are the easiest to perform (Silva et al., 2017). Most antioxidants are more susceptible to oxidation than anthocyanins that can be oxidized with present oxygen. Copigments that are rich in π electrons and metallic ions such as Mg, Al, and Fe will interact directly with anthocyanin molecules, retarding nucleophilic attack and oxidation of quinoidal base (Castaneda-Ovando et al., 2009; Shaked-Sachray, Weiss, Reuveni, Nissim-Levi, & Oren-Shamir, 2002; Zhu, Chen, Lou, Chen, Ye,  & Chen, 2020). However, the utilization of any compound to protect anthocyanins should consider their application to avoid the influence of the protector.

13.5 HEALTH BENEFITS AND APPLICATIONS OF ANTHOCYANINS FROM BAYBERRY FRUITS 13.5.1 ANTIOXIDANT CAPACITY OF ANTHOCYANINS FROM BAYBERRY FRUITS Antioxidant activity of anthocyanins has been proposed by many studies as a key mechanism for the prevention of many chronic diseases, including diabetes, tumor, and cardiovascular disease. High antioxidant capacities were reported for different varieties of Chinese bayberry fruits. The

Chinese Bayberry Fruits

245

antioxidant activity of different bayberry extracts showed significant positive correlation with their total phenolic content (61.6–256.9 mg/100 g FW), total flavonoids (13.6–117.6 mg/100 g FW), total anthocyanins (undetectable–76.2 mg/100 g FW), and C3G (undetectable–64. 8 mg/100 g FW), respectively, indicating the bayberry polyphenols may contribute to the high antioxidant capacities (Sun et al., 2013). The fruit color change from light to dark red among different varieties, the con­ tents of total phenolics, flavonoids, anthocyanins, C3G, and the antioxidant capacity all increased, and ‘Biqi’ variety contained the highest contents and antioxidant capacity (C3G accounting for 85% of the total antioxidant capacity) of all these components compared with other varieties. There is no anthocyanin content detected in ‘Baizhong’ varieties, and antioxidant content and antioxidant capacity are lowest in this variety.

13.5.2

PREVENTION OF DIABETIC DISEASES

Diabetes is an epidemic disease characterized as the inability to produce or improperly use insulin in the human. Recently, several studies have shown that anthocyanins-rich diet may decrease the incidence of diabetes, improve glucose disposal, and inhibit the development of diabetes-associated complications. In rodent models, C3G and delphinidin-3-glucoside shows a strong effect to stimu­ late high-level insulin secretion from pancreatic β cells in the presence of 4 and 10 mM/L glucose concentrations. This study discusses the protective effects of C3G-rich bayberry fruit extracts in oxidation stress injury of pancreatic β cells and their effects of hypoglycemic reduction in diabetic mice. Bayberry extracts from ‘Biqi’ are used for both in vitro and in vivo testing because of their high C3G content and high antioxidant capacity. The β cells would acquire pretreatment with bay­ berry fruit extract (containing 0.5 μM/L C3G) to prevent cell death, increase cellular viability, and decrease cell necrosis induced by mitochondrial reactive oxygen from 800 to 1200 μMl/ L H2O2. C3G and delphinidin-3-glucoside show high-level ability to stimulate insulin secretion from rodent pancreatic β cells in the presence of 4 and 10 mM/L glucose concentrations. The present study inves­ tigates the protective effects of C3G-rich bayberry fruit extracts against pancreatic β cells against oxidative stress–induced injury as well as their hypoglycemic effects in diabetic mice. Pretreatment of β cells with bayberry fruit extracts (containing 0.5 μg of C3G, 10 g) prevents cell death, increases cellular viability, and decreases mitochondrial reactive oxygen species production and cell necrosis induced by 800 or 1200 μM/ L H2O2. Bayberry fruit extracts dose-dependently upregulate pancre­ atic duodenal homeobox 1 gene expression, contributing to increased insulin-like growth factor II gene transcript levels and insulin protein in INS-1 cells. In addition, administration of bayberry fruit extracts (150 μg of C3G/10 g of body weight twice per day) significantly reduces blood glucose in streptozotocin-induced diabetic imprinting control region mice and increases the glucose tolerance in an oral glucose tolerance test (P < 0.05). These results suggest that bayberry fruit extracts may help prevent and control diabetes and diabetes-related complications (Zhang et al., 2011).

13.5.3

ANTITUMOUR ACTIVITY OF ANTHOCYANINS FROM BAYBERRY FRUITS

The red-colored Chinese bayberry fruits are a rich source of anthocyanins, especially cyanidin-3-glucoside (C3G), which has been well known by their anticancer activity in vitro and in vivo. Previous studies have demonstrated the in vitro anticancer activities of anthocyanins by promoting apoptosis and inhibiting cell cycle and cell invasion. Krüppel-like transcription factor 6 (KLF6) is a novel tumor suppressor gene, and it is involved in the pathogenesis of many cancers. It is discovered that C3G from Chinese bayberry could suppress SGC-7901 tumor xenografts growing through upregulating KLF6 gene expression. C3G treatments also show no toxic effects or other side effects compared with the positive control drug (Tegafur). These results might provide impor­ tant information about the possible mechanisms of C3G-induced antitumor activity against gastric adenocarcinoma in vivo and illustrate the potential application of food anthocyanins in cancer pre­ vention (Wang et al., 2016).

246

13.6

Asian Berries: Health Benefits

CONCLUDING REMARKS

Chinese bayberry (Myrica rubra Sieb. et Zucc.) is a perennial evergreen tree. Different parts (the leaves, bark, and fruits) of this plant are commonly used in traditional herbal medicine or folk recipes because of their various pharmacological activities such as the antioxidant, antibacterial, neuroprotective, and anticancer activities. As the number of bioactive compounds identified from bayberry extraction is increasing, the correlative researches of in vivo metabolism and bioavail­ ability, synergies, and competitive effects of bayberry fruit extracts are receiving more and more attention. The extensive studies should focus on (1) discovery, identification, and comprehensive utilization of the single nutrient and bioactive substances in the locally characteristic bayberry resources, (2) the mechanism and clinical verification of bioactive substances, such as C3G, in pre­ venting and treating human diseases, (3) biosynthetic pathway and genetic and environmental regu­ lation of quantity of important bioactive substances in bayberry fruits, and (4) targeting bioactive substances, developing the commercial treatment, deep processing, and developing pharmaceutical products from bayberry fruits.

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He, J., & Giusti, M. M. (2011). High-purity isolation of anthocyanins mixtures from fruits and vegetables – A novel solid-phase extraction method using mixed mode cation-exchange chromatography. Journal of Chromatography A, 1218(44), 7914–7922. doi: 10.1016/j.chroma.2011.09.005. Kapasakalidis, P. G., Rastall, R. A., & Gordon, M. H. (2006). Extraction of polyphenols from processed black currant (Ribes nigrum L.) residues. Journal of Agricultural and Food Chemistry, 54(11), 4016–4021. doi: 10.1021/jf052999l. Khoo, H. E., Azlan, A., Tang, S. T., & Lim, S. M. (2017). Anthocyanidins and anthocyanins: colored pigments as food, pharmaceutical ingredients, and the potential health benefits. Food & Nutrition Research, 61(­ 1), 1361779. doi: 10.1080/16546628.2017.1361779. Kostadinovik, S., Mirhosseini, H., & Bogeva, E. (2013). Isolation of anthocyanins by high-speed countercur­ rent chromatography and application of the color activity concept to different varieties of red grape pomace from Macedonia. Journal of Nutrition and Food Science, 3(6). Shaked‐Sachray, L., Weiss, D., Reuveni, M., Nissim‐Levi, A., & Oren‐Shamir, M. (2002). Increased anthocy­ anin accumulation in aster flowers at elevated temperatures due to magnesium treatment. Physiologia plantarum, 114(4), 559–565. Silva, S., Costa, E. M., Calhau, C., Morais, R. M., & Pintado, M. E. (2017). Anthocyanin extraction from plant tissues: A review. Critical Reviews in Food Science and Nutrition, 57(14), 3072–3083. doi: 10.1080/10408398.2015.1087963. Siriwoharn, T., Wrolstad, R. E., Finn, C. E.,  & Pereira, C. B. (2004). Influence of cultivar, maturity, and sampling on blackberry (Rubus L. hybrids) anthocyanins, polyphenolics, and antioxidant properties. Journal of Agricultural and Food Chemistry, 52(26), 8021–8030. doi: 10.1021/jf048619y. Sun, C., Huang, H., Xu, C., Li, X., & Chen, K. (2013). Biological activities of extracts from Chinese bayberry (Myrica rubra Sieb. et Zucc.): a review. Plant Foods for Human Nutrition, 68(2), 97–106. Vatai, T., Škerget, M., Knez, Ž., Kareth, S., Wehowski, M., & Weidner, E. (2008). Extraction and formulation of anthocyanin-concentrates from grape residues. The Journal of Supercritical Fluids, 45(1), 32–36. doi: 10.1016/j.supflu.2007.12.008. Vrhovsek, U., Masuero, D., Palmieri, L.,  & Mattivi, F. (2012). Identification and quantification of flavonol glycosides in cultivated blueberry cultivars. Journal of Food Composition and Analysis, 25(1), 9–16. doi: 10.1016/j.jfca.2011.04.015. Wang, Y., Zhang, X.-n., Xie, W.-h., Zheng, Y.-x., Cao, J.-p., Cao, P.-r., . . . Sun, C.-d. (2016). The growth of SGC-7901 tumor xenografts was suppressed by Chinese bayberry anthocyanin extract through upregu­ lating KLF6 gene expression. Nutrients, 8(10), 599. Zhang, B., Kang, M., Xie, Q., Xu, B., Sun, C., Chen, K., & Wu, Y. (2011). Anthocyanins from Chinese bay­ berry extract protect β cells from oxidative stress-mediated injury via HO-1 upregulation. Journal of Agricultural and Food Chemistry, 59(2), 537–545. doi: 10.1021/jf1035405. Zhang, W., Li, X., Zheng, J., Wang, G., Sun, C., Ferguson, I. B., & Chen, K. (2008). Bioactive components and antioxidant capacity of Chinese bayberry (Myrica rubra Sieb. and Zucc.) fruit in relation to fruit maturity and postharvest storage. European Food Research and Technology, 227(4), 1091–1097. doi: 10.1007/s00217-008-0824-z. Zhang, X. N., Huang, H. Z., Zhang, Q. L., Fan, F. J., Xu, C. J., Sun, C. D., . . . Chen, K. S. (2015). Phytochemical characterization of Chinese bayberry (Myrica rubra Sieb. et Zucc.) of 17 cultivars and their antioxidant properties. International Journal of Molecular Sciences, 16(6), 12467–12481. doi: 10.3390/ijms160612467. Zheng, W., & Wang, S. Y. (2003). Oxygen radical absorbing capacity of phenolics in blueberries, cranber­ ries, chokeberries, and lingonberries. Journal of Agricultural and Food Chemistry, 51(2), 502–509. doi: 10.1021/jf020728u. Zhu, Y., Chen, H., Lou, L., Chen, Y., Ye, X.,  & Chen, J. (2020). Copigmentation effect of three phenolic acids on color and thermal stability of Chinese bayberry anthocyanins. Food Science & Nutrition, 8(7), 3234–3242.

14

Haskap Berries (Lonicera caerulea L.) Phytochemical Constituents

and Health Benefits Hae-Jeung Lee and Anshul Sharma Gachon University

CONTENTS 14.1 14.2 14.3 14.4

Introduction ........................................................................................................................ 249

Methodology ....................................................................................................................... 250

Botanical Description and Status ....................................................................................... 250

Composition ........................................................................................................................ 252

14.4.1 Nutritional Composition........................................................................................ 252

14.4.2 Phytochemical Composition ................................................................................. 254

14.4.2.1 Phenolic Acids ...................................................................................... 254

14.4.2.2 Anthocyanins ........................................................................................ 254

14.4.2.3 Flavonoids ............................................................................................. 258

14.4.2.4 Other Phytochemicals........................................................................... 258

14.5 Bioavailability ..................................................................................................................... 258

14.6 Quality Profile of Haskap from Asian Regions .................................................................. 259

14.7 Use of Haskap as Commercial Products/Functional Foods ............................................... 259

14.8 Potential Health Benefits .................................................................................................... 259

14.8.1 Antioxidant Activity..............................................................................................260

14.8.2 Anti-Diabetic Activity........................................................................................... 263

14.8.3 Antiobesity Activity .............................................................................................. 263

14.8.4 Antitumor/Radioprotective Activity .....................................................................264

14.8.5 Anti-inflammatory Activity ..................................................................................264

14.8.6 Hepatoprotective Activity ..................................................................................... 265

14.8.7 Cardiovascular Disease Protective Activity.......................................................... 272

14.8.8 Antimicrobial Activity .......................................................................................... 273

14.8.9 Pulmonary Protective Activity ............................................................................. 273

14.9 Summary ............................................................................................................................ 273

References...................................................................................................................................... 274

14.1 INTRODUCTION Haskap (Lonicera caerulea L.) is an emerging plant with ample health benefits because of their phenolics, anthocyanins, and other antioxidants (Rupasinghe et al., 2015). In 1894, haskap origi­ nated as a horticultural crop in Russia (Hummer et  al., 2012); since then, the plant has been gaining popularity as a new superfood (Bors et al., 2012). L. caerulea is commonly called as haskap, blue honeysuckle, edible honeysuckle, honeyberry, and sweet-berry honeysuckle (Celli 249

250

Asian Berries: Health Benefits

et al., 2014). A native to Northern Hemisphere, L. caerulea is typically grown in cold temperate zones such as China, Korea, Japan, Siberia (Kamchatka Peninsula, Russia), Inner Mongolia, and some portions of North America and, lately, extensively cultivated in Poland, Czech Republic, Slovakia, USA, Canada, Lithuania, Belarus, and Slovenia (Chaovanalikit et al., 2004; Jin et al., 2006; Naugžemys et al., 2011; Ochmian et al., 2012; Senica et al., 2018; Thompson & Barney, 2007). The standard varieties of haskap include Lonicera edulis, Lonicera kamtschatica, Lonicera emphyllocalyx, Lonicera altaica, and Lonicera boczkarnikowae (Jurikova et al., 2012). In Korea, L. caerulea var. edulis has been reported from alpine regions, including mountain (Mt.) Bangtae, Mt. Halla, Mt. Jeombong, Mt. Seorak 1, Mt. Seorak 2, and Mt. Gyebang (Choi et  al., 2015; Hyun et  al., 2015). Haskap at Mt. Gyebang is found between 1,200 and 1,400 m altitude (Yang et al., 2014). The initial cultivation of the Japanese haskap dated back in 1967 by Hokkaido Prefectural Agriculture Experiment Station and a Farmers Co-operative in Chitose (Fu et  al., 2011). The variety L. caerulea var. emphyllocalyx Nakai has been commercially cultivated in Hokkaidǀ and northern Honshu and is commonly famous as haskap or hasukappu in the Japanese language among Ainu people (Miyashita et  al., 2009). The Japanese haskap thrives best in a moderate climate. In Japan, Hokkaidǀ and Aomori have been used for the commercial cultivation of the L. caerulea, with 90.2 tons production in 2012 (Iketani, 2016). In China, L. caerulea is mostly cultivated in the northeastern region, generally consisted of prov­ inces namely Xinjiang and Xeilongjiang, whereas L. edulis is found in the northwestern region (Plekhanova, 1999; Zhou et al., 2018). The wild variety of haskap is common in China, whereas cultivar ‘Beilei’ is the approved variety (S. Liu, L. You et al., 2018). Northeast China harvests 2,000 tons of haskap per year (Jie, 2006). However, recently, many varieties with different fruit flavor and size have been cultivated (Y. Wang, B. Li, J. Zhu et al., 2016). Likewise, L. caerulea var. kamtschatica variety has been grown in eastern (zhimolost) Russia (Caprioli et al., 2016). North America has recently begun the cultivation of important haskap cultivars, Indigo Gem, Borealis, and Tundra along with other important varieties (Rupasinghe et al., 2018). The wealth of literature has been published on the bioactivities of haskap berries and their phytochemical constituents (Jurikova et al., 2012; Rupasinghe et al., 2018). However, people have also started evaluating the health-promoting activities of stems, branches, and leaves of haskap (Hyun et al., 2015; Minami, Nakamura et al., 2019). Herein, we focus on the status, composition, and specifi­ cally the recently reported literature about bioactivities of haskap.

14.2

METHODOLOGY

A comprehensive literature search in Google Scholar, Pubmed, and Science direct databases was conducted using the following keywords: “Lonicera caerulea” and “haskap” or “blue honeysuckle” or “sweet berry honeysuckle” or “honeyberry” or “haskap and antioxidant, anti-inflammatory, pulmonary protecting, cardiovascular protecting, antimicrobial, antitumor, hepatoprotective, potential” or “Lonicera caerulea and health properties” to trace all appropri­ ate articles published between 2002 and August 2019, covering detailed morphology, nutri­ tional and biochemical composition, and potential health activities. In total, 83 references were included in the study, including 66 research articles, 12 review articles, and 5 articles from books as well as reports.

14.3

BOTANICAL DESCRIPTION AND STATUS

Lonicera is the largest genus (family Caprifoliaceae), including approximately 200 species (Becker & Szakiel, 2018). Haskap is of either diploid (2n = 18) or tetraploid form (2n = 36) and has a life cycle of 25–30 years (Miyashita et al., 2009; Svarcova et al., 2007). Haskap is a peren­ nial plant and typically reaches a height of 3 m, while leaves attain a width of 5 cm and a length of 8 cm (Becker & Szakiel, 2018). The leaves morphology consists of single-blade leaves with

Haskap Berries: Phytochemical Constituents

251

the opposite arrangement, oval to elongated shape, and 3–5 cm in length (Becker  & Szakiel, 2018) (Figure 14.1). Haskap flowers are small (~2 cm long), funnel shaped, and pale yellow to cream in color and bloom in early spring. Bumblebees generally pollinate the flowers; in Japan, blue orchard bees (Osmia sp.) are used. The flowers usually exist in temperatures between −8°C and −10°C (Thompson & Barney, 2007). Usually, the fruit of the genus Lonicera is small and inedible; however, the species of haskap has dark blue to purple edible (sour to sweet) fruits (Bors et al., 2012). Haskap fruits are soft and generally 2 cm long with an elongated, oval, or cylindrical shape, and weight of the fruits ranges from 0.3 to 2.0 g (Figure 14.1) (Hummer et al., 2012). It has been reported that around 17 species of these berry fruits are edible (Plekhanova et al., 1998; Rop et al., 2011). Haskap plants have the remarkable frost-resistant ability and can withstand extremely low (−46°C) temperature and a broad pH (5–8) range. They are also highly resistant to diseases and pests (Hummer et al., 2012; Ochmian et al., 2012). Fruits with access to pollinators are responsible for 90% of the production, while the fruits of isolated haskap plants have smaller, lighter, and reduced number of seeds (BoĪek, 2012).

FIGURE  14.1 Plant parts and berries of haskap. (a) Image by zoosnow from Pixabay, (b) Image by Alex Khaizeman from Pixabay.

252

14.4

Asian Berries: Health Benefits

COMPOSITION

14.4.1 NUTRITIONAL COMPOSITION It is widely acknowledged that the haskap is an emerging functional food with various dietary and health-promoting constituents, including sugars, organic acids, and macronutrients as major constituents (OszmiaĔski et al., 2016; Rupasinghe et al., 2012; Wojdyło et al., 2013). Minor constitu­ ents include minerals and vitamins (Rupasinghe et al., 2015; Senica et al., 2018). The nutritional composition may fluctuate across geographical locations and with diverse environmental factors. Table 14.1 summarizes the details of the nutritional components of haskap.

TABLE 14.1 Nutritional Composition of Haskap S. No 1

2

Constituents Sugars

Organic acids

Content

Cultivar/Genotypes

References

Glucose

0.8–3.4 g/100 g 0.1–0.6 g/100 g 3.6 g/100 g 626.04–1,129.35 mg/100 g

Rupasinghe et al. (2015) Wojdyło et al. (2013) OszmiaĔski et al. (2016) Senica et al. (2018)

Fructose

0.9–2.9 g/100 g 2.8 g/100 g 913.35–1,363.64 mg/100 g

Sucrose Sorbitol

Less than 0.2g/100 g 9.29–111.37 0.04–0.1

Citric acid

686.8–1,620.9 30–58% 430.90–807.36

Malic acid

185.89–411.34 mg/100 g 28–50% 192.95–386.76 mg/100 g

Fumaric acid

0.06–0.24 mg/100 g

Berry Blue, Tundra, Indigo Gem, Borealis, LC Czelabinka, Jolanta, Wotjek, Duet, Klon 44a, Klon 38a, Klon Ba, Klon Ca Wotjek Aurora, Honey Bee, Tundra, Borealis Berry Blue, Tundra, Indigo Gem, Borealis, LC Wotjek Aurora, Honey Bee, Tundra, Borealis Berry Blue, Tundra, Indigo Gem, Borealis, LC Aurora, Honey Bee, Tundra, Borealis Czelabinka, Jolanta, Wotjek, Duet, Klon 44, Klon 38, Klon B, and Klon C Czelabinka, Jolanta, Wotjek, Duet, Klon 44, Klon 38, Klon B, and Klon C Berry Blue, Tundra, Indigo Gem, Borealis, LC Aurora, Honey Bee, Tundra, Borealis Czelabinka, Jolanta, Wotjek, Duet, Klon 44, Klon 38, Klon B, and Klon C Berry Blue, Tundra, Indigo Gem, Borealis, LC Aurora, Honey Bee, Tundra, Borealis Aurora, Honey Bee, Tundra, Borealis

Subgroup

Rupasinghe et al. (2015) OszmiaĔski et al. (2016) Senica et al. (2018) Rupasinghe et al. (2015) Senica et al. (2018) Wojdyło et al. (2013)

Wojdyło et al. (2013) Rupasinghe et al. (2015) Senica et al. (2018)

Wojdyło et al. (2013) Rupasinghe et al. (2015) Senica et al. (2018)

Senica et al. (2018) (Continued)

Haskap Berries: Phytochemical Constituents

253

TABLE 14.1 (Continued) Nutritional Composition of Haskap S. No

Constituents

Subgroup Quinic acid

Content 57.89–81.26 mg/100 g 10–32% 27.81–77.89 mg/100 g

Schikimic acid 23.22–32.70 mg/100 g 0.57–0.95 mg/100 g Oxalic acid

72.07–97.2 mg/100 g

Phytic acid

254.09–472.45 mg/100 g

Tartaric acid

3

4

Macronutrients (%)

Minerals

1.28–7.56 mg/100 g Ascorbic acid 17.75–25.77 mg/100 g 22.5 mg/100 g Carbohydrates 10.2–15.6 0.86

Cultivar/Genotypes

References

Czelabinka, Jolanta, Wotjek, Duet, Klon 44, Klon 38, Klon B, and Klon C Berry Blue, Tundra, Indigo Gem, Borealis, LC Aurora, Honey Bee, Tundra, Borealis Czelabinka, Jolanta, Wotjek, Duet, Klon 44, Klon 38, Klon B, and Klon C Aurora, Honey Bee, Tundra, Borealis Czelabinka, Jolanta, Wotjek, Duet, Klon 44, Klon 38, Klon B, and Klon C Czelabinka, Jolanta, Wotjek, Duet, Klon 44, Klon 38, Klon B, and Klon C Aurora, Honey Bee, Tundra, Borealis Aurora, Honey Bee, Tundra, Borealis Kamtschatica Borealis, Tundra, Indigo Gem Kamtschatica

Wojdyło et al. (2013) Rupasinghe et al. (2015) Senica et al. (2018)

Protein

4.6–8.4 2.12

Borealis, Tundra, Indigo Gem

Kamtschatica

Fat

2.2–4.8 0.01

Borealis, Tundra, Indigo Gem

Kamtschatica

Fiber Ash

8.3 3.27–4.33 0.45

Kamtschatica

Borealis, Tundra, Indigo Gem

Kamtschatica

Calcium

1,030 mg/kg 47.7–52.7 mg/100 g fw 0.02% 863 mg/kg

Kamtschatica

Altaj, Amur, Amfora, Pojark,

and Sinoglaska Borealis, Tundra, Indigo Gem Kamtschatica

8.44–15.4 mg/100 g fw 1,020 mg/kg 10.5–12.3%

Altaj, Amur, Amfora, Pojark, and Sinoglaska Kamtschatica Borealis, Tundra, Indigo Gem

224.4–422 mg/100 g fw 324 mg/kg

Altaj, Amur, Amfora, Pojark, and Sinoglaska Kamtschatica

Sodium

Magnesium

Manganese Potassium

Wojdyło et al. (2013) Senica et al. (2018)

Wojdyło et al. (2013)

Wojdyło et al. (2013)

Senica et al. (2018) Senica et al. (2018) Caprioli et al. (2016) Rupasinghe et al. (2012) Caprioli et al. (2016) Rupasinghe et al. (2012) Caprioli et al. (2016) Rupasinghe et al. (2012) Caprioli et al. (2016) Caprioli et al. (2016) Rupasinghe et al. (2012) Caprioli et al. (2016) Caprioli et al. (2016) Jurikova et al. (2012) Rupasinghe et al. (2012) Caprioli et al. (2016) Jurikova et al. (2012) Caprioli et al. (2016) Rupasinghe et al. (2012) Jurikova et al. (2012) Caprioli et al. (2016) (Continued)

254

Asian Berries: Health Benefts

TABLE 14.1 (Continued) Nutritional Composition of Haskap S. No

Constituents

Subgroup Phosphorous

5

Vitamins

Copper Zinc Iron Vitamin C

Content 0.17%–0.24% 35.8–66.6 mg/100 g fw 124 mg/kg 203 mg/kg 128 mg/kg 3.2–32.1%

67.7–186.6 mg/100 g

Cultivar/Genotypes

References

Borealis, Tundra, Indigo Gem Altaj, Amur, Amfora, Pojark, and Sinoglaska Kamtschatica Kamtschatica Kamtschatica Czelabinka, Jolanta, Wotjek, Duet, Klon 44, Klon 38, Klon B, and Klon C Altaj, Amur, Amfora, Pojark, and Sinoglaska

Rupasinghe et al. (2012) Jurikova et al. (2012) Caprioli et al. (2016) Caprioli et al. (2016) Caprioli et al. (2016) Wojdyło et al. (2013)

Jurikova et al. (2012)

a *gene modifed types

14.4.2 PHYTOCHEMICAL COMPOSITION Haskap berries have fascinated the scientifc community for their distinct phytochemicals profle (Jurikova et al., 2012). These fruits are either consumed raw or processed forms such as juice, jams, pastries, ice cream, and dried forms (Celli et al., 2014). Haskap berries possess the most potent antioxidant activity than the reported berry commodities, such as blueberries, raspberries, strawberries, blackberries, bilberries, sea buckthorn berries, and black currants (Celli et al., 2014; Raudsepp et al., 2013; Rop et al., 2011). Previous reviews described the detailed description of chemical constituents of haskap berries (Becker & Szakiel, 2018; Jurikova et al., 2012). This chapter also recites the chemical constituents with new additions (Table 14.2). 14.4.2.1 Phenolic Acids Phenolic acids are nonfavonoid phytochemicals. On a structural basis, phenolic acids from haskap can be categorized into two types: (1) benzoic acid (C1–C6) derivatives with benzene ( basic unit) with a bonded carboxylic acid group and (2) cinnamic acid (C3 –C6) derivatives consisting of a benzene ring attached to cinnamic (propenoic acid) acids. The hydroxycinnamic acids constitute the major fraction of phenolic acids, primarily with m-coumaric acid, p-coumaric acid, chlorogenic acid, neochlorogenic acid, caffeic acid, 3,4-dimethoxycinnamic acid, and ferulic acid (Zadernowski et al., 2005). On the other hand, hydroxybenzoic acids constitute a relatively smaller fraction, including gallic acid (Gal; 3,4,5-trihydroxybenzoic acid) and 4-aminobenzoic acid (Gazdik et al., 2008). Several other less common constituents such as salicylic acid, protocatechuic acid, gentisic acid, vanillic acid, and hydroxycaffeic acid have been reported. 14.4.2.2 Anthocyanins Anthocyanins are the most prevalent vacuolar plant pigments, responsible for vivid colors of fruits and vegetables depending upon the structural composition and pH. Structurally, anthocyanins consist of basic favylium salts with cyanidin, petunidin, pelargonidin, delphinidin, malvidin, and peonidin, degree of methylation on the ring B, sugar molecules position, and OH group variations. To date, the potential health benefts of haskap have been attributed mainly to its anthocyanins (K hattab et al., 2016; Rupasinghe et al., 2018). Cyanidin 3-glucoside (C3G) is the most widespread anthocyanin responsible for numerous health benefts (Paredes-López et al., 2010).

Haskap Berries: Phytochemical Constituents

255

TABLE 14.2 Phytochemical Composition of Haskap S. No 1

2

Constituents Phenolic acid

Anthocyanins

Subgroup

Content (mg/100 g)

Cultivar

m-Coumaric acid

201.4

Sevast

p-Coumaric acid

98.7

Sevast

Gallic acid 4-Amino benzoic acid Chlorogenic acid

60 17 76.6–294 22.45–46.06

Altaj, Goluboje vereteno Altaj Goluboje vereteno Czelabinka, Jolanta, Wotjek, Duet Berry Blue, Tundra, Indigo Gem, Borealis, LC

Neochlorogenic acid

184.1–381.6 1.4–15.4 2.0–5.0

Caffeic acid

59.8 0.1–0.2

Wojtek, Zielona, Brazowa, Jolanta Czelabinka, Jolanta, Wotjek, Duet Berry Blue, Tundra, Indigo Gem, Sevast Berry Blue, Tundra, Indigo Gem, Borealis, LC

Salicylic acid

123.5

Sevast

Ferulic acid

3.69

Sevast

Protocatechuic acid

14.4 dw

Sevast

Gentisic acid

15.3

Sevast

Vanillic acid

2.1

Sevast

Hydroxycaffeic

5.1

Sevast

3,4-Dimethoxycinnamic 4.4

Sevast

Cyanidin-3-O-glucoside 67.7–649.0 1,649.4– 4,226.7 89.626– 91.340a 82.68a 40.13–56.93

Berry Blue, Tundra, Indigo Gem, Borealis, LC Wojtek, Zielona, Brazowa, Jolanta Wild, Beilei, Nos. 1, 2 – Aurora, Honey Bee, Tundra, Borealis

References Zadernowski et al. (2005) Zadernowski et al. (2005) Gazdik et al. (2008) Gazdik et al. (2008) Wojdyło et al. (2013) Rupasinghe et al. (2015), Khattab et al. (2016) Kusznierewicz et al. (2012) Wojdyło et al. (2013) Khattab et al. (2016) Zadernowski et al. (2005) Rupasinghe et al. (2015) Zadernowski et al. (2005) Zadernowski et al. (2005) Zadernowski et al. (2005) Zadernowski et al. (2005) Zadernowski et al. (2005) Zadernowski et al. (2005) Zadernowski et al. (2005) Rupasinghe et al. (2015), Khattab et al. (2016) Kusznierewicz et al. (2012) Y. Wang, J. Zhu et al. (2016) Liu et al. (2016) Senica et al. (2018) (Continued)

256

Asian Berries: Health Benefits

TABLE 14.2 (Continued) Phytochemical Composition of Haskap S. No

Constituents

Subgroup

Content (mg/100 g)

Cyanidin-3,5­ diglucoside

15.0–31.0 1.437%– 2.346%a 5.46a Fresh 61.4–152.2 1.18–3.68

Cyanidin-3-O­ rutinoside

10.0–64.9 4.76a Fresh 8.32–9.62

Pelargonidin-3-O­ glucoside

3.9–14.7 0.509–1.057a 1.33–4.82

0.20a Fresh 0.86a Fresh

–

0.055–0.337a 0.20a Fresh

Wild, Beilei, Nos. 1, 2 –

Peonidin-3-O-glucoside 3.0–25.0 3.088–3.912a 3.60a Fresh 8.55–13.94

Peonidin-3-O-rutinoside 0.48a Fresh Peonidin-3, 0.093–2.087a 5-dihexoside 18.05–26.06 Delphinidin-3-glucoside 1.163a

Flavonoids

References

Khattab et al. (2016) Y. Wang, J. Zhu et al. (2016) Liu et al. (2016) Kusznierewicz et al. (2012) Senica et al. (2018) Khattab et al. (2016), Rupasinghe et al. (2015) Liu et al. (2016) Senica et al. (2018) Berry Blue, Tundra, Indigo Rupasinghe et al. Gem, Borealis, LC (2015), Khattab Wild, Beilei, Nos. 1, 2 et al. (2016) Aurora, Honey Bee, Tundra, Y. Wang, J. Zhu Borealis et al. (2016) Senica et al. (2018) Aurora, Honey Bee, Tundra, Senica et al. (2018) Borealis Berry Blue, Tundra, Indigo Khattab et al. Gem (2016) Wild, Beilei, Nos. 1, 2 Y. Wang, J. Zhu – et al. (2016) Aurora, Honey Bee, Tundra, Liu et al. (2016) Borealis Senica et al. (2018) – Liu et al. (2016) Wild, Beilei, Nos. 1, 2 Y. Wang, J. Zhu Aurora, Honey Bee, Tundra, et al. (2016) Borealis Senica et al. (2018) Wild Y. Wang, J. Zhu et al. (2016) – Liu et al. (2016)

Pelargonidin-dihexoside 1.20–6.29

3

Cultivar

Delphinidin-3­ rutinoside Delphinidin-3-p­ coumaroylglucoside Cyanidin-3-hexoside­ catechin Quercetin

0.1–0.2

Quercetin-3-O­ glucoside

1.4–10.0 0.69–1.22

Berry Blue, Tundra, Indigo Gem Wild, Beilei, Nos. 1, 2 – Wojtek, Zielona, Brazowa, Jolanta Aurora, Honey Bee, Tundra, Borealis Berry Blue, Tundra, Indigo Gem, Borealis, LC – Aurora, Honey Bee, Tundra, Borealis

Liu et al. (2016)

Y. Wang, J. Zhu et al. (2016) Liu et al. (2016) Berry Blue, Tundra, Indigo Rupasinghe et al. Gem, Borealis, LC (2015) Berry Blue, Tundra, Indigo Rupasinghe et al. Gem, Borealis, LC (2015), Khattab Aurora, Honey Bee, Tundra, et al. (2016) Borealis Senica et al. (2018) (Continued)

Haskap Berries: Phytochemical Constituents

257

TABLE 14.2 (Continued) Phytochemical Composition of Haskap S. No

Constituents

Subgroup Quercetin-3-O­ rutinoside

4

Steroids

Content (mg/100 g) 6.2–34.0 7.51–28.98

Cultivar

Berry Blue, Tundra, Indigo Gem, Borealis, LC Aurora, Honey Bee, Tundra, Borealis Quercetin-3-O­ 0.1 Berry Blue, Tundra, Indigo galactoside 5.1–15.0 Gem, Borealis, LC 4.43-8.63 Wojtek, Zielona, Brazowa, Jolanta Aurora, Honey Bee, Tundra, Borealis Quercetin-rhamnoside 1.9–63.0 Czelabinka, Jolanta, Wotjek, Duet Quercetin-3-O­ 1.14–4.66 Aurora, Honey Bee, Tundra, vicianoside Borealis Kaempferol-3-O­ 0.09–0.26 Aurora, Honey Bee, Tundra, glucoside Borealis Kaempferol-3-O­ 0.09–0.45 Aurora, Honey Bee, Tundra, galactoside Borealis Catechin 1.7–5.4 fw Berry Blue, Tundra, Indigo 0.45–3.40 Gem, Borealis, LC 1.8–11.3 DM Aurora, Honey Bee, Tundra, Borealis Czelabinka, Jolanta, Wotjek, Duet Epicatechin 0.7–7.1 Berry Blue, Tundra, Indigo 1.55–13.25 Gem, Borealis, LC 22.2–136.1 Aurora, Honey Bee, Tundra, DM Borealis Czelabinka, Jolanta, Wotjek, Duet Procyanidins 228.6–512.0 Czelabinka, Jolanta, Wotjek, DM Duet Luteolin hexoside 0.18–0.36 Aurora, Honey Bee, Tundra, Borealis Luteolin-3-rutinoside 0.26–0.47 Aurora, Honey Bee, Tundra, Borealis 24­ 20.30–39.51; Czelabinka, Dlinnopłodna, Methylenecycloartanol 10.99, 43.90; Wołoszebnica, Sinogłaska; 7.15–49.20 Wojtek, Jolanta; Genotypes 30, 38, 44, and T Cycloart-23-ene-3,25­ 1.27–3.10; Czelabinka, Dlinnopłodna, diol 0.44, 0.89; Sinogłaska, Wołoszebnica; 0.36–1.12 Wojtek, Jolanta; Genotypes 30, 38, 44b, and T Sitosterol 0.25–4.59; Czelabinka, Dlinnopłodna, 3.01, 1.09; Sinogłaska, Wołoszebnica; 2.27-4.28 Wojtek, Jolanta; Genotypes 30, 38, 44, and T

References Rupasinghe et al. (2015), Khattab et al. (2016) Senica et al. (2018) Rupasinghe et al. (2015) Kusznierewicz et al. (2012) Senica et al. (2018) Wojdyło et al. (2013) Senica et al. (2018) Senica et al. (2018) Senica et al. (2018) Rupasinghe et al. (2015) Senica et al. (2018) Wojdyło et al. (2013) Rupasinghe et al. (2015) Senica et al. (2018) Wojdyło et al. (2013) Wojdyło et al. (2013) Senica et al. (2018) Senica et al. (2018) Becker et al. (2019)

Becker et al. (2019)

Becker et al. (2019)

(Continued)

258

Asian Berries: Health Benefits

TABLE 14.2 (Continued) Phytochemical Composition of Haskap S. No

Constituents

Subgroup

Content (mg/100 g)

Tremulone

0.64–1.09; 0.54; 0.04–0.25

5

Iridoids

Loganin-7-O-pentoside

0.30–0.46

6

Triterpenoids

α-Amyrin

6.51–11.77; 1.12, 40.37; 1.67–2.21

β-Amyrin

1.32–2.42; 0.43, 8.14 0.36–7.30

Friedelin (triterpene ketone) Oleanolic acid

14.15, 0.55

Ursolic acid

0.69–1.41; 0.64–2.88 0.69–2.48

a

0.28–1.22; 0.28, 0.16; 0.14–0.38

Cultivar

References

Czelabinka, Dlinnopłodnab, Sinogłaska, Wołoszebnica; Jolanta, Wojtekb; Genotypes 30b, 38, 44b, and T Aurora, Honey Bee, Tundra, Borealis Czelabinka, Dlinnopłodna, Sinogłaska, Wołoszebnica; Jolanta b*, Wojtek; Genotypes 30, 38b*44, and Tb* Czelabinka, Dlinnopłodna, Sinogłaska, Wołoszebnica; Jolanta b*, Wojtek; Genotypes 30, 38b, 44, and T b* Jolanta, Wojtek

Becker et al. (2019)

Senica et al. (2018)

Becker et al. (2019)

Becker et al. (2019)

Becker et al. (2019)

Czelabinka, Dlinnopłodna, Becker et al. (2019)

Sinogłaska, Wołoszebnica; Jolanta, Wojtek; Genotypes 30, 38, 44, and T Czelabinka, Dlinnopłodna, Becker et al. (2019)

Sinogłaska, Wołoszebnica; Jolanta, Wojtek; Genotypes 30, 38, 44, and T

Detected through mass spectrometry. b* Not detected.

14.4.2.3 Flavonoids Flavonoids are secondary metabolites with numerous biological functions, consisted of flavonols, flavones, and flavanols as their subclasses. Flavonols mainly consisted of quercetin and kaempferol derivatives. Luteolin derivatives constitute flavones. Flavanol is the most complex subclass with catechins, epicatechins (ECs), and procyanidins as constituents (Table 14.2). 14.4.2.4 Other Phytochemicals Recent studies by Becker et al. have identified steroids and triterpenoids using cuticular waxes of honeysuckle leaves (Becker et al., 2019). In addition, iridoids, a type of monoterpenoids, have also been described (Kucharska et al., 2017).

14.5

BIOAVAILABILITY

Bioavailability defines the extent to which a target nutrient is subjected to digestion, absorp­ tion, and metabolization via normal pathways (McGhie  & Walton, 2007). The informa­ tion to evaluate the bioavailability study of haskap or its extract is limited. Moreover, most of haskap’s health-promoting activities are linked to its major (80%–92%) anthocyanin, cyanidin-3-O-glucoside (C3G) (Kusznierewicz et al., 2012; Rupasinghe et al., 2018). Notably,

Haskap Berries: Phytochemical Constituents

259

only one human study investigated the effect of L. caerulea fresh berries involving healthy volunteers from the Czech Republic. The intervention involved consumption of 165 g fresh haskap berries for 1 week. The results showed no changes in hematological and biochemi­ cal tests and clinical safety profiles. In addition, the total antioxidant status of plasma was unchanged. However, significant increases in glutathione peroxidase (GPx) level, catalase in erythrocytes, and TBARS in both plasma and erythrocytes were reported. The anthocyanins did not show any adverse effects. However, liquid chromatography/mass spectrometry (LC/ MS) analysis identified phenolic acids, including benzoic, vanillic, protocatechuic, ferulic, 3-hydroxycinnamic, p-coumaric, isoferulic, and hippuric acids (metabolic end products) in the urine of healthy volunteers, indicating metabolic activity against haskap polyphenols in the colon (Heinrich et al., 2013).

14.6 QUALITY PROFILE OF HASKAP FROM ASIAN REGIONS In the Hokkaido region of Japan, people gave a viewpoint on haskap imported from China. As per the processors, the sweetness of the Chinese haskap was less compared with that of their Japanese counterparts (Lefol, 2007). Besides, Chinese haskap also suffered drawbacks such as inconsistent sizes, poor quality, and different maturing stages. On the other hand, Japanese haskap offers high cost of production compared with the Chinese haskap. Conversely, the bitterness of the Chinese haskap (Unimproved varieties) berries could be attributed to the presence of large amounts of anti­ oxidants and anti-carcinogenic compounds, making Chinese haskap a potential candidate for the health food industry (Lefol, 2007). From China, it has been reported that the haskap variety ‘Beilei’ showed no significant differ­ ence in the anthocyanins amount and free radical scavenging activity compared with wild varieties, however, in terms of taste and yield, Beilei performed well (Y. Wang, J. Zhu et al. 2016). Another study also showed that among seven varieties from China, Beilei showed the highest antioxidant activity, attributed to the presence of C3G. Besides, the fruit peel had higher amounts of anthocya­ nins than pulp (Wang et al., 2018). From Korea, our group evaluated the antioxidant potential of Korean and Chinese haskap. The Chinese haskap showed higher antioxidant potential than Korea (Lee et al., 2018). The different species of haskap may lead to a statistical difference in the polyphenols concentrations. Overall, differences in the phytochemical, morphological, and biochemical activities may be ascribed to the variations in the topographical factors and the species of haskap.

14.7 USE OF HASKAP AS COMMERCIAL PRODUCTS/FUNCTIONAL FOODS Many commercial products of haskap are available. In Japan (www.cropweek.com), and other countries, haskap has been commercially utilized for the synthesis of jams, beverages, candy, noodles, pastry, haskap chocolate jelly, wine, and tea as well as its frozen foam in yogurt and ice cream (www.kirkaberryfarms.ca/haskaps.html) (Fu et al., 2011). Berries can also be consumed in fresh and dried forms (Celli et  al., 2014). The commercial haskap companies from Japan include Hori, Morimoto, and Mitsuboshi (Lefol, 2007). Recently, Biohaskap® Vitality juice has got the Best New Product Award at BIOFACH 2019 (www.biohaskap.com). Nevertheless, the research by our group has recently been recognized by the Ministry of Food Science and Drug Safety, South Korea (www.nifds.go.kr), to use haskap as functional food. The research highlighted the use of haskap for the cure of nonalcoholic fatty liver disease (NAFLD) (Park, Yoo et al., 2019).

14.8 POTENTIAL HEALTH BENEFITS Various health-promoting activities of haskap have been summarized in Figure 14.2.

260

Asian Berries: Health Benefits

FIGURE 14.2 Health-promoting activities of haskap.

14.8.1

ANTIOXIDANT ACTIVITY

Both nonradical and radical reactive oxygen species (ROS) such as hydroxyl radical (OH−), superox­ ide radical anion ( OH 2Σ− ), hydrogen peroxide (H2O2), and reactive nitrogen species (RNS) including nitric oxide (NO) at low concentrations are vital in cellular signaling processes and for physiologi­ cal homeostasis. However, at high levels, both species cause damage to lipids, DNA, and proteins, alerting their metabolic pathways, leading to several degenerative diseases. Many studies reporting the antioxidant activities of haskap have been published (Celli et al., 2014). This section describes the antioxidant potential of haskap mainly from Asian countries (Table 14.3). Haskap (edulis) anthocyanins were shown to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), superoxide ( ΣO −2 ) anion, and •OH radicals by 30, 80, 1.43, and 0.03 μg/mL, respectively (Zhao et al., 2011). In another study, at con­ centrations (50 and 100 μg/mL), the DPPH scavenging ability of L. caerulea branches (LcB) was found to be 22.30 and 31.22 electron donation ability (EDA) %, while those values for scavenging ability of leaves (LcL) were 33.44 and 69.10 EDA%, respectively. In addition, Fe3+–Fe2+ transfor­ mation at 100–300 μg/mL ranged from 0.28 to 0.60 for LcB and 0.38 to 0.81 for LcL. High anti­ oxidant activity of LcL could be due to the presence of higher level of flavonoids compared with LcB (Hyun et al., 2015). In a different concept, a study from China evaluated the antioxidant activ­ ity of haskap berries using cyclic voltammetry (CV) along with DPPH, FRAP, and ABTS assays. Different extracts (dichloromethane, ethyl acetate, hexane, methanol, and water) were used, and the results showed scavenging in the range of 10.93–193.09, 43.84–495.01, 0.19–200.21 μM TE/g, and 17.23–24.83 Q1000, μC (under anodic current wave) using DPPH, FRAP, ABTS, and CV assays, respectively. Among all, the methanolic extract displayed the highest scavenging potential in all four assays (Zhao et al., 2015). Another investigation reported that the oxygen radical absorbance capacity (ORAC) scavenging values of the four cultivars of haskap were in the range of 52–68 μM TE/g fw. Wild variety (68 μM TE/g fw) displayed maximum activity followed by Beilei (64.7 μM TE/g fw), and two unidentified cultivars: No. 1 (55.7 μM TE/g fw) and No. 2 (52 μM TE/g fw) (Y. Wang, J. Zhu et al., 2016). Liu et al. showed the radical scavenging potential of wild blue honeysuckle berries using DPPH (72.12 μg/mL DM), ORAC (198.19 μM TE/100 mg DM), and cellular antioxidant activity (CAA; 14.75 μM QE/100 mg DM) assays (Liu et  al., 2016). Recently, our research group investigated the antioxidant potential of Korean (HBK) and Chinese (HBC) honey berry using HepG2 cell

China

China

China

China

L. caerulea

L. caerulea 

Wild L. caerulea berry

Blue honeysukle

Beilei, BLS, BKQE HSY-24, Haskap L-5 and wild

3.0–4.5 μg/mL

48.40 μg/mL DM

LcB (22.30 and 31.22 EDA %) LcL (33.44 and 69.10 EDA %) 10.93– 193.09 (μM TE/g)

South Korea

Wild, Beilei, Nos. 1*, 2*

30 μg/mL

Country

DPPH (IC50, mg/mL)

China

Cultivar/ Genotype

Lonicera caerulea var. edulis L. caerulea

Haskap Subspecies

595.57– 2,708.6 μM vit C equivalent/100 g fw

PSC

43.84–495.01

LcB0.28–0.60 nm LcL0.38–0.81 nm

FRAP (μM TE/g)

368– 497 μM TE/g

0.19– 200.21

80 μg/mL

ABTS (μM TE/g)

TABLE 14.3 Antioxidant Activities of Haskap: Chemical, In Vitro, and In Vivo Studies

198.19 

52–68

ORAC (μM TE/g DM) Hydroxyl 0.03 mg/mL

Superoxide 1.43 mg/mL

17.23–24.83

CV (Q1000, μC)

References

(Continued)

Y. Wang, J. Zhu et al. (2016) Liu et al. (2016)

Zhao et al. (2015)

Hyun et al. (2015)

Zhao et al. (2011)

HepG2 Cells – 14.75 μM QE/100 mg DM HepG2 Wang et al. 7.88–43.02 μM (2018) quercetin/100 μg sample

CAA

Haskap Berries: Phytochemical Constituents 261

S. Liu, L. You et al. (2018)

References

Lee et al. (2018)

ABTS, 2,2′-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid); CAA, cellular antioxidant activity; CAT, catalase; CV, cyclic voltammetry; DPPH, 1,1-diphenyl-2-picrylhydrazyl; EDA, electron-donating ability; FRAP, ferric-reducing antioxidant power; Gclc, glutamate-cysteine ligase catalytic subunit; HBC, honeyberry China; HBK, honeyberry Korea; HO-1, heme oxygenase-1; LcB, Lonicera caerulea branches; LcL, Lonicera caerulea leaves; MDA, malondialdehyde; Nqo1, NADPH:quinone oxidoreductase-1; ORAC, oxygen radical absorbance capacity; PSC, peroxyl radical scavenging capacity; ROS, reactive oxygen species; SD, Sprague–Dawley; SOD, superoxide dismutase; T-AOC, total antioxidant capacity; TMAO, trimeth­ ylamine N-oxide.

Haskap Cultivar/

Subspecies Country Genotype DPPH(%) Model/Dose Outcome L. caerulea L. South Korea HBK DPPH – 300 In vitro HepG2 Ĺ HO-1, Gclc, Nqo1 expression HBC μg/mL cells Ĺ SOD and CAT activities

HBK (3–300 μg/mL)

– 86.1%

HBC

– 92.96% L. caerulea China In vitro – Caco-2 Improved antioxidant status of colon and plasma berry cells (40 and 80 Ļ ROS, MDA levels polyphenol μg/mL) Ĺ T-AOC (LCBP) In vivo – SD rats Ļ TMAO levels in serum 80% acidic

ethanol (75, 150,

and 300 mg/kg),

12 weeks

TABLE 14.3 (Continued) Antioxidant Activities of Haskap: Chemical, In Vitro, and In Vivo Studies

262 Asian Berries: Health Benefits

Haskap Berries: Phytochemical Constituents

263

line. At 300 μg/mL, HBK and HBC displayed 86.1% and 92.96% DPPH scavenging activity, respec­ tively. In addition, the effect on Nrf2-dependent antioxidant genes such as glutamate cysteine ligase catalytic subunit (Gclc), NAD(P)H dehydrogenase [quinone] 1 (Nqo1), and heme oxygenase-1 (HO­ 1) was evaluated. In addition, HBK and HBC supplementation led to increasing mRNA expressions by 1.4, 1.8-fold, for HO-1, and 2.0, 2.5-fold for Nqo1, respectively. Moreover, higher expression of Gclc was observed for HBC. On the other hand, superoxide dismutase (SOD) and catalase (CAT) activities were 25.7 and 506.5 μm/mg protein for HBK as well as 33.7 and 802.2 μm/mg protein for HBC, respectively (Lee et al., 2018). Recent studies by Wang et al. described the extracellular and intracellular antioxidant activities of seven blue honeysuckle varieties using PSC, ABTS, DPPH, and CAA assays (Table 14.2). The scavenging potential for PSC, DPPH, ABTS, and CAA ranged from 595.57 to 2,708.6 μM vitamin C equivalent/100 g fw, 3.0–4.5 μg/mL, 368–497 μM TE/g, and 7.88–43.02 μM quercetin equivalent/100 μg of a sample, respectively (Wang et al., 2018). In a recent investigation, the antioxidant potential was evaluated using L. caerulea berry poly­ phenols (LCBPs) against Caco2 cells and Sprague–Dawley (SD) rats. LCBP-H (cholesterol micellar solution+80 μg/mL LCBP) significantly decreased ROS and malondialdehyde (MDA) levels, and increased total antioxidant capacity (T-AOC) levels in Caco-2 cells, compared with HC control. In rats, LCBP (75, 150, and 300 mg/kg) supplementation decreased MDA and ROS and increased T-AOC and TMAO levels (S. Liu, L. You et al., 2018).

14.8.2

ANTI-DIABETIC ACTIVITY

It has been reported that the inhibition of α-glucosidase and α-amylase enzymes contributes to type 2 diabetes (T2D) inhibition. A study assessed the postprandial inhibitory effect of the anthocyanin-rich haskap extract on hyperglycemic SD rats. After 30 min of haskap supplementa­ tion, rats were consecutively fed with sucrose (2g/kg body weight), resulting in inhibitory activity against sucrase. Furthermore, the administration of 1.5% or 3.0% of haskap for a long-term (4-week) in high-fat diet (HFD)–induced diabetic rats resulted in the lower blood glucose levels (Takahashi et al., 2014). Studies by Lee et al. suggest the effect of haskap and C3G (at a concentration of 1, 2.5, 5, and 10 μg/mL) on insulin release using INS-1 cell line. Both stimulated insulin release, while C3G increased insulin receptor (IR) phosphorylation and decreased PI3K and IRS-1 protein expres­ sion in the presence of glucose (Lee et al., 2016). Dietary polyphenols are active antidiabetic agents against α-glucosidase. In this line, Hyun et al. described the α-glucosidase inhibitory activity of L. caerulea branches (LcB) and leaf (LcL) extract. Notably, LcB showed strong inhibition (IC value 7.2 μg/mL) than LcL (Hyun et al., 2015). Recently, Zhang et al. used purified anthocyanins (9,913.40 μg, 63.16% pure) and anthocyanidins (1,320.77 μg, 58.04% pure) from 1 g of blue honeysuckle (freeze-dried) and evaluated their anti-α-glucosidase activity. Both anthocyanins and anthocyani­ dins inhibited α-glucosidase activity (as IC 50 value) with 0.188 and 0.025 mg/mL, respectively (Zhang et al., 2019). Our group described diabetes alleviating effects of BHBe at 400, 200, and 100 mg/kg on HFD-induced imprinting control region (ICR) mice. After 84 days of supplementation, BHBe resulted in lower levels of blood glucose, insulin, blood urea nitrogen (BUN), blood hemo­ globin A1c (HbA1c), and creatinine in a dose-dependent manner. In addition, BHBe decreased insulin- and glucagon-producing cells, as well as insulin/glucagon cell ratios (Sharma et al., 2019).

14.8.3

ANTIOBESITY ACTIVITY

The antiobesity effect of honeysuckle anthocyanins (HAs) was described in HFD-fed C57BL/6 mice. HA consumption for 8-week at 100 and 200 mg/kg dose lowered body weight gain by 17.1% and 24.1%, respectively. A reduction in serum and hepatic lipid levels improved liver functions, and significantly attenuated leptin and insulin levels, as well as increased adiponectin levels, were observed (Wu et al., 2013). Another study described the effect of blue honeyberry extract (BH – 400, 200, and 100 mg/kg) treatment (12-week) using HFD-fed ICR mouse model. The weight gain

264

Asian Berries: Health Benefits

was suppressed, and adipocyte histopathology was improved. The mRNA expressions of uncou­ pling protein 2 (UCP2) and adiponectin were upregulated, whereas C/ EBPα, C/EBPβ, SREBP1, and leptin levels were decreased. The effect was related to increasing 5‫ މ‬AMP-activated protein kinase (AMPK) phosphorylation (Chun et  al., 2018). In a similar study, BHe supplementation at 400, 200, and 100 mg/ kg for 84 days improved hepatic obesity in HFD-fed ICR mice by increasing adiponectin and adipose tissue UCP2 expression and downregulating CCAAT/enhancer-binding protein alpha/ beta (C/ EBPα/β), sterol regulatory element–binding protein-1c (SREBP-1c), and leptin levels (Kim et  al., 2018). Recently, our group shed lights on the antiadipogenic effect of L. caerulea extract (LCE; 0.25–1 mg/mL) using 3T3-L1 cells and mouse adipose–derived stem cells (MADSCs). Downregulated expressions of peroxisome proliferator–activated receptor gamma (PPARγ), C/EBPα, and SREBP-1c in both models advocated antiobesity effect (Park, Lee et al., 2019).

14.8.4 ANTITUMOR/RADIOPROTECTIVE ACTIVITY In a recent investigation, anthocyanin (ABL-2) from Beilei cultivar inhibited growth and promoted apoptosis along with the arrest of G2/M phase in the SMMC-7721 cell line. Also, in an in vivo attempt, survival of H22 tumor-bearing Kunming mice was improved by increasing antioxidase activity, by modulating cytokines (interferon-gamma [IFN-γ], interleukin-2 [IL-2], and tumor necrosis factor-alpha [TNF-α]) levels, and by decreasing lipid peroxidation (Table 14.4) (Zhou et al., 2018). Furthermore, the radio-protecting activity against the damaging effect of ionizing radiations using ICR mice has been elucidated. The animals were irradiated with 5 Gy whole-body 60 Coγ radiation. Haskap significantly upregulated SOD, glutathione peroxidase (GPx), and glutathione peroxidase (GSH) levels, whereas MDA level was reduced compared with control (Zhao et  al., 2012).

14.8.5

ANTI-INFLAMMATORY ACTIVITY

High production of proinflammatory mediators such as IL-6, TNF-α, IFN-γ, nitric oxide (NO), nitric oxide synthase (NOS), prostaglandin E2 (PGE2), and cyclooxygenase-2 (COX-2) frequently leads to the development of many chronic inflammatory diseases. Jin et al. (2006) described the anti-inflammatory properties of haskap fruit extracts on eye uveitis (uvea inflammation) using RAW264.7 murine macrophage cell line (in vitro) and lipopolysaccharide (LPS)-induced ocular inflammation using a Lewis rat model (in vivo). Haskap attenuated inflammation by reducing NO, TNF-α, and PGE2 levels and by inhibiting the activation of nuclear factor (NF)-κB dependent signaling pathway (Jin et al., 2006). Studies by Wu et al. (2015) investigated the anti-inflammatory effects of ethanolic blue honeysuckle extract (BHE) using adjuvant-induced arthritis SD rat model. BHE significantly reduced the levels of TNF-α, IL-6, and NO as well as an induced nitric oxide synthase (iNOS) and COX-2 enzyme (Wu et al., 2015). Recently, Wu et al. (2017) described the effect of haskap using LPS-induced paw edema in ICR mice and macrophage cell (RAW264.7) model. The haskap showed its effect by dually modulat­ ing inflammatory and antioxidant mediators. The C3G and EC-rich haskap extract attenuated the production of multiple inflammatory cytokines such as monocyte chemotactic protein-1 (MCP-1), TNF-α, IL-6, IL-4, IL-10, macrophage inflammatory protein (MIP)-1α, and IL-12 (p-70) by down­ regulating mitogen-activated protein kinase (MAPK) pathway. Correspondingly, enhanced expres­ sions were observed for nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and manganese-dependent superoxide dismutase (MnSOD) (Wu, Yano, Chen et al., 2017). Lee et al. (2018) demonstrated the related anti-inflammatory potential of HBK and HBC using RAW264.7 model. Decreased NO pro­ duction was reported in both HBK and HBC at 300 μg/mL (Lee et al., 2018).

Haskap Berries: Phytochemical Constituents

14.8.6

265

HEPATOPROTECTIVE ACTIVITY

NAFLD is a broader term describing the chronic liver disease, ranging from a simple form of hepatic steatosis (accumulation of lipids in the liver) to a most severe form known as nonalcoholic steatohepatitis (NASH), which is characterized by liver cell injury (hepatocellular ballooning) and inflammation (Younossi et al., 2018). Furthermore, it may lead to severe conditions such as cirrho­ sis, hepatocellular carcinoma (HCC), diabetes, and heart diseases (Samuel  & Shulman, 2018; Tilg et al., 2017). The oxidative stress–mediated lipid peroxidation is an important cause for the progression of NAFLD (Rolo et al., 2012). In an investigation, BHE (0.5% or 1%) caused a reduction in hepatic fat deposition and obesity in HFD-induced C57BL/6N mice. BHE attenuated glucose metabolism by reducing (dose-dependent) insulin, glucose, and homeostatic model assessment – insulin resistance (HOMA-IR) levels. Also, liver antioxidant activity was improved by upregulating Nrf2-mediated pathway (M. Liu et al., 2018). Wu et al. described that the supplementation of LCBPs (0.5% and 1%) for 45 days led to a significant reduction of lipid accumulation, lipid peroxidation, inflamma­ tory cell infiltration, and insulin resistance by suppressing proinflammatory cytokines production (Table 14.4), increasing the expression of Nrf2 and MnSOD, and downregulating the expression of forkhead box protein O1 (FoxO1) and HO-1 using HFD-fed C57BL/6N mice induced with carbon tetrachloride (CCl4) (Wu, Yano, Hisanaga et al., 2017). Our research group described the consump­ tion of three haskap extracts (BHe, BHw, and BHj) by CCl4-induced ICR mice that showed a posi­ tive liver-protecting effect. BHe (200 mg/kg) displayed a similar effect to silymarin (a flavonoid from Silybum marianum herb [milk thistle]; 100 mg/kg) by activating hepatic antioxidant defense system (Lee et al., 2019). In another study, LCBPs (75, 150, or 300 mg/kg) were capable of reducing total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TGs) as well as increasing high-density lipoprotein cholesterol (HDL-C) levels in hypercholesterolemic SD rats. The extract reduced lipid accumulation by downregulating the expressions of miR-33, miR-122, fatty acid synthase (FAS), SREBP-1c, SREBP2, and HMG-CoA reductase (HMGCR), whereas ATP-binding cassette A1 (ABCA1) and Niemann-Pick C1 (NPC1) (mRNA and protein) were upreg­ ulated. In addition, an upregulated expression was reported for PPARγ, cholesterol-7α-hydroxylase (CYP7A1), liver X receptor alpha (LXRα), and low-density lipoprotein receptor (LDLR), thereby improving cholesterol metabolism and reducing cholesterol accumulation (S. Liu, Z. Wu et al., 2018). In another study, LCBPs were capable of modulating gut bacteria in a fatty liver of HFD-induced C57BL/6N mice by downregulating IL-6, IL-2, TNF-α, and MCP-1 expressions (Wu et al., 2018). In addition, LCBPs decreased endotoxin levels in serum and liver homogenates. The endotoxin produced by gut bacteria might contribute to developing NAFLD (Kessoku et  al., 2017). These findings suggested that LCBPs modulated the gut bacteria, especially the ratio of Firmicutes to Bacteroidetes as analyzed by 16S rRNA gene sequencing of fecal microbiota (Wu et al., 2018). Zhou et al. (2016) showed that chlorogenic acid from haskap (variety edulis) modulated hepatic lipid dysregulation by reducing lipid accumulation, increasing fatty acid oxidation, and activat­ ing AMPK in endotoxin-induced female SD rats (Zhou et  al., 2016). It has been reported that AMPK activation protects against NAFLD and diet-induced obesity (Garcia et al., 2019). Studies by Kim et al. stated that consumption of blue honeyberry extract (BHe; 400 mg/kg for 84 days) by HFD-induced ICR mouse improved hyperlipidemia, hepatic steatosis (NAFLD), and obesity, simi­ lar to metformin (an AMPK activator) treatment. BHe upregulated SOD, GSH, and CAT levels and hepatic GK, G6Pase, and phosphoenolpyruvate carboxykinase (PEPCK) activities and decreased serum aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase(ALP), γ-glutamyltransferase (GGT), lactate dehydrogenase (LDH), and MDA levels. In addition, mRNA expressions of hepatic C/EBPα, C/EBPβ, SREBP1c, acetyl-CoA carboxylase1 (ACC1), and leptin were decreased, whereas expressions of AMPKα1, AMPKα2, adiponectin, and UCP2 were increased (Kim et al., 2018). Chun et al. (2018) reported similar observations in ICR mice by Korean HB (200 mg/kg) supplementation for 12 weeks. HB reduced lipid peroxidation by improving hepatic

Antidiabetic

Antiobesity

1

2

S. No

Health-

Promoting Activity

Methanolic extract

Aqueous

Aqueous

Blue honeysuckle (BH) extract

Blue honeysuckle extract (BHe)

Aqueous

Anthocyanins Anthocyanidins

Methanolic leaves (LcL) and branches (LcB)

Ethanolic

Extract

Honeysuckle anthocyanins (HA)

Blue honeysuckle fruits Blue honeyberry (BHB)

L. caerulea fruit L. caerulea extract and C3G isolated L. caerulea

Type

TABLE 14.4 Health-Promoting Activities of Haskap

HFD-induced diabetic SPF ICR mice; 400, 200 and 100 mg/kg; 84 days

HFD-induced C57BL/6 mice; 50, 100, or 200 mg/kg; 8 weeks

HFD-fed ICR mice; 400, 200, and 100 mg/kg; 12 weeks

HFD-fed ICR mice; 400, 200 and 100 mg/kg; 84 days

In vivo

In vivo

In vivo

Type 2 diabetes enzyme inhibition assay

Sucrose (2 g/kg BW) + HFD-fed SD rats; 300 mg/kg BW; 4 weeks INS-1 cells (Glucose-sensitive pancreatic cell line); 1, 2.5, 5 and 10 μg/mL Type 2 diabetes enzyme inhibition assay

Model/Dose/Duration

In vivo

In vitro

In vivo

Study

Design Outcomes

Inhibitory activity against α-glucosidase Anthocyanins (0.188 mg/mL) Anthocyanidins (0.025 mg/mL) Ļ blood glucose, insulin, HbA1c, BUN, and creatinine levels Ļ insulin-immunoreactive cells and glucagon­ immunoreactive cells, and insulin/glucagon ratio Ļ weight gain, serum/liver lipid profiles, leptin, and insulin Ĺ adiponectin level Improved hepatic function Ļ weight gain Ĺ UCP2 and adiponectin mRNA expressions Ļ C/EBPα, C/EBPβ, SREBP1, and leptin levels Ĺ AMPK activation Improved adipocyte histopathology Improved hepatic obesity Ļ C/EBPα, C/EBPβ, SREBP1c, and leptin Ĺ adiponectin and adipose tissue UCP2 expression

Insulin secretion activated Ĺ IR phosphorylation Ļ IRS-1, PI3K enzyme expression Strong inhibitory activity of LcB against α-glucosidase with IC value 7.2 μg/mL LcL least activity with IC value >200 μg/mL

Ļ blood glucose levels

References

(Continued)

Kim et al. (2018)

Chun et al. (2018)

Wu et al. (2013)

Sharma et al. (2019)

Zhang et al. (2019)

Hyun et al. (2015)

Takahashi et al. (2014) Lee et al. (2016)

266 Asian Berries: Health Benefits

5

Antiinfammatory

4

Haskap (LCBP) 75% ethanol

Ethanolic

In vivo

In vitro

In vitro In vivo

In vivo

In vivo

L. caerulea extract (BHE)

Lewis rats with LPS-induced endotoxin-induced uveitis (EIU); 1, 10, or 100 mg Adjuvant-induced arthritis in SD rats; 0, 75, 150, 300 μg/g bw; 28 days RAW264.7; 75, 150, or 300 μg/mL LPS-induced paw edema ICR mice; 300 mg/kg bw; 4 days RAW264.7; 3, 10, 30, 100, and 300 μg/mL HFD-induced C57BL/6N mouse; 0.5% or 1%; 45 days

In vivo

Ethanol/water/ hydrochloric acid (60:40:0.1)

Ethanol

ICR mice irradiated with 5 Gy whole-body 60 Coγ radiation, 50–200 mg/kg/bw/day; 14 days

In vitro In vivo

70% ethanol

Outcomes

References

Glucose, insulin, HOMA-IR, and lipid peroxidation Nrf2-mediated pathway

MCP-1, MIP-1α, TNF-α, IL-6, IL-4, IL-10, and IL-12(p-70) levels Nrf2 and MnSOD NO production at 300 μg/mL

infammatory cell infltration TNF-α, NO, PGE2 NF-κB activation TNF-α, IL-6, iNOS, and COX-2

Oxidative damage in liver SOD, GPx, and glutathione levels MDA level

(Continued)

Wu, Yano, Chen et al. (2017) Lee et al. (2018) M. Liu et al. (2018)

Wu et al. (2015)

Jin et al. (2006)

Zhao et al. (2012)

Inhibited adipogenesis Park, Lee PPARγ, C/EBPα, and SREBP1 in 3T3-L1 and et al. (2019) MADSC SMMC-772; 0.2 mg/mL Inhibited cell growth, induced apoptosis Zhou et al. H22 (4 × 105 cells in 0.2 ml PBS) SOD and GSH-Px (2018) transplanted Kunming mice, 50–200 MDA levels mg/kg bw/day; 15 days TNF-α, IL-6, and IFN-γ expression

Model/Dose/Duration 3T3-L1 cell line; MADSC from C57BL/6 mouse; 0.25-1 mg/mL

L. caerulea Beilei fruit Purifed anthocyanin (ABL-2) Anthocyanin from L. caerulea var. edulis (ALC) L. caerulea extract (BHE)

In vitro

Study Design

Aqueous

Extract

L. caerulea extract (LCE)

Type

Honeyberry Aqueous (HBK, HBC) Hepatoprotecting Blue Aqueous ethanol activity honeysuckle (75%) extract (BHE) 

Antitumor

3

S. No

HealthPromoting Activity

TABLE 14.4 (Continued) Health-Promoting Activities of Haskap

Haskap Berries: Phytochemical Constituents 267

S. No

Health-

Promoting Activity

BHe (BH solution extract), BHw (hot water), and BHj (juice) 80% acidic ethanol

Blue honeysuckle (BH)

L. caerulea var. Sterile saline edulis chlorogenic acid

Aqueous ethanol (75%)

75% aqueous ethanol

L. caerulea L. polyphenols

L. caerulea berry polyphenols (LCBP) extract LCBP

Extract

Type

TABLE 14.4 (Continued) Health-Promoting Activities of Haskap

In vivo

In vivo

In vivo

In vivo

In vivo

Study

Design Outcomes

Ļ lipid accumulation and lipid peroxidation Ļ G‐CSF, IL‐1α, IL‐1β, IL‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐10, MIP 1β, KC, TNF‐α, MCP‐1, IFN‐γ, IL‐12(p70), IL‐13, eotaxin, MIP‐1α, IL‐17, RANTES, and granulocyte– macrophage colony‐stimulating factor Ĺ Nrf2 and HO-1 levels Ļ FoxO1 and HO-1 CCl4‐induced acute liver damage Ĺ CAT, SOD and GSH level ICR mice; 200 mg/kg; 7 days Ļ AST, ALT, and MDA level, cleaved caspase‐3, NT, PARP, and 4‐HNE Strongest effects by BHe > BHw > BHj SD rats; 75, 150, or 300 mg/kg; Ļ hepatic TC, TG, and LDL-C levels, and 12 weeks miR-33, miR-122, FAS, SREBP-1c, SREBP2, and HMGCR expressions Ĺ HDL-C, PPARγ, CYP7A1, LXRα, and LDLR levels HFD-induced C57BL/6N mice; 0.5% Ļ IL-2, IL-6, MCP-1, TNF-α expression and or 1%; 45 days endotoxin production Gut bacterial ratio modulated Endotoxin-injected SD rats with lipid Ļ serum TG, FFA, and hepatic TGs, cholesterol metabolic disorder; 60 mg CGA/kg Ļ FAS and ACC activities bw; 28 days Ĺ phosphorylated AMPK, CPT-1, and β-oxidation

Model/Dose/Duration  HFD+ CCl4 induced C57BL/6N mice, 0.5% or 1%; 45 days

References

(Continued)

Zhou et al. (2016)

Wu et al. (2018)

S. Liu, Z. Wu et al. (2018)

Lee et al. (2019)

Wu, Yano, Hisanaga et al. (2017)

268 Asian Berries: Health Benefits

S. No

Health-

Promoting

Activity

LPS-induced chronic liver inflammation in SD rats; 50, 100, and 200 mg/kg; 4 weeks

LPS-induced inflammation in BRL-3A cells; 300 μg/mL; 4 h daily for two consecutive days

LPS-induced toxicity in BRL-3A rat liver cells; 100, 300 and 500 μg/ mL; 4 h daily

In vitro

In vitro

L. Acidified methanol caerulea berry (0.1%) Digested and undigested extract

L. caerulea

Acidified methanol (0.1%)

L. caerulea berry extract

In vivo

Aqueous

Blue honeysuckle (BH) extract

Acidified methanol (0.1%)

Model/Dose/Duration

HFD-fed ICR mice; 400, 200 and 100 mg/kg; 12 weeks

Aqueous

Blue honeysuckle extract (BHe)

In vivo

Study

Design

HFD-fed ICR mice; 400, 200 and 100 mg/kg; 84 days

Extract In vivo

Type

TABLE 14.4 (Continued) Health-Promoting Activities of Haskap

Outcomes

Ĺ CAT, SOD and T-AOC, AChE, IL-10, PARP, Bcl-2 level Ļ AST, ALT, TNF-α, NF-κB, caspase 3, cleaved caspase 3, cleaved PARP, and BAX levels

Improved hyperlipidemia, NAFLD, and hepatic obesity Ļ serum AST, ALT, ALP, GGT, LDH, MDA, ACC1 levels Ļ G6Pase and PEPCK activities Ĺ SOD, GSH, CAT, hepatic GK, and AMPKα1 and AMPKα2 levels Ļ hepatocyte hypertrophies Ļ AST, ALT, ALP, LDH, GGT, BUN, creatinine, MDA levels and G6Pase and PEPCK activities Ĺ SOD, GSH, CAT, and hepatic glucosidase activities Ĺ AMPK activation Ļ TLR2, TLR4 expression, IL-6, CRP, AST, and ALT levels Ĺ increased GSH level MAPK signaling inhibited Cell cycle normalized Ļ ROS production and liver peroxidation Ļ IL-1β, IL-6, AST, and ALT levels Ĺ GSH and AChE levels Normalized ATP synthetase, COX, and SDH activities

References

(Continued)

Wang et al. (2017)

Y. Wang, B. Li, Y. Ma et al. (2016)

Y. Wang, B. Li, J. Zhu et al. (2016)

Chun et al. (2018)

Kim et al. (2018)

Haskap Berries: Phytochemical Constituents 269

Cardiovascular protection

Antimicrobial activity

6.

7

S. No

Health-

Promoting Activity

L. caerulea

L. caerulea berry polyphenol (LCBP) LCBP In vitro

80% Acidic ethanol

Leaves (LcL) and branches (LcB) (methanolic extract)

In vitro In vivo

80% Acidic ethanol

In vivo

Ethanolic

In vitro In vivo

In vivo

75% ethanol

L. caerulea L. purified component (PLE) Haskap (var. edulis) L. caerulea fruit

In vitro In vivo

Study

Design

Alcoholic

25% ethanol

Extract

Honeyberry extract

Type

TABLE 14.4 (Continued) Health-Promoting Activities of Haskap

Model/Dose/Duration

Bacillus atrophaeus, Kocuria rhizophila, Micrococcus luteus, Staphylococcus epidermidis, Bacillus subtilis subsp. spizizenii, Klebsiella pneumoniae, Enterobacter cloacae, Salmonella enterica subsp. enterica, Pseudomonas aeruginosa

RAW264.7, 80 μg/mL

FFA-induced HepG2 cells;250, 500, and 1000 μg/mL HFD-fed ICR mice; 0.5%–1%; 6 weeks AML-12 cells; 31.25, 62.5, 125 μg /mL Ethanol-induced ALD in C57BL/6 mice; 80, 150 mg/kg; 11 days HFD-fed Wistar rats; 0.75 g/kg and 0.15 g/kg; 28 days Corn oil emulsion (12.5 mL/kg) HFD-fed SD rats; 300 mg/kg BW anthocyanins; 4 weeks Caco-2 cells, 80 μg/mL SD rats; 75, 150, and 300 mg/kg; 12 weeks

References

Ļ TC, TG, LDL-C, and lipoprotein levels Ĺ HDL-C level Ļ NPC1L1, ACAT2, and MTP Ĺ ABCG5 and ABCG8 Ļ intracellular cholesterol levels Ĺ SIRT1 and ABCA1 expression Ļ SREBP2 and miR-33 expression Inhibited Foam cell formation LcB most active against gram-positive bacteria LcL comparatively no or lower activity

Ļ AST, ALT, IL-1β, IL-6, TNF-α, and SREBP1 level Ĺ PPARα, SIRT1, AMPK phosphorylation, and F4/80 level inhibited caspase-1 Ļ TG and TC levels Ĺ HDL level Ļ postprandial serum TAG level

(Continued)

Hyun et al. (2015)

Liu et al. (2019)

S. Liu, Z. Wu et al. (2018)

Guang et al. (2004) Takahashi et al. (2014)

Zuo et al. (2019)

Ļ PPARγ, SREBP-1c, C/EBPα, FAS expressions Park, Yoo et al. (2019) Ĺ CPT-1 and PPARα expressions Ĺ ACC and AMPK signaling

Outcomes

270 Asian Berries: Health Benefits

Pulmonary protective activity

Extract

Acidified ethanolic

Acidified ethanolic

Blue honeysuckle extract (BHE)

BHE

L. caerulea var. Methanolic extract of emphyllocalyx stem, fruit (LCE) LCE Methanolic extract of stem, fruit, and leaf

Type

In vivo

In vitro In vivo

Study Design

IFN-γ, TNF‐α production differentiation of hematopoietic stem cells

Antibacterial and antibiofilm effects

Outcomes

Inhibited inflammation of lung and macrophage infiltration IL-1β, IL-6, TNF-α, MCP-1 levels NF-κB and iNOS expression Nrf2 and HO-1 expression SP-induced lung fibrosis in IL-4, Gata 3, and iNOS expressions C57BL/6; 100, 200, and 400 mg/kg; MAPK activation blocked 56 days Nrf2 and HO-1 expression

MH-S cell line; – 25, 50, 75, 150, 300, and 500 μg/mL SP-induced C57BL/6 mouse; 100, 200, and 400 mg/kg; 7 days

Streptococcus pyogenes 1529 strain injected to female Slc:ICR mice

Streptococcus pyogenes

Model/Dose/Duration

Zhao et al. (2019)

Minami, Takase et al. (2019) Minami, Nakamura et al. (2019) Zhao et al. (2018)

References

4‐HNE, hydroxynonenal; ABCA1, ATP-binding cassette transporter A1; ABCG5/8, ATP-binding cassette transporter subfamily G members 5 and 8; ACAT2, acyl coenzyme A: cholesterol acyltransferase 2; ACC, acetyl-CoA carboxylase; AChE, acetylcholine esterase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AMPK, 5, AMP-activated protein kinase; AST, serum aspartate transaminase; BAX, B cell lymphoma 2 (BCL-2)–associated X protein; Bcl2, B-cell lymphoma-2; BUN, blood urea nitrogen; C/EBPα/β, CCAAT/enhancer-binding protein-alpha/beta; CCl4, carbon tetrachloride; COX, cytochrome c oxidase; COX-2, cyclooxygenase-2; CPT1, carnitine palmitoyltransferase; CRP, C-reactive protein; CYP7A1, cholesterol-7α-hydroxylase; FAS, fatty acid synthase; FFA, free fatty acid; FoxO1, forkhead box protein O1; GGT, γ-glutamyltransferase; GPx, glutathione peroxidase; GSH-Px, glutathione peroxidase; HbA1c, blood hemoglobin A1c; HBC, honeyberry China; HBK, honeyberry Korea; HDL-C, high-density lipoprotein cholesterol; HFD, high-fat diet; HMGCR, HMG-CoA reductase; HO-1, heme oxygenase-1; HOMA-IR, homeostatic model assessment – insulin resistance; ICR, imprinting control region; IFN-γ, interferon-gamma; IL, interleukin; IR, insulin receptor; LDH, lactate dehydrogenase; LDL-C, low-density lipoprotein cholesterol; LDLR, low-density lipoprotein receptor; LXRα, liver X receptor-alpha; MADSCs, mouse adipose-derived stem cells; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemotactic protein-1; MDA, malondialdehyde; MIP-1α, macrophage inflammatory protein-1 alpha; MnSOD, manganese-dependent superoxide dismutase; MTP, microsomal triacylglycerol transport protein; NF, nuclear factor; NF-κB, NF-kappa B; NO, nitric oxide; NOS, NO synthase; NPC1L1, Niemann-Pick C1-like 1; NQO1, NADPH:quinone oxidoreductase-1; Nrf2, nuclear factor (erythroid-derived 2)–like 2; NT, nitrotyrosine; PARP-1, poly-(ADP-ribose) polymerase-1; PEPCK, phosphoenolpyruvate carboxykinase; PGE2, prostaglandin E2; PPARα, peroxisome proliferator–activated receptor-alpha; PPARγ, peroxisome proliferator–activated receptor-gamma; ROS, reactive oxygen species; SD, Sprague–Dawley; SDH, succinate dehydrogenase; SIRT1, Sirtuin 1; SOD, superoxide dismutase; SREBP-1c, sterol regulatory element–binding protein-1c; SREBP2, sterol regulatory element–binding protein 2; TAG, triacylglycerol; T-AOC, total antioxidant capacity; TC, total cholesterol; TG, triglyceride; TLR, Toll-like receptor; TNF-α, tumor necrosis factor-alpha; UCP2, uncoupling protein 2.

8

S. No

HealthPromoting Activity

TABLE 14.4 (Continued) Health-Promoting Activities of Haskap

Haskap Berries: Phytochemical Constituents 271

272

Asian Berries: Health Benefits

lipid metabolism, serum lipid contents, and hepatic enzymes activities. Authors reported compa­ rable effects for both HB treatment at 200 mg/kg and metformin (250 mg/kg) (Chun et al., 2018). In another research, L. caerulea berry extracts (LCBEs; 50, 100, and 200 mg/kg) were capable of pre­ venting hepatic inflammation via reduced expressions of C-reactive protein (CRP), IL-6, and inhibi­ tion of toll-like receptor (TLR) as well as MPAK signaling in LPS-induced chronic liver of SD rats. In addition, LCBEs also normalized hepatocytes’ cell cycle (Y. Wang, B. Li, J. Zhu et al., 2016). Y. Wang, B. Li, Y. Ma, et al. (2016) described the attenuating effects of haskap on LPS-induced liver inflammation in BRL-3A rat liver cells by inhibiting oxidative stress and maintaining ATP synthetase, succinate dehydrogenase (SDH), and cytochrome c oxidase (COX) activities and modulated the liver function by preventing an increase in AST and ALT levels and decrease in acetylcholine esterase (AChE) and GSH levels. In addition, expressions of IL-1β and IL-6 were significantly attenuated (Y. Wang, B. Li, Y. Ma et  al., 2016). Likewise, LCBE showed health-promoting activities against LPS-induced BRL-3A cells. The extract reversed the reduc­ tion of CAT, SOD, T-AOC, AChE, IL-10, poly-(ADP-ribose) polymerase (PARP) levels, and B cell lymphoma-2 (Bcl-2), whereas levels of AST, ALT, TNF-α, NF-κB, caspase 3, cleaved caspase 3, cleaved PARP, and BCL-2-associated X protein (BAX) were decreased (Wang et al., 2017). Our group has recently investigated the inhibitory effect of honeyberry extract (HBE) on NAFLD in free fatty acid–induced HepG2 cells and in HFD-fed ICR mice by downregulat­ ing expressions of PPARγ, SREBP-1c, C/EBPα, and FAS (Park, Yoo et al., 2019). In addition, expression of fatty acid oxidation genes, including carnitine palmitoyltransferase (CPT-1) and PPARα, was upregulated. The suggested mechanism increased AMPK and acetyl-CoA carbox­ ylase (ACC) phosphorylation and thus was capable of modulating AMPK and ACC signaling in both models (Park, Yoo et al., 2019). In a recent study, a purified component (PLE) from L. cae­ rulea was investigated on alcoholic hepatosteatosis using alcohol-induced C57BL/6 mice and AML12 cells. PLE mainly consisted of C3G (81%) and peonidin-3-rutinoside (8.8%) and 10.2% other components. PLE downregulated IL-1β, TNF-α, and TL-6 levels by reducing F4/80 level and hindering caspase-1 activity. In addition, the levels of PPARα and Sirtuin 1 (SIRT1) were upregulated, whereas SREBP1c level was suppressed by AMPK phosphorylation. In AML12 cells, PLE phosphorylated AMPK and suppressed the SREBP1c and F4/80 levels (Zuo et al., 2019).

14.8.7

CARDIOVASCULAR DISEASE PROTECTIVE ACTIVITY

Cardiovascular diseases (CVDs) have an increased mortality rate globally. It has been suggested that intake of anthocyanin-rich fruits can reduce the risk of CVDs. As far as haskap, many studies have shown positive activities. For example, in one study, alcoholic extract of L. edulis resulted in lower blood lipid levels in hyperlipidemic Wistar rats (Guang et al., 2004). Haskap (300 mg/kg B.W. anthocyanin) supplementation decreased postprandial serum triacylglycerol ( TAG) levels in SD rats. Rats were orally administered with corn oil emulsion (short-term) and high-fructose diet (long-term) (Takahashi et  al., 2014). In another study, the effect of LCBPs (75, 150, and 300 mg/kg) on cholesterol-lowering using in vitro (Caco-2 cells) and in vivo (high-cholesterol diet (HCD)-fed SD rats) models. LCBP treatment (150 and 300 mg/kg) sig­ nificantly reduced the TC, TG, and LDL-C levels and upregulated HDL-C level in rats. The cholesterol-lowering activity was attributed to downregulating and upregulating the expressions of Niemann-Pick C1-like 1 (NPC1L1), microsomal triacylglycerol transport protein (MTP), acyl-coenzyme A: cholesterol acyltransferase 2 (ACAT2), and ATP-binding cassette transporter subfamily G members 5 and 8 (ABCG5/8), respectively, at transcription and translation levels (S. Liu, L. You et al., 2018). LCBP-H (cholesterol micellar solution + 80 μg/mL LCBP) signifi­ cantly decreased ROS and MDA levels and increased total antioxidant capacity (T-AOC) levels in Caco-2 cells, compared with HC control. In rats, LCBP (75, 150, and 300 mg/kg) supple­ mentation decreased MDA and ROS levels and increased T-AOC and TMAO levels (S. Liu, L.

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You et al., 2018). The same group continued the study further to show the inhibitory effect of LCBPs (80 μg/mL) on ox-LDL- induced lipid accumulation in RAW264.7 cells (foam cell inhibi­ tion) by activating of SIRT1 and upregulating ATP-binding cassette transporter A1 (ABCA1; a cholesterol efflux gene) expression. They further tested three constituents (C3G, catechins, and chlorogenic acid); chlorogenic acid displayed the strongest effect in activating STRT1, leading to upregulating ABCA1 as well as downregulating miR-33 and sterol regulatory element–binding protein 2 (SREBP2) (Liu et al., 2019).

14.8.8

ANTIMICROBIAL ACTIVITY

Only a few studies reporting the antimicrobial activities of honeysuckle have been described. A study from Korea evaluated the antimicrobial activity of L. caerulea branches (LcB) and leaves (LcL) against gram-positive (Bacillus atrophaeus, Kocuria rhizophila, Micrococcus luteus, Staphylococcus epidermidis, Bacillus subtilis subsp. Spizizenii) and gram-negative (Klebsiella pneumoniae, Enterobacter cloacae, Salmonella enterica subsp. enterica, Pseudomonas aeru­ ginosa) bacterial species. Compared with LcL, LcB showed antimicrobial activity against gram-positive with maximum activity against B. subtilis ( MIC = 250 μg/mL) and Kocuria rhi­ zophila ( MIC = 250 μg/mL). Lower activity was reported for gram-negative bacteria (Hyun et al., 2015). Recently, L. caerulea var. emphyllocalyx (LCE) fruit extract has been described as an active inhibitor of Streptococcus pyogenes. In addition, LCE also showed antibiofilm effect (Minami, Takase et al., 2019). In continuing research, the authors stated the immunomodulatory effect of LCE against S. pyogenes infection in Slc: ICR mice. Out of three LCE (stem, fruit, and leaf) extracts, stems and leaves produced better results. At 500 μg/mL concentration, LCE significantly decreased the production of IFN-γ and TNF‐α in both splenocytes and mesen­ teric lymph nodal cells and enhanced the differentiation of hematopoietic stem cells (Minami, Nakamura et al., 2019).

14.8.9 PULMONARY PROTECTIVE ACTIVITY Zhao et al. (2018) reported that BHE (ethanolic; 400 mg/kg) extract inhibited silica particle (SP)– induced lung inflammation and macrophages infiltration in C57BL/6 mice. BHE supplementa­ tion alleviated IL-6, TNF-α, IL-1β, and MCP-1 levels in bronchoalveolar lavage fluid (BALF) in a dose-dependent manner. In addition, a reduction in c-Jun N-terminal kinase (JNK) and p38 phos­ phorylation, as well as NF-κB expression, was reported. Furthermore, in MH-S cell line, BHE decreased proinflammatory cytokines production, iNOS expression, and apoptosis, whereas this induced expressions of HO-1 and Nrf2 (Zhao et al., 2018). On a similar line, the ameliorating effect of BHE (100, 200, and 400 mg/kg) on SP-induced pulmonary fibrosis (C57BL/6 mice) was exhib­ ited by modifying pulmonary T helper (Th; Th1/Th17) immune responses, resulting in decreased Th2-related cytokine (IL-4) and Th2 transcription factor (Gata 3) mRNA levels. In addition, BHE stimulated Nrf2 and HO-1 levels, whereas this reduced iNOS level and thus modulated the MAPK signaling pathway (Zhao et al., 2019).

14.9 SUMMARY Considering an active link between phytochemicals and health, L. caerulea, known as haskap, is gaining more attention as a new superfood. It is a perennial plant belonging to the Caprifoliaceae family. Haskap berries are rich in phytochemicals, and recent advances investigating other plant parts have accelerated their worth on a large scale. The main health-promoting activities of this wonder plant have been attributed to the presence of anthocyanins. In this chapter, we mainly focus on the impending health benefits of haskap for the prevention and treatment of numerous chronic diseases.

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Additionally, the new phytochemical constituents have also been listed. A wealth of scientific data described in this chapter advocate that the haskap is a promising functional food. The point remains that the human intervention studies are required to confirm in vitro and experimental findings concerning the beneficial interactions among phytoconstituents of haskap and metabolic targets.

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Wang, Y., Li, B., Ma, Y., Wang, X., Zhang, X., Zhang, Q., Meng, X. (2016). Lonicera caerulea berry extract attenuates lipopolysaccharide induced inflammation in BRL-3A cells: oxidative stress, energy metabo­ lism, hepatic function. Journal of Functional Foods, 24: 1–10. Wang, Y., Li, B., Zhu, J., Zhang, Q., Zhang, X., Li, L., . . . Meng, X. (2016). Lonicera caerulea berry extract suppresses lipopolysaccharide-induced inflammation via Toll-like receptor and oxidative stress-associ­ ated mitogen-activated protein kinase signaling. Food & Function, 7(10): 4267–4277. Wang, Y., Xie, X., Ran, X., Chou, S., Jiao, X., Li, E., . . . Li, B. (2018). Comparative analysis of the polyphe­ nols profiles and the antioxidant and cytotoxicity properties of various blue honeysuckle varieties. Open Chemistry, 16(1): 637–646. Wang, Y., Zhu, J., Meng, X., Liu, S., Mu, J., Ning, C. (2016). Comparison of polyphenol, anthocyanin and anti­ oxidant capacity in four varieties of Lonicera caerulea berry extracts. Food Chemistry, 197: 522–529. Wojdyło, A., Jáuregui, P. N. N., Carbonell-Barrachina, A. A., Oszmianғski, J., Golis, T. (2013). Variability of phytochemical properties and content of bioactive compounds in Lonicera caerulea L. var. kamts­ chatica berries. Journal of Agricultural and Food Chemistry, 61(49): 12072–12084. Wu, S., He, X., Wu, X., Qin, S., He, J., Zhang, S., Hou, D.-X. (2015). Inhibitory effects of blue honeysuckle (Lonicera caerulea L.) on adjuvant-induced arthritis in rats: crosstalk of anti-inflammatory and antioxi­ dant effects. Journal of Functional Foods, 17: 514–523. Wu, S., Hu, R., Nakano, H., Chen, K., Liu, M., He, X., . . . Hou, D.-X. (2018). Modulation of gut microbiota by Lonicera caerulea L. berry polyphenols in a mouse model of fatty liver induced by high fat diet. Molecules, 23(12): 3213. Wu, S., Yano, S., Chen, J., Hisanaga, A., Sakao, K., He, X., . . . Hou, D.-X. (2017). Polyphenols from Lonicera caerulea L. berry inhibit LPS-induced inflammation through dual modulation of inflammatory and antioxidant mediators. Journal of Agricultural and Food Chemistry, 65(25): 5133–5141. Wu, S., Yano, S., Hisanaga, A., He, X., He, J., Sakao, K., Hou, D. X. (2017). Polyphenols from Lonicera caerulea L. berry attenuate experimental nonalcoholic steatohepatitis by inhibiting proinflamma­ tory cytokines productions and lipid peroxidation. Molecular Nutrition  & Food Research, 61(4): 1600858. Wu, T., Yu, Z., Tang, Q., Song, H., Gao, Z., Chen, W., Zheng, X. (2013). Honeysuckle anthocyanin supplemen­ tation prevents diet-induced obesity in C57BL/6 mice. Food & Function, 4(11): 1654–1661. Yang, J.-C., Hwang, H.-S., Lee, H.-J., Jung, S.-Y., Ji, S.-J., Oh, S.-H., Lee, Y.-M. (2014). Distribution of vascular plants along the altitudinal gradient of Gyebangsan (Mt.) in Korea. Journal of Asia-Pacific Biodiversity, 7(1): e40–e71. Younossi, Z., Anstee, Q. M., Marietti, M., Hardy, T., Henry, L., Eslam, M., . . . Bugianesi, E. (2018). Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nature Reviews Gastroenterology & Hepatology, 15(1): 11. Zadernowski, R., Naczk, M., Nesterowicz, J. (2005). Phenolic acid profiles in some small berries. Journal of Agricultural and Food Chemistry, 53(6): 2118–2124. Zhang, J., Sun, L., Dong, Y., Fang, Z., Nisar, T., Zhao, T., . . . Guo, Y. (2019). Chemical compositions and α-glucosidase inhibitory effects of anthocyanidins from blueberry, blackcurrant and blue honeysuckle fruits. Food Chemistry, 299: 125102. Zhao, H., Wang, Z., Cheng, C., Yao, L., Wang, L., Lu, W., . . . Ma, F. (2011). In-vitro free radical scavenging activities of anthocyanins from three berries. Journal of Medicinal Plants Research, 5(32): 7036–7042. Zhao, H., Wang, Z., Ma, F., Yang, X., Cheng, C., Yao, L. (2012). Protective effect of anthocyanin from Lonicera caerulea var. edulis on radiation-induced damage in mice. International Journal of Molecular Sciences, 13(9): 11773–11782. Zhao, J., Lin, Y., Zhao, Y., Wang, Y., Ning, C., Ma, Y., Meng, X. (2018). Polyphenol-rich blue honeysuckle extract alleviates silica particle-induced inflammatory responses and macrophage apoptosis via NRF2/HO-1 and MAPK signaling. Journal of Functional Foods, 46: 463–474. Zhao, J., Zang, J., Lin, Y., Wang, Y., Li, D., Meng, X. (2019). Polyphenol-rich blue honeysuckle extract allevi­ ates silica-induced lung fibrosis by modulating Th immune response and NRF2/HO-1 MAPK signaling. Journal of Functional Foods, 53: 176–186. Zhao, L., Li, S., Zhao, L., Zhu, Y., Hao, T. (2015). Antioxidant activities and major bioactive components of consecutive extracts from Blue honeysuckle (Lonicera caerulea L.) cultivated in China. Journal of Food Biochemistry, 39(6): 653–662. Zhou, L., Wang, H., Yi, J., Yang, B., Li, M., He, D., . . . Ni, H. (2018). Anti-tumor properties of anthocyanins from Lonicera caerulea ‘Beilei’fruit on human hepatocellular carcinoma: In vitro and in vivo study. Biomedicine & Pharmacotherapy, 104: 520–529.

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Zhou, Y., Ruan, Z., Wen, Y., Yang, Y., Mi, S., Zhou, L., . . . Wu, G. (2016). Chlorogenic acid from honeysuckle improves hepatic lipid dysregulation and modulates hepatic fatty acid composition in rats with chronic endotoxin infusion. Journal of Clinical Biochemistry and Nutrition, 58(2): 146–155. Zuo, A., Wang, S., Liu, L., Yao, Y., Guo, J. (2019). Understanding the effect of anthocyanin extracted from Lonicera caerulea L. on alcoholic hepatosteatosis. Biomedicine & Pharmacotherapy, 117: 109087.

15

Health Benefits of Haskap Berries (Lonicera caerulea L.) Rabie Khattab Imam Abdulrahman Bin Faisal University

H.P. Vasantha Rupasinghe and Marianne Su-Ling Brooks Dalhousie University

Giovana Bonat Celli The Whole Coffee Company

CONTENTS 15.1 15.2

15.3

15.4

Introduction ........................................................................................................................280

Chemical Composition of Haskap Berries ......................................................................... 281

15.2.1 Proximate Composition ........................................................................................ 281

15.2.1.1 Caloric Value and Dry Matter .............................................................. 281

15.2.1.2 Crude Proteins, Carbohydrates, Fibers, and Fat Contents ................... 285

15.2.1.3 Ash Content .......................................................................................... 285

15.2.1.4 Sugar Content ....................................................................................... 285

15.2.1.5 Organic Acids ....................................................................................... 285

15.2.2 Minor Components ............................................................................................... 286

15.2.2.1 Vitamins ............................................................................................... 286

15.2.2.2 Minerals ............................................................................................... 286

15.2.3 Secondary Metabolites ......................................................................................... 287

15.2.3.1 Polyphenols .......................................................................................... 292

15.2.3.2 Anthocyanins ....................................................................................... 293

15.2.3.3 Iridoids ................................................................................................. 294

15.2.4 Antioxidant Potential of Haskap Berries .............................................................. 295

Effect of Storage and Processing ........................................................................................ 296

15.3.1 Prefreezing Treatments and Frozen Storage Conditions ...................................... 297

15.3.2 Thawing Conditions.............................................................................................. 297

15.3.3 Juice Production.................................................................................................... 298

15.3.4 Drying ...................................................................................................................300

15.3.5 Extraction Methods............................................................................................... 301

Evidence for Haskap Berry Health Benefits .......................................................................302

15.4.1 Bioavailability of Haskap ..................................................................................... 303

15.4.2 Potential Human Health Benefits of Dietary Anthocyanins and C3G:

Evidence from Meta-analyses...............................................................................304

15.4.3 Cardiovascular Benefits ........................................................................................304

15.4.4 Anti-Inflammatory Effects ................................................................................... 305

15.4.5 Antidiabetic Effects ..............................................................................................306

15.4.6 Neuroprotective Effects ........................................................................................307

15.4.7 Chemopreventive Effects ......................................................................................308

15.4.8 Chemotherapeutic Effects.....................................................................................308

279

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Asian Berries: Health Benefits

15.4.9 Other Health Benefits ...........................................................................................309

15.4.10 Summary ..............................................................................................................309

15.5 Delivery Systems for Haskap Berry Extracts ..................................................................... 310

15.6 Concluding Remarks .......................................................................................................... 312

References ...................................................................................................................................... 312

15.1 INTRODUCTION The haskap berry (Lonicera caerulea L), also known as blue honeysuckle or honey berry (Figure  15.1), is native to northeastern Russia, China, Japan, and Canada (OszmiaĔski et  al., 2016). There is currently increasing interest in the haskap berry (L. caerulea L.) and its associated health-promoting properties, which can be attributed to its chemical composition and bioactive compounds present. Recent research findings support the potential of haskap berries as a new superfruit and a competitor to other highly nutritive and functional fruits, including blueber­ ries (Bors et  al., 2012; Khattab et  al., 2016a; Rupasinghe et  al., 2018). Haskap berries contain high levels of anthocyanins (ANCs), phenolic acids, flavanols, vitamin C, and other minor con­ stituents including magnesium, phosphorus, calcium, potassium, manganese, sodium, and iron (Hummer, 2006; Lefèvre et al., 2011; Rop et al., 2011; Rupasinghe et al., 2012; Khattab et al., 2016a; Rupasinghe et al., 2018). Phenolics from haskap berries have been associated with anti­ microbial, antifungal, anticancer, and antioxidant properties (Farcasanu et al., 2006; Gruia et al., 2008; Palikova et al., 2008). They protect cells against oxidative damage and inhibit the progress of various human diseases (Senica et al., 2018). Haskap berries can be processed into various products targeted toward the premium and gourmet food market, including frozen and dried berries, juice, wine, pastries, jams, chutneys, and powder, where the nutritional and health-related benefits are key marketing features, along with the pleasing taste (Chaovanalikit et al., 2004; Pigul, 2005). Although haskap berries have a high content of bio­ active compounds, the loss of these compounds in berry fruits during frozen storage and processing can be significant (Sadilova et al., 2006; Serpen et al., 2007; OszmiaĔski et al., 2009; Patras et al., 2010; Xu et al., 2012; Mirsaeedghazi et al., 2014; Khattab et al., 2015a, 2015b, 2016b, 2016c, 2017). These are important considerations if haskap products are targeted toward the functional food and health foods sectors, where the retention of the health-promoting bioactive compounds is desired.

FIGURE 15.1

Haskap berries on bush.

Health Benefits of Haskap Berries

281

Haskap berries have been used as a traditional medicine to treat various ailments (Anikina et  al., 1988; Thompson  & Barney, 2007). They have also been used as diuretic remedies and antiseptic agents, for throat and eye treatment (Jurikova et al., 2012) and for slowing the aging process (Lefol, 2007). There is increasing scientific evidence linking haskap berry compounds, particularly ANCs, and extracts to a range of positive health benefits for the treatment of diseases including diabetes, atherosclerosis, cardiovascular diseases (CVDs), neurodegenerative disease, gastrointestinal disorders, bacterial infection, and cancer (Svarcova et al., 2007; Heinrich et al., 2013; JurgoĔski et al., 2013; Vostálová et al., 2013; Celli et al., 2014; Rupasinghe et al., 2018). To increase the effectiveness of ANCs for the treatment of disease, gastrorententive systems have been proposed for the targeted delivery of ANCs and sustained release in the stomach (Celli et al., 2016c). Advantages include increased ANC stability in the acidic conditions of the stom­ ach and reduced concentration and frequency of dosage required for biological effect. In this chapter on the health benefits of haskap berries, we begin by outlining the chemical composition of haskap berries including macronutrients, micronutrients, and secondary metabolites associated with health-promoting benefits and antioxidant activity. This is followed by a discus­ sion of studies investigating the effect of frozen storage and processing conditions on the quality of haskap-related food and juice products, with emphasis on the bioactive content. Scientific evi­ dence is presented from recent research on the positive health benefits observed with haskap-related compounds for the potential treatment of many different diseases. Finally, a novel gastroretentive strategy for targeted delivery of ANCs is described.

15.2

CHEMICAL COMPOSITION OF HASKAP BERRIES

Haskap berries are a rich source of sugars (glucose, fructose) and dietary fibers (cellulose, hemicel­ lulose, pectin) but low in calories and fat. They have high contents of vitamin C, organic acids, and certain minerals and are abundant in polyphenols. They are also one of the highest natural sources of the health-promoting bioactive phytochemicals, ANCs (Khattab et al., 2016a). As the chemical composition of fruits determines their sensory attributes, nutritive values, and prohealth properties, it is important to investigate fruit composition using vigorous and accurate techniques, especially if these fruits are claimed to be part of the “functional” or “nutraceutical” foods. This chapter summarizes recent research conducted on the chemical composition of haskap berries including macronutrients, micronutrients, and secondary metabolites, putting more focus on those bioactive components with direct health-promoting effects.

15.2.1 PROXIMATE COMPOSITION The proximate chemical composition and the minor components of haskap berries have been exten­ sively investigated (Table 15.1) and are discussed in the following sections. 15.2.1.1 Caloric Value and Dry Matter The results of the chemical composition and estimated energetic value obtained for blue honey­ suckle fruits are shown in Table  15.1. Caprioli et  al. (2016) found that 100 g of haskap berries contained 87.67 g moisture and provided about 50 kcal. Similar to other berries, haskap is low in calories. SkupieĔ et al. (2007) found that haskap berry cultivar Zielona had a dry weight content of 12.65% and soluble solids content of 10.17%. The dry matter content was found to be 12.4%– 17.7% for Borealis, Indigo Gem (IG), and Tundra (T) varieties grown in Canada (Rupasinghe et al., 2012) and 13.8%–18.1% for Amur, Altaj, Sinoglaska, Amfora, and Pojark varieties grown in Czech Republic (Jurikova et al., 2012). Similarly, the average dry matter content of nine haskap varieties (Kamtschatica, Czelabinka, Duet, Jolanta, Wojtek, Klon 44, Klon 38, Klon B, and Klon C) grown in Poland was found to be 14.89% (Wojdyło et al., 2013). The corresponding value was 13%–17% for Slovenian haskap berries (Aurora variety) (Senica et al., 2018). Similar values were reported by Pokorná-Juríková and Matuškoviþ (2007) and Ochmian et al. (2008).

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TABLE 15.1 Chemical Composition of Haskap Berries: Proximate Analysis Constituent

Cultivar

Proximate Composition Energy Kamtschatica Dry matter Aurora Zielona, Czelabinka, Duet, Jolanta, Wotjek Borealis, Indigo Gem, Tundra Pojark and Turcz. ex Freyn Amur, Altaj, Sinoglaska, Amfora, Pojark Wojtek. Czarna, N Protein

Fat

Fibers Carbohydrates

Ash

Sugar Profile Sucrose

Fructose

Glucose

Location

Khabarovsk, Russia Slovenia Rajkowo and Skierniewice, Poland Saskatchewan, Canada Nitra, Slovakia

Content

References

0.50 kcal/g 13%–17% 12.7%–16.9%

Caprioli et al. (2016) Senica et al. (2018) SkupieĔ et al. (2007), Wojdyło et al. (2013)

12.4%–17.7%

Rupasinghe et al. (2012)

14.4%–20.3% 13.8%–18.1%

Pokorná-Juríková and Matuškoviþ (2007) Jurikova et al. (2012)

11.8%–13.9%

Ochmian et al. (2008)

2.1% 4.6%–8.4%

Caprioli et al. (2016) Rupasinghe et al. (2012)

0.01% 2.2%–4.8%

Caprioli et al. (2016) Rupasinghe et al. (2012)

8.34% 0.86% fw 10.2%–15.6%

Caprioli et al. (2016) Caprioli et al. (2016) Rupasinghe et al. (2012)

0.49%–0.64%

Wojdyło et al. (2013)

Kamtschatica Borealis, Indigo Gem, Tundra Kamtschatica Borealis, Indigo Gem, Tundra Kamtschatica Kamtschatica Borealis, Indigo Gem, Tundra Czelabinka, Duet, Jolanta, Wotjek Kamtschatica

Brno, Czech Republic Western Pomerania, Poland Khabarovsk, Russia Saskatchewan, Canada Khabarovsk, Russia Saskatchewan, Canada Khabarovsk, Russia Khabarovsk, Russia Saskatchewan, Canada Skierniewice, Poland Khabarovsk, Russia

Borealis, Indigo Gem, Tundra

Saskatchewan, Canada

Berry Blue, Borealis, Tundra, Indigo Gem, LC Aurora Berry Blue, Borealis, Tundra, Indigo Gem, LC Wojtek Unknown Aurora Berry Smart Blue, Indigo Gem, Indigo Treat, Morena, Tundra, Uspiech, Viola Czelabinka, Duet, Jolanta, Wotjek Wojtek Berry Blue, Borealis, Tundra, Indigo Gem, LC

Nova Scotia, Canada 0.90) between the phenolics and ANC concentration and the amount of DPPH scavenged (Khattab et al., 2016a). Similar results have been reported by other researchers (Wu et al., 2004; Jurikova et al., 2012) who found a linear correlation between total anti­ oxidant capacity and phenol content in blackberries (r = 0.96) and raspberries (r = 0.91). These find­ ings agree with those previously reported on other haskap berry varieties. Haskap berry extracts were found to scavenge 85% of DPPH as compared with 43%, 74%, and 51% for tomato (Lycopersicon esculentum Mill.), sea buckthorn (Hippophae rhamnoides L.), and the standard ascorbic acid, respec­ tively (Raudsepp et al., 2013). Rupasinghe et al. (2012) reported high antioxidant capacity of haskap berry crude extract as compared with blueberry. A higher antioxidant capacity of haskap extract was also demonstrated when compared with bog bilberry and raspberry (Zhao et al., 2011). Wojdyło et al. (2013) used two in vitro assays (ABTS and FRAP) to evaluate the potential antioxidant activ­ ity of haskap berries and found that the antioxidant activity was in the range of 12.65–49.73 mM Trolox equivalents (TE)/100 gdw. Thompson and Chaovanalikit (2003) measured the total antioxi­ dant capacity in 11 fruit samples of different subspecies of L. caerulea as ORAC with values of 18–104 μM TE per g of fresh weight and FRAP with values of 37–113 μM TE per g of fresh weight. The antioxidant capacity was correlated with both ANC content and TPCs. These berries seem to be prospective sources of health-supporting phytochemicals that exhibit beneficial antiadherence and chemoprotective activities; thus, they may have the potential for the prevention of neurodegenerative diseases and may provide protection against a number of chronic illnesses (Gazdik et al., 2008).

15.3

EFFECT OF STORAGE AND PROCESSING

Haskap berries are typically frozen within hours of harvest and stored frozen to extend their shelf life and allow for consumption and processing to occur independently from the growing season (Figure 15.3). In the following sections, we discuss the effect of storage and processing condition on the chemical composition and quality of haskap-related products, such as juice, dried berries, pomace, powder, and extracts.

FIGURE 15.3

Frozen haskap berries.

Health Benefits of Haskap Berries

297

15.3.1 PREFREEZING TREATMENTS AND FROZEN STORAGE CONDITIONS Despite the high content of bioactive components in haskap berries, the loss of these nutraceu­ ticals during frozen storage and consequent thawing is high, rendering the fruits and the end products containing only marginal amounts of these compounds of interest. Freezing causes cell rupture and division allowing reactions between enzymes and their substrates. Therefore, ANCs and other phenolic compounds may be degraded during freezing and more extensively during thawing due to their interaction with oxidative enzymes such as polyphenoloxidases. The effects of freezing and frozen storage on ANC content and phenolic profile of different kinds of berries have been investigated (Häkkinen et al., 2000; Mullen et al., 2002), and considerable ANC and ascorbic acid losses have been reported during frozen storage for several months (OszmiaĔski et al., 2009). In the study by Khattab et al. (2015a), the effect of steam blanching as a prefreezing treatment and freezing at −18°C and −32°C over 6 months on the antioxidant content and phenolic profile of haskap berries was investigated. Three haskap berry varieties grown in Nova Scotia, Canada, were studied: T, BB, and IG. Frozen storage at –18°C for 6 months reduced the TPC/total reducing capacity (TRC) by 37.08%–47.16%. TAC was also reduced, where the highest decrease was for BB (59.24%) followed by that of IG (46.34%). The DPPH scavenging activity was decreased by 26.78%– 30.86%. This reduction was attributed to the decrease in polyphenols and ANCs that are responsible for the antioxidant effect potential of haskap berries. In comparison, Chaovanalikit and Wrolstad (2004) found that more than 75% of ANCs in frozen cherries were destroyed after 6 months of stor­ age at −23°C. These results further agree with those reported for strawberries (Hartmann et al., 2008) and cherries (Poiana et al., 2010). Blanching prior to freezing improved the retention of bioac­ tive compounds and lower temperature storage (at −32°C) did not yield significant improvements as compared with the conventional frozen storage at −18°C. Khattab et  al. (2015a) concluded that blanching prior to freezing followed by frozen storage at −18°C is recommended for better retention of the bioactive components of haskap berries. Blanching before freezing has been used to inactivate enzymes that cause detrimental changes in color, flavor, and nutritive value during frozen storage (Fennema, 1982). Rickman et al. (2007) also reported that blanching appeared to prevent the degradation of phenolic antioxidants from oxida­ tion during storage and increased the amount of those antioxidants available to the human body. However, as water-soluble phenols may be leached into water during blanching, steam blanching is preferred, as water blanching can cause 20%–30% loss in the phenolic compounds in some veg­ etables (Puupponen-Pimia et al., 2003).

15.3.2 THAWING CONDITIONS Thawing is the last step in the freezing process before food consumption, processing, or cooking. The quality of frozen food is often more affected by the thawing process than by freezing, as a lon­ ger time is required for thawing (Kim et al., 2011) and studies have shown that the thawing method can affect the bioactive content in different berries (Häkkinen, 2000; OszmiaĔski et al., 2009). In commercial haskap juice production, frozen berries are thawed at room temperature. However, refrigerated thawing is an alternative, which would reduce the temperature to which the product is subjected and also increase the thawing time and potential loss of nutrient and bioactive compo­ nents. Microwave thawing is another method, which would require a shorter time and smaller space for processing (Virtanen et al., 1997). The effect of different thawing conditions on the content and phenolic profile of three has­ kap berry varieties was investigated by Khattab et al. (2015b). Thawing methods included thaw­ ing the frozen berries at room temperature (25°C ± 2°C) and refrigerated temperature (4°C) and using a domestic microwave oven. Frozen storage for 6 months followed by consequent thaw­ ing at the standard conditions (room temperature) caused 35.85%–44.73%, 32.14%–53.23%, and

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Asian Berries: Health Benefits

26.28%–29.57% reductions in the TPC/TRC, TAC, and DPPH scavenging activity, respectively. Refrigerated thawing did not significantly improve the retention of phenolic compounds and ANCs. After frozen storage for 6 months, there were no significant differences between room temperature and fridge thawing on the retention of phenolics. Despite the lower temperature, the longer time needed for thawing at 4°C might have contributed to the degradation of pheno­ lics. Microwave thawing could be achieved in significantly shorter time (17 min) as compared with room temperature (12 h) and refrigerated thawing (22 h) and could significantly improve the retention of phenolics, ANCs, and antioxidant potential. The corresponding reductions in the TPC/TRC, TAC, and DPPH scavenging activity were 24.97%–39.87%, 18.92%–47.22%, and 21.39%–27.29%, respectively. In comparison, Häkkinen (2000) studied the influence of different thawing methods including refrigerator (7°C for 16 h), room temperature (21°C for 1.5 h), and microwave oven (2–3 min) on the flavonol and phenolic acid contents of different Finnish berries including cranberry (Vaccinium oxycoccos), bog whortleberry (Vaccinium uliginosum), lingon­ berry (Vaccinium vitisidaea), and crowberry (Empetrum nigrum). Microwave thawing has been found to be the most effective for the retention of phenolic compounds and chosen as the best and most practical method. Further investigation into the industrial application of microwave thawing is recommended to maximize the retention of bioactive compounds in products manufactured from frozen haskap berries.

15.3.3 JUICE PRODUCTION The quality and bioactive content of haskap berry products is dependent on the process used, and there is potential to make improvements by modifying the processing steps and conditions. In juice production, depending on the extraction process parameters, pressed berries or pomace can be left as by-products. Pomace is typically composed of the pulp, peel, and seeds of the berry after the juice and water have been pressed (OszmiaĔski et al., 2016). Haskap juice can be produced commercially by pressing thawed berries using an expanding bladder. The resultant juice has a very intense flavor, so it is diluted with water and sugar is added to bring the Brix up to 12.5°. The juice is subsequently pasteurized and bottled, with a final pH of 3.5 and a density of 1.0. After being osmotically treated, the residual pressed berries are pressed again and then hot-air dried until a moisture content of 25% is reached. The liquid obtained from the second press is a by-product called “syrup” and is directed toward blended haskap-maple syrup products. In the study by Khattab et al. (2016b), the physicochemical characteristics of haskap prod­ ucts (juice, syrup, and pressed berries) obtained from a method based on a commercial conven­ tional process (process A) were compared with those of a simpler process (process B) (Figure 15.4). Process B merged the thawing and osmotic treatment into one step, followed by a one-time press, and would save a full day on the production line. The sugar solution from process B could be used for juice (after dilution and sugar adjustment) or blended directly with maple syrup. The results of this study showed that there was a negative correlation between the total soluble solids (TSS) and the titratable acidity (TA) which increased with the extraction yield. The TA was 1.89–2.18, 1.50–1.82, and 1.31 g citric acid/100 g in the juice (process A), syrup (process A), and sugar solu­ tion (process B), respectively. Extracting juice from the berries significantly reduced the content of individual ANCs in the resultant pressed product (Figures 15.5 and 15.6). The pressed berries from process B showed higher TAC than those of process A. Vitamin C content of the whole frozen fruit (89.11 mg/100 g fresh weight) decreased by 60.21%, 79.36%, and 82.18% in the juice, syrup, and pressed berries, respectively, in process A. Process B, however, could retain significantly more vitamin C in the obtained products. In another study, OszmiaĔski et al. (2016) analyzed the chemical composition and antioxidant activity of fresh haskap berry pomace from the pressing of whole intact fruit, crushed fruit, sepa­ rated skins, and flesh. They reported that the total polyphenol content in the fruit was 1,140 mg/ 100 g fw and was higher in all types of pomace, with the peel-based pomace having the greatest

Health Benefits of Haskap Berries

299

Fresh Haskap Berries

Frozen Storage Process A

Process B

Thawing (2-3 days)

Osmotic Treatment

First Pressing

Thawing (2-3 days)

Pressed Berries

Pressing

Juice

Osmotic Treatment Pressed Berries

Sugar Solution

Second Pressing

Pressed Berries

Syrup

FIGURE 15.4 Haskap juice extraction processes: conventional (process A) and simplified (process B).

FIGURE 15.5

Juice from thawed haskap berries.

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Asian Berries: Health Benefits

FIGURE 15.6 Pressed haskap berries – a by-product of juice production.

content with over four times as much as the fruit. Their results confirm that haskap berry ANCs and flavonols are located mainly in the peel and that peel-derived pomace is a good source of these biologically active compounds. In comparison, the content of phenolic acids did not differ as much between the various samples.

15.3.4

DRYING

The removal of moisture from foods by drying is a common method to reduce the weight and bulk of foods and improve their stability. However, nutrients that are susceptible to damage by heat, light, and oxygen are often degraded during this process (Santosa & Silvaa, 2008). Osmotic pretreatments using sucrose or other osmotic agents prior to air drying can improve the quality of dried fruits (Yadav & Singh, 2014). Conventional hot-air drying uses high temperatures; however, other drying techniques such as freeze drying (lyophilization) and refractance window drying (RWD) can be used to produce dried berry powders, where the lower temperatures allow a greater retention of nutrients. Khattab et al. (2017) studied the effect of air drying temperatures (60°C, 100°C, and 140°C) on the quality of the residual berries from different juice extraction processes. The conventional juice extraction (process A) was compared with a modified extraction (process B), which applied osmotic treatment during fruit thawing and used only one press extraction (Figure 15.4). The quality param­ eters investigated included moisture content, pressed berry yield, extraction loss, drying yield, TAC, vitamin C content, and the rehydration characteristics of the final dried berries. Pressing the berries to 70% juice yield resulted in a higher-pressed berry yield and better physicochemical quality in the pressed product. The TACs of pressed berries from extraction processes A and B were 24.62 and 33.03 mg C3G/g dw (Figure 15.7), and the vitamin C contents were 14.14 and 36.18 mg/100 g, respectively. Drying at 60°C until 25% moisture content was better than at higher temperatures, resulting in a better quality dried product. It achieved drying yields of 45.32% and 52.75%, TACs of 4.00 and 4.30 mg C3G/g dw vitamin C contents of 2.97 and 4.91 mg/100 g, and rehydration ratios of 2.22 and 2.37 from processes A and B, respectively. Process B with the one-step extraction was rec­ ommended for higher-pressed berry yield, higher drying yield, and enhanced quality of the pressed and dried products. It is also a more efficient process, in terms of time, cost, and energy. Khattab et al. (2017) also found that drying the berries to 25% moisture content at 60°C, 100°C, and 140°C reduced the individual ANCs by 73.85%–76.19%, 78.46%–80.95%, and 90.77%–95.40%, respectively. These results indicate the heat-labile nature of the ANCs (Jackman & Smith, 1996) that has been reported by other researchers. For example, Sadilova et al. (2006) observed that the ANC content in elderberry was very sensitive to thermal treatment, whereas after 3 h of heating at 95°C, only 50% of elderberry pigments were retained. Similar losses in raspberry purees were

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FIGURE  15.7 Anthocyanins and vitamin C contents of whole, pressed, and dried haskap berries, where dw = dry weight and fw = fresh weight. (Adapted from Khattab et al., 2016b; Khattab et al., 2017.)

reported by Ochoa et al. (1999). The overall stability of the major ANCs in haskap berries during storage and processing is summarized by the following trend (from most to least stable): peonidin 3-O-glucoside > pelargonidin 3-glucoside > cyanidin 3,5-diglucoside > cyanidin3-rutinoside > cyani­ din 3-glucoside (Khattab et al., 2016c). The effect of different preparation methods for producing freeze-dried haskap berry pow­ der from pomace was investigated by OszmiaĔski et al. (2016). Pomace was produced by press­ ing whole fruit, crushed fruit, separated skins, or flesh; the different types of pomace were then freeze-dried, milled, and analyzed for chemical composition and antioxidant activity. The powder products showed significantly higher antioxidant activity from pomace produced from peel > whole berries ≥ crushed berries than from flesh. They reported TPC in freeze-dried fruit of 12.29, 7.27 g/100 g dw for flesh-based pomace powder and 21.03 g/100 g dw for peel-based pomace powder. The authors concluded that products with higher bioactive content could be produced from a peel-based pomace in comparison with pomace derived from whole berries. In other study, RWD was investigated as a novel drying system to produce haskap powder, and the ANC retention and physicochemical properties of the powder were reported (Celli et al., 2016d). RWD is a technique that converts foods (as liquids or slurries) into flakes or powders within a typi­ cal residence time of 3–5 min (Nindo et al., 2007) where the product is dried as a thin film with excellent nutrient retention (Caparino et al., 2012). The different stages of haskap berry powder for­ mation by RWD are shown in Figure 15.18. Celli et al. (2016d) reported that the RWD powder had a solubility of 75.63% in water and retained 93% of ANCs from the original frozen fruits, as assessed by both high-performance liquid chromatography and the pH-differential method. This was consid­ ered comparable with what would be expected from freeze drying. Celli et al. (2016d) also identified three ANCs in frozen berries and RWD powder: cyanidin 3-glucoside, peonidin 3-glucoside, and cyanidin 3-rutinoside, which exhibited the lowest retention.

15.3.5

EXTRACTION METHODS

Conventional extraction methods typically require long time to complete and consume significant amounts of solvents. For technological, economical, and environmental safety issues, the research is currently geared toward the development of alternative extraction procedures using environmentally

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FIGURE  15.8 Haskap berry powder produced by refractance window drying: slurry application (a), film formation (b), flake formation (c).

friendly technologies and solvents. Examples of enhanced techniques include supercritical fluid extraction (SFE), pulsed electric fields, pressurized liquid extraction, and microwave- and ultrasound-assisted extraction (UAE) (Tsao & Deng, 2004; Garcia-Salas et al., 2010; Stalikas, 2010). The optimum conditions for the conventional extraction of haskap berry ANCs were found to be 69% (v/v) acidified ethanol, 1:15 solid–liquid ratio, and three times continuous extraction at 68°C for 90 min (Zenga et al., 2018) where 4.48 ± 0.03 mg/mL of ANCs were obtained. The use of ultrasound allows an efficient extraction of bioactive molecules in a shorter time and at lower temperatures than traditional methods such as maceration. Researchers have previously used ultrasound-assisted extraction to obtain phytochemicals from haskap berries (L. caerulea  L.) (Chaovanalikit et al., 2004; Bakowska et al., 2007; Zhao et al., 2011, 2012; Kusznierewicz et al., 2012). The UAE of ANCs from haskap berries was optimized using response surface methodol­ ogy (Celli et  al., 2015), and optimum conditions for the extraction were liquid/solid ratio 25:1 (mL/g), solvent composition of 80% ethanol, addition of 0.5% formic acid, and ultrasound bath temperature of 35°C for 20 min. Under these conditions, the TAC of 22.73 mg C3G equivalent/g dry weight (dw) was consistent with the predicted response of 22.45 mg C3G/g dw from the model. Over the past two decades, SFE using CO2 has been investigated as a possible replacement for the conventional solvent extraction (Friedrich et al., 1982; Bruhl & Matthaus, 1999; Barthet & Daun, 2002) owing to its numerous advantages (Friedrich & Pryde, 1984; Fattori et al., 1988). By using CO2, SFE could be utilized selectively to extract specific ingredients. To retrieve the bioactive polar compounds, a polar cosolvent such as ethanol can be added to the supercritical carbon dioxide (Dunford & Temelli, 1995). Extracts rich in ANC compounds were obtained from haskap berry pulp paste using supercritical carbon dioxide and water as cosolvent (Jiao  & Kermanshahipour, 2018). The highest total ANCs yield (52.7%) was achieved at 45 MPa, 65°C, 5.4 g water to 3.2 g berry pulp paste, and 15 min static and 20 min dynamic time. Different combinations of water and ethanol as cosolvent did not significantly affect the yield. The use of supercritical carbon dioxide and water as cosolvent achieved higher ANC extraction (52.7%) as compared with conventional extraction (38.3%) with improved antioxidant activity (89.8% versus 72.2%).

15.4

EVIDENCE FOR HASKAP BERRY HEALTH BENEFITS

In this section, the results from recent research studies are presented as evidence for the health ben­ efits associated with haskap berries and their extracts, some of which are summarized in Table 15.4. Many of the studies are focused on the ANCs, particularly cyanidin-3-O-glucoside (C3G), as it is

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TABLE 15.4 Some Potential Health Benefits Associated with Haskap Berries Benefits In vitro studies

Animal studies

Human studies

References

Reduction of biofilm formation and adhesion Antimicrobial properties

Palíková et al. (2008) Molina et al. (2019), Palíková et al. (2008), Raudsepp et al. (2013) Cytotoxic effect against hepatocellular carcinoma HepG2 and breast Pace et al. (2018) cancer MDA-MB-231 cells Anti-inflammatory properties Rupasinghe et al. (2015) Zhao et al. (2018) Hepatoprotective effects by inhibition of reactive oxygen species Park et al. (2017, 2019) and activation of antioxidant mechanisms Cytotoxic effect against hepatocarcinoma SMMC-7721 cells Luo et al. (2017) Protective effect against hepatitis induced by lipopolysaccharide Wang et al. (2016) Improvement of alcoholic steatohepatitis in AML12 cells treated Zuo et al. (2019) with ethanol Antioxidant properties Rupasinghe et al. (2012) Caprioli et al. (2016), Kucharska et al. (2017) Alleviation of silica particle-induced lung inflammation and Zhao et al. (2018) reduction of macrophage recruitment to the lungs Reduction of lipid peroxidation and activation of endogenous Wang et al. (2017) antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) Antidiabetic properties by ameliorating diabetic and related Sharma, Kim, Ku, Choi, and complications in high-fat diet animals Lee (2019) Amelioration of hepatosteatosis in simulated acute ethanol model Zuo et al. (2019) Attenuation of inflammation induced by lipopolysaccharide Wu et al. (2017a) Improvement of episodic memory and blood pressure (by Bell and Williams (2018) vasodilation)

the major bioactive compound in haskap berries. The potential health benefits include cardiovascu­ lar, anti-inflammatory, antidiabetic, chemopreventive, and chemotherapeutic effects, and these are further discussed in the following section.

15.4.1

BIOAVAILABILITY OF HASKAP

The bioavailability of bioactive compounds such as ANCs is the availability of the parent compound or its metabolites in the systemic circulation to exert its biological effects at the specific target sites. The bioavailability of ANCs including the major ANC of haskap berry, cyanidin-3-O-glucoside (C3G), has been extensively reviewed (Kamiloglu et al., 2015; McGhie & Walton, 2007; Miyazawa et al., 1999; Olivas-Aguirre et al., 2016). Human intervention studies have shown that the bioavail­ ability of ANCs is comparable with flavanols and flavones (Czank et al., 2013). Czank and colleagues reported the detection of phase II metabolites of C3G and cyanidin, their metabolite protocatechuic acid (PCA), phase II metabolites of PCA, and others such as phenylacetic acids, phenylpropenoic acids, and hippuric acid in serum, urine, and fecal samples of human subjects. After the intake of ANC-rich food, the initial metabolism takes place in the oral cavity where C3G is released from the food matrix. At pH 6–7, C3G exists predominantly in quinoidal-base form, and the glucose moiety improves its solubility. In the mouth, a portion of C3G could

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become cyanidin due to the deglycosylation catalyzed by β-glucosidase of oral microflora, saliva, and oral epithelium. Then, cyanidin can transform to PCA and cyanidin glucuronides (Mallery et al., 2011). Once C3G reaches the stomach, under the acidic condition of pH around 3, it becomes more stable as the flavylium cation. C3G is efficiently absorbed into the gastric wall and central circulation and then excreted into bile as an intact form and metabolites (Talavéra et al., 2003). The remaining C3G reaches the small intestine and exists as carbinol pseudobase, quinoidal base, and chalcone pseudobase and is absorbed into enterocytes through Na+-glucose transporter-1 (SGLT-1) and transported to the liver through portal circulation (Hassimotto et al., 2008). In the liver, C3G undergoes phase II metabolism forming methylated and glucuronidated metabolites (Kay et al., 2005) that enter the systemic circulation to exert their biological effects or to be metabolized and eliminated in the urine. C3G metabolites could be recycled through enterohepatic circulation (Fang, 2014). Finally, the unabsorbed C3G and its metabolites can be transformed into different metabotypes by colon microbiota in the colon (Hanske et al., 2017).

15.4.2 POTENTIAL HUMAN HEALTH BENEFITS OF DIETARY ANTHOCYANINS AND C3G: EVIDENCE FROM META-ANALYSES The review of recent meta-analyses suggests that dietary intervention of ANCs has health-promotional effects primarily toward cardiometabolic health. A literature search of MEDLINE and EMBASE for published studies up to August 2017 revealed that ANC supplementation had a significant effect on total cholesterol (TC) and low-density lipoprotein (LDL) cholesterol when the diet is supplemented with more than 300 mg ANC/day for more than 12 weeks. However, no significant effect of ANC supplementation was observed on weight, waist circumference, body mass index, and systolic and diastolic blood pressure (Daneshzad et al., 2019). Another systematic literature study of the impact of ANC intake on risk of fatal or nonfatal CVD showed that there was no relationship between ANC intake and the reduced risk of myocardial infarction (MI), stroke, or total CVD. However, the study provides evidence that specifically anthocyanidins reduce the risk of coronary heart disease and CVD mortality (Kimble et al., 2019). Data from 24 studies demonstrated that the consumption of ANC-rich food significantly improves vascular health, particularly concerning vascular reactivity measured by flow-mediated dilation (Fairlie-Jones et al., 2017). Furthermore, a meta-analysis of ran­ domized controlled trials based on online databases revealed the consumption of ANC significantly improved glycemic control and lipid support, which can further prevent and manage cardiometabolic diseases (Yang et al., 2017). As well, based on six studies involving 586 subjects, there is evidence that ANC supplementation has significant effects on total cholesterol (Liu et al., 2016). The results from 17 randomized controlled trials have demonstrated a significant reduction in lipid profile and inflammatory status with the supplementation of ANC (Shah & Shah, 2018). Based on systematic searches in databases up to January 2016 for relevant original stud­ ies on dietary ANC consumption, it was found that a 18% reduction of type 2 diabetes mellitus (T2DM) risk is associated with berry consumption (Guo et al., 2016). Overall, the outcome of these meta-analyses is in favor of the health-promotional effects of dietary ANCs.

15.4.3 CARDIOVASCULAR BENEFITS There is a strong link between fruits intake and reduced risk of CVD (Bazzano et  al., 2003; Rupasinghe et al., 2016; Thilakarathna & Rupasinghe, 2012). Hyperglycemia, dyslipidemia, obe­ sity, atherosclerosis, and hypertension are considered major risk factors for the development of CVD (Galassi et al., 2006). Interestingly, it has been reported that haskap has been used in folk medicine to lower blood pressure (Anikina et al., 1988), emphasizing the importance of exploring its potential role in cardioprotection. There are many preclinical animal studies demonstrating the benefits of haskap berry consumption. For example, haskap intervention suppressed the postprandial serum

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triacylglycerol (TAG) and glucose concentrations in rats following oral administration of corn oil emulsion, sucrose, or high-fat diet (Takahashi et al., 2014). Similarly, haskap berry supplementation showed normalized levels of plasma TAG and decreased levels of plasma non-HDL (high-density lipoprotein) cholesterol, which was significantly elevated by a high-fructose diet in Wistar rats (JurgoĔski et  al., 2013). An alcoholic extract of a haskap var. Edulis (Fly honeysuckle) reduced HDL, TAG, and cholesterol in high-fat diet–fed hyperlipidemic Wistar rats (Guang et al., 2004). Wild L. caerulea L. extract significantly reduced the body weight, normalized serum lipid levels, and reduced the liver total cholesterol and TAG levels in hypercholesterolemic rats. Moreover, the haskap supplement also enhanced the histopathological features of the liver tissues and aorta pecto­ ralis in rats fed with high-cholesterol diet (Liu et al., 2018b). The cardioprotective effects of haskap may be largely attributed to its major bioactive phytochemical, C3G. Chronic administration of C3G exerts an antihyperglycemic effect in streptozotocin-induced diabetic rats, where an increase in HDL cholesterol levels and a decrease in LDL cholesterol and glucose levels were observed (Nasri et  al., 2010). C3G has also shown similar antihyperglycemic effects through normalizing liver total lipids and TAG in mice fed with high-fat diet (Tsuda et al., 2003). Another study reported that the size of MI followed by coronary occlusion-induced regional ischemia was significantly reduced in hearts of rats fed with C3G-rich maize diet (Toufektsian et  al., 2008). C3G suppressed lipid peroxidation and oxidative stress– induced malondialdehyde in ischemic and reperfused rat heart, suggesting that C3G can alleviate tissue damages that consequently occur in myocardial ischemia and reperfusion (Amorini et al., 2003). C3G has also been shown to alleviate obesity-associated insulin resistance and fatty infiltra­ tion of the liver in mice fed with a high-fat diet, potentially through regulating Forkhead box protein O1 (FoxO1) activity that may reduce lipid accumulation by suppressing TAG synthesis (Guo et al., 2012a; Guo et al., 2012b). A random, double-blinded crossover study with 31 men between the ages 35 and 51 years (with screening blood pressure > 140/90 mm Hg) showed a reduced cardiovascular risk after the consump­ tion of 640 mg C3G-rich purified ANCs daily for 4 weeks. The supplementation increased HDL cho­ lesterol level in plasma of the prehypertensive and nondyslipidemic participants but did not affect markers of inflammation, endothelial dysfunction, or oxidative stress (Hassellund et al., 2013). Zhu and colleagues conducted a short-term crossover study (n = 12) and a long-term human intervention trial (n = 150) by giving a supplementation of 320 mg/day ANC extracted from berries. The observa­ tions revealed an overall enhancement of endothelial function with improvements in the serum lipid profile and decreased inflammation in the hypercholesterolemic individuals (Zhu et al., 2011). Their next investigation using a randomized, double-blind trial of 150 subjects with hypercholesterolemia confirmed the anti-inflammatory effects through reduced proinflammatory biomarkers such as high sensitivity C-reactive proteins, soluble vascular cell adhesion molecule-1, and plasma interleukin (IL)-1β after consumption of purified ANC mixture (320 mg/day) for 24 weeks (Zhu et al., 2013). Taken together, the C3G-containing haskap berries may exert cardioprotective effects by inter­ fering with glucose and lipid metabolism, favorably modulating dyslipidemia and upregulating eNOS expression to help maintain normal vascular function and blood pressure. Evidence reporting the health benefits of C3G against CVD is abundant.

15.4.4 ANTI-INFLAMMATORY EFFECTS Inflammation is generally the response of living tissues in host defenses against infectious agents or injuries. However, chronic inflammation, coupled with activated immune cells, could increase the risk of many chronic diseases, including atherosclerosis, diabetes, neurodegenerative disorders, and cancer (Libby, 2007). The prolong and excessive production of proinflammatory mediators such as interleukin-6 (IL-6), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), prosta­ glandin E2 (PGE2), nitric oxide (NO), nitric oxide synthase (NOS), and cyclooxygenase-2 (COX-2) often contributes to the progression of many chronic diseases. Rupasinghe et  al. showed that

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polyphenol-rich haskap berry extracts significantly inhibit the production of the major proinflam­ matory cytokines such as IL-6, TNF-α, and PGE2 as well as a COX-2 enzyme in lipopolysaccha­ ride (LPS)-stimulated human macrophages (Rupasinghe et al., 2015). Interestingly, the study also reported that the anti-inflammatory properties of haskap polyphenols are comparable with diclof­ enac, a COX-2 inhibitor drug. Similarly, Wu and colleagues showed that serum concentrations of IL-6, TNF-α, and NO were significantly reduced in adjuvant-induced arthritis Sprague–Dawley rats fed with haskap compared with the control group (Wu et al., 2015). Haskap significantly reduced the levels of NO, TNF-α, and PGE2 in the aqueous humor of LPS-induced uveitis rats (Jin et al., 2006). Most recently, Wu and colleagues have demonstrated that C3G and epicatechin-rich haskap extracts inhibit LPS-induced inflammation through modulating both inflammatory and antioxidant media­ tors such as nuclear factor erythroid 2–related factor 2 (Nrf2) (Wu et al., 2017a). In another study, Wang and colleagues reported evidence on the effect of haskap on LPS-induced hepatic inflam­ mation (Wang et  al., 2016a). Apart from maintaining energy metabolism and improving hepatic function, pretreatment with haskap also significantly reduced the production of IL-1β and IL-6 in rat liver BRL-3A cells. In addition to haskap, the anti-inflammatory effects of C3G have also been demonstrated. C3G significantly suppressed LPS-induced endothelial NOS (eNOS) and COX-2 expression (Wang et al., 2008). As well, C3G significantly suppressed LPS-stimulated TNF-α and IL-6 mRNA and protein expression and inhibited phosphorylation of nuclear factor kappa B (NF-κB) in LPS-stimulated macrophages (Zhang et al., 2010). C3G also reduced paws swelling and joint inflammation through regulating TNF-α and PGE2 levels in Freud’s adjuvant–induced arthritis in Sprague–Dawley rats (He et al., 2005).

15.4.5

ANTIDIABETIC EFFECTS

The prevalence of diabetes is rapidly increasing, in both developed and developing countries (Panchal & Jivarajani, 2017). T2DM is the most common type of diabetes, which causes a defi­ ciency in insulin-mediated glucose uptake in muscles and an impaired insulin action in the liver (Lin & Sun, 2010). The impact of T2DM includes long-term damage, dysfunction, and failure of various organs (Alberti & Zimmet, 1998) and increased risk of cardiovascular, peripheral vascu­ lar, and cerebrovascular disease (Alberti  & Zimmet, 1998). Among the recommendations of the World Health Organization (WHO) to reduce the risk of overweight and obesity-related diseases is the increased consumption of fruits, vegetables, legumes, whole grains, and nuts (WHO, 2016; Pods‫ܗ‬dek et  al., 2014). The inhibition of dietary carbohydrate oligosaccharide and disaccharide hydrolyzing enzymes such as pancreatic α-amylase and intestinal α-glucosidase (maltase) by poly­ phenols that are present in berries controls hyperglycemia and assists in preventing T2DM (Johnson et al., 2011; McDougall et al., 2008). The inhibition of these enzymes is useful for the management of T2DM as it decreases the rapid release of glucose into the blood by high glycemic index food (Pods‫ܗ‬dek et al., 2014). The antiglucosidase activity of ANCs has also been reported for colored fruits, including haskap (Mcdougall & Stewart, 2005). Among six tested fruits, haskap demonstrated the strongest α-glucosidase inhibitory activity with an IC50 value of 39.91 mg/mL (Pods‫ܗ‬dek et al., 2014). C3G, the major ANCs in haskap, has shown 1.8 times greater α-glucosidase inhibitory activity than cyanidin-3-O-galactoside indicating that anthocyanidin of haskap is superior to other berries in terms of antidiabetes activity (Bräunlich et  al., 2013). C3G can also inhibit intestinal β-fructosidase (sucrase) and pancreatic α-amylase (Akkarachiyasit et  al., 2010). The delaying digestion of disaccharides by inhibition of carbohy­ drate hydrolyzing enzymes is a therapeutic approach for controlling postprandial hyperglycemia in diabetic patients (Baron, 1998; Chiasson et al., 2004; Mcdougall & Stewart, 2005). As well, C3G directly stimulates the secretion of insulin from pancreatic β-cells (Jayaprakasam et al., 2005). The insulin released by the pancreas is stimulated by incretin hormones such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) (Gautier et al., 2005).

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Dipeptidyl peptidase-4 (DPP-4), a peptidase that inhibits incretins by cleaving N-terminal region of GLP-1 and GIP, is a new pharmacological target for T2DM treatment (Holst et al., 2009; Nauck et al., 2009). The results from computational docking analyses showed that C3G inhibits DPP-4 activity by binding their aromatic B-ring to the active sites of DPP-4 (Fan et al., 2013). C3G-rich fruit juice has a greater inhibitory effect against DPP-4 (Kozuka et  al., 2015). The insulin-like effect of C3G in human omental adipocytes is associated with the upregulation of adiponectin and glucose transporter type 4 (GLUT4), which is putatively caused by the increase of C3G-induced peroxisome proliferator–activated receptor-γ (PPARγ) activity (Scazzocchio et al., 2011). Therefore, future research should be directed to understand the role of haskap ANCs on the PPARγ-regulated mitochondrial biogenesis and homeostasis, leading to the energy balance of T2DM patients. AMP-activated protein kinase (AMPK) is an important factor for cellular energy balance and a potential therapeutic target for the prevention and treatment of T2DM (Hardie, 2008). Increased GLUT4 expression is regulated by the activation of AMPK through an insulin-dependent mecha­ nism (Hardie, 2008). Activation of AMPK by dietary polyphenols leads to suppression of hepatic gluconeogenesis and induction of fatty acid β-oxidation that both improve hepatic glucose utiliza­ tion and insulin sensitivity (Hwang et al., 2009; Rupasinghe et al., 2016). Enrichment of the diet with C3G-rich haskap extract has improved glucose homeostasis and insulin sensitivity in high-fat and high-sucrose fat–induced obese mice (Biswas et al., 2018). Another study showed that haskap supplementation improved glucose metabolism by increasing insulin sensitivity and attenuated oxi­ dative stress potentially by upregulating Nrf2-mediated pathway (Liu et al., 2018a).

15.4.6

NEUROPROTECTIVE EFFECTS

In general, berry consumption has been shown to decrease the occurrence of neurodegenerative dis­ ease by improving cognitive and motor function, especially in the aging population (Devore et al., 2012; Shukitt-Hale et al., 2008). The cognition-sparing effect of dietary ANCs is largely due to their abilities to inhibit the excessive production of ROS and proinflammatory mediators linked with neurodegenerative disorders (Bhullar  & Rupasinghe, 2013). A recently conducted double-blind, counterbalanced, crossover intervention study of 20 older adults aged between 62 and 81 years demonstrated that haskap berry supplementation (equivalent to 400 mg ANC/day/individual) sig­ nificantly improved of postprandial episodic memory and lowered diastolic blood pressure and heart rate. Haskap berries showed an impact on ameliorating age-related memory defects through improved vascular and metabolic health (Bell  & Williams, 2018). Lonicera japonica, also known as Japanese honeysuckle, protected primary rat cortical cells against glutamate-induced toxicity by both inhibiting NO production and maintaining superoxide dismutase (SOD) activity (Weon et al., 2011). Although direct evidence showing the neuroprotective effects of haskap berry is limited, the beneficial effects C3G on neuronal growth and survival have been reported extensively. C3G has been shown to alleviate ethanol-induced neuronal death through inhibiting the activity of glycogen synthase kinase 3β (GSK3β), a key neuronal apoptosis mediator (Chen et al., 2009; Ke et al., 2011). A C3G-rich fruit fraction inhibited lipopolysaccharide-induced microglial activation in mouse brain BV2 cells by suppressing proinflammatory enzymes such as NOS and COX-2 (Poulose et al., 2012). Dietary supplementation of C3G-rich extracts was shown to significantly reduce infarction vol­ ume and improved neurological functional outcome in mice subjected to cerebral ischemic damage (Kang et al., 2006; Min et al., 2011; Shin et al., 2006). Furthermore, C3G reversed cellular injury and improved hippocampal neuronal survival in senescence-accelerated mouse prone 8 (SAMP8) mice (Tan et al., 2014). Alzheimer’s disease is associated with the progressive accumulation of amyloid-beta (Aβ) aggre­ gates in the human brain (Murphy & LeVine, 2010). C3G significantly rescued Aβ-induced impair­ ment of learning and memory in mice (Qin et  al., 2013). C3G has also protected Aβ-mediated long-term potentiation deficits in hippocampal slices isolated from mouse (Wang et al., 2014). One

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plausible mechanism of action of C3G is the interaction with the aromatic residues in the core of amyloidogenic proteins, thus interfering with Aβ plaque formation (Porat et al., 2006). Interestingly, the efficacy of C3G may vary largely due to its ability to cross the blood–brain barrier (BBB) and localize in certain regions of the brain. For example, intravenous administration of C3G in anes­ thetized Wistar rats showed a positive correlation between plasma and brain C3G levels (Fornasaro et al., 2016). Overall, the significant neuronal bioactivity of C3G strongly suggests that whole has­ kap berry might exhibit similar or greater neuroprotection. However, human intervention studies remain to be investigated to achieve definite conclusions on the direct effect of whole haskap berries against neurodegenerative disorders.

15.4.7

CHEMOPREVENTIVE EFFECTS

Dietary polyphenols interfere with carcinogenesis by targeting multiple signaling pathways, thereby suppressing malignant cell proliferation and inducing apoptosis, while sparing survival pathways for normal cells. Strong scientific evidence exists on the protective effects of dietary polyphenols against DNA damage and failure of a repair mechanism that could ultimately lead to carcinogenesis (George et al., 2017). In general, ROS are constantly formed in aerobic cells, and oxidative stress is one of the major causes of DNA damage. At higher levels, ROS can perturb normal cellular func­ tions by damaging DNA, proteins, and lipids. Aerobic cells have several defensive enzymes, includ­ ing SOD, catalase (CAT), and glutathione peroxidase (GPx) to eliminate ROS, and nonenzymatic defensives such as glutathione and vitamin C to balance the oxidative stress in cells (Panieri  & Santoro, 2016). C3G-rich haskap extracts and pure C3G have been reported to protect cells from exogenous oxidative stress and prevent cellular damage in vitro (Svobodová et al., 2013; Vostálová et al., 2013; Zhao et al., 2012). For example, phenolic fractions of haskap significantly suppressed ROS produc­ tion and lipid peroxidation, while improving intracellular glutathione levels in human keratinocytes HaCaT exposed to UVA irradiation (Svobodová et  al., 2008). Pre- and posttreatment of HaCaT human keratinocytes with C3G-rich haskap berry extracts reduced ultraviolet B (UVB)–induced DNA single-strand break, DNA fragmentation, and UVB-induced cell apoptosis. The haskap extract also suppressed IL-6 expression and ROS generation, resulting in oxidative DNA damage in keratinocytes (Svobodová et al., 2009). Moreover, the oral administration of haskap berries to mice protects from the adverse effects of a single UVB exposure via modulation of antioxidant activity and reduction of DNA damage (Svobodová et al., 2013). Polyphenolic extracts from L. cae­ rulea var. Kamtschatica Sevast were reported to protect erythrocytes and lipid membrane against UVC-induced oxidative stress (Bonarska-Kujawa et al., 2014). Zhao and colleagues have shown the radioprotective effects of haskap var. edulis in ICR mice exposed to a sublethal dose of 5 Gy whole-body 60Coγ radiation (Zhao et  al., 2012). The SOD, GPx, and glutathione levels were upregulated in the haskap-treated group compared with the con­ trol group. Similarly, another study reported that C3G decreases UVB-induced phosphorylation of ataxia–telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and p53, DNA damage mark­ ers, in HaCaT human keratinocytes (Hu et al., 2016b). In addition, C3G also reduced ROS genera­ tion and induced apoptosis through suppressing B cell lymphoma-2–related X proapoptotic protein (Bax) levels and caspase-3 activity in C3G-treated HaCaT cells.

15.4.8

CHEMOTHERAPEUTIC EFFECTS

Dietary polyphenols have shown significant cytotoxic effects against various cancer types (Arumuggam et al., 2015; Fernando & Rupasinghe, 2013; Seeram et al., 2006; Sudan & Rupasinghe, 2014; Yi et al., 2005). Polyphenols are also effective as adjuvants that enhance the overall efficacy of conventional chemotherapeutics (Sak, 2012). C3G-rich haskap berry extracts and pure C3G have also been shown to arrest the cell cycle and/or induce apoptosis in cancer cells through various

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mechanisms both in vitro and in vivo. Haskap berry extracts (10–150 μg/mL dose) suppressed pros­ tate cancer cell proliferation and induced caspase-dependent apoptosis in multiple prostate cancer cell lines (Ali et al., 2017). Moreover, haskap berry extracts suppressed the migrating capacity and colony-forming ability of prostate cancer cells in vitro (Ali et al., 2017). Haskap berry extracts also showed cytotoxic effects against hepatocellular carcinoma HepG2, breast cancer MDA-MB-231 (Pace et al., 2018), and colon carcinoma HT-29 cells (Fan et al., 2011). Several studies have also shown anticancer effects of C3G on cancer cells. For example, pure C3G has suppressed the proliferation and invasion of A459 lung cancer cells (Ding et al., 2006). C3G has arrested cell cycle at G2/M phase by decreasing cyclin-dependent kinases, CDK1, CDK2, and cyclin B1 and D1 in Hs578T breast cancer cells (Chen et al., 2005). C3G has been shown to ameliorate the expression of ErbB2 receptor phosphorylation and its downstream effectors of focal adhesion kinase (FAK) and c-Src (FAK/C-Src) and activation of Jun N-terminal kinases that is nec­ essary for cell migration and invasion of breast cancer cells, MCF7 and MDA-MB-231 (Xu et al., 2010). C3G has also reduced the levels of matrix metalloproteinase (MMP)-9 and urokinase-type plasminogen activator (u-PA) in human hepatocarcinoma SKHep-1 cells and consecutive invasion and migration (Chen et al., 2006). C3G has effectively suppressed endothelial cell–specific vascular endothelial growth factor (VEGF)–induced migration of human umbilical code endothelial cells and human retinal microvascular endothelial cells (Tanaka et al., 2012). The apoptotic and anti­ metastatic effects of C3G against cancer cells suggest that C3G-rich haskap has great potential in killing malignant cells and keeping tumor cells localized.

15.4.9 OTHER HEALTH BENEFITS Substantial evidence from animal studies suggests the beneficial effects of haskap on liver-associated disorders. Nonalcoholic steatohepatitis (NASH) is a common fatty liver disease caused by the accumulation of fat in the liver of nonalcoholic patients, which leads to hepatic inflammation and damage. Dietary supplementation of haskap polyphenols (300 mg/kg body weight) has protected a mouse model displaying high-fat diet plus carbon tetrachloride–induced NASH (Wu et al., 2017b). The haskap intervention has significantly suppressed fat accumulation, inflammatory cell infiltra­ tion, lipid peroxidation, and insulin resistance in the aforementioned NASH mouse model. Oral administration of haskap reduced the release of hepatic enzymes, which are normally elevated in the inflamed or injured liver, supporting the protective effect of haskap on hepatocytes (Park et al., 2016). Moreover, a recent study also demonstrated the antiobesity and fatty liver, preventing prop­ erties of haskap in high-fat diet–fed mice (Kim et al., 2018). A haskap extract inhibited the inflam­ matory response and suppressed the lipid accumulation in the liver (Zuo et al., 2019). Furthermore, haskap berry polyphenols showed the ability to reduce triglyceride accumulation by downregulat­ ing hepatic lipid metabolic gene expressions in the liver cells of obese mice and ameliorate nonal­ coholic fatty liver disease (Park et al., 2019). Several studies have reported the antimicrobial effects of haskap. Adhesion to host tissues and biofilm formation are crucial steps for microbial colonization and infection. Haskap and berry extracts rich in polyphenols reduced the biofilm formation and artificial surface adhesion of human pathogenic microbial strains including Candida parapsilosis, Staphylococcus epidermidis, Enterococcus faecalis, and Streptococcus mutans (Palíková et al., 2008).

15.4.10 SUMMARY Scientific evidence shows that haskap berry possesses health-promotional and disease prevention properties through alleviating the risk factors of oxidative stress–induced chronic and metabolic diseases. In addition, evidence exists for the ability of haskap polyphenols for reducing the impact of chronic and acute inflammation. However, it is important to establish human clinical trials to fur­ ther validate the potency of haskap berry against these major chronic diseases. Preclinical evidence

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suggests that C3G-rich haskap extracts have significant potential in both chemoprevention and chemotherapy; however, future studies should employ more advanced preclinical models targeting specific cancer types and in vivo studies permitting oral administration of haskap berry. The effect of haskap polyphenols on the composition of the gut microbiome and the impact of consequent microbial metabolites need to be further understood. Health promotion effects of nonpolyphenol constituents such as iridoids have not been explored in detail and need to be investigated. Validating the health benefits of haskap berry products using properly designed human clinical trials will provide more evidence for promoting the consumption of haskap berries and their products for improved human health and well-being.

15.5

DELIVERY SYSTEMS FOR HASKAP BERRY EXTRACTS

Haskap berries are a rich source of ANCs, a class of secondary metabolites associated with several health benefits. For instance, a recent double-blind, crossover pilot study carried out by Bell  & Williams (2018) with 20 older adults has shown that a high concentration of ANC-rich haskap berry extract (containing 400 mg of total ANCs) resulted in significantly lower blood pressure, heart rate, and improved episodic memory than a lower concentration (200 mg) and a placebo. Nevertheless, our group has identified a considerable gap between the positive results associated with the con­ sumption of ANCs assessed by in vitro and animal studies and those from clinical trials. Potential reasons include the complex nature of plant extracts, with synergistic effects between compounds from various chemical groups (Rojo et al., 2012); excessive doses used in in vitro and in vivo studies that are not truthful representations of the usual amounts consumed through diet (Al-Awwadi et al., 2004; Hidalgo et al., 2012); extensive metabolism of ANCs in vivo, which could result in the forma­ tion of compounds with biological activity that are not naturally found in the plant source (Ferrars et al., 2014); and relatively low bioavailability in humans. Our group has further investigated the pharmacokinetics of ANCs based on published data to identify any biological-related explanation to the gap in knowledge (Celli et al., 2017a). Similar to pharmaceutical drugs, compounds ingested orally are subjected to absorption in the gastrointesti­ nal tract, distribution by the systemic circulation, metabolization, and excretion (or elimination). In most cases, the absorption of exogenous compounds (including ANCs) occurs in the small intestine, and it depends on factors associated with the food matrix (Charron et al., 2009), the compound itself (Ugalde et al., 2009), or physiological conditions. However, evidence has suggested that the stomach could play a role in the absorption of ANCs (Cai et al., 2011; Felgines et al., 2007; Passamonti et al., 2003), either by serving as an absorption site through bilitranslocase or by controlling their release to the initial portions of the intestine. Undeniably, the stomach is an interesting site for targeted delivery of ANCs. This is because the stability of these compounds is favored in the acidic conditions of the stomach (Stalmach et al., 2012; Woodward et al., 2011), where they are likely to be found in the stable flavylium cation form (Brouillard & Dubois, 1977). A proposed strategy for the delivery of ANCs is through gastroreten­ tive systems (Celli et al., 2016c), which differ from conventional platforms as they remain in the stomach for longer periods of time. In addition, sustained release would prevent the saturation of carriers involved in ANCs absorption (Fernandes et al., 2012; Kurilich et al., 2005; Talavéra et al., 2003), without the need to increase their concentration to result in a biological effect. Among differ­ ent classes of gastroretentive systems originally described for pharmaceutical drugs, our group has investigated the development of raft-forming and low-density (or floating) systems for the delivery of ANC-rich haskap berry extract. Raft-forming or in situ gelling systems are liquid formulations that gel in the body due to changes in pH (Kubo et al., 2004), such as the formation of alginate gel under acidic gastric environment. This gel (or continuous phase) is term raft. The formulations often include gas-generating compounds, such as carbonate salts, that will release CO2 gas in the stomach, which is entrapped within the gel matrix as it is forming in the body, thus reducing the system density. This will allow the delivery system to float

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above the gastric content (Tang et al., 2010), prolonging its residence time in the stomach. Ideally, this type of system should remain buoyant and in the stomach for up to 4 h (Strugala et al., 2012). In our study, we developed 13 formulations with varying amounts of sodium alginate as the hydro­ gel and sodium bicarbonate and calcium carbonate as the gas-generating compound, with further assessment in vitro. This type of system is expected to gel considerably fast (high gelling capacity) (Rajinikanth et al., 2007) to avoid premature emptying from the stomach. All formulations showed high gelling capacity in simulated gastric fluid (without enzymes), which means that the gelation occurred almost instantaneously upon contact with acidic medium and gels remained stable for more than 24 h when left undisturbed (Celli et al., 2016a). In addition, this type of gel needs to flow com­ pletely on the surface of the liquid and be held together as one mass when manipulated, which shows good coherence (Hampson et al., 2010). An example of the gel is presented in Figure 15.9. Next, we investigated the parameters that would affect gel weight, volume, and resilience. Both weight and volume were significantly affected by the concentration of alginate, bicarbonate, and carbonate. This is because calcium ions would contribute to alginate gel formation, whereas CO2 gas generated by both carbonate and bicarbonate would enable its flotation (Celli et  al., 2016a). Resilience was a measure of raft durability and stability under vigorous agitation, in a similar fash­ ion that is observed in the stomach (Hampson et al., 2010). An increase in gel weight and volume resulted in improved resilience (Celli et al., 2016a). The incorporation of haskap berry extract did not affect the gel properties. Release profile showed a biphasic pattern similar to what was noted by Rajinikanth et al. (2007), an initial burst up to 30 min in which a high amount of ANCs was released into the simulated gastric fluid, followed by a reduction in release. The release profile curves were fitted to the Peppas power law equation, and the suggested release mechanism was by Fickian dif­ fusion (Celli et al., 2016a). Floating (or low density) systems were another type of platform investigated by our group. These systems have lower density, which enables them to float above gastric contents as long as sufficient liquid is present in the stomach (Soppimath et al., 2001). Often, floating systems also contain effer­ vescent compounds (e.g., carbonate salts) that is generated during the preparation of the platform, not in situ as the previous example. Initially, a study was carried out to determine the parameters that would affect the encapsulation of ANC-rich haskap berry extract into calcium-crosslinked algi­ nate microparticles by the extrusion method (Figure 15.10). Results showed that an encapsulation efficiency of up to 63% could be achieved with this type of system, when the concentration of algi­ nate increased and allowed the particles to set in the gelling bath for shorter periods of time (Celli et al., 2016b). When floating alginate particles were prepared (with required adjustments on process­ ing conditions) using calcium carbonate as gas-generating compound, the encapsulation efficient

FIGURE 15.9 Example of gel formed under simulated gastric conditions (without enzymes); side (a) and top view (b).

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FIGURE  15.10 Calcium-crosslinked alginate microparticles encapsulated with haskap berry extract: fresh beads (a) and beads dried overnight at room temperature (b).

increased significantly than a control prepared without carbonate. The presence of carbonate reduced the density of the particles during preparation, in which nearly 100% floating capacity was achieved. After drying, low-density particles still exhibit significantly high floating capacity, whereas the con­ trol samples sank uniformly in the simulated gastric fluid. Similar to the raft system, the mechanism for release of ANCs from floating particles was most likely by diffusion, following a biphasic profile consisting of a burst phase followed by a reduction in release rate (Celli et al., 2017b).

15.6 CONCLUDING REMARKS Haskap berries are one of the richest sources of polyphenols, particularly in ANCs (C3G), in com­ parison with other “superfruits” such as blueberry. The high antioxidant activity corresponds to its chemical composition and bioactive content. Although the berries are typically frozen to extend their shelf life, the bioactive content of the berries can significantly decrease during frozen storage and thawing. Juice extraction parameters can affect the quality and chemical composition of the juice and also potential products from the remaining solids or pomace. Processing methods that reduce the over­ all processing time or temperature (e.g., RWD) and enhanced extraction techniques (e.g., UAE and SFE) can help retain bioactive content, which is important for the development of health-promoting functional foods and nutraceuticals. The body of scientific evidence for specific health benefits from haskap berries is growing, although more clinical trials are needed to confirm results from in vitro and in vivo tests. Further development of targeted delivery approaches for ANCs, such as gastroretentive systems, would improve anthocyanin bioavailability and effectiveness for treating disease.

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16

Haskap (Lonicera caerulea) Berries Shusong Wu Hunan Agricultural University

De-Xing Hou Kagoshima University

CONTENTS 16.1 Cultivars and Chemical Composition of Haskap (Lonicera caerulea) Berries ................. 327

16.1.1 Cultivars................................................................................................................ 328

16.1.2 Chemical Composition ......................................................................................... 328

16.2 Health Benefits of Haskap (Lonicera caerulea) Berries .................................................... 331

16.2.1 Antioxidant Activity ............................................................................................. 331

16.2.2 Anti-inflammatory Activity .................................................................................. 335

16.2.3 Amelioration of Glucose and Lipid Metabolism .................................................. 337

16.2.4 Anticancer Activity............................................................................................... 338

16.2.5 Modulation of Gut Microbiota .............................................................................. 338

16.3 Concluding Remarks .......................................................................................................... 339

References ...................................................................................................................................... 339

16.1 CULTIVARS AND CHEMICAL COMPOSITION OF HASKAP (LONICERA CAERULEA) BERRIES Haskap (Lonicera caerulea), also called blue honeysuckle, is a deciduous perennial shrub belong­ ing to the Caprifoliaceae family, which is native to the Northern Hemisphere (Caprioli et  al., 2016). In plant taxonomy, Lonicera belongs to Caprifoliaceae family, Dipsacales order, and Magnoliopsida class, as shown in Table 16.1. Native haskap (L. caerulea) plants grow from 0.8 to 3.0 m tall, but under cultivation the shrubs reach 1.0 m wide to 1.8 m tall. Their branches are dense and multibranched, and the backbone is not smooth. The new branches germinate from the surface or main stem, and branches are dark yellow-brown or dark reddish-brown. They usually have single leaf and are opposite-growing, with long elliptical to lanceolate shape, 2–7 cm long, and 1–2.3 cm wide. The leaf is pointed or obtuse, and the base is rounded, entire, and margin hairy. There are sparsely short villous on the surface of leaf, which sometimes is glabrous, or only vein is hairy. The flower is yellow-white, opposite-growing, lacted in axillary, or whorled on branches. Flowers are symmetrical left and right. The corolla is tubular, lobes are five, the tube base is shal­ low saccular, and stamens are 5. Blue honeysuckles are not self-pollinating so at least two single plants are required. They can begin fruiting within 1 year after planting, and after 3 years, almost 500 g of fruits can be acquired from one plant. Fruits become ripe very early in May or June in European conditions, but the fruit ripening period is from August to September in Asia and varies in different regions. Most wild Lonicera fruits grow in marshes, wetlands, and rivers between 700 and 1,800 m above sea level. The fruits are initially green; during the ripening process, the color changes from green to yellow and finally to dark blue. Matured fruit shapes are oval to long and 327

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TABLE 16.1 The Plant Taxonomy of Lonicera caerulea (Huxley et al., 1992) Lonicera caerulea Kingdom Subkingdom Superdivision Division Class Subclass Order Family Genus Species

Plantae Tracheobionta – vascular plants Spermatophyta – seed plants Magnoliophyta – flowering plants Magnoliopsida – dicotyledons Asteridae Dipsacales Caprifoliaceae Lonicera L. – honeysuckle Lonicera caerulea L. – sweetberry honeysuckle

dark navy blue to purple in color with blue waxy coating according to the genotype. The fruits can grow up to more than 2 cm long and weigh more than 1.5 g, but also the size and weight vary in dif­ ferent genotypes and regions. The berries grown in Russia are generally reported as 0.5 g, whereas Japanese cultivars can achieve 2.4 g, and the fruit weight of the Russian genotype honeysuckle grown in Oregon of the United States is varied from1 to 2 g (Celli et al., 2014). The flavor of ber­ ries is similar to that of bilberries or black currants, and it can vary from bitter to sweet (Svarcova et al., 2007). The berries of Haskap (L. caerulea) are widely harvested as edible berries in Russia, China, and Japan (Rop et al., 2011).

16.1.1 CULTIVARS Blue honeysuckle has been mentioned as a horticultural plant for the first time in 1894. By1998, the number of commercial cultivars in Russia has reached 60 and spread widely in private and com­ mercial gardens of Northern and Central Russia, in the Urals, Siberia, and the Far East (Plekhanova, 2000). They do not require special soil type, even wet sandy or loamy soil is suitable, pH can range from 5 to 7, and the organic proportion can be higher (Svarcova et al., 2007). Haskap (L. caerulea) can outstand frost resistance, since it is able to survive very low temperatures (up to −46°C) without being damaged (Caprioli et al., 2016). It is mainly distributed in the northern part of the Northern Hemisphere and widely distributed in Northeast China, in which the reserves of wild resources in Greater Khingan Mountains, Lesser Khingan Mountains, and Changbai Mountains are the largest. The primary cultivars of haskap (L. caerulea) around the world are as shown in Table 16.2; Russia has the most abundant haskap (L. caerulea) varieties.

16.1.2

CHEMICAL COMPOSITION

Fruits of haskap (L. caerulea) are small berries, rich in sugar, organic acids, minerals, vitamins, and various trace elements. They are delicious and edible and can also be processed into fruit wines, pulps, and fruit juices. Haskap (L. caerulea) berries are internationally recognized as a new small berry after blueberries, blackcurrants, and raspberries since they have high nutritional value and health functions. The nutritional characteristics of haskap (L. caerulea) berries are various in different areas. Table  16.3 shows the proximate composition, vitamin, animo acid, and min­ eral contents of haskap (L. caerulea) berries from eastern Russia, Czech Republic, China, and Canada, in which the highest protein content is 2.12% in fresh berries from eastern Russia, the maximum concentration of vitamin C is 37.3 mg/100 g from Dongning City of China, the berries

Lonicera altaica Pall (Holubec et al., 2019)

Lonicera baltica Pojark (Holubec et al., 2019)

Breed 6

Breed 7

Breed 5

Lonicera boczkarnikovae Plek (Gerbrandt et al., 2018) Czelabinka (Becker et al., 2019)

Wołoszebnica (Becker et al., 2019)

Breed 4

Zielona (SkupieĔ et al., 2007)

Czelabinka (Wojdyło et al., 2013)

Wojtek (Wojdyło et al., 2013)

Jolanta (Wojdyło et al., 2013)

Lonicera iliensis Pojark (Gerbrandt et al., 2018)

Sinogłaska (Becker et al., 2019)

Breed 3

Duet (Wojdyło et al., 2013)

Atut (Becker et al., 2019)

Poland

Freyn (Gerbrandt et al., 2018)

Dlinnopłodna (Becker et al., 2019)

Breed 2

Lonicera edulis Turcz (Gerbrandt et al., 2018)

Kuril Islands

Kamtschatica (Caprioli et al., 2016)

Russia

Breed 1

Cultivars

Asia

TABLE 16.2 Cultivars of Lonicera caerulea around the World

Pojark (PokornaJurikova & Matuskovic, 2007) Turcz. ex Freyn (PokornaJurikova & Matuskovic, 2007)

Slovakia

Pojark (Jurikova et al., 2012)

Amfora (Jurikova et al., 2012)

Sinoglaska (Jurikova et al., 2012)

Altaj (Jurikova et al., 2012)

Amur (Jurikova et al., 2012)

Czech Republic

Europe

Region

Morena (Auzanneau et al., 2018) Uspiech (Auzanneau et al., 2018) Viola (Auzanneau et al., 2018)

Indigo Treat (Auzanneau et al., 2018)

Tundra (Auzanneau et al., 2018)

Indigo Gem (Auzanneau et al., 2018)

Berry Smart Blue (Auzanneau et al., 2018)

Switzerland

Bees (Senica et al., 2018) Tundra (Senica et al., 2018)

Aurora (Senica et al., 2018) Borealis (Senica et al., 2018)

Croatia

Tundra (Rupasinghe et al., 2012)

Indigo Gem (Rupasinghe et al., 2012)

Borealis (Rupasinghe et al., 2012)

Canada

(Continued)

Boreal Blizzard Blue Moon (Rupasinghe (Becker et al., et al., 2018) 2019) Boreal Beauty Blue Bird (Rupasinghe (Becker et al., et al., 2018) 2019) Boreal Beast Blue Belle (Rupasinghe (Becker et al., et al., 2018) 2019)

Aurora (Joseph Blue Velvet et al., 2016) (Becker et al., 2019)

Honey Bee (Rupasinghe et al., 2018)

Indigo Gem (Rupasinghe et al., 2018)

Borealis (Rupasinghe et al., 2018)

North America

America

Haskap (Lonicera caerulea) Berries 329

Breed 15

Breed 14

Breed 13

Breed 12

Breed 11

Breed 10

Breed 9

Breed 8

Cultivars

Kuril Islands

Lonicera buschiorum Pojark (Holubec et al., 2019) Lonicera caerulea L. (Holubec et al., 2019) Lonicera edulis Turcz. ex Freyn (Holubec et al., 2019) Lonicera liensis Pojark (Holubec et al., 2019) Lonicera kamtschatica (Sevast.) Pojark (Holubec et al., 2019) Lonicera pallasii Ledeb (Holubec et al., 2019) Lonicera stenantha Pojark (Holubec et al., 2019) Lonicera turczaninovii Pojark (Holubec et al., 2019)

Russia

Asia Poland

TABLE 16.2 (Continued) Cultivars of Lonicera caerulea around the World

Slovakia

Czech Republic

Europe

Region

Switzerland

Croatia

North America

America Canada

330 Asian Berries: Health Benefits

Haskap (Lonicera caerulea) Berries

331

from Central Moravia of Czech Republic have a higher concentration of amino acids, and the ber­ ries from Khabarovsk of Russia have a higher concentration of minerals (Table 16.3). Although fatty acid is considered as the minor component of haskap (L. caerulea) berries, fatty acid methyl esters (FAMEs) analyzed by gas chromatography-flame ionization detector (GC-FID) has shown that the fatty acids in berries from eastern Russia is mostly represented by unsaturated fatty acids (93.9%, Table 16.4) with linoleic acid as the most abundant (66.8%), followed by oleic acid (22.8%), then by palmitic acid (5.3%), and by linolenic acid (2.9%). The phenolic fraction of haskap (L. caerulea) berries is considered as the main part that pos­ sesses multiple biological functions. According to a study that analyzed the phenolic and flavonoid contents of 12 kinds of haskap (L. caerulea) berries from Brno, Czech Republic, the phenolic con­ tent ranges from 6 to 9.03 g of gallic acid/kg of fresh mass, whereas the flavonoid content ranges from 3.06 to 4.01 g/kg of fresh mass (Table 16.5). Anthocyanins predominate the phenolic fraction of haskap (L. caerulea) berries and are closely correlated with the biological function. Table  16.6 shows the relative percentages of anthocya­ nins in haskap (L. caerulea) berries from Czech Republic, eastern China, and eastern Russia; cyanidin 3-glucoside is the main anthocyanin ranging from 83.3% to 87.4%, and other anthocy­ anins highlighted are cyanidin-3,5-diglucoside, cyanidin-3-rutinoside, delphinidin-3-glucoside, delphinidin-3-rutinoside, and peonidin-3-glucoside.

16.2 HEALTH BENEFITS OF HASKAP (LONICERA CAERULEA) BERRIES Haskap (L. caerulea) berries possess multiple biological functions, which are mainly due to the high phenolic content. The bioactive compounds of the berries are mainly found in the peel resi­ due. The content of phenolic compounds in the peel residue is 4.3 times that of fresh berries. After juicing, the peel has a higher dry matter content and has a higher sugar content and less organic acid content than fresh berries. Moreover, because of the low moisture content, it is easier to freeze the dried pomace than the fresh fruit. Dried pomace also exhibits higher antioxidant activity. The lyophilized pomace contains more than 21% phenolic compounds, whereas the polyphenol content in dried berries is only 12.3% and only about 7.3% in the pulp. In vitro, the peeled pomace had the strongest antioxidant capacity, whereas the dried pulp showed the worst (OszmiaĔski et al., 2015).

16.2.1 ANTIOXIDANT ACTIVITY The beneficial effects of haskap (L. caerulea) berries are considered to be mainly attributed to the antioxidant activities derived from the high content of polyphenols. Based on in vitro experi­ ments, extracts of haskap (L. caerulea) berries from China have a strong scavenging ability on free radicals such as 2,2-diphenyl-1-picrylhydrazyl and show a high ferric reducing antioxidant power (Zhao et al., 2015). The antioxidant activities of haskap (L. caerulea) berries are well cor­ related with the total phenolic and total anthocyanin contents (Rupasinghe et al., 2015), and the con­ tent of cyandin-3-glucoside is significantly positively correlated to free radical scavenging ability (Wang et al., 2018). The berry extracts are reported to exert antioxidant function in multiple models, such as improving the activity of antioxidant enzymes including superoxide dismutase, catalase (CAT), and glutathione peroxidase (GPx) in mouse liver and thus enhancing the body’s antioxidant capacity to reduce liver lipid peroxidation (Wang et al., 2017), enhancing the activities of GPx and CAT in human red blood cells (Heinrich et  al., 2013), reducing the consumption of glutathione and lipid peroxidation to attenuate inflammation in human gingival fibroblasts (Zdarilova et  al., 2010), and activating antioxidant response system to alleviate silica-induced pulmonary fibrosis (Zhao et  al., 2019). The mechanisms underlying the antioxidant properties of haskap (L. caeru­ lea) berries mainly include three aspects. First, phenolics of haskap (L. caerulea) berries extract can directly quench free radicals such as reactive oxygen species (ROS) (Zdarilova et al., 2010). Second, the phenolic compounds can activate antioxidant signaling such as upregulating nuclear

Moisture (%) Protein (%) Carbohydrate (%) Sugars (%) Total reducing sugar (%) Fat (%) Ash (%) Crude fiber (%) Energy (kJ) Total acid (g/kg) Vitamin C (mg/100 g) Nicotinamide (mg/100 g) Pantothenic acid (mg/100 g) Niacin (mg/100 g) Vitamin B1 (mg/100 g) Vitamin B2 (mg/100 g) Vitamin B6 (mg/100 g) Folic acid (mg/100 g) Vitamin B12 (mg/100 g) Vitamin A (mg/100 g) Vitamin K1 (mg/100 g) Carotene α (mg/100 g)

Components – – – – – – – – – – 12.10 1.87 1.40 0.47 0.16 0.02 0.02 0.02 0.02 ascorbic acid. Even after heating, ethanol extracts from amla had maintained both their superoxide anion and hydroxyl radical scavenging activities (Saito et al., 2008). In addition to antioxidant activity, amla fruit extracts are also reported to possess antibac­ terial activity against Staphylococcus aureus (Mayachiew  & Devahastin, 2008). Sakaguchi and Teetamu (2006) have obtained a patent for an antioxidative composition obtained from amla fruits using different solvent systems. The antioxidant phenolics reported in amla fruits are summarized in Table 17.5. Among the various phenolics isolated from the methanolic extract of amla fruits, geraniin showed the highest antioxidant activity of 4.7 and 65.7 μM of IC50 values of DPPH and lipid peroxi­ dation assay, respectively (Liu et al., 2008a). In a follow-up study, Liu et al. (2008b) have reported a total phenolics content in amla fruits ranging from 81.5 to 120.9 mg gallic acid equivalents (GAE)/g,

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Asian Berries: Health Benefits

TABLE 17.5 Type of Antioxidant Phenolics in Amla Fruits S. No 1 2 3 4 5 6 7 8

Name of Antioxidant Phenolic Gallic acid, methyl gallate, 1-O-galloy-glucoside Emblicanin A, emblicanin B Punigluconin, chebulanin, chebulagic acid, corilagin Mucic acid 3-O-gallate, mucic acid 1,4-lactone 2-O-gallate, mucic acid 1,4-lactone 5-O-gallate, mucic acid 1,4-lactone 3,5-di-O-gallate, 1,6-di-O-galloyl-glucoside Elaeocarpusin, furosin, geraniin, geraniinic acid Ellagic acid, ellagic acid-4-O-glucoside, ellagic acid-4-O-xyloside, ellagic acid-4-O-rahmnoside Isostrictinin, mallonin, tercatain, punicafolin, putranjivain A Phyllanemblinin A

Adapted from: Yang and Liu (2014), Rose et al. (2018).

flavonoids, 20.3 to 38.7 mg quercetin equivalents (QE)/g, and proanthocyanidins, 3.7 to 18.7 mg catechin equivalents (CE)/g. In the ethanol extract of air-dried amla fruit powder, cinnamic acid, quercetin, 5-hydroxymethylfurfural, gallic acid, β-daucosterol, and ellagic acid have been identified by Luo et al. (2009).

17.6

PROCESSED PRODUCTS AND POSTHARVEST STORAGE

The amla fruit being a hard-textured fruit can be transported to long distances without much loss in quality (Shankar, 1969). While studying the effect of different storage conditions on the shelf life of fresh amla fruits, Singh and Kumar (1997a) reported that the ascorbic acid content decreased with extended storage period under all storage conditions employed. However, only the modified atmo­ sphere storage in combination with zero-energy chamber proved to be the most effective in reducing the loss of fruit weight and ascorbic acid in fresh amla fruits. In a follow-up study, Singh and Kumar (1997b) reported the use of kinetin (150 ppm) to be the most effective (followed by 25 ppm of gib­ berellic acid treatment) in retaining highest ascorbic acid content during the postharvest storage of fresh amla fruits. Because of their acidic and astringent taste, fresh amla fruits are generally processed with the addition of sugar to prepare preserve, jam, beverages, or the use of salt to prepare pickle-like prod­ ucts. The processed products generally develop white specks on the surface and interior of the fruit, which adversely affects their acceptance by the consumers. The development of white specks is due to the interaction of calcium with mucic acid (D-galactaric acid) in the processed amla fruits (Premi et al., 1998). To control the development of white specks during the storage of amla fruits, Premi et al. (1999) recommended the steeping of fresh amla fruit segments in a solution of 10% sodium chloride and 0.04% potassium metabisulfite. To extend the shelf life of fresh amla fruits, the application of high electric field (HEF) technol­ ogy is feasible. To examine weight loss, rotting, ascorbic acid retention, and hunter color values, Bajgai et al. (2006) applied alternating current (AC) and direct current (DC) (HEF of 430 kV/m) for 2 h to the fresh amla fruits packed in open polyethylene pouches. Compared with control amla fruits, the HEF-treated fruits retained the better freshness and other quality parameters. Pathak et al. (2009) have extended the shelf life by about 3 weeks under ambient conditions (13.3°C, 65.6% RH) of storage, if injured fresh amla fruits are wax coated. The use of gunny bags, corrugated fiber­ board boxes (CFBs), wooden crates, and bamboo baskets with polyethylene liners or newspaper lin­ ers (NPLs) for storage of fresh amla fruits under ambient conditions (13.3°C, 65.6% RH) has been

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investigated by Singh et al. (2010). Compared with control, the lowest spoilage was obtained in fresh amla fruits packaged in CFBs with NPLs at the end of 13 days of storage period. Amla fruits are being used in folk medicine for centuries in India, and they are also processed into juice, beverages, powder, pickle, jam, preserve, and candy. The benefits of consuming an ancient India nutraceutical preparation (Chyawanprash) made from amla with a combination of 50 herbs, mineral oxides, honey, and sugar syrup have been discussed by Bajpai (2005). As the amla fruits are known to contain many health-promoting bioactive compounds (Rao & Juneja, 2005), a number of patents have recently been issued for their use as a health food (Kihira & Teetamu, 2006; Nirei, 2007; Krishan & Buecker, 2009). Because of better stability of natural antioxidants, dried amla fruit powder, which has higher antioxidant activity, was used in controlling the lipid oxidation in biscuits during storage (Reddy et al., 2005). Moreover, the sensory properties of biscuits were not affected by the addition of 1%–2% of natural antioxidant extracts from amla fruit powder. Blanching of whole amla fruits and then separating the segments for obtaining the highest con­ tent of soluble components as well as ascorbic acid in the juice has been suggested by Jain and Khurdiya (2002). Amla fruit as being one of the major ingredients, a number of mixed fruit juice beverages have been developed (Deka et al., 2001, 2004). A process to prepare a carbonated non­ alcoholic beverage using amla juice in combination with a few herbs has been obtained by Prakash (2009). During the preparation of amla preserve, the original amount of 624 mg/100 g of ascorbic acid content got reduced to 121 mg/100 g (Mehta & Tomar, 1979). The major spoilage organism in amla preserve has been identified as the heat-resistant spore-forming Bacillus cereus. A number of chemical preservatives, such as sulfur dioxide (692 ppm), sodium benzoate (800 ppm), sodium propionate (2800 ppm), or potassium sorbate (3,000 ppm), have been suggested for the control of this spoilage organism in amla preserve (Sethi & Anand, 1984). A few of the osmophilic microor­ ganisms, such as Eurotium repens and Saccharomyces bailii, have been identified in spoiled amla preserves, and these fungi could be controlled with the use of 100 ppm of sulfur dioxide (Garg & Yadav, 2007). During sun drying of fresh amla pulp to a powder having 9% moisture content, the original amount of ascorbic acid content of 766 mg/100 g in fresh amla fruit pulp was reduced by about 30% (Ramasastri, 1974), but during storage of this amla powder at room temperature for 48 weeks, the ascorbic acid content got reduced to 9.9 mg/100 g at the end of 48 weeks. Interestingly, with the use of a solar drier for obtaining amla fruit flakes, the retention of ascorbic acid was much higher (76.6%) (Verma & Gupta, 2004). The design specification and performance of solar drier, in com­ parison with an electric drier for handling about a 1-ton batch of amla pulp, have been investigated by Rathore et al. (2006). Pragati et al. (2003) obtained a minimal amount of browning in the fin­ ished product of amla powder with osmo-air drying, among the four different methods of drying, i.e., osmo-air drying, direct sun drying, indirect solar drying, and oven drying, used by them. A few workers have obtained a better quality (color, flavor, texture, and overall quality) in the ready-to-eat finished product from amla at the end of 6 months of storage at 7°C ± 2°C, if amla fruit shreds are soaked in sugar syrup of 70°Brix for 5 min just before drying in a hot-air cabinet drier to a final moisture content of 5% (Sagar & Kumar, 2006; Kumar & Sagar, 2009). The fluidized bed drying of amla fruits than the solar or hot-air tray drying methods resulted in better retention of ascorbic acid (Murthy & Joshi, 2007). The use of the latest technique of thermal-assisted high-pressure process­ ing (THPP) in the domain of 200–500 MPa on the physicochemical properties, bioactives, and anti­ oxidant activity of amla juice was found to significantly improve the ascorbic acid, total phenolics, and total antioxidant activity of amla juice (Raj et al., 2019).

17.7

OTHER MISCELLANEOUS USES OF AMLA FRUIT

Medicinal properties of various herbs have long been recognized to assist in the primary healthcare needs of the general public in many developing countries of the world. Triphala, a product contain­ ing amla fruit, has been analyzed using the neutron activation technique for estimating the efficacy

354

Asian Berries: Health Benefits

and safety of the mineral composition (Waheed & Fatima, 2013). According to their results, these herbs contained potassium as a major mineral, and amla fruits were found to be rich in cobalt, chromium, and sodium too. Recently, the use of green chemistry approach (plant extracts) to tackle the corrosion problems, especially to replace the metallic corrosion inhibitors, has attracted significant attention from scien­ tists. Verma et al. (2018) have reviewed the use of plant extracts (including amla fruit) as environ­ mentally sustainable and green corrosion inhibitors for metals and alloys under aggressive media conditions. This green chemistry approach, suggested by them, has reduced environmental risk, lesser costly, easy availability, high effectiveness for corrosion inhibition, and at the same time, being less detrimental to the environment than the toxic traditional synthetic corrosion inhibitors. Heavy metals are many environmental pollutants that are known to affect the growth and pro­ ductivity of field crops. Among these metals, chromium is a key metal pollutant that, when present in higher concentrations in soil, affects the crop growth and productivity adversely. Pandey et al. (2019) have used amla fruit extracts (at 5 mg/mL level), eliminated the chromium-induced decline in plant growth, increased biomass production and yield, reduced oxidative stress, and modulated the antioxidant enzyme activities. Kannaujia et al. (2019) have prepared amla fruit extract–stabilized biogenic silver nanoparticles as a plant growth promoter of wheat plants. According to their find­ ings, the use of phytochemicals–capped biogenic silver nanoparticles acted as better growth pro­ moters by delivering antioxidants during the early growth of wheat seedlings than the chemically synthesized silver nanoparticles. The preparation of palladium nanoparticles has an important con­ cern as some of the end products formed are known to be toxic by-products. Nasrollahzadeh et al. (2020) have recently reviewed the plant-based biosynthesis (used amla seeds also), characterization, mechanism of action, stability, and catalytic and microbial activities of palladium-based nanopar­ ticles. These biogenic Pd-based nanoparticles have attracted the attention of researchers due to their economic sustainability and green and friendly nature, without forming any toxic by-products. Another heavy metal, arsenic, in food or water, causes serious skin disease and cancer in humans. The poorest people suffer the most from arsenic poisoning, because of low intake of essential nutri­ ents in their diet. Sharma and Flora (2018) have reviewed the alternate methods, using herbal prepa­ rations (including amla fruit extracts) in alleviating the toxic effects of arsenic in the diet of such population. An interesting example of green chemistry is the use of amla fruit extracts as a natural reagent to determine the ferric iron in foods by simple flow injection spectrophotometric proce­ dure (Jaikrajang et al., 2018). Their findings were in close agreement with the traditional official ICP-OES technique at a 95% confidence level. As a green chemistry approach, many attempts have been made to develop plant-based insecti­ cides against mosquitoes. Pavela et al. (2019) have recently reviewed the plant-based, environmen­ tally friendly insecticides to control the growth and development of mosquito larvae and singled out 400 plant species; 29 of them had outstanding larvicidal activity (i.e., LC50 value below 10 ppm), against mosquito species. The chemical constituents responsible for this insecticidal activity have been attributed to a host of bioactives present in plants, such as sequiterpenes, triterpenes, alkaloids, alkamides, coumarins, flavonoids, sterols, xanthones, anthraquinones, acetogenonins, and many aliphatics. There is also a possibility of developing plant-based products to repel other insects of anthropod species. Just to replace the costly fossil fuels, Singh et al. (2018a) by using response sur­ face methodology have developed a lubricant from P. emblica seed oil, which exhibited excellent lubricant additive with minimum wear debris in the oil under usual use conditions.

17.8

FUTURE RESEARCH NEEDED

Herbal plants are being used by a large number of people of the world for thousands of years for various ailments. Amla fruits are known to be rich in many bioactive compounds that are respon­ sible for the medicinal benefits to cure various disease conditions, but the quality of ingredients, their efficacy, and safety are not always evaluated under strict experimental protocols. More work

Indian Gooseberry (Phyllanthus emblica)

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is needed to develop specific markers that could be used for evaluating the molecular and bio­ logical mechanisms of these phytochemicals in providing health benefits to the consumers of amla fruits. As some of these phytochemicals have been shown to affect the gene expression, the nutrig­ enomic and metabolomic approaches should be applied to study the effect of amla fruits on various genes and baseline metabolic profiles. More research is warranted on the synergistic as well as side effects, when these herbal plants are used in combination with each other. It is equally important to investigate the extent of health benefits that could be obtained if the isolated phytochemicals present in amla fruits are consumed as pure compounds. The hydrolyzable tannins are quite complex molecules, and their biological activities and physio­ logical effects have been investigated using animal models, with very few studies with humans. The colon microbial metabolites of ellagitannins, such as urolithins, are absorbed into tissues, where they are shown to exhibit estrogenic and antiestrogenic activities, which need to be further inves­ tigated in humans who consume amla fruits. Also, the antioxidant and antimicrobial activities of amla fruit phenolics should be explored for use as natural food preservatives. In the case of plant-based insecticides, more studies to examine their efficacy under field condi­ tions and the epidemiological impact potentially arising from mosquito vector control operations are needed, especially to apply these chemicals to control ticks and mites. In another upcoming area, more research is warranted to examine the stability, efficacy, and toxicity, if any, from the use of amla fruit–based metallic nanoparticles.

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Singh, S., Singh, A.K., Joshi, S.K., Bagle, B.G., More, T.A. (2010). Effect of zero energy cool chamber and post-harvest treatments on shelf-life of fruits under semi-arid environment of Western India. Part 2. Indian gooseberry fruits. Journal of Food science and Technology, 47(4): 450–453. Singh, P.L., Singh, S. (2011). Amla (Emblica officinalis) drying in solar-assisted heat pump dryer. International Energy Journal, 12: 253–262. Singh, V., Singh, P., Sharma, A., Choudhary, P., Singla, A., Singh, S.K. (2018a). Optimization of wear and friction characteristics of Phyllanthus emblica seed oil-based lubricant using response surface method­ ology. Egyptian Journal of Petroleum, 27: 1145–1155. Singh, S., Verma, V., Yadav, R., Singh, B. (2018b). Pharmacognostical study of amalaka (Emblica officinalis Gaertn.). Journal of Pharmacognosy & Phytochemistry, 7(3): 3476–3480. Srinivasan, M. (1944). Vitamin C in plants: Indian gooseberry (Phyllanthus emblica) – (A letter to the editor). Nature, 153: 684. Srinivasan, P., Vijaykumar, S., Kothandaraman, S., Palani, M. (2018). Anti-diabetic activity of quercetin extracted from Phyllanthus emblica L. fruit: In silico and in vivo approaches. Journal of Pharmaceutical Analysis, 8: 109–118. Srivastava, R.P., Srivastava, R.K. (1964). Chemical composition of fresh and dried aonla fruits. Science & Culture, 30(9): 446–447. Srivasuki, K.P. (2012). Nutritional and health care benefits of amla. Journal of Pharmacognosy, 3(2): 147–151. Steinberg, D., Parthasarathy, S., Carew, T.E., Khoo, J.C., Witztum, J.L. (1989). Beyond cholesterol: Modification of low-density lipoproteins that increase its atherogenicity. New England Journal of Medicine, 320: 915–924. Sugino, T., Yasuda, N., Hlahla, H., Teetamu, P.R., Aoi, N., Reka, R.J. (2006). Composition or inhibiting plate­ let aggregation caused by thrombin. Japanese Patent No.: JP 2006335705 A. Suryanarayana, P., Anil Kumar, P., Saraswat, M., Petrash, J.M., Reddy, B.P. (2004). Inhibition of aldose reductase by tannoid principles of Emblica officinalis: Implications for the prevention of sugar cataract. Molecular Vision, 10: 148–154. Taneja, S., Parmar, S.M.S., Williamson, D., Jain, B.L. (1982). Changes in ascorbic acid content of amla fruit (Emblica officinalis) after fungal infection. Science & Culture, 48(6): 225–226. Tayel, A.A., Shaban, S.M., Moussa, S.H., Elguindy, N.M., Diab, A.M., Mazrou, K.E., Ghanem, R.A., El-Sabhagh, S.M. (2018). Bioactivity and application of plant seeds’ extracts to fight resistant strains of Staphylococcus aureus. Annals of Agricultural Sciences, 63: 47–53. Tewari, R., Kumar, V., Sharma, H.K. (2019). Physical and chemical characteristics of different cultivars of Indian gooseberry (Emblica officinalis). Journal of Food Science & Technology, 56(3): 1641–1648. Thilaga, S., Nair, R.R., Kannan, M.R., Ganesh, D. (2017). RAPD markers for screening shoot gall maker (Betousa stylophera Swinhoe) tolerant genotypes of amla (Phyllanthus emblica L.). Journal of Genetic Engineering & Biotechnology, 15: 323–330. Tirawanchai, N., Homongkol, P., Chansriniyom, C., Somkasetrin, A., Jantaravinid, J., Kengkoom, K., Ampawong, S. (2019). Lipid-lowering effect of Phyllanthus emblica and Alpinia galangal extracts on HepG2 cell line. PharmaNutrition, 9: 100153. Tung, Y.T., Huang, C.Z., Lin, J.H., Yen, G.C. (2018). Effect of Phyllanthus emblica L. fruit on methionine and choline-deficiency diet-induced nonalcoholic steatohepatitis. Journal of Food  & Drug Analysis, 26(4): 1245–1252. Vadde, R., Radhakrishnan, S., Kurundu, H.E.K., Reddivari, L., Vanamala, J.K.P. (2016). Indian gooseberry (Emblica officinalis Gaertn.) suppresses cell proliferation and induces apoptosis in human colon cancer stem cells independent of p53 status via suppression of c-Myc and cyclin D1. Journal of Functional Foods, 25: 267–278. Vaithiyanathan, V., Mirunalini, S. (2013). Chemo preventive potential of fruit juice of Phyllanthus emblica Linn. (amla) against mammary cancer by altering oxidant/antioxidant status, lipid profile levels and estrogen/progesterone receptor status in female Sprague-Dawley rats. Biomedical  & Preventive Medicine, 3: 357–366. Variya, B.C., Bakrania, A.K., Patel, S.S. (2016). Emblica officinalis (Amla): A review for its phytochemistry, ethnomedicinal uses and medicinal potential with respect to molecular mechanisms. Pharmacological Research, 111: 180–200. Verma, K.K., Jain, A., Rawat, R. (1984). Titrimetric determination of ascorbic acid using chloranil. Journal of the Association of Official Analytical Chemists, 67(2): 262–265. Verma, K.K., Palod, S. (1983). Determination of ascorbic acid in fruits and pharmaceuticals by titration with thallium (III). Mikrochimica Acta, II(5–6): 361–367.

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Verma, R.C., Gupta, A. (2004). Effect of pre-treatments on quality of solar-dried amla. Journal of Food Engineering, 65(3): 397–402. Verma, C., Ebenso, E.E., Bahadur, I., Quraishi, M.A. (2018). An overview on plant extract as environmentally sustainable and green corrosion inhibitors for metals and alloys in aggressive corrosive media. Journal of Molecular Liquids, 266: 577–590. Vidhya Malar, H.L., Mettilda Bai, S.M. (2009). Hepato-protective activity of Phyllanthus emblica against paracetamol induced hepatic damage in Wister albino rats. African Journal of Basic & Applied Sciences, 1(1–2): 21–25. Waheed, S., Fatima, I. (2013). Instrumental neutron activation analysis of Emblica officinalis, Terminalia bellirica, and Terminalia chebula for trace element efficacy and safety. Applied Radiation & Isotopes, 77: 139–144. Walia, K., Boolchandani, R. (2015). Role of amla in Type-2 diabetes mellitis – A review. Research Journal of Recent Sciences, 4: 31–35. Wang, W., Liu, T., Yang, L., Ma, Y., Dou, F., Shi, L., Wen, A., Ding, Y. (2019a). Study on the multi-tar­ gets mechanism of triphala on cardio-cerebral vascular disease based on network pharmacology. Biomedicine & Pharmacotherapy, 116: 108994. Wang, Y.C., Huang, X.Y., Chiu, C.C., Lin, M.Y., Lin, W.H., Chang, W.T., Tseng, C.C., Wang, H.M. (2019b). Inhibitions of melanogenesis via Phyllanthus emblica fruit extract powder in B16F10 cells. Food Bioscience, 28: 177–182. Wankhar, D., Devi, R.S., Ashok, I. (2014). Emblica officinalis outcome on noise stress induced behavioral changes in Wistar albino rats. Biomedicine & Preventive Nutrition, 4: 219–224. Yadav, P. (2018). Amla (Emblica officinalis) – Medicinal food and pharmacological activity. International Journal of Academic Research & Development, 3(4): 53–55. Yadav, S.S., Singh, M.K., Singh, P.K., Kumar, V. (2017). Traditional knowledge to clinical trials: A review on therapeutic actions of Emblica officinalis. Biomedicine & Pharmacotherapy, 93: 1292–1302. Yamakami, Y., Morino, K., Takauji, Y., Kasukabe, R., Miki, K., Hossain, M.N., Ayusawa, D., Fujii, M. (2019). Extract of Emblica officinalis enhances the growth of human keratinocytes in culture. Journal of Integrative Medicine, 17: 141–146. Yang, B., Liu, P. (2014). Composition and biological activities of hydrolysable tannins of fruits of Phyllanthus emblica. Journal of Agricultural & Food Chemistry, 62: 529–541. Yokozawa, T., Kim, H.Y., Kim, H.J., Tanaka, T., Sugino, H., Okubo, T., Chu, D.C., Juneja, L.R. (2007). Amla (Emblica officinalis G.) attenuates age-related renal dysfunction by oxidative stress. Journal of Agricultural & Food Chemistry, 55(19): 7744–7752. Zhang, Y.J., Nagao, T., Tanaka, T., Yang, C.R., Okabe, H., Kouno, I. (2004). Antiproliferative activity of the main constituents from Phyllanthus emblica. Biological & Pharmaceutical Bulletin, 27: 251–255.

18 Characteristics, Therapeutic

Amla (Indian Gooseberry) Potential, and Its Value Addition M. Selvamuthukumaran Hindustan Institute of Technology and Science

CONTENTS 18.1 18.2 18.3

18.4

Introduction ........................................................................................................................364

Physicochemical Components of Amla .............................................................................. 365

Therapeutic Benefits of Amla ............................................................................................. 365

18.3.1 Hepatoprotective Activity ..................................................................................... 365

18.3.2 Antidiabetic Activity ............................................................................................ 366

18.3.3 Antipyretic and Analgesic Activities .................................................................... 366

18.3.4 Antiulcer Activity ................................................................................................. 366

18.3.5 Antioxidant Activity ............................................................................................. 366

18.3.6 Anti-inflammatory Activity .................................................................................. 367

18.3.7 Immunomodulatory Activity ................................................................................ 367

18.3.8 Cardioprotective Properties .................................................................................. 367

18.3.9 Anticancer Activity............................................................................................... 367

18.3.10 Other Applications ................................................................................................ 368

18.3.10.1 Hair Growth ..................................................................................... 368

18.3.10.2 Antivenom Activity .......................................................................... 368

18.3.10.3 Dental Problems............................................................................... 368

18.3.10.4 For Treating Skin-Related Problems ............................................... 368

18.3.10.5 For Whitening of Skin ..................................................................... 368

18.3.10.6 Headache Problems.......................................................................... 368

18.3.10.7 Nausea and Gonorrhea ..................................................................... 368

18.3.10.8 Constipation-Related Problems........................................................ 369

18.3.10.9 Diarrhea ........................................................................................... 369

18.3.10.10 For Cholesterol and Dyslipidemia Problems ................................... 369

18.3.10.11 Respiratory Problems....................................................................... 369

18.3.10.12 Enhancement of Memory Power...................................................... 369

Processed Value-Added Products from Amla .................................................................... 369

18.4.1 Amla Juice ............................................................................................................ 369

18.4.2 Amla RTS ............................................................................................................. 369

18.4.3 Amla Squash ......................................................................................................... 371

18.4.4 Amla Jam .............................................................................................................. 371

18.4.5 Amla Sauce ........................................................................................................... 372

18.4.6 Amla Pickle .......................................................................................................... 373

18.4.7 Amla Candy .......................................................................................................... 373

18.4.8 Amla Preserve ...................................................................................................... 375

363

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18.4.9 Amla Powder ........................................................................................................ 375

18.4.10 Amla Chyawanprash ............................................................................................. 377

18.4.10.1 Preparation of Amla Chyawanprash ................................................ 377

18.5 Conclusion .......................................................................................................................... 377

References ...................................................................................................................................... 379

18.1 INTRODUCTION Amla, scientifically known as Emblica officinalis, is a fruit berry popular for its nutritional and therapeutic health benefits. The berry belongs to the family Euphorbiaceae and is widely grown in Asian continents. It is native to India, and it is also cultivated in other countries such as Sri Lanka, Pakistan, China, and Malaysia. There are different names used to identify this fruit: it can be called as amla in India and Germany; in Nepal by amba; in China by an mole; and in Malaysia by popok melaka. In India, there are two different varieties of amla, which are in use: first one is of very big size (Figure 18.1), which contains more juice, is widely cultivated, and is available across the different sectors of market, and the another variety is of small and wild, which has its limited usage in Indian markets. It is basically a tree, which can reach up to the height of 9–17 m having trunk with branches that can spread across the tree. The tree yields fruits, which looks greenish yellow in color with spherical shape having both smooth and hard texture. The berry contains five to six vertical stripes, which adds up aesthetic appeal for the consumers to procure this fruit several times. Ripening of berries takes place in autumn; the berries are harvested manually with hands after climbing the tree. The fruit is highly astringent and sour and contains lot of fibres with health benefits. The ripening period in India starts for this fruit from November and ends in February. Amla fruits can be used in dried and fresh forms; leaves, flowers, seeds, and root bark can also be used to avail significant health effects. The diameter of the fruit grown under Indian conditions ranges from 19 to 24 mm with a height of 16–19 mm. The mesocarp portion of the fruit seems to be yellowish in color, and endocarp looks yellowish brown. Taste wise, the mesocarp of fresh fruit is acidic, and the dried fruit is acidic cum astringent. The leaves of this tree look pale green with 7–10 mm longer size. The leaves contain several polyphenolic constituents; to name a few, they have got array of ellagic acid, amlic acid, phyllanti­ dine, and gallic acid, which can add up more therapeutic values. The seeds of amla fruits resemble dark brown color and contain essential oil of up to 15%. The oil obtained from seed is the source of various fatty acids such as steric, palmitic, meristic, oleic, and linolenic acids. The tree bark is around 10–12 mm thick with grayish brown color appearance, which contains components such as tannin and proanthocyanidin. The root of this tree contains ellagic acid and lupeol. The berries are dried under sun for time duration of 2 weeks, and they are grounded well to give fine flowing powders; these powders are incorporated with coconut oil as well as yogurt, which can be used to eliminate residues of hair product from curly hair. The routine use of this mixture helps

FIGURE 18.1 Amla berry.

Amla (Indian Gooseberry)

365

the hair to prevent from its breakage; it is used for effective hair strengthening and also to enhance the shiny nature of the hair. It also further avoids the hair premature graying; it is also used for maintaining healthy skin to the greater extent.

18.2

PHYSICOCHEMICAL COMPONENTS OF AMLA

The pulp extracted from fruit is found to possess moisture content of 80%, protein content of 0.4%, fat content of 0.1%, fiber content of 3.3%, carbohydrate content of 14%, minerals such as calcium content of 0.04%, phosphorus content of 0.01%, iron content of 1.1 mg%, and vitamin B3 content of 0.3 mg%. The pulp is the richest source of water-soluble vitamin, i.e., vitamin C, which accounts for 500–600 mg%. The vitamin C is 20 times higher than juice obtained from orange fruit. The amla fruit is enriched with several minerals and amino acids as compared with apple. The various health-promoting phenolic components can be isolated form fruit, which includes ellagic acid, gal­ lic acid, quercetin, chebulinic acid, chebulagic acid, and kaempferol (Habib-ur-Rehman et al., 2007; El-Desouky et al., 2008).

18.3

THERAPEUTIC BENEFITS OF AMLA

The amla contains several therapeutic benefits that are shown in Figure 18.2. The clinical studies to justify therapeutic potential of amla are described in detail in the following.

18.3.1 HEPATOPROTECTIVE ACTIVITY The extracts of amla fruits are assessed for their hepatoprotective activity by feeding the rats with amla fruit extracts and chyawanprash extracts, i.e., processed product obtained from amla fruits (Kumar et al., 2012). The rats are induced to liver damage by using carbon tetrachloride. The liver is assayed for its various enzymatic activities tests, viz., glutamate–pyruvate transaminase, lipid

Antidiabetic activity

Antipyretic & Antianalgesic activity

Antiulcer activity

Antioxidant activity

Antiinflammatory activity

Anticancer activity

Hepatoprotective & Cardioprotective activity

FIGURE 18.2

Therapeutic potential of amla.

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Asian Berries: Health Benefits

peroxides, and alkaline phosphatase, which is an indicator of liver injury. Their results showed that feeding of amla extracts significantly reduced the various enzyme activity levels in the CCl4­ induced rats. It also further reduced the fibrosis induction in rat model, thereby reducing the levels of hydroxyl proline (Kumar et al., 2012).

18.3.2 ANTIDIABETIC ACTIVITY The amla fruit has got excellent antidiabetic activity. It has been observed from research findings that feeding of extracts at 100 mg/kg body weight had significantly reduced the sugar level in alloxan-induced diabetic rat groups within time interval of 4 h. Suryanarayana et al. (2004) reported that the tannoid constituents of amla extracts have suppressed the diabetic cataract in animal model. The aldose reductase is further prevented, which is a cause for diabetes, so that lifestyle diseases can be prevented to a greater extent (Treadway Linda, 1994). The quercetin is extracted from amla berries with the help of methanol, and it is fed at 25–75 mg/ kg of body weight to streptozotocin-induced diabetic rats. It has been observed that after 1 month of feeding amla quercetin, the blood and urine glucose levels are significantly reduced in diabetic rats with enhanced level of insulin in plasma projecting strong antidiabetic activity (Srinivasan et al., 2018).

18.3.3 ANTIPYRETIC AND ANALGESIC ACTIVITIES The extract obtained from amla fruit had shown potent antipyretic and analgesic activities. These effects are confirmed by oral supplementation of amla extracts of 500 mg/kg, which is obtained by ethanol and water extraction of amla fruits. The analgesic effects have been observed in acetic acid–induced mice model after successful feeding of amla extracts (Burkill, 1966). The leaf decoc­ tion is used for treating fever in Malaysia, and seed extracts are also used for treating some fevers (Nadkarni and Nadkarni, 1999).

18.3.4

ANTIULCER ACTIVITY

Srikumar et al. (2005) have observed that amla has the power to act against ulcer, which has been proved in rat model, which is fed with methanol extract obtained from source of amla, which has shown ulcer-preventive and ulcer-curative effects in rats, because of its mucosal factor’s offensive and defensive actions in the biological system.

18.3.5 ANTIOXIDANT ACTIVITY The free radicals play a major role in lifestyle-related diseases. The formation of radicals may lead to tissue injury and can cause diseases. The antioxidants play a major role in combating such lifestyle-related diseases. In albino rats, the triphala (amla-based herbal powder) was fed orally at 1 g/kg/body weight in order to study the effect of the drug over cold stress–induced albino rats; their studies show that the drug obtained from amla source is having the potent power to alleviate cold-induced stress (Scartezzini et al., 2006). The reduction of stress may be ascribed to presence of vitamin C content, which has more antioxidant activity (Rao et al., 2005). The amla extracts are fed to the streptozotocin-induced diabetic rats; the regular feeding of amla extracts has shown reduced oxidative stress in diabetic rats with enhanced body weight gain, because of possessing strong free radical scavenging activities of amla extracts (Panda and Kar, 2003). The amla extracts contain potent antioxidants such as tannins in hydrolysable forms and vitamin C. The tannin constituents of amla, viz., pedunculagin, emblicanin A, and emblicanin B exhibited potent antioxidant activity both in vitro and in vivo (Sairam et  al., 2002).

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DHC-1, a herbal preparation made from Emblica officinalis, Glycyrrhiza glabra, Syzygium aromaticum, and Mangifera indica, has been tested for its antioxidant activity by inducing isoproterenol-induced myocardial infarction and cisplatin-induced renal damage in rats. It has been found that the product has reduced the adverse effect against kidney and heart, has reduced hepatic lipid peroxidation, and has enhanced the antioxidant enzymes, viz., superoxide dismutase (SOD) and catalase (CAT) activities in product-fed rat groups (Chaudhuri, 2002).

18.3.6 ANTI-INFLAMMATORY ACTIVITY The berries are used in Asian continents, viz., India, Malaysia, and China for curing inflammatory diseases; similar to berries, the leaves are also used for anti-inflammatory and antipyretic treat­ ments. The preventive effect of various solvent extractions of amla extracts against polymorpho­ nuclear leukocyte (PMN) in human model is studied. The results show that methanolic extracts of amla of 50 μg/mL have prevented leukotriene B4–induced movement of PMNs to the rate of 90%. The leaves also show good anti-inflammatory activities based on movement of PMNs in human platelets during the time of clotting (Kumar et al., 2012).

18.3.7 IMMUNOMODULATORY ACTIVITY Immunity is very much essential for preventing diseases; therefore, one should have strong immune system. Perianayagam et al. (2004) fed triphala to albino rats for assessing the immunity by means of neutrophil functions. It has been observed that the neutrophil function is very much stimulated in the rats as a result of oral administration of triphala, and it has been further proved that the stress-induced suppression in neutrophil function is further minimized by administration of triphala.

18.3.8 CARDIOPROTECTIVE PROPERTIES Sancheti et al. (2005) studied the cardioprotective activity of amla on oxidative stress–induced rat model; their findings proved that the antioxidants present in amla homogenates protected the heart and reduced the stress ascribed to oxidation process in rat models.

18.3.9

ANTICANCER ACTIVITY

The research studies show that the amla is having chemopreventive effects. The amla con­ tains major antioxidants such as tannins and flavonoids, which have chemopreventive prop­ erties. Deep et  al. (2005) found that berry extract showed chemopreventive activity in 7,12-dimethylbenz(a)anthracene (DMBA)-induced Swiss albino mice. The consumption of triphala had led to chemoprevention as a result of antioxidant status increment in rats (Kaur et al., 2005). The presence of polyphenol, i.e., gallic acid in triphala had suppressed the cancer cell growth (Veena et al., 2006). Sandhya et al. (2006) further proved that triphala had potential effect in reducing cancer in the mice; therefore, their studies recommended the use of triphala as an anticancer drug. The effect has also been tested in various human cancer cell lines, which shows that the berry extract is having the ability to prevent L929 cells, based on dose-dependent manner, the IC50 value is 16.5 μg/mL, and the extract is successful in prohibiting cell proliferations in vitro (Rajeshkumar et al., 2003). Haque et al. (2001) observed that polyphenol source from amla had induced apoptosis in DLA and CeHa cell lines and it also further inhibited DNA topoisomerase I in Saccharomyces cerevisae. The medicinal formulations of amla fruit had demonstrated cancer-preventive effect among patients (Zhang et al., 2003). The phenolic fractions obtained from sources of berries, leaves, and root exhib­ ited strong antiproliferative activities (Srikumar et al., 2007).

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Asian Berries: Health Benefits

The amla is having the potent power to prevent skin cancer, which is shown in Swiss albino mice. The mice were induced with DMBA, and they were fed with amla; it has been observed that the amla has supressed tumor growth and has minimized the incidence of tumor as well as papillomas cumulative number, which further shows that amla is having chemopreventive effect against skin cancer (Deep et al., 2005).

18.3.10 OTHER APPLICATIONS 18.3.10.1 Hair Growth The berry-derived oil is found to possess excellent hair growth–promoting properties. The fruits in dried form can be used as an ingredient for preparing shampoo and oil for hair growth. The hair color can be nourished with this berry, and it is a commercially acceptable tonic for hair growth since age-old period. The berries can be sliced and soaked in water overnight, and the extracts can be used for hair nourishment, so that the glowing hair can be obtained by using such extracts of berries (Kumar et al., 2012). 18.3.10.2 Antivenom Activity It has been reported that the extracts obtained from amla fruits neutralize poison of Vipera russellii, proving the antivenom activity. The in vitro and in vivo studies showed that the berry extracts neutralize the hemorrhage and anti-inflammatory activities. There has been no formed precipitating band across the snake venom and the extract projecting excellent snake venom neutralizing capacity (Jayaweera, 1980). It is an excellent antidote; thereby, poison can be reduced to a greater extent in living organisms. 18.3.10.3 Dental Problems The amla roots are finely ground, and the ground powder is consumed 10 g two times a day to cure dental-related problems. The juices extracted from leaves can be further applied as ear drops, which can also cure the tooth-related problems indirectly (Kumar et al., 2012). 18.3.10.4 For Treating Skin-Related Problems The bark paste obtained from the amla tree is a potent source for curing sores and wound-related problems; 10 g of bark paste can be applied for a period of 2–3 days; leaf extract juice can also solve this issue; the restoration of dashes can lead to healing (Biswas et al., 2001). 18.3.10.5 For Whitening of Skin Singh et al. (2008) proved that the extracts obtained from berries can eradicate the skin pigments as well as lighten the skin from global consumers, viz., Asia, Middle East continents, and so on. The amla-based cosmetic products available in European markets are utilized for removing freckles and age-related spots in skin. 18.3.10.6 Headache Problems The paste obtained from amla fruits can be applied in forehead for curing cephalalgia. The insanity and fits can be cured by using the juice along with other ingredients (Suryanarayana et al., 2007); in Indonesia, the fruit can be applied on head for curing headache, and it is also being used for curing heat-caused dizziness problems. The berry pulp can be fortified in butter milk, and it can be applied as a natural coolant by applying on the head (Biswas et al., 2001). 18.3.10.7 Nausea and Gonorrhea The gonorrhea can be cured by consuming the bark juice with incorporated turmeric and honey ((Suryanarayana et al., 2007; Drury, 1873). The powder obtained from amla can be combined with honey and red sandal wood for stopping vomiting and nausea-related problems.

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18.3.10.8 Constipation-Related Problems The preserved fruits with either sugars or salt or in the form of pickles or fruits as such have a good laxative effect. The effect has been shown more in children; for children, the confectionery product is prepared from deseeded fruit pulp so that the constipation problem can be resolved (Kumar et al., 2012). 18.3.10.9 Diarrhea Diarrhea can be efficiently treated by using decoctions obtained from fruits. The fruit-based decoc­ tions can be incorporated into butter milk, and it can be given to people having dysentery problems. The decoction can be prepared form the roots, which is quiet astringent and can significantly cure diarrhea, when the extracts are taken along with fenugreek seeds (Suryanarayana et al., 2007). 18.3.10.10 For Cholesterol and Dyslipidemia Problems The research findings demonstrate that the amla is the potent source for reducing the cholesterol and LDL oxidation both in vitro and in vivo. The atherosclerosis and hypercholesterolemia can be prevented by consuming amla fruits (Anila and Vijayalakshmi, 2002). The flavonoid content of the fruit drastically reduces the serum and tissue lipid levels in hyperlipidemia-induced rats. The cho­ lesterol is eliminated to the greater extent (Thakur et al., 1989). 18.3.10.11 Respiratory Problems The lung inflammation can be consumed by using fresh fruits. The amla juice can be mixed up with honey and given for patients suffering from more cough, and it will also aid in good respiration, asthma, and other diseases (Suryanarayana et al., 2007). 18.3.10.12 Enhancement of Memory Power The research studies proved that the amla is having the potent power to enhance memory power in rats particularly both in young and in adult rats. The amla-based products can also cure Alzheimer’s disease and improve cognition, and it also aids in memory deficit reversal (Alam and Gomes, 2003).

18.4

PROCESSED VALUE-ADDED PRODUCTS FROM AMLA

The amla can be processed into wide variety of products, viz., juice, squash, RTS (ready-to-serve), jam, pickles, candy, preserve, and so on, such that the tart taste can be reduced and consumer accept­ ability can be increased. The detailed preparation of these products is described in the following.

18.4.1

AMLA JUICE

For preparing amla juice (Figure 18.3, Table 18.1), the berries are sorted and washed to remove the adhering surface microorganisms, seeds are removed, and segments are separated. For extraction of juice, water is added equally to the weight of the segments taken; the mixture is sent to the pulper for extraction of pulp; the pulp is filtered to get fine juice and boiled at 80°C to kill pathogenic microbes; potassium metabisulphite is added to prevent browning and also to kill microbes, filled in sterilized glass bottles, capped airtight, pasteurized at 82°C for 15–20 min, and cooled and stored until marketing.

18.4.2

AMLA RTS

The amla RTS (Figure 18.4) is a beverage product that can be especially prepared from extracted amla juice with added sugar to increase its palatability and consumer acceptability. The RTS can be fortified with lemon and ginger juice, which can reduce the tart taste and add up flavor to the final

370

FIGURE 18.3

Asian Berries: Health Benefits

Amla juice.

TABLE 18.1 Ingredients Used for Preparing Amla Juice Name of the Ingredient Amla Water Potassium metabisulfite

FIGURE 18.4 Amla RTS. RTS, ready-to-serve.

Quantity (g) 10 kg 10 L 65 g

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371

TABLE 18.2 Ingredients Used for Preparing Amla RTS Name of the Ingredient Amla juice Ginger juice Lemon juice Sugar Citric acid Water RTS, ready-to-serve

FIGURE 18.5

Quantity (g) 10 L 1L 2L 16 kg 220 g 77 L

Amla squash.

product. The desired amount of juice (Table 18.2) is mixed with sugar, citric acid, and water; the mixture is stirred well, blended with ginger and lemon juice, filled in sterilized glass bottle, capped airtight, pasteurized at 80°C for a period of 20 min, and cooled and stored until marketing. This beverage will enhance health for the body of consumers in addition to its nutritive value.

18.4.3 AMLA SQUASH For preparation of squash (Figure 18.5), sound berries are sorted, washed well, steam-blanched for 10–15 min, and deseeded; pulp is extracted, and the desired amount of pulp (Table 18.3) is mixed with strained acidified sugar syrup, which is prepared by boiling sufficient amount of sugar with cit­ ric acid; preservatives such as KMS/sodium benzoate are added; color is added for esthetic appeal; the mixture is filled hot in sterilized bottles, crown-capped to exclude air and also to increase the stability, cooled. and stored until its use. The squashes prepared from amla quench the thirst of the consumers and further add up the functional value to the consumers.

18.4.4 AMLA JAM It is a delicious product liked by children; it can also be used as bread spread. The jam (Figure 18.6, Table 18.4) can be produced by utilizing this medicinal berry. The berries are sorted and washed well; segments are separated and pulped; and the pulp is having tart taste; therefore, it can be

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TABLE 18.3 Ingredients Used for Preparing Amla Squash Name of the Ingredient Amla pulp Sugar Citric acid Water Sodium benzoate

FIGURE 18.6

Quantity (g) 10 kg 14 kg 250 g 10 L 23 g

Amla jam.

TABLE 18.4 Ingredients Used for Preparing Amla Jam Name of the Ingredient Amla pulp/guava pulp Sugar Citric acid Water Pectin

Quantity (g) 10 kg 7.5 kg 45 g 1.5 L 10 g

equally blended with guava pulp, which is the source of pectin. The blended pulp is boiled in stain­ less steel kettle, followed by addition of desired pectin and citric acid; the contents are boiled until total soluble solid (TSS) reaches 68.5°Brix. The jam is cooled slightly, filled into sterilized glass bottles up to brim, capped airtight, and stored.

18.4.5

AMLA SAUCE

Healthy sauce can be prepared from amla berries (Figure 18.7) as like tomato sauce. The ingredi­ ents used for preparing amla sauce are given in Table 18.5. The amla pulp is transferred to stainless

Amla (Indian Gooseberry)

FIGURE 18.7

373

Amla sauce.

TABLE 18.5 Ingredients Used for Preparing Amla Sauce Name of the Ingredient Amla pulp Sugar Salt Onion Garlic Chilli powder Cinnamon, cardamom, aniseed, cumin Mint and ginger

Quantity (g) 10 kg 750 g 100 g 500 g 50 g 50 g 100 g Optional

steel kettle, followed by addition of sugar and salt; the contents are boiled well; during this period, the spices along with onion and garlic are taken in muslin cloth, and they are tied tightly and dipped in boiling kettle. They are boiled well along with added spice bag; during this time, the bags are squeezed to release the spice extracts in the boiling contents, so that the spicy flavor is incorporated into the boiling sauce, until desired TSS reaches; the contents are transferred hot into sterilized glass bottles, filled up to the neck, crown-capped, and stored.

18.4.6

AMLA PICKLE

Amla pickle (Figure 18.8) goes well with curd rice especially in South Indian cuisine. The pickles are prepared by using ingredients shown in Table 18.6. The berries are sorted, cleaned well, and steam-cooked for 5–10 min; kept aside, the enlisted spices are fried in oil, followed by transferring the cooked amla; once again the amla is well cooked in oil for 5 more minutes and filled into steril­ ized glass jars; the contents are filled with sesame or edible oil, so that the oil can act as a natural preservative and the stability can be enhanced up to 8 months.

18.4.7 AMLA CANDY It is a confectionery kind of product liked by kids (Figure 18.9). The product is prepared from amla berries, which provide rich source of vitamin C to the consumers (Table 18.7). The amla

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Asian Berries: Health Benefits

FIGURE 18.8 Amla pickles.

TABLE 18.6 Ingredients Used for Preparing Amla Pickle Name of the Ingredient Amla Salt Turmeric powder Red chilli powder Fenugreek Clove Edible oil

FIGURE 18.9 Amla candy.

Quantity (g) 10 kg 1.5 kg 100 g 100g 300 g 50 No’s 3.5 L

Amla (Indian Gooseberry)

375

TABLE 18.7 Ingredients Used for Preparing Amla Candy Name of the Ingredient Amla Sugar Water Citric acid Potassium metabisulfite

Quantity (g) 10 kg 11 kg 5L 64 g 12 g

berries are sorted, washed well in water, and dipped in 2% salt cum alum solution for 24 h. They are soaked in cold water for 1 h, steam-blanched for 15 min, and deseeded; segments are sepa­ rated. The sugar is weighed, and its weight should be equal to the weight of the amla segments taken. The weighed sugar is divided into two portions. The first portion is boiled in acidified sugar syrup along with segments; preservatives are added; and segments are steeped in sugar syrup for 24 h. Second leftover sugar is divided into again three parts: the first part is mixed with sugar syrup containing amla segments and again steeped for 24 h, and the remaining two parts are mixed after every 24 h. The steeping is stopped until segments containing sugar syrup reach 75°Brix. The segments are washed well with water, and they are drained and dried using cabinet drier at 60°C for a period of 5–6 h. The pieces are packed in high-density polyethylene bags and stored under proper condition.

18.4.8

AMLA PRESERVE

It is a product prepared from sound amla berries, which are boiled under thick sugar syrup until they become soft and transparent (Figure 18.10, Table 18.8). The berries are sorted and washed in water followed by soaking them in salt and alum solution (2% each) for removing astringency taste; before soaking, they are pricked with fork or needle, so that after sugar addition, the berries may absorb sugar solution and prevent the product from spoilage. The berries are blanched to take utmost care that segments will not break during blanching; equal amounts of amla and sugar are taken; sugar is added to the fruit in alternate layers so that the berries can naturally absorb moisture present in surface of the fruit and convert the sugar into solution after 24 h of standing period. At this stage, the TSS of the sugar solution will be around 38°Brix. On the second day, the sugar syrup is boiled to increase its TSS to around 60°Brix; at this stage, to prevent syrup crystallization, the citric acid is added and boiled for 5–10 min and allowed to rest for another 24 h. Again on the third day, the process is carried out similarly by raising the sugar syrup strength to 70°Brix; after that, amla ber­ ries are allowed to steep in the sugar solution for a period of 1 week. During this time, the amla berries nicely absorb sugar syrup and become tender; the product is ready and is packed in airtight glass jar or containers for marketing.

18.4.9 AMLA POWDER In North India, it can be called as amla churna (Figure 18.11), which can be prepared from dried amla berries, which contain rich source of vitamin C. This powder can be prepared by crushing the dried segments of amla berries. This powder is widely used for preparing several Ayurvedic formu­ lations. It has hot excellent curative properties; it can be a potent source of medicines for digestion, boosting immune power, constipation, several skin ailments, and so on. The amla powder has quiet bitter taste, which can be reduced by incorporating with either honey or ginger or lemon during its consumption. This powder is also used in cosmetic preparations for nourishing long hair growth. It

376

Asian Berries: Health Benefits

FIGURE 18.10 Amla preserve.

TABLE 18.8 Ingredients Used for Preparing Amla Preserve Name of the Ingredient Amla Sugar Water Citric acid Salt Alum

FIGURE 18.11

Amla powder.

Quantity (g) 10 kg 10 kg 10 L 15 g 200 g 200 g

Amla (Indian Gooseberry)

377

is a good antioxidant that can relieve oxidative stress and calm down and relax the nerves. It con­ tains more amounts of tannins, flavonoids, and iron.

18.4.10 AMLA CHYAWANPRASH It is a popular Ayurvedic product made out of amla with several blended Indian herbal extracts, flavored with a variety of spices with incorporated sweeteners; finally the contents are boiled to give thick consistency (Figure 18.12, Table 18.9). This product is having the stability of around 1 year and is commercially available in Indian markets. 18.4.10.1 Preparation of Amla Chyawanprash Amla is the major ingredient for preparing chyawanprash. The amla berries are taken, washed well, transferred to muslin cloth; the cloth is tied well and placed in larger boiling pans; all the boiling herbal ingredients are taken in that pan; the contents are boiled for 2 h by addition of 24 L water; after that, the extracts are allowed to settle overnight; the next day, the amla berries are separated, and the boiled herbal extracts are filtered and kept aside. The tied muslin cloth is opened; the amla berries are inspected for their black color and deseeded; segments are separated and crushed well to a thick paste; the paste is allowed to pass through strainer of bigger size sieves, so that fiber can be separated. For preparing the product, the strained pulp is mixed with herbal extracts, stirred well, and boiled in iron vessel with incorporated ghee, followed by addition of sugar; once the sugar is fully dissolved, the sesame oil is added; Various flavoring spice ingredients are grinded to a fine powder and added to the boiling product followed by addition of honey and saffron. The boiling is stopped until thick consistency is achieved. The contents are slightly cooled, and after cooling, the product can be packed in either plastic or glass jars. The product is a rich source of several phytoconstituents; therefore, it boosts immunity, enhances antioxidant status of consumers, improves cognition, detoxi­ fies heavy metals, scavenges free radicals, and supports heart, liver, and skin health.

18.5

CONCLUSION

There is a lot of scope for adding value to the wonderful berry, i.e., amla; the industries can utilize such nature’s gift and convert the berry into several value-added processed food products in the future. The products can be made easily available to the consumers through commercialization, so that the consumers can enhance their health to a greater extent.

FIGURE 18.12 Amla chyawanprash.

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Asian Berries: Health Benefits

TABLE 18.9 Ingredients Used for Preparing Amla Chyawanprash Name of the Ingredient Amla Boiling Ingredients Vidarikand White Indian sandalwood Malabar nut Pyrethrum root Asparagus Brahmi Bael Chebulic myrobalan Lotus saffron Spikenard Gulancha tinospora Wild turmeric Nutgrass Clove Orris root Dhasmula (blended roots of shrubs and trees) Jivanthi Red spiderling Fig Ashwagandha Tulsi Curry leaf Indian tinospora Dried ginger Dried grapes Liquorice Kakadsinghi Flavoring Ingredients Indian long pepper Cinnamon Indian bay leaf Small cardamom Indian rose-chestnut Tabashir Saffron Honey Sugar Ghee Sesame seed oil

Quantity (g) 10 kg

100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g

200 g 100 g 40 g 40 g 40 g 300 g 4g 500 g 6 kg 500 g 500 g

Amla (Indian Gooseberry)

379

REFERENCES Alam, M.I. and A. Gomes, 2003. Snake venom neutralization by Indian medicinal plants (Vitex A. negundo and Emblica officinalis) root extracts. J Ethnopharmacol., 86(1): 75–80. Anila, L. and N.R. Vijayalakshmi, 2002. Flavonoids from Emblica officinalis and Mangifera indica – effec­ tiveness for dyslipidemia. J Ethnopharmacol., 79(1): 81–7. Biswas, N.R., S.K. Gupta, G.K. Das, N. Kumar, P.K. Mongre, D. Haldar and S. Beri, 2001. Evaluation of oph­ thacare eye drops-a herbal formulation in the management of various ophthalmic disorders. Phytother Res., 15(7): 618–20. Burkill, I.H., 1966. A Dictionary of the Economic Products of the Malay Peninsula, Vol. 1. Ministry of Agriculture and Co-operatives, Kuala Lumpur. Chaudhuri, R.K., 2002. Emblica cascading antioxidant: a novel natural skin care ingredient. Skin Pharmacol Physiol., 15(5): 374–80. Deep, G., M. Dhiman, A.R. Rao and R.K. Kale, 2005. Chemopreventive potential of Triphala (a composite Indian drug) on benzo(a)pyrene induced for stomach tumorigenesis in murine tumor model system. J Exp Clin Cancer Res., 24(4): 555–63. Drury, H., 1873. The Useful Plants of India; with Notices of Their Chief Medicinal Value in Commerce, Medicine and the Arts. Higginbotham and Co, Madras. El-Desouky, S.K., S.Y. Ryu and Y.K. Kim, 2008. A new cytotoxic acylatedapigeninglucoside from Phyllanthusemblica L. Nat Prod Res., 22(1): 91–5. H.U. Rehman, K.A. Yasin, M.A. Choudhary, N. Khaliq, A.R. Atta-ur-Rahman, M.I. Choudhary and S. Malik, 2007. Studies on the chemical constituents of Phyllanthusemblica. Nat Prod Res., 21(9): 775–81. Haque, R., B. Bin-Hafeez, I. Ahmad, S. Parvez, S. Pandey and S. Raisuddin, 2001. Protective effects of Emblica officinalis Gaertn. in cyclophosphamide treated mice. Hum Exp Toxicol., 20(12): 643–50. Jayaweera, D.M.A., 1980. Medicinal Plants used in Ceylon Part 2. National Science Council of Sri Lanka, Colombo. Kaur, S., H. Michael, S. Arora, P.L. Härkönen and S. Kumar, 2005. The in vitro cytotoxic and apoptotic activ­ ity of Triphala – an Indian herbal drug. J Ethnopharmacol., 97(1): 15–20. Kumar, A., A. Singh and J. Dora, 2012. Essentials perspectives for Emblica officinalis. Int J Pharm Chem Sci., 1 (1): 11–8. Nadkarni, K.M. and A.K. Nadkarni, 1999. Indian Materia Medica – With Ayurvedic, Unani-Tibbi, Siddha, Allopathic, Homeopathic, Naturopathic and Home Remedies, Vol. 1. Popular Prakashan Private Ltd., Bombay. ISBN No. 81-7154-142-9. Panda, S. and A. Kar, 2003. Fruit extract of Emblica officinalis ameliorates hyperthyroidism and hepatic lipid peroxidation in mice. Pharmazie, 58(10): 753–5. Perianayagam, J.B., S.K. Sharma, A. Joseph and A.J. Christina, 2004. Evaluation of anti-pyretic and analgesic activity of Emblica officinalis Gaertn. J Ethnopharmacol., 95(1): 83–5. Rajeshkumar N.V., M. Radahakrishna Pillai and R. Pillai, 2003. Induction of apoptosis in mouse and human carcinoma cell lines by Emblica officinalis polyphenols and its effect on chemical carcinogenesis. J Exp Clin Cancer Res., 22: 201–12. Rao, T.P., N. Sakaguchi, L.R. Juneja, E. Wada and T. Yokozawa, 2005. Amla (Emblica officinalis Gaertn.) extracts reduce oxidative stress in streptozotocin induced diabetic rats. J Med Food., 8(3): 362–8. Sairam, K., C.V. Rao, M.D. Babu, K.V. Kumar, V.K. Agrawal and R.K. Goel, 2002. Antiulcerogenic effect of methanolic extract of Emblica officinalis: an experimental study. J Ethnopharmacol., 82(1): 1–9. Sancheti, G., A. Jindal, R. Kumari and P.K. Goyal, 2005. Chemopreventive action of Emblica officinalis on skin carcinogenesis in mice. Asian Pac J Cancer Prev., 6(2): 197–201. Sandhya, T., K.M. Lathika, B.N. Pandey and K.P. Mishra, 2006. Potential of traditional ayurvedic formula­ tion, Triphala, as a novel anticancer drug. Cancer Lett., 231(2): 206–14. Scartezzini, P., F. Antognoni, M.A. Raggi, F. Poli and C. Sabbioni, 2006. Vitamin C content and antioxidant activity of the fruit and of the ayurvedic preparation of Emblica officinalis Gaertn. J Ethnopharmacol., 104(1–2): 113–8. Singh, D.P., R. Govindarajan and A.K. Rawat, 2008. High-performance liquid chromatography as a tool for the chemical standardisation of Triphala – an ayurvedic formulation. Phytochem Anal., 19(2): 164–8. Srikumar, R., N.J. Parthasarathy and D.R. Sheela, 2005. Immunomodulatory activity of Triphala on neutro­ phil functions. Biol Pharm Bull., 28(8):1398–403.

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Srikumar, R., N.J. Parthasarathy, E.M. Shankar, S. Manikandan, R. Vijayakumar, R. Thangaraj, K. Vijayananth, D.R. Sheela and U.A. Rao, 2007. Evaluation of the growth inhibitory activities of Triphala against common bacterial isolates from HIV infected patients. Phytother Res., 21(5): 476–80. Srinivasan. P., S. Vijayakumar, S. Kothandaraman and M. Palani, 2018. Anti-diabetic activity of quercetin extracted from Phyllanthus emblica L. fruit: in silico and in vivo approaches. J Pharm Anal., 8(2): 109–118. Suryanarayana, P., P.A. Kumar, M. Saraswat, J.M. Petrash and G.B. Reddy, 2004. Inhibition of aldose reduc­ tase by tannoid principles of Emblica officinalis: implications for the prevention of sugar cataract. Mol Vis., 10: 148–54. Suryanarayana, P., M. Saraswat, J.M. Petrash and G.B. Reddy, 2007. Emblica officinalis and its enriched tan­ noids delay streptozotocin-induced diabetic cataract in rats. Mol Vis., 13: 1291–7. Thakur, R.S., H.S. Puri and A. Husain, 1989. Major Medicinal Plants of India. Central Institute of Medicinal and Aromatic Plants, Lucknow. Treadway, L., 1994. Amla traditional food and medicine: herbal gram. J Amer Bot Coun., 31: 26. Veena, K., P. Shanthi and P. Sachdanandam, 2006. The biochemical alterations following administration of Kalpaamruthaa and Semecarpus anacardium in mammary carcinoma. Chem Biol Interact., 161(1): 69–78. Zhang, L.Z., W.H. Zhao, Y.J. Guo, G.Z. Tu, S. Lin and L.G. Xin, 2003. Studies on chemical constituents in fruits of Tibetan medicine Phyllanthus emblica. Zhongguo Zhong Yao Za Zhi., 28(10): 940–3.

19

Pepper Berries (Piper nigrum L.) – Drupes with Therapeutic and Nutraceutical Potential Vanshika Adiani and Prasad S. Variyar Bhabha Atomic Research Centre

CONTENTS 19.1 19.2

Introduction ........................................................................................................................ 381

Chemistry of Pepper ........................................................................................................... 382

19.2.1 Pepper Oleoresin ................................................................................................... 382

19.2.2 Volatile Essential Oils .......................................................................................... 383

19.2.3 Pungent Principles ................................................................................................ 384

19.2.4 Phenolic Compounds ............................................................................................ 385

19.3 Processing and Quality Characteristics .............................................................................. 386

19.4 Quality Evaluation .............................................................................................................. 387

19.4.1 Microbial Contamination and Insect Infestation .................................................. 387

19.4.2 Postharvest Preservation of Pepper Berries.......................................................... 388

19.5 Medicinal Properties and Health Benefits .......................................................................... 389

19.6 Nutraceutical Significance .................................................................................................. 389

19.6.1 Antioxidant Properties .......................................................................................... 390

19.6.2 Anticancer Activities ............................................................................................ 390

19.6.3 Anti-inflammatory Activities ............................................................................... 391

19.6.4 Digestive Activities ............................................................................................... 391

19.6.5 Bioavailability Enhancement Activities ............................................................... 391

19.6.6 Hepatoprotective Activities .................................................................................. 392

19.7 New Products Developments .............................................................................................. 393

19.8 Future Outlook .................................................................................................................... 394

References...................................................................................................................................... 395

19.1

INTRODUCTION

Pepper (Piper nigrum L.) plant is a perennial woody, climbing, and flowering vine belonging to the family Piperaceae. It reaches a height of 10 m (33 ft) by means of its aerial roots. The fruit, known as peppercorn, is dried and used as a spice and seasoning. The word pepper has its origin from the Sanskrit word pippali, meaning berry. It is about 5 mm (0.20 in.) in diameter when fresh and fully mature, is dark red in color, and contains a single seed, like all drupes. The fruit and the dried ground form may be described simply as pepper or more precisely as green pepper (dried unripe fruit), black pepper (cooked and dried unripe fruit), or white pepper (ripe fruit seeds). Pepper is often described as the “king of spices” and shares a place along with salt on most dinner tables. The spice has its origin in southern India and Sri Lanka but is now cultivated in other countries where 381

382

Asian Berries: Health Benefits

FIGURE 19.1

Green pepper berries.

uniform warm temperatures and moist soil conditions prevail. The plant requires a long rainy sea­ son and fairly high temperatures. It is sometimes grown interspersed within coffee and tea planta­ tions. It is propagated by stem cuttings that are planted near a tree or a pole to serve as a support. They start bearing the berries in 2–5 years (Figure 19.1) and may continue to do so for as long as 40 years (Devasahayam et al., 2015). Black pepper is one of the earliest spices known and is native to the Malabar Coast of India. It was widely cultivated in the tropics of Southeast Asia during early historic times where it became highly regarded as a condiment. It was an important trade commodity, often serving as a medium of exchange. In ancient Greece and Rome, taxes were levied in pepper. In the middle ages, the Venetians and the Genoese were the main distributors of pepper in Europe, and their monopoly prompted the search for an eastern sea route. In 1497, Portuguese explorer Vasco da Gama reached India, thereby opening the trade route for pepper, among many other spices. The plant is widely cultivated in Indonesia and also has been introduced into tropical areas of Africa and the Western Hemisphere. Over 100 cultivars of pepper (P. nigrum L.) are grown all over the world. Some popu­ lar varieties are listed in Table 19.1. The global pepper market is largely dominated by Vietnam, which contributes over 42% of the world’s pepper production and the largest share in exports. The global production of black pepper in 2018 is reported to be around 523,400 Mt of which Vietnam accounted for 218,000 Mt followed by India at 92,000 Mt and Brazil and Indonesia at 82,000 and 67,000 Mt, respectively. Cambodia has in recent years gained significant impact on the global supply of the commodity with an eight­ fold increase since 2013. The majority of the quality pepper finds its way to Europe and the United States, which is the prominent market for close to 30% of the world’s pepper output (Gulick, 2018).

19.2

CHEMISTRY OF PEPPER

19.2.1 PEPPER OLEORESIN Oleoresin can be defined as an organic extract containing the odor, flavor, and pungent principle of the spice. Organoleptic properties of oleoresin are principally determined by its essential oil (EO) and piperine content that in turn is influenced by the quality of the raw material used for its prepara­ tion. In general, the average yield of pepper oleoresin is 10%–13% containing 15%–20% volatile EO and 35%–55% piperine (Borges & Pino, 1993). Indian Malabar and Tellicherry black pepper as well as Lampong and Sri Lankan black pepper with high oleoresin content are generally more valued commercially (Nambudiri et al, 1975). Acetone, ethanol, or dichloroethane are the solvents gener­ ally used for preparation of oleoresin. However, as acetone and ethanol are both miscible in water, they can extract water-soluble nonflavor substances such as polysaccharides and gums. On the other hand, dichloroethane is water immiscible and free from the aforementioned disadvantages and is

Pepper Berries (Piper nigrum L.)

383

TABLE 19.1 Popular Varieties of Pepper Cultivar Karimunda Kottanadan Narayakodi Aimpiriyan Neelamundi Kuthiravalli Balankotta Kalluvalli Panniyur-1 Panniyur-2 Panniyur-3 Panniyur-4 Panniyur-5 Subhakara Sreekara Panchami Kuching Semongok Emas Semongok Perak Nyerigai Daun Lebar Vinh Linh Tieu Se Bragantina

Country of Origin Kerala, India Plains and hilly region, India Throughout India Plains and hilly region, India Hilly areas, India Kerala, India Kerala, India Kerala, India Suitable to all pepper-growing areas, India Kerala, India Kerala, India Kerala, India Suitable to all pepper-growing areas, India Kerala and Karnataka, India All pepper-growing areas, India Kerala, India Sarawak, Malaysia Sarawak, Malaysia Sarawak, Malaysia Sarawak, Malaysia Philippines Vietnam Vietnam Brazil

Distinguishing Attributes Good yielder High yielding, high quality Medium yield, small fruit High yielding, late maturing Moderate yielding, medium quality Long spikes, high quality Medium yielding, medium quality Medium yielding, medium quality Long spikes with large berries Shade tolerant Late maturing Sustains adverse condition Disease tolerant High quality Adapt to varying conditions Late maturing Most popular, high yielder Stable yield, even ripening Early yielding, disease resistant High yielding High yielding, high quality Stable yield, disease resistant Stable yield, early fruit setting Long spikes with large berries

thus the preferred solvent. Freshly prepared pepper oleoresin is dark green, viscous, heavy liquid with a strong aroma. Oleoresins overcome several disadvantages inherent in the use of dry pow­ dered spices such as presence of thermoresistant bacteria, nonuniformity of flavor quality, random intense spots distribution throughout the product, and loss of aroma components during storage. They are also convenient to use in food industries and institutional cooking. Well-made oleores­ ins overcome all the aforementioned drawbacks and additionally save transport and storage space. However, as the concentration is sometimes too high for suitable measurement and distribution in the food, they are dispersed in suitable bases, such as sugar or salt, which mix easily with the food (Govindarajan & Stahl, 1977)

19.2.2 VOLATILE ESSENTIAL OILS Plants of the piper species are rich in EOs. A wide variation in the content and distribution of chemical constituents of the EOs obtained from various parts of the plant such as fruits, seeds, leaves, branches, roots, and stems has been reported. In general, sesquiterpene hydrocarbons and oxygenated compounds constitute the most important chemical classes in fruits and aerial parts. The chemical composition is highly variable and depends on plant part, geographical differences, environmental conditions, and chemotypes (Mgbeahuruike et al., 2017; Thin et al, 2018). The outer skin of black pepper that accounts for 25% of the dry berry weight is composed mainly of fibers and EO cells. White pepper prepared by removal of outer skin has less fiber and lower EO contents. Thus, while the volatile oil content of black pepper ranges from 0.6% to 2.6%, the volatile content of white pepper usually ranges from 0.5% to 1.0%. The volatile oil generally obtained by

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Asian Berries: Health Benefits

steam distillation is water-white or pale greenish-gray mobile liquid that becomes viscous on aging. Its odor is described as fresh, dry-woody, and warm-spicy that lacks the pungency of the spice (Purseglove et al., 1981). Approximately 135 compounds have been identified in the EOs of black pepper by gas chromatography–mass spectrometry. Mono- and sesquiterpenes are the main con­ stituents of the oil, whereas the other chemical classes are present in lower amounts. Monoterpene hydrocarbons and sesquiterpene hydrocarbons range from 70%–80% to 20%–30% of the volatile oil constituents, respectively, with oxygenated constituents accounting for less than 4% of the oil. α-Pinene, β-pinene, and limonene are the major monoterpene hydrocarbons constituting up to 13%, 40%, and 32% of the monoterpene hydrocarbon fraction, whereas β-caryophyllene accounts for 22% of sesquiterpene hydrocarbons. Oxygenated compounds, although present in relatively small amounts, are known to play an important role in contributing to the organoleptic properties of the spice (Purseglove et al., 1981). Lower oil yields and low monoterpene contents have been reported from old damaged pep­ per corns, whereas those from trade wastes (husks, pinheads, etc.) yield a low cost product in the range of 0.6%–1.0% devoid of monoterpenes. The average yield of volatile oil obtained from green pepper is found to be approximately 0.43% (wet weight basis) although a wide variation in the content depending on the varieties used is noted. However, no significant qualitative and quantita­ tive changes have been observed in the volatile oil constituents between black and green pepper (Purseglove et al., 1981). Distribution of the volatile oil in different parts of the pepper plant showed that the stalk accounted for the lowest oil content (0.03%) followed by leaf (0.12%) and berries (0.43%) on a wet weight basis. Leaf oil was essentially constituted of sesquiterpenes with a very low content of monoterpenes and was devoid of pepper aroma while stalk was dominated by high boiling constituents that had a low peppery note (Bandyopadhyay & Variyar, 1988). Use of gas chro­ matography for the determination of total terpene hydrocarbons, total sesquiterpene hydrocarbons, and the ratio of sabinene + β-pinene + δ-3-carene to caryophyllene for use as indicators of pepper oil characteristics has been proposed as a good method for characterization of EOs from various sources (Govindarajan & Stahl, 1977).

19.2.3

PUNGENT PRINCIPLES

The major pungent principle of pepper has been identified as piperine ([1-[5-[1, 3-benzodioxol-5-yl]-1-oxo-2,4, pentadienyl]piperidine) that accounted for 95% of the total pungent alkaloids present in the spice. Other minor alkaloids identified include piperettine, piperyline, pipero­ lein A and B, and piperanine (Gorgani et al., 2017). Based on the pungency of series of piperine analogs, it was found that while methylenedioxy group is not required for pungency, an aliphatic side chain containing a minimum of four atoms with an aromatic ring preferably at the terminal position is neces­ sary. Unsaturation in the side chain is not essential for pungency, but the presence of one double bond or two will result in improved taste (pungency). Compounds containing an amino group could replace piperidine without a loss of pungency. The linkage between the acids and the basic moiety should be acylamide (Govindarajan & Stahl, 1977). Piperine and its isomers have been estimated based on colorimetric, spectrometric, and alka­ line hydrolysis–distillation–titration methods. The colorimetric method is considered unreli­ able because of its large standard deviation. The spectrophotometry is the simplest method and takes the least time. It is based on the absorption of piperine in the UV region with a maximum at 345 nm that also corresponds to the total absorption maximum of pepper extract at 345 nm. The method, however, registers a piperine content that is too small as it does not include the isomers of piperine in the determination. The Labruyere’s alkaline hydrolysis–distillation– titration that determines all the alkaloids of pepper is the most accurate and reliable method (least standard deviation and high reproducibility) and is recommended for the estimation of pungent components of pepper. The method involves solvent extraction of the pepper to obtain the active constituents, subjecting the extract to alkaline hydrolysis at optimum conditions,

Pepper Berries (Piper nigrum L.)

385

followed by distillation with water and titration of the liberated piperidine (Govindarajan  & Stahl, 1977). Separation of piperine and its isomers by gas chromatography has also been demonstrated. Total content of piperine in different varieties of black pepper has been shown to range from 4.10% to 5.73%, whereas that in white pepper has been found in the range of 5.12%–8.54%. Green pepper on the other hand has been reported to have a piperine content of 3.8%–5.6% (Purseglove et al., 1981).

19.2.4

PHENOLIC COMPOUNDS

The phenomenon of blackening is a favorable attribute during processing of black pepper as it contributes to the color, luster, and flavor of the spice. Phenolic compounds have been shown to be the major contributor to blackening. Blackening occurs during sun drying of green pep­ per berries either by an enzymatic process involving action of polyphenol oxidase (PPO) on phenolic substrates present in green pepper or via a nonenzymatic phenomenon. The enzy­ matic process has been reported to be mainly caused by the catalytic oxidation of two pheno­ lic compounds inherently present in green pepper, namely, 3,4-dihydroxyphenylethanolglucosi de (A) and 3,4-dihydroxy- 6-[N-ethylamino] benzamide (B) by the pepper PPO characterized as O-diphenolase/pyrocatechase (Variyar et al., 1988; Bandyopadhyay et al., 1990). These phenolic substances were completely oxidized by inherent O-diphenolase and were absent in black pep­ per. Based on their ability to act as substrates toward pepper PPO, the phenolic constituents in green pepper were broadly classified into two groups: “enzyme inactive” and “enzyme active.” The former group comprised of nine phenolic acids that were identified as protocatechuic, gentisic, p-hydroxy benzoic, vanillic, caffeic, syringic, ferulic, synapic, and salicylic acids. As black pepper is commercially graded based on the degree of blackness/browness, enzyme active phenolics were proposed to be the major compounds responsible for appearance of the finished product. A ratio of the concentration of compound B to A was suggested to provide a rough estimate of the degree of blackness/brownness of the finished pepper corns (Variyar, & Bandyopadhyay, 1994). Among the different Indian green pepper varieties studied, the ratio of B to A followed the order Karimunda > Wayanadan > Geerakamundi > Nadesan > Balankotta = Panniyur-1. The intensity of blackening of the final dried black pepper corns also correlated well with the observed results with the former three varieties having an intense black color, whereas the latter three turned to brownish-black on sun drying. Based on the aforementioned observations, a high-performance liquid chromatogra­ phy method that provides a fingerprint chromatogram of phenolic compounds in crude extract of green pepper varieties has been proposed that could be used for grading black pepper of commerce (Variyar & Bandyopadhyay, 1994). While the total phenolic content decreased by approximately 75% in black pepper, no consistent relationship was observed between the degree of blackening and the content of enzyme active phe­ nolic compounds. Heat treatment by blanching, prior to sun drying, deepened the color of pepper berries although PPO activity significantly decreased. Thus, nonenzymatic oxidation of phenolic compounds and chlorophyll a and b as well as vitamin C degradation were further proposed to contribute to blackening (Gu et al., 2018a). An exhaustive study based on nontargeted metabolomics using a combination of liquid chromatography–tandem mass spectrometry (LC-MS/MS) and mul­ tivariate statistical analysis was used to screen the phenolic and polyhydroxy compounds in black, white, and green pepper berries. A total of 186 phenolic and polyhydroxy compounds that included anthocyanins, proanthocyanidins, catechin derivatives, flavanones, flavones, flavonols, isoflavones, flavone C-glycosides, hydroxycinnamoyl derivatives, quinate and its derivatives, and flavonolignan were identified. Fresh green pepper berries had the highest content of phenolic compounds, whereas blanched sundried white pepper and direct sundried black pepper had the lowest phenolic content. The possible roles of nonenzymatic oxidation of phenolic compounds, as well as chlorophyll a and b, and enhanced oxidation reactions resulting from degradation of vitamin C during drying were proposed (Gu et al., 2018b).

386

Asian Berries: Health Benefits

Schulz and Herramann (1980) isolated and identified nine phenolic acids, namely, caffeic, feru­ lic, gentisic, p-hydroxy benzoic, protocatechuic, salicylic, synapic, syringic, and vanillic acids as their glycosides in black pepper. White pepper with a total phenolic content one-fifth of black pep­ per (673 mg/kg) was reported to be devoid of caffeic, synapic, gentisic, and salicylic acids. Voagen and Herrmann (1980) reported the presence of considerable concentration of glycosides of kaemp­ ferol (1,500 mg), rhamnetin (600 mg), and quercetin (350 mg) with lesser amounts of isorhamnetin glycoside (