Next-Generation Algae, Volume 2: Applications in Medicine and the Pharmaceutical Industry 1119857287, 9781119857280

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Next-Generation Algae

Scrivener Publishing 100 Cummings Center, Suite 541J Beverly, MA 01915-6106 Publishers at Scrivener Martin Scrivener ([email protected]) Phillip Carmical ([email protected])

Next-Generation Algae Volume II: Applications in Medicine and the Pharmaceutical Industry

Edited by

Charles Oluwaseun Adetunji Julius Kola Oloke Naveen Dwivedi Sabeela Beevi Ummalyma Shubha Dwivedi Daniel Ingo Hefft and

Juliana Bunmi Adetunji

This edition first published 2023 by John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA and Scrivener Publishing LLC, 100 Cummings Center, Suite 541J, Beverly, MA 01915, USA © 2023 Scrivener Publishing LLC For more information about Scrivener publications please visit www.scrivenerpublishing.com. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. Wiley Global Headquarters 111 River Street, Hoboken, NJ 07030, USA For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Limit of Liability/Disclaimer of Warranty While the publisher and authors have used their best efforts in preparing this work, they make no rep­ resentations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchant-­ ability or fitness for a particular purpose. No warranty may be created or extended by sales representa­ tives, written sales materials, or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further informa­ tion does not mean that the publisher and authors endorse the information or services the organiza­ tion, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Library of Congress Cataloging-in-Publication Data ISBN 978-1-119-85728-0 Cover image: Pixabay.Com Cover design by Russell Richardson Set in size of 11pt and Minion Pro by Manila Typesetting Company, Makati, Philippines Printed in the USA 10 9 8 7 6 5 4 3 2 1

Contents Preface xv 1 Discovery of Novel and Biologically Active Compounds from Algae M. Singh, N. Gupta, P. Gupta, Doli, P. Mishra and A. Yadav 1.1 Introduction 1.2 Microalgae-Derived Natural Products 1.3 Bioprospecting for New Algae 1.4 Therapeutically Essential Natural Products 1.5 Screening for Bioactive Constituents 1.6 Extraction Methods 1.7 Biosynthesis and Biological Activities 1.7.1 Antibacterial Action 1.7.2 Antifungal Action 1.7.3 Anti-Inflammatory Action 1.7.4 Antiprotozoal Action 1.7.5 Antioxidant Action 1.7.6 Antineoplastic (Anticancer) Action 1.7.7 Antiviral Action 1.7.8 Anticoagulant Action 1.7.9 Immunosuppressive Action 1.8 Conclusion References 2 Bioactive Compounds Synthesized by Algae: Current Development and Prospects as Biomedical Application in the Pharmaceutical Industry Preeti Mishra, Namrata Gupta, Monika Singh and Deeksha Tiwari 2.1 Introduction 2.2 Algal-Sourced Compounds of Medical Interest

1 2 3 4 6 7 9 11 15 17 18 19 20 21 24 25 25 26 27

41 42 43 v

vi  Contents 2.3 Microalgae with Potential for Obtaining Bioactive Compounds 2.3.1 Spirulina 2.3.2 Chlorella 2.3.3 Nostoc 2.3.4 Dunaliella 2.4 Bioactive Compounds from Cyanobacteria 2.5 Secondary Metabolites from Microalgae 2.5.1 Carotenoids 2.5.1.1 β-Carotene 2.5.1.2 Astaxanthin 2.5.1.3 Zeaxanthin and Lutein 2.5.1.4 Violaxanthin 2.5.1.5 Fucoxanthin 2.5.2 Polyunsaturated Fatty Acids 2.5.3 Proteins and Polypeptides 2.6 Biomass of Microalgae 2.6.1 Biomass Production 2.6.1.1 Cultivation 2.6.1.2 Harvesting 2.6.1.3 Biomass Dehydration 2.6.1.4 Extraction of Bioactive Compounds 2.7 Pharmaceutical Applications of Microalgae 2.8 Conclusion References 3 Bioactive Compounds Derived from Microalgae Showing Diverse Medicinal Activities D. Tiwari, P. Mishra and N. Gupta 3.1 Introduction 3.2 Microalgae with Anti-Inflammatory Activity 3.3 Microalgae with Immunomodulatory Activity 3.4 Microalgae Anticancer Activity 3.5 Potential of Microalgae in Quality Enhancement of Natural Products 3.5.1 Pharmaceutical Industry 3.5.2 Cosmetics and Personal Care 3.5.3 Food Industry References

44 46 47 49 50 51 55 55 55 57 58 59 59 60 61 62 62 62 63 64 66 66 71 72 77 78 81 82 85 87 87 87 88 90

Contents  vii 4 Application of Astaxanthin and Carotenoids Derived from Algae for the Production of Nutraceuticals, Pharmaceuticals, Additives, Food Supplement and Feed Abiola Folakemi Olaniran, Joshua Opeyemi Folorunsho, Bolanle Adenike Akinsanola, Abiola Ezekiel Taiwo, Yetunde Mary Iranloye, Clinton Emeka Okonkwo and Omorefosa Osarenkhoe Osemwegie 4.1 Carotenoids and Its Characteristics 4.1.1 Sources of Carotenoids 4.1.2 Production/Extraction of Carotenoids 4.2 Astaxanthin and Its Characteristics 4.2.1 Production/Extraction of Astaxanthin 4.2.2 Historical Perspective of Consumption of Alga as Food and Utilization in the Food Industry 4.3 Application/Utilization of Astaxanthin and Carotenoids in Different Sectors 4.3.1 Nutraceuticals 4.3.2 Food Additives, Supplements and Feed Formulation 4.3.3 Alga as a Potential Source of Astaxanthin and Food Supplement 4.3.4 Technological Application of Algae as Origins of Supplements and Bioactive Mixtures in Healthier Food Varieties and Drinks 4.3.5 Enriching Dairy Products with Algae 4.3.6 Algae as a Potential Healthy Protein and Fat Source 4.4 Future Perspective References 5 Production of Polyunsaturated Fatty Acids (PUFAs) and Their Biomedical Application Olorunsola Adeyomoye, Olugbemi T. Olaniyan and Charles O. Adetunji 5.1 Introduction 5.2 Polyunsaturated Fatty Acids 5.3 Production of Polyunsaturated Fatty Acids 5.4 Nanomedicine-Based Formulations Containing Polyunsaturated Fatty Acids 5.5 Biological and Medical Application of Polyunsaturated Fatty Acids 5.6 Metabolism of Polyunsaturated Fatty Acid

95

96 97 99 102 103 104 105 108 110 111 112 114 115 117 118 125 125 126 127 128 129 131

viii  Contents 5.7 Challenges and Issues of Production and Use of Polyunsaturated Fatty Acids 5.8 Conclusion References 6 Utilization of Algae and Their Anti-Proliferative and Anti-Inflammatory Activities Olorunsola Adeyomoye, Olugbemi T. Olaniyan and Charles O. Adetunji 6.1 Introduction 6.2 Physiology and Biochemistry of Algae 6.3 Algae Biocomposites 6.4 Techniques and Methods Involved in the Production of Algae Biocomposites 6.5 Antiproliferative Activities of Algae 6.6 Anti-Inflammatory Activities of Algae 6.7 Potential Health Benefits of Algae Biocomposites 6.8 Challenges and Issues Related to Algae Biocomposites Use 6.9 Conclusion References

132 133 133 139 140 141 141 143 144 144 146 147 147 148

7 Natural Compounds of Algae Origin with Potential Anticarcinogenic Benefits 153 Adewale Omowumi Oyeronke, Asowata-Ayodele Abiola Mojisola, Akomolafe Seun Funmilola and Adetunji Juliana Bunmi 7.1 Introduction 154 7.2 Progression, Predisposing Factors and Treatment of Cancer 156 7.2.1 Cancer Progression 156 7.2.2 Predisposing Factors to Cancer 157 7.2.3 Treatment of Cancer 157 7.3 Features of Microalgae 157 7.4 Sources of Microalgae 158 7.5 Fractions of Microalgae Species with Anticancer Properties 158 7.5.1 Carotenoid-Rich Extracts of Chlorella Species 158 7.5.2 Chaetoceros Calcitrans Ethyl Acetate and Ethanol Extracts 159 7.5.3 Amphidinium Carterae Organic Fractions 159 7.5.4 Methanolic Extracts from Amphidinium Carterae, Prorocentrum Rhathymum, Symbiodinium sp.,

Contents  ix Coolia Malayensis, Ostreopsis Ovata, Amphidinium Operculatum, and Heterocapsa Psammophila 160 7.5.5 Skeletonema Marinoi Hydrophobic Fraction 160 7.5.6 Canadian Marine Microalgal Pool Aqueous Extract 160 7.5.7 Chlorella Sorokiniana Aqueous Extract 161 7.6 Compounds with Anticarcinogenic Activities Isolated from Marine Microalgae 161 7.6.1 Polysaccharides 161 7.6.2 Phycocyanin 163 7.6.3 Chlorophyll 163 7.6.4 Polyunsaturated Aldehydes (PUAs) 164 7.6.5 Violaxanthin 164 7.6.6 Eicosapentaenoic Acid (EPA) 165 7.6.7 Stigmasterol 166 7.6.8 Fucoxanthin 166 7.6.9 Nonyl 8-Acetoxy-6-Methyloctanoate (NAMO) 167 7.6.10 Monogalactosyl Glycerols 168 7.6.11 Other Active Compounds from Microalgae with Anticarcinogenic Activities 168 7.7 Conclusion and Recommendation 168 References 169 8 Current Research on Algal-Derived Sulfated Polysaccharides and Their Antiulcer Bioactivities Abiola Mojisola Asowata-Ayodele, Adewale Omowumi Oyeronke, Akomolafe Seun Funmilola and Adetunji Juliana Bunmi 8.1 Introduction 8.1.1 Symptoms of Peptic Ulcer Disease 8.2 Treatment Using Synthetic Medicines 8.3 Natural Products Used in the Treatment of Peptic Ulcer 8.4 Antiulcer Products Developed from Algae 8.4.1 Phycocolloids 8.4.2 Fucoidan 8.4.3 Ulvans 8.4.4 Laminaran 8.4.5 Xylan and Porphyran 8.5 Conclusion References

177 178 179 181 183 184 186 188 189 190 191 193 193

x  Contents 9 Pharmacological and Antioxidant Attributes of Significant Bioactives Constituents Derived from Algae Juliana Bunmi Adetunji, Abigail Omotayo Agbolade, Omowumi Oyeronke Adewale, Ikechukwu P. Ejidike, Charles Oluwaseun Adetunji and Isreal Olu Oyewole 9.1 Introduction 9.1.1 Brown Algae 9.1.1.1 Fucoidan and Its Bioactivity 9.1.1.2 Benefits Derived from Fucoidan 9.1.1.3 Laminarin 9.1.1.4 Fucosterol 9.1.1.5 Saccharides 9.1.1.6 Phlorotannins 9.1.1.7 Dieckol 9.1.2 Red Algae 9.1.2.1 D-Isofloridoside 9.1.2.2 Phycoerythrin 9.1.3 Blue-Green Algae 9.1.3.1 Phycocyanin and Phycocyanobilin 9.1.4 Other Potential Applications of Algae 9.1.4.1 Antioxidant and Anti-Tyrosine Capabilities 9.2 Conclusion References 10 Utilization of Pharmacologically Relevant Compounds Derived from Algae for Effective Management of Diverse Diseases Olulope Olufemi Ajayi 10.1 Introduction 10.2 Algae in the Management of Some Diseases 10.2.1 Cancer 10.2.2 Inflammatory Bowel Disease 10.2.3 Osteoarthritis 10.2.4 Gastric Ulcers 10.2.5 Neurodegenerative Diseases 10.2.6 Diabetes Mellitus 10.2.7 Hypertension 10.2.8 Atherosclerosis 10.2.9 Kidney and Liver Diseases 10.2.10 Skin Diseases/Disorders 10.2.11 Uterine Leiomyomas

197

198 198 198 200 202 204 204 206 207 207 207 208 209 209 215 215 216 216

223 223 225 225 226 227 227 227 228 228 229 230 230 231

Contents  xi 10.2.12 Obesity 10.2.13 Tuberculosis 10.2.14 Asthma 10.2.15 Hepatitis 10.3 Xanthophylls 10.3.1 Astaxanthin 10.3.2 Fucoxanthin 10.3.3 Lutein and Zeaxanthin 10.3.4 Beta-Cryptoxanthin 10.3.5 Siphonaxanthin 10.3.6 Saproxanthin and Myxol 10.4 Alga Diterpenes 10.5 Conclusion References

232 234 235 236 236 236 237 237 237 238 238 238 239 239

11 Application of Algae in Wound Healing 251 Ebenezer I. O. Ajayi, Johnson O. Oladele and Abraham O. Nkumah 11.1 Introduction 252 11.1.1 Current Trends in the Design of Wound Dressings 253 11.2 Brown Seaweed Polysaccharides 256 11.2.1 Fucoidan 257 11.2.2 Alginate 258 11.2.3 Carrageenan 259 11.2.4 Red Seaweed Polysaccharides 260 11.2.5 Green Seaweed Polysaccharides 260 11.3 Mechanisms Underpinning the Wound Healing Effects of Algae 261 11.3.1 Hemostatic Activity 263 11.3.2 Immunomodulatory and Anti-Inflammatory Effects 264 11.3.3 Antioxidant Activity 267 11.3.4 Antifungal Activity 269 11.3.5 Antibacterial Properties 269 11.3.6 Wound-Healing Property of Algae and Cyanobacteria 271 11.4 Conclusion 274 References 274

xii  Contents 12 Application of Nanotechnology for the Bioengineering of Useful Metabolites Derived from Algae and Their Multifaceted Applications 285 Charles Oluwaseun Adetunji, Olugbemi T. Olaniyan, Inobeme Abel, Ruth Ebunoluwa Bodunrinde, Nyejirime Young Wike, Wadzani Dauda Palnam, Juliana Bunmi Adetunji, Phebean Ononsen Ozolua, Arshad Farid, Shakira Ghazanfar, Olorunsola Adeyomoye, Muhammad Akram, Chibuzor Victory Chukwu and Mohammed Bello Yerima 12.1 Introduction 286 12.2 Various Types of Nanoparticles Derived from Algae 287 12.3 Nanoparticles from Algae and the Key Role They Play in the Medical and Pharmaceutical Sectors 295 12.3.1 Anticancer Activity 298 12.4 Algae-Derived Nanoparticles and Their Key Role in the Cosmetics Industry 302 12.4.1 Algae-Derived Nanoparticles as Moisturizer 302 12.4.2 Algae-Derived Nanoparticles as Skin Sensitizing and Thickening Agents 302 12.4.3 Algae-Derived Nanoparticles as Anti-Aging Agents 303 12.4.4 Algae-Derived Nanoparticles as Antioxidant Agent 303 12.5 Algae-Derived Nanoparticles as Antibacterial Agent 303 12.6 Algae-Derived Nanoparticles as Antifungal Agent 306 12.7 Algae-Derived Nanoparticles as Antiviral Agent 306 12.8 Conclusion 307 References 307 13 Discovery of Novel Compounds of Pharmaceutical Significance Derived from Algae Charles Oluwaseun Adetunji, Muhammad Akram, Fahad Said Khan, Olugbemi T. Olaniyan, Babatunde Oluwafemi Adetuyi, Inobeme Abel, Ruth Ebunoluwa Bodunrinde, Juliana Bunmi Adetunji, Phebean Ononsen Ozolua, Nyejirime Young Wike, Wadzani Dauda Palnam, Arshad Farid, Shakira Ghazanfar, Olorunsola Adeyomoye, Chibuzor Victory Chukwu and Mohammed Bello Yerima 13.1 Introduction

321

322

Contents  xiii 13.2 Bioactive Compounds 13.3 Pharmacological Significance of Algae 13.3.1 Antioxidative Activity 13.3.2 Antihypertensive Activity 13.3.3 Anticoagulant Activity 13.3.4 Antiproliferation Activities 13.3.5 Immune-Stimulant Activity 13.3.6 Cholesterol-Lowering Activity 13.3.7 Anti-Inflammatory Activity 13.3.8 Anticancer Activity 13.3.9 Cancer Prevention Agent 13.3.10 Antidiabetic 13.3.11 Different Biomedical Activities 13.4 Research Results on Well-Studied Algal Strains 13.5 Conclusion and Future Recommendations References 14 Applications of Algae in the Production of Single-Cell Proteins and Pigments with High Relevance in Industry Juliana Bunmi Adetunji, Omowumi Oyeronke Adewale, Charles Oluwaseun Adetunji and Isreal Olu Oyewole 14.1 Introduction 14.2 Microalgae-Derived Single Cell Protein (SCP) 14.2.1 Dunaliella 14.3 Applications of SCP in Diets 14.4 Pigments Derived from Algae 14.4.1 Astaxanthin 14.4.2 Fucoxanthin 14.4.3 Carotenoids 14.5 Conclusion References

323 324 324 325 326 326 327 328 329 329 330 330 331 332 334 334 343 344 345 346 347 348 348 348 349 349 349

Index 353

Preface The application of both micro and macroalgae has been recognized as a next-generation biotechnological tool with the potential to mitigate various challenges faced by humankind globally, particularly in catering to the needs of an ever-increasing population. This companion volume includes valuable information on nanoparticle synthesis derived from algae and microalgae, as well as their diverse uses and applications in the production of products for pathogen diagnostics, pharmacological properties, and different drugs/medicines for human health. Algae has numerous medical attributes that could serve as a permanent replacement to all the challenges associated with synthetic drugs, such as high level of resistance, low effectiveness, and high cost. In recent years, there has been a significant shift in focus toward the application of algae, owing to its numerous applications in several sectors. Algae is a valuable resource for not only energy and food, but also conservation, environmental bioremediation, pharmacologically active substances, agriculture, new industrially important bio-products, and nutraceuticals. This book explains how the application of algae could help to resolve diverse human health challenges through the use of nutritional supplements, therapeutic proteins, vaccines, and algae-derived drugs. It also provides great detail about the biological activities of some natural drugs derived from algae, such as anticancer, antimicrobial, antiviral, antiulcer, antiinflamatory, antihyperlipidermia, antithrombic agents, anticoagulants, cardioverscular, antitumour, immunomdularory, and various antibiotics. Also, it describes the use of algae-derived antioxidants in the management of several health concerns such as inflammations, chronic disorders, and cardiovascular diseases. This second volume places special emphasis on the discovery of novel and biologically active compounds from algae. It covers a wide range of applications, including the use of astaxanthin and carotenoids derived from algae for the production of nutraceuticals, pharmaceuticals, additives, food supplements, and feed. The book also discusses the production xv

xvi  Preface of polyunsaturated fatty acids (PUFAs) and its biomedical applications, recent advancements in the research of sulfated polysaccharides from algal origin, and its antiulcer bioactivities. Other topics include the application of algae in wound healing, the use of nanotechnology for the bioengineering of useful metabolites derived from algae and their multifaceted applications, and the production of single-cell proteins and pigments with high relevance in industry. This book targets a diverse audience, including global leaders, industrialists, pharmaceutical and food industry professionals, innovators, policy makers, educators, researchers, and undergraduate and postgraduate students in various interdisciplinary fields such as medicine, pharmaceuticals, and biotechnology. The information contained in this book will be invaluable to anyone seeking to explore the potential applications of algae in various fields and to discover new biologically active compounds derived from algae for use in medicine, pharmaceuticals, and other related industries. I want to express my deepest appreciation to all the contributors who have dedicated their time and efforts to make this book a success. Furthermore, I want thank my coeditors for their effort and dedication during this project. Moreover, I wish to gratefully acknowledge the suggestions, help, and support of Martin Scrivener and others from Scrivener Publishing. Charles Oluwaseun Adetunji (Ph.D, AAS affiliate MNYA; MBSN; MNSM, MNBGN) Dean Faculty of Science, Edo State University, Uzairue, Nigeria March 2023

1 Discovery of Novel and Biologically Active Compounds from Algae M. Singh1*, N. Gupta2, P. Gupta3, Doli1, P. Mishra1 and A. Yadav1 Faculty of Pharmacy, RBS Engineering Technical Campus, Bichpuri, Agra, India 2 Faculty of Engineering & Technology, RBS Engineering Technical Campus, Bichpuri, Agra, India 3 Department of Chemistry, Faculty of Engineering and Technology, SRM Institute of Science and Technology, Modinagar, Ghaziabad, Uttar Pradesh, India 1

Abstract

The identification of new therapeutically active constituents from algae is generating growing attention due to the unique makeup of these organisms and the potential for widespread industrial use of these constituents. Recent study has concentrated on algae, which have a novel biochemical proclivity and a diverse variety of possible commercial uses, as a provider of novel biologically active constituents. The growing number of researchers are becoming interested in identifying novel physiologically active chemicals from algae, owing to its unique composition and the potential for vast commercial uses. It is very essential to identify the organisms of those species that produce bioactive secondary metabolites that could be a potential source for new drug development. A variety of constituents, such as carbohydrates, minerals, oil, proteins along with polyunsaturated fatty acids, are found in algae preparations. Additionally, biologically active constituents such as antioxidants (tocopherols or vitamin E), vitamin C and pigments (like phycobilins, carotenoids and chlorophylls) are found in algae preparations. These biologically active compounds possess different therapeutic properties, such as antimicrobial (antibacterial, antiviral, antifungal), antineoplastic, antioxidative and anti-­inflammatory properties. They also have the potential to be used as food by humans. Algae have been discovered to be a significant source of physiologically active chemicals that may be used in a variety of goods for animals, plants, cosmetics, and medicines, among other things. *Corresponding author: [email protected] Charles Oluwaseun Adetunji, Julius Kola Oloke, Naveen Dwivedi, Sabeela Beevi Ummalyma, Shubha Dwivedi, Daniel Ingo Hefft and Juliana Bunmi Adetunji (eds.) Next-Generation Algae: Volume II: Applications in Medicine and the Pharmaceutical Industry, (1–40) © 2023 Scrivener Publishing LLC

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2  Next-Generation Algae: Volume II Keywords:  Algae, biologically active compounds, therapeutic activities

1.1 Introduction Water occupies almost 70% of Earth’s surface. Therefore, it is a tremendous resource for the identification of novel/unique compounds with potential therapeutic uses. Over the last several decades, a vast variety of new chemicals obtained from marine creatures with pharmaceutically therapeutic benefits have been discovered. Because of this, marine resources are considered a promising source of new therapeutically active chemicals not only for the creation of active pharmaceutical ingredients but also for the development of food items [1]. The marine environment has a diverse range of fauna (sea hares, fishes, soft corals, sponges, nudibranchs, tunicates, sea slugs, bryozoans, echinoderms, shells, along with prawns) and flora (microorganisms such as micro/macroalgae, cyano- and actinobacteria, bacteria, fungi, halophytes). Among the most remarkable characteristics of marine life is the close connection that exists between different groups of creatures in order to enable them to adapt to the harsh and tough ocean circumstances that are significantly different than those that exist in a given ecosystem [2]. Phytoplankton (microalgae) have received tremendous interest nowadays because they are seen as a continuous raw material for producing a range of bioactive constituents. There are many different types of compounds that could be utilized in nutraceuticals, pharmaceuticals and as ingredients in some products like cosmetics. Some of these include terpenoids, amino acids, phycobiliproteins [3], fatty acids, chlorophylls, steroids, phenolic compounds, halogenated ketones, vitamins, and carotenes [4]. Photosynthetic microorganisms are known as cyanobacteria. They are Gram-negative and abundantly distributed throughout the environment. In various types of industries, including biofuel, nutrition, agriculture, and medicines, etc., they have a huge spectrum of biotechnological applications to offer [5]. Micro- and macroalgae (seaweeds), which make up the majority of marine algae, have possible potential use in different areas of biomedicine and marine pharmacology. Nowadays, tissue culture technologies are an up-and-coming area. As significant marine biological resources, algae are abundant on shallow, coastal, and backwater substrates and may be found in great quantities in shallow, coastal, and backwater habitats. It has also been discovered that algae may grow on a variety of solid objects such as rocks and stones as well as on dead corals, pebbles, and other small objects.

Novel-Biologically Active Compounds Discovery  3 A surprising amount of agar is produced by algae in intertidal and shallow water, with a total production of about 6000 tonnes of total agar yield. Investigations have demonstrated that unrefined and refined compounds generated from marine algae showed significant antimicrobial action in vitro against a broad range of both Gram-negative as well as Gram-positive pathogenic microorganisms and also showed in vivo activity [6]. In addition to being interesting as research targets, because of their potential therapeutic qualities, the natural significant bioactive chemicals derived from microalgae are anticipated to be commercialized in the next several years [3]. In the aquatic environment, marine algae, including dinoflagellates (uni­ cellular along with biflagellate organisms) and phytoplankton, are symbiotic in corals, seaweeds, and sea anemones, among other things. A wide variety of seaweeds are divided into four groups: Chlorophyta (means green algae), Rhodophyta (means Red algae), Phaeophyta (means Brown algae), and Cyanobacteria (certain filamentous Blue-green algae) [1]. Neoplasm (cancer, carcinoma or malignancy), diabetes, metabolic syndrome, obesity, chronic stress, stroke, immunological diseases and chronic respiratory sickness are all contributing to an increase in global morbidity and death. Dietary modification along with lifestyle modification are currently suggested as potential approaches to preventing or treating various ailments [3]. Furthermore, foods containing bioactive constituents may have the ability to behave as necessary nutrients. Antibiotics were formerly considered to be “magic bullets,” but by picking certain bacteria for treatment, they might end up becoming a contributing factor to the spread of illness [7].

1.2 Microalgae-Derived Natural Products Microalgae are microorganisms of only one cell in size that flourish in salt water. It also thrives in freshwater environments. Their diameter or length ranges from 3 to 10 millimeters, and they are available in a variety of forms and sizes. Microalgae include both bacterial and eukaryotic species, and the term “microalgae” applies to both [8]. Cyanobacteria are structurally comparable to bacteria in terms of their composition. They are classified as microalgae, however, because of the presence of chlorophyll and other photosynthesis-related compounds in their composition. Known as green algae due to the fact that they have the same quantities of chlorophyll-a and chlorophyll-b as green plants [9, 10], they have been studied extensively.

4  Next-Generation Algae: Volume II Microalgae produce biocompounds by utilizing light energy along with inorganic nutrients (nitrogen, phosphorus, carbon dioxide, and other elements) and are classified as autotrophic microorganisms. They include nutrients of great nutritional value, such as proteins, lipids, carbohydrates, polymers, and pigments, as well as medicinal properties. In recent research, it has been shown that microalgae may create a vast variety of chemical constituents (compounds) with diversified biological functions, including phycobilins, polysaccharides, polyunsaturated fatty acids, proteins, sterols, carotenoids, and vitamins, among other substances [10]. Phaeophyta, i.e., brown algae, are a well-known commercialized alginate source due to their brown color. Alginates are straight, long chains of amino acids. They consist of residues of the amino acids. Alginates are usually observed in toothpastes and ice creams, where they are employed as thickening agents, foam stabilizers, and preservatives. When taken orally, a low-density agonic acid gel formed from alginate salts operate as a “raft” that floats over the stomach content, similar to corresponding gelatine. As a result, stomach acid is prevented from refluxing into the esophagus. Therefore, sodium/magnesium salts of agonic acid are included in variety of compound antacid formulations, such as Favicon (Reckitt & Coleman) or Alicen (Rorer), among others [11]. There are wide variety of applications of algae, from biofuel production, in particular bioethanol, macroand microalgae fermentation, to enzyme extraction in the paper, textile, and detergent industries, and laboratory applications [12]. Bioactive constituents are substances that are physiologically active in the human body and have functional characteristics in the body. Many biologically active compounds that have the possibility of being used as useful components are being developed and manufactured, including polyphenols, phycocyanins, fatty acids, carotenoids, and other polyunsaturated compounds.

1.3 Bioprospecting for New Algae Numerous new compounds were found in marine algae during the previous six decades, and a vast variety of these chemicals have been shown to have intriguing biological activities [13]. When it comes to the isolation of new species, there are numerous obstacles to overcome; the dearth of information regarding the metabolite demands of growth genus, pH, need for consumption of certain nutrients (e.g., sulphate, nitrogen sources, and phosphorus), and other growth parameters, like crop density and temperature, among others. It is critical to understand the chemical interactions

Novel-Biologically Active Compounds Discovery  5 between strains that have not been thoroughly described or novel strains in order to maximize their production [3]. In 2009, Ou et al. [14] found that clinical studies are useful for concentrating efforts on extracting protective bioactive substances with specific therapeutic properties using various pharmacological models. The method of developing novel molecules as therapies, from preclinical validation through FDA clearance, is lengthy, laborious, and costly. A bioactive molecule with high therapeutic promise requires preclinical investigations, human clinical trials [14, 15], along with regulatory process permission from the FDA following post-trial for commercialization and marketing in the contemporary environment [15]. Keep in mind that not all of the medicines included in the database have been authorized by the Food and Drug Administration (FDA), but they are all recognized only after evaluation of biological action. In many other countries, medications are permitted for clinical use; however, in the United States, none of them have been approved. Animal and human clinical trials are conducted in order to evaluate the therapeutic property of the isolated constituents in various periods of development, using a variety of pharmacological models to do so [3]. Over 18,000 bioactive compounds have been identified to date. Despite this, only six drugs derived from marine sources have been clinically authorized and commercialized. Moreover, only a few algal isolates have been acknowledged clinically. Brentuximab vedotin, marketed under the trade name Adcetris, is, for instance, an antibody-drug combination made from bioactive molecules [16] derived from an algal source used to treat non-Hodgkin lymphoma [17]. Fucoidan extracts have anti-aging action on the human body in clinical double-blind trials [18]. Interestingly, the first antiviral algal component found from Eucheume/Chondrus, a red edible alga, is iota-carrageenan (Carragelose). Numerous derivatives of dolastatin have been developed and are being clinically investigated in EMA tests and by the FDA [19]. These derivatives names are glembatumumab vedotin, depatuxizumab mafodotin, and pinatuzumab vedotin. It has been revealed in clinical studies that EPA, coupled with DHA, are essential amino acids from marine macroalgae that have clinical use [20]. As feed additives and immunological boosters, Ocean Feed™ from macroalgae and Tasco™ from A. nodosum were already on the market [21]. There have been several well-publicized incidents in the UK of livestock and other animals being poisoned as a result of cyanobacteria contamination in their drinking water. Anabaena flosaquae is a plant that produces the alkaloid named anatoxin-a, which is a neurotoxin that depolarizes neuromuscular blocking and has both nicotinic and muscarinic action [5, 22].

6  Next-Generation Algae: Volume II

1.4 Therapeutically Essential Natural Products Marine organisms, which include both animals and plants, are the richest sources of bioactive constituents, which have a diverse variety of pharmacological actions, including free radical scavenging, anticancer, neuroprotective, analgesic, antimicrobial, and immunomodulatory properties, among other activities. Underwater drugs provide an alternate source for meeting the growing need for safe, effective, and low-cost medications, which is increasing in tandem with the world population’s dramatic rise. In developed nations, the disease of neoplasm (cancer) is among the most prevalent causes of mortality, whereas communicable infections are the main cause of mortality in impoverished (developing) nations. Despite the significant advances in neoplasm or tumor therapy that have occurred over the past three decades, there is still a pressing need for novel medicines in the field of cancer biology, particularly in the relatively untapped field of marine anticancer chemicals, to combat cancer [2]. Chlorella and Spirulina are the most common microalgae species found on the market, and they dominate the whole market. The first is a green microalga that includes microalgae and also macroalgae, part of the broad phylum of Chlorophyta [12]. Polyketides, alkaloids, cyanopeptides, isoprenoids and other metabolites are among the cyanobacterial natural products classified according to their metabolic origins. While much of the research was focused on toxicity, many studies also have revealed that cyanobacteria create chemicals of considerable pharmaceutical and biotechnological importance. Forty percent (40%) of lipopeptides, others are less than 10% (e.g., fatty acids, amino acids, amides, and macrolides) make up cyanobacterial compounds. Therefore, Cyanobacteria activity is dominated by lip peptides such as cytotoxic (41%), antitumor (13%), antiviral (4%), and antibiotics (12%). The remaining 18% of cyanobacterial activity includes antimalarial, antimitotic, and immunosuppressive agents, herbicides, antifeedant, and multi-drug resistance reversing agents, among others [23]. Various types of Blue-green algae are available in the market as organic algae nutraceuticals, as well as a source of pharmacologically important substances. Examples of such species are Spirulina, Chlorella and Aphanizomenon flos-aquae. Spirulina sp. is a kind of blue-green algae that is found in the ocean. Lipids, chlorophyll, protein, carotenoids, minerals, vitamins, and vibrant colors are all rich in this plant’s composition. Moreover, they might contain helpful probiotic components [24]. As well as other carotenoids, minerals (including Ca and Fe) and B vitamins

Novel-Biologically Active Compounds Discovery  7 (including B12), Spirulina sp. is a wonderful abundant source of potassium, calcium, magnesium, iron, selenium and zinc. As an added bonus, important component fatty acid is beneficial in potentiating hair and skin growth, regulating metabolism and maintaining bone health. It also ensures proper functioning of reproductive system. Numerous minerals and vitamins have powerful antioxidant activities that assist in the elimination of toxins from the environment and the prevention of diseases. Cyanobacteria have recently attracted the public’s curiosity due to their high concentrations of bioactive chemicals and their potential as dietary supplements. They also serve as a model for organisms belonging to some very promising categories of organisms in terms of the production of bioactive chemicals. Scientific evidence demonstrating bioactive chemicals produced from blue-green algae may show therapeutic promise in the treatment of illness and health issues in both human clinical trials and animal clinical studies [1]. Agars as well as carrageenan are generated from species of red algae. Both the agents are utilized as gelling, thickening, and emulsifying agents, and are the most significant products obtained from red algal species. Agar is also utilized as a microbiological culture medium, and agarose, which is a component of nutrient agar used for microbial growth, is also utilized in electrophoresis, immunodiffusion, and gel chromatography, among other applications. It’s also used as a hydrogel in the surgical dressing by Geistlich, which is another use. A carrageenan implant is a substance used in the pharmaceutical industry to induce inflammation in models for animal testing. It may be utilized to evaluate prospective anti-inflammatory and anti-arthritis medicines to produce edema. According to Blunden and Gordon [11], the gastrointestinal system of primates does not absorb high molecular weight carrageenans, and as a result, they are believed to be acceptable additions for human consumption, given that the molecular weight is tightly controlled.

1.5 Screening for Bioactive Constituents Multidisciplinary approaches are needed for the determination of bioactive constituents. The advancement of analytical and molecular methods is a critical ongoing process that is required as a precondition for the targeting of novel products by means of high-throughput strategies. Public and private interest has been growing over the last few decades, along with their investments in marine biotechnology, which has further increased the possibility of generating information and collecting huge amounts

8  Next-Generation Algae: Volume II of data to elaborate a better understanding of different cellular processes and mechanism of biological actions. Furthermore, marine biotechnology tends to utilize genomics, transcriptomics, proteomics, metabolomics, metagenomics, and metatranscriptomics, etc. [25], in conjunction with heterologous expression or genetic engineering to identify potential bioactive species and increase the required constituents/substances production [26]. A “test first” approach or an “isolate first” method is used in the screening process of natural goods, respectively. Natural bioactive components have been discovered using both approaches, and a recent trend has been to advocate the use of a fusion method, in which extracted or fractioned extracts are evaluated for the presence of biologically active elements. Bioassays are only utilized when the extracts demonstrate a high level of biological activity [27]. In order to isolate or identify fractions of extracts for chemical characterization, a number of techniques might be utilized. One of these techniques is liquid chromatography–mass spectrometry (LC-MS) and another is nuclear magnetic resonance (NMR). If novel constituents are identified, they should be refined as well as evaluated utilizing biological tests. Ideally, those tests should be reliable, repeatable, quick, cost-effective, simple to run, and sensitive, and should also be reproducible [3]. Microorganisms play an increasingly significant role in modern life, since they have evolved into essential components of a number of human life functions, such as digestion and food assimilation, among others. They also focus on human well-being by providing diversified foods, chemicals, and medications. Many microbiological pathogens, including fungi, protozoa, viruses, and bacteria, are responsible for serious illnesses despite the fact that effective management strategies are available. Bacteria and fungi are responsible for the spoilage of food goods [6]. De Vera et al. carried out experiments on more than 30 marine microalgae strains (haptophytas, dinoflagellates, chlorophyta, and heterokontophytas) in order to obtain extracts for evaluation of biological activity. As part of their research, they chose a number of intriguing samples for additional investigation of marine bioactive compounds. The unialgal isolates were kindly provided by the Oceanographic Center of Vigo. The available strains were cultured in the lab environment to determine their viability. Cell-free culture medium extract and biomass extract (two types) were produced from each strain. In order to get knowledge about antibacterial, antiproliferative, and anticancer (apoptotic) characteristics, these two extracts were further analyzed in order to get information on these qualities [28].

Novel-Biologically Active Compounds Discovery  9 As per the current scenario on the non-selective and unsystematic widespread use of antibiotics as antimicrobial agents, a new generation of antibiotic-­resistant and genetically modified microorganisms has emerged, posing a serious threat to the treatment of infectious diseases. The negative consequences and side effects of frequently used antibiotics, as well as the increasing prevalence of infectious diseases, have fueled the pursuit of novel antimicrobial agents from diversified sources from the marine environment [6].

1.6 Extraction Methods Various methods or techniques can be utilized to separate potential therapeutically or biologically active constituents from different varieties of algal biomass. Various extracting agents were utilized to extract soluble constituents derived from the microalgae matrix. The simplest approach is to separate algal powder using water or organic solvents for large-scale samples, with the latter being the preferred method. The extraction rates vary from 8 to 30% of the dry algal yield under these conditions [29]. New types of extraction methods, like enzymolysis and extraction aided by a microwave, have, however, recently emerged. The first has impressive impacts, with high catalytic effectiveness characteristics, high specificities, mild reactive and maximum efficiency [30]. Moreover, there were several advantages to using the latter technique, including shorter processing times, the use of less solvents, greater extraction rates, and the production of better low-cost products [31, 32]. Complementary to the investigation of soluble chemicals, cell-binding compounds (CBCs) that are attached to the cell wall and cannot be easily isolated by applying the conventional methods of isolation with aqueous solvents, are also being investigated. This could also limit the study of marine-derived active components and their potential industrial applications. Of interest is the enzyme digestion of algae, which produces high biological yields compared to water and organic extracts [33], and which exhibits improved biological activity. Michalak and Chojnacka reviewed an examination on the use of enzyme assistants using seaweed as an alternate approach for increasing the recovery of industrially valuable chemicals from the sea [30]. Recent extraction methods extract biologically active constituents without causing any loss of that activity. These are supercritical fluid extraction (SFE), ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE). Among others, enzyme-assisted extraction (EAE) and pressurized liquid extraction (PLE) also have the advantage of extracting

10  Next-Generation Algae: Volume II therapeutically active constituents. Moreover, this type of extraction method is distinguished by a larger yield of extraction, a shorter processing time, and, as a result, is more environmentally friendly as compared to previous extraction methods. The extraction of soxhlets, as well as liquid–­ liquid extraction along with solid–liquid extraction, are all examples of traditional extraction methods (SLE) [34]. Their primary disadvantage is the use of a huge quantity of solvents (many of which are hazardous) and the large amount of time needed for isolation [35]. Starting a decade ago, there seemed to be a substantial rise in the use of alternative techniques to replace traditional methods largely due to the numerous advantages of new extraction techniques. As per number of authors, new green technologies (e.g., higher yields) are superior to extraction by organic solvents, which incorporates the release of solvents. These solvents could be potentially hazardous for the environment and can also cause hydrothermal stress to extracts in terms of functional properties. The degradation of thermally labile compounds may also result from the high-temperature processing [36]. For instance, in the matter of the SFE utilization with carbon dioxide, the yield of the lipid removal was higher than in the case of Soxhlet solvent. Because of the numerous advantages of new extraction techniques, it has been noticed that they have increasingly been used to replace old approaches in recent years. The superiority of new green technologies (e.g., better yield) over extraction techniques by organic solvent has been demonstrated by numerous authors. As a result of hydrothermal stress, organic solvent extraction entails the release of potentially toxic solvents into the surrounding environment. The functional characteristics of the extracts were severely harmed as a result of the release of potentially hazardous solvents. High-temperature processing can cause the deterioration of components that are thermally labile as well as the degradation of other constituents [36]. For example, when utilizing SFE with CO2, the amount of lipid extracted from Sargassum hemiphyllum was greater than when using the Soxhlet solvent extraction technique with chloroform/methanol [37]. Tierney et al. discovered in their research that PLE was more efficient than standard SLE in the extraction of polyphenols using a water:acetone (20:80) mixture [38]. Denery et al. also had a parallel observation that compared to conventional solvent extraction techniques, PLE displayed more or equivalent carotenoids extraction abilities from Haematococcus pluvialis as well as Dunaliella salina [39]. Pasquet et al. examined extraction of pigment from two marine microalgae using two different approaches (one is cold and hot soaking and another is ultrasound-assisted extraction). Due to its high rate, uniform heating, reproducibility, and higher separation rates,

Novel-Biologically Active Compounds Discovery  11 MAE has been selected as the most effective pigment extraction technique [40]. Authors investigated the emerging green technologies (such as MAE, SFE and PLE) being more capable of replacing traditional organic solvent extractions. Extraction with SFE is one of the most widely utilized methods of extraction on an analytic and preparatory scale nowadays [41]. Aim of this chapter is to show the unique qualities of biologically active constituents and their wide applications obtained from algal biomass. The utilization of extracts from various algae is widely described in different areas of food, nutraceuticals and fuel manufacturing. It also explains the application in agriculture (plants and animal products) and cosmetics of algal extracts. New extraction techniques are widely used in several industries for obtaining algal extracts such as SFE, UAE, MAE, PLE, EAE, etc. These techniques protect against degradation of the bioactive constituents isolated from algae. Algae’s unique properties allow for a wide range of applications to be developed. They contain a high concentration of kilo grains (such as eicosapentaenoic acid, docosahexaenoic acid, β-linoleic acid) and in components such as polyunsaturated fatty acids (PUFAs) protein, minerals, carbohydrates, fats, oil, (e.g., docosahexaenoic acid, eicosapentaenoic acid along with γ-linoleic acid), in addition to the amount of bioactive constituents. These bioactive constituents are polyphenols, carotenoids, terpenoids, and tocopherols, which have antiviral, antibacterial, antifungal, antioxidative, anti-inflammatory, and antitumor activities. For plants, animals and human beings, algal extracts generated in solvent-free conditions or algal extracts obtained from minimal use of solvents are safe. These all are used in modern agriculture for three different categories: • Animals (feed additives), • Plants (bioregulators, biostimulants, fertilizers), and • Humans (food, cosmetics, pharmaceuticals) [31, 42].

1.7 Biosynthesis and Biological Activities Due to the influence of time-course and cohabitation on biological substances, biochemical pathways have been developed to the point where many microalgal lines now assemble a large number of distinct compounds. Despite the fact that secondary plant metabolites are more comprehensive than algae-derived metabolites, the diversity of secondary algal-­derived metabolites is orders of magnitude more than that of soil plants [5, 3]. Bioactive substances are additional nutritional components found in small

12  Next-Generation Algae: Volume II Table 1.1  Algae and cyanobacterial constituents with potential biological action. Name of microalgae

Bioactive compounds

Biological action

Reference

Arthrospira platensis (also known as Spirulina platensis)

PUFAs (n-3) fatty acids, oleic acid, linolenic acid, (vitamin E), phytol, palmitoleic acid, sulfated polysaccharide

Antiviral action

[46–49]

Botryococcus braunii

Carotenoids, linear alkadienes

Antioxidant action

[50, 51]

Antioxidant action

[52]

Caulerpa racemosa Polyphenols Chlorella ellipsoidea

Zeaxanthin, violaxanthin Anti-inflammatory action, anticancer action

[53, 54]

Chlorella minutissima

Eicosapentaenoic acid (EPA)

Antioxidant action, cholinesterases inhibitory action

[48]

Chlorella protothecoides

Zeaxanthin, canthaxanthin, lutein

Anti-inflammatory action, antifungal action

[55–57]

Chlorella pyrenoidosa

Sulfated polysaccharide, lutein

Antiproliferative action

[49]

Chlorella sp.

Carotenoids polyunsaturated fatty acids, sulfated polysaccharides, sterols

Immunostimulant action, antitumor action, antioxidant action

[49, 53, 58, 59]

Chlorella vulgaris

Canthaxanthin, peptide, Antioxidant action astaxanthin, oleic acid and antitumor action

[48, 53]

Chlorella zofingiensis

Lutein, astaxanthin

[60, 61]

Anti-inflammatory action

(Continued)

Novel-Biologically Active Compounds Discovery  13 Table 1.1  Algae and cyanobacterial constituents with potential biological action. (Continued) Name of microalgae

Bioactive compounds

Biological action

Reference

Cystoseira abies-marina, Halopitys incurvus

Polyphenols, neoantioxidants, and amino acids

Antimicrobial action and antioxidant action

[62]

Dunaliella salina

β-carotene (both trans and cis geometric isomers), oleic acid, palmitic acid, linolenic acid

Antioxidant action (restores the activity of hepatic enzymes)

[48–50, 63]

Dunaliella tertiolecta

7-Dehydroporiferasterol, Action on the ergosterol, nervous system Oxocholesterol acetate

[64]

Eucheuma spinosa

Different types of galactose units

Antioxidant action

[65]

Gelidium pusillum

R-phycocyanin and R-phycoerythrin

Hypocholesterolemic action, antioxidant action, antineoplastic action, antiinflammatory action, and hepatoprotective action

[66]

Haematococcus pluvialis

β-Carotene, oleic acid, astaxanthin, lutein, zeaxanthin, canthaxanthin

Antioxidant action

[46, 67, 68]

Himanthalia elongata Hormosira banksii

Polyphenols, polysaccharides

Antiviral action

[69, 70]

(Continued)

14  Next-Generation Algae: Volume II Table 1.1  Algae and cyanobacterial constituents with potential biological action. (Continued) Name of microalgae

Bioactive compounds

Biological action

Isochrysis galbana

Cholest-5-en-24-1,3(acetyloxy)-, and 3β-ol Ergost-5-en3β-ol, etc.

Antitubercular action [71]

Laurencia obtuse

Phenolic constituents

Antioxidant action

[72]

Lyngbya majuscula Lipopeptides

Antitumor action

[73, 74]

Nostoc ellipsosporum

Protein

Antiviral action

[75]

Nostoc spongiaeforme, Nostoc linckia

Borophycin and cryptophycin

Antibacterial action

[48, 76]

Nostoc sp. GSV 224

Cyclopeptide

Antineoplastic action

[77]

Saccharina japonica

Fucoxanthin, polyphenols, carotenoids, and phlorotannins

Antineoplastic action and antioxidant action

[78–80]

Sargassum muticum, Sargassum vulgare

Polyphenols, neoantioxidants, amino acids

Antimicrobial action and antioxidant action

[62]

Sargassum thunbergii

Polysaccharides

Antioxidant action and antidiabetic action

[81]

Scenedesmus bajacalifornicus

Polyphenols, flavonoids and alkaloids

Antioxidant action, [82] antidiabetic action, anti-inflammatory action

Scytonema varium Polypeptide constituents

Antiviral action

Reference

[83] (Continued)

Novel-Biologically Active Compounds Discovery  15 Table 1.1  Algae and cyanobacterial constituents with potential biological action. (Continued) Name of microalgae

Bioactive compounds

Biological action

Reference

Skeletonema marinoi

Nucleoside inosine

Antiepileptic action

[84]

Spirulina fusiformis

Phycobiliproteins, diacylglycerols

Antibacterial action

[48, 85]

Spirulina sp.

Polysaccharides phycocyanin, C-phycocyanin, phenolic acids, tocopherols

Antiviral action

[53]

Ulva prolifera

Polysaccharides

Antioxidant activity, [86] antihyperlipidemic action

Undaria pinnatifida

Neo-antioxidants, polyphenols, and amino acids

Antihyperlipidemic and antioxidative properties

[62]

amounts in foods. A variety of bioactive substances appear to provide health benefits. Microalgae and Cyanobacteria have been discovered to have a large number of physiologically active chemicals having antiviral, antibacterial, antifungal, and anticancer properties [43]. Phytoplankton (microalgae) are a diverse community of microscopic plants which also involve a diverse and wide range of physiological and biochemical characteristics, including up to 8–14% carotene, up to 50–70% protein (roughly equivalent to up to 50% protein in meat and 15–17% protein in wheat), over 40% glycerol, 30% lipids, and a notably higher concentration of water-­ soluble vitamins (B1, B2, B3, B6, B12) and fat-soluble vitamins (E, K, D) and others. A record of various bioactive constituents from algae along with cyanobacteria is presented in Table 1.1 [5, 22, 44, 45].

1.7.1 Antibacterial Action In order to protect themselves from other invading organisms, algons produce an immense variety of chemically active constituents which include terpenoids, phlorotannins, amino acids, phenolic compounds, steroids,

16  Next-Generation Algae: Volume II alkenes, cyclic polysulfide and halogenated ketones [87]. Furthermore, organic extracts made from Chaetoceros pseudocurvisetus and the diatoms Skeletonema costatum were looked into and found to have antitubercular activity towards Mycobacterium tuberculosis and Mycobacterium bovis by Lauritano and his team, and according to various researchers, in standard human cell lines, they were found to be nontoxic [88]. Phlorotannins (derived from Sargassum thunbergii) have been shown to inhibit the growth of Vibrio parahaemolyticus, as a result of which membranes are destroyed and insignificant cytoplasmic leakage takes place [60]. Various bacteria, including Candida albicans, Staphylococcus aureus, Aspergillus niger, and Escherichia coli, have been shown to be susceptible to Haematococcus pluvialis. The susceptibility noticed is because of the presence of propanoic and butanoic acid molecules in the bacteria [89]. Spirulina fusiformis consisting of phycobiliproteins showed strong antibacterial action towards Streptococcus pyogenes and Spirulina fusiformis phycobiliproteins [84]. Synechocystis sp. extracts containing fatty acids reduced the development of the bacteria Bacillus cereus and E. coli, C. coli, as well as C. albicans. The C-phycocyanin generated by Streptomyces platensis seems to suppress the spread of Pseudomonas aeruginosa, Salmonella enteritidis, S. aureus, E. coli, and Klebsiella pneumoniae in vitro [90]. Algal polysaccharides resembling fucoidan and laminar are demonstrated to possess antibacterial action against Staphylococcus aureus and E. coli strains. According to some previous reports, it has been proven to impede the formation of Helicobacter pylori biofilms in the mucosa of stomach [91] as well as the proliferation of H. pylori [86]. Kubota et al. discovered that the bioactive constituent amphidinolide Q, which comes from the Amphidinium sp. (symbiotic dinoflagellate), was effective towards the bacteria Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, as well as others [92]. Algal polysaccharides (fucoidan- and laminarin-like) showed efficient antimicrobial activity towards E. coli and Staphylococcus aureus strains. They have been shown to impede the production of Helicobacter pylori biofilms in the mucosa of stomach [93] and the growth of H. pylori [86]. The bioactive ingredient amphidinolide Q from the Amphidinium sp. was effective against the bacteria B. subtilis, S. aureus, and E. coli [92]. Pahayokolide A appeared to inhibit the formation of Bacillus megaterium and Bacillus subtilis, as well as exhibiting cytotoxic properties [94] derived from Lyngbya sp. [95]. Antibacterial activity against MRSA and vancomycin-resistant Enterococcus faecium (VRE) was demonstrated by the chemicals bromophycolide P and bromophycolide Q. These were separated from the Fijian red alga Callophycus serratus [96]. Neuraminidases A and B, two pyrone macrolides derived from the red alga Neurymenia

Novel-Biologically Active Compounds Discovery  17 fraxinifolia [97], were found to have antimicrobial efficacy towards MRSA and VRE. A compound found in Phaeodactylum tricornutum, EPA, palmitoleic and hexadecatrienoic acids, among others, could reduce the growth of bacteria such as B. cereus, S. aureus, S. epidermidis, MRSA, and others [98]. Bacillus subtilis, Micrococcus flavus, and Staphylococcus aureus growth have been shown to be effected and inhibited by fatty acids derived from Oscillatoria redekei containing dimorphecolic, coriolic, and linoleic acids [99]. Antibacterial activity towards E. coli, S. aureus and B. subtilis was also observed in lipid fractions from Chaetoceros muelleri. They were also known to contain unsaturated fatty acids [triglycerides and docosapentaenoic acid (DPA)]. Mendiola et al. discovered DPA was present in unsaturated fatty acid from Chaetoceros muelleri. Many antimicrobial compounds discovered from the Nostoc sp., including noscomin, which was acquired from the terrestrial Nostoc commune. It was shown to possess antibacterial action towards bacteria such as E. coli, B. cereus, and S. Epidermidis [100]. It has been suggested that Muscoride A, an alkaloid derived from the plant Nostoc muscorum, may have antibacterial action towards E. coli and B. subtilis [101].

1.7.2 Antifungal Action Antifungal activity of ethanolic fractions of Laurencia paniculata was investigated by Mickymaray and Alturaiki [102], which contained the sesquiterpene constituent aristolene in patients with bronchial asthma. The results revealed that the fractions had antifungal activity, particularly in patients with bronchial asthma. It was discovered that the compounds isolated from Microcystis aeruginosa demonstrated antifungal action, particularly towards the fungus Aspergillus. Hexadecanoic acid, methyl ester, and BHT were all found to be effective [102]. Shishido et al. and Marrez and Sultan identified scytophycin as a strong antifungal chemical from species of Nostoc, Scytonema, and Anabaena sp. [103, 104]. Researchers discovered other various types of antifungal chemicals called hassallidins from Nostoc sp. and Anabaena sp. The Amphidinium sp. as a symbiotic dinoflagellate [92], displayed antifungal efficacy towards Candida albicans due to derived chemical amphidinolide Q. Phycobiliproteins produced by Porphyridium aerugineum have the potential to provide resistance against Clostridium difficile. C. albicans is an acronym for Candida albicans [84]. Chlorococcum humicola growth was discovered to be inhibited by pigments and organic solvent extracts from C. humicola, such as chlorophyll a, carotene, and chlorophyll b, which were proven to be effective against the growth of the bacteria. There are many different types [4] such as Aspergillus flavus,

18  Next-Generation Algae: Volume II A. albicans, Aspergillus niger and others. When tested against Aspergillus candidus, nostofungicidine, which is produced from N. commune, showed significant antifungal effectiveness, according to the researchers [87]. The fatty acids having short chain produced from H. pluvialis were found to be effective against C. albicans in a laboratory setting [69]. Lipopeptides laxaphycin B along with laxaphycin C are obtained from species Anabaena laxa. These constituents displayed antifungal action towards C. albicans, Saccharomyces cerevisiae, Aspergillus oryzae, Penicillium notatum, and Trichophyton mentagrophytes [105]. Dahms et al. observed antifungal properties of fisherellin from Fischerella muscicola [106]. Ciguatoxin and okadaic acid are effective chemicals against fungi, synthesized by Giardia toxics and Prorocentrum lima, respectively [107]. In 2006, Washida et al. reported antimycotic activities. This action was shown by karatungiols. Its constituents were derived by the dinoflagellate Amphidinium [108]. Hassalldin A as well as Hassallidin B were obtained from Hassallia sp. displayed fungicidal properties [109] towards Acremonium strictum, Fusarium sp., Aspergillus sp., Ustilago maydis, Penicillium sp. and Cryptococcus neoformans. Hapalosiphon welwitschii as well as Westiella genus were also discovered to possess fungicidal agents such as N-methylwelwitindolinone C isocyanate and welwitindolinone A isonitrile [110].

1.7.3 Anti-Inflammatory Action Whenever something affects, irritates, or damages our body, we experience inflammation as a quick reaction. As part of this response, the body recognizes the agents accountable for the attack and gets a chance to neutralize them as soon as feasible. Pain, redness, swelling, and warmth are all symptoms of inflammation that usually occur at the infection site. The anti-inflammatory chemicals absorption aids in the prevention of illness and the speeding up of the healing process. It regulates the immune response of body to the infection. Anti-inflammatory medicines derived from microalgae are widely used nowadays. When combined in food or applied topically in cosmetics and other pharmaceutical products, they are protective to the body’s tissues. Researchers found that sulfurized polysaccharides as well as pigments [111] and PUFAs are the most important anti-inflammatory substances found in microalgae around the world [112]. The immunological response to many cyanobacterial polysaccharides can be improved by reacting through a variety of events, including reactive oxygen species, macroelectric phenomena, secreting chemo-­ cytokines and cytokines. As per J. K. Park et al., these are signalling inflammatory and immune responses. The introduction of reactive oxygen into

Novel-Biologically Active Compounds Discovery  19 various cyanobacterial polysaccharides is responsible for the activation of macrophage functions in the body. As a result of the chemical cytokines it secretes, cytokines can boost immune responses through a variety of mechanisms, including signaling immunological as well as inflammatory responses [113]. As a result, more types of cytokines were stimulated for further secretion [114]. Phycocyanine is one of the most essential cyanobacterial pigments and denotes a phycobiliprotein. It works as a photosynthetic antenna by the collection of light and energy. Phycocyanins recently became a highlight in medicine because they have various pharmaceuticals, like anti-­ inflammatory, antioxidant, and antineoplastic actions. The cytokines produced in greater quantities are responsible for anti-inflammatory action [115]. They inhibit the COX-2 enzyme which further inhibit the synthesis of prostaglandin E2 synthesis (PGE2). Based on its pharmacological action and distinctive properties, phycocyanin may be developed as a possible therapeutic agent against inflammation and neurodegenerative diseases. Different types of neurodegenerative diseases are Alzheimer’s disease (memory loss), Parkinson’s disease (personality disorder), Huntington’s disease, etc. [116]. The pigment scytonemin was discovered in cyanobacteria as a secondary metabolite containing an aromatic alkaloid [117, 118]. According to the literature, its anti-inflammatory function on another normal cell has been shown to have no harmful effects. Scytonemin has also been proposed for use in the creation of an anticancer treatment that inhibits the advancement of the cell cycle [5].

1.7.4 Antiprotozoal Action The antiprotozoal activity of several cyanobacterial compounds against the pathogenic parasite has been demonstrated in laboratory studies (e.g., malaria caused by Plasmodium falciparum, leishmaniosis caused by Leishmania donovani and sleeping sickness caused by Trypanosoma brucei). However, some cyanobacterial metabolites, particularly those that are effective against drug-resistant strains, also have antiprotozoal activity. The companeramides A and B are metabolites separated from the plant Leptolyngbya sp. which are cyclic depsipeptides. Now Hyalidium has been explored extensively by researchers [119]. These compounds, on the other hand, have antimalarial efficacy against three separate strains of Plasmodium falciparum which are chloroquine-resistant. Their therapeutic activity against a parasite is one hundred times lower than that of chloroquine, which limits their potential use as pharmaceutical agents in treating parasitic infections. Even so, some metabolites have shown a high

20  Next-Generation Algae: Volume II efficacy against the parasite, so they might replace antibiotic medications like dolastatins  and hoshinolactam for protozoal infections. Dolastatins are indeed natural marine peptides and Dolabella auricularia (sea hare) was identified as the first members of this family [22]. Antiprotozoal activity [120] against the Trichomonas vaginal, Entamoeba histolytica, Leishmania mexicana, Trypanosoma cruzi, Giardia intestinalis, etc., has been shown in Lobophora variegata extracts. Alkaloids of Cladophora crispate along with ethyl acetate constituents were found to possess antiprotozoal action towards protoscolices of Echinococcus granulosus hydatid cysts. The ethyl acetate constituents and alkaloids were isolated from the plant Cladophora crispate [121]. Inhibition of growth of Leishmania braziliensis was observed in algal extracts from the following species: Canistrocarpus cervicornis, Caulerpa cupressoides, Ochtodes secundiramea, Anadyomene saldanhae, Dictyota sp. and Padina sp. [122]. A polyanionic sulfated polysaccharide known as fucoidan [123], prevalent in numerous brown algae, was discovered. It has been shown to have an inhibitory effect on the intracellular amastigote part of Leishmania donovani. Dolabelladienetriol, derived from the Diktyota pfaffii plant, was tested for its leishmanicidal action against intracellular amastigotes. It was also found effective for antihuman immunodeficiency virus (HIV)-1 action. As HIV-1 has been shown to increase the amount of Leishmania parasites present in macrophages, dolabelladienetriol seems promising for chemotherapy of leishmaniasis. Elatol isolated from Laurencia dendroidea (the Brazilian red algae) [124], demonstrated antiprotozoal activity towards the amastigotes and trypomastigotes of T. cruzi species. The Sargassum hemiphyllum has constituent sargaquinoic (meroter­ penoid) [125], which was a powerful agent against in-vitro Plasmodium falciparum [126]. Fennel et al. discovered that, irrespective of its strong actions, dolastatins are not recognized as promising antiprotozoal drugs. Hoshinolactam is an aromatic metabolite of the lactam family produced by cyanobacteria. Separated from Oscillatoria sp., it demonstrated antiprotozoal action with IC50 equivalent (3.9 nM)  to the commercial medicinal product pentamidine (4.7 nM) against Trypanosoma brucei [127]. Therefore, it has the potential to be utilized as effective alternate medication for trypanosomiasis caused by Trypanosoma brucei. Marine algae have various bioactive compounds with antimalarial/antiprotozoal activities which still need to be explored.

1.7.5 Antioxidant Action The demand for algal foods is rising rapidly on the global platform, with excellent health benefits being used on the market as “functions or

Novel-Biologically Active Compounds Discovery  21 nutraceuticals.” It is possible to prevent oxidative damage in cells caused by bioactive chemicals through a process of active oxygen and scavenging free radicals, which can help prevent cancer [128]. Serious health conditions like atherosclerosis, cardiac ailments, strokes, tumors, neurodegenerative disorders, muscular degeneration, infant retinopathy, renal disease and age-related diseases, are caused by oxidative stress [129]. Different algal constituents  have antioxidant properties, in addition to anti-inflammatory, antibacterial and antiviral effects in reduction and/or disease prevention. These components are linolenic acid [130], cyanocyanine, oleic acid [46], B-12, vitamin E, palmitoleic acid [131], β-carotene, phycocyanin, zeaxanthin, etc. [55]. Inverted association with the consumption of fruit and vegetables was established in epidemiological studies. The antioxidant activity of these foods is attributed to this phenomenon [7]. Cyanobacterium phytochemicals along with pigments possess a active oxygen-free radical, or nitrogen scavenger, and therefore act as antioxidant. Often high oxygen levels and high irradiation are subjected to algae and cyanobacteria. These organisms tend to form an oxidative stress defense mechanism. The antioxidants found in microalgae (dimethyl sulfoniopropionate and mycosporic amino acids) have been identified and are extremely strong molecules that block ultraviolet light [132]. Skjånes et al. discovered that algae possess a variety of constituents that have antioxidant properties, including pigments, lipids, and polysaccharides [133].

1.7.6 Antineoplastic (Anticancer) Action The photosynthetic microbes, including cyanobacteria and algae, developed to survive and flourish in a hostile atmosphere on the biochemical basis of bioactive compounds and secondary metabolites. Separated secondary metabolites have a high therapeutic value, which is further enhanced for antineoplastic properties by active pharmacological ingredients [2]. Cyanobacteria strains, such as Oscillatoria, Nostoc, and Spirulina, generate a mixture of acetyl Co-A synthesis and anabolic pathways to produce cytotoxic lipopeptides [23]. Recently, researchers discovered that somocystinamide, a marine lipopeptide derived from the seaweed L. majuscula, can activate the apoptosis pathway and restrict the growth of numerous cancer cell lines, including leukaemia. Others are carcinoma, melanoma, neuroblastoma and myeloma [73]. Didemnin [74, 134], lyngbyabellins [135], and hectochlorin are examples of lipopeptides that have been discovered [136].

22  Next-Generation Algae: Volume II Cyclopeptide cryptophycin produced by Nostoc also has demonstrated a great potential for anticancer to multidrug-resistant cells because of their effects on the cytoskeletal protein tubulin. Besides which, the effects against solid tumors have been highly effective. The cancer suppression mechanism has been connected to binding of tubulin [137, 138], which results in the depolymerization of microtubules [139] and the instability of microtubules, which results in cell cycle arrest and apoptosis, among other things [1]. Similarly, apratoxin A, a natural compound derived from marine cyanobacteria, inhibits the transcription factor STAT3 [140], which prevents G1 cells from becoming cancerous and induces apoptosis in different cell types [141, 142]. Based on their adaptation to exposed anthropogenic environment, cyanobiological flora in freshwater ponds generates an unpleasant smell. These blooms (blooms are accumulation of algal cells to any point where they discolor the water) of blue-green algae cultivate in vast numbers and are harmful to all creatures because of their cyanotoxin content. However, the promising properties of these toxins as anticancer drugs have been demonstrated. The clinical effectiveness of different carcinomas has been demonstrated by microcystins, cryptophicins, anatoxin A and numerous peptide toxins [143, 144]. Successful clinical trials of cyanobacterial depsipeptides like dolastatin 15, including tasidotin, soblidotin and cemadotin, were carried out [1]. A significant element in chemotherapy is the mechanism by which cyanobacterial metabolites act on tumor cells. Cells are programmed for apoptotic cell death to die from stimulus by changed homeostasis caused by infections, oncogenic transformations, oxidants, abnormal proliferation, and so on. There is therefore a high pharmacological value for anticancer treatment for metabolite-­inducing apoptosis. A class of anticancer compounds known as cyanobacterial metabolites interconnect with molecular cell parts, such as DNA, protein kinases of the receiver and microtubules. Cell cycle controls protein synthesis. These interactions result in cell blockage [145], mitochondrial dysfunction, oxidative damage [146], and non-cascade activation [147]. Different pharmacoactive cyanobacterial constituents for powerful anticancer and apoptotic signalling have been tested. Calothrixin A revealed cell cycle G2 phase or M phase arrest in tumor cells of humans. Calothrixin A is a class of indolophenanthridine obtained from Calothrix [148]. So, phycobiliprotein (C-phycocyanin) was mentioned as scavenging peroxyl and phormidium radicals from both Lyngbya and Phormidium [149]. Besides the abovementioned apoptotic markers, the sodium concentration in the cells is enhanced apart from a few metabolites, such as the antillatoxins as lipopeptides separated from the majuscula [150], and the hermitamides [151]. In marine environment, microalgae growing covers

Novel-Biologically Active Compounds Discovery  23 nearly forty percent of worldwide economic output. Microalgal bloom natural products were already extensively investigated for bioactive antineoplastic compounds, polysaccharides, pigments, and secondary metabolites. Whereas C-phycocyanin and phycobiliprotein, from both Phormidium and Lyngbya, were discovered to neutralize, i.e., scavenging radicals of hydroxyl and peroxyl [149]. When cultures are cultivated under specified conditions, such as in specialized medium, at specific temperatures, and under specific light, microalgal extracts have been proven to be effective in anticancer (antineoplastic) research [88]. In general, carotenoids, such as lutein, alpha-carotene, beta-carotene, xanthene, lycopene, and other terpenes found in algae and cyanobacteria, are abundant because they are photosynthetic byproducts. As a scavenger for singlet electron species, carotenoids and various terpenoids play a crucial role. All these scavengers are therefore used to avoid the proliferation of cancer cells as an antioxidant. In various cancers, there are few reports of carotenoid cancer activity. However, some cases had inversely affected the development of carotenoids, which subsequently was detected due to smoking effects in individuals affected by lung cancer [80]. However, the neoplasm proliferation risk was reduced by dietary carotenoids studied by numerous authors [152]. Algae water extracts mainly constitute chemical molecules like alkalis, polysaccharides, polyphenols, polyunsaturated fatty acids (PUFAs), fatty lipids, glycoproteins, terpenoids and vitamins. Many of these constituents were examined and discovered as efficient in producing anticancer activity. The anticancer properties of a few secondary metabolites, including hormothamnione A from Chrysophaeum taylorii, hormothamnin A in Hormothamnion enteromorphoides, and malyngamide D in L. majuscule [153], have been discovered after extracts of these plants were made and tested on numerous cell lines. In addition to studies involving active molecules, a number of experiments were carried out to establish antineoplastic efficacy utilizing crude extracts, for example, extract of carotenoid [154], crude organic solvent extracts [155], polyunsaturated aldehydes [156], and chrysolaminarin (polysaccharide) [157]. It could be expected that in the current scenario of several tumor ailments, microalgae-based nanoformulated delivery systems must be marketable presently. In the literature, however, the above aspects have not been explored up to now for marketing nanoformulation or sustainable, efficient formulations and proper use of microalgae medications in nanomedicine-­based therapy. It might be good to test for the nanoformulation’s marketing in cancer therapy with diatoms and other microalgal specimens without the danger of experiencing any adverse effects in

24  Next-Generation Algae: Volume II order to determine their effectiveness. Studies in this area will provide future opportunities to exploit or capitalize on the possibilities of marine natural microalgal-­derived compounds with nanoformulated therapeutic activities [158].

1.7.7 Antiviral Action Despite the fact that successfully reported vaccine inventions have provided acquired immunity, current efforts to develop medications such as antivirals have expanded dramatically as new or re-emerging infectious diseases have evolved or reappeared. Because viral pathogenic organisms can rapidly manipulate genetic make-up at a time when a treatment strategy is witnessed that results in drug resistance, it is possible that this is the case [159]. Algae are one source, which are well-known for their many medicinal properties. Recently, researchers discovered that the Phormidium tenue and Lyngbya lagerheimeii cyanobacteria have antiviral activity towards HIV, also known as the human immunodeficiency virus. In the following years, researchers discovered and identified a novel family of anti-HIV protein known as Cyanovirin-N [75], which restricts the virus from fusing with the cell and, as a result, renders the viral particles inactive [160]. Afterwards, calcium spirulan, a new SP, was discovered in Spirulina platensis. It was reported to supress the reproduction of enveloped viruses of a large range, including the human cytomegalovirus [47], Herpes simplex virus type 1 (HSV-1), influenza A virus, mumps virus [161], HIV-1, and measles virus, among others [162]. The antiviral activity of spirulina-­like compounds derived from Arthrospira platensis polysaccharide fractions was enhanced against human cytomegalovirus (HSV-1), which was previously reported [163]. In vitro antiviral activity against human cytomegalovirus, also known as HSV-1, was enhanced by a class of spirulina-like compounds derived from Arthrospira platensis polysaccharide fractions [163]. Microcystis ichthyoblabe produce depsipeptides A and B, which have been demonstrated to impede the replication of the influenza A virus [164]. According to an article written by Vijayakumar and Menakha, Nostoflan, a virucidal medicine for HSV-1, was produced by Nostoc flagelliforme [142]. Scytovirin, when used in nanomolar quantities, binds to the HIV glycoprotein and causes the virus’ protein envelope to degrade [83]. For several decades, many polysaccharides identified in marine algae species were investigated and revealed to possess intriguing antiviral

Novel-Biologically Active Compounds Discovery  25 properties [165]. As an example, carrageenan, a compound derived from the red algae Gigartina skottsbergii, has been discovered to be therapeutically active against viruses (both non-enveloped and enveloped). Polysaccharide samples, such as calcium spirulan, nostaflan and naviculan from diatom Navicula directa, and galactan from red algae, have also been discovered to possess unexpected antiviral properties against HSV, DENV, HPV, HIV, and other viruses [165].

1.7.8 Anticoagulant Action Marine algae are very important bioactive constituent possessing an anticoagulant property among all other marine sources. Phlorotannins produced from marine algae have shown tremendous promise in biomedical research as anticoagulant medicines [166]. Prothrombin time, activated partial thromboplastin time, as well as thrombin time are all measured to assess whether or not a medication has anticoagulant action. According to the findings of a study, substances containing anticoagulant proteins and antiplatelet as well as fibrinolytic enzymes, have the potential to regulate endothelial cell activities and trigger the fibrinolysis system [167]. Natural and healthy anticoagulant compounds that generate phenolic constituents or phlorotannins are derived from, for example, Ecklonia stolonifera, Hizikia fusiformis, Ecklonia cava, Ecklonia kurome, Ishige okamurae, Eisenia bicyclis and S. thunbergii [166]. Brown algae’s fucoidan along with laminarans, red algae’s carrageenan, and green algae’s ulvan have all received a lot of interest in the sectors of food, cosmetics [168], and pharmaceuticals [169]. Due to their good health benefits, these oceanic algae are being explored as possible medicinal medication components.

1.7.9 Immunosuppressive Action Immunosuppressive medicines, notably T and B cells, have the tendency to suppress the immune system in a variety of ways. They are essential in order to increase the probability of surviving allogeneic organ transplantation by lowering immunological responses of the host. An immunosuppressive effect of SQDG (sulfolipids) derived from blue-green algae was observed in a study to be significant in a human-mixed lymphocyte reaction while having no influence on overall immune competence [170]. The bioactive protein found in Spirulina (blue-green algae) boosts the intestinal immune system by a variety of ways [171]. Spirulina has been studied

26  Next-Generation Algae: Volume II for its potential to protect the kidney from heavy metals and pharmaceuticals by lowering glucose and lipid levels in the blood [172]. Chlorella’s -1,3 glucan lowers blood cholesterol and free radicals [10]. Several immunosuppressive bioactive chemicals are found in blue-green algae. Our goal in this chapter is to reveal the biological production of bioactive compounds by various green algal species. This review confirms that a variety of green algae possess biological active compounds. The phytochemicals in these algae show various beneficial effects, which was supported by variety of the literature.

1.8 Conclusion Nature and its chemical equivalents are the principal sources of medicinal molecules used on a daily basis. Bioactive chemicals derived from algae have been widely studied for antibacterial, antioxidant, anti-inflammatory, anticoagulant, antiprotozoal, antitumor, and antiviral applications over the past several decades. In order to survive under severe environmental conditions, algae and cyanobacteria have developed natural defense mechanisms via the creation of bioactive compounds. Stress factors and oxidants were neutralized by bioactive molecules such as alkaloids and terpenoids. Extracted algal ingredients were examined for bioactivity against bacteria, fungus, viruses, and protozoa. In strict clinical tests, the proliferation of cancer was also inhibited. In light of microalgae’s shown ability to create bioactive compounds, these microorganisms have been thrust into the biotechnology limelight for potential uses in a wide range of fields of research, particularly the life sciences. Since the discovery of microalgal metabolites, which boost the body’s defense mechanisms, researchers have been studying the use of microalgal biomass in a variety of meals, pharmaceutical products, and medicinal items. The found chemicals and their activity in the prevention as well as cure of numerous ailments, as well as the ongoing search for other, as of yet unreported metabolites, are clearly in need of further investigation. Marine biotechnology in pharmaceuticals and food applications is an emerging sector that is globally encouraged by an increasing number of policy and financial instruments. Techniques for the optimization of culture conditions, harvesting and extraction methods, combined with recombinant techniques, have become essential to the majority of industrial models that may be put to achieving corporate use, especially in food and nutraceutical applications. The emergence of this sector has been

Novel-Biologically Active Compounds Discovery  27 facilitated by the growing consumer demand for healthy foods that also have beneficial effects on health, combined with a less stringent legislation than that which applies to pharmaceutical substances [12]. Furthermore, in extreme temperatures worldwide, there are several algal and cyanobacterial bases and the pressure has yet to be identified. With vigilance, the identification and cultivation of these species could discover some new chemical compounds which can produce high therapeutic effectiveness. Such pharmaco-active moieties may, as observed for dolastatin 10, also be modified into analogues to improve clinical utilization. Besides, at the same time, a combination of two or more medicinal compounds can contribute in identifying and evaluating antagonistic, additive, or synergistic effects by in-vitro testing and clinical trials on animals to decrease toxicity problems. The challenge is still there to optimize our ability to access, identify as well as exploit them while remaining cost-effective. The task for researchers is also to establish the circumstances in which the large proportion of these microbes could be cultivated in order to provide a reliable source of supply in the future.

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32  Next-Generation Algae: Volume II 64. Francavilla, M., Colaianna, M., Zotti, M., Morgese, M.G., Trotta, P., Tucci, P., Schiavone, S., Cuomo, V., and Trabace, L., Extraction, characterization and In Vivo neuromodulatory activity of phytosterols from microalga Dunaliella tertiolecta. Curr. Med. Chem. 19 , 18, pp. 3058-3067, 2012. 65. Herrero M., Cifuentes A. Alejandro, and Ibanez E., Phytochemical and Antioxidant Activities of Eucheuma spinosum as Natural Functional Food from North Sulawesi Waters, Indonesia. Asian Network for Scientific Information, 24, 1, pp. 132–138, 2021. 66. Mittal, R., Tavanandi, H.A., Mantri, V.A., and Raghavarao, K.S.M.S., Ultrasound assisted methods for enhanced extraction of phycobiliproteins from marine macro-algae, Gelidium pusillum (Rhodophyta). Ultrason. Sonochem. 38, pp. 92-103, 2017. 67. Thana, P., Machmudah, S., Goto, M., Sasaki, M., Pavasant, P., and Shotipruk, A., Response surface methodology to supercritical carbon dioxide extraction of astaxanthin from Haematococcus pluvialis. Bioresour. Technol. 99, pp. 3110–3115, 2008. 68. Spiller, G.A., and Dewell., A. Safety of an Astaxanthin-Rich Haematococcus pluvialis Algal Extract: A Randomized Clinical Trial. J. Med. Food, 6, 1, pp 51–56, 2003. 69. Dang, T.T., Van Vuong, Q., Schreider, M.J., Bowyer, M.C., Van Altena, I.A., and Scarlett, C.J., Optimisation of ultrasound-assisted extraction conditions for phenolic content and antioxidant activities of the alga Hormosira banksii using response surface methodology. J. Appl. Phycol. 29, 6, pp. 3161–3173, 2017. 70. Santoyo, S., Plaza, M., Jaime, L., Ibañez, E., Reglero, G., and Señorans, J., Pressurized liquids as an alternative green process to extract antiviral agents from the edible seaweed Himanthalia elongate. J. Appl. Phycol. 23, pp. 909– 917, 2011. 71. Prakash, S., Sasikala, S.L., and Aldous, V.H.J., Isolation and Identification of MDR–Mycobacterium Tuberculosis and Screening of Partially Characterised Antimycobacterial Compounds from Chosen Marine Micro Algae. Asian Pac. J. Trop. Med. 2010, 3, pp. 655–661, 2010. 72. Topuz, O.K., Gokoglu, N., Yerlikaya, P., Ucak, I., and Gumus, B., Optimization of Antioxidant activity and phenolic compound extraction conditions from red seaweed (Laurencia obtuse). J. Aquat. Food Prod. Technol. 25, pp. 414– 422, 2016. 73. Wrasidlo, W., Mielgo, A., Torres, V.A., Barbero, S., Stoletov, K., and Suyama, T.L., The marine lipopeptide somocystinamide A triggers apoptosis via caspase 8. Proc. Natl. Acad. Sci. U.S.A., 105, pp. 2313–2318, 2008. 74. Xu, Y., Kersten, R.D., Nam, S.J., Lu, L., Al-Suwailem, A.M., and Zheng, H., Bacterial biosynthesis and maturation of the didemnin anti-cancer agents. J. Am. Chem. Soc. 134, pp. 8625–8632, 2012. 75. Boyd, M.R., Gustafson, K.R., McMahon, J.B., Shoemaker, R.H., O’Keefe, B.R., and Mori, T., Discovery of cyanovirin-N, a novel humanimmunodeficiency

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36  Next-Generation Algae: Volume II 114. Schepetkin, I. A., and Quinn, M. T. Botanical polysaccharides: Macrophage immunomodulation and therapeutic potential. Int. Immunopharmacol., 6, 3, pp. 317-333, 2006. 115. Saini, M. K., Vaish, V., and Sanyal, S. N. Role of cytokines and Jak3/ Stat3 signaling in the 1,2-dimethylhydrazine dihydrochloride-induced rat model of colon carcinogenesis: Early target in the anticancer strategy. Eur. J. Cancer Prev., 22, 3, pp. 215-228, 2013. 116. Romay, C.H., Gonzalez, R., Ledon, N., Remirez, D. and Rimbau, V., C-phycocyanin: a biliprotein with antioxidant, anti-inflammatory and neuroprotective effects. Curr. protein pept. Sci.,  4, 3, pp. 207-216, 2003. 117. Stevenson, C. S., Capper, E. A., Roshak, A. K., Marquez, B., Eichman, C., Jackson, J. R., Mattern, M., Gerwick, W. H., Jacobs, R. S., and Marshall, L. A. The identification and characterization of the marine natural product scytonemin as a novel antiproliferative pharmacophore. J Pharmacol. Exp. Ther., 303, 2, pp. 858–866, 2002. 118. Stevenson, C. S., Capper, E. A., Roshak, A. K., Marquez, B., Grace, K., Gerwick, W. H., Jacobs, R. S., and Marshall, L. A. Scytonemin - A marine natural product inhibitor of kinases key in hyperproliferative inflammatory diseases. Inflammation Research, Journal of the European Histamine Research Society. 1, 2, pp. 112–114, 2002. 119. Vining, O. B., Medina, R. A., Mitchell, E. A., Videau, P., Li, D., Serrill, J. D., Kelly, J. X., Gerwick, W. H., Proteau, P. J., Ishmael, J. E., and McPhail, K. L. Depsipeptide companeramides from a panamanian marine cyanobacterium associated with the coibamide producer. Journal of Natural Products, 78, 3, pp. 413–420, 2015. 120. Vieira, C., Gaubert, J., De Clerck, O., Payri, C., Culioli, G., and Thomas, O.P., Biological activities associated to the chemodiversity ofthe brown algae belonging to genus Lobophora (Dictyotales, Phaeophyceae). Phytochem. Rev. 16, pp. 1–17, 2017. 121. Athbi, M.A., Al-Mayah, S.H., and Khalaf, A.K., Antiparasitic activity of the microalgae Cladophora crispata against the protoscolices of hydatid cysts compared with albendazole drug. Afr. J. Biotechnol. 13, pp. 3068–3080, 2014. 122. Bianco, É.M., De Oliveira, S.Q., Rigotto, C., Tonini, M.L., Da Rosa Guimarães, T., and Bittencourt, F., Anti-infective potential of marine invertebrates and seaweeds from the Brazilian coast. Molecules, 18, pp. 5761–5778, 2013. 123. Kar, S., Sharma, G., and Das, P.K., Fucoidan cures infection with both antimony-­susceptible and-resistant strains of Leishmania donovani through Th1 response and macrophage-derived oxidants. J. Antimicrob. Chemother. 66, 3, pp. 618–625, 2011. 124. Veiga-Santos, P., Pelizzaro-Rocha, K.J., Santos, A.O., Ueda-Nakamura, T., Filho, B.P.D., and Silva, S.O., In vitro anti-trypanosomal activity of elatol isolated from red seaweed Laurencia dendroidea. Parasitology, 137, pp. 1661– 1670, 2010.

Novel-Biologically Active Compounds Discovery  37 125. Afolayan, A.F., Bolton, J.J., Lategan, C.A., Smith, P.J., and Beukes, D.R., Fucoxanthin, tetraprenylated toluquinone and toluhydroquinone metabolites from Sargassum heterophyllum inhibit the in vitro growth of the malaria parasite Plasmodium falciparum. Z. Naturforsch, 63, pp. 848–852, 2008. 126. Lategan, C., Kellerman, T., Afolayan, A.F., Mann, M.G., Antunes, E.M., and Smith, P.J., Antiplasmodial and antimicrobial activities of South African marine algal extracts. Pharm. Biol. 47, 5, pp. 408–413, 2009. 127. Ogawa, H., Iwasaki, A., Sumimoto, S., Iwatsuki, M., Ishiyama, A., Hokari, R., Otoguro, K., O̅   Mura, S., and Suenaga, K., Isolation and total syn- thesis of hoshinolactam, an antitrypanosomal lactam from a marine cyanobacterium. Organic Letters, 19, 4, pp. 890–893, 2017. 128. Aditya, T., Bitu, G., and Mercy Eleanor, G., The role of algae in pharmaceutical development. Res. Rev.: J. Pharm. Nanotechnol. 4, pp. 82–89, 2016. 129. Pham-Huy, L.A., He, H., and Pham-Huy, C., Free radicals, antioxidants in disease and health. Int. J. Biomed. Sci. 4, 2, pp. 89–96, 2008. 130. Smee, D.F., Bailey, K.W., Wong, M.H., O’Keefe, B.R., Gustafson, K.R., and Mishin, V.P., Treatment of influenza A (H1N1) virus infections in mice and ferrets with cyanovirin-N. Antiviral Res, 80, pp. 266–271, 2008. 131. Harun, R., Singh, M., Forde, G.M., and Danquah, M.K., Bioprocess engineering of microalgae to produce a variety of consumer products. Renew. Sustain, Energy Rev. 14, pp. 1037–1047, 2010. 132. Mata, T.M., Martins, A.A., and Caetano, N.S., Microalgae for biodiesel production and other applications: a review. Renew. Sustain. Energy Rev. 14, 1, pp. 217-232, 2010. 133. Skjånes, K., Rebours, C., and Lindblad, P., Potential for green microalgae to produce hydrogen, pharmaceuticals and other high value products in a combined process. Crit. Rev. Biotechnol. 33, pp. 172–215, 2013. 134. Vera, M.D., and Joullie, M.M., Natural products as probes of cell biology: 20 years of didemnin research. Med. Res. Rev. 22, 2, pp. 102–145, 2002. 135. Maruthanayagam, V., Nagarajan, M., and Sundararaman, M., An insight into biological significance of marine cyanobacterial toxins in the recent decade. Cah. Biol. Mar. 54, pp. 221–238, 2013. 136. Pearson, L.A., Dittmann, E., Mazmouz, R., Ongley, S.E., D’Agostino, P.M., and Neilan, B.A., The genetics, biosynthesis and regulation of toxic specialized metabolites of cyanobacteria. Harmful Algae, 54, pp. 98-111, 2016. 137. Magarvey, N.A., Beck, Z.Q., Golakoti, T., Ding, Y., Huber, U., and Hemscheidt, T.K., Biosynthetic characterization and chemoenzymatic assembly of the cryptophycins. Potent anticancer agents from cyanobionts. ACS Chem. Biol, 1, 12, pp. 766–779, 2006. 138. Panda, D., Himes, R.H., Moore, R.E., Wilson, L., and Jordan, M.A., Mechanism of action of the unusually potent microtubule inhibitor cryptophycin 1. Biochemistry, 36, 42, pp. 12948–12953, 1997. 139. Ughy, B., Nagy, C.I., and Kós, P.B., Biomedical potential of cyanobacteria and algae. Acta Biol. Szeged, 59, pp. 203–224, 2015.

38  Next-Generation Algae: Volume II 140. Luesch, H., Yoshida, W.Y., Moore, R.E., Paul, V.J., and Corbett, T.H., Total structure determination of apratoxin A, a potent novel cytotoxin from the marine cyanobacterium Lyngbya majuscule. J. Am. Chem. Soc, 123, 23, pp. 5418–5423, 2001. 141. Liu, Y., Law, B.K., and Luesch, H., Apratoxin A reversibly inhibits the secretory pathway by preventing cotranslational translocation. Mol. Pharmacol. 76, 1, pp. 91–104, 2009. 142. Vijayakumar, S., and Menakha, M., Pharmaceutical applications of cyanobacteria-a review. J. Acute Med, 5, 1, pp. 15-23, 2015. 143. Kounnis, V., Chondrogiannis, G., Mantzaris, M.D., Tzakos, A.G., Fokas, D., and Papanikolaou, N.A., Microcystin LR shows cytotoxic activity against pancreatic cancer cells expressing the membrane OATP1B1 and OATP1B3 transporters. Anticancer Re, 35, 11, pp 5857-5865, 2015. 144. Geada, P., Gkelis, S.,Teixeira, J.,Vasconcelos,V.,Vicente, A.A., and Fernandes, B.,Cyanobacterial toxins as a high value-added product. Microalgae-Based Biofuels and Bioproducts, pp. 401–428, 2017. 145. Lodish, H., Berk, A., Matsudaira, P., Kaiser, C., and Krieger, M.S.M., Molecular Cell Biology. 6th edition. New York, 2008. 146. Kini, S., Bahadur, D., and Panda, D., Mechanism of anti-cancer activity ofbenomyl loaded nanoparticles in multidrug resistant cancer cells. J.  Biomed. Nanotechnol. 11, pp. 877–889, 2015. 147. Rai, A., Gupta, T.K., Kini, S., Kunwar, A., Surolia, A., and Panda, D., CXIbenzo-84 reversibly binds to tubulin at colchicine site and induces apoptosis in cancer cells. Biochem. Pharmacol, 86, 3, pp. 378-391, 2013. 148. Chen, B., You, W., Huang, J., Yu, Y., and Chen, W., Isolation and antioxidant property of the extracellular polysaccharide from Rhodella reticulate. World J. Microbiol. Biotechnol, 26, 5, pp. 833-840, 2010. 149. Lyngbya, P., Patel, S.S.A., Mishra, S., and Ghosh, P.K., Antioxidant potential of C-phycocyanin isolated from cyanobacterial species. Indian J. Biochem. Biophys, 43, pp. 25–31, 2006. 150. Li, W.I., Berman, F.W., Okino, T., Yokokawa, F., Shioiri, T., and Gerwick, W.H., Antillatoxin is a marine cyanobacterial toxin that potently activates voltage-gated sodium channels. Proc. Natl. Acad. Sci. U.S.A.   98, 13,  pp. 7599-7604, 2002. 151. De Oliveira, E.O., Graf, K.M., Patel, M.K., Baheti, A., Kong, H.S., and MacArthur, L.H., Synthesis and evaluation of hermitamides A and B as human voltage-gated sodium channel blockers. Bioorg. Med. Chem, 19, 14, pp. 4322-4329, 2011. 152. Torregrosa-Crespo, J., Montero, Z., Fuentes, J.L., García-Galbis, M.R., Garbayo, I., and Vílchez, C., Exploring the valuable carotenoids for the largescale production by marine microorganisms. Mar. Drugs, 16, 6, pp. 203, 2018.

Novel-Biologically Active Compounds Discovery  39 153. Gerwick, W.H., Roberts, M.A., Proteau, P.J., and Chen, J.L., Screening cultured marine microalgae for anticancer-type activity. J. Appl. Phycol, 6, pp. 143–149, 1994. 154. Kwang, H.C., Song, Y.I.K., and Lee, D.U., Antiproliferative effects of carotenoids extracted from Chlorella ellipsoidea and Chlorella vulgaris on human colon cancer cells. J. Agric. Food Chem. 56, pp. 10521–10526, 2008. 155. Nigjeh, S.E., Yusoff, F., Banu, N., Alitheen, M., Rasoli, M., and Keong, Y.S., Cytotoxic effect of ethanol extract of microalga, Chaetoceros calcitrans, and its mechanisms in inducing apoptosis in human breast cancer cell line. Biomed. Res. Int, pp. 1–9, 2013. 156. Miralto, A., Barone, G., Romano, G., Poulet, S.A., Ianora, A., and Russo, G.L., The insidious effect of diatoms on copepod reproduction. Nature, 402, pp. 173–176, 1999. 157. Kusaikin, M.I., Ermakova, S.P., Shevchenko, N.M., Isakov, V.V., Gorshkov, A.G., and Vereshchagin, A.L., Structural characteristics and antitumor activity of a new chrysolaminaran from the diatom alga Synedra acus. Chem. Nat. Compd. 46, pp. 1–4, 2010. 158. Bajpai, V. K., Shukla, S., Kang, S. M., Hwang, S. K., Song, X., Huh, Y. S., and Han, Y. K., Developments of cyanobacteria for nano-marine drugs: Relevance of nanoformulations in cancer therapies. Mar. Drugs, pp. 16-179, 2018. 159. Nijhuis, M., van Maarseveen, N.M., and Boucher, C.A., Antiviral resistance and impact on viral replication capacity: evolution of viruses under antiviral pressure occurs in three phases. Handb. Exp. Pharmacol, pp. 299–320, 2008. 160. Yang, F., Bewley, C.A., Louis, J.M., Gustafson, K.R., Boyd, M.R., and Gronenborn, A.M., Crystal structure of cyanovirin-N, a potent HIVinactivating protein, shows unexpected domain swapping. J. Mol. Biol. 288, pp. 403–412, 1999. 161. Mansour, H., Shoman, S., and Kdodier, M., Antiviral effect of edaphic cyanophytes on rabies and herpes-1 viruses. Acta Biol. Hung. 62, pp. 194–203, 2011. 162. Yakoot, M., and Salem, A., Spirulina platensis versus silymarin in the treatment ofchronic hepatitis C virus infection. A pilot randomized, comparative clinical trial. BMC Gastroenterol. 12, pp. 1-9, 2012. 163. Rechter, S., König, T., Auerochs, S., Thulke, S., Walter, H., and Dörnenburg, H., Antiviral activity of Arthrospira-derived spirulan-like substances. Antiviral Res, 72, 3, pp. 197-206, 2006. 164. Arment, A.R., and Carmichael, W.W., Evidence that microcystin is a thio-template product. J. Phycol. 32, 4, pp. 591–597, 1996. 165. Ahmadi, A., Zorofchian Moghadamtousi, S., Abubakar, S., and Zandi, K., Antiviral potential of algae polysaccharides isolated from marine sources: a review. Biomed. Res. Int. pp. 1–10, 2015. 166. Kim, S.K., and Wijesekara, I., Anticoagulant effect ofmarine algae. Adv. Food Nutr. Res. 64, pp. 235–244, 2011.

40  Next-Generation Algae: Volume II 167. Matsubara, K., Recent advances in marine algal anticoagulants. Curr. Med. Chem. Hematol. Agents, 2, pp. 13–19, 2005. 168. Lahaye, M., and Robic, A., Structure and function properties ofulvan, a polysaccharide from green seaweeds. Biomacromolecules, 8, 6, pp. 1765–1774, 2007. 169. Usman, A., Khalid, S., Usman, A., Hussain, Z., and Wang, Y., Algal polysaccharides, novel application, and outlook. Algae Based Polym. Blends, Compos. pp. 115-153, 2017. 170. Matsumoto, Y., Sahara, H., Fujita, T., Hanashima, S., Yamazaki, T., and Takahashi, S., A novel immunosuppressive agent, SQDG, derived from sea urchin. Transpl. Proc. 32, pp. 2051–2053, 2000. 171. Khan, Z., Bhadouria, P., and Bisen, P.S., Nutritional and therapeutic potential of Spirulina. Curr. Pharm. Biotechnol. 6, pp. 373–379, 2005. 172. Ambrosi, M.A., Reinehr, C.O., Bertolin, T.E., Costa, J.A.V., and Colla, L.M., Propriedades de saúde de Spirulina spp. Rev. Cienc. Farm Basica Apl, 29, 2, pp. 109-117, 2008.

2 Bioactive Compounds Synthesized by Algae: Current Development and Prospects as Biomedical Application in the Pharmaceutical Industry Preeti Mishra1*, Namrata Gupta2, Monika Singh1 and Deeksha Tiwari2 Faculty of Pharmacy, Raja Balwant Singh Engineering Technical Campus, Bichpuri, Agra, India 2 Faculty of Engineering, Raja Balwant Singh Engineering Technical Campus, Bichpuri, Agra, India 1

Abstract

Algae and microalgae are a diverse group of organisms that contain a diverse range of bioactive chemicals, including pigments, polyunsaturated fatty acids, polysaccharides, polyphenols, and other bio-algae and microalgae, as well as their bioactive chemicals, which have antioxidant, anti-inflammatory, anticancer, and antiobesity characteristics, making them ideal for use in food preparation. Because of the tremendous diversity of organisms and molecules, new effects and/ or compounds will inevitably be found in the future. A growing number of people are becoming interested in algae extracts as a result of their particular composition and potential for use in a wide variety of industrial applications. Algal biomass is transformed into extracts by the use of several extraction techniques. Microalgae bioactive compounds offer a variety of beneficial effects, including anti-inflammatory, antibacterial, and antioxidant capabilities. These bacteria also have the potential to promote health and reduce the risk of degenerative disorders, according to researchers. As outlined, the purpose of this chapter is to describe bioactive metabolites generated by microalgae and their potential uses in the pharmaceutical sector.

*Corresponding author: [email protected] Charles Oluwaseun Adetunji, Julius Kola Oloke, Naveen Dwivedi, Sabeela Beevi Ummalyma, Shubha Dwivedi, Daniel Ingo Hefft and Juliana Bunmi Adetunji (eds.) Next-Generation Algae: Volume II: Applications in Medicine and the Pharmaceutical Industry, (41–76) © 2023 Scrivener Publishing LLC

41

42  Next-Generation Algae: Volume II Keywords:  Microalgae, bioactive compounds, carotenoids, polyunsaturated fatty acids, protein and polypeptides

2.1 Introduction Algae are photosynthetic microorganisms that live and grow in water under hydroponic conditions, allowing them to absorb carbon in heterotrophic and mixotrophic environments [1]. The vast majority of algae are found in fresh or salt water, where they can either float freely (planktonic) or stick to the bottom of the water body. Algae may grow on rocks, soil, and plants as long as there is enough moisture present to support growth. Lichens can form when a small number of algae build tight interactions with fungus. The hairs of the South American sloth and the polar bears are remarkable algal habitats because they are so thick. Chlorophyll is a pigment found in algae (other types of chlorophyll, such as b, c, and/or d, may also be present), and algae employ photosynthesis to generate their food. Chlorophyll is found in the chloroplasts of algae, and it is responsible for the green color of many algae. However, some algae appear brown, yellow, or red because they contain additional pigments that, in addition to chlorophylls, conceal the green color [2]. Microalgae are unicellular micro­organisms that may be found in a variety of forms and sizes with a diameter or length ranging from 3–10 μm [3]. These bacteria are responsible for around 40% of all photosynthesis on the planet [4]. Changes in the external environment create changes in the internal environment in the metabolism of microalgae, and the reverse is true as well. When the culture conditions are altered, the presence or absence of specific nutrients, for example, might cause the production of specific compounds to increase or decrease. The bioactive chemicals synthesized by microalgae include proteins, fatty acids, vitamins, and pigments, all of which can be taken directly from primary metabolism or created indirectly through secondary metabolism [5]. These compounds possess antifungal, antiviral, antialgal, anti-enzymatic, and antibiotic capabilities, among other characteristics. Numerous chemicals in this group (cyanovirin, oleic acid, linolenic acid, palmitoleic acid, vitamin E, B12, β-carotene, phycocyanin, lutein, and zeaxanthin) possess antibacterial and anti-inflammatory characteristics, and as a result, can decrease and prevent sickness [3]. Most microalgae collect bioactive compounds in their biomass; but, in rare instances, these metabolites are expelled into the surrounding media and are referred to as exometabolites. Bioactive metabolites derived from microalgae are of great relevance in the creation of novel

Bioactive Compounds Synthesized by Algae  43 products for the medical, pharmaceutical, cosmetic, and food industries. Further research into these bioactive compounds is required to validate their beneficial effects on humans, their environmental degradability, and their effects when used in animal experiments [3]. Marine algae are rich in bioactive nutraceuticals (carbohydrates, proteins, minerals, fatty acids, antioxidants, and pigments), which are beneficial to human health. Natural secondary metabolites are produced by algae in response to environmental factors, including biotic (plants, microorganisms) and abiotic factors such as temperature (pH, salinity, and light intensity). Simple, cost-effective, and long-term recovery options include novel solid-liquid and liquid-liquid extraction techniques (e.g., supercritical, high pressure, microwave, ultrasonic, enzymatic) and novel solid-liquid separation techniques [6]. Because marine microalgae and cyanobacteria contain a diverse range of chemical substances, they have the potential to be used in biological applications that have health benefits. Microalgae can be used to produce a wide range of chemicals, including polyphenols, polyunsaturated fatty acids (PUFA), and phytosterols. In contrast, unexplored natural sources of bioactive components are gaining a great deal of research since they have the potential to lead to the identification of new molecules or bioactivities [5].

2.2 Algal-Sourced Compounds of Medical Interest Throughout the last few decades, marine algae have attracted considerable attention as a potential source of potential renewable energy. There are more than 8000 unique types of marine algae species that have been identified throughout the world, according to recent research [6–8]. Primary and secondary metabolites (e.g., polysaccharides, proteins, amino acids, dietary fibre, essential fatty acids) found in seaweeds, including pigments, phytosterols, polyphenols, terpenoids, carotenoids, tocopherols, minerals, and vitamins, have been shown to have antiviral, antihelminthic, antifungal, and antibacterial properties in laboratory studies. Based on their mechanistic differences, physiologically active compounds found in marine algae can be divided into two categories [8, 9]: (i) non-absorbed high-­ molecular materials and (ii) absorbed low-molecular materials, both of which have direct effects on human homeostasis; and absorbed low-­molecular materials. These substances are now widely used in both fresh and processed foods, and they have gained prominence in nutritional science as a result of their potential pharmacological applications

44  Next-Generation Algae: Volume II as antioxidants, anti-inflammatories, anti-proliferative (including tumor suppression), antithrombotic, anticoagulant, antihypertensive, antidiabetic, and cardioprotective agents (among other things). The development of novel drugs generated from macroalgal biocompounds that are clinically viable and commercially available in cancer, infection, diabetes, and neuroprotective formulations has emerged as a rapidly expanding pharmacological specialty. Furthermore, various research studies aiming at the prevention of diabetes and neurological disorders (neurodegeneration), as well as the reduction of oxidative stress in the central nervous system, have proved the antidiabetic and neuroprotective properties (CNS) of seaweed biocompounds. Seaweed-based antidiabetic and neuroprotective compounds, on the other hand, are still in their infancy, and more studies and discoveries are needed in this field [6].

2.3 Microalgae with Potential for Obtaining Bioactive Compounds Because microalgae are such a varied group of heterogeneous microorganisms with such a vast range of coloration, shapes, and cell features, the issue of marine biotechnology involves their manipulation as well as the modification of other microbes [3]. Microalgae species are in the hundreds, and only a tiny proportion of those are known to exist in collections throughout the globe, and only a few hundred of them are believed to have been studied for the presence of chemicals in the algae’s biomass. Only a handful of these plants are commercially produced in large quantities [10]. They have potential applications in a range of biotechnological industries because of their undiscovered diversity, including the production of biochemicals used in food, medicine, cosmetics, and pharmaceutical products as well as the energy industry [11]. Microalgae are a naturally occurring source of biologically active compounds that have a wide range of potential uses in a variety of fields. These compounds have piqued the interest of both academia and industry in recent years, owing to their potential applications in a variety of life science disciplines. Their uses range from biomass production for food and feed to the manufacture of bioactive chemicals for use in the medical and pharmaceutical industries [10]. Because of the wide diversity of microalgae available and recent developments in genetic engineering, this group of microorganisms is one of the most promising sources of new commodities and applications [3].

Bioactive Compounds Synthesized by Algae  45 As autotrophic microorganisms, microalgae are capable of developing and synthesizing biocompounds with high aggregated nutritional value and therapeutic functions. These biocompounds include lipids, proteins, carbohydrates, pigments, and polymers, all of which are derived from light energy and inorganic nutrients (carbon dioxide, nitrogen, phosphorus, etc.). Recently discovered microalgae may produce a wide range of Table 2.1  The most important bioactive chemicals derived from microalgae [3]. Microalgae

Bioactive compounds

Spirulina sp.

Polysaccharides

Spirulina platensis

Phycocyanin, C-phycocyanin, Phenolic acids, tocopherols (vitamin E), neophytadiene, phytol, PUFAs (n-3) fatty acids, oleic acid, linolenic acid, palmitoleic acid

Spirulina fusiformis

Diacylglycerols

Haematococcus pluvialis

Astaxanthin, lutein, zeaxanthin, canthaxanthin, lutein, β-carotene, oleic acid

Chlorella sp.

Carotenoids, sulfated polysaccharides, sterols, PUFAs (n-3) fatty acids

Chlorella vulgaris

Canthaxanthin, astaxanthin, peptide, oleic acid

Chlorella minutissima

Eicosapentaenoic acid (EPA)

Chlorella ellipsoidea

Zeaxanthin, violaxanthin

Dunaliella salina

trans-β-carotene, cis-β-carotene, β-carotene, oleic acid, linolenic acid, palmitic acid

Dunaliella

Diacylglycerols

Botryococcus braunii

Linear alkadienes (C25, C27, C29, and C31), triene (C29)

Chlorella zofingiensis

Astaxanthin

Chlorella protothecoides

Lutein, zeaxanthin, canthaxanthin

Chlorella pyrenoidosa

Lutein, sulfated polysaccharide

Nostoc linckia and Nostoc spongiaeforme

Borophycin

Nostoc sp.

Cryptophycin

46  Next-Generation Algae: Volume II chemical substances with varying biological activities, including carotenoids, phycobilins, polyunsaturated fatty acids, proteins, polysaccharides, vitamins, and sterols. Carotenoids and phycobilins are phytochemicals that have biological activity [3, 12]. Microalgal components have been shown to have antimicrobial, antiviral, anticoagulant, antienzymatic, antioxidant, antifungal, anti-­inflammatory, and anticancer activities, among other qualities [3]. The most important bioactive chemicals derived from microalgae are listed in Table 2.1 [3].

2.3.1 Spirulina Spirulina is a member of the Cyanophyta (prokaryotic cyanobacteria) that first appeared more than three million years ago, resulting in the formation of the current oxygen atmosphere. Spirulina (Arthrospira) has played an important part in the management of the planetary biosphere. A GRAS (generally recognized as safe) accreditation was granted to spirulina by the FDA (Food and Drug Administration) in 1981 [13]. According to the FDA, spirulina can be lawfully sold as a food or food supplement without compromising human health [14]. Spirulina contains significant amounts of essential polyunsaturated fatty acids and phenolic compounds, as well as high protein content and digestibility, making it a nutritious addition to any diet [15]. As a result of characteristics such as its high nutritional value and the presence of active biocompounds, this microbe is one of the most extensively examined microalgae on the planet. Table 2.2 shows the bioactive compounds extracted from Spirulina. Spirulina is a green leafy vegetable with a protein content ranging from 50 to 70% (w/w) [3], carbohydrate content from 10 to 20% (w/w), and lipid content from 5 to 10% (w/w). This microalga has high concentrations of vitamins B1, B2, B12, and E (especially vitamin B12). It also includes significant amounts of pigments, minerals, and oligo-elements (between 6 and 9% by dry weight of the biomass), with iron, calcium, magnesium, phosphorus, and potassium being the most abundant. Because of its antioxidant properties, this microalga has been employed in the production of pigments in several research studies throughout the years. There are several different carotenoids found in Spirulina. These include β-carotene, tocopherols, phycocyanin, and phycoerythrin. Spirulina also includes a range of additional components such as tocopherols, phycocyanin, and phycoerythrin [16]. Researchers have discovered that cyanobacteria can synthesize intracellular and extracellular compounds that have antibacterial, antifungal, antiviral, anticancer, anti-HIV, anti-inflammation, antioxidant, antimalarial,

Bioactive Compounds Synthesized by Algae  47 Table 2.2  Bioactive compounds extracted from Spirulina genus. Microalga

Bioactive compounds

Concentration (%, w/w)

Spirulina fusiformis

C-phycocyanin

46.0

Spirulina platensis

C-phycocyanin

9.6

Spirulina platensis

Allophycocyanin

9.5

Spirulina sp.

C-phycocyanin

17.5

Spirulina sp.

Allophycocyanin

20.0

Spirulina platensis

Phenolic

0.71

Spirulina platensis

Terpenoids

0.14

Spirulina platensis

Alkaloids

3.02

Spirulina maxima

Phenolic

1.29

Spirulina maxima

Flavonoids

0.46

herbicidal, and immunosuppressive properties, as well as immunosuppressive compounds. The therapeutic potential of Spirulina has been demonstrated in several research studies. Its applications include the treatment of hyperlipidemia, cancer, HIV, diabetes, obesity, and hypertension, as well as the enhancement of immune response in kidney protection against heavy metals and medications, as well as the lowering of glucose and lipid levels in the blood. It is also used in the prevention of diabetes and obesity [3].

2.3.2 Chlorella Spirulina and Chlorella produce between 3,000 and 4,000 tons of micro­ algal biomass each year, accounting for the vast majority of the market for microalgal biomass [17]. In the Chlorophyta family, the eukaryotic genus Chlorella sp. contains unicellular green microalgae that are eukaryotic [18]. This microalga was discovered by the Japanese, who are accustomed to eating algae and who use it as a nutritious supplement to enhance their diet. Chlorella is a superfood that is strong in chlorophyll, proteins, polysaccharides, vitamins, minerals, and essential amino acids, amongst other beneficial compounds. The alga is composed of 53% (w/w) protein, 23% (w/w) carbohydrate, 9% (w/w) lipids, and 5% (w/w) minerals and oligo-­ elements [14].

48  Next-Generation Algae: Volume II These nutrient concentrations may be manipulated by manipulating the culture conditions. This microalga’s biomass also contains a high concentration of B vitamins, notably B12, which are required for the synthesis and repair of red blood cells. Chlorella, like Spirulina, has received GRAS certification from the Food and Drug Administration, suggesting that it may be consumed as food without posing a health risk provided it is grown in an acceptable environment with adequate sanitation and manufacturing practices [14, 19]. [Table 2.3 shows the bioactive compounds extracted from Chlorella]. Chlorella contains a variety of bioactive chemicals that have medicinal properties. Chromolaria has been proven to have cancer-fighting and anticoagulant characteristics in experimental studies. It has also been shown to have antibacterial, antimicrobial, antioxidant, and antihyperlipidemic capabilities, as well as a hepatoprotective function and immunostimulatory activity [3]. Chlorella possesses functional properties that might be ascribed to several antioxidant compounds found in the algae. Free radicals have been demonstrated to be inhibited by antioxidants such as lutein, α-carotene, β-carotene, ascorbic acid, and α-tocopherol, among other substances. Some of these compounds are important not just as natural colorants or additives, but they may also be beneficial in the prevention of cancer and the treatment of macular degeneration [18, 20]. The most major bioactive element in Chlorella is β-1,3 glucan, which is a powerful immune stimulant that decreases free radicals and cholesterol levels in the bloodstream. Gastric ulcers, sores, and constipation have all Table 2.3  Bioactive compounds extracted from the microalgae of the Chlorella genus. Microalga

Bioactive compounds

Concentration (%, w/w)

Chlorella protothecoides

Lutein

4.60

Chlorella zofingiensis

Astaxanthin

1.50

Chlorella vulgaris

Phenolic

0.20

Chlorella vulgaris

Terpenoids

0.09

Chlorella vulgaris

Alkanoids

2.45

Chlorella minutissima

Phytol

2.70

Chlorella minutissima

Phenol

1.81

Bioactive Compounds Synthesized by Algae  49 been proven to be alleviated by the use of this substance. Besides having anticancer properties, it has also been proven to have prophylactic properties against atherosclerosis and hypercholesterolemia [21].

2.3.3 Nostoc Nostoc is a Cyanophyta microalga that forms spherical colonies that come together as filaments. It is a member of the Nostocaceae family of Cyanophyta and belongs to the Nostocaceae genus. The filament of this microalga is made up of heterocysts, each of which has a pattern of homogeneous cells and a regular spacing between cells between them [22]. Heterocysts fix nitrogen from the atmosphere to produce amino acids for microalgal biomass. Heterocysts form in the lack of a nitrogen supply during microalgal culture, allowing the cells to develop without being constrained by the nutrient’s limitation [23]. Microalgal biomass from the Nostoc genus has been used in medicine and as a nutritional supplement due to its high protein, vitamin, and fatty acid content (Table 2.4). When this microalga is used to treat fistula and some types of cancer, it proves to be quite beneficial [24]. Previous studies have shown that biomass is anti-inflammatory and that it also aids with digestion, blood pressure management, and immune system stimulation. Several studies have revealed that Nostoc can produce compounds that are antibacterial, antiviral, and anticancer. Large-scale Table 2.4  Bioactive compounds extracted from the Nostoc genus. Microalga

Bioactive compounds

Concentration (%)

Nostoc sp.

Phycocyanin

20.0 (p/p)

Nostoc muscorum

Phenolic

0.61 (p/p)

Nostoc muscorum

Terpenoids

0.10 (p/p)

Nostoc muscorum

Alkaloids

2.30 (p/p)

Nostoc muscorum

Phycobilins

0.0229 (p/v)

Nostoc humifusum

Phenolic

0.34 (p/p)

Nostoc humifusum

Terpenoids

0.10 (p/p)

Nostoc humifusum

Alkaloids

1.65 (p/p)

Nostoc humifusum

Phycobilins

0.0031 (p/v)

50  Next-Generation Algae: Volume II production has been motivated by these discoveries, and it has enormous economic potential as a result of its nutritional and therapeutic properties [25]. It has been demonstrated that cyanovirin, which is a possible protein molecule produced by the Nostoc microalga, is useful in the treatment of HIV and Influenza A (H1N1) [3]. Many essential fatty acids, such as linoleic, α-linolenic, γ-linolenic, octadecatetraenoic, and eicosapentaenoic acid, are present in Nostoc, and these polyunsaturated fatty acids (PUFAs) are found in Nostoc. Because essential fatty acids are precursors of prostaglandins, the pharmaceutical sector has taken an interest in them [26].

2.3.4 Dunaliella Dunaliella is a green, unicellular halotolerant microalga that belongs to the Chlorophyceae family. This microalga is frequently investigated because of its ability to withstand harsh environmental conditions, its physiological characteristics, and the numerous biotechnological uses it has. For example, Dunaliella is an excellent source of carotenoids, glycerol, lipids, and several other useful substances, such as enzymes and vitamins [27, 28]. Table 2.5 shows the bioactive compounds extracted from Dunaliella. When grown in settings with high salinity, light, and temperature, as well as nutrition limitation, this microalga may generate up to 14% of its dry weight in natural β-carotene [29]. Furthermore, in addition to β-carotene,

Table 2.5  Bioactive compounds extracted from the microalgae of the Dunaliella genus. Microalga

Bioactive compounds

Dunaliella salina

β-Carotene

Dunaliella salina

All-trans-β-carotene

Dunaliella salina

All-trans-zeaxanthin

Dunaliella salina

All-trans-lutein

Dunaliella tertiolecta

Sterols

Dunaliella salina

Sterols

Bioactive Compounds Synthesized by Algae  51 microalga is high in protein and essential fatty acids, and is considered safe to ingest as indicated by its GRAS designation [14]. A variety of biological activities are exhibited by the compounds found in the Dunaliella biomass, including antioxidant, antihypertensive, bronchodilatory, analgesic, muscle relaxant, hepatoprotective, and antiedemal effects. Microalgal biomass can also be employed directly in food and medicinal formulations, as well as in other applications [14, 30]. Researchers Chang and colleagues discovered that Dunaliella cells contained antibiotic compounds. According to these researchers, the crude extract of this microalga significantly prevented the development of bacteria such as Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, and Enterobacter aerogenes, among others. Dunaliella microalga was shown to have antibacterial action against a variety of pathogens that are important to the food industry, including Escherichia coli, Staphylococcus aureus, Candida albicans, and Aspergillus niger, according to another investigation [28, 31]. Under optimum growth conditions, Dunaliella may be pushed to create roughly 400 mg of β-carotene per square meter of growing area, which is equivalent to approximately 400 mg of vitamin A [3]. The cultivation of Dunaliella to produce β-carotene has been carried out in several countries, including Australia, Israel, the United States of America, and China. Earlier this year, Pentapharm announced the introduction of a substance derived from Dunaliella that has a high potential to stimulate cell proliferation and enhance the energy metabolism of the skin (Basel, Switzerland). New pilot plants are being built in India, Chile, Mexico, Cuba, Iran, Taiwan, Japan, Spain, and Kuwait, among others [29].

2.4 Bioactive Compounds from Cyanobacteria Microalgae have a large number of bioactive chemicals that may be extracted and used in a variety of applications. As key sources of value-­ added chemicals with medicinal and nutritional significance, they have emerged as important sources of protein. Cyanobacteria or blue-green algae were once considered to be laboratory curiosities or nuisance agents in water bodies, but today they are recognized as an important component of integrated nutrient management in agriculture [32, 33] and as a source of pigments, vitamins, phycocolloids, immunodiagnostic agents, and therapeutics, as well as a source of biofuel [34, 35].

52  Next-Generation Algae: Volume II In recent years, screening methods have been used to find several bioactive metabolites generated by cyanobacteria and algae. These screening procedures used target organisms that were not related to the organisms that the metabolites evolved for. Numerous biological activities and chemical structures of these substances have been discovered, and they have an impact on a wide variety of biochemical processes within cells (mainly directed against photosynthetic processes). Chemicals of this type are probably associated with the management and succession of algal and bacterial populations, and can operate as natural herbicides or biocontrol agents in certain situations [36]. A cyanobacterium with algicidal and bactericidal characteristics should be predicted in light of the co-occurrence of these organisms in aquatic natural communities, where an inhibitory interaction between producers and competitors within the same ecosystem has been seen to occur. In lakes with the presence of cyanobacterial water blooms, Chrost observed a significant drop in the number of Gram-positive bacteria, and the generation of antibiotic compounds may be one of the factors contributing to these phenomena. Natural herbicides are bioactive allelochemicals produced by cyanobacteria that have been classified as algicides. Because they are aimed against photosynthesis (photosystem II), they are referred to as natural herbicides. In both prokaryotic cyanobacteria and eukaryotic algae, light-dependent activities are unique, and they are therefore logical targets for a bioactive producing organism while competing with other similar organisms (the targets) in the same habitat [36]. Many different types of cyanobacteria generate substances that are typically regarded to be secondary metabolites, which are molecules that are not required for general metabolism or growth of the organism and are only found in certain types of cyanobacteria. Cyanobacteria, such as Microcystis, Anabaena, Nostoc, and Oscillatoria, create a wide range of secondary metabolites, some of which are toxic to humans. Many key marine cyanobacterial compounds, including dolastatin, cryptophycin, and curacin A, have been identified and are now undergoing preclinical or clinical testing as anticancer medicines. Dolastatin, cryptophycin, and curacin A are examples of such molecules [37]. Because a large number of secondary metabolites are strong poisons, they can cause health issues in animals and people when the producer organisms are found in large numbers in bodies of water. Different compounds, such as cytotoxic (41%), antitumor (13%), antiviral (4%), antibiotics (12%), and the remaining 18% activities include antimalarial, antimycotics, multidrug resistance reversers, antifeedant, herbicides, and immunosuppressive agents; aside from the immune effect, blue-green

Bioactive Compounds Synthesized by Algae  53 algae have been shown to improve metabolism. Cyanobacterial lipopeptides are derived from cyanobacteria [38]. A prolific producer of almost 800 distinct bioactive secondary metabolites, cyanobacteria are a major source of nonribosomal peptide synthetase (NRPS) or mixed polyketide synthetase (PKS)–NRPS biosynthesis, with the majority deriving from NRPS biosynthesis [39, 40]. Several studies have demonstrated that they are effective against viruses and tumors as well as against bacteria and HIV. In addition, they are used as a food additive in several countries. Cyanobacteria are a primitive and varied collection of microorganisms that share traits with both bacteria and algae. They are a basic but primitive and diverse group of microorganisms that share characteristics with both bacteria and algae. Their capacity to survive as a group in a diverse variety of environments has been linked to their distinct physiological characteristics as well as their strong adaptation ability under a diverse range of environmental circumstances. Cyanobacteria have long been known for their ability to produce a wide range of compounds, including polysaccharides, lipids, and proteins as well as vitamins and sterols, as well as enzymes, medicines, and other fine chemicals, and the demand for these compounds is rising. There have been significant developments in the identification of bioactive chemicals from cyanobacteria, and their relevance in agriculture and industry has been reviewed in this compilation. Spirulina platensis, a cyanobacterium, has a high concentration of nutrients such as proteins, vitamins, minerals, carbs, and linolenic acid, among others. It is garnering more and more attention, not only for its food-related characteristics but also for its potential to be used in the creation of medicinal products [36]. Hydroperoxy-unsaturated fatty acids, particularly hydroperoxy-linoleic acid or linolenic acid, have been discovered in algae to be the intermediate compound of physiological bioactives that are involved in chemical defense or wound healing; for example, jasmonic acid, n-hexanal, 2(E) and 3(Z)-nonenal, 2(E) and 3(Z)-hexenal, in contrast to higher plants. Many bioactive chemicals are produced by Anabaena species, the majority of which are lipopeptidases with antibacterial, antialgal, anticancer, anti-­ inflammatory, cytotoxic, and enzyme-inhibiting properties [36]. Fatty acids, tetraamine, spermine, and piperazine derivatives are produced by Oscillatoria species, and these compounds have antibacterial action. According to some researchers, the Omega-3 fatty acids present in the oils of some cold-water marine fish are responsible for a reduction in the prevalence of coronary heart disease. Most likely, these fatty acids come from plankton in the food chain, which is where they originated. In this

54  Next-Generation Algae: Volume II study, several leptosins were isolated from the marine alga Leptosphaeria sp. and their biological activity was assessed. Of them, leptosin M was found to be highly hazardous to human cancer cell lines. In vitro and in vivo, the sulphated polysaccharide of the red microalga Porphyridium sp. has shown high-order antiviral efficacy against herpes simplex virus (HSV-1 and 2), indicating that it is a potential therapeutic agent [36]. Phormidium sp., a cyanobacterium, has been shown to impede the development of a variety of Gram-positive and Gram-negative bacterial strains, yeasts, and fungi, according to research. The same is true for the plant Lyngbya majuscula, which generates a wide range of chemicals, including nitrogen-containing compounds, polyketides, lipopeptides, cyclic peptides, and countless more. This group of chemicals has a wide range of biological activities, including protein kinase C activators and tumor promoters, inhibitors of microtubulin assembly, antibacterial and antifungal agents, and sodium channel blockers. With each passing year, the number of bioactive chemicals extracted or synthesized from Nostoc species grows exponentially. Cryptophycins are anticancer drugs that have been identified from Nostoc strains found in the wild. Recently, it was discovered that the carbolinium alkaloid nostocarboline (Figure 2.1), which was isolated from the Nostoc plant, functions as a cholinesterase inhibitor, which is an enzyme that has been targeted in the treatment of Alzheimer’s disease [36]. Norharmane (Figure 2.2), a chemical derived from the Nodularia harveyana plant, has recently been shown to have anti-cyanobacterial activity C1 I

–

+

N N H

Figure 2.1  Nostocarboline.

N N H

Figure 2.2  Norharman.

CH3

Bioactive Compounds Synthesized by Algae  55 against both filamentous and unicellular cyanobacteria and has the potential to be employed in the prevention of hazardous algal blooms [41].

2.5 Secondary Metabolites from Microalgae 2.5.1 Carotenoids Microalgae generate a large number of high-value carotenoids [42], which are the most diversified and widely distributed pigments, and are often tinted yellow, orange, or red [43]. Carotenoids are a class of terpenoid pigments that are derived from a 40-carbon polyene chain, which is considered to be their molecular backbone. This molecular backbone provides the carotenoids with a unique molecular structure as well as the associated chemical properties, which include light-absorption features that are essential for photosynthesis, to which they are linked. According to their structural characteristics, carotenoids are divided into two groups: xanthophylls or oxycarotenoids (such as astaxanthin [H. pluvialis], zeaxanthin [P. cruentum], and others) and carotenes (α- and β-carotenes [D. salina], lutein [C. pyrenoidosa], and lycopene) [44]. Carotenes are compounds that do not have any substituents (or even oxygen) in their chemical structure. Whether they are strict hydrocarbon carotenoids, xanthophylls, or oxycarotenoids, they all include –OH groups (hydroxycarotenoids: zeaxanthin from P. cruentum, lutein from C. pyrenoidosa), =O groups (ketocarotenoids: canthaxanthin from C. striolata, echinenone from B. braunii, S. platensis (astaxanthin from H. pluvialis). Although there are over 400 carotenoids discovered so far, just a few of them on the market: β-carotene, astaxanthin, and to a lesser degree lutein, lycopene, fucoxanthin, and bixin. Carotenoids play an important role in human nutrition by reducing the risk of certain diseases by providing provitamin A and preventing cerebrovascular and age-related macular degeneration diseases. Carotenoids are also important in the prevention of cerebrovascular and age-related macular degeneration diseases. It has also been established that pigments, including astaxanthin, β-carotene, lutein, neoxanthin, and zeaxanthin, have scavenging properties, with astaxanthin claiming to have the greatest impact of all the carotenoids studied [45].

2.5.1.1 β-Carotene Three primary microalgae species are needed to produce β-carotene (also known as β, β-carotene): Dunaliella, Spirulina maxima, and Haematococcus.

56  Next-Generation Algae: Volume II It has been employed in a variety of food and beverage items, and is one of the most widely used food colorants on the market. Animal feed has also benefited from the inclusion of beta-carotene as a source of vitamin A. The intestinal enzyme β-carotene 15,15′-monooxygenase catalyzes the conversion of provitamin A carotenoids to retinal, which is essential for vision. As previously observed, absorption of β-carotene-1 decreases the chance of developing age-related macular degeneration (AMD) (Table 2.6). In the health sector, natural β-carotene is chosen over synthetic β-carotene because it has a blend of cis and trans isomers, the latter of which has anticancer potential [42, 44]. The demand for β-carotene as a pro-vitamin A (retinol) in multivitamin formulations has risen significantly in recent years [44, 46]. The antioxidant β-carotene can quench singlet oxygen (1O2*) by electron energy transfer. It can also help to prevent eye disorders such as cataracts and night blindness from developing. Furthermore, β-carotene prevented the nuclear translocation of the NF-kB p65 protein component as well as

Table 2.6  Carotenoids from microalgae [42]. Carotenoids

Source

Therapeutic Activities

β-carotene

D. salina

Antioxidant, Provitamin A, Agerelated macular degeneration (AMD), Liver fibrosis, anti-inflammatory

Astaxanthin

H. pluvialis, C. zofigiensis, C. Vulgaris

Antitumoral, anti-oxidant, anti-inflammatory

Lutein

D. salina, C. pyrenoidosa, C. protothecoids

Age-related macular degeneration (AMD), Atherosclerosis, retinal neural damage

Zeaxanthin

D. salina, P. cruentum, C. protothtcoids

Antioxidant, Maculopathy, cataracts, anti-inflammatory

Violaxanthin

D. tertiolecta, C. ellipsoidea

Anti-inflammatory, anti-cancer

Fucoxanthin

P. tricornutum

Antioxidant, anti-inflammatory, anti-cancer

Bioactive Compounds Synthesized by Algae  57 IkBa phosphorylation and degradation, which resulted in the inhibition of inflammatory cytokines when administered both in vivo and in vitro. β-carotene has been used to treat a variety of conditions, including asthma, cardiovascular disease, and erythropoietic protoporphyria. It has also been shown to lower the chance of developing a variety of malignancies, including breast cancer and lung cancer. As it turns out, feeding DO11.1.0 mice with both additional β-carotene-1 and vitamin E increases the activity of Th1 cells in their spleen cells, which is a good thing. It was shown that oral administration of β-carotene-1 to BALB/c mice that had been inoculated with OVA reduced the titer of specific IgE and IgG1 antibodies and inhibited the antigen-induced anaphylactic reaction by reducing blood histamine levels [42].

2.5.1.2 Astaxanthin The chemical formula of Astaxanthin is C40H52O4. Although both natural and synthetic astaxanthin exists, the natural astaxanthin is mostly found in esterified form, whilst the synthetic equivalent is found in free form [47]. It is derived from Haematococcus pluvialis, Chlorella zofingiensis, Chlorella vulgaris, and Chlorococcum sp., which are the most important sources of astaxanthin. The levels gathered by the green alga H. pluvialis outstrip those collected by any other known source, amounting to up to 4–5% of dry weight in some cases. Due to its superior antitumor activity compared to β-carotene and other carotenoids, the red pigment astaxanthin produced by microalgae has attracted considerable attention. Astaxanthin is a carotene derivative that is more potent than β-carotene and other carotenoids [42]. The pigment Astaxanthin is responsible for the pinkish look of aquatic fish and shrimp, as well as the color of their skin. It displays several-fold greater effective antioxidant activity than carotene and vitamin E, making it the most powerful antioxidant among the carotenoids in this category. The combination of astaxanthin with vitamin C may also have a synergistic impact, recharging astaxanthin after it has scavenged ROS via its terminal rings, which appears to be the last scavenger of ROS. Astaxanthin-2 supplementation has been shown to have anticancer effects in the post-initiation phase of carcinogen-induced colon and oral cancer models, respectively. When used with aspirin, it has the potential to increase antibody production, anti-aging, sun-protective, and antiinflammatory properties. It reduces the oxidation of low-density

58  Next-Generation Algae: Volume II lipoprotein (LDL) cholesterol while increasing the production of high-­ density lipoprotein (HDL) cholesterol and adiponectin. The 3S,3′S geometrical isomer of astaxanthin was shown to be more readily absorbed than the other geometrical isomers, even though it is found in lower concentrations in a racemic mixture [48].

2.5.1.3 Zeaxanthin and Lutein Microalgae are an important source of the antioxidants zeaxanthin and lutein, which exist naturally in the environment. D. salina, C. protothecosis, and Spirulina are the primary sources of lutein and zeaxanthin, respectively. In addition, two more bioproducts, namely, lutein-3 and zeaxanthin, are becoming increasingly essential in the nutraceutical sector as time passes [42, 43]. The yellow xanthophylls or oxycarotenoid lutein has two cyclic end groups (one β-ionone ring and one ɛ-ionone ring) that are responsible for its color. Zeaxanthin-4, which is structurally similar to lutein and accumulates in the central retina, on the other hand, is structurally distinct. In conclusion, evidence from epidemiological and intervention studies using lutein and zeaxanthin supports the notion that nutrients and health are linked in the prevention of agerelated cataracts and maculopathy. Two lutein-containing products, Aztec Marigold and Tagetes, have recently been introduced to the market in the United States [42]. Lutein-3 is useful as a pigment in animal tissue (for coloring chicken skin and egg yolks), as a food coloring (for coloring egg yolks), cosmetics, and medicinal items since it has a high nutritional value and a low toxicity level [49]. Some studies have found that lutein has anti-­ inflammatory properties against endotoxin-induced uveitis (EIU) by inhibiting Ik-degradation and the subsequent production of proinflammatory mediators such as NO, TNF-α, IL-6, PGE2, MCP-1, and MIP-2. Both zeaxanthin and lutein have been shown to have a significant role in the maintenance of normal visual function in laboratory animals [50]. Given that these organs are particularly vulnerable to oxidative damage, lutein and zeaxanthin shield the eyes from the effects of antioxidants, a fact that makes them extremely useful substances [51]. Oncologists discovered that lutein from the plant C. ­vulgaris has anticancer properties when tested on a human colon cancer cell line (HCT-116) and that eating lutein-rich foods lowers the chance of developing cancer [52].

Bioactive Compounds Synthesized by Algae  59

2.5.1.4 Violaxanthin Violaxanthin is a naturally occurring xanthophyll with an orange color that may be found in a variety of microalgae. D. tertiolecta and C. ellipsoidea are two of the most important suppliers of this product. The anti-inflammatory properties of violaxanthin isolated from C. ellipsoidea, which were mediated through regulation of the NF-kB and MAPK pathways, show that C. ellipsoidea has significant promise as a therapy option for inflammatory illnesses [42]. Researchers from Soontornchaiboon et al. revealed that the antioxidant violaxanthin suppressed the generation of NO and PGE2 in RAW 264.7 cells in a dose-dependent manner. The impact of violaxanthin-5 on NO and PGE2 synthesis was similar to the effects of carotenoids, which have been proven to decrease NO production. For example, β-carotene, lutein, and fucoxanthin have all been found to suppress NO production. Furthermore, violaxanthin-5 inhibited the synthesis of PGE2 as well as the expression of COX-2 at the mRNA and protein levels. Violaxanthin, an organic natural component that is not synthesized, may thus be a safe and effective anti-inflammatory drug that might be utilized for medicinal purposes, according to the researchers [53]. The antioxidant violaxanthin had a strong anti-proliferative effect on MCF-7 breast cancer cells and generated biochemical alterations that were characteristic of early apoptosis.

2.5.1.5 Fucoxanthin Fucoxanthin is a carotenoid pigment with a golden-brown color that was originally discovered in 1914 and is one of the most abundant carotenoids found in marine sources [54]. It may be found in a variety of microalgae classifications, including bacilophytes, bolidophytes, chrysophytes, silicoflagellates, pinguiophytes, and brown microalgae phaehytes. Bacilophytes are the most common type of microalgae. Because of its health advantages, such as its anticancer and antiobesity properties, as well as its anti-inflammatory and antioxidant properties, and its ability to prevent cerebrovascular illnesses, fucoxanthin is receiving a lot of attention right now [55, 56]. Additionally, fucoxanthin has been shown in a mouse model to be harmless, despite its ability to raise the blood levels of both HDL and non-HDL cholesterol. Several investigations revealed that fucoxanthin displayed cytotoxicity against several human colon cancer cell lines by causing apoptosis and cell cycle arrest in the cells tested. When compared to other carotenoids, it had a greater effect on the viability of colon

60  Next-Generation Algae: Volume II cancer cell lines (DLD-1, Caco-2, and HT-29) than the other carotenoids. Furthermore, fucoxanthin prevented the induction of mouse colon carcinogenesis by 1,2-dimethylhydrazine in an in-vivo study. It has also been claimed that fucoxanthin can block the development of duodenal and skin cancers, as well as the development of liver tumors in mice. It has been discovered that its anticancer efficacy is mediated by cell cycle arrest, antioxidant activity, and apoptosis induction [55]. In lipopolysaccharide-induced RAW264.7 macrophages, fucoxanthin-6 prevented the activation of the nuclear factor-κB (NF-κB) by reducing Iκβ-a degradation and the nuclear translocation of the p50 and p65 proteins. In vitro, both fucoxanthin and its metabolite fucoxanthinol displayed antioxidant activity as free radical scavengers and as a singlet oxygen quencher, demonstrating that they are powerful antioxidants [56].

2.5.2 Polyunsaturated Fatty Acids Animals and higher plants do not synthesize polyunsaturated fatty acids (PUFAs) with more than 18 carbons because the enzymes required for this process are not present in these species. Microalgae are the principal producers of polyunsaturated fatty acids (PUFAs) in the oceanic environment, even though fish oil is the conventional source of PUFAs. More recent research has concentrated primarily on the synthesis of polyunsaturated fatty acids (PUFAs), which are employed as nutritional supplements and medicinal agents. Almost all polyunsaturated fatty acids (PUFAs) derived from primary producers can be transformed by bioconversions as they go up the food chain, a process known as trophic upgrading. According to recent findings, the amounts of essential fatty acids in human diets are associated with the development of atherosclerosis and coronary heart disease (CHD) [42]. The presence of omega-3 PUFAs in the brain has been demonstrated to be important physiological components of the total amount of lipid present in the brain, in addition to their beneficial effects against cardiovascular system disorders and their protective actions against uncontrolled cellular proliferation. Several neurological activities, such as neurogenesis and neurotransmission, as well as the protection of the brain against oxidative stress-induced cerebral damage, rely on omega-3 PUFAs for their survival. Furthermore, it has been demonstrated that long-chain N-3 PUFAs, such as docosahexaenoic acid (DHA-8, C22:6n-3) and eicosapentaenoic acid (EPA7, C20:5n-3), have significant therapeutic potential for a variety of ailments, including cancer, atherosclerosis, rheumatoid arthritis, Alzheimer’s disease, and psoriasis, among others [42]. EPA-7 is critical in the development and regulation of physiology in humans and higher animals because it is

Bioactive Compounds Synthesized by Algae  61 a precursor to a series of eicosanoids that are key in the development and regulation of physiology. A class of hormone-like substances known as eicosanoids includes thromboxane (TX), prostaglandins (PG), and leukotrienes (LT), whereas arachidonic acid (AA, 20:4 N-6) and EPA are precursors of eicosanoid compounds. TX is a hormone-like substance produced by the body. The omega-3 fatty acids EPA-7 and DHA-8 have been shown to lower circulating levels of CRP, TNF-α, IL-6, and IL-1, suggesting that they may be involved in anti-­inflammatory activities. The administration of C. vulgaris extracts orally to tumor-bearing mice resulted in a considerable increase in phagocyte generation and quality, resulting in significantly longer life. PUFAs have a vital function in the fluidity of membranes, cellular metabolism, transport, and as precursors of eicosanoid receptors. In the 1980s, fish oil was the most important source of polyunsaturated fatty acids (PUFAs). The depletion of fish supplies and contamination of fish have prompted the usage of other sources of protein [42].

2.5.3 Proteins and Polypeptides The use of algae as an alternative nutritional source goes back to World War II when the ingestion of microalgae and seaweeds became more popular as a means of overcoming dietary protein deficiency [57]. Microalgae are regarded as prospective sources for the creation of both elementary proteins and therapeutic peptides and proteins because of their high protein content. These algal proteins are of good quality and equivalent in nutritional value to traditional vegetable proteins, according to extensive analysis and nutritional studies. Arthrospira, Chlorella, and D. salina have all been used in human nutrition meals as a result of their high protein content and nutritional value, respectively. Spirulina cells have a great nutritional value as well as a high digestibility, owing to their high protein content and the abundance of minerals they contain. All 20 proteinogenic amino acids are synthesized by microalgae, which means they can be used as a novel supply of necessary amino acids for human nutrition. The development of recombinant DNA and hybridoma technologies made it feasible to generate huge quantities of proteins that behave as medications, which are referred to as drugs, biologics, or biopharmaceuticals, in a controlled environment (BFs). Previous studies have found that possible vaccine candidates have been created by viruses and bacteria, as well as by malaria and other communicable illnesses, or that they have been explored for nonviral diseases in the laboratory. Researchers have discovered that phycobiliproteins from marine cyanobacteria and red algae are capable of exhibiting anticancer, anti-inflammatory, immunomodulatory,

62  Next-Generation Algae: Volume II antioxidant, hepatoprotective, and neuroprotective properties [42]. When compared to the limited secondary metabolites generated by ribosomal proteins, cyanobacterial polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS) create a wide variety of bioactive secondary metabolites that are beneficial to the host. The hybrid polyketide-polypeptide structural class of molecules is distinguished by several distinctive structural characteristics, including the integration of modified amino/ hydroxyl acids, heteroaromatic ring systems, and polyketide-derived units with extended lengths. The extended polyketide-derived units can be either linear or undergo cyclization to produce a common scaffold, such as the pyrrolidone rings in the Jamaicamides, which serve as a scaffold for other compounds. For example, a unit of acetate is employed in the extension of several amino acids, such as Gly, Phe, Pro, and Ala, to make them longer. Apratoxin A-45 is a mixed peptide–polyketide natural product derived from the polyketide synthase/nonribosomal peptide synthase pathway of the cyanobacterial secondary metabolite. It is a cytotoxin that is derived from the apratoxin family of toxins. This cytotoxin is well-known for its ability to cause cell cycle arrest in the G1 phase as well as apoptosis [58, 59].

2.6 Biomass of Microalgae The large-scale production of algal biomass is crucial for a variety of businesses. With the evolution of technology in this field, a variety of ways for developing microalgae-based products and downstream processes have been developed [60].

2.6.1 Biomass Production Cultivation, harvesting, and biomass dehydration are all steps in the development of microalgae biomass, and each one is important.

2.6.1.1 Cultivation The open-pond and closed-photobioreactor (PBR) technologies have been developed for the production or cultivation of algal biomass in a controlled environment. Waterways found naturally in the environment (ponds, lakes, and lagoons) and man-made ponds are the two forms of open pond production available (circular and raceway). In comparison to the PBR, the open pond is a less costly method of producing significant amounts of

Bioactive Compounds Synthesized by Algae  63 algal biomass on a large scale. When it comes to manufacturing, the PBR system provides a good and well-controlled closed culture system that prevents contamination by molds, bacteria, protozoa, and other microalgae. To make use of the free energy sources given by the sun, it is usually put outside. PBR is available in three configurations: tubular (TPBR), vertical column (VCPBR), and flat-plate PBR (FP-PBR) [60].

2.6.1.2 Harvesting To separate microalga biomass from the culture medium or harvest it, four procedures are used: biomass aggregation (flocculation and ultrasonication), flotation, centrifugation, and filtering. Combinations of two or more techniques are occasionally used to increase the efficacy of a single strategy. The microalgae harvesting process is influenced by a variety of elements, including the density of the algae, their size, and the final products that are required. a) Biomass aggregation: The flocculation technique, which involves adding flocculants to the medium, such as multivalent cations and cationic polymers, to aid in neutralizing the cells’ surface charge, is used to aggregate microalgae cells together to create a bigger particle known as a floc. The two types of flocculating agents are chemical flocculants and biological flocculants. Iron and aluminum salts are widely used as chemical flocculants because they are inexpensive and easily accessible. Acrylic acid and chitosan are two biopolymer bioflocculants that are often used in industry. The use of ultrasonic technology facilitates the harvesting of algal biomass by causing aggregation, which is followed by improved sedimentation. As a result of the fact that ultrasonic harvesting does not generate shear stress on the biomass even though it is used continuously, the valuable metabolites are kept [60]. b) Flotation: Micro-air bubble disperser technology is a technique that uses a micro-air bubble disperser to float algal cells on the water’s surface without the use of chemicals. It is cost-effective to use this technology because it has low operating expenses, a simple operating method, and a high biomass collection rate. c) Centrifugation: It is the recovery of algal biomass from culture media by the use of gravitational force, which is

64  Next-Generation Algae: Volume II accomplished by the use of a centrifuge. Because of the significant energy input and the need for continual maintenance, this process is quick, easy, and effective; but the cost can soon escalate. When a substantial gravitational force is applied, this method produces internal cell damage that results in the loss of essential nutrients, which is another issue with this method. d) Filtration: A technique for isolating algal biomass from a liquid culture medium that involves the use of a porous membrane with varying particle sizes is described here. Conventional, microfiltration and ultrafiltration are the three procedures that may be used to put it into action (isolation of metabolites). Small size microalgae (>70 μm) such as Coelastrum and Spirulina are collected by the use of conventional filtering methods. To capture microalgae that are smaller than a bacterium, microfiltration and ultrafiltration are employed in conjunction with each other [60].

2.6.1.3 Biomass Dehydration Algae biomass is processed to the next phase as soon as it is removed from the growth medium to minimize rotting or prolong the shelf-life of the biomass. The three most popular types of drying or dehydration procedures are sun-drying, spray-drying, and freeze-drying. The following are the final elements on which the strategy will be based: a) Sun-drying: This is the traditional method of lowering the moisture content of paddy that involves laying the grains out under the sun to dry them. When the grains are exposed to solar radiation, they become hotter as well as warmer than the surrounding air, increasing the rate at which water evaporates off the grains [61]. Sun drying, on the other hand, is typically labor-­ intensive and has a limited capacity. With this approach, it is also difficult to maintain temperature control, and grains can quickly get overheated, resulting in cracked grains and poor milling quality. At night or during heavy rain, it is also impossible to dry by the sun. The cost of this approach is the least expensive when compared to the other two options on the table. This approach relies solely on solar energy, which has downsides in terms of weather conditions, long drying

Bioactive Compounds Synthesized by Algae  65 times, and the amount of drying space that must be set aside for drying. Microalgae drying in the sun is an unpredictable process, and as a result, overheating may occur along with alterations in texture, color, and flavor. b) Spray-drying: This is a technique for rapidly drying a liquid or slurry into a dry powder by using a hot gas to accelerate the drying process. Heat-sensitive things, such as foods and medicines, or items that require a highly consistent and small particle size are among the items for which this method of drying is preferred [62]. In most cases, the air is used as the heated drying medium; however, nitrogen may be used in the case of flammable liquids, such as ethanol, or in the case of products that are sensitive to oxygen. It is wellknown that spray-drying is a method of particle manufacturing that entails the transformation of a fluid substance into dried particles by making use of a gaseous hot drying medium. Spray-drying has significant advantages in the manufacture of medical devices, as demonstrated below. In reality, the creation of microspheres and microcapsules for use in drug delivery systems is a very typical occurrence [62]. A thin spray of suspension droplets in continuous contact with hot air in a large vessel is used to create dry powder in this process, which is similar to the previous one. There are various advantages to using this technology, including the capacity to operate continuously, the fineness of the powder produced, and the ability to retain a high-quality product by drying it quickly after production. Even though some algal constituents, such as pigments, may deteriorate substantially over time, and the technique is expensive, this technology is frequently utilized for high-value operations because of its efficiency [62]. c) Freeze-drying or Lyophilization: Using a vacuum to remove any water or other solvents from a completely frozen sample, the ice can transition straight from a solid-state to a vapor state without passing through a liquid state [63]. This method of drying microalgae is only employed in the laboratory due to the high expense of large-scale manufacture. Freeze-drying is a direct dehydration process for frozen materials that uses sublimation to accomplish dehydration. Before freeze-drying, the microalgae are frozen to solidify the material contained therein. Low temperatures are used

66  Next-Generation Algae: Volume II to gradually reduce the moisture content of the micro­algae, while still maintaining the product’s firm structure and high quality [63].

2.6.1.4 Extraction of Bioactive Compounds Microalgae are made up of a range of different components, including carbohydrates, lipids, proteins, minerals, and a variety of other substances. The algal biomass will be pretreated to release the bioactive component that has been stored in the cells. This bioactive component will be used for a variety of purposes, including biofuels/energy production and agricultural use. Several methods, including physical, mechanical (bead milling, homogenization, microwave, ultrasonic, and pulsed electric field) and chemical (solvent, acid, and alkali) lysis, as well as biological extraction, will be used to lyse the cell walls and extract the essential components (enzymes). The pretreatment procedure that is utilized is dictated by the final goods that are intended to be produced [60].

2.7 Pharmaceutical Applications of Microalgae In addition to being a source of bioactive components, microalgae have the potential to be employed in medicinal formulations. A large number of studies have demonstrated that microalgae compounds are beneficial in the prevention and treatment of diseases such as diabetes, obesity, cardiovascular disease, cancer, inflammation, Alzheimer’s disease, depression, and bacterial, fungal, and viral infections. Despite this, there are currently just a handful of microalgae with pharmacological potential that have been identified. Because of limited extraction yields and high production costs, the commercialization of several micro­algaederived bioactives has been delayed. Furthermore, several micro­algae species have the potential to induce major side effects, such as allergic reactions and the accumulation of heavy metals and toxins. Microalgaederived purified compounds and enriched extracts for approval and commercialization require a strong emphasis on good manufacturing practices in cultivation, harvesting, extraction, and purification, as well as controls to limit toxins and impurities. This is especially true for the production of enriched extracts. a) Cardioprotective properties: The cardioprotective benefits of several microalgae-derived compounds have been

Bioactive Compounds Synthesized by Algae  67 investigated. Antioxidant properties of carotenoids have been demonstrated, and these properties are critical in preventing cell damage caused by free radicals, which have been associated with chronic cardiovascular disease and stroke. There has been some research on the cardioprotective properties of certain microalgae that generate a large number of carotenoids in this area. For example, D. salina microalgae may produce up to 10–13% β-carotene, which is protective against atherosclerosis in both rats and humans in laboratory studies. Polyunsaturated fatty acids (PUFAs), notably omega-3 fatty acids, such as DHA, EPA, and α-linoleic acid (ALA), are another kind of bioactive having cardioprotective properties. DHA and EPA are particularly beneficial in this regard. It has been demonstrated that they can reduce blood cholesterol levels and improve hypertension. DHA is the only PUFA that is now commercially available among these fatty acids. It is currently prohibitively expensive to synthesize purified EPA obtained from various microalgae, such as Porphyridium purpureum and Isochrysis galbana, for commercial use [60]. b) Anticancer properties: In recent years, there has been a significant amount of research on the anticancer potential of bioactives derived from microalgae. Microalgae pigments, such as astaxanthin, beta-carotene, lutein, violaxanthin, and fucoxanthin, have the greatest potential to be marketed as medicines due to their established uses as nutraceuticals and cosmetics, as well as increased demand for dietary supplements. Astaxanthin is the most widely studied microalgae pigment. While astaxanthin and β-carotene are already commercially produced from the microalgae Dunaliella salina and Haematococcus pluvialis, other carotenoids such as lutein and fucoxanthin are gaining popularity. It has been discovered that the diatom microalgae Phaeodactylum tricornutum, which is grown in a pilot-scale photobioreactor and can be considered a commercially viable source of fucoxanthin, can be used to extract fucoxanthin. Fucoxanthin has been extracted from these algae and is a commercially viable source of fucoxanthin [60]. β-carotene: Astaxanthin and β-carotene are produced by Dunaliella and Haematococcus, respectively, which are both

68  Next-Generation Algae: Volume II important sources of carotenoids in the environment. Using human colon cancer cell lines, revealed that β ­ -carotene pigments significantly reduced the multiplication of the cells [15]. Astaxanthin: The microalgae Haematococcus pluvialis, Chlorella zofingiensis, and Chlorococcum sp. are the primary sources of astaxanthin in the environment. Palozza et al. investigated the effect of astaxanthin in the development of cell lines and discovered that this carotenoid can suppress the proliferation of human CRC (colorectal cancer) cell lines, which was previously unknown. Astaxanthin, lutein, and zeaxanthin were shown to be the most effective carotenoids for preventing cell proliferation. The antioxidant astaxanthin has been proven in multiple studies to have an inhibitory impact on apoptosis (programmed cell death). As a result, astaxanthin generated from microalgae has gained popularity and should be an excellent choice for both cancer prevention and chemotherapy. Lutein: In general, dietary carotenoid consumption, particularly lutein consumption, is thought to be associated with a lower risk of developing a variety of malignancies (colon, lung, and breast). Lithium-containing algae Dunaliella salina, Chlorella sorokiniana, and Chlorella protothecoides are the primary producers of lutein, which has been shown to have an antiproliferative impact on the human colon cell line HCT-116 in studies. Low doses of dietary lutein (0.002% and 0.02%) have also been shown to be effective in preventing the development of breast cancer [60]. Fucoxanthin: For cancer prevention, the carotenoid fucoxanthin is the most promising of the carotenoid family of pigments. Cancer cells (Caco-2, HT29, and DLD-1), prostate cancer PC-3 cells, and HL-60 leukemia cells were all shown to have antiproliferative properties when the compounds were tested. The antioxidant fucoxanthin is showing tremendous promise as a cancer-fighting agent as a result of these findings [64]. Fucoxanthin is rapidly gaining popularity as a result of its numerous health benefits, which include cancer

Bioactive Compounds Synthesized by Algae  69 prevention, weight reduction, anti-inflammatory properties, antioxidant properties, and the prevention of cerebrovascular disease. It has been demonstrated that fucoxanthin is harmless in a mouse model and that it also increases levels of both HDL and non-HDL cholesterol in the bloodstream [60]. Phycobiliproteins: Scientists have discovered that phycobiliproteins, which are pigments present in red algae and cyanobacteria, have advantageous fluorescence qualities. A few examples of common phycobiliproteins are phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC). This group of pigments is composed of hetero-­ oligomers that are grouped into complexes known as “phycobilisomes” within the producing cells (Cyanophyta) or chloroplasts (Rhodophyta)  of the cells that generate the pigments. Long used as natural colors, phytobiliproteins are now widely used in nutraceuticals and other biotechnological applications, as well as in cosmetics products [65]. Another possible anticancer medicine is phycocyanin, a protein pigment that belongs to the phycobiliprotein family of proteins. Scientists have recovered phycocyanins from the microalgae Spirulina platensis, which is used in commercial production, but they have also recovered them from other cyanobacteria, including Limnothrix sp. c) Antiviral properties: Microalgae may potentially include bioactives with antiviral action, which are yet to be discovered. For example, antiviral lectin proteins such as cyanovirin (CV-N) and scytovirin (SVN) have been identified to work against viruses. Cyclic peptides are also known to have antiviral properties [60]. d) Antimicrobial properties: The emergence of antibiotic resistance in humans as a result of the widespread use of antibiotics in clinical practice highlights the urgent need for the discovery of new antibacterial compounds. Microalgae contain antibiotics that have a broad and efficient antibacterial action against a variety of microorganisms. The antimicrobial activity of these bacteria is enhanced by their ability to produce substances such as polyunsaturated fatty acids, acetogenins, halogenated aliphatic compounds, terpenoids,

70  Next-Generation Algae: Volume II sterols, sulfur-containing heterocyclic compounds, carbohydrates, terpenoids, sterols, and halogenated aliphatic compounds [3]. The amount of fat present in microalgae extracts has an impact on their antibacterial efficacy. Due to their capacity to produce compounds such as α- and β-ionone and βcyclocitral, as well as neophytadiene and phytol, microalgae have demonstrated significant antibacterial activity. The antimicrobial activity of microalgae against human pathogens such as E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis has been related to several fatty acids, including γ-linolenic acid, eicosapentaenoic acid, hexadecatrienoic acid, docosahexaenoic acid, and palmitoleic acid [60]. e) Anti-inflammatory properties: As a protective mechanism against acute infections, inflammation is essential because it allows invading bacteria to be detected and eliminated. In contrast, if it develops as a chronic and subclinical condition, and if the process is not managed over time, the active immune system can cause tissue damage and upregulate chronic disease states such as cardiovascular disease, Alzheimer’s disease, inflammatory bowel disease, and obesity, among others [42]. The anti-inflammatory activities of many bioactive compounds produced from marine life have been proved to be potent and mechanistically intriguing, and marine cyanobacteria play a crucial part in the production of this class of molecules (for instance, anti-inflammatory bis-bromo indoles from Rivularia sp.). Micro-algal phytosterols and their secondary metabolites are potential anti-inflammatory medicines, and the synergistic effect of these compounds should be taken into consideration while optimizing their respective roles. Phospholipid polysaccharides, which have a pharmacological effect such as anti-inflammatory or immunomodulating qualities, are generated by algae such as Phaeodactylum, Porphyridium, and C. stigmatophora, among other species [42]. f) Antioxidant properties: The desire for a natural substance that is both safe and effective as an antioxidant is increasing across the world. This is due to the rising need for marketed items such as food, medicines, and cosmetics that

Bioactive Compounds Synthesized by Algae  71 reduce oxidative damage to living cells and prevent degradation. This is especially true in the food industry. It is thought that oxidative damage caused by ROS, such as the hydroxyl radical (HO•), superoxide anion, and hydrogen peroxide (H2O2), can cause atherosclerosis, cataracts, muscular dystrophy, rheumatoid arthritis, neurodegeneration, cancer, and aging. Given the health concerns and hazards associated with synthetic antioxidants such as butylated hydroxytoluene (BHT) and propyl gallate (PG), antioxidants derived from natural products are urgently needed. BHT is an example of such an antioxidant. In their natural environments, photoautotrophs are subjected to high levels of oxidative and radical stress, which they must overcome to survive. In microalgae, antioxidant molecules, such as carotenoids, phenolic compounds, fatty acids, tocopherol, flavonoids, and particularly abundant alkaloids, play an important role in the regulation of the oxidative process [42].

2.8 Conclusion Algae can generate natural compounds that have vital biological activities, which are otherwise unavailable. Natural product research in this subject is still in its early stages, even though significant research on high-value compounds obtained from microalgae has been published. Great-value microalgae compounds are in high demand due to their applicability in a wide range of industrial applications including pharmaceutical, nutraceutical, cosmeceutical, animal feed, biological waste treatment, and other fields of science and technology. This chapter discussed the significance of microalgae as distinct sources of bioactive chemicals and the value of microalgae as a source of bioactive chemicals. We have utilized the algaebased chemicals astaxanthin and fucoxanthin as examples of intriguing algae-based compounds with health and economic promise, and we have emphasized the immense potential for identifying novel bioactive molecules in microalgae. Identifying novel compounds with improved activity in newer and older microalgae species represents a fruitful research approach for advancing the field of biotechnology in general and biomedical applications in particular, demonstrating their significant potential for biotechnological and biomedical applications.

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74  Next-Generation Algae: Volume II 33. Kronick, M.N., The use of phycobiliproteins as fluorescent labels in immunoassay. J. Immunol. Meth.; 92: pp: 1-13, 1986. 34. Prasanna, R. et al., Evaluation of Tolypothrix germplasm for phycobilibrotein content. Folia Microbiol; 48(1): pp: 59-64, 2003. 35. Shahzad, I. et al., Algae as an alternative and renewable resource for biofuel production. The boil (Ejournal for life sciences); 1(1): pp: 16-32, 2010. 36. Priyadarshani, I. and Rath, B., Bioactive Compounds from Microalgae and Cyanobacteria’ Utility and Applications. Int J Pharm Sci Res. 3(10); pp: 41234130, 2012. 37. Newman, D.J. and Cragg, G.M., Marine natural products and related compounds in clinical and advanced preclinical trials. Nat. Prod. 67: pp: 12161238, 2004. 38. Burja, A.M. et al., Marine cyanobacteria-a prolific source of natural products. Tetrahedron; 57: pp: 9347–9377, 2001. 39. Welker, M., von Dohren, H., Cyanobacterial peptides—Nature’s own combinatorial biosynthesis. FEMS Microbiol Rev., 30(4) pp:530–563, 2006. 40. Tan, L.T., Bioactive natural products from marine cyanobacteria for drug discovery. Phytochemistry. 68(7) pp: 954–979, 2007. 41. Blom, J.F. et al., Potent algicides based on the cyanobacterial alkaloid nostocarboline. Org. Lett. 8: pp: 737–740, 2006. 42. Bule, M.H. et al., Microalgae as a source of high-value bioactive compounds.’ Frontiers in Bioscience-Scholar, 10, 2, pp: 197-216, 2018. 43. Gong, M. andBassi, A., Carotenoids from microalgae: A review of recent developments.’ Biotechnol. Adv., 34, pp: 1396–1412, 2016. 44. Guedes, A.C., Amaro, H.M. and Malcata, F.X., Microalgae as Sources of Carotenoids. Mar. Drugs 9, pp: 625–644, 2011. 45. Safafar, H. et al., Carotenoids, Phenolic Compounds and Tocopherols Contribute to the Antioxidative Properties of Some Microalgae Species Grown on Industrial Wastewater. Mar. Drugs 13, pp: 7339–7356, 2015. 46. Vaz, B. da S. et al., Microalgae as a new source of bioactive compounds in food supplements. Curr. Opin. Food Sci., 7, pp: 73–77, 2016. 47. Liu, J. et al., Chlorella zofingiensis as an Alternative Microalgal Producer of Astaxanthin: Biology and Industrial Potential. Mar. Drugs 12, pp: 3487–3515, 2014. 48. Raposo, M. F. de J., de Morais, A. M. M. B. and de Morais, R. M. S. C., Carotenoids from Marine Microalgae: A Valuable Natural Source for the Prevention of Chronic Diseases. Mar. Drugs, 13, pp: 5128–5155, 2015. 49. Perez-Garcia, O. et al., Heterotrophic cultures of microalgae: Metabolism and potential products. Water Res., 45, pp: 11–36, 2011. 50. Jin, E. et al., Xanthophylls in microalgae: From biosynthesis to biotechnological mass production and application, 2003. Retrieved from https://www. scienceopen.com/document?vid=26f51e8f-c5c7-44a1-b9df-8001f5205277

Bioactive Compounds Synthesized by Algae  75 51. Sun, Z. et al., Microalgae as a Source of Lutein: Chemistry, Biosynthesis, and Carotenogenesis. In Microalgae Biotechnol, Eds: C. Posten & S. F. Chen, Springer International Publishing Switzerland, pp: 37–58, 2016. 52. Parveen, S. and Nadumane, V., Algae as Sources of Anticancer Compounds. IJBPAS 5, pp: 2257–2277, 2016. 53. Soontornchaiboon, W., Joo, S.S. and Kim, S.M., Anti-inflammatory Effects of Violaxanthin Isolated from Microalgae Chlorella ellipsoidea in RAW 264.7. Macrophages. Biol. Pharm. Bull 35, pp: 1137–1144, 2012. 54. Stonik, V. and Stonik, I., Low-Molecular-Weight Metabolites from Diatoms: Structures, Biological Roles and Biosynthesis. Mar. Drugs 13, pp: 3672–3709, 2015. 55. Mikami, K. and Hosokawa, M., Biosynthetic Pathway and Health Benefits of Fucoxanthin, an Algae-Specific Xanthophyll in Brown Seaweeds. Int. J. Mol. Sci., 14, pp: 13763–13781, 2013. 56. Plaza, M., Cifuentes, A. and Ibáñez, E., In the search of new functional food ingredients from algae. Trends Food Sci. Technol 19, pp: 31–39, 2008. 57. Nagarajan, D. et al., Recent insights into biohydrogen production by microalgae – From biophotolysis to dark fermentation.’ Bioresour. Technol 227, pp: 373–387, 2017. 58. Tan, L.T., Pharmaceutical agents from filamentous marine cyanobacteria. Drug Discov Today 18, pp: 863–871, 2013. 59. Vijayakumar, S. and Menakha M., Pharmaceutical applications of cyanobacteria—A review. J. Acute Med 5, pp: 15–23, 2015. 60. Balasubramaniam, V. et al., Isolation of Industrial Important Bioactive Compounds from Microalgae’ Molecules. 26(4): pp: 943, 2021. 61. ‘Sun drying’ (n.d.). Retrieved from http://www.knowledgebank.irri.org/ step-by-step-production/postharvest/drying/traditional-drying-systems/ sun-drying#:~:text=Sun%20drying%20is%20a%20traditional,water%20 evaporating%20from%20the%20grains. 62. Santos, D. et al., Spray Drying: An Overview; Biomaterials: Physics and Chemistry’ - New Edition, 2017. 63. Hilgedick, A., Introduction to freeze drying, 2020. Retrieved from https:// www.labconco.com/articles/introduction-to-freeze-drying 64. Tsianta, A., Pharmaceutical Applications of Eukaryotic Microalgae, 2019. Retrieved from https://repository.ihu.edu.gr/xmlui/bitstream/handle/11544/ 29668/a.tsianta.pdf?sequence=1 65. Santhosh, S., Dhandapani R. and Hemalatha N., Bioactive compounds from Microalgae and its different applications- a review. Advances in Applied Science Research, 7(4) pp: 53-158, 2016. Retrieved from www.pelagiaresearchlibrary.com

3 Bioactive Compounds Derived from Microalgae Showing Diverse Medicinal Activities D. Tiwari*, P. Mishra and N. Gupta Faculty of Pharmacy, RBS Engineering Technical Campus, Bichpuri, Agra, India

Abstract

Many diseases are becoming resistant to drugs. With this in mind, researchers are searching for cheap and effective drugs for their cure. Cyanobacteria can help attain this goal. Biologically active compounds are derived from algae and marine organisms. These natural compounds possess diverse biological activities like anthelmintic, antibacterial, antifungal, antimalarial, antiprotozoal, antituberculosis, and antiviral activities. These biologically active compounds are created with a variety of molecular targets in mind and thus may potentially contribute to several pharmacological classes. The synthesis of natural products and their analogues provide effective structural modifications on the parent compounds, which may be useful in the discovery of potential new drug molecules with different biological activities. Natural organisms have developed complex chemical defense systems by repelling or killing predators, such as insects, microorganisms, animals, etc. These defense systems have the ability to produce large numbers of diverse compounds which can be used as new drugs Cyanobacteria are photosynthetic prokaryotic microorganisms. Since ancient times, cyanobacteria have been used as food and fodder. However, they are wellknown for producing toxins and fouling ponds, as they form water bloom. Various studies are currently being done to also prove their potency in medical science. Their strength is that they produce various metabolites that are antibacterial, antifungal, antimalarial, antitumor, antialgal, antiviral, UV protectants, inhibitors of enzymes, hepatotoxins and neurotoxins.

*Corresponding author: [email protected] Charles Oluwaseun Adetunji, Julius Kola Oloke, Naveen Dwivedi, Sabeela Beevi Ummalyma, Shubha Dwivedi, Daniel Ingo Hefft and Juliana Bunmi Adetunji (eds.) Next-Generation Algae: Volume II: Applications in Medicine and the Pharmaceutical Industry, (77–94) © 2023 Scrivener Publishing LLC

77

78  Next-Generation Algae: Volume II Keywords:  Cyanobacteria, hepatotoxins, prokaryotic

3.1 Introduction Algae are eukaryote photosynthetic organisms with polyphyletic and paraphyletic characteristics ranging from unicellular to multicellular. Algae can be classified into six phyla based on scientific classification: Chrysophyta (diatoms), Chlorophyta (green algae), Euglenophyta (euglenoids), Phaeo­ phyta (brown algae), Pyrrophyta (dinoflagellates), Pyrrophyta (dinoflagellates), and Rhodophyta (red algae). By using DNA sequencing technique, algae are classified into ten major phyla: Heterokontophyta, Glaucophyta, Euglenophyta, Cryptophyta, Haptophyta, Rhodophyta, Dinophyta, Chlorophyta (green algae) and the prokaryotic Cyanophyta and Prochlorophyta [1, 2]. Each class of algae is known to produce a broad range of secondary metabolites with different biological mode of action; but just a small subset of these compounds has been discovered, screened and isolated. The rapid growth rate of algae, combined with their extensive spectrum of secondary metabolites, makes them potentially prolific sources of highly bioactive metabolites. Diverse algae are currently being used for production of food, pharmaceuticals, cosmetics, fertilizers, and biofuels [3–5]. Algae are photoautotrophic organisms, either prokaryotic or eukaryotic, capable of absorbing nitrogen and phosphorus from the medium into biomass during their growth, and the produced biomass can subsequently be transformed to various bioproducts following an apposite process [6] (Table 3.1). The biochemical composition, specifically the Table 3.1  Biochemical compounds of microalgae. Algae

Proteins (%)

Carbohydrates (%)

Anabaena cylindrica

43–56

25–30

Aphanizomenon flos-aquae

62

23

Chlamydomonas reinhardtii 48

17

C. pyrenoidosa

57

26

C. vulgaris

51–58

12–17

Dunaliella salina

57

32

Dunaliella bioculata

49

4

Diverse Medicinal Activities of Microalgae  79 protein and carbohydrate of microalgae, make them attractive in producing several value-added compounds. The ratio of biochemical components or the ratio of proteins/carbohydrates/lipid of algae is species specific. For example, Spirulina maxima is an excellent source for protein (60–71% w/w), Porphyridium cruentum is a rich source of carbohydrates (40–60%), and Scenedesmus dimorphus is 40% lipids [7]. Plants, fruits, and seaweeds have long been used to extract antioxidant components such as phenolic compounds, pigments, polysaccharides, and carotenoids [8]. Various techniques, such as the 1,1-diphenyl-2-­ picrylhydrazyl radical (DPPH•) scavenging assay, hydroxyl/­superoxide radicals (•OH/O2•) scavenging assay, and ferric chloride scavenging assay, have been used to validate relative polysaccharides and pigment compounds as effective antioxidants in ferric-reducing antioxidant power (FRAP) or lipid peroxidation inhibition capacity assay [9, 10]. A great number of new efficient natural antioxidants continue to be discovered among the vastness of marine biotopes. In fact, the marine environment spans at least three-quarters of the Earth’s surface, and many microbes living on it have evolved extremely clever growth and adaptation tactics. As a result, marine creatures provide an unlimited supply of antioxidant biomolecules, including I enzymes (catalase and superoxide), (ii) exopolysaccharides, (iii) carotenoids, and (iv) peptides [11, 12]. The constant growth of international markets asking for more natural antioxidant biomolecules has to be correlated with the industrial expansion of marine microorganisms for high scale-up industrial processes to produce better yields of selected marine antioxidants. Consequently, the goal of this chapter is to provide an overview of recent developments in the field of antioxidant biomolecules generated from marine microorganisms, such as bacteria and microalgae, as well as their present development for potential industrial applications in nutraceutical, cosmeceutical, and pharmaceutical fields. Microalgae are known to be excellent sources of pigments, lipids, including omega-3 fatty acids, vitamins, toxins and other chemicals [13, 14]. Recent studies have shown that various microalgae may have antiproliferative and anticancer activities [15]. Key qualities (e.g., easy to culture and fast-growing) are commonly used to select microalgal strains for potential biotechnological applications. In addition, when growing a microalga, numerous elements must be considered, such as suitable circumstances (e.g., light, temperature, air bubbling, nutrients, salinity, and pH) and the possible presence of associated bacteria. Algal bioactivity may be owing to bacterial presence if the strain is not axenic. Isolation and cultivation are still problematic for a number of species observed in natural samples or detected in meta-genomics data sets; however, when compared with some

80  Next-Generation Algae: Volume II of the other most widely used marine creatures, especially macroorganisms, mass production of microalgal species is easier. They can be mass cultivated in photobioreactors and have quick generation times (doubling time of 5–8 h for some species), allowing them to overcome issues related with overutilization the depletion of marine resources and the adoption of damaging collection techniques. Moreover, for a large number of species, genetic alteration procedures are available to boost their potential as active chemical sources. The most common pathway for drug discovery from microalgae includes their culture in small or large volumes (e.g., using photobioreactors), pellet concentration (through centrifugation or filtration), and chemical analysis. Extraction of microalgal pellets can be done using a variety of methods to locate the desired metabolites [16] and screening the extracts for different bioactivities (e.g., antioxidant, anti-­ inflammatory, antimicrobial and anticancer testing). Bioactivity-guided fractionation allows the active fraction to be identified, along with the likelihood of active compound. Since marine microalgae are often easier to grow than other microorganisms, there is the possibility of obtaining a constant source of marine natural products (MNPs) to meet the demands of the food, nutraceutical and cosmeceutical market. They do, in fact, represent a renewable and underutilized resource for drug discovery. Aside from the compound discovery pipeline mentioned, other methodologies for microalgae have been employed, such as identifying enzyme pathways involved for the synthesis of bioactives (through genomics and transcriptomics analyses “or” proteomics and metabolomics investigations). To activate production of the largest spectrum of metabolites or enhance the production of selected ones, microalgae are first cultivated under control or stressful conditions such as altering light exposure, salinity, or nutrient content [17, 18]. When the culturing parameters are modified, a strategy known as “One Strain Many Compounds” (OSMAC), the same species may produce different metabolites and have diverse bioactivities. The RNA, DNA, and proteins extracted from the microalgal cells may then be extensively sequenced and examined with bioinformatics tools in order to undergo drug discovery with in-silico approaches. In-silico identification is then confirmed/implemented by bioactivity screening, heterologous expression, genetic engineering and/or chemical synthesis in order to produce higher amounts of the metabolite of interest and meet industrial demand.

Diverse Medicinal Activities of Microalgae  81

3.2 Microalgae with Anti-Inflammatory Activity Anti-inflammatory properties were previously found for various marine diatoms, such as Porosira glacialis, Attheya longicornis [18], Cylindrotheca closterium, Odontella mobiliensis, Pseudo-nitzschia delicatissima [17] and Phaeodactylum tricornutum for the dinoflagellate Amphidinium carterae [19]. Furthermore, the green algae Dunaliella bardawil and Dunaliella tertiolecta [20] analyzed the release of tumor necrosis factor (TNF), one of the primary inflammatory effectors [21] in lipopolysaccharide (LPS)stimulated monocytic leukemia cells (THP-1). In their study on LPSinduced RAW macrophages, Samarakoon et al. found that the suppression of nitric oxide (NO) generation (percent) was a good indicator of anti-­ inflammatory efficacy [19]. In a double-blind placebo-controlled randomized clinical trial, seventy patients affected by non-alcoholic fatty liver disease (NAFLD) were recruited to test the effects of Chlorella vulgaris 300 mg tablets commercially available as Algomed®; the supplements, which contained 98% C. vulgaris powder, 1% separating agent, and 1% plant-based magnesium stearate, were used in the experiment. For eight weeks, patients were given four C. vulgaris pills each day. For patients treated with C. vulgaris, the pro-inflammatory cytokine TNF-α was found to be substantially lower in the blood of those who took the supplements [22]. Finally, Lavy et al. showed the protective effects of spray-dried D.  bardawil powder in rats, which inhibited acetic acid-induced small intestinal inflammation [20], whereas Caroprese et al. demonstrated that a combination of phytosterols from D. tertiolecta reduced the cytokine production in a sheep model of inflammation [23]. With the exception of the final example, nothing is known about the chemicals responsible for the anti-inflammatory action seen in the trials. In addition, there are some studies reporting the anti-inflammatory activity of pure compounds isolated from both marine and freshwater micro­ algae: the carotenoids lutein and astaxanthin, and fatty acids. Ingebrigtsen et al. studied EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), as well as sulphated polysaccharides (sPS) [18]; and Lauritano et al. used an OSMAC (one strain many compounds) approach, changing culturing condition parameters, such as nutrients, light, and temperature, in order to trigger anti-inflammatory activity [17]. They discovered that the investigated species were active only under certain circumstances, emphasizing the necessity of choosing the right parameters to boost microalgal extract bioactivity. Similarly, they found that chemical

82  Next-Generation Algae: Volume II stimuli (e.g., low oxygen, NaCl, and nutritional deprivation) increased the synthesis of anti-inflammatory molecules (carotenoids, fatty acids, and sulphated polysaccharides) from some microalgal species (e.g., Chlorella zofingiensis, Coccomyxa onubensis, Cromochloris zofingiensis, Dunaliella salina, D. tertiolecta, Haematococcus pluvialis, Nannochloropsis oceanica, Pavlova lutheri, P. tricornutum, and Spirulina platensis).

3.3 Microalgae with Immunomodulatory Activity Several microalgae have been shown to exhibit immunomodulatory activity in humans and other species (e.g., sheep and mice), but the chemicals that cause this activity are often still unknown. As reported, dried algae and raw extracts were active against various immune cells. Cutignano et  al. [24] examined raw methanolic extracts and fractions derived from them (designated A–E) from a variety of microalgal species (Alexandrium andersoni FE108, Alexandrium tamarense FE107, Chaetoceros calcitrans FE20, Chaetoceros socialis FE17, Ditylum brightwellii (T. West) Grunow, Dunaliella salina FE209, Skeletonema costatum RCC1716, S. dohrnii FE82, S. marinoi FE65, S. pseudo-costatum FE25, Skeletonema sp. KS5 and Thalassiosira weissflogii 1336) on human peripheral blood mononuclear cells (PBMCs). Immunostimulant activity in this study was considered as induction of IL-6 in PBMCs cells. They found that raw methanolic extracts were active for S. costatum and S. dohrnii. Fractionation allowed for these two species to identify the active fractions. Triglycerides rich fraction (named fraction E) was active for S. marinoi. The nucleoside-rich fraction (called fraction B) was active for S. dohrnii, while the S. pseudo-costatum fraction was inactive. Fractionation also assisted in the identification of active fractions for additional species whose raw methanolic extracts were not active. This is most likely because fractions had less salt (mostly NaCl) than primary extracts. The glycolipidic and phospholipidic fraction (called fraction C) was active for A. The nucleosides-rich fraction (called fraction B) of the tamarense was active for D. Fraction A, which was rich in salina, ammino acids, and saccharides, the glycolipidic and phospholipidic fraction (fraction C) and the fraction rich in free fatty acids and sterols (called fraction D). Other extracts produced from microalgae have been evaluated for their immunostimulatory properties. Polysaccharide extracts (aqueous extracts) from Chlorella stigmatophora were evaluated evaluated in vitro and in vivo in an animal model study. Specifically, BALB/c mice were injected with

Diverse Medicinal Activities of Microalgae  83 5 mg of polysaccharide extracts per kg of body weight in order to assess phagocytic activity in vitro and in vivo utilizing macrophages from the peritoneal cavity. Polysaccharide extracts of 5 mg or 10 mg per kg of body weight were administered into C57BI mice two days before or after the injection of sheep red blood cells (SRBC). This experiment was performed in order to test the activation on SRBC [25]. Polysaccharide extracts from C. stigmatophora were able to trigger phagocytic activity in macrophages from the peritoneal cavity in both tests. At 150 g/ml, Euglena gracilis glucans (also known as paramylon) activated NK cells and raised levels of TNF- and IL-6, two pro-inflammatory mediators of TNF-α and IL-6 [26, 27]. Sulfated exopolysaccharides from Gyrodinium impudicum KG03 (at 0.1–10 g/ml) elicited a cellular response in peritoneal macrophages in in-vitro mouse models [28] (Table 3.2). Sulphated polysaccharides isolated from Tribonema sp. have also been discovered to increase macrophage cell survival and cytokine expression. The authors discovered that cell viability was only enhanced in the presence of preservatives of 25 µg/mL of sulfated polysaccharides, while the cytokine expression increased with sulfated polysaccharide treatment at 12.5–200 µg/mL. Park et al. tested the immunostimulatory activity of Thraustochytriidae sp. on human lymphocyte B-cells. They discovered that polysaccharides from this alga could stimulate cell proliferation but not cytokine production when tested at 103 to 109 w/v. The in-vivo immunostimulatory efficacy of Chlorella vulgaris has also been tested in double-blind placebo-controlled randomized clinical studies. Sixty participants were selected and allocated to one of two groups: placebo or chlorella. The active component of the pills tested was dried C. vulgaris, which was added to the diet of the participants at a rate of 5 g per day. All of the participants were advised to continue living their normal lives and eating their normal diets. The NK activity of PBMCs derived from treated patients was enhanced by a C. vulgaris-supplemented diet, which also raised blood levels of INF-, IL-1, and IL-12 [29]. Immunostimulatory action has also been linked to microalgae dietary supplementation. Indeed, feeding mice commercially available spraydried preparations of D. salina (369 or 922.5 mg of algal extract per kg of body weight) enhanced NK and macrophage activation as well as leukemic mouse survival rates [30]. Tetraselmis chuii (50 or 100 g of lyophilized alga per kg of dry food) in gilthead seabream (Sparusaurata L) increased the expression of numerous immune system genes, including T-cell receptor (TCR) beta, major histocompatibility complex genes, and IgM. The majority of these chemicals function as vaccine adjuvants, enhancing immune response by activating APCs [31] (Table 3.2).

84  Next-Generation Algae: Volume II Table 3.2  Immunomodulatory chemicals are synthesized or have immunomodulatory effects in marine microalgae. PBMC stands for human peripheral blood mononuclear cells, NA stands for not available, and w/v stands for weight/volume. Mechanism/ organism and target cells (or model)

Microalgae

Extract/fraction/ compound

Active concentration

Alexandrium tamarense

Total Extract/ Fractions

NA

Activation of IL-6/Human PBMC

Chaetoceros calcitrans

Fractions

NA

Activation of IL-6/Human PBMC

Chaetoceros socialis

Total extract

NA

Activation of IL-6/Human PBMC

Chlorella Crude polysaccharide 5 or 10 mg/kg stigmatophora extracts

Chlorella vulgaris

Diet supplementation/ commercially available pills

Activation of phagocytic activity SRBC/Mouse

Diet Improvement of supplementation/ NK activity commercially and serum available pills level of INF-γ, IL-1β and IL-12/Human trials

Dunaliella salina Diet supplementation 369, and 922.5 mg/ of commercially kg available spraydried preparations

Mice NK and Macrophage activation/ In-vivo mice model

Euglena gracilis

Activation of NK cells and improvement+ in inflammatory mediator/ Human PBMC

β-Glucans

150 µg/mL

(Continued)

Diverse Medicinal Activities of Microalgae  85 Table 3.2  Immunomodulatory chemicals are synthesized or have immunomodulatory effects in marine microalgae. PBMC stands for human peripheral blood mononuclear cells, NA stands for not available, and w/v stands for weight/volume. (Continued)

Active concentration

Mechanism/ organism and target cells (or model)

Microalgae

Extract/fraction/ compound

Gyrodinium impudicum

Sulfated 0.1–10 µg/mL exopolysaccharides

Macrophage activation/ Murine

Skeletonema costatum

Total Extract/ Fractions

Activation of IL-6/Human PBMC

NA

We have summarized the algae which have shown immunostimulatory activity. Pure compounds from microalgae with immunomodulatory activity will be discussed in the next section.

3.4 Microalgae Anticancer Activity The significant medical and pharmacological qualities of microalgae have gotten a lot of attention around the world. Furthermore, several studies have been conducted on the usefulness of microalgae metabolites in the treatment of various human diseases, with the growing of algae-derived compounds gaining much interest in the pharmaceutical industry [32]. Recently, the anticancer properties of some algae-derived resources have been found to modulate several cellular mechanisms such as cellular cytotoxication, downregulate invasion of tumor cells, and enhancement of cancer cells apoptosis [33, 34]. Many cellular and molecular investigations have suggested the potent natural antimalignant activity of algae-derived compounds [35, 36]. Another example is fucoxanthin, a carotenoid found in microalgae, diatoms, and brown seaweeds that has shown significant anticancer effects through malignant cell growth inhibition and the promotion of potent anticancer properties via growth by preventing malignant cell growth, stimulating cancer suppressor genes, and arresting cell cycles, without affecting tumor cell apoptosis [37, 38]. Data on algae-­derived

86  Next-Generation Algae: Volume II anticancer resources might help researchers learn more about a new effective tumor therapy for humans. Bioactive substances having hypotensive, hypoglycemic, and hypolipidemic properties have been derived from microalgae and marine micro­ organisms. Bioactive compounds also lower blood pressure. Because of its high incidence and link to increased morbidity and death, hypertension is a global health issue. It is the most significant risk factor for cardiovascular disease, outnumbering cigarette smoking, dyslipidemia, and diabetes [39], and is thought to be responsible for 54% of all strokes and 47% of all heart attacks, and is a main cause of ischemic heart disease worldwide [40]. There have been reports of peptides with antihypertensive action [41]. Peptides from several species of microalgae, including Chlorella vulgaris, Chlorella ellipsoidea, and Spirulina platensis (Arthrospira platensis) have demonstrated antihypertensive activity. Many research studies have shown that consuming Chlorella lowers blood pressure [42]. A hendecapeptide (Val-Glu-Cys-Tyr-Gly-Pro-AsnArg-Pro-Gln-Phe) with angiotensin I-converting enzyme (ACE) inhibitory action was identified from the pepsin hydrolysate of proteins from C. vulgaris [43]. The isolated hendecapeptide’s inhibitory kinetics demonstrated a noncompetitive binding mechanism, indicating that it has a significant level of ACE inhibitory action. ACE is a hypertension-converting enzyme that accelerates the conversion of angiotensin I to the powerful vasoconstrictor angiotensin II [44] and inactivates the vasodilator bradykinin The biochemical features of this isolated hendecapeptide, together with the low cost of microalgae protein, make it an appealing option for developing a high-value product for BP management [43]. The alcalase-­ proteolytic hydrolysate of C. ellipsoidea protein likewise had substantial ACE-inhibitory activity and was fractionated into three molecular weight ranges, with the Fr range being the most active fraction of less than 5 kDa showing the highest ACE-inhibitory activity. The peptide Val-Glu-Gly-Tyr (467.2 Da) was found to be an effective ACE inhibitor. The isolated peptide has ACE-I inhibitory properties and was a competitive inhibitor of ACE. In spontaneously hypertensive rats, oral treatment of the isolated peptide can considerably lower systolic blood pressure. Oral administration of C. vulgaris peptidic fractions into spontaneously hypertensive rats resulted in significant antihypertensive effects. The peptides (Ile-Val-Val-Glu, Val-Val-Pro-Pro-Ala, Ala-Phe-Leu, AlaGlu-Leu, and Phe-Ala-Leu) extracted from C. vulgaris pepsin hydrolysates were shown to inhibit ACE-I [64].  Chlorella is thought to reduce blood pressure via modulating the rennin–angiotensin system. Furthermore, S. platensis hydrolysate and purified peptide fractions Ile-Ala-Pro-Gly,

Diverse Medicinal Activities of Microalgae  87 Ile-Ala-Glu, and Val-Ala-Phe have been demonstrated to have ACE-I inhibitory effects [45, 46]. The high amount of branched and aromatic amino acids in marine proteins, such as Ile, Val, Phe, and Tyr, has been reported to be responsible for the ACE-I inhibitory activity. Moreover, branched amino acid residues at N-terminal positions and aromatic amino acid residues at C-terminal positions in the substrates or competitive inhibitors could also be preferred for anti-ACE activity [46].

3.5 Potential of Microalgae in Quality Enhancement of Natural Products Natural products are defined as products with natural origins, such as animals, plants, or microbes, that have not undergone any processing or treatment other than a simple preservation method [47]. It is possible to incorporate high-value bioactive chemicals derived from microalgae biomass into the vast majority of consumer products on a regular basis to increase their nutritional and functional value.

3.5.1 Pharmaceutical Industry Microalgae have drawn a great deal of interest in the pharmaceutical industry in recent years for their potential therapeutic applications. Various research investigations have shown that bioactive chemicals derived from microalgae biomass have therapeutic qualities such as antibacterial, antiviral, antifungal, antitumor, and neuroprotective effects [48, 49]. Anticancer medicines, particularly those that are poorly water-soluble, are delivered in a targeted manner. Diatomaceous earth microparticles, which are derived from marine microalgae, are utilized in cancer targeted therapy, such as colorectal cancer. The compounds were coated with vitamin B12, a tumor-targeting drug, before being loaded with cisplatnum or cisplatin, an anticancer agent, for delivery of the cancer drug to the targeted site to kill cancer cells [50].

3.5.2 Cosmetics and Personal Care Microalgae produce substances that have an endless number of possible skin advantages, such as improving blood circulation, moisturizing the skin, triggering cell renewal and metabolism, strengthening skin resistance, and having an anti-inflammatory effect. Oral care, skin care, sun

88  Next-Generation Algae: Volume II care, hair care, decorative cosmetics, body care and perfumes are among the personal care products. Skin care products range from facial cleanser, toner, scrub, essential oil, moisturizer, and masks to restore, protect, and regenerate the skin with various functions such as hydration, brightening, pore-tightening, and anti-aging. Skin care products made from microalgae biomass are a topic of discussion. For over 30 years, the Daniel Jouvance company has formulated all of its products with microalgae as part of their Algo [2] product line, including face care, body care and personal care. For example, Daniel Jouvance’s ALGO[2]IODE has a high concentration of marine iodine from Thalassiosira to help postpone the creation of new adipose cells, resulting in a 7-day slimming cure; while. Dunaliella salina microalgae, which is high in marine glycerin, has been used in the their AQUACEANE line as a potent moisturizing ingredient that keeps skin hydrated for a long time. ÉCLACÉANE from Noctiluca microalgae, which releases visible and shimmering light on the ocean’s surface, provides a fresh source of radiance to the skin (Table 3.3). Furthermore, certain cosmetics made from microalgae have been commercialized. PEPHA®-TIGHT, for example, is a patented pure, oneof-a-kind natural skin firming and highly purified biotechnologically manufactured extract from N. oculate microalgae to be used in anti-aging creams [51].

3.5.3 Food Industry The Food and Drug Administration (FDA) in the United States considers algae-based goods or additives to be safe [52]. Microalgae-based culinary ingredients include cookies, biscuits, pasta, and dairy products, to name a few [53]. Bioactive substances from microalgae can be added to dairy products for health advantages. For example, Arthrospira sp. in flora can help probiotics thrive in fermented milk and yogurts by promoting their development and viability [54]. Apart from yogurt, Chlorella sp. has also been used in cheese to increase hardness and springiness while lowering meltability and cohesiveness [55]. Arthrospira platensis phycocyanin extracts have been introduced in the baking of cookies to increase fiber and protein content [56]. The addition of H. pluvialis to cookies helps to lower the glycemic response while also increasing antioxidant capacity [57], and the protein and antioxidant content of cookies is increased by integrating C. vulgaris, T. suecica, A. platensis, and P. tricornutum. Pigments and carotenoids from microalgae can be used as food additives as colorings and thickeners [58]. Dunaliella sp., Chlamydomonas sp., Chlorella sp.,

Diverse Medicinal Activities of Microalgae  89 Table 3.3  Properties of microalgae for application in skin care products. Microalgae

Function

Monodus sp., Thalassiosira sp., Chaetoceros sp., and Chlorococcum

Integrated into anti-aging products for collagen stimulation

Phaeodactylum tricornutum

Improves skin elasticity and firmness as well as protects skin against UV exposure

Chlamydocapsa sp. (snow algae)

Hydrates skin following exposure to dry climates, UV radiation or the cold

Spirulina platensis

Protects against free radicals’ harmful activity in the dermis and epidermis

Chlorella vulgaris and Spirulina maxima

Produce vitamins that help to get rid of dark circles, refresh and purify the skin

Aurantiochytrium sp., Thraustochytrium sp., and Schizochytrium sp.

Produce squalene that is used in moisturizing lotions to provide ideal skin properties by stimulating emollient and antistatic actions

Cyanidioschyzon sp., Synechocystis sp., and S. platensis generate carotenoids that are used as a food colorant. Arthrospira sp. has also been employed as an enrichment element in extruded snack foods [59]. Microalgae such as Spirulina platensis can be employed as a protein element in drinks, such as sports nutrition beverages, as S. platensis proteins contain all the amino acids needed to increase the athletes’ endurance and performance [60]. Microalgae can also be incorporated into pasta, which is a prominent food in the diets of Europeans and Asians. It has no effect on pasta cooking or texture [61], and a high microalgae concentration in pasta improves stickiness and maintains elasticity. C. vulgaris and A. maxima can be put into fresh spaghetti to boost the nutritious content [61]. Pasta made with D. vlkianum and I. galbana contains -3 PUFAs and antioxidants that may have health benefits [62]. Another fascinating feature of microalgae like I. galbana, P. tricornutum, C. calcitrans, M. subterraneus, S. obliquus, N. gaditana, C. cohnii, and T. pseudonana is their ability to synthesize lipids (fatty acids) in the food industry [63].

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92  Next-Generation Algae: Volume II lymphocytes by upregulating pro-inflammatory factors. Food Sci. Nutr. 5, 205–214, 2017. 28. Bae, S.Y., Yim, J.H., Lee, H.K., Pyo, S., Activation of murine peritoneal macrophages by sulfated exopolysaccharide from marine microalga Gyrodiniumimpudicum (strain KG03): Involvement of the NF-kappa B and JNK pathway. Int. Immunopharmaco. 6, 473–484, 2006. 29. Kwak, J.H., Baek, S.H., Woo, Y., Han, J.K., Kim, B.G., Kim, O.Y., Lee, J.H., Beneficial immunostimulatory effect of short-term Chlorella supplementation: Enhancement of natural killer cell activity and early inflammatory response (randomized, double-blinded, placebo-controlled trial). Nutr. J. 11, 53, 2012. 30. Chuang, W.C.,Ho, Y.C.,Liao, J.W., Lu, F.J., Dunaliella salina Exhibits an Antileukemic Immunity in a Mouse Model of WEHI-3 Leukemia Cells. J. Agric. Food Chem., 62, 11479–11487, 2014. 31. De Jesus Raposo, M.F., De Morais, A.M., De Morais, R.M., Marine polysaccharides from algae with potential biomedical applications. Mar. Drugs. 13, 2967–3028, 2015. 32. Shanab, S.M., Mostafa, S.S., Shalaby, E.A., Mahmoud, G.I., Aqueous extracts of microalgae exhibit antioxidant and anticancer activitie. Asian Pac. J. Trop. Biomed., 2  pp. 608-615, 2012. 33. Lee, J.C., Hou, M.F., Huang, HW., Chang, FR., Yeh, C.C., Tang, J.Y., Chang, H., Marine algal natural products with anti-oxidative, anti-­inflammatory, and anti-cancer properties. Cancer Cell Int., 13, pp. 55-61, 2013. 34. Farooqi, A.A., Butt, G., Razzaq, Z., Algae extracts and methyl jasmonate anti-cancer activities in prostate cancer: choreographers of ‘the dance macabre’ Cancer Cell Int., 12 (1) (2012), pp. 50-56, 2012. 35. Talero, E., GarciaMaurimo, S., Avila, J., Roman, A., Rodriguez Luna, A., Alcaide, V., Motilva., Bioactive compounds isolated from microalgae in chronic inflammation and cancer. Mar. Drugs, 13 , pp. 6152-6209, 2015. 36. Kumar, S.R., Hosokawa, M., Miyachita, K., Fucoxanthin: a marine carotenoid exerting anti-cancer effects by affecting multiple mechanisms. Mar. Drugs, 11, pp. 5130-5147, 2013. 37. Takahashi, K., Hosokawa, M., Kasajima, H., Hatanaka, K., Kudo, K., Shimoyam, a,N., Miyash, K., iAnticancer effects of fucoxanthin and fucoxanthinol on colorectal cancer cell lines and colorectal cancer tissues. Oncol. Lett., 10, pp. 1463-1467, 2015. 38. Peng, J., Yuan, JP., Wu, CF., Wang, J.H., 2011. Fucoxanthin, a marine carotenoid present in brown seaweeds and diatoms: metabolism and bioactivities relevant to human health. Mar. Drugs, 9, pp. 1806-1828, 2011. 39. Kannel, W.B., & Higgins, M., Smoking and hypertension as predictors of cardiovascular risk in population studies. Journal of Hypertension, 8, S3–S8, 1990.

Diverse Medicinal Activities of Microalgae  93 40. Lawes, C.M., Vander Hoorn, S. & Rodgers, A., International Society of Hypertension.Global burden of blood-pressure-related disease. Lancet, 371, 1513, 2008. 41. Norris, R., Harnedy, P.A. & FitzGerald, R.J. Antihypertensive peptides from marine sources. In: Bioactive Compounds from Marine Foods: Plant and Animal Sources. (edited by B. Hernandez-Ledezma & M. Herrero) Pp. 27–56, Hoboken, NJ, USA: John Wiley & Sons, 2013. 42. Kim, S.K., & Kang, K.H., Medicinal effects of peptides from marine microalgae. Advances in Food and Nutrition Research, 64, 313–323, 2011. 43. Sheih, I.C., Fang, T.J. & Wu, T.K., Isolation and characterization of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from the algae protein waste. Food Chemistry, 115, 279–284, 2009. 44. Skeggs, L.T., Kahn, J.R. & Shumway, N.P., The preparation and function of the hypertension-converting enzyme. Journal of Experimental Medicine, 103, 295–299, 1956. 45. Suetsuna, K. & Chen, J.R., Identification of antihypertensive peptides from peptic digest of two microalgae, Chlorella vulgaris and Spirulinaplatensis. Marine Biotechnology, 3, 305–309, 2001. 46. He, H.L., Chen, X.L., Wu, H., et al., High throughput and rapid screening of marine protein hydrolysates enriched in peptides with angiotensinI-­ converting enzyme inhibitory activity by capillary electrophoresis. Bioresource Technology, 98, 3499–3505, 2007. 47. Gurnar, O., Drug of natural origin a text book of Pharmacognosy 4th revised edition. Sweden: aporekarsocieteten, 15-23, 1999. 48. Barkia, I., Saari, N., Manning, S.R., Microalgae for high-value products towards human health and nutrition. Mar. Drugs 17 (5), 304, 2019. 49. Najdenski, H.M., Gigova, L.G., Iliev, I.I., Pilarski, P.S., Lukavsky, J., Tsvetkova, I.V., Ninova, M.S., Kussovski, V.K., Antibacterial and antifungal activities of selected microalgae and cyanobacteria. Int. J. Food Sci. Tech. 48 (7), 1533– 1540, 2013. 50. Delasoie, J., Rossier, J., Haeni, L., Rothen-Rutishauser, B., Zobi, F., Slowtargeted release of a ruthenium anticancer agent from vitamin B 12 functionalized marine diatom microalgae. Dalton Trans. 47 (48), 17221–17232, 2018. 51. Sumathy, B., Kim, E.-K., Effect of Marine Cosmeceuticals on the Pigmentation of Skin, pp. 63–66, 2011. 52. Wijnands, J.H., Bremmers, H.J., van der Meulen, B.M., Poppe, K.J., An economic and legal assessment of the EU food industry’s competitiveness. Agribusiness: An Int. J. 24 (4), 417–439, 2008. 53. Caporgno, M.P., Mathys, A., Trends in microalgae incorporation into innovative food products with potential health benefits. Front. Nutr. 5, 2018. 54. Varga, L., Szigeti, J., Kovacs, R., Foldes, T., Buti, S., Influence of a Spirulinaplatensis biomass on the microflora of fermented ABT milks during storage (R1). J. Dairy Sci. 85 (5), 1031–1038, 2002.

94  Next-Generation Algae: Volume II 55. Cho, E., Nam, E., Park, S., Keeping quality and sensory properties of drinkable yoghurt with added Chlorella extract. Korean J. Food Nutr. 17 (2), 128– 132, 2004. 56. Singh, P., Singh, R., Jha, A., Rasane, P., Gautam, A.K., Optimization of a process for highfibre and high protein biscuit. J. Food Sci. Technol. 52 (3), 1394–1403, 2015. 57. Hossain, A., Brennan, M.A., Mason, S.L., Guo, X., Zeng, X.A., Brennan, C.S., The Effect of astaxanthin-rich microalgae “Haematococcuspluvialis” and whole meal flours incorporation in improving the physical and functional properties of cookies. Foods 6 (8), 57, 2017. 58. Mourelle, M.L., Gomez, C.P., Legido, J.L., The potential use of marine microalgae and cyanobacteria in cosmetics and thalassotherapy. Cosmetics 4(4), 46, 2017. 59. Lucas, B.F., de Morais, M.G., Santos, T.D., Costa, J.A.V.,. Spirulina for snack enrichment: Nutritional, physical and sensory evaluations. LWT- Food Sci. Technol., 90, 270–276, 2018. 60. Gubanenko, G., Naimushina, L., Zykova, I., Spirulina as a protein ingredient in a sports nutrition drink. 4th International Conference on Innovations in Sports,Tourism and Instructional Science (ICISTIS 2019). Atlantis Press, 2019. 61. Fradique, M., Batista, A.P., Nunes, M.C., Gouveia, L., Bandarra, N.M., Raymundo, A., Incorporation of Chlorella vulgaris and Spirulina maxima biomass in pasta products. Part 1: preparation and evaluation. J. Sci. Food Agric. 90 (10), 1656–1664, 2010. 62. De Marco, E.R., Steffolani, M.E., Martinez, C.S., Leon, A.E., Effects of spirulina biomass on the technological and nutritional quality of bread wheat pasta. LWT-Food Sci. Technol. 58 (1), 102–108, 2014. 63. Klok, A., Lamers, P., Martens, D., Draaisma, R., Wijffels, R., Edible oils from microalgae: insights in TAG accumulation. Trends Biotechnol. 32 (10), 521– 528, 2014. 64. Murakami, Y. (ed.), Stress Intensity Factors Handbook. In 2 Volumes. pp. 1456, Oxford, Pergamon Press, 1987.

4 Application of Astaxanthin and Carotenoids Derived from Algae for the Production of Nutraceuticals, Pharmaceuticals, Additives, Food Supplement and Feed Abiola Folakemi Olaniran1*, Joshua Opeyemi Folorunsho1, Bolanle Adenike Akinsanola1, Abiola Ezekiel Taiwo2, Yetunde Mary Iranloye1, Clinton Emeka Okonkwo3 and Omorefosa Osarenkhoe Osemwegie1 Landmark University SDG 12 (Responsible Consumption and Production Group), Department of Food Science and Microbiology, Landmark University, Omu-Aran, Kwara State, Nigeria 2 Faculty of Engineering, Mangosuthu University of Technology, Durban, South Africa 3 Department of Food Science and Technology, College of Agriculture and Veterinary Medicine, United Arab Emirates University (UAEU), AI Ain, United Arab Emirates 1

Abstract

The importance of astaxanthin and carotenoids for the production of nutraceuticals, pharmaceuticals, additives, food supplement and feed cannot be overemphasized due to several health benefit among which are anti-inflammatory and immune booster properties in people and animals. They are a major natural source of pigments and are high in antioxidants that have been documented to be more competent compared with vitamins C, E, carotene, lutein, lycopene, zeaxanthin, etc. Therefore, it’s vital to explore the use of astaxanthin and carotenoids from algae; hence, this chapter discusses the potentials of optimizing astaxanthin and carotenoids from macro- and microalga cells. Utilization of astaxanthin and carotenoids from algae must be extended beyond serving as a food colorant by leveraging its powerful antioxidant potentials as a scavenger using microencapsulation *Corresponding author: [email protected]; [email protected] Charles Oluwaseun Adetunji, Julius Kola Oloke, Naveen Dwivedi, Sabeela Beevi Ummalyma, Shubha Dwivedi, Daniel Ingo Hefft and Juliana Bunmi Adetunji (eds.) Next-Generation Algae: Volume II: Applications in Medicine and the Pharmaceutical Industry, (95–124) © 2023 Scrivener Publishing LLC

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96  Next-Generation Algae: Volume II to reduce oxidation damage which is germane in treatment of several documented health challenges. Hence, considering the future prospects of maximizing astaxanthin and carotenoids produced from algae as a major raw material in manufacturing of nutraceuticals, pharmaceutical products, and nutritional supplements on an industrial scale for the global market is a progressive phase towards attaining sustainable technology and agriculture that is eco-friendly and supports healthy living of humans and animals. Keywords:  Algae, bioactive compounds, carotenoids, astaxanthin, food supplement, health, sustainable consumption, nutraceuticals

4.1 Carotenoids and Its Characteristics Carotenoids are naturally occurring chemicals. Photosynthetic organisms, as well as certain fungi and bacteria, biosynthesize them. Carotenoids (CTN) cannot be biosynthesized by the great majority of animals, but they may be absorbed into the diet and structurally changed afterwards. Certain invertebrate species, like hemipteran and dipteran insects and mites, have been shown to manufacture carotenoid [1]. Carotenoids are a cluster of over 1000 pigments that occur naturally and synthesized by several organisms like plants, photosynthetic bacteria, fishes and microalgae [2]. They range from red to orange to yellow pigments that give the characteristic color to tomatoes, canaries, carrots, pumpkins, flamingos, shrimps, salmon, lobsters, among others. They serve two key roles in algae and plants: they provide, via non-photochemical quenching, photoprotection, and they absorb light energy in photosynthesis [3, 4]. Based on the presence of oxygen, CTN are categorized into Xanthophylls (contains oxygen) and Carotenes (pure hydrocarbons with no oxygen). CTNs are formed from isoprene that consists of 40 carbon atoms, making them derivates of tetraterpenes. They are the dominant color pigments in leaf coloration during the autumn season for about 25% plant species. Their structure allows for different biological activities, including plant coloration, cell communication, photosynthesis and photoprotection. The length of CTN compound plays a part in plants pigmentation, and the span of polyene tails shows which wavelength of sunrays the plant absorbs. They are lipophilic in nature as a result of the presence of aliphatic chains that are unsaturated, which can be seen in some fatty acids. Depending on the presence of bile salts and fats, these fat-soluble compounds are adsorbed in human beings [4].

Astaxanthin and Carotenoids Derived from Algae  97

4.1.1 Sources of Carotenoids Carotenoids are regarded as one of the most common and widely distributed naturally occurring lipid-dissolvable pigments, despite having little natural presence. They are found in fruits, flowers, leaves and seeds. In terrestrial animals, they’re primarily localized in fatty tissues like abdominal fat and egg yolk [5]. Bacteria and algae, which can manufacture a broad range of carotenoid structures, have a particularly vast diversity. Natural sources of carotenoids include plants, microalgae, bacteria, and fungus. Carotenoids are produced by a variety of bacteria and accumulate in the chromatophores, which are connected to the membrane (cytoplasmic membrane). Carotenoids, when in the form of beta-carotene, are found almost exclusively in Halobacterium spp., resulting in a brilliant red cell suspension orange-pigmented, Gram-negative, rod-shaped, aerobic motile by peritrichous flagella, and astaxanthin-enriched Paracoccus carotinifaciens isolated from soil [6]. This organism produces carotenoids, primarily astaxanthin, but not bacteriochlorophyll. Q-10 is the ubiquinone system. Several fungal families, in comparison to other species, are able to generate and store exceptionally large amounts of carotenoids intracellularly, a trait shared by some microalgae. Several fungal phyla, including Ascomycetes, related species, Basidiomycetes, and also the Deuteromycetes, generate significant quantities of carotenoids in contrast to other organisms such as algae, bacteria, plants, and mammals [7]. Microalgae, to the contrary, proffer a quality source of carotenoid colors, polyphenols, vitamins, fats, and proteins. Algae are basic unicellular creatures (microalgae) that can be found alone or in colonies, and they can also be found in very sophisticated pluricellular tissues (macroalgae) [8]. Microalgae cultivation has certain distinct benefits over traditional plant-required sources, including a quicker cultivation time, processing time, and harvesting cycle, and also the possibility to be cultivated on waste products. Despite the advantages noted, cost-effective and largescale synthesis of carotenoids cultivated from algae is still extremely difficult during production, development and downstream extraction [9]. As a result, an integrated/merged bioprocessing strategy employing microalgae must address both upstream microalgae production and downstream carotenoids harvesting and extraction. The presence of stiff cell walls in many algae species creates challenges since it inhibits full recovery of bioactive chemicals [8, 10]. Carotenoids are responsible for the wide-ranging colors in leaves and flowers, as well as providing a diversity of smells in plants. Carotenoids are completely expressed in many flowers because they are usually found

98  Next-Generation Algae: Volume II aggregated in chromoplasts, which are usually in the absence of chlorophyll pigment [11]. Carotenoids found in terrestrial plants are mostly yellow and red xanthophylls like capsanthin, the popular lutein, violaxanthin, zeaxanthin, or neoxanthin, which are all part of the usual xanthophyll cycle and are regularly involved in photosynthetic protection [12]. Several variables including genotype, maturation period, growth method with weather conditions, and also processing, influences the composition and kinds of prospective carotenoids in the respective plants. Different classes and quantities of carotenoids can be found in different sections of the same plant. Fruit peel, for example, is usually higher in carotenoids than fruit pulp [13]. Carotenoids are divided into two sets based on their structures: hydrocarbon carotenoids and xanthophylls. Carotenoids with polar properties are known as carotenes and include alpha-carotene, gamma-­carotene, beta-carotene, phytofluene, lycopene and phytoene [14]. Xanthophylls are known to be more polar than chlorophylls and include oxygen at the carboxy-, hydroxy-, keto-, methoxy-, and epoxy- positions. Zeaxanthin, lutein, astaxanthin, and fucoxanthin are examples of xanthophylls. Carotenoids are also categorized by the presence and absence of ring groups at the chain’s ends [3, 16]. Xanthophylls are yellow pigmentations found to occur in nature that include oxygen atoms. Lutein is a dihydroxy-carotene composed of carotenoids with a hydroaromatic structure and an alcohol group. It has hydroxyl groups on both of its beta-ionone rings. It is a pigment that gives chicken fats, and their products, including feathers and egg yolk, their color [10]. The yellow-pigmented lutein reported in plants is said to be an organic colorant that may be found on the leaves of green crops like spinach and black pepper. It’s most commonly seen in fatty acid covalent connections. Lutein and zeaxanthin have identical chemical formulae; in other words, they can be said to be isomers, but are not referred to as stereoisomers. The situation of the two-fold bond within the terminal ring differs between the two. A popular prevalent carotenoid alcohol discovered in nature is zeaxanthin. It’s a pigment that gives maize and several plants their color. Picrocrocin, which gives saffron its flavor and scent, is formed when zeaxanthin breaks down. The alcohol group in the beta-cryptoxanthin carotenoid has a hydro-­aromatic structure with an OH group in the first ring. The second ring is the beta-ionone, which may be used to make one molecule of vitamin A. Beta-cryptoxanthin is a naturally occurring carotenoid pigment usually found in vegetables and fruits like mandarin, bell pepper, and sweet potato, which offers various health advantages [16]. Beta-cryptoxanthin and beta-carotene are related. Beta-cryptoxanthin was given only one OH group. Although beta-carotene may be found in

Astaxanthin and Carotenoids Derived from Algae  99 great amounts in a diverse fruits and vegetables, beta-cryptoxanthin is only found in a few dietary sources but in high concentrations. Carotenes are carotenoids with hydroaromatic rings. These rings can be found at both ends of the four isoprene molecules,. Each carotene molecule has two hydroaromatic rings as a result. These hydrocarbons have three ionone rings which are known as ionospheric rings. There are two types of ionone rings: alpha- and beta-ionone rings [15]. Alpha- with beta-­ionone rings have one double bond and are closed rings. In beta-ionone rings and pseudo rings, the location of the double bond is not the same. Two double bonds make up the pseudo-ionone ring. Carotene is an eight-­isoprene-unit biochemically produced terpene. Carotenoids are vitamin A precursors that are metabolized to vitamin A in the human body. One molecule of vitamin A is generated from alpha-carotene and gamma-­carotene, whereas two molecules of vitamin A are created from beta-carotene. Between them, there are four molecules of polymerized isoprene. Vitamin A is made up of half of an alpha-carotene molecule. A beta-ionone ring and two isoprene molecules are linked to one end of vitamin A. Carrots, squash, tomatoes, bell peppers, and dark green-colored vegetables are high in alpha-carotene, the second most prevalent type of carotene [17]. The provitamin beta-carotene is fat-soluble. Vitamin A is the active form. The beta-ionone ring is present on both ends of beta, which distinguishes it from alpha. Vitamin A is created when beta-carotene is split into two molecules. It may be found in cereals, also in fruits, vegetables and oils. One molecule of vitamin A is generated when the molecule is divided, with ending the beta-ionone ring and pseudo-ionone ring at the other side. Lycopene is a carotenoid with an olefinic structure [18]. Vegetables, watermelons, grapefruit, and rosehip are all high in lycopene. Because lycopene dissolves in oil, it is absorbed more readily by the digestive system when oils are present. Phytoene, a 40-carbon and 3-conjugated double-bond molecule with a symmetric structure, acts as an intermediary in the synthesis of phytoene carotenoids. Phytofluene is a carotenoid pigmentation found in tomatoes and other plants that are orange in hue [19].

4.1.2 Production/Extraction of Carotenoids Recovery of important chemicals from several natural origins follows analytical chemistry principles, and is widely known to be done in five stages: (i) macroscopic preprocessing, (ii) macro- and micromolecules differentiation, (iii) segmentation, (iv) purification, and lastly, (v) product creation. This approach was created to optimize the output of the specific targeted mixtures, meet the manufacturing step-by-step processing needs, separate

100  Next-Generation Algae: Volume II the valuable substances from any hazardous compounds and impurities found, minimize functionality loss during the processing stage, and assure the end product’s food grade nature. Macroscopic pretreatment, in particular, attempts to modify the water, solids, and lipids content, activate or deactivate enzymes, moderate the microbial burden, and ultimately enhance matrix permeability [20]. A wet milling phase is required when the substrate is a vegetable or fruit byproduct so as to expedite and raise the output of the subsequent extraction stages. This is accomplished by inducing swellings and softness of tissue, which allows for greater extractant integration within the food matrix. If the substrate is wastewater, on the other hand, concentration is used with the goal of eliminating water and raising the content of useful components [21]. Alcohol precipitation is the most common technique for separating smaller chemicals, either acids or ions, from macromolecules, such as nutritional fibers, pectin and hydrocolloids in the second recovery step, which are accumulated in the insoluble alcohol residue. The subsequent phase in downstream processing is of utmost importance, and must be standardized to details, with many techniques applied to the target molecules and physical properties (i.e., solubility or its volatile nature). Contingent on the form of the original substrate and the specific compounds, many traditional and new methods have been utilized for this goal. The most common and extensively utilized approach is solvent extraction. This occurs because it is highly convenient, as the solvent acts as a physical carrier for transferring important chemicals across multiple physical phases while maintaining their physicochemical properties (e.g., volatile nature or solubility) [22]. Carotenoids must be extracted from fruits and vegetables in a simple, quick, and low-cost manner for commercial use. The employment of traditional extraction methods, on the other hand, has its drawbacks.  Not only did these tactics endanger human health and also the environment, they also jeopardized the extraction process.  Long extraction times and an enormous volume of solvent were also required. Furthermore, traditional technologies have a number of flaws that limit their application in actuality. Membrane technologies (such as ultrafiltration) need more energy, whilst others have a high operating cost [23, 24]. The drawbacks of traditional techniques might be addressed by employing new (usually non-thermal) technologies, dubbed emergent technologies. Shortening process time and residence times to enhance the heat as well as mass transfer, improve the superiority of product, control the reactions (Milliard), enhance functionality (which also protects from environmental stresses), and elongate preservation are all benefits of these technologies that are currently being researched and, in many instances, used

Astaxanthin and Carotenoids Derived from Algae  101 in industries involved with food. Radio-frequency drying, cold plasma treatment, ultrasound-assisted extraction (UAE), electro-osmotic dewatering, high-voltage electrical releases, homogenization, pulsed electric field, and nanoencapsulation are the most current evolving technologies being experimented on in the comprehensive field of food science. High hydrostatic pressure, for example, enhances the mass exchange rate of carotenoids during extraction by aiding plant cell penetrability and allows molecules to diffuse. MAE works by utilizing microwaves to increase pressure on the cell wall caused by moisture evaporation within the cells [22]. Microwave energy permeates the biomaterials, then they interact with molecules that are polar, such as water, to generate heat. A rise in pressure causes the physical characteristics of the sample tissues to be disrupted, and the rise in permeability of the matrix lets the extraction solvent penetrate more deeply, improving carotenoids recovery. The microwave’s impact is determined by the ionic solvent’s dielectric susceptibility and the solid plant matrix. Because H2O has a relatively high dielectric constant, moistening all plant samples with it can enhance recovery of carotenoid [22]. The rehydrated biomaterial can also interact more effectively with the energy of a microwave to generate heat, swell within the cytoplasm, and finally rupture, releasing carotenoids into the solvent. It’s important to choose the right ionic solvent for the job. Solubility, dissipation, and dielectric constant variables are three important aspects to consider when choosing a solvent. In comparison to non-polar solvents like hexane, which has a relatively low dielectric constant, high dielectric constant solvents like water, ethanol, and methanol can absorb microwave radiation significantly [25]. The relationship between the solvent and the microwave may be modulated using solvent mixes. The efficiency of a microwave-heated liquid is represented by the dissipation factor. Despite its excessive dielectric constant, water possesses a poor dissipation factor, making it ineffective in heating up the moisture within the sample. As a result, even without using water as the ionic solvent, microwave can aid in the extraction of carotenoids [21]. As a pretreatment, enzymatic treatment is generally executed preceding traditional solvent extraction; hydrolytic enzymes such as cellulase and pectinase are generally used by enzyme-assisted extraction in plant. They disrupt the cell wall’s structure to enable effective extraction and release of bioactive substances. When compared to commercial enzymes, enzymes have been found to produce a carotene that is high in carotene concentration with a shorter processing time. The breakdown of the structure of the cell wall generally speeds up metabolic transformations, which results in unappealing colors and flavors. These variations, in contrast, were not

102  Next-Generation Algae: Volume II obvious in the plant matrices treated using enzymes. Because the extractants formed by cellulase and pectinase, such as carotenoids, are fastened to proteins, the pigment structure that is highly unsaturated remains stable. The enzyme concentrations used for plant pretreatment vary from 0.01% to 0.1% (w/w). The presence of water is required for enzymatic hydrolysis to occur. Excessive water, on the other hand, causes the development of an aqueous phase that inhibits the solvent from interaction with carotenoids and hence inhibits the extraction time process. Agitation is crucial in enzymatic treatment because it allows enzyme to diffuse from the aqueous into the solid stage, which improves cell wall lysis and increases extraction yield [21, 26].

4.2 Astaxanthin and Its Characteristics Astaxanthin (AXT) is a red pigment belonging to the Xanthophyll family, which are oxygenated carotenoids derivatives from lycopene basically in plants. Although it is classified as a Xanthophyll, it is currently used to describe compounds of carotenoid composed of oxygen-containing components [27]. AXT is also one of the main pigments and secondary metabolites synthesized naturally by a number of yeasts, bacteria, and microalgae. It is also commonly found in salmon (Pacific) fish and gives it the characteristic pink color. With the chemical formula C40H54O4, molecular mass 596.841 g/mol; the red to orange color of AXT is due to the elongated conjugated chain (which are interchanging single and double) at the center of the compound. The antioxidant properties of ATX and other carotenoids are linked to its double-bond conjugated chains which result in a section of decentralized electrons which are released to minimize oxidizing reacting molecule [28]. Algae,  salmon, yeast,  shrimp, and crayfish are all natural sources of astaxanthin. The majority of commercial astaxanthin comes from Phaffia yeast, Haematococcus spp., and chemical production. One of the greatest sources of astaxanthin that occur naturally is Haematococcus pluvialis.  The greatest astaxanthin level in the raw Oncorhynchus class was investigated to be in the range of 25–39 mg/kg meat in salmon; however, low source of astaxanthin content was recorded in chum salmon. The concentration of astaxanthin in farmed Atlantic salmon meat was reported to be 5–9 mg/ kg flesh. Microorganisms with the highest percentage of astaxanthin per dry weight include: Haematococcus pluvialis (about 3.5%), Chlorococcum (0.2%) and Neochloris wimmeri (0.6%). Xanthophyllomyces dendrorhous, a Phaffia yeast, produces 100 percent non-esterified astaxanthin, which is

Astaxanthin and Carotenoids Derived from Algae  103 beneficial since it can be easily absorbed and does not need to be hydrolyzed in the fish’s system. Unlike bacteria-derived and synthetic astaxanthin, the yeast-derived astaxanthin is mostly comprised of the (3R, 3’R)-form, which is a common astaxanthin source in nature [29].

4.2.1 Production/Extraction of Astaxanthin Astaxanthin is a lipophilic molecule that dissolves in a variety of solvents and oils. Astaxanthin is extracted using edible oils, solvents, acids, and enzymatic techniques. Astaxanthin accumulates in Haematococcus encysted cells. The pigment Astaxanthin was recovered from Haematococcus using various treatments using acids, with HCL recovering up to 75% of the pigment [12]. When cystic cells were introduced to acetone (40% acetone) for 2 min at 80 °C, then cellulose, kitalase, and acetone powder, about 70% of the astaxanthin was recovered. The use of sonication to treat hydrochloric acid at different temperatures for between 15 and 30 min yielded a high astaxanthin output. In another investigation, astaxanthin was extracted from Haematococcus using vegetable oils (maize, olive, soybean and grape seed). The astaxanthin in the cell was retrieved from the oils when the culture was combined with oils, with olive oil yielding the greatest recovery of 93%. Under acidic conditions, Astaxanthin (about 1.3 mg/g) was recovered from the yeast Phaffia rhodozyma [30]. Microwave-aided extraction at around 75 °C for about 5 min yielded 75% of astaxanthin; nevertheless, the astaxanthin concentration in acetone extract was high. When introducing supercritical fluid extraction using ethanol and sunflower plant oil as co-solvent, the yield of astaxanthin from Haematococcus was 80–90%. Astaxanthin was isolated many times using different solvents, then collected and vaporized using a rotary evaporator. The extract was then dissolved again into a solvent, and the absorbance of the extract was measured between 475–480 nm to determine the astaxanthin concentration. The extract may also be tested for astaxanthin quantity and identification using high-pressure liquid chromatography (HPLC) and mass spectra (MS) [16]. Currently, most astaxanthin production takes place outside of the tropics, owing to the excessive lumination strengths and temperatures necessary for astaxanthin production in the red stage, that would be unprofitable inside; nevertheless, most of its production takes place outside of the tropics [6]. Because of the high light expenditures, the red stage might account for about 60% of the power expenses in indoor production. Only Fuji Chemicals use an all-indoor manufacturing method. In its BioDome™ system, Fuji Chemicals abandoned H. pluvialis cultivation outside. Due to unfavorable conditions for astaxanthin synthesis in outdoors temperate

104  Next-Generation Algae: Volume II zones, commercial manufacture of astaxanthin derived from H. pluvialis is limited, and only in-house culture is possible. Aragreen, based in Gloucestershire, was looking at producing astaxanthin from H. pluvialis, however, the firm went bankrupt in 2017. There are a variety of industrial cultivation methods, with the majority seeking to use a more sustainable manufacturing approach. In the instance of Cyanotech, H. pluvialis is cultivated inside in the production stage under carefully standardized and supervised growth conditions before being moved to open ponds in the red stage for astaxanthin induction [30]. AlgaTechnologies uses photovoltaic cells and performs their entire process outside in photobioreactors (PBRs) to make use of natural sunshine. Algalif, on the other hand, uses light-­ emitting diodes to harness geothermal energy for a complete industrial process indoors (LEDs). The majority of firms are focusing on phototrophic culture, while Fuji Chemicals, for example, is looking into mixotrophic cultivation. The encasement process culminates in the production of spores during astaxanthin production, which can take between around 3–5 days (cysts). The aplanospores are collected with the help of gravity and further concentrated using ultracentrifugation after culture and astaxanthin induction. The biomass is subsequently dried using spray-­drying, which is more cost-effective than freeze-drying or drum-drying [31]. An extraction method is used to break down the cell walls of dried thickwalled aplanospores and make all astaxanthin accessible. Because the walls of the aplanospores are resistant to digestion by animals (in feed supplement application) and humans (in nutraceutical applications), they must be disturbed in order for astaxanthin to become accessible. In the extraction process, care must be taken to prevent oxygen exposure and increased temperatures, both of which can degrade astaxanthin and can result in process losses. On a commercial basis, supercritical fluid extraction (SFE) using CO2 (ScCO2) is the most popular method for extracting astaxanthin. The dried end-product is generally treated with an effective preservative before being delivered to feed producers to be included in designed feed [6].

4.2.2 Historical Perspective of Consumption of Alga as Food and Utilization in the Food Industry Algae have been utilized as food for humans since ancient times. For instance, in the Eastern part of the world and the Pacific region of Asia, eating algae has long been an accepted custom. However, in Western nations, the attention garnered by food products from algae has only greatly expanded in recent years, to the degree that diverse palatable species of algae are presently usually advertised for use in the European Community

Astaxanthin and Carotenoids Derived from Algae  105 and the American continent [32]. Acceptance of algae has additionally expanded as of late, particularly among veggie lovers, because of their utilization in connoisseur cooking as extra dishes, toppings, and ingredients on café set menu these days. Algae suggest intriguing possibilities for the creation of inventive foods or to potentially foster novel products in order to mitigate the shifting perceptions of consumers, opening doors to new food products in the food industry, which requires ceaseless novelty [33]. Moreover, there will probably be a surge in the utilization of algae related to the requirement for extra food sources as the population keeps on developing. Algae has been found to be utilized as a supplement in feed for animals and as part of manures. The nourishing properties of algae are a significant wellspring of supplements like vital nutrients and minerals, nourishing fiber, crucial unsaturated fats and good proteins [34]. Constituents from marine algae like hydrocolloids (carrageen, alginate and agar) likewise afford mechanical benefits in industrial food processes whenever utilized as emulsification, gelling and thickening agents during food and drink manufacturing. As of late, attention to marine algae has been on the rise because it is seen as the origin of bioactive elements having numerous uses in the improvement and creation of functional food sources [35]. The developing understanding of the connection between food and wellbeing is prompting new bits of knowledge about the impact of food elements on physical capacity and wellbeing. The logical result of this is the public’s interest for solid, nutritious food sources with extra health-advancing capacities, like functional food sources [36]. Algae can be incorporated into products as algal extracts of selected elements or, alternatively, pieces of dried algae can be crushed and utilized in the manufacture of food and drinks. In addition to being utilized as single-cell proteins, lately, microalgae are likewise projected as plants with living-cells required in creating different valuable biochemicals and bioenergies utilized in poultry, production of food, hydroponics, and drug companies because of the presence of various helpful compounds. Generally, the pattern towards expanding a healthy interest for algal items on a worldwide basis originated from a more noteworthy focus on wellbeing and more extensive utilization of food additives [37].

4.3 Application/Utilization of Astaxanthin and Carotenoids in Different Sectors Astaxanthin and carotenoids provide wide benefits in various fields and are applied in several areas such as pharmacology, food industry and feed

106  Next-Generation Algae: Volume II production. Carotenoids have lengthy conjugated double bonds in their polyene chains, which function as antioxidants by dousing oxygen and scavenging free radicals to stop any domino effect. Carotenoids’ natural advantages might be linked to its antioxidant capabilities, which are ascribed to their physicochemical interactions with different cell membranes. Carotenoids containing an oxo functional group have greater antioxidant activity without contributing to pro-oxidation. Comparing astaxanthin to other carotenoids, including lutein, lycopene, beta-­carotene, and alpha-carotene, astaxanthin exhibited greater antioxidant activity [38]. With Haematococcus biomass as a major source of astaxanthin, the antioxidant enzymes like peroxidase, catalase, and thiobarbituric acid reactive materials were shown to be high in rat liver and blood plasma. In rats, astaxanthin, followed by beta-carotene, and lutein, provided the highest protection against free radicals. Astaxanthin has a unique chemical structure with the presence of hydroxyl and keto moieties on each ionone ring, which contribute to its strong antioxidant capabilities. Astaxanthin has ten times the antioxidant capabilities of zeaxanthin, canthaxanthin, lutein, and beta-carotene, and a hundred times more antioxidant activity than alpha-tocopherol. Astaxanthin’s polyene chain ends trap free radicals in the cell membrane, while its terminal ring also scavenges free radicals on the cell membrane’s inner and outer surfaces. Antioxidant enzymatic undertakings were measured in rabbit serum after astaxanthin was added to their diet, and superoxide dismutase and also the enzyme thioredoxin reductase activity were increased, while paraoxonase activity was suppressed in the oxidatively induced rabbits. When astaxanthin was administered to ethanol-induced gastric ulcer rats, the levels of antioxidant enzymes rose. This proves the effects of astaxanthin as an oxidant and its application in antioxidant pharmaceutical drugs [24]. Astaxanthin can also be used as an anti-inflammatory drug for hypertension, type 2 diabetes,  hypercholesterolemia, and obesity, which are among the chronic conditions exacerbated by inflammation. It has a strong anti-inflammatory impact that may be linked to its antioxidant properties and leads to different physiological formations that improve cardiovascular function. Atherosclerosis is a degenerative, chronic illness that affects large- and medium-caliber supply arteries. Atherogenesis, the first stage of the process, is marked by the accumulation and formation of low-density lipoprotein (LDL) inside the vascular wall subendothelial layer, which is sensitive to irritation interceded by natural and varied resistance responses [18]. Astaxanthin’s anti-inflammatory properties have been demonstrated in the prevention of atherosclerosis. The main damage associated with single-molecule patterns recognized by macrophages are those generated

Astaxanthin and Carotenoids Derived from Algae  107 by oxidation of LDL, which are liable for the initiation of the inflammatory flow involving the discharge of cytokines and chemokines, which acquire more resident vascular macrophages and monocytes from blood. Not only has astaxanthin been proven to reduce oxidative stress in T cells, but it has also been demonstrated to modulate their function. In scientific research on young healthy females, supplementation using astaxanthin induced mitogen-lymphoproliferation which increased the subpopulation of T lymphocytes devoid of altering the number of T killer/helper cells. It also improved the reaction to tuberculin, a marker of T lymphocyte function. Using an animal model of nonalcoholic steatohepatitis, astaxanthin decreased T-helper and T-killer cell migration in the liver, reducing inflammation and insulin resistance [39–41]. When compared to fish oil alone, 45 days of supplementation with the fish oil having astaxanthin (with a body mass of 1 mg/kg) substantially reduced T-cell proliferation in reaction to mitogens and RONS production. Astaxanthin substantially reduced the activation of T lymphocytes produced by phytohemagglutinin in several in-vitro tests with peripheral mononuclear cells from individuals with different allergic inflammatory diseases. As a result, astaxanthin has been demonstrated to have a distinct modulatory impact on T cells, either enhancing their overall immunological response or reducing potentially harmful activation of the immune system. However, the function of astaxanthin-mediated T lymphocyte regulation in the development of cardiovascular illnesses and its risks are yet unknown. Recent research on potential applications of astaxanthin as antidiabetic drug has been reported. Although prediabetes may be managed by diet and other ways to keep blood sugar in check, the inflammation and oxidative stress induced by elevated sugar in the blood pose a risk for cardiovascular disease. Hyperglycemia causes oxidative stress, which harms the body’s tissues and cells. Inflammation is one of several pathogenic processes of type-II diabetes, and after administering astaxanthin in type 2 diabetes patients, it was discovered that astaxanthin can lower IL-6 levels in patients over time. Reversing prediabetes represents a hugely untapped possibility for preventing diabetes, which lowers the cardiovascular disease burden [18, 41, 42]. Notwithstanding the evident nutritional worth of carotenoids, the unequivocal proof of their relevance in promoting health is exceedingly difficult owing to the complexity of the nutritional intakes and the human system. However, multiple data from various research (epidemiological and lab research) indicate that the use of carotenoids as part of a regular diet may have health advantages [43]. The fast growth of cancer cells

108  Next-Generation Algae: Volume II characterizes tumor development. Cancer cells multiply, which encourages invasion, migration, and adhesion to target tissue. These stages permit the tumor cell to acquire the phenotypic of a metastatic cell. Cell proliferation is controlled by signaling pathways like the MAPK (mitogen-activated protein kinase) and PI3K (phosphatidylinositol 3-kinases) flows, which are triggered by growth factors and adhesion proteins [40]. Higher levels of beta-carotene  and total carotenoids in the blood have the strongest correlations to a reduced risk of breast cancer. Breast tumors that are estrogen receptor-negative (ER-) have a stronger link than those that are estrogen receptor-positive (ER+). In the case of ER- malignancies, circulating alpha-carotene also appears to be protective. Because certain carotenoids have been shown to suppress the development of both ER+ and ER- breast cancer cells in the lab, it’s conceivable that their effect in human research is simply overwhelmed by the hormone effects that are dominant in ER+ tumors [38]. In meta-analyses for the AICR/WCRF study, dietary consumption and blood stages of beta-carotene and other carotenoids were linked to a diminished risk of lung cancer. Current and previous smoking intensity and duration were factored into the studies. However, because tobacco smoking lowers blood beta-carotene levels, the link between lower beta-carotene levels and increased risk may involve some residual tobacco confounding. High-dose beta-carotene supplements, on the other hand, are thought to raise the menace of lung cancer among current and past smokers [11, 38, 40, 44].

4.3.1 Nutraceuticals The term “nutraceutic” comes from the English words “nutrition” and “pharmaceutics.” The phrase describes products made for dietary supplements, herbal extracts, specialized diets, and also processed meals including cereals, sauces, and drinks that are utilized for more than just the usual nourishment. Nutraceutical products are regulated as medicines, food additives, and dietary supplements in the United States. The phrase isn’t always defined the same way in different nations, although it’s commonly described as a food-derived product marketed in medical forms that aren’t normally connected with food [45]. A substance that has physiological advantages or protects against several chronic illnesses is described as a nutraceutical product. These products can be used to enhance health, slow down the aging process, deter chronic illnesses, prolong life, and also maintains the body’s function and structure [46]. Nutraceuticals are arranged in a variety of ways depending on the user’s preference. However, dietary fibers, pre- and probiotics, fatty acids that are

Astaxanthin and Carotenoids Derived from Algae  109 polyunsaturated (PUFA), antioxidants, polyphenols, and carotenoids are all examples of traditional techniques of defining nutraceuticals. Carotenoids constitute a wide group of lipophilic tetraterpenoids that are made up of long, thin molecules in a chromophoric system with a high degree of conjugation. They are classified as carotenes or xanthophylls (hydrocarbons containing oxygen) and are produced by condensation of isoprenyl units. They are one of several common and widespread yellow to orange to red pigments found in nature. Fruits, vegetables, algae, fungus, and some bacteria produce carotenoids as secondary micronutrients. Nature synthesizes about 100 million tons of carotenoids, according to estimates. Primary and secondary carotenoids are two types of carotenoids. Primary carotenoids, such as beta-carotene, neoxanthin and violaxanthin, are required by several plants during their photosynthetic processes, while secondary carotenoids, such as gamma-carotene, beta-cryptoxanthin, zeaxanthin, antheraxanthin and capsanthin, are found in fruits and flowers. The purple color of Proteobacteria, the greenish to brown color of Phaeophyceae, the yellowish color and texture of birds’ feathers, the orange coloration of carrots; or the autumn foliage of different deciduous trees, the pigmentation in fungi, mosses, algae, bacteria, and different marine animals or crustacean exoskeletons, night vision of vertebrates, and even the human skin coloration are all examples of carotenoids’ wide distribution [18, 26]. Moran and Jarvik [43] found that three different species of arthropods: Myzus persicae (green peach aphids), Tetranychus urticae (two-spotted spider mites), and Acyrthosiphon pisum (pea aphids), all have the ability to synthesize carotenoids after horizontal transfer of carotenogenic genes from fungi. Also, it was hypothesized that these genes may be found in the genomes of other arthropods, and that the carotenoid biosynthetic gene could be one of them. Humans, on the other hand, are unable to manufacture carotenoids from scratch and must obtain them from their food, as carotenoids are required for a variety of biological activities. Only approximately 40 of them are found in a regular human diet, with 20 of them being located in human blood and tissues. Carotene, lycopene, lutein, and cryptoxanthin account for about 90% of the carotenoids present in the human body and food. Human absorption and digestion of dietary carotenoids is a complicated process that may vary based on an individual’s genetic makeup. Although the dynamics of carotenoids inside the human body remain mostly unknown, human plasma contains significant quantities of zeaxanthin, lycopene, lutein, beta-cryptoxanthin, beta-carotene, and alpha-carotene. Although carotenoids are mostly stored in adipose tissues and the liver, significant per gram concentrations of carotenoids accumulate in the adrenal gland, lungs, corpus luteum, and testes. Human plasma

110  Next-Generation Algae: Volume II and tissue contain significant amounts of the colorless carotenoids, phytoene and phytofluene.

4.3.2 Food Additives, Supplements and Feed Formulation The most common way that humans utilize astaxanthin is as a dietary supplement, yet, as of 2018, there has been no sufficient data from scientific studies to indicate that it changes the risk of illness or human health, and it is still under investigation. In 2018, the EFSA (European Food Safety Authority) requested scientific evidence on the safety of astaxanthin from dietary supplement producers. Astaxanthin is mostly utilized as a pigment in aquaculture and nutritional supplements in the food sector, as well as in nutraceuticals and medicines [47]. This carotenoid pigment is well recognized as an aquaculture feed additive responsible for impacting the pinkish-red color of the meat of salmon, ornamental fish, trout, shrimp, crayfish and lobsters, bringing about better quality and acceptability by customers. The aquaculture industry’s continuing expansion has resulted in a huge need for astaxanthin pigment. Apart from that, astaxanthin has anti-inflammatory and immune system boosting properties when used as a human dietary supplement [47]. Because of its large unsaturated molecular structures, astaxanthin is particularly sensitive to heat, strong light, and oxidative conditions. When astaxanthin is exposed to a variety of these circumstances during the preparation and storage of feed, it may lose its nutritional and biologically beneficial characteristics. Astaxanthin must thus stay stable when added to various feed compositions in order to achieve maximum effectiveness. Milling, mixing, extrusion, pelletizing, and drying are all steps in the feed manufacturing process. It has been hypothesized that milling has no effect on the stability of astaxanthin [21]. In fact, the ability to disintegrate or disturb microalgal cells by grinding seems to be the only significant factor in maximizing intracellular astaxanthin consumption. The machinery utilized, the residence duration, and the amount of heat produced all have a role in astaxanthin degradation during milling. Feed mixing is necessary to achieve consistent nutrient distribution, resulting in a uniform nutrient component in each fish pellet as a result of the formulation. Because homogenization may introduce air into the mixture, resulting in undesired carotene oxidation [48], using a vacuum mixer to curtail air exposure as a method of eliminating air entering the mixture is a smart way to proceed. In addition, secondary antioxidants have been shown to improve the oxidative stability in dietary carotenoids during feed production. Extrusion, on the other hand, is used in feed processing to increase starch (gelatinization)

Astaxanthin and Carotenoids Derived from Algae  111 and protein digestibility while reducing food component degradation [49]. It’s also a great way to make floating or sinking pellets by adjusting the recipe. Extrusion technique, on the other hand, includes significant amounts of mechanical shear, heat, pressure, and moisture, most all of which are likely to affect the stability of carotenoid pigments [50].

4.3.3 Alga as a Potential Source of Astaxanthin and Food Supplement The green single-celled freshwater alga, Haematococcus pluvialis, has been found to be an intense maker of astaxanthin and a primary hotspot for human utilization. For many years in the Asian nations, the green miniature algae have been utilized as a food origin or nutritive enhancement. These days, all the world consumes them for their health advantages. The green algae Haematococcus pluvialis, Chlorella vulgaris (Chlorophyceae), Cyanobacteria Spirulina (maxima) and Dunaliella salina are probably the most biotechnologically significant microalgae, and are generally utilized and popularized predominantly as human nourishment enhancements and as added substances in feed for animals [29]. There is a blue-green alga known as Spirulina platensis. This alga is acquiring overall acceptance as a supplement for food, being perhaps the most nourishing food known by man. Alga has been demonstrated to be an astounding origin of colors [51, 52], proteins nutrients and phenolics [53] and polyunsaturated fatty acids [54]. Currently, Spirulina has been noted to be significantly utilized for the extraction of the blue photosynthetic color, especially phycocyanin [55]. Dunaliella salina is another significant microalga under current development. This species was developed as an origin of beta-carotene and as the photosynthetic shade. Beta-carotene is utilized as a vitamin C enhancement and an orange color. Currently, the microalgal business is overwhelmed by Spirulina and Chlorella [56, 57], fundamentally on account of their ease of cultivation as well as their high nutritive worth and protein content. The energy source of these algae is showcased as pills, fluids and containers; and utilized for nutrition enhancement. A practical oil food, plenteous in antioxidants and unsaturated fats, pigmented with colors (carotenoids), separated with CO2 that is supercritical, was delivered from a microalga, Chlorella vulgaris, with its prospective utilization in the food business particularly for seafood derivatives [20]. Microalgal pigments have profitable uses as an unprocessed food coloring and beautifying constituent. Some microalgae contain noteworthy measures of carotene (other than beta-carotene). Various shades of color

112  Next-Generation Algae: Volume II are also seen in microalgae. Usually, beta-carotene is utilized as a pigmenting substance (specifically applied in giving the yellow pigment to margarine), as a food added substance to upgrade the shade of the tissue of fish and the yolk of eggs, and to work on the wellbeing and productiveness of cows fed with grains. The hidden capability of miniature algae as a wellspring of food coloring is restricted, though, on the grounds that algal derivative food coloring isn’t photostable and the color will generally blanch with cooking. Nonetheless, regardless of this restriction, the possible market for miniature alga derivative food coloring is tremendous [12]. For instance, Dunaliella salina produces significant beta-carotene, and it is cultivated to serve as an origin of photosynthetic color. β-Carotene, other than being one of the main food colorants as recently referenced, has solid antioxidant amplitude which assists in the intervention of the harmful impacts of free radicals ensnared in different ailments like many types of gastrointestinal malignant growth, joint inflammation, or untimely maturing brought about by bright radiation [58]. Furthermore, β-carotene may well upgrade resistance contrary to different nonresistant ailments. Nevertheless, β-carotene has been expressed to be capable of functioning as a pro-oxidant during the course of peroxidation of lipids, when both oxygen strain and carotenoid consolidation is high [59].

4.3.4 Technological Application of Algae as Origins of Supplements and Bioactive Mixtures in Healthier Food Varieties and Drinks The presence of different supplements and bioactive mixtures from algae in a food could be a source of natural functional enrichment; likewise, the mechanical fortuity offered by algae in the reformulation of food products. This explains algae’s effectiveness as normal constituents in reformulation procedures for quality drinks and food production, as well as functional food varieties [60]. The improvement of functional food sources offers tremendous opportunities in the food and drink industry. The utilization of algae as elements in quality food and drink inventions offers fascinating potential outcomes. Novel healthy foods should be visible as a chance to provide for the needs of consumers, and furthermore to refresh suggestions (concerning supplement and dietary objectives) [61]. Algae, a food variety of low-energy, consists of a varied assortment of compounds with helpful wholesome and mechanical properties. It also contains bioactive substances with possible diverse utilitarian properties when consolidated into the frameworks of drink and food sources.

Astaxanthin and Carotenoids Derived from Algae  113 Contrasted with the consideration of algae extracted mixtures, the utilization of the entire algae in drinks and food manufacturing offers a method for concurrently connecting various parts (dietary fiber, protein, minerals, nutrients, carotenoids, polyphenols, and so forth) in a way that doesn’t include the arduous, costly and ecologically unpleasant sanitization and extraction procedures that are required whenever these parts are utilized independently. An extensive assortment of algae has been utilized for numerous purposes in food (dairy, meat, pastry shop, fish, pasta and different items) and drinks preparations. The inclusion of these algae in food such as meat foods unlocks fascinating new possibilities for the utilization of algae in the plan of better muscle-based food products, as well as the chance of conquering mechanical issues related with low-salt foods [36]. In addition, there has been a significant amount of attention on water algae as useful food sources to decrease certain health risks, and the epidemiological evidence linking routine algae utilization to a lower risk of heart diseases. Although a few parts of algae like polyoses (alginates, carrageenates, and agar) have been utilized for innovative drives in the food business for quite some time, as of late, they have been utilized more as texturing and bulking mediators (because of their physicochemical properties), especially in the production of food sources with low calories. Algae is utilized in the definition of better meat items to beat mechanical issues (connected with water and fat restricting properties and surface) related with low-salt issue. Complete algae can be added to food sources to exploit their wholesome, functional and mechanical properties. As a sole constituent, the consolidation of entire algae inclines toward the synchronous existence of various constituents (protein, minerals, polyphenols, nutrients, dietary fiber, carotenoids, and so on), together with their advantageous health impacts as well as innovative benefits. Algae are appropriate regular specialists for conveying bioactive substances in an extensive assortment that conceivably possess multifarious useful characteristics in connection with the frameworks of food and drink sources [62]. Functional parts of algae have been utilized in the detailed arrangement of specific substances or as components of elements like vegetable concentrates, flours, food fibers, spices and flavors, probiotics, plant and marine oils, and so on. Likewise, they are useful in upgrading dietary benefit, providing medical advantages and conferring helpful innovative and tangible qualities in light of various issues related to reformulation processes [63]. The alga U. pinnatifid is being considered for further development for its water-restricting characteristics in burgers, thereby helping with issues connected with lower salt content [53]. Ocean spaghetti (Himanthalia elongata), an eatable earthy colored alga, has been utilized as a utilitarian fixer

114  Next-Generation Algae: Volume II in various meat items; 5% of H. elongata has been utilized in the process of redesigning low-fat or low-salt sausages with firmer and chewier textures and improved water- and fat-restricting characteristics, which very well may be utilized in other lower salt items in order to forestall quality imperfections because of a low salt fixation. The incorporation of red alga (Porphyra umbilicalis) in meat items raises the content of protein, likewise the strata of a few amino acids like glycine, alanine, tyrosine, arginine, valine, serine and phenylalanine. Laminaria japonica (1–5%) has been utilized as a replacer of fat in low-fat pork patties with practically no unfavorable effects observed in the nature of the meat by consumers. Integration of L. japonica improved textural and physical characteristics of meat patties, expanded the fiber of the diet, and diminished cooking failures [64]. Kappaphycus alvarezii is known to have constructive outcomes in enhancing minerals, prompting a decrease in the problems associated with cooking, and an expansion in patties’ taste [65]. Prabhasankar et al. [66] examined the impact of U. pinnatifida introduced at various levels (0–30%) on the tangible, cooking, nourishing and biofunctional nature of pasta, announcing that pasta consisting of about 20% algae had satisfactory tactile qualities and enhanced biofunctional characteristics. Pasta containing added algae brought about superior amino corrosive and unsaturated fat outlines, higher entire phenolic substance and cell reinforcement activity as well as developed fucoxanthin and fucosterol material.

4.3.5 Enriching Dairy Products with Algae Dairy products have similarly been enriched with algae. For instance, individuals lacking casein-debasing catalysts can’t reingest calcium from dairy products, hence foster some sort of hypocalcemic reaction. The incorporation of calcium-rich green algae could expand its focus in items from dairy. Algae have been consolidated into various sorts of cheese. Curds (simple and economically created) were produced with 3, 9 and 15% respectively from L. japonica and U. pinnatifida, the cheddar with 0.5% Chlorella was preferred (ideal tactile boundaries) to the 1% Chlorella, and enhanced the surface and color [67]. Additionally, Heo et al. [68] explored the impact of adding 0–2.0% of Chlorella powder on sensory characteristics, development of lactic acid bacteria and maturing speed of Appenzeller cheddar. Higher lactic acid bacteria were observed in the cheddar with added algae than in the control.

Astaxanthin and Carotenoids Derived from Algae  115

4.3.6 Algae as a Potential Healthy Protein and Fat Source Microalgae stood out as a capable sustainable substitute of protein source. Several proteins, amino acids and peptides from Algal have been reported to have beneficial effects to combat binding of mineral, programmed cell death, cell differentiation of cancer cells, metastasis, anti-human immunodeficiency virus activities, inhibition of human platelet accumulation and lipase activities reticence among others [60]. Microalgae is an important origin of ω-3 PUFAs. Dunaliella, Arthrospira, Haematococcus, Chlorella, Schizochytrium, Crypthecodinium cohnii and Porphyridium cruentum have GRAS status. Tetraselmis chuii also could serve as a seafood seasoning operator; as well as Odontella aurita, the diatom is now being eaten as additive in food rich in EPA. Some food derivatives of microalgae showcased DHA from C. cohnii, β-carotene from Dunaliella, and the blue colorant phycocyanin from Arthrospira among others [69]. For example, the cost of items such as phycocyanin, astaxanthin, and β-carotene ranges from hundreds to thousands of euros for every kg contingent upon their cleanliness; also, the top tier they draw in the marketplace caused industries to be extremely desirous of them. Then again, entire microalgae cells as food enhancements are available under 40 e for every kg [55]. Likewise, products derived from microalgae with biofunctional compounds that cost more than normal food enhancements can probably expand on their commercial possibility. The greater marketing costs would permit taking care of greater expenses resulting from new propagation and handling machineries. Other than supplying supplements and energy for organism upkeep, development, and active work, food varieties can be a vehicle for conveying bioactive mixtures with health advantages. Among the various sorts of compounds derived from microalgae, the ones having antioxidant abilities are likely the most intriguing ones for modern applications. Aside from being a decent origin of proteins, Arthrospira, Chlorella and Nannochloropsis have been accounted for as significant wellsprings of oligosaccharides or polysaccharides, as they are projected as potential prebiotic competitors. Other derived compounds from microalgae and the entire cells have been utilized as food constituents with various intents. Khemiri et al. [70] and Niccolai et al. [71] noticed constructive outcomes on the techno-­utilitarian as well as the antioxidant characteristics of food emulsions following addition of certain microalgae species. Gels continue to be proposed as a means to give significant compounds derived from microalgae. Batista et al. [72] added a few animal types of microalgae into

116  Next-Generation Algae: Volume II gels to work on their construction as well as a method for giving cell reinforcements and certain ω-3 PUFAs to possible customers. Comparable investigations introducing other species of microalgae were accounted. Chlorella has been effectively introduced into cheeses and yogurts [73]. Cookies and biscuits have been found to be applicable classifications to convey constituents derived from microalgae such as astaxanthin. Motives involve great acknowledgment of taste, flexibility, advantageous ingesting because of their simplicity of protection and conveyance, outward form and surface. Though addition of Chlorella vulgaris in cookies has proven to be an operator for pigmentation and possibly as an antioxidant and food enhancement, incorporation of Isochrysis galbana furnished cookies with ω-3 PUFAs valuable for human health. Likewise, addition of phycocyanin concentrates and entire A. platensis to formulate cookies provided cookies with possible health advantages, improved protein and fiber constituents [53, 72]. Batista et al. [72] reported improved nourishing and healthy support capability of cookies, thus expanding protein and antioxidants elements, by introducing Tetraselmis suecica, A. platensis, Phaeodactylum tricornutum and C. vulgaris. Addition of Haematococcus pluvialis into cookies also brought down the glycemic reaction and expanded the limit in antioxidant levels. Another product similar to cookies is bread, which is likewise generally eaten. Some years back, a few researchers declared microalgae from astaxanthin integration into bread in order to improve its nourishing characteristics. In previous years, prior to being added to white bread produced from wheat, Dunaliella was recommended as a protein supplement in bread. In order to build its protein content, Arthrospira and a bleached extract acquired from this species were additionally integrated in bread. Likewise, other species of microalgae were utilized in bread. Ongoing accounts similarly referenced the incorporation of microalgae in bread without gluten; incorporation of Arthrospira altogether expanded protein constituent and further developed quality of bread because of the existence of a few fundamental amino acids contrasted with bread which is non-­enhanced. Comparable advantages were seen when Arthrospira was utilized as constituents in extruded appetizers [74, 75]. Another product that astaxanthin is generally acknowledged for and incorporated into is pasta. Addition of both Arthrospira maxima and C. vulgaris improved the healthful constituents of new spaghetti, with an all-around acceptability of the product by the panel set up for the sensory evaluation. Pasta likewise provided a means to convey ω-3 PUFAs and antioxidants with latent health advantages. In spite of the fact that

Astaxanthin and Carotenoids Derived from Algae  117 Arthrospira expanded the protein ingredients, protein edibility diminished as the microalgae constituent expanded [76, 77]. The techno-useful properties of astaxanthin regulate the appropriateness of added substances in food items. For example, properties like frothing, gelation, emulsifying, fat, and water retention limits are accounted for in some hydrolysates and proteins of microalgae. Spirulina platensis showed antiviral activity against different infections, such as human cytomegalovirus, herpes simplex and measles, by obstructing their entrance because of the existence of sulfur-containing polysaccharide. Microalgae obviously express a likely capability of addressing the requirements for extra sustainable nutrition arrangements for the populace. The extravagance of composites in microalgae can foster a food industry that is algal based, and concerned with delivering and using microalgae for creative utilitarian food items. Other than the constituent of protein and adjusted profiles of the amino acids, consolidation of microalgae into food varieties could prompt possible advantages for human health because of the bioactive mixtures that are present in some species of microalgae. The use of items produced from microalgae-inferred or microalgae as substitutes for food isn’t inexpensive yet, essentially because of the short TRL as well as absence of the shrinking economy for the propagation of microalgae and its handling [78].

4.4 Future Perspective The utilization of bioactive compounds as food components has become necessary due to rising health concerns and lifestyle changes. Carotenoids have a bright future in this regard. Cancers, high blood pressure, diabetes, cardiovascular, digestive, hepatic, neurological, and skin disorders have showed positive signs when  astaxanthin is introduced. Its antioxidant capabilities are utilized to protect sick cells from oxidative damage. Astaxanthin’s high antioxidant effects and biological stability have been demonstrated in several studies and have applications in numerous fields. Astaxanthin is a nutrient that can penetrate cell membranes and has positive impacts on cells. It is utilized as a targeted medicine to scavenge free radicals at specific places via a carrier; as a powerful antioxidant to shield cells from oxidation damage, and so on. Astaxanthin has prospective application value in treating diabetes which is based on these benefits. As a result, the utilization of carotenoids as a nutraceutical, pharmaceutical agent, and nutritional supplement in the worldwide market might be a positive move for humanity. Though acceptability and restriction are challenges that

118  Next-Generation Algae: Volume II must be overcome; when these hurdles are cleared, integrating astaxanthin and carotenoid from microalgae as components of food won’t just give therapeutic advantages, but will likewise add to further developing issues connected with supportability, considering the developing populace and our present eating regimen, propensities, and wellbeing.

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5 Production of Polyunsaturated Fatty Acids (PUFAs) and Their Biomedical Application Olorunsola Adeyomoye1*, Olugbemi T. Olaniyan2 and Charles O. Adetunji3 Department of Physiology, University of Medical Sciences, Ondo City, Nigeria 2 Laboratory for Reproductive Biology and Developmental Programming, Department of Physiology, Rhema University, Aba, Nigeria 3 Applied Microbiology, Biotechnology and Nanotechnology Laboratory, Department of Microbiology, Edo State University Uzairue, Iyamho, Nigeria 1

Abstract

Polyunsaturated fatty acids (PUFAs) are a unique class of food constituents that serve a variety of biological functions. Because of a growing awareness of their potential to improve diet and human health, these compounds have received worldwide attention in recent years. Microbial cell biotechnologies are an important solution for supplying these biomolecules in sufficient quantities, as well as supplying PUFA-rich aquafeed, superfoods, and medical formulations. Their therapeutic and pro-health effects have already been demonstrated in a variety of diseases, including cancer, neurological disorders, cardiovascular disease, diabetes, obesity, and others. The various classes of PUFAs are discussed in this chapter, as well as their production and therapeutic potential in natural food sources and through genetically engineered microalgae cultivation. Keywords:  Polyunsaturated fatty acids, microbial cell biotechnologies, human health, food sources, cancer

5.1 Introduction Polyunsaturated fatty acids (PUFAs) made from hydrocarbon chains are known to contain two or more bonds. They are of benefit to human health *Corresponding author: [email protected]; [email protected] Charles Oluwaseun Adetunji, Julius Kola Oloke, Naveen Dwivedi, Sabeela Beevi Ummalyma, Shubha Dwivedi, Daniel Ingo Hefft and Juliana Bunmi Adetunji (eds.) Next-Generation Algae: Volume II: Applications in Medicine and the Pharmaceutical Industry, (125–138) © 2023 Scrivener Publishing LLC

125

126  Next-Generation Algae: Volume II and are also present in plants. They are also concentrated in microbes and a large collection of PUFAs are made through microbial engineering [1]. Polyunsaturated fatty acids are classified in line with their chemical composition which include conjugated fatty acids, methylene-interrupted polyenes, and other PUFAs. They could as well be divided into two groups according to their length of carbon backbone. These include short-chain with 18 carbon atoms and long-chain with 20 or more carbon atoms. Arachidonic and docosahexaenoic acids are precursors to dietary polyunsaturated fatty acids, linoleic acid, and linolenic acid [2]. Polyunsaturated fatty acids have been linked to enhanced neurological and cognitive development [3]. PUFAs have been shown to reduce blood triglyceride levels through increase in oxidation of fatty acids by PPAR activation or by reducing SREBP-1 activity, which inhibits lipogenesis [4]. Dietary PUFAs cause lipid oxidation and decrease insulin resistance through the activation of PPAR-α, which also results in reduction in hepatic steatosis [5]. Evidence from human and animal studies has shown that PUFAs play a role in prevention and management of diabetes, obesity, cancer, and heart diseases [6]. Corn, safflower oils, flaxseed, fish and soy are sources of PUFAs.

5.2 Polyunsaturated Fatty Acids At least two bonds are present in the backbone of polyunsaturated fatty acids. They also have essential substances which are responsible for properties of the oils. Ingredients such as essential acids and those that are responsible for the different properties of dried oils are contained in them [7]. The type of PUFA is determined largely by the shape of the first double bond in relation to the methyl end of the fatty acid. Double bonds are usually of cis configuration. The trans fatty acids consist of approximately 2–5% of fatty acids present in soluble milk and meat, and 5–20% of fatty acids present in hydrogenated fats [8]. PUFAs possess amphipathic properties, which means they possess a head (hydrophobic) and tail (hydrophilic). Hexadecatrienoic acid, alpha-linolenic acid, stearidonic acid, tetracosapentaenoic acid, and heneicosapentaeno acid are some examples of omega-3-series PUFAs. Omega-6 series includes eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, adrenic acid, tetracosapentaenoic acid, and docosapentaenoic acid. Mead acid, erucic acid, oleic acid, eicosenoic acid, and nervonic acids are all part of the omega-9 series. Rumenic acid, calendic acid, parinaric acid, and others are conjugated fatty acids. PUFAs are precursors of body metabolites. Arachidonic acid, for example, is a precursor to prostanoids series 2 and leukotrienes

Production of PUFAs and Their Biomedical Application  127 series 4. In addition, the metabolism of eicosapentaenoic acid and docosahexaenoic acid produces a series of prostanoids 3 and leukotrienes series 5 [9]. Many of these compounds possess anti-inflammatory, antiplatelet, and antiarrhythmic properties, according to research [10]. Other functions include maintaining cell membrane fluidity, reducing macrophage release of proinflammatory cytokines, enhancing vascular endothelial cell functions, and reducing triglyceride synthesis in the liver [11].

5.3 Production of Polyunsaturated Fatty Acids Polyunsaturated fatty acids from fish oil, particularly arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, are valuable dietary supplements with numerous health benefits. PUFAs production from fish oil had decreased in the last decade due to overfishing and ocean pollution. Some microalgae species have also been identified as ideal producers of LC-PUFAs because they naturally accumulate large amounts of LC-PUFAs by using CO2 and light energy [12]. A growing set of genetic tools is also available for the creation of high-performance strains of micro­algae, with increased efficiency in the production of fatty acids and their derivatives. Triacylglycerols accumulate in microalgae which are mainly composed of monounsaturated fatty acids and saturated fats [13]. The red microalga of Porphyridium cruentum TAGs shows high arachidonic acid and eicosapentaenoic acid content. This algae variability was impaired at lower temperatures with lower levels of eicosapentaenoic acid and monogalactosyldiacylglycerol. In contrast, Green TAGs can grow at low temperatures, with lots of arachidonic acids. An arachidonic acid was stored at 25 °C for 12 hours. This labeled arachidonic acid was transferred from TAGs to polar lipids with increased yield at low temperatures. The use of microalgal in producing polyunsaturated fats has continued to yield promising results. Phaeodactylum tricornutum, a genetically modified micro­algae, have been shown to accumulate PUFAs with arachidonic acid and a docosahexaenoic acid content of 18.98 g/mg and 9.15 g/mg, respectively [14]. Eicosapentaenoic acid levels reached a maximum of 85.35 g/mg. Arachidonic acid and eicosapentaenoic acid can both be formed from triacylglycerides, while docosahexaenoic acid is made only in phospholipids. The combination or expression of many enzymes has the potential to produce stable PUFA [15]. PUFA is also found in vegetable seeds, fish oil, and filamentous fungi. Mortierella alpina, a filamentous fungus, is capable of producing triacylglycerols that are rich in arachidonic acids, with concentrations of up to 20 g/l [16]. For the exploitation of genes of M cells alpina

128  Next-Generation Algae: Volume II strains has shown great promise in PUFA biosynthesis and specification of the activities of their enzymes [16].

5.4 Nanomedicine-Based Formulations Containing Polyunsaturated Fatty Acids Omega-3 polyunsaturated fatty acids are a dietary component and have been linked to the prevention of heart disease, inflammation, and cancer [17]. A multidisciplinary approach has been developed to produce omega 3-containing nanoformulations with the aim of protecting these fatty acids from degradation, thereby increasing their availability, bioactivity and transport to tissues [17]. These nanoformulations were created to provide omega-3-PUFA in combination with other medications. Omega 3-PUFAs are obtained from diet due to the inability of the body to synthesize them. The increased oxidative stability of ω-3-PUFAs, along with their decreased bioavailability and bioactivity in targeted tissues, is a barrier to determining their therapeutic potential. ω-3-PUFA-rich-in-water nanoemulsion formulation was used to enhance the delivery of 17-­β-­estradiol and C6 ceramide payload to adult blood cells [18]. ω-3-PUFA-rich nanoemulsion, as well as CER given nanoemulsion have been shown to inhibit the growth factor that stimulates cell proliferation with increase in pro-­apoptotic caspase 3/7 activity. The mitogen-induced protein kinase signal reduced VSMC proliferation while improving endothelial cell increase in the loaded 17-β-estradiol nanoemulsion. The antiproliferation effect of 17-β-estradiol and C6 ceramide laden nanoemulsions was more pronounced in VSMCs than in the endothelial cells. These biological applications of ω-3-PUFA, C6 ceramide, and 17-β-estradiol experiments of nanoemulsion are acceptable for the approach developed in animal models of occlusive vasculopathies. During experiment on atherosclerosis, the impact of novel ω-3-fatty-rich, 17-β-estradiol, CREKA-peptide-modified nanoemulsion system were tested [19]. The 17-β-estradiol nanoemulsion technology in combination with CREKA-peptide-modified nanoemulsion was shown to enhance cellular nitrate/nitrite in endothelial cells, which indicates an improved production of nitric oxide. The systematic administration of this nanoemulsion in ApoE-/-mice feeding on a high-fat diet developed problems related to occlusive atherosclerotic vasculopathy. The 17-β-estradiol and ω-3-­ polyunsaturated fatty acid constituents of a nano delivery system have been found to be biocompatible, and act as a measurement of body weight, plasma levels of alanine aminotransferase/aspartate aminotransferase, and

Production of PUFAs and Their Biomedical Application  129 assessing the histology of the liver and kidneys, at therapeutic doses. The distribution profile and pharmacokinetic of ­17β-estradiol in wild-type C57BL/6 mice, and system delivery using cationic or CREKA-peptidemodified omega-3-fatty acid oil containing a nanoemulsion system was investigated [20]. When administered, 17-β-estradiol was shown to accumulate in the heart, aorta, liver, and kidneys. In the wild version of C57BL/6 mice, the CREKA-peptide-modified nanoemulsion system was found to be the best 17-β-estradiol system management vehicle.

5.5 Biological and Medical Application of Polyunsaturated Fatty Acids Polyunsaturated fatty acids form various components of fats because of their unique properties and function in the diet. Their therapeutic potential has been demonstrated in many inflammatory and autoimmune disorders which include changes in lipid production, gene expression, and signal transduction [21]. Aurantiochytrium sp. strain was used to remove dissolved organic carbon and nitrogen in boiling bean sprouts and miso processing during which PUFA was produced. This strain removed 52% of dissolved organic carbon and 37% of dissolved nitrogen in boiling bean contaminated water, thereby producing a biomass that contains 137 mg/g of fatty acids, which also include 96.2 mg/g of docosahexaenoic acid [22]. The growth of this strain of microalgae in stagnant polluted water also caused increase in biomass that contains 147.6 mg/g of fatty acids, which include docosahexaenoic acids, as well as 47% removal of dissolved carbon and 55% removal of dissolved nitrogen. These results from numerous findings showed that microalgae are capable of producing polyunsaturated fatty acids when used in removing organic substances from polluted wastewater. The applications of these polyunsaturated fatty acids have widely been reported. Omega-3 fatty acids have been reported to influence dopaminergic and serotonergic pathways in the central nervous system. Omega-3 fatty acids are produced and used as treatment for many neurological disorders [23]. The application of omega-3 fatty acids in the management of schizophrenia, psychiatric disorder, anxiety disorder, major depressive disorder, attention deficit hyperactivity disorder, psychiatric disorders, obsessive-compulsive disorder, post-traumatic stress disorder, autism spectrum disorders, and eating disorders was shown by data generated on the efficacy and tolerance of omega-3 fatty acids in psychiatric disorders published between 1980 and 2019 [23]. Omega-3-fatty acids

130  Next-Generation Algae: Volume II have shown great effectiveness in the treatment of depressive symptoms in patients with severe bipolar depression. During early stages of schizophrenia, in addition to antipsychotic treatment, some success was recorded, but not in the chronic stages of psychosis following fatty acid administration. Minimal beneficial effects of omega-3 fatty acids in attention deficit hyperactivity disorder were seen, and positive effects on major BPD symptoms were reported in a few trials. The anticancer properties of linolenic acid and dihomo-linolenic acid have been investigated, including their alteration of cell apoptotic changes and inhibition of cell proliferation [24]. Furthermore, human studies have revealed a relationship between high fish oil consumption and a lower risk of colon, prostate, and breast cancer, even though some other studies have not found a significant link. Despite this, the available epidemiological evidence, when combined with the effects caused by fatty acids on animal cancer and cell culture models, has encouraged the production of clinical therapeutic interventions that incorporate n-3 PUFAs in cancer prevention and treatment, in addition to nutritional support. Polyunsaturated fatty acids have resulted in loss of weight and balance of immune system in the cancer patient. The biotransformation of omega-3 and omega-6 PUFA ​​ produces a series of lipid metabolites that are capable of bringing about cellular processes. The lipid metabolites present in omega-6 PUFA have been linked to a variety of biological properties, which include the development of a variety of clinically approved medications, including latanoprost, which is an essential medication [25]. In addition, evidence suggests that omega-3 PUFA may simply act as competing substrates for enzyme biotransformation and reducing the production of omega-6 PUFA-derived lipid metabolites [26]. The effects of different dietary concentrations of omega-3 and omega-6 PUFAs on brown adipose tissue in type 2-induced diabetic mice was investigated. At lower concentrations, these fatty acids significantly increased the mass of brown adipose tissue by 67.55% and 60.49% respectively. In addition, they decreased total cholesterol, low-density lipoprotein, fasting blood sugar, and triglyceride, and increased sugar tolerance [27]. The assessment of qRT-PCR showed that low omega-6 and omega-3 PUFA activated brown adipose tissue with increased expression of Ucp1, PPAR-α, cAMP, GLU1, HSL, LPL, lipid and glucose metabolism in diabetic mice. Many other applications of PUFAs, particularly omega-3 and omega-6 ​​PUFAs, have been reported in the literature. The aforementioned therapeutic potentials of PUFA were based on research conducted on animal and human experimental models. Therefore, incorporating them in the diet may have health benefits and help prevent the development of many diseases.

Production of PUFAs and Their Biomedical Application  131

5.6 Metabolism of Polyunsaturated Fatty Acid Polyunsaturated fatty acid metabolism was analyzed using isolated cells from cardiomyocytes, lymphocytes, fibroblast as well as hepatocyte of mice [28]. Dietary and hormonal factors were shown to affect the activation of fatty acids, which determines whether the fatty acids have been cleared, chain-longated, directly esterified, or oxidized [29]. In both mice and humans, short-term and long-term regulation of dietary fats have been investigated. There are important differences in fatty acid esterification, oxidation, and fatty acid formation in lipoproteins. This discrepancy is said to be due to at least changes in protein concentrations that bind intracellular fatty acids in the liver [30]. The modification of the peroxisomal retroconversion of unsaturated fatty acids to its homologues is said to play a role in regulating fatty acid formation in phospholipids bilayer membrane. Many patients with various disorders of lipids do not have peroxisomes or are deficient in peroxisomes. The beta-oxidation of very-long-chain acids is affected in a few peroxisomal disorders. The strength of fibroblasts in people with Zellweger disease, peroxisomal disorders, X-linked adrenoleukodystrophy and adrenoleukodystrophy to degrade fatty acids varies. In the brain, docosahexaenoic acid is rich in omega-3 fatty acids which regulates very important functions and acts as precursor of bioactive metabolites [31]. The presence of docosahexaenoic acid in the brain is regulated by a number of processes that leads to its utilization and metabolism. Docosahexaenoic acid in the brain is absorbed and re-digested in the membrane phospholipids, leading to the distribution in the brain. During neurotransmission, docosahexaenoic acid is released from membrane phospholipids and converted to bioactive metabolites that controls signaling pathways that are important for synaptogenesis, neuroinflammation, and cell survival, and also help in the treatment of neurological disorders. In the liver of mice, metabolism of double bonds delta-3 fatty acids was investigated [32]. Subcellular fractionation tests showed the presence of peroxisomal and mitochondrial delta 3, the activity of delta-2-enoyl-CoA isomerase following the introduction of delta-3-trans-dodecenoic acid into the liver. These observation in line with previous finding of peroxisomal 2, 4-dienoyl-CoA reductase, suggest that peroxisomes contain all necessary beneficial enzymes needed for beta-oxidation of unsaturated fatty acids. Peroxisomes can oxidize a variety of substrates, which include (poly) estoy-free unsaturated estoy-CoA esters. Furthermore, in peroxisomes and mitochondria, beta-oxidation of unfilled enoyl-CoA esters involves chain shrinkage, and also the use of metabolizable double carbon-to-carbon

132  Next-Generation Algae: Volume II bonds [33]. Auxiliary enzymes such as delta 3, delta 2-enoyl-CoA isomerase, 2,4-dienoyl-CoA reductase, 2-enoyl-CoA hydratase 2 or 3-hydroxyacyl-CoA epimerase, and delta 3,5 delta 2,4-dienoyl CoA isomerase are necessary for fatty acid metabolism in addition to beta-oxidation spiral enzymes. There are different isoforms of these enzymes and they can be found in various subcellular compartments that include mitochondria, peroxisomes, and endoplasmic reticulum [34]. Subcellular distribution of delta 3,5, delta 2,4-dienoylCoA isomerase was demonstrated in rat liver peroxisomes. The peroxisomal and mitochondrial isomerase was shown to have the same specificity and are homologous, as evidenced by the antisera produced against the peroxisomal enzyme [35]. Peroxisomes contain all the necessary enzymes required for beta oxidation of fatty acids with unique numbers of bonds and NADPH-dependent 2,4-dienoyl-CoA reductase which removes the double bonds by reduction process [49].

5.7 Challenges and Issues of Production and Use of Polyunsaturated Fatty Acids There has been remarkable progress over the past few decades in the production and use of polyunsaturated fatty acids. However, new evidence has emerged stating that there are challenges concerning the bioavailability and use of PUFA in aquatic environments and humans [36]. The content of PUFA in microalgae has become an important measure of the quality of algae nutrients for consumers. Cryptophyceae and Dinophyceae have been shown to have increased levels of eicosapentaenoic acid and docosahexaenoic acid, making them ideal as food for zooplankton [37]. In contrast, Chlorophyceae, a green algae and cyanobacteria, did not contain long-chain PUFAs but high levels of ALA, a short-chained fatty acid with low nutritional value [38]. This implies that not all algae serve as a source of nutrients for aquatic organisms, so some of them are not a source of high-quality food. Eicosapentaenoic acid and docosahexaenoic acid play an important biochemical and physiological role in humans and many animals. All vertebrates and most invertebrates require these long-chain PUFAs. Most animals do not have the capacity to combine eicosapentaenoic acid and docosahexaenoic acid in the short chain, so they have to get these long-chain PUFAs into their diet. In addition, people who do not eat fish cannot have their main source of eicosapentaenoic acid and docosahexaenoic acid as fish. Other long-chain PUFA dietary sources that provide eicosapentaenoic acid and docosahexaenoic acid with a limited or

Production of PUFAs and Their Biomedical Application  133 near value should be available to such individuals [39]. Polyunsaturated fatty acids have been shown to be more prone to oxidation during heating. Therefore, in the literature, the breakdown of long-chain PUFAs in the diet has been reported during cooking as well as other therapeutic modalities.

5.8 Conclusion Polyunsaturated fatty acids are commonly found in a variety of dietary sources with significant health benefits. Microalgae cultivation has continued to accumulate these compounds in many areas. In addition, these polyunsaturated fatty acids have been used as part of the formulation for the management of several diseases. Recent advances in the use of biotechnologies in the cultivation of microalgae can improve the efficiency of production and utilization of these compounds [40–48].

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6 Utilization of Algae and Their Anti-Proliferative and Anti-Inflammatory Activities Olorunsola Adeyomoye1*, Olugbemi T. Olaniyan2 and Charles O. Adetunji3 Department of Physiology, University of Medical Sciences, Ondo City, Nigeria 2 Laboratory for Reproductive Biology and Developmental Programming, Department of Physiology, Rhema University, Aba, Nigeria 3 Applied Microbiology, Biotechnology and Nanotechnology Laboratory, Department of Microbiology, Edo State University Uzairue, Iyamho, Nigeria

1

Abstract

Algae  are mostly aquatic, photosynthetic, and nucleus-bearing organisms without the true roots, stems, leaves, and specialized multicellular reproductive systems found in  plants. Many contain chemical constituents  used in cosmetics, pharmaceuticals, the food industry, manure, and different food supplements. Their metabolites have been utilized to treat a variety of cancers due to their cytotoxic, antiproliferative, and apoptotic properties. They have been shown to possess anti-inflammatory phytochemicals that can be employed as treatments or in the development of structural analogs with strong anti-inflammatory properties. Algae also have additional uses, such as the development of algae-based nanocomposites for heavy metal removal and the formation of biopolymers for diverse materials. This chapter introduces algae, its properties, and the possible health benefits of algae biocomposites. Keywords:  Algae, nanocomposite, antiproliferative, anti-inflammatory, biopolymers

*Corresponding author: [email protected]; [email protected] Charles Oluwaseun Adetunji, Julius Kola Oloke, Naveen Dwivedi, Sabeela Beevi Ummalyma, Shubha Dwivedi, Daniel Ingo Hefft and Juliana Bunmi Adetunji (eds.) Next-Generation Algae: Volume II: Applications in Medicine and the Pharmaceutical Industry, (139–152) © 2023 Scrivener Publishing LLC

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6.1 Introduction Algae are groups of photosynthetic organisms that range from unicellular microalgae, such as Chlorella, Prototheca and Diatoms, to multicellular forms such as giant kelp. They are found in aquatic environment and can synthesize organic compounds such as carbohydrates, fats and proteins. Many of them are devoid of stem, xylem and phloem seen in land plants. Seaweeds are marine algae, such as Rhodophyta, Phaeophyta and Chlorophyta macroalgae, which are multicellular in nature. Algae are also found in freshwater, examples of which include Spirogyra and stonewort. Other algae groups include Zygnematales, euglenoids, cryptomonads, chrysophytes, dinoflagellates, and cyanobacteria [1]. Many of them have the capacity to live in extreme weather conditions and acidic environments. They are primary producers with different colors and are members of other eukaryotes such as the viruses, bacteria and fungi. They are present in snow environment and help in melting of snow packs and for the surveillance of climate change [2]. Algae have been shown to increase the production of secondary metabolites such as astaxanthin and purpurogallin, which help protect chloroplast and nucleus from the cellular damage from ultraviolet radiation. Algae have several applications in the food industry due to their chemical composition [3]. Algae can act as food supplements and can be added to meat product such as sausages, pasties and frankfurters. It is also added to cereal products to improve their quality. Fermented dairy products such as milk, yogurt, cheese and cream have all been reported to contain algae [4]. Evidence has shown that cosmetic products produced from algae may act as an anti-aging agent used for skin whitening and reducing pigmentation [5]. Some algae have also been used in formulating cosmetic products that are used for skin thickening and moisturizing. Marine algae, such as the seaweeds, have been reported to be useful in producing novel antidiabetic agents due to their bioactive composition [6]. The brown and green algae have been shown to exhibit antidiabetic potentials by regulating blood glucose level through inhibition of carbohydrate hydrolyzing enzymes, insulin sensitivity and glucose uptake. Bioremediation and biodegradation of wastewater contaminants have been studied extensively using microalgae with resultant increase in biofuels production [4]. In addition to the aforementioned applications, algae biofuels are being considered as third generation biofuels. Several studies have investigated the use of macro- and microalgae in producing biofuels for economic sustainability [7]. Some of these biofuels include biosyngas, bio-oil, biodiesel, and biohydrogen.

Utilization of Algae  141

6.2 Physiology and Biochemistry of Algae Algae have the ability to produce toxins that can harm humans and animals, but they also provide a lot of benefits. They support fisheries and other aquatic habitats and are a source of marine food webs. They are photosynthetic, meaning they take in CO2 and produce oxygen. Eukaryotic algae can be found in either a haploid or diploid form. The life cycle of most multicellular eukaryotic algae includes a sexual component, sometimes with two or even three phases [8]. Cell division and cell separation are required for asexual reproduction in unicellular algae. Asexual or sexual unicellular phases, as well as fragmentation, are used in the reproduction of colonial and multicellular algae [9]. Many eukaryotic microalgae (for example, many diatoms and green algae) are known to reproduce sexually. Blue-green algae have a high amount of metabolic complexity despite their simplicity. Their photosynthetic system, for example, is similar to that of higher plants. Although the mechanism of these algae’s respiration is unknown, many studies have described the effect of light on this process in vivo [10, 11]. Light reduces oxygen intake at low levels but stimulates it at high intensities. Chlorophyll enhances this effect, while photosystem II inhibitor 3,4-dichlorophenyldimethyl-urea inhibits the stimulation [12]. Algae develop a variety of metabolites to assist them in dealing with the harsh conditions of the sea. These compounds have recently attracted a lot of interest for the identification of medicinally beneficial drugs, especially those with potential anticancer properties, due to their structural diversity and distinctiveness [12]. Included among the beneficial activities of algae are their selectivity in inhibiting cancer cell proliferation over normal cells, ability to kill cancer cells via non-apoptotic signaling pathways, ability to bypass MDR-related efflux pumps, and efficacy in vivo in relevant pre-­ clinical studies [13].

6.3 Algae Biocomposites Biocomposites are materials composed of at least two distinct constituent materials, both natural and synthetic, that are combined to produce a new material with superior performance to the individual constituent materials. Because of the environmental crisis and the issue of sustainability, there has been an increase in interest in using natural resources to reinforce other materials in recent years [14]. Algae is one of these natural resources that can be used to strengthen diverse materials [15]. A red algae (Gelidium

142  Next-Generation Algae: Volume II Elegance) fiber was investigated as a biocomposite reinforcement. The extracting and bleaching of red algae fiber was effective for both removing mucilage materials and fiberization of red algae fiber. With a maximum thermal decomposition temperature of 359.3 °C, bleached red algae fiber (BRAF) exhibited very similar crystallinity to cellulose as well as higher thermal stability [16]. Poly(butylene succinate) (PBS) biocomposites reinforced with BRAF were fabricated using a compression molding method with varying BRAF contents, and their mechanical and thermal properties were investigated. With increasing BRAF content, the storage modulus of PBS matrix and thermomechanical stability improve noticeably, with a maximum storage modulus and the lowest coefficient of thermal expansion value at 50 wt% fiber loading. This suggests that red algae fiber can be used effectively as a reinforcement for materials, contributing to the development of environmentally friendly biocomposites. In a study to improve the oxidative stability of docosahexaenoic acid (DHA) algae oil, various antioxidants such as ascorbyl palmitate (AP), vitamin E (VE), phytic acid (PA), and polyphenols (PH), were combined [17]. The oxidative stability of DHA algae oil containing 80 mg/kg AP, 80 mg/kg VE, 40 mg/kg PA, and 80 mg/kg PH was significantly improved. In addition, DHA algae oil containing the optimal composite antioxidant had a longer shelf life than the control sample. Chromium oxidation states are considered biologically and environmentally relevant based on their stability in the presence of water and oxygen. Chromium-containing compounds are mutagenic and carcinogenic when inhaled and may be carcinogenic if consumed in large quantities orally [18]. The high reductive Fe content and specific surface area of algae-based Fe/C nanocomposite have been shown to have a high capacity for hexavalent chromium (Cr(VI)) removal [19, 20]. Because of the stabilizing property of EPS in algae, the use of extracellular polymeric substances (EPS) has been shown to improve the product composition and efficiency of Cr(VI) removal. As a result, Cr(VI) was efficiently removed by an algae-based Fe/C nanocomposite, indicating its potential application in environmental remediation. Evidence has shown that the development of synthetic nanoparticles has several hazardous implications. In order to produce a newer environmentally friendly nanoparticles, the use of green methods is inevitable. Several novel approaches for nanoparticle synthesis have been explored using natural resources such as the blue-green algae [21]. Many of the bionanocomposites produced have several applications in medicine, catalysis, sensors and bioremediation. Algae with hydroxyl, amino, and peptide functional groups have also been employed to boost the adsorption capability of a layered double hydroxyl material. The Cr(VI) removal effectiveness of these algae-prepared composites was improved,

Utilization of Algae  143 as well as the stability and reusability over multiple cycles. The creation of a diatom-FeOx composite has been demonstrated to be a new sorbent for the removal of arsenic from water [22]. The primary function of diatom was to immobilize iron-oxide on the composite in order to achieve successful bioremediation.

6.4 Techniques and Methods Involved in the Production of Algae Biocomposites Natural fiber composites have continued to be in high demand. Natural fiber composites made from highly marketed fibers like flax, hemp, and sisal have grown to represent a significant part of polymeric composites. However, there has been minimal focus on expanding natural fiber composites to accommodate new sources of emerging natural reinforcements, such as recycled algal fibers, with a number of environmental advantages [23]. Where traditional mineral and synthetic-based materials can be substituted with green composite materials, there are tremendous prospects. It’s crucial to understand green composites’ production procedures and optimize process parameters before they can be employed to make a variety of products. Injection molding, extrusion, thermoforming, and compression molding are some of the most prevalent technologies. Cooling is the most time-consuming part of the injection molding process which can be improved on by using conformal cooling systems or copper molds with high thermal conductivity [24]. Conformal cooling channels are often aligned with the injection-molded product’s geometry, they tend to extract more heat and remove it more uniformly than traditional cooling systems. Cooling channels are created by drilling in copper mold inserts, and heat removal is aided by copper’s high thermal conductivity coefficient, which is many times that of steel. Designing optimal cooling systems is a difficult task; proper design necessitates injection molding simulations, but the accuracy is determined by the precision of the input parameters and boundary conditions. Extrusion is a process in which a substance is forced to flow through an aperture or die, resulting in plastic deformation. The material takes on the die’s cross-sectional profile, and if the material has the right characteristics, that shape is preserved in the final extrudate [25]. Thermoforming procedure starts with an extruded sheet of plastic. The procedure entails heating the plastic sheet to a soft or pliable temperature range. The sheet is then stretched against a single-sided mold that is kept cool. Compression molding is a technique in which the preheated molding

144  Next-Generation Algae: Volume II material is first placed in an open, heated mold cavity. The mold is closed with a top force or plug member, and pressure is applied to force the material into contact with all mold surfaces, while maintaining heat and pressure until the molding material cures [26]. Some of the other methods that could be employed include melt mixing, solvent casting and hot molding.

6.5 Antiproliferative Activities of Algae Evidence suggests that marine algae have cytotoxic, antiproliferative, and apoptotic effects, which help to manage cancer pathologies. Petalonia fascia, Jania longifurca, and Halimeda tuna culture extracts were prepared to test their effects on the mouse neuroblastoma cell line, NA2B [27, 28]. The survival and proliferation of NA2B cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after treatment with algae extracts. The neurotoxicity-screening test (NST) showed significant neurite inhibition with moderate damage at IC50 dilutions of the extracts, implying that the algae tested may have potential for use in cancer treatment. The effects of three macroalgae (Ulva reticulata, Sargassum wightii, Gracilaria sp.) were determined against cervical carcinoma cells using MTT bioassay method. At concentrations of 1–50 M, the extracts of U. reticulata exhibited antimicrobial activity against Pseudomonas aeruginosa (6.00 mm) and potential antiproliferative activity against HeLa cells (IC50 37 mol/L) [28]. Consumption of marine algae has been linked to a lower incidence of cancer in countries that traditionally consume marine products. The effects of water-soluble sulfated polysaccharides (hydrocolloids) isolated from the red seaweed Laurencia papillosa on the human breast cancer cell line MCF-7 were investigated in a study [29]. For cells exposed to the polysaccharides for 24 hours, the results showed a significant inhibition of MCF-7 cell viability in a dose-dependent manner. This indicates that L. papillosa SPs could be a promising candidate for breast cancer prevention and treatment.

6.6 Anti-Inflammatory Activities of Algae Many physiological and pathological processes are characterized by infla­ mmation. The pathological aspects of many types of inflammation, in contrast to the physiological processes, are well documented. Infection and tissue injury are the most common causes of inflammation. These are both adverse conditions that trigger inflammation and cause the recruitment of

Utilization of Algae  145 leukocytes and plasma proteins to the affected tissue site [30]. Tissue stress also causes an adaptive response known as para-inflammation, which responds to tissue-macrophages and is an intermediate between a basal homeostatic state and a classic inflammatory response. Para-inflammation may be responsible for the chronic inflammatory conditions that are often associated with human diseases [31]. Neuroinflammation plays a role in the development and progression of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Natural anti-inflammatory compounds, such as algae, are promising candidates for developing effective therapeutic strategies. These algae contain antioxidants, proteins, vitamins, minerals, soluble dietary fibers, polyunsaturated fatty acids, polysaccharides, sterols, carotenoids, tocopherols, terpenes, phycobilins, phycocolloids, and phycocyanins. In a study involving a high-fat diet-induced obese mouse model and bone marrow-derived macrophages, researchers looked into the potential anti-obesity and anti-inflammatory effects of four types of domestic brown seaweeds (BMDM) [32]. For 16 weeks, male C57BL/6N mice were fed a low-fat diet (LFD), a high-fat diet (HFD), or an HFD containing Undaria pinnatifida, Laminaria japonica (LJ), Sargassum fulvellum, or Hizikia fusiformis (HF). When BMDM from mice fed HFDs with seaweeds were compared to BMDM from mice fed HFDs with LPS stimulation, they showed differential regulation of pro-inflammatory cytokines such as IL-1 and IL-6. Although seaweed consumption did not prevent long-term HFD-induced obesity in C57BL/6N mice, it did reduce insulin resistance (IR) and pro-inflammatory cytokine circulation. Brown seaweeds Laminaria japonica (LJ) and Hizikia fusiformis (HF) are known to have a variety of health-promoting properties. A study was conducted using both in-vitro and in-vivo models to investigate the anti-­inflammatory activities of LJ and HF [33]. In vitro, C2C12 myotubes, mouse-derived skeletal muscle cells, were treated with LF or HF extracts, and in vivo, muscle tissues from C57BL/6N mice fed a high-fat diet supplemented with 5% LF or HF for 16 weeks were used. LJ and HF induced phosphorylation of protein kinase B and AMP-activated protein kinase in both in-vivo and in-vitro skeletal muscle models. In lipopolysaccharide-stimulated C2C12 myotubes, LJ and HF significantly decreased tumor necrosis factor-alpha; whereas both extracts increased interleukin (IL)-6 and (IL)-10 production, which suggests that they could be useful therapeutic agents for reducing associated inflammation. Ephedra nebrodensis (Ephedraceae) is a plant with numerous biological functions. In traditional medicine, it is used to treat respiratory problems and hepatic pathologies. In a study in mice, the anti-inflammatory properties of two hydro-alcoholic extracts of E. nebrodensis were evaluated in vitro and in vivo [34]. The anti-inflammatory

146  Next-Generation Algae: Volume II activity of the extracts was determined in vitro and in vivo using bovine serum albumin denaturation and croton oil-induced ear edema, respectively. In vivo, the extracts have strong anti-inflammatory effects and reduce ear edema by 70.37% ± 2.00% and 72.22% ± 1.94%. This validates the herb’s traditional use in the treatment of various diseases.

6.7 Potential Health Benefits of Algae Biocomposites Macroalgae are a large type of photosynthetic algae that live in the ocean’s intertidal zone. Recent research suggests that glycans generated from macroalgae have a lot of potential for keeping the gut microbiome and immune system healthy [35]. The human gut bacteria have developed unique arsenals for utilizing a wide range of macroalgal glycans and producing a wide range of oligosaccharides. These oligosaccharides interact with immune cell receptors and are also available for microbial fermentation, allowing them to play an important role in gut homeostasis. The demand for marine and freshwater algae and related products is growing over the world. Algae may become one of the most important foods for nutritional purposes in the near future. They include high-quality protein and other bioactive compounds that could help with obesity and diabetes management. Algae contain fiber, polyphenols, ω-3 PUFAs, and bioactive molecules with potential antidiabetic activity. Carbohydrate digestion and absorption, blood glucose, gastrointestinal neurohormone secretion, glycosylation products, and insulin resistance have all been shown to be influenced by whole algae, algal extracts, or its isolated compounds [36]. Seaweeds, for example, have recently been discovered to be potential sources of beneficial metabolites and bioactive components with a variety of physiological actions. Seaweed polysaccharides, for example, have been shown to have anticoagulant, anti-inflammatory, antioxidant, anticarcinogenic, and antiviral properties [37]. Sulfated polysaccharides, which interact with surfaces and cellular proteins, are abundant in seaweeds and contribute to the majority of their biological action. Consumption of seaweeds may be considered as a means to boost immunological responses, according to several toxicological assessments and clinical trials. Polysaccharides from marine microalgae and seaweeds have been demonstrated to have immunomodulatory activities in humans and animals [38]. As a result, these polysaccharides can act as immunosuppressive molecules in the treatment of inflammatory disorders like autoimmune diseases and sepsis, as well as inducing immunological responses to cancer and other infectious diseases. Marine algae have recently been incorporated into synthetic polymers, which is being

Utilization of Algae  147 investigated as an alternate approach for manufacturing grafts because they are a plentiful and inexpensive supply of biopolymers. As an alternative to porcine-derived membranes, a unique composite patch was developed by combining Sargassum vulgare powders (SVP) with polylactide (PLA) [39]. A modified solvent casting process was used to create SVP-PLA composite patches. Human osteoblasts (HOBs) and osteosarcoma cells (SaOS-2) were planted on tidy PLA and SVP-PLA patches after extensive material characterization to test cytocompatibility. HOBs cultivated on SVP-PLA composite had stronger cytocompatibility and proliferation than those cultured on clean PLA, according to MTT and BrdU tests. Cell proliferation was significantly reduced in SaOS-2 cells grown on SVP-PLA. These composite patches have an antiproliferative effect on SaOS-2 cells while having minimal influence on HOB adhesion or proliferation.

6.8 Challenges and Issues Related to Algae Biocomposites Use Researchers are interested in using algae-based biopolymers as one of the alternative options to create a sustainable circular economy around the world. Microalgae biopolymer has several advantages over other feedstocks, including its autotrophic complex, which reduces greenhouse gas emissions, quick growth ability with flexibility in a variety of settings, and the capacity to compost, which provides greenhouse gas credits [40]. The challenges faced in the circular economy, issues regarding the scale-up and operational cost of microalgae production, and the life cycle assessment of algal-based biopolymer are all part of the algae bioeconomy’s future prospects and challenges [41]. 

6.9 Conclusion Algae have been shown to have several biological activities, some of which include immunomodulatory, antitumor, antiviral, antioxidant, and hypolipidemic activities. Their anti-inflammatory and antiproliferative potential have made them useful in the protection against cancer cells. In addition, they offer a wide range of health benefits, which makes them a vital bioresource. These algae have important applications in producing biopolymers which have a variety of applications such as environmental remediation, adsorbents, and antioxidants [42–50]. Although there are conventional

148  Next-Generation Algae: Volume II methods of manufacturing these algae-biopolymers, efforts are ongoing to develop novel approaches to improve the yield and efficiency of these biopolymers.

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150  Next-Generation Algae: Volume II on the NA2B cell line. Biotechnic & Histochemistry : Official Publication of the Biological Stain Commission, 93(1), 59–69, 2018. 28. Palanisamy, S. K., Arumugam, V., Rajendran, S., Ramadoss, A., Nachimuthu, S., Peter D, M., & Sundaresan, U. Chemical diversity and anti-proliferative activity of marine algae. Natural Product Research, 33(14), 2120–2124, 2019. 29. Ghannam, A., Murad, H., Jazzara, M., Odeh, A., & Allaf, A. W. Isolation, Structural characterization, and antiproliferative activity of phycocolloids from the red seaweed Laurencia papillosa on MCF-7 human breast cancer cells. International Journal of Biological Macromolecules, 108, 916–926, 2018. 30. Medzhitov R. Origin and physiological roles of inflammation. Nature, 454(7203), 428–435, 2008. 31. Barbalace, M. C., Malaguti, M., Giusti, L., Lucacchini, A., Hrelia, S., & Angeloni, C. Anti-Inflammatory Activities of Marine Algae in Neurodegenerative Diseases. International Journal of Molecular Sciences, 20(12), 3061, 2019. 32. Oh, J. H., Kim, J., & Lee, Y. Anti-inflammatory and anti-diabetic effects of brown seaweeds in high-fat diet-induced obese mice. Nutrition Research and Practice, 10(1), 42–48, 2016. 33. Kang, S. Y., Kim, E., Kang, I., Lee, M., & Lee, Y. Anti-Diabetic Effects and Anti-Inflammatory Effects of Laminaria japonica and Hizikia fusiforme in Skeletal Muscle: In Vitro and In Vivo Model. Nutrients, 10(4), 491, 2018. 34. Hamoudi, M., Amroun, D., Baghiani, A., Khennouf, S., & Dahamna, S. Antioxidant, Anti-inflammatory, and Analgesic Activities of Alcoholic Extracts of Ephedra nebrodensis From Eastern Algeria. Turkish Journal of Pharmaceutical Sciences, 18(5), 574–580, 2021. 35. Singh, R. P., Bhaiyya, R., Khandare, K., & Tingirikari, J. Macroalgal dietary glycans: potential source for human gut bacteria and enhancing immune system for better health. Critical Reviews in Food Science and Nutrition, 62(6), 1674–1695, 2022. 36. Bocanegra, A., Macho-González, A., Garcimartín, A., Benedí, J., & SánchezMuniz, F. J. Whole Alga, Algal Extracts, and Compounds as Ingredients of Functional Foods: Composition and Action Mechanism Relationships in the Prevention and Treatment of Type-2 Diabetes Mellitus. International Journal of Molecular Sciences, 22(8), 3816, 2021. 37. Tanna, B., & Mishra, A. Nutraceutical Potential of Seaweed Polysaccharides: Structure, Bioactivity, Safety, and Toxicity. Comprehensive Reviews in Food Science and Food Safety, 18(3), 817–831, 2019. 38. Geetha Bai, R., & Tuvikene, R. Potential Antiviral Properties of Industrially Important Marine Algal Polysaccharides and Their Significance in Fighting a Future Viral Pandemic. Viruses, 13(9), 1817, 2021. 39. Veziroglu, S., Ayna, M., Kohlhaas, T., Sayin, S., Fiutowski, J., Mishra, Y. K., Karayürek, F., Naujokat, H., Saygili, E. I., Açil, Y., Wiltfang, J., Faupel, F., Aktas, O. C., & Gülses, A. Marine Algae Incorporated Polylactide Acid Patch: Novel Candidate for Targeting Osteosarcoma Cells without Impairing the Osteoblastic Proliferation. Polymers, 13(6), 847, 2021.

Utilization of Algae  151 40. Mal, N., Satpati, G., Raghunathan, S., & Davoodbasha, M. Current strategies on algae-based biopolymer production and scale-up. Chemosphere, 289, 133178, 2022. 41. Devadas, V. V., Khoo, K. S., Chia, W. Y., Chew, K. W., Munawaroh, H., Lam, M. K., Lim, J. W., Ho, Y. C., Lee, K. T., & Show, P. L. Algae biopolymer towards sustainable circular economy. Bioresource Technology, 325, 124702, 2021. 42. Adetunji C.O., Anani O.A. Utilization of Microbial Biofilm for the Biotransformation and Bioremediation of Heavily Polluted Environment. In: Panpatte D.G., Jhala Y.K. (eds) Microbial Rejuvenation of Polluted Environment. Microorganisms for Sustainability, vol 25. Springer, Singapore, 2021a. https://doi.org/10.1007/978-981-15-7447-4_9 43. Okeke N.E., Adetunji C.O., Nwankwo W., Ukhurebor K.E., Makinde A.S., Panpatte D.G. A Critical Review of Microbial Transport in Effluent Waste and Sewage Sludge Treatment. In: Adetunji C.O., Panpatte D.G., Jhala Y.K. (eds) Microbial Rejuvenation of Polluted Environment. Microorganisms for Sustainability, vol 27. Springer, Singapore, 2021. https://doi. org/10.1007/978-981-15-7459-7_10 44. Adetunji C.O., Anani O.A. Bioaugmentation: A Powerful Biotechnological Techniques for Sustainable Ecorestoration of Soil and Groundwater Contaminants. In: Panpatte D.G., Jhala Y.K. (eds) Microbial Rejuvenation of Polluted Environment. Microorganisms for Sustainability, vol 25. Springer, Singapore, 2021b. https://doi.org/10.1007/978-981-15-7447-4_15 45. Charles Oluwaseun Adetunji, Osikemekha  Anthony Anani, Olugbemi T. Olaniyan, Abel Inobeme, Frances N. Olisaka, Eseosa Oluwadamilare Uwadiae, Omoregbe Nosa Obayagbona, Recent Trends in Organic Farming. In: Soni R., Suyal D.C., Bhargava P., Goel R. (eds) Microbiological Activity for Soil and Plant Health Management. Springer, Singapore, 2021a. https://doi. org/10.1007/978-981-16-2922-8_20 46. Charles Oluwaseun Adetunji, Olugbenga Samuel Michael, Ajit Varma, Julius Kola Oloke, Oseni Kadiri, Muhammad Akram, Ruth Ebunoluwa Bodunrinde, Areeba Imtiaz, Juliana Bunmi Adetunji, Khuram Shahzad, Aditi Jain, Benjamin Ewa Ubi, Noshiza Majeed, Phebean Ononsen Ozolua, Frances N Olisaka, Recent Advances in the Application of Biotechnology for Improving the Production of Secondary Metabolites from Quinoa. In: Varma A. (eds) Biology and Biotechnology of Quinoa. Springer, Singapore, 2021b. https://doi.org/10.1007/978-981-16-3832-9_17 47. Charles Oluwaseun Adetunji, Olugbenga Samuel Michael, Wilson Nwankwo, Kingsley Eghonghon Ukhurebor, Osikemekha Anthony Anani, Julius Kola Oloke, Ajit Varma, Oseni Kadiri, Aditi Jain, Juliana Bunmi Adetunji Quinoa, The Next Biotech Plant: Food Security and Environmental and Health Hot Spots. In: Varma A. (eds) Biology and Biotechnology of Quinoa. Springer, Singapore, 2021c. https://doi.org/10.1007/978-981-16-3832-9_19 48. Charles Oluwaseun Adetunji, Olugbenga Samuel Michael, Oseni Kadiri, Ajit Varma, Muhammad Akram, Julius Kola Oloke, Hamda Shafique,

152  Next-Generation Algae: Volume II Juliana Bunmi Adetunji, Aditi Jain, Ruth Ebunoluwa Bodunrinde, Phebean Ononsen Ozolua, Benjamin Ewa Ubi, Quinoa: From Farm to Traditional Healing, Food Application, and Phytopharmacology. In: Varma A. (eds) Biology and Biotechnology of Quinoa. Springer, Singapore, 2021d. https://doi. org/10.1007/978-981-16-3832-9_20 49. Adetunji C.O., Roli O.I., Adetunji J.B. Exopolysaccharides Derived from Beneficial Microorganisms: Antimicrobial, Food, and Health Benefits. In: Mishra P., Mishra R.R., Adetunji C.O. (eds) Innovations in Food Technology. Springer, Singapore, 2020. https://doi.org/10.1007/978-981-15-6121-4_10 50. Olaniyan O.T., Adetunji C.O. Biological, Biochemical, and Biodiversity of Biomolecules from Marine-Based Beneficial Microorganisms: Industrial Perspective. In: Adetunji C.O., Panpatte D.G., Jhala Y.K. (eds) Microbial Rejuvenation of Polluted Environment. Microorganisms for Sustainability, vol 27. Springer, Singapore, 2021. https://doi.org/10.1007/978-981-15-7459-7_4

7 Natural Compounds of Algae Origin with Potential Anticarcinogenic Benefits Adewale Omowumi Oyeronke1*, Asowata-Ayodele Abiola Mojisola2†, Akomolafe Seun Funmilola3† and Adetunji Juliana Bunmi1† Department of Biochemistry, Faculty of Basic and Applied Sciences, Osun State University, Osogbo, Osun State, Nigeria 2 Department of Biological Sciences, Faculty of Science, University of Medical Sciences, Ondo, Ondo State, Nigeria 3 Department of Biochemistry, Faculty of Science, Ekiti State University, Ado Ekiti, Ekiti State Nigeria 1

Abstract

Cancer continues to be a menace to public health, with the increasing rise in cancer cases demanding effective therapies with minimal side effects. Generally, typical treatment of various diseases started shifting towards natural products, especially those of plant origin, several years back. The reason is not unrelated to the fact that various conventional anticancer drugs, for example, kill rapidly dividing cells otherwise regarded as cancer cells but the normal cells are also affected, which eventually leads to the frequently reported side effects. Of the various plants that have been utilized in treatment of numerous diseases, algae happen to be the most underexplored for various medicinal properties. Recently, various bioactive components have been isolated from different algae, especially microalgae, for treatment of various diseases, including viral infections, diabetes, asthma, inflammation and cancer to mention a few. Their anticarcinogenic properties are the most striking features of these bioactive components found in microalgae. This chapter focuses on natural compounds isolated from microalgae which are engaged in the management of different cancers and also discusses the reported principles of antiproliferative properties of these compounds.

*Corresponding author: [email protected]; [email protected] † These authors have the same contribution to this work. Charles Oluwaseun Adetunji, Julius Kola Oloke, Naveen Dwivedi, Sabeela Beevi Ummalyma, Shubha Dwivedi, Daniel Ingo Hefft and Juliana Bunmi Adetunji (eds.) Next-Generation Algae: Volume II: Applications in Medicine and the Pharmaceutical Industry, (153–176) © 2023 Scrivener Publishing LLC

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154  Next-Generation Algae: Volume II Keywords:  Microalgae, natural products, antioxidants, anticancer therapy, oxidative stress, inflammation, proliferation, DNA mutation

7.1 Introduction Algae are defined as an enormous, diverse group of photosynthetic organisms that are not fundamentally strictly associated, but belonging to varying evolutionary ancestors. They vary from unicellular microalgae, which include Chlorella and Diatoms, to multicellular types, including giant kelp, which is enormous brown alga that can reach 50 m length. Algae are mostly marine and autotrophic, and lack many of the distinct cell and tissue types, such as stomata, xylem, and phloem, the features which distinguish plants on the land [1]. Algae have ability to grow in isolation or alongside other organisms, and can withstand a wide array of contrary environmental conditions [2]. Algae could be categorized in two ways: i) based on colors, they can be categorized mainly into green algae (Chlorophyta), red algae (Rhodophyta) and brown algae (Phaeophyta); and ii) based on size, they can be classified into macroalgae or microalgae. Macroalgae are large-sized multicellular algae known as seaweeds and are evident to the bare eye, whereas microalgae are single-celled microscopic algae, that can be either prokaryotic or eukaryotic [3]. Algae are special plants which are endowed with great varieties of medicinal properties as a result of the general byproducts derived from them. These byproducts have been reported to take care of many diseases due to their medicinal effects, including anticoagulation [4], antiasthmatic and anti-inflammatory activities [5], antidiabetic and antioxidant activities, antiviral activity [6], antiallergic activity [7], and anticancer activity to mention a few. Of the several diseases reported globally, cancer happens to be the most common and serious one. Reports have it as the leading cause of death which overwhelms the public [8]. It is established that one in every six deaths are caused by cancer [3]. Death by cancer is increasing yearly with the largest proportion being recorded in the low-income population [9]. Over 200 different types of cancer exist [8] that affect different parts of the human body. The European Cancer Observatory reported the estimates of four types of cancer that are most common in the European Union in 2012: colon cancer cases were 342,137, lung cancer (plus trachea with bronchus cancer) cases were 309,589, breast cancer cases were 358,967 and there were 82,075 cases of skin melanoma [10]. Some of these cancers have

Anticarcinogenic Compounds of Algae Origin  155 been discovered to eventually spread into other tissues through metastasis, which is usually deadly [8]. As a result of this great threat, significant consideration has been given toward elimination of cancer [11]. The first line of treatment to cure cancers has usually been chemotherapy, which encompasses utilization of chemicals to constrain the growth or damage of cancerous cells [3]. While several chemotherapeutic drugs can succeed in expanding the survival time of patients, there are extensive side effects associated with them and these frequently compromise the value of the lives of affected patients. These side effects include vomiting, canker sores, baldness, nausea, diarrhea, loss of appetite and fatigue. Therefore, new anticancer agents are constantly searched for from various resources. Exploration of natural agents for their active anticancer components with minimal side effects has provided an impressive strategy for anticancer drug development [9]. Almost 60% of the drugs utilized in medical research, including oncology, have been reported to have their foundation from natural bases, and about a third of those frequently traded are either natural compounds or products obtained from them. In recent times, there has been increased interest in marine bioprospecting; and effective natural products, including terpenes, steroids, alkaloids, polyketides, etc., are now being assessed from marine sources. Presently, there are four anticancer agents obtained from seven marine-derived drugs available on the market [8]. Moreover, 23 out of close to 26 marine natural yields available in clinical trials are anticancer agents [12]. Of all the aquatic organisms, algae have recently been explored for various medicinal activities, especially anticarcinogenic activities. A lot of natural antitumor combinations have been generally reported to be produced by algae, which have now made them important targets in cancer therapy in the last few decades [9]. The type of algae that is more commonly reported for high anticarcinogenic constituents are microalgae. Phytochemicals derived from microalgae have been reported to possess additional prospective biological properties compared with the terrestrial source (plant phytochemicals) [13, 14]. Microalgae contain a collection of phenolic compounds which are discovered to be quite distinct compared to numerous plant classes, including fruits, vegetables and pharmaceutical plants [15]. These unique constituents have actually been earlier reported for their anticarcinogenic activities which could at least in part justify the anticarcinogenic effects of microalgae. The mechanism through which microalgae elicit their effects of anticarcinogenic activity is multifaceted due to the fact that their substantial mechanical multiplicity involves various connections. Microalgae act as

156  Next-Generation Algae: Volume II an excellent source of numerous beneficial derivatives, including carbon compounds, which have application in medical, cosmetics, and pharmaceuticals industries [16]. Additionally, many other components which exist in microalgae, including proteins, lipids, antioxidants, vitamins and polysaccharides [9], perform diverse beneficial roles in living systems. Microalgae also increase natural killer-cell activity, thereby improving the host’s defense system, and enhance stimulation of the immune system, which prevents growth of cancer cell [3]. Consequently, it is being postulated that microalgae will be a significant causative feature in the establishment of anticancer therapy. This chapter briefly discusses cancer progression, predisposing factors and treatment. It also discusses features of microalgae as new sources of products for cancer treatment and attempts to elucidate different microalgae-derived natural products with anticancer activities and various cancers that can be treated by them. In addition, possible mechanisms for the activities of these products are discussed.

7.2 Progression, Predisposing Factors and Treatment of Cancer 7.2.1 Cancer Progression Cancer happens to be the most common serious disease with a higher mortality rate than malaria, HIV/AIDS, and tuberculosis combined [17]. Overall, cancer grew from 7.56 million of global deaths reported in 2008 to 10.21 million deaths in 2020 [18]. Developing countries had more cancer cases compared to developed countries [19]. Development of cancer by a normal cell have been linked to certain characteristics, including resistance to the signals which control cell proliferation, resistance to apoptosis, ability to avoid replicative senescence and evade differentiation thereby overcoming the limitations on proliferation, genetic instability, ability to invade surrounding tissues and ability to metastasize [20, 21]. During development of cancer, cancer cells overcome irreversible arrest of cell proliferation and altered cell function, which is replicative cell senescence, and become “immortalized,” which implies continuous and indefinite cell division [22]. Development of cancer takes many years to occur and comprise numerous advancements. The initial abnormal cell first undergoes certain modifications that allow it to propagate considerably more rapidly than normal cells in similar tissue; this ability is then transmitted to succeeding newer cells [19, 20, 23].

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7.2.2 Predisposing Factors to Cancer The vast majority of cancers occur by chance as a result of genetic mutations in the genomes of cancer cells which can occur as a consequence of inheritance and metabolic reactions. Other predisposing factors include cigarette smoking, radiation, diet, environmental pollutants, alcohol, oxidative stress, obesity and physical inactivity and infectious organisms such as viruses. These factors might act sequentially or in combination to initiate or activate cancer cells [20, 23, 19].

7.2.3 Treatment of Cancer There are several treatment strategies for cancer, including radiation therapy, surgery, targeted therapy, chemotherapy, hormone therapy, stem cell transplant, immunotherapy, precision therapy, etc. Although these treatment strategies can extend the lives of patients, the significant side effects associated with them are numerous and there is usually a trade-off with the quality of life of the affected individual [3]. Therefore, looking for more alternative approaches to cure cancer with minimal or no side effects continues to be of great importance for establishing novel agents for cancer therapy in the continuing war against this menacing disease [8]. The substantial number of numerous drugs of natural origin used in the management of diseases, especially cancer, testify to the benefits derived from marine organisms, especially algae. The type of algae that is more commonly reported for high anticarcinogenic constituents are microalgae, which contain a collection of phenolic compounds that are discovered to be quite distinct compared to many other plant species [15]. Microalgae are now being explored for their various natural compounds with anticarcinogenic properties.

7.3 Features of Microalgae Microalgae are known to be photosynthetic and microscopic organisms, that form the main component of phytoplankton in fresh and marine water. They have the capacity to acclimatize to extremely adverse conditions from mild to very harsh environments, including icy environments and hydrothermal outlets [3]. They contribute approximately 40% of global output [24]. They have short generation times and have populated nearly all biotopes [25, 26]. Microalgae may be simply cultured in photobioreactors (such as 100,000 L biodigesters) to acquire a large organic matter and

158  Next-Generation Algae: Volume II signify a renovatable reserve for drug detection. They utilize energy from sun and carbon fixation, thereby moderating greenhouse gas properties with the elimination of byproducts from phosphorous and nitrogen, which may constitute pollution as determined by their quantities [27]. Microalgae are useful in aquatic drug detection due to their anabolic plasticity and this can activate the generation of numerous products with potential uses in different biotechnology sectors, including health, energy, food, biomaterials and environment [25, 26]. Currently, research on the therapeutic effects of microalgae has increased due to the efficacy of their intrinsic active constituents against a number of pathogens [28–30]. Microalgae are mostly composed of various amounts of lipids, carbohydrates, proteins, and nucleic acids; the level of these compounds may differ depending on vitamin, mineral, and polysaccharide constituents [8].

7.4 Sources of Microalgae Microalgae are large clusters of autotrophic phytoplankton that can be categorized according to the source from which they are obtained as either freshwater microalgae or maritime microalgae. Freshwater microalgae are those found in freshwater environments; they can be separated microscopically and majorly into 10 phyla [3]. Additionally, there are negligible phyla, which have minimal effect on freshwater milieu, namely Haptophyta (five species), Prasinophyta (13 species), Eustigmatophyta (three species), Raphidophyta and Glaucophyta (two species each) have also been reported [31]. Maritime microalgae are regarded as an example of the significant primary bases for the newly discovered beneficial genes and metabolites.

7.5 Fractions of Microalgae Species with Anticancer Properties As of now, over 30,000 deep-rooted microalgae species exist. Fractions of several of these species were recounted for their capacity to prompt cell growth arrest in various cancer cell lines, thereby eliciting anticarcinogenicity.

7.5.1 Carotenoid-Rich Extracts of Chlorella Species Carotenoid-rich extracts of Chlorella species, including C. ellipsoidea and C. vulgaris, were assessed for their antiproliferative abilities against

Anticarcinogenic Compounds of Algae Origin  159 a human colon carcinoma cell line (HCT116). The species generally are known to be a rich salable basis of carotenoids such as lutein, astaxanthin, zeaxanthin and β-carotene. The result of this study showed antiproliferation and pro-apoptotic abilities of the two Chlorella species [32].

7.5.2 Chaetoceros Calcitrans Ethyl Acetate and Ethanol Extracts Nigjeh et al. and Goh et al. worked on ethanol extract and ethyl acetate extract respectively from Chaetoceros calcitrans (the planktonic diatom). They used various breast cancer cell models, including MCF-7; MDA-MB-231 (adenocarcinoma); MCF-10A (breast epithelial cancer cells); and mouse breast carcinoma (4T1). They also used peripheral blood mononuclear cells (PMBC), cervix epithelial carcinoma (HeLa), liver HCC (HepG2), human lung adenocarcinoma (A549), human prostate carcinoma (PC-3), human ovarian adenocarcinoma (Caov3) and human colon adenocarcinoma (HT-29). Nigjeh et al. observed interesting antiproliferative outcome of Chaetoceros calcitrans ethanol extract which was comparable to tamoxifen and also observed a significant rise in the Bax, with the caspases 3 and 7 transcripts which are proapoptotic protein. Goh et al., who tested some other extracts (like hexane, dichloromethane and methanol), found C. calcitrans crude ethyl acetate extract to have cytotoxic properties against the MDA-MB-231 cancer cell line. The lethal effect was found to be precise against cancer cells as an assessment on fibroblasts, which are nontumorigenic cell line and showed no cytotoxic ability [33–35].

7.5.3 Amphidinium Carterae Organic Fractions Samarakoon et al. confirmed several portions from the dinoflagellate Amphidinium carterae extract to have antiproliferative efficacy in cells such as HL-60 (human promyelocytic leukemia cells), B16F10 (mouse melanoma tumor cells), with A549 (adenocarcinomic human alveolar basal epithelial cells). The authors also used a mouse monocyte macrophage cell line to perform cytotoxicity tests (RAW 264.7). The chloroform fraction of A. carterae had the strongest activity and caused a considerable decrease in the HL-60 cells viability [33].

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7.5.4 Methanolic Extracts from Amphidinium carterae, Prorocentrum rhathymum, Symbiodinium sp., Coolia malayensis, Ostreopsis ovata, Amphidinium operculatum, and Heterocapsa psammophila Eleven different benthic dinoflagellate strains (Prorocentrum rhathymum, Coolia malayensis strain 1, Amphidinium carterae, Symbiodinium sp., Ostreopsis ovata strain 1, Amphidinium operculatum strain 1, Ostreopsis ovata strain 2, Coolia malayensis strain 2, Coolia malayensis strain 3, Heterocapsa psammophila and Amphidinium operculatum strain 2) were obtained from Jeju Island off the coast of Korea in 2011, and then cultured to monitor RAW264.7 and HL-60 cells. In the study, Ostreopsis ovata 1 and Amphidinium operculatum 1 were the only extracts found to hinder the growth of HL-60 cells significantly [36].

7.5.5 Skeletonema marinoi Hydrophobic Fraction Lauritano et al. studied the role of several species of microalgae (32 in number) obtained with water-acetone (1:1) mixture and additionally separated with Amberlite RXAD16N resin using acetone as the resin eluent to attain a non-polar fraction. The results showed that the non-polar portions from Skeletonema marinoi, Alexandrium minutum, Alexandrium tamutum and Alexandrium andersoni repressed the progression of melanoma cell line (A2058) significantly. Further study showed that Alexandrium species were lethal on a normal lung fibroblast (MRC-5) cell line. Strains of Skeletonema marinoi (FE6 and FE60 strains from the Adriatic Sea) were verified on an A2058 cell line, assay was carried out in normal and cancer cells to check cytotoxicity. The outcome revealed that the FE60 strain was the only one active against A2058 and this occurred solely after cultured in nitrogen-starvation conditions [25].

7.5.6 Canadian Marine Microalgal Pool Aqueous Extract Antiproliferative and anti-colony-forming activities of a fresh aquatic microalgal material (dried powder) from Canada was investigated on proliferation of different cancer cells, virtually all of which were significantly repressed at a measure of 5 mg/mL. The anti-colony activity of the extract revealed successful inhibition of colony creating capacity of all the tested cancer cells tested with the extract at the most minimal measure (0.5 mg/ mL) tested [37].

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7.5.7 Chlorella sorokiniana Aqueous Extract In many countries, Chlorella sp. biomass, which is mainly produced in Asia, is listed among the widely used dietary supplements. A study on the role of diatom Chlorella sorokiniana (marine strain) extracted with hot water, was carried out on lung adenocarcinoma cell lines (A549 and CL1-5). Results of cell viability revealed a dose-related suppression on the two cell lines tested. The authors also verified the principle of function of C. sorokiniana extract by employing analysis by flow cytometry to ascertain a probable process of cell apoptotic/cycle arrest. It was found that cell cycle arrest was not involved during the period of the study but a significant upsurge in population of sub-G1 phase was detected, an indication of significant apoptosis. Protein expression of different types of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) was elevated from the two cell lines beyond 24 hours introduction to the extract of microalgal. Caspases 3 and 9 activation indicated mitochondrial pathway as the main apoptotic pathway involved. Furthermore, the ratio of Bax/Bcl-2 was elevated beyond 24 hours of exposure, confirming increased apoptosis [38].

7.6 Compounds with Anticarcinogenic Activities Isolated from Marine Microalgae In the last section outstanding and exciting anticarcinogenic activities were displayed by various fractions obtained from several species of microalgae. However, the activities of these fraction character may not be unrelated to their constituent bioactive molecules. Active compounds with anticancer capabilities are one main group of highlighted compounds from microalgae; therefore, some of the natural bioactive compounds with anticancer activities extracted from microalgae are discussed below.

7.6.1 Polysaccharides Polysaccharides are complex carbohydrate structures which are made from several monosaccharide molecules joined by diverse glucosidic linkages [39]. Polysaccharides are organic bioactive compounds possessed by plants but they generally appear in microalgae and also in animals, microorganisms and other different plants [40, 41]. Unique functional properties of polysaccharides have been linked to their structure organized by the building blocks [70].

162  Next-Generation Algae: Volume II Polysaccharides obtained from Spirulina species and Ecklonia cava were asserted to enhance excision, unscheduled DNA synthesis, radiation-damaged DNA repair activity and also significantly exhibited enhanced activity of endonuclease, DNA production of sarcoma 180, ascetic hepatoma cells, and inhibited propagation of ascitic hepatoma cells of mice [42, 43]. Polysaccharide from Spirulina platensis prevented tumor attack and spread [44]. It was detected that the mechanism of anticarcinogenic ability of this acidic polysaccharides in S. platensis included tumoricidal property associated with macrophage-tumor necrosis factor (TNF). It was also found that polysaccharides from Spirulina reduced growth of glioma cell (murine RSV-M) past partial modulation of interleukin-17 generation and suppression of angiogenesis [45]. Polysaccharides which were extracted from Japanese kelp, such as Hijikia fusiforme, Undaria pinnatifida, Laminaria japonica and Eisenia bicyclis, have been documented as future antigenotoxic compounds [46]. Fucoidans (Figure 7.1) (sulfated polysaccharides rich in fucose obtained mostly in brown seaweeds) were reported for their antiproliferative activities in Lewis lung adenocarcinoma affected mice through a yet unknown mechanism [47]. Anticancer activity of these substances on human HS-Sultan cells also included ERK and caspase pathways [48]. Fucoidans are also reported to enhance immunomodulatory capacity that confines tumor cells diffusion and growth by facilitating the damage of the tumors past responses of NK cell and type 1 T helper (Th1) cells [49]. This polysaccharide consists of fucose and a few galactose units. The anticancer activities of fucoidans were confirmed to be associated with their molar masses and sulfate contents. It is documented that the anticancer ability of fucoidans is increased when they are hydrolyzed under gentle conditions [50]. Fucoidans are confirmed to enhance Smac/Diablo and cytochrome c discharge by mitochondria as well as enhance mitochondrial membrane permeability [51].

H3C

HO H3C

O -O SO 3

OSO3-

O OSO3-

Figure 7.1  The structure of Fucoidan.

O

Anticarcinogenic Compounds of Algae Origin  163

7.6.2 Phycocyanin Phycocyanin is a pigment-protein complex from the light-harvesting phycobiliprotein family, along with phycoerythrin and allophycocyanin [52]. It is consistently used as a color-enhancing agent in dairy and dietary products, including beverages, candies, gums, jellies; and also in maquillages, including eyeliners, eye shadows and lipsticks in Japan and China [53]. Phycocyanin (Figure 7.2) isolated from algae has been reported for its anticarcinogenic activities against different types of cancers [54]. The phycocyanin isolated from S. platensis is said to possess radical scavenging and antioxidant properties and exhibits anticancer activity against squamous cell carcinoma. It exhibits inflammatory and anticancer properties and also prompts in-vitro apoptosis in HeLa cells by stimulating cell death enzymes, caspases 2, 3, 4, 6, 8, 9, and caspase 10 [55]. The selenium-­enriched phycocyanin isolated from Spirulina platensis also showed strong anticarcinogenic properties against MCF-7 and A375 cancer cells where it stimulated apoptosis via nuclear compression and buildup of sub-G1 cells and DNA disintegration in both cells [56]. Phycocyanin also improves immune response by stimulating function of macrophage, interleukin-I assembly and phagocytosis. This ability is what contributes to its importance in treatment and prevention of all types of cancers [57]. COOH

COOH H3C

H 3C H3C

O

CH3

H3C

N H

N H

N

H3C

N H

O

Figure 7.2  The structure of phycocyanin.

7.6.3 Chlorophyll Chlorophyll and related compounds like carotene and lutein isolated from different strains of algae have accounted for antiproliferative bioactivity both in vitro and in vivo. Tumor preventive impacts of chlorophyll (Figure 7.3) with subordinates is widely focused on, particularly their in-­ vitro anticarcinogenic impact on several ecological and dietary mutagens. Chlorophyll-a and chlorophyllin have displayed huge importance in the

164  Next-Generation Algae: Volume II O

H H3C

CH3 CH3

N

N MGII N

N CH3

H3C CH3

CH3

H3C

CH3 2

O O

O

O OCH3

Figure 7.3  The structure of chlorophyll.

instigation of ornithine decarboxylase in fibroblasts on mouse skin created via a tumor enhancer with in-vitro cell culture tests [26, 58].

7.6.4 Polyunsaturated Aldehydes (PUAs) Three polyunsaturated aldehydes were isolated in the aquatic diatoms Thalassiosira rotula, S. costatum and P. delicatissima. These compounds, 2-trans-4-trans-7-cis-decatrienal, 2-trans-4-cis-7-cisdecatrienal and 2-trans4-trans-decadienal (Figure 7.4), were found to have anticarcinogenic activity on the human colon adenocarcinoma (Caco-2) cell line [59] and on A549 and COLO 205 [60]. They showed anticarcinogenicity through apoptosis and were found to be nontoxic in normal cell lines. CHO

2E,4Z,7E-decatrienal CHO

2E,4Z-octadienal

CHO

CHO

2E,4E,7Z-decatrienal CHO

2E,4E-decadienal CHO

2E,4Z,7-octatrienal

2E,4Z-heptatrienal

Figure 7.4  The structure of polyunsaturated aldehydes.

7.6.5 Violaxanthin Anticancer monitoring of the extracts obtained in Dunaliella tertiolecta was performed in diverse lines of cancer cells: MDA-MB-231, A549, MCF-7 and LNCaP. The extracts preparation was by several solvents with

Anticarcinogenic Compounds of Algae Origin  165 H3C

CH3

CH3

H 3C

CH3

O

O HO

OH

CH3

CH3

CH3

H3C

CH3

Figure 7.5  The structure of violaxanthin.

different polarities; these include ultrapure water, ethanol and dichloromethane. Dichloromethane extract was the only one that revealed remarkable action on MCF-7 cells. A subfraction of this extract was then made using RP-HPLC examination and separation, the subfraction was later recognized to be violaxanthin (Figure 7.5) with 95% purity. Further analysis with violaxanthin indicated early apoptosis. Cytotoxicity tests are yet to be achieved in regular human cell lines [61].

7.6.6 Eicosapentaenoic Acid (EPA) Marine diatom Cocconeis scutellum extracts were screened on MB-MDA468, BT20, COR, LNCaP, JVM2 and BRG-M [62]. The results, though not entirely published, showed extract from C. scutellum had extra activity on BT20. The fractionation of the most active cell line (diethyl ether extract) from C. scutellum formed three portions having different capabilities. Out of the three portions, only one significantly reduced the viability of the treated cell line. Fragmentation of DNA was further determined on this portion. Results of its composition indicated 4-methylcholesterol (2.3%) and fatty acids (81.7%). It was eventually decided that the activity of this OH O

Figure 7.6  The structure of Eicosapentaenoic Acid (EPA).

166  Next-Generation Algae: Volume II portion was specifically due to its fatty acid subfractions, these fatty acids were identified to be eicosapentaenoic acid (EPA, Figure 7.6) [62]. This is because EPA was the only compound in the fraction that was established for its ability to stimulate apoptosis [63]. The fraction also activated caspases 3 and 8 by Western blot analysis. It has not been established whether EPA is the individual influence associated with death of BT20 cells or if cordial relationship exists among different compounds within the same portion [62].

7.6.7 Stigmasterol Stigmasterol (Figure 7.7) is one of the major sterols in plasma membranes of plant cells, and plays a major role in sustaining the structure and physiology of cell membranes [64]. Stigmasterol was isolated from Navicula incerta extracts via chromatographic techniques [65]. The antiproliferative role of this compound was assessed on HepG2. A dose-dependent trend was observed in the cytotoxicity induced by this compound on HepG2 cells, though this has not been confirmed on normal human cells. Another compound, having structures like phytosterol and double bonds between C-5 and C-22 positions, which is also found in stigmasterol, was confirmed to induce apoptosis [66]. Generally, all results of the assays carried out specified that stigmasterol has an enormous apoptotic induction ability, which is probably through mitochondrial intrinsic apoptotic signaling pathway [8].

H H H

H

HO

Figure 7.7  The structure of stigmasterol.

7.6.8 Fucoxanthin Fucoxanthin (Figure 7.8), a xanthophyll having molecular formula C₄₂H₅₈O₆, is established as an accessory pigment in aquatic micro- and macroalgae. Fucoxanthin is one of the most observed substances in these organisms, and is a key carotenoid from brown algae [67]. The fucoxanthin lethality was

Anticarcinogenic Compounds of Algae Origin  167

O HO

C

O

O HO

O

Figure 7.8  The structure of fucoxanthin.

evaluated in rats but no obvious toxicity was shown within the 28-day period; therefore, it is considered as a safe pharmaceutical ingredient. Fucoxanthin exhibited a solid anticarcinogenic role in HL-60 cells and apoptotic induction in these cells. Cell viability of this compound was also assessed in cell lines of colon cancer, including DLD-1, Caco-2, and HT-29, and fucoxanthin produced significant reduction in tested cell lines viability; however, there is no report on normal human cells [68]. It is also established that fucoxanthin is part of the best active antiproliferative compounds among the 15 types of carotenoids tested.

7.6.9 Nonyl 8-Acetoxy-6-Methyloctanoate (NAMO) The anticarcinogenicity of NAMO (Figure 7.9) isolated in Phaeodactylum tricornutum was tested on some cell lines: HL-60, A549 and B16F10 [33]. In this study, NAMO was reported to be solely active on HL-60 cells at the doses tested. These authors did not test the toxic action of this compound on normal human cells. Mechanism of action of NAMO was reported to include induction of DNA lesion and enhanced apoptotic body development. Arrest of cell cycle and sub-G1 phase cell compilation was also detected to arise with NAMO in a dose-related way [8]. Regression of Bcl-x, an anti-apoptotic, activation of Bax, a pro-apoptotic protein, and an enhancement in the appearance of p53 proteins and caspase-3 were also reported to complement the mechanism of action of NAMO [8, 33].

OH O O HO

Figure 7.9  The structure of nonyl 8-acetoxy-6-methyloctanoate.

168  Next-Generation Algae: Volume II

7.6.10 Monogalactosyl Glycerols Monogalactosyl glycerols (Figure 7.10) are forms of glycerol isolated from Phaeodactylum tricornutum. Two different monogalactosyl glycerols were isolated from diatom Phaeodactylum tricornutum in the genus Phaeodactylum [69]. These authors tested the isolated monogalactosyl glycerols on W2 and D3 (immortal mouse epithelial cells). W2 is a wild-type cell line, whereas, the apoptotic ability has been inactivated via gene manipulation in D3 cells. In the present study, the monogalactosyl glycerols significantly induced apoptosis in the wild-type cell as the treatment enhanced cell death rate, while in the D3 cells, since the gene responsible for apoptotic function has been deleted in these cells, the growth rate increased significantly. The results proved that the isolated compounds induced apoptosis in the W2 cell line [69].

HO HO

O

OH O O OH

O O O

Figure 7.10  The structure of monogalactosyl glycerols.

7.6.11 Other Active Compounds from Microalgae with Anticarcinogenic Activities There are other active compounds isolated from microalgae which show significant anticarcinogenic capacities, including pheophytin [70, 71], carotenoids, siphonaxanthin [68, 72], stypodiol diacetate [73], glycoprotein [73, 74], meroditerpenoids [75], stypotriol triacetate [76], yessotoxins [77], elatol [78], cannabinoids [79], and monoterpenes [80], both in vitro and in vivo.

7.7 Conclusion and Recommendation Plants remain an important part of health maintenance. Traditional plantbased remedies are still very important to people in impoverished nations, and they are also leading to the development of new medication candidates. However, scientific testing of therapeutic herbs is required to justify their usage. The high mortality rate among cancer patients continues to indicate the present low efficacy of medicines in cancer treatment. Substances formed chemically continued to be the focus of cancer research for many

Anticarcinogenic Compounds of Algae Origin  169 years. Natural products or a combination of diverse phytochemicals have only begun being studied for cancer treatment in the last few decades. This chapter emphasized the promising and effective function of regular algae-derived products in the fight against cancer. These products are cytotoxic to both common and uncommon cancer cells and even in some animal models. Most of their cytotoxic activities involve the mechanism of apoptotic induction by regression of anti-apoptotic Bcl-x protein, activation of pro-apoptotic Bax protein, and enhancement in function of caspase-3 and p53 proteins; the major apoptotic pathway involved is intrinsic with the employment of mitochondria in the process. Mechanisms of initiation for damage of DNA and arrest of cell cycle via gathering of sub-G1 phase cells also complement the mechanism of action of these products. The limitation of most of the reported studies lack sufficient data from normal cells to ascertain non-cytotoxic effects of these compounds on normal tissues. However, this work shows the potential capacity of microalgae-derived natural products to combat the public menace known as cancer. It is recommended that these compounds be studied on normal cells to determine their safety for use. Further in-vivo studies are also recommended to be carried out on these impressive compounds.

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174  Next-Generation Algae: Volume II 60. Sansone C., Braca A., Ercolesi E., Romano G., Palumbo A., Casotti R., Francone M. and Ianora A. Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells. PLoS One. 9(7), pp e101220, 2014. 61. Pasquet V., Morisset P., Ihammouine S., Chepied A., Aumailley L., Berard, J.B., Serive B., Kaas R., Lanneluc I., Thiery V. and Lafferriere M. Antiproliferative activity of violaxanthin isolated from bioguided fractionation of Dunaliella tertiolecta extracts. Marine Drugs. 9(5), pp 819-831, 2011. 62. Nappo M., Berkov S., Massucco C., Di Maria V., Bastida J., Codina C., Avila C., Messina P., Zupo V. and Zupo S. Apoptotic activity of the marine diatom Cocconeis scutellum and eicosapentaenoic acid in BT20 cells. Pharmaceutical Biology. 50(4), pp 529-535, 2012. 63. Chajès V., Sattler W., Stranzl A. and Kostner G. M. Influence of n-3 fatty acids on the growth of human breast cancer cells in vitro: Relationship to peroxides and Vitamin-E. Breast Cancer Research and Treatment. 34(3), pp 199-212, 1995. 64. Ferrer A., Altabella T., Arró M. and Boronat A. Emerging roles for conjugated sterols in plants. Progress in Lipid Research. 67, pp 27-37, 2017. 65. Kim Y. S., Li X. F., Kang K. H. Ryu, B. and Kim S. K. Stigmasterol isolated from marine microalgae Navicula incerta induces apoptosis in human hepatoma HepG2 cells. BMB Reports. 47(8), pp 433, 2014. 66. Ryu B., Li Y., Qian Z.J., Kim M.M. and Kim S. K Differentiation of human osteosarcoma cells by isolated phlorotannins is subtly linked to COX-2, iNOS, MMPs, and MAPK signaling: implication for chronic articular disease. Chemico-Biological Interactions. 179(2-3), pp 192-201, 2009. 67. Kong Z.L., Kao N.J., Hu J.Y. and Wu, C.S. Fucoxanthin-rich brown algae extract decreases inflammation and attenuates colitis-associated colon cancer in mice. J. Food Nutr. Res. 4(3), pp 137-147, 2016. 68. Takahashi K., Hosokawa M., Kasajima H., Hatanaka K., Kudo K., Shimoyama N. and Miyashita K. Anticancer effects of fucoxanthin and fucoxanthinol on colorectal cancer cell lines and colorectal cancer tissues.  Oncology Letters. 10(3), pp 1463-1467, 2015. 69. Andrianasolo E.H., Haramaty L., Vardi A., White E., Lutz R. and Falkowski P. Apoptosis-inducing galactolipids from a cultured marine diatom, Phaeodactylum tricornutum.  Journal of Natural Products. 71(7), pp 11971201, 2008. 70. Athukorala Y., Kim K. N. and Jeon Y. J. Antiproliferative and antioxidant properties of an enzymatic hydrolysate from brown alga, Ecklonia cava. Food and Chemical Toxicology. 44(7), pp 1065-1074, 2006. 71. Pangestuti R. and Kim S. K. Biological activities and health benefit effects of natural pigments derived from marine algae. Journal of Functional Foods. 3(4), pp 255-266, 2011. 72. Ganesan P., Noda K., Manabe Y., Ohkubo T., Tanaka Y., Maoka T., Sugawara T. and Hirat T. Siphonaxanthin, a marine carotenoid from green algae,

Anticarcinogenic Compounds of Algae Origin  175 effectively induces apoptosis in human leukemia (HL-60) cells. Biochimica et Biophysica Acta (BBA)-General Subjects. 1810(5), pp 497-503, 2011. 73. Farooqi A.A., Butt G. and Razzaq Z. Algae extracts and methyl jasmonate anti-cancer activities in prostate cancer: choreographers of ‘the dance macabre. Cancer Cell International. 12(1), pp 50, 2012. 74. Ramos A.L., Torello C.O. and Queiroz M.L. Chlorella vulgaris modulates Immunomyelopoietic activity and enhances the resistance of tumor-bearing mice. Nutrition and Cancer. 62(8), pp 1170-1180, 2010. 75. Areche C., San-Martin A., Rovirosa J., Munoz M. A., Hernández-Barragán A., Bucio M.A. and Joseph-Nathan P. Stereostructure reassignment and absolute configuration of isoepitaondiol, a meroditerpenoid from Stypopodium flabelliforme. Journal of Natural Products. 73(1), pp 79-82, 2009. 76. Areche C., Vaca I., Labbe P., Soto-Delgado J., Astudillo L., Silva M., Rovirosa J. and San-Martin A. Biotransformation of Stypotriol triacetate by Aspergillus niger. Journal of Molecular Structure. 998 (1-3), pp 167-170, 2011. 77. Pang M., Gao C.L., Wu Z.X., Lv N., Wang Z.L., Tang X.X. and Qu P. Apoptosis induced by yessotoxins in Hela human cervical cancer cells in vitro. Molecular Medicine Reports. 3(4), pp 629-634, 2010. 78. Campos A., Souza C. B., Lhullier C., Falkenberg M., Schenkel E. P., Ribeiro‐ do‐Valle R.M. and Siqueira J. M. Anti‐tumour effects of elatol, a marine derivative compound obtained from red algae Laurencia microcladia. Journal of Pharmacy and Pharmacology. 64(8), pp 1146-1154, 2012. 79. Guzman M. Cannabinoids: potential anticancer agents. Nature Reviews Cancer. 3(10), pp 745, 2003. 80. De la Mare J.A., Lawson J.C., Chiwakata M.T., Beukes D.R., Edkins A.L. and Blatch G.L. Quinones and halogenated monoterpenes of algal origin show anti-proliferative effects against breast cancer cells in vitro.  Investigational New Drugs. 30(6), pp 2187-2200, 2012.

8 Current Research on Algal-Derived Sulfated Polysaccharides and Their Antiulcer Bioactivities Abiola Mojisola Asowata-Ayodele1*, Adewale Omowumi Oyeronke2, Akomolafe Seun Funmilola3† and Adetunji Juliana Bunmi2† Department of Biosciences and Biotechnology, Faculty of Science, University of Medical Sciences, Ondo, Ondo State, Nigeria 2 Department of Biochemistry, Faculty of Basic Medical Science, Osun State University, Osogbo, Nigeria 3 Department of Biochemistry, Ekiti State University, Ado Ekiti, Nigeria

1

Abstract

Peptic ulcer disease has been characterized as a stomach ulcer that affects almost the entire thickness of the mucous membrane. Natural therapies for the treatment of gastrointestinal ulcers have been developed using medicinal plants and animals. Due to the presence of its metabolites with high amounts of bioactive compounds, phytoplankton has been shown to be beneficial in drug development. Seaweeds are one of the roots from which botanical extracts are made, which make them highly useful as a natural product in daily life. More nutritional compositions of the aforesaid organisms especially need to be researched for the total eradication of Helicobacter pylori so that peptic ulcers no longer afflict the population. Keywords:  Alginates, antiulcer, natural products, algae, peptic ulcer, polysaccharides, seaweeds

*Corresponding author: [email protected]; [email protected] † This authors contributed equally to this work. Charles Oluwaseun Adetunji, Julius Kola Oloke, Naveen Dwivedi, Sabeela Beevi Ummalyma, Shubha Dwivedi, Daniel Ingo Hefft and Juliana Bunmi Adetunji (eds.) Next-Generation Algae: Volume II: Applications in Medicine and the Pharmaceutical Industry, (177–196) © 2023 Scrivener Publishing LLC

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178  Next-Generation Algae: Volume II

8.1 Introduction The term “gastritis” refers to an inflammation or abscess that begins in the mucous membrane of the stomach and extends across the muscularis mucosae, generally characterized by various phases of coagulation [1]. Falco [2] defined duodenal ulcer (DU), pyloric canal ulcer (PCU), gastrointestinal ulcer (GU), and postoperative ulcer at the surgical anastomosis site as a spectrum of illnesses. Helicobacter pylori, a bacterium that lives in the acidic environment of the stomach and colonizes the gastric mucosa, has been blamed for all ulcer-related disorders since its discovery. The bacterium has a spiral-like form and flagella, allowing it to travel continuously across the stomach’s acid and mucous membranes [2]. H. pylori may attach epithelial cells to the plasma membrane of stomach epithelial cells, destroying their cytoskeleton components. Without urease, colonizing the mucosa with this substance and protecting the germs from hydrochloric acid would be impossible [1]. Urease, a cytoplasmic enzyme that acts intracellularly and is affine to gastric mucin, is abundant in microbial cells. This enzyme activates neutrophils and monocytes, causing the generation of various pro-inflammatory cytokines as well as the formation of nitric oxide and oxygen radicals [2–5]. The majority of ulcer sufferers have abdominal discomfort. The pain which usually does not radiate is localized in the epigastrium. The signs, in reality, are neither unusual nor uncommon. Back pain might indicate that an ulcer has migrated rearward or that the discomfort is pancreatic in nature. Patients may feel a burning or hunger discomfort that builds up over one to two hours, then gradually subsides. Certain drugs, such as antacids, may provide brief relief. Meals, according to some, aggravate the discomfort of stomach ulcers while alleviating the agony of duodenal ulcers [6]. As a result, people with stomach ulcers avoid eating and lose a lot of weight, but those with duodenal ulcers are unaffected. Although these tendencies are prevalent, it is crucial to recognized that they are not specifically characteristic of a disease condition. The nature of the symptoms makes distinguishing between helpful ulcers and stomach neoplasms difficult. Despite the fact that H. pylori is a documented cause of a variety of gastric disorders, there is much controversy regarding whether it performs any function in the pathogenesis of non-stomach infections (Figure 8.1) [3]. Others argue that H. pylori does not cause ulcers in sick people, but that the bacteria disturb the stomach and duodenum’s safe secretions, causing the

Algal-Derived Sulfated Polysaccharides  179 H. pylori Gastric acid Pepsin NSAIDS

Mucosa

Necrotic cells

Peptic ulcer (Microscopic view)

Submucosa Muscularis externa

Blood vessel

Figure 8.1  Pathogenesis of peptic ulcer disease [7].

acid to leak out [8]. However, given the use of various traditional therapies, this virus offers a severe challenge for medical study; it seems doubtful that this pathogen can be killed in the human body system [4]. Due to the intricacies of peptic ulcer disease, a new strategy for removing the causal factor is always needed. The purpose of this chapter is to evaluate current breakthroughs in the work of sulfate polysaccharides in marine species, as well as their potential antiulcer bioactivities.

8.1.1 Symptoms of Peptic Ulcer Disease The disease’s clinical manifestations are categorized as minor, moderate, or severe [5], as described below: • Mild: Not more than three stools in a day, with or without blood stain, and a regular erythrocyte sedimentation rate (ESR). • Moderate: More than four stools in a day with minor systematic interruption. • Severe: Bloody bowel movements more than six times a day with more than 30 symptoms of systemic interruption such as tachycardia, fever, anemia, or an elevated erythrocyte sedimentation rate [5]. In addition to the above-mentioned symptoms, peptic ulcer disease may be exacerbated by the following factors.

180  Next-Generation Algae: Volume II a) Hypercalcemia Hypercalcemia is a state of elevated body calcium levels. The hypersecretory condition of stomach acid reported in Zollinger-Ellison syndrome patients is directly related to hypercalcemia. Intravenous calcium infusion causes stomach acid hypersecretion in healthy participants, according to [9]. Calcium has also been demonstrated to induce gastrin secretion directly from gastrinomas in vivo and in vitro [5]. b) Genetic Variables The etiology of infectious disorders is heavily influenced by genetic differences. First-degree relatives of ulcer patients should have a greater rate of improving ulcer infection than the general population. Duodenal ulcers affect about 20% to 50% of individuals with a good family history; sufferers of ulceration in the stomach frequently identify clusters of afflicted relatives [7]. c) Smoking The development of ulcer disease, complications, mortality, recurrences and delayed healing are all strongly linked to cigarette smoking. Ulcer disease is two times more common in smokers than it is in non-smokers. Peptic ulcer disease is caused by a combination of causes such as the close link between H. pylori and smoking cigarettes. This is because cigarettes may make people more vulnerable, reduce protective components in the gastrointestinal mucosa, or create a more favorable environment in which bacteria can thrive [1]. d) Anxiety Because psychology’s place in the world isn’t well understood, several studies have shown conflicting results on the relevance of psychological elements in peptic ulcer disease pathophysiology and natural history. Acute stress causes a rise in anxiety, blood pressure and heart rate; only individuals with ulcers in the duodenum have shown substantial increases in basal acid production as a consequence of acute stress [7]. Ryan said the disease doesn’t have a distinct “ulcer type” personality. Mentally, ulcer patients are no different from the normal population, but their levels of stress are higher. There is also no indication that the likelihood of developing an ulcer is related to a person’s work and lifestyle [10]. e) Diet and Alcohol Animal studies have shown that alcohol damages the stomach mucosa, the amount of absolute ethanol administered seems to be one of the determining factors. Pure ethanol is lipid-soluble and causes severe, open mucosal injury. Mucosal damage may occur at doses of less than 10% ethanol since most individuals do not consume complete ethanol. Low quantities of ethanol (5%), on the other hand, may increase stomach acid output while

Algal-Derived Sulfated Polysaccharides  181 greater concentrations inhibit it. While this is intriguing from a physiological standpoint, it has no bearing on the cause of ulcers [5]. f) Gastrinoma (Zollinger-Ellison Syndrome) Irritable bowel syndrome has been linked to certain foods and beverages. No conclusive assertion has been made on the link between a specific diet and ulcer disease. Cola, decaffeinated, caffeinated beverages or milk and beer have not been linked to an increased risk of ulceration in epidemiological research. Other than avoiding items that cause discomfort, dietary changes are unnecessary [5]. Gastrinoma is a kind of cancer that affects the intestines (ZollingerEllison syndrome). Abnormally located peptic ulcers, such as in the jejunum, severe oversecretion of stomach acid, and a pancreatic gastrinoma are all symptoms of Zollinger-Ellison syndrome. Gastrinoma affects the pancreas in around half of all individuals. Another 20% of patients have it in the peri-pancreatic lymph nodes, stomach, ovary, liver, or small bowel mesentery [5]. Only 0.1% of all duodenal ulcer illnesses are caused by Zollinger-Ellison syndrome. As part of multiple neoplasia syndromes, one-fourth of the patients have this disorder. Because gastrin is trophic to the gastrointestinal mucosa, people with gastrinoma may have intractable ulcer disease. Endoscopy or X-ray may reveal gastric hypertrophy. Sufferers may present with diarrhea (with accompanied constipation) as well. Diarrhea as a result of lipase inactivation by acid and reflux in the gastroesophageal region are possible side effects. In 75% of patients, these symptoms are episodic [7]. H. pylori affects roughly 20% of those under the age of 40 and 50% of those over the age of 60 in general. It is not recorded in young children; but is predicted for those of poor socio-economic position. Immigration is to blame in certain western nations for the isolated, densely populated places where it is more likely to occur.

8.2 Treatment Using Synthetic Medicines Inhibiting stomach acid production, increasing gastro-protection to scavenge reactive oxygen species and eliminating H. pylori are all part of the contemporary strategy for gastroduodenal ulcer treatment. Although proton pump inhibitors (lansoprazole, omeprazole), receptor blockers (famotidine, ranitidine, etc.), and antibiotics (amoxillin, clarithromycin, tetracycline, and others) are currently used for efficient management of the condition [11], there is currently no cure for it. However, the standard treatment relies on reducing aberrant inflammation in the colon lining,

182  Next-Generation Algae: Volume II which reduces symptoms such as diarrhea, rectal bleeding, and stomach discomfort. Therapy is tailored to the severity of the condition; as a result, treatment is tailored to each person [5]. To decrease inflammation and improve symptoms, most people with early stage or severe inflammatory bowel disease are given corticosteroids such as dexamethasone [5]. After one year, over a quarter of patients with ulcerative colitis who need steroid treatment become steroid-dependent, and nearly all experience steroid-­ related side effects [5]. Other immunomodulators (azathioprine and 6-mercaptopurine) that impact the immune system and amino-salicylates to reduce inflammation are available. Various classes of pharmacological drugs have been shown to be beneficial in the treatment of acid-induced peptic diseases: Acid-suppressive agents such as lanzoprazole and omeprazole; antacids such as magnesium trisilicate and aluminum hydroxide; antagonist of histamine H2-receptor such as ranitidine and cimetidine; cytoprotective agents such as sucralfate and analogs of prostaglandin such as misoprostol; anticholinergic agents such as pirenzepine; and antimicrobials (H. pylori eradication). Ranitidine is a reversible antagonist of H2 receptor known to be highly competitive and usually used for the treatment of peptic ulcers, ZollingerEllison syndrome, gastroesophageal reflux disease, and other conditions. Mahmoud found a link between oral and intravenous ranitidine infusion and severe allergic responses such as toxic epidermal necrolysis, bronchospastic reaction, and exanthematous pustulosis [10]. The acute form of ranitidine responses involves mechanisms that are both non-immunological and immunological, according to the literature. Ranitidine-induced anaphylaxis is a multi-organ clinical condition marked by the fast development of cardiovascular and respiratory symptoms as the first life-threatening recognized signs in the majority of patients [10]. Scientists have highlighted the urgent need for a novel antiulcer medication derived from natural sources to replace the presently utilized pharmaceuticals of questionable safety and effectiveness. Medicinal plants, herbs, spices, fruits and other natural products are considered potential sources of supply to regulate and manage various inflammatory bowel diseases [5]. A large number of functional foods, secondary metabolites and medicinal plants with antiulcer properties have been documented [10, 12]. Between natural sources, red algal components have a complicated method for protecting against gastrointestinal ulcers that require therapy. Overcoming treatment resistance to H. pylori is a critical healthcare problem, since eradication of the agent is difficult in 15–20% of instances. In actuality, after 3–5 years, the effectively treated individuals were re-infected

Algal-Derived Sulfated Polysaccharides  183 with H. pylori. As a result, finding innovative strategies to combat this illness is critical. The current materials indicate that research into the use of marine algal polysaccharides for treating Helicobacter infections is promising since these substances have bidirectional activity and are non-adhesive, non-toxic, immunomodulatory, non-oxidant and non-inflammatory. Polysaccharides derived from algae have a significant bactericidal action and may remove biofilms generated by microorganisms because of the potency of polysaccharides produced from algae, the products now accessible in the literature are primarily synthetic. Since obtaining specimens with a standard structure is still a difficulty, there are no recognized medications based on such substances [5]. Seaweed extracts and polysaccharides are frequently utilized in other nations. Several authors recommend adding algae-related sulfated polysaccharides to useable food items for patients with Helicobacter pylori infection. Polysaccharides, like sulfate, offer a number of advantages, including a large supply of raw materials, a lower level of technological expertise in separation and purification operations, high pharmacological activity, and low toxicity rates [4]. Brown algae fucoidans from sulfated polysaccharides have also been identified as the source of novel drugs with similar actions, demonstrating sufficient potential when combined with biomedical schemes for the treatment of infection caused by H. pylori [4].

8.3 Natural Products Used in the Treatment of Peptic Ulcer A wide range of medicinal herbs are used to cure ulcer-related disorders, following are a few of them: • Centella asiatica is a highly popular medicinal plant used in Asia. It is known as gotu kola in China, and it is said to have existed over 2,000 years ago as “wonder elixirs of creation.” In rats, C. asiatica leaf extracts of about 50 to 250 mg/ kg dose demonstrated a good gastroprotective effect against indometacin-induced ulcers [1]. • Baccharis dracunculifolia, sometimes known as green propolis, is a Brazilian plant. Its essential oil is high in both oxygenated and non-oxygenated terpenes, and it has been shown to have antiulcer effects in rats; lowering total area of

184  Next-Generation Algae: Volume II lesion, the index of lesion, and lesion percentage. It also lowers the quantity of gastric juice and overall acidity of lesion as well as the volume of total acidity and gastric juice [1]. • Baccharis trimera: The anti-secretory and antiulcer action of B. trimera is known as carqueja in Brazil. The aqueous extract has previously been tested in a pylorus tourniquet and stress-induced ulcer model. • Helichrysum gymnocephalum blooms at 25 to 200 mg/kg in Wistar rats with indomethacin-induced severe ulceration reveals the extracts’ gastro-protective and anti-­inflammatory properties, despite its lack of antioxidant activity. It is argued that triterpene alcohols should be present based on these findings. • Tanacetum larvatum is an endangered annual plant that grows in unstable regions of Montenegro, Croatia, and Albania. The phenolic extracts generated from T. larvatum blooming aerial parts showed anti-inflammatory effectiveness dose-dependently, with somewhat fewer gastrointestinal lesions in ulcerated Wistar rats treated with indomethacin at 200 mg/kg. These effects might be due to different components of the plant extracts inhibiting the transcription factor NF-B from binding to DNA [2]. The maritime ecosystem, which spans around 70% of the entire surface of the Earth, is home to half of the world’s biodiversity. Many pharmacologically active compounds, such as phytochemicals, sulfated polysaccharides, polyunsaturated fatty acids, proteins, bioactive peptides, sterols, etc., are abundant in the aquatic environment due to its vast abundance [9]. These marine bioactive compounds come from a variety of places, such as seaweed, algae, champignons, and other aquatic plants.

8.4 Antiulcer Products Developed from Algae Many aquatic species are important in phytomedicine because they contain significant bioactive secondary metabolites that may aid in the creation of novel pharmacological drugs. Algae are a diverse category of autotrophic organisms that range from single to multicellular. They are main producers and nutritional sources for a large number of people. Microalgae, such as blue-green algae, dinoflagellates and bacillariophyta, and macroalgae, such as seaweeds, are the two primary varieties [13].

Algal-Derived Sulfated Polysaccharides  185 Gamal is a green, brown, and red alga that grows in the water. Brown (Phaeophyceae), red (Rhodophyceae), and green (Chlorophyceae) algae are classified as seaweeds based on their pigmentation [14]. Both biodiversity and chemical diversity abound in the marine environment. Marine organisms have identified a large number of new metabolites with powerful pharmacological effects in recent years. Seaweeds, also known as microalgae, are thought to be a rich source of biologically active compounds that are used for therapeutic and medical purposes. The rising number of novel compounds produced by macroalgae is promoting marine science as a viable study topic for medication development [11, 15]. Seaweeds are used for a variety of purposes such as for animal and human consumption. Potential anti-ulcerogenic and anti-inflammatory drug development has lately focused on investigating the efficacy of extracts isolated from medicinal plants that are more active and safer to use. Because they are regarded to be the true producers of biologically active chemicals from marine resources, algae have been acknowledged in pharmacology as bringing chemical and pharmacological novelty and variety [9]. Natural chemicals obtained from edible algae may be hygienic to use as anti-ulcerogenic and anti-inflammatory therapy in the stomach. This is because algae contain physiologically active chemicals such as phlorotannins, polyunsaturated fatty acids, carotenoids, flavonoids, and phenolic acids, as well as sterols and polysaccharides. Polymers composed of monosaccharide units are known as polysaccharides, which are joined together by glycosidic linkages. Hot water extraction, alkaline extraction, and fermentation have all been used to remove and produce polysaccharides [16]. A significantly larger field of polysaccharide formation is also ensured by readily accessible resource reports. These polymers are found in macroalgae cell walls and vary in terms of the composition of the monomeric units, degree of polymerization, glycosidic linkages and sequence of the sugar residue. Sulfate esters are connected to polymeric units of fucose, rhamnose, galactose, and glucose in some of the polysaccharides. Polysaccharides of this kind have a variety of functional and structural activities, as well as defense roles in plants. Polysaccharide bioactivities and pharmacological characteristics are said to be intimately connected to their molecular configurations [16]. Alginate, fucoidans, agar, ulvans, laminaran and carrageenans are among the polysaccharides found in brown and green macroalgae [17, 18]. By comparison with other algal groups, red algae are the most significant source of a variety of physiologically active metabolites. They are abundant in many sections of the world’s coastal locations, providing a great and unexplored prospect for novel therapeutic supplies. Red algae

186  Next-Generation Algae: Volume II have been found to contain active compounds that can help to increase food inflammation and ameliorate food tract inflammation, avoid or treat oxidative stress-induced stomach ulcers and cancers, reduce inflammatory activity by blocking the development of inflammatory mediators, and trigger stomach and colon cancer cell apoptosis [9].

8.4.1 Phycocolloids The name phycocolloid refers to three primary compounds recovered from brown and red seaweeds (alginate, carrageenan, and agar, respectively) [6]. Phycocolloids like alginic acid, agar, and carrageenan are the major components of red and brown algal cell walls. They are found in many kinds of seaweed cell walls and may be removed using hot water. Polymers comprising chemically modified sugar molecules, like agar and carrageenan galactose or organic acids like mannuronic acid and algal glucuronic acid in alginates, are examples of such phycocolloids. Humans and other animals may safely ingest most phycocolloids, as they are usually used to prepare meals, including puddings, ready-mix cakes, and dairy toppings. a) Alginates Alginates (Figure 8.2) are a kind of sugar that may be found in brown seaweed that is used to make alginates, whereas red seaweed is used to make agar and carrageenan. Both acid and salt forms of algins/alginates are accessible. An alginic acid or linear polyuronic is usually the acid form, while the salt form is a major composition of the cell wall in brown seaweed, accounting for nearly 40–47% of the dry weight of algal biomass [6]. Anionic polysaccharides are alginates. They combine covalently (1–4) linked -D-mannuronate with the C5 epimer-L-guluronate to form linear blocks. The most common commercial alginate phaeophytes are Laminaria,

OOH

O O

OH

O

O

HO

O O

HO O

Om

Figure 8.2  Chemical structure of Alginates.

n

Algal-Derived Sulfated Polysaccharides  187 Mycrocystis, and Ascophyllum. Other small sources may include Durvillaea, Sargassum, Lessonia, Turbinaria, and Ecklonia [19]. Alginates or alginic acids which are usually collected from brown seaweeds are used in ice cream to prevent the formation of ice crystals (produce a smooth texture). Alginates have been used in folklore medicine to heal wounds and stomach ulcers [20]. Alginate also lowers cholesterol levels in the blood. Alginic acid has been shown to block mast cell degranulation and hyaluronidase, both of which are implicated in allergic responses [8]. In various countries, including Germany, United States, Japan, Canada, Belgium, etc., patents protect the treatment of gastroduodenal ulcers and gastritis with the use of alginic acid and other compounds derived from it, as well as the use of alginates as antiulcer treatments [6]. In humans, certain medicines containing alginate have proven to be efficiently able to prevent acid reflux after eating, duodenal ulcers and bile acid aggregation. Examples of such medicine include “Gaviscon” made of sodium alginate, sodium bicarbonate, and calcium carbonate; “Algitec” made of sodium alginate H2 antagonist, and “Gastralgin” made of sodium alginate, alginic acid, and aluminum hydroxide [20]. In children aged 4 to 15, clinical trials demonstrated that sodium alginate lowers inflammation, improves stomach mucous membrane regeneration, eradicates mucous membrane Helicobacter pylori colonies, and normalizes resistance in nonspecific mucous membrane [6]. A “suspension of polaprezinc-­sodium alginate” as a high-performance combination for treating severe gingivostomatitis (cold sores) aggravated by hemorrhagic erosions and ulcers. In functional gastroenterology, mixtures of alginates, alginic acid and antacids are used to treat epigastric burning and gastroesophageal reflux [6]. b) Carrageenans Carrageenans (Figure 8.3) are often employed as a food additive, but they’ve also been utilized in animal clinical trials to develop pleurisy and colon ulcers. Carrageenans function in the formation of colonic ulcers is controversial, and it seems to be highly reliant on the chemical properties of the carrageenan [6]. Carrageenan from red algae is a linear SP made up of 3,6-anhydro-D-galactose and D-galactose [23]. c) Agar Agar (Figure 8.4) is a polysaccharide combination made up of agarose and agropectin that has structural and functional characteristics that are comparable with carrageenans. It’s made from red sea algae like Gelidium spp. and the species Gracilaria [6].

188  Next-Generation Algae: Volume II OH

HO

O O

OSO3-

O

O

OSO3HO

OSO3

Figure 8.3  Chemical structure of Carrageenan.

OH O

HO O O

O H

OH

HO

O

OH

n

Figure 8.4  Chemical structure of Agar.

8.4.2 Fucoidan Fucoidan (Figure 8.5) is a sulfated polysaccharide that is usually found in marine brown algae, sea urchin jelly coat shells and sea cucumber body walls. It’s most typically found in the brown seaweed cell walls, but not in other higher plants or algae and is mostly made up of sulphated L-fucose, with less than 10% other monosaccharides. Fucus vesiculosus, a type of brown algae fucoidan, was the focus of much of the study on its biological activities [8]. It was discovered that fucoidan from Fucus vesiculosus has substantial biological effects on mammalian cells. Turbinaria conoides, Cladosiphono kamuranus, Fucus vesiculosus, Undaria pinnatifida and Laminaria japonica have all been shown to contain fucoidan [21]. Another researcher found that algal fucoidans had either alternating (13)- and (14)-linked-L-fucopyranosyl residues or repetitive (13)-­ linked-L-fucopyranosyl residues in their homofucose backbone chains. In addition to sulfonate- and acetyl-groups, as well as L-fucopyranosyl, D-glucuronopyranosyl, and algal polysaccharides, some additional sugar

Algal-Derived Sulfated Polysaccharides  189 H3C

HO H3C

O -O SO 3

OSO3-

O OSO3-

O

Figure 8.5  Chemical structure of Fucoidan.

residues of the backbone’s L-fucose units occur in O-2 and/or O-4 [22]. Seaweeds were the first kind used to isolate fucoidans with backbones. Fucoidans from Fucus evanescens, Fucus distichus, and Ascophyllum nodosum have a second backbone type. Cladosiphon okamuranus from fucoidan was shown to be more effective than F. vesiculosus in curing ulcers. The pathogen Helicobacter pylori is only found in humans. It infects human stomach epithelial cells and has been associated with significant upper gastrointestinal illnesses. According to a research study, fucoidan (1.5–4.5 mg/kg•day) alleviated non-ulcer dyspepsia symptoms, as judged by a structured review. During this investigation, no major side effects from fucoidan were identified. The authors claim that taking fucoidan on a regular basis helps with non-ulcer dyspepsia [8]. Ex-vivo experiments with mice with H. pylori-induced gastritis revealed that the brown algae fucoidans, F. evanescens, Cladosiphon okamuranus, and F. vesiculosus, relieved the condition in people. Besednova found that fucoidans significantly reduced H. pylori cycle adherence to the mucous membrane in the stomach mucosa. At pH 2.0 and pH 4.0, brown algae fucoidan from C. okamuranus decreased H. pylori adhesion to the gastric mucosa in pigs, but the other two fucoidans utilized in these trials only inhibited attachment at pH 2.0. H. pylori adherence to the head was not inhibited by non-sulfated polysaccharides (dextran and mannan) or carboxylated polysaccharides [4].

8.4.3 Ulvans The term “ulvan” is derived from the original terms ulvin and/or ulvacin, which were introduced by Kylin to describe various fractions of water-­ soluble sulfated polysaccharides Ulva lactuca. Ulvans are highly char­ged

190  Next-Generation Algae: Volume II COONa

H3C O

O O HO OH

HO

OH

Figure 8.6  Chemical structure of Ulvan.

sulphated polyelectrolytes containing a common disaccharide ingredient; the aldobiuronic acid, [4)-D-glucuronic acid-(14)-L-rhamnose3-sulfate-(1]. Iduronic acid is also a sugar component. Ulvans have a molecular weight ranging from 189–8,200 KDa [6]. Ulvans (Figure 8.6) are sulfated polysaccharides that are highly branched and water soluble, with uronic acid, rhamnose, and xylose as their primary sugar monomers. Ulva rigida, Monostroma sp. and Ulva lactuca are all probable sources of marine ulvans [21]. Ulvans are also sources of iduronic acid, which is utilized to make heparin component analogues for antithrombotic procedures. Ulvan has been reported to be used for treating stomach ulcers and as an anti-influenza medication [21]. Due to its anti-thrombotic properties, it is used to diagnose gastric ulcers, stomach ulcers, and as an anti-influenza agent [8]. Another study found that seaweeds had strong wound healing, antiulcer and hepatoprotective properties. In investigations [12, 13], it was discovered that L. papillosa provided the most protection against stomach ulcers (81%) followed by Gracilaria crassa (76%) when compared to the usual medicine ranitidine (90%).

8.4.4 Laminaran Laminaran (Figure 8.7) is a linear polysaccharide glucan that is made up of 13- and 16-glucose residues, with a ratio of 13:16 of roughly 3:1. It is usually found in the fronds of the Laminaria saccharina plant, and to a lesser extent in the plants Ascophyllum, Fucus, and Undaria. The amount of material varies according to the season and environment, but it may account for up to 32% of the dry weight. Laminaran does not bind or mold other laminarans, nor does it produce a viscous solution. Its principal use seems to be in medical and pharmaceutical applications [6].

Algal-Derived Sulfated Polysaccharides  191 HOH2C HO

O H2C HO

O HO

O

n

O HO

O

OH

Beta-1,3

m Beta-1,6

Figure 8.7  Chemical structure of Laminaran.

8.4.5 Xylan and Porphyran Xylan has not yet been shown to be economically viable and just a few uses have been identified. One of the 3- and 4-linked-d-galactosyl residues in its disaccharide units, porphyran, is comparable to agarose; however, some of the residues in porphyran are 6-sulfate [23]. Porphyra species have a sulphated polysaccharide known as porphyran (Figure 8.8), which is part of the galactan complex. Porphyran is a high-quality dietary fiber that is chemically related to agar [6]. Porphyran is a sulfated polysaccharide found in the red algae Porphyra vietnamensis, which may also be found in the red seaweeds. Porphyran includes numerous major components that have a significant impact, mostly through inhibiting Helicobacter pylori development [11]. To extract and identify bioactive molecules that may have benefits in the inflammatory

(a)

HO

OMe O

O

OH O

O OH

OH

(b)

HO

n

O

OR O

O

SO3–

O

OH

Figure 8.8  Chemical structure of Porphyran.

O OR

n

192  Next-Generation Algae: Volume II O H

H

O OH HO

HO

Taondiol

O

HO

H

OH

H

Isoepitaondiol O

OH O

O

HO

Stypodiol Stypoldione O

Sargaquinone

O O HO

Sagaol

Figure 8.9  Chemical structure of isolated secondary metabolites from marine alga T. atomaria [21].

and therapeutic domains, further chemical study on the P. vietnamenis aqueous and alcoholic fractions is necessary. Among natural sources, red algal components have a complicated mechanism that demands attention for protecting against stomach ulcers. Porphyra is one of these algae that contains several important components, especially via controlling H. pylori, which has a significant impact on pathogen proliferation [11]. In MS/MS analysis of the methanolic extract of Gracilaria changii (MeOHGCM), red algae extracts revealed the presence of methyl10-hydroxyphaeophorbide and 10-hydroxypheophytinea, recognized chlorophyll proteins, and a number of unknown molecules [9]. During U937 cell differentiation, treatment with 10 g/ml MeOHGCM6 extract significantly reduced the degree of IL-6 gene expression and TNF-α reaction with inhibitory activity that is comparable to that of a standard drug (betamethasone). No cytotoxic effects have been detected in cells treated with the 10 g/ml MeOHGCM6 extract. When MeOHGCM6 extract was supplied to rats at a rate of 500 mg/kg bw, the entire ethanol-induced stomach

Algal-Derived Sulfated Polysaccharides  193 lesions scale was reduced by > 99 percent (p 0.05). This shielding effect was the same as that produced by OMP. The pH of the stomach mucus fell in a dose-dependent manner from 5.51 to 3.82, with substantial increases. The authors of various studies claimed that the mass spectrometric standardized methanolic extract of Gracilaria changii had gastroprotective, anti-inflammatory, and anti-ulcerogenic effects [6, 9, 11, 12]. In the future, more research into the extract’s active constituent and mode of action is required. According to a recent study on algae antiulcer activity, the aqueous extract of Turbinaria conoides reduced gastric ulceration to 50.98 from 99.85% at a dosage of 500 mg.kg-1 [24]. Seaweed acetone extracts from Gracilaria crassa, Laurencia papillosa, and Turbinaria ornata showed antiulcer, wound-healing and hepatoprotective properties. Chemical structure of isolated secondary metabolites from marine alga of Turbinaria species include sagaol, Sargaquinone, stypoldione, Stypodiol which showed in Figure 8.9. In comparison to Laurencia papillosa, G. crassa was notable since it displayed noticeable and remarkable wound-healing and hepatoprotective activity, as well as strong antiulcer activity. G. crassa has the potential to be of biomedical significance.

8.5 Conclusion Algae are a diverse category of aquatic photosynthetic creatures that make up around 10% of the plant kingdom. Based on their biological structure, they are classified as macroalgae (red, green, and brown seaweed) and microalgae (tiny seaweed) (blue-green algae; normally unicellular organism). The three major forms of photosynthetic algae mentioned in this work are generally fat-soluble producing organisms, which from ancient times have been used for treating different ailments in humans. Since algae are scientifically proven for the antibiotics activities they exhibit, scientists have highlighted the urgent need for a novel antiulcer medication derived from natural sources to replace the presently utilized pharmaceuticals of questionable safety and effectiveness.

References 1. Sharifi-rad M., Valere P., Fokou T., Id F.S., Martorell M., Ademiluyi A. O. and Salehi B., Antiulcer Agents: From Plant Extracts to Phytochemicals in Healing Promotion. 23, 17, Pp. 1-35, 2018. https://doi.org/10.3390/ molecules23071751.

194  Next-Generation Algae: Volume II 2. Falcão HD. S., Leite JA., Barbosa-Filho J., M., De Athayde-Filho, PF., Chaves M., Moura, MD., Batista LM., Gastric and duodenal antiulcer activity of alkaloids: A review. 13 5, Pp. 3198–3223, 2008. https://doi.org/10.3390/ molecules13123198 3. Kirsner JB., Blossoming of gastroenterology during the twentieth century. World Journal of Gastroenterology, 10, 2004. https://doi.org/10.3748/wjg.v10. i11.1541 4. Besednova NN., Zaporozhets TS., Somova LM., & Kuznetsova TA,. Review: “prospects for the use of extracts and polysaccharides from marine algae to prevent and treat the diseases caused by Helicobacter pylori”. Helicobacter, 20 2, Pp. 89–97, 2015. https://doi.org/10.1111/hel.12177 5. Awaad AS., El-meligy RM., and Soliman GA., Natural products in treatment of ulerative colitis and peptic ulcer. J. Saudi Chem. Soc., 17 1, Pp. 101–124, 2013. https://doi.org/10.1016/j.jscs.2012.03.002 6. Usman A., Khalid S., Usman A., Hussain Z., and Wang Y., Algal Polysaccharides, novel application, and utlook. Algae Based Polymers, Blends, and Composites. Chem. Biotech and Materials Sci., Pp. 115–153, 2013. https://doi.org/10.1016/B978-0-12-812360-7.00005-7 7. Ryan AJ., Peptic ulcer disease: Introduction. Postgrad. Med., 63 4, Pp. 81, 1978. 8. Nagaoka M., Shibata H., Kimura-takagi I., and Hashimoto S., Anti-ulcer effects and biological activities of polysaccharides from marine algae. Biofactors 12 1, Pp. 267–274, 2000. http://dx.doi.org/10.1002/biof.5520120140 9. Shu M., Appleton D., Zandi K., and Abubakar S., Ulcerogenic effects of red algae Gracilaria changii (Gracilariales, Rhodophyta) extract. BMC Complementary and Alternative Medicine 2013, 13, 6, Pp. 1-13, 2013. http:// www.biomedcentral.com/1472-6882/13/61 10. Mahmoud AH., Farrag EM., and Fayed DB., Antiulcer activity of Citharexylum quadrangular jacq leaves (ethanolic extract) on experimentally induced gastic ulceration in rats, Med. 5 2, Pp. 145–159, 2016. 11. Bhatia S., Sharma K., Sharma A., Nagpal K., and Bera T., Anti-inflammatory, analgesic and antiulcer properties of Porphyra vietnamensis. Avicenna J. Phytomedicine, 5 1, Pp. 69–77, 2015. https://doi.org/10.22038/ajp.2014.3758 12. Pati MP., Sharma S., Das, Nayak L., and Panda CR., Uses of seaweed and its application to human welfare: A review. Int. J. Phar. and Pharma Sci., 8 10, Pp. 12–20, 2016. https://doi.org/10.22159/ijpps.2016v8i10.12740 13. Kovac DJ., Simeunović JB., Babic OB., Misan AC., and Milovanovic IL., Algae in food and feed. Food and Feed Res., 40 1, Pp. 21–31, 2013. 14. Akira S., Toll-like receptor signaling and its inducible proteins. Microbiol Spectrum. 4 6, Pp. 447-453, 2017. 15. Bhatia S., Rathee P, Sharma K, Chaugule BB., Immunomodulation effect of sulphated polysaccharide (porphyran) from Porphyra vietnamensis. Int. J. Bio. Macromol., 57, 1, Pp. 50–56, 2013.

Algal-Derived Sulfated Polysaccharides  195 16. Wang Z., Xie J., Shen M., Nie S., and Xie, M., Sulfated modification of polysaccharides: Synthesis, characterization and bioactivities. Trends in Food Science and Technology, 74, Pp. 147–157, 2018. https://doi.org/10.1016/j. tifs.2018.02.010. 17. Thomas NV., and Kim SK., Potential pharmacological applications of polyphenolic derivatives from marine brown algae. Environmental Toxicology and Pharmacology, 32, 3. Pp. 325–335, 2011. https://doi.org/10.1016/j. etap.2011.09.004 18. Olasehinde TA., Mabinya LV., Olaniran AO., and Okoh AI., Chemical characterization of sulfated polysaccharides from Gracilaria gracilis and Ulva lactuca and their radical scavenging, metal chelating, and cholinesterase inhibitory activities. International Journal of Food Properties, 22, 1, Pp. 100– 110, 2019. https://doi.org/10.1080/10942912.2019.1573831 19. Alassali, A., Cybulska, I., Brudecki, G. P., Farzanah, R., & Thomsen, M. H., Advanced Techniques in Biology & Medicine Methods for Upstream Extraction and Chemical Characterization of Secondary Metabolites from Algae Biomass, 4(1), 1–16, 2016. https://doi.org/10.4172/2379-1764.1000163 20. Gamal AE., Biological importance of marine algae. Saudi Pharmaceutical J., 18(1), 18, 1, Pp. 1–25, 2010. Retrieved from https://doi.org/10.1016/j. jsps.2009.12.001 21. Shahidi F., and Rahman MJ., Bioactives in seaweeds, algae, and fungi and their role in health promotion. J. Food Bioactives, 2, 1, 2018. https://doi. org/10.31665/jfb.2018.2141 22. Çakir AŞ., Ozyilmaz A., Demirci S., and Şükran ÇA., A study on the rich compounds and potential benefits of algae: A review. The Pharma Innovation Journal, 6, 12, Pp. 42–51, 2017. Retrieved from www.the pharmajournal.com 23. Kim, J. H., Lee, J. E., Kim, K. H., & Kang, N. J., Beneficial effects of marine algae-derived carbohydrates for skin health. Marine Drugs, 16, 11, pp. 1–20, 2018. https://doi.org/10.3390/md16110459. 24. Murugan A., and Senthil L., Antiulcer, wound healing and hepatoprotective activities of the seaweeds Gracilaria crassa, Turbinaria ornata and Laurencia papillosa from the southeast coast of India, Brazilian J. Pharm., 49, 4 pp. 669673, 2013.

9 Pharmacological and Antioxidant Attributes of Significant Bioactives Constituents Derived from Algae Juliana Bunmi Adetunji1*, Abigail Omotayo Agbolade1, Omowumi Oyeronke Adewale1, Ikechukwu P. Ejidike2, Charles Oluwaseun Adetunji3 and Isreal Olu Oyewole1 Department of Biochemistry, Faculty of Basic and Applied Sciences, Osun State University, Osogbo, Nigeria 2 Department of Chemical Sciences, Faculty of Science and Science Education, Anchor University, Lagos, Nigeria 3 Department of Microbiology, Biotechnology and Nanotechnology Laboratory, Edo University Iyamho, Edo State, Nigeria 1

Abstract

Algae are marine or freshwater phytosynthetic microscopic organisms with fast growing rate. Moreover, reports have revealed that they have diverse bioactive substances which have proven potent in enhancing nutrients in food and feed, promote good health, and serve to maintain economic sustainability in industries over the coming years. Algae have been documented to be a reservoir for several secondary metabolites such as terpenoid, taondiol, stypodiol, isoepitaondiol; and hydroxycinnamic and cinnamic acids. In this chapter, the chemical structure of some bioactive constituents used in the formulation of high-valued therapeutic drugs and their benefit are documented. Also, some natural bioactives like fucoidan, phycocyanin, phycocyanobilin, laminarin, fucosterol, saccharides, phlorotannins, dieckol, D-isofloridoside and phycoerythrin from algae were revealed. Applications of these bioactive constituents as immune booster, neuroprotective, anti-inflammatory, antioxidants, anticancer, wound healing, antimicrobial, etc., were also stated. Keywords:  Algae, natural bioactives, pharmacological, antioxidant, health *Corresponding author: [email protected]; [email protected] Charles Oluwaseun Adetunji, Julius Kola Oloke, Naveen Dwivedi, Sabeela Beevi Ummalyma, Shubha Dwivedi, Daniel Ingo Hefft and Juliana Bunmi Adetunji (eds.) Next-Generation Algae: Volume II: Applications in Medicine and the Pharmaceutical Industry, (197–222) © 2023 Scrivener Publishing LLC

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9.1 Introduction As a potent nutrient source of food and feed and a health-promoting substance, algae will continue to be a part of sustainable industrial development with economic benefits in coming years. For instance, the commercially available, naturally rich antioxidant astaxanthin, which is derived from microalgae (Haematococcus pluvialis), has diverse applications in industries like nutraceuticals, cosmetics, food, and aquaculture. Also, astaxanthin has been documented to be significant in decreasing oxidative stress, thereby maintaining the state of a person’s health. Therefore, astaxanthin will continue to play a part in potent microalgal products in high demand for future use [1]. Furthermore, a complex polysaccharide called fucoidan (FUC) produced by brown marine algae has L-fucose and sulfate functional constituents with therapeutic benefits in the management and treatment of many human disorders. Due to its numerous chemical structures and antioxidant potentials, it is used to formulate substances with high pharmacological properties to manage and treat inflammation, cancer, diabetes, viral and neurodegenerative situations [2]. Meanwhile, fucoidan involvement in alterations of intracellular signaling pathways, ROS generation regulation, and cell survival and death processes maintenance contributes to disease prevention via the immunomodulatory and antioxidant capability of FUC [2]. It was documented that FUC medicinal potential is due to the abundant bioactive constituents with an enhanced confirmational structure that contributes to the therapeutic potency of drugs synthesized from FUC [2]. Fucoidan is mostly sourced from brown algae and it is an extremely hygroscopic negatively charged polysaccharide. Reports by Salehi et al. documented that A. nodosum, M. pyrifera, L. digitata, and F. vesiculosus leaves are high in FUC [3]. It was gathered that FUC could be soluble in water and acids and advantageous in hepatocytes damage and urinary system failure protection [4]. Fucoidan was reported to be a multifunctional marine polymer by Huang et al., which is often extracted from seaweed through solvent, acid, and high-temperature extraction [5]. This extracted bioactive is strategically deployed in the prevention, management, and therapeutic intervention of diseases [2].

9.1.1 Brown Algae 9.1.1.1 Fucoidan and Its Bioactivity Huang et al. reported that fucoidan (FUC) derived from algae has lots of biological activities that can serve as an antioxidant, anti-inflammatory,

Antioxidant and Pharmacological Capability of Algae  199 anticoagulant, immunomodulatory, etc. [6]. The authors demonstrated the efficacy of fucoidan on microbiome and inflammation in high-fat-­dietinduced obese mice. FUC0.5(50mg/(kg.d) and FUC2.5(250mg/(mg.d) of FUC were fed to mice for 35 days and it was revealed that the bioactive substance in FUC suppressed the obesity in mice and restored tissue damage via decline in the body mass index and weight, enhanced organ index, liver steatosis, decreased body weight, and small intestine structure enhancement. Huang and colleagues also observed that FUC treatment caused a decline in tumor necrosis factor-α, triacylglycerol, LDL-c, lipopolysaccharides, cholesterol, and bile acid; but stimulated high-density lipoprotein cholesterol [6]. The authors revealed that it enhanced gut microbiota structure and caused an abundance increase in Shannon diversity index, Faecalibacterium prausnitzii, and evenness as examined via PCR and gradient gel electrophoresis denaturation. Used as a functional food, FUC could protect against obesity and modulate the microbiota of the gut [6].

9.1.1.1.1 Fucoidan Structure

An active substance derived from brown algae extracellular matrix is a fucose-enriched sulphated polysaccharide referred to as fucoidan (FUC). Moreover, FUC derived from diverse brown algae species consists of sulphate and L-fucose groups with small mannose, xylose, rhamnose, glucuronic acid, galactose, glucose, and arabinose [7]. The synthesized fucoidan is named after the composition of the monosaccharides, such as galactofucan (galactose and fucose), rhamnogalactofucan (rhamnose, galactose, and fucose) and rhamnofucan (rhamnose and fucose), and therefore determines the variation in the structure of the seaweeds. Coronado-Reyes et al. found that there are two forms of fucoidan: one with repeated units of L-fucopyranose with 1→3 linkage and one with alternating and recurring units of L-fucopyranose with 1→3 and 1→4 linkage [8]. The fucoidan obtained from brown alga (Sargassum thunbergia) was used as a source of glucuronomannan oligosaccharides (Gs) and the sulfated molecule was derived by sulfation to give sulphated glucuronomannan oligosaccharides (SGs). The authors found that the sulfation process as observed through NMR techniques revealed in this increasing order Man-C6 > Man-C4 > Man-C1R > GlcA-C3 > Man-C3 > GlcA-C2. Furthermore, there was an assessment of superoxide and OH radical scavenging activity, DPPH as well as the reducing power, but all have higher degree of polymerization which enhanced their reactivity with the exception of the hydroxyl radical quenching activity [9]. Their result

200  Next-Generation Algae: Volume II also revealed an increased sulfate content, low reducing power activity and DPPH quenching power, while the opposite was seen in superoxide radical quenching activity. In all, Gs and SGs antioxidant contents are higher when compared with fucoidan and as such could be a key source of antioxidants [9]. Also, Sargassum horneri serves as a fucoidan source and the products derived from enzymatic transformation was over 20 kDa. Fucoidan was hydrolyzed by recombinant fucoidanase FFA1, and its fraction of higher molecular weight was fractionated with anion-exchange chromatography. Meanwhile, three diverse molecular weights (63−138 kDa) of sulfated polysaccharides were obtained [10] while NMR spectroscopy was used to analyze the structures resulting in branched polysaccharide with repeating →3-alpha-L-Fucp(2SO3−)-1→4alpha-L-Fucp(2,3SO3−)-1→ fragment backbone and alpha-L-Fucp-1→2-­ alpha-L-Fucp-1→ or alpha-L-Fucp-1→3-alpha-L-Fucp(4SO3−)-1→ units side chains linked to the Carbon 4. Interestingly, the F3 fragment differs in its side chain and molecular weight from other FUC fragments with radiosensitizing and anticancer activities [10]. The interaction existing between chitosan/fucoidan nanoparticles (CTS-FUC-NP) was established using Fucus evanescens algae. The structure of the nanonized compound was ascertained by NMR spectroscopy. Moreover, about 55% of other fucoidan structures were contained therein, which include a long-sulfated sequence of α-L-­fucopyranose fragments at Carbon 2. Furthermore, the influence of the FUC/CTS ratio on the nanoparticles’ zeta potential and size was examined. The 3D model structure of the FUC and CTS regular section was then carried out using molecular docking to show whether the polymer occupies the complex exterior depending on their ratio [11]. The authors also reported that the thermodynamic parameters of the fucoidan-chitosan binding process indicate that changes in FUC and CTS molecules conformation were observed during the effective binding interaction [11].

9.1.1.2 Benefits Derived from Fucoidan 9.1.1.2.1 Neuroprotective

Kim et al. reported on the neuroprotective role of sulfated fucoidan derived from brown algae on cerebral ischemia (CI) and its mechanism of action via gerbil model of CI, which revealed that after 5 min, CI caused the removal of pyramidal neurons in the hippocampal cornu ammonis 1 (CA1) [12]. Treatment with 25 and 50 mgkg-1 FUC was done via i.p./day

Antioxidant and Pharmacological Capability of Algae  201 .

for a period of 5 days prior to tGCI. The result showed that 50 mg/kg FUC pretreatment decreased hyperactivity of CI-induction and guide CA1 pyramidal neurons against CI [12]. There was also inhibition of astrocytes activations and microglia in the ischemic CA1 region by 50 mgkg-1 FUC pretreatment. Consequently, a significant reduction of superoxide anion radical and 4-hydroxy-2-noneal produced by 50 mg/kg FUC was seen in the ischemic CA1 region with corresponding increase in superoxide dismutase 1 and 2 (SOD1 and SOD2) expressions prior and after CI in the CA1 pyramidal neurons. Interestingly, treatment with SODs inhibitor (diethyldithiocarbamate) with the FUC-treated gerbils, however, suppressed the FUC-mediated neuroprotection. It was concluded that protective role of FUC from CI is via the decrease in oxidative stress with corresponding increase of SODs and activated glial cells attenuation by FUC results in protection of CI [12].

9.1.1.2.2 Immune Booster

In another study, Jiang and colleagues isolated fucoidan from Stichopus chloronotus (Sc), known as sea cucumber, and used it as a traditional tonic food in southern China because of its high nutritive value [13]. It is the key bioactive polysaccharide in the plant which has proven to be effective in immune activation when studied on RAW264.7 cells. The authors observed that Fucoidan isolated from Sc could activate RAW264.7 cells through stimulation of TNF-alpha, Interleukins-6, NO, and Interleukins-10 production. Toll-like receptors 4 and 2 (TLR4 and TLR2) were observed from the RT-PCR analysis, which assist fucoidan-Sc enhancement in the downstream NF-κB signal pathway [13]. More so, the observed molecular weight and chemical structure revealed Fucoidan-Sc’s major role in NO production enhancement. The study found that FUC-Sc degraded product with molecular mass 113.1 × 104 Da activities were more than of the intact FUC-Sc resulting from the peak chain length. Jiang et al. therefore found that the immunostimulating activity of FUC-Sc could be through activation of TLR2/4 in the NF-κB pathway [13]. The pharmaceutical and food industries have seen polysaccharide derived from marine algae as a potent therapeutic source. The substance consists of a sulfate group and L-fucose that contributes to its excellent bioactive function as anticoagulant, antitumor, antithrombotic, anti-inflammatory, immunoregulatory, and antiviral [14]. Fucoidan has been established to be protective to gastrointestinal tract, bone health, angiogenesis, and eases metabolic syndrome.

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9.1.1.2.3 Anti-Inflammatory

Zhu et al. isolated different Mw fucoidan from Holothuria tubulosa (Ht-FUC) and then investigated Ht1/2/3/4 chain conformation in metabolic inflammation via a co-cultured medium of macrophages and adipocytes in vitro [15]. The authors confirm the efficacy of Ht-FUC in vivo using obese mice maintained on a high-fat with sucrose diet (HFD w/Suc). However, the result revealed Ht-FUC’s capability in enhancing co-cultured macrophage M2 phenotypic polarization via the activation of PPARγ. Ht-FUC also degrades lipids present in the adipocytes of the co-culture through TLR4/ NF-κB-dependent pathway inhibition [15]. In-vivo study of Ht-FUC established the reduction in serum inflammation concentration lessened liver Kupffer cells M1/M2 polarization as well as inflammatory infiltration of adipose tissue epididymal. Ht-FUC was documented to have an ameliorative metabolic function on macrophage polarization and lipid catabolism lysis through improving FFA-induction in the co-cultured system and obese mice [15].

9.1.1.2.4 Antidiabetic

Another potential source of fucoidan bioactive is Ecklonia maxima. The compound FUC which is sulfated polysaccharides was documented to have several health benefits like in diabetes. In 2020, Daub et al. worked on hot water extraction of FUC from Ecklonia maxima and obtained 6.89% FUC containing L-fucose and sulfate (4.45 ± 0.25% and 6.01 ± 0.53%) with about 10 kDa molecular weight obtained from the water extraction [16]. The authors also reported that the integrity and structure of the obtained FUC are analogous to the previously reported one. The activities of carbohydrate digestive enzymes (alpha-amylase and α-glucosidase) were also investigated, and the result revealed that FUC at 0.27–0.31 mg.ml-1 IC50 range is a strong mixed-type inhibitor of α-glucosidase and a stronger antidiabetic than acarbose in managing type 2 diabetes [16].

9.1.1.3 Laminarin The brown marine algae dispersed among numerous seas in the world was observed to contain a high level of bioactive constituents which include ώ-3 fatty acids (FAs), polysaccharides, polyphenols, and carotenoids. Reports have shown that laminarin (LAM) formed through glucose monomers linkage byα-1→3 and β-1→6-glucosidic bonds is the stored form of carbohydrate mainly present in brown algae. LAM and LAM oligosaccharides are made up of 2–10 monomers and they have various biological activities

Antioxidant and Pharmacological Capability of Algae  203 and prebiotic properties [17]. Also, LAM and LAM oligosaccharides are important precursors for the production of bioethanol, which consists of lots of glucose monomers. It could be documented that brown-algae-­ derived LAM and LAM oligosaccharides have numerous uses in medicine, cosmetics, food, and bioenergy fields [17].

9.1.1.3.1 Laminarin Quantification

Becker et al. documented that marine alga releases several glycans with enormous biological functions as carbon and energy requirement for heterotrophic microbes [18]. The glycan organic matter was analyzed after being hydrolyzed to measurable monosaccharides. Also, to quantify the laminarin in the organic matter, glycan was digested via the use of selective enzymes. The data gathered from environmental metaproteome showed that the active enzymes carbohydrate derived from marine flavobacteria as substrates for algal-glucan laminarin hydrolysis, which yielded glucose and oligosaccharides as a product of digestion. The cloning of new glycoside hydrolases (GHs) from Formosa bacteria, two of which are endo-1→3-glucanases from GH16 and GH17 families, with that from GH30 an exo--1→6-glucanase, were then expressed, purified, and characterized [18]. The significant strain of Hel1_33_131 GH30 derived from Formosa sp. hydrolyzes the 1,6-glucose side chains, while -1,3-glucose of the laminarin was hydrolyzed by Formosa agariphila GH17A and GH16A strain. The authors observed that specificity profiling of FaGH17A and FbGH30 with the glucan oligosaccharides and polysaccharides library revealed the enzyme high specificity but FaGH16A caused complex glucans and -1,4-glucose hydrolysis. It was then resolved that the two strain above are more specific for laminarin quantification.

9.1.1.3.2 Benefit Derived from Laminarin 9.1.1.3.2.1 Antioxidant

Brown alga (Sargassum thunbergia) was exploited by Hu et al. and was claimed to have an oligosaccharides compound having high proportion of bioactives. Beta-1,3-glucanase derived from bacteria around marine water can serve as a tool that degrades LAM present in brown algae cell wall [19]. Beta-1,3-glucanase (laminarinase), MaLamNA was cloned, expressed and characterized by the authors from Microbulbifer sp. ALW1 in marine bacterium. Phylogenetically, ALW1 shows a clear difference of glycoside hydrolase families of a characterized laminarinase. However, expression and purification of 57.3  kDa recombinant laminarinase was heterologously done on Pichia pastoris GS115 cells [19]. The hydrolytic function

204  Next-Generation Algae: Volume II of MaLamNA was exerted against LAM and its highest activity revealed at pH 4.5–5.5 and 45 °C respectively, while it has extreme adaptability against high acidic and alkaline pH upon exposure. It was found that dithiothreitol (reducing agent) addition could boost MaLamNA activity significantly. The product obtained from MaLamNA hydrolysis of laminarin exhibited great antioxidant activities when compared with the undigested LAM. The above activity of MaLamNA support it use as industrial resources in LAM bioresource development [19].

9.1.1.4 Fucosterol Fucosterol derived from the brown algae Eisenia bicyclis has various benefits such as anticancer, antioxidant, and antidiabetic potentials. Interestingly, Mo et al. examined the protective functions of derived fucosterol pretreatment on acute liver necrosis induced in mice by Concanavalin A (ConA) with its molecular mechanisms of action [20]. Acute liver necrosis in BALB/c mice was induced with ConA (25 mgkg-1) while doses of 25, 50, and 100 mgkg-1 of fucosterol prepared with 2% DMSO were administered daily orally. The authors measured apoptosis, liver necrosis, and autophagy linked inflammatory cytokines at 2, 8, and 24 h. Fucosterol was found to have reduced liver marker enzymes, necrosis of the hepatic and ­interleukins-6, TNF-α, and interleukins-1β induced apoptosis. However, fucosterol was also noticed to suppress apoptosis as well as autophagy through Bcl-2 by upregulation, and reduced the level of functional Beclin-1 and Bax. In addition, NF-κB and P38 MAPK signaling reduction was accompanied by activation of PPARγ. All the authors observed that attenuation of injury of the liver caused by ConA is via P38 MAPK/ PPARγ/NF-κB signaling suppression, which contributed to its potency as therapeutic agent in liver injury management or treatment [20].

9.1.1.5 Saccharides 9.1.1.5.1 Alginate Oligosaccharides

In another experiment, Li et al. studied the beneficial potentials of extracted alginate oligosaccharides from Sargassum species on β-glucan isolated from Sparassis latifolia and was characterized via nuclear magnetic resonance spectroscopy [21]. It was used to refine sodium alginate from three species of brown marine algae, S. fusiforme, S. fulvellum, and S. horneri, and it was found that mannuronate produces guluronate (M/G) ratio (0.64 to 1.38). The fractions of oligosaccharides derived were solid

Antioxidant and Pharmacological Capability of Algae  205 fraction (SF), ethanol fraction (EF), and liquid fraction (LF), which were obtained from acid hydrolysis but analyzed through high-performance anion exchange chromatography using a pulsed amperometric detector and spectra of Fourier transform infrared (FTIR) spectroscopy. The S. fusiforme solid fraction gave the highest hydrolysate but low LF, while the M/G ratio was consistently on the high side [21]. The SF of S. fusiforme and LF of S. horneri were chosen for elicitation on S. latifolia, resulting in high yield of β-glucan ranging from 56.01 ± 3.45% to 59.74 ± 4.49%, respectively, in the stalk. Also, S. latifolia aqueous extract had antioxidant activities. Total polyphenol content, superoxide dismutase activity and 2,2’-Azino-bis(3ethylbenzthiazoline-6-sulfonate) radical quenching were triggered by alginate. Therefore, the extracted alginate oligosaccharides from brown algae could serve to improve mushroom nutritional value [21].

9.1.1.5.2 Polysaccharides

Polysaccharides possess anti-inflammatory, antioxidant and immunomodulatory potential. However, sulfated polysaccharide (PS) derived from the brown algae Turbinaria ornata was obtained at doses of 2.5, 5, 10 mgkg-1bw for 7 days prior to systemic inflammation induction with 10 mgkg-1 i.p. lipopolysaccharide (LPS). Thereafter, samples were subjected to molecular, biochemical and histopathological assessment. Some biochemical as well as molecular parameters, like AST, γGT, GSH, CK-MB, SOD, Grx, NFκB, LPO, IL1β, PI3k, IL6, IL10, iNOS, and Akt, were identified to determine PS mode of actions. The pretreatment with PS caused a significant suppression of the activities of CK-MB, AST and γGT raised by LPS in serum, while the level of mRNA, LPO, Grx, and IL6 were also reduced in the heart [22]. Upon induction with LPS a significant decline was seen in the activities of GSH and SOD. The PS also reduced IL6, thioredoxin (Trx), and elevated IL10 mRNA measurement in the heart, which supports its antioxidant and anti-inflammatory role. The PS also modulate the proinflammatory markers (IL1β and NFκB), oxidative marker (iNOS), and pPI3k/pAkt expressions majorly in the cardiac region, hence contribute to the immunomodulatory potency. Furthermore, an improvement was seen in the inflammatory pathology of the heart tissue when compared with the LPS control, as observed in histopathology analysis. Bhardwaj et al. documented that Turbinaria ornata macroalgae PS prevented LPS-induced inflammation systematically in the cardiac tissue, which could be attributed to the presence of glucopyranose and fucopyranose subunits [22].

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9.1.1.5.3 Heteropolysaccharide

In 2019, Zhang and colleagues reported that an upsurge in oxidative stress aggravates the aging process, hence the need to work on potential ways to reverse oxidative damage to cells in order to promote longevity [23]. The authors conducted their study on isolated Sargassum fusiforme heteropolysaccharide (SFPS) from brown algae in mice to suppress oxidative damage in their aging process. However, it was claimed that no report exists demonstrating the ability of SFPS to extend organism lifespan. Also, the SFPS component that enhanced the antioxidant activity responsible for the mechanism action was also not documented. The in-vitro radical scavenging assays carried out in this study revealed that fractions II of SFPS (SP2) exhibit strong antioxidant potentials [23]. Also, the survival rate of D. melanogaster exposed to stress was enhanced by a diet containing SP2 through significant improvement and reduced deposit of triacylglycerol at older age. The activities of some antioxidant enzymes like SOD, glutathione peroxidase (GSH-Px), and CAT were boosted by SP2 with a decline in oxidized glutathione (GSSG) and malondialdehyde (MDA) level in old flies. In addition, nuclear factor erythroid-2 like 2 expression levels and its downstream target genes was upregulated by SP2 while revealing a decline in Kelch-like ECH-associated protein 1 expression in old flies [23]. The high rate of survival of D. melanogaster treated with SP2 supplement under heat stress depends on the Nrf2/ARE route and the antioxidant potentials of SP2, though this process was inhibited by the addition of Nrf2/ARE pathway-specific inhibitors. It was concluded that SP2 derived from SFPs could improve aging in D. melanogaster through the Nrf2-mediated antioxidant signaling route [23].

9.1.1.6 Phlorotannins Phlorotannins were isolated from a species of brown algae called Ishige foliacea, which has anticancer, antioxidant, and anti-inflammatory potentials. Um et al. established the protective potential of phlorotannin-rich fraction derived from I. foliacea (PFRI) for neurons in mice induced with memory impairment using scopolamine. The authors administered supplementation containing 50 or 100 mgkg-1 PRFI for 42 days to scopolamine-induced memory impaired mice. The PRFI supplemented diets suppressed the activity of acetylcholinesterase and decreased lipid peroxidation levels with a corresponding increase in SOD activity and glutathione concentrations in the brains of mice [24]. PRFI triggers the regulation of brain-derived neurotrophic factor and tropomyosin receptor kinase B expression level,

Antioxidant and Pharmacological Capability of Algae  207 and extracellular cyclic AMP-response element-binding protein (CREB) and signal-regulated kinase (ERK) phosphorylation of hippocampus and the cerebral cortex involved in neuroplasticity. Consequently, PRFI is suggested to hinder memory impairment induced by scopolamine via ERKCREB-BDNF pathway regulation and oxidative scavenging [24].

9.1.1.7 Dieckol A phlorotannin called dieckol, which is extracted from the brown algae Ecklonia cava, has antioxidant potential. Kang et al. described the antioxidant thermostability of dieckol at different temperatures of 30, 60, and 90 °C for a week with DPPH and OH radical quenching activities with respect to its standard (ascorbic acid) [25]. The authors investigated apoptotic body formation and reactive oxygen species quenching activity of dieckol with DCF-DA assay, flow cytometry, propidium iodide and nuclear staining. Exposure of dieckol for a week revealed the stability of radical quenching activities of DPPH and OH- radicals. Dieckol also showed its protective potential against H2O2-induced apoptosis in Vero cells. Meanwhile, ascorbate revealed a decline in intracellular ROS and radical scavenging activities at 60 and 90 °C for the 4th and 3rd days, respectively [25].

9.1.2 Red Algae 9.1.2.1 D-Isofloridoside Yang et al. studied the biologically active constituents of Laurencia undulata, an edible red alga [26]. D-isofloridoside (DIF), an active compound with matrix metalloproteinases (MMP) and antioxidant inhibitory properties having molecular weight 940.68Da, was derived from L. undulata. The authors examined the processes used by DIF in the effective management of tumor metastasis and angiogenesis in human vascular endothelial cell (HUVEC) and HT1080 cell. DIF decreased the MMP-2/9 activity with inhibition of hypoxia-inducible factor-1α (HIF-1α) gene through mitogen-activated protein kinases and PI3K/AKT downstream regulation routes. In turn, vascular endothelial growth factor (VEGF) was downregulated in CoCl2-induced HT1080 cell [26]. The bioactive component DIF also suppressed VEGF receptor-2 expression, activated apoptosis, regulated PI3K/AKT, MAPK, NF-κB signal downstream routes, and ­platelet-derived growth factor (PDGF) production in VEGF-induced HUVEC was downregulated. It was then suggested that DIF could be a potent antitumor functional food utilized for suppressing tumor angiogenesis [26].

208  Next-Generation Algae: Volume II

9.1.2.2 Phycoerythrin Phycobiliproteins (e.g., phycoerythrin) are a group of important pigments isolated from red and blue-green algae. Reports revealed that food-derived bioactive constituents have the capability of antagonizing dysregulated targets in cellular signaling routes, hence exhibiting antineoplastic capabilities. Therefore, extraction of biologically active components from algae and the pharmaceutical activity determination will assist in deducing and predicting their specific molecular targets and also establish their safety or toxicity in normal tissues. Phycoerythrin (R-PE)-rich protein with hepatoprotective potential was isolated from Portieria hornemannii and examined in vitro on HepG2 cells using H2O2 as inducing agent and in vivo on Wistar hepatocellular carcinoma (HCC) rats. However, a dose-dependent decline was notice in cell viability of HepG2 after H2O2 induction but its effect was reverse with R-PE-rich extract treatment [27]. The enzymatic, non-enzymatic antioxidants and the liver marker enzymes studied in the in-vivo experiment were greatly suppressed in the induced HCC male Wistar rats after exposure to a carcinogen (N-diethylnitrosamine (DEN)). Interestingly, R-PE-rich protein extract treatment caused reversal of the parameters in the carcinoma rats. Moreover, alternative food supplements are beneficial in nutritional therapy because of their chemo-preventive effects against the development of cancer [27]. Phycoerythrin is a pigment protein extracted from Grateloupia filicina a red alga which is a potent target that could be used in the management and treatment of neurological disorders. In this experiment, astrocytes were treated with phycoerythrin extracted from G. filicina (PEGf), it was thereafter assessed for its antioxidative ability with H2O2. Hence, PEGf was suggested to be effective on astrocytes’ viability and proliferation downregulated under oxidative stress, which in turn stimulate H2O2 [28].

9.1.2.2.1 Anticancer Potential

Gracilaria pygmaea serves as a potent source of antioxidants in the southwest of Iran. In 2019, Hashkavayi et al. determined its antioxidant content using phenolic content, and DPPH radical scavenging ability with the voltammetric method [29]. The authors also carried out alteration of surface carbon screen-printed electrode and electrochemical sensors production with gold nanoparticles synthesis. The ability of the red algae extract to suppress oxidizing agent was carried out with the use of ferrocene as the target species. The phenolic and antioxidant content was evaluated by

Antioxidant and Pharmacological Capability of Algae  209 electrochemical sensor to be 2.24 and 0.8 mgg-1. The extract was shown to suppress radical and oxidizing compounds, while its anticancer activity and effect were determined on MCF-7, HT-29, SKMES-1 and SKOV3 cell lines with MTT assay [29].

9.1.3 Blue-Green Algae 9.1.3.1 Phycocyanin and Phycocyanobilin Zheng et al. studied the antioxidative and renal protective potentials of phycocyanin derived from blue-green algae (Spirulina platensis), and its chromophore phycocyanobilin, whose structure looks like that of biliverdin, was examined on type 2 induced diabetes in mice and rodents [30]. The experiment was carried out by pretreatment with 300 mg/kg phycocyanin for 10 weeks through oral route to guard against nephron mesangial expansion and albuminuria in diabetes mice and rodents, and a normalized fibronectin and tumor growth factor expression was seen. The bioactive compound phycocyanin also ameliorated NAD(P)H oxidase expression, and urinary and nephritic oxidative stress markers. Furthermore, administration of 15 mg/kg phycocyanobilin for 14 days through oral route caused a suppression of NADPH-dependent superoxide release in nephritic mesangial cultured cells. It was concluded that phycocyanin and phycocyanobilin supplement could be a potent therapeutic approach in diabetic nephropathy prevention [30]. A dietary inclusion capable of reducing the cholesterol level is Spirulina platensis. The alga is also a rich in constituents of tetrapyrrolic compounds, a key antioxidant and antiproliferative agent like bilirubin molecule. A study done by Koníčková et al. evaluated the potency of tetrapyrroles derived from S. platensis as an anticancer agent [31]. The antiproliferative efficacy of phycocyanobilin (PCB) and chlorophyllin were also examined on numerous cancer cell lines of human and xenotransplanted exposed mice. S. platensis therapeutics efficacy was examined on mitochondrial production of free radicals and glutathione redox status. The experimental therapeutics in a dose-dependent manner triggered a significant decrease in pancreatic cancer cell lines proliferation in vitro [31]. Blue-green algae are key constituents in the production of nutritional supplements because they contain active and non-protein substances with biological activity. Spirulina was documented to have numerous antioxidants like ascorbic acid, carotenoids and flavonoids, which exert a protective role against oxidative damage to cells. However, polyphenolic compounds identified in blue-green algae were established to protect and

210  Next-Generation Algae: Volume II

Table 9.1  Antioxidant compounds derived from algae. S/N

Class of algae

Species of algae

Antioxidant derived

Function/uses

References

1.

Rhodophyta (Red algae)

Osmunda spectabilis sp. Porphyra perforate sp.

Beta-carotene

Antioxidants, anticancer, antitumor use as pharmaceutical additives and food supplements

[33]

Stenogramme interrupta sp.

Carrageenans isolated from cystocarpic and tetrasporophytic plants Carraguard, a carrageenan-based microbiocide

Antiherpetic activity

[34]

Restricts in-vitro expression of HIV and all sexually transmitted diseases

[35]

Chondrus crispus sp.

Ascorbic acid

Wound healing and aids collagen production, supports immune system; Use in pharmaceutical additives and food supplements

[36]

Mastocarpus stellatus sp.

Ascorbic acid

Wound healing and aids collagen production; supports immune system

[36]

(Continued)

Antioxidant and Pharmacological Capability of Algae  211

Table 9.1  Antioxidant compounds derived from algae. (Continued) S/N

2.

Class of algae

Phaeophyta (Brown algae)

Species of algae

Antioxidant derived

Function/uses

References

Kappaphycus alvarezii sp.

Vitamin A

Cell growth and development (wound healing properties), supports vision, supports immune system

[37]

Corallina sp.

Polysaccharides possessing antiviral activities

Strong antiviral activity against herpes simplex virus 1 and 2

[38]

Taonia atomaria sp.

Terpenoids Taondiol Stypodiol Isoepitaondiol Hydroxycinnamic and cinnamic acids

Antimicrobial, anticonvulsant, antioxidant, anesthetic, antiinflammatory, antiseptic, anticancer, antitubercular; disinfecticide; and antiParkinson’s activity

[39]

Sargassum sp.

Ascorbate

Antioxidant to protect cellular components from free radical damage

[40]

(Continued)

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Table 9.1  Antioxidant compounds derived from algae. (Continued) S/N

3.

Class of algae

Chlorophyta (Green algae)

Species of algae

Antioxidant derived

Function/uses

References

Padina arborescens sp. Sargassum patens sp.

Fucoidan-derived polysaccharide from brown algae

Strong antiviral activity towards herpes simplex virus 1 and 2 (HSV type 1 & 2) Antiviral activity towards RSV Anti-inflammatory compounds

[38, 41–43]

Laminaria sp.

Phlorotannins

Anticancer, antitumor activities

[44–46]

Halimeda sp. Caulerpa sp.

Catechin Epicatechin Gallate Flavonoids

Polyphenols with anticardiovascular and anticancer properties; Promotes brain function; improves digestion

[47]

Dunaliella salina sp.

Beta-carotene

Antioxidants, anticancer, antitumor use as pharmaceutical additives and food supplements

[48]

(Continued)

Antioxidant and Pharmacological Capability of Algae  213

Table 9.1  Antioxidant compounds derived from algae. (Continued) S/N

Class of algae

Species of algae

Antioxidant derived

Function/uses

References

Chlorella vulgaris sp.

Ascorbic acid; source of potential vitamins like water and fatsoluble, e.g., A, B1, B2, B6, C, E, nicotinate, biotin, folic acid and pantothenic acid

The algae contain Chlorella Growth Factor derived from Chlorella RNA/DNA. It triggers cellular regeneration/ repair, aids collagen production, and supports immune system Has preventive action on disorders like gastric ulcers, atherosclerosis, hypercholesterolemia, tumor development and constipation

[49]

Haematococcus pluvialis sp.

Carotenoids, astaxanthin

Powerful antioxidant properties against free radicals and oxidative stress; antiulcer activities

[51]

Bryopsis sp.

Kahalalide F (KF)

Anticancer and antitumor properties

[52–54]

[50]

(Continued)

214  Next-Generation Algae: Volume II

Table 9.1  Antioxidant compounds derived from algae. (Continued) S/N

Class of algae

Species of algae

Antioxidant derived

Function/uses

References

4.

Blue-green algae

Spirulina (Arthrospira)

High protein content

Anti-hyperlipidemia, nephroprotective, antihypertensive, growth of intestinal Lactobacillus, and elevated serum glucose suppression Suppresses HIV progression in human T-cells, Langerhans cells in the lymphatic systems and other organs, peripheral blood mononuclear cells; and boosts immune system

[49, 55]

Cardioprotective and cholesterol lowering potentials

[58]

Mucopolysaccharides

[56, 57]

Antioxidant and Pharmacological Capability of Algae  215 recover neuronal cells’ functions via brain-derived neurotrophic factor (BDNF) production in the brain glial cells [32]. In preparing Spirulina platensis aqueous extract, all the protein component was removed to have a protein-free extract, whose effect was investigated on C6 glioma cells in the BDNF gene transcription. It was noticed that the protein-free extract stimulates the levels of BDNF mRNA via heme oxygenase-1 expression in the glioma cells. The observed results then suggest that protein-free Spirulina extract could control the brain function indirectly via glial cell activity [32]. The antioxidant potential of algae is shown in Table 9.1.

9.1.4 Other Potential Applications of Algae 9.1.4.1 Antioxidant and Anti-Tyrosine Capabilities Baek et al. evaluated 11 Sargassum species inhabiting the coasts of Korea to determine their antioxidant and anti-tyrosine capabilities [59]. The author found that the flavonoid content was from 2208–8233 × 10-2 mg quercetin equivalent/g dw, total phenolic content ranges from 2057–8897 × 10-2 mg gallic acid equivalent/g dry weight (dw), and anti-tyrosinase activity between 1330-12630 × 10 -2 mg kojic acid equivalent/dw. However, S. hemiphyllum and S. miyabei Yendo had high total antioxidant content, though S. miyabei Yendo contain tangible amounts of total flavonoid and phenolic contents. Moreover, S. fillicinum and S. miyabei Yendo revealed the highest anti-tyrosinase activity. Strong antioxidant capability was noticed in two derived meroterpenoid compounds from S. miyabei Yendo and S. serratifolium, and so it could be mobilized for commercial usage [59]. In another study carried out on Halopteris scoparia biological activities and the in-vivo toxicity of the various solvent extractions was carried out, along with phytochemical screening and antioxidant determination of the fractions. The cytotoxic activities of the extracts were tested on cell lines of breast adenocarcinoma (MCF7), colon colorectal adenocarcinoma (CaCo2), and cervical adenocarcinoma (HeLa) using MTT assay method. The total RNAs obtained for the gene expression analysis of the cell lines were evaluated by high-tech equipment (Real Time Ready Human Apoptosis Panel 96). Halopteris scoparia methanol extract toxicity and irritation effects were determined by the acute lethal dose (LD50) test and hen’s egg test using chorioallantoic membrane (HET-CAM) assessment respectively. It was revealed from the results that the extract had 3320 ± 1.41  × 10-2 mg GAE/g and 126 ± 0.95  × 10-2 mg QE/g total phenolic and flavonoid contents. The n-hexane extract in DPPH and methanol extract in ABTS+ showed high antioxidant activity while the MTT assay of the three extracts

216  Next-Generation Algae: Volume II resulted in a significant reduction in cell viability, especially in HeLa [60]. After treatment with the three extracts, the authors examined the apoptotic gene expressions; pro-apoptotic genes in both caspase-independent and caspase-dependent intrinsic and extrinsic pathways increased. In all, edible H. scoparia showed no irritation and toxicity in vivo, and could be used as a natural antioxidant for apoptotic/cytotoxic activities in human cancers [60].

9.2 Conclusion Algae as a reservoir of bioactive substances with numerous health benefits could serve as an alternative source of bioactive in the development of potent therapeutics that can be used to manage and treat numerous disorders and enhance the function of the immune system.

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218  Next-Generation Algae: Volume II α-glucosidase. International Journal of Biological Macromolecules, 2020. https://doi.org/10.1016/j.ijbiomac.2020.02.161 17. Huang, Y., Jiang, H., Mao, X., & Ci, F., Laminarin and Laminarin Oligosaccharides Originating from Brown Algae: Preparation, Biological Activities, and Potential Applications. Journal of Ocean University of China, 2021. https://doi.org/10.1007/s11802-021-4584-8 18. Becker, S., Scheffel, A., Polz, M. F., & Hehemann, J. H., Accurate Quantification of Laminarin in Marine Organic Matter with Enzymes from Marine Microbes. Applied and environmental microbiology, 83(9), e0338916, 2017. https://doi.org/10.1128/AEM.03389-16 19. Hu, Q., Yin, X., Li, H., Wang, X., Jiang, Z., Li, L., Ni, H., Li, Q., & Zhu, Y. Characterisation of a novel laminarinase from Microbulbifer sp. ALW1 and the antioxidant activity of its hydrolysates. International Journal of Food Science and Technology, 2021. https://doi.org/10.1111/ijfs.15041 20. Mo, W., Wang, C., Li, J., Chen, K., Xia, Y., Li, S., Xu, L., Lu, X., Wang, W., & Guo, C. Fucosterol protects against concanavalin a-induced acute liver injury: Focus on P38 MAPK/NF-κ B pathway activity. Gastroenterology Research and Practice, 2018. https://doi.org/10.1155/2018/2824139 21. Li, J., Kim, Y. W., Wu, Y., Choi, M. H., & Shin, H. J., Alginate-derived elicitors enhance β-glucan content and antioxidant activities in culinary and medicinal mushroom, sparassis Latifolia. Journal of Fungi, 2020. https://doi. org/10.3390/jof6020092 22. Bhardwaj, M., Mani, S., Malarvizhi, R., Sali, V. K., & Vasanthi, H. R., Immunomodulatory activity of brown algae Turbinaria ornata derived sulfated polysaccharide on LPS induced systemic inflammation. Phytomedicine, 2021. https://doi.org/10.1016/j.phymed.2021.153615 23. Zhang, Y., Xu, M., Hu, C., Liu, A., Chen, J., Gu, C., Zhang, X., You, C., Tong, H., Wu, M., & Chen, P., Sargassum fusiforme Fucoidan SP2 Extends the Lifespan of Drosophila melanogaster by Upregulating the Nrf2-Mediated Antioxidant Signaling Pathway. Oxidative Medicine and Cellular Longevity., 2019. https://doi.org/10.1155/2019/8918914 24. Um, M. Y., Lim, D. W., Son, H. J., Cho, S., & Lee, C., Phlorotannin-rich fraction from Ishige foliacea brown seaweed prevents the scopolamine-induced memory impairment via regulation of ERK-CREB-BDNF pathway. Journal of Functional Foods, 2018. https://doi.org/10.1016/j.jff.2017.10.014 25. Kang, M.-C., Kim, E.-A., Kang, S.-M., Wijesinghe, W. A. J. P., Yang, X., Kang, N.-L., & Jeon, Y.-J., Thermostability of a marine polyphenolic antioxidant dieckol, derived from the brown seaweed Ecklonia cava. ALGAE, 2012. https://doi.org/10.4490/algae.2012.27.3.205 26. Yang, S., Xiao, Z., Lin, L., Tang, Y., Hong, P., Sun, S., Zhou, C., & Qian, Z. J., Mechanism Analysis of Antiangiogenic d -Isofloridoside from Marine Edible Red algae Laurencia undulata in HUVEC and HT1080 cell. Journal of Agricultural and Food Chemistry, 2021. https://doi.org/10.1021/acs. jafc.1c05007

Antioxidant and Pharmacological Capability of Algae  219 27. Senthilkumar, N., Thangam, R., Murugan, P., Suresh, V., Kurinjimalar, C., Kavitha, G., Sivasubramanian, S., & Rengasamy, R., Hepato-protective effects of R-phycoerythrin-rich protein extract of Portieria hornemannii (Lyngbye) Silva against DEN-induced hepatocellular carcinoma. Journal of Food Biochemistry, 2018. https://doi.org/10.1111/jfbc.12695 28. Jung, S. M., Park, J. S., Shim, H. J., Kwon, Y. S., Kim, H. G., & Shin, H. S., Antioxidative effect of phycoerythrin derived from Grateloupia filicina on rat primary astrocytes. Biotechnology and Bioprocess Engineering, 2016. https://doi.org/10.1007/s12257-016-0369-0 29. Hashkavayi, A. B., Hashemnia, S., Osfouri, S., & Zarei, S., Electrochemical Study of Antioxidant Capacity of Gracilaria Pygmaea Macro-Algae Based on the Green Synthesis of Gold Nanoparticles: Assessment of Its Cytotoxic Effect on Four Cancer Cell Lines. Journal of The Electrochemical Society, 2019. https://doi.org/10.1149/2.0951912jes 30. Zheng, J., Inoguchi, T., Sasaki, S., Maeda, Y., Mccarty, M. F., Fujii, M., Ikeda, N., Kobayashi, K., Sonoda, N., & Takayanagi, R., Phycocyanin and phycocyanobilin from spirulina platensis protect against diabetic nephropathy by inhibiting oxidative stress. American Journal of Physiology - Regulatory Integrative and Comparative Physiology, 2013. https://doi.org/10.1152/ ajpregu.00648.2011 31. Koníčková, R., Vaňková, K., Vaníková, J., Váňová, K., Muchová, L., Subhanová, I., Zadinová, M., Zelenka, J., Dvořák, A., Kolář, M., Strnad, H., Rimpelová, S., Ruml, T., Wong, R. J., & Vítek, L. Anti-cancer effects of blue-green alga Spirulina platensis, a natural source of bilirubin-like tetrapyrrolic compounds. Annals of Hepatology, 2014. https://doi.org/10.1016/ s1665-2681(19)30891-9 32. Morita, K., Itoh, M., Nishibori, N., Her, S., & Lee, M. S., Spirulina NonProtein Components Induce BDNF Gene Transcription via HO-1 Activity in C6 Glioma Cells. Applied Biochemistry and Biotechnology, 2015. https://doi. org/10.1007/s12010-014-1300-9 33. Nadine S., Garcia-mendoza E. and Pacheo-Ruiz I., Carotenoid composition of marine red algae. Journal of Phycology 42(6):1208-1216, 2006. 34. Cáceres, P. J., Carlucci, M. J., Damonte, E. B., Matsuhiro, B. and Zúñiga, E. A., Carrageenans from Chilean simples of Stenogramme interrupta (Phyllophoraceae): Structural analysis and biological activity. Phytochemistry 53: 81-86, 2000. 35. Huskens, D., Schols, D., Algal lectins as potential HIV microbicide candidates. Mar Drugs. Jul;10(7):1476-1497, 2012. doi: 10.3390/md10071476. Epub 2012 Jul 10. PMID: 22851920; PMCID: PMC3407925. 36. Lohrmann N.L., Logan B.A. and Johnson A.S., Seasonal acclimatization of antioxidants and photosynthesis in Chondrus crispus and Mastocarpus stellatus, two co-occurring red algae with differing stress tolerances. Biol. Bull., 207: 225-232, 2004.

220  Next-Generation Algae: Volume II 37. Kumar K.S., Ganesan K. and Subba Rao P.V., Antioxidant potential of solvent extracts of Kappaphycus alvarezii Doty: an edible seaweed. Food. Chem., 107: 289-295, 2008. 38. Zhu, W. Ooi, V. E. C., Chan, P. K. S. and Ang, Jr. P. O., Inhibitory effect of extracts of marine algae from Hongkong against Herpes simplex Viruses. In: Proceedings of the 17th International seaweed Symposium. A. R. O. Chapman, R. J. Anderson, V. J. Vreeland, I. R. Davison, eds., Oxford University Press, Oxford, pp.159-164, 2003. 39. Nahas R., Abatis D., Anagnostopoulou M.A., Kefalas P., Va- gias C. and Roussis V., Radical-scavenging activity of Aegean Sea marine algae. Food. Chem., 102: 577-581, 2007. 40. Garcia-Casal M.N., Ramirez J., Leets I., Pereira A.C. and Quiroga M.F., Antioxidant capacity, polyphenol content and iron bioavailability from algae (Ulva sp., Sargassum sp. and Porphyra sp.) in human subjects. Br. J. Nutr., 101: 79-85, 2009. 41. Malhotra, R., Ward, M., Bright, H., Priest, R., Foster, M.R., Hurle, M., Blair, E. and Bird, M., Isolation and characterisation of potential respiratory syncytial virus receptor(s) on epithelian cells. Microbes Infect., 5: 123-133, 2003. 42. Prabhu, S., Vijayakumar S., Praseetha, P., Cyanobacterial metabolites as novel drug candidates in corona viral therapies: A review. Chronic Dis Transl Med. Mar 31;8(3):172-183, 2022. doi: 10.1002/cdt3.11. PMID: 35572950; PMCID: PMC9086949. 43. Huicheng Y., Mingyong Z., Shiyuan D., Zunying L. and Ruixue Li., Antipoliferative activity of phlorotannin extracts from brown algae luminaria japonica Aresch. Chinese Journal of Oceanology and Limnology 28, 122-130, 2010. 44. Jaswir, I. and Monsur, H. A., Anti-inflammatory compounds of macroalgae origin: a review. J. Med. Pl. Res., 5 (33): 7146-7154, 2011. 45. Bhakuni, D. S. and Rawat, D. S., Bioactive Marine Natural Products. Springer, New Delhi, India, 2005. 46. Catarino, M.D., Fernandes, I., Oliveira, H., Carrascal, M., Ferreira, R., Silva, A.M.S., Cruz, M.T., Mateus, N., Cardoso, S.M. Antitumor Activity of Fucus vesiculosus-Derived Phlorotannins through Activation of Apoptotic Signals in Gastric and Colorectal Tumor Cell Lines. Int J Mol Sci., 22(14):7604, 2021. 47. Devi K.P., Suganthy N., Kesika P. and Pandian S.K., Bio-protective properties of seaweeds: in vitro evaluation of antioxidant activity and antimicrobial activity against food borne bacteria in relation to polyphenolic content. BMC Complement Altern. Med., 8: 38, 2008. 48. Rad F. A., Aksoz N., Hejazi M. A., Effect of salinity on cell growth and β-carotene production in Dunaliella sp. isolates from Urmia Lake in northwest of Iran. African Journal of Biotechnology 10(12), pp. 2282-2289, 2011. 49. Becker, W., Microalgae in human and animal nutrition. In: Handbook of microalgal culture. A. Richmond, ed. Blackwell, Oxford, pp. 312-351, 2004.

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10 Utilization of Pharmacologically Relevant Compounds Derived from Algae for Effective Management of Diverse Diseases Olulope Olufemi Ajayi

*

Department of Biochemistry, Edo State University Uzairue, Edo State, Nigeria

Abstract

Algae are a diverse group of organisms containing various pharmacological compounds. These compounds include pigments, such as phycobilins, chlorophylls and carotenoids; polysaccharides, such as galactans, fucoidans and alginic acids; proteins and fatty acids among others. These pharmacological compounds exert anti-inflammatory, antioxidant, antiviral, antibacterial effects. etc., which are the basis of their exploration as alternatives in the treatment of diseases. This is contingent on the adverse side effects of conventional chemotherapeutic agents. In this study, the pharmacological potentials of compounds derived from algae in the treatment of certain diseases are discussed. There is evidence that these compounds provide safer and more effective alternatives to the conventional drugs in managing diseases. Keywords:  Algae, fucoidan, alginic acids, astaxanthin, fucoxanthin

10.1 Introduction Algae are an assorted group of organisms that possess numerous bioactive components. Their significance is based on their pigments, which are categorized into carotenoids, phycobilins and chlorophylls, to which their numerous biological benefits are attributed [1]. The following algae have been reported to possess antioxidant properties: Macrocystis pyrifera, Email: [email protected]

*

Charles Oluwaseun Adetunji, Julius Kola Oloke, Naveen Dwivedi, Sabeela Beevi Ummalyma, Shubha Dwivedi, Daniel Ingo Hefft and Juliana Bunmi Adetunji (eds.) Next-Generation Algae: Volume II: Applications in Medicine and the Pharmaceutical Industry, (223–250) © 2023 Scrivener Publishing LLC

223

224  Next-Generation Algae: Volume II Porphyra haitanensis, Bryopsis plumose, Laminaria ochroleuca, Bifurcaria bifurcata, Rhodella reticulata, Ecklonia cava among others [2]. Among others, the following algae were reported to possess anti-inflammatory properties: Ulva lactuca, Chlamydomonas hedleyi, Ishige okamurae, Undaria pinnatifida and Eisenia bicyclis [2]. Oxidative stress and inflammation are connected and are linked with diseases [3]. Oxidative stress induces transcription factors which could enhance the expression of genes related to inflammation [4]. Evidence indicates that oxidative stress-induced inflammation is related to the pathobiology of chronic ailments [4]. Algal polysaccharides have been reported for their antioxidative and anti-inflammatory properties, which enhance their therapeutic effects. The most common brown algal polysaccharides are fucoidans and alginic acids. Laminarans are also reportedly present in brown algae. Fucoidans are sulphated branched polysaccharides usually found in brown algal cell wall. They are biodegradable with highly minimal toxicity. Alginic acids are linear copolymers made up of β-D-mannuronic and α-L-guluronic acid residues [5]. They account for between 18 and 40% of brown algae mass [6]. Laminarans are made up of β1-3-linked–D-glucose residues. They account for between 10 and 35% of certain brown algae [7]. Red algae contain structural polysaccharides, including galactans such as carrageenans, agars, porphyrin and xylan. Carrageenans are sulphated polysaccharides containing galactose moieties and its derivatives. They are the most researched algal polysaccharides [7]. The most prominent green algal polysaccharide is the ulvan, which contains xylose, glucuronic acid, rhamnose and sulphate [7]. Apo-9-fucoxanthionone extracted from Sargassum muticum brown algae showed anti-inflammatory effect via mechanisms involving suppressing nitric oxide and prostaglandin E2 production, as well as suppressing IkB-α in macrophages [8]. This could be a potent therapy for inflammatory illnesses. Antineoplastic effect Sargassum muticum methanolic extract has been reported [9]. Reduction in angiogenesis coupled with hindered breast cancer growth was observed in the study. The antiproliferative and antioxidative effects of chlorophyta loaded on albumin nanoparticle on MCF7 and HepG2 cell lines have been reported [10]. This was evidenced by elevated levels of caspases 8 and 9 that suggest apoptosis with the possible involvement of the granzyme pathway. The principal bioactive constituent of this algae are phytocyanins. The study suggested chlorophyta could be a potent anticancer therapy specific for breast and liver cancers [10].

Algae-Derived Compounds for Disease Management  225 Phlorotannins are phenolic compounds derived from brown algae. Major sources include Ascophyllum nodosum and Fucus vesiculosus. Their antioxidant as well as anti-inflammatory properties are contributory to their significant therapeutic outcomes. In a study, phlorotannins inhibited benzo(α)pyrene-induced toxicity by inhibiting P2X7 receptor [11]. This is suggestive of its cancer chemopreventive potentials.

10.2 Algae in the Management of Some Diseases 10.2.1 Cancer Algae exhibit antioxidant mechanisms which have also been attributed to their antineoplastic effects; including activation of apoptosis, triggering nonspecific immune system, enhancement of the activity of natural killer cell, halting of angiogenesis and cell growth, particularly at G1 phase [12]. Evidence showed that consumption of seaweed enhanced the activities of superoxide dismutase and glutathione peroxidase enzymes [12]. Ethanolic extract of Gracillaria corticata inhibited breast cancer growth via apoptotic induction [12]. Certain studies have attributed algal anticancer effects to fucoidan. Fucoidan exerts its antineoplastic effects via the following mechanisms: suppression of neoplastic cells’ proliferation by altering mitosis, activation of apoptosis, inhibition of VEGF expression, thereby inhibiting angiogenesis and activation of immune system, hence, boosting the efficacy of natural killer and T cells [13]. Fucoidan elicited apoptosis in colon cancer cells which is controlled via both receptor-dependent apoptotic and mitochondrial death-dependent pathways [14]. The stimulation of apoptosis in HT-29 cancer cells by fucoidan was also reported in a different study. This was by mechanisms involving downregulation of IGF-IR, a major component of IRS-1/P13K/AKT pathway [15]. Apoptosis was induced in MCF-7 cells by fucoidan via the induction of chromatin condensation as well as fragmentation of nuclear interstitial DNA. Apoptosis could also be induced in MCF-7 cells via caspase 8-­ reliant pathway [16]. Inhibiting the expression of apoptosis-related gene by fucoidan could also be a means of inducing apoptosis [17]. It also reduces the transcriptional repressors, including Snail, Slug and Twit. This also suggests the anti-metastatic potentials of fucoidan. Apoptotic initiation in cancer cell can be via reactive oxygen species’ production. A report

226  Next-Generation Algae: Volume II showed apoptotic induction in hepatocellular carcinoma cells by reactive oxygen species-mediated mitochondrial pathway [13]. There is evidence indicating the ability of fucoidan to inhibit cancer cell proliferation. The proliferation of hepatoma cell lines BEL-7402 and LM3 treated with fucoidan was inhibited via the p38MAPK/ERK pathway. The activation of P13K was inhibited by fucoidan, this led to the activation of MAPK and the inhibition of ERK [13]. Report also showed the anti-­ leukemic effect of fucoidan via inhibition of the cell cycle. The treatment of NB4, a human acute myeloid leukemia cell, with fucoidan resulted in the activation of CIP1, P21 and WAF1, which led to cell cycle arrest [13]. Bladder cancer (T24) cells were inhibited by fucoidan in a reported study. Cancer cells’ viability was reduced via mechanisms involving triggering of G1 cell cycle arrest [13]. In a study in which bladder cancerous cells were treated with fucoidan, tumor growth was suppressed as evidenced by elevated level of cyclin-dependent kinase inhibitor1 (p21WAF1) [13]. Fucoidan effectively suppressed the growth of melanoma B16 cell transplanted mice. Fucoidan suppressed VEGF expression; hence, inhibiting angiogenesis [18].

10.2.2 Inflammatory Bowel Disease Inflammatory bowel disease (IBD) is typified as a group of chronic state of inflammation of the intestines without a known cause. Dysbiosis and oxidative stress have been associated with IBD. It is a complex disorder in which about 160 genes have been implicated [7]. Marine algae have been exploited for their therapeutic effects, particularly in the management of IBD. Of importance is the anti-inflammatory effect as well as the ability of algal polysaccharides to protect the gastrointestinal tract (GIT). This is because of their non-absorption in the upper part of the GIT, resistance to gastric fluid and the activities of the digestive enzyme [7]. Reports showed interleukin-17 (IL-17) has the primary pathogenetic factor of IBD. IL-17 promotes both pro-inflammatory cytokines, IL-11, IL-12, IL-23, IL-6, and anti-inflammatory cytokines IL-10 and TGF-β [7, 19, 20]. Immune modulation potential of brown algae polysaccharides makes them suitable as an alternative to conventional therapies in the management of IBD [6].

Algae-Derived Compounds for Disease Management  227

10.2.3 Osteoarthritis Osteoarthritis (OA) describes a progressive degenerative disorder of the joints. The pain experienced by individuals with OA is due to inflammation of the joints. Mediators of pro-inflammation, including IL-1β, reactive oxygen species and nitric oxide, are characteristic of OA [21]. Actinotrichia fragilis neutralized free radicals generated in OA in a report. This is attributed to sulfated algal polysaccharides, antioxidant amino acids (including glutamic acid, methionine, glycine and cysteine) and histidine, an anti-inflammatory compound [21]. This is the basis of the anti-inflammatory effects of A. fragilis [21]. Fucoidan extracted from Undaria pinnatifida significantly diminishes degeneration of cartilage and bones in rheumatoid arthritic mice [22].

10.2.4 Gastric Ulcers The gastroprotective and anti-inflammatory potentials of methanolic extract of Gracilaria changii were reported. These properties were attributed to pheophytin, as this extract contains methyl 10-hydroxyphaeophorbide a and 10-hydroxypheophytin. Reduction of gastric acidity and alteration of overproduction of macrophage-mediated pro-inflammatory cytokines are plausible mechanisms [23].

10.2.5 Neurodegenerative Diseases Neuroinflammation is implicated in the pathobiology of neurodegenerative diseases. Ideally, neuroinflammation of neuro-immune cells ­(astrocytes and microglia) is a mechanism of defense aimed at protecting the central nervous system from trauma, injuries and infections. Conversely, protracted neuroinflammation has a potential of damaging the neurons [24]. Glial toll-like receptor (TLR) stimulation sustains neuroinflammation in neurodegenerative disorders. The activation of TLR4 has been linked with Alzheimer’s disease [24]. Algal bioactive components are being explored to target pathways of neuroinflammation in the management of neurodegenerative diseases [24]. Impediment of pro-inflammatory enzymes, mitogen-activated protein kinase (MAPK) pathway modulation and activation of NK-κB are some anti-inflammatory mechanisms of marine algae [24].

228  Next-Generation Algae: Volume II In a report, astaxanthin ameliorated neuronal dysfunctions and degeneration consequent on subarachnoid hemorrhage induced in mice. Furthermore, the expressions of NF-κB, IL-1β, TNF-α were downregulated [25]. In another study, astaxathin reduced the activities of neuronal nitric oxide synthase (nNOS), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) [26]. Floridoside extracted from Laurencia undulata impeded nitric oxide and reactive oxygen species’ generation as well as reduced the activities of iNOS mRNA and protein as well as COX-2 [27]. Phlorofucofuroeckol B obtained from Ecklonia stolonifera impeded IκB-α/NF-κB and Akt/ERK/ JNK pathways [28]. Glycoprotein isolated from Undaria pinnatifida inhibited Beta-secretase 1, AChE and BChE activities as well as enhances neurite extension [29]. Dieckol obtained from Ecklonia cava downregulated p-38/NFκB pathway [30]. Also, 8,8’-bieckol, dieckol and eckol from Ecklonia cava in another study exhibited antioxidant and antiapoptotic effects [31]. Fucosterol isolated from Padina australis inhibited Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE) activities. It also impeded pro-inflammatory cytokines’ expression [32]. Isolates from Sargassum polycystum, Padina australis and Caulerpa racemosa also diminished the expression of pro-­ inflammatory mediators [33].

10.2.6 Diabetes Mellitus A high proportion of water and dietary fiber in algae makes them ideal for diabetics. Furthermore, the colonic fermentation of undigested fiber enhances the production of short-chained fatty acids that are of immense benefit to health. Additionally, the ease of digestibility of algal starches to disaccharides is beneficial [34]. Diabetic complications were reduced in mice administered fucoidan from Saccharina japonica. This was via the hypoglycemic effect of fucoidan. Furthermore, levels of TNF-α and IL-6 were also reduced in the animal model [35]. In another study, IL-6 and TNF-α levels were lessened in diabetic mice treated with fucoidan and fucoxanthin [36].

10.2.7 Hypertension Oxidative stress is involved in the pathogenesis of hypertension. Proinflammatory effects of hypertension boost free radical production, which may eventually reduce endothelial nitric oxide production [37].

Algae-Derived Compounds for Disease Management  229 Inflammation is the basis of endothelial disorder and ensuing endothelial damage [38]. Reports have shown the antioxidant effects of some algae which also exhibit antihypertensive effects. IQP, an angiotensin converting enzyme inhibitor isolated from Spirulina platensis, showed a high free radical scavenging potential [39]. Furthermore, peptide isolated from Chlorella. vulgaris effectively moped-up superoxide [40]. In another study, Chlamydomonas reinhardtii lowered systolic blood pressure in hypertensive rats [41]. In another study, Phe-Glu-lle-His-Cys-Cys (FEIHCC) isolated from Isochrysis zhanjiangensis impeded NF-κB, Akt and MAPK pathways, hence inhibiting inflammation as well as inducing Nrf2 pathway upon angiotensin II application [42]. Activation of Nrf2 minimized the expression of mediators of inflammation, including COX-2 and NO [43].

10.2.8 Atherosclerosis The pathogenesis of atherosclerosis starts with vascular endothelial cell dysfunction and, thereafter, the development of atherosclerotic lesions [44]. Dyslipidemia is an aetiologic factor of atherosclerosis. Fucoidan inhibited hypertriglyceridemia and hypercholesterolemia in apolipoprotein E-deficient mice while elevating the plasma concentration of HDL cholesterol [45]. The elevation of plasma HDL level was by repressing the gene that codes for fatty acid synthase and acetyl-CoA carboxylase in liver fat biosynthesis [46]. Fucoidan also inhibited liver 3-hydroxy-3-methyl-glutaryl CoA reductase activity [44]. Mediators of pro-inflammation were reduced while anti-inflammation mediators were increased by fucoidan. The level of IL-6 was reduced in apoE-knockout mice while the level of IL-10 was increased in mice fed with fucoidan [44]. Fucoidan has also been reported to dissolve thrombus; hence, protecting against atherosclerosis. Formation of thrombus has been implicated in vascular diseases, including stroke [47]. Fucoxanthin inhibited the production of oxidized LDL-induced endothelial damage [44]. Fucoxanthin derived from Sargassum inhibited angiotensin-converting enzyme activity [48]. In another study, seaweed phloroglucinol inhibited arachidonic acid-­ mediated platelet aggregation as well as the formation of thromboxane B2 [49]. This is another antithrombotic effect of algae.

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10.2.9 Kidney and Liver Diseases Extract of Galaxaura oblongata was reported to exert a protective effect against liver and kidney injuries in mice. This was via reduction of oxidative stress and proinflammatory cytokines, including NF-kB [50]. Sargassum polycystum extract assuaged hepatic and renal damages in diabetic rats at 150 mg/kg [51]. Dunaliella salina reduced liver inflammation and fibrosis in study animals. This was attributed to matrix metalloproteinase-9 stimulation and concomitant reduction of tissue inhibitors of metalloproteinase [52]. A report showed lower hepatic fat accumulation in rats fed with Spirulina maxima-supplemented diet. Reduced LDL cholesterol was also observed in these animals contrasted with animals in the control group [53]. Blue-green algae also inhibited nonalcoholic fatty liver disease (NAFLD) development via mechanisms involving reduction in liver lipogenesis, inhibition of lipid peroxidation and enhancing antioxidant enzymes’ activities [54]. A study on human subjects illustrated a converse relationship between the consumption of algae and NAFLD [55].

10.2.10 Skin Diseases/Disorders Phlorotannins extracted from Corallina pilulifera and administered to human dermal fibroblast cells reduced ultraviolet (UV)-stimulated oxidative stress in addition to the expression of gelatinases. Phlorotannins are matrix metalloproteinase (MMP) inhibitors [56]. The anti-inflammatory potential of Nannochloropsis oculata on RAW 264.7 cells was characterized with reduction of iNOS in addition to COX-2 expression [57]. In another study, sulfated polysaccharides isolated from Porphyridium impeded polymorphonuclear leucocyte migration and erythema development [58, 59]. Reactive oxygen species (ROS) are implicated in skin’s aging. They activate MAPK that elicits transcription factor activator protein-1 phosphorylation that leads to the upregulation of MMP, which ultimately results in skin collagen degradation and its attendant skin aging [60]. Collagen is essential for the sustenance of structural integrity of tissues. Wrinkling of the skin is a consequence of deficit of type 1 collagen as well as expression of MMP-1 and elastase. This leads to the degradation of collagen and elastin. Inflammation occurs upon skin exposure to UV radiation. This causes alteration in microvascular structure, vasodilation, leucocyte transendothelial

Algae-Derived Compounds for Disease Management  231 movement and loss of plasma protein [61]. Usually, elevated ROS generation in skin inflammation is protective of host cells and is aimed at destroying the infective microbe [62]. Inflammatory response is a function of exposure to light’s specific wavelength. For instance, exposure to UVB light elicits inflammatory responses in the keratinocytes via mediators; prostaglandin E2, nitric oxide, iNOS, TNF-α, COX-2 and cytokines. There is now evidence that algal extracts exhibit anti-inflammatory effects. Extracts from Ecklonia (E) kurome and E. cava inhibited nitric oxide stimulation [30, 63]. Pro-inflammatory cytokines were inhibited by extracts of Porphyridium spp. [2]. Extended exposure to UV radiation can initiate DNA mutations which alter oncogenic functions, i.e., activating the oncogenes and inactivating the tumor suppressor genes. This eventually results in cancer. Furthermore, UV radiation alters DNA repair mechanisms by reducing DNA repair time frame as well as inhibiting the activities of DNA repair enzymes [64].

10.2.11 Uterine Leiomyomas Uterine leiomyomas (ULs) are nonmalignant tumors of the uterine tissues that affect about 25% of reproducing women [65]. They are usually symptom-free in a majority of women; there are, however, indications that about 20% with UL have symptoms, including pelvic pain, recurrent urination and extreme uterine bleeding, thus reducing their quality of life [65]. Altered fertility was reported in some individuals with ULs [66]. Hormonal, genetic and immunological factors have been implicated in ULs growth. Uterine leiomyomas are characterized by an enormous pileup of extracellular matrix (ECM), particularly transforming growth factor-β, which enhances the accumulation of ECM components [67]. Recent studies have focused on the preventive and anti-inflammatory potentials of certain polyphenolic compounds in the management of ULs. This is considered a safer alternative to conventional therapies. In a study, significant reduction was observed in UL cells treated with 0.5 mg/ ml of fucoidan. Cell cycle arrest at the subG1 phase was also observed. Downregulation of proteins associated with ECM, including vimentin and fibronectin, were also observed [68]. In another study, fucoidans impeded TGF-B1-dependent epithelial mesenchymal transition (EMT) via ERK pathway [69, 70]. Levels of connective tissue growth factor and fibronectin were also reduced by fucoidans [70].

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10.2.12 Obesity Obesity is a global health challenge and a significant risk factor of cardiovascular diseases. It is defined as BMI ≥ 30.00kg/m2. The annual mortality is about 2.8 million adults [71]. Sedentary lifestyle, genetic vulnerability, and high-caloric diet consumption are factors implicated in its etiology [72]. Therefore, therapies, including consistent reduction in caloric diet intake and physical activity, appear to be effective in enhancing weight reduction. However, consistency in maintaining a healthy lifestyle can be quite challenging for a lot of people; hence, the need for effective chemotherapies with minimal adverse effects. Currently, phytochemicals are at the center of research focus for affordable and safe alternatives to conventional anti-obesity drugs [72]. Seaweeds are known sources of dietary fiber which are recognized for delaying the rate of gastric emptying as well as plummeting rate of food consumption. There is evidence of the anti-obesity potential of some algae. There was a reduction in energy expenditure in a population of overweight and obese females after being fed a diet supplemented with drinks rich in alginate-pectin. This was attributed to the mechanism involving enhanced satiety [73]. The appetizing effect of alginate was examined among study participants of various BMI categories. When sodium alginate was administered to participants for 7 days, the study outcome showed a significant reduction in caloric intake. This suggests its anti-obesity potential [74]. There is evidence that fucoxanthin has the potential for weight reduction. In a study using rats and mice, weight reduction was observed in animals fed fucoxanthin in comparison to the controls [75]. In a separate study, fucoxanthin and its derivative fucoxanthinol inhibited the activity of pancreatic lipase and reduced plasma triglyceride level [76]. Another metabolite of fucoxanthin, amarouciaxanthin A, was observed to effectively restrain the growth of white adipose tissue [77]. Xanthigen reduced adipocyte lipid accumulation by the downregulation of principal proteins of transcription factors in adipogenesis [78]. This also suggests its anticancer potential. Spirulina is a cyanobacterium known for its numerous health benefits, including maintenance of caloric homeostasis. It is rich in the antioxidants phycocyanin, beta-carotene and the tocopherols. Gamma-linolenic acid is the principal polyunsaturated fatty acid it contains [71]. There is evidence of weight loss potential of Spirulina, which is attributed to its phytochemicals, including phycocyanin. Phycocyanobilin present in phycocyanin inhibits NADPH oxidase, an enzyme associated with induction of oxidative stress

Algae-Derived Compounds for Disease Management  233 in the cells of adipose tissues. The inhibition of this enzyme results in the anti-inflammatory effects of Spirulina [71]. In a study involving 52 individuals that were obese, considerable weight loss was observed after they were fed 2 g of spirulina/day along with a low caloric diet for 3 months. In the study, low levels of a high-sensitivity C-reactive protein and triglycerides were also observed [79]. In another study of 62 obese individuals, reduced level of total cholesterol and elevated level of HDL cholesterol were observed after been fed a diet containing 1 g of spirulina for 3 months [80]. In a study of 50 obese individuals placed on antihypertensive therapy, the administration of 2 g of spirulina for 84 days led to a reduction in their BMI and waist circumference [81]. Inhibition of infiltration of macrophages to adipocytes, oxidative stress and fat accumulation in the liver are plausible anti-obesity mechanisms of spirulina. Due to its high content of gamma-linolenic acid, Spirulina inhibits accumulation of cholesterol. The presence of niacin also enhances dyslipidemic conditions [71]. There was a considerable diminution in blood pressure in hypertensive patients that took 2 g spirulina within 12 weeks [82]. The anti-obesity potentials of macroalgae have been reported. In a study, obese mice were administered Codium cylindricum, a green microalga with high concentration of siphonaxanthin. A considerable reduction in body weight and mRNA levels of lipogenic pathway genes were outcomes of the study [83]. Reported obesity-reducing mechanisms of green microalgae include reduction in adipogenesis and GIT absorption as well as enhanced oxidation of fatty acid in the adipose tissue, particularly the white type. For red microalgae, additional mechanism is enhanced thermogenesis in brown adipose tissue [84]. In brown algae, enhanced lipid mobilization within adipose tissue as well as an increase in oxidation of fatty acids in the skeletal muscle are the reported mechanisms [84]. Phlorotannins are polyphenolic compounds found in brown seaweeds [85]. They have also been reported to possess anti-obesity capacity via the mechanisms described above, i.e., prohibition of differentiation of adipocytes and pancreatic lipase inhibition. A report showed the ability of phlorotannins to inhibit peptidyl prolyl cis/trans isomerase Pin1, which boosts triglyceride uptake [86]. Therefore, inhibition of Pin1 can be targeted while treating obesity-dependent ailments [85]. Phlorotannins can also inhibit protein tyrosine phosphatase 1B, which relates to adverse insulin signal transduction [87]. Eckol has also been reported to be a potent anti-obesity compound. Algal docosahexaenoic acid has been reported to exert a favorable effect on the lipid profile of overweight and obese individuals. Its potency at a

234  Next-Generation Algae: Volume II lower concentration (2g/day) is equivalent to conventional DHA supplementation at 4g/day for 42 days [88]. Momentous increase was observed in the levels of particle sizes of HDL and large LDL and, conversely, reduction was observed in the levels of VLDL and IDL, which indicates positive effects on cardiovascular health [88].

10.2.13 Tuberculosis Tuberculosis is a bacterial infection which adversely affects the lungs and other body organs. As of 2018, about 10 million people had tuberculosis, out of which about 1.5 million people passed away [89]. Delayed diagnosis and noncompliance with therapies have been reported to exacerbate the infection [90]. The principal challenge in the treatment of tuberculosis is multidrug-resistant strains which often contributes significantly to its mortality [91]. Antibacterial-resistant infections account for almost 700,000 annual deaths [92]. Antibacterial effects of algae have been described. Extracts of two microalgae, Chaetoceros pseudocurvisetus and Skeletonema costatum, were reported to possess anti-tuberculosis capacity [93]. The antibacterial effects of fucoidans derived from Fucus vesiculosus L. have been eported. This is via plausible mechanisms, including the binding of sulphated polysaccharide to the surface of bacteria, hence resulting in bacterial cell damage and nutrients leakage. Another mechanism is fucoidan’s ability to trap nutrients, thereby hindering bacterial cells access to nutrients [92]. Nanotechnology has greatly enhanced the effectiveness of disease treatment. This is because of its many merits, including improved drug bioavailability, minimal side effects, and reduction in the frequency of drug administration due to regulated and consistent drug release to target site [89]. Anti-tuberculosis drug loaded on polypeptide micelles and alginate nanocarrier was reported to be effective against Mtb H37Rv in comparison with unloaded drug. In another report, amikacin and moxifloxacin co-loaded on poly(lactic-co-glycolic acid and alginate significantly hampered the growth of Mtb h37Ra cells as compared to individual drug. Alginate-dependent nanoparticles significantly enhanced the antibacterial effects of some drugs with minimal toxicity to key organs in another report [89]. Anti-tuberculosis pulmonary drug delivery shows the potential of effective pulmonary tuberculosis treatment. Reduction in systemic drug toxicity and specific site targeting of the drug are possible merits of this route of drug administration. In a study, inhalable microparticles dependent on

Algae-Derived Compounds for Disease Management  235 fucoidan alongside isoniazid and rifabutin significantly impeded mycobacterial cell growth. This suggests fucoidan as a potential drug vehicle for effective TB treatment [90].

10.2.14 Asthma Asthma is a widespread respiratory inflammatory ailment worldwide with a growing prevalence due to increased environmental pollution attributed to industrialization. Asthmatic patients are allergic to pollutants, pollens, mites and cold [94]. Shortness of breath, wheezing, tightness of the chest and dry cough are frequent symptoms. Allergens further increase the sensitivity and stimulation of the epithelial cells of the trachea, thus enhancing secretion of mucous which could block the airway. The levels of inflammation mediators, including TNF-alpha and IL-4, are elevated in asthmatic patients, which are produced by swollen macrophages and activated T cells of the lungs. The release of these cytokines further enhances the inflammation of the epithelial cells of the trachea as well as the production of mediators of inflammation, including eotaxins, cytokines and additional chemokines, that culminate in cell damage, inflammation and alteration of the airway, which is the basis of worsening breathing inconveniences experienced by asthmatic patients [94]. It is also pertinent to note that Th2 and ILC2 play a significant role in the pathobiology of asthma as their activation and excessive production have been implicated in massive production of IL-13, IL-4 and IL-5 [95], which aid lung infiltration of eosinophils and mast cell induction. Production of tracheal goblet cell by Th2 also aggravates mucous production capable of blocking the airways [96]. Deposition of collagen in the lungs contingent on sustained inflammation coupled with oxidative damage also caused a breathing challenge in asthmatic patients [97]. Mechanisms targeting reduction of free radicals’ generation, inflammation and expression of Th2 are currently being explored in the management of asthma. Current conventional therapies appear ineffective, hence the need for more effective alternative treatments. Extract of Eucheuma cottonii exerted anti-inflammatory, reduction in mucin synthesis and pro-inflammatory cytokine production according to a study [98]. Another alga, Eisenia arborea rich in phlorotannins, has been reported to exert anti-allergic effects [99]. A report showed the initiation of ERK1/2 and NF kB as well as suppression of Akt in platelet-derived growth factor-activated airway smooth muscle cells by oligo-fucoidan [100]. Inhibitory effects of fucoxanthin on pro-inflammatory cytokines have been reported [101, 102]. In another study, fucoxanthin inhibited

236  Next-Generation Algae: Volume II production of inflammatory cytokines with reduction in histamine and IgE levels in asthmatic lab animals [31, 103]. There is evidence that fucoxanthin ameliorated oxidative stress in asthmatic mice [94]. In a study, oligo-fucoidan boosted the levels of IL-10 and interferon–y and also ameliorated the Treg/Th17 and Th1/Th2 imbalance [31]. It therefore provides an alternative adjuvant in the treatment of asthma.

10.2.15 Hepatitis Lectin extracted from blue-green and red algae inhibited the growth of hepatitis C virus. This antiviral property was attributed to scytovirin and griffithsin, proteins with exceptional ability to bind several carbohydrate moieties concurrently [104].

10.3 Xanthophylls Xanthophylls consist of zeaxanthin, astaxanthin, fucoxanthin and ­β-cryptoxanthin [1].

10.3.1 Astaxanthin Chlorella zofingiensis, Haematococcus pluvialis and Chlorococcum sp. are principal sources of astaxanthin [1]. The antineoplastic effects of astaxanthin are reported. They include anti-proliferation, anti-oxidative, anti-­ inflammatory, anti-metastatic and induction of apoptosis [105]. The selective anti-proliferative impact of astaxanthin on cancerous cells and not on normal cells has been reported [106]. The comparison of the astaxanthin with capsanthin, bixin and beta-­ carotene showed that astaxanthin was more effective than other carotenoids [107]. Astaxanthin reduced cyclin D1 and proliferating cell nuclear antigen expression in oral cancer cells [108]. Astaxanthin induced mitochondrial-apoptosis via hampering of anti-­ apoptotic mediators; p-Bcl-2-associated death promoter and pro-survival Bcl-2 [109]. Astaxanthin inhibited free radical production by augmenting the activities of superoxide dismutase and catalase [110]. In a study, astaxanthin impeded NF-ΚB level, which led to inhibition of pro-inflammatory cytokines’ production [105]. Astaxanthin reduced mRNA and protein of MMP-2 and MMP-9, indicating its anti-invasion, anti-migration and anti-metastatic properties [108]. Astaxanthin also upregulated gap junctional intracellular communication while enhancing the expression Cx43 protein [105].

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10.3.2 Fucoxanthin Fucoxanthin is a carotenoid found in marine algae with potent antioxidant properties. Its peculiar structure consisting of an allenic bond and 5,6-monoepoxide distinguishes it from other carotenoids [111]. In a report, fucoxanthin boosted nuclear factor erythroid 2-related factor 2 (Nfr2) expression in ocular tissues. This was evidenced in/by the elevated activity of SOD as well as reduction in the level of malondialdehyde contingent on pathogen-associated molecular pattern (PAMP)-induced uveitis suggestive of inhibition of oxidative stress [111]. Upon the liberation of Nrf2 into the nucleus of cells, it fastens to the antioxidant response elements of DNA to boost the activities of antioxidant enzymes [111]. The ability of fucoxanthin to protect against hyperglycemic-induced diabetic retinopathy and the deleterious effects of 4-hydroxynonenal has also been reported. The report illustrated that fucoxanthin enhanced catalase activity, hence, reducing oxidative stress [112].

10.3.3 Lutein and Zeaxanthin The primary source of lutein is microalgae [113]. It is a potent antioxidant. Lutein as well as zeaxanthin are primarily found in Scenedesmus spp., Spirulina spp. and Chlorella spp. [1]. Galdieria sulphuraria and Dunaliella salina are also rich sources of lutein [114]. Lutein impeded the high proliferation rate of cells induced by ultraviolet radiation [115]. The antioxidant and anti-inflammatory potentials of zeaxanthin have been reported [1]. Of note are reports of its photoprotection abilities [116, 117]. The antineoplastic mechanism of zeaxanthin in uveal melanoma (UM) includes induction of apoptosis, inhibition of incursion and cancerous cells migration via reduction in MMP-2 level [118]. Lutein and zeaxanthin inhibited oxidative stress in retina. Hence, it has the potential of inhibiting diabetic retinopathy. Additionally, neuroprotection in diabetic retina by lutein has been reported [119]. Lutein and zeaxanthin also inhibited lipid peroxidation in epithelial cell [119].

10.3.4 Beta-Cryptoxanthin Beta-cryptoxanthin is an oxygenated carotenoid with similar structure to beta-carotene. Its potent pro-vitamin A effect, antioxidant, anti-obesity and anti-inflammatory effects are well known [1, 120]. Intake of β-cryptoxanthin reduced the risk of having type 2 diabetes mellitus, according to a reported study conducted in Finland [121]. A study

238  Next-Generation Algae: Volume II carried out in Japan showed a considerably lower level of ­β-cryptoxanthin in participants NAFLD. Administration of β-cryptoxanthin to these participants lowered serum levels of markers of hepatic damage [122]. β-cryptoxanthin enhanced insulin sensitivity in non-alcoholic steatohepatitis (NASH)-induced mice. It also prevented hepatic fat accumulation as well as inhibiting non-alcoholic fatty liver (NAFL) via improving insulin resistance [123]. It also reduced inflammation and the production of macrophages [123].

10.3.5 Siphonaxanthin Siphonaxanthin is a carotenoid found in Codium fragile, Umbraulva japonica and Caulerpa lentillifera. There is evidence of its potent anticancer effects on leukemia in comparison with fucoxanthin [124]. This was attributed to the presence of an extra OH group on carbon-19 and absence of epoxide in its structure. In a report, siphonaxanthin reduced the degranulation of mast cell via the modulation of lipid rafts, thus showing its anti-­ inflammatory effect [125].

10.3.6 Saproxanthin and Myxol These carotenoids are known for their antioxidant potential. Additionally, myxol enhanced biomembranes by inhibiting oxygen permeation [1]. Saproxanthin and myxol have been reported to possess potent antioxidant potentials, which are attributed to their ability to inhibit lipid peroxidation [120]. Saproxanthin can be converted to myxol by 2’-hydroxylase [120]. A report showed higher antioxidant activities of saproxanthin and myxol than zeaxanthin and beta-carotene [126]. The neurotoxicity effect of 1-glutamate was impeded by these carotenoids [127].

10.4 Alga Diterpenes There is evidence that diterpenes obtained from algae exert antiviral effects on viruses, including hepatitis virus and HIV among others [128]. Certain microalgae have been reported to contain phytofurans and phytoprostanes, which are derived from alpha linolenic acid. Gracilaria longissima is such a typical alga. In a report, phytofurans and phytoprostanes extracted from Gracilaria longissima exerted anti-inflammatory effect in endothelial cells by inhibiting IL-6 and ICAM levels [129].

Algae-Derived Compounds for Disease Management  239 The biologic effects of diterpenes have been reported [68]. As at 2017, about 233 diterpenes were detected in Dictyota spp., the majority of which came from Dictyota dichotoma. Compound 204, a bicyclic diterpene, was reported to exhibit anti-inflammatory effects via mechanisms involving inhibition of nitric oxide production [130].

10.5 Conclusion Evidence from this study indicates the numerous health-boosting effects of pharmacological compounds derived from algae. Furthermore, these compounds appear to be more effective and safer in comparison with conventional drugs. Therefore, they are better alternatives to conventional drugs in the management of diseases.

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248  Next-Generation Algae: Volume II 105. Zhang L. and Wang H., Multiple Mechanisms of Anti-Cancer Effects Exerted by Astaxanthin. Mar. Drugs, 13, pp 4310-4330, 2015. 106. Song XD., Zhang JJ., Wang MR., Liu WB., Gu XB. and Lv CJ., Astaxanthin induces mitochondria-mediated apoptosis in rat hepatocellular carcinoma CBRH-7919 cells”. Biol. Pharm. Bull. 34, pp 839–844, 2011. 107. Zhang X., Zhao WE., Hu L., Zhao L. and Huang J., Carotenoids inhibit proliferation and regulate expression of peroxisome proliferators-­activated receptor gamma (PPARgamma) in K562 cancer cells”. Arch. Biochem. Biophys. 512, pp 96–106, 2011. 108. Kowshik J., Baba AB., Giri H., Deepak Reddy G., Dixit M. and Nagini S., Astaxanthin inhibits JAK/STAT-3 signaling to abrogate cell proliferation, invasion and angiogenesis in a hamster model of oral cancer. PLoS ONE, 9, e109114, 2014. 109. Kavitha K., Kowshik J., Kishore TK., Baba AB. and Nagini S. Astaxanthin inhibits NF-kappaB and Wnt/beta-catenin signaling pathways via inactivation of Erk/MAPK and PI3K/Akt to induce intrinsic apoptosis in a hamster model of oral cancer. Biochim. Biophys. Acta 1830, pp 4433–4444. 110. Franceschelli S., Pesce M., Ferrone A., de Lutiis MA., Patruno A, Grilli A., Felaco M and Speranza L. Astaxanthin treatment confers protection against oxidative stress in U937 cells stimulated with lipopolysaccharide reducing O2- production. PLoS ONE 2014, 9, e88359. 111. Chen S-J., Lin T-B., Peng H-Y., Lin C-H., Lee A-S., Liu H-J., Li C-C. and Tseng K-W., Protective Effects of Fucoxanthin Dampen Pathogen-Associated Molecular Pattern (PAMP) Lipopolysaccharide-Induced Inflammatory Action and Elevated Intraocular Pressure by Activating Nrf2 Signaling and Generating Reactive Oxygen Species. Antioxidants, 10, 1092, 2021. https:// doi.org/10.3390/antiox10071092 112. Chiang Y-F., Chen H-Y., Chang Y-J., Shih Y-H., Shieh T-M., Wang K-L. and Hsia S-M., Protective Effects of Fucoxanthin on High Glucose and 4-Hydroxynonenal (4-HNE)-Induced Injury in Human Retinal Pigment Epithelial Cells”. Antioxidants, 9, pp 1176, 2020. 113. Lin J., Lee D., Chang J., Lutein production from biomass: Marigold flowers versus microalgae”. Biores. Technol., 184, pp 421–428, 2015. 114. Sun Z., Li T., Zhou ZG. and Jiang Y. Microalgae as a source of lutein: Chemistry, biosynthesis, and carotenogenesis. In Advances in Biochemical Engineering/Biotechnology; Springer: Heidelberg, Germany, Volume 153, pp. 37–58, 2016. 115. González S., Astner S., An W., Goukassian D and Pathak MA., Dietary Lutein/ Zeaxanthin Decreases Ultraviolet B-Induced Epidermal Hyperproliferation and Acute Inflammation in Hairless Mice. J. Invest. Dermatol. 2003, 121, pp 399–405, 2003. 116. Stahl W. and Sies H., Bioactivity and Protective Effects of Natural Carotenoids. Biochim. Biophys. Acta - Mol. Basis Dis., 1740, pp 101–107, 2005.

Algae-Derived Compounds for Disease Management  249 117. Ma L. and Lin XM., Effects of Lutein and Zeaxanthin on Aspects of Eye Health”. J. Sci. Food Agric., 90, pp 2–12, 2010. 118. Bi M-C., Hose N., Xu C-L., Zhang C., Sassoon J. and Song E., Nonlethal Levels of Zeaxanthin Inhibit Cell Migration, Invasion, and Secretion of MMP-2 via NF-κB Pathway in Cultured Human Uveal Melanoma Cells. Journal of Ophthalmology, 2016, Article ID 8734309, 8 pages, 2016 http:// dx.doi.org/10.1155/2016/8734309. 119. Scripsema NK, Hu D-N and Rosen RB., Lutein, Zeaxanthin, and meso-Zeaxanthin in the Clinical Management of Eye Disease. Journal of Ophthalmology, Volume 2015, Article ID 865179, 13 pages http://dx.doi. org/10.1155/2015/865179, 2015. 120. Gammone MA., Riccioni G. and D’Orazio N., Marine Carotenoids against Oxidative Stress: Effects on Human Health. Marine Drugs, 13, pp 6226-6246, 2015. 121. Montonen J., Knekt P., Järvinen R. and Reunanen A., Dietary antioxidant intake and risk of type 2 diabetes. Diabetes Care, 27, pp 362–366, 2004 122. Matsuura B., Miyake T., Yamamoto S., Furukawa S. and Hiasa Y., Usefulness of beta-cryptoxanthin for non-alcoholic fatty liver diseases. J.  Food Nutr. Disord., 5, pp 1000196, 2016. 123. Nishino A., Maoka T. and Yasui H., Preventive Effects of β-Cryptoxanthin, a Potent Antioxidant and Provitamin A Carotenoid, on LifestyleRelated Diseases—A Central Focus on Its Effects on Non-Alcoholic Fatty Liver Disease (NAFLD). Antioxidants, 11, pp 43. https://doi.org/10.3390/ antiox11010043, 2022. 124. Sugawara T., Ganesan P., Li Z., Manabe Y. and Hirata T., Siphonaxanthin, a Green Algal Carotenoid, as a Novel Functional Compound. Mar. Drugs, 12, pp 3660–3668, 2014. 125. Manabe Y., Hirata T. and Sugawara T., Suppressive Effects of Carotenoids on the Antigeninduced Degranulation in RBL-2H3 Rat Basophilic Leukemia Cells. J. Oleo Sci., 63, pp 291–294, 2014. 126. Shindo K., Kikuta K., Suzuki A., Katsuta A., Kasai H., Yasumoto-Hirose M., Matsuo Y. and Takaichi S., Rare carotenoids, (3R)-saproxanthin and (3R,2’S)myxol, isolated fromnovel marine bacteria (Flavobactaeriaceae) and their antioxidative activities. App. Microbiol. Biotechnol., 74, pp 1350-1357, 2007. 127. Shindo K., Kimura M. and Iga M., Potent antioxidant activity of cacol, a sesquiterpene contained in Cacalia delphiniifolia Sleb et Zuc. Biosc. Biotechnol. Biochem., 68, pp 1393-1394, 2004. 128. Wardana PA., Aminah NS., Rosyda M., Abdjan MI., Kristanti AN., Tun KNW., Choudhary MI. and Takaya Y. Potential of diterpene compounds as antivirals, a review. Heliyon 7, e07777, 2021.

250  Next-Generation Algae: Volume II 129. Sánchez SM., Domínguez-Perles R., Montoro-García S., Gabaldón JA.,   Guy A., Durand T., Oger C., Ferreres F. and Gil-Izquierdo A., Bioavailable phytoprostanes and phytofurans from Gracilaria longissima have anti-­ inflammatory effects in endothelial cells. Food Funct., 11, pp 5166-5178, 2020. 130. Zhao M., Cheng S., Yuan W., Dong J., Huang K., Sun Z. and Yan P., Further new xenicanes from a Chinese collection of the brown alga Dictyota plectens. Chem. Pharm. Bull., 63, pp 1081–1086, 2015.

11 Application of Algae in Wound Healing Ebenezer I. O. Ajayi1*, Johnson O. Oladele2 and Abraham O. Nkumah3 1

DC&ONID, Department of Biochemistry, Faculty of Basic and Applied Sciences, College of Science, Engineering and Technology, Osun State University, Osogbo, Osun State, Nigeria 2 Biochemistry Unit, Department of Chemical Sciences, Faculty of Sciences, Kings University, Ode Omu, Osun State, Nigeria 3 Phytochemistry & Phytomedicine (molecular unit), Phytomedicine Research Group, Department of Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria

Abstract

Wounds in living tissues, such as pressure ulcers or chronic skin wounds, pose a major threat to the healthcare system, especially in the treatment or management of some pathological conditions, including spinal cord injury, diabetes, etc. Such wounds have a significant recurrence rate and are only partly successfully treated with the present medical approaches and technologies. Invasive wound infections pose a more serious threat to open wounds and can result in amputation and additional impairment. It takes a multidisciplinary approach to create novel, nontoxic, noninvasive, and more potent treatments. Thus, there is a growing global interest in finding natural products that promote skin regeneration. A natural source of intriguing bioactive chemicals with potential for use in applications for wound healing is algae. Due to their many biological activities, such as the ability to inhibit secretion of inflammatory cytokines, antioxidant properties, and anti­ microbial activities which are vital in the wound healing process, algae and other products, including polysaccharides, polyunsaturated fatty acids, and microalgal carotenoids, may be beneficial in wound management and healing applications. This chapter presents comprehensive information on wound healing processes, possible beneficial effects of algae as wound-healing agents, and mechanisms underpinning the therapeutic wound-healing effects of algae.

*Corresponding author: [email protected] Charles Oluwaseun Adetunji, Julius Kola Oloke, Naveen Dwivedi, Sabeela Beevi Ummalyma, Shubha Dwivedi, Daniel Ingo Hefft and Juliana Bunmi Adetunji (eds.) Next-Generation Algae: Volume II: Applications in Medicine and the Pharmaceutical Industry, (251–284) © 2023 Scrivener Publishing LLC

251

252  Next-Generation Algae: Volume II Keywords:  Algae, wound healing, natural products, biomaterials

11.1 Introduction Wound cicatrization is a dynamic and complex process involving inflammation, proliferation, and remodeling, which are a set of important biochemical and physiological phenomena [1]. Wounds and scarring can arise from accidents, injuries, hazards, burns, ulcers (such as the diabetic foot ulcer, Buruli ulcer), lesions (such as the cutaneous leishmaniasis lesions), bumps, cancer, among many others. In recent years, algae polysaccharides (PS) have drawn attention for diverse uses due to their distinct structure similar to the extracellular matrix of humans, including their biological activity, high biocompatibility, biodegradability, low toxicity, reproducibility, considerable moisturizing and swelling ability, and colloidal properties. Algae PS have found a wide range of applications in regenerative medicine technologies, including the composition and technique of wound dressings. Hydrogel-forming polysaccharides fall into this category, so have other polysaccharide gels, such as agarose, alginate, carrageenan, ulvan, starch, porphyran, and native (nano-)cellulose. Natural anionic polysaccharides can also be used, and their gelation mechanisms make them suitable. A wide range of wound dressings with many possible applications have been developed from PS. Impressively clever generation of PS-based materials for drug delivery systems (DDS), implantable medical devices, organ and tissue transplant modulation are now developed using the latest advances in polymer manufacturing technology, including intelligent PS hydrogels that respond to changes in temperature, pH, pressure, bioelectricity and biomagnetism that promote wound healing [2]. The new innovative approach appreciates the physicochemical and biological properties required for PS and synthetic polymers. It also offers the opportunity to employ tissue engineering techniques in reconstructive and transplant surgery. These smart materials can modulate their conformation in the wound environment without causing biological harm, thus rendering them very attractive to biosensor monitoring, target-specific molecular recognition, metabolic control mechanisms, diagnosis, and theranostics, apart from drug delivery. The purpose of tissue engineering is to grow viable cell populations with the desired spatial arrangement by three-dimensional (3D) bioprinting based on biocompatible materials (combination of different biopolymers and synthetic polymers) that can contain living cells.

Application of Algae in Wound Healing  253 Three-dimensional bioprinting is a rapidly developing trend in modern biomedicine used to create viable biological structures in fascinating and useful spatial configurations. The final product of 3D bioprinting is implanted in the body and fully dissolves and renovates the host tissue within a few months. This technology represents an advanced breakthrough in the medical field in areas such as reconstruction and transplant surgery, implantable medical devices, and controlled drug delivery [3]. The most studied and advantageous compounds that can be used in these systems are sulfated PS from a wide variety of algae (brown algae alginate and fucoidan, red algae carrageenan, and ulvans from green algae).

11.1.1 Current Trends in the Design of Wound Dressings An injury is damage to tissues and/or organs characterized by the destruction of the integrity of the system (skin and mucous membrane, for instance) [4]. There is no universal classification system for wounds. However, various approaches to wound classification (by etiology, localization, type of damage, depth, degree of complexity, degree of infection, etc.) characterize wounds to provide their adequate treatment. The following factors are of greatest importance in the evaluation of wounds: the circumstance (nature and etiology) of the injury, the time of its occurrence, whether the injury is acute or chronic, and the depth of damage to the skin and underlying tissues [5]. Understanding the fundamental mechanisms of wound healing, as well as knowledge of the type and function of the available dressing materials, allows a systemic strategy for the choice of dressings to be put together in the individual patient`s treatment plan. Wound repair or healing is a complicated process involving multiple overlapping stages that lead to the repair and restoration of damaged skin anatomy and function [6]. This biological process is highly organized and consists of various forms of cell renewal such as collagen formation, epithelialization, and tissue remodeling. The majority of authors distinguish four stages of wound healing: hemostasis (exudate coagulation and blood coagulation) which  occurs immediately after injury, inflammation (proteas 1–5 days after trauma, release of reactive oxygen species (ROS), cytokines, and growth factors, proliferation (granulation and angiogenesis, days 6–14) and remodeling (contraction, epithelialization, and scar tissue formation, days 15–16). Since the inflammatory stage coincides with the hemostasis stage, some of the authors combine inflammation and hemostasis in the early stages [7]. Hemostasis, necrotic tissue rejection, wound drainage, tissue nutrition improvement, and infection control are the goals of Phase 1-2 treatment.

254  Next-Generation Algae: Volume II Wound dressings with hemostatic, protective, and absorbent properties are used to ensure that wound exudate is drained scientifically. Wound dressings with appropriate agents (anti-inflammatory agents, antibiotics, preservatives, anesthetics, proteolytic enzymes, and more) are used to regulate the inflammatory response [8]. The main objectives of proliferative therapy are the protection of granules, the stimulation of reparative processes by growth factors and the prevention of infections. At the granulation stage, non-traumatic moisturizing dressings containing antibiotics can be used. At the remodeling stage, treatment should be aimed at stimulating the repair process and epithelialization and preventing the formation of hypertrophic, keloid, and atrophic scars. Non-traumatic moisturizing dressings, ointments, and gels with low osmotic activity are used for this purpose. Wound dressings with regenerative properties are used to stimulate epithelial growth. Bandages that allow delivery of growth factors and bioactive substances (BAS) are used to promote tissue regeneration at the site of the local wound. It is also important to adhere to the core requirements of wound dressings at all stages of treatment. Optimal gas exchange, moist wound environment, biocompatibility, and avoidance of allergic reactions, irritation, and burning pain are to be ensured. There are a wide variety of materials for wound care (healing). Appropriate wound dressings and patient management plans should be selected taking into account several factors, including the patient’s general health, wound complexity and its etiology, and the stage of wound healing [9]. Except for predominantly closed surgical wounds, a single type of wound care is rarely used to treat chronic or non-healing open acute wounds. Complex (with damage to internal organs, large blood vessels, large nerve stems or bones) and compound wounds (with simultaneous damage to various organs in several anatomical areas, such as wounds in the thoracoabdominal region) are complex and need treatment. In this regard, according to Intisar et al. [10], modern applied wound dressings must be multifunctional. Analysis of many studies has made it possible to summarize the basic requirements for modern wound dressings, viz. adsorption, helps remove excess wound exudate and related toxic compounds; hemostatic effect as needed, they are involved in gas exchange and need to ensure high moisture on the wound surface; elasticity and vapor permeability while impermeable to microorganisms; insulation, possibility of application without additional fixation; biocompatibility, they must be sterilized and easily decomposed after use; they should also be strong and essential, but concise and cheap.

Application of Algae in Wound Healing  255 The use of bio- and synthetic polymers in the form of hydrogels, thin films (membranes), nanofibers, wafers, foams, and sponges is currently trending in wound dressing design. Hydrogel coatings are one of the structural types of wound dressings that deserve special attention. Hydrogels are a promising biomaterial for biomedical applications. They’ve been used in a variety of medical applications, including hemostatic wound dressings, drug delivery, tissue engineering, and biosensors. Hydrogel dressings are believed to have the properties of a complete wound dressing material. Hydrogels are 3D structures (scaffolds) made of hydrophilic polymer chains with corresponding structures and properties. The presence of a 3D polymer skeleton gives the hydrogel the mechanical properties of a solid, including lack of fluidity, shape, strength, and the ability to retain properties such as plasticity and elasticity. Hydrogels resemble the extracellular matrix of the skin, including collagen and elastin fibers, glucose aminoglycans and proteoglycans, non-­ collagen structural proteins, and mineral constituents, allowing them to perform various functions characteristic of the extracellular matrix. Because hydrogels are usually transparent, they can be used to monitor the wound’s condition without having to remove the bandage. The use of hydrogel dressing helps maintain a moist environment for clean and healthy granulation tissue, facilitating self-digestive wound resection of wounds with necrotic tissue such as eschars. Hydrogels can be applied to pressure ulcers, skin lacerations, surgical wounds, and burns. These dressings are suitable for wounds with minimal to moderate exudate production [10, 11]. Over the last 15 years, many studies have focused on the development of topical drug delivery materials using multifunctional compounds that not only promote stabilization and controlled drug delivery to the target but also provide biocompatibility. As a drug delivery system (DDS, e.g., antibacterial or anti-inflammatory agents, proteolytic enzymes, growth factors, BAS), hydrogels have received a lot of attention, primarily due to their highly porous structure and gradual release [11]. Hydrogel-based DDS includes nano- or micro-sized particles, nanofibers, microspheres, and microneedles. Nano and microparticles have numerous advantages, including the ability to deliver drugs via various routes of administration, the ability to adjust particle size and surface properties, and the ability to deliver drugs to the desired target in a controlled and long-term manner. The development of carriers in the form of particulates has created new opportunities for the development of DDS with improved pharmacokinetic and pharmacodynamic properties [8].

256  Next-Generation Algae: Volume II Nanofibers have a high surface-area-to-volume ratio and are considered suitable substrates when highly porous structures are required. Unlike traditional rigid porous structures, nanofibers are a dynamic system that can change the size and shape of pores to form flexible or rigid crosslinked structures. Nanofibers are typically used for drug encapsulation and sustained release, Top of Form as well as in tissue-engineering technologies such as, in particular, 3D bioprinting [12]. New strategies, such as the use of new polymer materials that combine the desired physicochemical and biological properties, represent the current advancements in the advancement of new-generation materials to design wound dressings, DDS, and implantable structures. Natural biopolymers derived from marine organisms that are biocompatible and biodegradable could be developed using this method. The most noteworthy of them are algae PS (alginates, fucoidans from brown algae, carrageenans from red algae, and ulvans from green algae).

11.2 Brown Seaweed Polysaccharides The brown algae comprising the class Phaeophyceae, are a widespread group of large algae that abundantly grow in cold seawater at northern latitudes, although some species grow naturally in freshwater and brackish water [13]. Brown algae contain fucoxanthin, a carotenoid pigment, which suppresses other pigments to give plants their characteristic brown color. Brown algae plants range in size from a few millimeters to about 70  m, demonstrating a variety of structures and compositions among species within the same algae class. Large brown algae are composed of complex and dynamic cell walls rich in polysaccharides such as alginate (also alginic acid and algin), fucoidan (also fucoidin and fucan), laminarin (also laminaran), and cellulose [14], which also contains polyphenols, proteins, glycoproteins, phlorotannins (sulfated phenolic compounds), halogens such as iodine, minerals such as sodium, potassium, calcium, and magnesium [2]. The relative content of the various compounds varies from species to species and is highly dependent on seasonal, environmental, and regional factors. Alginate appears to form a significant portion of all brown algae, although the laminarin and fucoidan appear to be more common in some species, such as the genus Laminaria and Fucus spp., than in others [2]. The presence of free sugars such as glucose, fructose, and sucrose in brown algae is not important as monosaccharides are quickly converted into D-mannitol polysaccharides [15]. For example, glucose content is seasonal

Application of Algae in Wound Healing  257 and primarily binds to beta-glucan laminarin. It acts as an intracellular storage polysaccharide. Deniaud-Bouët et al. [16] pointed out that the cell wall of brown algae is composed of fucose, which in turn contains sulfated polysaccharides (FCSPs), which are important crosslinked glycans that connect cellulose microfibrils placed in layers parallel to the cell surface, forming a scaffold for cells. They went further to mention that short fragments of hemicellulose are intermediates between cellulose and crosslinked FCSP, but the composition of these proposed hemicellulose fragments are undefined. These structures are embedded in the alginate matrix. The cell wall matrix is supposed to be composed primarily of alginate, proteins, polyphenols (related to these two groups of compounds), and iodine. Mixed-bound cellulose and/or glucans, which make up a small proportion ( 95%) [82]. As a result, K562 cells exposed to 50 M C-phycocyanin for up to 48 hours had 49% fewer multiplications overall. Additionally, cytometric stream analysis of cells treated with 25 and 50 Mof C-phycocyanin for 48 hours revealed, independently, 14.11% and 20.93% of cells in the G0/G1 stage. The truth is that the transfer of cytochrome c from the mitochondria to the cytoplasm confirmed the formation of apoptotic bodies as a result of the involvement of C-phycocyanin in K562 cells [82]. When C-phycocyanin from S. platensis was rewarded in human hepatocarcinoma (HepG2) cell lines, several studies revealed that there was a reduced articulation of medicine 1 obstruction proteins (MDR-1). Additionally, ROS and COX-2-intervened pathways were found to be related to NF-B and AP-1 [69].

13.3.5 Immune-Stimulant Activity The various useful large scale and microalgal segments are mostly responsible for the medical benefits. As a result, proteins and peptides are becoming popular as supplements and as possible tools for improving health. Bioactive auxiliary metabolites can cause and animate human wellbeing aspects, according to later in vitro and in vivo studies. Ex vivo testing using enzymatic hydrolysis can be used to identify additional bioactive proteins as peptides obtained from green growth proteins. Furthermore, the majority of peptides are likely released and processed during gastrointestinal digestion because they are encoded in the main algal proteins [51]. Healthy growth Pancreatic protein hydrolyzes C. vulgaris at a rate of 20 AU/g compound/substrate with a pH of 7.5 and a temperature of 45 °C for a period of 4 hours [62]. Exams showed that after using depleted Balb/c mice,

328  Next-Generation Algae: Volume II he experienced both an innate and explicit immune response. Additionally, oral administration of a protein hydrolyzate (500 mg/kg) was provisionally finished between 8 and 3 days at a fixed time, and there was a discernible increase in the lymphocyte pool of 128% in comparison to the benchmark group of mice (pb0.01). In order to assess hematopoiesis, the amount of leukocytes in peripheral blood and the bone marrow’s cellularity were restored. The practical mobility of macrophages was also substantially increased. In fact, the T-incited counteracting agent reactions and recovery of postponed type extreme touchiness responses (DTH) were among the revitalized humoral and cell invulnerable capacities as well [62]. On murine splenocytes, the protease catalyst concentrate of E. cava stimulated the immune system in vitro. As it was discovered, E. cava hydrolyzate significantly increased the proliferative effect of splenocytes, including lymphocytes, monocytes, and granulocytes, when administered to ICR mice. Additionally, compared to untreated controls, more CD4 + T cells, CD8 + T cells, and CD45R/B220 + B cells were present overall. Additionally, there was a reduction in the production of Th-1-type cytokines, such as TNF-an and IFN-y, as well as mRNA articulation. As a result, cytokines of the Th-2 sort, such as IL-4 and IL-10, were improved [20].

13.3.6 Cholesterol-Lowering Activity It has been proven that consuming phytosterols and their esters has a cholesterol-lowering effect, resulting in a 10% to 15% decrease in serum levels of LDL-X, one of the primary risk factors for IHD in persons with hypercholesterolemia [61]. Patients who received antihypercholesterolemic drugs such statins [65] and fibrates [68] experienced a significantly more drastic decline. Phytosterols obtained from microalgae were also observed to have a cholesterol-lowering effect; this was accomplished by reducing the assimilation of cholesterol from meals and reducing endogenous cholesterol produced by the gastrointestinal system. The statement of the intestinal quality ACAT2, which is responsible for cholesterol intake in the gut [24], is restrained by schizosterol removal [58]. The ability of this concentrate to lower cholesterol is also linked to hepatic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, a substance ­ involved in the synthesis of cholesterol. The LDL-C receptor is stimulated by Schizochytrium sterols, which promotes the removal of plasma cholesterol from the circulatory system. According to studies, hamsters fed 0.06 and 0.3 g of schizophirrium sterol extract per kilogram of food had their cholesterol levels drop by 19.5% and 34%, respectively. The schizochitria sterol extricate’s bioactivity was

Novel Compounds Derived from Algae  329 just as impressive as that of the positive benchmark group given yararl-sitosterol, a phytosterol that is already added to foods like margarine and vegetable oils as beneficial additives. The mechanism of action of different phytosterols differs. Extricate of blue-green growth lipid, cooperative kind of Nostoc. Sphaeroids Kützing (N. collective) suppress the synthesis of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and the expression of LDL receptor in human hepatoma cell lines. However, in a previous research, this species was accounted for to be described by the proximity of campesterol, p-sitosterol, and clionasterol [72]. The lipid of intrigue was not recognized in this examination. By reducing mRNA articulation in the gastrointestinal system and competing with cholesterol for the NPC1L1 carrier, B-action sitosterol’s to lower cholesterol is achieved.

13.3.7 Anti-Inflammatory Activity By preventing the production of particularly provocative cytokines (TNF-an) and COX-2 articulation, ergosterol isolated from edible mushrooms can quell the ferocious responses of LW-incited RAW264.7 macrophages in vitro [54]. Chlorella ulgaris [91] and Dunalliella tertiolecta have been reported to contain ergosterol, which exhibits similar systems of activity by reducing the reaction caused by LPS [28]. In addition to ergosterol, mice by 0-tetradecanoylphorbol-13-acetic acid derivation (TPA: solid tumor advertiser) prompted by ergosterol, 7-dehydroporiferetherol peroxide, and 7-oxo-cholesterol (TPA: solid tumor advertiser) showed a strong calming action with 2-0.7 mg/ear as a half inhibitory portion [91]. The microalga Dunaliella tertiolecta is thought to be a potential source of phytosterol for the future because it has recently been linked to ergosterol, 7-dehydroporiferasterol, and ergosterol peroxide [34]. A synergistic system of some microalgae phytosterols has been reported in some studies to increase the natural mobility of another phytosterol For instance, sheep fringe blood mononuclear cells (PBMC) were stimulated to produce a combination of ergosterol and 7-dehydroporiferasterol (both from Dunalliella tertiolecta), which was more effective than ergosterol alone in the same focus [28]. Synergistic effects should always be taken into account when streamlining effectiveness because microalgal phytosterols and other metabolites are promising potential calming agents.

13.3.8 Anticancer Activity Several studies have suggested that phytosterols may act naturally to inhibit tumor growth [23]. For instance, human colorectal cancer cells

330  Next-Generation Algae: Volume II were cytostatically affected by ergosterol [54]. Ergosterol peroxide inhibited the growth of Walker 256 carcinosarcoma cells and MCF-7 human bosom ­adenocarcinoma cells in vitro [48]. 2 mol of ergosterol peroxide from Chlorella vulgaris effectively prevented tumor movement (77% decrease) in mice when TPA and 7,12-dimethylbenz (an) anthracene were present (DMBA: immunosuppressor and tumor initiator). According to one theory, the biosynthetic polyamine chemical ornithine decarboxylase (ODC), which is produced by TPA, is inhibited by these bioactive sterols. Hepatocarcinoma (HepG2) cells were severely damaged by a stigmasterol isolated from the navicula incerta, and this stigmasterol is effective at triggering apoptosis by increasing the proapoptotic Bax and p53 quality’s guiding principle and inhibiting the activity of the antiapoptotic Bcl-2 quality [52]. Separated from the earthy-hued Sargassum carpophyllum growth, fucosterols and oxygenated fucosterol shown cytotoxicity against various disease cell lines [87].

13.3.9 Cancer Prevention Agent The ability of phytosterols from microalgae to prevent cancer has received scant scientific support. In any event, phytosterols from various sources have been taken into account to provide a beneficial result. For instance, stigmasterol from Butea monosperma strip revealed the highest proxidative content of 5.2 mg per kg of food daily due to an increase in lipid peroxidation (the primary cause of cell damage) and the activity of glutathione, catalase, and superoxide dismutase (SOD), endogenous cancer prevention agents [71]. Additionally, mice poisoned with carbon tetrachloride (CCl4) and given food containing 30 mg of fucosterol per day for seven consecutive days showed a notable drop in blood transaminases and an increase in free radicals. SOD, catalase, and glutate peroxidation are retaining proteins that are individually 33.89%, 21.56%, and 39.34% [56].

13.3.10 Antidiabetic A characteristic complexity of diabetes mellitus is diabetic retinopathy (DR). The underlying cause of the vision loss associated with this condition is close to the oxidative damage inflicted by high glucose levels on the epithelial cells of the retinal pigment epithelium (RPE). In RPE cells, superoxide dismutase (SOD) is thought to be the primary protective protein against oxidative damage. In addition, RPE cells are protected from oxidative damage by the tripeptide glutathione (GSH), which is present in

Novel Compounds Derived from Algae  331 RPE cells. Therefore, it is crucial to protect RPE cells by maintaining adequate centralizations of SOD and GSH as molecules in order to protect the retina from oxidative damage. A layer of retinal cells known as EPR plays a significant role in the development and regulation of photoreceptors in the retina of vertebrates. RPE cells function as a vehicle and capacity for materials in the typical retina, including retinadeide, phagocytosis separated from the external photoreceptor sections, light-blocking attempts, the elimination of free radicals, the union of cytokines, and the development of the retinal blood hindrance [26]. To maintain the integrity and proper functioning of the human retina, the reliability of the EPR is crucial [92]. RPE cells are typically isolated in the G1 stage after being refreshed and sorted. RPE cells release SOD and GSH as a protective measure in response to oxidative damage [47].

13.3.11 Different Biomedical Activities Numerous researchers have discovered that phytosterols derived from microalgae have antibacterial and anti-diabetic effects. The second most frequent cause of death in humans is tuberculosis, which is contagious and spread through the air. Sasikala Prakash [73] Microalgal isochrysis galbana (ergost-5-en-3-ol and cholest-5-en-24-1,3-derived from ­24-oxocholesterol) (with acetyloxy) Amikacin at a concentration of 50 g per ml, 200 g per ml with pyraminamide, and rifambicin at a concentration of 40 g per ml are safe anti-tuberculosis medications in comparison to other medications. Additionally, 0.5 g per ml disconnected from earthy green growth. In a comparable study, sargassum ringgoldianum was found to be as effective at suppressing M. tuberculosis H37Rv growth as the anti-tuberculosis drug rifampicin. Microalgae Micromonas aff. pusilla [88] and saringosterol both have unidentified natural actions. Diabetes is a chronic illness that manifests as severe entanglements like high glucose and hypoglycemia. Streptozotocin-induced antidiabetic effects in diabetic mice have been observed in fucosterols contained in Pelvetia siliquosa green growth. Additionally, a few microalgae species, such as Chrysoderma sp. Additionally, Olisthodiscus luteus [59]; However, there is currently no evidence to support the usefulness of these fucosterols. The pharmaceutical industry has much more potential for phytosterols [34] obtained from microalgae because of the high yearly microalgal lipid yield. This is because it not only addresses the issue of growing global interest, but it also provides increasingly popular alternatives due to the side effects associated with manufactured medications.

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13.4 Research Results on Well-Studied Algal Strains Numerous studies on widely studied types of green growth bioactive mixtures, such as Arthrospira, Botryococcus braunii, Chlorella vulgaris, Dunaliella salina, Haematococcus pluvialis, and Nostoc, have been conducted. These mixtures have been found to have antibacterial and antiviral properties as well as calming, fiery, anticoagulant, and antienzyme properties. The extraction of bioactive mixtures from these microalgae is the basis for these tests [26]. Because Nostoc includes proteins, minerals, and unsaturated fats, it is used as a feedstock, pharmaceutical, and nutritional supplement. The use of this bacterium in the treatment of fistulas and some types of cancer regulates its clinical estimation [38]. Generally speaking, Nostoc’s biomass has been recognized as a soothing agent and has also been shown to aid in absorption, manage circulatory strain, and strengthen the immune system. Cyanobine, a protein atom that Nostoc species may transport, has shown promising results in the treatment of HIV and influenza A(H1N1) [43]. Additionally, Nostoc species include polyunsaturated unsaturated fats (PUFAs), which include basic unsaturated fats as linoleic, -linolenic, -linolenic, octadecatetraenoic, and eicosapentaenoic corrosive [53]. Basic unsaturated fats, which precede prostaglandins and are extremely enthused by the pharmaceutical industry, are essential. Chlorella is a very frequent green growth that has been discovered by traditional Japanese ocean growth buyers, who frequently eat it and value it as a dietary supplement. In addition to chlorophyll, proteins, and polysaccharides, chlorella also includes 53% (w/w) protein, 23% (w/w) starches, 9% (w/w) lipids, and 9% (w/w) lipid atomic segments. rich in essential amino acids, minerals, and nutrients. The following components and 5% (w/w) of minerals [35]. By adjusting the settings under which they develop, these supplement focuses can be changed. The B12 complex is also rich in B12, which is essential for platelet formation and recovery after injury. Chlorella, like spirulina, has a GRAS certification from the FDA, which means that, when grown in the right conditions and with the use of proper sanitation and assembly procedures, it has the potential to influence human health. It frequently serves as nourishment without being given [58]. Dunaliella is a single-cell, salt-tolerant green microalga that has received significant attention for its potent pharmacological mixtures. Due to its unique physiology, this microalgae is frequently regarded as an exceptional scenario with several biotechnological uses. Other bioactive mixtures, including as carotenoids, glycerol, lipids, chemicals, and minerals, are abundant in Dunaliella. This microalgae is a substantial natural source

Novel Compounds Derived from Algae  333 of beta-carotene and can produce up to 14% of its dry weight when grown under supplement limitations, under salty, bright, and hot conditions [35]. In addition to P-carotene, this microalgae is abundant in proteins and fundamental unsaturated fats that can be consumed safely, as evidenced by the recognition of GRAS. The biomass blends made from Dunaliella exhibit a variety of natural properties, including hepatoprotective, antihypertensive, bronchodilator, pain reliever, muscle relaxant, and anti-­carcinogenic effects. Additionally, microalgal biomass can be used directly in food and pharmaceutical formulations [58]. Polysaccharides are a group of highly valuable additives that have uses in the industries of medicine, food, cosmetics, tissues, stabilizers, and emulsifiers [21]. Sulfated polysaccharides, which are sulfate esters included in microalgal polysaccharides, offer unique clinical applications [25, 26]. The activation and modification of macrophages are essential for the therapeutic effect. The area, level of sulfation, and sugar synthesis of sulfur polysaccharides are used to identify their natural movement [49]. According to some, microalgae are responsible for the antibacterial properties of lipid fragments. The potential mix of compounds in microalgae, such as and -ionones, p-cyclotitral, neoplastic dienes, and phytol, gives rise to their antibacterial properties. Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, -linolenic corrosive, eicosapentaenoic corrosive, and the lipid structure of microalgae hexadecatrienoic corrosive are a few examples of human pathogens that exhibit antibacterial activity. This antibacterial property of microalgae is due to the likely arrangement of mixtures, including phytol 55, neoplastic dienes, and -ionone, and -cyclocitral. Pseudomonas aeruginosa, S. aureus, E. coli -linolenic corrosive, eicosapentaenoic corrosive, hexadecatrienoic corrosive, docosahexaenoic corrosive, palmitoleic corrosive, lauric corrosive, oleic, lactic corrosive, and arachidonic corrosive are all attributed to antibacterial effect against human pathogens. Carotenoids have a wide range of potential benefits for human health, including the enhancement of waterfalls and the treatment of degenerative diseases like macular degeneration. These mixtures function as anti-cancer agents and lessen the oxidative damage caused by ROS. Studies have shown that increased phenol use lowers the frequency of degenerative diseases. Microalgae phenolic mixtures that can combat free radicals have been identified [1]. Medicines play a vital role in enhancing the character of the population and its future. Every year, enormous amounts of drugs are used in human and veterinary medicine to cure illnesses, bacterial infections, stress, and other disorders, prevent pregnancy, and promote the growth of aquaculture

334  Next-Generation Algae: Volume II and homesteads substance for waste water treatment. This is because to its effectiveness in removing toxic elements from wastewater and potential for natural goodwill [63].

13.5 Conclusion and Future Recommendations This chapter has provided detailed information on the upcoming sections on the most recent developments in the discovery of novel compounds of pharmaceutical significance derived from algae, including bioactive compounds, pharmacological significance of algae, antioxidative activity, antihypertensive activity, anticoagulant activity, antiproliferation activities, immune-stimulant activity, cholesterol-lowering activity, anti-­ inflammatory activity, anticancer activity, and cancer prevention.

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Novel Compounds Derived from Algae  339 53. Stolz, P., & Obermayer, B. Manufacturing microalgae for skin care. Cosmetics Toiletries, 120, 99–106, 2005. 54. Subhashini, J., Mahipal, S. V. K., Reddy,M. C., Reddy, M. M., Rachamallu, A., & Reddanna, P. Molecular mechanisms in C-Phycocyanin induce apoptosis in human chronic myeloid leukemia cell line-K562. Biochemical Pharmacology, 68, 453–462, 2004. 55. Suetsuna, K., & Chen, J. R. Identification of antihypertensive peptides from peptic digests of two microalgae, Chlorella vulgaris and Spirulina platensis. Marine Biotechnology, 3, 305–309, 2001. 56. Suetsuna, K., Maekawa, K., & Chen, J. R. Antihypertensive effects of Undaria pinnatifida (wakame) peptide on blood pressure in spontaneously hypertensive rats. The Journal of Nutritional Biochemistry, 15, 267–272, 2004. 57. Wang, T., Jonsdottir, R., Kristinsson, H. G., Hreggvidsson, G. O., Jonsson, J.  O., Thorkelsson, G., et al. Enzyme-enhanced extraction of antioxidant ingredients from red algae Palmaria palmate. LWT-Food Science and Technology, 43, 1387–1397, 2010. 58. Xie PY, Zhang XM, Matsukura M, Fujii I, Ito K, Zhao J, Shinohara M. Effect of Tangkangle on expression of superoxide dismutase in cultured rabbit retinal pigment epithelial cells under hyperglycemia condition. Int. J. Ophthalmol., 7, 1584–1586, 2007. 59. Yasukawa, K.; Akihisa, T.; Kanno, H.; Kaminaga, T.; Izumida, M.; Sakoh, T.; Tamura, T.; Takido, M. Inhibitory effects of sterols isolated from Chlorella vulgaris on 12-O-tetradecanoylphorbol-13-acetate-Induced inflammation and tumor promotion in mouse skin. Biol. Pharm. Bull. 19, 573–576, 1996. 60. Zarbin MA. Age-related macular degeneration: review of pathogenesis. Eur. J. Ophthalmol., 8, 199–206, 1998. 61. Adeleke, D. A., Olajide, P. A., Omowumi, O. S., Okunlola, D. D., Taiwo, A. M., & Adetuyi, B. O. Effect of Monosodium Glutamate on the Body System. World News of Natural Sciences, 44, 1-23, 2022. 62. Adetuyi B.O,  Oluwole E.O Dairo J.O. Chemoprotective Potential of Ethanol Extract of  Ganoderma Lucidum  on Liver and Kidney Parameters in Plasmodium Beghei-Induced Mice, International Journal of Chemistry and Chemical Processes (IJCC). 1(8).:29-36, 2015. 63. Adetuyi B.O., Oluwole E.O., Dairo J.O. Biochemical effects of shea butter and groundnut oils on white albino rats, International Journal of Chemistry and Chemical Processes (IJCC). 1(8).:1-17, 2015. 64. Adetuyi B.O.,  Dairo J.O., Didunyemi M.O. Anti-Hyperglycemic Potency of Jatropha Gossypiifolia in Alloxan Induced Diabetes. Biochem Pharmacol (Los Angel) 4(5):193, 2015.  65. Adetuyi, B. O., Omolabi, F. K., Olajide, P. A., Oloke, J. K. Pharmacological, Biochemical and Therapeutic Potential of Milk Thistle (Silymarin): A Review World News of Natural Sciences 37:75-91, 2021. 66. Adetuyi, B. O., & Farombi, E. O. 6‐Gingerol, an active constituent of ginger, attenuates lipopolysaccharide‐induced oxidation, inflammation,

340  Next-Generation Algae: Volume II cognitive deficits, neuroplasticity, and amyloidogenesis in rat.  Journal of Food Biochemistry, 45(4), e13660, 2021. 67. Adetuyi, B. O., Adebayo, P. F., Olajide, P. A., Atanda, O. O., Oloke, J.  K. Involvement of Free Radicals in the Ageing of Cutaneous Membrane. World News of Natural Sciences, 43, 11-37, 2022. 68. Adetuyi, B. O., Adebisi, O. A., Adetuyi, O. A., Ogunlana, O. O., Toloyai, P-B., Egbuna, C., Uche, C. Z., Khan, J., Adumanya, O. C., Patrick-Iwuanyanwu, K.C. Ficus exasperata Attenuates Acetaminophen-Induced Hepatic Damage via NF-κB Signaling Mechanism in Experimental Rat Model BioMed Research International, 2022. 69. Adetuyi, B. O., Adebisi, O. A., Awoyelu, E. H., Adetuyi, O. A., Ogunlana, O. O. Phytochemical and Toxicological effect of Ethanol extract of Heliotropium indicum on Liver of Male Albino Rats. Letters in Applied NanoBioscience (LIANB) 10(2):2085-2095, 2020. https://doi.org/10.33263/ LIANBS102.20852095 70. Adetuyi, B. O., Odine, G. O., Olajide, P. A., Adetuyi, O. A., Atanda, O. O., Oloke  J. K. Nutraceuticals: role in metabolic disease, prevention and treatment, World News of Natural Sciences 42, 1-27, 2022. 71. Adetuyi, B. O., Ogundipe, A. E., Ogunlana, O. O., Egbuna, C., Estella, O. U., Mishra, A. P., Akram, M., & Achar, R. R. (2022). Banana Peel as a Source of Nutraceuticals.  Food and Agricultural Byproducts as Important Source of Valuable Nutraceuticals.1st ed. Springer, Berlin. 400 p, 2020.  72. Adetuyi, B. O., Okeowo, T. O., Adetuyi, O. A., Adebisi, O. A., Ogunlana, O., Oretade, O. J., Marraiki, N., Beshbishy, A. M., Welson, N. N., & Batiha, G. E. Ganoderma lucidum from red mushroom attenuates formaldehydeinduced liver damage in experimental male rat model.  Biology  9(10), 313, 2020. https://doi.org/10.3390/biology9100313 73. Adetuyi, B. O., Olajide, P. A., Awoyelu, E. H., Adetuyi, O. A., Adebisi, O. A., Oloke, J. K. Epidemiology and Therapeutic measure for COVID-19; A review. African Journal  of Reproductive Health June 2020 (Special Edition on COVID-19); 24 (2):142, 2020. https://www.ajrh.info/index.php/ajrh/article/ view/2300 74. Adetuyi, B. O., Olajide, P. A., Omowumi, O. S., Odine, G. O., Okunlola, D. D., Taiwo, A. M., & Opayinka, O. D. Blockage of Alzheimer’s gene: Breakthrough effect of Apolipoprotein E4. African Journal of Advanced Pure and Applied Sciences (AJAPAS), 26-33, 2022. 75. Adetuyi, B. O., Toloyai, P. Y., Ojugbeli, E. T., Oyebanjo, O. T., Adetuyi, O. A., Uche, C. Z., Olisah, M. C., Adumanya, O. C., Jude, C., Chikwendu, J. K., Akram, M., Awuchi, C.G., Egbuna, C. Neurorestorative Roles of Microgliosis and Astrogliosis in Neuroinflammation and Neurodegeneration.  Scicom Journal of Medical and Applied Medical Sciences 1(1):1-5, 2021. 76. Adetuyi, B.O., Olajide, P. A., Oluwatosin, A., Oloke, J. K. Preventive Phytochemicals of Cancer as Speed Breakers in Inflammatory

Novel Compounds Derived from Algae  341 Signaling.  Research Journal of Life Sciences, Bioinformatics, Pharmaceutical and Chemical Sciences 8 (1) 30-61, 2022. 77. Adewale, G. G., Olajide, P. A., Omowumi, O. S., Okunlola, D. D., Taiwo, A.  M., & Adetuyi, B. O. Toxicological Significance of the Occurrence of Selenium in Foods. World News of Natural Sciences, 44, 63-88, 2022. 78. Awoyelu, E. H., Oladipo, E. K., Adetuyi, B. O., Senbadejo, T. Y., Oyawoye, O. M., Oloke, J. K. Phyloevolutionary analysis of SARS-CoV-2 in Nigeria. New Microbes and New Infections  36.  100717, 2020.  https://doi.org/10.1016/j. nmni.2020.100717 79. Batiha, G. B., Awad, D. A., Algamma, A. M., Nyamota, R., Wahed, M. I., Shah, M. A., Amin, M. N., Adetuyi, B. O., Hetta, H. F., Cruz-Marins, N., Koirala, N., Ghosh, A., & Sabatier, J. Diary-derived and Egg White Proteins in Enhancing Immune System against COVID-19.  Frontiers in Nutritionr. (Nutritional Immunology) 8:629440, 2021. 80. Didunyemi, M. O., Adetuyi, B. O., & Oyewale, I. A. Inhibition of lipid peroxidation and in-vitro antioxidant capacity of aqueous, acetone and methanol leaf extracts of green and red Acalypha wilkesiana Muell Arg. Int J Biol Med Res. 11(3):7089-7094, 2020. 81. Didunyemi, M. O.,  Adetuyi, B. O., Oyebanjo, O. O. Morinda lucida attenuates acetaminophen-induced oxidative damage and hepatotoxicity in rats,  Journal of Biomedical sciences, 2019. https://www.jbiomeds.com/­ biomedical-sciences/morinda-lucida-attenuates-­acetaminopheninducedoxidative-damage-and-hepatotoxicity-in-rats.php?aid=24482 82. Farombi, E. O., Abolaji, A. O., Adetuyi, B. O., Awosanya, O., & Fabusoro, M. Neuroprotective role of 6-Gingerol-rich fraction of Zingiber officinale (Ginger) against acrylonitrile-induced neurotoxicity in male Wistar rats. Journal of Basic and Clinical Physiology and Pharmacology, 30(3), 2019. 83. James-Okoro, P. O., Iheagwam, F. N., Sholeye, M. I., Umoren, I. A., Adetuyi, B. O., Ogundipe, A. E., Braimah, A. A., Adekunbi, T. S., Ogunlana, O. E., & Ogunlana, O. O. Phytochemical and in vitro antioxidant assessment of Yoyo bitters World News of Natural Sciences 37:1-17, 2021. 84. Nazir, A., Itrat, N., Shahid, A., Mushtaq, Z., Abdulrahman, S. A., Egbuna, C., Adetuyi, B. O., Khan, J., Uche, C. Z, Toloyai, P. Y. Orange Peel as a Source of Nutraceuticals.  Food and Agricultural Byproducts as Important Source of Valuable Nutraceuticals. 1st ed. Springer, Berlin. 400 p, 2022. 85. Ogunlana, O. O., Adetuyi, B. O., Adekunbi, T. S., Adegboye, B. E., Iheagwam, F. N., Ogunlana, O. E. Ruzu bitters ameliorates high–fat diet induced non-alcoholic fatty liver disease in male Wistar rats.  Journal of Pharmacy and Pharmacognosy Research 9(3), 251-26, 2021. 86. Ogunlana, O. O., Adetuyi, B. O., Rotimi, M., Esalomi, I., Adeyemi A., Akinyemi, J., Ogunlana, O., Adetuyi, O., Adebisi, O., Okpata, E., Baty, R., & Batiha, G. Hypoglycemic Activities of Ethanol Seed Extract of Hunteria umbellate  (Hallier F.) on Streptozotocin-induced Diabetic Rats.  Clinical Phytoscience, 7 (1), 1-9, 2021.

342  Next-Generation Algae: Volume II 87. Ogunlana, O. O., Ogunlana, O. E., Adekunbi, T. S., Adetuyi, B. O., Adegboye, B. E., & Iheagwam, F. N. Anti-inflammatory Mechanism of Ruzu Bitters on Diet-Induced Nonalcoholic Fatty Liver Disease in Male Wistar Rats. Evidence-Based Complementary and Alternative Medicine, 2020 https:// doi.org/10.1155/2020/5246725 88. Ogunlana, O. O., Ogunlana, O. E., Popoola, J. O., Adetuyi, B. O., Adekunbi, T.  S., David, O. L., Adeleye, O. J., Udeogu, S. A., & Adeyemi, A. O. Twigs of Andrographis paniculata (Burn. F) Nees attenuates Carbon Tetrachloride (CCl4) Induced Liver Damage in Wistar Albino Rats. RASAYAN Journal of Chemistry 14(4): 2598-2603, 2021. 89. Ogunlana, O. O.,  Adetuyi, B. O., Esalomi, E. F., Rotimi, M. I., Popoola, J. O., Ogunlana, O. E., & Adetuyi, O. A. Antidiabetic and Antioxidant Activities of the Twigs of Andrograhis paniculata on Streptozotocin-Induced Diabetic Male Rats. BioChem. 1(3):238-249, 2021. 90. Olajide, P. A., Adetuyi, O. A., Omowumi, O. S. & Adetuyi, B. O. Anticancer and Antioxidant Phytochemicals as Speed Breakers in Inflammatory Signaling. World News of Natural Sciences 44, 231-259, 2022. 91. Olajide, P. A., Omowumi, O. S., Okunlola, D. D., & Adetuyi, B. O. Deadly Pandemia: Monkeypox Disease, a Case Study. African Journal of Advanced Pure and Applied Sciences (AJAPAS), 34-37, 2022. 92. Olajide, P. A., Omowumi, S. O., & Odine, G. O. Pathogenesis of Reactive Oxygen Species: A Review. World News of Natural Sciences 44:150-164, 2022.

14 Applications of Algae in the Production of Single-Cell Proteins and Pigments with High Relevance in Industry Juliana Bunmi Adetunji1*, Omowumi Oyeronke Adewale1, Charles Oluwaseun Adetunji2 and Isreal Olu Oyewole1 Department of Biochemistry, Faculty of Basic and Applied Sciences, Osun State University, Osogbo, Nigeria 2 Department of Microbiology, Biotechnology and Nanotechnology Laboratory, Edo State University Uzairue, Edo State, Nigeria 1

Abstract

Algae serve as a source for diverse types of chlorophyll. It is a major constituent of the ecosystem, specifically marine and freshwater habitats. Nutritionally, algae have been established to have high protein content when compared to conventional sources of protein. In industry, biotechnology tools have been used to produce many nutritional supplements from microalgae. Also, microbial dried cells or biomass, also known as single-cell protein (SCP), were isolated from microalgae like Chlorella, Spirulina, and Dunaliella, which are the most common ones. The supplements produced from these algae have nutraceutical potential for diverse health-related issues. The biomass consists of vitamins, lipids, minerals, carbohydrates, and proteins; and its cells also produce some amino acids useful for animal and human consumption. This chapter addresses the production of SCP from microalgae and its application in the diet, along with highlighting some beneficial pigments, like astaxanthin, fucoxanthin, carotenoid, etc., derived from algae and their commercial application. Keywords:  Algae, single-cell proteins, pigments, industry, astaxanthin, fucoxanthin and carotenoid pigments

*Corresponding author: [email protected]; [email protected] Charles Oluwaseun Adetunji, Julius Kola Oloke, Naveen Dwivedi, Sabeela Beevi Ummalyma, Shubha Dwivedi, Daniel Ingo Hefft and Juliana Bunmi Adetunji (eds.) Next-Generation Algae: Volume II: Applications in Medicine and the Pharmaceutical Industry, (343–352) © 2023 Scrivener Publishing LLC

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344  Next-Generation Algae: Volume II

14.1 Introduction The scarcity of food and protein caused by the ever-increasing population requires drastic measures to sustain future growth. Due to the this, there is a need for an alternative novel protein source to meet the growth in population. Hence, algae have been documented as resource-efficient and natural protein feed and food constituents, known as single-cell protein (SCP), to support those derived from bacteria and yeast microbes [1]. Over the years, proteins have been sourced from animal-based products like beef and turkey, which give an equivalent of 50–60% protein content [2]. Interestingly, proteins have also been derived from plant materials and as such are referred to as vegetable proteins with a high nutrient composition like fibers, minerals, vitamins, and antioxidants due to less environmental value and increased sustainability when compared with the animal-based proteins [3]. It was also documented that insects have served as an important protein source with about 60% protein content on average; hence, insect farming has led to a decrease in the ecological footprint as a result of its low impact on deforestation, soil fertility, and water content [4]. Moreover, there is evidence that algae food and feed products can be developed commercially because they’re natural and have health benefits resulting from the variety of nutrients and bioactive constituents present in them [5]. It was also documented that algae biomass serves as a sustainable protein source with a high protein content of about 70% of microalgae. Bleakley and Hayes documented that close to 47% of specific red seaweed species have high protein levels compared to other seaweeds in which the protein level is usually between 9–22% [6]. The authors also considered microalgae as a feasible protein source with diverse essential amino acids of interest similar to those present in animal protein [7, 8]. Consequently, algae which serve as protein derived from vegetables have numerous nutrients and biologically active constituents that support nutraceutical food products. Algae cultivation is mostly performed on small land from agricultural byproducts to support the circular economy. De Mendonça et al. documented that alga can extract carbon dioxide in about 10 to 50 times greater amounts than the plants on land, thus it serves as a potential food source [9]. Algae contain low protein digestibility like a whole cell or single-cell protein in their unprocessed form because the composition and structure of the cell wall consists of a high percentage of fibers and polyphenols [10]. Moreover, the single cell protein (SCP) is an alternative source of the

Single-Cell Proteins and Pigments from Algae  345 traditional protein derived from animal feeds, without any adverse effects on humans [11].

14.2 Microalgae-Derived Single Cell Protein (SCP) Single cell proteins are dried cell bioprotein derived from organisms like algae, fungi, yeast, and bacteria. Reports have shown that fungi and bacteria have great potential in producing high protein content as a result of their chemical structure composition. However, carbohydrates, nucleic acids, fats, minerals, and vitamins have also been documented to contain high protein components, and hence could serve as SCP with high essential amino acids such as lysine, methionine, and threonine. It is essential to research an alternative for protein replacement due to the expensive nature of the available protein from sources like soybeans and fish. Reports have shown that SCP is a dietary product of high quality derived from wastes [12]. This alternative source of protein is documented to have a less detrimental effect. However, the focus for over a decade now has been the exploration of single-product from microalgal while the biomass-derived after extraction could also be used for valorization [13]. Microalgae like Chlorella and Spirulina with low protein and lipids production are also utilized in higher-value production of pigments, fatty acids, and phycocyanin. Besides which, Bratosin et al. documented that SCP contains other macro- and micronutrients like lipids, minerals, nucleic acid, carbohydrates, vitamins, and other major amino acids [11]. The SCP serves as a significant substitute for proteins derived from food sources like fish and soybean products that are extremely expensive. In 2022, Janssen et al. established that the protein content of microalgae is much higher than that of chicken, peanuts, soy flour, skimmed milk, and fish [14]. Though Dunaliella, Galdieria, and Spirulina are exceptional, Galdieria shows high sulfur-containing essential amino acid contents. Bogale documented that SCP derived from microorganisms are mainly from dead cells, dry microbial cells as well as total proteins isolated from pure microbe cell culture from numerous microorganisms like filamentous fungi, yeast, and algae that serve as an alternative protein source for animals and humans [15]. However, microorganisms make use of waste and cost-effective feedstock as the carbon energy source for their growth to release protein concentrate, biomass, or amino acids. This becomes necessary due to the hike in the world population and shortage of protein worldwide, which could eventually lead to nutrient deficiency. It was

346  Next-Generation Algae: Volume II stated that SCP provides a very promising protein source that can satisfy the world shortage of food while the population increases, by using diverse energy sources for the growth of the microorganism [16]. For many years, the nutritional supplements derived from microalgae have been the major focus of microalgal biotechnology research. The biomass of Spirulina, Dunaliella, and Chlorella have great commercial value [17] and are often used as nutraceuticals due to their numerous health benefits. SCP contains mainly proteins, vitamins, lipids, carbohydrates, minerals (trace minerals), and salts because the cells can generate all essential amino acids useful to man and animals. Najafpour stated that large algae, being a source of SCP, have promising potential for protein supplements for the world’s food shortage due to the ever-increasing population [16]. Most single cell-derived proteins are commonly from microalgae Chlorella Vulgaris or Arthrospira platensis [18]. However, it was established that diverse species of microalgae are often whole cells, but as of today, the EU has documented that to attain a substantial position with other developed nations in the development of microalgae-based food and feed sector, it is essential to maximize protein volume and content production, reduce the cost of production and also there must be a high range of EFSAapproved microalgae suitable for food applications [18, 19].

14.2.1 Dunaliella Microalgae like Dunaliella have been identified as a natural protein source as far back as the 1950s; it has numerous applications, particularly as feed, high-value products, and food for humans [20]. The algae Dunaliella also produce a unique β-carotene that is commercially relevant through the process of hypercarotenogenesis [21]. The process of hypercarotenogenesis from dried biomass of Dunaliella started in the 80s in countries like the USA, Israel, Australia, and later China and India joined the β-­ carotene production process from microalgae [21]. In the US, Dunaliella is claimed to be Generally Regarded as Safe (GRAS) by the US Food and Drug Administration (FDA) for cosmetic products, animal and human nutrition, and food coloring [20]. Dunaliella biomass protein content and composition were performed using a snapshot approach under strict conditions and specific growth stages [1]. The assessment revealed that 50–80% protein is present in the dried biomass of Dunaliella [1, 22]. The Dunaliella microalgae do not have rigid cellulosic cell walls, and as such, animals and humans digest it readily.

Single-Cell Proteins and Pigments from Algae  347 Gifuni et al. reported the co-production of microalgae with protein and vitamin nutritional benefit derived from Dunaliella [13]. Dunaliella biomass protein can be improved through the co-production of functional proteins resulting in high digestibility protein, and carotenoids with coloring, immune-stimulating, and antioxidant functions embedded in products like poultry, ornamental fish feed, feed, pet food, and ornamental bird feed [23].

14.3 Applications of SCP in Diets Application of the derived SCP from microalgae depends on minerals, nucleic acid, enzymes, vitamins, and amino acids composition, which complements the relatively cheap protein content when compared to that from animals and some plant sources [24]. Meanwhile, the amino acid profiles are excellent, which contributes to their nutritional acceptability to conventional protein sources [25]. Algae produce about 40% SCP after processing, which supports the findings of Ferreira et al. that the essential amino acids present in microbes are determined by the growth media and the substrate utilized [26]. Interestingly, bacterial and yeast microorganisms replicate within a short period of 5–15 min but for algae and mold species their replication is between 2–4 h. Nasseri et al. also documented that due to the fast-growing rate of microorganisms yielding a large quantity of biomass from algae within 3–6 h, bacteria: 30 min to 2 h, yeast: 40 min to 3 h [17]. Cyanocobalamin (B12) vitamin has been reported to be abundant in algae and bacteria while other vitamins in SCP include pantothenic acid (B5), thiamine (B1), pyridoxine (B6), riboflavin (B2), niacin choline, folic acid (B9), para-aminobenzoic acid, inositol and biotin (B7) [27]. Also, the production processes do not depend on the climatic and environmental conditions contributing to its availability throughout the year [28]. Also, Banach et al. have reported on the inclusion of macroalgae in the human diet for years, and as such more macroalgae have been approved by EFSA [29]. However, the production in Europe is far less than that obtainable in Asia. Meanwhile, macroalgae provide an essential component of the Asian diet due to their protein composition, which includes Porphyra tenera (44%), Chondrus crispus (20%), Ulva lactuca (29%), Undaria pinnatifida (29%), Palmaria palmata (19%), and Fucus serratus (17%). The growing interest in algae-containing products has, however, triggered the EU’s effort to increase the cost-effective production of cultivated macroalgae,

348  Next-Generation Algae: Volume II instead of harvested ones, to boost their incorporation in food and feed markets and to improve their food safety [19, 30].

14.4 Pigments Derived from Algae 14.4.1 Astaxanthin In 2010, Dragos et al. reported that most red-colored pigments seen in most marine organisms and freshwater are derived from astaxanthin and have great benefits to human and aquaculture health [31]. It was documented that green microalgae called Haematococcus pluvialis could accumulate high quantities of astaxanthin during a stress condition. Dragos and co-author performed an experiment in 2010 that established that astaxanthin synthesis induced via a change in light intensity through increased irradiance coupled with continuous illumination, resulted in algal biomass astaxanthin content approximated to be 5.7 mg · g -1 per dry biomass. Under light stress conditions, the biomass composition was significantly changed, and increased the astaxanthin content by approximately 10 times along with a decrease of protein amount and increase of carbohydrates content [30]. Raza et al. documented that astaxanthin (AST), a known red pigment of carotenoid, provides the high-quality keto-carotenoid pigment used in livestock, therapeutics, food, and nutraceuticals [31]. Astaxanthin could be sourced from algae but other sources include crustaceans, fish, and birds. It is of great benefit in animal nutrition when used as a pharmaceutical agent. Astaxanthin was also reported to have several biological properties which include anticarcinogenic, antihypertensive, antioxidant, and anti-obesity. Both humans and animals have also used astaxanthin as an immunomodulator in health sustainability [31].

14.4.2 Fucoxanthin Fucoxanthin is a xanthophyll pigment in most micro and macro marine algae. Moreover, brown macroalgae belonging to the Phaeophyceae family have been established to be the key source of fucoxanthin (a chloroplast accessory pigment responsible for its observed colour). Consequently, species of microalgal like Dunaliella salina, Spirulina platensis, Haematococcus pluvialis, Chlorella spp., Scenedesmus spp., Chlorococcum spp., Nannochloropsis spp., and Botryococcus braunii were reported to have abundant carotenoid that is applied in nutraceutical, food, pharmaceutical

Single-Cell Proteins and Pigments from Algae  349 products and feed [32]. Gupta et al. stated that carotenoid antioxidants and green cell photoprotector production can be scaled up in algae using light-harvesting apparatus [33]. Moreover, phycocyanin, a natural blue pigment used in food and cosmetics, are products of eukaryotic algae, cryptophytes, rhodophytes, and cyanobacterium, which are mostly used as colorants due to the lack of natural stable blue pigment in nature [34, 35].

14.4.3 Carotenoids Carotenoids are lipophilic pigments found in plants and algae. Moreover, the novel microalga Chlorococcum sp. is a target candidate for the production of high-value carotenoid phytoene with cosmeceutical, nutraceutical, and pharmaceutical applications [34]. In 2019, Laje and colleagues conducted a study on the reduction of phytoene desaturase (PDS) activity with the pigment-inhibiting herbicide 1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]pyridin-4-one (fluridone), that caused excess accumulation of phytoene in the characterized microalgal strain Chlorococcum sp. (UTEX B 3056). After post-incubation with fluridone, phytoene levels were measured at ~33 ug/mg cell tissue, as opposed to non-detectable levels in control cultures.

14.5 Conclusion Single cell proteins derived from algae have proven to have higher protein content than the conventional protein sources, and hence could serve as an alternative source of protein to feed the ever-increasing population. Also, the pigments isolated from algae have also provided some beneficial supports to the food and pharmaceutical industries.

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Index Additives, 95, 96, 105, 107, 110 Agars, 7 Algae, 95–97, 99–107, 109–111, 113, 115–118, 140, 322, 324 Algae and cyanobacterial constituents with potential biological action, 12–15 Algae metabolites, 286 lipids, 286, 288 polysaccharides, 298, 301, 302, 307 proteins, 287, 288, 290, 292, 296 Algae respiration, 141 Algae-based biopolymer, 147 Algal, 198, 203 Alginate, 204–205, 258, 259 Amino acids, 343–347 Amphipathic properties, 125 Animal protein, 344 Antiarrhythmic properties, 127 Antibacterial action, 15 Antibacterial properties, 269–271 Antibiotics, 9 Anticancer action, 21 Anticancer activity, 289, 295 Anticarcinogenic, 146 Anticoagulant action, 25 Antidiabetic activity, 146 Antifungal action, 17 Antifungal activity, 269 Antiherpetic, 210 Anti-inflammatory, 139, 144, 147 Anti-inflammatory action, 18 Antineoplastic action, 21

Antioxidant action, 20 Antioxidant activity, 267, 268 Antiproliferative, 144 Antiprotozoal action, 19 Antipsychotic treatment, 130 Antiviral action, 24 Apoptosis, 204, 207, 215 Apoptotic, 144 Asexual reproduction, 141 Astaxanthin, 198, 213, 216, 236 Astrocytes, 201, 208 Atherosclerosis, 229 Autoimmune disease, 129, 146 Beta oxidation, 131, 132 Beta-cryptoxanthin, 237 Bioactive compounds, 322, 334 Bioactive compounds from cyanobacteria, 51 Bioactive constituents, 2–4, 6, 7, 11, 15 Bioactive metabolites, 287, 295 Bioactive substances, 11 Biocomposite, 141 Biodegradation, 140 Bioethanol, 203 Biofuel, 2, 4, 140, 286, 294 Biomass, 344 Biomolecules, 125 carotene, 286, 287, 303 lutein, 286, 287 oleic acid, 286, 287 phycocyanin, 287, 295, 296, 302 Bioprospecting for new algae, 4

353

354  Index Bioremediation, 140 Biosynthesis and biological activities, 11 Bipolar depression, 130 Brown seaweed polysaccharides, 256, 257 Cancer, 224–226, 236, 237 Carrageenan, 259, 260 Cell proliferation, 130, 147 Chlorella, 6, 12, 26 Chlorophyta, 3, 6, 8 Cholesterol, 199, 209, 213–214 Circular economy, 147 Compounds with anticarcinogenic, 161 anticarcinogenic activities, 168 chlorophyll, 163–164 eicosapentaenoic acid (EPA), 165–166 fucoxanthin, 166–167 monogalactosyl glycerols, 168 nonyl 8-acetoxy-6-methyloctanoate (NAMO), 167 other active compounds from microalgae, 168 phycocyanin, 163 polysaccharides, 161–162 polyunsaturated aldehydes (PUAs), 164 stigmasterol, 166 violaxanthin, 164–165 Compression modelling, 142 Cosmetics and personal care, 87 Current trends in the design of wound dressing, 253–256 Cyanobacteria, 2, 3, 5–7 Cyanobacterial constituents, 12 Cytotoxic, 143 Diabetes mellitus, 228 Dinoflagellates, 3, 8 Dopaminergic pathway, 129

Eicosapentaenoic acid, 132 Endothelial cells, 128 Environmental, 147 Extraction methods, 9 advantages of new extraction techniques, 10 microwave, 9 polyunsaturated fatty acids (PUFAs), 11 solid–liquid extraction, 10 supercritical fluid extraction, 9 techniques, 9 Extrusion, 143 Features of microalgae, 157–158 Food and drug administration, 5 Food industry, 88 Fossil fuel, 286, 287 Fractions of microalgae species with anticancer properties, 158 Amphidinium carterae organic fractions, 159 Canadian marine microalgal pool aqueous extract, 160 carotenoid-rich extracts of chlorella species, 158–159 Chaetoceros calcitrans ethyl acetate and ethanol extracts, 159 Chlorella sorokiniana aqueous extract, 161 methanolic extracts from amphidinium carterae, 160 Skeletonema marinoi hydrophobic fraction, 160 Fucoidan, 197–202, 212, 216–217, 257, 258 Fucosterol, 197, 204 Fucoxanthin, 228, 229, 232, 235–238 Functional groups, 142

Index  355 Gastrinoma (Zollinger-Ellison syndrome), 181 antiulcer products developed from algae, 184 natural products used in the treatment of peptic ulcer, 183 treatment using synthetic medicine, 181–183 Glucanase, 203 Glycoside hydrolases, 203 Green growth, 322–327, 329, 331, 332 Green seaweed polysaccharides, 260, 261 Hemostatic activity, 263, 264 Hydrophilic, 126 Hydrophobic, 126 Hypertension, 228 Hypolipidemic, 147 Immunomodulatory, 146 Immunomodulatory and antiinflammatory effects, 264–267 Immunosuppressive action, 25 Inflammation, 129 Injection molding, 143 Laminarin, 197, 202–204 laminarinase, 203 Lipid metabolites, 131 Lipoproteins, 131 Liquid chromatography–mass spectrometry (LC-MS), 8 Lutein, 237 Mechanisms underpinning the wound healing effects of algae, 261, 262 Microalgae, 3, 4, 6, 8–10, 12–15, 18, 21–23, 26, 198, 217, 220–221, 323, 324, 326, 328–333 Microalgae anticancer activity, 85

Microalgae with anti-inflammatory activity, 81 Microalgae with bioactive compounds, 44 chlorella, 47 dunaliella, 50 nostoc, 49 spirulina, 46 Microalgae with immunomodulatory activity, 82 Microbial cell, 345 Microorganisms, 8 Nanocomposite, 142 Nanoformulation, 128 Natural bioactive components, 8 Natural fiber, 143 Neoplasm (cancer), 3, 6, 23 Neuroinflammation, 131, 145 Nuclear magnetic resonance (NMR), 8 Nutraceutical, 343–344, 346, 348–349 Nutrient, 344–345 Obesity, 232, 233 Omega-3 polyunsaturated fatty acid, 128 Operational cost, 147 Peroxisomal disorder, 131 Phaeodactylum tricornutum, 127 Phaeophyta, 3, 4 Pharmaceutical applications of microalgae, 66 anticancer properties, 67 anti-inflammatory properties, 70 antimicrobial properties, 69 antioxidant properties, 70 antiviral properties, 69 cardioprotective properties, 66 Pharmaceutical industry, 87 Pharmaceuticals, 322, 323, 331–334

356  Index Phycocolloids, 186–187 agar, 187 alginates, 186–187 carrageenans, 187 fucoidan, 188 laminaran, 190 ulvans, 189 xylan and porphyran, 191–193 Phycocyanin, 209 Phycoerythrin, 197, 208 Phytoplankton, 2, 3, 15 Polymeric composite, 143 Polyphenols, 344 Polyunsaturated fatty acids, 60 Porphyridium cruetum, 127 Post-traumatic stress disorder, 129 Potential of microalgae in quality enhancement of natural products, 87 Progression, predisposing factors and treatment of cancer, 156 cancer progression, 156 predisposing factors to cancer, 157 treatment of cancer, 157 Proteins and polypeptides, 61 Receptor, 201, 206–207 Red pigment, 348 Red seaweed polysaccharides, 260 Rhamnose, 199 Rhodophyta, 3

Saproxanthin, 238 Seaweed, 198–199, 216, 218, 220 Secondary metabolites from microalgae, 55 carotenoids, 55 β-carotene, 55 astaxanthin, 57 fucoxanthin, 59 violaxanthin, 59 zeaxanthin and lutein, 58 Single cell protein, 344–349 Siphonaxanthin, 233, 238 Sources of microalgae, 158 Spirulina, 6, 7, 12, 15, 16, 21, 24, 25 Symptoms of peptic ulcer disease, 181 anxiety, 181 diet and alcohol, 181 genetic variable, 181 hypercalcemia, 181 smoking, 181 Synaptogenesis, 131 Therapeutic, 198, 201, 204, 209, 216 Thermoforming, 143 Tyrosine, 215 Vasculopathies, 128 Wound-healing property of algae and cyanobacteria, 271–273 Zeaxanthin, 237, 238

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