Marine Phenolic Compounds: Science and Engineering 0128235896, 9780128235898

Table of contents :
Front Cover
Marine Phenolic Compounds: Science and Engineering
Copyright
Contents
Contributors
Foreword
Preface
Section 1: Chemical characterization and classification
Chapter 1: Clean and green analytical techniques
1. Introduction
2. Sample preparation
2.1. Sample pre-treatment
2.2. Extract obtention
2.3. Isolation of compounds from extracts
3. Chromatographic separation approaches: Instrumental methods
4. Advanced mass spectrometry
4.1. Ionization source
4.2. Analyzer
4.2.1. Quadrupole
4.2.2. Ion trap
4.2.3. Time of flight
4.2.4. FT-ICR
4.2.5. Orbitrap
4.2.6. Tandem mass analyzers
4.3. Detector
5. Nuclear magnetic resonance approaches
6. Conclusions
References
Chapter 2: Marine phenolic compounds: Sources, commercial value, and biological activities
1. Introduction
2. Sources of marine phenolic compounds
2.1. Macroalgae
2.2. Phytoplankton
3. Marine phenolics of interest
4. Commercial value of marine phenolics
5. Biological activities and applications
5.1. Biological activities and applications against non-communicable diseases
5.1.1. Antidiabetic and antiobesity
5.1.2. Cardioprotective
5.2. Antimicrobial and antiviral activity
5.3. Other biological activities
5.3.1. Antioxidant
5.3.2. Anti-inflammatory
5.3.3. Neuroprotective
5.3.4. Antiallergic
5.3.5. Photoprotective
5.3.6. Antiaging
6. Conclusions
Acknowledgments
References
Further reading
Chapter 3: Marine natural bromophenols: Sources, structures, main bioactivities, and toxicity
1. Introduction
2. Sources, structures, bioactivities, and toxicity of BPs
2.1. Anticancer activity
2.2. Antidiabetic and anti-obesity activity
2.3. Antioxidant activity
2.4. Antimicrobial activity
2.5. Anti-inflammatory activity
2.6. Anti-neurodegenerative disease activity
2.7. Enzyme inhibitory activity
2.8. Possible toxicological effects of BPs
3. Conclusions
Acknowledgment
References
Section 2: Extraction and purification
Chapter 4: Marine phenolics: Extractions at low pressure
1. Introduction
2. Phenolic classes
2.1. Phenolic acids
2.2. Phlorotannins
2.3. Bromophenols
2.4. Flavonoids
2.5. Phenolic terpenoids
2.6. Mycosporine-like aminoacids (MAA)
3. Phenolic compounds extraction methods
3.1. Pre-treatment
3.2. Extraction methods
3.2.1. Classical methods
3.2.2. Modern extraction methods
Ultrasound-assisted extraction (UAE)
Microwave assisted extraction (MAE)
Enzyme-assisted extraction (EAE)
3.2.3. Green solvent extraction
Subcritical water extraction (SWE)
Ionic liquids
3.2.4. Other techniques
3.3. Extraction problems and future developments
4. Purification, quantification, and characterization
5. Conclusions
Acknowledgments
References
Further reading
Chapter 5: Extraction of marine phenolics using compressed fluids
1. Introduction
2. Supercritical fluid extraction (SFE)
2.1. Theoretical and practical fundamentals of SFE
2.2. Phenolic compounds extraction using SFE
2.2.1. Macroalgae
2.2.2. Microalgae
3. Gas expanded liquid extraction (GXLs)
3.1. Theoretical and practical fundaments of GXLs
3.2. Phenolic compounds extraction using GXLs
4. Pressurized liquid extraction (PLE)
4.1. Theoretical and practical fundamentals of PLE
4.2. Phenolic compounds extraction using PLE
4.2.1. Macroalgae
4.2.2. Microalgae
5. Biorefinery based on the use of compressed fluids
5.1. Marine biorefineries
5.1.1. Macroalgae
5.1.2. Microalgae
6. Conclusions
Acknowledgments
References
Chapter 6: Purification and fractionation of crude seaweed extracts by adsorption-desorption processes
1. Introduction
2. Resin purification (RP): Description and applications in seaweed extracts
3. Adsorption preparative liquid chromatography (APLC): Description and applications to seaweed extracts
4. Design, operating, and response variables of RP and APLC processes
4.1. Design and operating variables
4.2. Response variables
5. Adsorption-desorption mechanism
5.1. Preliminary assays in batch systems
5.2. Preliminary assays in column systems
6. Mathematical modeling of APLC: An option for optimal design
7. Conclusion and perspectives
References
Section 3: Bioavailability and bioactivity
Chapter 7: Interactions with other macromolecules
1. Phlorotannin-polysaccharide interactions
2. Phlorotannin-protein interactions
2.1. Noncovalent interactions
2.2. Covalent interactions
3. Comparison between terrestrial and marine tannin interactions with macromolecules
3.1. Mechanism of interactions
3.2. Influence of tannin structure
3.3. Influence of protein structure
3.4. Influence of polysaccharide structure
4. Conclusions
References
Chapter 8: Recent advances in the encapsulation of marine phenolic compounds
1. Introduction
2. Encapsulation techniques
2.1. Chemical encapsulation
2.1.1. Complexation
2.2. Lipid-based structured delivery vehicles: Liposomes
2.3. Physical encapsulation
2.3.1. Drying
2.3.2. Spray-drying
2.3.3. Freeze-dried liposomes
3. Applications of encapsulated microalgae-derived products
3.1. Functional foods
3.1.1. Biomass capsules as functional food ingredients
3.1.2. Encapsulated microalgae extract as functional food ingredients
3.2. Cosmetics
4. Conclusions
References
Chapter 9: Bioaccesibility and bioavailability of marine polyphenols
1. Introduction
2. Algae marine polyphenols: Source and their occurrence
3. Gastrointestinal stability of marine polyphenols: In vitro approaches
4. Bioavailability of marine polyphenols in humans
5. Metabolism and metabolic processes
6. Factors affecting marine polyphenol bioavailability: Influence of the food matrix, dose, and interindividual differences
6.1. Food matrix
6.2. Dose
6.3. Interindividual differences
7. Biomarkers of marine phenolic intake
Acknowledgments
References
Chapter 10: Antioxidant capacity of seaweeds: In vitro and in vivo assessment
1. Antioxidant capacity as a relevant health parameter
2. Antioxidant capacity techniques
3. Current evidence on the antioxidant capacity of seaweeds
3.1. Direct antioxidant capacity measurement
3.2. Antioxidant capacity in cell cultures
3.3. Antioxidant capacity in animal models
3.4. Antioxidant capacity in clinical studies
4. Other effects of seaweeds in oxidative stress modulation
5. Perspectives
References
Chapter 11: Gut microbiota and marine phenolics
1. Introduction
2. Structural classification of marine polyphenols
2.1. Phlorotannins
2.2. Bromophenols
2.3. Simple phenolic acids
2.4. Flavonoids
3. Properties and activities of phenolics
3.1. Antioxidant activity
3.2. Anti-inflammatory activity
3.3. Anti-diabetic properties
3.4. Antibacterial activity
4. Digestion and metabolism of polyphenols
5. Gut microbiota
5.1. Nutrient absorption and metabolites synthesis
5.2. Protection against pathogens and mucosal gut barrier
5.3. Normal microbiota composition and dysbiosis
5.4. Disease and gut microbiota composition
6. Metabolization of marine phenolics by the gut microbiota
7. Prebiotic role of seaweed compounds
8. Conclusions
References
Chapter 12: Marine phenolics: Classes, antibacterial properties, and applications
1. Phenolics and marine resources
2. Marine phenolics
2.1. Classes and sources
2.1.1. Macroalgae
Phenolic acids
Flavonoids
Bromophenols
Phlorotannins
2.1.2. Microalgae
2.2. Antibacterial properties
2.3. Industrial applications and future perspectives
Acknowledgments
References
Section 4: Health and diseases prevention
Chapter 13: Impact of phlorotannins on cardiovascular diseases
1. Introduction
2. Phlorotannins
3. Underlying pathologies with impact on CVDs
4. Evidence of protective effects of phlorotannins on CVDs
4.1. Dyslipidemia
5. Endothelial function
6. Conclusions
Acknowledgments
References
Chapter 14: Immune system: Inflammatory response
1. Introduction
2. Arterial hypertension (HTN) and inflammation
3. Biotic drivers of inflammation
4. Therapeutic alternatives for the treatment of the inflammatory process
5. Phenolic compounds present in marine algae and their anti-inflammatory effect
6. Phlorotannins and therapeutic effect against oxidation
7. Phlorotannins and their therapeutic effect against aging and neuroprotection
8. Phenolic compounds in seaweed and its antihypertensive effect
9. Phlorotannins trends
10. Conclusion
References
Chapter 15: Effects of marine phenolics on diabetes, obesity, and metabolic syndrome
1. Introduction
2. Pathophysiology of diabetes
3. Antidiabetic effects of marine phenols
3.1. Effect of marine phenolics on postprandial glycemia and glucose levels
3.2. Effect of marine phenolics on β-cell function
3.3. Effects of marine phenolics on insulin sensitivity
3.4. Effect of marine phenolics on gut microbiota in diabetes
4. Pathophysiology of obesity
5. Antiobesity effects of marine phenols
5.1. Effect of marine phenolics on lipid levels
5.2. Effect of marine phenolics on adipogenesis
5.3. Effect of marine phenolics on the ``browning´´ of the adipose tissue
5.4. Effect of marine phenolics on inflammation, oxidative stress, and GM
6. Pathophysiology of the metabolic syndrome
7. Effects of marine phenols on metabolic syndrome
8. Conclusions
Acknowledgments
References
Chapter 16: Neurodegenerative diseases
1. Introduction
2. Alzheimer's disease
3. Parkinson's disease
4. Perspectives
References
Chapter 17: Applications of seaweed polyphenols in food
1. Introduction
2. Incorporating polyphenols in food
2.1. Algae-based snack
2.2. Meat products
2.3. Starchy food
3. Relevance of sensorial properties and food neophobia
3.1. Sensorial properties
3.2. Food neophobia and algae-based foodstuff development
4. A comprehensive approach to a sustainable industry
5. Conclusions
Acknowledgments
References
Index
Back Cover

Citation preview

MARINE PHENOLIC COMPOUNDS: SCIENCE AND ENGINEERING

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MARINE PHENOLIC COMPOUNDS: SCIENCE AND ENGINEERING Edited by

JOSE RICARDO PEREZ-CORREA Professor of Chemical and Bioprocess Engineering Department at Pontificia Universidad Cato´lica de Chile, Santiago, Chile

RAQUEL MATEOS Tenured Scientist at the Institute of Food Science, Technology and Nutrition (ICTAN) belonging to the Spanish National Research Council (CSIC), Spain

HERMINIA DOMI´NGUEZ Professor, University of Vigo, Spain

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-12-823589-8 For information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Susan Dennis Acquisitions Editor: Gabriela Capille Editorial Project Manager: Lena Sparks Production Project Manager: Paul Prasad Chandramohan Cover Designer: Christian Bilbow Typeset by STRAIVE, India

Contents Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

Section 1 Chemical characterization and classification Chapter 1 Clean and green analytical techniques . . . . . . . . . . . 1

 Francisco Javier Leyva-Jimenez, M. Elena Alan˜o´n, Marı´a del Carmen Villegas-Aguilar, A´lvaro Ferna´ndez-Ochoa, Alejandro Rojas-Garcı´a, Patricia Ferna´ndez-Moreno, David Arra´ezRoma´n, Marı´a de la Luz Ca´diz-Gurrea, and Antonio Segura-Carretero 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Chromatographic separation approaches: Instrumental methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Advanced mass spectrometry . . . . . . . . . . . . . . . . . . . . 5 Nuclear magnetic resonance approaches . . . . . . . . . . . 6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 2 Marine phenolic compounds: Sources, commercial value, and biological activities . . . . . . . . . . . . 47 Pilar Fallas Rodrı´guez, Laura Murillo-Gonza´lez, Evelyn Rodrı´guez,  and Ana M. Perez 1 2 3 4

Introduction . . . . . . . . . . . . . . . . . . . . . Sources of marine phenolic compounds Marine phenolics of interest . . . . . . . . . Commercial value of marine phenolics .

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5 Biological activities and applications . 6 Conclusions . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . Further reading . . . . . . . . . . . . . . . . . . .

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Chapter 3 Marine natural bromophenols: Sources, structures, main bioactivities, and toxicity . . . . . . . . . . . . . . . . . . . . . 87 Hui Dong, Poul Erik Hansen, Songtao Dong, Dimitrios Stagos, Xiukun Lin, and Ming Liu 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Sources, structures, bioactivities, and toxicity of BPs 3 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Section 2 Extraction and purification Chapter 4 Marine phenolics: Extractions at low pressure . . . . . . . . 115 Joa˜o Cotas, Diana Pacheco, Pedro Monteiro, Ana M.M. Gonc¸alves, and Leonel Pereira 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Phenolic classes . . . . . . . . . . . . . . . . . . . . . . . . 3 Phenolic compounds extraction methods . . . . . 4 Purification, quantification, and characterization 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Further reading . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 5 Extraction of marine phenolics using compressed fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Lidia Montero, Ba´rbara Socas-Rodrı´guez, Jose Antonio Mendiola, and Elena Iba´n˜ez 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Supercritical fluid extraction (SFE) . . . . . . . . . . . . 3 Gas expanded liquid extraction (GXLs) . . . . . . . . . 4 Pressurized liquid extraction (PLE) . . . . . . . . . . . . 5 Biorefinery based on the use of compressed fluids 6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 6 Purification and fractionation of crude seaweed extracts by adsorption-desorption processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Marı´a Salome Mariotti-Celis, Pamela Raquel Rivera-Tovar,  Nils Leander Huama´n-Castilla, and Jose Ricardo Perez-Correa

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Resin purification (RP): Description and applications in seaweed extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Adsorption preparative liquid chromatography (APLC): Description and applications to seaweed extracts . . . . . . . . 4 Design, operating, and response variables of RP and APLC processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Adsorption-desorption mechanism . . . . . . . . . . . . . . . . . . . 6 Mathematical modeling of APLC: An option for optimal design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Conclusion and perspectives . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Section 3 Bioavailability and bioactivity Chapter 7 Interactions with other macromolecules . . . . . . . . . . . . . 219 Xuwei Liu and Carine Le Bourvellec 1 Phlorotannin-polysaccharide interactions . . . . . . . 2 Phlorotannin-protein interactions . . . . . . . . . . . . . 3 Comparison between terrestrial and marine tannin interactions with macromolecules . . . . . . . . . . . . . 4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 8 Recent advances in the encapsulation of marine phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 Wendy Franco, Migdalia Caridad Rusindo Arazo, and Sergio Benavides 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Encapsulation techniques . . . . . . . . . . . . . . . . . . . . . . . . . 3 Applications of encapsulated microalgae-derived products 4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 9 Bioaccesibility and bioavailability of marine polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

 Salud Ca´ceres-Jimenez, Jose Luis Ordo´n˜ez-Dı´az,  Jose Manuel Moreno-Rojas, and Gema Pereira-Caro

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Algae marine polyphenols: Source and their occurrence . 3 Gastrointestinal stability of marine polyphenols: In vitro approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Bioavailability of marine polyphenols in humans . . . . . .

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5 Metabolism and metabolic processes . . . . . . . . . . . . . . . . . . . 6 Factors affecting marine polyphenol bioavailability: Influence of the food matrix, dose, and interindividual differences . . . . . 7 Biomarkers of marine phenolic intake . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 10 Antioxidant capacity of seaweeds: In vitro and in vivo assessment . . . . . . . . . . . . . . . . . . . . . . . . . 299   H. Sa´nchez-Ayora and J. Perez-Jim enez

1 Antioxidant capacity as a relevant health parameter . . . . . 2 Antioxidant capacity techniques . . . . . . . . . . . . . . . . . . . . 3 Current evidence on the antioxidant capacity of seaweeds 4 Other effects of seaweeds in oxidative stress modulation . 5 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 11 Gut microbiota and marine phenolics . . . . . . . . . . . . . . . 343  Urrutia, and Daniel Garrido Samantha Nu´n˜ez, Arles 1 2 3 4 5 6

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Structural classification of marine polyphenols Properties and activities of phenolics . . . . . . . Digestion and metabolism of polyphenols . . . Gut microbiota . . . . . . . . . . . . . . . . . . . . . . . . Metabolization of marine phenolics by the gut microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Prebiotic role of seaweed compounds . . . . . . . 8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 12 Marine phenolics: Classes, antibacterial properties, and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 Cla´udia Lea˜o, Manuel Simo˜es, and Anabela Borges 1 Phenolics and marine resources 2 Marine phenolics . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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Section 4 Health and diseases prevention Chapter 13 Impact of phlorotannins on cardiovascular diseases . . . 395 So´nia J. Amarante, Marcelo D. Catarino, Artur M.S. Silva, and Susana M. Cardoso 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Phlorotannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Underlying pathologies with impact on CVDs . . . . . . . . . 4 Evidence of protective effects of phlorotannins on CVDs . 5 Endothelial function . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 14 Immune system: Inflammatory response . . . . . . . . . . . . . 415 Diana Del Juncal-Guzma´n, Carlos Eduardo Camacho-Gonza´lez, Francia Guadalupe Lo´pez-Ca´rdenas, Sonia Guadalupe Sa´yago-Ayerdi, and Jorge Alberto Sa´nchez-Burgos 1 2 3 4 5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arterial hypertension (HTN) and inflammation . . . . . . . . . . . . . Biotic drivers of inflammation . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutic alternatives for the treatment of the inflammatory process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenolic compounds present in marine algae and their anti-inflammatory effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contents

6 Phlorotannins and therapeutic effect against oxidation . . . 7 Phlorotannins and their therapeutic effect against aging and neuroprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Phenolic compounds in seaweed and its antihypertensive effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Phlorotannins trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 15 Effects of marine phenolics on diabetes, obesity, and metabolic syndrome . . . . . . . . . . . . . . . . . . . . . . . . 431 Esther Garcı´a-Dı´ez, Marı´a A´ngeles Martin, and Sonia Ramos 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Pathophysiology of diabetes . . . . . . . . . . . . . . . . . 3 Antidiabetic effects of marine phenols . . . . . . . . . 4 Pathophysiology of obesity . . . . . . . . . . . . . . . . . . 5 Antiobesity effects of marine phenols . . . . . . . . . . 6 Pathophysiology of the metabolic syndrome . . . . . 7 Effects of marine phenols on metabolic syndrome 8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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434 435 437 446 448 461 461 464 464 464 464

Chapter 16 Neurodegenerative diseases . . . . . . . . . . . . . . . . . . . . . 473 Raquel Mateos 1 Introduction . . . . . . 2 Alzheimer’s disease . 3 Parkinson’s disease . 4 Perspectives . . . . . . References . . . . . . . . .

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Chapter 17 Applications of seaweed polyphenols in food . . . . . . . . 495 Javier Parada 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Incorporating polyphenols in food . . . . . . . . . . . . . . . 3 Relevance of sensorial properties and food neophobia 4 A comprehensive approach to a sustainable industry . 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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495 496 500 504 507 508 508

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513

Contributors ´ n Department of Analytical Chemistry and Food Science ˜o M. Elena Alan and Technology; Regional Institute for Applied Scientific Research (IRICA), Area of Food Science, University of Castilla-La Mancha, Ciudad Real, Spain ´ nia J. Amarante LAQV-REQUIMTE, Department of Chemistry, So University of Aveiro, Aveiro, Portugal Migdalia Caridad Rusindo Arazo Dietetics School, Faculty of Medicine, Universidad Finis Terrae, Pedro de Valdivia, Providencia, Santiago, Chile David Arra´ez-Roma´n Department of Analytical Chemistry, University of Granada, Granada, Spain Sergio Benavides Research Center in Agri-food and Applied Nutrition, Adventist University of Chile, Chilla´n, Chile Anabela Borges LEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto; ALiCE—Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias; DEQ—Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal nez Department of Agroindustry and Food Quality, Salud Ca´ceres-Jime Andalusian Institute of Agricultural and Fisheries Research and Training (IFAPA); Department of Food Science and Food Technology, Campus Rabanales, Ed. Darwin-anexo University of Co´rdoba, Co´rdoba, Spain Carlos Eduardo Camacho-Gonza´lez Division of Graduate Studies, National Technological Institute of Mexico/Instituto Tecnolo´gico de Tepic, Tepic, Nayarit, Mexico Susana M. Cardoso LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, Aveiro, Portugal Marcelo D. Catarino LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, Aveiro, Portugal Joa˜o Cotas University of Coimbra, MARE—Marine and Environmental Sciences Centre, Department of Life Sciences, Calc¸ada Martim de Freitas, Coimbra, Portugal Marı´a de la Luz Ca´diz-Gurrea Department of Analytical Chemistry, University of Granada, Granada, Spain

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Contributors

Marı´a del Carmen Villegas-Aguilar Department of Analytical Chemistry, University of Granada, Granada, Spain Diana Del Juncal-Guzma´n Division of Graduate Studies, National Technological Institute of Mexico/Instituto Tecnolo´gico de Tepic, Tepic, Nayarit, Mexico Hui Dong Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao; Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, China Songtao Dong Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao; Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, China Patricia Ferna´ndez-Moreno Department of Analytical Chemistry, University of Granada, Granada, Spain ´ lvaro Ferna´ndez-Ochoa Department of Analytical Chemistry, A University of Granada, Granada, Spain Wendy Franco Chemical Engineering and Bioprocess; Health Science Department, Nutrition and Dietetics, Pontifical Catholic University of Chile, Macul, Chile Esther Garcı´a-Dı´ez Department of Metabolism and Nutrition, Institute of Food Science and Technology and Nutrition (ICTAN-CSIC), Ciudad Universitaria, Madrid, Spain Daniel Garrido Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Cato´lica de Chile, Santiago, Chile Ana M.M. Gonçalves University of Coimbra, MARE—Marine and Environmental Sciences Centre, Department of Life Sciences, Calc¸ada Martim de Freitas, Coimbra, Portugal; Department of Biology and CESAM, University of Aveiro, Aveiro, Portugal Poul Erik Hansen Department of Science and Environment, Roskilde University, Roskilde, Denmark Nils Leander Huama´n-Castilla School of Agroindustrial Engineering, Universidad Nacional de Moquegua, Moquegua, Peru ˜ ez Laboratory of Foodomics, Institute of Food Science Elena Iba´n Research (CIAL, CSIC-UAM), Madrid, Spain Carine Le Bourvellec INRAE, Avignon University, UMR408 SQPOV, Avignon, France

Contributors

Cla´udia Lea˜o LEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto; ALiCE—Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, Porto, Portugal nez Department of Analytical Chemistry Francisco Javier Leyva-Jime and Food Science and Technology; Regional Institute for Applied Scientific Research (IRICA), Area of Food Science, University of Castilla-La Mancha, Ciudad Real, Spain Xiukun Lin Department of Pharmacology, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, China Ming Liu Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao; Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, China Xuwei Liu INRAE, Avignon University, UMR408 SQPOV, Avignon, France; College of Food Science, South China Agricultural University, Guangzhou, China ´ pez-Ca´rdenas Division of Graduate Studies, Francia Guadalupe Lo National Technological Institute of Mexico/Instituto Tecnolo´gico de Tepic, Tepic, Nayarit, Mexico  Mariotti-Celis School of Nutrition and Dietetics, Faculty Marı´a Salome of Medicine, Universidad Finis Terrae, Santiago, Chile ´ ngeles Martin Department of Metabolism and Nutrition, Marı´a A Institute of Food Science and Technology and Nutrition (ICTAN-CSIC), Ciudad Universitaria; Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Instituto de Salud Carlos III (ISCIII), Madrid, Spain Raquel Mateos Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Spanish National Research Council (CSIC), Madrid, Spain Jose Antonio Mendiola Laboratory of Foodomics, Institute of Food Science Research (CIAL, CSIC-UAM), Madrid, Spain Pedro Monteiro University of Coimbra, MARE—Marine and Environmental Sciences Centre, Department of Life Sciences, Calc¸ada Martim de Freitas, Coimbra, Portugal Lidia Montero Applied Analytical Chemistry; Teaching and Research Center for Separation, University of Duisburg-Essen, Essen, Germany

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 Manuel Moreno-Rojas Department of Agroindustry and Food Jose Quality, Andalusian Institute of Agricultural and Fisheries Research and Training (IFAPA); Foods for Health Group, Instituto Maimo´nides de Investigacio´n Biom edica de Co´rdoba (IMIBIC), Co´rdoba, Spain Laura Murillo-Gonza´lez Food Engineering School, Guanacaste Campus, University of Costa Rica, Liberia, Guanacaste, Costa Rica ´n ˜ ez Department of Chemical and Bioprocess Engineering, Samantha Nu School of Engineering, Pontificia Universidad Cato´lica de Chile, Santiago, Chile  Luis Ordo´n ˜ez-Dı´az Department of Agroindustry and Food Quality, Jose Andalusian Institute of Agricultural and Fisheries Research and Training (IFAPA), Co´rdoba, Spain Diana Pacheco University of Coimbra, MARE—Marine and Environmental Sciences Centre, Department of Life Sciences, Calc¸ada Martim de Freitas, Coimbra, Portugal Javier Parada Institute of Food Science and Technology, Faculty of Agricultural and Food Sciences, Austral University of Chile, Valdivia, Chile Leonel Pereira University of Coimbra, MARE—Marine and Environmental Sciences Centre, Department of Life Sciences, Calc¸ada Martim de Freitas, Coimbra, Portugal Gema Pereira-Caro Department of Agroindustry and Food Quality, Andalusian Institute of Agricultural and Fisheries Research and Training (IFAPA); Foods for Health Group, Instituto Maimo´nides de Investigacio´n Biom edica de Co´rdoba (IMIBIC), Co´rdoba, Spain rez National Center of Food Science and Technology (CITA), Ana M. Pe , University of Costa Rica, Ciudad Universitaria Rodrigo Facio, San Jose Costa Rica  Ricardo Pe rez-Correa Chemical and Bioprocess Engineering Jose Department, School of Engineering, Pontificia Universidad Cato´lica de Chile, Santiago, Chile rez-Jime nez Department of Metabolism and Nutrition, Institute of J. Pe Food Science, Technology and Nutrition (ICTAN-CSIC), Madrid, Spain Sonia Ramos Department of Metabolism and Nutrition, Institute of Food Science and Technology and Nutrition (ICTAN-CSIC), Ciudad Universitaria, Madrid, Spain Pamela Raquel Rivera-Tovar Chemical and Bioprocess Engineering Department, School of Engineering, Pontificia Universidad Cato´lica de Chile, Santiago, Chile Evelyn Rodrı´guez National Center of Food Science and Technology (CITA), University of Costa Rica, Ciudad Universitaria Rodrigo Facio, , Costa Rica San Jose

Contributors

Pilar Fallas Rodrı´guez National Center of Food Science and Technology (CITA), University of Costa Rica, Ciudad Universitaria Rodrigo Facio, San Jose, Costa Rica Alejandro Rojas-Garcı´a Department of Analytical Chemistry, University of Granada, Granada, Spain H. Sa´nchez-Ayora Department of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Madrid, Spain Jorge Alberto Sa´nchez-Burgos Division of Graduate Studies, National Technological Institute of Mexico/Instituto Tecnolo´gico de Tepic, Tepic, Nayarit, Mexico Sonia Guadalupe Sa´yago-Ayerdi Division of Graduate Studies, National Technological Institute of Mexico/Instituto Tecnolo´gico de Tepic, Tepic, Nayarit, Mexico Antonio Segura-Carretero Department of Analytical Chemistry, University of Granada, Granada, Spain Artur M.S. Silva LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, Aveiro, Portugal ˜ es LEPABE—Laboratory for Process Engineering, Manuel Simo Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto; ALiCE—Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias; DEQ—Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal Ba´rbara Socas-Rodrı´guez Laboratory of Foodomics, Institute of Food Science Research (CIAL, CSIC-UAM), Madrid, Spain Dimitrios Stagos Department of Biochemistry and Biotechnology, School of Health Sciences, University of Thessaly, Biopolis, Larissa, Greece s Urrutia Department of Chemical and Bioprocess Engineering, Arle School of Engineering, Pontificia Universidad Cato´lica de Chile, Santiago, Chile

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Foreword We eat tasty structures that have proven to be safe and provide pleasure as well as benefits to our health and well-being. The fact that natural structures are composed of molecules, as we are, was not realized until the late 1700s. Thereafter, several famous chemists became interested in discovering important chemical species in foods. For example, the Swedish scientist Carl Wilhelm Scheele isolated and identified some organic acids present in a wide variety of fruits and berries. By the mid-19th century, scientists like the German chemist Justus von Liebig started to classify major food constituents into carbohydrates, fats, and proteins. By the early 20th century, the links between some nutritional deficiencies and ailments such as beriberi, rickets, and scurvy became evident. This started the era of micronutrients, molecules that need to be present in our foods in small amounts as their presence is essential for health. Interestingly, the role of major food components in providing energy for body functioning and molecules for body growth and repair were studied later, particularly in relation to protein-calorie malnutrition in developing countries. Why all this preamble? Because this book is about a special kind of molecular species in foods that at present attract unique attention in the field of food science and nutrition. Moreover, to make lay people aware that we eat molecules and whether we perceive them as colors, tastants, and aromas while eating dishes, their ultimate function is to be metabolized and incorporated into our bodies. Some of these molecules, together with some vitamins, essential amino acids, and essential fatty acids, cannot be synthesized by us and have to be part of our diets. Phenolic compounds belong to a type of chemical species that are present in small quantities in plants and are essential for their interactions with the environment. They possess a range of complex structures and are classified as bioactive compounds in foods, meaning that they exert beneficial effects on human health beyond their possible role as nutrients. Bioactive compounds are believed to protect human health against some chronic degenerative diseases. They exhibit low bioavailability in the upper digestive tract, but once in the colon, these compounds interact with the microbiota, releasing metabolites whose action in our bodies is yet to be fully assessed. Phenolic compounds in foods

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contribute to color, bitterness, astringency, flavor, aroma, and stability against oxidation. In recent decades, marine macroalgae have attracted the attention of food scientists and food technologists as an abundant, economic, and sustainable source of edible raw materials. Seaweeds are actively studied as culinary ingredients, sources of tasty and functional ingredients, and for their positive effects on health. Many phenolic compounds found in marine algae have antioxidant properties and antiviral activity. Although some epidemiologic studies have reported protective associations between phenolic compounds and some chronic ailments, further research needs to be done to demonstrate causality and reveal the exact mechanisms of the possible effects. The book Marine Phenolic Compounds: Science and Engineering is quite timely as it provides a comprehensive overview of the science and technology of marine phenolics. Among the topics covered are analytical techniques, technological developments for their extraction and purification, uses in foods as antioxidants and antimicrobial agents, their fate during digestion, possible roles in preventing or alleviating neurodegenerative diseases, obesity, diabetes, and metabolic syndrome, and potential applications in new foods that promote health and well-being. The publisher Elsevier Inc., the editors, and the authors of the chapters must be congratulated for making available to the food science community a much-needed text on the fascinating subject of marine phenolic compounds.  Miguel Aguilera Jose Zapallar, Chile

Preface Marine organisms represent an extremely diverse worldwide resource offering high-value metabolites with potential commercial applications for their nutritive, technological, and pharmacological properties. Phenolic compounds in the oceanic environment and marine organisms are attracting growing interest since some chemical structures are unique and not found in terrestrial sources. These compounds are mainly synthesized by marine organisms to protect themselves against biotic and abiotic stressors. Previous research has shown potent antimicrobial, antioxidant, antitumoral, and anti-inflammatory activity; therefore, they have the potential to protect us from several diseases. Nevertheless, the chemical and functional characterization of marine phenolics is far from complete and remains challenging, given their complex and diverse structures, the scarcity of chemical standards, and the many biological activities they present. Many conventional and innovative techniques have been applied to obtain marine phenolic extracts that can be used to develop innovative products, particularly in the pharmaceutical, nutraceutical, and cosmetic fields. Both the relevance and potential of marine phenolics, at a time when there is increasing awareness of the importance of diet for healthy aging, encouraged us to bring together the latest advances in marine phenolics from the hand of well-known experts in this field. This book offers a brief overview of the main sources of marine phenolics and the different analytical techniques for their chemical characterization. Additionally, the interaction of marine phenolics with other macromolecules and the gut microbiota, as well as their bioavailability and biotransformation, is discussed. The specific bioactivities of marine phenolics that are useful to face prevalent pathologies such as cardiometabolic processes (obesity, diabetes, and metabolic syndrome) and neurodegenerative diseases are described in depth. These basic science aspects are complemented with technological chapters, describing the newest results regarding extraction, purification, and encapsulation of marine phenolics for the industrial production of functional ingredients and foods. This book is intended for undergraduate and graduate students and researchers working in universities, R&D institutes,

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and innovative companies. This book will benefit many disciplines such as food science and engineering, nutrition, pharmaceutical sciences, chemistry, biotechnology, and chemical engineering. Let this book encourage the R&D community to continue working on understanding and exploiting the true potential of these phytochemicals, as powerful as they are mysterious.

Jos e Ricardo P erez-Correa Pontificia Universidad Cato´lica de Chile, Santiago, Chile

Raquel Mateos Institute of Food Science, Technology and Nutrition (ICTAN, CSIC), Madrid, Spain

Herminia Domı´nguez University of Vigo, Ourense, Spain

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SECTION

Chemical characterization and classification

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Clean and green analytical techniques

1

Francisco Javier Leyva-Jim
eneza,b, M. Elena Alan˜o´na,b, Marı´a del Carmen Villegas-Aguilarc, A´lvaro Ferna´ndezOchoac, Alejandro Rojas-Garcı´ac, Patricia Ferna´ndezMorenoc, David Arra´ez-Roma´nc, Marı´a de la Luz Ca´diz-Gurreac, and Antonio Segura-Carreteroc a

Department of Analytical Chemistry and Food Science and Technology, University of Castilla-La Mancha, Ciudad Real, Spain. bRegional Institute for Applied Scientific Research (IRICA), Area of Food Science, University of CastillaLa Mancha, Ciudad Real, Spain. cDepartment of Analytical Chemistry, University of Granada, Granada, Spain

1. Introduction A rigorous evaluation of potential bioactive compounds, i.e., marine phenolics, requires constant innovation in sustainable technologies, chromatographic materials, and analytical approaches to achieve early identification and accurate quantification of every candidate able to show benefits in human health. The main improvements in analytical chemistry have taken place since 1900 where instrumental analysis has progressively been the dominant section in this field. In fact, analytical chemistry is considered an engine of evolution for other areas of chemistry. However, the development of chemical products and sustainable procedures that minimize the use and generation of hazardous substances has been conducted in order to achieve the idea of green analytical chemistry. This concept emerged in 2000 with the premise of making smart design of environmentally friendly and cheap approaches (Filippou et al., 2017). In this sense, numerous analytical methods and techniques have been developed although it is important to introduce the sustainable development concept to analytical chemistry laboratories to achieve conscientious analytical methods (Tobiszewski et al., 2010). There are stages that are unavoidable; for this reason, choosing clean analytical methodologies is highly recommended. Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00003-0 Copyright # 2023 Elsevier Inc. All rights reserved.

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Chapter 1 Clean and green analytical techniques

Focusing on bioactive compounds, such as phenolic compounds from marine sources, there are organisms that can synthesize these compounds providing good sources for its revalorization. Among the huge diversity of marine organisms, algae are the most common candidates; in fact, they refer to phytoplankton (microalga), macroalga, and seaweed with more than 11,000 different known species in total ( Jimenez-Lopez et al., 2021). Concerning the chemical structure, phenolic compounds are characterized by a common structure based on one aromatic phenolic ring with hydroxyl substituents. However, the substituents can be highly polymerized comprising a wide number of heterogeneous structures that need specific methodologies for their recovery. In this chapter, we will shortly encompass the main milestones of clean and green analytical techniques related to the obtention of bioactive marine compounds.

2. Sample preparation Sample preparation takes a key role in each analytical procedure, which depends on the sample properties including complexity of the source or phytochemical nature. Overall, in green analytical procedures, there are numerous steps involved and some of them are unavoidable; thus, it is crucial to find and set the more environmentally benign methodologies. The analytical procedure can be summarized in four crucial steps, which involve (Tobiszewski et al., 2010) (a) pre-treating the original source to increase the analyte recovery; (b) extracting and concentrating the analytes from pre-treated matrix and reducing the interferences between matrices and analytes; (c) drying or removing the solvent; and (d) separating and isolating the analytes contained in the extracts. These stages should be optimized to reduce time, solvent and energy consumption, and residue production with the purpose of achieving environmentally friendly methods demanded from green analytical chemistry. For instance, marine products or by-products represent a relevant source of bioactive compounds that can be recovered in order to incorporate them into food, cosmetic, and pharmaceutical products to improve human health. In this sense, compounds such as polyphenols, polysaccharides, carotenoids, or polyunsaturated fatty acids and monounsaturated fatty acids (PUFAs and MUFAs) are present in marine products, including macro- and microalgae, fungi, or echinoderms (Alvarez-Gomez, 2016; Kumari et al., 2018; Mamelona et al., 2007), which possess a wide range of bioactivities such as

Chapter 1 Clean and green analytical techniques

antioxidant, anti-inflammatory, neuroprotective, or antidiabetic activities (Lajili et al., 2016; Thomas and Kim, 2011; Zhong et al., 2007). In order to achieve an efficient recovery of these compounds and their isolation, it is necessary to know the nature of the bioactive compounds contained in the natural source. This information is required to choose a suitable pre-treatment (drying, grinding…), to define an extraction technique, and to establish the analytical conditions to separate, isolate, and quantify the target compounds, including solvents and derivatizations that may be used in these processes. All these crucial steps are described in detail next.

2.1 Sample pre-treatment With the purpose of obtaining green extracts to analyze, separate, and isolate bioactive compounds from natural sources, it is necessary to carry out several steps before analytical assays, considering solvent interactions, sample residues, or chemicals used to isolate the target compounds. For these reasons, the selection of pre-treatment and extraction techniques takes a relevant role to achieve the expected results during isolation, characterization, and quantitation by analytical platforms. In the pretreatment of the sources, it is necessary to consider three steps. The first consists of washing thoroughly with water several times after the recollection for removing impurities (Michalak and Chojnacka, 2014). Some samples have a high water content (marine sources, fruits…); for this reason, a previous drying step is mandatory to improve the preservation of fresh material and the concentration of target analytes. In this sense, three different drying methods could be applied: chemical, physical, or physicochemical in order to avoid the degradation of compounds and enhance their preservation. This step is not a green stage in any analytical procedure since it generates chemical residues, and requires energy to reach the specified vacuum and temperature (Płotka-Wasylka, 2018). After that, it is convenient to perform a grinding step (manual or mechanical) to reduce the particle size, which increases particle/solvent contact area, hence improving the analyte recovery (Crampon et al., 2011). Finally, the dried powder obtained is sieved through a sieve with a specific mesh size to attain the appropriate powder size. Implementing this pretreatment pretends to enhance the extraction yield since it usually facilitates the bioavailability of the analytes. Furthermore, several approaches are used to increase this bioavailability including mechanical, thermal, physical, chemical, and enzymatic treatments such as autoclaving, alkaline and acidic hydrolysis, high-pressure homogenization, sonication, osmotic

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Chapter 1 Clean and green analytical techniques

shocks, or microwave. All of them are used to increase the disruption of cell walls and increase the release of bioactive compounds. For instance, enzymatic treatments with cellulase or protease may be applied to improve the extraction of analytes that are contained inside the cell by disrupting the cell wall or releasing the compounds that are bounded to this wall (Deniaud-Boue¨t et al., 2014). In the case of marine sources such as algae, this step may enhance the recovery of targeted analytes to a large extent since many of these analytes are bounded to the cell wall or form polymers with variable complexity (Sa´nchez-Camargo et al., 2016). Additionally, sonication involves the production of bubbles at high temperatures and pressures that may produce the breakage of the cell wall, enhancing the bioavailability of compounds (Varo et al., 2019). Moreover, microwaves can be performed without solvents, so the microwaves penetrate directly into the cell, increasing the temperature of the water in the cell. The microwave heat evaporates the water in the cells, causing cells to swell and break, which enables the release of the target compounds
nez et al., 2019; Xiao et al., 2012). Another approach (Leyva-Jime intended to increase the bioavailability of target compounds is chemical lysis, which involves the use of acid or alkalis (sulfuric acid or sodium hydroxide) to hydrolyze the natural source into constituent molecules (Lee et al., 2010). These steps may be applied before or combined with extraction procedures, and their selection can be determinant for the extraction yield. It is necessary to remark that some of these approaches may leave several chemicals in the sample that can interact during analytical characterization tests requiring different processes such as centrifugation, precipitation, or filtration, to remove them.

2.2 Extract obtention Ideally, extraction procedures should not be applied, but most of the time, they are unavoidable. In addition, further separation and isolation steps are often required. Extract preparation frequently involves the use of solvents and other chemicals, which generate high amounts of harmful residues. Indeed, conventional methods require liquid-liquid or liquid-solid extractions with an organic solvent, usually followed by cleanup and preconcentration using other solvents. Being non-green processes, these may be optimized. These conventional extraction methods are time-consuming and costly in terms of solvents and energy, and their yields are limited; consequently, these are inefficient methods. For instance, Soxhlet extraction is a robust conventional method to obtain low polarity compounds, although it needs

Chapter 1 Clean and green analytical techniques

considerable amounts of organic solvents and long extraction times (Ramluckan et al., 2014). Currently, several methods have been developed and applied to achieve faster and more efficient and selective extractions, requiring much less solvents. These are innovative or green technologies since they are environmentally friendly, requiring less solvent, energy, and time than conventional methods. Conventional and innovative extraction techniques have been applied to obtain enriched extract with bioactive compounds from marine sources (see Table 1). For instance, supercritical fluid extraction (SFE) has received growing interest as a sustainable technology that allows the attainment of extracts without any solvent residue, since in most cases, it applies only carbon dioxide. This solvent is subjected to pressures and temperatures above 73.8 bar and 31.1°C, respectively, conferring it liquid-like solvent solubility power and gas-like diffusivity increasing its extracting power. After the extraction process, carbon dioxide becomes volatile and it is automatically removed from the extracts, reducing the energy consumed in the following
nez et al., 2020). The non-polar nature of drying steps (Leyva-Jime carbon dioxide enhances the selective extraction of analytes such as fatty acids, some vitamins, and carotenoids being broadly applied to obtain bioactive ingredients from microalgae (Abrahamsson et al., 2012), algae (Fabrowska et al., 2016), or sea cucumber (Zakharenko et al., 2020). Moreover, pressurized liquid extraction (PLE) is another innovative extraction technology, which applies a combination of high pressures and temperatures to the solvents and samples, enhancing the extraction of a wide variety of compounds with high yield efficiency (Howard and Pandjaitan, 2008). Although this technique applies high temperature and pressure and consequently its energy demand is high, it needs low extraction times and solvent consumptions compared to conventional methods. Indeed, several studies have revealed its utility to obtain phenolic compounds and polysaccharides from different algae (Saravana et al., 2016; Sumampouw et al., 2021) or microalgae ( Jaime et al., 2005). As mentioned above, microwave-assisted extraction (MAE) may be used to enhance the recovery of analytes from different sources allowing its application without solvents but also can be used with different polar solvents. The heat transfer of this method facilitates the obtention of extracts enriched in thermosensitive compounds since the sample heating is fast (Azmir et al., 2013). Extractions with co-solvents such as ethanol can improve phlorotannin yields from Fucus vesiculosus (Amarante et al., 2020), and flavonoids and phenolic acid yields from brown algae (Yuan et al., 2018).

7

Table 1 Extraction of bioactive compounds from marine species. Extraction technique Maceration

Solvents/ enzymes

Bioactive compounds

Gelidium corneum, Gelidium pusillum, Porphyra umbilicalis, Halopithys incurva, Gracilariopsis longissima, Hydropuntia cornea, Ulva rotundata

Water (100%) Ethanol (100%) Ethanol (50%) Methanol (20%)

Lipids, carbohydrates, UV photoprotectors

Talaromyces purpureogenus

Hexane Petroleum ether Diethyl ether Chloroform Dichloromethane Ethyl acetate

Phenolics, alkaloids, flavonoids, steroids, terpenoids

Stylissa carteri

Ethanol

Flavonoids, triterpenoids, steroids

Species

Results

Bioactivity

References

Higher extraction yield with MeOH 20%. Significant interactions between “solvent” and “antioxidant capacity method” Species with the highest antioxidant capacity were Gracilariopsis longissima and Hydropuntia cornea Maximum antiproliferative activity was observed in ethyl acetate extract, while maximum antioxidative activity was found in hexane fungal extract Ethyl acetate extract induced membrane apoptosis and cytotoxicity in HeLa cells Cytotoxic activity in breast cancer cells Potential antitumor activity in breast cancer cells

Antioxidant

A´lvarezGo´mez et al. (2016)

Antiproliferative and antioxidative

Kumari et al. (2018)

Anticancer

Bashari et al. (2019)

Maceration/ sonication and SPE

Phyla Dinophyta, Heterokontophyta, Haptophyta, Chlorophyta

Methanol

–

Maceration and liquidliquid partition (LLP)

Himanthalia elongata

Methanol: water 60% (v/v)

Phenolics

Solventsolvent partition

Gracilaria edulis

Methanolhexane (70%) Methanolchloroform (70%) Methanol-ethyl acetate (70%)

Phenolics, alkaloids, flavonoids

9% of the extracts showed 80%–100% of apoptotic activity. 15% of all dinoflagellates yielded extracts that showed high antiproliferative activity Overall, most of the extracts were inactive (antimicrobial activity) against the species studied 8 Phenolic compounds with potential antioxidant capacity were identified The purified fractions showed higher antioxidant capacity than the reference ascorbic acid High antioxidant and enzyme inhibitory potential in the ethyl acetate fraction Presence of several potent antidiabetic compounds Strong hypoglycemic and antiglycation potential

Antimicrobial, antiproliferative, and apoptotic potential

de Vera et al. (2018)

Antioxidant

Rajauria et al. (2016)

Antioxidant, hypoglycemic potential

Gunathilaka et al. (2019)

Continued

Table 1 Extraction of bioactive compounds from marine species—cont’d Extraction technique

Species

Solvents/ enzymes

Bioactive compounds

Haliclona exigua

Ethyl acetate

–

Rhodomela confervoides

Methanol/ chloroform (2:1 v/v)

Phenolics

SPE

Haliotis tuberculata coccinea

Fatty acids, alkaloids, terpenes, flavonoids

PLE

Laminaria ochroleuca

Ethyl acetate Hexane Acetone Chloroform Ethanol Methanol Methanoldichloromethane (1:2) Hexane Ethyl acetate Ethanol Ethanol: water Water

Soxhlet extraction

Gracilaria chilensis, Callophyllis concepcionensis, Macrocystis pyrifera, Scytosiphon lomentaria, Ulva sp., and Enteromorpha compressa

Fatty acids and phenols

Phenolics and dietary fibers

Results

Bioactivity

References

Secondary metabolites of sponges exhibited significant bactericidal activity Sponges are an excellent source for the discovery of novel antioxidant Significant associations between the antioxidant potency and the total phenolic content Ethyl acetate extract showed cytotoxic activity on cancer cell lines A375, MBA-MD 231, HeLa, and MCF7 and antimicrobial activity

Antibacterial, antioxidant, antiinflammatory

Bhimba et al. (2013)

Antioxidant

Wang et al. (2009)

Anticancer, antimicrobial, anthelmintic activity

Tortorella et al. (2021)

PLE provided a great extraction efficiency of fatty acids achieving a low ratio of o6/o3 The main constituents of the extract were hydroxycinnamic and hydroxybenzoic acids and flavonoids. The fiber extracted comprised up to 50% of dry weight

Antioxidant

Otero et al. (2019)

Antioxidant

Sanz-Pintos et al. (2017)

EAE

Sargassum boveanum and angustifolium, Padina gymnospora, Canistrocarpus cervicornis, Feldmannia irregularis

Viscozyme AMG 300 L Celluclast Termamyl Ultraflo L Flavourzyme Alcalase Neutrase

Phenolics

UAE

Ascophyllum nodosum, Laminaria hyperborea

Water

Phenolics

MAE

Saccharina japonica

Ethanol: water (50%–70%)

Phenolics

Sargassum vestitum

Ethanol: water (30%–70%)

Phenolics

Gracilaria mammillaris

Ethanol (2%–8%) CO2

Phenolics and carotenoids

SFE

The extract with higher antioxidant capacity was obtained from S. boveanum, S. angustifolium, and F. irregularis. Flavourzyme provided a higher number of extracts with antimicrobial activity Both extracts presented similar antioxidant capacity and antimicrobial effects against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella typhimurium MAE provided an efficient method to obtain enriched extracts in phlorotannins with high anticancer properties MAE achieved better efficiency in terms of phenolic extraction compared to conventional extraction methods SFE was a selective method to obtain enriched extracts in carotenoids

Antioxidant and antimicrobial

Habeebullah et al. (2020)

Antioxidant and antimicrobial

Kadam et al. (2015)

Inhibitory effects on HepG2 cancer cells

He et al. (2013)

Antioxidant

Dang et al. (2018)

Antioxidants

Ospina et al. (2017)

12

Chapter 1 Clean and green analytical techniques

As MAE, ultrasound-assisted extraction (UAE) represents a rapid, simple, and ecological process that is in consonance with green chemistry criteria since it can be used without solvents or a reduced volume of solvents improving the recovery of interesting compounds. “Solvent-free” UAE has been performed to obtain lipids (PUFAs and MUFAs) from Nannochloropsis oculata, preserving the quality of fatty acid but extracting lower amount than conventional extraction. However, the simplicity and solvent free of this technique make it more economic and hence a suitable alternative to the scale-up process (Adam et al., 2012). Considering the green analytical chemistry principle “it is better to prevent waste than to treat or clean up waste after it is generated” (Anastas and Eghbali, 2010), the best option is to perform solventfree extractions [SFE, MAE, UAE (Adam et al., 2012; Molino et al., 2018; Natarajan et al., 2015)], followed by a reduction of green solvents, such as water-ethanol or eutectic solvents, consumed applying techniques that increment pressure or agitation as PLE (Otero et al., 2018). For this reason, the miniaturization of classical extraction methods is also considered in green chemistry; for example, liquid-phase microextraction (LPME), hollow-fiber liquid-phase microextraction (HF-LPME), and single-drop microextraction (SDME) are also interesting options to recover the analyte from several sources in a greener way that can be combined with innovative extraction technologies. Indeed, solid-phase SFE has been applied to extract bioactive phenolics from different microalgal and cyanobacterial strains (Klejdus et al., 2009), as well as PLE or UAE, followed by an SPE, was used to extract several phenolic compounds, mostly cinnamic acid and benzoic acid derivatives (Klejdus et al., 2017). Moreover, natural deep eutectic solvents (NADESs) are promising green solvents to recover bioactive compounds from natural sources since they are eco-friendly, non-toxic, biodegradable organic com˜ o´n et al., 2020), which may perform pounds and cheap solvents (Alan relative selective extractions of analytes. For instance, L-lactic acid, betaine, choline chloride, malic acid, glucose, and glycerin mixed at several moles ratios achieved the extraction of phlorotannins from brown algae in a conventional extraction method (Obluchinskaya et al., 2019), but also, they may be applied as solvents in innovative extraction techniques (Ca´diz Gurrea et al., 2017). Despite the elevated investment costs of these technologies, they are in accordance with the green analytical chemistry principles, as shown in Table 2, allowing an efficient extraction yield, more selective extraction, less production of residues and green solvent applications and consequently their use as a relevant alternative to reach environmentally friendly analytical processes in the future.

Table 2 Sample preparation and extraction techniques according to green analytical chemistry principles.

Freeze drying, grinding Liquid-liquid extraction Liquid-phase microextraction (LPME) Hollow-fiber liquid-phase microextraction (HF-LPME) Single-drop microextraction (SDME) Maceration Ultrasoundassisted extraction (UAE) Microwaveassisted extraction (MAE) Supercritical fluid extraction (SFE) Pressurized liquid extraction (PLE)

Energy consumption

Solvent usage (organic or green)

Timeconsuming

Selectivity

Waste generation

Solvent reusability

Automation

Environmentally friendly

High

None

High

–

Low

–

Yes

No

High

Organic

High

Low

Medium

No

No

No

Low

Organic

Low

Medium

Low

No

Yes

Yes

Low

Organic

Low

High

Low

No

Yes

Yes

Low

Organic

Low

Medium

Low

No

Yes

Yes

High Medium

Both Both

High Low

Medium Medium

High Low

No No

Yes No

No Yes

Medium

Both

Low

Medium

Low

No

Yes

Yes

High

Both

Medium

High

Low

Yes

No

Yes

High

Both

Low

Medium

Low

No

Yes

Yes

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Chapter 1 Clean and green analytical techniques

2.3 Isolation of compounds from extracts In many cases, the extraction procedure is not sufficient to isolate or separate the targeted analytes from the original matrix since extracts still are complex mixtures of compounds that may interact with each other giving signals, which may lead to erroneous interpretations. In this sense, purification or isolation techniques achieve to separate a complex mixture into a simpler one by applying several techniques, such as filtration, SPE, and chromatographic techniques. For these reasons, purification steps are required to achieve a suitable analytical signal detectable by analytical platforms, which enhance the characterization and the quantitation of targeted analytes. In this sense, it is probable that SPE is the isolation method of the first choice when polar analytes are involved since it can be coupled with HPLC platforms and it may have low solvent consumption (Koel and Kaljurand, 2006). In addition, SPME has been proposed as an alternative to classical extraction techniques since it integrates sampling, extraction, isolation, and concentration of analytes in a single step. The system consists of a fused silica fiber coated with a specific sorbent material, which is set according to the affinity with the targeted compound. Despite being an organic solvent-free technique, it has some limitations related to the extraction of polar and low volatility analytes including technical limitations such as the fragility of the fiber (Omena et al., 2019). Furthermore, stir bar sorptive extraction (SBSE) and rotating disk sorptive extraction (RDSE) have been developed as alternatives to SPME, which consists of sorptive element which is maintained in rotation by stirring (Baltussen et al., 1999; Richter et al., 2009). These methodologies, coupled with LC or GC, have provided a high sensitivity and reproducibility to isolate fatty acids from cyanobacteria ( Jin et al., 2021), and odorous compounds such as geosmin from marine water (Benanou et al., 2003). Although the 8th principle of green chemistry recommends eliminating derivatization, it is still difficult to fulfill in analytical chemistry since, occasionally, the filtration or purification of analytes contained in extracts is not enough to obtain a suitable analytical signal, making this step unavoidable. Derivatization consists of synthesis or modification of analytes to increase their analytical detection and involves an increment of hazardous reagents, solvents and energy and the waste generated during analytical procedure (Keith et al., 2007). The greener alternatives to classic organic solvents for derivatizations whose use may be prioritized in these processes are (1) water; (2) bio-derived solvents and deep eutectic solvents; and (3) supercritical fluids or

Chapter 1 Clean and green analytical techniques

ionic liquids (Lavilla et al., 2014). Nevertheless, few derivatization reactions may be performed in an aqueous medium (Naik et al., 2004). On the other hand, regardless of the investment costs of this technique, reaching supercritical state of a fluid implies the compression of the solvent and consequently the consumption of energy, which may represent a negative factor from the standpoint of green chemistry. The last greener alternatives are ionic liquids, which have attracted attention in the last years since they are extremely low volatile, non-flammable, and thermally stable. Moreover, they can be used during extractions and microextractions and even separation techniques (GC and LC) being used simultaneously with derivatization steps. However, its high solubility in water, its persistence in the environment, and its toxicity reduce its suitability toward a green solution to derivatization procedures (Thuy Pham et al., 2010). For these reasons, some studies showed that the current trends lead to miniaturization, automation, and faster, simpler procedures reducing the use of solvents and reagents as well as the generation of residues (Rosenfeld, 2003, 2011). The efforts are intended to eliminate or reduce organic solvents by applying SPME or LPME techniques, which may be used simultaneously with extraction processes. Moreover, greener derivatization includes some instrumental settings, such as pre-column/ in-capillary derivatization with liquid chromatography (LC) or capillary electrophoresis (CE). The miniaturization of the derivatization process has developed pre-column to improve the detection of fatty acids previously extracted from different algal species (Araya et al., 2021; Jiao et al., 2021). On the other hand, the injection in the port of both sample and derivatization agents, during gas chromatography (GC) analysis, simplifies, especially combined with SPME, the preparation of the sample and reduces the generation of wastes. One of the most used technologies to characterize odorous compounds is headspace solid-phase microextraction (HS-SPME) that implies the injection of the volatile compounds that are in the headspace on the SPME fiber. The greener approach has been used to analyze geosmin and 2-methylisoborneol from cyanobacteria (Alghanmi et al., 2018; Oh et al., 2017) or volatile compounds from brown, red, and green macroalgae (Bravo-Linares et al., 2010). Some advantages and inconveniences of traditional and greener derivatization methods are compiled in Table 3. Because the derivatization process is usually unavoidable to reach a suitable signal in analytical procedures that imply nonenvironmentally friendly methodologies, it feels necessary the search for greener alternatives at this stage. The most relevant

15

16

Chapter 1 Clean and green analytical techniques

Table 3 Degree of compliance with green analytical chemistry (GAC) principles of traditional and green derivation methods. Compliance with GAC

Methodology

Advantages

Drawbacks

Liquid-phase microextraction (LPME)

Extremely simple, affordable, and almost solvent-free. Fast phase transportation and large extracting phase capacity. High degree of selectivity and enrichment Extraction and analyte concentration performed with high efficiency and in one single step, being able to remove most of the matrix interferences; low time-consuming and cost; quite low amount of solvents and sorbents required. It is possible to reuse the fiber Low time- and energy-consuming, and quite great simplicity. Low amounts of samples, solvents, and materials are needed, so false positives or overestimations can be avoided. Ideal for volatile compounds. In agreement with green analytical chemistry The sample is not directly in contact with the extracting solvent. Effective and low-cost technique. Enhanced solvent stability and no carry-over effects between each extraction, promoting reproducibility. Pores improve selectivity by avoiding large molecules

Difficulties in the stabilization of the liquid extracting phase

Yes

Low availability of stationary phases and fiber coatings; low selective and thermal stable fibers; short number of adequate sorption materials. Relatively low recovery of analytes and low precision of determinations, limiting qualitative and quantitative analysis Rapid loss of adsorptive capacity caused by fiber aging or abrasion (Dennenl€ohr et al., 2020)

Yes

Membrane barrier causing a decrease in extraction rate and an increase in extraction time; air bubbles on the HF leading to reduce the transport rate and the reproducibility; pore blockage after adsorption of hydrophobic substances on fiber surface in real samples. Also, due to the fact that the HF segments are cut manually, it is common to find variations in length Drop volume fluctuation and instability are enhanced by intense operational conditions

Yes

Solid-phase microextraction (SPME)

Headspace solid-phase microextraction (HS-SPME)

Hollow-fiber liquid-phase microextraction (HF-LPME)

Single-drop microextraction (SDME)

Quite simple and eco-friendly procedure, do not carry wastes from an extraction to a different one. Can be fully automated

Yes

Yes

Chapter 1 Clean and green analytical techniques

17

Table 3 Degree of compliance with green analytical chemistry (GAC) principles of traditional and green derivation methods—cont’d Compliance with GAC

Methodology

Advantages

Drawbacks

Stir bar sorptive extraction (SBSE)

Combination of extraction and concentration of analytes in a single step; significant simplicity, robustness, and sample cleanup ability; solventless, fast, and environmentally friendly technique. Low toxic waste generation, and improved sensitivity and selectivity High analytical quality; significant robustness, sensitivity, and accuracy. Can be automated

The extraction conditions need to be hardly supervised. Restriction on its application due to a lack of stir bar coatings with defined affinity. Also, low interaction with certain analytes

Yes

Selectivity could be improved. It is limited to its application to biological materials (serums, plasmas, saliva, etc.)

Yes

Rotating disk sorptive extraction (RDSE)

factor that should be changed is the solvents used prioritizing the application of water, bio-derived solvents, NADESs, supercritical fluids, or ionic liquids, which are conveniently incorporated into derivatization steps. Moreover, the reduction of energy consumption is an aspect, which is improving by combining different methodologies, shortening the preparation of the sample, and reducing the steps involved during this process. However, these approaches, which are applied in marine samples but may be extrapolated to other natural sources, represent the beginning to achieve green analytical chemistry since there is room for improvement.

3. Chromatographic separation approaches: Instrumental methods Awareness of the complexity and the overwhelming number of marine phenolics, an advanced instrumentation, and new analytical strategies are required to carry out more comprehensive and effective analytical approaches. One of the most popular and versatile analytical techniques is chromatography, which is commonly used for separating a mixture of chemical substances into its individual components, so that the individual components can be thoroughly analyzed.

18

Chapter 1 Clean and green analytical techniques

There are many types of chromatography, e.g., liquid chromatography, gas chromatography, ion-exchange chromatography, and affinity chromatography, but all of these share the same basic principles of chromatography. Basically, a mixture composed of several analytes is applied onto a surface or stationary phase where analytes are separating from each other while eluting thanks to a mobile phase. But, in the last years, there has been a growing interest in the development of high-throughput, robust, and sensitive chromatographic methods. Consequently, new types of stationary phases, columns, and instrumentations have been developed with the aim of shortening analysis times and obtaining higher resolutions and sensitivities, regardless of the application. Focusing attention on the analysis of phenolic compounds from the marine environment, high-performance liquid chromatography has primarily been used as separation technique. Highperformance liquid chromatography, commonly known as HPLC, is capable of separating the components of a mixture based on the different distribution between a liquid mobile phase and a stationary phase that may be a liquid or a solid. The mixture previously dissolved in a suitable solvent is entrained by the mobile phase and forced to flow through a chromatographic column containing the stationary phase under high pressures of up to 400 atm. The separation takes place in the chromatographic column according to the affinities between stationary and mobile phases. The individual components of the mixture migrate through the column at different rates because they are retained to a varying degree by interactions with the stationary phase. The chromatographic analysis can operate in two modes: The reversed phase employs a non-polar stationary phase and a polar mobile phase, while the normal phase employs a polar stationary phase and a less polar mobile phase. The choice of operating in reversed phase or normal phase will be determined, basically, by the nature of the target compounds. The use of HPLC offers a number of advantages, as the possibility of managing different mobile-phase gradients makes HPLC compatible with the analysis of compounds of a wide range of polarity. In addition, it allows the analysis of thermolabile compounds and eliminates the derivatization step for non-volatile compounds, resulting in a versatile, efficient, and fast analytical technique. For a successful separation of marine phenolics by HPLC, a good choice of the operational parameters involved should be made (columns, mobile phases, pressure, flow…). Different solvent systems, extraction temperatures, and particle size affect the phenolic content. It is important to bear in mind that one

Chapter 1 Clean and green analytical techniques

of the main drawbacks found in the analysis of marine phenolics is that extracts are often highly concentrated in other metabolites (polysaccharides, proteins…), which may hinder the accurate determination of phenolic compounds that are usually in lower concentrations, or even cause damages in the HPLC equipment. To solve this problem, a fractionating stage is required before the HPLC analysis. This fractionating step is optional but, in the analysis of phenolic compounds from macroalgae, is very useful for preparative column chromatography (CC) in normal-phase, reversed-phase, and size-exclusion separation modes to remove impurities (Santos et al., 2019). Silica gel as stationary phase is the column packing mostly used for the determination of phlorotannins, phenolic acids, flavones, aurones, and coumarins in a normal phase. Reversed-phase column chromatography (RP-CC) is less common, although some applications have been reported and Sephadex LH-20 is used in the fractionation of phlorotannins and coumarins from macroalgal extracts by sizeexclusion chromatography (SEC-CC). Each marine specimen contains different phenolic compounds of diverse nature and structure with diverse linkages, functional groups, reactivity, and sizes, which implies that the wide variability in structures may have different solubilities based on the structures of polyphenols (Santos et al., 2019). In this sense, the normal phase is preferred for certain purposes like the separation of isoflavones in macroalgae species (Klejdus et al., 2010) or analytical characterization of sulfated coumarins in marine green macroalga (Hartmann et al., 2018), both applications performed with a diisopropylcyanopropylsilane stationary phase (Zorbax SB-CN column). However, reversed-phase HPLC is the most frequently employed method for marine phenolic separation. The reversed phases became popular not only for increased resolution of many organic compounds, but also for the shift in mobile-phase composition, from non-polar to polar solvents—often water or aqueous mixtures. The most common reverse stationary phase is C18/ODS, which is composed of packed silica-bonded octadecyl chains with a particle size of 3.5–5 μm on columns of diverse internal diameters and lengths. These chains provide a hydrophobic moiety that retains the more hydrophobic compounds. Elutions of marine target compounds from reversed phases have been performed using water, methanol, or acetonitrile in different combinations. Although water to acetonitrile has been the most frequently used eluent, water-tomethanol gradients have also been used with success, which is an advantage since methanol is relatively easy to evaporate and is much cheaper than acetonitrile. Nonetheless, the addition of trifluoroacetic acid, formic acid, or acetic acid is commonly used for

19

20

Chapter 1 Clean and green analytical techniques

the achievement of greater chromatographic resolution. In this sense, several phenolic compounds (gallic acid, 4-hydroxybenzoic acid, catechin hydrate, epicatechin, catechin gallate, epicatechin gallate, epigallocatechin, epigallocatechin gallate, and pyrocatechol) from nine commercial algal food products were chromatographically separated on a C18 column in reversed phase using water and acetonitrile acidified with acetic acid (Machu et al., 2015). The same stationary phase enables the separation of 22 phenolic acids, 17 flavonoids, 11 other polyphenols, and 4 lignans from eight seaweed species with a mobile phase consisting of acidified water with acetic acid (98:2) and a mixture of acetonitrile, water, and acetic acid (50.0:49.5:0.5) (Zhong et al., 2020). Similarly, 84 different phenolic compounds of diverse nature and structure (phenolic acids, flavonoids, and phlorotannins up to octamers) from macroalgal species were also separated on a C18 column with 1% aqueous formic acid and acetonitrile (Olate-Gallegos et al., 2019). Phenolic acids such as quinic, hydroxybenzoic, rosmarinic, p-coumaric, and ferulic acid derivatives and flavonoids such as acacetin, hispidulin, gallocatechin, and cypellocarpin C together with various phlorotannins were also separated on a C18 column using water and methanol, both acidified with acetic acid (2.5%), as mobile phase (Agrega´n et al., 2017). Other authors have also performed the separation of phenolic compounds of diverse nature and structure on a C18 column from seaweeds, thermoresistant freshwater green microalgal, brown macroalgal, and marine actinobacterial strains, with diverse operational conditions on the analytical procedure (Bulut et al., 2019; Grina et al., 2020; Karthik et al., 2013; Yuan et al., 2019). Another reversed stationary phase commonly used in marine separation approaches is C8 column composed of packed silica-bonded octyl chains. This type of column is indicated for analytes with medium hydrophobicity or when a less hydrophobic phase is required for optimal retention. Selectivity of C8 column is similar to C18 one with predictable elution order but less retention. Lower hydrophobicity allows compounds to elute faster, enabling faster separations. This stationary phase has been used for the separation and determination of zosteric acid, a natural sulfated phenolic capable of preventing the settlement of marine organisms and protecting crops from fungal diseases from Zostera seagrasses (Achamlale et al., 2009a). The analysis of some claimed bioactive compounds such as rosmarinic and chicoric acids from Zostera and Cymodocea seagrasses, respectively, was also performed on a C8 column (Achamlale et al., 2009b; Grignon-Dubois and Rezzonico, 2013). In the mentioned applications, the pore size of the stationary phase was 175 A with a particle size of 5 μm. The length of the column was 250 and 4.6 mm

Chapter 1 Clean and green analytical techniques

in inner diameter, and the mobile phase consisted of a gradient of water to methanol, both acidified with trifluoroacetic acid 0.1%. Among the marine phenolics, the determination of phlorotannins and dehydro-oligomer derivatives of phloroglucinol units (1,3,5-trihydroxybenzene) is probably one of the most challenging and troublesome tasks due to their high susceptibility to oxidation and similarities in physical properties despite having broad structural and molecular weight diversities (Isaza Martı´nez and Torres Castan˜eda, 2013; Parys et al., 2007). The choice of the appropriate chromatographic separation method is not clear (Ford et al., 2019). Some authors claim the reversed-phase HPLC, but the high polarity of phlorotannins causes them to elute very fast due to the lack of inter-action with the non-polar stationary phase (Koivikko et al., 2007). Vissers et al. detected and separated phlorotannins from Laminaria digitata, after the NP-flash CC, with polymerization degrees between 2 and 18 using an ethylene bridged hybrid (BEH, Waters) C18 column (Vissers et al., 2017). This type of column was also preferred for the separation of phlorotannins from Sargassum palladium (Ye et al., 2009) and Ascophyllum nodosum extracts (Allwood et al., 2020). In contrast, other authors argue for the normal-phase liquid chromatography as the more appropriate approach for retaining phlorotannins. In this regard, Koivikko et al. demonstrated a superior performance in the normal phase for the separation of phlorotannins from Fucus vesiculosus compared with reversed phase (Koivikko et al., 2007). However, poor hydrophilic analyte solubility in normal-phase liquid chromatography eluents can promote adsorption onto the silica stationary phase, compromising peak shape and resulting in long retention times (Steevensz et al., 2012). Moreover, the poor ionization efficiency in non-polar solvents will drive a sensitivity reduction in their further detection (Koivikko et al., 2007). However, in the last years, there has been a growing interest in the development of high-throughput, robust, and sensitive chromatographic methods for marine phenolic separation. New implementations have been developed in an attempt to obtain higher resolutions and shorter analysis times. In this sense, new types of stationary phases and columns have been developed. Hydrophilic interaction liquid chromatography (HILIC) columns have been proposed as variants of normal-phase HPLC. HILIC employs a polar stationary phase with semi-aqueous/polar organic mobile phases. Water fraction (5%–50%) forms a liquid layer on the stationary phase that facilitates partitioning of analytes from the high organic mobile phase to the hydrophilic stationary phase, offering a selectivity based on electrostatic interactions, dipole-dipole interactions, cation/anion exchange, and

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hydrogen bonding (Alpert, 1990). The implementation of HILIC for the profiling of phlorotannins in brown macroalgae (Fucus vesiculosus, Pelvetia canaliculate, Fucus spiralis, Ascophyllum nodosum, and Saccharina longicruris) seems to be an appropriate choice due to the strong interaction of phlorotannins with polar stationary phase, which can be eluted with reversed-phase-type eluents (Steevensz et al., 2012). A column composed of bridged ethylene hybrid particles with trifunctional amide ligands (BEH amide, 1.7 μm, 2.1 mm  100 mm) was demonstrated to be an effective separation support, particularly for low-molecularweight phlorotannins, when the gradient was ranged between 5% and 35% aqueous phase. However, for larger phlorotannins ( 1200 Da), this separation mode was not effective in resolving highly retained compounds. In addition, novel fluids such as ionic liquids (ILs) offer a new approach to be used in liquid chromatography as mobile-phase additives and as functional groups of stationary phases (Ho et al., 2014). Ionic liquids can be added as modifiers to solvents in reversed-phase liquid chromatography to adjust the eluent strength reducing tailing issues. However, ionic liquids have not been still applied to chromatography methodology for marine phenolic analysis, except in the extraction stages for isolation purposes (Martins and Ventura, 2020). Other strategies to reduce the chromatographic runtimes such as the use of monolithic columns or high-temperature liquid chromatography columns have not yet been implemented in the separation of marine phenolics for unsuitability or thermal degradation issues. On the other hand, novel strategies in analytical instrumentation have also been applied in the field of marine phenol determination. In this respect, the ultra-high-performance liquid chromatography (UHPLC) provides faster analysis and better chromatographic efficiency as well as resolution by means of higher pressure together with smaller internal diameter columns packed with sub-2 μm particles. The inconvenience of this type of column is that it demands a more careful sample preparation to avoid high back pressure or column clogging. There are a wide range of columns, but the methods used for marine phenol applications preferred the use of reversed-phase C18 and C8 columns as in the case of the conventional liquid chromatography. Although columns based on sub-2 μm fully porous particles have been more frequently used, columns packed with fused core particles have gained attention. One of the main applications of UHPLC has been the analysis of phlorotannins. For this purpose, a superficially porous silica particle column with a solid silica core and a porous silica outer layer with a C18 bonded phase applied (Poroshell 120 EC-C18 column, 2.7 μm,

Chapter 1 Clean and green analytical techniques

2.1  50mm) has been employed. The separation of 42 different molecular weights of phlorotannins with diverse degree of polymerization from various species of brown algae was performed on this column together with a gradient of aqueous solution to acetonitrile as mobile phase (Li et al., 2017). However, separation on C18 phases is based on hydrophobic interaction, so highly polar molecules such as phlorotannins are expected to show limited retentions or resolutions. Pentafluorophenyl (PFP) phase seems to provide a combination of different separation/retention mechanisms including polar interactions, which reinforce its suitability for highly polar aromatic phlorotannins (Tenorio-Rodrı´guez et al., 2019; Tierney et al., 2014). In this sense, PFP non-endcapped silica-based column (1.8 μm particle size, 2.1  100 mm) was able to increase efficiency in separating phlorotannin isomer for oligomers between polymerization degrees of 3–16 phloroglucinol units (Heffernan et al., 2015; Kirke et al., 2017; Tierney et al., 2014). The use of hydrophilic interaction columns also implemented the UHPLC analytical platform for phlorotannin determination. A rapid profiling of phlorotannins in brown seaweeds based on their polymerization degree was achieved by means of a UPLC BEH amide column (1.7 LiChrospher diol-5 μm, 2.1  100 mm), capable of handling up to 15,000 psi, with a flow rate of 400 μL per min (Steevensz et al., 2012). A recent instrument development to improve the separation of complex matrices is the multidimensional chromatography (2D LC or LC x LC) whose dimensions are based on different separation mechanisms. In this sense, the separation and characterization of phlorotannins from brown algae Cystoseira abiesmarina were performed by comprehensive two-dimensional liquid chromatography (Montero et al., 2014). In this research, the authors performed the first separation using a hydrophilic interaction diol bonding-phase column (LiChrospher diol-5 (5 μm, 150  1.0 mm)) coupled with the second dimension in reverse phase where two columns were tested: a C18 partially porous column (2.7 μm, 50  4.6 mm) and a pentafluorophenyl partially porous particle column (2.6 μm, 50  4.6 mm). Thanks to the first dimension, phlorotannin separation was achieved according to the polymerization degree, while in the second dimension, separation was attained in terms of relative hydrophobicity. More than 50 phlorotannins containing 5 to 17 phloroglucinol units were separated by this approach showing better performance when C18 column in the second dimension was used instead of PFP column. Another type of chromatography used in analytical chemistry for separating phenolic marine compounds is gas chromatography (GC). Sample is vaporized in the injected port and, thanks to a carrier gas, is passed through a stationary phase. The requirement for

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the use of this technique is that target compounds must be vaporized without thermal decomposition. For that reason, the use of gas chromatography is an analytical technique limited to the analysis of phenolic compounds in comparison with liquid chromatography. However, this analytical platform attains detection limits down to the low nanogram level being preferred, in some cases, for the analysis of semi-volatile phenolic compounds. The use of GC has been reported in bibliography for some marine applications such as the separation and detection of coumarin and flavones together with fatty acids on extracts from brown algae, Padina Tetrastromatica (Uma Maheswari et al., 2018), for flavonoid determination of a solitary marine invertebrate animal, Phallusia nigra (Asayesg et al., 2021), and for some endocrine-disrupting chemicals of phenolic nature in the marine environments (Almeida et al., 2007). In all cases, the separation was performed in a non-polar column composed of (5%-phenyl)-methylpolysiloxane of 30 m in length, 0.25 mm in inner diameter, and 0.25 or 0.5 μm in film thickness, using helium as carrier gas. The gas chromatography approach seemed to be also appropriate for the determination of bioactive methoxylated brominated diphenyl ethers of marine sponges or for the screening of bioactive compounds from marine endophytic fungi whose separation was also performed in a non-polar column (5%-phenyl)-methylpolysiloxane (Haraguchi et al., 2011; Parthasarathy et al., 2020). Other chromatographic techniques have also been applied for the analysis of phenolic compounds from the marine environment. For instance, thin-layer chromatography (TLC), which consists of an adsorbent layer as stationary phase placed on a close chamber with various solvents as mobile phase, has often been applied to separate phlorotannins and other phenolic compounds from seaweeds and other marine species (Achamlale et al., 2009b; Agatonovic-Kustrin et al., 2019; Agatonovic-Kustrin and Morton, 2018; Isaza Martı´nez ˜eda, 2013; Kim et al., 2009). This chromatographic and Torres Castan separation is usually performed in normal-phase silica gel of diverse dimensions eluted with different mixtures of solvents composed of dichloromethane and methanol or n-hexane, ethyl acetate, and acetic acid. However, this technique is not recommended when dealing with complex matrices/extracts.

4. Advanced mass spectrometry Mass spectrometry techniques can be coupled with both liquid chromatography and gas chromatography, for which the parameters and instruments used are different depending on the chromatography selected. Because the use of liquid chromatography

Chapter 1 Clean and green analytical techniques

is more widespread for the study of marine phenolic compounds, in this section, we are going to focus on the different parameters to be selected in LC-coupled mass spectrometry. Any mass spectrometer consists of three main components, which are ionization source, analyzer, and detector (Steinmann and Ganzera, 2011).

4.1 Ionization source Ionization of the analyte occurs at the ion source. These ions are then passed to the mass analyzer, and their resolution is carried out based on their mass/charge ratio (m/z). Ions impact a detector more frequently to produce a signal that is recorded. In this sense, the mass spectrum is a graph of the relative abundance of ions versus their m/z (Haag, 2016). Due to the polarity of phenolic compounds, in most of the works in which these types of compounds have been analyzed in marine organisms, the ionization selection method is electrospray ionization (ESI). ESI is a technique in which a gentle ionization is carried out; that is, little fragmentation of the compounds occurs. Using this technique, ionization is achieved by applying a high electrical charge to the sample needle. The needle has an inner part for the LC eluent and an outer part for a nebulizer gas (e.g., nitrogen). A fine aerosol is produced, in which the size of the droplets continuously decreases due to the evaporation of the solvent. When the Rayleigh limit is reached, ions are formed by a process called “Coulomb explosion” and enter the analyzer due to a difference in potential or pressure (Soler et al., 2008). The ESI ionization source has been used in many studies in which phenolic compounds present in marine organisms were analyzed. Thus, for example, phenolic compounds from extracts of the brown algae Ascophyllum nodosum, Bifurcaria bifurcata and Fucus vesiculosus were analyzed by LC-DAD-ESI-MS/MS (Agrega´n et al., 2017). Phenolic compounds from algae of the genera Ulva sp., Caulerpa sp., Codium sp., Dasya sp., Grateloupia sp., Centroceras sp., Ecklonia sp., and Sargassum sp. were also analyzed using ESI as an ionization source (Zhong et al., 2020). In another study, Ecklonia stolonifera Okamura extracts were analyzed with a validated HPLC-ESI-MS method. The major phlorotannins found were dieckol, eckol, and phlorofucofuroeckol A, the first one being the most abundant (Goo et al., 2010). Another source of ionization that is also widely used is atmospheric pressure chemical ionization (APCI). The working mode of the APCI ionization source is similar to the ESI, but the charge is applied through a corona pin, which is located at the outlet of a heated tube. This source is used more frequently for non-polar

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compounds, and like ESI, it can work in positive and negative modes (Soler et al., 2008). Recently, the search for technical improvements has been carried out to achieve greater efficiency in relation to the ionization source. An example of this improvement is the multimode ionization source that uses ESI and APCI simultaneously, alternating between positive and negative polarity, all within a single chromatographic run. The advantage is the possibility to analyze a wide range of compounds, regardless of
rez-Ferna´ndez their functionality, polarity, or thermal stability (Pe et al., 2017). Compounds such as carotenoids, phenolics, and flavonoids present in different microalgae (Phaeodactylum tricornutum, Nannochloropsis gaditana, Nannochloropsis sp., and Tetraselmis suecica) were identified by using APCI as a source of ionization (Haoujar et al., 2019). Another study in which the identification of phenolic compounds in different types of algae isolated from the Arabian Sea of Karnataka Coast is carried out using this type of ionization source is the one carried out by Sushanth and Rajashekhar (Sushanth and Rajashekhar, 2015). In addition to these two widely used ionization techniques, more and more new techniques are appearing, and a more recent development is atmospheric pressure photoionization (APPI). It is an alternative to traditional ESI and APCI techniques for compounds that are poorly ionized with them. APPI is based on the interaction of a photon beam produced by a krypton or xenon lamp with vapors formed by the nebulization of a liquid solution (Hanold et al., 2004). In the APPI technique, it is necessary to use a doping substance, a preferably ionized substance that acts as an intermediate between photons and analytes. In this sense, solvents such as toluene, anisole, or acetone are generally used, most of which ultimately lead to the formation of positively charged compounds (Marchi et al., 2009).

4.2 Analyzer There are many different analyzer designs available. Besides the main function of the analyzer, the resolution of ions of different m/z, various analyzers are also capable of trapping and storing ions. Depending on the type of analyzer used, they can carry out very different functions. The most common types of analyzers incorporated in today’s equipment are quadrupole, time of flight (TOF), ion trap, and Fourier transform (FT) (ion cyclotron [ICR] and Orbitrap) analyzers, in addition to the numerous combinations or hybrids of these.

Chapter 1 Clean and green analytical techniques

The choice of the mass analyzer depends on several factors and experimental considerations, among which the following stand out: the desired m/z range to be analyzed, the mass of the analyte, the ability of the analyzer to interact with the ion source of the mass spectrometer, the required resolving power of the analyzer, and the required detection limit (Haag, 2016). The main mass analyzers used for the analysis of marine phenols are described below.

4.2.1 Quadrupole The quadrupole mass analyzer continues to be one of the most widely used mass analyzers, due to its low cost, durability, reliability, and compact design. It is often used in tandem, as in triple quadrupole mass spectrometers, or in combination with other analyzers, such as TOF (Ca´diz-Gurrea et al., 2020). The basic operation of this type of analyzer is based on the discrimination and filtering of different m/z ions. Its structure consists of four cylindrical rods arranged parallel to each other. The opposing rods are electrically connected, and a radio frequency (RF) potential is applied. A direct current (DC) potential is superimposed on this RF potential. This combination of RF and DC potential allows the ions to oscillate as they pass through the quadrupole in the z-direction. Thus, depending on the DC potential and the frequency of the RF field, only ions of a certain m/z will have stable trajectories. Ions with unstable trajectories will collide with the bars and be filtered out. By varying the DC and RF potentials, ions of different m/z can be scanned through the quadrupoles (March, 1997). Quadrupole-type analyzers have fairly fast duty cycles and require a continuous flow of ions, and this allows them to be coupled with both GC and LC (Ucl
es et al., 2017). The main disadvantages of the quadrupole analyzer are its limitation to the mass range and low resolution, which makes it an insensitive tool for analyzing high-molecular-weight compounds that may not form multi-charged ions or complex mixtures of compounds with similar masses. In the study of phenolic compounds present in marine organisms, the quadruple has been widely used. In this sense, this technique was used in a study for the analysis of phenolic acids from two freshwater algae (Anabaena doliolum and Spongiochloris spongiosa) and from food products of marine macroalgae Porphyra tenera (nori) and Undaria pinnatifida (wakame) (Onofrejova´ et al., 2010). In another work, this type of analyzer is used to identify phenolic compounds extracted by MAE in important species of

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brown algae (Ascophyllum nodosum, Laminaria japonica, Lessonia trabeculata, and Lessonia nigrescens) (Yuan et al., 2018). Three quadrupole analyzers are placed in sequence in a triple quadrupole mass spectrometer. In the first quadrupole, the ions that are going to pass to the second quadrupole are selected, which functions as a collision cell to fragment the ions, which finally pass to the third quadrupole to obtain the mass spectrum (Kasperkiewicz and Pawliszyn, 2020). The use of this type of analyzer is also used for the research of marine phenols in which greater precision is needed. Thus, Petroutsos et al. (2007) used the triple quadrupole to study the toxicity and metabolism of p-chlorophenol in the marine microalgae Tetraselmis marina (Petroutsos et al., 2007). Another example of the use of this type of analyzer is the one used for the identification and characterization of phenolic antioxidant compounds from brown Irish seaweed Himanthalia elongate (Rajauria et al., 2016).

4.2.2 Ion trap This type of analyzer is a modification of the quadrupole type analyzer. The 3D ion trap has been widely used; however, recently, the 2D linear ion trap has become more popular due to its numerous advantages over 3D traps. The 3D traps are formed by two plates of hyperbolic electrodes facing each other, and between them, there is a hyperbolic ring electrode. The ions are trapped between the electrodes by using an oscillating RF field and a superimposed DC electric field. These types of equipment serve as analyzers since the ions that have different m/z are expelled from the trap in a selected way by varying the RF potential. In relation to 2D traps, these are equivalent to quadrupoles but with the use of a potential field at each end of the quadrupole so that the ions are trapped within it (O’Hair, 2006). One of the main drawbacks of this type of analyzer is its lowresolution power, which is why the combination of this type of analyzer with others is also frequent. This type of analyzer is also useful for the study of marine phenols; for example, in a study that studied the anti-inflammatory properties of phenolic compounds and crude extract from Porphyra dentata, the ion trap was used as a mass spectrometry analyzer (Kazłowska et al., 2010). It was also used for the analysis of phlorotannins extracted from the brown alga Fucus vesiculosus (Wang et al., 2012) and in the case of the marine microalgae Tetraselmis marina the study of 2,4-dichlorophenol (Petroutsos et al., 2008).

Chapter 1 Clean and green analytical techniques

4.2.3 Time of flight TOF analyzers consist of a flight tube and an accelerating grid that allows the ions to accelerate from the ionization source to the MS detector. Since the initial kinetic energy (Ek) of the ions and the length of the flight tube (d) do not vary and remain constant, the mass of the ions depends on the time it takes for the ions to be detected after the initial acceleration (time of flight). To increase the resolution in such analyzers, methods such as delayed extraction can be used. After the formation of the ions, delayed extraction introduces a small delay in the electrical pulse of the acceleration network before the ions are accelerated. This small delay allows the ions formed after ionization to equilibrate and have a more uniform mean momentum before acceleration (Boesl, 2017). Studies such as the one carried out by Nakai et al. (2006) use this type of analyzer to study phenols of marine origin; in this case, phlorotannins as radical scavengers from the extract of Sargassum ringgoldianum were analyzed (Nakai et al., 2006). TOF was also used for the characterization of laminarin from Irish brown seaweeds Ascophyllum nodosum and Laminaria hyperborean (Kadam et al., 2015).

4.2.4 FT-ICR In FT-ICR analyzers, the ions are introduced from the ionization source into a magnetic field. Then, these ions experience a Lorentz force, which makes them acquire a circular motion in a plane perpendicular to the magnetic field. The ions introduced to the magnetic field are initially not in phase and normally have very small orbits, making it impossible to detect them. In order to achieve detection, these ions are excited with a limited frequency sweep of a broadband RF field. This excitation allows the ions to be placed in a higher cyclotron band, which allows their detection. The cyclotron frequencies of the ions are proportional to their m/z. Among the main advantages of FT-ICR analyzers, their high mass precision and resolving power stand out (Ghaste et al., 2016). This type of analyzer has also been used for the study of compounds present in algae; thus, it has been used for the study of polyphenols in Sargassum sp. (Powers et al., 2019).

4.2.5 Orbitrap It is similar to the FT-ICR analyzer, but unlike those that use a magnetic field to induce the oscillations of the ions, the Orbitrap analyzers use an electric field to induce these oscillations. One of

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the main advantages of this type of analyzer is its high-resolution power when the m/z is higher (Scigelova et al., 2011). The Orbitrap-type analyzer has been used to search and characterize new phenolic compounds present in Posidonia oceanica (Astudillo-Pascual et al., 2021) and Sargassum cristaefolium algae (Saraswati et al., 2020).

4.2.6 Tandem mass analyzers Mass spectrometers that use two or more consecutively placed mass analyzers are called tandem mass spectrometers. In this type of analysis, the first analyzer selects the ions of a specific m/z, these ions are subjected to collision-induced dissociation, and the resulting product ions are analyzed using a second analyzer. Some examples of this type of analyzer are the triple quadrupole, Q-TOF, and the TOF/TOF (de Hoffmann, 1996). Due to the greater power of analysis and the better resolution that tandem mass analyzers reach, they are widely used in research work in the study of phenolic compounds. In this sense, for the study of phenols of marine origin, the triple quadrupole (Bogolitsyn et al., 2019; Sandsdalen et al., 2003), the Q-TOF ( Jimenez-Lopez et al., 2021; Zhong et al., 2020), and the TOF/ TOF ( Jin et al., 2015; Karthik et al., 2016) have been widely used.

4.3 Detector The last key point to consider in mass spectrometry is the detector. MS detectors are responsible for recording the current or charge produced by the ions. Traditional analog (Faraday) and electron multiplier (EM) detectors have been used for decades (Koppenaal et al., 2005). In most modern detectors, this detection is carried out using microchannel plate (MCP) technology. The MCP is formed by a thin plate perforated by small tubes or slots of about 6 to 10 μm in diameter. When an electric field appears, each channel acts as an electron multiplier, and whenever it is hit by a photon or ion, an electronic cascade begins. The electrons then exit the channel at the opposite end, and the resulting current is measured at an anode. After the electronic cascade, the MCP has to recharge its charge before it can detect another signal (Langstaff, 2002). Other types of detectors that have been traditionally used are the photoplate detector, Faraday detector, EM detectors, Daly detector, and image-current detection (Koppenaal et al., 2005). In the development of advanced mass spectrometry, both the ionization source and the analyzer have been studied and

Chapter 1 Clean and green analytical techniques

progress has been made in equipment with greater analysis power; on the contrary, ion technology has received less attention. Furthermore, detector technology is often sparsely or not mentioned in MS texts and reviews. Therefore, it has remained an area of rather underestimated importance, despite the fundamental need to “see” the ions better. In this regard, as there are continuous advances in ionization and separation techniques and the development of increasingly sensitive methods, detector technology will have to keep pace and revolutionary new approaches will be required.

5. Nuclear magnetic resonance approaches Nuclear magnetic resonance (NMR) is a very powerful and robust technique that represents one of the most important analytical techniques for the structural elucidation of chemical compounds. NMR stands out for being a non-invasive, non-destructive technique and for requiring a small amount of samples. Furthermore, no extra steps are needed for sample preparation, derivatization, or prior separation, which implies that it does not require the use of reagents as well as large amounts of solvents. Specifically, a small volume less than 1 mL of deuterated solvents, such as D2O, chloroform-D, or dimethyl sulfoxide-d6 (DMSO-d6), is typically used. For all these reasons, this technique can be considered as the one with the best characteristics in terms of clean and green analytical techniques. In addition, the high reproducibility or the analysis rapidity is another important advantage of this technique (Emwas, 2015). This technique has the ability to analyze solid, semi-solid, and liquid samples allowing to obtain a metabolic profile as well as structural and/or quantitative information (Laghi et al., 2014). All the compounds having a concentration level higher than the NMR detection limits can be detected in a simple measurement, without the need for prior separation. In contrast, one of its main limitations is its lower sensitivity, especially compared to mass spectrometry. This fact means that NMR is not as powerful, especially for the detection of secondary compounds present in low concentrations, compared to the applications performed by MS. Despite this weakness, various NMR-based applications have been carried out in recent years to study phenolic compounds such as structure elucidation, phenolic profiling, foodomic studies, authentication, and quality control (Ferna´ndez-Ochoa et al., 2021; Laghi et al., 2014; Parlak and Guzeler, 2016). Briefly, this technique is based on the measurement of the resonance of magnetic nuclei (e.g., 1H, 13C, 31P…) when a strong

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magnetic field is applied. Considering the natural abundance of the different magnetic nuclei, 1H NMR has been widely used, representing a powerful technique for the study of phenolic compounds. In general, this technique has presented two different types of applications for the analysis of marine phenolics, depending on whether the matrices analyzed are crude extracts or isolated compounds. One of the main applications is to determine the phenolic compound profile in natural extracts. This type of application is based on the analysis of the region of aromatic compounds in the 1H NMR spectra, comprised between 4.5 and 6.5 ppm
rantola et al., 2006). In this regard, this technique has also been (Ce applied for the profiling of phenolic compounds in marine extracts or for knowing quickly the presence or absence of certain compounds. For example, Gager et al. determined by 1H NMR the phenolic fingerprint of seven brown marine macroalgal extracts (Laminaria ochroleuca, Ascophyllum nodosum, Bifurcaria bifurcata, Fucus serratus, Halidrys siliquosa, Alaria esculenta, and Himanthalia elongata) (Gager et al., 2020). They mainly focused on exploring the region around 6 ppm for the determination of the phlorotannin profile, since the marine species are known to be rich in these compounds. In another example, Powers et al. used 1H NMR analysis to estimate the relative phenolic percentage in Sargassum natans considering the signals between 5.8 and 7.2 ppm (Powers et al., 2019). Apart from 1H NMR, 13C NMR has also been used for the analysis of phenolic compounds from marine species although this technique has a lower sensitivity due to the lower natural abundance of 13C. In relation to the previous example of Sargassum natans, 13C NMR was also used in that study for the relative estimation of phenolic compounds considering the signal region between 90 and 160 ppm (Powers et al., 2019). The direct measurement of the backbone structures of organic compounds is one of the main advantages of 13C NMR (Clendinen et al., 2014). Although some of the studies use it to ensure the purity of extracts or for profiling analysis (Santos et al., 2019), 13C NMR has been widely used in combination with 1H NMR. For example, Li et al. identified seven phlorotannins and three sterols in brown alga Ecklonia cava using 1 H NMR and 13C NMR (Li et al., 2009). Both techniques can be used independently or using a two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) technique. 2D-NMR techniques can combine the same (e.g., 1H-1H-COSY, 1H-1H-ROESY, and 1 H-1H-NOESY) or different (e.g., heteronuclear multiple-quantum correlation (HMQC) and heteronuclear multiple-bond correlation (HMBC)) atomic nuclei. These techniques have the advantage of

Chapter 1 Clean and green analytical techniques

obtaining structural information about the correlations between neighboring nuclei (Hatzakis, 2019). In this regard, these 2D-NMR techniques have been used in several studies for the structural elucidation of marine compounds. For example, Lee et al. used HMQC-NMR and HMBC-NMR in combination with 1 H NMR and 13C NMR for the characterization of phlorotannin compounds in Ecklonia stolonifera (Lee et al., 2012). In another study, Zhou et al. applied different NMR modes (1H-1H-COSY, HSQC, HMBC, 1H NMR, and 13C NMR) to identify phlorotannin compounds in the marine alga Ecklonia maxima. These examples show the complementarity of the different modes, which allow to have more structural information for a successful structural elucidation (Zhou et al., 2019). Since NMR is the most powerful technique for the structural elucidation of chemical compounds, it has been widely used to characterize compounds isolated from marine extracts. These isolated compounds are usually previously fractionated from the extracts using a purification process based on silica gel column flash chromatography, thin-layer chromatography, or semipreparative HPLC (Hartmann et al., 2018; Yoon and Cho, 2018). For example, 17 phenolic polyketides were isolated from two marine-derived fungal strains of Aspergillus unguis using a vacuum liquid chromatography and a semi-preparative HPLC (Van Anh et al., 2022). Since three of these compounds were not previously known, they were characterized using 1H NMR and 13C NMR. This is a clear example of the potential of this analytical technique for the elucidation of new compounds. However, it is important to note that in this type of study to discover new compounds, NMR has also been used in combination with other techniques such as HPLC-MS (Li et al., 2009; Van Anh et al., 2022). In this regard, the compounds that cannot be identified by using MS are subsequently isolated for their characterization by NMR. In summary, NMR is the technique that has been widely used for the structural elucidation of phenolic compounds in marine species, especially when these are previously isolated. In addition, this technique has also been used for phenolic profiling analyses, although generally in a complementary way to MS analysis.

6. Conclusions Since marine organisms are becoming an attractive and sustainable source of bioactive compounds with health benefits, such as phenolics, innovative analytical procedures with minimum environmental impact are necessary. These new procedures aim to reduce waste, energy, solvents, reagents, and instrumentation.

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Current trends in eco-friendly sample treatments are focused on different strategies such as miniaturization, automation, and faster, simpler procedures for the reduction of the use of solvents and reagents as well as the generation of residues. In this sense, chromatographic and spectrophotometric parameters have been optimized by applying fast methods and robust platforms and using different solvents, separation temperatures, and particle and column sizes. It is worth to note that due to the public interest in sustainable approaches, the research in this field is extremely active. This chapter has shown the most relevant advancements made in sample treatment, chromatographic separation, and mass detection, highlighting the different requirements of green analysis. Thus, in the future, the influence of each step in the analytical procedure should be considered carefully to study valuable compounds from natural sources such as marine phenolics.

References Abrahamsson, V.; Rodriguez-Meizoso, I.; Turner, C. Determination of Carotenoids in Microalgae Using Supercritical Fluid Extraction and Chromatography. J. Chromatogr. A 2012, 1250, 63–68. https://doi.org/10.1016/j.chroma. 2012.05.069. Achamlale, S.; Rezzonico, B.; Grignon-Dubois, M. Evaluation of Zostera detritus as a Potential New Source of Zosteric Acid. J. Appl. Phycol. 2009a, 21 (3), 347–352. https://doi.org/10.1007/s10811-008-9375-8. Achamlale, S.; Rezzonico, B.; Grignon-Dubois, M. Rosmarinic Acid from Beach Waste: Isolation and HPLC Quantification in Zostera detritus From Arcachon Lagoon. Food Chem. 2009b, 113 (4), 878–883. https://doi.org/10.1016/j. foodchem.2008.07.040. Adam, F.; Abert-Vian, M.; Peltier, G.; Chemat, F. “Solvent-Free” Ultrasound-Assisted Extraction of Lipids From Fresh Microalgae Cells: A Green, Clean and Scalable Process. Bioresour. Technol. 2012, 114, 457–465. https://doi.org/10.1016/j. biortech.2012.02.096. Agatonovic-Kustrin, S.; Morton, D. W. Quantification of Polyphenolic Antioxidants and Free Radical Scavengers in Marine Algae. J. Appl. Phycol. 2018, 30 (1), 113–120. https://doi.org/10.1007/s10811-017-1139-x. Agatonovic-Kustrin, S.; Kustrin, E.; Gegechkori, V.; Morton, D. W. HighPerformance Thin-Layer Chromatography Hyphenated With Microchemical and Biochemical Derivatizations in Bioactivity Profiling of Marine Species. Mar. Drugs 2019, 17 (3), 1–14. https://doi.org/10.3390/md17030148. Agrega´n, R.; Munekata, P. E. S.; Franco, D.; Dominguez, R.; Carballo, J.; Lorenzo, J. M. Phenolic Compounds From Three Brown Seaweed Species Using LC-DAD–ESI-MS/MS. Food Res. Int. 2017, 99, 979–985. https://doi.org/ 10.1016/j.foodres.2017.03.043. ˜ o´n, M. E.; Ivanovi
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Marine phenolic compounds: Sources, commercial value, and biological activities

2

Pilar Fallas Rodrı´gueza, Laura Murillo-Gonza´lezb, ereza Evelyn Rodrı´gueza, and Ana M. P
a

National Center of Food Science and Technology (CITA), University of Costa
Costa Rica. bFood Rica, Ciudad Universitaria Rodrigo Facio, San Jose, Engineering School, Guanacaste Campus, University of Costa Rica, Liberia, Guanacaste, Costa Rica

1.

Introduction

Phenolic compounds are phytochemicals with a characteristically high antioxidant activity. In plants, their main function is protection against ultraviolet radiation and pathogens. However, in recent years, phenolic compounds have become relevant in the human diet, and their reported health benefits continue to increase the attention worldwide from consumers and food manufacturers (Chojnacka et al., 2018; Hayes, 2015; Manach et al., 2004). Polyphenols can be found in plants, fruits, vegetables, cereals, and beverages such as coffee, tea, and wine. Their concentration and bioavailability depend on many factors, such as the dietary source form, their resistance to environmental conditions, genetics, and extraction and stabilization technology (Chojnacka et al., 2018). Marine sources such as seaweeds, seagrasses, and sponges are rich in polyphenolic compounds. Many of these marine phenolics are proven more biologically active than their terrestrial plant equivalents (Nagayama et al., 2002), making them suitable for meeting the industrial and commercial goals of providing better and healthier products. The increasing worldwide interest in the potential of marinerelated bioactive compounds for different products and largescale applications is evidenced by a large number of scientific Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00005-4 Copyright # 2023 Elsevier Inc. All rights reserved.

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Chapter 2 Marine phenolic compounds

recently published review papers (Barba, 2017; da Silva Vaz et al., 2016; Hamed et al., 2015; Jesumani et al., 2019; Lomartire et al., 2021; Mateos et al., 2020; Morais et al., 2021; Negara et al., 2021; Sˇimat et al., 2020; Sivak et al., 2018).

2.

Sources of marine phenolic compounds

According to Mateos et al. (2020), the main sources of marine natural products are phytoplankton, microorganisms (bacteria, fungi), sponges, macroalgae (blue, red, brown, and green), seawater, mollusks, coelenterates, and bryozoans; as well as other marine organisms that may ingest them throughout the food chain. These bioactive compounds, which may be either toxic for predators or an attraction for catching prey, are mainly a product of the harsh conditions that marine organisms have long been facing (Lu et al., 2021). For example, seaweeds grow in rapidly changing locations and conditions where sunlight and oxygen levels fluctuate, demanding a higher defense against oxidative stress. These changing conditions explain the variability in concentrations of phenolics from marine environments across different seasons (Getachew et al., 2020). Marine phenolics are mostly isolated from macro- and microalgae; as mentioned before, they can also be found in other organisms such as seagrasses and sponges. The most widely studied sources of marine phenolics are algae, but other sources are also cited. The term “microalgae” refers to photosynthetic organisms that can be either prokaryotic (mainly cyanobacteria) or eukaryotic.

2.1

Macroalgae

According to Zhao et al. (2018), the following are examples of the most studied macroalgae sources of bioactive polyphenolics, including some examples of various genera in which these compounds have been identified. Brown seaweeds from the Ochrophyta family have been extensively investigated as sources of polyphenolic compounds of interest, mainly phlorotannins (Mahmoud et al., 2021). These algae are multicellular and macroscopic and can accumulate between 30% and 50% of their dry weight in phenolic compounds, depending on environmental conditions (Gager et al., 2021). Some examples of brown algae genera are Cystoseira, Pelvetia, Ecklonia, Laminaria, Sargassum, Eisenia, Ascophyllum, Fucus, Bifurcaria, and Ishige.

Chapter 2 Marine phenolic compounds

Red algae belong to the Rhodomelaceae family and are a source of terpenoids, particularly bromophenols, which have also been frequently linked to potential antidiabetic effects. Examples of red algae genera are Laurencia, Grateloupia, Polysiphonia, Rhodomela, Hypnea, Gelidium, Odonthalia, Symphyocladia, and Kappaphycus. Green algae belong to the Chlorophyta family. Some examples of green algae genera are Dasycladus, Cladophora, and Ulva. Also referred to as green seaweeds, they are researched for their content of bromophenols and flavonoids (Cotas et al., 2020). In addition, some nontypical phenolics, such as coumarins and vanillic acid derivatives, have been identified in green seaweeds (FreilePelegrı´n and Robledo, 2013).

2.2

Phytoplankton

Microalgae consist of unicellular eukaryotic organisms that produce antioxidant molecules mainly as a stress response (Mateos et al., 2020). Phenolic content in this group is low compared to macroalgae; however, studies on structural elucidation mainly focused on freshwater-grown species such as Arthrospira, Anabaena, Nostoc Cylindrospermum, and Botryococcus. Among the identified compounds are phloroglucinol, phenolic acids, flavones and isoflavones, quercetin, several bromophenols, and certain cyanotoxins (Gager et al., 2021). Other phytoplankton sources of marine phenolics are diatoms, dinoflagellates, and green
nez-Lo´pez et al., 2021). yellow-brown flagellates (Jime Diatoms, seawater, and marine sediments are different types of phytoplankton. The presence of phenolics in diatoms is attributed to the release of free phenolics from phytoplankton. However, phenolics have also been found in seawater, probably due to reactions with lignin coming from melting glaciers. In seawater, compounds such as vanillin, vanillic acid, acetovanillone, and p-coumaric acid have been detected (Mateos et al., 2020). Seagrasses are a source of simple and sulfated phenolic acids. These compounds are researched due to their scavenging activity, resulting in anti-inflammatory, antitumoral, hypocholesterolemic, anticoagulant, antiviral, and antimicrobial activities (Jim
enez-Lo´pez et al., 2021). Sponges and other microorganisms such as fungi have been widely studied in the past 50 years. It has been recently discovered that some bioactive compounds from sponges come from microbial symbionts (Romano et al., 2017). For example, several new phenolic C-glycosides and aglycones with anti-inflammatory activity were identified in marine-derived Aspergillus grown on

49

50

Chapter 2 Marine phenolic compounds

the surface of the brown algae Saccharina cichorioides f. sachalinensis (Wen et al., 2020).

3.

Marine phenolics of interest

Marine phenolic compounds range from phenolic acids and other polyphenolic compounds with relatively simple chemical structures to more complex structures, such as bromophenols and phlorotannins, not found in terrestrial sources (Iba´n˜ez et al., 2012; Mateos et al., 2020). The following are the main groups of phenolic compounds of marine origin researched due to their potential bioactivity and uses, as shown in Fig. 1. Tannins comprise hydrolyzable tannins (derived from simple phenolic acids), flavonoid-based condensed tannins (from flavins and catechins), and phlorotannins, of which eckol, dieckol,

Fig. 1 Classification of naturally occurring marine phenolic compounds: primary sources, reported bioactivity, and some applications.

Chapter 2 Marine phenolic compounds

triphloroethol A, and eckstolonol are some prominent examples (Jim
enez-Lo´pez et al., 2021). Phlorotannins are highly hydrophilic compounds, isolated mostly from brown algae (mainly from the Laminariaceae family), with a wide range of molecular sizes (up to several thousand Da). They are composed of phloroglucinol units and are classified according to the different ways they are linked (Thomas and Kim, 2011). Phloroglucinol is a benzenetriol that exists as two tautomers in equilibrium: one phenol-like and another ketone-like. They are among the most studied marine phenolics due to their unique structures (Freile-Pelegrı´n and Robledo, 2013). Also, they are exclusive of marine origin and are present in high percentages within their sources, given that they are necessary for the develop
nez-Lo´pez et al., 2021). If the ment and growth of cell walls (Jime linkage is aryl ether (C-O-C), they are called phloroethols and fuhalols, and they are called fucols when the linkage is phenyl (C-O). Eckols are those with a benzodioxin linkage (Getachew et al., 2020). Bromophenols are common to various marine sources and can be found mainly in red macroalgae and to a lesser degree in brown and green, and also in cyanobacteria. Also, they can pass to other species throughout the marine food chain. Thus, they have been detected in many kinds of seafood (Mateos et al., 2020). Their relevance lies in the fact that a halogenated structure often confers a stronger biological activity (Freile-Pelegrı´n and Robledo, 2013). Phenolic terpenoids are present in brown and red macroalgae and produced by terrestrial organisms, and they are a group of interest in drug development. Reported sources of marine terpenoids are mangroves, seagrasses and seaweeds, marine sponges, corals, sediments, and fungi. The types of terpenoids identified from marine sources are sesquiterpenoids, diterpenoids, meroterpenoids, sesterterpenes, and triterpenoids (Gozari et al., 2021). They are commonly found in the Sargassaceae (as meroterpe
neznoids) and Rhodomelaceae families (Cotas et al., 2020; Jime Lo´pez et al., 2021). Flavonoids are low-molecular-weight molecules that are not exclusive to terrestrial plants, being also present in marine sources. This category includes (in order of abundance in marine sources) flavones, flavonols, flavanones, anthocyanins, and isoflavones. Most of the flavonoids isolated from marine sources come from seagrasses and halophytes and have been investigated for their pharmacological activities (Martins et al., 2019). Some of the simple phenolics of marine origin are flavonoids, catechins, and phenolic acids, such as benzoic, ferulic, rosmarinic, and cinnamic (Mateos et al., 2020). Additionally, gallic acid,

51

52

Chapter 2 Marine phenolic compounds

epicatechin, epigallocatechin, catechin gallate, protocatechuic acid, chlorogenic acid, caffeic acid, and vanillic acid have been found in marine species (Getachew et al., 2020). Hard to find natural compounds, such as colpol (Colpomenia), coumarins with the same skeleton as cinnamic acid (present in green algae), and C6-CN marine phenolics, are present in marine species. Among the less studied phenolics in marine species are lignans
nez-Lo´pez et al., 2021). and lignins (Jime

4.

Commercial value of marine phenolics

The Infinium Global Research Report estimated that the global market for marine-derived compounds in 2018 was over USD 10,500 million and was expected to rise to USD 22,000 million by 2025, an annual growth rate of 11.3% for the 2019–25 period. North America was the primary consumption market for marine-derived compounds, with around 48% in 2017; Europe was the second largest region with a market share of 28% (Medgadget, 2019). This market includes some types of bioactive compounds related to positive health effects and potential uses in food, pharmaceutical, and medical applications. These bioactive compounds comprise protein and peptides, polysaccharides, ω-3 polyunsaturated fatty acids (PUFA), enzymes, phenolic compounds, pigments, and vitamins. According to the Food and Agriculture Organization (FAO, 2020), seaweeds dominate the global production of farmed aquatic algae. In 2018, as shown in Table 1, farmed seaweeds in the world represented 97.1% of the total 32.4 million tons of the sum of wild-harvested (capture sector) and cultivated aquatic or marine algae (aquaculture sector), for a total amount of USD 13.3 billion (FAO, 2020). Three genera constitute almost 75% of the global production of seaweeds, Laminaria, Eucheuma spp., and Gracilaria (Table 1). Seaweed farming is done in a few countries, mainly in Asia’s Eastern and Southern regions. In 2015, the leading producers in decreasing volume order were Chile, China, Norway, and Japan for wild species; and China, Indonesia, Japan, South Korea, and the Philippines for cultured species (FAO, 2018). Seaweed or marine algae extracts are commercialized as liquid, powder, or flake products. They can be used in different applications, such as food and beverages, cosmetics and personal care, pharmaceutical or health care, agriculture, and horticulture. These extracts can be produced in mixtures derived from red, brown, and green algae. The global production of marine algae,

Chapter 2 Marine phenolic compounds

Table 1 Global farmed and wild algae production. Species

Weight in tons

Japanese kelp (Laminaria japonica) Eucheuma seaweeds (Eucheuma spp.) Gracilaria seaweeds (Gracilaria spp.) Wakame (Undaria pinnatifida) Nori (Porphyra spp.) Elkhorn sea moss (Kappaphycus alvarezii) Laver (Porphyra tenera) Fusiform sargassum (Sargassum fusiforme) Spiny eucheuma (Eucheuma denticulatum) Chilean kelp (Lessonia nigrescens)c Lessonia trabeculatac Arthrospira spp. (Spirulina)c North Atlantic rockweed (Ascophyllum nodosum)c Leisterc Giant kelp n.e.i (Macrocystis pyrifera)c Skottberg’s gigartina (Gigartina skottsbergii)c Tangle (Laminaria digitata)c North European Kelpc Bull kelp (Durvillaea antarctica)c Green laver Codium fragile Mazzaella laminarioidesc Gelidium seaweedsc Chondracanthus chamissoic Caulerpa seaweeds Bright green nori Gracilaria gracilis (Warty gracilaria) Haematococcus pluvialis Others Others Othersc

11,448,300a 9,237,001a 3,454,001a 2,320,000a 2,017,001a 1,597,000a 855,000a 268,001a 174,001a 115,311b 72,071b 69,001a 44,203b 41,077b 31,959b 27,327b 12,509b 10,489b 9441b 6748b 3895b 3013b 2707b 2199b 1219b 1000b 634b 200b 940,002a 804,131b 598,374b

n.e.i, not elsewhere identified. a FAO (2020). The state of world fisheries and aquaculture 2020. Sustainability in action. b FAO (2018). The global status of seaweed production, trade, and utilization. c Wild production.

53

54

Chapter 2 Marine phenolic compounds

or seaweed, has risen around 300% in the last decades, from 10.6 million tons in 2000 to 32.4 million tons in 2018. Seaweed and other algae exports from China alone were reported at more than USD 43.75 million that year. The three main countries for Chinese exports were Japan, United States, and Thailand, representing 28%, 20.2%, and 12% of the exports, respectively (Table 2; Trend Economy, 2021). Market research results have shown that North America will become the largest market for seaweed extracts between 2020 and 2027 because of the growing demand for liquid seaweed extracts in the horticulture and agriculture industries (Data Bridge Market Research, 2021). The bioactive potential of marine algae is still underexploited; the medicinal properties of macroalgae have been mainly associated with traditional and folk medicines. However, in recent years, various industries from different branches (fuel, varnish, textile, paints, plastics, cosmetics, pharmaceutical, and food, to name a few) have been focused on discovering and developing compounds derived from marine algae (Barbosa et al., 2014). The increasing demand for algal products results from a greater consumer focus on health and the broader use of food additives (Wells et al., 2017). Therefore, the food, medical, and cosmetic industries are, in fact, the strongest players in algal product commercialization. In 2014, about 28.5 million tons of seaweed (macroalgae) and other algae were harvested for direct consumption or further food processing, traditionally in Japan, Korea, and China. Because of their proven antimicrobial and antioxidant activity, the food industry is

Table 2 Seaweeds and other algae exports from China in 2018. Country

% Share

Amount (USD million)

Japan United States Thailand Russia Mexico Portugal Malaysia Korea Hong Kong

28.0 20.2 12.6 5.78 3.04 1.83 1.48 1.33 1.16

17.4 12.2 7.68 3.51 1.85 1.11 0.903 0.813 0.708

Source: Trend Economy. China Export Seaweeds & Other Algae, Fresh/Chilled/Frozen/Dried, Whether or Not Ground, 2021. https:// trendeconomy.com/data/h2?commodity ¼ 121220&reporter ¼ China&trade_flow ¼Export,Import&partner ¼ World&indicator¼TV, YoY&time_period ¼2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019,2020 (accessed November 22, 2021).

Chapter 2 Marine phenolic compounds

exploiting seaweed compounds as natural and safe alternatives to stabilize and conserve the intrinsic quality and nutritional value of foods, as well as to develop innovative nutraceutical and functional food products (Eom et al. (2012); Gupta and Abu-Ghannam, 2011). Phlorotannin and bromophenol extracts from brown and red algae have been applied for or patented (Tables 3 and 4) in many cosmetic, food, pharmaceutical, agricultural, and animal feeding markets. In the cosmetic industry, seaweed phenolic compounds ^ ve
e®, prohave been patented as natural UV sunscreens (Aethic So ® duced by Aethic in London, United Kingdom, from Porphyra umbilicalis) (Cardozo et al., 2007) and antiaging agents (ECKLEXT® BG, produced by NOF Group). An Ulva compressa extract containing flavonoids, tannins, polysaccharides, and acrylic acid, and a Fucus vesiculosus extract (Bladderwrack Extract) containing fucoidan and phlorotannins, are produced by Natural Solution in Flemington, NJ, United States (Cotas et al., 2020). Therefore, the interest of the cosmetic industry in the many properties of algae products is pushing forward a growing research area focusing on new phenolic compounds and cosmetic uses.

5.

Biological activities and applications

As mentioned before, there is an increasing interest from industries and consumers in marine phenolics due to the numerous health benefits and biological properties associated with the treatment of non-communicable diseases (Table 5), antimicrobial activity (Table 6), and others, such as antioxidant, anti-inflammatory, neuroprotective, antiallergic, and photoprotective properties (Table 7; Agatonovic-Kustrin et al., 2016; Agatonovic-Kustrin and Morton, 2018; Bahadoran et al., 2013; Capozzi et al., 2001; Cotas et al., 2020; Dai and Mumper, 2010; Manach et al., 2004; Onofrejova´ et al., 2010; Rosello´-Soto et al., 2015; Vimala and PoonghuzhaliIn, 2017). The potential for using marine phenolics as a functional ingredient is now noticeably clear for many industrial applications (Wijesinghe and Jeon, 2012).

5.1

Biological activities and applications against non-communicable diseases

5.1.1

Antidiabetic and antiobesity

One of the major diseases affecting civilization is diabetes. The World Health Organization (WHO) estimates that by 2030, diabetes will be the seventh leading cause of death worldwide (Mathers

55

Table 3 Patent applications for products containing phlorotannin extracts from brown algae. Country, application number, and publication number

Raw material

Product

Uses and applications

Ecklonia kurome

Antibacterial agent based on phlorotannins

Bactericide against various disease-causing bacteria in water, prophylactic or therapeutic agents

Japan, JP2002083316A, JP2003277203A

Ecklonia cava

A composition for inhibiting the activity of matrix metalloproteinase comprising phlorotannin isolated from sea algae

Korea, KR20060064390A, KR20080005711A

Ecklonia stolonifera Okamura

Functional food with recovery effect on blood composition and function

Ecklonia stolonifera

A skin-protective extract

A composition including phlorotannin compounds such as dieckol and bieckol to prevent and treat diseases related to the matrix metalloproteinase Pharmaceutical uses, food composition effective in recovering blood composition and function worsened by chemotherapy, surgery, and old age and stress. Functional cosmetics, functional foods, or pharmaceutical compositions for skin protection. Extracts containing phlorotannin compounds such as exostolonol and florofucopuroekol A

Inventors and year Nagayama, Kiminori; Hirayama, Izumi; Nakamura, Takashi; Iwamura, Yoshitoshi; Ginnaga, Akihiro (2002) Kim, Se Kwon; Kim, Moon Moo (2008)

South Korea, KR1020110003566A, KR101266889B1

Lee, Haeng-woo; Shin, Hyeol Cheol; Kim, Seongho; Hyun, Jong-Hoon (2012)

South Korea, KR20080100945A, KR101155512B1

Kim, Hyeung Rak; Kim, Jong Oh; Seo, Ho Chan; Kim, A Reum; Park, Kyoung Eun; Park, Ji Young; Choi, Jae Sue; Byun, Dae Seok (2012)

Bifurcaria bifurcata, Ascophyllum nodosum

Ecklonia cava, Dictyopteris prolifera Okamura, Dictyota dichotoma Lamouroux, Sargassum horneri C. Agardh, Sargassum patens C. Agardh, Ishige okamurae Yendo Ecklonia cava

Ecklonia cava, Eisenia, or Ecklonia species

Fucus or Ascophyllum genus

Ecklonia cava, Eisenia bicyclis, Ecklonia cava, Ecklonia kurome, Ecklonia stolonifera, Ecklonia maxima, Ecklonia radiata, Eisenia arborea

An antioxidant aqueous extract containing phlorotannins obtained by a continuous assisted ultrasound A purified extract of phlorotannins from brown algae

Phlorotannin extract with high antioxidant activity obtained by using supercritical carbon dioxide extraction A composition containing eckol for inhibiting cancer stem cell growth Phlorotannin extract as a stimulant for mycorrhizal and rhizobial symbioses Beverage with phlorotannin, ginseng extract, and caffeine

Ingredient for cosmetic and food applications

Spain, ES2441469B2, WO2014167162A1

Sineiro Torres, Jorge; Sa´nchez Guerrero, Marivel; Nu´n˜ez Garcı´a, Marı´a Jos
e (2013)

Pharmaceutical uses: aqueous ethanolic extract having affinity to gammaaminobutyric acid (GABA) receptors, which can induce sedation and sleeping effects. Phlorotannin extracts can be used as tablets or injections Cosmetic application

South Korea, KR1020130118015A, KR101592251B1

Cho, Sueng Mok; Han, Dae Seok; Kim, Dong Soo; Kim, Ji Young; Yang, Hye Jin; Yoon, Min Seok; Kwon, Sang Oh; Lee, Eung Joo; Yeo, Kyung Mok (2013)

South Korea, KR1020060063450A, KR20080004758A

Byun, Sang Yo; Yoo, Byoung Sam; Kim, Ju Ho; Ham, Young Min; Jeong, Dong Jin; Song, Young Keun; Kim, Hyung Bae (2013) Lee, Su Jae; Kim, Min Jung; Hyun, Kyung Hwan; Lee, Nam Ho; Hyun, Jin Won (2013) Briand, Xavier; Salamagne, Sylvie C
eline (2016)

Pharmaceutics: anticancer adjuvant

South Korea, KR20120053265A, KR101309538B1

Plant fertilizing product

France, FR2016052127W, WO2017032954A1 South Korea, KR20170050392A, KR101845287B1

Beverage used for improving concentration and reducing fatigue, preventing sleepiness, relieving hangover symptoms, reducing headaches, and improving motor abilities

Lee, Haeng Woo; Cheon, Hyun Cheol; Kim. Seong Ho; Shin. Hyeon Cheol; Kim, Ju Hee; Oh, Jae Keun (2018)

Continued

Table 3 Patent applications for products containing phlorotannin extracts from brown algae—cont’d

Raw material

Product

Uses and applications

Ecklonia or Eisenia species

An animal food containing phlorotannin

Fucaceae, Himanthaliaceae, Durvillaeaceae, Laminariaceae, Alariaceae, and Sargassaceae families

Antiviral composition

Animal food active ingredient containing phlorotannin extract with antioxidant and inflammation-improving effects Phlorotannins or the extract as an antiviral agent

Country, application number, and publication number

Inventors and year

Korea, KR102192712B1, WO2019147044A1

Cheon, Hyun Cheol; Kang, Moon Seok; Kim, Seong Ho; Lee, Haeng Woo; Yun, Jin Suk (2019)

Great Britain, GB2019053439W, WO2020115489A1

Evans, Huw; Plummer, Christopher; Luck, Matias; Mcinerney, Rose Elizabeth Piercy; Burns, Lauren Mairead (2020)

Table 4 Patent applications for products containing bromophenol extracts from red algae. Country, application number, and publication number

Raw material

Product

Uses and applications

Sargassum and more preferably from Sargassum siliquastrum Odonthalia corymbifera

Additive composition containing bromophenol extracts from marine algae to feed aquaculture animals Bromophenol composition for the control of harmful fungi

Improving organoleptic quality (flavor) in animal foods

United States, US89069504A, US20050011464A1

Fungicidal compositions for agriculture

Polysiphonia morrowii Harvey

Composition with bromophenols having antiviral activity against fish pathogenic viruses

Rhodomela confervoides

Seaweed bromophenol compounds containing amide or sulfone substituted groups

Animal food additive to prevent fish from being infected with viral hemorrhagic septicemia virus (VHSV), infectious pancreatic necrosis virus (IPNV), and infectious hematopoietic necrosis virus (IHNV) Food and cosmetic additives to delay or prevent oxidative deterioration of edible oils, oily foods, and grease cosmetics

South Korea, KR20070018063A, KR100896268B1 South Korea, KR1020060130195A, KR100838621B1

Rhodomelaceae plants, more preferably Odonthalia corymbifera Symphyocladia latiuscula

Bromophenol-based compounds harmful to Grampositive and Gram-negative bacteria

Antimicrobial, antibiotic, fungicide, preservative compositions

Seaweed bromophenol compound extract as an antioxidant in food oils

Food additive to delay or prevent the oxidation of edible oil related to free radical causes, to extend the shelf life

Inventors and year Chung Hau Yin; Ang Put O.; Woo Ying Shiu Norman; Ma Wing Chi Joyce (2005) Oh Ki Bong; Shin Jong Heon (2008) Kang, So Young; Oh, Myuong Joo; Kim, Seok Ryel (2008)

China, CN200910013880 A, CN101475509 A, CN101643442A South Korea, KR20080027192A, KR100896268B1

Bingui Wang; Ke Li; Xiaoming Li (2009)

China, CN200710010110A, CN101177382B

Wang, Bingui; Duan, Xiaojuan; Li, Xiaoming (2010)

Lee Hyi Seung; Shin Hee Jae; Shin Jong Heon; Oh Ki Bong; Park Heung Sik (2009)

Continued

Table 4 Patent applications for products containing bromophenol extracts from red algae—cont’d

Raw material

Product

Uses and applications

Rhodomela confervoides

Purified seaweed bromophenol urea compounds with activity against free radicals

Polysiphonia urceolata

Bromophenol compound capable of eliminating free radical activity Bromophenol-based compounds harmful to pathogenic Gram-positive and Gram-negative bacteria and harmful fungi Bromophenol compound extract for inhibiting inflammation

Food additive. Bromophenol composition, which can be used in food oils to prevent or delay the oxidation caused by free radicals and extend the shelf life of food products. Food additive as an antioxidant in food oils

Odonthalia corymbifera

Polysiphonia morrowii

Antimicrobial and antifungal compositions for pharmaceutical products

Anti-inflammatory compound for drugs, food, and cosmetic compositions

Country, application number, and publication number

Inventors and year

China, CN200810139114A, CN101643442A

Wang Bingui; Li Ke; Li Xiaoming (2010)

China, CNA2007100137305A, CN101177383A South Korea, KR20110076819A, KR101279822B1

Wang Bingui; Li Ke; Li Xiaoming (2010)

South Korea, KR1020170134546A, KR101805452B1

Lee Hyi Seung; Park Seung Il; Oh Ki Bong; Susan E. Matthews; Elise Bouthenet (2013) Soo Jin Heo; Youn Kyung Choi; Eun A Kim; Do Hyung Kang (2017)

Chapter 2 Marine phenolic compounds

61

Table 5 Marine phenolic compounds concerning non-communicable diseases. Phenolic compound group

Source

Species

References

Brown seaweed

Leathesia marina (Lyngbye) Decaisne Leathesia nana Setchell and N.L. Gardner Dysidea sp. Pseudoceratina sp.

Cotas et al. (2020)

Anticancer activity

Bromophenols

Marine sponge Red seaweed

Hydroxybenzoic acids

Brown seaweed

Phenolic terpenoids

Brown seaweed

Phlorotannins

Brown seaweed

Dong et al. (2020) Mateos et al. (2020) Dong et al. (2020)

Laurencia nipponica Yamada Neorhodomela aculeata (Perestenko) Masuda Odonthalia corymbifera (S.G. Gmelin) Greville Rhodomela confervoides (Hudson) P.C. Silva Symphyocladia latiuscula (Harvey) Yamada Vidalia colensoi (Hooker f. and Harvey) J. Agardh Ecklonia cava Kjellman Ishige okamurae Yendo

Dong et al. (2020)

Stypopodium flabelliforme Weber Bosse Acanthophora spicifera (M. Vahl) Børgesen Ascophyllum nodosum (Linnaeus) Le Jolis Ecklonia cava Kjellman

Cotas et al. (2020)

Dong et al. (2020) Cotas et al. (2020) Cotas et al. (2020)

Dong et al. (2020)

Cotas et al. (2020) Jesumani et al. (2019) Jesumani et al. (2019)

Cotas et al. (2020) Corona et al. (2016) Chojnacka et al. (2018), Cotas et al. (2020), FreilePelegrı´n and Robledo (2013), and Kim and Himaya (2011), Kim, Thomas and Li et al. (2011), Mateos et al. (2020), Thomas and Kim (2011), Wijesinghe and Jeon (2012) Continued

62

Chapter 2 Marine phenolic compounds

Table 5 Marine phenolic compounds concerning non-communicable diseases—cont’d Phenolic compound group

Source

Species

References

Ecklonia kurome Okamura Ecklonia maxima (Osbeck) Papenfuss Ecklonia stolonifera Okamura Eisenia bicyclis (Kjellman) Setchell Eisenia arborea Areschoug Eucheuma cottonii Weber Bosse Fucus vesiculosus Linnaeus Hizikia fusiformis (Harvey) Okamura Hydropuntia edulis (S.G. Gmelin) Gurgel and Fredericq Ishige okamurae Yendo Kappaphycus sp. Laminaria japonica Areschoug Sargassum muticum (Yendo) Fensholt Sargassum thunbergii (Mertens ex Roth) Kuntze Undaria pinnatifida (Harvey) Suringar

Chojnacka et al. (2018) and Freile-Pelegrı´n and Robledo (2013) Mateos et al. (2020)

Symphyocladia latiuscula (Harvey) Yamada Grateloupia elliptica Holmes Laurencia similis K.W. Nam and Y. Saito Odonthalia corymbifera (S.G. Gmelin) Greville

Dong et al. (2020)

Chojnacka et al. (2018) and Freile-Pelegrı´n and Robledo (2013) Chojnacka et al. (2018), Freile-Pelegrı´n and Robledo (2013), and Mateos et al. (2020) Chojnacka et al. (2018) Cotas et al. (2020) Mateos et al. (2020) Freile-Pelegrı´n and Robledo (2013) Cotas et al. (2020)

Freile-Pelegrı´n and Robledo (2013) Cotas et al. (2020) Freile-Pelegrı´n and Robledo (2013) and Kim, Thomas and Li et al. (2011) Mateos et al. (2020) Freile-Pelegrı´n and Robledo (2013)

Freile-Pelegrı´n and Robledo (2013)

Antidiabetic activity

Bromophenols

Marine sponge Red seaweed

Cotas et al. (2020) and Hayes (2015) Cotas et al. (2020) Cotas et al. (2020) and Dong et al. (2020)

Chapter 2 Marine phenolic compounds

63

Table 5 Marine phenolic compounds concerning non-communicable diseases—cont’d Phenolic compound group

Flavonoids Phenolic terpenoids Phlorotannins

Source

Green seaweed Brown seaweed Brown seaweed

Species

References

Rhodomela confervoides (Hudson) P.C. Silva Symphyocladia latiuscula (Harvey) Yamada Ulva prolifera O.F. M€uller Sargassum serratifolium (C. Agardh) C. Agardh Ascophyllum nodosum (Linnaeus) Le Jolis Ecklonia cava Kjellman

Cotas et al. (2020), Dong et al. (2020), and Mateos et al. (2020)

Ecklonia kurome Okamura Ecklonia stolonifera Okamura Eisenia bicyclis (Kjellman) Setchell Eisenia arborea Areschoug Fucus distichus Linnaeus Fucus vesiculosus Linnaeus Ishige foliacea Okamura Ishige okamurae Yendo Lessoniaceae sp. Padina pavonica (Linnaeus) Thivy Sargassum patens C. Agardh

Cotas et al. (2020)

Cotas et al. (2020) Cotas et al. (2020) Cotas et al. (2020) Barbosa et al. (2014), Chojnacka et al. (2018), Cotas et al. (2020), Kim and Himaya (2011), Mateos et al. (2020), Thomas and Kim (2011), and Wijesinghe and Jeon (2012) Chojnacka et al. (2018) Chojnacka et al. (2018), Cotas et al. (2020), Kim and Himaya (2011), Mateos et al. (2020), and Thomas and Kim (2011) Chojnacka et al. (2018), Cotas et al. (2020), and Thomas and Kim (2011) Chojnacka et al. (2018) Cotas et al. (2020) Cotas et al. (2020) Cotas et al. (2020) and Mateos et al. (2020) Chojnacka et al. (2018), Kim and Himaya (2011), and Thomas and Kim (2011) Mateos et al. (2020) Cotas et al. (2020) Cotas et al. (2020)

Antiobesity activity

Bromophenols

Red seaweed

Laurencia nipponica Yamada

Dong et al. (2020)

Continued

64

Chapter 2 Marine phenolic compounds

Table 5 Marine phenolic compounds concerning non-communicable diseases—cont’d Phenolic compound group

Phlorotannins

Source

Brown seaweed

Species

References

Neorhodomela aculeata (Perestenko) Masuda Odonthalia corymbifera (S.G. Gmelin) Greville Polysiphonia morrowii Harvey Symphyocladia latiuscula (Harvey) Yamada Ecklonia cava Kjellman Ecklonia cava Kjellman Ecklonia stolonifera Okamura Eisenia bicyclis (Kjellman) Setchell Fucus distichus Linnaeus Ishige okamurae Yendo

Dong et al. (2020)

Leathesia marina (Lyngbye) Decaisne Avrainvillea rawsonii (Dickie) M. Howe Ulva lactuca Linnaeus

Cotas et al. (2020)

Cotas et al. (2020) and Dong et al. (2020) Cotas et al. (2020) and Dong et al. (2020) Cotas et al. (2020) and Dong et al. (2020)

Cotas et al. (2020) Mateos et al. (2020) Mateos et al. (2020) Mateos et al. (2020) Mateos et al. (2020) Mateos et al. (2020)

Cardiovascular activity

Bromophenols

Phlorotannins

Brown seaweed Green seaweed Green seaweed Red seaweed Brown seaweed

Odonthalia corymbifera (S.G. Gmelin) Greville Ecklonia cava Kjellman Ecklonia kurome Okamura Ecklonia stolonifera Okamura Eisenia bicyclis (Kjellman) Setchell Eisenia arborea Areschoug Ishige okamurae Yendo

Cotas et al. (2020) Mateos et al. (2020) Cotas et al. (2020) Chojnacka et al. (2018), Cotas et al. (2020), and Mateos et al. (2020) Chojnacka et al. (2018) Chojnacka et al. (2018), Cotas et al. (2020), and Thomas and Kim (2011) Chojnacka et al. (2018) Chojnacka et al. (2018) Chojnacka et al. (2018)

Chapter 2 Marine phenolic compounds

Table 6 Antimicrobial and antiviral activity. Phenolic compound group

Source

Species

References

Kappaphycus sp. Polysiphonia morrowii Harvey Rhodomela confervoides (Hudson) P.C. Silva Symphyocladia latiuscula (Harvey) Yamada Ecklonia cava Kjellman Ishige okamurae Yendo Ecklonia cava Kjellman

Dong et al. (2020) Sivak et al. (2018)

Antimicrobial activity

Bromophenols

Red seaweed

Hydroxybenzoic acids

Brown seaweed

Phlorotannins

Brown seaweed

Ecklonia kurome Okamura Ecklonia stolonifera Okamura Eisenia bicyclis (Kjellman) Setchell Eisenia arborea Areschoug Ishige okamurae Yendo Laminaria japonica Areschoug

Cotas et al. (2020)

Dong et al. (2020)

Jesumani et al. (2019) Jesumani et al. (2019) Chojnacka et al. (2018), Cotas et al. (2020), and Jesumani et al. (2019) Chojnacka et al. (2018) Chojnacka et al. (2018) Chojnacka et al. (2018) and Jesumani et al. (2019) Chojnacka et al. (2018) Chojnacka et al. (2018) and Freile-Pelegrı´n and Robledo (2013) Cotas et al. (2020)

Antibacterial activity

Bromophenols

Green seaweed

Red seaweed

Avrainvillea nigricans Decaisne Cymopolia barbata (Linnaeus) J.V. Lamouroux Vidalia colensoi (Hooker f. and Harvey) J. Agardh

Cotas et al. (2020) Cotas et al. (2020)

Cotas et al. (2020)

Continued

65

66

Chapter 2 Marine phenolic compounds

Table 6 Antimicrobial and antiviral activity—cont’d Phenolic compound group

Source

Species

References

Phenolic terpenoids

Brown seaweed

Cotas et al. (2020)

Phlorotannins

Brown seaweed

Stypopodium flabelliforme Weber Bosse Ecklonia cava Kjellman Ecklonia kurome Okamura Ecklonia stolonifera Okamura Eisenia bicyclis (Kjellman) Setchell Eisenia arborea Areschoug Fucus vesiculosus Linnaeus Hizikia fusiformis (Harvey) Okamura Laminaria japonica Areschoug Sargassum thunbergii (Mertens ex Roth) Kuntze Undaria pinnatifida (Harvey) Suringar

Chojnacka et al. (2018), Cotas et al. (2020), Eom et al. (2012), and Freile-Pelegrı´n and Robledo (2013) Chojnacka et al. (2018), Cotas et al. (2020), Eom et al. (2012), and Freile-Pelegrı´n and Robledo (2013) Chojnacka et al. (2018), Eom et al. (2012), and FreilePelegrı´n and Robledo (2013) Chojnacka et al. (2018), Eom et al. (2012), and FreilePelegrı´n and Robledo (2013) Chojnacka et al. (2018) Cotas et al. (2020) Freile-Pelegrı´n and Robledo (2013) Freile-Pelegrı´n and Robledo (2013) Freile-Pelegrı´n and Robledo (2013) and Shannon and AbuGhannam (2019) Freile-Pelegrı´n and Robledo (2013)

Antifungal activity

Bromophenols

Red seaweed

Leathesia nana Setchell and N.L. Gardner Odonthalia corymbifera (S.G. Gmelin) Greville Symphyocladia latiuscula (Harvey) Yamada Vidalia colensoi (Hooker f. and Harvey) J. Agardh

Dong et al. (2020)

Cotas et al. (2020)

Cheung et al. (2014)

Cotas et al. (2020)

Chapter 2 Marine phenolic compounds

Table 6 Antimicrobial and antiviral activity—cont’d Phenolic compound group Phlorotannins

Source

Species

References

Brown seaweed

Cystoseira nodicaulis (Withering) M. Roberts Cystoseira usneoides (Linnaeus) M. Roberts Fucus spiralis Linnaeus Fucus vesiculosus Linnaeus

Cheung et al. (2014) and Lopes et al. (2013)

Polysiphonia morrowii Harvey Symphyocladia latiuscula (Harvey) Yamada Stypopodium zonale (J.V. Lamouroux) Papenfuss Ecklonia cava Kjellman

Cotas et al. (2020) and Dong et al. (2020)

Cheung et al. (2014) and Lopes et al. (2013) Cheung et al. (2014) and Lopes et al. (2013) Cotas et al. (2020)

Antiviral activity

Bromophenols

Red seaweed

Phenolic terpenoids

Brown seaweed

Phlorotannins

Brown seaweed

Ecklonia kurome Okamura Ecklonia stolonifera Okamura Eisenia bicyclis (Kjellman) Setchell Eisenia arborea Areschoug Hizikia fusiformis (Harvey) Okamura Ishige okamurae Yendo Laminaria japonica Areschoug Sargassum thunbergii (Mertens ex Roth) Kuntze Undaria pinnatifida (Harvey) Suringar

Cotas et al. (2020)

Cotas et al. (2020)

Chojnacka et al. (2018), Cotas et al. (2020), Freile-Pelegrı´n and Robledo (2013), Mateos et al. (2020), Thomas and Kim (2011), and Wijesinghe and Jeon (2012) Chojnacka et al. (2018) and Freile-Pelegrı´n and Robledo (2013) Freile-Pelegrı´n and Robledo (2013) Freile-Pelegrı´n and Robledo (2013) Chojnacka et al. (2018), Cotas et al. (2020), and FreilePelegrı´n and Robledo (2013) Chojnacka et al. (2018) Freile-Pelegrı´n and Robledo (2013) Chojnacka et al. (2018), Freile-Pelegrı´n and Robledo (2013), and Mateos et al. (2020) Freile-Pelegrı´n and Robledo (2013) Freile-Pelegrı´n and Robledo (2013)

Freile-Pelegrı´n and Robledo (2013)

67

68

Chapter 2 Marine phenolic compounds

Table 7 Other biological activities. Phenolic compound group

Source

Species

References

Brown seaweed

Ecklonia cava Kjellman

Chojnacka et al. (2018), Cotas et al. (2020), Freile-Pelegrı´n and Robledo (2013), Kim and Himaya (2011), Thomas and Kim (2011), and Wijesinghe and Jeon (2012) Chojnacka et al. (2018) and Freile-Pelegrı´n and Robledo (2013) Chojnacka et al. (2018), Cotas et al. (2020), Freile-Pelegrı´n and Robledo (2013), and Thomas and Kim (2011) Chojnacka et al. (2018) and Freile-Pelegrı´n and Robledo (2013) Chojnacka et al. (2018), Cotas et al. (2020), Kim and Himaya (2011), and Thomas and Kim (2011) Freile-Pelegrı´n and Robledo (2013)

Antiallergic activity

Phlorotannins

Ecklonia kurome Okamura Ecklonia stolonifera Okamura

Eisenia bicyclis (Kjellman) Setchell Eisenia arborea Areschoug Hizikia fusiformis (Harvey) Okamura Ishige okamurae Yendo Laminaria japonica Areschoug Sargassum thunbergii (Mertens ex Roth) Kuntze Undaria pinnatifida (Harvey) Suringar

Chojnacka et al. (2018) and Freile-Pelegrı´n and Robledo (2013) Freile-Pelegrı´n and Robledo (2013) Freile-Pelegrı´n and Robledo (2013) Freile-Pelegrı´n and Robledo (2013)

Anti-inflammatory activity

Bromophenols Flavonoids

Red seaweed Red seaweed

Hydroxybenzoic acids

Brown seaweed

Phenolic terpenoids Phlorotannins

Red seaweed Brown seaweed

Polysiphonia morrowii Harvey

Dong et al. (2020)

Neoporphyra dentata (Kjellman) L.-E. Yang and J. Brodie Ecklonia cava Kjellman Ishige okamurae Yendo

Cotas et al. (2020)

Gracilaria opuntia Durairatnam

Cotas et al. (2020)

Carpodesmia tamariscifolia (Hudson) Orellana and Sanso´n

Cotas et al. (2020)

Jesumani et al. (2019) Jesumani et al. (2019)

Chapter 2 Marine phenolic compounds

Table 7 Other biological activities—cont’d Phenolic compound group

Source

Species

References

Cystoseira nodicaulis (Withering) M. Roberts Ecklonia cava Kjellman

Jesumani et al. (2019)

Ecklonia kurome Okamura Ecklonia stolonifera Okamura Eisenia bicyclis (Kjellman) Setchell Eisenia arborea Areschoug Fucus spiralis Linnaeus Laminaria japonica Areschoug Treptacantha nodicaulis (Withering) Orellana and Sanso´n

Chojnacka et al. (2018), Cotas et al. (2020), Kim and Himaya (2011), and Wijesinghe and Jeon (2012) Chojnacka et al. (2018) and Jesumani et al. (2019) Chojnacka et al. (2018) Chojnacka et al. (2018) and Jesumani et al. (2019) Chojnacka et al. (2018) and Sugiura et al. (2013) Cotas et al. (2020) Cotas et al. (2020) and Kim and Himaya (2011) Cotas et al. (2020)

Antioxidant activity

Bromophenols

Green seaweed

Red seaweed

Capsosiphon fulvescens (C. Agardh) Setchell and N.L. Gardner Chaetomorpha moniligera Kjellman Cladophora rupestris (Linnaeus) K€utzing Codium fragile (Suringar) Hariot Ulva australis Areschoug Ulva clathrata (Roth) C. Agardh Ulva compressa Linnaeus Ulva flexuosa Wulfen Ulva intestinalis Linnaeus Ulva linza Linnaeus Polysiphonia morrowii Harvey Polysiphonia stricta (Mertens ex Dillwyn) Greville Polysiphonia urceolata (Lightfoot ex Dillwyn) Greville

Cotas et al. (2020)

Cotas et al. (2020) Surget et al. (2017) Surget et al. (2017) Cotas et al. (2020) Cotas et al. (2020) Cotas et al. (2020) Cotas et al. (2020) Cotas et al. (2020) Cotas et al. (2020) Dong et al. (2020) Cotas et al. (2020) Dong et al. (2020) Continued

69

70

Chapter 2 Marine phenolic compounds

Table 7 Other biological activities—cont’d Phenolic compound group

Hydroxybenzoic acids

Source

Species

References Dong et al. (2020)

Red seaweed Brown seaweed

Rhodomela confervoides (Hudson) P.C. Silva Symphyocladia latiuscula (Harvey) Yamada Caulerpa spp. Capsosiphon fulvescens (C. Agardh) Setchell and N.L. Gardner Chaetomorpha moniligera Kjellman Ulva australis Areschoug Ulva clathrata (Roth) C. Agardh Ulva compressa Linnaeus Ulva flexuosa Wulfen Ulva intestinalis Linnaeus Ulva linza Linnaeus Acanthophora spicifera (M. Vahl) Børgesen Ecklonia cava Kjellman Ishige okamurae Yendo

Green seaweed Hydroxycoumarins

Green seaweed

Phenolic terpenoids Phenols

Red seaweed Brown seaweed

Red seaweed

Cladophora rupestris (Linnaeus) K€utzing Codium fragile (Suringar) Hariot Cladophora rupestris (Linnaeus) K€utzing Codium fragile (Suringar) Hariot Gracilaria opuntia Durairatnam Ascophyllum nodosum (Linnaeus) Le Jolis Fucus vesiculosus Linnaeus Sargassum mangarevense (Grunow) Setchell Sargassum siliquastrum (Mertens ex Turner) C. Agardh Gelidiella acerosa (Forsska˚l) Feldmann and Hamel

Cotas et al. (2020) and Dong et al. (2020) Cotas et al. (2020) Cotas et al. (2020)

Cotas et al. (2020) Cotas Cotas Cotas Cotas Cotas Cotas Cotas

et et et et et et et

al. (2020) al. (2020) al. (2020) al. (2020) al. (2020) al. (2020) al. (2020)

Jesumani et al. (2019) Jesumani et al. (2019) Surget et al. (2017) Surget et al. (2017) Surget et al. (2017) Surget et al. (2017) Cotas et al. (2020) Lopez-Padron et al. (2020) Lopez-Padron et al. (2020) Lopez-Padron et al. (2020) Lopez-Padron et al. (2020) Lopez-Padron et al. (2020)

Chapter 2 Marine phenolic compounds

71

Table 7 Other biological activities—cont’d Phenolic compound group

Phlorotannins

Source

Brown seaweed

Species

References

Kappaphycus alvarezii (Doty) L.M. Liao Palmaria palmata (Linnaeus) F. Weber and D. Mohr Rhodomela confervoides (Hudson) P.C. Silva Ascophyllum nodosum (Linnaeus) Le Jolis Ecklonia cava Kjellman

Lopez-Padron et al. (2020)

Ecklonia kurome Okamura Ecklonia stolonifera Okamura

Polyphenols

Green seaweed

Eisenia bicyclis (Kjellman) Setchell Eisenia arborea Areschoug Fucus spiralis Linnaeus Fucus vesiculosus Linnaeus Himanthalia elongata (Linnaeus) S.F. Gray Ishige okamurae Yendo Laminaria japonica Areschoug Ulva lactuca Linnaeus

Lopez-Padron et al. (2020) Lopez-Padron et al. (2020) Qu
eguineur et al. (2013) Chojnacka et al. (2018), Cotas et al. (2020), Kim and Himaya (2011), Lopez-Padron et al. (2020), Mateos et al. (2020), Shannon and AbuGhannam (2019), Thomas and Kim (2011), and Wijesinghe and Jeon, 2012 Chojnacka et al. (2018) and Thomas and Kim (2011) Chojnacka et al. (2018) and Kim and Himaya (2011) Chojnacka et al. (2018), Lopez-Padron et al. (2020), and Thomas and Kim (2011) Chojnacka et al. (2018) Shannon and Abu-Ghannam (2019) Barbosa et al. (2014) and Cotas et al. (2020) Qu
eguineur et al. (2013) Kim and Himaya (2011) Cotas et al. (2020) Lopez-Padron et al. (2020)

Neuroprotective activity

Bromophenols Flavonoids Phenolic terpenoids Phlorotannins

Red seaweed Brown seaweed Brown seaweed Brown seaweed

Symphyocladia latiuscula (Harvey) Yamada Turbinaria ornata (Turner) J. Agardh Dictyopteris undulata Holmes Ecklonia cava Kjellman

Cotas et al. (2020) and Dong et al. (2020) Cotas et al. (2020) Cotas et al. (2020) Barbosa et al. (2014), Chojnacka et al. (2018), Cotas et al. (2020), and Mateos et al. (2020) Continued

72

Chapter 2 Marine phenolic compounds

Table 7 Other biological activities—cont’d Phenolic compound group

Source

Species

References

Ecklonia maxima (Osbeck) Papenfuss Ecklonia stolonifera Okamura Eisenia bicyclis (Kjellman) Setchell Eisenia arborea Areschoug Ishige foliacea Okamura Ishige okamurae Yendo

Barbosa et al. (2014), Cotas et al. (2020) and Mateos et al. (2020) Barbosa et al. (2014) and Cotas et al. (2020) Barbosa et al. (2014), Cotas et al. (2020), and Mateos et al. (2020) Chojnacka et al. (2018) Mateos et al. (2020) and Cotas et al. (2020) Barbosa et al. (2014) and Chojnacka et al. (2018)

Symphyocladia latiuscula (Harvey) Yamada Vidalia obtusiloba (Mertens ex C. Agardh) J. Agardh Dactylosiphon bullosus (D.A. Saunder) Santian˜ez, K.M. Lee, S.M. Boo and Kogame Ecklonia cava Kjellman

Dong et al. (2020)

Photoprotective activity

Bromophenols

Phlorotannins

Red seaweed

Brown seaweed

Ecklonia kurome Okamura Ecklonia stolonifera Okamura Eisenia bicyclis (Kjellman) Setchell Eisenia arborea Areschoug Halidrys siliquosa (Linnaeus) Lyngbye Ishige okamurae Yendo

Dong et al. (2020) Cotas et al. (2020)

Chojnacka et al. (2018), Cotas et al. (2020), Jesumani et al. (2019), Kim and Himaya (2011), Thomas and Kim (2011), and Wijesinghe and Jeon (2012) Chojnacka et al. (2018) Chojnacka et al. (2018), Jesumani et al. (2019), and Thomas and Kim (2011) Chojnacka et al. (2018) Chojnacka et al. (2018), Jesumani et al. (2019), and Le Lann et al. (2016) Kim and Himaya (2011)

Chapter 2 Marine phenolic compounds

and Loncar, 2006). Metabolic disorders associated with this disease and the resulting complications can lead to other dangerous diseases. It has been argued that the current treatments for diabetes are insufficient and have undesirable side effects; therefore, finding and introducing additional methods involving the use of supplements or nutraceuticals is needed. The main mechanisms attributed to marine polyphenols are the modulation of glucose-induced oxidative stress and the inhibition of starch-digestion enzymes. Nwosu et al. (2011) found that extracts high in polyphenols obtained from Palmaria, Alaria, and Ascophyllum inhibited α-amylase activity in vitro, with dieckol being the most effective component. The inhibition of starchdigestion enzymes, such as amylase and glucosidase, also represents an opportunity to reduce glucose levels. In China, marine algae have long been used for treating diabetes (Jarald et al., 2008). Marine phenolics, such as phlorotannins from brown algae and bromophenols from red algae, are involved in various antidiabetic enzyme inhibitory and complementary mechanisms. There is compelling evidence regarding the antidiabetic activity of several marine polyphenols, although most of the studies are in vitro or in vivo with animals and not with humans, as desirable. Recently, a study on extracts with free and bound phenolic molecules such as luteolin, genistein, rosmarinic acid, and salvianic acid, obtained from Padina tetrastromatica grown in India, showed α-amylase and amyloglucosidase inhibitory activity (as well as antioxidant activity); thus, this brown alga is an option for food and pharma applications for diabetes treatment and prevention (Naveen et al., 2021). This promising antidiabetic activity has led to the development of polyphenol-rich extracts from seaweeds as alternative drugs to treat the disease (Mateos et al., 2020). An example is the commercial formulation of Ascophyllum nodosum and Fucus vesiculosus phlorotannins as the brand InSea2™ (Rimouski, QC, Canada), which promotes a 90% reduction of postprandial blood glucose. Also, Mannas® All (Botamedi Inc., Seoul, Korea) contains marine algae extracts (Seanol®), and it is promoted to control blood glucose and cholesterol after meals and reduce peak insulin secretion. Seapolynol™ extract (Botamedi Inc., Seoul, Korea) is another food supplement from Ecklonia cava containing phlorotannins, which has an antidiabetic effect on insulin sensitivity in type 2 diabetes (Table 8; Jeon et al., 2015). Considering examples like the ones mentioned above and given that diabetes is closely related to diet, marine phenolics would be a promising natural alternative against diabetes through

73

Table 8 Commercial applications of marine phenolics. Commercial brand

Company

Marine phenolic compound

Mannas All

Botamedi Inc.

Phloroglucinol (MOP™)

Mannas™Well

Botamedi Inc.

Phloroglucinol (MOP™)

HealSea™

Diana Naturals Inc.

Phloroglucinol (Phytonutriance®)

ID-alG™ Dr. Sinatra

Bio Serae Laboratories S.A. Simple Healthy LLC

Phlorotannins (Ascophyllum nodosum extract) Phlorotannins (Seanol™)

iSeanol®

Botamedi Inc.

Phlorotannins (Seanol™)

NeuroSeanol

Botamedi Inc.

Phlorotannins (Seanol™)

Smart 7®Jelly

Botamedi Inc.

Phlorotannins (Seanol™)

Vritol™

Botamedi Inc.

MD Sun Cream Sa´lvora InSea2™

®

Application

Description of the product Prevents diabetes complications, lowers blood sugar and cholesterol Indicated for weight loss and cholesterol management Indicated for cardiovascular health

Phlorotannins (Seanol™)

Food supplement Food supplement Food supplement Food supplement Food supplement Food supplement Food supplement Food supplement Cosmetic

Seanol inside Inc. Cetalga Extract Sl

Phlorotannins (Seanol™) Phlorotannins

Cosmetic Cosmetic

Innovatic Inc.

Polyphenols

Food supplement

Indicated for weight control Promotes heart health Improves blood flow and nutrient supply to the brain Improves memory, movement, and learning Boosts mental and physical energy Promotes blood circulation, improving focus, memory, and mental flexibility UV sun protection, including UVA, UVB, and UVC light Antiaging effects and protection of elastin and hyaluronic acid Reduces glycemic stress

Chapter 2 Marine phenolic compounds

their application in pharmaceutical, as well as in functional and nutraceutical food products (Kim and Himaya, 2011; Thomas and Kim, 2011; Wijesinghe and Jeon, 2012). Some marine phenolics are promoted as mechanisms for inhibiting enzyme activity, adipocyte differentiation and proliferation, and glucose consumption (Mateos et al., 2020). Among them is the use of natural bioactive products such as Mannas™ (Botamedi Inc., Korea), which contains Ecklonia cava extract, promoted for weight control (Table 8) (www.botamedihk.com).

5.1.2

Cardioprotective

Marine phenolics, specifically phlorotannins, are being explored by the market as supplements to treat several cardiac diseases such as arteriosclerosis and hypertension. Some of these products containing phlorotannins are HealSea™ (produced by Diana Naturals in Rennes, France), Id-alG™ (produced by Bio Serae in Bram, France), and Seanol™ (produced by LiveChem in Jeju-do, South Korea, and distributed by Simple Health, Maitland, United States under the name of Dr. Sinatra). Seapolynol™ (produced by Botamedi Inc., Seoul, Korea), which the EFSA has authorized, is a food supplement containing dieckol and other polyphenols from Ecklonia cava with promising results as an antihyperlipidemic and cardioprotective agent against doxorubicininduced cardiotoxicity (Table 8; Cotas et al., 2020). Studies demonstrated that phlorotannins positively affect hypertension treatments and that flavonoid-enriched diets improve endothelial function and lower blood pressure. For example, based on ex vivo and in vivo experiments reported by Park et al. (2017) in spontaneously hypertensive rats, the sargachromenol D from Sargassum siliquastrum demonstrated high potential to become a drug for blood pressure control in instances where it cannot be controlled by conventional combinatorial therapy. Also, other cardioprotective mechanisms related to the inhibitory power of angiotensin-converting enzyme (ACE) and lipoxygenase enzyme are being described for phlorotannins (Go´mez-Guzma´n et al., 2018).

5.2

Antimicrobial and antiviral activity

Some marine phenolic compounds such as phlorotannins and bromophenols are being discovered as promising therapeutic metabolites in pharmacotherapy to prevent and treat immunodeficiency disease.

75

76

Chapter 2 Marine phenolic compounds

Some examples are diphlorethohydroxycarmalol isolated from Ishige okamurae, 6,60-bieckol isolated, and 8,400 -dieckol isolated from Ecklonia cava, which have demonstrated effects over acquired immunodeficiency syndrome (HIV/AIDS) by inhibiting enzyme activities (Mateos et al., 2020). In addition, Symphyocladia latiuscula bromophenol 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether is another compound that has demonstrated an antiviral effect against various types of herpes simplex type 1; in this case, the behavior of the antiviral activity was comparable with acyclovir in skin lesions (Cotas et al., 2020; Park et al., 2005). Bromophenols isolated from Polysiphonia morrowii, the 3-bromo-4,5-dihydroxybenzyl methyl ether (BDME) and 3-bromo-4,5-dihydroxybenzaldehyde (BD), demonstrated antiviral activity against the infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV), which are two aggressive fish pathogenic virus (Cotas et al., 2020). In addition, the same authors reported that polyphenols obtained from Fucus vesiculosus inhibited both HIV-1-induced syncytium formation and HIV-1 reverse transcriptase enzyme activity and that dieckol from Ecklonia cava can interfere in the viral replication mechanism of SARS-CoV.

5.3

Other biological activities

5.3.1

Antioxidant

Phenolic compounds are among the most well-known and important groups of compounds with antioxidant properties (Agatonovic-Kustrin and Morton, 2018; Dai and Mumper, 2010; Gupta and Agrawal, 2007; Stengel et al., 2011; Tas¸ et al., 2015). The antioxidant activity of phenolic compounds is primarily related to the stabilization and binding of free radicals, reducing properties, the inhibition of certain enzymes, and the ability to chelate metal ions (Li et al., 2011). Marine phenolics are dominant contributors to antioxidant activity. Phlorotannins from brown algae are the main classes of antioxidant phenolic compounds found in marine algae. Nevertheless, other marine phenolic compounds such as bromophenols, flavonoids, and phenolic terpenoids are found to be strong antioxidant agents. The antioxidant activity of bioactive algal compounds has been ascribed to their reactive oxygen species’ scavenging ability, quenching singlet oxygen, reducing power, and chelating ability (Andrade et al., 2013; Maharana et al., 2015). It has been reported that marine phenolics with high antioxidant activity are promising candidates for potent antimicrobial

Chapter 2 Marine phenolic compounds

agents that could replace synthetic chemical products, representing promising new horizons for the pharmaceutical industry. Also, their use in the food, cosmetic, and therapeutic industries is a promising alternative to synthetic antioxidants given the low cost, high compatibility with dietary intake, and lack of harmful effects on the human body oxygen species’ scavenging ability, quenching singlet oxygen, reducing power, and chelating ability (Andrade et al., 2013; Maharana et al., 2015; Wijesinghe and Jeon, 2012). For example, in a study on Aspergillus versicolor, 22 phenolics were obtained (six of them new aspergilols). Most of them showed antioxidant activity, and four exhibited radical scavenging activity (Wu et al., 2016).

5.3.2

Anti-inflammatory

Marine polyphenols have been described as one of the most potent anti-inflammatory and anticarcinogenic compounds among the existent bioactive compounds from marine resources. The carcinogenic activity of seaweed was indirectly verified using a clinical trial where a modest improvement in DNA damage was observed in an obese group after consuming 100 mg/day for eight weeks of a (poly) phenol-rich extract of the brown algae Ascophyllum nodosum. Nevertheless, there are no human studies to directly confirm these biological properties (Mateos et al., 2020). It has been found that polyphenol-rich extracts, as well as isolated phlorotannins and bromophenols, can inhibit cancer cell proliferation and play significant roles as anticancer metabolites, performing in different phases of cancer evolution such as proliferative signaling, metastasis, cell cycle, resistance to cell death, evasion, angiogenesis, and the evasion of growth suppressors (Cotas et al., 2020). Also, several marine compounds such as fucoxanthin, polyphenols, and other antioxidants, including phlorotannins, iodine, and sulfated polysaccharides, such as fucoidan, participate in diverse mechanisms for cancer therapy, one of which is the acti
rrezvation of apoptosis, which is programmed cell death (Gutie Rodrı´guez et al., 2018; Jiang and Shi, 2018; Namvar et al., 2012). Angiogenesis is the formation of new blood vessels, which facilitate cancer invasion. Fucodiphloroethol G from Ecklonia cava has inhibited this process in an angiogenesis-induced cellular model (Li et al., 2011). An Ecklonia cava extract consisting of 58  1% of phlorotannins (500 mM) has potent inhibitory activity against MMP 2,9 expression in HT1080 cells. Interestingly, this inhibition was more potent than the commercially available MMP inhibitory drug doxycycline (10 mg/mL) (Kim and Himaya, 2011).

77

78

Chapter 2 Marine phenolic compounds

Also, it has been demonstrated that phlorotannins isolated from Ecklonia cava algae can inhibit enzymes. The impact of these phlorotannins on the matrix metalloproteinase (MMP) inhibition plays a vital role in diseases such as chronic inflammation, wrinkles, osteoporosis, arthritis, periodontal disease, and cancer (Li et al., 2011). These findings show that this edible brown alga is a good option to include in the human diet and develop chemoprevention pharmacological products (Kim and Himaya, 2011).

5.3.3

Neuroprotective

Bromophenols and phlorotannins have been identified as important components for neuroprotection and treatment of psychological disorders and neurodegenerative diseases such as Parkinson’s and Alzheimer (Cotas et al., 2020). This bioactivity is associated with the main Alzheimer’s disease factors, such as ligand to neurosensors, inhibition of amyloid enzyme activity, antineuroinflammatory activity, inhibition against the monoamine oxidases, and inhibition of secretase and acetylcholinesterase (AChE). Additionally, these compounds may play a key role in the suppression of the overproduction of intracellular ROS, reduction of intracellular Ca2 + levels and apoptosis, reduction of the expression and release of nitric oxide, prostaglandin E2, interleukin-1β, and tumor necrosis factor-α in microglial cells, scavenging of reactive carbonyls, and inhibition of the formation of advanced glycation end products (AGEs) (Barbosa et al., 2014). Some commercial products containing Ecklonia cava extract focus on neuroprotective functions, such as Seanol®, iSeanol®, NeuroSeanol, and Smart 7® Jelly (from Botamedi Inc.), promoted as products that improve memory and blood flow. Also, Vritol™ cream is promoted as support for the brain’s well-being by massaging the head and neck (Table 8; www.botamedihk.com). Cotas et al. (2020) mention that zonarol can be studied further to verify if it is a useful therapy for neurodegenerative diseases. It is a sesquiterpene derived from Dictyopteris undulata that protects neuronal cells from oxidative stress.

5.3.4

Antiallergic

Phlorotannins or phlorotannin extracts from edible brown algae have shown promising potential in antiallergic therapy in vivo and in vitro models (Kim and Himaya, 2011). In vitro studies have shown that extracts rich in phlorotannins isolated from the brown algae Eisenia arborea, Eisenia bicyclis, Ecklonia kurome, and Ecklonia cava can prevent allergic and

Chapter 2 Marine phenolic compounds

inflammatory reactions because they are capable of inhibiting hyaluronidase, the enzyme involved in allergic reactions. These studies demonstrated that some crude phlorotannin extracts have a stronger inhibitory effect on hyaluronidase than some known inhibitors, such as catechins, sodium cromoglycate, and epigallocatechin gallate (Thomas and Kim, 2013).

5.3.5

Photoprotective

Marine polyphenols can be considered active compounds for skin protection and the treatment of skin diseases. Phlorotannins can be potential natural inhibitors of enzymes involved in processes like skin aging, where collagen is degraded by increased matrix metalloproteinase (MMP) regulation. In vitro studies show that phenolic compounds from algae Corallina pilulifera have photoprotective activity against UV radiation and prevent expressions of MMPs in human dermal fibroblast cells. Also, it has been shown that phlorotannins can be inhibitors of tyrosinase, an enzyme that catalyzes the rate-limiting pigmentation stage; thus, phlorotannins may play a significant role in the skin pigmentation process. In addition, in vitro studies on mouse skin models have confirmed the efficacy of brown alga phenolic compounds in protecting against harmful UV radiation and their potential anticancer activity (Thomas and Kim, 2013). Commercial products, which contain Ecklonia cava extracts, such as MD Sun Cream SPF 50 (Seanol®) (Table 8), are reported to protect from UVA, UVB, and UVC rays (www.botamedihk.com).

5.3.6

Antiaging

There are various marine polyphenols with identified free radical scavenging functionality in cells, suitable for application in antiaging products. Additionally, compounds such as phloroglucinol and eckol extracted from other seaweeds can inhibit tyrosinase activity or reduce melanin production. For example, a polyphenolic extract from Sargassum presented attenuation of photodamage effects in an in vitro study of cells irradiated with UVA light (Wang et al., 2021). A recent study reported increased marine ingredients (mostly from macro- and microalgae) for antiaging products in the past decade. The main marine ingredients in antiaging products were Laminaria digitata, Kappaphycus alvarezii, and Chondrus crispus. These are used not only for the beneficial effects of the phenolic molecules present but also due to the hydrating and firming effects of other lipids and polysaccharides (Resende et al., 2021).

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Regarding the antiaging functionality, the commercial brand Sa´lvora (Cetalga Extract Sl) produces antiaging cosmetic creams containing Ascophyllum nodosum extracts (Table 8; www. salvora.eu).

6.

Conclusions

There is a growing interest within the global market (particularly in North America) for phenolic compound products due to their increasingly known potential for preventing and treating several non-communicable diseases. Other relevant functionalities include antimicrobial, antioxidant, and anti-inflammatory effects. Commercial extracts used for producing food supplements and cosmetics promote antiobesity, antidiabetic, cardioprotective, photoprotective, neuroprotective, and antiaging activity. Many research studies, patents, and industrial applications (mostly originated and offered in Asia and Europe) highlight the potential of seaweeds to produce marine phenolic compounds and extracts in large quantities. Phlorotannins and bromophenols are the most studied marine phenolics. Nevertheless, many underexploited marine sources with valuable phenolic molecules are yet to be analyzed and understood.

Acknowledgments Vice-Rectorate of Research, University of Costa Rica (project grant 735-C0-455), Ing. Mauricio Villegas (Proinnova, University of Costa Rica), and Lic. Sara Cascante (SIBDI, University of Costa Rica) for technical help.

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Further reading Lee, S. H.; Jeon, Y. J. Anti-Diabetic Effects of Brown Algae Derived Phlorotannins, Marine Polyphenols Through Diverse Mechanisms. Fitoterapia 2013, 86 (1), 129–136. https://doi.org/10.1016/j.fitote.2013.02.013. MarketWatch. Seaweed Extracts Market Outlook; 2021. https://www.marketwatch. com/press-release/seaweed-extracts-market-outlook-2021-detailed-industryanalysis-overview-growth-opportunities-top-companies-and-global-forecast2027-2021-08-17. Accessed November 22, 2021.

Marine natural bromophenols: Sources, structures, main bioactivities, and toxicity

3

Hui Donga,b, Poul Erik Hansenc, Songtao Donga,b, Dimitrios Stagosd, Xiukun Line, and Ming Liua,b a

Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China. b Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, China. cDepartment of Science and Environment, Roskilde University, Roskilde, Denmark. d Department of Biochemistry and Biotechnology, School of Health Sciences, University of Thessaly, Biopolis, Larissa, Greece. eDepartment of Pharmacology, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, China

Abbreviations A549 ABTS AChE AD BACE1 BChE Bel-7402 B16F10 BPs CA ChEs CPT-1B DPPH ED50 FABP3 G6PD GMPS GSK-3β HbA1c HCT-116 HELF hMAO-A

human lung adenocarcinoma epithelial cell line 2,20-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt acetylcholinesterase Alzheimer’s disease β-site amyloid precursor protein cleaving enzyme 1 butyrylcholinesterase human hepatoma cell line malignant melanoma cell line bromophenols carbonic anhydrase cholinesterases carnitine palmitoyltransferase 1B 1,1-diphenyl-2-picryl hydrazyl median effective dose fatty acid binding protein 3 glucose 6-phosphate dehydrogenase guanosine monophosphate synthetase glycogen synthase kinase-3β hemoglobin A1c human colorectal carcinoma cell line human embryo lung fibroblast cell line human monoamine oxidase-A

Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00017-0 Copyright # 2023 Elsevier Inc. All rights reserved.

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IC50 IgE IL-6 IMPDH MIC NF-κB Nrf2 PBDEs PD PGE2 PON1 PTP1B RAW 264.7 ROS SAR STAT1 TEAC UVB Vero

half-inhibitory concentration immunoglobulin E interleukin-6 inosine monophosphate dehydrogenase minimum inhibitory concentration nuclear factor kappa light chain enhancer of activated B cells nuclear factor erythroid 2-related factor 2 polybrominated diphenyl ethers Parkinson’s disease prostaglandin E2 paraoxonase-1 tyrosine phosphatase 1B leukemia cells in mouse macrophage reactive oxygen species structure activity relationships signal transducer and activator of transcription 1 trolox equivalent antioxidant capacity ultraviolet B African green monkey kidney cells.

1. Introduction Bromophenols (BPs) are common marine secondary metabolites, mainly isolated from marine algae (Gamal, 2010; Wijesekara et al., 2011), including red algae (Cherian et al., 2019; Choi et al., 2018; Paudel et al., 2019a; Wang et al., 2015; Xu et al., 2012a), green algae (Flodin and Whitfield, 1999a; Laney, 1994; Mcconnell et al., 1982; Park et al., 1992; Sun et al., 1983), and brown algae (Chung et al., 2003; Green et al., 1993; Xiu et al., 2004; Xu et al., 2004a; Yao, 2004). Some BPs are also found in other marine organisms, such as sponges (Ciminiello et al., 2000; Fu et al., 1995; Hanif et al., 2007; Hattori et al., 2002; Popov, 2001; Yao, 2004), ascidians (Carroll et al., 1999; Lindsay et al., 1998; Rudi et al., 2000), mussels (Malmvarn et al., 2005), polychaetes (Whitfield et al., 1999), and marine proteobacteria (Agarwal et al., 2014). Structurally, BPs include one or more benzene rings and contain a different number of hydroxyl, bromine, and other groups. The first two BPs were isolated from the red alga Rhodomela larix in 1967 (Katsui et al., 1967). Since then, many BPs have been isolated from various marine organisms, and their bioactivities have been investigated thoroughly, as has been reviewed previously (Dong et al., 2020; Liu et al., 2011a). The ecological functions of marine BPs are possibly chemical defense and deterrents against other marine organisms (Kicklighter et al., 2004). BPs are biosynthesized in the presence of bromoperoxidases, laccase, bromide, bromate, and hydrogen peroxide. The biosynthetic pathways in the marine organisms of natural BPs are not well known, with only a few reports illustrating the molecular

Chapter 3 Marine nature bromophenols

genetic basis and bromate effects for the production of BPs (Agarwal et al., 2014; Flodin and Whitfield, 1999b; Lin et al., 2015; Moore, 2017). For example, Lindqvist et al. published an apparent correlation between the concentration of pigments in Ceramium tenuicorne and the BP levels (Lindqvist et al., 2017). They suggested an association with the photosynthetic activity via bromoperoxidase working as a scavenger for the formed hydrogen peroxide (Dahlgren et al., 2015; Liu et al., 2011a). Nowadays, in the field of food and pharmaceutical agents, marine BPs have attracted much attention due to their potential health benefits, such as antimicrobial, anticancer, antioxidant, and antidiabetic activities (Dong et al., 2020; Liu et al., 2011a). Therefore, the isolation and testing for the biological activity of BPs found in natural products is still an important research area. Additionally, recent studies focus on the design and synthesis of BP’s derivatives with novel functionalities (Akbaba et al., 2013a; Balaydın et al., 2012; Boztas et al., 2019; Dong et al., 2020; Feng et al., 2017; Ho et al., 2012; Li et al., 2018; Lin et al., 2014) and on unveiling the mechanisms underlying their bioactivity and toxicity.

2. Sources, structures, bioactivities, and toxicity of BPs 2.1

Anticancer activity

One of the most interesting activities of marine BPs is that these compounds could inhibit the growth of cancer cell lines and the in vivo growth of tumors. For example, BPs isolated from the brown algae Leathesia nana, 1.1–1.6 (Scheme 1), which share the 2,3-dibromo-4,5-dihydroxybenzyl unit, inhibit the viability of several human cancer cell lines (Shi et al., 2009; Xu et al., 2004b), and the Leathesia nana extract, rich in BPs, could inhibit the growth of Sarcoma 180 tumors in vivo (Shi et al., 2009). The in vitro anticancer mechanisms of 1.2 are related to the modulation of β1-integrin/FAK signaling (Wu et al., 2015). Detailed anticancer activity of compound 1.6 revealed that this compound induces mitochondrial apoptosis in cancer cells and inhibits topoisomerase I (Liu et al., 2012). Moreover, compounds 1.2 and 1.6 also showed anti-angiogenic properties via inhibition of tyrosine kinase, endothelial nitric oxide synthase (Wang et al., 2015), and VEGF signaling systems (Qi et al., 2015).

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Scheme 1 Chemical structures of BPs with anticancer activity.

Chapter 3 Marine nature bromophenols

3-Bromo-4,5-dihydroxy benzoic acid methyl ester (1.7) and 3-bromo-4,5-dihydroxy-benzaldehyde (1.8), obtained from the red alga Rhodomela confervoides, are cytotoxic against several cancer cell lines, including KB, Bel-7402, and A549 (IC50, 12.5–40.1 μM) (Han et al., 2005). Lanosol butenone (1.9), isolated from red algae Osmundaria colensoi, inhibits the proliferation of human leukemia cells (IC50 ¼ 8.0 μM) (Popplewell and Northcote, 2009). 3-bromo-4,5-dihydroxybenzylalcohol (1.10), isolated from the tropical green algae Avrainvillea nigricans, is cytotoxic to KB cells with IC50 of 47 μM (Colon et al., 1987). Compounds 1.11, 1.12, and 1.13, isolated from the red algae Polysiphonia lanosa, showed potent cytotoxicity against DLD-1 and HCT-116 cell lines with IC50 ranging from 1.32 to 14.6 μM. The phenylethanol and phenylethanol sulfate BPs 1.14–17 show moderate cytotoxicity against several human cancer cell lines (Ma et al., 2006). Comparing the IC50 value of 1.14 with that of 1.15, it seems that the sulfate group is dispensable. Interestingly, the activity increases after bromination in 1.16, suggesting the importance of Br for anticancer activity. Polybrominated diphenyl ethers (1.18–20), isolated from the marine sponge Phyllospongia dendyi, inhibit the polymerization of tubulin in vitro (IC50 29.6, 33.5, and 20.9 μM, respectively) and inhibit the maturation of oocytes (IC50 3.6, 4.2, and 4.2 μM, respectively) (Yao, 2004). However, in NBT-T2 rat bladder epithelial cells, 1.18–19 show low cytotoxicity (IC50 > 15 μg/mL), while 1.21 and 1.22 are moderate cytotoxic with IC50 values of 2.8 and 8.5 μg/mL, respectively (Hanif et al., 2007). In addition to targeting tubulin, 1.18 and 1.19 could also inhibit some enzymes, such as inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate synthetase (GMPS). BPs 1.23–25 also show similar inhibition against the enzyme (Fu et al., 1995). Spongiadioxins A (1.26), B (1.27), and C (1.28), isolated from the Australian marine sponge Dysidea, inhibit the division of fertilized eggs of the sea urchin Strongylocentrotus intermedius (IC50 ¼ 4.8, 1.1, and 5.7 μM, respectively). Spongiadioxins A and B are also cytotoxic against mouse Ehrlich carcinoma cells (ED50 ¼ 29 and 15.5 μg/mL, respectively) (Popov, 2001; Utkina et al., 2002).

2.2

Antidiabetic and anti-obesity activity

In folk medicine, marine algae have been used for a long time (Jarald et al., 2008), and BPs derived from marine algae have been reported to possess antidiabetic activities, acting as inhibitors against both tyrosine phosphatase 1B (PTP1B), α-glucosidase,

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Scheme 2 BPs with antidiabetic and anti-obesity activity.

and aldose reductase, which are all usual drug targets for the treatment of diabetes. BPs 2.1–2.4 (Scheme 2), isolated from the red alga Rhodomela confervoides, which contain a 2,3dibromo-4,5-dihydroxybenzyl unit and are highly brominated, inhibit PTP1B activity with IC50 of 2.4, 1.7, 1.5, and 0.84 μM, respectively; in diabetic rats, the Rhodomela confervoides extracts could decrease the blood glucose level (Shi et al., 2008a). Molecular docking studies and surface plasmon resonance studies have been performed to reduce the risk of false positives (Luo et al., 2020a).

Chapter 3 Marine nature bromophenols

Moreover, BP 2.3 (Scheme 2), isolated from the red alga Odonthalia corymbifera, is a dual inhibitor of PTP1B (Xu et al., 2016) and α-glucosidase (Liu et al., 2011b). In vivo, BP 2.3 could decrease the blood glucose, HbA1c, and triglyceride levels and downregulate the bodyweight (Xu et al., 2016). BPs 2.5 and 2.6 (Scheme 2), isolated from the marine alga Symphyocladia latiuscula, showed antidiabetic potential by inhibiting PTP1B and α-glucosidase enzymes (Paudel et al., 2019b). Compound 2.7 (Scheme 2), isolated from the red alga Rhodomela confervoides, competitively inhibited PTP1B, activated insulin signaling, and ameliorated insulin resistance (Guo et al., 2020; Xu et al., 2018). Both in normal and insulinresistant cells, compound 2.7 also increased the glucose uptake (Guo et al., 2020; Xu et al., 2018). In vivo, BP 2.7 could reduce the blood glucose level in streptozotocin-induced diabetic mice (Guo et al., 2020); thus, 2.7 represents a potential candidate for further development as an antidiabetic agent. BP 2.8 (Scheme 2), isolated from the red algae Rhodomela confervoides, showed inhibition against PTP1B with an IC50 value of 1.7 μM. At the cellular level, BP 2.8 could sensitize insulin activity and ameliorate palmitic acid-induced insulin resistance (Luo et al., 2020b). In addition, BP 2.8 significantly enhanced the mRNA level related to fatty acid oxidation, including carnitine palmitoyl transferase 1B (CPT-1B) and fatty acid-binding protein 3 (FABP3) (Luo et al., 2020b). Moreover, other studies showed BPs 2.9–2.15 (Scheme 2), isolated from marine Rhodomelaceae algae, Odonthalia corymbifera, and Grateloupia elliptica, inhibit the activity of α-glucosidase (Kim et al., 2008; Kurihara et al., 1999a, 1999b). In the present series of BPs, bis (2,3,6-tribromo-4,5-dihydroxybenzyl) ether (2.9) is the most potent α-glucosidase inhibitor (IC50 ¼ 0.03 μM) (Kurihara et al., 1999a), while 2,4-dibromophenol (2.14) is the weakest one, with an IC50 value of 110.4 μM. It seems that the α-glucosidase inhibition has a close relationship with a degree of bromination in these BPs. In addition to inhibiting PTP1B and α-glucosidase, some BPs also inhibit the activity of aldose reductase, which is the first enzyme of the polyol pathway contributing to the formation of fructose and the development of diabetes complications (Suzen and Buyukbingol, 2003). BPs 2.16–2.20, from the red algae Symphyocladia latiuscula (Scheme 2), have aldose reductase inhibitory activity and potential application in treating eye and nerve damage in T2DM patients (Wang et al., 2005). Besides inhibiting aldose reductase, additional in vivo experiments reported that BP 2.17 showed promising antidiabetic activities (Shi et al., 2012), also via highly selective inhibition against PTP1B (Shi et al., 2012).

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In addition to the antidiabetic activity, some marine BPs also have the potential to treat obesity. For example, BP 2.21 (Scheme 2), isolated from red algae Polysiphonia morrowii, could regulate the expression of adipogenic transcription factors, the activation of AMP protein kinase in 3T3-L1 adipocytes, inhibit the adipogenesis, inhibit the intracellular lipid accumulation, and decrease the triglyceride level, thus showing the potential to treat obesity (Ko et al., 2019).

2.3

Antioxidant activity

A growing body of research indicates that BPs have potential antioxidant activity; for example, BPs 3.1–3.11 (Scheme 3) isolated from the red algae Symphyocladia latiuscula were reported to possess 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities (Choi et al., 2000; Duan et al., 2007). Compound 3.2 has the highest activity with an IC50 value of 7.5 μM, while compound 3.11 shows the lowest, with an IC50 value of 24.7 μM (Choi et al., 2000; Duan et al., 2007). The antioxidant activity closely relates to the hydroxyl group numbers in the molecules (Duan et al., 2007); meanwhile, their common 3,4-dihydroxy-2,5,6-tribromobenzyloxy unit may be another relevant factor for their DPPH radical scavenging activities. BPs 3.12–3.21 (Scheme 3), isolated from the red algae Polysiphonia urceolata, also show scavenging activity toward DPPH radicals. BP 3.16 (Scheme 3) was isolated in several kinds of marine red algae species, including Rhodomela confervoides, Polysiphonia morrowii, and Polysiphonia urceolata. BP 3.16 could scavenge DPPH free radicals (IC50 value is 20.3 μM) (Fan et al., 2003; Kim et al., 2011; Li et al., 2008). Further investigations at the cellular level showed that BP 3.16 activated ERK- and Aktmediated Nrf2 signaling and thereof upregulated HO-1 expression in keratinocytes, which contribute significantly to the cytoprotective effects against oxidative stress (Ryu et al., 2019). In addition to scavenging DPPH radicals, BP 3.16 also scavenged hydroxyl and alkyl radicals. BP 3.16 could inhibit H2O2-induced lipid peroxidation, cell death, and apoptosis by inhibiting ROS production in Vero cells and zebrafish embryos (Cho et al., 2019). BP 3.16 also increased the reduced glutathione level via Nrf2 activation (Kim et al., 2017), thereby protecting human keratinocytes from UVBinduced oxidative stress (Hyun et al., 2012). BP 3.16 protects the skin via the Nrf2/HO pathway (Ryu et al., 2019). BPs 3.18 and 3.19, with four hydroxyl groups in the molecules, are the most active (IC50 values of 6.8 and 6.1 μM, respectively). However, BP 3.17, with one hydroxyl substituent, is the least active

Chapter 3 Marine nature bromophenols

Scheme 3 BPs with antioxidant activity.

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(IC50 ¼ 35.8 μM) (Li et al., 2007, 2008); Thus, this further confirmed that hydroxyl groups are potent contributors to the antioxidant activity of the compound. Conjugation in the chemical sense is another significant factor for the antioxidant activity, as seen by comparing 3.19 with 3.15 since the former has conjugation in the dihydrophenanthrene skeleton. However, bromination may not be a determining factor by comparing the IC50’s of 3.5 and 3.15, also 3.18 and 3.19. Bromination slightly decreases the antioxidant activity in the case of 3.5 and 3.15; however, bromination slightly increased it for 3.19 vs 3.18. BPs containing nitrogen (3.22–3.26), obtained from red algae Rhodomela confervoides, also showed radical scavenging activity in the DPPH and ABTS model (Li et al., 2012). BPs (3.27–3.30), obtained from red algae Vertebrata lanosa, were reported to have antioxidant activity at the cellular level by detecting the cellular lipid peroxidation level, where compound 3.28 has the most potent activity (Olsen et al., 2013). BPs 3.31–3.36 (Scheme 3), isolated from the marine red algae Rhodomela confervoides, could scavenge DPPH and ABTS free radicals (Li et al., 2011). Some new nitrogen-containing BPs were isolated from the marine red algae Rhodomela confervoides, such as BPs 3.37–3.39 (Scheme 3). These BPs showed strong scavenging activity against DPPH free radicals, with IC50 values from 5.22 to 23.60 μM, while exhibiting moderate activity against ABTS free radicals, with Trolox equivalent antioxidant capacity (TEAC) values from 3.11 to 3.58 mM (Li et al., 2012). Another nitrogen-containing BP 3.40–3.45 (Scheme 3), isolated from the marine red alga Rhodomela confervoides, effectively scavenged DPPH free radicals (Li et al., 2021). (R)-rhodomelin A (3.46, Scheme 3) isolated from the red algae Rhodomela confervoides had potential antioxidant activity mainly evaluated by DPPH (IC50 ¼ 3.82 μM) and TEAC assays (IC50 ¼ 4.37 mM) (Li et al., 2018). New compounds 3.47–3.56 with antioxidant activity are also isolated from Ceramium sp. (Zhang et al., 2020) and Avrainvillea amadelpha (Hawas et al., 2021).

2.4

Antimicrobial activity

The discovery of novel antibacterial compounds has been ongoing for many years, and various marine BPs possess promising antibacterial activity. For example, five BPs (4.1–4.5, Scheme 4) with antibacterial activity were isolated from the marine alga Rhodomela confervoides. Among these BPs, compound 4.5 is the most potent with the minimum inhibitory concentration (MIC) less than 70μg/mL when tested against Gram-positive and Gram-negative

Chapter 3 Marine nature bromophenols

Scheme 4 BPs with antimicrobial activity.

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bacteria (Fan et al., 2003). Compound 4.5 (Scheme 4) also possessed antifungal activity against several phytopathogenic fungi, such as Botrytis cinerea, Valsa mali, and Fusarium graminearum (Liu et al., 2014). Mechanically, compound 4.5 inhibited the spore germination of the mycelial growth and disrupted the cell membrane of Botrytis cinerea, indicating that BPs could also be developed as antifungal agents (Liu et al., 2014). Lanosol methyl ether (4.6), lanosol butenone (4.7), and rhodomelol (4.8) were isolated from the New Zealand red alga Osmundaria colensoi and showed antibacterial activity against the MC155 strain of Mycobacterium smegmatis (IC50 7.8, 26.2, and 28.1 μM, respectively) (Popplewell and Northcote, 2009). Lanosol ethyl ether (4.9) shows little antimicrobial activity, with a mean bacteriostatic and fungistatic MIC of 0.27  0.07 mg/mL and mean bactericidal and fungicidal MIC of 0.69  0.15 mg/mL. 2-(20 ,40 dibromophenoxy)-4,6-dibromophenol (4.10), isolated from the marine sponge Dysidea granulosa (Bergquist), exhibits broadspectrum antibacterial activity, with a MIC of 0.117–2.5 μg/mL against Gram-positive bacteria and 0.5–2 μg/mL against Gramnegative bacteria (Shridhar et al., 2009). BP 4.11, similar to 4.10 but with an extra hydroxyl group, is active against Gram-positive bacterium Bacillus subtilis. The primary SAR showed that the phenolic hydroxyls are important for the antibacterial activity; the methylated form of 4.11 showed no activity (Hanif et al., 2007). According to the antimicrobial disc diffusion test, the aldehyde 4.12, isolated from the marine red alga Kappaphycus sp. (Scheme 4), inhibits the growth of both Gram-positive and Gram-negative bacteria. BP 4.12 showed potent inhibition of Pseudomonas fluorescens and Staphylococcus aureus (Rajasulochana et al., 2012). In addition, 4.3, 4.4, and 4.6, together with 4.13, 4.14, and 4.15 (Scheme 4), showed inhibition against crop fungus. These BPs could reduce the appressorium formation on rice plants via inhibition of isocitrate lyase (ICL), which is highly expressed in appressorium-mediated plant infection (Lee et al., 2007). Symphyocladin G (4.16), a BP compound derived from the marine red alga Symphyocladia latiuscula, possessed antifungal activity against Candida albicans (Xu et al., 2012b). Moreover, isolated from the red alga Symphyocladia latiuscula, compounds 4.17 and 4.18 inhibit Candida albicans with MIC values of 25 and 12.5 μg/mL, respectively (Xu et al., 2014); the bromobenzyl methyl sulphoxide (4.19) possesses weaker antifungal activity (MIC ¼ 37.5 μg/mL) (Xu et al., 2013). Obtained from the marine red alga Symphyocladia latiuscula, compounds 4.20 (Scheme 4)

Chapter 3 Marine nature bromophenols

revealed antimicrobial activities against Candida albicans with MIC values in the range of 10 to 37.5 μg/mL (Xu et al., 2014). Interestingly, 3-bromo-4,5-dihydroxybenzyl methyl ether (4.21) and 3-bromo-4,5-dihydroxybenzaldehyde (4.22) inhibit fish pathogenic viruses, such as hematopoietic necrosis virus and pancreatic necrosis virus (Kim et al., 2011), while compounds 4.23–25 inhibit wild-type herpes simplex type 1 (HSV-1), phosphonoacetic acid-resistant HSV-1 (APr HSV-1), and thymidine kinase-deficient HSV-1 (TK HSV-1) strains. In HSV-1 strain 7401H-infected mice, oral administration (20 mg/kg) of 4.23 for 6–10 days delayed skin lesions and decreased the number of virus particles in the skin and brain (Park et al., 2005).

2.5

Anti-inflammatory activity

In recent years, the anti-inflammatory activity of BPs has attracted significant attention. Vidalols A (5.1) and (5.2), obtained from the Caribbean marine red alga Vidalia obtusiloba, were reported to have anti-inflammatory effects. These compounds reduced the edema swelling of the mouse ear by inhibiting the enzyme phospholipase A2 (bee venom PLA2) (Wiemer et al., 1991). Immunoglobulin E (IgE), an important target for atopic dermatitis, induces mast cells to produce inflammatory mediators, including various cytokines. 3-Bromo-4,5-dihydroxybenzaldehyde 5.3 (Scheme 5), isolated from the red alga Polysiphonia morrowii, alleviated IgE-mediated inflammatory responses in mouse and macrophage models, indicating their potential for treating allergic inflammatory diseases (Kang et al., 2017). Further mechanism studies illustrated that BP 5.3 reduced the IgE level, inhibited the production of interleukin-6 (IL-6), and down-regulated the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription 1 (STAT1) pathways (Kang et al., 2017). Interestingly, in injured hearts, 5.3 also reduced CD68+ macrophages, M1 and M2 macrophage infiltration, inhibited the phosphorylation of NF-κB, suppressed the secretion of pro-inflammatory cytokines, showed myocardial protection via the Akt-PGC1a-Sirt3 pathway, and finally improved cardiac function recovery ( Ji et al., 2018; Qin et al., 2018). Bis (3-bromo-4,5-dihydroxybenzyl)ether 5.4 (Scheme 5), isolated from the red alga Polysiphonia morrowii, significantly decreased lipopolysaccharide (LPS)-induced NO, PGE2, and pro-inflammatory cytokines released in RAW 264.7 macrophage cells via inhibiting the ERK signaling pathway (Choi et al., 2018).

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Scheme 5 BPs with anti-inflammatory and anti-neurodegenerative disease activity.

2.6

Anti-neurodegenerative disease activity

Alzheimer’s disease (AD) and Parkinson’s disease (PD), characterized by a progressive loss of specific neuronal cells, are the most prevalent and deadly neurodegenerative diseases. Some marine BPs have been reported to have potential anti-AD activities. For example, BPs 6.1–6.3 (Scheme 5), isolated from Symphyocladia latiuscula (Harvey) Yamada, inhibit several anti-AD drug-target enzymes, including ChEs, BACE1, and GSK-3β. Among these BPs, compound 6.3 exhibited the most potent inhibitory activity against AChE, BChE, and BACE1 with IC50 values of 2.66, 4.03, and 2.32 μM, respectively. In addition, BPs 6.1–6.3 inhibited more than 50% of the self-induced Aβ25–35 aggregation (Paudel et al., 2019a), which may be another mechanism contributing to their anti-AD activity. Concerning anti-PD activity, BPs 6.1–6.3 are also inhibitors of human monoamine oxidase-A (hMAO-A) (Paudel et al., 2020), which catalyzes the inactivation of multiple neurotransmitters in PD treatment. Furthermore, BPs 6.1–6.3 were also good dopamine D3/D4 receptor agonists (Paudel et al., 2020). Considering that AChE, BChE, BACE1, hMAO-A, and dopaminergic receptors have a close relationship to AD and PD, these BPs have the potential to develop

Chapter 3 Marine nature bromophenols

new drugs. In addition, BPs 6.4–6.6 (Scheme 5) containing a 4-phenylbutenone moiety, isolated from the red alga Rhodomela confervoides, also inhibited ChEs. BPs 6.4–6.6 inhibited AChE with Ki values from 19.02  6.15 to 32.38  8.01 pM, while inhibiting BChE with Ki values from 8.013  3.06 to 13.28  0.07 pM (Bayrak et al., 2017). BP 6.7 isolated from algae, ascidians, and sponges reduced inflammation in lipopolysaccharide-induced acute liver injury (Yang et al., 2020, 2021; Zhang et al., 2018). All the research discussed above shows that natural marine BPs are excellent candidates for treating neurodegenerative diseases such as AD and PD.

2.7

Enzyme inhibitory activity

In addition, BPs also showed inhibition against other kinds of enzymes. BPs 7.1 and 7. 2 (Scheme 6), isolated from Symphyocladia latiuscula, inhibit tyrosinase with IC50 values of 10.78  0.19 and 2.92  0.04 μM, respectively. BPs 7.1 and 7.2 also decrease the tyrosinase expression levels and melanin content and inhibit the intracellular tyrosinase activity in B16F10 cells (Paudel et al., 2019c). A newly discovered function of marine BPs is their inhibition of Carbonic anhydrases (CA), which are key targets for clinically used diuretics, anticonvulsants, and anti-glaucoma drugs. The naturally occurring BPs (7.3–7.4) were reported to inhibit CA, and some of their derivatives have been designed to enhance this inhibition (Akbaba et al., 2013a; Balaydın et al., 2012; Boztas et al., 2015; Nar et al., 2013), BPs 7.5 and 7.6 (Scheme 6), obtained from red algae Symphyocladia latiuscula, had weak inhibitory activity on human carbonic anhydrase (hCA) II with IC50 values of 86.4 38.29 μM, respectively. However, the derivative 7.7 (Scheme 6) had a more potent activity with an IC50 value of 0.7 μM (Balaydın et al., 2012). BP 7.8 (Scheme 6), isolated from the Caribbean red algae Vidalia obtusiloba, inhibited hCA I, II, IV, and VI, with Ki values of 12.24, 1.13, 1.84, and 3.41 μM, respectively. BP 7.9 (Scheme 6), isolated from the red alga Symphyocladia latiuscula (Choi et al., 2000), also inhibited hCA I, II, IV, and VI, with Ki values of 1.67, 0.56, 1.08, and 0.59 μM, respectively (Balaydın et al., 2012). In general, the natural BPs may be candidates as potent CA inhibitors and thus protect the body from several diseases related to CA disorders. Some BPs inhibit G6PD, which is a critical enzyme in the pentose phosphate pathway and a potential target for treating obesity and cancer. For example, BPs 7.10–12 (Scheme 6), isolated from three rhodomelaceae algae species (Laurencia nipponica,

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Scheme 6 BPs with enzyme inhibitory and possible toxicological activity.

Chapter 3 Marine nature bromophenols

Polysiphonia morrowii, and Odonthalia corymbifera), inhibited the activity of G6PD. Among them, BP 7.12 was the strongest inhibitor with an IC50 value of 0.85  0.1 μM (Mikami et al., 2013). BP 7.13 (Scheme 6) was isolated from the algae Odonthalia corymbifera, Neorhodomela aculeata, and Symphyocladia latiuscula and found to inhibit prokaryotic Leuconostoc mesenteroides G6PD with an IC50 value of 321  18 μM. However, the inhibitory effect of BP 7.13 on eukaryotic Saccharomyces cerevisiae G6PD was weak. This specificity was possibly due to the difference in the recognition site of the hydrophobic alkyl group on the side chain of this compound (Mikami et al., 2016). Thrombin is the ultimate proteinase of the coagulation cascade, which makes it an attractive target for treating a variety of cardiovascular diseases. (+)-3-(2,3-dibromo-4,5-dihydroxyphenyl)-4-bromo-5,6-dihydroxy-1,3-dihydro-isobenzofuran (7.14) (Scheme 6), isolated from the brown alga Leathesia nana, inhibits the thrombin activity both in vitro and in vivo (Shi et al., 2008b, 2010). Rawsonol (7.15), a brominated diphenyl methane derivative and isolated from the green alga Avrainvillea rawsoni, could inhibit the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Carte et al., 1989). HMG-CoA reductase is the target of the widely available cholesterol-lowering drugs acting as the key enzyme in cholesterol biosynthesis. Other BPs 7.16–20, isolated from Indo-Pacific Dysidea sponges, has been observed to inhibit the activity of 15-lipoxygenase with IC50 values of 1.3, 2.5, 15, 7.4, 7.4 μM, respectively (Fu et al., 1995). 15-lipoxygenase is involved in human atherosclerotic lesions. The derivatives with two phenolic hydroxyls (7.16–17) are more active than those with only one hydroxyl (7.18–20) (Fu et al., 1995). The anti-allergic activity of 3-bromo-4,5-dihydroxybenzaldehyde, 2.21 from Polysiphonia morrowii Harvey, was tested on mouse bone marrow mast cells cultured with immunoglobulin (Ig)E/bovine serum and stimulated with albumin (Han et al., 2020). Psammaplin P (7.21) was isolated from the marine sponge Aplysinella rhax and was suggested as a new precursor for synthesizing new antiparasitic agents (Oluwabusola et al., 2020).

2.8

Possible toxicological effects of BPs

BPs have several bioactivities that may be useful in defending against certain diseases. However, we should keep in mind that not all the natural BPs and their derivatives show beneficial health effects, and some of them are suspected of harming human and animal health and the environment. For example, hydroxylated polybrominated diphenyl ethers (OH-PBDEs, 8.1–8.7, Scheme 6)

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were discovered in macro marine alga Ceramium tenuicorne and blue mussels Mytilus edulis (Malmvarn et al., 2005). These PBDEs may induce endocrine disorders, neurological toxicities, and genotoxicity, and high concentrations of specific PBDE isomers may cause adverse effects in sensitive humans such as young children, indigenous peoples, and fish consumers. Besides, since they are hard to degrade, they may cause environmental pollution (He et al., 2008; Ji et al., 2011; Wit, 2002). Despite BPs showing promising cytotoxicity against various tumor cells, BPs are not selective, which is a limitation they share with current anticancer drugs. BPs are also cytotoxic to normal cell lines, such as the human embryo lung fibroblasts (HELF) (Han et al., 2005; Xu et al., 2004c), limiting their application in vivo. In addition, some synthetic BPs are toxic, disturbing cellular € ver Ca2+ signaling in neuroendocrine cells (PC12) (Hassenklo et al., 2006) and interfering with the steroidogenic pathway, reproduction, and embryo development in zebrafish (Deng n et al., 2010). Furthermore, some et al., 2010; Norman Halde BPs enhanced the risk of cardiovascular disease by inhibiting bPON1 (Akbaba et al., 2013b) and affecting the pharmacokinetics of other agents by targeting UDP-glucuronosyltransferases (Wang et al., 2020). Whether natural BPs share this toxicity needs further research.

3. Conclusions Marine organisms, mainly marine algae, are the primary source of natural BPs. The biological activities of these compounds have been extensively studied, mainly focused on anticancer, antidiabetic, anti-obesity, antioxidant, antimicrobial, anti-inflammatory, anti-neurodegenerative, and enzyme inhibitory activities. BPs will continue to enlarge the chemical library by discovering new natural BPs and developing new BP derivatives, improving the opportunity to discover new drugs. It should also be reminded that marine BPs can display unwanted toxicity to different organisms, including human beings.

Acknowledgment This research was funded by the National Key Research and Development Program of China (2017YFE0195000).

Chapter 3 Marine nature bromophenols

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Paudel, P.; Wagle, A.; Seong, S. H.; Park, H. J.; Jung, H. A.; Choi, J. S. A New Tyrosinase Inhibitor From the Red Alga Symphyocladia latiuscula (Harvey) Yamada (Rhodomelaceae). Mar. Drugs 2019c, 17 (5), 295. Paudel, P.; Park, S. E.; Seong, S. H.; Jung, H. A.; Choi, J. S. Bromophenols From Symphyocladia latiuscula Target Human Monoamine Oxidase and Dopaminergic Receptors for the Management of Neurodegenerative Diseases. J. Agric. Food Chem. 2020, 68, 2426–2436. Popov, A. M. Spongiadioxins A and B, Two New Polybrominated Dibenzo-pDioxins From an Australian Marine Sponge Dysidea dendyi. J. Nat. Prod. 2001, 64 (2), 151–153. Popplewell, W. L.; Northcote, P. T. Colensolide A: A New Nitrogenous Bromophenol From the New Zealand Marine Red Alga Osmundaria colensoi. Tetrahedron Lett. 2009, 50 (49), 6814–6817. Qi, X.; Liu, G.; Qiu, L.; Lin, X.; Liu, M. Marine Bromophenol Bis(2,3-Dibromo-4,5Dihydroxybenzyl) Ether, Represses Angiogenesis in HUVEC Cells and in Zebrafish Embryos Via Inhibiting the VEGF Signal Systems. Biomed. Pharmacother. 2015, 75, 58–66. Qin, S. G.; Tian, H. Y.; Wei, J.; Han, Z. H.; Zhang, M. J.; Hao, G. H.; Liu, X.; Pan, L. F. 3-Bromo-4,5-Dihydroxybenzaldehyde Protects Against Myocardial Ischemia and Reperfusion Injury Through the Akt-PGC1α-Sirt3 Pathway. Front. Pharmacol. 2018, 9, 722. Rajasulochana, P.; Krishnamoorthy, P.; Dhamotharan, R. Isolation, Identification of Bromophenol Compound and Antibacterial Activity of Kappaphycus sp. Int. J. Pharm. Bio. Sci. 2012, 3, 173–186. Rudi, A.; Evan, T.; Aknin, M.; Kashman, Y. Polycitone B and Prepolycitrin A: Two Novel Alkaloids From the Marine Ascidian Polycitor africanus. J. Nat. Prod. 2000, 63 (6), 832–833. Ryu, Y. S.; Fernando, P. D. S. M.; Kang, K. A.; Piao, M. J.; Zhen, A. X.; Kang, H. K.; Koh, Y. S.; Hyun, J. W. Marine Compound 3-Bromo-4,5-Dihydroxybenzaldehyde Protects Skin Cells Against Oxidative Damage Via the Nrf2/HO-1 Pathway. Mar. Drugs 2019, 17 (4), 234. Shi, D.; Xu, F.; He, J.; Li, J.; Fan, X.; Han, L. Inhibition of Bromophenols Against PTP1B and Anti-Hyperglycemic Effect of Rhodomela confervoides Extract in Diabetic Rats. Chin. Sci. Bull. 2008a, 53 (16), 2476–2479. Shi, D.; Li, J.; Guo, S.; Han, L. Antithrombotic Effect of Bromophenol, the AlgaDerived Thrombin Inhibitor. J. Biotechnol. 2008b, 136 (Suppl. 1), S579. Shi, D.; Li, J.; Guo, S.; Su, H.; Fan, X. The Antitumor Effect of Bromophenol Derivatives In Vitro and Leathesia Nana Extract In Vivo. Chinese J. Oceanol. Limnol. 2009, 27 (2), 277–282. Shi, D.; Li, X.; Li, J.; Guo, S.; Su, H.; Fan, X. Antithrombotic Effects of Bromophenol, an Alga-Derived Thrombin Inhibitor. Chinese J. Oceanol. Limnol. 2010, 28 (1), 96–98. Shi, D. Y.; Li, J.; Jiang, B.; Guo, S. J.; Su, H.; Wang, T. Bromophenols as Inhibitors of Protein Tyrosine Phosphatase 1B With Antidiabetic Properties. Bioorg. Med. Chem. Lett. 2012, 22 (8), 2827–2832. Shridhar, D. M.; Mahajan, G. B.; Kamat, V. P.; Naik, C. G.; Parab, R. R.; Thakur, N. R.; Mishra, P. D. Antibacterial Activity of 2-(20 ,40 -Dibromophenoxy)-4,6Dibromophenol From Dysidea granulosa. Mar. Drugs 2009, 7 (3), 464–471. Sun, H. H.; Paul, V. J.; Fenical, W. Avrainvilleol, a Brominated Diphenylmethane Derivative With Feeding Deterrent Properties From the Tropical Green Alga Avrainvillea longicaulis. Phytochemistry 1983, 22 (3), 743–745. Suzen, S.; Buyukbingol, E. Recent Studies of Aldose Reductase Enzyme Inhibition for Diabetic Complications. Curr. Med. Chem. 2003, 10 (15), 1329–1352.

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Utkina, N. K.; Denisenko, V. A.; Virovaya, M. V.; Scholokova, O. V.; Prokof’eva, N. G. Two New Minor Polybrominated Dibenzo-P-Dioxins From the Marine Sponge Dysidea dendyi. J. Nat. Prod. 2002, 65 (8), 1213–1215. Wang, W.; Okada, Y.; Shi, H.; Wang, Y.; Okuyama, T. Structures and Aldose Reductase Inhibitory Effects of Bromophenols From the Red Alga Symphyocladia latiuscula. J. Nat. Prod. 2005, 68 (4), 620–622. Wang, S. Y.; Wang, L. J.; Jiang, B.; Wu, N.; Li, X. Q.; Liu, S. F.; Luo, J.; Shi, D. Y. AntiAngiogenic Properties of BDDPM, A Bromophenol From Marine Red Alga Rhodomela confervoides, With Multi Receptor Tyrosine Kinase Inhibition Effects. Int. J. Mol. Sci. 2015, 16 (6), 13548–13560. Wang, F. G.; Wang, S.; Yang, K.; Liu, Y. Z.; Yang, K.; Chen, Y.; Fang, Z. Z. Inhibition of UDP-Glucuronosyltransferases (UGTs) by Bromophenols (BPs). Chemosphere 2020, 238, 124645. Whitfield, F. B.; Drew, M.; Helidoniotis, F.; Svoronos, D. Distribution of Bromophenols in Species of Marine Polychaetes and Bryozoans From Eastern Australia and the Role of Such Animals in the Flavor of Edible Ocean Fish and Prawns (Shrimp). J. Agric. Food Chem. 1999, 47 (11), 4756–4762. Wiemer, D. F.; Idler, D. D.; Fenical, W. Vidalols A and B, New Anti-Inflammatory Bromophenols From the Caribbean Marine Red Alga Vidalia obtusaloba. Experientia 1991, 47 (8), 851–853. Wijesekara, I.; Pangestuti, R.; Kim, S. K. Biological Activities and Potential Health Benefits of Sulfated Polysaccharides Derived From Marine Algae. Carbohydr. Polym. 2011, 84 (1), 14–21. Wit, C. A. D. An Overview of Brominated Flame Retardants in the Environment. Chemosphere 2002, 46 (5), 583–624. Wu, N.; Luo, J.; Jiang, B.; Wang, L.; Wang, S.; Wang, C.; Fu, C.; Li, J.; Shi, D. Marine Bromophenol Bis (2,3-Dibromo-4,5-Dihydroxy-Phenyl)-Methane Inhibits the Proliferation, Migration, and Invasion of Hepatocellular Carcinoma Cells Via Modulating Beta1-Integrin/FAK Signaling. Mar. Drugs 2015, 13 (2), 1010–1025. Xiu, L. X.; Xiao, F.; Fu, H. S.; Jie, L. Z.; Li, J. H.; Jian, G. S. A New Bromophenol From the Brown Alga Leathesia nana. Chin. Chem. Lett. 2004, 015 (6), 661–663. Xu, X. L.; Xiao, F.; Song, F. H.; Zhao, J. L.; Han, L. J.; Yang, Y. C.; Shi, J. G. Bromophenols From the Brown Alga Leathesia nana. J. Asian Nat. Prod. Res. 2004a, 6 (3), 217–221. Xu, X.; Song, F.; Wang, S.; Li, S.; Xiao, F.; Zhao, J.; Yang, Y.; Shang, S.; Yang, L.; Shi, J. Dibenzyl Bromophenols With Diverse Dimerization Patterns From the Brown Alga Leathesia nana. J. Nat. Prod. 2004b, 67 (10), 1661–1666. Xu, N.; Fan, X.; Yan, X.; Tseng, C. K. Screening Marine Algae From China for Their Antitumor Activities. J. Appl. Phycol. 2004c, 16 (6), 451–456. Xu, X. L.; Yin, L. Y.; Wang, Y. H.; Wang, S. Y.; Song, F. H. A New Bromobenzyl Methyl Sulphoxide From Marine Red Alga Symphyocladia latiuscula. Nat. Prod. Res. 2012a, 27 (8), 723–726. Xu, X.; Piggott, A. M.; Yin, L.; Capon, R. J.; Song, F. Symphyocladins A–G: Bromophenol Adducts From a Chinese Marine Red Alga, Symphyocladia latiuscula. Tetrahedron Lett. 2012b, 53 (16), 2103–2106. Xu, X.; Yin, L.; Wang, Y.; Wang, S.; Song, F. A New Bromobenzyl Methyl Sulphoxide From Marine Red Alga Symphyocladia latiuscula. Nat. Prod. Res. 2013, 27 (8), 723–726. Xu, X.; Yin, L.; Gao, J.; Gao, L.; Song, F. Antifungal Bromophenols From Marine Red Alga Symphyocladia latiuscula. Chem. Biod. 2014, 11 (5), 807–811. Xu, F.; Wang, F.; Wang, Z. H.; Lv, W. S.; Wang, W.; Wang, Y. G. Glucose Uptake Activities of Bis (2, 3-Dibromo-4, 5-Dihydroxybenzyl) Ether, A Novel Marine Natural Product From Red Alga Odonthaliacorymbifera With Protein Tyrosine Phosphatase 1B Inhibition, In Vitro and In Vivo. PLoS One 2016, 11 (1), e0147748.

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Xu, Q.; Luo, J.; Wu, N.; Zhang, R. S.; Shi, D. Y. BPN, a Marine-Derived PTP1B Inhibitor, Activates Insulin Signaling and Improves Insulin Resistance in C2C12 Myotubes. Int. J. Biol. Macromol. 2018, 106, 379–386. Yang, F.; Cai, H. H.; Feng, X. E.; Li, Q. S. A Novel Marine Halophenol Derivative Attenuates Lipopolysaccharide-Induced Inflammation in RAW264.7 Cells Via Activating Phosphoinositide 3-Kinase/Akt Pathway. Pharmacol. Rep. 2020, 72 (4), 1021–1031. Yang, F.; Cai, H.; Zhang, X.; Sun, J.; Feng, X.; Yuan, H.; Zhang, X.; Xiao, B.; Li, Q. An Active Marine Halophenol Derivative Attenuates Lipopolysaccharide-Induced Acute Liver Injury in Mice by Improving M2 Macrophage-Mediated Therapy. Int. Immunopharmacol. 2021, 96, 107676. Yao, X. S. Isolation and Characterization of Polybrominated Diphenyl Ethers as Inhibitors of Microtubule Assembly From the Marine Sponge Phyllospongia dendyi Collected at Palau. J. Nat. Prod. 2004, 67 (3), 472–474. Zhang, Y. L.; Feng, X. E.; Chang, R. R.; Zhang, L. H.; Xiao, B. G.; Li, Q. S.; Hao, X. L. Therapeutic Effects of 5,20 -Dibromo-2,40 ,50 -Trihydroxydiphenylmethanone (LM49) in an Experimental Rat Model of Acute Pyelonephritis by Immunomodulation and Anti-Inflammation. Int. Immunopharmacol. 2018, 62, 155–164. Zhang, Y.; Glukhov, E.; Yu, H.; Gerwick, L.; Dorrestein, P.; Gerwick, W. Monomeric and Dimeric Bromophenols From the Red Alga Ceramium Sp. with Antioxidant and Anti-Inflammatory Activities. Phytochemistry 2020,.

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SECTION

Extraction and purification

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Marine phenolics: Extractions at low pressure

4

Joa˜o Cotasa, Diana Pachecoa, Pedro Monteiroa, Ana M.M. Gonc¸ alvesa,b, and Leonel Pereiraa a

University of Coimbra, MARE—Marine and Environmental Sciences Centre, Department of Life Sciences, Calc¸ada Martim de Freitas, Coimbra, Portugal. b Department of Biology and CESAM, University of Aveiro, Aveiro, Portugal

1. Introduction Seaweeds are a promising source of bioactive compounds for biotechnological applications, with the potential to provide natural alternatives to a wide range of industries (Cotas et al., 2020a, 2020b; Garcı´a-Poza et al., 2020). Among bioactive algal molecules, phenolic compounds are synthesized as secondary metabolites. Ecologically, these molecules play a role on plants and seaweeds defense mechanisms (Dimitrios, 2006; Wang et al., 2009), and have demonstrated interesting health-beneficial properties for humans and animals, namely, their bioactive qualities such as antioxidant, antiviral, and antibacterial properties (Cotas et al., 2020a, 2020b). In general, phenolic chemicals are defined as molecules with hydroxylated aromatic rings (Cotas et al., 2020a, 2020b; Domı´nguez, 2013; Mekini
c et al., 2019). In seaweeds, phenolic compounds act as protective agents against biotic and abiotic stressors, such as ultraviolet radiation and herbivores (Audibert et al., 2010). In this context, seaweeds produce a variety of phenolic compounds, which differ in molecular size and chemical structure, such as phenolic acids, phlorotannins, bromophenols, flavonoids, phenolic terpenoids, and mycosporine-like amino acids (MAAs). However, concentration of these metabolites varies according to the phylum of the algal species. For instance, phlorotannins are widely present in brown seaweeds (Phaeophyceae), while phenolic acids, bromophenols, flavonoids, phenolic Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00015-7 Copyright # 2023 Elsevier Inc. All rights reserved.

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terpenoids, and MAAs seem to have a dominant presence in green (Chlorophyta) and red (Rhodophyta) seaweeds (Corona et al., 2017; Go´mez-Guzma´n et al., 2018; Heo et al., 2005; Wells et al., 2017). Seaweed phenolic compounds received much attention because of their bioactivities and wide range of health-promoting benefits (Lopes et al., 2013; Mekini
c et al., 2019; Wijesekara et al., 2011). Due to their structural similarities and tendency to react with other chemicals, phenolic compounds are extremely difficult to extract effectively on an industrial scale (Mekini
c et al., 2019). On a lab scale, highly pure extracts of these chemicals have been obtained (Fairhead et al., 2005). There are several extraction and purification methodologies that can be employed for phenolic compounds; however, these methodologies must be selected according to the seaweed species and the target compound for extraction. The most popular and widely used technique is traditional extraction, often known as solvent extraction or solid-liquid extraction (SLE). This technique can be accomplished through several methodologies, including Soxhlet refluxing, by boiling the sample and solvent with or without stirring for a set period, or maceration with constant stirring. Several solvents, such as methanol, ethanol, acetone, ethyl acetate, trichloromethane, or a mixture of these chemicals with different polarities, namely, ethanol, acetone, acetonitrile, and methanol at different mixing proportions can be utilized (Catarino et al., 2019; Getachew et al., 2020; Lopes et al., 2012; Pantidos et al., 2014; Parys et al., 2010; Vijayan et al., 2018). However, traditional extraction techniques are time-consuming, energydemanding, and use large amounts of solvent. Hence, the upscaling of these techniques is unpractical at an industrial scale, due to high costs and environmental impact (Ojha et al., 2020). As a result, different innovative and emergent extraction technologies were developed to tackle these classic method’s limitations. The emerging new techniques innovate on the extraction energy or mechanism, such as ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and enzymeassisted extraction (EAE). Nevertheless, new techniques can also innovate on extreme process conditions such as pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE). Other innovative techniques like pulsed electric field-assisted extraction (PEF) and ohmic heating are also efficient for phenolic compound extraction, but they are not commonly applied (Garcia-Vaquero et al., 2020; Rodrigues et al., 2015). Although these newly emerging technologies show potential regarding the increase of phenolic

Chapter 4 Marine phenolics: Extractions at low pressure

compound extraction efficiency, reducing extraction time, improving extract quality, and reducing hazardous waste generation and its associated environmental impact, they also face some challenges, particularly in terms of optimization and industrial application (Getachew et al., 2020). Overall, the development of new extraction techniques for phenolic compounds is a new hot topic nowadays, due to the high potential of these interesting molecules as a substitute of synthetic chemicals (Cotas et al., 2020a, 2020b; Getachew et al., 2020). In this work, we review the phenolic content found in seaweed species and explore conventional and modern extraction methodologies used to obtain these bioactive compounds.

2. Phenolic classes Seaweed phenolic compounds are metabolites chemically characterized by the presence of hydroxylated aromatic rings in the molecules (Cotas et al., 2020a, 2020b; Mekini
c et al., 2019; Swanson, 2003). These phytochemicals show a wide variety of chemical structures, ranging from simple moieties to high molecular polymers. It is assumed that the synthetic pathways for the production of these phytochemicals is through the shikimate or the acetate pathways (Bravo, 2009; Dai and Mumper, 2010; Shahidi and Naczk, 2014). Phenolic molecules are regarded by the presence of an aromatic ring with one or more hydroxyl groups. These structures have broad structural variability, ranging from simple molecules such as phenolic acids to further complex polyphenolic polymers, characterized by a wide range of molecular sizes (126–650 kDa) and chemical structures (Arnold and Targett, 2002; Bilal Hussain et al., 2019; Freile-Pelegrı´n and Robledo, 2013; Maqsood et al., 2014; Mukherjee, 2019; Rocha-Santos and Duarte, 2014; Santos et al., 2019). “Phenol” is a term for a substructure that has one phenolic hydroxyl group; catechol and resorcinol are characterized by two phenolic hydroxyl groups (benzenediols); and pyrogallol and phloroglucinol are characterized by three hydroxyl groups (benzenetriols) (Santos et al., 2019).

2.1

Phenolic acids

Phenolic acids (PA) are compounds normally found attached to other molecules like simple or complex carbohydrates, organic acids, and bioactive substances like flavonoids or terpenoids. The presence of a single phenolic acid ring, and at least a functional

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carboxylic group, is what distinguishes them. These are usually classified by the number of carbons bound to the chain, being further classified as C6-C1 for hydroxybenzoic acid (HBA; with one carbon chain linked to the phenolic ring), C6-C2 for acetophenones and phenylacetic acids (with two carbon chains linked to the phenolic ring), and C6-C3 for hydroxycinnamic acids (HCA; with three carbon chains linked to the phenolic ring). Furthermore, these acids can be split into numerous categories based on the identity, quantity, and location of the acyl residue: (1) mono-esters of caffeic, ferulic, and p-coumaric; (2) di-, tri-, and tetra-esters of caffeic acids; (3) mixed di-esters of caffeic-ferulic acid or caffeic-sinapic acids; and (4) mixed esters of caffeic acid with dibasic aliphatic acids, such as oxalic or succinic (Liwa et al., 2017; Luna-Guevara et al., 2018; Pietta et al., 2003). Cinnamic acids can also condense with molecules other than quinic acid, such as rosmarinic and malic acids, as well as aromatic amino acids and choline (Liwa et al., 2017; LunaGuevara et al., 2018; Pietta et al., 2003). Several studies show the existence of PAs in seaweeds. However, these research are scarce, and most of them focus on phenolic characterization and extraction using conventional extractions methods (Liwa et al., 2017; Luna-Guevara et al., 2018; ManciniFilho et al., 2009; Pietta et al., 2003).

2.2

Phlorotannins

Due to their intriguing bioactive capabilities, phlorotannins have received more attention than other seaweed phenolics (Arnold and Targett, 2002). Phlorotannins are oligomers of phloroglucinol, exclusive to brown seaweeds (Fig. 1). Usually located in cellular structures called physodes, these compounds have functioned as primary and secondary metabolites. Phloroglucinol is the monomeric unit of phlorotannins that are metabolized by the acetate-malonate (polyketide) pathway, which occurs in the Golgi apparatus. Phloroglucinol residues bind through C-C and/ or C-O-C residues and form polymeric molecules consisting of phloroglucinol, ranging from 10 to 100 kDa. Such molecular heterogeneity is attributed to the structural variability of the chemical linkage between phloroglucinol molecules and the hydroxyl groups. Consequently, phlorotannins can be subdivided in six groups that are defined according to the nature of the structural linkage: (1) phlorethols (aryl-ether bonds); (2) fucols (aryl-aryl bonds); (3) fucophlorethols (ether or phenyl linage); (4) eckols (dibenzo-1,4-dioxin linkages); (5) fuhalols (ortho-/para-arranged

Chapter 4 Marine phenolics: Extractions at low pressure

OH

HO

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OH

HO

HO

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OH HO

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B

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O HO

OH

O

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OH HO

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HO O

O

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F

Fig. 1 Chemical structures of phlorotannins: (A) phloroglucinol; (B) tetrafucol A; (C) tetraphlorethol B; (D) fucodiphlorethol A; (E) tetrafuhalol A; and (F) phlorofucofuroeckol.

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ether bridges containing an additional hydroxyl group on one unit); and (6) carmalols (dibenzodioxin moiety) (Achkar et al., 2005; Imbs and Zvyagintseva, 2018; Santos et al., 2019). Furthermore, within each class, monomer binding to the phloroglucinol ring can occur at various locations, resulting in structural isomers in addition to conformational isomers. As such, due to the increasing complexity of the classification of these molecules, other criteria for classification are used, for which linear phlorotannins are classified for the C-C/C-O-C oxidative couplings with two terminal phloroglucinol residues, or branched phlorotannins, bound to three or more monomers.

2.3

Bromophenols

Bromophenols are secondary metabolites involved in ecological functions of defense and deterrence, with a wide array of biological activities. These phenolic compounds were first isolated from the red algae Neorhodomela larix and were later found to be ubiquitous in all taxonomical groups of seaweeds (Liu et al., 2011; Stengel et al., 2011). The presence of phenolic groups with different degrees of bromination is what distinguishes bromophenols from other phenolic compounds. Bromination is the introduction of bromine ions in molecules and occurs through a bromoperoxidase. Bromoperoxidase activity and isolation were characterized in seaweed. Bromination of phenols through bromoperoxidases leads to the formation of bromophenols, but there is not a defined brominated phenol that leads to the formation of found bromophenols (Cotas et al., 2020a, 2020b).

2.4

Flavonoids

Flavonoids are phenolic compounds with a heterocyclic oxygen attached to two aromatic rings that vary depending on the degree of hydrogenation (Bilal Hussain et al., 2019; Mukherjee, 2019). Flavonoids such as rutin, quercetin, and hesperidin were detected in various species belonging to the phylum Chlorophyta, Rhodophyta, and Ochrophyta-Phaeophyceae (Santos et al., 2019). Some of the flavonoid compounds discovered are restricted to seaweeds, such as hesperidin, kaempferol, catechin, and quercetin (Yoshie-Stark and Hsieh, 2003). Isoflavones, such as daidzein or genistein, occur in the red seaweed Chondrus crispus and Porphyra/Pyropia spp. and in brown seaweeds such as Sargassum muticum and Sargassum vulgare. A high number of flavonoid

Chapter 4 Marine phenolics: Extractions at low pressure

glycosides have been discovered in the brown seaweed Durvillaea antarctica, Lessonia spicata, and Macrocystis pyrifera (formerly known as Macrocystis integrifolia) (Santos et al., 2019). However, studies in the seaweed flavonoid content and bioactivities are still scarce (Freile-Pelegrı´n and Robledo, 2013).

2.5

Phenolic terpenoids

Phenolic terpenoids have been identified and described in brown and red seaweeds (Stengel et al., 2011). Brown seaweed phenolic terpenoids have been identified as meroditerpenoids, supplementary distributed in plastoquinones, chromanols, and chromenes, almost exclusively found in Sargassaceae (Phaeophyceae). These meroditerpenoids are identified by a polyprenyl chain bound to a hydroquinone ring moiety (Reddy and Urban, 2009). Diterpenes and sesquiterpenes were identified and described in Rhodomelaceae (Rhodophyta), and a macrolide formation under secondary cyclization was found in the red seaweed Callophycus serratus (bromophycolides) (Lane et al., 2009).

2.6

Mycosporine-like aminoacids (MAA)

Mycosporine-like amino acids (MAA) are a clutch of UV-absorbing compounds present in Cyanobacteria, micro- and macroalgae, with the function of reducing UV-induced cellular damage (Cardozo et al., 2007; Carreto and Carignan, 2011; Llewellyn and Airs, 2010; Stengel et al., 2011; Wada et al., 2015). These compounds were first identified in fungi and assumed to have a role in UV-induced sporulation (Llewellyn and Airs, 2010). These compounds were detected in species from the phylum Rhodophyta. There is some debate on the detection of MAA’s in seaweeds belonging to green and brown seaweeds species (Stengel et al., 2011).

3. Phenolic compounds extraction methods Several studies point the bioactivities of algal phenolic compounds (Duan et al., 2007; Liu et al., 2018; Machu et al., 2015; Maqsood et al., 2013; Nwosu et al., 2011). However, as discussed above, there are various subclasses of seaweed phenolic compounds; thus, there are various extraction methods for each class, according to the molecules’ chemical structure and polarity (Cotas et al., 2020a, 2020b). Upon choosing the extraction method, target seaweed species must be determined, and the class of phenolic compound is targeted. Identifying where the compounds for extraction

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are—intracellular or extracellular—is essential to define the proper methodologies for extraction, and isolation, for further accurate assessment of their bioavailability and bioactivities (D’Archivio et al., 2010). The development of products/solutions based in seaweed phenolic compounds lies heavily in optimized practical and analytical methods (Fig. 2). Phenolic compounds’ solubility and separation capabilities are also influenced by their chemical structural variations, having an influence on their polarity level, conjugation, and interactions with the sample matrix. Because of their structural composition, high molecular weight phenolics are frequently insoluble and more stable whereas others are prone to oxidation, and are thermolabile or volatile (Alara et al., 2021; Robards, 2003).

3.1

Pre-treatment

Fresh collected seaweed biomass is generally subjected to a cleaning process, for the removal of stones, sand, epiphytes, or other impurities. After that, the algal biomass could be used fresh, dried, or freeze-dried (Stengel and Connan, 2015). Several studies evidence the advantages of freeze-drying, as it maintains the integrity of the biomolecules and allows better extraction yields (Badmus et al., 2019; Charles et al., 2020; Michalak, 2018). Milling or grinding algal biomass is advantageous, as reducing particle size increases exposure area between the algal biomass and the extraction solvent, increasing extraction yield (Michalak and Chojnacka, 2014). Usually, there is a pre-extraction process to avoid the co-extraction of pigments or fatty acids (Santos et al., 2019). Extraction with low-polar solvents such as n-hexane (Koivikko et al., 2007), n-hexane:acetone (Onofrejova´ et al., 2010), n-hexane:ethyl acetate (Sugiura et al., 2006), or dichloromethane (Hartmann et al., 2018) has shown to be effective in improving phenolic compound extraction yield. Extraction of phlorotannins from the brown seaweed Fucus vesiculosus with a pretreatment with acetone:water has demonstrated higher yields than using ethanol or methanol (7:3) (Koivikko et al., 2005).

3.2

Extraction methods

Extraction methodologies are broad and variable. Traditional extraction techniques include maceration, Soxhlet-extraction, solid-liquid, and liquid-liquid extractions. Usually, the extraction methodologies involve the utilization of organic solvents

Chapter 4 Marine phenolics: Extractions at low pressure

Fig. 2 Diagram of seaweed phenolic extraction process at low pressures.

(e.g., hexane, petroleum ether, cyclohexane, ethanol, methanol, acetone, benzene, dichloromethane, ethyl acetate, chloroform). The solvent used in the extraction process should ideally be non-toxic and inexpensive (Michalak and Chojnacka, 2014). From an industrial point of view, ethanol is favored as an extraction solvent due to its lower cost (Stengel and Connan, 2015).

3.2.1

Classical methods

Maceration is a classical method, where compounds are extracted by submerging the algal biomass in an appropriate solvent, followed by agitation at room temperature, either continuous or sporadic (Kim et al., 2013; Olejar et al., 2015). The percolation method is identical to maceration because it includes putting the milled material in a closed system and gradually dropping the solvent from the top en route to the bottom. Filtration is not necessary in percolation, since the percolator systems include filters that only allow the solvent containing the extract to pass through. The disadvantages of the percolation method approach are comparable to those with the maceration method

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(time-consuming and high solvent volumes), on top of the uncertain extraction of phenolic compounds, due to the solubility, sample size, and extraction time (Alara et al., 2021; Kaufmann and Christen, 2002; Sticher, 2008). The classical Soxhlet extraction method performed in a Soxhlet apparatus has several advantages. As it is a continuous process, the solvent can be recycled and is less time- and solvent-consuming than maceration and percolation techniques. Additionally, its simplicity in operation and the ability of process scale-up make this technique one of the most frequently used industrially (Michalak and Chojnacka, 2014; Ospina et al., 2017; Ramluckan et al., 2014). However, the extraction solvent is constantly heated at its boiling point, which can damage thermolabile compounds and compromise extraction and analysis (Freile-Pelegrı´n and Robledo, 2013). Nevertheless, these methods are not efficient and environmental friendly due to their high reliability on organic solvents (Dai and Mumper, 2010). Overall, conventional extraction usually involves the use of a large volume of solvents, and has longer extraction times and high temperatures. Such harsh extraction conditions can lead to oxidation and hydrolysis of the phenolic compounds. Moreover, the upscaling of this technology at an industrial level would be difficult, owing to practicality, energetic, economic, and environmental considerations (Ojha et al., 2020). Thus, to overcome the challenges associated with the method, several emerging extraction technologies are introduced (Getachew et al., 2020).

3.2.2

Modern extraction methods

Traditional methods employed for extracting bioactive compounds are time-consuming and have low extraction efficiencies. To overcome these disadvantages, novel technologies for extraction of biologically active compounds from marine algae have been investigated. These new techniques are microwave-assisted extraction (MAE) (Rodriguez-Jasso et al., 2011; Topuz et al., 2016), ultrasound-assisted extraction (UAE) (Ummat et al., 2020), enzymatic-assisted extraction (EAE) (Wijesinghe and Jeon, 2012), and green solvent extraction, which comprises subcritical water extraction (SWE) and ionic liquid extraction (Habeebullah et al., 2021; Kadam et al., 2015a, 2015b; Wijesinghe and Jeon, 2012). Also, there are other new techniques which are still being exploited, so there is scarce information available. These methods have evolved over time to improve the extraction efficiency and sustainability. Currently, ultrasound- and microwave-assisted extractions are low cost and feasible at

Chapter 4 Marine phenolics: Extractions at low pressure

large-scale industrial settings (Iban˜ez et al., 2012). These techniques induce a mechanical strain on the cells causing cellular membrane disruption, facilitating liberation of the target compounds to the solvent media (Vinatoru, 2001). Ultrasound-assisted extraction (UAE) Ultrasound-assisted extraction (UAE) uses acoustic cavitation to increase cell wall permeability and disrupt cell walls, reducing size particle and enhancing the surface-contact between the solvent and the target compound (Rajbhar et al., 2015). Sound waves (frequency between 20 and 2000 kHz) migrate through the medium and induce pressure variations. The generated acoustic cavitations grow and collapse and transform the sound waves in mechanical energy, which causes disruption of cellular structures and integrity (Hahn et al., 2012; Ying et al., 2011). In some cases, UAE is also employed as a pretreatment method before extraction, usually through immersion of the sample in an ultrasonic bath or through utilization of an ultrasound probe instrument (Klejdus et al., 2010). This technique is a low-cost, straightforward solution, which may be employed at small and large scale (Shirzad et al., 2017). The great advantage of this extraction or pre-treatment technique is that UAE offers a quicker and better extraction of phenolic compounds with reduced chemical degradation of the compounds, in comparison to other extraction processes (Vinatoru, 2001). Because of that, in the recent years, there is an increasing application of the UAE method to phenolic compounds from seaweeds (Dahmoune et al., 2014; Qun et al., 2017; Shirzad et al., 2017). Microwave assisted extraction (MAE) Microwave-assisted extraction (MAE) involves the utilization of microwave radiation to heat solvents in contact with a sample. Algal cell wall is highly susceptible to microwave irradiation, and rapid internal heating leads to cellular disruption, releasing target compounds to the solvent (Yuan et al., 2018). In MAE, microwaves induce the vibration of water molecules within the cell, increasing the temperature of intracellular liquids. This is followed by water evaporation that exerts pressure on the cell walls. As the cell wall breaks down, the intracellular contents are released into the medium (Hahn et al., 2012). Hydrogen bonds are disrupted in MAE, and dissolved ions increase the penetration of the solvent into the matrix, facilitating the extraction of active compounds (Routray and Orsat, 2012). In the literature, it is indicated that MAE is a more economic option compared to

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supercritical fluid extraction (Kadam et al., 2013). In the literature, this method can be used to extract polysaccharides (fucoidan), iodine, bromine, pigments (fucoxanthin), docosahexaenoic acid, phenols, phytosterols, and phytol from algae biomass (Michalak and Chojnacka, 2014). MAE and UAE can also be applied simultaneously (Cravotto et al., 2008). Both technologies used either alone or combined could significantly improve the extraction rate and yield and reduce costs compared to conventional extraction processes (Kadam et al., 2013). Enzyme-assisted extraction (EAE) Enzyme-assisted extraction (EAE) is characterized by the use of enzymes to disrupt cell wall structures and interior storage compounds stability, resulting in a release of the intracellular compounds to the medium (Wang et al., 2010). The complex structure of seaweeds cell wall, consisting of mixtures of sulfated and branched polysaccharides with several proteins and ions associated, is a challenge for the efficient extraction of bioactive contents (Domozych, 2019; Wijesinghe and Jeon, 2012). EAE has demonstrated the potential to effectively release secondary metabolites, maintain properties of bioactive extracts, and improve overall extraction yields (Gil-Cha´vez et al., 2013; Rodrigues et al., 2015). One of the advantages of this technique is the selectivity of the compounds, especially vital on fragile and unstable substances. In aqueous solutions, enzymes can transform insoluble compounds to water-soluble molecules in specific instances (Heo et al., 2005). In this case, enzymatic hydrolysate can be enriched in phenolic compounds, after that, there is a need to sub-fraction to obtain a purified extract or compound (Lee et al., 2012). For the extraction of biologically active compounds from algae biomass, application of a well-defined mixture of enzyme is necessary, since algal cell walls are chemically and structurally more heterogeneous than other cells, in specific, vascular plants (Wijesinghe and Jeon, 2012). EAE is performed under moderate conditions, which protect the biologically active compounds from their degradation (Hahn et al., 2012). The advantage of EAE is that for the algal cell disruption, common enzymes in the food industry (neutral and alkaline protease, α-amylase, cellulase, and pepsin) can be used (Liang et al., 2012; Wijesinghe and Jeon, 2012). For these enzymes, optimal experimental conditions should be determined (temperature and pH) (Liang et al., 2012). In the work

Chapter 4 Marine phenolics: Extractions at low pressure

of Kadam et al. (2015a, 2015b), a list of enzymes and their optimal conditions for EAE of bioactive compounds from marine algae is presented. EAE has shown potential to improve extraction yield, to release phenolic compounds metabolites, and to maintain bioactive properties of the extracts (Rodrigues et al., 2015). Several studies have reported a higher extractability of bioactive compounds from several brown algae and considered EAE a sustainable, solvent-free, low-cost, fast, and efficient process. Nevertheless, it must be applied in combination with another technique to isolate the phenolic compounds from the crude extract (Hardouin et al., 2014; Heo et al., 2003).

3.2.3

Green solvent extraction

With the urgent demand for sustainable technology, alternative green solvents are being highly exploited, for the replacement of conventional usage of toxic solvents. Techniques such as subcritical water extraction (SWE) and ionic liquids (ILs), in particular, have been successfully applied in several food and medical extracting methods (Du et al., 2007; Tang et al., 2012; Vo Dinh et al., 2018). Subcritical water extraction (SWE) Among these techniques, SWE has currently received much attention as an alternative extraction methodology for secondary metabolites in plants and algae (Iban˜ez et al., 2003; Ong et al., 2006; Plaza et al., 2013). Under regular conditions, water has a high polarity, not suitable for the removal of organic compounds from raw materials (Ong et al., 2006). However, under subcritical environments where the temperature and pressure are significantly increased, a significant reduction in the dielectric constant (ε) of water occurs (ε ¼ 80 at 25°C to ε ¼ 27 at 250°C and 50 bar) (Ong et al., 2006; Teo et al., 2010). This phenomenon results in a noteworthy increase in diffusivity, which allows water to act like an organic solvent and extract bioactive compounds. Overall, SWE is a clean and green process, with utilization of nonflammable and non-toxic solvents, fast reaction times and excellent extraction capacities, making SWE a preferable technique for the extraction of valuable products (Iban˜ez et al., 2003; Mustafa and Turner, 2011). Ionic liquids Ionic liquids (ILs) have recently demonstrated potential capabilities in extraction of bioactive compounds from natural resources. The physicochemical properties of IL, such as negligible

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vapor pressure, high thermal and electrochemical stability, wide range dissolving properties and strong miscibility with aqueous substances, are attractive for extracting functional bioactive compounds (Han and Row, 2010). Different techniques have already been associated with IL, such as microwave and ultrasound complex systems, liquid-liquid micro-extraction, IL-based silica’s and polymers (Lo´pez-Darias et al., 2011; Lou et al., 2012; Tang et al., 2012). These ILs are simple molten salts types that are in a liquid state at or near room temperature and have relatively significant portion of organic cations and inorganic anions. Ionic liquids provide potential benefits over conventional organic solutions, such as a low melting point, a wide range of liquid temperature, low vapor pressure, and specialized solvent characteristics, which makes them highly effective for extraction of phenolic compounds (Niu et al., 2009). In recent years, IL-based extraction methods have been explored to extract plant phenolic compounds (Cla´udio et al., 2012; Du et al., 2009; Lou et al., 2012). However, information about the ILs-based methodology for the extraction of seaweed phenolic compounds remains scarce (Getachew et al., 2020; Han et al., 2011; Martins and Ventura, 2020; Vo Dinh et al., 2018).

3.2.4

Other techniques

Other extraction techniques such as pulsed electric field extraction, centrifugal partition extraction, surfactants-mediated extraction, and extraction using green solvents similar to ILs (deep eutectic solvents) are being explored for the extraction of phenolic components from seaweeds (Getachew et al., 2020). In pulsed electric field extraction (PEF) methodology, high voltages (kV range) are utilized in short time pulses (only nanoor microseconds) with the main goal of electro-permeabilizing and disrupting seaweed cell membranes, to speed up the extraction rate (Kotnik et al., 2015). This extraction technique is widely explored in plant phenolic compounds (Ameer et al., 2017; Rocha et al., 2018; Yan et al., 2017). There is a relatively little information on the application of PEF on seaweeds; nevertheless, since this approach has been demonstrated to be efficient in the extraction of phenolic compounds from terrestrial plants, presumably it can be reproduced for extracting seaweed metabolites (Getachew et al., 2020). Centrifugal partition extraction (CPE) is a centrifugal-fieldbased multi-stage liquid-liquid extraction technique. The partition coefficients between the two liquid phases are used to extract

Chapter 4 Marine phenolics: Extractions at low pressure

the target compounds, which can be more sensible when compared with other extraction techniques (Nagaosa and Wang, 2003). This extraction technique is not new, as it is a fractioning technique already applied in the seaweed phenolic compounds isolation and purification step (Lee et al., 2013, 2014, 2017). Furthermore, Anae¨lle et al. (2013) applied this technique to extract phenolic compounds from brown seaweed Sargassum muticum and compared the yield and activity of the extracts with two green high-pressure techniques, supercritical fluid extraction (SFE), and pressurized liquid extraction (PLE). The result obtained demonstrated that CPE obtained higher yields of total phenolic content than the other two techniques. Moreover, the total phenolic compounds concentration in CPE was twice that of the PLE extract. The use of surfactants in surfactant-mediated extraction (SME) is also a potential developing method for phenolic compound extraction (Sharma et al., 2015). Surfactants can produce monomolecular layers on a liquid’s surface, lowering the interfacial tension between two liquids and enabling them to mix (forming a homogeneous solution). This could permit SME to be applied for extraction and isolation of phenolic compounds with a wide range of polarities and with simple or complex chemical struc€ mu € s¸ Yılmaz et al., 2019). Gu € mu € s¸ Yılmaz et al. (2019) comtures (Gu pared SME with EAE and PLE for the separation of total phenolic compounds and phlorotannins from Lobophora variegata, a brown seaweed. The authors reported that SME obtained more yield of total phenolics and phlorotannins than EAE and PLE. Deep eutectic solvent (DES) is a liquid extraction technique based on two or more Lewis acids and bases or Brønsted-Lowry acids and bases, showing lower freezing point than its constituents. The establishment of DES is based in the complexation of a halide salt, which serves as both a hydrogen-bond acceptor and a hydrogen-bond donor (HBD). Some DES liquids are hypothesized to have a physical structure identical to ILs. DESs are typically distinct from ILs in terms of the raw sources and the chemical synthesis process. Henceforth, the applications of their chemical features vary in many ways (Zainal-Abidin et al., 2017). DESs are already widely applied to extract plants phenolic compounds (Dai et al., 2013; Ruesgas-Ramo´n et al., 2017; Wei et al., 2015a, 2015b).

3.3

Extraction problems and future developments

As described in Table 1, there are several phenolic compounds with potential and interesting bioactivities that hold promise for applications in several industries, such as pharmaceuticals,

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Table 1 Phenolic compounds found in seaweeds, its extraction technique, and biological activity. Phenolic compounds

Species

Phylum/ class

Extraction technique

Phlorotannins

Ascophyllum nodosum

P

Solid phase

Ecklonia cava

P

E. cava

P

Avrainvillea nigricans

C

Cymopolia barbata

C

Rhodomela confervoides

R

Symphyocladia latiuscula

R

Ulva compressa (formerly known as Enteromorpha compressa), U. australis (formerly known as U. pertusa), Capsosiphon fulvescens, Chaetomorpha moniligera Ulva flexuosa, U. clathrata, Ulva linza Ulva prolifera

C

Heating (12 h, 85°C) Silica gel chromatography column Vacuum chromatography Silica gel chromatography column Chromatography on silica gel Column chromatography Heating (60°C, 2 h)

Bromophenols

Flavonoids

Alsidium corallinum

C C R

Ultrasound assisted extraction Ultrasound assisted extraction Maceration

Solvent

Bioactivities

Ref.

First extraction with methanol and then with chloroform and ultra-pure water (1:1) 50% aqueous ethanol

Anti-diabetic

Nwosu et al. (2011)

Anti-obesity

MeOH

Antiviral

Oh et al. (2019) Ahn et al. (2004)

Diethyl ether: ethyl acetate (3:1) Diethyl ether

Antimicrobial Antibacterial

Ethanol

Antitumor

Methanol

Antioxidant

Ethanol

Antioxidant

Methanol

Antioxidant

Ethanol

Anti-diabetic

Ethanol-water mixture (70:30 v/v)

Hepatoprotective

Colon et al. (1987) Estrada et al. (1987) Lijun et al. (2005) Choi et al. (2000) Cho et al. (2010)

Farasat et al. (2014) Yan et al. (2019) Ben Saad et al. (2017)

Phenolic Terpenoids

Mycosporinelike aminoacids (MAA)

Stypopodium flabelliforme

P

Gracilaria opuntia

R

Laurencia dendroidea (formerly known as Laurencia scoparia) Callophycus serratus Amphiroa crassa Gracilaria domingensis

R

Thin layer chromatography

R

Chondrus crispus, Mastocarpus stellatus, Palmaria palmata

R

Reversed-phase (C18) HPLC Maceration and heating (50  5°C, 8 h) Ultrasound assisted extraction

R, Rhodophyta; C, Chlorophyta; P, Phaeophyceae.

R

Normal phase vacuum liquid chromatography Heating (60–70°C, 3 h)

2:1 CH2Cl2/MeOH

Anticancer

Sabry et al. (2005)

Ethyl acetate: methanol (1:1)

Antioxidant; antiinflammatory Anthelmintic

Makkar and Chakraborty (2018) Davyt et al. (2001)

Antimalarial

Stout et al. (2010) Torres et al. (2018)

Three times with dichloromethane for 1 day each time MeOH and MeOH:DCM (1:1, 1:2) Methanol (10%; w/v)

Methanol

Antioxidant

Antiproliferative and antioxidant

Athukorala et al. (2016)

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Chapter 4 Marine phenolics: Extractions at low pressure

nutraceuticals, and food. Nevertheless, there are various extraction factors that have an impact in the compound bioavailability and extraction success. Optimized methods can vary according to the algae used, time of the year, season, and reproductive status of the target algae. Additionally, the correct selection of the extraction solvent, temperature, pressure, static time, and number of cycles are variables with a direct influence on the total phenolic yield and rate (Kadam et al., 2013). When applying subcritical extraction at temperatures of 100°C and 200°C, it was demonstrated that the total phenolic yield rises (Plaza et al., 2010). This phenomenon might be explained by the increase in mass transfer due to the higher solubility of the cellular membrane and due to the rise in temperature (Huie, 2002). In this study, the cited trend was verified to all the seaweeds assessed, namely, Sargassum vulgare, Sargassum muticum, Undaria pinnatifida (Phaeophyceae), Porphyra spp., and Halopithys incurva (Rhodophyta) (Plaza et al., 2010). However, high temperatures could lead to polymerization or oxidation of some phenolic substances. In some cases, a combination of various extraction procedures may be required to enhance the seaweed phenolic extraction. This happens when a single extraction technique is not enough to effectively extract the phenolic compounds from the seaweed biomass. As a result, assimilation of multiple extraction techniques can be applied to have greater efficiency, as described before. However, the integration of various methodologies is still in the beginning. In the actual mindset, there is a need for “green” methodologies. As such, it is necessary to have more studies and research to choose and understand the most prominent extraction techniques for the recovery of high yield and quality seaweed phenolic compounds, in a sustainable way.

4. Purification, quantification, and characterization Following the extraction process is necessary to proceed with the isolation, characterization, and quantification of the target compound. Several methodologies can be applied, according to the targeted compound to isolate. The study of the extracted phenolic compounds is affected by their raw source, extraction and purification techniques employed, sample particle size, storage conditions, and the presence of interfering substances in extracts, such as fatty acids or pigments (Shahidi and Naczk, 2014).

Chapter 4 Marine phenolics: Extractions at low pressure

Typically, the quantification of phenolic content is performed by colorimetric methods, namely, Folin-Ciocalteu, Folin-Denis, or Prussian blue assays (Mekini
c et al., 2019). The most widely used assay for phenolic compounds’ valuation is Folin-Ciocalteu—the redox reaction with the reagent Folin-Ciocalteu—that allows the spectrophotometric quantification assessment of phenolic compounds. However, this technique has some limitations, such as the interference of non-phenolic-reducing compounds (Shahidi and Naczk, 2014). Currently, isolation of phenolic compounds is achieved through preparative chromatography techniques, such as column chromatography, high-performance liquid chromatography (HPLC), reversed-phase high-performance liquid chromatography (RPHPLC), or thin-layer chromatography (TLC). Furthermore, these chromatographic methods have evolved in order to be employed for separation, isolation, purification, identification, and quantification of different phenolic compounds (Mekini
c et al., 2019). HPLC is an automated analytical methodology and allows separation, purification, and characterization of a wide range of ˇ uvela et al., 2019). It presents several advanchemical samples (Z tages due to its speed, low amount of extract sample requirements, and the user friendliness of the operating equipment (Santos et al., 2019). HPLC research reports that by this technique it is possible to identify, isolate, and quantify nine different phenolic compounds (gallic acid, 4-hydroxybenzoic acid, catechin hydrate, epicatechin, catechin gallate, epicatechin gallate, epigallocatechin, epigallocatechin gallate, pyrocatechol) in brown edible seaweeds (Phaeophyceae)—Eisenia bicyclis (formerly Eisenia arborea f. bicyclis), Sargassum fusiforme (formerly Hizikia fusiformis), Saccharina japonica (formerly Laminaria japonica), Undaria pinnatifida—and in red edible seaweeds (Rhodophyta)—Palmaria palmata and Neopyropia tenera (formerly Porphyra tenera) (Machu et al., 2015). RP-HPLC, in which the analysis requires a non-polar stationary ˇ uvela et al., 2019), phase and a polar hydro-organic mobile phase (Z is a technique that increases the compounds retention using the hydrophobicity of the solutes, hydrophobicity of the stationary phase, and/or with the polarity of the mobile phase (Moll, 1969; Snyder, 1974). In this technique, the separation of compounds is accomplished through the partitioning process and the adsorption of the compounds (Rafferty et al., 2007). For example, a study shown with the red seaweed Rhodomela confervoides, RP-HPLC allowed to identify and characterize bromophenols such as 3-bromo-4,5-dihydroxy benzoic acid methyl ester and 3-bromo-4,5-dihydroxy-benzaldehyde (Lijun et al., 2005).

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Thin-layer chromatography (TLC) is a methodology for compound identification and isolation, in which the stationary phase is a layer of a fine-particle sorbent, usually silica. This layer is placed on a closed chamber and the extract sample is applied in the lower side of the layer. Inside this chamber is the mobile phase, characterized by a mixture of solvents that differ according to the target compounds and their characteristic polarity. The distance covered by the compound is then marked for the calculation of the retention factor (Rf ), allowing the compound identification (Sherma and Fried, 2005). In fact, using this method in a dichloromethane/methanol/water (65:35:10, v/v/v) solvent system, researchers isolated phlorotannins (phlorofucofuroeckol, dieckol, and dioxinodehydroeckol) from Ecklonia cava subsp. stolonifera (formerly Ecklonia stolonifera) (Phaeophyceae) (Kim et al., 2009). However, the lack of sensitive processes to separate phenolic compounds with different molecular weight and its isomers leads to the coupling of liquid or gas chromatography and mass spectrometry, enabling a better characterization of phenolic compounds (Ge et al., 2020; Imbs and Zvyagintseva, 2018). For instance, gas chromatography-mass spectrometry (GC-MS) permitted the identification of coumarin and flavones on crude extracts of Padina tetrastromatica (Phaeophyceae) (Maheswari et al., 2018). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is an alternative procedure for the identification and structural characterization of biomolecules. This approach has been used in the past to identify the presence of phloroglucinol derived from the brown seaweed Sargassum wightii (Karthik et al., 2016). Recently, quantitative nuclear magnetic resonance (qNMR) has been effective to identify and quantify several metabolites (Nerantzaki et al., 2011; Pauli et al., 2005). In general, NMR spectrum is derived by measuring Fourier transformed signals and converting them to radio-frequency impulses. As a result, NMR has a lower mass sensitivity than other spectroscopic approaches € mich and Singh, 2018). (Blu

5. Conclusions This chapter reviews seaweed phenolic compounds, conventional extraction methodologies, and low-pressure extraction methodologies for the extraction of seaweed phenolic compounds. The widely applied conventional methods such as Soxhlet and maceration can be scaled up easily. However, they have

Chapter 4 Marine phenolics: Extractions at low pressure

shown several limitations: (i) limited extraction yields; (ii) high use of extraction solvents (some of them are toxic, harmful, or noxious); (iii) long extraction times; and (iv) high amounts of extraction residues. Thus, currently, there is an active search for new extraction methods, which demonstrated promising results at laboratory and pilot scale; however, they are still under development for extracting seaweed phenolic compounds. For these novel extraction methods, it is necessary to analyze their efficacy and efficiency to ensure high recovery yields and high-quality extracts. Establishing first which target compound to extract is important to choose the correct methodology to achieve high recoveries of the target compounds. To confirm the results obtained with these novel extraction techniques, adequate characterization methods should be applied.

Acknowledgments This work was financed by national funds through the FCT—Foundation for Science and Technology, I.P., within the scope of the projects UIDB/04292/2020 granted to MARE—Marine and Environmental Sciences Centre and UIDP/ 50017/2020 + UIDB/50017/2020 (by FCT/MTCES) granted to CESAM—Centre for Environmental and Marine Studies. Joa˜o Cotas thanks to the European Regional Development Fund through the Interreg Atlantic Area Program, under the project NASPA 523 (EAPA_451/2016). Diana Pacheco thanks to PTDC/BIA-CBI/ 31144/2017-POCI-01 project-0145-FEDER-031144-MARINE INVADERS, co-financed by the ERDF through POCI (Operational Program Competitiveness and Internationalization) and by the Foundation for Science and Technology (FCT, IP). Pedro Monteiro thanks to the project MENU—Marine Macroalgae: Alternative recipes for a daily nutritional diet (FA_05_2017_011) funded by the Blue Fund under Public Notice No. 5—Blue Biotechnology which co-financed this research. Ana M. M. Gonc¸alves acknowledges University of Coimbra for the contract IT057-18-7253.

Conflict of interest The authors declare no conflict of interest.

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Further reading Shimadzu. Principles of MALDI-TOF Mass Spectrometry: SHIMADZU (Shimadzu Corporation); 2020. https://www.shimadzu.com/an/lifescience/maldi/ princpl1.html (accessed 2020–04 -14).

Extraction of marine phenolics using compressed fluids

5

Lidia Monteroa,b, Ba´rbara Socas-Rodrı´guezc, Jose Antonio Mendiolac, and Elena Iba´n˜ezc a

Applied Analytical Chemistry, University of Duisburg-Essen, Essen, Germany. Teaching and Research Center for Separation, University of Duisburg-Essen, Essen, Germany. cLaboratory of Foodomics, Institute of Food Science Research (CIAL, CSIC-UAM), Madrid, Spain b

1. Introduction Phenolic compounds are secondary metabolites commonly found in terrestrial sources like vegetables, fruits, herbs, and microorganisms; however, marine organisms, particularly macro- and microalgae, are considered a novel source of these interesting compounds. There are several theories about the role that phenolic compounds play in these organisms, but the most accepted one is related to their protective function against biotic and abiotic stresses that occur in the competitive and harsh marine environment. These stresses are mainly UV radiation, scarcity of nutrients, predators, or extreme environmental changes. Under these situations, marine organisms like macro- and microalgae produce phenolic compounds as the defense, with UV protection and antioxidant activities (Getachew et al., 2020; Jimenez-Lopez et al., 2021). Consequently, the interest in marine organisms is increasing, since they are a novel source of bioactive compounds for nutritional and medicinal applications (Silva et al., 2020). Phenolics found in macroalgae can be classified into simple and polymeric phenolic compounds. The most common simple phenolics are phenolic acids like gallic acid, protocatechuic acid, benzoic acid, cinnamic acid, hydroxybenzoic acid, caffeic acid, p-coumaric acid, chlorogenic acid, and sinapic acid. Additionally, macroalgae contain different families of flavonoids such as flavanols, flavones, isoflavones, anthocyanins, flavanones, and flavan3-ols, particularly catechin, epicatechin, epigallocatechin, and Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00007-8 Copyright # 2023 Elsevier Inc. All rights reserved.

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catechin gallate. Besides, brominated compounds called bromophenols are also typical of macroalgae, especially red and green algae (Getachew et al., 2020; Jimenez-Lopez et al., 2021). Polymeric phenolic compounds are the most abundant phenolics in macroalgae. These polymeric compounds are called phlorotannins and their structure consists of the union of several phloroglucinol units. Depending on how the phloroglucinol moieties are linked to each other, phlorotannins are classified into phlorethols (ether bonds), fucols (phenyl bonds), fucophlorethols (ether and phenyl linkages), fuhalols (ether bonds plus additional hydroxyl groups), and eckols and carmalols (dibenzodioxin linkage). Due to the enormous variety of possible bonds and polymerization degrees (126 Da–650 kDa) of these compounds, their chemical structure is complex and heterogeneous. Interestingly, phlorotannins have been exclusively described in brown algae, providing the highest total phenolic content (TPC) among macroalgae and, therefore, the highest antioxidant activity (Jimenez-Lopez et al., 2021; Mateos et al., 2020; Silva et al., 2020). The antioxidant activity of microalgae has also been correlated to the phenolic compound content (Gam et al., 2020; Goiris et al., 2012; Haoujar et al., 2019; Jerez-Martel et al., 2017; Safafar et al., 2015). In these organisms, only simple phenolic compounds have been reported, mostly phenolic acids and derivatives (gallic acid, protocatechuic acid, syringic acid, caffeic acid, chlorogenic acid, feruloylglucaric acid, p-coumaroyl tyrosine, or caffeoylcoumaroyl-quinic acid) and flavonoids such as catechin, epicatechin, kaempferol, apigenin-rutinoside, quercetin, and rhamnosyl hexosyl-methyl-quercetin (Haoujar et al., 2019; Jerez-Martel et al., 2017). Seagrasses are also marine organisms containing phenolic compounds but have been less explored. Some phenolic compounds found in seagrasses are rosmarinic acid, caffeic acid, and chicoric acid (Mateos et al., 2020). From the human health point of view, the interest in the phenolic compounds found in marine organisms is due to the enormous potential functional activities associated with them. Phenolic compounds are antioxidant agents and this free radical scavenging property is related to the protection of several diseases. In particular, phenolic compounds from algae have been tested as anti-inflammatory, antitumoral, hypocholesterolemic, anticoagulant, antiviral, antimicrobial, antidiabetic, and antiobesity agents (Getachew et al., 2020; Jimenez-Lopez et al., 2021; Montero et al., 2018a). Regarding the nutritional composition of macro- and microalgae, they are complex organisms whose main compounds are polysaccharides, proteins, lipids, minerals, and vitamins.

Chapter 5 Extraction of marine phenolics using compressed fluids

Phenolic compounds are minor constituents of these organisms (Silva et al., 2020); consequently, their efficient extraction is challenging. The extraction process is a crucial step to obtain a fraction with high phenolic content and high quality. Efficient extraction of phenolic compounds from algae is required to determine their chemical composition and associated biological properties and commercially to produce foods, pharmaceutics, and cosmetics (Getachew et al., 2020). Traditionally, the extraction of phenolic compounds from algae has been carried out using conventional extractions techniques like solid-liquid extraction (SLE) and liquid-liquid extraction (LLE). SLE involves different extraction methods like Soxhlet extraction, hydrodistillation, maceration, decoction, infusion, pressing, or percolation (Grosso et al., 2015; Sosa-Herna´ndez et al., 2018). These methods are based on the use of organic solvents like methanol, ethanol, acetone or ethyl acetate, and water or aqueous-organic mixtures to increase the polarity of the recovered compounds (Getachew et al., 2020). In general, in SLE, different sequential processes happen during the extraction. First, the solvent should penetrate in the matrix, then, the compounds of interest should be solubilized in the solvent and transported from the solute to the outside of the matrix; then, the extracted compounds should migrate from the surface of the matrix into the bulk solution. Finally, separating the extract from the residue by filtration, decantation, or clarification is necessary (Grosso et al., 2015). This process is slow and, in general, involves using large amounts of organic solvents. Besides, these SLE usually require high temperatures and long extraction times, producing degradation of the phenolic compounds. On the other hand, LLE is a method commonly used to purify phenolic compounds from a liquid extract of the algae that usually contains lipids, proteins, and polysaccharides that disrupt the analysis of the phenolic fraction. In this method, the raw extract is partitioned with different non-miscible solvents, usually petroleum ether, hexane, cyclohexane, isooctane, toluene, benzene, diethyl ether, dichloromethane, isopropanol, or chloroform for the removal of lipids (Shanmugam et al., 2021), and ethanol and acetone for the precipitation of proteins and carbohydrates, respectively (Montero et al., 2016). In general, these methods have several drawbacks like high costs and environmental impact due to the need for large amounts of solvent (that require evaporation) and long extraction times. Additionally, the compounds of interest suffer degradation and hydrolysis; it is difficult to separate the extract from the residue, and the selectivity and yields are low (Getachew et al., 2020; Shanmugam et al., 2021; Sosa-Herna´ndez et al., 2018).

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Novel extraction techniques are required to reduce the environmental impact and the drawbacks mentioned above, providing enhanced efficiency in costs, time, extraction yield, and selectivity. Equally important is the application of generally recognized as safe (GRAS) or bio-based solvents. Therefore, traditional extraction techniques are being substituted by emergent processes like enzyme-assisted extraction (EAE), microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), compressed fluids extraction techniques such as supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE), as well as the extraction with deep eutectic solvents and ionic liquids (ILs) (Getachew et al., 2020; Grosso et al., 2015; Herrero et al., 2013a; Kadam et al., 2013). Among all these emerging techniques, this chapter is focused on those applying compressed fluids, i.e., supercritical fluid extraction (SFE), gas expanded liquid extraction (GXL), and pressurized liquid extraction (PLE), to isolate phenolic compounds from marine organisms.

2. Supercritical fluid extraction (SFE) 2.1 Theoretical and practical fundamentals of SFE Among the different emerging technologies for the extraction of marine bioactive compounds, supercritical fluid extraction (SFE) has played an important role because of its green and sustainable characteristics as well as the effectiveness achieved in the extraction of this kind of compounds. SFE, developed by Hannay and Hogarth in 1880 (Hannay and Hogarth, 1880), is based on the application of an extraction solvent under supercritical conditions, that is, at a temperature and pressure above its critical point, at which the fluid shares the characteristics of liquids and gases at the same time. Under these conditions, the physicochemical properties of any fluid change significantly, presenting a density similar to a liquid, whereas its viscosity is similar to a gas. Other aspects such as diffusivity, dielectric constant, or surface tension are also modified. Additionally, such properties can be customized by modifying the temperature and pressure within the supercritical region, which change smoothly without abrupt modifications (Getachew et al., 2020). Despite the difficulties that could involve reaching the critical point of solvents, the required equipment for SFE is not particularly complex, although suitable materials are essential to withstand the high pressures required. As can be seen in Fig. 1, the

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Fig. 1 Typical diagram of the equipment used for SFE, GXL, and PLE to extract marine phenolics and other bioactive products. Reprinted with permission from Springer from Bueno, M.; Gallego, R.; Mendiola, J. A.; Iba´n˜ez, E., Downstream Green Processes for Recovery of Bioactives from Algae. In Grand Challenges in Algae Biotechnology; Springer, 2019; pp. 399–425.

equipment is constituted by (i) a solvent source; (ii) a highpressure pump to reach the critical point: this pump can be preceded by a cooler to bring the gaseous components into the liquid state; (iii) a heat exchanger, (iv) an extraction chamber, where pressure is established and maintained by a backpressure regulator or restrictor located at the exit; and (v) a vial to collect the final extract. Regarding the extraction mode, solid samples can be extracted in dynamic or static modes or even using a combination of both. Under static conditions, the supercritical fluid is placed in the extraction vessel and kept in contact with the sample for a given time; then, the extract is released through the backpressure regulator to the vial or trapping vessel. On the other hand, in the dynamic process, the supercritical fluid, obtained by mixing carbon dioxide with the selected modifier, continuously enters the extraction cell, flowing through the sample to the collectors. When both methods are combined, a static extraction is applied for a

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given time, followed by a dynamic extraction; an additional pump is necessary for the dynamic extraction to drive the co-solvent or modifier (Belwal et al., 2020). The main advantage of using supercritical fluids as extraction agents is the increase of the mass transfer due to the low viscosity and liquid-like solvating characteristics. These properties allow the supercritical fluid to penetrate the matrix, which is not easily accessible for liquids. In this sense, it is essential to know the behavior of the selected extraction agent and to control the extraction parameters to increase the extraction efficiency and reduce the cost and solvents consumption (Getachew et al., 2020). Many solvents used as supercritical fluids have been intended for specific applications; however, most of them are highly reactive, toxic, corrosive, and environmentally questionable (Padrela et al., 2018). In this sense, the most commonly used extraction solvent in SFE is supercritical carbon dioxide (ScCO2) since it presents low critical temperature (31.1°C) and low critical pressure (72.8 bar), which allows reaching the supercritical state easily. In addition, this solvent is inexpensive, non-toxic, non-flammable, recyclable, available at high purity, and easily removable after extraction. Hence, ScCO2 is a food-grade and green chemistry solvent applicable in food and pharmaceutical processes (Belwal et al., 2020; Zhou et al., 2021). CO2 is a linear molecule without a net dipole; that is, why ScCO2 is ideal for the extraction of non-polar compounds whereas for polar substances, such as phenolic compounds, the addition of co-solvents is usually necessary (Zhou et al., 2021). Nevertheless, it should be remarked that thanks to its capacity to establish specific acid/base, dispersion, induced dipole or quadrupole solute-solvent interactions, as well as considering its low molecular weight, CO2 is also able to dissolve low-polar compounds to some extent (Padrela et al., 2018). Operational parameters such as extraction temperature, pressure, and solvent flow rate can also affect the extraction efficiency, and as previously indicated, they should be optimized depending on the matrix and the target analytes in each specific study. The increment of the pressure increases the solvent density, raising the solvating power and improving the extraction efficiency. Increasing pressures lead to an exponential increment of the solubility around the critical point (higher densities). However, if the pressure is too high, it could compact the extraction bed and decrease the diffusivity, reducing the efficiency of the process. As a general rule, a component with high vapor pressure has higher solubility in a supercritical medium. Other important aspects influencing the solubility of components in the SCF are polarity and molecular weight. In SFE using CO2, the component solubility is lowered as the polarity, or the

Chapter 5 Extraction of marine phenolics using compressed fluids

molecular weights of the solutes are increased. The effect of temperature is more complex and should be carefully tuned for each solute or mixture. When the temperature increases (at constant pressure), two opposite effects occur: the CO2 solvent power is reduced (by decreasing the density), and the solutes vapor pressures increase, facilitating their transfer to the supercritical phase. The net effect on solubility in the supercritical solvent depends on the operating pressure. Near the critical pressure, the effect of the fluid density is predominant; thus, a moderate increase in temperature leads to a significant decrease in the fluid density and, therefore, to a decrease in solubility. At high pressures, the increase in the vapor pressure prevails; thus, the solubility increases with temperature, resulting in the retrograde behavior of the solubility. These aspects are specific for each solute; when a mixture of compounds needs to be extracted, such trends should be thoroughly evaluated to reach a compromise. The influence of the flow rate should also be highlighted. An increase of the solvent flow rate enhances mass transfer and decreases extraction time; however, too high a flow rate can create channels inside the matrix and reduce the extraction effectiveness of the solvent (Getachew et al., 2020). Based on the discussion above, it can be inferred that a careful evaluation of each specific application is essential to achieve adequate extraction conditions. The non-polar nature of ScCO2 makes its application even more complex for the extraction of phenolic compounds since polar co-solvents or modifiers such as ethanol are required in most cases, as it has been widely described in the literature (Getachew et al., 2020).

2.2 Phenolic compounds extraction using SFE Due to the above advantages, SFE has also been applied to extract phenolic compounds from marine products, including macro- and microalgae in marine media. This method is preferred over conventional procedures since it uses generally recognized as safe (GRAS) solvents, with low or no toxicity, providing reduced extraction times and avoiding compounds degradation. However, as shown in Table 1, the number of applications is reduced compared to other green techniques due to the low efficiency of SFE to extract polar compounds.

2.2.1 Macroalgae Macroalgae constitute the most widely studied marine matrix for the extraction of phenolic compounds using SFE, including different species such as brown seaweeds (Phaeophyta) like

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Table 1 Some remarkable examples of the extraction of phenolic compounds from marine sources using SFE.

Algae Macroalgae

Undaria pinnatifida (brown seaweed) Sargassum muticum (brown seaweed) Sargassum muticum (brown seaweed) Saccharina japonica and Sargassum horneri (red seaweed) Gracilaria mammillaris (red seaweed) Undaria pinnatifida (brown seaweed)

Phenolic compound

Extraction solvent

SFE Extraction time and Extraction number temperature Pressure of cycles

Extraction yield

Bioactivity

ScCO2 + 3% of ethanol as cosolvent ScCO2 + 12% of ethanol as cosolvent Total phenolic ScCO2 + 10% content of ethanol as cosolvent Total phenolic ScCO2 + 1 mL/min content of ethanol as cosolvent

49.85°C

30 MPa

50 min

5% dry weight

60°C

15.2 MPa

90 min

3.45% dry weight

Antioxidant activity

50°C

20 MPa

40 min

4% dry weight

Antioxidant activity

45°C

25 MPa

2h

Total phenolic ScCO2 + 8% content of ethanol as cosolvent Phenolic, ScCO2 + ethanol compounds as cosolvent and gallic acid (see comments)

60°C

30 MPa

240 min

40°C

27.58 MPa 60 min (static extraction) +10 min (dynamic extraction)

Total polyphenol content Total phenolic content

1%–1.25% (w/w)

Reference Roh et al. (2008)

Antioxidant, antibacterial, antihypertensive activity 3.03% (w/w)

Comment

Antioxidant activity

Comparison with SLE, CPE and PLE was carried out Comparison with conventional solvent extraction Comparison with conventional solvent extraction

Anae¨lle et al. (2013)

Comparison with Soxhlet extraction

Sivagnanam et al. (2015)

5 mL of ethanol as cosolvents (static extraction) + 4.5 mL of ethanol (dynamin elution). Comparison with conventional extraction

Ospina et al. (2017)

Conde et al. (2015) Kumar et al. (2020)

Microalgae

Sargassum wightii and Turbinaria conoides (brown seaweed) Phormidium valderianum

Isochrysis galbana

Total phenolic ScCO2 + 6% content of ethanol as cosolvent

50°C

25 MPa

1h

Total phenolic content

50°C

50 MPa

48°C

34.8 MPa

60 min (static extraction) +30 min (dynamic extraction) 2h

80°C

20 MPa

6h

Total phenolic ScCO2 + 8% of content ethanol as co-solvent Aurantiochytrium Total phenolic sp. content

Antioxidant activity

Comparison with conventional extraction

Yin et al. (2019)

3.97% (w/w)

Antioxidant activity

Comparison with Soxhlet extraction

Chatterjee and Bhattacharjee (2014)

4.67% (w/w)

Antioxidant activity

Optimization by BBD

13.4% (w/w)

Antioxidant activity

Eluate was collected in a vial containing ethanol. Comparison with Soxhlet extraction.

RuizDomı´nguez et al. (2020) De Melo et al. (2020)

BBD, Box-Behnken; CPE, centrifugal partition extraction; PLE, pressurized liquid extraction; ScCO2, supercritical carbon dioxide; SLE, solid liquid extraction.

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Undaria pinnatifida (Roh et al., 2008), Sargassum muticum (Anae¨lle et al., 2013; Conde et al., 2015), Sargassum wightii and Turbinaria conoides (Kumar et al., 2020), or Saccharina japonica and Sargassum horneri (Sivagnanam et al., 2015), as well as red seaweeds (Rhodophyta) like Gracilaria mammillaris (Ospina et al., 2017). Although marine seaweeds are exposed to marine stress conditions including abrupt temperature changes, radiation, bacterial infection, desiccation, osmotic stress or even grazing, epiphytism, or inter- and intra-communication, they present exceptional stability against oxidation since they synthesize and reserve different protective compounds in their bodies. These algae produce and accumulate phenolic acids, mostly phlorotannins, secondary defense metabolites with different structures and polarities. The antioxidant properties of these compounds make them ideal natural antioxidants to be used in different foods to replace the usually applied synthetic compounds, which are highly volatile, thermally unstable, and cancerigenous (Anae¨lle et al., 2013; Ospina et al., 2017). Especially remarkable is the application of red seaweeds with this purpose. Ospina et al. (2017) carried out an interesting evaluation of the antioxidant capacity of the Colombian red seaweed, Gracilaria mammillaris, using SFE. Different extraction conditions were evaluated applying a central composite design in which extraction pressure was tested in the range of 10–30 MPa, temperature between 40°C and 60°C, and the percentage of co-solvent (ethanol) in the range of 2%–8%, using in all cases, an extraction time of 240 min. Results were tested based not only on the total phenolic content and antioxidant capacity but also on the total content of carotenoids. The total content of phenolic compounds, evaluated by a modified Folin-Ciocalteu procedure, indicates that the % of co-solvent was the most significant parameter followed by pressure and temperature; the interaction between factors did not seem to be statistically significant. Similar results were found when extraction yield was evaluated. The best results were obtained when 30°C, 30 MPa, and 8% of ethanol were applied, reaching a yield of 3.03% (w/w) and total phenolic content of 3.79 mg gallic acid equivalents (GAE)/g. This data suggest that the increase of the co-solvent favors the extraction capacity of SFE toward these compounds, although yields obtained were still lower than those achieved using Soxhlet at reduced pressure (two times higher than SFE under optimal conditions). However, it is worth mentioning that Soxhlet extract did not provide the highest content of phenolic compounds, and, in addition, the extract showed low protection against oxidation. Moreover, SFE under optimal conditions

Chapter 5 Extraction of marine phenolics using compressed fluids

did not show significant differences in terms of antioxidant activity compared to the synthetic antioxidant 3,5-di-tert-butyl4-hydroxytoluene (BHT). As previously indicated, brown seaweeds (Phaeophyta) have been the type of macroalgae most evaluated by SFE. The recognized antioxidant, antimicrobial, antiviral, antidiabetic, antiinflammatory, antiviral, and antitumor properties of these marine resources make them attractive matrices for their use in food, pharmaceutical, or biotechnology (Kumar et al., 2020; Yin et al., 2019), although the revalorization of macroalgae waste has also been studied using SFE (Yin et al., 2019). Regarding the application of SFE for the extraction of brown seaweeds, different strategies have been applied with temperatures in the range of 40–60°C, pressures between 15.2 and 30 MPa, percentages of ethanol as co-solvent between 3% and 12% and times ranging from 10 to 120 min, using dynamic or combinations of dynamic and static extraction. Yin et al. (2019) demonstrated the suitability of such a combination to extract phenolic and other bioactive compounds from Undaria pinnatifida and compared the results obtained with the application of conventional extraction using acetone, water, ethanol, methanol, and mixtures. With this aim, 5 g of lyophilized algae were located inside the extraction cell together with 5 mL of ethanol, and static SFE was performed during 60 min at 27.58 MPa and 40°C, respectively. After that, the outlet valve was opened and the dynamic extraction was performed for 10 min pumping the ScCO2 at 1 mL/min to maintain the same conditions of pressure and temperature, and the extract was collected in a 4.5 mL ethanol trap by bubbling the CO2 stream. The total phenolic content obtained with SFE was slightly lower than that obtained with acetone but higher than that obtained with the other solvents. The best results were achieved using conventional extraction with methanol, acetone, and ethanol. However, it should be remarked that those differences were lower than 20 mg GAE/g whereas the extraction time was considerably shorter for SFE (70 min against 24 h) and the sample/organic solvent ratio was 10 times higher in conventional extractions. Another study using Undaria pinnatifida was developed by Roh et al. (2008); in this work, a simpler SFE was performed using the dynamic mode for 50 min at 49.85°C and 30 MPa. High extraction yields (around 5% (w/w)) were achieved, highlighting the suitability of SFE compared to conventional procedures in terms of sustainability and effectiveness. The advantages of SFE over conventional extractions (with organic and GRAS solvents) have also been demonstrated with other seaweed species (Conde et al., 2015; Kumar et al., 2020; Sivagnanam et al., 2015). Particularly remarkable is the work

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developed by Kumar et al. (2020) evaluating the extraction efficiency of SFE using ScCO2 with 6% of ethanol as co-solvent and comparing them with solid-liquid extraction using water and mixtures with 60% and 40% of ethanol for the extraction of phenolic and other bioactive compounds from two different marine brown seaweeds (Sargassum wightii and Turbinaria conoides). Conventional extractions were performed using 5 g of powdered seaweed and 100 mL of solvent by stirring for 1 day at 650 rpm. SFE was carried out using 100 g of macroalgae, a ScCO2 flow of 30 g/min, 6% ethanol, 50°C, and 25 MPa for 1 h. Results were compared in terms of total phenolic content and antioxidant activity, among others. Data associated with the total phenolic compounds showed the highest values when SFE was applied to Turbinaria conoides, which agrees with previous studies. However, it should be highlighted that the phenolic content obtained with this method was considerably higher than a previous study reported by Sivagnanam et al. (2015) in which other species, Saccharina japonica and Saccharina horneri, were evaluated using similar conditions. Kumar et al. explained such differences in terms of the different chemical composition of each species. The highest antioxidant activity was found when conventional extraction with 40% of ethanol was applied. These results point out the strong influence of the solvent nature on the extraction of the target polar compounds since the solvent with the highest water content provided the best results. Comparison with other green extraction techniques has been carried out by Anae¨lle et al. (2013) that compared the extraction of phenolic compounds from Sargassum muticum using (i) SFE with ScCO2 and ethanol as co-solvent; (ii) conventional extraction using mixtures of hexane/water, ethanol/water, and ethyl acetate/water; (iii) centrifugal partition extraction (CPE) with ethyl acetate/water (5/5, v/v); and (iv) pressurized liquid extraction (PLE) with ethanol/water at different compositions. The higher total phenolic contents were found when the ethyl acetate/water mixtures were applied in both conventional and CPE (highest content). On the contrary, lower contents were obtained with the less polar mixtures, hexane/ethanol in the classical method, and ScCO2-ethanol in SFE. This study highlights the relevance of the solvent nature on the extraction efficiency and points out the most significant limitation of the application of SFE for the extraction of phenolic compounds from natural resources. This fact necessitates further studies to improve yields and harness the environmental advantages of SFE.

Chapter 5 Extraction of marine phenolics using compressed fluids

2.2.2 Microalgae As occurs with macroalgae, microalgae are considered a valuable source of bioactive compounds for the food and pharmaceutical industries. The potential of these bioactives on the prevention of numerous widely extended diseases has increased the interest in the study of microalgae. The evaluation of antioxidants activity and phenolic compounds has played a notable role due to the known benefits that these types of compounds provide to human health (Chatterjee and Bhattacharjee, 2014). Applications of SFE to marine microalgae extractions are less documented than macroalgae since they are less varied and abundant. SFE using GRAS solvents in microalgae extractions, an alternative to conventional procedures, avoids toxic solvents and produces safer extracts that can be added to food and pharmaceutical products without further and more complex treatments. Besides, the solvent can be easily removed at the end of the procedure, reducing the required operating steps (Chatterjee and Bhattacharjee, 2014). SFE has been used to extract phenolic compounds from valuable marine microalgae species such as Phormidium valderianum (Chatterjee and Bhattacharjee, 2014), Isochrysis galbana (Ruiz-Domı´nguez et al., 2020), and Aurantiochytrium sp. (De Melo et al., 2020), among others (Table 1). In general terms, the conditions applied do not significantly differ from those employed for the extraction of macroalgae. Temperatures in the range of 48–80°C and pressures between 20 and 50 MPa have been utilized. Higher extraction times were necessary for these applications, with periods around 1.5–6 h. No co-solvent was used (Chatterjee and Bhattacharjee, 2014; De Melo et al., 2020) due to the specific influence of each matrix and its interaction with the analytes of interest. The thorough assessment of the conditions applied in the different extraction processes constitutes one of the most significant parts of the developed works due to their influence on extraction effectiveness. An interesting example is a work carried out by Ruiz-Domı´nguez et al. (2020), in which SFE was applied to extract phenolic compounds, as well as other bioactive compounds from Isochrysis galbana microalgae. A Box-Behnken design was performed to optimize the extraction procedure, varying extraction temperature (40–60°C), pressure (20–40 MPa), and % of co-solvent (0–8% ethanol) and maintaining the rest of the conditions without further modifications (ScCO2 flow rate: 7.2 g/min; time: 120 min). Response surface plots obtained fixing the concentration of ethanol at 8% (see Fig. 2A) showed a clear increase

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Fig. 2 Response surface plots of the combined effect of pressure and temperature on (A) total phenolic content and (B) antioxidant activity, applying a fixed percentage of ethanol as co-solvents (8% of ethanol). Taken from Ruiz-Domı´nguez, M. C.; Cerezal, P.; Salinas, F.; Medina, E.; Renato-Castro, G., Application of Box-Behnken Design and Desirability Function for Green Prospection of Bioactive Compounds from Isochrysis galbana. Appl. Sci. 2020, 10 (8), 2789 reprinted with the permission of MDPI under the terms and conditions of the Creative Commons Attribution license.

of the total phenolic content when the pressure was raised at intermediate temperature. Optimal conditions were selected at 38.4 MPa and 48°C, which provided a total phenolic content of 133.9 mg GAE/g biomass. The authors remarked that these data are better than previous results using conventional solvents to extract phenolic compounds from Isochrysis galbana. The antioxidant activity was also studied (see Fig. 2B), obtaining the best values when 40 MPa, 51.6°C, and 4.9% of ethanol were applied. It is striking that a reduction of the % of polar co-solvent increases the antioxidant activity. This increment could be related to

Chapter 5 Extraction of marine phenolics using compressed fluids

extracting other less polar bioactive compounds with antioxidant activity, such as carotenoids. Even though this result is better than previously reported studies using conventional methods, it is worth mentioning that the authors discussed the relevance of the co-solvent on the extraction yield, while suggesting the possible incorporation of water mixed with ethanol as modifiers in the extraction process using ScSO2. Chatterjee and Bhattacharjee (2014) and De Melo et al. (2020) contributed significantly to the SFE extraction of phenolic compounds (without polar co-solvents) from Phormidium valderianum and Aurantiochytrium sp., respectively. In the first case, the authors carried out a combined static + dynamic extraction for a total period of 90 min, whereas in the second study, a dynamic procedure was applied for 6 h and the extract was collected on ethanol. Both approaches were compared with Soxhlet using n-hexane (8 h), in the first study, and n-hexane and dichloromethane (6 h), in the second one. In both comparisons, the extraction yields obtained were similar using SFE and Soxhlet. However, as the authors highlighted, when Soxhlet is used, traces of toxic solvents are present in the final extract limiting its direct use in foods and pharmaceuticals. In addition, from a sustainable point of view, the consumption of organic solvents is considerably higher in this approach than in SFE in which just very little or no ethanol was necessary.

3. Gas expanded liquid extraction (GXLs) 3.1 Theoretical and practical fundaments of GXLs This kind of solvent is midway between supercritical fluids and pressurized liquids in many aspects. Normally an organic solvent that dissolves, partially or totally, a compressible gas or a supercritical fluid can form a gas expanded liquid (GXL). The reader must note the way it is written since, in this case, the main component is the liquid solvent containing a certain amount of gas or supercritical fluid. The gas phase is ordinarily carbon dioxide, ethylene or ethane. Nevertheless, CO2 is mainly used in GXL for economic, safety, and environmental advantages, forming the so-called carbon dioxide expanded-liquids, CXLs. GXLs can be considered switchable solvents since they can be easily turned back to a pure solvent by lowering the pressure or removing the gas phase. GXLs can also be defined as “a type of switchable solvent that is half way from pressurized liquids to supercritical fluids by increasing the amount of compressed CO2.” (Herrero et al., 2017)

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The advantages of using GXLs are similar to those described for supercritical fluids, such as higher gas diffusivity and enhanced transport properties than organic solvents, but the extraction conditions using GXL can be milder than those required for SFE. It is also interesting the possibility offered by GXL of introducing a gas or a fluid such as supercritical CO2 producing multiple immiscible phases with enhanced fluidity. Different types of GXL can be obtained according to the experimental settings used, mainly, pressure and CO2 molar fraction. It is important to remark that the partition of GXL described by Jessop and Subramaniam (2007) depends on the solubility of the gas in the liquid phase. • One-phase GXL: the gas is highly soluble in the liquid phase, so its physicochemical properties exhibit enormous variations. In this regard, the most common mixtures are those with CO2 or ScCO2 and ethanol, methanol, and hexane as liquid phases (compounds with high affinity to CO2). • The liquid phase can dissolve only small quantities of gas: consequently, minor changes in some properties can be observed compared to the liquid solvent (polarity or volume expansion). Nevertheless, viscosity can change significantly in some liquid phases like certain liquid polymers, oils, and ionic liquids. • Two-phases GXL: the gas phase is not entirely soluble in the liquid; hence, there are two different phases in the mixture (tiny bubbles in the liquid). The changes in physicochemical parameters are between the other types of GXL. Mostly used GXLs are composed of water and CO2, but the pH is highly affected in this case, so buffers can be used. The same mixture of liquid and gas phases can be in any of the two types mentioned above, mainly depending on the temperature and pressure of the system. The study of phase equilibrium in GXL is relatively complicated; therefore, approximations like Monte Carlo and molecular-dynamics simulation are the most precise methods to predict changes in volume expansion, viscosity, pressure-composition, and pressure-density; in some cases, even better than standard PR-EOS (Peng Robinson Equation of Estate) thermodynamic modeling (Laird et al., 2009). Significant changes in the GXLs’ physicochemical properties can be observed with the extraction temperature, pressure, and solvent composition (gas:liquid molar ratio). Therefore, these conditions have to be carefully chosen for each extraction. Effects of processing conditions on GXL parameters related to extractions are the following: a. Density: It is a critical property when dealing with extraction processes since it is strongly related to transport phenomena.

Chapter 5 Extraction of marine phenolics using compressed fluids

The temperature, pressure, and composition of the mixture (molar ratios) affect density. In one-phase GXL, density is commonly enhanced by increasing pressure or decreasing temperature. Nonetheless, changes in two-phase GXL densities are not easy to predict. For example, in a fixed volume chamber, an expansion of the gas phase (adding gas phase) increases the density of the liquid phase. Increasing the temperature has two effects: it expands the gas phase and reduces the liquid phase density. In contrast, a slight pressure increase may reduce the mixture’s density due to enhanced gas solubility. b. Volume expansion: It is the parameter that best defines GXLs; that is, the volume of a neat liquid solvent is expanded by adding gas phase and pressure. The volume expansion is associated with pressure, contrary to pure liquids, pure gases, or supercritical fluids. GXL volume expansion at a given temperature can be defined as the “fraction V=V 0, where V0 corresponds to the volume of 1 mole of the pure solvent at 1 atm at the chosen temperature, and V corresponds to the molar volume of the mixture.” The volume change depends on the solubility of the gas phase in the organic solvent and is caused by the dissolution of CO2 into the liquid phase, expanding its volume. When the temperature is increased, the gas forms a new phase, yielding a two-phase GXL. c. Polarity: Unquestionably, polarity is a key parameter for solubility prediction, attending to the principle “like dissolves like.” In GXL, polarity can be modified mainly by changing the gas:liquid molar ratio. If the CO2 ratio increases in the liquid phase, the GXL polarity is reduced, but the acidity increases; this is relevant for extracting phenolic compounds with hydroethanolic mixtures. When dealing with one-phase GXL, the gas:liquid molar ratio can be increased even without adding gas to the mixture, for example, by decreasing temperature or increasing pressure. Several strategies exist to predict theoretically polarity changes like Electronic Transition Energy and Kamlet-Taft solvatochromic parameters or even software like COSMO-RS. These methods provide reasonable estimations to choose the appropriate GLX for the target compounds to be extracted from natural matrices. Some GXL show insignificant volumetric expansion, which is usually connected with gas sorption; for instance, it is well known that in water/CO2 mixtures, CO2 dissolution is associated with the carbonic acid equilibria in solution and, therefore, to pH changes. These changes can be estimated using the HunterSavage equation (Hunter and Savage, 2008). This acidified

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GXL, also called carbonated subcritical water, is a more environmentally friendly alternative to inorganic acids like hydrochloric or sulfuric acids (Dhamdere et al., 2012; Gallina et al., 2017). The equipment needed for extraction using GXL can be the same as the one described in Section 2 for SFE. But the design can be less demanding since pressures used in GXL extraction are, usually, below 10 MPa. Typically, the preparation of GXL consists of adding the liquid phase and the gas or supercritical gas simultaneously to the extraction vessel, although it can be done sequentially. The differences in compressibility of both phases must be considered when using two-phase GXL; therefore, the second option is preferred to avoid problems during mixing and pumping. As seen, GXL is composed of CO2 and alcohols or bio-based solvents. They can be considered as green solvents according to the principles of Green Chemistry. Their components are often not produced ex profeso, but they are byproducts of other processes, presenting high biodegradability rates.

3.2 Phenolic compounds extraction using GXLs To date, the list of applications of GXL is not as extensive as the other techniques presented in this chapter, although its growth is expected in the next few years. Two main advantages should be highlighted: efficiency (low thermal degradation and high mass transfer rates) and effectiveness (facility to procure compounds of interest selectively from marine matrices). The tunability of GXL physicochemical properties (polarity, density, viscosity, and diffusivity) achieved by minor variations in the working conditions allow increasing selectivity, mass transfer, and solubility of phenolics. These variations lead to more sustainable processes since the extractions are faster, require less solvent and energy, and produce lower degradation of thermolabile compounds. GXL extraction is one of the greenest options to obtain phenolic compounds from marine organisms and their industrial byproducts. To prove the possibilities of GXL to extract phenolic compounds, the solubility, thermal degradation, partitioning coefficient, and mass transfer resistance of some pure compounds have been studied: a polyphenol (curcumin) and a flavonoid (quercetin). Two green organic solvents with different polarity (ethanol and ethyl lactate) were tested by Cunico et al. (2020). These authors examined the effects of the amount of CO2 (10, 30, and 50 mol%) and temperature (35°C, 50°C, and 70°C) at a

Chapter 5 Extraction of marine phenolics using compressed fluids

fixed pressure (10 MPa). The highest solubility for both, curcumin and quercetin, was achieved with ethanol at 30 mol% CO2 and 70° C, while the minimum solubility was obtained with ethyl lactate (35°C and 10% mol CO2). These authors found higher thermal degradation with ethyl lactate despite its lower optimal temperature, possibly due to the lower percentage of CO2 used. Hence, CO2 is a relevant parameter to consider in the GXL extraction of thermolabile phenolic compounds. For quercetin, only the mixture CO2-expanded ethanol (10 and 50 mol%) was used between 35°C and 50°C, and 10–30 MPa (Cunico et al., 2017). The authors found that the solubility was enhanced at 50°C by adding only 10 mol% CO2, while the pressure showed an insignificant influence in the tested range. Therefore, adding 10 mol% of CO2 in the mixture can reduce the amount of organic solvent, and consequently, it is an excellent starting point to achieve greener phenolic compounds extraction. To date, the only applications of phenolic compounds extraction from marine sources using GXL are those described in Section 5 of this chapter (Biorefinery).

4. Pressurized liquid extraction (PLE) 4.1 Theoretical and practical fundamentals of PLE PLE was described by Richter et al. in 1996 for the first time. In this publication, a home-made pressurized system for extracting polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and total petroleum hydrocarbons from reference materials was developed (Richter et al., 1996). Since then, the use of PLE as an extraction technique has been increasing considerably to extract many valuable compounds, and there are commercial equipment available in the market. This technique has been presented as an alternative to conventional extraction techniques. Some of the commercially available equipment that can be found at the time of writing this chapter (October 2021) are ASE by Thermo-Dione (https://www.thermofisher.com/order/catalog/product/083114), Speed Extractor by Buchi (https://www.buchi.com/en/products/ instruments/speedextractor-e-914-e-916), and PLE by FMS Inc. (https://www.fms-inc.com/sample-prep/#PLE). PLE is also known as accelerated solvent extraction (ASE), pressurized fluid extraction (PFE), enhanced solvent extraction (ESE), or high-pressure solvent extraction (HPSE). The key to the effectiveness of this extraction technique relies on the use of solvents at high temperatures but applying pressures high enough to maintain the solvents in the liquid state (below the

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critical point). Under these conditions, the physicochemical characteristics of the solvents suffer modifications that improve the extraction process. For instance, the viscosity and the solvent surface tension are reduced while the solubility of the target analytes increases, improving the mass transfer. Besides, the pressure applied in the method could enhance the matrix disruption, assisting, even more, the penetration of the solvent and increasing the mass transfer rate. All these factors allow better penetration of the solvent into the matrix, reducing the extraction time and improving the extraction yield of the process in comparison to conventional techniques (Herrero et al., 2013a; Sa´nchezCamargo et al., 2017). The current commercially available PLE equipment is fully automatized at different scales. For analytical scale, there are different equipment as cited above; besides, the use of PLE at industrial scale is possible using an equipment like the SWE by Tournaire (https://www.tournaire.fr/en/products-services/ equipment/solutions/sub-critical-water-extraction/) or the Ethanol Extractor by (https://www.cedarstoneindustry.com/productsservices/ethanol-extraction/). In static extraction, the most common mode, the process starts with loading the sample into the extraction cell. The extraction cell should be adapted to resist high temperatures and pressures. Then the cell is placed in the oven, and a pump introduces the solvent into the cell and maintains the established pressure in the system. Then the cell is heated, and the conditions are maintained during the selected extraction time. When the extraction is finished, the solvent is purged from the extraction cell with nitrogen gas (EsquivelHerna´ndez et al., 2017). The extraction solvent selected for the extraction is the main parameter directly affecting the selectivity and efficiency of the PLE process. In theory, all the solvents used in conventional extraction techniques can be used in PLE to extract the target compounds with a wide range of polarities. However, due to the physicochemical modifications suffered by solvents at high temperatures and pressures, the affinity of the solvent for the solutes is also modified. This fact allows avoiding the use of toxic solvents like chloroform, hexane, or dichloromethane for the extraction of non-polar compounds and substituting them with more environmentally friendly solvents like ethanol, D-limonene, ethyl acetate, or even water. Water under PLE conditions (subcritical water) modifies its properties; for example, the dielectric constant is reduced from ε ¼ 78 at ambient conditions to 27 at 250°C and 5 MPa. This value is similar to the dielectric constant of organic solvents like methanol (ε ¼ 33) and ethanol (ε ¼ 24) at 25°C.

Chapter 5 Extraction of marine phenolics using compressed fluids

Besides, the surface tension and the viscosity decrease and the diffusivity of water improves. Consequently, subcritical water acts as an organic solvent which is able to extend the range of extracted compounds (Gallego et al., 2018; Montero et al., 2021). The use of PLE with subcritical water is called subcritical water extraction (SWE), pressurized hot water extraction (PHWE), or superheated water extraction (SHWE). Therefore, PLE is considered as a green extraction technique due to the possibility of using GRAS solvents with the same extraction abilities as hazardous organic solvents. Besides the selected solvent, other relevant parameters affect PLE extraction and should be considered for the optimization of the PLE process to maximize the recovery of the target compounds. The most relevant factors are temperature and time. Although pressure is necessary to maintain the liquid state of the solvents and can have an influence on the matrix disruption, several authors have shown that the impact of the pressure in the extraction yield is not significant (Herrero et al., 2013b, 2015; Sun et al., 2012), and typically, pressures between 1 and 20 MPa are used in PLE (Getachew et al., 2020; Osorio-Tobo´n, 2020; Sa´nchez-Camargo et al., 2017). On the other hand, the temperature has a strong influence on the extraction recovery of target compounds. The increase in the temperature is related to disruptions of intermolecular forces in the matrix such as van der Waals forces, hydrogen bonds and dipole attractions, and the modifications already mentioned in the physicochemical characteristics of the solvents. However, this parameter should be carefully optimized since very high temperatures can lead to the thermal degradation of thermolabile compounds. Temperatures applied in the literature for PLE extraction of phenolic compounds vary between 40°C and 275°C (Osorio-Tobo´n, 2020). The extraction time is defined in PLE as the effective time or period in which the solvent is in direct contact with the matrix at the established temperature and pressure, and depends on the type of extraction strategy (static or dynamic). In static PLE, the most common one, the solvent is in contact with the matrix under the optimized conditions in the closed extraction cell, and the saturation of the extraction solvent determines the extraction time. The optimal extraction time should be the minimum time required to maximize the transfer of the compounds to the extraction solvent, avoiding any degradation process. That way, typical static extraction times for obtaining bioactives in PLE vary between 5 and 20 min. In the dynamic PLE extraction, the pressurized solvent at high temperature is continuously flowing through the extraction cell. In this mode, the extraction is favored since the saturation of the solvent is not achieved; however, much more solvent is

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consumed, and the extraction time may be longer depending on the solvent flow rate. An in-between approach consists of sequential static extraction cycles, where a fresh solvent is introduced into the cell after the previous cycle has finished. With these repetitive extraction cycles, the efficiency and the extraction recoveries may be increased (Sa´nchez-Camargo et al., 2017). Due to the complex individual and synergic effects of all the parameters that affect the PLE extraction (extraction solvent, temperature, and extraction time), usually, experimental designs are used for the optimization of the extraction factors to maximize the extraction of total phenolics, antioxidant capacity, or specific phenolic compounds. Therefore, the advantages of using PLE in comparison to conventional extraction techniques are the reduction of solvent volumes and extraction times, the use of environmentally friendly solvents for the extraction of a wide variety of compounds, the protection of the compounds against oxidation due to the absence of oxygen, and the automation of the process achieving high extraction yields (Esquivel-Herna´ndez et al., 2017; Getachew et al., 2020; Sosa-Herna´ndez et al., 2018).

4.2 Phenolic compounds extraction using PLE The extraction of phenolic compounds from marine sources by PLE has been reported for macro- and microalgae. In particular, the recovery of these compounds from macroalgae has been explored extensively. The PLE methods for the extraction of different phenolic compounds from macro- and microalgae developed in the literature are summarized in Table 2.

4.2.1 Macroalgae The development of PLE extraction methods in macroalgae has been evaluated mainly in brown algae due to their higher content of phenolic compounds. Besides, as mentioned above, many applications have been developed targeting phlorotannins, the main phenolic compounds present in brown macroalgae. Therefore, most of the PLE methods found in the literature are optimized for brown algae. In particular, PLE has been used for the extraction of phlorotannins and phenolic compounds from brown algae with different functional activities. For example, the anti-hyaluronidase activity of the brown algae Padina pavonica was evaluated using two different extraction solvents, ethyl acetate and water. Although both solvents provided extracts with high anti-hyaluronidase activity, the extract obtained with ethyl

Table 2 PLE methods for the extraction of phenolic compounds from marine sources.

Algae Padina pavonica (brown seaweed) Gelidium sp., Mazzaella laminarioides, Nothogenia sp., Pyropia sp. (red seaweed), and brown Durvillaea antarctica and Lessonia spicata (brown seaweeds) Fucus vesiculosus (seaweed)

Phenolic compound

Extraction solvent

Extraction temperature

PLE Extraction time and number Pressure of cycles

Total phenolic content Total phenolic content

Water

60°C

15 MPa

Ethanol

Gallic, protocatechuic, gentisic acids Laminaria Total phenolic ochroleuca (brown content seaweed) Phlorotannins Sargassum muticum (brown seaweed) Saccharina Total phenolic longicruris, content Ascophyllum nodosum (brown seaweeds), and Ulva lactuca (green seaweed)

Yield and TPC

Bioactivity

Comment

Reference

120°C

10 min 2 cycles 10.3 MPa 20 min

Anti-hyaluronidase activity TPC (highest value Antidiabetic activity in Durvillaea antarctica): 7.4 mg GAE/g dw

Sun et al. (2012) Boisvert et al. (2015)

Ethanol/water, 58.56:41.44, v/v

137.18°C

1 MPa

4.68 min

Antioxidant activity

Otero et al. (2019)

Ethanol/water, 50:50, v/v

160°C

10 MPa

10 min

Yield: 31.16% TPC: 36.9 mg GAE/g dw TPC: 173.65 mg GAE/g dw

Ethanol/water, 95:5, v/v

160°C

10.3 MPa 20 min

TPC: 148.97 mg PGE/g dw

Ethanol

50°C

10.3 MPa 5 min

TPC: 50.2 mg GAE/g dw

Antiproliferative activity against colon cancer cells Antibacterial activity against Escherichia coli, Micrococcus luteus, and Brochothrix thermosphacta

Osorio-Tobo´n (2020) Montero et al. (2016) Fayad et al. (2017)

Continued

Table 2 PLE methods for the extraction of phenolic compounds from marine sources—cont’d

Phenolic compound

Extraction solvent

Extraction temperature

PLE Extraction time and number Pressure of cycles

Lobophora variegatea (brown seaweed) Sargassum muticum (brown seaweed)

Total phenolic content and total phlorotannin content Total phenolic content

Ethanol/water, 95:5, v/v

100°C

10 MPa

Ethanol/water, 95:5, v/v

160°C

10.3 MPa 20 min

Sargassum muticum (brown seaweed) Cystoseira abiesmarina, Undaria pinnatifida, Sargassum muticum (brown seaweeds), and Chondrus crispus (red seaweed)

Total phenolic content

Ethanol/water, 75:25, v/v

120°C

10.3 MPa 20 min

Phenolic acids: gallic, protocatechuic, p-hydroxybenzoic, chlorogenic, vanillic, caffeic, syringic, p-coumaric, ferulic, salicylic acids and, vanillin, 3,4-dihydroxybenzaldehyde and phydroxybenzaldehyde Phlorotannins

Methanol/water, 80:20, v/v

130°C

13 MPa

10 min

(1) Water (2) Methanol (3) Dichloromethane

40°C

10 MPa

(1) 10 min, 3 static cycles (2) 5 min (3) 5 min

5 MPa

5 min

Algae

Fucus vesiculosus (brown seaweed)

Saccharina japonica (brown seaweed)

Chlorogeni, gentisic, Water +0.5 M [C4C1im] 175°C protocatechui, caffei, [BF4] p-hydroxybenzoi, syringic and vanillic acids

20 min

Yield and TPC

Bioactivity

Comment

TPC: 96.14 mg GAE/g dw Extraction yield: 21.9% TPC: 94.0 mg GAE/g dw TPC: 4.5% of the alga dw

Montero et al. (2018b) Antioxidant activity

Sumampouw et al. (2021)

Antioxidant activity

Anae¨lle et al. (2013) UAE-PLE-m-SPE platform

TPC: 39.55 mg GAE/g dw

Reference

Antioxidant, antimicrobial, and antiproliferative activity against pancreas cancer cells Antioxidant activity

O’sullivan et al. (2013)

del Pilar Sa´nchezCamargo et al. (2016) Heavisides et al. (2018)

Microalgae

Durvillaea incurvata and Lessonia spicata (brown seaweeds) Ascophyllum nodosum, Fucus vesiculosus, and Fucus serratus (brown seaweeds)

Phlorotannins

Water +15% glycerol

150°C

10.3 MPa 5 min

TPC: 95.24 mg PGE/g dw

Antioxidant and antidiabetic activity

Klejdus et al. (2017)

Total phenolic content

Ethanol/water, 80:20, v/v

100°C

6.9 MPa

Antioxidant and antiproliferative activity against colon cancer cells

Pacheco et al. (2020)

Chlorella sp.

Water

163°C

Antioxidant activity

Water

170°C

Haematococcus pluvialis

Total phenolic content Total phenolic content Total phenolic content

Water

200°C

0.1– 5 min 4.0 MPa 10.3 MPa 20 min

TPC Ascophyllum nodosum: 50.2 mg GAE/g dw Fucus vesiculosus: 99.5 mg GAE/g dw Fucus serratus: 28.0 mg GAE/g dw TPC: 58.73 mg GAE/g dw TPC: 55.61 mg GAE/g dw Yield: 30%

Heffernan et al. (2014) Tierney et al. (2013) Cha et al. (2010)

Phaeodactylum tricornutum

Total phenolic content

Ethanol

50°C

10.3 MPa 20 min

Chlorella sp.

5 min

Spirulina platensis Total phenol content Ethanol

115°C

15 min

Chlamydomonas sp.

Total phenolic content

Ethanol

180°C

10.3 MPa 20 min

Chlorella vulgaris

Total phenolic content

Ethanol/water, 90:10, v/v

160°C

10.3 MPa

Extraction Yield: 24.06% TPC: 42.16 mg GAE/g dw

Antioxidant and Pretreatment antimicrobial activity to disrupt the cell wall: three freezing-smashingthawing cycles Antioxidant activity

Antioxidant activity

Yield: 30% TPC: 59.63 mg GAE/g extract Yield: 42% TPC: 16 mg GAE/g dw

Antioxidant activity

Antioxidant activity

Rodrı´guezMeizoso et al. (2010) Zakaria et al. (2017); Zakaria et al. (2020) Jaime et al. (2005) Santoyo et al. (2006)

[C4C1im][BF4], 1-butyl-3-methylimidazolium tetrafluoroborate; GAE, gallic acid equivalents; PGE, phloroglucinol equivalents; PLE, pressurized liquid extraction; TPC, total phenolic content; UAE, ultrasoundassisted extraction; m-SPE, micro solid phase extraction.

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acetate presented higher content on phenolic compounds. However, the water extract revealed higher anti-hyaluronidase activity, probably due to the synergic activity of sulfated polysaccharides, which are easily extracted with water due to their high polarity (Fayad et al., 2017). In the PLE extraction of phenolic compounds with bioactivity from marine sources, ethanol and ethanol/water mixtures have been reported as the most effective extraction solvent (Table 2) achieving higher TPC and bioactivities such as antioxidant, antiproliferative, antidiabetic, or antimicrobial activities (Anae¨lle et al., 2013; Boisvert et al., 2015; Montero et al., 2016; O’sullivan et al., 2013; Otero et al., 2019; Pacheco et al., 2020; Sa´nchez-Camargo et al., 2016; Sumampouw et al., 2021). The advantages of these solvents are their similar polarity to the target phenolic compounds and their compliance with the European standard regulations for food product. Besides, the use of PLE with ethanol/water solvents makes scaling up the process more feasible providing good productivity and high efficiency (Anae¨lle et al., 2013). Otero et al. evaluated the effect of four solvents with different polarities, namely, hexane, ethyl acetate, ethanol, and ethanol/ water (50:50, v/v) for the extraction of phenolic compounds from the brown algae Laminaria ochroleuca. The extraction carried out using ethanol/water mixture at 160°C gave rise to the fraction with the highest recovery of phenolic compounds, with a TPC value of 173.65 mg GAE/g extract, while the extract obtained with hexane resulted in a TPC of 6 mg GAE/g extract (Otero et al., 2019). Although ethanol/water mixtures have been shown as very efficient PLE solvents for the recovery of phenolic compounds, due to the complex composition of macroalgae, these mixtures can extract other compounds that interfere in the determination and identification of phenolic compounds. In these cases, additional purification steps are required. For example, a pure extract consisting of phlorotannins from Sargassum muticum algae collected along the North Atlantic coast was obtained using an optimized PLE method using ethanol/water (95:5, v/v) at 160°C followed by an LLE with dichloromethane to remove lipid compounds, and precipitation of carbohydrates and proteins with ethanol and acetone, respectively. That way, a pure phlorotannin fraction was obtained and it was possible to identify a complex mixture of phlorotannins, including hydroxyfuhalos for the first time in this algae. Besides, the antiproliferative activity of these PLE extracts was tested against a colon cancer cell line (HT-29) showing a good cytotoxicity potential (Montero et al., 2016). In another work, three fractionation steps of the algae extract were

Chapter 5 Extraction of marine phenolics using compressed fluids

carried out with PLE: the initial step consisting of a water-rinsing phase to remove salts and water-soluble compounds at 40°C, a second PLE step was performed using methanol followed by a dichloromethane extraction to cover a wide range of polarities in the extracted compounds. In this case, methanol and dichloromethane fractions were pooled together and the bioactivities of the different compounds extracted with both solvents were analyzed. The main groups of compounds extracted under these conditions were phlorotannins, phosphatidylcholine, betain, lipids, chlorophylls, and carotenoids. In this study, the seasonal variability of phenolic compounds and bioactivity of the PLE extract of the algae Fucus vesiculosus was evaluated, being in the summer season the one that promoted the highest phlorotannin content in the alga as well as the highest antioxidant and apoptotic activity against pancreas cancer cells, correlating these bioactivities to the phlorotannin content (Heavisides et al., 2018). Another alternative for the sample clean-up and pre-concentration of the phenolic compounds consisted of the coupling of micro solid-phase extraction (μ-SPE) after PLE. With this strategy, extracts can be directly analyzed by liquid chromatography to identify pure phenolic compounds. Applying this platform, fourteen phenolic acids (gallic acid, protocatechuic acid, 3,4-dihydroxybenzaldehyde, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, phydroxybenzaldehyde, caffeic acid, syringic acid, vanillin, p-coumaric acid, ferulic acid, and salicylic acid) were identified and quantified in the macroalgae Cystoseira abies-marina, Undaria pinnatifida, Sargassum muticum, and Chondrus crispus (Klejdus et al., 2017). However, novel solvents and additives have been studied to enhance the recovery of phenolic compounds from marine sources. An innovative study explored the use of SWE with ionic liquids (ILs) for the extraction of different phenolic compounds from the brown algae Saccharina japonica. 1-Butyl-3-methylimidazolium tetrafluoroborate [C4C1im][BF4] was selected as ILs. The two more influencing parameters in the extraction of phenolic compounds were the concentration of ILs and the temperature. The optimal conditions for the ILs-SWE process were achieved using 0.5 M of [C4C1im][BF4] in pure water at 175°C. At these conditions, chlorogenic acid, gentisic acid, protocatechuic acid, caffeic acid, and p-hydroxybenzoic acid as well as minor compounds such as syringic acid and vanillic acid were extracted. The recovery values of these phenolic compounds using ILs in the SWE were up to 7.67 times higher compared to normal SWE, and the TPC was 19-fold higher than conventional SLE. The rich phenolic content of the extract obtained in the ILs-SWE alternative was correlated with the high antioxidant

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activity observed in this extracted fraction of the algae (Dinh et al., 2018). The use of 15% glycerol diluted in water has also been considered for the extraction of phlorotannins from the two seaweeds Durvillaea incurvata and Lessonia spicata as well as the study of their antioxidant and anti-hyperglycemic activities. Glycerol is a food-grade cheap additive, considered environmentally friendly. The addition of glycerol into the aqueous extraction solvent produced an effective extraction of polar and moderately polar compounds. Moreover, in this study, the impact of the anatomical part of the algae was pointed out. The PLE extract of the holdfasts of the algae showed the highest phenolic content (95.24 mg PGE/g dw) and the greatest antioxidant and antidiabetic activities (Erpel et al., 2021). However, in spite of the advantages described for the extraction of phenolic compounds by PLE, some authors have reported better results with conventional SLE compared to PLE. Heffernan et al. evaluated the effect of PLE and SLE on the extraction of phenolic compounds of different seaweeds (Fucus serratus, Laminaria digitata, Gracilaria gracilis, and Codium fragile) and the results revealed that SLE with cold water was able to obtain a higher TPC (81.17 mg GAE/g sample) compared to PLE with water at 120°C (Heffernan et al., 2014). In the same way, Tierney et al. described higher phenolic compound recoveries for SLE using water and ethanol/water (80:20, v/v) as extraction solvent than the corresponding PLE extracts (142.81 and 124.0 mg PGE/g sample, respectively) (Tierney et al., 2013). In these studies, the authors argued that the high temperatures and pressures applied in PLE do not improve the recovery of phenolic compounds and consequently the bioactivity of the extracts. However, although in these works different solvents for the phenolic compound extraction were compared, other parameters like temperature and extraction time that significantly affected the extraction were not optimized, so perhaps the optimization of these two parameters could make the difference in the extraction yield of phenolic compounds when compared with conventional SLE.

4.2.2 Microalgae The extraction of phenolic compounds from microalgae applying PLE has not been studied as profoundly as the PLE extraction in macroalgae, probably due to the interest of microalgae in other important compounds such as polyunsaturated fatty acids, carbohydrates, and carotenoids (Gallego et al., 2018). However, relevant studies have developed green PLE methods for the extraction of bioactive phenolic compounds

Chapter 5 Extraction of marine phenolics using compressed fluids

from microalgae. The most studied microalgae in this term are Chlorella sp. (Zakaria et al., 2017, 2020) and Spirulina platensis (Jaime et al., 2005; Santoyo et al., 2006). Other green PLE methods for the extraction of the phenolic compounds have been developed for the microalgae Chlorella vulgaris (Cha et al., 2010), Chlamydomonas sp. (Montero et al., 2018b), and Haematococcus pluvialis (Rodrı´guez-Meizoso et al., 2010). For Spirulina platensis, the best PLE conditions for the extraction of phenolic compounds have been described using ethanol as pure extraction solvent at 115°C and 15 min of extraction time. Under these conditions, antioxidant PLE extracts have been obtained (Jaime et al., 2005; Santoyo et al., 2006). Zakaria et al. optimized the SWE process to maximize the extraction of phenolic compounds and antioxidant activity from Chlorella sp. The optimal parameters achieved for this extraction were established at 163°C and 5 min of extraction time, obtaining TPC values of 58.73 mg GAE/g (Zakaria et al., 2017). In a more recent study, the kinetics of the SWE extraction of phenolic compounds from Chlorella sp. was described by the same authors who reported that the SWE extraction followed first-order kinetics and that the extraction was strongly dependent on the temperature. To carry out this study, SWE at temperatures between 100°C and 250° C as well as extraction times from 5 to 20 min were considered. The constant rise in temperature was observed to exponentially increase the solubility of the phenolic compounds in the water solvent and, therefore, the mass transfer and diffusion rates. Regarding the extraction time, the maximum extraction yield was achieved in the first 5 min of extraction. The study of the combination of both parameters revealed that maintaining 5 min as extraction time, the increase of the temperature from 100°C to 175°C enhances 2.6-folds the extraction of phenolic compounds. This significant increase was related to the reduction of the polarity and dielectric constant of water at high extraction temperatures and pressures. The relationship between the extraction yield and the water dielectric constant as a function of the temperature was determined. It was observed that the dielectric constant of water decreases with temperature while the extraction yield of phenolic compounds increases. Microalgae cell wall is another characteristic affecting PLE extraction of phenolic compounds; for instance, Haematococcus pluvialis present a thick cyst-like cell wall that encloses the cytoplasm and the target compounds making them more inaccessible and therefore hampering their extraction. To enhance the extractability of the bioactives from Haematococcus pluvialis previous to the SWE, Rodrı´guez-Meizoso et al. evaluated four different

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methods of cell wall disruption, namely, ultrasonic bath, freezingthawing cycles, Ultraturrax homogenization, and freezingsmashing-thawing cycles. Among these four methods, the best extraction yields were obtained by treating the sample under three freezing-smashing-thawing cycles (Rodrı´guez-Meizoso et al., 2010). PLE is therefore presented as a feasible and versatile technique for the extraction of phenolic compounds from marine sources, making possible the tuning of the extraction conditions to recover phenolic-rich fractions from a wide variety of macro- and microalgae species.

5. Biorefinery based on the use of compressed fluids Biorefinery was defined by the International Energy Agency as the sustainable processing of biomass into a spectrum of bio-based products (food, feed, chemicals, materials) and bioenergy (biofuels, power, and/or heat). This class of “refinery” is equivalent to the classical petroleum refining, where several types of fuels and products are achieved from the same raw material (crude oil). Initially, biorefineries were developed to produce biofuels (bioenergy) and biochemicals to mitigate climate change. Biomass energy and material recovery are maximized if a biorefinery approach is considered, where many technological processes are jointly applied to different kinds of biomass feedstock for producing a wide range of bioproducts. Many biorefinery pathways, from feedstock to products, can then be established, according to the different types of feedstock, conversion technologies, and products (Cherubini, 2010). Ideally, a biorefinery is a highly energy-efficient and zero-waste (almost) production process. So the “waste” outcomes can be considered coproducts and could be reallocated for value-added use or recycling processes. In this way, the biorefinery optimizes the consumption of resources and minimizes leftovers, thus maximizing the profitability and benefits (Ferreira, 2017). To set up a biorefinery process, initial biomass availability, potential use, and characteristics need to be considered, along with their advantages and disadvantages (Mabee et al., 2005). Almost any biomass (forest, aquaculture, agriculture, and byproducts) can be used on a biorefinery, for example, grass, biosolids, bagasse, waste paper, microalgal culture, sawdust, animal wastes, aquatic plants, and algae, among many others (Demirbas, 2005). In this sense, it is necessary a detailed and accurate characterization of

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the starting biomass, intermediates, and outcomes to understand how the individual biomass components and reaction products act together during each phase in the process. Ferreira (2017) classified biorefineries according to biomass feedstock in two big groups: lignocellulosic or marine biorefineries. This section focuses on marine biorefineries based on marine biomass such as aquatic plants, macroalgae (e.g., seaweed), and microalgae. Due to the wide variety of marine biorefineries, not all the biorefineries included here are focused on marine phenolics, but it is worth talking about them to give the reader a comprehensive view.

5.1 Marine biorefineries 5.1.1 Macroalgae On the one hand, there are biorefineries in which more remarkable outcomes are obtained through fermentation processes, providing valuable exploitation of the remaining byproducts. The polysaccharides xylose, glucose, and rhamnose of Ulva lactuca biomass were hydrolyzed enzymatically and used as a substrate for the fermentative production of butanol, ethanol, acetone, and 1,2-propanediol, whereas a protein-enriched extracted portion was considered for use in the animal feed (Bikker et al., 2016). This type of enzymatic processing was also used with Saccharina latissima (Marinho et al., 2016), where hydrolysis generated succinic acid. At the same time, the solid hydrolysate was rich in phenolic compounds, and a concentrated macro- (Ca, K, Na, Mg, P, N, and Fe) and micronutrients solid residue that can be used as fertilizer. Economic assessment for this biorefinery demonstrated that for the analyzed scenarios, the critical product’s price (succinic acid) could be lowered significantly by coproducing high-volume moderate-value fertilizers and lowvolume high-value phenolic antioxidants. Considering the high-pressure processes that are the core of this chapter, we can find a couple of downstream processes for the valorization of Sargassum muticum. In both examples, there is first a supercritical CO2 fucoxanthin extraction, then polysaccharides extraction, such as alginate or fucoidan, and, lastly, antioxidant phenolics extraction by autohydrolysis, such as phlorotannins (P erez-Lo´pez et al., 2014). Authors concluded that the most efficient biorefinery approach from an economic point of view is the one that obtains only alginate and antioxidants. Including the supercritical fucoxanthin extraction as the initial step incurs high electricity consumption that drifts into

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environmental burdens. Nevertheless, Balboa et al. (2015) optimized the process (see Fig. 3) using only green processes and techniques (microwave drying, SFE, and membrane microfiltration) besides green and safe solvents (ethanol, ethyl acetate, water, and CO2), making the whole process more economically efficient. Sargassum muticum (Sm) Fitting out sample Drying Grinding E CSE

SC-CO2 R

Products 1, 2

R Autohydrolysis

Alginate Discarded

AH-L Precipitation

EM

Drying

MF

Product 3 Diafiltration Drying

Concentration + Diafiltration L-L extraction

Drying Product 5

Product 4 Product 6

Fig. 3 Flowchart of the biorefinery platform described by Balboa et al. (2015) for Sargassum muticum. Description: ethanol conventional solvent extraction (CSE); SC-CO2, supercritical CO2 extraction; MF, membrane microfiltration; L-L, liquidliquid extraction; E, crude ethanolic extract; R, exhausted Sargassum muticum; AH-L, autohydrolysis liquor; EM, exhausted autohydrolysis material; Product 1, fucoxanthin-enriched SC-CO2 fraction from E; Product 2, fucoxanthin-enriched SC-CO2 extract from Sargassum muticum; Product 3, freeze- or spray-dried autohydrolysis liquors from ovSm; Product 4, diafiltrated and freeze-dried permeate from membrane microfiltrated autohydrolysis liquors; Product 5, spraydried retentate from the concentration of microfiltered autohydrolysis liquors; Product 6, pooled ethyl acetate fractions of permeates generated by previous membrane diafiltration. Reprinted with the permission of MDPI under the terms and conditions of the Creative Commons Attribution license.

Chapter 5 Extraction of marine phenolics using compressed fluids

5.1.2 Microalgae As seen above, much research has been focused on microalgae as the starting biomass to obtain high-value compounds employing green solvents under pressurized conditions. This research can set the basis for designing multistep biorefineries. For example, Gilbert-Lo´pez et al. (2017) optimized a biorefinery protocol using several green extraction stages to achieve different valuable fractions from Scenedesmus obliquus. These steps included SFE, GXL, and PLE, using supercritical CO2, a mixture of ethanol + supercritical CO2 and, finally, water, respectively. Thus, they could effectively fractionate triglycerides, photosynthetic pigments such as lutein, β-carotene, and chlorophylls, as well as proteins and phenolic compounds. Analogous research was done with Isochrysis galbana (Gilbert-Lo´pez et al., 2015), but in this case, two PLE steps were used, one with ethanol and another with water, to maximize the phenolic compounds’ recovery. It is important to note that in both processes, a single cell was loaded with dried microalgae powder, starting the extraction sequence with the less polar solvent and subsequently increasing the polarity of the extractant. This way, fucoxanthin, polar lipids, phenolic compounds, and other bioactive compounds with high antioxidant activity were recovered in various fractions during the whole biorefinery process, leaving an exhausted minerals-rich residue that could be used as fertilizer.

6. Conclusions In this chapter, fundamentals on the use of compressed fluid technologies such as SFE, GXLs extraction, and PLE have been introduced and discussed, focusing on isolating phenolic compounds from marine biomasses. Compared to conventional procedures, many applications showing the advantages of these processes have been developed targeting phenolic compounds from macro- and microalgae. The extraction efficiency depends mainly on the composition of the biomass and the applied extraction conditions. Therefore, careful optimization of the parameters involved in the extraction processes is needed. Considering the enormous variety of macro- and microalgae species and compositions, together with the fact that environmental conditions affect its final content on bioactives, this field of research can be seen as almost inexhaustible. New compounds (including phenolics) and their associated bioactivities are expected to be discovered, which could be employed as functional ingredients for foods, nutraceuticals, cosmeceuticals, and pharmaceuticals.

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On the other hand, compressed fluids processes have demonstrated its feasibility to work under a unique platform for biorefinery of marine biomasses; in this sense, sequential processes should be developed to obtain all the valuable fractions from the starting biomass while minimizing the energy requirements. In any case, economic and feasibility studies are needed to design a profitable biorefinery without forgetting the requirement for a life cycle assessment (LCA) of the whole process to determine its environmental impact. New research to come includes testing new bio-based solvents under pressurized conditions to widen the applicability of the described technologies. The use of new solvents such as natural deep eutectic solvents (NADES) under compressed fluid conditions is still in its infancy, but significant advances in this field are expected to develop even more sustainable processes. Process integration combining compressed fluids technologies and biotechnological processes is also expected to increase not only as biomass pretreatment but also to produce new and more valuable compounds.

Acknowledgments The authors would like to thank project AGL2017-89417-R (Spanish Ministry of Science and Innovation, Spain). B.S.-R. would like to acknowledge the Spanish Ministry of Science, Innovation and Universities for her “Juan de la Cierva” postdoctoral grant.

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Ospina, M.; Castro-Vargas, H. I.; Parada-Alfonso, F. Antioxidant Capacity of Colombian Seaweeds: 1. Extracts Obtained from Gracilaria mammillaris by Means of Supercritical Fluid Extraction. J. Supercrit. Fluids 2017, 128, 314–322. Otero, P.; Lo´pez-Martı´nez, M. I.; Garcı´a-Risco, M. R. Application of Pressurized Liquid Extraction (PLE) to Obtain Bioactive Fatty Acids and Phenols from Laminaria ochroleuca Collected in Galicia (NW Spain). J. Pharm. Biomed. Anal. 2019, 164, 86–92. rez-Correa, J. R.; Mariotti-Celis, M. S.; Erpel, F.; Pacheco, L. V.; Parada, J.; Pe Zambrano, A.; Palacios, M. Bioactive Polyphenols from Southern Chile Seaweed as Inhibitors of Enzymes for Starch Digestion. Mar. Drugs 2020, 18 (7), 353. Padrela, L.; Rodrigues, M. A.; Duarte, A.; Dias, A. M.; Braga, M. E.; de Sousa, H. C. Supercritical Carbon Dioxide-Based Technologies for the Production of Drug Nanoparticles/Nanocrystals–A Comprehensive Review. Adv. Drug Deliv. Rev. 2018, 131, 22–78. rez-Lo´pez, P.; Balboa, E. M.; Gonza´lez-Garcı´a, S.; Domı´nguez, H.; Feijoo, G.; Pe Moreira, M. T. Comparative Environmental Assessment of Valorization Strategies of the Invasive Macroalgae Sargassum muticum. Bioresour. Technol. 2014, 161, 137–148. Richter, B. E.; Jones, B. A.; Ezzell, J. L.; Porter, N. L.; Avdalovic, N.; Pohl, C. Accelerated Solvent Extraction: A Technique for Sample Preparation. Anal. Chem. 1996, 68 (6), 1033–1039. ˜ ez, E. Rodrı´guez-Meizoso, I.; Jaime, L.; Santoyo, S.; Sen˜ora´ns, F. J.; Cifuentes, A.; Iba´n Subcritical Water Extraction and Characterization of Bioactive Compounds from Haematococcus pluvialis Microalga. J. Pharm. Biomed. Anal. 2010, 51 (2), 456–463. Roh, M.-K.; Uddin, M. S.; Chun, B.-S. Extraction of Fucoxanthin and Polyphenol from Undaria Pinnatifida Using Supercritical Carbon Dioxide with Co-Solvent. Biotechnol. Bioprocess Eng. 2008, 13 (6), 724–729. Ruiz-Domı´nguez, M. C.; Cerezal, P.; Salinas, F.; Medina, E.; Renato-Castro, G. Application of Box-Behnken Design and Desirability Function for Green Prospection of Bioactive Compounds from Isochrysis galbana. Appl. Sci. 2020, 10 (8), 2789. Safafar, H.; Van Wagenen, J.; Møller, P.; Jacobsen, C. Carotenoids, Phenolic Compounds and Tocopherols Contribute to the Antioxidative Properties of some Microalgae Species Grown on Industrial Wastewater. Mar. Drugs 2015, 13 (12), 7339–7356. ˜ ez, E.; Cifuentes, A.; Herrero, M. Bioactives Sa´nchez-Camargo, A.d. P.; Iba´n Obtained from Plants, Seaweeds, Microalgae and Food by-Products Using Pressurized Liquid Extraction and Supercritical Fluid Extraction. In Green Extraction Techniques: Principles, Advances and Applications; Vol. 76; Elsevier, 2017; p. 27. Sa´nchez-Camargo, A.d. P.; Montero, L.; Stiger-Pouvreau, V.; Tanniou, A.; ˜ ez, E. Considerations on the Use of EnzymeCifuentes, A.; Herrero, M.; Iba´n Assisted Extraction in Combination with Pressurized Liquids to Recover Bioactive Compounds from Algae. Food Chem. 2016, 192, 67–74. ˜ ez, E.; Jaime, L. FuncSantoyo, S.; Herrero, M.; Senorans, F. J.; Cifuentes, A.; Iba´n tional Characterization of Pressurized Liquid Extracts of Spirulina Platensis. Eur. Food Res. Technol. 2006, 224 (1), 75–81. Shanmugam, M.; Ganesan, A. R.; Rajauria, G. Extraction Technologies to Recover Dietary Polyphenols from Macro-and Microalgae. In Recent Advances in Micro and Macroalgal Processing: Food and Health Perspectives; 2021; pp. 163–187. Silva, A.; Silva, S. A.; Carpena, M.; Garcia-Oliveira, P.; Gullo´n, P.; Barroso, M. F.; Prieto, M.; Simal-Gandara, J. Macroalgae as a Source of Valuable Antimicrobial Compounds: Extraction and Applications. Antibiotics 2020, 9 (10), 642.

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Sivagnanam, S. P.; Yin, S.; Choi, J. H.; Park, Y. B.; Woo, H. C.; Chun, B. S. Biological Properties of Fucoxanthin in Oil Recovered from Two Brown Seaweeds Using Supercritical CO2 Extraction. Mar. Drugs 2015, 13 (6), 3422–3442. Sosa-Herna´ndez, J. E.; Escobedo-Avellaneda, Z.; Iqbal, H.; Welti-Chanes, J. Stateof-the-Art Extraction Methodologies for Bioactive Compounds from Algal Biome to Meet Bio-Economy Challenges and Opportunities. Molecules 2018, 23 (11), 2953. Sumampouw, G. A.; Jacobsen, C.; Getachew, A. T. Optimization of Phenolic Antioxidants Extraction from Fucus vesiculosus by Pressurized Liquid Extraction. J. Appl. Phycol. 2021, 33 (2), 1195–1207. Sun, H.; Ge, X.; Lv, Y.; Wang, A. Application of Accelerated Solvent Extraction in the Analysis of Organic Contaminants, Bioactive and Nutritional Compounds in Food and Feed. J. Chromatogr. A 2012, 1237, 1–23. Tierney, M. S.; Smyth, T. J.; Hayes, M.; Soler-Vila, A.; Croft, A. K.; Brunton, N. Influence of Pressurised Liquid Extraction and Solid–Liquid Extraction Methods on the Phenolic Content and Antioxidant Activities of Irish Macroalgae. Int. J. Food Sci. Technol. 2013, 48 (4), 860–869. Yin, S.; Shibata, M.; Hagiwara, T. Extraction of Bioactive Compounds from Stems of Undaria pinnatifida. Food Sci. Technol. Res. 2019, 25 (6), 765–773. Zakaria, S. M.; Kamal, S. M. M.; Harun, M. R.; Omar, R.; Siajam, S. I. Subcritical Water Technology for Extraction of Phenolic Compounds from Chlorella sp. Microalgae and Assessment on its Antioxidant Activity. Molecules 2017, 22 (7), 1105. Zakaria, S. M.; Kamal, S. M. M.; Harun, M. R.; Omar, R.; Siajam, S. I. Extraction of Phenolic Compounds from Chlorella sp. Microalgae Using Pressurized Hot Water: Kinetics Study. Biomass Convers. Biorefin. 2020, 1–9. Zhou, M.; Ni, R.; Zhao, Y.; Huang, J.; Deng, X. Research Progress on Supercritical CO 2 Thickeners. Soft Matter 2021, 17 (20), 5107–5115.

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Purification and fractionation of crude seaweed extracts by adsorption-desorption processes

6

Marı´a Salom
e Mariotti-Celisa, Pamela Raquel Rivera-Tovarb, e Ricardo Nils Leander Huama´n-Castillac, and Jos
P
erez-Correab a

School of Nutrition and Dietetics, Faculty of Medicine, Universidad Finis Terrae, Santiago, Chile. bChemical and Bioprocess Engineering Department, School of Engineering, Pontificia Universidad Cato´lica de Chile, Santiago, Chile. cSchool of Agroindustrial Engineering, Universidad Nacional de Moquegua, Moquegua, Peru

1. Introduction Seaweeds, or marine macroalgae, have been recognized as “natural functional foods” since their consumption is associated with the prevention of many human diseases. They contain nutrients and molecules that exert relevant health effects, such as anticancer, antidiabetic, anti-inflammatory, and antiviral bioactivities. Polyphenols are the main contributors to these properties in marine macroalgae due to their antioxidant, metal-chelating, and protein-inhibition activities (Baldrick et al., 2018; Barbosa et al., 2019; Corona et al., 2016). The structure of marine macroalgae polyphenols varies from simple molecules, such as phenolic acids, flavonols, and flavanols, to the more complex phlorotannins, which consist of polymeric structures made up of units of phloroglucinol (1,3,5trihydroxybenzene), typically isolated from marine brown algae such as Durvillaea antarctica and Durvillaea incurvata (Erpel et al., 2020, 2021; Mateos et al., 2020; Santos et al., 2019). Seaweed polyphenols contain one or more phenolic rings, which may be halogenated to confer different and often stronger biological activities (Cabrita et al., 2010; La Barre et al., 2010). Halogenated polyphenols, like bromophenols, have been detected mainly in Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00009-1 Copyright # 2023 Elsevier Inc. All rights reserved.

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red algae such as Ceramiales, Gelidiales, and Corallinales, but these polyphenols are also present in brown and green seaweeds (Choi et al., 2012). Extraction, purification, and isolation processes should be applied alone or combined to obtain seaweed polyphenol extracts with different degrees of purity. Extraction is the first fundamental process, where the compounds of interest are transferred from the natural matrix into the solvent. Typically, the nature of the solvent, extraction temperature and time, solvent/solid ratio, and particle size are optimized to obtain high concentrations of polyphenolics in the seaweed extracts (Erpel et al., 2020). However, this process is unselective since the crude extracts contain large amounts of other compounds such as carbohydrates and lipoidal material (Erpel et al., 2020, 2021; Pacheco et al., 2020; Parada et al., 2019). Additionally, seaweed extracts contain heavy metals, questioning their use as raw materials for obtaining natural generally recognized as safe (GRAS) additives (Erpel et al., 2020, 2021; Roleda et al., 2019). Thus, integrating subsequent purification processes to eliminate undesirable compounds is required. The purification stage aims to separate non-polyphenolic compounds (difficult to handle in food and nutraceutical applications) from the crude extract to achieve a concentrated polyphenolic extract (Muzaffar et al., 2015). Resin purification (RP) is a solid-phase extraction method that typically removes carbohydrates, organic acids, and other polar non-polyphenolic compounds such as 5-hydroxymethylfurfural (5-HMF). RP involves adsorption and desorption with macroporous resins such as amberlite, XAD-2, XAD-7, and HP-20; they have been used successfully to concentrate seaweed polyphenolic extracts (Dai and Mumper, 2010; Huaman-Castilla et al., 2019). Specific polyphenols and polyphenol fractions can be obtained from purified extracts in the isolation or fractionation process. Presently, adsorption preparative liquid chromatography (APLC) has become essential for fractionating high-value compounds on laboratory and industrial scales (Gu et al., 2006). Specifically, APLC with cross-linked dextran, silica, and agarose gel has been used for seaweed polyphenols isolation obtaining high purity fractions (Wang et al., 2013; Zou et al., 2008). APLC, unlike RP, uses microporous adsorbents as stationary phase, which provides more selective interactions with each polyphenol (adsorbates) of the seaweed extract. During the adsorption-desorption separation processes (i.e., RP and APLC), a liquid (mobile phase) is pumped through a bed of porous particles (stationary phase or adsorbent). Those

Chapter 6 Purification and fractionation of crude seaweed extracts

compounds (adsorbates) dissolve in the mobile phase and interact differentially with the stationary phase, resulting in adsorbates moving through the stationary phase at different speeds, eventually leading to their separation. Adsorbates diffuse in and out of the stationary phase particles, undergo thermodynamic interactions, or form transient chemical bonds until they finally leave the column (He et al., 2004). The type of interaction that governs the separation of compounds by RP and APLC depends on the physical and chemical properties of the stationary phase, the adsorbate, and the mobile phase (Qi et al., 2007). Currently, different studies have evaluated the best operational conditions to obtain both purified and isolated seaweed polyphenols. However, many of them do not consider how the solvent used in the previous extraction stage can affect the recovery of these bioactive compounds. Furthermore, it has not been defined yet how the intermolecular interactions between the seaweed polyphenols, solid-phase, and solvent can improve the selectivity and efficiency of these processes. This chapter compiles current RP and APLC applications to obtain seaweed polyphenol extracts. It discusses practical considerations regarding the solid phase, the design, and operating variables to maximize the seaweed polyphenol extracts’ yield and purity (response variables). Finally, the adsorption-desorption mechanisms behind RP and APLC are discussed as an alternative for establishing the operating range of the most relevant process factors.

2. Resin purification (RP): Description and applications in seaweed extracts Different extraction processes such as hot pressurized liquid extraction, ultrasound assisted extraction, and atmospheric extraction, among others, allow obtaining extracts rich in seaweed polyphenols. However, these separation methods are not selective, requiring a subsequent stage to remove interfering or unwanted compounds (Soquetta et al., 2018; Vilcanqui et al., 2021). RP, which uses macroporous resins, is one of the most frequently applied techniques to purify crude extracts obtained from vegetable matrices (Sun et al., 2013). This process is low cost, environmentally friendly, and simple to scale up, operate, and regenerate (Silva et al., 2013). RP consists of four stages: adsorption (A), washing (B), desorption (C), and regeneration (D) (Fig. 1). First, the target compounds (e.g., seaweed polyphenols) are retained in the packed column

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Chapter 6 Purification and fractionation of crude seaweed extracts

Fig. 1 RP of seaweed polyphenols.

according to their affinity with the resin during the adsorption step. Then, the resin is washed with distilled water to remove unwanted compounds (amino acids, sugars reducing, and heavy metals). In the third stage, an eluent (hydroethanolic mixture) is used to recover the polyphenols retained in the resin. Finally, the resin is regenerated (Buran et al., 2014; Lin et al., 2012; Silva et al., 2013). Macroporous resins are made of styrene-divinylbenzene (SDVB) or acrylic-based polymers (aliphatic ester or phenolformaldehyde) with different polarities. Their surface area can vary between 100 and 1000 m2/g, with pore diameters from ˚ (Table 1) (Li et al., 2010; Wang et al., 2013). 38 to 600 A Depending on the physicochemical characteristics of the macroporous resin, different compounds can be separated. Styrene-divinylbenzene polymer is a nonpolar resin with aromatic groups that can present a heterogeneous surface area and a range of pore diameters. These characteristics allow this resin to adsorb nonpolar substances of variable chemical structures and molecular weights, such as seaweed polyphenols (Sunresin, 2021).

Chapter 6 Purification and fractionation of crude seaweed extracts

Table 1 Characterization of macroporous resins.

Resin XAD-7HP XAD-761 XAD-16 XAD-1180 FPX-66 HP-20 XAD-2 SP-825

Chemical structure Aliphatic acrylic ester Phenolformaldehyde Styrenedivinylbenzene Styrenedivinylbenzene Styrenedivinylbenzene Styrenedivinylbenzene Styrenedivinylbenzene Styrenedivinylbenzene

Polarity

Surface area (m2/g)

Pore diameter (A)

Polar

500

450

Polar

200

600

Non-polar

800

150

Non-polar

700

400

Non-polar

700

200–250

Non-polar

600–690

510

Non-polar

330

90

Buran et al. (2014) and Sandhu and Gu (2013) Buran et al. (2014) and Sandhu and Gu (2013) Huaman-Castilla et al. (2019) Kim et al. (2014)

Non-polar

1000

38

Kim et al. (2014)

Nonpolar macroporous resins (polystyrene-divinylbenzene) have been applied to purify phlorotannins from crude seaweed extracts. Kim et al. (2014) found in a batch system that the HP-20 macroporous resin (25°C) reached a higher phlorotannin adsorption capacity (57.8 mg/g) than other styrenedivinylbenzene macroporous resins such as XAD-2 (20.1 mg/g) and SP-825 (38.6 mg/g). They mentioned that the phlorotannins adsorption capacity of a given resin is related to its physical characteristics, such as surface area and pore diameter. Several authors have highlighted that the HP-20 resin presents a higher surface area than the XAD-2 resin, which would increase the contact surface between phlorotannins and resin, favoring their interactions both at the external surface and inside the resin pores. Interestingly, the SP-825 resin presented a lower adsorption capacity of phlorotannins than the HP-20 resin, despite having a larger surface area and smaller pore sizes. In this sense, if the ˚ ), the adsorption of resin presents extremely small pores (200 76.6 >200 99.3 12.7

a

Moon et al. (2011). Eom et al. (2013).

b

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Chapter 7 Interactions with other macromolecules

groups, i.e., both hydroxyl groups and aromatic rings are able to bind proteins as in the case of terrestrial tannins (Le Bourvellec and Renard, 2012). For example, 8,80 -bieckol that contains 11 hydroxyl groups has a higher inhibitory activity against BACE1 than dieckol and eckol that contain 10 and 6 hydroxyl groups, respectively ( Jung et al., 2010). However, the flexibility and steric hindrance of the phlorotannins may also influence their inhibitory activity. Hence, the inhibitory activity of phlorofucofuroeckol A and dieckol is lower compared to 7-phloroeckol, whereas their molecular weights are higher (Eom et al., 2013) (Table 1). The presence of a hinder group at C-7 position and the formation of intra- and intermolecular hydrogen bonds between hydroxyl groups may cause conformational changes in the molecules that reduce their inhibitory effects. Most enzyme kinetic studies using either Michaelis-Menten, Lineweaver-Burk, or Dixon plots to determine the inhibitory activities of different phlorotannins (i.e., phlorofurofucoeckol, 7-phloroeckol, dieckol, fucofuroeckol, dioxinodehydroeckol) have shown that they exhibit non-competitive or mixed-type (Barbosa et al., 2020; Eom et al., 2012; Jung et al., 2010; Kang et al., 2012; Lee et al., 2009; Manandhar et al., 2019; Moon et al., 2011; Park et al., 2018; Wijesinghe et al., 2011; Yoon et al., 2009) and competitive (Kim et al., 2019; Moon et al., 2011; Park et al., 2018) inhibition against different enzymatic activities. Dieckol can act as a competitive (Moon et al., 2011) or non-competitive inhibitor (Lee et al., 2009) against α-glucosidase. This discrepancy could be due to the purity of both the enzyme and the phlorotannins, the environment in which they are stored, the experimental procedures, or to the method used to determine the kinetic parameters, i.e., Lineweaver-Burk for Lee et al. (2009) or Dixon plots for Moon et al. (2011). Hence, most of the phlorotannins analyzed perform a non-competitive or mixed-type activity, i.e., they preferentially bind to a wide region of the enzyme other than the active site. The binding of the phlorotannins to the protein may cause modifications in both its structure and shape, thus the modified enzyme is no longer able to bind correctly with the substrate. Molecular docking simulation is also used to clarify the binding sites and confirm the underlying mechanism, i.e., competitive and non-competitive inhibition and the type of bonds, by which the different phlorotannins inhibit enzymes. The docking simulation confirms the non-competitive ( Jung et al., 2010; Kang et al., 2012; Manandhar et al., 2019; Park et al., 2018) or the competitive (Kim et al., 2019; Park et al., 2018) inhibition determined by enzyme kinetic studies. A non-competitive inhibitor of tyrosinase

Chapter 7 Interactions with other macromolecules

eckol displayed six hydrogen bonds with six amino acids, and phlorofucofuroeckol-A displayed eight hydrogen bonds with eight amino acids. Moreover, electrostatic interactions at histidine and glutamine residues also stabilize the complex formed between the phlorotannins and the enzyme (Manandhar et al., 2019). Hence, as a competitive inhibitor of tyrosinase 2-phloroeckol and 2-O-(2,4,6-trihydroxyphenyl)-6,60 -bieckol is docked at the active site, 2-phloroeckol displays six hydrogen bonds with three amino acids and 2-O-(2,4,6-trihydroxyphenyl)-6,60 -bieckol displayed seven hydrogen bonds with eight amino acids. Both phlorotannins interact with the same amino acids, i.e., His85 and Asn260 (Kim et al., 2019). Moreover, in all cases, hydrophobic interactions stabilize the interactions and allow for positioning phlorotannins in the catalytic or the allosteric site of the enzyme (Kim et al., 2019; Manandhar et al., 2019). Although many studies have defined the parameters influencing the interactions between phlorotannins and proteins, an in-depth study of the impact of both the structure and the conformation of phlorotannins (position and number of hydroxyl groups and O-bridge linkages) and proteins (amino acid composition, size, secondary/tertiary structure, and shape) will be necessary to better understand the factors that govern these interactions and their biological activities.

2.2

Covalent interactions

Under similar experimental conditions, in contrast to terrestrial tannins (e.g., proanthocyanidins, prosetinidin, and gallotannins), which only bond non-covalently to proteins, some phlorotannins in marine plants (e.g., Carpophyllum maschalocarpum, Ecklonia radiata, and Lobophora variegata) can form covalent bonds with proteins by oxidation (Stern et al., 1996). At high pH, covalent bonds may be formed between the oxidized phlorotannin and the protein, as phenolics are more easily oxidized under these conditions. Stern et al. (1996) found that seaweed phlorotannin spontaneously oxidized and reacted with proteins to form dark complexes resistant to dissolution. For example, a light brown precipitate resistant to solubilization was obtained in an alkaline solution when Carpophyllum phlorotannins were incubated with protein. It can be presumed that stable covalent compounds were formed, since the precipitate cannot be redissolved by protein denaturants (e.g., sodium dodecyl sulfate) or by hydrogen bond-breaking solvents (e.g., dimethylformamide). When a mild reducing agent (e.g., dithiothreitol and β-mercaptoethanol) is added to the complex solution, the

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precipitates formed by the phlorotannins and proteins are readily dissolved in the dimethylformamide, meaning that under these conditions, non-covalent complexes are formed. These reducing reagents are effective over a wide pH range for a wide range of proteins and phlorotannins. These results demonstrated the significance of oxidation in interactions between phlorotannins and proteins to form covalent complexes. Structural differences between phlorotannins are key to their sensitivity to oxidation, which may affect interactions with proteins. The phlorotannins from Carpophyllum are more easily oxidized than other sources (e.g., Ecklonia and Lobophora). The mixture of phlorotannins found in Carpophyllum included fuhalol- and hydroxyphenol-type phlorotannin, which have an ortho hydroxy group on the terminal unit. The strong electrondonating hydroxyl group adjacent to the terminal unit enhances the sensitivity to oxidation on other hydroxyl groups. While phlorethol-type phlorotannin from Ecklonia did not contain any hydroxyl groups in the ortho orientation and is less sensitive to oxidation. Therefore, phlorotannins with an ortho-substituted hydroxyl group in one or more rings are likely to be the most susceptible to oxidation. Aryl-aryl-linked fucol-type phlorotannins are the least susceptible to oxidation because the aryl group has a weaker electron-donating group than the aryl ether (Stern et al., 1996). Thus, the environmental conditions causing the oxidation of phlorotannins are different from those of terrestrial tannins but result in the formation of covalent bonds with proteins.

3. Comparison between terrestrial and marine tannin interactions with macromolecules We will focus below on comparing the structure-activity relationships between terrestrial and seaweed tannins for macromolecules binding.

3.1

Mechanism of interactions

Tannins, whether of terrestrial or marine origin, have strong interactions with proteins and polysaccharides and involve both reversible, i.e., non-covalent bonds implying hydrogen bonding, hydrophobic interactions, and Van der Waals forces, and irreversible, i.e., covalent bonds caused after polyphenol activation by oxidation or nucleophilic addition, interactions (Le Bourvellec and Renard, 2012; Stern et al., 1996; Vissers et al., 2017).

Chapter 7 Interactions with other macromolecules

The reaction between terrestrial tannins and proteins involves three main stages. The first is binding to protein by hydrogen bonds and hydrophobic interactions leading to the formation of soluble complexes. Then, these soluble complexes aggregate by self-association and then precipitate via colloid formation. The kinetic and colloidal consequences of polysaccharides are different as the binding is rapid, and the interactions do not lead to aggregate formation (Renard et al., 2017). To our knowledge, with phlorotannins, no in-depth study of their ability to form soluble aggregates or colloids with both proteins and polysaccharides has been carried out. It can be assumed that the same complexation mechanisms will be implemented between phlorotannins and proteins or polysaccharides. Although non-covalent reversible tannin complexes have been studied, there are still some reasons to extend our work to include covalently stabilized complexes. At alkaline pH, phenol is readily oxidized to form radicals and quinones, which may react with proteins to form stable adducts. Even at low pH, phenols can spontaneously form radicals or react with biological radicals or reactive oxygen species to form phenol radicals, which can eventually be quenched by reaction with proteins. Early investigators determined that the quinone formed by tannin oxidation reacts with the side chains of lysine (amine) and cysteine (thiol) but did not covalently bind to other amino acids such as histidine, arginine, tryptophan, or amide functional groups (Mason and Peterson, 1965). To prevent spontaneous oxidation of tannins and inhibit covalent complex formation, tannin complexation studies are generally performed at weakly acidic to neutral pH. However, contrary to terrestrial tannins, phlorotannins can spontaneously form covalent adducts with bovine serum albumin even under weakly acidic conditions (Stern et al., 1996). That is, orthodiphenolic compounds are generally considered more susceptible to the spontaneous oxidation than phenolic compounds with meta-orientation, but phlorotannins derived only from brown marine algae are more susceptible to spontaneous formation of oxidation products than both proanthocyanidins and hydrolyzable tannins. Therefore, for phlorotannins, covalent and noncovalent binding may occur simultaneously. Whereas with proteins, the formation of addition products between terrestrial tannin o-quinones and nucleophiles (amino acids, peptides, proteins) has been monitored by different techniques, with polysaccharides only indirect evidence for the formation of covalent bonds between terrestrial tannins and polysaccharides are observed (Le Bourvellec and Renard, 2012; Renard et al., 2017). With phlorotannins for both proteins and

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polysaccharides, only indirect evidence, such as protein resistance to solubilization (Stern et al., 1996) and structure of adhesive (Vreeland et al., 1998), allow to favor the existence of covalent bonds. Although the interactions between terrestrial tannins and macromolecules have been intensively studied over the past 60 years, an in-depth understanding of all the mechanisms underlying the interactions between marine tannins and macromolecules is still lacking. These very interesting phenolic natural products can be further examined with other macromolecules regarding covalent or non-covalent interactions.

3.2

Influence of tannin structure

Some key structural characteristics namely molecular weight and the presence of hydroxyl groups and aromatic ring that influence the ability of both condensed tannins, hydrolyzable tannins, and phlorotannins to interact with macromolecules are commune. For terrestrial tannins, such as proanthocyanidins, the larger the molecular weight or the degree of polymerization, the more strongly they interact with proteins and polysaccharides. Because the addition of aromatic and hydroxyl groups enhanced hydrophobic interactions and hydrogen bonds (Le Bourvellec and Renard, 2012; Liu et al., 2020). However, for phlorotannin, the situation may not be the same. Phlorofucofuroeckol A and dieckol have a lower inhibitory activity than 7-phloroeckol, whereas their molecular weights are higher (Eom et al., 2013). Higher polymerization of larger phlorotannins may lead to steric hindrance and thus reduce their reactivity. The flexibility and conformation of tannins are also important factors, which led flexible tannins to bind more strongly to macromolecules than the more rigid molecules. For example, gallotannins are highly flexible and can easily alter their conformation by intramolecular rotation, while the intergalloyl covalent bonds in ellagitannins limit their ability to change their conformation, thus reducing their protein affinity (Le Bourvellec and Renard, 2019). Moreover, the type of linkage between the phloroglucinol units (e.g., ether bonds, phenyl bonds or both, and dibenzo-p-dioxin connection) could also influence their flexibility and hence their binding for macromolecules such as for condensed and hydrolysable tannins. Phlorotannins’ complexing ability may vary according to their chemical structure (position and number of hydroxyl groups and O-bridge linkages); however, their conformation and flexibility have not been studied yet.

Chapter 7 Interactions with other macromolecules

3.3

Influence of protein structure

Protein size, conformation, secondary/tertiary structure, and amino acid composition significantly influence protein interactions with terrestrial tannins. Thus, proteins that strongly bind to terrestrial tannins have a high basic residues content, high proline content, are large and hydrophobic, and have an open and flexible structure (Bordenave et al., 2014; Le Bourvellec and Renard, 2012). However, for phlorotannins, it seems to be different. Contrary to terrestrial tannins, phlorotannins affinity is higher for bovine serum albumin, a model of globular protein, than β-casein, an open random coil protein (Vissers et al., 2017). The difference observed could be due to the hydrophobicity/hydrophilicity of the protein but also to the conformational flexibility of the phlorotannins. At present, many studies have focused on the biological activities of phlorotannins (Meng et al., 2021), but there is no in-depth study on the effect of both the structure and the conformation of proteins that drive their interactions with phlorotannins. Understanding the biological properties of phlorotannins requires information about the characteristics of proteins that interact with them. Studies of marine tannin-protein interactions have lagged behind studies of terrestrial tannin-protein interactions. Therefore, this area needs more attention.

3.4

Influence of polysaccharide structure

Like proteins, interactions between tannins and cell walls or polysaccharides depend on the physical, chemical, and macromolecular features of polysaccharides. The cell wall and polysaccharide characteristics that affect their interaction capacity with terrestrial tannins are their botanical origin, the surface area and the porosity, the molecular weight, the conformation and flexibility, the degree of esterification of pectins, the presence of side chains and the branching ratios, and the presence in the cell wall of other molecules (Bordenave et al., 2014; Jakobek, 2015; Jakobek and Mati c, 2019; Le Bourvellec and Renard, 2012; Liu et al., 2020; Renard et al., 2017; Zhu, 2017). Land plant cell walls are structurally diverse depending on the composition, size distribution, shape, charge, extractability, and combination of their constituent components. They constitute a complex porous material consisting of a cellulose microfibril network, tethered by hemicelluloses, embedded in an amorphous matrix constituted mostly of pectins (Carpita and Gibeaut, 1993). Brown algae cell wall shares features with land plant cell walls but also exhibits unique characteristics. The brown algae cell wall is composed of a

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cellulose microfibril network, occurring at low levels, embedded within an alginate and fucose-containing sulfated polysaccharides (FCSP) gel that forms the greater part of the cell wall, FCSP acting as cross-linkers between cellulose microfibrils. In addition, brown algal cell walls contain short-chain hemicelluloses that might act as intermediate cross-linkers between FCSP and cellulose microfibrils and phlorotannins (Deniaud-Boue¨t et al., 2014). Alginates or alginic acids are linear anionic polymers made of β-(1 ! 4)-D-mannuronate (M) and α-(1 ! 4)-L-guluronate (G), which are C-5 epimers, arranged in blocks along the polysaccharide chain. Alginates are a form of three different blocks: two types of homopolymeric region, L-guluronic acid blocks (GG) that form “egg-box” junctions with calcium, D-mannuronic acid blocks (MM) that do not gel with calcium, and heteropolymeric region of D-mannuronic (M) and L-guluronic acid blocks (DeniaudBoue¨t et al., 2014). Fucoidans are sulfated heteropolysaccharides containing α-L-fucose residues that comprise highly sulfated homofucan molecules together with a variety of highly branched polysaccharides, ranging from high-uronic acid, low-sulfatecontaining polymers with a significant proportion of xylose, galactose, mannose, and rhamnose. Some α-L-fucose residues are also acetylated (Deniaud-Boue¨t et al., 2014). As such, alginates and fucoidans may therefore be seen as structural analogs to polysaccharides like pectins and hemicelluloses in land plants as a function of their structures. It can be supposed that their chemical and physical features, i.e., monosaccharide composition, M/G ratio, distribution of M and G unit along the chain, glycosidic bonds, degree of polymerization, branching site and degree of branching, degree of sulfation, molecular weight, shape, flexibility, and conformation, will drive their interactions with phlorotannins as in the case of interactions between pectins or hemicelluloses and terrestrial tannins. However, only a few studies dealing with interactions between phlorotannins and alginates and fucoidans are found (Abraham et al., 2021; Park et al., 2019), and no in-depth studies on the effect of the structural diversity of brown cell walls and their components like cellulose, alginates, and fucoidans have been performed.

4. Conclusions Although some features of phlorotannin-protein interactions have been studied, many important questions about interactions with other macromolecules remain unanswered. Hence, little data are available in the literature about the phlorotannins’ structure-activity relationship as in the case of terrestrial tannins.

Chapter 7 Interactions with other macromolecules

The most satisfactory data concerning complexation between phlorotannins and proteins have been obtained using enzyme inhibition with a characterization of IC50 and kinetic analyses via the Michaelis-Menten, Lineweaver Burk, or Dixon plots. In comparison, a huge range of techniques have been used to study the interactions between terrestrial tannins and macromolecules because interactions are complex, and no single technique can provide all the information required (Bordenave et al., 2014; Jakobek, 2015; Jakobek and Mati c, 2019; Le Bourvellec and Renard, 2012; Liu et al., 2020; Renard et al., 2017; Zhu, 2017). With polysaccharides, the most satisfactory data have been obtained during elucidation of the cell wall and adhesive structure and composition (Bitton et al., 2006, 2007; Bitton and Bianco-Peled, 2008; Vreeland et al., 1998; Vreeland and Laetsh, 1990) and studies of encapsulation of phlorotannins within biomaterial matrices (Abraham et al., 2021). As for terrestrial tannins, the biological properties of the phlorotannins are related to the presence of both the benzene ring skeleton, the hydroxyl groups, the linkages, and their degree of polymerization. However, as far as we know, no data are available in the literature regarding their flexibility and conformation in solution. Hence, as for terrestrial tannins, the interactions between phlorotannins and macromolecules, i.e., protein and polysaccharides, are based on various mechanisms and are affected by numerous factors, i.e., intrinsic and extrinsic. A schematic overview of the different non-covalent bonds and potential bind sites of interactions between phlorotannins and protein (A) and polysaccharide (B) is visualized in Fig. 1. By exploring more types of phlorotannins with different degrees of polymerization, bond type, and branching, a wider understanding of the interactions can be gained. Only a limited number of molecules have been examined, and many macromolecules, e.g., proteins, carbohydrates, and lipids, have been neglected. Moreover, more in-depth research is still needed on the separation, purification, and characterization of the structure and conformation of the phlorotannins. The use of purified, structurally defined phlorotannins is essential for the exploration of the biological activities as their structure greatly affects their physical and chemical properties. Understanding interactions between phlorotannins and both proteins and polysaccharides will allow better understand their biological activities and design extraction and purification protocols to further use them as food ingredients or nutraceuticals with targeted functions. Given the lively interest in marine tannins in human health and disease and their importance in the beverage and food industry, it is likely that tannin-macromolecule interactions will continue to be an active area of investigation in the future.

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O

A

R

H N

O

N H

protein R NH

N N H

OH HO

HO

NH

H

OH

H O HO

HO

O

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H

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HO H O O H

HO

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H

OH

H O

hydrophobic interaction

O

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HO

OH HO

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(R)

O

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(R) O

H3C

(S) OR

O

O

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(S) O

O polysaccharide alginate

(S) O

(S) OR

O

(S)

(S) OR

O

(S)

CO

O– OH

H3C

O–

HO

OH

O

hydrogen bond

(S) polysaccharide fucoidans R = H or sulfate

Fig. 1 Potential non-covalent molecular interactions between phlorotannins (tetrafucol A, i.e., a fucol-type phlorotannins) and protein (A) or polysaccharide (alginate and fucoidans) (B).

Chapter 7 Interactions with other macromolecules

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Recent advances in the encapsulation of marine phenolic compounds

8

Wendy Francoab, Migdalia Caridad Rusindo Arazoc, and Sergio Benavidesd a

Chemical Engineering and Bioprocess, Pontifical Catholic University of Chile, Macul, Chile. bHealth Science Department, Nutrition and Dietetics, Pontifical Catholic University of Chile, Macul, Chile. cDietetics School, Faculty of Medicine, Universidad Finis Terrae, Pedro de Valdivia, Providencia, Santiago, Chile. d Research Center in Agri-food and Applied Nutrition, Adventist University of Chile, Chilla´n, Chile

1.

Introduction

As stated in previous chapters, phenolic compounds (PCs) are ubiquitous phytochemicals that are produced in both terrestrial plants (Condezo-Hoyos et al., 2021) and marine macroalgae (seaweeds) (Fernando et al., 2020; Mekini
c et al., 2019; Mahendran et al., 2021). Although their natural function is to act as the defense system of these organisms, PCs possess great potential from both the nutritional and therapeutic standpoints. Recently, there has been a growing interest in PCs exclusively found in marine species such as phlorotannins and bromophenols (Savaghebi et al., 2020). Several research topics related to marine PCs have included their biological activity (Mahendran et al., 2021), metabolism (Ma et al., 2020; Matacchione et al., 2020), isolation methods (Gangopadhyay et al., 2016; Sun et al., 2021), and characterization techniques (Lucci et al., 2017). However, despite all these practical advantages and the current societal needs and challenges (health, well-being, or environmental protection), many limitations make it difficult for phenolic compounds of marine origin to be commercially available. Currently, there is a growing industrial interest in using these marine compounds in pharmaceutical, food, feed, and cosmetic applications (Martı´nez-Abad et al., 2013), but their use faces some challenges. First, a bottleneck for upscaling the extraction Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00011-X Copyright # 2023 Elsevier Inc. All rights reserved.

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technologies is mainly associated with the process and yield optimization; large volumes of organic solvents are used in traditional extraction methods, as well as long extraction times, low extraction yields, and degradation of extracted compounds during the process (Casado et al., 2020; Dzah et al., 2020; Piccolella et al., 2019). Once a marine extract is obtained, subsequent steps must be tackled by a transdisciplinary team to develop commercial applications, which should define the delivery format, study different bioactivities, perform preclinical validations, and carry out consumers’ acceptance tests (Rotter et al., 2021). Typically, PCs extracts cannot be directly incorporated into foods or pharmacological matrices because their bioactive compounds are fragile in the environment; they are mainly sensitive to light, oxygen, extreme pH, and high temperatures (Marisa Ribeiro et al., 2020; Massounga Bora et al., 2018). For example, phlorotannins are very sensitive to dissolved oxygen, light, temperature, and time during their storage (Cassani et al., 2020). Additionally, phenolic compounds can interact with other food or pharmaceutical matrix components, such as proteins, lipids, or complex carbohydrate macromolecules, thus reducing their potential beneficial effects (Zhang et al., 2014). The protection and stabilization of the PCs are required to increase their bioaccessibility and bioavailability, expanding their application and commercialization. The most used and effective technology for PCs’ preservation is encapsulation, where the bioactive compound gets trapped within a polymeric matrix (de Sousa et al., 2018) that acts as a semi-permeable barrier (Mourtzinos and Goula, 2019). This chapter provides an overview of current advances related to methodology and carrier systems used to encapsulate marine polyphenols and discusses their effectiveness, limitations, and applications.

2.

Encapsulation techniques

Encapsulation is a “packaging” technique in which a bioactive agent is incorporated into a polymer matrix or coating material (Benavides et al., 2016; Lengyel et al., 2019) to form a capsule. According to size, capsules can be classified as nanoparticles (1 to 100 nm) or microparticles (100 to 1000 nm). There are currently two major structures for encapsulation at the micrometric level: microencapsulation and microspherification (Zanetti et al., 2018). A microcapsule (Fig. 1) has a bioactive core and an outer shell consisting mainly of the polymer used (Benavides et al., 2020). A microsphere, on the other hand (Fig. 1), is a solid matrix where the bioactive component is evenly distributed throughout the matrix (Benavides et al., 2016; Martı´n et al., 2015). The

Chapter 8 Recent advances in the encapsulation

Microcapsule

Microsphere Polymer

Bioactive compound

Fig. 1 Encapsulating matrices.

bioactive agent should be solubilized or emulsified in the biopolymer solution before structuring the microspheres to achieve a homogeneous distribution in the containment matrix. The purpose of encapsulating bioactive compounds can vary according to the specific application (Fig. 2). For instance, purposes may include protection, trapping, flavor masking, compatibility stabilization, and controlled release of encapsulated compounds. On the other hand, not all encapsulation techniques and carriers are appropriate for all active ingredients and purposes (Chaware et al., 2020), and therefore the encapsulating method as well the shell coating material have to be carefully studied and selected. Microencapsulation techniques can be classified into three main methods: physical, chemical, and physicochemical (Fig. 3). In the first method, the encapsulating matrix is structured using physical processes, mainly dehydration or cooling. In the second method, the encapsulating matrix components are subjected to chemical interactions between them or with the bioactive agent. Finally, in the physicochemical methods, chemical interactions together with physical processes allow the formation of the encapsulating matrix (Ye et al., 2018). Microencapsulation methods reported for marine polyphenols or extracts include complexation, liposome entrapment, and drying; these applications are summarized in Table 1.

2.1

Chemical encapsulation

2.1.1

Complexation

Complexation refers to the entrapment of a “ligand” (i.e., encapsulated compound) within a cavity-bearing “substrate” (i.e., carrier material or coating) via chemical interactions, e.g.,

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Chapter 8 Recent advances in the encapsulation

Coating CH3

CH3

OH

Protect against adverse environmental factors

CH3

CH3 OH

OH

CH3

CH3

CH3

CH3

CH3 OH

CH3

Controlled release OH

CH3 CH3 CH3

CH3 OH

CH3

CH3

CH3

Bioactive compound

CH3

Microcapsule Fig. 2 Encapsulation advantages.

Physical

Fig. 3 Encapsulating methods classified according to the type of capsule formation.

• • • •

Spray-drying Spray-cooling Freeze drying Thermoplastic extruction • Co-extrution

Chemical • Ionic gelation • Complex coacervation • Liposome entrapment

Physicochemical • Interfacial polymerization • Molecular inclusion

van der Waals forces, hydrogen bonds, or hydrophobic effects. These complexes can be prepared by simple mixing, kneading, coprecipitation, or antisolvent precipitation (Hosseini et al., 2021a). Complexation has been extensively studied during the last few years to encapsulate active ingredients ( Jeyakumari et al., 2016). For this methodology, understanding the interaction between the polyphenols and other constituents of the matrix is essential and represents a challenge since the complexes formed might affect the bioactive compound bioavailability. Due to the significant amount of aliphatic and phenolic hydroxyl radicals, tannins often interact with carbohydrates and proteins (Bacelo et al., 2016), forming non-covalent hydrophilic or hydrophobic complexes with proteins, mainly when hydrophobic forces are relevant. Tannins are generally attached to proline residues through

Table 1 Literature review of encapsulation systems for marine phenolics. Encapsulation method Natural source Complexation

Bioactive/coating material Major findings

Phlorotannins/soybean protein Laminariaceous brown algae (Eisenia bicyclis, Ecklonia cava, and Ecklonia kurome)

Laminaria digitata

Phlorotannins/b-casein (random coil) and bovine serum albumin (globular)

Brown algae

Phlorotannins/ polyvinylpyrrolidone nanoparticles

Sporophyll from Undaria pinnatifida brown algae

Phlorotannins/Scomberomorus Niphonius myofibrillar protein

• The phlorotannins showed a significant radical scavenging activity against the superoxide anion and DPPH with IC50 values of 6.5–8.4 mM and 12–26 mM, respectively • The phlorotannins and soybean protein complex showed a DPPH-radical scavenging activity four times stronger than the lyophilized soybean protein extract • Complete protein resolubilization was associated with minor PhT resolubilization, 24% for the PhT/b-casein combination (pH 7), and 12% for PhT/BSA (pH 2) • The charge state of the protein mainly governs the resolubilization of the phlorotannin-protein complexes at pH deviating from pI • Phlorotannins encapsulated by PVP nanoparticles showed a slow and sustained release in simulated gastrointestinal fluids • PPNPS treatment decreased the ROS production by 12 and 18% at 6.25 and 12.5 mg/mL, respectively • Phlorotannin extract increased the gel strength and cooking yield in a dosedependent manner. The highest gel strength (308.43
8.12 mNcm) and cooking yield (76.16
1.40%) were obtained at 625 mmol/g protein PTE

References Shibata et al. (2008)

Vissers et al. (2017)

Bai et al. (2020)

Jiang et al. (2020)

Continued

Table 1 Literature review of encapsulation systems for marine phenolics—cont’d Encapsulation method Natural source Liposomes

Bioactive/coating material Major findings

Spirulina Strain LEB-18 and Methanolic and ethanolic Chlorella pyrenoidosa extracts/soy lecithin

Spirulina sp. LEB-18

Spirulina LEB-18

Brown seaweed Sargassum boveanum

Methanolic extract/soybean asolecthin and dimyristoylphosphatidylcholine Methanolic extract/soy and rice lecithin

Methanolic extract/soybean lecithin

• Liposomes with ethanolic extracts of Spirulina LEB-18 and Chlorella pyrenoidosa showed higher encapsulation efficiency with 92.97 and 96.40%, respectively • Ethanolic extracts showed to be more stable during 21 days of storage than methanol extracts • The encapsulated extract showed higher antifungal activity (90% vs. 74%) and a slower release profile than its free form • The phenolic compounds were protected during gastric digestion for their subsequent release in the small intestine • The encapsulation of the phenolic extracts in soy and rice lecithin liposomes showed an increase of the bioaccessibility values from 31.65 to 35.83 and 45.89%, respectively • The entrapment efficiency of phenolic compounds for the optimal conditions was 45.5%, and high zeta potential (37.3 to 50.7 mV), and narrow particle size (86.6 to 118.7 nm) of the nanoliposomes indicated high stability of the formulation during storage • The nanoliposomes encapsulation of algal extract can protect the encapsulated material against thermo-oxidative decomposition • After encapsulation in nanoliposomes, the antioxidant activity of the phenolic compounds in the algae extract decreased but remained at an acceptable level

References de Assis et al. (2014)

Pagnussatt et al. (2016) Machado et al. (2019)

Savaghebi et al. (2020)

Red algae Kappaphycus alvarezii

Spray-drying

Methanolic extract/folic acidPEG-DSPE conjugate

• PEGylated liposome enhanced the delivery of the K. alvarezii extract in MCF-7 cells with IC50 of 81 mg/mL for the cytotoxicity MTT assay Tetraselmis chuii microalga Biomass/maltodextrin, gum • Beta-carotene (80%–92%) and phenolic comarabic, chitosan, and gelatin pounds (46%–81%) were preserved in the microencapsulated microalgae after 3 months of storage at 25 °C • Maltodextrin at 130 °C was found to be the best wall material Brown algae Sargassum Phlorotannins/maltodextrin, • Phlorotannin content and antioxidant activiserratum glucose, and saccharose ties were affected by the spray drying conditions • The optimal spray drying condition (10% carrier-to-solution ratio, 0.8 bar compressed air pressure, 10 mL/min liquid feed speed, and 110 °C inlet temperature) provides the highest phlorotannin content and antioxidant activity Brown seaweed Aqueous extract/polysaccharides • Whey protein and gelatin exhibited the best EE (87.11% and 86.59%, respectively), while (Saccharina japonica) (dextrin, maltodextrin, lactose, the polysaccharides presented EE efficiency and gum arabic) and proteins below 40% (whey protein, gelatin, and • The smallest particles were observed in polysodium caseinate) saccharides, while proteins showed the largest particles Brown seaweed Extract powder/maltodextrin • The stability of phlorotannin compounds was (Sargassum plagyophyllum) improved with the freeze-drying process with maltodextrin • The maltodextrin-dried powder has a higher phlorotannin content (113.06 mg of phloroglucinol) than dried powder without maltodextrin (37.10 mg of phloroglucinol)

Baskararaj et al. (2020)

BonillaAhumada et al. (2018)

Cuong (2020)

Nkurunziza et al. (2021)

Anwar et al. (2018)

Continued

Table 1 Literature review of encapsulation systems for marine phenolics—cont’d Encapsulation method Natural source Freeze-dried/ liposomes

Sea fennel (Crithmum maritimum)

Brown algae

Electrospinning

Brown algae

Bioactive/coating material Major findings

References

Ethanolic extract/soy lecithin

Alema´n et al. (2019)

• The liposomes showed an entrapment efficiency of 65.6% and 49.1% for the aqueous extract and the ethanolic extract, respectively • Higher antioxidant activity was found in the liposomes loaded with the ethanolic extract Phlorotannins/polyethylene oxide • Phlorotannins-loaded nanofibers effectively and sodium alginate decreased the cell count drastically from 6.20 to 3.28 log CFU/g at 4 °C and from 8.80 to 2.53 log CFU/g at 25 °C • Nanofibers improved the shelf life of preserved chicken for a prolonged period Phlorotannins/Momordica • Cold plasma treatments of the phlorotanninscharantia polysaccharide loaded nanofibers increased their release efficiency (by 23.5 and 25% for 4 and 25 ° C, respectively) • Nanofibers improved their antibacterial and antioxidant activities

Surendhiran et al. (2019)

Cui et al. (2020)

Chapter 8 Recent advances in the encapsulation

conjugation of CH-π, which are then reinforced by hydrogen bonds between the carbonyl groups of the peptide bonds, flanking the proline residue and the phenolic hydroxyl groups (Vissers et al., 2017). Understanding the interaction between polyphenols and other matrix constituents remains a challenge; hence, developing complexation encapsulation methods is difficult. Few studies have applied this technique to encapsulate marine PCs (like phlorotannins) using protein/polysaccharide complexes; very little is known about the effect of these complexes on the bioavailability of the bioactive compound. Cassani et al. (2020) stated that the complexes formed between tannins and carbohydrates, or proteins, are poorly digested in the upper intestinal tract because of steric hindrance. They reach the colon almost unaltered, where they serve as substrates for the microbial community, resulting in readily absorbable metabolites. Shibata et al. (2008) prepared a complex of the phlorotannin crude extract with soybean proteins as a functional food ingredient. The phlorotannin-soybean protein complex had a potent DPPH-radical scavenging activity, higher than the soybean protein only. Bai et al. (2020) used a solid dispersion technique to encapsulate phlorotannins in a high molecular weight polyvinylpyrrolidone (PVP) shell. The water-soluble carrier (PVP) was able to form nanoparticles through complexation that were an effective oral delivery vehicle and protected from oxidative skin damage. Jiang et al. (2020) enhanced the gel properties of minced mackerel Scomberomorus niphonius myofibrillar protein by using phlorotannin extracts (PTE) from sporophyll of Undaria pinnatifida at different levels (0, 25, 125, 625 μmol/g protein), under ultraviolet A (UVA) irradiation. UVA irradiation and the addition of phlorotannin extracts induced the formation of protein-phenolic complexes, promoting protein crosslinking.

2.2

Lipid-based structured delivery vehicles: Liposomes

Liposomes are one of the most researched encapsulation techniques because they are effective drug delivery vehicles to administrate nutrients and pharmaceutical drugs (Benavides et al., 2012; Bozzuto and Molinari, 2015; Massounga Bora et al., 2018). The technique involves the formation of lipid vesicles from aqueous dispersions of amphiphilic molecules, e.g., polar lipids, which tend to produce at least one lipid bilayer structure (Mohammadabadi and Mozafari, 2018). Liposomes are typically spherical (Fig. 4), with sizes varying from nanometers (less than 200 nm) to micrometers; the vesicles formed may contain single or multiple layers of

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Hydrophobic bioactive

Fig. 4 Liposome structure.

Hydrophilic bioactive

Lipid bylayer

Aqueos core

amphiphilic polymolecular membranes (Taylor et al., 2005; Vieira et al., 2020). Liposomes can be formed using several methods, including mechanical dispersion, detergent depletion, and solvent depletion (Mohammadabadi and Mozafari, 2018). The main advantages of liposomes are the possibility of encapsulating both lipophilic and hydrophilic compounds and their versatility in terms of size and number of layers. Besides, liposomes can enhance particular bioactive properties, such as antifungal and anticytotoxic (Machado et al., 2019; Prakash et al., 2018), antioxidant (Gonza´lez-Reza et al., 2018), and anticancer properties (Baskararaj et al., 2020). Liposomes can augment the stability of PCs by protecting them from extreme pH, temperatures, and ion concentrations (Mozafari et al., 2008). According to Pagnussatt et al. (2016), the interaction between the phenolic extract from Spirulina sp. LEB-18 and phosphatidylcholine-based liposomes led to the highest antifungal activity compared to the free extract, and the active principles encapsulated were released slower than the free phenolic extract. The authors stated that the phenolic compounds promoted ordering and disordering effects in specific regions of the phosphatidylcholine liposomes, thus increasing their bioactivity. In another study (Machado et al., 2019), phenolic extracts from Spirulina LEB-18 were encapsulated in liposomes with soy (S-SL) and rice lecithin (S-RL) as encapsulating agents by using the reverse phase evaporation technique. Liposomes exhibited high encapsulation efficiency (88.28% and 97.35% for S-RL and S-SL, respectively) and sizes between 250 and 291 nm. Bioaccessibility values increased in the soy and rice lecithin liposomes compared to the free phenolic extracts from 31.65% to 35.83% and 45.89%, respectively. The encapsulated phenolic compounds extracted

Chapter 8 Recent advances in the encapsulation

from the algae were protected during gastric digestion, allowing bioactive compounds to be released in the small intestine. These results provide essential information about the design of liposome-marine polyphenol formulations and their effect on in vitro gastrointestinal digestion. Baskararaj et al. (2020) encapsulated a polyphenol-rich extract from Kappaphycus alvarezii with folate conjugated PEGylated liposome to target the overexpressed folate receptors in human adenoma MCF-7 breast cancer cells. PEGylated liposomes were found to be nanometric (140 nm diameter), and this helped to increase the cellular uptake efficiency. The PEGylated liposomes loaded with the phenolic extract inhibited cell growth and induced apoptosis. Thus, use of liposomes was demonstrated to be an effective strategy for cancer treatment through site-specific delivery of phenolic compounds, reducing toxicity and multidrug resistance (MDR). The research group of Savaghebi et al. (2019) studied the Iranian macroalgae of the Sargassum genus, especially Sargassum boveanum, which is considered a rich, natural, fresh, and economical mixture of bioactive substances, like meroterpenoids, phlorotannins, and fucoxanthins (Lim et al., 2019). First, the feasibility of the algal extract encapsulation in nanoliposomes was evaluated; in addition, the nanoliposome stability, release behavior, and antioxidant activity of free and entrapped extract were studied (Savaghebi et al., 2019). The nanoliposomes exhibited a high level of stability under storage conditions with zeta potential values that varied between 37.3 and  50.7 mV. The systems showed a controlled release of phenolic compounds at different pH values, where the release of phenolic compounds burst at pH ¼ 3. Although the antioxidant activity of the formulated nanoliposomes showed a decrease in the EC50 values for DPPH, ABTS, and FRAP (267.7, 453.9, and 152.6 ppm) compared to the free algal extract, it maintained an acceptable level. Even with these promising studies, commercial applications of liposomes are still under review due to their potential toxicity associated with the formation of organic solvents’ residues and their low physical and chemical stability (Hosseini et al., 2021a, 2021b).

2.3

Physical encapsulation

2.3.1

Drying

Drying techniques have been used to encapsulate different compounds for many years. The methodology has several advantages allowing mass production, such as low costs, simple

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operation, and flexibility. Two major drying techniques are used for encapsulation: spray-drying and freeze-drying.

2.3.2

Spray-drying

In order to produce a spray-dried capsule, the bioactive compound needs to be dissolved or dispersed into an aqueous solution that contains the coating polymer. The mixture is then dried with a counter-current hot air stream, yielding a fine powder with particle sizes ranging from 1 to 50 μm. The size, morphology, encapsulation efficiency, and loading capacity of the resulting particles depend on the dryer operating conditions and the properties of the coating material. Moreover, this material determine under which conditions (mainly pH and temperature) the bioactive compound is released; hence, a good selection is critical. Many studies have applied spray-drying to protect and ensure the proper delivery of marine bioactive compounds, especially microalgae. Bonilla-Ahumada et al. (2018) encapsulated Tetraselmis chuii, using a Maltodextrin-Gum Arabic (60:40) complex (MGC), chitosan (3%) and gelatin (2%). Although MGC microcapsules had moistures above 5%, after 3 months of storage at 25 °C, 80% of the phenolics and 90% of the β-carotene were preserved. The antioxidant activity of the stored microcapsules was 6.2
0.2 (μg/g ascorbic acid equivalent). The microcapsules, which dissolved quickly in cold water and showed a bright green color, were attractive for rotifers and larval fish. Nkurunziza et al. (2019) analyzed the use of different polymers (dextrin, maltodextrin, lactose, Arabic gum, whey protein, gelatin, and sodium caseinate) for the spray-drying encapsulation of supercritical water polyphenolic extracts of Saccharina japonica. The extract showed a varied polyphenol composition, where p-hydroxybenzoic acid was the most abundant (40%). The best encapsulation efficiency was observed for the whey protein and gelatin encapsulation with yields of 87.1% and 86.6%, respectively. The capsules formed with gelatin showed the highest p-hydroxybenzoic acid concentration (28 mg/g), while the capsules formed with whey protein were less concentrated (22 mg/g). The protein microcapsules were the largest (average size of 143 μm), while the microcapsules formed with the polysaccharides were 9 to 11 times smaller. The highest antioxidant activity was observed for the whey protein microcapsules with DPPH values of 7.07
0.03mg TE/g and 22.7
0.34 mg TE/g for ABTS. Given these results, the authors concluded that both gelatin and whey are interesting coating materials for the encapsulation of the polyphenol extracts. Moreover, both polymers have the GRAS (generally recognized as safe) status, which

Chapter 8 Recent advances in the encapsulation

allows future functional food applications. However, more studies are needed to determine the release of the bioactives under different processing and storage conditions. The spray-dryer operating conditions (flow rate, inlet/outlet temperature, polymer/bioactive relation, and pressure) are essential since they impact the encapsulation efficiency and the loading capacity. Xuan (2021) studied the optimal conditions for encapsulating phlorotannins extracted from Sargassum serratum, reporting the following optimum conditions: 10% of phlorotannin extract, 0.8 bar, a feed flow rate of 10 mL/min, and 110 °C inlet temperature. The antioxidant activity of the resulting microcapsules reached the highest values: 4.35
0.01 g ascorbic acid equivalent/100 g, 9.39
0.02 g FeSO4 equivalent/100 g reducing powder, and 70.02%
0.26 free radical scavenging activity. The powder formed had a phlorotannin content of 2.29
0.01 g of phloroglucinol equivalent/100 g, about 5% of moisture, and 100% of solubility degree (Cuong, 2020). Optimizing spray-drying conditions is challenging when working with polyphenol extracts since they are susceptible to temperature damage. Moreover, the dryer conditions affect the feasibility of microparticles’ mass production (scaling-up); therefore, optimization of the operation is critical.

2.3.3

Freeze-dried liposomes

As mentioned before, liposome encapsulation is a suitable method to protect bioactive compounds against pH, temperature, and the presence of ions that might affect their stability. However, aqueous liposomes are susceptible to losing their stability due to fusion, aggregation, and sedimentation, which results in the entrapped bioactive compound release (Sharma and Sharma, 1997). Freeze-drying can enhance the stability of the liposomes; although it has been barely explored for marine extracts’ encapsulation, a longer shelf-life due to water activity loss can be achieved (Liapis and Bruttini, 2020). Alema´n et al. (2019) proposed the use of freeze-dried liposomes to encapsulate phenolic compounds extracted from sea fennel (Crithmum maritimum). This edible halophile alga is abundant in the Atlantic and Mediterranean sea coast and is rich in polyphenols and antioxidants, especially chlorogenic acid and vitamin C (Nabet et al., 2016). The dried capsules can be industrially produced, with reasonable costs, and easily added as food ingredients. For the encapsulation, aqueous and ethanolic extracts, combined with glycerol and semi-purified soybean phosphatidylcholine (PC), were frozen at 80 °C and then lyophilized. The

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encapsulation efficiency was 65.6% and 49.1% for the aqueous and ethanolic extracts, respectively. Although chlorogenic acid was the most abundant polyphenol in the ethanolic extracts, most compounds, including rutin and rosmarinic acid, ended up on the surface of the liposome. The release of these surface polyphenols is fast, producing a shock effect at the desired site, enhancing the bacterial control in pharmaceuticals, cosmetics, and foods; however, a prolonged therapeutic effect is not feasible. The solids’ concentration of the aqueous extracts affected the size of the dried liposomes, varying from 171.1 nm to 311.4 nm when the extract concentrations were 5 and 64% (with respect to PC weight), respectively. Since the partially purified soy polyphosphates as encapsulating polymers are considered GRAS, the liposomes obtained might be suitable for food supplementation. However, more studies are necessary to evaluate the impact of the encapsulated phenols as functional ingredients.

3.

Applications of encapsulated microalgaederived products

Due to their bioactive attributes, phenolic compounds extracted from marine sources could be exploited to produce and enrich several products. However, the direct use of the extracts or bioactive compounds faces several challenges, including forming a uniform matrix and ensuring that they do not impair odor and flavor. In that sense, some studies have researched the potential of including microencapsulated marine extracts and compounds in food products and cosmetic applications. The following lines and Table 2 describe current applications.

3.1

Functional foods

Functional foods are natural or processed foods containing bioactive compounds that, when consumed, exert a beneficial health effects different from nutritional attributes (Fernando et al., 2020). These foods are usually produced by incorporating ingredients that prevent diseases or enhance overall well-being (Steinhart, 2006). There is increasing interest in using naturally derived bioactives as functional ingredients, where marinederived products stand out despite several factors limiting their commercial use. Some limitations are their high sensitivity to processing conditions (mainly temperature), short shelf-life, the release of odor and flavors, limited uptake and bioavailability, lack of compatibility with the food matrix, and degradation in the

Chapter 8 Recent advances in the encapsulation

253

Table 2 Different applications of marine polyphenolic compounds through encapsulation systems. Applications

Systems

Ingredient

References

Functional foods

Coloring/antioxidant Yogurt Noodles-like pasta 3D printed cookies

Biomass Biomass Biomass Biomass, extract Extracts

Gouveia et al. (2006) Niizawa et al. (2019) Zen et al. (2020) Vieira et al. (2020)

Mayonnaise

Cosmetics

Pharmaceuticals

Chicken meat preservation Cookies Skin care product (patch) Skincare product (UV protection) Facial cleaner emulsion Skincare product (photodamage protection) Tissue engineering (scaffold) Antifusarium

Phlorotannin Biomass Extract Biomass Extract Extract

Savaghebi et al. (2020) and Savaghebi et al. (2021) Surendhiran et al. (2019) S¸ahin (2020) Byeon et al. (2017) Hama et al. (2012) Liu et al. (2020) Hu et al. (2019)

Extract Extract

Jung et al. (2013) Pagnussatt et al. (2016)

gastrointestinal tract (Vieira et al., 2020). Encapsulation of these ingredients can improve the chance of developing successful commercial products.

3.1.1

Biomass capsules as functional food ingredients

Marine algae biomass is an excellent source of several bioactive compounds (Fernando et al., 2020; Wijesekara et al., 2011). Research has been conducted to explore the feasibility of direct microalgae incorporation in bakery, milk-derived, pasta, and vegetarian food products (Lafarga, 2019); however, interactions with the food matrix have limited their use. From the sensory standpoint, which is very important in food formulations, a disadvantage of this type of inclusion is the sensory effect (flavor and color) that might affect the overall perception and acceptance of the enriched food products. Moreover, the bioactive compounds derived from the marine sources must resist food processing conditions (pH, temperature, presence of light, and oxygen) to exert their beneficial effect. For example, phenolic compounds can be oxidized and react with amino acids, forming insoluble complexes, decreasing the nutritional profile of the food product

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Chapter 8 Recent advances in the encapsulation

YSMA

YSM

YS

YC

(Lafarga, 2019). Additionally, bioaccessibility and bioavailability should be ensured so that the bioactive metabolites are absorbed in the right place. Consequently, it is advisable to microencapsulate marine biomass for the formulation of food products. Encapsulated Spirulina platensis and Haematococcus pluvialis have been studied as food functional ingredients among the microalgal species. The first is widely recognized as a protein and antioxidant source, while the second is rich in the carotenoid astaxanthin, a natural colorant with antioxidant properties (Niizawa et al., 2019). Spray-dried S. platensis was added into yogurt formulations using encapsulation in pure maltodextrin and maltodextrin complexed with citric acid. Better encapsulation performance was observed with a maltodextrin-citric acid complex as a coating material, reporting 75% yield and 0.48 g S. platensis per g of microcapsules (da Silva et al., 2019). A significant increase in the antioxidant activity was observed with biomass inclusion. The yogurt, formulated with the encapsulated microalgae, showed EC50 of 9, 13, and 15 mg/mL for the maltodextrin complex encapsulation, free biomass addition, and pure maltodextrin encapsulation, respectively. The DPPH scavenging activity (2.1
0.1 μg/mL) of the yogurt with encapsulated biomass was higher than that with free microalgae (0.93
0.03 μg/mL). As expected, the protein content of the formulated yogurts increased by 0.9/100 g of fresh yogurt compared to plain yogurt. However, the protein concentration decreased with time, and after 7 days of cold storage, it was similar to plain yogurt. The inclusion of the encapsulated microalgae in the maltodextrincitric acid complex within the yogurt resulted in a more homogenous matrix. But, as expected, the yogurt color was altered and presented an opaque green color (Fig. 5); the microcapsules’ incorporation partially masked the odor.

Fig. 5 Different yogurt formulations, prepared with S. platensis encapsulated in a maltodextrin-citric acid complex (YSMA), maltodextrin only (YSM), free dry biomass (YS). YC represents plain yogurt. Adapted from da Silva et al. (2019).

Chapter 8 Recent advances in the encapsulation

Encapsulated S. platensis has also been incorporated into dry food products. Zen et al. (2019) used spray-dried alginate S. platensis microcapsules to fortify noodles-like pasta. The encapsulation efficiency was 87.6%, and the capsules contained high amounts of microalgae. The moisture and water activity of the fortified noodles were similar to plain noodles and within the Brazilian standard for this type of food (Zen et al., 2020). After cooking, the encapsulated biomass fortified noodles showed higher moisture, water absorption, firmness, and hardiness than plain noodles and free biomass fortified noodles; however, only 37.8% of the antioxidant activity was conserved after cooking. The color was affected by the inclusion of the free and encapsulated biomass, and the encapsulated biomass fortified noodles (fresh and cooked) presented green dots. The characteristic odor of this microalga was partially masked by encapsulation, and the panelists that tasted the pasta in a sensory analysis were still able to perceive fish-like odors in the formulated noodle. Nevertheless, the aroma was less intense than free biomass fortified noodles, where the overall acceptance of the encapsulated biomass fortified noodles was very similar to the plain noodles (88%).

3.1.2

Encapsulated microalgae extract as functional food ingredients

A few studies have been associated with encapsulated algae extracts as functional food ingredients. Vieira et al. (2020) studied the inclusion of S. platensis as free biomass, free extract, and encapsulated antioxidant extract to fortify 3D printed cookies. The extracts were encapsulated in alginate using a vibrational extrusion technique. The authors reported that the inclusion of the encapsulated extract reduced the cookie dough viscosity and thus lowered hardness, probably due to the water molecules present in the calcium-alginate network. After baking, the microencapsulated-enriched cookies lost more water and were smaller than the other fortified cookies without losing their hardness after 30 days of storage at room temperature. Interestingly, the antioxidant activity of the cookies with microencapsulated extracts remained high during storage, with ORAC values similar to the freshly prepared cookies with the free extract. The addition of the extracts affected the color of the dough and cookies, although those with microcapsules showed a less opaque tone. The acceptance and the characterization of other sensory attributes were not reported (Şahin, 2020). The bioactive compounds found in marine products can also be used as natural food preservatives for spoilage control.

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Savaghebi et al. (2021) developed a nano-liposome system to encapsulate Sargassum boveanum algae extracts. The antibacterial activity of the microcapsules was tested against common Gram-positive and Gram-negative bacteria. The encapsulated extracts could inhibit both types of bacteria, but greater inhibition was observed for the Gram-negative. Furthermore, the minimum inhibitory concentrations (MIC) to control Pseudomonas aeruginosa (0.937 mg/mL), Bacillus cereus (0.469 mg/mL), and Staphylococcus aureus (0.469 mg/mL) were 50% lower than the concentrations reported for the crude extracts. In the same study, Sargassum boveanum extracts encapsulated in nano-liposomes were included as an ingredient for mayonnaise formulation at a concentration of 1000 mg/kg. Over a 4 month storage period at room temperature, the samples with the nano-liposomes showed higher microbial stability when compared with samples without any additive or samples with the free extract. The mayonnaise with nano-liposomes showed a stable microbial shelf-life, with lower than 250 CFU/g of total viable microbial counts, less than 100 CFU/g of yeast and mold, and less than 7 CFU/g of lactic acid bacteria (LAB). Moreover, the protective effect of the encapsulated extracts was slightly lower than benzoic acid, a commercial food additive. Benzoic acid is an aromatic carboxylic acid, widely used in food applications as a preservative against spoilage microorganisms, including molds. It is a substance recognized as GRAS (generally recognized as safe), but its use in food products should not exceed 0.1% (United States Food and Drug Administration, 2022). Preservation of fresh foods is challenging, especially meats, prone to microbial deterioration and a transmission vector for food-borne bacteria. In particular, chicken meat has been associated with Salmonella enteritidis, responsible for salmonellosis that affects millions of people worldwide (Wessels et al., 2021). Surendhiran et al. (2019) studied the feasibility of using encapsulated phlorotannins in an alginatepolyethylene oxide (PEO) complex blended with nanofibers to control this bacterium in chicken meat. In vitro assays showed that S. enteritis cultures were 99.99% controlled after 12 h of exposure to the encapsulated bioactive extract. In addition, a microbiological challenge test was conducted by submerging 1 g chicken samples wrapped with the nanofibers enriched with encapsulated phlorotannins in a fresh culture of the bacterium. The samples were stored at 4 and 25 °C for 7 days. The nanofibers could control the bacterium growth under both conditions and even reduce the initial concentration by approximately 4 and 6 logs at 4 and 25 °C, respectively. Sensory analysis showed

Chapter 8 Recent advances in the encapsulation

that treated samples were not significantly affected, maintaining the sensorial quality (Surendhiran et al., 2019). In another study, mayonnaise was prepared with nanoliposome brown algae extracts. The addition of the encapsulated extract did not significantly affect the mayonnaise color, maintaining a white-yellowish color, while the mayonnaise formulated with the free extract showed a bright green color. Other factors affecting the sensory quality are the taste and the odor of the food product once the extracts are added. The mayonnaise samples formulated with nano-liposome had acceptance scores close to 4 on a scale from 1 to 5, indicating that encapsulation retarded the release of the characteristic odor and flavor of the microalgae extracts (Savaghebi et al., 2020).

3.2

Cosmetics

Formulating skincare products is a highly relevant business, with a market size forecasted by 2025 of 183 billion dollars and compound annual growth of 4.4% (Release et al., 2021). The consumer demands have challenged this industry to include naturally-derived bioactive compounds that prevent skin damage, commonly induced by free radicals such as reactive oxygen species (de Silva et al., 2017). Among the several natural compounds included in skincare formulations, marine-derived bioactives are an exciting option, particularly microalgae-derived products. Microalgae can produce many compounds to cope with the different stresses they are exposed to in their natural habitat (Fernanda Pessoa, 2012). However, the studies of these bioactives are still incipient, focusing on astaxanthin and C-phycocyanin which show potent antioxidant activities (Saide et al., 2021). A limiting factor for including these bioactives is their poor water solubility and chemical degradation, resulting in low bioavailability (Hosseini et al., 2021b). To cope with these challenges, encapsulation of purified bioactives, extracts, or plain biomass might be suitable. Hama et al. (2012) encapsulated Haematococcus pluvialis biomass in liposomes and evaluated their effect against UV-induced damage using a topical application before UV exposure. The authors reported that the application reduced UV-induced damage, prevented collagen degradation, and inhibited melanin production; this indicates that encapsulation is a feasible technique to incorporate poor water-soluble bioactives in skincare products. In another study, nanoparticles produced by emulsification and solvent evaporation, loaded with extracts from Haematococcus pluvialis encapsulated in polylactic-co-glycolic acid (PLGA),

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were studied for their photodamage protective effect using human keratinocyte cells (HaCaT) (Hu et al., 2019). The nanoparticles’ yield was reported as 96.42
0.73%, and the loading capacity was 7.19
0.12%. In vitro analysis with HaCaT cells exposed to UVB radiation showed that the encapsulated extracts were more effective against reactive oxygen species and showed low cytotoxicity. Moreover, the nanoparticles were able to restore the mitochondrial membrane potential, indicating that they are feasible ingredients for the formulation of skincare products. Castangia et al. (2016) encapsulated in hyalurosomes’ phycocyanin extracted from Klamath algae. These capsules were composed of phospholipid vesicles immobilized with hyaluronan sodium salt or PEG. The vehicles produced were homogeneous in both formulations, with sizes smaller than 150 nm. The encapsulation yields were 65% and 61% for the hyalurosomes and PEG hyalurosomes, respectively. In vitro permeability studies showed that the hyalurosomes increased the bioactive extract permeation and deposition in the deeper skin layer. In addition, the liposomes were able to increase the antioxidant activity of the bioactive extract when applied to stressed human keratinocyte cells. Recently, skin patches have gained attention for cosmetic formulations since they can deliver active ingredients directly to the skin. Byeon et al. (2017) developed a patch embedded with Spirulina extract/alginate and supported in polycaprolactone nanofibers prepared by electrospinning. The patch components did not show toxicity in human keratinocyte cell-based examinations. Wetting of the dry patch resulted in a 30 min release of the extract. To our knowledge, this is the first time a cyanobacterium has been used as part of a cosmetic patch, with promising results for skincare.

4.

Conclusions

Although encapsulation studies of marine-bioactives have been limited, this is an excellent option to protect their bioactivity and maintain or increase their antioxidant properties compared to the free compounds. Spray-drying and liposome entrapment have shown better results among the different available encapsulation techniques. However, the latter is difficult to scale up for industrial purposes, while spray-drying represents a cost-efficient and scalable technique. In terms of encapsulation performance, the selection of the coating polymer strongly impacts the yield, encapsulation efficiency, and loading capacity. Some applications have been reported in which the encapsulated compounds have

Chapter 8 Recent advances in the encapsulation

been included in food products, used as antibacterial, and supplements for skincare with promising results for increasing antioxidant activities, control of food-borne bacteria, and protection against UV radiation. The encapsulation of marine polyphenols and extracts shows immense potential for developing bioactive ingredients for the food, pharmaceutical, and cosmetic industry. However, research is still in its infancy, and more studies are necessary to standardize encapsulation parameters and test them in a broad spectrum of applications.

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Bioaccesibility and bioavailability of marine polyphenols

9

Salud Ca´ceres-Jim
eneza,b, Jos
e Luis Ordo´n˜ez-Dı´aza, a,c Jos
e Manuel Moreno-Rojas , and Gema Pereira-Caroa,c a

Department of Agroindustry and Food Quality, Andalusian Institute of Agricultural and Fisheries Research and Training (IFAPA), Co´rdoba, Spain. b Department of Food Science and Food Technology, Campus Rabanales, Ed. Darwin-anexo University of Co´rdoba, Co´rdoba, Spain. cFoods for Health Group,
Instituto Maimo´nides de Investigacio´n Biomedica de Co´rdoba (IMIBIC), Co´rdoba, Spain

Abbreviations ADME BSI CBG COMT FAO HMW LD LMW LPH MCT NMR OPLS-DA SPE SULT TPC UGT UHPLC-HR-MS UP

absorption distribution metabolism and excretion biomarkers of seaweed intake cytosolic β-glucosidase cytosolic catechol-O-methyltransferases Food and Agriculture Organization high molecular weight laminaria digitate low molecular weight lactase phlorizin hydrolase monocarboxylate nuclear magnetic resonance orthogonal partial least squares discriminant analysis seaweed polyphenol extract sulfotransferases total phenolic content uridine-50 -diphosphate glucuronosyltransferases ultra-high-performance liquid chromatography coupled to high-resolution-mass spectrometry Undaria pinnatifida

1. Introduction Seaweed has been a part of the Asian diet for centuries, and in recent decades, its consumption has increased in Western countries as a result of increased awareness of its beneficial effects (Pereira, 2015). According to FAO, the world production of algae Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00008-X Copyright # 2023 Elsevier Inc. All rights reserved.

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(mainly macroalgae) has increased significantly, from 4 million tons in 1990 to 34.7 million tons in 2019. Currently, this production represents 30% of the world aquaculture production of all species (Cai et al., 2021; Food and Agriculture Organization of the United Nations (FAO), 2020). Marine resources are known to contribute to a good nutritional status, algae being a rich source of proteins, minerals (calcium, iodine, phosphorus, potassium, and sodium, mainly), vitamins (A, C, D, and E), and dietary fiber (Lomartire et al., 2021; Shannon and Abu-Ghannam, 2019). Algae are also characterized by a low lipid content, while being enriched in polyunsaturated fatty acids (Pereira, 2015). Likewise, the interest that this marine resource arouses lies in a wide spectrum of non-essential compounds called bioactive or phytochemical compounds, related to activities that potentially promote human health. Standing out among these are phenolic compounds, which constitute a wide and diverse group of secondary metabolites ( Jimenez-Lopez et al., 2021). Recently, the study of marine phenolics has attracted great interest. However, research on phenolic compounds in marine sources is less advanced than those in land plants (Mateos et al., 2020). Both terrestrial plants and algae have been reported to share some groups of phenolic compounds, such as flavonoids or phenolic acids. However, there is one group of phenolic compounds found solely in algae, namely, phlorotannins (oligomers and polymers of phloroglucinol units) (Mateos et al., 2020; Shannon et al., 2021). Another characteristic phenolic group of algae is bromophenols, which can also be found in other marine sources such as sponges, ascidians, mussels, or marine proteobacteria (Agarwal et al., 2014; Dong €rn et al., 2020; Hattori et al., 2001; Lindsay et al., 1998; Malmva et al., 2005). Bioactive compounds such as bromophenols and phlorotannins present potential health benefits against several human diseases due to their potential antioxidant (Agrega´n et al., 2018; Kang et al., 2013), antidiabetic (Lee et al., 2009, 2016), antihypertensive (Kim et al., 2020), anti-inflammatory (Dong et al., 2019; Jung et al., 2013), anticancer (Abdelhamid et al., 2019; Kim et al., 2015; Sadeeshkumar et al., 2017), antiviral (Artan et al., 2008), antimicrobial (Maadane et al., 2017; Rajauria et al., 2012), and prebiotic (Charoensiddhi et al., 2017) activities. These findings are mainly based on in vitro studies and in vivo animal assays (Erpel et al., 2020). However, there is a lack of clinical intervention studies that analyze the human metabolism transformation whereby conjugated (methylated, sulfated, or glucuronide) metabolites and microbial phenolic acid catabolites derived from marine phenolic compounds are formed in the gastrointestinal tract or the liver. Nowadays, there is increasing evidence that these metabolites

Chapter 9 Bioaccesibility and bioavailability of marine polyphenols

are beneficial to human health, so studies aiming to determine the bioavailability of bioactive compounds are important to fully understand their bioactivity and, therefore, the real effects of phlorotannins or bromophenols in humans (Erpel et al., 2020). To exert these effects, the bioactive compounds present in marine sources must be bioaccessible and to some extent bioavailable. To date, little information is available regarding the bioavailability of marine polyphenol, with only three in vivo studies reporting data on the absorption, metabolism, and excretion of phenolic compounds present in marine sources. For instance, assessments have been performed on the microbial metabolites, also known as catabolites, derived from seaweed phenolic compounds excreted in the urine (Baldrick et al., 2018; Corona et al., 2016; Xi et al., 2020) and plasma (Baldrick et al., 2018; Corona et al., 2016) of healthy and overweight volunteers after brown algae intake. Understanding the degree of biotransformation and conjugation of marine polyphenols during their passage through the gastrointestinal tract and the liver is relevant in order to understand their ability to act as effective bioactive molecules in target tissues (Corona et al., 2017).

2. Algae marine polyphenols: Source and their occurrence • Source: microalgae and macroalgae (seaweed) There are more than 11,000 different species of algae divided into two groups: microalgae and macroalgae or seaweed ( Jimenez-Lopez et al., 2021). - Microalgae are a highly diverse group of unicellular eukaryotic organisms (Mateos et al., 2020) that have been proposed as an alternative source of natural antioxidants due to their metabolic diversity and adaptive flexibility compared to higher plants (Li et al., 2007; Safafar et al., 2015). However, the phenolic compounds present in microalgae tend to be less studied than those in terrestrial plants (Sansone and Brunet, 2019). Simple phenolic acids such as hydroxybenzoic and hydroxycinnamic acids have been reported to be the major families identified in microalgae (Mateos et al., 2020). These phenolic acids include 3,4-dihydroxybenozic acid, chlorogenic, 30 ,40 -dihydroxycinnamic acid, 3,4,5trihydroxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, and 40 -hydroxy-30 -methoxycinnamic acid, among others ( Jerez-Martel et al., 2017; Scaglioni et al., 2019). - Macroalgae, also known as seaweed, are grouped into three classes according to their pigment content: red seaweeds

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(Rhodophyceae), green seaweeds (Chlorophyceae), and brown seaweeds (Phaeophyceae) (Tanna and Mishra, 2018). Within these classes of macroalgae, brown algae are those with the highest content of phenolic compounds (Heffernan et al., 2015; Montero et al., 2017). The total polyphenolic content of brown seaweed is around 12%–14% of its dry mass, the content in red and green algae being much lower (maximum 5% of dry mass) (Holdt and Kraan, 2011; Shannon et al., 2021). In fact, brown algae are the most studied in terms of the bioavailability of phenolic compounds, possibly due to the presence of phlorotannins. Indeed, there is a family of brown algae called Fucaceae, which is the most dominant algae family along Northern Hemisphere shorelines (Catarino et al., 2017). • Polyphenols: Simple phenolics, bromophenols, and phlorotannins - Simple phenolics. They are mainly made up of two phenolic groups, hydroxycinnamic acids and hydroxybenzoic acids. The presence of these compounds has mainly been reported in brown algae (Mateos et al., 2020). Among hydroxycinnamic acids, it is worth highlighting 40 hydroxycinnamic acid (I), 30 ,40 -dihydroxycinnamic acid (II), 40 -hydroxy-30 -methoxycinnamic acid (III), chlorogenic acid (IV), and sinapic acid (V) ( Jimenez-Lopez et al., 2021). On the other hand, hydroxybenzoic acids include 3,4,5trihydroxybenzoic acid (VI), p-hydroxybenzoic acid (VII), 4-hydroxy-3-methoxybenzoic acid (VIII), and 3,5dimethoxy-4-hydroxybenzoic acid (IX), among others ( Jimenez-Lopez et al., 2021) (Fig. 1, Structures I-IX). - Bromophenols. They are secondary metabolites found in red, green, and brown seaweed (Mateos et al., 2020), where the red variety is the major natural source of these bioactives (Cotas et al., 2020; Liu et al., 2011). They are composed of one to five phenol groups with varying degrees of bromination (Cotas et al., 2020; Shannon et al., 2021). Specifically, tri-bromophenols are the most common metabolites found in seaweeds, followed by di- and mono-bromophenols (Chung et al., 2003; Shannon et al., 2021). Examples of these bromophenol groups are shown in Fig. 1 (Structures X, XI, and XII). Compared to phlorotannins, bromophenols are present in limited amounts in algae, so fewer studies have been conducted on their isolation and characterization (Cotas et al., 2020), despite red algae being the subject of extensive research.

Chapter 9 Bioaccesibility and bioavailability of marine polyphenols

269

SIMPLE PHENOLICS Hydroxycinnamic acids O

O

CH3

II

I

III

O

HO OH

OH

O OH

HO

HO

HO

4’-Hydroxycinnamic acid

4’-Hydroxy-3’-methoxycinnamic acid

3’,4’-Diydroxycinnamic acid OH

HO

C

IV O

V

OCH3 OH

HO

O HO

OH OH

Chlorogenic acid

OCH3

Sinapic acid

OH

Hydroxybenzoic acids O

OH

O

O

OH

VII

VI

COOH

IX

VIII

OH

HO

HO

H3CO

OH

OCH3

OCH3

OH

OH

OH

p-Hydroxybenzoic acid

3,4,5-Trihydroxybenzoic acid

4-Hydroxy-3-methoxybenzoic acid

3,5-Dimethoxy-4-hydroxybenzoic acid

BROMOPHENOLS OH

OH

OH

X

Br

Br

2-Bromophenol

XI

Br

Br

XII

Br

Br

2,6-Dibromophenol

2,4,6-Tribromophenol

PHLOROTANNINS OH

OH O

HO

OH O

HO

OH

OH

OH

OH

OH

O

OH

XIII

OH

OH

XIV

XVII

Trifuhalol A Fuhalol OH

O OH HO

OH HO

HO

OH O

O HO

OH OH HO OH

XVI

XV

OH

OH

O

OH

HO

OH

OH

O

OH

HO

O

OH

HO

OH

OH

Tetraphlorethol C

Difucol

Phlorethol

Fucol

Fucophlorethol A Eckol

Fucophlorethol

Fig. 1 Chemical structures of the main marine polyphenols: simple phenolics, bromophenols, and phlorotannins.

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Chapter 9 Bioaccesibility and bioavailability of marine polyphenols

- Phlorotannins. Compared with other polyphenols produced by seaweeds, phlorotannins are the most studied group. They are found in brown seaweeds (Francisco et al., 2020), Eisenia, Ecklonia, and Ishige genera being the main sources of bioactives (Erpel et al., 2020; Rosa et al., 2020). These secondary metabolites are characterized by a unique structure that is not found in terrestrial plants (Cotas et al., 2020; Freile-Pelegrı´n and Robledo, 2013). They are composed of phloroglucinol units (1,3,5trihydroxybenzene) and four groups of phlorotannins exist depending on the linkages between these phloroglucinol units, which are fuhalols (XIII) and phlorethols (XIV) (with ether linkages), fucols (XV) (with phenyl linkages), fucophlorethols (XVII) (with phenyl and ether linkages), and eckols (XVI) (with dibenzodioxin linkages) (Rosa et al., 2020; Singh and Sidana, 2013). Examples of these phlorotannins groups are shown in Fig. 1 (Structures XIII-XVII). Consequently, phlorotannins constitute a very heterogeneous group of compounds, which present a wide range of molecular sizes (126 Da to 650 KDa), commonly found in the 10–100 kDa range (Freile-Pelegrı´n and Robledo, 2013; Steevensz et al., 2012). It is believed that the activity of these compounds depends on their molecular weight; the greater the degree of polymerization, the greater its activity (Kirke et al., 2019; Meng et al., 2021). Compared with phenolic compounds from terrestrial plants, phlorotannins from brown seaweeds have better antioxidant activity (Balboa et al., 2013; Meng et al., 2021). Several in vitro and in vivo in animal studies have analyzed their bioactivity, showing their potential pharmacological and food applications. However, metabolomic studies and clinical trials focusing on the biological response of phlorotannins in the body are scarce (Erpel et al., 2020; Meng et al., 2021).

3. Gastrointestinal stability of marine polyphenols: In vitro approaches To exert their health effects, marine polyphenols must be available to a certain extent in the human body. Therefore, their biological effects, as with those of other dietary bioactive compounds, are closely related to the different steps of their metabolic fate. These steps include release from the food matrix by the action of the digestive enzymes or bacterial degradation, thus becoming bioaccessible, followed by partial absorption and

Chapter 9 Bioaccesibility and bioavailability of marine polyphenols

further transformation. Thus, bioaccessibility is defined as the fraction of a specific compound, in this case, a phenolic compound, that is released from the food matrix during digestion (bioaccessible fraction) and is capable of being absorbed by intestinal cells (Lorenzo et al., 2019). Therefore, the real content of the phenolic compound accessible to the human body may be different from that ingested (Francisco et al., 2020). Absorption is mainly influenced by their chemical structure and to a lesser extent by genetic, culinary, or environmental characteristics (Scalbert and Williamson, 2000). Most phenolic compounds found in food form complex structures (linked to acyl, ester, or glycoside groups, or even to other phenolic compounds) that cannot be absorbed. In general, only polyphenols in their aglycone form can be absorbed, so these complex structures must be hydrolyzed by intestinal enzymes or by colonic microbiota in order to be absorbed (Manach et al., 2004). After food intake, phenolic compounds are usually stable to the hydrolytic activity of saliva (Rodriguez-Mateos et al., 2014). Then, during gastric digestion, the release of polyphenols from the food matrix is promoted, and phenolic acids can be absorbed in their free form in the stomach without prior hydrolysis through monocarboxylate transporters (MCT), which are also involved in their intestinal absorption (Konishi et al., 2006; Lafay et al., 2006; Zhao et al., 2004). Subsequently, structural modifications of phenolic compounds continue to take place in the small intestine due to the action of pH and enzymes so that the compounds/metabolites produced can be absorbed (Corona et al., 2014). Polyphenols in their aglycone form, and to a lesser extent, a few glycosides can be absorbed in the intestinal mucosa (Manach et al., 2004; Sallam et al., 2021). Passive diffusion or active transport (glucose-dependent transporter, SGLT-1) is involved in aglycone absorption. Depending on the enzyme which carries out glycoside cleavage—either lactase phlorizin hydrolase (LPH) or cytosolic β-glucosidase (CBG)—one or another absorption mechanism will occur (Day et al., 2000; Gee et al., 2000). The absorbed fraction in the upper gastrointestinal tract is relatively small, as studies with ileostomy volunteers have shown. Therefore, approximately 90% of phenolic compounds in food pass unaltered to the colon (Dufour et al., 2018). These compounds that are unabsorbed or re-excreted in the bile reach the colon, where they are metabolized by the action of the colonic microbiota into simpler compounds, such as low molecular weight phenolic acids, which may be absorbed (Corona et al., 2016; Manach et al., 2004).

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Overall, there are a vast number of studies focused on the bioaccessibility of phenolic compounds of terrestrial plants; however, seaweed polyphenol studies are limited. Table 1 shows a summary of the in vitro and in vivo studies assessing the transformation of seaweed polyphenols through the gastrointestinal tract. For the first time, Corona et al. (2016) carried out an in vitro gastrointestinal digestion and colonic fermentation of a seaweed polyphenol extract from Ascophyllum nodosum, a brown seaweed, followed by a dialysis step, to simulate absorption into the human body. Some phlorotannins such as hydroxytrifuhalol A, the CdOdC dimer of phloroglucinol, diphlorethol/difucol, and 7-hydroxyeckol were identified in the digested and fermented samples. A total of seven compounds corresponding to in vitroabsorbed metabolites were reported in the dialyzed samples. The full characterization of these seaweed polyphenol metabolites was not possible due to the complexity of phlorotannin analysis and the lack of commercially available standards, which implies a limited capability for method development, especially for the analysis of digested material or biological fluids (plasma or urine) (Corona et al., 2016). The same authors (Corona et al., 2017) also observed a significant decrease in the total phenolic content (TPC) of three polyphenol extracts from brown seaweed (A. nodosum) after in vitro digestion. Particularly, the TPC of the high molecular weight (HMW) fraction (>10KDa) was marginally affected by in vitro digestion, whereas they were much more reduced during colonic fermentation (86.5% of the initial levels) than the low molecular weight (LMW) fraction. Similar results were obtained more recently by Catarino et al. (2021), which observed a decrease in the TPC of both a crude extract and a phlorotannin-purified fraction from Fucus vesiculosus (brown seaweed) after in vitro gastrointestinal digestion. In addition, the purified fraction experienced higher phlorotannin degradation than the crude extract. On the other hand, Huang et al. (2020) reported that the TPC of seven polyphenol seaweed extracts increased after in vitro digestion. This release of bioactive compounds from several vegetables after gastric and intestinal digestion was also observed by Lafarga et al. (2019). Studies conducted on seaweed (Catarino et al., 2021), lettuce (Ketnawa et al., 2020), and other vegetables such as green pepper, tomato, or zucchini (Lafarga et al., 2019) reported that the TPC decreased in the intestinal phase compared to the gastric phase. The gastric phase, characterized by an acidic pH (pH 2–4), favors polyphenol stability and their release from the food matrix (Hu et al., 2017) due to the breakdown of the bonds maintained with dietary components such as proteins, fiber, or carbohydrates (Alminger et al., 2014;

Table 1 Summary of the in vitro and in vivo studies assessing the transformation of the seaweed polyphenols through the gastrointestinal tract.

Model

Study design Phenolic compound/food Sample Seaweed polyphenol extract Digested (SPE) rich in phlorotannins from samples Two sequential a brown seaweed Ascophyllum stages: gastric nodosum and small intestinal phases 10 g SPE/HMW Seaweed polyphenol extract Digested fraction/LMW (SPE) from A. nodosum (brown samples seaweed) + 2 fractions (HMW, fraction >10KDa and LMW, 1–10 KDa) Two sequential stages: gastric and small intestinal phases

In vitro GID 10 g SPE

Three sequential stages: oral, gastric and small intestinal phases 1 g of dried sample Three sequential stages: oral, gastric, and small intestinal

Catabolites/ metabolites detected

Metabolomic platform

Findings

Reference

Hydroxytrifuhalol A CdOdC dimer of phloroglucinol Diphloretol/difucol 7-hydroxyeckol

LC-MS/MS utilizing ESI Tentative identification of metabolites

–

NP-HPLC-DAD analysis TPC of HMW fraction was less Corona et al. affected by in vitro digestion (2017) (reduction: 40.4%) compared to Folin-Ciocalteau SPE (reduction: 81.7%) and LMW fraction (reduction: 64.7%) LC-MS/MS utilizing ESI Tentative identification of metabolites

Hydroxytrifuhalol A C-O-C dimer of phloroglucinol Diphloretol/difucol 7-hydroxyeckol –

Extract from Fucus spiralis (brown seaweed)

Digested samples

Fucus vesiculosus (brown seaweed) Extract from F. vesiculosus (brown seaweed)

– Digested samples + Dialyzed samples (permeate and retentate)

Corona et al. (2016)

Folin-Ciocalteu

Total polyphenol bioaccessibility: 43.7%

Francisco et al. (2020)

2,4dimethoxybenzaldehyde (DMBA) colorimetric method

Total phlorotannin bioaccessibility: 14.1% Total phlorotannin bioaccessibility: 2.0%

Catarino et al. (2021)

Continued

Table 1 Summary of the in vitro and in vivo studies assessing the transformation of the seaweed polyphenols through the gastrointestinal tract—cont’d

Model

Study design Phenolic compound/food Sample

Catabolites/ metabolites detected

Metabolomic platform

–

HPLC/ESI-MS analysis

Findings

Reference

phases + Dialysis (48 h at room temperature) 1 g of dried sample Two sequential stages: gastric and small intestinal phases

Digested Undaria pinnatifida (UP) samples Sargassum thunbergii (ST) Sargassum fusiforme (SF) Laminaria japonica (LJ) Gracilaria lemaneiformis (GL) Sargassum siliquastrum (SS) Sargassum kjellmanianum (SK)

Gastric phase: bound phenolic Huang et al. content > free phenolic content (2020)

HPLC-DAD Folin-Ciocalteau

Intestinal phase: bound phenolic content—free phenolic content no significant difference TPC of all seaweeds increased from 4.16–17.24 mg GAE/g before simulated digestion to 4.08–40.37 mg GAE/g after GID, especially in the intestinal phase Sargassum thunbergia (brown seaweed) presented the highest TPC (40.37 mg GAE)/g) and Laminaria japonica (brown seaweed) the lowest one (15.24 mg GAE/g) after GID

In vitro From digested Fermentation and dialyzed samples

Seaweed polyphenol extract Fermented (SPE) rich in phlorotannins from samples a brown seaweed A. nodosum

15 mL fecal slurry (1:10, w/v) 24 h From digested Seaweed polyphenol extract Fermented extracts (SPE) from A. nodosum (brown samples seaweed) + 2 fractions (HMW, >10KDa and LMW, 1-10KDa) 15 mL fecal slurry (1:10, w/v) 24 h

7-hydroxyeckol

LC-MS/MS utilizing ESI Tentative identification of metabolites

Corona et al. (2016)

–

NP-HPLC-DAD analysis TPC of HMW fraction was significantly reduced during colonic fermentation, up to Folin-Ciocalteau 86.5% of the initial levels.

Corona et al. (2017)

Hydroxytrifuhalol A CdOdC dimer of phloroglucinol Diphloretol/difucol 7-hydroxyeckol

Dialysis

Fermented From retentate F. vesiculosus (brown samples seaweed) + Extract from samples F. vesiculosus (brown Fecal slurry of seaweed) 2% (v/v) 48 h From digested Seaweed polyphenol extract Dialyzed (SPE) rich in phlorotannins from samples samples a brown seaweed A. nodosum Overnight at 4 ° C against water (4 L) + remove +2 additional hours

TPC of SPE and LMW fraction were further reduced to 99.5% and 92.5%, respectively. LC-MS/MS utilizing ESI Tentative identification of metabolites

Real-time PCR –

Positive effect on the growth of Catarino et al. certain bacteria from the human (2021) gut, producing potentially changes in the growth of Enterococcus spp.

Seven phlorotannin LC-MS/MS utilizing ESI Presence of seven phlorotannin Corona et al. metabolites metabolites (in vitro-absorbed (2016) metabolites)

Continued

Table 1 Summary of the in vitro and in vivo studies assessing the transformation of the seaweed polyphenols through the gastrointestinal tract—cont’d

Model

Study design Phenolic compound/food Sample

Metabolomic platform

Findings

Reference

Corona et al. TPC: 0.0118–7.757 mg/mL Hydroxytrifuhalol A RP-HPLC analysis (2016) CdOdC dimer of LC-MS/MS utilizing ESI Few phlorotannin metabolites phloroglucinol were excreted at early time 7-hydroxyeckol points (0–8 h) Two unknown Most of phlorotannin metabolites metabolites were mainly Phase II phlorotannin excreted at later times points derivatives (8–24 h) HMW phlorotannins are poorly absorbed in the upper gastrointestinal tract, suggesting colonic metabolism Presence of phase II phlorotannin derivatives (glucuronides and sulfates metabolites) Phase II phlorotannin RP-HPLC analysis TPC: 0.15–33.52 mg/mL Seaweed polyphenol extract Plasma derivatives Few phlorotannin metabolites (SPE) rich in phlorotannins from can be absorbed in the upper a brown seaweed A. nodosum Collection: 0, gastrointestinal tract, which 1, 2, 3, 4, 6, 8 first appeared in plasma at and 24 h after early time points (2–4 h) SPE capsule Most of phlorotannin intake metabolites were mainly excreted at later times points (6–24 h) HMW phlorotannins are poorly absorbed in the upper

Seaweed polyphenol extract Urine (SPE) rich in phlorotannins from a brown seaweed A. nodosum Collection: 0, 0–8 and 24 healthy 8–24 h after volunteers SPE capsule 400 mg SPE intake capsule intake

Human study Single-dose design

Catabolites/ metabolites detected

gastrointestinal tract, suggesting colonic metabolism Presence of phase II phlorotannin derivatives (glucuronides and sulphates metabolites)

Laminaria digitate (LD) Three-way crossover study Undaria pinnatifida (UP) 20 healthy volunteers 5 g of Laminaria digitate (LD)/5 g of Undaria pinnatifida (UP)/ Control meal

Urine

Hydroxyldihydrocoumarin

Collection: baseline, 1.5, 3 and 24 h after SPE intake

One-week washout phase between each meal Seaweed polyphenol extract Urine Placebo(SPE) rich in phlorotannins from controlled a brown seaweed A. nodosum 24 h crossover Collection: design

Xi et al. (2020)

LC-ESI-QTOF-MS

Tentative identification of hydroxyl-dihydrocoumarin, presented as a specific metabolite for UP Major excretion after 3 h post consumption Absorption takes place mainly in the upper gastrointestinal

UHPLC-HR-MS Pyrogallol/ phloroglucinol sulfate Dioxinodehydroeckol

Tentative identification of the compounds mentioned

Baldrick et al. (2018)

TPC: 0.001–4.140 mmol Continued

Table 1 Summary of the in vitro and in vivo studies assessing the transformation of the seaweed polyphenols through the gastrointestinal tract—cont’d

Model

Study design Phenolic compound/food Sample 80 healthy volunteers Body mass index 25 kg/m2 400 mg SPE capsule/day for 8 weeks

weeks 0, 8, 16, and 24

Catabolites/ metabolites detected glucuronide Fucophloroetholglucuronide Diphlorethol sulfate CdOdC dimer of phloroglucinolsulfate CdOdC-dimer of phloroglucinol Hydroxytrifurahol A-glucuronide

Metabolomic platform

Findings

Reference

Interindividual variability: Low excretors (25% of the volunteers): urinary excretion 2 mmol

GID, gastrointestinal digestion; GAE, equivalents of gallic acid; LC-MS/MS, liquid chromatography-tandem mass spectrometry; HPLC/ESI-MS, high-performance liquid chromatography/electrospray ionization-mass spectrometry; HPLC-DAD, diode-array detection. NP-HPLC-DAD, normal phase-high-performance liquid chromatography-diode-array detection. RP-HPLC, reverse phase-high-performance liquid chromatography. LC-ESI-QTOF-MS, liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry; Real-Time PCR, real-time polymerase chain reaction.

Chapter 9 Bioaccesibility and bioavailability of marine polyphenols

Lucas-Gonza´lez et al., 2018). In the intestinal phase, characterized by a neutral-alkaline environment, polyphenols are susceptible to degradation (Hu et al., 2017). Therefore, losses in the TPC are observed after the intestinal phase. In addition to pH, other factors play a part, such as interactions with other food components or enzymatic activity (Alminger et al., 2014; Ketnawa et al., 2020; Kroll et al., 2003). However, other studies carried out on seaweeds (Huang et al., 2020) and lotus leaf (Su et al., 2018) showed an increase in TPC after the intestinal phase compared to the gastric phase. The study by Huang et al. (2020) examined seven brown, red, and green seaweeds, among which Sargassum thunbergia (brown seaweed) presented the highest TPC (40.37 mg equivalents of gallic acid (GAE)/g) and Laminaria japonica (brown seaweed) the lowest (15.24 mg GAE/g) in the intestinal phase. Among phenolic compounds, they evaluated both free and bound phenolics and observed that the free phenolic content in Sargassum thunbergia increased 6.3 times between the gastric phase (1.95 mg GAE/g) and the intestinal phase (12.27 mg GAE/g). Moreover, they observed that, in the gastric phase, the TPC was mainly composed of bound phenolics, while later, in the intestinal phase, both free and bound phenolic content were balanced since this phase enhanced the dissolution of free phenolics from seaweeds (Huang et al., 2020). Moreover, Francisco et al. (2020) evaluated the total polyphenol bioaccessibility of Fucus spiralis (brown seaweed). In fresh seaweed, the TPC before and after in vitro digestion was 0.016  0.002 and 0.007  0.001 mmol of gallic acid equivalents (EGA)/g, respectively, which represents 43.7% bioaccessibility. It means that slightly less than half of the initial phenolic content will be released from the food matrix and probably absorbed by intestinal cells. Another study reported lower bioaccessibility percentages in F. vesiculosus (14.1%) (Catarino et al., 2021), which indicates that only a small fraction of phlorotannins initially present in seaweed are likely to be absorbed in the upper gastrointestinal tract. Therefore, most phlorotannins tend to accumulate in the large intestine, where they are subjected to the action of the gut microbiota (Catarino et al., 2021). Similar results have been reported in terrestrial plants such as purple radish (12.1%), kale (14.1%), or white grape (13%) (Lingua et al., 2019; Tomas et al., 2021). Bioaccessibility is evaluated by in vitro experiments that mimic human digestion, colonic fermentation, and the absorption of phenolic compounds (Lorenzo et al., 2019). Thus, these studies shed light on the mechanisms of action of a dietary

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Chapter 9 Bioaccesibility and bioavailability of marine polyphenols

polyphenol, which must be demonstrated by in vivo experiments in humans (D’Archivio et al., 2010).

4. Bioavailability of marine polyphenols in humans To exert their biological functions in vivo, the degree of biotransformation of phlorotannins during their passage through the gastrointestinal tract, colon, liver, and cells acquires great importance (Corona et al., 2016, 2017). As previously mentioned, information about bioaccessibility is based on in vitro studies, the first step for learning about the mechanisms of action of a dietary polyphenol, but it must be demonstrated by in vivo experiments in humans (D’Archivio et al., 2010). How marine phenolic compounds affect the human organism depends on a crucial factor, namely, their bioavailability (Nova et al., 2020). Bioavailability is defined as the fraction of the ingested compound or its metabolite that reaches the systemic circulation to exert a biological effect on target tissues (D’Archivio et al., 2010). This concept includes several mechanisms, such as absorption, distribution, metabolism, and excretion (ADME process) of bioactive components/metabolites within the body (Carbonell-Capella et al., 2014; Kara
s et al., 2016) (Fig. 2). The bioavailability of phenolic compounds is largely determined by their molecular weight and degree of glycosylation and esterification. The literature has shown that the higher the degree of polymerization, the lower the absorption of tannins in the upper gastrointestinal tract (Kara
s et al., 2016; Montero et al., 2017; Serrano et al., 2009). The results obtained by Corona et al. (2016) support this fact since they observed that, after the intake of a capsule rich in phlorotannins by healthy volunteers, most of the phlorotannin metabolites were later present in their urine and plasma, which shows their low absorption in the small intestine (Corona et al., 2016). Phenolic bioavailability is evaluated by intervention studies where single-dose designs are the most common approach. These intervention studies focus on a single intake of a portion of food containing the phenolic compounds under study (D’Archivio et al., 2010). The fate of these compounds in humans, in this case, marine phenolic compounds and their metabolites, is evaluated by measuring their plasma concentration and/or urinary excretion after a marine source intake from a group of volunteers (Baldrick et al., 2018; Corona et al., 2016; Xi et al., 2020) (Table 1). Very little information is available in the literature about the bioavailability of seaweed phenolic compounds in humans.

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Fig. 2 General scheme of the absorption, distribution, metabolism, and excretion (ADME) of bioactive compounds.

A summary of the ADME of seaweed polyphenols in humans is shown in Fig. 3. The first study to shed light on the bioavailability of phlorotannins in humans was the one by Corona et al. (2016). In this work, a human intervention study was carried out in which 24 healthy participants (18–65 years old) were fed a single dose of a seaweed polyphenol capsule (SPE) containing 101.89 mg of polyphenols composed of HMW phlorotannins (obtained from fresh A. nodosum, a brown seaweed). Blood and urine samples were collected 24 h after the SPE capsule intake. Although the analysis of phlorotannins is challenging because of the high range of molecular weights present and because of the lack of available standard compounds, a total of 18 phlorotannins metabolites were detected but not identified or quantified in plasma and urine after SPE capsule intake. The metabolites were mainly excreted at later time points (6–24 h) after SPE intake, indicating a predominant large intestinal metabolic transformation. HMW phlorotannins were poorly absorbed in the upper gastrointestinal tract; hence, a large fraction reached the colon where they were metabolized into low molecular-weight derivatives by the action of the

Fig. 3 Summary of the bioavailability of seaweed phenolic compounds in humans.

Chapter 9 Bioaccesibility and bioavailability of marine polyphenols

colonic microbiota, and then absorbed and finally excreted by urine. These results are consistent with a later in vitro study (Corona et al., 2017), which reported that the TPC of HMW fraction (>10 KDa) was less affected by in vitro digestion, being more significantly reduced during colonic fermentation. The analysis of the plasma and urine showed that after the SPE capsule consumption, the plasma concentration of phlorotannins and their metabolites ranged from 8.72105 to 0.062 mmol, while between 0.001 and 0.2658 mmol were found in urine. These results are expressed as phloroglucinol equivalents, the calibration curve of phloroglucinol being used to quantify all the phlorotannins due to the lack of phlorotannin standards. Moreover, the ranges of the total phlorotannin metabolite concentrations reported in both biological fluids showed large interindividual differences, attributed to differences in gut microbial ecology and the expression of metabolizing enzymes (Selma et al., 2009; Toma´s-Barbera´n et al., 2016). Another human intervention study that evaluated the bioavailability of seaweed polyphenols was carried out by Baldrick et al. (2018). The study followed a placebo-controlled crossover design with eighty overweight healthy volunteers (30–65 years old) divided into two groups of forty, each starting by taking either a 400 mg SPE capsule or a 400 mg maltodextrin placebo daily for eight weeks. Then, volunteers underwent an eight-week washout phase and, subsequently, continued with the opposite treatment to the one they initially received for another eight weeks. Blood samples were collected before and after each phase (weeks 0, 8, 16, and 24) and 24-h urine samples were taken at each time point (weeks 0, 8, 16, and 24). An untargeted analysis was performed on the urine and plasma samples to determine the identity of the polyphenol metabolites excreted in urine and plasma after SPE capsule intake. The metabolomic analysis showed that the human urine and plasma metabolome of the participants was modified after the 8 weeks of supplementation with SPE capsules compared with baseline urine and plasma metabolic profiles. Moreover, this modification was mainly due to the appearance in the urine or plasma of specific metabolites associated with SPE capsule intake, tentatively identified as pyrogallol/phloroglucinol sulfate, hydroxytrifurahol A-glucuronide, dioxinodehydroeckol glucuronide CdOdC dimer of phloroglucinolsulphate, CdOdC-dimer of phloroglucinol, fucophloroetholglucuronide, diphlorethol sulfates, and dioxinodehydroeckol glucuronide. These two last phlorotannin metabolites were also reported by Corona et al. (2016), as previously mentioned,

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although hydroxytrifurahol A-glucuronide was only identified in its unconjugated form. Based on the studies in which polyphenol metabolites have been identified in seaweed, the total amount of metabolites excreted in urine varies largely among volunteers, ranging from 0.001 to 4.140 mmol (Baldrick et al., 2018), highlighting that there is interindividual variability in the metabolization and transformation of seaweed polyphenols. More recently, Xi et al. (2020) conducted a three-way crossover study in which twenty healthy volunteers (aged 28.8  5.4) were given, in a randomized order, 5 g of Laminaria digitate (LD), 5 g of Undaria pinnatifida (UP) or a control meal, with a one-week washout phase between each meal. In this study, urine samples at four time points (baseline, 1.5, 3, and 24 h afterwards) and plasma samples at seven time points (20 min before the meals, 20, 40, 60, 90, 120, and 180 afterward) were collected and analyzed using an untargeted approach to discover biomarkers of seaweed consumption. The authors reported the presence of urine metabolites tentatively identified as hydroxyl-dihydrocoumarin, derived from seaweed polyphenols, and two glucuronide forms of loliolid, derived from seaweed carotenoids. In this study, none of the phlorotannin derivatives previously mentioned by Baldrick et al. (2018) were identified, probably due to differences in the instrumentation and method used for measuring seaweed compounds or in the quantity and presentation (capsule or fresh) of seaweed intake (Xi et al., 2020).

5. Metabolism and metabolic processes The metabolism of marine phenolic compounds in the body is considered extremely complex and extensive (Meng et al., 2021) in which glucosidase enzymes, phase I enzymes, including cytochrome P450, and phase II enzymes (glucuronosyltransferases, sulfotransferases) carry out their enzymatic activity (Catarino et al., 2017). Currently, very few human studies have been published based on the metabolism and bioavailability of seaweed polyphenols (Table 1). Thus, phlorotannins are commonly considered to behave in a similar way to phenolic compounds in terrestrial plants (Catarino et al., 2017). In the upper gastrointestinal tract (stomach and small intestine), phenolic compounds are exposed to structural modifications due to the action of pH and phase I enzymes (oxidases, reductases, hydrolases), resulting in aglycone release. Specifically, a glycoside is cleaved by lactase phlorizin hydrolase (LPH), which is presented in the brush border of the

Chapter 9 Bioaccesibility and bioavailability of marine polyphenols

small intestine epithelial cells. The compounds/metabolites produced can be absorbed, further metabolized by phase II enzymes in enterocytes (prior to passage into the bloodstream) and in the liver, distributed throughout the tissues, and finally, eliminated by renal excretion (Corona et al., 2014). In the gut barrier and to a greater extent in the liver, conjugated reactions take place to produce glucuronides, O-methylated, and sulfate derivatives, or even combinations of these (Manach et al., 2004). These reactions are catalyzed by endoplasmic uridine-50 -diphosphate glucuronosyltransferases (UGT), cytosolic catechol-O-methyltransferases (COMT), and sulfotransferases (SULT), respectively (del Rio et al., 2013). Conjugation mechanisms are highly efficient, so conjugated forms predominate over free aglycones in plasma, which are generally absent or in low concentrations (D’Archivio et al., 2010). Furthermore, conjugation, as a metabolic detoxification pathway, leads to greater solubility and higher molecular weight of the conjugated compounds, thus promoting their biliary and urinary excretion (D’Archivio et al., 2010; Day et al., 2000). One study reported the presence of phase II phlorotannin derivatives in urine and plasma (glucuronides and sulfates metabolites) after SPE capsule intake by healthy volunteers (Corona et al., 2016). Thus, these phlorotannin metabolites could tentatively be considered to be responsible for the health effects associated with seaweed consumption. Phenolic compounds unabsorbed in the small intestine, together with those previously absorbed, conjugated in the enterocytes and/or in the liver, are re-excreted in the bile and returned to the small intestine, reaching the colon (Manach et al., 2004). There, they are subjected to structural modifications by colonic microbiota, composed of 1012 microorganisms per gram of gut content (Grgi
c et al., 2020). Colonic microbiota present huge catalytic and hydrolytic activity, leading to the degradation of phenolic compounds into simpler phenolic molecules that can be absorbed and further metabolized in the liver due to the conjugation process. The catalytic activity of bacterial enzymes involves hydrolysis, dehydroxylation, demethylation, isomerization, decarboxylation, and/or reduction reactions which allow for the hydrolysis of glycosides, glucuronides, sulfates, amides, esters, polymers, and lactones as well as the fragmentation of their structure and the fission of the phenolic rings (Corona et al., 2014; Selma et al., 2009). These simple phenolic compounds formed in the colon (catabolites) are sometimes more biologically active than their parent molecules (Selma et al., 2009).

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6. Factors affecting marine polyphenol bioavailability: Influence of the food matrix, dose, and interindividual differences Marine polyphenol bioavailability may be affected by the food matrix, doses and/or interindividual differences, and therefore, their bioactivity will also be affected (Bohn et al., 2015; Manach et al., 2017).

6.1 Food matrix The influence of the food matrix in the bioavailability of phenolic compounds has been the subject of many studies. The food matrix is a complex system in which phenolic compounds can interact synergistically or antagonistically with other dietary components such as macronutrients (proteins, carbohydrates, and/or lipids) (Aguilera, 2019). Phlorotannins are characterized by different linkages, functional groups, reactivities, and molecular weights, so they probably interact differently with these macronutrients (Ford et al., 2020). Moreover, more indirect effects of the diet on various parameters of gut physiology (pH, intestinal fermentation, biliary excretion, transit time, etc.) may have consequences on the absorption of polyphenols. Little information about phlorotannins bioavailability starting from food matrices is available in the literature, thus it has been considered that these seaweed compounds behave in a similar way to tannins from terrestrial plants (Catarino et al., 2017). Tannins can interact strongly with dietary and/or endogenous proteins (Meng et al., 2021; Stern et al., 1996), and this can affect their bioavailability. It has been reported that the higher the molecular weight of the phenolic compound, the greater its ability to form bonds with proteins (Buitimea-Cantu´a et al., 2018), and also, the higher the degree of polymerization, the lower the absorption of tannins in the upper gastrointestinal tract (Kara
s et al., 2016; Montero et al., 2017; Serrano et al., 2009). Consequently, HMW phlorotannins (up to 100 kDa) are more inclined to form bonds with proteins (Shannon et al., 2021) and to reach the colon, where they will be subjected to colonic microbiota, resulting in simpler absorbable compounds (Corona et al., 2016). More specifically, in the studies by Corona et al. (2016) and Baldrick et al. (2018), healthy and overweight volunteers ingested an SPE capsule to determine the fate of seaweed polyphenols. To obtain this capsule, first polyphenols were extracted from brown seaweed, and then, the resultant SPE was encapsulated.

Chapter 9 Bioaccesibility and bioavailability of marine polyphenols

Encapsulation strategies are used to enhance bioavailability by improving polyphenols apparent solubility, minimizing their interactions with other dietary compounds, and achieving targeted gut release. However, the use of encapsulation strategies in these studies has a limitation since the bioaccessibility and bioavailability of the polyphenols extracted and/or encapsulated may differ greatly from those of the same polyphenols present in a food matrix (Bohn, 2014).

6.2 Dose Phenolic compound bioavailability is evaluated by intervention studies, single-dose designs being the most common approach. These studies have observed a transient increase in the concentration of seaweed phenolics in the blood, reflecting the organism’s ability to take up these compounds from the food matrix and subsequently absorb them (D’Archivio et al., 2010). Furthermore, the literature also reports differences in the absorption of phenolic compounds and, therefore, different effects on the organism. A possible explanation for this may be the administration of different doses of phenolic compounds (Bohn, 2014). Regarding phenolic compounds from seaweeds, none of the studies available in the literature has evaluated a dose-response relationship, which would be of interest in future research. In general, any phenolic compound subject to metabolism before being absorbed will be affected by the dose administered. It has been reported that higher doses result in decreased fractional absorption compared to lower ones. At lower doses, absorption appears to be linear (Bohn, 2010, 2014). Higher doses could also affect other saturable mechanisms such as the cleavage of glycosides into aglycones in the gut barrier in order to be absorbed (Bohn, 2014). In addition, the primary site of phlorotannin metabolism could differs according to the dose administered. Generally, at higher doses, phenolic compounds tend to be primarily metabolized in the liver, and at lower doses, they are probably first metabolized in the small intestine and then further modified in the liver (Scalbert and Williamson, 2000).

6.3 Interindividual differences Phenolic compound bioavailability can also be influenced by interindividual characteristics such as age, genetics, health, and/or lifestyle (Nova et al., 2020). These large interindividual differences have been attributed to variations in gut microbiota

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composition and the expression of phase II metabolizing enzymes, resulting in different phenolic compound metabolic profiles and, thus, different physiological responses (Cassani et al., 2020; Toma´s-Barbera´n and Espı´n, 2019). Gut microbiota play an important role in the metabolism of phenolic compounds in terms of their biotransformation into bioactive metabolites (Toma´s-Barbera´n et al., 2016). The interindividual variation that exists in the microbiota composition is reflected in the variability observed in phenolic metabolites excreted in biological fluids (Bingham, 2006) after seaweed intake. Corona et al. (2016) reported substantial differences among twenty-four volunteers regarding the profile of phlorotannin metabolites observed in both plasma and urine samples after one SPE capsule intake. For instance, the TPC (phlorotannins and their metabolites) ranged from 0.011 to 7.757 μg/mL (8.72105–0.062 mmol) in the plasma and 0.15 to 33.52 μg/mL (0.001–0.2658 mmol) in the urine. These interindividual variations agree with those observed in other in vivo studies where other dietary polyphenols such as black tea (Pereira-Caro et al., 2017a), grape pomace (Castello et al., 2018), orange juice (Pereira-Caro et al., 2017b), cranberry (Feliciano et al., 2017), or a capsule containing a mix of berries, fruits, and vegetables (Bresciani et al., 2017) were consumed. In addition, Baldrick et al. (2018) compared different metabolic profiles of the urine samples of seventy-eight volunteers, observing that the total seaweed metabolite concentration in the urine samples varied greatly (0.001–4.140 mmol). Considering that these differences were due to gut microbiota composition, volunteers were stratified into three groups based on the amount of seaweed metabolite absorbed (high, medium, or low excretors). Low excretors (25% of total volunteers) were characterized by a urinary seaweed metabolite excretion below 0.5 mmol; medium excretors (55% of the population) by urinary excretion of 0.5–2 mmol; and high excretors (the remaining 20% of the population), the urinary excretion was greater than 2 mmol (Baldrick et al., 2018). Another way to stratify individuals is by their ability to produce specific polyphenol-derived gut microbiota metabolites (metabotypes) (Toma´s-Barbera´n et al., 2016). For instance, individuals can be stratified by their ability to produce equol (gut microbiota metabolite derived from soy isoflavones) into equol producers and equol non-producers (Setchell et al., 2002). In one study, researchers reported that equol administration only exerted vascular benefits in those volunteers who belonged to the equol producer metabotype (Hazim et al., 2016). As mentioned above, interindividual variation in phase II (conjugation) enzymes may also influence phenolic compound

Chapter 9 Bioaccesibility and bioavailability of marine polyphenols

bioavailability. For example, UDP-glucuronosyltransferases are characterized by a wide polymorphic expression pattern which could result in high interindividual variability in seaweed phenolic compound glucuronidation (Manach et al., 2004). The statistical significance of certain interventions is sometimes limited by the high interindividual variability observed (Toma´s-Barbera´n et al., 2016). On the other hand, as high interindividual variability in phenolic compound metabolism could result in different physiological responses, personalized treatment strategies based on an individual metabotype should be developed (Bolca et al., 2013).

7. Biomarkers of marine phenolic intake Food intake biomarkers are objective measures related to actual intake estimation, unlike common tools such as food frequency questionnaires, food diaries, or 24-h recalls, which often provide random and systematic errors (Pratico` et al., 2018). These biomarkers allow us to evaluate the relationship between the beneficial effects for the body and seaweed intake; hence the importance of conducting studies focused on the determination of their levels. Two methods can be used to identify biomarkers of seaweed intake (BSIs), the targeted and untargeted approaches. In both approaches, the metabolites excreted in biological fluids are measured to see if they increase after seaweed intake. Targeted approaches focus on identifying known metabolites of interest. On the other hand, with untargeted approaches, it is possible to discover and measure unknown metabolites (new biomarkers) present in biofluids and related to seaweed intake since they are based on their metabolic profile of biofluids (Rothwell et al., 2018). To this end, powerful analytical techniques are used, such as nuclear magnetic resonance (NMR) or ultra-high-performance liquid chromatography (UHPLC) coupled to high-resolution mass spectrometry (HRMS) (Xi et al., 2020). Few candidates proposed as BSIs have been identified in urine samples from healthy and overweight volunteers fed with seaweed polyphenol extracts. For instance, Baldrick et al. (2018) carried out an untargeted analysis to identify phlorotannin metabolites in urine after SPE capsule intake during eight weeks, which could be clear biomarkers. An unsupervised PCA and an HCA were carried out with the urine data obtained, showing the stratification into two groups (G1, G2) of volunteers that shared a common excretion metabolite profile before and after the SPE capsule intake. Subsequently, thanks to the two supervised

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OPLS-DA models, the urine metabolic profile after the eight-week treatment (SPE capsule intake) was shown to differ from that found before treatment in both groups, enabling the tentative identification of several potential BSIs including pyrogallol/phloroglucinol sulfate (G1), dioxinodehydroeckol glucuronide (G1,G2), fucophloroethol-glucuronide (G2), diphlorethol sulfate (G2), CdOdC dimer of phloroglucinol-sulfate (G2), CdOdC-dimer of phloroglucinol (G2), and hydroxytrifurahol A-glucuronide (G1). All these compounds are phlorotannin metabolites and are therefore associated with seaweed intake, except for pyrogallol/phloroglucinol sulphate, which is not a clear a BSI since it can be found in plasma or urine after the intake of berry fruits (Pimpa˜o et al., 2014), nuts (Tulipani et al., 2011), red-fleshed apple (Yuste et al., 2019), or tea (Pereira-Caro et al., 2017a; van Duynhoven et al., 2014). Xi et al. (2020) conducted another untargeted analysis, tentatively identifying for the first time a urine metabolite as a possible BSI, namely, hydroxyl-dihydrocoumarin. On the other hand, in vivo studies (Baldrick et al., 2018; Corona et al., 2016; Xi et al., 2020) have only evaluated brown seaweeds, not red or green ones, so it could be said that the proposed biomarkers are specific to the first group. One BSI tentatively identified as hydroxyl-dihydrocoumarin was presented as a metabolite specific to one of the two types of brown seaweed studied by Xi et al. (2020), namely, Undaria pinnatifida (UP). The intensity observed in its excretion curve increased markedly after UP intake. Nevertheless, further studies are needed to confirm the specificity associated with the brown seaweed previously mentioned, as well as further studies of red and green seaweeds since their specificity is currently not so clear. Some authors also propose the use of combined biomarkers as a solution to the lack of specificity of individual biomarkers for certain food sources € rdeniz et al., 2016). Researchers (Dragsted et al., 2018; Gu emphasize the need for further research to show that proposed biomarkers follow the validation criteria (plausibility, doseresponse, time-response, robustness, reliability, stability, analytical performance, and reproducibility) (Dragsted et al., 2018). Xi et al. (2020) identified a plausible phenolic compound that had an appropriate time-response after seaweed intake, but more work is required to validate the other criteria. In general, more cross-sectional studies, larger cohort studies, and dose-response relationships related to seaweed intake, and the influence of storage, food matrix, and food processing on phenolic compounds needs to be evaluated to determine whether the proposed BSIs meet all the validation criteria mentioned above (Xi et al., 2020; Xi and Dragsted, 2019).

Chapter 9 Bioaccesibility and bioavailability of marine polyphenols

Acknowledgments
nez is supported by a predoctoral fellowship funded by the S. Ca´ceres-Jime ˜ ez was supported by Spanish Ministry of Universities (FPU20/03549). J.L. Ordo´n a research contract funded by the project PP.AVA.AVA2019.037. G. Pereira-Caro was supported by a research contract funded by the project RTI2018-096703J-I00 (09/2019-10/2021) and by a research contract “Programa Emergia 2020” funded by the Secretarı´a General de Universidades, Investigacio´n e Innovacio´n, Junta de Andalucı´a (from 11/2021-ongoing).

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Antioxidant capacity of seaweeds: In vitro and in vivo assessment

10

H. Sa´nchez-Ayora and J. P
erez-Jim
enez Department of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Madrid, Spain

Abbreviations AAPH ABTS ARE CAT CUPRAC DCF DCFH DCFH-DA DMPD DPPH EPR FRAP GPx GR GSH GST HAT MDA NEAC ORAC ROS SET SOD TOSC TRAP

2,20 -azobis(2-methylpropionamidine) dihydrochloride 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) antioxidant response element catalase cupric reducing antioxidant capacity dichlorofluorescein dichloro-dihydro-fluorescein dichloro-dihydro-fluorescein diacetate N,N-dimethyl-p-phenylenediamine dihydrochloride 2,2-diphenyl-1-picrylhydrazyl electron paramagnetic resonance ferric reducing antioxidant power glutathione peroxidase glutathione reductase reduced glutathione glutathione-S-transferase hydrogen atom transfer malondialdehyde nonenzymatic antioxidant capacity oxygen radical absorbance capacity reactive oxygen species single electron transfer superoxide dismutase total oxyradical scavenging capacity total radical-trapping antioxidant parameter

Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00006-6 Copyright # 2023 Elsevier Inc. All rights reserved.

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1. Antioxidant capacity as a relevant health parameter Oxidative status is a basic physiological feature in living organisms, including humans. It is referred to the equilibrium that takes place between, on the one hand, the production of free radicals (molecules with an unpaired electron) and, on the other hand, the presence of antioxidant substances. Free radicals, mainly corresponding to several oxygen species but also derived from nitrogen, are constantly originated from physiological processes, including cell metabolism, and they also come from the exposure to external agents, such as radiation or smoking. While antioxidant defenses are a combination of both endogenous systems (mainly superoxide dismutase and catalase enzymes, together with the glutathione system) and antioxidant compounds derived from the diet (mostly, but not exclusively, vitamin C and E, carotenoids, and phenolic compounds). The term oxidative stress has been used for decades in order to define the alteration of the balance between oxidant and antioxidant agents in the body, leading to an excess of free radicals, which cause the oxidation of biomolecules (lipids, proteins, nucleic acids) and it is involved in the pathophysiology of several chronic diseases, such as cardiometabolic diseases, cancer, or neurological disorders (Barnham et al., 2004; Ndisang et al., 2014; Reuter et al., 2010). Recently, it has been suggested the dissociation of the term oxidative stress into two concepts, i.e., oxidative eustress and oxidative distress (Sies, 2020). Thus, the term oxidative eustress would be a “positive” mild oxidative stress, being applied when there is low exposure to free radicals, which are directed toward specific targets. This activates adaptive responses of redox signaling, and it allows to ensure a proper status of this defense system. In contrast, in a situation of oxidative distress, there is a high exposure to free radicals directed toward unspecific targets, and this originates a disrupted redox signaling linked to conditions of physiopathology and, ultimately, disease. The interest in oxidative stress as a key process involved in chronic pathologies has led to the development of several methodologies for its assessment. In this context, the concept of total antioxidant capacity measurement emerged some decades ago (Pellegrini et al., 2020). Basically, it consists in the determination, either in an extract from a natural product, supernatants of cell cultures, or biological fluids, of the overall ability of that medium in order to counteract increased oxidative stress. As described later, several antioxidant capacity techniques have been developed with specific characteristics. In the case of biological fluids,

Chapter 10 Antioxidant capacity of seaweeds

the concept of non-enzymatic antioxidant capacity (NEAC) has been suggested in order to highlight that the determination assesses the contribution of those antioxidants different to endogenous antioxidant systems of enzymatic nature (Serafini et al., 2011). In this chapter, the current evidence on the antioxidant capacity of seaweeds will be discussed, including data from direct seaweed extracts, cell cultures treated with those extracts, preclinical studies, and clinical studies. Additionally, current evidence on the effect of seaweeds in some complementary measures of oxidative status will be presented. Nevertheless, some previous considerations on the ability of antioxidant capacity as a proper biological marker should be introduced, since this concept has been widely discussed. Due to the straightforward character of antioxidant capacity determinations, these techniques quickly gained high popularity and were extended to the field of natural products. This led to the publication of rankings of antioxidant capacity and, in many cases, for both research and industry, to establish a direct association between antioxidant capacity measured in food extracts and derived biological actions, or to incorrectly use the term “superfoods,” as recently reviewed (Pellegrini et al., 2020). This kind of direct association was criticized based on several aspects, e.g., the influence of aspects such as the solvents used or the interference with other food compounds in the obtained
rez-Jime
nez and Saura-Calixto, 2006); the interference results (Pe of a controversial constituent, uric acid, in the antioxidant capacity values obtained in biological samples (Mikami and Sorimachi, 2017); or the intrinsic problems to in vitro emulate such an intricate process as the absorption, disposal, and biological action of dietary antioxidants (Serafini and Del Rio, 2004). Nowadays, it is known that oxidative status regulation is a complex matter, and direct associations between antioxidant capacity and health effects should be avoided. However, this does not mean there is no association at all between antioxidant capacity determination and potential biological activities. In particular, after developing databases on food antioxidant capacity and their applications in multiple cohorts, significant associations have been found between diet antioxidant capacity (considered as the sum of the antioxidant capacity provided by all foods consumed in a diet) and several health outcomes. Thus, the first study with this approach reported an inverse association between this parameter and plasma C-reactive protein (Brighenti et al., 2005). From this, several observational studies have been performed. Just to mention some of them, a Swedish study on more than 30,000 subjects found a significant decrease in the risk of

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stroke (Colarusso et al., 2017) when the higher the antioxidant capacity was, or a French study on more than 60,000 middle-aged women found the same tendency, in this case regarding the risk of developing type 2 diabetes (Mancini et al., 2018). Overall, some recent reviews on observational studies on dietary antioxidant capacity and several health outcomes have found, for instance, significant inverse correlations between dietary antioxidant capacity and all-cause mortality analyzing 41 studies involving more than half a million subjects (Jayedi et al., 2018), or with fasting blood glucose or blood pressure after the evaluation of 16 studies (Mozzafari et al., 2018). Finally, it must not be disregarded that the lack of association sometimes found between antioxidant capacity results and in vivo biological effects may not be only due to the limitations of antioxidant capacity techniques, but also to the complexity of the matter, with aspects such as the influence of other food constituents affecting the final absorption of antioxidant compounds (De Camargo et al., 2019). Indeed, in the field of phenolic compounds, there is an increasing interest in the aspects affecting the inter-individual variability observed in many clinical trials on the topic (Manach et al., 2017). In this context, antioxidant capacity evaluation should be considered an additional tool in the complex evaluation of the biological activity of dietary antioxidants, which does not have an unequivocal association with beneficial effects but does not either lack any association with them. At the same time, a last general consideration is pertinent: it is known that, at high doses, antioxidants may become prooxidants, as it was clearly shown in the study with β-carotene and lung cancer (Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group, 1994); thus, despite the relevance of achieving high dietary antioxidant capacity intakes, it must be kept in mind that, above certain levels, they do not have beneficial effects, but the opposite ones.

2. Antioxidant capacity techniques Due to the complexity of the oxidative stress process and the biological responses to it, where dietary antioxidants are able to block oxidative stress by multiple procedures, there is not a single way to emulate it, so several antioxidant capacity assays have been developed based on the different mechanisms of action or using several molecular probes. From a mechanistic point of view, they are divided into two wide categories: single electron transfer (SET) and hydrogen atom transfer (HAT) reactions; both

Chapter 10 Antioxidant capacity of seaweeds

303

Fig. 1 Scheme of the two mechanisms of reaction in antioxidant capacity methods: SET (single electron transfer) and HAT (hydrogen atom transfer). A, antioxidant; C, compound.

processes are depicted in Fig. 1. The results in both cases are the stabilization of the free radical, while the antioxidant molecule remains with an unpaired electron; however, in SET mechanism, this takes place by a two-step process, with the subtraction of an electron from the free radical followed by that of a proton from the antioxidant, while HAT is a one-step process, involving the exchange of a hydrogen atom from the free radical to the antioxidant molecule. Regarding thermodynamic aspects, the ionization potential of the antioxidant is the most important parameter in SET reactions, while for HAT mechanism of action, the most relevant parameter is the bond dissociation enthalpy. Besides this basic mechanism of the reaction, there are other underlying chemical aspects in these methods, such as steric hindrance, chemical targets, and additional initiators, which have not been explored in detail for all antioxidant capacity techniques (Apak et al., 2016a). Regarding the molecules used to originate oxidative stress in these assays, there is the problem that, by their own nature, free radicals are unstable very reactive molecules. So it is not easy to perform a standardized laboratory technique based on the use of those free radicals present in the human body. For this reason, some assays are based on the use of synthetic free radicals that are more stable than real reactive oxygen species (ROS). Such is the case of DPPH (2,2-diphenyl-1-picrylhydrazyl) or ABTS (2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)). Also, peroxyl radicals which are indeed present in biological fluids have been used in these assays, since they have a relatively longer half-life (10 s) compared, for instance, to that of hydroxyl radicals, of 109 s (Pryor, 1986). A last general consideration regarding antioxidant capacity measurements is that some of them are end-point methods, so

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they measure the final stage of a particular reaction, while others have a kinetic approach to the way the reaction takes place. Based on these general considerations, these are the most common antioxidant capacity techniques currently used: – FRAP (ferric reducing antioxidant power) (Benzie and Strain, 1996). This is an end-point method based on a SET reaction. For a better understanding of it, it must be considered that antioxidant compounds are, indeed, reducing agents, so it measures the ability of the ensemble of antioxidants present in a sample to transform the ferric ion into its ferrous forms in the presence of tripyridyltriazine tridentate ligand, leading to the formation of a colored complex with Fe(II) with a change of color measured by spectrophotometry at 595 nm. The original method developed the reaction for a few minutes, although an adaptation was suggested, showing the need to extend it up to 30 min (Pulido et al., 2000). Although this method does not directly use free radicals, which has been signaled as a drawback, it is known that, in vivo, the transformation of Fe(III) to Fe(II) is involved in the lipid oxidation process (Braughler et al., 1986). An extension of this method called CUPRAC, cupric reducing antioxidant power (Apak et al., 2004), was later developed. It is based on the ability to reduce Cu(II) to Cu (I) in the presence of a selective Cu(I)stabilizing ligand, neocuproine (2,9-dimethyl-1,10-phenanthroline). Modifications for both methods have been recently suggested (Jones et al., 2020; Suktham et al., 2020), including coupling with chromatography techniques in order to simultaneously identify the compounds responsible for the observed antioxidant capacity. – DPPH. In this kinetic method, both SET and HAT mechanisms may take place, depending on the environment (Foti et al., 2004). It is based on the reaction of the DPPH radical with an antioxidant able to neutralize it, originating the DPPHhydrazine, and leading to a change of color from purple to pale yellow, which is monitored at 515 nm (Brand-Williams et al., 1995). The coupling of this method to high performance liquid chromatography (HPLC) has been reported (Chandrasekar et al., 2006). Additionally, in 2019, a modification of this method was suggested. It was based in combination with EPR (electron paramagnetic resonance) in order to reduce the interference of pigment in the results (Yeo and Shahidi, 2019). In this sense, it should be signaled that the presence of borates, widely distributed in plants, has been shown to affect DPPH results (Chen et al., 2020); in the same sense, the influence of some specific seaweed constituents in any of the antioxidant capacity techniques may not be disregarded. One of the main criticisms of this method is based on a

Chapter 10 Antioxidant capacity of seaweeds

N-centered free radical, while O-centered free radicals reflect better the oxidation processes in foods or in biological systems (Apak et al., 2016b). – ABTS. In this method, the limits between SET and HAT mechanisms of reaction are neither clear (Apak et al., 2016a). It uses the green ABTS•+ free radical, generated from ABTS molecule by the use of H2O2/peroxidase, MnO2, or persulfate. This free radical is quenched by antioxidant molecules with free-radical scavenging capacity, leading to a transparent solution (Re et al., 1999). The color modification is measured at 734 nm. Although it was developed as an end-point method and is the most common way to perform it, a kinetic approach was also developed
nez and Saura-Calixto, 2008). As it happens with (P
erez-Jime DPPH assay, this technique is based on a N-centered free radical, with the limitations mentioned already. – ORAC. In contrast with the previous ones, this end-point method is exclusively based on a HAT mechanism (Prior et al., 2005). In this case, the peroxyl radicals cause a decrease in the signal emitted by a fluorescent molecule (Cao et al., 1993). At the beginning, the fluorescent molecule used in this method was β-phycoerythrin, but it had some limitations that were solved with the change to fluorescein (Ou et al., 2001). The method is based on the retardation observed in the fluorescence decrease by the presence of antioxidants; although, as stated previously, it is based on the use of peroxyl radicals present in living organisms, it uses as intermediate molecular probes absent in the human body. It is not the aim of this chapter to provide a whole list of antioxidant capacity techniques, but it should be mentioned that other methods exist, such as TRAP (total radical-trapping antioxidant parameter), LDLs (low-density lipoproteins) oxidation assay, TOSC (total oxyradical scavenging capacity), DMPD (N,N-dimethylp-phenylenediamine dihydrochloride) radical scavenging assay, or crocin bleaching assay. Due to the differences between all these methodologies, it has been usually recommended to use at least two different methods, to have an overview of this complex matter. And, precisely due to these differences, the same results may not be obtained by the selected antioxidant capacity methodologies (Apak et al., 2016a). Nevertheless, this aspect has been criticized by some authors, who wondered to what extent data provided by several methodologies for the sample analysis may offer more confusion instead of clarity (Pellegrini et al., 2020). All of the samples where these techniques may be applied can be used in aqueous-organic extracts from natural products, and the DPPH method is especially useful for non-polar extracts— CUPRAC has also been used for this purpose. While FRAP,

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CUPRAC, ABTS, and ORAC techniques are also applied in biological samples, DPPH method has not shown to be helpful for this kind of sample. Some specific considerations should also be considered when these methods are used either to characterize natural products or to evaluate antioxidant capacity in biological samples. In the first case, the common way of applying these methods is to perform an optimized extraction with aqueous-organic solvents, carrying out the determination in the derived supernatant, where all dietary antioxidants are supposed to be transferred. Nevertheless, there is cumulative evidence on the existence of the non-extractable polyphenols (NEPP), i.e., compounds of either high molecular weight or small phenolic structures associated with the food matrix, all of them remaining in the residues of these extractions and needing specific hydrolysis procedures to be released. Thus, a comprehensive assessment of antioxidant capacity requires the determination not only in extraction supernatants, but also in
rez-Jime
nez et al., 2008). This is biologthe associated residues (Pe ically relevant, since the partial bioavailability to these compounds, through colonic microbiota transformation, has been
rez-Jime
nez shown, as well as their specific health effects (Pe et al., 2013). As regards the determination of antioxidant capacity in biological fluids, it must be considered that factors such as the level of intake of dietary antioxidants, short or long-term exposure, sampling times, or simultaneous intake of food affecting antioxidant metabolic fate are aspects that may influence antioxidant capacity values (Pellegrini et al., 2020). The results of these techniques may be expressed in multiple ways. But the most common unit for the expression of antioxidant capacity results is Trolox equivalents. Trolox (6-hydroxy-2,5,7,8tetramethyl chroman-2-carboxylic acid) is a synthetic watersoluble analog of vitamin E. A calibration curve is commonly performed with Trolox for each one of the methods described earlier, and the results for the different samples are interpolated in that curve. Indeed, ABTS method is many times referred to as TEAC (Trolox Equivalent Antioxidant Capacity), but this may be a confusing term since the other methods are also commonly expressed as Trolox equivalents. Here, the term “equivalent” is relevant, since it implies that values are providing the estimation of a capacity, i.e., the amount of a given sample that exhibits the same behavior as a certain Trolox concentration, but they do not mean the exact concentration of specific compounds (Pellegrini et al., 2020); indeed, structure-activity relationships have been reported for many antioxidant compounds and not all dietary antioxidants exhibit the same antioxidant capacity (Santos et al., 2021). For

Chapter 10 Antioxidant capacity of seaweeds

instance, flavonoids need to accomplish several structural features in order to exhibit a high antioxidant capacity: (i) the presence of a 30 ,40 -dihydroxy structure in the B ring, (ii) the presence of a 2,3-double bond in conjunction with the 4-oxo group in the heterocycle, and (iii) the presence of 3- and 5-hydroxyl groups in the A ring together with a 4-oxo function in the A and C rings (RiceEvans et al., 1996). The other most common way of expressing results for antioxidant capacity methods is EC50, which corresponds to the amount of extract/fluid needed to reduce to 50% the original amount of free radical. In this case, it must be noticed that, in contrast to the expression of results as Trolox equivalents, the lower the EC50 value, the higher the antioxidant capacity is.

3. Current evidence on the antioxidant capacity of seaweeds 3.1

Direct antioxidant capacity measurement

Seaweeds are known to contain a wide variety of antioxidant compounds, as detailed in other chapters of this book; this is due to the need to possess several defense systems against the hard conditions (light, salinity, temperature) where these materials are grown. Thus, a wide number of studies have explored the antioxidant capacity of several dozens of seaweeds, directly measured in extracts from the samples. Table 1 shows the taxonomical classification of 78 seaweed species that were identified in a bibliography search on antioxidant capacity of seaweed extracts. The first remarkable fact is that brown seaweeds (Phaeophyceae class) were the most studied ones, with 38 species, followed by red seaweeds (Bangiophyceae and Florideoceae classes), with 30 species, and then green seaweeds (Ulvophyceae class), with 10 species. The most studied order was Fucales, in brown seaweeds, with 21 species. At family level, Sargassaceae (brown seaweeds) was the most studied one, with 15 species, and it included Sargassum, the most studied genus, with 11 species. Since seaweeds are extended worldwide, samples from many countries have been collected for studying antioxidant capacity. A bibliography search with the terms “antioxidant capacity” or “antioxidant activity” and “seaweed*” in the period 2000–2020 showed the geographical origin of these studies. They came from a total of 56 countries, being India, South Korea, and Brazil the ones with the highest number of publications, above 30. China,

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Table 1 Taxonomical classification of seaweed species where antioxidant capacity in derived extracts has been determined. Phylum

Class

Order

Family

Genus

Species

Chlorophyta

Ulvophyceae

Bryopsidales

Caulerpaceae

Caulerpa

Codiaceae

Codium

Ulvales

Hallimedaceae Cladophoraceae Valoniaceae Ulvaceae

Hallimeda Cladophora Valoniopsis Enteromorpha Ulva

Dictyotales

Dictyotaceae

Dictyota Padina

C. racemosa C. lentillifera C. intertextum C. intricatum H. opuntia C. vagabunda V. pachynema E. intestinalis U. fasciata U. lactuca D. friabilis P. australis P. pavonica P. tenuis P. tetrastromatica S. asperum S. marginatumse Z. tournefortii A. nodosum C. abies C. barbata C. indica F. spiralis F. vesiculosus H. elongata B. bifurcata N. zanardinii S. acinarium S. cinctum S. cristaefolium S. duplicatum S. horneri S. ilicifolium S. muticum S. polycystum S. tenerrimum S. vachellianum S. vulgare

Cladophorales

Ochrophyta

Phaeophyceae

Fucales

Fucaceae

Spatoglossum Stoechospermum Zonaria Ascophyllum Cystoseira

Fucus Himanthaliaceae Sargassaceae

Himanthalia Bifurcaria Nizamuddinia Sargassum

Chapter 10 Antioxidant capacity of seaweeds

309

Table 1 Taxonomical classification of seaweed species where antioxidant capacity in derived extracts has been determined—cont’d Phylum

Class

Order

Ishigeales Laminariales

Rhodophyta

Bangiophyceae Florideophyceae

Bangiales Ceramiales

Corallinales Gracilariales

Gelidiales

Family

Genus

Species

Treptacantha Turbinaria

T. abies T. conoides T. decurrens I. okamurae L. digitata L. saccharina E. cava E. stolonifera E. radiata L. trabeculata P. dioica S. hypnoides C. lentillifera C. racemose Centroceras sp. C. intricatum C. intertextum Dasya sp. H. incurva L. dendroidea L. trabeculate P. stricta S. latiuscula J. rubens A. anceps C. vagabunda G. beckeri G. corticata G. fergusonii G. pristoides G. spinosum P. capillacea H. spinella C. crispus K. alvarezii E. cottonii E. denticulatum D. dichotomus Grateloupia sp.

Ishigiceae Laminariaceae

Ishige Laminaria

Lessoniaceae

Ecklonia

Bangiaceae Callithanmiaceae Caulerpaceae

Lessonia Porphyria Spyridia Caulerpa

Ceramiaceae Codiaceae

Centroceras Codium

Delesseriaceae Rhodomelaceae

Dasya Halopithys Laurencia Lessonia Polysiphonia Symphyocladia Jania Amphiroa Cladophora Gracilaria

Corallinaceae Lithophyllaceae Cladophoraceae Gracilariaceae

Gigartinales

Gelidiaceae Pterocladiaceae Cystoclonaceae Gigartinaceae Solieriaceae

Gelidium Pterocladiella Hypnea Chondrus Kappaphycus Eucheuma

Hallymeniales

Hallymeniaceae

Dermocorynus Grateloupia

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Chapter 10 Antioxidant capacity of seaweeds

Indonesia, Malaysia, Spain, Ireland, and Iran were the countries where the number of papers was between 26 and 10, respectively. And in the rest of the countries, there were less than 10 papers published on the topic during the last 20 years. The influence of geographical origin on the antioxidant capacity of seaweeds has not been yet explored in detail, particularly taking into account that a study in Chilean seaweeds with different geographical distributions found that their UV stress tolerance—and consequent effects in different parameters, including antioxidant capacity— did not follow linear latitudinal or central-edge gradients, while it was affected by among-site variability of solar intensities (Veliz et al., 2020). An overview of the methods used to evaluate seaweed antioxidant capacity in a selection of recent studies is shown in Table 2. Most studies applied more than one antioxidant capacity method, as recommended for evaluating this parameter, although some of them only used one technique. DPPH is largely the most used method for assessing seaweed antioxidant capacity, while FRAP and ABTS are similarly used, and only some studies applied ORAC technique. Few studies included some specific antioxidant capacity techniques, such as CUPRAC (Mahomoodally et al., 2020); β-carotene bleaching assay (Belkacemi et al., 2020), which is commonly used for assessing the antioxidant capacity of carotenoids; or other ones evaluating the capture of specific oxidant molecules, such as hypochlorous acid (Pimentel et al., 2020) or peroxyl radicals (Jesumani et al., 2020). Overall, studies on the antioxidant capacity of seaweed extracts obtained remarkable values, with potential biological relevance. Although no reference values exist for antioxidant capacity (such as range meaning low, medium, or high values), for instance, Himanthalia elongata provided values which were similar to those obtained for vitamin C (Rajauria, 2019). At the same time, due to the different mechanisms of action involved in each antioxidant capacity method, some seaweeds provided rather different values depending on the selected methodologies. This happened in a study with a selection of brown, green, and red seaweeds, where some of them showed antioxidant capacity by ABTS method but not in DPPH test (Zhong et al., 2020). However, usually, the same tendencies were observed for the different antioxidant capacity methods, for instance, with a significant correlation between DPPH and FRAP results (Zubia et al., 2009). Taking all studies on seaweed antioxidant capacity together, a problem arises; due to all the different extractions and antioxidant methodologies applied, it is a hard task to establish a rank of the samples with the highest and the lowest antioxidant capacity and this is

Chapter 10 Antioxidant capacity of seaweeds

311

Table 2 Antioxidant capacity techniques in selected recent studies on the antioxidant capacity of seaweed extracts. Seaweed

FRAP

ABTS

Ulva lactuca, Ulva fasciata, Enteromorpha intestinalis, Jania rubens, Gelidium spinosum Ulva lactuca, Stoechospermum marginatum Cystoseira barbata Silvetia compressa, Cystoseira osmundacea, Ecklonia arborea, Pterygophora californica, Egregia menziesii Sargassum polycystum, Sargassum duplicatum

DPPH

Saeed et al. (2019)

X X X

Anjali et al. (2019) Cadar et al. (2019) Mu´zquiz de la Garza et al. (2019)

X

X

Gracilaria beckeri, Gelidium pristoides, Ecklonia maxima, Ulva rigida Ascophyllum nodosum Turbinaria conoides Cystoseira barbata Eucheuma cottonii

X

X

X

X

Johnson et al. (2019) Manandhar et al. (2019) Olasehinde et al. (2019) Poole et al. (2019) Sari et al. (2019) Trifan et al. (2019) Prasasty et al. (2019) Rajauria (2019)

X X X X X X

X

X

Caulerpa racemosa

X X

X

Fucus vesiculosus Fucus spiralis

X

X

X

X

References

X

Ecklonia stolonifera

Himanthalia elongata, Laminaria saccharina, Laminaria digitata Padina australis, Padina tetrastromatica, Padina tenusis, Nizimuddinia zanardinii, Sargassum ilicifolium, Sargassum cristaefolium, Sargassum tenerrimum, Dictyota friabilis, Cystoseira indica, Stoechospermum marginatum Bifurcaria bifurcata Ascophyllum nodosum, Laminaria digitata Caulerpa racemosa, Caulerpa lentillifera Lessonia trabeculata Kappaphycus alvarezii Codium intricatum Eucheuma denticulatum

ORAC

X X X X X X X

Sharifian et al. (2019)

X

X

X X

X

X

X

Silva et al. (2019) Walsh et al. (2019) Yap et al. (2019) Yuan et al. (2019) Arau´jo et al. (2020) Arguelles (2020) Balasubramaniam et al. (2020) Belkacemi et al. (2020) Corsetto et al. (2020) Francisco et al. (2020) Continued

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Table 2 Antioxidant capacity techniques in selected recent studies on the antioxidant capacity of seaweed extracts—cont’d Seaweed

FRAP

Fucus spiralis

ABTS

DPPH

ORAC

References

X

X

X

Ascophyllum nodosum

X

X

Sargassum vulgare

X

Freitas et al. (2020) Garcia-Vaquero et al. (2020) Ibrahim et al., 2022 Lee et al. (2020) Ismail et al. (2020)

X

Sargassum horneri Turbinaria decurrens, Padina pavonica, Sargassum muticum, Sargassum acinarium, Pterocladia capillacea, Ulva lactuca Sargassum vachellianum

X X X

X

X

Jesumani et al. (2020) Mahomoodally et al. (2020) Nho et al. (2020) Pimentel et al. (2020) Pozharitskaya et al. (2020) Raja et al. (2020) Saraswati et al. (2020) Vasanthi et al. (2020) Zhong et al. (2020)

X

Vega et al. (2020)

X X

Alkhalaf (2021) Fonseca et al. (2021)

Halimeda sp., Spyridia hypnoides, Gracilaria fergusonii, Amphiroa anceps Ecklonia cava Porphyra dioica

X

X

X

X

X X

X X

Fucus vesiculosus

X

X

Gracilaria corticata, Halimeda opuntia Sargassum cristaefolium

X X

X X

Sargassum polycystum, Sargassum tenerrimum, Sargassum cinctum Ulva sp., Caulerpa sp., Codium sp., Dasya sp., Grateloupia sp., Centroceras sp., Ecklonia sp., Sargassum sp. Codium intertextum, Caulerpa racemosa, Ulva rigida, Pterocladiella capillacea, Hypnea spinella, Dermocorynus dichotomus, Halopithys incurva, Laurencia dendroidea, Fucus spiralis, Treptacantha abies Chondrus crispus Cystoseira abies, Zonaria tournefortii

X

X X

X

X X

X

FRAP, ferric reducing antioxidant power; ABTS, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH, 2,2-diphenyl-1-picrylhydrazyl; ORAC, oxygen radical absorbance capacity.

Chapter 10 Antioxidant capacity of seaweeds

mainly possible when several samples have been analyzed in the same study, ensuring all conditions were similar. In that situation, brown seaweeds exhibited more antioxidant capacity than red and green species (Zhong et al., 2020). Although antioxidant capacity determines the joint action of the whole combination of antioxidant compounds, there is an interest in identifying the compounds that contribute most to this activity. In this way, studies in several species have found correlations between antioxidant capacity and total polyphenol content: Bifurcaria bifurcata, Cystoseira tamariscifolia, Fucus ceranoides, and Halidrys siliquosa (Zubia et al., 2009); Ulva lactuca, Enteromorpha intestinalis, and Cladophora vagabunda (Sirbu et al., 2019); and Turbinaria decurrens, Padina pavonica, Sargassum muticum, Sargassum acinarium, Pterocladia capillacea, and Ulva lactuca (Ismail et al., 2020). In these cases, polar extracts, mainly with methanol, ethanol, or water, were obtained, and in some cases, the authors identified the combination that provided the highest antioxidant capacity: 60% methanol was more effective than combinations between 20% and 80% methanol for extracting antioxidants in Himanthalia elongata (Rajauria et al., 2013); ethanol as compared to warm water (Prasasty et al., 2019); ethanol or 70% ethanol in comparison with water, cyclohexane, or ethyl acetate (Freitas et al., 2020); and 70% acetone also resulted in a valid combination as compared to methanol or water (Trifan et al., 2019). In these polar extracts, there was also a contribution of sulfated polysaccharides, particularly in those seaweeds containing fucoidan, a structure constituted by sulfated fucoses; fucoidanrich extracts have shown antioxidant capacity in several studies (Jesumani et al., 2020; Pozharitskaya et al., 2020). Moreover, other less studied seaweed polysaccharides have also shown antioxidant capacity: alginates, laminarin, carrageenan, agar, and ulvan (Liu and Sun, 2020). Factors affecting the antioxidant capacity of seaweed polysaccharides were reviewed (Liu and Sun, 2020): generally, low molecular weight increases antioxidant capacity since the structure is less compacted and there are more hydroxyl and amine groups which are exposed; sulfate group presence also affects antioxidant capacity, not only due to the content of these constituents but also due to their position in the structure. It should be kept in mind that some of these aspects may be affected by the extraction method, so this should be optimized in order to reduce losses of antioxidant capacity. And the last contributors to the antioxidant capacity of polar extracts may be certain peptides; thus, protein hydrolysates from Porphyra dioica also exhibited antioxidant capacity (Pimentel et al., 2020).

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In contrast, other studies explored the antioxidant capacity derived from apolar extracts, since there are antioxidant constituents which are preferentially extracted by this kind of solvents, such as carotenoids, phytosterols, or terpenoids (Belkacemi et al., 2020). For evaluating the antioxidant capacity of these constituents, several solvents have been tested: chloroform (Belkacemi et al., 2020); ethyl acetate (Freitas et al., 2020; Sharifian et al., 2019); and a mixture of hexane, diethyl ether, and chloroform (Rajauria et al., 2019). Indeed, in some cases, these compounds provided more antioxidant capacity than phenolic extracts; for instance, chloroform extracts yielded more antioxidant capacity than methanol or water extracts from Caulerpa racemosa (Yap et al., 2019). These polar extracts were sometimes obtained as whole extracts but in some cases an additional fractionation was performed (Saraswati et al., 2020). Overall, the antioxidant capacity of these apolar compounds is measured by DPPH and β-carotene bleaching assay (also called β-carotene-linoleic acid system). Nevertheless, the mechanisms of reactions of these two methods are rather different, and indeed, in some cases their results did not show a correlation (Zubia et al., 2009). Independently of the compounds contributing to antioxidant capacity, several studies have reported a dose-response relationship in antioxidant capacity, i.e., the more concentrated the extracts were, the highest the antioxidant capacity was, showing a consistency in the obtained results. This has been observed, for instance, in polyphenol extracts from Eucheuma cottonii (Prasasty et al., 2019); polyphenol and fucoidan extracts from Ulva lactuca and Stoechospermum marginatum (Jesumani et al., 2020); fucoidan extract from Fucus vesiculosus (Pozharitskaya et al., 2020); and polyphenol extract from Chondrus crispus (Alkhalaf, 2021). Besides traditional chemical extractions by the combinations of solvents already mentioned, other studies have explored strategies such as the use of enzymes for extracting antioxidant compounds. Thus, the antioxidant capacity of Ecklonia radiata was measured after using several carbohydrases (Viscozyme, Celluclast, and Ultraflo) and proteases (Alcalase, Neutrase, and Flavourzyme), alone or intensified with microwave heating. The highest antioxidant capacity values were obtained when performing microwave-assisted extraction with Viscozyme between 5 and 30 min (Charoensiddhi et al., 2015). In the same way, other technologies, such as sub- and supercritical fluid extractions, have been tested for extracting antioxidants from seaweeds. Supercritical carbon dioxide was compared with the use of conventional solvents (water, 60%, 40%, and absolute ethanol) for extracting

Chapter 10 Antioxidant capacity of seaweeds

antioxidants from the brown seaweeds Sargassum wightii and Turbinaria conoides, finding that supercritical extraction provided significantly higher antioxidant capacity values evaluated by FRAP assay than traditional extractions (Kumar et al., 2020). Additionally, the authors performed a principal component analysis that also showed a clear separation between supercritical fluid extraction and solvent extraction. Similarly, a central composite analysis was designed for evaluating antioxidant capacity in Gracilaria mammillaris from the Colombian Caribbean coast subjected to supercritical CO2 extraction with ethanol as co-solvent (Ospina et al., 2017). The parameters tested were pressure (10, 20, and 30 MPa), temperature (40°C, 50°C, and 60°C), and co-solvent concentration (2%, 5%, and 8%), and the highest antioxidant capacity was obtained for 30 MPa, 60°C, and 8% co-solvent. For producing extracts with antioxidant capacity but enriched in apolar antioxidant compounds, several edible oils were tested as co-solvents in supercritical CO2 extraction (sunflower oil, soybean oil, and canola oil), obtaining the best results for sunflower oil (Saravana et al., 2017). Finally, supercritical fluid extraction may also be used for obtaining extracts enriched in specific compounds, such as fucoxanthin from Saccharina japonica and Sargassum horneri (Sivagnanam et al., 2015). In all the mentioned studies, the usual procedure for evaluating seaweed antioxidant capacity is obtaining, by chemical or enzymatic procedures, an extract where it is assumed that all antioxidants have been transferred and, therefore, total antioxidant capacity is being assessed. However, as previously mentioned, those extracts only contain extractable polyphenols (EPP), while
rez-Jime
nez NEPP remain in the corresponding residues (Pe et al., 2013). NEPP are present in seaweeds and for instance, a study with Chilean seaweeds reported that their contribution to total antioxidant capacity was quite different depending on the sample, ranging from no contribution in Scytosiphon sp. to be the only contributors to total antioxidant capacity in Gracilaria sp. and Callophyllis sp. (Sanz-Pintos et al., 2017). Similarly, another study with seven seaweeds found that the antioxidant capacity derived from NEPP was about tenfold higher by ABTS assay and sixfold higher by FRAP assay than that associated with EPP (Huang et al., 2021). More studies dealing with the antioxidant capacity of NEPP in edible seaweeds should be performed, in order to provide a comprehensive overview of the antioxidant capacity of these materials. All the mentioned studies evaluated the antioxidant capacity of extracts (or residues, in the case of NEPP) directly obtained from seaweeds. But there is a tendency in the field of food bioactive

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compounds for evaluating different bioactivity parameters after performing in vitro digestion, since food compounds are extensively transformed after intake and circulating compounds in the human body, as well as their associated activities, may be rather different to those present in the original samples. In this way, a study performed an in vitro digestion (including a step with intestinal enzymes followed by in vitro colonic fermentation) of Ascophyllum nodosum, reporting some interesting results (Corona et al., 2017). It was observed that, as regards the original extracts, the antioxidant capacity (by ABTS assay) of high molecular weight phenolic compounds was higher than that of the whole extract or the fraction of low molecular weight phenolic compounds. Intestinal digestion drastically decreased (more than 70%) the antioxidant capacity of the whole extract and low molecular weight phenolic compounds, although that of high molecular weight phenolic compounds was only decreased by 24%, meaning they remained nearly intact during their passage throughout the digestive tube. After colonic fermentation, the antioxidant capacity of the whole extract and low molecular weight phenolic compounds had an additional decrease, indicating some microbial transformation to less antioxidant compounds. And, in that step, the antioxidant capacity of high molecular weight phenolic compounds was clearly reduced, up to 82% of the original value, showing these compounds may also be used as subtract of colonic microbiota. Despite this clear decrease in antioxidant capacity after colonic fermentation, the whole extract and the two fractions (low and high molecular weight phenolic compounds) were able to significantly decrease DNA damage in intestinal epithelial cells stimulated with hydrogen peroxide. The authors suggested that this indicated that antioxidant capacity was not related to this effect, although it may also be that, even when antioxidant capacity was much lower than in the initial sample, the compounds still were concentrated enough to perform that preventive effect on DNA damage. Another study carried out the in vitro digestion of Fucus spiralis showing that the decrease in antioxidant capacity after the digestion was 50% in FRAP assay and 60% in ORAC assay, thus indicating that about half of the antioxidant capacity of the original matter was bioaccessible (Francisco et al., 2020). And a last study evaluated the antioxidant capacity (by FRAP and ABTS assays) of EPP and NEPP fractions from seven seaweeds in the original matter and after in vitro gastric and intestinal digestion, showing contradicting results: in some cases, the digestion increased the antioxidant capacity, while in other cases it decreased it (Huang et al., 2021). Overall, this shows that more studies of

Chapter 10 Antioxidant capacity of seaweeds

this kind should be performed in order to have a closer idea on the behavior of seaweed antioxidants under physiological conditions. A final aspect to be mentioned is that, besides the evaluation of antioxidant capacity in seaweed extracts, some studies have explored the effects of the incorporation of seaweeds in different food products on this parameter. Thus, significant increases in antioxidant capacity have been reported after incorporating Sirophysalis trinodis and Polycladia myrica to corn snacks (2% and 4%) or Sargassum wightii to fish jerky (0.3% and 5%) (Nova et al., 2020).

3.2

Antioxidant capacity in cell cultures

Cell cultures are a common model for measuring antioxidant capacity. Although they are an in vitro approach for assessing the modulation of oxidative status, they are closer to real physiological conditions than antioxidant capacity determinations performed in a test tube. Thus, these models have also been used for assessing seaweed antioxidant capacity. The most common way to evaluate antioxidant capacity in cell cultures is the cellular antioxidant assay (CAA), where the nonfluorescent probe 20 ,70 -dichlorodihydrofluorescein (DCFH) is oxidized by peroxyl radicals, generating the fluorescent molecule dichlorofluorescein (DCF). Higher antioxidant concentrations in the cell medium will decrease the formation of this fluorescent molecule, meaning therefore a reduction in ROS generation (Wolfe and Liu, 2007). Other authors use as fluorescent probe 20 ,70 -dichlorohydrofluorescein diacetate (DCFH-DA), which indeed was used more than 20 years ago for evaluating the antioxidant capacity of Fucus evanescens immersed in seawater (Collen and Davison, 1997). Similar to antioxidant capacity methods performed directly in extracts, determination of antioxidant capacity in cell cultures has benefits and drawbacks, which have been reviewed by several authors (Lo´pez-Alarco´n and Denicola, 2013; Wardman, 2007; Wolfe and Liu, 2008). Several seaweed species have been tested for antioxidant capacity in different cell culture models. Some studies were performed with extracts containing a mixture of antioxidant compounds, while in other cases, concentrates of some substances, such as antioxidant polysaccharides, were tested (Liu and Sun, 2020). Regarding the cell models, hepatic cells and different neuron cell lines have been the most evaluated ones. Regarding effects on hepatic oxidative damage, the human hepatoma cell line HepG2 has been used for evaluating several seaweeds. For instance, a significant decrease in ROS generation was observed

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when phlorotannin-rich extracts from Ascophyllum nodosum and Himanthalia elongata were submitted to gastrointestinal digestion and then transferred to HepG2 cells where oxidative stress had been induced by treatment with tert-butyl hydroperoxyde (Queguineur et al., 2013). In another study, oxidative stress was induced by AAPH (2,20 -azobis(2-methylpropionamidine)dihydrochloride); in this case, ROS generation was inhibited up to 60% when extracts from Fucus vesiculosus were added (Corsetto et al., 2020). A remarkable aspect is that effects on ROS generation may also depend on the conditions used for extract preparation. Thus, a study with Turbinaria tricostata from Mexico evaluated the potential protective effect of water and salt (calcium chloride) extracts in a situation where hydrogen peroxide was used to induce oxidative stress in HepG2 cells. It was found that while salt extracts were able to significantly decrease ROS generation from concentrations of 0.125 mg/mL, the water extract needed a concentration of 1 mg/mL for exerting a beneficial effect. And at a concentration of 2 mg/mL, although both extracts inhibited ROS generation, that of the salt extract was significantly more pronounced than the one observed for water extract (Chale-Dzul et al., 2015). Thus, these aspects should be considered when comparing results from studies where different extraction conditions were used. Several studies have used different brain cell lines for evaluating the potential antioxidant capacity of seaweed extracts. In this way, an assay with an extract from Halimeda incrassata in mouse hypothalamic cells subjected to oxidative stress induced by methyl-mercury found that the treatment significantly decreased ROS levels after 24 h of exposure to methyl-mercury (Linares et al., 2004). Similarly, in a study with a human dopaminergic neuronal cell line, where oxidative stress was induced by rotenone, dieckol was able to significantly decrease ROS accumulation (Cha et al., 2016). In two neuron cell lines (PC-12 and SH-SY5Y) where oxidative stress was induced by AAPH, both extracts from Diecklonia cava and isolated dieckol were able to protect cells from increased oxidative stress (Nho et al., 2020). Antioxidant capacity was also measured in neuron cells where toxicity had been induced by β-amyloid protein, in order to emulate Alzheimer’s disease. While the exposure to that protein significantly decreased antioxidant capacity, this was significantly increased when cells were treated with fucoxanthin (Zhao et al., 2015). Cell culture models have also been used to elucidate further the molecular mechanism by which seaweeds might modulate oxidative stress. Thus, a study with extracts from Ulva lactuca (Wang et al., 2013) showed that these extracts, and particularly

Chapter 10 Antioxidant capacity of seaweeds

some unsaturated fatty acids isolated from them, were able to regulate Nrf2, a nuclear transcription factor involved in the activation of the antioxidant response element (ARE) what ultimately increases the expression of phase II detoxifying enzymes. The authors observed that there was an increased activation, but not expression, of Nrf2. These effects were further validated in a murine model supplemented with an extract enriched in one of these fatty acids, a new keto-type C18 fatty acid. In line with this, a study with isolated fucoxanthin found that it was able to provide neuroprotection due to a decrease in the ROS levels, together with the stimulation of the Nrf2-ARE pathway (Zhang et al., 2017). Finally, it should be mentioned that, besides in vitro studies, it is also possible to determine antioxidant capacity in ex vitro assays. Thus, the effects of 17 seaweed species on ROS generation were evaluated in liver homogenates, identifying 3 of them which were able to significantly decrease ROS generation: Ulva pertusa, Symphyocladia latiuscula, and Ecklonia stolonifera (Hye et al., 2004).

3.3

Antioxidant capacity in animal models

In contrast to the wide body of evidence on the antioxidant capacity of seaweed extracts and the multiple studies performed in cell cultures, studies on animal models on the effects of seaweed on in vivo antioxidant capacity are nearly inexistent. Although, as discussed in Section 4, many preclinical studies have evaluated the effect of seaweed supplementation on oxidative status by other markers, we identified only five studies directly evaluating antioxidant capacity (Gheda et al., 2021; Godard ˜ ez et al., 2012; Liu et al., 2019; Tong et al., 2009; Go´mez-Ordo´n et al., 2015). Three of these studies (Gheda et al., 2021; Go´mez˜ ez et al., 2012; Tong et al., 2015) were performed in rats, Ordo´n one in hamsters (Godard et al., 2009), and one in mice (Liu et al., 2019). While in one of the cases no disease or physiological ˜ ez et al., condition was induced in the animals (Go´mez-Ordo´n 2012), the other studies consisted of animal models for senescence (Liu et al., 2019), type 2 diabetes (Gheda et al., 2021), insulin resistance (Tong et al., 2015), or atherosclerosis (Godard et al., 2009). And regarding the studied seaweeds, three of the assays were focused on the green seaweeds Ulva lactuca, Ulva rigida, Capsosiphon fulvescens, and Enteromorpha prolifera (Godard et al., 2009; Liu et al., 2019; Tong et al., 2015), one on the red seaweed Mastocarpus stellatus (Go´mez-Ordo´n˜ez et al., 2012) and two ones on the brown seaweeds Cystoseira compressa, Undaria pinnatifida, and Hizikia fusiforme (Gheda et al., 2021;

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Chapter 10 Antioxidant capacity of seaweeds

Tong et al., 2015). The main findings of these studies regarding antioxidant capacity (other biochemical markers were measured, depending on the specific objectives) will be exposed. High-cholesterol-fed hamsters were used as an animal model of atherosclerosis where the effect of Ulva rigida was assessed (Godard et al., 2009). In particular, 36 animals were divided into 3 groups: control, supplementation with the seaweed, or supplementation with an aqueous extract from the seaweed, both subjected to freeze-drying. The dose was adjusted, replacing cellulose in the control diet with an equivalent dietary fiber amount provided by Ulva rigida. Plasma antioxidant capacity was measured by ABTS method, finding that the two supplemented groups had a significant increase (about 50%) compared to the control group. No differences were observed depending on whether the whole seaweed or the aqueous extract was provided. ˜ ez et al., 2012) was developed in The next study (Go´mez-Ordo´n two groups of Wistar rats (n ¼ 6), one used as control and the other supplemented with dried Mastocarpus stellatus. In this case, no significant modifications were found in serum antioxidant capacity, measured by FRAP assay, either in the original value or in that obtained after subtracting uric acid content—a procedure recommended by some researchers due to the controversial character of this contributor to antioxidant capacity. In contrast, a significant increase (about 30%) was found in cecal antioxidant capacity in supplemented animals. The authors attributed it to the presence in this seaweed of sulfated polysaccharides, specifically kappa-/iota-hybrid carrageenan, with in vitro antioxidant capacity. These compounds arrive intact to the colon and would generate this increase in antioxidant capacity. Another study was focused on SAMP8 mice, a model of accelerated senescence (Liu et al., 2019). Forty animals were divided into four groups: a control one and three groups supplemented with resveratrol (a polyphenol found in the plant kingdom), oligosaccharides from Ulva lactuca, or oligosaccharides from Enteromorpha prolifera. Supplementations were always 150 mg/kg weight day. Additionally, there was a group of SAMR1 mice, which did not show accelerated senescence. SAMP8 mice showed significantly lower antioxidant capacity values than SAMR1 mice, while the three supplementations restored this parameter to original levels. Interestingly, this was found not only in serum samples but also in the brain. Additionally, the authors found some correlations between antioxidant capacity values and some features of intestinal microbiota, which deserve further exploration: direct correlation with the phylum Verrucomicrobiaceae and inverse

Chapter 10 Antioxidant capacity of seaweeds

correlation with specific bacteria belonging to the phyla Verrucomicrobiaceae, Streptococcaceae, Christensenellaceae, Pseudomonaceae, and Lachnospiraceae. A high-fat diet was induced for induced insulin resistance in rats in a study, where three seaweeds, i.e., Capsosiphon fulvescens, Undaria pinnatifida, and Hizikia fusiforme were tested (Tong et al., 2015). Forty rats were divided into four groups: a control group and three high-fat-fed groups, where three of them received each one of the dried seaweeds at a dose of 400 mg/kg weight day for 8 weeks. Plasma antioxidant capacity was measured by ABTS assay. At the end of the study, a significant decrease in this parameter was observed in the rats fed with the high-fat diet compared to the control diet, while the supplementation with any of the seaweeds restored the values to those found in the control group. In the most recent study (Gheda et al., 2021), the authors explored the effect of a phlorotannin extract from Cystoseira compressa on an animal model of type 2 diabetes. Forty animals were divided into four groups, two ones used as control and two ones with diabetes induced by intraperitoneally administering streptozotocin. A control group and a diabetic group were supplemented with the seaweed extract, at a dose of 60 mg/kg weight day for 4 weeks. Hepatic antioxidant capacity was measured by a specific procedure, where depletion of hydrogen peroxide by total antioxidants was determined. This parameter was found to be significantly increased in both supplemented groups as compared to their non-supplemented counterpart.

3.4

Antioxidant capacity in clinical studies

In the case of clinical trials, current evidence for the effects of seaweed supplementation is even more limited than for animal models, concerning not only the direct evaluation of antioxidant capacity, but also the determination of other markers of the oxidative status. Indeed, only one study explored plasma antioxidant capacity after supplementation with seaweed (Allsopp et al., 2016) and the other two evaluated other markers of oxidative status (Baldrick et al., 2018; Kang et al., 2012). In the first study (Allsopp et al., 2016), 40 healthy subjects received either a placebo bread or a bread enriched with a dose of 5 g/day of the red seaweed Palmaria palmata for 28 days. Plasma antioxidant capacity was measured by FRAP assay, although no significant modification was observed after supplementation. Similarly, no modifications were observed in DNA damage, either in basal values or in those obtained after stimulation with hydrogen peroxide.

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The second clinical trial (Kang et al., 2012) was focused on subjects with high endogenous levels of γ-glutamyltransferase, which has been associated with either high alcohol intake, liver damage, or increased oxidative stress. A total of 48 participants received either a placebo or fermented Laminaria japonica for 4 weeks. Together with an improvement in the levels of γ-glutamyltransferase, a significant decrease in lipid oxidation and a significant increase in endogenous antioxidant systems were observed. In the last clinical trial (Baldrick et al., 2018), 80 overweight or obese subjects received either a 400 mg capsule containing 100 mg of polyphenols from the brown seaweed Ascophyllum nodosum and 300 mg maltodextrin or a 400 mg maltodextrin placebo control capsule daily for 8 weeks. Antioxidant capacity was not measured in this study, but the authors evaluated the effects on the oxidative status by measuring peroxide concentration and DNA damage. No significant differences in peroxide concentration were observed after the treatment. Regarding DNA damage, measured by the comet assay, a significant decrease in these values was observed in obese subjects but not in those ones exhibiting overweight. When additional DNA damage was induced by hydrogen peroxide, no difference was observed as compared to the placebo treatment, either in the whole group or in the subgroups of subjects with obesity or overweight. At the same time, some phlorotannin metabolites were found in urine, but the wide variability in the excretion values for the subjects avoided establishing them as exposure markers. Despite these effects regarding oxidative status, both clinical trials found significant improvements in markers of inflammation after seaweed supplementation. Overall, these results show the differences that there may be between in vitro or preclinical studies and clinical trials, as well as the need to develop clinical trial studies covering the diversity of seaweeds which have shown promising results in the studies performed so far.

4. Other effects of seaweeds in oxidative stress modulation As discussed in the previous section, sometimes the effects of seaweeds on oxidative stress are not measured in terms of antioxidant capacity, but evaluating the modifications in other markers of oxidative status. Here, some of the main results from preclinical studies using these approaches will be discussed (the only available data from clinical trials where shown previously). In particular, the most common way of measuring the total effect

Chapter 10 Antioxidant capacity of seaweeds

of circulating antioxidants, besides direct antioxidant capacity determination, is the assessment of endogenous antioxidant defense systems. As described earlier, it has been shown that phenolic compounds and, specifically, phlorotannins, are able to behave, not only as direct antioxidants but also as indirect ones, since they stimulate the expression of antioxidant enzymes due to their interaction with the transcription factor Nrf2, linked to the antioxidant response (Park et al., 2019). Some studies with cell cultures have evaluated the levels of these endogenous antioxidant systems. For instance, fucoxanthin, at physiological concentrations of 1–20 μM, was able to increase GSH (reduced glutathione) levels in a cell model of iron-induced oxidation (Liu et al., 2011). Similarly, diphlorethohydroxycarmalol, a polyphenol isolated from Ishige okamurae, activated NRF2 and increased the mRNA expression of antioxidant enzymes in a human embryonic kidney cell line subjected to methylglyoxal-induced oxidative stress (Cha et al., 2018). Also, some studies have focused on the effects of seaweed antioxidant polysaccharides, such as alginate, fucoidan, laminarin, or ulvan, finding significant increases in endogenous antioxidant systems in different cell models (Liu and Sun, 2020). Nevertheless, the determination of antioxidant enzymes has been performed specially in animal studies. An overview of the several dozens of articles dealing with preclinical studies with seaweed supplementation where antioxidant enzymes were measured is shown in Table 3. A first general remark is the fact that, as observed for in vitro studies, due to the wide diversity of seaweeds, it is difficult to see a repetition of the analyzed seaweeds. Thus, although several studies have been performed for species such as Ulva lactuca, Gracilaria birdiae, or Sargassum polycystum, there is only one study for many other species. There are also differences in the products provided: some studies were focused on the whole dried seaweed, other ones provided extracts aimed to obtain either phlorotannin or polysaccharide concentrates, and some of them supplemented the animals with isolated compounds, such as some of the antioxidant polysaccharides previously mentioned or the phlorotannin dieckol. These studies covered a wide diversity of pathologies or physiological conditions: gastrointestinal pathologies (gastric mucosal gastric injury, delayed gastrointestinal motility, ulcerative colitis), cardiometabolic disorders (diabetes, insulin resistance, hyperlipidemia), toxicity in several organs (liver damage, kidney, mitochondria), cancer (breast, liver, testicles, prostate), cognitive aspects (memory impairment, amnesia), or other disorders (hyperoxaluria). But the most remarkable aspect from Table 3 is the fact that there

323

Table 3 Reported effects from animal studies on the effects of supplementation with seaweeds or derived products on antioxidant defense systems.

Supplementation

Animal model

Ulva reticulata (extract)

Acetaminophen-induced hepatic damage in rats HCl and ethanol-induced gastric mucosal damage in rats Acetaminophen-induced hepatic damage in rats Acetaminophen-induced hepatic damage in rats Ethylene glycol-induced hyperoxaluria in rats Cyclosporin A-induced mitochondrial alterations in rats Hyperlipidemic-fed rats

Sargassum polycystum (extract) Sargassum polycystum (extract) Sargassum polycystum (extract) Fucus vesiculosus (polysaccharide extract) Sargassum wightii (polysaccharide extract) Eucheuma cottonii Hizikia fusiformis (polysaccharide extract) Kappaphycus alvarezii, Caulerpa lentillifera, Sargassum polycystum Himanthalia elongata in restructured meat

Ethanol-induced toxicity in rats

Gracilaria birdiae (polysaccharide extract) Laminaria japonica (fucoidan) Halimeda opuntia

Study duration

SOD

CAT

GSH

15 days

"

"

"

15 days

"

"

"

15 days

"

"

28 days

"

"

21 days

"

35 days

"

Hyperlipidemic-fed rats

Hyperlipidemic-fed rats

35 days

Naproxen-induced gastrointestinal damage in rats Ab (1-4)-induced memory impairment in rats CCl4-induced liver damage in rats

2 days Acute study Acute study

GPx

"

"

15 days

Acute study 112 days

GR

" " "

" "

" "

"

"

" "

"

"

"

#

GST

References Raghavendra et al. (2004a) Raghavendra et al. (2004b) Raghavendra et al. (2004c) Raghavendra et al. (2005) Veena et al. (2007) Josephine et al. (2007) Wresdiyati et al. (2008) Choi et al. (2009) Matanjun et al. (2010) Schultz Moreira et al. (2011) Silva et al. (2012) Gao et al. (2012) De Olivera e Silva et al. (2012)

Low molecular weight fucoidan Ecklonia cava (dieckol) Dieckol Gracilaria birdiae (polysaccharide extract) Padina tetrastromatica (extract) Turbinaria decurrens (fucoidan) Ulva linza, Lessonia trabeculata Ulva lactuca (polysaccharide extract) Kappaphycus alvarezii, Sargassum polycystum (mixture of extracts) Capsosiphon fulvescens, Hizikia fusiforme, Undaria pinnatifida Sodium alginate

Gracilaria changii Ecklonia cava (dieckol) Hypnea musciformis Ulva lactuca (polysaccharide extract) Pyropia columbina (added to extruded maize products)

Cisplatin-induced delayed gastrointestinal motility in rats C57Bl/KsJ-db/db, type 2 diabetic mice CCl4-induced kidney damage in rats TNBS-induced colitis in rats CCl4-induced liver damage in rats Ethanol-induced intoxication in rats Hyperlipidemic and hyperglycemic-fed rats

35 days

"

"

14 days

"

"

"

"

"

Acute study 3 days Acute study 60 days

#

#

#

"

"

"

"

#

28 days

4 weeks

"

"

56 days

"

"

Hyperlipidemic-fed rats

56 days

"

Monosodium-glutamate-induced liver damage in obese and diabetic mice Hyperlipidemic-fed rats

112 days

Tong et al. (2015) "

"

Miyazaki et al. (2016)

"

Chan et al. (2016) Sadeeshkumar et al. (2016) Balamurugan et al. (2017) Abd-Ellatef et al. (2017) Cian et al. (2018)

56 days

"

"

105 days

"

"

112 days

"

"

"

70 days

"

"

"

60 days

Song et al. (2012) Kang et al. (2013a) Kang et al. (2013b) Brito et al. (2014) Gnana Selvi et al. (2014) Meenakshi et al. (2014) Ramı´rezHiguera et al. (2014) Sathivel et al. (2014) Dousip et al. (2014)

"

D-Galactosamine-induced liver damage in rats Hyperlipidemic-fed rats

N-Nitrosodiethylamine-induced liver cancer in rats DMBA-induced breast cancer in rats DMBA-induced breast cancer in rats Growing rats

"

"

"

" "

" "

"

"

Continued

Table 3 Reported effects from animal studies on the effects of supplementation with seaweeds or derived products on antioxidant defense systems—cont’d

Supplementation

Animal model

Codium fragile (polysaccharide extract) Fucoidan

Alloxan-induced oxidative damage in diabetic rats Acetaminophen-induced toxicity in rats Contrast-induced kidney damage in rats CCl4-induced oxidative damage in rats Hyperlipidemic-fed rats Aspartame-induced toxicity in rats Diazinon-induced oxidative damage in rats Alloxan-induced oxidative damage in diabetic rats Scopolamine-induced amnesic mice Monosodium-glutamate-induced testicular and prostatic damage in rats Acetic acid-induced ulcerative colitis in mice Cisplatin-induced kidney damage in rats Thioacetamide-induced liver injury in mice

Sargassum fusiforme (polysaccharide extract) Bryothamnion triquetrum (extract) Saccharina japonica Sargassum vulgare (extract) Laminaria japonica (fucoidan) Kappaphycus alvarezii (carrageenans) Sargassum ilicifoium, Padina tetrastromatica (extract) Ulva lactuca (extract)

Gracilaria caudata (polysaccharide extract) Ulva fasciata (extract) Fucus serratus (fucoidan)

Study duration

SOD

CAT

30 days

"

"

7 days

"

"

5 days

"

GSH

GR

GPx "

"

56 days 21 days

" "

" "

28 days

"

"

"

"

45 days

"

"

"

"

"

"

7 days "

"

"

Acute study 10 days 42 days

"

" "

"

" "

References Kolski et al. (2018) Wang et al. (2018) Dai et al. (2019)

"

20 days

45 days

GST

"

De Vidal Novoa et al. (2019) Jo et al. (2019) Ibrahim et al., 2022 Abdel-Daim et al. (2020) Sanjivkumar et al. (2020) Yende et al. (2021) Helal et al. (2021) Dutra et al. (2021) Sohail et al. (2021) Tsai et al. (2021)

Only significant modifications as compared to the alteration-induced animals are shown. SOD, superoxide dismutase; CAT, catalase; GSH, reduced glutathione; GR, glutathione reductase; GPx, glutathione peroxidase; GST, glutathione-S-transferase.

Chapter 10 Antioxidant capacity of seaweeds

was a nearly unanimous tendency toward restoration in antioxidant defense systems, damaged by the different experimental approaches sued when seaweed supplementation was performed. Thus, a total of six molecules were measured (SOD, superoxide dismutase; CAT, catalase; GSH; GPx, glutathione peroxidase; GR, glutathione reductase; GST, glutathione-S-transferase), although only some studies included the whole battery of determinations. But, independently of whether a single molecule or the six of them were measured, significant increases compared to altered animals were found in most studies. This indicates the ability of seaweeds to exert as in vivo antioxidants and, therefore, agrees with in vitro results, in that case, measured as antioxidant capacity. It should be highlighted that some commercial drugs, such as glibenclamide, and oral antihyperglycemic agents, also originate, as a consequence of their pharmacological action, an increase in the activity of these antioxidant systems (Sanjivkumar et al., 2020), showing the biological implications of the modifications in these parameters observed in preclinical studies with seaweeds. Additionally, it is relevant to determine not only the modifications in antioxidant enzymes but also in the molecular mechanisms that activate this response; thus, a study in growing rats where Pyropia columbina was added to extruded maize products found a significant increase in the expression of the transcription factor Nrf2 in both proximal colon and distal colon, an effect concomitant to the increased expression of GR and CAT (Cian et al., 2018). In contrast to other studies, this one found a decrease in SOD levels in the animals supplemented with seaweed. The authors suggested that the difference in the expression of the antioxidant systems, with some of them enhanced while that one was decreased, might indicate a situation where hydrogen peroxide removal is higher than the formation. Interestingly, the stimulation of endogenous antioxidant systems has been shown, in some of the reported studies, to cause an improvement in the oxidation of biomolecules. For instance, in a study in rats, ethanol and chloroform extracts of Ulva fasciata decreased lipid peroxidation in the kidney where damage had been induced by cisplatin (Sohail et al., 2021). Similarly, sulfated polysaccharides from Gracilaria caudata significantly decreased malondialdehyde (MDA) in the inflamed mucosa of a mice model for ulcerative colitis (Dutra et al., 2021). Or a study in a rat model of liver cancer supplemented with dieckol from Ecklonia cava found a significant decrease in other markers of lipid oxidation (conjugated dienes, lipid hydroperoxides) as well as in protein oxidation, measured as carbonyl groups (Sadeeshkumar et al., 2016).

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These effects on the oxidation of biomolecules are biologically relevant, since it is known that oxidative damage of proteins, lipids, or DNA is a factor involved in the progression of several chronic diseases. For this reason, some of the studies described in Table 3 evaluated, together with the effects on oxidative status, markers of cardiometabolic risk (Jo et al., 2019; Sanjivkumar et al., 2020), cancer (Abd-Ellatef et al., 2017) or cognitive function (Yende et al., 2021), finding that the ability of seaweeds to modulate oxidative response may be related to the improvements observed in those parameters. Moreover, increased oxidation also affects other physiological functions, such as the regulation of thyroid hormones, explored in a study on aspartame-induced toxicity in rats (Ibrahim et al., 2022) or semen quality, a reason by which some animal studies on the antioxidant capacity of seaweeds have focused on reproductive parameters (Helal et al., 2021). A relevant aspect of being elucidated is how long after seaweed intake, the biological effects from their bioactive compounds are active. In that way, a study where toxicity was induced by acetaminophen in rats observed that the significant improvement in endogenous antioxidant systems, particularly in GSH levels, that was observed 24 h after supplementation with fucoidan did not remain in later measurements (Wang et al., 2018). Finally, it should be mentioned that studies in different breeding species, with an animal nutrition approach, also found a beneficial effect of seaweed supplementation in terms of oxidative studies. In this way, goat kids were supplemented with Sargassum spp. at a dose either of 2.5% or 5% of feed during 70 days (Angulo et al., 2020). CAT activity was significantly increased in the rumen, liver, and intestinal mucus at both doses, while it was significantly decreased in jejunum, cecum, and colon also with both doses. Regarding SOD, significant increases with both doses were found in the rumen, muscle, and jejunum, as well as in abomasum, liver, kidney, and colon for 5% dose. The authors attributed that the observed effects were higher for SOD activities than for CAT activities to the presence in the tested seaweeds or minerals, which are cofactor for this enzyme. Another study evaluated the effect of the supplementation with Sargassum latifolium (at doses of 2% and 4% of the feeding diet) in sheep subjected to heat stress-induced toxicity, a situation that caused multiple alterations in these animals (Ellamie et al., 2020). A significant improvement was observed with both doses in plasma antioxidant capacity, SOD, and CAT enzymes. And a study with late-phasing laying hens evaluated the effect of the supplementation with polysaccharides from Enteromorpha at three different concentrations on

Chapter 10 Antioxidant capacity of seaweeds

reproductive parameters and oxidative status (Guo et al., 2020). Polysaccharide supplementation quadratically improved serum SOD activity and the liver CAT activity and linearly enhanced jejunal CAT activity.

5. Perspectives Data analyzed earlier show a sustained body of evidence on the ability of edible seaweeds to modulate oxidative status, a relevant aspect in the prevention or modulation of a wide number of pathologies. Nevertheless, some aspects to be further explored during the next years emerge from this review of the literature on the topic. Regarding the products analyzed, the diversity of seaweeds makes that, although several dozens of them have been analyzed, in many of them there is a single available study, while many other species have not yet been tested for their antioxidant potential. This means that a comprehensive approach toward the general antioxidant effects of seaweeds should be made, particularly because these effects may come from several bioactive constituents present in different proportions in seaweeds. Thus, a first consideration would be to extend the evaluation of the antioxidant potential of edible seaweeds. Another relevant aspect is regarding the products tested in the existing studies, since they go from those ones performed with isolated compounds, to other ones using extracts or to the cases where the whole seaweed was evaluated. Of course, the ranges of concentrations of bioactive compounds in each case are very different, and this should be taken into account when evaluating the results, particularly regarding their practical implications. In this sense, it is relevant to state that extract production would decrease the content of some undesirable compounds of seaweeds or present above the recommendations (such as certain heavy metals) but, at the same time, it implies industrial processing that would have an economic impact on the final price of seaweed-derived products (as compared to those ones based on the use of the intact seaweed as an ingredient). A final consideration of the samples analyzed is that determinations on whole seaweeds are performed in dried samples from practical processing and analytical aspects. Nevertheless, although seaweeds are consumed in a dried form in some preparations, on other ones, they are consumed as fresh products where, obviously, the amount of bioactive compounds and associated antioxidant capacity is much lower. This is an important point that should be kept in mind when translating findings from

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the antioxidant capacity (and other markers) to nutritional recommendations of seaweed consumption. Regarding study design, as shown previously, there is a high accumulation of data on the antioxidant capacity of extracts, and a substantial number of studies exploring cell cultures or animal models. Nevertheless, some aspects, such as the contribution of NEPP to total antioxidant capacity or the determination of bioaccessibility/bioavailability of seaweed antioxidant capacity, have been scarcely explored. And the highest limitation appears in the deficient number of clinical trials dealing with the antioxidant capacity of seaweeds. Thus, efforts during next year should try to cover especially this knowledge gap, developing more clinical trials on seaweed supplementation and which included, among other chemical biomarkers, the evaluation of the effects on oxidative status (measured as antioxidant capacity but also as oxidative damage to biomolecules). And observational studies might also provide valuable information on the effects of seaweed consumption on oxidative status, although, for this, edible seaweeds should be included in food frequency questionnaires, where they are now commonly missing. Overall, the incorporation of these aspects in seaweed research may help to provide a step forward in a near future on the knowledge of these products to reverse increased oxidative stress as an altered physiological process underlying several chronic diseases.

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Suktham, T.; Jones, A.; Acquaviva, A.; Dennis, G. R.; Shalliker, R. A.; Soliven, A. Better Than Bench Top. High Speed Antioxidant Screening via the Cupric Reducing Antioxidant Capacity Reagent and Reaction Flow Chromatography. Microchem. J. 2020, 152, 104348. Tong, T.; Ko, D. O.; Kim, B. S.; Ham, K. S.; Kang, S. G. Beneficial Effect of Seaweed on High-Fat Diet-Induced Oxidative Stress and Insulin Resistance in Rats. Food Sci. Biotechnol. 2015, 24, 2185–2191. Trifan, A.; Vasincu, A.; Luca, S.; Neophytou, C.; Wolfram, E.; Opitz, S.; Sava, D.; Bucur, L.; Cioriu, B.; Miron, A.; Aprotosaie, A. C.; Cioanca, O.; Hancianu, M.; Jitaeranu, A.; Constantinou, A. Unravelling the Potential of Seaweeds From the Black Sea Coast of Romania as Bioactive Compounds Sources. Part I: Cystoseira barbata (Stackhouse) C. Agardh. Food Chem. Toxicol. 2019, 134, 110820. Tsai, M. Y.; Yang, W. C.; Lin, C. F.; Wang, C. M.; Liu, H. Y.; Lin, C. S.; Lin, J. W.; Lin, W. L.; Lin, T. C.; Fan, P. S.; Hung, K. H.; Lu, Y. W.; Chang, G. R. The Ameliorative Effects of Fucoidan in Thioacetaide-Induced Liver Injury in Mice. Molecules 2021, 26, 1937. Vasanthi, C.; Appa Rao, V.; Narendra Babu, R.; Sriram, P.; Karunakaran, R. In-Vitro Antioxidant Activities of Aqueous and Alcoholic Extracts of Sargassum Species—Indian Brown Seaweed. J. Food Process. Preserv. 2020, 44, e14877. Veena, C. K.; Josephine, A.; Preetha, S. P.; Varalakshmi, P. Beneficial Role of Sulfated Polysaccharides From Edible Seaweed Fucus vesiculosus in Experimental Hyperoxaluria. Food Chem. 2007, 100, 1552–1559. ´ lvarez-Go´mez, F.; Gu € enaga, L.; Figueroa, F.; Go´mez-Pinchetti, J. AntioxiVega, J.; A dant Activity of Extracts From Marine Macroalgae, Wild-Collected and Cultivated, in an Integrated Multi-Trophic Aquaculture System. Aquaculture 2020, 522, 735088. Veliz, K.; Chandia, N.; Bischof, K.; Thiel, M. Geographic Variation of UV Stress Tolerance in Red Seaweeds Does Not Scale With Latitude Along the SE Pacific Coast. J. Appl. Phycol. 2020, 56, 1090–1102. Walsh, P.; McGrath, S.; McKelvey, S.; Ford, L.; Sheldrake, G.; Clarke, S. The Osteogenic Potential of Brown Seaweed Extracts. Mar. Drugs 2019, 17, 141. Wang, R.; Paul, V. J.; Luesch, H. Seaweed Extracts and Unsaturated Fatty Acid Constituents From the Green Alga Ulva lactuca as Activators of the Cytoprotective Nrf2-ARE Pathway. Free Radic. Biol. Med. 2013, 57, 141–153. Wang, Y. Q.; Wei, J. G.; Tu, M. J.; Gu, J. G.; Zhang, W. Fucoidan Alleviates Acetaminophen-Induced Hepatotoxicity via Oxidative Stress Inhibition and Nrf2 Translocation. Int. J. Mol. Sci. 2018, 19, 4050. Wardman, P. Fluorescent and Luminescent Probes for Measurement of Oxidative and Nitrosative Species in Cells and Tissues: Progress, Pitfalls, and Prospects. Free Radic. Biol. Med. 2007, 43, 995–1022. Wolfe, K. L.; Liu, R. H. Cellular Antioxidant Activity (CAA) Assay for Assessing Antioxidants, Foods, and Dietary Supplements. J. Agric. Food Chem. 2007, 55, 8896–8907. Wolfe, K. L.; Liu, R. H. Structure-Activity Relationship of Flavonoids in the Cellular Antioxidant Activity (CAA) Assay. J. Agric. Food Chem. 2008, 56, 8404–8411. Wresdiyati, T.; Hartanta, A. B.; Astawan, M. A. D. E. The Effect of Seaweed Eucheuma cottonii on Superoxide Dismutase (SOD) Liver of Hypercholesterolemic Rats. HAYATI J. Biosci. 2008, 15, 105–110. Yap, W.; Tay, V.; Tan, S.; Yow, Y.; Chew, J. Decoding Antioxidant and Antibacterial Potentials of Malaysian Green Seaweeds: Caulerpa racemosa and Caulerpa lentillifera. Antibiotics 2019, 8, 152. Yende, S. R.; Arora, S. K.; Ittadwar, A. M. Antioxidant and Cognitive Enhancing Activities of Sargassum ilicifolium and Padina tetrastromatica in Scopolamine Treated Mice. J. Biol. Active Prod. Nat. 2021, 11, 11–21.

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Yeo, J.; Shahidi, F. Critical Re-Evaluation of DPPH Assay: Presence of Pigments Affects the Results. J. Agric. Food Chem. 2019, 67, 7526–7529. Yuan, Y.; Zheng, Y.; Zhou, J.; Geng, Y.; Zou, P.; Li, Y.; Zhang, C. Polyphenol-Rich Extracts From Brown Macroalgae Lessonia trabeculate Attenuate Hyperglycemia and Modulate Gut Microbiota in High-Fat Diet and StreptozotocinInduced Diabetic Rats. J. Agric. Food Chem. 2019, 67, 12472–12480. Zhang, L.; Wang, H.; Fan, Y.; Gao, Y.; Li, X.; Hu, Z.; Ding, K.; Wang, Y.; Wang, X. Fucoxanthin Provides Neuroprotection in Models of Traumatic Brain Injury via the Nrf2-ARE and Nrf2-Autophagy Pathways. Sci. Rep. 2017, 7, 46763. Zhao, X.; Zhang, S.; An, C.; Zhang, H.; Sun, Y.; Li, Y.; Pu, X. Neuroprotective Effect of Fucoxanthin on β-Amyloid-Induced Cell Death. J. Chin. Pharm. Sci. 2015, 24, 467–474. Zhong, B.; Robinson, N.; Warner, R.; Barrow, C.; Dunshea, F.; Suleria, H. LC-ESIQTOF-MS/MS Characterization of Seaweed Phenolics and Their Antioxidant Potential. Mar. Drugs 2020, 18, 331. Zubia, M.; Fabre, M. S.; Kerjean, V.; Le Lann, K.; Stiger-Pouvreau, V.; Fauchon, M.; Deslandes, E. Antioxidant and Antitumoural Activities of Some Phaeophyta From Brittany Coasts. Food Chem. 2009, 116, 693–701.

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Gut microbiota and marine phenolics

11

Samantha Nu´n˜ez, Arl
es Urrutia, and Daniel Garrido Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Cato´lica de Chile, Santiago, Chile

1. Introduction The marine ecosystem is a rich source of bioactive compounds such as phenolics, carbohydrates, vitamins, minerals, and polyunsaturated fatty acids (PUFA). Most of them have proven beneficial for human health and potentially useful for medical applications (Nova et al., 2020). This chapter focuses on the polyphenols produced by seaweed or macroalgae and their impact on the gut microbiota. Seaweeds are the primary producers and a rich source of bioactive molecules, so they are vital in the marine ecosystem. Seaweeds are exposed to harsh environmental conditions such as light and nutrients limitations, and infections by pathogens and parasites. In response, they produce polyphenols as secondary metabolites to protect them against UV radiation, predators, and temperature variations (Cotas et al., 2020; Leandro et al., 2020). Phenolic compounds are secondary metabolites found in both marine and terrestrial organisms, and those metabolites from terrestrial sources have been extensively studied (Besednova et al., 2020; Liu et al., 2011; Lopez-Santamarina et al., 2020; Manach et al., 2004). Meanwhile, marine polyphenols are produced by three relevant groups of marine macroalgae, named by the color of the thallus: red (Rhodophyta), brown (Phaeophyceae), and green (Chlorophyta) algae (Leandro et al., 2020), and recent studies demonstrated their relevance in human health (Cherry et al., 2019; Paudel et al., 2019). Improvement in the extraction and purification techniques allows researchers to choose the most valuable seaweeds according to total Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00010-8 Copyright # 2023 Elsevier Inc. All rights reserved.

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polyphenols for farming and production processes (Huang et al., 2021; Kim et al., 2005; Sabeena Farvin and Jacobsen, 2013). As mentioned, in the last decade, polyphenols have become a compelling subject of study (Mateos et al., 2020) due to their beneficial properties for biomedical applications as anti-diabetic, anti-inflammatory, antimicrobial, antitumoral, and antioxidant. Additionally, the consumption of phenolic compounds has been demonstrated to modulate the gut microbiota composition (GM) (Catarino et al., 2021). These dietary polyphenols are poorly absorbed in the small intestine, reaching the colon almost unchanged where they are transformed by the GM, revealing a potential prebiotic use (Catarino et al., 2021; Duda-Chodak et al., 2015).

2. Structural classification of marine polyphenols These diverse compounds have one or two aromatic rings linked to a hydroxyl substituent (Kalogianni et al., 2020; Kawabata et al., 2019), and the total molecular size varies in a wide range (126–650 kDa) from simple molecules to complex polymers (P
erez et al., 2016). Four main groups of available marine phenolic compounds can be described as follows (Mateos et al., 2020) and detailed in Table 1.

2.1 Phlorotannins They are the most studied marine phenolics and the structural components of the cell wall are exclusively found in brown algae (Arnold and Targett, 2002) with content up to 20% in dry mass (Mekini
c et al., 2019). They are highly hydrophilic oligomers formed by monomers of an acetate/malonate pathway product called phloroglucinol. They contain additional halogen or
rez et al., 2016), hydroxyl groups (Arnold and Targett, 2002; Pe and according to their structural linkage and their diversity of the structure, they are classified into six groups: phlorethols (aryl-ether bonds), fucols (aryl-aryl bonds), fucophlorethols (ether or phenyl linkage), eckols (dibenzo-1,4-dioxin linkage), fuhalols (ortho/para-arranged ether bridges and an additional hydroxyl group on one unit), and carmalols (dibenzodioxin moiety) (Cotas et al., 2020).

Table 1 Structural classification and properties of marine phenolics. Classification

Structures

Phlorotannins

HO

OH HO

HO OH

O

HO

OH

HO

HO

OH HO

B. Fucols

A. Phlorethols OH

OH

HO HO

HO

OH

OH O

HO

HO

OH

O O

OH

HO

Properties

References

Modulation of the gut microbiota Antioxidant Antimicrobial activity Antiinflammatory

Ahn et al. (2015), Choi et al. (2010), Cotas et al. (2020), Kazłowska et al. (2010), Kong et al. (2009), Mateos et al. (2020), Nagayama et al. (2002), and Vinet and Zhedanov (2011)

OH

O

C. Fucophlorethols

OH

D. Eckols OH OH

OH

O

OH

OH

O OH

OH

OH

O

OH OH

HO

E. Fuhalols (tetrafuhalols) OH O

O

HO

OH

O OH

OH

HO OH

F. Carmalols Continued

Table 1 Structural classification and properties of marine phenolics—cont’d Classification

Structures

Bromophenols

Br

OH

HO

Br

Br

A. 2-Bromophenol

Properties

References

Anti-diabetic Antioxidant Antimicrobial activity

Chung et al. (2003), Cotas et al. (2020), Oh et al. (2008), Paudel et al. (2019), Sun et al. (2017), Xu et al. (2003), Xu et al. (2012), and Xu et al. (2018)

Antioxidant Antimicrobial activity

Mancini-Filho et al. (2009), Senthilkumar and Sudha (2012), and Shanura Fernando et al. (2016)

Modulation of the gut microbiota Antioxidant Antimicrobial activity Anti-diabetic

Al-Saif et al. (2014), Cotas et al. (2020), Ozdal et al. (2016), Yan et al. (2019), and Yonekura-Sakakibara et al. (2019)

B. 2,6-Dibromophenol Simple phenolic acids

O

O HO

HO

OH

OH HO

HO OH

A. Hydroxybenzoic acid (gallic acid)

B. Hydroxycinnamic acid (caffeic acid)

Flavonoids O

O

O

O

A. Flavones

B. Flavonols

OH

O

OH

C. Flavan-3-ol

Chapter 11 Gut microbiota and marine phenolics

2.2 Bromophenols It is well known that these compounds are produced by red algae, green algae, brown algae (Chung et al., 2003; Liu et al., 2011; Oh et al., 2008), and even by some sponges (Utkina et al., 2002). Although the synthesis pathway of these compounds has not been clarified yet (Cotas et al., 2020), some authors have suggested that tyrosine and phenylalanine are bromophenol precursors (Peng et al., 2005). Bromophenols are structured by a phenyl ring with different degrees of bromination (Cotas et al., 2020; Liu et al., 2011; Mateos et al., 2020).

2.3 Simple phenolic acids These compounds can be found in green seaweed (Gupta and Abu-Ghannam, 2011), brown seaweed (Agrega´n et al., 2017), and some red algae (Xu et al., 2015). Simple phenolic acids are formed by one aromatic ring with at least one carboxylic acid group. These phenolics can be classified according to the number of carbon chains attached to the phenolic ring (Cotas et al., 2020; Mateos et al., 2020): hydroxybenzoic acids and acetophenones contain one carbon chain, phenylacetic acids contain two, and hydroxycinnamic acids contain three (Cotas et al., 2020). The hydroxybenzoic acids include gallic acid, p-hydroxybenzoic acid, vanillic acid, and syringic acid, while hydroxycinnamic acids include caffeic, ferulic, sinapic, and p-coumaric acids (Cotas et al., 2020; Mateos et al., 2020; Shanura Fernando et al., 2016).

2.4 Flavonoids Although flavonoids found in marine sources are scarce, an increasing number of studies demonstrate their presence in red seaweed, green seaweed, brown seaweed (Rajauria et al., 2016; Santos et al., 2019; Yarnpakdee et al., 2019), and microalgae (Yonekura-Sakakibara et al., 2019). Their structure is characterized by an heterocyclic oxygen bound to two aromatic rings, and they are classified as flavones, flavonols, flavan-3-ol, anthocyanins, and proanthocyanidins (Cotas et al., 2020). Although there are discussions regarding the path synthesis for flavonoids in algae, Yonekura-Sakakibara, Higashi, and Nakabayashi concluded that naringenin is the precursor of all flavonoids (YonekuraSakakibara et al., 2019).

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3. Properties and activities of phenolics Marine polyphenols’ properties are related to algal survival
rez et al., 2016) and have demonunder ecological pressures (Pe strated multiple beneficial effects such as antitumoral, antimicrobial, anti-diabetic, anti-inflammatory, and antioxidant (Cotas et al., 2020; Kim et al., 2015; Ko et al., 2013; Lee et al., 2017; Lopez-Santamarina et al., 2020; Mateos et al., 2020; Singh et al., 2019; Table 1). Most of these activities can be used for biotechnological applications and proposed as candidates for several health treatments.

3.1 Antioxidant activity The high oxidative stress level occurs for a concentration imbalance of redox molecules and triggers metabolic and inflammation-related diseases in humans (Wang et al., 2021). Cotas et al. (2020) showed that phlorotannins, bromophenols, and flavonoids exhibited high scavenging activity against free radicals reducing the level of oxidative stress. Devi et al. determined that a rich-polyphenol extract from the brown seaweed Gelidiella acerosa exhibited high scavenging activity against peroxide, hydroperoxide, and lipid peroxyl, similarly as butylated hydroxytoluene (BHT), a synthetic antioxidant used in food preservation (Devi et al., 2008). Sadati et al. found a positive correlation between phenolic content and antioxidant activity in the brown seaweeds Sargassum swartzii, Cystoseira myrica, and Colpomenia sinuosa (Sadati et al., 2011). Additionally, Mancini-Filho et al. found that salicylic, cinnamic, gallic, and caffeic acids in extracts from Halimeda monile (green algae) helped hepatic recovery from injuries induced by treatment with tetrachloromethane in rats, which evidenced the antioxidant activity of marine phenols in mammals models, and their positive influence in our mechanisms for the liver repairment by scavenging free radicals (Mancini-Filho et al., 2009).

3.2 Anti-inflammatory activity The triggering of inflammation is a multifactor process where molecules change their function according to cellular signaling cues. For example, marine phenolics demonstrated their anti-inflammatory activity when nitric oxide (NO) is in an abnormal concentration, acting as an antiinflammatory molecule (Kazłowska et al., 2010). Kazłowska et al. showed that an extract

Chapter 11 Gut microbiota and marine phenolics

from Porphyra dentata (red algae) decreased concentrations of nitric oxide when acting as a pro-inflammatory cytokine in a macrophages model of leukemia (RAW 264.7) (Kazłowska et al., 2010). The anti-inflammatory mechanisms are the downregulation of nitric oxide (NO) expression, direct scavenging, or inhibition of an enzyme related to NO production (Lopes et al., 2012). Other studies have demonstrated that phlorotannins promoted the downregulation of the TLR-4-NF-κB-MMP-9 signaling axis on human in vitro breast and ovarian cancer (Ahn et al., 2015; Kong et al., 2009; Lee et al., 2020), revealing a possible biomedical application.

3.3 Anti-diabetic properties Due to the increasing concern for diabetes and obesity, several scientists are working on therapeutical alternatives for their treatment, and some marine phenolics demonstrated promising results. Several studies have found the anti-diabetic properties of bromophenols (Paudel et al., 2019), flavonoids (Yan et al., 2019), and phlorotannins (Catarino et al., 2019). They act mainly by reducing the glucose blood levels, inhibiting hepatic gluconeogenesis, and reducing the activity of digestive enzymes such as α-amylase and α-glucosidase (Lee et al., 2017; Lee and Jeon, 2013; Lin et al., 2019). The downregulation of diabetes-related genes had been observed in high-fat diet and diabetic mice models. When those models were treated with extracts of Nitzschia laevis (with only 13% of phenols) and Enteromorpha prolifera, the transport and metabolism of the carbohydrates were reduced, as well as the enzymatic activity of phosphatidylinositol 3-kinase (Lin et al., 2019; Nagayama et al., 2002). Finally, in the liver, Enteromorpha prolifera suppressed c-Jun N-terminal kinase and restored Srebp1c (Nagayama et al., 2002).

3.4 Antibacterial activity The antibacterial activity is one of the most remarkable properties of marine phenolics and has been widely studied. Crude phlorotannin extracted from Ecklonia kurome and Ecklonia cava displayed antibacterial activity against Staphylococcus aureus, Streptococcus pyogenes, Bacillus cereus, Campylobacter fetus, Campylobacter jejuni, Escherichia coli, and Salmonella typhimurium, among others (Al-Saif et al., 2014; Xu et al., 2003). Alcoholic, aqueous, and organic extraction fractions from Ulva reticulata, Caulerpa occidentalis, Cladophora socialis, Dyctiota

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ciliolate, and Gracilaria dendroides, containing flavonoids, such as rutin, quercetin, and kaempferol, demonstrated antibacterial activities against Escherichia coli and Pseudomonas aeruginosa. Gracilaria dendroides presented the highest content of flavonoids and the highest antibacterial activity (Al-Saif et al., 2014). Moreover, some bromophenols such as bis(2,3dibromo-4,5-dihydroxy benzoyl) ether from the red algae Rhodomela confervoides and 3,30 ,5,50 -tetrabromo-2,20 ,4,40 tetrahydroxydiphenylmethane from red alga Odonthalia corymbifera present the most significant antibacterial activity (MIC < 70 μg/mL) against Staphylococcus aureus and Escherichia coli (Xu et al., 2003), and can inhibit the growth of Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Proteus vulgaris, and Salmonella typhimurium (Oh et al., 2008), respectively. An extract from the sponge Phyllospongia papyracea rich in 2-(30 ,50 -dibromo-20 -methoxyphenoxy)-3,5-dibromophenol demonstrated antibacterial activity against Bacillus subtilis, Campylobacter jejuni, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae (Sun et al., 2017). An alcoholic extract of the green seaweed Chaetomorpha linum (gallic acid content of 672.3 mg/L) displayed antibacterial activity against pathogenic bacterial strains such as Staphylococcus aureus, Bacillus cereus, Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, and Salmonella typhimurium (Senthilkumar and Sudha, 2012). Specific metabolites such as phenolic compounds are produced for defense against herbivores and infections of pathogenic bacteria (Abu-Ghannam and Rajauria, 2013; Leandro et al., 2020). Some authors associate the antibacterial activity of phenolics with their ability to induce changes in the bacterial cell membrane, disturbing its function and finally affecting bacterial growth (Singh et al., 2019). In contrast, other authors attribute this property to the interaction of phenols with bacterial proteins and enzymes (Lopes et al., 2012). An interesting proposed mechanism is that the phenolics’ antimicrobial activity is due to the inhibition of transpeptidation and cellular peptidoglycan synthesis (Bajpai, 2016). The above is related to Gram-positive bacteria being more sensitive than Gram-negative to phenolic compounds due to the difference in their wall composition (Cardona et al., 2013). Even though the antibacterial activity is evident and many marine polyphenols serve as antibacterial agents, their action mechanism has not been clarified yet (Kalogianni et al., 2020). Marine phenolics such as phlorotannins, bromophenols, simple phenolic, and flavonoids could become a possible treatment by inhibiting the growth of pathogenic bacteria in infectious

Chapter 11 Gut microbiota and marine phenolics

diseases. Also, this antibacterial property could be helpful to decelerate or inhibit the growth of some bacteria in the GM, preventing intestinal diseases (Duda-Chodak et al., 2015; Marı´n et al., 2015; Nazzaro et al., 2019).

4. Digestion and metabolism of polyphenols As we exposed before, marine phenolics could be a promising and valuable preventive or therapeutic strategy against certain diseases. However, the chemical structures of the majority of phenolic compounds are complex, and they could be considered xenobiotics in the gastrointestinal (GI) tract. Their bioavailability is very low compared to micro- and macronutrients. The absorption of marine phenolic compounds at the small intestine is just 10% of the total intake, with the simplest structures (Singh et al., 2019). Many marine polyphenols are glycosylated derivatives bound to monosaccharides such as glucose, galactose, rhamnose, ribulose, arabinopyranose, or arabinofuranose, with limited bioavailability. Therefore, the absorption of phenolic compounds in the small intestine is a multi-step process: first, deglycosylation or cleavage of the glycoside bond by the microbial community in the small intestine; later, these products (aglycones) are transported by passive diffusion into the enterocyte; then, the phenolic aglycones pass into the hepatocyte where they are conjugated into a series of water-soluble metabolites such as methyl, glucuronide, and sulfates by several chemical reactions involved in the biotransformation of xenobiotics. These reactions are catalyzed by phase I enzymes (reduction, oxidation, and hydrolysis) such as cytochrome P450 (CYP), hydroxylases, epoxidases, and hydrolases and phase II enzymes (conjugation) such as glutathione S-transferases, sulfotransferases, methyltransferases, and N-acetyltransferases (Croom, 2012). Finally, the metabolic products are released into the circulatory system reaching a systemic distribution (Cardona et al., 2013). After partial absorption in the small intestine, about 90% of the total intake of polyphenols reach the colon almost unchanged, as aglycones and glycones (Fig. 1). They are retained for a long time in the colon (Kawabata et al., 2019; Ozdal et al., 2016), where a complex microbial community, the GM, resides (Yuan et al., 2019). Here the microbial community metabolizes them, generating a wide range of low-molecular-weight phenolic acids (Corona et al., 2016) better absorbed than the original phenolic compounds (Toma´s-Barbera´n and Espı´n, 2019).

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Circulatory system

Polyphenols and its catabolites

Bioconversion by the Gut Microbiota

Phase I and II metabolism Simple phenolic structures

~10%

Phenolic compounds

Complex phenolic structures

Small Intestine

Bacterial enzymatic reactions: i) Hydrolysis. ii) Cleavages. iii) Reductions.

Excretion

Large intestine

Fig. 1 Absorption and metabolization of marine phenolics in the gastrointestinal tract. At the small intestine, phase I and II enzymes perform reduction, oxidation, hydrolysis, and conjugation reactions into the enterocyte and the liver. Then, at the large intestine, the gut microbiota degrades the polyphenols, as glycone or aglycone, into simpler structures.

5. Gut microbiota The gut microbiota (GM) of a human adult is 1.3 times higher than the total somatic cell number of the host. It contains around 1014 bacterial cells (Sender et al., 2016), also including viruses, fungi, and protozoa. These microorganisms coexist and possess a symbiotic connection with the host (Singh et al., 2019). The intestinal microbiota is one of the most diverse, complex, densest, and heterogeneous environments. It shows a concentration gradient; low abundances in the stomach and duodenum of around 101 to 103 bacteria per gram, continuing with the jejunum and ileum with 104 to 107 cells per gram, and finally, the highest density of 1011 to 1012 cells per gram in the colon (Sekirov et al., 2010). The colonization of the GI tract occurs immediately after birth. The development and maturation of the GM composition during infancy are influenced by several factors: mode of delivery, type of feeding, medication usage, the mother’s age, diet, metabolic status, family genetics, and even the family lifestyle (Jandhyala et al., 2015; Milani et al., 2017; Rinninella et al., 2019). Increasing evidence shows that the infant gut microbiota has a long-term impact on health, and infancy offers an early window to promote proper health and development (Relman, 2012).

Chapter 11 Gut microbiota and marine phenolics

The gut microbial community plays a crucial role in developing an appropriate intestinal immune response, nutrient absorption, synthesis of metabolites, protection against pathogens, the maintenance of the gut mucosal barrier integrity, and energy homeostasis (Mills et al., 2019; Petersen and Round, 2014; Walsh et al., 2018).

5.1 Nutrient absorption and metabolites synthesis Most of the absorption of the nutrients occurs in the stomach, and the small intestine; the other portion that represents around 10% to 30% of the total nutrient intake reaches the colon, where the microbial community is responsible for the degradation of the undigested carbohydrates and proteins (Krajmalnik-Brown et al., 2012). Members of the Bacteroidetes and some Bifidobacterium genera express enzymes such as glycosyltransferases, glycoside hydrolases, and polysaccharide lyases that allow them to be the primary organisms that participate in carbohydrate metabolism (Hidalgo-Cantabrana et al., 2014; Jandhyala et al., 2015). The saccharolytic bacterial fermentation results in beneficial metabolites. For example, the fermentation by Bacteroides, Roseburia, Bifidobacterium, Faecalibacterium, and Enterobacteria produce shortchain fatty acids (SCFAs). The most common SCFAs are acetic acid, propionic acid, and butyric acid (in a 3:1:1 M ratio) (Markowiak-Kope
c and S´lizewska, 2020; Rowland et al., 2018), which display essential physiological effects in the GI tract and beyond. Also, other metabolites such as lactate, fumarate, and succinate are produced by bacterial fermentation, and they are extensively metabolized by other bacteria, resulting in crossfeeding interactions (Rowland et al., 2018). The nutritional interaction between the GM, such as cross-feeding of essential molecules synthesized by a subpopulation, plays a crucial role in maintaining the whole ecosystem’s homeostasis and proper operation (Seth and Taga, 2014). Butyrate is produced majorly by Firmicutes species, including Faecalibacterium prausnitzii, Roseburia spp., and Eubacterium spp. (Vital et al., 2014). This molecule is the primary energy source of colonocytes and has anti-cancer properties inducing the apoptosis of colon cancer cells (Rowland et al., 2018). It stimulates mucin production, inhibits inflammation, and decreases oxidative stress (Hamer et al., 2008). Propionate is also an energy source for epithelial cells, and it can reduce adiposity by reducing hepatic glucose production (Frost et al., 2014). De Vadder et al. attributed this phenomenon to the conversion of propionate to glucose by 

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intestinal gluconeogenesis, promoting beneficial effects on energy homeostasis (De Vadder et al., 2014). The most abundant SCFA is acetate, an essential cofactor for the growth of other bacteria; in the human body, acetate participates in the metabolism of cholesterol and lipogenesis (Rowland et al., 2018). Also, it has been suggested that acetate has a direct role in regulating appetite (Frost et al., 2014). The gut bacterial community also plays a vital role in synthesizing vitamins contributing to the total pool (Jandhyala et al., 2015). Vitamins, which humans cannot synthesize, are precursors of many essential enzymes in all cells, so they must be obtained from foods (LeBlanc et al., 2013) or by bacterial synthesis. Some bacteria, such as Bifidobacterium, synthesize vitamin K and some water-soluble vitamins B (Jandhyala et al., 2015; LeBlanc et al., 2013).

5.2 Protection against pathogens and mucosal gut barrier The gut microbiota also plays an essential role against pathogens by inducing the synthesis of antimicrobial proteins (AMP) such as cathelicidins, C-type lectins, and defensins by the host Paneth cells (Jandhyala et al., 2015). Paneth cells are specialized epithelial secretory cells, located in the small intestine, whose function is to maintain the immune homeostasis and control the microbiota growth by the secretion of AMP (Bevins and Salzman, 2011; Stahl et al., 2018). The mucosal gut barrier is the front line of the innate host defense, and it is constituted of mucin glycoproteins (Jandhyala et al., 2015). Globet cells produce mucin glycoproteins and other bioactive molecules. Together, they form a very viscous extracellular layer (Kim and Ho, 2010). In the colon, the inner layer is denser and does not contain any microorganisms, while the outer layer is thinner and provides nutrients to the commensal microbial community (Johansson et al., 2011). The outer layer is dynamic because it is quickly eliminated and degraded by microorganisms providing significant energy. For that reason, its thickness varies depending on the bacteria present (Johansson et al., 2011; Kim and Ho, 2010). The intestinal immune system plays an integral part in maintaining the beneficial nature of the intestinal host-microorganism relationship by producing the gut mucosal barrier and by the secretion of antimicrobial proteins (Hooper, 2009). The GM can

Chapter 11 Gut microbiota and marine phenolics

induce these mechanisms of the intestinal immune system due to the crosstalk established between the host and the microbial community.

5.3 Normal microbiota composition and dysbiosis A healthy microbiota is characterized by its remarkable resilience to environmental changes, often returning to its original €ckhed et al., 2012). Many factors can alter the composition (Ba colon environment, such as genetics, age, and antibiotics. These alterations can disrupt the gut microbiota, causing dysbiosis, which can be categorized into three types: (i) loss of beneficial microbial organisms, (ii) increased pathobionts or potentially harmful microorganisms, and (iii) loss of overall microbial diversity (Petersen and Round, 2014). The most common factors that cause this GM disruption are diet, sedentary lifestyle, stress, and drugs (Singh et al., 2019). In a normal condition, the GM is mainly composed of strict anaerobes belonging to the phyla Firmicutes and Bacteroidetes. They could represent up to 90% of the GM (Rinninella et al., 2019; Sekirov et al., 2010). Other important phyla are Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobia (O’Callaghan and Corr, 2019). The GM composition of each person is different, unique, and dynamic since it varies with age, diet, and various environmental factors that the host is exposed to (Grigor’eva, 2021). Generally, Firmicutes are more abundant than Bacteroidetes, and this proportion has been demonstrated to have an essential role in intestinal homeostasis (Ahmed et al., 2019; Magne et al., 2020; Sutoyo and Atmaka, 2020). Firmicutes are the main phylum of the butyrate-producing bacteria (Hamer et al., 2008), contributing to the maintenance of the mucosal gut barrier. Firmicutes include Gram-positive and some Gram-negative bacteria, comprising genera such as Faecalibacterium, Clostridium, Roseburia, Ruminococcus, Lactobacillus, and Enterococcus, among several others. Clostridium could represent 95% of the phylum. Some authors have found a relationship between an imbalance in Firmicutes abundance and inflammatory diseases, obesity, and diabetes (Huang et al., 2018). The phylum Bacteroidetes includes Gram-negative bacteria such as Bacteroides, Prevotella, Sphingobacterium, Tannerella, Parabacteroides, and Alistipes (Rinninella et al., 2019), where the more predominant genera are Bacteroides and Prevotella. Different studies have shown the complexity and diversity of this phylum. The members of the Bacteroidetes phylum share an outstanding ability to utilize polysaccharides due to the significant

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number of carbohydrate-active enzymes they encode (Mills et al., 2019). Some researchers related low abundance of Bacteroidetes to obesity and concluded that their relative abundance seems relevant to the host’s health (Johnson et al., 2017). Actinobacteria are Gram-positive bacteria and include the genus Corynebacterium, Bifidobacterium, and Atopobium, where Bifidobacterium species are dominant (Rinninella et al., 2019). Although this phylum represents a low percentage of the total microbial community, increasing interest has been focused on this genus in the last decades due to its butyrate production ability, recognized as beneficial microorganisms, and some used as probiotics (Binda et al., 2018; Quin et al., 2018). Proteobacteria are represented by the genus Escherichia, Shigella, Desulfovibrio, and Bilophila (Rinninella et al., 2019), and these members are in low abundance in a healthy microbiota. The abnormal expansion of Proteobacteria is related to inflammation, invasion of exogenous pathogens, and dysbiosis. For example, metabolic disorders (obesity and diabetes mellitus) often include an increased prevalence of Proteobacteria (Shin et al., 2015). Verrucomicrobia includes the Akkermansia genera (Rinninella et al., 2019). Akkermansia muciniphila, the sole member of this phylum identified in humans, is a mucus-degrading bacteria that colonize the human gut early (Xu et al., 2020). In inflammatory bowel disease, appendicitis, and obesity, Akkermansia muciniphila is less abundant than a healthy GM. This health-promoting bacterium can maintain the gut barrier function and present antiinflammatory and immunostimulant properties (Fujio-Vejar et al., 2017; Mills et al., 2019).

5.4 Disease and gut microbiota composition Because maintaining the balance of the intestinal ecosystem seems essential for the normal function of the human body, many therapeutic strategies have been designed to accomplish an appropriate Firmicutes/Bacteroidetes (F/B) ratio (Stojanov et al., 2020). Firmicutes and Bacteroidetes are the most abundant bacterial phyla in the human gut (Mills et al., 2019; Rinninella et al., 2019), and the Firmicutes/Bacteroidetes (F/B) ratio is a health biomarker related to gastrointestinal homeostasis (Ahmed et al., 2019; Magne et al., 2020; Stojanov et al., 2020). Variations of this ratio are associated with several diseases such as diabetes (Salazar et al., 2020), obesity (Magne et al., 2020; Yang et al., 2020), and inflammatory bowel disease (Contijoch et al., 2019; Milani et al., 2017; Ott et al., 2004).

Chapter 11 Gut microbiota and marine phenolics

Obesity is considered a multifactorial disease with a significant impact on public health worldwide, where genetic, endocrine, environmental, and psychosocial factors can contribute to excessive weight gain (Indiani et al., 2018). Several experimental studies showed that an increment of Firmicutes and a decrement of Bacteroidetes in the GI tract are related to obesity (Flint, 2011; Ignacio et al., 2016; Lo´pez-Cepero and Palacios, 2015; Magne et al., 2020), while other studies have shown opposite findings. Hence, it has been concluded that this association depends on the population, age group, genders, and environmental factors (Stojanov et al., 2020; Vaiserman et al., 2020). Notwithstanding, most of the studies support that the increment of Firmicutes is closely related to obesity. Firmicutes bacteria have a better capacity to ferment and metabolize carbohydrates, promoting a higher SCFA production and leading to more energy harvesting (Sutoyo and Atmaka, 2020). Also, members of this phylum can metabolize lipids, promoting its development and increasing its abundance in a high-fat diet (Stojanov et al., 2020). Ley et al. showed that a loss of body weight in obese people correlated positively with an increment of the relative abundance of Bacteroidetes (Ley et al., 2005). Diabetes mellitus (DM), an increasing worldwide pathology, is a metabolic disorder characterized by the inability of the body to properly maintain blood sugar homeostasis leading to fluctuations between hyperglycemia and hypoglycemia (Ahmed et al., 2019). Increasing evidence has demonstrated changes in the gut microbiota composition or function in type 2 diabetic patients (Larsen et al., 2010; Woldeamlak et al., 2019). This microbial dysbiosis is related to the loss of richness and diversity (Sohail et al., 2017). Some experimental studies exposed a relationship between a lower relative abundance of Firmicutes and a higher relative abundance of Bacteroidetes and Proteobacteria with diabetes (Larsen et al., 2010). While other studies establish a relationship between diabetes, obesity, and increased F/B ratio (Ahmed et al., 2019). The therapeutic strategy of lowering the F/B ratio may be beneficial in managing obesity and obesity-related DM (Ahmed et al., 2019; Stojanov et al., 2020). Inflammatory bowel disease is a group of intestinal disorders characterized by inflammation in the small and large intestine. This pathology includes two types of inflammatory diseases: ulcerative colitis and Crohn’s disease (Morgan et al., 2012; Stojanov et al., 2020). The patients with inflammatory bowel disease (IBD) present an increment of Proteobacteria abundance and decrement of Firmicutes (Contijoch et al., 2019), showing a reduced F/B ratio (Morgan et al., 2012; Papa et al., 2012;

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Stojanov et al., 2020; Walters et al., 2014). The depletion of Firmicutes leads to a decrement of the SCFA production, which affects the inhibition of the inflammatory response and the maintenance of the gut mucosal barrier (Hamer et al., 2008; Stojanov et al., 2020; Vital et al., 2014). According to the discussion above, the GM composition, especially the ratio of the two major phyla, could indicate the host health given the metabolic, inflammatory, and energy regulation processes that Firmicutes and Bacteroidetes perform.

6. Metabolization of marine phenolics by the gut microbiota Degradation of polyphenols by bacterial enzymes in the small intestine mainly involves O-deglycosylation reactions. In the colon, relevant reactions catalyzed by the gut microbiota are mainly (i) hydrolysis, such as deglycosylations and ester hydrolysis; (ii) cleavages, such as C-cleavage of the aromatic ring, delactonization, and demethylation; (iii) and reductions, such as dehydroxylation and double-bound reduction (Fig. 1). These bacterial enzymatic reactions produce low-molecular-weight compounds such as hydroxyphenylpropionic acid and hydroxyphenylacetic acids (Charoensiddhi et al., 2020a). The bacterial enzymatic degradation of some terrestrial phenolic compounds has been described in detail. For example, Bacteroides distasonis, Bacteroides uniformis, Bacteroides ovatus, Enterococcus casseliflavus, and Eubacterium ramulus participate in the breakdown of the glycosidic bond (glycosidase activity), thanks to R-rhamnosidase, β-glucosidase, and β-glucuronidase enzymes (Selma et al., 2009). Genera such as Clostridium, Bacteroides, and Eubacterium can break out the aromatic ring of polyphenols (Rechner et al., 2004). Another example is the demethylation reaction of the phenolic compounds, which could be carried out mainly by strains of Eubacterium limosum, Eubacterium callanderi, Streptococcus, Lactobacillus, Clostridium, Butyribacterium methylotrophicum, and Peptostreptococcus productus (Selma et al., 2009). The complete mechanisms of these enzymatic reactions are not entirely understood. However, it is expected that these enzymatic reactions for marine phenolics are not so different from those described for terrestrial polyphenols.

Chapter 11 Gut microbiota and marine phenolics

7. Prebiotic role of seaweed compounds Seaweed consumption is mainly concentrated in East Asian countries and is not extensively observed in Western countries (Murai et al., 2021; White and Wilson, 2015). Not only in the Asian market but also worldwide, the consumption of marine phenolics, as an ingredient with industrial applications, has increased due to its content in bioactive compounds with multiple beneficial properties associated with benefits in human health (Charoensiddhi et al., 2020b; Corona et al., 2016; Lopez-Santamarina et al., 2020). One of the strategies to revert and control diseases related to intestinal dysbiosis could be the consumption of prebiotics to improve the host health through the selective promotion or inhibition of bacterial metabolism (Fong et al., 2020; Tsai et al., 2019) to reach a healthy F/B ratio. An expert consensus in 2017 defined prebiotics as any substrate that can selectively enrich beneficial microorganisms to modify the interactions in the gastrointestinal tract and produce health benefits to the host (Gibson and Roberfroid, 1995; Gurpilhares et al., 2019; Tsai et al., 2019). This actual definition could include other compounds such as PUFAs and polyphenols (Gibson et al., 2017). Increasing evidence has supported the benefits of the consumption of algae compounds as prebiotics. For example, phenolic compounds are prebiotic candidates because they can interact with GM, resulting in benefits for metabolic health and the gastrointestinal system (Alves-Santos et al., 2020), as it is described below. Extracts from seaweeds demonstrated anti-diabetic properties by modulating host gene expression (Guo et al., 2019; Yan et al., 2019). Otherwise, in the same studies and several more, the researchers also demonstrated the role of marine phenolic compounds like flavonoids, phlorotannins, and bromophenols in regulating the GM community, mainly by decreasing the Firmicutes/ Bacteroidetes ratio in dysbiosis cases (Catarino et al., 2021; Kazłowska et al., 2010; Lin et al., 2019; Lopez-Santamarina et al., 2020; Manach et al., 2004; Ozdal et al., 2016; Va´zquez-Rodrı´guez et al., 2021; Wanyonyi et al., 2017; Yan et al., 2019). Extracts rich in polyphenols from Nitzschia laevis and Enteromorpha prolifera have been shown to modulate the GM composition. The experimental investigations were developed in diabetic model mice. After 8 weeks of oral treatment, the diabetic group presented lower blood glucose levels and presented a reduction in the F/B ratio, which was increased in the diabetic mice (Guo et al., 2019; Lin et al., 2019). These are not the only

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studies in the diabetic mice model. Yuan et al. treated diabetic mice with a rich extract from Lessonia trabeculata. After the treatment, the F/B ratio was restored to normal levels, showing an increment of some genus of Bacteroidetes, specifically Odoribacter, Parabacteroides, and Alistipes. Also, the treatment with the rich polyphenol extract increased the number of Lachnospiraceae (Firmicutes). However, it did not show an increment of the genus Firmicutes. The benefits of the extract were attributed to the increment of butyrate-producing bacteria (Odoribacter, Alistipes, and Lachnospiraceae) (Yuan et al., 2019) because butyrate plays an essential role in gut homeostasis, the maintenance of colonic epithelial tissues, and the prevention of insulin resistance and high fat-diet induced obesity (McNabney and Henagan, 2017). The gut modulation property also has been demonstrated in rats with metabolic syndrome (obesity). Wanyonyi et al. supplemented the high-fat diet of the mice with the whole red algae Kappaphycus alvarezii. The algae consumption demonstrated positive effects on the gut microbiota of the obese mice promoting Bacteroidetes and Bifidobacterium and decreasing Firmicutes compared to the obese control group, yielding weight loss (Wanyonyi et al., 2017). On the other hand, studies in gastrointestinal in vitro simulations have been developed. Catarino et al. evaluated a crude extract and an ethyl acetate fraction with phlorotannins content from the brown algae, Fucus vesiculosus. Both samples revealed an increment of Enterococcus spp. abundance and the ethyl acetate fraction displayed an increment in Lactobacillus spp. and Bifidobacterium ssp. abundances, but after 24 h, these microorganisms progressively decreased (Catarino et al., 2021), pointing out the importance of regular consumption of phenolics to achieve long-lasting effects in the microbial community. SCFA production in both cases showed that the crude fraction enhanced propionate production. In contrast, the ethyl acetate fraction enhanced butyrate production. Charoensiddi et al. obtained similar results, where several extracts from Ecklonia radiata were evaluated for 24 h in an in vitro anaerobic fermentation system containing a human fecal inoculum. The extraction with smaller phlorotannins content (3.5 g/100 g dry weight) showed a higher prebiotic potential by stimulating the total SCFA production (63.4 μmol/mL). The lowest SCFA production (50.7 μmol/mL) was the polysaccharide fraction with the highest phlorotannins content (4.5 g/100 g dry weight). They attributed this event to the promoting effect of the extract to Bifidobacterium and Lactobacillus genus, both beneficial bacteria in the gastrointestinal tract used as healthy microbiota

Chapter 11 Gut microbiota and marine phenolics

biomarkers (Charoensiddhi et al., 2016). However, the impact of the total phenolic content in SCFA production is not mentioned. A recent study evaluated the prebiotic effect of a hydroethanolic extract of polysaccharides and phlorotannins from Silvetia compressa. The extract was tested on in vitro human colonic simulations, and it was compared with an inulin treatment. It was found that the total count of bacteria increased after 8 h, compared to the negative control but was slightly less compared to the inulin treatment. However, after 24 h, there was no difference between the inulin treatment and the hydroethanolic extract. At the microbiota composition level, the treatment with the extract caused a decrease in Firmicutes and an increment of Bifidobacterium (18%) compared with the negative control. Also, the experimental study showed an increment of SCFA after 48 h compared with the negative control but slightly less compared to the inulin treatment. The best levels of SCFA were produced with the hydroethanolic extract of phlorotannins and polysaccharides, where the production has an increment of 78% acetic acid, 81% propionic acid, and 4.09% butyric acid compared to the negative control (Va´zquez-Rodrı´guez et al., 2021). The potential of consuming seaweed extract as prebiotics lies mainly in the increment in SCFA production through stimulation of beneficial bacteria as Odoribacter, Parabacteroides, Alistipes, Bifidobacterium, and Lactobacillus. Most studies usually show the benefits of polyphenols-rich extracts that contain polysaccharides, lipids, and dietary fiber; hence, it is impossible to distinguish the benefits of the whole algae extract from the benefits of the specific components within. For example, polysaccharides, the major seaweed component, present many beneficial properties: promote the specific gut microbial population, activate the fatty acids receptors to reduce body weight, reduce luminal pH to inhibit pathogens, among other benefits (Cherry et al., 2019). Therefore, it is essential to investigate the impact of isolated marine phenolics on the microbial community, although this is rather difficult since very few isolated marine phenolics are commercially available. Charoensiddhi et al. evaluated the prebiotic potential of different extract fractions with different polyphenol contents. They observed that the phlorotannins-enriched (PF) fraction (13.4 g/100 dry weight) showed low levels of short-chain fatty acids (SCFA) in in vitro anaerobic fermentations compared to all other seaweed fractions. The crude extract, which contained lower amounts of phenolics (4.5 g/100 g dry weight) than the PF fraction, showed the highest SCFA production. A similar result

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was exposed by the same author in 2016 (Charoensiddhi et al., 2016). Being a parameter to consider in future studies, how does the concentration of polyphenols (dose) affect GM? In the study, the authors mentioned that the low SCFA production could be explained by the antibacterial activity of some marine phenolics like phlorotannins. They attribute the antibacterial activity of the PF extract to a decrease in the abundance of Enterococcus, a member of the Firmicutes phylum, which could explain the reduced F/B ratio observed in the in vitro model (Charoensiddhi et al., 2017). This reduction could also explain the restoration of the F/B ratio in other studies, where the dysbiosis models presented an increased ratio. The prebiotic potential of marine phenolics is a world to discover and may have more applications than those reviewed in this chapter, such as treating inflammatory bowel disease. Terrestrial phenolics have already been tested in a pilot clinical study with IBD patients, revealing promising results by reducing biomarkers of inflammation and modulating the intestinal microbiota. The treated patients presented higher fecal butyric acid production and an increased abundance of Lactobacillus reuteri, Lactobacillus spp., Lactobacillus lactis, and Lactobacillus plantarum (Kim et al., 2020). Compiling the investigations carried out to date, it is concluded the use of marine phenolics as potential prebiotic lies in the restoration of the Firmicutes/Bacteroidetes ratio, the increment of short-chain fatty acids, and the possible applications of the antimicrobial activity against pathogenic bacteria.

8. Conclusions Phenolic compounds are an exciting group of marine metabolites with multiple properties and biotechnological applications. The different beneficial properties of polyphenols for human purposes have been widely studied. A remarkable property is their ability to modulate the intestinal microbial composition, which leads to a potential prebiotic effect. The colonic digestion of phenolic compounds depends on the unique GM composition and the polyphenol structure (Jandhyala et al., 2015). Unveiling the associations between polyphenols modulation of GM dysbiosis with health conditions and bacterial digestion of marine phenolics are of utmost importance. Human clinical trials are needed to develop safe and effective health treatments since almost all discoveries regarding the bioactivities of phenolic compounds come from in vitro or murine

Chapter 11 Gut microbiota and marine phenolics

model experiments. Nevertheless, consumption of foods rich in dietary phenolics should be encouraged, given their potential health benefits. Finally, these benefits largely depend on the structure and bioavailability of these compounds and the GM composition of each host (De Bruyne et al., 2019; Nova et al., 2020).

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Marine phenolics: Classes, antibacterial properties, and applications

12

Cla´udia Lea˜oa,b, Manuel Simo˜esa,b,c, and Anabela Borgesa,b,c a

LEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, Portugal. b ALiCE—Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, Porto, Portugal. c DEQ—Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

1. Phenolics and marine resources Plants produce a wide range of low molecular weight organic compounds that can be grouped into primary and secondary metabolites as well as hormones (Erb and Kliebenstein, 2020). While substances resulting from primary metabolism are essential for plant growth/development and cell maintenance (de Reis Giada, 2013; Ruchika et al., 2018), those produced from secondary metabolism are specific and specialized substances that evolved from biosynthetic pathways of the primary metabolism and mediated plant-environment interactions (Table 1; de Reis Giada, 2013; Pott et al., 2019). These last ones are multifunctional metabolites crucial for plant defenses against external stresses (e.g. UVB and oxidative damage, desiccation) and attacks (e.g. pathogens, insects, and herbivores) (Machmudah et al., 2017; Pandey and Rizvi, 2009; Saltveit, 2009; Shahidi and Ambigaipalan, 2015). Phenolics are the most predominant secondary metabolites present in plants (Ayad and Akkal, 2019; de Reis Giada, 2013; Lin et al., 2016; Minatel et al., 2017). Among the pathways involved in the biosynthesis of plants, phenolics are shikimate, phenylpropanoid, and flavonoid pathways (Patil and Masand, 2018). They play a crucial role in the maintenance of the quality of plant-based foods (vegetables, fruits, cereals, and beverages), as they are responsible for their organoleptic properties, like color, flavor, odor, bitterness, and astringency (Cheynier, 2012; Pandey and Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00013-3 Copyright # 2023 Elsevier Inc. All rights reserved.

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Table 1 Products of primary and secondary metabolism of plants. Primary Metabolism Lipids Proteins Carbohydrates Nucleic acids Amino acids Organic acids

Reference(s) de Reis Giada (2013), Maeda (2019), and Bowsher (2019) de Reis Giada (2013) and Bowsher (2019) de Reis Giada (2013), Maeda (2019), and Bowsher (2019) de Reis Giada (2013), Maeda (2019), and Bowsher (2019) Maeda (2019) and Bowsher (2019) Maeda (2019)

Secondary metabolism Phenolic compounds Alkaloids

Reference(s)

Steroids

Hussein and El-Anssary (2019) and Guerriero et al. (2018) Hussein and El-Anssary (2019), Guerriero et al. (2018), and Schaller (2010) Schaller (2010)

Saponins

Hussein and El-Anssary (2019)

Terpenes

Hussein and El-Anssary (2019), Guerriero et al. (2018), and Schaller (2010) Ji and Mo (2011)

Mycosporinelike amino acids

Rizvi, 2009). Furthermore, plant phenolic metabolites can also act as pollination attractants (Lin et al., 2016; Zhang et al., 2016). Recently, phenolic compounds have also won great interest due to their antioxidant power as well as their antibacterial, antifungal, and anticarcinogenic activities (de Reis Giada, 2013; Herrero et al., 2013; Lin et al., 2016). That is why multiple potential applications in the food, cosmetic, and pharmaceutical industries have been ascribed (Pokorny´, 1991; Sharma et al., 2020). Considering their natural healing capabilities, a greater concern to include sources of phenolic compounds in human and animal diets is of potential interest. Therefore, it is obvious that, as primary sources of these bioactive molecules, plant-based foods can significantly decrease disease risk development. Although phenolics from terrestrial sources are far more studied, these compounds are also produced by marine organisms like algae (macroalgae and microalgae) (Anku et al., 2017; FreilePelegrin and Robledo, 2014). On our planet, there are around 72,500 algae species described so far. Of the total algae identified, 20% are macroalgae and the remaining 80% are microalgae (Barsanti and Gualtieri, 2014). Added to phenolics, these organisms are believed to be a rich source of many other compounds, most of which are pharmacologically bioactive (Gnanavel et al., 2019). Interestingly, macroalgae and microalgae might be almost a continuous and unlimited source of phenolic compounds as,

Chapter 12 Marine phenolics

besides the marine environment, they can be obtained from the waste of many industrial sectors and easily cultivated in bioreactors at a large scale, respectively (Bin Li et al., 2007; Lourenc¸oLopes et al., 2020). The need to search and explore marine sources for the assembly of phenolic compounds comes from the concern of people about the use of synthetics. For instance, the demand for products containing natural additives has grown in recent years, mainly in the food industries. However, it is of note that the majority of these natural additives are extracted from terrestrial sources. In order to maintain the balance of the ecosystems and avoid the overexploitation of land resources, the exploitation of marine sources might be a potential solution (Pokorny´, 1991). In this regard, macroalgae and microalgae have already started to be explored for industrial applications; however, they contain many other unexplored potentials.

2. Marine phenolics Phenolic compounds are divided into different classes according to their chemical structure, commonly comprising aromatic rings and hydroxyl groups (Machmudah et al., 2017; Saranraj et al., 2019). A phenolic compound containing one aromatic ring is a phenolic acid, whereas a phenolic compound comprising more than one aromatic ring is a polyphenol (Cheynier, 2012; Minatel et al., 2017). Polyphenols include flavonoids, tannins, stilbenes, coumarins, lignins, and lignans (Ayad and Akkal, 2019; Saranraj et al., 2019). Their antioxidant power arises from the phenolic rings and hydroxyl groups (Minatel et al., 2017). In the marine environment, marine organisms such as coral, algae, and sponges are naturally exposed to predators, lack of space, UV radiation, temperature, salinity, and nutrient availability. This hostile and competitive environment led them to produce secondary metabolites, which can provide protection from other
rez et al., 2016). competitive organisms (Mateos et al., 2020; Pe Among the secondary metabolites produced by marine organisms, phenolic compounds are the ones that appealed to great interest, largely due to their biological properties (Mateos et al., 2020). Although some classes of phenolics are common to both terrestrial and marine organisms, some of them were found exclusively in marine organisms, such as phlorotannins and bromophenols (Kirke et al., 2019; Mateos et al., 2020). For example, bromophenols can be found in macroalgae (red, brown, and green algae) and cyanobacteria. Among these, red algae are the major

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source of marine bromophenols (Bidleman et al., 2019; Mateos et al., 2020). Instead, phlorotannins are exclusively produced by brown algae (Freile-Pelegrin and Robledo, 2014; Heffernan et al., 2015). Besides these exclusive classes of phenolics, flavonoids and phenolic acids are also found in the previously mentioned macroalgae (Zangrando et al., 2019).

2.1

Classes and sources

2.1.1

Macroalgae

Macroalgae are essential to life in marine environments, and in addition to their function as animal and human feed, they also act as fertilizers (Cotas et al., 2020). Besides, macroalgae are many times used for applications in the cosmetic industry and agriculture (Bidleman et al., 2019). They are divided into three different classes, according to their pigments: red algae (Rhodophyta), green algae (Chlorophyta), and brown algae (Phaeophyceae) (Cotas et al., 2020; Freile-Pelegrin and Robledo, 2014; JimenezLopez et al., 2021; Santos et al., 2019; Whitfield et al., 1999). Macroalgae are rich in phenolic compounds that include different classes, namely simple phenolic acids, flavonoids, bromophenols, and phlorotannins. Phenolic acids Phenolic acids are molecules with a phenolic ring with at least one carboxylic acid group. According to the number of carbon chains, these compounds are divided into three groups: hydroxybenzoic acids, acetophenones/phenylacetic acids, and hydroxycinnamic acids. Hydroxybenzoic acids have one carbon chain attached to the phenolic ring, C6-C1 type. Instead, acetophenones/phenylacetic acids belong to C6-C2 type, with two carbon chains attached to the phenolic ring. C6-C3 type phenolics are hydroxycinnamic acids with three carbon chains attached to the phenolic ring (Alvarez-Suarez et al., 2013; Cotas et al., 2020; Jimenez-Lopez et al., 2021; Liwa et al., 2017; Luna-Guevara et al., 2018). Hydroxybenzoic acids present in macroalgae include, among others, gallic acid, gentisic acid, p-hydroxybenzoic acid, syringic acid, chlorogenic acid, and vanillic acid (Fig. 1; Alvarez-Suarez et al., 2013; Cotas et al., 2020; Luna-Guevara et al., 2018). From the phenolic acids of this group, gallic acid is the most abundant phenolic in macroalgae. Even though higher concentrations were detected for green and red algae than for brown algae (Mateos et al., 2020). However, others might be found in high

Chapter 12 Marine phenolics

O

O

OH

HO OH

O

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OH

O OH

(2)

OH

O

O

OH

(1)

O

OH

OH

(3)

(4)

Fig. 1 Chemical structure of hydroxybenzoic acids: (1) 4-hydroxybenzoic acid; (2) 3,4,5-trihydroxybenzoic acid (gallic acid); (3) 4-hydroxy-3-methoxybenzoic acid (vanillic acid); and (4) 4-hydroxy-3,5-dimethoxybenzoic acid (syringic acid).

concentrations. For example, a study with brown algae detected high concentrations of gallic acid, chlorogenic acid, syringic acid, vanillic acid, and gentisic acid (Kumar et al., 2018). Mekini
c et al. (2019) found gallic acid and 4-hydroxybenzoic acid in several species of brown macroalgae. Hydroxycinnamic acids from macroalgae include caffeic, ferulic, sinapic, and p-coumaric acids (Fig. 2). However, these phenolics are usually subdivided into smaller groups according to the number and position of their acyl groups (Cotas et al., 2020): • mono-esters of ferulic, p-coumaric, and caffeic acids • di-esters, tri-esters, or tetra-esters of caffeic acid • blended di-esters of caffeic and ferulic acids and caffeic and sinapic acids • blended esters of caffeic acid and dibasic aliphatic acids, such as oxalic or succinic O

OH

O

HO OH

(1)

OH

O

O OH

(2)

OH

O

O

O OH

(3)

OH

OH

(4)

Fig. 2 Chemical structures of hydroxycinnamic acids: (1) 4-hydroxycinnamic acid (p-coumaric acid); (2) 3,4-dihydroxycinnamic (caffeic acid); (3) 3-methoxy-4-hydroxycinnamic acid (ferulic acid); and (4) 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid).

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Caffeic, ferulic, and p-coumaric acids were detected in the brown macroalgae Sargassum vulgare, in similar concentrations as well as in the green algae Dasycladus vermicularis. Besides these compounds, chlorogenic and sinapic acids are also found in the green macroalgae (Jimenez-Lopez et al., 2021). Flavonoids Flavonoids are molecules with a heterocyclic oxygen attached to two aromatic rings. Their structure also depends on the levels of hydrogenation. They can be divided into six subgroups, namely: flavanols, flavones, anthocyanins, flavonols, flavanones, and isoflavones (Fig. 3; Bilal Hussain et al., 2019). Epicatechin, catechin, catechin gallate, and epigallocatechin are the most abundant flavonoids in green, red, and brown algae. They were found at high concentrations in several brown algae species, including Eisenia bicyclis, Sargassum fusiforme, and Saccharina japonica (Jimenez-Lopez et al., 2021; Santos et al., 2019). However, a few more can be found such as morin, kaempferol, hesperidin, myricetin, rutin, quercitrin, and cirsimaritin (Santos et al., 2019). Flavonoids were detected in several species of green (Nitella hookeri, Ulva clathrata (Roth), Ulva linza Linnaeus, Ulva flexuosa Wulfen, and Ulva intestinalis Linnaeus), red (Jania rubens, Corallina mediterranea, Pterocladia capillacea, and Acanthophora spicifera), and brown algae (Sargassum sp. and Padina sp.)

O O

O

+

OH OH

(1)

OH

(2)

O

(3) O

O

O

O

O

O

(4)

(5)

(6)

Fig. 3 Chemical structure of flavonoids: (1) flavan-3-ols; (2) anthocyanins; (3) flavonols; (4) flavones; (5) flavanones; and (6) isoflavones. Based on Kopustinskiene, D. M.; Jakstas, V.; Savickas, A.; Bernatoniene, J. Flavonoids as Anticancer Agents. Nutrients 2020, 12 (2), 457.

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(Farasat et al., 2014; Markham and Porter, 1969; Mohy El-Din and El-Ahwany, 2016; Ruslin et al., 2018; Zeng et al., 2001). However, there are some contradictions in this regard, and the studies concerning macroalgae flavonoids are scarce. Flavonoids’ metabolic pathways have been extensively studied in terrestrial plants, and they are mainly synthetized through phenylpropanoid biosynthetic pathway. Interestingly, the genes encoding the enzymes chalcone isomerase and chalcone synthase, in the phenylpropanoid biosynthetic pathway, responsible for flavonoids synthesis are not present in some macroalgae genera, such as Ostreococcus, Chlamydomonas, Klebsormidium, and Micromonas (Cotas et al., 2020; Yonekura-Sakakibara et al., 2019). The explanation for this phenomenon might be related to the time and season of harvest and the geographical location (Cotas et al., 2020). Bromophenols Bromophenols have been identified as being largely responsible for the taste of fish, mollusks, crustaceans, and prawns (Freile-Pelegrin and Robledo, 2014; Whitfield et al., 1999). Some of these compounds include 2-bromophenol, 4-bromophenol, 2,4dibromophenol, 2,6-dibromophenol, and 2,4,6-tribromophenol (Fig. 4). In a study with 87 samples of red, brown, and green Australian marine algae, bromophenol compounds were found in all of these samples. Furthermore, all of the samples had 2,4-dibromophenol and 4,6-tribromophenol. Instead, 2,6-dibromophenol was found in 98% of the samples, 2-bromophenol in 91% of the samples, and 4-bromophenol in 72% of the samples (Whitfield et al., 1999). Bromophenols are produced by macroalgae in the presence of bromide and hydrogen peroxide through an organic substrate-bromination

OH

OH

OH Br Br

Br

Br

(1)

OH

(2)

Br Br

Br

(3)

OH Br

Br

(4)

(5)

Fig. 4 Chemical structures of bromophenols: (1) 2-bromophenol; (2) 4-bromophenol; (3) 2,4-dibromophenol; (4) 2,6-dibromophenol; and (5) 2,4,6-tribromophenol. Based on Oliveira, A. S.; Silva, V. M.; Veloso, M. C. C.; Santos, G. V.; Andrade J. B. D. Bromophenol Concentrations in Fish From Salvador, BA, Brazil. An. Acad. Bras. Cienc. 2009, 81 (2), 165–172.

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reaction. This process is mediated by bromoperoxidases present in
n et al., 1994; Flodin and brown, green, and red algae (Colle Whitfield, 1999; Liu et al., 2011). Phlorotannins Phlorotannins play important roles in macroalgae as primary and secondary metabolites (Jimenez-Lopez et al., 2021). They are important structural components of the cell walls in brown algae (Cotas et al., 2020; Freile-Pelegrin and Robledo, 2014; Heffernan et al., 2015; Jimenez-Lopez et al., 2021). Plus, phlorotannins play an essential role in the protection against UV radiation and herbivory (Coll
en et al., 1994; Sardari et al., 2020). Phlorotannins are believed to absorb a portion of UVB spectrum. In fact, macroalgae that inhabit areas of high sun exposure are the ones with higher phlorotannin content. Some studies showed that phlorotannin content increases with the increasing UVB radiation (Halm et al., 2011; Heo et al., 2009; Pavia et al., 1998). Furthermore, phlorotannins may be inhibitors of the digestive enzymes of mollusks and bacteria as a survival strategy (Imbs and Zvyagintseva, 2018). These components can be exclusively found in macroalgae and are complex polymers of phloroglucinol (1,3,5-trihydroxybenzene), synthetized through acetate-malonate (polyketide) pathway (Cotas et al., 2020; Jimenez-Lopez et al., 2021). Phlorotannins are the most studied polyphenol in macroalgae due to their unique structure and also the fact that they are not found in terrestrial plants (Freile-Pelegrin and Robledo, 2014; Heffernan et al., 2015; Kirke et al., 2019). Phlorotannins comprise up to about 25%–30% of the dry weight of brown algae (Freile-Pelegrin and Robledo, 2014; Koivikko et al., 2005; Sardari et al., 2020; Singh and Sidana, 2013). Even though their concentration varies according to the external conditions like the time of harvest, environment, nutrient availability, and light exposure (Freile-Pelegrin and Robledo, 2014; Sardari et al., 2020). Phlorotannins are hydrophilic compounds formed by the polymerization of phloroglucinol units. These units can bind through C-C type or C-O-C type residues, creating phloroglucinol oligomers. Different linkages between phloroglucinol units and the multiple hydroxyl groups lead to heterogeneous molecular weights that may range between 126 Da and 650 kDa (Eom et al., 2012; Goiris et al., 2012; Jimenez-Lopez et al., 2021; Safafar et al., 2015; Wijesekara and Kim, 2010). However, most of them are usually found in a range of 10 and 100 kDa (FreilePelegrin and Robledo, 2014). Phlorotannins can be divided into

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379

four groups according to the linkage type: fuhalols and phlorethols holding an ether linkage, fucols holding a phenyl linkage, fucophlorethols holding ether and phenyl linkage, along with eckols and carmalols having a dibenzo-dioxin linkage (Fig. 5; Cotas et al., 2020; Imbs and Zvyagintseva, 2018; Jimenez-Lopez et al., 2021; Santos et al., 2019).

OH

OH OH

HO

HO

O

OH

(1) HO OH

OH

HO OH

OH

OH

OH

OH

O

O OH

HO

HO

HO

OH

OH HO

OH

OH

(3)

OH HO

(2)

O

OH HO OH

HO

OH O

O

OH HO

HO

O

HO

O

OH

HO

OH HO

OH

OH

HO

(5)

OH HO

OH

OH

OH

(4)

HO

OH

O O O HO O HO

O HO

OH

(6)

Fig. 5 Chemical structures of phlorotannins: (1) phloroglucinol; (2) tetrafucol a; (3) tetraphlorethol b; (4) fucodiphlorethol; (5) tetrafuhalol a; and (6) phlorofucofuroeckol. Based on Cotas et al., J. Seaweed Phenolics: From Extraction to Applications. Mar. Drugs 2020, 18 (8) 384.

OH

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2.1.2

Microalgae

Similar to macroalgae, phenolic compounds were also identified in microalgae. In fact, microalgae are an almost inexhaustible source of natural antioxidants and offer more biodiversity than terrestrial plants (Bin Li et al., 2007). Flavonols, flavones, and isoflavones were detected in several lineages of microalgae, namely, Rhodophyta, Chlorophyta, Ochrophyta, Cyanobacteria, and Haptophyta (Goiris et al., 2012; Wijesekara and Kim, 2010; YonekuraSakakibara et al., 2019). Safafar et al. (2015) (Eom et al., 2012) evaluated the phenolic composition of six microalgae from different classes, Phaeodactylum sp. (Bacillariophyceae), Nannochloropsis spp. and Nannochloropsis limnetica (Eustigmatophyceae), Chlorella spp., Dunaliella spp., and Desmodesmus spp. (Chlorophyta). Only simple phenolic acids were found, namely, cinnamic acid, salicylic acid, ferulic acid, caffeic acid, gallic acid, p-coumaric acid, 2,5-dihydroxybenzoic acid, and 3,4-dihydroxybenzoic acid. The hydroxycinnamic acids (ferulic acid and p-coumaric acid) were found in all of the algae from Chlorophyta class, Chlorella spp., Dunaliella spp., and Desmodesmus spp. They were also found in Nannochloropsis sp., being the most prevalent compounds within the analyzed species. Gallic acid, salicylic acid, and cinnamic acid were present in three of the six microalgae species, while 3,4-dihydroxybenzoic acid was only present in two of the six microalgae species. Differently, 2,5-dihydroxybenzoic acid was not found in any of the species of microalgae studied (Eom et al., 2012; Safafar et al., 2015). Four microalgae from different classes were also screened for their phenolic compounds content, Phaeodactylum tricornutum (Bacillariophyceae), Nannochloropsis gaditana (Eustigmatophyceae), Nannochloris sp. (Trebouxiophyceae), and Tetraselmis suecica (Chlorodendrophycea). Among all the phenolic compounds found, caffeic acid, p-coumaroyl tyrosine, and dimethoxyflavone were detected in three of the four microalgae species. Protocatechuic acid was the only phenolic compound present in all of the four analyzed microalgae species. Caffeic acid was the most abundant phenolic in Phaeodactylum tricornutum and Tetraselmis suecica. Instead, the most frequent phenolics in Nannochloropsis gaditana and Nannochloris spp. were quercetin and caffeoylcoumaroyl-quinic acid, respectively (Haoujar et al., 2019). In a study with Spirulina maxima, the phenolics predominantly detected were phenolic acids (ferulic acid, caffeic acid, p-hydroxybenzoic acid, chlorogenic acid, gallic acid, cinnamic acid, and vanillic acid). However, some flavonoids were also found

Chapter 12 Marine phenolics

(quercetin, genistein, eugenol, kaempferol, chrysin, galangin, and pinostrobin) (Abd El-Baky et al., 2009).

2.2

Antibacterial properties

In the past years, multidrug-resistant bacteria are emerging, causing persistent infections that are harder to treat (Church and McKillip, 2021; Rossolini et al., 2014; Ventola, 2015). This may have devastating economic and social consequences, as they cause around 700,000 death and imply a spent of $100 trillion per year, both in developed and underdeveloped countries (Church and McKillip, 2021). Unfortunately, antibiotic resistance is getting worse with time and the medical/scientific community urge for new quick and effective solutions (Rossolini et al., 2014; Ventola, 2015). Interestingly, among the recognized bioactive properties of phenolic compounds are their antibacterial and antibiofilm activities (Martillanes et al., 2017). These potentialities are very appreciated, particularly in an era in which we are faced with the silent and growing crisis of antibiotic resistance. In this sense, a question arises: “Could phenolic compounds derived from marine organisms be a possible alternative to the use of antibiotics?” The answer is yes. In effect, the antibacterial properties of several phenolic compounds derived from marine sources, such as algae, have been explored, and the results proved to be promising, as specified in the studies presented below. Phlorotannins showed to be effective against both Gramnegative and Gram-positive bacteria, including aerobic and anaerobic. Although Gram-negative bacteria are less susceptible to phlorotannins than the Gram-positive ones, mainly due to the physiological differences in the outer membrane, these compounds are able to cause cell death (Besednova et al., 2020). It has been demonstrated that the lipophilic characteristics of phlorotannins enhance their antibacterial activity. This can be related to the fact that they are able to bind to bacterial proteins, interacting with cell membrane and enzymes, through H-bonds and hydrophobic interactions (Besednova et al., 2020; Nagayama et al., 2002; Venkatesan et al., 2019). Consequently, they cause irreversible changes in the membrane by inducing cell lysis and the coagulation of intracellular contents (McDonnell, 2007). Silva et al. (2020) verified that phlorotannins from Fucus vesiculosus have antibacterial activity against Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa (Silva et al., 2020). Phlorofucofuroeckol, another phlorotannin from Eisenia bicyclis, showed antibacterial activity against methicillin-resistant

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Staphylococcus aureus (MRSA) (Eom et al., 2012, 2014; Safafar et al., 2015). Similar to the previous one, these compounds cause cell membrane damage, leading to the leakage of intracellular content and, as a result, death. The suppression of genes related to antibiotic resistance is also reported as a mode of action of phlorotannins (Eom et al., 2012; Silva et al., 2020; Safafar et al., 2015). Another recent study reported that phlorotannins also inhibit quorum sensing (QS) activity, as well as disrupt biofilm formation and reduce the production of virulence factors of Pseudomonas aeruginosa (Tang et al., 2020). Nagayama et al. (2002) assessed the bactericidal activity of phlorotannins against several Gram-positive and Gram-negative bacteria and proved that phlorotannins are effective against all the bacterial strains tested (Staphylococcus aureus, including MRSA, Streptococcus pyogenes, Bacillus cereus, Campylobacter fetus, Campylobacter jejuni, Escherichia coli, Salmonella enterica serotype Enteritidis, Salmonella typhimurium, and Vibrio parahaemolyticus) (Nagayama et al., 2002). Another study with phlorotannins isolated from the diatoms Cymbella spp. evaluated their inhibitory power against several Gram-positive (Staphylococcus aureus, Corynebacterium diphtheriae) and Gram-negative bacteria (Escherichia coli, Proteus mirabilis, Klebsiella pneumonia, Salmonella typhi, and Pseudomonas aeruginosa). The isolated phlorotannins showed positive outcomes against all the seven bacterial strains tested, being Pseudomonas aeruginosa and Corynebacterium diphtheriae the most susceptible ones, and Proteus mirabilis the least susceptible. It has been suggested that phlorotannins’ mode of action may be related to their ability to inactivate enzymes and cell envelope transport proteins or even prevent cell adhesion (Al-Mola, 2009). Li et al. (2011) also reported the ability of phlorotannins from Ascophyllum nodosum to reduce the prevalence of Escherichia coli in bovine feces. Ascophyllum nodosum phlorotannins’ also showed antibiofilm activity by preventing biofilm formation of two Shiga toxin-producing strains of Escherichia coli within 24 h of incubation. Besides phlorotannins suppressing biofilm formation, they did not totally stop bacterial growth, since after 72 h, both strains were able to overcome the inhibitory effect of phlorotannins, and the biofilm characteristics were found to be close to the control. The authors proposed that this activity results from their ability to inhibit cell growth and exopolysaccharides production. They also suggest that phlorotannins may suppress QS activity, preventing biofilm formation (Besednova et al., 2020). Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermis are some of the pathogens that usually infect

Chapter 12 Marine phenolics

the human skin. These bacteria enter the skin epidermis and secret toxins, causing infection, which leads to pimples and abscesses formation. Studies revealed that phlorotannins might be a natural solution for acne treatment. Effectively, phlorotannins from Eisenia bicyclis showed inhibitory activity against acne produced by the mentioned bacterial species (Jesumani et al., 2019). Another study found out that flavonoids have effective antibacterial activity against Propionibacterium acnes. Besides, cinnamic acid, caffeic acid, ferulic acid, chlorogenic acid, and gallic acid also showed effective antibacterial activity against this bacterium (Działo et al., 2016). As stated previously, brown algae seem to contain higher flavonoid content than green and red algae (Alghazeer et al., 2017). Thus, in a study from Alghazeer et al. (2017), two brown algae, Cystoseira compressa and Padina pavonica, were evaluated for their antibacterial activity against a number of Gram-positive and Gram-negative bacterial strains. Flavonoids from Cystoseira compressa showed antibacterial activity against most of the tested Gram-positive (Staphylococcus aureus and Bacillus cereus) and Gram-negative (Salmonella enterica and Enterohemorrhagic Escherichia coli (EHEC)) bacteria. However, Salmonella enterica was the one that showed the highest susceptibility to the isolated flavonoids. Regarding Padina pavonica, a very weak effect against Salmonella enterica and Bacillus cereus was observed. No activity was found against EHEC. Although less effective than the flavonoids from Cystoseira compressa, those from Padina pavonica showed some antibacterial activity against Staphylococcus aureus (Alghazeer et al., 2017). Maftuch and coworkers (Maftuch et al., 2016) verified that flavonoids from Gracilaria verrucosa also have some antibacterial activity against several bacterial species (moderate effect: Aeromonas hydrophila, Pseudomonas aeruginosa, and Pseudomonas putida; weak effect: Vibrio harveyi and Vibrio alginolyticus). In other work, several flavonoids that include flavone, 6-aminoflavone, 6-hydroxyflavone, apigenin, chrysin, curcumin, daidzein, fisetin, genistein, luteolin, phloretin, and quercetin were tested for their antibacterial potential against the pathogen Acinetobacter baumannii. The authors found that five of the 12 tested flavonoids showed positive results (genistein, quercetin, fisetin, and phloretin) (Raorane et al., 2019). Additionally, based on a molecular docking analysis, they suggest that the mode of action of the tested flavonoids against Acinetobacter baumannii can be related to BfmR binding (Raorane et al., 2019). Bromophenols have been found abundantly in marine organisms, such as red algae, brown algae, and sponges,

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and they are also known for their antibacterial activity. Rajasulochana et al. (2012) studied the antibacterial activity of the bromophenol (E)-3-(2,3-dibromo 4, 5-dihydroxyphenyl)-2methylpropenal against four bacterial species, namely Pseudomonas fluorescens, Staphylococcus aureus, Vibrio cholerae, and Proteus mirabilis. They found that this bromophenol has good antibacterial activity against Pseudomonas fluorescens and Staphylococcus aureus, and moderate activity against Vibrio cholerae (Rajasulochana et al., 2012). In another study, six bromophenols, namely 4-(2-aminoethyl)-2,6-dibromophenol (1), 2,3-dibromo-4,5-dihydr-oxybenzyl alcohol (2), 2,3-dibromo-4,5-dihydroxybenzylmethyl ether (3), 2,20 ,3,30 -tetrabromo-4,40 ,5,50 -tetra-hydroxydiphenylmethane (4), 2,20 ,3-tribromo-30 ,4,40 ,5-tetrahydroxy-60 -hydroxymethyl diphenylmethane (5), and 3-bromo-4-(2,3-dibromo-4,5-dihydroxybenzyl)5-methoxymethylpyrocatechol (6) were tested against Gram-positive (Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus) and Gram-negative (Proteus vulgaris, Salmonella typhimurium, and Escherichia coli) bacteria. They found that bromophenols from 1 to 3, which are mono-phenolic compounds, have no antibacterial activity against any bacteria assayed. Instead, bromophenols from 4 to 6, which are di-phenolic compounds, showed good antibacterial effects against all bacteria, except against Escherichia coli (Oh, 2008). Liu et al. (2011) evaluated the antibacterial capability of other five distinct bromophenols namely 3-bromo-4-(2,3-dibromo-4,5-dihydroxyphenyl) methyl-5-(hydroxymethyl)-1,2-benzenediol (1), 3-bromo-4(2,3-dibromo-4,5-dihydroxyphenyl) methyl-5-(ethoxymethyl)-1,2benzenediol (2), 3-bromo-4-(2,3-dibromo-4,5-dihydroxyphenyl) methyl-5-(methoxymethyl)-1,2-benzenediol (3), 4,40 -methylenebis (5,6-dibromo-1,2-benzenediol) (4), and bis(2,3-dibromo-4,5-dihydroxybenzyl)ether (5) against several bacterial strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa. Bromophenol number 4 and 5 were the ones with the best antibacterial activity, showing low minimum inhibitory concentration values for most of the bacterial strains. While bromophenol 1 did not show antibacterial activity for any bacteria, bromophenol 2 showed antibacterial activity against Staphylococcus aureus strains and bromophenol 3 against Staphylococcus aureus and S. epidermidis strains (Liu et al., 2011). Overall, it is believed that phenolic compounds can cause cell lysis by affecting membrane permeability, enzymes inhibition, and several metabolic pathways. Their antibacterial activity may also be related to the number of hydroxyl groups and the degree of polymerization (Nagayama et al., 2002; Silva et al., 2020).

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2.3

Industrial applications and future perspectives

In the past years, consumer awareness regarding synthetic additives used in many industries has increased (Martillanes et al., 2017). Mostly, in the food industry, the concern about the impact of non-natural additives on health leads people to search for alternatives (Jacobsen et al., 2019; Martillanes et al., 2017). A possible solution is the choice of natural sources without forgetting the sustainability issue. In this direction, phenolic compounds were primarily introduced to the food industry due to their coloring and preservation (e.g. prevention of the lipid oxidation and increases of the products shelf-life) properties (Albuquerque et al., 2021; Suleria et al., 2015). However, they have multiple potential properties, which makes them suitable for applications in other industries, such as pharmaceutical, cosmetic, and textile (Albuquerque et al., 2021; Getachew et al., 2020; Pokorny´, 1991; Sharma et al., 2020). For now, applications of industrial phenolics are diverse and in progress. Thus, consumers demand led to the need to look for new sources of phenolics beyond these terrestrial (Shanura Fernando et al., 2016). Although marine sources are underexplored, they show to be a potential source of phenolic compounds (Shanura Fernando et al., 2016). For that reason, phenolic compounds from marine organisms like algae (macroalgae and microalgae) have gained great interest in industrial applications. Phenolic compounds like gallic acid, catechins, and epicatechins are effective in hyperpigmentation treatment, as they are potent tyrosinase inhibitors (Guillerme et al., 2017). In addition to whitening properties, phenolic compounds showed skin wrinkle reduction. That is why they can be useful for the cosmetic industry (Albuquerque et al., 2021). Phlorotannins are also recognized for their skin-whitening and anti-wrinkling properties, as well as for promoting hair growth, which makes them suitable for cosmetic industry applications. They also show a strong ability to suppress metabolic diseases, such as obesity, diabetes, and heart diseases, being important for the nutraceuticals industry (Fernando et al., 2021). Phlorotannins, phenolic acids, and flavonoids are recognized for their antibacterial activity against acne-producing bacteria, namely, Propionibacterium acnes, Staphylococcus aureus, and S. epidermis. Thus, they might as well be used in cosmeceutical formulations (Choi et al., 2014; Działo et al., 2016). Phlorotannins proved to increase mineralization, collagen and total protein synthesis, and alkaline phosphatase activity in

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human osteosarcoma cells (MG-63 cells) (Gwladys et al., 2017). Indeed, studies suggest that phenolic compounds regulate osteoblast differentiation and osteosarcoma differentiation (Gwladys et al., 2017; Karadeniz et al., 2015). Karadeniz et al. (2015) found out that phlorotannins are highly effective in improving cell growth and differentiation, considering them successful osteoblastogenesis enhancers as well as osteoblasts differentiators (Karadeniz et al., 2015). Therefore, these natural compounds can be suitable for pharmaceutical application in the treatment of bone diseases, as they potentiate bone mineralization. By promoting cell differentiation and adhesion, these compounds can also be used as nanocoatings to functionalize biomaterials (Co´rdoba et al., 2015; Gwladys et al., 2017). In fact, studies showed that bioceramic-based medical scaffolds supplemented with marine phenolic compounds resulted in the enhancement and improvement of bone tissue regeneration as well as in the increase of bone density (Yeo et al., 2012). Overall, phenolic compounds from algae origin can serve to treat and prevent several diseases, including as anti-infective agents. They can be used in cosmeceutical and pharmaceutical formulations with diverse functions (Albuquerque et al., 2021). However, there are many gaps, which implies intensive studies before reaching industrial-scale applications. Epidemiological studies must be performed in order to assess what types of macro and microalgae present better results (Go´mez-Guzma´n et al., 2018). Besides, although phenolic compounds seem to be safe, long-term and high-dose intake must be studied in order to avoid unexpected side effects (Albuquerque et al., 2021; Go´mezGuzma´n et al., 2018; Shanura Fernando et al., 2016).

Acknowledgments This work was financially supported by: Base Funding—UIDB/00511/2020 of LEPABE funded by national funds through the FCT/MCTES (PIDDAC); Project Biocide_for_Biofilm—PTDC/BII-BTI/30219/2017—POCI-01-0145-FEDER-030219; Germirrad—POCI-01-0247-FEDER-072237, funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalizac¸a˜o (POCI) and by national funds (PIDDAC) through FCT/MCTES; Project AlgaValor, from the Portugal 2020 program (grant agreement nº POCI-01-0247-FEDER035234; LISBOA-01-0247-FEDER-035234; ALG-01-0247-FEDER-035234);—Project “HealthyWaters—Identification, Elimination, Social Awareness and Education of Water Chemical and Biological Micropollutants with Health and Environmental Implications,” with reference NORTE-01-0145-FEDER-000069, supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Anabela Borges thanks the FCT for the financial support of her work contract through the Scientific Employment Stimulus—Individual Call—[CEECIND/ 01261/2017].

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Yeo, M.; Jung, W.-K.; Kim, G. Fabrication, Characterisation and Biological Activity of Phlorotannin-Conjugated PCL/β-TCP Composite Scaffolds for Bone Tissue Regeneration. J. Mater. Chem. 2012, 22 (8), 3568–3577. Yonekura-Sakakibara, K.; Higashi, Y.; Nakabayashi, R. The Origin and Evolution of Plant Flavonoid Metabolism. Front. Plant Sci. 2019, 10, 943. Zangrando, R.; et al. Free Phenolic Compounds in Waters of the Ross Sea. Sci. Total Environ. 2019, 650, 2117–2128. Zeng, L. M.; et al. Flavonoids From the Red Alga Acanthophora spicifera. Chinese J. Chem. 2001, 19 (11), 1097–1100. Zhang, F. P.; Yang, Q. Y.; Zhang, S. B. Dual Effect of Phenolic Nectar on Three Floral Visitors of Elsholtzia rugulosa (Lamiaceae) in SW China. PLoS One 2016, 11 (4).

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Health and diseases prevention

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Impact of phlorotannins on cardiovascular diseases

13

So´nia J. Amarante, Marcelo D. Catarino, Artur M.S. Silva, and Susana M. Cardoso LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, Aveiro, Portugal

1. Introduction Cardiovascular diseases (CVDs) are medical conditions associated with pathologic changes that affect the heart and/or the blood vessels, which include hypertensive heart disease, coronary artery disease, rheumatic heart disease, congenital heart disease, thromboembolic disease, and venous thrombosis, among others (World Health Organization, 2011). According to World Health Organization (WHO), CVDs are the number one cause of death globally since 2000, with an estimated number of 17.9 million deaths per year, of which heart attack and stroke are responsible for approximately 8.9 and 6.2 million deaths, respectively (World Health Organization, 2020). The identification of the main causes and prevention strategies of CVDs are critical steps to counteract such statistics. The underlying disorder mechanisms vary depending on the specific CVD disease, but in most cases, these are mostly caused by atherosclerosis or thrombosis, which can lead to the obstruction of the blood vessels, reducing the normal blood flow rate (Rafieian-Kopaei et al., 2014). Atherosclerosis is characterized by a large accumulation of lipids on the blood vessels wall, originating from an atheroma plaque, and thrombosis occurs when a thrombus (an unattached mass that results from the partial rupture of the atheroma plaque) travels through the bloodstream and creates the blockage of a blood vessel (Badimon et al., 2012; Libby et al., 2002). The associated risk factors of atherosclerosis and thrombosis are classified Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00014-5 Copyright # 2023 Elsevier Inc. All rights reserved.

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into two main groups: the non-modifiable risk factors, which include genetics, age, and sex; and the modifiable risk factors, comprising unhealthy dietary habits, smoking, alcohol abuse, and lack of exercise. In addition, as a consequence of these risk factors, there are usually other underlying pathologies, such as diabetes, hypercholesterolemia, hypertension, and obesity (World Hearth Federation, 2022). The best strategy to prevent CVDs is to improve and/or treat behavior risk factors, as well as the underlying biological pathologies with impact on CVDs. Epidemiological evidence suggests an association between the consumption of polyphenol-rich vegetables and the reduction in the prevalence of CVDs, a fact that may be related to the anti-inflammatory, antithrombotic, and vasodilatory effects of polyphenols (Bahadoran et al., 2013).

2. Phlorotannins Among natural phenolic compounds, marine algal phenolics, particularly phlorotannins, have drawn much attention from the scientific community over the recent years, due to their versatile biological activities and potential industrial applications, including the prevention of CVDs. Indeed, over the past three decades, the Japan has recorded the longest life expectancy as well as the world’s lowest rates of CVDs, which can be attributed not only to their favorable economy and health systems, but also to their dietary patterns which include a regular consumption of macroalgae, particularly brown (Yamori et al., 2001; Zava and Zava, 2011). Phlorotannins are a group of phenolic compounds biosynthesized exclusively by brown macroalgae, playing an important structural role, since they are present in the cell wall, and help to protect brown algae from stress conditions such as UV radiation and herbivorous species (Geiselman and McConnell, 1981; Go´mez and Huovinen, 2010; Koivikko et al., 2005). The accumulation of phlorotannins in seaweeds may reach as much as 15% DW, depending on the macroalgae species and a number of factors, such as salinity, seasonality, nutrient availability, tides, exposure to waves, light, and temperature (Imbs and Zvyagintseva, 2018). Chemically, these compounds are built via polymerization of phloroglucinol (1,3,5-trihydroxybenzene) units, forming structures that may range from simple molecules of 126 Da to very large and complex polymers (Catarino et al., 2017), which according to the nature of the structural linkages, can be characterized into

Chapter 13 Impact of phlorotannins on cardiovascular diseases

Fig. 1 Representation of the different classes of phlorotannins.

different subclasses: phlorethols, containing only ether linkages; fucols, containing only C-C linkages; fucophlorethols, containing either ether and C-C linkages; and eckols, containing a dibenzodioxin linkage. Within the subclass of phlorethols, there are also the fuhalols, which are ether-linked phlorotannins that possess at least one additional hydroxyl group. Likewise, eckols with additional hydroxy groups are known as carmalols (Fig. 1; Pal Singh and Bharate, 2006).

3. Underlying pathologies with impact on CVDs The risk of developing CVDs is related to the aggravation and progression of a continuous unhealthy behavior associated with modifiable risk factors that usually lead to inflammation and oxidative stress (Siti et al., 2015). As mentioned, some underlying pathologies with impact on CVDs, as a result of risk factors, include atherosclerosis, obesity, diabetes mellitus, and hypertension (World Hearth Federation, 2022). Likewise, pathologies related to atherosclerosis, such as dyslipidemia, represent one of the main causes of cardiovascular events. Moreover, it is also important to understand the role of the heart and blood vessels,

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as well as their interaction with the subjacent inflammation process related to CVDs, which clearly vary with the endothelial function (Pigozzi et al., 2011; Virani et al., 2020). Dyslipidemia represents a major risk factor for CVDs and is characterized by the high concentration and accumulation of lipids, including phospholipids, triglycerides, and/or cholesterol, in the bloodstream (Klop et al., 2013). The abnormal presence of lipids can be related to hyperlipidemia and/or hyperlipoproteinemia, with the first characterized by an increase of lipids, namely cholesterol and triglycerides, while an increase in lipoproteins, mainly of low-density lipoprotein (LDL), is typically found in the latter (Kuo, 1994). The diagnosis of this type of disease is performed through the monitoring levels of triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the blood (Jellinger et al., 2017). Furthermore, dysfunction in the vascular endothelial system is a hallmark of vascular diseases, usually regarded as a key early event in the development of atherosclerosis, and is also linked to hypertension and thrombosis (Briasoulis et al., 2012). The endothelium is a squamous tissue formed by one single layer of cells and is the integrant surface in the interior of the heart and blood vessels (Grassi et al., 2011). The main function of these interface barriers is the control of different substances that flows into the bloodstream, like white blood cells. Moreover, endothelial cells are involved in other main vessel functions, such as blood clotting (thrombosis and fibrinolysis), inflammation and angiogenesis, and are responsible for regulating blood pressure through vasoconstriction and vasodilation of the blood vessels (Flammer et al., 2012; Tousoulis, 2005). The endothelial dysfunction is characterized by an imbalance in the production and consumption of NO• (which is usually deficient) and the consequent impairment of vasodilation and vasoconstriction functions. This creates an environment that favors the activation of platelet and leukocyte adhesion, as well as an increase in the production of pro-inflammatory factors and reactive oxygen species (Sun et al., 2020). The endothelial dysfunction can be accessed through numerous biomarkers, such as cell adhesion molecules or transcription factors. Cell adhesion molecules including E-selectin, VCAM-1, and ICAM-1, usually overexpressed during episodes of endothelial dysfunction, are important proteins located on the endothelial cell surfaces that regulate the adhesion and migration of immune cells (Schram and Stehouwer, 2005). Likewise, overexpression of transcription factors, such as AP-1 and NF-κB, is a frequent manifestation of endothelial dysfunction and is accountable for the settlement of an

Chapter 13 Impact of phlorotannins on cardiovascular diseases

abnormal inflammatory state (Csiszar et al., 2008). Another important marker of endothelial dysfunction is the increased levels of asymmetric dimethylarginine, an analog of L-arginine that interferes with the normal L-arginine-stimulated nitric oxide synthesis and is one of the main causes of the decreased production of NO• ( Jay Widmer and Lerman, 2014).

4. Evidence of protective effects of phlorotannins on CVDs The cardioprotective effects of phlorotannins are a subject that has been steadily gaining ground over the recent years. Indeed, these compounds have been shown to exert a notable impact on the control of hyperlipidemia, blood pressure, chronic inflammation, and oxidative stress (Murray et al., 2018). The following subsections summarize the recent advances in the research on phlorotannins´ potential to prevent CVDs according to their capacity to interfere with different risk factors that contribute to their development.

4.1

Dyslipidemia

One of the main treatments of dyslipidemia involves a pharmacological approach that is followed by uncomfortable and dangerous side effects, like hepatic and/or rhabdomyolysis. Thus, to reduce such impacts on the patients’ lifestyle, the search for more natural-based treatments as an alternative to synthetic drugs is growing (Chu et al., 2015; Pahan, 2006). To fulfill the consumers’ demand, recent research for plant-based compounds has included marine algae, since their consumption in Eastern societies is claimed to be associated with the prevention of CVDs pathologies (Gavrilova and Gavrilov, 2012; Miyagi et al., 2003; Willcox et al., 2009). Table 1 summarizes relevant studies focusing on the ability of enriched extracts and/or isolated phlorotannins to counteract the hyperlipidemic state in animal models and humans. Among them, the dietary supplementation of hyperlipidemia-induced mice with a high-molecular-weight (HMW) phlorotannin extract with MW > 1.2
104 from Sargassum thunbergii, or with a polyphenol extract (Seapolynol) from Ecklonia cava, for 19 days or 4 weeks, respectively, was reported to decrease TC, TG, and LDL-C levels in the serum of hyperlipidemic mice (Wei et al., 2011; Yeo et al., 2012). Likewise, studies on high-fat diet-induced

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Table 1 Selected studies of the effect of phlorotannin extracts and isolated phlorotannins in dyslipidemia, as measured in vitro and in vivo. Extract or compound

Model

HMW phlorotannin Hyperlipidemic ext. from Sargassum Kunming mice thunbergii

Conditions

Intragastric administration of 200 mg/kg bw over 19 days HFD-fed ICR mice Oral Seapolynol™ administration of (Ecklonia cava 1.25–5 mg/mouse commercial ext) over 4 weeks EtOH 70% ext. from HFD-induced obese Oral gavage with Ecklonia cava C57BL/6 mice 100 and 500 mg/kg/day 5 times/week over 12 weeks EtOAc fract of the HFD-induced obese Oral gavage with MeOH ext. from C57BL/6 mice 200 mg/kg/day Ecklonia cava over 8 weeks Commercial ext. from Overweighted human Oral Ecklonia cava subjects administration of 72 or 144 mg twice a day over 12 weeks EtOH ext. from Subjects with Oral Ecklonia cava TC > 200 mg/dL and administration of LDL-C > 110 mg/dL 200 mg twice a day over 12 weeks Sprague Dawley rats Oral gavage with EtOH ext. and 100–250 mg/kg/ with HFD or phlorotannin-rich EtOAc and n-BuOH poloxamer 407day over 3 days fract from Ecklonia induced hyperlipidemia stolonifera 3T3-L1 adipocytes 12.5–50 mg/mL Phlorotannin-rich over night subfraction isolated from the EtOAc fract of EtOH ext. from Fucus distichus

Effect

Refs.

# TC, TG, and LDL-C levels in the Wei et al. serum (2011)

# TC, TG, and LDL-C levels in the Yeo et al. serum and # body weight gain (2012)

# Body weight gain, adipose tissue mass, hepatic fat deposition, insulin resistance, and the plasma TC, TG, and leptin/adiponectin ratio # Serum TG, ALT, and AST, and # body weight gain, total fat mass, and peripheral fat pad # Serum TC, LDL-C, and TC/HDL ratio, and # BMI, body fat ratio, waist circumference, waist/hip ratio and AI; " serum HDL-C only on high-dose subjects # Serum TC and LDL-C

Eo et al. (2015)

Park et al. (2012) Shin et al. (2012)

Choi et al. (2015)

# Serum TG, TC, and LDL-C, and Yoon et al. # AI; " serum HDL-C (2008)

# Lipid accumulation and " free Kellogg et al. glycerol; " mRNA expression of (2015) UCP-1 and adiponectin, and # of leptin

Chapter 13 Impact of phlorotannins on cardiovascular diseases

401

Table 1 Selected studies of the effect of phlorotannin extracts and isolated phlorotannins in dyslipidemia, as measured in vitro and in vivo—cont’d Extract or compound

Model

Conditions

Effect

# Liver TC and TG, # serum TC, TG and FFA, and # body weight gain; " serum HDL-C 3T3-L1 adipocytes # Lipid accumulation; 6,60 6,60 -Bieckol, 6,80 0 bieckol, 8,8 -bieckol, bieckol # expression of FAS and AAC at mRNA level and dieckol, and PFE-A expression of PPARg, C/EBPa, from Eisenia bicyclis and SREBP-1c at protein and mRNA level 3 T3-L1 preadipocytes 12.5–100 mM over # Adipogenesis and TC Dieckol, 24 h accumulation; dieckol # protein phloroglucinol, expression of PPARg and dioxinodehydroeckol, C/EBPa and PFE-A from Ecklonia stolonifera # Serum TC, TG, and LDL-C and Dieckol commercial HFD-fed ICR mice Oral administration of # body weight gain;" serum 1.25–5 mg/mouse HDL-C over 4 weeks Sprague Dawley rats Oral gavage with # Serum TG, TC, and LDL-C Dieckol and eckol 10 and 20 mg/kg/ more efficiently than lovastatin with HFD or from Ecklonia and # AI; dieckol " serum HDL-C day over 3 days poloxamer 407stolonifera induced hyperlipidemia Dieckol-rich ext. from C57BL/KsJ-db/db Ecklonia cava mice

Supplementation with 0.5 mg/g feed for 6 weeks 10–50 mg/mL for 8 days

Refs. Lee et al. (2012)

Kwon et al. (2015)

Jung et al. (2014)

Yeo et al. (2012)

Yoon et al. (2008)

ext, extract; fract, fraction #, decreased; ", increased; HFD, high fat diet; HMW, high molecular weight; TC, total cholesterol; TG, triglycerides; LDL-C, low-density lipoprotein cholesterol; ALT, alanine transaminase; AST, aspartame transaminase; BMI, body mass index; HDL-C, high-density lipoprotein cholesterol; AI, atherosclerotic index; FFA, free fatty acid; PPARg, peroxisome proliferator-activated receptor g; C/EBPa, CCATT/enhancer-binding protein a; SREBP-1c, sterol regulatory element-binding protein-1c; FAS, fatty acid synthase; AAC, acetyl-CoA carboxylase; UCP-1, uncoupling protein 1; EtOH, ethanol; EtOAc, ethyl acetate, n-BuOH, n-butanol; PFE-A, phlorofucofuroeckol A.

C57BL/6 mice revealed promising results when using Ecklonia cava phlorotannin extracts (Eo et al., 2015; Park et al., 2012). In particular, Eo and co-workers demonstrated that the oral supplementation with doses of 500 mg/kg/day or 100 mg/kg/day, for a period of 12 weeks, were both able to reduce TG and TC levels in plasma, as well as the atherogenic index (Eo et al., 2015). In turn, Park et al. found that, for a dosage of 200 mg/kg of phlorotannin extract of Ecklonia cava, the only effect observed was

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Chapter 13 Impact of phlorotannins on cardiovascular diseases

the reduction in cholesterol levels, while TG remained unchanged (Park et al., 2012). Positive effects of oral supplementation with algae extracts were also described in human trials (Choi et al., 2015; Ding et al., 2020; Shin et al., 2012). As reported by Shin and co-workers, the dietary supplementation with a phlorotannin extract from Ecklonia cava origin, at a low dose (72 mg-ECP/ day) or high dose (144 mg-ECP/day) for 12 weeks, resulted in a significant decrease in TC (7.1 and 9.3%, respectively) and LDL-C (10 and 14.3%, respectively) levels, as well on the TC/HDL-C ratio and the atherogenic index, compared with the placebo group (Shin et al., 2012). In another study, Choi and co-workers reported that the oral administration of an Ecklonia cava extract (400 mg/day, for 12 weeks) reduced TC levels by 2.8% and the LDL-C level by 11.1%, in comparison with the placebo group (P ¼ 0.039, 0.030, respectively) (Choi et al., 2015). Importantly, the ability of purified fractions and/or isolated phlorotannins to counteract dyslipidemia has also been attested, allowing to further consolidate the promising abilities of these compounds in relation to this disorder (Kellogg et al., 2015; Yoon et al., 2008). Along with an ethanolic crude extract of Ecklonia stolonifera, two phlorotannin-rich partitioned fractions (EtOAc and n-BuOH) were shown to significantly reduce TG, TC, and LDL-C levels and the atherogenic index in hyperlipidemic rats (Yoon et al., 2008). Additionally, Kellogg and co-workers reported a decrease in lipid accumulation in 3T3-L1 adipocytes of 77.5  0.03%, caused by a EtOAc subfraction from Fucus distichus origin (Kellogg et al., 2015). Promising results have also been achieved for the isolated phlorotannins phloroglucinol, eckol, dieckol, dioxinodehydroeckol, and phlorofucofuroeckol A, fucophloroethols, 6,60 bieckol, 6,80 -bieckol, and 8,80 -bieckol, since they revealed the ability to reduce lipid accumulation, TC, TG, and LDL-C at a cellular level, and on animal models as well (Jung et al., 2014; Kellogg et al., 2015; Kwon et al., 2015; Lee et al., 2012; Yeo et al., 2012; Yoon et al., 2008). A supplementation diet of C57BL/KsJ-db/db mice (i.e., a diabetic mouse strain, characterized by extreme obesity and early onset of hyperglycemia) with dieckol at 0.5/100 g feed for 6 weeks was shown to reduce plasma and hepatic TG levels more effectively than the drug rosiglitazone (1.38 mmol/L and 1.71 mmol/g vs 1.40 mmol/L and 1.84 mmol/g, respectively). Likewise, a dieckol-supplemented diet on db/db mice was reported to reduce plasma and liver TC (4.79 mmol/L and 2.58 mmol/g vs 6.39 mmol/L and 3.76 mmol/g, respectively) and plasma free fatty acids when compared to the control group (Lee et al., 2012).

Chapter 13 Impact of phlorotannins on cardiovascular diseases

At the cellular level, the phlorotannins, phloroglucinol, eckol, dieckol, dioxinodehydroeckol, and phlorofucofuroeckol A, were demonstrated to effectively inhibit adipogenesis in a concentration range of 12.5–100 μM and inhibit the cellular TG accumulation in 3T3-L1 adipocytes in a 92%, 58%, 15%, 32.5%, and 95%, respectively, with phloroglucinol, eckol, and phlorofucofuroeckol A, showing inhibition of TG accumulation in a dose-dependent manner with IC50 values of 22.43, 35.72, and 17.86 μM, respectively (Jung et al., 2014). Moreover, five isolated phlorotannins from Eisenia bicyclis, namely 6,60 -bieckol, 6,80 -bieckol, 8,80 -bieckol, dieckol, and phlorofucofuroeckol A, were shown to inhibit lipid accumulation during adipocyte differentiation in a dosedependent manner in 3T3-L1 cells, being this effect close to 60% at 50 μg/mL (Kwon et al., 2015). Overall, it can be noticed that phlorotannins can counteract lipid accumulation and therefore improve the dyslipidemia state, which will prevent the development of CVDs. However, to further understand which underlying mechanisms are responsible for this improvement, researchers have investigated a wide spectrum of modulators and biological pathways. Some authors claim that the lipid regulation mechanism of phlorotannins can occur through their ability to increase LDL receptor protein (LDL-R) levels in liver tissue. This hypothesis is supported by the fact that the treatment of Kunming mice with a phlorotannin extract did not inhibit the expression of HMG-CoA reductase genes (Wei et al., 2011). However, in another study, a polyphenol extract from Ecklonia cava origin, as well as isolated dieckol, revealed the ability to significantly inhibit the HMG-CoA reductase activity in vitro by approximately 78% and 61%, respectively (Yeo et al., 2012). More recently, the hepatic lipid metabolism of C57BL/6 mice was demonstrated to be ameliorated by phlorotannins treatment through activation of AMP-activated protein kinase (AMPK) and Sirtuin 1 (SIRT1) modulators (Eo et al., 2015). Additionally, enriched phlorotannin extracts were shown to increase not only the phosphorylation of AMPK but also the mRNA expression of adipogenic genes in adipose tissue of C57BL/6 mice, namely proliferator-activated receptor γ (PPARγ), CCAAT/enhancerbinding protein α (C/EBPα), sterol regulatory element-binding transcription factor 1 (SREBP-1c), and fatty acid synthase (FAS) (Park et al., 2012). Phlorotannins have also been reported to improve lipid metabolism in 3T3-L1 adipocyte cells, by increasing adiponectin and UCP-1 and decreasing leptin mRNA expression (Kellogg et al., 2015), while the phlorotannins, phloroglucinol, eckol, dieckol, dioxinodehydroeckol, and phlorofucofuroeckol A, were found to modulate the expression of adipocyte marker genes in the same

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Chapter 13 Impact of phlorotannins on cardiovascular diseases

cell line by decreasing the expression of PPARγ and C/EBPα (Jung et al., 2014). Similarly, incubation of 3T3-L1 with 6,60 -bieckol isolated from Eisenia bicyclis suppressed the mRNA expression and protein levels of PPARγ, C/EBPα, and SREBP-1c and decreased mRNA expression levels of FAS and acyl-coA carboxylase (Kwon et al., 2015).

5. Endothelial function The link between endothelial dysfunction and CVDs is unquestionable and well established, particularly in the occurrence of atherosclerosis. In this field, phlorotannins have demonstrated great potential as their antioxidant and anti-inflammatory activities may valuably contribute to re-establishment of the endothelium homeostasis and prevent additional cardiovascular complications (Table 2).

Table 2 Selected studies of the effect of phlorotannin extracts and isolated phlorotannins in endothelial parameters, as measured in vitro and in vivo. Extract or compound EtOH extract from Ecklonia stolonifera, Ecklonia cava, P. siliquosa, H. fusiforme and U. pinnatifida EtOAc fraction of Ecklonia stolonifera EtOH extract Phlorotannin fractions with 3 kDa from a MeOH extract of Fucus spiralis

MeOH extract of Fucus spiralis

Model

Conditions

Effect

Refs.

ACE inhibitory activity

Incubation with 163.93 mg/mL

Inhibitions of enzymatic activity about 26% (Hizikia fusiforme) and maximum (Ecklonia cava)

Jung et al. (2006)

ACE inhibitory activity

Incubation with serial dilutions

# Enzymatic activity (IC50 ¼ 17 mg/mL)

Jung et al. (2006)

ACE inhibitory activity

Incubation with 200 mg/mL

Paiva et al. (2016)

ACE inhibitory activity

Incubation with 200 mg/mL

# Of enzymatic activity proportional to the concentration of phlorotannins in each fraction (17, 7 and 89% for 3 kDa, respectively) 80% inhibition of enzymatic activity

Paiva et al. (2016)

Chapter 13 Impact of phlorotannins on cardiovascular diseases

405

Table 2 Selected studies of the effect of phlorotannin extracts and isolated phlorotannins in endothelial parameters, as measured in vitro and in vivo—cont’d Extract or compound

Model

Conditions

Effect

Refs.

ACE inhibitory activity

Incubation with serial dilutions

# Enzymatic activity (IC50 ¼ 0.96–1.31 mg/mL)

Wijesinghe et al. (2011)

ACE inhibitory activity

Incubation with serial dilutions

# Enzymatic activity (IC50 ¼ 1.5–3.0 mM)

Wijesinghe et al. (2011)

ACE inhibitory activity

Incubation with serial dilutions

# Enzymatic activity (IC50 ¼ 13–410 mM)

Jung et al. (2006)

HG-induced HUVECs

10–50 mg/mL for 20 h

Lee et al. (2010)

Eckol and dieckol from Eisenia bicyclis

In vitro: TNFa-activated HUVECs In vivo: Male IRC mice

In vitro: incubation with serial dilutions 10 min prior to TNF-a In vivo: oral administration of 50 mg/kg bw 1 h prior to tail transection

Eckol and dieckol from Eisenia bicyclis

In vitro: LPSactivated HUVECs In vivo: ale ICR mice injected with 0.7% acetic or CMC acid injection In vitro: LPS and HMGB1stimulated HUVECs

In vitro: incubation with 10 mM min prior to LPS In vivo: oral administration of 10 mM/ mouse 6 h prior to 0.7% AA injection or CMC

# Glucose-induced toxicity, TBARS, and expression of iNOS, COX.2, and NF-kB In vitro: prolonged aPTT and PT and # thrombin and FXa activities and protein expression; # PAI-1 production and PAI1/t-PA ratio In vivo: " anticoagulant effect In vitro: # barrier disruption and transendothelial migration of leukocytes In vivo: # AA-induced hyperpermeability and CMC-induced leukocytes migration In vitro: # LPS-induced HMGB1 release; # HMGB1-mediated barrier disruption, expression of

Organic extracts (EtOH, EtOAc, CHCl3, Et2O, Hex) from Ecklonia cava Phloroglucinol, triphlorethol A, eckol, dieckol, and dioxinodehydroeckol from Ecklonia cava Dioxinodehydroeckol, eckol, phloroglucinol, diecol, and triphlorethol A from Ecklonia stolonifera Dieckol from Ecklonia cava

Eckol, dieckol and phloroglucinol from Eisenia bicyclis

In vitro: incubation with serial dilutions 6 h prior to LPS or HMGB1 stimulation In vivo:

Kim et al. (2012a)

Hoon Kim et al. (2012)

Kim et al. (2012b)

Continued

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Chapter 13 Impact of phlorotannins on cardiovascular diseases

Table 2 Selected studies of the effect of phlorotannin extracts and isolated phlorotannins in endothelial parameters, as measured in vitro and in vivo—cont’d Extract or compound

PPB from Ecklonia cava

PPB from Ecklonia cava

Model

Conditions

Effect

In vivo: Female ICR mice injected with 0.7% acetic or CMC acid injection

treatment with 20 mM 1 h prior to 0.7% acetic or carboxymethylcellulose acid injection

HFD-induced obese C57BL/ 6N male mice and HCSDinduced hypertensive mice PA-BSA-treated monocytes, ECs and VSMCs

Oral administration of 2.5 mg/kg/day of PPB over 4 weeks

CAMs, and adhesion/ trans-endothelial migration of leukocytes to endothelial cells In vivo: # AA inducedhyperpermeability and CMC-induced leukocytes migration Improve blood circulation with # of CAMs expression, EC death, VSMC proliferation, and migration, blood pressure, and lipoprotein and cholesterol levels # Monocyte migration, levels of inflammatory macrophage differentiation; protected against monocyteassociated EC death, by pPI3K-pAKT, pAMPK and pERK; # casp levels and monocyte-associated VSMC proliferation and migration

Incubation with 2.5 mg/ mL and 0.25 mM PA-BSA for 48 h

Refs.

Son et al. (2019)

Oh et al. (2018)

#, decreased; ", increased; EtOH, ethanol; EtOAc, ethyl acetate; MeOH, methanol; Et2O, diethyl ether; Hex, hexane; ACE, angiotensinconverting enzyme; HG, high glucose; HUVECs, human umbilical vein endothelial cells; TNF-a, tumor necrosis factor a; CMC, carboxymethylcellulose; AA, acetic acid; CAMs, cell adhesion molecules; HMGB1, high mobility group 1; LPS, lipopolysaccharide; FXa, activated factor X; aPTT, activated partial thromboplastin time; PT, prothrombin time; PAI-1, plasminogen activator inhibitor-1; t-PA, tissue plasminogen activator; PPB, pyrogallol-phloroglucinol-6,6-bieckol; HFD, high fat diet; HCSD, high cholesterol and salt diet; EC, endothelial cells; VSMC, vascular smooth muscle cells; pPI3K, phosphor-phosphoinositide 3-kinase; pAKT, phosphor-protein kinase B; pAMPK, phosphor-activated protein kinase; pERK, phosphor-extracellular-signal-regulated kinase; casp, caspases; PA-BSA, bovine serum albumin-palmitate saturated fatty acid complex; PAg, platelet aggregation; TXB2, thromboxane B2; ROS, reactive oxygen species; COX, cyclooxygenase; p38-MAPK, p38 mitogen-activated protein kinases.

As mentioned earlier, one of the main functions of endothelium is to control blood pressure, which is regulated by the reninangiotensin-aldosterone system and its activation results in the conversion of angiotensin I to angiotensin II, being the latter a potent vasoconstrictor. Numerous authors have screened the

Chapter 13 Impact of phlorotannins on cardiovascular diseases

ability of phlorotannin-enriched extracts or purified fractions and/or purified phlorotannins to inhibit angiotensin-Iconverting enzyme (ACE) as an alternative treatment for hypertension. For example, Jung et al. showed that ethanolic extracts of Ecklonia stolonifera, Ecklonia cava, Pelvetia siliquosa, and Undaria pinnatifida, at a concentration of 163.93 μg/mL, could inhibit more than 50% of the activity of ACE. Moreover, different purified fractions from Ecklonia stolonifera also showed ACE inhibitory properties in the order n-butanol > n-hexane > ethyl acetate, with IC50 values ranging between 17 and 92 μg/mL ( Jung et al., 2006). Similarly, different extracts from Ecklonia cava were shown to inhibit ACE in the following order: chloroform > hexane > ethyl acetate > diethyl ether > ethanol, with IC50 values between 0.96 and 1.31 mg/mL. The inhibitory capacity of a methanolic extract of Fucus spiralis (TPC value of 212.7 mg PE/g DW extract) was also shown to be above 80% at a concentration of 200 μg/mL (Paiva et al., 2016; Wijesinghe et al., 2011). In addition, isolated phlorotannins from Ecklonia stolonifera, namely eckol, phlorofucofuroeckol A, and dieckol, were proved to inhibit the ACE activity, with IC50 values of 71, 13, and 34 μM, respectively ( Jung et al., 2006). While Wijesinghe and co-workers reported values of 1.5–3.0 mM for phloroglucinol, triphlorethol A, eckol, dieckol, and eckstolonol isolated from Ecklonia cava. Interestingly, the authors concluded that the effect of dieckol on ACE was caused by a non-competitive inhibition (Wijesinghe et al., 2011). Apart from regulating blood flow, the endothelium also controls the migration of different substances into the bloodstream, many of which are related to inflammatory processes normally associated with CVDs. Phlorotannins have already been shown to have great antioxidant and anti-inflammatory properties, so it is feasible to hypothesize that they can improve endothelial function by releasing controlled substances. For instance, dieckol was reported to protect glucose-induced HUVECs cells against oxidative stress, by lowering, dose-dependent manner, ROS generation, lipid peroxidation, and NO levels through the decrease of iNOS, COX-2, and NF-kB protein overexpression (Lee et al., 2010). Additionally, Kim and co-workers demonstrated that eckol and dieckol displayed anticoagulant properties, once they were able to prolong activated partial thromboplastin time (aPTT), prothrombin time (PT) and significantly reduced thrombin and activated factor X (FXa) activities and proteins expression, as well as the production of PAI-1 and the PAI-1/t-PA ratio in TNF-αactivated HUVECs cells. In vivo experiments further revealed that eckol and dieckol promote anticoagulation activities in ICR mice,

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as they prolonged tail bleeding time compared with the control (Kim et al., 2012a). The same research group also reported that, at the concentration of 10 μM, eckol and dieckol were able to reduce the barrier disruption and transendothelial migration of leukocytes in lipopolysaccharide (LPS)-mediated HUVECs cells, as well as to suppress acetic acid-induced hyperpermeability and carboxymethylcellulose-induced leukocytes migration in ICR mice. In addition, they effectively inhibited the binding of monocytes to HMGB1-stimulated endothelial cells (Hoon Kim et al., 2012). In a different project, the protective effects of phloroglucinol, eckol, and dieckol were found to occur via inhibition of LPS-induced HMGB1 release, HMGB1-mediated barrier disruption, expressions of cell adhesion molecules (CAMs), and the adhesion/transendothelial migration of leukocytes in LPS and HMGB1-induced HUVECs cells. These phlorotannins also suppressed acetic acidinduced hyperpermeability and carboxymethylcellulose-induced leukocytes migration in ICR mice (Kim et al., 2012b). Other isolated phlorotannins were shown to have the ability to modulate endothelial dysfunction. Among them, phloroglucinol was described as an effective suppressor of AA-induced platelet aggregation and thromboxane B2 (TXB2) production, as well as a good inhibitor of ROS production, which were decreased by 53% at a concentration of 10 μM and showed an IC50 of 13.8 μM, in AA-induced platelets. The inhibition of ROS production resulted from the inhibition of COX-1 and COX-2 by 74% and 72%, respectively, at a concentration of 50 μM. Moreover, phloroglucinol reduced ERK and p38 MAPK phosphorylation because of the antiplatelet effect of this compound and inhibition of COX-1. Supporting these results, ex vivo experiments on AA-induced ICR mice also showed a dose-dependent decrease in platelet aggregation (by 57% and 71% for 2.5 and 5 μmol/mouse, respectively), and an increased anticoagulatory capacity (Chang et al., 2012). In the study performed by Son and co-workers, the authors demonstrated the benefits of dietary supplementation with the phlorotannin pyrogallol-phloroglucinol-6,6-bieckol, in HFDinduced obese C57BL/6 N mice and HCSD-induced hypertensive mice. The oral administration of this compound at a dose of 2.5 mg/kg/day, for 4 weeks, significantly improves blood circulation, including a reduction in adhesion molecules expression, endothelial cell (EC) death, excessive vascular smooth muscle cells (VSMC) proliferation and migration, blood pressure, and lipoprotein and cholesterol levels (Son et al., 2019). Moreover, pyrogallol-phloroglucinol-6,6-bieckol significantly inhibited in vitro monocyte migration by reducing inflammatory macrophage differentiation and its related molecular factors. It also

Chapter 13 Impact of phlorotannins on cardiovascular diseases

protected against monocyte-associated endothelial cell death by increasing the phosphorylation of PI3K-AKT and AMPK, decreasing caspase levels, and reducing monocyte-associated vascular smooth muscle cell proliferation and migration by decreasing the phosphorylation of ERK and AKT (Oh et al., 2018).

6. Conclusions In conclusion, the data gathered so far evidence that brown algae phlorotannins and phlorotannin-rich extracts are unanimously acknowledged for their effects on critical steps involved in the pathogenesis of cardiovascular diseases, particularly acting as modulators of multiple biochemical mechanisms underlying the manifestation of dyslipidemia and endothelial dysfunction. These compounds are, therefore, endowed with high versatility and capacity to promote and improve cardiovascular health status by interfering with the abnormal expression of different hormones, enzymes, adhesion molecules, transcription factors, and other important proteins and biomarkers that may impair the homeostasis of the cardiovascular system. As such, phlorotannins can be viewed as valuable naturally occurring alternatives with great pharmacological and economic interest for developing novel therapeutic strategies in the field of CVDs.

Acknowledgments Thanks to project PTDC/BAA-AGR/31015/2017, “Algaphlor—Brown algae phlorotannins: From bioavailability to the development of new functional foods,” co-financed by the Operational Program for Competitiveness and Internationalization—POCI, within the European Regional Development Fund (FEDER), and Science and Technology Foundation (FCT), through national funds. Thanks to the University of Aveiro (UA) and the Science and Technology Foundation/Ministry of Education and Science (FCT/MEC) for funding the Associated Laboratory for Green Chemistry (LAQV) of the Network of Chemistry and Technology (REQUIMTE) (UIDB/50006/2020), through national funds and, where applicable, co-financed by FEDER, within Portugal 2020.

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Immune system: Inflammatory response

14

Diana Del Juncal-Guzma´n, Carlos Eduardo Camacho-Gonza´lez, Francia Guadalupe Lo´pez-Ca´rdenas, Sonia Guadalupe Sa´yago-Ayerdi, and Jorge Alberto Sa´nchez-Burgos Division of Graduate Studies, National Technological Institute of Mexico/ Instituto Tecnolo´gico de Tepic, Tepic, Nayarit, Mexico

1.

Introduction

Inflammation in humans is known to be derived from various human health disorders, including cancer, obesity, diabetes mellitus II, neurodegenerative and cardiovascular diseases, arthritis, stress, lack of sleep, poor eating habits, and high blood pressure (Song et al., 2019; Yahfoufi et al., 2018). Specifically, it has been shown that inflammation can contribute to the development of arterial hypertension, which is classified as a high-risk factor in the development of cardiovascular diseases, considered one of the main causes of morbidity and mortality in the world, due to exhaustion of antioxidant defenses and accumulation of inflammatory markers in vascular tissues (Farooqui et al., 2021; Orejudo et al., 2019). The immune system is known as a natural homeostatic defense system of the body, which involves the activation of the immune response (Kaur et al., 2020), thus inducing the initial phase of acute inflammation with beneficial but limited effects. If the acute inflammation continues, chronic inflammation occurs, generating tissue damage (Kaur et al., 2020). The immune system is made up of various types of cells that together generate two main types of immunity: (1) innate immunity, which is considered the first line of defense against foreign agents, consisting of detrital cells, monocytes, macrophages, granulocytes, and natural killer (NK) cells; these include genetically controlled mechanisms that protect the host from pathogens (Cho et al., 2016); and (2) adaptive immunity, which includes T and B cells which are Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00001-7 Copyright # 2023 Elsevier Inc. All rights reserved.

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responsible for secreting antibodies and producing different types of cytokines, activating different immune cells and cytotoxic destruction of damaged cells or tissues, having a crucial role in maintaining human health (Li et al., 2019; Shakoor et al., 2021). Activated macrophages play an important role in the host’s immune reactions, protecting it from external agents through phagocytosis, directly eliminating the threat, and favoring the presence of antigens for the subsequent activation of the immune response. At the same time, nitric oxide synthase (iNOS) is induced in cells to activate the production of nitric oxide (NO), which is associated with the activation of immune processes (Kaur et al., 2020; Li et al., 2019), secreting pro-inflammatory cytokines such as interleukin beta (IL-b), interleukin 6 (IL-6), and interleukin 10 (IL-10). Cytokines intervene in various biological functions; hence, their deregulation is associated with chronic inflammation and autoimmune disorders (Kaur et al., 2020). The tumor necrosis factor alpha (TNF-a), considered a biomarker that regulates the secretion of other cytokines, is considered a therapeutic target (Burgos-Edwards et al., 2019; Villago´mezRodrı´guez et al., 2019). Cyclooxygenase-II (COX-2), mainly expressed during inflammation (Chen et al., 2019; Ribeiro et al., 2019; Xu et al., 2017), is a key enzyme in the metabolism of arachidonic acid that contributes to the formation of prostaglandins, prostacyclins, and thromboxanes, among others (Wang et al., 2019).

2.

Arterial hypertension (HTN) and inflammation

The inflammatory process has been strongly associated with arterial pressure, defined as the product of cardiac output and peripheral resistance; thus, arterial hypertension is associated with high cardiac output or high peripheral resistance (Saxena et al., 2018). Arterial hypertension (HTN) is considered the second leading cause of kidney failure, which can in turn be complicated by injuries to the cardiovascular, brain, and kidney systems (Orejudo et al., 2019); however, its etiology is still not completely clear and a matter of debate (Farooqui et al., 2021). Renal dysfunction is associated with an increase in sodium reabsorption and glomerular hyperfiltration, playing a key role in the development of the disease. Additionally, renal dysfunction leads to an increase in blood pressure, which causes an increase in the activity of the renal sympathetic nerve, angiotensin II, and aldosterone

Chapter 14 Immune system: Inflammatory response

(antinatriuretic hormones). This process culminates in the activation of innate and adaptive immune cells invading the body’s tissues. A high production of inflammatory cytokines is generated by this invasion, favoring cardiovascular and tissue injuries, which stimulate the formation of antigens that triggers an immune response on the infiltration of macrophages and T lymphocytes, favoring the release of inflammatory mediators (Hall et al., 2021). Loperena (2018) reported that a high salt intake favors histological lesions, promoting the differentiation of macrophages and monocytes in kidneys with AHT, with a marked reduction of NO and a greater release of IL-6. On the other hand, oxidative stress leads to the production of reactive oxygen species (ROS) and is considered essential in the development of HTN as it is an initiator of renal and vascular inflammation, where T cells become key by producing TNF-α, favoring vascular alterations in a NO-dependent manner in macrophages (Guzik and Touyz, 2017). In hypertension, ROS promote endothelial dysfunction that includes apoptosis, angiogenesis, and inflammation that affect membrane permeability by releasing cytochrome C and some caspases, promoting the adhesion of inflammatory cells (Sorriento et al., 2018). The endothelium of the blood vessels produces NO on a regular basis, which acts as a relaxant at the same time that it produces constricting endothelin; both mechanisms are the main regulators of blood pressure and vascular tone (Saxena et al., 2018). In hypertension, there is a deregulation of the endothelial tone, altering the production of NO, favoring its bioavailability. This deregulation inhibits vascular remodeling and regulation of the renin-angiotensin-aldosterone system. Additionally, endothelial tone deregulation favors the pro-inflammatory and prothrombotic constriction of endothelial origin, and the release of thromboxane. It also transforms the growth factor B, making the NO pathway one of the most important regulatory mechanisms against hypertension (Di Giosia et al., 2018).

3.

Biotic drivers of inflammation

Particulate matter (PM) is a hazardous air pollutant that contains transition metals (e.g., Fe, Cu, Cr, Ce, Ni, and Zn), organic compounds, and polycyclic aromatic hydrocarbons (PAHs) (Chen et al., 2015). Recently, MP has been suggested as an oxidant, causing inflammation, and lung diseases when inhaled for a prolonged period of time through oxidative stress. This process causes an imbalance between ROS, such as superoxide anion

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(O•2), hydrogen peroxide (H2O2), hydroxyl radical (OH), and antioxidant enzymes (Bai et al., 2016). Previous studies evidence that uninterrupted oxidative stress plays a critical role in driving DNA damage, cell death, and inflammation (Bai et al., 2016; Crobeddu et al., 2017; Fiordelisi et al., 2017; Chen et al., 2019). Lung epithelial cells are the main site where oxidative stress persists when exposed to MP (Crobeddu et al., 2017), especially alveolar. Type II epithelial cells (CEA II) are known to be highly susceptible to oxidative damage, and many antioxidant enzymes are specially localized in CEA II to maintain lung function, even in the presence of airborne toxicants (Dalrymple et al., 2016; Mishra et al., 2018). Manganese superoxide dismutase (SOD-Mn), catalase (CAT), and glutathione peroxidase (GPx) in CEA II are the most important antioxidant enzymes to defend the lung epithelium against inhaled oxidants (Ighodaro and Akinloye, 2018). The generation of intracellular ROS begins with the rapid uptake of oxygen and proceeds through a series of reactions such as the activation of NADPH oxidase and the production of superoxide radical anion (O•2) during oxidative reactions. O•2 is then rapidly converted to H2O2 by SOD, and H2O2 is finally reduced to H2O by CAT and GPx (Pisoschi et al., 2021). This antioxidant system (e.g., SOD, CAT, and GPx) maintains cellular redox homeostasis against rapid ROS generation (Rahman and Adcock, 2006).

4.

Therapeutic alternatives for the treatment of the inflammatory process

Pharmacological treatments to counteract inflammation are generally “governed” by nonsteroidal anti-inflammatory drugs (NSAIDs); however, its use can cause side effects such as gastritis and ulcers, due to suppression of cytokines, increased blood pressure, and cardiovascular events (Grosser et al., 2017). These side effects triggered the search for natural compounds that help treating inflammatory processes (de Souza et al., 2019). Several studies confirmed that phenolic compounds (PCs) from various sources have anti-inflammatory, anticancer, antiviral, and antioxidant activities, among others (Srinivasan et al., 2020). Epidemiological, clinical, and nutritional studies strongly support that dietary polyphenols play an important role in human health. Regular consumption of polyphenols has been associated with a lower risk of suffering from different diseases related to inflammation, among other conditions. Various studies with PCs have shown they are effective on controlling the response of the immune system, since they could

Chapter 14 Immune system: Inflammatory response

suppress pro-inflammatory cytokines, influencing dendritic cells, T and NK cells (Shakoor et al., 2021). Thus, PCs could have a potential role in improving the immune response by activating macrophages, while inducing the production of antibodies activated by β cells (Srinivasan et al., 2020), which could decrease NO production, TNF-α, and IL-6. Additionally, PCs have immunostimulatory effects against allergic reactions and autoimmune diseases (Shakoor et al., 2021). Seaweeds, traditionally used as functional foods (GomezZavaglia et al., 2019), contain many bioactive compounds beneficial for the human health, such as polysaccharides, carotenoids, alginate, carrageenan, and phenolics. The highest proportion of PCs contained in green and red algae are bromophenols, phenolic acids, and flavonoids. In particular, phlorotannins (PTs, phenolic acids), a group of complex polymers of phloroglucinol (FG, 1,3,5trihydroxybenzene), are the dominant PCs found in brown marine algae only. These phytochemicals have attracted a lot of attention, because, similar to other PCs, they are bioactive with many potential benefits.

5.

Phenolic compounds present in marine algae and their anti-inflammatory effect

Seaweed extracts and bioactive components, specifically phenolic acids, have shown strong activity against inflammation. This activity is associated with the inhibition of key inflammatory pathways such as NF-KB, which leads to suppression of NO, TNF-α, IL-6, IL-1β, iNOS, and COX-II, as well as other routes of inflammation as illustrated in Table 1. For example, brown algae contain large amounts of PCs, some with antioxidant activity that function as ROS terminators, and others with metal ion chelation activity that are capable to reduce oxidative stress (Kojima-Yuasa, 2018). However, there is little information on the antioxidant mechanism of brown algae against MP-induced oxidative stress. Sargassum horneri (SHE) is a brown seaweed in East Asia, rich in vitamins, amino acids, polysaccharides, and PCs (Herath et al., 2019). Although SHE has been identified as having antioxidant, antitumor, and anti-inflammatory activities, studies on its antioxidant mechanisms are limited and incomplete (Herath et al., 2019; Lee et al., 2020). In some studies, the ethanolic extract of SHE was found to contain 67.58 mg GAE/g of total polyphenols and contributed to antioxidant activity through free radical scavenging and metal ion chelation (Lee et al., 2020).

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Table 1 Anti-inflammatory effects of phenolic compounds present in marine algae.

Alga

Associated compounds

Ishige foliacea

Octaphlorethol A

Eisenia arborea Chondrus Crispus Eisenia bicyclis

Florofucofuroechol B

Porphyria dentata

Catechin, gallic acid, and p-coumaric acid Phloroglucinol, eckol, florofucofuroechol A, and dioxinodehydroeckol Hesperidin, rutin, and catechol

Laurencia undulata

NR

Ishige okamurae

Diphlorethohydroxycarmalol

Ecklonia stolonifera

Florofucofuroechol A and B

Ecklonia cava

Dieckol

Anti-inflammatory effect

References

Inhibition of pro-inflammatory cytokines, mitochondria-activated protein kinase, and stimulated NF-kB pathways in CpG macrophages and dendritic cells Inhibitory effect on histamine release

Manzoor et al. (2013)

Significant inhibition against bovine serum albumin denaturation compared to aspirin Inhibition of LPS-induced NO production in RAW 264.7 cells Inhibition of NO production in LPS-stimulated RAW 264.7 cells. Catechol was shown to be a potent suppressor of upregulation of the iNOS promoter and NF-kB enhancer Reduced IL-4 and IL-5 concentration by 70% and 85%, respectively, and led to an 83.5% reduction in TNF-a secretion Downregulation of iNOS and COX-II expression and NF-kB activation in human umbilical vein endothelial cells and RAW 264.7 cells. Inhibition of NO production by downregulating iNOS and prostaglandin E2 production in LPS-stimulated RAW 264.7 cells Inhibition of LPS-induced NO production in murine BV2 microglia

Sugiura et al. (2006) Alkhalaf (2021) Jung et al. (2013) Kazłowska et al. (2010)

Jung et al. (2009a, 2009b) Heo et al. (2010) Lee et al. (2012) Jung et al. (2009a, 2009b)

NR, not reported.

One study hypothesizes that SHE may moderate oxidative stress and transition metal chelation against PM in lung epithelial cells. The authors investigated the effect of PM and antioxidant efficacy of SHE in MLE-12 (lung alveolar epithelial cell line, type II) (Kim et al., 2021). It has been suggested that SHE polyphenols could regulate the apoptosis of these cells.

Chapter 14 Immune system: Inflammatory response

Previous studies observed a high antioxidant potential of SHE and identified gallic acid as one of its main phenolic compounds (Herath et al., 2020; Lee et al., 2020). Gallic acid is known to scavenge radicals by stabilizing the resulting phenolic radicals with hydrogen bonding or extended electron delocalization (Bai et al., 2020; Rajan and Muraleedharan, 2020). Furthermore, compared to the in vitro antioxidant activities of SHE, half of the maximal efficacy of SHE is in peroxide scavenging activity (83.92 μg/mL), reducing power (188.59 μg/mL), chelating effect of metal ions (412.50 μg/mL), superoxide dismutase activity (469.36μg/mL), DPPH free radical scavenging (550.23 μg/mL), and OH radical scavenging activity (1434.90 μg/mL). An ethanol extract of SHE containing polyphenols attenuated the oxidative stress induced by PM through elimination and transition of ROS and metal chelation, avoiding or attenuating a possible inflammatory effect via ROS (Kim et al., 2021).

6.

Phlorotannins and therapeutic effect against oxidation

According to what was indicated above, phlorotannins (PTs) are interesting PCs, common in marine algae and with no presence in terrestrial plants. PTs are very hydrophilic compounds, and their molecular size ranges between 126 Da and 650 kDa. Among the most abundant PTs are fuhalols, phlorethols, fucoles, fucophlorethols, eckols, and carmalols, which are generated by linking several phloroglucinol units. Eckol is a highly studied PT that has shown a strong regulatory activity on the expression of HO-1 in animal models by activating the biological marker Nrf2 (Yang et al., 2014). Dieckol can suppress the iNOS gene involved in inflammatory processes, showing at low concentrations satisfactory results in raw cells (in vitro model) (Rajan et al., 2021). Ecklonia cava is one of the edible brown algae with the highest concentration of phlorotannins (Cho et al., 2022; Heo et al., 2022); other phlorotannin-rich algae are Eisenia bicyclis and Ecklonia kurome. These algae contain high amounts of eckol, phlorofucofuroeckol A, dieckol, 8,80 -bieckol, eckstolonol, triphloroethol A, and phloroglucinol. These phlorotannins have shown on in vitro models higher antioxidant activity than catechinepigalocatechin-3-gallate, even better activity than powerful antioxidants such as ascorbic acid and α-tocopherol (Fernando et al., 2022). Other algae with high antioxidant activity and rich in PT are Ulva pertusa, Symphyocladia latiuscula, Ecklonia stolonifera, and Ascophyllum nodosum.

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Red (Botryocladia sp. and Gracilaria sp.) and green algae (Caulerpa sertularioides and Codium sp.) contain other PCs with high antioxidant activity (Mahendran et al., 2021) such as gallic acid, protocatechuic acid, p-hydroxybenzoic acid, gentisic acid, catechin, p-coumaric acid, and cinnamic acid, to name a few (Alkhalaf, 2021).

7.

Phlorotannins and their therapeutic effect against aging and neuroprotection

In addition to enzymatic activity, PTs have shown beneficial effects against neurodegenerative diseases. In studies carried out with Ecklonia cava extracts rich in PT, a significant reduction in the production of H2O2 and oxidative stress was found in human neuroblastoma SH-SY5Y (Cha et al., 2016). The primarily responsible for these activities has been found to be phloroglucinol (PG) (Shen and Qi, 2021), which additionally reduced ROS and lipid peroxidation. Furthermore, PG increased the protein expression and activities of CAT and GPx and reversed Nrf2 reduction in the nucleus, highlighting its beneficial properties in vivo. Another interesting biological effect of PG is the inhibition of the activity of the enzymes AChE and butyrylcholinesterase (Shin et al., 2021), which reduce the β protein levels and the number of amyloid plaques in the brain of mouse models of AD. Shrestha et al. (2020) observed that Aβ1-42-induced ROS levels were reduced after treatment with Ecklonia radiata PG extracts on in vivo assays. All the studies mentioned above could lead to the assumption that the beneficial effects of PT can be attributed to their purifying and antioxidant activities. Especially, PG studies have indicated that it has an electron-rich component that could function as a free radical stabilizer (Mahendran et al., 2021). Furthermore, its ability to activate UPS-related pathways, such as the Nrf2/ARE pathway, reinforces the possibility of acting as a potent antiaging and neuroprotective agent through UPS modulation.

8.

Phenolic compounds in seaweed and its antihypertensive effect

It has been reported that there is a positive effect in patients with high blood pressure (HBP), one of the main risk factors for cardiovascular diseases, when they have consumed wakame (Undaria pinnatifida) (Hata et al., 2001). In different experimental HBP models, it has been shown that PCs generate an

Chapter 14 Immune system: Inflammatory response

antihypertensive effect, where the presence of alginates and hydrolyzed peptides can contribute to these effects (Go´mezGuzma´n et al., 2018). Marine algae such as Undaria pinnatifida, Palmaria palmata, Porphyra columbina, and Porphyra yezoensis, among others, contain antioxidant PCs with an antihypertensive activity similar to that observed in PCs from terrestrial plants (Go´mez-Guzma´n et al., 2018). In this context, it has been shown that PCs can act on the angiotensin-1 enzyme, a metalloprotease (Zn) that catalyzes the conversion of angiotensin-1 to angiotensin-11, which is a potent vasoconstrictor involved in the pathogenesis of HBP. Among natural polyphenols, PTs stand out as potent ACE inhibitors by forming complexes with proteins or glycoproteins. Phlorofucofuroeckol A, dieckol and eckol in extracts of Ecklonia cava and Ecklonia stolonifera have shown an angiotensin inhibitory activity higher or equal to captopril (Go´mez-Guzma´n et al., 2018). A similar inhibitory activity against ACE has been observed with some PTs discovered in other species of marine algae such as Lomentaria catenata, Lithophyllum okamurae, Ahnfeltiopsis flabelliformis, and Fucus spiralis (Go´mez-Guzma´n et al., 2018). Although isolated PTs or PT-rich extracts show strong activity at room temperature (25–30°C), their activity decreases when they are exposed to higher temperatures (60°C), as in the case of phenolic extracts from Ulva rigida (Bourguiba et al., 2017).

9.

Phlorotannins trends

Fig. 1 represents a search of the most recent research (2015 to 2021) relating PTs and inflammation. Current research on PTs is focused on identifying these compounds in more species of marine algae, mainly brown algae, and relating oxidative stress with anti-inflammatory response and its consequent effect on diseases such as metabolic syndrome. Bioavailability of PTs on the human body and their effect on the microbiota are also highly active research topics.

10.

Conclusion

Marine organisms, mainly algae, store a large number of PCs that have similar or higher anti-inflammatory activity than those from terrestrial sources. Scientific evidence indicates that there is greater stability of these compounds and prevalence of their biological activity under normal environmental conditions; however, in extreme conditions, its activity can be significantly reduced.

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Fig. 1 Trends of investigation in PT from marine algae.

Nowadays, the application of seaweed PCs as functional ingredients for developing therapeutic products (preventive, palliative, and prophylactic) with beneficial human health effects has become viable and feasible. These facts, together with the growing consumer demand for products of natural origin, have triggered great interest in the food, nutraceutical, and pharmaceutical industries since they recognize algae as an ideal natural matrix

Chapter 14 Immune system: Inflammatory response

to extract PCs. In addition, seaweeds aquaculture is growing fast and becoming more optimized than cultivation of terrestrial plants, reducing costs and simplifying its production process.

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rez, L.; Jime
nez-Aspee, F.; Theoduloz, C.; SchmedaBurgos-Edwards, A.; Martı´n-Pe Hirschmann, G.; Larrosa, M. Anti-Inflammatory Effect of Polyphenols From Chilean Currants (Ribes magellanicum and R. punctatum) After In Vitro Gastrointestinal Digestion on Caco-2 Cells: Anti-Inflammatory Activity of In Vitro Digested Chilean Currants. J. Funct. Foods 2019, 59 (June), 329–336. https:// doi.org/10.1016/j.jff.2019.06.007. Cha, S. H.; Heo, S. J.; Jeon, Y. J.; Park, S. M. Dieckol, an Edible Seaweed Polyphenol, Retards Rotenone-Induced Neurotoxicity and α-Synuclein Aggregation in Human Dopaminergic Neuronal Cells. RSC Adv. 2016, 6 (111), 110040–110046. Chen, G. L.; Fan, M. X.; Wu, J. L.; Li, N.; Guo, M. Q. Antioxidant and AntiInflammatory Properties of Flavonoids From Lotus plumule. Food Chem. 2019, 277, 706–712. https://doi.org/10.1016/j.foodchem.2018.11.040. Cho, B. O.; Yin, H. H.; Park, S. H.; Byun, E. B.; Ha, H. Y.; Jang, S. I. Anti-Inflammatory Activity of Myricetin From Diospyros lotus Through Suppression of NF-κB and STAT1 Activation and Nrf2-Mediated HO-1 Induction in LipopolysaccharideStimulated RAW 264.7 Macrophages. Biosci. Biotechnol. Biochem. 2016, 80 (8), 1520–1530. https://doi.org/10.1080/09168451.2016.1171697. Cho, C. H.; Lee, C. J.; Kim, M. G.; Ryu, B.; Je, J. G.; Kim, Y.; Lee, S. H. Therapeutic potential of phlorotannin-rich Ecklonia cava extract on methylglyoxal-induced diabetic nephropathy in in vitro model. Mar. Drugs 2022, 20 (6), 355. https:// doi.org/10.3390/md20060355. Crobeddu, B.; Aragao-Santiago, L.; Bui, L. C.; Boland, S.; Squiban, A. B. Oxidative potential of particulate matter 2.5 as predictive indicator of cellular stress. Environ. Pollut. 2017, 230, 125–133. € ttner, A.; Dalrymple, A.; Ordon˜ez, P.; Thorne, D.; Walker, D.; Camacho, O. M.; Bu Meredith, C. Cigarette smoke induced genotoxicity and respiratory tract pathology: evidence to support reduced exposure time and animal numbers in tobacco product testing. Inhal. Toxicol. 2016, 28 (7), 324–338. De Souza, M. C.; Vieira, A. J.; Beserra, F. P.; Pellizzon, C. H.; No´brega, R. H.; Rozza, A. L. Gastroprotective Effect of Limonene in Rats: Influence on Oxidative Stress, Inflammation and Gene Expression. Phytomedicine 2019, 53, 37–42. https://doi.org/10.1016/j.phymed.2018.09.027.

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Di Giosia, P.; Giorgini, P.; Stamerra, C. A.; Petrarca, M.; Ferri, C.; Sahebkar, A. Gender Differences in Epidemiology, Pathophysiology, and Treatment of Hypertension. Curr. Atheroscler. Rep. 2018, 20 (3). https://doi.org/10.1007/s11883-018-0716-z. Farooqui, Z.; Mohammad, R. S.; Lokhandwala, M. F.; Banday, A. A. Nrf2 Inhibition Induces Oxidative Stress, Renal Inflammation and Hypertension in Mice. Clin. Exp. Hypertens. 2021, 43 (2), 175–180. https://doi.org/10.1080/10641963. 2020.1836191. Fernando, I. P. S.; Lee, W.; Ahn, G. Marine Algal Flavonoids and Phlorotannins; an Intriguing Frontier of Biofunctional Secondary Metabolites. Crit. Rev. Biotechnol. 2022, 42 (1), 23–45. Fiordelisi, A.; Piscitelli, P.; Trimarco, B.; Coscioni, E.; Iaccarino, G.; Sorriento, D. The mechanisms of air pollution and particulate matter in cardiovascular diseases. Heart Fail. Rev. 2017, 22 (3), 337–347. Go´mez-Guzma´n, M.; Rodrı´guez-Nogales, A.; Algieri, F.; Ga´lvez, J. Potential Role of Seaweed Polyphenols in Cardiovascular-Associated Disorders. Mar. Drugs 2018, 16 (8), 250. Gomez-Zavaglia, A.; Prieto Lage; Jimenez-Lopez, C.; Mejuto, J. C.; Simal-Gandara, J. The potential of seaweeds as a source of functional ingredients of prebiotic and antioxidant value. Antioxidants 2019, 8 (9), 406. Grosser, T.; Ricciotti, E.; FitzGerald, G. A. The Cardiovascular Pharmacology of Nonsteroidal Anti-Inflammatory Drugs. Trends Pharmacol. Sci. 2017, 38 (8), 733–748. https://doi.org/10.1016/j.tips.2017.05.008. Guzik, T. J.; Touyz, R. M. Oxidative Stress, Inflammation, and Vascular Aging in Hypertension. Hypertension 2017, 70 (4), 660–667. https://doi.org/10.1161/ HYPERTENSIONAHA.117.07802. Hall, J. E.; Mouton, A. J.; da Silva, A. A.; Omoto, A. C. M.; Wang, Z.; Li, X.; do Carmo, J. M. Obesity, Kidney Dysfunction, and Inflammation: Interactions in Hypertension. Cardiovasc. Res. 2021, 117 (8), 1859–1876. https://doi.org/ 10.1093/cvr/cvaa336. Hata, Y.; Nakajima, K.; Uchida, J.; Hidaka, H.; Nakano, T. Clinical Effects of Brown Seaweed, Undaria pinnatifida (Wakame), on Blood Pressure in Hypertensive Subjects. J. Clin. Biochem. Nutr. 2001, 30, 43–53. https://doi.org/10.3164/ jcbn.30.43. Heo, S.-J.; Hwang, J.-Y.; Choi, J.-I.; Lee, S.-H.; Park, P.-J.; Kang, D.-H.; Oh, C.; Kim, D.-W.; Han, J.-S.; Jeon, Y.-J.; Kim, H.-J.; Choi, I.-W. Protective Effect of Diphlorethohydroxycarmalol Isolated From Ishige Okamurae Against High GlucoseInduced-Oxidative Stress in Human Umbilical Vein Endothelial Cells. Food Chem. Toxicol. 2010, 48 (6). https://doi.org/10.1016/j.fct.2010.02.025. Heo, S. Y.; Jeong, M. S.; Lee, H. S.; Park, W. S.; Choi, I. W.; Yi, M.; Jung, W. K. Dieckol Induces Cell Cycle Arrest by Down-Regulating CDK 2/Cyclin E in Response to p21/p53 Activation in Human Tracheal Fibroblasts. Cell Biochem. Funct. 2022, 40 (1), 71–78. Herath, K. H. I. N. M.; Cho, J.; Kim, A.; Kim, H. S.; Han, E. J.; Kim, H. J.; Jee, Y. Differential modulation of immune response and cytokine profiles of Sargassum horneri ethanol extract in murine spleen with or without Concanavalin A stimulation. Biomed. Pharmacother. 2019, 110, 930–942. Herath, K. H. I. N. M.; Kim, H. J.; Mihindukulasooriya, S. P.; Kim, A.; Kim, H. J.; Jeon, Y. J.; Jee, Y. Sargassum horneri extract containing mojabanchromanol attenuates the particulate matter exacerbated allergic asthma through reduction of Th2 and Th17 response in mice. Environ. Pollut. 2020, 265. https:// doi.org/10.1016/j.envpol.2020.114094. Ighodaro, O. M.; Akinloye, O. A. First line defence antioxidantssuperoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): their fundamental role in the entire antioxidant defence grid. Alexandria J. Med. 2018, 54, 287–293.

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Jung, W.-K.; Choi, I.; Oh, S.; Park, S.-G.; Seo, S.-K.; Lee, S.-W.; Lee, D.-S.; Heo, S.-J.; Jeon, Y.-J.; Je, J.-Y.; Ahn, C.-B.; Kim, J. S.; Oh, K. S.; Kim, Y.-M.; Moon, C.; Choi, I.-W. Anti-Asthmatic Effect of Marine Red Alga (Laurencia undulata) Polyphenolic Extracts in a Murine Model of Asthma. Food Chem. Toxicol. 2009a, 47 (2). https://doi.org/10.1016/j.fct.2008.11.012. Jung, W.-K.; Heo, S.-J.; Jeon, Y.-J.; Lee, C.-M.; Park, Y.-M.; Byun, H.-G.; Choi, Y. H.; Park, S.-G.; Choi, I.-W. Inhibitory Effects and Molecular Mechanism of Dieckol Isolated From Marine Brown Alga on COX-2 and iNOS in Microglial Cells. J. Agric. Food Chem. 2009b, 57 (10). https://doi.org/10.1021/jf9003913. Jung, H. A.; Jin, S. E.; Ahn, B. R.; Lee, C. M.; Choi, J. S. Anti-Inflammatory Activity of Edible Brown Alga Eisenia bicyclis and Its Constituents Fucosterol and Phlorotannins in LPS-Stimulated RAW264.7 Macrophages. Food Chem. Toxicol. 2013, 59. https://doi.org/10.1016/j.fct.2013.05.061. Kaur, S.; Bansal, Y.; Kumar, R.; Bansal, G. A Panoramic Review of IL-6: Structure, Pathophysiological Roles and Inhibitors. Bioorg. Med. Chem. 2020, 28 (5), 115327. https://doi.org/10.1016/j.bmc.2020.115327. Kazłowska, K.; Hsu, T.; Hou, C.-C.; Yang, W.-C.; Tsai, G.-J. Anti-Inflammatory Properties of Phenolic Compounds and Crude Extract From Porphyra dentata. J. Ethnopharmacol. 2010, 128 (1). https://doi.org/10.1016/j.jep.2009.12.037. Kim, H. J.; Herath, K. H. I. N. M.; Dinh, D. T. T.; Kim, H. S.; Jeon, Y. J.; Kim, H. J.; Jee, Y. Sargassum Horneri Ethanol Extract Containing Polyphenols Attenuates PM-Induced Oxidative Stress Via ROS Scavenging and Transition Metal Chelation. J. Funct. Foods 2021, 79, 104401. Kojima-Yuasa, A. Biological and pharmacological effects of polyphenolic compounds from Ecklonia cava. In Polyphenols: Mechanisms of Action in Human Health and Disease; Academic Press, 2018; pp. 41–52. Lee, M.-S.; Kwon, M.-S.; Choi, J.-W.; Shin, T.; No, H. K.; Choi, J.-S.; Byun, D.-S.; Kim, J.-I.; Kim, H.-R. Anti-Inflammatory Activities of an Ethanol Extract of Ecklonia stolonifera in Lipopolysaccharide-Stimulated RAW 264.7 Murine Macrophage Cells. J. Agric. Food Chem. 2012, 60 (36). https://doi.org/10.1021/jf3022018. Lee, J. H.; Kim, H. J.; Jee, Y.; Jeon, Y. J.; Kim, H. J. Antioxidant potential of Sargassum horneri extract against urban particulate matter-induced oxidation. Food Sci. Biotechnol. 2020, 6, 855–865. https://doi.org/10.1016/j.jep.2019.112363. Li, W.; Ye, S.; Zhang, Z.; Tang, J.; Jin, H.; Huang, F.; Yang, Z.; Tang, Y.; Chen, Y.; Ding, G.; Yu, F. Purification and Characterization of a Novel Pentadecapeptide From Protein Hydrolysates of Cyclina sinensis and Its Immunomodulatory Effects on RAW264.7 Cells. Mar. Drugs 2019, 17 (1), 1–16. https://doi.org/ 10.3390/md17010030. Loperena, R.; et al. Hypertension and increased endothelial mechanical stretch promote monocyte differentiation and activation: roles of STAT3, interleukin 6 and hydrogen peroxide. Cardiovasc. Res. 2018, 114, 1547–1563. https://doi. org/10.1093/cvr/cvy112. Mahendran, S.; Maheswari, P.; Sasikala, V.; jaya Rubika, J.; Pandiarajan, J. In Vitro Antioxidant Study of Polyphenol From Red Seaweeds Dichotomously Branched gracilaria Gracilaria edulis and Robust Sea Moss Hypnea valentiae. Toxicol. Rep. 2021, 8, 1404–1411. Manzoor, Z.; Mathema, V. B.; Chae, D.; Kang, H.-K.; Yoo, E.-S.; Jeon, Y.-J.; Koh, Y.-S. Octaphlorethol A Inhibits the CpG-Induced Inflammatory Response by Attenuating the Mitogen-Activated Protein Kinase and NF-κB Pathways. Biosci. Biotechnol. Biochem. 2013, 77 (9). https://doi.org/10.1271/bbb.130299. Mishra, V.; Banga, J.; Silveyra, P. Oxidative stress and cellular pathways of asthma and inflammation: therapeutic strategies and pharmacological targets. Pharm. Therap. 2018, 181, 169–182. Orejudo, M.; Rodrigues-Diez, R. R.; Rodrigues-Diez, R.; Garcia-Redondo, A.; Santos-Sa´nchez, L.; Ra´ndez-Garbayo, J.; Cannata-Ortiz, P.; Ramos, A. M.;

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Effects of marine phenolics on diabetes, obesity, and metabolic syndrome

15

Esther Garcı´a-Dı´eza, Marı´a A´ngeles Martina,b, and Sonia Ramosa a

Department of Metabolism and Nutrition, Institute of Food Science and Technology and Nutrition (ICTAN-CSIC), Ciudad Universitaria, Madrid, Spain. b Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Instituto de Salud Carlos III (ISCIII), Madrid, Spain

Abbreviations ACC ACS1 ADP A-FABP AGE AKT ALP ALT AMPK aP2 ASCL1 AST AT ATGL ATP BAT Bax Bcl-2 Bcl-xL BD BMI BMP4

acetyl-CoA carboxylase acetyl-CoA synthetase adenosine diphosphate adipocyte fatty acid-binding protein advanced glycation end products protein kinase B alkaline phosphatase aspartate aminotransferase AMP-activated protein kinase adipocyte protein 2 achaete-scute homolog 1 alanine aminotransferase adipose tissue adipose triglyceride lipase adenosine triphosphate brown adipose tissue Bcl-2-associated X protein b-cell lymphoma 2 B-cell lymphoma-extra large bromophenol body mass index bone morphogenetic protein 4

Marine Phenolic Compounds: Science and Engineering. https://doi.org/10.1016/B978-0-12-823589-8.00016-9 Copyright # 2023 Elsevier Inc. All rights reserved.

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BP BUN BW CAT CD C/EBP CHOP Cho Cidea COX-2 CRE CPT1 CRP DGAT DM DPHC eIF2α ERK FABP FAS FATP1 F/B FFA FIS1 GK GLUT GM GOT G6Pase GPT GPx GSH HbA1c HDL HFD HMGB1 HMGCoA HO-1 HOMA-β HOMAIR HSL ICAM

blood pressure blood urea nitrogen body weight catalase cluster of differentiation CCAAT/enhancer-binding protein CCAAT-enhancer-binding protein homologous protein cholesterol cell death-inducing DNA fragmentation factor-like effector A cyclooxygenase 2 creatinine carnitine palmitoyltransferase C-reactive protein diacylglycerol acyltransferase diabetes mellitus diphlorethohydroxycarmalol eukaryotic initiation factor 2 alpha extracellular signal-regulated kinase fatty acid-binding protein fatty acid synthase fatty acid transport protein-1 Firmicutes/Bacteroidetes ratio free fatty acid mitochondrial fission 1 protein glucokinase glucose transporter gut microbiota glutamic oxaloacetic transaminase glucose 6 phosphatase glutamic pyruvic transaminase glutathione peroxidase glutathione glycated hemoglobin high-density lipoprotein high-fat diet high-mobility group box 1 3-hydroxyl-methyl glutaryl coenzyme A reductase heme oxygenase-1 homeostatic model assessment of beta cell homeostatic model assessment of insulin resistance hormone-sensitive lipase intercellular adhesion molecule

Chapter 15 Effects of marine phenolics on diabetes, obesity, and metabolic syndrome

IL IR IRE1 IRS JNK KLF4 LAMP2 LC3 LDL LNE LPAAT LPL LPS MCP-1 MDA MetS MGL MNF1 NF-κB iNOS ObR PDK4 PEPCK PERK PGD2 PI3K PKA PRDM16 PPAR Pref-1 PTP1B RAGE Rb RCDB ROS S100β SIRT-1 SOC3 SOD SREBP1 STAT STZ Tbx1

interleukin insulin resistance inositol-requiring enzyme 1 insulin receptor substrate c-jun N-terminal kinase Kr€ uppel-like factor lysosome-associated membrane protein 2 microtubule-associated proteins 1A/1B light chain 3B low-density lipoprotein polyphenolic extract from Lessonia nigrescens lysophosphatidic acid acyltransferase lipoprotein lipase lipopolysaccharide monocyte chemoattractant protein-1 malondialdehyde metabolic syndrome monoacylglycerol lipase mitochondrial nucleoid factor 1 nuclear factor kappa B inducible nitric oxide synthase long form of the leptin receptor pyruvate dehydrogenase lipoamide kinase isozyme 4 phosphoenolpyruvate carboxykinase PKR-like ER kinase prostaglandin D2 phosphoinositide 3-kinase protein kinase A PR domain-containing 16 peroxisome proliferator-activated receptor preadipocyte factor-1 protein tyrosine phosphatase 1B receptor for advanced glycation end products retinoblastoma protein randomized controlled double blind reactive oxygen species S100 calcium-binding protein sirtuin-1 suppressor of cytokine signaling 3 superoxide dismutase sterol regulatory transcription factor 1 signal transducer and activator of transcription streptozotocin T-box 1

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T-Cho T2DM TG TLR Tmem26 TNF UCP-1 WAT WC WHO Xbp1 ZO-1

1.

total cholesterol type 2 diabetes mellitus triglycerides toll-like receptor transmembrane protein 26 tumor-necrosis factor uncoupling protein-1 white adipose tissue waist circumference World Health Organization X-box-binding protein 1 zonula occludens-1

Introduction

The prevalence of metabolic diseases is reaching pandemic levels at present, especially in developed countries (Saklayen, 2018; WHO, 2021a, 2021b). Actually, pathologies such as type 2 diabetes mellitus (T2DM), obesity, and metabolic syndrome (MetS) are the most common chronic diseases in almost all countries and constitute a worldwide health burden (Saklayen, 2018; WHO, 2021a, 2021b). This high incidence has been associated with the current lifestyle, characterized by sedentarism and unbalanced diets. In this regard, dietary interventions are recognized as the most efficient approach for preventing or delaying diabetes, obesity, and metabolic syndrome. The pathogenesis of T2DM, obesity, and MetS is very different, but dysregulation of key metabolic signaling pathways (i.e., lipid and glucose homeostasis, insulin route, etc.), as well as enhanced oxidative stress and inflammation, is present in all these diseases (Ghaben and Scherer, 2019; Hurrle and Hsu, 2017; Martı´n et al., 2016; Martı´n and Ramos, 2021; Van Treuren and Dodd, 2020). In addition, recently the alteration of the gut microbiota (GM) has also been highlighted because of its potential contribution to these pathologies. Under these conditions, seaweeds, which are rich in phenolic compounds, are attracting the interest of scientists because of their potential beneficial effects against different noncommunicable chronic diseases, including T2DM, obesity, and MetS (Mateos et al., 2020). Indeed, epidemiological studies have shown that countries, where seaweeds are regularly consumed, have a significantly lower incidence of obesity and dietary-related diseases (Shannon and Abu-Ghannam, 2019). In this chapter, an overview of the chemopreventive activity of seaweed phenolic compounds with special emphasis on the molecular actions of these components on key signaling pathways

Chapter 15 Effects of marine phenolics on diabetes, obesity, and metabolic syndrome

in T2DM, obesity, and MetS is provided. In addition, the scarce evidence existing on the link between these natural compounds and the mentioned metabolic diseases based on human clinical trials is described.

2.

Pathophysiology of diabetes

Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia as a consequence of defects in insulin secretion and/or insulin action (Kharroubi and Darwish, 2015). According to World Health Organization (WHO), DM can be considered an epidemy due to the high number of people affected by this disease (diagnosed and undiagnosed) and the number of deaths caused (WHO, 2021c). In 2014, there were 422 million cases in the world, four times more than in 1980 (WHO, 2021a), and it is supposed to continue rising to 578 million by 2030 and 700 million by 2045 (Saeedi et al., 2019). Four types of DM have been described by American Diabetes Association; however, T2DM is the most common pathology among these diseases because it represents 90%–95% of the cases (Association AD, 2021). Many factors increase the risk of developing T2DM, such as age, sedentary lifestyle, unbalanced diet, smoking, gestational diabetes mellitus, genetics, and obesity, especially metabolically unhealthy obesity, the strongest risk factor (Association AD, 2021; Bellou et al., 2018; Gbadamosi and Tlou, 2020). In addition, it is worth to mention that this disease is commonly associated with several complications such as heart failure, hypertension, retinopathy, infections, nephropathy, etc. (Forbes and Cooper, 2013). However, many cases of T2DM could be prevented with lifestyle changes, including maintaining healthy body weight, eating a healthy balanced diet, and staying physically active (Asif, 2014). T2DM causes elevated blood glucose levels resulting from tissue insulin resistance (IR) and insulin-producing pancreatic β-cell dysfunction. During IR, β-cells maintain glucose homeostasis by increasing the insulin output and only when cells cannot release sufficient hormones to compensate this imbalance, the glucose concentration rises, leading to dysfunction of β-cells and/or death (Cerf, 2013). Both mechanisms contribute to the T2DM pathogenesis, but the β-cell damage usually results from chronic IR (Cerf, 2013; Galicia-Garcia et al., 2020). Indeed, all these complex and interconnected mechanisms related to IR and β-cell injury are also responsible for the damaging effects in other tissues, the so-called complications associated with T2DM, namely cardiovascular

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disease, nephropathy, neuropathy, retinopathy, blindness, infections, etc. (Forbes and Cooper, 2013). The main function of insulin is to regulate blood glucose levels promoting hexose transport into target cells, playing a major role in the energetic metabolism ( Johnson and Olefsky, 2013). Insulin is a hormone controlled by multiple factors, being glucose metabolism the most important aspect (Fu et al., 2013). Under physiological conditions, glucose is internalized into β-cells through the glucose transporter-2 (GLUT-2). Then, the glucose catabolism increases the adenosine triphosphate (ATP)/adenosine diphosphate (ADP) ratio, and the intracellular calcium concentration is enhanced due to the membrane depolarization through the involvement of ATP-sensitive K+ channels and Ca2+ channels. Next, the granules of insulin fusion with the membrane and the hormone pass into the plasma to exert its activity on the target tissues. At molecular level, insulin binds to its receptor (insulin receptor), which belongs to the superfamily of receptor tyrosine kinases, and induces the Tyrphosphorylation of the insulin receptor substrates (IRS)-1 and -2 and the phosphatidylinositol-3 kinase (PI3K) pathway activation. The stimulation of this route mostly controls metabolic functions, i.e., stimulation of GLUT-4 translocation, glycogen and protein synthesis, and adipogenesis (Fu et al., 2013; Johnson and Olefsky, 2013; Rachdaoui, 2020). During T2DM, chronic hyperglycemic exposure promotes IR and compromises β-cell survival (Galicia-Garcia et al., 2020). In line with this, β-cell dysfunction impairs the calcium mobilization, which in turn is involved in the insulin secretion, the insulin gene expression is decreased, apoptotic signals are stimulated, and the pro-inflammatory state, already present in IR, is aggravated (Galicia-Garcia et al., 2020). In addition, sustained hyperglycemia promotes IR, which refers to a decrease in the metabolic response of insulin-sensitive tissues, mainly the liver, skeletal muscle, and adipose tissues. Different molecular alterations can contribute to IR, such as a decrease in the number of insulin receptors or their activity, increased Ser/Thr-phosphorylated IRS or insulin receptor levels with a diminution in values of those Tyr phosphorylated, an enhancement in the Tyr-phosphatase activity, an increment in the IRS degradation, or defects in both expression and function of GLUT (Fu et al., 2013; Johnson and Olefsky, 2013; Rachdaoui, 2020). Indeed, GLUT-4, responsible for the transport of glucose into the peripheral tissues, diminishes its translocation into the membrane during IR (Fu et al., 2013; Hurrle and Hsu, 2017). Furthermore, some signaling pathways are also altered due to the oxidative stress provoked by the

Chapter 15 Effects of marine phenolics on diabetes, obesity, and metabolic syndrome

hyperglycemia, and these alterations can also provoke IR. Indeed, during T2DM, the glucotoxicity and lipotoxicity conditions promote the oxidative stress leading to an increased reactive oxygen species (ROS) intracellular generation (Fu et al., 2013; GaliciaGarcia et al., 2020), being β-cells especially vulnerable to the enhanced levels of ROS because of their low intrinsic level of antioxidant enzymes (Lenzen, 2008). Elevated ROS production also inhibits the insulin signaling in peripheral tissues, such as skeletal muscle and adipose tissues (Hurrle and Hsu, 2017). In the liver, which is also involved in glucose and lipid metabolism, insulin promotes glycogen synthesis and lipogenesis and inhibits glucose production. However, during IR conditions, the hepatic metabolism is not well-regulated and therefore, excessive glucose and lipid production occurs (Santoleri and Titchenell, 2019). In addition, in T2DM, the pro-inflammatory state, already present in IR, is aggravated (Galicia-Garcia et al., 2020), and GM alterations have also been reported (Van Treuren and Dodd, 2020).

3.

Antidiabetic effects of marine phenols

Marine algae are known to be rich in important secondary bioactive metabolites, which can effectively be used to manage various metabolic diseases, including T2DM (Nova et al., 2020). Among these components, phenolics such as bromophenolic compounds, phenolic acids, and flavonoids, as well as phlorotannins are gaining recognition for their reported antidiabetic activity (Murray et al., 2018). There is accumulating evidence, from both in vitro and animal models, that phenolic compounds present in marine algae may attenuate postprandial glycemic responses and fasting hyperglycemia and improve insulin secretion and sensitivity in T2DM. The likely mechanisms involved in these actions include inhibition of starch-digesting enzymes α-glucosidase and α-amylase, an increase of insulin release from the pancreatic β-cells, inhibition of hepatic glucose output, an increase of glucose uptake in the insulin-sensitive tissues, and modulation of GM (Table 1).

3.1

Effect of marine phenolics on postprandial glycemia and glucose levels

Postprandial hyperglycemia plays an important role in the development of T2DM and its complications associated, being its tight control essential in the treatment of diabetes and the prevention of its cardiovascular complications (Inzucchi et al., 2015). Accordingly, one of the therapeutic approaches to ameliorate

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Chapter 15 Effects of marine phenolics on diabetes, obesity, and metabolic syndrome

Table 1 Antidiabetic activity of marine phenolicsa. Cell/Animal model

Treatment

Main outcomes

Reference

KK-Ay type 2 diabetic mice

High polyphenol extract from Ecklonia stolonifera (1%) daily for 4 weeks

Iwai (2008)

STZ-induced type 2 diabetic mice

Acute administration of diphlorethohydroxycarmalol from Ishige okamurae (100 mg/kg body weight) together with starch (2 g/kg body weight) Acute administration of dieckol from Ecklonia cava (100 mg/kg body weight) together with starch (2 g/kg body weight) Acute administration of phlorofucofuroeckol A from Ecklonia cava (10 mg/kg body weight) together with starch (2 g/kg body weight) Acute administration of 2,700 phloroglucinol-6,60 -bieckolc from Ecklonia cava (10 mg/kg body weight) together with starch (2 g/kg body weight) Dieckol-rich extract from Ecklonia cava (690 mg of polyphenols) daily for 12 weeks 45–60 years, 73 adults (prediabetics)

# Plasma glucose and insulin " glucose tolerance # lipid peroxidation in plasma and liver # Postprandial blood glucose level

# Postprandial blood glucose level

Lee et al. (2010)

# Postprandial blood glucose level

You et al. (2015)

# Postprandial blood glucose level

Lee et al. (2017)

# Postprandial blood glucose level # Plasma insulin

Lee and Jeon (2015)

# Lipid peroxidation, # ROS, " CAT, " SOD, "GPx # Bax, "Bcl-2, and " caspase-3 expression # Lipid peroxidation, # ROS, " CAT, " SOD, "GPx # Bax, "Bcl-xL, and " caspase-3 expression

Lee et al. (2012a)

Glucose levels

STZ-induced type 2 diabetic mice

STZ-induced type 2 diabetic mice

STZ-induced type 2 diabetic mice

RCDB, placebocontrolled

Heo et al. (2009)

b-cell function

Rat INS-1 b-cells

Rat pancreatic RINm5F b-cells

INS-1 b-cells were preincubated with glucose (30 mM) for 48 h and then incubated with dieckol (35 or 70 mM) for 24 h RINm5F b-cells were treated for 3 h with octaphlorethol A (12.5 mg/mL or 50 mg/mL) followed by incubation with 10 mM STZ for 24 h.

Lee et al. (2013)

Chapter 15 Effects of marine phenolics on diabetes, obesity, and metabolic syndrome

439

Table 1 Antidiabetic activity of marine phenolics—cont’d Cell/Animal model

Treatment

Main outcomes

Reference

STZ-induced type 1 diabetic rats

Ecklonia cava extracts (3 or 5%) daily for 4 weeks

Kim and Kim (2012)

STZ-induced type 2 diabetic rats

Sargassum polycystum extracts (150 or 300 mg/kg body weight) daily for 22 days Sargassum hystrix extracts (200, 300, and 400 mg/kg body weight) daily for 15 days

# Plasma glucose, "plasma insulin " glucose tolerance # TG, # LDL, " HDL # Plasma glucose # pancreas, liver, and renal damage # Plasma glucose # TG, no effect on cholesterol, LDL, and HDL # Pancreas damage # Plasma glucose, #HOMA-IR, " HOMA- b, ¼plasma insulin, total cholesterol and HDL, # pancreas damage

Gotama et al. (2018)

STZ-induced type 2 diabetic rats

STZ-induced type 2 diabetic rats

Sargassum oligocystum extracts (150 and 300 mg/kg body weight) daily for 30 days

Motshakeri et al. (2014)

Akbarzadeh et al. (2018)

Insulin sensitivity

C57BL/-KsJ-db/ db type 2 diabetic mice

Ishige okamurae extract (0.5%) daily for 6 weeks

C57BL/-KsJ-db/ db type 2 diabetic mice

Dieckol rich extract from Ecklonia cava (0.5%) daily for 6 weeks

C57BL/-KsJ-db/ db type 2 diabetic mice

Octaphlorethol A rich extract from Ishige foliacea (5 mg/kg body weight) daily for 5 weeks

High-fat/highsucrose diet and STZ-induced type 2 diabetic mice

Flavonoid-rich extract from Enteromorpha prolifera (150 mg/kg body weight) daily for 4 weeks

STZ-induced type 1 diabetic rats and C2C12 skeletal muscle cells

Polyphenol-rich extract from Ecklonia cava (300 mg/kg body weight) daily for 3 weeks to diabetic rats Polyphenol-rich extract from Ecklonia cava (50–300 mg/mL) for 1 h to C2C12 skeletal muscle cells

# Plasma glucose, #HbA1c, " glucose tolerance, # HOMA-IR, " GK, # G6Pase, # PEPCK, " glycogen level in liver # Plasma glucose, #HbA1c, " glucose tolerance, # HOMA-IR, # TG, " HDL, #FFA, #cholesterol, " GK, # G6Pase, # PEPCK, and " glycogen level in liver # Plasma glucose, "glucose tolerance, # HOMA-IR, #G6Pase, and # PEPCK in liver; " AMPK, " AKT, and " GLUT-4 in muscle # Plasma glucose, "glucose tolerance # liver and renal damage " IRS1, " PI3K, "AKT and #JNK1/2 expression in liver # Plasma glucose; "plasma insulin, # pancreas damage in diabetic rats " AMPK and " AKT in C2C12 skeletal muscle cells

Min et al. (2011)

Lee et al. (2012b)

Lee et al. (2016)

Yan et al. (2019)

Kang et al. (2010)

Continued

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Chapter 15 Effects of marine phenolics on diabetes, obesity, and metabolic syndrome

Table 1 Antidiabetic activity of marine phenolics—cont’d Cell/Animal model

Treatment

Main outcomes

Reference

C57BL/-KsJ-db/ db type 2 diabetic mice Alloxan-induced diabetic zebrafish

Dieckol-rich extract from Ecklonia cava (10 and 20 mg/kg body weight) daily for 14 days Dieckol-rich extract from Ecklonia cava (1 mg per g body weight) during 90 min

Kang et al. (2013)

Rat myoblast L6 cells

Differentiated L6 cells were treated for 2 h with octaphlorethol A-rich extract from Ishige foliacea (6.25, 12.5, 25, and 50 mM)

# Plasma glucose, #plasma insulin, " CAT "SOD, " GPx in liver " AMPK and " AKT in skeletal muscle # Plasma glucose # G6Pase and #PEPCK in liver " AKT but not AMPK in skeletal muscle " Glucose uptake " GLUT-4 translocation " IRS1, " AKT, and " AMPK

# Plasma glucose, "glucose tolerance, " PI3K, and # JNK expression in liver " Barnesiella and # Clostridium and Alistipes # Plasma glucose, #HOMA-IR, # TG, # LDL, " CAT, " SOD, " GSH, # liver damage " Odoribacter, " Muribaculum, and " Parabacteroides # Plasma glucose, "glucose tolerance, " PI3K and # JNK expression in liver, # liver damage " Akkermansia, # Turicibacter, # Alistipes # Plasma glucose, "glucose tolerance, " PI3K, " AKT, and # JNK1/2 expression in liver " Alisties, " Lachnospiraceae, " Odoribacter, # Akkermansia

Zhao et al. (2018)

Kim et al. (2016)

Lee et al. (2012c)

Gut microbiota

High-fat/highsucrose diet and STZ-induced type 2 diabetic mice

Polyphenol-rich extracts from Lessonia nigrescens (75, 150, and 300 mg/kg body weight) daily for 4 weeks

High-fat diet and STZ-induced type 2 diabetic rats

Polyphenol-rich extracts from Lessonia trabeculate (200 mg/kg body weight) daily for 4 weeks

High-fat/highsucrose diet and STZ-induced diabetic mice

Ethanol extract from Enteromorpha prolifera (300 mg/kg body weight) daily for 28 days

High-fat/highsucrose diet and STZ-induced diabetic mice

Water-ethanol extract from Enteromorpha prolifera (300 mg/kg body weight) daily for 28 days

Yuan et al. (2019)

Lin et al. (2018)

Yan et al. (2019)

AKT, protein kinase B; AMPK, AMP-activated protein kinase; Bax, Bcl-2-associated X protein; Bcl-2, b-cell lymphoma 2; Bcl-xL, B-cell lymphoma-extra large; CAT, catalase; FFA, free fatty acids, GK, glucokinase; GLUT, glucose transporter; GPx, glutathione peroxidase; G6Pase, glucose 6 phosphatase; GSH, glutathione; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; HOMA-b, homeostatic model assessment of beta-cell; HOMA-IR, homeostatic model assessment of insulin resistance; IRS, insulin receptor substrate; JNK, c-jun N-terminal kinase; LDL, low-density lipoprotein; PEPCK, phosphoenolpyruvate carboxykinase; PI3K, phosphoinositide 3-kinase; RCDB, randomized controlled double blind; ROS, reactive oxygen species; SOD, superoxide dismutase; STZ, streptozotocin; TG, triglycerides. a The arrow indicates an increase (") or decrease (#) in the levels of the different parameters analyzed.

Chapter 15 Effects of marine phenolics on diabetes, obesity, and metabolic syndrome

postprandial hyperglycemia involves the retardation of glucose absorption following a meal. Dietary saccharide digestion in the small intestine largely occurs via the action of two key enzymes, α-amylase and α-glucosidase. Both enzymes hydrolyze carbohydrates into monosaccharides that are absorbed into the bloodstream, resulting in an elevation of blood glucose concentration (Murugan et al., 2015). Inhibition of both α-amylase and α-glucosidase activities may greatly reduce postprandial hyperglycemia following carbohydrate intake and could be an effective strategy for the control of glucose levels in diabetics. In this sense, one of the most well-known properties of marine phenolics on carbohydrate metabolism is the inhibition of these carbohydrate enzyme activities. Recently, very few reviews have summarized the numerous in vitro studies indicating the ability of seaweed extracts to inhibit α-glucosidase and/or α-amylase (Bermano et al., 2020; Erpel et al., 2020; Gunathilaka et al., 2020; Mateos et al., 2020). For instance, phlorotannins extracted from brown seaweeds such as Ascophyllum nodosum (Lordan et al., 2013), Ecklonia cava (Lee et al., 2009), Laminaria digitata (Zaharudin et al., 2018), Undaria pinnatifida (Zaharudin et al., 2019), Sargassum serratifolium (Ali et al., 2017), Alaria marginata, and Fucus distichus (Kellogg et al., 2014) have demonstrated to inhibit α-glucosidase and/or α-amylase activities. Importantly, the concentration ranges were similar to acarbose, the main clinically glucosidase inhibitor drug utilized to reduce postprandial glucose. Likewise, a small number of studies have also demonstrated an inhibitory enzymatic activity in extracts from red (Eucheuma denticulatum) and green (Halimeda macroloba) seaweeds (Balasubramaniam et al., 2016; Chin et al., 2015). More importantly, this antidiabetic activity has been confirmed in few in vivo studies performed with type 2 diabetic animal models. Thus, a high polyphenol extract obtained from the brown algae Ecklonia stolonifera showed strong inhibition of α-glucosidase in vitro, and the ingestion of the extract to non-insulin-dependent diabetic mice moderated the elevation of plasma glucose levels after the oral administration of maltose (Iwai, 2008). Similarly, the phlorotannin diphlorethohydroxycarmalol isolated from the brown algae Ishige okamurae exerted a profound inhibitory effect on α-glucosidase and α-amylase enzymes and delayed the absorption of dietary carbohydrates in the intestine of streptozotocin (STZ)-induced diabetic mice, resulting in the suppression of increased blood glucose levels after a meal in diabetic mice (Heo et al., 2009). Similar results were found with the phlorotannins dieckol (Lee et al., 2010), phlorofucofuroeckol A (You et al., 2015), and 2,700 -phloroglucinol-6,60 -bieckol (Lee et al., 2017),

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being all of their constituents from the brown algae Ecklonia cava. The administration of these phlorotannins together with orally soluble starch significantly delayed the absorption of carbohydrates and suppressed the postprandial hyperglycemia in STZ-induced diabetic mice. Altogether, these results indicate that marine phenolics may inhibit the action of carbohydratedigesting enzymes in vitro and reduce postprandial hyperglycemia in animals, providing an effective antidiabetic option to prevent the development or progression of T2DM. However, to date, there is no human intervention examining the effect of marine polyphenols on postprandial glycemia in diabetic patients. Only one study has evaluated this effect in 73 prediabetic adults with high fasting blood glucose levels (Lee and Jeon, 2015). In this sense, supplementation with a dieckol-rich extract of Ecklonia cava (690 mg of polyphenols) for 12 weeks significantly improved the postprandial blood glucose in prediabetic individuals.

3.2

Effect of marine phenolics on b-cell function

Pancreatic β-cell survival and function play a key role in the pathogenesis of T2DM; therefore, both improving β-cell function and increasing β-cell mass are important for preventing and delaying the progression of T2DM. Although the exact mechanism underlying β-cell destruction remains unknown, it has been suggested that the oxidative stress induced by hyperglycemia is one of the major factors contributing to β-cell death in diabetes (Yaribeygi et al., 2020). In this sense, the antioxidant properties of marine phenolics can help to preserve β-cells from the damaging effects of hyperglycemia, thus improving glucose homeostasis in diabetes. Accordingly, it has been shown that dieckol, the main phlorotannin from Ecklonia cava, was able to protect rat insulinoma INS-1 β-cells against high glucose conditions by reducing ROS-induced oxidative stress and apoptosis (Lee et al., 2012a). Pretreatment of pancreatic INS-1 β-cells with dieckol resulted in increased expression levels and activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) that were associated with reduced ROS generation and enhanced β-cell survival. Similar results were found with the phenolic compound octaphlorethol A isolated from the marine algae Ishige foliacea (Lee et al., 2013). Pretreatment of rat insulinoma RINm5F pancreatic β-cells with 12.5 μg/mL or 50 μg/mL of octaphlorethol A increased the activities of antioxidant enzymes, preventing thus the enhanced generation of ROS

Chapter 15 Effects of marine phenolics on diabetes, obesity, and metabolic syndrome

induced by STZ treatment. Once again, the phlorotannin pretreatment protected pancreatic β-cells against apoptosis under STZtreated conditions, and it was associated with an increased antiapoptotic B-cell lymphoma-extra large (Bcl-xL) expression and reduced both proapoptotic Bax and cleaved caspase-3 expressions. More importantly, this pancreatic protective effect has been confirmed in animal models of diabetes. Thus, the daily supplementation with Ecklonia cava extracts (3 or 5%) to type 1 STZinduced diabetic rats prevented the loss of β-cell mas induced by STZ and stimulated insulin secretion by the remaining β-cells (Kim and Kim, 2012). As a result, the treatment with the phenolic extract significantly ameliorated hyperglycemia in diabetic animals. Likewise, in STZ-induced type 2 diabetic rats, the supplementation with extracts from the seaweed Sargassum polycystum (150 or 300 mg/kg bw) (Motshakeri et al., 2014) or Sargassum hystrix (200, 300, and 400 mg/kg bw) (Gotama et al., 2018) induced a hypoglycemic effect that was closely associated with the prevention of the pathological damage found in the pancreatic islets of diabetic animals. More recently, Akbarzadeh et al. (2018) have also found that STZ-induced diabetic rats supplemented with an extract from Sargassum oligocystum (150 and 300 mg/kg bw) reduced the fasting blood glucose concentration and regenerated damaged pancreatic β-cells. Overall, these outcomes strongly indicate the positive impact of marine phenolic extracts against glucotoxicity and support their use to protect pancreatic β-cells against the damage caused by the oxidative stress associated with diabetes.

3.3

Effects of marine phenolics on insulin sensitivity

IR is a condition characterized by impaired insulin response of target tissues, causing a state of transient and unpredictable hyperglycemia and hyperinsulinemia (Samuel and Shulman, 2012). The liver plays a major role in maintaining the balance of glucose homeostasis, via adjusting the equilibrium among glycogenesis and glycolysis of stored glycogen, as mentioned (Santoleri and Titchenell, 2019). Thus, IR results in excessive glucose production in the liver, contributing to fasting and postprandial hyperglycemia. In this sense, it has been described that dietary supplementation with a polyphenol-rich methanol extract of Ishige okamurae (Min et al., 2011), a dieckol extract from Ecklonia cava (Lee et al., 2012b) or the phlorotannin octaphlorethol A isolated from Ishige foliacea (Lee et al., 2016) significantly suppressed the increase in the fasting blood glucose level of db/db

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diabetic mice. This hypoglycemic effect was closely related to changes in the hepatic glucose metabolism such as the enhancement of glucokinase (GK) activity and the reduction of glucose-6phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) activities, leading to an increased hepatic glycogen level. More recently, Yan et al. (2019) have shown that the administration of a flavonoid-rich extract from Enteromorpha prolifera was able to regulate the glucose metabolism in the liver of type 2 diabetic mice via the activation of IRS1/PI3K/protein kinase B (AKT) pathway and the inhibition of c-Jun N-terminal kinase (JNK)1/2. These findings strongly support that marine phenolics may alleviate IR in diabetic mice by improving glucose metabolism and hepatic glucose uptake in the liver through the insulin signaling pathway. Skeletal muscle is the major tissue for glucose uptake and disposal, accounting for approximately 70% of glucose uptake from blood; hence, it plays an important role in energy balance regulation (Evans et al., 2019). In the skeletal muscle, IR leads to a decrease in glucose uptake and utilization (mainly in glycogen synthesis) and is a major risk factor for T2DM. Kang et al. (2010) found that dietary supplementation with a polyphenolrich extract from the brown alga Ecklonia cava reduced plasma glucose levels and increased insulin concentration in STZinduced type 1 diabetic rats. The same polyphenol-rich extract was able to activate both AMP-activated protein kinase (AMPK) and PI3K/AKT signaling in C2C12 skeletal muscle cells, suggesting that the mechanism of action seems to be mediated by the activation of both signaling pathways. Likewise, the hypoglycemic effect of purified dieckol from Ecklonia cava in db/db type 2 diabetic mice was strongly associated with increased phosphorylated levels of AMPK and AKT in the muscle tissues of these animals (Kang et al., 2013). Similar results were found in a zebrafish model of alloxan-induced diabetes in which dieckol attenuated blood glucose levels via AKT activation in the skeletal muscle (Kim et al., 2016). Interestingly, the activation of PI3K/AKT and AMPK induced by the phenolic octaphlorethol A isolated from Ishige foliacea has been related to increased GLUT-4-mediated glucose uptake and insulin sensitivity in both L6 rat muscle cells (Lee et al., 2012c) and in the muscles of type 2 diabetic mice (Lee et al., 2016). In addition, a number of in vitro studies (Zhou et al., 2017) have shown the inhibitory activity of several marine phenolics against protein tyrosine phosphatase 1B (PTP1B), the major negative regulator of insulin signaling. However, at present, there is no study showing this effect in in vivo models of diabetes.

Chapter 15 Effects of marine phenolics on diabetes, obesity, and metabolic syndrome

3.4

Effect of marine phenolics on gut microbiota in diabetes

During the last years, GM has emerged as a key regulator of human health and disease (Van Treuren and Dodd, 2020). GM plays a significant role in many specific functions, including the maintenance of the gut mucosal barrier, development of the immune system, and production of several nutrient-derived metabolites with the potential to affect human metabolic function (Martı´n and Ramos, 2021). As a result, the alteration of the microbial composition (known as dysbiosis) may lead to the development of many pathologies, including metabolic diseases such as T2DM (Scheithauer et al., 2020). Even though microbiota composition can be influenced by numerous external factors, several studies have demonstrated the strong influence of diet and dietary components on the structure and function of GM and their effects on health (Ramos and Martı´n, 2021). In this sense, functional compounds from marine algae have been shown to regulate the balance of GM and improve glucose metabolism (Bermano et al., 2020). The ability of bioactive compounds from seaweed to affect GM has been tested in both animals and humans; however, only few studies have examined marine phenolic-induced GM changes in diabetes. Zhao et al. (2018) investigated the antidiabetic effect of a polyphenolic extract from Lessonia nigrescens (LNE) in type 2 diabetic mice and its relationship with GM modulation. The supplementation with the extract promoted the glucose uptake through PI3K activation and JNK inhibition in the liver, thus reducing hyperglycemia. Notably, LNE treatment regulated intestinal microbiota in mice by enriching the amount of beneficial bacteria Barnesiella (Bacteroidetes phylum) and reducing the abundance of Clostridium species (Firmicutes phylum), which are negatively correlated with fasting glucose, insulin, and plasma triglycerides (TG). In addition, the supplementation of a high-fat diet (HFD) and STZ-induced diabetic rats with a polyphenol extract from Lessonia trabeculata for 4 weeks lowered the fasting blood glucose and positively affected the dysbiosis regulation of the microbial ecology (Yuan et al., 2019). After feeding polyphenol extracts, the abundance of Odoribacter (butyrateproducing bacteria) and Muribaculum (related with reduced insulin resistance) genera were increased in diabetic animals. Similar results were found with polyphenol-rich extracts from Enteromorpha prolifera that significantly decreased the fasting blood glucose and improved the fasting glucose tolerance in high-fat/high-sucrose diet and STZ-induced diabetic mice (Lin et al., 2018; Yan et al., 2019). Specifically, the supplementation

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with an ethanol extract from E. prolifera significantly increased the relative abundance of Akkermansia, which correlated with ^ et al., 2016) improved body weight and glucose tolerance (Anhe and decreased proportion of Alistipes and Turicibacter (Lin et al., 2018). Likewise, the treatment with a water-ethanol extract from E. prolifera significantly enriched the abundance of Lachnospiraceae and Alisties, which were positively correlated with the release of intestinal hormones for regulating insulin release (Yan et al., 2019). In summary, experimental data indicate that seaweed consumption may have beneficial effects on T2DM by modulating glucose homeostasis, insulin sensitivity, β-cell function, and GM composition. However, more research is required, especially in humans, to clearly demonstrate the boundaries of algae against this disease.

4.

Pathophysiology of obesity

Obesity is a multifactorial and chronic disease defined by WHO as an excess or abnormal fat accumulation in the white adipose tissue (WAT) and in important peripheral organs and tissues resulting in an impairment of health (Ali et al., 2014; WHO, 2021d). This pathology constitutes a major public health issue due to its prevalence, which is reaching pandemic levels, as well as due to its high contribution to a decline in both quality of life € her, 2019). According to WHO, in 2016, and life expectancy (Blu more than 1.9 billion adults (18 years old) were overweight (body mass index [BMI] 25 kg/m2), and 650 million were obese (BMI 30 kg/m2), and more worryingly, 39 million children (