Glucosinolates: Properties, Recovery, and Applications [1 ed.] 012816493X, 9780128164938

Table of contents :
Cover
List of author's published works
Glucosinolates: Properties, Recovery, and Applications
Copyright
Contributors
Preface
1 . The dilemma of ``good'' and ``bad'' glucosinolates and the potential to regulate their content
1.1 Introduction
1.2 Ecological role of glucosinolates
1.3 Potential antinutritional, undesired, and beneficial properties of GSLs and relative degradation products
1.4 Breeding to reduce antinutritional and undesired GSLs/ITCs
1.4.1 Glucosinolate analysis
1.4.2 Glucosinolate biosynthesis: an overview
1.4.3 Breeding Brassica crops for low glucosinolate content
1.5 Glucosinolate biofortification: breeding to selectively increase beneficial GSL/ITC
1.6 Preharvest factors influencing the concentration of GSL and the potential for agronomic biofortification
1.7 Postharvest and processing factor influencing the concentration of GSLs and their decomposition
1.8 Conclusions and future prospects
References
2 . Biosynthesis and nutritious effects
2.1 Introduction
2.2 Biosynthesis of glucosinolates
2.2.1 General biosynthesis of GLs
2.2.1.1 The synthesis of GLs in plants
2.2.1.2 Side-chain modifications
2.2.2 Biosynthesis of indole glucosinolates
2.2.3 Biosynthesis of aliphatic glucosinolates
2.3 The effects of plant in vitro culture conditions on biosynthesis and levels of GLs
2.3.1 The effects of phytohormones
2.3.2 Effects of level of cell differentiation
2.3.3 The effects of elicitors
2.3.4 The effects of metabolic engineering strategies
2.4 Effects of nutrition and other factors on levels of glucosinolates in plants
2.4.1 The effects of potassium
2.4.2 The effects of sulfur
2.4.3 The effects of nitrogen
2.4.4 The effects of other factors
2.5 Conclusion
Acknowledgments
References
3 . Enzymatic activities behind degradation of glucosinolates
3.1 Introduction
3.2 Myrosinase structure and function
3.3 The glucosinolates-myrosinase defense system
3.4 Products of GL hydrolysis and mechanism of their formation
3.4.1 Nitriles and epithionitriles
3.4.2 Thiocyanates
3.4.3 Isothiocyanates
3.4.4 Indolic compounds
3.4.5 Oxazolidine-2-thiones
3.5 Methods of myrosinase activity determination
3.5.1 General remarks
3.5.2 Sample preparation
3.5.3 Monitoring of reactants during hydrolysis of GLs
3.6 Concluding remarks
References
4 . Glucosinolates and metabolism
4.1 Myrosinase, a key enzyme in glucosinolates metabolism
4.2 Human metabolism of glucosinolates
4.3 Gut metabolism
4.3.1 Isothiocyanates
4.3.2 Nitriles
4.4 Hepatic metabolism
4.5 Future perspectives
4.6 Plant metabolism of glucosinolates
4.6.1 Sulfur metabolism
4.6.2 Metabolism
4.6.3 REDOX regulation of metabolism
4.6.4 Transport in glucosinolate metabolism
4.7 Conclusion
References
Further reading
5 . Different sources of glucosinolates and their derivatives
5.1 Introduction
5.2 General properties and structure of glucosinolates
5.3 Different sources of glucosinolates
5.4 Selected glucosinolate degradation products and their properties
5.4.1 Sulforaphane
5.4.2 Sinigrin and progoitrin
5.4.3 Phenethyl thiocyanate (gluconasturtiin)
5.5 Classes of glucosinolates
5.5.1 Aliphatic glucosinolates
5.5.2 Indole glucosinolates
5.5.3 Aromatic glucosinolates
5.6 Glucosinolates in selected plants
5.6.1 Rocket (Eruca sativa and Diplotaxis tenuifolia)
5.6.2 Mustard seed Brassica juncea L. (syn. Sinapis juncea L.)
5.6.3 Capers (Capparis sicula)
5.6.4 Nasturtium (Tropaeolum majus)
5.6.5 Rapeseed (Brassica napus L. and Brassica rapa L.)
5.6.6 Spider plant (Cleome or Gynandropsis spp.)
5.6.7 Turnip (Brassica rapa)
5.6.8 Moringa (Moringa oleifera Lam.)
5.6.9 Horseradish (Armoracia rusticana)
5.6.10 Upland cress (Barbarea varna)
5.6.11 Outside the order Brassicales
5.6.11.1 Papaya (Carica papaya)
5.7 New and/or alternative methods to increase the glucosinolates contents in plants
5.7.1 Increasing the desired glucosinolate component
5.7.2 Metabolism of different sources
5.7.3 Metabolic engineering approaches
5.7.4 Effect of gut microflora
5.7.5 Sustainable sources/by-products
5.8 Conclusions
References
6 . Processing and cooking effects on glucosinolates and their derivatives
6.1 Introduction
6.2 Postharvest storage and packaging conditions
6.3 Industrial nonthermal technologies
6.4 Industrial thermal technologies
6.5 Culinary treatments
6.6 Processed food ingredients enriched in GLS
6.7 Conclusions
References
7 . Analysis of glucosinolates content in food products
7.1 Introduction
7.2 Glucosinolates and isothiocyanates effect on human health
7.3 Analysis of glucosinolates in food
7.3.1 Glucosinolates stability during processing
7.3.2 Glucosinolates extraction methods
7.3.2.1 Conventional extraction methods
7.3.2.2 Emergent extraction methods
High pressure-assisted extraction
Pulsed electric field-assisted extraction
Ultrasounds combined with microwave-assisted extraction
Supercritical carbon dioxide-assisted extraction
7.3.3 Glucosinolates detection methods
7.3.4 Glucosinolates quantification methods
7.3.5 Isolation
7.3.6 Purity of analytical standards: water content by NMR
7.3.7 Extinction coefficients
7.3.8 Labeled internal standards
7.4 Conclusions
Acknowledgments
References
8 . Recovery techniques, stability, and applications of glucosinolates
8.1 Introduction
8.2 Recovery strategies
8.3 Conventional technologies
8.4 Nonthermal technologies
8.4.1 Ultrasound and microwave-assisted extraction
8.4.2 High pressure processing
8.4.3 Supercritical fluids and pressurized hot water extraction
8.4.4 Pulsed electric fields
8.5 Stability of GLs and ICs during preservation and storage
8.6 Applications of GLs and ICs
8.7 Commercialization aspects
8.8 GLs in animal nutrition
8.9 Conclusions
References
9 . Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates
9.1 Introduction
9.2 Discovery and existence of sulforaphane and sulforaphene
9.3 Analysis and recovery of sulforaphane and sulforaphene
9.4 Procedures and analysis data
9.5 Purification
9.5.1 Macroporous resin
9.5.2 Preparative high-performance liquid chromatography
9.5.3 High-speed countercurrent chromatography
9.6 Anticarcinogenic activity of sulforaphane and sulforaphene
9.6.1 In vitro inhibition of cancer cells
9.6.2 The anticarcinogenic mechanism of sulforaphane and sulforaphene
9.6.3 Acute toxicity testing in mice
9.6.4 Pharmacokinetics in mice
9.6.5 In vivo anticancer effects of sulforaphene
9.7 Conclusions
References
Index
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Back Cover

Citation preview

List of author’s published works 1. Carotenoids: Properties, processing and applications (2019). Eds by Galanakis, C.M., Elsevier Inc. ISBN: 9780128170670. 2. Lipids and Edible Oils: Properties, Processing and Applications (2019). Eds by Galanakis, C.M., Elsevier-Academic Press. ISBN: 9780128171059. 3. Innovations Strategies in Environmental Science (2019). Eds by Galanakis, C.M., Elsevier Inc. ISBN: 9780128173824. 4. Valorization of fruit processing by-products (2019). Eds by Galanakis, C.M., Elsevier Inc. ISBN: 9780128171066. 5. Trends in Non-alcoholic Beverages (2019). Eds by Galanakis, C.M., Elsevier Inc. ISBN: 9780128169384. 6. Nutraceuticals and Natural Product Pharmaceuticals (2019). Eds by Galanakis, C.M., Elsevier Inc. ISBN: 9780128164501. 7. Food Quality and Shelf-life (2019). Eds by Galanakis, C.M., Elsevier Inc. ISBN: 9780128171905. 8. Dietary Fiber: Properties, Recovery & Applications (2019). Eds by Galanakis, C.M. Elsevier Inc. ISBN: 9780128164952. 9. Proteins: Sustainable Source, Processing and Applications (2019). Eds by Galanakis, C.M. Elsevier Inc. ISBN: 9780128166956. 10. The Role of Alternative and Innovative Food Ingredients and products in consumer wellness (2019). Eds by Galanakis, C.M. Elsevier Inc. ISBN: 9780128164532. 11. Trends in Personalized Nutrition (2019). Eds by Galanakis, C.M., Elsevier-Academic Press. ISBN: 978012816403. 12. Sustainable Water and Wastewater Processing (2019). Eds by Galanakis, C.M. & Agrafioti, E. Elsevier Inc. ISBN: 9780128161708. 13. Saving Food: Production, Supply Chain, Food Waste and Food Consumption (2019). Eds by Galanakis, C.M., Elsevier-Academic Press. ISBN: 9780128153574. 14. Innovations in traditional foods (2019). Eds by Galanakis, C.M., Elsevier-Woodhead Publishing. ISBN: 9780128148877. 15. Separation of functional molecules in food by membrane technology (2018). Eds by Galanakis, C.M., Elsevier-Academic Press. ISBN: 9780128150566. 16. Sustainable meat production and processing (2018). Eds by Galanakis, C.M., ElsevierAcademic Press. ISBN: 9780128148747. 17. Polyphenols: properties, recovery and applications (2018). Eds by Galanakis, C.M., Elsevier-Academic Press. ISBN: 9780128135723. 18. Sustainable recovery and reutilization of cereal processing by-products (2018). Eds by Galanakis, C.M., Elsevier-Woodhead Publishing. ISBN: 9780081021620. 19. Sustainable food systems from agriculture to industry: improving production and processing (2018). Eds by Galanakis, C.M., Elsevier-Academic Press. ISBN: 9780128119358. 20. Handbook of coffee processing by-products: sustainable applications (2017). Eds. by Galanakis, C.M., Elsevier-Academic Press. ISBN: 9780128112908. 21. Handbook of grape processing by-products: sustainable solutions (2017). Eds. by Galanakis, C.M., Elsevier-Academic Press. ISBN: 9780128098707. 22. Olive Mill Waste: Recent advances for the Sustainable Management (2017). Eds. by Galanakis, C.M., Elsevier-Academic Press. ISBN: 9780128053140. 23. Nutraceutical and functional food components: effects of innovative processing techniques (2017). Eds. by Galanakis, C.M., Elsevier-Academic Press. ISBN: 9780128052570. 24. Innovation Strategies for the food industry: tools for implementation (2016). Eds. by Galanakis, C.M., Elsevier-Academic Press. ISBN: 9780128037515. 25. Food Waste Recovery: Processing Technologies & Techniques (2015). Eds. by Galanakis, C.M., Elsevier-Academic Press. ISBN: 9780128003510.

GLUCOSINOLATES: PROPERTIES, RECOVERY, AND APPLICATIONS

Edited by

CHARIS M. GALANAKIS Research & Innovation Department, Galanakis Laboratories, Chania, Greece Food Waste Recovery Group, ISEKI Food Association, Vienna, Austria

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 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. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-816493-8 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Charlotte Cockle Acquisition Editor: Nina Rosa de Araujo Bandeira Editorial Project Manager: Laura Okidi Production Project Manager: Prem Kumar Kaliamoorthi Cover Designer: Matthew Limbert Typeset by TNQ Technologies

Contributors Elisabete M.C. Alexandre QOPNA & LAQV-REQUIMTE, Chemistry Department, University of Aveiro, Aveiro, Portugal; Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal Nieves Baenas Institute for Nutritional Medicine, Molecular Nutrition Group, University Medical Center Schleswig-Hosltein Campus Lübeck, Lübeck, Germany Agnieszka Bartoszek Department of Food Chemistry, Technology and Biotechnology, Faculty of Chemistry, Gdansk University of Technology, Gdansk, Poland M. Elena Cartea Group of Genetics, Breeding and Biochemistry of Brassicas, Misión Biológica de Galicia (MBG-CSIC), Pontevedra, Spain Ibrahim Guillermo Castro-Torres Colegio de Ciencias y Humanidades, Plantel Sur, Universidad Nacional Autónoma de México (UNAM), Ciudad de México, México Víctor Alberto Castro-Torres Instituto de Química, UNAM, Ciudad de México, México Li Cheng State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, P.R. China Antonio de Haro Bailón Institute of Sustainable Agriculture, Spanish National Research Council, Campus Alameda del Obispo, Córdoba, Spain Francesco Di Gioia Department of Plant Science, Pennsylvania State University, Pennsylvania, PA, United States Miguel Ángel Domínguez-Ortiz Instituto de Ciencias Básicas, Universidad Veracruzana, Xalapa de Enríquez, Veracruz, México Isabel C.F.R. Fereira Centro de Investigação de Montanha (CIMO), Campus de Santa Apolónia, Bragança, Portugal Marta Francisco Group of Genetics, Breeding and Biochemistry of Brassicas, Misión Biológica de Galicia (MBG-CSIC), Pontevedra, Spain

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Charis M. Galanakis Research & Innovation Department, Galanakis Laboratories, Chania, Greece; Food Waste Recovery Group, ISEKI Food Association, Vienna, Austria Minerva Hernández-Lozano Facultad de Química Farmacéutica Biológica, Universidad Veracruzana, Xalapa de Enríquez, Veracruz, México Hao Liang State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, P.R. China Sílvia A. Moreira QOPNA & LAQV-REQUIMTE, Chemistry Department, University of Aveiro, Aveiro, Portugal; Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal Diego A. Moreno Phytochemistry and Healthy Foods Lab, Department of Food Science and Technology, CEBAS-CSIC, Murcia, Spain Elia Brosla Naranjo-Rodríguez Departamento de Farmacia, Facultad de Química, UNAM, Ciudad de México, México Karol Parchem Department of Food Chemistry, Technology and Biotechnology, Faculty of Chemistry, Gdansk University of Technology, Gdansk, Poland Spyridon A. Petropoulos Department of Agriculture, Crop Production and Rural Environment, University of Thessaly, Volos, Greece Anna Piekarska Department of Food Chemistry, Technology and Biotechnology, Faculty of Chemistry, Gdansk University of Technology, Gdansk, Poland José Pinela Centro de Investigação de Montanha (CIMO), Campus de Santa Apolónia, Bragança, Portugal Manuela Pintado Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal Carlos A. Pinto QOPNA & LAQV-REQUIMTE, Chemistry Department, University of Aveiro, Aveiro, Portugal Jorge A. Saraiva QOPNA & LAQV-REQUIMTE, Chemistry Department, University of Aveiro, Aveiro, Portugal Z. Tacer-Caba Department of Food and Nutrition, University of Helsinki, Helsinki, Finland

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María Tortosa Group of Genetics, Breeding and Biochemistry of Brassicas, Misión Biológica de Galicia (MBG-CSIC), Pontevedra, Spain Quan V. Vo Department of Natural Sciences, Quang Tri Teachers Training College, Quang Tri Province, Viet Nam Kai Wan State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, P.R. China Qipeng Yuan State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, P.R. China

Preface Glucosinolates are a group of sulfur- and nitrogen-containing glycosides (e.g., b-D-thioglucose and sulfonated oxime moieties) found in abundance in Cruciferous plants. They include thioglucosides, characterized by side chain with varying aliphatic, aromatic, and heteroaromatic carbon skeletons. Glucosinolates get converted into various degradation products (e.g., isothiocyanates, thiocyanates, indoles), when vegetables containing them are cut or chewed because during this process they come in contact with the enzyme myrosinase which hydrolyzes them. These compounds are considered as highly bioactive secondary metabolites with significant effects against various types of cancer and carcinogenesis, either in intact form or after their enzymatic or nonenzymatic transformation in isothiocyanates and indolic compounds. Because of the noted properties, they have attracted the interest of scientific community due to their healthy properties as bioactive compounds and potential natural antimicrobial ingredients. In addition, understanding of glucosinolates’ biosynthesis pathway is a key factor to improve yield and nutritional properties of crops. The effectiveness of glucosinolates and their derivatives depends on preserving their stability, bioactivity, and bioavailability during handling, extraction, and processing. For instance, the content of glucosinolates and their hydrolysis products considerably subjected to changes after harvest due to the effect of storage conditions and industrial treatments, as well as the different cooking methods utilized by consumers. The preservation of these bioactive compounds during processing of crucifers vegetables is a particular interest to maintain the health properties of these products. The metabolism of glucosinolates in Brassicaceae/Cruciferous plants has been well studied; however, relatively little research has been conducted in humans. The first step of the glucosinolates degradation is gut, where different chemical reactions are carried out depending on the processing that cruciferous have before consumption. In any case, different researchers have investigated these issues, whereas the development of applications in functional food and nutraceutical industries has attracted great interest. Food Waste Recovery Group (www.foodwasterecovery.group of ISEKI Food Association) is organizing training and development actions in the food science and technology field, including basic theories (e.g., “The Universal Recovery Strategy”), teaching activities (reference modules,

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e-courses, training workshops, and webinars), expert databases, news channels (social media pages, videos, and blogs), and an open innovation network, aiming at bridging the gap between academia and food industry. Besides, the group has published books dealing food waste recovery technologies, valorization of certain food processing by-products (e.g., generated from olive, grape, cereals, coffee, meat industry), sustainable food systems, innovations in the food industry and traditional foods, nutraceuticals and nonthermal processing, and targeting functional compounds such as polyphenols, proteins, carotenoids, and dietary fiber. Following these efforts, this book aims to cover properties and health effects of glucosinolates and isothiocyanates in view of the new trends in recovery procedures (technologies and plant by-products as initial sources) and applications. The ultimate goal is to support the scientific community, professionals, and enterprises that aspire to develop industrial and commercialized applications. This book consists of nine chapters. Chapter 1 provides an overview of the beneficial effects of glucosinolates, while special focus is given on those cases where adverse effects and toxicities have been reported. Moreover, the means by which plant content in glucosinolates could be regulated to increase nutritional value of plant products and minimize toxicities risk is presented. There are more than 120 types of glucosinolates in Cruciferous plants which depending on their content and chemical structure may exhibit toxic, antinutritional, or beneficial effects to human health. Although glucosinolates in general are considered as “good” metabolites, there are cases where they are associated with toxicity effects, mainly regarding the hypertrophy of thyroid gland and the induction of goiter. Chapter 2 revises the biosynthesis of glucosinolates in the consideration of the newly discovered redox regulation of the pathway and the role of various transcription factors as well as the contribution of their intercellular and intracellular. The effects of plant in vitro culture conditions and nutritious supplies on the biosynthesis, quality, and quantity of glucosinolates in plants have also been highlighted. There are many studies focusing on the investigation of the biosynthesis and metabolism of glucosinolates. The transcriptional regulation of this pathway is well described; however, cofactors and forming intermediates have still been controversial and need further investigation. Myrosinase (thioglucosidase, EC 3.2.1.147) is the enzyme responsible for the hydrolysis of glucosinolates. In plant tissue, myrosinase and glucosinolates are sequestered in separate cellular compartments. As a result of cell

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disruption, e.g., after pathogen attack or on chopping or grinding during food preparation, the myrosinase comes into contact with glucosinolates and catalyzes the hydrolysis of thioglucosidic bond in glucosinolates structure. Consequently, glucose is cleaved off and an unstable aglucond thiohydroximate-O-sulfatedbecomes released. Chapter 3 presents information on the myrosinase structure, its enzymatic activity, and the role of additional protein factors involved in the glucosinolates metabolism. In addition, methods to determine the activity of myrosinase in plant material are discussed. Chapter 4 discusses the metabolism of glucosinolates, the influence of bacteria in the intestine, and the degradation chemicals of these metabolites. Metabolism of glucosinolates in plants has been well studied, but relatively little research has been conducted in humans. The first step of the glucosinolates degradation is gut, where different chemical reactions are carried out depending on the processing that cruciferous have before consumption. Metabolism produces various components, such as isothiocyanates, nitriles, oxazolidine-2-thiones, and indole-3-carbinols, by the action of myrosinase enzyme or intestinal microbiota. Chapter 5 provides an overview for the different and less common sources of glucosinolates. Vegetables such as broccoli, Brussels sprouts, cabbage, collards, kale, capers, turnip, daikon, mustard, radish, watercress, and wasabi or rapeseed or leaf rape belong to Brassicaceae. Not only cruciferous species but also noncruciferous species such as dicotyledonous angiosperms in families such as Capparaceae, Limnanthaceae, and Resedaceae have been reported to contain one or more of the >120 known glucosinolates. This chapter summarizes recent applications and methods to increase the glucosinolates in selected plants and highlights potential valorization of respective vegetable processing by-products. Glucosinolates are thermolabile compounds, being necessary to develop new methods for their preservation, such as freeze-drying, or modified atmosphere packaging, as well as new methodologies for their safe and efficient extraction. In Chapter 6, the effects of postharvest storage, industrial nonthermal and thermal treatments, and domestic culinary methods on glucosinolates and isothiocyanates contents have been denoted. The effect of postharvest processing on myrosinase activity and epithiospecifier proteins has been described as factors affecting glucosinolates breakdown products formation. In addition, applications of cruciferous by-products

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rich in glucosinolates in the food industry have been referred. On the other hand, Chapter 7 focuses on analytical tools to conduct accurate determinations of glucosinolates content of foods. The stability, extraction by different methods, detection and quantification procedures, isolation, determination of purity, extinction coefficients, and the availability of stable isotope labeled internal standards are reviewed. Chapter 8 revises the conventional and emerging technologies for the recovery of glucosinolates and isothiocyanates from different sources. The stability of glucosinolates and isothiocyanates during preservation is discussed in spite of their application in different industries, giving emphasis on critical commercialization aspects. Chapter 9 discusses the extraction and purification methodologies for the recovery of sulforaphane and sulforaphene. Sulforaphane and sulforaphene are two phytochemicals of cruciferous vegetables that have been widely researched over the last years as potential chemopreventive compounds. These compounds do not exist intact in vegetables but are formed from glucoraphanin after hydrolysis with myrosinase. Considering their potential applications, stability and formulation of sulforaphane and sulforaphene are also reviewed in line with their chemopreventive and anticarcinogenic properties. Conclusively, this book addresses food scientists, technologists, and chemists working on food applications and food processing as well as those new product developers, researchers, and professionals who are interested in the development of innovative products and functional foods. It could be utilized by University libraries and Institutes all around the world as a textbook and ancillary reading in undergraduates and postgraduate level multidiscipline courses dealing with food chemistry, food science and technology, and food processing. I would like to express my gratitude to all authors for accepting my invitation, following editorial guidelines, and meeting the timelines. I consider myself fortunate to have had the opportunity to collaborate with different experts around the world, e.g., colleagues from China, Finland, Germany, Greece, Mexico, Poland, Portugal, Spain, United States, and Vietnam. I would also like to acknowledge the acquisition editor Megan Ball, the book manager Katerina Zaliva, and Elsevier’s production team for their assistance during editing and publication. Last but not least, I have a message for all the readers of this textbook. Big collaborative book projects of hundreds thousands words may contain some errors or gaps, so any

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instructive comments or even criticism are always welcome. Please do not hesitate to contact me anytime to discuss current and emerging issues of glucosinolates’ properties, recovery, and applications. Charis M. Galanakis Food Waste Recovery Group ISEKI Food Association Vienna, Austria [email protected] Research & Innovation Department Galanakis Laboratories Chania, Greece [email protected]

CHAPTER 1

The dilemma of “good” and “bad” glucosinolates and the potential to regulate their content Francesco Di Gioia1, José Pinela2, Antonio de Haro Bailón3, Isabel C.F.R. Fereira2, Spyridon A. Petropoulos4 1 2

Department of Plant Science, Pennsylvania State University, Pennsylvania, PA, United States; Centro de Investigação de Montanha (CIMO), Campus de Santa Apolónia, Bragança, Portugal; 3Institute of Sustainable Agriculture, Spanish National Research Council, Campus Alameda del Obispo, Córdoba, Spain; 4Department of Agriculture, Crop Production and Rural Environment, University of Thessaly, Volos, Greece

Contents 1.1 Introduction 1.2 Ecological role of glucosinolates 1.3 Potential antinutritional, undesired, and beneficial properties of GSLs and relative degradation products 1.4 Breeding to reduce antinutritional and undesired GSLs/ITCs 1.4.1 Glucosinolate analysis 1.4.2 Glucosinolate biosynthesis: an overview 1.4.3 Breeding Brassica crops for low glucosinolate content 1.5 Glucosinolate biofortification: breeding to selectively increase beneficial GSL/ITC 1.6 Preharvest factors influencing the concentration of GSL and the potential for agronomic biofortification 1.7 Postharvest and processing factor influencing the concentration of GSLs and their decomposition 1.8 Conclusions and future prospects References

1 3 4 11 15 15 18 20 22 29 31 31

1.1 Introduction Glucosinolates (GSLs) are a group of sulfur- and nitrogen-containing glycosides found exclusively in the Capparales order, although they are present in abundance mostly in Cruciferous plants, including many important vegetable species of the Brassica genus such as broccoli, cabbage, Glucosinolates: Properties, Recovery, and Applications ISBN 978-0-12-816493-8 https://doi.org/10.1016/B978-0-12-816493-8.00001-9

Copyright © 2020 Elsevier Inc. All rights reserved.

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Glucosinolates: Properties, Recovery, and Applications

and cauliflower among others (Fahey et al., 2001). More than 120 different GSLs have been identified so far, with many of them being species-specific (Agerbirk and Olsen, 2012; Chen and Andreasson, 2001; Kwang et al., 2010), while GSL polymorphism is a very common phenomenon among the plant species containing such compounds (Kim et al., 2017; Juergen Kroymann et al., 2005; Mithen et al., 2010). Moreover, depending on their chemical structure and the amino acids they are derived from, they can be classified into three classes, including aliphatic, aromatic, and indole GSLs (Redovnikovic et al., 2008). The main ecological role of GSLs is plant defense, because when plant tissues are damaged they are hydrolyzed by thioglucosidases into isothiocyanates (ITCs) which are toxic to herbivores and pathogens (Wittstock and Halkier, 2002). Moreover, to avoid self-toxicity, plants have evolved a compartmentalization mechanism where GSLs and hydrolyzing enzymes are located in different cell structures and come in contact only after tissue damage (Sirikantaramas et al., 2008). However, the importance of GSLs is even higher from an anthropocentric point of view because most of these compounds have been associated with many beneficial effects to human health, such as anticancer, antibacterial, antidiabetic, antiobesity, antifungal, antioxidant, and antimutagenic activities among others (Giacoppo et al., 2015; Raiola et al., 2018; Vig et al., 2009). Apart from beneficial effects, GSLs and their by-products have been also considered to be potentially toxic against nontarget organisms, including animals, humans, and soil arthropods (Assayed & Abd El-Aty, 2009; Speijers, 1995; van Ommen et al., 2012). Most of the toxicity studies refer to animal models with feeding doses that exceed the normal human consumption on a daily basis, while scarce epidemiological evidence in humans and toxicological data for safety regulations is available (Latté et al., 2011; Speijers, 1995). Special interest has been given to progoitrin and indolylic GSLs that decompose to goitrin and thiocyanates, respectively (Felker et al., 2016). Several studies have highlighted toxicity incidences, such as goitrogenic and mutagenic effects of ITCs (Eisenbrand and Peter, 2016; Wiesner et al., 2014), while other hydrolysis by-products such as nitriles, thiocyanates, goitrins, epithionitriles, and cyanides have been also associated with adverse activities (Cipollini and Gruner, 2007; Felker et al., 2016; Kupke et al., 2016). Of special interest is the case of Carica papaya, a species which is unique for containing both “good” (glucotropaeolin) and “bad” (cyanogenic glucosides) compounds with beneficial and adverse health effects, respectively (Bennett et al., 1997; Bolarinwa et al., 2016; Olafsdottir

The dilemma of “good” and “bad” glucosinolates

3

et al., 2002; Williams et al., 2013). Moreover, some GSL decomposition products have been attributed with both beneficial and adverse effects, e.g., epithionitriles which may cause liver and kidney toxicity in mammals (Kupke et al., 2016), while at the same time they may exhibit cancerpreventive/therapeutic properties (Hanschen et al., 2015). Considering the health effects of GSLs and by-products, there is great research interest toward modulating its content through breeding techniques to increase beneficial compounds and decrease antinutritional factors content (Bell and Wagstaff, 2017; Ishida et al., 2014; Nour-Eldin et al., 2017). Besides common breeding tools, including conventional breeding strategies or modern techniques such as molecular genetics, biofortification is another promising sustainable approach for modulating GSL content (Augustine and Bisht, 2015; Park et al., 2011). GSL content is greatly affected by growing conditions, ontogeny, and genotype among other preharvest factors (Bloem et al., 2007; Schreiner et al., 2012). Postharvest conditions may also play pivotal role in GSL content and bioactive properties with food processing techniques and storage conditions being the most noticeable ones (Jones et al., 2010; Kapusta-Duch et al., 2016; Song and Thornalley, 2007). Therefore, preand postharvest treatments all together interact and may feature important means for regulating GSL content and increase nutritional value and beneficial health effects of the end products. In this chapter, an overview of the beneficial effects of GSLs is presented, while special focus is given on those cases where adverse effects and toxicities have been reported. Considering the contradictory reports regarding the effects of GSLs and its by-products, the dilemma of “good” and “bad” GSLs will be further analyzed. Moreover, the means by which plant content in GSLs may be regulated to increase nutritional value of plant products and minimize toxicities risk is also presented.

1.2 Ecological role of glucosinolates The main role of GSLs is plant defense against various pests and pathogens through a reaction well known as “mustard oil bomb.” During this reaction, GSLs are hydrolyzed on tissue fracture by myrosinases, while a great variation exists on the hydrolysis products depending on genotype, plant or pests specifier proteins (SPs), the chemical structure of GSLs, and the conditions where the reaction takes place (Agerbirk and Olsen, 2012; Kuchernig et al., 2011). Moreover, a significant environment
genotype

4

Glucosinolates: Properties, Recovery, and Applications

interaction exists which further diversifies content and composition of GSLs in cruciferous plants (Brown et al., 2002; Farnham et al., 2004). However, although the hydrolysis products are usually toxic against various enemies (mostly herbivores) and contribute to plant’s defense mechanisms, there are various bacteria, fungi, and insects that have developed evading mechanisms through detoxification or biochemical escape from hydrolysis reaction (Humphrey et al., 2016; Winde and Wittstock, 2011). Apart from biotic stress, GSLs are also highly involved in plant responses to abiotic stressors, because their content increases under salinity, drought, and high temperature conditions (Justen et al., 2013; Radovich et al., 2005; Yuan et al., 2010). The importance of GSLs for plants under stress conditions may be further supported through the evidenced organ or tissue allocation and redistribution of such compounds (Martínez-Ballesta et al., 2013). Another important aspect of GSLs role in plant physiology is their use as sulfur and nitrogen pools that can be utilized after enzymatic hydrolysis for biosynthetic purposes (Redovnikovic et al., 2008). Enzymatic degradation of GSLs and bioactivities of hydrolysis products has been intensively reviewed during the last decade, while there is also nonenzymatic, thermal, and mammalian intestinal degradation to consider regarding the potential health effects of the various by-products. For example, thermal processing (e.g., cooking) is responsible for myrosinase inactivation; therefore, further reactions after human consumption are due to gut microflora (Bones and Rossiter, 2006).

1.3 Potential antinutritional, undesired, and beneficial properties of GSLs and relative degradation products After ingestion of GSL-containing plants, most of these compounds are metabolized in the gut lumen, while a small fraction is absorbed in their intact form through the gastrointestinal mucosa. As illustrated in Fig. 1.1, when these foods are consumed without processing, GSLs are hydrolyzed by myrosinase in the proximal part of the gastrointestinal tract producing a molecule of glucose and an unstable aglycone whose reorganization results in the release of sulfate ion and formation of various metabolites, including ITCs, thiocyanates, nitriles, epithionitriles, and oxazolidine-2-thiones (Barba et al., 2016). Although ITCs are the main products from the GSL-myrosinase system, nitriles/epithionitriles and thiocyanates are formed under influence of SPs (e.g., broccoli, cabbage, and Brussels sprouts contain an epithiospecifier protein and garden cress (Lepidium sativum L.) presents a

The dilemma of “good” and “bad” glucosinolates

5

Tissue damage

Myrosinase + Glucose

HO

Unstable intermediate

Glucosinolate

Acidic pH, Fe , SPs

Epithionitriles

Nitriles

SO

Neutral pH

Oxazolidine-2-thiones

Isothiocyanates

Thiocyanates

Figure 1.1 Myrosinase-promoted hydrolysis reaction of glucosinolates and their different breakdown products. SPs, specifier proteins.

thiocyanate-forming protein) (Ishida et al., 2014). An acidic pH and presence of ferrous ions also promotes the formation of nitriles, whereas the existence of a terminal double bond in the GSL side chain leads to epithionitriles (Barba et al., 2016). In addition, ITCs can split into thiocyanate ion and indole-3-carbinol. However, when these foods are cooked before consumption, myrosinase is denatured and GSLs transit to the colon where they are hydrolyzed by the intestinal microbiota (Barba et al., 2016). Therefore, the bioavailability of GSLs is affected by the ingestion of myrosinase-lacking foods (Dinkova-Kostova and Kostov, 2012). As shown in Table 1.1, several GSL-containing plants are commonly used as foods or spices. However, based on their toxic properties or pungent taste, GSLs are often classified as antinutritional factors. In fact, GSLs and their degradation products are precursors of “bad” compounds with goitrogenic activity in humans and animals, including oxazolidine-2thiones (e.g., progoitrin, and its myrosinase-induced degradation product, goitrin, and glucoconringin) and thiocyanate ions, which may interfere with thyroxine production, drastically reducing iodine supply to the thyroid gland, and resulting in the development of goiter and other associated problems (Felker et al., 2016; Vanetten, 1969). The thiocyanate ion is a competitive inhibitor of the sodium/iodide symporter located on the

Crop

Antinutritional/undesired and/or beneficial/desired effects

Glucobrassicin

Broccoli, Brussels sprouts, papaya, cabbage, mustard, woad, kale, cauliflower

Precursor of BITC, indole-3carbinol (which inhibits NF-kB), and thiocyanate ion. Bitter taste

Gluconapin

Broccoli, Brussels sprouts, kale, cabbage Cabbage, mustard, rape, watercress, wasabi Broccoli, cauliflower, brassicas, and kale

Bitter taste

Gluconasturtiin Glucoraphanin

Glucotropaeolin

Garden cress, papaya

Progoitrin

Broccoli, Brussels sprouts, cabbage, cauliflower, kale, Siberian kale, rapeseed

Sinigrin

Broccoli, Brussels sprouts, collard, cabbage, cauliflower, mustard seeds, garlic mustard, kale, horseradish

Precursor of PEITC. Toxicity to many organisms Precursor of sulforaphane and an isothiocyanate. Anticarcinogenic affects Chemopreventive effects. Unpalatable flavor Bitter/pungent taste. After ingestion, it is converted into goitrin, which decreases the thyroid hormone production. Long-term ingestion associated with goiter formation Precursor of AITC. Bitter/pungent taste

References

Bell et al. (2018); Olafsdottir et al. (2002); van Doorn et al. (1998); Wieczorek et al. (2017); Williams et al. (2013) Bell et al. (2018) IARC (2004) Augustine and Bisht (2015); Bischoff (2016); IARC (2004) IARC (2004); Olafsdottir et al. (2002); Williams et al. (2013) Bischoff (2016); Fahey et al. (2001); Langer and Michajlovskij (1969); van Doorn et al. (1998); Wieczorek et al. (2017)

Bischoff (2016); Fahey et al. (2001); IARC (2004); van Doorn et al. (1998); Wieczorek et al. (2017)

Glucosinolates: Properties, Recovery, and Applications

Compound

6

Table 1.1 Glucosinolates and isothiocyanates found in Brassica and other crops and respective antinutritional/toxic and/or beneficial effects.

Brussels sprouts, cauliflower, horseradish, kale, wasabi

BITC

Garden cress, papaya

Sulforaphane

Broccoli, Brussels sprouts, cabbage

Bitter/pungent taste. Antimicrobial and antiinflammatory activities. Multiple chemopreventive effects. Capacity to inhibit microRNA expression and activate transcription factor Nrf2. Apoptosis inducer in human prostate, colon, and leukemia cancer cells Chemopreventive effects. Apoptosis inducer in human prostate, colon, and leukemia cancer cells Chemopreventive effect by inducing phase 2 detoxication enzymes, blocking the cell cycle, and promoting apoptosis. Central nervous system protector. Inhibitory effect for urease from Helicobacter pylori

Atsumi and Saito (2015); Fahey et al. (2001); Gründemann and Huber (2018); Patten and DeLong (1999); Uematsu et al. (2002); Whitty and Bjeldanes (1987); Xiao et al. (2003); Yuesheng Zhang (2010)

Kassie et al. (2002); Olafsdottir et al. (2002); Patten and DeLong (1999); Tang (1971); Williams et al. (2013); Xiao et al. (2003) Bischoff (2016); Dinkova-Kostova et al. (2007); Fahey et al. (2013); Kushad et al. (1999); Zhang et al. (1992)

The dilemma of “good” and “bad” glucosinolates

AITC

7

8

Glucosinolates: Properties, Recovery, and Applications

basolateral membrane of the thyroid follicular cell that can induce hypothyroidism (Dai et al., 1996; Tonacchera et al., 2004). Therefore, overconsumption of cruciferous vegetables with high progoitrin contents such as Siberian kale (Brassica napus), collard (Brassica oleracea), Brussels sprouts, and rapeseed (B. napus and Brassica rapa) can cause goiter (Table 1.1), a phenomenon that has been associated with the development of endemic goiter in certain regions (Felker et al., 2016; Michajlovskij et al., 1969; Vanderpas, 2003). In turn, minor amounts of goitrogenic GSLs have been reported in broccoli (Felker et al., 2016). As goitrin has the ability to inhibit thyroxinogenesis (i.e., thyroid hormone biosynthesis), it has been used in the development of drugs for thyrotoxicosis (Liener, 2003). Benzyl isothiocyanate (BITC), a product of the enzymatic hydrolysis of glucotropaeolin that can be found in high amounts in garden cress and papaya (Table 1.1), is one of the most studied ITCs with regard to cancer chemoprevention (Kassie et al., 2002; Tang, 1971). In papaya, glucotropaeolin levels are high in the latex, new leaves, and seeds; flesh fruit contains considerably low concentrations, but as glucotropaeolin imparts an unpalatable flavor to fruit, attempts to increase its levels in the edible fraction is inadvisable (O’Hare et al., 2009). BITC has been studied for its capacity to induce chemoprotective Phase I and II biotransformation enzymes, antiproliferative action against cancer cell growth, and anthelmintic effects (Basu and Haldar, 2009; Kermanshai et al., 2001; Mitsiogianni et al., 2018; Tan et al., 2010). However, cyanogenic glycosides (e.g., prunasin and tetraphyllin B) have also been reported in papaya (Bennett et al., 1997; Olafsdottir et al., 2002; Williams et al., 2013), which offer a potential source of highly toxic hydrogen cyanide (HCN). The postmortem process of cyanogenesis1 is initiated by any decompartmentation, resulting in the contact of cyanogenic glycosides and hydrolytic enzymes (b-glucosidases and hydroxynitrile lyases), whose hydrolysis produces unstable hydroxynitriles that decay to HCN and a carbonyl compound (Selmar, 2010). Interestingly, Carica species are rare examples of taxa in which GSLs and cyanogenic glycosides cooccur, both being derived from the same amino acid, phenylalanine (Olafsdottir et al., 2002). Another exception is garlic mustard (Alliaria petiolata), a highly invasive weed known to contain not only the “good” GSL sinigrin but also large amounts of alliarinoside, a g-hydroxynitrile glucoside structurally related to cyanogenic glucosides that 1

The term “cyanogenesis” means not only the synthesis or presence of a cyanogenic glycoside but the enzymatic hydrolysis producing free HCN and other compounds.

The dilemma of “good” and “bad” glucosinolates

CYP79B2

Phenylalanine

CYP71E1

E,Z-Phenylacetaldoxime

9

CYP71E1

R,S-Phenylacetonitrile

R,S-Mandelonitrile

CYP83B1

Glucosinolates

Cyanogenic glycosides

S-(Benzohydroxymoyl)-L-cysteine

Figure 1.2 Schematic pathway of the separation point in the biosynthesis of glucosinolates and cyanogenic glycosides starting from the same precursor.

confers resistance to specialized (GSL-adapted) herbivores (Frisch et al., 2015; Haribal et al., 2001; Renwick et al., 2001). This specie also releases HCN after tissue disruption suggesting the presence of cyanogenic glucosides (Cipollini and Gruner, 2007). These features are viewed as a third line of defense of the plant, with GSLs and thiocyanate-forming protein being the first and second lines, respectively (Frisch et al., 2015). As illustrated in Fig. 1.2, GSLs and cyanogenic glycosides share a common biosynthetic pathway at the early stages. Both groups of compounds are derived from amino acids and have aldoximes as intermediates (Bak et al., 1998). The biosynthesis of GSLs includes a phase of chain elongation of amino acids, conversion of the precursor amino acid via aldoximes into GSLs, and further modifications (such as hydroxylations, desaturations, and glycosylations) of the resulting GSLs (Dewick, 1984; Selmar, 2010). Similarly to the biosynthetic pathway of cyanogenic glucosides, aldoximes are produced by cytochrome P450 enzymes located in the endoplasmic reticulum (Bak et al., 1998). First, the conversion of phenylalanine to E,Z-phenylacetaldoxime is catalyzed by CYP79B2. The oxime-producing enzyme is the same in both cases (Fig. 1.2), which raises the question why these compounds cooccur in so few species. Then, in course of cyanogenic glycoside biosynthesis, CYP71E1 converts E,Z-phenylacetaldoxime into R,Smandelonitrile. This aldoxime-metabolizing P450 enzyme catalyzes a dehydration of the oxime to form a nitrile, followed by a hydroxylation to a hydroxynitrile and a glycosylation to a cyanogenic glycoside (Clausen et al., 2015; Selmar, 2010; Vetter, 2000; Wittstock and Halkier, 2000). On the other hand, the biosynthetic pathway of GSLs also includes the formation of E,Z-phenylacetaldoxime from phenylalanine but not R,S-phenylacetonitrile

10

Glucosinolates: Properties, Recovery, and Applications

(Fig. 1.2). In this case, an aci-nitro compound is produced by the oximemetabolizing enzyme CYP83B1 (Selmar, 2010). Therefore, GSLs and cyanogenic glycosides can only coexist if the species harbors both CYP83B1 and CYP71E1 enzymes, or if the pathway is different from the traditional ones (Clausen et al., 2015; Frisch et al., 2015). GSLs are compounds characterized by producing mustard oils (i.e., a mixture of ITCs, thiocyanates, and related nitriles) and responsible for the characteristic, sharp, and bitter taste of Brassicaceae plants (Table 1.1). Considering the thiocyanate N]C]S group presented in ITCs has the ability to interact with bitter taste receptors and odor receptors, it has been suggested that ITCs contribute more to the overall bitterness and pungency than GSLs (Bell et al., 2018; Wieczorek et al., 2017). In addition, the high amounts of hydrogen sulfides in the ITC volatile fraction also play a crucial role in odor formation (Wieczorek et al., 2017). Actually, dimethyl disulfide and dimethyl trisulfide have been recognized as aroma components in raw and cooked Brassica vegetables (Engel et al., 2002; Maruyama, 1970). In Brussels sprouts, for example, sinigrin, progoitrin, gluconapin, and glucobrassicin constitute the major fraction of the total GSL content (van Doorn et al., 1998). According to sensorial studies, while sinigrin and gluconapin were considered quite bitter, progoitrin and glucobrassicin were evaluated as bitter only by a small number of panelists (Wieczorek et al., 2017). In turn, sinigrin and neoglucobrassicin (a glucobrassicin derivative) were identified as responsible for the bitterness of cooked cauliflower (Engel et al., 2002). It was also confirmed that allyl isothiocyanate (AITC), dimethyl trisulfide, dimethyl sulfide, and methanethiol are key odorants of cooked cauliflower “sulfur” odors. Therefore, all these compounds may determine the potential acceptance of cooked cauliflower. Almost all the biological effects of GSLs can be attributed to their hydrolytic products. Among them, ITCs are the most studied and important from a nutritional point of view, being responsible for distinct bioactivities and health-promoting effects (Callaway et al., 2004; Zhang et al., 2006). In broccoli, glucoraphanin, gluconapin, and glucobrassicin are major GSLs (Shahidi et al., 2011). Regarding ITCs commonly used for health benefits (Table 1.1), AITC derives from the hydrolysis of sinigrin in cabbage, mustard, and horseradish, BITC from glucotropaeolin in red cabbage, phenethyl isothiocyanate (PEITC) from gluconasturtiin in watercress and wasabi, and sulforaphane from glucoraphanin in broccoli, cauliflower, kale, and Brassica (IARC, 2004). These ITCs have shown interesting chemopreventive effects against several chronic and degenerative diseases,

The dilemma of “good” and “bad” glucosinolates

11

including different types of cancer, neurodegeneration, cardiovascular diseases, and diabetes (Fimognari et al., 2012). PEITC is a highly promising cancer chemopreventive agent capable of inducing cell cycle arrest in Caco2 colon cells, apoptosis in human prostate cancer PC-3 cells, and antiangiogenic effect in animal models (Thejass and Kuttan, 2007; Xiao and Singh, 2002). It also shows antiinflammatory and immunomodulatory properties (Fimognari et al., 2012). AITC displays antimicrobial activity against a wide spectrum of pathogens (Lin et al., 2000), as well as antiinflammatory and multiple chemopreventive effects (Yuesheng Zhang, 2010). This is the predominant mustard oil found in wasabi, horseradish, Brussels sprouts, cabbage, cauliflower, and kale (Fahey et al., 2001; Kushad et al., 1999; Sultana et al., 2002; Uematsu et al., 2002). AITC and BITC are also potent in inducing apoptosis in human prostate, colon, and leukemia cancer cells (Patten and DeLong, 1999; Xiao et al., 2003). Among the GSLs studied for their health effects, glucoraphanin has capacity to induce the formation of Phase II detoxification enzymes that protect against carcinogens (Kensler et al., 2005). In turn, sulforaphane inhibits the proliferation of cancer cells through the induction of cell cycle arrest and apoptosis and protects the central nervous system (Gamet-Payrastre, 2006). This organosulfur compound also shows inhibitory effects for urease from Helicobacter pylori, a common cause of gastroduodenal inflammation (Fahey et al., 2013). In addition, its potential to be used as a therapeutic agent to alleviate metabolic disorder and protect against renal damage and pain associated with diabetes has been encouraged (Dinkova-Kostova and Kostov, 2012). In human intervention studies, broccoli sprouts and extracts have been used as delivery vehicles of glucoraphanin and sulforaphane (after enzymatic hydrolysis), which have ability to protect against environmental toxins by enhancing their detoxification (Dinkova-Kostova and Kostov, 2012). However, the doses exhibiting chemopreventive effects should be considered. This is because BITC and PEITC, for example, present genotoxic potential at doses endowed with a chemopreventive effect (Fimognari et al., 2012).

1.4 Breeding to reduce antinutritional and undesired GSLs/ITCs To be successful, a plant breeding program depends on the following: i) the existence of available genetic variability for the targeted character and

12

Glucosinolates: Properties, Recovery, and Applications

ii) The development of adequate methods of selection to ensure the identification and selection of the best genotypes for this character. To look for genetic variability, breeders are screening world collections of cultivated material as well as germplasm collections of species related to those they wish to improve, with the aim of finding the gene pools for the targeted character. Brassicaceae family includes many plant species domesticated thousands of years ago for a wide range of uses: oil, condiment, vegetables, medicinal, lubricant, etc. (Gómez-Campo and Prakash, 1999). In particular, some species belonging to Brassica genus comprise very common crops cultivated across the world for food, feed, and industrial uses: B. napus (oilseed and meals), B. rapa (oilseed, vegetable, forage), B. oleracea (vegetable, forage), Brassica nigra (condiment), Brassica juncea (condiment, vegetable) and Brassica carinata (oilseed, biofumigation, biodiesel). The genetic relationship between these Brassica species was established by Nagaharu (1935), describing natural allopolyploidization events using three ancestral diploids: B. rapa (A genome), B. nigra (B genome), and B. oleracea (C genome), and derived allotetraploids: B. juncea (AB genome), B. napus (AC genome), and B. carinata (BC genome; Fig. 1.3). Originally, Brassica oil was considered inferior in quality to other vegetable oils because of its high content in undesirable fatty acids as eicosenoic acid and erucic acid (Fenwick and Heaney, 1983; Vaughan et al., 1976). After oil extraction, the utilization of the seed meal as protein supplement, with a well-balanced amino acid composition, was limited due to the presence of GSLs, identified as responsible of low palatability, toxic effects, and nutritional disorders in animals and birds (Tripathi and Mishra, 2007). Nowadays, Brassica species are among the most important crops in the world after the success of plant breeders in developing double-zero oilseed varieties (with zero erucic acid and low GSL content) and vegetable cultivars with high content in specific GSLs that have a protective effect against cancer (Downey et al., 1969; Fahey et al., 2001). As has been described in previous chapters, GSLs are present in about 16 families of the order Capparales, including Brassicaceae (Fahey et al., 2001). The GSL molecule consists of two parts, a common glycone moiety and a variable aglycone side chain (R group) derived from a corresponding amino acid. GSLs can be classified into three groups: aliphatic (derived from Me, Ala, Val, Leu, Ile), indole (derived from Trp), and aromatic (derived from Phe,Tyr).

The dilemma of “good” and “bad” glucosinolates

13

B.rapa (A, n=10)

AA

B.napus (AC, n=19)

CC

B.oleracea (C, n=9)

AAC

AABB

BBCC

B.carinata (BC, n=17)

B.juncea (AB, n=18)

BB

B.nigra (B, n=8)

Figure 1.3 Genomic relationship between diploid and amphidiploid Brassica species. (Modified from Nagaharu, U., 1935. Genome analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. Japanese Journal of Botany 7, 389e452.)

A wide variability in the profile and composition of GSLs can be found among species, varieties, physiological state, organ tissue, and environmental conditions (Brown et al., 2003). Generally, a single plant species contains up to 4 different GSLs in significant amounts, while as many as 15 different GSLs can be found in the same plant. Previous reviews of the major GSLs present in economically important species of the Brassicaceae family (Table 1.2) were carried out by Rosa et al. (2010), Fahey et al. (2001), Verkerk et al. (2009), and Bell and Wagstaff (2017). The key factor for determining the GSL composition and concentration in Brassicaceae is the genetic background, but environmental conditions and plant development also influence GSL content. All these factors have to be considered by breeders aiming to obtain new varieties of Brassica crops

14

Glucosinolates: Properties, Recovery, and Applications

Table 1.2 Trivial name, chemical class, systematic name, and abbreviations of the most common GSLs present in Brassica crops. Trivial name Chemical classdsystematic name Abbreviation Aliphatic (3C carbon chain length)

Glucoiberverin Glucoiberin Sinigrin

3-Metylthiopropyl glucosinolate 3-Methylsulphinylpropyl glucosinolate 2-Propenyl glucosinolate

GIV GIB

4-Methylthiobutyl glucosinolate 4- Methylsulphinylbutyl glucosinolate 3-Butenyl glucosinolate R-2-Hydroxy-3-butenyl glucosinolate S-2-Hydroxy-3-butenyl glucosinolate

GER GRA GNA PRO

5-Methylthiopentyl glucosinolate 5-Methylsulphinylpentyl glucosinolate 4-Pentenyl glucosinolate 2-Hydroxy-4-pentenyl glucosinolate

GBE GAL

SIN

(4C carbon chain length)

Glucoerucin Glucoraphanin Gluconapin Progoitrin Epiprogoitrin

E-PRO

(5C carbon chain length)

Glucoberteroin Glucoalyssin Glucobrassicanapin Gluconapoleiferin

GBN GNL

Indole

Glucobrassicin 4Hydroxyglucobrassicin 4Metoxyglucobrassicin Neoglucobrassicin

3-Indolymethyl glucosinolate 4-Hydroxy-3-indolymethyl glucosinolate 4-Methoxy-3-indolymethyl glucosinolate N-Methoxy-3-indolymethyl glucosinolate

GBS 4-OHGBS

2-Phenylethyl glucosinolate Benzyl glucosinolate

GST GTL

4-OMGBS NGBS

Aromatic

Gluconasturtin Glucotropaeolin

with high content of beneficial GSL and low content of antinutritional GSL (Sønderby et al., 2010). Similarly to other characters related with quality components (fatty acids, fiber, etc.), conventional breeding for GSLs does not require a deep

The dilemma of “good” and “bad” glucosinolates

15

understanding of the biochemical pathways leading to final GSL composition and concentration. After the implementation of analytical methods for GSLs allowing fast, accurate, and inexpensive analysis of germplasm collections and segregating populations, plant breeders can select the best genotypes carrying out the desired GSL profile and content to obtain crops with high food quality and make conclusions about the genetic control of the GSL content (Downey et al., 1969; Josefsson and Appelqvist, 1968; Love et al., 1990; Velasco et al., 1999).

1.4.1 Glucosinolate analysis Different methods have been developed for the analysis of the GSL content and composition in Brassicaceae species, according to the need for quantitative or qualitative information, analytical speed, and analytical accuracy. A simple, fast, and cost-effective method for quantitative analysis of GSL is necessary for analyzing the total GSL content in germplasm collections and segregating populations normally used in plant breeding work. Palladium test (Thies, 1982) and Glucose test tape (McGregor and Downey, 1975) are the most common methods used for quantitative determination of GSL content in plant breeding. Reverse phase high-performance liquid chromatography with gradient system is the reference method for the accurate and reliable analysis of GSL content and composition in Brassicaceae samples (Bjerg and Sørensen, 1987) but requires expensive equipment, specialized personnel, and is time consuming. Near infrared reflectance spectroscopy is widely used in breeding oilseed and vegetable Brassica for fast, low cost, and nondestructive analysis of total and individual GSL content in seeds and green tissues (Font et al., 2005; Hernández-Hierro et al., 2012; Sen et al., 2018).

1.4.2 Glucosinolate biosynthesis: an overview The knowledge of the biochemical basis of GSL biosynthesis provides an explanation of the diversity and relationships of different GSL found in Brassicaceae species. In the last decades, the biosynthetic pathway of GSLs has been elucidated in detail in Arabidopsis thaliana, and many of the corresponding genes have been identified and cloned in this model species. As the GSL biosynthetic pathway has been rather well conserved in the Brassicaceae family, the advances in Arabidopsis research on GSLs have been crucial in the knowledge about the molecular basis of the genetic control of GSL in economically important oilseed and vegetable Brassica crops. On the basis of

16

Glucosinolates: Properties, Recovery, and Applications

this knowledge, genetic engineering allows changes in the levels and types of GSL in Brassica crops by acting at different steps of the biosynthetic pathway of GSLs. On the other hand, understanding the molecular genetics control of GSL biosynthesis facilitates the design of molecular markers to be used in plant breeding programs (Wittstock and Halkier, 2002). Briefly, the biosynthesis of GSLs comprises three independent stages: (1) some amino acids are elongated by one or several methylene groups, (2) the precursor aminoacids are converted into the parent GSLs, and (3) the parent GSLs are subjected to secondary modifications (oxygenations, hydroxylations, alkenylations, and methoxylations). Previous review of the molecular genetics and biochemistry of GSLs biosynthesis can be found in Verkerk et al. (2009), Sønderby et al. (2010), Hirani et al. (2012), Ishida et al. (2014), Velasco et al. (2016), and Raiola et al. (2018). As the major GSLs found in the most important Brassica crops are derived from the methionine or phenylalanine, many biochemical and genetic studies on GSL biosynthesis have focused on these compounds. For aliphatic GSLs, two independent pathways determine the final GSL profile and content in Brassica crops (Fig. 1.4): a) elongation of the aliphatic side chain, through the activity of genes at GSL-ELONG loci, leading to 3C (propyl), 4C (butyl), or 5C (pentyl) type of GSLs, and b) modifications of the aliphatic side chain: firstly, the methylthioalkyl GSLs are converted into methylsulphinylalkyl GSLs by the activity of genes at GSL-OX locus; secondly, the methulsulphinylalkyl GSLs are converted into alkenyl GSLs by enzymes controlled by genes located at GSL-ALK locus; and, finally, alkenyl GSLs are modified to hydroxyalkenyl GSLs by enzymes at GSL-OH locus. Side chain modifications of indole GSLs in Brassica species occur through hydroxylations and methoxylations catalyzed by enzymes of the CYP81F family. In Arabidopsis, the biosynthesis of indole GSLs is controlled by transcription factors (TFs) of the MYB family. These TFs play a role in biotic and abiotic stresses by altering GSLs levels (Ishida et al., 2014). The activity and interaction of the genes regulating the total amount with those determining the side chain elongation and chain modifications will determine the final GSL content and profile. Much of the genomic research for traits related to GSLs biosynthesis and metabolism has been conducted in species such as A. thaliana and B. oleracea, and it is possible to identify orthologous genes for biosynthesis, transcriptional regulation, and environmental response in other species.

The dilemma of “good” and “bad” glucosinolates

17

Methionine GSL-ELONG

homo-methionine GSL-PRO

3-methylthiopropyl (glucoibervirin)

GSL-OX

3-methylsulphinylpropyl (glucoiberin)

GSL-ALK

2-propenyl (sinigrin)

GSL-ELONG

di-homo-methionine CYTp450

4-methylthiobutyl (glucoerucin)

GSL-OX

4-methylsulphinylbutyl (glucoraphanin)

GSL-ALK

3-butenyl (gluconapin)

GSL-OH

2-hydroxy-3-butenyl (progoitrin)

tri-homo-methionine CYTp450

5-methylthiopentyl (glucoberteroin)

GSL-OX

5-methylsulphinylpentyl (glucoalyssin)

GSL-ALK

4-pentenyl (glucobrassicanapin)

GSL-OH

2-hydroxy-4-pentenyl (gluconapoleiferin)

Figure 1.4 General scheme for glucosinolate core structure and side chain modification step for 3C, 4C, and 5C aliphatic glucosinolates. Loci names of enzymes catalyzing specific steps are also indicated. (Adapted from Magrath, R., Bano, F., Morgner, M., Parkin, I., Sharpe, A., Lister, C., et al., 1994. Genetics of aliphatic glucosinolates. I. side chain elongation in Brassica napus and Arabidopsis thaliana. Heredity 72(3), 290e299; Mithen, R., Clarke, J., Lister, C., Dean, C., 1995. Genetics of aliphatic glucosinolates. 3. Side-chain structure of aliphatic glucosinolates in Arabidopsis thaliana. Heredity 74, 210e215; Verkerk, R., Schreiner, M., Krumbein, A., Ciska, E., Holst, B., Rowland, I., et al., 2009. Glucosinolates in Brassica vegetables: the influence of the food supply chain on intake, bioavailability and human health. Molecular Nutrition & Food Research 53(Suppl. 2), 219e265; Velasco, P., Cartea, M.E., González, C., Vilar, M., Ordás, A., 2007. Factors affecting the glucosinolate content of kale (Brassica oleracea acephala Group). Journal of Agricultural and Food Chemistry 55 (3), 955e962; Hirani, A.H., Li, G., Zelmer, C.D., McVetty, P.B.E., Asif, M., Goyal, A., 2012. Molecular genetics of glucosinolate biosynthesis in Brassicas: genetic manipulation and application aspects. In: Goyal, A. (Ed.), Crop Plant. IntechOpen, pp. 189e216.)

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Glucosinolates: Properties, Recovery, and Applications

Unfortunately, because of the complexity of the Brassica genome and the long life cycle of commercial crops, important resources have to be allocated to generate breeding populations for adequate plant selection for this phytochemical health trait.

1.4.3 Breeding Brassica crops for low glucosinolate content The seed of wild-type B. napus contains high erucic acid content (above 40% of the total fatty acids) and high GSL content (above 100 mmol/g). This represented an important obstacle for its utilization. The screening of germplasm collections of oilseed Brassicas for low GSL content in the seeds led to the discovery of the cultivar “Bronowski,” a Polish spring rape (B. napus) with a very low level of GSLs in seeds (12 mmol/g defatted dry matter) (Josefsson and Appelqvist, 1968). Selected lines derived from this cultivar were used as gene sources for the development of double-zero rapeseed varieties (zero erucic acid in the oil, low GSL in the seed meal). Genetic analysis from the segregating populations involving crosses between Bronowski and Target B. napus lines indicated that as many as 11 recessives alleles were involved in the metabolic block in the biosynthesis of the main GSLs that resulted in the low contents in progoitrin, gluconapin, and glucobrassicanapin. Three loci were involved in the control of gluconapin, four loci were indicated for progoitrin, and four to five loci control glucobrassicanapin content. Inheritance was determined by maternal genotype rather than by the embryo genotype (Konrda and Stefansson, 1970). The rapeseed varieties developed by Canadian breeders having low erucic acid content in the oil (less than 2%) and low GSL content in the seed meal (less than 15 mmol/g defatted dry matter) have been named as “canola” (Canadian oil low acid), allowing a dramatic increase in the production of oil for human consumption and defatted seed meal for livestock farming. Bronowski has been the only low GSL donor for all current B. napus canola cultivars worldwide. The discovery of genetic stock of summer turnip rape with low GSL content and the transfer of “Bronowski” genes through interspecific crosses allowed the development of double-zero B. rapa varieties (Downey et al., 1969; Jönsson, 1977). The first two canola-quality varieties Tower (B. napus) and Candle (B. rapa) were registered in 1997. Love et al. (1990) accomplished the development of low GSL B. juncea (genotype 1058), through an interspecific cross between an Indian B. juncea line and a “Bronowski” B. rapa line, followed by selection for GSL content in BC1F2 and BC1F3 generations. Progeny of 1058 plant grown at

The dilemma of “good” and “bad” glucosinolates

19

different locations in Canada contained less than 10 mmol/g meal, but poor fertility. Arid and Amulet varieties were registered in 2002 in Canada as canola-quality B. juncea with high drought and heat resistance. Double-zero cultivars of B. carinata have not been developed yet, but some progress in the reduction of GSL content has been made after intraspecific breeding and chemical mutagenesis (EMS 1% v/v) and selection of mutant lines with reduced GSL content in M4 generation (Velasco et al., 1999). Chavadej et al. (1994) reduced the indole GSL content of B. napus seeds by transforming canola plants with a gene that encodes tryptophan decarboxylase. Transgenic plants accumulated tryptamine, while a correspondingly low level of tryptophan-derived indole GSL was produced in all plant parts. Particularly, the indole GSL content of mature seeds from transgenic plants was only 3% of that found in nontransformed seeds. Liu et al. (2012) used RNA interference (RNAi) to silence MAM (methylthioalkylmalate synthases) gene family in B. napus canola and B. napus rapeseed, resulting in the reduction of aliphatic GSLs and total GSL content, thus increasing the meal quality of B. napus cultivars. The silencing of GSL-ELONG gene family significantly induced the production of sinigrin (C3), not normally detectable in this Brassica species. Hirani et al. (2013) modified the aliphatic GSL of a resynthesized B. napus by backcross with a Chinese cabbage line. The resynthesized B. napus line (from a cross between B. rapa and B. oleracea) was backcrossed with B. rapa recurrent parent. The replacement of the functional BraGSLELONGþ allele of B. rapa with the nonfunctional GSL-ELONG-from B. oleracea lead to the identification of genotypes from the BC3F2 progenies of the recurrent parent that carried the GSL-ELONG-gene. These genotypes showed a significant reduction in the content of the 5C aliphatic GSLs (gluconapoleiferin, glucoalyssin, and glucobrassicanapin). The Chinese cabbage lines with reduced 5C aliphatic GSLs could be used for manipulation of GSLs profiles in B. napus, B. juncea, and other B. rapa accessions. Priyamedha et al. (2014) selected 11 yielding double low B. juncea lines from among a pool of 1200 lines in F7 generation, derived from three crosses involving double low donors and high yielding varieties. Among the selected lines, two lines (BPRQ 2-1-5 and BPRQ 2-2-11) were highlighted in terms of oil quality and yield performance and were chosen as potential new canola B. juncea varieties. Seeds of A. thaliana and of Brassica crops import GSLs from maternal tissues. In Arabidopsis, the GSL transporters AtGTR1 and AtGTR2 are essential for the accumulation of GSLs in seeds, and the mutation of the

20

Glucosinolates: Properties, Recovery, and Applications

gene encoding these transporters eliminated GSLs from A. thaliana seeds. Based on this fact, Nour-Eldin et al. (2017) successfully reduced the GSLs levels in seeds by 60%e70% in B. rapa and B. juncea by mutating one out of 7 and 4 of 12 GTR orthologs. This transport engineering approach may find wide application for reducing the GSL content in the seeds and for the enhancement of the nutritional value of Brassica crops.

1.5 Glucosinolate biofortification: breeding to selectively increase beneficial GSL/ITC Biofortification is based on the improvement of crops through the utilization of nutrient-rich fertilizers, breeding, or plant engineering strategies to produce and/or accumulate nutritionally important molecules, such as GSLs (Gómez-Galera et al., 2010; Raiola et al., 2018). In the early 1990s, Zhang et al. (1992) and Zhang et al. (1994), put in evidence for the first time the ability of some ITC, derived from the hydrolysis of GSLs, to inhibit tumor formation in several animal systems. In particular, sulforaphane, ITC derived from glucoraphanin, was highlighted as a potent inducer of phase II enzymes involved in cancer detoxification. For this reason, sulforaphane has been extensively investigated due to its potent cancer-preventive effects and apoptosis induction in cancer cells. Based on the current knowledge about the potential health-promoting effects of some GSLs, the increase of aliphatic GSLs such as glucoraphanin, glucoiberin, and sinigrin, and aromatic GSLs such as glucotropaeolin and gluconasturtin should be targeted for breeding Brassica species for beneficial GSLs. Selection against progoitrin (goitrogenic effect) and against some indole GSLs should be also considered, as they have been found to act in Phase I enzyme system related with procarcinogenesis activation (Bell and Wagstaff, 2017; Verkerk et al., 2009). The most comprehensive example of breeding Brassica for increasing beneficial GSL/ITC crops is the development and commercialization of “Beneforté” broccoli that consistently accumulates a threefold higher level of glucoraphanin than standard broccoli cultivars grown in the same environment. In a first step, wild B. oleracea species were extensively screened for GSLs composition, and accessions from Brassica villosa bivoniana with high glucoraphanin content were selected and crossed with a standard broccoli cultivar (Faulkner et al., 1998). In a second step, the mapping of QTLs in segregating backcross populations derived from these F1 hybrids led to the identification of a major QTL on linkage group 2 that

The dilemma of “good” and “bad” glucosinolates

21

determined the concentration of methionine-derived GSLs (Mithen et al., 2003). Thirdly, high quality F1 cultivars (breeding line 428-11-69) containing the high glucoraphanin trait were developed, and extensive field studies in United States, Mexico, Spain, Italy, and United Kingdom were carried out to ensure consistence in the high glucoraphanin phenotype over several years in different environments (Traka et al., 2013). All the high glucoraphanin hybrids possessed an introgressed B. villosa segment which contained a Myb28 allele. The introgression of this allele enhanced sulfate assimilation and specifically channeled the additional sulfur to glucoraphanin. In addition to this high concentration of glucoraphanin, these selected hybrids have enhanced conversion of GSLs into ITC through a reduction in nitrile production. Looking for sources of aliphatic GSLs in Sicilian Brassica wild species and local cultivars, Branca et al. (2002) found a great variability for GSL composition between the wild species surveyed, and crops of the same species (B. oleracea, B. rapa, Brassica macrocarpa, and Brassica rupestris). They also found Sicilian local cultivars of broccoli and colored cauliflower containing glucoraphanin that could be useful to initiate a breeding program for functional food development. Divergent mass selection has been used to generate groups of individuals with extreme values for a specific GSL, and with the same genetic background. Sotelo et al. (2016) reported the first results of divergent selection for the three major GSLs (sinigrin, glucoiberin, and glucobrassicin) in leaves of B. oleracea var. acephala. After three cycles of divergent selection, the content of sinigrin and glucoiberin increased 52.5% and 77.7% and decreased 51.9% and 45.3%, respectively. The divergent selection for glucobrassicin content was only successful for reducing the concentration (39.0%). Liu et al. (2012) demonstrated that it is possible to produce B. napus with high content in glucoraphanin and low content in progoitrin. The canola cultivar “Westar” of B. napus was transformed with fragments from the B. oleracea GSL-ALK gene, and they used RNA interference of the GSL-ALK gene family to silence the entire GSL-ALK gene family. As a consequence, progoitrin was reduced by 65% and glucoraphanin was increased to 42.6 mmol/g in seeds of B. napus transgenic plants. Zang et al. (2008) modified aliphatic GSL biosynthesis in Chinese cabbage (B. rapa) by introducing three Arabidopsis cDNAs, MAM1, CYP79F1, and CYP83A1 using Agrobacterium tumefaciens-mediated transformation. GSL contents of the transgenic plants were compared with those of the wild type. The different transgenic lines exhibited dissimilar GSL profiles. In the

22

Glucosinolates: Properties, Recovery, and Applications

MAM1 line M1-1, accumulation of aliphatic GSLs gluconapin and glucobrassicanapin significantly increased. However, CYP79F1-2 and CYP79F1-3 transgenic lines showed a decrease in the levels of gluconapin and glucobrassicanapin and an increased level of 4-OH glucobrassicin. Seo et al. (2016) transferred three BrMYB28 genes into two different Chinese cabbage inbred lines using Agrobacterium-mediated transformation. All transgenic plants showed markedly increased contents of both aliphatic and indolic GSLs compared with nontransgenic plants (Table 1.3).

1.6 Preharvest factors influencing the concentration of GSL and the potential for agronomic biofortification The GSL profile of cruciferous plants is the outcome of a balance between GSL biosynthesis and metabolism, which are determined by a complex interaction between genetic and environmental factors (Engelen-Eiges et al., 2005; Farnham et al., 2004). While plant genotype seems to determine mostly the qualitative GSL profile, environmental conditions are strong determinants of the quantitative GSL profile (Table 1.4). Climatic conditions such as solar radiation, photosynthetic period, temperature, and relative humidity concurrently contribute to determine synthesis and metabolism, and thus the accumulation of GSLs. According to Justen et al. (2013), high temperatures throughout the growing period (21e34 C) increased GSL accumulation in both roots and shoots of two turnip cultivars through the regulation of TF BrMYB. Similarly, in rutabaga (B. napus L. ssp. rapifera Metzg.) Johansen et al. (2016) observed enhanced progoitrin content and higher bitter taste at 21 C compared with 9 C; in cabbage, temperatures over 30 C induced stress and caused higher accumulation of GSLs (Radovich et al., 2005). Such results were consistent with the lower GSL content observed in kale at lower temperatures (Velasco et al., 2007). However, analyzing the temperature effect on multiple accessions of A. thaliana, Kissen et al. (2016) observed that in most accessions moderate and pronounced chilling temperatures led to higher levels of GSLs, and especially of aliphatic GSLs, although the temperature effect on GSL content was accession-dependent. Other authors observed that low or high temperatures enhanced GSL as compared with moderate temperatures (Charron and Sams, 2004; Pereira et al., 2002), suggesting that GSL biosynthesis is stimulated by thermal stresses. Schonhof et al. (2007b) also observed that the effect of the temperature on GSL content is mediated by the radiation level. In broccoli grown in greenhouse under different

The dilemma of “good” and “bad” glucosinolates

23

Table 1.3 Summary of the loci/genes involved in the biosynthesis or production of GSLs in some Brassicaceae cited in this chapter. Crop Loci/genes Function References

Arabidopsis thaliana

GSL-ELONG/ MAMs

Control of sidechain length

A. thaliana

GSL-ALK

A. thaliana

GSL-OH/AOPs

Brassica napus

GSL-PRO

B. napus

Two loci

B. napus

Four QTLs

B. napus

Nine QTLs

Brassica juncea B. juncea

Seven unlinked loci

B. juncea

J16Gsl4 GSL-PRO

B. juncea

BjuA.GSL-Elong.a

B. juncea

BjuA.GSL-Elong.c

B. juncea

CYP79F1

Brassica oleracea

GSL-OX

Control of sidechain desaturation Control of sidechain hydroxylation Control the presence or absence of propyl GSL Control hydroxylation of alkenyl GSLs Control seed GSL accumulation Control of major GSLs in seeds Control of low GSL content Control the base level GSL production Control the C3 elongation pathway Control the C4 elongation pathway Control the C5 elongation pathway Control the synthesis of C3 GSLs Control oxidation of

BjuA.Myb28.a

Kroymann et al. (2001); Magrath et al. (1994) Mithen and Campos (1996); Mithen et al. (1995) Konrda and Stefansson (1970) Kliebenstein et al. (2001); Mithen and Campos (1996); Parkin et al. (1994) Parkin et al. (1994)

Howell et al. (2003)

Liu et al. (2016)

Sodhi et al. (2002) Bisht et al. (2009)

Bisht et al. (2009)

Bisht et al. (2009)

Bisht et al. (2009)

Sharma et al. (2016)

Giamoustaris and Mithen (1996) Continued

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Glucosinolates: Properties, Recovery, and Applications

Table 1.3 Summary of the loci/genes involved in the biosynthesis or production of GSLs in some Brassicaceae cited in this chapter.dcont'd Crop

Loci/genes

Function

References

methylthio to methysufinil alkyl GSL B. oleracea

GSL-PRO

B. oleracea

GSL-ELONG

Brassica rapa

BrGTR1 þ BrGTR2

Control synthesis of C3 GSLs Control synthesis of C4 GSLst Regulates GSLs transport from maternal tissues to seeds

Li et al. (2001)

Li et al. (2001)

Nour-Eldin et al. (2017)

Table 1.4 Summary of the effects of preharvest factors on qualitative and quantitative glucosinolate profile of Brassicaceae crops. Preharvest Crop factors Effect on GSL/ITC References

Brassica oleracea var. italica, Eruca sativa Arabidopsis thaliana

Germination temperature

Growing temperature

B. oleracea var. acephala B. oleracea

Brassica napus ssp. rapifera

Photosynthetic light period

Higher germination temperature increased GSL content Moderate and pronounced chilling temperatures increased GSL content Low temperature decreased GSL content Low and high temperatures enhanced GSL content compared with intermediate temperatures 24 h of natural light reduce GSL content compared with 12 h photoperiod

Ragusa et al. (2017)

Kissen et al. (2016)

Velasco et al. (2007) Charron and Sams (2004); Pereira et al. (2002)

Mølmann et al. (2018)

The dilemma of “good” and “bad” glucosinolates

25

Table 1.4 Summary of the effects of preharvest factors on qualitative and quantitative glucosinolate profile of Brassicaceae crops.dcont'd Crop

B. oleracea var. italica

Preharvest factors

Preharvest light spectral properties

B. oleracea var. italica

Atmospheric CO2

B. napus

Drought stress

B. oleracea var. capitata

B. oleracea var. italica

Salinity Stress

Raphanus sativus Brassica rapa

B. oleracea var. italica

Sulfur fertilization

B. napus Tropaeolum majus B. oleracea var. capitata

Nitrogen fertilization

Effect on GSL/ITC

References

Supplemental far-red and red þ far-red light reduced GSL content Supplemental blue light increased GSL content Increased levels of CO2 (685 e820 ppm) increased aliphatic and total GSL content Early and late drought stress increased GSL content Drought stress during head development increased total and indole GSL Increased indolic and total GSL content

Steindal et al. (2016)

Increased total GSL content Decrease of aliphatic GSLs with chloride salt, increase of indolic and aromatic GSLs with Na2SO4 Increased methionine-derived and total GSL content

Decreased GSL content

Kopsell and Sams (2013); Kopsell et al. (2014) Schonhof et al. (2007c)

Jensen et al. (1996)

Radovich et al. (2005)

Di Gioia et al. (2018a); Guo et al. (2014) Yang et al. (2015) Aghajanzadeh and Ziaiifar (2018)

Rangkadilok et al. (2004); Schonhof et al. (2007a); Yang et al. (2015) Zhao et al. (1994) Bloem et al. (2007) Rosen et al. (2005) Continued

26

Glucosinolates: Properties, Recovery, and Applications

Table 1.4 Summary of the effects of preharvest factors on qualitative and quantitative glucosinolate profile of Brassicaceae crops.dcont'd Crop

Preharvest factors

Effect on GSL/ITC

B. oleracea var. italica B. napus B. oleracea var. italica

Schonhof et al. (2007a) Selenium fertilization

B. oleracea

B. napus Eruca sativa, Diplotaxis tenuifolia, Diplotaxis erucoides

References

Decreased GSL content No or inconsistent effect on GSL content

Methyl jasmonate Growing system

Increased GSL content Soilless cultivation increased total GSL content

Zhao et al. (1994) Mahn (2017); Robbins et al. (2005) Ávila et al. (2014); Doughty et al. (1995); McKenzie et al. (2017) Doughty et al. (1995) Di Gioia et al. (2018a)

temperature and radiation levels, Krumbein et al. (2010) observed an increase of glucoraphanin (aliphatic GSL) with decreasing temperature and increasing radiation, while glucobrassicin (indole GSL) increased in broccoli grown under high temperatures (>18 C) and low radiation levels (31.1
C), CO2 transits to the liquid phase, with a density of z 0.47 g/cm3, behaving as a lipid-soluble fluid and gas, which can be adjusted according to the biomass specificities (compounds to be extracted), according to the CO2 phase diagram. Temperature also plays a major role on supercritical fluid extraction, as it influences the density of the gases and, thus, the solubility of the compounds to be extracted (lower temperatures result in higher densities and increase the extractability of the compounds) (Patil et al., 2018) and that is why higher operation temperatures (for the same pressure) may not result in higher extraction yields. Dunford and Temelli (1996) studied the effect of supercritical CO2 on myrosinase activity and its impact on glucosinolates degradation in canola flakes and seeds, by combining temperature (35e75
C), pressures (21.4e62.1 MPa), extraction times (up to 5 h), and moisture content (up to 20% w/w). The results showed that supercritical fluid extraction at 75
C, 62.1 MPa for 5 h at 20% of moisture (w/w) inactivated the myrosinase in canola flakes and seeds of about 90 and 44%, respectively. Interestingly, it was reported a significant decrease in glucosinolates content in canola seeds, while in canola flakes the degradation was minimal, allowing to conclude that, for efficient extraction of whole glucosinolates, prior inactivation of myrosinase in canola flakes is not necessary. Li et al. (2010) also evaluated the feasibility of supercritical extraction, using CO2 on the extraction of glucosinolates, namely allyl isothiocyanates from wasabi (Wasabia japonica) leaves, roots, and stems, and showed that, at

Analysis of glucosinolates content in food products

231

25 MPa and 35
C, the autolysis of isothiocyanates was inhibited, contrarily to what would be observed in aqueous extraction systems. In a more recent study, Mirofci (2014) studied the influence of pressure, temperature, CO2 flow rate, and cosolvent on the extractability of total glucosinolates from arugula (Eruca sativa leaves) and found out that the extraction yields were the highest (among the studied conditions) when the extraction conditions were set to 300 bar, 45
C, 7% wt cosolvent (water), allowing to obtain a yield of 21.71%, while with ethanol and methanol the extraction yields were, respectively, 5.54 and 5.56%. Generally, an extraction temperature increase allowed to obtain a higher extraction yield; contrarily, rising the pressure level led to reduced amounts of glucosinolates. When compared with conventional Soxhlet extraction, the authors reported higher extraction yields to the supercritical fluid extraction, mainly due to the degradation of glucosinolates as a consequence of the high temperatures used in the traditional technique.

7.3.3 Glucosinolates detection methods Intact glucosinolates can be difficult to analyze, and only recently their study was possible when starting to use hydrolysis to produce forms more compatible with chromatography (Michinton et al., 1982; Clarke, 2010). Analytical determinations can now be performed to the desulfoglucosinolates or intact-glucosinolates forms. Intact glucosinolates can be converted into desulfoglucosinolates via sulfatase or into isothiocyanates via myrosinase. To detect the desulfoglucosinolates, one must use an official method proposed by the European Union, the ISO 9167-1 (1992) and an AOCS method Ak 1e92 (AOCS, 1998). HPLC-UV and GC-FID (flame ionization detector) methods can also be applied to detect and analyze desulfoglucosinolates (Hrncirík et al., 1998). For example, to use the HPLC-UV method one needs to know the relative response factor for the desulfoglucosinolates under study. These factors can be calculated using the response of individual purified standards under UV assay, being the majority of them continuously transcribed from original works. Nevertheless, there are already three official methods to obtain the necessary response factors: (1) EEC 1864/90 1990 (EEC, 1990), (2) ISO 9167-1 1992 (ISO, 1992), and (3) Ak 1e92 AOCS 1998 (AOCS, 1998). In fact, there are several studies indicating their own values for these factors, being necessary a more rigorous approach for the update and documentation of the different experimental procedures to obtain a certain value.

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Glucosinolates: Properties, Recovery, and Applications

On the other hand, to detect the isothiocyanates, GC-MS can be a good method for the more volatile compounds or using the derivatization of the desulfoglucosinolates to trimethylsilyl (TMS) ethers (ISO, 1992; AOCS, 1998; Clarke, 2010). There are several methods available for total intact-glucosinolates detection, such as thymol, palladium, or ELISA methods, as also the enzymatic release of glucose, among others. Nevertheless, as one can see in Table 7.2, HPLC is still the most accurate technique for deestimation of total and individual glucosinolates, nonetheless having some disadvantages such as the expensive equipment and the high volume of solvents needed (Li et al., 2017; Mawlong et al., 2017). For so, the development of a simple and low cost method was optimized by Mawlong et al. (2017) for a faster and economical estimation of total glucosinolates in oilseed Brassica. This method consists on the mixture of the defatted seed cake with 80% methanol, being centrifuged after keeping overnight at room temperature. The supernatant is collected after centrifugation and mixed again with 80% methanol, distilled water sodium tetrachloropalladate. The absorbance is then read at 425 nm, after incubation at room temperature for 1 h, and the estimation is predicted by a mathematical formula. The authors defend that this method can be used for an initial screening for which estimate of a matrix has high/low content in glucosinolates. The content of glucosinolates can be estimated by HPLC-MS using different standards, such as benzyl glucosinolate, sinigrin hydrate, and glucobrassicin (Bell et al., 2017; Hassini et al., 2017; Li et al., 2017). Zhong et al. (2012) studied the glucosinolates content in different parts of maca using HPLC. The extracts obtained with 70% methanol were subjected to adsorbing, enzymatic hydrolysis, and analysis through HPLC-MS. The main glucosinolates detected were two aromatic compounds, benzyl glucosinolates (about 75% of the total content) and methoxybenzyl glucosinolates. Bell et al. (2015) evaluated liquid chromatographyemass spectrometry (LC-MS) to obtain glucosinolates content for 35 rocket accessions and commercial varieties, being able to detect 13 different intact glucosinolates. LC-MS analysis was performed in the negative ion mode, photodiode array detector, and mass trap spectrometer, and the separation of samples was achieved with a C18 column. ESI was carried out at atmospheric pressure in negative ion mode and the identification of the compounds was performed at 229 nm, using their nominal mass and characteristic fragment ions. A similar method was used by Hassini et al. (2017) to analyze the intact glucosinolates content in broccoli sprouts, who used an HPLC-DAD system to

Table 7.2 Studies on detection and quantification of glucosinolates in food products. Method of analysis

Maca

Benzyl glucosinolates and methoxy-benzyl glucosinolates Total glucosinolates

Arabidopsis thaliana

Total glucosinolates

HPLC-DAD

Broccoli florets and collard Indian mustard Rocket salad

Total glucosinolates and its degradation products Total glucosinolates

HPLC-UV-DAD

Total glucosinolates

Spectroscopy (at 425 nm) HPLC-MS

Broccoli sprouts

Intact glucosinolates

HPLC-DAD

Chinese cabbage and pak choi

Total glucosinolates

HPLC

Rocket salad

Particularities of each study

Reference

HPLC-MS

70% methanol as solvent for extraction; further analyses of antifatigue effects

Zhong et al. (2012)

LC-MS

70% methanol as solvent for extraction; photodiode array detector, and mass trap spectrometer (229 nm) Identification and quantification by comparing retention times and UV spectra (229 nm) with known standards Aqueous extracts blanched and boiled, to study the effect of cooking on bioactive compounds content 80% methanol as solvent and homogenization of defatted seed cake 70% methanol as solvent for extraction; photodiode array detector, and ion trap spectrometer evaluation by principal component analysis 70% methanol as solvent for extraction; determination according to UV spectra at 227 nm Stir-frying experiments and effect of high temperatures on glucosinolates content

Bell et al. (2015)

Kissen et al. (2016) Radosevic et al. (2017) Mawlong et al. (2017) Bell et al. (2017)

Hassini et al. (2017) Nugrahedi et al. (2017)

233

Compound(s)

Analysis of glucosinolates content in food products

Source

Continued

234

Table 7.2 Studies on detection and quantification of glucosinolates in food products.dcont'd Compound(s)

Broccoli Radish

Sinigrin, glucobrassicin Total glucosinolates

Broccoli sprouts

Glucoraphanin and glucoerucin

Spider plant

Individual desulfoglucosinolates

Broccoli and radish sprouts Broccoli

Total glucosinolates Total glucosinolates

UHPLC-QqQMS/MS HPLC

Mustard seed

Isothiocyanates

HPLC-DAD

Sinigrin

HPLC-DAD

UPLCTQDMS/MS UHPLCQTRAP/MS/ MS LC-MS (for intact glucosinolates) and HPLC-DAD (for isothiocyanates) UHPLC-DAD

Particularities of each study

Reference

70% methanol as solvent for extraction; further analyses of antimicrobial activity Quantification by principal component analysis

Hinds et al. (2017) Maldini et al. (2017)

High hydrostatic pressure processing effect on final concentration and conversion to isothiocyanates

Westphal et al. (2017)

Identification by comparing retention times and UV spectra (229 nm) with known standards Further analysis of urine for biomarkers concentration Effect of selenium application on glucosinolates final content Reaction between the benzene 1,2-dithiol and the isothiocyanates that yields the compound 1,3-benzodithiole-2-thione; determination at 365 nm Determination at 365 nm

Omondi et al. (2017) Baenas et al. (2017) Mckenzie et al. (2017) Cools and Terry (2018)

Glucosinolates: Properties, Recovery, and Applications

Method of analysis

Source

Analysis of glucosinolates content in food products

235

identify the compounds according to their UV spectra and order of elution at 227 nm. These authors observed that independently of the primed seeds used, the level of individual glucosinolates did not change significantly, being obtained similar concentrations for the seven different compounds identified. Instead of using only one method to quantify total intact glucosinolates, Cools and Terry (2018) used two different techniques to obtain more accurate results relatively to isothiocyanates and sinigrin compounds. For isothiocyanates, it was necessary to promote the reaction between the benzene 1,2-dithiol and the isothiocyanates that yields the compound 1,3benzodithiole-2-thione in a total isothiocyanate concentrationedependent manner. The concentration of this last compound was then analyzed by HPLC-DAD at 365 nm. On the other hand, sinigrin extract was analyzed with no further treatment also by HPLC-DAD assay.

7.3.4 Glucosinolates quantification methods The quantification of glucosinolates can be of great interest because it helps to easily determine different sources for active components. Hinds et al. (2017) studied various broccoli cultivars using ultra performance liquid chromatographytandem mass spectrometry (UPLCTQD-MS/ MS) to determine their glucosinolates content. Sinigrin (100.2e244.3 mg/ g of DW) was found to be the most abundant glucosinolates present within the majority of broccoli (Brassica oleracea) cultivars investigated, followed by glucobrassicin (13.4e49.9 mg/g of DW) (Hinds et al., 2017). Radish can also be a great source of glucosinolates, and Maldini et al. (2017) analyzed and quantified the occurrence of glucosinolates in different parts of radish. The identification was performed by ultra-high performance liquid chromatography-triple quadrupole/linear ion trap tandem mass spectrometry (UHPLC-QTRAP/MS/MS); and the quantitative results were analyzed by principal component analysis (PCA). These authors observed that the glucosinolate with higher concentration was glucoraphenin representing about 87% of dry weight of total glucosinolates content, followed by glucobrassicin, glucoraphasatin, and glucoraphanin (Westphal et al., 2017) (153 mg/100 g, 149 mg/100 g, and 141 mg/100 g FW, respectively) in fruits. Relatively to the glucosinolate content in flowers, glucoraphasatin (3 mg/100 g FW) was the most predominant compound, while for leaves the compounds with higher concentration were glucobrassicin, 4-hydroxyglucobrassicin, and glucoraphasatin (145 mg/100 g, 27 mg/100 g, and 24 mg/100 g FW,

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Glucosinolates: Properties, Recovery, and Applications

respectively). In roots, the major glucosinolates was glucoraphasatin (56 mg/100 g FW). PCA allowed to discriminate the fruit samples from the other parts of the plant for the majority of glucosinolates, indicating that the fruits are the better source for these compounds (Maldini et al., 2017). As was stated above, glucosinolates themselves are not known to be bioactive, being necessary their conversion to isothiocyanates for achieving bioactivity through the glucosinolatemyrosinase system. Thereby, it is interesting not only to analyze and quantify total glucosinolates’ content in food but also to study their isothiocyanates content. Westphal et al. (2017) studied the predominant glucosinolates in broccoli and broccoli sprouts (glucoraphanin and glucoerucin), which can be hydrolyzed into the isothiocyanates sulforaphane and erucin, respectively. In this study, the authors predicted that, if the broccoli sprouts were treated by HP, the processing would lead to the decompartmentalization and enzymatic conversion of glucosinolates into isothiocyanates. According to that prediction, more glucosinolates were converted into isothiocyanates after HPP at 400e600 MPa, 3 min, 30
C, because the sprouts submitted to HP presented higher isothiocyanate content (3.05e3.45 mmol/g) than the raw, untreated sprouts (0.57 mmol/g). For quantification, glucosinolates content was analyzed by LC-MS, and isothiocyanates content was analyzed by HPLC using a photodiode array detection (Westphal et al., 2017). Relatively to desulfoglucosinolates quantification, as stated in the previous section of this chapter, it is necessary to use the response factor relatively to some external standard, such as sinigrin or 2-propenyl glucosinolates (Clarke, 2010; Omondi et al., 2017). Omondi et al. (2017) studied the extraction and quantification of glucosinolates from spider plant using a UHPLC-DAD device and observed that 3-hydroxypropyl glucosinolates were the main glucosinolates present, followed by the aliphatic methyl glucosinolates (glucocapparin), the 2-hydroxy-2-methylbutyl glucosinolates (glucocleomin), and the methyl glucosinolate. Desulfoglucosinolates were identified using their respective retention times and UV spectra by comparison with known standards. Concerning their quantification at 229 nm, it was considered the response factor of glucosinolates relative to 2-propenyl glucosinolate, used as external standard (Omondi et al., 2017). These authors also concluded that glucosinolates quantification can be different depending on the growth environment for each plant; for example, for the same spider plant species, if it had grown in the greenhouse or in the field, there would be higher concentration of total and

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237

indole glucosinolates in the plants that have grown in the field. These differences may be due to the different climate conditions, e.g., higher irradiation intensities, especially of UVB radiation (Mewis et al., 2012; Omondi et al., 2017). Although it is important to quantify glucosinolates content in certain foods, it is also of great importance to know their bioavailability after consumption. Baenas et al. (2017) studied the bioavailability of glucosinolates from broccoli and radish sprouts after a 7-day-crossover study with 14 women. The analyses were performed by UHPLC-QqQ-MS/MS in the urinary excretion, and some compounds were studied as biomarkers, such as sulforaphene, sulforaphane-N-acetyl-L-cysteine, and 3,30 -diindolylmethane, because they are easily excreted through urine. These authors concluded that, even after repeated dosage of sprouts to the subjects, it was not observed an increase of the biomarkers in the urine, meaning a safe consumption of cruciferous foods, because no accumulation of isothiocyanates nor indoles was observed during the first 12 h after ingestion for a 7-day intervention (Baenas et al., 2017). Besides the need to study the bioavailability of these compounds after ingestion, also the cooking process of cruciferous foods can affect the glucosinolates concentration on such foods. Nugrahedi et al. (2017) studied the effect of stir-frying, a traditional cooking method from Asia, on the content of glucosinolates from Chinese cabbage, as also as boiling for cooking of pak choi. These authors concluded that, due to the high temperature used for frying, the myrosinase enzyme is readily inactivated right after the first minutes of cooking, and, for so, intact glucosinolates are retained and Chinese cabbage maintains also its nutritional value. On the other hand, pak choi was subjected to boiling water for an extended period of time, being observed much lower values of glucosinolates, a clear consequence of the high thermal degradation rate of these compounds and its leaching into the cooking water (Nugrahedi et al., 2017). Radosevic et al. (2017) also studied the effect of cooking treatment (blanching in tap water for 5 min at 60
C and boiling in tap water for 5 min at 100
C) of broccoli florets and wild-type collard on glucosinolates and its degradation products concentration. The determination of total glucosinolates was carried out by HPLC-DAD according to the ISO (1992), and their detection and identification was performed by UV-DAD peak spectra analyses, while glucosinolates degradation products, such as glucobrassicin, were identified and quantified according to UV spectra (190 e370 nm), retention times, and calibration curves of standards (indolca-3-carbinol, indole-3-acetonitrile, 3,30 -diindolylmethane). These authors observed

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Glucosinolates: Properties, Recovery, and Applications

similar values of glucosinolates after blanching (compared with the initial value of fresh vegetables), indicating that myrosinase was still active. Nevertheless, after boiling only about 20% and 35% glucosinolates remained in broccoli and collard, respectively (Radosevic et al., 2017). These results reflect the fragile glucosinolates stability during processing, already discussed in Section 7.3.1 of this chapter, indicating that during thermal processing of these food products, the concentration of glucosinolates may be reduced primarily through leaching into the cooking water, but also due to enzyme action or even thermal breakdown (Wu et al., 2017). In addition to the bioavailability after ingestion and the effect of processing on glucosinolates concentration in food products, the environmental factors can also greatly affect its content. Kissen et al. (2016) studied how environmental factors affect the concentration and composition of glucosinolates in Arabidopsis thaliana grown at 9e21
C. The desulfoglucosinolates were separated by HPLC, monitored by UV DAD, identified through their retention times, and quantified by absorbance at 229 nm (by comparison with standard and respective response factors). The main results showed that chilling temperatures (9 and 15
C) allowed obtaining higher concentration values for total glucosinolates.

7.3.5 Isolation Some specific plants rich in glucosinolates are used to isolate analytical standards, such as lead acetate (Pb(OAc)2) and barium acetate (Ba(OAc)2), which are frequently used to precipitate protein and free sulfate, respectively (Clarke, 2010). Therefore, there is a need for isolation and purification methods for the industrialization usage of glucosinolates. After glucosinolates extraction and centrifugation, a purification step is usually required, which generally involves an anion-exchange step in a DEAE Sephadex A-25 column. A typical procedure for intact glucosinolates extraction consists in their elution with 0.5 M of potassium sulfate and their collection into absolute ethanol. After a centrifugation step, glucosinolates should be dried and redissolved in hot methanol to separate the insoluble salts. Then, after another centrifugation step, glucosinolates are recrystallized with cold ethanol and dried (De Graaf et al., 2015; Förster et al., 2015). To clean up and isolate intact glucosinolates, florisil solid-phase extraction is commonly used with application in methanol/dichloromethane/hexane, washing with dichloromethane/hexane and elution with

Analysis of glucosinolates content in food products

239

methanol/ethyl acetate (Clarke, 2010). The resulting crystallized glucosinolates mixture is redissolved in ultrapure water and fractionated with a Dionex HPLC system equipped with a fraction collector which can collect single peaks. The fractions are dried and dissolved in ultrapure water or buffer to be analyzed by mass spectrometry to confirm the identity of individual glucosinolates (De Graaf et al., 2015; Förster et al., 2015). The anion exchange on a styrene-divinylbenzene copolymeric anion exchanger gives an additional dimension for separation, with high selectivity and elution based in glucosinolates hydrophobicity. For that, the    inorganic anions SO2 4 , NO3 , ClO3 , and ClO4 should be used in sequence to the high-speed countercurrent chromatography relying solely on the partition coefficient of the solute between the stationary and mobile phases that can be used to separate structurally and chromatographically similar glucosinolates (Elfakir et al., 1994; Clarke, 2010).

7.3.6 Purity of analytical standards: water content by NMR The elution of glucosinolates from ion-exchange chromatographic resins (and its isolation) with potassium sulfate results in white crystalline salts of potassium, that might be not pure. HPLC-UV is often used to measure the organic content, as purities higher than 95% are generally accepted. As glucosinolates are reported to be quite hydroscopic, it is difficult to evaluate the amount of crystallization water in each standard (Tian et al., 2013). According to Mohn et al. (2007), an internal standard of trimethoxybenzene (0.333 mM) in 10% d4-methanol in deuterium oxide (D2O) can be used to dissolve 1000  4 mg of solid glucosinolates (3 mM) in an NMR test tube (Mirofci (2014) used deuterated chloroform (CDCl3) as solvent for the analysis of glucosinolates in arugula (E. sativa) leaves). The aforementioned authors were able to analyze both nonaromatic and aromatic glucosinolates using this procedure. For nonaromatic glucosinolates, it was possible to compare the anomeric hydrogen at 4.5 ppm with the area of nine methyl protons from the trimethoxybenzene, while for aromatic glucosinolates, the region between 7 and 8 ppm was integrated and divided by the number of originating protons. The real content was between 99% sinigrin/glucotropaeolin, 77% gluconapin, to 17% for 4hydroxyglucobrassicin. In a more recent study, De Graaf et al. (2015) suspended 1 mg of glucosinolates extracts (isolated from Noccaea caerulescens) in D2O or CDCl3 for the isothiocyanates, to a final concentration lower than 0.1 M.

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Glucosinolates: Properties, Recovery, and Applications

7.3.7 Extinction coefficients A feasible indicator of the purity of organic substances relies on the molar extinction coefficients, allowing introducing correction factors between natural products and the reference values. For example, at a wavelength 224 nm, the N-hydroxysulfate moiety of glucosinolates has an extinction coefficient of 7000 M1cm1. Indole, alkenyl, and aryl side chains have absorbances of low intensities (at 250e280 nm) (Troyer et al., 2001). The determination of extinction coefficients is a procedure that requires precision and high accuracy. For example, at 227 nm, the extinction coefficients values for pure sinigrin were reported to range from 7140 to 7662 M1cm1. The authors Stoin et al. (2007) also argued that the standard error associated with the individual values was minimal (