Phenolic Acids: Composition, Applications and Health Benefits : Composition, Applications and Health Benefits [1 ed.] 9781619420403, 9781619420328

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Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Phenolic Acids: Composition, Applications and Health Benefits : Composition, Applications and Health Benefits, Nova Science Publishers, Incorporated,

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Phenolic Acids: Composition, Applications and Health Benefits : Composition, Applications and Health Benefits, Nova Science Publishers,

BIOCHEMISTRY RESEARCH TRENDS

PHENOLIC ACIDS

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

COMPOSITION, APPLICATIONS AND HEALTH BENEFITS

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BIOCHEMISTRY RESEARCH TRENDS

PHENOLIC ACIDS COMPOSITION, APPLICATIONS AND HEALTH BENEFITS

SERGI MUNNÉ-BOSCH Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

EDITOR

Nova Science Publishers, Inc. New York Phenolic Acids: Composition, Applications and Health Benefits : Composition, Applications and Health Benefits, Nova Science Publishers,

Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data Phenolic acids : composition, applications, and health benefits / editor, Sergi Munni-Bosch. p. ; cm. Includes bibliographical references and index. ISBN:  (eBook) I. Munni-Bosch, Sergi. [DNLM: 1. Hydroxybenzoic Acids--chemistry. 2. Hydroxybenzoic Acids--therapeutic use. QU 98] 547'.2--dc23 2011042420

Published by Nova Science Publishers, Inc. † New York Phenolic Acids: Composition, Applications and Health Benefits : Composition, Applications and Health Benefits, Nova Science Publishers,

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To Leonor Alegre, for excellent advice during my early career steps To my wife Marta, for our past, ongoing and future projects

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Contents

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Preface

ix

Chapter I

Fruits and Vegetables: A Rich Source of Phenolic Acids C. Proestos, A. E. Koutelidakis, M. Kapsokefalou and M. Komaitis

Chapter II

Phenolic Compounds in Staple Plants F. Edi-Soetaredjo, S. Ismadji and Y-H. Ju

Chapter III

Phenolic Acids in Plant Cell Walls: Composition and Industrial Applications I. Zarra, G. Revilla, J. Sampedro and E. R. Valdivia

Chapter IV

p-coumaric Acid Production from Lignocelluloses S. Y. Ou, J. W. Teng, Y. Y. Zhao and J. Zhao

Chapter V

Antioxidant Activity of Phenolic Acids: Correlation with Chemical Structure and in vitro Assays for Their Analytical Determination G. Cirillo, O. I. Parisi, D. Restuccia, F. Puoci and N. Picci

Chapter VI

Chapter VII

Phenolic Acid Composition in Food Systems: Sample Preparation and Analytical Aspects R. N. Cavalcanti, M. A. Rostagno and M. A. A. Meireles Phenolic Acids as Additives in the Food Industry M. L. R. Giada

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1

15

33

63

73

97

125

viii Chapter VIII

Chapter IX

Chapter X

Contents Phenolic Acids from Microbial Metabolism of Dietary Flavan-3-ols María Boto-Ordóñez, Mireia Urpi-Sarda, María Monagas, Sara Tulipani, Rafael Llorach, Montse Rabassa-Bonet , Mar Garcia-Aloy, María Isabel Queipo-Ortuño, Ramon Estruch, Francisco Tinahones, Begoña Bartolomé and Cristina Andres-Lacueva The Potential Role of Phenolic Acids in Tea and Herbal Teas in Modulating Effects of Obesity and Diabetes E. Joubert, C. J. F. Muller, D. De Beer, R. Johnson, N. Chellan and J. Louw Salicylates and Cancer L. A. J. Mur and P. Elwood

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Index

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147

173

213 229

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Preface This book aims at introducing the non-specialist as well as the professionals in the fields of chemistry, biochemistry, biotechnology, food science and biomedical sciences into the biochemistry, applications and health benefits of an important group of natural products, phenolic acids. The book shows how phenolic acids display a potent antioxidant capacity and therefore health promoting effects and describes the phenolic acid composition in fruits and vegetables. The book also covers industrial applications related to phenolic acids. Furthermore, the book discusses the application of phenolic acids as food additives, since the preparation of foods with a high content of phenolic acids can lead to a reduction in the use of synthetic additives, resulting in healthier foods that can be included in the functional foods group. The book is of interest to specialists in the field but also to graduate or postgraduate students that are interested in this particular group of natural products. Chapter I – In recent years many scientific studies have emphasized on the importance of bioactive compounds such as phenolic acids and their biological role in human diet. A plethora of plant based foods have been assessed for their antioxidant and antimicrobial properties in vitro, which have been linked with the presence of phenolic acids and other bioactive compounds. There is a tendency of replacing synthetic antioxidants such as BHT and BHA with plant antioxidants such as phenolic acids. Phenolic acids have antioxidant properties and can protect against a number of degenerative diseases (e.g. cancer, cardiovascular diseases) in which reactive oxygen species (e.g. hydroxyl radicals, peroxy radicals, superoxide ions) are involved. Phenolic acids are derivatives of benzoic and cinnamic acids and are present in all plant based foods. They occur in both free and bound forms. The purpose of this chapter is to give an overview of some of the phenolic acids present in plant foods and discuss their important antioxidant capacity. Chapter II – A staple food is consumed regularly. It is a dominant constituent in the diet and is a major supplier of energy and nutrients. Staple foods vary from place to place, but are typically inexpensive, starchy foods that supply one or more of the three macronutrients needed for human health: carbohydrate, protein and fat. Staple foods mostly are from cereals such as wheat, barley, rye, maize, rice, or starchy root vegetables such as potato, yam, taro, cassava, sago (derived from the pith of the sago palm tree), and fruits such as breadfruit and plantains. The minor compositions of staple foods are minerals (phosphorous, potassium, sodium, calcium, magnesium, iron, zinc, etc.), vitamins and phenolic compounds. Rice, wheat, maize,

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Sergi Munné-Bosch

barley, and rye are the dominant staple foods in the world. A number of studies have been carried out to investigate the phenolic compounds in those staple foods that are described in this chapter. Phenolic acids are secondary metabolites widely distributed in the plant kingdom and are second only to flavonoids in terms of their dominance. Cereals, including barley, oats and wheat, are known to contain a wide range of phenolic acids that belong to benzoic and cinnamic acid derivatives. These include ferulic, caffeic, phydroxybenzoic, protocatechuic, p-coumaric, vanillic and syringic acids. This chapter also provides phenolic content data in other staple plants such as barley, rye, potato, yam and sago. The composition and amounts of phenolic compounds in staple plants are important for their contribution to color, sensory attributes and nutritional and antioxidants properties of foods. These natural antioxidants are known to exhibit a wide range of biological effects including antibacterial, antiviral and anti-inflammatory, anti-allergic, antithrombotic and vasodilatory activities. As the world population is increasing, the amount of staple food production and consumption is also increasing; therefore staple plants together with vegetables and fruits have an important role in fulfilling human need for antioxidants. Moreover, the increasing number of staple plant byproduct such as bran, is a potential source of phenolic compounds. Chapter III – Phenolic compounds in plants show not only a very diverse structure but also a wide range of functions. In this chapter we focus on phenolic acids associated with plant cell walls and their role as structural components, as well as their industrial applications. Plant cell walls are made of cellulose fibrils embedded in a matrix of polysaccharides, structural proteins, enzymes and phenolics. In the walls surrounding growing cells (primary walls) phenolic acids, such as ferulic acid and coumaric acid, are involved in the cross linking of cell wall polysaccharides. In addition, the secondary walls of cells that have stopped growing and have differentiated are typically impregnated with lignin which strengthens and dehydrates the wall. Thus, phenolic compounds have a key role not only in the primary wall but also in the secondary one as the main component of lignin. Besides extensibility, matrix cross-links are also likely to determine wall digestibility. In secondary walls lignin provides mechanical support and a water-impermeable surface essential for the evolution of terrestrial vascular plants. However, the presence of lignin leading to lignocellulosic biomass recalcitrance is a bottleneck for the industrial use of plants. Lignin not only reduces digestibility but also interferes with the pulping process in the paper industry and with saccharification (enzymatic bioconversion of cellulose and other wall polysaccharides into fermentable sugars). The different approaches developed for the modification of plant biomass through manipulation of phenolics will be discussed. Chapter IV – p-coumaric acid is a hydroxycinnamic acid with many physiological actions, including antioxidant, antimicrobial, antimutagenic, anxiolytic, analgesic, sedative, and immunoregulatory activities. It is widely used in the chemical, food, health, cosmetic, and pharmaceutical industries. p-coumaric acid exists in plants in two states, soluble and insoluble. Soluble p-coumaric acid can be found in a wide variety of foods of plant origin, including fruits, vegetables, and cereals. Insoluble p-coumaric acid constitutes a major component of lignocelluloses, mainly esterified with lignin, in straws, cobs, sugarcane bagasse, and maize cob, and is present at much higher levels than soluble p-coumaric acid. This chapter will review the production of p-coumaric acid for their application in the food or

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Preface

xi

pharmaceutical industries. Production of p-coumaric acid from sugarcane bagasse will be discussed in detail. Chapter V – Phenolic acids are a subclass of a larger category of plant metabolites commonly referred to as “phenolics” possessing a carboxylic acid functionality. In general, the term phenolics encompasses approximately 8000 naturally occurring compounds, all of which possess one common structural feature, a phenol (an aromatic ring bearing at least one hydroxyl substituent). The naturally occurring phenolic acids contain two distinguishing constitutive carbon frameworks: the hydroxycinnamic and hydroxybenzoic structures. Although the basic skeleton remains the same, the numbers and positions of the hydroxyl groups on the aromatic ring create the variety. Recent interest in phenolic acids stems from their potential protective role, through ingestion of fruits and vegetables, against oxidative damage-related diseases due to their antioxidant activity (AOA). This property is related with the ability to scavenge free radicals, donate hydrogen atoms or electron, or chelate metal cations. The molecular structure, referred as structure–activity relationships SAR (including substituents on the aromatic ring, numbers and positions of the hydroxyl groups in relation to the carboxyl functional group, esterification, glycosylation) affects the antioxidant properties.With the current upsurge of interest in the function, measurement of efficacy and use of natural antioxidants has received much attention. There is a great multiplicity of the methods used for the determination of AOA, which can be broadly classified in two groups. In the first group, the degree of inhibition of lipid peroxidation is measured by using lipid or lipoprotein substrate under standard conditions; while in the second the radical scavenging ability is determined. Moreover, a number of assays have been introduced for determining the total antioxidant activity, intended as the cumulative capacity of food compounds to scavenge free radicals. In the discussion, phenolic acids structure as well as SAR will be considered. The various in vitro methods used for the determination of antioxidant activity, with their merits and limitations, will be also presented. Chapter VI – Increasing scientific evidence supports that some phytochemicals naturally present in the diet may play a role on the prevention of important diseases, such as cardiovascular diseases, cancer and some neurodegenerative disorders. Among them, polyphenols are one of the most studied phytochemicals because of their relative abundance in foods and their antioxidant properties. Phenolic acids are simple phenolic compounds which are present in high amounts in coffee, tea, cocoa, grape and other fruits. However, depending of the sample, the profile and concentration of phenolic acids can be very different. Moreover, environmental and genetic factors can influence the chemical profiles of the sample. In general, for the determination of phenolic acids, they need to be isolated from the sample matrix in a series of steps beginning with extraction, followed by isolation and purification to obtain a clean extract rich in phenolic compounds that are later analyzed. There are several “modern” sample preparation techniques that are currently being used for the extraction of phenolics from different sample types, such as supercritical fluid extraction, pressurized liquid extraction and solid phase extraction. On the other hand, several analytical methods can be used for the determination of the phenolic profile of the samples in which HPLC is the most widespread technique. The current research focus of HPLC separations is being directed towards highly efficient methods that allow faster separations to be achieved. This chapter intends to highlight the main extraction, separation, and identification techniques and methods used for the determination of the phenolic acid composition of different food samples.

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Sergi Munné-Bosch

Chapter VII – Phenolic acids are among the most numerous and widely distributed group of plant secondary metabolites. Like other plant phenolic compounds, they have received increasing interest in recent years, once scientific research has shown that the consumption of vegetables or beverages rich in those substances may reduce the risk of developing several diseases. However, beyond the nutritional interest, such substances are also of technological interest. They can act as natural antioxidants present in food. Additionally, many of these substances have antimicrobial activity and can retard the deterioration of food due to the action of microorganisms. Thus, the preparation of foods with a high content of such substances leads to a reduction in the use of synthetic additives, resulting in healthier foods that can be included in the functional foods group. In this chapter, phenolic acids sources as well as their physicochemical, antioxidant and antimicrobial properties are discussed. Additionally, foods with potential for the application of these substances as additives are presented. Chapter VIII – Flavan-3-ols are polyphenols present in the diet in monomeric, oligomeric and polymeric forms, but their bioactivity and in vivo health effects remain unclear due to their complex metabolism. According to the degree of polymerization, monomeric flavan-3ols can be absorbed in the small intestine, whereas oligomers and polymers need to be biotransformed by the colonic microbiota before absorption. This latter gives rise to a wide number and variety of phenolic acids which may be responsible for the health effects derived from flavan-3-ol consumption rather than the original phenolic forms found in foods. Although in vitro studies have revealed that some bacteria are able to catabolise certain class of polyphenols, the identification of human colonic bacteria with capacity to catabolise flavan-3-ols is in its early stages. However, in the last decade a great progress has been achieved in the identification of phenolic acids derived from the catabolism of flavan-3-ols by gut microbiota. The link between consumption of flavan-3-ols food sources and those metabolites found in vivo, with related health effects is still a difficult challenge due to the huge variability in colonic biotransformation found among individuals, and other factors such as the own structural diversity of these polyphenols and food matrix that add a further variability in catabolism. Studies performed with isolated phenolic compounds in a colonic environment may help us to identify colonic bacteria involved in catabolism and understand their activity in the colon, and set up a link to circulating metabolites found in vivo. Although the biological relevance of microbial metabolites remains largely unknown, evidences related to their antioxidant, anti-inflammatory and anti-proliferative activities and cytotoxicity are starting to be accumulated. This chapter aims to give an insight into the phenolic acids formed by the colonic catabolism of dietary flavan-3-ols, including tentative metabolic pathways, potential microbial groups/species involved in their catabolism, plasma and urine concentrations found after in vivo consumption, and specific bioactivities. All these aspects may help us better understand the complexity of the colonic catabolism of flavan-3-ols and the role of phenolic acids in health effects derived from the consumption of flavan-3-ol rich sources. Chapter IX – Obesity and diabetes have reached global epidemic proportions both in the developed and developing world. Major contributing factors are a sedentary lifestyle and diets rich in saturated fats and sugar. This chapter will provide general background on obesity and diabetes in terms of their prevalence and burden to society, the link between insulin resistance, obesity and type 2 diabetes and the use of natural products as alternatives to pharmaceutical products. Current evidence indicates that phenolic acids may play a

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Preface

xiii

preventative and protective role against risk factors associated with obesity and diabetes. Phenolic acids, shown to have a beneficial effect on carbohydrate digestion, glucose absorption and metabolism, insulin secretion and lipid metabolism, amongst others, are present in plant foods and beverages consumed as part of the daily diet. The focus will fall on green and black teas (Camellia sinensis), the South American herbal tea, mate (Ilex paraguariensis), German chamomile (Matricaria chamomilla) and the South African herbal teas, rooibos (Aspalathus linearis) and bush tea (Athrixia phylicoides) as sources of phenolic acids, and specifically gallic acid, ferulic acid and chlorogenic acid. Insight will be provided into their intake through ingestion of these teas, as well as their bioavailability and mechanisms of action associated with anti-obesity and anti-diabetic effects. Chapter X – Aspirin is an acetylated form of a naturally occurring plant phenolic salicylic acid and both have analgesic, anti-pyretic and anti-inflammatory pharmaceutical properties. The taking of prophylactic aspirin is an established means of reducing the incidence of cardiovascular events such as heart attacks or strokes. This property of aspirin arises from its inhibition of cyclo-oxygenase (COX) to reduce the prostaglandin thromboxane which regulates platelet aggregation. Salicylate may not be a significant inhibitor of COX activity. Recent evidence indicates that aspirin can reduce the incidence and development of certain types of cancer, particularly colorectal cancer. This is likely to arise from COX-dependent and COX-independent mechanisms. COX derived prostaglandins will promote an inflammatory response which is a contributory factor to tumourigenesis, thus COX inhibition will reduce the incidence of cancer. COX-independent mechanisms will also include responses to salicylate as aspirin is readily metabolized to salicylate in the human body. These mechanisms include 1) DNA mismatch repair 2) the initiation of tumour cell death (apoptosis) through mitochondrial dysfunction and the release of pro-apoptotic proteins coupled to the suppression of anti-apoptotic proteins of the Bcl-2 class; 3) reducing capillary formation around the tumour by suppressing VEGF expression and 4) lowering metalloproteinase activity to suppress metastasis. In plants salicylate also influences mitochondrial function and hence this organelle is a cross-kingdom target for salicylate function. Thus, dietary salicylate could have a nutraceutical role now being revealed by modern studies on the prophylactic effects of aspirin. This chapter will demonstrate how a cross-kingdom understanding of salicylate action can facilitate major clinical advances.

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In: Phenolic Acids Editor: Sergi Munné-Bosch

ISBN: 978-1-61942-032-8 © 2012 Nova Science Publishers, Inc.

Chapter I

Fruits and Vegetables: A Rich Source of Phenolic Acids C. Proestos1, A. E. Koutelidakis2, M. Kapsokefalou2 and M. Komaitis2,* 1

Food Chemistry Laboratory, Department of Chemistry, National and Kapodistrian University of Athens, Panepistimioupolis Zografou, 15771, Athens, Greece 2 Unit of Human Nutrition, Laboratory of Food Chemistry and Analysis, Department of Food Science and Technology, Agricultural University of Athens, Athens, Greece

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Abstract In recent years many scientific studies have emphasized on the importance of bioactive compounds such as phenolic acids and their biological role in human diet. A plethora of plant based foods have been assessed for their antioxidant and antimicrobial properties in vitro, which have been linked with the presence of phenolic acids and other bioactive compounds. There is a tendency of replacing synthetic antioxidants such as BHT, and BHA with plant antioxidants such as phenolic acids. Phenolic acids have antioxidant properties and can protect against a number of degenerative diseases (e.g. cancer, cardiovascular diseases) in which reactive oxygen species (e.g. hydroxyl radicals, peroxy radicals, superoxide ions) are involved. Phenolic acids are derivatives of benzoic and cinnamic acids and are present in all plant based foods. They occur in both free and bound forms. The purpose of this chapter is to give an overview of some of the phenolic acids present in plant foods and discuss their important antioxidant capacity.

Phenolic Acids in Plant Foods Phenolic acids are plant metabolites widely spread throughout the plant kingdom [1]. Currently there is much scientific interest for their potential protective role, through ingestion *

E-mail address: [email protected].

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2

C. Proestos, A. E. Koutelidakis, M. Kapsokefalou et al.

of infusions, fruits and vegetables, against oxidative stress-related diseases (e.g. coronary heart disease, stroke, and cancers). Phenolic acids include both benzoic acid and cinnamic acid derivatives (Figures 1 and 2).

Figure 1. Chemical structures of (a) hydroxybenzoic acids: p-hydroxybenzoic acid, R1=H, R2=H; gallic acid, R1=OH, R2=OH, and (b) ellagic acid.

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Figure 2. Chemical structure of three common hydroxycinnamic acids: p-coumaric acid, R1=H; caffeic acid, R1=OH; ferulic acid, R1=OCH3.

Hydroxybenzoic acids have a general structure of C6-C1 derived directly from benzoic acid. Variations in the structures of individual hydroxybenzoic acids lie in the hydroxylations and methylations of the aromatic ring. Four acids occur commonly: p-hydroxybenzoic, vanillic, syringic, and protocatechuic acid [1]. They may be present in soluble form conjugated with sugars or organic acids as well as bound to cell wall fractions, e.g. lignin. A common hydroxybenzoic acid is also salicylic acid (2-hydroxybenzoate). Gallic acid is a trihydroxyl derivative which participates in the formation of hydrolysable gallotannins. Its dimeric condensation product (hexahydroxydiphenic acid) and related dilactone, ellagic acid, are common plant metabolites. Ellagic acid is usually present in ellagitannins as esters of diphenic acid analogue with glucose. The four most widely distributed hydroxycinnamic acids in fruits are p-coumaric, caffeic, ferulic and sinapic acids. Hydroxycinnamic acids usually occur in various conjugated forms, the free forms being artefacts from chemical or enzymatic hydrolysis during tissue extraction. The conjugated forms are esters of hydroxyacids such as quinic, shikimic and tartaric acid, as well as their sugar derivatives. The hydroxybenzoic acid content of edible plants is generally very low, with the exception of certain red fruits, black radish, and onions, which can have amounts of several tens of milligrams per kilogram fresh weight [2]. Tea is an important source of gallic acid: tea leaves may contain up to 4.5 g/kg fresh wt [3]. Furthermore, hydroxybenzoic acids are components of complex structures such as hydrolyzable tannins (gallotannins in mangoes and ellagitannins in red fruit such as strawberries, raspberries, and blackberries) [4].

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Table 1. Phenolic acids content of fruits consumed in Scottland [7]

Moisture (%) Phenolic acid (free) Gallic Protocatechuic p-hydroxybenzoic Gentisic Caffeic Vanillic Syringic p-coumaric Ferulic Sinapic

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Salicylic Gallic Protocatechuic p-hydroxybenzoic Gentisic

Raspberry 84.4

5.73 ± 0.53 3.83 ± 0.83 33.31 ± 6.54 n.d. 2.33 ± 0.62 24.58 ± 6.32 107.51 ± 47.28 n.d. 74.33 ± 17.20 36.89 ± 10.01 7.64 ± 4.77 1669.25 ± 256.24 273.26 ± 6 8.81 676.31 ± 76.59 n.d.

Vanillic

63.95 ± 8.05 94.34 ± 26.65

Syringic

5.91 ± 3.07

Caffeic

Gooseberry 86.4

Blackcurrant 79.0

Strawberry 87.9

Banana 73.9

Apple 85.2

1.27 ± 0.50

5.63 ± 0.64

27.22 ± 2.13

10.45 ± 1.70

n.d.

4.33 ± 0.97

1.67 ± 0.29 39.09 ± 2.07 193.91 ± 19.88 30.85 ± 2.10

0.02 ± 0.03 0.16 ± 0.02

0.11 ± 0.04 3.37 ± 0.84 34.09 ± 18.62

2.01 ± 0.40

n.d.

n.d

13.23 ± 1.45

3.73 ± 0.38

n.d.

n.d.

n.d.

15.01 ± 2.54

98.46 ± 3.73

0.30 ± 0.01 0.21 ± 0. 10

n.d.

n.d.

n.d.

n.d.

19.83 ± 11.7 9

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

62.09 ± 5.44

43.59 ± 15.63 389.80 ± 100.52 120.75 ± 19.75 41.28 ± 6.30 354.51 ± 60.82

999.18 ± 82.06 355.91 ± 53.78 259.16 ± 16.41 77.37 ± 6.87 537.23 ± 58.43

567.89 ± 28.82 450.30 ± 27.54

n.d

n.d.

Pear 84.5

Grapes 69.4

Orange 88.8

n.d

0.17 ± 0.03

n.d

0.45 ± 0.24

1.67 ± 0.68

2.53 ± 1.19

0.48 ± 0.15

1.20 ± 0.60

17.41 ± 6.32

n.d

n.d

n.d

0.20 ± 0.12

n.d

n.d

7.03 ± 1.04

1.00 ± 0.82

n.d

n.d

n.d

4.86 ± 1.35

n.d

n.d

2.53 ± 0.76

n.d

n.d

6.65 ± 2.74

1.10 ± 0.64 11.65 ± 10.39

n.d.

n.d

n.d

n.d

30.06 ± 3.99

n.d.

13.42 ± 18.38

0.96 ± 0.63

n.d

17.28 ± 2.96

n.d.

n.d.

n.d

0.37 ± 0.20

n.d

2.37 ± 0.61

1416.04 ± 443.09 76.15 ± 17.59 429.52 ± 58.12 89.79 ± 17.80 130.97 ± 61.85

3.22 ± 1.42

0.44 ± 0.15 28.47 ± 5.51

0.45 ± 0.23

1.29 ± 0.26

n.d

2.65 ± 0.59

21.17 ± 2.3 2

1.79 ± 0.98

n.d

6.94 ± 1.35

15.12 ± 7.71

5.81 ± 3.81

2.99 ± 0.31

0.92 ± 0.29

2.81 ± 0.48

0.40 ± 0.20

3.63 ± 0.46 13.74 ± 1.39

n.d. 1.62 ± 0.69 0.64 ± 0.54 n.d.

n.d 2.29 ± 0.99 1.77 ± 0.40

48.21 ± 6.07

83.40 ± 6.22

47.38 ± 10.36

1.04 ± 1.12

5.21 ± 1.14

7.98 ± 1.03

0.22 ± 0.21

n.d.

10.27 ± 1.25

n.d.

3.54 ± 6.92

n.d

33.03 ± 11. 16

n.d

0.59 ± 0.41

ultco/detail.action?docID=3017830.

Table 1. (Continued)

Moisture (%) p-coumaric Ferulic Sinapic Salicylic

Raspberry 84.4 224.64 ± 37.62 127.74 ± 12.85 42.89 ± 9.33 25.13 ± 5.57

Gooseberry 86.4 504.81 ± 62. 83

Blackcurrant 79.0 591.31 ± 58.18 120.96 ± 13.99

Strawberry 87.9 1107.61 ± 142.65 121.79 ± 29.90

n.d.

37.34 ± 4.69

n.d.

n.d.

55.76 ± 9.41

93.17 ± 9.57

67.27 ± 5.37

Banana 73.9 3.47 ± 3. 33 40.07 ± 7.98 0.27 ± 0. 53 3.93 ± 0. 68

Apple 85.2 2.00 ± 0.55 0.56 ± 0.17 6.34 ± 4.93 6.73 ± 1.53

Pear 84.5

Grapes 69.4 16.31 ± 5.9 7

Orange 88.8 20.89 ± 10.7 6

0.10 ± 0.05

n.d

30.01 ± 2.79

34.87 ± 32. 92

2.93 ± 1.66

28.86 ± 2.94

1.93 ± 0.59

9.50 ± 1.36

16.97 ± 1.95

5.84 ± 3.28

Values are specified on a dry weight basis in mg kg−1 and are given as mean ± standard deviations (n = 3). Not detected = n.d.

Table 2. Phenolic compounds including phenolic acids in fruits and vegetables (FAO, 2000) [8] Source Apple

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Apricot Avocado Banana Cacao Coffee beans Eggplant Grape Lettuce Mango Mushroom Peach Pear Plum Potato Sweet potato Tea

Phenolic Compounds chlorogenic acid (flesh), catechol, catechin (peel), caffeic acid, 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxy benzoic acid, p-cresol, 4-methyl catechol, leucocyanidin, p-coumaric acid, flavonol glycosides isochlorogenic acid, caffeic acid, 4-methyl catechol, chlorogenic acid, catechin, epicatechin, pyrogallol, catechol, flavonols, p-coumaric acid derivatives 4-methyl catechol, dopamine, pyrogallol, catechol, chlorogenic acid, caffeic acid, DOPA 3,4-dihydroxyphenylethylamine (Dopamine), leucodelphinidin, leucocyanidin catechins, leucoanthocyanidins, anthocyanins, complex tannins chlorogenic acid, caffeic acid chlorogenic acid, caffeic acid, coumaric acid, cinnamic acid derivatives catechin, chlorogenic acid, catechol, caffeic acid, DOPA, tannins, flavonols, protocatechuic acid, resorcinol, hydroquinone, phenol tyrosine, caffeic acid, chlorogenic acid derivatives dopamine-HCl, 4-methyl catechol, caffeic acid, catechol, catechin, chlorogenic acid, tyrosine, DOPA, p-cresol tyrosine, catechol, DOPA, dopamine, adrenaline, noradrenaline chlorogenic acid, pyrogallol, 4-methyl catechol, catechol, caffeic acid, gallic acid, catechin, Dopamine chlorogenic acid, catechol, catechin, caffeic acid, DOPA, 3,4-dihydroxy benzoic acid, p-cresol chlorogenic acid, catechin, caffeic acid, catechol, DOPA chlorogenic acid, caffeic acid, catechol, DOPA, p-cresol, p-hydroxyphenyl propionic acid, p-hydroxyphenyl pyruvic acid, m-cresol chlorogenic acid, caffeic acid, caffeylamide flavanols, catechins, tannins, cinnamic acid derivatives

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Caffeic acid, free and esterified, is generally the most abundant phenolic acid and represents between 75% and 100% of the total hydroxycinnamic acid content of most edible plants and fruits. Hydroxycinnamic acids are found in all parts of plant foods, although the highest concentrations are seen in the outer parts of ripe fruit and in the leaves of plants. Apples, for example, contain chlorogenic acid and small quantities of other hydroxycinnamic acids, in the skin of certain red varieties [9, 10]. Phenolic acids, are directly involved in the response of plants to different types of stress contributing to healing by lignification of damaged areas. They have antimicrobial properties, and their concentrations may increase after infection [2,6,11]. With the current state of knowledge, it is extremely difficult to determine for each family of plant products the key variables that are responsible for the variability in the content of each phenolic acid and the relative weight of those variables. A lot of research is made, for example, determination of the p-coumaric acid content of >500 red wines showed that genetic factors were more important than was exposure to light or climate [12]. In general, phenolic acid concentrations decrease during ripening, whereas anthocyanin concentrations increase. Although very few studies directly addressed this issue, the phenolic content of vegetables produced by organic or sustainable agriculture is certainly higher than that of vegetables grown without stress, such as those grown in conventional or hydroponic conditions. This was shown recently in strawberries, blackberries, and corn [13]. Storage may also affect the content of phenolics that are easily oxidized. Oxidation reactions result in the formation of more or less polymerized substances, which lead to changes in the quality of foods, particularly in color and organoleptic characteristics. Such changes may be beneficial (as is the case with black tea) or harmful (browning of fruit) to consumer acceptability. Methods of culinary preparation also have a marked effect on the polyphenol content of foods. Simple peeling of fruit and vegetables can eliminate a significant portion of phenolics because these substances are often present in higher concentrations in the outer parts than in the inner parts. Cooking may also have a major effect. Onions and tomatoes lose between 75% and 80% of their initial phenolic content after boiling for 15 min, 65% after cooking in a microwave oven, and 30% after frying [14]. Steam cooking of vegetables, which avoids leaching, is preferable. Potatoes contain up to 190 mg chlorogenic acid/kg, mainly in the skin [15]. Extensive loss occurs during cooking, and no remaining phenolic acids were found in French fries or freeze-dried mashed potatoes [12]. Production of fruit juice often involves clarification or stabilization steps specifically aimed at removing certain phenolic compounds responsible for discoloration and haze formation. Manufactured fruit juices thus have low this content. The pectinolytic enzymes used during such processing also hydrolyze the esters of hydroxycinnamic acid [16]. Because of the wide range of existing phenolic compounds such as phenolic acids and the considerable number of factors that can modify their concentration in foods, no reference food-composition tables (as they exist for other micronutrients such as vitamins) have yet been reported. Only partial data for certain polyphenols, such as flavonols and flavones, catechins, and isoflavones, have been published on the basis of direct food analysis [17,18] or bibliographic compilations [19,20]. Since March 2003, a database in which the flavonoid contents of 225 selected foods were compiled from 97 bibliographic sources has been available on the US Department of Agriculture website [21].

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Dietary Intake of Phenolic Acids Only partial information is available on the quantities of phenolic acids that are consumed daily. These data have been obtained after hydrolysis of their ester, ether, or acetal bonds either to structural components of the plant (cellulose, proteins, lignin), or to larger polyphenols (flavonoids), or smaller organic molecules (e.g., glucose, quinic, maleic, or tartaric acids) or other natural products (e.g., terpenes) [22] in the foods most widely consumed by humans. Consumption of hydroxycinnamic acids may vary highly according to coffee consumption. Some persons who drink several cups per day may ingest as much as 500–800 mg hydroxycinnamic acids/d, whereas subjects who do not drink coffee and who also eat small quantities of fruit and vegetables do not ingest >25 mg/d [12]. A German study estimated daily consumption of hydroxycinnamic acids and hydroxybenzoic acids at 211 and 11 mg/d, respectively. Caffeic acid intake alone was 206 mg/d, and the principal sources were coffee (which provides 92% of caffeic acid) and fruit and fruit juices combined (source of 59% of p-coumaric acid) [23]. Caffeic acid intake alone was 206 mg/d, and the principal sources were coffee (which provides 92% of caffeic acid) and fruit and fruit juices combined (source of 59% of p-coumaric acid) [20]. Intake of phenolic acids ranged from 6 to 987 mg/d in Germany [20]. Polyphenol content is expressed usually as the amount provided by a food serving, hence the consumption of one particular food, such as coffee for hydroxycinnamic acids, clearly appears to be capable of markedly changing the total polyphenol intake. Because phenolic compounds intake is difficult to evaluate by using dietary questionnaires, biomarkers for phenolic exposure would be very useful. Despite the scarcity of studies performed on the bioavailability of hydroxycinnamic acids, when ingested in the free form, these compounds are rapidly absorbed from the small intestine and are conjugated and, in particular, glucuronidated in the same way that flavonoids are [12,22]. However these compounds are naturally esterified in plant products, and this impairs their absorption. Human tissues (intestinal mucosa, liver) and biological fluids (plasma, gastric juice, duodenal fluid) do not possess esterases capable of hydrolyzing chlorogenic acid to release caffeic acid [2426]. This has also been observed in rats [27,28]. Only the colonic microflora would be capable of carrying out this hydrolysis, and some of the bacterial strains involved have been identified [29]. Consequently, as observed for flavonoid glycosides that must be hydrolyzed by the microflora, the efficiency of absorption of phenolic acids is markedly reduced when they are present in the esterified form rather than in the free form [25,27,30]. In patients who have undergone colonic ablation, caffeic acid was much better absorbed than was chlorogenic acid: 11% and 0.3% of the ingested doses were excreted in urine, respectively [25]. Similarly, when chlorogenic acid was given by gavage to rats, no intact compound was detected in plasma in the following 6 h, and the maximum concentrations of metabolites obtained after administration of caffeic acid in the same conditions were 100-fold those of the metabolites (various glucuronidated or sulfated derivatives of caffeic and ferulic acids) obtained after chlorogenic acid administration [27]. Surprisingly, the plasma concentrations were maximal only 30 min after gavage, which may seem inconsistent with hydrolysis of chlorogenic acid in the cecum. The same observation was made in a human study. When volunteers ingested coffee containing high amounts of esterified phenolic acids but no free caffeic acid, the peak plasma concentration of caffeic acid was observed only 1 h after ingestion of the coffee [31]. In this study, the alkaline hydrolysis of coffee showed that chlorogenic acid represented only

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30% of the bound caffeic acid. Thus, a possible explanation is that other forms of caffeic acid present in coffee may have been hydrolyzed in the upper part of the gut. Furthermore, the modes of administration used in both studies, ie, direct stomach intubation in the rat study and ingestion of coffee alone by fasted volunteers in the second study, might allow a rapid transit to the colon and explain the rapid kinetics of appearance of plasma metabolites. However, these 2 studies raise doubt about the total inability of the tissues to hydrolyze esterified phenolic acids.

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Metabolism and Intestinal Absorption Despite the scarcity of studies performed on the bioavailability of hydroxycinnamic acids, when ingested in the free form, these compounds are rapidly absorbed from the small intestine and are conjugated and, in particular, glucuronidated in the same way that flavonoids are [12, 32]. However these compounds are naturally esterified in plant products, and this impairs their absorption. Human tissues (intestinal mucosa, liver) and biological fluids (plasma, gastric juice, duodenal fluid) do not possess esterases capable of hydrolyzing chlorogenic acid to release caffeic acid [33-35]. Apart from being esterified to simple acids, hydroxycinnamic acids can be bound to polysaccharides in plant cell walls. The esterification of ferulic acid to arabinoxylans in the outer husks of cereals is a typical example. Although free ferulic acid is reported to be rapidly and efficiently absorbed (up to 25%) from tomatoes in humans [36], its absorption after ingestion of cereals is expected to be much lower because of this esterification. Ferulic acid metabolites recovered in the urine of rats represent only 3% of the ingested dose when ferulic acid is provided as wheat bran, whereas the metabolites represent 50% of the dose when ferulic acid is provided as a pure compound [37]. Another study showed that feruloyl esterases are present throughout the entire gastrointestinal tract, particularly in the intestinal mucosa, and that some of the ester bonds between ferulic acid and polysaccharides in cell walls may thus be hydrolyzed in the small intestine [28]. However, the role of feruloyl esterases seems to be very limited, and absorption occurs mainly in the colon after hydrolysis by enzymes of bacterial origin. The bioactivity of aqueous extracts of aromatic plants performed in vivo animal models is recently reported. The ingestion of the aforementioned aqueous infusion increases total antioxidant capacity (TAC) of plasma and specific organs [38] and iron fortificants may exert oxidative activity on colon tissue homogenates (CTH), depending on the antioxidant capacity of this infusion received with mice diet [39]. The susceptibility of mice colon to oxidation induced by the retentates of iron in vitro digests was evaluated by the thiobarbituric acid reactive substances (TBARS) method [40]. The antioxidant capacity and the decrease of susceptibility to oxidation of plasma and tissues after administration of infusions may be explained by the phenolic compounds (including phenolic acids) that the ingested infusions contain [41].

Antioxidant Capacity of Phenolic Acids The antioxidant capacity of phenol acids and their esters depends on the number of hydroxyl groups in the molecule that would be strengthened by steric hindrance [1]. The

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electron withdrawing properties of the carboxylate group in benzoic acids has a negative influence on the H-donating abilities of ths hydroxybenzoates. Hydroxylated cinnamates are more effective than benzoate counterparts [1]. A variety of tests expressing antioxidant capacity has been suggested. The tests can be categorized into two groups: assays for radical scavenging ability and assays that test the ability to inhibit lipid oxidation under accelerated conditions. The antioxidant reactions involve multiple steps including the initiation, propagation, branching, and termination of free radicals. The antioxidants which inhibit or retard the formation of free radicals from their unstable precursors (initiation) are called the “preventive” antioxidants, and those which interrupt the radical chain reaction (propagation and branching) are the “chain-breaking” antioxidants [42]. Test systems that evaluate the radical scavenging ability of antioxidants aim to simulate basic mechanisms involved in lipid oxidation by measuring either the reduction of stable radicals or radicals generated by radiolysis, photolysis, or the Fenton reaction [43, 44]. The DPPH (Figure 3) radical is one of the few stable organic nitrogen radicals, commercially available, which bears a deep purple color. This assay is based on the measurement of the reducing ability of antioxidants toward DPPH . The ability can be evaluated by measuring the decrease of its absorbance. The widely used decoloration assay was first reported by Brand-Williams and co-workers [45]. This antioxidant assay is based on measurement of the loss of DPPH color at 517 nm after reaction with test compounds [46] and the reaction is monitored by a spectrometer. Experiments were carried out according to the method of Blois [44] with a slight modification. The Trolox Equivalent Antioxidant Capacity Assay (TEAC) assay was first reported by Miller and Rice-Evans in 1993 and later improved [47]. In the improved version, ABTS -, the oxidant, was generated by persulfate oxidation of 2,2-azinobis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS2-) (Figure 4). Specifically, of ABTS ammonium is dissolved in water and treated with potassium persulfate, and the mixture was then allowed to stand at room temperature for 12-16 h to give a dark blue solution. This solution was diluted with ethanol or buffer (pH 7.4) until the absorbance reached 0.7 at 734 nm. The resulting solution is mixed with the sample. The absorbance was read at 30°C, 1, 4, and 6 min after mixing at 30°C.

Figure 3. The structure of DPPH radical. The DPPH radical is one of the few stable organic nitrogen radicals, commercially available, which bears a deep purple color. The DPPH assay is based on the measurement of the reducing ability of antioxidants toward this radical.

The difference of the absorbance reading is plotted versus the antioxidant concentrations to give a straight line. The concentration of antioxidants giving the same percentage change of absorbance of the ABTS - as that of 1 mM Trolox is regarded as TEAC. The TEAC assay is very simple and has been used by many researchers for measuring antioxidant capacity, and

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TEAC values of many compounds and food samples are reported. The TEAC values for pure antioxidant compounds do not show clear correlation between TEAC values and the number of electrons an antioxidant can give away.

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Figure 4. Antioxidant capacity is based on the scavenging ability of antioxidants to the long-life radical anion ABTS. In this assay, ABTS - is oxidized by peroxyl radicals or other oxidants to its radical cation, ABTS +, which is intensely coloured (λmax = 734 nm), and antioxidant capacity is measured as the ability of test compounds to decrease the colour (ABTS 2- colourless) reacting directly with the ABTS + radical.

The TEAC values of ascorbic acid (1.05), R-tocopherol (0.97), glutathione (1.28), and uric acid (1.01) are almost the same, although glutathione can normally donate one electron (to form oxidized glutathione) whereas the others are two-electron reductants. Ferulic acid (1.90) and p-coumaric acid (2.00) have comparable TEAC values. However, caffeic acid has a TEAC value of 1.00 even though its structure is similar to that of ferulic acid. The TEAC value difference between quercetin (3.00) and kaempferol (1.00) is also rather surprising as they have similar chemical structures [47].The FRAP assay was originally developed by Benzie and Strain [48] to measure reducing power in plasma, but the assay subsequently has also been adapted and used for the assay of antioxidants in botanicals [49-53]. The reaction measures reduction of ferric 2,4,6-tripyridyl-s-triazine (TPTZ) to a colored product (Figure 5) [54].Accelerated test systems mainly include lipids, which rapidly oxidize in order to simulate a long induction period in a short time. To accelerate oxidation, an increase in temperature is often used. Rancimat test was used which is an assay that tests the ability to inhibit lipid oxidation under accelerated conditions. Samples of sunflower oil (3.5g) containing 2% w/w ground material were subjected to oxidation at 110 °C (air flow 20 l/h). The standard compounds (0.02% addition) were also examined. Induction periods, IP (h), were recorded automatically. The protection factors (PF) were calculated according to the following formula: (PF = IPextract / IPcontrol ) [55]. Total phenolic content was measured by the FolinCiocalteu assay [56]. Results were expressed as mg of gallic acid/ g dry sample. The Folin-Ciocalteu assay has for many years been used as a measure of total phenolics in natural products, but the basic mechanism is an oxidation/reduction reaction and, as such, can be considered another antioxidant method.

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C. Proestos, A. E. Koutelidakis, M. Kapsokefalou et al.

Figure 5. Reaction for FRAP assay. The FRAP assay was used for the assay of antioxidants in herbs. The reaction measures spectrometrically reduction of ferric 2,4,6-tripyridyl-s-triazine (TPTZ) to a colored product under specific conditions (pH=3,6, T=37 oC, λ=595 nm ).

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The original Folin-Ciocalteu method developed by Folin [57] originated from chemical reagents used for tyrosine analysis in which oxidation of phenols by a molybdotungstate reagent yields a colored product with max at 745-750 nm. The method is simple, sensitive, and precise. However, the reaction is slow at acid pH, and it lacks specificity. Singleton and Rossi [58] improved the method with a molybdotungstophosphoric heteropolyanion reagent that reduced phenols more specifically; the max for the product is 765 nm. They also imposed mandatory steps and conditions to obtain reliable and predictable data: (a) proper volume ratio of alkali and Folin-Ciocalteu reagent; (b) optimal reaction time and temperature for color development; (c) monitoring of optical density at 765 nm; and (d) use of gallic acid as the reference-standard phenol. Lack of standardization of methods can lead to several orders of magnitude difference in detected phenols. Hence, continued efforts to standardize the assay are clearly warranted.

Conclusion Hydroxycinnamic acid compounds occur most frequently as simple esters with hydroxy carboxylic acids or glucose. Hydroxybenzoic acid compounds are present mainly in the form of glucosides. Furthermore, phenolic acids may occur in food plants as esters or glycosides conjugated with other natural compounds such as flavonoids, alcohols, hydroxyfatty acids, sterols, and glucosides. The absorption and bioavailability of phenolics in humans are controversial. Data on these aspects of phenolic acids are scarce and merely highlight the need for extensive investigations of the handling of phenolics by the gastrointestinal tract and their subsequent absorption and metabolism. Generally, the analytic studies of phenolic compounds in foods that have been conducted to date provide a good indication of their distribution. Fruits and beverages such as tea and other plant obtained infusions, red wine, and coffee constitute the principal sources of phenolic compounds, but vegetables, leguminous plants, and cereals are also good sources. Phenolic concentrations in foods vary according to numerous genetic, environmental, and technologic factors. The main tasks ahead are identifying the plant varieties that are the richest in phenolic substances such as phenolic acids or other, improving growing methods, and limiting losses during the course of industrial processing and domestic cooking. The health effects of these compounds depend on both their respective intakes and their bioavailability, which can vary greatly. Finally, hydroxycinnamic

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acids are found in a wide variety of plant foods, often at high concentrations, but esterification decreases their intestinal absorption. As a general rule, the metabolites of phenolic compounds are rapidly eliminated from plasma, which indicates that consumption of plant products on a daily basis is necessary to maintain high concentrations of metabolites in the blood.

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Rice-Evans, C.A., Miller, N.J. and Paganga G. Free Rad. Bio.l Med., 20, 933 (1996). Shahidi F, Naczk M. Food phenolics, sources, chemistry, effects, applications. Lancaster, PA: Technomic Publishing Co Inc., (1995). Tomas-Barberan, F.A. and Clifford, M.N. Dietary hydroxybenzoic acid derivatives and their possible role in health protection. J. Sci. Food Agric., 80, 1024 (2000). Clifford, M.N. and Scalbert A. J. Sci. Food Agric., 80:1118 (2000). Clifford, M.N. J. Sci. Food Agric., 79:362 (1999). Macheix, J.-J., Fleuriet, A. and Billot, J. Fruit phenolics. Boca Raton, FL: CRC Press (1990). Russell, W.R., Scobbie, L., Labat, A., Duncan, G.J. and Duthie, G.G. Food Chem., 115,100 (2009). www.fao.org/waicent/faoinfo/agricult/ags/Agsi/enzymefinal/enzymaticbrowning.html, FAO (2000). Guyot, S., Marnet, N., Laraba, D., Sanoner, P. and Drilleau, J.-F. J. Agric. Food Chem., 46, 1698 (1998). Sanoner, P., Guyot, S., Marnet, N., Molle, D. and Drilleau, J,-F. J. Agric. Food Chem., 47, 4847 (1999). Parr, A.J. and Bolwell, G.P. J. Agric. Food Chem., 80, 985 (2000). Clifford, M.N. J. Sci. Food Agric., 80, 1033 (2000). Asami, D.K., Hong, Y.J., Barrett, D.M. and Mitchell, A.E. J. Agric. Food Chem., 51,1237 (2003). Crozier, A., Lean, M.E.J., McDonald, M.S. and Black, C. J. Agric. Food Chem., 45, 590 (1997). Friedman, M. J. Agric. Food Chem., 45, 1523 (1997). Macheix, J.J. and Fleuriet, A. Phenolic acids in fruits. In: Rice-Evans C, Packer L, eds. Flavonoids in health and disease. New York: Marcel Dekker, Inc, 1998:35–59. Hertog, M.G.L., Hollman, P.C.H. and Katan, M.B. J. Agric. Food Chem., 42, 2379 (1992). Arts, I.C.W., van de Putte, B. and Hollman, P.C.H. J. Agric. Food Chem., 48, 1746 (2000). Reinli, K. and Block, G. Nutr. Cancer, 26, 123 (1996). Radtke, J., Linseisen, J. and Wolfram, G. Z .Ernahrungswiss., 37, 190 (1998). US Department of Agriculture. USDA database for the flavonoid content of selected foods. March 2003. Internet: http://www.nal.usda.gov/ fnic/foodcomp/ (accessed 20 May 2003).

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[22] Middleton, E., Kandaswami, C. and Theoharides, T.C. Pharmacol. Rev., 52, 673 (2000). [23] Radtke, J., Linseisen, J. and Wolfram, G. Z. Ernahrungswiss., 37, 190 (1998). [24] Cremin, P., Kasim-Karakas, S. and Waterhouse, A.L. J. Agric. Food Chem., 49, 1747 (2001). [25] Plumb, G.W., Garcia-Conesa, M.T., Kroon, P.A., Rhodes, M., Ridley, S. and Williamson, G. J. Sci. Food Agric., 79, 390 (1999). [26] Olthof, M.R., Hollman, P.C.H. and Katan, M.B. J. Nutr. 131, 66 (2001). [27] Rechner, A.R., Spencer, J.P., Kuhnle, G., Hahn, U. and Rice-Evans, C.A. Free Radic. Biol. Med., 30, 1213 (2001). [28] Azuma, K., Ippoushi, K., Nakayama, M., Ito, H., Higashio, H. and Terao, J. J. Agric. Food Chem., 48, 5496 (2000). [29] Andreasen, M.F., Kroon, P.A., Williamson, G. and Garcia-Conesa, M.T. J. Agric. Food Chem. 49, 5679 (2001). [30] Couteau, D., McCartney, A.L., Gibson, G.R., Williamson, G. and Faulds, C.B. J. Appl. Microbiol., 90, 873 (2001). [31] Gonthier, M.P., Verny, M.A., Besson, C., Rémésy, C. and Scalbert, A. J. Nutr., 133, 1853 (2003). [32] Cremin, P., Kasim-Karakas, S. and Waterhouse, A.L. J. Agric. Food Chem. 49, 1747 (2001). [33] Plumb, G.W., Garcia-Conesa, M.T., Kroon, P.A., Rhodes, M., Ridley, S. and Williamson, G. J. Sci. Food Agric., 79, 390 (1999). [34] Olthof, M.R., Hollman, P.C.H. and Katan, M.B. J. Nutr. 131, 66 (2001). [35] Rechner, A.R., Spencer, J.P., Kuhnle, G., Hahn, U. and Rice-Evans, C.A. Free Radic. Biol. Med., 30, 1213 (2001). [36] Nardini, M., Cirillo, E., Natella, F. and Scaccini, C. J. Agric. Food Chem., 50, 5735 (2002). [37] Bourne, L.C. and Rice-Evans, C. Biochem. Biophys. Re.s Commun., 253, 222 (1998). [38] Koutelidakis, A.E., Argyri, K., Serafini, M., Proestos, C., Komaitis, M., Pecorari, M., and Kapsokefalou, M. Basic Nutr. Invest., 25, 453 (2009). [39] Koutelidakis, A.E., Serafini, M. and Komaitis, M. Food Chem., 120, 895 (2010). [40] Matsingou, T.C., Petrakis, Ν., Kapsokefalou, M. and Salifoglou, A. J. Agric. Food Chem.,51, 6696 (2003). [41] Proestos C, Boziaris IS, Nychas GJE, Komaitis M. Food Chem., 95, 664-671 (2006). [42] Ou, B., Hampsch-Woodill, M. and Prior, R.L. J. Agric. Food Chem., 49, 4619 (2001). [43] Blois, M.S. Nature, 181, 1199 (1958). [44] Madsen, H.L., Nielsen, B.R., Bertelsen, G. and Skibsted, L.H. Food Chem., 57, 331 (1996). [45] Brand-Williams, W., Cuvelier, M.E. and Berset, C. Lebensm. Wiss. Technol., 28, 25 (1995). [46] Bondet, V., Brand-Williams, W. and Berset, C. Food Sci. Technol., 30, 609 (1997). [47] Huang, D., Ou, B. and Prior, R.L. J. Agric. Food Chem.,53,1841 (2005). [48] Benzie, I.F. and Strain, J. J. Anal. Biochem., 239, 70 (1996). [49] Benzie, I.F.F. and Szeto, Y.T. J. Agric. Food Chem., 47, 633 (1999). [50] Gil, M.I., Tomas-Barberan, F., Hess-Pierce, B., Holcroft, D. and Kader, A. J. Agric. Food Chem., 48, 4581 (2000).

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[51] Ou, B., Huang, D., Hampsch-Woodill, M., Flanagan, J.A. and Deemer, E.K. J. Agric. Food Chem., 50, 3122 (2002). [52] Proteggente, A.R., Pannala, A.S., Paganga, G., Van Buren, L., Wagner, E., Wiseman, S., Van De Put, F., Dacombe, C. and Rice-Evans, C.A. Free Rad. Res., 36, 217 (2002). [53] Pellegrini, P., Serafini, M., Colombi, B., Del Rio, D., Salvatore, S., Bianchi, M. and Brighenti, F. J. Nutr., 133, 2812 (2003). [54] Benzie, I.F.F. Clin. Biochem., 29, 111 (1996). [55] Exarchou, V., Nenadis, N., Tsimidou, M., Gerothanasis, I.P., Troganis, A., Boskou, D. J. Agric. Food Chem., 50, 5294 (2002). [56] Kähkönen, M.P., Hopia, A.I., Heikki, J.V., Rauha, J.-P., Pihlaja, K., Kujala, T.S., and Heinonen, M. J. Agric. Food Chem., 47, 3954 (1999). [57] Folin, O. J. Biol. Chem., 73, 672 (1927). [58] Singleton, V.L. and Rossi, J.A. Am. J. Enol. Viticult., 16, 144 (1965).

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In: Phenolic Acids Editor: Sergi Munné-Bosch

ISBN: 978-1-61942-032-8 © 2012 Nova Science Publishers, Inc.

Chapter II

Phenolic Compounds in Staple Plants F. Edi-Soetaredjo1,2*, S. Ismadji2 and Y-H. Ju1 1

Department of Chemical Engineering, National Taiwan University of Science and Technology, 43, sec. 4. Keelung Rd., Taipei, Taiwan 2 Department of Chemical Engineering, Widya Mandala Surabaya Catholic University, Kalijudan 37, Surabaya 60114, Indonesia

Abstract

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A staple food is consumed regularly. It is a dominant constituent in the diet and is a major supplier of energy and nutrients. Staple foods vary from place to place, but are typically inexpensive, starchy foods that supply one or more of the three macronutrients needed for human health: carbohydrate, protein and fat. Staple foods mostly are from cereals such as wheat, barley, rye, maize, rice, or starchy root vegetables such as potato, yam, taro, cassava, sago (derived from the pith of the sago palm tree), and fruits such as breadfruit and plantains. The minor compositions of staple foods are minerals (phosphorous, potassium, sodium, calcium, magnesium, iron, zinc, etc.), vitamins and phenolic compounds. Rice, wheat, maize, barley, and rye are the dominant staple foods in the world. A number of studies have been carried out to investigate the phenolic compounds in those staple foods that are described in this chapter. Phenolic acids are secondary metabolites widely distributed in the plant kingdom and are second only to flavonoids in terms of their dominance. Cereals, including barley, oats and wheat, are known to contain a wide range of phenolic acids that belong to benzoic and cinnamic acid derivatives. These include ferulic, caffeic, p-hydroxybenzoic, protocatechuic, pcoumaric, vanillic and syringic acids. This chapter also provides phenolic content data in other staple plants such as barley, rye, potato, yam and sago. The composition and amounts of phenolic compounds in staple plants are important for their contribution to color, sensory attributes and nutritional and antioxidant properties of foods. These natural antioxidants are known to exhibit a wide range of biological effects including antibacterial, antiviral and anti-inflammatory, anti-allergic, antithrombotic and *

E-mail addresses: [email protected]; [email protected]; [email protected].

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F. Edi-Soetaredjo, S. Ismadji and Y-H. Ju vasodilatory activities. As the world population is increasing, the amount of staple food production and consumption is also increasing; therefore staple plants together with vegetables and fruits have an important role in fulfilling human need for antioxidant. Moreover, the increasing number of staple plant byproduct such as bran, is a potential source of phenolic compounds.

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Introduction to Phenolic Compounds in Plants Various studies suggest that phenolic acids are absorbed in humans and their antioxidant activity may reduce the risk of coronary heart diseases, cancers, and aging process. A phenolic compound contains an aromatic ring bearing one or more hydroxyl substituents. Phenolic compounds range from simple phenolic molecules to highly polymerized molecules. Most naturally occurring phenolic compounds are present as conjugates with mono- and polysaccharides, linked to one or more of the phenolic groups and may also occur as functional derivatives such as esters and methyl esters [1]. Though such structural diversity results in the wide range of phenolic compounds that occur in nature, phenolic compounds can basically be categorized as flavonoids, phenolic acids, tannins (hydrolysable and condensed), stilbenes and lignans [2]. Flavonoids can be further classified into anthocyanins, flavones, isoflavones, flavanones, flavonols and flavanols. Phenolic acids consist of two subgroups, the hydroxybenzoic and hydroxycinnamic acids. Hydroxybenzoic acids include gallic, p-hydroxybenzoic, protocatechuic, vanillic and syringic acids, which have in common the C6-C1 structure. Hydroxycinnamic acids are aromatic compounds with a three-carbon side chain (C6-C3), caffeic, ferulic, p-coumaric and sinapic acids being the most common representatives. Tannins are relatively high molecular weight compounds, which may be subdivided into hydrolysable and condensed tannins based on flavan-3-ols(-)-epicatechin and (+)-catechin. A main representative stilbene is resveratrol, which is mostly in the form of glycosylated trans isomers. Lignans are produced by oxidative dimerisation of two phenylpropane units. [2, 3]. Natural phenolic compounds accumulate in different plant tissues and cells during ontogenesis and under the influence of various environmental stimuli. Phenylpropanoid and flavonoid compounds usually accumulate in the central vacuoles of guard cells and epidermal cells as well as subepidermal cells of leaves and roots. Some compounds were found to be covalently linked to plant cell walls, others occur in waxes or on the external surfaces of plant organs [4, 5]. Plant cell wall phenolics consist of two groups of compounds: (1) lignin, the polymer of monolignol units, linked by oxidative coupling; and (2) low molecular weight hydroxycinnamic acids, that are bound to various cell wall components and are involved in cross-linkages [5]. Phenolic acids are the major phenylpropanoid components in cereals, and different levels of these phenolics are found in different fractions of cereals. In different cereals, the starchy endosperm contains low levels, whereas the outer layers of the grain (pericarp, aleurone layer, and germ) contain the highest. The most abundant cinnamic acid derivative is ferulic acid. Ferulic acid and p-coumaric acid (the second most abundant phenolic acid) are mostly concentrated in the aleurone layer and in the pericarp. Both ferulic acid and p-coumaric acid are associated with cell wall constituents through linkage, especially arabinoxylans and lignin. The arabinose side chains of the arabinoxylans are substituted at O-5 by phenolic

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Phenolic Compounds in Staple Plants

acids. Ferulic acid is known to be esterified to arabinoxylans in grasses and cross bridges between arabinoxylans are possible by peroxidation to form ferulic acid dimmers [6]. Phenolics are important compounds in the sensory and nutritional qualities of plants, which are produced by secondary plant metabolites [7, 8]. Phenolic acids have been proposed as bridging molecules between wall polymers (e.g. between polysaccharides and polysaccharides and between polysaccharides and lignin). Bound phenolic acids in wheat straw walls have been fractionated into alkali-labile and acid-labile components by successive alkaline and acid treatments. The acid-labile linkage was identified as ferulic acid [9]. Alkalilabile and acid-labile phenolic acids were also found in rice and maize stem internodes cell walls [10], with the acid-labile phenolic acid covalently linked through an aryl ether linkage to lignin and ester linked to polysaccharide [4-6, 9, 11]. Phenolic compounds are essential for the growth and reproduction of plants, and are produced as a response for defending injured plants against pathogens [2, 3, 12-15].

Staple Plants A staple food is consumed regularly. It is a dominant constituent in the diet and is a major supplier of energy and nutrients. Staple foods vary from place to place, but are typically inexpensive starchy foods that supply one or more of the three macronutrients needed for human health: carbohydrate, protein and fat. Staple foods mostly are from cereals such as wheat, barley, rye, maize, rice, or starchy root vegetables such as potatoes, yams, taro, cassava, sago (derived from the pith of the sago palm tree), and fruits such as breadfruit and plantains.

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Table 1. Composition of some staple foods [18] Product

Water (%)

Protein (%)

Fat (%)

Carbohydrate (%)

Wheat

14

12.7

2.2

63.9

Flour (plain)

14

9.4

1.3

77.7

Bran

8.3

14.1

5.5

26.8

Germ

11.7

26.7

9.2

44.7

Bread (white sliced)

40.4

7.6

1.3

46.8

Raw macaroni

9.7

12.0

1.8

75.8

Rye (grain and wholemeal)

15

8.2

2.0

75.9

Rice (raw white)

11.4

7.3

3.6

85.8

Oatmeal

8.2

11.2

9.2

66.0

Rice is the predominant staple food for 17 countries in Asia and the Pacific, nine countries in North and South America and eight countries in Africa. Rice, wheat, and maize share the largest portion as the world’s dietary energy, 20%, 19%, and 5%, respectively [16]. In the year of 2000, The World Bank estimated the world population will increase by more than 1.2 billion people between 1998 and 2018.

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Agricultural development has progressed enormously to provide enough food to the increasing world population. The world production of maize, wheat and rice in 2009 are 817,110,509 Mt; 681,951,838 Mt; and 678,688,289 Mt, respectively [17]. The major composition of staple foods are carbohydrate (~90%), protein (~13%), and fat(~10%) depending of the type of staple food (Table 1, [18]). Staple foods also contain minerals (nitrogen, phosphorous, potassium, sodium, calcium, magnesium, iron, zinc, etc.), vitamins and phenolic compounds [19-31]. The compositional differences among varieties or cultivars can be very significant for macronutrients, micronutrients and bioactive nonnutrients [32].

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Phenolic Compounds in Staple Plants Phenolic compounds are universally distributed in plants such as leave, trunk, root, seed, and fruit; and usually are bounded within plant tissues. A number of studies have investigated the composition of phenolic compounds in staple plants, which found that the processing of staple food results in the production of by-products, rich in phenolic compounds [33]. Bound phytochemicals are the major contributors to the total antioxidant activity: 90% in wheat, 87% in corn, 71% in rice, and 58% in oats [34]. The fraction of free and bound phenolics, flavonoids and total antioxidant activity of several staple foods are shown in Table 2. Staple foods are consumed daily, their by-products are good sources of phenolic compounds, and they have been explored as source of natural antioxidants [35]. Grains contain unique phytochemicals that complement those in fruit and vegetables when consumed together. For instance, various classes of phenolic compounds such as derivatives of benzoic and cinnamic acids, anthocyanidins, quinones, flavonols, chalcones, flavones, flavanones, and amino phenolic compounds can be found in grain. Grains contain tocotrienols and tocopherol, and rice contains significant amount of oryzanols. Some of these phytochemicals such as ferulic acid and diferulates are predominantly found in grains but are not present in significant quantities in some fruits and vegetables. Phenolic compounds present in grain have antioxidant properties associated with health benefits of grain and grain products [34]. Table 2. Fraction of free and bound phenolics, flavonoids and total antioxidant activity of several staple foods [34]

Corn Wheat Oats Rice

Phenolic Content (%)

Flavonoid content (%)

Total antioxidant activity (%)

Free

Bound

Free

Bound

Free

Bound

15 25 25 38

85 75 75 62

9 7 39 35

91 93 61 65

13 10 42 29

87 90 58 71

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Phenolic Compounds in Staple Plants Rice (Oryza Sativa L.)

Whole rice is made up of the endosperm, the germ and the bran of the grain. The endosperm represents about 80% of the whole grain, while the germ and the bran vary among different grains, but remaining bran usually comprises about 10% [21]. Table 3. Phenolic compounds in rice

Rice bran Light brown bran Purple-red bran

Pigmented rice Brown rice

Red rice

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Black rice

White rice (polished rice)

Phenolic Compound 5 mg /g (FAEa) 3.1-3.8 mg/g (GAEb) 8.6-45.4 mg/g (GAE)

Free phenolic: 0.0226-0.2275 mg/g (GAE) Bound phenolic: 0.0177-0.259 mg/g (GAE) Total phenolic: 0.138-0.6376 mg/g (GAE) Total phenolic: 3.4537 mg/g (FAE) Total phenolic: 5.1558 mg/g (FAE)

Free phenolic: 0.017-0.0189 mg/g (GAE) Bound phenolic: 0.00330.0272 mg/g (GAE) Total phenolic: 0.0556-0.2722 mg/g (GAE)

Identified compound Protocatechuic acid p-hydroxybenzoic acid p-coumaric acid Syringic acid Vanillic acid Caffeic acid Gallic acid Ferulic acid Vanillin Protocatechuic acid Syringic acid Gallic acid Ferulic acid p-coumaric acid Guaiacol p-Cresol o-Cresol 3,5-Xylenol p-hydroxybenzoic acid Vanillic acid Caffeic acid Sinapinic acid Protocatechuic acid Syringic acid Gallic acid Ferulic acid p-coumaric acid Guaiacol p-Cresol o-Cresol 3,5-Xylenol

Reference [27, 37, 39]

[21, 28, 29, 31, 40]

[29, 31, 40]

Rice (Table 3) contains a large spectrum of phenolic compounds including derivatives of benzoic and cinnamic acids, which are mainly ferulic acid and diferulates. In addition, rice contains anthocyanins, anthocyanidins and polymeric proanthocyanidins, which are also known as condensed tannins [21, 28, 31]. Since phenolic compounds mainly exist in cereal bran layer rather than in endosperm, whole grain rice thus possesses potential antioxidant activities which are much higher than that of its milled rice counterpart. In addition to possessing good quality protein, high fiber, and vitamin contents, pigmented rice varieties have the potential to promote human health because they contain antioxidative compounds that have the ability to inhibit the formation or to reduce the

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F. Edi-Soetaredjo, S. Ismadji and Y-H. Ju

concentrations of reactive cell-damaging free radicals. It has been confirmed that antioxidative activities are higher in pigmented than in non-pigmanted rice cultivars, suggesting that color of the pericarp may be important for predicting antioxidant effects in human diet [36]. Black rice has been considered as highly nutritional due to the presence of high level of anthocyanins, and phenolic acids [29, 31]. Total phenolic compounds in pigment and brown rice samples are higher than those in non-pigmented and polished rice samples [29]. The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging activity in three rice cultivars differing in pericarp colour (white, black and red) has been studied and it was found that coloured rice has notably higher radical-scavenging activity than white rice; the polymeric procyanidins being the major components responsible for that activity [37]. Cell wall materials may survive gastrointestinal digestion. However, colonic microflora digestion seems capable of releasing the bulk of ferulates, that may interact with target tissues, before or after absorption. The intake of bran or pure ferulic acid leads to different pharmacokinetics. Bound ferulic acid is slowly released by intestinal enzymes, being progressively absorbed, thus maintaining and an almost constant serum concentration for up to 24 h accompanied by a decrease in urinary excretion, while free ferulic acid in serum decreases down to zero after 4.5h [38]. Many varieties of rice are grown in various environments which lead to large differences in nutrient composition including phenolic compounds. The nutrition lost is affected by milling, cooking and soaking process. Milling of rice removes the outer layer of grain where most fats and phenolic compounds are concentrated. Therefore as greater amount of rice bran is removed from grain during milling and polishing, more phenolic compounds are lost (Table 3).

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Wheat (Triticum Aestivum L.) Wheat is one of the major cereals in the world. It is the main ingredient in a wide range of products such as bread, noodles, cakes, biscuits, cookies. Wheat kernel is composed of endosperm (81-84%), bran (14-16%), and germ (2-3%). Wheat endosperm is composed mostly of starch and protein, whereas bran and germ are rich in dietary fiber, minerals and phytochemicals. Phytochemicals such as phenolic acids play important roles in nutrition and health for human. Phenolic acids in cereal grains exist in free, soluble conjugate and insoluble bound forms (Table 4). The fact that phenolic acid content in wheat correlates well with its antioxidant activity suggests that the predominant source of antioxidant in wheat is phenolic compounds. Phenolic acids in cereals primarily occur in bound form, and are strongly associated with cell wall polysaccharides. There are two main groups of phenolic acids in cereal bran: benzoic and cinnamic acid derivatives. A number of studies using different varieties of wheat found that ferulic acid, p-coumaric and vanillic acids are the major phenolic acids in whole wheat flour, while syringic and protocatechuic acid only exist in small amount [41, 42]. A number of studies have reported that most phenolic acids in wheat grains are in the bound form and exist in bran associated with cell wall materials. These phenolic acids are covalently bound and concentrated in the cell walls of various grain tissues especially the aleurone and the pericarp-seed coat where they are esterified to the arabinose side groups of arabinoxylans. Phenolic acids present in cell wall play an important role in the cross-linking

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Phenolic Compounds in Staple Plants

of polysaccharides with other cell-wall components, including lignin through ester and ether bonds, and also in the cross-linking of polysaccharide chains [43-45]. The bound phenolic contents are significantly higher than the free phenolic acid contents in all wheat varieties, indicating that major phenolic acids in whole wheat are not extractable by aqueous ethanol but can be released upon alkaline or acid hydrolysis [41, 42, 44]. Table 4. Phenolic compounds in wheat

Whole wheat flour

Waxy grain

White flour

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Red wheat bran

White wheat bran

Phenolic compound Free phenolic acid 0.1966-0,2602 mg/g flour (GAE) Bound phenolic acid 0.5146-0.6804 mg/g flour (GAE) Total Phenolic acid 0.7112-0.9406 mg/g flour (GAE) Free phenolic acid 1.01 mg/g flour (FAE) Bound phenolic acid 2.40 mg/g flour (FAE) Free phenolic acid 0.28 mg/g flour (FAE) Bound phenolic acid 0.34 mg/g flour (FAE) Free phenolic acid 0.570-0.633 mg/g bran (GAE) Bound phenolic acid 3.201-3.396 mg/g bran (GAE) Free phenolic acid 0.458-0.562 mg/g bran (GAE) Bound phenolic acid 2.799-3.326 mg/g bran (GAE)

Identified Compound

Ferulic acid p-coumaric acid Vanillic acid Syringic acid Protocatechuic acid

Reference [23, 41]

[46]

[46]

p-hydroxybenzoic acid Vanillic acid Caffeic acid Syringic acid p-coumaric acid Ferulic acid Salicylic acid p-hydroxybenzoic acid Vanillic acid Syringic acid p-coumaric acid Ferulic acid trans-Cinnamic acid

[42]

[42]

Conventionally wheat grain fractionation is performed by using dry milling which leads to the separation of flour and semolina (from the starchy endosperm) from germ and bran which are recovered either in a single fraction or as two separate fractions, depending on the milling process. Valuable nutritional constituents including micronutrients, phytochemicals and fiber are particularly concentrated in the germ and bran fractions [45]. An alternative gradual milling was developed to reduce nutrition lost. Roller milling with or without debranning has been reported to increase the amount of phenolic compounds and improve antioxidant activity of pearled wheat fractions. Waxy (amylase-free) wheat milled from the outer parts of grain contain significantly higher amount of phenolic and exhibits significantly higher antioxidant capacity than milled whole grain [46]. Wheat variety with a high level of

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F. Edi-Soetaredjo, S. Ismadji and Y-H. Ju

antioxidant activity should have the potential for use as an excellent dietary source of antioxidants for disease prevention and health promotion since phenolic acids play an important role in combating oxidative stress in human body by maintaining a balance between oxidants and antioxidants. However, many factors such as genotypes and growing conditions were reported to have a significant effect on the phenolic acid content and antioxidant properties of wheat [41, 47].

Maize (Zea Mays L.) Corn is an important crop worldwide. Several studies have reported the identification and quantification of phenolic compounds (Table 5) from yellow corn including phydroxybenzoic acid, vanillic acid, protocatechuic acid, syringic acid, p-coumaric acid, ferulic acid, caffeic acid and sinapic acid. Purple cobs and seeds of purple corn are important sources of anthocyanins, including cyaniding-3-gluciside, pelargonidin-3-glucoside, and peonidin-3-glucoside [48]. Table 5. Phenolic compounds in maize

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Young yellow corn grain

Mature yellow corn grain

Yellow maize flour

Phenolic compound Free phenolic acid 0.177-0.330 mg/g dry base (GAE) Soluble glycoside-bound phenolics 0.229-0.575 mg/g dry base (GAE) Soluble ester-bound phenolics 0.280-0.631 mg/g dry base (GAE) Insoluble cell-wall-bound 2.761-3.179 mg/g dry base (GAE) Total phenolics 3.654-4.298 mg/g dry base (GAE) Free phenolic acid 0.0617 mg/g dry base (GAE) Soluble glycoside-bound phenolics 0.1635 mg/g dry base (GAE) Soluble ester-bound phenolics 0.2547 mg/g dry base (GAE) Insoluble cell-wall-bound 2.331 mg/g dry base (GAE) Total phenolics 2.811 mg/g dry base (GAE) Free phenolics: 0.25 mg/g dry weight (GAE) Bound phenolics 1.09 mg/g dry weight (GAE) Total phenolics 1.34 mg/g dry weight (GAE)

Identified compound p-coumaric acid Ferulic acid Vanillic acid

Reference [51]

Protocatechuic acid and its derivatives Gallic acid Ferulic acid and derivative p-coumaric acid and its derivative Catechin

[53, 54]

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Phenolic Compounds in Staple Plants

White maize flour

Blue maize flour

Red maize flour

Corn bran

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Corn fiber

Phenolic compound Free phenolics: 0.29 mg/g dry weight (GAE) Bound phenolics 1.39 mg/g dry weight (GAE) Total phenolics 1.68 mg/g dry weight (GAE) Free phenolics: 0.28 mg/g dry weight (GAE) Bound phenolics 1.14 mg/g dry weight (GAE) Total phenolics 1.42 mg/g dry weight (GAE) Free phenolics: 0.28 mg/g dry weight (GAE) Bound phenolics 1.12 mg/g dry weight (GAE) Total phenolics 1.40 mg/g dry weight (GAE) Ferulic acid: 28-31 mg/g Diferulic acid: 6.8-32 mg/g p-coumaric acid: 3-4 mg/g Total phenolics: 55 mg/g Ferulic acid: 1.02-18.5 mg/g p-coumaric acid: 3-4 mg/g

Identified compound

Reference

Ferulic acid p-coumaric

[50, 55]

Ferulic acid p-coumaric Dehydrodiferulic acid

[55, 56]

The distribution of total ferulic acid in seed were estimated to be 5.820 mg/g in the combined pericarp and aleurone layer, 0.067 mg/g in endosperm, and 0.309 mg/g in embryo [49]. Maize bran is mainly composed of matrix polysaccharides and cellulose which originate in the pericarp tissues. The matrix polysaccharides, which represent more than 50% of the wall, are heteroxylans made primarily of xylose and arabinose units with minor amounts of galactose and glucuronic. In addition, cinnamic acids represent up to 4-5% by weight of the destarched maize bran. These are mainly ferulic acid (3-methoxy-4-hydroxycinnamic acid) with some p-coumaric acid (4-hydroxycinnamic acid). All these acids are esterified to the wall polymers [50]. Recent studies have shown that main nutrient, phytochemicals, antioxidant activity, and certain physical parameters in yellow corn change greatly during various stages of corn growth. Immature seeds possess high concentrations of reducing sugars but low concentrations of starches and total lipids. They have high content of protein with high biological activity (albumin and globulin) and antioxidant activity. Consequently, immature corn grain can be used as a raw material for the development of whole grain foods [51, 52].

Barley (Hordeum Vulgare) Barley is widely consumed due to its positive dietary and properties. Barley meals and fractions are now gaining renewed interest as ingredients for the production of functional

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foods (pastas, baked products) due to its content of bioactive compounds, such as β-glucans and tocols. Moreover, there are several classes of compounds in barley that have a phenolic structure, such as benzoic and cinnamic acid derivatives, proanthocyanidins, quinones, flavonols, chalcones, flavones, flavanones, and amino phenolic compounds. Phenolic compounds in cereals can be in free or bound forms. The free phenolic compounds are proanthocyanidins or flavonoids, whereas the bound phenolic compounds are ester-linked to cell wall polymers and mainly consist of ferulic acid and its oxidatively coupled dimmers [57, 58]. The flavanols exist as monomers (mainly catechin and gallocatechin) or as polymers (proanthocyanidins). Proanthocyanidins are known to have antioxidant activity and health benefits. In barley, proanthocyanidin oligomers have been detected. The main proanthocyanidins detected are two dimers (procyanidin B3 and prodelphinidin B3) and four trimers (Procyanidin C2, prodelphinidin C2, and two other prodelphinidins) [59].

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Rye (Secale Cereale L.) Rye is second to wheat as the most commonly used grain in the production of bread. Most cereal grains are commonly used infractionated or refined form, whereas rye is traditionally consumed as wholemeal products in the Nordic countries. Rye products are particularly known for their high level of dietary fibre. Rye grains also have a beneficial composition of fatty acids and protein, and their high amounts of bioactive compounds have been suggested to contribute to positive health effects. Bioactive compounds, such as lignans, phenolic acids (Table 7), phytosterols, minerals, tocotrienols and other vitamins, are mainly concentrated in germ and in outer layers of the kernel [62]. The flavor of rye products, composed of volatile and non-volatile compounds, is derived from raw materials and process-induced changes. With respect to the flavor, the impact of the grain variety is more dominant than the cultivar. Several chemical compounds influence the perceived flavor. Among non-volatile compounds, at least phenolic compounds, free amino acids and fatty acids may affect the flavor characteristics of rye. Non-volatile phenolic compounds, such as phenolic acids and avenanthramides, are known to influence the flavor of oat. Some phenolic compounds were reported to contribute to the bitter taste of whole meal rye bread [12, 62]. Phenolic acids are hydroxylated derivatives of benzoic and cinnamic acids. Hydroxycinnamic acids are more common than hydroxybenzoic acids and consist chiefly of p-coumaric, caffeic, ferulic and sinapic acids. Ferulic acid is the most abundant hydroxycinnamic acid in rye, followed by sinapic acid and couramic acid. Among the benzoic acid derivatives, only small amounts of vanillic acid, syringic acid and hydroxybenzoic acid exist in rye grain and its products. Most of these compounds are concentrated in the outermost aleurone layers and bran of the grain. Hemicellulose is a matrix of polysaccharides present in almost all plant cell walls, which contains many different sugar monomers in its structure, such as glucose, xylose, arabinose, mannose, galactose and rhamnose. Hemicelluloses, mainly arabinoxylans and β-glucans, represent a major group of cell wall polysaccharides in rye. [63, 64].

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Phenolic Compounds in Staple Plants Table 6. Phenolic compounds in barley

Barley flour

Phenolic compound Total phenolics: 0.68 mg/g (GAE)

Whole barley flour

Total phenolic acids: 0.612-1.346 mg/g Total phenolics: 0.966-1.873 mg/g

Barley straw

Total phenolic 1,730 mg/g

Identified compound Flavonols Hydroxycinnamic acids o-Diphenols Procyanidins Prodelphinidins Catechin Procyanidin B3 Prodelphinidin B3 Trimeric proanthocyanidins Ferulic acid 5-5’-Diferulic acid 8-O-4’-Diferulic acid 8-5’-Diferulic acid Triferulic acid p-hydroxybenzoic acid p-Hydrobenzaldehyde Vanillic acid Syringic acid Vanillin Syringaldehyde Acetovanillone p-coumaric acid Ferulic acid Sinapic acid

Reference [57, 58, 60]

[59]

[61]

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Table 7. Phenolic compounds in rye

Whole rye grain

Rye flour

Rye bran

Phenolic compound

Identified compound

Total non-bound phenolics 0.014 mg/g (GAE) Total phenolic acids 0.653 mg/g (GAE) Total non-bound phenolics 0.009-0.013 mg/g (GAE) Total phenolic acids 0.036-0.046 mg/g (GAE) Total non bound phenolics 0.099 mg/g (GAE) Total phenolic acids 1.272 mg/g (GAE)

Sinapic acid Syringic acid Vanillic acid Ferulic acid Caffeic acid p-OH-benzoic acid Veratric acid

Referen ce [62, 6466]

Ferulic acid is linked to the o-5 position of the arabinofuranose substituents in the arabinoxylans and may also be linked by ester and ether bonds to lignin [65]. Ferulic acid units esterified to arabinoxylan chain can undergo dimerisation by peroxidase-catalysed oxidative coupling. Adjacent arabinoxylan chain can thereby be linked through diferulic acid bridges. These oxidatively coupled products are called diferulic acids or ferulic acid

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dehydrodimers and are present in various forms (resulting from 5-5-, 8-o-4-, 8-5-, and 8-8coupling events). The most abundant ferulic acid dehydromer in rye is β-4-[(E)-2-carboxyvinyl]-2methoxyphenyl-4-hydroxy-3-methoxycinnamic acid (8-o-4’-diferulic acid) [65]. Ferulic acid is also found in steryl ferulates, which are ferulic acid esters of sterols. These compounds are present in high content in rye and wheat bran [63, 64].

Other Staple Plants In addition to the major staple plants discussed above, there are other staple plants that have been a major proportion of energy and nutrient sources. Examples of theses staple plants are oat, starchy root vegetables such as potato, yam, taro and cassava and sago starch derived from the pith of sago palm tree. These staple plants are popular in certain countries. Oat (Avena sativa), which ranks sixth in world cereal production, differ from most other cereals in that it is consumed almost exclusively in whole grain products. Oat in the form of oatmeal is a popular breakfast in The United States, Britain, and is called sowans in Scotland. Root crops are probably the most important staple foods for poor smallholders all over the world. Sweet potato (Ipomoea batatas L.) and cassava (Manhiot esculenta Crantz) are prominent root crops throughout the tropics, and taro (Colocasia esculenta Schott) and yam (Dioscorea spp) are also important [19]. Yam is a major dietary source in certain African countries and Chinese yam is consumed in considerable amount in Taiwan due to its nutritional values and unique taste [20].

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Table 8. Phenolic compounds in various other staple plants

Whole oat grain

Potato Yam Sweet potato

Sago

Phenolic compound Free phenolics: 0.05-0.1422 mg/g Conjugated phenolics: 0.22-0.364 mg/g Bound phenolics: 0.1307-0.6404 mg/g Total phenolics: 0.4594-1.0158 mg/g

Total phenolics: 0.344-0.50 mg/g (GAE) Total phenolics: 0.15-0.20 mg/g (GAE) Total phenolics: 1.927-11.59 mg/g (GAE)

Phenolic compound: 0.20.9% of sago pith

Identified compound 4-Hydrobenzoic acid Vanillic acid Syringic acid Syringaldehyde Caffeic acid 2,4-Dihydrobenzoic acid Sinapic acid Ferulic acid p-coumaric acid 2-Hydroxycinnamic acid Catechin Catechol Catechin Caffeic acid Catechin Epicatechin Caffeic acid Ferulic acid Catechin Epicatechin

Reference [68]

[69] [19, 20] [19, 67, 70]

[26, 71]

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The composition and amounts of phenolic compounds including flavonoids (e.g. anthocyanins, flavonols or flavanols) and phenolic acids (e.g. cinnamic acids, or cinnamic acid esters of some staple foods are presented in Table 8. Phenolic compounds are important for their contribution to color, sensory attributes and nutritional and antioxidant properties of these staple foods. Given the increasing interest in natural products, the food industry will most likely pay increased attention to these root crop species. In Japan, sweet potato has been shown to contain highly acylated anthocyanins, which confer increased stability to this innocuous natural dye. Some varieties are now commercially exploited as a source of natural colorants and antioxidants by food and beverage industry [19, 67].

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Benefits of Phenolic Acids Antioxidants are believed to contribute to health benefits through several possible mechanisms such as by quenching free radicals, chelating transition metals, reducing peroxides and stimulationg antioxidative enzyme defense. Natural antioxidants are known to exhibit a wide range of biological effects including anti-bacterial, antiviral, antiinflammatory, anti-allergic, antithrombotic and vasodilatory activities [72]. The antioxidant activity of phenolic compounds is mainly attributed to their redox properties, which allow them to act as reducing agents, hydrogen donors and quenchers of singlet oxygen. In addition, they may also possess metal chelation properties. Phenolic compounds possess aromatic structure along with hydroxyl substituent which enable them to protect human tissues from damages caused by oxygen or free radicals, and consequently reduce the risk of various diseases, and offer beneficial effect against cancer, cardiovascular disease, diabetes, and Alzheimer’s disease [15, 27, 73-76]. However, the antioxidant activity is dependent on the structure of the molecule. Every phenolic acid compound has specific number and position of hydroxyl groups on the aromatic ring. Furthermore, the presence of a CH=CH-COOH group in the cinnamic acid derivatives gives higher antioxidant activities than the COOH group in benzoic acids. Also, phenols with o- or p-dihydroxylic group as well as alkoxyl phenols containing one free and one alkylated hydroxyl group (usually methoxy) are effective antioxidants [77]. Thus, for the cinnamic acid derivates, caffeic acid has a higher antioxidant activity than ferulic acid, which has a higher antioxidant activity than p-coumaric acid. The different antioxidant activities in different fractions of the grain (bran) are because different phenolic acids are unevenly distributed in the layers of the bran. [59]. Phenolic acids are secondary metabolites widely distributed in the plant kingdom and are second only to flavonoids in terms of their dominance. Cereals such as barley, oat and wheat, are known to contain a wide range of phenolic acids that belong to benzoic and cinnamic acid derivatives. These include ferulic, caffeic, p-hydroxybenzoic, protocatechuic, pcoumaric, vanillic and syringic acids. Ferulic acid is a phenolic antioxidant. Compare to other naturally occurring antioxidants, such as gallic acid, caffeic acid, malvidin, delphinidin, catechin, epicatechin, rutin and quercetin, ferulic acid has been shown as the most efficient in inhibiting lipid and protein oxidation in a lecithin/liposome oxidation system. Ferulic acid also possesses antimicrobial activity against spoilage and pathogenic micro-organisms. Because of

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its antioxidant and antimicrobial properties, ferulic acid has great potential for use in food industry as a preservative [55]. Ferulic acid, sinapic acid and the dimer 8-o-4-diferulic acid in rye extract were reported to exert antioxidant activity to inhibit LDL (low density lipoprotein) oxidation in vitro [78].

Conclusion Phenolic compounds are universally distributed in plants such as leave, trunk, root, seed, and fruit; and usually are bounded within plant tissues. The composition of phenolic compounds in staple plants have been studied by numerous researchers and staple plants are a potential source of phenolic compounds. This is important because there has been increasing demand of natural phenolic compounds in recent years. Phenolic compounds are secondary metabolites widely distributed in the plant kingdom. Phenolic compounds in cereals can be in free or bound forms. The free phenolic compounds are proanthocyanidins or flavonoids, whereas the bound phenolic compounds are ester-linked to cell wall polymers and mainly consist of ferulic acid and its oxidatively coupled dimmers. Ferulic acid and diferulates are predominant phytochemicals in grains that are not present in significant amount in some fruits and vegetables. Phenolic acids play important roles in nutrition and health for human. Phenolic acids are absorbed in humans and their antioxidant activity may reduce the risk of coronary heart diseases, cancers, and aging process.

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In: Phenolic Acids Editor: Sergi Munné-Bosch

ISBN: 978-1-61942-032-8 © 2012 Nova Science Publishers, Inc.

Chapter III

Phenolic Acids in Plant Cell Walls: Composition and Industrial Applications I. Zarra, G. Revilla, J. Sampedro and E. R. Valdivia Departamento de Fisiología Vegetal, Facultad de Biología, Campus Vida, Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain

Abstract Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Phenolic compounds in plants show not only a very diverse structure but also a wide range of functions. In this chapter we focus on phenolic acids associated with plant cell walls and their role as structural components, as well as their industrial applications. Plant cell walls are made of cellulose fibrils embedded in a matrix of polysaccharides, structural proteins, enzymes and phenolics. In the walls surrounding growing cells (primary walls) phenolic acids, such as ferulic acid and coumaric acid, are involved in the cross linking of cell wall polysaccharides. In addition, the secondary walls of cells that have stopped growing and have differentiated are typically impregnated with lignin which strengthens and dehydrates the wall. Thus, phenolic compounds have a key role not only in the primary wall but also in the secondary one as the main component of lignin. Besides extensibility, matrix cross-links are also likely to determine wall digestibility. In secondary walls lignin provides mechanical support and a waterimpermeable surface essential for the evolution of terrestrial vascular plants. However, the presence of lignin leading to lignocellulosic biomass recalcitrance is a bottleneck for the industrial use of plants. Lignin not only reduces digestibility but also interferes with the pulping process in the paper industry and with saccharification (enzymatic bioconversion of cellulose and other wall polysaccharides into fermentable sugars). The different approaches developed for the modification of plant biomass through manipulation of phenolics will be discussed.



E-mail: [email protected].

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Introduction Phenolic compounds are broadly distributed in the plant kingdom and are the most abundant secondary metabolites of plants, with more than 8,000 structures currently known, ranging from simple molecules such as phenolic acids to highly polymerized substances such as lignins and tannins. Although interest in their study was based initially on their well-known antioxidant features and their role in disease treatment [1,2], they also play a variety of functions both in the normal development of plant tissues and in responses to physical injury, infections or other stresses in plants [3,4]. These compounds can also act as signaling molecules in the initiation of legume-rhizobia symbioses and the establishment of arbuscular mycorrhizal symbioses [5]. Ferulic acid and pcoumaric acid affect rhizobial growth [6] and phenolic acids present in the root nodule of legumes stimulate the production of the plant hormone auxin, indole-3-acetic acid (IAA) in Rhizobium [7]. The term “phenolic” can be defined chemically as a substance which possesses an aromatic ring bearing a hydroxyl substituent or a functional derivative (esters, methyl ethers, glycosides, etc.) [8]. A major class of phenolic compounds are phenolic acids. The name “phenolic acids” in general, describes phenols that possess at least one carboxylic-acid functionality. They can be divided into two classes: hydroxybenzoic acids and hydroxycinnamic acids (Figure 1). The former are derivatives of benzoic acid, such as gallic acid, and the latter derivatives of cinnamic acid, such as coumaric, caffeic and ferulic acid. Phenolics are uncommon in bacteria, fungi and algae. Bryophytes are regular producers of phenolics and it is in the vascular plants that the full range of phenolics is found [8]. Phenolic acids found in plant cell walls and lignin have a unique chemical structure of C6-C3 (phenylpropanoid type) that is essentially absent in other living organisms [9], whereas those of microbial origin are of the form C6-C1 (phenylmethyl type) [10].

Figure 1. Structure of hydroxybenzoic acids (C6-C1 type phenolics), and hydroxycinnamic acids (C6C3 type).

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The Cell Wall The wall surrounding plant cells is a characteristic feature of plants. Despite its name, the “wall” is not a static structure that encloses the cell, but rather a highly dynamic organelle whose structure is closely related to the function of different cells and tissues [11,12]. Cell walls provide mechanical support, determine the cell shape and act as a barrier against biotic and environmental aggressions. They also provide the plasticity necessary for cell expansion and plant growth [13], and play a pivotal role in mediating responses to external stimuli [14]. All growing cells have a thin cell wall between 50 and 200 nm thick, able to sustain cell expansion and known as the primary cell wall. Although all primary walls share similar features, we can distinguish between type I walls typical of most plants (dicotyledons, some monocotyledons and gymnosperms) and type II walls present in grasses and related plants (commelinoid monocotyledons) [11,15]. The primary cell walls consist of cellulose microfibrils embedded in a matrix of polysaccharides (up to 90%), glycoproteins (10-20%) and a small amount of phenols (mainly ferulic acid). The matrix polysaccharides can be grouped into two major classes, pectins and cross-linking glycans. In type I walls xyloglucan is the principal polymer that interlocks the microfibrils, whereas in type II this role is taken by glucuronoarabinoxylan. Another difference is that type II walls have a lower content of pectins [16]. The overall structure is similar in both cases, with a multipolymer network of cellulose and cross-linking glycans embedded in pectins. In the primary cell walls, phenolic acids are minor compounds, but they are present in most plant groups, for example, in mosses [17], monocotyledons [18-23], dicotyledons [2427] and gymnosperms [28]. The content and composition of hydroxycinnamic acids can vary between different organs, tissues or stages of development. Hydroxycinnamates, such as ferulic acid, sinapic acid and p-coumaric acid, appear ester-linked to plant cell wall polymers and may act as cross-links between polysaccharides [29,30]. Such coupling reactions, catalyzed by peroxidases, decrease the wall extensibility and digestibility [31]. Once cell expansion ceases, in some cell types new layers are deposited between the primary cell wall and the membrane to create a secondary cell wall [15]. These secondary walls are generally enriched in cellulose, while the pectin content decreases [15]. Crosslinking glycans are typically xylan in angiosperms and mannans in gymnosperms. Many secondary walls are lignified, with mature walls reaching levels of 20-30% lignin in dicots and 7-15% in grasses [32].

Association of Phenolics Acids with Cell Wall Polysaccharides Type II walls typically contain larger amounts of hydroxycinnamic acids (0.5-1.5% dry weight) than type I walls [32,33]. These hydroxycinnamic acids are mainly ferulic, pcoumaric and sinapic acid. Initially, it was thought that these phenolic acids were esterbonded only to lignin [34], but further studies showed that they were also attached to wall polysaccharides, arabinoxylans in type II walls and pectins in type I walls. In grasses a proportion of the -L-arabinofuranose residues of arabinoxylans are substituted at position 5 with o-feruloyl ester groups (Figure 2). The linkage between ferulic

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acid and arabinoxylans was established with the identification of low-molecular-weight carbohydrate esters of ferulic acid. These compounds are released from walls treated with mild acid [35,36] or digested with polysaccharide hydrolyzing enzymes without any esterase activity, such as Driselase [36,37]. With this approach feruloylated arabinoxylan di-, tri- or oligosaccharides have been isolated from cell walls of Italian ryegrass [19], wheat bran [38], barley aleurone [18,39], barley endosperm [40], maize shoot [41], bamboo shoot [37,42], sugar cane bagasse [43], Festuca arundinacea cell cultures [44,45], wild rice flour [46] and maize bran [36]. Similarly p-coumaroylated arabinoxylan tri- and tetrasaccharide have also been isolated, although in lesser amounts, from barley straw [47] and growing bamboo [48], and a pcoumaroylated monosaccharide has been isolated from maize bran [36]. Bunzel et al. [46] identified sinapic acid in cell walls of wild rice grain, although isolation of defined sinapyloligosaccharides was not achieved. Finally, in growing bamboo a feruloylated xyloglucan disaccharide has been characterized with the site of o-feruloylation in position 4 of the Dxylopyranosyl residue [48]. In addition to grasses, a feruloyl trisaccharide has been characterized from pineapple fruit digested with Driselase, showing that in this species ferulic acid is ester-linked to arabinoxylan in the same way as in Poaceae [49]. In non commelinoid monocotyledons, ferulate has been identified in white and green asparagus spears [50] and a small amount of ferulic acid was found in onion bulb walls [51].

Figure 2. Structure of ferulic acid esterified to arabinan (A) and to arabinoxylan (B). Detail of arabinosyl ferulate (C).

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Whithin dicots, ferulic acid has been found in ‘core’ Caryophyllales species ester-linked to the side chain arabinans and galactans of the pectic polysaccharide rhamnogalacturonan I [52]. The ferulic acid is ester-linked at O-2 or O-3 of arabinofuranose residues in arabinans and at O-6 of galactopyranose residues in galactans. The first feruloyl-oligosaccharides characterized were isolated from cell walls of rapidly growing cell suspension cultures of spinach digested with Driselase [53]. Later, these and other oligosaccharides have been isolated and characterized from spinach leaves [27], sugar beet pulp [35,54,55], carnation stems [56] and amaranth seeds [57]. Other studies have identified ferulate in primary cell walls of glassworth stem [58] and quinoa [59]. Moreover, enzymatic hydrolysis of cell walls from sugar beet roots allowed the isolation of two new feruloylated oligosaccharides, an arabinotriose and an arabinotetraose esterified by two ferulic acid residues. Their structure was elucidated by mass spectrometry (MS) showing that the extra feruloyl groups are linked at O-5 of arabinose residues, in addition to the known O-2 position [60]. Regarding other dicots outside the Caryophyllales, low concentrations of ferulic acid have been found in carrot root [61,62] and tobacco stems [63], but the polysaccharides to which it is linked are unknown. Recently, in cell walls of black cottonwood, Gou et al. [64] have shown the presence of p-coumarate and ferulate. Both phenolics display a different tissue-specific developmental pattern of accumulation. Thus, while p-coumarate predominantly accumulates in young leaves, ferulate was mainly detected in the stem. Finally, ferulic acid and p-coumaric acid have also been identified in pine hypocotyl cell walls [28] and in primary cell walls of different organs in several gymnosperms species [65]. Besides lignin monomers, other cell wall phenolics have also been detected in cell walls. A good example is the presence of p-hydroxybenzoic acid in carrot roots. In this material, phydroxybenzoic acid is the predominant phenolic ester and appears to be attached to the branched pectic polysaccharides, possibly to either arabinose or galactose [56].

Feruloylation of Wall Polysaccharides It is assumed that the process of polysaccharide feruloylation occurs within the protoplast, probably in the Golgi apparatus [66-69]. However Yoshida-Shimokawa et al. [70], in an enzyme extract of suspension cultured rice cells, have detected feruloyl transferase activity not only in the cytosolic fraction, but also in the ionically bound fraction which they consider derived from the cell wall. The most frequently suggested donor substrate for polysaccharide feruloylation is feruloyl-CoA [68,70,71]. Nevertheless, Obel et al. [69] suggested feruloyl-glucose as a substrate for intracellular synthesis of feruloylated arabinoxylan, whereas synthesis of feruloylated proteins appears to use feruloyl-CoA as a donor. In apical root segments of wheat seedlings incubated with 14Ccinnamate, Mastrangelo et al. [72] detected the presence of 14C-feruloyl-polymers in the wall after 2 minutes of 14Ccinnamate feeding, while polysaccharide secretion took longer. This would indicate that feruloylation can occur in the wall. Furthermore the accumulation of 14C-feruloyl-polymers was resistant to treatment with brefeldin A (BFA). This compound leads to the disappearance of a distinct Golgi apparatus and to the complete disruption of the secretory system in plant cells [73]. This result further supports feruloylation in the wall and suggests the presence of a

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mechanism for the secretion of hydroxycinnamoyl precursors independent of polysaccharide secretion. Despite the isolation and characterization of feruloyl polysaccharides from many different plant materials, the feruloyl transferases responsible for the formation of the ester links between ferulates and cell wall polysaccharides have not yet been identified. Recently, Mitchell et al. [74] proposed that a family of 12 rice genes from Pfam family PF02458, which is part of the CoA-acyl transferase superfamily, encodes enzymes that transfer feruloyl residues onto arabinoxylan. They based this hypothesis on the high level of expression of this family in grasses, in contrast with a very low level of expression in dicots. In addition the rice genes in this family show high coexpression with other rice genes putatively involved in arabinoxylan synthesis. Based on these predictions, Piston et al. [75] studied the effect on cell wall ferulates of the RNAi down-regulation of rice members of Pfam family PF024458. Their results support a role for these proteins as arabinoxylan feruloyl transferases.

Cross-Linking between Hydroxycinnamate Polysaccharides

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Hydroxycinnamate Dimers Ferulate esters can be cross-linked via oxidative phenol coupling mediated by peroxidases and hydrogen peroxide [32] and/or photochemical processes [29]. The first ferulate dimer was isolated in small amounts from the water-insoluble pentosans of wheat endosperm in 1976 by Markwalder and Neukom [30], and identified as 5–5 coupled (referred to as diferulic acid) (Figure 3). For over 20 years this was the only known ferulic acid dehydrodimer. Diferulic acid has been identified in Italian ryegrass shoots [76], barley grain [77], rice endosperm [78] and avena coleoptiles [21]. In 1991, Ishii [79] isolated and characterizated a diferuloyl arabinoxylan hexasaccharide from an enzymic hydrolyzate of bamboo shoot cell walls, showing that arabinoxylans are covalently cross-linked via diferulic acid. Further studies found that other dimers besides the 5-5 coupled were also present in primary walls [80]. Seven dehydrodimers of ferulic acid were identified by nuclear magnetic resonance (NMR) spectroscopy in extracts of saponified cell walls from leaf blades of cocksfoot and switchgrass, as well as suspension-cultures of maize. All of them arise from oxidative coupling of ferulate esters in cell walls, which can happen in different configurations (8-5, 8-8, 8-O-4, 4-O-5 and 5-5) (Figure 3). The 4-O-5 coupled dehydrodimer is present in some of these samples at a very low concentration (only traces were detected). The amounts of 8-5, 8-O-4 and 8-8 dimers match or exceed those of the only previously reported dimer, the 5-5 coupled dehydrodimer [80]. Later, the 4-O-5 diferulic was identified in grains of maize, wheat, spelt and rice and showed that it is present approximately 70-100 times lower concentration that the sum of 8-5-coupled diferulic acids, the major diferulic acids in the investigated fibers [81].

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Figure 3. Structure of ferulic acid dimers.

Since the publication of Ralph’s group work [80], diferulates have been identified in many grasses such as wheat stems [82], maize coleoptiles [22], Italian ryegrass leaves [83] and different cereal grains [84,85]. In addition to grass, diferulates were identified in other monocotyledons such as Chinese water chestnut [86], chufa [87], pineapple and banana fruit [88], as well as asparagus [89].In dicots, diferulates have been characterized in sugar-beet pulp [90,91], carrot root [61] and mint suspension cultures [92]. In pine hypocotyls, Sánchez et al. [28] identified 8-8 coupled diferulates as the isomer present in the highest amount, while the 5-5 isomer was not detected in this material. In cell walls of suspension–cultured mint cells, dehydrodimers of caffeic acid have also been detected [93]. Although present in small amounts, sinapates can dimerize via radical coupling reactions to produce sinapate dehydrodimers. Thus, sinapic acid homodimers (two isomers of 8–8-coupled lactones) were identified in some cereal grains such as wild rice and spelt, but not in others such as oat or millet [94,95]. These findings suggest that oxidative dehydrodimerization of the available polysaccharide hydroxycinnamate esters is a general mechanism for cross-linking plant cell wall polysaccharides.Dimerization of ferulates and p-coumarates via photochemical processes leads to the formation of truxilic and truxinic acids (Figure 3). Cyclodimers, probably resulting from the photodimerization of p-coumaric, were identified in Italian ryegrass [96] and spear grass leaves [97]. Setaria and pangola leaves contain dimers derived from ferulic acid [97] and these have also been found in pine hypocotyls [28]. More recently, 18 cyclodimers made up of ferulic and/or p-coumaric were identified by GC-MS in maize stover [98].

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Hydroxycinnamate Oligomers In 2003 two groups isolated and identified a 5-5/8-O-4 coupled triferulic acid from maize bran [99,100]. Afterwards the presence of triferulates has been detected in other plant species and organs such as wheat bran [101], rye, wild rice, sugar beet and asparagus [29]. Recently four ferulate dehydrotrimers (5-5/8-O-4, 8-O-4/8-O-4, 8-8(aryltetralin)/8-O-4 and 8-O-4/8-5dehydrotriferulic acids) have been isolated from maize stover and characterized by UV spectroscopy, MS and NMR [98]. The authors suggest that these dehydrotrimers (or higher ferulates) can contribute to the formation of cross-linking networks in the cell walls.

Formation of Cross-Links Between Polysaccharides

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Feruloyl residues can form covalent bonds with each other by oxidative coupling, in the presence of peroxidase and hydrogen peroxide (Figure 4). This oxidation enables the formation of cross-links between cell wall polysaccharides, via diferulate and oligoferulate bridges This process contributes to wall assembly and promotes tissue cohesion, while restricting cell expansion [102,103]. In addition this process is widely assumed to render the cell wall recalcitrant to digestion, inhibiting the rate and extent of enzymatic hydrolysis of cell wall carbohydrates by ruminant microbes and fungal enzymes. Similarly, it makes cell walls recalcitrant to enzymatic saccharification prior to fermentation in biofuel production [104]. Dimerization or oligomerization also play an important role in the textural and processing properties of plant foods [87].

Figure 4. Arabinoxylan cross-linking. 1: ester linked FA toAX. 2: FA ether linked to lignin. 3: 5-5 FA dimer. 4: arabinose- lignin. Modified from [4]. Arabinoxylan chains are represented at the top and the bottom of figure.

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Recently, Mélida et al. [105] have observed higher dimerization levels in maize cell cultures habituated to dichlobenil (DCB) than in non-habituated ones, through the use of [14C] cinnamate, a ferulate precursor. This process probably strengthens the cellulose-deficient walls. The oxidative coupling of polysaccharide-sterified feruloyl groups could occurs partly in the protoplast (presumably within Golgi vesicles) and partly in the apoplast following secretion. Fry et al. [68] found, in maize suspension-cultures fed with 14Ccinnamate, that diferulate formation takes place in the protoplasm in young cultures and shifts to the cell wall in older cultures. In addition, the incorporation of 14Cferulic acid in the polymeric fraction of suspension-cultured wheat suggests that different dimers are synthetised intracellularly and extracellularly. In particular intracellular dimer formation appeared to be confined to 8-5diferulic acid [69]. However these results are contradicted by recent studies of the kinetics of diferulate radio-labeling with 14Ccinnamate in maize and spinach cell suspension cultures [106]. In this work six putative diferulate isomers were detected whose synthesis at least began intraprotoplasmically. On the other hand, evidence for oxidative coupling of feruloyl groups in the apoplast was found in cultured maize cells fed with a radiolabelled feruloyloligosaccharide 5-O-feruloyl--L-arabinofuranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-Dxylose. During binding to the wall, the [14C]feruloyl groups were converted to [14C]dehydrodiferulates and larger coupling products [107]. Although the results presented above show that oxidative coupling of feruloyl residues occurs in vivo, they do not indicate whether or not dehydrodiferuloyl residues act as interpolysaccharide bridges (true cross-links) rather than intra-polysaccharide bridges (creating looped molecules). To distinguish between both possibilities, Burr and Fry [103] studied the timing and stability of cross-links in soluble extracellular arabinoxylans, using maize cell suspension cultures that slough some of their wall-related polysaccharides, mainly arabinoxylans, into the culture medium. Their results provide evidence that the oxidation of the feruloyl ester side-chains of arabinoxylans enables the formation of cross-links between the polysaccharides, mainly via oligoferulate bridges rather than via diferulates. They also propose that, after the oxidative coupling, ether-like bonds are formed between ferulates and polysaccharides via quininemethide intermediates. Previous experiments with mature oat internodes and maize cell suspensions undergoing lignification had demonstrated the presence of ether bonds between ferulate and lignin [108,109]. However, these bonds cannot play the same role in nonlignified maize cells walls [103]. It is widely assumed that feruloyl coupling products are always involved in wall tightening and therefore in the cessation of cell growth. The fact that young, rapidly growing maize cells, secrete into their walls arabinoxylan molecules that already contain diferuloyl bridges [68] conflicts with that “dogma”. Fry [68,102] argues that the cross-linked arabinoxylan secreted by these cells would be expected to hydrogen-bond loosely to the most recently deposited cellulose microfibrils. It could thus be a mechanism to prevent excessive hydrogen-bonding between microfibrils and keep high cell wall extensibility. In contrast, older maize cells, whose growth rate is slower, secrete more individual arabinoxylans chains than can hydrogen-bind to cellulose, tethering neighbouring microfibrils and thus thightening the wall.

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Role of Peroxidases It is generally considered that peroxidases are responsible for oxidative coupling of feruloyl residues. The peroxidases not only generate free radical intermediates of ester-linked feruloyl residues, but may also generate the hydrogen peroxide needed for the reaction from various hydrogen donors [32]. One of the consequences of ferulate cross-linking between matrix polysaccharides can be the stiffening of the cell walls and, consequently, the cessation of cell elongation. This mechanism has been proposed for dicots [31] and monocots [21]. There are many reported examples of negative correlations between the rate of cell expansion and the amount of diferulic acids bound to cell wall polysaccharides [21,28,110-112]. A similar relationship between apoplastic peroxidase activity and cell wall stiffening has been found in many different studies [28,24,113-119]. In oat cell walls, the increase of peroxidase activity preceded the accumulation of diferulic acid [120]. It is well known that feruloylated arabinoxylans and pectins can be cross-linked in vitro by incubation with peroxidase and hydrogen peroxide [121,122]. Primary maize cell walls become cross-linked by oxidative coupling of ferulic acids monomers after treatment with hydrogen peroxide [109,123]. Also, a feruloyl-arabinoxylan trisaccharide infiltrated into the walls of maize cells was oxidatively coupled without the addition of exogenous hydrogen peroxide [107]. However, the identification of diferulates and oligoferulates in the cell wall, and the ability of peroxidase activities to catalyze their formation in vitro, does not automatically prove that peroxidase are responsible in vivo. Alternative possibilities are that this reaction is catalyzed by oxidases (‘laccases’) or even that it takes place non-enzymically [102]. To gain new information about the oxidative coupling of feruloyl groups in vivo, Burr and Fry [33] used non-lignifying maize cell cultures to study arabinoxylan cross-linking in vivo and in vitro. Previous work had shown that maize cell-suspension cultures secrete some of their wall polysaccharides into the medium, where they remain soluble [124]. Feeding maize cell-cultures with 3Harabinoxylans in the presence of 20 mM KI, a peroxide scavenger, Burr and Fry [33] showed that in vivo cross-linking was H2O2-dependent, supporting the role of apoplastic peroxidases in this process. The inhibition of cross-linking cannot be attributed to a toxic effect on cells of 20 mM KI, which had previously been tested in loblolly pine cell-suspension culture [125]. The results of Burr and Fry [33] show that the cross-linking of polysaccharides is age-related. The lack of cross-linking in younger cultures is probably due to the presence of a low molecular weight inhibitor [105] or to high levels of ascorbate in the apoplast [126]. A function for apoplastic ascorbic acid in preventing the dimerization of phenolics at the same time as scavenging hydrogen peroxide has been proposed in dicots [127-129] and pine [126]. The regulation of the cross-linking of cell wall polysaccharides by the ratio ascorbate/hydrogen peroxide in the apoplast has been proposed by Pedreira et al. [116]. The formation of cross-linkages in wall polysaccharides catalyzed by apoplastic peroxidases requires the local provision of hydrogen peroxide. However, although hydrogen peroxide has been detected and quantified in the apoplast of pine hypocotyls [130], the mechanism responsible for its production has not yet been determined. In cereals, a generator of reactive oxygen species (ROS) is the apoplastic enzyme oxalate oxidase (OXO; EC 1.2.3.4) initially designated as "germin" [131]. In germinating wheat embryos, oxalate oxidase accumulates in cells that have ceased to expand [132]. It has also been demonstrated

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that callus induction by auxin, which prevents cell expansion, is associated with a rapid accumulation of this activity in the walls [133]. Very recently, Wakabayashi et al. [134] showed the presence of OXO activity in wheat shoot walls. Moreover, addition of oxalate caused an increased in the amounts of diferulic acid isomers comparable to addition of hydrogen peroxide. This result suggests that cell wall OXO promotes peroxidative crosslinking of wall polysaccharides and contributes to the cessation of cell elongation.

Hydroxycinnamates and Lignins

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Lignin Cross-Linking to Polysaccharides Lignins are complex polymers of phenylpropanoid residues, mainly derived from three phydroxycinnamyl alcohols that are referred to as monolignols: p-coumaryl, coniferyl and sinapyl alcohol. They respectively give rise to the p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units of the lignin polymer. The monolignols are C6–C3 phenylpropanoids and differ from each other only by their degree of methoxylation [15,135,136]. The term “lignin” does not refer to a defined structure but rather denominates a group of polymers with a structure controlled by their polymerization conditions [135]. Lignins from different taxa, tissues, cell types, cell wall layers or environmental conditions vary in monomeric composition, types of linkages between monomers and organization of monomers in the polymer [15,135]. These differences influence the strength and pulping properties of woody tissues [137]. The most common types of linkage are β-O-4-, β-5- (mainly from monomeroligomer couplings), 5-5-, 5-O-4- (from oligomer-oligomer couplings), β-β- and minor β-1couplings [95,136]. In addition to its role in the formation of cross-links between wall polysaccharides, ferulates are also implicated in the cross-linking of wall polysaccharides to lignin (Figure 4). Covalent cross-linking between wall polymers is a physiologically significant strategy contributing to the termination of wall extensibility, wall strengthening, and the blocking of pathogen penetration [138]. In 1985, Scalbert et al. [139], identified bound-phenolic acids in lignin preparation isolated from wheat straw. p-coumaric acid was mainly ester-linked whereas most ferulic acid was ether-linked to lignin. Isolated lignins were associated with arabinoxylans suggesting that ferulic acid ether-linked to lignin can form cross-bridges to polysaccharides through an ester linkage. Later, this hypothesis was supported by results obtained by Lam et al. [140] and Iiyama et al. [141] in mature and maturing wheat internodes. Analysis of lignin-polysaccharide complexes from extract-free wheat and phalaris internodes have shown that all the etherified ferulic acid present is also esterified, presumably to polysaccharides, although lignin-lignin diferulyl bridges could not be excluded [142]. Lapierre et al. [143] have provided evidence that typical lignin structures are tightly associated with maize bran heteroxylan extracted by alkali and extensively purified, supporting the occurrence of covalent linkages between heteroxylan chains and lignin structures. However, they could not determine whether these linkages existed ab initio and/or were formed during the alkaline extraction of heteroxylan. The cross-linking, mediated by ferulates, between polysaccharides and lignins, strengthens the cell wall, protects against pathogen invasion while decreasing the enzymatic degradability [144].

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Role of Ferulate in Lignification Ferulate participation in lignification has been proposed to explain the identification of ferulate-monolignol cross-coupled dimers. Jacquet et al. [145] isolated and characterized two isomers of β-O-4-crossed dimers from wheat and oat straws. Later, in addition to these dimers, the 8-5- and 8-β-crossed dimers have been identified in ryegrass [146], and cereal grains (barley, millet, oat, rice, rye, spelt, wheat and wild rice) [147]. Using feruloylated primary walls of maize artificially lignified by supplying exogenous monolignols and a source of hydrogen peroxide, Grabber et al. [109,148], have also demonstrated that ferulate and diferulates are able to cross-couple with coniferyl alcohol, forming mostly β-O-4- and 8β- cross-coupled structures. Recently, the study of the products of radical coupling reactions between ethyl ferulate, a simple model for feruloyl polysaccharides in planta, and coniferyl alcohol allowed the identification of a greater variety of cross-coupling products [149]. All these results support the hypothesis that ferulate (and also dehydroferulates and oligomers of ferulate) esterified to polysaccharides may function as nucleation sites for lignification, at least in grasses [146]. Recently, Ralph [150] has reviewed the role of ferulate in lignification. This author suggests that if ferulates are incorporated into lignins by reactions that typify lignification, they should be also considered as lignin monomers, in addition to “classic” monolignols.

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Presence of p-Coumarates on Lignins Besides ferulates, grass species contain significant amounts of p-coumarate esters acylating lignin [20,151,152]. The amount of p-coumarate found in grass lignins greatly exceeds the amount of a p-coumarylated monosaccharide that has been isolated from maize bran [36] as well as the amounts of cyclodimers resulting from the photodimerization of pcoumarate that have been identified in some grasses [96,97]. It thus seems that most of the pcoumarate in grasses is present in lignin [152]. The accumulation of p-coumarate in maize walls occurs in the lignin fraction and is shuttled out to the wall as sinapyl alcohol pcoumarate esters [153]. A strong relationship has been found between lignification and pcoumaroylation of lignin monomers during maize stem development [154]. Although pcoumarate can be attached to coniferyl or sinapyl alcohol lignin residues, there is a strong preference in maize (90%) for sinapyl alcohol [153]. In contrast with ferulates, p-coumarates do not enter into radical coupling reactions in order to become incorporated into the lignin. Although p-coumarates will undergo radical coupling in vitro, there is no evidence that they do so in grass cell walls [150]. Recently, Hatfield et al. [155] studied the capacity of peroxidases from maize cell walls to oxidize cell wall phenolic components. They found that p-coumarate is oxidized rapidly while sinapyl alcohol was poorly oxidized. The addition of p-coumarate enhances the rate of synapyl alcohol oxidation. Their results support the role of p-coumarate as providing a radical transfer mechanism for radical coupling of sinapyl alcohol into the lignin polymer. Furthermore, Hatfield et al. [156] have identified the p-coumaroyl transferase responsible for acylation of lignins in a range of C3 and C4 grasses. This enzyme utilizes p-coumarateCoA as activated donor and sinapyl alcohol as acceptor to produce sinapyl p-coumarate

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conjugates that are transported to the cell wall. It can also use coniferyl alcohol as donor, but the activity of the donor ferulate-CoA was negligible.

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Phenol Occurrence Although cell walls are a characteristic of all plants, they are also present in other organisms. Most bacteria as well as algae and fungi are also surrounded by an extracellular structure. The macromolecular composition is characteristically different among the different evolutionary lineages [157]. A cell wall based on polysaccharides is found in eukaryotic groups. The basic polysaccharide components of plant cell walls are cellulose and hemicellulose. The presence of additional components confers distinct characteristics to cell walls, which have played significant roles for the survival of these lineages through the course of evolution [158]. The Archaeplastida, a monophyletic group comprising the Glaucophyta, the Rhodophyta and the Chloroplastida (green algae and land plants), are characterized by carbohydrate-rich cell walls. Cell wall modifications enabled diversification and adaptation to different ecological roles and were part of the major events in the evolution of this group, such as multicellularity, terrestrialization and vascularization. During the course of evolution the intense competition for resources in the oceans, in addition to changes in the atmosphere, resulted in a gradual shift from aquatic to terrestrial habitats within the group of green algae that gave rise to land plants. Lignification is considered a key acquisition in this evolutionary transition. Lignin, a complex crosslinked aromatic heteropolymer, embeds cellulose microfibrils creating a stiff secondary wall. The mechanical support that lignin lends to walls prevents the collapse of conductive vessels and allowed plants to develop deeper roots with access to permanent sources of water, as well as to grow taller enhancing light capture and thereby primary productivity. It is for this reason that lignins are found in all vascular plants, although there are differences among the different groups. H and G lignins are ubiquitous but S lignins are characteristic of Lycopods and certain Angiosperm groups [159]. Bryophytes resemble the earliest land plants, growing in moist land where water is close to the surface. They do not possess a specialized water transport structure, because simple diffusion seems to be enough for the short distance that water needs to travel [160]. Some phenolic compounds, but not lignin, have been found in bryophytes. Ligrone et al. [161], using two polyclonal antibodies against synthetic lignin-like polymers entirely made of guaiacyl or guaiacyl-syringyl precursors [161], found that all the bryophytes tested gave a positive reaction with the labeling ranging from very weak to intense in certain cell types. Electron microscopy showed that labeling was evenly distributed in the cell walls including the middle lamella and the cell corners. However, bryophytes did not present reaction to the Wiesner test which detects coniferyl and sinapyl aldehydes that have not been reduced to alcohols and are incorporated into lignins. The lack of response to the Wiesner test reflects the absence of cinnamyl aldehydes but the presence of other related lignols cannot be excluded. Moreover, moss species possess cell wall autofluorescence, which is considered an indication of the presence of polyphenolic material [162]. Although the presence of true lignin in bryophytes remains controversial, the presence of lignin-like polymers and polyphenolic material in bryophytes must be accepted.

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Components corresponding to lignin units have also been found in a red alga (Rodophyta) [163]. Calliathron cheiloaporoides fronds calcify encasing cells in calcium carbonate and have decalcified joints named genicula that allow fronds to bend when struck by waves. Cell walls of genicular cells undergo deposition of secondary walls. Wall analysis showed the presence of components derived from monolignols corresponding to H, G and S units, the same components that characterize lignin in land plants. It thus seems reasonable to think that wall phenolics or lignin-like materials are not exclusively related with water conducting systems since they may be present in aquatic organisms as well as in non vascular plants. The fact that in these latter groups phenolic material is not associated with vascular tissues suggests that it can fulfil other structural functions or confer protection against microorganisms or UV radiation. Lignification may have originated as a mechanism to reinforce certain walls of specific cells and was only later coopted for the strengthening of tracheids. Figure 5 shows the appearance of phenolic material in different lineages. Since monolignol synthesis is exceptionally complex, it seems unlikely that Calliarthon and land plants evolved monolignol synthesis fully independently. It is more reasonable to think that the relevant pathways such as phenylpropanoid biosynthesis and polymerization by peroxidase-catalyzed oxidation, may be deeply conserved, having evolved before the divergence of red and green algae. If this hypothesis is correct, we can expect to find conserved enzymatic pathways, and potentially evidences of lignification among the multitude of evolutionary intermediates [161]. A recent confirmation of this idea is the discovery of peroxidases able to oxidize S monolignols in Bryophytes lacking lignin or xylem tissue [164].

Figure 5. Simplified phylogeny of Archaeplastida highlighting the occurrence of phenolic material associated with cell walls. The arrow indicates the origin of plastids by primary endosymbiosis. Close circles denote the appearance of lignins and open circles of aromatic compounds. H, G, and S are the three lignin types.

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Phenylpropanoid Biosynthesis The shikimic pathway starts from the condensation of eritrose-4-phosphate and phosphoenolpyruvic acid mediated by 3-desoxy-arabinoheptulosonato-7P synthase, and leads to the formation of aromatic compounds, such as phenylalanine, which is the precursor for monolignols.Phenylpropanoids are synthesized from phenylalanine by the action of phenylalanine ammonia lyase and cinnamate 4-hydroxylase (C4H), which result in the formation of p-coumarate (Figure 6). p-Coumarate is further activated by the formation of a coenzyme A-thioester, in some cases hydroxylation and methoxylation of the aromatic ring, and finally reduction of the carboxylic group to alcohol. These are the main reaction leading to the formation of monolignols, the precursors of lignin. The three different monolignols are coumaryl, coniferyl and sinapyl alcohol and they become respectively H, G and S type subunits when incorporated into lignin [165,166].

From Phenylalanine to Monolignols: Enzymes Involved

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Although the shikimic pathway has been located in plastids [166], there are no conclusive evidences about the cellular localization of the phenylpropanoid pathway. The main reactions of phenylpropanoid pathway are shown in Figure 6.

Figure 6. A scheme of monolignol biosynthesis pathways. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, hydroxycinnamate-CoA/hydroxyferuloyl-CoA ligase; HCT, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase; C3H, 4-hydroxycinnamate 3-hydroxylase; CCoAOMT, S-adenosyl-methionine caffeoyl-CoA/5-hydroxyferuloyl-CoA-Omethyltransferase; CCR, hydrxycinnamoyl-CoA:NADPH oxidoreductase; CA5H, coniferylaldehyde 5hydroxylase; AldOMT, 5-hydroxyconiferyl aldehyde o-methyltransferase; CAD, hydrxycinnamoyl alcohol dehydrogenase; CALDH, coniferylaldehyde dehydrogenase; SALDH, sinapylaldehyde dehydrogenase; R, shikimic or quinic acid. Monolignol precursors for lignin are in a box. Cell wall bound phenolic acids are in a dotted box.

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Although there are some controversies as to whether lignin biosynthesis in all plants follows the same metabolic pathway, most of the genes and reactions involved have been characterized in various plant species. The initial reactions of the pathway until the formation of p-coumaryl-CoA are mandatory and provide the precursor for all subsequent branches leading to the synthesis of different metabolites in addition to monolignols, such as flavonoids, stilbenes, catechins, phenylpropanoid esters and others [166].

PAL

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The deamination of phenylalanine to form cinnamic acid is catalyzed by the phenylalanine ammonia-lyase (PAL), and is the gateway that channels the metabolic flow from the primary metabolism towards the synthesis of phenolics. PAL enzymes are coded by small multigene families in all plant species studied. Arabidopsis and poplar, for example, have four and five genes respectively, while rice has nine [167]. A close relationship between gene expression, enzyme activity and increases in phenolic compounds under different stimuli has been found in several species [165]. In addition, PAL is regulated by a negative feedback control exerted at the level of C4H activity. If cinnamic acid production is greater than utilization, both PAL transcription and enzyme activity are inhibited [168]. Furthermore, it has been suggested that PAL and C4H enzymes associate to form a metabolon (i.e. multienzyme complex). Tight coupling between both enzymes could maintain a low cinnamic acid level that would avoid feed-back inhibition. Moreover, if different PALs have different subcellular localizations, as is the case for PAL1 and PAL2 in tobacco, this could be the basis for the canalization of the metabolic flow towards the synthesis of different phenylpropanoids [169].

Hydroxylation More than sixteen cytochrome P450 monooxygenases are involved in the biosynthesis of different phenylpropanoid classes. The reactions carried out by P450 monooxygenases involve oxygen and use NAD(P)H as cofactor. They are highly exothermic and channel the metabolic flow irreversibly into specific branches [170]. The hydroxylation of the aromatic ring in the synthesis of monolignols is catalyzed by three independent enzymes of the P450 family. Cinnamate 4-hydroxylase (C4H) hydroxylates cinnamate in the general phenylpropanoid pathway, while coumaryl 3-hydroxylase (C3H) and coniferylaldehyde 5-hydroxylase (CA5H) participate in the synthesis of coniferyl and sinapyl alcohols (Figure 6). Genes from the CYP73 family code for C4H enzymes, which have been shown to be highly specific for cinnamate. These proteins are bound to the endoplasmic reticulum [171]. The association of C4H enzymes with some specific PALs in multienzyme complexes would thus channel the phenylpropanoid flow towards the secretion pathway. C3H enzymes are responsible for the 3-hydroxylation of coniferyl and sinapyl alcohols. The mechanism of this reaction remained elusive until the involvement of ester intermediates was suggested. Using genetic and bioinformatic approaches three different groups [172-174] identified a member of the CYP98 family as the P450 enzyme most likely responsible for the

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C3H activity in the phenylpropanoid pathway. This enzyme does not metabolize neither free p-coumaric, nor its glucose or CoA esters, or its corresponding aldehyde or alcohol. On the other hand it converts efficiently the 5-O-shikimate or 5-O-quinate esters of p-coumaric into the corresponding caffeic acid conjugates. Finally the 5-hydroxylation present in sinapyl alcohol can occur on coniferylaldehyde or coniferyl alcohol, but not on free ferulic acid [170,174]. Therefore, the enzyme involved must be named coniferylaldehyde 5-hydroxylase (CA5H) and not ferulic acid 5-hydroxylase (F5H), as it was formerly known. The CA5H plays a central role in the regulation of the metabolic flux into the sinapyl route.

Activation In many species, 4-coumarate coenzyme A ligase (4CL) genes exist as a family of multiple members with different expression patterns. In addition different 4CL proteins have distinct substrate specificities and this probably provides the basis for the control of the different branches of phenolic synthesis, such as flavonoids or lignin, in a highly compartimentalized metabolism [175].

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CAD and CALDH/SALDH Cinnamyl alcohol dehydrogenases (CAD) catalyze the production of multiple cinnamyl alcohols from their corresponding cinnamyl aldehydes, the last step forming the three monolignols, coumaryl, coniferyl and sinapyl alcohols. The cinnamyl aldehydes can also be the precursors of hydroxycinnamic acids. The prevalent pathway to ferulic and sinapic acid proceeds from 4-coumarate-CoA, producing coniferylaldehyde and sinapylaldehyde. Instead of being reduced to coniferyl and sinapyl alcohols, these aldehydes are then oxidized to ferulic and sinapic free acids by a bifunctional coniferylaldehyde/sinapylaldehyde dehydrogenase (CALDH/SALDH) [176].

CCR The reduction of cinnamoyl-CoA esters to cinnamoyl aldehydes by cinnamoyl-CoA reductase (CCR) is a key metabolic step in the formation of monolignols. This enzyme can act on two different substrates. Reduction of p-coumaryl-CoA leads to formation of coumaryl alcohol, while reduction of feruloyl-CoA leads to either coniferyl or sinapyl alcohol. The affinity of CCR against different substrates may be important for the distribution of the pathway flux towards the synthesis of different phenolic compounds. When recombinant CCR from aspen was coupled with CCoAOMT, coniferylaldehyde was formed from caffeoylCoA, suggesting that both are neighboring enzymes. However, at least in vitro, they require very different pH environments suggesting that compartmentalization is necessary [177].

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Methyl Transferases Methylation can be present at the 3- and 5-positions on the aromatic ring of monolignols. The 3- and 5-methylations occurs at different metabolic stages. The 3-methylation is on the caffeoyl-CoA ester and is catalyzed by the caffeoyl-CoA 3-O-methyltransferase (CCoAMT) [178]. The 5-methylation occurs on the 5-hydroxyconiferyl aldehyde and is catalyzed by the 5-hydroxyconiferyl aldehyde o-methyltransferase (AldOMT) [179].

Compartmentalization

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The biosynthesis of phenylpropanoids involves multiple enzymes and it is currently unknown how they are organized to facilitate the flow of metabolites through the pathway. It is generally assumed that hidroxycinnamic acids and monolignols are synthesized in the cytosol. While hydroxycinnamic acids are incorporated into the Golgi apparatus to be esterified to non-cellulosic polysaccharides [66-69], monolignols are exported through the plasma membrane for lignin biosynthesis. The symplastic monolignols may be exported directly to the cell wall through diffusion or by active transport. Alternatively, monolignols may be glycosylated and stored in vacuoles. Glucosylation reduces lipophilicity of small molecules as well as diffusion across membranes. The stored glucosides could then be transported to the cell wall and hydrolyzed before they are polymerized to form lignin [180]. Glycosylation of monolignols catalyzed by UDP-glucose glucosyltransferases has been demonstrated [181,182]. Additionally, the specific glucosidases necessary to release monolignols have been characterized from a survey of different gymnosperms and angiosperms species [180]. The presence of both monolignol glucosyltransferase and the correspondent glucosidases suggests the existence of a system to regulate the storage and mobilization of monolignols for cell wall lignification.

Transport The transport mechanism of monolignols across membranes is not known and more than one mechanism may be involved in the transport of monolignols and their glucosides. Although the possibility of passive diffusion cannot be excluded, specific membrane transporters seem necessary. The existence of membrane monolignol ABC transporters has been postulated [183,184] but the evidence is not conclusive. Furthermore, in addition the direct export of monolignols across the plasma membrane the role of the secretory pathway needs to be elucidated.

Transcription Factors The synthesis of monolignols and phenolic acids are strictly regulated during plant development, both in space and time. In the last years, a wide range of transcription factors controlling these specific pathways have been characterized. Several transcription factors from the MYB and LIM families have been shown to interact with promoters of genes

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involved in monolignol synthesis [185,186]. More specifically, EgMYB2 was cloned from Eucalyptus and the recombinant protein bound specifically to cis-regulatory regions of the promoters of two monolignol biosynthetic genes, cinnamoyl-CoA reductase and cinamylalcohol dehydrogenase, which contain MYB consensus binding sites [187].

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Cell Wall Integration After export of the lignin monomers into the cell wall they are oxidized and polymerized to lignin. The monolignols are oxidized by peroxidases or laccases producing free radicals which react among them to form new linkages [188-193]. The classical theory develop by Freudenberg [194] proposes that free radicals produced by oxidation of monolignols are coupled in a combinatorial way [195]. Any monolignol free radical present in the cell wall is capable to enter in a combinatorial coupling process to the extent allowed by structural compatibility. The process is influenced by pH, temperature, ionic environment, monolignol supply, hydrogen peroxide concentration, peroxidase activity and the matrix in general. It is difficult demonstrate the occurrence of such mechanism in vivo, but it has been replicated in mimetic systems under different conditions including the presence of polysaccharides [196]. An alternative hypothesis has also been proposed, the dirigent protein model. The dirigent protein would act as a template forming a defined primary sequence of lignin polymer [197]. Numerous genes coding for putative dirigent proteins have been tentatively identified but only one of them has been well characterized. The action of this protein results in the formation of an 8-8’ linked coniferyl alcohol dimer [198,199]. However, for the dirigent protein hypothesis to gain acceptance it would be necessary to demonstrate that lignin has a defined sequence, in addition to the characterization of the different proteins involved [195]. The random coupling of monolignols leading to metabolic plasticity could be an advantage in the defense against pathogen attack. The lack of regularity in its structure poses a problem to the evolution of hydrolytic enzymes. Plant evolution could thus have resulted in the production of lignin without a very precise structure under a careful regulation of the supply of its precursors, as a mechanism of defense against the battery of existing pathogens.

Cell Wall Phenols and Biomass Recalcitrance As described above, plant cell walls contain phenolic material associated with polysaccharides which contributes to their structure. The presence of phenolic compounds in the cell walls confers new properties to the plant cells. The hydroxycinnamic acids associated with polysaccharides contribute to the mechanical strength of the primary walls, as well as the secondary ones. The diphenyl bridges between individual polysaccharide chains contribute to growth cessation at the same time as they increase the mechanical strength. Furthermore, the presence of lignin in secondary walls confers to plant cells the necessary mechanical strength to grow taller and transport water and nutrients from the deepest roots to the higher branches, while its chemical stability contributes to the defense against pathogens. As previously mentioned the appearance of phenolics in plants was one of the main milestones in their

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evolution. On the other hand, from an anthropocentric point of view, phenolics can constitute a problem for the exploitation of the plants. Plant biomass is a source of renewable carbon which can be used not only as food but also for the production of different chemicals, biofuels, etc. However the strong association of lignin with cellulose microfibrils and hemicelluloses make its industrial deconstruction a high energy consuming process. Cell walls are the main component of plant biomass. This lignocellulosic complex is a source for different applications: forage, pulping, biofuels, etc. The common problem for all these uses is the recalcitrance of the biomass, considered as the resistance that it poses to deconstruction with microbes and enzymes. Different approaches are being considered to overcome such recalcitrance, which can be divided into two groups: modifications in the structure of the lignocellulosic complex and improvements in the efficacy of the degrading enzymes. The modification of the phenolic composition of the lignocellulosic biomass has been attempted by genetic engineering techniques (see [200] for a review). The downregulation of PAL, C4H, 4CL, HCT, C3H, CCoAOMT or CCR have important effects on lignin content. For example, down regulation of 4CL decreased in 45% the lignin content in Populus tremuloides [201]. Reduction in monolignol synthesis is associated with changes in the pools of soluble phenolics [202-205]. If the lignin content is too low, plants present a pleiotropic effect on growth and development often attributed to a dysfunction of the vascular system [206,207]. It is not necessary to reduce the lignin content to modify the properties of lignocellulosic biomass, changes of its monomeric composition or H/G/S ratio, can significantly modify its recalcitrance. HCT- [208] or C3H-down regulated [209] plants present lignin enriched in H units, and in Medicago this approach produced a material that is easily digested [210]. Furthermore, down regulation of CA5H results in lignin enriched in G units while its overexpression results in lignins almost entirely composed of S units, resulting in poplar plants that are digested and pulped more easily [211,212]. However, analysis of a set of transgenic alfalfa lines with defects in nearly all steps of monolignol synthesis has demonstrated that S/G composition, unlike lignin levels, has no strong effects on saccharification efficiency in this species [213,214]. The possibility of modifying the composition of lignin in terms of H/G/S ratio or in the levels of minor units demonstrates the extraordinary plasticity of the polymerization process. An increase in the incorporation of units different from the classical ones can occur when the main flow of metabolites in the phenylpropanoid pathway is altered. For example, when CAD is downregulated a reduction in the production of coniferyl and sinapyl alcohols was observed and the incorporation of their correspondent aldehydes into lignin significantly increased [136,215]. The flux from coniferyl alcohol precursors towards sinapyl alcohol requires the activity of CA5H for the hydroxylation at position 5 to produce 5-hydroxyconiferylaldehyde, which is then methylated by COMT also at position 5. Therefore, both enzymes are necessary to determine the resulting S/G ratio of lignins. However, as the previous hydroxylation at position 5 is irreversible, downregulation of COMT has a novel effect. The conversion of 5hydroxyconiferylaldehyde to sinapylaldehyde is blocked but it can still be reduced to 5hydroxyconiferyl alcohol by CAD. This novel monomer is exported to the cell wall where, like other monolignols, it becomes incorporated into lignin [216,217].

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In vivo monolignols are methylated exclusively at position 3/5 but not at the 4 position. The hydroxyl at position 4 is necessary for the oxidative coupling of phenoxy radicals to form polymers. Therefore, Bhuiya and Liu [218] have created a 4-O-methyltransferase to substitute the hydroxyl at position 4 and interfere with the polymerization. By site-saturation mutation of a cDNA encoding a methyltransferase from Clarkia breweri an enzyme able to methylate at position 4 was obtained. Experiments carried out in vitro with this enzyme demonstrate that the presence of the methoxy group at position 4 impairs oxidative coupling. This result opens a new way to engineer lignin structure. The analysis of lignin mutants using transcriptomic and metabolomic approaches has revealed that modification of the expression of individual genes in the phenylpropanoid biosynthesis pathway has important effects on plant metabolism beyond phenolics. Changes in the polysaccharide fraction of cell walls, such as a reduction in xylan synthesis, have been found when lignin levels in Populus were reduced by CCR downregulation [219,220]. Furthermore, CCR downregulation in Nicotiana tabacum increases starch metabolism and photorespiration [221]. In addition to down or upregulation of lignin biosynthesis, the incorporation of phenolics from other pathways into lignin biosynthesis has been shown to be effective for efficient deconstruction of the lignocellulosic complex. The incorporation into lignin of some compounds with easily broken linkages can reduce the recalcitrance of the lignocellulosic material. Following this approach, maize cell suspensions were fed coniferyl ferulate, which has an ester interunit linkage (Figure 7).

Figure 7. Polymerization of coniferyl ferulate to introduce alkali labile linkages into lignin. Ester linkages facilitates lignin depolymerization by alkali treatment.

This ferulate monolignol ester was incorporated into lignin. Although it is stable under in vivo conditions, when the cell walls of the maize suspension were treated with aqueous NaOH, the ester linkages were broken and the efficiency of lignin extraction was significantly

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improved [222]. A similar effect can be expected in hardwood, softwood and herbaceous dicotiledoneous plants. This experimental approach opens the option of redesigning lignins through the incorporation of new units, resulting in the modification of lignin properties. The challenge now is the production of this labile “lignol units” in vivo. An alternative approach to modify lignin extractability is to introduce tyrosine rich peptides into the lignin macromolecule. Poplar plants have been obtained that express in lignifying tissues a transgene encoding a high tyrosine-peptide sorted to apoplast. The presence of the aromatic ring of tyrosine allows the formation of linkages between tyrosine residues and the monolignol units of the lignin. Some transgenic plants did not show phenotypic anomalies but their cell walls were more susceptible to protease digestion than wild type plants [223]. The ability of xylophagous organisms to degrade lignocellulosic biomass has been exploited as a source of enzymes to deconstruct plant biomass. Feruloyl esterase genes have also been found in plants [224-226] but their function remain to be elucidated. A role in modifying the crosslinking between arabinan in stomata guard cells has been proposed [227,228]. In any case most of the studies about feruloyl esterases have been focused on the application of esterases of microbial origin to modify the digestibility or the saccharification of plant cell walls. In Lolium multiflorum, a ferulate esterase gene from Aspergillus niger was targeted to the vacuole under the control of a constitutive rice promoter. Transgenic plants expressing ferulate esterase showed reduced levels of monomeric and dimeric ferulates and increased biomass digestibility, compared with nontransformed plants [83]. The heterologous ferulate esterase was targeted to the vacuole to be released after cell disruption. This approach should prevent the enzyme from affecting the living plant, while still contributing to wall digestion upon homogenization of the material. However, some plants with high level of esterase showed reduced levels of ferulates. Similar approaches using thermostable enzymes that are inactive at growth temperatures could be useful in increasing the potential digestibility of cell walls without affecting the plant development, as it has been demonstrated for thermostable cellulase [229] and xylanase [230].

Dietary Implications Phenolics released from dietary fiber are one of the effective sources of protection against colon tumour development [231]. The hydroxycinnamic and dehydrodiferulic acids released from wheat bran by esterases from the colon intestine can be absorbed into the circulatory system [232,233]. However, the amount of ferulic acid released by the gut microorganisms in a standard diet is below the threshold considered to have a chemopreventive effect [234]. The increase in ferulate content of dietary fiber would increase its availability in the gut for release by the intestinal flora. An increase in the amount of ferulic acid of maize starch by chemical coupling has been obtained [235]. On the other hand, transgenic wheat expressing a ferulic esterase from Aspergillus niger in endosperm presented a low amount of ferulic acid as compared with wild-type grains, but the proportion of diferulic to total ferulic acid was increased [236]. The presence of a large and diverse number of bioactive phenolic compounds in foods of plant origin gives them a great potential in terms of health benefits, including the prevention

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of some diseases. A systems biology approach opens new opportunities for the study of the mechanisms of action of these compounds at different levels. Therefore a multidisciplinary cooperation would be necessary to get new insight into healthy foods.

Conclusions Genetic engineering of phenolic compounds associated with plant cell walls, either linked to noncellulosic polysaccharides or polymerized into lignin, makes it possible to modify their levels and degree of association with plant biomass, improving their value for specific uses (healthy foods, forage digestibility, saccharification, etc.). However we cannot ignore the potential limitations of this approach. Engineered plants can show alterations in their development, productivity, biotic and abiotic resistance, and so on. To cope with these problems, it is necessary to advance our knowledge of the biology of the whole plant.

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In: Phenolic Acids Editor: Sergi Munné-Bosch

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Chapter IV

p-coumaric Acid Production from Lignocelluloses S. Y. Ou, J. W. Teng, Y. Y. Zhao and J. Zhao Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China

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Abstract p-coumaric acid is a hydroxycinnamic acid with many physiological actions, including antioxidant, antimicrobial, antimutagenic, anxiolytic, analgesic, sedative, and immunoregulatory activities. It is widely used in the chemical, food, health, cosmetic, and pharmaceutical industries. p-coumaric acid exists in plants in two states, soluble and insoluble. Soluble p-coumaric acid can be found in a wide variety of foods of plant origin, including fruits, vegetables, and cereals. Insoluble p-coumaric acid constitutes a major component of lignocelluloses, mainly esterified with lignin, in straws, cobs, sugarcane bagasse, and maize cob, and is present at much higher levels than soluble pcoumaric acid. This chapter will review the production of p-coumaric acid for their application in the food or pharmaceutical industries. Production of p-coumaric acid from sugarcane bagasse will be discussed in detail.

Introduction Phenolic acids have received considerable attention as potentially protective factors against cancer and heart disease, in part because of their potent antioxidative properties and their ubiquity in a wide range of commonly consumed foods of plant origin [1]. They also influence the flavor, taste, and color of foods [2]. Phenolic acids can be divided into two groups according to their chemical structures, hydroxybenzoic and hydroxycinnamic acids, with the later more common [2]. p-coumaric acid (Figure 1) is one of most common 

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hydroxycinnamic acids in plants, and is easily identified by UV absorbance maxima (max) at 226 and 312 nm [3]. It shows antioxidant, antimicrobial, antimutagenic, anxiolytic, sedative, analgesic, and immunoregulatory activities. In this chapter, its occurrence, physiological functions, and preparation will be discussed. The p-coumaric acid content of plants can be divided into soluble and insoluble fractions. The soluble molecules can exist in free or bound states. Bound p-coumaric acid can form esters with other small molecules of sugars, aliphatic alcohols, phenols, phenolic acids, and alkaloids [1]. These bound p-coumaric acid derivatives are stored in vacuoles and can be extracted with hot water, alcohol, or organic solvents, and identified after acid or basic hydrolysis [3].

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Figure 1. Structure of p-coumaric acid.

Figure 2. Lignin/phenolic–carbohydrate complex from corn cell walls and its cleavage with alkali [8].

Soluble p-coumaric acid exists at low levels in plants. Mattila and Hellstrom measured the soluble p-coumaric acid content in 20 potato varieties and in 45 vegetables and found that Phenolic Acids: Composition, Applications and Health Benefits : Composition, Applications and Health Benefits, Nova Science Publishers,

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contents ranged from undetectable to 9.7 mg/100 g fresh weight [4]. Yu et al. found that soluble p-coumaric acid contents ranged from 1.68 to 34.38 g/g dry weight in 25 barley varieties [2], while other cereal foods, including rice, rye, and wheat contain amounts similar to barley [5-7]. Compared to bound p-coumaric acid, free p-coumaric acid is present at much lower levels in plants [1,6]. Table 1. Contents of p-coumaric acid in some cereal straws and cobs Families

Plant species (parts)

Gramineae

Saccharum officinarum ( bagasse) Triticum sestivum (straw) Triticum sestivum (flour) Hordeum Vulgare (straw) Secale cereale (straw) Oryza sativa (straw) Oryza sativa (flour) Avena sativa (straw) Avena sativa (flour) Zea mays (straw) Zea mays (stover) Zea mays (flour) Fragaria daltoniana (fresh fruit) Pyrus communis (fresh fruit) Prunus persica (fresh fruit) Malus domestica (fresh fruit) Arachis hypogaea (flour) Phaseolus vulgaris (flour) Vigna radiata (flour) Pisum satiuum (flour) Lens culinaris (flour) Vicia fuba minor (flour) Cajanus cajun (flour) Lupinus albus (flour) Phaseolus lunatus (flour) Cicer arietinum (flour) Vigna unguiculata (flour) Glycine max (seed) Cocos nucife (seed) Gossypium hirsutum (seed) Sesamum indicum (seed) Linum usitatissimum (seed) Brassica oleracea (dry leaf) Brassica hirta (seed) Brassica campestris (seed) Helianthus annuus (seed) Carthamus tinctoriwsa (flower) Spinacia oleracea (dry leaf) Citrus reticulate (fruit, dry base) Citrus tankan (fruit, dry base) Citrus grandis (fruit, dry base) Solanum tuberosum (fresh tuber)

Rosaceae

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Leguminosae

Palmae Malvaceae Pedaliaceae Linaceae Cruciferae Compostae Chenopodiaceae Rutaceae

Solanaceae

p-coumaric acid content (mg/kg) 17600 4200 trace 2800 2900 2600 1.3 3100 2.0 14100–17000 4000 18.9 9~37 38.7 112.1 369.2 1193–1347 13–124 18 12 11 16 trace 9 49 trace 74 94 54 39 57 49 2 22 trace 56 209 2.1 69.2 162 10.1 0.32–1.1

Reference [9] [10] [11] [12] [12] [12] [11] [12] [11] [13-14] [14] [11] [15] [20] [20] [20] [11,17] [16-17] [16] [16] [16] [16] [16] [16] [16] [16] [16] [17] [17] [17] [17] [17] [17] [17] [17] [17] [17] [18] [19] [19] [19] [21]

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The fraction linked to lignocelluloses is called insoluble p-coumaric acid. The typical linkage type is illustrated in Figure 2. Saponification in 1 M NaOH at 30ºC for 18 to 24 h is often employed to cleave the ester linkage and release p-coumaric acid [9]. In contrast to soluble p-coumaric acid, the insoluble fraction is present at much higher concentrations in plants, mainly in cereal straws (Table 1), so this insoluble fraction is a valuable potential source for the isolation of p-coumaric acid. The absorption, bioavailability, and hepatic elimination of p-coumaric acid are all very high. The intestinal absorption efficiency of p-coumaric acid is 100 times higher than that of gallic acid in Caco-2 cells, and the relative bioavailability of p-coumaric acid against gallic acid was 70-fold greater in humans [22]. Konishi et al. administered p-coumaric acid and gallic acid (100 mol/kg in 10% propyleneglycol) by gastric intubation and found that the serum concentration of p-coumaric acid in the portal vein peaked at 10 min after dosing at a concentration of 165.7 mol/L and then decreased rapidly (t1/2=15.9 min), while gallic acid peaked at 60 min, reached only 0.71 mol/L, and decreased slowly thereafter (t1/2=24.1 min). Thus, the rapid pharmacokinetics of p-coumaric make it a good functional ingredient due to low accumulation in the body. Cells are highly susceptible to attack by free radicals and reactive oxygen species that can lead to a series of diseases, such as atherosclerosis, cancer, cataracts, and macula lutea [23]. Intake of antioxidants has demonstrated protective effects against these diseases. p-coumaric acid was shown to efficiently scavenge free radicals and to retard lipid peroxidation. In an in vitro study by Ou et al. [24], p-coumaric acid showed similar efficiency to trans-ferulic acid against linoleic acid peroxidation. Zang et al. [25] used electron spin resonance in combination with spin trapping techniques to determine the capacity of p-coumaric acid to scavenge reactive oxygen species (ROS) and to alter LDL oxidation, and found that pcoumaric acid effectively scavenged ·OH, the most reactive and damaging of endogenously generated free radicals. In this same study, oral administration of p-coumaric acid (317 mg/day) for 30 days significantly inhibited LDL oxidation and reduced LDL cholesterol levels in serum. p-coumaric acid can effectively prevent oxidation of low density lipoprotein and prevent atherosclerosis [26]. Chen et al. used an in vitro model of human low density lipoprotein (LDL) peroxidation to study free radical-induced damage to biological membranes and the protective effects of hydroxycinnamic acid derivatives. The results demonstrated that p-coumaric acid was more effective against both 2-amidinopropane hydrochloride (AAPH)-induced and Cu2+-induced LDL peroxidation than -tocopherol but less effective than caffeic acid, chlorogenic acid, sinapic acid, or ferulic acid [26]. Abdel-wahab et al. found that p-coumaric acid could protect rat heart against doxorubicin (DOX)-induced oxidative stress. Doxorubicin is a quininecontaining anticancer antibiotic that is widely used to treat different types of human neoplastic diseases such as hematopoietic, lymphoblastic, and solid tumors, but its clinical value has been hampered by its dose-limiting cardiotoxicity [27]. Their findings suggest that p-coumaric acid may be an important adjuvant therapy in cancer management by increasing the tolerable dose of DOX. p-coumaric has been shown to possess inhibitory activity on the growth of Staphylococcus aureus and Bacillus subtilis, with a lowest inhibition concentration of 250

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µg·mL-1[28]. Bodini et al. used the quorum sensing technique to test the inhibitory effect of p-coumaric acid on Chromobacterium violaceum 5999, Agrobacterium tumefaciens NTL4, and Pseudomonas chlororaphis [29], and found that p-coumaric acid fully inhibited quorum sensing responses, but showed no influence on cell viability. The antimicrobial mechanism of p-coumaric acid against spoilage or pathogenic bacteria is not clear. For plant pathogenic bacteria, such as Dickeya dadantii, Li et al. found that p-coumaric acid inhibited bacteria by repressing expression of genes encoding the type III secretion system (T3SS) [30], a major virulence factor in many gram-negative bacterial pathogens. Whether p-coumaric acid, like its closely-related derivative ferulic acid, inhibits bacteria growth through inhibition of arylamine N-acetyltransferase is currently unknown [23]. Expression of tyrosinase contributes to skin pigmentation. Nongenetic factors, including hormonal changes, chronic inflammation, ageing, and ultraviolet (UV) light, affect skin pigmentation by stimulating expression of this enzyme [31]. p-coumaric acid can inhibit skin pigmentation. An et al. used -melanocyte stimulating hormone (-MSH) to stimulate melanoma B16/F10 cells to produce melanin, and found that p-coumaric acid inhibited cellular melanogenesis [31] more effectively than arbutin and other structurally similar compounds, including 3-(4-hydroxyphenyl) propionic acid, cinnamic acid, and caffeic acid. Kim et al.[32] found that p-coumaric acid suppressed the functional activity of indoleamine 2, 3-dioxygenase (IDO), which catalyses oxidative catabolism of tryptophan, and significantly rescued IDO-dependent T cell suppression. Thus, p-coumaric acid suppresses a key enzyme mediating T cell suppression and induction of immune tolerance by tumors. p-coumaric acid has also shown analgesic, sedative, and anxiolytic effects (19). Yoon et al. found that coumaric acid had anxiolytic-like effects in the elevated plus maze (EPM) test and attributed this anxiolytic-like action to effects on the monoaminergic neurotransmitter system (serotonergic, dopaminergic, or noradrenergic system) [33]. Yu et al. found that pcoumaric acid significantly increased the number of entries into and the time spent in the open arms of the EPM by mice [34]. Therefore, production of p-coumaric acid is of interest for the pharmaceutical and food industries.

Preparation of p-coumaric Acid from Lignocelluloses As discussed above, soluble p-coumaric acid exists at a very low level in plants, while cereal straws or cobs show great potential as convenient sources for preparation of pcoumaric acid due to their much high contents (albeit bound to lignocelluloses). Thus, two processes are required to prepare p-coumaric acid from straws or cobs: release of p-coumaric acid from lignocelluloses (hydrolysis) and subsequent recovery of p-coumaric acid from the hydrolysates. Two methods, enzymatic (feruloyl esterase) and alkaline hydrolysis, can be used to release p-coumaric acid from lignocelluloses. Feruloyl esterases (FAEs; E.C. 3.1.1.73), also called cinnamoyl esterases or cinnamic acid hydrolases, are the enzymes (at least four types are available) responsible for cleaving the ester-link between polysaccharides and monomeric or dimeric ferulic acid, which liberate

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phenolic acids (ferulic acid and p-coumaric acid) and their dimers from naturally occurring hemicelluloses and pectins [35]. Enzymatic methods result in fewer impurities in the hydrolysates compared to alkaline hydrolysis. However, the shortcomings of this method are obvious. First, each cinnamoyl esterase has its own specificity in regard to the release of specific cinnamic acids [36]. For example, a feruloyl esterase from Pseudomonas fluorescens sp Cellulosa releases not only ferulic acid but also p-coumaric acid, sinapic acid, and caffeic acid, while a feruloyl esterase from Aspergillus niger can release only ferulic and sinapic acids, but not p-coumaric or caffeic acid [36]. Second, the release efficiency is low and substrate-dependent. Faulds et al. used two A. niger esterases to release ferulic acid from lignocelluloses and found that less than 1% of alkali-extractable ferulic acid was released from de-starched maize bran even in the presence of carbohydrase mixtures [37]. Benoit et al. found that only 16% of alkaliextractable p-coumaric acid was extracted from wheat straw by purified feruloyl esterase from A. niger [38]. Third, the enzyme activity is relative low and no commercial cinnamoyl esterases have been available until recently [35]. Alkaline hydrolysis using 1 mol/L NaOH or KOH can release all esterified p-coumaric acid from straws or cobs. 1% NaOH in a solid-liquid ratio of 1:10 could release most of pcoumaric acid from sugarcane bagasse at 40 °C within 4 h [39]. The main shortcoming of this method is that other components, such as polysaccharides and lignin were also dissolved by alkaline treatment [40]. Sugarcane bagasse hydrolysis by NaOH

Utrafiltration Recycle of NaOH

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Decoloration by powdered activated charcoal

Adsorption to the anion macroporous resin

Recycle of resin Desorption from the anion macroporous resin Recycle of ethanol Concentration and crystallization

Product

Figure 3. Flow chart for the production of p-coumaric acid from sugarcane bagasse.

Technology for the recovery of p-coumaric acid from alkaline hydrolysates is illustrated in Figure 3, including ultrafiltration to remove polysaccharides and part of the soluble lignin, decoloration, adsorption using anion exchange chromatography, desorption of p-coumaric acid from the resin, concentration, and crystallization [40].

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Figure 4. The FT-IR spectra of the brown substances (proved to be lignin monomers) adsorbed to the resin.

Polysaccharides and lignin should be removed before adsorption, as they can influence pcoumaric acid adsorption to the resin and its subsequent desorption from the resin and further crystallization. Ultrafiltration using a membrane with 5000 to 20000 Da molecular cutoff has proven to effectively remove all of the dissolved polysaccharides and most of the lignin. However, lignin monomers (Figure 4) were also produced during alkaline hydrolysis [40], which could significantly affect the color of the final product and cannot easily be removed by ultrafiltration. Thus, decoloration by powdered activated charcoal (which was found adsorbs p-coumaric acid under acidic condition but not under basic condition) was necessary. After these pretreatments, anion exchange chromatography can effectively recover p-coumaric acid from the alkaline hydrolysates and purify it after desorption and crystallization. It should be noted that desorption is also a key step in this technology. In contrast to the situation for inorganic anions, acid agents could not desorb any p-coumaric acid from the resin, possibly because of its hydrophobicity. Ethanol at a concentration of more than 60% in hydrogen chloride fulfils this task effectively. This characteristic of the anion resin for adsorption and desorption of pcoumaric acid is useful for further purification of the product, as dilute acid can be used to wash out some impurities before desorption. If the second decoloration step was untaken after desorption, the purity of the final product was increased from 82.1% to 99.2% (Ou et al., unpublished data).

Conclusion As other phenolic compounds, p-coumaric acid has a high interest for the pharmaceutical and food industries. Therefore, it is important to obtain it form plants that contain this compound at high levels. However, it is usually bound to lignocellulases and therefore not easy to obtain. Here, the two methods available, enzymatic and alkaline hydrolysis used to

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release p-coumaric acid from lignocelluloses are presented, as well as the technology currently available nowadays to recover this important compound from alkaline hydrolisates.

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Mattila, P. and Kumpulainen, J. J. Agric. Food Chem., 50, 3660 (2002). Yu, J., Vasanthan, T., Temelli, F. J. Agric. Food Chem., 49, 4352-4358 (2001). Robbins, R.J. J. Agric. Food Chem., 51, 2866 (2003). Mattila, P. and Hellstrom, J. J. Food Comp. Anal., 20, 152 (2007). Sosulski, F., Krygier, K. and Hogge, L. J. Agric. Food Chem., 30, 337 (1982). Zhou, Z.K., Robards, K., Helliwell, S and Blanchard, C. Food Chem., 87, 401 (2004). Andreasen, M.F., Christensen, L.P., Meyer, A.S., Hansen, A. J. Agric. Food Chem., 48, 2837 (2000). Buranov, A.U. and Mazza, G. Ind. Crops Prod., 28, 237 (2008). Xu, F., Sun, R.C., Sun, J.X., Liu, C.F., He, B.H. and Fan, J.S. Anal. Chim Acta., 552, 207(2005). Pan, G.X., Bolton, J.L. and Leary, G.J. J. Agric. Food Chem., 46, 5283 (1998). Shahidi, F. and Naczk, M. Phenolics in Food and Nutraceuticals. CRC Press, Boca Raton, 2004. Salomonsson, A.C., Theander, O. and Aman, P. J. Agric. Food Chem., 26, 830 (1978). Topakas, E., Kalogeris, E., Kekos, D., Macris, B.J. and Christkopoulos, Eng. Life Sci., 4, 283 (2004). Eylen, D.V., Dongen, F.V., Kabel, M. and de Bont, J. Bioresource Tech., 102, 5995(2011). Hakkinen, S.H. and Torronen, A.R. Food Res. Intern., 33, 517 (2009). Luthria, D.L. and Pastor-Corrales, M.A. J. Food Comp. Anal., 19, 205 (2006). Sosulski, F.W., Dabrowski, K.J. J. Agric. Food Chem., 32, 131 (1984). Huang, H.M., Johanning, G.L. and O’Dell, B.L. J. Agric. Food Chem., 34, 48 (1988). Wang, Y.C., Chuang, Y.C. and Hsu, H.W. Food Chem., 106, 277 (2008). Gorinstein, S., Martin-Belloso, O., Lojek, A., Ciz, M., Soliva-Fortuny, R., Park, Y.S., Caspi, A., Libman, I. and Trakhtenberg, S. J. Sci, Food Agric., 82, 1166 (2002). Meändez, C.D.M.V.; Delgado, M.A.R., Rodriguez, E.M.R. and Romero, R.D. J. Agric. Food Chem., 52, 1323 (2004). Konishi, Y., Hitomi, Y. and Yoshioka, E. J. Agric. Food Chem., 52, 2527 (2004). Ou, S.Y. and Kwock, K.C. J. Sci Food Agric., 84, 1261 (2004). Ou, S.Y., Luo, Y.L., Huang, C.H. and Jackson, M. Innov. Food Sci. Emer. Tech., 10, 253 (2009). Zang, L.Y., Cosma, C., Gardner, H., Shi, X.L., Castranova, V. and Vallyathan, V. Am. J. Physiol. Cell Physiol., 279, C954 (2000). Cheng, J.C., Dai, F., Zhou, B., Yang, Y. and Liu, Z.L. Food Chem., 104, 132 (2007). Abdel-wahab, M.H., El-mahdy, M.A. and Abd-ellan, M.F. Farmacol. Res., 48, 461 (2003). Chen, H.F. A Dictionary for Active Ingredients of Plant, 2nd volume. Beijing: China Medical Science Press. p. 386-388 (2001).

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[29] Bodini, S.F., Manfredini, S., Epp, M., Valentini, S. and Santori, F. Isrim Scarl., 49, 551 (2009). [30] Li, Y., Peng, Q., Selimi, D., Wang, Q., Charkowski, A.O., Chen, X. and Yang, C.H. Appl. Environ. Microbiol., 75, 1223 (2009). [31] An, S.M., Lee, S.I., Choi, S.W., Moon, S.W. and Boo, Y.C. British J. Dermatol., 159, 292 (2008). [32] Kim, S. and Jeongy, J.D. Intern. Immunopharm., 7, 805 (2007). [33] Yoon, B.H., Choi, J.W., Jung, J.W., Shin, J.S., Hyeon, S.Y. and Cheong, J.H. Yakhak Hoeji, 49, 437 (2005). [34] Yu, H.S., Lee, S.Y. and Jang, C.G. Pharm. Biotech. Behav., 87, 164 (2007). [35] Ou, S.Y., Zhang, J., Wang, Y. and Zhang, N. Enzyme Res., doi:10.4061/2012/848939 (2011)). [36] Yu, P.Q., McKinnon, J.J., Maenz, D.D., Racz, V.J. and Christensen, D.A. J. Chem. Technol. Biotechnol., 79, 729 (2004). [37] Faulds, C.B., Kroon, P.A., Saunier, L., Thibault, J.F. and Williamson, G. Carhohydr. Polymers, 27, 187 (1995). [38] Benoit, I., Navarro, D., Marnet, N., Rakotomanomana, N., Lesage-Meessen, L., Sigoillot, J. and Asther, M. Carhohydr. Polymers, 341, 1820 (2006). [39] Ou, S.Y., Luo, Y.L., Huang, C.H. and Jackson, M. Production of coumaric acid from sugarcane bagasse.Innovative Food Sci. Emer. Tech., 10, 253 (2009). [40] Zhao, J., Ou, S.Y., Ding, S.H. and Wang, Y. Clin. Eng. Res., Design, In press (2011).

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In: Phenolic Acids Editor: Sergi Munné-Bosch

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Chapter V

Antioxidant Activity of Phenolic Acids: Correlation with Chemical Structure and in vitro Assays for Their Analytical Determination G. Cirillo*, O. I. Parisi, D. Restuccia, F. Puoci and N. Picci Department of Pharmaceutical Sciences, University of Calabria, Arcavacata di Rende (CS), Italy

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Abstract Phenolic acids are a subclass of a larger category of plant metabolites commonly referred to as “phenolics” possessing a carboxylic acid functionality. In general, the term phenolics encompasses approximately 8000 naturally occurring compounds, all of which possess one common structural feature, a phenol (an aromatic ring bearing at least one hydroxyl substituent). The naturally occurring phenolic acids contain two distinguishing constitutive carbon frameworks: the hydroxycinnamic and hydroxybenzoic structures. Although the basic skeleton remains the same, the numbers and positions of the hydroxyl groups on the aromatic ring create the variety. Recent interest in phenolic acids stems from their potential protective role, through ingestion of fruits and vegetables, against oxidative damage diseases due to their antioxidant activity (AOA). This property is related with the ability to scavenge free radicals, donate hydrogen atoms or electron, or chelate metal cations. The molecular structure, referred to as structure–activity relationships SAR (including substituents on the aromatic ring, numbers and positions of the hydroxyl groups in relation to the carboxyl functional group, esterification, glycosylation) affects the antioxidant properties.With the current upsurge of interest in the function, measurement of efficacy and use of natural antioxidants has received much attention. There is a great multiplicity of the methods used for the determination of AOA, which can be broadly classified two groups. In the first group, the degree of inhibition of *

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G. Cirillo, O. I. Parisi, D. Restuccia et al. lipid peroxidation is measured by using lipid or lipoprotein substrate under standard conditions; while in the second the radical scavenging ability is determined. Moreover, a number of assays have been introduced for determining the total antioxidant activity, intended as the cumulative capacity of food compounds to scavenge free radicals. In the discussion, phenolic acids structure as well as SAR will be considered. The various in vitro methods used for the determination of antioxidant activity, with their merits and limitations, will be also presented.

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Introduction Antioxidants are substances which counteract free radicals and prevent the damage caused by them. These can greatly reduce the adverse damage due to oxidants by crumbling them before they react with biologic targets, preventing chain reactions or preventing the activation of oxygen to highly reactive products [1-4]. In recent years, antioxidants have gained a lot of importance because of their potential use in food, pharmaceutical, and cosmetic industries. This interest is rooted in the cumulative evidence that connects oxidative stress with numerous degenerative disorders ranging from premature aging, prostaglandinmediated inflammatory processes, cancer, and a long series of diseases in which free radicals are implicated [5]. In addition, many states implement very rigorous regulations on the use of food preservatives, so that they only allow the use of natural antioxidants. Although not many antioxidants are listed in pharmacopoeias, extensive research is being carried out globally on these agents, and most of them have been proven pharmacologically active. Antioxidants can be classified into two major groups: enzymatic and non-enzymatic antioxidants. Some of these antioxidants are endogenously produced, including enzymes, low molecular weight molecules and enzyme cofactors [6]. Among non-enzymatic antioxidants, many are obtained form dietary sources. Dietary antioxidants can be classified into various classes, with polyphenols as the largest class [7]. Polyphenols consist of phenolic acids and flavonoids. The other classes of dietary antioxidants include vitamins, carotenoids, organosulfural compounds and minerals are listed in Figure 1. Phenolic acids are a subclass of a larger category of metabolites commonly referred to as “phenolics”. They are synthesized in plants and microorganisms by specific metabolic pathways (Figure 2). The name phenolic acid, in general, describes phenols possessing one carboxylic functionality and, when describing plant metabolites, it refers to a distinct group of organic acids containing two distinguishing constitutive carbon frameworks: the hydroxycinnamic and hydroxybenzoic structures [8]. Hydroxybenzoic acids include gallic, p-hydroxybenzoic, protocatechuic, vanillic and syringic acids, having in common the C6–C1 structure. Hydroxycinnamic acids, on the other hand, are aromatic compounds with a three-carbon side chain (C6–C3), with caffeic, ferulic, p-coumaric and sinapic acids being the most common [9] (Figure 3). Although the basic skeleton remains the same, the numbers and positions of the hydroxyl groups on the aromatic ring create the variety and in many cases.

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Figure 1. Classification of Antioxidants.

Figure 2. Biosynthesis of the main groups of phenolic compounds.

Both classes of acid compounds are widely distributed in the plant kingdom. In particular caffeic, p-coumaric, vanillic, ferulic, and protocatechuic are acids present in nearly all plants while other acids are found in selected foods or plants (e.g., gentisic, syringic) [10]. Phenolic acids, hydroxylated derivatives of benzoic acid, are quite common in the free state as well as combined as esters or glycosides (gallic acid) while phenolic acids derived from cinnamic acid (coumaric, caffeic, ferulic acid) are widely distributed and occur rarely in the free state and are usually found as esters of organic acid or glycosides or are bound to protein and other cell wall polymers [11].

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Figure 3. Chemical structures of phenolic acids.

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Influence of the Chemical Structure on Antioxidant Features of Phenolic Acids and Derivatives By definition, the antioxidant activity is the capability of a compound to inhibit or prevent oxidative degradation [12]. In a broad sense, antioxidants can be classified based on chemical nature, as preventive antioxidants, scavenging, or chain breaking, antioxidants [13]. Preventive antioxidants act by binding to and sequestering oxidation promoters and transition metal ions, which contain unpaired electrons and accelerate formation of free radicals. Scavenging or chain breaking antioxidants act by presenting themselves for oxidation at an early stage in the free radical chain reaction and give rise to low energy products that are unable to propagate further. Moreover, based on their mode of action, the antioxidants can be classified as primary, secondary or co-antioxidants. The primary antioxidants are the compounds able to donate a hydrogen atom rapidly to a radical, forming a new radical, which is more stable. The secondary antioxidants are compounds that can react with the initiating radicals (or inhibit the initiating enzyme), or reduce the oxygen level (without generating reactive radical species). These can retard the rate of radical initiation reaction by elimination of initiators. This can be accomplished by deactivation of high energy species (e.g., singlet oxygen), absorption of UV light, scavenging of oxygen (thus reducing its concentration), chelation of metal catalyzing free radical reaction, or by inhibition of enzymes, such as peroxidases, NADPH oxidase, xanthine oxidase, etc. Considering phenolics, their antioxidant activity is due to their ability to scavenge free radicals, donate hydrogen atoms or electron, or chelate metal cations. As well as system

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characteristics and hydrophobicity/hydrophilicity the structure of phenolic compounds is one of the key determinants of their radical scavenging and metal chelating activity, and this is referred to as structure–activity relationships (SAR) [14-19]. To this regard, attention has focused on the role and mechanism of several simple phenolic acids as inhibitors of oxidative processes. It has been concluded that, although there are several mechanisms, the predominant mode is believed to be radical scavenging via hydrogen atom donation and that the antioxidant activity is reasonably related with the substitutions on the aromatic ring and the structure of the side chain [20-24]. In general, the activity of phenolic acids and their esters improves as the number of hydroxyl and methoxy groups increases, the number of OH groups being more important [25, 26]. Moreover, the electron-withdrawing properties of the carboxylate group in benzoic acids have a negative influence on the H-donating abilities of the hydroxy benzoates implying that hydroxylated cinnamates are more effective than benzoate counterparts [27-29]. This aspect has been systematically investigated by comparing the capacity of four derivatives of benzoic acid and their four homologous derivatives of cinnamic acid [30]. The couples of compounds differed for the kind of aromatic substitution (p-hydroxy, p-hydroxymethoxy, p-hydroxydimethoxy, dihydroxy) and the antioxidant activity was measured using (i) a competition kinetic test, to measure the relative capacity to quench peroxyl radical and (ii) the in vitro oxidative modification of human low-density lipoprotein (LDL), initiated by 2,2′-azobis(amidinopropane) dihydrochloride or catalyzed by Cu(II). Obtained data confirmed that the antioxidant efficiency of monophenols is strongly enhanced by the introduction of a second hydroxy group and is increased by one or two methoxy substitutions in position ortho to the -OH group. In all systems, the greatest antioxidant capacity of hydroxycinnamic acid derivatives was linked to the presence of the propenoic side chain, instead of the carboxylic group of benzoic acid derivatives. Moreover, the conjugated double bond in the side chain showed a stabilizing effect by resonance on the phenoxyl radical, thus enhancing the antioxidant activity of the aromatic ring. As for the influence of the aromatic substitution, in the kinetic test the antioxidant activity increased in the sequence p-hydroxy N-trans-caffeoyltyramine ≥ N-trans-caffeoylL-cysteine methyl ester > caffeic acid > Trolox > ferulic acid. This order showed that the antioxidative activity of the molecules depended not only on the hydroxyl groups or catechol rings but also on the solubility, hydrophobicity (or partition coefficient, log P), and stability of the compounds. These results confirmed the paradoxical behavior of antioxidants in lipid emulsions. Caffeic acid phenethyl ester and N-trans-caffeoyl-β-phenethylamine were the most active compounds in this emulsion system within the series of caffeic acid amide and ester analogues showing highest log P values. However, when comparing two compounds of comparable lipophilicity, the structural factors predominated in determining the activity of a specific compound. N-trans-caffeoyldopamine, although having lower log P value but with an additional hydroxyl group in the structure, exhibited stronger antioxidative activity than Ntrans-caffeoyltyramine, as well as caffeic acid vs ferulic acid. The fact that N-trans-caffeoylL-cysteine methyl ester did not have the highest potential activity in the emulsion system, as

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it does in the radical scavenging assay, has been related with (a) its low affinity for partitioning between the lipid and aqueous phases (lower value of log P), (b) its ability to form the hydroperoxide-adducts, (c) the rapid oxidation of the sulfhydryl (-SH) group to the disulfide (-S-S-) group [50, 51]. On the other hand, Trolox C showed stronger activity at the initial stage of peroxidation, but then it rapidly losed activity. This may be explained by its high partitioning affinity in the emulsion system and by its ability to undergo one-electron oxidation to its corresponding phenoxyl radical which is then converted, through subsequent reactions, to a ketodiene intermediate and finally to the stable Trolox C quinone [52]. In addition, Trolox C has only one hydroxyl group [53]. The effects of structural characteristics of the carbon side chain (-COOH, -CHO, CH2OH, -CH3, and -COOC2H5) on the antioxidant performance of ferulic acid were studied [54]. These compounds, except for ferulic acid (4-hydroxy-3-methoxycinnamicacid, coniferic acid), were ethyl ferulate (4-hydroxy-3-methoxyethyl cinnamate), coniferyl aldehyde (4hydroxy-3-methoxycinnamaldehyde), coniferyl alcohol (4-hydroxy-3-methoxycinnamyl alcohol), and isoeugenol (2-methoxy-4-propylidene-phenol) and were examined using DPPH radical scavenging assay, oven test in bulk oils, and tests in oil/water emulsions. It was found that all of the tested compounds possessing one hydroxyl group in the aromatic ring seemed to have similar activity. However, when the overall kinetics of the reaction was taken into account the effect of the different characteristic groups at the end of the carbon side chain was revealed. The relative order of the scavenging activity toward the DPPH radical was isoeugenol ~ coniferyl alcohol » ferulic acid ~ coniferyl aldehyde ~ ethyl ferulate. These differences in activity among tested compounds have been ascribed to electronic phenomena more than to steric hindrance effects. Electron-donating groups, (e.g., -CH3 and -OH), when present in the aromatic ring, increase the ease of hydrogen atom abstraction. Groups with electron-withdrawing properties (e.g., -COOH, -CHO, and -COOR) have the opposite effect. The effect of such properties of substituents seems to be transmitted to the aromatic centre through extended conjugation in the side chain. Once again, in the o/w emulsion autoxidation, lipophilicity of the phenols was the determining factor because the least polar compounds bearing CH3 and COOC2H5 were the most effective ones. The better performance of isoeugenol in comparison to that of ferulic acid in a dispersed system can be expected as, when the affinity to lipid phase predominates and differences in the polarity within the group of components are important, the ultimate performance of the antioxidant depends to a lesser extent on the electronic phenomena of the characteristic groups.

Measurement of the Antioxidant Activity Several methods to measure the antioxidant activity have been published in the past 20 years, and the discussion about the real meaning and the usefulness of this kind of measurement is still open [55-60]. In addition to the traditional methods based on fat oxidation monitoring [61], conjugate diene formation [62], and measure of thiobarbituric reactive substances [64], the use of colored radical compounds was largely adopted as a result of low cost and good reproducibility of the developed procedures [65-67]. An antioxidant is a substance that when present at low concentrations, compared to those of the oxidisable substrate, significantly delays, or inhibits, oxidation of that substrate [68].

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Some of the mechanisms by which antioxidants act include: removing O2 or decreasing local O2 concentrations, removing catalytic metal ions, removing key ROS, e.g. O2•- and H2O2, scavenging initiating radicals, e.g. •OH, RO•, RO2-, breaking the chain of an initiated sequence, quenching or scavenging singlet oxygen, enhancing endogenous antioxidant defences by up-regulating the expression of the genes encoding the antioxidant enzymes, repairing oxidative damage caused by radicals, increasing elimination of damaged molecules and not repairing excessively damaged molecules in order to minimise introduction of mutations [69]. The effectiveness of an antioxidant is measured by monitoring the inhibition of oxidation of a suitable substrate under standard conditions and measuring the extent of oxidation (as end point) by chemical, instrumental, or sensory methods. Hence, essential features of any method for such measurement would be a suitable substrate, an oxidation initiator and an appropriate measure of end point [13]. In general, the methodologies for antioxidant measurement could be divided into two main categories: Direct and Indirect assays (Tables 1 and 2.). Table 1. Direct Measurement of Antioxidant Activity

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DIRECT MEASUREMENT OF ANTIOXIDANT ACTIVITY ANTIOXIDANT TOTAL ANTIOXIDANT ACTIVITY CONCENTRATIONS Dietary

Endogenous

Against Biological Oxidants

Vitamins E,C Flavonoids Carotenoids

SOD CAT GSH GSHPx

Hydroxyl Radical (HO•) Peroxyl Radical (ROO•) Hydrogen Peroxide (H2O2) Superoxide Radical Anion (O2•−) Singlet Oxygen (1O2) Hypochlorous Acid (HOCl) Nitric Oxide Radical (NO•) Peroxynitrite (ONOO−)

Against Non-Biological Oxidants Folin-Ciocalteu Assay DPPH• Radical ABTS•+ Radical Cation FRAP Assay

Table 2. Indirect Measurement of Antioxidant Activity INDIRECT MEASUREMENT OF ANTIOXIDANT ACTIVITY MEASUREMENT OF FREE RADICALS

MARKERS OF OXIDATIVE STRESS Lipid Peroxidation

DNA Damage

TBARS/MDA 4-HNE Lipid Hydroperoxides

8-ohdG

Protein Peroxidation Nitrotyrosine Protein Carbonyls

ESR Chemiluminescence H2O2

Direct Measurement of Antioxidant Activity Direct methods for measuring antioxidant activity include the analytical determination of dietary and endogenous antioxidants concentrations and several assays for measuring overall reducing capacity [69].

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Determination of Antioxidant Concentrations The human body is equipped with antioxidant defenses, including enzymatic and nonenzymatic systems, protecting itself from the potentially deleterious effects of ROS. Some of the most important dietary antioxidants providing protection against ROS include vitamin E, vitamin C, carotenoids such as α- and β-carotene, lycopene and lutein, and flavonoids such as quercetin, phloridzin and catechins; regarding the endogenous antioxidants, they include superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSHPx) enzymes, glutathione (GSH), uric acid and plasma proteins such as albumin, ceruloplasmin, transferrin-lactoferrin and bilirubin. High performance chromatography (HPLC) is a highly accurate and sensitive technique usually performed in order to measure dietary antioxidants [70-73], plasma proteins with antioxidant properties such as albumin [74], levels of both the reduced and oxidised forms of GSH [75]. However, this method presents drawbacks such as expensive equipment and time consuming extraction procedures, moreover many of these antioxidants are very unstable ex vivo and precautions are necessary to prevent the degradation of he samples. Measurement of endogenous antioxidant enzymes, including GSHPx, SOD and CAT, is generally performed by spectrophotometric assay [76-78], for which inexpensive commercial kits are available, while enzymatic methods are employed for most proteins, including albumin [79].

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Determination of Total Antioxidant Activity In literature, a large number of simple and convenient in vitro assays for the measurement of total antioxidant activity are reported. These methods are designed to evaluate overall antioxidant capacity as an index of the ability to contrast the action of free radicals involved in oxidative stress. These assays could be divided into two main categories depending on the type of the oxidant species [80]: scavenging capacity assays against biological oxidants (ROS and RNS); scavenging capacity assays against non-biological radicals.

Scavenging Capacity Assays against Biological Oxidants Hydroxyl Radical (HO•) Scavenging Activity Hydroxyl radicals are characterized by high reactivity and several in vitro assays are available for the evaluation of scavenging activity against these species. Most of them are based on Fe3+ + EDTA + H2O2 + ascorbic acid system to generate HO• radicals that attack the sugar 2-deoxy-D-ribose used as target. As a consequence, the sugar is degraded into a series of fragments, some or all of which react upon heating with thiobarbituric acid at low pH to give a pink [81]. A HO• scavenger inhibits the formation of oxidation products by acting as a competitor with deoxyribose for HO• radicals. In particular, the antioxidant may interfere with the HO• generation system by reacting directly with H2O2 or by chelating the metal ion. In this way, the performance of the deoxyribose assay without EDTA allows the identification of compounds which chelate metal ions [82, 83]. In this case, iron (III) ions are chelated by deoxyribose causing “site specific”

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hydroxyl radical damage, and when the test substances are iron chelating agents the hydroxyl radical damage of deoxyribose is inhibited. Peroxyl Radical (ROO•) Scavenging Activity Peroxyl radicals (ROO•) are formed during lipid oxidation chain reactions. The available methods for evaluation of ROO• scavenging activity act by measuring the ability of an antioxidant to scavenge peroxyl radicals by hydrogen atom transfer reactions. These assays are based on a competitive mechanism in which antioxidants, which inhibit or retard the oxidation process, compete for the radical species with target molecules. The assay system has three main components: thermolabile azo-compound, oxidizable target and antioxidant compound. The thermolabile azo-compound yields carbon-centered radicals (R•) reacting fast with O2 to generate ROO• radicals. Some of the most frequently employed peroxyl radical sources are the water-soluble 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) and the lipid-soluble 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN). The ORAC assay (Oxygen Radical Absorbance Capacity) is one of the most common methods for evaluation of ROO• scavenging capacity and it is based on the decrease of fluorescence intensity of the target/probe along time. Peroxyl radical are generated by thermal decomposition of AAPH in aqueous buffer and the presence of the antioxidant inhibits the decay of fluorescence [83]. Initially, the protein isolated from Porphyridium cruentum, βphycoerythrin (β-PE), was used as the fluorescent target/probe which reacts with ROO• to form a non-fluorescent product [84]. However, this molecule presents some limitations and thus it has been replaced by the synthetic non-protein fluorescein [85]. Lipophilic compounds were also quantified by ORAC assay using organic media, AMVN as a lipophilic peroxyl radical source and 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a, 4a-diaza-s-indacene3-undecanoic acid (BODIPY 581/591 C11) as a fluorescent target/probe [86]. The Total Radical Trapping Antioxidant Parameter (TRAP) assay was introduced by Wayner et al. [87] with the aim to evaluate the antioxidant status of human plasma. In this assay, the human plasma represents the target while the oxidation of plasma material is monitored by measuring the oxygen consumed during a controlled ROO• peroxidation reaction induced by the thermal decomposition of an azo-compound. The measurement is based on the induction time (lag phase) corresponding to the time period between the beginning of the assay and the beginning of the oxidation of the target molecules. Through the comparison between the lag phase duration, in which oxidation is inhibited by the antioxidants in the plasma, and the lag phase of Trolox, it is possible to quantify the antioxidant capacity of plasma. However, the antioxidant capacity profile after the lag phase is totally ignored. This assay was later improved using (β-PE) as the fluorescent target/probe, and the ability of the plasma to protect this compound from oxidation was fluorimetrically monitored [88]. In crocin bleaching test, the antioxidant ability to protect the carotenoid derivative crocin from oxidation induced by ROO• radicals is evaluated [89]. The reaction is initiated by the addition of AAPH and the bleaching rate (absorbance decrease/time) of crocin is monitored at 443 nm during 10 min. Antioxidants compete with crocin for ROO•, and the degree of inhibition of crocin oxidation depends on the antioxidant capacity of tested samples. The quantification of antioxidant capacity is based on the ratio of initial crocin bleaching rates in absence and in presence of antioxidants, A microplate-adapted crocin bleaching assay based on the inhibition percentage at a fixed time instead of kinetic analysis was also reported [90].

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In another assay for the determination of peroxyl radical scavenging activity, low-density lipoproteins (LDL) are employed as oxidizable target/probe [91-93]. The reaction is initiated by thermal decomposition of AAPH or by a transition element, such as Cu(II), and followed by the formation of conjugated dienes determined spectrophotometrically at 234 nm after HPLC separation [94].

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Hydrogen Peroxide (H2O2) Scavenging Activity Hydrogen peroxide is naturally produced under physiological conditions in human body as a by-product of oxidative metabolism. One of the most common methods for assessing the scavenging capacity against this molecule is based on the intrinsic absorption of H2O2 in the UV region [95, 96]. As the H2O2 concentration is decreased by scavenger compounds, the absorbance value at 230 nm is also decreased. Usually a “blank” measurement is required because of the interference of some samples that absorb at this wavelength. Other method employs horseradish peroxidase (HRP), which uses H2O2 to oxidize scopoletin into a nonfluorescent product [97]. In the presence of antioxidants, the oxidation of scopoletin is inhibited and the scavenging reaction can be fluorimetrically monitored. Another fluorimetric assay is based on homovanillic acid (HVA), whose fluorescent biphenyl dimer is more stable than scopoletin [98]. In this case, the presence of an antioxidant prevents the oxidation of homovanillic acid. Another assay is based on the inhibition of the absorbance increase due to formation of Ti–H2O2 complex [99] measured spectrophotometrically at 410 nm [100]. Superoxide Radical Anion (O2•−) Scavenging Activity Superoxide radical anion (O2•−) is produced as a result of the donation of one electron to oxygen and it arises either from several metabolic processes or following oxygen activation by irradiation [101]. XOD/hypoxanthine system is employed as source of superoxide anion radical in several assays for determination O2•− scavenging capacity that is measured by reaction with α-keto-γmethiolbutyric acid (KMBA). The oxidation of KMBA produces ethylene, which is monitored by gas chromatography [102, 103] The scavenging capacity against this radical can also be measured by using electron spin resonance (ESR) spectrometry [104, 105]. The radical is trapped by 5,5-dimethyl-1-pyrrolineN-oxide (DMPO), and the resultant DMPO-OOH adduct is detected by ESR. Singlet Oxygen (1O2) Scavenging Activity Singlet oxygen (1O2) is an excited state of molecular oxygen that has no unpaired electrons and it is known to be a powerful oxidizing agent, reacting directly with a wide range of biomolecules [106]. Due to its decay to the lower energy ground state, 1O2 emits characteristic phosphorescence at 1270 nm. One of the methods is based on the measurement of the decrease in intensity of luminescence from self-emission of 1O2 in the presence of antioxidant compounds [107, 108]. A more sensitivity method is based on monitoring the scavenging of singlet oxygen delayed fluorescence of tetra-tert-butylphthalocyanine was developed [109].

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Hypochlorous Acid (HOCl) Scavenging Activity In the analytical methods for in vitro determination of HOCl scavengers, this oxidant is obtained from the enzymatic system myeloperoxidase/H2O2/Cl− or by acidifying commercial sodium hypochlorite to pH 6.2 with sulphuric acid [110]. One of the assays for the determination of hypochlorous acid scavenging activity is based on the oxidation of 5-thio-2-nitrobenzoic acid (TNB) by HOCl into 5,5-dithiobis(2nitrobenzoic acid) (DTNB). The absorbance decrease, due to oxidation of TNB to DTNB, is inhibited by antioxidant compounds and measured after a fixed time period at 412 nm [111]. However, it was observed that compounds containing free thiol groups, such as cysteine and glutathione, could interfere in this method. To overcome these limitations, another method was developed [112]. In this protein carbonyl assay, the bovine serum albumin (BSA) carbonyl content increases due to oxidation by HOCl and it is inhibited in the presence of antioxidants.

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Nitric Oxide Radical (NO•) Scavenging Activity Nitric oxide (NO•) is an endogenously-synthesized free radical first characterized as a component of endothelial-derived relaxation factor. This radical plays a central role in regulating different processes, such as vascular physiologic and cellular homeostasis as well as critical intravascular free radical and oxidant reactions. The Griess reaction is used for in vitro determination of NO• scavenging capacity and the nitric oxide remaining after reaction with the sample is measured as nitrite [113]. Nitrate may also be formed, thus it should be reduced to nitrite prior to determination and for this purpose NADH-dependent nitrate reductase could be employed, with elimination of interference of NADH by addition of lactate dehydrogenase and pyruvate [114]. The chromophoric azo derivative formed from nitrite after Griess reaction is then measured spectrophotometrically at 540 nm. Standard curves were generated using sodium nitrite and results were expressed as percentage change from control response. Peroxynitrite (ONOO−) Scavenging Activity In vitro assays for evaluation of the peroxynitrite scavenging activity usually depend on tyrosine nitration [115, 116] or on dihydrorhodamine 123 (DHR) [117]. In the first method, the formation of 3-nitrotyrosine is detected spectrophotometrically along with the unreacted tyrosine after HPLC separation. The concentration of 3-nitrotyrosine is measured after incubation of tyrosine with different concentrations of ONOO− scavengers and the inhibition percentage of 3-nitrotyrosine formation is determined. The second method is based on the oxidation of nonfluorescent DHR by peroxynitrite to the fluorescent rhodamine 123. In presence of ONOO− scavengers the fluorescence intensity is lower than that of the control and the inhibition percentage of DHR oxidation is assessed. Peroxynitrite radical could be also synthesized from sodium nitrite/H2O2 acidified with HCl and the residual H2O2 was removed by passing the solution through granular MnO2, and fluorescein could be used as ONOO- detecting agent because it is bleached by hydroxyl, peroxyl radicals and by peroxynitrite and hypochlorite. In all cases, the bleaching can be prevented by antioxidants, so the degree of protection may be a measure of the antioxidant activity of a sample [118]..

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Scavenging Capacity Assays against Non-Biological Oxidants Determination of the Disposable Phenolic Groups The Folin-Ciocalteu phenol reagent is used to obtain a crude estimate of the amount of disposable phenolic groups [119]. Phenolic compounds undergo a complex redox reaction with phosphotungstic and phosphomolybdic acids present in the Folin-Ciocalteu reactant. The color development is due to the transfer of electrons at basic pH to reduce the phosphomolybdic/ phosphotungstic acid complexes to form chromogens, in which the metals have a lower valence. Another useful test is the molybdate assay [120]. The assay is based on the reduction of Mo(VI) to Mo(V) by the flavonoid in the polymer with the subsequent formation of a green phosphate/Mo(V) complex at acid pH.

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Scavenging Activity on 2,2'-Diphenyl-1-Picrylhydrazyl (DPPH•) Radicals The DPPH• radical is a stable organic free radical with an absorption maximum band around 515-528 nm. This radical is commercially available and it is a useful reagent for evaluation of antioxidant properties of compounds in organic media, moreover, it does not have to be generated before the assay. In the DPPH• assay, the antioxidants reduce the DPPH• radical to a yellow-colored compound, diphenylpicrylhydrazine, and the extent of the reaction will depend on the hydrogen donating ability of the antioxidants [121]. The scavenging activity of the tested samples is measured as the decrease in absorbance of the DPPH• and the results are reported as the antioxidant concentration (EC50) that is necessary to decrease by 50% the initial DPPH• concentration or as percent inhibition of DPPH• radicals. Scavenging Activity on 2,2′-Azinobis-(3-Ethylbenzothiazoline-6-Sulphonate) (ABTS•+) Radical Cation The long lived radical cation chromophore 2,2′-azinobis-(3-ethylbenzothiazoline-6sulphonate) (ABTS•+) is characterized by absorption maxima at 414, 645, 734, and 815 nm. The assay is based on the decolourization strategy in order to prevent the interference of antioxidant compounds with radical formation, making the assay more reliable and less susceptible to artifacts. The sample to be tested is added after generation of a certain amount of ABTS•+ radical cation and the remaining ABTS•+ concentration after reaction with antioxidant is then quantified [122]. Different strategies have been used for ABTS•+ generation, reaction time applied, detection wavelength used for monitoring the reaction. ABTS•+ radical cation can be generated by chemical reaction using manganese dioxide [123] AAPH [123], or potassium persulfate [122], by enzymatic reaction using metmyoglobin [125] or horseradish peroxidase [126], or by electrochemical generation [127]. Regarding the wavelength of detection, the determination at 734 nm is preferred because the interference from other absorbing components and from sample turbidity is minimized [128]. The ABTS•+ radical is soluble in water and organic solvents, thus it is possible to use this assay for determining of antioxidant capacity of both hydrophilic and lipophilic compounds.

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Ferric Reducing Antioxidant Power (FRAP Assay) The FRAP assay measures the ability of antioxidants to reduce the ferric 2,4,6-tripyridyls-triazine complex [Fe(III)-(TPTZ)2]3+ to the intensely blue coloured ferrous complex [Fe(II)(TPTZ)2]2+ in acidic medium [129, 130]. FRAP values are calculated by measuring the absorbance increase at 593 nm and relating it to a ferrous ions standard solution or to an antioxidant standard solution (e.g. ascorbic acid).

Indirect Measurement of Antioxidant Activity These assays are based on the measurement of a damage induced by free radical and its reduction by the action of a specific antioxidant molecule. Several different substrates are used for this purpose and the degradation products can act as bio-markers for the antioxidant action.

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Lipid Peroxidation Oxidative damage can be initiated in lipids (lipid peroxidation). Free radicals such as the hydroxyl radical (•OH), the alkoxyl radical (RO•) and peroxyl radical (ROO•), initiate lipid peroxidation of Polyunsaturated fatty acids (PUFAs), such as linoleic acid, linolenic acid, arachidonic acid, and various ω-3 fatty acids [131-133]. During peroxidation, lipids produce various so-called secondary oxidation products, which can be used as markers for indicating the extension of peroxidation. One of the most important marker is malonaldehyde (MA), which is easy detected after reaction with thiobarbituric acid (TBA) to form an adduct spectrophotometrically detectable (Figure 4). However, TBA reacts with many different carbonyl compounds formed from lipid peroxidation and the resulting TBA adducts absorb at the same UV wavelength than MATBA. Thus, total carbonyl compounds reacted with TBA came to be called TBA reacting substances (TBARS). TBARS can be detected by UV-Vis spectrometry, High-Performance Liquid Chromatography (MA/HPLC) or Gas Chromatography (GC) [135-143].

Figure 4. Malonaldehyde-Thiobarbituric acid Adduct.

Another assay is based on the use of thiocyanate. During the linoleic acid oxidation, peroxides are formed. These compounds oxidize Fe2+ to Fe3+. The later Fe3+ ions form complex with SCN−, which have maximum absorbance at 500 nm. Therefore, high absorbance indicates high linoleic acid oxidation, while lower absorbance indicates a higher level of antioxidant activity [144].

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In a different model system, β-carotene undergoes rapid discoloration in the absence of an antioxidant, which results in a reduction in absorbance of the test solution with reaction time. This is due to the oxidation of linoleic acid that generates free radicals (lipid hydroperoxides, conjugated dienes and volatile byproducts) which attack the highly unsaturated β-carotene molecules in an effort to reacquire a hydrogen atom. When this reaction occurs, the β-carotene molecule loses its conjugation and, as a consequence, the characteristic orange colour disappears. The presence of antioxidant avoids the destruction of the β-carotene conjugate system and the orange colour is maintained [145, 146].Another system is based on the spectrophotometric measurement of conjugated diene formation at 234 nm. Conjugated dienes, indeed, are readily formed on PUFAs by the action of ROS and oxygen. The major drawback of this method is that many biological and natural compounds have significant absorbance around 234 nm, which, consequently, interferes with absorption by a conjugated diene [147].A different a marker of oxidative stress, are the isoprostanes, and this detection methods was found to be 20 times more sensitive than the measurement of TBARS. Detectable levels of isoprostanes can be found in all normal animal and human biological fluids (including plasma, airway lining fluid, urine, bile, gastric juice, synovial fluid and cerebrospinal fluid), and, after oxidant injury, these levels are dramatically increased. The most common methods of quantifying isoprostanes are gas chromatography– mass spectrometry (GC-MS) and enzyme immunoassay [148].A further marker for antioxidant power evaluation is 4-hydroxy-2-nonenal (4-HNE), which is produced from arachidonic acid, linoleic acid or their hydroperoxides in situations of high oxidation condition. 4-HNE is measured by HPLC and ELISA [149].

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DNA Damage The oxidative damage of DNA leads to the formation of modified DNA bases, crosslinking between DNA bases, coupling of DNA bases to proteins and strand breaks. The most commonly measured marker of DNA oxidation is 8-hydroxydeoxyguanosine (8-ohdG), which is formed by 8-hydroxylation of guanine after oxidative attack on DNA. 8-ohdG is released by exonucleases after DNA repairing [150].

Protein Peroxidation The oxidative damage of protein carries out to the modification of the aminoacidic residue in the backbone, with, tyrosine, histidine, tryptophan, cysteine, proline, arginine and lysine being the most susceptible of attack [151]. In particular, tyrosine can be attacked by RNS with formation of 3-nitrotyrosine (detectable immunologically or by GCMS or HPLC techniques) or by HOCl and HOBr with the generation of chlorotyrosine and bromotyrosine, respectively, both measurable by GC-MS [152, 153]. Attack on the others mentioned aminoacid results in the production of protein carbonyls and other amino acid modifications detectable by using atomic absorption spectroscopy, fluorescence spectroscopy or HPLC following reaction with 2,4dinitrophenylhydrazine [154].

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Measurement of Free Radical Concentration The direct measurement of free radicals is a difficult approach to determine the antioxidant power of a tested antioxidant because of the highly reactive nature of free radicals [69]. Anyway, some specific technique have been developed in this direction. One of the most important techniques is the Electron spin resonance spectroscopy (ESR), which measures the absorption of energy as a result of the interaction of various free radicals with an applied external magnetic field. With this methodology free radical such as ascorbyl and nitric oxide radicals could be easily determined [155]. Chemiluminescence is another method of measuring free radicals. This method is related to the emission of light in the wavelengths in the near-infrared and infrared regions, which occurs as a consequence of a series of electron transfer reactions in the sample. Two main probes are used for this purpose, lucingenin for the detection of superoxide anion and luminol for the analysis of the others ROS [156]. Finally, several colorimetric and fluorimetric assays are developed for the detection of hydrogen peroxides [157].

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In: Phenolic Acids Editor: Sergi Munné-Bosch

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Chapter VI

Phenolic Acid Composition in Food Systems: Sample Preparation and Analytical Aspects R. N. Cavalcanti, M. A. Rostagno* and M. A. A. Meireles LASEFI, Department of Food Engineering, School of Food Engineering, University of Campinas, Campinas, SP, Brazil

Abstract

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Increasing scientific evidence supports that some phytochemicals naturally present in the diet may play a role on the prevention of important diseases, such as cardiovascular diseases, cancer and some neurodegenerative disorders. Among them, polyphenols are one of the most studied phytochemicals because of their relative abundance in foods and to their antioxidant properties. Phenolic acids are simple phenolic compounds which are present in high amounts in coffee, tea, cocoa, grape and other fruits. However, depending of the sample, the profile and concentration of phenolic acids can be very different. Moreover, environmental and genetic factors can influence the chemical profiles of the sample. In general, for the determination of phenolic acids, they need to be isolated from the sample matrix in a series of steps beginning with extraction, followed by isolation and purification to obtain a clean extract rich in phenolic compounds that are later analyzed. There are several “modern” sample preparation techniques that are currently being used for the extraction of phenolics from different sample types, such as supercritical fluid extraction, pressurized liquid extraction and solid phase extraction. On the other hand, several analytical methods can be used for the determination of the phenolic profile of the samples in which HPLC is the most widespread technique. The current research focus of HPLC separations is being directed towards highly efficient methods that allow faster separations to be achieved. This chapter intends to highlight the main extraction, separation, and identification techniques and methods used for the determination of the phenolic acid composition of different food samples.

*

Email: [email protected].

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R. N. Cavalcanti, M. A. Rostagno and M. A. A. Meireles

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Introduction Plants produce huge amounts of metabolites in order to better adapt to the climatic conditions, to protect themselves from microbial attack, and resist to both biotic and abiotic stresses [1]. These metabolites are also known as phytochemicals and can be specific for a given plant species or cultivar. Phytochemicals may also be essential components of human nutrition and health and are present in the human daily diet through fruit and vegetables; beverages such as fruit juices, green or black tea, coffee; beans and grains. There are several phytochemical classes, including polyphenols (flavonoids, phenolic acids, tannins, stilbenes, coumarins and lignans), carotenoids, phytosterols, alkaloids, terpenes and sulfur-containing compounds (sulfides and glucosinolates) [2]. Phenolic compounds are one of the main classes of secondary metabolites in plants and are derived from phenylalanine or, to a lesser extent, from tyrosine [3]. Chemically, phenolics comprise compounds that possess at least one aromatic ring bearing one or more hydroxyl groups [4] (Figure 1). Phenolic compounds may be divided in at least 10 different classes depending on their chemical structure which basically includes phenolic acids (simple phenols) and polyphenols (complex phenols), depending on the number of phenol subunits attached to it [5]. Phenolic acids possess just one phenol subunit comprising low molecular weight compounds. Other polyphenols may possess two or more phenol subunits including intermediate (flavonoids) or high (hydrolysable or condensed tannins, stilbenes, and lignans) molecular weight compounds [4]. Currently, the increasing demand for high quality products has favored the use of natural products by the cosmetic, pharmaceutical and food industries. Phenolics have many industrial applications such as natural colorants and preservatives for foods, or in the production of paints, paper, and cosmetics [6]. Diets rich in phenolic compounds have received enormous attention among consumers, scientists, and industries due to their potential health-promoting effects [7, 8], including the prevention of various degenerative disorders such as cardiovascular diseases and cancer [9, 10]. Several properties of phenolic compounds have been reported, including antimicrobial [11, 12, 13], antiinflamatory [14], antiallergic, enzyme inhibition [15], antimutagenic, anticarcinogenic activities [16-18], but the most studied properties of phenolic compounds is their antioxidant activity [15, 19-21].

Figure 1. Classification of major groups and sub-groups of phenolic compounds found in food systems. Phenolic Acids: Composition, Applications and Health Benefits : Composition, Applications and Health Benefits, Nova Science Publishers,

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Phenolics are known to have antioxidant capacity and may act as a defense against oxidative stress [22]. The antioxidant activity of phenolics is related with its chemical structure that confers them redox properties. Polyphenols and phenolic acids play an essential role in adsorbing and neutralizing reactive oxygen species (ROS), quenching singlet and triplet oxygen, or decomposing peroxides. ROS derived from oxidation processes are an important part of the defense mechanisms against infection, but excessive generation of free oxygen radicals may cause damages. When there is an imbalance between ROS and antioxidant defense mechanisms, the ROS lead to the oxidative modification in cellular membranes or intracellular molecules and result in the peroxidation of membrane lipids, leading to the accumulation of lipid peroxides [15]. This oxidative stress has been linked to aging and the development of cancer, atherosclerosis and neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease [23]. Therefore, antioxidants, such as phenolic compounds, are considered as possible protective agents, reducing the oxidative damage from ROS in the human body and retarding the progress of many chronic diseases as well as the oxidation of low-density lipoproteins (LDL), which is thought to play an important role in atherosclerosis [15]. Although most of the evidence of the antioxidant activity of phenolic compunds is based on in vitro studies, increasing evidence indicates they may act in ways beyond the antioxidant functions in vivo [22]. Phenolic acids are a diverse group of aromatic secondary plant metabolites that are widely distributed throughout the plant kingdom. Because of their almost universal distribution, phenolic acids are an integral part of the human diet [4]. The term “phenolic acids”, in general, designates phenols that possess one carboxylic acid functionality. However, when referring to plant metabolites, it represents a distinct group of organic acids [18]. Two classes of phenolic acids can be distinguished: hydroxybenzoic (HBA) and hydroxycinnamic acids (HCA) (Figure 2). The hydroxycinnamic acids are aromatic compounds with a three-carbon side chain (C6C3), which p-coumaric, caffeic and ferulic acids are the most frequently forms. On the other hand, the hydroxybenzoic acids have in common the C6-C1 structure which includes phydroxybenzoic, gallic and ellagic acids, being presented mainly in the form of glucosides [6]. Furthermore, phenolic acids can occur in plant foods conjugated with other natural compounds, such as flavonoids, alcohols, hydroxyl-fatty acids, sterols and glucosides [24]. Phenolic acids are widely represented in plant kingdom and their distribution may be influenced by species, cultivar, and environmental conditions. They are mainly localized in the cell wall of plants and their main sources are fruits and vegetables [25]. They clearly play a role both in the interactions between the plant and its biotic or abiotic environment and in the organoleptic and nutritional qualities of fruits, vegetables, and derived products (Fleuriet and Macheix, 2003). Therefore, highly efficient and sensitive techniques are necessary for the determination of the composition of these compounds in foods [25]. In view of the efforts done in the last decade for the evaluation of phenolic acid composition in foods and its relation the potential biological effects, it is of ultimate importance to have reliable, accurate and selective methods for the quantification of these compounds. Most analytical methods used for the determination of phenolic acids in natural products consist of four basic steps: sampling, sample preparation, analysis and data interpretation. However, actual procedures used in each step will depend of the sample and target analytes. Liquid samples, for instance, are usually treated differently than solid samples. Achieving reliable and accurate compositional data is a direct consequence of the

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whole process and any errors or biases may be carried over between procedures, resulting in poor performance of the analytical method [26].

(HCA)

(HBA)

Hydroxybenzoic acid (HBA)

Name R1

R2

R3

R4

Gallic acid

H

OH

OH

OH

Salicylic acid

OH

H

H

H

Protocatechuic acid

H

OH

OH

H

Syringic acid

H

OCH3

OH

OCH3

Gentisic acid

OH

H

H

OH

Veratric acid

H

OCH3

OCH3

H

Vanillic acid

H

OCH3

OH

H

p-Hydroxybenzoic acid

H

H

OH

H

Benzoic acid

H

H

H

H

Hydroxycinnamic acid (HCA)

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Name R1

R2

R3

R4

Ferulic acid

H

OCH3

OH

H

Caffeic acid

H

OH

OH

H

Sinapic acid

H

OCH3

OH

OCH3

o-Coumaric acid

OH

H

H

H

m-Coumaric acid

H

OH

H

H

p-Coumaric acid

H

H

OH

H

Cinnamic acid

H

H

H

H

Figure 2. Structure of the most common naturally occurring phenolic acids: hydroxybenzoic (HBA) and hydroxycinnamic acids (HCA).

Sample Preparation Sample preparation is of great importance to any reliable determination and is defined as all necessary steps before actual analysis of sample components [18]. Sample preparation may have several purposes depending of the sample. It may be used to isolate the analytes of interest and to improve the selectivity, detectability, reliability, accuracy, and repeatability of the analysis. Sample preparation is usually a multi-procedure step and may include procedures such as sample pre-treatment, extraction, isolation and/or pre-concentration [27]. There are several methods and techniques which can be used in the sample preparation part of the determination of polyphenols and phenolic acids. However, due to the wide polarity range, acidity, number of hydroxyl groups and aromatic rings, concentration levels, and complexity of the matrix, there is no universal sample preparation procedures.

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Figure 3. Main sample preparation and analytical instrumental techniques used in the determination of polyphenols and phenolic from plant, food products and biological matrices.

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Thus, it is more appropriate to choose the optimal preparation method for a specific matrix taking into account the chemical structure and properties of the compounds to be analyzed [18]. In Figure 3, the main methods of sample pretreatment, extraction, clean-up, identification and quantification of phenolic acids in plant matrices are presented. Table 1 shows a survey of recent reports dealing with the determination of phenolic acids in food systems.

Sample Pre-Treatment Procedures Prior to the extraction of polyphenols and phenolic acids samples should be pre-treated properly [22]. These sample pre-treatment methods may include physical or chemical procedures. Solid samples are usually subjected to milling, grinding, and homogenization, which may be preceded by air-drying or freeze-drying. Liquid samples are usually filtered or centrifuged, after which they are either directly injected into the analysis system or the analytes are isolated through additional steps using appropriate techniques. Fruits and their derivates have minimal manipulation requirements [18]. Some of sample pre-treatments procedures such as drying, lyophilization, or freezing usually intended to prevent potential degradation of phenolic compounds due to high moisture content aids enzyme activities. Heating and exposure to light and oxygen may affect the phenolic composition in many cases; therefore high-temperature processes should be avoided as much as possible [18, 22]. Phenolic acids are frequently presented as insoluble bound complexes coupled to polymers via ester and glycosidic links and are not directly extractable via organic solvents [27]. Hydrolysable tannins are derivatives of gallic acid (3,4,5 trihydroxyl benzoic acid). Gallic acid is esterified to a core polyol, and the galloyl groups may be further esterified or oxidatively crosslinked to yield more complex hydrolysable tannins [6]. Phenolic acids are

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usually released using enzymatic reactions, acid hydrolysis, basic hydrolysis, or both prior to extraction procedures [28].

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Table 1. Examples of works about techniques used in sample preparation and instrumental analysis for the identification and quantification of phenolic acids in food systems Analytes Chlorogenic acid

Sample Leaves and flowers of Hypericum perforatum.

Sample preparation Pre-treatment: samples were dried and pulverized Extraction: UAE with MeOH. Post-extraction: extracts were filtered and diluted with water

Caffeic acid and ferulic acid

Propolis

Ferulic acid, pcoumaric acid, vanillic acid, and caffeic acid

Wine

Gallic, caffeic, sinapic, p-coumaric, chlorogenic, 3,4,5trimethoxy

Cramberry juice

Pre-treatment: -. Extraction: UAE with MeOH. Post-extraction: extracts were were centrifugated; diluted with MeOHrunning buffer and filtered. Pre-treatment: -. Extraction: extraction with diethyl ether. Post-extraction: separation of the ether layer, evaporation to dryness; dissolved in MeOH. Pre-treatment: acid hydrolysis (adjusted to pH 2.0 with 2.0 M HCl). Extraction: MSPD with Sep-Pak C18 cartridge to clean and fractionate free phenolic acids.

cinnamic acids

Vanillic and syringic acids

Wine

Pre-treatment: adjustment at pH 2.0. Extraction: extraction with diethyl ether. Post-extraction: the organic layer was evaporated to dryness and re-dissolved in MeOHH2O2 (1:1).

Instrumental analysis Technique: CZE Capillary/voltage: fluorinated ethylenepropylene copolymer, 16 cm x 0.3 mm. Solvent: 25 mM b-hydroxy4morpholinopropanesulphoni c acid – 50 mM tris(hydroxymethylamino)methane – 65 mM H3BO3 – 0.2% 2hydroxyethylcellulose – 20% MeOH (pH 8.3 – 8.7). Detector: UV Technique: CE Capillary/voltage: 60 cm (50 cm) x 75 µm/ 23 kV Solvent: 50mM borate (pH 9.2) Detector: UV

Reference [97]

Technique: CE Capillary/voltage: 57 cm (50 cm) x 375 µm/20 kV. Solvent: 150 mM boric acid (pH 8.5) – 50 mM SDS – 5% MeOH. Detector: UV

[99]

Technique: HPLC Column: Eclipse XDR-C RP (150 mm x 4.6 mm, 5 m) Solvent:

[76]

H2O-acetic acid (97:3 v/v) (solvent A); MeOH (solvent B); flow rate: 0.9 – 1.0 mL/min. Detection: UV Technique: HPLC. Column: Nova-Pak C18 (150 X 3.9 mm, 4 m) Solvent: MeOH-acetic acidH2O (10:2:88, v/v) (solvent A); B: MeOH-acetic acidH2O (90:2:8, v/v) (solvent B); flow rate: 1.0 mL/min. Detection: UV

[76]

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Phenolic Acid Composition in Food Systems Analytes Gallic, protocatechuic, gentisic, chlorogenic, 4hydroxybenzoic, 3hydroxybenzoic, syringic, vanillic, caffeic, p-coumaric, ferulic, salicylic acids

Sample Leaves of yacon (Smallanthus sonchifolius).

Sample preparation Pre-treatment: -. Extraction: i) Soxhlet with MeOH, chlorophyll removal with petroleum ether; acidification and extraction with ethyl acetate; ii). Decoction in water under reflux; iii) Infusion of the leaves with boiling water for 20 min. Post-extraction: i) evaporation; ii) freezedrying. Pre-treatment: -. Extraction: reflux of plant material in MeOH; pH adjustment to 7.0 – 7.2 with 5% NaHCO3. Post-extraction: SPE (quaternary amine); final elution with 0.2 M H3PO4–MeOH (1:1, v/v).

Chlorogenic, protocatechuic, phydroxybenzoic, caffeic, vanillic, syringic, pcoumaric, ferulic acids.

Eleutherococcus senticosus

Gallic, protocatechuic, chlorogenic, caffeic, p-coumaric acids.

Teas, mate, instant coffee, soft drink, energetic drink.

Pre-treatment: grinding/degasification Extraction: UAE Conditions: 1g; 50-100% MeOH; 25 mL (x3); 60 oC; 30 min (x3) Post-extraction: centrifugation, filtration

Gallic, caffeic, ferulic, sinapic, and p-coumaric acids

Rapeseed (Brassica napus)

Ferulic, caffeic, and chlorogenic acids.

Green coffee beans

Pre-treatment: grinding Samples were extracted by SLE with methanolwater (1:1, v/v) under shaking at room temperature. Post-extraction: centrifugation and filtration. Pre-treatment: grinding. Extraction: SLE using MeOH-H20 (4:6). Post-extraction: filtration through a 0.47 mm membrane. Injection samples were prepared by making a 1:1 dilution of the above extract in the 4:6 methanol:water solution.

Protocatechuic, phydroxybenzoic, vanillic, syringic, ferulic, caffeic, and p-coumaric.

Wine

Pre-treatment: acid hydrolysis. Extraction: LLE under stirring using diethyl ether.

Instrumental analysis Technique: HPLC Column: Tessek Separon SGX C18 (250 x 4 mm, 5 m)/KH2PO4 (25 mm, pH 3.0)/ACN (90:10 or 80:20, v/v). flow rate: 1.0 mL/min. Detection: Amperometric (+550 mV to +1200 mV versus SCE)

Reference [101]

Technique: HPLC Column: ODS-Hypersil C18 (200 x 4.6 mm, 5 µm) (A); Symmetry C18 (250 x 4.6 mm, 5 µm) (B). Solvent: MeOH-acetic acidH2O (23:1:77, v/v/v) (A); MeOH-0.001M H3PO4 (23:77, v/v) (B); flow rate: 1 mL/min. Detector: UV and PDA. Technique: HPLC Column: Kinetex C18 (100 x 4.6 mm, 2.6 um) Solvent: water (1% phosphoric acid) (A) acetonitrile (1% phosphoric acid) (B) Gradient elution, 2.2 mL.min-1, 55 °C, Detection: DAD and FL Technique: HPLC Column: Discovery RP-C18 150 mm x 4.6 mm i.d., 5 μm Solvent: 2% acetic acid in water, pH 3.2 (A) and methanol (B); flow rate of 1 mL/min. Detector: DAD

[102]

Technique: HPLC Column: Waters X-Terra MS C18 19 x 50 mm id, 5 μm. Solvent: 50 mM stock solution (17.6 mM phosphate, 5% methanol and 70 mM SDS) at pH 2.50. Detector: UV Technique: CE Capillary/voltage: Solvent: 50 mM stock solution (17.6 mM phosphate, 5% methanol and 70 mM SDS) at pH 2.50. Detector: DAD Technique: HPLC Column: Pelliguard LC-18 (5 x 4.6 mm i.d., 40 μm).

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Table 1. (Continued) Analytes

Sample

Gallic, 4aminobenzoic, phydroxybenzoic, vanillic, caffeic, syringic, gentisic, pcoumaric, ferulic, and chlorogenic acids. Caffeic and chlorogenic acds.

Commercial beer, red wine and brandy

Gallic, phydroxybenzoic, gentisic, pcoumaric, vanillic, ferulic, syringic, and caffeic acids

Plant extracts

Benzoic acid, pcoumaric acid, 3,4dimethoxycinnamic acid, ferulic acid, isoferulic acid, caffeic acid

Propolis.

Pre-treatment: -. Extraction: LLE with 70% EtOH at room temperature. Post-extraction: filtration and evaporation to dryness.

Ferulic acid, pcoumaric acid, 4hydroxy-3methoxy- benzoic acid, 4hydroxybenzoic acid

Roots and seedlings of Lupinus albus

Pre-treatment: Grinding Extraction: UAE with 80% aqueous MeOH for 30 min. Post-extraction: filtration under vacuum and elution from SCX and C18 with MeOH.

C. glaziovii

Sample preparation Post-extraction: Centrifugation; purification using SPE; evaporated to dryness; and diluted in synthetic wine (EtOH, water, NaOH, and tartaric acid mixture) prior to be injected. Pre-treatment: dilution of samples 1 :10 with water containing 0.3% acetic acid and subsequent filtration with nitrocellulose membrane 0.45 µm pores. Pre-treatment: drying and grinding. Extraction: maceration with 5% ((plant:solvent ratio; w/v) using aqueous ethanol (20, 50, and 80%, v/v) at 4 and 25°C. Post-extraction: the extracts were filtered through a 0.45 µm HVLP membrane. Extraction: ABE, UAE followed by reflux in aqueous MeOH containing HCl, for 2 h. Post-extraction: extracts were filtered and extracted with ethyl acetate; evaporation of the organic layer after removal of moisture with anhydrous Na2SO4 was performed.

Instrumental analysis Solvent: A (formic acid in water, 2.5% v/v): B (methanol). Detector: DAD

Reference

Technique: HPLC Column: Synergi 4 m Fusion-RP 80 C18 (250 mm x 4.60 mm, id) Solvent: 10 mM NaH2PO4 plus 10 mM Na2HPO4 with 3% or 10% of MeOH. Detector: amperometric Technique: HPLC Column: Zorbax C HP C18 column (150mm × 4.6mm, 5 µm) Solvent: acetonitrile (A)– 1.0% acetic acid (B) with a flow rate of 1mL/min. Detector: UV and DAD

[77]

Technique: GC. Column: CP-Sil 8 capillary column, 30 m x 0.32 mm id, 0.25-µm film thickness. Carrier gas: Gradient: temperature program: from 70°C to 135°C at 2°C/min, held for 10 min, to 220°C at 4°C/min, held for 10 min, to 270°C at 3.5°C/min, held for 20 min. Detection: MS. Technique: GC. Column: capillary column 20 m x 0.30 mm id, 0.1-lm film thickness. Carrier gas: 15% phenyl85% methylpolysiloxane Gradient: column temperature programme: 40 °C, at 15 °C/min to 390 °C, held for 20 min. Detection: MS Technique: GC Column: DB-5 capillary column, 30 m x 0.25 mm id. Carrier gas: Gradiente: temperature programme: 200°C for 2 min, at 5°C/min to 300°C, held for 12 min. Detector: MS.

[73]

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Phenolic Acid Composition in Food Systems Analytes Benzoic, ohydroxybenzoic, cinnamic, mhydroxybenzoic, phydroxybenzoic, phydroxyphenyl acetic, phthalic, 2,3dihydroxybenzoic, vanillic, ohydroxycinnamic, 2,4dihydroxybenzoic, p-coumaric, ferulic, caffeic, sinapic acids Trimethoxybenzoic, 4-hydroxybenzoic, vanillic, quinic, chlorogenic and rosmarinic acids

Sample Cramberry fruit

Sample preparation Pre-treatment: ground in distilled-deionised water; acidified with1 N HCl to pH 2. Extraction: extracted with diethyl ether. Post-extraction: separation of ethereal phase with 5% NaHCO3, acidification with 1 N HCl to pH 2 and extraction with ether; evaporation of ethereal extract to dryness.

Instrumental analysis Technique: GC. Column: DB-5 capillary column, 30 m x 0.35 mmid, 0.25-µm film thickness. Gradient: column temperature programme: 80°C for 1 min, to 120°C at 5°C/min, to 240°C at 108C/min, to 2808C at 208C/min, held for 5 min. Detector: MS

Reference [71]

Orange, grapefruit, and lemon juices

Pre-treatment: evaporated to dryness at 50-60 °C; hydrolysed with TFA for various periods of times. Extraction: Post-extraction: evaporation to dryness

[74]

Caffeic, ferulic, vanillic, sinapic, protocatechuic, 4hydroxybenzoic, pcoumaric, syringic, and gallic acids

Vitis vinifera

Caffeic, benzoic, chlorogenic, gallic, protocatechuic, vanillic, syringic, and coumaric acids

Labisia pumila

Pre-treatment: freezedrying. Extraction: UAE and stirring with MeOH for 15 min and 24 hs, respectively. Post-extraction: centrifuged, and evaporated; reconstituted in bi-distilled water; SPE (Isolute C8 SPE column) eluted with ethyl acetate; evaporated to dryness. Pre-treatment: drying and grinding. Extraction: UAE with hexane at 30 °C. Post-extraction: the extracts were filtered, dried and diluted in 60% MeOH. Methanolic supernatant was filtered and evaporated to dryness. The extract was dissolved in MeOH (170 ml), loaded onto a preconditioned SPE column and eluted with 40%, 60%, 80% and 100% MeOH. The four fractions were concentrated in oven overnight at 40 °C. All samples were filtred with 0.2 µm nylon membrane filter prior to injection.

Technique: GC. Column: BPX5 capillary column, 30m x 0.25 mm id, 0.25-µm film thickness. Gradient: column temperature program: 150°C, held for 2 min, to 330°C at 10°C/min, held for 7 min. Detector: MS Technique: GC. Column: HP-5 MS capillary column, 30 m x 0.25 mm, 250-µm thickness. Gradient: column temperature program: 70°C for 5 min, to 130°C at 15°C/min, to 160°C at 4°C/min, for 15 min, to 300°C at 10°C/ min, held for 15 min. Detector: MS. Technique: UPLC Column: Acquity column C18 (150 mm x 4.6 mm, 1.7 µm) Solvent: A (water with 0.1% formic acid) and solvent B (CH3CN); flow rate was 0.25 ml/min. Detector: ESI-MS/MS

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Table 1. (Continued) Analytes 4-hydroxybenzoic, vanillic, caffeic, syringic, pcoumaric, ferulic and sinapic acids.

Sample Wheat (Triticum turgidum L. var. durum and Triticum aestivum L.)

Gallic, protocatechuic, chlorogenic, caffeic acids

Eucommia ulmodies

Chlorogenic, pcoumaric, gallic, caffeic acids

Propolis

Chlorogenic and pcoumaric acids

Apple peel and pulp

Caffeic, chlorogenic, procatechinic, syringic, ocoumaric, pcoumaric, ferulic, cinnamic and transcinnamic acids

Olive oil mill waste

Sample preparation Pre-treatment: sterilization, drying, grinding, acid and alkaline hydrolysis. Extraction: free phenolic acids extraction by SLE under shaking using aqueous ethanol 80%. Bond phenolic compounds extraction by SLE under stirring using 10% glacial acetic acid in water (85:15, v/v). Post-extraction: centrifugation, evaporation to dryness, reconstitution in MeOH. Pre-treatment: drying, grinding, defatted with ether, evaporation. Extraction: MAE with water under reflux Post-extraction: filtration through 0.45 µm microporous membrane Pre-treatment: -. Extraction: PLE using Ethanol:water:HCl (70:25:5 include 0.1 g BHQ) at 1500 psi and 40 °C for 15 min. Post-extraction: extracts were evaporated to dryness, reconstituted in 2 mL EtOH:H2O (1:1, v/v) and filtered through a 0.45 μm PTFE filter. Pre-treatment: samples were homogenized with a solution of 0.1 g/mL ascorbic acid and lyophilized. Extraction: ASE with methanol at 40 °C and 1000 psi during 5 min. Post-extraction: filtered through a 0.45 µm nylon membrane and diluted to 10 mL of a aqueous methanol solution with 0.1% of hydrochloric acid and filtered through a 0.45 µm PTFE filter. Pre-treatment: pre-treated sub-product. Extraction: SFE was performed at 40 °C, 350 bar and for 60 min using supercritical CO2 as solvent.

Instrumental analysis Technique: UPLC Column: Agilent ZORBAX Eclipse Plus C18 (30 mm x 2.1 mm, 1.8 µm). Solvent: water containing 0.1% TFA (solvent A) and acetonitrile containing 0.1% TFA (solvent B) Detector: DAD

Reference [105]

Technique: HPLC Column: Dalian C18 column (250 mm x 4.6 mm i.d.; 5 µm). Solvent: MeOH-H2O-glacial acetic acid (20:80:1, v/v) at flow rate of 1 mL/min. Detector: UV Technique: HPLC Column: ACE 5 C18 A11608 (250 mm x 4.6 mm, 4 μm). Solvent: 3% acetic acid in water (A); 3% acetic acid, 25% acetonitrile and 72% water (B) Detector: DAD

[42]

Technique: HPLC Column: Nova-Pak C18 (300 mm x 3.9 mm i.d., 4 µm). Solvent: A (acetic acid/water, 10:90, v/v) and B (methanol); flow rate of 0.8 mL/min. Detector: DAD.

[47]

Technique: Column: Shimadzu Pathfinder® AS silica 100 (150 mm x 4.6 mm id, 5 µm)

[57]

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Sample

Sample preparation Post-extraction: The extract was diluted to a constant volume of 3 ml in methanol.

Protocatechuic, gallic, syringic acids

Defatted mill grape seeds (DMGS) and commercial concentrate of complex phenols and tannins (CPT) from grape seeds

Pre-treatment: pre-treated subproduct. Extraction: SFE using CO2 with EtOH or MeOH (2, 5, 10 and 15%) as modifiers at 40°C in 20 and 30 MPa. Post-extraction:

Instrumental analysis Solvent: 0.01% acetic acid in water (A) versus methanol-acetonitrile-acetic acid (95:5:1 v/v/v) (B). Detector: UV Technique: HPLC Column: Spherisorb ODS2, 150 mm x 4.6 mm id, 5 µm Solvent: A (water/formic acid, 95.5/4.5) and solvent B (H3CN/solvent A, 10/90) Detector: MS

Reference

[56]

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Abbreviations: PTFE (polytetrafluoroethylene). Id (inside diameter). ASE (accelerated solvent extraction).

Acid hydrolysis method involves treating the plant extract or the food sample itself with inorganic acid at reflux or above reflux temperatures in aqueous or alcoholic solvents. Acid concentrations range from 1 to 2 N, usually using HCl and the reaction times range from 30 min to 1 h. However, aqueous HCl is reported to promote degradation of hydroxycinnamic acids. Basic hydrolysis is normally carried out with NaOH in concentrations ranging from 2 to 10 N. Incubation times can reach up to 16 h and sometimes the reactions are carried out in the dark as well as under an inert atmosphere as argon or nitrogen gas [29, 30]. Basic or acid hydrolysis is sometimes performed to free bound phenolics that have not yet been hydrolyzed. Basic or acid hydrolysis of ellagitannins yields hexahydroxydiphenic acid, which spontaneously lactonizes to ellagic acid [31]. In food samples, this reaction is commonly used in order to detect and quantitate ellagitannins as ellagic acid equivalents [32, 33, 27]. Likewise the process of hydrolysis, enzymatic treatments has been used as sample pretreatment to release phenolic acids before the step of analytical identification [34]. Enzymatic reactions have been reported to release phenolic acids. Enzymes such as pectinases, cellulases, and amylases are employed for the degradation of carbohydrate linkages. Andreasen et al. [35] discussed and compared several different enzyme preparations for the release of phenolic acids from the cell wall of rye grains [18]. Yu et al. [34] reported that a sequential acid, α-amylase, and cellulose hydrolysis might be applicable to the release of phenolic acids from barley.

Extraction and Clean-up Methods Among the procedures of sample preparation extraction is the main step for the recovery and isolation of bioactive phytochemicals from plant materials prior to instrumental analysis. This procedure is influenced by the chemical nature of the target compound, the extraction method employed, sample particle size, as well as the presence of interfering substances. Additional steps may be used if the removal of unwanted phenolics and non-phenolic substances such as waxes, fats, terpenes, and chlorophylls is necessary [18]. Solvent extraction, as a function of the biomass status may be classified into liquid–liquid extraction or solid–liquid extraction [6]. Liquid–liquid extraction (LLE) and solid–liquid extraction

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(SLE) are the most commonly used procedures prior to analysis of polyphenols and simple phenolics in plants. They are the most widely used techniques, mainly because of their ease of use, efficiency, and wide-ranging applicability [18]. In liquid–liquid extraction (LLE), a liquid matrix initially containing one or more solutes is thoroughly mixed with an immiscible or nearly immiscible liquid solvent. The solvent exhibits preferential affinity or selectivity towards one or more of the components in the feed and has different density. Two streams result from this contact: the extract, which is the solvent rich solution containing the desired extracted solute, and the raffinate, the residual feed solution containing little solute. Extraction becomes a very useful tool if a suitable extraction solvent is chosen. For the separation of phenolic compounds, liquid–liquid extraction is frequently used with industrial liquid by-products, such as those resulting from the beverage industry [6]. Solid–liquid extraction (SLE), or leaching, can be defined as a mass transport phenomenon in which solids contained in a solid matrix migrate into a solvent brought into contact with the matrix. Mass transport phenomena can be enhanced by changes in concentration gradients, diffusion coefficients or boundary layer [36]. It is extensively used to recover many important food components: sucrose in cane or beets, lipids from oilseeds, proteins in oilseed meals, phytochemicals from plants, functional hydrocolloids from algae and phenolic compounds from plants, fruits, vegetables, etc. [6]. Several techniques have been used for the extraction of polyphenols and phenolic acids in plants, food products, and biological matrices as presented in Table 3. Conventional solvent extraction such as maceration, steam extraction, Soxhlet, and agitated bed extraction (ABE) are the most employed techniques. Currently there is growing interest in efficient and environmentally friendly methods of extraction obtaining high quality products at ever lower costs. The desirable features of “green” extraction methods are small solvent consumption, short extraction time and high extraction yield. In this context, innovative extraction methods such as pressurized liquid extraction (PLE), supercritical fluid extraction (SFE), ultrasoundassisted extraction (UAE), and microwave-assisted extraction (MAE) are currently being evaluated as alternatives to conventional techniques [27]. In most solvent-based extraction methods, relatively polar solvents such as alcohols (methanol, ethanol), acetone, diethyl ether, and ethyl acetate are used. This is because the pKa values of plant phenolics vary from 8 to 12 and oil:water partition coefficients range from 6 × 10−4 to 1.5. However, very polar phenolic acids (benzoic, cinnamic acids) are not efficiently extracted completely with pure organic solvents, and mixtures of alcohol–water or acetone– water are recommended. Less polar solvents (dichloromethane, chloroform, hexane, benzene) are suitable for the extraction of nonpolar extraneous compounds (waxes, oils, sterols, chlorophyll) from the plant matrix. Other factors, such as pH, temperature, sample-to-solvent volume ratio, and the number and time intervals of individual extraction steps, also play an important role in the extraction procedure. [27] and constitute other important method variables which can affect extraction efficiency. Ultrasound-Assisted Extraction Ultrasound-assisted extraction (UAE) involves acoustic vibrations that are applied to the sample enhancing the extraction yield. The power of the ultrasonic extraction technique lies in the cavitation phenomenon. The cavitation bubbles, which are formed and compressed with the passage of ultrasound waves, collapse generating high local temperatures and pressures. This phenomenon enhances the penetration of solvent into the sample matrix, improves the

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contact between solid and liquid phase surfaces and accelerates mass transfer rates [29]. Traditionally, UAE is performed in static mode, but dynamic mode is also possible. As with other extraction techniques, dynamic extraction is advantageous in several respects. The analytes are removed as soon as they are transferred from the solid matrix to the solvent, and the continuous exposure to fresh solvent enhances the transfer of analytes from the sample matrix to the solvent. Several applications involving UAE have been published, including both static and dynamic UAE [37, 38]. A recent example of the use of UAE is the extraction of phenolic compounds from Satsuma mandarin (Citrus unshiu Marc.) peels [38]. They observed that optimal ultrasound conditions were different depending of target analytes. These differences were partly attributed to both the chemical structures of phenolic acids and the combination effects of ultrasonic variables. The contents of seven phenolic acids and two flavanone glycosides in extracts obtained by UAE were significantly higher than in extraction obtained by a conventional maceration method. In this study, increasing the extraction time and temperature increased the extraction yields. However, phenolic acids may be degraded at elevated temperatures and prolonged extraction times. Extractions for 20 min at 40°C have been reported to reduce yields of p-coumaric acid, ferulic acid, and p-hydroxybenzoic acid by 35.3 to 48.9%. [27]. On another report [26], three sequential UAE with different solvents were used for the determination of several phenolic acids and other organic compounds in teas, mate and instant coffee by high-performance liquid chromatography. The use of three extraction steps and different solvent polarity were used with the objective of extracting a wide range of phenolic compounds and to achieve quantitative recoveries of target analytes. Dugo et al. [39] also used UAE as a sample preparation for the analysis of phenolic acids in mate extracts, including gallic acid, chlorogenic acid and caffeic acid among other polyphenols and alkaloids. The method consisted of using methanol at high temperature (75°C) for 180 min. However, such high temperature and long extraction time may cause degradation of some polyphenols and therefore this aspect should be considered when treating samples with this extraction method. In general, UAE can be considered an efficient extraction technique for phenolic acids and an attractive alternative to conventional methods since it can be easily used to “upgrade” conventional soaking extraction or Soxhlet methods in order to reduce solvent consumption and processing time. There are several reports of the use of ultrasound to improve the extraction efficiency of other techniques, including microwave-assisted extraction, pressurizeliquid extraction, supercritical fluid extraction and solid-phase extraction. It can be expected that applications of combinatory techniques for the extraction of phenolic acids from different matrixes will be the focus of research in the near future. Microwave-Assisted Extraction Microwave-assisted extraction (MAE) is a process that uses microwave energy and solvents to extract sample components from a matrix [40]. Since the organic solvent and sample are subjected to radiation from a magnetron, the solvent (or the sample) must be dielectric in character. The main advantages of MAE are attributed to the fast and localized heating which usually is translated in higher extraction efficiency. Elevated temperatures and the associated high mass-transfer rates are often essential when the goals are fast extractions. The highly localized temperature and pressure can cause selective migration of target

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compounds from the material to the surroundings at more rapid rate and with similar or better recoveries compared with conventional extraction, with the main advantages of reducing both extraction time and solvent consumption [40]. MAE can be used in a static or a dynamic mode, or a combination of the two modes. The main factors affecting the extraction process are the power of the microwave irradiation, the liquid/solid ratio, the solvent flow rate, and the irradiation time [27]. Many reports have been published on the application of MAE of for the extraction of phenolic acids from different natural products. Spigno and Faveri, [40] studied the extraction of several polyphenols, including phenolic acids, from black tea powder using microwaves and reported that a simplified mass transfer model was capable of predicting experimental data. Pan et al., [41], developed an extraction method for polyphenols from green tea leaves using microwaves. Several extraction variables, including ethanol concentration (0–100%, v/v), MAE time (0.5–8 min), liquid/solid ratio (10:1–25:1 mL/g), pre-leaching time (0–90 min) and different solvents were investigated to optimize the extraction method. They also observed that MAE was more efficient and produced higher percentages of polyphenols and caffeine in extracts than the conventional extraction methods studied. Li et al. [42] used a factorial design to optimize the extraction of gallic acid, protocatechuic acid, chlorogenic acid and caffeic acid from Eucommia ulmodies. They studied the effect of several extraction variables and observed that methanol produced a higher recovery than pure water. The optimal extraction conditions were a ratio of methanol:water:glacial acetic acid in the solvent mixture (2:8:0.3, v/v), 50% microwave power and 30 s irradiation at a solvent:sample ratio of 10 (mL/g). The solvent used is one of the most important components of the extraction method since the extractability of different solvents depends mainly on the solubility of the compound in the solvent, the mass transfer kinetics of the product, and the strength of the solute/matrix interactions. Nevertheless, in MAE heating rate is the most important factor in extraction efficiency. Thus, the solvent must have high dielectric constant (which measures the efficiency in which the absorbed microwave energy can be converted into heat inside a material when an electric field is applied) and high dielectric loss constant (which describes the polarizability of the molecule to an electric field, and then measures the ability of a material to store electro-magnetic radiation) allowing a faster heating rate under microwave radiation [43, 44]. Due to the values of these constants, water is regarded as a good solvent for MAE. Furthermore, addition of water to other solvents that are commonly employed for the extraction of plant bioactive compounds (ethanol, methanol, acetone and ethyl acetate) can be exploited to increase polarity indices, and then to increase the mixture’s dielectric constant. Also the addition of salts to the mixture can increase the heating rate, since besides dipole-orientation, the ion conductivity is the main origin of polarization and corresponds to losses into heat in dielectric heating. Another important concept, known as power penetration, defines the distance an incident electro-magnetic wave can penetrate beneath the surface of a material as the power decreases to 1/e of its power at the surface. Power penetration of the solvent will influence the heating rate, but it should be considered in combination with the sample size [45]. Altogether, microwaves can be seen as an attractive technique for the extraction of phenolic acids. It is also noteworthy that microwaves can be used in combination with other techniques to provide a more efficient and homogenous heating source. There are reports of the combination of microwave heating with Soxhlet, UAE or SPE for the extraction of other sample components which allowed use of milder extraction conditions and reduced extraction

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time while consuming less organic solvents and producing comparable (and validated) results with a reference method [26]. Although this approach was used for the extraction of other sample components, it can be expected that it may be explored to enhance the performance of the available methods for the extraction of phenolic acids from different sample matrixes in the future. Pressurized Liquid Extraction Pressurized liquid extraction (PLE) consists of using liquid solvents at high temperatures and pressures and has been widely explored for the extraction of natural products. Liquid solvents at elevated temperatures and pressures achieve higher yields and better efficiency compared with extractions at or near room temperature and atmospheric pressure because of the enhanced solubility and mass-transfer effects and the disruption of surface equilibrium. Moreover, PLE methods usually are faster and require fewer amounts of organic solvents than conventional extraction with a high degree of automation [18, 26]. In addition, the sample is retained in light- and oxygen-free environment which turns this technique a powerful tool in the nutraceutical industry. The main variables that influence extraction efficiency in PLE are solvent composition, solid-to-solvent ratio, temperature, particle size distribution, and the number of extraction cycles [46]. As in conventional techniques, the solvent plays an important role in the overall efficiency of the extraction method. There are several applications of pressurized liquids for the extraction of phenolic acids and polyphenols from natural products. Alonso-Salces et al. [47] used PLE for the determination of polyphenolic profiles of apple varieties. Main polyphenols present were flavan-3-ols and hydroxycinnamic acids, representing between 86 and 95% of total polyphenols present in the samples. PLE was also used for the extraction of caffeic and chlorogenic acids (among other polyphenols) from honey. Best extraction conditions were 40°C, 1500 psi, ethanol:water:HCl; (70:25:5, v/v/v) containing 0.1% tert-butylhydroquinone (tBHQ) as solvent, three extraction cycles within 15 min, and a cell size of 11 mL [48]. Palma et al. [46] developed a new method of pressurized-fluid extraction coupled in-line with solidphase extraction for the extraction of phenolic compounds from grapes. Five different solvents have been assayed using different extraction pressures and temperatures. Using two extraction stages with two different solvents, water and methanol, quantitative recovery of assayed compounds was achieved. While only gallic acid was found distributed in both extracts, cinnamic esters like caftaric acid, cis and trans-coutaric acids were found only in the methanolic extract. Beside of organic solvents traditionally used in PLE such as methanol, ethanol, acetone, diethyl ether, ethyl acetate, and isopropyl alcohol, water has been increasingly used in the extraction of polyphenols and phenolic acids from plant matrices due to its high polarity, dielectric character, and swell capacity [49, 50].Extraction using pressurized water is carried out using water above its boiling point and sufficiently pressurized to maintain it in a liquid state, and thus, is usually referred as pressurized hot water extraction (PHWE) or subcritical water extraction (SWE). PHWE can be performed in a static or dynamic manner, or a combination of the two modes and its efficiency is affected by temperature and extraction time. The water under pressure varies significantly its polarity with temperature. The polarity of water can be tuned by the temperature; when the temperature is increased, the apparent polarity of water is decreased substantially. For relatively polar analytes, quantitative extraction can be obtained already at 100°C, whereas for less polar compounds, temperatures

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up to 250–300°C should be used [27]. Another example of the influence of the solvent composition is given by the extraction of phenolic acids from black cohosh [51]. In this case, addition of water to all solvents was found to increase the extraction by causing the plant material to swell, thus allowing the solvent to penetrate the solid particles more easily. Maximum extraction efficiency was achieved at 90°C with water–methanol (40:60, v/v) as the solvent. Particle size distribution was observed to play an important role in the extraction. There was an almost threefold increase in the extraction efficiency as the particle size decreased from greater than 2.00 mm to less than 0.25 mm [27]. It is clear that PLE offers an excellent technique for the extraction of phenolic acids from natural products. Besides of its high efficiency, the possibility of using water instead of organic solvents increases its attractiveness. The combination of different techniques with PLE may also be expected since there is increasing evidence that this strategy can enhance extraction processes. Furthermore, the on-line coupling of PLE and other instrumental techniques has also been shown to be a viable option and therefore further investigation in this area can be expected. Supercritical Fluid Extraction A pure substance is considered a supercritical fluid when its temperature and pressure are greater than their critical values, Tc and Pc, respectively [52]. The main characteristics of supercritical fluids are liquid-like density, gas-like viscosity and surface tension, and intermediate values of diffusivity. These properties allow faster penetration of the solvent which combined with the high solvating power (due to high density) facilitates mass transfer enabling a more efficient extraction than conventional extraction methods. The solvent commonly used in extraction processes in the industry is carbon dioxide because of its several advantages: it is inert, non-flammable, stable, nontoxic, noncorrosive [53] and it is available in large quantities, with low cost and high purity. Due to all these characteristics, supercritical fluid extraction (SFE) may be an environmentally compatible alternative to conventional extraction methods that use large amounts of organic solvents. Furthermore, SFE methods are usually faster and selective, with a high degree of automation possible. Additionally, the low critical point (31.1°C and 7.38 MPa), allied to the absence of light and air during the extraction may avoid degradation of thermo labile compounds which can take place with traditional extraction techniques [6]. Supercritical fluid extraction (SFE) provides relatively clean extracts, free from certain degradation compounds which may emanate from lengthy exposure to high temperatures and oxygen. There are several reports about the application of supercritical fluids for the extraction of phenolic compounds from natural products. Usually, due to the polar nature of phenolic acids it is necessary to add a modifier to carbon dioxide. Depending on the sample and target analytes, the best modifier may not be the same. In the case of SFE of phenolic acids, methanol was a much more effective modifier than acetonitrile, acetone, water or dichloromethane [54]. Palma et al. [55] also used methanol (20%) as modifier for the extraction of phenolic compounds in grape seeds. Using pure CO2, only fatty acids, aliphatic aldehydes and sterols were extracted while with 20% methanol-modified CO2, epicatechin and gallic acid were effectively extracted. Because SFE is performed without exposure to light and air, the antioxidant properties of the extracts could be conserved. Other reports dealing with SFE of phenolic compounds from different types of samples can be found in the literature, such as grape seeds [56], olive oil [57], rosemary [50] and others. This technique

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can also be combined with other sample preparation techniques such as solid phase extraction, which has been recently reported for the extraction of phenolic acids from freshwater microalgae and selected cyanobacterial species [58]. Altogether, SFE is an interesting alternative to conventional methods although method development and the achievement of quantitative recoveries may require extensive knowledge of the technique and other aspects that may influence extraction efficiency. Solid Phase Extraction Solid-phase extraction (SPE) is one of the simplest, most versatile and effective methods of sample preparation. SPE is a low cost technique that uses pre-packed, disposable cartridges where target compounds such as phenolic acids are separated from other species by applying the sample mixture to an appropriate chosen solid sorbent and selectively eluting the desired components [59]. Several authors have carried out SPE as clean-up procedures of crude plant extracts and other sample types. Applications for the isolation and pre-concentration of phenolic acids from beer [60], honey [59], wine [61] and other plants [62]. The main advantages over other techniques is that SPE is faster and more reproducible, and fairly clean extracts are obtainable, emulsion creation is avoided, and smaller volumes of toxic solvents are applied. Furthermore, in some cases, more than one single-functional group SPE cartridge is used, or two or more SPE cartridges with different sorbents could be combined [63]. New generation sorbents are now also commercially available with hydrophilic–lipophilic properties which enable the simultaneous separation of both polar and less polar compounds. Very simple SPE assay is required for all acidic and basic analyte isolation from the crude plant extract and high recoveries are common for this simple procedure. The great advantage of SPE is the possibility of combining the SPE procedure, online, with the HPLC equipment and realizing the so-called direct sample analysis (DSA). This means that the “crude” extract of plant material is injected directly into this SPE/HPLC system [64]. Furthermore, SPE is one of the techniques with greater potential to be used in combination with other extraction techniques, such as PLE and SFE, or hyphenated with the analysis system, such as HPLC. Usually, when SPE is combined or hyphenated with another extraction technique, high selectivity and much cleaner samples can be obtained [26]. Matrix Solid-Phase Dispersion Matrix solid-phase dispersion (MSPD) is a sample preparation applied to many plant matrices. The technique is based on manually blending the solid or semisolid sample in a mortar with a suitable solid-phase material, such as C8- and C18-bonded silica, silica gel, or sand [64]. The samples are usually dried with anhydrous sodium sulphate or freeze-dried before blending with the MSPD sorbent. As in SPE, MSPD is a multi-residual assay which consists of matrix homogenization with a solid silica phase placed into a short column. Analytes and matrix interferences are retained on the mixed solid-phase material with completely new separation characteristics. Specific elution allows one to obtain analytes after the elimination of matrix compounds via washing steps. This technique combines homogenization, cellular disruption, extraction, fractionation, and purification in a single process. MSPD has many advantages over other sample preparation methods such as: dispersion of sample over a large surface area, low consumption of solvents in comparison with liquid extraction, and as SPE, it is a technique that can also be automated. The application of MSPD as the preparation step before HPLC analysis of various

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analytes in fruits, vegetables, herbs, and other plants has been reviewed [65]. This technique has been tested for isolation and concentration of phenolic acids from plants [64, 66], grapes [67], tea [68] and fruit juices [69], among other sample types. One of the key advantages of MSPD is that it combines sample clean-up and extraction into a single step, which may reduce errors derived from sample manipulation. Additionally, it is a relatively simple technique that may provide excellent results depending on the sample being analyzed.

Separation, Identification and Quantification of Phenolic Acids

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The last step in the analytical determination of sample components is usually carried out by instrumental analysis. Separation and identification of phenolic acids can be performed by several instrumental techniques. Gas chromatography-mass spectrometry (GC-MS) [70-75] and liquid chromatography (LC) coupled to diode-array (DAD), electrochemical (ECD), and particularly tandem mass spectrometry (ESI-MS/MS) [26, 76-80] are the most commonly used analytical methodologies reported for analysis of polyphenols and phenolic acids. There are several available methods for the analysis of phenolic acids and other polyphenols using these techniques and recent reviews can be found in the literature [6, 81, 82]. Usually, GC methodologies provide higher resolution than LC methodologies but require a laborious sample preparation step which usually involves isolation of metabolites by liquid-liquid extraction (LLE) or other extraction procedures followed by derivatization. In contrast, for LC analysis the sample preparation step is simpler since some samples such as beverages and wine may be diluted without a previous isolation step and provides a very sensitive method for quantification of target compounds when coupled to MS/MS [83]. Thin-Layer Chromatography Thin-layer chromatography (TLC) has been used in phenolic analysis since the early 1960s and plays a significant role in the determination of phenolic acids in natural products. It is especially useful for the rapid screening of plant extracts for pharmacologically active substances prior to detailed analysis by instrumental techniques because of its capacity for high sample throughput. In most cases, TLC entails using silica as stationary phase and plates are developed with either a combination of 2-(diphenylboryloxy)ethylamine and polyethylene glycol or with AlCl3. Detection is mainly performed using UV light at 350 365 or 250–260 nm or with densitometry at the same wavelengths. [18]. Quantitation generally is not the main goal of TLC studies. However, densitometry is used in several studies to achieve this goal. A rapid high-performance TLC densitometric method has been proposed for the simultaneous quantification of gallic and ellagic acids in herbal raw materials. The method was validated for precision, repeatability and accuracy [18]. Although this technique has not seen significant advances over the time it remains a useful tool for the identification of sample components. Due to its simplicity and low cost it will always be an alternative to more sophisticated identification techniques in the screening of complex samples. Capillary Electrophoresis and Capillary Electrochromatographic Methods Capillary electrophoresis (CE) is an advantageous technique applicable for the separation and quantification of low to medium molecular weight polar and charged compounds, being often faster and more efficient than the corresponding HPLC separations [6]. The

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electromigration modes primarily used are capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Detection is usually performed with UV, but electrochemical and MS detectors are also used [84]. CE has become a versatile analytical tool for the determination of a wide variety of polyphenols and phenolic acids in different types of samples due to its high resolution power, high separation efficiency, short analysis time and low consumption of sample and reagents. However, the major limitations of CE, compared to other techniques like GC or HPLC, is its low sensitivity in terms of solute concentration, and low reproducibility compared to chromatographic techniques, which is caused by the short optical path-length of the capillary used as detection cell and also by the small volumes that can be introduced into the capillary [85]. Moreover, with the development of lab-on-a-chip CE-MS systems using microfluids there is a growing potential for this technique to compete with HPLC as an efficient analytical tool [86]. Gas Chromatography Many volatile compounds are directly amenable to analysis by gas chromatography, a technique of unsurpassed separation capacity. In particular, when combined with mass spectrometry it offers high sensitivity and selectivity. One chemical characteristic of the OH group in phenolic compounds is the hydrogen bonding capability, which increases the melting point. Consequently, the significant concern with this technique is the low volatility of phenolic compounds. Despite these shortcomings, gas chromatography is one of the main chromatographic techniques employed for the analysis of phenolic acids in plants. Preparation of samples for GC may include the removal of lipids from the extract, liberation of phenolics from ester and glycosidic bonds by alkali, acid, or enzymatic hydrolysis. Traditionally, analysis in the gas phase requires a chemical derivatization step, in addition to sample extraction, isolation, and clean-up. Hyphenation of chromatographic and spectroscopic methods is important in analytical chemistry and is of great value in modern natural product analysis. But early work with phenolic derivatives was typically performed with flame ionization detection (FID). Mass spectrometry later became widespread [18]. High-Performance Liquid Chromatography In the last 20 years, high-performance liquid chromatography (HPLC) has been the analytical technique that has dominated the separation and characterization of phenolic compounds. Due to the relatively high-molecular mass and intrinsic features of hydrophobic flavonoid aglycones and hydrophilic flavonoid glycosides, the overwhelming majority of chromatographic methods in the literature fall in the realm of HPLC and related technologies. HPLC techniques offer a unique tool to separate simultaneously a wide range of analytes together with their possible derivatives or degradation products. In many cases, they enable the determination of low concentrations of analytes in the presence of many other interfering and coeluting components. There are many advantages dictating the widespread use of HPLC in the analysis of phenolic compounds in plant-derived and biological matrices, such as sample preparation requirements, speed, sensitivity and the wide range of column chemistries available. However, separation of a great number of analytes can be a difficult task, requiring a complex gradient of a mixture of solvents and a long column. Separation of a given analyte mixture is influenced by the number of target compounds, column dimensions and characteristics (particle size, chemistry, etc.), temperature, mobile phase flow rate and composition, among other factors. The analysis of a few compounds is

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usually achieved in short times with relatively simple methods. As the complexity of the sample increases, it is more difficult to separate the sample components and therefore analysis time increases proportionally. In general, separation of a large number of polyphenols can be achieved in relatively short times when employing optimal chromatographic conditions. There are several HPLC methods capable of separating large number of polyphenols from complex samples. Separation is usually carried using water and methanol or acetonitrile, with small amounts of acid (formic, acetic, phosphoric or trifluoroacidic acids) as mobile phase. The use of acids to modify the mobile phase is intended to enhance chromatographic separation and resolution and improve peak shape. Elution is most commonly performed in gradient mode and isocratic elution is generally used for the separation of only a few compounds. Gradient elution is generally used because there is a higher resolution and separation efficiency requirement when analyzing complex samples. Conventional microparticulate RP-C18 columns with 5 µm particles are the most used stationary phase for the analysis of phenolic acids. Usually, relatively long columns (250 mm) are necessary in order to separate a greater number of sample components in complex matrices. Shorter columns can be used depending on the number of target analytes and efficiency of the column. Higher chromatographic performance can be achieved with columns of smaller particles (1.7-3.5 µm), allowing shorter columns to be used. By using shorter columns, analysis time can be reduced, which implies lower cost and environmental impact of the analysis. The negative aspect of columns with smaller particles is the higher column back-pressure generated. Therefore, to take full advantage of sub-2 µm particles stationary phases, systems that can withstand higher pressures are required. Separations using sub-2 µm particles stationary phases are usually termed ultra high performance liquid chromatography (UPLC). Some UPLC separations of phenolic acids can be found in the literature. Recently, a UPLC method was developed for the separation of 30 different phenolics compounds and caffeine present in tea. Separation of 30 compounds was achieved in less than 19 min using a short column (100 mm) with a sub-2 µm particles stationary phase [87]. Also recently, Zhao et al. [88] reported separation of 68 phenolics and alkaloids in teas. Such fast separations of large number of different types of compounds clearly indicate the huge potential of UPLC for a comprehensive analysis of food components. However, in both cases very low flow-rates (0.4-0.5 mL/min) were used due to the high backpressure generated by the small particle column. Since pressure is one of the main limitations in HPLC, to reduce the impact of particle size on the column pressure drop new stationary phases are being developed. One of the recent developments in column technology is particles with a porous shell fused to a solid core (fused-core particle) which improves chromatographic performance while generating lower pressure than conventional stationary phases. The enhanced chromatographic performance is attributed to the improved mass transfer of analytes and smaller diffusion paths than conventional particles resulting in faster separation with narrower peaks and better overall resolution. Also, due to their characteristics, resolving capacity is maintained even at relatively high flow-rates [26]. Illustratively, separation of 19 phenolics, and alkaloids in several complex samples was achieved in less than 5 minutes [26] using conventional HPLC instrumentation. Clearly fused-core particle columns have a great potential for fast separations of a large number of phenolic compounds. It can be expected that they will be increasingly used to improve chromatographic performance in terms of resolution, speed and

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organic solvent consumption of current chromatographic systems. The increase in chromatographic performance is also linked to the number of compounds that can be simultaneously determined in a single run, which can be expected to increase as stationary phase technology continues to advance. Therefore, HPLC will remain the technique of choice regarding the analysis of phenolic acids since it can deliver very fast separations and provide accurate information about a large number of sample components simultaneously. Supercritical Fluid Chromatography Supercritical fluid chromatography (SFC) is an emerging chromatographic technique used in the separation and identification of natural compounds. What differentiates SFC from other chromatographic techniques such as GC and HPLC is the use of a supercritical fluid as the mobile phase. Supercritical fluid chromatography is more versatile than high performance liquid chromatography, more cost-efficient, user friendly, better resolution and faster analysis times than general liquid chromatographic methods. The instrumentation that is required for supercritical fluid chromatography is versatile because of its multi-detector compatibility [6]. Despite the great potential for the separation of phenolic compounds from natural products, SFC has seen very few applications. Kamangerpour et al. [89], for example, used supercritical fluid chromatography for the separation and identification of eight polyphenols in grape seed extract. Carbon dioxide modified with methanol, which contained less than 1% (w/w) citric acid as a secondary additive, served as the mobile phase. Various components in the extract could be identified by retention time and ultraviolet spectral comparison with a synthetic mixture of polyphenols. The limited number of reports of SFC for phenolic compounds is due to the relative polarity of carbon dioxide, which is seen as a replacement of normal phase separations. However, other applications for polar compounds has shown that SFC can be used with excellent results in terms of speed, resolution and more importantly, solvent consumption. In this case, it will require the use of modifiers, such as methanol, to modify the relative polarity of the mobile phase and allow adequate retention of polar compounds. However, the most important aspect in SFC is the stationary phase used. In this context, a higher potential of SFC for the separation of phenolic compounds in general and phenolic acids in particular can be expected with the recent developments in column technology. In this context, sub-2 µm particles stationary phases are showing promising results in SFC because one of the main disadvantages of this type of column is the backpressure generated. Since modified carbon dioxide as mobile phase produces a much lower system pressure, much higher linear flow-rates can be used leading to much faster separations. On the other hand, new stationary phases specifically designed for SFC were recently introduced and the number of available chemistries is increasing and thus the possibilities for the separation of phenolic compounds. With these advances in column technology, it can be expected that SFC will be capable of HPLC-like performance for analytical separation of polyphenols with the inherent advantages of supercritical fluids. These new and improved stationary phases also demand that many reverse-phase separations be revised to SFC, including polyphenols in natural products, due to solvent saving and chromatographic performance.

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Detectors Once sample components are separated from each other, it is still necessary to correctly identify and accurately quantitate them. Different detectors can be used in the analysis of phenolic acids in natural products depending of the chromatographic technique. However, since the multiple conjugate bonds make phenolic compounds strong chromophores with high UV absorption (Table 2), ultraviolet/visible (UV/VIS) and photodiode array (PDA) are the primary detectors employed for detection and quantitation [18]. The employment of these reliable detectors also contributes to the wide spread use of HPLC as a separation technique. Detection is usually carried out in the 240-260 nm range because of UV absorbance maxima. However, some phenolic acids also have high absorbance at higher wavelenghts and therefore differential detection wavelengths can be used to selectively detect some phenolic acids. For example, Rostagno et al. [26] used three different wavelengths (240, 260 and 320 nm) for the detection of phenolic acids, flavan-3-ols, flavonoids and alkaloids from several sample types. Identification of compounds is usually carried out by comparison of retention times with authentic standards. In the case of PDA detectors, it is possible to contrast absorption spectra of eluted compounds and reference standards providing further evidence about the identity of eluted peaks. Additionally, absorption spectra can be combined with retention parameters to identify unknown compounds and to measure peak purity by spectral homogeneity and indicate co-elution of other sample components. Although most often retention time and UV absorption spectra are used as identification tools, UV absorption spectra of phenolic compounds are often very similar, and the possibility of unambiguous identification does not exist. However, UV and diode array detectors can be coupled on-line with other detectors which are used as complementary information to increase reliability of identification or quantitation. Table 2. UV Absorbance Maxima (ìmax) of Selected Phenolic Acids. Adapted from Rebecca J. Robbins, Phenolic Acids in Foods: An Overview of Analytical Methodology, J. Agric. Food Chem. 2003, 51 (10), 2866-2887 Phenolic Acid Gallic Protocatechuic Gentisic Caffeic Vanillic Syringic p-coumaric Feruilic

UV ìmax (nm) 272 260, 295 239, 332 240, 294, 326 261, 294, 320 276, 328 312 236, 295

Other methods have also been used for the detection of phenolics, including electrochemical, colorimetric, and mass spectrometry detectors [18]. After UV detectors, mass spectrometry is without doubt the second most used technique for the analysis of phenolic compounds, especially because the molecular structure of the phenolic compounds makes them fairly easy to ionize by ESI and APCI. In general, sensitivity is greater with negative ionization, but this is highly compound specific. However, quantitation by LC-

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UV/VIS is more reliable than LC-MS, if the separation is satisfactory [27]. When using massselective detection methods, the characteristic mass fragments are used to confirm the identity of the compounds. This differs from other methods, such as HPLC–UV/VIS, electron capture detection (ECD), or use of fluorescence detectors in which peak identity cannot be confirmed with certainty. Electrochemical detection has been used with a series of voltametric or colorimetric detectors but has had limited use in the analysis of phenolic acids. Fluorescence detection has also had limited application since few phenolic compounds, such as flavan-3ols, have fluorescence properties. The hyphenation of several detectors on-line is one of the most useful tools in the separation, quantitation and identification of phenolic compounds in natural products. In fact, different detectors (fluorescence, MS, etc.) and multiple UV wavelengths is the best approach for the analysis of phenolic acids, which combined provide reliable and accurate data about the compounds present in the sample.

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Recent Trends in Sample Preparation and Detection Systems Reliable and robust hyphenated analysis systems such as HPLC-MS and GC-MS are among the most important developments in analytical chemistry. However, it is only recently that analytical instrumentation is taking advantage of the hyphenation of different techniques. It can be expected that the complexity of analytical systems will increase as multiple on-line coupling with different separation and detection techniques are used. Such trend already can be seen with the coupling of several detectors on-line (UV, IR, MS, NRM, etc.) and the development of multidimensional chromatographic techniques (GCxGC, LCxLC, SFCxGC, etc.) [26]. Although application of such advanced tools for the analysis of natural products is still rare, it is feasible to assume that they will ultimately be part of the analytical methodology for the determination of phenolic compounds in complex samples. Comprehensive two-dimensional LC (LC × LC) is particularly suitable for the analysis of complex samples where phenolic acids are present. In LC × LC the sample is subjected to two individual separations, resulting in a tremendous increase in resolving power. In most of these applications, RPLC has been connected to another RPLC method (RPLC × RPLC), or with hydrophobic interaction chromatography (RPLC × HILIC) or with normal phase liquid chromatography (NPLC × RPLC) [27]. Another clear trend in methods development is towards on-line systems that integrate the sample preparation, separation and detection. The primary benefit of on-line systems is the elimination of sample manipulation and derived errors. In addition, exposure to toxic solvents, high level of automation and reduced solvent consumption. Illustratively, extraction methods coupled on-line to liquid chromatography (LC) of phenolic compounds include SFE [35,90,91], dynamic microwave-assisted extraction [92], continuous-flow liquid membrane extraction [93], subcritical water extraction [94] and SPE [95,96]. It can be expected that combination and/or hyphenation of different sample preparation and analysis/detection techniques will provide comprehensive and detailed information about phenolic acids distribution in natural products even at low concentrations and in the context of a complex sample, where several other compounds are present simultaneously.

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Conclusions Determination of phenolic acids in natural products can be a challenging task due to their natural diversity and the complexity of the samples were they are present. Several aspects have to be considered in order to achieve reliable data about their concentration and distribution in natural products. Although sample preparation is as important as the analysis step, it is an often overlooked part of the overall analytical methodology. One of the main aspects that should be carefully controlled is analyte quantitative recovery during extraction. In this aspect, authors are increasingly using short sequential extraction methods, where different solvents are used. It can also be observed that despite the great advances in sample preparation technology, outdated techniques and methods are still in use. There are several modern sample preparation techniques available that can be used for the extraction of phenolics which has been proved to be highly superior to conventional techniques. However, others factors have to be considered such as the cost, operating costs, complexity of method development and level of automation. Static UAE extraction is fast, but it is labor-intensive. MAE and PLE offer different advantages and disadvantages. While MAE is capable of extracting multiple samples simultaneously in a short time, additional cleanup is required to remove the sample matrix from the analyte-containing solvent, after cooling of the sample vessels. PLE allows multiple samples to be extracted sequentially in an automated system, but the instrumentation is relatively expensive. In the analysis of phenolic acids UPLC and fastLC methods provide faster and more efficient separation in comparison with conventional HPLC analyses. The benefit of UPLC and fast HPLC methods is that the analysis time can be decreased without sacrificing the separation efficiency. Consequently, the number of compound classes that can be simultaneously analyzed increases. On the other hand, multidimensional separations, such as LC × LC, are more complex but can be an extremely useful tool in the identification of a large group of sample components otherwise impossible by conventional HPLC or UPLC. Finally, integration of different techniques and procedures in one single step are increasingly used in natural product analysis and can be expected to remain the focus of intensive research in the next years. The development of new techniques and procedures are likewise expected to continue increasing, and current methodology should be updated in terms of efficiency, speed, accuracy and reproducibility.

Acknowledgments The authors acknowledge financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

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Chapter VII

Phenolic Acids as Additives in the Food Industry M. L. R. Giada* Department of Basic and Experimental Nutrition, Institute of Nutrition, Health Sciences Center, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

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Abstract Phenolic acids are among the most numerous and widely distributed group of plant secondary metabolites. Like other plant phenolic compounds, they have received increasing interest in recent years, once scientific research has shown that the consumption of vegetables or beverages rich in those substances may reduce the risk of developing several diseases. However, beyond the nutritional interest, such substances are also of technological interest. They can act as natural antioxidants present in food. Additionally, many of these substances have antimicrobial activity and can retard the deterioration of food due to the action of microorganisms. Thus, the preparation of foods with a high content of such substances leads to a reduction in the use of synthetic additives, resulting in healthier foods that can be included in the functional foods group. In this chapter, phenolic acids sources as well as their physicochemical, antioxidant and antimicrobial properties are discussed. Additionally, foods with potential for the application of these substances as additives are presented.

Introduction Phenolic acids are one of the most numerous and widely spread groups of plant secondary metabolites [1] and take part in several functions in plants, such as in antioxidation and defensive or signal compounds, being essential to their physiology and cellular metabolism [2]. These compounds are usually found in plants as esters. They are also found in the glycoside form, joined to sugars and accumulating in the vacuoles, or bound to proteins and

*

E-mail addresses: [email protected].

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other cell wall polysaccharides generating insoluble stable complexes. The existence of such compounds in a free form in plant tissues also occurs, though it is less common [3]. Phenolic acids can be split in two large groups: benzoic and cinnamic acids, as well as their derivatives. These substances are characterized by having one benzenic ring, one carboxylic group and one or more hydroxyl and/or methoxyl groups in the molecule. The benzoic acids have seven carbon atoms (C6-C1) and are the simplest phenolic acids found in nature. The cinnamic acids have nine carbon atoms (C6-C3) [4]. The general formulas and names of the main benzoic and cinnamic acids are found in Figure 1 (A) and (B), respectively. (Paragraph) In the benzoic acids group (Figure 1A) protocatequinic, vanillic, syringic, gentisic, salicylic, p-hydroxybenzoic and gallic acids stand out. Among the cinnamic acids (Figure 1B), the p-coumaric, ferulic, caffeic and sinapinic are the most commonly found in nature [5]. Cinnamic acids are usually associated with a cyclic acid-alcohol, such as the quinic acid, producing isochlorogenic, neochlorogenic, cryptochlorogenic and chlorogenic acids [4]. Figure 2 shows the chemical structure of chlorogenic acid.

Figure 1. General formulas and names of benzoic (A) and cinnamic (B) acids.

It is known that our body cells need oxygen to convert nutrients into energy. However, the use of oxygen produces free radicals in our body, which are highly reactive molecules and can oxidize our cells. In addition, environmental pollution, ultraviolet radiation, smoking and alcohol also produce free radicals in our body. Excessive production of free radicals or the lack of antioxidants may lead to the development of a great variety of diseases, such as atherosclerosis, cancer, AIDS [6], cataract [7, 8] and osteoporosis [9], among others. Like all other plant phenolic compounds, phenolic acids have received increasing attention in recent years because scientific studies have shown that the vegetables and beverages consumption rich in such substances can reduce one’s risk of developing many different types of diseases.

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The phenolic acids may constitute about a third of the phenolic compounds in the human diet [10] and have antioxidant activity, which enables them to protect our body against free radicals [7]. In some cases, those compounds have also been used for therapeutic purposes due to their pharmacological properties [11].

Figure 2. Chlorogenic acid chemical structure.

Occurrence Phenolic acids are often found in fruits and other edible vegetables, in addition to infusions such as coffee and tea [12, 13]. Table 1 shows some foods that are phenolic acids sources.

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Table 1. Phenolic acids food sources Phenolic acid

Source

Reference

Mango, Cereals, Black currant, Clove buds Strawberry, Raspberry, Blackberry Cereals, Strawberry Date, Raspberry, Cinnamon bark, Clove buds

[14,16-18] [14] [16,18] [15,17,18]

Apple, Plum, Grape, Tomato, Artichoke, Peanut, Orange, Pineapple, Coffee Coffee, Citrus fruits, Apple, Pear, Artichoke, Aubergine, Peach, Cherries Plum, Spinach Aubergine, Cereal grains, Tomato, Asparagus, Pea, Citrus fruits Apple, Pear, Chicory, Artichoke, Potato, Corn flour, Wheat flour, Rice flour, Oat flour, Cider, Coffee

[3,18,19]

Hydroxybenzoic acids Gallic acid Ellagic acid p-hydroxybenzoic acid Protocatechuic acid Hydroxycinnamic acids Caffeic acid Chlorogenic acid p-coumaric acid Ferulic acid Sinapic acid

[3,21,22] [18,24] [18,20] [18]

Hydroxybenzoic acids are commonly present in edible vegetables as components of complex structures such as the gallotannins, mango hydrolysable tannins, and ellagitannins of Phenolic Acids: Composition, Applications and Health Benefits : Composition, Applications and Health Benefits, Nova Science Publishers,

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berries such as strawberries, raspberries and blackberries [14]. (No paragraph) However, they may also present themselves in free form. The date is a good source of free (2.61-12.27 mg/100 g) and bound (6.84-30.25 mg/100 g) phenolic acids. Four free phenolic acids (among them, protocatechuic, vanillic, and syringic acids) and nine bound phenolic acids (among them, gallic, protocatechuic, p-hydroxybenzoic, vanillic, and syringic acids) were identified in the date [15]. Free or bound hydroxybenzoic acids are also found in cereals such as maize, barley, wheat, oat, rice and rye [16]. On the other hand, in some herbs and spices such as the cinnamon bark, clove buds, anise, star anise, dill, fennel, caraway and parsley, the hydroxybenzoic acid glycosides are characteristic [17]. Hydroxycinnamic acids are more often found than the hydroxybenzoic acids. In fruits, they are distributed in all parts. However, the highest concentrations are found in the outer part of the ripe fruit. The caffeic acid, in both free and esterified forms, is usually the most abundant phenolic acid and represents between 75% and 100% of the hydroxycinnamic acids total content in most fruits, such as apples, plums and grapes [18]. Other sources are oranges, pineapples, tomatoes, artichokes, peanuts and coffee [19]. The ferulic acid is another hydroxycinnamic acid commonly found in vegetables, both in free form as in covalently linked to lignin and other biopolymers. Ferulic acid is the most abundant hydroxycinnamic acid present in cereal grains, which constitute the main food type where it can be found. The ferulic acid content of wheat grain is approximately 0.8 to 2 g kg-1 dry weight, which can represent up to 90% of the grain total polyphenols. It is found mainly in the outer parts of the grain. (No paragraph) Aubergines [18], tomatoes, asparagus, peas and citrus fruits [20] are other sources of ferulic acid. Chlorogenic acid can be found in many types of fruit, for example, peach [21]. Wang et al. [22], studying the antioxidant polyphenols from tart cherries, identified chlorogenic acid as one of the main antioxidants in those fruits. (No paragarph) High concentrations of the chlorogenic acid can also be found in coffee, in about 7% of grains dry weight [10]. It is the key substrate for enzymatic oxidation leading to the browning reaction of the grain [23]. On the other hand, Bergman et al. [24], studying the chemical identity of a variety of antioxidant compounds present in the aqueous extract of spinach leaves, showed, for the first time, the presence of p-coumaric acid derivatives as one of the main antioxidant components. However, besides the nutritional interest, phenolic acids generate technological interest since they can also act as food natural antioxidants. Thus, a wide range of agro-industrial byproducts and non-edible vegetables have been studied as sources of antioxidants, potentially safer to replace artificial antioxidants and with a lower cost to obtain them, in addition to high efficiency regarding natural antioxidants (ascorbic acid, tocopherols) that have been employed in the food industry. As it can be seen in Table 2, phenolic acids have been found in several agroindustrial byproducts, such as cereal hulls, coconut husk, other vegetables and fruits residues (skins, peels, seeds and pomace), waste waters and blanching waters [5]. (spacing) Different phenolic acids, such as chlorogenic acid, p-coumaric acid, ferulic acid and sinapic acid, were characterised and identified not only in almod seed but also in its skin, shell and hull [25]. Studying the antioxidant phytochemicals in hazelnut kernel and its by-products (skin, hard shell, green leafy cover, and tree leaf), Shahidi et al. [26] demonstrated that gallic acid, caffeic acid, p-coumaric acid, ferulic acid and sinapic acid were present in all studied samples. However, the by-products extracts exhibited stronger activity than the kernel. The p-

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coumaric and ferulic acids were also identified as the two major hydroxycinnamic acids found in complex cell walls of oat hull [27].

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Table 2. Agro-industrial by-products and phenolic acids found in them Agro-industrial by-products Almond hulls

Phenolic acid Chlorogenic acid, p-coumaric, Ferulic acid, Sinapic acid

Reference [25]

Hazelnut by-products (skin, hard shell, green leafy cover, and tree leaf) Oat and barley hulls Pigmented rice husks Buckwheat hulls Dried coconut husks Almond skins

Gallic acid, Caffeic acid, p-coumaric acid, Ferulic acid, Sinapic acid

[26]

Ferulic acid and p-coumaric acid Ferulic acid and p-coumaric acid Protocatechuic acid p-hydroxybenzoic acid p-hydroxybenzoic acid, Protocatechuic acid, Ferulic acid, Sinapic acid, p-coumaric acid and Chlorogenic acid Ellagic acid p-hydroxybenzoic acid

[27,28] [29] [30] [31] [25]

Gallic acid Ferulic acid Sinapic acid Chlorogenic acid, Caffeic acid and Ferulic acid Chlorogenic acid and Caffeic acid Chlorogenic acid

[34] [35] [36] [37] [38]

Gallic acid and Protocatechuic acid p-coumaric acid Gallic acid and Caffeic acid Hydroxycinnamic acids Neochlorogenic acid

[39] [40] [41] [42] [43]

Boysenberry and Blackberry seeds Pumpkin hull-less seed, skin, oil cake meal, dehulled kernel and hull Grape seeds and skins Sour orange peel Potato peel Apple pomace Blackcurrant and chokeberry pomaces Bayberry pomace Sugarcane pomace Olive pomace Olive mill waste water Artichoke blanching water

[32] [33]

On the other hand, investigating the effect of barley variety and growth year on ferulic and p-coumaric acids, Du and Yu [28] verified that the whole barley seed contained higher ferulic acid concentration than p-coumaric acid. The authors also demonstrated that the concentration of ferulic acid in the barley hull is higher than in whole seed. In the sixteen barley varieties studied, the ferulic acid percentage in hull and dehulled seed ranged from 38 to 70% and 30 to 62%, respectively. Ferulic acid and p-coumaric acid were also found as the major phenolic acids in the free fraction of pigmented rice husks, whereas vanillic acid was the dominant phenolic acid in the free fraction of normal rice husks. In contrast, p-coumaric acid was found at high concentrations in the bound form of both pigmented and normal rice husks [29]. Five antioxidant compounds were isolated from buckwheat hulls. One of them was identified as protocatechuic acid [30]. Dey et al. [31] showed that mesocarpic husk materials can form an alternative source of p-hydroxybenzoic acid. Bushman et al. [32] identified the ellagitannins and free ellagic acid as the major phenolics detected in five caneberry species, being approximately 3-fold more abundant in blackberries and boysenberry seeds than in raspberries. Pericin at al. [33] found that the p-hydroxybenzoic acid was the dominant phenolic compound in pumpkin, with 34.7%, 52.0%, 51.4%, 67.4% and 51.8% in the hull-less seed, oil cake meal, skin, and dehulled kernels and hulls,

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respectively, based on total phenolic acid content. Most phenolic acids were present in the bound (esterified and insoluble) form, from 50.6% in skin to 84.1% in the hull-less seed. Grape seeds and skins are also suitable raw materials for the extraction of natural antioxidants, as they have high content of gallic acid [34]. In a study about the antioxidant activity and phenolic composition of methanol extract and methanol extract hydrolyzed by alkaline hydrolysis of citrus peels and seeds, Bocco et al. [35] observed that the hydrolyzed extract of sour orange peel was the richest sample in phenolic acids, especially ferulic and sinapic acids. Nara et al. [36] showed that the total amount of chlorogenic acid and caffeic acid in the free-form was highly correlated with the potato peel antioxidant activity. On the other hand, ferulic acid was identified as the active radical scavenging compound in the bound-form from the peel. (spacing) Phenolic acids have also been found in pomace. Evaluating eleven different cider apple pomaces (six singlecultivar and five from the cider-making industry) for their phenolic and antioxidant composition, García et al. [37] found the chlorogenic and caffeic acids among the major phenols identified in the samples. Baranowski et al. [38] found a high level of chlorogenic acid in the blackcurrant and chokeberry pomaces. Studying the phenolic compounds and antioxidant property of pomaces of five bayberry cultivars, Zhou et al. [39] demonstrated that the gallic acid and protocatechuic acid are the dominant phenolic acids in the studied samples. Additionally, other phenolic acids as the p-hydroxybenzoic, vanillic, caffeic, p-coumaric and ferulic acids were also found in the samples. However, chlorogenic acid was only detected in one of the cultivars. By alkaline hydrolysis at 30° C for 4 h, Ou et al. [40] showed that the main component of the purified sugarcane pomace hydrolysate was the p-coumaric acid. Cioffi et al. [41], studying the phenolic compounds and antioxidant activity of two samples of virgin olive oil and olive pomace, found that all samples were quite similar from both the qualitative and quantitative viewpoint, and that caffeic acid and gallic acid were among the major simple phenolic constituents in olive oil pomace. (Paragraph) Waste waters and blanching waters have also been investigated for phenolic acids. Mulinacci et al. [42] demonstrated that the commercial olive oil waste water is rich in polyphenols, among them, caffeic acid. Llorach et al. [43] found a higher content of phenolics (11.3 g of phenolics/100 mL) in the artichoke blanching water than in the raw artichoke (9.9 g of phenolics/100 mL) and blanched (thermally treated) artichoke (10.3 g of phenolics/100 mL). (No paragarph) The main compound identified in the blanching water was the neochlorogenic acid.The search for new natural antioxidants to replace the artificial ones applied in food has also fostered research on non-food plants. Table 3 shows some non-food plants, as well as the phenolic acids found in them. (Paragraph) Among the different non-food plants that have been studied with regards to their phenolic compounds, leaves have received special attention. Seven different phenolic acids (gallic acid, catechinic acid, pyrocatechol, caffeic acid, coumaric acid, ferulic acid and benzoic acid) can be found in Populus sp. (poplar) leaves [44]. The benzoic acid, salicylic acid, p-hydroxybenzoic acid, vanillic acid, 3,4-dihydroxybenzoic, syringic acid, ferulic acid and caffeic acid as well as the cis and the trans-p-coumaric acid were identified in unrolled and rolled Ctenanthe setosa leaves [45]. Among the phenolic derivatives from Croton xalapensis L. leaves, gallic acid has been found [46]. Studying the biologically active compounds from Cacalia hastata leaves, Olennikov et al. [47] isolated two compounds that were identified as chlorogenic and gallic acids. In their comparative phytochemical investigation of the Thalictrum species, Vladimir-Knežević and Nikolić [48] identified, among others, the chlorogenic, isochlorogenic and caffeic acids in

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leaf methanolic extracts. Masuda et al. [49], evaluating the antioxidant activity of seashore plants leaves extracts, verified that the extracts from the Excoecaria agallocha and Terminalia catappa showed remarkably potent activity in all assay system studied. The HPLC analysis of both extracts indicated the presence of the same antioxidant that was identified as the ellagic acid. (spacing) Phenolic acids can also be found in other parts of plants besides the leaves. Achenbach et al. [50] found gallic acid as one of the main phenolic constituents of the Bauhinia manca bark. Table 3. Non-food plants and phenolic acids found in them Non-food plants Populus sp. leaves Ctenanthe setosa leaves Croton xalapensis leaves (No underline) Cacalia hastata leaves Thalictrum species leaves Excoecaria agallocha and Terminalia catappa leaves Bauhinia manca bark Eucalyptus globulus bark Mimusops elengi bark

Phenolic acid Gallic acid, Catechinic acid, Pyrocatechol, Caffeic acid, coumaric acid, Ferulic acid and Benzoic acid p-hydroxybenzoic acid, Syringic acid, Ferulic acid, Caffeic acid and p-coumaric acid Gallic acid

Reference [44]

Caffeic acid, Chlorogenic acid and Gallic acid Chlorogenic acid, Isochlorogenic acid and Caffeic acid Ellagic acid

[47] [48] [49]

Gallic acid Caffeic acid Gallic acid esters

[50] [51] [52]

[45] [46]

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In the water extract of the Eucalyptus globulus bark, Santos et al. [51] identified twenty nine phenolic compounds, caffeic acid being one of them. Seven new gallic acid esters were isolated from the ethanolic extract of the Mimusops elengi L. (Sapotaceae) bark [52].

Physicochemical Properties Physicochemical properties, such as physical state, colour, odor, specific gravity, solubility, melting point, boiling point and stability, are commonly applied to characterise a substance. Table 4 summarizes some physicochemical properties of the main phenolic acids found in plants. Because of their hydrophilic nature, phenolic acids cannot be properly used in oil-based systems, which is an important issue in industrial applications. However, these substances can be modified by esterification or alkylation with long chain fatty acids or alcohols to improve their lipophilicity. Several studies have been developed on chemical and enzymatic esterification of phenolic compounds. The new molecules obtained should preserve their original functional properties and could be used as multifunctional emulsifiers in the food and other products [53]. (spacing) Phenolic acids are also sensitive, unstable and susceptible to degradation. (No paragraph) The major degradation factors are presence of oxygen and light [54]. Nevertheless, other factors can influence the degradation of such compounds. It is known that sinapic acid oxidize to thomasidioic acid, 2,6-dimethoxy-pbenzoquinone and 6-hydroxy-5,7-dimethoxy-2-naphthoic acid under alkaline conditions [55]. These substances could compromise the nutritional quality and functional properties of food products [56]. (No paragraph) Caffeic acid and other catechols readily autoxidize in the

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presence of oxygen and transition metal catalysts to form reactive oxygen species, which include the superoxide radical and hydrogen peroxide. The rate of formation of hydrogen peroxide depends on oxygen tension, pH, ambient temperature and transition metal availability. In particular, Mn2+ has been shown to catalyze the autoxidation of phenolic compounds and thus increase hydrogen peroxide formation [57]. 4-Vinylguaiacol, a major off-flavor in citrus products, was found in stored model solutions of orange juice added of ferulic acid, and its content increased with time and temperature. (No paragraph) Vanillin, another ferulic acid degradation product, was also detected in the samples after incubation at 35 and 45°C, but only low levels occurred at 25°C. Incubation of the samples under a nitrogen atmosphere rather than air or adding butylated hydroxytoluene (BHT) did not affect 4-vinylguaiacol levels even though nonenzymic browning products, and optical density values decreased. Cu2+ ions accelerated browning but reduced 4-vinylguaiacol levels [58]. In a study for the stability of caffeic, chlorogenic, ferulic, and gallic acids as well as other polyphenols in a model system to pH in the range 3-11, Friedman and Jürgens [59] demonstrated with the aid of ultraviolet spectroscopy that caffeic, chlorogenic and gallic acids were not stable at high pH values and that the pH and time-dependent spectral transformations were not reversible. By contrast, chlorogenic acid was stable at an acid pH, to heat, and to storage when added to apple juice. Chethan and Malleshi [60], investigating the suitable solvents for polyphenols extraction from millet seed coat fraction and the stability of the compounds during changes of pH and temperature, observed that the phenolic contents (6.4%) of the HCl-methanol extract remained constant at highly acidic to near neutral pH (6.5) but decreased gradually to 2.5 % as the alkalinity increased to pH 10. The increase in pH resulted in precipitation of some of the extracted matter, and this increased from 4% to 40% of the extracted matter, as the pH increased from 1 to 10. Yet, the polyphenol contents of the extract were stable to the changes in the temperature of the extract. Fractionation of the polyphenols extracted by HPLC showed that the phenolics were derivatives of benzoic acid (gallic acid, protocatechuic acid, and p-hydroxybenzoic acid) and cinnamic acid (p-coumaric acid, syringic acid, ferulic acid and trans-cinnamic acid). However, in the higher alkaline condition (pH 10), only gallic acid and protocatechuic acid were detected. On the other hand, evaluating the stability of eight phenolic compounds of five commercial grape extracts after different thermal treatments (pasteurisation HTST and LTLT, cooking, baking and sterilisation), Davidov-Pardo et al. [61] found that gallic acid showed a greater tendency to increase in the studied treatments compared to the control. Chen et al. [62], investigating the effects of different drying temperatures (50, 60, 70, 80, 90 and 100°C) on changes in the flavonoid, phenolic acid and antioxidative activities of the methanol extract of citrus fruit peels, found that caffeic acid and p-coumaric acid contents in the 100°C dried sample also increased by around 5 and 2 folds, respectively, compared to those of a fresh sample. In this study, the authors concluded that the heating treatment might release some low molecular weight phenolic compounds from the samples. The gallic acid is a hydroxybenzoic acid with two functional groups in the same molecule, hydroxyl groups and a carboxylic acid group. They can yield numerous esters and salts including digallic acid. Conventionally, gallic acid is obtained by acid hydrolysis of tannins, which are esters of the digallic acid [63]. Alternatively, it can be produced by enzymatic hydrolysis using spent broths from Penicillium glaucum or Aspergillus niger, which contain tannase [64]. Some esters of gallic acid [propyl (E310), octyl (E311), and dodecyl (E312) gallates] have been widely used as antioxidants in food [53].

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Table 4. Physicochemical properties of the main phenolic acids found in plants

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Phenolic acid

Molecular weight

Physical State and Colour

Melting Point (°C)

Hydroxybenzoic acids Gallic acid

170.12

White to beige powder

250°C

Ellagic acid

302.19

360°C

p-hydroxybenzoic acid Protocatechuic acid (No italic) Hydroxycinnamic acids Caffeic acid

138.12

Gray to slightly beige crystalline powder White to off-white powder

154.12 (No italic)

White to brownish crystal powder (No italic)

180.16

Off-white to brown crystalline solid

211213°C

Chlorogenic acid

354.31

White to off-white solid

208°C

p-coumaric acid (No italic)

164.16 (No italic)

Off-white to tan powder (No italic)

Ferulic acid

194.18

Sinapic acid

224.21

Off-white to beige, slightly yellow powder Yellow-brown crystalline powder

210213°C (No italic) 174°C

213214°C 199°C

203205°C

Solubility in water

Stability

Reference

1 g/ 87 mL water or 3 mL boiling water Slightly soluble in water Soluble in about 125 parts water Soluble in 50 parts water (No italic)

Unstable to light and high pH. Hygroscopic Stable under normal conditions

[59,63,64]

Stable under normal conditions

[63,66]

Sparingly soluble in cold water. Freely soluble in hot water Hemihydrate form soluble in water at 25°C about 4%. Much more soluble in hot water Slightly soluble in cold water. Soluble in hot water (No italic) trans-Form soluble in hot water Chloride form very soluble in water

Unstable to high pH. Turn from yellow to orange in alkaline solutions

[59,63]

Unstable to high pH. Forms a black compound with iron

[59,63]

Stable under normal conditions (No italic)

[63]

Iron catalyzes its oxidation to a dilactone Air oxidation in aqueous solutions at pH 7–10 causes darkening of the system color

[63,20]

Discolors in air (No italic)

[63,65]

[63, 67] (No italic)

[55]

ultco/detail.action?docID=3017830.

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The ellagic acid is a hydroxybenzoic acid that has generated commercial interest in recent years due to its properties and potential food applications. The ellagic acid has four rings representing the lipophilic domain, four phenolic groups and two lactones, which can act as hydrogen-forming sides and electron acceptors, respectively, and represent the hydrophilic domain. Commercial ellagic acid is obtained by chemical extraction with acid-methanol mixtures as solvents. Usually, concentrated HCl or H2SO4 are used to hydrolyze the ellagitannin from plant sources. Several attempts have been made to develop a bioprocess to produce ellagic acid through fermentation technology, providing a less contaminant, less expensive and less aggressive product, with high yields, than chemical extraction provides [65]. The p-hydroxybenzoic acid is a hydroxybenzoic acid whose esterification with the corresponding alcohol in the presence of an acid catalyst produces parabens, a group homologous series of p-hydroxybenzoic acid esterified at the C-4 position. As the chain lenght of the ester group of parabens increases, water solubility decreases. As a consequence, usually the lower esters (methyl and propyl) are the practical choices for use of parabens in food. Parabens are generally resistant to hydrolysis in water (hot and cold) and in acidic solutions and are stable in air. The resistance of parabens to hydrolysis is increased by an increase in its alkyl chain length. However, appreciable hydrolysis occurs at pH above 7. Parabens occur naturally in food and are also widely used as preservatives in them [66]. The protocatechuic acid is a hydroxybenzoic acid that can be obtained by the alkaline fusion of vanillin [60]. Through the alkaline fusion, the pigment quercetin also breaks up into protocatechuic as well as phloroglucinol and oxalic acids [67]. Protocatechuic acid extracted from hibiscus is a natural antioxidant that has been used as food additive [68]. A bioprocess to produce protocatechuic acid by fermentation technology has been also developed [69]. The caffeic acid is a constituent of plants that probably occurs only in the conjugated forms, e. g. chlorogenic acid (C16H18O9). In this way, this hydroxycinnamic acid is obtained by acid hydrolysis of chlorogenic acid [63]. Chlorogenic acid can be isolated from green and roasted coffee by organic solvents (chloroform, dichloromethane, hexane, methanol, etc.) [70, 71]. It has been also extracted from Eucommia ulmoides leaves and honeysuckle flowers and marketed as food additive by different companies, such as Lukee High-Tech Co. Ltd, Changsha Sunfull Bio-Tech Co. Ltd and others. Similarly, caffeic acid has been used as food additive. The p-coumaric acid is a hydroxycinnamic acid that is also of considerable interest for food applications. As a result, different sources and extraction methods have been studied to optimize the obtainment of p-coumaric acid. Mussatto et al. [72], investigating the pcoumaric and ferulic acids extraction by alkaline hydrolysis of brewer's spent grain, demonstrated that the best alkaline hydrolysis conditions consisted in using a 2% NaOH concentration, at 120 °C for 90 min. Under these conditions, a liquor containing 145.3 mg/L ferulic acid and 138.8 mg/L p-coumaric acid was obtained. These values corresponded to 9.65 mg ferulic acid and 9.22 mg p-coumaric acid per gram of solubilized lignin. In their study for supercritical extraction of phenolic compounds from Baccharis dracunculifolia, Piantino et al. [73] found that p-coumaric acid extraction yield was better by conventional methods using organic solvents (ethanol and methanol) than by supercritical CO2. The ferulic acid was first applied in food in 1975. This hydroxycinnamic acid is one of the most well known phenolic acids with regards to its physicochemical characteristics. The cis-ferulic acid does not crystallize, but forms a yellow oil with a UV maximum at 316 nm in

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ethanol. Colorless orthorhombic needles of the trans-ferulic acid can be crystallized from hot water. Ferulic acid is a strong dibasic acid. The first proton dissociation produces the carboxylate anion, while the second proton dissociation generates a phenolate anion. The high degree of phenolate anion resonance stabilization across the entire conjugated molecule markedly increases its acidity in comparison to similar phenolic acids. The trans-ferulic acid strongly absorbs in the UV range with absorption maxima at 284 nm and 307 nm in aqueous solution, pH 6.0. It also exhibits strong fluorescence. It may undergo slow thermal decarboxylation in water to form 4-vinylguaiacol. One hour heating at pH 4.0 and 100°C resulted in a 5.2% decarboxylation. Iron catalyzes its oxidation to a dilactone [20]. Ferulic acid 100% extracted and purified from rice bran of Oryza sativa L. was obtained and can be applied as food additive and active ingredient in health food. The dried product contains a minimum of 98.0% ferulic acid and is a white or light yellowish brown crystalline powder. It has no smell. The product has maximum values of absorption spectrum at wavelength 236 nm and 322 nm in ethanol solution [74]. Ferulic acid can be obtained either by chemical synthesis or by biological transformation [75]. Sinapic acid is a hydroxycinnamic acid that can be obtained from black mustard seeds [61] and rapeseed/canola [55]. It is found as sinapine, the choline ester of sinapic acid. Sinapine is widely distributed among the family Brassicaceae [76]. It has been extracted by organic solvents (methanol, hexane) [77, 78] and is easily hydrolyzed to sinapic acid by the enzyme sinapine esterase [79]. On the other hand, concern for safety and consumer preference for more natural foods has resulted in a high demand for natural additives, which can extend the shelf life of processed and unprocessed food. As a result, phenolic acids are increasingly of interest in the food industry because they have antioxidant activity and can prevent lipid oxidation, preserving the quality of food.

Antioxidant Power Research has shown that phenolic acids and their derivatives have a high antioxidant activity, mainly caffeic and chlorogenic acids. It is known that, although other characteristics also contribute to the antioxidant activity of phenolic acids and esters, such activity is usually determined by the number and positions of hydroxyl groups relative to the carboxyl functional group present in the same molecule [5]. Monohydroxybenzoic acids with hydroxyl in the ortho- or para- position relative to the carboxyl group do not have antioxidant activity. On the other hand, m-hydroxybenzoic acid does have antioxidant activity. The antioxidant activity of the benzoic acids is raised due to the increase on the degree of hydroxylation, which occurs with the trihydroxylated gallic acid that shows a high antioxidant activity. However, the replacement of hydroxyl groups of positions 3- and 5- by methoxyl groups, such as syringic acid, reduces this activity [80]. In general, the hydroxycinnamic acids are more effective than benzoic acids [81]. The increased activity of hydroxycinnamic acids is due to the -HC =CH-COOH group, which ensures higher -H donating capacity and stabilization of the radical than the -COOH group in hydroxybenzoic acids.

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Thus, p-coumaric and sinapinic acids are more effective antioxidants than benzoic acid derivatives, such as protocatequinic, syringic and vanillic acids, due to the double bond of the -HC= H-COOH group, which participates in the stabilization of the radical through unpaired electron displacement resonance, while the benzoic acid derivatives do not exhibit this characteristic. The ferulic acid hydroxyl present in ortho- position with the electron-donating methoxyl group increases the radical phenoxyl stability as well as the antioxidant efficiency. The presence of a second hydroxyl in the ortho- or para- position also increases the antioxidant activity of cinnamic acids. The caffeic acid, which has this same feature, has an antioxidant activity higher than that of the ferulic acid [82]. Therefore, phenolic acids mainly comprise the category of free radicals antioxidant scavengers, blocking the chain reaction. Due to the molecular conformation, the phenoxyl radicals formed are quite stable intermediates and do not easily start a new chain reaction. These intermedite radicals work by reacting with other free radicals, ceasing the propagation reactions [83]. However, depending on their chemical structures, the phenolic acids can also act as chain breakers [84] and metal chelating agents [85]. Esterified phenolic acids may also have a high antioxidant activity. Propyl gallate has an antioxidant power very similar to that of gallic acid. Caffeic and dihydrocaffeic acids n-alkyl esters (methyl, ethyl, and propyl) have significant antiradical scavenging activity compared with (±)--tocopherol. Esterification of ferulic acid increases its antioxidant activity when measured in a bulk oil system at 40 and 90C [53].

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Antimicrobial Properties The control of microorganisms’ growth in food by using synthetic additives such as currently approved antibiotics is a cause for concern due to the potential development of resistence. Several studies have been published indicating that phenolic acids might be potent agents as natural food additives in order to prevent food contamination and deterioration by microorganisms. Already in 1992, Bowles and Miller [86] tested the caffeic acid activity against Clostridium botulinum spores and found that at 0.78 and 3.25 mM caffeic acid was able to inhibit the Clostridium germination for 6 and 24 h, respectively, with > 100 mM required to render spores nonviable. Caffeic acid concentrations ≥ 50 mM reduced 80C spore thermal resistance. Sporostatic activity was retained when tested in commercial meat broths, and 5.0 mM caffeic acid delayed toxigenesis. The authors concluded that caffeic acid has potential as a food additive to inhibit growth of C. botulinum, and reduce thermal processing requirements of heat sensitive foods. From this time, several other studies on the antimicrobial properties of phenolic acids have been developed. Using the disk diffusion method, Fernández et al. [87] demonstrated the potent antibacterial activity of the phenolic acids fractions of aerial part of Scrophularia frutescens and S. sambucifolia (Scrophulariaceae) against Gram-positive and Gram-negative bacteria. The authors isolated the ferulic, isovanillic, p-hydroxycinnamic, p-hydroxybenzoic, syringic, caffeic, gentisic and protocatechuic acids from S. frutescens and ferulic, p-coumaric, vanillic, p-hydroxibenzoic and syringic acids from S. sambucifolia. They concluded that since

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phenolic acids have been shown in the literature to exert an antibacterial effect, the presence of these substances explains the antibacterial property found for both studied samples. Comparing the antimicrobial activity of five free phenolic acids (procatechuic, caffeic, pcoumaric, ferulic and chlorogenic acids) isolated from Ginkgo biloba L. leaves by broth dilution test, Ellnain-Wojtaszek and Mirska [88] demonstrated that the studied acids had various antimicrobial activity. The acids acted at a wide range of concentrations: from 0.312 mg/mL to 5.0 mg/mL. The minimal inhibitory concentration and minimal bactericidal concentration values for cocci were within the widest scope of concentrations (0.312 mg/mL5.0 mg/mL) but that range was narrower for fungi (0.6 mg/mL-5.0 mg/mL) and bacilli (1.25 mg/mL-5.0 mg/mL). p-coumaric acid showed the highest antibacterial activity. This acid was markedly active against cocci, inhibiting their growth at concentration of 0.312 mg/mL. The bactericidal effect appeared at the concentration twice the concentration needed for the bacteriostatic effect, and at 0.625 mg/mL the acid was fungicidal. Bacilli were more resistant to p-coumaric acid than cocci and were inhibited at the concentrations twice as high as (minimal bactericidal concentration: 1.25 mg/mL). Chlorogenic acid was the weakest antimicrobial agent. In contrast to the other acids, this acid was bactericidally active only against Gram-negative bacilli (minimal bactericidal concentration: 2.5 mg/mL) and was inactive against cocci and fungi. The other phenolic acids had similar activity spectrum. They were active against staphylococci (minimal inhibitory concentration and minimal bactericidal concentration: 1.25 mg/mL and 2.5 mg/mL) and fungi (minimal bactericidal concentration: 2.5 mg/mL). Moreover, micrococci were susceptible to protocatechuic acid (minimal bactericidal concentration: 0.612 mg/mL) and bacilli E. coli were sensitive to ferulic acid (minimal bactericidal concentration: 1.25 mg/mL). Aljadi and Yusoff [89], isolating and identifying phenolic acids in two Malaysian floral honeys with antibacterial properties, found that the phenolic fractions of gelam and coconut honeys showed potent antibacterial activities against two strains of standard bacteria and two strains of pathogenic bacteria, including Escherichia coli ATCC25922, and Stapylococcus aureus ATTC 25923, Methicillin-resistant Staphylococcus aureus and Methicillin-sensitive Staphylococcus aureus. The authors identified the gallic, caffeic, and benzoic acids in both samples. However, gelam honey contained the ferulic and cinnamic acids as additional phenolic acids. Applying a broth dilution method, Wen et al. [90] also studied several phenolic acids including chlorogenic acid and the hydroxycinnamic acids, caffeic acid, p-coumaric acid and ferulic acid with regards to their activity against five strains of Listeria monocytogenes. The authors found that the minimum inhibitory concentrations ranged between 0.20% and 0.27% (w/vol) for the hydroxycinnamic acids, but chlorogenic acid was ineffective at 1.0% (w/vol). Mixtures of the acids usually exhibited synergic antilisterial effects in a checkerboard assay. Growth experiments conducted at pH 4.5, 5.5 and 6.5 revealed a strong relationship between pH and activity. All the hydroxycinnamic acids were bactericidal at pH 4.5 and bacteriostatic at higher pH. On the other hand, chlorogenic acid inhibited growth of L. monocytogenes only at pH 6.5. Verifying the antimicrobial activity against select food-borne pathogens by phenolic antioxidants enriched in cranberry pomace by solid-state bioprocessing and using the food grade fungus Rhizopus oligoporus, Vattem et al. [91] found that for E. coli 0157:H7 inhibition correlated with the pomace extracts corresponding to highest ellagic acid concentration.

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Almajano et al. [92] investigated the effect of pH on the antimicrobial activity of oil-inwater emulsions containing caffeic acid and found that the antibacterial properties of caffeic acid was pH dependent. A concentration of 0.41% (w/w) caffeic acid was sufficient to inhibit the growth of some of the studied microorganisms in the pH range of 5 to 7. However, nearneutral pH concentrations higher than 0.4% were needed to inhibit Listeria monocytogenes, E. coli, Staphylococcus aureus, and others microorganisms in the medium. Assessing the selective antimicrobial activity of catechin, chlorogenic acid and phloridzin from fruits against three marker pathogenic bacteria, one probiotic bacterium, two yeasts and one food spoilage fungus by the turbidity assay, Muthuswamy and Rupasinghe [93] found that the growth of pathogenic bacteria, Escherichia coli O157:H7, Listeria innocua and the food spoilage fungus, Penicillium chrysogenum, were inhibited by all the phenolics at 25 mM but the growth of food spoilage yeast Saccharomyces cerevisiae was inhibited only by chlorogenic acid and phloridzin. Additionally, chlorogenic acid showed a greater inhibitory effect on opportunistic pathogen, Candida albicans, than that of catechin and phloridzin. Studying the phenolic acid contents of kale extracts and their antibacterial activities by the agar well diffusion method, Ayaz et al. [94] identified and quantified nine phenolic acids in leaves and ten in seeds of kale (black cabbage). Ferulic and caffeic acids were the most abundant phenolic acids in the leaves and sinapic acid in the seed. Free, ester (methanolsoluble), glycoside and ester-bound (methanol-insoluble) phenolic acid contents were highly correlated with their antimicrobial property assays. All of the fractions were effective against Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and (most strongly) Moraxella catarrhalis. Chao and Yin [95] evaluated the antibacterial effects of roselle calyx aqueous and ethanol extracts as well as protocatechuic acid against food spoilage bacteria Salmonella typhimurium DT104, Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus in ground beef and apple juice and found that the minimal inhibitory concentrations of roselle calyx aqueous and ethanol extracts and protocatechuic acid against these bacteria were in the range of 112–144, 72–96, and 24–44 μg/mL, respectively. The authors also demonstrated that the antibacterial activity of roselle calyx ethanol extract and protocatechuic acid was not affected by heat treatments from 25 to 75°C and from 25 to 100°C, respectively. They concluded that these two samples might be potent agents as food additives to prevent contamination from the studied bacteria. Isolating four diterpenoid tanshinones and three phenolic acids from the crude ethanol extract of the cultured hairy roots of Salvia miltiorrhiza Bunge by bioassay-guided fractionation and identifying the compounds by physicochemical and spectrometric analysis, Zhao et al. [96] found tanshinone IIA, tanshinone I, cryptotanshinone, dihydrotanshinone I, rosmarinic acid, caffeic acid, and danshensu. These compounds were evaluated to show a broad antimicrobial spectrum of activity on test microorganisms, including eight bacterial and one fungal species. The results indicated that the major portion of the antimicrobial activity was due to the presence of not only tanshinones but also of phenolic acids in S. miltiorrhiza hairy roots. Determining the effect of phenolic compound mixtures on the viability of Listeria monocytogenes in meat model, Vaquero et al. [97] found that at 20°C 100 mg/L of gallic acid and protocatechuic acid, then gallic acid and caffeic acid, and rutin and quercetin mixtures decreased the growth of L. monocytogenes, as compared to the control. The inhibitory effect of gallic acid and protocatechuic acid mixtures increased at the concentration of 200 mg/L.

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However, gallic acid and caffeic acid, and rutin and quercetin were the most effective mixtures since after 14 days of incubation no viable cells of L. monocytogenes were detected. Recently, Ozcelik et al. [98] evaluated the in vitro antiviral, antibacterial and antifungal activities of alkaloids, flavonoids, and phenolic acids. The authors tested the antiviral activity of the compounds against DNA virus herpes simplex type 1 and RNA virus parainfluenza (type-3). Antibacterial activity was studied against following bacteria and their isolated strains: Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis, although they were screened by microdilution method against two fungi: Candida albicans and Candida parapsilosis.The results showed that not only atropine but also gallic acid showed potent antiviral effect, while all of the samples exerted robust antibacterial effect. Thus, the preparation and obtention of foods with high content of these antioxidant and antimicrobial substances can lead to a reduction in the use of synthetic additives, resulting in healthier foods that may be included in the group of functional foods.

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Food Applications The organic compounds oxidation processes are a major cause of reduced shelf life of processed food products and raw materials in general. Among the main oxidation reactions in food products there is the lipid oxidation. The main problems arising from lipd oxidation are sensory changes due to the development of unpleasant odors and flavors, called rancidity, with a consequent decrease in the nutritional quality caused by the destruction of vitamins and safety caused by the formation of potentially toxic secondary compounds [99]. For this reason, foods containing significant amounts of polyunsaturated fatty acids have contributed to the need of using antioxidants to prevent oxidation, preserve flavor and odor, as well as avoid vitamins destruction. The most widely used antioxidants are butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tert-butylhydroquinones (TBHQ). In some cases, the synergistic action of the citric acid is essential. These synthetic antioxidants tend to stabilize the fatty acids in foods by reacting with free radicals, blocking the propagation phase of lipid oxidation and chelating metal ions. However, the safety of some commercial antioxidants has been questioned, since they may favor mutagenic and carcinogenic effects [100]. On the other hand, except when microorganisms are cultivated by selective inoculation or by controlled conditions for food production, their multiplication on or in foods is the main cause of food deterioration. The microorganisms that are mainly involved in food deterioration are bacteria, fungi and yeasts. They may act on all food constituents by fermenting sugars and hydrolyzing starches and cellulose as well as by hydrolyzing fats and producing rancidity or by digesting proteins and producing putrid and ammonia-like odors. They may also act on food constituents by forming acid and producing a taste of sour in food as well as by producing gas and making food foamy or by forming pigments. A few microrganisms may still produce toxins and give rise to foodborne illnesses. When food is contaminated under natural conditions, several types of organisms may be present at the same

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time. Such mixed organisms contribute to a complex of simultaneous or sequential changes which may include acid, gas, putrefaction and discoloration [101]. Therefore, phenolic acids have been studied as natural additives to replace artificial ones which have been used in the food industry. Table 5 summarizes some phenolic acids, and food in which they have been investigated with success as natural antioxidant and/or antimicrobial additives. As it can be seen in Table 5, phenolic acids have been investigated as additives in different foods for preventing lipid peroxidation and protecting from oxidative damage as well as preventing microbial deterioration. Dziedzic and Hudson [102], assessing the efficiency of some phenolic acids as antioxidants in lard by the Rancimat method, found that protocatechuic, caffeic, ferulic, gallic and sinapic acids showed a good activity. Papadopoulos and Boskou [103] investigated the antioxidant activity of each phenolic acid contained in the polar fraction of refined olive oil in the same oil by the method of Shall at 60C, in the absence of light. The peroxide value was determined as an index of the oxidative process. The results showed that caffeic acid had higher activity than BHT, while for protocatechuic and syringic acids the activity was lower than BHT. On the other hand, o-coumaric, p-coumaric, p-hydroxybenzoic and vanillic acids showed low or no antioxidant power.

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Table 5. Phenolic acids and food in which they have been investigated as natural antioxidant and/or antimicrobial additives Food Lard

Effect Antioxidant

Olive oil

Antioxidant

Soybean oil

Antioxidant

Cod liver oil Cooked beef, chicken and pork

Antioxidant Antioxidant

Commercial meat broths Ground beef and apple juice Lean meat

Antimicrobial Antimicrobial

Candy, chewing gum, gummy, snacks, cookies, chocolate, wafer, jelly, drink, green tea, banana and others

Antioxidant

Orange

Antimicrobial

Antimicrobial

Phenolic acid Protocatechuic acid, Caffeic acid, Ferulic acid, Gallic acid and Sinapic acid Caffeic acid, Protocatechuic acid, Syringic acid Sunflower seed aqueous extract (chlorogenic acid, caffeic acid and quinic acid) Caffeic acid Canola acetone 70% extract (sinapic acid, ferulic acid and p-hydroxybenzoic acid) Caffeic acid Roselle calyx ethanol extract (protocatechuic acid) Gallic and caffeic acids mixture, Gallic and protocatechuic acids mixture Ferulic acid

Reference [102] [103] [104]

[105] [106]

[86] [95] [97] [74]

[20]

Studying the antioxidant capacity of the striped sunflower seed aqueous extract by the Rancimat method at 110C, Giada [104] demonstrated that this extract, mainly composed of chlorogenic acid (12.88%), had an antioxidant activity comparable to the synthetic antioxidant BHA. Leonardis et al. [105] evaluated the chlorogenic acid and its metabolites,

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caffeic acid and quinic acid, as potential antioxidants for fish oils. In this study, all three chemicals were separately added to cod liver oil and tested by Rancimat method at 80C or 100C. The results showed that caffeic acid was the best antioxidant. Its antioxidant power was directly proportional to the dose added, but only within the concentration range of 0.005– 0.050 wt-%. Quinic acid did not show any effect on cod liver oil oxidation and chlorogenic acid had a weak antioxidant activity. Determining the phenolic acids composition and antioxidant activity of canola acetone 70% extracts in cooked beef, chicken and pork, Brettonnet et al. [106] found that the canola extract contained sinapic (99.7%), ferulic (0.28%) and p-hydroxybenzoic acids (0.07%). The crude polyphenol extracts (15 or 100 mg gallic acid equivalents/kg meat) from canola meal reduced the formation of 2-thiobarbituric acid-reactive substances (TBARS) in pre-cooked beef (66–92%), pork (43–75%) and chicken (36–70%). On the other hand, caffeic acid and protocatechuic acid as well as gallic and caffeic acids mixture or gallic and protocatechuic acids mixture showed good results for meat [97], meat products [86, 95] and apple juice [95] as natural additives with antimicrobial activity. Among the phenolic acids, ferulic acid is one of the best investigated as food additive. It is known that ferulic acid has health-promoting properties and low toxicity [74]. LD50 conducted using mouse model deduced to be 857 mg/kg [73]. This phenolic acid can be absorbed and easily metabolized in the human body as well as have many physiological functions, including antioxidant, antimicrobial, anti-inflammatory, anti-thrombosis, and anticancer activities. It also protects against coronary disease, lowers cholesterol and increases sperm viability. Thus, ferulic acid is now widely used in the food industry. It has also been reported to preserve oranges [20] and to prevent discoloration of green tea as well as to prevent black color due to oxidation of the banana and, consequently, reduce bacterial contamination. In addition, it is used as the raw material for the production of vanillin and preservatives, as a cross-linking agent for the preparation of food gels and edible films, and as an ergogenic ingredient in sports foods [72]. Cao et al. [107] studied the mechanical properties of gelatin films cross-linked, respectively, by ferulic acid and tannin acid. The authors found that the two natural cross-linking phenolic acids had cross-linking effects on gelatin film and that the maximal mechanical strength of gelatin film could be obtained when the pH value of film-forming solution was 7 for ferulic acid as cross-linked agent, or was 9 as for tannin acid. (No paragraph) The cross-linking agents could also decrease swelling ratios of the films but there were no obvious effects on water vapor permeability. The properties of films treated by tannin acid could become better after stored for more than 90 days, while storage time had little effect on ferulic acid-modified films. The role of ferulic acid in preparing edible films from soy protein isolate was determined by Ou et al. [108]. (No paragraph) The results showed that an optimal concentration of ferulic acid increased the tensile strength, elongation percentage at break and antioxidant activity of films for preservation of fresh lard. The optimal concentration for ferulic acid in film forming solution is 100 mg/100 g. Moreover, the properties of the film were further improved when ferulic acid was oxidized by hydrogen peroxide. Another potential application for phenolic acids in the food industry is the use of these compounds on the reduction of methanol content in wine processing. Hou et al. [109] verified that adding gallic acid or coumaric acid with commercial pectic enzyme inhibited the increase of methanol production. In addition, when 0.2 mg/L of gallic acid or coumaric acid was

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added, the amount of total phenolic acid released from commercial pectic enzyme + gallic acid or commercial pectic enzyme + coumaric acid groups became higher than commercial pectic enzyme group by approximately 466 and 539 mg/L, respectively. Authors also verified that the values of lightness, red content, yellow content and total pigment increased in the presence of gallic acid or coumaric acid with commercial pectic enzyme, suggesting that adding these phenolic acids into winemaking process is a potential method for reducing methanol content, improving wine quality, as well as increasing healthy compounds in wine production.

Conclusion Many food and non-food plants are phenolic acids sources. By-products of food processing, such as seeds, peels and pomace, are promising for obtaining phenolic acids for food industry applications. These substances have already been added to some foods with a different degree of success. It is difficult to predict the effects of phenolic acids in different food systems because the same phenolic acid can have different effects in different food matrices. Phenolic acids may be important as food additives with less possible side effects and undesirable characteristics than are observed in the synthetic antioxidants currently used in food products.

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In: Phenolic Acids Editor: Sergi Munné-Bosch

ISBN: 978-1-61942-032-8 © 2012 Nova Science Publishers, Inc.

Chapter VIII

Phenolic Acids from Microbial Metabolism of Dietary Flavan-3-ols María Boto-Ordóñez1,2,3, Mireia Urpi-Sarda2,3, María Monagas4, Sara Tulipani1, Rafael Llorach1, Montse Rabassa-Bonet1 , Mar Garcia-Aloy1, María Isabel Queipo-Ortuño3,5, Ramon Estruch2,3, Francisco Tinahones3,5, Begoña Bartolomé4 and Cristina Andres-Lacueva1 1

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Nutrition and Food Science Department, XaRTA-INSA. Pharmacy Faculty, University of Barcelona, Av Joan XXIII, s/n, Barcelona, Spain 2 Department of Internal Medicine, Hospital Clinic, Institut d'Investigació Biomèdica August Pi i Sunyer (IDIBAPS), University of Barcelona, Villarroel 170, 08036 Barcelona, Spain 3 CIBER 06/03: Fisiopatología de la Obesidad y la Nutrición, Instituto de Salud Carlos IIIm Spain 4 Instituto de Investigación en Ciencias de la Alimentación (CIAL), CSIC-UAM, Madrid, Spain 5 Servicio de Endocrinología y Nutrición, Hospital Clínico Virgen de la Victoria, Málaga, Spain

Abstract Flavan-3-ols are polyphenols present in the diet in monomeric, oligomeric and polymeric forms, but their bioactivity and in vivo health effects remain unclear due to their complex metabolism. According to the degree of polymerization, monomeric flavan-3-ols can be absorbed in the small intestine, whereas oligomers and polymers need to be biotransformed by the colonic microbiota before absorption. This latter gives rise to a wide number and variety of phenolic acids which may be responsible for the health effects derived from flavan-3-ol consumption rather than the original phenolic forms 

E-mail addresses: [email protected].

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María Boto-Ordóñez, Mireia Urpi-Sarda, María Monagas et al. found in foods. Although in vitro studies have revealed that some bacteria are able to catabolise certain class of polyphenols, the identification of human colonic bacteria with capacity to catabolise flavan-3-ols is in its early stages. However, in the last decade a great progress has been achieved in the identification of phenolic acids derived from the catabolism of flavan-3-ols by gut microbiota. The link between consumption of flavan-3ols food sources and those metabolites found in vivo, with related health effects is still a difficult challenge due to the huge variability in colonic biotransformation found among individuals, and other factors such as the own structural diversity of these polyphenols and food matrix that add a further variability in catabolism. Studies performed with isolated phenolic compounds in a colonic environment may help us to identify colonic bacteria involved in catabolism and understand their activity in the colon, and set up a link to circulating metabolites found in vivo. Although the biological relevance of microbial metabolites remains largely unknown, evidences related to their antioxidant, anti-inflammatory and anti-proliferative activities and cytotoxicity are starting to be accumulated. This chapter aims to give an insight into the phenolic acids formed by the colonic catabolism of dietary flavan-3-ols, including tentative metabolic pathways, potential microbial groups/species involved in their catabolism, plasma and urine concentrations found after in vivo consumption, and specific bioactivities. All these aspects may help us better understand the complexity of the colonic catabolism of flavan3-ols and the role of phenolic acids in health effects derived from the consumption of flavan-3-ol rich sources.

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Introduction Flavan-3-ols are the most common group of flavonoids found in the diet, and, in view of their presence in food as functional ingredients, also one of the most commonly groups linked to health benefits. The group includes a wide number of molecules of different chemical structure and degree of polymerization that may vary depending on the source. This variation is reflected in the number of structural units, hydroxylation pattern, stereochemistry of C2 and C3 of the central ring, type interflavan linkages and possible esterification with other polyphenols (i.e. galloylation) [1]. Those chemical characteristics have a profound impact on the absorption and metabolism, and hence in their functionality in our organism. Monomers and oligomers may be absorbed at the small-intestine level, undergoing glucuronidation and methylation in the enterocyte. The portal vein drives them to the liver, where a new process of methylation, glucuronidation and glycination takes place. From the liver, new metabolites are then released to various tissues and organs, before being excreted through urine or turning back to the intestine for enterohepatic circulation. Procyanidins and some oligomeric forms are not absorbed in the small intestine and reach the colon where they are metabolized by intestinal bacteria to more simple metabolites, phenolic acids. Phenolic acids, with different patterns of hydroxylation and methylation and side-chain length, have shown diverse bioactivities and in certain cases could be more active than their precursors. These metabolites generated by microbiota are also absorbed and metabolized in the same way as monomers once they reach the liver, and they are also found in biofluids such as urine or plasma in their simple or conjugated form [2-4]. A small fraction of phenolic acids is also eliminated through the faeces and found in faecal waters. Most of the bacteria responsible for these changes in flavan-3-ols remains unknown, because of the number of species (concentrations of 1012 microorganisms per gram gut content) and interindividual variety,

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which could be affected by age, diet, environment and phylogeny [5-7], the complex structural features of these polyphenols and their own antimicrobial activity, but the number of studies evaluating the bacterial enzymes that change flavan-3-ols into more active components is of current interest [8, 9].

Sources of Flavan-3-ols Flavan-3-ols are widely distributed in foodstuffs such as fruits, red wine, beer and nuts, as well as in herbs, being the richest sources and the most widely studied tea and chocolate [1, 10]. Their presence in foods ranges from monomers such as (+)-catechin, (-)-epicatechin, (-)epigallocatechin, (-)-epicatechin-3-O-gallate, (-)-epigallocatechin-3-O-gallate and (+)gallocatechin to procyanidins (dimers, trimers or other polymers). Flavan-3-ol classification and their main food sources are described in Table 1. Table 1. Flavan-3-ols distribution in foods Flavan-3-ols Monomers

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(+)-Catechin

(-)-Epicatechin

(-)-EGC

(-)-ECG

Flavan-3-ols (-)-EGCG

(+)-Gallocatechin

Food

Mean

Min-Max

Red wine Black tea Chocolate Cocoa, powder Strawberry Red Grape Peach, peeled Apple cider Red wine Green tea Chocolate Cocoa, powder Red Grape Peach, peeled Cherry, sweet Apple Cider Broad beans Green tea Black tea Broad beans Pecan Nuts Plums Green tea Peppermint tea Grape black Carob, flour Strawberries Food Kiwi Green tea Black tea Hazelnuts Raspberries Plum Red wine

6.81 mg/100 ml 2.45 mg/100 ml 20.50 mg/100 mg FW 107.75 mg/100 g FW 6.36 mg/100 g FW 5.46 mg/100 g FW 5.47 mg/100 g FW 5.56 mg/100 g FW 3.78 mg/100 ml 7.93 mg/100 ml 70.36 mg/100 g FW 158.30 mg/100 g FW 5.24 mg/100 g FW 7.97 mg/100 g FW 7.78 mg/100 g FW 28.67 mg/100 g FW 22.51 mg/100 g FW 19.68 mg/100 ml 7.19 mg/100 ml 14.03 mg/100 g FW 5.60 mg/100 g FW 13.06 mg/100 g FW 7.50 mg/100 ml 9.24 mg/100 ml 1.68 mg/100 g FW 30.06 mg/100 g FW 0.15 mg/100 g FW Mean 0.08 mg/100 g FW 27.16 mg/100 ml 9.12 mg/100 ml 1.10 mg/100 g FW 0.54 mg/100 g FW 0.48 mg/100 g FW 0.08 mg/100 ml

1.38- 39 mg/100 ml 0 -17.08 mg/100 ml 0.75-50mg/100 mg FW 61-202 mg/100 mg FW 1.57-18.7 mg/100 mg FW 0.82-8.94 mg/100 mg FW 0.53-19.6 mg/100 mg FW 0-58.04 mg/100 mg FW 0-16.50 mg/100 ml 0-73.89 mg/100 ml 32.74-125 mg/100 mg FW 63-330 mg/100 mg FW 0.7-8.64 mg/100 mg FW 0.6-16.4 mg/100 mg FW 5.4-9.5 mg/100 mg FW 0-141mg/100 mg FW -0.01-100 mg/100ml FW 0.006-51 mg/100ml FW ---0.1-64.2 mg/100 ml FW 3.72-14.76 mg/100 ml FW 0.17-2.81 mg/100 mg FW --Min-Max 0-0.2 mg/100 g FW 0.57-271.4 mg/100 ml FW 0-67.9 mg/100 ml FW ---0-0.42 mg/100 ml

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[11-13]

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María Boto-Ordóñez, Mireia Urpi-Sarda, María Monagas et al. Table 1. (Continued)

Flavan-3-ols

Food Redcurrant Green tea Black tea Broad bean

Mean 1.28 mg/100 g FW 2.26 mg/100 ml 14.01 mg/100 ml 9.68 mg/100 g FW

Min-Max 1.22-1.35 mg/100 mg FW 0-15.69 mg/100 ml 0-59.2 mg/100 ml --

Red wine Cocoa powder Peach, peeled Plum, fresh Broad bean Red wine Cocoa powder Apple, cider Plum, fresh Broad bean Red wine Barley grain Quince jelly Red wine Custard apple Broad bean Plum, fresh Apple, dessert Custard apple Red wine Plum, fresh Apple dessert Green tea

4.14 mg/100 ml 112 mg/100 g FW 25.77 mg/100 g FW 8.84 mg/100 g FW 11.26 mg/100 g FW 4.97 mg/100 ml 71.57 mg/100 g FW 19.60 mg/100 g FW 5.20 mg/100 g FW 12.08 mg/100 g FW 9.47 mg/100 ml 10.90 mg/100 g FW 29.38 mg/100 g FW 7.29 mg/100 ml 2.48 mg/100 g FW 18.47 mg/100 g FW 1.59 mg/100 g FW 0.97 mg/100 g FW 0.82 mg/100 g FW 0.27 mg/100 ml 4.69 mg/100 g FW 3.76 mg/100 g FW 0.63 mg/100 ml

2.15-14 mg/100 ml -0.7-68.7 mg/100 mg FW --0.43-9 mg/100 ml 13-262 mg/100 mg FW 5.6-87.5 mg/100 mg FW --0-11.96 mg/100 ml 8.8-14.2 mg/100 mg FW 4.3-81.9 mg/100 mg FW 0.08-11. mg/100 mg FW ---0.18-1.9 mg/100 mg FW ------

Chocolate Plum, fresh Apple juice Plum, fresh Apple dessert Custard apple Red wine Beer

26.00 mg/100 g FW 10.01 mg/100 g FW 29.97 mg/100 ml 7.73 mg/100 g FW 1.37 mg/100 g FW 0.97 mg/100 g FW 6.71 mg/100 ml 0.3 mg/100 ml

13-44 mg/100 mg FW -19.9-40 mg/100 mg FW -0.43-2.4 mg/100 mg FW ----

Blueberries Cranberries Plums Blueberries Cranberries Plums Blueberries Cranberries Plums

25.7 mg/100 g FW 70.3 mg/100 g FW 58 mg/100 g FW 27.8 mg/100 g FW 62.9 mg/100 g FW 58 mg/100 g FW 266.4 mg/100 g FW 233.5 mg/100 g FW 57.3 mg/100 g FW

----------

Procyanidins dimers

B1

B2

B3 B4

B5

B7

[11, 12, 14]

Procyanidins trimers

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C1

Trimer EEC T2 C2 >Trimers 4-6 mers

7-10 mers

>10 mers

(-)-EGC, Epigallocatechin; (-)-ECG, Epicatechin-3-gallate; (-)-EGCG, Epigallocatechin-3-gallate; FW, Fresh Weight.

In contrast to the main classes of flavonoids which are present as glycosides, the aglycones (+)-catechin and (-)-epicatechin are present in foods such as cocoa powder (61-330 mg/100 mg FW), tea (0–74 mg/100 mL FW) red wine (0–39 mg/100 mL FW), nuts (0–4 mg/100 mg FW), beer (0–10 mg/100 mL FW) and fruits (0-58.04 mg/100 mg FW). Brewed

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tea is a rich source of flavan-3-ols, with (+)-catechin and (-)-epicatechin being present in high amounts (6.8–395 mg/100 g FW) [11, 15]. (Epi)catechins are present in the majority of foods, while gallo(epi)catechins and galloyled monomers such as (+)-gallocatechin, (-)-epicatechin 3-gallate, and (-)-epigallocatechin 3-gallate are only found in certain fruits, such as plums, apples, berries, red grapes and peaches, and normally at very low concentrations (less than 1 mg/100 g FW) [13]. On the other hand, proanthocyanidins, also known as condensed tannins, are widely distributed and are the second most abundant natural phenolic compounds after lignins, in terms of their presence and distribution. They are oligomeric and polymeric flavan-3-ols that are usually found as procyanidins (i.e. oligomers and polymers constituted by (epi)-catechin). The proportion of monomers, dimers, trimers and polymers of total flavan-3-ols ingested following an American-style diet has been estimated to account for 7.1, 11.2, 7.8 and 73.9% respectively, being apples (32.0%), followed by chocolate (17.9%) and grapes (17.8%) the major sources of procyanidins [14].In the Spanish diet, total flavan-3-ol content was found to range from 10 mg to 50 mg/100 g FW in fruits such as plums, apples and cherries. Among the beverages, the highest flavan-3-ol levels were determined in green and black teas (43.8 and 26.8 mg/100 mL of infusion, respectively), followed by wine, mainly red wine (2.66 mg/100 mL), but also rosé and white wines (2.20 and 0.2 mg/100 mL respectively). Finally, a few vegetables contained flavan-3-ols at very low concentrations (below 1.5 mg/100 g FW) [11, 13].

Bioavailability

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Flavan-3-ols: Absorption and Metabolism The bioavailability of flavan-3-ols is affected by different factors, such as the degree of polymerization and galloylation, and the matrix effect in which the compound is delivered. Therefore, beneficial effects are largely dependent on the precursor consumed. Most polyphenols present in food are conjugated (esters, glycosides or polymers), which means that they cannot be absorbed in their native form and they must be hydrolyzed in the gut by enzymes or by the colonic microflora to become more simple components [10]. However, monomeric flavan-3-ols do not need to be hydrolyzed, and are readily absorbed in the small intestine and extensively metabolized to monoglucuronides, sulfates, methyl ethers or combined derivatives. This conjugation occurs in the small intestine and liver and the most common conjugation positions are the hydroxyl groups at C-5 and C-7 (A ring), C3´ and C-4´ (B ring), and C-3 (C ring). The enzymes responsible for glucuronidation in the small intestine are uridine 5´-diphosphate glucuronosyl transferases (UGTs), especially UGT1, present in the luminal part of the endoplasmatic reticulum, cytosol sulfotransferases and catechol-O-methyltransferase (COMT) for sulfation and methylation in the liver, respectively. The formed metabolites are released to the systemic circulation from where they may be distributed to tissues or be excreted through urine and bile. Some of the glucuronides and sulfates excreted in the liver return back to the intestine to be reabsorbed, a phenomenon known as enterohepatic circulation, which may lead to a longer presence of polyphenols within the body, thereby continuing to have beneficial effects (Figure 1) [9, 10].

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María Boto-Ordóñez, Mireia Urpi-Sarda, María Monagas et al.

Figure 1. Schematic diagram of bioavailability of flavan-3-ols in organism after its ingestion. After ingestion, flavan-3-ols reach the small intestine where monomers and dimers could be absorbed through enterocytes and may suffer methylation and glucuronidation. Metabolites reach the liver where may be glucuronidated, methylated or sulfated before being liberated to systemic circulation for its latter distribution to organs and excretion by urine. Oligomers and polymers that are not absorbed in intestine arrive intact to colon where are metabolized by colonic microbiota releasing phenolic acids, that could be absorbed, metabolized, distributed and excreted by faeces. Metabolites reach the liver from portal vein, where may be glucuronidated, methylated or sulphated before being liberated to systemic circulation for its latter distribution to organs and excretion by urine.

Microbial Catabolism: From Flavan-3-ols to Phenolic Acids The colon is considered to be a complex organ where metabolism of polyphenols is particularly significant due to the fact that around 90–95% of dietary polyphenols are not absorbed in the small intestine and arrive intact to colon, where they are metabolized by microbiota or excreted in their intact form [16]. In the case of flavan-3-ols, particularly proanthocyanidins, greater knowledge of biotransformation by the colonic microbiota is needed for a better understanding of the bioavailability and bioactivity described in in vivo studies [17, 18]. Deprez et al. first demonstrated that only 9–22% of the radioactivity of a 14Cradiolabel proanthocyanidin polymer was present in the metabolite pool after in vitro fermentation by human microbiota, and that ethyl acetate soluble metabolites represented 2.7% of the initial radioactivity, demonstrating the extensive catabolism by intestinal bacteria [19]. More recently, in an in vivo study performed with ileostomy patients, ileostomy fluids were recovered after ingestion of green tea, and it was found that approximately 70% of the ingested monomeric flavan-3-ols from green tea could pass from the small to the large

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intestine, including 33% corresponding to the intact parent compounds [20] .Enzymatic reactions start from microbial glycosidases and esterases, then microbial glucuronidases and sulfatases deconjugate the phase II metabolite from the enterohepatic circulation enabling reuptake [21]. Demethylation, isomerization and fission reactions, among others, may also be achieved by the human intestinal microbiota before being absorbed, producing several derivatives of phenylvaleric, phenylpropionic, phenylacetic and benzoic acids with different patterns of hydroxylation, depending on the degree of polymerization-condensed catechins [17, 18]. Firstly, it was thought that procyanidins could be depolymerized into bioavailable monomers because of the acidic conditions in the stomach [22], but this could not been proven by in vivo studies [23]. However, the possible depolymerization of dimeric structures into monomeric units by the gut microbiota, firstly proposed by Groenewould et al. [24] has been recently confirmed, however it represents less than 10% in the case of procyanidin B2 [25]. In the same study 5-(2,4-dihydroxyphenyl)-2-eno-valeric acid arising exclusively from the catabolism of dimeric procyanidins by microbiota was identified, together with other derivatives from the A-ring of the upper unit [25]. Figure 2 summarizes the metabolic pathways for microbial catabolism of flavan-3-ols. In the case of galloylated monomeric flavan-3-ols (ECG and EGCG), the microbial catabolism usually starts with the rapid cleavage of the gallic acid ester moiety by microbial esterases, giving rise to gallic acid which is further decarboxylated into pyrogallol. Subsequent reactions involve the reductive cleavage reaction of the heterocyclic C-ring resulting in the formation of diphenylpropan-2-ols, which are later converted by lactonization into 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone if the precursor is (epi)catechin, or 5-(3´,4´,5´-trihydroxyphenyl)-γ-valerolactone if the precursor is (epi)gallocatechin [7]. The next stage is the fission of the valerolactone ring leading to hydroxyphenylvaleric acids: 5-(3´,4´-dihydroxyphenyl) valeric acid and/or 4-hydroxy-5(3´,4´-dihydroxyphenyl) valeric acid [7, 26]. In addition, it has been proposed that an interconversion between the 4-hydroxy-5- (3´,4´dihydroxyphenyl) valeric acid and 5-(3´,4´-dihydroxyphenyl)-γ-valerolactones forms, may exist with a strong tendency to be displaced through the former molecule [25]. Hydroxyphenylvaleric acids undergo β-oxidation of the side chain resulting in 3,4dihydroxyphenylpropionic and 3,4-dihydroxybenzoic acids through a successive loss of carbon atoms [7]. The α-oxidation of 3,4-dihydroxyphenylpropionic acid leading to phenylacetic acids has been described for monomeric and dimeric procyanidins. However, Appeldoorn et al. suggested that this may exclusively arise from the cleavage of the upper unit of dimeric procyanidins, being 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone derived from lower units [27]. Dehydroxylation of 3,4-dihydroxylated phenolic acids at C-4´ and, preferentially, at C-3´, results in 3 and 4-monohydroxylated phenolic acids, respectively. In the case of (epi)-gallocatechins dehydroxylation at C-5 results in the 3,4-dihydroxylated form which could be further dehydroxylation at C-4 and C-3 [9, 28]. Phenolic acids that are formed during microbial catabolism of flavan-3-ols in the gut may suffer further changes by microbial metabolism, as has been observed for caffeic and ferulic acids [18]. These findings were first described when caffeic acid was transformed either to 4ethylcatechol via decarboxylation and reduction, or to 3-(3-hydroxyphenyl)-propionic acid after incubation with fecal microflora [29]. The same authors observed the production of metabolites in urine after ingestion of caffeic acid, when germ-free rats were infected with human microbial species [30]. Ferulic

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acid was shown to be transformed to 3-(3-hydroxyphenyl)-propionic acid in rodents and ruminants [18]. In the liver and kidney, phenolic acids may undergo different reactions involving glycine conjugation, dehydrogenation, hydroxylation and methylation, releasing glycinates, monoglucuronide and monosulfate conjugates, which are later found in urine and plasma [17, 28].

Figure 2. Metabolic pathway for microbial catabolism of flavan-3-ols. Reactions of demethylation, isomerization and fission reactions, among others, may be achieved before being absorbed, producing several phenolic acids with different patterns of hydroxylation, depending on their precursors. (1) (epi)catechin; (2) dimeric procyanidin; (3) 1-(3´, 4´-dihydroxyphenyl)-3-(2´´,4´´,6´´-trihydroxyphenyl) propan-2-ol; (4) 5-(2,4-dihydroxy)-phenyl-2-ene-valeric acid; (5) 5-(3-hydroxyphenyl)-γ-valerolactone; (6) 5-(3,4-dihydroxyphenyl)-γ-valerolactone; (7) 4-hydroxy-5-(3,4-dihydroxyphenyl)-valeric acid; (8) 4-hydroxy-5-(3-hydroxyphenyl)-valeric acid; (9) 3-hydroxyphenyl valeric acid; (10) 3,4(dihydroxyphenyl)-valeric acid; (11) 3-hydroxyphenylpropionic acid; (12) 3,4dihydroxyphenylpropionic acid; (13) m-coumaric acid; (14) isoferulic acid; (15) ferulic acid; (16) pcoumaric acid; (17) caffeic acid; (18) 3-hydroxybenzoic acid; (21) protocatechuic acid; (20) vanillic acid; (21) 4-hydroxybenzoic acid; (22) 3-hydroxyhippuric acid; (23) 4-hydroxyhippuric acid; (24) 3,4dihydroxyphenylacetic acid; (25) 3-hydroxyphenylacetic acid; (26) 3-methoxy-4-hydroxyphenylacetic acid.

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Table 2. Degree of excretion and presence in urine and plasma of main metabolites derived from microbial metabolism of flavan-3-ol in human studies

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Metabolite vanillic acid 4-hydroxy-5- (3,4-dihydroxyphenyl) valeric acid 3´-methoxy-4´-hydroxyphenyl valerolactone 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone sulfate 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone glucuronide 3´-methoxy-4´-hydroxyphenylvalerolactone glucuronide protocatechuic acid vanillic acid 4-hydroxybenzoic acid 3-hydroxybenzoic acid 4-hydroxyhippuric acid p-coumaric acid caffeic acid ferulic acid m-coumaric acid isoferulic 3,4-dihydroxyphenylacetic acid 4-hydroxy-3-methoxyphenylacetic acid 3-hydroxyphenylacetic acid phenylacetic acid 3,4-dihydroxyphenylpropionic acid 3-hydroxyphenylpropionic acid 5-(hydroxyphenyl)-γ-valerolactone 5-(dihydroxyphenyl)-γ-valerolactone 5-(dihydroxyphenyl)-γ-valerolactone glucuronide 5-(dihydroxyphenyl)-γ-valerolactone sulfate 5-hydroxymethoxyphenyl)-γ-valerolactone glucuronide

Nº Subjects

10 humans

Source d

Urine c

Plasma c

Identified Identified Identified

----

40 g cocoa powder dissolved in 250 2 metabolites identified ml whole milk or water 2 metabolites identified

2 humans

10 capsules almond skin extract

[Continued]

[Continued]

--

Referenc e

[31]a

--

Identified

--

Medium Medium Medium Low High Low Medium Low Low Medium Medium High Medium Medium Medium Low Low High

High High High ND Low Low Medium Low Low Low Medium Medium Medium High High High ND ND

Medium

ND

High

Medium

Low

Low

[2]a

[2]a

ultco/detail.action?docID=3017830.

Table 2. (Continued)

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Metabolite 5-(hydroxymethoxyphenyl)-γ-valerolactone sulfates protocatechuic acid vanillic acid 4-hydroxybenzoic acid 3-hydroxybenzoic acid 4-hydroxyhippuric acid 3-hydroxyhippuric acid p-coumaric acid caffeic acid ferulic acid m-coumaric acid 3,4-dihydroxyphenylacetic acid 3-methoxy-4-hydroxyphenylacetic acid 3-hydroxyphenylacetic acid phenylacetic acid 3,4-dihydroxyphenylpropionic acid 3-hydroxyphenylpropionic acid 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone gallic acid 4-methylgallic acid 3-methylgallic acid 1,3,5-trimethoxybenzene benzoic acid 3-(4-hydroxyphenyl) propionic acid 3,4-dihydroxyphenylpropionic acid 3,4-dihydroxybenzoic acid vanillic acid ferulic acid 4-ethylphenol 3-phenylpropionic acid 2-methoxyphenylacetic acid

Nº Subjects

42 humans

[Continued]

12 humans

Source d

Urine c

Sulf_ 1:Medium; Sulf_2: Low Medium Medium* Medium Low 40 g/day cocoa Medium powder with 250 mL skimmed milk High Low Treatment period: Low 4 weeks Low Low Low Medium Medium* Medium [Continued] Low Low Low* a) --; b)Low* a) --; b)Low* a) --; b)Medium * a)High*; b) High* 200 mg of the pure --flavonoids: (a) epicatechin -and (b) (-)-EGCG -------

Plasma c

Referenc e

ND Medium Medium Medium ND Low Low Low Low Low ND Low ND Low* Medium Low Low Low* a) Low*; b)Low* a) Low*; b)Low* a) Low*; b)Low* a) High*; b)High* a) High; b) High a) Medium; b) Medium a) Medium; b) Medium a) Low; b) Low a) Low; b) Low a) Low; b) Low a) Medium; b)High a) Low; b) Medium a) Medium; b) High

[7]b

[7]b

[32]b

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Metabolite 4-ethylbenzoic acid protocatechuic acid gallic acid vanillic acid 4-hydroxybenzoic acid syringic acid benzoic acid 3-hydroxybenzoic acid 2,4-dihydroxybenzoic acid 3,5-dihydroxybenzoic acid salicyluric acid hippuric acid ferulic acid 3,4-dihydroxyphenylacetic acid 4-hydroxyphenylacetic acid homovanillic acid 3-hydroxyphenylacetic acid 2-hydroxyphenylacetic acid phenylacetic acid mandelic acid 3-hydroxybenzoic acid 4-hydroxybenzoic acid 4-hydroxyhippuric acid hippuric acid vanillic acid ferulic acid 3,4-dihydroxyphenylacetic acid 3-hydroxyphenylacetic acid Phenylacetic acid 3,4-dihydroxyphenylpropionic acid 3-hydroxyphenylpropionic acid

Nº Subjects

20 humans

11 humans

[Continued]

protocatechuic acid 21 humans vanillic acid

Source d

Urine c -Low Low* Medium Low Low Low Medium * Low 4 g of black tea solids Low Treatment period: Low* 1 week High * Low Low* Medium Medium Medium * Low Low Low Low 80 g chocolate Medium Medium High High * High * Medium * [Continued] High * Medium * Low Low* 40 g cocoa powder a) Medium*; b) Medium* dissolved in 250 ml (a) water or (b) a) Medium*; b) whole milk Medium*

Plasma c a) Medium; b) High -------------------------------

Reference

[33]b

[34]b

[34]b

-[3]b --

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Table 2. (Continued) Metabolite 4-hydroxybenzoic acid 3-hydroxybenzoic acid 4-hydroxyhippuric acid hippuric acid p-coumaric acid caffeic acid ferulic acid m-coumaric acid 3,4-dihydroxyphenylacetic acid

Nº Subjects

Source d

3-methoxy-4-hydroxyphenylacetic acid 3-hydroxyphenylacetic acid phenylacetic acid

[Continued]

[Continued]

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3,4-dihydroxyphenylpropionic acid 4-hydroxyphenylacetic acid vanillic acid hippuric acid 3-hydroxyphenylacetic acid 3-hydroxyhippuric acid 4-hydroxyhippuric acid homovanillic acid 3-(3.hydroxyphenyl)-hydroxypropionic acid syringic acid 3-(3-Hydroxyphenyl) propionic acid phenylacetic acid 4-hydroxybenzoic acid 4-hydroxymandelic acid pyrocatechol pyrogallol 4-hydroxybenzoic acid 5-(3´,4´,5´-trihydroxyphenyl)-γ-valerolactone

26 humans

Cellulose capsules with a polyphenol-rich mix of red wine and red grape juice extracts Treatment period: 4 weeks

5 humans

300 mL of green tea

Urine c a) Low*; b) Low* a) Low*; b) Low a) Low*; b) Low* a) High*; b) High* a) Low*; b) Low* a) Low*; b) Low a) Medium *; b) Low a) Low; b) Low a) Low*; b) Low a) Medium*; b) Medium* a) Medium; b) Medium a) High*; b) High* a) Medium*; b) Medium* Identified Identified Identified Identified Identified Identified Identified Identified Identified Identified --Identified Low*

Plasma c ----------

Medium*

--

a) Medium*; b) Medium* a) Medium; b) Medium

Reference

----

[3]b

-----------Identified Identified ----

[35]a

[36]b

--

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Metabolite 4-hydroxyphenylacetic acid 3-methoxy-4-hydroxyphenylacetic acid hippuric acid 3-(3-hydroxyphenyl)-3-hydroxypropionic acid 5-(3´,5´-dihydroxyphenyl)-γ-valerolactone 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone 5-(3´,4´,5´-trihydroxyphenyl)-γ-valerolactone 5-(3´,4´,5´-trihydroxyphenyl)-γ-valerolactone 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone 5-(3´,5´-dihydroxyphenyl)-γ-valerolactone 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone glucuronide 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone sulfate 5-(3´,4´,5´-trihydroxyphenyl)-γ-valerolactone 5-(3´,4´,-dihydroxyphenyl) valerolactone 5-(3´,5´,-dihydroxyphenyl)-valerolactone 5-(3´,5´-dihydroxyphenyl)-γ-valerolactone glucuronide 5-(3´,4´,5´-trihydroxyphenyl)-γ-valerolactone sulfate 5-(4´-methoxy-3´,5´-dihydroxyphenyl) valerolactone sulfate 4´-O-methyl-5-(3´,4´,5´-trihydroxyphenyl)-γvalerolactone-methyl-sulfate-3´-sulfate 5-(3´,5´-dihydroxyphenyl)-γ-valerolactone sulfate 5-(3´,5´-dihydroxyphenyl)-γ-valerolactone sulfate 5-(3´,4´,5´-trihydroxyphenyl)-γ-valerolactone glucuronide 5-(3´,5´-dihydroxyphenyl)-γ-valerolactone glucuronide

Nº Subjects

Source d

[Continued]

[Continued]

4 humans

20 mg/kg green tea solids

1 humans

200 mg of pure ()-EGCG, (-)-EGC or (-)-epicatechin

3 men

Urine c High Medium* High Medium* High High Low High Medium Medium

Plasma c ----Low Identified Identified ----

Identified

--

Identified 200 ml reconstituted green Identified tea (from 3 g tea Identified solids) Identified

--

Identified Identified

[Continued]

[Continued]

-----

Reference [36]b

[37]a [37]a

[38]a

--

Identified

--

Identified

--

Identified

--

Identified

--

Identified

--

Identified

--

[38]a

a

Study with no statistical analysis. Study with statistical analysis. * Compound that significantly increased after the intervention Concentrations categorized in low, medium and high (classified within a same study in percentiles). When no category is given, metabolites are not quantified. d If treatment period is not described, the study was carried out with a single dose of polyphenols or food source. (-)-EGCG, (-)-Epigallocatechin gallate, (-)-EGC, (-)-Epigallocatechin. b c

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Principal Phenolic Acids Found in Plasma and Urine The phenolic acids present in biofluids after consumption of flavan-3-ols depend on several factors, such as food source, food matrix and the compounds that are delivered in these foods. The phenolic acid concentration in plasma or urine after consumption of food rich in flavan-3-ols or pure compounds is shown in Tables 2, 3 and 4.

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Human Studies Sources of flavan-3-ol that have been used in humans studies include cocoa powder, chocolate, almond skins, black and green tea, and red wine and red grape juice. In addition, ()-epicatechin, (-)-epigallocatechin, and (-)-epigallocatechin-3-O-gallate have also been tested (Table 2). After intake of cocoa powder with milk, main phenolic acids identified in plasma were: protocatechuic, vanillic, 4-hydroxybenzoic, and phenylacetic acids. Besides these metabolites, 4-hydroxyhippuric, 3-hydroxyhippuric, 4-hydroxy-3-methoxyphenylacetic, and 3-hydroxy-phenylacetic acids, most of them arising from further liver/kidney metabolism after absorption from the colonocytes acid were also found in urine in medium concentration range, with the exception of 3-hydroxyhippuric acid which was present in high concentration [7]. Only 3-hydroxyphenylacetic and vanillic acids were significantly changed in urine and plasma after the intake of cocoa powder for one month [7]. Similarly, Rios et al. found that vanillic together with ferulic and 3-hydroxy-phenylacetic acids, as one of the main phenolic acids in urine significantly changing after a single intake of chocolate in humans. 3,4-Dihydroxyphenylacetic and phenylacetic acids (found in the medium concentration range), and 3-hydroxyphenylpropionic acid (found in the low concentration range), were also characteristic phenolic acids of the intake of chocolate flavan3-ols [34]. However, differences in the profile of the urinary excretion of phenolic acids were found when cocoa was consumed with water instead of milk, indicating that the food matrix has a profound influence in microbial metabolism of flavan-3-ols [3]. After a single intake of almond skin polyphenols, main phenolic acids identified in plasma were: protocatechuic, vanillic, 4-hydroxybenzoic, phenylacetic, 3,4dihydroxyphenylpropionic, and 3-hydroxyphenylpropionic acids. In urine, main metabolites include those derived from subsequent hepatic metabolism in the form of methoxylated, sulfated or glycinated derivates, including: 4-hydroxyhippuric and 4-hydroxy-3methoxyphenyl-acetic acids. In the case of red wine and red grape juice, other phenolic acids such as 4-hydroxymandelic acid were also identified by Grün et al. [35]. The profile of phenolic acids was slightly different after the intake of pure (-)-epicatechin or (-)-epigallocatechin-3-O-gallate, since 1,3,5-trimethoxybenzene was detected in the highest concentration in both plasma and urine. In addition to this metabolite, gallic, 4-methyl-gallic, 3-methyl-gallic acids, derived from the hydrolysis of the gallic acid moiety from (-)epigallocatechin-3-O-gallate, significantly changed after consumption of this compound in both type of biological samples [32].

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Gallic acid was also found as one of the phenolic acids significantly changing after consumption of black tea solids for 1 week [33]. Other metabolites showing significant differences included the following ones, which were found in different concentration ranges: high (hippuric acid); medium (3-hydroxybenzoic and 3-hydroxyphenylacetic acids), and low (salicyluric and 3,4-dihydroxyphenylacetic acids) [33]. Pyrogallol and pyrocatechol, derived from the further decarboxylation and dehydroxylation of gallic acid, are among other metabolites derived from galloylated flavan3-ols of tea [36].

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Animal Studies For animal studies, diets supplemented with (+)-catechin, (-)-epicatechin-3-O-gallate, procyanidin B3 and C2, polymeric flavan-3-ol, and wine powder have been used (Table 3 and Table 4). The influence of the degree of polymerization (DP) on microbial phenolic acid was studied by Gonthier et al. [28]. Although some metabolites were found in the same concentration range in rats fed either (+)-catechin, dimers, trimeric or polymers, changes for some metabolites such as protocatechuic, vanillic, 4-hydroxybenzoic, 3-hydroxybenzoic, 4hydroxyhippuric, and 3-hydroxyhippuric acids were only significant after the intake of (+)catechin and dimeric procyanidins, indicating an influence of polymeric proanthocyanidins in the extent of microbial degradation. Other metabolites such as 3,4-dihydroxyphenylacetic, 3-hydroxyphenylacetic, and 3,4dihydroxyphenylpropionic acids only appear to change after the intake of procyanidin B3, whereas others (m-coumaric, 3-hydroxyphenylpropionic, and 3-hydroxyphenyl valeric acids) significantly changed independently of the DP [39]. The influence of different doses (low and high) of red wine powder in comparison to (+)catechin in the formation of microbial phenolic acids was also studied in rats. The urinary levels of some metabolites such as vanillic and p-coumaric acids appeared to significantly increase after the intake of the high dose of red wine powder, whereas other including 3hydroxy-benzoic, 3-hydroxy-hippuric and 3-hydroxyphenyl-propionic acids tended to increase after the intake of (+)-catechin, indicating that not only the dose but the flavan-3-ols source and structural features influence the catabolism of these compounds by gut microbiota [28]. In addition to all the phenolic acids described above, phenyl--valerolactones and 4hydroxy-5-(phenyl)-valeric acid derivatives are considered as characteristic metabolites derived from flavan-3-ols. Although the quantification of these compounds has been limited by the lack of the appropriate standard, both humans and animal studies have a allowed great advances in the identification of tri-, di- and monohydroxylated forms of phenyl--valerolactones with different conjugation profile (glucuronidated, sulfated and methoxylated forms) after the intake of flavan-3-ols rich sources such as cocoa, almond skins, and green tea, or after supplementation of purified compounds such as (+)-catechin and (-)-epicatechin-3-O-gallate [2, 26, 31, 36-38, 40].

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Table 3. Degree of excretion and presence in urine and plasma of main metabolites derived from microbial metabolism of flavan-3-ol in animal studies Metabolite protocatechuic acid vanillic acid

Nº Subjects

Source d

4-hydroxybenzoic acid 3-hydroxybenzoic acid 4-hydroxyhippuric acid 3-hydroxyhippuric acid hippuric acid p-coumaric acid ferulic acid m-coumaric acid 3,4-dihydroxyphenylacetic acid 3-hydroxyphenylacetic acid 3,4-dihydroxyphenylpropionic acid

25 male Wistar rats

0.1% (w/w) catechin diet (a) procyanidin dimer B3 diet (b) trimer C2 diet (c) polymer diet (d) Treatment period: 5 days

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3-hydroxyphenylpropionic acid 3,4-dihydroxyphenylvaleric acid 3-hydroxyphenylvaleric acid vanillic acid 4-hydroxybenzoic acid 3-hydroxybenzoic acid 4-hydroxyhippuric acid 3-hydroxyhippuric acid hippuric acid p-coumaric acid caffeic acid ferulic acid 3,4-dihydroxyphenylacetic acid 3-hydroxyphenylacetic acid phenylacetic acid 3,4-dihydroxyphenyl-propionic acid 3-hydroxyphenyl-propionic acid 5-(4-hydroxy)-3-hydroxyphenylvaleric acid

32 male Wistar rats

(a)0.25 g/100 g wine powder, (b) 0.50 g/100 g wine powder, (c) 0.12 g/100 g catechin Treatment period: 8 days

Urine c Reference a) Low*; b) Low*; c) Low ;d) Low a) Medium*; b) Medium; c) Medium; d) Medium a) Medium*;b) Medium*; c) Medium; d) Medium a) Low *;b) Low*;c, d) ND a) Medium; b) Medium*; c) Medium; d) Medium a) Medium *;b) Low*;c, d) ND a) High; b) High; c) High; d) High a) Low; b) Low; c) Low ;d) Low [39]b a ) Low; b,c,d) ND a) Medium*; b) Medium*; c) Low*; d) Low* a) Low; b) Medium*; c) Low; d) Low a) Low; b) Medium*; c) Medium; d) Medium a) Low; b) Low*; c,d, e: ND a) Medium*;b) Medium*;c) Medium*;d)medium* a) Low*;b,c,d,) ND a) Low*; b) Low*; c) Low* ;d) ND a) Medium*; b) High*; c) Medium a) Medium*; b) Medium*; c) Medium* a) Low*; b) Low*; c) Medium* a) Medium; b) Medium; c) Medium a) Low*; b) Low*; c) Medium* a) High*; b) High*; c) High a) Low*; b) Medium*; c) Low a) Low*; b) Low*; c) ND [28]b a) Low*; b) Low*; c) Low* a) Low; b) Low*; c) Low* a) Medium*; b) Medium*; c) Low a) High; b) High; c) High a) Low*; b) Low*; c) Low* a) Medium*; b) Medium*; c) High* Identified

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Metabolite

Nº Subjects

3-hydroxyphenylvaleric acid

Source d hyperlipidemic with catechin supplementation

Urine c

Reference

Identified

Treatment period: 6 weeks hippuric acid 4-hydroxy-hippuric acid methoxy-hydroxyphenyl valerolactone dihydroxyphenyl valerolactone glucuronide methoxy-hydroxyphenyl valerolactone glucuronide methoxy-hydroxyphenyl valerolactonesulfate m-coumaric acid 3-(3-hydroxyphenyl)-propionic acid 4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid 5-(3´,4´,-dihydroxyphenyl)-valerolactone

48 male Wistar rats

Normolipidemic or hyperlipidemic with catechin supplementation Treatment period:6 weeks

Identified Identified Identified Identified

[40]a

Identified Identified

6 male Wistar rats

100 mg/kg of body weight (-)-ECG

Free and conjugated: high Free and conjugated: high

[26]a

Free and conjugated: medium Free and conjugated: medium

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a

Study with no statistical analysis. b Study with statistical analysis. * Compound that significantly increased after the intervention. c Concentrations categorized in low, medium and high (classified within a same study in percentiles). When no category is given, metabolites are not quantified. d If treatment period is not described, the study was carried out with a single dose of polyphenols or food source. (-)-ECG, (-)-Epicatechingallate, ND: not detected.

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Table 4. Degree of excretion and presence in plasma of main metabolites derived from microbial metabolism of flavan-3-ol in animal studies Metabolite

Nº Subjects

hippuric acid

32 male Wistar rats

Source d 0.25 g-0.50 /100 g wine powder or 0.12 g/100 g catechin

Plasma c

Main

Reference

[28]b

Treatment period: 8 days 3-(3-hydroxyphenyl) propionic acid 4-hydroxy-5-(3,4dihydroxyphenyl) valeric acid 5-(3´,4´,-dihydroxyphenyl) valerolactone

Free and conjugated: high 6 male Wistar rats

100 mg/kg of body weight (-)-ECG

Free and conjugated: medium

[26]b

Free and conjugated: high

a

Study with no statistical analysis. Study with statistical analysis. * Compound that significantly increased after the intervention c Concentrations categorized in low, medium and high (classified within a same study in percentiles). When no category is given metabolites are not quantified. d If treatment period is not described, the study was carried out with a single dose of polyphenols or food source. (-)-ECG, (-)-Epicatechingallate, ND: not detected.

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b

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165

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Intestinal Microbiota Involved in the Metabolism of Flavan-3-ols The differences in microbial metabolites found in samples from in vitro fermentation studies or after flavan-3-ol ingestion could be partly attributed to differences in the individual microbiota, suggesting that metabolites and pathways could be different, depending on the microbiota composition [6]. Few studies have analyzed, identified and characterized bacteria with the ability to catabolize flavan-3-ols and, therefore, the bacteria involved in the catabolism of flavan-3-ols remains largely unknown. The inhibitory effects of proanthocyanidins and structural features of flavan-3-ols as complex non-planar molecules could explain the difficulties in establishing specific bacteria [9]. Most of the findings made in this field come from in vitro studies with selected bacteria from either the human gut or fecal suspensions [27, 41-43]. Eubacterium spp strain SDG-2 was found to be able to cleave the C-ring of (3R)- and (3S)-flavan-3-ols to give 1,3diphenylpropan-2-ol derivatives, as well as to produce p-dehydroxylation reactions in the Bring of (3R)-flavan-3-ols, after incubation with (-)-epicatechin, (-)-catechin, (-)epigallocatechin and (-)-gallocatechin. The same bacteria were also found to be able to metabolize other related compounds such as taxifolin to caffeic acid and mhydroxyphenylpropionic acid [44]. In a recent study, the intestinal human bacteria involved in the conversion of catechins were isolated and characterized as Eggerthella lenta and Flavonifractor plautii (formerly Clostridium orbiscindens). Eggerthella lenta rK3 reductively cleaved the heterocyclic C-ring of both (-)-epicatechin and (+)-catechin, giving rise to 1-(3,4-dihydroxyphenyl)-3-(2,4,6trihydroxyphenyl) propan-2-ol, and Flavonifractor plautii aK2 further converted the former metabolite into 5-(3,4-dihydroxyphenyl)-γ-valerolactone and 4-hydroxy-5-(3,4dihydroxyphenyl) valeric acid [8]. It was suggested that individual species catalyze only single steps in the degradation pathway, which implies an even more complex situation in metabolites such as procyanidins or other polymers.

Bioactivity of Phenolic Acids There are few studies in humans linking phenolic acids from microbial metabolites with health effects after in vivo consumption of flavan-3-ols, instead most of them have been carried out on cell cultures or animal models. To get a complete understanding of the role of microbiota in the release of phenolic acids and bioactivity, further studies are needed.

Effects of Phenolic Acids on Gut Microbiota Phenolic acids released to the medium by microbial metabolism in colon may exert a protective effect by regulation of the microbial species. Prebiotic actions have been demonstrated in vitro [41], in which incubation of (-)-epicatechin or (+)-catechin with fecal bacteria led to the generation of 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone, 5-phenyl-γvalerolactone and 3-phenylpropionic acid.

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The growth of selected microflora was affected, resulting in an increase in the growth of the Clostridium coccoides–Eubacterium rectale group, Bifidobacterium spp. and Escherichia coli, as well as a significant inhibitory effect on the growth of the C. histolyticum group, more significantly in the case of catechin than epicatechin [41]. Similarly, it has been reported that phenolic acids produced from the microbial degradation of flavan-3-ols such as 3-O-methyl gallic, gallic, caffeic, 4-hydroxyphenylpropionic, phenylpropionic and 4-hydroxyphenylacetic acids, were able to protect against the growth of several pathogenic and non-beneficial intestinal bacteria, including Clostridium perfringens, Clostridium difficile and Bacteroides spp., but to a lesser extent affected the growth of some beneficial bacteria such as Lactobacillus spp. and Bifidobacterium spp [45]. The tentative mechanisms that will explain their antimicrobial activity for flavanols have been elucidated as membrane interaction, enzyme inhibition, reactive oxygen generation and inhibition of virulence factors [21]. A link between the number and position of substitutions in the benzene ring of the phenolic acids and the saturated side-chain length and the antimicrobial activity was also described, and they were more active that their corresponding precursors. Changes in intestinal bacteria and pathogen growth can be affected by phenolic acids from the microbial degradation of dietary phenolic compounds by affecting the epithelium at the site of conversion and may also affect colonic microbiota locally [46]. In this line, an effect on cell surface structures was observed when incubating with berry extracts inhibited the growth of Gram-negative but not Gram-positive bacteria, weakening Salmonella, which may be specifically related to dihydroxylated forms [47, 48]. Evidence related to the in vivo effects of polyphenols on the intestinal microbiota is still scarce. First studies conducted in both humans and animals (pigs and chickens) have revealed an increase in Lactobacillus and a decrease in Enterabacteriaceae after the administration of monomeric flavan-3-ols from green tea [49]. Smith et al. later also showed that animals rats feed with a tannin-rich diet significantly decreased the Clostridium leptum cluster and increased the growth Bacteroides group [50]. Similarly, Dolara et al. found that rats feed with red wine polyphenols had significantly lower levels of Clostridium spp, and higher levels of Lactobacillus spp [51]. More recently, a significant increase in the numbers of Bifidobacteria and/or Lactobacillos together with a significant decrease in the numbers of Bacteriodes and Clostridium have been reported in rat fed blackcurrant extract powder [52]. Viveros et al. also found that birds fed grape pomace concentrate and grape seed extract had higher populations of Escherichia coli, Lactobacillus, Enterococcus, and Clostridium in the cecal digesta than the control group [53].

Anti-Inflammatory Activity Larrosa et al. recently proved that the polyphenol metabolites, hydrocaffeic, dihydroxyphenyl acetic and hydroferulic acid, derived from colon microbiota, provided the best inhibition of prostaglandin E2 production in cancer cells of fibroblast (CCD-18) stimulated with IL-1. The same metabolites had an anti-inflammatory effect in rodents. The effect of hydrocaffeic acid was investigated in in vivo experiments with rats using a dextran sodium sulfate (DSS)-induced colitis model, and it was found to be the most potent metabolite for reducing the expression of cytokines TNF-, IL-1, IL-8, as well as the levels of malonaldehyde and oxidative damage to DNA in the distal colon mucosa [54].In an in vitro

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study the ability of the compounds to inhibit the release of arachidonic acid and the production of nitric oxide (NO) by lipopolysaccharide (LPS)-stimulated murine macrophages was evaluated with (-)-5-(3´,4´,5´-trihydroxyphenyl)-γ-valerolactone, which inhibited NO production by 50% at 20 μM [55].Other studies involving inflammatory molecules showed that phenolic acids presenting 4-hydroxy-3-methoxy substitution and a one-carbon side chain (vanillic acid and its derivatives) and three-carbon side chain (cinnamic, o-, m- and pcoumaric acid, and caffeic acid) inhibited cytokine-induced prostanoid biogenesis in human colonic fibroblasts by up to 81% in the case of vanillin and 75% in the case of coniferyl alcohol [56], and it was also shown that dihydroxylated phenolic acids incubated peripheral blood mononuclear cells (PBMC) stimulated with LPS, significantly inhibiting the production of pro-inflammatory cytokines TNF-, IL-1, IL-6, but none are affected by monohydroxylated phenolic acids [57].

Effects of Phenolic Acids on Anti-Thrombotic Activity Rechner et al. demonstrated the anti-trombotic activity of phenolic acids using a mixture of polyphenols and some derived microbial metabolism in different tests related to platelet aggregation, P-selectin expression on resting platelets, the effect on TRAP-induced platelet activation and epinephrine-stressed platelets. In those tests the most significant activity was performed by dihydrocaffeic acid (3,4-dihydroxyphenyl propionic acid), dihydroferulic acid (4-hydroxy-3-methoxyphenylpropionic acid) and 3-hydroxyphenylpropionic acid [58].

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Anti-Proliferative Activity, Cytotoxicity and Inhibition of Specific Enzymes (-)-5-(3´,4´,5´-trihydroxyphenyl)-γ-valerolactone and (-)-5-(3´,4´-dihydroxyphenyl)-γvalerolactone and their methoxy-derivatives were first assessed for their ability to inhibit the growth of a panel of immortalized and malignant human cell lines, the former being the more active component for human esophageal squamous cell carcinoma cells (KYSE150), human colon adenocarcinoma cells (HT-29 and HCT-116), immortalized human intestinal epithelial cells (INT-407) and an immortalized rat intestinal epithelial cell line (IEC-6) [55]. Karlsson et al. demonstrated that fecal samples containing microbial phenolic acids inhibited cyclooxygenase-2 (COX-2) protein levels in colon cancer cells (HT-29) induced with TNF-α in a range between 14.7±15% and 67 ± 6% depending on the concentration and dose [59]. Similar effects have been proved for 3,4-dihydroxyphenylacetic acid and 3-(3,4dihydroxyphenyl)-propionic acid in human adenoma cells LT97 for which a reduction in COX-2 gene and protein expression has been observed [60] .In another study in which LNCaP prostate cell line, HCT116 colonic cell line, and IEC6 normal intestinal epithelial cell line were incubated with phenolic acids from human microbial fermentations, 3,4dihydroxyphenylacetic acid presented anti-proliferative activity of in the two former cell lines [61]. Protocatechuic acid is a very active metabolite and its cytotoxic capacity has been tested on HepG2 hepatocellular carcinoma cells by stimulating the c-Jun N-terminal kinase (JNK) and p38 subgroups of the mitogen-activated protein kinase (MAPK) family [62]. Similar pathways haven been proved in human gastric adenocarcinoma cells b [63].Finally, the

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María Boto-Ordóñez, Mireia Urpi-Sarda, María Monagas et al.

neuroprotective effects of protocatechuic acid on retenone- induced apoptosis in PC12 cells by improving apoptosis/necrosis have also been described [64].

Other Effects Due to their phenolic nature, phenolic acids are related to other effects such as oxidative stress, antioxidant activity and modulation of lipid metabolism [9]. 3,4-Dihydroxyphenyl acetic is linked to oxidative stress [65, 66] and antioxidant activity, acting as a radical scavenging activity against DPPH in cultured rat hepatocytes [67]. Unno et al. proved that antioxidant potential measured by TEAC, of epicatechin and its bacterial metabolite 5-(3´,4´dihydroxyphenyl)-γ-valerolactone in comparison with L-ascorbic acid. (-)-Epicatechin showed almost double the capacity, of 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone, however the latter one showed stronger antioxidant activity than L-ascorbic acid [68].

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Conclusions Although the health benefits of flavan-3-ols have been described in numerous intervention studies, the direct relationship between the flavan-3-ol bioavailability and activities such as antioxidant, anticarcinogen, cardiopreventive, antimicrobial or neuroprotective has not yet been fully established in vivo. Over the past years, the recognition that the microbiota plays a fundamental role in the bioavailability of flavan-3-ols, has led to the consideration that microbial phenolic acids may be partly responsible for their in vivo health effects. Although great advances have been made in the determination of metabolic pathways and identification of phenolic acids in biological samples, understanding the role of these microbial metabolites in the health benefits derived from flavan-3-ols is still a very difficult task. Considering the interaction existing between flavan-3-ols and gut microbiota, more interest should be focused on microbiota-related diseases affecting the host immune system, such intestinal inflammatory diseases and infections, obesity and allergies, among others. In order to obtain greater knowledge in this field, the link between bacterial composition, phenolic acids and health benefits should be made by combining the complex information from human intervention studies with that from animals and in vitro mechanistic studies. These studies would reveal specific bacterial groups and phylum involved in the catabolism of flavan-3-ol as well as new potential bioactive microbial metabolites that could then be correlated with in vivo health effects. All this information will finally provide the means to adopt new nutritional strategies to increase the bioavailability and health effects of flavan-3-ols, for example, by redirecting or shifting the microbiota towards a more active microbial ecosystem.

Acknowledgments This work has been funded by AGL2006-14228-C03-01/02, AGL2009-13906-C02-01 and AGL2010-10084-E, CONSOLIDER INGENIO 2010 Programme and FUN-C-FOOD

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(CSD2007-063) from the Ministry of Science and Innovation (MICINN) of Spain and The CIBEROBN, an initiative of the Instituto de Salud Carlos III. MBO would like to thank the FPU 2009 predoctoral fellowship from the Spanish Ministry of Education, MUS to the postdoctoral program Sara Borrell CD09/00134, ST to the postdoctoral fellowship for the mobility of foreign researchers to Spain from the Ministry of Science and Innovation, R.Ll. thanks to the Ramon y Cajal program from the Ministry of Science and Innovation and Fondo Social Europeo and MGA the predoctoral FI-DGR fellowship from the Generalitat de Catalunya’s Agency for Management of University and Research Grants (AGAUR).

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In: Phenolic Acids Editor: Sergi Munné-Bosch

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Chapter IX

The Potential Role of Phenolic Acids in Tea and Herbal Teas in Modulating Effects of Obesity and Diabetes

1

E. Joubert1,2*, C. J. F. Muller3, D. De Beer1, R. Johnson3, N. Chellan3 and J. Louw3

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Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC), Infruitec-Nietvoorbij, Stellenbosch, South Africa 2 Department of Food Science, Stellenbosch University, Stellenbosch, South Africa 3 Diabetes Discovery Platform, Medical Research Council (MRC), Cape Town, South Africa

Abstract Obesity and diabetes have reached global epidemic proportions both in the developed and developing world. Major contributing factors are a sedentary lifestyle and diets rich in saturated fats and sugar. This chapter will provide general background on obesity and diabetes in terms of their prevalence and burden to society, the link between insulin resistance, obesity and type 2 diabetes and the use of natural products as alternatives to pharmaceutical products as background to current evidence indicating that phenolic acids may play a preventative and protective role against risk factors associated with obesity and diabetes. Phenolic acids, shown to have a beneficial effect on carbohydrate digestion, glucose absorption and metabolism, insulin secretion and lipid metabolism, amongst others, are present in plant foods and beverages consumed as part of the daily diet. The focus will fall on green and black teas (Camellia sinensis), the South American herbal tea, mate (Ilex paraguariensis), German chamomile (Matricaria chamomilla) and the South African herbal teas, rooibos (Aspalathus linearis) and bush tea (Athrixia phylicoides) as sources of phenolic acids, and specifically gallic acid, ferulic acid and chlorogenic acid. Insight will be provided into their intake through ingestion of

*

E-mail addresses: [email protected]; [email protected]; [email protected]; Rabia.Johnson@ mrc.ac.za; [email protected]; [email protected].

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E. Joubert, C. J. F. Muller, D. De Beer et al. these teas, as well as their bioavailability and mechanisms of action associated with antiobesity and anti-diabetic effects.

Introduction

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The health-promoting properties of tea, especially green tea, and herbal teas have received much attention in recent years due to the potential cardio-protective and anti-cancer effects of the antioxidants that they contain. The focus fell largely on flavonoids as the bioactive phytochemicals responsible for the therapeutic effects of these beverages [1-8]. There is a growing body of scientific evidence showing the potential of polyphenols in the prevention of and protection against risk factors associated with obesity and diabetes [9-11]. However, the pharmacological actions of phenolic antioxidants are not limited to their free radical scavenging and metal chelating properties. Their modulatory role in cell signalling cascades [12] and gene expression [13,14] underscores their potential preventative and protective roles against cardiovascular complications, cancer and diabetes. Only low concentrations are required to affect cell signalling cascades compared to those needed for an antioxidant effect in the cell [15]. Minor phenolic acid constituents in teas and herbal teas, especially when consumed regularly as part of the diet, could therefore become important in ameliorating the effects of obesity and diabetes. The potential benefits of these phenolic compounds for moderating risk factors associated with obesity and diabetes are the focus of this chapter. Furthermore, phenolic acids could potentially exert a synergistic effect in combination with the commercial anti-diabetic drug, metformin, leading to lower doses and thus reducing the side effects and toxicity associated with this drug [16].

Obesity The term obesity is derived from the Latin ‘obesus’ which means fat or plump and/or the Greek ‘ob-edere’ meaning over-eating. In essence it is true that no-one becomes obese in times of famine. However, the causes of obesity are complex and it is an over-simplification to attribute it solely to gluttony [17]. Obesity is defined using the body mass index (BMI), which reflects body weight (BW) in kilograms relative to height in meters squared. A BMI of 18.5 to 24.9 is regarded as normal, 25 – 29.9 as over-weight and above 30 as obese [18,19]. Obesity has become one of the greatest public health challenges of the 21st century as reported by the World Health Organisation (WHO) [19]. It is estimated that globally over 1.5 billion individuals are overweight, of which 200 million men, 300 million women and 43 million children under the age of five are obese. These figures are expected to increase to 2.3 billion overweight adults, of which over 700 million could be obese by 2015 if the current trends continue. The dramatic increase in the incidence of obesity, particularly amongst children in the developed and developing world, is associated with economic prosperity, diet, urbanisation and lifestyle changes. The ever-increasing availability of refined foods with high fat and sugar content and a sedentary lifestyle are the predominant factors contributing to the epidemic

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[20]. Other complicating factors relating to obesity and unhealthy eating habits include emotional disposition, stress, depression, boredom, smoking and socio-economic factors [21]. Obesity is associated with a reduction in antioxidant status, which together with chronic inflammation increases the risk of developing chronic conditions such as hypertension, gout, rheumatism, arthritis, dyslipidaemia, insulin resistance, thrombotic tendencies and coronary artery disease. The risk of developing cardiovascular disease is increased by 23%, type 2 diabetes (T2D) by 44% and certain types of cancer by between 7 – 41%. WHO estimates that 2.8 million adults die each year from being overweight or obese [19,22].

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Diabetes Diabetes is a complex, multi-systemic, chronic disease caused by inherited and/or acquired deficiency in the production of insulin by the pancreas, or by the ineffectiveness of the insulin produced. Such a deficiency results in increased concentrations of glucose and lipids in the blood, which in turn damage many of the body's systems, in particular the blood vessels and nerves. There are two principal forms of diabetes mellitus. With type 1 diabetes (formerly known as insulin-dependent diabetes) the pancreas fails to produce insulin which is essential for survival. This form develops most frequently in children and adolescents. T2D (formerly named non-insulin-dependent diabetes) results from the body's inability to respond properly to insulin produced by the pancreas. This type of diabetes is much more common and accounts for around 90% of all diabetes cases worldwide. It occurs most frequently in adults, but is being noted increasingly in adolescents as well [23-25]. In T2D, lifestyle rather than genetic disposition is a major contributor to the development of the disease. As with obesity, diet, especially during the formative years, could predispose the progeny to developing T2D in later life [26]. Recently, some genes have been consistently associated with increased risk for T2D in certain populations. However, both types of diabetes are complex diseases caused by mutations in more than one gene, as well as by environmental factors [27]. The prevalence of diabetes has reached epidemic proportions. WHO predicts that developing countries will bear the brunt of this epidemic in the 21st century. An estimated 285 million people, corresponding to 6.4% of the world's adult population, will live with diabetes in 2010. The number is expected to grow to 438 million by 2030, corresponding to 7.8% of the adult population. Much of the forecasted increase in diabetes is likely to be in developing countries due to population growth, ageing, increased urbanisation, unhealthy diets, obesity and sedentary lifestyles [23,25]. Diabetes has become a major cause of premature illness and death in most countries, mainly through the increased risk of cardiovascular disease. Each year, diabetes-related causes account for 3.8 million deaths; equivalent to one death every ten seconds and claiming the lives of as many people as HIV/AIDS [25,28]. The long-term complications of the disease are the major causes of morbidity and mortality. These include retinopathy leading to blindness, nephropathy leading to end-stage renal disease, neuropathy leading to lower extremity amputations and cardiovascular disease leading to heart attacks and stroke [24,25,28-31].

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Link between Insulin Resistance, Obesity and T2D The link between obesity and the development of T2D revolves around insulin resistance and the ability of the pancreatic β-cells to compensate for the resultant increased insulin required to maintain normoglycaemia. In obese individuals the abundance of adipose tissue contributes directly to the loss of insulin sensitivity particularly in the liver, muscle and adipose tissue. Adipose tissue affects insulin signalling by releasing non-esterified fatty acids (NEFAs), metabolically active hormones, as well as pro-inflammatory cytokines [26,32,33]. In obese and T2D individuals the increased NEFA plasma concentrations are responsible for aggravating the insulin resistance associated with these conditions [26]. At the cellular level, insulin resistance is associated with increased accumulation of intramuscular fatty acids [34,35]. Increased serum free fatty acids (FFAs) are associated with hepatic insulin resistance and increased accumulation of intracellular fatty acid metabolites including diacylglycerol. Diacylglycerol is a potent allosteric activator of protein kinase C (PKC) that increases the phosphorylation of the serine residues while simultaneously decreasing tyrosine phosphorylation of the insulin receptor substrate-1. Subsequent attenuation of insulin signalling via phosphatidylinositol 3 kinases (PI3K) results in insulin resistance [36,37]. Retinol-binding protein-4 (RBP-4), a protein secreted by adipocytes, is increased in obese mice and humans. RBP-4 causes insulin resistance by inhibiting PI3K signalling in skeletal muscle and stimulates hepatic gluconeogenesis by activating phosphoenolpyruvate carboxykinase (PEPCK) [26,38]. In addition, adipocytes secrete factors that induce inflammation and perpetuate insulin resistance via cytokine signalling using tumour necrosis factor-α, interleukin-6 and monocyte chemo-attractant protein-1 (MCP-1) [26,38,39]. The progression from insulin resistance to T2D is related to pancreatic β-cell function and the maintenance of adequate β-cell mass. β-Cell dysfunction is an early manifestation of T2D before glucose intolerance is present. The failure of the β-cell to respond to its secretagogues, particularly glucose, results in decreased glucose uptake by the muscle and liver, which is further perpetuated by the failure of insulin to suppress glucagon-stimulated glucose release from the liver. The resultant glucotoxicity due to hyperglycaemia is damaging to the β-cells [26]. Glucotoxicity and inflammation, which culminate in oxidative stress, are implicated in an imbalance in the proliferation/apoptosis ratio causing a reduction in functional β-cell mass [40].

Plant Extracts as Alternative Therapeutic Agents Obesity is a chronic metabolic condition requiring long-term treatment. At present only one drug, Orlistat, is approved for long-term therapy by the Federal Drug Administration (FDA). However, Orlistat is associated with gastrointestinal (GI) side effects and lately with more serious liver disease [41]. Another FDA approved obesity drug, Sibutramine, has recently been withdrawn from the US market due to an increased risk of heart attack or stroke [41,42]. Currently, the major classes of oral drugs used to treat T2D include metformin, sulfonylureas, thiazolidinediones and α-glucosidase inhibitors. These drugs have different

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modes of action, which include increased insulin sensitivity and improved hepatic glucose control for metformin and the thiazolidinediones, increased insulin secretion by sulfonylureas and decreased glucose absorption from the gut by α-glucosidase inhibitors [43]. As monotherapies these drugs are ineffective at preventing the progression of T2D over the longterm. The choice of which drug to use is often complicated by their side-effects that include GI disturbances (metformin and α-glucosidase inhibitors), hypoglycaemia (sulfonylureas), weight gain and liver- and cardio-toxicity (thiazolidinediones) [26,43,44]. A new class of diabetic drug that augments the effect of glucagon like peptide-1 (GLP-1), an incretin hormone involved in post-prandial glucose homeostasis, shows great promise in the treatment of T2D. GLP-1 regulates glucose levels by enhancing insulin synthesis and secretion, suppressing glucagon secretion, delaying gastric emptying and promoting satiety [45,46]. It also has a protective effect on β-cells [45]. GLP-1 has a very short circulating halflife before being cleaved by the enzyme dipeptidyl peptidase IV (DPP-4). These new drugs are thus either DDP-4 resistant GLP-1 analogues or DDP-4 inhibitors [47,48]. Their longterm effects and safety still need investigation [49-51]. When monotherapies are not effective, combination therapies are required, yet they exacerbate the side-effects of the drugs with hypoglycaemia a major concern [52]. Natural products as alternative to or in combination with conventional oral T2D drug therapies are rapidly gaining popularity, because of the favourable risk benefit profile of these products. Natural products could be beneficial as adjunctive therapy to improve insulin sensitivity [53]. A plant extract used in combination with metformin and sulfonylurea enhanced the hypoglycaemic activity of these drugs in a rat model [54]. In T2D humans the effects of combining conventional drugs with natural products are, however, largely unknown [53,55].

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Tea and Herbal Teas Consumption, Production and Processing Black and Green Teas (Camellia sinensis L., Family Theaceae) Tea is the most consumed beverage in the world. Its annual production in 2009 was estimated at 3.9 million tonnes, of which 2.4 and 1.2 million tonnes were black and green teas, respectively [56]. Processing of black tea, essential for the development of its sensory character, basically entails withering of the leaves after harvest, size reduction and leaf cell disruption through various means, fermentation and firing [57]. Oxidation of the flavanols during fermentation leads to the formation of theaflavins and thearubigins, amongst others. Changes in phenolic composition introduced by the fermentation process have been reviewed previously [58-60]. Green tea, an unfermented product, is mostly consumed in China and Japan. Its per capita worldwide consumption in the late 1990s was estimated at ca 120 mL brewed tea per day [61]. The small-leaf variety C. sinensis var. sinensis is used in Japan to produce green tea [62]. This variety is cultivated in temperate regions and contains less polyphenols than the large-leaf variety, C. sinensis var. assamica, cultivated in tropical and subtropical regions and used for black tea production. With the processing of green tea the aim is to prevent oxidation of the polyphenols. This is achieved by inactivating the enzymes, particularly polyphenol oxidase, by steaming or pan-frying (exposure to dry heat) the fresh

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leaves immediately after harvest [63]. In Japan steaming is mostly favoured, while pan-frying is used in China. By harvesting the leaves of shaded, semi-shaded or unshaded tea plants, and by varying the degree of firing after drying, different types of Japanese green tea are obtained [62]. Gyokuro, a mildly fired tea, is for example produced from shaded plants. Similarly, Matcha is produced from shaded plants, but the withering step is excluded and the dried leaves are ground before firing. The beverage is prepared by suspending the ground leaves in hot water, which is then agitated well with a bamboo brush. Yerba Mate (Ilex paraguariensis St. Hill, Family Aquifoliaceae) Processed leaves and fine branches of yerba mate (Ilex paraguariensis St. Hill var. paraguarensis), a native forest tree of South America, which is also cultivated to meet demand, are widely consumed in Argentina, Chili, Paraguay, Uruguay and Brazil [4,64,65]. The high level of caffeine in yerba mate has made it popular as a stimulant since preColombian times [3,4]. Argentina is the largest producer, followed by Brazil and Paraguay [65]. Uruguay has the highest per capita consumption of yerba mate at 7 kg/person/year [66]. It is gaining in popularity in the United States of America (USA) and Europe as herbal tea, functional ingredient and food flavouring [4,65,67]. Yerba mate has GRAS (generally recognised as safe) status in the USA [67]. The raw plant material is processed into different types of products of which consumption predominates in certain regions of South America [68]. Processing for the production of green yerba mate usually involves blanching of the leaves to inactivate enzymes, drying, milling and ageing (forced or natural for up to a year) [65,69]. It is used to prepare the beverages, ‘chimarrão’ and ‘tereré’ [3,4], with hot and cold water, respectively [68]. Most of the yerba mate produced, is processed and consumed as chimarrão [3], with daily consumption ranging from 1.5 to 6 L [70]. The cold water drink is consumed mainly in Paraguay [4]. Preparation of these beverages entails packing either dry or moistened leaves into a gourd made from the dry and hollowed out fruit of Lagenaria vulgaris Ser. [4,66], which is frequently topped up with water as the infusion is consumed. The infusion is sucked up through a special metal straw with a strainer at the bottom [3,4,65]. Roasting of green mate at 160°C for ca 12 min produces mate, which is brewed similarly to other herbal teas [71]. German Chamomile (Matricaria chamomilla L., Family Astereaceae) Matricaria chamomilla L., also commonly known as German chamomile, is an annual herbaceous plant native to Europe and the north-western part of Asia and North Africa [72]. Some confusion exists about its correct botanical classification. It is also known as Matricaria recutita L. and Chamomilla recutita (L.) Rauschert [73]. Traditionally used for medicinal purposes, the flowers of chamomile are also in demand as a herbal tea [74]. For a high quality product the stem and leaf content should be as low as possible [75]. According to McKay and Blumberg [76], chamomile is one of the most commonly consumed single ingredient herbal teas. In 2006, chamomile, together with peppermint and rooibos, made up ca 60% of the consumed herbal and fruit infusions in Germany [77]. It is estimated to be consumed world-wide at a rate of more than 1 million cups per day [7]. Currently ca 20,000 ha of chamomile is cultivated mainly in Argentina, Egypt and Europe to supply the demand [78]. Hungary is the major producer of plant biomass [74].

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Rooibos (Aspalathus linearis (Brum.F) Dahlg., Family Fabaceae) The herbal tea, rooibos, is produced from Aspalathus linearis (Brum.f.) Dahlg., a fynbos plant endemic to the Cape Floristic region of South Africa. It grows naturally in the Cederberg area encompassing the Citrusdal, Clanwilliam and Nieuwoudtville regions, situated in the western parts of the Western Cape Province of South Africa [5]. Currently ca 36,000 ha are under cultivation in this area. Rooibos had no commercial value at the beginning of the 20th century, but its popularity slowly grew over the years. The beverage does not contain caffeine, which together with its image as a ‘healthy’ beverage contributes to its popularity. In South Africa the product is now well established, enjoying popularity amongst an estimated 10.9 million households. Rooibos is also sold world-wide, with Germany, the Netherlands, the United Kingdom, Japan and the USA representing 86% of the export market in 2010 (data supplied by South African Rooibos Council, 2011). Rooibos was initially consumed as a strong, hot brew with milk and sugar added. Preparation entailed boiling the leaves and stems in water, whereafter the brew was kept at low heat. Today, most of the rooibos is sold in tea bag form and an infusion prepared in the same manner as black tea, i.e. infusing one tea bag (ca 2 g) per cup with freshly boiled water for 2 – 5 min to release flavour and colour, which is then served hot, with or without milk and sugar according to taste. In summer the rooibos infusion is also enjoyed cold, usually with lemon juice and sugar [5]. The popular form of rooibos is the traditional ‘fermented’ product. The plant material, comprising the stems and leaves, is harvested during the hot summer months and early autumn (January to April) by topping the whole bush to a height of ca 45 cm. Processing entails cutting the shoots into small pieces, which are then placed in heaps to ‘ferment’ at ambient temperature, usually for 12-14 h, before being sundried. Shredding the shoots initiates enzymatic oxidation of the polyphenols, leading to rapid browning [79]. The ‘fermentation’ process is accelerated by adding water to the heap, mechanical bruising of the plant material and aeration of the heap. Fermentation leads to the formation of its characteristic red-brown colour and slightly sweet to honey-like flavour [80]. Bush Tea (Athrixia phylicoides DC., Family Asteraceae) Athrixia phylicoides DC. is an aromatic indigenous South African shrub growing in the wild in mountainous areas [81]. It is commonly known as bush tea (English), Umtshanela (Zulu), Icholocholo (Xhosa), Sephomolo (Sotho) and Luphephetse (Swazi) [5]. The dried leaves and fine twigs of this plant have traditionally been used by the Khoi and Zulu people as a herbal tea and medicinal drink. Current usage is limited to regions where the plant is found naturally and small amounts are traded informally in the Gauteng region of South Africa, although potential for commercialisation has been shown [82]. Traditional processing of the plant material (fine twigs and leaves) involves drying and breaking the twigs into smaller pieces. The tea decoction is prepared by boiling a handful of the dried plant material in approximately 1.5 L of water for 5 min, followed by straining [82].

Sources of Phenolic Acids A wide variety of phenolic acids belonging to both the hydroxybenzoic and hydroxycinnamic acid groups are found in infusions prepared from C. sinensis, yerba mate, German chamomile, rooibos and bush tea (Table 1).

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Camellia sinensis L. The polyphenol content of C. sinensis teas mostly comprises a number of flavanols, although flavonols and phenolic acids are also present [83-85]. The hydroxybenzoic acids, including gallic acid, protocatechuic acid, vanillic acid, galloylquinic acids and galloylated glucose derivatives, are the main phenolic acids in C. sinensis teas [83-85,87,94,96]. These teas are also a source of unesterified hydroxycinnamic acids (caffeic acid, ferulic acid and pcoumaric acid), mono-p-coumaroylquinic acids and mono- and di-caffeoylquinic acids [8385,87,94]. Other tentatively identified compounds in C. sinensis teas include galloylglucose and caffeoylmalic acid [84]. Quantitative data for phenolic acids in C. sinensis infusions indicate that gallic acid is the major phenolic acid in black tea (up to 33 mg/L), while in green tea the quantified phenolic acids are present in similar concentrations ( 1g daily)

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such as taken for analgesic and anti-inflammatory efficacy will inhibit both COX-1 and COX2 and thus suppress the production of both TXA2 and pro-inflammatory prostaglandins. Much of the recently re-ignited interest in prophylactic aspirin has focused on evidence which suggests a role in reducing the risks of cancer. Although very substantive support for this role has been recently published [24, 25], this only represented the culmination of a long series of suggestive studies. Previously, epidemiological data have suggested aspirin exerted an anti-tumour effect on colorectal but also oesophageal, gastric, lung, breast and prostate cancer [26]. Studies show that the incidence of colorectal cancers in people regularly taking NSAIDs was reduced by around 40%. Given that the risk of colorectal cancer in the Developed World is ~ 4-5% for men and 2.5-3% for women, if aspirin effects could be reproduced at a whole population level, this would represent a major benefit to public health. Familial adenomatous polyposis (FAP) is an inherited condition where an increased incidence of colorectal cancers is preceded by adenomas (initially benign epithelial outgrowths which form a polyp) formation [27]. The genetic basis of FAP lies within the adenomatous polyposis coli (Apc), whose protein product interacts with the extracellular matrix protein -catenin and the cytoskeleton, indicating a role in cellular adhesion [28, 29]. Ademona formation represents an initial stage in many forms of colorectal cancer and thus aspirin effects on adenomas numbers and size in FAP patients are often used as a surrogate for effects on cancer. Early studies have shown that colon tumours contained high concentrations of prostaglandins [30] and increased COX-2 activity was noted in the majority of colorectal tumours [31]. Such observations inspired experimental studies where different tumour types were transplanted into animals and the effects of NSAIDs examined [31]. Nonisozyme-specific COX NSAIDs inhibitors meclofenamate, indomethacin, prioxicam and the COX-2 specific inhibitor Sulindac reduced tumour growth [31]. Aspirin and other NSAIDs also reduced the incidence, number and size of carcinogen-induced tumours in rats. In a conclusive study polyp formation was examined in Apc mutant mice (Apcwhere COX-2 expression had been abolished (Ptgs2-/-) [32]. Compared to Apccontrols the ApcPtgs2-/- lines exhibited significantly reduced polyp numbers and size, thereby substantiating a link between COX-2 inhibition by NSAIDs and their observed effects on polyposis [33]. Epidemiological studies of FAP patients showed that NSAIDs were effective in reducing the polyp formation with lower incidences of between 30-50% [34, 35]. The length of period of aspirin prophylaxis required to reduce risk was either 10 [36] or > 20 [37] years. Clinical trial results indicated a requirement for persistent NSAID treatment because adenomatous polyps will re-growif NSAID treatment is interrupted [38]. After such studies pressing questions remained regarding the lowest effective dose, the age at which to recommend the taking of prophylactic NSAIDs and the treatment time [39]. For example, based on epidemiological observations Garcia Rodriguez et al. [34] suggest daily doses > 300 mg for 1 year to reduce adenomatous polyps. Against this, studies have shown that lower doses have been effective at reducing polyp formation [40]. Considering the best time to start prophylactic aspirin taking, most cancers develop after 60 and develop from premalignant growths which arise between 40-50 years of age. As the time between a cell going cancerous and a patient presenting with a diagnosable tumour is 5-10 years, so aspirin prophylaxis should commence at about 40-45 years of age. If aspirin is to be taken at such an early age the possible risks of such long term treatment require fuller assessment, particularly to identify individual cases where adverse effects could be more substantial [39]. It should

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also be noted that certain trials have suggested that NSAIDs have unclear effects. Follow-up studies of two large US trials had given inconsistent evidence of an effect of aspirin upon cancer. In the US Physicians’ Health Study of 22,000 men randomised to 325 mg aspirin or placebo on alternate days there was a non-significant excess in colorectal cancer in men given aspirin in a five-year follow-up study [41]. On the other hand, a ten-year follow up of the 40,000 women in the Women’s Health Study given aspirin on alternate days provided no evidence of an overall reduction in the incidence of total cancer, breast cancer or colon cancer [42]. However, as Cook et al. observed, lung cancer deaths had been reduced in these two trials by 22% and 18% respectively [42]. Yet both follow-ups may have been underpowered both because the participants in the trials were relatively young, with average ages 53 and 55 respectively and relatively few subjects aged over 65 years in either trial, and all patients with cardiovascular disease excluded. In addition, aspirin had been taken on alternate days in the US trials, rather than daily as in the trials followed-up by Rothwell et al. [24, 25] Other studies also suggest experimental design plays a major role in the conclusions of a study, particularly the length of observation. A good example of this was provided by a recent study of the effects of aspirin on people with Lynch Syndrome; hereditary nonpolyposis colorectal cancer [HNPCC]. This is a genetically dominant disease leading to a high risk of cancer in the lower bowel as well as other sites. Lynch Syndrome arises from mutations in DNA repair genes and is the cause of 5% of colon cancers [43]. In an initial paper, researchers failed to note any effect of daily aspirin doses of 600 mg on colon tumourigenesis after 29 months in a population of 937 people in 43 centres [44]. However, when reexamining the same patient cohort during a 10 year follow-up period and focusing on the incidence of cancers rather than adenomas; significant reductions were detected. In this follow-up study the effects of aspirin were first seen after 3 years and interestingly this was around a year after patients could be expected to cease taking prophylactic aspirin. No increase in bleeding events was also observed but this could reflect the relatively low age of people in the trails (mean = 45 years old) [45, 46]. The comprehensive long term assessment of aspirin in randomized trials demanded in the international consensus document [39] was in fact published in the following year. A group led by Prof. Peter Rothwell from Oxford (UK) followed the outcome of four randomized trials in the UK, Sweden and the Netherlands originally intending to focus on vascular events and were based on daily low-dose aspirin [75-300 mg] with one high dose [500 mg] trial [24, 25]. Data from eight trials showed that starting about five years after daily low-dose aspirin had commenced, there was a reduction of one third in all cancer (Hazard ratio [HR] 0.66; 95% confidence limits [CI] 0.50, 0.87). Aspirin reduced the 20-year risk of gastrointestinal cancers (HR 0.65; CI 0.54, 0.78) and all solid tumours [HR 0.80; 95%CI 0.72, 0.88], but probably not haematological cancers (HR 1.03; CI 0.74, 1.43). Crucially observations indicated that the incidences of cancer were not further reduced if doses of aspirin greater than 75 mg were taken. The reductions were maintained for 20 years in three trials with adequate data, and benefit increased with duration of aspirin taking, and with increasing age. The reduction by aspirin appeared to be greater in subjects with tumours which overexpressed COX-2 (HR 0.39; CI 0.20, 0.76), while there appeared to be no reduction in the subjects with weak or absent COX-2 expression [47] Such positive outcomes beg the question as to how effective aspirin could be against other cancers. Lung and breast cancers are leading sources of cancer-related mortality. COX2 is up-regulated in a range of solid tumours including lung and breast carcinomas. COX-2 is

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over-expressed in many lung cancers; adenocarcinoma, squamous cell carcinomas, bronchiolar-alveolar carcinomas and lymph-node metastases [48]. In an experimental model mice were exposed to the carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone the incidence of lung cancer was reduced by aspirin as well as selective COX-2 inhibitors Sulindac and NS398. Cell growth was inhibited in Lewis lung carcinoma lines by the COX-2 inhibitor Celecoxib [49]. In lung cancer CL1-0 and A549 cells aspirin acted powerfully with troglitazone [peroxisome proliferator-activated receptor gamma agonists] to cause growth inhibition and G(1) arrest [50]. These encouraging results have not yet been substantiated by clear results from epidemiological data. One group has examined the impact of long-term aspirin therapy aspirin on survival post potentially curative surgery for lung cancer. Survival after resections for non-small cell lung cancer remains poor but significantly increases survival following resection for potentially curative non-small cell lung cancer [51]. A study in Washington State [USA] examined the effect of a range of NSAIDs on the total incidence of lung cancer and specific morphologies after a five year follow-up. This concluded that there was a small reduced risk of lung cancer, which was strongest for adenocarcinoma [52]. Similar positive results have been reported for breast cancer. In vitro studies of breast cancer cells have shown that aspirin inhibits their growth and decreases the invasiveness of breast cancer cells [53, 54]. Metastatic breast cancer cells also show markedly increased COX-2 activity suggesting that aspirin could both decrease the risk of breast cancer death and distant recurrence among women with stage I to III breast cancer. In a recent epidemiological study Holmes et al., examined the responses of 4,164 female registered nurses in the Nurses' Health Study who were diagnosed with breast cancer [55]. Here aspirin use was associated with a decreased risk of breast cancer death which did not differ significantly with cancer stage, menopausal status, body mass index, or oestrogen receptor status. Thus, aspirin, use was associated with a decreased risk of distant recurrence and breast cancer death.

Mechanisms of Aspirin and Salicylate Action Decisions regarding the proper use of prophylactic aspirin for cancer in individual cases would be enormously facilitated by a fuller cytological and molecular understanding of its mechanism of action. A persistent inflammatory response has recently been added to the established “hallmarks of cancer” presumably through the production of reactive oxygen species (ROS) which would contribute to DNA damage and oncogene activation [56]. Thus, suppression of COX-generated prostaglandin production is undoubtedly one target for aspirin but a wide range of data indicates that it by no means the only one. Questions remain regarding the relative importance of COX-inhibition to aspirin action in cancer and of the role of salicylate as a facet of aspirin effects. In a now classical study one mechanism of aspirin action was the inhibition of COX activity through irreversible trans-acetylation of a serine within the enzyme’s active site [22]. Several attempts have been made to show salicylate inhibition of purified COX-2 but various IC50 values have been reported ranging from 30 M to 1.5mM [57] and the effectiveness of salicylate seems to be dependent on the concentration of arachidonic acid [58]. This could indicate that salicylate may be acting as a competitive inhibitor, thus it is questionable

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whether the concentration of serum salicylate would be sufficient for this to significantly affect COX activity [31]. Transcriptional studies have also yielded equivocal results with reports that salicylate either decreases, has no effect or even increases COX expression [57]. On balance, COX would seem to be a major target for aspirin and other NSAIDs but this remains to be established for salicylate. Nevertheless, salicylate is likely to be a major source of COX-independent effects of aspirin. Intrinsic to oncogene activation by pro-inflammatory events is DNA damage. Cells have DNA proof-reading enzymes which can repair/suggest errors which may be compromised in certain tumours [59]. Aspirin treatment of HCT116+ and SW480 cell lines resulted in an increase in mismatch repair proteins, and was linked to either cell cycle arrest or cell death [60]. As none of these cell lines expressed COX-2, this demonstrated the importance of a COX-2 independent mechanism for aspirin. Pro-inflammatory transcription factors could represent such a COX-independent target. Perhaps most importantly, both salicylate and aspirin have been shown to inhibit the activation of Nf-kB via targeting the activity of the activating kinase IKK[61, 62]. Nf-kB is a key transcription factor in the regulation of a large number of genes that contribute to the inflammatory response including cytokines [57]. However, both salicylate and aspirin must have other molecular targets since they retain their anti-inflammatory role in Nf-kB knockout mice [63]. Another important target for salicylate and aspirin could be Activator Protein 1 (AP-1) complex which is made up of the oncogenes fos and jun and is involved in initiating the cell cycle and also contributing to the inflammatory response. In the mouse epidermal JB6 P11-1 cells culture system, aspirin or salicylate inhibited UVB-induced AP-1 activity in a dose-dependent manner. UVB irradiation also induced a rapid increase in AP-1 activity in the skin of transgenic mice which could be reduced by topical application of aspirin [64]. Both aspirin and salicylate also function by inhibiting cell division and promoting cell death by apoptosis – a genetically regulated process of cell suicide [65-69]. Loss of apoptotic capacity is another important feature of the cancerous cell. Apoptosis is a highly regulated process where cysteine-aspartic proteases known as “caspases”, play vital roles. Caspases may be activated by release of a range of internal mitochondrial proteins including cytochrome c. Cytochrome c release is initiated by the formation of specific channels formed by pro-apoptotic proteins such as Bax. Increased COX-2 expression has been linked to the inhibition of apoptosis [32], stimulation of angiogenesis and tumour metastasis [34, 35]. In a key experimental study Tsuiji and DuBois generated a COX2 over-expressing transgenic rat line which was resistant to apoptosis but this effect was abolished with the application of the NSAID Sulindac [70]. COX-derived products have been shown to induce anti-apoptotic Bcl-2 and the Bcl-2 like protein Mcl-1 [71] which would be antagonized by aspirin. All of these observations notwithstanding, apoptosis was unaffected in cell lines with reduced COX-2 so that its relative importance in suppressing apoptosis must be questioned [72]. Non-COX dependent aspirin effects of apoptosis include promoting the release of cytochrome c [73] and increasing the half-life of Bax, via suppression of proteasome mediated proteolysis of this, and other proteins. In addition, aspirin was also shown to perturb mitochondrial membrane potential which would also facilitate release of cytochrome c with concomitant activation of caspase-9 and caspase-3 [74]. Other suggested pro-apoptotic mechanisms include the conversion of sphingomyelin to pro-apoptotic ceramide [75] and the induction of 15-lipoxygenase whose product, 13-S-hydroxyoctadecadienoic acid, can induce cell death [76]. As with the inflammatory responses, it is difficult to assess how far such proapoptotic effects are aspirin specific or are shared by salicylate. The data showing a salicylate

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effect on p38 mitogen activated protein kinases [MAPK] are much more compelling. MAPK are classically activated in response to growth factors but the p38 MAPK are more responsive to stress stimuli. p38 MAPK is activated by numerous extracellular mediators of inflammation and crucially, also contributes to apoptosis. One mechanism involves p38 MAPK phosphorylation of the anti-apoptotic Bcl-xL and Bcl-2 proteins so that they are unable to suppress the release of pro-apoptotic components [77]. Unlike most other signalling components, salicylate activates rather than inhibits p38 MAPK activity thereby promoting apoptosis [78, 79]. In a novel observation, salicylate was observed to suppress necrosis; a non-programmed cell death, which typically occurs within solid tumours. Necrosis results in cell membrane rupture and the release of pro-inflammatory contents thereby increasing the probability of oncogene activation [80]. In A549 lung adenocarcinoma cells, both aspirin and salicylate suppressed necrosis favour a process of self-degradation known as autophagy [81] . Beyond tumour initiation, angiogenesis (the establishment of a blood capillary supply to the tumour) and metastasis (spread of cancerous cells) are major features in cancer development. Aspirin will block the effects of vascular endothelial growth factor (VEGF) a key player in angiogenesis [82, 83]. This, in part, has been associated with the COX-products prostaglandin E2 and E2 [84, 85]. Cancer cell invasiveness has been linked to activation of the matrix metalloproteinases (MPPs) which are required to digest the extracellular matrix and basal membrane. COX-2 inhibition by NSAIDs had been shown to reduce MPP expression and thus should reduce cancer cell spread [86].

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Salicylate Action in Plants Given the efficacy salicylate action in humans, it is worth considering the action of salicylate in plants for possible parallels. The role of SA in plant defence against disease has been very well characterized by many groups over the last few decades [87]. Initial studies utilized aspirin to examine salicylate action in plants and this was shown to induce gene expression for many pathogenesis-related proteins [PRP] which have come to be accepted markers for increased resistance to pathogens in plants. Subsequently, SA has become associated with tolerance to heat stress and in responses to chilling [88, 89]. Exploitation of the model species Arabidopsis thaliana and particularly the analysis of mutant derivatives has been instrumental in elucidating SA biosynthesis and signalling pathways. Biochemical data suggested that SA was formed from benzoic acid via 2hydroxylation, but genetic evidence showed that the major route was via the chorismate pathway, following the formation of iso-chorismate and its spontaneous hydrolysis to yield pyruvate and SA [90]. Thus, the key isochorismate synthase gene was first targeted in an enhanced disease susceptibility (eds5) and the SA-induction deficient 2 (sid2) mutant. Induction of SA biosynthesis requires many protein elements, all of which are defined by mutation. These include Enhanced Disease Susceptibility 1 (EDS1), Phytoalexin Deficient 4 (PAD4) and Senescence-Associated Gene 101 (SAG101) which are activated following recognition of certain pathogens. EDS1-PAD4 and SAG101 are required to interact to initiate SA biosynthesis [91, 92] but the exact role of this complex remains to be clarified. It is also the case that there are a large number of other initiators of SA biosynthesis as would be expected given the central role of SA in plant responses to stress [93].

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Many SA responsive signal transduction pathways converge on Nonexpressor of PR genes (NPR1). In non-stressed cell NPR1 is cytoplasmically located but upon the imposition of oxidative stress or nitric oxide generation [94, 95] localizes to the nucleus to bind to and activate a series of transcription factors TGA2, NIMIN1 and WRKY58 to induce gene expression [95-97]. Another site of SA action is the mitochondrion. Addition of SA uncoupled mitochondrial electron transport in tobacco cells, diverting electron flow from Complex 1 (NADH dehydrogenase) to the ubiquinone pool [98]. This enhanced ROS generation at the mitochondrion which resulted in the release of cytochrome c. This represents a striking parallel with the mammalian system and specifically suggests that salicylates could act in human cell apoptosis by increasing ROS levels. To further substantiate a role for ROS, SA affects another plant oxidative burst generating complex, NADPH oxidase. The plant NADPH oxidase exhibits high sequence homology to the cytochrome b558 component of its mammalian neutrophil equivalent [99] and is similarly regulated by calcium [100] and monomeric GTP binding proteins [101]. Modulation of plant NADPH oxidase by SA is a key mechanism for increased oxidative stress and therefore accelerated cell death [102]. Other targets are the important anti-oxidant enzymes catalase and ascorbate peroxidase whose activity has been shown to be inhibited by SA [103] [104]. This inhibition was based on the co-ordination of SA with the enzymatic heme-centres so that this is likely to also occur in mammalian systems [105]. Based on these plants paradigms and preliminary mammalian studies, we suggest that it is likely that salicylates act to elevate ROS levels within mammalian systems to promote apoptotic mechanisms. Salicylates have been shown to uncouple oxidative phosphorylation in mammalian mitochondria [106, 107] and can be accompanied by the formation of a membrane permeability transition (MPT) pore and the release of pro-apoptotic proteins including cytochrome c [106]. This study also established that the ortho-hydroxyl group on SA played a key role in inducing oxidative stress by interacting with the 2[Fe-S] cluster of complex I. As this is ortho-hydroxyl group is not present on aspirin this could be a salicylate specific action.

Are Salicylates Nutriceuticals? If salicylate action is beneficial to human health, could they be considered to be a component of a healthy mixed diet? In most plant species, SA does not accumulate to high levels (an exception is rice where baseline levels of ~ 16ug g fresh weight (FW)-1 have been reported [108]) except when undergoing biotic or abiotic stress when there is significant de novo synthesis of SA. SA rises have been most well-characterised following pathogenic challenge where concentrations at the site of infection rise to 4-8 g g FW-1 from baseline levels of ~0.5 g g FW-1 (e.g.[109, 110]). Subsequently, there is a whole plant accumulation of SA, where levels are in the order of ~1 g g FW-1. Such systemic increases in SA are more pronounced during abiotic stresses such as chilling and heating where levels can reach ~6 g g FW-1 [88, 89]. As plants would be expected to have undergone some form of abiotic or biotic stress, it follows that salicylate should be present in fruit and vegetable produce. Interestingly, in a sixty-year cohort study of 4,000 subjects, fruit intake during childhood

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reduced the risk of all cancers in adulthood, with an odds ratio of 0.62 [0.43-0.90] in the quarter of subjects with the highest fruit consumption. Meta-analyses have confirmed these findings, and in a review of 206 human and 22 animal studies, there was a consistent protective effect of high fruit and vegetable consumption for cancer of a range of cancers, including those arising in the stomach, oesophagus, lung, and colon [111, 112]. The levels of salicylate are significantly increased in humans with a vegetarian diet, and may be equivalent to patients consuming 75-150 mg aspirin per day [113]. Such levels are equivalent to those of low dose ingestion of aspirin for which beneficial medical effects have been noted [114]. However, the actual daily intake may be as low as 2mg day-1, which is likely to be too low to have significant effects [115]. In this context, it should be noted that current agricultural practice will tend to produce plants with lower salicylate levels. By growing plants in pesticide-rich and or environmentally cosseted [for example, large glasshouses] conditions those stresses which promote salicylate synthesis are avoided and thus could be removing a valuable nutriceutical in the human diet.

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Aspirin are We at a “Put-it-in-theWater Moment”? Aspirin represents an excellent example of how a botanically active compound has been exploited and developed within a medical context to have a considerable impact. It also offers the possibly of showing how cross-kingdom studies could throw some real insights into molecular actions. As described in this chapter, the epidemiological studies coupled with improved mechanistic understanding of both aspirin and salicylate makes a compelling argument for the prophylactic ingestion of low dose aspirin to reduce the risks of cancer. Should prophylactic aspirin be taken by all over 45 year olds? This question was recently amusingly put as a “should we put it in the water moment” by Professor Gordon McVie of the European Institute of Oncology [116]? Clearly, at an individual level due care must be exerted and consultation with the personal doctors is advised. There were too many individual instances where taking aspirin would be delirious; most obviously in individuals with a peptic ulcer or any bleeding disorders such as haemophilia or if allergic to NSAIDs. Against this, the general public must be made aware of the potential benefits of taking aspirin to suppress cancers so they can make informed choices.

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Index

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A absorption spectra, 118 absorption spectroscopy, 90 abstraction, 78, 82 acarbose, 200 access, 45 acetic acid, 34, 102, 103, 104, 106, 107, 110, 160, 217 acetone, 108, 110, 111, 112, 140, 141 acetonitrile, 103, 104, 106, 107, 112, 116 acetylation, 220 acidic, 69, 89, 113, 132, 134, 153 acidity, 100, 135, 217 active compound, 81, 224 active site, 220 active transport, 50 acylation, 44 adaptation, 45 additives, vii, x, 125, 135, 136, 139, 140, 141 adenocarcinoma, 167, 220, 222 adenoma, 167 adenomatous polyposis coli, 218 adhesion, 218 adipocyte, 191, 197 adiponectin, 197, 201, 202 adipose, 176, 185, 193, 195, 196, 197, 198, 201, 202 adipose tissue, 176, 185, 193, 195, 196, 197, 198, 201 adjunctive therapy, 177, 203 adjustment, 102, 103 adolescents, 175 adrenaline, 4 adrenoceptors, 198 adsorption, 68, 69 adulthood, 224 adults, 175 adverse effects, 218

Africa, 17, 173, 179, 206 agar, 138 age, 42, 149, 174, 218, 219 aggregation, 217 aging process, 16, 28 agriculture, 5 Agrobacterium, 67 AIDS, 126 albumin, 23, 84, 195 alcohols, 10, 43, 45, 48, 49, 52, 64, 99, 108, 131 aldehydes, 45, 49, 52, 112 alfalfa, 52 algae, 34, 45, 108 alkaline hydrolysis, 6, 67, 68, 69, 106, 130, 134 alkalinity, 132 alkaloids, 64, 98, 109, 116, 118, 139 alkylation, 131 alternative hypothesis, 51 alters, 191 amine, 103 amino, 18, 24, 90, 199 amino acid(s), 24, 90 ammonia, 47, 48, 139 ammonium, 8 amylase, 21, 107, 186, 195, 197, 200, 202 analgesic, viii, x, 63, 64, 67, 213, 215, 217, 218 angiogenesis, 216, 221, 222 anthocyanin, 5 antibiotic, 66 anti-cancer, 141, 174 anti-inflammatory agents, 217 antioxidant activity (AOA), ix, 73 antioxidative activity, 78, 79, 81 antipyretic, 214 apoptosis, xi, 168, 176, 192, 194, 195, 197, 213, 216, 221, 223 apoptotic mechanisms, 221, 223 apples, 128, 151

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230

Index

aqueous solutions, 133 Arabidopsis thaliana, 222 ARC, 173 Argentina, 178 arginine, 90 argon, 107 aromatic compounds, 16, 46, 47, 74, 99 aromatic rings, 100 arrest, 216, 220, 221 artery, 188, 190 arthritis, 175 ascorbic acid, 9, 42, 84, 89, 106, 128, 168 Asia, 17, 178 asparagus, 36, 39, 40, 128 assessment, 78, 218, 219 atherosclerosis, 66, 99, 126 atmosphere, 45, 107, 132 atmospheric pressure, 111 atoms, 126 ATP, 192 attractant, 176, 199 automation, 111, 112, 119, 120

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B Bacillus subtilis, 66, 138, 139 back pain, 214 bacteria, 34, 45, 67, 136, 137, 138, 139, 148, 152, 165, 166 bacterial pathogens, 67 bacterial strains, 6 bacteriostatic, 137 bacterium, 138 base, 22, 65 Bcl-2 proteins, 222 beef, 138, 140, 141 beer, 104, 113, 149, 150 Beijing, 70 Belgium, 204 beneficial effect, x, 27, 151, 173, 192, 193, 201, 202, 203, 214 benefits, vii, 18, 24, 27, 54, 148, 168 benign, 218 benzene, 108, 166 beverages, x, 10, 98, 114, 125, 126, 151, 173, 174, 178, 187, 189 bile, 90, 151, 187, 190 bilirubin, 84 bioactive compounds, vii, 1, 24, 110 bioassay, 138 bioavailability, x, 6, 7, 10, 66, 151, 152, 168, 174, 187, 190, 191, 202, 203 biochemistry, vii, 205

bioconversion, viii, 33 biofuel, 40 biological activity, 23 biological fluids, 6, 7, 90 biological samples, 160, 168 biologically active compounds, 130 biomarkers, 6 biomass, viii, 33, 52, 54, 55, 107, 178 biomolecules, 86 biopolymers, 128 biosynthesis, 46, 47, 48, 50, 53, 61, 195, 200, 202, 216, 222 biotechnology, vii biotic, 35, 55, 98, 99, 223 birds, 166 black tea, x, 5, 98, 110, 151, 157, 161, 173, 177, 179, 180, 185, 187, 188, 205 bleaching, 78, 85, 87 bleeding, 217, 219, 224 blindness, 175 blood, 11, 175, 186, 191, 192, 194, 195, 196, 198, 200, 202, 216, 222 blood vessels, 175 BMI, 174 body fat, 201 body mass index (BMI), 174, 220 body weight, 163, 164, 174, 195, 196, 199 bonding, 41, 115 bonds, 6, 21, 25, 41, 80, 115, 118 boredom, 175 boric acid, 102 bowel, 219 branching, 8 Brazil, 97, 125, 178 breakdown, 201 breast cancer, 219, 220 breast carcinoma, 219 Britain, 26 by-products, 18, 108, 128, 129

C Ca2+, 196, 200 cabbage, 138 CAD, 47, 49, 52 caffeine, 110, 116, 178, 179 calcium, vii, 15, 18, 46, 223 calcium carbonate, 46 calyx, 138, 140 cancer, vii, ix, xi, 1, 27, 63, 66, 74, 97, 98, 99, 121, 126, 166, 174, 175, 213, 214, 216, 218, 219, 220, 222, 224 cancer cells, 166, 220

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Index cancer death, 220 cancerous cells, 222 capillary, xi, 104, 105, 115, 213, 216, 222 capsule, 189 carbohydrate, vii, x, 15, 17, 18, 36, 45, 64, 107, 173, 186, 200 carbohydrate metabolism, 186 carbohydrates, 40 carbon, ix, 16, 52, 73, 74, 82, 85, 99, 112, 117, 126, 153, 167 carbon atoms, 126, 153 carbon dioxide, 112, 117 carbon-centered radicals, 85 carboxyl, ix, 73, 77, 79, 135 carboxylic acid(s), ix, 10, 73, 78, 99, 132, 214 carcinogen, 218, 220 carcinoma, 188, 220 cardiovascular disease(s), vii, ix, 1, 27, 97, 98, 121, 175, 219 carotene, 84, 90 carotenoids, 74, 84, 98 cascades, 174 caspases, 221 catabolism, 67, 148, 152, 153, 154, 161, 165, 168, 191 catalyst, 134 cataract, 126 cation, 9, 88 CCR, 47, 49, 52, 53 cDNA, 53 cecum, 6 cell culture, 36, 41, 42, 165 cell cycle, 216, 221 cell death, xi, 192, 213, 216, 221, 223 cell division, 221 cell invasiveness, 222 cell line(s), 167, 188, 192, 200, 221 cell size, 111 cell surface, 166 cell wall polysaccharides, viii, 20, 24, 33, 38, 39, 40, 42, 126 cellular energy, 192 cellular homeostasis, 87 cellulose, viii, 6, 23, 33, 35, 41, 45, 52, 107, 139 ceramide, 221 cerebrospinal fluid, 90 ceruloplasmin, 84 challenges, 174, 192 chemical, viii, ix, 2, 9, 10, 24, 34, 51, 54, 63, 76, 79, 81, 83, 88, 97, 98, 99, 101, 107, 109, 115, 126, 127, 128, 131, 134, 135, 136, 148 chemical characteristics, 148 chemical stability, 51, 81

231

chemical structures, 9, 63, 79, 109, 136 chemicals, 52, 141 chicken, 140, 141 childhood, 223 children, 174, 175 China, 63, 70, 177 chloroform, 108, 134 chlorogenic acid, x, 4, 5, 6, 7, 66, 79, 103, 104, 109, 110, 111, 126, 128, 130, 132, 134, 135, 137, 138, 140, 173, 182, 183, 184, 185, 187, 189, 191, 192, 193, 197, 199, 200, 201, 202, 203 chlorophyll, 103, 108 cholesterol, 66, 141, 185, 186, 193, 195, 196, 199, 200, 201, 202 choline, 135 chromatograms, 185 chromatographic technique, 115, 117, 118, 119 chromatography, 68, 69, 84, 86, 90, 114, 115, 117, 119 chronic diseases, 99 circulation, 148, 151, 152, 153 classes, 18, 24, 34, 35, 48, 74, 75, 98, 99, 120, 150, 176 classification, 149, 178, 204 cleanup, 120 cleavage, 64, 153 climate, 5 CO2, 106, 107, 112, 134 cocoa, ix, 97, 150, 155, 156, 157, 160, 161 coding, 51 coenzyme, 47, 49, 185, 193, 195, 199, 201 coffee, ix, 6, 10, 97, 98, 103, 109, 127, 128, 134, 189, 191, 199, 201, 202, 203 colitis, 166 colon, 7, 54, 148, 152, 165, 166, 167, 187, 188, 190, 191, 193, 200, 218, 219, 224 colon cancer, 167, 219 color, viii, 5, 8, 10, 15, 20, 27, 63, 69, 88, 133, 141 colorectal cancer, xi, 213, 218, 219 commercial, 68, 84, 87, 107, 130, 132, 134, 136, 139, 141, 174, 179, 198, 200, 203, 217 compatibility, 51, 117 competition, 45, 77 complement, 18 complexity, 100, 116, 119, 120, 148 complications, 174, 175 composition, vii, viii, ix, 5, 15, 18, 20, 24, 27, 28, 35, 43, 45, 52, 97, 99, 101, 111, 112, 115, 130, 141, 165, 168, 177 condensation, 2, 47 conductivity, 110 conjugated dienes, 86, 90 conjugation, 82, 90, 151, 154, 161, 190

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232 consensus, 51, 219 constituents, 16, 21, 130, 131, 139, 174, 185, 190, 214 Constitution, 61 consumers, 98 consumption, viii, x, 6, 11, 16, 108, 109, 110, 113, 115, 117, 119, 125, 126, 147, 160, 165, 177, 178, 186, 214, 224 contaminant, 134 contamination, 136, 138, 141 control group, 166, 185 controversial, 10, 45 controversies, 48 COOH, 27, 77, 82, 135, 136 cooking, 5, 10, 20, 132 cooling, 120 cooperation, 55 coronary artery disease, 175 coronary heart disease, 2, 16, 28 correlation(s), 9, 42 cosmetic, viii, 63, 74, 98 cosmetics, 98 cost, 82, 112, 113, 114, 116, 117, 120, 128, 204 coumaric acid, viii, 2, 4, 5, 6, 9, 16, 22, 23, 27, 33, 34, 35, 37, 63, 64, 65, 66, 67, 68, 69, 71, 102, 103, 104, 105, 106, 109, 128, 129, 130, 132, 134, 137, 141, 154, 155, 156, 158, 161, 162, 163, 167, 180 coumarins, 98 covalent bond, 40 COX-dependent, xi, 213, 216 COX-independent mechanisms, xi, 213, 216 CPP, 199, 201 CPT, 107 critical value, 112 crop(s), 22, 26, 27 crops, 26 crystalline, 133, 135 crystallization, 68, 69 cultivars, 18, 20, 130 cultivation, 179, 206 culture, 41, 42, 200, 221 culture medium, 41 cycles, 111 cyclooxygenase, 167 cyclo-oxygenase (COX), x, 213, 216, 217 cysteine, 81, 87, 90, 221 cytochrome, 48, 216, 221, 223 cytokines, 166, 176, 221 cytoskeleton, 218 cytotoxicity, 148

Index

D damages, 27, 99 database, 5, 11, 169 deaths, 175, 219 decay, 85, 86 deconstruction, 52, 53 defects, 52 defence, 222 defense mechanisms, 99 deficiency, 175 degradation, 76, 84, 89, 101, 107, 109, 112, 115, 131, 161, 165, 166, 186, 187, 190, 191, 202, 222 Denmark, 203 deoxyribose, 84 Department of Agriculture, 5, 11, 169 depolymerization, 53, 153 deposition, 46 depression, 175 derivatives, vii, viii, 1, 2, 4, 6, 11, 15, 16, 18, 19, 20, 22, 24, 27, 34, 64, 66, 75, 77, 78, 79, 80, 81, 101, 115, 126, 128, 130, 132, 135, 136, 151, 153, 161, 165, 167, 180, 181, 184, 185, 217, 222 desorption, 68, 69 destruction, 90, 139 detectable, 89, 90 detection, 88, 90, 91, 115, 118, 119 detection techniques, 119 detoxification, 188 developing countries, 175 diabetes, x, 27, 173, 174, 175, 187, 191, 193, 202, 203, 204 diacylglycerol, 176 dielectric constant, 110 dienes, 90 diet, vii, ix, x, 1, 7, 15, 17, 20, 54, 97, 98, 99, 127, 147, 148, 149, 151, 162, 166, 173, 174, 175, 185, 186, 194, 195, 196, 198, 199, 203, 214, 223 dietary fiber, 20, 54 diffusion, 45, 50, 108, 116, 136, 138, 190, 191 diffusivity, 112 digestibility, viii, 33, 35, 54, 55 digestion, x, 20, 40, 54, 173, 200 dihydroxyphenylalanine, 4 dimerization, 41, 42 direct measure, 91 diseases, vii, ix, x, 1, 2, 27, 55, 66, 73, 74, 97, 125, 126, 168, 175 dispersion, 113 displacement, 136 disposition, 175 dissociation, 78, 135 distilled water, 105

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233

Index distribution, 10, 23, 49, 99, 111, 112, 119, 120, 149, 151, 152 divergence, 46 diversification, 45 diversity, 16, 120, 148 DNA, xi, 83, 90, 139, 166, 213, 216, 219, 220, 221 DNA damage, 220, 221 DNA repair, 90, 219 doctors, 224 DOI, 30, 58, 59, 95 dominance, viii, 15, 27 donors, 27, 42 dopamine, 4 dopaminergic, 67 dosing, 66, 190 down-regulation, 38 drugs, 176, 177, 185, 198, 200, 203 drying, 101, 103, 104, 105, 106, 132, 178, 179

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E ecological roles, 45 ecosystem, 168 editors, 225 Egypt, 178, 214 electric field, 110 electromigration, 115 electron(s), ix, 8, 9, 66, 73, 76, 77, 79, 80, 81, 82, 86, 88, 91, 119, 134, 136, 216, 223 electrophoresis, 114 ELISA, 90 elongation, 42, 43, 141 elucidation, 203 emission, 86, 91 employment, 118 emulsions, 81, 82, 138 encoding, 53, 54, 67, 83 endosperm, 16, 19, 20, 21, 23, 36, 38, 54 end-stage renal disease, 175 energy, vii, 15, 17, 26, 52, 76, 86, 91, 109, 110, 126, 185, 192 engineering, 55 environment, 51, 99, 111, 148, 149 environmental conditions, 43, 99 environmental factors, 175 environmental impact, 116 environmental stimuli, 16 environments, 20, 49, 78 enzyme immunoassay, 90 enzymes(s), viii, 5, 7, 20, 27, 33, 36, 37, 38, 40, 42, 44, 48, 49, 50, 51, 52, 53, 54, 67, 68, 74, 76, 83, 84, 90, 98, 101, 107, 135, 141, 149, 151, 166,

177, 178, 186, 188, 190, 191, 193, 194, 195, 196, 200, 201, 202, 216, 217, 220, 221, 223 epidemic, x, 173, 174, 175 epinephrine, 167 epithelial cells, 167 epithelial transport, 188, 191 epithelium, 166, 191 equilibrium, 111 equipment, 84, 113 erythrocytes, 198 ESI, 105, 114, 118 ESR, 83, 86, 91 ester, 6, 7, 17, 21, 22, 24, 25, 28, 35, 36, 37, 38, 40, 41, 42, 43, 48, 50, 53, 66, 67, 78, 79, 80, 81, 101, 115, 134, 135, 138, 153, 191 ester bonds, 7 ethanol, 8, 21, 80, 104, 106, 108, 110, 111, 134, 135, 138, 140 ethers, 34, 151 ethyl acetate, 103, 104, 105, 108, 110, 111, 152 ethylene, 86, 102 ethylene-propylene copolymer, 102 eukaryotic, 45 Europe, 178, 214 evaporation, 102, 103, 104, 105, 106 evidence, ix, x, 41, 43, 44, 50, 74, 97, 99, 112, 118, 173, 174, 185, 213, 216, 217, 218, 219, 222 evolution, viii, 33, 45, 51, 52 excretion, 20, 152, 155, 160, 162, 164, 187, 189, 195 experimental design, 219 exploitation, 52 export market, 179 exposure, 5, 6, 101, 109, 112, 119, 177 extracellular matrix, 218, 222 extraction, ix, 2, 43, 53, 84, 97, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 119, 120, 130, 132, 134, 184 extracts, 7, 38, 102, 104, 105, 106, 109, 110, 111, 112, 113, 114, 128, 131, 132, 137, 138, 141, 158, 166, 185, 186, 191, 203, 214

F Fairbanks, 208 families, 48, 50 famine, 174 FAS, 193, 195, 196, 197, 199, 200, 201 fasting, 186, 197, 201, 202 fat, vii, 15, 17, 18, 82, 174, 185, 191, 197, 200, 201 fatty acids, 24, 89, 99, 112, 131, 139, 176, 193, 201 FDA, 176, 205 fermentation, 40, 134, 152, 165, 177, 179, 180, 185 fermentation technology, 134

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234 ferrous ion, 89 fever, 214 fiber(s), 19, 21, 23, 38, 54 fibroblasts, 167 film thickness, 104, 105 films, 141 filtration, 103, 104, 106 financial, 120 financial support, 120 fish, 141 fish oil, 141 fission, 153, 154 fitness, 214 flame, 115 flavonoids, viii, 6, 7, 10, 15, 16, 18, 24, 27, 28, 48, 49, 74, 84, 98, 99, 118, 139, 148, 150, 156, 174, 184, 187 flavonol, 4 flavor, 24, 63, 132, 139 flavour, 179 flexibility, 80 flour, 20, 21, 22, 23, 25, 36, 65, 127, 149 flowers, 102, 134, 178, 184, 186 fluid, ix, 6, 7, 90, 97, 108, 109, 111, 112, 117, 122 fluid extract, ix, 97, 108, 109, 111, 112, 122 fluorescence, 85, 86, 87, 90, 119, 135 food additive, vii, 134, 135, 136, 138, 141, 142 food additives, vii, 138, 142 food industry, 27, 28, 128, 135, 140, 141, 142 food intake, 192 food production, viii, 16, 139 food products, 101, 108, 131, 139, 142 food spoilage, 138 foodborne illness, 139 Ford, 58 formation, xi, 2, 5, 8, 19, 38, 39, 40, 41, 42, 43, 47, 48, 49, 51, 54, 76, 79, 80, 82, 84, 86, 87, 88, 90, 132, 139, 141, 153, 161, 177, 179, 186, 196, 198, 213, 216, 218, 221, 222, 223 formula, 9 fragments, 84, 119 free radicals, ix, 8, 20, 27, 51, 66, 73, 74, 76, 84, 90, 91, 126, 127, 136, 139 freezing, 101 freshwater, 113 fructose, 195 fruits, vii, viii, ix, 2, 3, 4, 5, 11, 15, 16, 17, 18, 28, 63, 73, 97, 99, 108, 114, 127, 128, 138, 149, 150, 151 functional food, vii, x, 24, 125, 139 fungi, 34, 45, 137, 139 fungus, 137, 138 fusion, 134

Index

G gallic acid, x, 2, 4, 9, 10, 27, 34, 66, 75, 77, 80, 101, 105, 109, 110, 111, 112, 126, 128, 130, 131, 132, 135, 136, 138, 139, 141, 153, 156, 157, 160, 161, 173, 180, 184, 185, 187, 188, 189, 192, 194, 200, 203 gastrointestinal tract, 7, 10, 217 gel, 113 gene expression, 48, 174, 186, 192, 196, 198, 202, 203, 222, 223 genes, 38, 48, 49, 50, 51, 53, 54, 67, 83, 175, 185, 186, 191, 194, 197, 200, 201, 219, 221, 223 genetic engineering, 52 genetic factors, ix, 5, 97 genome, 216 genus, 215 Germany, 6, 178, 179, 214 germination, 136 glucagon, 176, 177, 198 gluconeogenesis, 176, 198, 200, 202 glucose, x, 2, 6, 10, 24, 37, 49, 50, 173, 175, 176, 177, 180, 186, 187, 189, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203 glucose tolerance, 195, 197, 199, 202 glucose tolerance test, 199, 202 glucosidases, 50, 186 glucoside, 22 glucosinolates, 98 GLUT4, 187, 192, 193, 194, 195, 196, 198, 199, 200, 202 glutathione, 9, 84, 87, 193, 195, 196, 199 glycans, 35 glycerol, 193, 195, 196, 199 glycine, 154 glycogen, 186, 187, 195, 196, 198, 200 glycol, 114 glycolysis, 186 glycoproteins, 35 glycoside, 22, 125, 138, 214 glycosylation, ix, 73 gout, 175 GRAS, 178 grass(s), 17, 35, 36, 38, 39, 44 Great Britain, 214 Greece, 1 green alga, 45, 46 group work, 39 growth, 17, 23, 34, 41, 51, 52, 54, 66, 129, 136, 137, 138, 166, 167, 220, 222 growth factor, 222 growth rate, 41 growth temperature, 54

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Index Guangzhou, 63 guanine, 90 guard cell, 16, 54

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H habitats, 45 half-life, 177, 214, 216, 221 harvesting, 178 haze, 5 healing, 5 health, vii, viii, 10, 11, 18, 20, 22, 24, 27, 28, 54, 63, 98, 135, 141, 147, 148, 165, 168, 174 health effects, 10, 24, 147, 165, 168 health promotion, 22 heart attack, x, 175, 176, 213, 214 heart disease, 2, 63 heating rate, 110 height, 174, 179 heme, 223 hemicellulose, 45 hepatocellular carcinoma, 167 hepatocytes, 168, 195, 198, 200 herbal medicine, 215 herbal teas, x, 173, 174, 178, 180, 185, 187, 203 herpes, 139 herpes simplex, 139 hexane, 105, 108, 134, 135 high density lipoprotein, 199 high fat, 174, 185, 186, 195, 196, 199 histidine, 90 HIV, 175 HIV/AIDS, 175 homeostasis, 177, 200 homogeneity, 118 homovanillic acid, 86, 157, 158 hormone, 34, 177, 214, 215 hormones, 176 host, 168 human, vii, viii, xi, 1, 6, 15, 16, 17, 19, 20, 22, 27, 28, 66, 77, 84, 85, 86, 90, 98, 99, 127, 141, 148, 152, 153, 155, 165, 167, 168, 188, 190, 191, 194, 202, 213, 214, 216, 223 human body, xi, 22, 84, 86, 99, 141, 213, 214 human diet, vii, 1, 20, 99, 127, 224 human health, vii, 15, 17, 19, 223 Hungary, 178 Hunter, 225 hydrogen, ix, 27, 38, 40, 41, 42, 44, 51, 69, 73, 76, 78, 81, 82, 85, 88, 90, 91, 115, 132, 134, 141 hydrogen abstraction, 78 hydrogen atoms, ix, 73, 76 hydrogen chloride, 69

hydrogen peroxide, 38, 40, 42, 44, 51, 91, 132, 141 hydrolysis, 2, 6, 7, 21, 37, 40, 64, 67, 68, 102, 103, 107, 115, 130, 132, 134, 160, 222 hydroperoxides, 90 hydrophilicity, 77 hydrophobicity, 69, 77, 81 hydroquinone, 4 hydroxyacids, 2 hydroxycinnamic acid, viii, 2, 5, 6, 7, 11, 16, 23, 24, 34, 35, 49, 50, 51, 63, 66, 77, 79, 80, 81, 99, 100, 107, 111, 128, 129, 134, 135, 137, 179, 180, 184, 185 hydroxyl, vii, ix, 1, 7, 16, 27, 34, 53, 73, 74, 77, 79, 80, 81, 82, 85, 87, 89, 98, 99, 100, 126, 132, 135, 136, 151, 214, 217, 223 hydroxyl groups, ix, 7, 27, 73, 74, 77, 79, 80, 81, 98, 100, 132, 135, 151 hyperglycaemia, 176, 186, 192, 200 hypertension, 175, 193 hypocotyl, 37 hypothalamus, 192 hypothesis, 38, 43, 44, 46, 51, 202

I identification, ix, 22, 36, 42, 44, 84, 97, 101, 102, 107, 114, 117, 118, 119, 120, 148, 161, 168 identity, 118, 119, 128 IL-8, 166 ileostomy, 152, 191 ileum, 190 image, 179 images, 215 immune system, 168 improvements, 52 impurities, 68, 69 in vitro, vii, ix, 1, 7, 28, 42, 44, 49, 53, 66, 74, 77, 78, 84, 87, 99, 139, 148, 152, 165, 167, 168, 185, 186, 188, 190, 191, 192, 200, 202, 203 in vivo, 7, 41, 42, 51, 53, 99, 147, 152, 153, 165, 166, 168, 188, 192, 194, 195, 196, 197, 198, 199, 200, 202, 203 incidence, x, 174, 213, 214, 217, 218, 219, 220 India, 92, 205 individuals, 148, 174, 176, 203, 217, 224 Indonesia, 15 induction, 9, 43, 67, 85, 197, 221, 222 induction period, 9 induction time, 85 industrial processing, 10 industry(s), viii, 27, 33, 63, 67, 69, 74, 98, 108, 111, 112, 130, 214 ineffectiveness, 175

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Index

infection, 5, 99, 223 inflammation, 67, 175, 176, 201, 222 inflammatory disease, 168 inflammatory responses, 221 ingest, 6 ingestion, ix, x, 1, 6, 7, 73, 152, 153, 165, 173, 185, 187, 188, 190, 224 ingredients, 23, 148 inhibition, ix, x, 42, 48, 66, 73, 76, 83, 85, 86, 87, 88, 98, 137, 166, 186, 192, 195, 197, 200, 201, 213, 218, 220, 221, 222, 223 inhibitor, x, 42, 202, 213, 216, 218, 220 initiation, xi, 8, 34, 76, 213, 222 injury, 34, 90 inoculation, 139 INS, 193, 196, 200 insulin, x, 173, 175, 176, 177, 186, 187, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202 insulin resistance, x, 173, 175, 176, 186, 187, 201 insulin sensitivity, 176, 177, 192, 197 insulinoma, 192, 194 integration, 120 interference, 86, 87, 88 intervention, 159, 163, 164, 168 intestinal flora, 54 intestine, 7, 54, 148, 151, 152, 193, 200 intravenously, 199 ionization, 115, 118 ions, vii, 1, 84, 89, 132 IR spectra, 69 iron, vii, 7, 15, 18, 84, 133 irradiation, 86, 110 isolation, ix, 36, 37, 38, 66, 97, 100, 107, 113, 114, 115 isomerization, 153, 154 isomers, 16, 39, 41, 43, 44 isozyme, 199, 200, 201 Italy, 73

J Japan, 27, 177, 179 joints, 46

K kaempferol, 9 kidney, 154, 160, 193, 199, 202 kidneys, 187, 190, 191, 214 kinetics, 7, 41, 82, 110 KOH, 68

L labeling, 41, 45 lactate dehydrogenase, 87 Lactobacillus, 166 lactoferrin, 84 large intestine, 153 later life, 175 LCAT, 199 LC-MS, 119, 185 LDL, 28, 66, 77, 86, 99, 186, 196, 197, 199, 201, 202 leaching, 5, 108, 110 lead, vii, 5, 10, 20, 66, 99, 126, 139, 151, 187, 201, 217 Leahy, 226 lecithin, 27, 79, 199, 202 legume, 34 leptin, 194, 197, 198, 201 liberation, 115 lifestyle changes, 174 light, 5, 45, 67, 91, 101, 111, 112, 131, 133, 135, 140, 192, 195 lignans, 16, 24, 98 lignin, viii, 2, 6, 16, 17, 21, 25, 33, 34, 35, 37, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 61, 63, 68, 69, 128, 134 linoleic acid, 66, 80, 81, 89, 90 lipid metabolism, x, 168, 173, 191, 192, 201 lipid oxidation, 8, 9, 85, 135, 139 lipid peroxidation, ix, 66, 74, 89, 140, 192, 193, 196 lipid peroxides, 99, 202 lipids, 9, 23, 89, 99, 108, 115, 175, 186, 194, 200, 202 lipolysis, 192 liquid chromatography, 109, 114, 115, 116, 117, 119 liquid phase, 109 liquids, 111 Listeria monocytogenes, 137, 138 liver, 6, 7, 140, 141, 148, 151, 152, 154, 160, 176, 177, 186, 187, 188, 190, 191, 195, 197, 198, 199, 200, 201, 202, 214 liver cells, 187 liver disease, 176 localization, 47 low-density lipoprotein, 77, 86, 99, 185, 202 luminescence, 86 lung cancer, 219, 220 Luo, 30, 70, 71, 91, 143 lutein, 84 lycopene, 84 lymph, 220 lymphoma, 192, 195

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Index lysine, 90

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M macronutrients, vii, 15, 17, 18 macrophages, 167, 201 magnesium, vii, 15, 18 magnetic field, 91 magnitude, 10 majority, 115, 151, 218 management, 66 manganese, 88 manipulation, viii, 33, 101, 114, 119 mass, 37, 90, 108, 109, 110, 111, 112, 114, 115, 116, 118, 174, 176, 220 mass spectrometry, 37, 90, 114, 115, 118 materials, 20, 38, 46, 107, 129, 206 matrix, viii, ix, 23, 24, 33, 35, 42, 51, 97, 100, 101, 108, 109, 110, 113, 120, 148, 151, 160, 187, 216, 222 matrix metalloproteinase, 216, 222 matrixes, 109, 111 matter, 132 Matua, 210 MCP, 176, 197, 199, 201 MCP-1, 176, 197, 199, 201 measurement, ix, 8, 55, 73, 82, 83, 84, 85, 86, 89, 90 meat, 136, 138, 140, 141 mechanical properties, 141 media, 85, 88 medical, 224 medicine, 214 melanin, 67 melanocyte stimulating hormone, 67 melanoma, 67 mellitus, 204 melting, 115, 131 membrane permeability, 216, 223 membranes, 50, 66, 99 Metabolic, 154 metabolic dysfunction, 203 metabolic pathways, 74, 148, 153, 168, 188, 191 metabolism, x, 10, 48, 49, 53, 86, 125, 147, 148, 152, 153, 155, 160, 162, 164, 165, 167, 173, 187, 191, 203 metabolites, viii, ix, x, 1, 2, 6, 7, 11, 15, 17, 27, 28, 34, 48, 50, 52, 73, 74, 98, 99, 114, 125, 140, 148, 151, 152, 153, 155, 159, 160, 161, 162, 163, 164, 165, 166, 168, 176, 187, 189, 190, 191 metabolized, xi, 141, 148, 151, 152, 213 metal ion(s), 83, 84, 139 metalloproteinase, xi, 213 metals, 88

metastasis, xi, 213, 216, 221, 222 metformin, 174, 176, 177, 196, 198, 200, 202 methanol, 103, 104, 106, 107, 108, 109, 110, 111, 112, 116, 117, 130, 132, 134, 135, 138, 141 methodology, 91, 119, 120 methylation, 50, 148, 151, 152, 154, 188, 191 mice, 7, 67, 176, 185, 186, 193, 195, 196, 197, 198, 200, 201, 202, 218, 220, 221 microbiota, 147, 148, 152, 153, 161, 165, 166, 168 micronutrients, 5, 18, 21 microorganisms, x, 46, 54, 74, 125, 136, 138, 139, 148 microscopy, 45 microsomes, 198, 202 microwave heating, 110 microwave radiation, 110 microwaves, 110 middle lamella, 45 migration, 109 milligrams, 2 Ministry of Education, 169 mitochondria, 201, 216, 223 mitogen, 167, 216, 222 mixing, 8 model system, 90, 132, 196, 202 models, 7, 79, 165, 196 modifications, 45, 52, 90 moisture, 101, 104 moisture content, 101 molecular mass, 115 molecular oxygen, 86 molecular structure, ix, 73, 118 molecular weight, 16, 42, 74, 98, 114, 132 molecules, 6, 16, 17, 34, 41, 50, 64, 74, 77, 81, 83, 85, 90, 99, 126, 131, 148, 165, 167 monomers, 24, 37, 42, 43, 44, 51, 69, 148, 149, 151, 152, 153 monosaccharide, 36, 44 Moon, 71, 92 morbidity, 175 mortality, 175, 219 mRNA, 192, 194, 197, 198, 201, 202 mucosa, 6, 7, 166, 190 multidimensional, 119, 120 multiplication, 139 mutant, 218, 222 mutation(s), 53, 83, 175, 219, 222 myocardial infarction, 217

N Na+, 186 Na2SO4, 104

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Index

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NAD, 48 NADH, 87, 186, 223 Native Americans, 214 natural compound, 10, 90, 99, 117 natural food, 135, 136 necrosis, 168, 176, 216, 222 nephropathy, 175 Netherlands, 179, 219 neurodegenerative diseases, 99 neurodegenerative disorders, ix, 97 neuropathy, 175 neurotransmitter, 67 neutral, 132, 138 nicotinamide, 199 nitric oxide, 87, 91, 167, 223 nitrite, 87 nitrogen, 8, 18, 107, 132 nitrogen gas, 107 NMR, 38, 40 non-enzymatic antioxidants, 74 normal development, 34 North Africa, 178 NSAIDs, 218, 220, 221, 222, 224 nuclear magnetic resonance, 38 nucleation, 44 nucleus, 223 nurses, 220 nutraceutical, xi, 111, 213 nutrient(s), vii, 15, 17, 18, 20, 23, 26, 51, 126 nutrition, 20, 21, 28, 98

O obesity, x, 168, 173, 174, 175, 176, 185, 186, 187, 191, 192, 193, 194, 195, 197, 201, 203 oceans, 45 ODS, 103 oesophageal, 218 OH, 2, 25, 66, 77, 82, 83, 89, 115, 181 oil, 9, 78, 80, 82, 106, 108, 129, 130, 131, 134, 136, 138, 140, 141, 215 oilseed, 108 oligomerization, 40 oligomers, 24, 44, 147, 148, 151 oligosaccharide, 41 olive oil, 112, 130, 140 oncogenes, 221 operating costs, 120 opportunities, 55 optical density, 10, 132 organ, 152 organelle, xi, 35, 213 organic compounds, 109, 139

organic solvents, 64, 88, 101, 108, 111, 112, 134, 135 organism, 148, 152 organs, 7, 16, 35, 37, 40, 148, 152 osteoporosis, 126 overweight, 174, 175, 204 overweight adults, 174 oxalate, 42 oxidation, 7, 8, 9, 10, 27, 40, 41, 44, 46, 51, 66, 76, 78, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90, 99, 128, 133, 135, 139, 141, 153, 177, 179, 191, 192, 197, 201, 202 oxidation products, 84, 89 oxidative damage, ix, 73, 83, 90, 99, 140, 166 oxidative stress, 2, 22, 66, 74, 84, 90, 99, 168, 176, 192, 193, 194, 195, 200, 202, 203, 223 oxygen, 27, 48, 66, 74, 76, 83, 85, 86, 90, 99, 101, 111, 112, 126, 131

P Pacific, 17 pain, 214 paints, 98 Pakistan, 28 pancreas, 175, 196, 200 Paraguay, 178 parallel, 223 participants, 219 partition, 78, 80, 81, 108 pathogenesis, 222 pathogens, 17, 51, 137, 222 pathways, 46, 47, 50, 53, 165, 167, 188, 192, 194, 203, 217, 222, 223 PBMC, 167 peptic ulcer, 224 peptidase, 177 peptide(s), 54, 177, 195, 198 perfusion, 190 peripheral blood, 167 peripheral blood mononuclear cell, 167 permeability, 141 peroxidation, 17, 66, 82, 85, 89, 99 peroxide, 42, 86, 132, 140 peroxynitrite, 87 pesticide, 224 petroleum, 103 pH, 8, 10, 49, 51, 84, 87, 88, 102, 103, 105, 108, 132, 133, 134, 135, 137, 138, 141 pharmaceutical, viii, x, 63, 67, 69, 74, 98, 173, 213, 214 pharmacokinetics, 20, 66 phenol, ix, 4, 7, 10, 38, 73, 77, 82, 88, 98

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Index phenoxyl radicals, 136 phenylalanine, 47, 48, 98 phosphate, 47, 88, 103, 193, 195, 196, 199, 200 phosphoenolpyruvate, 176, 193, 196, 199 phospholipids, 202 phosphorescence, 86 phosphorous, vii, 15, 18 phosphorylation, 176, 192, 201, 216, 222, 223 photolysis, 8 phycoerythrin, 85 phylum, 168 physicochemical characteristics, 134 physicochemical properties, 131 Physiological, 29, 196 physiology, 125 phytosterols, 24, 98 PI3K, 176, 192, 193, 194, 195, 196, 200, 202 pigmentation, 67 pigs, 166 pith, vii, 15, 17, 26 placebo, 219 plant based foods, vii, 1 plant growth, 35 plant kingdom, viii, 1, 15, 27, 28, 34, 75, 99 plants, viii, xi, 2, 5, 7, 10, 15, 16, 17, 18, 26, 28, 33, 34, 35, 45, 46, 48, 51, 52, 54, 55, 63, 64, 66, 67, 69, 74, 75, 98, 99, 108, 113, 114, 115, 125, 130, 131, 133, 134, 142, 178, 213, 214, 215, 216, 222, 223 plasma membrane, 50 plasma proteins, 84 plasticity, 35, 51, 52 platelet aggregation, x, 167, 213, 217 platelets, 167 playing, 191 polar, 82, 108, 111, 112, 113, 114, 117, 140 polarity, 82, 100, 109, 110, 111, 117 polarizability, 110 polarization, 110 pollution, 126 polymer, 16, 35, 43, 44, 51, 88, 152, 162 polymerization, 43, 46, 52, 53, 147, 148, 151, 153, 161 polymerization process, 52 polymers, 17, 23, 24, 28, 35, 37, 43, 45, 53, 75, 101, 147, 149, 151, 152, 161, 165 polyp(s), 218 polypeptide, 195 polyphenols, ix, 5, 6, 74, 97, 98, 100, 101, 108, 109, 110, 111, 114, 115, 116, 117, 128, 130, 132, 147, 148, 149, 151, 152, 159, 160, 163, 164, 166, 167, 174, 177, 179, 184, 190, 199, 201 polysaccharide chains, 21, 51

239

polysaccharide(s), viii, 7, 16, 17, 20, 21, 23, 24, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 51, 53, 55, 67, 68, 69, 126 polyunsaturated fat, 139 polyunsaturated fatty acids, 139 pools, 52 poor performance, 100 population, viii, 16, 17, 175, 183, 194, 197, 218, 219 population growth, 175, 194, 197 portal vein, 66, 148, 152, 187, 188, 190 potassium, vii, 8, 15, 18, 88 potassium persulfate, 8, 88 potato, vii, viii, 4, 15, 26, 27, 64, 130 potential benefits, 174, 224 PPAR ligands, 201 precipitation, 132 preparation, vii, ix, x, 5, 43, 64, 67, 97, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 113, 114, 115, 119, 120, 125, 139, 141, 199, 201 preservation, 141 preservative, 28 prevention, ix, 22, 54, 97, 98, 174, 193 primary antioxidants, 76 principles, 122 pro-apoptotic proteins, xi, 213, 216, 221, 223 probability, 222 probe, 85, 86 probiotic, 138 producers, 34 professionals, vii pro-inflammatory, 167, 176, 216, 218, 221, 222 proliferation, 176 proline, 90 promoter, 54 propagation, 8, 136, 139 prophylactic, x, 213, 218, 219, 220, 224 prophylaxis, 217, 218 prosperity, 174 prostaglandin thromboxane, x, 213 prostaglandins, xi, 213, 216, 217, 218 prostate cancer, 218 proteasome, 216, 221 protection, 9, 11, 46, 54, 84, 87, 174, 185, 192 protective factors, 63 protective role, ix, x, 1, 73, 173, 174 protein kinase C, 176, 195, 199 protein kinases, 222 protein oxidation, 27 proteins, xi, 6, 37, 38, 48, 49, 51, 84, 90, 108, 125, 139, 191, 213, 216, 221, 222, 223 proteolysis, 221 protoplasm, 41 Pseudomonas aeruginosa, 139

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PTFE, 106, 107 public health, 174, 218 pulp, 37, 39, 106 pure water, 110 purification, ix, 69, 97, 104, 113 purity, 69, 112, 118

Q quality of life, 203 quantification, 22, 85, 99, 101, 102, 114, 161 quercetin, 9, 27, 84, 134, 138 quinone(s), 18, 24, 79, 81, 82

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R radiation, 109, 110, 126 radical formation, 88 radicals, vii, ix, 1, 8, 9, 27, 53, 66, 74, 76, 78, 83, 84, 85, 87, 88, 89, 91, 99, 126, 136 radio, 41 raw materials, 24, 114, 130, 139 reactant, 88 reaction time, 10, 88, 90, 107 reactions, 5, 8, 35, 39, 44, 47, 48, 74, 82, 85, 87, 91, 102, 107, 136, 139, 153, 154, 165 reactive oxygen, vii, 1, 42, 66, 99, 132, 166, 200, 216, 220 reactivity, 77, 84 reading, 8, 216, 221 reagents, 10, 115 receptors, 191 recognition, 168, 222 recovery, 67, 68, 107, 110, 111, 120, 190, 191 recurrence, 220 red wine, 5, 10, 104, 149, 150, 151, 158, 160, 161, 166 reducing sugars, 23 regulations, 74 relaxation, 87 relevance, 148 reliability, 100, 118 repair, xi, 213, 216, 221 reproduction, 17 requirements, 101, 115, 136 researchers, 8, 28, 169, 219 resection, 220 residues, 35, 37, 38, 40, 41, 42, 43, 44, 54, 128, 176 resistance, 52, 55, 134, 176, 222 resolution, 114, 115, 116, 117 resorcinol, 4 resources, 45

response, xi, 5, 17, 45, 87, 186, 200, 213, 216, 217, 220, 221, 222 resveratrol, 16 reticulum, 48, 151 retinopathy, 175 Rhizopus, 137 Rhodophyta, 45 ribose, 84 rice husk, 129 rings, 81, 134 risk(s), x, 16, 27, 28, 121, 125, 126, 173, 174, 175, 176, 177, 216, 217, 218, 219, 220, 224 risk benefit, 177 risk factors, x, 173, 174 RNA, 139 RNAi, 38 rodents, 154, 166, 186, 191, 201 room temperature, 8, 103, 104, 111 root, vii, 15, 17, 18, 26, 27, 28, 34, 37, 39 roots, 16, 37, 45, 51, 138

S safety, 135, 139, 177, 217 salicylates, 214, 215, 223 Salmonella, 138, 166 salts, 110, 132, 216 saturated fat, x, 173 saturation, 53, 190 scarcity, 6, 7 scavengers, 77, 81, 87, 136 science, vii scope, 137 SCX, 104 secondary metabolites, viii, ix, x, 15, 27, 28, 34, 98, 125 secrete, 41, 42, 176 secretion, x, 37, 41, 48, 67, 173, 177, 190, 192, 194, 196, 200, 202 sedative, viii, 63, 64, 67 sedentary lifestyle, x, 173, 174, 175 seed, 18, 20, 23, 28, 65, 117, 128, 129, 132, 138, 140, 166 seedlings, 37, 104 selectivity, 100, 108, 113, 115 sensing, 67 sensitivity, 86, 115, 118 serine, 176, 220 serum, 20, 66, 87, 176, 185, 186, 194, 195, 196, 197, 200, 201, 214, 221 serum albumin, 87, 196 shape, 35, 116 shelf life, 135, 139

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Index shoot(s), 36, 38, 43, 38, 179 showing, 36, 37, 38, 81, 117, 161, 174, 184, 202, 221, 224 side chain, 16, 37, 74, 77, 78, 79, 80, 81, 82, 99, 153, 167 side effects, 142, 174, 176, 203 signal transduction, 223 signalling, 174, 176, 192, 194, 217, 222 silica, 106, 113, 114 Sinai, 205 skeletal muscle, 176, 192, 197, 202 skeleton, ix, 73, 74 skin, 5, 67, 128, 129, 155, 160, 215, 221 small intestine, 6, 7, 147, 148, 151, 152, 186, 187, 190, 191 smoking, 126, 175 society, x, 173 sodium, vii, 15, 18, 87, 113, 166, 186, 190, 217 solid matrix, 108, 109 solid phase, ix, 97, 113 solid tumors, 66 solubility, 81, 110, 111, 131, 134 solution, 8, 87, 89, 90, 103, 106, 108, 135, 141 solvents, 107, 108, 109, 110, 111, 112, 113, 115, 119, 120, 132, 134 South Africa, x, 173, 179, 206, 208, 214 South America, x, 17, 173, 178 SP, 97 Spain, 33, 147, 169 specialists, vii species, vii, 1, 27, 36, 37, 40, 42, 44, 45, 48, 49, 50, 52, 65, 66, 76, 84, 85, 98, 99, 113, 129, 130, 131, 132, 138, 148, 153, 165, 186, 200, 214, 215, 216, 220, 222, 223 specific gravity, 131 spectroscopy, 38, 40, 90, 91, 132 sperm, 141 spin, 66, 86, 91 spore, 136 Sprague-Dawley rats, 185 squamous cell, 167, 220 squamous cell carcinoma, 167, 220 stability, 27, 41, 79, 80, 81, 131, 132, 136, 187 stabilization, 5, 78, 79, 81, 135, 136 stable complexes, 126 stable radicals, 8 standard deviation, 4 standardization, 10, 217 staphylococci, 137 staple foods, vii, 15, 17, 18, 26, 27 starch, 20, 26, 53, 54 state(s), viii, 5, 63, 64, 74, 75, 86, 111, 131, 137, 186 sterilisation, 132

sterols, 10, 26, 99, 108, 112 stimulant, 178 stimulation, 201, 221 stock, 103 stomach, 7, 153, 187, 190, 214, 217, 224 stomata, 54 storage, 50, 132, 141, 187 stress, 5, 99, 175, 200, 214, 215, 222, 223 stroke, 2, 175, 176, 217 structural characteristics, 82 structural protein, viii, 33 structure, viii, ix, 2, 8, 9, 16, 24, 27, 33, 34, 35, 37, 43, 45, 51, 52, 53, 73, 74, 77, 81, 92, 98, 99, 101, 126, 127, 148, 214 structure–activity relationships, ix, 73, 77 style, 151 subgroups, 16, 167 substitution(s), 77, 79, 80, 166, 167 substrate(s), ix, 37, 49, 68, 74, 80, 82, 83, 89, 128, 176 sucrose, 108, 186 sugar beet, 37, 40 sugarcane, viii, 63, 68, 71, 130 suicide, 221 sulfate, 155, 159, 163, 166 sulfonylurea, 177 sulfur, 98 Sun, 29, 30, 59, 70, 91 supplementation, 161, 163, 193, 199, 200, 201 supplier, vii, 15, 17 suppression, xi, 67, 185, 213, 220, 221 surface area, 113 surface tension, 112 survival, 45, 175, 220 susceptibility, 7, 222 suspensions, 41, 53, 165 Sweden, 219 swelling, 141 switchgrass, 38 synergistic effect, 174, 185, 203 synovial fluid, 90 synthesis, 37, 38, 41, 46, 48, 49, 50, 52, 53, 135, 177, 186, 201, 217, 223 synthetic antioxidants, vii, 1, 139, 142

T T cell, 67 Taiwan, 15, 26 tannins, 2, 4, 16, 19, 34, 98, 101, 107, 121, 127, 132, 151 target, xi, 20, 84, 85, 86, 99, 107, 109, 112, 113, 114, 115, 116, 185, 202, 203, 213, 220, 221

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242 taxa, 43 techniques, ix, 52, 66, 90, 91, 97, 99, 100, 101, 102, 108, 109, 110, 111, 112, 113, 114, 115, 117, 119, 120 technology(s), 69, 70, 116, 117, 120, 122 temperature, 9, 10, 51, 101, 104, 105, 108, 109, 111, 112, 115, 132, 179 tensile strength, 141 tension, 132 terpenes, 6, 98, 107 testing, 217 therapeutic effects, 174 therapeutics, 187 therapy, 66, 176, 220 thermal decomposition, 85, 86 thermal resistance, 136 thermal treatment, 132 thiazolidinediones, 176 thrombosis, 141, 217 tissue, 2, 7, 37, 40, 46, 176, 185, 201, 202 TNF, 166, 167 TNF-α, 167 tobacco, 37, 48, 223 tocopherols, 128 total cholesterol, 185, 196, 199 toxic effect, 42 toxicity, 141, 174, 177, 192 transcription, 48, 50, 191, 192, 202, 216, 221, 223 transcription factors, 50, 191, 192, 202, 216, 221, 223 transferrin, 84 transformation(s), 132, 135 transgene, 54 transition metal, 27, 76, 132 transition metal ions, 76 translocation, 192, 194 transport, 45, 50, 51, 108, 190, 201, 216, 223 transportation, 187 treatment, 34, 37, 42, 53, 68, 100, 101, 102, 103, 104, 105, 106, 107, 132, 159, 163, 164, 176, 177, 185, 186, 187, 193, 201, 202, 218, 221 treatment methods, 101 trial, 218, 219 triglycerides, 193 tryptophan, 67, 90 tumors, 67 tumour growth, 218 tumours, 216, 218, 219, 221, 222 type 1 diabetes, 175 type 2 diabetes, x, 173, 175 tyrosine, 4, 10, 54, 87, 90, 98, 176

Index

U UK, 121, 206, 213, 215, 219 ultrasound, 108, 109 United, 26, 29, 178, 179 United Kingdom, 179 United Nations, 29 United States, 26, 178 urban, 217 urbanisation, 174, 175 uric acid, 9, 84 urine, 6, 7, 90, 148, 151, 152, 153, 155, 160, 162, 188, 190, 191 Uruguay, 178 USA, 55, 60, 61, 62, 121, 122, 123, 178, 179, 204, 206, 220, 226, 227, 228 USDA, 11, 169 UV, 40, 46, 64, 67, 76, 86, 89, 102, 103, 104, 106, 114, 115, 118, 119, 134 UV absorption spectra, 118 UV light, 76, 114 UV radiation, 46 UVB irradiation, 221

V vacuole, 54 vacuum, 104 valence, 88 variables, 5, 108, 109, 110, 111 varieties, 5, 10, 18, 19, 20, 21, 27, 64, 111, 129 vascular endothelial growth factor (VEGF), 216, 222 vascular system, 52 vascularization, 45 vasodilatory activities, viii, 16, 27 vegetables, vii, viii, ix, x, 2, 4, 5, 6, 10, 15, 16, 17, 18, 26, 28, 63, 64, 73, 98, 99, 108, 114, 125, 126, 127, 128, 151, 190 VEGF expression, xi, 213 very low density lipoprotein, 199 vessels, 45, 120 viscosity, 112 vitamin C, 84, 199, 202 vitamin E, 84, 199, 202 vitamins, vii, 5, 15, 18, 24, 74, 139 VLDL, 199 volatility, 115

W Wales, 213 wall polysaccharides, viii, 33, 35, 42, 43

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243

Index worldwide, 22, 175, 177

X xylem, 46

Y yeast, 138, 195, 200 yield, 101, 108, 132, 134, 222

Z zinc, vii, 15, 18 Zulu, 179

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Washington, 220 waste, 106, 128, 129, 130 waste water, 128, 129, 130 water, viii, 8, 33, 38, 39, 45, 46, 51, 64, 80, 82, 85, 88, 102, 103, 104, 105, 106, 107, 108, 110, 111, 112, 116, 119, 129, 130, 131, 133, 134, 135, 138, 141, 155, 157, 160, 178, 179, 182, 183, 184, 185, 186, 187, 189, 191, 198, 224 water vapor, 141 wavelengths, 91, 114, 118, 119 web, 144 weight gain, 177, 200 Western Cape Province, 179 WHO, 174, 175, 204 wild type, 54 workers, 8 World Bank, 17 World Health Organisation, 174, 204

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