Antioxidants in food and biology Facts and fiction Also in the Oily Press Lipid Library: Volume 19. Lipids: Structure, Physical Properties and Functionality Written by Kare Larsson, Peter Quinn, Kiyotaka Sato and Fredrik Tiberg Volume 18. Lipid Oxidation Written by Edwin N. Frankel Volume 17. Bioactive Lipids Edited by Anna Nicolaou and George Kokotos Volume 16. Advances in Lipid Methodology – Five Edited by Richard O. Adlof Volume 15. Lipid Analysis (third edition) Written by William W. Christie Volume 14. Confectionery Fats Handbook Written by Ralph E. Timms Volume 13. Lipids for Functional Foods and Nutraceuticals Edited by Frank D. Gunstone Volume 12. Lipid Glossary 2 Written by Frank D. Gunstone and Bengt G. Herslöf Volume 11. Lipids in Nutrition and Health: A Reappraisal Written by Michael I. Gurr Volume 10. Lipid Oxidation Written by Edwin N. Frankel Volume 9. Trans Fatty Acids in Human Nutrition Edited by Jean Louis Sébédio and William W. Christie Volume 8. Advances in Lipid Methodology – Four Edited by William W. Christie Volume 7. Advances in Lipid Methodology – Three Edited by William W. Christie Volume 6. Waxes: Chemistry, Molecular Biology And Functions Edited by Richard J. Hamilton (out of print) Volume 5. Lipids: Molecular Organization, Physical Functions and Technical Applications Written by Kåre Larsson Volumes 1– 4. Out of print Woodhead Publishing in Food Science, Technology and Nutrition Antioxidants in food and biology Facts and fiction EDWIN N. FRANKEL University of California, California, USA Oxford Cambridge Philadelphia New Delhi Published by Woodhead Publishing Limited, 80 High Street, Sawston, Cambridge CB22 3HJ, UK www.woodheadpublishing.com www.woodheadpublishingonline.com Woodhead Publishing, 1518 Walnut Street, Suite 1100, Philadelphia, PA 19102-3406, USA Woodhead Publishing India Private Limited, G-2, Vardaan House, 7/28 Ansari Road, Daryaganj, New Delhi – 110002, India www.woodheadpublishingindia.com First published by The Oily Press, 2007 Reprinted by Woodhead Publishing Limited, 2012 © PJ Barnes & Associates, 2007; © Woodhead Publishing Limited, 2012 The author has asserted his moral rights This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. 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Trademark notice: Product or corporate names may be trademarks or registered trade- marks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-9552512-0-7 (print) ISBN 978-0-85709-790-3 (online) This book is Volume 20 in The Oily Press Lipid Library Typeset by Ann Buchan (Typesetters), Middlesex, UK Printed by Lightning Source Preface The field of antioxidants has expanded over the past six decades into a wide variety of multidisciplinary areas that affect foods and health. This book conveys the complexity of antioxidant chemistry by providing an appreciation of the various phenomena that affect oxidation and its inhibition in foods and biological systems. By emphasizing mechanistic aspects of antioxidants and lipid oxidation, this book also attempts to sort out facts from fiction, by identifying the many problem areas requiring further research to improve our understanding of complex antioxidant effects and to stimulate better designed methodology and dietary studies for the future. The introductory Chapter 1 provides an overview of past, present and future aspects to initiate readers into the broad interdisciplinary fields of antioxidants in foods and biology. There is a vast basic literature on how antioxidant structures affect activity in solutions, but our knowledge on how these structural effects apply to multiphase foods and biological systems is limited. Knowledge on the sites of antioxidant action in foods and biological systems is necessary for a better understanding of their effects on their stability and susceptibility to oxidation. In foods, the activities of antioxi- dants are often difficult to predict and control, because their interactions with metal–protein complexes may either inhibit or promote oxidation. In biology, the activity of antioxidants is even more difficult to predict on the basis of in vitro studies, because interfacial interactions occur between dif- ferent cellular sites and the complex effects of enzyme cofactors and inhibitors, and immune systems. Chapter 2 deals with the classical chemistry necessary to understand more fully how antioxidants operate and the main aspects of the mechanisms of lipid oxidation and antioxidants. In addition to inhibiting the initiation and the propagation of oxidation, other multiple effects of antioxidants are dis- cussed, including inhibiting the decomposition of hydroperoxides, inactivating prooxidant metals, reducing hydroperoxides and scavenging oxygen. Due to the multiplicity of factors influencing antioxidants’ activities in complex foods and biological systems, the common use of artificial and non-relevant azo initiators to evaluate antioxidants is discouraged, because it may be misleading. Chapter 3 presents details on how the activity of antioxidants is affected at the interface of complex multiphase lipid systems. This chapter introduces the concept of interfacial antioxidation that depends on the partition of antioxidants v vi PREFACE between the aqueous phase, lipid phase and surfactant-enriched interface in foods and biological systems, and the colloidal chemistry of different types of emulsions affecting activity. Knowledge on the sites of antioxidant and prooxidant actions in multicomponent systems is essential to predict more successfully their activity in complex foods and biological systems. Chapter 4 discusses the problems of evaluating the activities of antioxidants in foods and biological systems. Because antioxidant activity is strongly affected by the physical composition of the target systems, valid methods to evaluate antioxidants require the control of a multitude of parameters. A judicious choice of several methods is also necessary to determine the effects of different products of lipid oxidation. The many important questions are discussed for the careful choice of antioxidant protocols in foods and biological systems. This chapter ends with recommended protocols based on several substrate properties for valid antioxidant evaluations. Chapter 5 on antioxidants in different foods deals with their interactions between food lipids, proteins and sugars, synergistic effects of phospholipids, and plant and beverage sources of phenolic compounds. This chapter includes information on the natural antioxidants in vegetable oils, milk, meat, fish and cereal products, special foods, herbs and spices. Plant polyphenols constitute the most important dietary antioxidants evaluated by a multitude of in vitro tests. The many analytical problems are discussed regarding the widespread use of one-dimensional methods to evaluate multifunctional food and biological antioxidants, and the caution required in making nutritional recommendations based on the so-called antioxidant capacity values of foods. Chapter 6 covers extensive worldwide research on biological antioxidants, generally based on the hypothesis that the health of an individual is influenced by the efficiency of various protection systems against oxidant damage. The nutritional approach to antioxidant therapy is, however, poorly understood due to the multiple interacting factors that relate degenerative diseases to diet and to oxidation. Because of a lack of reliable biomarkers of oxidative stress, animal and human feeding studies have produced controversial and mixed results. Although there is extensive evidence that diets high in fruits and vegetables rich in phenolic antioxidants are associated with a lower incidence of cardiovascular disease, very few studies have shown that flavonoids are directly responsible for health effects in the diet. Because of complexities in the behavior of natural phenolic antioxidants in different systems, the true impact of oxidation processes in biological tissues is controversial. Results of most in vitro and in vivo studies to assess the effects of phenolic antioxidants in biological systems are extremely difficult to interpret, because questionable methodology has been used to measure oxidation and the oxidative susceptibil- ity of polyunsaturated lipids and other biological targets. The chapter ends with the many questions that remain to be researched to understand and predict the effectiveness of phenolic antioxidants better in various biological systems. PREFACE vii Chapter 7 covers the renewed attention in biochemistry on the Maillard browning reaction, developed early in food chemistry, and on the interactions of reducing sugars, proteins and lipid secondary oxidation products. The complex biological modification of proteins by glucose in blood known as glycation and glycosylation involves oxidative and non-oxidative processes, producing low-molecular weight aldehydes that may initiate cardiovascular diseases and are implicated in age-related chronic diseases, including obesity, diabetes and renal disorders. The cross-linking between proteins and carbo- hydrates generates lifelong products of advanced glycation end products (AGEs) at later stages of the Maillard reactions, contributing to tissue degen- eration. Advanced glyco-sylation has been shown to affect a number of proteins and has been implicated in the pathogenesis of several diabetes and age-related diseases. Glycation products that are formed in heated foods and a number of recently developed inhibitors are discussed in detail to control their formation in biological systems. The final chapter, Chapter 8, addresses the important question that con- cerns food scientists and nutritionists today, as to whether additional or more effective natural phenolic antioxidants are needed in our diet to reduce oxidative stress from dietary and environmental factors, and the risk of cardiovascular disease. There is much in vitro evidence supporting a possi- ble beneficial role for polyphenols in preventing cardiovascular disease and cancer. Because oxidative damage is involved in atherosclerosis and other degenerative diseases, antioxidants have been generally thought to contrib- ute to cardiovascular protective effects. However, intervention trials with vitamin E and different phytochemicals produced confusing results. The beneficial nutritional effects of fruits and vegetables have been tied up to increased levels of antioxidants in the body. However, very few studies provide direct evidence that the benefits of eating fruits and vegetables are actually due to in vivo antioxidant activity. Many phenolic compounds rec- ognized for their antioxidant activity in vitro might have different and additional in vivo properties. Discussions include the evidence for several non-antioxidant activities of vitamin E, flavonoids and other phenolic com- pounds. Looking at future research, the nutritional and health properties of plant foods are examined, with a list of the many unsettled questions that deserve additional research using more relevant and reliable bioassays for clarification of the interactions between dietary polyphenols and health ef- fects. Another section discusses recent claims for the health benefits of organic compared to conventional plant foods. These claims have been diffi- cult to prove due to the complex environmental and agricultural factors known to influence the contents of phenolic compounds in plants, and be- cause very little reliable data have been published on their corresponding antioxidant activity. A final section deals with nanotechnology, a new devel- opment in food technology that deals with extremely small structures that viii PREFACE have unique, novel and potentially useful functional properties caused by modified interfacial phenomena with potential applications for antioxidants. The author gratefully acknowledges the invaluable editorial work of Frances Daniel. Edwin Frankel Department of Food Science and Technology University of California Davis, California 95616 USA [email protected] April 2007 CHAPTER 1 Introduction to antioxidants The field of antioxidants has expanded over the past six decades into a wide variety of multidisciplinary areas that have an impact on food chemists and biochemists. This introductory overview strives to convey the complexity of antioxidant chemistry, by providing an appreciation of the various phenomena that affect oxidation and its inhibition by antioxidants in multiphase foods and biological systems. Much confusion has developed as a result of a poor understanding of the role of complex interfacial interactions in heterogeneous food and biological systems, the limitations in the methodology applied in this field, poorly designed dietary studies of antioxidant supplementation, and exaggerated claims of their health benefits in the diet. This book will attempt to sort fact from fiction, by emphasizing the mechanistic aspects of antioxidants and lipid oxidation and by identifying the many problem areas needing further research to improve our understanding of complex antioxidant effects, and to stimulate better designed studies for the future. A. Past aspects The development of rancidity in foods containing polyunsaturated lipids has occupied the attention of food chemists and technologists for more than six decades. Much progress has been achieved in controlling lipid oxidation by applying principles of improving oxidative stability in packaging and the use of antioxidants. However, serious problems of oxidation continue in some foods because of: 1. Increasing emphasis on the use of polyunsaturated vegetable oils 2. Including oils containing long-chain omega-3 polyunsaturated fatty acids from fish and algae 3. Limiting the use of synthetic antioxidants 4. Concern over the consequences of using partial hydrogenation of poly- unsaturated vegetable oils to lower their susceptibility to oxidation 5. Increasing the practice of iron fortification of cereals and infant foods. Oxidation of polyunsaturated lipids not only produces undesirable rancid odors and flavors, but can also decrease the nutritional quality and safety of foods by the formation of secondary oxidation products in foods after cook- ing, processing and storage. Adding and exploiting the properties of antioxidants in foods are effective methods for the control of lipid oxidation. 1 2 ANTIOXIDANTS IN FOOD AND BIOLOGY The current applications of antioxidants in foods are often empirical, how- ever, and a better basic understanding of their mode of action is required to predict their activity in different food systems and when they are stored in different environments. 1. Natural versus synthetic antioxidants In the past two decades, the use of natural antioxidants has attracted special interest because of the possible, but poorly established, hazardous effects of synthetic antioxidants, and because of the worldwide trend against the use of regulated food additives. It should be made clear that most toxicological studies of synthetic food antioxidants have been carried out at concentrations representing several hundred times the average human consumption of these additives. The possible hazards from the presence of these materials in foods therefore may have been exaggerated. Recent evidence suggests that results from animal cancer tests at high intakes cannot be used to predict absolute human risks accurately when consumed at normal levels. The actions of food processors who remove antioxidants from their formulations are also economically motivated. The Food and Drug Administration (FDA) in the United States and other international regulatory agencies require extensive and expensive testing of food antioxidants to meet safety standards. Because such studies have become costly, many companies have eliminated the development and use of synthetic antioxidant additives to save on testing and reformulation costs. The elimination of synthetic antioxidants from many foods may not have been justified, however. In some cases, these synthetic antioxidants provide potential benefits in controlling cancer and biologically harmful oxidation reactions in the body. Overall, the deleterious effects of lipid oxidation products and reactive oxygen species may be greater than the possible hazards from the synthetic antioxidants used to inhibit their formation in foods. There is, therefore, an obvious need for studies that would more accurately estimate the benefits versus risks in the uses of synthetic antioxidants in foods. On the other hand, because natural antioxidants have been generally recognized as safe, their possible health risks have not been carefully investigated. Many studies of plant extracts evaluated for their antioxidant activities contained mixtures of several known and unidentified compounds without standardiza- tion. The possible health risks of these crude natural antioxidant mixtures have not been carefully analysed. However, the benefits from the protective effects of antioxidants against the hazards of reactive oxygen species in the body are more convincingly established than the toxicological hazards of these food additives. The advantages of food antioxidants may therefore outweigh their disadvantages.