Antioxidants in Food and Biology. Facts and Fiction-Oily Press (2007)

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

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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
enfrankel@ucdavis.edu
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.
INTRODUCTION TO ANTIOXIDANTS 3
2. Lipid peroxidation in vivo

The inaccurate but generally accepted term lipid peroxidation refers broadly to
oxidation and the oxidative deterioration of various biological systems. Ac-
cording to the well-recognized Free Radical Theory, in the absence of adequate
antioxidant protection, free radicals and reactive oxygen species cause damage
to lipids, proteins, lipoproteins and DNA, either by direct attack or by initiating
oxidation of polyunsaturated fatty acids (PUFA) in tissue membranes, causing
pathological changes contributing to degenerative diseases such as atheroscle-
rosis and cancer. According to the type of initiators and oxidants, several
mechanisms of antioxidant protection are known, including:
1. Inhibiting free radicals and reactive oxygen species and the formation of
hydroperoxides and aldehyde decomposition products
2. Preventing initiation and decomposition of hydroperoxides by chelating
and binding metals
3. Protecting biological tissues from lipid oxidation by several important
antioxidant enzymes.
Both animals and human subjects have been used to study the effects of
dietary antioxidant supplementation and lipid peroxidation in vivo. Studies
with experimental animals have included the testing of tissues obtained after
consumption of diets:
1. Deficient in vitamins E and C or antioxidants
2. High in saturated fats and/or cholesterol known to promote atheroscle-
rosis
3. Containing oxidized or heated fats, excess iron or other prooxidants.
Studies with humans have been largely limited to the analyses of blood com-
ponents, plasma lipoproteins, red blood cell membranes, breath, urine and
cultured cells.
3. Tocopherols and vitamin E antioxidants

Tocopherols are the most important natural antioxidants in vegetable oils and
the majority of plant foods. The tocopherols are not particularly effective
antioxidants, especially in PUFA-containing vegetable oils and in fish oils, and
foods containing trace metals. With natural soybean oil containing relatively
large levels of a mixture of α-, γ- and δ-tocopherols, the removal of 30 to 40%
of these tocopherols during processing does not generally affect the oxidative
stability, provided that adequate protection is afforded by chelation of metals
with citric acid. The γ- and δ-tocopherols are generally more effective in vitro
antioxidants than α-tocopherol, which is also less stable and more easily
oxidized. The early literature on the relative antioxidant effectiveness of
various tocopherol homologs is, however, controversial due to the varying

conditions, lipid substrates and methods used to test activity.
Nutritionally, α-tocopherol or vitamin E is recognized as one of the most
important lipid-soluble, chain-breaking antioxidants to prevent in vivo lipid
peroxidation. The biological activity of vitamin E was defined previously as
α-tocopherol equivalents equal to 1 mg α-tocopherol, 2 mg β-tocopherol,
10 mg γ-tocopherol and 3.3 mg α-tocotrienol (National Research Council,
1989). The discovery of a specific α-tocopherol transfer protein, which regu-
lates the concentration of vitamin E in blood plasma, accounts for the
bioavailability of α-tocopherol and the relatively low or lack of bioactivity of
the other γ-, and δ-tocopherol homologs that are prevalent in plant foods and
generally consumed by the public. The recognition of this biological specificity
led to the revision of tocopherol equivalents and now only α-tocopherol is
defined as vitamin E.
The relationship between the vitamin E activity and its antioxidant proper-
ties in the body has been debated for several decades. Other physiological
non-antioxidant activities have now been identified for vitamin E that include
the regulation of signal transduction pathways and cell proliferation, though
they are not yet fully described. γ-Tocopherol from plant sources has received
some attention in the literature, because it behaves as a powerful nucleophile
that reacts with nitrous oxides and protects lipids and DNA in vitro, though this
activity has not been demonstrated in vivo. The antioxidant activity of
γ-tocopherol has been suggested to be beneficial in reducing the production of
mutagens in the digestive tract, and thus may contribute to a lowering in risk of
colon cancer.
The new vitamin E dietary reference intake (DRI) recommended by the Food
and Nutrition Board, Institute of Medicine, in 2000, is 15 mg per day for adults,
with an upper tolerable limit of 1000 mg per day. There remain many difficult
questions to consider regarding these recommendations:
• How reliable is the new vitamin E DRI for the entire population?
• Does increased PUFA consumption in our diets, especially n–3 PUFA from
fish raise the requirement for vitamin E?
• Do oxidized food lipids in our diet raise the vitamin E requirements?
• Do dietary phenolic antioxidants decrease or increase the vitamin E require-
ments? Are they synergistic or antagonistic to vitamin E?
• How important are the non-antioxidant effects of vitamin E, such as signal
transduction and cell proliferation?
• Would the elderly population or individuals suffering from chronic disease
benefit from increased intake of vitamin E?
There is a current debate on whether vitamin E requirements are either not
high enough or too high. Some scientists recommend higher vitamin E supple-
mentation as part of a heart-healthy program. Other scientists consider that the
new DRI of 15 mg per day may be too high if individuals routinely consume
aspirin for its anti-platelet activity. Aspirin and vitamin E supplements com-
monly used in combination may increase the incidence of bleeding and
tendency to hemorrhagic strokes. According to some scientists, until the
physiological (non-antioxidant) functions of vitamin E are better understood,
the present DRI remains the best available recommendation.
4. Ascorbic acid or vitamin C

Ascorbic acid has multiple functions as a water-soluble antioxidant, a strong
reducing agent, a prooxidant and a metal chelator. Many of these effects act in
complicated combinations depending on conditions and the food or biological
system. In aqueous systems containing metal ions, ascorbic acid can promote
oxidation by reducing metals into the more catalytically active lower valence
state. If metals are inactivated by effective chelation, ascorbic acid acts as an
antioxidant, especially at higher concentrations. As with tocopherols, ascorbic
acid can exhibit different antioxidant effects even in the same food or biologi-
cal system, depending on the method and conditions used for testing its
activity. Ascorbic acid is readily oxidized into dehydroascorbic acid, according
to mechanisms that depend on pH, metals, oxygen pressure and water activity.
At physiological pH, ascorbic acid is ionized by donating a hydrogen atom to
form anionic ascorbate, which is in turn oxidized to dehydroascorbic acid
through a resonance stabilized ascorbyl radical. Ascorbyl palmitate and other
esters are used as antioxidants in vegetable oils, and may act synergistically
with tocopherols.
The recommended daily allowance (RDA) for vitamin C is 70 to 90 mg per
day, with an upper tolerable limit of 2 g per day. Among several recognized
biological functions, vitamin C is a cofactor for enzymes in the biosynthesis of
collagen, carnitine and neurotransmitters. Ascorbic acid is an efficient quencher
of reactive oxygen and nitrogen species, including superoxide, hydroperoxyl
radicals, singlet oxygen, nitrogen dioxide and hypochlorous acid. In many
systems, ascorbate exhibits a strong synergistic effect in the presence of vitamin
E. According to one mechanism, this synergistic effect is explained by the ability
of ascorbate to regenerate tocopherol from the tocopheroxyl radicals produced
during oxidation (Chapter 2.C). Another mechanism for this synergism involves
the metal chelating effect of ascorbic acid in protecting tocopherols and other
phenolic antioxidants that are readily inactivated by catalytic metals. Acting as a
peroxide destroyer, ascorbic acid may also reduce hydroperoxides into stable and
relatively benign hydroxy derivatives. Although both ascorbic acid and dehy-
droascorbic acid are reported to be good in vitro biological antioxidants, ascorbic
acid is effective in interrupting lipid peroxidation and protecting vitamin E. Other
in vivo biological activities of vitamin C include scavenging reactive oxidants in
activated leucocytes, lung and gastric mucosa, and inhibition of lipid peroxidation.
Since ascorbic acid is readily regenerated from dehydroascorbate in vivo, it is

difficult to predict the exact functional requirements as an antioxidant. As for
vitamin E, the optimal intake of vitamin C for health promotion and prevention
of chronic diseases is not well established, nor is the amount of vitamin C required
by children, pregnant women and older adults.
B. Present aspects
1. Interfacial phenomena
In heterophasic food and biological systems, the activity of antioxidants is
significantly changed in part by their tendency to partition between the aqueous
phase, the lipid phase and the surfactant-enriched environment. The net
effectiveness of antioxidants is in turn not only determined by their localization
in different phases, but also by other factors, including the colloidal properties
of the substrates, the conditions and the stages of oxidation used as endpoints,
the type and physicochemical state of the lipid substrate and its degree of poly-
unsaturation, the presence and types of initiators, emulsifiers and other
surface-active components, and their interactions. Antioxidant activity is thus
strongly affected by the physical composition of the target system, and the
relative activity of antioxidants of different polarity varies significantly in
different multiphase systems (Figure 1.1). For these reasons, valid methods for
the evaluation of antioxidants in complex foods and biological systems require
the precise control of several parameters affecting oxidation, and a judicious
choice of several methods to determine the effects of different products of lipid
oxidation.
Oil/water emulsion
Bulk oil
Hydrophilic
Lipophilic
antioxidants
LDL particle Lecithin

liposome
Bilayer
Figure 1.1. Antioxidant action in different lipid systems. Relative partition of hydrophilic and
lipophilic antioxidants in four different multiphase systems.
The activity of even single antioxidants varies in different bulk and multiphase
emulsion systems. In oil-in-water emulsions, hydrophilic antioxidants are
generally less effective than lipophilic antioxidants, whereas in bulk oil
systems, hydrophilic antioxidants are more effective. This interfacial phenom-
enon that is unrelated to the redox activity of the molecules has been explained
by the relative affinities of the antioxidants toward the air–oil interfaces in bulk
oil and the water–oil interfaces in emulsions. Research has now established that
oxidation itself is promoted at different sites within a heterogeneous food or
biological matrix. In essence, oxidation proceeds most rapidly where catalysts
tend to concentrate. Not surprisingly, the most effective antioxidants are those
that tend to concentrate preferentially in these same sites. In bulk oil systems,
hydrophilic antioxidants are apparently more protective against oxidation by
being oriented in the air–oil interfaces. The lipophilic antioxidants are less
protective by remaining in solution in the oil phase where they are present at
low concentrations. In contrast, in the oil-in-water emulsions, the lipophilic
antioxidants are oriented at the oil–water interface by virtue of their surface
activity, and are more protective against oxidation than the hydrophilic antioxi-
dants, which are dissolved and diluted in the water phase and become less
effective. Although these differences in interfacial behavior are evident for
antioxidants with great differences in polarity, they may not be obvious for
antioxidants with smaller differences in polarity. Interactions of antioxidants
with other surface-active components may counteract the effect of antioxidant
partitioning. These interactions are, however, poorly understood (see Chapter 5).
Although there is a vast literature on how antioxidant structures affect
activity in solutions, there is only limited knowledge on how these structural
effects apply in heterophasic systems, and the resulting partitioning in different
phases. Antioxidants may also be distributed into surfactant/emulsifier-rich
interfacial layers in heterophasic food emulsions. The partitioning properties of
a particular antioxidant depend not only on the chemical structure, and relative
polarity of the antioxidant, but also vary according to the lipid substrates,
surfactants, pH, temperature and the composition of the phases. Knowledge
about the sites of prooxidant and antioxidant action in multicomponent systems
is therefore necessary for better prediction of their effects on the oxidative
stability of complex foods and biological systems. Foods of improved quality
may be developed if the concentration, associations and driving forces of
antioxidants and prooxidants can be controlled in multiphase systems.
Significant differences can be expected in the antioxidant activity between
liposome and micelle systems, and how metal catalysts influence them. Metal-
catalysed lipid oxidation is thus greatly accelerated by adding a surfactant, by
changing from a liposome, composed of lecithin, to a micelle system, com-
posed of surfactant, by increasing the accessibility of the interface to the metal
prooxidant catalysts. It is not yet clear, however, why vitamin E is readily
oxidized in LDL and in micelles, but is more stable in liposome systems.
The critical role of trace metals in lipid oxidation and in biological cytotox-
icity, and their effects on antioxidant activity and stability of foods, has led to
important advances in the principles of metal chelation, especially in multipha-
sic systems. Reactions of oxygen radicals are complex, and the ways they
interact with antioxidants are poorly understood. This complexity is particu-
larly evident in foods and living tissues as a result of the compartmentalization
of multiple reaction environments. In general, the activity of any antioxidant is
significantly compromised in the presence of trace metals, and can therefore be
enhanced in the presence of suitable metal chelators. In the same way that
antioxidants are influenced by various properties of the food matrix, metal
chelators can either inhibit or promote oxidation, depending on reaction
conditions, their relative concentration and the colloidal properties of the
substrate. Although water-soluble chelators are usually effective against free
metal ions in aqueous media, in contrast, hydrophilic antioxidants are ineffec-
tive in the same aqueous systems where they can accelerate oxidation by
reducing metals to the more active lower valence state. In multiphase systems,
metal catalysis would be expected to occur in the interfaces between the lipid
and water phases (Figure 3.4). The interaction between various antioxidants
and metal protein complexes is often difficult to predict and may result in either
inhibition or promotion of oxidation. More research is needed to understand
more fully the chemical mechanisms of different metal protein complexes in
different reaction environments. In biology, the relative activity of antioxidants
is very difficult to predict on the basis of in vitro studies, because of wide
differences in interfacial interactions between different cellular localization
and the complex multiple effects of enzyme cofactors and inhibitors, and
immune systems.
2. Oxidant–antioxidant balance
A competition occurs in vivo between oxidative damaging and protective
processes that depends on many factors, including the polyunsaturated fatty
acid composition of tissue lipids, and the presence of various prooxidants,
antioxidants and defense systems. According to the oxidant–antioxidant bal-
ance hypothesis, to achieve optimum health, the damaging effects of reactive
oxygen species and lipid oxidation products must be counteracted by an
adequate supply of antioxidants or defense and repair systems. If antioxidant
protection is insufficient, an imbalance occurs, referred to as oxidative stress,
with resulting tissue damage and increased susceptibility to diseases.
Oxygen radicals or oxidants generated in vivo are now recognized as
mediators of various degenerative and inflammatory diseases, including rheu-
matoid arthritis, diabetes, cancer, cataract formation, immune and brain
dysfunctions, and the universal problems of aging. The relative toxicity of
oxygen radicals is also influenced by the antioxidants present in tissues that
ensure the removal of excess oxidants. Many kinds of tissue disruptive injuries
may initiate free radical reactions, particularly lipid peroxidation, by destroy-
ing the protective cellular membrane separation between oxidizable lipids and
prooxidant metals, by inactivating cellular antioxidants or by liberating damag-
ing metal ions from metal binding proteins in cells.
Extensive studies are now being carried out worldwide to test the oxidant–
antioxidant balance hypothesis. At the center of this hypothesis is the assumption
that the health of an individual is influenced by the efficiency of various
protection systems against repetitive and accumulated oxidant damage. The
sources of stress and tissue injuries resulting from oxidant damage may range
from viral infections, trauma, inflammation, cigarette smoke or environmental
pollution. Requirements of antioxidants will vary acutely in relation to the
oxidative cellular damage or oxidant stress, and would be therefore very
difficult to establish for a general population.
Evidence is accumulating that the principles being established for food
antioxidants may reflect and may ultimately be used to predict the ability of
these molecules to inhibit and control the adverse effects of biologically
harmful oxidation reactions in the body. Several animal feeding and epidemio-
logical studies have shown that antioxidants such as vitamin E can act as
antiatherogens, and that an increased intake of antioxidants may well, under
certain circumstances, be associated with decreased risk of cardiovascular
disease. As research progresses, however, conflicting results have failed to
develop convincing support for a simple relationship between the intake of
nutrients that exhibit antioxidant activity and the risk or incidence of chronic
diseases. While diets of fruits and vegetables containing high proportions of
antioxidants afforded protection from coronary heart disease in most studies,
the supplementation of experimental diets with individual phenolic antioxi-
dants produced mostly negative results. The correct nutritional approach to
antioxidant therapy is apparently unknown due to a poor mechanistic under-
standing of the multiple interacting factors that relate such diseases to diet and
to oxidation. There is also a lack of understanding on how antioxidants interact
in mixtures with other food components. The most glaring gap is the lack of any
reliable biomarkers of oxidative stress. Better-designed feeding studies may
provide a sounder mechanistic basis for improved antioxidant therapies, but it
is clear that there are significant obstacles yet to be overcome.
3. Oxidation of low-density lipoproteins (LDL)

There is now good evidence from in vitro and in vivo studies that the oxida-
tion of LDL is involved in the genesis and cause of atherosclerosis and
cardiovascular disease. Various degrees of oxidative modification have been
achieved by exposing LDL to endothelial or smooth muscle cells, or
macrophages found in arterial lesions. Minimally oxidized LDL increases
adherence of monocytes and expression of a scavenger receptor. More exten-

sively oxidized LDL increases its binding to the scavenger and other receptors
on the arterial macrophages that lead to foam formation, fatty streak lesions,
and ultimately to thrombosis and myocardial infarction. On this basis, the
hypothesis has been formulated that, by protecting LDL against oxidation,
antioxidants partially prevent or delay the progression of atherosclerosis and
vascular diseases.
Many complex biological effects have been reported for LDL oxidized in
vitro, but very few have been evaluated in vivo. The oxidative modification of
LDL in vitro either by cells or by copper catalysts can be effectively inhibited
by a large number of natural and synthetic antioxidants, including vitamin E,
butylated hydroxytoluene (BHT), and a wide variety of phytochemicals (see
Figures 2.5 and 2.6). Although antioxidants have been thoroughly demon-
strated to inhibit LDL oxidation and atherogenesis in many studies with animal
models, their anti-atherosclerotic effectiveness cannot be predicted on the
basis of ex vivo testing. Results from studies of even the oxidative susceptibility
of LDL isolated from subjects before and after antioxidant consumption have
been highly variable. These discrepancies may be due to variation in the
solubility of antioxidants and in the methods used to isolate LDL. Improved
methodology is required to clarify the complex mechanism of in vivo oxidative
modification of LDL.
Nutritional recommendations based on the ratio of LDL to HDL in blood
are generally based on the assumption that LDL high in cholesterol content is
atherogenic and that HDL is desirable because of its effects on reverse
transport of cholesterol and its antioxidant effect toward LDL under certain
conditions. The simple assumption that all LDL and HDL in circulation and
sampled by routine diagnostics are the same is clearly not true. Both LDL and
HDL are relatively heterogeneous populations of lipoprotein particles vary-
ing in composition, and in biological properties during blood circulation.
Considerable research has focused on, for example, the observation that
subsets of LDL can be isolated from humans that differ in their susceptibility
to oxidation. In more comprehensive epidemiological observations, those
individuals whose LDL tend to be of the more susceptible class are more
likely to exhibit heart disease. Recent studies indicate also that HDL can be
atherogenic when it is obtained from diseased subjects afflicted with inflam-
mation. The standard test for LDL/HDL ratios in blood used as a prognosis
for the risk of coronary heart disease may therefore not apply to the general
population and cannot be reliable for samples obtained from diseased sub-
jects. New measures of inflammation based on carbon-reactive proteins are
now being advocated as a more reliable diagnostic test for the risk of coro-
nary heart disease. Preventive treatments to prevent inflammation may
therefore be more effective in some individuals than the traditional treatments
to reduce blood cholesterol.
4. Phytochemicals
There is now a large body of evidence showing that diets high in fruits and
vegetables, which are rich in antioxidants, are associated with a lower inci-
dence of cardiovascular disease. This benefit is usually presumed to be related
to the levels of vitamins E and C and β-carotene, but the potential biological
effects of flavonoid compounds are generally ignored. The estimated daily
intake of flavonoids ranges from less than 25 mg to 1 g. Flavonoids are plant
secondary metabolites found in many fruits and vegetables, many of which are
potent antioxidants when tested in vitro. The scientific literature continues to
debate whether these compounds provide some health benefits when consumed
at effective levels as part of a varied diet. The flavonoid class of natural
antioxidants represents an important group of phytochemicals and includes
more than 5000 compounds thus far identified. The flavonoids may be impor-
tant in reducing the risk of atherosclerosis, as they also have been shown to
affect plasma lipids and inhibit platelet aggregation. Recently, the occurrence
of relatively high levels of polyphenolic antioxidants in fruits, vegetables and
certain beverages, especially red wine and tea, has received special attention
because of the potentially protective effect against damage from biological
oxidants. On this basis, strong recommendations have been made to include in
the diet more phenolic antioxidants from fruit and vegetables. Most studies
have focused on the activities of these compounds in widely consumed foods
and beverages, such as fruits, vegetables, legumes, chocolate, tea, wine and
olive oil, yet relatively little is known about the absorption and metabolism of
these compounds.
In addition to their antioxidant activity, dietary phytochemicals are thought
to have several beneficial activities by behaving as antibacterial and antiviral
agents, inducing or inhibiting several key enzymes affecting immune function
and thrombosis. Attempting to allocate recommendations for absolute daily
intakes of phytochemicals is difficult because of the large number of com-
pounds included, and because since they are not essential nutrients, there is no
distinctive deficiency syndrome nor physiological role required in food.
Flavonoids and anthocyanins are the most important natural antioxidants in
fruits and vegetables. Considerable research has been published on the flavo-
noid compounds of grapes and wine due to their importance to grape color and
taste, but they also are being studied in other plant foods. These flavonoids are
found in many fruits and vegetables in a wide variety of structures and
specificities. Green tea has also attracted much attention because of the
presence of unusually high levels of epigallocatechin gallate, and its physi-
ological effects. Marked variations are found in the antioxidant activity of
green tea and berry extracts in different lipid systems. More research is needed
to relate the studies showing antioxidant activities of flavonoids to their
potential health effects in the diet.
Although the pharmacological activities of various flavonoid compounds

have long been known, their nutritional significance is still controversial. The
enormous multiplicity of these ubiquitous compounds has made their system-
atic study very difficult. Known properties of the flavonoids include free
radical scavenging, strong antioxidant activities in preventing LDL oxidation,
inhibition of hydrolytic and oxidative enzymes (phospholipase A2,
cyclooxygenase, lipoxygenase), and anti-inflammatory actions. However,
present knowledge on the great diversity of phytochemicals and their biologi-
cal effects is incomplete. Several poorly established issues remain, including
the extent to which these plant antioxidants are absorbed in the body, and their
molecular mechanisms of protection in vivo and of disease intervention. There
are serious gaps in our understanding of the mechanism of how polyphenolic
compounds can influence disease risk in humans. Recent evidence suggests
that lipid hydroperoxides from dietary sources rise in blood during the post-
prandial state. Polyphenolic antioxidants in food and wine may thus provide
protection against damage to blood components by inhibiting this prandial
formation of hydroperoxides and in particular by decreasing the oxidative
susceptiblility of LDL oxidation.
5. Antioxidant testing
The literature on the activity of natural antioxidants in plant extracts in
protecting foods from oxidation is difficult to interpret because of the diverse
testing systems, methods and conditions used for oxidation. Oxidation in
complex foods and biological materials in general is largely initiated by the
combination of traces of metal catalysts and the presence of hydroperoxides.
The primary hydroperoxide products of lipid oxidation are highly susceptible
to decomposition, especially in the presence of metals. The aldehydes formed
by hydroperoxide decomposition are recognized as the main source of rancid-
ity in oxidized food lipids. Aldehydes also interact with biologically active
proteins, enzymes and lipoproteins, and produce toxicity at elevated levels.
While an attractive solution, natural antioxidants have been especially
difficult to evaluate in oils and food emulsions due in part to the complex
interfacial phenomena involved. In heterogeneous food systems the physical
properties, such as solubility and partition of the compounds between the
aqueous and lipid phases, can become crucial in determining antioxidant
activity. Thus, for example, the lipophilic antioxidants α-tocopherol, ascorbyl
palmitate and the methyl ester of carnosic acid (the active antioxidant in
rosemary extracts) are more effective in an oil-in-water emulsion system than
in bulk oil, while the opposite trend was found for the corresponding hy-
drophilic antioxidant derivatives Trolox (the carboxylic acid analog of
α-tocopherol), ascorbic acid and carnosic acid. These water-soluble anti-
oxidants often behave as prooxidants in aqueous systems by reducing catalytic
metals to the more active lower valence state. Interfacial phenomena are
apparently keys to a better understanding of antioxidant action in complex
foods and biological materials (see Figure 4.1). In the evaluation of natural
antioxidants, varied results can be obtained by methods measuring products at
different stages of lipid oxidation. For example, the effects of antioxidants in
inhibiting hydroperoxide formation should be distinguished from their effects
in preventing hydroperoxide decomposition. The decomposition of different
hydroperoxides into various aldehydes varies significantly with types of oils
and with temperature of oxidation.
Given these complexities of the behavior of natural antioxidants in relatively
simple food models, it is not surprising that the true impact of oxidation
processes in biological tissues is very controversial because of the questionable
methodology used to measure lipid oxidation. Results of most in vitro and in
vivo studies to assess the effects of oxidation and antioxidation processes in
biological systems are impossible to interpret, because questionable methodol-
ogy has been used to measure lipid oxidation and the oxidative susceptibility of
lipids containing polyunsaturated fatty acids.
In response to attempts to simplify the oxidation and antioxidative protection
processes, several non-specific assays have been introduced recently to esti-
mate a total antioxidant capacity of biological tissues and fluids. Even if in
principle one could imagine that the complexity of oxidation could be simpli-
fied to a small number of surrogate markers, these methods are not accurate
because they do not reproduce the actual events of oxidation in living tissues,
but instead many are based on artificial synthetic free radical initiators and on
non-physiological oxidation endpoints. The clinical utility of these tests is
therefore at best questionable. To learn about the real effects of antioxidants, it
is important to obtain specific chemical information on what substrate(s) are
protected and what products of oxidation are inhibited. Several specific assays
are needed to elucidate how oxidation products act in the complex multi-step
mechanism of lipid oxidation in foods and cause damage in biological tissues.
The problems of evaluating antioxidant activity in various complex food and
biological systems continue to be a serious barrier to progress in this field.
6. Nutritional effects of food ingredients

Recommendations to increase substantially the consumption of polyunsatu-
rated lipids in the diet require that we examine carefully the possible nutritional
consequences of such a change, including oxidative damage from reactive
oxygen species. In considering dietary changes, it is appropriate to look at all
of the variables related to oxidation. To illustrate this point, oxidation of LDL
in circulating blood has been implicated in the etiology of coronary heart
diseases. There is evidence that diets rich in oleic acid and antioxidants
increase the oxidative stability of LDL and may reduce coronary disease. Some
of the nutritional variables that affect lipid oxidation in LDL could be allevi-
ated either by consuming more antioxidants, or by increasing the amounts of
oxidatively stable oleic acid in our diet. Although in healthy subjects an
important major antioxidant defense is the prevention of transition metals
catalysing the generation of reactive oxygen species, excessive iron supple-
mentation in the diet may overload this defense system, especially in ill or
elderly individuals. The oxidative susceptibility of polyunsaturated lipids in
the diet may therefore be considered a risk factor. We need to improve our diet
by reducing or minimizing the risk factors associated with oxidative deteriora-
tion of polyunsaturated dietary lipids that may also be aggravated by excessive
iron supplementation. Flavonoid antioxidants may represent a positive poten-
tial in our diet and require further research to improve our understanding of
their mechanism of action.
7. Effect of antioxidants on aging

A large body of literature has accumulated in support of the free radical theory
of aging, postulating that life span is influenced by the relative accumulating
damage from reactive oxygen species and the resulting oxidative stress. The
damage can result either from an increase in oxidant generation, or from a
decrease in antioxidant defenses. Thus, the ultimate outcome of oxidative
stress is a function of the generation of oxidants, antioxidant defenses, and
repair of oxidative damage.
Cellular defenses include various antioxidant enzymes that inactivate reac-
tive oxygen species, antioxidant compounds that scavenge radicals, and
metal-binding proteins. Interactions between oxidants, antioxidants and vari-
ous repair systems are still very difficult to study experimentally. Many cell
repair mechanisms have been suggested. For example, phospholipases react
selectively with oxidized lipids, proteases with oxidized proteins and
glycosylases with oxidized nucleic acids. Because of the multiplicity of
enzymatic and non-enzymatic mechanisms of defenses, the measurements of
antioxidant activities have led to many confusing and conflicting results. There
is a variety of antioxidant enzymes known to act either synergistically or in
series of consecutive pathways. For example, the hydrogen peroxide produced
by superoxide dismutase is removed by catalase and glutathione peroxidase
(Chapter 2.F.2). The protective mechanism of glutathione reductase involves
reduction of oxidized glutathione (GSSG) into glutathione (GSH), to maintain
a high ratio of GSH/GSSG as required in normal cells. The age-related effects
of antioxidants can be ambiguous because when defenses are induced in
response to stress, a higher level of antioxidant enzymes may be the negative or
positive feedback consequence of either better protection or of an increase in
oxidative stress. Measurements of the susceptibility of various biological
systems to induced oxidation may only be considered as an indicator of
multiple antioxidant defenses. In general, accumulating evidence in the litera-

ture suggests that, on aging, cells may become less efficient in preventing
oxidative damage, and in repairing the resulting damage. When oxidative
damage occurs, either positive feedback can arise from the antioxidant defenses,
or negative feedback resulting from the malfunction of the oxidatively
damaged molecules.
C. Future aspects
Lipid oxidation proceeds by a complex sequence of reactions affected by a
multitude of factors that become extremely difficult to unravel in real food and
biological systems. These systems are multi-phased and controlled by complex
colloidal phenomena affecting different sites of oxidation and antioxidants. In
interpreting the effects of prooxidant and antioxidant compounds, we must
consider their effective and dynamic concentrations in different phases.
Many questions remain to be explored before we can better predict the
effectiveness of antioxidants in various systems.
• What are the relevant initiators of lipid oxidation?
• How are oxidants and antioxidants partitioned in different phases?
• What are the relevant levels of oxidation that should be inhibited by the
antioxidants?
The application of natural antioxidants in foods and biological systems has
received considerable interest because of their presumed safety and potential
nutritional and therapeutic effects. The evidence for their benefits is weakened,
however, by the lack of reliable chemical information on their effects in
inhibiting lipid oxidation in critical food and biological systems. Our under-
standing of the effects of antioxidant compounds can only be improved if more
specific methodology is used for evaluation under conditions and endpoints
relevant to foods and biological systems.
Although plant antioxidants in fruits and vegetables have clear protective
effects against coronary heart disease and cancer, the molecular basis of
protection is not understood. A better knowledge of the mechanism of antioxi-
dants will require more systematic research, with methods that can provide
specific chemical information directly related to oxidative modifications of
foods and biological systems. The failure of several epidemiological studies to
demonstrate a causative relationship between a low intake of antioxidants and
coronary heart disease has been variously attributed to the confounding influ-
ence of risk factors such as smoking, serum cholesterol, hypertension and
obesity. Negative results of many human dietary interventions testing the
effects of antioxidant supplementation may be due to poorly designed feeding
trials, based on unreliable or insufficient number of relevant endpoints. The
presence in food of hydroperoxides and lipid oxidation secondary products
known to be cytotoxic and mutagenic may also account for the ineffectiveness
of dietary studies using low levels of antioxidants. Nutritional studies will
remain unreliable until we better understand the molecular basis of the complex
mechanisms of antioxidant therapy, with more valid methods to measure
antioxidant activity in humans. More useful results may be derived from basic
studies focused on specific chemical effects and on several nutritionally
relevant biomarkers and endpoints. Many claims that have been made for the
health benefits from dietary antioxidants may be challenged until more physio-
logically valid biomarkers are used in the study of degenerative diseases that
slowly develop with age.
Many questions on the health effects of flavonoids and anthocyanins need to
be clarified by further research. Although in vitro studies have contributed
considerable mechanistic insights on the antioxidant actions of the aglycone
and glycosylated derivatives, very little is known about the antioxidant activi-
ties of the glycoside, glucuronide, sulfated and methylated metabolites (Figure
6.2), identified in blood after consumption of red wine and grapes. Other
questions that need to be clarified include how the partition of these metabolites
between aqueous and lipid environments affect their biological functions.
Based on the premise that free radical mediated damage could be controlled
by adequate antioxidant defenses, many exaggerated claims have been made
that a proper intake of antioxidant nutrients and supplements may have
nutritional value expressed vaguely as life-enhancing quality. However, many
dietary studies of antioxidant supplementation with animals and humans have
led to negative results. An important question is whether the effect of dietary
supplementation with selected antioxidants could be even tested in complex
organisms such as humans. Designing suitable in vivo models to investigate
properly the effects of antioxidants on oxidation of biological systems and on
aging is a formidable challenge that requires a much better understanding of the
biochemical mechanism of age and other related oxidative stress and defenses.
Vitamin E and other antioxidants are known to regulate the ways in which
oxidized LDL induces gene expression in endothelial cells and activation of
DNA binding to transcription factors, cell receptors, adhesion molecules, and
enzymes involved in cholesterol metabolism. These effects are apparently
related to the pro-inflammatory conditions associated with the initiation of
atherosclerosis. Future research on the molecular mechanisms controlling
genes sensitive to vitamin E, phytochemicals and other antioxidants may lead
to a better understanding of the initiation, causes and prevention of cardio-
vascular disease.
Essential antioxidant vitamin E and vitamin C as well as non-antioxidant
compounds in the diet have a wide variety of biochemical and physiological
effects that are distinct from their actions as chain-breaking antioxidants.
Oxidation cannot be simply inferred to be either the cause or effect in
experiments showing a significant effect of antioxidants on a physiological or
pathophysiological process. Many recent studies have inaccurately ascribed

oxidative mechanisms to events that are altered by antioxidants, when in fact
the effects are unrelated to oxidation per se. It is well known that vitamin E
affects cellular processes from gene transcription to membrane function by
mechanisms that do not involve oxidation directly. Therefore, studies to
understand the multiple functions of antioxidants in vivo must take into account
very creative controls to ensure that their mechanisms of action relate indeed to
oxidation.
Various phytochemicals also alter the induction of genes that influence the
metabolism of a variety of foods and pharmaceuticals. Therefore, consumption
of compounds that are thought to affect oxidation as antioxidants can also
influence metabolism in a much more general way. The interactions of diet and
drugs have been shown both to attenuate and exaggerate the actions of drugs.
Therefore, nutrient interactions are also to be expected, as the complexity of
antioxidant nutrient mixtures is explored and understood more fully.
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CHAPTER 2
Chemistry of antioxidation
Most unsaturated organic compounds react with oxygen when exposed to air,
heat or light. This oxidation has undesirable effects on flavors and odors,
nutritional properties and safety of lipid-containing foods. The use of various
antioxidants is an important method for the control of oxidation in foods and
biological systems, where free radical reactions are now implicated in the
development of many degenerative diseases. To understand better how antioxi-
dants operate, it is necessary to discuss first the main aspects of the mechanism
of lipid oxidation. The general topics of lipid peroxidation and biological
antioxidants were introduced in Chapter 1. The next chapter will deal with the
effect of lipid oxidation at the interface of complex multiphase lipid systems on
the activity of various types of antioxidants.
A. Free radical mechanisms

The oxidation of unsaturated lipids is generally a free radical chain reaction that
includes three processes: initiation, propagation and termination.
1. Initiation
Unsaturated lipids (LH) produce free radicals (L·) by hydrogen abstraction in
the presence of various initiators, including light, heat, peroxides or
hydroperoxides and transition metals (1):
––——➤ L· + ·H
Initiator
LH (1)
The formation of lipid free radicals by the decomposition of hydroperoxides
(LOOH) is much more energetically favorable than by the direct reaction of
lipids with oxygen. Hydroperoxides accumulated in small amounts as initial
products of oxidation are the most important source of initiation relevant to
antioxidation. Hydroperoxides dissociate either thermally to produce alkoxyl
radicals (LO·) (2), or catalytically by metals of variable valency in redox
reactions, producing alkoxyl radicals (3), and peroxyl radicals (LOO·) (4):
LOOH ––——➤ LO· + ·OH

Heat
(2)
LOOH + M+ ––——➤ LO· + OH– + M++ (3)
LOOH + M ––——➤ LOO· + H + M
++ + +
(4)
21
The simple thermal decomposition of hydroperoxides (2) seldom occurs

because it is extremely difficult to prepare lipids free of trace metals. The more
significant metal catalysed decomposition of hydroperoxides is more favorable
via reaction (3) than reaction (4) because the metal is about 10 times more
catalytically active in the lower valence state.
Hydroperoxides and carbonyl compounds introduced during processing can
also produce free radicals by direct photolysis.
LOOH ––——➤ LO· + ·OH

hν
(5)
>C=O ——➤ [>C=O*] 1
——➤ [>·C –·O]3 ——➤ >·C–OH + L· (6)
2. Propagation
In the presence of atmospheric air pressure, lipid radicals readily react with
molecular oxygen to produce peroxyl radicals (LOO·) (7).
L· + O 2 LOO· (7)
This addition reaction is extremely fast approaching diffusion control (k 109.5
5
M–1sec–1) and its activation energy is estimated to be zero. Although the reverse
reaction (–7) is usually unimportant, it can become significant with increasing
temperatures when oxygen becomes less soluble in lipids, less available and a
limiting factor in lipid oxidation.
The peroxyl radicals react with the substrate by the slow (k6 102 M–1sec–1) and
rate-controlling hydrogen transfer reaction (8) to form the primary
hydroperoxide (LOOH) products.
LOO· + LH ——➤ LOOH + L· (8)
Because this reaction is slow, the peroxyl radicals selectively abstract the most
weakly bound hydrogen atom from mono- and poly-unsaturated fatty acids.
The oxidizability of unsaturated fatty acids can thus be related to the ease of
abstraction of allylic hydrogens. Since methyl linoleate (18:2) is about 40 to 50
times more oxidizable than methyl oleate (18:1), the bis-allylic hydrogen on
carbon-11 in linoleate must be also more easily abstracted than the mono-
allylic hydrogens on carbon-8 and carbon-11 of oleate. Because methyl
linolenate (18:3) is about twice as oxidizable than linoleate, the oxidizability of
other polyunsaturated fatty acids increases approximately two fold for each
bis-allylic group. Thus the relative oxidation rates are 1, 2, 3, 4 and 5 for 18:2,
18:3, 20:4, 20:5 and 22:6, respectively.
3. Termination
When radicals accumulate to a sufficient level, they eventually interact to
terminate two chains and form stable molecular products. In the uninhibited
CHEMISTRY OF ANTIOXIDATION 23
oxidation (in the absence of an antioxidant) under atmospheric conditions, the

most important peroxyl radicals self-react by the Russell mechanism (9),
involving a tetroxide intermediate producing the corresponding carbonyl and
alcohol.
2 LOO· ——➤ [LOOOOL] ——➤ >C=O + O2 + >CH–OH (9)
There is evidence that the oxygen produced by this reaction is activated in the
singlet state.
Antioxidants at low concentrations interfere with chain propagation, chain
initiation and chain termination. In the inhibited oxidation, termination (10)
occurs through the reaction of peroxyl radicals with a chain-breaking phenolic
antioxidant (AH), by interrupting the chain reaction by hydrogen transfer to
produce a phenoxy radical A· that is too stable to continue the chain by reaction
(8).
LOO· + AH LOOH + A· (10)
Effective chain-breaking antioxidants produce a relatively stable phenoxy
radical A·, by the presence of bulky groups near the hydroxyl group, which
reacts more rapidly with the peroxyl radical LOO· than with the lipid substrate
LH. The antioxidant radical will either react again with a peroxyl radical to
form a stable peroxide (11), or react with another antioxidant radical to form a
dimer (12).
A· + LOO· ——➤ LOOA (11)
2 A· ——➤ A–A (12)
To break the free radical chain effectively the structure of an active antioxi-
dant is designed to produce a phenoxyl radical in which the unpaired electron
is delocalized around the aromatic structure and is stabilized by high resonance
energy (Figure 2.1). The activity is enhanced by bulky tributyl or electron-
donating substituents. Reactions (11) and (12) producing the antioxidant
hydroperoxide LOOA and antioxidant dimers A–A are supported by product
studies with different trialkylphenols varying in stability according to their
substituents (see Section B.2).
Less effective chain-breaking antioxidants may produce phenoxyl radicals
that are less resonance stabilized and can act as chain carriers by the hydrogen
chain transfer reaction (13), thus reinitiating the chain.
A· + LH ——➤ AH + L· (13)
Under certain conditions, antioxidants may have prooxidant activity, either
by the chain transfer reaction (13) producing lipid radicals, or by the reverse
reaction (–8) regenerating peroxyl radicals. The prooxidant reactions may
occur with less hindered antioxidants at high concentrations or at elevated
Figure 2.1. Resonance stabilization of phenoxy radicals from tri-substituted phenols.
temperatures, such as frying, or in the presence of metal catalysts or other

oxidation promoters such as free fatty acids. Antioxidants can also inhibit the
initiation steps (2), (3) and (4) by reacting with the alkoxyl and the peroxyl
radicals, and the decomposition of hydroperoxides by reacting with the alkoxyl
radicals produced by the homolytic cleavage reaction (14).
LOOH ——➤ LO· + ·OH (14)
The alkoxyl radicals from reaction (14) undergo cleavage to produces
aldehydes and other decomposition compounds, which contribute to rancidity
in foods and biologically damaging reactions with enzymes, proteins and
lipoproteins. Antioxidants may react with alkoxyl radicals either directly by
hydrogen donation to yield hydroxy compounds and stable antioxidant radicals
(15), or by a termination reaction between antioxidant radicals and alkoxyl
radicals (16).
AH + LO· ——➤ LOH + A· (15)
A· + LO· ——➤ LOA (16)
The antioxidant inhibition of hydroperoxide decomposition in reaction (15),
producing stable hydroxy lipid derivatives is important, because it diminishes
the aldehyde and other volatile products contributing to rancidity and biologi-
cal damage.
B. Classes of antioxidants
Antioxidants may be divided into two types, according to their mode of action
in inhibiting either the initiation or the propagation of oxidation. Many
antioxidants may also inhibit the decomposition of hydroperoxides and act as
oxygen scavengers.
1. Initiation inhibitors
Compounds that inhibit initiation include metal inactivators or chelators,
hydroperoxide destroyers and ultraviolet stabilizers. They are also known as
preventive antioxidants.
a. Metal inactivators. These compounds function by removing or chelating

metal catalysts to change their redox potential and inhibit reactions (3) and (4).
They may also inhibit the decomposition of hydroperoxides by preventing their
complexation with catalytic metal ions. The most common metal inactivators
used in vegetable oils and food lipids include citric acid, phosphoric acid and
ethylenediaminetetraacetic acid (EDTA) (Figure 2.2). The metal chelators
have to be selected carefully to avoid the activation of metals by altering their
redox potential. Although ascorbic acid may act as a metal chelator, under
certain conditions it has other effects and can be converted into a potent
prooxidant at low concentrations relative to the metal content. Chelators in
Figure 2.2. Structures of common metal chelators.

Table 2.1. Effect of EDTA and gallic acid on lipid oxidation in mayonnaise containing
16% fish oila
Samples TAGOOH CEOOH Fishy flavor Fishy aroma
Control 7.86 0.34 2.8 2.0

+ Emulsifier (Em)b 6.87 0.26 3.0 1.7
+ Gallic acid 3.13 0.19 3.4 2.5
+ Gallic acid + Em 2.49 0.13 3.0 2.1
+ EDTA 1.86 0.18 0.3 0.3
+ EDTA + Em 1.60 0.13 0.1 0.2
From Jacobsen et al. (2001)

a
Mayonnaise contained by weight 16.0% sand eel oil, 64% rapeseed oil, 10.4% water, 4.0% vinegar,
0.3% salt, 1.0% sugar, 0.1% potassium sorbate, 4.0% egg yolk, 0.15% emulsifier (diacetyltartaric acid
ester of mono- and diglycerides of fatty acids). Gallic and EDTA (in the form of calcium disodium
ethylenediaminetetraacetic acetate) (Em) added at the rate of 200 ppm. Analyses of hydroperoxy
triacylglycerol (TAGOOH), hydroperoxy cholesterol esters (CEOOH) and evaluations for fishy flavor
and aroma (sensory scale of 0 to 9) were made after 3 weeks storage at 20°C.
b
plus extra emulsifier
which oxygen atoms bind to the metal tend to prefer the oxidized forms of iron
or copper and decrease their redox potential.
Metal chelators and metal binding proteins are often more effective oxida-
tion inhibitors than chain-breaking antioxidants in n–3 PUFA-containing
foods, because they are particularly susceptible to oxidation and hydroperoxide
decomposition catalysed in the presence of metal contaminants. Thus EDTA
proved to be effective in inhibiting the oxidative and flavor deterioration of
mayonnaise containing fish oil, but gallic acid did not (Table 2.1). The effect of
metal chelators such as EDTA and metal-binding proteins such as lactoferrin
also proved to be particularly effective in inhibiting the formation and decom-
position of hydroperoxides in food systems supplemented with iron for
nutritional purposes.
The effects of iron and EDTA were tested in an emulsion containing fish oils
(Table 2.2). Although in the absence of added iron, 100 μM EDTA almost
completely inhibited the oxidation of the fish oil emulsion, in the presence of
100 or 200 μM of Fe2+, EDTA significantly promoted the oxidation. However,
EDTA effectively inhibited oxidation of these emulsions on increasing the
molar ratios of EDTA to iron (2:1 and 4:1). Therefore EDTA was an effective
iron chelator in these emulsions, when added at equivalent or higher molar
concentrations than those of iron. This prooxidant effect can be explained by
the preferential chelation of EDTA with Fe3+ relative to Fe2+. Because the
formation constant (log of equilibrium constant) (17) of EDTA:Fe3+ is larger
(Kf = 25.1) than of EDTA:Fe2+ (Kf = 14.3), in a mixture of Fe3+ and Fe2+, EDTA
Fen+ + Y4– FeYn–4 (17)
Kf = [FeY ]/[Fe ][Y4–]
n–4 n+
Table 2.2. Effect of iron and EDTA on increase in peroxide values of oil-in-water
emulsions containing fish oila
Emulsified mackerel oil Peroxide value (meq/kg)

24 h 48 h 72 h
Control 36.1 91.3 132

+ 100 μM Fe 49.8 109 157
+ 200 μM Fe 60.8 127 181
+ 100 μM EDTA 0.0 0.9 5.3
+ 100 μM Fe + 100 μM EDTA 54.6 169 246
+ 200 μM Fe + 100 μM EDTA 86.8 247 296
From Frankel et al. (2002).

a
Emulsions contained 5% oil and 1% lecithin as emulsifier.
Analyses for peroxide values were made after storage at 40°C.
can thus promote iron catalysed oxidation if used at insufficient concentrations

by favoring complex formation with Fe3+ over Fe2+, thus shifting the ratio in
favor of the more catalytically active Fe2+. To achieve proper inhibition of lipid
oxidation it is important therefore to use an excess molar concentration with
EDTA. The same precaution is necessary with ascorbic acid to avoid prooxidant
effects.
Various proteins inhibited lipid oxidation in different test systems, including
liposomes, fatty acids, oils and emulsions, by their capacity to bind or chelate
metal ions. The relative antioxidant potency of phenolic compounds in liposomes
was mediated to different extents by the presence of protein. At high relative
metal concentrations, however, these phenolic complexes can also promote
lipid oxidation.
Lactoferrin is an iron transport non-heme glycoprotein, present in human
milk and many biological secretions, which can effectively inhibit the oxida-
tion of various lipid systems through its iron-binding capacity. However, this
protein can also have prooxidant activity depending on the lipid system and its
concentration relative to the concentration of metal ions. Lactoferrin effec-
tively inhibited oxidation and increased the oxidative stability of infant formula
supplemented with iron (Table 2.3). Because of its antimicrobial properties,
lactoferrin may also be a useful additive in other foods supplemented with iron.
Other known proteins that chelate or inactivate transition metals found in
human serum include transferrin, which binds iron, and albumin, which binds
copper. Ceruloplasmin inhibits lipid oxidation apparently by oxidizing Fe2+ to
the catalytically less active Fe3+.
b. Hydroperoxide destroyers. These compounds are mainly reducing

agents that convert hydroperoxides into stable hydroxy products. Ascorbic
acid and tocopherols at more than stoichiometric concentrations can reduce
Table 2.3. Inhibition of hydroperoxide formation and decomposition by lactoferrin (LF)

in whey based infant formula supplemented with different levels of iron (Fe)a
Additives % Inhibition
Fe (μ M) LF (μ M) Hydroperoxides Hexanal Fe:LF ratio
0 12.5 61.9 72.0 0:12.5

0 12.5 92.1 95.2 0:25.0
88 12.5 18.7 27.8 7.0:1.0
88 25.0 65.4 77.7 3.5:1.0
88 44.0 94.7 95.8 2.0:1.0
172 12.5 17.5 33.9 13.8:1.0
172 25.0 48.0 76.8 6.9:1.0
172 86.0 94.4 92.5 2.0:1.0
220 12.5 23.2 65.9 17.6:1.0
220 25.0 64.8 87.9 8.8:1.0
220 110.0 101 97.8 2.0:1.0
From Satué-Gracia et al. (2000).

a
Analyses for hydroperoxides and hexanal were made after oxidation at 40°C.
hydroperoxides to produce stable hydroxy compounds in low yields. Sodium

borohydride has been used to reduce hydroperoxides accumulating during
vegetable oil processing, but has not been adopted commercially. Several
phosphorus and sulfur reducing compounds have been used in industrial olefin
applications, but they are not suitable for foods.
c. Ultraviolet stabilizers. Compounds that inhibit photo-induced lipid

oxidation include ultraviolet absorbers, which act by transferring light energy
without the formation of free radicals. Certain pigments and carbon black have
been used to stabilize industrial polyolefins against light exposure, but they are
not suitable for foods.
2. Propagation inhibitors
The most widely used phenolic antioxidants react generally with peroxyl
radicals according to the following stoichiometry (18):
n LOO· + inhibitor ——➤ stable products (18)
The value of n, referred to as the stoichiometric factor, is defined as the number
of radicals trapped by each molecule of antioxidant. This factor can be
determined either kinetically or by structure studies of the initial oxidation
products of the phenolic antioxidants. Kinetically the rate of oxidation is
determined in the presence of radicals that are produced solely by added
synthetic diazo initiators [e.g. α, α-azobisisobutyronitrile (AIBN)], which
decompose slowly to generate known quantities of radicals in a steady and

constant rate by reaction (19).
A-N=N-A ——➤ 2 A· + N2 (19)
The azo radicals (A·) react rapidly with oxygen to give peroxyl radicals (AO2·),
which attack lipids by abstraction of hydrogen atoms to form lipid radicals
leading to lipid hydroperoxides (20).
A· + O2 ——➤ AO·2 + LH ——➤ AOOH + L· (20)
The rate of initiation (Ri) is measured by determining the induction period (IP)
during which the oxidation is inhibited with a known concentration of an
antioxidant (AH), such as α-tocopherol which has a known n value of 2.
Ri = n [AH]/IP
Under these artificial initiation conditions, any antioxidant that can cause an
induction period is considered to react only with propagating radicals. From the
length of the induction period and the known rate of production of initiating
radicals, the number of free radicals reacting with each antioxidant molecule
may be calculated. An n value of 2 corresponding to the two peroxyl radicals
trapped by reactions (10) and (11) is found for many phenolic antioxidants
including various tocopherol homologs. Less active antioxidants such as
hydroquinones and tocoquinones have n values of less than 0.5.
This kinetic approach may be useful for studying free radical oxidation in
simple homogeneous model systems. This approach assumes the existence of
a stationary concentration of reaction intermediates, which is only true if an
initiator is added to the system and if it is the only initiator present. Further-
more, the added azo initiators are artificial systems that produce a large flux of
peroxyl radicals, thereby promoting the propagation phase leading to
hydroperoxide formation and minimizing their decomposition. In the absence
of an added initiator, the radical species and hydroperoxides change in concen-
tration continuously, especially during the induction period.
In contrast to metals that catalyse the initiation as well as the decomposition
of hydroperoxides, the artificial azo compounds used as initiators in kinetic
studies have no effect on the decompostion of hydroperoxides. Therefore, for
lipid oxidation in complex foods where the presence of trace metals and
hydroperoxides is generally unavoidable, artificial azo initiators are not rel-
evant and using them to evaluate antioxidants kinetically may be misleading.
C. Structure-activity relationships
Products and ESR studies provide more direct evidence than the kinetic
approach for the stoichiometry of lipid oxidation inhibited by phenolic anti-
oxidants. Thus, various other phenolic compounds behaved like α-tocopherol,
Figure 2.3. Formation of peroxy-cyclohexadienones and stilbene-quinone by oxidation of 2,6-di-tert-

butyl-4-methyl phenol (BHT) (Bickel and Kooyman, 1953).
in having a stoichiometric factor n = 2 or less. Both the peroxide products

(LOOA) of reaction (11) and the dimers (A-A) of reaction (12) were identified
from 2,4-dimethyl-6-t-butyl phenol and from less hindered phenols containing
more methyl than tert-butyl substituents (Figure 2.3). ESR studies substanti-
ated that the phenoxyl radical of BHT is formed by abstraction of the phenolic
hydrogen. The stability of the phenoxyl radical increases with the number of
ortho tert-alkyl groups, with a half-life of several minutes at room temperature.
The stilbene-diquinone is the end product of BHT formed via the most stable
‘galvinoxyl’ radical because the electron is delocalized equally in both aroma-
tic rings.
The effect of structures on antioxidant activity of substituted phenolic
compounds can be generalized as follows:
a. Electron-donating groups such as methyl and methoxy in the 2 and 4
positions significantly increase activity, whereas electron-attracting
groups decrease activity.
b. Aryl groups in the 2 and 4 positions that delocalize the unpaired electron
of the intermediate radical increase antioxidant activity.
c. Branched alkyl groups such as tertiary butyl in the 2 and 6 positions
increase antioxidant activity, but increasing branching in the 4 position
decreases activity (Figure 2.4).
Figure 2.4. Relative antioxidant activity of tri-substituted phenols for the oxidation of gasoline (Scott,
1965).
1. Inductive effects
Electron-donor substituents increase activity, whereas electron-acceptor
substituents decrease it. There is a linear relationship between antioxidant
activity and the redox potential of phenolic antioxidants. By attracting or
donating electrons from the phenolic ring, substituent X changes the energy of
the transition state of the peroxyl radical that is an electron acceptor, making
the phenoxy ring partially positive (adapted from Scott, 1993).
R
δ–
X δ+ O H O OR
Electron-donating substituents in the 4 position thus increase antioxidant

activity by decreasing the redox potential of phenols and changing the energy
of the transition state. Effective electron-releasing groups such as alkoxy and
alkyl produce the most favorable effects.
2. Steric effects
Phenolic antioxidants were most effective in inhibiting oxidation of petroleum
when all the ortho and para positions are substituted and when one or two ortho
substituents is a branched group such as tert-butyl (Figure 2.4). When the para
substituent is methyl, ethyl or n-butyl, the phenol is more effective than when
it is tert butyl. Apparently, increasing branching in the para position decreases
the ability of the phenoxyl radical to trap a second alkyl peroxyl radical.
With heavily hindered phenolic compounds (e.g. BHT, Figure 2.5), the
extent of inhibition is directly related to the antioxidant concentration. With
less hindered phenolic compounds (e.g. α-tocopherol, Figure 2.6), inhibition
increases and goes through a maximum with increasing concentration. This
decrease in activity at high antioxidant concentrations can be attributed to the
Figure 2.5. Structures of synthetic antioxidants.
participation of chain transfer reactions (–10) and (13). The effectiveness of

antioxidants depends therefore on the balance between the rates of inhibition
reactions (10) and (11) and the rates of chain transfer reactions (–10) and (13).
These transfer reactions are reduced by steric hindrance and are generally
completely suppressed by two bulky ortho substituents. When these transfer
reactions do not occur, the rate of oxidation is related to the ratio of lipid
concentration to that of the antioxidant concentration.
d[O2]/dt = k8 ki[LH]/k10[AH]
where k8 is the propagation reaction (8), ki is the initiation reaction (1) and k10
the inhibition reaction (10). With less effective antioxidants, transfer reactions
(–10) and (13) become important at high concentrations of phenolic com-
pounds (e.g. α-tocopherol) and at elevated temperatures (e.g. frying). The
effectiveness of phenolic antioxidants is also related to the stability of the
phenoxy radicals as influenced by delocalization (Figure 2.1) and by the
absence of oxidizable sites. The efficiency of an antioxidant will increase via
the presence of a labile A–H bond and the stability of the resulting phenoxyl
radical. The activities observed with different phenolic antioxidants vary
markedly with different oxidizing lipid substrates (Lipid Oxidation, 2nd ed,
2005, Chapter 9).
The activity of phenolic antioxidants is also affected by their initial oxidation
products. Some of these products have similar antioxidant activity, via the
same hydrogen donor property of the parent compound. Oligomeric deriva-
tives of partially oxidized phenolic compounds are also effective antioxidants.
The antioxidant activity of rosemary extracts is due to carnosic acid and
carnosol as main components and to rosemarinic acid as a minor component.
The structure of carnosic acid consists of three six-membered rings including
a dihydric phenolic ring and a free carboxylic acid (Figure 2.6). Carnosol is a
derivative of carnosol containing a lactone ring. Rosmarinic acid has two six-
membered rings including a dihydric phenolic ring, an ester group and a free
carboxyl group. Although carnosic acid and carnosol are readily oxidized,
reduced and isomerised into quinone and lactone products (Figures 5.4 and
5.5), they retain antioxidant activity.
Figure 2.6. Structures of natural antioxidants.
D. Synergistic antioxidant systems

Many antioxidant combinations are known to impart more protection against
lipid oxidation than the sum of the activities of the components used separately.
Effective synergistic inhibition can be achieved if both initiation and propaga-
tion are suppressed. Three kinds of synergisms can be observed.
1. Homosynergism
This arises between two antioxidants having the same mechanism. An effective
synergistic combination is achieved with a mixture of chain-breaking antioxi-
dants of different relative reducing capacity. The synergistic combination of
ascorbic acid (AscH) and α-tocopherol (α-TOH, Figure 2.6) in lipid systems
has long been known, and has received much attention in biological systems.
Considerable evidence has been obtained in support of a mechanism involving
the reduction of α-tocopheroxyl radicals (α-TO·) by ascorbic acid to regener-
ate α-tocopherol.
α-TOH + LOO· ——➤ α-TO· + LOOH (21)

AscH + α-TO· ——➤ α-TOH + Asc (22)
The one-electron reduction potential (Eo/mV) of α-tocopherol is 500 and that
of vitamin C is 282. This mechanism thus requires that the synergist is a
stronger reducing agent and has a lower oxidation potential than that of the
antioxidant. Although the lipid-soluble vitamin E is located in biological
membranes, the more polar tocopheroxyl radical formed by reaction (21)
becomes oriented at the membrane–water interface where it becomes accessi-
ble to the water-soluble ascorbate allowing reaction (22) to occur, thus recycling
α-tocopherol.
Synergism is also observed between two chain-breaking antioxidants of
different activities, such as BHA and BHT or BHA and propyl gallate (Figure
2.5). This synergism may be explained by an increase in oxidative stability of
the antioxidants by the mutual protection of the antioxidant radicals in a
mixture. Another type of synergism is observed with mixtures of phenolic
compounds and lecithin polyols and amino acids. These synergistic compo-
nents may act as metal scavengers. Lecithin may also behave by its emulsifying
properties to improve the contact between the polar phenolic antioxidants and
the less polar lipid substrate.
2. Heterosynergism
This arises between two antioxidants having different mechanisms. The com-
bination of a preventive metal inactivator such as citric acid and a chain
breaking antioxidant such as BHA, BHT or TBHQ is commonly used in foods
(Figure 2.5). Thus, vegetable oils containing varying concentrations of α-, γ-
and δ-tocopherol mixtures are effectively stabilized by added citric acid. In
these combinations, synergism may also arise from the protective effect of
citric acid against the metal catalysed oxidation of the tocopherol mixtures.
Phenolic antioxidants also have synergistic properties in reinforcing the anti-
oxidant properties of lactoferrin in various lipid systems supplemented with
iron for nutritional purposes. This concept was later expanded in biology to
suggest a hierarchy of redox systems interacting according to their respective
redox potentials. Ascorbic acid may also protect α-tocopherol by metal inacti-
vation in metal catalysed oxidations.
3. Autosynergism
This arises when one antioxidant has different functions. These kinds of
antioxidants are more effective and assume more practical and theoretical
significance in chemistry and biology. Flavonoids provide classical examples
of multiple inhibiting functions in the same molecule, especially in foods and
biology. Flavonoids scavenge free radicals, deactivate metals by complex

formation, inhibit enzymatic generation of free radicals, inhibit oxidation of
membranes and LDL, prevent damage from cellular proliferation, promote
synthesis of prostacyclin and nitrous oxide and optimize blood flow through
the arterial system, and have anticancer potential (Chapters 5 and 6). In
industrial applications, multiple functional antioxidants include sulfur-con-
taining hindered phenolic compounds used as stabilizers at high temperatures,
as UV stabilizers and metal deactivators.
E. Inhibition of photosensitized oxidation

Hydroperoxides can be produced from unsaturated lipids by photo-induced
oxidation in the presence of oxygen, visible light and photosensitizers, such as
chlorophyll, hemeproteins and riboflavin. There are two types of sensitizers for
photosensitized oxidation. Type I sensitizers such as riboflavin react in the
triplet state (3Sens) with lipids by hydrogen atom or electron transfer to form
radicals which can react with oxygen (23). In contrast to free radical oxidation,
this photosensitized reaction is not inhibited by chain-breaking antioxidants.
hν
3
Sens + LH ——➤ [Intermediate] + O2 ——➤ Hydroperoxides + Sens (23)
Type II sensitizers, such as chlorophyl and methylene blue in the triplet state,
interact with oxygen by energy transfer to give singlet oxygen (1O2), which
reacts further with unsaturated lipids (24). This type of photosensitized oxida-
tion is also not inhibited by chain-breaking antioxidants.
hν
3
Sens + O2 ——➤ [Intermediate] + 1Sens ——➤ 1O2
(24)
+ LH ——➤ Hydroperoxides
Carotenoids are the most important inhibitors of type II photosensitized
oxidation. They protect unsaturated lipids against photosensitized oxidation by
interfering with the activation of triplet oxygen to singlet oxygen. This quench-
ing effect takes place by an energy transfer mechanism from singlet oxygen to
carotene. By a similar energy transfer mechanism, carotenoids also react with
the triplet state of the excited sensitizers. α-Tocopherol is highly reactive
towards singlet oxygen and inhibits photosensitized oxidation by quenching
singlet oxygen and by forming stable addition products.
Chemical quenching by reaction with singlet oxygen to produce stable
products can be distinguished from physical quenching without undergoing
chemical changes. The rate of physical quenching by β-carotene approaches
diffusion rate (1.5 × 1010 M–1sec–1), and is much greater than that for α-tocopherol
(2.6 ×108 M–1sec–1). On the other hand, the rate of chemical quenching for
α-tocopherol (6.6 × 106 M–1sec–1) is much greater than that for β-carotene
(0.8 × 102 M–1sec–1). δ-Tocopherol (2.6 × 106 M–1sec–1) and γ-tocopherol
(0.7 × 106 M–1sec–1) present in many plant oils at much greater concentrations
than α-tocopherol are less efficient chemical singlet oxygen quenchers than
α-tocopherol.
The inhibition of photosensitized oxidation by β-carotene is complicated,
because it is highly susceptible to photo-induced oxidation and is quickly
destroyed in the presence of free radicals or hydroperoxides. To be effective in
unsaturated lipids exposed to light, β-carotene must be protected by a chain-
breaking antioxidant. The effectiveness of β-carotene as an inhibitor of
photosensitized oxidation in unsaturated lipids (and most likely in biological
systems) depends to a large extent on its protection against oxidation by natural
tocopherols, which also can reinforce the activity of β-carotene. However, in
the absence of a chain-breaking antioxidant, β-carotene is rapidly oxidized and
behaves as a prooxidant at high concentrations via the formation of free radical
inducing oxidation products.
β-Carotene and other carotenoids such as lycopene have been classified as
antioxidants because they behave as relatively weak free radical scavengers at
low oxygen pressures. However, the dual effect of β-carotene and other
carotenoids as strong singlet oxygen quenchers and weak free radical scaven-
gers has led to much confusion and controversy in the biochemical and
nutrition literature. Because carotenoids are highly susceptible to oxidation,
they can behave as prooxidants at atmospheric pressure and under non-
physiological conditions, especially in the absence of antioxidant protection.
β-carotene + LOO· ——➤ β-carotene· (25)
β-carotene· + O2 ——➤ β-carotene -OO· (26)
In vitro studies showed that the antioxidant activity of β-carotene is greatly
influenced by the type of initiators and interactions with other antioxidants.
Apparently, without proper protection from vitamin E, vitamin C or other
antioxidants, β-carotene and other carotenoids may promote rather than retard
lipid oxidation. This prooxidant activity could be due to the oxidation products
of β-carotene acting as free radical promoters of lipid oxidation. On the other
hand, β-carotene showed synergistic activity by increasing the antioxidant
activity of α-tocopherol, when used in combination during storage of vegetable
oils exposed to light.
F. Antioxidant enzymes in food systems

Enzymes have been exploited as antioxidants in various food applications to
remove oxygen from packaged products, to neutralize reactive oxygen species
(see Section G) and to reduce lipid hydroperoxides into benign hydroxy
products. For any food application, the enzymes are required to be stable in the
presence of the lipids under processing and storage conditions. They must not
Figure 2.7. Oxidation of β-D-glucose by glucose oxidase-catalase system (to reduce the resulting
hydrogen peroxide).
require nor generate toxic or unpleasant compounds that may affect the safety
or sensory properties of foods.
Glucose oxidase coupled with catalase is used commercially to remove
oxygen from foods. By catalysing the oxidation of D-glucose, oxygen is
removed by glucose oxidase to produce 2-δ-gluconolactone and hydrogen
peroxide (Figure 2.7). The gluconolactone is spontaneously hydrolysed to
D-gluconic acid and the hydrogen peroxide can be removed by catalase. This
glucose oxidase system has been applied in fruit juices, mayonnaise and salad
dressings to increase shelf life and prevent off-flavor development.
Superoxide dismutase reacts with superoxide, an active species of oxygen
(see Section F) to produce oxygen and hydrogen peroxide. This enzyme
retarded the hemin-catalysed oxidation of model linoleic acid in suspensions.
When used in conjunction with catalase to remove hydrogen peroxide,
superoxide dismutase had antioxidant activity in a milk fat model system
oxidized with iron. This system was not effective, however, in food emulsions
containing corn oil. Glutathione S-transferase catalysed the reduction of lino-
leic acid hydroperoxides to hydroxy derivatives and inhibited the
copper-stimulated oxidation of arachidonate in the presence of glutathione.
However, the usefulness of these enzyme systems was not demonstrated in
food systems.
G. Inhibition of biological oxidation

The oxidation of unsaturated lipids proteins and DNA can be induced by a
number of readily oxidizable substances, referred to as reactive oxygen species,
in addition to the free radicals generated from unsaturated lipids in the presence
of redox metal catalysts. These oxidant species are responsible for the toxic
effects of oxygen in the body. They include singlet oxygen, superoxide
radicals, hydrogen peroxide, hydroxyl radicals and hypochlorous acid. A
number of extracellular and intracellular antioxidant systems also referred to in
the biological context as repair systems are known to inactivate effectively
these reactive oxygen species and oxidants.
1. Metal binders and chelators

Metal chelation requires the participation of two or more oxygen, nitrogen or
sulfur atoms on the same biomolecule capable of binding with a metal. Because
iron and copper are efficient catalysts of biological oxidation that may be toxic
to cells, low concentrations of these metal catalysts must be controlled by
various type of chelation or by protein binding. Living organisms are thus well
protected against the cytotoxic effects of excess iron, by decreasing the
intracellular pool of free iron with a transport protein called transferrin and a
storage protein called ferritin. Transferrin is a glycoprotein carrier molecule
that under normal conditions is about 30% loaded with iron. No free iron salts
are thus expected in the blood plasma of healthy individuals. Cells may also
contain small amounts of free iron that may catalyse the formation of hydroxyl
radicals.
Metal chelation by plasma proteins is generally regarded as one of the most
important defense mechanisms against oxidation. Albumin can inhibit lipid
peroxidation by binding copper ions. In addition to its metal binding capacity,
albumin is also a good scavenger of peroxyl radicals (by reacting with the
sulfhydryl groups of its amino acids components) and of hypochlorous acid.
Under oxidative stress, small amounts of free iron may be released and
mobilized from ferritin and other protein binders to catalyse free radical
reactions. Oxidative stress can also be induced by superoxide radical (O–2·), by
hydrogen peroxide (H2O2) or by hydroxyl radicals (·OH). These reactive
oxygen species can be removed by a sequence of antioxidant enzymes.
2. Antioxidant enzymes in biological systems

Superoxide dismutase (SOD) catalyses the dismutation of superoxide radicals
produced in aerobic cells by one-electron reduction of oxygen through the
cellular electron transport chain of mitochondria chloroplasts and phagocyte
cells. Superoxide can act either as a reducing agent or as a weak oxidizing
agent. Superoxide reduces the iron in cytochrome c from Fe3+ to Fe2+ and
oxidizes ascorbic acid. The cells are protected against the damage from excess
superoxide by superoxide dismutases that catalyse the dismutation of superoxide
by reaction (27) to produce hydrogen peroxide, which is removed by catalase

and glutathione peroxidase.
2 O–2· + 2H+ ——
SOD
➤ H2O2 + O2 (27)
Several SOD enzymes have been characterized, including the copper-zinc-
SOD in mammalian tissues, fish and plant tissues, while the manganese-SOD
and the iron-SOD arise in higher organisms and bacteria. The hydrogen
peroxide produced by the dismutation of O–2· in the presence of SOD is removed
via reaction (28) catalysed by catalase.
2 H2O2 ——➤ 2 H2O + O2 (28)
Catalases contain an Fe3+ proporphyrin group that catalyses peroxidase-type
reactions. Catalase activity is elevated in the liver and erythrocytes of animals
and provides biological protection by removing hydrogen peroxide from the
cell.
Glutathione peroxidase provides an important protective mechanism against
lipid peroxidation in vivo. This enzyme reacts with hydrogen peroxide within
cells by using glutathione (GSH), a tripeptide of glutamic acid, cysteine and
glycine, as a hydrogen donor. Two GSH molecules condense by reaction (29),
producing oxidized glutathione GSSG through the -SH groups of cysteine.
H2O2 + 2 GSH ——➤ GSSG + H2O (29)
Glutathione reductase reduces GSSG back to GSH to maintain a ratio of
GSH/GSSG greater than 10 in normal cells. Glutathione peroxidase reduces
lipid and steroid hydroperoxides to form stable hydroxy lipids that do not
decompose to form alkoxyl radicals and cell-damaging aldehydes.
Myeloperoxidase oxidizes chloride ions in the presence of hydrogen perox-
ide to produce hypochlorous acid (HOCl), a powerful oxidant generated at sites
of inflammation in activated phagocytic cells. Bacteria are killed by the strong
oxidizing action of HOCl on proteins, especially the SH-containing amino
acids and other biological molecules, such as ascorbic acid.
3. Non-enzymatic reducing agents

Ascorbic acid is regarded as the most effective water-soluble antioxidant in
plasma. A multitude of reactive oxygen species are reduced by ascorbic acid,
including singlet oxygen, superoxide, hydrogen peroxide, hydroperoxyl radi-
cals, hydroxyl radicals and hypochlorous acid to give semidehydroascorbate.
Hydroperoxides are also reduced by ascorbic acid into stable hydroxy lipids.
However, at low concentrations in the presence of catalytic metals, ascorbic
acid can become a prooxidant by reducing Fe3+ to Fe2+ and Cu2+ to Cu+ by redox
cycling. This prooxidant action is generally overcome in vivo by maintaining
higher concentration ratios of ascorbic acid to catalytic metals.
Uric acid (Figure 2.2) is a water-soluble antioxidant that inhibits lipid

peroxidation by tightly binding iron and copper ions into inactive forms, and by
scavenging various oxidants such as hydroxyl radicals, peroxyl radicals,
singlet oxygen and hypochlorous acid. By complexing with iron, uric acid
stabilizes ascorbic acid in human serum. Ubiquinol-10 (or coenzyme Q10) is a
lipid-soluble antioxidant, which may act like ascorbic acid in regenerating
vitamin E by reducing the vitamin E radicals produced during lipid peroxidation.
4. Radical chain breakers

α-Tocopherol (vitamin E) is the major lipid-soluble antioxidant in membranes
and lipoproteins, acting by scavenging free radicals (superoxide and hydroxyl
radicals) by reacting with nitric oxide and by deactivating singlet oxygen.
Vitamin E reacts with lipid peroxyl and alkoxyl radicals by donating a labile
hydrogen and terminating the lipid peroxidation by reactions (10), (11), (15)
and (16). The resulting α-tocopherol radical is stabilized by electron delocal-
ization around the phenol ring structure. To be effective in vivo, the α-tocopherol
radicals are reduced back to α-tocopherol by ascorbic acid [reactions (21) and
(22)] or other reducing agents such as cysteine or glutathione. This synergistic
effect apparently takes place at the membrane interface, as the hydrophilic
reducing agents are in solution in the aqueous phase and would be oriented at
the membrane interface, because of their affinity for the phospholipids at the
surface.
5. Singlet oxygen quenchers

In the presence of biological sensitizers such as chlorophylls, riboflavin and
derivatives bilirubin, retinal and porphyrins, a large number of biologically
important compounds are damaged by singlet oxygen, including polyunsaturated
lipids, α-tocopherol, DNA, cholesterol, β-carotene and proteins. Relatively
low concentrations of β-carotene are effective in preventing lipid oxidation by
singlet oxygen, provided it is protected by antioxidants. Other compounds,
such as α-tocopherol, amino acids and glutathione, behave as weak quenchers
by oxidation with singlet oxygen (Section E).
On one hand, the combination of β-carotene and α-tocopherol inhibited lipid
oxidation in a liver microsome model system more significantly than the sum
of the individual inhibitions. This synergistic effect suggests a reinforcing
interaction between the two components as efficient radical trapping antioxi-
dants. On the other hand, supplementation with β-carotene in vivo and in vitro
did not inhibit the oxidation of LDL induced either with copper or with the
artificial azo initiator AAPH. By itself, β-carotene supplementation promoted
the in vivo oxidation of LDL by shortening the lag phase using copper as
catalyst. However, in combination β-carotene and α-tocopherol apparently
mutually interact to protect each other from oxidation.
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CHAPTER 3
Antioxidant action in multiphase systems
Lipid oxidation is generally not well understood in systems in which the fat is
dispersed in emulsion systems, because of their complexity and the influence of
a multitude of additional factors that affect different types of antioxidants.
Interfacial oxidation is a ‘surface’ reaction between phases that depends on the
rate of oxygen diffusion and its interactions with unsaturated lipids, metal
initiators, radical generators and antioxidants, which are distributed according
to their relative surface activity in different compartments of colloidal food and
biological systems. This interfacial oxidation affects a large number of foods
which exist in the form of emulsions. The classical mechanism of inhibited
lipid oxidation does not explain changes in antioxidant activity between
solutions and multiphase emulsion systems. In these systems, interfacial
antioxidation depends on the partition of antioxidants between the aqueous
phase, lipid phase and surfactant-enriched interface in food and biological
systems. The relative effectiveness of various antioxidants is determined by
many factors, including localization of antioxidants in different phases, the
colloidal properties of the substrates, the conditions of oxidation, and the stages
of oxidation. The type and physicochemical state of the lipid substrate, and its
degree of unsaturation, play an important part, as well as the presence and types
of initiators, such as transition metals, other components, and their possible
interaction. Antioxidant activity is thus strongly affected by the physical
composition of the test system, and the relative activity of antioxidants of
different polarity varies significantly according to the multiphase systems
involved.
A. Multiphase colloidal systems

Food emulsions consist of oil and water phases, in which one phase is dispersed
in the other in the form of droplets stabilized by surface-active emulsifiers
forming a protective film to prevent aggregation (Figure 3.1). On one hand, oil-
in-water emulsions consist of oil droplets dispersed in a continuous water
phase, stabilized by an emulsifier containing both hydrophilic and lipophilic
components. Common food emulsions include milk and salad dressing that are
stabilized by surface-active phospholipids, such as lecithin, and proteins, such
as casein or bovine serum albumin (BSA). On the other hand, water-in-oil
emulsions consist of water droplets dispersed in a continuous oil phase,
stabilized by more water-soluble emulsifiers with a stronger hydrophilic and
43
Figure 3.1. Four different emulsion types (from Lipid Oxidation, 2nd ed, 2005, Figure 10.1, p. 261).
weaker lipophilic group, such as monoglycerides. Common water-in-oil emul-

sions include butter and margarine. Micelles consist of free fatty acids dispersed
in water by having the polar carboxyl group oriented mainly in the water phase
and the non-polar fatty tail lying mainly in the oil phase. Although free fatty
acid micelles are uncommon in foods consisting mainly of triacylglycerols, in
small amounts micellar free fatty acids can decrease the oxidative stability of
foods by solubilizing metal contaminants. During frying of potatoes, the
presence of free fatty acids and steam can seriously decrease the stability of the
oil by micellar solubilization of any metal contaminants. Liposomes form
spontaneously when phospholipids are dispersed in water in the presence of oil.
These colloidal systems consist of bilayers between the water phase and the oil
phase separated by a monolayer.
Emulsifiers at the interface surrounding the droplets inhibit lipid oxidation
by providing protection against the penetration and diffusion of metal catalysts.
Higher concentrations of emulsifier can thus increase the oxidative stability of
emulsified lipids by providing tighter packing at the oil–water interface and
prevent diffusion of oxidation initiators. As food emulsifiers, proteins can
either retard or promote lipid oxidation in oil-in-water emulsions. At certain
concentrations, when proteins are dispersed in the water phase, they may either
scavenge free radicals or be preferentially oxidized and retard lipid oxidation.
However, in the presence of metals, some milk proteins may promote lipid
oxidation. In liposome systems, the lipid bilayers of membranes containing
ANTIOXIDANT ACTION IN MULTIPHASE SYSTEMS 45
Water
Aqueous layer
Oily layer
Oil Oil
Water
Interface
Figure 3.2. The interfacial region is a narrow membrane between the phases consisting of a mixture of
oil, water and emulsifier (from Lipid Oxidation, 2nd ed, 2005, Figure 10.5, p. 264).
phospholipids are protected against oxidation by lipophilic antioxidants such

as α-tocopherol.
The various constituents of an emulsion are distributed according to their
polarity and surface activity between the different phases, including the oil
phase, the water phase and the interface. The interfacial region is a narrow
membrane between the phases, consisting of a mixture of oil, water and
emulsifier (Figure 3.2). Non-polar constituents are partitioned in the oily
phase, the polar constituents mainly in the aqueous phase, and the surface-
active amphiphilic constituents in the interface. Surface-active constituents
that tend to concentrate at the interface include proteins, phospholipids,
emulsifiers and insoluble solid particles. This oil–water-emulsifier mixture
becomes more chemically reactive at the interface due to the increased surface
created by spreading into a thin film membrane.
1. Effect of antioxidants
Antioxidant activity varies significantly in different multiphase systems, accord-
ing to their colloidal distribution and the lipid substrates. The behavior of
different types of antioxidants in emulsions, referred to as the polar paradox,
is based on the observation that non-polar antioxidants are more effective in
polar lipid systems in emulsions, while polar antioxidants are more effective in
non-polar lipid systems (Figure 4.1, Table 3.1, ref 1, 2). In bulk oil systems,
hydrophilic antioxidants are generally more effective than lipophilic antioxi-
dants, whereas in oil-in-water emulsions, lipophilic antioxidants are more
effective. Thus, the order of activity of α-tocopherol versus Trolox and
ascorbic acid versus ascorbyl palmitate is reversed when comparing bulk oil
and emulsified systems. In marked contrast to bulk triglycerides and methyl
linoleate, bulk linoleic acid and emulsified linoleic acid, Trolox was more
active than α-tocopherol. This result was explained by the unique tendency of
Table 3.1. Effect of lipid systems on relative activities of antioxidants
Lipid systems Antioxidant trendsa Ref.b
Bulk corn oil (CO) Trolox > α-tocopherol, ascorbic acid > ascorbyl palmitate (1)
CO/W emulsion α-Tocopherol > Trolox ≈ ascorbyl palmitate > ascorbic acid
Bulk CO, MeLo Trolox > α-tocopherol
Bulk, Lo ac and
emulsions (2)
CO/, MeLo/W α-tocopherol > Trolox
emulsions
Bulk CO CA ≈ rosmarinic acid ≈ α-tocopherol > carnosol (3)
CO/W emulsion α-tocopherol > carnosol > CA > rosmarinic acid
Bulk CO, Methyl carnosate > CA > α-tocopherol (4)
O/W emulsion
Bulk CO, CA > carnosol > α-tocopherol
MeLo/emulsion
CO, Lo ac/W α-tocopherol > CA ≈ carnosol (5)
emulsion
Bulk Lo ac α-tocopherol > carnosol > CA
Bulk corn oil EGC ≈ EGCG ≈ ECG > GA > PG > EC > C
CO/W emulsions Tea catechin, GA, PGc (6)
Soy lecithin liposomes EGCG > EC » PG > catechin » ECG > EGC » GA
Lecithin liposome BHT > BHA > PG > TBHQ > GA (7)
DLPC liposome Trolox = α-tocopherol (8)
PC liposome EG > EC > quercetin > α-tocopherol (9)
Solutiond α-tocopherol >> quercetin > EC = EG
SDS– micelles α-tocopherol = ascorbyl palmitate > Trolox > ascorbic acid (10)
HDTBr+ micelles Ascorbic acid > Trolox > ascorbyl palmitate > α-Tocopherol
Salmon oil emulsion Methyl gallate > gallamidee > gallic acid (11)
Bulk CO Trolox > MeCA > GA ≈ PG(13)CA > α-tocopherol
CO/W emulsion PG > Trolox > CA > α-tocopherol > MeCA > GAc (12)
W/CO emulsion MeCA > CA » Trolox > α-tocopherol > PG c, GAc
Lecithin liposome BHA ≈ BHT > Trolox > α-tocopherol > TBHQ > caffeic acid
Triolein/water BHA ≈ BHT > α-tocopherol > Trolox c ≈ (13)
emulsion TBHQ c > caffeic acidc
Liposomes Ascorbic acidc = prooxidant in all liposomes (14)
(PC, PS, PA) α-tocopherolc = prooxidant in PC and PA, antioxidant in PS
a
See structures in Figures 2.5 and 3.3, and Tables 3.5 and 3.8.
b
References: (1) Frankel et al. (1994), (2) Huang et al. (1996a), (3) Frankel et al. (1996a), (4) Huang et
al. (1996b), (5) Hopia et al. (1996), (6) Huang and Frankel (1997), (7) Porter et al. (1989), (8) Barclay
and Vinqvist (1994), (9) Terao et al. (1994), (10) Pryor et al. (1988), (11) Mei et al. (1999), (12) Schwarz
et al. (2000), (13) Nenadis et al. (2003), (14) Gal et al. (2003).
Abbreviations: GA, gallic acid; PG, propyl gallate; EGCG, epigallocatechin gallate; C, catechin; EC,
epicatechin; ECG, epicatechin gallate; EGC, epigallocatechin; BHT, tert-butylhydroxytoluene; BHA,

tert-butylhydroxyanisole; TBHQ, tert-butyl hydroquinone; DLPC, dilinoleyl phosphatidylcholine; PC,
phosphatidylcholine; PS, phosphatidylserine; PA, phosphatic acid; EG, epicatechin gallate; SDS,
sodium dodecyl sulfate; HDTBr+, hexadecyltrimethyl ammonium bromide; MeCA, methyl carnosate;
CA, carnosic acid.
c
Compounds underlined are prooxidant.
d
hexane–isopropanol (1:1, by vol).
e
Gallamide has an amide (CONH2) substituent instead of a carboxyl group (COOH) in the 1-position of
gallic acid.
f
Emulsifiers: SDS–, PHLC, Brij 58 are respectively anionic, zwitterionic and non-ionic.
linoleic acid to form mixed micelles and the emulsifier Tween 20, where Trolox
is more active than in oil-in-water emulsion. We also found that in a corn oil-
in-water emulsion Trolox partitions mainly in the water phase (see Section B).
For this reason, in the evaluation of antioxidants, linoleic acid cannot be used
as a representative model system for foods, since antioxidant behavior in
linoleic acid micelles will be significantly different from that in food emulsions
composed mainly of triglycerides.
The interfacial behaviour of antioxidants between bulk oil and emulsion
systems was further confirmed with the observations that the hydrophilic
rosemary compounds carnosic acid (CA) and rosmarinic acid (see Figure 2.6)
were better antioxidants in bulk oils than the corresponding lipophilic antioxi-
dants carnosol (Table 3.1, ref 3). This order of activity was reversed in
emulsified corn oil systems. On the other hand, when carnosic acid was
compared to methyl carnosate, the less polar methyl ester was more active in
both bulk and emulsified corn oil triglycerides, and both were more active than
α-tocopherol (Table 3.1, ref 4). This comparison is complicated, however,
because carnosic acid was less stable than methyl carnosate and α-tocopherol
in both bulk and emulsified corn oil. In comparing antioxidant activities in
different lipid systems, carnosic acid was more active than carnosol, followed
by α-tocopherol in both bulk corn oil and methyl linoleate. Although in bulk
linoleic acid, α-tocopherol and carnosol were both more active than carnosic
acid, in linoleic acid emulsions carnosol and carnosic acid had similar activities
and both were less active than α-tocopherol (Table 3.1, ref 5). Carnosic acid
may have also lost activity by partitioning more easily into the water phase.
Further complications arise from the observation that both carnosic acid and
carnosol are readily decomposed during oxidation into quinone and lactone
products, which exhibit antioxidant activity (Lipid Oxidation, 2nd ed, 2005,
Figure 9.13, p. 242).
The polar and hydrophilic green tea catechin gallates, flavonoids, propyl
gallate and gallic acid (Figure 3.3) showed similar behavior in being active
antioxidants in bulk corn oil, but prooxidant in the corresponding corn oil-in-
water emulsion (Table 3.1, ref 6). In bulk corn oil, the highly polar EGC, EGCG
and ECG were the most active, followed by gallic acid, propyl gallate, and the
less polar epicatechin and catechin were the least active. The prooxidant
Figure 3.3. Structures of flavonoids and phenolic acids.
activity of tea catechins in emulsions can be explained by the high reducing

properties of these water-soluble and polar antioxidants in converting ferric
iron into the more catalytically active ferrous state. In contrast to oil-in-water
emulsion, the polar tea catechin gallates were active antioxidants in a lecithin
liposome, where EGCG was the best antioxidant, followed by epicatechin,
propyl gallate, catechin, ECG, EGC and gallic acid. Also, in a liposome system,
the lipophilic BHA, BHT and propyl gallate were much more active antioxi-
dants in protecting polar phospholipids than the hydrophilic TBHQ, caffeic and
gallic acids (Table 3.1, ref 7).
The activity of antioxidants is increased by the electrostatic attraction
between the lipid surface and antioxidants. The charge of phospholipid
liposomes can thus affect the activity of charged antioxidants. The negatively-
charged Trolox was much more active in the liposome prepared with
dilinoleoyl-phosphatidylcholine (DLPC) at pH 4, because it was positively
charged due to electrostatic attraction. At pH 7, α-tocopherol and Trolox had
about the same antioxidant activity (Table 3.1, ref 8). However, Trolox had no
antioxidant activity at pH 11, at which the liposome was neutral and zwitterionic
(±). Although in PC liposomes, ECG and EC in green tea were more active
antioxidants than quercetin, and α-tocopherol, the order of activity was re-
versed in solution of hexane and isopropanol (Table 3.1, ref 9).
The lipophilic α-tocopherol and its hydrophilic analog Trolox behave quite
differently when in linoleic acid micelles rather than PC liposomes. Linoleic
acid and SDS form mixed micelles in the aqueous phase in which the polar
antioxidant Trolox equilibrates more rapidly and becomes more effective than
in liposomes. The charge of fatty acid micelles affects the activity of charged
and uncharged antioxidants. Because ascorbic acid and Trolox are negatively
charged, they were more effective antioxidants in a micelle system of linoleic
acid stabilized with the positively charged emulsifier hexadecyltrimethyl-
ammonium bromide (HDTBr+), and much less effective when the system was
stabilized with a negatively charged emulsifier, such as SDS– (Table 3.1, ref
10). These charged antioxidants have an ionized carboxylate group which
causes it to be repelled by the negatively charged SDS micelles.
In salmon oil emulsions stabilized with non-ionic Brij emulsifier at pH 7.0,
the structurally similar phenolic galloyl derivatives inhibited lipid oxidation in
the order: Me gallate > gallamide > gallic acid (Table 3.1, ref 11). However, at
pH 3, these galloyl antioxidants were prooxidant in this salmon oil emulsion. In
SDS-stabilized salmon oil emulsions, the galloyl derivatives were less effec-
tive compared to the Brij-stabilized emulsions. Differences in antioxidant
activity were related to an increase of prooxidant activity of iron at low pH.
In comparing different lipid systems, the polar Trolox was the most active in
bulk corn oil, followed by methyl carnosate, gallic acid, which had similar
activity to propyl gallate and carnosic acid, and α-tocopherol was the least
active (Table 3.1, ref 12). In corn oil-in-water emulsions, the trend changed
with propyl gallate being the most active, followed by Trolox, carnosic acid,
α-tocopherol, methyl carnosate, and gallic acid became prooxidant. In the
corresponding water-in-oil emulsions, methyl carnosate became the most
active, followed by carnosic acid, which had the same activity as α-tocopherol,
and both propyl gallate and gallic acid became prooxidant. Oil-in-water and
water-in-oil emulsions as well as bulk oils showed different behaviors on
oxidation. In addition to their inhibiting effects on hydroperoxide formation
shown on Table 3.1, all emulsions produced more hexanal than the bulk corn
oil, suggesting that in aqueous emulsions proton catalysis promotes the decom-
position of hydroperoxides. The most polar gallic acid and propyl gallate were
either inactive or prooxidant in both types of emulsions, but were relatively
active in bulk oil. The increased activity of these polar hydrophilic antioxidants
in bulk oils is consistent with the interfacial phenomenon that they accumulate
due to their insolubility at the oil–air interface where oxidation takes place
(Figure 4.2). The less polar lipophilic antioxidants such as α-tocopherol are
diluted in the main oil phase and show diminished activity.
In a lecithin liposome, BHA and BHT were the most effective antioxidants,
followed by Trolox, α-tocopherol, TBHQ and caffeic acid, which could be
prooxidant depending on concentration (Table 3.1, ref 13). In a triolein-in-
water emulsion, BHA and BHT were more active antioxidants than
α-tocopherol, but Trolox, TBHQ and caffeic acid became prooxidant. In
comparing different liposomes, ascorbic acid was prooxidant in all liposomes
(PC, PS and PA), while α-tocopherol showed antioxidant activity in
phosphatidylserine and prooxidant activity in phosphatidylcholine and
phosphatidic acid (Table 3.1, ref 14). The low polarity of BHA and BHT and
their effectiveness at low concentrations on one hand, and the size of the
molecules for α-tocopherol and Trolox on the other, were considered critical
factors for a good performance in dispersed systems. When considering the
wide variation in antioxidant activities according to the multiple complex
factors and interfacial phenomena involved, it is not surprising that predicting
the activity of antioxidants in different multiphase systems is extremely
difficult and remains an empirical process.
To obtain further evidence in support of the interfacial phenomenon of
antioxidant activity in emulsions, the effect of different emulsifiers was
investigated on the activity of phenolic antioxidants varying in polarity in bulk
oil (Table 3.2). Polar emulsifiers CGS (mixture of Cetheareth-15 and glyceryl
stearate) and PGMS (polyglyceryl glucose methyl distearate) increased the
antioxidant activity of α-tocopherol, decreased the activity of propyl gallate
and gallic acid, but had no effect on the activity of Trolox. Similar trends were
noted for the inhibition of hexanal, except for Trolox, which showed a small
decrease in activity after addition of the polar emulsifiers. The increased
activity of α-tocopherol in bulk corn oil caused by the polar emulsifiers is
attributed to its partial solubilization and increase in surface accessibility. A
similar increase in the antioxidant activity of α-tocopherol in bulk oil is
observed on the addition of polar phospholipids. These results support the
interfacial partition hypothesis that polar antioxidants are oriented at the air–oil
Table 3.2. Effect of emulsifier addition on antioxidants in bulk oila
Antioxidants Without CGS PGMS

Inhibition of hydroperoxides, %
Tocopherol 11 55 32
Trolox 58 60 60
Propyl gallate 67 29 12
Gallic acid 64 47 44
Inhibition of hexanal, %
Tocopherol 8 74 58
Trolox 77 69 71
Gallic acid 77 44 44
a
From Schwarz et al. (2000).
Abbreviations: CGS, Cetheareth-15 + glyceryl stearate, PGMS, Polyglyceryl glucose methyl distearate.
Structures:
OCOC17H35
O C17H35 CH2 (OCH2-CH2)15 OH
HO OCH3 +
C17H35OCO C15H31 CH2 (OCH2-CH2)15 OH
+ +
OR,H OR,H C17H35 COOCH2
RO O O
H HOCH
n
R = Stearic acid; n = 1-4 HOCH2
Polyglyceryl glucose methyl Cetheareth-15 and glyceryl stearate

distearate (PGMS) (CGS)
(HLB value = 11.5) (HLB value = 12.1)
HLB: Hydrophilic–lipophilic balance
interface due to their insolubility and surface activity. The addition of polar
emulsifiers CGS and PGMS causes higher solubilization of the polar antioxi-
dants in the non-polar oil system (Figure 4.2).
2. Effect of metal catalysts

In bulk oils systems, the hydrophilic free metals catalyse oxidation by becom-
ing oriented in the air–oil surface and the oil–air interface in air bubbles
produced by agitation during processing (Figure 3.4). In emulsions and
liposomes, the metals may dissolve in the aqueous phases and become oriented
in either the oil–water or phospholipid–water interfaces, where they have an
affinity for the hydrated layer around the droplets.
Figure 3.4. Metal catalysis in emulsion and liposome systems (from Lipid Oxidation, 2nd ed, 2005,
Figure 10.7, p. 272).
The effects of chelators may be very complex in emulsion systems, because

they vary in their metal affinities and charge, and in their relative solubility and
partition between the lipid and water phase. In aqueous systems, ethylenedi-
aminetetraacetic acid (EDTA) can reduce lipid oxidation by changing the
location and by reducing the redox capacity of iron. However, EDTA may also
promote oxidation if its concentration is too low, because it changes the ratio of
Fe3+ to Fe2+ in favor of the more catalytically active Fe2+.
3. Effect of proteins
Lipid oxidation in emulsions is mainly influenced by the properties of the
interface. When proteins are used as emulsifiers in foods, they can be oxi-
datively modified by Michael addition reactions, producing protein adducts
with aldehyde-containing fatty acids derived from lipid oxidation (Figure 3.5).
Figure 3.5. Michael addition and Schiff base adducts by interaction of 4-hydroxy-2-nonenal with
amino acid residue of a protein (From Lipid Oxidation, 2nd ed, 2005, Figure 13.7, p. 410).
Proteins can also inhibit lipid oxidation by adsorption in emulsions to improve

their oxidative stability, by protecting the interface against the prooxidant
effects of increased interfacial area of emulsions.
a) Interactions between polyphenols and proteins. The antioxidant activity

of phenolic compounds can be significantly influenced by complex interactions
with proteins. Four types of interactions between phenolics and proteins
include hydrogen bonding, hydrophobic, covalent and non-covalent interactions.
Phenolic hydroxyl groups can readily donate hydrogen to form hydrogen bonds
with the amide carbonyl of peptide backbone of proteins. Hydrophobic
interactions occur by complex formation with proline residues. Covalent
interactions may occur between several phenolic molecules of relatively small
molecular weight (180–700 Da) and proteins of much larger molecular weight
(14000–350000 Da) at multidentate sites. Non-covalent binding may also
control a large part of interactions, but both of these interactions are likely to
take place simultaneously. Non-covalent interactions may take place by
π-bonding, hydrogen bonding, and hydrophobic or ionic interactions.
Although interactions with proteins often decrease the antioxidant activity
of phenolic compounds, the protein–phenol complexes produced have not
been well characterized. The main reactive residues of proteins are nucle-
ophilic side chains provided by lysine, tryptophan and cysteine. The
protein–polyphenol complex formation is generally strongest below the
isoelectric point of the proteins at which electrostatic repulsion between
proteins is minimized. The resulting derivatization of phenolic compounds
results in significant changes in antioxidant activity due to changes in their
solubility and interfacial properties relative to the food substrates to be pro-
tected. Antioxidant activity based on the TEAC assay is decreased when
quercetin is derivatized with various ratios of BSA (Table 3.3). With higher
concentrations of quercetin, reactive quercetin quinones are formed in increas-
ing covalent binding with BSA. The BSA–quercetin derivatives retain
antioxidant capacity varying according to the degree of protein derivatization.
The derivative with highest amount of bound quercetin (2 parts BSA: 1 part
quercetin) showed 79% inhibition relative to that of free quercetin. In another
study of interactions between green tea catechins and β-casein, the TEAC
antioxidant capacity of different flavonoids was masked to different degrees
(Table 3.4). The relative contribution of tea catechins to the masking of TEAC
antioxidant activity decreased in the order: epigallocatechin gallate (26%),
epicatechin gallate (19%), epicatechin (15%), epigallocatechin (11%) and
gallic acid (1.2%). Unfortunately, the TEAC assay only measures antiradical
activity that does not necessarily reflect a direct relationship to antioxidant
activity in the presence of a lipid and protein substrates to be protected (see
Chapter 4).
A postulated mechanism of interaction involves the oxidation of quercetin to
Table 3.3. Relationship between covalent bound quercetin (Q) in BSA–quercetin

mixtures and their relative antioxidant TEAC activitya
Mixtures Bound Q (µg Q/mg BSA) TEAC (mM/l)

Free Q Bound Q
Control – 0.5 –
BSA-Q 20:1 8 4.3 2.1
BSA-Q 10:1 11 4.8 2.8
BSA-Q 7:1 12 5.0 3.2
BSA-Q 5:1 15 5.5 3.8
BSA-Q 2:1 18 5.9 4.6
a
From Rohn et al. (2004). TEAC values were approximated from original figure of barograms.
TEAC, Trolox equivalent antioxidant capacity
Table 3.4. Masking of the TEAC antioxidant activity of green tea by β-caseina
Polyphenols TEAC value % Masking
Green tea 7.3 14

Catechin 3.9 4.5
Epicatechin 13 15
Epicatechin gallate 15 19
Epigallocatechin 29 11
Epigallocatechin gallate 32 26
Gallic acid 1 1.2
a
From Arts et al. (2002). TEAC: mmol/g.
TEAC, Trolox equivalent antioxidant capacity
the corresponding quinone reacting with an amino residue of BSA, to produce

a covalently bound phenol–BSA derivative that has antioxidant activity (Fig-
ure 6.5). A second oxidation produces a BSA–quercetin quinone complex,
which may cross-link further BSA molecules, resulting in polymerization.
Similar polymerization was shown with other proteins, including myoglobin,
lysozyme, whey and soy proteins and chymotrypsin.
b) Emulsion systems. Food proteins derived from milk fractions are

commonly used to impart physical and oxidative stability to food emulsions.
Unfortunately, the vast literature in this field is difficult to interpret, owing to
the wide range of experimental conditions used, including lipid surfactants,
buffers, pH, antioxidants, metal chelators and methods to evaluate lipid and
protein oxidation (Table 3.5). Additional confusion was caused by the use of
questionable methods to measure lipid oxidation, such as TBARS, and oxidative
stability methods, such as ORAC (see Chapter 4). Whey and other food
proteins can inhibit lipid oxidation by a number of mechanisms, including
chelating prooxidant metals, repelling anionic metals by controlling pH to form
cationic charges, decreasing interactions between lipid hydroperoxides and

metals, and reducing free radicals by sulfhydryl groups.
The activity of whey proteins in inhibiting lipid oxidation has been generally
attributed to chelation of transition metals by the constituents, lactoferrin and
albumin, and to the free radical scavenging activity of residue amino acids
including tyrosine and cysteine. Sulfhydryl (SH) groups are known to scavenge
free radicals and contribute significantly to the antioxidant activity. Thus, the
activity of the high molecular weight (HMW) fraction was reduced by blocking
the SH groups with N-ethylmaleimide (Table 3.5, ref 1). Additional antioxidant
activity of the HMW fraction of whey was attributed to the free radical
scavenging activity of amino acids, and to iron chelation.
The rate of lipid oxidation was promoted in BSA-stabilized emulsions by
increasing the interfacial surface area (Table 3.5, ref 2). By decreasing the size
of the oil droplets, the number of lipid molecules per droplet diminishes and the
amount of surface-active compounds adsorbed at the interface increases.
Emulsification to increase the interfacial area thus favored diffusion of oxygen
and metal catalysts and accelerated oxidation at the initial stages of oxidation.
Apparently, protein adsorbed at the interface may have restrained this prooxidant
effect. At later stages of lipid oxidation, however, the concentration of
hydroperoxides was not affected by the droplet size of the emulsion. The
decomposition products hexanal and pentane were detected only at later stages
and were also not affected by the oil droplet size.
Lactoferrin is an iron-binding glycoprotein in milk that has antioxidant and
several biological properties, including iron absorption, especially for breast-
fed infants, and inhibition of pathogenic bacteria. This protein inhibits
iron-catalysed lipid oxidation by chelating free iron (Lipid Oxidation, 2nd ed,
2005, Table 11.11, p. 322), and can be used as a supplement to prevent
oxidation in iron-containing infant formula (Chapter 5.E.4). Several phenolic
compounds (α-tocopherol, ferulic acid, coumaric acid, tyrosol), and natural
phenolic extracts from different extra virgin olive oils reinforced the antioxi-
dant activity of lactoferrin in oil-in-water emulsions and liposomes (Table 3.5,
ref 3, Table 3.8).
Caseinophosphopeptides are phosphorylated peptides from milk containing
unique anionic clusters that can be used as natural metal chelators, without
being influenced by denaturation. By binding transition metals, enriched
caseinophosphopeptides inhibited the formation of lipid oxidation in corn oil-
in-water emulsions at pH 3.0 and 7.0 as determined by lipid hydroperoxides
and hexanal (Table 3.5, ref 4). However, casein hydrolysates were more
effective in inhibiting lipid oxidation than the enriched caseinophosphopeptides
at equal phosphorus content. Whether or not the greater activity of casein
hydrolysates is due to free radical scavenging was not established.
Proteins can be used to produce highly stable cationic oil-in-water emulsions
at pH 3.0 to repel cationic iron. In salmon oil-in-water emulsions stabilized
56
Table 3.5. Effect of proteins on lipid oxidation of oil-in-water emulsions
Lipid Emulsifier/Protein Buffer pH Inhibitors Conditions Methods Ref.a
Salmon oil Tween-20 Phosphate 7.0 Whey proteins 20°C Peroxides, TBARS, (1)
HMW fraction ORAC
Sunflower oilb BSA None 4.3 BSA 47°C O2 uptake, CD, (2)
pentane, hexanal
Corn oilb, Lecithin Phosphate 6.6 Phenolics, lactoferrin 30°C CD, fluorescence (3)
lecithin
Corn oil Brij 35 Imidazole/acetate 3.3, Caseino-phospho- 37°C Peroxides, hexanal (4)
7.0 peptides
Salmon oil Proteins Imidazole/acetate 3.0 Whey protein isolate 4, 20, 37°C Peroxides, propanal (5)
Corn oil Proteins Imidazole/acetate 3.0 Casein, whey, soy 37°C Peroxides, hexanal (6)
protein
Sunflower oilb BSA None 4.3 EDTA, Isoeugenol 47°C CD, hexanal (7)
α-toc, Trolox
Fish oil Casein None – Rosemary, EVOO 30°C CD, fluorescence (8)
ANTIOXIDANTS IN FOOD AND BIOLOGY
Soybean oil Casein Phosphate 7.4 Alkylamines, PC 37°C O2 uptake, GC (9)

Triglyceridesb decrease in substrate
Menhaden oil Proteins Imidazole/acetate 3.0, Whey, soy protein, 20°C Peroxides, propanal (10)
7.0 Na caseinate
Algae oil Whey proteins Imidazole/acetate 3.0, EDTA, citrate, poly- 37°C Peroxides, propanal (11)
7.0 phosphates
Algae oil Whey proteins Imidazole/acetate 3.0, Grape seed extract 37°C Peroxides, propanal (12)
7.0
Rapeseed oil Whey proteins None 4.6 Berry juices 40°C CD, TBARS, (13)
Cu sulfate protein carbonyls,
fluorescence
Rapeseed oil Whey proteins None 4.6 Anthocyanins 37°C CD, hexanal, protein (14)
Cu acetate carbonyls,
fluorescence
Menhaden oil Brij 35 Phosphate 7.0 β-Lactoglobulin 20°C Peroxides, TBARS (15)
Sunflower oilb BSA None 6.5 BSA, Na caseinate 50°C O2 uptake, CD, (16)
volatiles
a
References: (1) Tong et al. (2000), (2) Lethuaut et al. (2002), (3) Medina et al. (2002), (4) Diaz et al. (2003), (5) Hu et al. (2003a), (6) Hu et al. (2003b), (7) Cuvelier
et al. (2003), (8) Medina et al. (2003), (9) Hirose et al. (2003), (10) Faraji, et al. (2004), (11) Hu et al. (2004a), (12) Hu et al. (2004b), (13) Viljanen et al. (2005a),
(14) Viljanen et al. (2005b), (15) Elias et al. (2005), (16) Villiere et al. (2005).
Abbreviations: BSA, bovine serum albumin; CD, conjugated dienes; EVOO, extra virgin olive oil; PC, phosphatidylcholine; HMW fraction, high-molecular weight
ANTIOXIDANT ACTION IN MULTIPHASE SYSTEMS
fraction; TBARS, thiobarbituric acid reactive compounds; α-toc, α-tocopherol; ORAC, oxygen radical absorbance capacity.
b
Stripped of tocopherols.
57
with whey protein isolate (WPI), lipid oxidation was effectively inhibited at pH
values below the protein’s isoelectric point (pI), at which the emulsion droplets
were positively charged, compared to that at pH values above the pI, at which
the emulsion droplets were negatively charged (Table 3.5, ref 5). At pH 3, the
oxidative stability decreased in the order of β-lactoglobulin ≥ sweet whey > α-
lactoglobulin ≥ WPI. The oxidative stability of different protein-stabilized
corn oil-in-water emulsions decreased in the order: casein > WPI > soybean
protein isolate (SPI) (Table 3.5, ref 6). The degree of positive charge on the
protein-stabilized emulsion droplets was not the only factor affecting the
inhibition of lipid oxidation, because the charge of the emulsion droplets
(WPI > casein ≥ SPI) did not parallel oxidative stability. Other mechanisms to
explain the differences in oxidative stability of the protein-stabilized emulsions
may include differences in interfacial film thickness, protein-chelating proper-
ties, and differences in free radical-scavenging amino acids.
The oxidation of oil-in-water emulsions prepared with stripped sunflower
oil, stabilized by BSA, was strongly inhibited in the presence of EDTA and
isoeugenol (Table 3.5, ref 7). A suggested mechanism of inhibition involves
inactivation of metallic ions by EDTA or by the protein, resulting in synergism
reinforcement of antioxidant properties of isoeugenol. A similar significant
antioxidant synergism was shown by rosemary extracts rich in carnosic acid
with fish proteins and phenolics from extra virgin olive oil (Table 3.5, ref 8).
The extract of extra virgin olive oil was more active than the rosemary extract
in inhibiting oxidation of fish oil-in-water emulsions. Partition studies showed
high adsorption of phenolic compounds on fish muscle emulsions. The
synergism between fish proteins and rosemary extracts was attributed to the
protection of fish proteins against the oxidation of carnosic acid.
In milk casein emulsions, stearylamine and other alkylamines in the pres-
ence of phosphatidylcholine (PC) strongly inhibited the copper-catalysed
oxidation of soybean oil triglycerides (Table 3.5, ref 9). Unfortunately, oxida-
tion was measured coarsely by oxygen absorption and decrease in triglyceride
substrate by GC. The antioxidant effect could be explained by the electrostatic
repulsion between the positively charged stearylamine and positively charged
copper ion at the interface. These positive effects were not observed in the
absence of PC, suggesting the importance of PC interactions with charged
components at the oil–water interface.
Although WPI, SPI and sodium caseinate (CAS) can inhibit lipid oxidation
by producing a positive charge at the interface of emulsion droplets, only a
fraction actually adsorb to the emulsion droplets, while the rest remain in the
continuous phase. Washed emulsions prepared to remove WPI from the
continuous phase, by repeated centrifuging and re-suspending the emulsion,
were less oxidatively stable than unwashed emulsions at pH 7.0, suggesting
that continuous phase proteins inhibited oxidation. The oxidative stability of
emulsions containing different proteins in the continuous phase decreased in
the order SPI > CAS > WPI, on the basis of hydroperoxide and headspace
propanal formation (Table 3.5, ref 10). Iron-binding studies showed that the
chelating ability of the proteins decreased in the order CAS > SPI > WPI. The
free SH groups of both WPI and SPI were apparently involved in their
antioxidant activity. Therefore, continuous phase proteins could be an effective
means of protecting emulsions from oxidative deterioration.
The oxidative stability of algae oil-in-water emulsions containing ω-3
polyunsaturated fatty acids could be improved by modifying the interface of
the emulsion droplets to decrease transition metal–lipid interactions by lower-
ing the pH below the pI of the protein at which the emulsion droplets are
cationic. At concentrations equal or greater than 1 μM, EDTA increased the
oxidative stability of WPI-stabilized emulsions, at pH 3.0 and 7.0 (Table 3.5,
ref 11). Neither citrate nor polyphosphates were effective, however, at inhibit-
ing lipid oxidation at these concentrations. EDTA can thus increase the
oxidative stability of oil-in-water emulsions containing WPI without affecting
their physical stability.
In algae oil-in-water emulsions stabilized with WPI, catechin and ascorbic
acid were found to be prooxidative at pH 3.0 and ineffective at pH 7.0 (Table
3.5, ref 12). In contrast, the grape seed extract inhibited both lipid hydroperoxide
and propanal formation at pH 3.0 and 7.0. The superior antioxidant activity of
the grape seed extract may be due to the presence of oligomeric procyanidins,
which are better antioxidants than their monomeric counterparts. Grape seed
extracts were recently recognized in the USA as GRAS (generally recognized
as safe) and thus represent a potentially useful natural food antioxidant.
The antioxidant activities of anthocyanins and derivatives from blackcur-
rants, raspberries and lingonberries were compared in a whey protein-stabilized
rapeseed oil emulsion (Table 3.5, ref 13). Blackcurrant anthocyanins were the
most potent antioxidants for both lipid and protein oxidation. This activity was
attributed to the combination of delphinidin and cyanidin glycosides. In a
similar whey protein-stabilized emulsion, raspberry juice provided better
overall antioxidant protection against lipid and protein oxidation compared to
blackberry juice (Table 3.5, ref 14). The antioxidant activity of berry juices
towards lipid oxidation increased with increasing concentration of berry juices.
Red berry juice anthocyanins, and other phenolic compounds acted as antioxi-
dants by improving the oxidative stability of whey protein emulsions.
As shown previously (Table 3.5, ref 10), proteins dispersed in the continuous
phase of oil-in-water emulsions are effective inhibitors of lipid oxidation
reactions. The oxidative stability of cysteine, tryptophan and methionine resi-
dues was markedly inhibited by low concentrations of β-lactoglobulin
incorporated into the continuous phase of a menhaden oil-in-water emulsion
(Table 3.5, ref 15). β-Lactoglobulin also significantly inhibited lipid oxidation.
Cysteine oxidized before tryptophan in β-lactoglobulin, and both protein
residues oxidized before lipid oxidation could be detected. However, no
oxidation of the methionine residues of β-lactoglobulin was observed, appar-

ently because it may have been less exposed to the protein surface than the
protein residues. The antioxidant activity of these proteins may involve both
free radical scavenging by amino acid residues and chelation of catalytic
transition metals, but the precise mechanism remains unclear. Further research
is needed to develop more effective applications of food proteins as inhibitors
of oxidation in food emulsions.
The stabilizing effects of BSA and sodium caseinate (NaCas) were com-
pared in stripped sunflower oil-in-water emulsions with similar and stable
droplet size distributions (Table 3.5, ref 16). At pH of 6.5 above the isoelectric
point of both proteins, the oxidative stability of the emulsions depended on
metal availability. In the absence of EDTA and when metal ions were freely
available, oxygen uptake, conjugated dienes and volatile compounds devel-
oped faster in sodium caseinate stabilized emulsions than in those prepared
with BSA. This effect was attributed to the chelating properties of NaCas and
to electrostatic interactions attracting some metal ions at the interface where
they could initiate lipid oxidation. When metal were inactivated by the pres-
ence of EDTA, oxidation was delayed to a greater extent in emulsions made
with sodium caseinate than in BSA stabilized emulsions. Under these condi-
tions, the metal chelating activity of NaCas was fully expressed.
c) Liposome systems. Proteins can strongly bind with flavonoid antioxidants

in foods, and generally can increase the oxidative stability of liposomes. Thus,
the addition of BSA increased the oxidative stability of a lecithin liposome and
changed the relative activity of flavonoids and phenolic acids (Tables 3.6 and
3.7, ref 1). In the absence of BSA, the relative activities of phenolic compounds
showed the following trend: ferulic acid > epicatechin > catechin > malvidin
> caffeic acid ~ quercetin > propyl gallate. In the presence of BSA, the relat-
ive lipid oxidation inhibition activity of malvidin increased, followed by that
of rutin and ferulic acid, whereas the activity of epicatechin and catechin
decreased, followed by propyl gallate, caffeic acid and quercetin. The highly
polar gallic acid and delphinidin were prooxidant. Protein oxidation inhibition
activity was highest for ferulic acid, followed by rutin, malvidin and catechin.
BSA may improve the antioxidant activity of malvidin, rutin and ferulic acid
by enhancing their adsorption to the liposome interface undergoing oxidation.
The activities of phenolic compounds containing anthocyanins, ellagitannins
and proanthocyanidins, from raspberry, bilberry and blackcurrant were com-
pared in a lactalbumin–liposome system (Table 3.7, ref 2). Lipid oxidation was
best inhibited by the phenolics in lingonberries and bilberries, followed by
phenolics in blackcurrant and raspberries, whereas bilberries and raspberries
best inhibited protein oxidation. In lingonberries, proanthocyanidins in dimeric
and trimeric forms were the most active constituents for inhibition of both lipid
and protein oxidation. In bilberries and blackcurrants, anthocyanins were most
effective in inhibiting hexanal formation and protein carbonyls. A mechanism

was postulated for the inhibition of protein oxidation by berry phenolics,
involving interaction between aromatic rings of the polyphenols and aromatic
residues of tyrosine and tryptophane of the protein.
The effect of different proteins on the antioxidant activity of anthocyanins
and other phenolic compounds varied in liposome systems (Table 3.7, ref 3).
Thus, casein, lactalbumin and BSA influenced the relative antioxidant activi-
ties of berry phenolic compounds on both lipid and protein oxidation in a
lactalbumin–lecitin liposome. Casein and lactalbumin inhibited conjugated
diene hydroperoxides more effectively than lactalbumin. Casein was the most
stable in this liposome system. The formation of protein carbonyls was greater
in the liposome containing lactalbumin than those containing casein or BSA.
Procyanidins and ellagic acid were better antioxidants than anthocyanins on
the basis of lipid oxidation. Glycosylation of cyanidin with one glucose
increased the inhibition of protein oxidation more effectively than glycosylation
with two or three glucose molecules.
These phenolic compounds may inhibit liposome oxidation by a mechanism
involving covalent and non-covalent interactions of tryptophan protein residues
and lipid decomposition aldehydes, producing protein carbonyl compounds.
These protein modifications may also involve quinones, formed by oxidation
of the corresponding phenolic compounds. Various milk proteins exhibited
different oxidative stability on the basis of loss of tryptophan and formation of
protein carbonyl compounds.
Lactoferrin is a non-heme glycoprotein in mammalian milk that inhibits lipid
oxidation by binding tightly two ferric ions to produce a stable iron complex
(Figure 3.6). Lactoferrin inhibits oxidation more efficiently in liposomes than
in emulsions (Lipid Oxidation, 2nd ed, 2005, Table 10.8, p. 275). Various
phenolic antioxidants showed synergistic effects by reinforcing the inhibitory
activity of lactoferrin in emulsions and liposomes (Table 3.8). The highest
synergistic effects were observed with the phenolic extracts from extra virgin
olive oils rich in polyphenols added to lactoferrin. This synergistic effect was
higher in liposomes than in emulsions. Thus, the calculated synergism was 6%
and 49% between 1 μM lactoferrin and 100 μM ferulic acid respectively in the
emulsions and liposomes, and 126% between lactoferrin and phenolic extracts
from extra virgin olive oil. Therefore, the activity of lactoferrin in preventing
lipid oxidation could be improved by using it in combination with natural
phenolic antioxidants.
Proteins can also increase the oxidative stability of phenolic antioxidants.
Thus, in the presence of BSA, sunflower oil-in-water emulsions containing
epigallocatechin gallate, caffeic acid and Trolox were much more stable during
storage than controls without BSA. During storage of solutions containing
BSA and antioxidants, the structure of BSA changed, with loss of fluorescent
tryptophan groups, and formation of a BSA protein antioxidant adduct with
62
Table 3.6. Effect of bovine serum albumin (BSA) on inhibition (%) of lipid and protein oxidation on the antioxidant activity of phenolic
compounds (5 µM) in lecithin liposome (PC)a
Inhibition of
hydroperoxides Lysine loss Protein carbonyls
Structures R Antioxidants
PC PC + BSA PC PC + BSA
O
HO H Gallic acid –34.6 –23.8 –35.9 –15.7
OR
HO
OH
C3H7 Propyl gallate 5.6 –6.8 –25.2 –9.3
O
RO H Caffeic acid 19.2 4.7 –7.0 –4.2
OH
HO
OH Me Ferulic acid 54.6 57.8 97.5 40.9
OH
OH H Delphinidin –18.6 –28.3 –37.4 –31.9
HO O+
OR
Me Malvidin 23.7 40.7 57.1 21.5
OH
OH
OH
OH
– (+)-Catechin 44.0 18.5 21.5 5.9
HO O
OH
– Epicatechin 47.5 16.0 11.7 –1.2
OH
OH
OH
H Quercetin 19.1 16.5 6.8 2.0
HO O
OR Rutinose Rutin 23.3 31.7 83.3 10.8

OH
O
a
From Heinonen et al. (1998b) and Lipid Oxidation, 2nd ed, 2005, Table 10.13, page 283. Liposome contained 0.8% by wt PC, oxidized at 37°C, with 3 µM cupric
acetate, in 25 mM succinate buffer at pH 4.7, without or with 0.16% BSA.
ANTIOXIDANT ACTION IN MULTIPHASE SYSTEMS
63
Table 3.7. Effect of phenolic antioxidants on lipid and protein oxidation of liposomes
Proteins Conditions Inhibitors Methods Ref.a
BSA 37 °C Phenolics, grape extracts, CD, hexanal, trypto- (1)

Cu acetate wine phan, protein carbonyl
BSA, 37 °C Anthocyanin, procyanidins, CD, hexanal, (2)
lactalbumin, Cu acetate ellagic acid fluorescence
casein
Lactalbumin 37 °C Berry phenolics CD, hexanal, (3)
Cu acetate fluorescence
a
References: (1) Heinonen et al. (1998), (2) Viljanen et al. (2004a), (3) Viljanen et al. (2004b).
Table 3.8. Effect of α-tocopherol and other phenolic compounds on the inhibition of
lipid oxidation by lactoferrin in oil-in-water emulsions and liposomesa
Lipid systems Inhibition, %
Hydroperoxides Fluorescence
Emulsions
1 µM lactoferrin –5.1 2.4
500 µM α-tocopherol 68 30
100 µM extra virgin olive oil (EVO) 71 60
1 µM lactoferrin + 500 µM α-tocopherol 83 46
100 µM ferulic acid 71 21
1 µM lactoferrin + 100 µM ferulic acid 71 25
1 µM lactoferrin + 100 µM tyrosol 30 18
1 µM lactoferrin + 100 µM EVO 77 55
Liposomes
1 µM lactoferrin 2.5 2.1
5 µM α-tocopherol 78 45
5 µM EVO 28 12
1 µM lactoferrin + 5 µM α-tocopherol 83 52
5 µM ferulic acid 53 35
1 µM lactoferrin + 5 µM ferulic acid 78 61
1 µM lactoferrin + 5 µM tyrosol 70 21
1 µM lactoferrin + 5 µM EVO 83 27
a
Medina et al. (2002). Oil-in-water emulsions contained 10% corn oil stripped of tocopherols and 1%
lecithin in 25 mM phosphate buffer at pH 6.6. Oxidation was carried out at 30°C and measured by
conjugated diene hydroperoxideds at 234 nm and fluorescence at 345/416 nm. Liposomes contained 1%
lecithin in 25 M phosphate buffer at pH 6.6 and were oxidized the same way as emulsions.
radical-scavenging activity. The stabilizing and synergistic effect of protein–

polyphenolic antioxidant adducts formed during storage is explained by their
concentration at the oil–water interface because of the surface-active nature of
the protein.
Figure 3.6. Diagramatic structure of lactoferrin.
d. Special emulsions. Several techniques have been developed to modify

the interface of oil-in-water emulsions to improve its oxidative stability. Fish
oil-in-water emulsion droplets coated by multiple layers of emulsifiers including
corn syrup solids, lecithin and chitosan (a cellulose-like biopolymer) produced
emulsions with cationic thick membranes that were more oxidatively stable
than emulsions coated by lecithin alone (Table 3.9). Mixtures of EDTA and
tocopherols were as effective as mixed tocopherols in increasing the oxidative
stability of liquid and freeze-dried emulsions prepared with lecithin and
chitosan. However, the combination of EDTA and mixed tocopherols was not
more effective than EDTA alone, suggesting that control of prooxidant metals
was the most important factor. These results suggest that controlling the
prooxidant activity of transition metals with EDTA is more important than free
radical scavenging by tocopherols. Spray-drying tuna oil-in-water emulsion
droplets, coated with a lecithin and chitosan multilayer system to produce
thick, cationic emulsion droplet interfacial membranes, produced similar
protective effects. The coating of emulsions with high moisture was also more
oxidatively stable, apparently due to the formation of browning Maillard
reaction products. The production of emulsion droplets coated with lecithin
and chitosan could thus afford stabilization of oxidatively labile fish lipids, and
increase the effectiveness of metal chelators for use in a variety of food
products and ω-3 fatty acid ingredients for functional foods.
B. Partition
In multiphase food and biological systems, the effectiveness of antioxidants is
influenced by their partition between the lipid phase, the water phase and the
interface, according to their chemical structures and polarity as a function of pH
and the nature of the surfactant. The partitioning behavior of different phenolic
antioxidants varies in oil-in-water emulsions, according to their solubilization
equilibrium as affected by interactions between oils and emulsifiers. In corn
66
Table 3.9. Oxidative stability of special emulsions of liquid and dried tuna oil with multilayered membranes at 37°Ca,b
Types of emulsions PV (5d)a Freeze-dried emulsions PV (9d)a Spray-dried or bulk oil PV (10d)b
Primary (lec) 18 Primary 30 Spray-dried >10

Primary + 20% CSS 14 Secondary 6 + MT >10
Secondary (lec + chit) 5 Control 8 11% RH 8
+ CSS 4 + EDTA 2 33% RH 15
Secondary 5 + MT 4 52% RH 5
+ 100 ppm MT 4 + EDTA + MT 2 11% RH + MT + EDTA 5
Secondary 4.5 – – 33% RH 7
+12 µM EDTA 3 – – 52% RH 3
+ MT 4 – – Bulk oil 150
+ MT + EDTA 3 – – + MT 80
+ EDTA 2.5 – – – –
a
Klinkesorn et al. (2005a)
b
Klinkesorn et al. (2005b).
All peroxide values were estimated from Figures in the references. Oil-in-water emulsions were prepared by electrostatic deposition producing droplets coated by
multiple layers of emulsifiers. Primary emulsion prepared after first sonication + additives, secondary emulsion after second sonication + additives.
Abbreviations: PV, peroxide values; d, days; lec, lecithin; chit, chitosan; CSS, corn syrup solids; MT, mixed soybean tocopherols (α, γ, δ); EDTA, disodium
ethylenediaminetetraacetic acid; RH, relative humidity.
Table 3.10. Partition of antioxidants in Tween 20 corn oil-in-water emulsionsa
Antioxidants Aqueous phase Oil phase Surfactant phase Partition

(%) (%) (%) (Po/w)
O
MeO
OH
HO
49 9 43 0.74
OH
Ferulic acid
O
HO
OH
HO
55 1.5 44 0.11
OH
Caffeic acid
O
HO O C 3H 7
HO
43 10 48 0.89
OH
Propyl gallate
O
HO OH
HO
68 2 30 0.12
OH
Gallic acid
R
HO
Me 27 24 50 3.6
R
O COOH
R
Trolox
OH
OH
HO O
48 0.9 51 0.08
OH
OH
Catechin
a
From Schwarz et al. (1996) and Lipid Oxidation, 2nd ed, 2005, Table 10.17, p. 291.
oil-in-water emulsions, the concentration of antioxidants in the aqueous phase

decreased in the order: gallic acid > caffeic acid > ferulic acid ~ catechin >
propyl gallate > Trolox > (Table 3.10). The concentration of Trolox in the
water phase decreased at different rates according to its affinity toward the
emulsifier Tween-20. The other hydrophilic antioxidants catechin, propyl
gallate and gallic acid also favored the interface (30–51%), which is signifi-
cantly more enriched with emulsifier than the oil phase (1–10%). The
concentration of Trolox in the water phase of different emulsions also varied
according to the lipid, decreasing in the order: egg phosphatidylcholine (80%),
linoleic acid (42%), corn oil (36%), methyl linoleate (31%).
The partitioning properties of ethyl gallate and gallic acid were studied in
four different emulsions prepared with anionic dodecylsulfate (SDS–), cationic
cetyltrimethylammonium bromide (CTAB+), non-ionic polyoxyethylene 20
cetyl ether (Brij 58) and zwitterionic partially hydrolysed lecithin (PHLC±)
(Table 3.11). The proportion of ethyl gallate increased in the order:
PHLC < SDS < Brij 58 < CTAB, in the lipid phase, and decreased in the same
order in the aqueous phase. With the anionic SDS emulsions, increasing the
concentrations of SDS from 1 to 2% increased the proportion of ethyl gallate
associated with the emulsifier phase and decreased it in the oil phase. In
contrast, with PHLC emulsions, although increasing the concentration of
HPLC also increased the proportion of ethyl gallate in the emulsifier phase, it
had no effect in the oil phase. With the cationic CTAB emulsions, the high
concentration of gallates in the emulsifier phase was attributed to the interac-
tions of the negative bromide counterion acting as hydrogen bond acceptors.
With the non-ionic Brij 58 emulsions, the bulky and polar polyoxyethylene
chains resulted in the highest solubilization of the antioxidant by hydrogen
bond formation. With the zwitterionic PHLC emulsions, the much lower
solubilization capacity observed for ethyl gallate accounts for the relatively
lower decrease solubilzed by the oil. Free gallic acid showed the highest
solubilization capacity in the aqueous phase. The significant charge and polar
effects of the emulsifiers on the solubilization of the antioxidant are apparently
related to hydrophobic interactions and hydrogen bonds.
Emulsions prepared with different types of emulsifiers affected the effec-
tiveness of gallic acid and esters of different chain length and polarity (Table
3.12). Except for the increase in inhibition of hydroperoxide formation be-
tween gallic acid and methyl gallate, the order of antioxidant activity followed
significantly different trends in emulsions of sodium dodecyl sulfate (SDS–),
polyoxyethylene 20 cetyl ether (Brij 58) and partially hydrolysed lecithin
(PHLC±). In SDS– emulsions, the activity increased in the order: gallic
acid < octyl gallate < butyl gallate < propyl gallate < ethyl gallate = methyl
gallate. In Brij 58 emulsions, the antioxidant activity decreased from methyl to
propyl gallate, and then increased for butyl and octyl gallate. In PHLC
emulsions, antioxidant activity increased with increasing alkyl chain length,
except for octyl gallate which was less active than propyl and butyl gallates.
The antioxidant activity exhibited a non-linear relationship with decreasing
polarity of the gallates in SDS–, Brij 58 and PHLC emulsions. Since differences
in polarity did not account for these results, other factors may be considered. In
SDS– emulsions, hydrophobic interactions and reduced mobility with increasing
Table 3.11. Effect of different emulsifiers on the partition behaviors of ethyl gallate and
gallic acid in oil-in-water emulsionsa
Emulsion phases Proportion of antioxidant in emulsion phases, %
SDS CTAB Brij58 PHLC
Ethyl gallate
Emulsifier 49 95 66 40
Oil 2 0.1 5 6
Water 49 5 29 55
Gallic acid
Emulsifier <1 55 22 6
Oil trace trace trace trace
Water >99 56 78 94
a
Stöckmann et al. (2000).
Emulsions prepared with 20% corn oil stripped of tocopherols in acetate buffer, (pH 5) and 1%
emulsifier. See Table 3.11 for antioxidant activities.
Abbreviations: SDS, sodium dodecyl sulfate; CTAB, cetyltrimethylammonium bromide; Brij 58,
polyoxyethylene 20 cetyl ether; PHLC, partially hydrolysed lecithin.
Table 3.12. Effect of emulsifier types on inhibition of hydroperoxides by gallate

derivatives in corn oil-in-water emulsionsa
Antioxidants Inhibition of hydroperoxides, %
SDS– Brij58 PHLC
Gallic acid –14 7.2 40

Methyl gallate 96 86 73
Ethyl gallate 96 70 74
Butyl gallate 56 73 79
Octyl gallate 13 73 77
a
Stöckman et al. (2000). See Table 3.11 for emulsion preparation and abbreviations.
chain length may lower the diffusion of gallates and decrease their activity. In
Brij 58 emulsions, the antioxidant activity may be affected by increased
solubility of the emulsifier molecules, resulting in deeper penetration of into
the interface. In PHLC± emulsions, the antioxidant trend followed the polar
paradox, with higher antioxidant activity correlating with increased chain
length and decreasing polarity. The polar paradox may therefore be limited to
emulsions based on phospholipids. The relatively low antioxidant activity of
anionic gallic acid is expected from its water solubility and reducing capacity
in the aqueous phase by converting Fe3+ into the more catalytically active Fe2+
ions. Therefore, the lipid phase solubilization capacity of different emulsifiers
in oil-in-water emulsions depends not only on the relative charge and polarity
of the antioxidants, but also other properties including hydrogen donation,

hydrophobic interactions and structural properties. How these complex inter-
actions influence antioxidant activity is still not well understood.
The association or affinity of the antioxidants can explain the order of the
decrease in partition in the water phase between oil–water mixtures and the
corresponding Tween 20 emulsions. A significant proportion of Tween 20 in
corn oil emulsions was partitioned in the oil–water interface (26–68%), and
also formed micelles (32–74%) that do not reside in the interface. In Tween
20 micelles, the ether oxygen of the polyoxyethylene chain takes part in
hydrogen bond formation. The low antioxidant activity of hydrophilic anti-
oxidants such as rosmarinic acid, catechin, propyl gallate, gallic acid and tea
catechins was attributed to their partition into the Tween 20 micelles and
into the aqueous phase. The prooxidant activity of highly hydrophilic tea
catechin gallates in Tween 20 emulsions was also attributed to the reduction
of Fe3+ to more catalytically active Fe2+ when solubilized in the aqueous
phase (Table 3.1, ref 6).
Antioxidants may also be distributed into surfactant/emulsifier-rich interfa-
cial layers in multiphase food emulsions. The partitioning properties of a
particular antioxidant depend not only on the chemical structure and relative
polarity of the antioxidant, but also vary according to the lipid substrates,
surfactants, pH, temperature and the composition of the phases. In a mayon-
naise based on rapeseed and fish oils, α-tocopherol and various polar
antioxidants (Trolox, ferulic acid, caffeic acid, propyl gallate, gallic acid and
catechin) partitioned between the oil, aqueous, precipitate and emulsion phases
according to their structures and polarity (Table 3.13). The distribution of
antioxidants varied significantly. The majority of non-polar α-tocopherol
(94%) was present in the oil phase, and the remaining portions were in the
Table 3.13. Partition of antioxidants in different phases separated from mayonnaisea
Antioxidants Oil phase Aqueous Phase 2 Precipitate Emulsion phase
α-Tocopherol 94 0 1.9 3.8

Trolox 83 5.7 7.0 4.3
Ferulic acid 76 16 6.2 2.4
Caffeic acid 19 50 26 5.7
Propyl gallate 45 24 24 7.0
Gallic acid 0 80 18 2.4
Catechin 0 59 35 6.8
a
From Jacobsen et al. (1999).
Mayonnaise prepared from rapeseed oil (64%), fish oil (16%), distilled water (9.2%), egg yolk (4%),
vinegar (4%), lemon juice (1.2%), sugar (1%), NaCl (0.3%), water-dispersible 20% tocopherol mixture
(0.2%), and potassium sorbate (0.1%). Mayonnaises were frozen at –40°C and then separated by
centrifugation into oil and water phases, and the water phase was further separated by ultracentrifugation
into aqueous phase 2, precipitate, and emulsion phase.
Figure 3.7. Partition of phenolic compounds.
precipitate and emulsion phase and none in the aqueous phase. The more polar
Trolox was distributed mainly in the oil phase (83%), with more in the aqueous
phase (5.7%), and the remainder in the precipitate (7%) and emulsion phase
(4.3%). The polar antioxidants gallic acid (80%) and catechin (59%) were
found mainly in the aqueous phase and none in the oil phase. After ultracen-
trifugation, large proportions of polar antioxidants were found in the emulsion
phase (7–35%) and the precipitate (2–7%). This distribution indicated entrap-
ment of these polar antioxidants at the oil–water interface in mayonnaise. The
antioxidants tested were classified according to their partition coefficients
(Po/w) determined in unbuffered oil–water mixtures (20:80 w/w) in increasing
polarity: Trolox (Po/w ~3.5), propyl gallate, ferulic acid (Po/w 0.7–0.9), and
gallic acid, caffeic acid, catechin (Po/w 0.1) (Figure 3.7).
In rapeseed emulsions containing 2% whey protein emulsions, the polar
berry anthocyanins partitioned mainly into the aqueous phase (69–73%), and
the rest was associated with the proteins located in the interfacial oil/water
environment. In this phase, whey proteins act as emulsifiers, forming viscous
layers surrounding the oil droplets. The polar anthocyanins were thus located
favorably for antioxidant action towards protein oxidation (Table 3.5, ref 13,
14). The presence of the lipid decreased the share of anthocyanin in the aqueous
phase. Thus, the structure of food affects the antioxidant activity by influencing
the partitioning of the antioxidant.
C. Summary
The relative activities of various antioxidants vary widely according to the site
of action in different systems, the charge and the solubility of components in
micelle compared to liposome systems and solutions. Antioxidant activity is

thus significantly affected by the physical state of different multiphase systems.
The oxidative stability of food lipids varies according to their colloidal
location, because of their exposure to prooxidant metals and phenolic antioxi-
dants varying widely in relative polarity and affinity in different phases of
foods and biological systems. The relative activities of lipophilic and hy-
drophilic antioxidants vary between bulk oil systems and oil-in-water emulsions.
In most emulsions, polar antioxidants such as Trolox and ascorbic acid acted as
prooxidants. In contrast to polar antioxidants, α-tocopherol was much more
active in emulsions than in bulk systems. The partitioning of antioxidants
according to their relative polarity, surface accessiblity and the interaction of
emulsifiers with antioxidants are important parameters that determine their
antioxidant activity in lipid-containing systems. Knowledge about the sites of
prooxidant and antioxidant action in multi-component systems is essential to
predict better the oxidative stability of complex foods and biological systems.
Foods of improved quality may be developed if the association and driving
forces of prooxidants and antioxidants can be controlled in multiphase systems.
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CHAPTER 4
Antioxidant protocols for foods and biological
systems
The antioxidant activity of natural antioxidants in foods and biological systems

has been evaluated by a great multiplicity of methods, which have received
much attention in the past decade. Many different antioxidant protocols have
been used to evaluate food and biological antioxidants. Unfortunately, variable
and inconsistent results have been obtained, depending on the methods and
conditions used to test activity. Many different substrates, system compositions
and analytical methods have been employed to evaluate the effectiveness of
antioxidants. In complex foods, however, antioxidants inhibit lipid oxidation
by a multi-step mechanism that requires more than one specific evaluation
method. When testing natural antioxidants, it is important to consider the food
system composition, the type of food, the mode of accelerating oxidation, the
methods used to assess oxidation, and how to quantify activity. Antioxidant
effectiveness is also determined by the heterogeneous nature of the system, the
physicochemical state of lipid substrate, the degree of unsaturation and the
presence and types of initiators, notably metal catalysts and their possible
interaction. Because most natural antioxidants are multifunctional, a reliable
antioxidant protocol requires the measurement of more than one property
relevant to foods. Each evaluation should be carried out under various condi-
tions of oxidation, using methods to measure primary and secondary products
of oxidation.
For the careful choice of antioxidant protocols, several important questions
should be addressed in food systems:
1. What are the true protective properties of antioxidants?
2. What is the antioxidant protecting against?
3. What substrates are oxidized and what products are inhibited?
4. What is the location of the antioxidant in the system?
5. What is the effect of other interacting components?
6. What conditions are relevant to food applications?
7. What is the mechanism of protection?
8. What is the accessibility of substrates to antioxidants and prooxidants?
Several protocols used to test antioxidants in foods consist of oxidizing a
lipid substrate under standard conditions with or without an added initiator, and
evaluating the activity by various appropriate methods to determine how much
oxidation is inhibited at a relevant end point (Table 4.1). The validity of some
77
Table 4.1. In vitro antioxidant assays with lipid substrates to determine activity of food and biological antioxidantsa
78
Oxidizing species Solvents Conditions, Measurement and Antioxidants Ref.b

inducers quantification tested
Tween emulsified linoleic Buffer pH 7.0 25°C in cuvette, Rate of β-carotene destruction Several (1)
acid with β-carotene different inducers (nmole/min)
Tween emulsifed linoleic acid Buffer pH 7.0–7.4 37°C, dark, 16 h, TBARS assay, conjugated dienes Phenolics, (2)
ferrous sulfate (234 nm) or HPLC anthocyanins
SDS emulsified Linoleic acid Buffer pH 7.4 37 or 40°C, minutes, Conjugated dienes (234 nm) Several (3, 4)
emulsion AAPH Kinh/Kpc
Methyl linoleate Hexane/2-propanol/ 37°C, AMVN Methyl linoleate hydro- Buckwheat (5)
ethanol peroxides by HPLC compounds
Methyl linoleate Dodecane 110°C, O2 bubbling Residual methyl linoleate by GC Phenolics, (6)
spices
PC liposomes with Buffer pH 7.0 23°C in cuvette, Decrease in relative Tart cherry (7)
phenylpropionic acidd with NaCl ~ 20 min, FeCl2 fluorescence intensity compounds
Human low-density PBSe 2–several hours, Cu2+, Conjugated dienes (234 nm), Several (8, 9)
lipoprotein (LDL) metmyoglobin hexanal, induction time,
50% oxidation, % inhibition
LDL or membrane phospho- PBSe 37°C, 35 min–2 h, Rate of PnA fluorescence Phenolics (10)
lipids with PnA incorporatedf decrease
AAPH or AMVN or H2O2

a
References: (1) Kanner et al. (1994), (2) Tamura and Yamagami (1994), (3) Pryor et al. (1993), (4) Foti et al. (1996), (5) Watanabe (1998), (6) Cuvelier et al. (1992),
(7) Wang et al. (1999b), (8) Frankel et al. (1992, 1995), (9) Miller and Paganga (1998), (10) Laranjinha et al. (1992, 1994).
b
Abbreviations: TBARS, thiobarbituric acid reactive substances; SDS, sodium dodecylsulfate; AAPH, 2,2'-azobis(2-amidinopropane) dihydrochloride; AMVN,
2,2'-azobis(2,4-dimethylvaleronitrile); PC, phosphatidylcholine.
c
Kinh , rate constant for reaction of the linoleic acid peroxyl radical with the antioxidant; Kp propagation rate for linoleic acid oxidation.
d
Liposomes are a mixture of 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphocholine and 3-p-[p-(6-phenyl)-1,3,5-hexatrienyl] phenylpropionic acid probe.
e
PBS is phosphate buffered (10 mM, pH 7.4) saline (100 mM).
f
PnA, cis-parinaric acid, is a fluorescent 18-carbon conjugated tetraene fatty acid.
ANTIOXIDANT PROTOCOLS FOR FOODS AND BIOLOGICAL SYSTEMS 79
of the currently used methods may be questioned because the data obtained
may be difficult to interpret. To evaluate antioxidants, it is essential to measure
several parameters of oxidation. Although many natural antioxidants such as
tocopherols and flavonoids inhibit both the formation and decomposition of
hydroperoxides, many studies use only one method to measure primary oxida-
tion products such as peroxide value or conjugated dienes. The choice of
substrate greatly influences the degree of unsaturation. The oxidation condi-
tions, analyses and the type of kinetics and mechanism of antioxidants are all
important parameters that strongly affect their evaluation. Misleading data can
be obtained in many ill-defined test systems, by neglecting important
compositional and interfacial phenomena concerning the charge and solubility
of multiple components in food systems that strongly affect antioxidant per-
formance (Section C).
Because many antioxidants are multifunctional, their activity and mecha-
nism dominating in a particular test system depend on the oxidation conditions
affecting the kinetics of oxidation and the resulting products. The effectiveness
of antioxidants in complex heterogeneous foods and biological systems is
affected by many factors, including the partitioning properties of the antioxi-
dants between lipid and aqueous phases, and their interface between them, the
oxidation conditions, and the physical state of the oxidizable substrate. Of
particular importance are the conditions used to accelerate oxidation by raising
the temperature, by using metal catalysts or other types of initiators, by
increasing surface and by exposing to light. Finally, antioxidant protocols must
be carefully designed by considering the specificity of methods employed to
analyse the progress of oxidation and choosing a proper end point based on
protecting effects in foods.
A. Food and biological oxidation methods

The effectiveness of antioxidants in protecting foods against oxidative deterio-
ration is very dependent on complex phenomena determined by the relative
physical states of the lipid substrates, the conditions of oxidation, the methods
used to follow oxidation and the stages of oxidation. In foods, antioxidant
effectiveness is evaluated by measuring inhibition of oxidation of a suitable
substrate under appropriate and standard conditions, and by choosing a proper
method to determine an end point of oxidative spoilage. The methods used to
evaluate antioxidants in foods are complicated by many factors:
(i) The use of different lipid substrates has a significant impact on the
activity of various antioxidants according to their hydrophilic or lipo-
philic nature.
(ii) Solubility and partition properties affect the activity of antioxidants in
heterogeneous systems where they are distributed differently between
Figure 4.1. Interfacial phenomenon in antioxidant activity: bulk oil versus emulsion systems
(from Lipid Oxidation, 2nd ed, 2005, Figure 10.5, p. 277).
Bulk oil +
Bulk oil polar emulsifiers
Solubization of hydrophilic
antioxidants in oil phase:
● Hydrophilic > ● Lipophilic Lipophilic > Hydrophilic
Figure 4.2. Effect of polar emulsifiers on partition of hydrophilic antioxidants.
aqueous phase, lipid phase with surfactant and micelles (Figures 4.1
and 4.2).
(iii) Different results can be obtained at different temperatures because the
mechanism of oxidation and hydroperoxide decomposition, and the
solubility of oxygen change with temperature.
(iv) Different methods used to follow oxidation can give varying results
according to the different effects of antioxidants on formation of
hydroperoxides and their decomposition. Relative antioxidant
efficiencies vary markedly from one oxidizing lipid substrate to an-
other. In the same lipid substrate, the relative activities of antioxidants
often depend on the antioxidant concentrations.
The activity of natural antioxidants is greatly affected by complex interfacial
phenomena in emulsions and multi-component foods. The methodology for
evaluating natural antioxidants must be carefully interpreted, depending on
whether oxidation is carried out in bulk oils or in emulsions, and which
analytical method is used to determine the extent and end point of oxidation. To
understand and predict better how natural antioxidants may protect foods
against oxidation, the following complex questions need to be carefully
considered for the judicious choice of antioxidant evaluation protocols:
a) What are the protective properties of antioxidants?
b) What substrates are oxidized and what products of oxidation are inhib-
ited?
c) In a multiphase food system, is the antioxidant located where oxidation
takes place?
d) Are there any other interacting components that may affect the results?
e) What conditions are relevant to real-life applications?
Meaningful interpretation of antioxidant action thus requires:
a) Specifying the oxidizing substrate protected
b) Measuring the correct extent of oxidation and inhibition
c) Choosing an appropriate end point of oxidation
d) Determining any possible adverse prooxidant effects from the anti-
oxidants by using a range of concentrations.
Each antioxidant evaluation should thus be carried out under various condi-
tions of oxidation, using several methods to measure different products of
oxidation related to real food quality. There cannot be a short cut approach to
determining the activity of antioxidants. Various testing protocols should
include:
a) A suitable substrate (triacylglycerols or phospholipids) in bulk, emul-
sions, or liposomes systems
b) Relatively mild conditions of oxidation (below 60°C) to minimize
changes in mechanism due to oxygen solubility, and avoiding artificial
azo initiators that are not relevant to either food or biological oxidation
c) Analyses of both initial and decomposition products at several time
periods to include the initiation and early stages of propagation phases
of oxidation
d) Sensory evaluations for foods, vegetable and fish containing linolenic
acid and long-chain polyunsaturated fatty acids that produce fishy
responses at very low levels of oxidation
e) Different levels of antioxidants compared at the same molar concentra-
tions of active components
f) Calculations based on induction period, or percent inhibition or rates of
hydroperoxide formation or decomposition, or antioxidant concentra-
tion required to obtain an appropriate level of inhibition.
The use of artificial thermolabile azo compounds, such as the water-soluble
AAPH and lipid-soluble AMVN, are very popular among researchers inter-
ested in the kinetics of lipid oxidation, because they can be readily decomposed
Table 4.2. Various lipid peroxidation protocols used to evaluate natural polyphenolsa
Antioxidants Substrates Conditions Methods Ref.b
Quercetin PC liposomes Fe-ascorbate TBARS (1)

Caffeic acid
Kaempferol
Catechins LDL+VLDL Cu2+, 37°C Conjugated dienes (2)
Tea catechins
Catechin Rat liver microsome Fe-ascorbate TBARS (3)
Tea catechins homogenate
Myricetin
Epicatechin Diluted blood plasma Cu2+ Fragmentation of (4)
Tea catechins APO B-100
Epicatechin Human plasma AAPH TBARS (5)
Rutin
Tea catechins
Quercetin Rat liver microsome AAPH, 37°C TBARS (6)
Catechin homogenate
Rutin
a
From Roginsky and Lissi (2005).
b
References: (1) Plumb et al. (1997), (2) Vinson and Dabbagh (1998), (3) van der Sluis et al. (2000), (4)
Hashimoto et al. (2000), (5) Lotito and Fraga (2000), (6) Silva et al. (2002).
into free radicals at known rates that can be controlled. Unfortunately, these
artificial initiators are not relevant to either foods or biological systems, where
lipid oxidation is initiated by redox transition metals. An important inhibition
mechanism of lipid oxidation by natural antioxidants and flavonoids involves
the inactivation of transition metal catalysts by chelation and complex forma-
tion (Chapter 2). Although the kinetics of metal catalysis are complex and
difficult to reproduce, they represent the actual problems of lipid oxidation in
foods and biological systems. For these very reasons, reliable evaluations of
antioxidants require replicate testing on a multitude of systems and conditions
that are relevant to foods and biology.
A wide variety of biological protocols have been used to evaluate natural
antioxidants (Table 4.2). Crude substrates vary from PC liposomes, lipoproteins,
liver microsomes, blood plasma and crude liver homogenates. Oxidation
conditions include iron-ascorbate, copper and AAPH. The TBA method re-
mains one of the most popular in biology, even though this method has been
severely criticized for its limitations and is flawed by analytical artifacts (Lipid
Oxidation, 2nd ed, 2005, Chapter 5, pp. 108–110, 416–417). Other methods
include conjugated dienes, TEAC and LDL APO-B fragmentation. To under-
stand the multiple functions of antioxidants more fully, better protocols are
needed to measure specific products of lipid oxidation and their interactions
with other cell components.
B. Antiradical methods
The potential health benefits of phytochemicals in fruits and vegetables has led
to an explosion of research into the antioxidant properties of polyphenolic
compounds, especially the flavonoids, which constitute an estimated 8000
different compounds. Many simplistic one-dimensional methods have been
developed, using a broad range of conditions, oxidants, methods to measure
and end points of oxidation. This diversity of methodology used to evaluate
natural antioxidants from plant extracts and pure phenolic compounds has led
to widely conflicting results that are very difficult to interpret.
Several protocols have been developed for measuring the free radical trap-
ping, or ‘antiradical’ ability of antioxidants, using a wide variety of radical
generating systems and methods to analyse oxidation and end points of oxida-
tion. The terms antioxidant capacity or antiradical capacity are now commonly
used for testing antioxidants in foods and various plant extracts, including
biological samples. Many antiradical methods have been published, but there is
much confusion in understanding and interpreting the significance of results and
possible biological implications. Because these methods are unspecific and one-
dimensional, they cannot be used to study the multiple protection mechanisms
known for natural antioxidants. They do not take into account the complex
multi-step mechanism of phenolic antioxidants (Chapter 2), their multiple
actions in complex foods and biological systems, partitioning effects, and the
significant effect of different substrates on antioxidant effectiveness. Several
commonly used free radical assays (Table 4.3) are described below. Many of
these methods have been modified and the published results have added to the
difficulty of interpreting comparative data, increasing the confusion in the field.
1. DPPH assay
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical is one of the earliest syn-
thetic radicals used in a substrate-free assay to study the effects of structures on
the activity of phenolic antioxidants (Table 4.3, ref 1–5, Table 4.4). Commer-
cially available DPPH serves as both the oxidizing radical to be reduced by the
antioxidant (AH) and as the colored indicator for the reaction:
DPPH· + AH ——➤ DPPH-H + A·
The effect of antioxidants in decreasing the absorption of DPPH at 517 nm is
measured spectrophotometrically in a methanol solution until the absorbance
reaches a steady state. Assay time may vary from 5–20 minutes to about 8 hours
(Table 4.4). Antiradical efficiency is usually based on the amount of antioxidant
and the time necessary to reach the steady state to 50% of the initial DPPH
concentration. Because of its simplicity, this assay is widely used to determine
the ‘antiradical efficiency’ of polyphenolic compounds, and of different wines,
Table 4.3. Selected antiradical protocolsa
84
Free radical reactant Solvent Conditions inducers Measurements Antioxidants Ref.b

DPPH Methanol Decay at 515 nm, EC50 or Several phenols, (1–5)
1/EC50 × TEC50c wines
ABTS·+ (TEAC) PBS H2O2 and metmyoglobin ABTS·+ decay at 734 nm Plant extracts, juices (6–9)
Trolox reference and wines
Linoleic acid (TRAP) PBS pH 7.4 Plasma 37°C in O2 electrode O2 uptake, induction period Vitamin E, urate, (10)
ABAP plasma, Trolox reference ascorbate, -SH
β-carotene, linoleic acid Aqueous Tween 20/40 55°C Bleaching of β-carotene Plant extracts, (11)
absorption at 470 nm flavonoids
β-PE (ORAC) Buffer pH 7.0 37°C, cuvette AAPH β-PE fluorescence decay Plant extracts (12–16)
Trolox reference
ORACFLd Phosphate buffer, 37°C, AAPH Fluorescein Trolox reference Tea, berry and grape (17–19)
pH 7.4 Automated system extracts
Superoxide O·2 – e Buffer pH 7.4 Phenazine methosulfate- NBT reduction, % inhibiton Phenolic extracts (20–22)
NADH, NBT or IC50e
Fe3+-TPTZ (FRAP) Aqueous 4–8 min Absorbance at 593 nm Fruit juices (23–25)
Fe3+ reduced
a
Abbreviations: ABAP and AAPH, 2,2'-azobis(2-amidinopropane) dihydrochloride; ABTS, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate); AMVN, 2,2´-
azobis(2,4-dimethylvaleronitrile); DPPH, 2,2-diphenyl-1-picrylhydrazyl; NBT, nitroblue tetrazolium; PBS, phosphate saline buffer; PC, phosphatidylcholine;
β-PE, β-phycoerythrin; SDS, sodium dodecylsulfate; TBARS, thiobarbituric acid reactive substances; TBTZ, 2,4,6-tripyridyltriazine.
b
References: (1) Blois (1958), (2) Brand-Williams et al. (1995), (3) Sanchez-Moreno et al. (1998), (4) Sanchez-Moreno et al. (1999a,b), (5) Wang et al. (1999a),
(6, 7) Miller et al. (1993, 1995), (8) Simonetti et al. (1997), (9) Re et al. (1999), (10) Wayner et al. (1985), (11) Koleva et al. (2002), (12–14) Cao et al. (1993, 1996,
1997), (15) Wang et al. (1996), (16) Wang and Cao (1997), (17) Ou et al. (2001), (18) Prior et al. (2003), (19) Prior et al. (2005), (20) Robak and Gryglewski (1988),
(21) Costantino et al. (1992), (22) Basaga et al. (1997), (23) Benzie and Strain (1996), (24) Gardner et al. (2000), (25) Nilsson et al. (2005).
c
EC50 is efficient concentration of antioxidant to decrease initial [DPPH] by 50%, TEC50 is time needed to reach steady state at EC50.
d
A modification of this assay named ORACFL-LIPO (Huang et al., 2002) was made to assay lipophilic antioxidants by using a solvent mixture of 1:1 acetone:water
containing 7% randomly methylated β-cyclodextrin as ‘solubility enhancer’.
e
Phenazine methosulfate and NADH are used to generate superoxide anions non-enzymatically; IC50 is the phenol concentration required to inhibit NBT reduction
by 50%.
grape juices, grape and plant extracts. The decay slope and the absorbance level
reached by the remaining DPPH radical vary significantly with different types
and concentrations of antioxidants. Phenolic compounds are generally very
active in scavenging DPPH radicals. Phenolic compounds react strongly with
DPPH free radicals to produce o-quinone intermediates by H-abstraction,
followed by disproportionation and often by dimer formation (Table 4.3, ref 5).
Many variations in protocols of the DPPH assay have been published to
improve interpretation of results in a wide variety of natural plant sources and
extracts (Table 4.4). The reaction duration, the ratio of antioxidants to the
radical DPPH and the solvent varied widely. The solvent used affected the
relative activity of different polar phenolic compounds, with higher values for
commonly used ethanol than for tert-butyl alcohol. Although the reaction of
quercetin with DPPH in ethanol or methanol produces an initial increase
followed by a decrease in antiradical activity, in aqueous alcoholic mixtures the
antiradical activity decreases. The DPPH chromogen may be also limited by
interference at wavelengths higher than 515 nm used for the assay, where the
activity is underestimated by interference near the visible region with samples
of plant materials.
This assay is further limited because it does not use a substrate, and thus
provides no information on the protective activity of antioxidants towards
foods or biological systems. The DPPH radicals interact with other alkyl
radicals and the time response curve to reach a constant value is not linear with
different ratios of antioxidant:DPPH. Because the DPPH radicals are artifi-
cially generated, this assay is not relevant to real food lipid radicals. This assay
also cannot be used in blood because plasma proteins are precipitated in the
solvent using a mixture of ethanol and methanol. Like many other antiradical
protocols using artificial radicals, the DPPH radical is relatively more stable
than peroxyl radicals produced by lipid oxidation.
2. TEAC or ABTS assay

This indirect assay uses another artificial metastable radical cation, 2,2'-
azinobis(3-ethylbenzothiazoline 6-sulfonate) (ABTS·+), produced continuously
by reacting the ferryl myoglobin radical generated from metmyoglobin, H2O2
and peroxidase (Table 4.3, ref 6–9, Table 4.5). The method consists of
monitoring the decay of the ABTS radical caused by the addition of phenolic
antioxidants. Because the ABTS radical is formed during the incubation with
the test compound, the activity measured is due not only to prevention of the
ABTS radical formation but also to the scavenging of this radical. The activity
of antioxidants in scavenging the ABTS radical cation is measured by the
decrease in its absorbance at 734 nm. The Trolox equivalent antioxidant
activity (TEAC) reflects the amount of Trolox (mM) producing the same
activity as 1 mM of the test compound. This result is consistent with some
86
Table 4.4. Different parameters affecting the DPPH assay
Antioxidants Duration Antioxidant/DPPH ratio Solvent Ref.a
Phenolic compounds Variable Variable 80% aqueous methanol (1)

Flavonoids 5 min Constant Methanol (2)
Caffeic acids, Rosmarinic ac. 20 min Variable Ethanol, t-butyl alcohol (3)
Plant extracts 15 min Constant Ethanol (4)
Phenolic compounds, Apple extracts 30 min Constant 80% aqueous ethanol (5)
Ferulic acid derivatives 30 min Variable Ethanol (6)
Extra virgin olive oils 15 min Variable Methanol (7)
Dietary supplements Variable Variable Methanol (8)
Sorghum extracts 8 hours Constant Methanol (9)
Polyphenols 20 min Constant Methanol (10)
Dry beans 20 min Constant Ethanol (11)
a
References: (1) Fukumoto and Mazza (2000), (2) Burda and Oleszek (2001), (3) Nenadis and Tsimidou (2002), (4) Koleva et al. (2002), (5) Kim et al. (2002), (6)
Kikuzaki et al. (2002), (7) Lavelli (2002), (8) Davalos et al. (2003), (9) Awika et al. (2003), (10) Pinelo et al. (2004b), (11) Heimler et al. (2005).
interaction occurring between antioxidants and ferryl myoglobin in the original

assay. Although some authors suggested the use of ascorbic acid instead of
Trolox as reference, such a step would be questionable in applications of this
method to fruit extracts containing varying natural levels of ascorbic acid.
To minimize the interaction between antioxidants and ferryl myoglobin in
the original assay, the TEAC assay was modified by generating the relatively
long-lived ABTS radical by direct oxidation with potassium persulfate or
manganese dioxide prior to reaction with the test antioxidants (Table 4.5, ref 4,
7, 8). In another modification, a commercial peroxidase was used at an optimal
wavelength of 414 nm, to avoid interference from colored compounds in plant
materials in citrus juice and wines, and to improve detection (Table 4.5, ref 9).
Further modifications of the TEAC test to avoid overestimation of antiradical
activity involves separating the production from the scavenging of the radicals,
by using a more stable N,N-dimethyl-p-phenylenediamine (DMPD·+) radical.
The TEAC assay was applied to the evaluation of total antioxidant activity of
various fruit juices and drinks, where vitamin C is a major antioxidant (Table
4.5). For this reason, the application of this assay to wine phenolics becomes
confounded by ascorbic acid, which is commonly used to inhibit browning
during wine fermentation (Table 4.5, ref 9).
Despite recent improvements and increased use, the TEAC assay has several
limitations. Like the DPPH assay, the ABTS radical chromogen may be limited
by sample interference of pigmented components of plant extracts, juices and
wines, causing the activity to be significantly underestimated. Another limita-
tion is that the TEAC assay only measures reactivity toward the artificial ABTS
radical and does not test antioxidants for their inhibition of oxidation. The
TEAC value is considered to reflect the relative ability of hydrogen- or
electron-donating antioxidants to scavenge the ABTS radical. This value
corresponds to the stoichiometric factor, n, defined as the number of radicals
trapped by each molecule of antioxidant (see Chapter 2, reaction 18), and does
not describe direct reactivity. Kinetic studies indicate that the ABTS radical
reacts non-specifically with any aromatic OH-group, including those that do
not contribute to antioxidant activity. The literature shows wide variations in
TEAC values for phenolic compounds of different structures.
Since this test does not involve a substrate, it does not provide information
on the relative protective properties of antioxidants and the nature of damaging
products of oxidation. The ABTS radical has poor selectivity for hydrogen
donors and cannot be used to correlate with the structures of different phenolic
antioxidants. The TEAC assay can measure the ability of phenolic compounds
to scavenge the artificial ABTS radical, but may not reflect their antioxidant
activity by other mechanisms effective in complex food lipids or biological
substrates, such as metal chelation (Figure 2.2), and interfacial antioxidant
effects arising from partitioning in multiphases of compounds of different
polarity (Figure 3.1).
88
Table 4.5. Different conditions and variations of the TEAC assay
Antioxidants Reagents Absorption Matrix Standards Ref.a

measured, time
Phenols, apple juice ABTS + H2O2 + A734 nm Lag phase Phoshate buffer, pH 7.4 Trolox, ascorbate (1)
constituents metmyoglobin
Plant materials, ABTS + H2O2 + A734 nm 5 min Phosphate buffer, pH 7.5 Trolox (2, 3)
vegetable soups HR peroxidase
Flavonoids, carotenoids, ABTS + K A734 nm AUC Ethanol or buffer, pH 7.4 Trolox (4)
plasma persulfate
Phenolics, α-tocopherol, ABTS + ABAP A734 nm 6, 10 min Phosphate buffer saline Trolox, ascorbate (5, 6)
carotenoids, ascorbic acid (AAPH) pH 7.4
Phenolic compounds, ABTS + K A734 nm 1 min Ethanol, olive oils Trolox (7)
α-tocopherol persulfate
Tea components ABTS+ K A734 nm 30 min Ethanol + water Trolox (8)
persulfate
Wine anthocyanins ABTS + A734 nm 6 min Phosphate buffer, pH 7.4 Trolox (9)
microperoxidase
a
References: (1) Miller et al. (1995), (2) Cano et al. (1998), (3) Arnao et al. (2001), (4) Re et al. (1999), (5) van den Berg (1999, 2000), (6) Kim et al. (2002), (7)
Pellegrini et al. (2001), (8) Arts et al. (2002, 2004a), (9) Borkowski et al. (2005).
Abbreviations: ABTS, 2,2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid; HR peroxidase, horse radish peroxidase; AUC, area under curve (% inhibition vs
concentration in μM); ABAP and AAPH, 2,2'-azobis(2 amidinopropane) dihydrochloride.
3. Linoleic acid TRAP assay

The total radical trapping antioxidant parameter (TRAP) assay was developed to
measure ‘total antioxidant capacity’ of plasma or serum (Table 4.3, ref 10). This
assay uses peroxyl radicals generated by the azo initiator 2,2'-azobis(2-
amidinopropane hydrochloride) (ABAP, also abbreviated as AAPH) to oxidize
plasma antioxidants, and the oxidation is monitored by oxygen absorption. The
induction period is compared to that of Trolox used as reference. This method
was later modified by adding linoleic acid as an oxidizable lipid substrate, before
oxidation with ABAP. Like the previous antiradical methods, the use of an
artificial water-soluble azo compound as a radical generator does not provide a
useful estimate of the important protective activities of metal chelators and
lipophilic antioxidants, such as vitamin E and flavonoids. To overcome the
major problem of an unsteady electrode end point, several modifications were
developed by using luminol-enhanced chemiluminescence as end point. How-
ever, the measurement of TRAP activity may be invalidated because free radical
production would have to be sufficiently extensive to disturb the steady-state
level of antioxidants. The use of either plasma or serum presents a problem due
to the instability of antioxidants in the treatment and storage of blood samples. In
general biological applications, antioxidants would have to disrupt the
compartmentalization protection afforded by cell membranes.
4. β-Carotene bleaching method

This widely used old method is based on the competitive bleaching of β-
carotene during oxidation of linoleic acid in aqueous emulsion systems and
measurement of the decay in absorption at 470 nm (Table 4.3, ref 11, 12). The
addition of antioxidants causes a delay in the β-carotene decay. This method is
simple and sensitive, but not specific and precise; it is subject to interference
from other pigments, reducing agents and oxidizing agents present in plant
extracts. Linoleic acid also forms micelles in aqueous systems, which can
significantly affect the relative activities of antioxidants of different polarity
and partitioning properties compared to their behaviour in emulsions based on
triglycerides (Chapter 3). The catalytic effect of metals is also accelerated in
micelle systems converting flavonoid compounds into prooxidants.
The competitive crocin (a natural pigment with strong visible absorption)
bleaching test is a more recent protocol, related to β-carotene bleaching by the
radicals generated by AAPH. This method has many problems which affect the
reliability of the results. Many phytochemicals and food pigments may inter-
fere with the bleaching of crocin, resulting in variable lag phase.
5. ORAC assay
In the oxygen radical absorbance capacity (ORAC) method, the highly
fluorescent proteins phycoerythrin (β-PE) and R-phycoerythrin (R-PE), de-

rived from red algae containing a photoreceptor pigment, are used as the
substrate of oxidative damage (Table 4.3, ref 12–19, Table 4.6). The effect of
an antioxidant in quenching the oxidation of β-PE and R-PE induced by AAPH
is quantified by calculating the area under the time recorded fluorescence curve
(AUC) and expressed as ‘antioxidant capacity’ in μmoles of Trolox equiva-
lents. The decrease in fluorescence is not linear with time, and the shape of
fluorescence decay curves differ in the presence of different antioxidants and
with different concentrations of the same antioxidant. The calculation using the
differences in AUC between a sample and the blank is used, therefore, to
circumvent erroneous assumptions of linearity and response similarities. In
some application of this method, the inhibition of β-PE oxidation is evaluated
from the midpoint of the assay. However, the ORAC method is based on the
assumption that the oxidative deterioration of the fluorescent proteins can
simulate food and biological substrates. Furthermore, the effect of oxidation of
the photoreceptor portion of β-PE and R-PE on fluorescence measurements
does not necessarily reflect the extent of antioxidant protection afforded
against oxidative damage of the protein itself. In a modification of the ORAC
method, the fluorescent protein PE was replaced by fluorescein, because it is
more stable and less reactive with polyphenols (Table 4.3, ref 16–18; Table 4.6,
ref 5–8). Significantly higher ORAC values were produced with fluorescein
than with PE, due to complex formation of polyphenols with the fluorescent
protein PE.
The antioxidant activities of many juices and fruit extracts were reported as
ORAC values in μmoles of Trolox equivalents. Representing the combination
of both inhibition time and extent of inhibition, this value cannot be compared
with other methods for quantifying antioxidants using inhibition time, or per
cent inhibition, or per cent inhibition at a given time.
In principle, measuring the effects of antioxidants by the integrated areas
under the decay curves including the total oxidation period can be misleading,
because it does not distinguish between the initial and propagation phases that
are more significant in relation to oxidative deterioration of foods and biologi-
cal damage. The relative activity of many antioxidants can often change at
different stages of lipid oxidation. For example, α-tocopherol is known to be
prooxidant at the initial phase of oxidation and to be antioxidant at later stages
of oxidation. The mechanism of oxidation in the ORAC method is probably
more complex than generally assumed, because some of the peroxyl radicals
derived from the artificial radical from AAPH can react with each other, instead
of reacting with the fluorescent probe and antioxidants. This problem cast some
doubt on the validity of the ORAC protocol. Furthermore, like many of the
other antiradical methods, the use of Trolox as a reference compound is not
justified, because it is not structurally related to any of the polyphenolic
compounds found as sources of antioxidants in foods or in biological systems.
Table 4.6. Different conditions and variations of the ORAC assay (Standard: Trolox)
Antioxidants Reagents Absorption measured, time Matrix temp. Ref.a
Ascorbate, urate, glutathione, plasma, R-PE + AAPH, Fluorescence, 575 nm, Phosphate buffer, (1)
proteins, DNA Cu2+-ascorbate AUC, 12 min pH 7.0, 37°C
α-tocopherol, vitamin C, β-carotene, β-PE + AAPH Automated fluorescence decay, Phoshate buffer, (2)
uric acid, bilirubin, fruit extracts em 515 nm, ex 493 nm pH 7.0, 37°C
Tea, vegetables extracts (aqueous, β-PE + AAPH, Automated fluorescence decay, Phoshate buffer, (3)
acetone), flavonoids H2O2 – Cu2+,CuSO4 em 515 nm, ex 493 nm pH 7.5
Phenolics, anthocyanins, ascorbate, R-PE +AAPH Automated fluorescence, AUC, Phoshate buffer, (4)
small fruits em 515 nm, ex 493 nm pH 7.5
Tea, blueberry, grape skin, grape seed, Fluorescein + AAPH Automated fluorescence, AUC, PBS buffer, (5)b
juice, biological fluids, vegetables ORACFL em 515 nm, ex 493 nm pH 7.4, 37°C
Tocopherols, tocotrienols, DTBMP, Fluorescein + AAPH Automated fluorescence decay, Phosphate buffer, (6)b
γ-oryzanol H-ORACFL em 515 nm, ex 493 nm Ph 7.0 + β-cyclo-
L-ORACFL dextrin (7%) in
acetone–water
Phenolic compounds, wines Fluorescein or Fluorescence microplate Phosphate buffer, (7)b
β-PE + AAPH reader, AUC pH 7.0, 7.4
ORACFL
Fruits, vegetables, nuts, spices, grains, Fluorescein + AAPH Fluorescence microplate reader Phosphate buffer, (8)b
other foods, cocoa and chocolate products H-ORACFL pH 7.4, 37°C
L-ORACFL
a
References: (1) Glazer (1990), (2) Cao et al. (1993), Wang et al. (1996), (3) Cao et al. (1995,1996,1997), (4) Kalt et al. (1999), (5) Ou et al. (2001, 2002), (6) Huang
et al. (2002), (7) Davalos et al. (2004), (8) Prior et al. (2003), Wu et al. (2004), Gu et al. (2006).
ANTIOXIDANT PROTOCOLS FOR FOODS AND BIOLOGICAL SYSTEMS
Abbreviations: AAPH, 2,2'-azobis(2 amidinopropane) dihydrochloride; AUC, area under curve; DTBMP, 2,6-di-tert-butyl-4-methyl phenol; ex, excitation ; em,
emission ; ORACFL, ORAC based on fluorescence ; PE, phycoerythrin.
b
H-ORACFL, hydrophilic ORAC based on fluorescence, L-ORACFL, lipophilic ORAC based on fluorescence.
91
Since the original ORAC assay was limited to hydrophilic antioxidants, it

was further modified to measure both lipophilic and hydrophilic anti-
oxidants, by using a 1:1 mixture of acetone and water containing 7%
methylated β-cyclodextrin to solubilize the antioxidants (Table 4.6, ref 6, 8).
The use of such a solvent mixture is questionable, however, because it
cannot properly represent the interfacial relationship that operates in hetero-
geneous food and biological systems (Chapter 3). However, the modified
ORAC assay has been used extensively to evaluate the lipophilic and hy-
drophilic antioxidant capacities of a wide range of foods, including fruits,
vegetables, nuts, spices, cocoa and chocolate products. For all the reasons
discussed, the widely used automated and non-automated ORAC protocol is
of questionable validity.
6. Superoxide anion scavenging assays

Although the superoxide radical anion (O·2 –) cannot directly initiate lipid
oxidation, these assays use O·2 – scavenging, because in the presence of metal
ions the highly reactive hydroxyl radical, ·OH, can be generated by the Fenton
reaction (Table 4.3, ref 20–22). However, the scavenging of O·2 – is not the only
mechanism for inhibition of lipid oxidation in either biological or lipid food
systems. Therefore, phenolic compounds in plant extracts having O·2 – scaveng-
ing properties are not necessarily effective in preventing lipid oxidation. The
same argument may apply in the measurement of inhibitory effects on xanthine
oxidase, which generates superoxide radicals. In addition to these shortcom-
ings, measurements of O·2 – scavenging should be interpreted with caution,
because no equilibrium can be achieved when superoxide radicals are gener-
ated continuously during the test.
7. FRAP assay
The ferric reducing antioxidant power (FRAP) assay measures directly the
ability of antioxidants to reduce a ferric tripyridyltriazine complex (Fe3+-
TPTZ) to the ferrous complex (Fe2+-TPTZ) at low pH (Table 4.3, ref 23, 24).
Except for the difference in pH, this assay is related to the TEAC assay run at
neutral pH, because it is based on the redox potential of the ferric complex. The
resulting blue color measured spectrophotometrically at 593 nm is taken as
linearly related to the total reducing capacity of electron-donating antioxidants.
The main disadvantage of this approach are that the measured reducing
capacity reflects not necessarily antioxidant activity but total antioxidant
concentration. Since the method does not include an oxidizable substrate, no
information is provided on the protective properties of antioxidants.
C. Comparison of antiradical methods with in vitro LDL oxidation

The relative antioxidant activity of many phenolic compounds varies widely
according to different testing methods. The antioxidant capacities of
polyphenolic compounds based on their stoichiometric factor, defined as the
number of radicals trapped by each molecule of antioxidant (Chapter 2.B.2),
showed only poor correlation between the DPPH, ABTS and ORAC methods
(Table 4.7). Stoichiometric values calculated for tea catechins EGC and EGCG
determined by the DPPH and TEAC assays did not correlate with the ORAC
assay. Some studies also showed differences in relative activities between the
individual phenolic compounds. The relative activities did not always corre-
spond to the number of hydroxyl groups and other polar groups per molecule.
With the oxidation of methyl linoleate in bulk, caffeic acid was the most
Table 4.7. Relative activities of phenolic compounds by different methods on the basis
of calculated stoichiometric factorsa
Methods Relative activities (number of OH groups) Ref.b
DPPH EGCG(8) > ECG(7) > EGC(5) > GA(3+)c > EC(4) ≈ C(4) (1)
EGCG(8) > EC(4) > EGC(5) > C(4) > GA(3+)c (2)
K(4) > Rut(10) > Q(4) > Myr(5) (3)
CA(3+) > α-T(1) > SA(2+) ≈ FA(2+) > ferulates > p-CoumA(1+) (4)
ABTS ECG(7) > EGCG(8) ≈ Q(4) > EGC(5) > Myr(5) > GA(3+)c (5)
> EC(4) > C(4) ≈ Rut(10) > CA(3+)c
Q(4) > C(4) > EC(4) > Myr(5) > GA(3+)c ≈ Rut(10) > CA(3+)c (6)
Q(4) > FA(2+)d > C(4) > Rut(10) > CA(3+) > Trol(1+)e > Chl(5+)f (7)
ORAC Q(4) > K(4) > C(4) > EC(4) > GA(3+)c > Rut(10) (8)
Me Lo, CA(3+) > α-T(1) > SA(2+) > FA(2+) > p-CoumA(1+)
Bulk, 40°C
OSI, 90°C CA(3+) > SA(2+) ≈ α-T(1) ≈ FA(2+) ≈ p-CoumA(1+) (4)
LA EtOH- α-T(1) > FA(2+) > p-CoumA(1+) >> SA(2+)* g > CA(3+)*g
buffer, 40°C
a
From Roginsky and Lissi (2005). See Chapter 2.B.2.
Abbreviations: C, catechin; CA, caffeic acid; Chl, chlorogenic acid; p-CoumA, p-coumaric acid; EC,
epicatechin; ECG, epicatechin gallate; EGC, epigallocatechin; EGCG, epigallocatechin gallate; FA,
ferulic acid; ferulates, several esters of ferulic acid; GA, gallic acid; K, kaempferol; LA, linoleic acid; Me
Lo, methyl linoleate; Myr, myricetin; OSI, oxidative stability index; Q, quercetin; Rut, rutin; SA, sinapic
acid; α-T, α-tocopherol; Trol, Trolox.
b
References: (1) Nanjo et al. (1999), (2) Gardner et al. (1998), (3) Burda and Oleszek (2001), (4)
Kikuzaki et al. (2002), (5) Rice-Evans et al. (1996), (6) Ishige et al. (2001), (7) Nilsson et al. (2005), (8)
Guo et al. (1997).
c
3+ = 3 OH groups + 1 COOH group
d
2+ = 1 OH group + 1 OMe group + 1 COOH group
e
1+ = 1 OH group + 1 COOH group
f
5+ = 5 OH groups + 1 COOH group
g
* = prooxidant activity
active antioxidant, showing a clear induction period, followed by α-tocophe-

rol. Sinapic and ferulic acid were weak antioxidants while p-coumaric acid was
slightly prooxidant. Ferulate esters were less active than ferulic acid, in
agreement with the polar paradox, with polar antioxidants being more active in
the non-polar bulk methyl linoleate substrate than the non-polar esters. With
linoleic acid in an ethanol buffer solution at pH 7, α-tocopherol and ferulic acid
were the most active antioxidants, and p-coumaric acid the least active, while
sinapic and caffeic acid were prooxidant. The prooxidant activity of the highly
polar sinapic and caffeic acids and low activity of p-coumaric acid can be
attributed to their solubility in aqueous systems, where they can strongly reduce
the metals to the more catalytically active lower valence state.
There is no agreement between the relative activity of various flavonoids,
anthocyanins, hydroxycinnamic acids and tea catechins for the in vitro inhibi-
tion of copper-induced human LDL oxidation compared to the total antioxidant
capacity, measured by the TEAC, ORAC, FRAP, β-carotene bleaching and
liposome oxidation methods (Table 4.8). On one hand, among phenolic com-
pounds, catechin showed the highest activity for inhibiting LDL oxidation by
the FRAP assay; on the other hand, quercetin was the most active as determined
by the TEAC, ORAC, β-carotene bleaching and liposome oxidation methods
(Table 4.8, ref 1–5). Rutin, the 3-rutinose glycoside of quercetin, had the lowest
antioxidant value by the ORAC method. Although both LDL and liposome
oxidations were induced by copper, rutin showed good antioxidant activity for
LDL and was prooxidant by the liposome oxidation method. Contrasting
results were also reported for the antioxidant potentials of anthocyanins,
hydroxycinnamates and tea catechins for the inhibition of human LDL oxida-
tion in vitro and their activities by the TEAC assay (Table 4.8, ref 1, 2, 6, 8, 9).
Similar discrepancies in relative antioxidant activities were observed for
anthocyanins by the ORAC and β-carotene bleaching methods, and
hydroxycinnamates by the LDL oxidation, TEAC, ORAC and FRAP assays
(Table 4.8, ref 2, 3, 5, 7–9). For tea catechins, relative activities were signifi-
cantly different according to two methods, with epigallocatechin gallate being
most active by the liposome oxidation method, while epicatechin gallate was
most active by the TEAC method.
These data emphasize that the ranking of antioxidant activity is strongly
dependent on the test system and on the substrate to be protected by the
antioxidants. Polyphenolic flavanols may inhibit LDL oxidation by several
mechanisms in addition to free radical scavenging. In contrast, the TEAC and
ORAC assays only measure radical scavenging activity in aqueous systems.
The FRAP assay measures reducing capacity without an oxidizable substrate
whereas the β-carotene bleaching method uses free linoleic acid as substrate in
an aqueous emulsion system (Section B, 2, 4, 5). The liposome oxidation
method uses lecithin as substrate. The discrepancy in ranking of antioxidants
shown in Table 4.8 can be explained not only by the multiplicity of mechanisms
Table 4.8. Relative antioxidant activity of selected phenolic compounds by different

methods
Phenolics Inhibition TEAC ORAC FRAP β -carotene Liposome

5 μM (%) of mM μM ECc bleachingd oxidationd
GAE LDL Trolox Trolox μ mol/l
oxidationa
Ref (1)b Ref (2)b Ref (3)b Ref (4)b Ref (5)b Ref (5)b
Catechin 74.9 2.40 2.49 192 – –

Myricetin 68.1 3.12 – – – –
Epicatechin 67.6 2.50 2.36 – – –
Rutin 67.6 2.42 0.56 162 13 –41
Gallic acid 63.3 3.01 1.74 142 – –
Quercetin 61.4 4.72 3.29 92 49 37
Ellagic acid 36.6 – – – – –
Sinapic acid 35.1 – – – – –
α-Tocopherol 32.6 0.97 – – – –
Tea catechins Ref (6)b,e
Epigallocatechin – 4.75 – – – 82.0
gallate
Epicatechin – 2.50 – – – 80.2
Epicatechin gallate – 4.93 – – – 59.6
Epigallocatechin – 3.82 – – – 22.2
Anthocyanins Ref (7)b Ref (8)b
Cyanidin 79.4 4.42 2.2 68 85
Delphinidin 71.8 4.44 1.8 – –
Malvidin 59.3 2.06 2.0 72 99
Pelargonin 39.0 1.30 1.1 68e 87 e,f
Hydroxycinnamates Ref. (9)b Ref. (3)b
Caffeic acid 96.7 1.26 2.23 139 – –
Chlorogenic 90.7 1.2 – – – –
Ferulic acid 24.3 1.90 1.33 319 – –
p-Coumaric acid 24.5 2.22 1.09 – – –
a
Copper-catalysed oxidation of human LDL monitored by hexanal determination by headspace gas
chromatography (Frankel et al., 1992).
b
References: (1) Teissedre et al. (1996), (2) Rice-Evans et al. (1996), 3) Cao et al. (1997), (4) Pulido et
al. (2000), (5) Hassimotto et al. (2005), (6) Huang et al. (1997), (7) Satué-Gracia et al. (1997), (8) Wang
and Cao (1997), (9) Meyer et al. (1998).
c
EC1 = concentration of antioxidant with reducing ability equivalent to1 mmol/l FeSO4.7H2O
d
50 μM expressed as gallic acid equivalents (GAE). Values expressed as % inhibition.
e
Contained 8 mg soya lecithin/ml water, oxidized at 37°C with 10 μM cupric acetate.
f
Values are for pelargonidin.
effective for these polyphenolic compounds, but also by the influence of the
interfacial properties of multiphase LDL particles compared to the aqueous or
methanol test solutions used for the TEAC, ORAC and FRAP assays, linoleic
acid emulsion system in the β-carotene bleaching method, and lecithin in the
liposome oxidation method.
Although the indirect DPPH, TEAC and ORAC methods for evaluating
antiradical activity are rapid and useful, they can only reflect the ability of
phenolic compounds to scavenge stable free radicals. Not only are the radical
sources in the various modification of the DPPH, TEAC, ORAC and FRAP
assays not biologically or nutritionally relevant, but their results do not provide
quantitative information on the oxidation process, on the initial and decompo-
sition products, or their antioxidant protection.
The indirect DPPH, TEAC and ORAC tests show generally poor correlation
with the results of direct antioxidant tests in protecting real foods against
oxidation. It is therefore questionable whether the results of these indirect
methods provide useful information on the real protective properties of antioxi-
dants in inhibiting oxidation processes. The data from indirect tests are also
poorly reproducible and require considerable effort to standardize. Any appli-
cations of the indirect tests should always include determinations of total
phenol contents of plant sources, and comparative data from direct antioxidant
tests based on methods for determining products of oxidation.
D. Recommended protocols
We have seen in this survey that the effectiveness of antioxidants is strongly
dependent on the test system, the physical states of the lipid substrates, the
conditions of oxidation, the oxidizing substrate, the localization of anti-
oxidants, and the method employed to evaluate oxidation and the stages of
oxidation. The activity of antioxidants is greatly affected by complex inter-
facial phenomena in emulsions and multi-component foods according to their
hydrophilic or lipophilic character. The methodology for evaluating natural
antioxidants must be therefore carefully interpreted according to the system,
and the analytical method used to determine the extent and end point of
oxidation.
Each antioxidant evaluation should be carried out under various conditions
of oxidation, using several methods to measure different products of oxidation
related to real food quality or critical biological reactions. There cannot be a
short cut approach to determining the activity of antioxidants. Various testing
protocols should consider the following parameters:
(1) Substrates. Use substrates relevant to foods and biological systems,

including triacylglycerols and phospholipids, in bulk, emulsions or liposome
systems. Free fatty acids should be avoided because they form micelles and
antioxidants behave differently in these than in triacylglycerols. To evaluate
protein oxidation, lipid emulsions should also contain a suitable food protein,
such as BSA, casein or whey proteins.
(2) Conditions. Test under various oxidation conditions including different

temperatures (below 60°C), metal catalysts or surface exposures. Select

conditions to simulate real food or biological systems as closely as possible,
depending on the application.
(3) Analyses. Measure relatively low levels of oxidation (below 1%), and
include both initial products (hydroperoxides, peroxide value, conjugated
dienes) and secondary decomposition products (carbonyls, volatile compounds).
(4) Concentrations. Compare antioxidants at the same molar concentration

of active components, using structurally related reference compounds. Consider
carefully the concentration ratios of catalytic inducers:antioxidants and
antioxidant:substrates. With crude plant extracts, the total phenol contents and
compositional data are required to compare samples.
(5) Calculations. Quantify on the basis of induction period, per cent inhibition
or rates of hydroperoxide formation or decomposition, I50 (antioxidant
concentration to achieve 50% inhibition) and T50 (time to reach 50% inhibition).
Because of the complexity of real foods, accelerated test systems are difficult
to standardize and each antioxidant test should be calibrated for each lipid or
food. Accelerated oxidation conditions should be close to the storage condi-
tions under which the food is to be protected. Ultimately, antioxidants should
be evaluated on the food itself.
In biological systems, phenolic compounds can participate in several anti-
oxidant defenses, including preventing oxidant formation, scavenging
activated oxidants, reducing active intermediates and inducing repair sys-
tems. To improve our understanding of these complex interactions in
different systems, the use of non-specific and one-dimensional antiradical
assays for antioxidant capacity would be unsafe, because they do not provide
information on the food and biological target(s) protected. A better approach
is to measure specific products of oxidation in both relevant in vitro and in
vivo biological systems.
The large amount of effort expended in testing new natural antioxidants
emphasizes the need for improved test methods. Several currently used meth-
ods and model systems (Tables 4.2–4.7) may not evaluate the true protective
effects of antioxidants, and the data obtained can be confounded by many
factors, including the composition of the test system, the substrate to be
protected and the mode of inducing oxidation. In simplified model systems,
interfacial phenomena may be overridden when interpreting antioxidant mecha-
nisms and activity that appear strongly influenced by complex interfacial and
phase distribution properties. When testing antioxidant activity of potential
food antioxidants or bioactive compounds, the first aim may be to develop a
model system, where basic chemical principles can be deduced. On the other
hand, the true effectiveness of antioxidants cannot be properly assessed unless
the conditions (i.e. the complexity of the system) are as close as practically
possible to the conditions under which protection against autoxidation is
required. Targeting of antioxidants to prevent particular free radical formation
steps and oxidative deterioration processes requires detailed understanding of
the mechanisms of oxidation. Specific lipid model systems should mimic the
food or physiological target systems to be protected as closely as possible.
There are various sources and types of oxidation and we should first define the
targets of oxidation – lipids, protein, DNA – before selecting methods for
assessment of the protective properties of antioxidants under the conditions of
their potential action and use. The total antioxidant capacity of phytochemicals
based solely on one property, such as the scavenging ability toward artificial
radicals assessed by antiradical methods, provides no information on which
lipid or other substrate is protected. There cannot be a short cut approach to
evaluating antioxidants. In view of the wide divergence of results of natural
antioxidants in foods and biological systems, more valid guidelines and assay
protocols are urgently needed to bring some order to the present chaos in this
important field. Our understanding of the effects of antioxidant compounds can
only be improved if more specific methodology is used, capable of defining
which products are formed and inhibited by antioxidants, depending on condi-
tions, systems and targets of protection.
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CHAPTER 5
Food antioxidants
As a major form of deterioration, lipid oxidation constitutes a significant factor

in the useful storage of food products. The changes due to oxidation occur at
different stages of food preparation, from the raw materials to processing,
packaging, storage, cooking, and various retail or large scale applications. In
processed foods, an important factor is the formation of non-enzymatic brown-
ing materials at elevated temperatures by the interactions of sugars or lipid
oxidation products with proteins and amino acids. These compounds inhibit
oxidation, and their formation is influenced by the water activity, which affects
the quality of foods and their nutritional value. Moisture in foods also affects
the effectiveness of antioxidants and metal chelators. Precautions are therefore
necessary to reduce changes in the water activity during preparation and
storage of processed foods susceptible to lipid oxidation, by using packaging
materials of low moisture permeability, or vacuum packaging.
A. Interactions of lipids with proteins and sugars

The association of potent antioxidant activity with the development of brown-
ing reaction products and the interaction of oxidized lipids and their carbonyl
decomposition products with proteins and amino acids has long been known.
The reactions between amino acids and sugars were first reported by Maillard
in 1912. Sugars and carbonyl compounds interact with amino acids or proteins
in a sequence of complex reactions known as the Maillard reaction, or non-
enzymatic browning. The strongly reducing browning products contribute to
the stabilization of foods against lipid oxidation. Hundreds of browning
reaction products are formed by a series of consecutive and parallel oxidations,
reductions and aldol condensations (Lipid Oxidation, 2nd ed, pp. 311–315). If
foods of intermediate moisture contents are heated at sufficiently high tempera-
tures, the rate of the browning reaction can be accelerated with the formation of
Maillard reaction products that inhibit lipid oxidation effectively. This process
is a useful way of stabilizing certain foods like evaporated milk, baked foods
and heat extruded cereals.
The antioxidant properties of Maillard reaction products are attributed to the
formation of reductone (enaminone) structures that have both strong reducing
and metal complexing properties. The interactions between sugars and amines
produce N-glycosides and Amadori rearrangement compounds with strong
antioxidant activity. Melanoidins, more complex macromolecular pigments of
105
Figure 5.1. Formation of imines Schiff bases by addition of protein amine residue to the carbonyl
function of reducing carbohydrates forming an Amadori product from glucose and a Heyns product from
fructose.
unknown structures, are also produced, which have varying molecular weights
and solubility.
Carbonyl groups in carbohydrates react with amino groups in proteins and
undergo rearrangements, producing enediols to form Amadori or Heyns ad-
ducts (Figure 5.1) Amadori adducts rearrange under anaerobic conditions to
more reactive dicarbonyl compounds by reverse Aldol reactions. Reductones
are produced by heating reducing sugars that retain a carbonyl group in the
vicinity of an enediol group. Ascorbic acid is a reductone characterized by
strong reducing properties under acidic conditions and at low temperatures.
Basic amino acids (histidine, lysine and arginine) produce the most effective
antioxidant products with sugars. A range of these Maillard interaction prod-
ucts of amino acids and sugars have antioxidant activity that may be useful in
food processing in retarding oxidation in heated foods, cereals and milk
products.
Model systems are often used to study the antioxidant activity of browning
reaction products, including binary mixtures of sugars and amino acids or
proteins, oxidized lipids and proteins, and pyrrolized phospholipids (Table 5.1).
Unfortunately, many papers in the literature have used non-specific and
insensitive methods to measure the effect of browning. These methods are not
useful for model food systems, because of the interference from complex
Table 5.1 Model systems used to evaluate antioxidant activity of browning reaction productsa
Model system Conditions Antioxidant activity Oxidation method Ref.b
Glucose–histidine 100°C, 5 h Linoleic acid oxidation O2 uptake, PV, MRP volatiles (1)
Glucose–glycine 40°C, 40 h, aw 0.23–0.82 Reducing power K ferricyanide test (2)
Sucrose–lysine 100°C, 6 h Linoleic acid oxidation O2 uptake, PV, MRP volatiles (3)
Glucose/fructose–lysine 86–159°C, 45–119 min Linoleic acid emulsion TBA, O2 uptake, DNA breaking (4)
BSA–ribose–MeLo-OOH 25–120°C, pH 4, 7, 10 Soybean oil oxidation TBARS (4)
Glucose–lysine–starch 100°C, 90 min Radical quenching ABAP, crocin bleaching (5)
β-lactoglobulin glycated with sugars 60°C Antiradical activity DPPH (5)
Casein–glucose or fructose or ribose 55°C, 28 days Radical and OH radicals DPPH (6)
quenching
Glucose–lysine or arginine or glycine 100°C, 60 min LDL oxidation Cu induced CD at 234 nm (7)
Pyrroles 37°C, 30 h Linolenic acid emulsion Hemoglobin-benzoylleuco (7)
methylene blue
Sugars–sulfur compound–amino acid 103°C, 14 h Inhibition of polyphenol (8)
FOOD ANTIOXIDANTS
O2 uptake by polargaphy
oxidase
Sugar–lysine 121°C, 1 h Intracellular oxidation DPPH, ORAC (9)
Histidine–glucose 120°C, 10–30 min ORAC Phycoerythin fluorescence (10)
a
Expanded table from Lipid Oxidation, 2nd ed, Table 11.6, p. 314.
b
(1) Lingnert and Eriksson (1981), (2) Eichner (1981), (3) Kim and Harris (1989), (4) Wijiewickreme and Kitts(1997), (4) Alaiz et al. (1999), (5) Mastrocola and
Munari (2000), (5) Chevalier et al. (2001), (6) Jing and Kitts (2002), (7) Dittrich et al. (2003), (7) Hidalgo et al. (2003), (8) Billaud et al.(2005), (9) Kitts and Hu
(2005), (10) Yilmaz and Toledo (2005).
Abbreviations: PV, peroxide value; MRP, Maillard reaction products; BSA, bovine serum albumin; MeLo-OOH, methyl linoleate hydroperoxides; TBARS,
thiobarbituric reactive substances; ABAP, 2,2'-azobis(2-amidinopropane) dichloride; DPPH, 2,2-diphenyl-1-picrylhydrazyl; LDL, low-density lipoproteins; CD,
conjugated dienes; ORAC, oxygen radical absorbance capacity.
107
interaction products involved in the browning reaction products. Linoleic

acid is also not a suitable model for lipids because it produces micelles that
oxidize differently from triglycerides and with polar hydrophilic antioxi-
dants (Lipid Oxidation, 2nd ed, pp. 286–289). Thermal processing can affect
the antioxidant activity of food components through complex interactions
with lipid oxidation products, ascorbic acid and polyphenolic compounds,
and can undergo further changes during storage. Foods such as tomato juice,
wines, coffee, tea, nuts and oxidized phospholipids develop various complex
interactions that can either increase or decrease the antioxidant activity of
browning reaction products (Table 5.2). Thermal processes can either de-
crease the antioxidant activity of foods and beverages by the degradation of
natural polyphenolic antioxidants, or increase their antioxidant activity by
the formation of reducing browning reaction products. For example, the
prolonged heat treatment of tomato juice results in an overall increase in
antioxidant potential due to formation of browning reaction products. The
roasting of coffee and peanuts produce browning reaction compounds with
antioxidant activity.
The addition of various phospholipids to soybean oil increased the oxidative
stability of the oil at 60°C (Table 5.3). Phosphatidylcholine (PC) was the most
efficient inhibitor of oxidation, followed by phosphatidylethanolamine (PE),
while phosphatidylinositol (PI) was inactive. Although protection observed for
PC and PI decreased after oxidation, the antioxidant activity of slightly
oxidized PE increased initially (between 24 and 48 h of oxidation) and then
decreased (between 54 and 96 h of oxidation). The antioxidant activity of PE
was completely lost after further oxidation for 196 h as a result of fatty acid
oxidation. The initial increase in antioxidant activity of slightly oxidized PE
was attributed to the formation of carbonyl compounds reacting with the
primary amine group of PE, to produce pyrrolized phospholipids by amino-
carbonyl reactions (Figure 5.2). Unfortunately, using TBARS to measure
oxidation of soybean oil supplemented with phospholipids is questionable,
because this method is notoriously unspecific and unreliable. Reducing brown-
ing reaction products are known to interfere with the TBA color reaction (Lipid
Oxidation, 2nd ed, pp. 108–110).
Generally, in plant foods containing high levels of phenolic compounds such
as tea, apple products and wine, the antioxidant activity resulting from complex
interactions can increase initially and decrease with storage time. In many of
these studies, the effect of thermal processing is measured by a wide variety of
unspecific antioxidant protocols, producing results that are difficult to inter-
pret. Since the browning reaction products are multifunctional, more reliable
protocols are needed to measure more than one activity relevant to foods,
including initial oxidation and decomposition products of lipids and proteins.
In biology, the Maillard browning reaction products are involved in the
formation of advanced glycation end products (AGEs), derived from reactive
Table 5.2. Antioxidant activity of browning reaction products in foods and beverages
Food systems Conditions Antioxidant activity Oxidation method Ref.a
Tomato juice 95°C, 0–30 h Radical (ABAP) quenching Rancimat, O2 uptake (1)
White and red wines Vintages: 1995, 1996, 1973 Radical (DPPH) quenching O2 uptake (2)
Coffee (green, roasted) Roasting 100–210°C β-Carotene-Lo acid emulsion Carotene absorbance (3)
Peanuts (defatted) 180°C, 0–60 min Linoleic acid emulsion, DPPH PV -(Fe-thiocyanate) (4)
Starch, glucose, lysine, soybean oil 100°C, 90 min ABAP initiated oxidation Rate of crocin bleaching (5)
Coffee, cocoa, teab Standard beverage LDL oxidation Cu or AAPH Lag time, CD at 234 nm (6)
Roasted coffee Roasting 225–240°C, 3 min Radical (ABTS·+) quenching Decolorization at 734 nm (7)
Roasted coffee residues Medium roasted coffee Oxidation of protein Liposome oxidation (TBA), (8)
beans DPPH
Tomato pulp, puree, paste 30, 40, 50°C, 3 months Xanthine oxidase, Lo acid (Cu) Ethane, CD at 234 nm (9)
FOOD ANTIOXIDANTS
Oxidized PE in soybean oil 60°C, 24–48 h TBARS Induction period (10)

a
(1) Anese et al. (1999), (2) Manzocco et al. (1998), (3) Daglia et al. (2000), (4) Mastrocola and Munari (2000), (5) Huang et al. (2001) (6) Richelle et al. (2001),
(7) Del Castillo et al. (2002), (8) Yen et al. (2005), (9) Lavelli and Giovanelli (2003), (10) Hidalgo et al. (2005).
Abbreviations: AAPH and ABAP, 2,2'-azobis(2-amidinopropane) dichloride; DPPH, 2,2-diphenyl-1-picrylhydrazyl; LDL, LDL, low-density lipoproteins; Lo,
linoleic; CD, conjugated dienes; ABTS, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid); PE, phosphatidyl ethanolamine.
b
Antioxidant activity determined by the lag phase of LDL oxidation for a standard cup varied in the range: 292–948 min for coffee, 217–444 min for cocoa, 186–
338 min for green tea and 67–277 min for black tea. Addition of milk did not alter the antioxidant activity.
109
Figure 5.2. Formation of pyrrolized phospholipids by amino-carbonyl reactions (from Hidalgo et al.,
2005).
Table 5.3. Effect of phospholipids before and after oxidation at 60°C on

oxidative stability of soybean oila
Additions (200 ppm) Oxidative stability

Induction period (days)
Before oxidation After oxidation
None 11.7 –
PC 13.7 13.0
PE 13.5 14.3
PI 12.0 11.9
BHT 15.3 –
a
From Hidalgo et al. (2005).
PC = phosphatidylcholine, PE = phosphatidylethanolamine, PI = phosphatidylinositol,
BHT = butylatedhydroxytoluene.
carbonyl compounds produced by both lipid oxidation and glycation of proteins

that induce atherosclerosis and diabetes (Chapter 7).
B. Synergism of phospholipids
Phospholipids have multiple functions, as effective metal chelators and
FOOD ANTIOXIDANTS 111
emulsifying agents, and as synergists though reinforcing the antioxidant activ-

ity of phenolic compounds. In food emulsions, lecithin may improve interactions
between the polar phenolic antioxidants and less polar lipid substrates through
its surface activity. Phospholipids in foods and crude vegetable oils develop
browning reaction products after thermal treatments which contribute to their
antioxidant activity. The brown color reversion occurring after deodorization
of improperly degummed vegetable oils is due to the formation of pyrrolized
phospholipids (Figure 5.2). A mixture of α-tocopherol with phosphatidyl-
ethanolamine (PE) exhibits higher antioxidant activity than a mixture with
phosphatidylcholine (PC). This difference is attributed to improved regenera-
tion of the tocopheroxyl radical produced by oxidation to tocopherol by easier
hydrogen transfer from the amino group to the radical, resulting in effective
synergistic protection against lipid oxidation. In general, the presence of a
strong acid and proton generating function is important for this synergism.
Synergism is also specific for certain classes of phospholipids, PE being a
much more active synergist than PC and phosphatidylinositol (PI) having no
synergistic activity. In addition to reinforcing the antioxidant activity of
tocopherols, PC and PE also have synergistic effects through increasing the
antioxidant activity of flavonoids in food lipids. PE is also a potent synergist
with synthetic antioxidants such as propylene glycol, BHA, BHT and TBHQ at
elevated temperatures (above 80°C), but it has very little synergistic action at
lower temperatures. Phospholipids may thus have a tendency of producing
reducing browning materials that can act as food antioxidants when heated at
the elevated temperatures.
C. Plant and beverage sources of phenolic compounds

Fruits, vegetables and beverages are major sources of phenolic compounds in
the diet that contribute to their in vitro antioxidant activity. Wide variations of
total phenol contents are observed in different fruits, vegetables, beverages and
juices (Table 5.4). These literature data vary widely according to the methods
of extraction, processing, concentrations, storage and the empirical analytical
methods used for total phenols. The method of tea preparation greatly affects
their catechin gallate contents and distribution. The variability of wine phenolics
is particularly affected by a multitude of factors, including grape varieties and
maturity, growth and soil conditions, wine technologies and processing. Ex-
pressed as gallic acid equivalents, the total phenol contents of beverages
decreased in the order: red wine > rosé wine > white wine > green tea > black
tea > instant coffee > ground coffee. Other sources of plant phenolics in agri-
cultural by-products include hulls from rice, nuts, and oats, peels of citrus and
fruits, and residues from the olive and grape industries.
In addition to plant foods, beverages provide a significant amount and
variety of antioxidants that may play a part in synergistic mixtures. The most
112
Table 5.4. Total phenolic contents of fruits, vegetables, beverages and juices (gallic acid equivalents)a
Fruit Total phenols Vegetables Total phenols Beverages Total phenols Juices Total phenols
Apple 296 Broccoli 102 Black tea 62–163 Apple 339

Blackberry 27–555 Cabbage 55 Green tea 61–200 Grapefruit 535
Blueberry 171–961 Carrot 56 Instant coffee 146–151 Orange 755
Peach 85 Cucumber 20 Ground coffee 53–57 Prune 441
Plums 174–375 Spinach 91 Red wines 1600–4200 Pineapple 358
Raisins 400 Tomato 68b White wines 191–854 Red grape 1728
Raspberry 114–178 Onions 73–76 Rosé wines 340–1304 White grape 519
Red grapes 201–221 Red wines 558c
Strawberry 160–290 White wines 15c
Rosé wines 17c
a
Adapted from Balasundram et al. (2006). As mg gallic acid/l.
b
As mg ferrulic acid/l.
c
From Auger et al. (2004). French wines, mean concentrations of catechin and procyanidins in mg/l.
Table 5.5. Antioxidant capacity and total phenolic contents of plant foods and
beveragesa
Foods Total phenol contentb Antioxidant capacityc

FRAP ABTS
Foods
Nuts 894 45 34
Fruits 538 26 10
Vegetables 287 10 7
Legumes 155 9 6
Cereals 107 2 0.2
Beverages
Coffee 340 2270 1330
Red wine 160 1200 1100
Tea 76 600 630
White wine 20 150 180
Beer 60 110 80
Orange juice 50 520 250
a
From Saura-Calixto and Goñi (2006).
b
mg/100 g dry matter edible parts.
c
Plant foods: Trolox equivalents/g dry matter edible part
Beverages: µmol Trolox equivalents/100 ml
FRAP = ferric reducing antioxidant power
ABTS = 2,2'-azinobis(3-ethyl-benzothiazoline-6-sulfonate) free radical scavenging capacity.
widely consumed non-alcoholic beverages include coffee, fruit juices and

colas, while the main alcoholic beverages include wine and beer. Evaluations
of the so-called ‘dietary antioxidant capacity’ of plant foods based on two
antiradical protocols, FRAP (ferric reducing antioxidant power) and ABTS
(2,2'-azinobis(3-ethyl-benzothiazoline-6-sulfonate) (Chapter 4), showed
approximately the same trends as the total phenol content by the Follin–
Ciocalteu method (Table 5.5). The relative phenolic contents, reducing and
antiradical capacity of plant foods decreased in the order: nuts > fruits >
vegetables > legumes > cereals. The relative inhibition of LDL oxidation
showed the same trend for fruits, vegetables and cereals. For beverages, the
trends for total phenolic contents, reducing and antiradical capacity values
agreed only for: coffee > red wine > tea >> cola. Small variations were noted
for white wine, rosé wine and beer.
Significant flavonoid components of cocoa and chocolate products include
catechins and procyanidins (consisting of monomers and oligomers) that are
known to have potent antioxidant activities. Berry extracts rich in antioxidants
derived from bilberry and blackcurrant contribute to the antioxidant properties
of fruit beverages, and when mixed with milk. Blends of berry with low-fat
milk or low-fat fermented milk were shown to have significantly greater
antioxidant activity and presumably a longer shelf life than plain milk.
The antioxidant activity of a standard cup based on the lag phase of LDL
Table 5.6. Antioxidant activity of polyphenolic beverages based on in

vitro LDL oxidationa
Beverages LDL oxidative stability

(increase of lag phase, min)
Coffee 292–948
Cocoa 217–444
Tea, green 186–338
Tea, black 67–277
Tea, herbal 6–78
a
From Richelle et al. (2001). Coffee made with 7% soluble coffee, cocoa as 3%, tea as
one tea bag per 220 ml hot water. LDL oxidation with copper sulfate based on
conjugated monitored at 234 nm.
oxidation decreased in the order: coffee > cocoa > green tea > black tea, and
herbal tea (Table 5.6). These trends vary widely according to many factors
(Chapter 4). In estimates made of the antioxidant capacity based on plant foods
and beverages consumed in the daily Spanish diet, polyphenols appear to
represent the main dietary antioxidants from beverages (68%), followed by
vegetables (20%), nuts and legumes (8%). In the Mediterranean diet, rich in
vegetables and fruit, French consumption of 180 ml of red wine (containing
558 mg/l of catechin and procyanidins, Table 5.4) gives a mean daily intake of
100 mg of these phenolic compounds.
Nutritional recommendations based on the dietary antioxidant capacity
values of foods should be tempered by the many analytical problems from the
widespread use of one-dimensional methods to evaluate multifunctional food
and biological antioxidants. Although commonly consumed beverages such as
coffee and teas contain polyphenols with significant in vitro antioxidant
activity, much more research is needed to understand the absorption and their
in vivo activity. The low bioavailability of plant polyphenols may raise further
questions on the validity of dietary recommendations for plant antioxidants
(Chapter 6). Such recommendations may be premature until we improve our
understanding of the metabolism and in vivo activity of the complex
phytochemicals in the diets.
D. Vegetable oils
The fatty acid composition, natural antioxidants and minor constituents are the
major factors that determine the oxidative stability of edible vegetable oils. The
addition of synthetic antioxidants such as BHA, BHT and TBHQ to stabilize
vegetable oils has been generally limited in the past few years by the notion that
natural antioxidants, such as extracts of plants, rosemary and flavonoids, are
more desirable on the basis of poorly defined nutritional claims. Because of
legal requirements in most countries, synthetic antioxidant additives are either
not permitted in vegetable oils, or used at significantly lower concentrations

(not exceeding 100–200 ppm) than extracts of natural antioxidants (250–
500 ppm of crude extracts). However, these natural extracts cannot be labeled
as sources of natural antioxidants because of legal requirements for safety
testing. Pure components isolated from rosemary extracts (carnosic acid,
carnosol, rosemarinic acid), flavonoids (tea catechins), and olive oil phenolics
have attracted much interest, but they have not been legally approved for food
use because of the cost of animal testing. The literature on antioxidant testing
is also difficult to evaluate because of limited or no information on the
composition of crude extracts, the use of inappropriate reference compounds,
comparisons made at different temperatures of oxidation, and on the basis of
weight percent rather than molar percent of compounds of widely different
molecular weights.
1. Salad and fish oils

A variety of synthetic and natural antioxidants have been added to vegetable
oils containing variable amounts of α-, δ- and γ-tocopherols to improve their
oxidative stability in different food applications. Unfortunately, the literature is
confusing and difficult to interpret because of the variety of stability methods
used under a wide range of conditions (Table 5.7). The effectiveness of
antioxidants is very dependent on the conditions of oxidation, the methods used
to determine oxidation and the level of oxidation. It is thus impractical to
compare the results of tests evaluating synthetic and natural antioxidants at
temperatures that vary so widely, between room temperature and Rancimat at
120°C, and under frying conditions. Tocopherols are depleted at different rates
in vegetable oils, according to their fatty acid composition. Complex oxidation
products from tocopherols include tocoquinone, and epoxy derivatives (Figure
5.3) that can affect the stability of vegetable oils.
Rosemary extracts and constituents, carnosic acid, carnosol and rosmarinic
acid, were effective in inhibiting hydroperoxide formation in corn, peanut and
fish oils, when tested in bulk. The rosemary extract and constituents were more
active in bulk corn, peanut and fish oils than in bulk soybean oil, apparently
because soybean oil had a higher concentration of natural tocopherols which
appear to have a negative effect on the antioxidant activity of rosemary
constituents (Lipid Oxidation, 2nd ed, p. 240). In contrast to bulk oils, these
rosemary compounds were either inactive or prooxidant in the corresponding
oil-in-water emulsions. These results were explained by the interfacial phe-
nomenon (Figure 1.1), by increasing the activity of hydrophilic rosemary
extract and carnosic acid in bulk oil where they are more protective by being
oriented in the air–oil interface. However, in the oil-in-water emulsions where
the hydrophilic rosemary compounds remain in the water, they become less
effective in the oil–water interface where oxidation takes place.
Table 5.7. Effect of antioxidants on oxidative stability of vegetable oilsa,b
Vegetable oils Antioxidantsb Conditions Ref.c
Corn Tocopherols, ascobyl palmitate, 45°C (1)

ascorbic acid
Corn oil, Soybean, Rosemary extract, carnosic acid, 40, 60°C (2a,b)
Peanut, Fish carnosol
Cottonseed Thyme and clove oils Room temp. (3)
Cottonseed Extracts of oat hulls, TBHQ 30, 60°C (4)
Soybean Frying conditions
Cottonseed Extracts of oregano 63°C (5)
Olive Olive phenolics AOM, 97.8°C (6)
Olive Olive phenolics,d phenolic 60°C (7)
acids,d α-tocopherold
Olive, Sunflower Rosemary, BHA 63 and 120°C (8)
Olive, Linseed Tocopherols Rancimat (120°C) (9)
Peanut Essential oils 60°C (10)
Rapeseed, Sunflower Rosemary and sage extracts 40, 60°C (11)
Rapeseed Old tea leaves 60°C (12)
Canola Green tea catechins 95°C (13)
Soybean Carnosic acid, carnosol, Rancimat (100°C) (14)
BHT, BHA, TBHQ
Sunflower Caffeic acid, esculetin, fraxetinb 25 and 100°C (15)
a
From Yanishlieva and Marinova (2001).
b
See structures in Figures 5.3, 5.7, and in Lipid Oxidation, 2nd ed, Figure 9.1 (p. 210), Figure 9.12, (p.
238) and Figure 12.14, (p. 380).
c
(1) Cort (1974), (2a, b) Frankel et al. (1996), (3) Farag et al. (1989), (4) Tian and White (1994a, b), (5)
Lagouri and Boskou (1996), (6) Nergiz (1991), (7) Satué et al. (1995), (8) Wanasundra and Shahidi
(1998), (8) Gamel and Kintsakis (1999), (9) Wagner and Elmadfa (2000), (10) Maestri et al. (1996), (11)
Nguyen et al. (1999), (12) Zandi and Gordon (1999), (13) Chen and Chan (1996), (14) Richheimer et al.
(1996), (15) Yanishlieva and Marinova (1996).
d
Olive phenolics, phenolic acids and α-tocopherol were added to a commercial refined, bleached and
deodorized olive oil containing initially 8 ppm total phenols as gallic acid equivalents.
Rosemary extracts in combination with the synthetic antioxidant BHT

showed synergistic activity when used in mixtures in soybean oil. Various
phenolic compounds found in tea, spices, thyme, clove, oregano, sesame and
oats were reported to have a wide range of antioxidant activities in corn,
cottonseed, soybean, peanut, canola, sunflower and fish oils by improving their
oxidative stability under a wide range of conditions. The literature in this field
is difficult to interpret because of the wide diversity of methods used to
evaluate lipid oxidation carried out at different temperatures.
Figure 5.3. Oxidation products of α-tocopherol (from Pazos et al., 2005).
2. Olive oils
Virgin olive oils prepared by cold-pressing or refined olive oils, also referred
to as ‘pure’, are the main edible fats in the Mediterranean diet recognized for
their health benefits. Although olive oils are generally considered stable to
oxidation because of their high oleic acid content (76–80%), total polar
phenols (200–1500 mg/kg) and tocopherols (100–300 mg/kg), they are still
susceptible to oxidation due to their polyunsaturated fatty acids (5–9%), and
the presence of minor constituents including chlorophylls (9–20 mg/kg),
carotenoids (up to 10 mg/kg) and metal impurities (Fe 0.5–3 and Cu 0.001–
0.2 mg/kg). Despite their relatively high total phenolic contents compared to
other vegetable oils, the quality of stored commercial olive oils can vary widely
(Lipid Oxidation, 2nd ed, Tables 8.6 and 8.11, pp. 196, 205), as may be
expected from their variable initial peroxide values ranging from 0.4 to
33 meq/kg. The oxidative stability of a commercial refined, bleached and
Table 5.8. Composition and effect of storage on quality properties of extra virgin olive
oilsa
Composition/properties Greece Greece Italyb Spain Spain

(1)b (2)c (1)b (2)d
C18:1 76.2 76–77 78.0 80.3 –

C18:2 7.1 5.8–9.2 7.6 3.6 –
α-Tocopherol, mg/kg (Initial) 158 169–210 208 145 100–172
Stored: ~15% 20–58% 35% 15% 3–33%
β-Carotene, mg/kg (Initial) 1.1 1.0–2.7 1.7 1.3 –
Stored: ~30% 0.8–1.7 ~38% ~20% –
Chlorophyll, mg/kg (Initial) 12 9.1–23 14 14 –
Stored: 24 months ~80% 7.7–20.4 ~80% ~80% –
Acidity, % oleic (Initial) 0.58 0.28–0.54 0.55 0.38 0.25–0.78
PV, meq/kg (Initial) 9.6 6.4–12.9 8.7 7.9 7.4–11.4
Stored: ~165 24.1–36.8 ~50 ~8 5.8–8.0
Conjugated diene, K232nm (Initial) 2.05 1.56–1.63 1.61 1.84 1.6–2.1
Stored: ~7.5 3.64–5.14 ~5 ~2.5 1.4–1.9
Total polar phenolics, mg/kg (Initial) 340 98–209 734 603 715–1195
Stored: ~30% 31–38% ~40% ~15% 18–19%
a
Values with symbol ~ are estimated from original Figures.
b
Hrncirik and Fritsche (2005). Stored for 50 days at 60°C and induction periods = 40, 46 and 88 days for
Greece (Koroneiki), Italy (Coratina) and Spain (Picual). Values for stored samples are estimated from
Figures as residual %. Total polar phenolics were determined by HPLC.
c
Psomiadou and Tsimidou (2002). Stored for 24 months in the dark at room temperature. Total phenols
were determined on the polar fraction (60:40 MeOH:water) by the Folin-Ciocalteu test.
d
Brenes et al. (2001). Samples stored for 340 days at 30°C. Total phenolic compounds are expressed as
μM. Total polar phenolics were determined on the dimethylformamide fraction by HPLC.
deodorized olive oil, containing only 8 ppm of total phenols (as gallic acid
equivalents), was increased by adding the natural phenolic compounds ex-
tracted from extra virgin olive oils, pure phenolic acids, and α-tocopherol
(Table 5.7, ref 7). Phenolic extracts of olive oil (50–100 ppm) inhibited
hexanal formation, measured by static headspace GC (up to 100%), much more
effectively than hydroperoxide formation, measured by peroxide values (up to
54%). Similar trends were observed for added caffeic, vanillic, cinnamic and
ferulic acids. Although α-tocopherol was very effective in inhibiting hexanal
formation, it was only effective in inhibiting peroxide formation initially with
100 ppm (after 3 days of oxidation), but was prooxidant with 500 ppm after 11
and 15 days of oxidation. Similar results were obtained for corn oil and other
natural antioxidants. Therefore, results can vary significantly with many
antioxidants, depending on their concentrations, the level of oxidation used as
end point, and the method used to determine lipid oxidation (peroxide value
versus volatile decomposition products) (Chapter 4.D).
Oxidative stability studies of representative extra virgin olive oils from
Greece, Italy and Spain showed induction periods varying from 40 to 88 days
at 60°C, with peroxide values ranging from 6 to 37 meq/kg after storage at
room temperature between 11 and 24 months, and between 8 to 165 meq/kg

after storage at 60°C for 50 days (Table 5.8). Storage under different conditions
resulted in wide ranges of depletion of α-tocopherol (3– 58%), β-carotene (20–
38%), chlorophyll (80%) and total polar phenolic compounds (18–38%). Total
polar phenolic compounds varied widely according to the methods of extrac-
tion, detection, processing and conditions of storage.
3. Frying oils
The use of vegetable oils for frying is an important application involving very
complex sequences of reactions that affect the quality of fried foods (Lipid
Oxidation, 2nd ed, Chapter 12). Many synthetic antioxidants, such as BHT,
propyl gallate and TBHQ, are sometimes used to stabilize vegetable oils used for
frying. Although these antioxidants decompose to varying degrees during
frying, they are partially retained and absorbed by the frying foods depending on
the turnover rate with make-up fresh vegetable oil used in the frying process. The
shelf life of potato chips is extended by adding TBHQ and BHT. Natural
tocopherol mixtures in vegetable oils can stabilize fried foods after storage. The
level of residual tocopherols in the fat absorbed by the fried foods would be
expected to increase by more rapid rates of fat turnover during frying. Although
heating to 180°C, corresponding to frying temperature, readily decomposes
α-tocopherol, the resulting tocopherol quinone products could potentially have
protective antioxidant activity under frying conditions (Table 5.9). However, no
detailed information is currently available in the literature on the antioxidant
activity of tocopherol quinones and semi-quinones under actual frying conditions.
Although carnosic acid is lost during oxidation and under frying conditions,
the resulting oxidation products have antioxidant activity. Rosemary extracts
are particularly active as antioxidants at the elevated temperatures in frying
fats. Peanut and palm oils are stabilized by rosemary antioxidants during
frying, and their activity is carried over into fried foods. Rosemary and sage
extracts were equally effective in retarding deterioration of palm olein during
Table 5.9. Loss of α-tocopherol and formation of α-tocoquinone in soybean and

sunflower oils by heating at 180°Ca
Oils α -Tocopherol Heating Tocoquinoneb

(ppm) 5h 10 h 20 h 30 h (ppm)
Soybean (SBO) 138 28 28 79 93 50

SBO + α-tocopherol 1175 69 79 99 100 200
Sunflower (SUN) 829 6 78 100 100 100
SUN + α-tocopherol 1128 21 80 98 100 150
a
Rennick and Warner (2006). (See structure of α-tocoquinone in Figure 5.3).
b
After heating for 20 hours.
Figure 5.4. Products of oxidation, reduction and isomerization of carnosic acid (from Masuda et al.,
2002).
Figure 5.5. Products of oxidation, reduction and isomerization of carnosol (from Masuda et al., 2005).
frying of potato crisps (Lipid Oxidation, 2nd ed, p. 378). Rosemary extracts
may be of special interest because the active component carnosic acid is readily
oxidized, reduced and isomerized into quinone and lactone products (Figure
5.4) that retain antioxidant activity under artificial conditions (Lipid Oxidation,
2nd ed, Figure 9.13, p. 242). Whether or not related products may be formed
under frying conditions that retain effectiveness remains to be established.
Recent studies showed that carnosol, a major phenolic constituent of sage and
rosemary, was readily converted into antioxidant products by heating the o-
quinone derivative in aqueous solvents (Figure 5.5). Small amounts of rosmanol
and its quinone derivative were also identified. The results indicated that
rosmanol can reduce carnosol quinone back to the carnosol precursor which is
active as an antioxidant.
Virgin olive oil contains complex mixtures of natural phenolic compounds
that decompose to varying degrees during frying of potato slices (Figure 5.6).
Tyrosol (p-HPEA) and its derivatives (p-HPEA-EDA and p-HPEA-EA) were
more thermally stable than hydroxytyrosol (3,4-DHPEA) and its derivatives
(3,4-DHPEA-EDA and 3,4-DHPEA-EA). The phenolic extract from the olive
oil used for frying has not lost all its antioxidant activity and also showed weak
prooxidant activity. Quality evaluations of the fried potatoes are needed,
however, to determine how much of the olive phenolic compounds is absorbed
and to predict their storage stability more successfully.
TBHQ is converted by thermolysis into several types of dimers, which retain
activity before they are completely decomposed at elevated temperatures and
under frying conditions. Other synthetic quinones such as rosmariquinones and
ubiquinones are known to have antioxidant properties. However, no information
Figure 5.6. Phenolic compounds in virgin olive oil (initial and final concentrations after 12 fryings, in
mg/kg) (from Gómez-Alonso et al., 2003).
is available on the degradation and activity of the oxidation products formed

under actual frying conditions, and whether or not they may have any carry-
over antioxidant effect that contributes antioxidant protection to the frying
foods.
At elevated temperatures, tocopherols are depleted more slowly in polyun-
saturated fats than more saturated fats (Lipid Oxidation, 2nd ed, p. 376). With
the current trend towards avoiding hydrogenated fats containing nutritionally
undesirable trans isomers, the use of less polyunsaturated fats such as high-
oleic or mid-oleic sunflower or canola oils, and high-oleic soybean oil, may
result in greater losses of tocopherols. Supplementation with either TBHQ or
rosemary antioxidants may improve the shelf life of fried foods. The general
use of methyl silicone as an anti-polymerization additive may also reduce the
loss of antioxidants at the elevated temperatures of frying, and may
synergistically reduce the thermal decomposition of tocopherols.
E. Milk products
The extent of lipid oxidation in dairy products depends on the fatty acid
composition of the raw milk, the balance between pro- and antioxidants and
enzyme activity, which are influenced by the feeding, breed, age and health of
the cows. After milking, the rapid cooling, processing and packaging condi-
tions, storage temperature and light exposure also influence lipid oxidation in
dairy products. The oxidative quality of dairy products has generally been
evaluated by determining peroxide value, thiobarbituric acid reactive sub-
stances, anisidine value or sensory analyses. More sensitive and reliable
methods are based on headspace volatile analyses, because flavor deterioration
is observed in milk products at very low levels of oxidation, usually below
peroxide values of 1.
1. Tocopherols
The oxidative stability of milk correlates well with its α-tocopherol level
(average of 25 μg/g milkfat), and especially in the lipids of the fat globule
membrane (44 μg/g of fat globule membrane). Supplementing the ration of
animals or direct addition with various forms of tocopherols provide an
effective control measure against lipid oxidation in milk.
2. Phospholipids
The phospholipids concentrated in the fat globule membrane in milk act as
synergists by reinforcing the antioxidant activity of tocopherols (see Section
B). Thus, solvent-extracted milkfat containing phospholipids is much more
stable to oxidation than milkfat free of phospholipids, obtained by melting
churned butter into butter oil. The buttermilk resulting from churning is a good
source of antioxidant-active phospholipids. The stabilizing effect of phospho-
lipids is generally attributed either to their direct metal scavenging properties,
or to their synergistic protective interaction with tocopherols.
3. Ascorbic acid
According to its concentration, ascorbic acid can have either prooxidant or
antioxidant activity. Combinations of ascorbic acid and copper show either
prooxidant effects at relatively low levels, or antioxidant effects at relatively
high concentrations of ascorbate. Several mechanisms may explain these
effects, including conversion of cupric ions to the more active cuprous state,
formation of an active copper–ascorbic acid–oxygen complex catalyst, sparing
lipid oxidation by preferential oxidation of ascorbic acid and depleting the
available oxygen, and reduction of hydroperoxides by ascorbate into stable and
innocuous allylic alcohols. Ascorbic acid acts as an antioxidant at the normally
low copper concentrations in milk. However, during storage, the concentration
of ascorbic acid decreases continuously and is depleted by consuming dis-
solved oxygen.
Other reducing thiol compounds may also have dual pro- and antioxidant
effects in the presence of copper, similar to those of ascorbic acid. The ligands
associated with copper and iron can have a profound effect on their catalytic
activities.
4. Other antioxidants
Several synthetic antioxidants (BHA, BHT, propyl gallate) and metal chelators
(citric acid, phosphoric acid and salts of EDTA) are effective in inhibiting lipid
oxidation, but the use of these compounds is not legally permitted in dairy
products in the USA and other countries.
Lactoferrin is a glycoprotein found in bovine milk that is 22% saturated with
iron, which inhibits lipid oxidation by binding two ferric atoms very tightly and
reversibly (Figure 3.6). Each iron atom is coordinated with four protein ligands
(2 tyrosine, 1 aspartic acid and one histidine) and one carbonate anion (HCO3–)
acting as a bidentate ligand. Together with transferrin, lactoferrin play a key
role in regulating the levels of iron in biological fluids. Compared to human
milk, infant formulas are more susceptible to lipid oxidation because they are
supplemented with greater amounts of iron and do not contain lactoferrin.
Commercially available bovine lactoferrin isolated from cheese whey inhibited
lipid oxidation in corn oil-in-water emulsions and lecithin liposome systems
(Lipid Oxidation, 2nd ed, Table 10.8, p. 275). Lactoferrin was a better iron
chelator in the liposome than in the emulsion systems. When added to infant
formulas, lactoferrin inhibited lipid oxidation in the absence and presence of
different amounts of supplemented iron (Lipid Oxidation, 2nd ed, Table 11.11,
p. 322). Lactoferrin inhibited hydroperoxide and hexanal formation in a
concentration-dependent manner. Lactoferrin is also useful in foods for its
antimicrobial activity in reducing the availability of iron to bacteria.
Addition of human lactoferrin to infant formula or human milk inhibits lipid
oxidation in human milk supplemented with iron. The presence of more iron
and the absence of lactoferrin in infant formula compared with human milk
apparently results in greater lipid oxidation. The formation of an iron–lactoferrin
complex may be more resistant against thermal denaturation than lactoferrin
alone. Lactoferrin inhibits iron-catalysed lipid oxidation by chelating free iron.
Therefore, iron-containing infant formula and supplemented with lactoferrin
may provide an effective way to prevent lipid oxidation.
5. Other components of milk

Milk is protected from lipid oxidation by casein and other proteins capable of
binding and inactivating copper and iron. Casein inhibits lipid oxidation by
binding copper and by protecting the oil–water interface around the fat globule
membrane against oxidation. For this reason, homogenized milk has improved
oxidative stability though resurfacing the fat globules with casein which
provides a protective membrane. Heating milk products to release free sulfhydryl
groups in proteins is another method of increasing the oxidative stability of
dairy products.
6. Addition to other beverages containing antioxidants

The effect of milk added to other beverages has attracted much interest
recently, since coffee and chocolate have been shown to be rich sources of
flavonoid antioxidants. When milk is added to coffee, a significant proportion
of chlorogenic acid in coffee becomes bound to dairy proteins. Although no
significant effect is reported on the antioxidant activity as measured by the
antiradical DPPH assay, more reliable testing methods are needed before we
know the true effect of milk protein interactions, not only with chlorogenic acid
but also with other important flavonoid components found in coffee and
chocolate.
F. Meat products
Muscle tissues contain a complex mixture of lipid-soluble (α-tocopherol,
ubiquinone) and water-soluble antioxidants (ascorbate, histidine-dipeptides),
and enzymes (superoxide dismutase, catalase, glutathione peroxidase). Lipid
oxidation products can react with proteins to cause protein oxidation via
peroxyl radical intermediates. Protein radicals are produced that can cross-link
to produce a protein network. Carbonyl complexes due to protein–lipid inter-

actions are formed rapidly with lysine residues reacting with aldehydes to
produce polymers. Phenolic compounds can also inhibit protein–lipid interac-
tions in meat.
Lipid oxidation in meats can be effectively controlled by adding various
natural antioxidants and phenolic compounds derived from spice extracts, by
vitamin E supplementation of animal diets, and by processing of cured meat
with sodium nitrite. Various natural antioxidant mixtures of tocopherols,
ascorbyl palmitate and citric acid show synergistic effects in stabilizing cooked
and frozen meat. Synthetic antioxidants, BHA, TBHQ, propyl gallate (Chapter
2) and combinations with citric acid, ascorbic acid or phosphates are also
effective in retarding lipid oxidation in meat. A number of metal chelators
(EDTA, sodium pyrophosphate, tripolyphosphate, citric acid), and reducing
agents (ascorbic acid, isoascorbic acid and their salts, sulfur dioxide) are used
to improve the sensory quality and color stability of stored meat products.
Supplementation of vitamin E in the diet has a significant effect in increasing
the oxidative and color stability of poultry, beef and pork products. Dietary
incorporation of α-tocopherol is more effective in stabilizing meat than exog-
enous addition of this antioxidant.
Rosemary extracts containing potent antioxidants, including carnosic acid
and carnosol, are effective in stabilizing cooked pork, beef, chicken and turkey
meat. In cooked ground beef, rosemary extracts are effective in controlling the
development of warmed-over flavor during storage. Many plant extracts and
spices containing flavonoids and polyphenols reduce lipid oxidation and flavor
deterioration, either when incorporated in the animal diets or when added
directly to various ground meat products. However, the flavor associated with
these plant extracts when added directly may limit their usefulness in muscle
foods. Furthermore, many current reports in the literature are difficult to
interpret because no information is given on the amounts of active components
in crude extracts. For example, pure Trolox and ascorbic acid are reported to be
more effective than various commercial crude extracts of rosemary, but such
comparisons made at different concentrations expressed in weight percent are
not valid without compositional information on the active ingredients carnosic
acid and carnosol. Another complication is the ready thermal oxidation of
carnosic acid into antioxidant active products which have not been completely
identified (Lipid Oxidation, 2nd ed, Figures 9.13 and 12.4).
Phytic acid and carnosine (histidine-containing dipeptide), obtained from
cereal and meat by-products, are effective inhibitors of lipid oxidation by
several mechanisms, including metal inactivation and free radical quenching.
Uric acid obtained from the decomposition of adenosine triphosphate in muscle
also inhibits lipid oxidation by the same mechanisms. The importance of uric
acid as an endogenous antioxidant in muscle foods is not clear, however.
Various protein concentrates from soybeans, cottonseed and peanuts inhibit
Table 5.10. Effect of natural extracts on oxidation of pre-cooked pork pattiesa
Extracts Levels (ppm) TBARS Hexanal Vitamin E
% decreaseb % increaseb
Rosemary 200 69 77 83
Grape skin 50 47 2 2
Green tea 200 46 34 23
Coffee 200 11 8 16
a
From Nissen et al. (2004). Meat patties were mixed with water, salt and extracts, and stored in
polyethylene bags at 4.5°C for 10 days.
b
% decrease or increase relative to control without additives.
lipid oxidation in muscle foods. In addition to their iron binding activity, these
crude extracts contain complex polyphenolic flavonoids that have potent
antioxidant activity.
Other factors affecting lipid oxidation in animal tissues include diet, process-
ing and additives (salt, nitrite, spices and antioxidants). Rosemary extracts are
very efficient against deterioration due to lipid oxidation and tocopherol
degradation in minced, pressure-processed chicken breast, after refrigerator
storage and during cooking. Rosemary also protects tocopherols against degra-
dation in pressurized chicken breast during chill storage and subsequent
cooking. A comparison of different antioxidant extracts showed the following
decreasing trend in efficiency based on TBARS and hexanal: rosemary > grape
skin extract ≈ green tea > coffee (Table 5.10). Rosemary also provided the
most protection against loss of vitamin E, followed by green tea and coffee, but
showed no protection from a grape skin extract. The positive effect of extracts
towards vitamin E may be due to the interaction of polyphenols in regenerating
this antioxidant from its radical produced after oxidation. Rosemary extract
also proved to be superior in protecting dehydrated chicken meat. Tea
polyphenols were shown to protect meat during frying. Green and black tea was
used in coating meat on both sides to inhibit the formation of mutagenic
compounds during frying.
Minced meat is commonly mixed with spices to enhance flavor and with
different proteins from soya or milk to enhance texture and as emulsifiers to
increase water-holding capacity. Plant extracts and spices inhibit lipid oxida-
tion in cooked meat products. Proteins and peptides are also beneficial in
inhibiting oxidation in cooked meat. The addition of whey protein concentrate
and soya protein isolate inhibits lipid and oxymoglobin oxidation in cooked
meat balls.
Rapeseed meal provides a rich source of phenolic compounds that are potent
antioxidants used for food, cosmetic and pharmaceutical preparations. Rapeseed
and pine bark were shown to have antioxidant properties by inhibiting the
oxidation of lipids and proteins in meat (Table 5.11). When added to cooked
Table 5.11. Plant phenolic antioxidants in cooked pork meat pattiesa
Plant materials Phenolic composition (μ g/g) Total phenols Inhibition of

(mg/100 g meat) (μ g/g) oxidation (%)
Lipid Protein
Rapeseed meal (RM) – 80 42
282 mg
RM extracts
29 mlb Sinapine, 2861 + sinapic acid, 275 4751 94 61
29 mlc Sinapine, 2381 + sinapic acid, 275 5885 91 72
4.7 mld Vinylsyringol, 463 + sinapic acid, 22 785 84 58
Pine bark extract Phenolic acid derivatives, 70 + 762 98 64

11 ml flavonoids, 336 + lignan glycosides, 83
Sinapic acid, – – 89 60
23.5 mg
Vinylsyringol, – – 100 78
23.5 mg
a
From Vuorela et al. (2005). Lipid oxidation based on hexanal; protein oxidation based on protein
carbonyls. Rapeseed meal obtained after pressing at high temperature and pressure.
b
Aqueous ethanolic (70%) extract. Structures: see Figure 5.7, p. 133.
c
Enzyme assisted extract.
d
Phenolic extract.
pork, these plant materials inhibited lipid and protein oxidation. Lipid oxida-
tion was determined by measuring the formation of hexanal by static headspace
gas chromatography. Protein oxidation was estimated spectrophotometrically
by the formation of protein carbonyls using the 2,4-dinitrophenylhydrazone
derivatives at 370 nm. At certain concentrations, rapeseed and pine bark
materials were effective in preventing lipid oxidation between 80 and 98%, and
protein oxidation between 42 and 72%. The main phenolic compounds in
rapeseed meal extracts include sinapic acid and its derivative sinapine, choline
ester of sinapic acid. The phenolic extract containing a mixture of sinapic acid
and vinylsyringol shows a higher antioxidant activity than pure sinapic acid,
which may be due either to synergistic interactions or to the relatively greater
lipophilicity of vinylsyringol. These phenolic compounds can also protect
cooked meat from oxidative deterioration by acting as metal chelating agents
and inactivating the iron (II) released from myoglobin. Several other phenolic
compounds also present in pine bark may contribute to antioxidant activity,
including lignans and catechins. Rapeseed meal and pine bark extracts could
thus be useful supplements for increasing the antioxidant protection of meat
products.
In the interest of producing meat with a more favorable n–6:n–3 PUFA
balance, pigs and ruminants can be fed linseed and grass rich in 18:3. However,
adverse effects are observed on meat quality, on the basis of myoglobin
oxidation and flavor, when the concentrations of 18:3 approach 3% of neutral

lipids or phospholipids. DHA can also be increased by feeding fish oil, but
adverse flavor and color changes are observed if the DHA levels are too high.
Grazing also provides antioxidants including vitamin E, which maintain PUFA
levels in meat and prevent quality deterioration during processing and display.
G. Fish products
Lipid oxidation is retarded in fish by synthetic antioxidants (BHA, BHT,
TBHQ), natural antioxidants (tocopherols, flavonoids) and metal chelators
(EDTA, ascorbate, phosphate, citrate, carnosine). Ascorbate acid retards the
oxidation of herring during frozen storage, but may promote oxidation in
cooked fish. Ascorbate may become prooxidant by decomposing accumulated
hydroperoxides in stored fish to produce lipid radicals which promote oxida-
tion mediated by hemoglobin. Flavonoids are effective antioxidants in
prolonging the shelf life of ground fish. EDTA and antioxidants inhibit
enzyme-catalysed lipid oxidation (superoxide dismutase, catalase and
peroxidases) by removing iron and reducing hydrogen peroxide. Antioxidants
are more effective in minced fish where they become more readily incorporated
with the oxidizable lipids than in whole fish.
Various antioxidants were tested in an assortment of fish products, fish oils
and emulsions by several methods under a wide range of conditions (Table
5.12). Unfortunately, many oxidations studies commonly used the TBA test to
evaluate antioxidants in fish and muscle foods. This test is notoriously unspe-
cific and unreliable in these complex foods, where much interference would be
expected from fish proteins and interaction products causing fluorescence
(Lipid Oxidation, 2nd ed, pp. 108–110). Rosemary extracts and active constitu-
ents, carnosic acid and carnosol, effectively inhibited lipid oxidation based on
conjugated diene hydroperoxides, propanal and pentenal, but promoted oxida-
tion in the corresponding emulsions based on hydroperoxides.
Many studies showed beneficial antioxidant effects, with green tea and tea
catechins showing activities that compared favorably with α-tocopherol and
synthetic antioxidants, BHT, BHA and TBHQ. Green tea extract containing
chlorophyll was prooxidant in fish oils (blubber and menhaden oils) oxidized
at 65°C (Table 5.12, ref 3). After removal of chlorophyll by column
chomatography, the antioxidant activity of the green tea extract (at levels
higher than 200 ppm) was higher than that of BHA, BHT and α-tocopherol, and
lower than of TBHQ. Unfortunately, this study used in addition to the unreli-
able TBA method, the weight gain method, which is one of the least sensitive
ways to measure lipid oxidation. Polyphenols extracted from extra virgin olive
oil were effective in inhibiting lipid oxidation in canned tuna at concentrations
higher than 400 ppm (Table 5.12, ref 4). At this concentration, the olive oil
extract was as effective as a 1:1 mixture of BHT and BHQ. However, at a lower
Table 5.12. Effect of antioxidants on oxidative stability and methods to evaluate lipid oxidation of fish products
Fish products Antioxidants Conditions Methods Ref.a
Fish oil + DHA and oil-in-water Rosemary extract, carnosol, 40°C CD, propanal, pentenal (1)
emulsion carnosic acid
Ground mackerel Green tea, tea catechins 4°C TBARS, volatiles (2)
BHT, BHA, TBHQ
Fish oils Green tea extract (GTE) 65°C Weight gain, PV, TBARS (3)
GTE – chlorophyllb
Canned tuna Polyphenol extract of extra 40, 100°C PV, static headspace GC
virgin olive oil (4)
Whiting and mackerel Tea catechins, α-tocopherol 4°C TBARS (5)
Whiting and mackerel Tea catechins 4°C, light exposure TBARS (6)
Horse mackerel fillets Citric acid, ascorbic acid –20°C TBARS, fluorescence (7)
Menhaden oil-in-water emulsion Protein isolates, Na caseinate 20°C Hydroperoxides, propanal (8)
Mackerel muscle fish oil, Grape polyphenols, propyl –10°C, 30°C, 40°C PV, CD, CT, DPPH, TBARS, (9)
emulsions gallate fluorescence
FOOD ANTIOXIDANTS
Minced and horse mackerel Grape extract, propyl gallate –10°C Depletion of α-tocopherol, (10)
ubiquinone-10, glutathione
Blue sprat Tea extracts 25°C Lipoxygenase, linoleic acid (11)
emulsion
Menhaden oil Dispersed sugars, polyols Fluorescent lighting, 60°C PV, volatiles (12)
a
(1) Frankel et al. (1996b), (2) He and Shahidi (1997), (3) Wanasundra and Shahidi (1998), (4) Medina et al. (1999), (5) Tang et al. (2001a), (6) Tang et al. (2001b),
(7) Aubourg et al. (2004), (8) Farji et al. (2004), (9) Pazos et al. (2005a), (10) Pazos et al. (2005b), (11) Seto et al. (2005), (12) Faraji and Lindsay (2005).
Abbreviations: BHT, butylated hydroxytoluene; BHA, butyl hydroxyanisole; CD, conjugated dienes; CT, conjugated trienes; DPPH, 2,2-diphenyl-1-picrylhydrazyl;
GC, gas chomatography; PV, peroxide value; TBARS, thiobarbituric acid reactive substances; TBHQ, tert-butyl hydroquinone.
b
Green tea extract containing chlorophyll was prooxidant. After removal of chlorophyll, its antioxidant activity was higher than that of BHA, BHT and α-tocopherol,
and lower than that of TBHQ.
129
concentration of 100 ppm, the olive oil extract promoted hydroperoxide forma-
tion and decomposition. The polyphenol extract was more effective in canned
tuna packed in brine than in refined olive oil. The higher activity in brine was
explained by better partition of the polyphenols toward the polar water–fish oil
interface. Phenolic extracts from grape pomace were as effective as propyl
gallate in fish muscle during frozen storage (Table 5.12, ref 10).
For minced fish muscle, tea catechins were reported to be more effective than
α-tocopherol, when tested at the same weight concentration. However, such
comparisons based on weight rather than molar concentrations are misleading,
because complex mixtures of catechin gallates have different molecular weights
from pure α-tocopherol used as a reference.
A soaking pretreatment of mackerel fish fillets with aqueous solutions of
citric acid and ascorbic acid effectively inhibited lipid oxidation during frozen
storage. A mixture of citric and ascorbic acids produced the best inhibition of
lipid oxidation with whole fish during frozen storage. Other antioxidant
systems shown to be effectively in inhibiting oxidation of fish lipids during
frozen storage included protein isolates, sodium caseinate, grape polyphenols
and dispersed sugars and polyols. Among grape phenolic compounds, flavanol
monomers were more effective in oil systems than oligomeric procyanidins and
glycosylated flavonols. Flavanol oligomers were the most potent inhibitors of
oxidation in emulsions and in frozen fish muscle.
Grape polyphenols and propyl gallate, added to minced mackerel muscle and
mackerel fillets before freezing, inhibited the depletion of endogenous α-toco-
pherol, ubiquinone-10 and total glutathione. Phenolic compounds were effective
in delaying lipid oxidation in frozen mackerel fillets by spraying and glazing,
in the decreasing order of antioxidant efficiency propyl gallate > hydroxytyrosol
>procyanidins, corresponding with their reducing power, but not with their
chelating capacity. Washing the fillets with water prior to spraying phenols
increased the antioxidant activity of grape procyanidins synergistically and
changed the relative antioxidant efficiency to propyl gallate ≈ procyanidins
> hydroxytyrosol. This change was attributed to improved distribution of the
procyanidins onto the fillet surface by residual water remaining on the fillets
surface after washing.
The treatment of raw and cooked trout muscle with sodium tripolyphosphate,
added after cooking, significantly reduced lipid oxidation. Cooking enhanced
the antioxidant activity of sodium tripolyphosphate, sodium citrate and EDTA.
In the presence of added copper, only EDTA prevented lipid oxidation. These
results suggest that phosphates are good metal chelators, provided cooking has
eliminated phosphatase hydrolysis.
Variable amounts of preformed hydroperoxides in cod muscle significantly
affected the function of ascorbic acid as either a prooxidant or an antioxidant.
Since EDTA had no effect on the hemoglobin mediated lipid oxidation in
washed cod muscle, low-molecular weight iron apparently did not contribute to
Table 5.13. Oxidation of α-tocopherol in mackerel minced muscle during storage at

4°Ca
Analyses Oxidation, days

0 3 7 11
α-Tocopherol 3.7 0.95 0.59 0.65
TQ 0.05 0.68 0.72 0.76
TQE1 0.03 0.09 0.09 0.10
TQE2 0.01 0.21 0.18 0.19
Peroxides 1.9 18 65 44
a
From Pazos et al. (2005c). Analyses by HPLC-atmospheric pressure CI-MS.
Abbreviations: TQ, α-tocopherolquinone, TQE1, 5,6-epoxy-α-tocopherolquinone, TQE2, 2,3-epoxy-
α-tocopherolquinone.
this oxidation. Ascorbate, on the other hand, was more effective in inhibiting
hemoglobin mediated lipid oxidation. Thus, by decomposing accumulating
lipid hydroperoxides to reactive lipid radicals, residual ascorbate may shift
from an antioxidant to a prooxidant. Ascorbate thus increased the lipid perox-
ide content in washed cod muscle and accelerated hemoglobin-mediated lipid
oxidation
In post-mortem fish, the distribution of α-tocopherol and its oxidized
products were significantly affected by the extent of oxidation produced (Table
5.13). The oxidation products of α-tocopherol in chilled and frozen fish muscle
were identified by HPLC-atmospheric pressure chemical ionization-mass
spectrometry, as α-tocopherolquinone, 5,6-epoxy-α-tocopherolquinone, and
2,3-epoxy-α-tocopherolquinone. Caffeic acid, hydroxytyrosol, and propyl
gallate (100 ppm) inhibited the depletion of α-tocopherol. These phenolic
compounds apparently reduced lipid oxidation by synergistically regenerating
endogenous α-tocopherol from its oxidized forms.
Fish oil emulsions could also be stabilized against oxidation by incorporat-
ing proteins into their continuous phase. With menhaden oil-in-water emulsions,
added whey protein isolate (WPI), soy protein isolate (SPI) and sodium
caseinate (CAS), only a fraction of proteins adsorb to the emulsion droplets,
with the rest remaining in the continuous phase. Unwashed emulsions were
more oxidatively stable than when WPI was removed by repeated centrifuga-
tion from the continuous phase of the emulsions (Table 5.14). The oxidative
stability of emulsions containing different proteins in the continuous phase
decreased in the order SPI > CAS > WPI, on the basis of both hydroperoxide
and headspace propanal formation. Iron-binding studies showed that the
chelating ability of the proteins decreased in the order CAS > SPI > WPI. The
free sulfhydryls of both WPI and SPI may be involved in their antioxidant
activity. Proteins can therefore protect n–3 PUFA containing emulsions by
incorporating them into the continuous phase of emulsions.
Table 5.14. Effect of whey protein isolate (WPI) on oxidation of menhaden oil-in-water
emulsions at pH 7 and 20°Ca
Added WPI, % Protein in continuous Hydroperoxides after Propanal after

phase (μ g/ml) 11 days (μ mol/ml) 14 days (μ mol/ml)
Unwashed
0.5 512 0.4 0.02
1.0 1383 0.1 0.02
Washedb
0.5 5.0 11 0.7
1.0 11 9 0.6
a
From Faraji et al. (2004).
b
Emulsions washed by repeated centrifuging and resuspending the concentrated emulsion with buffer
solution.
After so many years of research on the complex chemistry of lipid oxidation

in foods, it is distressing to know that the most unreliable TBA test is still
commonly used in this field. To improve our understanding of the effects of
antioxidants in inhibiting the sequence of complex oxidation processes in
multiphase meat and fish products, several improved methods more reliable
than the TBA test are required to evaluate lipid oxidation. A minimum of two
methods is recommended to determine the initial hydroperoxides formed as
precursors and their aldehydes produced by decomposition. Since it is now
possible to determine key aldehydes that have extremely low theshold values in
the ppb range from n–3 PUFA, there is really no justification for relying on
TBARS to measure lipid oxidation in meat and fish oil products.
H. Cereal products
Cereals are more stable than other foods, because they are low in total fat (2–
5%) and contain relatively high levels of natural tocopherols (20–50 ppm α-, β-
and γ-tocopherols). The lipids in wheat flour become more susceptible to
oxidation because the tocopherol content decreases significantly during stor-
age. Added synthetic antioxidants such as BHA, TBHQ and natural antioxidants
such as rosemary extracts are effective in prolonging the shelf life of dry cereal
products. The presence of natural flavonoid antioxidants in oats and other
cereals is also known to reduce rancidity problems.
Antioxidant products formed during baking by the browning or Maillard
reaction can also stabilize cereals (Section A). In roasted cereal products, the
antioxidant activity varies according to the level of browning reaction products
generated. The extracts of roasted wheat germ and roasted press cake from
wheat germ processing were the most active when tested with stripped (toco-
pherol-free) maize oil oxidized at 50°C (Lipid Oxidation, 2nd ed, Table 11.25,
p. 348). Ground and roasted hazelnut and sweet almond showed comparable
Figure 5.7. Structures of different food antioxidants and inhibitors of lipid oxidation.
antioxidant activity, while a coffee extract had the highest antioxidant activity.
When the antioxidant activity of these extracts, based on inhibition of conju-
gated diene hydroperoxide formation, was compared with their antiradical
activity towards 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH), the results
were not strictly correlated.
Oat phenolic compounds are a mixture of free phenolic acids (9 mg/kg),
soluble phenolic esters (21 mg/kg) and insoluble phenolic acids (58 mg/kg),
phenolic glycosides as well as flavonols and polyphenols, phytic acid, tocols
(15–48 mg/kg of α-tocotrienol and α-tocopherol) and avenanthamides
(N-cinnamoylanthanilate alkaloids) (Figure 5.7). The stability of oat food
products is controlled well by endogenous antioxidants. However, oat becomes
unstable as soon as it is ground or flaked, if it is not treated with steam to
inactivate lipase and lipoxygenase before flaking. Oat flour increased stability
when added to fats and mayonnaise, and when dusted over food products.
Methanol extracts of oats have anti-polymerization effects, on the basis of
lower high-molecular weight polar compounds formed at frying temperature of
180°C. This effect was attributed to a purified fraction of the oat methanol
extract containing Δ5-avenasterol (Figure 5.7).
The antioxidant activity of cereal products and different fractions has been
reported on the basis of a wide variety of in vitro tests, including β-carotene
bleaching, oxidation of LDL, ORAC, oxidative stability of methyl linoleate,
liposome, and vegetable oils at 60°C and under Rancimat and frying tempera-
ture measuring peroxide value, hexanal, TBARS, DPPH, TOSC, ABTS, ORAC,
reducing power, iron chelation, and DNA strand breaking (Table 5.15). With
oat extracts, the total phenolic content significantly correlated with antioxidant
activity based on β-carotene bleaching, LDL oxidation and ORAC. With
uncooked whole grains of corn, wheat, oats and rice, the same correlation was
obtained between total phenol content and antioxidant activity based on the
total oxyradical scavenging capacity (TOSC) assay. With wheat and flour
extracts, no correlation was observed between total phenol content and radical
scavenging capacity for DPPH and ABTS. Whole grains and wheat flours vary
widely in total phenol contents with sorghum having the highest content,
followed by millet, rye, barley, hard and soft wheat (Table 5.16). In contrast to
other studies, cereals show similar correlations between total phenol contents
and antioxidant capacity by the DPPH radical scavenging method and the
ABTS (also known as TEAC) method.
With ethanolic extracts of different wheat germ (WG), hazelnut (H), almond
(A) and coffee (C), the results of radical scavenging effects measured by the
DPPH assay (C > H > WG > A) did not agree with those obtained by acceler-
ated oxidation with stripped maize oil and based on conjugated diene
hydroperoxides at 50°C (C > WG ~ H ~ A). More stable oat cereals products
were obtained by adding antioxidants (benzoin, catechin, chlorogenic acid,
ferulic acid and quercetin) prior to extrusion, which significantly decreased
total phenolic compounds.
Alcohol extracts of oat groat and flour have strong antioxidant activity, but
50% degradation of antioxidant phenolic compounds occurs during extrusion.
Since heat-generated browning reaction products may be even more active as
antioxidants than natural phenolic compounds, thermal processing of cereal
products should be aimed at optimizing the time and temperature profile on the
basis of valid antioxidant evaluations of the finished products. Phytochemicals
provide antioxidant protection against oxidation in extruded cereal foods.
Mixtures of de-germed yellow cornmeal with antioxidant-rich food materials
(ascorbic acid, quercetin, onion powder, potato peelings or wheat bran) showed
improved shelf life after extrusion on the basis of headspace GC analyses of
hexanal and other volatile indicators of oxidation.
It is not surprising that different results for antioxidant activity were obtained
Table 5.15. Antioxidants in cereal products and in vitro methods used to evaluate antioxidant activity
Cereal products Antioxidants Methods Ref.a
Oat fractions Phenolic acids LA/β-carotene, LDL oxidation, ORAC (1)

Roasted wheat germ, nuts, coffee Total phenol by Folin-Ciacalteu CD of stripped maize oil, DPPH (2)
Oats Phenolic acids, tocols, avenanthamides DPPH, ORAC, LDL, MeLo oxidation, (3)
LA/β-carotene, oil stability
Corn, wheat, oats, rice Ferulic acid (free, bound), total flavonoid TOSC (4)
Wheat extracts Total phenol by Folin-Ciacalteu DPPH, ABTS (5)
Extruded oat cereals Benzoinb, catechin, chlorogenic acid, Peroxide value, hexanal (6)
ferulic acid, quercetin
Sorghum flour extract Total phenol by Folin-Ciacalteu DPPH,LA/β-carotene (7)
Barley, pearl millet, rye, sorghum Total phenol by Folin-Ciacalteu DPPH, ABTS (8)
Wheat, rye, buckwheat extracts Hydroxycinnamic acid derivatives TEAC, ABTS, TBARS /liposome, ORAC (9)
FOOD ANTIOXIDANTS
Soft and hard wheats Total phenol by Folin-Ciacalteu TEAC, DPPH, red power, ORAC, Fe chelation,
Rancimat, DNA strand breaking (10)
a
(1) Emmons et al. (1999), (2) Krings and Berger (2001), (3) Peterson (2001), (4) Adom and Liu (2002), (5) Yu et al. (2002), (6) Viscidi et al. (2004), (7) Kamath
et al. (2004), (8) Ragaee et al. (2006), (9) Gallardo et al. (2006), (10) Liyana-Pathirana and Shahidi (2006).
Abbreviations: LA, linoleic acid; CD, conjugated dienes; ORAC, oxygen radical absorbance capacity; TOSC, total oxyradical scavenging capacity; DPPH, 2,2-
diphenyl-1-picrylhydrazyl radical; ABTS, 2,2'-azino-di(3-ethylbenzthiazoline sulfonate); TBARS, thiobarbituric acid reactive compounds.
See structures of flavonoids in Figure 2.6 and Lipid Oxidation, 2nd ed, Figure 9.14, p. 243.
b
Benzoin = 2-hydroxy-1,2-diphenylethanone.
135
Table 5.16. Antioxidant activities and total phenol contents of wheat flours and whole
grain cerealsa
Cereals DPPH ABTS Total phenols

(μ mole/g) (μ mole/g) (GAE, μ g/g)
Soft wheat 4.2 8.3 501

Hard wheat 4.3 8.8 562
Barley 21 15 879
Rye 12 13 1026
Millet 24 21 1387
Sorghum 196 52 4128
a
From Ragaee et al. (2006).
Abbreviations: DPPH, 2,2-diphenyl-1-picrylhydrazyl radical; ABTS, 2,2'-azino-di(3-ethylbenzthiazoline
sulfonate); GAE, gallic acid equivalents.
with many heterogeneous cereal extracts specially prepared with different

properties (Table 5.15). The use of such diverse antioxidant assays based on
widely different protocols, presumed to be relevant to foods or biological
systems, further confounded the results.
I. Special foods, fruits, plant extracts, herbs and spices

The so-called Mediterranean diet has attracted interest because of its recog-
nized health benefits, which may be partly due to the high content of plant
flavonoids, including flavones, flavonols, flavan-3-ols, flavanones, antho-
cyanidins and isoflavones. Herbs and spices are commonly used in traditional
Mediterranean cuisine and contribute to the intake of flavonol, flavone and
phenolic acids.
Used for many years for culinary purposes to improve food flavor and for
medicinal use, spices and herbs are of increasing interest as sources of
antioxidants. Extracts of spices show a wide range of antioxidant activity when
added to foods, vegetable oils and emulsions. However, the published results
are difficult to evaluate, because of a lack of reliable compositional data and of
standardized testing at arbitrary concentrations based on weight (dry or fresh),
rather than on known molar concentrations of active ingredients. The anti-
oxidant activity of various herbs has been of interest in determining their
potential nutritional value. Culinary herbs showed a wide range of antioxidant
activity based on their antioxidant capacity by thee common methods (Table
5.17). There are large differences in total antioxidant capacity in various
culinary herbs. However, the trends in relative antioxidant activity are signifi-
cantly different as measured by FRAP (ferric reducing antioxidant power),
ORAC (oxygen radical absorbance capacity) and DPPH methods. There is,
however, better agreement between the results of the FRAP method and total
phenol contents, because both methods are based on reducing capacity. On this
Table 5.17. Total antioxidant activity of herbs
Commercial herbs Total phenolsa FRAPb ORACc DPPHd
Clove – 465 – –
Allspice – 102 – –
Cinnamon – 98 – –
Sage 23–26 96–104 12 10–12
Rosemary 28–32 67 19 11–14
Thyme 12–14 64 13 4.8–6.1
Marjoram 12e 54 72 –
Oregano 18–24 45 92 7–8
Basil 11–12 31 14 2.1–2.5
Dill 3.1e 16 29 –
Chives 1.1e 7 9.2 –
Caraway 1.1e 4.5 11 –
Parsley 1.1e 4 11 –
Coriander 3.1e 2–3 22 –
Garlic 1.0e 2.1 7.5 –
a
Folin: mg gallic acid equivalent (GAE)/g dry weight (Cosio et al., 2006).
b
FRAP (ferric reducing antioxidant power): mmol/100g (Dragland et al., 2003).
c
ORAC (oxygen radical absorbance capacity): mmol Trolox equivalents/g fresh weight (Zhen and Wang.
2001).
d
DPPH (2,2-diphenyl-1-picrylhydrazyl radical): 1/IC50 mol DPPH/g dry weight (Cosio et al., 2006).
e
mg gallic acid equivalent (GAE)/g fresh weight (Zhen and Wang. 2001).
basis, among the dried culinary herbs listed, clove, allspice, cinnamon, sage,
rosemary and thyme contain relatively high levels of antioxidants.
Food processing may also affect the antioxidant activity of essential oils
containing spices. Essential oils containing clove and thyme protect α-toco-
pherol by heat induced losses. Essential oils also exhibit good antioxidant
properties and could be efficiently used to control lipid oxidation during food
processing. Extracts from basil, cinnamon, clove, nutmeg, oregano and thyme
showed good antioxidant properties when stored at room temperature. These
essential oils also showed good protective activity toward α-tocopherol in
virgin olive oil, after heating.
In summary, plant polyphenols appear to constitute the most important
dietary antioxidants by a multitude of in vitro tests. However, the biological
effects of these compounds are not well established (see Chapter 6). The
bioavailability of these phenolic compounds is presently an area of intense
research. Although significant beneficial effects may be expected because of
their relatively high intake and their antioxidant activities, the evidence is
accumulating that, in vivo, these phenolic compounds have a variety of
biological ‘non-antioxidant’ activities that require further evaluation for their
nutritional effects.
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CHAPTER 6
Antioxidants in biology
In the past few years, the literature on antioxidants in foods and biology has
exploded with accumulating evidence that they may contribute to the known
nutritional benefits of fruits, vegetables and beverages containing antioxidants.
Unfortunately, significant variations in test results have created much confu-
sion in this field (Chapter 5). Phenolic antioxidants in fruits and beverages may
have protective effects against coronary heart disease and other degenerative
diseases, but the mechanism of protection is not well understood. In addition to
their antioxidant radical scavenging activity, many health benefits from dietary
flavonoids and other natural phenolic compounds may be dependent on a
multitude of ‘non-antioxidant’ activities, discussed in Section H. They may
increase the capacity of endogenous antioxidant defenses; they appear to affect
intracellular metabolism by regulating various signaling pathways in cellular
survival, growth and differentiation; they serve as ligands for transcription
(transfer of genetic information) factors and alter protease activity; they may
also control the survival or death of genes and signal transduction (interactions
with cell signaling) pathways. In biology, flavonoids have recently been
described as ‘signaling molecules’, in reference to their cellular neuroprotective,
cardioprotective and chemopreventive properties.
Vitamin E has also been recently considered to have activities beyond being
an essential requirement, depending on the biological context. In addition to its
well-known antioxidant activity, vitamin E can also have prooxidant activity
and non-antioxidant functions. The prooxidant activity of vitamin E is usually
observed under in vitro conditions induced by artificial free radical initiators,
such as AAPH. The non-antioxidant functions of vitamin E include monocyte
and endothelial cell adhesion, platelet adhesion and aggregation, formation of
inflammatory mediators, uptake of oxidized LDL, and cytokine production.
Like flavonoids, vitamin E is also now considered to be a signaling molecule by
regulating gene expression and thus contributing to the prevention of athero-
sclerosis and cancer.
The term antioxidant has now assumed such a broad meaning in biology that
it has lost its traditional chemical definition as discussed in Chapter 2. Accord-
ingly, ‘biological antioxidants’ now include repair systems, such as iron
transport proteins, antioxidant enzymes, factors affecting vascular homeostasis
(vessel equilibrium), signal transduction (transfer of genetic material), and
regulation of gene expression of detoxifying enzymes. Some of the biological
activities attributed to various plant extracts containing flavonoids may in fact
143
Figure 6.1. Oxidant–antioxidant balance hypothesis (from Lipid Oxidation, 2nd ed, Figure 13.1, p.
398). AA, arachidonic acid; SOD, superoxide dismutase; GSH, glutathione; Me, metals; Se, selenium.
have little to do with antioxidant activity. Such activities include anti-allergic,

anti-hemorrhagic, anti-mutagenic, anti-tumor, anti-platelet activities,
immunomodulation, oral hygiene and interactions with specific receptors.
A large number of epidemiological studies have supported the ‘antioxidant
hypothesis’, by showing that the reduced risk of developing cardiovascular
disease and cancer in people consuming a diet rich in fruits and vegetable foods
can be explained by the presence of antioxidant nutrients, such as vitamin E,
vitamin C, β-carotene, selenium and flavonoids. The rationale for this hypoth-
esis is based on the well known effects of antioxidants in preventing the
formation of and scavenging free radicals that disrupt the ‘oxidant–antioxi-
dant’ balance between reactive oxygen species and antioxidants/repair systems,
leading to oxidative stress, which is implicated in the development of tissue
injury, causing cardiovascular disease, cancer, diabetes, and the universal
problems of aging (Figure 6.1). However, a number of intervention trials have
failed to show consistent benefits from the use of antioxidant supplements in
cardiovascular disease and cancer. Too many of these trials were based on the
administration of a single antioxidant at relatively high doses. Many vitamin
nutrients such as vitamin E and vitamin C are known to have prooxidant
activity when tested in vitro at high concentrations. Testing any benefits of
β-carotene is especially problematic, because this generally regarded ‘nutrient’
is a poor antioxidant under normal conditions and usually behaves as a
prooxidant in vitro, unless it is protected synergistically in mixtures with
vitamin E. β-Carotene is however, one of the best known singlet oxygen
quenchers, but whether or not this activity is important in biology is not well
established and controversial. This raises the question whether or not synergistic
mixtures of antioxidant and preventive nutrients may account for the nutritional
ANTIOXIDANTS IN BIOLOGY 145
benefits of fruits and vegetables in the diet. The systematic testing of mixtures
of antioxidants is evidently needed, with improved bioassays to obtain better
evidence for the benefits of phenolic compounds in fruits and vegetables.
A. Biological antioxidant defense systems

A number of extracellular and intracellular antioxidant systems are known to
inactivate reactive oxygen species and oxidants effectively. These systems
include: singlet oxygen quenchers (e.g. lycopene and β-carotene), metal bind-
ers (transferrin, ferritin, albumin), superoxide scavengers (superoxide
dismutases, SOD), enzymatic peroxide destroyers (glutathione peroxidase,
catalase), non-enzymatic peroxide destroyers (ascorbic acid, uric acid,
ubiquinol-10), and radical chain breakers (α-tocopherol). High-density lipo-
protein (HDL) particles can also transport antioxidant enzymes such as
acetylhydrolase and paraoxonase, which can break down oxidized lipids and
neutralize their pro-inflammatory effects. These protection and intracellular
defenses systems are effective as biological networks working synergistically
against a multitude of reactive oxygen and nitrogen species, phagocytes,
arachidonate metabolites, hydroperoxides, aldehydes and metal catalysts.
According to the oxidant–antioxidant balance hypothesis (Figure 6.1), for
optimum health, a balance is required between the oxidizing species and the
antioxidants or repair systems. If there is an imbalance due to excess oxygen
species and insufficient protection from antioxidants or repair systems, the
resulting oxidative stress will cause tissue damage and susceptibility to dis-
eases. Reactive oxygen and nitrogen species produced by several stimuli are
now recognized as mediators of various inflammatory diseases, including
rheumatoid arthritis, diabetes, cancer, cataract formation, immune and brain
dysfunctions, lung diseases and aging.
Flavonoid constituents of plants have been recognized for their nutritional
value, generally attributed to their antioxidant activities. The present knowl-
edge is incomplete, however, on the great diversity of plant phenolic compounds
or phytochemicals and their multiple biological effects. Although there is
accumulating evidence that very small amounts of these plant antioxidants are
absorbed in human blood as metabolites (Lipid Oxidation, 2nd ed, Table 13.11,
p. 439), their molecular mechanisms of protection in vivo and of disease
intervention are not well established. There is inconsistent and conflicting
evidence that supplements of antioxidant vitamins such as vitamins E and C
and flavonoids can reduce the incidence and severity of cancer and degenera-
tive heart disease in humans. Studies of supplementations with individual
flavonoid compounds have thus far shown negative or mixed results. More
reliable markers are needed of in vivo oxidative damage to lipids, proteins and
DNA. It is possible that mixtures of nutrients in fruits and vegetables may
interact to produce more important health benefits than the effect of any
individual flavonoid compound studied separately. This subject is now under

intensive investigation around the world.
B. Antioxidant enzymes
Enzymes that degrade superoxide and hydroperoxides can be included among
important intracellular antioxidants, acting by controlling either the formation
of free radicals and activated oxygen species or inhibiting their reactions with
bioactive nutrients. The main antioxidant enzymes that require cofactors
include superoxide dismutases and catalases, and the selenium-dependent
glutathione peroxidases (GPx) in animals and ascorbate peroxidases (Apx) in
plants. Antioxidant enzymes can be induced, inhibited or activated endo-
genously, and play important functions in aerobic cells. Synergistic interactions
of antioxidants involve a network of sequential degradation of hydroperoxides
and free radicals and mutual protection of enzymes. This network plays an
important role in regulating protein expression and activity at the transcrip-
tional or post-translational levels.
1. Superoxide dismutases (SOD)

These enzymes include Cu/Zn-SOD and Mn-SOD that catalyse the one-
electron dismutation of superoxide into hydrogen peroxide and oxygen (1).
2 O2·– + 2 H+ ——➤ H2O2 + O2 (1)
SODs are present in cells at sufficient levels to detoxify superoxide by
inhibiting the formation of oxygen complexes with transition metals and
release of free iron, of peroxynitrite, and the Fenton reaction producing
hydroxyl or alkoxyl radicals (Lipid Oxidation, 2nd ed, p. 394).
2. Catalase
This porphyrin-containg enzyme decomposes hydrogen peroxide by two-
electron dismutation into oxygen and water (2).
2 H2O2 ——➤ H2O + O2 (2)
Catalase can protect cells when hydrogen peroxide diffuses freely through
membrane, but hydrogen peroxide is mostly degraded by glutathione
peroxidases.
3. Glutathione peroxidases (GPx)

These selenoenzymes catalyse the reduction of hydroperoxides to alcohols (3)
and hydrogen peroxide to water (4) by co-oxidation of glutathione (GSH).
ROOH + 2GSH ——➤ ROH + H2O + GSSH (3)

H2O2 + 2GSH ——➤ 2 H2O + GSSH (4)
Phospholipid hydroperoxides of membrane bilayers are directly reduced by a
monomeric Se-GPx (PHGPx), which is partially bound to membranes.
4. Antioxidant network
Antioxidant enzymes interact in an efficient network that plays an important
role in regenerating reducing cofactors and reinforcing their mutual protection.
For example, the classical synergistic biphasic α-tocopherol–ascorbic acid
mixture occurs by reducing the tocopherol phenoxy radicals (α-TocO·) pro-
duced by polyunsaturated lipid peroxyl radicals (LOO·) with ascorbic acid
(AscH), to regenerate free α-tocopherol (α-TocOH) and ascorbyl radicals
(Asc·) (Figure 6.2). In phospholipids liposome, the polar tocopherol radicals
would become oriented towards the aqueous phase of the bilayer interface and
available for reduction by ascorbic acid and other aqueous reducing compounds.
The regeneration of tocopherol in phospholipids membranes is also coupled
to the oxidation of GSH in water. The tocopherol radicals are efficiently
reduced by ascorbate at the water–phospholipids membrane interface. This
mechanism for tocopherol regeneration, also referred to as free radical translo-
cation, slows down the consumption of α-tocopherol by using GSH for redox
recycling of ascorbate. The resulting glutathione disulfide (GSSG) is similarly
reduced to glutathione (GSH) at the expense of the reduced form of nicotina-
mide adenine dinucleotide phosphate (NADPH) by glutathione reductase (7).
GSSG + NADPH + H+ ——➤ 2 GSH + NADP+ (7)
Part of the GSH produced by these protective mechanisms reacts with SH
groups of proteins (PSH) to produce mixed protein disulfides (PSSG). The
resulting PSSG undergoes a thiol-disulfide exchange to regenerate PSH with a
GSH-containing reductase.
Gene transcription can also be regulated by redox-sensitive systems by GPx,
PHGPx and the thio-redox system. The antioxidant enzymes may prevent the
oxidative inactivation of phosphatases involved in dephosphorylation. They
can also down-regulate 5-lipoxygenase activity in leucocytes, and SOD activ-
ity increases the stability of nitric oxide. These complex enzymatic antioxidant
protection systems clearly have broad implications in biology which need
further research.
Figure 6.2. Tocopherol–ascorbate redox cycle.

C. Inhibition of LDL oxidation and coronary heart disease by

antioxidants
One hypothesis for the initiation of LDL oxidation is that is is the result of a
local tissue deficiency of antioxidants. The term oxidized LDL may be confus-
ing because native plasma LDL particles are complex, highly heterogeneous
mixtures of lipid components, consisting of multiple subpopulations varying in
degrees of oxidation and contents of antioxidants, including minor amounts of
tocopherols, carotenoids and ubiquinol.
Antioxidants such as butylated hydroxytoluene (BHT) and vitamin E can
inhibit atherosclerosis in some experimental animal models (rabbits, monkeys
and hamsters) that can be exposed to nutritional distress. In humans, epidemio-
logical studies demonstrated that high intake of vitamin E and the resulting
high blood levels of vitamin E correlate with reduced coronary disease.
Flavonoids can provide benefits indirectly by helping to prevent diseases by
protecting from the damage that they inflict. They can also protect biomolecules
(lipids, proteins, DNA) from free radicals. On one hand, essential vitamins E
and C are not effective when tested for their inflammatory effects. On the other
hand, non-essential plant nutrients such as flavonoids may protect against heart
disease, but the mechanism for such protection is not well established. There is
currently growing evidence to support the potential anti-atherogenic effects of
antioxidants as preventive measures for slowing atherosclerosis by inhibiting
LDL oxidation in vivo. Although LDL is well protected against oxidation in
blood plasma by an adequate supply of endogenous antioxidants and metal-
binding proteins, this protection may not be adequate in the arterial wall, where
the oxidative modification of LDL is induced by endothelial cells.
Another hypothesis is that endogenous antioxidants may be depleted within
the arterial sub-endothelial space where oxidation takes place. The oxidative
modification of LDL by endothelial cells can be completely inhibited in the
presence of sufficient vitamin E or other antioxidants such as BHT. In addition
to the lipid-soluble antioxidants associated with LDL (α- and γ-tocopherol, α-
and β-carotene, lycopene, ubiquinol-10), human plasma contains antioxidant
proteins and enzymes and water-soluble enzymatic and non-enzymatic per-
oxide destroyers, including glutathione. The non-enzymatic oxidation inhibitors
(metal-binding proteins, uric acid and ascorbic acid) are more important in
extracellular fluids than the enzymatic antioxidants (superoxide dismutase and
glutathione peroxidase). Plasma proteins containing thiol groups are also
active inhibitors through trapping aqueous peroxyl radicals in plasma. Vitamin
E is one of the most abundant lipid-soluble antioxidants in plasma and LDL that
may protect the lipids of LDL particles.
In vitro studies show that antioxidants react differently in LDL, and their
effectiveness varies greatly according to their concentration and the types of
oxidant used. When added to isolated LDL, vitamin C inhibits oxidation
induced by endothelial cells and macrophages. Vitamin C may inhibit LDL

oxidation by scavenging water-soluble peroxyl radicals and by regenerating
α-tocopherol present in LDL. Vitamin E added in vitro inhibits LDL oxida-
tion by endothelial cells and macrophages more effectively than oxidation
catalysed by copper. Supplementation in vivo with hydrophilic antioxidants
such as vitamin C and flavonoids cannot be tested with isolated LDL, be-
cause they are removed from LDL when separated from plasma. Flavonoids
may inhibit the in vitro oxidation of LDL by a multiplicity of mechanisms,
including scavenging reactive oxygen species (superoxide, oxidized lipids,
oxysterols), binding iron or copper catalysts, and protecting α-tocopherol
present in LDL. Flavonoids are also known to inhibit lipoxygenase and
cyclooxygenase enzymes that play key roles in eicosanoid synthesis. The
evidence supports the possible use of antioxidants for reducing the suscepti-
bility of LDL to oxidation, but the precise mechanism of how atherosclerosis
is initiated in vivo needs clarification before antioxidants can be used as a
mode of intervention to prevent this disease.
The current approach for evaluating the oxidative susceptibility of LDL,
prepared from subjects before and after consumption of diets containing
antioxidants, is limited by the methods used to separate lipoproteins. This
methodology removes water-soluble plasma constituents that include phenolic
acids and polyphenols. Methods for evaluating antioxidant capacity using
artificial free radical initiators and non-biological targets (see Chapter 4) in
blood plasma are also non-specific and carried out under non-physiological
conditions, because they do not reflect the true in vivo situation. These ex vivo
methods used with isolated blood plasma are not physiologically relevant
because they cannot replicate initiation of LDL oxidation occurring in the
subendothelial space. As discussed in Section G, a very small proportion (in
μmolar levels) of flavonoid compounds are absorbed in blood, and any
colorimetric measure of antioxidant capacity becomes questionable. Further-
more, as discussed in Section H, flavonoids are now known to have a multitude
of biological activities, in addition to antioxidant protection and metal scav-
enging, that do not involve free radical scavenging activities. These
‘non-antioxidant’ properties include cell signaling, platelet aggregation, va-
sodilation and macrophage biology.
Polyphenols affect cell-signaling mechanisms by modulating the proper-
ties of endothelial cells, platelets, macrophages, intestinal cells and smooth
muscle cells. Unfortunately, in vitro studies with cells do not reflect the
conditions in vivo and the levels that are likely to be achieved in diets
containing flavonoids. Although in many in vitro studies flavonoids inhib-
ited LDL oxidation stimulated by macrophages, it is difficult to separate
their effect on macrophages directly from those on LDL. However, it is now
generally accepted that polyphenolic compounds in fruits and vegetables, red
wine and green tea have, in addition to their antioxidant activities, a multi-
tude of cellular regulation of vascular functions including modulation of

platelet aggregation, macrophage functions and endothelial cell functions
that affect cardiovascular disease.
Although the administration of antioxidants in the diets of several experi-
mental animals can inhibit LDL oxidation ex vivo and retard the progression of
atherosclerosis, a large number of human clinical trials have shown mixed
results. Many confusing factors in these human trials can be attributed to the
lack of reliable quantitative in vivo specific biomarkers of lipid oxidation and
antioxidant activity, poor selection of human subjects, invalid and arbitrary
bases for antioxidant dose selection.
D. In vitro versus in vivo studies

Many studies have shown the beneficial effects of antioxidants in inhibiting
atherosclerosis in animals. Clinical trials with human subjects demonstrated
that dietary supplementation with vitamin E produced significant enrichment
of this vitamin in LDL and ex vivo protection of LDL against oxidation induced
by copper ions, endothelial cells and smoking. A large number of studies
showed that natural phenolic antioxidants reduce the oxidative modification of
LDL in vitro, and a few animal studies showed that they could inhibit
experimental atherosclerosis induced under oxidative stress.
Phenolic compounds may also have biological effects independent of their
inhibition of LDL, depending on the test system and conditions. The effect of
antioxidants on the ex vivo oxidizability of LDL by free metals cannot be
assumed to be causally related to in vivo susceptibility to atherosclerosis and
oxidative damage. Studies with animal models of atherosclerosis have demon-
strated the antioxidant effect of vitamin E and phenolic compounds in delaying
the progress of this disease. However, human clinical trials of antioxidants
have given inconsistent and mixed results. Although the accumulation of
circulating oxidized LDL in the artery and the development of lesions within a
few months has been demonstrated in various animal models on special diets,
human lesions develop much more slowly over periods of decades. Therefore,
the results from animal testing of antioxidants do not agree with those from
human studies because they are based on significantly different end points. The
same divergence may apply to studies comparing in vitro versus in vivo testing
of antioxidants being based on different end points of oxidative damage.
Flavonoid antioxidants from fruits and vegetables may be important in
regulating eicosanoids through their ability to inhibit phospholipase,
cyclooxygenase and lipoxygenase, which play a key role in the arachidonate
cascade (Lipid Oxidation, 2nd ed, p. 425). By down-regulating cyclooxygenase
and lipoxygenase, flavonoids and other phenolic compounds in fruits and
vegetables may reduce thrombotic tendencies and inflammatory reactions in
the body. By preventing or reducing the collecting of monocytes in vessel
Table 6.1. In vitro versus in vivo tests effects of antioxidants
Test In vitro In vivo Ref.a
Platelet aggregation Quercetin required 250–2500 Quercetin containing (1)

LDL oxidation μmol/l foods produced plasma
IC50: 2–20 μmol/l concentration not
higher than 1 μmol/l
LDL oxidation Vit C and flavonoids: effective Vit C and flavonoids have (2)
with cells, macrophages, Cu, no effect. Vit E improved
AAPH. Vit E more effective LDL resistance to oxidation
with cells, macrophages than with cells and copper;
Cu, prooxidant with AAPH prooxidant with AAPH
LDL oxidation Coffee, cocoa, tea, cocoa Human consumption of (3–5)
powder inhibit LDL oxidation flavanol-rich cocoa product
inhibited LDL oxidation
Tumor size and Inhibition of urokinase by Drinking 9 cups of green (6)
cancer in mice green tea tea produced only
EGCG required > 1mmol/l. ~ 1 μmol/l EGCG in plasma.
a
References: (1) De Whalley et al. (1990), (2) Frei (1995), (3) Waterhouse et al. (1996), (4) Kondo et al.
(1996), (5) Richelle et al. (1999), (6) Jankun et al. (1997)
Abbreviations: IC50, concentration required for 50% inhibition; AAPH, 2,2'-azobis(2-amidinopropane)
dihydrochloride; EGCG, epigallocatechin gallate.
walls, these phenolic compounds interfere with the immune response and with
platelet aggregation causing blood clotting.
Although flavonoids may have broad metabolic and physiological effects,
little is known about their in vivo antioxidant activity in humans. The great
difficulty of conducting in vivo experimental work to demonstrate the activity
of plant antioxidants has added to the uncertainty of their nutritional benefits.
Many studies are currently aimed at clarifying the bioavailability of plant
flavonoids from different foods (see Section G). How the food matrix and
various flavonoid–nutrient interactions affect absorption and metabolism of
flavonoid antioxidants and the mechanisms by which they affect human health
and disease is largely unknown.
In vitro studies generally employed concentrations of phenols that far exceed
the in vivo concentrations that might be reached in the body (Table 6.1, ref 1).
Thus, in vitro studies showed that much higher concentrations of quercetin are
required to inhibit platelet aggregation (250 to 2500 μmol/l) and LDL oxida-
tion (I50 of 2–20 μmol/l) than can be achieved with quercetin-containing foods
that in most cases never exceed 1 μmol/l. Therefore, dietary quercetin will not
be expected to affect platelet aggregation in vivo, because effective concentra-
tions would never be reached in plasma. Furthermore, quercetin is metabolized
in plasma as methoxy, glucuronide and sulfate derivatives (Figure 6.3) that
have significantly lower or no antioxidant activity than the parent quercetin
(Section G.3.a).
Figure 6.3. Metabolites of quercetin. By blocking phenolic hydroxyl groups, these metabolites have
diminished or lost antioxidant activity.
On one hand, in vitro studies also showed that vitamin C and flavonoids
were effective inhibitors of LDL oxidation induced with cells, macrophages,
copper and AAPH. On the other hand, vitamin E was more effective with
cells and macrophages than with copper, but it was a prooxidant with AAPH.
Corresponding ex vivo studies showed that the hydrophilic vitamin C and
flavonoids have no effect, because they partitioned into the aqueous phase
and were removed during the preparation of LDL. In contrast, vitamin E
supplementation improved LDL resistance to oxidation with cells and cop-
per, but was prooxidant with AAPH. These results emphasize the problems of
using the popular but questionable and misleading artificial azo dye AAPH to
induce LDL oxidation in vitro (Table 6.1, ref 2). Moreover, consumption of
flavonol-rich cocoa products showed effective inhibition of LDL oxidation
(Table 6.1, ref 3–5).
Inhibition of urokinase (a transphosphorylation enzyme) was shown to
decrease tumor size and cause remission of cancer in mice. Inhibition of this
enzyme in vitro by green tea epigallocatechin gallate (EGCG) required concen-
trations greater than 1 mmol/l. This concentration is too high a level to be
achieved in plasma because drinking 9 cups of green tea produced only about
1 μmol/l of EGCG. Therefore, the effect of tea catechins will not be expected
to occur in vivo, because an effective inhibitory concentration will not be
reached in plasma (Table 6.1, ref 6).
Table 6.2. Metabolites of quercetin and catechina–e
Phenolics Glycosides Phenolic acids
Quercetin Methoxy 3,4-OH benzoic acid

Monogluronides 3,4-diOH benzoic acid
Diglucuronides 3,4-diOH phenylacetic acid
Methylmonoglucuronides 3-OH phenylpropionic acid
Methyldiglucuronides 4-OH-3-methoxyphenyl-
propionic acid
Glucosedisulfate Ferulic acid, isoferulic acid
Sulfate OH/diOH ferulic acid
Hippuric acid, OH hippuric acid
Vanillic acid
Catechin Methoxy Phenylacetic acids
Sulfate Phenylpropionic acids
Glucuronides
Sulfoglucuronides
Epicatechin Non-conjugated –
Sulfate
Glucuronide
Epigallocatechin Non-conjugated –
Sulfate
Glucuronide
Epigallocatechin gallate Non-conjugated –
Tea catechin gallates Sulfate –
Glucuronide
a
Rice-Evans (2001), Spencer et al. (2001), Rechner et al. (2001, 2002), Kroon et al. (2004).
b
Hollman and Arts (2000), Hollman (2001).
c
Donovan et al. (1999).
d
Lee et al. (1995).
e
Hong and Mitchell (2004).
Some 21 quercetin metabolites and a complex mixture of phenolic acids

were identified in urine from human subjects consuming cooked onions (Table
6.2). Phenolic acids can be formed by oxidation of flavonoids mediated by
peroxidases. Incorporation of oxygen into the flavonoid C-ring followed by
enzymatic degradation of the intermediates leads to ring fission reactions and
the formation of complex mixtures, including mono- and dihydroxy benzoic
acids, phenylacetic and phenylpropionic acids. Further enzymatic degradation
of quercetin results in oligo- and polymerization reactions. Two isomers of
sulfate quercetin glucosides found in urine suggest that many quercetin
glucosides in onions are absorbed intact and metabolized into sulfate deriva-
tives. These quercetin glucoside metabolites could therefore be used as
biomarkers for the consumption of quercetin-containing fruits and vegetables.
Studies supplementing human diets high in flavonoid foods or with isolated
flavonoids correlated positively with urinary or plasma levels of specific
Figure 6.4. Postprandial oxidative stress. Increased susceptibility of organism to oxidative damage
after consumption of a meal rich in lipid and/or carbohydrates (adapted from Sies et al., 2005).
flavonoids, after hydrolysis. Subjects consuming cooked onions excreted

mainly diglucuronides, monoglucoside sulfate, monoglucuronides, and methyl
monoglucuronide conjugate of quercetin (Figure 6.4).
In contrast to quercetin, the metabolites from catechin, epicatechin and
galloyl tea catechins were less complex and included varying amounts of the
free aglycones. Much less information is available on the low molecular-
weight phenolic acids excreted from catechins and their gallic acid derivatives
in green tea than from those quercetin, which is more readily broken down by
colonic microflora. Short-term studies with animal models showed by various
biomarkers that catechins in green tea provide protection against various forms
of cancer and other degenerative diseases. Some biomarkers were not affected,
however, and long-term feeding studies are needed.
The bioavailability of flavonoids varies widely among the diverse dietary
polyphenols that produce complex mixtures of metabolites in different tissues.
Recent dietary studies with human have shown that plasma and tissues are
generally exposed to conjugated forms of flavonoids and seldom to the
aglycone forms, except for green tea catechins, which are already conjugated as
gallic acid derivatives. The metabolites formed from quercetin and catechins,
including glucuronides, sulfate, and methylated forms of their functional
groups (Figure 6.3), are expected to be differently distributed in tissues and
have different biological activities from those of their corresponding aglycones.

Although these metabolites can be enzymatically deconjugated in certain
tissues, for a better understanding of the nutritional benefits of flavonoids,
much more information is needed on their bioavailability and bioactivity of
different metabolites as they are distributed in different sites of the body (see
Section G.3).
E. Postprandial oxidative stress

When consuming a diet rich in triglycerides, a significant part of the day is
spent in the postprandial state (after a big meal) that lowers clearance rates of
chylomicron remnants (Lipid Oxidation, 2nd ed, Figure 13.2, p. 401) and
prolongs a hyperlipidemic state, causing vascular injury that initiates athero-
sclerosis. For these reasons, postprandial hyperlipemia (or hypertriglyceridemia)
is now considered to be a risk factor enhancing oxidative stress and susceptibil-
ity to atherosclerosis. Endothelial dysfunction observed after consuming a
high-fat meal causes oxidative stress by depleting antioxidant enzymes
(glutathione peroxidase) and increasing excretion of oxidation products.
Flavonoids in the diet can therefore offer direct antioxidant and other beneficial
functions within the gastrointestinal (GI) tract, before absorption into plasma.
Incomplete absorption of phenolic compounds in the diet results in higher
concentrations in the GI tract that may have a beneficial effect by binding
prooxidant metals, scavenging reactive oxygen species, and inhibiting oxyge-
nase enzymes. Foods rich in flavonoids can protect against the prooxidant
effects of gastric conditions by scavenging reactive oxygen species from the
diet produced by mixtures of iron and ascorbate, prooxidant heme proteins, and
by inhibiting lipid oxidation and decomposition products and the resulting
cytotoxic aldehydes.
The postprandial oxidative stress occurring after the consumption of a
meal rich in lipids and/or carbohydrates increases the susceptibility of the
organism to oxidative damage, endothelial dysfunction, atherosclerosis, and/
or diabetes (Figure 6.4). In hypertriglyceridemic and hyperglycemic subjects,
endothelial dysfunction occurring in the postprandial state contributes to
oxidative stress that modulates cardiovascular risk for atherosclerosis,
diabetes and obesity. The postprandial increase of oxidized and oxidizable
polyunsaturated lipids in the diet can further increase plasma levels of lipid
hydroperoxides and the susceptibility of LDL to oxidation. The postprandial
modification of LDL increases the negatively charged subfraction LDL–, an
oxidatively modified form of LDL which contains lipid oxidation products
and denatured apoprotein B-100. The resulting apoB becomes unfolded and
becomes partitioned inside the core of LDL particles and contributes to
increased susceptibility to oxidation.
Consuming a meal rich in polyphenols from red wine, grape seeds,
proanthocyanidin-rich grape seed extracts, green tea, and cocoa minimizes

postprandial oxidative stress. Red wine included with a meal prevents the
postprandial increase of plasma hydroperoxides and increases the resistance of
LDL to oxidation by increasing the antioxidant levels in plasma. Procyanidins in
red wine and grapes are rich sources of antioxidants, which prevent the
postprandial increase in oxidants in plasma by inhibiting lipid oxidation of foods
when absorbed in the digestive tract. The inhibition of lipid oxidation in meals
may thus provide a mechanism which explains the beneficial effects of anti-
oxidants in decreasing postprandial oxidative stress. Dietary polyphenols protect
endothelial functions not only by acting as antioxidants, but also by modulating
signaling molecules, a non-antioxidant activity discussed in Section H.
Dietary flavonoids from fruits, vegetables and beverages can provide more
direct antioxidant protection in the GI tract, where they are present in the
stomach and the intestine lumen at much higher concentration before absorp-
tion. After absorption into plasma, flavonoids are rapidly metabolized and
reach levels below 1 μmol/l. Flavonoids can therefore protect the stomach by
scavenging reactive species derived from the diet or from activated phagocytes
by ascorbate in the presence of iron, hydroperoxides and aldehydes from
polyunsaturated lipids exposed to gastric acids and other toxins. Dietary
flavonoids can also chelate and effectively inhibit the prooxidant effects of iron
in the colon, and may provide protection against postprandial oxidative stress
after consumption of a meal rich in lipid and/or carbohydrates (Figure 6.4).
Polyphenols may thus decrease the susceptibility of LDL to oxidation resulting
from oxidized fats and sugars. Such antioxidant protection would be particu-
larly important to hyperlipidemic and hyperglycemic subjects.
Red wine and grape polyphenols restrict the postprandial increase of lipid
hydroperoxides and the susceptibility to LDL oxidation when they are con-
sumed together with a meal containing oxidized and oxidizable lipids. When
red wine was consumed after a high-fat meal, plasma oxidative stress was
significantly decreased without changing postprandial lipemia. After a fatty
meal was taken with red wine, plasma concentrations of vitamin E, sulfhydryl
groups, uric acid and total plasma antioxidant capacity were increased, but the
total ascorbic acid decreased (Lipid Oxidation, 2nd ed, Table 13.13, p. 442).
Postprandial LDL obtained after the wine-meal was more resistant to oxidation
than after a control ethanol-meal. Red wine also increased the resistance of
postprandial LDL to oxidation and protected vitamin E but not vitamin C.
Similarly dietary flavanols in cocoa drinks lowered plasma levels of F2-
isoprostanes (Lipid Oxidation, 2nd ed, pp. 41–43, 416, 418), which are
regarded as good indicators of in vivo products of lipid oxidation and more
specifically from the oxidation of arachidonic acid. Wine polyphenolic com-
pounds and other dietary antioxidants may therefore attenuate the risk of
coronary heart disease by reducing the harmful prooxidant cytotoxic effects of
hydroperoxides in fat-rich foods.
In the postprandial phase, dietary polyphenols may thus further support the
oxidative hypothesis of atherosclerosis in tipping the ‘oxidant–antioxidant’
balance (Figure 6.1) to the antioxidant side during the postprandial phase.
Dietary antioxidants in wine, tea and cocoa can favorably affect vascular
response by reversing endothelial dysfunction and reducing the susceptibility
of LDL oxidation. A mixture of polyphenolic compounds from plants may thus
be effective in protection against the oxidative effects in the postprandial state
when consumed with diets high in fats and sugars. A major form of antioxidant
defense in human plasma is the prevention of iron and other transition metal
ions from promoting the generation of reactive oxygen species. Iron deficiency
was even advocated as a more effective and practical antioxidant treatment
than supplementation with either synthetic or natural antioxidants.
Whether or not there is a link between obesity and oxidative stress due to
hyperlipidemia/hyperglycemia is an important question that remains to be
researched. Since high levels of lipids and LDL are associated with coronary
artery disease, more research is needed to elucidate the multiple factors
contributing to the susceptibility of some individuals to inflammation and
vascular disease.
Upon ingestion, dietary flavonoids can undergo complex biological interac-
tions that may obscure any systemic ‘antioxidant’ effects. Misleading and
confusing results for biological antioxidant activity can be obtained if biomarkers
and products of lipid peroxidation are either removed by various repair systems
or metabolized. Furthermore, if flavonoids provide protection within the GI tract
before they are absorbed, their effects in the diet may be due to protection against
cancer caused by gastric oxidation in the stomach and colon. The GI tract is
considered a rich source of reactive oxygen and nitrogen species, prooxidant
mixtures of iron and ascorbate, leading to lipid peroxidation and formation of
cytotoxic aldehydes. Flavonoids can therefore scavenge these reactive species,
chelate prooxidant transition metals and their strong reducing properties may
alleviate the damaging effect of iron in the colon. However, one cannot overlook
the benefits of fruits and vegetables in the diet that can also be derived from other
phytochemicals, which may either add to or reinforce the effects of flavonoids.
F. Prooxidant chemistry of phenolic antioxidants

Although α-tocopherol is generally considered as an important biological
lipid-soluble antioxidant present in human LDL, it has been shown to have both
anti- and prooxidant properties towards LDL oxidation. As a prooxidant,
α-tocopherol accelerates LDL oxidation under in vitro conditions in the
presence of a variety of radical promoters, such as artificial azo compounds
(RN=NR), transition metals or cells containing transition metals. This prooxidant
activity of α-tocopherol (α-tocOH) is attributed to the formation of α-tocophe-
rol phenoxy radicals (α-tocO·), by reacting either with lipid peroxyl radicals
produced by oxidation of polyunsaturated lipids, or in the presence of a radical

oxidant or Cu2+ (6), (7) (Thomas and Stocker, 2000).
L· + O2 ——
➤ LOO· + α-tocOH ——
➤ α-tocO·+ LH ——
➤ α-tocOH + L· (6)
α-tocOH + Cu2+/radical oxidant ——➤ α-tocO· + LH ——

➤ α-tocOH + L· (7)
According to this ‘tocopherol-mediated peroxidation’ mechanism, the α-

tocopherol phenoxy radical acts as a chain-transfer agent from the aqueous
phase into the LDL lipid phase to produce lipid hydroperoxides. This mecha-
nism assumes that LDL particles contain a lipid core in which tocopherol
radicals remain long enough to be separated from the peroxyl radicals in the
aqueous phase. However, this mechanism is based on in vitro experiments
using either artificial sources of diazo radical initiators, or relatively high Cu2+
concentrations that cannot be considered physiologically relevant. Whether or
not α-tocopherol can have prooxidant effects in vivo, in the presence of co-
antioxidants such as ascorbic acid (see Section B.4), remains to be established.
More direct evidence is required on the types of oxidants that actually initiate
atherosclerosis in the body in vivo.
Peroxidation reactions (6) and (7) are considered to be applicable to lipid
dispersions, and can be inhibited when mixed with co-antioxidants such as
ascorbic acid and ubiquinol-10 to regenerate α-tocopherol by an antioxidant
network analogous to that described in Section B.4. However, by increasing the
levels of α-tocopherol under conditions of oxidative stress, the levels of
α-tocopherol radicals may increase to the point where they can no longer be
scavenged by co-antioxidants. Tumor promotion by vitamin E has also been
attributed to increased formation of α-tocopherol radicals.
Flavonoids and other strong reducing agents such as ascorbic acid in the diet
can promote oxidative damage in the presence of transition metals, which
could be liberated from metalloproteins after an injury or under pathological
conditions during a degenerative disease. Under these conditions in which
transition metals are no longer bound efficiently, flavonoid compounds could
promote oxidative damage by reducing any free metals into the very active low-
valence state and catalyse free radical oxidations. Therefore, flavonoids would
be beneficial as preventive agents only before oxidative damage is initiated, but
once this damage reaches more advanced stages of oxidation, they may
aggravate and promote further damage. The same analogy can be made with
oxidizable food lipids. Although antioxidants have beneficial activity by
preventing lipid oxidation before it occurs in fresh foods, they could promote
oxidation with food lipids already oxidized, especially in the presence of
contaminating metals, which accelerate the decomposition of antioxidants.
Flavonoids can also exert prooxidant chemistry in the presence of glutathione
(GSH) redox cycling and peroxidase activities, in promoting lipid oxidation. The
proposed mechanism proceeds by oxidation with the formation of semiquinone
radicals and oxidized quinone intermediates from flavonoid containing the

pyrogallol group (Section G.3). After reacting the electrophilic flavonoid
quinone with GSH, and converting it to the corresponding thiyl (GS·) radical, a
disulfide radical anion is generated that rapidly reduces oxygen to superoxide
anion radicals. Whether or not toxic health effects can be deduced from this
prooxidant chemistry determined under in vitro conditions is a matter of
conjecture. Although the potential toxic effects of these reaction intermediates
are generally unknown, the question remains about dose response effects to attain
beneficial versus toxic effects of phytochemicals in functional food ingredients.
There is more evidence that, at higher doses and under certain conditions,
other sources besides vitamin E, including vitamin C, carotenoids and flavonoids
used as ingredients in functional foods, may exhibit prooxidant activities of
possible toxicological concern. High levels of vitamin C in the presence of Fe3+,
producing catalytically active Fe2+, is a well known potent oxidant for the
production of reactive oxygen species, including superoxide anions and hy-
droxyl radicals. The prooxidant activity of vitamin C is also responsible for its
apoptosis-inducing activity (cell death), and is inhibited by catalase, N-
acetylcysteine and glutathione, but promoted by H2O2, Cu2+ and iron chelators.
Vitamin C has been found to be ineffective in cancer chemoprevention because
of its prooxidant activities, perhaps because free iron is generated in this
disease. If this hypothesis is correct, the use of a non-reducing iron chelator
may be more effective than either vitamin C or flavonoids.
For medicinal applications, it is important to appreciate that weak antioxi-
dants can become prooxidant under certain conditions. Methyl gallate, for
example, becomes prooxidant at pH 7.4 in the presence of Fe2+ at low levels and
can cause oxidative damage to deoxyribose. Other compounds containing the
pyrogallol group can promote the formation of superoxide anion with prooxidant
activity. However, when the methyl gallate moiety is part of the larger structure
of tannins, then it becomes active as an antioxidant. In general, polymeric
polyphenols show no prooxidant activity. Other factors become important in
polyphenols such as complex formation with proteins that become indigestible,
retention in the gastrointestinal tract and transport to different tissues.
Many health claims for phenolic compounds have been made from epide-
miological studies using fruits and vegetables, as well as virgin olive oil.
However, the many attempts made to determine the actual beneficial ingredi-
ents in these diets have been largely unsuccessful. The possible toxic prooxidant
effects observed for these ingredients should temper many of the health claims
made for the vitamins and flavonoid contents of functional foods. The high
concentrations of phenolic ingredients that may be taken in ‘nutritional’
supplements may pose toxicological concerns, because they may be aggra-
vated under conditions of oxidative stress in pathological conditions such as
degenerative disease or aging, in which metal binding proteins become less
effective and generate damaging free iron.
G. Bioavailability, absorption and pharmacokinetic studies

In pharmacology, the term bioavailability deals with how much a preparation
ingested orally reaches circulation over time (or a compound measured as area
under the curve, AUC). This amount represents the proportion of the com-
pound that is absorbed from the gastrointestinal tract. In nutrition, this concept
assumes a broader meaning to include compounds that are absorbed, distrib-
uted in tissues, and become bioactive depending on the relative degrees of
metabolism and excretion. Lipophilic compounds are generally not directly
excreted in urine, and appear as water-soluble or hydrophilic metabolites. The
time course of excretion is much longer for highly lipophilic compounds than
for hydrophilic compounds. This time course therefore depends on their
metabolism to more hydrophilic conjugates excreted through urine or bile.
Many compounds that have antioxidant activity and reducing properties in
vitro may not be necessarily absorbed to protect in the body, but can limit
oxidative damage from reactive oxygen species in the GI tract, protect intesti-
nal epithelial cells against oxidation, and reduce oxidative damage of DNA.
The nutritional and antioxidant value of food components will therefore not
only depend on their relative concentrations, but also on their stability in the
food matrix, the conditions of processing, storage and cooking. The eventual
health effects of foods will thus depend on how much will be absorbed,
distributed in different tissues and utilized in the body.
1. Tocopherols
All the tocopherol homologs (α, β, γ and δ) are apparently absorbed in the GI
tract. While α-tocopherol is specifically incorporated into plasma proteins by
the α-tocopherol transfer protein (TTP), some of the other tocopherols end up
in the bile and may have a beneficial effect in the GI tract. γ-Tocopherol was
superior to α-tocopherol in scavenging reactive oxygen and nitrogen species.
More recently, α-tocopherol was shown to have many other properties in
addition to its long established antioxidant activity. α-Tocopherol can also
have prooxidant as well as non-antioxidant activities. Because α-tocopherol
has prooxidant effects, its protective role in preventing LDL oxidation has been
questioned. However, these prooxidant effects have been observed under
rather artificial in vitro conditions in the presence of azo initiators and rela-
tively high concentrations of metals, which are not physiologically relevant
(see Section E).
In enterocytes (intestinal cells), all forms of free tocopherols are incorpo-
rated into chylomicrons and transported into blood circulation in the same way
as the food ingested. In hepatocytes (liver cells), the very low-density
lipoproteins (VLDL) are enriched with α-tocopherol by TTP (Lipid Oxidation,
2nd ed, Figure 13.2, p. 401), and carried to peripheral tissues together with
triglycerides and cholesterol. The simultaneous intake of fat stimulates bile

flow and secretion of lipase to allow micelle formation, which is affected by
various food components including fiber content. The absorption and distribu-
tion of vitamin E may also be affected by the level of oxidative stress in an
individual and may depend on the other available nutrients such as vitamin C
and flavonoids.
Vitamin E deficiency studies with rats showed a much larger synergistic
effect when accompanied by selenium deficiency on differential liver gene
expression. Tocopherols also regulate several genes implicated in glutathione-
S-transferase that protects cells against lipid hydroperoxides, with lipid uptake
and atherosclerosis, modulation of extracellular proteins, adhesion and inflam-
mation, and cell signaling and regulation. By lowering the uptake of oxidized
lipoproteins and decreasing foam cell formation, vitamin E plays a protecting
role against atherosclerosis. In animals on vitamin E deficient diets, α-tocophe-
rol increases the expression of the scavenger receptor activity associated with
inhibition of plaque formation.
More recent studies suggest that the simple well-known antioxidant function
of α-tocopheol against free radical damage cannot explain several other
biological effects of this vitamin. There is evidence that α-tocopheol may
protect against LDL oxidation by other mechanisms involving inhibition of
protein kinase C (PKC), which triggers the release of reactive oxygen species
that promote lipid oxidation. α-Tocopherol has important non-antioxidant
functions in regulating many cellular effects, by preventing the accumulation
of macrophages in circulating LDL to form atherosclerotic plaques by down-
regulating the oxidized LDL scavenger receptors, by inhibiting adhesion to
human monocyte-endothelial cells, platelet adhesion and aggregation.
α-Tocopherol inhibits PKC activity by causing dephosphorylation by acti-
vating a protein phosphatase, and by increasing collagenase in human skin
fibroblasts involved in aging. α-Tocopherol also regulates vascular homeostasis
(balance of physiological functions by chemical composition) by mediating
nitrous oxide production required for normal vascular function.
γ-Tocopherol is apparently more effective than α-tocopherol in decreasing
platelet aggregation, and scavenging peroxynitrites. There is increasing in vitro
evidence for the potential beneficial effect of γ-tocopherol in the attenuation of
atherosclerosis, and for its anti-cancer effects in inhibiting human cancer cell
progression and proliferation. Serum γ-tocopherol concentrations are signifi-
cantly lower in coronary heart disease patients than in healthy control patients.
γ-Tocopherol has a unique function in scavenging reactive nitrogen species,
and is more potent than α-tocopherol in inhibiting cyclooxygenase in cell
systems. γ-Tocopherol is also considered to be involved in the management of
inflammatory and cardiovascular diseases, Generally, most intervention hu-
man trials investigating the effect of vitamin E supplements using α-tocopherol
may lead to a decrease in γ-tocopherol in plasma. Since γ-tocopherol is the most
abundant homolog in plant seeds that we consume as vegetable oils, supple-

ments of mixed tocopherols may be most desirable to contribute to the body’s
antioxidant defenses. There is recent evidence that supplements of mixed γ-, δ-,
and α-tocopherols are more potent than α-tocopherol alone in inhibiting
human platelet aggregation. We still do not know the mechanisms by which
different dietary tocopherols function in atheroschlerosis.
2. Ascorbic acid (vitamin C)

The beneficial health effects of ascorbic acid have been traditionally attributed
to its antioxidant activity, based on its ability to scavenge free radicals in
aqueous biological compartments. Ascorbic acid is, however, an unstable
micronutrient, which varies widely in levels in different foods according to
harvesting, processing and storage conditions. On one hand, many studies have
demonstrated that endogenous lipids in human plasma are efficiently protected
against hydrophilic reactive oxygen species by ascorbate when tested in vitro.
On the other hand, vitamin C can act synergistically as a co-antioxidant by
regenerating vitamin E from its oxidized products. It can also alleviate the
prooxidant activity of vitamin E under certain conditions that induce the
oxidation of LDL. There is clear in vitro evidence that depletion of vitamin C
in plasma leads to the formation of hydroperoxides from triglycerides,
phospholipids and cholesterol. Conversely, at higher concentrations, vitamin C
prolongs the lag phase of oxidation of these lipids.
The in vivo activities of vitamin C are not well understood, because they are
much more difficult to evaluate. In human studies, short-term and long-term
vitamin C supplementation increased the concentration of ascorbate in plasma,
which increased resistance to lipid peroxidation when tested by the increases in
lag phase in the formation of cholesterol ester hydroperoxides ex vivo. Increase
in plasma ascorbate levels varied widely among human subjects taking vitamin
C supplementation. Clinical trials demonstrated that vitamin C supplements
are safe for most adults at levels below or equal to 2 g per day. Models of
vitamin C bioavailability indicate that a 100 mg dose leads to 80% absorption,
which decreases with higher doses. Pharmacokinetic studies with healthy men
and women showed concentrations of vitamin C as a function of dose reaching
plasma concentrations not exceeding 70–80 μmol/l. This tight control of
plasma vitamin C may be due to avoid harmful prooxidant effects at higher
concentrations.
Although healthy subjects have an excess of iron-binding capacity, much
caution is required regarding unhealthy subjects with tissue injury and human
disease. If the iron binding capacity is compromised in unhealthy subjects, the
combination of any unbound free iron and vitamin C would be extremely toxic,
through the potential generation of hydroxyl radicals produced by the Fenton
reaction (Lipid Oxidation, 2nd ed, p. 394) that would be very damaging to
Figure 6.5. Tocopherol–ascorbate–flavonoid redox cascade.
tissues and DNA bases. Whether or not vitamin C also has in vivo beneficial
non-antioxidant effects in humans, as has been demonstrated for vitamin E and
flavonoids (Section H), remains to be determined.
3. Flavonoids
Flavonoids present in foods mostly as glycoside conjugates are more hy-
drophilic than the parent aglycones. These glycoside conjugates may require
enzymatic cleavage of the sugar moiety by glycosidases before absorption. The
phenolic moieties may be derivatized enzymatically into glucuronides or
sulfates, which are more readily transported in the blood and excreted in bile or
urine than the parent aglycones. Therefore, the bioavailability of flavonoids
will be determined by a multitude of factors, including solubility, metabolic
fate and endogenous and exogenous biotransformation, and interactions with
other components of the diet.
Much attention has been given to the possible health benefits of flavonoids
derived from their strong antioxidant activities based on a large number of in
vitro studies. Flavonoids represent a multitude of complex molecules with
multiple chemical and biological activities (Figure 3.3 and Lipid Oxidation,
2nd ed, pp. 242–248). Chemically, in addition to their powerful ability to
scavenge radicals and inactivate prooxidant metals, flavonoids show synergistic
activity by participating in the redox cascade, extending the previously de-
scribed tocopherol–ascorbic acid cycle (Figure 6.2) by reducing the ascorbate
radicals (Asc·) with flavonoids (Flav), which have a lower redox potential, to
regenerate ascorbate (Figure 6.5). According to this redox cycle, α-tocopherol
radicals can be recycled back to their native vitamin E form by ascorbate. The
resulting ascorbate radicals can be regenerated to their native vitamin C by a
flavonoid that has a stronger reducing potential. These interactions between
redox antioxidants will partition according to their structural polarities be-
tween the lipid and the aqueous interface of biomembranes.
Biologically, flavonoids bind with metal sites of proteins, with enzymes and
with ApoB of LDL. Flavonoids thus inhibit several enzyme activities
(telomerase, cyclooxygenase, lipoxygenase, xanthine oxidase, matrix metallo-
proteinase and sulfotransferase). Flavonoids also affect signal transduction
(redox signaling) pathways, and platelet aggregation. For example, the regula-
tion of signal transduction in cells under toxic conditions may be induced by
quercetin after activation by oxidation–reduction to form reactive oxygen
species, via semiquinone and quinone intermediates (Figure 6.6). Depending
Figure 6.6. Oxidation reduction of quercetin (adapted from Metodiewa et al., 1999 and Awad et al.,
2000).
on concentration and free radical source, quercetin can thus have both anti-
oxidant and/or prooxidant activities. Quercetin, like α-tocopherol, has
prooxidant activity when transition metals are available and may cause harmful
mutagenic activity. Therefore, quercetin can have a concentration-dependent
cellular effect that is either protective or cytotoxic, but the active form of
quercetin in vivo is still unknown.
The o-quinone derivative produced by oxidation of quercetin can react with
the nucleophilic side chains of proteins including lysine, cysteine, and tryp-
tophan to form a protein–quercetin derivative (Figure 6.7). The derivative of
bovine serum albumin (BSA) and quercetin was shown to have reduced
antioxidant activity of about 79% compared to quercetin. A BSA–quercetin
quinone produced by secondary oxidation forms cross-links of protein mol-
ecules and further polymerization with loss of antioxidant activity. Reactions
of plant phenols with proteins and enzymes can therefore diminish the antioxi-
dant activity of phenolic compounds. In the same way, the antioxidant activity
of flavonoids from chocolate, cocoa, green and black tea is significantly
decreased when they interact with milk proteins. Similar interactions between
plasma proteins and phenolic compounds have been suggested to take place in
vivo, but the role of these interactions is still not clear and requires further
research.
The beneficial effects of flavonoids in foods can be demonstrated by their
protection against DNA oxidation. A meal of fried onions resulting in elevated
levels of quercetin and other flavonoid glycosides in plasma can be shown to
increase resistance to lymphocyte DNA oxidation in vitro with H2O2. Similar
protective effects can be demonstrated after consumption of soya milk rich in
Figure 6.7. Reaction of quercetin with proteins after quinone formation (adapted from Rohn et al.,
2004).
phytoestrogens and isoflavones (see Section G.3.c). The results of such studies
must be interpreted with caution, however, because flavonoids are metabolized
in plasma and their concentrations do not necessarily reflect their levels in
cells. Oxidative damage of DNA also cannot be interpreted as a marker of
cancer risk.
Many human studies have now been carried out to determine the
bioavailability of flavonoids contained in foods, to understand better the
absorption and eventually the in vivo activity of these compounds. The
glycosylation of polyphenols influences their hydrophilic properties and their
chemical and biological role in disease prevention, by influencing their distri-
bution and diffusion across biological membranes. Because some glycosides
are primarily absorbed from the small intestine, their concentrations in plasma
will be expected to be higher than the flavonoids that reach the colon, which are
degraded by microorganisms into phenolic acids (Figure 6.8). For this reason,
quercetin glycosides are more bioavailable than the quercetin aglycone or
quercetin rutinoside [(-6-O-rhamnosyl)-glucoside]. The bioactivity of phe-
nolic compounds depends on their biotransformation that occurs in the small
intestine and the liver, and the corresponding metabolites absorbed in the
colon. Flavonoid glycosides must be deglycosilated by epithelial β-glucosidases
before absorption in the liver and other tissues. The resulting aglycones are
further conjugated into methylated, sulfated and glucorinide derivatives by
Figure 6.8. Metabolism and conjugation of dietary flavonoids (adapted from Scalbert and Williamson,
2000).
metabolizing enzymes. Polyphenols that are absorbed, metabolized in the liver,

and excreted in the bile and back to the small intestine as glucuronides, reach
the colon, where microflora enzymes catalyse deglycosylation and ring fission
to produce a mixture of phenolic acids. Rutin (quercetin rutinoside), the major
flavonoid glycoside in tea, is poorly bioavailable because it is not enzymatically
deglycosilated and absorbed in the small intestine and reaches the colon where
it is rapidly degraded by microflora.
The concentrations of flavonoids absorbed through the GI tract into the
plasma are very low, in the micromolar range, because they are rapidly
metabolized to methylated, glucuronidated and sulfate derivatives by several
enzymes in the small intestine. Dietary flavonoid glucosides can also under-
go deglycosylation in the small intestines and liver. Many biotransformations
of absorbed flavonoids occur in the liver, including methylation, sulfation
and glucuronidation. These metabolites have a significantly diminished anti-
oxidant activity compared to the original aglycones, by blocking one or more
phenolic hydroxyl groups of catechols responsible for radical scavenging
activity (Figure 6.3). Bacterial enzymes catalyse the hydrolysis of
metabolites to produce phenolic acids by ring cleavage and decarboxylation
of some phenolic acids. Many metabolites are also produced from flavan-3-
ols by enzymes of the microflora of the large intestine, including
3,4-dihydroxyphenylacetic acid and 3-hydroxyphenylacetic acid. The arom-
atic phenolic acids formed in the colon, representing about one third of the
dietary phenols, have diminished reducing and antioxidant activity com-
pared to the original flavonoids, representing the remaining two-thirds. At
the wide range of doses used, varying from 20 to 1000 mg, the phenolic
compounds were metabolized into complex derivatives that have reduced

antioxidant activity by blocking one or two reactive hydroxyl groups respon-
sible for radical scavenging properties (Figure 6.3). The relative degree of
conjugation and deconjugation during absorption of phenolic compounds in
different tissues varies according to the dose administered and the metabolic
sites (Figure 6.8). Metabolism by gut microflora may also influence the
bioactivity of polyphenols in foods.
a. Quercetin and catechins. With intakes of flavonoids ranging from less

than 1 mg to 688 mg aglycones, the resulting concentration of metabolites in
plasma varies widely (less than 0.5 to 5 μmol/l) (Table 6.3). The bioavailability
of quercetin varies according to food sources, and the types of glycosides
present. Quercetin is more bioavailable from onions containing glycosides
than from apples and tea containing rutin and other glycosides. After consuming
dietary sources of quercetin-rich foods, absorption of quercetin glucosides was
moderately rapid, reaching peak plasma concentrations in blood of 0.65 and
0.74 μmol/l with onions, and 0.3 μmol/l with apples, with corresponding times
of maximum concentration (Tmax) of 2.9 and 0.7 hours, and half-life of
elimination of 17 and 28 hours, respectively (Table 6.3, ref 1, 2). Because the
elimination of quercetin metabolites is relatively slow, they could accumulate
in plasma, especially after repeated intakes and supplementation. There is also
great variability in absorption among individuals. While with pure quercetin
absorption reached a maximum of 0.42 μmol/l and a Tmax of 0.5 hour, with the
corresponding quercetin rutinoside, absorption reached lower maximal
concentrations of 0.18 and 0.3 μmol/l and longer Tmax of 6 and 9 hours (Table
6.3, ref 2–4). The quercetin glucosides in onions containing mainly quercetin-
β-glucosides (340–347 mg/kg), were more bioavailable than the quercetin
glycosides (containing a mixture of quercetin galactosidess and xylosides) in
apples (20–36 mg/kg) and of pure quercetin-3-rutinoside (30%). The
bioavailability of the rutinoside was only 20% of that of the glucoside. The
peak concentration (Cmax) and the time to reach it were much greater for
quercetin glucoside than for quercetin rutinoside. The sugar moiety is therefore
an important factor in the bioavailability of quercetin glucosides. The relatively
long time of elimination suggest that repeated consumption of quercetin-
containing foods may contribute to the accumulation of this flavonoid in blood.
The absorbed quercetin was chiefly methylated in the 3'-position, and entirely
conjugated with glucuronic acid and sulfate. The metabolites 3'-o-
methylquercetin and conjugated derivatives inhibited the copper-induced LDL
oxidation by about half as much as that of quercetin.
The bioavailability of catechins also varied significantly among sources and
the resulting metabolites (Table 6.3, ref 3, 5–9). The gallic derivatives of
catechin are absorbed less than the aglycone precursors. Except for EGCG,
which is found in the free form in high proportions, the other catechins and
168
Table 6.3. Bioavailability of flavonoids in human studiesa
Sources Flavonoids Dose Tmax plasma Plasma conc. AUC Elimination T1/2 Ref.b
(h) (μ mol/l) (μ mol/l) (h)
Fried onions Quercetin (Q) eq 64 mg 2.9 0.65 – 16.8 (1)

Onions Q eq 68 mg 0.7 0.74 7.7 28 (2)
Apples Q eq 107 mg 2.5 0.3 3.5 23 (2)
Pure compound Q 0.14 mg/kgc 0.5 0.15–0.42 – – (3)
Pure compound Q rutinoside 190 mg 6.0, 9.3 0.18, 0.3 3.7, 3.3 28 (2, 4)
Pure compound Q-4'-glucoside 150 mg <0.5 3.5 18.8 21.6 (4)
Pure compound Catechin 0.36 mg/kgc 0.5 0.14–0.49 – – (3)
Red wine Catechin 35 mg 1.5 0.091 0.36 3.1 (5)
Red wine Catechin 35 mg 1.44 0.077 0.31 3.2 (6)
Dry green powder EGCG 105 mg 2 0.14–0.31 – – (7)
Green tea extract EGCG 225, 375 mg – 0.66, 4.3 – – (8)
EGC 7.5, 12.5 mg 0.03, 0.14 at 1.5 h
Black tea Catechins 0.3 g 2.2 0.17 0.53 6.9 (9)
Cocoa, chocolate EC 220 mg 2 4.924.77 – – (10)
Chocolate EC 82164 2–2.6 0.380.7 1.53.7 1.9–2.3 (11)
Pure compound EGCG 688 mg 2.9 1.3 12.1 3.9 (12)
a
From Manach et al. (2005). Tmax = time at maximum concentration, AUC = area under curve, T1/2 = half-life of elimination, EGCG = epigallocatechin gallate,
EGC = epigallocatechin, EC = epicatechin.
b
References: (1) Hollman et al. (1996), (2) Hollman et al. (1997), (3) Goldberg et al. (2003), (4) Hollman et al. (1999), (5) Donovan et al. (2002), (6) Bell et al. (2000),
(7) Unno et al. (1996), (8) Nakagawa et al. (1997), (9) Van Het et al. (1998), (10) Baba et al. (2000), (11) Richelle et al. (1999), (12) van Amelsvoort et al. (2001).
c
Body weight.
epicatechin are extensively conjugated in human plasma as methyl, glucuronides

and sulfate derivatives (Table 6.3, ref 10, 11, 12).
Many intervention studies were based on questionable measures of lipid
oxidation, including the notoriously unspecific TBA test for urinary
‘malonaldehyde’ (MDA) and plasma lipid oxidation, and the misleading
tests for antioxidant capacity (Lipid Oxidation, 2nd ed, pp. 108–110, 435–
439) that use artificial azo initiators, including the popular ‘antiradical
activity’ tests, DPPH, ABTS (or TEAC), TRAP and ORAC, and unspecific
biomarkers such as electrophoretic mobility of LDL and measurements of
tryptophan residues by fluorescence (Table 6.4). Quercetin affected some
cancer markers and antioxidant biomarkers claimed to be in vivo (Table 6.4,
ref 1–3). Catechins and other polyphenols increased plasma antioxidant ca-
pacity, assessed by many questionable antiradical activity tests, decreased
plasma lipid peroxides, conjugated dienes and phospholipids
hydroperoxides, plasma isoprostane, increased plasma ascorbate concentra-
tion, and increased the resistance of LDL to oxidation (Table 6.4, ref 4–11).
Many of these biomarkers for in vivo activity have not been validated for
cardiovascular disease and carcinogenesis and in long-term studies. Before
intervention studies of flavonoids can be properly designed and improved, a
better understanding of their bioavailability is required. Tyrosol and hydr-
oxytyrosol decreased LDL oxidation ex vivo, but promoted postprandial
oxidative stress (Table 6.4, ref 12).
Quercetin glucuronides are metabolites of quercetin in humans. The activity
of different quercetin glucuronides in inhibiting xanthine oxidase and
lipoxygenase varies according to the conjugation position (Table 6.5). Querce-
tin-3-sulfate, quercetin-3-glucuronide, and quercetin-7-glucuronide are
significantly more active in inhibiting xanthine oxidase than the corresponding
3'- and 4'-glucuronides and 3'-methylquercetin, which are moderate inhibitors
of lipoxygenase activity. Although the bioactivity of these quercetin metabolites
can vary according to the conjugation position, the aglycone quercetin remains
the most active inhibitor of both enzymes.
This trend corresponds approximately with their in vitro antioxidant activity,
showing significant decrease in activity when the ortho 3',4'-dihydroxy posi-
tions are conjugated. On one hand, xanthine oxidase plays a crucial role in gout
by producing uric acid from xanthine, and in oxidative stress. On the other,
lipoxygenase induces LDL oxidation and participates in the early stages of
atheroschlerosis. These metabolites could exert physiological effects, perhaps
through synergistic effects with other components from fruits and vegetables.
The rapid postprandial absorption of quercetin can be explained by absorption
of the glycosides and rapid conjugation. Hydrolysis of glycosides during initial
digestion in the stomach would produce free quercetin in the proximal part of
the intestine. Although the antioxidant activity of quercetin is decreased by
glucuronidation or sulfation, the metabolites formed could still exert
Table 6.4. Selected intervention studies with flavonoid-containing foodsa
170
Substances given Principal phenol Dose Biomarkers affected Biomarkers not affected Ref b
Onions Quercetin 200 g + Resistance to erythrocyte RBC TBARS, Urinary MDA, (1)
Superoxide dismutase TBA-MDA adducts
Fried onions Quercetin 50 mg + 6% plasma antioxidant Susceptibility of LDL to (2)
capacity oxidation
Supplement Quercetin 30 mg + Oxidative resistance Plasma TG, HDL or LDL, (3)
of LDL cholesterol,vit E or vit C
Green tea Catechins 5g + Plasma vit C Plasma, β-carotene, vit E, (4)
uric acid
Green tea extract Catechins 254 mg –40% decrease in – (5)
catechins phospholipid-OOH
Black tea Catechins 750 ml tea + LDL oxidation lag time, Total cholesterol, TG, ApoB (6)
CD, TBARS, peroxides
Cocoa Procyanidins, 500 mg –Lower blood pressure Heart rate, plasma (7)
catechins polyphenols cholesterol, TG, glucose
Chocolate Procyanidins (PC), 320 mg PC, + Small increase plasma Plasma 8-isoprostane (8)
catechins 104 mg EC antioxidant activity (TBARS)
High-flavanol Flavan-3-ols 187 mg –Lower plasma F2-iso- No effect on plasma F2-iso- (9)
cocoa drink prostane when combined prostane without phys. exercise
with phys. exercise
Red wine PC, anthocyanins, 375 ml Decrease plasma lipid LDL or HDL cholesterol, (10)
quercetin peroxide plasma TG

Grape seed Proanthocyanidins 300 mg Decrease lipid hydro- – (11)
extract peroxides in chylomicrons
Olive oil Tyrosol, 366, 164, –LDL oxidation ex vivo Olive oil promoted post- (12)
hydroxytyrosol 2.7 mg/kg prandial oxidative stress
a
From Williamson and Manach (2005). Abbreviations: MDA, malonaldialdehyde; LDL, low-density lipoproteins; HDL, high-density lipoproteins; TG, triglycerides;
Vit, vitamin; -OOH, hydroperoxides; ApoB, apoprotein B; PC, procyanidins; EC, epicatechin.
b
(1) Boyle et al. (2000), (2) McAnlis et al. (1999), (3) Chopra et al. (2000), (4) Gomikawa and Ishikawa (2002), (5) Nakagawa et al. (1999), (6) Ishikawa et al. (1997),
(7) Taubert et al. (2003), (8) Wang et al. (2000), (9) Wiswedel et al. (2004), (10) Nigdikar et al. (1998), (11) Natella et al. (2002), (12) Covas et al. (2006).
Table 6.5. Effect of quercetin conjugates in inhibiting xanthine oxidase and

lipoxygenasea
Quercetin conjugates Inhibition Ki (μ M)

Xanthine oxidase Lipoxygenase
Quercetin (Q) 0.2 2.8

3'-methylquercetin 0.25 4.8
Q-3-sulfate 78 16
Q-3-glucuronide 160 60
Q-7-glucuronide 100 6.0
Q-4'-glucuronide 0.25 8.4
Q-3'-glucuronide 1.4 6.5
a
Day et al. (2000). Ki inhibition constant (mixed competitive and non-competitive inhibition).
See Table 8.4 for catalytic activity toward LDL oxidation.
physiological non-antioxidant effects, possibly in synergism with other com-

ponents from fruits and vegetables (Section H).
The metabolism of catechin was investigated after consumption of red wine
by analysing metabolites in human plasma (Lipid Oxidation, 2nd ed, Table
13.11, p. 439). After consumption of one serving of red wine, catechin was
found in plasma to be present almost exclusively as methylated, glucuronide
and sulfate conjugate metabolites. Less than 2% free catechin was detected one
hour after consumption of wine, and no free catechin after 3–4 hours. Maxi-
mum levels of total catechin (91 nmol/l for red wine, and 81 nmol/l for
de-alcoholized red wine), were reached 1.5 h after consumption, and varied
widely among individuals. Catechin in grapes and wine is therefore absorbed
to varying degrees, but rapidly metabolized and conjugated. In another study,
after consumption of apples, the half-life of elimination (E1/2) for quercetin was
found to be significantly larger (E1/2 = 23) than for catechin after consumption
of red wine (E1/2 = 3.1). Human absorption studies also showed that glucuro-
nide and sulfate metabolites were found in plasma after the consumption of
green tea rich in flavan-3-ols (and chocolate rich in epicatechin).
Absorption of tea catechins increased with increasing derivatization with
gallic acid. More than 80% of the tea catechins were conjugated in the plasma
and urine. Thus, after taking decaffeinated green tea powder, maximum plasma
concentrations (Cmax) after 1.4–2.4 h were obtained of 190 ng/ml with
epicatechin (EC), 550 ng/ml with epigallocatechin (EGC), and 326 ng/ml with
epigallocatechin gallate (EGCG) (Table 6.6). The Cmax values increased from
1.5 to 3.0 g and levelled off at 4.5 g. The half-life of EC and EGC (2.5–3.4 h)
was lower than that of EGCG (5.0–5.5). EGC and EC were eliminated in the
urine within 8 hours, but not EGCG. Consumption of chocolate rich in
epicatechin displayed similar pharmacokinetics, with shorter elimination times
than those for green tea. Epicatechin from chocolate reached a maximum
Table 6.6. Bioavailability of catechins with various doses of green tea powdera
Epicatechin Epigallocatechin Epigallocatechin gallate

(EC) (EGC) (EGCG)
Tea (g)
Tmax Cmax T1/2 Tmax Cmax T1/2 Tmax Cmax T1/2
(h) (ng/ml) (h) (h) (ng/ml) (h) (h) (ng/ml) (h)
1.5 1.4 55 5.7 1.4 148 2.7 1.6 119 5.5

3.0 1.8 189 3.4 1.8 508 2.8 2.4 326 5.0
4.5 1.8 190 3.2 1.3 550 2.5 2.7 321 4.9
a
From Yang et al. (1998). Study made with 18 human volunteers consuming decaffeinated green tea
solids dissolved in 500 ml of hot water. HPLC analyses of plasma and urine were carried out on free
catechins after treating samples with β-glucuronidase and sulfatase.
For abbreviations see Table 6.3.
concentration of 100 ng/ml within 2 hours, and was rapidly eliminated with 4
hours.
These results suggest that the beneficial effects of flavonols in plant foods,
red wine and green tea may depend on daily consumption to sustain useful
levels of these compounds in plasma or other parts of the body. Therefore,
phenolic antioxidants in fruits and beverages may have a protective effect
against coronary heart disease and cancer, but the mechanism of protection is
not understood. There is a significant gap in our knowledge concerning the
relative absorption and the activities of these natural antioxidants in the body
and their bioavailability, and a lack of reliable biomarkers to test their activity
in vivo. Although recent evidence indicates that small amounts of flavonoid
antioxidants are absorbed as various metabolites in the blood of humans, their
activities in vivo are unknown. The possible cardiovascular benefits of an
increased intake of vitamin E and other phytochemicals are therefore not yet
well established.
Although considerable evidence has been published on the in vitro antioxi-
dant activities of flavonoid-containing foods, in vivo effects reported in many
intervention studies were not consistent. Much confusion and mixed results
were reported from human intervention studies with flavonoid-rich diets, on
the basis of questionable protocols and unreliable biomarkers erroneously
claimed to measure lipid peroxidation in vivo. The discrepancy between in
vitro and in vivo studies may be partly due to the partial absorption and
metabolism of flavonoids in blood, and partly due to the use of a wide variety
of questionable and inappropriate biomarkers (Table 6.4). The biological
effects of the flavonoid metabolites are not well understood, mainly because of
lack of reliable in vivo testing protocols. Although the metabolites would be
expected to have different biological properties and may be less biologically
active than the corresponding aglycones from which they are derived, the
biological activities of metabolite mixtures formed in different tissues in the
Table 6.7. Normalized pharmacokinetic data from 97 bioavailability studiesa
Phenolic Plasma Tmax Cmax AUC Urinary Elimination

compounds (h) (μ mol.h/l) (μ mol.h/l) excretion E1/2
(% of intake) (h)
Quercetin 1.1 1.5 9.8 2.5 18

glucosides
Rutin 6.5 0.20 2.9 0.7 20
(Epi)catechin 1.8 0.40 1.1 19 2.5
EGC 1.4 1.1 2.0 11 2.3
EGCG 2.3 0.12 0.5 0.06 3.5
Anthocyanins 1.5 0.03 – 0.4 –
Proanthocyanidin 2.0 0.02 – – –
dimers
Chlorogenic acid 1.0 0.26 – 0.3 –
Caffeic acid 1.4 1.0 – 11 –
Ferulic acid 2.0 0.03 – 28 –
Gallic acid 1.6 4.0 – 38 1.3
a
From Manach et al. (2005). Mean values calculated to supply 50 mg aglycone equivalent.
See abbreviations in Table 6.3.
plasma body are not well established and understood. Deconjugation of

metabolites may also occur in some tissues and change their biological activity
and relative absorption in different parts of the body (Figure 6.8).
b. Polyphenols and phenolic acid derivatives. Normalized pharmacokinetic

data of pure phenolic compounds from 97 human studies (Table 6.7) showed
that quercetin glucosides are better absorbed than rutin, and both have
significantly higher elimination times than the catechins. Galloylation of
epicatechin markedly reduced absorption, making EGC significantly better
absorbed than EGCG. Anthocyanins and proanthocyanidin dimers are apparently
poorly absorbed, but the metabolites may not have been included in the
normalized data. Gallic acid was the best absorbed and the most excreted of all
the other phenolic compounds. Urinary excretion values used to estimate the
minimal absorption rate ranged from 0.06 to 38% of intake, showing the great
variability in the bioavailability of polyphenols. Although catechins and gallic
acid are rapidly eliminated, some of their metabolites may possess longer half-
lives.
Anthocyanins were previously considered to be poorly absorbed in the
blood, when analysed as the aglycones under conditions of ‘saturation meta-
bolic pathways’ that would not be expected to occur after normal consumption
of fruits, vegetables, juices or wine. More recent bioavailability studies were
made possible by HPLC-MS and NMR analyses of anthocyanin-3-glucosides
and metabolites (Table 6.8). A human feeding study using chokeberry extract
rich in cyanidin-3-glucosides (721 mg) showed that they were rapidly absorbed
Table 6.8. Bioavailability of cyanidin 3-glucosides and metabolites in human subjects

consuming chokeberry extracta
Anthocyanins Quantity Cmax AUC T1/2

(nmol/l) (h) (nmol.h/l) (h)
Cyanidin 3-galactosideb 24 2.5 67 <1.35

Cyanidin glucuronideb 15 2.0 91 –
Cyanidin 3-arabinosideb 8.9 3.5 54 1.67
Peonidin 3-galactosidec 3.8 4.0 31 –
Methyl cyanidin glucuronidesd 13 2.5 34 –
Methyl cyanidin glucuronidesd 33 2.5 100 –
a
From Kay et al. (2005). Chokeberry extract was chosen because it contained exclusively cyanidin 3-
glycosides, and the oral dose of 721mg was equivalent to about 120–230 g fresh weight of whole berries.
OR2
OH
HO O
OR 1
OH
b
Structures: R1 = galactose, glucuronic acid, arabinose; R2 = H
c
R1 = H; R2 = CH3
d
R1/R2 = glucuronic acid/CH3
and extensively metabolized. Cumulative concentrations of total anthocyanins

aglycones and metabolites detected in the serum of 377 nmol.h/l (AUC)
reached a maximum concentration of 96 nmol/l within 2.8 hours. The anthocy-
anin aglycone represented only 32% of the total anthocyanins in the serum, and
32.5% in the urine. The remaining 67.5% occurred as conjugated metabolites
identified as glucuronidated and methylated derivatives. Two other metabolites
were identified as methylated cyanidin glucuronides by MS but had different
HPLC and TLC characteristics (Table 6.8). Glucuronidation was the major and
methylation the second commonly observed pathway for anthocyanin metabo-
lism, but it may also produce phenolic acids.
These bioavailability data demonstrate great variability for different phenol-
ic compounds, as well as among the different studies. For most compounds, the
maximum concentration values also varied ten-fold. These variations can be
attributed to many factors including the food matrix, inter-individual differ-
ences in absorption and metabolic and absorption efficiencies.
c. Isoflavones. These phytoestrogens found only in soybean products have

generated much interest among nutritionists motivated by considerable published
data supporting their potent and significant biological activities. Many soybean-
fortified food products have now been classified as functional foods in support
of claims for their hormonal and non-hormonal effects relevant to human
Table 6.9. Pharmacokinetic data of soy isoflavones in human studies.
Isoflavones Tmax (h) T½ max (h) Cmax (µmol/l) AUC (µmol.h/l)
Gluc O
O
4.5 3.2 0.34 2.6
Gluc
Daidzein glucuronidea
Gluc O
6.0 8.4 0.50 8.0

OH O
Gluc
Genistein glucuronidea
SO4 O
O
4.5 3.1 0.24 1.7
SO4
a
Daidzein sulfate
SO4 O
OH O
4.5 5.7 0.14 1.6
SO4
Genistein sulfatea
Pure daidzein (Da)b 50 – 0.53 6.2 Da + 7 E
Pure gensitein (Ge)b 4.2 – 0.53 8.9
a
From Shelnutt et al. (2002): Study was carried out on 6 men and 6 women consuming a soy protein drink
containing 0.6 mg Da and 1.0 mg Ge/kg body weight. Analyses carried out by LC-MS of aglycones
obtained after hydrolysis with glucuronidase and sulfatase.
T½ max = apparent half-life to Cmax, AUC = area under curve, E = equol (4',7-isoflavandiol).
b
From Zubik and Meydani (2003): dose, 16 mg Da and 14 mg Ge.
disease. There is, however, limited information regarding the bioavailability,

metabolism and clinical effectiveness of a multitude of dietary supplements to
justify the claims for health benefits generally attributed to isoflavones or their
metabolites.
All soybean isoflavones are efficiently absorbed from the intestinal tract, but
they exhibit significant differences in the pharmacokinetics between the
glycosides of daidzein containing one hydroxyl group in the A and B rings, and
genistein containing two hydroxyl groups in the A ring and one in the B ring.
The two isoflavone conjugation sites (7 on the A ring, and 4' on the B ring), can
be either sulfated or glucuronidated, resulting in a mixture of mono- and

diconjugates and mixed conjugates with one site glucuronidated and one site
sulfated (Table 6.9). The glucuronides and sulfate conjugates of daidzein were
cleared from plasma faster (apparent half-life of 3.1 and 3.2 h) than the
corresponding genistein conjugates (apparent half-life of 5.7 and 8.4 h). The
genistein glucuronides (Cmax 0.50 μmol/l) were more bioavailable than the
daidzein glucuronides (Cmax 0.34 μmol/l), as indicated by their significantly
higher total concentration in circulating blood. Another study found no differ-
ence in the absorption efficiency (mean time to reach maximum plasma
concentration) for the daidzein and genistein aglycones. However, the produc-
tion of ‘equol’ (4',7-isoflavandiol), a bacterial estrogenic metabolite of daidzein
present mainly as glucuronides, was significant after ingestion of daidzein.
Equol was shown to be more estrogenic than its precursor daidzien in in vitro
and animal studies. Great inter-individual variability was also shown in the
capacity to produce equol. The sulfate conjugates of daidzen represented about
25% of the total equivalent in plasma compared to about 10 % for the sulfate
conjugates of genistein. The maximum concentration of daidzen sulfate was
higher than that of genistein sulfate.
Significant differences in isoflavone metabolism were reported between
animal models and humans. Metabolic profiles of women were more similar to
those of pigs than those of rats. The metabolites in human plasma included 75–
78% glucuronides, 20–24% sulfates and only 1% aglycones (Table 6.10). In rat
serum, however, genistein glucuronides and sulfates were found in about equal
proportions; for daidzein, 68% were glucuronides and 25% were sulfates.
Greater proportions of glucuronides (86–87%) than sulfates (13–14%) were
excreted in human urine and only traces of aglycones. In contrast, in rats, much
larger proportions of aglycones (41–46%), followed by glucuronides (42–
47%) and sulfates (12%) were excreted in urine. Also, rats and monkeys
Table 6.10. Isoflavone aglycones and conjugates in human plasma and animal serum
and urinea
Plasma/serum Urine
Isoflavones
Women Rats Women Rats
% Daidzein
Aglycone 1.4 7.3 0.3 41
Glucuronide 75 68 86 47
Sulfate 24 25 14 12
% Genistein
Aglycone 1.2 4 0.1 47
Glucuronide 78 50 87 42
Sulfate 20 46 13 12
a
From Gu et al. (2006). Data obtained from 6 women and 7–9 rats after consuming diets containing
soybean protein isolates.
Table 6.11. Phenolic acid metabolites formed in urine from subjected ingesting
supplementsa
Supplements/Metabolites Main metabolitesb (mol/100 mol supplement)
Chlorogenic acid supplement

Chlorogenic acid 1.7
Hippuric acid 49.5
DiOHphenylpropionic acid 1.7
DiOHcinnamic acid 1
MethoxyOHcinnamic acid 0.8
DiOHbenzoic acid 0.3
Hydroxycinnamic acid 0.3
Quercetin rutinoside supplement
OHphenyl acetic acid 36.1
MethoxyOHphenyl acetic acid 7.8
DiOHphenylacetic acid 5.0
Black tea catechins supplement
Hippuric acid 42.8
OHphenyl acetic acid 1.1
Gallic acid 0.2
DiOHphenylacetic acid 0.2
a
Olthof et al. (2003). Supplements ingested for 7 days by 20 subjects. Metabolic pathways are shown in
Figures 6.7, 6.8, 6.9.
b
Values determined after deconjugation by β-glucuronidase/arysulfatase.
produced significant amounts of equol, whereas the human subjects in this

study produced none of this metabolite and were apparently non-producers of
equol. However, other studies have shown wide differences in the capacity of
humans to produce equol, as well as a gender difference, with more women
than men producing equol after consuming soy for several weeks.
The health significance of isoflavone metabolites is still unclear, because
major differences are found in their metabolism among individuals, and
degradation by gut microorganisms. The results of metabolic studies most
often carried out with animal models must be interpreted with caution, because
they often cannot simulate what happens with humans. More detailed informa-
tion is needed on the biological effects of metabolites produced by human gut
microorganisms to determine the bioavailability of isoflavones.
d. Metabolism of phenolic compounds. Although dietary phenolic com-

pounds are strong antioxidants in vitro, the methylated, sulfate and glucuronide
metabolites (Figure 6.3), formed after circulation in the body, have much
reduced and perhaps no antioxidant activity. Much work is now focusing on
elucidating the sites at which phenolic metabolites are produced in the body.
Chlorogenic acid, tea phenols and quercetin-3-rutinosides are extensively
metabolized in humans. About half of the ingested chlorogenic acid and tea
178
Figure 6.9. Metabolism of chlorogenic acid (adapted from Olthof et al., 2003).
phenols were recovered in urine as hippuric acid, and 36 mol% of the quercetin-
3-rutinoside as hydroxyphenyl acetic acid (Table 6.11). Two-thirds of the
ingested chlorogenic acid is hydrolysed into caffeic acid and quinic acid by
colonic microflora (Figure 6.9). The caffeic acid moiety of chlorogenic acid is
then dehydroxylated by bacterial action in the colon, and converted mainly to
benzoic acid by β-oxidation. The quinic acid moiety of chlorogenic acid is
dehydroxylated to cyclohexane carboxylic acid, and then aromatized into
benzoic acid, either by colonic bacteria or after absorption into body tissues.
The resulting benzoic acid is converted to hippuric acid by reacting with
glycine.
The catechins in black tea were metabolized mainly to hippuric acid (Table
6.11). The catechins are partly absorbed and excreted in urine as 3'-
methoxycatechin (Figure 6.10). Catechins and derived procyanidins reaching
the colon are cleaved by bacterial action to produce valerolactones, which are
then sequentially metabolized to phenylpropionic acid, followed by benzoic
acid, and excreted in urine as hippuric acid. Valerolactones are also found in the
urine.
Quercetin-3-rutinoside is poorly absorbed in the small intestine of humans,
and more than 80% is transported into the colon where it is metabolized by
microflora as hydroxyl- and dihyhydroxyphenyl acetic acids (Figure 6.11).
After absorption in the liver, methylation takes place to produce 3-methoxy-4-
hydroxyphenylacetic acid, which is excreted in the urine (Table 6.11).
These studies show that chlorogenic acid, tea phenols and quercetin rutinoside
produce a complex mixture of phenolic acid metabolites that are excreted in
human urine. Dietary phenolic compounds are thus extensively metabolized
into compounds of lower antioxidant activity before they enter circulation. The
in vivo antioxidant activity of these mixtures is probably lower than the
antioxidant activity measured by the multitude of in vitro assays published in
the literature (Table 6.4). However, these metabolites may have beneficial non-
antioxidant functions (see Section H), which have not been determined from
lack of appropriate bioassays. These studies should also be extended to other
more common flavonoids generally consumed in fruits, vegetables and wines.
4. Methodology
Antioxidants have been commonly evaluated by determining their relative
concentrations in blood after supplementation. However, these analyses may
be confounded by the up-regulation of antioxidant enzymes in response to
oxidative stress. Antioxidants have also been evaluated on the basis of their
reactivity toward peroxyl radicals and their inhibition of hydroperoxide forma-
tion. Many studies have produced mixed results by claiming in vivo antioxidant
activity of phenolic compounds in plant extracts, fruits, and vegetables, red
wine and chocolate products. However, many non-specific assays of biological
180
Figure 6.10. Metabolism of catechin (adapted from Olthof et al., 2003).

ANTIOXIDANTS IN BIOLOGY
181
Figure 6.11. Metabolism of quercetin (adapted from Olthof et al., 2003).

tissues and fluids are based on the relative reactivity of phenolic compounds
toward artificial radicals and may be confounded by complex serum and
plasma components.
The relative antioxidant activity of many phenolic compounds varies
widely according to different testing methods. Methods using artificial radi-
cal model systems provide no information on which biological targets are
protected by the antioxidants. Results vary widely according to the relative
activity of phenolic compounds in scavenging peroxyl, hydroxyl or
superoxide radicals. To determine the real effects of natural antioxidants, it
is important to obtain specific chemical information about which oxidation
products are inhibited. Several specific assays are needed to elucidate prod-
ucts causing oxidative damage in biological tissues. The metabolites
identified in human feeding studies of flavonoids should also be tested for
‘non-antioxidant’ function such as anti-platelet, anti-mutagenic, anti-tumor,
anti-inflammation, anti-hemorrhagic activities and interactions with specific
receptors (see Section H).
Any significant dietary lipid oxidation would be expected to diminish
greatly the effectiveness of dietary antioxidants, especially if administered
together with highly oxidizable polyunsaturated fats. Postprandial impairment
of endothelial vasoactivity was prevented by the administration of vitamins C
and E or the presence of antioxidant-rich foods in meals. The confounding
effect of oxidized lipids may also account for the negative effects found in
many epidemiological studies of dietary antioxidants, because giving antioxi-
dant supplements will not overcome problems of lipid oxidation products. If
the levels of lipid-derived hydroperoxides and aldehyde decomposition prod-
ucts were sufficiently elevated to cause foods to become rancid, consumers
would reject them. However, many non-volatile polar secondary lipid oxida-
tion products occurring in fried and thermally abused foods may not be
detected readily by taste and may cause adverse biological effects if absorbed
by humans. The health implications of polar oxidation products in deep-fat
fried foods for human nutrition have concerned nutritionists for many decades
(Lipid Oxidation, 2nd ed, Chapter 12, pp. 355–389). These potentially serious
dietary problems arising from lipid oxidation are not yet clear, however, and
deserve much more research.
H. ‘Non-antioxidant’ activities of phenolic compounds

Much attention has been recently given to the non-antioxidant functions of α-
tocopherol, including inhibition of monocyte-endothelial cell adhesion, platelet
adhesion and aggregation, formation of inflammatory mediators of
cyclooxygenase-2 and 5-lipoxygenase, and inhibition of specific scavenger
receptors. Other reported non-antioxidant activities of vitamin E include
intracellular signaling functions by regulating protein kinase in smooth muscle
cells, modulating expression of scavenger reception in smooth muscle cells and

monocyte forming macrophages, and the α-tocopherol transfer protein in-
volved in lipoproteins. Several genes are also regulated by tocopherols, including
those involved in uptake and degradation of tocopherols (α-tocopherol transfer
protein and glutathione transferase), genes that modulate lipid uptake, athero-
sclerosis, and extracellular proteins, and genes affecting adhesion and
inflammation. Many of these effects are now considered not to be due to the
free radical scavenging activity of α-tocopherol, but rather to other specific
effects unrelated to its antioxidant properties.
Flavonoids, like tocopherols, can have a multitude of beneficial activities
that cannot be attributed to antioxidant properties, but rather to non-antioxidant
activities that do not involve free radical inhibition. The anti-inflammatory
properties of flavonoids, for example, can be related to their inhibition of a
series of enzymes that produce eicosanoids by a complex cascade of reactions
catalysed by cyclooxygeanes, and leukotrienes catalysed by lipoxygenases
(Lipid Oxidation, 2nd ed, Figures 13.11 and 13.12, pp. 425, 426). Flavonoids
can also stimulate the immune system for defense of the body by the production
of antibodies through the cytokine production assisted by protein phosphoki-
nase cascades. Flavonoids also prevent the synthesis of prostaglandins that
suppress T-cells (mediators of immune response). Immune cells communicate
by chemical signals called cytokines consisting of either proteins or eicosanoids,
which are stimulated by flavonoids. Flavonoids are thus involved in regulating
a complex network of inflammatory cytokines that regulate the synthesis of
acute phase proteins (tumor growth factors).
Flavonoids also stimulate the synthesis of interferons, a family of antiviral
proteins synthesized after a viral infection. Interferons are proteins synthesized
by cells to form an important line of defense against viral infection. They
stimulate protein phosphokinase by phosphorylating and preventing protein
synthesis, by blocking cell growth and proliferation.
Whether or not the conversion of flavonoids into non-antioxidant deriva-
tives is due to a protective mechanism against the well-known xenobiotic
activities of free polyphenolic flavonoids is not clear. The rapid oxidation of
flavonoids into semiquinones and quinones (Figure 6.6) may or may not
have beneficial health effects. The biological effects of the flavonoid
metabolites are thus not well understood, mainly because of lack of reliable
in vivo testing protocols. Mixed and confusing results have been reported
from human intervention studies with flavonoid-rich diets, on the basis of
questionable protocols (Table 6.4), and unreliable biomarkers claimed to
measure lipid peroxidation in vivo. The biological activities of metabolite
mixtures formed in different tissues in the body are not well established and
need more focus than the original flavonoid aglycones from which they are
derived.
I. Dietary recommendations
One important question that concerns nutritionists is whether we need addi-

tional or more effective natural antioxidants in our diet to reduce oxidative
stress, from either excessive dietary polyunsaturated lipids or from other
environmental factors. Although intervention studies have shown that the use
of antioxidants does not directly reduce the risk of cardiovascular disease, there
is a general consensus based mainly on epidemiological studies that we should
increase the intake of vegetable, fruit and whole grains (rich in antioxidants).
Western populations have tended to consume high amounts of polyunsatu-
rated fats that are susceptible to oxidation. On one hand, excessive levels of
polyunsaturated fatty acids in the diet, without providing sufficient protection
from antioxidants and other inhibitors, can promote LDL oxidation and upset
the oxidant–antioxidant balance (Section A, Figure 6.1) in favor of cellular
prooxidants and promote coronary heart disease. On the other hand, diets rich
in oleic acid including the Mediterranean diet have clear advantages in not only
lowering serum cholesterol but also in potentially decreasing the oxidative
susceptibility of LDL.
The prevention of LDL oxidation by antioxidants has been viewed as an
important potential means of decreasing the risk of heart disease. Plant phe-
nolic antioxidants in fruits and beverages (wine, grapes, grape juice and tea)
may have a protective effect against coronary heart disease and cancer, but the
molecular mechanism of protection is not understood. There is now intensive
interest in developing plant sources of polyphenolic antioxidants for foods and
biological systems, and for their possible health benefits. There is, however, a
significant gap in our knowledge on the relative absorption and in the activities
of these natural antioxidants in the body and their bioavailability, and a lack of
reliable biomarkers to test their activity in vivo (see Sections G and H).
Although recent evidence indicates that very small amounts of flavonoid
antioxidants are absorbed as various metabolites in the blood of humans, their
activities in vivo are unknown. The possible cardiovascular benefits of in-
creased intake of vitamin E and other natural polyphenolic compounds in
plants are not yet well established.
The nutritional benefits of vitamin E may lie beyond its well-established in
vitro antioxidant properties and may not necessarily deal with free radical
biochemistry. Vitamin E appears now to have important functions in cellular
signaling and gene expression. Like xenobiotics, vitamin E is metabolized by
β-oxidation and degradation into carboxyethyl chromans that are conjugated
with glucuronic acid or sulfate before elimination into urine. The physiological
function of vitamin E appears therefore to be due to its ability to interfere with
signaling cascades required in the expression of specific genes. The failure of
feeding trials with high-dose of vitamin E has now been explained by its
possible behavior as a foreign substance in lowering the efficacy of therapy
with other essential drugs. Flavonoids like vitamin E have been considered as
typical xenobiotics that are metabolized and thus excreted in urine. Although
high levels may be toxic, at lower levels they can be beneficial by raising the
levels of antioxidant enzymes. Therefore, flavonoid rich sources such as red
wine, tea, fruits and vegetables are nutritionally beneficial, but high-dose
supplements may not be protective.
The nutritional benefits from the Mediterranean diet are generally attributed
to olive oil containing up to 80% oleic acid, α-linolenic acid, small amount of
meat and dairy products containing saturated fats, and natural antioxidants
from fruits, vegetables and legumes. An adequate supply of natural antioxi-
dants is also necessary to prevent peroxidation of polyunsaturated lipids. There
are many unanswered questions, however. It would seem imprudent to make
dietary recommendations to the public before the mechanisms of polyunsatu-
rated lipid nutrition, in vivo activity of antioxidants, and in vivo lipid peroxidation
are better understood.
Although much progress has been recently made on the relative absorption
and pharmacokinetic data obtained on dietary flavonoids, there is still a
significant gap in our knowledge concerning the activities of these natural
polyphenolic compounds in the body and their bioavailability, due to a lack of
reliable biomarkers to test their activity in vivo. The recent evidence indicates
that small amounts of flavonoid compounds are absorbed and excreted as
various complex metabolites in the blood and urine of humans, but their
activities in vivo are diverse and not well established in other tissues. The
biological effects of the flavonoid metabolites are not well understood, mainly
because of a lack of reliable in vivo testing protocols. Much confusion and
mixed results have been reported from human intervention studies with flavo-
noid-rich diets, on the basis of questionable protocols and unreliable biomarkers
claimed to measure lipid peroxidation in vivo. The biological activities of
metabolite mixtures formed in different tissues in the body are not well
established and need more attention. In future research we need to improve
protocol methods and bioassays for better evaluation and optimization of the in
vivo effects of polyphenolic compounds in biological systems (see Chapter 4).
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CHAPTER 7
Browning and glycation reaction products in
biology
The products formed by the interactions of reducing sugars, proteins and lipid
secondary oxidation products have attracted renewed attention in biology.
These complex interactions are based on the chemistry of the Maillard brown-
ing reaction, which has been developed in food chemistry since the early 1900s
in connection with the effects of thermal processing on the quality of foods
(Chapter 5.A). The field of Maillard reaction products in vivo has now grown
dramatically in the biomedical literature in relation to the progression of
diabetes, atherosclerosis, carcinogenesis and aging. More research has been
aimed at evaluating, on one hand, the risks of consuming Maillard reaction
products in the diet, and on the other, the possible benefits from their antioxi-
dant effects in the body. Although the relationship between diabetes and
cardiovascular disease has long been recognized, the mechanism is poorly
understood. The biological modification of proteins by glucose in blood may
involve oxidative and non-oxidative processes, producing low-molecular weight
aldehydes that may initiate cardiovascular diseases. These complex biological
reactions, known as glycation, are also implicated in age-related chronic
diseases, including obesity, diabetes and renal disorders. The glycation reac-
tions accompanied by oxidation reactions are also referred to as glycoxidation.
The cross-linking between proteins such as collagen and carbohydrates
generates life-long cumulative glycosylation products of advanced glycation
end products (AGEs), at advanced stages of the Maillard reactions, contribut-
ing to tissue degeneration. AGEs consist of a complex heterogeneous mixture
of compounds that have significant prooxidant and pro-inflammatory actions
in the body. AGEs are produced by a succession of non-enzymatic rearrange-
ment reactions initiated with glucose- or fructose-derived Amadori and Heyns
products respectively (Figure 7.1, which is the same as Figure 5.1, repeated
here for the reader’s convenience). Further interactions proceed with the
formation of reactive species that cross-link irreversibly with terminal amino
acids of proteins. The chemical pathways leading to mixtures of AGEs have not
been well established, mainly because not all the complex heterogeneous
interactions products involved have been isolated and characterized. Although
the precise structures of the major AGEs formed in vivo have not been well
characterized, advanced glycosylation has been shown to affect a number of
proteins, and the resulting enhanced AGE formation has been implicated in the
pathogenesis of several diabetes and age-related diseases.
193
Figure 7.1 Formation of imines Schiff bases by addition of protein amine residue to the carbonyl
function of reducing carbohydrates forming an Amadori product from glucose and a Heyns product from
fructose.
Major factors in the pathology of atherosclerosis and diabetes include

advanced stages of lipoxidation and glycosylation reactions initiated by the
disturbance of carbohydrate and lipid metabolism. Age-related diseases are in
turn attributed to aging of proteins that accumulate in the vascular wall and are
characterized by accelerated rate of protein turnover. The current working
hypothesis relates degenerative diseases and oxidative stress to inflammation
disorders. Increasing evidence supports the contribution of glycation reactions
to oxidative stress, gene expression factors, and ultimately to inflammation and
injury. Oxidative stress and glycation products are thus significantly increased
in diabetes and aging organisms. Nutritional hypotheses are proposed on this
basis that foods containing advanced glycation and lipid oxidation products
may overwhelm the natural biological antioxidant defenses. The causes and
sources of all AGEs and their properties are, however, not well established and
the methodology for determining these products is limited. More information
is needed on whether or not dietary AGEs in foods pose risks to the vascular
system resulting in diabetes and aging, and on the structure-specific effects of
AGEs in the human body.
A. Biological antioxidant activity of Maillard reaction products

The effects of biological antioxidants in reducing cardiovascular diseases are
BROWNING AND GLYCATION REACTION PRODUCTS 195
Figure 7.2. Formation of aminoreductones during the Maillard reaction of D-glucose. From Dittrich et
al. (2003).
generally attributed to their effect in inhibiting the oxidation of LDL. Anti-

oxidant active Maillard browning reaction products in foods consist of
heterogeneous and complex mixtures of aminoreductones derived from inter-
actions of reducing sugars and amino acids (Figure 7.2). Similar related
Maillard reaction products formed in green and roasted coffee and in model
systems of glucose and lactose with amino acids have in vitro and ex vivo
protective activities in inhibiting copper-induced oxidation of human LDL
(Table 7.1). Maillard reaction mixtures of glucose–glycine and glucose–
arginine showed significant increase in antioxidant activity in inhibiting the
oxidation of LDL relative to control mixtures. However, the most active
aminohexose reductone had only about half as much antioxidant activity as
ascorbic acid. Maillard reaction products can therefore play a role in preventing
the oxidation of LDL in vitro. More advanced glycation products increase
together with lipid peroxidation products, causing advanced oxidative modifi-
cation of LDL in blood circulation of patients with diabetes (Section B.1).
B. Advanced lipoxidation and glycation end products

In atherosclerosis, metal- or enzyme-catalysed lipoxidation is implicated in the
complex multi-step oxidative modification of LDL by reactive aldehydes,
produced by decomposition of lipid hydroperoxides reacting with the apoB of
LDL, resulting ultimately in plaque formation in arteries that restrict blood
circulation (Chapter 6). Reactive carbonyl compounds produced by decompo-
sition of lipid hydroperoxides include a complex mixture of dicarbonyl
Table 7.1. Inhibition of copper-induced oxidation of LDL by Maillard reaction

mixturesa
Maillard reaction products (MRP) Relative increase in lag phase
Glycine + glucose MRP 2.1–6.5

Lysine + glucose MRP 1.4–7.5
Arginine + glucose MRP 1.1–5.7
R1
HC N
R2
C OH
O C
CH3
3-OH-4-(morpholino)-3-buten-2-one,
5 μM 2.5
10 μM 3.2
Aminohexose reductone,b
5 μM ca 1
10 μM 1.3
Ascorbic acid, 5 μM 6.3
a
From Dittrich et al. (2003). LDL oxidized with 5 mM CuSO4; antioxidant activity based on increase in
lag phase of formation of conjugated diene absorption at 234 nm, relative to a negative control of LDL
+ CuSO4.
b
A C4-aminoreductone and aminohexose reductone (NR1R2 = piperidino).
compounds and 4-hydroxynonenal, which form adducts with lysine, cysteine

and histidine residues of proteins.
AGEs accumulate during aging, hyperlipidemia, hyperglycemia and diabe-
tes. Glucose and glucose-modified proteins also produce low-molecular weight
aldehydes that form adducts with lysine and arginine residues, resulting in the
formation of Schiff bases and Amadori rearrangements with AGEs. As with lipid
oxidation, the formation of AGEs is also catalysed by transition metals. Reactive
dicarbonyl precursors produce intermediates by inter- and intramolecular cross-
linking and reactive oxygen species that generate AGEs. The dicarbonyl
compounds that are found at elevated levels in diabetics include glycoaldehyde
and intermediates formed during anaerobic glycolysis, 1- and 3-deoxyglucosone
(1-DG or 3-DG), which may react further by reverse aldol reactions to give
glyoxal (OHC–CHO), or methylglyoxal (MG). MG is also formed after phos-
phate elimination from triose phosphates, intermediates from carbohydrate
metabolism. All glycoxidation products increase in tissue proteins in diabetes
and during normal aging. However, the contribution of AGEs and secondary
decomposition products of lipid oxidation to the chemical modification and
cross-linking of proteins in the vascular wall is still not well established.
The dicarbonyl or α-oxoaldehyde precursors derived from lipid oxidation

and carbohydrate metabolism are involved in the process termed carbonyl
stress. Carbonyl species such as the 9- and 13-oxo-octadecadienoic acids
derived from the corresponding hydroperoxides of linoleate may also act as
intermediates in the metal-catalysed process of carbonyl stress. Covalent
modification by glycation may play a significant role in the oxidative modifi-
cation of LDL particles in plasma and in the artery wall, and thus contribute to
the significant increased levels of atherogenesis in diabetic patients.
Accelerated atherosclerosis in diabetics is explained by a complex mecha-
nism, involving interactions between reducing sugars and proteins or
lipoproteins in arterial walls and the modification of apoB in LDL leading and
its oxidation (Chapter 6). AGE formation involves similar non-enzymatic
pathways as those established for the Maillard reactions in foods between
reducing sugars, proteins and lipid degradation products (Lipid Oxidation, 2nd
ed, Chapter 11.B.3, pp. 312–315). The sugars form glycosylation products
known as Schiff bases with reactive amino groups of proteins in circulation or
on cell walls that rearrange to form Amadori-type intermediate products
(Figure 7.1).
1. Biological effects of AGEs

The stable AGEs comprise a large mixture of protein adducts that are formed
after glycoxidation consisting of a series of chemical rearrangements and
protein modifications combining glycation and oxidation reactions. The non-
enzymatic glycosylation of serum albumin in hyperglycemia may also modify
its metal binding ability, producing increased levels of free iron and copper in
blood that contribute to the complications of diabetes.
Nε-(Carboxymethyl)lysine (CML) is a predominant advanced glycation end
product formed in vivo. In protein containing foods, CML is a cross-linked
AGE product formed from the Amadori product Nε-fructose-lysine, which is in
turn derived from the imine Schiff base produced by interaction of a sugar and
proteins containing reactive ε-amino groups (Figure 7.3). In biology, CML can
be produced in different ways either by starting from oxidized Nε-fructose-
lysine, or by the reaction of dicarbonyl compounds with the lysine chain. CML
is also formed during the copper-catalysed oxidation of PUFA in the presence
of protein.
During copper-catalysed oxidation of LDL in vitro, the CML content
increases together with conjugated dienes. The presence of increasing AGE-
modified forms of LDL (AGE-LDL) has been characterized in the circulation
of patients with diabetes or renal insufficiency. The AGE binding with LDL
ApoB protein and interfering with its receptor uptake may provide an important
mechanism for the accelerated atherogenesis often exhibited by diabetics.
CML is also formed during incubation of ribonuclease with linoleate and
Figure 7.3. Formation of Nε-(carboxymethyl)lysine (CML) by carbohydrate degradation with cleav-

age of β-dicarbonyl compounds.
arachidonate. The formation of glyoxal during incubation of ribonuclease with

arachidonate suggests that lipid oxidation producing other dicarbonyl decom-
position compounds is accompanied by glycoxidation. CML may thus be a
useful marker of oxidative stress and protein damage in aging, atherosclerosis
and diabetes. In addition to CML, other glycoxidation products include
Nε-(carboxyethyl)lysine (CEL), and dimers of deoxyglucosone-lysine, glyoxal-
lysine, and methyl glyoxal-lysine (Figure 7.4). Because CML has been identified
as a product of both carbohydrate and lipid oxidation reactions, this AGE
product is considered as a potentially useful biomarker of oxidative stress and
damage of tissue proteins.
Glycosylation of proteins and lipoproteins interferes with several enzymes,
receptor functions, and especially with the normal physiology of LDL parti-
cles. Glycosylation of the apoB and phospholipids components of LDL promotes
its oxidative modification, and results in significant impairment of LDL-
receptor uptake by decreasing its in vivo clearance. The resulting glycated LDL
particles become recognized by the non-specific scavenger receptor in human
macrophages, thus promoting intracellular accumulation of cholesterol, lead-
ing to atherosclerosis in diabetic subjects. Hyperglycemia activates the protein
kinase C system, causing diabetic complications by increasing the formation of
diglycerides acting as a cellular co-factor from glycolytic intermediates.
Histological evidence of AGEs accumulating in different tissues and in vitro
studies showed that AGEs are derived from complex interactions responsible
for oxidative stress and vascular damage in atherosclerosis and in diabetes. In
this connection, the cross-linking between proteins is considered to be an
important process causing tissue damage.
Hyperglycemia associated with diabetes may also lead to the formation of
DNA-glycation adducts, including N(2)-(1-carboxyethyl)deoxyguanosine, a
major non-enzymatic glycation product of DNA. Glycated DNA causes muta-
tion in bacterial cells that may lead to DNA damage in vivo. Cellular interactions
with AGE-binding proteins or AGE receptors on cell surfaces result in increase
Figure 7.4 Other glycation products detected in tissue proteins: CEL, glyoxal-lysine dimers (GOLD),
and methylglyoxal-lysine dimers (MOLD). From Saraiva et al. (2006).
in lipid peroxidation products and receptor-mediated release of superoxide

anions and pro-inflammatory cytokines. Feeding studies comparing ‘Control
diet’ with egg white cooked with fructose, ‘AGE-diet’ and egg white without
fructose showed increase of AGE levels in serum from the AGE-diet in both
healthy and diabetic subjects.
Animal feeding studies to determine the risk/benefit balance of consuming
AGEs have generally been based on high doses of AGE mixtures that may be
too complex to evaluate their physiological effects. Liver function and antioxi-
dant effects suggested that AGE containing proteins were not toxic. There is,
however, evidence that the physiological effects of AGEs are significantly
influenced by whether or not the test animals are on calorie restricted diets.
Studies on the effect of dietary AGEs may also be confounded by limited
reliable data available on the AGE content of foods, and on absorption and
metabolism of AGEs in the diet.
2. Glycation products in foods

In vitro and in vivo studies may support the contribution of foods to AGEs in
tissues. Thus, AGEs and reactive oxygen species are significantly increased in
diabetic and other disorders related to aging. There is a significant correlation
between ingested and circulating AGEs in humans which may be the result of
inactivating the natural antioxidant defense systems in the body. The formation
of AGEs is markedly increased in diabetes, and is associated with markers of
systemic inflammation such as C-reactive proteins.
Diet-derived AGEs formed during cooking by heating may also be formed
continuously in the body. Accordingly, excessive AGE consumption in current
diets may impose inappropriate oxidative stress, which may promote diabetes
and cardiovascular diseases. Because AGE production is implicated in the
Table 7.2. CML contents of selected foodsa

Foods AGEb (KU/g or /ml)
Fats
Olive oil 120
Butter 265
Mayonnaise 94
Proteins
Chicken, broiled (15 min) 58
Chicken, fried (15 min) 61
Beef, broiled (15 min) 60
Beef, boiled (1 h) 22
Tuna, broiled (10 min) 51
Tuna, roasted (40 min) 6
Egg, fried 27
Egg yolk, boiled 12
Cheese, American 87
Carbohydrates
Bread 0.54
Milk, whole 0.05
Infant formulac 4.86
a
From Goldberg et al. (2004).
b
Immunoreactivity based on Nε-(carboxymethyl)lysine (CML), assessed by enzyme-
linked assay based on monoclonal antibody.
c
‘Enfamil’ (Mead Johnson).
disorders of diabetes and aging, the CML content was determined in different
foods, and the effect of heat treatments was used as marker of glucose–protein
or glucose–lipid interactions (Table 7.2). The AGE contents of foods support
the hypothesis that they are influenced by the nutrient composition, tempera-
ture and method of heating foods during cooking. On the basis of an anti-CML
monoclonal antibody assay using only one AGE marker, these preliminary data
may be of only relative interest. They showed that lipid-containing foods had
the highest and the carbohydrate group the lowest content of AGEs. Higher
values were also observed for broiled and fried meat than for boiled meat.
Higher AGE values were found in samples broiled or grilled for shorter
cooking times than in samples boiled in liquid media for longer periods. The
relatively high content of AGEs in a few common foods is produced by dry-
heat treatments of protein- and lipid-rich foods. Unfortunately, the methodology
required to analyse AGEs and the standardization of reference marker com-
pounds have not been well defined and generally accepted in this field because
AGEs consist of complex mixtures, which vary significantly with the degree of
heat treatment. Simpler and more practical methods are needed to survey the
AGE content of a much wider sampling of foods.
Glycosylation and lipid oxidation are promoted by the same factors which
are important for the production of edible fats, including the degree of heat in
the absence of moisture, and the presence of metals. The AGE content of infant
formula is also found to be significantly higher than that of human or cow’s
milk. Studies with human volunteers showed that most of the AGEs observed
in urine are derived from the diet. Animal and human studies also showed that
AGE-rich foods produced increases in plasma levels of AGEs. Therefore,
food-derived AGEs are directly absorbed into circulation and may contribute to
the AGE pool. These diets may thus constitute a special risk factor for
individuals susceptible to cardiovascular and kidney damage. Much more work
is needed, however, to clarify the metabolic fate and to evaluate the risk or any
benefit from ingestion of each AGE compound.
3. Analyses of glycation products

The structures of several advanced food- and biological-derived AGEs have
been analysed by immunochemical methods, using antibodies specific for
certain AGE structures, GC-MS, HPLC-MS and HPLC with fluorescence
detection after protein hydrolysis. Immunochemical methods are limited to
specific reference glycation compounds used as markers. Although these tests
might provide a rough indicator for the content of glycated products in foods,
and in human plasma and urine, they cannot clarify the chemical structures of
individual AGEs. GC and HPLC methods have limited sensitivity and require
hydrolysis steps that are sufficiently mild to prevent formation of artifacts.
More advanced methods using electrospray ionization (ESI) and matrix-
assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS) afforded, for the first time, the analyses of large molecules
such as proteins and their modified glycation derivatives in vivo and in vitro.
These techniques can be used to determine a mass increase of intact glycated
proteins. The MALDI-TOF-MS technique was used to detect specific AGEs by
peptide mapping to investigate the formation of adduct compounds of AGE,
produced by incubating lysozyme with different amounts of glucose (AGE-
lysozyme). The reactions of lysozyme with glucose and reactive sugar
degradation products and dicarbonyl compounds were thus identified (Figure
7.5). AGE-lysozyme showed an imidazolone product, and CEL- and CML-
lysozyme adduct modifications determined by their relative mass increase Δm
(Table 7.3).
Quantitative analyses by LC-MS/MS showed that pasteurized and sterilized
milk are rich sources of heat-stable glycation products, including CML and
CEL (Table 7.4). The CML and CEL protein residue in raw milk increased
three-fold after pasteurization, while the CML protein residue increased six-
fold and the CEL increased three-fold after sterilization. The corresponding
increase in the CML free adduct was 46% after pasteurization and 71% after
sterilization, but the CEL did not change significantly. These results suggest
that glycation adducts and AGE residues in heated food may have the most
Figure 7.5. Reactions of lysozyme with D-glucose and 3-deoxyglucosone and methylglyoxal. From
Humeny et al. (2002). Δm = increase in mass.
Table 7.3. Mass spectrometry of intact and glycated lysozymea
Reactants Mass (m/z) Mass Advanced Glycation reactions

increase glycation end
(Δ m) products (AGEs)
Lysozyme 14313
+ glucose 14371 +58 Da CML-lysozyme Carboxymethylation
+ methylglyoxal 14385 +72 Da CEL-lysozyme Carboxyethylation
+ 3-deoxyglucosone 14457 +144 Da 3DG-lysozyme Arginine
modificationb
+ methylglyoxal Max 15000 +687 Da MG-lysozyme MG modification
Glycated lysozyme Ca 15000 +687 Da AGE-lysozyme Amadori
modification
a
From Humeny et al. (2002). See Figure 7.5.
CML, Nε-(carboxymethyl)lysine; CEL, Nε-(carboxyethyl)lysine; 3DG, 3-deoxyglucosone; MG,
methylglyoxal.
b
Likely modification of an arginine residue with imidazolone (Figure 7.5).
impact on AGE exposure for non-diabetic subjects, if glycation free adducts are
not cleared from the body as a result of degenerative diseases.
Pentosidine is a fluorescent protein formed by bridging arginine and lysine
residues to produce a cross-link molecule that includes an imidazo-pyridinium
ring via a pentose intermediate (Table 7.5) derived from aged collagen. The
Table 7.4. LC-MS/MS analyses of protein glycation adduct residues and free adducts in
cow’s milk (nM)a
Sources AGE Raw Pasteurized Sterilized
Protein residue CML 337 877 2066

CEL 662 1809 1537
Free adduct CML 147 214 252
CEL 140 147 160
a
Adapted from Ahmed et al. (2005b).
CML: Nε-(carboxymethyl)lysine; CEL: Nε-(carboxyethyl)lysine
Table 7.5. Concentration of pentosidine in foodsa
Foods Concentration (mg/kg protein)
Sterile milk 0.1–2.6

Condensed milk 0.3–0.6
Bread crust 0.4–2.6
Roasted coffee 11–40
a
From Belitz et al. (2004).
Pentosidine is formed by bridging arginine and lysine residues via a pentose
intermediate.
N H NH2
N OH
N O
N H NH2
OH
O
Pentosidine
concentration of pentosidine is low in condensed milk (0.3–0.6 mg/kg), and

sterile milk (0.1–2.6 mg/kg), and higher in bread crust (0.4–2.6 mg/kg) and
roasted coffee (11 to 40 mg/kg). Pentosidine has been used as a glycoxidation
marker for advanced Maillard reaction in diabetes, aging and uremia. Other
AGE markers include C-reactive markers, lactate/pyruvate ratio, glycated
hemoglobin and collagen, changes in gene expression in endothelial cells and
macrophages.
C. Inhibition of AGE formation

The pathology associated with atherosclerosis and diabetes occurs only in
advanced stages, where the levels of reactive oxygen species exceed the
antioxidant protective capacity of cells to repair oxidative damage. In foods,
although antioxidants are efficient in preventing oxidation during early stages,
Table 7.6. AGE and glycation inhibitorsa
Inhibitors Properties Referencesb
AGE inhibitors
Aminoguanidine Nucleophilic compound with multi anti-AGE (1)
effects in vitro, in animals and humans with (2–3)
mixed clinical results resulting in abandoned
trials
Pyridoxamine Antilipidemic, antioxidant and AGE trapping (3–5)
Antioxidants, free radical trapping agents and metal chelators
Carnosine Antioxidant and anti-cataract agent, inhibits (6–8)
(β-alanyl-histidine) AGE cross-linked peptide, inhibits protein
modification by AGEs, traps lipid oxidation
products and aldehydes
Flavonoid-rich herbal extracts In vitro inhibition of enzyme inactivation by (9)
methylglyoxal, suppress fluorescent AGEs
Curcumin Chain-breaking antioxidant, anti-AGE and (10–12)
metal chelating properties
a
Condensed and expanded from Monnier (2003).
b
(1) Thornalley et al. (2003), (2) Brownlee (1994), (3) Metz et al. (2003b), (4) Khalifah et al. (1999), (5)
Onorato et al. (2000), (6) Babizhayev et al. (1994), (7) Munch et al. (1997), (8) Hipkiss et al. (1998, 2000,
2001), (9) Gugliucci and Menini (2002), (10) Masuda et al. (1999), (11) Masuda et al. (2001), (12) Jain
et al. (2006).
they are ineffective in more advanced stages of oxidation when they become
degraded and lose free radical scavenging activity. Similarly, in biology,
antioxidants are more effective in controlling oxidative deteriorations in the
earliest stages of degenerative diseases, but not when advanced secondary
oxidation products accumulate. Thus, the increase in decomposition products
of lipid oxidation in diabetes and hyperlipidemia may contribute to the in vivo
development of this disease and other disorders of aging. AGE formation and
oxidative stress resulting from glycosylation cause reduced levels of glutath-
ione content, vitamin E and vitamin C in plasma and tissues of diabetic
subjects. Therefore, hyperglycemia compromises antioxidant defenses in the
body by reduced glutathione and antioxidant vitamins E and C.
In advanced stages of oxidative deterioration, inhibitors of decomposition
reactions are expected to be more effective than antioxidants that scavenge free
radicals. Therefore, effective inhibitors of AGE formation include aldehyde
trapping agents, such as aminoguanidine and pyridoxamine, reducing com-
pounds, such as ascorbic acid, and chelating agents, which may prevent the
chemical degradation of proteins by decomposition products of lipid oxidation
in vitro (Table 7.6, ref 1–6). Several adducts of pyridoxamine formed by
incubation in vitro with PUFA have been identified in the urine of diabetic and
hyperlipidemic rats treated with pyridoxamine.
Figure 7.6. AGE inhibitors: aminoguanidine, pyridoxamine, dimethyl-3-phenacyl thiazonium chlo-

ride, curcumin.
Various antiglycation agents investigated for the inhibition of AGEs may act
by blocking carbonyl groups or reducing sugars, Amadori products, and
3-deoxyglucosones (Table 7.6, ref 7–13, Figure 7.6). They include anti-
oxidants or free radical scavenging antioxidants, metal chelators, carbonyl
traps and multifunctional inhibitors. Enzymes referred to as Amadoriases are
reported to catalyse deglycation. The results are, however, complex and
difficult to interpret in view of the poorly established biological significance of
AGEs formation and multiple pathways leading to diabetic complications in
humans.
1. Antioxidant therapy
Antioxidants may protect against free radicals derived from glycation, and
chelators may inhibit autoxidation of glucose and Amadori products by remov-
ing or inactivating transition metal catalysts. In biological systems, the presence
of advanced lipid oxidation products may be expected to lower the effective-
ness of antioxidants in biological tissues. Although α-tocopherol is known to
act as a prooxidant at high concentrations on the basis of hydroperoxide
formation, it is more effective in inhibiting aldehyde formation resulting from
the decomposition of hydroperoxides (Lipid Oxidation, 2nd ed, Chapter 4, p.
67). Since the main initiators of AGEs are dicarbonyls derived from decompo-
sition of lipid hydroperoxides and carbohydrates, free radical scavengers may
not be effective as antioxidants, but could inhibit aldehyde formation if they are
present at sufficiently elevated concentrations.
Different pathways for the inhibition of AGEs include the application of
cellular AGE-specific receptors, glutathione/glutathione peroxidase acting as a
cellular ‘antioxidant’ coenzyme system by reducing superoxide, promoting the

conversion of methylglyoxal to D-lactate, and the use of diets low in
glycoxidation products with reduced reactive carbonyls and their precursors.
N-Acetylcysteine inhibits LDL oxidation, and dietary glutathione suppresses
oxidative stress in vivo and prevents diabetic complications. These inhibitions
of AGEs support the hypothesis that AGE mixtures include components from
both oxidative and non-oxidative mechanisms. Antioxidants such as vitamin E
can also inactivate protein kinase C by inhibiting the formation of diglycerides.
Although the administration of diets containing multi-antioxidants can inhibit
early diabetic symptoms in rats, vitamin C improves vasodilation in diabetic
patients. However, low doses of vitamin E appear to fail to alter the risk of
diabetes in human patients.
Several flavonoid-rich herbal extracts containing antioxidant constituents
were also shown to inhibit free radicals and pentosidine, used as a glycoxidation
marker for advanced Maillard reaction in diabetes, aging and uremia (Table
7.6, ref 10). Curcumin and other phenolic compounds found in turmeric have
potent antioxidant and anti-inflammatory activities (Table 7.6, ref 11–13).
These dietary antioxidants were shown to be effective in directly scavenging
reactive oxygen species formed in animals during hyperglycemia, and in
inducing antioxidant enzymes (glutathione peroxidase and superoxide
dismutase). Indirectly, curcuminoids also have a protective role through the
redox regulation of glutathione. Experiments with diabetic animals showed
that curcumin supplementation can suppress cataract development and cross-
linking of collagen, promote wound healing and lower blood lipids and glucose
levels. With erythrocyte cell models treated with high levels of glucose to
simulate diabetes, curcumin prevents protein glycosylation and lipid
peroxidation.
The consumption of diets with low AGE content may constitute an important
preventive measure. However, the presence of advanced secondary lipid
oxidation products in arterial plaques in the presence of vitamins E and C in the
vascular wall may indicate that these antioxidant defenses are inadequate.
Furthermore, free radical antioxidants are known to be ineffective in oxidized
fats containing decomposition and advanced lipid oxidation products. In fact,
several clinical trials showed generally negative results on the effects of
antioxidants in diabetic patients.
2. AGE inhibitors
By trapping dicarbonyl compounds, aminoguanidine and pyridoxamine inhibit
the cross-linking of aortic and skin collagen and retard the development of
diabetic complications in diabetic animals (Figure 7.6, Table 7.6). These AGE
inhibitors are nucleophilic compounds effective in scavenging α,β-dicarbonyl
intermediates in AGE formation. Glyoxal and methyl glyoxal may originate
from Amadori or Schiff base intermediates, formed either during glycolysis or

from ascorbic acid and threonine. They have significant effects on dyslipidemia
(a disorder of lipoprotein metabolism, manifested by elevation of total choles-
terol, LDL and triglyceride, and a decrease in HDL) in diabetes. A large number
of AGE inhibitors can chelate metals, but the exact mechanism of how metals
mediate diabetetic complications is not well established. Both aminoguanidine
and pyridoxamine are potent inhibitors of nitric acid synthase and may either
catalyse or inhibit copper-dependent lipid peroxidation in vitro.
Most recent evidence supports a close relationship between atherosclerosis
and diabetes, through the accumulation of oxidized lipoproteins in the vascular
wall producing reactive carbonyl intermediates that cause cross-linking of
proteins. The same oxidative modifications of lipoproteins that contribute to
atherogenesis are accelerated in diabetes by further reactive carbonyls pro-
duced by oxidative modification and glycation of lipoproteins. Therefore, the
AGE inhibitors, aminoguanidine and pyridoxamine, known to trap carbonyl
intermediates, may also be useful in preventing atherosclerosis.
a) Aminoguanidine. This nucleophilic agent (Table 7.6, ref 1–3) traps reac-
tive dicarbonyl intermediates (glyoxal and glycoaldehyde, deoxyglucosone),
which may be elevated in diabetes through metabolic imbalance. Amino-
guanidine is also a good carbonyl scavenger which inhibits lipid peroxidation
and the formation of reactive oxygen species. It has bi-functional centers
consisting of the hydrazine group –NHNH2 and the guanidine group –NH–
C(=NH)–NH2, and acts by trapping the α,β-dicarbonyl glycation agents that
produce AGEs with arginine and lysine residues (Figure 7.6). Other physiological
dicarbonyl compounds scavenged by aminoguanidine are formed endogenously
by fragmentation and dehydration of hexoses and Amadori products, and can
be absorbed from ingested food and beverages. Aminoguanidine can also act
as an antioxidant by decreasing apoptosis, reactive oxygen species, lipid
peroxidation in cells and in animals, and copper-catalysed oxidation of
ascorbate. Aminoguanidine therapy decreases age-related disorders in rats.
In addition to trapping reactive dicarbonyls, preventing cross-linking forma-
tion, aminoguanidine was reported to have additional multiple effects by
inhibiting lipid peroxidation, catalase and oxidant induced apoptosis, and
lowering total and LDL cholesterol and triglycerides in diabetic subjects
independent of glucose control. However, at high concentrations
aminoguanidine can result in adverse diabetic complications through scaveng-
ing other beneficial carbonyl compounds by forming adducts with pyruvate
and glucose, and by inhibiting nitrous oxide synthase and decreasing protein
nitrosation. This inhibitor may also deplete essential carbonyls such as vitamin
B6 (pyridoxal-5-phosphate).
b) Pyridoxamine. This inhibitor traps carbonyl intermediates in lipid

peroxidation and prevents the modification of lysine residues, and the formation
of CML and adducts of lysine with malonaldehyde and hydroxynonenal during
copper oxidation of LDL in vitro (Table 7.6, ref 4–6). Pyridoxamine scavenges
toxic carbonyl products of glucose and lipid degradation, and traps reactive
oxygen species. This inhibitor is also reported to be more active than
aminoguanidine in preventing diabetic complications in animals. Its multiple
effects include trapping lipid oxidation products and dicarbonyl compounds,
metal-chelating properties, lowering lipids in diabetic and obese animal
models, inhibiting dyslipidemia, and decreasing chemical modification of skin
collagen. Along with its safety, pyridoxamine is claimed as a promising drug
for the treatment of diabetes and chronic conditions resulting from the
oxidative reactions and carbonyl compounds responsible for pathogenicity.
However, because these multiple effects are observed simultaneously, it is not
possible to identify the primary site of action of this AGE inhibitor and to
predict its activity in vivo.
Pyridoxamine was shown to be more active than aminoguanidine by inhib-
iting the conversion of Amadori intermediates to AGEs in the absence of excess
free sugar or reversibly bound Schiff base sugar.
The autoxidation of linoleate and arachidonate in the presence of pyridox-
amine (PM) produces several adducts, including hexanol-PM and
nonanediol-PM monamide that are detected in the urine of PM treated diabetic
and obese rats.
The 6-dimethylamino analog of PM (dmaPM) increased the radical trapping
ability of pyridoxyamine phenolic groups and may prove to be more useful in
limiting non-enzymatic protein modification associated with chronic age-
related diseases. Both dmaPM and Trolox prevented intermolecular protein
Table 7.7. Inhibition of N ε-(carboxymethyl)lysine (CML) and protein-cross-linking

during sugar glycation
Inhibitors Conc. mM % Protein cross-linkeda Relative CMLb

+ M buffer – M buffer
Control 0 40 100 5.5

Trolox 5 14 41 36
Trolox 1 16 – –
dmaPM 5 15 15 20
dmaPM 1 19 – –
PM 5 18 5.9 4.5
PM 1 27 – –
a
Culbertson et al. (2003a): 7 days ribose glycation 10 mg/ml bovine serum albumin (BSA) + 50 mM
ribose in PO4 buffer (pH 7.4).
b
Culbertson et al. (2003b): 21 days glucose glycation 10 mg/ml BSA + 250 mM glucose at 37°C in air;
% of + M buffer, not treated to remove trace metals, –M buffer, treated to remove metals with Chelex-
100 resin for 24 h, control = 44.2 mmol CML/mol lysine (BSA).
cross-linking more effectively than PM (Table 7.7). Therefore, dmaPM com-

bines the multiple functions of a carbonyl trap, metal ion chelator and
radical-trapping antioxidant. These results show that radical trapping provides
better cross-link prevention than carbonyl group trapping by PM alone. On the
one hand, PM was shown to trap keto-acid decomposition products during
linoleic acid oxidation. On the other hand, dmaPM may prevent protein cross-
linkage modification as well as break radical chain reactions and lead to more
lipid oxidation intermediates.
c. Other AGE inhibitors. AGE-protein cross-linking breakers have been

developed as therapeutic agents to disrupt AGE-derived cross-links in proteins,
and reverse the resulting cardiovascular stiffening caused by diabetes and other
age-related chronic diseases. Various AGE cross-links have been isolated by
in vitro glycation and identified in vivo. Lysyl–arginine is produced by
oxidative rearrangement of Amadori products; lysysl–lysyl is produced by
interactions of two lysines with two molecules of glyoxal and methyl glyoxal
and deoxyglucusone; and bis-lysine amide is produced interactions of bovine
serum albumin and glyoxal.
The first reported AGE-protein cross-linking breaker, phenyl thiazolium
bromide, was unstable in physiological buffers and was replaced by the more
stable and highly potent dimethyl-3-phenacyl thiazolium chloride (known as
ALT-711) (Figure 7.6). Oral administration of this AGE cross-linking breaker
has the ability to reverse already formed AGE cross-links, and to prevent cardiac
stiffening in aged animals. Whether the effect of ALT-711 is due to the inhibition
of AGE formation or breaking of AGE cross-links has not been well established.
Another approach to the inhibition of AGE formation involves the prepara-
tion of post-Amadori intermediates by protein glycation with ribose, which is
more reactive than glucose, and subsequent conversion to AGE-free glycated
proteins. This conversion of Amadori intermediates to AGEs in absence of
excess free or bound sugar is referred to as post-Amadori reaction and used to
evaluate putative Amadori inhibitors and termed ‘Amadorins’.
Carnosine (β-alanyl-L-histidine), normally present in human tissues at rela-
tive high concentrations (up to 20 mM), is known for its multiple activities as
an antioxidant, metal chelator, anti-tumor agent, aldehyde scavenger, anti-
ageing agent, and is claimed to be a potential non-toxic modulator of diabetes.
This dipeptide can protect proteins against cross-linking aldose, ketose hexose
and triose sugars and dialdehydes produced by lipid peroxidation. Carnosine
prevents the modification of proteins induced by methylglyoxal, and the AGE
product derived from a lysine–methylglyoxal adduct of proteins. By delaying
the aging of cultured human fibroblasts, carnosine is proposed to react with
protein carbonyl groups in vivo, and to have anti-glycation activity in rat
studies. Such reactions suggest carnosine’s possible role in controlling diabetes
and age-related diseases.
Table 7.8. Effect of green tea catechins on AGEs formationa
Tea catechins Concentration (μ M) Inhibition (%)
OH
OH
O
HO OH 5 33
10 47
O-G
OH
(–)-epigallocatechin-3-gallate
OH
OH
HO O
OH 5 42
O-G
10 48
OH
(–)-gallocatechin-3-gallate
OH
HO O
OH 5 38
10 49
O-G
OH
(–)-epicatechin-3-gallate
OH
OH
O
HO
OH 5 15
10 20
OH
OH
(–)-epigallocatechin
OH
OH
HO O
OH 5 40
OH
10 40
OH
(+)-gallocatechin
(–)-epicatechin 5 19
10 23
(+)-catechin 5 28
10 37
a
Nakagawa et al. (2002).
In addition to the well-known potent antioxidant activity of green tea

catechins (gallocatechin, epigallocatechin, catechin, epicatechin, epigallo-
catechin gallate, gallocatechin gallate and epicatechin gallate; see Figure 3.5),
these mixtures are reported to protect against free radical-, glucose- and
fructose-mediated albumin damage in vitro (on the basis of fluorescence
measurements used as indices of protein damage by free radicals) (Table 7.8).
The gallic acid catechin derivatives were significantly more effective than the
non-gallic acid containing catechin and epicatechin, indicating that the galloyl
substituents play an important protein protective activity. The catechin and
catechin derivatives were more active than the epicatechin and epicatechin
derivatives. Differences between steric structures play a more important part in
AGE formation than the presence of the 3 or 2 hydroxy groups in the B ring.
This protective action against protein oxidation and glycation was shown to be
greatly affected by chemical structure in in vitro studies, but the mechanisms
were not established.
All AGE inhibitors are effective metal chelators. In studies of copper-
catalysed autoxidation of ascorbate, AGE inhibitors such as aminoguanidine
and pyridoxamine were very effective copper chelators. Therefore, in many in
vitro studies inhibition of AGE formation the chelating or antioxidant activity
of AGE inhibitors may play a more significant role than their carbonyl trapping
activities. The capacity of plasma to protect against iron-induced lipid
peroxidation was shown to be compromised in diabetic patients by significant
decreased concentrations of transferrin, ceruloplasmin and albumin and result-
ing decreased iron-binding capacity. However, this effect could not be explained
by glycation of transferrin and higher non-transferrin-bound iron levels.
Animal studies suggested that the effects of AGEs in diets may be more
injurious to vascular and kidney tissues than those caused by hyperglycemia or
hyperlipidemia. Dietary AGEs restriction may therefore suppress diabetes and
cardiovascular disease in the same way as demonstrated by marked caloric
restrictions in animals. Food-derived AGEs were shown to have protein–
protein cross-linking activity and suppression of antioxidant reserves based on
reduction of GSH (glutathione)/GSSG (oxidized glutathione) ratio and in-
creased vascular cell injury.
Improved and more reliable methods are required to analyse and detect
AGEs to understand more successfully their role in the pathogenesis of
atherosclerosis and diabetes and their complications. Confirmation of animal
studies in humans is also required, together with carefully controlled dietary
intervention studies on the effects of antioxidants in reducing AGEs.
Information on the impact of the Maillard reaction on human health is
growing rapidly. Research into aging and diabetes has assumed an increasingly
broad scope to include a common goal in disease and nutrition. The develop-
ment of multiple diabetic complications in multifactorial disorders of aging
could be delayed by scavenging toxic AGEs and reducing their accumulative
damage. The details of molecular damage by oxidative and carbonyl stress on

cells and progression of various degenerative diseases is still not well under-
stood. More research is needed to establish the physiology and fate of Maillard
browning reaction products absorbed from the diet or produced and eliminated
in vivo. Potentially, using AGE inhibitors to delay pathology of age-related
diseases might contribute to a prolonged and healthier lifespan. Although
aminoguanidine therapy has demonstrated that inhibition of AGE formation
may delay the onset of diabetic complications, the toxicity of this treatment
prevented its clinical application. For this reason, the safety of pyridoxamine
makes this inhibitor a more promising therapeutic agent. However, the mode of
action of pyridoxamine in vivo is still unclear.
D. Future research
Advances in new methodology continue to improve the characterization of
browning reaction products. Novel approaches include the use of carbon-13
labeled carbohydrates and radiolabeled substrates, together with GC-MS tech-
niques, to elucidate the mechanism of formation of specific AGE compounds.
Much more research is required to identify the metabolic fate of specific Maillard
reaction products and evaluate their risks or benefits, if any. The new techniques
of proteomics are being applied in this field to analyse modifications of proteins
as affected during food processing. Personalized nutrition is also being devel-
oped on the basis of the proteomics of the browning reaction in foods and
biological systems. More research is needed to clarify the nutritional benefits and
risks of consuming browning reaction products and their effects on metabolism
in the body. The pharmacological applications of AGE inhibitors may be
expanded to include atherosclerosis, cancer and neurodegenerative disease.
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CHAPTER 8
Future perspectives
An important question that concerns nutritionists today is whether we need

additional or more effective natural antioxidants in our diet, to reduce oxidative
stress from either excessive dietary polyunsaturated lipids or from other
environmental factors. Although intervention studies have shown that the use
of antioxidants does not reduce the risk of cardiovascular disease, there is a
general consensus, mainly based on epidemiological studies, that we should
increase the consumption of vegetables, fruits and whole grains that are rich in
antioxidants, but such studies are often confounded and not convincing. The
confounding effect of oxidized lipids may also account for the negative and
mixed effects found in many epidemiological studies of dietary antioxidants,
because giving antioxidant supplements may not overcome problems of lipid
oxidation products in foods. These potential dietary problems due to lipid
oxidation are not yet clear, however, and deserve much more research. To
understand the nutritional effects of thermally oxidized fats more fully, much
more detailed information is required on the specific lipid oxidation products
responsible for their effect during postprandial lipemia (Chapter 6). The
marketing of nutritional supplements containing a concentrate of flavonoid
mixtures at relatively high doses may be questionable, because these products
exceed the level that one could reasonably received in most diets rich in fruits
and vegetables.
Polyphenolic compounds are present in most plant foods in a broad diversity
of structures and concentrations, varying according to genetic factors and
environmental conditions. Their levels in foods and beverages vary widely.
Red wine, apple and orange juices, and legumes are particularly rich in
polyphenolic compounds. The literature on antioxidants has exploded in the
past decade, as a result of accumulating evidence that they may add nutritional
benefits to foods and beverages containing phenolic compounds. There is
much in vitro evidence supporting a possible beneficial role for polyphenols in
preventing cardiovascular disease and cancer. The beneficial nutritional effects
of fruits and vegetables have been linked to increased levels of antioxidants in
the body. However, plants contain an abundance of other potentially nutritive
constituents besides antioxidants, including fiber, phytates, organic phos-
phates and a multitude of phenolic compounds with non-antioxidant activities.
In fact, very few studies have provided direct evidence that the benefits of
eating fruits and vegetables are actually due to in vivo antioxidant activity.
Many phenolic compounds recognized for their antioxidant activity in vitro
may have different and additional in vivo properties.
217
Studies of atherosclerosis with animal models have demonstrated the anti-

oxidant effect of vitamin E and phenolic compounds in delaying the progress
of this disease. However, human clinical trials of antioxidants have given
inconsistent and mixed results. Studies of disease with animal models do not
necessarily represent the same disease process and risk in humans. Although
the accumulation of circulating oxidized LDL by the artery and the develop-
ment of lesions within a few months has been demonstrated in various animal
models, human lesions develop much more slowly over periods of decades.
Therefore, the results of animal testing of antioxidants do not agree with those
of human studies, because they are based on significantly different end points.
The same divergence may apply to studies comparing in vitro versus in vivo
testing of antioxidants being based on different end points of oxidation.
A. Tocopherols
Although α-tocopherol (Chapter 6.G.1) is one of the most studied natural
phenolic antioxidants, many questions arise about the proposed nutritional
benefits, if any, of γ- and δ-tocopherol. In addition to α-tocopherol’s well-
known chain-breaking properties, non-antioxidant functions have been reported,
such as acting as a signaling molecule in regulating gene expression, and in
preventing cancer and atherosclerosis. The in vivo antioxidant activity of
α-tocopherol in providing beneficial nutritional effects is not generally accepted.
α-Tocopherol may not be effective in controlling lipid peroxidation under in
vivo conditions, because of the high reactivity of lipid peroxy radicals under
these conditions. In fact, the reported negative prooxidant effects of α-tocophe-
rol are not biological relevant when observed under certain conditions using
artificial azo initiators such as AAPH and other oxidants. These non-physi-
ological prooxidant effects have been perhaps over-emphasized and exaggerated
in the current literature (Chapter 6.F).
Because the protective role of α-tocopherol in preventing atherosclerosis by
acting as a biological antioxidant has been challenged by its activity in
mediating peroxidation (alas in the presence of artificial azo initiators such as
AAPH), its beneficial activity has now been attributed to several non-antioxi-
dant functions. Many effects of tocopherols beyond their chain-breaking
activity have now been recognized. In early stages of atherogenesis,
α-tocopherol is effective in inhibiting the action of protein kinase in vascular
smooth muscle cells (Table 8.1, ref 1, 2). The resulting inhibition of smooth
muscle cell proliferation is apparently due to the stimulation of phosphatase
2A, an enzyme that catalyses dephosphorylation and inactivation of protein
kinase. Monocyte-endothelial adhesion is significantly reduced by down-
regulation of intracellular and vascular cell adhesion molecules (ICAM-1,
ICAM-2) (Table 8.1, ref 3). α-Tocopherol also inhibits platelet adhesion and
aggregation, which contribute to arterial thrombosis. The mechanism proposed
FUTURE PERSPECTIVES 219
Table 8.1. Non-antioxidant activities of phenolic compoundsa
Phenolics Inhibition, down-regulation and other effects Ref.b
α-Tocopherol Inhibits protein kinase C in vascular smooth muscle (1, 2)

cells and cell proliferation
Inhibits monocytes-endothelial cell adhesion (3)
Inhibits platelet adhesion and aggregation (4)
Inhibits COX-2 and 5-LOX that promote (5, 6)
inflammation
Inhibits scavenger receptors SR-A and CD36 (7, 8)
of oxidized lipoproteins
γ-Tocopherol Inhibits peroxynitrite, PGE2, total nitrate/nitrite, (9–11)
TNFα, inflammation-mediated damage
Flavonoids (tea, wine, Effects on signal transduction pathways mediated (12–18)
plant phenolics) by growth factors (EGC, PDGF), UV, and
inflammatory mediators (LPS); inhibition of COX,
LOX, xanthine oxidase, metalloproteinase,
sulfotransferase and platelet aggregation
Green tea catechins Inhibit UV-induced skin damage (immuno- (19)
suppression, photoaging, inflammatory dermatoses,
photocarcinogenesis)
Quercetin and metabolites Cellular uptake of quercetin (Q) and its in vivo (20)
metabolites (methyl Q, and Q glucuronide) and
generation of Q-GSH
Q metabolites (urine) Modulate glucuronosyltransferase, monooxygenase, (21)
and glutathione S-transferase
a
From Schneider (2005) and Halliwell et al. (2005).
b
References: (1) Boscoboinik et al. (1991), (2) Tasinato et al. (1995), (3) Cachia et al. (1998), (4) Mabile
et al. (1999), (5) Eligini et al. (1999), (6) Linton and Fazio (2004), (7) Riciarelli et al. (2000), (8) Devaraj
et al. (2001), (9) Jiang et al. (2001), (10) Huang and Appel (2003), (11) Jiang and Ames (2003), (12)
Laughton et al. (1991), (13) Isemura et al. 1999), (14) Schewe et al. (2001), (14) Wiseman et al. (2001),
(15) Marchetti et al. (2001), (16) Van Hoorn et al. (2002), (17) Sadik et al. (2003), (18) Murphy et al.
(2003), (19) Katiyar et al. (2001), (20) Spencer et al. (2003, 2004), (21) Hong and Mitchell (2004).
Abbreviations: COX-2, cyclooxygenase-2; EGC, epigallocatechin; GSH, glutathione; LPS,
lipopolysaccharide; 5-LOX, 5-lipoxygenase; PDGF, platelet-derived growth factor; SR-A, scavenger
receptor-A; CD36, scavenger receptor; PGE2, prostaglandin E2; TNFα, tumor necrosis factor-α; UV,
ultraviolet.
for this activity involves the anti-platelet action of vitamin E by inhibiting

phosphorylation of endothelial nitric oxide synthase and increasing the release
of nitric oxide (Table 8.1, ref 4).
Atherosclerosis is an inflammatory disease that develops by the expression
of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) in activated
macrophages, which contribute to the synthesis of inflammatory mediators
such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) (Table 8.1, ref 5, 6).
Inhibition of either COX-2 or 5-LOX could attenuate the inflammation compo-
nent of atherosclerosis. α-Tocopherol inhibits the uptake of oxidized LDL into
macrophages by down-regulating mRNA (messenger ribonucleic acid) and

protein expression of the scavenger receptors SR-A and CD36 (Table 8.1, ref
7, 8). These receptors function by recognizing and scavenging oxidized lipo-
proteins and transform macrophages into foam cells.
γ-Tocopherol inhibits the activity of 5-LOX in monocytes by reducing the
levels of 5-LOX metabolites, PGE2, total nitrate/nitrite, TNFα (tumor necrosis
factor), and attenuates the damage of inflammation in atherosclerosis (Table
8.1, ref 9–11). Therefore, there is increasing interest in recommending vitamin
E supplements that comprise a mixture of tocopherol homologs rather than
pure α-tocopherol.
B. Flavonoids
Flavonoids (Chapter 6.G.3) possess a multitude of antioxidant activities. As
chain-breaking antioxidants, they scavenge radical species. As hydrogen do-
nors, they inhibit lipid oxidation by regenerating α-tocopherol. As metal
chelators, they inhibit free radical formation and preserve their own free radical
scavenging activity. Flavonoids have other important functions beyond their
antioxidant properties in protecting DNA from oxidative damage, inactivating
carcinogens and xenobiotics (foreign biologically active compounds), and
inhibiting enzymes that promote carcinogenesis. Flavonoids thus exhibit a
variety of biological reactions and processes that are considered nutritionally
as non-essential components in our diet. Their beneficial values must be
therefore viewed according to particular contexts that should be defined for
different age groups. Misleading nutritional claims should be avoided if they
do not define the specific benefits and contexts in which they work.
Flavonoids in tea, wine and other plant phenolics have multiple biological
activities, including effects on signal transduction pathways mediated by
growth factors, UV and mediators of inflammation, inhibiting cyclooxygenases
and lipoxygenases, xanthine oxidase, metalloproteinase, sulfotransferase and
platelet aggregation (Table 8.1, ref 12–18). Green tea catechins may act by
inhibiting UV-induced skin damage by protection against immunosuppression,
photoaging, inflammatory dermatoses, and photocarcinogenesis (Table 8.1, ref
19). Although not critically evaluated, some commercial skin lotions contain-
ing tea phenolics and flavonoids claim to have protection benefits against sun
exposure. Cellular uptake of quercetin and metabolites led to the generation of
glutathione adducts of quercetin (Table 8.1, ref 20). The potential protective
effects of these quercetin adducts, however, remain unclear.
Much evidence in the literature supports the many potential health benefits
of flavonoids from plant foods, based on their bioactivity in vitro (Table 8.2).
Although much of this evidence suggests that flavonoids have a protective
effect against cardiovascular disease and some types of cancer by decreasing
oxidative damage, a number of intervention trials failed to show consistent
Table 8.2. Sources and potential health effects of flavonoidsa
Phenolic compounds Selected sources Biological effects
Quercetin, kaempferol, Onions, apple, tea, Antioxidant and antimutagenic activities

catechin red wine, cocoa, Decrease: total cholesterol, LDL
chocolate oxidation, tumor, platelet aggregation,
eicosanoids
Increase: HDL
EC, EGC, ECG, EGCG Green/black tea, Antioxidant/antimutagenic/
cocoa, chocolate anticarcinogenic activities
Decrease: tumors, apoptosis, LDL
oxidation, platelet aggregation
Resveratrol Grapes, red wine, Decrease: LDL oxidation, platelet
peanuts aggregation, thrombosis, eicosanoid
synthesis
Antioxidant/ antimutagenic/
anticarcinogenic activities, antitumor
and antiestrogen activities.
Tyrosol, hydroxytyrosol, Extra virgin olive oil Antioxidant activity
oleoeuropeine, caffeic Decrease LDL oxidation
acid, coumaric acid
a
Adapted from Kris-Etherton et al. (2002).
Abbreviations: LDL, low-density lipoproteins; HDL, high-density lipoproteins; EC, epicatechin; EGC,
epigallocatechin; ECG, epicatechin gallate; EGCG, epigallocatechin gallate.
beneficial effects from the use of antioxidant supplements. Unfortunately, too

many of these trials were based on the administration of a single antioxidant at
relatively high doses. Putative mechanisms include inhibition of LDL oxida-
tion in vitro and in cell culture, platelet aggregation and adhesion,
anti-thrombotic effects, vasorelaxation by modulation of nitric oxide produc-
tion by vascular endothelium, and favorable alteration of eicosanoid synthesis.
On one hand, flavonoids may exert direct antioxidant effects by scavenging
radical species, inhibit lipid peroxidation by recycling α-tocopherol, chelate
prooxidants, prevent free radical formation and protect against carcinogenesis.
On the other, beyond their antioxidant activities, flavonoids may interfere with
carcinogenesis by protecting DNA from oxidative damage, by deactivating
carcinogens, by inhibiting the expression of mutated genes and the activity of
enzymes that promote carcinogenesis, and by detoxifying xenobiotics.
Olive oil and vegetable oil phenolics were shown to reduce the oxidizability
of LDL in the postprandial state, apparently as a consequence of its high
concentration of oleate, which is about 40 times more stable to oxidation than
linoleate (Lipid Oxidation, 2nd ed, 2005, Chapter 2). The tocopherol content of
olive and other vegetable oils may also contribute to their antioxidant effects.
The phenolic content of extra virgin olive oil averaging about 500 mg/l
provides high antioxidant activity in vitro, with hydroxyltyrosol being one of
Table 8.3. Activity of methylated derivatives of catechin on inhibition of LDL

oxidation catalysed by coppera
Catechin derivatives EC50 (μ M min–1) Lag time (min)
Control – ~80
OMe
OMe
B
HO O
A C 3.8 ~110
OH
OH
3',4'-dimethyl
OH
OH
B
HO O
A C 0.68 ~160
OH
OH
Catechin
OH
OH
B
MeO O
A C 0.63 ~180
OH
OMe
5,7-dimethyl
OH
OMe
B
HO O
A C 15 –
OH
OH
3'-methyl
OMe
OH
B
HO O
A C 12 –
OH
OH
4'-methyl
3',4',5,7-tetramethyl > 100 –
a
From Cren-Olivé et al. (2003). Copper catalysed oxidation of human LDL using 1.6 μM CuSO4 in PBS
buffer at 37oC.
Oxidation followed by formation of conjugated dienes.
Lag times estimated from Figure 1 in the paper of Cren-Olivé et al. (2003).
the most active components (Table 6.4, ref 12). Soy foods provide also rich
sources of isoflavones with phytoestrogenic activity that reduce total and LDL
cholesterol levels, and lower blood pressure in selected human populations
(Chapter 6.G.3.c).
1. Catechin
In red wine and grapes, catechin was shown to protect LDL from oxidation in
vitro. A comparison of the in vitro activity of different methylated catechin
analogs for the copper-catalysed oxidation of LDL oxidation showed that
dimethylation of the B ring had stronger effects in decreasing activity than
dimethylation of the A ring (Table 8.3). The 5,7-dimethylated catechin was in
fact slightly more active than catechin. The mono-methylated analogs on the B
ring were even less active than the corresponding dimethylated derivatives.
Since the mono- and dimethylated metabolites had values of EC50 varying from
0.63 to 15 μM min–1, the question is whether sufficient concentrations can be
absorbed in the body to act as effective antioxidants. Previous studies showed
that catechin from red wine (Table 6.3) is absorbed to varying degrees, with the
maximum concentration of plasma reaching only 0.091 μmol/l of catechin, but
rapidly conjugated into methylated, sulfates and glucuronide metabolites.
Therefore, the experiments of Table 8.3 showed that much higher concentra-
tions are required to increase the resistance of LDL to in vitro oxidation than
can be achieved in the plasma. On one hand, these results may support a non-
antioxidant mechanism for the protective effects of catechin and flavin-3-ols
(Chapter 6.G.3.a). On the other hand, no published data are available on
whether or not a higher steady state concentration of catechin metabolites could
be achieved in human plasma, after daily consumption of one or more glasses
of red wine, as presumably is the practice where the French Paradox was first
reported. For individuals with risk factors for atherosclerosis, most notably
high levels of circulating LDL, it may be that consuming catechin and other
sources of flavonoids could protect LDL from the deteriorative oxidation
reactions associated with atherosclerosis. The in vivo activity of phenolic
compounds as antioxidants is still not well understood.
2. Quercetin
This phenolic antioxidant represents an important plant component in human
diets, ranging from 2 to 250 mg/kg wet weight in fruits, 0 to 100 mg/kg in
vegetables, 4–16 mg/l in red wine, 10–25 mg/l in tea, and 3–13 mg/l in fruit
juices. Ingestion of 98 mg quercetin from apples resulted after 2.5 hours in a
maximum concentration of 300 nmol/l in hydrolysed plasma (Lipid Oxidation,
2nd ed, 2005, Table 13.11, p. 439). The activity of the conjugated metabolites
for the inhibition of LDL oxidation catalysed by copper was significantly
Table 8.4. Inhibition of copper-catalysed oxidation of LDL by quercetin

derivativesa
Quercetin derivatives (0.5 μ M) Lag phase (min)
Control 45
Quercetin (Q) 310
Q-glucuronides 90
Q-3-O-sulfate 110
Q-3'-O-methyl 90
a
Manach et al. (1998). Measured by conjugated diene formation.
diminished compared to precursor quercetin aglycone (Table 8.4). At the same

concentration of 0.5 μM, the lag period of conjugated diene formation de-
creased from 310 min for quercetin, to 110 min for quercetin 3-O-sulfate, to
90 min for quercetin glucuronides and 3'-O-methylquercetin (also called
isorhamnetin). Although the hydroxylated 3-position of the sulfate and
3'-position of the methyl derivatives were specified, the corresponding
hydroxylated position of the glucuronide substituent was not. Unfortunately,
the 3-O-sulfate derivative substituted on the C ring cannot be compared with
the 3'-O-methyl derivative substituted on the B-ring, which would be expected
to have a more significant effect on antioxidant activity. In comparing the
activity of quercetin glucuronides towards lipoxygenase, the 3'-isomer on the
B-ring was an active inhibitor, but the 3-isomer on the C-ring was not active.
Lipoxygenases play a part in the initial events of atherosclerosis by inducing
LDL oxidation.
The derivatization of quercetin observed after absorption would be expected
to modify its hydrophilic/lipophylic interfacial properties in plasma and their
resulting physiological effects, as discussed in Chapter 3. Hydrophilic flavonoids
and other plant phenolic compounds may also be more active in vivo by being
more protective in plasma because they cannot be properly evaluated by copper
oxidation of isolated LDL ex vivo. The ultracentrifugation preparation of LDL
would be expected to wash out hydrophilic antioxidants and change their
interfacial properties, as previously observed when vitamin C was compared
with vitamin E (Lipid Oxidation, 2nd ed, 2005, Table 13.6, p. 422). Attempts
were made to minimize this problem by diluting serum used in the copper-
catalysed oxidation to evaluate the effect of postprandial lipemia on
atherosclerosis (Lipid Oxidation, 2nd ed, 2005, Table 13.14, p. 443).
More recent studies on the intracellular metabolism of quercetin and its
methyl metabolites revealed significant uptake into skin cell fibroblast (Table
8.1, ref 20). Uptake of quercetin led to the in vivo formation of physiological
metabolites, including the glutathione quercetin adduct and its oxidation
(Figure 8.1). The 3'-O-methyl quercetin metabolite was readily oxidized into
3'-O-methyl quercetin-5-quinone methide and quinone derivatives, due to the
FUTURE PERSPECTIVES
225
Figure 8.1. Cellular conversion of quercetin into its O-methylated metabolites in fibroblasts and its postulated modulation of signaling pathways (adapted from
Spencer et al., 2004).
specificity of peroxidases toward the 4'-hydroxy group on the B-ring. Forma-

tion of the glutathione adduct of quercetin may occur either by the catalytic
action of glutathione S-transferase, or by nucleophilic addition of SH to
quercetin quinone. The formation of oxidation products of 4'-O-methyl querce-
tin was not observed, apparently because oxidation is hindered by the lack of
conjugating hydroxyl groups. Although quercetin and its methylated metabolites
are suggested to be protective against oxidatively induced cellular damage in
vivo, the precise mechanism remains to be established. After consumption of
cooked onions, a major source of quercetin, a large number of complex
metabolites were identified in human urine (Table 8.1, ref 21). The seven
quercetin metabolites included quercetin-monoglucuronides, -methyl mono-
glucuronides, -glucuronide sulfate, -diglucuronides, -methyl diglucuronides,
-glucoside sulfate and methyl quercetin. These metabolites can modulate
enzymes such as glucuronosyltransferase, monooxygenase and glutathione
S-transferase.
3. Tea catechins
The complex mixtures of catechin and catechin gallates in green tea have
received much attention, because of the many nutritional health effects claimed
for this tea, which is considered the most consumed beverage in the world. In
addition to a reduction in the risk of cardiovascular disease and cancer, it is
claimed to offer an anti-hypertensive effect, body weight control, antibacterial
and antiviral activity, skin protection against solar ultraviolet radiation, to
promote oral health, and to have anti-fibrotic and neuro-protective properties.
The relative antioxidant activity of the gallic acid derivatives of green tea
shows widely differing trends according to the different assays and lipid
systems used (Table 8.5). The broad variation in relative antioxidant trends
obtained between the antiradical assays TEAC, DPPH, superoxide and LDL
oxidation (Table 8.5, ref 1–5), reflects a multitude of analytical problems,
including evaluations that were not made at the same molar concentrations, and
the use of artificial chromogenic radicals that have no physiological relevance,
and do not reflect the true protective properties of antioxidants. The activity of
multifunctional tea catechins thus depends on a multitude of factors, including
the colloidal properties of the substrates, the conditions of oxidation, the
localization of antioxidants in different phases, the stages of oxidation, the
system composition, the presence or absence and the type of oxidizable
substrate, the mode of accelerating oxidation, and the methods used to assess
oxidation, as discussed in Chapter 4, Sections B and C. In addition to structural
differences based on polarity according to the number of hydroxyl groups per
molecule, the relative activities are greatly influenced by their interfacial
properties between phases in emulsions and liposomes, compared to bulk fish
and vegetable oils (Table 8.5, ref 6). The prooxidant activity of these highly
Table 8.5. Relative antioxidant activities of green tea catechinsa
Assay Relative activities Ref.
TEAC ECG > EGCG > EGC > GA > EC ≈ C (1)

LDL EC = C = ECG = EGCG > EGC > GA
DPPH EGCG > ECG > EGC > EC (2–4)
DPPH EGCG > EC > EGC > C > GA
DPPH EGCG > ECG > EGC > GA > EC ≈ GA
Superoxide EGCG, ECG, EGC > carnosol, carnosic acid
Fish oils ECG > EGCG > EGC > EC (5)
Corn oil EGC > EGCG ~ ECG > GA > PG > EC ~ C (6)
Emulsion Prooxidants
Liposome EGCG > EC > EGC > ECG > C
References: (1) Salah et al. (1995), (2) Chen and Ho (1995), (3) Gardner et al. (1998), (4) Nanjo et al.
(1999), (5) Wanasundra and Shahidi (1996), (6) Huang and Frankel (1997).
Abbreviations: see Table 8.2 for tea catechins; C, catechin; DPPH, 2,2'-diphenyl-1-picrylhydrazyl;
TEAC, Trolox equivalent antioxidant activity; GA, gallic acid.
polar catechin gallates, including gallic acid, in oil-in-water emulsions is

attributed to their partition into the aqueous phase, where they can effectively
reduce metals into the more catalytically active lower valence state. The
increased activity of green tea catechins in a lecithin liposome rather than an
emulsion system was explained by their greater affinity with the polar surface
of the lecithin lamellar bilayer (Lipid Oxidation, 2nd ed, 2005, Chapter 9, D.4,
p. 242).
The major polyphenol in green tea, EGCG, was shown to have significant
preventative effects against photocarcinogenesis and phototoxicity in mouse
models. The effects of topical application of EGCG to human skin before UV
irradiation were tested on antioxidant enzymes (Table 8.6). UV exposure to
human skin resulted in a decrease in total glutathione level and glutathione
peroxidase activity, and an increase in catalase activity. Pre-treatment with
EGCG restored the UV-induced decrease in GSH levels and afforded protec-
tion against glutathione peroxidase. The elevated level of catalase after UV
exposure indicated conversion of the increased H2O2 to water and oxygen.
EGCG also inhibited UV-induced epidermal lipid peroxidation, which is
detrimental to cell membrane structure and functions (loss of fluidity, in-
creased ion permeability and rupture of cell membrane). The increase in
catalase activity by UV exposure indicates that skin cells activated their
defense system to protect them from the adverse effects of UV irradiation.
There is further evidence that the 3'-O-methylated derivatives of epicatechin
produced in vivo have strong cytoprotective effects. The neuroprotective effect
of EGCG against oxidative stress-induced cell death was attributed to the
activation of protein kinase and partly mediated within signaling pathways.
Table 8.6. Effect of EGCG on reduced glutathione, glutathione peroxidase and catalase
activities on UV-induced effects on epidermis of human skina
Treatment GSH GSH peroxidase Catalase
Control (non-UV) 100 100 100

EGCG control 102 97 98
UV 24 h 65 46 145
EGCG + UV 24 h 82 (26)b 72 (56)b 123c
a
From Katiyar et al. (2001). Data presented as percentage of control.
b
Percent increase on EGCG treatment.
c
Increase in catalase activity.
EGCG and other green tea catechins also play an important role in inducing
apoptosis (cell death) in cancer cells, and may be useful in treating different
cancers, where activation of the signaling pathways influence tumor survival
and growth. EGCG and other flavonols may also play a role in preventing UV
and light induced skin damage, with greater protective effects when applied
topically than when given orally. These recent findings support the view that
flavonoids can have various beneficial effects on cells through their modula-
tion of protein and lipid kinase signaling cascades, which are independent of
their potential antioxidant activities.
C. Nutrition studies
Because oxidative damage is involved in atherosclerosis, antioxidants have
been generally thought to contribute to cardiovascular protective effects.
However, intervention trials with vitamin E and phytochemicals produced
confusing results. In addition to phenolic antioxidants, plant foods contribute
a multitude of constituents that may impart health benefits, including fibers, B
vitamins, folic acid and agents that modulate nitric oxide production. Supple-
mentation trials with vitamin E produced mixed results, showing benefits as
well as no protective effects. In some studies, the intake of vitamin E necessary
to exert cardiovascular benefits was higher than can be obtained from the diet.
Although no effect was reported in feeding studies with teas on the ex vivo
oxidation of LDL, some mixed results were reported on red wine. Most studies
of ex vivo LDL oxidation used copper to initiate oxidation, but other oxidants
could also be involved, including heme proteins, lipoxygenase, peroxynitrite
and hypochloride, as well as endothelial cells. These studies are also difficult
to interpret because, as discussed above, the hydrophilic flavonoids may wash
out from LDL during preparation. The most recent literature does not generally
support direct antioxidant effects of dietary phenolic compounds.
The health effects of flavonoids depend greatly on their intake and
bioavailability, which vary greatly according to individuals, the plant sources,
food processing and storage. The very low concentrations (nmolar levels) of
monomeric flavonoids found in human plasma after consumption may indicate
that they are either poorly absorbed or rapidly eliminated as metabolites
(Chapter 6.G.3.a). However, flavonoids can be extensively bound to plasma
proteins. Although these proteins would have a lower binding affinity towards
the conjugated metabolites than the corresponding aglycones, a significant
level of binding would be expected by the much larger concentration of plasma
proteins (mmolar levels) than the flavonoid metabolites (nmolar levels). A
daily consumption of these plant products may be required to maintain benefi-
cial levels of metabolites in blood. If some polyphenol metabolites accumulate
in cells of different tissues, they may vary in structures from those identified in
plasma. Although such structural differences could be determined in animal
studies, important differences probably exist between animals and humans and
their metabolisms of plant phenolics. Since much more research on the
bioavailability of polyphenolic compounds in humans has focused on the
aglycones rather than their corresponding metabolites, future research will
require more emphasis on the many known metabolites, and at the same ranges
of concentrations found in blood. Clinical studies will also require better
biomarkers, used under experimental conditions relevant to human nutrition
and health.
D. Activities in the gastrointestinal (GI) tract

Much evidence has accumulated recently that suggests flavonoids and various
polyphenols could provide antioxidant protective effects in the GI tract before
absorption (Chapter 6.E). This hypothesis is supported by the fact that greater
concentrations of phenolic compounds are found in the stomach and intestine
lumen than in the plasma. Polyphenols may also decrease the susceptibility of
LDL to oxidation resulting from oxidative stress of postprandial oxidized fats
and sugars. Such antioxidant protection would be considered particularly
important in hyperlipidemic and hyperglycemic subjects. The protective ef-
fects of plant foods can also be attributed to other components in foods (e.g.
fibers, B vitamins, folic acid and agents that modulate nitric oxide).
The GI tract can be exposed to a range of reactive oxygen, chlorine, and
nitrogen species from the diet and from phagocytes, mixtures of ascorbate and
iron, heme proteins, lipid hydroperoxides, aldehydes and isoprostanes, nitrite
converted to HNO2 by gastric acid, H2O2 in beverages, and bacteria and toxins.
Flavonoids and other phenolic compounds can therefore have a direct effect by
scavenging reactive oxygen species and chelate iron in the GI tract. They could
inhibit the catalytic effect of heme proteins and enhance the activities of
antioxidant enzymes in the GI tract. α-Tocopherol is selectively separated from
plant mixtures in the diet by the specific α-tocopherol transfer protein in the
liver. This selective action leads to a concentration of γ- and δ-tocopherols, in
the GI tract. Although γ- and δ-tocopherols are more active antioxidants in

vitro, they may have beneficial protective effects in the GI tract. γ-Tocopherol
is also known to scavenge reactive oxygen and nitrogen species better than α-
tocopherol, and to inhibit cyclooxygenase.
Postprandial hyperlipemia is a known risk factor for atherosclerosis. The
postprandial increase of lipid oxidation products could adversely affect the
oxidant–antioxidant balance (Chapter 1.B.2) and increase the susceptibility of
LDL to oxidation. Wine and grape seed proanthocyanidins were shown to
minimize the postprandial oxidative stress by inhibiting the increase of plasma
lipid hydroperoxides and increasing the antioxidant levels in plasma. These
effects resulted in enhancing the resistance of LDL to oxidation ex vivo. The
content of lipid peroxides in chylomicrons was significantly lower after a meal
supplemented with grape seed extract, compared to the control meal. There-
fore, supplementation of a meal with grape seed extracts minimizes postprandial
oxidative stress by decreasing the oxidants and increasing the antioxidant
levels in plasma, resulting in increased resistance of LDL to oxidation.
Wine usually taken with meals is considered a key element of Mediterranean
diets. The evidence supports a significant protective effect of wine against
cardiovascular disease by decreased oxidative damage to blood plasma
lipoproteins. Diets containing lipid oxidation products may also be protected
by red wine, as shown by relative increased plasma concentration of vitamin E,
sulfhydryl groups, uric acid and total antioxidant capacity compared to the
control (Lipid Oxidation, 2nd ed, 2005, Table 13.13, p. 442). Postprandial LDL
was more resistant to oxidation after consuming red wine with a high-fat fried
meal. Wine phenolics and other polyphenols can therefore attenuate the risk of
atherosclerosis by reducing the negative effects of lipid oxidation products in
fat-rich foods. Apparently, wine procyanidins are active in inhibiting lipid
oxidation in foods in the digestive tract and in preventing the postprandial
increase of oxidants in plasma.
Olive oil phenolics may also contribute to the nutritional benefits of Mediter-
ranean diets. The plasma antioxidant capacity of healthy volunteers increased
significantly after consuming an oral load of extra virgin olive oil (Table 8.2).
These results indicate that phenolic compounds in olive oil are absorbed and
may exert a significant protective effect in vivo in the postprandial phase.
Although the attenuation of postprandial oxidative stress by polyphenols may
be considered to be a true antioxidant activity, the mechanism for this effect is
still unclear.
The present evidence indicates that varying small amounts of these flavonoids
are absorbed in human blood as metabolites, and their bioavailability changes
widely according to dietary sources. These metabolites have a significantly
lower antioxidant activity than the original aglycones, by blocking one or more
phenolic hydroxyl groups responsible for radical scavenging activity (Table
8.4). A limited number of short-term intervention studies showing biological
effects of polyphenols are unconvincing, because few of them showed a dose-

response relationship and many showed mixed results. The health effects and
their mechanisms of action in vivo activity as antioxidants are still not well
understood. The biological effects of flavonoids in the GI tract and other tissues
require much further research.
E. What is an antioxidant?
The large amount of effort expended in testing new natural antioxidants
emphasizes the need for improved test methods and their standardization. The
great diversity of methods developed to evaluate antioxidants has created much
confusion in the field (Chapter 4). The broad terminology used in the literature
in reference to antioxidants has become especially confusing. Significant
differences are reported in antioxidant testing results between animal and
human studies, and between in vitro and in vivo effects.
The term antioxidant has become so loosely defined in biology as to be all
but unusable. By the traditional chemical definition, an antioxidant is a
molecule that slows a free radical chain reaction propagating the oxidation of
lipids. In biological contexts, the critical use of the antioxidant term should
include molecules that are protected from oxidation, and the resulting damage
that is prevented. The various steps of atherosclerosis are diverse, complex and
separated in time and location. In the most recognized process, considerable
research has shown that the development of atherosclerosis proceeds by the
accumulation of LDL particles within subendothelial macrophages, after they
are chemically modified by oxidation of the polyunsaturated lipids within these
particles. According to this mechanism of disease, one possible means of
preventing or delaying the processes of atherosclerosis is to protect LDL from
oxidation. However, many other complex enzymatic and non-enzymatic modi-
fications are now recognized which may not involve lipid oxidation, and
therefore free radical scavenging antioxidants can be ineffective.
The term antioxidant has a well-defined meaning in chemistry and food
science, on the basis of its radical-chain breaking mechanism, and inhibition of
initiation of free radicals. However, this term has assumed such a broad
meaning in biology, nutrition and health sciences, that it has lost its traditional
chemical definition. Accordingly, biological antioxidants now include repair
systems such as iron transport proteins, antioxidant enzymes, and factors
affecting vascular homeostasis (vessel equilibrium), signal transduction (trans-
fer of genetic material) and regulation of gene expression of detoxifying
enzymes. Some of the biological ‘antioxidant’ activities attributed to various
plant extracts and flavonoids (e.g. anti-allergic, anti-hemorrhagic, anti-muta-
genic, anti-tumor, anti-platelet activities, immunomodulation, oral hygiene,
and interactions with specific receptors) may in fact have little to do with
antioxidant activity. Many health benefits of flavonoids may be actually
dependent on additional non-antioxidants activities, including inhibition or

promotion of detoxification enzymes, inhibition of cell division, redox regula-
tion, transfer of genetic material and determinants, and neuroprotection (balance
between cell death and survival).
Similar non-antioxidant activities are indicated for the effect of α-tocophe-
rol on genetic changes, intracellular signaling, and control of smooth muscle
cell proliferation (Table 8.1). Therefore, much more research is required to
understand better the nutritional and biological benefits of the very complex
mixtures of polyphenolic compounds found in fruits, vegetables and bever-
ages, and the complex interactions that may occur with other nutrients in plants.
Information on bioavailability and metabolism of polyphenolic compounds is
still scarce.
F. Future research
1. Nutritional and health properties of plant foods

Much more research is required to understand better the multitude of nutri-
tional and biological benefits of the very complex mixtures of polyphenolic
compounds found in fruits, vegetables and beverages, and the complex interac-
tions that may occur with other nutrients in plants. Although information on the
bioavailability and metabolism of polyphenolic compounds is still scarce, it is
advancing slowly. The relative antioxidant activity of many phenolic com-
pounds varies widely according to different testing methods. Antioxidant
protocols commonly using either artificial radical model systems or lacking a
relevant substrate (Chapter 4.B, Table 4.2) provide no useful information on
which food or biological targets are protected by the antioxidants. To deter-
mine the real effects of natural phenolic compounds, it is therefore important to
obtain specific chemical information on which oxidation products are inhibited
from lipids, proteins, carbohydrates and DNA. Several specific bioassays are
required to elucidate products causing oxidative damage in biological tissues.
Antioxidants from fruits and vegetables need to be optimized to determine if
they play a significant role in preventing LDL and other biological oxidations
in vivo, and to elucidate their mechanism of protection.
The prevention of LDL oxidation by antioxidants has been viewed as an
important potential means of decreasing the risk of heart disease. Plant
phenolic antioxidants in fruits and beverages (wine, grapes, grape juice and
tea) may have a protective effect against coronary heart disease and cancer,
but the molecular mechanism of protection is not understood. There is now
intensive interest in developing plant sources of polyphenolic antioxidants
for foods and biological systems, and for their possible health benefits. Only
recently has there been research providing new knowledge on the relative
absorption and activities of these natural antioxidants in the body and their
bioavailability, but there is a lack of reliable biomarkers to test their activity

in vivo (Chapter 6).
Although recent evidence indicates that small amounts of flavonoid antioxi-
dants are absorbed as various metabolites in the blood of humans (Chapter
6.G.3), new knowledge on their activities in vivo is just beginning to emerge.
The possible cardiovascular benefits of increased intake of vitamin E and other
natural plants antioxidants are not yet well established. The nutritional benefits
from the Mediterranean diet are generally attributed to olive oil rich in oleic
acid, α-linolenic acid and natural antioxidants from fruits and vegetables. An
adequate supply of antioxidants from fruits and vegetables is also necessary to
prevent oxidation of polyunsaturated lipids.
There are still many unsettled questions that deserve additional research
using more relevant and reliable bioassays. The mechanism of antioxidant
protection is not well understood in foods and biological systems. Future
research is needed to clarify the interactions between dietary polyphenols and
health effects, and to obtain answers to the following crucial questions in food
nutrition and biology:
(a) How to improve protocol methods to measure health benefits with

better and non-invasive biomarkers of cardiovascular and degenera-
tive disease? How to evaluate LDL oxidation at the lipid–aqueous
interface where hydrophilic and amphiphilic polyphenolic compounds
may have their protective activity? What is their mechanism of protec-
tion (in the absence of possible confounding effects from oxidized
dietary lipids), and disease intervention?
(b) How can the phytochemical effects of mixtures of bioactive phenolics
be determined, to establish their potential role as synergists or co-
antioxidants in the nutrition of complex foods?
(c) How can bioassays be improved to allow the systematic testing of
mixtures of antioxidants and to obtain better evidence for the benefits
of phenolic compounds in fruits and vegetables?
(d) How can specific health effects be better defined between foods rich in
polyphenols and polyphenol antioxidant supplements?
(e) Can mechanisms of food processing be developed to permit more
efficient release of dietary components from the plant matrix and
improve their bioavailability?
(f) Can new biomarkers be developed on the basis of genomics and
proteomic technologies?
(g) What are the nutritional properties of the great diversity of
phytochemicals found in plant foods?
(h) What are the interactions and synergies between polyphenolic antioxi-
dants and other nutrients in food plants?
(i) Can health compositions in foods be optimized for the majority of the
population? Should the nutritional compositions be optimized to pro-

vide specific benefits for different segments of the population, such as
babies, adolescents, mature and aged people?
The bioavailability and absorption of antioxidants in human blood as

metabolites, their molecular mechanisms of protection in vivo and of disease
intervention are now subjects under intensive investigation around the world.
The development of reliable biomarkers that relate better to degenerative
disease presents one of the most difficult challenges in this field.
Future research will require more emphasis on the many known metabolites
of flavonoids, and at the same ranges of concentrations found in blood. Clinical
studies will also require better biomarkers used under experimental conditions
relevant to human nutrition and health.
Using the term antioxidant as a key word in literature searches leads to a
large and unmanageable number of misleading and confusing answers. To
continue using the term antioxidant in reference to the very diverse biological
functions of plant flavonoids and related phenolic compounds may be impru-
dent, because many of these activities have little or nothing to do with
antioxidant properties. Nutritional claims regarding antioxidant supplements
can be also unjustified until we can understand their benefits for humans more
fully. With so many unanswered questions, the conclusion can be made that it
would be imprudent to make dietary recommendations before the mechanisms
of in vivo activity of antioxidants are better understood.
As a result of early nutritional findings that saturated fats have adverse
effects on the LDL/HDL ratio in blood, Western populations have tended to
consume large amounts of polyunsaturated fats that are susceptible to oxida-
tion. Excessive levels of polyunsaturated fatty acids in the diet, without
providing sufficient protection from antioxidants and other inhibitors, can
promote LDL oxidation and upset the oxidant–antioxidant balance described
in Chapter 6, in favor of cellular prooxidants and promote coronary heart
disease. On one hand, recommendations to increase vegetable and fish oils
containing n–3 PUFA in our diet should also be tempered by their high
susceptibility to oxidation and decomposition. Metal chelators are generally
more effective in inhibiting oxidation of n–3 PUFA oils than phenolic anti-
oxidants (Lipid Oxidation, 2nd ed, 2005, Chapter 11.E). On the other hand,
diets rich in oleic acid including the Mediterranean diet have clear advan-
tages in not only lowering serum cholesterol, but also in decreasing the
oxidative susceptibility of LDL. Additional research is needed to optimize
the levels of dietary n–6 and n–3 PUFA required to lower plasma LDL
without increasing the oxidizability of LDL, and determining whether or not
antioxidants from fruits and vegetables can prevent LDL and other biologi-
cal oxidations in vivo. It would seem imprudent to make dietary
recommendations before the mechanisms of polyunsaturated lipid nutrition,
in vivo activity of antioxidants, and in vivo lipid peroxidation are better

understood.
It is unclear whether or not the conversion of flavonoids into ‘non-antioxi-
dant’ derivatives is due to a protective mechanism against the well-known
xenobiotic activities of free polyphenolic flavonoids. The rapid oxidation of
flavonoids into semiquinones and quinones may or may not have beneficial
health effects. As discussed in Chapter 6, the biological effects of the flavonoid
metabolites are thus not well understood, mainly because of a lack of reliable
in vivo testing protocols. The metabolites formed from quercetin and catechins,
including glucuronides, sulfate, and methylated forms of their functional
groups, are expected to be differently distributed in tissues and have different
biological activities to their corresponding aglycones. Although these
metabolites can be enzymatically deconjugated in certain tissues, for a better
understanding of the nutritional benefits of flavonoids, much more information
is needed on their bioavailability and bioactivity of different metabolites as
they are distributed in different sites of the body.
In future research, we also need to improve protocol methods to evaluate
better and optimize the in vivo effects of polyphenolic compounds in biological
systems. If the metabolites produced after absorption of flavonoids in the body
have diminished antioxidant activity, we need to develop appropriate tests to
determine how their non-antioxidant in vivo activities can be beneficial to
health, including their anti-inflammatory, anti-mutagenic, anti-tumor, and
anti-platelet activities. A number of intervention trials have failed to show
consistent benefits from the use of antioxidant supplements on cardiovascular
disease and cancer. Unfortunately, too many of these trials were based on the
administration of a single phenolic compound at relatively high doses. Future
studies of health compositions in foods should also be designed to provide
specific benefits for different segments of the population (young, mature or
aged people). Human intervention studies need to be re-designed to resolve
previous conflicting results by using separate biomarkers to distinguish between
healthy subjects and diseased individuals. In addition to the flavonoids in the
diet, nutritionists should also focus on the biological activities of the mixtures
of metabolites present in the body tissues after absorption.
The new study of ‘nutrigenomics’, applying genomic (study of all the genes
and their biological/physiolgical interactions) tools to integrate nutritional
effects on gene regulation, may clarify in the future how food nutrients affect
the whole human body in health and resistance to disease. Recent studies in
nutrition support the concept that the general traits of each subject is uniquely
susceptible to dietary factors, controlled by their genetic polymorphism (indi-
vidual characteristics) associated with a multitude of different effects on lipid
oxidation and antioxidant activity of food components. Therefore, nutritionists
should specifically design their future studies on the beneficial effects of
phenolic antioxidants by determining gene polymorphism from blood samples
of individual humans of different age and social-economic environments. This

approach may lead to the development of individualized diets containing the
proper food antioxidants for optimum health.
The potential beneficial effects of flavonols in plant foods, red wine and
green tea may depend on daily consumption to sustain useful levels of these
compounds in plasma or other parts of the body. Therefore, phenolic antioxi-
dants in fruits and beverages may have a protective effect against coronary
heart disease and cancer, but the mechanism of protection is not understood.
There is a significant gap in our knowledge on the relative absorption and in the
activities of these natural antioxidants in the body and their bioavailability, and
a lack of reliable biomarkers to test their activity in vivo. Although recent
evidence indicates that small amounts of flavonoid antioxidants are absorbed
as various metabolites in the blood of humans, their activities in vivo are
unknown. The possible cardiovascular benefits of increased intake of vitamin
E and other phytochemicals are therefore not yet well established.
2. Organic versus conventional plant foods

In the production of foods, variations in agricultural practices including
organic and sustainable agriculture have recently received much interest, with
the goal of improving their nutrition value and their environmental impact. In
contrast to conventional agriculture, organic farming is aimed at a) avoiding
crops that are genetically engineered, irradiated or fertilized with hazardous
waste (sewage sludge), (b) prohibiting the use of synthetic pesticides and
herbicides, (c) using disease-resistant cultivars, and (d) providing plant nutri-
ents by crop rotation, cover crops and animal manure. Sustainable agriculture
practices are designed to promote the environmental health and economical
state of farming regions by controlling animal pollution, recycling farm waste
and crop rotation.
If plant phenolic compounds can be regarded as important secondary
metabolites which determine the nutritional values of fruits and vegetables,
their higher concentrations in organically grown products are generally consid-
ered to make these more nutritionally beneficial than conventionally grown
products. In early studies, this difference has been considered either anecdotal
or insignificant. Although many claims have been made generally that organic
foods contain higher levels of phenolic compounds than conventional fruits
and vegetables, only recently have more reliable analytical data suggested that
organically produced fruits and vegetables may be more beneficial to human
health than conventionally produced crops. The total phenolic contents were
significantly higher from marionberries (or blackberries), corn, apple pulps,
strawberries and tomatoes grown by organic agricultural methods compared
with conventionally methods, but not from plums, apple peels and bell peppers
(Table 8.7). Ascorbic acid content was also higher for the organic corn,
Table 8.7. Total phenolic compounds in conventional and organically grown fruits and
vegetables
Foods Total phenols Ascorbic acid Ref.a

(mg/100g fresh weight) (mg/100g fresh weight)
Conventional Organic Conventional Organic
Marionberry 400 620 – – (1)

Corn 25 40 2.1 3.5
Plumsb 121 88 2.0 1.6 (2)
Apple pulp 0.09c 0.29d – – (3)
Apple peel 1.51c 1.47d – –
Strawberry 148e 186f 419e 515f (4)
Tomatoesg 43.4 59.8 14.0 24.8 (5)
Bell peppersg 58.7 59.9 51.8 55.5
a
References: (1) Asami et al. (2003), (2) Lombardi-Boccia et al. (2004), (3) Veberic et al. (2005), (4)
Olsson et al. (2006), (5) Chassy et al. (2006).
b
Quercetin (30.2 vs 19.6 mg tannic acid/kg) was higher in conventional plums, but myricetin (1.1 vs 0.9
mg/kg) and kampferol (1.7 vs 0.6 mg/kg) were higher in organic plums.
c
Average values from 8 cultivars grown in Austria and Slovenia; integrated: use of chemicals against
pests and disease.
d
Average values from 11 cultivars grown in Austria and Slovenia; organic: no synthetic chemicals used.
e
Average values from 4 cultivars grown in Sweden, corrected values based on dry weight.
f
Average values from 3 cultivars grown in Sweden, corrected values based on dry weight.
g
Values for tomatoes are averages of 2 cultivars and 3 subplots in 2003, and for bell peppers, averages
of 2 cultivars in 2003, 2004, 2005.
strawberries, tomatoes and bell peppers, but not for plums. When post-harvest
processing treatments were compared, higher total phenols were consistently
found in frozen and freeze-dried samples of marionberry, strawberry and corn
compared to the air-dried samples (Table 8.7, ref 1). Although the total
phenolic and quercetin content of plums was higher in conventional plums, the
myricetin and kaempferol content were higher in the organic plums (Table 8.7,
ref 2). Because of the significantly higher content of phenolics in the peel than
in the pulp of apples, they should not be peeled before consuming (Table 8.7,
ref 3). The ratio of ascorbate to dehydroascorbate was significantly higher in
the organically grown strawberries (Table 8.7, ref 4). The year-to-year varia-
bility and the varieties of tomatoes significantly affected their quercetin
contents (Table 8.7, ref 5). In addition to the common organic versus conven-
tional agricultural practices, many complex environmental and agricultural
factors are known to influence the contents of potential antioxidants in plants,
which are difficult to control. The type of soil management, growing technol-
ogy, cropping system differences, the response of plants to stress exposure and
the genotype source apparently influences the concentration of phenolic com-
pounds in food plants.
Most studies on the effect of organic versus conventional agriculture have
thus far focused on detailed composition analyses of plant extracts for total and
individual phenols, and for ascorbic acid, but very little reliable data have been
published on their corresponding antioxidant activity. Unfortunately, many
authors continue to make the misleading assumption that compositional analy-
ses of phenolic compounds can be equated to antioxidant activity, without
carrying out the more difficult and demanding evaluations of antioxidant
activity by reliable protocols (Chapter 4). Polyphenolic compounds are only
part of a wealth of secondary phytochemicals accumulated by plants by
genetics and environmental oxidative stress factors for the protection of plants.
Under these conditions, many other secondary plant chemicals and factors may
influence the antioxidant activity of plant extracts that must be tested in
addition to the detailed analyses of polyphenolic compounds and ascorbic acid.
3. Food nanotechnology
This new development in food technology deals with structures smaller than
100 nanometers that have unique, novel and potentially useful functional
properties caused by modified interfacial phenomena. Although several possi-
ble uses of nanotechnology have already been developed in the chemical and
pharmaceutical industries in the past few years, only a few food applications
have been reviewed in the literature. Antioxidants as functional food ingredi-
ents could be delivered in a variety of different polarities, molecular weights
and physical properties capable of release in foods under controlled conditions,
and designed for their protection against losses by oxidation. Delivery systems
based on nanotechnology have been developed with various multicomponent
systems. Free fatty acids and surfactants in the presence of water produce
mixed micelle systems that can either promote their oxidation with trace
metals, or inhibit it with antioxidants. Microemulsions or swollen micelles can
be prepared by encapsulating non-polar antioxidants such as tocopherols to
improve their water solubility in emulsions and reduce lipid oxidation. Non-
polar antioxidants may also be incorporated into multifunctional systems by
solubilizing within either the core or the surface of emulsifier micelles, lecithin
liposomes, or multiple emulsions (Chapter 3).
Liposomal nanocapsules have been successfully used to encapsulate protei-
nase for cheese ripening, vitamin D supplementation in dairy products,
lactoferrin to inhibit lipid oxidation (Chapter 5.E.4), antioxidants including
phosvitin, ascorbic acid, α-tocopherol, vitamin D, and nicin, a polycyclic
peptide antibiotic used for food preservation (Table 8.8). Nanoemulsions can
be prepared with high-pressure homogenizers, membrane extrusion
homogenizers, in the presence of special surfactants or polymers. Nanoparticles
with a wide range of applications can be prepared by special techniques (salting
out, micro-emulsification, and nanoprecipitation) using various biopolymers.
Functional foods can thus be designed to contain antioxidants incorporated into
nanoparticulates that can be better dispersed into different foods. However,
Table 8.8. Applications of liposomal nanocapsules in foodsa
Applications Vehicle Active agents Ref.b
Cheese ripening Small unilamelar vesicle (SUV) Proteinase protection (1, 2)

Antioxidant PC liposome PC + cholesterol Ascorbic acid (3)
Stabilization PC liposomes Lactoferrin (4)
Dairy products Liposome encapsulation Vitamin D (5)
Antioxidant PC liposome Phosvitinc (6)
Antioxidant PC liposome Vitamin E (7)
Antiobiotic PC liposome Nisind (8)
a
Taylor et al. (2005).
b
References: (1) Kirby et al. (1987), (2) Alkhalaf et al. (1988), (3) Kirby et al. (1991), (4) Huang et al.
(1999), (5) Banville et al. (2000), (6) Lee et al. (2002a), (7) Lee et al. 2002b), (8) Laridi et al. (2003).
c
Phosvitin is a phosphorylated glycoprotein found in egg yolk.
d
Nisin is a polycyclic peptide of 34 amino acids residues used as a food preservative.
safety aspects need to be considered, because it is not yet clear if the new
materials containing nanoparticles could cause environmental and health
problems.
Future research is therefore needed to develop food nanostructures for
delivery of antioxidants by testing different food matrices, biopolymers,
multiphase systems and emulsions to enhance food stability, flavor, acceptabil-
ity and their nutritive value. These nanoparticulates containing antioxidants are
claimed to have improved bioavailability and dispersibility in foods, but much
more testing is required. By improving our understanding of the mechanism of
delivery systems, various specific antioxidants may be designed for special
food applications.
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Glossary
Advanced Derived from reactive carbonyl compounds produced by lipid

glycation oxidation and glycation of proteins
end products
Aglycone Parent flavonoid compound that does not contain a carbo-
hydrate group
Aminoreductones Compounds derived from interactions of reducing sugars and
amino acids
Antigens Foreign substance producing immune reaction or response
Anti-platelet Reduction or inhibition of platelets accumulation in blood
activity
Antiradical Radical scavenging property
capacity
Apoptosis Cell death
Arthritis Inflammation of joints
Atherogenesis A long series of events that lead to development of athero-
sclerosis
Atheroma Acute thrombotic complications
Atherosclerosis Condition of decreased blood flow in vessels in which the inner
space is diminished by thickening of the vessel wall by deposi-
tion of cholesterol and fat
Autoimmune Immune to self antigens
Bacteriophage Virus that can dissolve or kill microorganisms
Cardiovascular Refers to the heart (cardio) and blood vessels (vascular) and the
system for distributing blood, oxygen and nutrients to various
tissues
Chelation Complexing metals by multi-site coordination
Chylomicrons Microscopic particles of fat found in plasma
Core aldehydes (also known as aldehydo glycerides, or 2½ glycerides): alde-
hydes attached to a glyceride or phospholipid
C-reactive protein Normal plasma protein that increases significantly in concen-
tration by infection, and inflammation
Crocin A natural pigment with strong visible absorption
Cyclooxygenase Enzyme catalysing the formation of prostaglandins from ara-
chidonic acid
Cytochrome Respirator enzyme that undergoes reduction and oxidation
Cytokines Tumour growth factor
GLOSSARY 245
Cytoplasm Cell substance without the nucleus

Cytotoxicity Toxicity to cells
Degumming Vegetable oil process to remove phospholipids by hydration
Diabetes Metabolic disorder marked by excessive urination
Diazo initiators A-N=N-A used to generate artificial aromatic A · radicals (see
Chapter 2.B.2)
Dyslipidemia Disorder of lipoprotein metabolism in diabetes
Eicosanoids Oxygenated derivatives of arachidonic acid
Endocytosis Uptake of material into a cell by the formation of a membrane-
bound vesicle
Endothelium Layer of flat cells lining blood vessels
Enterocytes Intestinal wall cells
Epitope Group recognized by an antibody formed in the presence of a
foreign substance
Erythrocytes Red blood cells
Ex vivo Out of the cells
Ferritin Storage protein that bind iron in blood
Genomics Study of genes and biological interactions
Glutathione Tripeptide of glutamic acid, cysteine and glycine acting as a
strong hydrogen donor
Glycation Binding of proteins by glucose in blood
Glycoxidation Glycation reactions accompanied by oxidation reactions
Hemoglobin Red respiratory protein of erythrocytes
Hepatic Acting on or in the liver
Hepatocytes Liver cells
Homeostasis Vessel equilibrium to balance physiological functions by chemi-
cal composition
Homolog Belonging to a series of organic compounds differing by
constant increment (CH2 or CH3)
Hyperchole- Condition of elevated levels of serum cholesterol
sterolemia
Hyperglycemic Producing excessive levels of sugars in blood
Hyperlipidemic Producing excessive levels of lipids in blood
Hypertriglyceri- Producing excessive levels of triglycerides in blood
demic
Immunochemical Chemical reactions from antigens and antibody stimulation of
tissues
Immune Defensive network of protective response against a pathogenic
agent or antigen
Immune Reduction of immune response
suppression
Interfacial Surface oxidation between phases
oxidation
Interferons Proteins synthesized by cells for defense against viral infection

Intima Innermost layer of blood vessels or arteries
In vitro Outside living organism
In vivo Within living organism
Ischemia Localized tissue anemia due to obstruction of arterial blood
Isoprostanes Cyclic oxidation products of arachidonic acid
Kinase Enzyme that can activate another enzyme
Leucocytes White or colorless nucleated blood cells
Leucotriene Eicosanoid formed by the action of lipoxygenase on arachi-
donic acid
Lipid peroxidation Oxidation and oxidative deterioration of biological systems
Lipemia Presence of abnormal amount of lipid in blood
Lipofuscin Brown pigment granules lipid-containing residues of lyso-
somal digestion
Liposomes Multiple layers produced by phospholipids dispersed in water
Lymphocyte White blood cell formed in lymphoid tissue
Lysosome Cytoplasmic particle containing hydrolysing enzymes
Macrophages White blood mononucleated cells that scavenge and digest
bacteria
Melanoidins Macromolecular pigments produced by heat that of varying
molecular weights and solubility
Micelles Colloidal structures produced by free fatty acids dispersed in
water
Microphages Small leucocytes which ingest chiefly bacteria
Microsomes Granules in protoplasm with minute cellular structure
Mitochondria Cellular organelles found outside nucleus
Monocyte Large mononuclear white blood cell
Myeloperoxidase A peroxidase occurring in leucocytes
Myocardial Necrotic heart muscle resulting from failure of local blood
infarction supply
Neutrophil Finely granular white cell (leucocyte) that is the chief phago-
cyte of blood
Nucleophile Anion capable of reacting with a positive center of a molecule
Organelle A structurally discrete component of a cell
Oxyhemoglobin Hemoglobin in combination with oxygen
Oxysterols Oxidized products of cholesterol
Phagocyte Cell that ingests bacteria, foreign particles, other cells and
consumes debris
Pathophysiological Physiology of diseases
Pharmacokinetic Pharmacological dynamics of ingested nutrients
Phytochemicals Chemicals derived from plants
Polymorphism Individual characteristics
Postprandial state After a big meal
GLOSSARY 247
Prostacyclin Antithrombotic prostaglandin produced by endothelial cells of

blood vessels
Prostaglandin Oxygenated derivative of arachidonic acid that contain a five-
membered ring
Prostanoids Metabolites of arachidonic acid
Postprandial Occurring after a meal
Redox Reduction-oxidation (oxido-reduction)
Reductones Browning reaction products formed by heating reducing sugars
Rheumatoid Inflammation on a joint
Rheumatoid Inflammation of muscles, tendons, joints, bones or nerves
arthritis
Signaling Molecules responsible for cellular neuroprotective,
molecules cardioprotective and chemopreventive properties
Signal Transfer of genetic material by redox signaling
transduction
Stoichiometric The number of radicals trapped by each molecule of anti-
factor oxidant
T-cells Mediators of immune response
Thrombosis Formation of blood clot of platelets and blood coagulation
proteins within a blood vessel that restricts the flow of blood
Thromboxane Prostaglandin intermediate causing platelet aggregation and
contraction of muscles
Thrombus Clot of blood formed within blood vessel remaining attached to
place of origin
Thrombotic Tendency for blood clotting
tendencies
α-Tocopherol Regulates the concentration of vitamin E in blood plasma
transfer protein
Transcription Transfer of genetic information
Transduction Transfer of genetic properties between microorganisms medi-
ated by bacteriophages
Transferrin Transport protein that binds iron in blood
Urokinase Transphosphorylation enzyme
Vasoactivity Condition to stimulate blood vessels
Vasoconstrictor Constricts blood vessels causing increase in blood pressure
Vasodilation Dilation of blood vessels causing decrease in blood pressure
Vesicle A closed membrane shell, derived either physiologically (bud-
ding) or mechanically (by sonication)
Xenobiotic Foreign biologically active compounds
Abbreviations
AAPH 2,2'-azobis-(2-amidinopropane) dihydrochloride

ABAP 2,2'-azobis(2-amidinopropane hydrochloride)
ABTS 2,2'-azinobis-(3-ethylbenzothiazoline 6-sulfonate)
AGEs advanced glycation end products
AIBN α,α-azobisisobutyronitrile
AMVN 2,2'-azobis-(2,4-dimethylvaleronitrile)
ApoB apolipoprotein B
AUC area under curve
BHA butylated hydroxyanisole
BHT butylated hydroxytoluene
BSA bovine serum albumin
Cmax maximum plasma concentrations
CD conjugated dienes
CE cholesterol esters
CEL (carboxyethyl)lysine
Ch cholesterol
CI-MS chemical ionization-mass spectrometry
CML (carboxymethyl)lysine
COX-2 cyclooxygenase-2
DLPC dilinoleoyl-phosphatidylcholine
DNA deoxyribonucleic acid
DPPH 2,2-diphenyl-1-picrylhydrazyl
DRI dietary reference intake
E1/2 half-life of elimination
EC epicatechin
ECG epicatechin gallate
EDTA ethylenediaminetetraacetic acid
EGC epigallocatechin
EGCG epigallocatechin gallate
ESR electron spin resonance
EVOO extra virgin olive oil
FRAP ferric reducing antioxidant power
GA gallic acid
GAE gallic acid equivalents
GC gas chromatography
GI gastrointestinal
ABBREVIATIONS 249
GPx glutathione peroxidases

GSH glutathione
GSSG oxidized glutathione or glutathione disulfide
HDL high-density lipoprotein
HDTBr hexadecyltrimethylammonium bromide
HPLC high-performance liquid chromatography
IC50 antioxidant concentration required for 50% inhibition
LDL low-density lipoprotein
5-LOX 5-lipoxygenase
LTB4 leukotriene B4
MALDI-TOF-MS matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry
MDA malonaldehyde
MeLo-OOH methyl linoleate hydroperoxides
mRNA messenger ribonucleic acid
MRP Maillard reaction products
NADPH nicotinamide adenine dinucleotide phosphate
ORAC oxygen radical absorbance capacity
PA phosphatidic acid
PC phosphatidylcholine
PE phosphtidylethanolamine
β-PE β-phycoerythrin
PG phosphatidylglycerol
PGE2 prostaglandin E2
PHGPx phospholipids hydroperoxide glutathione peroxidases
pI isoelectric point
PI phosphatidylinositol
PKC protein kinase C
PS phosphatidylserine
PUFA polyunsaturated fatty acids
n–3 PUFA PUFA with the last double bond located three carbons
from the end of the fatty acid
PV peroxide value
RDA recommended dietary allowances
RNA ribonucleic acid
ROS reactive oxygen species
SDS sodium dodecylsulfate
SH sulfhydryl group
SOD superoxide dismutases
SPI soy protein isolate
T50 time to reach 50% inhibition
Tmax times to reach maximum concentration
TBARS thiobarbituric acid reactive substances
TBHQ tertiary butylhydroquinone

TEAC Trolox equivalent antioxidant capacity
TG triglycerides
TNFα Tumor necrosis factor-α
TOSC total oxyradical scavenging capacity
TRAP total radical trapping antioxidant parameter
TTP tocopherol transfer protein
VLDL very low-density lipoprotein
WPI whey protein isolate
Index
AAPH 89, 90, 143, 152, 218 metabolites 171, 179, 180, 222, 223
ABTS assay see TEAC 113 cells 149, 150, 152
absorption 155, 156, 160, 165, 167, 172, 173, signal transduction 149, 163
184, 232–235 cereal products 132–136
AGE (advanced glycation end product) 193, cerulplasmin 27
196–200 chain transfer reactions 32
AGE-protein 209 chlorogenic acid 177–179
aging 14, 159, 193, 194, 200, 206 chlorophyl 35
albumin 27, 38 chocolate 11, 171
Amadori products 193, 194, 196, 197, 207, 209 cholesterol 3
anthocyanins 11, 173, 174 reverse transport 10
antiatherogens 9, 148 citric acid 25, 34
antioxidant(s) 4, 6, 7, 9, 12, 16, 25, 29, 31, 32, cleavage
77, 111, 121, 122, 143, 144, 147, 209, 231, homolytic 24
234 coffee 195
activity 152, 177 colon cancer 4
browning reaction products 193–195 collagen 5
mixtures 144, 145 colloidal properties 6, 8, 43
multifunctional 77–79 phenomena 15
prooxidant activity 23, 144 systems 43
ascorbic acid (vitamin C) 5, 12, 25, 27, 33, 39, compartmentalization 8
45, 46, 106, 162, 236–238 complexes
synergism 34, 162 metal–protein 8
ascorbyl palmitate 5, 12, 45 copper ions 38
atherosclerosis 3, 9, 10, 150, 154, 161, 193–195, cupric/cuprous 39
197, 198, 207 corn oil/emulsion 47, 49
coronary heart disease 13
biovailability 151, 160, 162, 172–175, 228, 232, cysteine 40
233, 235, 236 cytochrome c 38
bioassays 145, 179, 185, 232, 233, 235
biomarkers 9, 16, 169, 172, 183, 184, 229, 233– deconjugation 173
235 dehydroascorbic acid 5, 6
bulk oil 7, 45, 72 diabetes 154, 193, 194, 196–200, 204, 206–208
butylated hydroxyanisole (BHA) 21, 46 dietary recommendations 184, 217
butylated hydroxytoluene (BHT) 10, 30, 46 diseases 3, 4, 9, 145, 236
DNA 4, 16, 37, 145, 148, 160, 163–165, 220,
cancer 2, 151, 152, 193 221
green tea, urokinase inhibition 152 DPPH assay 83–86, 93
carnosic acid 12, 32, 46, 47, 119
oxidized, reduced, isomerized 120, 121 eicosanoids 149, 150, 183, 221
carnosol 32, 46, 120 emulsifier 6, 43, 47
carbonyl compounds 22, 197, 198, 205, 207 emulsions, 26, 43–47, 49, 52, 58, 238
carboxymethyl lysine (CML) 197–202, 208 oil-in-water 6, 7, 43, 50, 72
carboxyethyllysine (CEL) 198, 199, 201 phospholipids 43, 44
β-carotene bleaching method 84, 89, 94, 95 proteins 44, 54, 56
carotenoids 11, 35, 36, 40 special 65, 66
catalase 14, 146 water-in-oil 43, 49, 50
catechins 46, 167, 171, 179 enzymes 14, 37, 39, 147, 163, 152, 165, 166, 183
251
antioxidant 14, 36, 146–148 gastrointestinal (GI) tract 155, 157, 160, 166,
catalase 14, 37, 39, 227 229, 231
cyclooxygenases (COX) 12, 149, 150, 183, glutathione (GSH) 14, 39, 40, 146, 148, 205, 227
219, 220 disulfide (GSSG) 14, 147
glutathione peroxidase 14, 39, 155, 205 glycation/glycoxidation 193, 194, 196–212
glutathione transferase 37, 161, 183 grapes 11, 16
lipoxygenases (LOX) 12, 147, 149, 150, 169,
171, 183, 219, 220 health, 2, 8
protein kinase161, 183, 206, 218, 227 HDL 10, 145
superoxide dismutase 37–39 Heyns products 193, 194
xanthine oxidase 169, 171, 220 high-density lipoproteinssee HDL
epicatechin 47, 49, 53, 60, 94, 227 hydrogen reactions 1, 22, 24
epidemiology 15, 184, 217 hydrogen peroxide 38, 146
ethylenediaminetetraacetic acid (EDTA) 25–27, hydroperoxides 12, 27, 39, 147
52 decomposition 12, 13, 21, 22, 28, 29
ex vivo 10, 149, 150, 152, 162, 169, 195, 224, formation 13, 21, 22, 28, 29
228, 230 hydrophilic–lipophilic balance 51
excretion 160, 173 hydroxyl nonenal 52, 196, 208
hyperglycemia 154, 157, 196
fats/fatty acids hypertriglyceridemia/hyperlipidemia 154, 157,
saturated 3, 185 196, 204, 230
monounsaturated 22 hypochlorous acid 5, 38, 39
polyunsaturated 1, 3, 8, 22, 182, 184, 185, 234
thermally oxidized 217 inflammation 10, 150, 183, 193, 194, 199, 219
fatty esters initiation 3, 6, 25, 29, 32
methyl oleate 22, 221 diazo initiators 28, 81, 218
methyl linoleate 22, 221 initiators 13, 15
methyl linolenate 22 azo 29, 158, 160, 169
ferritin 38, 145 interactions, food antioxidants 105, 106, 108,
fish products 128–132 109, 111
antioxidants 128–131 AGEs (advanced glycation products) 108,
oils 3, 26, 115 195, 196
oil emulsion 26, 49, 131 browning products 105–108, 111
metal chelators 130 lipids with proteins and sugars 105
protein isolate 131, 132 lipid oxidation products 108
flavonoids 11, 16, 35, 48, 94, 145, 163, 164, 170, phospholipids 108, 110, 111
219, 220, 228–231 reductones 105, 106
absorption 145, 149, 165, 185, 229, 235 interface, 7, 50
bioavailability 154, 163, 165, 229 interfacial oxidation 43, 45
deglycosilation 165, 166 interfacial properties 1, 7, 8, 45, 50, 224, 226
glycosides 16, 163, 165 phenomena 6, 7, 12, 13, 50, 79, 96, 97, 115
metabolites 166, 167, 183, 229, 230, 234, 235 intervention studies 170, 172, 230, 234, 235
pharmacokinetics 173, 175, 185 in vitro 3–5, 8, 9, 13, 36, 40, 97, 137, 144, 149–
prooxidant effects 47, 158 152, 158–163, 169, 172, 176, 184, 195, 198
protection of GI tract 155, 156 199, 201, 204, 207, 217, 218, 223, 231
protection against DNA oxidation 164 in vivo 3, 4, 6, 8, 9, 12, 13, 16, 17, 40, 97, 114,
quinone formation 159, 183 137, 145, 148–152, 158, 162–165, 169, 172,
reactions with proteins 164, 229 179, 183–185, 197, 198, 201, 209, 217, 218,
foam formation 9 220, 221, 223, 224, 230–236
foods, 1, 2, 96, 105, 174, 233, 237 iron, 1, 26–28, 38, 39, 159
organic 236–238 isoflavones 174–176
plant 113, 233, 236
FRAP assay 92, 94, 95, 113 lactoferrin 26–28, 65
free radical reactions 3, 21 LDL oxidation 6, 13, 16, 93–95, 143, 148, 154,
frying 24, 32, 119, 182 161, 184, 195, 197, 219, 221–223, 228, 230–
234
gallic acid/esters 26, 46–50, 69, 173 AGE-LDL/glycated LDL 197, 198
INDEX 253
inhibition 148, 149, 157, 195, 196, 206 minor constituents 117
lecithin 6, 7 oxidative stability 116–118
leukotrienes 183, 219 phenolic compounds 117, 118, 230
linoleic acid 47, 89, 93, 94 onions, cooked 153, 154
lipid oxidation 1, 12, 13, 21, 28 ORAC assay 89–95
products 6, 182, 230 OSI method 93
lipid peroxidation 2, 4, 5 oxidant–antioxidant balance 8, 9, 144, 145, 157,
lipids 6, 13, 14 184, 194, 230, 234
lipoproteins 9, 10 oxidation 1, 23
liposomes 6, 7, 27, 49, 50, 60, 61, 238 biological 9, 194
oxidation 94, 95 photosensitized 35, 40
low-density lipoproteins (see LDL) oxidative stress 8, 9, 144
oxidizability 9, 22, 29
macrophages 9, 10, 149, 150, 152, 161, 183, 198, oxygen 8, 25, 35, 38, 89
203, 220, 231 singlet 5, 35, 38
Maillard reaction 105, 108, 193, 194
MRP (Maillard reaction products) 193, 195–
partition 6, 7, 15, 16, 65, 67–72, 79, 83, 163
197, 206
peroxide destroyers 5, 145
meat products 124, 200
phenolic acids 48, 153, 166, 173, 177, 179
antioxidants 124–128
phenolic compounds 46, 111–114, 166, 184, 237
mechanisms 3, 13, 23, 29, 77
absorption 155, 165
Mediterranean diet 114, 117, 136, 184, 185, 230,
metabolism 177, 179
233, 234
phosphatidylcholine (PC) 49
metabolites 16, 151, 152, 165–167
liposomes 6, 7, 27, 49
catechin, quercetin, tea 153–155
phospholipase 12, 14, 150
metal(s) 14
phospholipids 40, 106, 108, 110, 111, 147
binders 38, 145
phosphoric acid 25
catalysts 7, 12, 24–26, 51, 160
phytochemicals 10, 11
chelation 3, 5, 8, 25, 26, 38, 52, 82, 207, 234
pine bark phenolics 126, 127
ions 5, 9, 22
platelet aggregation 11, 151
iron 26, 157
polar paradox 45, 69, 94
reducing by flavonoids 158
polyphenols 173, 232
trace 3, 8
postprandial state 12, 154–156, 169, 182, 229,
methodology 13, 29, 108, 114, 149, 179, 182,
230
231, 232
prooxidant chemistry 7, 15, 157, 226
antiradical methods 83–92, 97, 98, 169, 226
vitamin C 159, 162
browning reaction products 109
vitamin E 157, 159, 160
comparison of methods 93–96
propyl gallate 46, 47, 49
specific 13, 15 prostaglandins 183, 219
recommended protocols 96–98 proteins 4, 10, 53–55, 59, 198
methyl carnosate 46, 47, 49 lactoferrin 55, 56, 64
methylene blue 35 metal–binding/chelation 14, 27, 55, 58, 59
micelles 7, 44, 89, 161
phenolic interactions 53, 59, 61–64
Michael addition 52
sulfhydryl groups 55, 59
milk products 122–124
protocols 6, 77–82, 84, 96, 183, 185, 233, 235
monocytes 150
recommended protocols 96–98
nanotechnology (food) 238–239 PUFA 4, 26, 234
nitrous oxide 4, 5
non-antioxidant activities 4, 5, 143, 144, 149, quercetin 46, 54, 151, 153, 163–165, 171, 219–
156, 171, 179, 182, 183, 218, 219, 223, 231, 221, 223, 224
232, 235 absorption 167, 173
nutrigenomics 235 bioavailability 167, 168
BSA complex 53
obesity 157, 193 gallic derivatives 167–169
olive oil 11, 117, 118, 233 metabolites 153, 167–171, 179, 181, 224–226
thermal decomposition products 121 rutin 166, 173
radicals 23, 29, 38, 182 TEAC assay 85–88, 93–95

rapeseed meal phenolics 126 termination 22, 23
cooked pork patties 127 tertiarybutylhydroquinone (TBHQ) 34, 46
reactive oxygen species 2, 3, 14, 37, 38, 154, 229 testing 2, 10
redox cycle/cascade 147, 163 tocopherols 3, 160
potential 31, 34 α 3, 4, 12, 29, 32–35, 45–47, 160, 161, 205,
reducing agents 39, 40 218–220
reductones 195 transfer protein 160, 183, 229
resonance stabilization 23, 24 γ 3, 4, 34, 160, 161, 219, 220, 229, 230
riboflavin 35 δ 3, 4, 34, 111, 115, 122, 160–162, 218, 229,
rosemary extract 32, 46, 47, 115 230
synergism with BHT 116 oxidation products 29, 115, 117, 131
rosemarinic acid 32, 46 tocotrienols 4
transcription factors 16, 17
Schiff base 52, 106, 194, 196, 197, 207 transferrin 27, 38
sensitizers 35, 40 Trolox 12, 45–47, 49, 83, 89, 90, 208
singlet oxygen quenchers 144, 145
structure-activity 29, 31 ubiquinol-10 40
substituents 23–31 ultraviolet stabilizers 28
superoxide 159 uric acid 40
assay 84, 92
dismutase 14, 146, 147 vegetable oils 1, 3, 114, 221
supplementation 16, 179, 233, 234 synthetic antioxidants 114
prooxidant effects 159 natural antioxidants 115
vitamin C 162 stability 116
surfactant 6, 7 very-low density lipoproteins (VLDL) 160
stoichiometric factor 28–30 vitamins
synergism 4, 5, 33, 34, 40, 147, 169 vitamin C 3, 5, 6, 11, 34, 36, 149, 152, 161,
systems 162, 182, 204, 206
heterophasic 7 vitamin E 3, 4, 6, 9–11, 16, 17, 34, 36, 40,
multiphase 6, 7, 8 148–150, 161, 182, 184, 204, 206, 218, 220,
228, 233
TBA test, TBARS 132, 169 inhibition of LDL oxidation 148
tea, green 11, 152, 154, 171, 172, 179, 210, 219, supplementation 4, 5, 145, 152, 228
220 wine 11, 12, 16, 83, 87, 108, 111, 113, 114, 149,
catechins 46–49, 94, 95, 220, 226–228 155–157, 171–173, 179, 184, 185, 220, 223,
gallates 47, 94, 152, 172, 228 228, 230, 232, 236

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Antioxidants in food and biology

Facts and fiction

Oxford Cambridge Philadelphia New Delhi

Woodhead Publishing, 1518 Walnut Street, Suite 1100, Philadelphia,

First published by The Oily Press, 2007

© PJ Barnes & Associates, 2007; © Woodhead Publishing Limited, 2012

Trademark notice: Product or corporate names may be trademarks or registered trade-

British Library Cataloguing in Publication Data

ISBN 978-0-9552512-0-7 (print)

Typeset by Ann Buchan (Typesetters), Middlesex, UK

between the aqueous phase, lipid phase and surfactant-enriched interface in

Chapter 7 covers the renewed attention in biochemistry on the Maillard

have unique, novel and potentially useful functional properties caused by

The author gratefully acknowledges the invaluable editorial work of Frances

The current applications of antioxidants in foods are often empirical, how-

1. Natural versus synthetic antioxidants

2. Lipid peroxidation in vivo

3. Tocopherols and vitamin E antioxidants

various tocopherol homologs is, however, controversial due to the varying

4. Ascorbic acid or vitamin C

Since ascorbic acid is readily regenerated from dehydroascorbate in vivo, it is

LDL particle Lecithin

3. Oxidation of low-density lipoproteins (LDL)

adherence of monocytes and expression of a scavenger receptor. More exten-

Although the pharmacological activities of various flavonoid compounds

6. Nutritional effects of food ingredients

7. Effect of antioxidants on aging

multiple antioxidant defenses. In general, accumulating evidence in the litera-

pathophysiological process. Many recent studies have inaccurately ascribed

A. Free radical mechanisms

LOOH ––——➤ LO· + ·OH

The simple thermal decomposition of hydroperoxides (2) seldom occurs

LOOH ––——➤ LO· + ·OH

oxidation (in the absence of an antioxidant) under atmospheric conditions, the

Figure 2.1. Resonance stabilization of phenoxy radicals from tri-substituted phenols.

temperatures, such as frying, or in the presence of metal catalysts or other

a. Metal inactivators. These compounds function by removing or chelating

Figure 2.2. Structures of common metal chelators.

Samples TAGOOH CEOOH Fishy flavor Fishy aroma

Control 7.86 0.34 2.8 2.0

From Jacobsen et al. (2001)

Emulsified mackerel oil Peroxide value (meq/kg)

Control 36.1 91.3 132

From Frankel et al. (2002).

can thus promote iron catalysed oxidation if used at insufficient concentrations

b. Hydroperoxide destroyers. These compounds are mainly reducing

Table 2.3. Inhibition of hydroperoxide formation and decomposition by lactoferrin (LF)

0 12.5 61.9 72.0 0:12.5

From Satué-Gracia et al. (2000).

hydroperoxides to produce stable hydroxy compounds in low yields. Sodium

c. Ultraviolet stabilizers. Compounds that inhibit photo-induced lipid

decompose slowly to generate known quantities of radicals in a steady and

Figure 2.3. Formation of peroxy-cyclohexadienones and stilbene-quinone by oxidation of 2,6-di-tert-

in having a stoichiometric factor n = 2 or less. Both the peroxide products

Electron-donating substituents in the 4 position thus increase antioxidant

Figure 2.5. Structures of synthetic antioxidants.

participation of chain transfer reactions (–10) and (13). The effectiveness of

Figure 2.6. Structures of natural antioxidants.

D. Synergistic antioxidant systems

α-TOH + LOO· ——➤ α-TO· + LOOH (21)

biology. Flavonoids scavenge free radicals, deactivate metals by complex

E. Inhibition of photosensitized oxidation

F. Antioxidant enzymes in food systems

G. Inhibition of biological oxidation