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CHAPTER ONE

Antioxidants in Food: Content,


Measurement, Significance,
Action, Cautions, Caveats, and
Research Needs
Iris F.F. Benzie1, Siu-Wai Choi
Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon,
Hong Kong
1
Corresponding author: e-mail address: htbenzie@polyu.edu.hk

Contents
1. Introduction: Antioxidants in FoodTheir Role and Importance
1.1 Basic concepts of oxidation and the role of antioxidants
1.2 Antioxidants: Types and action
1.3 A closer look at ascorbic acid: A key antioxidant
1.4 Human antioxidant defense and dietary influences
2. Measuring Total Antioxidant Content of Food
2.1 Basic principles, notes on calibration, units, and confusion
2.2 Limitations of the total antioxidant content approach: Cautions
and caveats
3. The FRAP Assay and Its Modified Form (FRASC) for Ascorbic Acid
3.1 Basic principles
3.2 The FRAP assayProcedure in brief
3.3 Application of the FRAP assay in food science
3.4 A note on the nonuric acid FRAP value of blood plasma and its relevance
to nutritional science
3.5 The modified FRAP assay (FRASC) for total antioxidant and ascorbic acid
measurement
4. Why Is the Antioxidant Content of Food of Interest?
4.1 Basic concepts
4.2 Antioxidants and health: The evidence and potential impact
5. Antioxidants in Food: Enigmas and Evolutionary Aspects
6. Mechanisms of Action: Redox Issues, Phytohormesis, and Colonic Microbiota
6.1 Redox tone changes as a driving force for cytoprotection and the biological
sense of restricted bioavailability of dietary antioxidants: The redox-sensitive
KEAP-1Nrf2 signaling pathway
6.2 Colonic microflora and metabolism of food antioxidants: Moving into new
territory of the microbiome

Advances in Food and Nutrition Research, Volume 71


ISSN 1043-4526
http://dx.doi.org/10.1016/B978-0-12-800270-4.00001-8

2014 Elsevier Inc.


All rights reserved.

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Iris F.F. Benzie and Siu-Wai Choi

7. The Story So Far


8. Concluding Remarks and Research Needs
Acknowledgments
References

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Abstract
There are a multitude of antioxidants in foods, especially in foods of plant origin. Higher
intake of antioxidant-rich foods is clearly associated with better health and functional
longevity. The specific agents and mechanisms responsible are not yet clear, but there
is convincing evidence that including more plant-based, antioxidant-rich foods, herbs,
and beverages in the diet is effective in promoting health and lowering risk of various
age-related diseases. The content of some individual antioxidants, such as vitamin C, in
food can be measured, but it is not feasible to attempt to measure each antioxidant
separately, and methods have been developed to assess the total antioxidant content
of foods. One of the most widely used methods is the ferric reducing/antioxidant power
(FRAP) assay, which is relatively simple, quick, sensitive, and inexpensive to perform.
There are many published studies that have used the FRAP assay, and these have generated a very large database of total antioxidant content of foods that can help guide
food choices for increased antioxidant intake. The FRAP assay has also been used to
assess the bioavailability of antioxidants in foods and to investigate the effects of growing conditions, storage, processing, and cooking method on the total antioxidant content of food. The test can be employed as a quality control check device, and to detect
adulteration of food. Furthermore, in a modified form (FRASC), the assay can measure
ascorbic acid content almost simultaneously with the total antioxidant content of the
sample. In this chapter, basic concepts of oxidation and the role of antioxidants, as well
as the types and action of different antioxidants in foods will be reviewed briefly, and the
underpinning concepts and evidence for health benefits of increased intake of dietary
antioxidants will be discussed, with some focus on vitamin C, and also in the context of
our evolutionary development. The basic concepts and limitations of measuring total
antioxidant content of food will be presented. The FRAP assay and the modified version
FRASC will be described, and the total antioxidant content (as the FRAP value) of a range
of foods will be presented. Finally, issues of bioavailability and redox balance will be
discussed in relation to the biological significance and molecular action of antioxidants
in foods, some caution and caveats are presented about overcoming biological barriers
to absorption of antioxidant phytochemicals, and research needs to further our understanding in the important area of food, antioxidants, and health will be highlighted.

1. INTRODUCTION: ANTIOXIDANTS IN FOODTHEIR


ROLE AND IMPORTANCE
1.1. Basic concepts of oxidation and the role
of antioxidants
In chemical terms, oxidation is the addition of oxygen to or the removal of
hydrogen or an electron from a molecule. A biological antioxidant can be

Antioxidants in Food

defined as a substance that prevents the oxidation of an important biomolecule, such as a fatty acid, DNA, or a protein, by a reactive oxygen species
(ROS) (Ames, 1998; Halliwell & Gutteridge, 2007). ROS are partially
reduced or energized forms of oxygen (Table 1.1). Some ROS contain
an unpaired electron in an orbital and are sometimes referred to as free radicals; others, such as hydrogen peroxide and singlet oxygen, are nonradical
species, but their reactivity is nonetheless greater than ground-state molecular oxygen, allowing them to oxidize biomolecules they interact with.
Some ROS have important physiological functions (Ames, 1998;
Halliwell & Gutteridge, 2007). For example, nitric oxide is a vasodilator,
hydrogen peroxide is a key intracellular signaling molecule, and superoxide
and hydrochlorous acid play an important role in our innate immune defense
(Wink et al., 2011). However, an excess of ROS can lead to uncontrolled or
indiscriminate oxidation of important biomolecules. This deleteriously
affects their structure and function and is known as oxidative stress
(Ames, 1998; Halliwell & Gutteridge, 2007). The imbalance that results
in oxidative stress can be caused by excessive production of ROS, or to a
deficit in the antioxidants that oppose ROS and their damaging oxidative
effects (Fig. 1.1).

1.2. Antioxidants: Types and action


In an oxygenic environment, the threat of oxidative stress is ubiquitous and
continuous for all organisms, but the photosensitizing plants that are responsible for our oxygenic environment are exposed to particularly high
Table 1.1 Types of ROS and relative reactivity
ROS
Redox couple

Hydroxyl radical

E0 (mV)

HO%, H/H2O

2310

1600

Alkoxyl radical

RO%, H /ROH

Peroxyl radical

ROO%,

H /ROOH

1000

GS%/GS

920

Glutathiyl radical
Tocopheroxyl radical
Hydrogen peroxide

480

TO%, H /TOH

H2O2, H /H2O,
%

Ascorbate radical

Asc , H /AscH

Superoxide radical

O2/O%2_

One-electron reduction potentials at pH 7.0.


Source: Halliwell and Gutteridge (2007).

HO%

320
282
330

Iris F.F. Benzie and Siu-Wai Choi

Figure 1.1 The concept of antioxidant/oxidant imbalance and the development of


oxidative stress.

intracellular levels of oxygen and ROS and have evolved specialized defense
factors to deal with the high oxidant challenge and protect plant structures
and tissues (Benzie, 2000). These factors are referred to as antioxidants.
The best-known antioxidant is vitamin C (ascorbic acid), but there are
various others, and many are unique to the plant kingdom. These include
vitamin E, a group of eight tocopherols and tocotrienols, and the very
large families of flavonoids and carotenoids (Benzie, 2005; Buettner,
1993; Gey, 1998; Tam et al., 2005). Indeed, there are thousands of different
antioxidants in plants, and they work in different ways (Table 1.2). Some
antioxidants prevent the generation of ROS, some are enzymes that destroy
ROS, some are small water-soluble molecules that act as reducing (hydrogen
or electron-donating) agents to neutralize free radicals, and some absorb
electrons or excess energy from ROS and dissipate this within their
complex lipophilic structure (Ames, 1998; Halliwell & Gutteridge, 2007).
Overall, the effect is to oppose ROS action and limit oxidative stress.

1.3. A closer look at ascorbic acid: A key antioxidant


Vitamin C (ascorbic acid) is present in high concentration in many plants
(Benzie & Wachtel-Galor, 2009; Frei et al., 1989; Gey, 1998;
Halliwell & Gutteridge, 2007; Halvorsen et al., 2002). The content of this
water-soluble antioxidant is particularly high in fruits, but it is found in various plant parts and aids repair and growth of plants and the ripening of seeds.

Antioxidants in Food

Table 1.2 Plant-derived antioxidant types, action, and sources


Antioxidant
Action
Mechanism
Sources

Ascorbic acid
(vitamin C)

Scavenging Sacrificial interaction


of ROS
by replaceable or
recyclable substrates to
scavenge and destroy
ROS

Fruits and vegetables,


particularly
strawberries, citrus,
kiwi, Brussels sprouts,
cauliflower, some
Chinese green
vegetables

Vitamin E (a, b,
d, g) isomers of
tocopherols and
tocotrienols

Quenching Absorption of electrons


of ROS and and/or energy
chain
breaking

Green leafy vegetables


(e.g., spinach); nuts,
seeds, especially wheat
germ; vegetable oils,
especially sunflower

Carotenoids
(hundreds)

Chain
breaking

Orange/red fruits and


vegetables (e.g., carrot,
tomato, apricot, melon,
pumpkin), green leafy
vegetables, peppers

Flavonoids (large
range of different
types)

Scavenging Sacrificial interaction


of ROS

Chain breaking at low


partial pressures of
oxygen, complements
action of vitamin E

Berries, apples, onions,


tea, red wine, some
herbs (parsley, thyme)
citrus fruits, grapes,
cherries

Source: Halliwell and Gutteridge (2007).

Most animals, fish, and reptiles can synthesize vitamin C in liver or kidney,
but humans have lost the ability to make ascorbic acid, even though we have
retained an absolute requirement for it (Benzie, 2000, 2003; Benzie &
Strain, 2005; Frei et al., 1989). Ascorbic acid is known to be needed for collagen synthesis in humans, and severe deficiency results in scurvy, which is
nowadays rare but cases are nonetheless still reported (Doll & Ricou, 2013).
Humans must obtain a regular and adequate dietary supply to maintain
plasma and tissue levels, and the best dietary sources are fruits and vegetables
(Frei et al., 1989). In the past, the daily recommended intake of vitamin
C was set at a level (30 mg/day) that would prevent overt deficiency
(Newton et al., 1983). However, accumulated epidemiological and experimental evidence indicates that the role of ascorbic acid in the human body is
not restricted to collagen synthesis and that it has an important role in health
maintenance overall (Benzie, 1999). Ascorbic acid is recognized as a major

Iris F.F. Benzie and Siu-Wai Choi

player in human antioxidant defense (Benzie, 1999, 2000). Current dietary


recommendations are for at least 70 mg/day for women, 100 mg/day for
men, with higher levels advised in pregnancy and for smokers (Carr &
Frei, 1999). These levels of intake are recommended to attain optimal status for health, though this has not been clearly defined. Human cells, especially white blood cells, and some organs, most notably the eye, contain
millimolar concentrations of ascorbic acid (Choy et al., 2001, 2003). Plasma
ascorbic acid concentrations in the fasting state range quite widely, from
<10 (worryingly low) to 100 mmol/l in apparently healthy adults (Choi
et al., 2004). Tissue and plasma levels are maintained by, and so reflect, dietary intake of vitamin C, and the plasma concentration of ascorbic acid can
act as a marker of fruit and vegetable intake (provided no supplements are
used) (Choi et al., 2004). A concentration <10 mmol/l indicates very low
intake and severe deficiency, even if signs of scurvy are not clearly present
(Levine et al., 2011). Plasma levels at 12 h after ingestion of a large dose (1 g
or more) of vitamin C can approach 200 mmol/l, but peak levels are limited
by restricted gastrointestinal absorption of a large dose and by urinary loss of
ascorbic acid when the plasma concentration exceeds the renal threshold of
100 mmol/l (Levine et al., 2011). Therefore, most of a single, large dose is
not retained in the body, and regular, modest doses (500 mg or less) are likely
to be more effective in enhancing the ascorbic acid status of the body. In
large prospective trials, it has been shown that healthy people with higher
fasting plasma concentrations of food-derived ascorbic acid (i.e., not from
supplements) have significantly lower risk of incident diabetes, cancer,
stroke, heart failure, and all cause mortality in the ensuing years (Fig. 1.2;
Harding et al., 2008; Pfister et al., 2011). It has been estimated that for every
20 mmol/l increase in fasting plasma ascorbic acid concentration, there is a
9% relative reduction in risk of incident heart failure and a 29% decrease in
risk of incident type 2 diabetes (Harding et al., 2008; Pfister et al., 2011).
Plant-based food is the primary source of ascorbic acid in human plasma.
However, it must be noted that the protective agent may not be vitamin
C itself. It could be something very closely associated with vitamin C in
plant-based foods, or it could be that the health benefits are due to the interactive effects of the various antioxidants present in plant-based foods.

1.4. Human antioxidant defense and dietary influences


The human body is exposed to ROS on a continuous basis, and we have an
effective antioxidant defense system that has evolved to help deal with the

Antioxidants in Food

Hazard ratio for incident heart failure

1.2

1.0

0.8

0.6

0.4

0.2

0.0
0

Quartiles of plasma vitamin C or dietary fruit and vegetable intake


Error bars indicate 95% Cl
Plasma vitamin C concentration
Self-reported dietary fruit and
vegetable intake

Figure 1.2 Risk for incident heart failure decreases with increased plasma vitamin C in
subjects observed over a period of 16 years. Data taken from Pfister et al. (2011).

threat of oxidant challenge (Benzie, 2000, 2003). The system is complex


and, as with plants, there are different types of antioxidants that prevent generation of, divert, or destroy ROS (Benzie & Wachtel-Galor, 2009). Our
intrinsic antioxidant defense is highly effective but not wholly adequate,
and dietary input of antioxidants is needed (Benzie, 2003; Benzie &
Wachtel-Galor, 2009; Norat et al., 2014). Plant-based foods and beverages,
such as fruits, vegetables, tea, coffee, spices, and herbs, are the main source of
antioxidants in the human diet (Benzie & Wachtel-Galor, 2009; Halvorsen
et al., 2006). It is noted that the only dietary-derived antioxidants that have
been shown to be essential for human health are the water-soluble vitamin C
and the lipophilic vitamin E (which in human tissues and structures is mainly
in the form of a-tocopherol). Still, conceptually at least, the other antioxidants that evolved to deal with oxidant challenge in plants could have a role
to play in defending human tissues from this same challenge, augmenting our
endogenous antioxidant system in the prevention of oxidative stress. To
what extent they do this and their contribution to health outcomes are

Iris F.F. Benzie and Siu-Wai Choi

not yet clear. Plant-derived antioxidants including the catechins and some
anthocyanins, carotenoids, xanthophylls, and other members of the vitamin
E family are found in human plasma (Table 1.3), and the antioxidant capacity
of human plasma increases after intake of polyphenol-rich dietary agents
such as tea and coffee (Benzie et al., 1999; Garca-Alonso et al., 2006;
Hukkanen et al., 2006; Molan et al., 2008; Othman et al., 2007; Seeram
et al., 2008). However, the very low (nanomolar) concentrations of the
nonessential plant-derived antioxidants have made it difficult to study
Table 1.3 Plant-derived antioxidants and their typical concentrations in fasting plasma
Concentration (mM) Food source

Ascorbic acid

1060

Fruits, particularly kiwi fruits,


strawberries

Vitamin
E (a-tocopherol)

1640

Wheatgerm, sunflower seeds, nuts

Epicatechin

0.005

Tea, chocolate, wine

Epicatechin gallate

0.041

Epigallocatechin
gallate

0.016

Epigallocatechin

Undetectable

Catechin

Undetectable

Quercetin

0.001

Apple, onion, red grape, leafy green


vegetables

Anthocynanin

0.046

Aubergine, blackberry, blackcurrant

Lutein

0.160

Green leafy vegetables, spinach, kale

Zeaxanthin

0.050

Wolfberry, egg yolk, corn, paprika

Lycopene

0.377

Tomatoes, guava, grapefruit,


watermelon

-carotene

0.354

Sweet potato, carrots, kale, butternut


squash

Phytoene

0.069

Most fruits and vegetables

Phytofluene

0.044

Most fruits and vegetables

Isoflavones

0.428

Soya, chickpea, peanut, alfalfa

Source: Benzie et al. (1993), Halliwell and Gutteridge (2007), Harding et al. (2008), and Engelmann
et al. (2011).

Antioxidants in Food

individual nonessential antioxidants in relation to their bioavailability,


bioactivity, and metabolism in human experimental trials, and most of the
evidence for their health benefits has come from epidemiological, foodbased studies. This is changing with the adoption of hyphenated technology
platforms, particularly liquid chromatography with tandem mass spectrometry (LCMS/MS), which provide highly specific and sensitive measurement methods for many individual plant-derived antioxidants in foods,
plasma, and other biological materials. Still, the antioxidant fingerprint or
profiles of foods are far from complete as it is not feasible to measure each
and every antioxidant in a complex mixture such as food. In the face of this
limitation, a widely adopted approach is to determine the total antioxidant
content of food and of blood plasma following intake of antioxidant-rich
food (Bartoz, 2003). Several methods have been developed for measuring
total antioxidant content. In Section 2, basic principles of total antioxidant
content measurement and limitations of this approach are discussed, and
details of one of the most widely used methods, the ferric reducing/antioxidant power (FRAP) assay, along with the total antioxidant content (as the
FRAP value) of a range of foods are presented.

2. MEASURING TOTAL ANTIOXIDANT CONTENT


OF FOOD
2.1. Basic principles, notes on calibration, units,
and confusion
The different methods that have been developed to measure total antioxidant content of food use one of two basic principles and can be described as
direct or indirect methods (Bartoz, 2003). The basic principle of a
direct method is to measure the reductive action of all redox-active (electron
donating) antioxidants present as they reduce a component, added to the
reaction mixture in excess, with the production of a change in an indicator
signal. The signal can be a change in absorption of light (spectrophotometric methods) or in an electrochemical signal, such as current flow. The magnitude of the change in signal is directly related to the combined reducing
action of the electron-donating antioxidants in the mixture, and the signal
change can be quantified with reference to the signal change induced by a
calibrator, such as pure ascorbic acid, run in parallel. This electron transfer
principle is applied in, for example, the FRAP assay and in the assay referred
to as Cuprac in which cupric ions are reduced to cuprous ions (Benzie &

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Iris F.F. Benzie and Siu-Wai Choi

Strain, 1996a). The basic principle of indirect (lag-phase) methods is to add a


free radical generator to the reaction mixture and to time how long it takes
before the antioxidants present in the reaction mixture are exhausted, and an
indicator molecule begins to be reduced by the radicals that by that stage act
unopposed on the indicator molecule. The reduction of the indicator molecule produces a change in signal, such as an absorbance change. The indicator molecule can be a dye that changes color when oxidized, but an
unsaturated fatty acid can be used, with the change in spectral signal related
to the formation of conjugated dienes within the oxidized lipid (Bartoz,
2003). The lag-phase approach is most commonly used in the forms of
the oxygen radical absorption capacity test and the ABTS radical test
(Bartoz, 2003).
To translate changes in signal to total antioxidant content in direct
methods, the lag time is translated into a measure of antioxidant action with
reference to the lag time of a known amount of a purified antioxidant, most
commonly Trolox as calibrator (standard). This is a water-soluble analogue of a-tocopherol, and the most commonly used unit for reporting total
antioxidant activity is mmol/l (or mmol/l or mmol/g depending on the sample type) of Trolox Equivalents (TE though different terminologies are
used to express results). Solutions of Trolox can be used in direct reductive methods also, but solutions of pure ascorbic acid are often used because
ascorbic acid is inexpensive and highly soluble in water, and so some
researchers use units of ascorbic acid equivalents (AAE). These units
(TE and AAE) should be interchangeable because one molecule of Trolox
and ascorbic acid can each donate two electrons, and therefore, in theory,
they have antioxidant equivalence. However, while Trolox in solution
is fairly stable, we have found it is far less easy to dissolve than the highly
soluble ascorbic acid. For this reason, ascorbic acid solutions of greater accuracy can be prepared. Yet, ascorbic acid, while fairly stable in its solid form, is
unstable in aqueous solution. Therefore, total antioxidant activity results
obtained using either Trolox-based or ascorbic acid standards can be overestimated because of, in one case, poor solubility, leading to lower actual
concentrations of standards than the nominal concentrations and, in the
other, degradation of the ascorbic acid in aqueous solution if these are
not prepared immediately before use. Such problems can be detected by
close monitoring of the signal given by the standards, which should be consistent across different test runs and batches of standards. A further complication is that in direct reductive methods, the stoichiometric factor (which as
noted is 2.0 in the case of both ascorbic acid and Trolox) is often applied to

Antioxidants in Food

11

give a result that reflects the number of electrons transferred during the reaction. In this case the total antioxidant activity result is twice that of the
result expressed in either ascorbic acid equivalents or Trolox equivalents. A further complication is that stoichiometric factors of antioxidants,
including Trolox and ascorbic acid, vary with concentration in some
methods. Moreover, while results on purified antioxidants such as ascorbic
acid can match fairly well across the different methods, results on complex
mixtures, such as food extracts or blood plasma, from the different assays do
not agree well because different antioxidants react differently in the different
test systems and reaction conditions. This makes it very difficult to make
meaningful comparison of total antioxidant results from studies that have
used different methods of measurement. It is easier to compare data obtained
using the same method, but even then results from different studies are often
difficult to compare directly because of differences in sample handling, standardization procedure, reaction conditions (time, temperature, pH), solvent
used, and the units used to express results. Various units, such as TEAC
units, TAC units, TAA units, TRAP units, TE, AAE, radical
scavenging, or radical absorbance units appear in the literature, and
some results are presented simply as absorbance or fluorescence units,
the lag time or the time taken to change the signal by a particular amount
(Bartoz, 2003). These variations in measuring principle and method
settings, calibration, and activity units reported cause confusion and make
it difficult to compare or combine results of different studies unless
sufficient information is given on how the results were obtained in terms
of: sample type and preparation; method and calibrator used; reaction time
used; the signal measured, and how that was translated into the result
presented (Benzie & Wachtel-Galor, 2013).

2.2. Limitations of the total antioxidant content approach:


Cautions and caveats
The basic concepts and principles of measuring total antioxidant content are
generally quite simple, but the underlying chemistry of the various methods
is complex, and as noted there are differences in measuring principles and
setting, calibrators, and reporting units to consider, confound, and confuse.
Each of the methods currently available has its own limitations, for example,
thiol groups do not react well or quickly in the FRAP assay so that GSH and
protein make very little contribution to the FRAP value, while results of
proteinaceous samples (meat, plasma) are overwhelmingly due to protein

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thiol groups, especially when the reaction time is prolonged to an hour or


more (Bartoz, 2003; Benzie & Strain, 1996a). Currently, there is no universally accepted reference method and even with the same method, reaction
conditions can vary greatly between laboratories. Furthermore, it must be
understood that the whole concept of measuring total antioxidant activity
by a chemical test has serious limitations (Bartoz, 2003). A clear understanding of what each method measures is needed for the result to have some
meaning for nutrition, food, and health science. None of the methods measures all antioxidants present in a mixture. The tests are limited to measuring
the effect of redox-active antioxidants, and so do not measure the catalytic
action of superoxide dismutase (SOD) and glutathione peroxidase (GPx), or
the preventive action of ferritin. As noted, some are not sensitive to thiols,
while others furnish results that are determined largely by the action of thiol
groups in proteins present in the sample (Bartoz, 2003). The tests are all
in vitro tests and most if not all the reaction conditions are very far from
the physiological. Results on an extract of food may have little in the way
of physiological significance, as antioxidants may work in quite a different
manner when present in a mixture or in a living system, as opposed to in purified form or in vitro. There are also issues of bioavailability and biotransformation to consider. Dietary antioxidants are often not absorbed well, and
what is estimated to be contained in a food or the diet overall is unlikely
to reflect what enters the circulation (Davey et al., 2000). Those that are
absorbed may undergo rapid conjugation in the liver and renal clearance.
Antioxidants that are not absorbed may be extensively metabolized by the
colonic microbiota. For example, ring scission metabolites of catechins
(flavan-3-ols) in green tea are formed by colonic metabolism and are found
in the circulating plasma several hours after ingestion of green tea (Del Rio
et al., 2010). Therefore, data on the in vitro total antioxidant content of
foods are of limited value in relation to physiological relevance. Still,
despite its limitations, the measurement of total antioxidant content of
foods has become a standard approach in nutritional and food science in
regard to the potential health benefits or value of different foods and food
extracts or supplements (Ashfari et al., 2007; Bartoz, 2003; Benzie & Szeto,
1999; Benzie et al., 1999; Chung et al., 2001; De et al., 2008; Dragsted
et al., 2004; Duthie et al., 2006; Fernandez-Pachon et al., 2005; GarcaAlonso et al., 2006; Lotito & Frei, 2006; Ma et al., 2008; Rabovsky
et al., 2006; Serafini et al., 2003; Smet et al., 2006; Wachtel-Galor,
Szeto, Tomlinson, & Benzie, 2004). Indeed, a quick search of the literature
with key words of antioxidant and food reveals 19,677 papers

Antioxidants in Food

13

published between 2008 and 2013 (Pubmed accessed August 2013). Many
packaged foods now include a measure of total antioxidant content on
their labels and marketing materials. Therefore, it is important for food scientists and nutritionists to understand the concepts and limitations outlined
above, and to generate and use data with these in mind.
In summary, different methods exist to measure the total antioxidant
content of foods, but there is no universally accepted reference method; there
are important caveats to the basic concept of measuring total antioxidant
content of food. Caution is needed in using the results. To compare results
from different analyses, it is important to understand what each method measures, how it is calibrated (standardized), and what the reported units of content or activity represent. It is important also to understand the limitations of
the method, and where possible to adopt a standard operating procedure for
the method. In this way, results obtained on different materials and from different research groups can be compared, integrated, and used to aid our
understanding of the role and value of total antioxidant activity in nutritional, food, and health sciences. In Section 3, the focus is on one widely used
method, the FRAP assay. This method has low cost and high sensitivity, is
quick and relatively simple to perform, and has been used to generate a rich
database of total antioxidant content of a wide variety of foods. As will be
described, the method has been used also to assess bioavailability of reductive
antioxidants in foods and the effect of processing and cooking on the antioxidant content of foods, and it can be used to monitor batch-to-batch variation
and detect adulteration of foods. In addition, the method in a modified form
can be used to measure ascorbic acid in the sample almost simultaneously with
its total antioxidant content (Benzie & Strain, 1997).

3. THE FRAP ASSAY AND ITS MODIFIED FORM (FRASC)


FOR ASCORBIC ACID
3.1. Basic principles
The FRAP assay uses the reduction of the pale yellow-colored triazinecomplexed ferric (Fe3) to its ferrous (Fe2) form as the signal, or
indicator, reaction in this direct method to measure total reductive antioxidant action. The 2,4,6-tripyridyl-s-triazine (TPTZ)Fe2 complex is
deep blue in color, and the reduction-driven change in absorbance at
593 nm is the signal measured. A wide range of sample types can be tested
in the FRAP assay, and the assay can be used successfully in a simple manual
version that requires little in the way of specialized equipment, in a

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semiautomated version using a microplate reader, or adapted to run in fully


automated mode using a user-defined program in a biochemical analyzer.
Reagents are stable and of low toxicity, sensitivity and precision of the
method are high, stoichiometric factors of reacting antioxidants are constant,
and the test is robust in that small differences in reaction conditions do not
markedly affect results. In a modified version known as the FRASC assay,
both the total antioxidant activity and the ascorbic acid (vitamin C) concentration of the test sample can be measured virtually simultaneously by
employing ascorbic oxidase to destroy ascorbic acid in one of a pair of samples run in parallel (Benzie & Strain, 1997, 1999; Szeto & Benzie, 2002). In
addition, the contribution of uric acid, a possible confounder in the measurement of total antioxidant activity of plasma, can be removed by simple
calculation (if the uric acid concentration is known), providing the nonuric
acid FRAP value (Benzie & Strain, 1996b; Benzie & Szeto, 1999). This is
useful when using the FRAP assay with plasma samples to investigate
absorption of antioxidants (Bartoz, 2003).

3.2. The FRAP assayProcedure in brief


For detailed technical procedures, please refer to Benzie and Strain (1996a).
The basic protocol given here can be employed in a simple manual method
and can be translated by the user into a semiautomated microplate method or
a program designed for a fully automated method on a biochemical analyzer.
In brief, the method simply involves the addition of an aliquot of the sample
of interest to a measured volume of working FRAP assay reagent. This
reagent contains ferric chloride and TPTZ in acetate buffer. The working
reagent is prepared just before use by mixing 300 mmol/l acetate buffer
(pH 3.6), 10 mmol/l TPTZ in 40 mmol/l hydrochloric acid, and
20 mmol/l ferric chloride in a ratio of 10:1:1. The recommended relative
amounts of sample and reagent are 1:30. The sample must be in liquid form.
The solvent/diluent is usually water, but can be methanol, ethanol, or hexane if lipophilic antioxidants are of interest. After 4 min (if the reaction is
carried out at 37  C), the absorbance at 593 nm is measured. If the reaction
mixture is kept at room temperature, a longer reaction time (8 min) is recommended to align results with those obtained with a 4-min reaction time at
37  C values. A reagent blank is used to zero the spectrophotometer at
593 nm (or as close to that as possible if wavelength selection is based on
filters). The absorbance of the sample/reagent mixture is used to calculate

15

Antioxidants in Food

the FRAP value with reference to the absorbance change given by a calibrator (standard) run in parallel with the test samples. This can be a freshly prepared aqueous solution of pure ascorbic acid. However, because ascorbic
acid in solution is unstable, the recommended calibrator is an aqueous solution of known Fe2 concentration (prepared from ferrous sulfate). Ferrous
sulfate is soluble in water up to 2 mmol/l, and the solution is stable for at
least 1 week. The recommended concentration of the calibrator is
1000 mmol/l (1 mmol/l). A range of calibrators of different concentrations
can be prepared if a calibration line is desired, but the doseresponse is highly
linear and reproducible (under fixed conditions), and in our laboratory, we
use a single-point calibration at 1000 mmol/l.
It is recommended that results are expressed as the FRAP value, that is,
the FRAP value, in mmol/l. This is calculated as follows:
4 min A593 nm of test sample reaction mixture
4 min A593 nm of Fe2 standard reaction mixture
 Fe2 standard concentration mmol=l
It is noted that the FRAP assay is run as a fixed point assay, but that the
reaction is not completed at 4 min. Some antioxidants and their metabolites
continue to react slowly beyond the 4-min reaction time, and so the absorbance at 593 nm continues to increase, though the rate of further change is
slow. If so desired, the reaction time can be extended for specific samples,
but a standard 4 min (at 37  C) reaction time window is recommended to
facilitate comparison of results from different studies. In regard to samples,
various biological fluids (plasma, serum, urine, or saliva) can be tested without
extraction. If urine is tested, it should be run neat and diluted 1/5 and 1/10.
Foods, herbs, and spices can be extracted in various solvents, such as in cold
water, hot water, methanol, ethanol, acetone, or hexane. It has been shown
that the FRAP assay is less susceptible to solvent effects than other tests of total
antioxidant capacity (Perez-Jimenez & Saura-Calixto, 2006). If the extract is
turbid or highly colored, it is recommended that a sample blank is run. This is
done by measuring the absorbance at 593 nm of the extract in working
reagent minus TPTZ, and subtracting this absorbance from that of the
complete reagent/sample mixture. It is noted that the FRAP value of
plant-based materials can be very high, and additional predilution of the
extract is often needed.

16

Iris F.F. Benzie and Siu-Wai Choi

3.3. Application of the FRAP assay in food science


The FRAP assay has been applied widely in nutritional science. Apart from
measuring the total antioxidant content of various foods, the FRAP assay
has been used also to explore absorption of antioxidants from foods, such as
soya milk, cocoa, and tea, and to investigate the effect of processing and
cooking on the antioxidant content of foods. It is well recognized that transport to market, storage, and cooking practices affect the content of labile
antioxidants in foods, and the World Health Organization (WHO) has taken
this information into account in their recommendations for vitamin and
mineral requirements in human nutrition. WHO recommendations for
cooking foods containing labile antioxidants are to steam or stir fry. If water
is used in the cooking of vegetables, it may be advisable to also consume the
cooking water, as it contains antioxidants released from the food (WachtelGalor et al., 2008). The FRAP assay has also been used as part of a quality
control system in the agri-food industry, and to assess the effect of genetic
variation, season, growing conditions, and storage on the total antioxidant
content of foods. For example, total antioxidant content of blueberries of
the same cultivar grown in the same field can vary by up to 25% depending
on the harvesting year, and variation of up to 47% in total antioxidant content is seen in different cultivars grown in the same area and harvested in the
same year (Dragovic-Uzelac et al., 2010). Some representative FRAP values
of different foods are given in Table 1.4, and some illustrative data on the
effects of cultivar, season, processing, and cooking on the antioxidant content of some foods are shown in Table 1.5.

3.4. A note on the nonuric acid FRAP value of blood plasma


and its relevance to nutritional science
The FRAP assay has been used to investigate the changes in plasma antioxidant content after ingestion of foods and beverages, including apples, fruit
juices, and teas, results being used to explore bioavailability of redoxactive antioxidants in the diet (Benzie & Szeto, 1999; Benzie et al., 1999;
Duthie et al., 2006; Lotito & Frei, 2004). However, it is important to note
that there is an increase in plasma uric acid following ingestion of some dietary agents. Uric acid is an endogenous compound formed from the breakdown of purines and generally contributes 5060% of the total antioxidant
activity of plasma. However, following ingestion of purine-rich red meat or
fructose-rich foods and beverages, a postingestion increase in uric acid (and
hence antioxidant content) of plasma is seen (Benzie & Strain, 1996b;

17

Antioxidants in Food

Table 1.4 Representative FRAP values of various foods


Total
Per average serving
Food and food type antioxidant (mmol)

References

Tea (1% w/v infusion)

Green tea

10,000

2500

Benzie and Szeto (1999)

Oolong tea

5000

1250

Black tea

3500

875

Orange juice

1500

375

Apple juice

1100

275

Blueberry juice

4300

1075

Grapefruit juice

8220

2050

Pear juice

7430

1858

Pineapple juice

5160

1209

Tropical juice

6150

1538

Grape juice

120,000

30,000

Prune juice

10,000

2500

Tomato juice

48,000

12,000

Cranberry juice

92,000

23,000

Red wine

25,000

6250

Benzie and Strain (1999)

Rose wine

7220

1805

Pellegrini et al. (2003)

White wine

3000

750

Benzie and Strain (1999)

Beer

0.137

0.034

Halvorsen et al. (2006)

Cola

0.046

0.012

Cows milk

300

75

Soya milk

750

187.5

Coffee (expresso)

129,380

15,526

Coffee
(decaffeinated)

93,010

23,253

Fruit juices

Seeram et al. (2008)

Pellegrini et al. (2003)

Carlsen et al. (2010)

Wine

Other beverages

Pellegrini et al. (2003)

Continued

18

Iris F.F. Benzie and Siu-Wai Choi

Table 1.4 Representative FRAP values of various foodscont'd


Total
Per average serving
Food and food type antioxidant (mmol)
References
Fruits

Apple (green)

6.3

1146.6

Apple (red)

4.2

764.4

Strawberry

15.9

2337.3

Blackberry

22.0

3168

Blueberry

10.8

1555.2

Orange

9.4

1231.4

Pear

4.1

680.6

Kiwifruit

8.2

565.8

Pineapple

6.0

996

Lemons

10.4

72.8

Banana

2.28

269.04

Cherries

8.1

1190.7

Peach

1.5

225

Papaya

1256

Szeto and Benzie (2002)

Szeto and Benzie (2002)

Halvorsen et al. (2002)

Honeydew melon 2.27

304.18

Grapes (black)

11.09

1663.5

Grapes (green)

3.25

487.5

Mango

5.06

834.9

Cranberries

32.9

4836.3

Plums

13.3

2008.3

Grapefruit

8.2

1049.6

Watermelon

0.4

48.8

Guava

30

4950

Patthamakanokporn
et al. (2008)

Onion

2.4

168

Halvorsen et al. (2002)

Pepper (green)

2.6

192.4

Vegetables (raw)

19

Antioxidants in Food

Table 1.4 Representative FRAP values of various foodscont'd


Total
Per average serving
Food and food type antioxidant (mmol)
References

Mushrooms
(white)

3.9

374.4

Tomato

2.4

436.8

Potato

1.4

298.2

Broccoli

3.9

577.2

Cabbage

3.5

311.5

Lettuce

4.94

439.66

Peppers

20.98

1552.52

Carrots

1.06

64.66

Cauliflower

3.5

374.5

Carlsen et al. (2010)

Cucumber

0.71

36.92

Pellegrini et al. (2003)

Courgettes

3.33

273.06

Wachtel-Galor et al.
(2008)

Vegetables (raw)

Kidney beans (can) 2.7

496.8

Peas (can)

1.2

220.8

Baked beans (can) 2.4

441.6

Pellegrini et al. (2003)

Halvorsen et al. (2006)

Snack foods

Chocolate (dark)

42

1848

Chocolate (milk)

30.8

1355.2

Walnut

230

26,910

Peanut

19.7

2304.9

Pecans

27.4

3205.8

Almond

4.1

479.7

Hazelnut

9.4

1099.8

Macadamia nuts

5.9

690.3

Sesame seeds

0.6

17.4

Pistachio

14.3

1673.1

Blomhoff et al. (2006)

Continued

20

Iris F.F. Benzie and Siu-Wai Choi

Table 1.4 Representative FRAP values of various foodscont'd


Total
Per average serving
Food and food type antioxidant (mmol)
References

Ice cream
(chocolate)

430

Ice cream (vanilla) 0.5

43

Yoghurt (plain)

0.4

68

Popcorn

4.6

138

Crisps

7.8

234

Pretzels

11

660

Butter

7.3

109.5

Margarine

13.8

207

Olive oil

153

2065.5

Sesame oil

803

10,840.5

Peanut oil

133

1795.5

Corn oil

100

1350

Sunflower oil

108

1458

Canola oil

400

5400

Cheddar cheese

0.62

17.36

Parmesan cheese

1.02

28.56

Mozarella cheese

0.64

17.92

Ginger

215.7

431.4

Halvorsen et al. (2002)

Garlic

8.0

16

Carlsen et al. (2010)

Cinnamon

176.5

353

Halvorsen et al. (2002)

Turmeric

156.8

313.6

Mustard seeds

105.3

210.6

Curry powder

99.8

199.6

Pepper (black)

44.5

89

Oils

Carlsen et al. (2010)

Cheung et al. (2007)

Halvorsen et al. (2006)

Herbs and spices

21

Antioxidants in Food

Table 1.4 Representative FRAP values of various foodscont'd


Total
Per average serving
Food and food type antioxidant (mmol)
References
Breakfast items

All Bran

15.6

468

Bran flakes

42.9

1287

Corn flakes

12.3

369

Shredded wheat

2.3

69

Rice Krispies

8.6

258

Egg muffin

0.9

27

Oat puff

21.1

633

Special K

15.6

468

Weetabix

13

390

3.2

NA

Halvorsen et al. (2006)

Carlsen et al. (2010)

Miscellaneous

Oat flour

Muffin (blueberry) 4.6

519.8

Cookie (chocolate) 17.2

430

Doughnut (plain)

1.5

169.5

Egg (whole)

0.4

45.2

Cheeseburger

1.1

125.4

Chicken nugget

2.0

2.0

Fish burger

1.3

182

French fries

3.3

396

Hamburger

1.4

1.4

Milkshake (vanilla) 1.2

360

French bread

1.7

39.1

Wheat bread

3.4

78.2

White bread

1.6

36.8

White rice
(cooked)

0.3

55.8

Halvorsen et al. (2006)

Continued

22

Iris F.F. Benzie and Siu-Wai Choi

Table 1.4 Representative FRAP values of various foodscont'd


Total
Per average serving
Food and food type antioxidant (mmol)
References

Spaghetti (cooked) 0.2

28

Oatmeal (cooked) 0.8

120

Honey

1.4

19.6

French salad
dressing

4.4

61.6

Italian salad
dressing

0.8

11.2

Ranch salad
dressing

3.7

51.8

Thousand Island
dressing

0.7

9.8

Tomato ketchup

4.1

57.4

Units given for beverages are mmol/l and mmol/g fresh wet weight for other foods.

Chung et al., 2001; Dragsted et al., 2004; Duthie et al., 2006; Lotito & Frei,
2004). In one case, this is due to the degradation of purines in red meat. In
the case of fructose, the increase in uric acid is due to increased degradation
of hepatic AMP. In contrast, a postingestion increase in ascorbic acid can
lead to increased renal excretion of uric acid and thereby lower its plasma
concentration. Therefore, purine- or fructose-driven postingestion
increases in uric acid can be misinterpreted as signaling absorption of antioxidants from food, and ascorbic acid absorption can be masked by its effects
on uric acid (and thereby on total antioxidant content). In such cases, it
would be useful to be able to remove the contribution of uric acid from the
total antioxidant content of plasma. This can be done very simply with the
FRAP value. The stoichiometric factor of uric acid in the FRAP assay is 2.
Provided the plasma uric acid concentration is known (and this can be measured using commercially available kit methods), the plasma FRAP value can
be corrected for the contribution of uric acid by subtracting twice the uric
acid concentration (in mmol/l) from the FRAP value (in the same units)
(Benzie & Strain, 1996a, 1996b, 1997; Benzie et al., 1999). By taking this
approach, nutritional science studies of total antioxidant bioavailability of
foods can be performed without being confounded by postingestion changes
in plasma uric acid concentration (Fig. 1.3). For bioavailability and biokinetics

23

Antioxidants in Food

Table 1.5 Effect of different cooking methods, growing season, storage conditions and
genotype on total antioxidant content (as the FRAP value) of plant foods
Total antioxidant
Food
content
References
(a) Effect of cooking method

Cauliflower

Wachtel-Galor et al.
(2008)

Raw

2.8

Microwaved

4.9

Boiled

6.8

Steamed

8.7

Cabbage
Raw

3.5

Microwaved

1.5

Boiled

2.5

Steamed

3.9

Broccoli
Raw

3.9

Microwaved

5.4

Boiled

7.2

Steamed

13.1

Artichoke

Ferracane et al.
(2008)

Raw

56.9

Boiled

524.2

Steamed

705.0

Fried

430.4

Carrot

Miglio et al. (2008)

Raw

6.8

Boiled

14.5

Steamed

12.3

Fried

32.5
Continued

24

Iris F.F. Benzie and Siu-Wai Choi

Table 1.5 Effect of different cooking methods, growing season, storage conditions and
genotype on total antioxidant content (as the FRAP value) of plant foodscont'd
Total antioxidant
Food
content
References

Courgette
Raw

27.9

Boiled

63.2

Steamed

59.2

Fried

79.7

Garlic

Gorinstein et al.
(2008)

Raw

12.0

Blanched

10.5

Boiled

7.4

Fried

10.9

Onion (white)
Raw

23.2

Blanched

22.1

Boiled

16.5

Fried

22.9
Adlia Lemos et al.
(2013)

Potato
Fresh

670

Baked

510

Boiled

790

Microwaved

610

Steamed

670

(b) Effect of storage

Guava

Storage at 5  C

Fresh

16.2

Day 3

15.2

Patthamakanokporn
et al. (2008)

25

Antioxidants in Food

Table 1.5 Effect of different cooking methods, growing season, storage conditions and
genotype on total antioxidant content (as the FRAP value) of plant foodscont'd
Total antioxidant
Food
content
References

Day 6

18

Day 10

22
Storage at 4  C

Storage at
25  C

Fresh

3.2

3.2

Day 2

2.75

2.75

Day 4

2.9

2.8

Day 7

2.95

3.25

Day 9

2.15

2.25

Day 11

3.0

2.9

Day 14

2.9

3.35

Day 17

2.5

3.25

Day 22

2.15

2.2

Day 30

3.75

Currants

Storage at 4  C

Storage at
25  C

Fresh

2.5

2.5

Day 2

2.25

1.25

Day 4

2.5

2.25

Day 7

1.4

Day 9

1.5

Day 11

2.75

Storage at 4  C

Storage at
25  C

Fresh

1.8

1.8

Day 2

2.35

2.75

Day 4

1.8

3.25

Strawberry

Raspberry

Piljac-Zegarac and
Samec (2011)

Piljac-Zegarac and
Samec (2011)

Piljac-Zegarac and
Samec (2011)

Continued

26

Iris F.F. Benzie and Siu-Wai Choi

Table 1.5 Effect of different cooking methods, growing season, storage conditions and
genotype on total antioxidant content (as the FRAP value) of plant foodscont'd
Total antioxidant
Food
content
References

Storage at 4  C

Storage at
25  C

Fresh

1.75

1.75

Day 2

1.5

1.45

Day 4

1.4

1.05

Day 7

1.3

1.1

Day 9

1.5

1.5

Day 11

1.2

1.3

Day 14

2.1

1.8

Day 17

1.3

1.7

Day 22

1.6

Day 30

1.5

Sour cherry

egarac and
Piljac-Z
Samec (2011)

egarac and
Piljac-Z
Samec (2011)

Storage at 4 C

Storage at
25  C

Fresh

1.1

1.1

Day 2

0.75

0.8

Day 4

0.75

0.75

Day 7

0.8

Day 9

0.85

Day 11

0.6

Day 14

1.4

Day 17

1.2

Storage at 4  C
(with cling film)

Giacalone and
Storage at
25  C (covered Chiabrando (2013)
in film
impermeable to
oxygen)

Fresh

7.8

7.8

Day 5

10.8

9.6

Cherry

Cherry

27

Antioxidants in Food

Table 1.5 Effect of different cooking methods, growing season, storage conditions and
genotype on total antioxidant content (as the FRAP value) of plant foodscont'd
Total antioxidant
Food
content
References

Day 10

7.8

Day 15

8.5

Mandarin

9.0
6.2


Storage at 10 C
(packed in boxes)

Fresh

6.14

Day 2

5.74

Day 4

5.80

Day 7

5.91

Day 9

5.80

Shen et al. (2013)

Storage at 1  C
and 15 kPa

Storage at 1  C Chen et al. (2013)


and 55 kPa

Fresh

1.2

2.4

Day 2

1.5

3.52

Day 4

1.5

2.88

Day 7

1.14

Day 9

1.5

3.68

Day 11

1.9

4.32

Bayberry

(c) Effect of different cultivars

Tomato species

Lenucci et al. (2006)

Cherubino

0.0035

Cherelino

0.003

Coralino

0.0034

Corbus

0.0045

LS203

0.0024

Lycorino

0.00235

Minired

0.0030

Noami

0.0024

Piccadilly

0.0023
Continued

28

Iris F.F. Benzie and Siu-Wai Choi

Table 1.5 Effect of different cooking methods, growing season, storage conditions and
genotype on total antioxidant content (as the FRAP value) of plant foodscont'd
Total antioxidant
Food
content
References

Rubino top

0.0025

Sakura

0.0026

Salentino

0.0022

Sharon

0.0021

Shiren

0.0032

HLY02

0.0026

HLY13

0.003

HLY18

0.004

Kalvert

0.0029

Durian species

Toledo et al. (2008)

Mon Thong

5.22

Chani

4.642

Kan Yao

4.094

Pung Mance

4.498

Kradum

3.948

Raspberry
genotypes

Tosun et al. (2009)

ERZ1

235

ERZ2

176

ERZ4

180

ERZ6

214

ERZ7

210

ERZ10

163

Apple species

Wojdyo et al. (2008)

Alwa

1.0

Bramleys
Seeding

3.6

Cortland

1.12

29

Antioxidants in Food

Table 1.5 Effect of different cooking methods, growing season, storage conditions and
genotype on total antioxidant content (as the FRAP value) of plant foodscont'd
Total antioxidant
Food
content
References

Delbarestivale 1.56
Discovery

3.68

Elise

0.736

Fameuse

1.24

Fialka

5.04

Freedom

1.304

Geneva Early 3.06


Getmanskoje 2.268
Jonafree

1.196

Julyred

2.648

Kosztela

5.12

Meris

3.88

Odra

1.292

Piekna z
Herrnhut

4.804

Rajka

1.292

Rubin

0.644

Sunrise

2.46

Teremok

1.872

Zimnieje
Limonnoje

1.38

(d) Effect of harvest time

Strawberry
(harvest month)

Pineli et al. (2012)

May

22

July

39

September

30
Continued

30

Iris F.F. Benzie and Siu-Wai Choi

Table 1.5 Effect of different cooking methods, growing season, storage conditions and
genotype on total antioxidant content (as the FRAP value) of plant foodscont'd
Total antioxidant
Food
content
References

Noni (harvest
month)

Unripe

Medium
ripe

Sub- Mature Iloki Assanga et al.


mature
(2013)

February
March

1600

1750

1450

1550

MayJune

750

2500

2300

2050

November

550

560

3450

3750

Herbs (harvest
time)

Summer Marjoram Thyme


savory

July

105

100

112

August

79

104

82

September

66

89

86

Fresh

Stored at
4  C for
1 day

Stored at 4  C
for 1 week

March

3.5

1.5

1.0

May

5.0

1.1

0.9

July

3.0

1.1

1.1

September

3.1

2.0

2.0

Fresh

Stored at
4  C for
1 day

Stored at 4  C
for 1 week

January

10.6

9.6

8.0

March

7.2

7.1

6.9

May

12.0

11.0

9.8

July

13.0

12.0

11.0

September

12.8

12.8

12.4

November

6.0

6.0

5.9

Curly Endive
(harvest time)

Rocket salad
(harvest time)

Vabkova and
Neugebauerova
(2012)

Venneria et al.
(2012)

Venneria et al.
(2012)

31

Antioxidants in Food

Table 1.5 Effect of different cooking methods, growing season, storage conditions and
genotype on total antioxidant content (as the FRAP value) of plant foodscont'd
Total antioxidant
Food
content
References

Fresh

Stored at
4  C for
1 day

Stored at 4  C
for 1 week

March

2.1

1.7

1.7

May

4.5

1.2

1.4

July

2.6

1.3

1.3

September

2.6

1.5

1.0

November

1.7

1.7

1.6

Endive (harvest
time)

Venneria et al.
(2012)

Units are mmol/g fresh wet weight, with exception of herbs, which are mmol/g dry weight.

1250

Total antioxidants (M)

1000

750
Uric acid
Ascorbic acid
Other antioxidants

500

250

0
Fasting total
FRAP

After meal total


FRAP

Fasting
non-UA FRAP

After meal
non-UA FRAP

Figure 1.3 Concept of nonuric acid FRAP value.

testing, it is desirable to monitor changes in the plasma and urine concentrations of specific antioxidants, such as epigallocatechin gallate, ascorbic acid or
zeaxanthin, or their metabolites. However, this is not always possible, and
exploring postabsorption changes in the FRAP or the nonuric acid FRAP
value offers a useful initial screening approach if specific antioxidants in the
food are not known or for some reason cannot be measured.

32

Iris F.F. Benzie and Siu-Wai Choi

3.5. The modified FRAP assay (FRASC) for total antioxidant


and ascorbic acid measurement
Many foods, especially fruits and fruit juices, are rich in ascorbic acid, and in
food science, it is useful to measure this in order to advise consumers on
which foods and how much of each is needed to reach the recommended
daily intake of 70100 mg (Chung et al., 2001; Levine et al., 1999, 2011).
Strawberries and kiwi fruits (also known as Chinese gooseberry) have high
ascorbic acid content, and the daily requirement can be met by 45 strawberries or 12 kiwi fruits. However, some fruits, including apples and pears,
have low ascorbic acid content. Therefore, the daily intake of ascorbic acid
can vary greatly depending on the type of fruit eaten, and even people who
take their five-a-day could have lower than recommended intakes.
The FRASC assay represents a simple, one-step modification of the FRAP
assay that permits the measurement of ascorbic acid in the same sample and at
the same time as the FRAP assay (Benzie & Strain, 1996a; Benzie et al., 1999).
FRASC has been validated against a reference HPLC method for ascorbic acid
(Chung et al., 2001). In brief, the procedure is to prepare two matching aliquots of each test sample. To one aliquot is added a small volume of a 4 unit/l
solution of ascorbic oxidase (ao). The recommended volume is 40 ml enzyme
solution added to exactly 100 ml of sample. This very quickly destroys all
ascorbic acid present. To the other aliquot, a matching volume of water is
added. The paired samples are then run in the FRAP assay as described above,
but an additional absorbance reading is taken at 1 min for both members of the
pair. The 1-min absorbance of the aliquot treated with water represents the
action of all the redox-active antioxidant present, including ascorbic acid,
while the 1-min absorbance of the enzyme-treated aliquot represents the
action of all the redox-active antioxidant except for ascorbic acid. The difference between these two absorbances is due to the action of ascorbic acid alone
and is used to obtain the ascorbic acid concentration, which is obtained as
follows:
Ascorbic acid AA concentration of test sample mmol=l

Sample 1 min A593 nm ao  A593 nm ao


1 min A593 nm standard
 AA concentration of standard mmol=l

The 4-min value of the aliquot to which water (not enzyme) is used to
obtain the FRAP value. To compensate for the dilution effect of the

Antioxidants in Food

33

addition of water to the samples, it is important that the calibrator be


treated the same way, that is, 40 ml of water should be added to exactly
100 ml of calibrator. If this is not done, the test results will be overestimated by 30%. For calibration, ascorbic acid solutions can be used,
but the usual Fe2 calibrator used for the FRAP assay works well, provided its concentration is translated into AAE for calculation of ascorbic
acid concentration. Remembering that one ascorbic acid molecule can
reduce two Fe3 to two Fe2, it follows that the signal (absorbance) given
by a 1000 mmol/l Fe2 calibrator in the FRAP assay is equivalent to the
signal of reductive activity that would be given by a 500 mmol/l solution
of ascorbic acid.
In summary, the FRAP assay offers a speedy, inexpensive, and flexible
tool to measure the combined activity of redox-active antioxidants in a
food, or in plasma or urine following ingestion of a food. The results
can be corrected easily for the contribution of uric acid. A modified form
of the assay, FRASC, can be used to measure ascorbic acid concentration at
the same time as the FRAP value. The FRAP assay is very widely used
worldwide, and there now exists a rich database of FRAP values of thousands of foods, beverages, spices, and herbs, and these can be used to guide
dietary choices for enhanced antioxidant intake. In Section 4, the reasons
why this is thought to be beneficial and the supporting evidence are
reviewed briefly.

4. WHY IS THE ANTIOXIDANT CONTENT OF FOOD


OF INTEREST?
4.1. Basic concepts
The concept that enhanced optimal antioxidant defense of the body lowers
risk of disease and slows biological aging is an attractive one and implies
that increased intake of antioxidant-rich foods can promote healthy aging
and lower risk of chronic degenerative disease. The concept derives from
the observation that key biomolecules undergo deleterious changes in
form and function owing to oxidation by ROS, and that antioxidants
oppose such changes, and thereby decrease the oxidative stress that
is believed to be core to the aging process and to the etiology of chronic
degenerative diseases, including cardiovascular disease, cancer, and
dementia (Ames, 2006; Benzie, 2005; Frei, 2004; Gey, 1998; Kaliora
et al., 2006; World Cancer Research Fund/American Institute for

34

Iris F.F. Benzie and Siu-Wai Choi

Cancer Research, 2007). This is a simplistic view, but optimal or enhanced


antioxidant defense achieved by increased intake of antioxidant-rich foods
may confer benefit in relation to lowering risk of chronic age-related
disease (Ames, 2006; Benzie, 2005; Frei, 2004; Gey, 1998; Kaliora &
Dedoussis, 2007; World Cancer Research Fund/American Institute for
Cancer Research, 2007).
Many different types of antioxidant supplements are commercially available, but supplementation trials with purified antioxidants, such as ascorbic
acid, a-tocopherol, or b-carotene, have not demonstrated beneficial effects,
and current recommendations for health promotion are for increased intake
of foods that are rich in antioxidants, not for supplements (Lampe, 1999;
Packer & Cadenas, 2007). The specific agents and mechanisms in foods that
are responsible for health benefits are not yet clear, but there is strong and
convincing evidence that including more plant-based, antioxidant-rich
foods, herbs, and beverages in the diet is effective in promoting health
and lowering risk of various age-related diseases (Benzie & WachtelGalor, 2009; Duthie et al., 2006; Ma et al., 2008; Packer & Cadenas,
2007; Tam et al., 2005; Thomas et al., 2006; Wachtel-Galor, Tomlinson,
Benzie, 2004).

4.2. Antioxidants and health: The evidence and potential


impact
The global population is aging, and the burden of chronic disease is increasing, bringing socioeconomic problems across the world. Therefore, there is
intense research interest into cost-effective strategies for health promotion.
Clearly, one attractive strategy is to increase the antioxidant status of the
body. Endogenous antioxidants such as SOD, glutathione and bilirubin cannot be easily or purposefully modulated, but higher intake of antioxidants in
the diet offers a potential route to higher defense and better protection
against oxidative stress. Information on the type, action, content, stability,
bioaccessibility, and bioavailability of antioxidants in food are important
and complex issues. The evaluation of foods and the design of new methods
of food production, processing, and storage and the production of functional and designer foods for health promotion through enhanced antioxidant content or effects are the focus of much research in the agri-food
sector. Therefore, the impact of research in measuring and modulating antioxidants in food has high potential impact on the commercial field as well as
on the area of preventive healthcare.

Antioxidants in Food

35

Vegetarians who consume a well-balanced diet (avoiding iron and


B vitamin deficiencies) have less chronic disease than omnivores and lead
longer, healthier lives (Benzie & Wachtel-Galor, 2009). Intake of vitamin
C from food and plasma vitamin C concentrations are inversely correlated
with risk of all cause mortality, incident heart disease, stroke, type 2 diabetes,
and dementia (Cooper et al., 2012; Harding et al., 2008; Jeurnink et al.,
2012; Leenders et al., 2013). Also, those whose habitual diet is rich in
plant-based, high antioxidant content foods have better glycaemic control,
lower inflammation, and less oxidative stress (Kashyap et al., 2005; Ma et al.,
2008; Thomas et al., 2006). Still, while high intake of antioxidant-rich foods
is associated with better health, the underlying mechanisms are not yet clear,
and a role for the nonessential dietary-derived antioxidants remains to be
established. Polyphenols are of special interest because it is known that some,
in particular, the catechins from green tea, enter the plasma fairly rapidly
after ingestion, although in small amounts (Del Rio et al., 2010; Fung
et al., 2013). Furthermore, supplementation with green tea is associated with
less oxidation-induced DNA damage and enhanced repair of these potentially mutagenic lesions (Han et al., 2011; Ho et al., 2013). This supports
the large body of observational data showing that those who include green
tea regularly in their diet have less cancer and other diseases associated with
oxidative stress (Benzie et al., 1999; Han et al., 2011; Ho et al., 2013; Molan
et al., 2008).
Overall, the observational and experimental data are sufficiently strong
and consistent to be able to recommend high intake of antioxidant-rich
foods as a public health measure to promote healthy aging, even though
the mechanisms of action remain unclear. The molecular action of antioxidants is an area of intense research interest but is very challenging to study.
Cell culture studies allow probing of gene activation and protein expression
in response to exposure to antioxidants, but results, especially when performed using immortalized cancer cells and very high doses of purified
antioxidants, have very limited relevance to the normal physiological setting
within the human body. Animal studies are useful as they offer a biosystem
approach, but most animals make their own vitamin C and are likely to have
quite different absorption and metabolism patterns for food-derived antioxidants compared to humans, and to have very different colonic microbiota.
Human studies are much more difficult because samples that can be collected
from healthy human subjects are very limited in type and volume. For studying effects at the biochemical level, blood plasma is most often used. For
studying effects in relation to signaling pathways, gene expression, and

36

Iris F.F. Benzie and Siu-Wai Choi

protein production, the most commonly used type of nucleated cell is the
peripheral lymphocyte. It is not known how representative these samples
are of other fluids and cells, but recent human studies throw some light
on how dietary-derived antioxidants work at the molecular level, and biochemical data are providing insight into the metabolic fate of dietary antioxidants. These studies may lead to the answer as to why, given that we
have an endogenous antioxidant defense system, we still require regular
input of dietary-derived, plant-based antioxidants for health maintenance.
In Section 5, some recent human studies of metabolism and action of antioxidants in food are reviewed briefly, and current thinking in regard to the
possible molecular action of redox-active antioxidants or their metabolites is
discussed.

5. ANTIOXIDANTS IN FOOD: ENIGMAS AND


EVOLUTIONARY ASPECTS
The human body expends valuable resources in producing a range of
antioxidants, and these are effective against the various ROS to which our
vital biological components are continually and unavoidably exposed. Still,
we have an absolute need for at least two antioxidants (vitamins C and E) that
the human body cannot synthesize and that we must obtain from food in
regular and adequate amounts. This reliance on dietary input is an enigma,
especially for vitamin C. With very few exceptions, all animals can manufacture vitamin C. One of these exceptions is homo sapiens. Our ancestors
could synthesize this antioxidant, and human cells still contain the gene
for the enzyme (L-gulono-g-lactone oxidase) that transforms glucose into
ascorbic acid, but it is highly mutated and inactive (Lachapelle & Drouin,
2011). Therefore, during human evolutionary development, our ancestors
lost the ability to manufacture a substance that was and remains essential for
health. Evolutionary theory tells us that the loss of this synthetic ability caused no physiological disadvantage; otherwise, it would not have become
prevalent. Indeed, because this has become a ubiquitous characteristic of
all humans, the initial silencing mutation of the gene must have conferred
some biological advantage. This is likely to have been a metabolic saving
of resources needed to manufacture something that was needed but supplied
in other ways, in this case in the diet. The Paleolithic diet of our huntergatherer ancestors is estimated to have provided a rich supply of ascorbic
acid, along with other antioxidants (Benzie, 2003; Cordain et al., 2002).

37

Antioxidants in Food

Therefore, it is likely that the mutation led to a metabolic saving without a


biological cost or downside.
A comparison of the estimated antioxidant content of the Paleolithic diet
and the modern-day diet is shown in Fig. 1.4. It can be seen that the intake of
plant-based antioxidants that our biological systems were habituated to during our evolutionary development was high. This level of intake is likely to
still be what our biological systems have developed to deal with and perhaps
require for optimal antioxidant defense and biological function. As noted,
the evidence is clear and strong that habitual diets that are rich in plant-based
foods are beneficial for health, and it is suggested that aligning our modernday antioxidant intake more closely to that of the hunter-gatherer may be an
effective strategy for health, even if the optimal doses, key bioactives, and
molecular action of antioxidants in plant-based foods remain unclear. Still,
it does not necessarily follow that health benefits of antioxidants in the diet
continue to accrue with higher and higher doses. Indeed, there is evidence
that very high doses of vitamin E, vitamin C, and polyphenols in pure form

Paleolithic diet

Micronutrient

Modern diet

604

Vitamin C (mg)

59115

33

Vitamin E (mg)

5.26.0

357

Folate (mg)

208317

5560

Carotenoids (mg)

18462048

87

Iron (mg)

9.517.2

Copper (mg)

11.3

43

Zinc (mg)

7.113.6

Paleolithic diet

Modern day diet

Figure 1.4 A closer look at the micronutrient intakes in the Paleolithic and modern-day
diet reveals that hunter gatherers consumed up to fivefold more vitamin C and vitamin E,
and at least double the amount of carotenoids. Data from Benzie (2003); figure courtesy of
SL Choi.

38

Iris F.F. Benzie and Siu-Wai Choi

do not lower risk of disease and may in fact be harmful. For example, very
high doses of epigallocatechin gallate have been found to damage liver and
kidney, and high-dose vitamin E and carotene were found to increase risk of
lung cancer (Byers, 2008; Lambert et al., 2010; Russell, 2002).
It is worth noting that the human body treats the polyphenol antioxidants from plant-based foods as xenobiotics. Their absorption is very low,
and there is rapid conjugation and excretion of those that are absorbed. Consequently, the plasma concentration of all polyphenols (including the catechins, quercetin, and the anthocyanins) is only 1 mmol/l, even with a diet
that provides up to 1 g per day of polyphenols from fruits and vegetables
(Manach et al., 2004; Packer & Cadenas, 2007). This compares to plasma
concentrations of 80 and 40 mmol/l, respectively, for ascorbic acid
and a-tocopherol, the daily inputs of which are 200 and 10 mg in
healthy diets. The severely restricted access of polyphenols is intriguing
and paradoxical, given the health benefits of polyphenol-rich, plant-based
diets. Recent evidence in two main areas gives clues as to solve the enigma
and explain why this may be central to the mechanisms by which diets rich in
antioxidant phytochemicals exert their beneficial effects. One area of recent
research is in redox chemistry and molecular effects of antioxidants, and the
other in relation to the symbiotic relationship between the human body and
its colonic microbiota.

6. MECHANISMS OF ACTION: REDOX ISSUES,


PHYTOHORMESIS, AND COLONIC MICROBIOTA
The mechanisms by which antioxidants in food drive health benefits
are not clearly established. Some effects are likely to be due to antioxidant
action, but the concepts that all effects are due to direct antioxidant effects
and that more is better are overly simplistic. The very limited bioavailability of most phytochemicals is unlikely to be an accidental physiological
occurrence. If more efficient absorption of phytochemicals from food was
of physiological benefit and led to improved reproductive success, then
mutations that enhanced absorption would have been retained and become
prevalent. The fact they have not implies that either these phytochemicals
are not needed or that they are needed in low levels only. Indeed, our biological restrictions that effectively limit bioavailability suggest that high levels
are to be avoided. This is paradoxicalbut by considering redox issues,
some sense of the biological value of restricted bioavailability of phytochemicals and the molecular action of these is emerging. In addition, recent

Antioxidants in Food

39

evidence points to the colonic microflora as playing a potentially important


symbiotic role in phytochemical metabolism.

6.1. Redox tone changes as a driving force for cytoprotection


and the biological sense of restricted bioavailability of
dietary antioxidants: The redox-sensitive KEAP-1Nrf2
signaling pathway
Protein-based antioxidants, such as enzymes, in food are likely to be destroyed during cooking and the digestive process. The antioxidants most relevant to nutritional science are redox-active substances that can destroy or
neutralize ROS by quenching or electron donation. These include vitamins
C and E, and the polyphenol family that contains catechins, quercetin, and
the deeply colored anthocyanins. Chocolate, teas, wines, berries and other
fruits, and leafy green vegetables are good sources of these redox-active antioxidants (Benzie & Wachtel-Galor, 2010; Halvorsen et al., 2006; Serafini
et al., 2003). During their reductive action, these antioxidants become
reduced, and all redox-active antioxidants have the potential to act as
prooxidants. Ascorbic acid and epigallocatechin gallate, for example, can
generate the ROS hydrogen peroxide in vitro, and it has been shown that
this ROS generation is dose dependent (Ho et al., 2013). At high concentration, this prooxidant activity leads to in vitro cytotoxic effects of a dietary
antioxidant, such as vitamin C, or an antioxidant-rich food or beverage, such
as tea (Chai et al., 2003). Whether this prooxidant effect occurs in vivo is a
topic of intense research interest. It is noted that there is no evidence that
antioxidant-rich foods (as opposed to purified supplements) induce oxidative stress or have other deleterious effects on human health. Furthermore,
if confirmed to occur in vivo, prooxidant effects would be limited by the very
limited bioavailability of most dietary-derived antioxidants and their rapid
metabolism and excretion. However, frequent but subtle prooxidant
changes in intracellular redox tone, or balance, could occur following dietary input of phytochemicals, and there is growing evidence that these small
prooxidant waves underlie the health protective effects of regular intake of
antioxidant-rich foods (Benzie & Wachtel-Galor, 2010; Han et al., 2011).
This does not preclude direct antioxidant effects by some food components
in some situations, but the prooxidant activity could lead to an array of
endogenous redox-sensitive cytoprotective adaptations that have widespread beneficial effects for health (Benzie & Wachtel-Galor, 2010; Lee
et al., 2013; Surh et al., 2008). This concept of prooxidant phytochemicals
triggering redox-sensitive cytoprotective adaptations has been termed

40

Iris F.F. Benzie and Siu-Wai Choi

redox hormesis and phytohormesis (Davinelli et al., 2012). There is a


growing body of evidence from cell culture and animal studies that many
phytochemicals, including allium compounds, sulforaphane, curcumin,
epigallocatechin gallate, lycopene, quercetin, and resveratrol, induce
changes in cellular signaling pathways that trigger these protective adaptations (Lee et al., 2013). The phytohormetic effect is believed to be mediated
mainly via the redox-sensitive KEAP-1Nrf2 signaling pathway, but there is
emerging evidence that there are phytochemical-induced effects on kinases
and DNA methylation also (Benzie & Wachtel-Galor, 2010; Lee et al.,
2013; Surh et al., 2008).
Nuclear factor erythroid-2-related factor-2 (Nrf2) is a transcription
factor that in resting cells is mainly bound in an inactive form to
cysteine-rich Kelch-like ECH-associated protein-1 (Keap-1) (Lee et al.,
2013; Surh et al., 2008). Keap-1 promotes ubiquitination and proteolysis
of bound Nrf2. Release of Nrf2 from its suppressor protein allows Nrf2
to translocate to the nucleus. The mechanism by which Nrf2 is released
from Keap-1 is currently unclear, but there is evidence that oxidation or
chemical modification of key cysteine residues in Keap-1 leads to stabilization of Nrf2, proteolysis of Keap-1, and release of stabilized Nrf2 into
the cytoplasm (Lee et al., 2013). However, phosphorylation of serine or
threonine residues in Nrf2 is suggested to occur, inducing release of Nrf2
from its suppressor. It is also suggested that phosphorylation of tyrosine141
residue in Keap-1 enhances its stability and Nrf2 binding, while dephosphorylation promotes degradation of Keap-1, with stabilization and release
of Nrf2 (Lee et al., 2013). Therefore, oxidative action, direct chemical
modification, and effects on kinases and phosphorylases may all be involved
in phytochemical action (Lee et al., 2013).
After release, stabilized Nrf2 translocates to the nucleus and combines
with Maf protein and other transcription factors, and binds to the regulatory
cis-element in the 50 -flanking promoter regions of genes that encode for various cytoprotective and stress opposing factors. The regulatory regions of
such genes are referred to as antioxidant response elements (ARE) or electrophile response elements. The products of the Nrf2/ARE-activated genes
include antioxidant and detoxifying enzymes, hOGG1, the enzyme that catalyzes the first step in repair of oxidation-induced lesions to DNA, and heme
oxygenase-1, which has antioxidant and antiinflammatory effects (Bach,
2005; Soares & Bach, 2008). This phytohormetic redox-associated concept
is outlined in Fig. 1.5. Overall, the regular, subtle push of mild prooxidant effects of antioxidant phytochemicals could induce powerful

41

Antioxidants in Food

Basal state (Nrf2 ubiquitinated and degraded)

ROS
Ub

Rb
x

Keap1

Ub

Ub Ub

Endogenous or
exogenous
sources

Ub

Nrf2

E2

CuI3

Dietary-derived phytochemicals may


act as mild prooxidants in vivo

(prooxidant changes in redox-tone)


P13k
HS - CyS

Cytoplasm

Induced
Nrf2

CyS - SH

JNK
Keap1
Causes Nrf2 phosphorylation of
serine/threonine on Nrf2

Nrf2

Keap1

Release of Nrf2

CyS - CyS
Keap1

Keap1
Kinases modulate nuclear
import and export

Nrf2

Postinduction

Nrf2

Enters nucleus
Nrf2
ction

leu
Nuc

Postindu

Nrf2

Maf

ARE

RTGACnnnGC

Keap1

Enters nucleus
Transcription of

HMOX-1,
hOGG1,
GST,
NQO1,
XRCC1, etc.

Figure 1.5 Nrf2 activation via ROS from endogenous sources, or dietary phytochemicals. Under basal conditions, Keap1 anchors Nrf2 within the cytoplasm in the cul
3Rbx1E2 complex where it is targeted for ubiquitination and degradation to maintain
low intracellular levels of Nrf2. When cells are exposed to changes in redox tone to a
prooxidant state, either through endogenous oxidants, or dietary-derived prooxidants,
cysteine residues on Keap1 become oxidized, and Nrf2 is released from the Keap1cul
3Rbx1E2 complex. Nrf2 is now free to translocate into the nucleus, where, along with
other transcription factors, (Maf ) bind to the ARE, leading to transcription of cytoprotective genes. Once redox homeostasis is reached, Keap1 travels into the nucleus
and dissociates Nrf2 from the ARE, and the Keap1Nrf2 complex is exported out of
the nucleus where it binds with cul 3Rbx1E2 and Nrf2 is degraded. An alternative
mechanism of ARE induction is that Nrf2 inducers phosphorylate Nrf2, allowing its
release from the Keap1cul 3Rbx1E2 complex and subsequent translocation into
the nucleus.

prosurvival signals that bring substantial health benefits in the long term,
such as the lower risk of various age-related disease and better quality of life
seen with plant-rich diets, such as vegetarian and Mediterranean diets
(Benzie & Wachtel-Galor, 2009, 2010; Bonaccio et al., 2013; World
Cancer Research Fund/American Institute for Cancer Research, 2007).
Evidence to date for this concept is largely from cell culture and animal
studies. Data from human nutritional studies are needed before it can be
confirmed. However, if confirmed there are important implications for diet
and health. This phytohormesis concept does not rule out some direct

42

Iris F.F. Benzie and Siu-Wai Choi

antioxidant effects of dietary antioxidants but ascribes far more widespread


health-related effects to prooxidant changes that are insufficient to cause
damage, but that induce a type of oxidative polishing or conditioning
of the cell that enhances its survival and defense. However, it is noted that
the effects, if confirmed, are driven by small amounts of phytochemicals, and
this brings into question the advisability of overcoming natural barriers that
limit absorption of phytochemicals. Advances in nanodelivery systems have
been used to enhance absorption of phytochemicals and nutraceuticals, and
microencapsulation technology can prevent destruction of antioxidants
within the gastrointestinal tract, increasing bioaccessibility of antioxidants
(Huang & Chang, 2009; Neves et al., 2013; Souto et al., 2013). These technologies apply mainly to production of supplements, but caution is needed
in their development and use. Overcoming the physiological barriers that
limit bioavailability of phytochemicals in the human body may not be helpful for health, and indeed may do more harm than the good no doubt
intended. Increasing absorption of redox-active phytochemicals could lead
to large changes in redox tone that the cells cannot adapt to and overcome,
with damage and cytotoxic effects ensuing rather than cytoprotection.
A further point worth considering is the fate of phytochemicals left
unabsorbed in the gut. Recent findings indicated that these may play an
important role in the health effects of antioxidant-rich diets. This aspect
of antioxidants in food is discussed briefly in the next section.

6.2. Colonic microflora and metabolism of food antioxidants:


Moving into new territory of the microbiome
The absorption of antioxidant phytochemicals from the human gastrointestinal tract is severely restricted. Even in the case of ascorbic acid, which is
the most soluble and best absorbed antioxidant, absorption is limited. If
the dose is low, absorption can approach 100%, but as the dose increases
the relative amount that is absorbed decreases (Davey et al., 2000). Consequently, the increase in plasma ascorbic acid following ingestion of several
grams of vitamin C is not significantly larger than that seen after taking a few
hundred milligrams (Benzie & Strain, 1997).
Many foods and beverages, especially of plant origin, have a high and
varied content of antioxidants, and most of the ingested antioxidants in every
meal or drink are left behind and arrive in large amounts in the large intestine. Previously, if considered at all, these were believed to serve no purpose,
representing only nonabsorbable and unwanted remnants of food. It was
then suggested that these unabsorbed phytochemicals could benefit colonic

Antioxidants in Food

43

health, perhaps by acting as antioxidants in situ or by acting as probiotics,


enhancing conditions for the more desirable types of colonic microflora.
However, the microbial inhabitants of the colon (the microbiota) are
increasingly recognized as our partners in metabolizing unabsorbed phytochemicals into absorbable and possibly bioactive molecules (Del Rio et al.,
2010; Leone et al., 2013). The bulk of the unabsorbed phytochemicals are
polyphenolic compounds, which includes anthocyanins, flavonols, and
flavanols. In the past few years, many colonic metabolites of flavan-3-ols
(catechins) from tea have been identified in human plasma (Del Rio
et al., 2010). Ring scission products of catechins appear in plasma at seven
or more hours after ingestion of tea, and if they are included in the calculation of overall bioavailability of tea polyphenols, the figure is substantially
elevated in some individuals (Crozier et al., 2009; Del Rio et al., 2010). It is
not yet known if these colonic metabolites are bioactive. Furthermore,
interindividual variation is very high, and these ring scission products of
tea polyphenols are not found in all subjects. The variation in response is
suggested to be due to differences in the colonic microbiome. The microbiome is not a constant and can be changed with changes in diet, antibiotics,
prebiotics, and probiotics (Leone et al., 2013). Furthermore, the colonic
microbiome of the breast-fed baby is different from that of the formulafed baby, and the first imprinting of the neonatal microbiome seems to
be difficult to change and to have an impact on risk of diseases later in life
(Zivkovic et al., 2011). Overall, there is increasing awareness that we have a
symbiotic relationship with the microbial inhabitants of our lower intestine,
and modifying the microbiome could affect the metabolism, bioactivity, and
absorption of unabsorbed antioxidants from food. This forms a new and
challenging area of research in food, nutrition, and health science.

7. THE STORY SO FAR


In summary, some key points in relation to antioxidants in food and
the concepts and cautions discussed in this chapter are:
1. The health benefits of antioxidant-rich diets are clear, but they are
related to intake of whole foods and do not apply to supplementation
with pure antioxidants.
2. Plant-based diets contain thousands of different antioxidant phytochemicals, but whether there are only few bioactives of importance
or if there is a role for each or if there is important interaction between
them is not known.

44

Iris F.F. Benzie and Siu-Wai Choi

3. It is neither possible nor feasible to measure the content of each individual antioxidant in food, and measurement of the total antioxidant
content of foods has become a widely adopted approach.
4. There is now a large body of data on the total antioxidant content of
foods, and this can be used in dietary planning for increased antioxidant
intake.
5. All the currently available tests that measure total antioxidant content
of foods and biological fluids are in vitro tests, and phytochemicals may
act quite differently in an in vivo system.
6. Vitamin C (ascorbic acid) is known to be vital for human health, and
vitamin E (mainly a-tocopherol in the human body) plays a key role in
protecting lipid components and structures, but even so, the recommended daily intakes of these are quite low (100 and 10 mg,
respectively), their absorption is limited, and the human body does
not make or store either of these antioxidants.
7. The great majority of the daily intake of antioxidant phytochemicals
in healthy plant-rich diets is formed from the polyphenols and carotenoids families, but as yet there is no established role for any of the
thousands of members of these families within the human body.
8. It is believed that the benefits of dietary-derived antioxidants relate at
least in part to their antioxidant properties, but this is not necessarily the
case, as many are extensively metabolized during and after absorption,
and as noted they may not act as antioxidants in vivo, even if they do so
in in vitro test systems.
9. Despite forming the majority of the dietary load of antioxidant phytochemicals, most (indeed almost all) of the polyphenols and carotenoids ingested are not absorbed, and even the absorption of vitamins
C and E is limited.
10. The physiological and biochemical systems of the contemporary
human body have evolved through millions of years.
11. Our current dietary requirements are likely to be very similar to those
of our evolutionarily successful ancestral prototype, the huntergatherer hominids of Paleolithic times, whose diet was rich in plantbased foods, such as fruits, seeds, and nuts.
12. During our evolution, we lost the metabolic ability to synthesize vitamin C, but we retain a somewhat paradoxical metabolic need for it.
13. This paradox can be resolved as follows: the high dietary input of
ascorbic acid in the plant-based ancestral diet met physiological needs,

Antioxidants in Food

14.

15.
16.

17.
18.

19.

20.

45

making the endogenous source redundant, and the blocking of the synthesis of ascorbic acid represented a metabolic cost saving and thereby
conferred a biological benefit, leading to the silencing mutation
becoming prevalent and then ubiquitous within our species.
Despite very large amounts of antioxidants in the ancestral diet, there
was no evolution of efficient absorption systems for polyphenols and
carotenoids, which form the bulk of antioxidant phytochemicals in
the diet; nor has any clear physiological role been identified for these
phytochemicals.
It is a characteristic of the human body that access of most antioxidant
phytochemicals is severely restricted, and there is extensive metabolism
and rapid excretion of most of those that are absorbed.
A reasonable suggestion is that the human body has no need for high
amounts of such phytochemicals, and indeed that restricted access
and rapid metabolism and excretion may well safeguard against toxicity
of an overload of xenobiotics, especially given that antioxidant phytochemicals, including ascorbic acid, epigallocatechin, quercetin, and resveratrol, have prooxidant properties and generate hydrogen peroxide
in vitro.
Though yet to be confirmed in vivo, the prooxidant activity of antioxidant phytochemicals may be the key to their molecular mechanism
of action.
Small prooxidant changes in intracellular redox tone, induced by phytochemicals, may trigger the Keap-1/Nrf2ARE signaling pathway,
with subsequent activation of an array of cytoprotective adaptations,
such as increased antioxidant defense, improved DNA repair and
detoxification systems, and it is this phytohormesis that may be
responsible for most of the health benefits of antioxidant-rich foods.
Food technology tools and strategies that are designed to overcome the
evolved physiological barriers that restrict bioavailability and drive
rapid excretion of antioxidant phytochemicals must be regarded with
caution, as these could lead to large prooxidant swings in redox tone
that overwhelm adaptive responses in the cell and induce cytotoxicity,
mutation, or cell death.
Unabsorbed antioxidant phytochemicals can be metabolized by colonic
microbiota into absorbable and possibly valuable ring scission products;
however, their bioactivity and the effect of varying the microbiota on
the profile and action of colonic metabolites are not known.

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Iris F.F. Benzie and Siu-Wai Choi

8. CONCLUDING REMARKS AND RESEARCH NEEDS


Many foods, especially plant-based foods, are rich in redox-active
antioxidants, and high intake of such foods benefits health. Advances in
hyphenated technologies such as LCMS/MS have facilitated the detection
and measurement of individual antioxidants in foods and biological fluids,
but there are thousands of antioxidants in foods, and levels in human plasma
are often very low, making detailed measurement of individual dietaryderived antioxidants an unrealistic task. The concept of measuring the
total antioxidant content (or activity, capacity, or power) has become
widely accepted and applied in nutritional and food science research. There
are several methods available. Provided the method of measurement is
objective and reproducible, and its procedures and limitations are understood, this can offer a useful approach in biomonitoring and environmental
studies, as well as in the agri-food and nutrition industries for product evaluation, development, and quality control. Indeed, the total antioxidant
capacity of foods, herbs, and food supplements is now a common feature
of their marketing profile. The FRAP assay is a widely adopted, relatively
simple, quick, and inexpensive method, and a very large amount of data
on the FRAP values of a wide range of foods and the effect of processing,
seasonal growth, and cooking on this are available and can be used to guide
dietary choices for increased antioxidant intake. However, more research is
needed into the role of nonessential dietary-derived antioxidants, especially the polyphenols, and the molecular mechanisms by which food antioxidants promote healthy aging. One area of promise is in relation to
molecular effects of antioxidants on intracellular redox tone and the expression of cytoprotective redox-sensitive genes. Another is in the symbiotic
effect of colonic microbiota on metabolism and the overall bioavailability,
action, and effect of food-derived antioxidants, and the way in which differences in the microbiome can be used to influence the health benefits
of antioxidants in food.

ACKNOWLEDGMENTS
The authors are grateful to The Hong Kong Polytechnic University for financially supporting
our work.

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47

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