Chapter 1

Antioxidant Measurement and Applications:

An Overview
1 2
Fereidoon Shahidi and Chi-Tang Ho
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1
Department of Biochemistry, Memorial University of Newfoundland,
St. John's, Newfoundland A1Β 3X9, Canada
Publication Date: March 12, 2007 | doi: 10.1021/bk-2007-0956.ch001

2
Department of Food Science, Rutgers, The State University of New Jersey,
65 Dudley Road, New Brunswick, NJ 08901

Antioxidants are added to fats, oils and fatty foods to prevent

their oxidative deterioration. Antioxidants also protect the
integrity of cellular structures and macromolecules from
damage due to free radicals. Carotenoids and phenolic
compounds are major dietary antioxidants. Because of the
importance of antioxidant potential in foods and dietary
supplements, it is necessary to have good and reliable methods
for measuring antioxidant activity. Various in vitro and in vivo
methods for assessing antioxidant activities are discussed.

Introduction

Autoxidation occurs widely in fats, oils and lipid-containing foods, and

causes food quality deterioration with concomitant generation of loss of
nutrients, unpleasant flavors, and even potentially toxic substances. Among the
methods for preventing oxidation, addition of antioxidants is the most effective,
convenient and economical one (7).
Antioxidants are also important to human health. Antioxidant protection
from damage due to free radicals is vital for the integrity of cellular structures

2 © 2007 American Chemical Society

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and macromolecules (2,3). As we age, the system which utilizes antioxidants for
our defense and protection also declines, and can be aggravated by the presence
of various oxidative stresses caused by pollution, exercise, smoke exposure and
radiation. This defense system operates through a series complex networks
between vitamins C and E, carotenoids, zinc, copper, selenium, and magnesium-
dependent enzyme antioxidants as well as other phytonutrients, which together
perform highly involved recycling and regeneration reactions to optimize free
radical protection. Deficiencies in any of the mentioned necessary components
could potentially lead to a severely compromised defense system (4,5). Owing to
the incomplete efficiency of our endogeneous defense systems, dietary
antioxidants are needed to overcome the oxidative damage (5).
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Publication Date: March 12, 2007 | doi: 10.1021/bk-2007-0956.ch001

Dietary Antioxidants

Carotenoids and phenolic compounds are major dietary antioxidants. Both

of these groups of compounds contain hundred of members (6).
Carotenoids are natural, fat-soluble pigments that provide bright coloration
to plants and animals and act as antioxidants, which include the possibility of
providing vitamin A activity. One defining characteristic of carotenoids is the
chemical structure of what is often considered their backbone molecule, a 40-
carbon polyene chain. The polyene backbone consists of a pattern of conjugated
double bonds, which allows the carotenoids to take up excess energy from other
molecules (7). This characteristic may be responsible for the antioxidant activity
seen in biological carotenoids. In addition to scavenging free radicals, other
health benefits related to this observed antioxidative activity include protection
from sunburn and inhibition of the development of certain types of cancers (<S-
10).
β-Carotene is the most common carotenoid in food and the most potent of
the provitamin A carotenoids. β-Carotene is believed to have antioxidant
activity. It has been shown to exhibit radical- trapping behavior only at partial
pressure of oxygen significantly less than that in normal air (77). Such low
oxygen partial pressures are found in most tissues under physiological
conditions. At higher oxygen pressure it loses the antioxidant activity and shows
a pro-oxidant effect (77).
Lutein and its isomer, zeaxanthin, are yellow pigments that belong to the
classes of non-provitamin A carotenoids. Unlike other carotenoids, hydroxyl
groups are substituted on the ring structures at the end of the conjugated double
bond chains of lutein and zeaxanthin. Lutein is naturally occurring and found
predominantly in dark green, leafy vegetables such as spinach and kale.
Zeaxanthin gives corn its yellow color. There is a growing body of evidence
(including in vivo, in vitro and epidemiological studies) supporting the claim that

In Antioxidant Measurement and Applications; Shahidi, F., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
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lutein and zeaxanthin contribute to health and delay age related macular
degeneration of the eyes and, to a lesser extent, cancers and heart diseases (72-
14). The evidence for the role of lutein and zeaxanthin in eye health is very
strong because of their exclusive presence in the ocular tissues and the high
numbers of epidemiological studies that have been conducted. With a high
accumulation in the macula of the eye, the area of highest visual acuity, lutein
and zeaxanthin are proposed to have the ability to filter out harmful blue light,
while at the same time acting as antioxidants to quench potentially damaging
reactive oxygen species (ROS; 75).
Lycopene, a carotenoid found in tomatoes, watermelon, papaya, apricot,
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orange and pink grapefruit, exhibits antioxidant and anticancer activities (76).
About 80% of lycopene is consumed through tomatoes and tomato-related
products. Numerous studies have suggested reduced risk of prostate cancer from
Publication Date: March 12, 2007 | doi: 10.1021/bk-2007-0956.ch001

the consumption of processed tomato products (77). Although, these beneficial

health effects of lycopene are thought to be due to its antioxidant properties,
evidence is accumulating to suggest other mechanisms of action like hormone
and immune system modulation (18). Lycopene is the most abundant carotenoid
in human plasma, which may imply its elevated level of importance in the human
body compared with other carotenoids, such as β-carotene and lutein (19).
Phenolic compounds occurring commonly in foods may be classified into
simple phenols, hydroxybenzoic and hydrocinnamic acid derivatives, flavonoids,
stibenes, lignans and hydrolysable as well as condensed tannins (20). Phenolics
in foods may occur in the free, esterified, etherified and insoluble-bound forms.
The most abundant phenolic compounds in food are flavonoids. Flavonoids
are present in edible fruits, leafy vegetables, roots, tuber bulbs, herbs, spices,
legumes, tea, coffee, cocoa, chocolate and red wine. They can be classified into
seven groups: flavones, flavanones, flavonols, flavanonols, isoflavones,
flavanols (catechins) and anthocyanidins. In general, the leaves, flowers and
fruits or the plant itself contain flavonoid glycosides, woody tissues contain
aglycones, and seeds may contain both (20).
As a result of their ubiquity in plants, flavonoids are an integral part of the
human diet. It is estimated that the average American's daily intake of the
consumption of flavonols is close to 20-25 mg/day (21).
Almost all flavonoids possess several common biological and chemical
properties: (a) antioxidant activity, (b) the ability to scavenge ROS, (c) the
ability to scavenge electrophiles, (d) the ability to inhibit nitrosation, (e) the
ability to chelate metals, (f) the potential to produce hydrogen peroxide in the
presence of certain metals and (g) the capability to modulate certain cellular
enzyme activities (22). It appears that diets rich in flavonoids may protect
against cardiovascular diseases, neurodegenerative disorders and some forms of
cancer.

In Antioxidant Measurement and Applications; Shahidi, F., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
 5

Antioxidant Measurement

The need to measure antioxidant activity is well documented; these are

carried out for meaningful comparison of foods or commercial products and for
provision of quality standards for regulatory issues and health claims (23). There
are numerous methods for measureing antioxidant activity; these may be
classified into two categories. The first category measures the ability of
antioxidants in inhibiting oxidation in a model system by monitoring the
associated changes using physical, chemical or instrumental means. Radical
scavenging assays include methods based on hydrogen atom transfer (HAT) or
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electron transfer (ET) mechanisms. Oxygen radical absorbance capacity (ORAC),

total radical trapping antioxidant parameter (TRAP) and crocin bleaching assays
are the major methods that measure H A T while Trolox equivalent antioxidant
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capacity (TEAC), ferric reducing antioxidant antioxidant power (FRAP) and 2,2-
diphenyl-l-picrglhydrozyl (DPPH) assays represent ET-based methods.
Extensive relevant reviews are provided in the literature (23-25) as well as in this
volume (26).
It is interesting to note that D P P H radical is used to test antioxidant activity
by its ability to abstract hydrogen atoms from polyphenols (27). Another stable
radical, tris(2,4,6-trichloro-3,5-dinitrophenyl)methyl radical, was developed as a
good sensor to test the activity of polyphenols measuring their capacity to
participate in electron transfer reactions (27).
Antioxidant activity of a compound can also be evaluated in different cell
culture assay for the prevention of carcinogenesis. Because oxidative D N A
damage is considered to be relevant in carcinogenic process, one can evaluate
the possible anticarcinogenic effect of dietary antioxidants by determining their
effect on 12-Otetradecanoylphorbol 13-acetate (TPA)-inducing ROS generation,
H 0 scavenging, H 0 -induced apoptosis, xanthine oxidase activity, and
2 2 2 2

lipopolysaccharide (LPS)-inducing N O generation. Details are discussed in this

volume (28).

Bioavailability of Dietary Antioxidants

Bioavailability is the degree to which a drug, nutrient, dietary supplement or

nutraceutical is available to the body. Bioavailability is influenced by how much
of a substance is absorbed and circulated in the human body. Problems with
bioavailability arise when trying to elucidate exactly what dose brings about the
desired physiological response. Manufactures producing 500 mg vitamin C pills
cannot claim that 500 mg of the vitamin are taken in and used by the body.

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ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
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There are variations between different human subjects and their uptake of
certain food-based chemicals. This means that two people taking the same dose
could actually absorb different amounts of the same compound. One might only
see the effect of 200 mg of a 500 mg pill while the other might see the effect of
100 mg. This disparity is due to variability in absorption, distribution,
metabolism and excretion of the bioactives abbreviated as A D M E . In addition,
it is sometimes the case that the ingested chemical is not the final bioactive
agent. Many molecules enter the digestive system in one form only to be broken
down into smaller metabolites that interact through absorption. Science has yet
to identify many of these breakdown reactions sufficiently to understand how
these reactions affect the bioavailability of compounds.
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Studies have shown that 11% of caffeic acid and trace amounts of
chlorogenic acid, present in coffee, are found in urine indicating that they failed
Publication Date: March 12, 2007 | doi: 10.1021/bk-2007-0956.ch001

to be fully absorbed through the gut barrier (29). The exact fate of the remaining
caffeic acid is unknown. This is the problem of bioavailability and it translates to
nearly every aspect of nutraceuticals.
While it is difficult to say for sure how much of a compound will be taken in
and used by an organism, there is some strong evidence showing how low some
of the uptakes can be. In a study using chlorogenic acid, only 1.7% of it was
recovered unchanged in the urine. In these cases, the colon could play a larger
role in the metabolism of polyphenols (30).
Polyphenols reaching the colon can be broken into smaller metabolites by
colonic microbiota, the bacteria found in the colon (30). These bacteria are able
to break down phenols, allowing absorption of these smaller metabolites by the
kidneys, liver and other organs. Later, these smaller metabolites may find their
way into the urine. Without a clear understanding of their chemical nature, we
cannot screen for them in the urine. The same study also concluded that a large
part of the ingested polyphenols will probably never enter the peripheral
circulation as smaller metabolites (30).
The problem of reduced antioxidant activity found in smaller metabolites of
larger parent compounds increases the uncertainty of bioavailability studies. A n
organism is not likely to absorb an entire dose, and it is likely that the compound
will be broken down into smaller, unidentified compounds. Further studies are
warranted to identify these compounds. Considering these variables, it is very
difficult to predict the total effect of an antioxidant on host cells.

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ACS Symposium Series; American Chemical Society: Washington, DC, 2007.