8

Introducing natural antioxidants

Dr Honglian Shi, Cornell University Medical College and Dr Noriko
Noguchi and Professor Etsuo Niki, Utsunomiya University

8.1 Introduction
The importance of the antioxidants contained in foods is well appre-
ciated for both preserving the foods themselves and supplying essential
antioxidants in vivo. With increasing experimental, clinical and epidemio-
logical data which show the beneficial effects of antioxidants against oxida-
tive stress-induced degenerative and age-related diseases, cancer and
ageing, the importance and role of antioxidants have received renewed
attention.
We are protected from oxidative stress by various antioxidants
which have different functions. Some are enzymes and proteins and
others are small molecule antioxidants. Foods are important as an essen-
tial source of such antioxidants, components and trace elements. In
addition, numerous synthetic antioxidants have been developed and
some of them have been used in practice as, for example, food additives,
supplements and drugs. The phenolic compounds such as vitamin E and
flavonoids are typical antioxidants. Numerous phenolic compounds
have been also synthesised: 2,6-di-tert-butyl-4-methylphenol known as
BHT is one of the most popular synthetic antioxidants. It is generally
accepted, however, that natural antioxidants are more potent, efficient
and safer than synthetic antioxidants. For example, a-tocopherol is the
most active form of vitamin E and natural 2R,4’R,8’R-a-tocopherol is
more potent than synthetic racemic a-tocopherol primarily because
a-tocopherol transfer protein selectively recognises natural a-tocopherol.
As such, natural antioxidants are more favourably accepted than synthetic
antioxidants.
 148 Antioxidants in food

8.2 Categorising natural antioxidants

Table 8.1 shows the antioxidants which constitute the defence system in
vivo. As shown, there are several lines of defence. The first defence line is
to inhibit the formation of active oxygen species and free radicals by seques-
tering metal ions, reducing hydroperoxides and hydrogen peroxide and to
quench superoxide and singlet oxygen. The radical-scavenging antioxidants
function as the second line defence. Vitamin E and vitamin C are major
lipophilic and hydrophilic radical-scavenging antioxidants. They scavenge
radicals and inhibit chain initiation or break chain propagation. Polyphe-
nolic compounds may also work as important radical-scavenging antioxi-

Table 8.1 Defence systems in vivo against oxidative damage

1. Preventive antioxidants: suppress the formation of free radicals.

(a) Non-radical decomposition of hydroperoxides and hydrogen peroxide:
catalase decomposition of hydrogen peroxide
2H2O2 Æ 2H2O + O2
glutathione peroxidase (cellular) decomposition of hydrogen peroxide and
free fatty acid hydroperoxides
H2O2 + 2GSH Æ 2 H2O + GSSG
LOOH + 2GSH Æ LOH + H2O+GSSG
glutathione peroxidase (plasma) decomposition of hydrogen peroxide and
phospholipid hydroperoxides
PLOOH + 2GSH Æ PLOH + H2O + GSSG
phospholipid hydroperoxide decomposition of phospholipid hydroperoxides
glutathione peroxidase
peroxidase decomposition of hydrogen peroxide and
lipid hydroperoxides
LOOH + AH2 Æ LOH + H2O + A
H2O2 + AH2 Æ 2H2O + A
glutathione-S-transferase decomposition of lipid hydroperoxides
(b) Sequestration of metal by chelation:
transferrin, lactoferrin sequestration of iron
haptoglobin sequestration of haemoglobin
haemopexin stabilisation of haem
ceruloplasmin, albumin sequestration of copper
(c) Quenching of active oxygens:
superoxide dismutase (SOD) disproportionation of superoxide
2O2•- + 2H+ Æ H2O2 + O2
carotenoids, vitamin E quenching of singlet oxygen
2. Radical-scavenging antioxidants: scavenge radicals to inhibit chain initiation and break
chain propagation.
hydrophilic: vitamin C, uric acid, bilirubin, albumin
lipophilic: vitamin E, ubiquinol, carotenoids, flavonoids
3. Repair and de novo enzymes: repair the damage and reconstitute membranes lipase,
protease, DNA repair enzymes, transferase.
4. Adaptation: generate appropriate antioxidant enzymes and transfer them to the correct
site at the correct time and in the correct concentration.
 Introducing natural antioxidants 149

dants.The third-line of defence is the repair, de novo and clearance of oxida-

tively damaged lipids, proteins and DNA. Various enzymes such as lipases,
proteases and DNA repair enzymes are responsible for such defence. There
is another defence mechanism in which appropriate antioxidants are pro-
duced and transferred to the correct place at the correct time and in the
correct amounts.
Foods are essential for supplying the above mentioned antioxidants,
components and trace elements. For example, glutathione peroxidases play
a pivotal role in reducing hydroperoxides and hydrogen peroxide. Various
types of glutathione peroxidases have been found and they and selenopro-
tein contain selenium as an essential metal ion. We obtain selenium mostly
from vegetables and any soil which contains insufficient selenium causes
selenium deficiency, such as in Keshan disease. We take phenolic antioxi-
dants from foods, fruits and drinks such as tea.

8.3 Potency of natural antioxidants

As endogenous antioxidants synthesised by aerobes (e.g. SOD, catalase,
GSH) do not completely prevent damage by reactive species in vivo,1 effi-
cient repair systems are needed to reduce the damage and humans must
also obtain antioxidants from the diet. There is currently a considerable
amount of interest in dietary antioxidants as bioactive components of food.
The physiological role of some of these, such as vitamin E and vitamin C,
is well established. The interest in flavonoids has increased in recent years
because of their ubiquitous presence as antioxidants in food.
Flavonoids are diphenylpropanes that commonly occur in plants and are
frequently components of human diet. They are consumed in relatively high
quantities in our daily food. The main source of flavonoids is vegetables,
fruits and beverages. For example, the content of quercetin glycoside in
outer leaves of lettuce could be as high as 237 mg/kg fresh weight, and the
content of kaemferol glycoside in kale could be 250 mg/kg fresh weight.2,3
It has been found that flavonoids and other polyphenols possess anti-
tumoral, anti-allergic, anti-platelet, anti-ischemic, and anti-inflammatory
activities. Epidemiological evidence for the importance of flavonoids in
reducing mortality from coronary heart disease was provided by the
Zutphen Elderly Study.4 The role of dietary phenolic antioxidants in vivo,
protecting against cancer, has also been underlined by some epidemiolog-
ical studies.5,6 In these studies, flavonoids and other dietary compounds have
been mentioned as statistically beneficial and protective against carcino-
genesis.4 Most of these biological effects are believed to come from their
antioxidant properties. Flavonoids can exert their antioxidant activity by
inhibiting the activities of enzymes including xanthine oxidase, myeloper-
oxidase, lipoxygenase and cyclooxygenase,7,8 by chelating metal ions,7,9–12 by
interacting with other antioxidant such as ascorbate,13 and most importantly,
150 Antioxidants in food

by scavenging free radicals. This chapter introduces the free radical scav-
enging potency of flavonoids in food, their scavenging effect on reactive
oxygen species and reactive nitrogen species, the inhibiting effect on lipid
peroxidation, assessment of their potency as hydrogen-donating antioxi-
dants, and their activity–structure relationships.

8.3.1 Ability to scavenge reactive oxygen species

Because of the importance of reactive oxygen species such as the superox-
ide anion and the hydroxyl radical in biological environments and in human
health and disease, the reactivity of flavonoids toward these radicals has
been extensively studied. There are generally two superoxide anion sources
used: the enzymatic (hypo)xanthine-xanthine oxidase (X/XO) system and
non-enzymatic sources such as the phenazine methosulphate-NADH
system or potassium superoxide. Quercetin, myricetin and rutin are the
flavonoids that were tested in the earlier studies. They quenched superox-
ide anions generated from either the X/XO system or from non-enzymatic
sources.14–16 Besides pure substances, an extract from Ginkgo biloba (GBE)
showed SOD activity and scavenges superoxide anions produced from a
non-enzymatic system. Further research showed that the scavenging ability
depends on the chemical structures of flavonoids.17,18 When the enzymatic
system is used in the study, the scavenging effect may come directly from
the radical-quenching effect or/and the enzyme-inhibiting effect. The struc-
ture–activity relationship of flavonoids as inhibitors of xanthine oxidase and
as scavengers of the superoxide radical was recently reported by Cos et al.19
It was found that the hydroxyl groups at C-5 and C-7 and the double bond
between C-2 and C-3 was essential for a high inhibitory activity on xanthine
oxidase. For a high superoxide scavenging activity, on the other hand, a
hydroxyl group at C-3¢ in ring B and at C-3 was essential. According to
their effect on xanthine oxidase and as superoxide scavengers, flavonoids
are classified into six groups: 1, superoxide scavengers without inhibitory
activity on xanthine oxidase; 2, xanthine oxidase inhibitors without any
additional superoxide scavenging activity; 3, xanthine oxidase inhibitors
with an additional superoxide scavenging activity; 4, xanthine oxidase
inhibitors with an additional pro-oxidant effect on the production of super-
oxide; 5, flavonoids with a marginal effect on xanthine oxidase but with a
pro-oxidant effect on the production of superoxide, and finally; 6, flavonoids
with no effect on xanthine oxidase or superoxide.
The hydroxyl radical is more reactive than the superoxide anion and is
therefore more harmful to biological samples. Most flavonoids possess a
high reactivity with the hydroxyl radical. For instance, (+)-catechin, (-)-epi-
catechin, 7,8-dihydroxy flavone, and rutin scavenge the hydroxyl radical at
100–300 times more than mannitol, a typical hydroxyl radical scavenger.17
The reactivity of flavonoids toward hydroxyl radical is generally much
higher than that toward superoxide anion. The reaction rate constants of
 Introducing natural antioxidants 151

quercetin and kaempferol with the hydroxyl radical are 4.3 ¥ 109 M-1 s-1 and
4.6 ¥ 109 M-1 s-1, respectively, which are about four orders higher than those
with the superoxide anion.20 Some of the compounds from Indian medici-
nal plants behaved as scavengers of the hydroxyl radical in the deoxyribose
degradation assay, with a calculated rate constant for kaempferol-3-O-
galactoside of 1.55 ¥ 1010 M-1 s-1.21 Baicalin reacts with the hydroxyl radical
and the superoxide anion at the rate constants 7.7 ¥ 1011 M-1 s-1 and 3.2 ¥
106 M-1 s-1, respectively.22
Flavonoids also react with singlet oxygen (1O2). Flavonoids in green
tea such as (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin
(EGC) and (-)-epicatechin (EC)) have been shown to scavenge 1O2.23 Tour-
naire et al.24 studied the reactivity of 13 selected flavonoids (from the
flavonol, flavone, flavanone and flavane families) with 1O2 and tried to estab-
lish a structure–activity relationship. They found that the efficiency of the
physical quenching is mainly controlled by the presence of a catechol
moiety on ring B, whereas the structure of ring C (particularly the presence
of a hydroxyl group activating the double bond) is the main factor
determining the efficiency of the chemical reactivity of these compounds
with 1O2.
Flavonoids inhibit the production of reactive oxygen species in cells.
Certain flavonoids slow down O2 consumption (respiratory burst) of stim-
ulated human neutrophils (PMNs) by its inhibitory action on NADPH-
oxidase, the enzyme responsible for the reduction of O2 to the superoxide
anion. Consequently, superoxide anions and hydrogen peroxide production
are significantly decreased when the PMNs stimulation is carried out in the
presence of flavonoids. Some flavonoids are able to reduce significantly the
activity of myeloperoxidase contained in neutrophils. This enzyme, secreted
into the intra- and extracellular medium, catalyses the oxidation of chloride
(Cl-) by H2O2 to yield strong oxidants such as HOCl.

8.3.2 Ability to interact with reactive nitrogen species

Although nitric oxide (NO) is relatively unreactive per se, it may become
potentially harmful once its concentration overwhelms its neurotransmitter
and second messenger function and in particular when it reacts with the
superoxide anion generating peroxynitrite (ONOO-), which is a very reac-
tive oxidant for most of the biological molecules. Some studies have demon-
strated that flavonoids can efficiently quench reactive nitrogen species. The
ONOO- scavenging activity of some of the flavonoids was found to be 10
times higher than that of ebselen, an efficient peroxynitrite scavenger.25–27
Anthocyanidins are potent scavengers of NO and ONOO- as described
by van Acker et al.28 and by Haenen et al.25 respectively. Pycnogenol, a
procyanidin-rich extract from pine bark, significantly decreased in a dose-
dependent fashion the accumulation of nitrite after spontaneous decomposi-
tion of sodium nitroprusside, thus acting as a nitric oxide radical scavenger.29
152 Antioxidants in food

Morin, quercetin, or catechin may attenuate SIN-1 induced cytotoxic to cul-

tured porcine aortic endothelial cells.30 On the other hand, flavonoids can
modulate the NO production level by regulating the expression of inducible
nitric oxide synthase (iNOS).31,32 Certain flavonoids have been reported to
inhibit NO production in lipopolysaccharide-activated RAW 264.7 cells, and
their inhibitory activity was suggested to be due to reduction of iNOS
enzyme expression. Quercetin at 0.1 mM inhibited lipopolysaccharide-
dependent production of iNOS mRNA and decreased NO release.

8.3.3 Effect on the lipid peroxyl radical and lipid peroxidation

As hydrogen-donating antioxidants, flavonoids directly scavenge lipid
peroxyl radicals.23,33 As clinical and biochemical evidence shows that low-
density lipoprotein (LDL) oxidation is a crucial event in the pathogenesis
of atheriosclerosis, the protective effect of flavonoids on LDL oxidation has
been an interesting subject. De Whalley et al. have shown that quercetin,
morin, fisetin and gossypetin with the IC50 at 1–2 mM inhibited the oxidative
modification of human LDL induced by macrophages and delayed the
depletion of endogenous a-tocopherol.34 It has been known that red wine
contains a considerable amount of flavonoids and experimental results
showed that it could inhibit porcine LDL oxidation induced by copper ion
or AAPH.35 Wine diluted 1000-fold containing 10 mM total phenolics inhib-
ited LDL oxidation significantly more than a-tocopherol.36 It was suggested
that wine polyphenols seem to act by their antioxidant activity rather than
by metal chelating, and it has been reported that the antioxidant activity of
human serum was elevated for several hours after wine consumption.37
Studies in humans provide direct evidence that regular and long-term con-
sumption of red wine, but not of ethanol, inhibited LDL oxidation in
vivo.38,39 It is suggested that red wine intake may reduce atherosclerosis and
morbidity and mortality from coronary heart disease. These studies have
provided a plausible explanation for the ‘French paradox’.40

8.3.4 Assessment of antioxidant potential of flavonoids

For measuring the antioxidant potentials of flavonoids, either the total
antioxidant activity (TAA), or the trolox equivalent antioxidant activity
(TEAC) has been extensively used.41–44 The TEAC method is to compare
the ability of a hydrogen-donating antioxidant to scavenge a radical gener-
ated from the reaction of 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS) with a ferrylmyoglobin radical species41–44 or potassium per-
sulphate,45 with that of trolox. The value of TEAC may be considered as a
stoichiometric number because TEAC for trolox was set at 1.0. To test the
efficacy of a certain substance as a radical scavenger, clear evidence may
come from the determination of reaction rate constants with specific radi-
cals. Although the stoichiometric number determines the duration of the
 Introducing natural antioxidants 153

inhibition period or lag time, the rate constant can give the extent of inhi-
bition in oxidation. Two methods including both inhibition period and
extent of inhibition in oxidation are used in studying the potential of natural
antioxidants such as flavonoids. Descriptions of them follow.

8.3.4.1 ORAC method

The oxygen radical absorbance capacity (ORAC) assay developed by Cao
and co-workers46,47 provides a new way of evaluating potential antioxidant
activity. The method uses an area-under-curve technique and thus combines
both inhibition time and inhibition degree of free radical action by an
antioxidant into a single quantity, while other similar methods use either
the inhibition time at a fixed inhibition degree or the inhibition degree at
a fixed time as the basis for quantifying the results.41,48–50 In addition, differ-
ent free radical (lipid peroxyl radical, hydroxyl radical, superoxide anion
etc.) generators can be used in the ORAC assay.

8.3.4.2 Galvinoxyl method51

Galvinoxyl is a rather stable radical and, while it accepts an electron or
hydrogen radical to become a stable and diamagnetic molecule, can be oxi-
dised irreversibly. Because of its odd electron, galvinoxyl shows a strong
absorption band at 428 nm (in ethanol), its solutions appearing yellow in
colour (at low concentration). As the electron is paired off, the absorption
vanishes and the resulting decolorisation is stoichiometric with respect to
the number of electrons taken up. Taking advantage of the colour change
of galvinoxyl in the presence of an antioxidant, the dynamics of the antio-
xidant activity, hydrogen-donating activity, can be easily measured in a
simple and similar manner. Galvinoxyl can be used not only to measure the
stoichiometric number of active phenolic hydrogens of a substance, but also
to determine the rate constant for related antioxidants. Furthermore, the
method can be used to determine and compare the antioxidative activity of
hydrogen-donating compounds, either pure substances or mixtures. The
second-order rate constant of GBE has been compared with other pure
substances on the molar basis of active hydroxyl groups.
Some results from the three methods, TEAC, ORAC and galvinoxyl, are
listed in Table 8.2. Although the radical sources are different, the efficacies
of flavonoids are comparable. The stoichiometric number in the reaction
with galvinoxyl decreases in the following order: myricetin > quercetin >
catechin > kaempferol > a-tocopherol. The total reactive rate constant
decreases in the order of myricetin > quercetin > a-tocopherol > kaempferol
> catechin. The average reactive potential of active hydroxyl groups studied
decreased in the order myricetin > a-tocopherol > quercetin > GBE =
kaempferol > catechin.The ORACLOO gives the order myrecetin > quercetin
•

> kaempferol. The TEAC order is quercetin > myricetin > catechin >
kaempferol > a-tocopherol. In the ORAC and galvinoxyl study, myricetin
was the strongest antioxidant among the substances tested, while the TEAC
154 Antioxidants in food

OH

OH O
B O
O
A C O

O O
O
H
O H

(a) (b) (c)

8.1 Antioxidant activity–structure relationship of flavonoids.

value of myricetin was less than that of quercetin. This indicates that
the TEAC value may not completely reflect the potential of an
antioxidant.

8.3.5 The structure–activity relationships

As mentioned above, the antioxidant activity of flavonoids depends on their
chemical structure. Generally, there are three structure groups in the
determination, the free radical scavenging and/or antioxidative potential of
flavonoids (Fig. 8.1): (a) a catechol moiety of the B-ring, (b) the 2,3-double
bond in conjugation with a 4-oxofunction of a carbonyl group in the C-ring
and (c) presence of hydroxyl groups at the 3 and 5 positions. Quercetin pos-
sesses all the three structure groups and thus usually gives a higher anti-
oxidant potential than kaempferol, which has no catechol moiety in the
B-ring. From our study (Table 8.2) and others, the presence of a hydroxyl
group at 5¢ in the B-ring increases the antioxidant potential significantly.

Table 8.2 Comparison of antioxidant potential determined by three methods,

galvinoxyl,51 ORAC52 and TEAC42

Galvinoxyl ORACLOO· TEAC

n k2b k21b

myricetin 4.5 1.1 ¥ 106 2.4 ¥ 105 4.3 3.1

quercetin 4.0 5.9 ¥ 103 1.5 ¥ 103 3.3 4.7
kaempferol 1.9 2.1 ¥ 103 1.1 ¥ 103 2.7 1.3
catechin 3.0 1.5 ¥ 103 5.0 ¥ 102 – 2.2
a-tocopherol 1.0 2.4 ¥ 103 2.4 ¥ 103 – 1.0
GBE 1.1 ¥ 10-4 a
0.13c 1.2 ¥ 103 – –
a
mol active hydroxyl groups/g
b
second-order rate constant for molecule (k2) and per active hydrogen (k2¢) in M-1 s-1
c
(g l-1)-1 s-1
 Introducing natural antioxidants 155

The stoichiometric number of myrecetin is only 0.5 greater than that of

quercetin but the total reactive potential (k2) and the average reactive
potential (k2¢) are two orders higher than those of quercetin.This is in agree-
ment with other reports indicating that the antioxidant activity of myricetin
is more potent than quercetin in emulsions, oils, liposome and LDL.

8.4 Future trends

There are now increasing reports which suggest the non-classical role and
function of antioxidants which are not explained by the inhibition of oxi-
dation alone. For example, there is a general consensus that vitamin E exerts
a broad range of effects that promote vascular homeostasis,53 by, for
example, inhibiting protein kinase C activity, proliferation of smooth muscle
cells and adhesion of inflammatory cells to be endothelial cells. Further-
more, it has been shown that some antioxidants are capable of inducing
phase II defence enzymes such as quinone reductase and glutathione S-
transferase. The physiological role of such functions is a matter of future
study.

8.5 Sources of further information

Basu T K, Temple N J and Carg M L, Antioxidants in Human Health and Disease,
Wallingford, CABI, 1999.
Cadenas E and Packer L, Handbook of Antioxidants, New York, Marcel Dekker,
1996.
Frei B, Natural Antioxidants in Human Health and Disease, San Diego, Academic
Press, 1994.
Ong A S H, Niki E and Packer L, Nutrition, Lipids, Health, and Disease, Champaign,
AOCS Press, 1995.
Packer L and Cadenas E, Handbook of Synthetic Antioxidants, New York, Marcel
Dekker, 1997.
Packer L and Ong A S H, Biological Oxidants and Antioxidants. Molecular Mecha-
nisms and Health Effects, Champaign, AOCS Press, 1998.
Papas A M, Antioxidant Status, Diet, Nutrition, and Health, Boca Raton, CRC Press,
1999.

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