8 Antioxidant Properties

and Mechanisms of
Tea Polyphenols
Xiaochun Wan, Daxiang Li, and Zhengzhu Zhang

Contents

8.1 Introduction.................................................................................................. 131

8.1.1 Catechins.......................................................................................... 132
8.1.2 Flavonol and Glycosides................................................................... 132
8.1.3 Anthocyanidin and Leucoanthocyanidin.......................................... 134
8.1.4 Phenolic Acids and Depsides............................................................ 135
8.2 Structural Characteristics of Tea Polyphenols............................................. 135
8.3 Antioxidant Properties and Mechanisms of Tea Polyphenols..................... 139
8.3.1 Antioxidant Properties of Tea Polyphenols...................................... 140
8.3.2 Antioxidant Mechanisms of Tea Polyphenols.................................. 141
8.3.2.1 Scavenging Reactive Oxygen Species................................. 141
8.3.2.2 Chelating Metal Ions to Prevent Oxidation......................... 153
8.3.2.3 Regulation Enzymes or Genes Related to Oxidation or
Antioxidation....................................................................... 153
8.4 Conclusions.................................................................................................. 155
References............................................................................................................... 155

8.1 Introduction
The tea bush and in particular its young leaves contain a high concentration of poly-
phenols and oxidative enzymes, thus the young leaves are better for tea manufacture.
Tea polyphenols, previously called tea tannins, are also known as tea flavonoids.
Among the polyphenols in fresh tea leaves, catechins are the predominant form of
polyphenols, which account for 12–24% of the dry weight. Besides catechins, flavo-
nol, and their glycosides, anthocyanidin and leucoanthocyanidin, phenolic acids and
depsides are also present. Their typical concentrations are shown in table 8.1.1 These
phenolic compounds are directly or indirectly associated with the characteristics of
tea, including its color, taste, and aroma.

131

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 132 Tea and Tea Products: Chemistry and Health-Promoting Properties

Table 8.1
Polyphenols in young tea shoots and their typical contents
Polyphenols Content (dry weight basis)
Total 18–36%
Flavan-3-ols (catechins) 12–24%
Flavonol and glycosides 3–4%
Anthocyanins and leucoanthocyanidin 2–3%
Phenolic acids and depsides ~5%
Source: Wan, X. C., Huang, J. Z., and Shen, S. R., Eds. 2003. Tea biochemistry, 3rd ed., 9–
20, 180–94. Beijing: China Agriculture Press. With permission.

8.1.1 Catechins
3' Catechins are a group of flavonoids (fig-
2' 4'ure 8.1) belonging to flavan-3-ols. In
B fresh tea leaves, the principal catechins
8 1
O 2 5'
7
are (–)-epicatechin (EC), (–)-epigal-
6' locatechin (EGC), (–)-epicatechin gal-
A C
6 3 late (ECG), (–)-epigallocatechin gallate
5 4 (EGCG), catechin (C), and gallocatechin
(GC) (figure 8.2) (1). EGCG is the most
Figure 8.1 Basic flavonoid structure.
abundant catechin, followed by EGC,
ECG, and EC (table 8.2).
Catechins are responsible for the
astringent taste and strength of green tea infusion. During green tea manufacture,
most catechins and other polyphenols are preserved owing to the inactivation of the
endogenous enzymes by dry heating or steaming at the initial step. Green tea quality
correlates positively with the concentration of polyphenols. However, a high con-
centration of polyphenols or catechins, which makes the infusion strongly bitter and
astringent, is not necessarily required for high-quality green tea. High-quality green
tea is characterized by high contents of free amino acids with appropriate concentra-
tions of catechins and caffeine.2

8.1.2 Flavonol and Glycosides

In fresh tea leaves, a total of 20 flavonols and glycosides have been reported.3,4 The
flavonols found in fresh tea leaves include kaempferol, quercetin, and myricetin (fig-
ure 8.3), which account for 0.14–0.32%, 0.27–0.48%, and 0.07–0.2% of the dry weight
of tea leaves, respectively.1 Flavonol glycosides are flavonols bound to a monosac-
charide (e.g., glucose, rhamnose, galactose, arabinose) or disaccharide (e.g., rutin-
ose). The principal flavonol glycosides are rutin, quercetin glycoside. and kaempferol
glycoside (figure 8.4). Their contents in tea are, respectively, 0.05–0.15%, 0.2–0.5%,
and 0.16–0.35% of dry weight.1 Flavonol and its glycosides are often bright yellow
and thought to contribute to the yellowish green color of green tea liquor.1 Moreover,
some flavon-3-ol glycosides induce a silky, mouth-drying, and mouth-coating sensa-

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 Antioxidant Properties and Mechanisms of Tea Polyphenols 133

OH
3' OH OH
2' 4'
B
8 5' OH
OH O
7 2 R1
A C 6' OH
3 OH O
6 R
5 OR2 Galloyl = D OH
4
OH O
OH OH

OH

1. R1 = R2 = H Epicatechin 5. R = H Catechin

2. R1 = OH, R2 = H Epigallocatechin 6. R = OH Gallocatechin

3. R1 = H, R2 = galloyl Epicatechin gallate

4. R1 = OH, R2 = galloyl Epigallocatechin gallate

Figure 8.2 Structures of major catechins.

Table 8.2
Catechins in young tea leaves
Catechins Contenta (dry weight base)
(–)-Epicatechin (EC) 1–3%
(–)-Epigallocatechin (EGC) 3–6%
(–)-Epicatechin gallate (ECG) 3–6%
(–)-Epigallocatechin gallate (EGCG) 8–12%
(+)-Catechin (C) 1–2%
(+)-Gallocatechin (GC) 3–4%
a Data adapted from www.tocklai.net/teachem/index.htm.

R1

OH

OH O
R2

OH

OH O

1. R1 = R2 = H Kaempferol

2. R1 = OH, R2 = H Quercetin

3. R1 = R2 = OH Myricetin

Figure 8.3 Structures of major flavonols.

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 134 Tea and Tea Products: Chemistry and Health-Promoting Properties

OH OH

OH OH

OH O OH O

OGlc Rha ORha

OH O OH O
Rutin
Glc = Glucosyl, Rha = Rhamnosyl Quercetin glycoside

OH

OH O

OGlc

OH O

Kaempferol glycoside

Figure 8.4 Structures of major flavonol glycosides.

tion with extremely low taste thresholds, ranging from 0.001 to 19.8 μmol/l.5 Other
studies demonstrate that flavanol-3-glycosides not only impart a velvety astringent
taste sensation to the oral cavity, but also contribute to the bitter taste of tea infusions
by amplifying the bitterness of caffeine.6

8.1.3 Anthocyanidin and Leucoanthocyanidin1

Leucoanthocyanidins (flavan-3,4-diols) are distributed widely in plants. In fresh tea
leaves, leucoanthocyanidins, which account for 2–3% of the dry weight of fresh tea
leaves, are the principal leucocyanidins and leucodelphinidins present (figure 8.5a).
Leucoanthocyanidins are colorless and easy to be oxidized and condensed to pig-
ments during tea fermentation. They are precursors for the important classes of
colored anthocyanidins and anthocyanins (anthocyanidin glycosides). Anthocyani-
dins, including pelargonidin, cyanidin, delphinidin, and tricetinidin (figure 8.5b),
account for ~0.01% of the dry weight of tea leaves, whereas they reach up to 1.0%
in purple tea shoots, which give undesirable bitterness and color to tea quality.
Anthocyanidins are not very stable, and usually glycosylation transfers a glycoside
(usually glucose) to position 3 of the anthocyanidin to form a stable anthocyanin.
With heat or acidic hydrolysis, anthocyanins release anthocyanidins. Anthocyanins
are localized in the cell vacuole. As cellular pH increases, the anthocyanins’ struc-
tural changes produce red, purple, or blue colors of many blooms, fruits, leaves, and
stems of plants.

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 Antioxidant Properties and Mechanisms of Tea Polyphenols 135

OH R1

OH OH

+
OH O OH O
R R2

R3
OH OH

OH OH OH

1. R = H Leucocyanidin 1. R1 = R2 = R3 = H Pelargonidin
2. R = OH Leucodelphinidin 2. R1 = OH, R2 = R3 = H Cyanidin

3. R1 = R2 = OH, R3 = H Delphinidin

4. R1 = R3 = OH, R2 = H Tricetinidin

(a) (b)

Figure 8.5 Structures of major leucoanthocyanidins and anthocyanidins.

8.1.4 Phenolic Acids and Depsides1

Phenolic acids are a diverse group that includes hydroxybenzoic and hydroxycin-
namic acids and depsides, the latter being intermolecular esters formed from two
or more molecules of the same or different phenolic benzoic acids. In fresh tea
leaves, phenolic acids and depsides account for about 5% of the dry weight of young
tea leaves. Phenolic acids are mainly gallic acid, chlorogenic acid, and theogallin
(figure 8.6), which account for 0.5–1.4%, 0.3%, and 1–2% of the dry weight of tea
leaves, respectively, whereas depsides are mainly ellagic acid and m-digallic acid as
trace compounds. In tea plants, phenolic acids are precursors of catechin gallates. In
manufactured tea, phenolic acids associated with other polyphenols contribute to the
astringent taste of tea liquor.

8.2 Structural Characteristics of Tea Polyphenols

During the tea fermentation process, polyphenols located within the vacuoles of the
intact leaf cells are released and oxidized catalytically with polyphenol oxidases
located in cytoplasm. Polyphenol oxidase can use any of the catechins as a substrate
to form complex polyphenolic constituents. The catechins in fresh tea leaves undergo
enzymatic and chemical oxidation leading to oxidized, condensed, and polymerized
polyphenols known as theaflavins and thearubigins, which contribute to the color
and taste of liquors of black tea. The oxidative fermentation of catechins results in
the development of appropriate flavor and color. It will cause a darkening of the leaf
and a decrease in astringency. Theaflavins account for 1–3% of the dry weight of
black tea. Thearubigins are by far the major components of black tea extract. They
constitute as much as 10–20% of the dry weight of black tea.1
Catechins are characterized by di- or trihydroxyl group substitution of the B-
ring and meta-5,7-dihydroxyl substitution of the A-ring of the flavonoid structure

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 136 Tea and Tea Products: Chemistry and Health-Promoting Properties

OH
OH
O O OH
OH
HO O O
OH
HOOC HOOC
HO COOH OH OH
HO HO
HO OH OH
Gallic acid Theogallin Chlorogenic acid

Figure 8.6 Structures of major phenolic acids.

(figure 8.1).1 Catechins possess a 1,3-dihydroxy phenyl group (A-ring) and an o-

dihydroxy phenyl group (B-ring). Both groups can be oxidized, with the o-dihydroxy
phenyl group being more susceptible. Oxidation can be mediated with oxidase and
metal ions, and occurs autocatalytically at pH values of approximately 6.0 and above.
One-electron oxidation generates a free radical that can couple with other radicals
to form C–C or C–O bonds from which theasinensins (C–C bond formation) and
related derivatives are probably derived. O-Dihydroxy phenyl rings and o-trihydroxy
phenyl rings can, through two one-electron oxidations, lead to the highly reactive
o-quinones. These species can react further via intermolecular cycloaddition reac-
tions to form products that contain a benzotropolone ring system and have intense
yellow/orange colors. These are the basic structural requirements and mechanisms
for the formation of dimeric flavonoid-derived pigments.7 In some conditions, the
trihydroxyl group of galloyl moiety (D-ring) can be oxidized as the way of the trihy-
droxyl group of the B-ring to produce a benzotropolone skeleton by its reaction with
the dihydroxyl group of the B-ring of another molecule.8–10
A recent study further proves that the trihydroxy group of the B-ring of catechins
is more active and could be oxidized to several types of catechin dimers. It has dem-
onstrated that enzymatic oxidation of epigallocatechin affords three quinone dimers,
dehydrotheasinensin C, proepitheaflagallin, and an unnamed symmetrical quinone
dimer. Dehydrotheasinensin C has a hydrated cyclohexenetrione structure and its
oxidation-reduction dismutation reaction yields black tea polyphenols, theasinensins
C and E, and desgalloyl oolongtheanin, while proepitheaflagallin is decomposed
upon heating to afford epitheaflagallin and hydroxytheaflavin (figure 8.7).11
Theaflavins possess a hydroxyl-substituted benzotropolone ring system, which
is formed principally from oxidative coupling of one o-dihydroxy group and an o-
trihydroxy group of the B-ring in another. The major theaflavins (figure 8.8) in black
tea are theaflavin (TF), theaflavin-3-gallate (TF-3-G), theaflavin-3'-gallate (TF-3'-G),
and theaflavin-3,3'-digallate (TF-3,3'-DG)1; their precursors and contents in black tea
are shown in table 8.3, and their formation mechanism is shown in figure 8.9.12 Com-
pared with EGCG or other catechins, theaflavins have higher molecular weights,
have more phenol groups, and possess a benzotropolone moiety. It is reasonable that
these compounds have similar biological activities to their precursor catechins. It has
been shown that theaflavins are more active than EGCG in inhibiting 12-O-tetradec-
anoylphorbol-13-acetate (TPA)-induced mice ear edema.12

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8082.indb 137
OH OH
HO HO

HO HO
OH O OH O OH
OH OH OH
HO OH HO OH
OH OH + OH
HO O
HO O HO O
OH
OH OH OH
H OH
HO OH OH OH
O O
OH OH OH
HO HO Theasinensin C Theasinensin E
O
OH
OH
O HO
OH 1 OH OH HO
OH OH OH OH
EGC
OH HO
Dehydrotheasinensin C
OH O
HO HO O O
O OH
H OH
OH HO + HO
O O O
O O
O OH
OH H O
HO HO OH
O OH O
O ×2 OH OH
O
Desgalloyl oolongtheanin Dehydrotheasinensin E
OH OH
OH HO OH
O
EGC quinone O

OH
OH An unnamed dimer OH
OH HO OH
OH OH OH
O
Antioxidant Properties and Mechanisms of Tea Polyphenols

HO O
OH O
HO O
OH OH
HOOC O
HO +
O O
HO O
HO O OH OH
H OH
O OH
O OH OH
OH
OH
OH
Epitheaflagallin Hydroxytheaflavin
Proepitheaflagallin

Figure 8.7 Enzymatic oxidation products of epigallocatechin. (From Matsuo, Y., Tanaka, T., and Kouno, I. 2006. A new mechanism for oxidation of
137

epigallocatechin and production of benzotropolone pigments. Tetrahedron 62:1–10. With permission.)

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 138 Tea and Tea Products: Chemistry and Health-Promoting Properties

OH
OR2

OH
HO O
OH

HO O O

OH
OR1
OH

R1 R2

Theaflavin TF H H

Theaflavin-3-gallate TF-3-G Galloyl H

Theaflavin-3΄-gallate TF-3΄-G H Galloyl

Theaflavin-3, 3΄-digallate TF-3, 3΄-DG Galloyl Galloyl

Figure 8.8 Major theaflavins in black tea.

Table 8.3
Theaflavins in black tea
Content
Precusors Theaflavins (dry weight base)
EC + EGC TF 0.2–0.3%
EC + EGCG TF-3-G
1.0–1.5%
ECG + EGC TF-3'-G
ECG + EGCG TF-3,3'-DG 0.6–1.2%

Theasinensins (biflavanols) could be formed from the paired condensation of

two gallocatechins, EGCG and EGC, during tea processing. Their mechanism of
formation is shown in figure 8.7. Theasinensins are a group of colorless substances,
and are reactive compounds that may rearrange to form other undefined polymers.13
Thearubigins are the major oxidation products of catechins during fermentation.
They could also be partly produced by further oxidation of theaflavins. The possible
formation mechanism of thearubigins (TRs) is shown in figure 8.10.14 It is notewor-
thy that up to 75% of catechins in tea leaves may ultimately find their way into
thearubigins during black tea processing. Thearubigins are heterogeneous groups of
orange-brown phenolic pigments. However, due to the difficulty encountered in their
separation, the chemistry of thearubigin is poorly understood.15 Spectra of TRs by
analysis of matrix-assisted laser desorption ionization (MALDI) with a linear time-
of-flight (TOF) mass spectrometry have shown that some TRs are polymers of cat-
echins in which the 3-OH group is more or less esterified with gallic acid; others are
derivatives of dimers of catechins for which the gallate moiety has been condensed
with “B-ring” catechins to form a benzotropolone skeleton.16

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 Antioxidant Properties and Mechanisms of Tea Polyphenols 139

O O
OH
O
OH
[O] X O

X OH
X
OH O
OH O
X
OH
O
[O]
[O] -CO2

X OH OH
X O

X OH

X OH

Figure 8.9 Scheme of the formation mechanism of theaflavins. (From Sang, S., Lambert,
J. D., Tian, S., Hong, J., Hou, Z., Ryu, J., Stark, R. E., Rosen, R. T., Huang, M. T., Yang, C. S.,
and Ho, C.-T. 2004. Enzymatic synthesis of tea theaflavin derivatives and their anti-inflam-
matory and cytotoxic activities. Bioorg. Med. Chem. 12:459–67. With permission.)

Thearubigins

PPO/O2 Thearubigins
Catechins Catechin quinones
POD/H2O2
POD/H2O2
Theaflavins Thearubigins
PPO/O2
Gallocatechins Gallocatechin quinones
POD/H2O2
Thearubigins
PPO: polyphenol oxidase; POD: peroxidase

Figure 8.10 Scheme of the possible formation mechanism of thearubigins. (From Finger,

A. 1994. In-vitro studies on the effect of polyphenol oxidase and peroxidase on the formation
of polyphenolic black tea constituents. J. Sci. Food Agric. 66:293–305. With permission.)

8.3 Antioxidant Properties and

Mechanisms of Tea Polyphenols
Free radicals are very important both in food systems and in biological systems. In
food, the process of lipid auto-oxidation and development of rancidity involves a
free radical chain mechanism proceeding via initiation, propagation, and termina-
tion steps. This lipid peroxidation process is responsible for the development of off-
flavors and undesirable chemical compounds in food. In vivo, free radical–initiated
auto-oxidation of cellular membrane lipids can lead to cellular necrosis and is an

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 140 Tea and Tea Products: Chemistry and Health-Promoting Properties

accepted important factor of a variety of pathological conditions, such as cancer,

cardiovascular disease, and even aging.17
The potential health benefits associated with tea consumption have been par-
tially attributed to the antioxidant properties of tea polyphenols.18

8.3.1 Antioxidant Properties of Tea Polyphenols

The reduction potentials of catechins have shown that gallocatechins have lower
redox potentials than simple catechins; catechins have lower redox potentials than
catechin gallates (table 8.4). The 5'-OH group in the B-ring of gallocatechins and
catechins without gallate ester into the C-ring is thought to have a better antioxidant
activity. Differences in redox potential between catechins and epicatechins were also
observed with all epi forms having significantly lower values. The reduction poten-
tials of theaflavins are similar to those of simple catechins and higher than those of
gallocatechins (table 8.4). Their potentials are similar, although gallate esterifica-
tion tends to increase the redox value. TF had the lowest first reduction potential;
however, the TF gallates have higher antioxidant activity than TF in the lipid phase,
demonstrating that redox potential is an indicator, but not a perfect one, of antioxi-
dant activity.15
The structural features thought to be responsible for antioxidant activity of tea
polyphenols are the o-dihydroxy catechol (3',4'-OH) arrangement on the B-ring,
either di- or trihydroxy-substituted catechins or flavonols, and the C2–C3 double
bond in the C-ring conjugated with a C4 carbonyl group found in flavonols. This
structure also allows electron delocalization, conferring high reactivity to quench
free radicals.18 The antioxidant activity of catechins is determined by the B-ring
catechol structure and is further enhanced in gallocatechins by the 5'-hydroxyl group
on the B-ring. Various structure-antioxidant activity studies have concluded that the

Table 8.4
Redox potentials of catechins, gallocatechins, and theaflavins
Component First redox potential vs. SCE (V)
Epigallocatechin (EGC) 0.09
Gallocatechin (GC) 0.13
Epigallocatechin gallate (EGCG) 0.14
Gallocatechin gallate (GCG) 0.15
Epicatechin (EC) 0.19
Epicatechin gallate (ECG) 0.20
Catechin (C) 0.20
Theaflavin (TF) 0.16
Theaflavin-3-gallate (TF-3-G) 0.20
Theaflavin-3'-gallate (TF-3'-G) 0.19
Theaflavin-3,3'-digallate (TF-3,3'-DG) 0.19
Note: SCE, saturated calomel electrode.
Source: Balentine, D. A., Wiseman, S. A., and Bouwens, L. C. M. 1997. The chemistry of tea
flavonoids. Crit. Rev. Food Sci. Nutr. 37:693–704. With permission.

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 Antioxidant Properties and Mechanisms of Tea Polyphenols 141

presence of a gallate group in the 3-position and a trihydroxy B-ring plays the most
important role in the free radical scavenging abilities of catechins.19–21 Antioxidant
function is also thought to be promoted by C5, C7 dihydroxylation on the A-ring.15
Generally, the A-ring is very insensitive to oxidation, and therefore unlikely to par-
ticipate directly in the antioxidant reactions. However, Zhu et al.22 reported that ring
A of catechins may also serve as an antioxidant site. The functional group of cat-
echins for antioxidant activity is shown in figure 8.11. It is worthy to note that there
is a synergistic effect on antioxidation among catechins as well as catechins with
vitamin E23 or Trolox.24
It should be pointed out that catechins may undergo auto-oxidation and behave
like prooxidants under certain conditions. A recent review by Yang et al. 25 points out
that EGCG is unstable in cell culture medium, with a half-life of less than 30 min due
to its auto-oxidation. Dimers as well as superoxide radicals and hydrogen peroxide
are formed. Many of the reported biological activities of EGCG could be caused by
these reactive oxygen species. For example, EGCG can cause the apoptosis of H661
lung cancer cells through the hydrogen peroxide produced. Reactive oxygen species
produced during auto-oxidation of EGCG could lead to the inhibition of epidermal
growth factor receptor (EGFR) phosphorylation and EGFR protein degradation in
human esophageal squamous cell carcinoma KYSE 510 cells.25

8.3.2 Antioxidant Mechanisms of Tea Polyphenols

There are three proposed mechanisms by which tea polyphenols act as antioxidants.
These mechanisms are explained in the following sections.

8.3.2.1 Scavenging Reactive Oxygen Species

Tea polyphenols have been shown to scavenge reactive oxygen species, such as
superoxide radical, singlet oxygen, hydroxyl radical, nitric oxide, nitrogen dioxide,
and peroxynitrite, which may play important roles in carcinogenesis.
Catechins can trap peroxyl radicals and thus suppress radical chain reactions and
terminate lipid peroxidation.26 Catechins also inhibit metmyoglobin-initiated per-
oxidation of low-density lipoproteins (LDLs) and the consumption of α-tocopherol.19
Among tea catechins, EGCG is most effective in reacting with most reactive oxygen

OH R = H, Catechol group;
R = OH, Pyrogallol group
OH
B
OH O
R OH
A C
Meta-5,7- O C OH Galloyl group
D
dihydroxyl group
OH O
OH

Figure 8.11 The functional group of catechins for antioxidant activity.

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 142 Tea and Tea Products: Chemistry and Health-Promoting Properties

species.18 Yang et al.27 reported that the centers of scavenging reaction of EGCG are
B-, D-, and A-rings, and each EGCG traps six superoxide anions (O2·–) or hydroxyl
radicals (·OH) in vitro as reflected in the electron spin resonance (ESR), whereas EC
only traps two free radicals. The scavenging mechanism of EGCG is as follows:

O2·– (·OH) + EGCG → H2O2 + EGCG·

5O2·– (·OH) + EGCG· → Nonradical products

A number of studies have shown that the antioxidative properties of theaflavins

manifest themselves in their abilities to scavenge reactive oxygen species and to
inhibit their generation.28 Theaflavins also show strong antioxidant activity against
lipid oxidation as detected in rabbit erythrocyte ghost system29 and rat liver homog-
enates30 against LDL oxidation in mouse macrophage cells,31 in preventing DNA
oxidative damage in cell-free systems,29 in the inhibition of xanthine oxidase and
suppression of intracellular reactive oxygen species in HL-60 cells, and through
H2O2 scavenging ability.32

8.3.2.1.1 DPPH
The DPPH (2,2-diphenyl-1-picrylhydrazyl) system offers a stable radical-generating
procedure. It is sensitive enough to detect active principles at low concentrations. The
antioxidant process of catechins is thought to be divided into the following two stages:

DPPH· + AH ↔ DPPH-H + A·
H· + X· → Nonradical materials

AH is the antioxidant, A· is the antioxidant radical, and X· is another radical

species or the same species as A·. Although the first stage is a reversible process,
the second stage is irreversible and produces stable radical termination compounds.
Structural information about these nonradical products would provide important
information about antioxidant mechanism studies.33
Sang et al.33 reported that catechin and epicatechin in the DPPH oxidant sys-
tem could be oxidized to compounds 3 and 4 and compounds 5 and 6, respectively.
From the elucidation of the chemical structures of these compounds, the antioxidant
mechanisms of catechin and epicatechin were proposed. As shown in figure 8.12, an
initial one-electron oxidation of catechin on the B-ring by a DPPH radical generates
a catechin (or epicatechin) phenoxyl radical. This phenoxyl radical can be tautomer-
ized to the corresponding o-quinone, which is then subjected to nucleophilic attack
by the reactive C-8 (or C-6) carbon of another catechin unit in a Michael-type addi-
tion reaction to the quinone B-ring to form compounds 3 and 4 or 5 and 6. This
suggests that the configuration of position 3 for catechin and epicatechin does not
affect the oxidative reaction. Both the B-ring and A-ring are the principal sites of
antioxidant activity of catechin and epicatechin in the DPPH oxidant system.33 How-
ever, the antioxidant mechanism of gallocatechin and epigallocatechin was different
from that of catechin (or epicatechin) in the DPPH oxidant system. As shown in fig-
ure 8.13, attacked with DPPH radical, epigallocatechin (or its ester) phenoxyl radical
was generated and tautomerized to o-quinone. This quionone attacks the C-2' carbon

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8082.indb 143
O O OH
OH OH HO O
OH
R2
H
R R1
H
OH OH
H
HO HO HO O
OH
R2
R1
O O OH
OH H 3: R1 = OH, R2 = H 5: R1 = H, R2 = OH
HO O OH
O H
OH HO O
OH OH
R2 DPPH R2
R1
OH R1
OH
1: R1 = OH, R2 = H 2: R1 = H, R2 = OH O OH
(+)- catechin (–)-epicatechin O OH
OH HO
H
O
H
R
OH H OH R1 R2
Antioxidant Properties and Mechanisms of Tea Polyphenols

O
O
H
OH
OH
4: R1 = OH, R2 = H 6: R1 = H, R2 = OH

Figure 8.12 Proposed scavenging mechanism of epicatechin and catechin to 2,2-diphenyl-1-picrylhydrazyl. (From Sang, S., Cheng, X., Stark, R.
E., Rosen, R. T., Yang, C. S., and Ho, C.-T. 2002. Chemical studies on antioxidant mechanism of tea catechins: Analysis of radical reaction products of
catechin and epicatechin with 2,2-diphenyl-1-picrylhydrazyl. Bioorg. Med. Chem. 10:2233–37. With permission.)
143

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8082.indb 144
144

OH

OH OH HO O
DPPH DPPH H OH OH OH

OH OH Keto-enol
OH OH tautomerism OR OH
H OH
H O OH
OH OH
O H O OH HO O
EGC OH
EGCG
OH OH
OR OH
OH OH OH
R = Galloyl Theasinensin A
R=H Theasinensin C

Figure 8.13 Proposed scavenging mechanism of epigallocatechin and epigallocatechin gallate to 2,2-diphenyl-1-picrylhydrazyl. (From Zhu, N.,
Wang, M., Wei, G. J., Lin, J. K., Yang, C. S., and Ho, C.-T. 2001. Identification of reaction products of (–)-epigallocatechin, (–)-epigallocatechin gallate
and pyrogallol with 2,2-diphenyl-1-picrylhydrazyl radical. Food Chem. 73:345–49. With permission.)
Tea and Tea Products: Chemistry and Health-Promoting Properties

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 Antioxidant Properties and Mechanisms of Tea Polyphenols 145

of another epigallocatechin (or its ester) to form theasinensin A or C. This suggests

that the trihydroxyphenyl B-ring, rather than the gallate moiety, is the active site of
antioxidant reaction in catechins. Moreover, the trihydroxyphenyl B-ring is more
active than the dihydroxyphenyl B-ring in the DPPH oxidant system.17
Jhoo et al.34 reported that the oxidation of theaflavin in either DPPH or the per-
oxidase–hydrogen peroxide model system leads to the generation of theanaphthoqui-
none as the major oxidation product. The proposed radical scavenging mechanism
of theaflavin is shown in figure 8.14. Theanaphthoquinone is formed through one-
electron oxidation. This indicates that the benzotropolone moiety of theaflavin is the
active site for scavenging of radicals.34

8.3.2.1.2 Peroxyl Radical

Sang et al.35 studied the antioxidant mechanism of epicatechin upon reaction with a
peroxyl radical generated by thermolysis of the initiator 2,2'-azo-bisisobutyronitrile
(AIBN). The progress of the reaction was thought to proceed via the following stages:

(1) Radical generation

Δ
RN = NR → 2R· + N2

2R· + 2O2 → 2ROO·

(2) Radical trapping

ROO· + AH ↔ ROOH + A·
OH O O OH– O
O O O O–
R R R R
OH
OH –e OH –e OH
+ OH
–H+ –H

R OH R O H R O O
R
Theaflavin

R OH
R OH OH
R R O–
O [O] –
OH –CO2 COO COOH
OH OH
R O
R OH
R O O
R

Figure 8.14 Proposed scavenging mechanism of theaflavin to 2,2-diphenyl-1-picrylhy-

drazyl and hydrogen peroxide. (From Jhoo, J., Lo, C., Li, S., Sang, S., Ang, C. Y. W., Heinze,
T. M., and Ho, C.-T. 2005. Stability of black tea polyphenol, theaflavin, and identification of
theanaphthoquinone as its major radical reaction product. J. Agric. Food Chem. 53:6146–50.
With permission.)

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 146 Tea and Tea Products: Chemistry and Health-Promoting Properties

(3) Radical termination

A·+ X· → Nonradical material

AIBN decomposes thermally to yield alkyl radicals (R·), which then react rap-
idly with oxygen to generate peroxyl radicals (ROO·). AH is the phenolic antioxi-
dant, A· is the antioxidant radical, and X· is another radical species or the same
species as A·. Although the second stage is a reversible process, the third stage is
irreversible and produces stable radical termination compounds. Structural infor-
mation about these nonradical products can lead to the elucidation of antioxidant
mechanisms. The chemical structures of the reaction products (compounds 2–5) of
epicatechin with alkylperoxyl radicals from AIBN in a homogeneous solution sug-
gest the antioxidant mechanisms for epicatechin illustrated in figure 8.15. Epicate-
chin is proposed to react with peroxyl radicals by a single-electron transfer followed
by deprotonation from the hydroxyl group of the B-ring to form a resonance pair.
If the reaction is initiated at the hydroxyl group of C-3', compounds 2 and 4 should
be formed; conversely, compound 5 should be formed if the reaction is initiated at
the hydroxyl group of C-4'. Compound 3 could be formed by further oxidation of
compound 4. These results suggest that the B-ring is the initial site for formation of
reaction products. Both the B-ring and A-ring exhibit antioxidant activity observed
for epicatechin in the peroxyl radical oxidant system.35
Valcic et al.36,37 studied the reaction of EGCG and EGC with peroxyl radicals
generated upon thermolysis of the azo initiator 2,2'-azobis(2,4-dimethylvaleroni-
trile) (AMVN) to produce several oxidation products. The antioxidant progress of
the reaction is similar to that of AIBN described above. The antioxidant pathway is
illustrated as figure 8.16. The B-rings of EGC and EGCG are transformed into ring-
opened unsaturated dicarboxylic acid moieties. The oxidation products also include
a seven-membered B-ring anhydride and a symmetrical dimer and an unsymmetrical
dimer. These changes occur solely in the B-ring of EGCG or EGC, and suggest that
the principal site of antioxidant reaction in EGCG and EGC is the trihydroxyphenyl
B-ring, regardless of the presence of a 3-galloyl moiety. The antioxidant mechanism
involves an initial one-electron oxidation of EGCG or EGC by a peroxyl radical that
generates a EGCG or EGC phenoxyl radical. This phenoxyl radical either reacts with
a second peroxyl radical to form an unstable AMVN adduct and cleaves to com-
pounds 3 and 8, or attacks a second EGCG or EGC molecule to form a dimer radical,
then forms an adduct with a second peroxyl radical, and cleaves and rearranges to
compounds 4, 6, and 7 with loss of an AMVN-derived alcohol fragment. Compound
3 undergoes hydrolysis and decarboxylation to produce compound 2.36,37

8.3.2.1.3 Hydrogen Peroxide

As an important oxidant, hydrogen peroxide (H2O2) can normally be produced from
many physiological sources in the aerobic environment of mammalian cells and
tissues. For example, H2O2 is generated during NADH oxidation by cell wall per-
oxidase, a process that can be stimulated by monophenolic compounds. Catechins
exhibit a strong capacity for scavenging hydroxyl radicals and suppressing cytotoxic-
ity induced by H2O2.22

8082.indb 146 6/2/08 9:56:57 AM

8082.indb 147
O OH
OH HO O
OH OH
HO O –e
OH
OH –H+ OH
OH
O OH
OH
OH OH HO
HO
1 Epicatechin
OH O
HO O
O
OH H
O OH HO
OH H HO
OH OH
HO
O HO
OH OH
4 O OH 5
OH
OH HO
O
OH HO O
[O] OH

OH 2 OH OH
O
OH O
O OH OH H
O OH O
O O O
HO O
O OH
O O
O O
OH
Antioxidant Properties and Mechanisms of Tea Polyphenols

O O
H O O O O OH
OH
OH
OH
OH OH 3
OH
OH

Figure 8.15 Scavenging mechanism of EC to peroxyl radicals. (From Sang, S., Tian, S., Wan, H., Stark, R. E., Rosen, R. T., Yang, C. S., and Ho, C.-
T. 2003. Chemical studies of the antioxidant mechanism of tea catechins: Radical reaction products of epicatechin with peroxyl radicals. Bioorg. Med.
Chem. 11:3371–78. With permission.)
147

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8082.indb 148
148

OH HO
OH O
8: R = H
3: R = G
O
HO HO OH
O OH O
O HO
OR +R'OO
OR
OH 1(EGCG): R = G O
OH
5(EGC): R = H OH O
+R'OO
R2O
+R'OO +R'OO
HO O OH
O OH OH HO
O O OH
OH OH
HO HO OH
O
O 6: R1 = R2 = H
4: R1 = R2 = G R1O
O O OH
OG OH
OH
2 HO OH
OH
O O OH G = Galloyl =
7 O
OH
HO OH OH O R' = (CH3)2CHCH2C(CN)(CH3)

Figure 8.16 Oxidation products of epigallocatechin and epigallocatechin gallate with peroxyl radicals. (Adapted from data in Valcic, S., Muders, A.,
Jacobsen, N. E., Liebler, D. C., and Timmermann, B. N. 1999. Antioxidant chemistry of green tea catechins. Identification of products of the reaction of
(–)-epigallocatechin gallate with peroxyl radicals. Chem. Res. Toxicol. 12:382–86; Valcic, S., Burr, J. A., Timmermann, B. N., and Liebler, D. C. 2000.
Antioxidant chemistry of green tea catechins. New oxidation products of (–)-epigallocatechin gallate and (–)-epigallocatechin from their reactions with
peroxyl radicals. Chem. Res. Toxicol. 13:801–10.)
Tea and Tea Products: Chemistry and Health-Promoting Properties

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 Antioxidant Properties and Mechanisms of Tea Polyphenols 149

EGC and EGCG oxidized in the H2O2 oxidant system were studied by Zhu et
al.22 It was demonstrated that the oxidation products are formed by the oxidation and
decarboxylation of the A-ring in the catechin molecule, as shown in figure 8.17. This
study provides unequivocal proof that the A-ring of EGCG and EGC may also serve
as an antioxidant site. However, the oxidant mechanism remains unclear.22 Theafla-
vin-3,3'-digallate may be oxidized with hydroxyl radicals generated by hydrogen per-
oxide and produce two A-ring fission products as illustrated in figure 8.18.38 For the
oxidant mechanism, the possible initial step is the attack by the hydroxyl radical on
the A-ring. The A-ring radical then undergoes a series of further reactions, including
cleavage of the A-ring. If the hydroxyl radical attacks the A-ring of the flavan-3-ol
connected to the benzene moiety of the benzotropolone group, 2 will be formed;
conversely, 3 should be formed if the reaction is initiated at the A-ring of the flavan-
3-ol connected to the tropolone part of the benzotropolone group. It is noteworthy
that the initial site for the formation of these two major reaction products is the A-
OH
OH

OH O
HOOC OH
OH
H2O2 OH
HOOC O
HO
O OH OH
O
OH OH
O OH
OH
OH OH
O H2O2
OH HOOC O OH
EGCG
OH
HOOC O
OH
O
OH

OH
OH
OH
OH
O OH
HOOC
HO H2O2
O OH HOOC
OH

OH
OH
EGC

Figure 8.17 Oxidation products of epigallocatechin gallate and epigallocatechin with

hydrogen peroxide. Zhu, N., Huang, T. C., Yu, Y., LaVoie, E. J., Yang, C. S., and Ho, C.-T.
2000. Identification of oxidation products of (–)-epigallocatechin gallate and (–)-epigallocat-
echin with H2O2. J. Agric. Food Chem. 48:979–81. With permission.)

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8082.indb 150
150

OH
O OH
OH OH
OH O
OH O
O OH OH
OH
O OH
OH O OH
HOOC
HO O
OH OH
O O
HO HOOC
OH O O O
+
H2O2
OH OH
HO O OH
HO
O OH O OH
OH HOOC
O
OH OH
OH OH HOOC O O
O OH OH OH
OH O O
OH OH
1
Theaflavin 3, 3'-digallate 2 3

Figure 8.18 Oxidation products of theaflavin-3,3'-digallate with hydrogen peroxide. (From Sang, S., Tian, S., Jhoo, J. W., Wang, H., Stark, R. E.,
Rosen, R. T., Yang, C. S., and Ho, C.-T. 2003. Chemical studies of the antioxidant mechanism of theaflavins: Radical reaction products of theaflavin 3,3'-
digallate with hydrogen peroxide. Tetrahedron Lett. 44:5583–87. With permission.)
Tea and Tea Products: Chemistry and Health-Promoting Properties

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 Antioxidant Properties and Mechanisms of Tea Polyphenols 151

ring, not the benzotropolone group or the gallate group, in the hydrogen peroxide
oxidant system.38

8.3.2.1.4 Superoxide Anion

Superoxide anion radicals are generated in living cells by a single-electron reduc-
tion of oxygen under physiological conditions, and play harmful roles as precursors
of more reactive oxygen species, contributing to the pathological processes of many
diseases. Studies by Nanjo et al.39 and Unno et al.40 have shown that the presence
of at least an o-dihydroxyl phenyl group and a galloyl moiety at the 3-position was
important in maintaining the scavenging ability of the superoxide anion.

8.3.2.1.5 Hydroxyl Radical

The hydroxyl radical is a highly reactive free radical (diffusion rate limited) that can
react with most living organisms. Hydroxyl radicals damage lipids, polypeptides,
proteins, and DNA.41 Hanasaki et al.42 reported that (+)-catechin and (–)-epicatechin
display a hydroxyl radical scavenging effect 100–300 times superior to that of man-
nitol, a typical hydroxyl radical scavenger. The hydroxyl radical scavenging activity
of these compounds was investigated in a photolysis of the H2O2 system. It was found
that their ability to scavenge hydroxyl radicals decreased in the order of ECG > EC
> EGCG >> EGC.43

8.3.2.1.6 Nitrite Ion

Under acidic pH, nitrite ions are converted to nitrous acid (pKa = 3.25), which
decomposes to afford a range of species according to the following equations:

NO2— + H+ ↔ HNO2
HNO2 + H+ ↔ NO+ + H2O
NO+ + NO2— ↔ N2O3

The acid-promoted reaction of EGCG and nitrite ions is initiated by one-elec-

tron oxidation of EGCG via the NO2 generated by decomposition of nitrous acid,
as detailed above. Disproportionation or further oxidation of the resulting semiqui-
none might then lead to the formation of the quinone with concomitant conversion
to nitric oxide (NO), as illustrated in figure 8.19. It is worth noting that quinone
1a, when exposed to excess nitrite ions in an acidic environment, is susceptible to
nitrosation through competing pathways involving nucleophilic sites on the A- and
B-rings. This indicates the potential role of epigallocatechin quinones in the protec-
tive effects of tea polyphenols against nitrite-induced gastric cancer.44

8.3.2.1.7 Peroxynitrite
Peroxynitrite (ONOO –) is a cytotoxic species generated by the reaction between
superoxide and nitric oxide (NO), which is a very strong oxidant and can cause
oxidation of cell membrane protein and lead to cell damage and diseases. It can also
generate hydroxyl radicals and NO2 under acidic conditions. Pannala et al.45 found
that the scavenging effect of ECG and EGCG on ONOO – was more pronounced
than that of EC and EGC. Catechins were also found to protect from peroxynitrite-

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8082.indb 152
152

OH O
O O

HO HO
EGCG O O O
O
NO2 +2H+ NOH
Path a NO
OR OR
NO+H2O OH -H+
O OH 2a OH OH
OH
O
O
HO NOH
O O
HO
O O O O
NO+ –H+ O
NO+
b Path b
OR
1a -H+
HON OR
OH O OH HON OR
NO+ O 5 O 4a
R = Galloyl = OH

OH

Figure 8.19 Proposed scavenging mechanism of epigallocatechin gallate to nitrite ions. (From Panzella, L., Manini, P., Napolitano, A., and d’Ischia,
M. 2005. The acid-promoted reaction of the green tea polyphenol epigallocatechin gallate with nitrite ions. Chem. Res. Toxicol. 18:722–29. With
permission.)
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 Antioxidant Properties and Mechanisms of Tea Polyphenols 153

induced modification of critical amino acids of apolipoprotein B-100 of LDL, which

contribute toward its surface charge.45

8.3.2.2 Chelating Metal Ions to Prevent Oxidation

Catechins can chelate metal ions, mainly Fe and Cu, which are catalysts of free radi-
cal reactions, because of their vicinal dihydroxy or trihydroxy structures, and thus
prevent the generation of free radicals. Green and black tea polyphenols reduced
cell-mediated low-density lipoprotein oxidation induced by Cu2+ in vitro, which is
proposed to contribute to the prevention of atherosclerosis and other cardiovascular
diseases.18
Guo et al.43 found that EGC, EGCG, ECG, or EC could protect synaptosomes
from the damage of lipid peroxidation in part through their iron-chelating activity.
The chelated ratios of EGC, EGCG, ECG, and EC with iron (III) were 3:2, 2:1, 2:1,
and 3:1, respectively.
Sugihara et al.46 reported the effects of EC, EGC, ECG, and EGCG on lipid per-
oxidation induced by either ferrous, copper, or vanadium ions in normal and α-lino-
lenic acid–loaded (LNA-loaded) cultured rat hepatocytes. Each catechin displayed
a marked variation in its antioxidative potency depending on the added metal ion
species. However, butylated hydroxytoluene (BHT), a typical lipid radical scavenger,
exhibited a similar antioxidative potency with all metal ions. These findings suggest
that the metal-chelating property of catechins may play a major role in determining
antioxidative activity in cultured hepatocytes.46
Both catechins and theaflavins can chelate iron (III). O’Coinceanainn et al.47
have shown that theaflavins can chelate iron (III) to form a complex, subsequently to
be oxidized to o-quinone and converted to dehydrotheaflavin, theanaphthoquinone,
and polymers, as illustrated in figure 8.20.47

8.3.2.3 Regulation Enzymes or Genes Related

to Oxidation or Antioxidation
Tea polyphenols perform their antioxidant activity through regulation enzymes or
genes related to oxidation/antioxidation. Tea polyphenols also enhance the expression
of intracellular endogenous antioxidants such as glutathione, glutathione reductase,
glutathione peroxidase, glutathione-S-reductase, catalase, and quinine reductase.48,49
Lin et al.50 found that long-term oral feeding of green tea leaves to Wistar rats resulted
in the enhancement of the activities of superoxide dismutase (SOD) activity in serum
and phase II enzyme, and glutathione S-transferase (GST) and catalase in liver. A
recent publication51 reports that Ang II- and pressure-overload-mediated cardiac
hypertrophy was attenuated by EGCG with the suppression of ROS generation and
NADPH oxidase expression. Yu et al.52 reported that tea polyphenols may regulate
antioxidant response element (ARE)–mediated phase II enzyme expression through
a mitogen-activated protein kinase C pathway. Recently, a well-documented review
demonstrated that dietary polyphenols, including tea polyphenols, could stimulate
antioxidant transcription and detoxification defense systems through ARE with
the coordination of endogenous antioxidants involving glutathione and its related
enzymes to execute their antioxidative abilities in biological systems.53 Kuzuhara et

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8082.indb 154
154

R OH R O
R O
Fe
OH 2Fe3+ O Theabenzoquinone High molecular
R O O weight oxidation
R O
1 2 R products
O
OH Fe
O
Theaflavin OH 3

OH OH
R O
H
O
O OH O OH
HO HO
OH OH R O
A
O OH O O OH Theanaphthoquinone
O
HO O O HO O O HO

R=
OH OH
O OH
OH OH
OH
C B
Dehydrotheaflavin Dehydrotheaflavin

Figure 8.20 Proposed oxidation products of theaflavin with iron. (From O’Coinceanainn, M., Bonnely, S., Baderschneider, B., and Hynes, M. J. 2004.
Reaction of iron(III) with theaflavin: Complexation and oxidative products. J. Inorg. Biol. 98:657–63. With permission.)
Tea and Tea Products: Chemistry and Health-Promoting Properties

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 Antioxidant Properties and Mechanisms of Tea Polyphenols 155

al.54 reported that EGCG could bind directly to single-stranded 18 mers of DNA and
RNA, and double-stranded (AG-CT) oligomers of various nucleotide lengths. These
indicate that tea polyphenols may regulate the expression of related genes through
direct binding.
An experiment on human leukemia cells HL-60 proved that TF-3,3'-DG,
TFMG, TF, and EGCG with TF-3,3'-DG most effectively inhibited the prooxidative
enzyme–xanthine oxidase activity, which catalyzes oxidation of hypoxanthine and
xanthine to uric acid, accompanied by oxygen reduction to superoxide radical and
hydrogen superoxide.32 Moreover, it was shown that EGCG influenced free radi-
cal generation through reduction of NADPH–cytochrome P-450 reductase activity.55
Tea polyphenols also inhibited the activity of cyclooxygenase COX-2 and 5-, 12-, and
15-lipoxygenase, enzymes participating in enzymatic lipid peroxidation in human
colon mucosa and colon tumor tissues.56 Studies on RAW 264.7 mice macrophages
revealed that theaflavins, in particular TF-3,3′-DG, effectively inhibit the activation
of transcription factor NFκB, preventing expression of an inducible nitric oxide syn-
thase (iNOS) gene in mRNA and, as a consequence, contributing to a decrease in the
synthesis of inducible nitric oxide synthase to prevent NO generation.57

8.4 Conclusions
Tea polyphenols, especially catechins and theaflavins, can execute their antioxidant
activities principally through scavenging free radicals, chelating transition metal
ions, and modulating oxidant/antioxidant enzymes or genes. The main sites of anti-
oxidant action of catechins are the catechol or pyrogallol group of the B-ring, the
meta-5,7-dihydroxyl group of the A-ring, and the galloyl group of the D-ring. The
main antioxidant sites of theaflavins are similar to those of catechins. However, the
benzotropolone skeleton of theaflavins participates in the antioxidant activity. The
main sites of antioxidant action of tea polyphenols depend on the oxidant used. Dif-
ferent oxidants can result in distinctly different oxidation products.
It should be pointed out that catechins may display prooxidant activity under
certain conditions, in particular, in the presence of copper ion (II) or ferric ion (III).
Antioxidant/prooxidant activity of polyphenols is dependent on many factors, such
as metal-reducing potential, chelating behavior, pH, solubility characteristics, bio-
availability, and stability in tissues and cells.58
Future studies will need to clarify further the antioxidant mechanisms of tea
polyphenols, especially thearubigins.

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