Antioxidant effects of resveratrol and its analogues against the free-radical-induced peroxidation of linoleic acid in micelles

Article abstract of DOI:10.1002/1521-3765(20020916)8:18<4191::AID-CHEM4191>3.0.CO;2-S

The antioxidant effect of resveratrol (3,4′,5-trihydroxy-trans-stilbene) and its analogues, that is, 4-hydroxy-trans-stilbene (4-HS), 3,5-dihydroxy-trans-stilbene (3,5-DHS), 4,4′-dihydroxy-trans-stilbene (4,4′-DHS). 3,4-dihydroxy-trans-stilbene (3,4-DHS), 3,4,5-trihydroxy-trans-stilbene (3,4,5-THS) and 3,4,4′-trihydroxy-trans-stilbene (3,4,4′-THS), against the peroxidation of linoleic acid has been studied in sodium dodecyl sulfate (SDS) and cetyltrimethyl ammonium bromide (CTAB) micelles. The peroxidation was initiated thermally by a water-soluble azo initiator 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), and the reaction kinetics were studied by monitoring the formation of linoleic acid hydroperoxides. The synergistic antioxidant effect of these compounds with atocopherol (vitamin E) was also studied by following the decay kinetics of atocopherol and the reaction intermediate, the a-tocopheroxyl radical. Kinetic analysis of the antioxidant process demonstrates that these compounds are effective antioxidants in micelles used either alone or in combination with atocopherol. The antioxidative action involves trapping the propagating lipid peroxyl radical and reducing the atocopheroxyl radical to regenerate atocopherol. It was found that the antioxidant activity of resveratrol analogues depends significantly on the position of the hydroxyl groups, the oxidation potential of the molecule and the reaction medium. Molecules with ortho-dihydroxyl and/or para-hydroxyl functionalities possess high activity.

Full text of DOI:10.1002/1521-3765(20020916)8:18<4191::AID-CHEM4191>3.0.CO;2-S

FULL PAPER
Antioxidant Effects of Resveratrol and its Analogues against the
Free-Radical-Induced Peroxidation of Linoleic Acid in Micelles
Jian-Guo Fang, Man Lu, Zhi-Hua Chen, Hui-He Zhu, Yan Li, Li Yang,
Long-Min Wu, and Zhong-Li Liu*[
a]
Abstract: The antioxidant effect of res-
veratrol (3,4',5-trihydroxy-trans-stil-
bene) and its analogues, that is, 4-hy-
droxy-trans-stilbene (4-HS), 3,5-dihy-
droxy-trans-stilbene (3,5-DHS), 4,4'-
tor
2,2'-azobis(2-methylpropionami-
onstrates that these compounds are
effective antioxidants in micelles used
either alone or in combination with a-
tocopherol. The antioxidative action in-
volves trapping the propagating lipid
peroxyl radical and reducing the a-
tocopheroxyl radical to regenerate a-
tocopherol. It was found that the anti-
oxidant activity of resveratrol analogues
depends significantly on the position of
the hydroxyl groups, the oxidation po-
tential of the molecule and the reaction
medium. Molecules with ortho-dihy-
droxyl and/or para-hydroxyl functional-
ities possess high activity.
dine) dihydrochloride (AAPH), and
the reaction kinetics were studied by
monitoring the formation of linoleic acid
hydroperoxides. The synergistic antiox-
idant effect of these compounds with a-
tocopherol (vitamin E) was also studied
by following the decay kinetics of a-
tocopherol and the reaction intermedi-
ate, the a-tocopheroxyl radical. Kinetic
analysis of the antioxidant process dem-
dihydroxy-trans-stilbene
(4,4'-DHS),
,4-dihydroxy-trans-stilbene (3,4-DHS),
,4,5-trihydroxy-trans-stilbene (3,4,5-
3
3
THS) and 3,4,4'-trihydroxy-trans-stil-
bene (3,4,4'-THS), against the peroxida-
tion of linoleic acid has been studied in
sodiumdodecyl sulfate (SDS) and cetyl-
trimethyl ammonium bromide (CTAB)
micelles. The peroxidation was initiated
thermally by a water-soluble azo initia-
Keywords: antioxidation ¥ kinetics
¥
lipids ¥ peroxidation ¥ resveratrol
sclerosis and ageing.[10±12] Resveratrol has been reported to be
a good antioxidant against the peroxidation of low-density
Introduction
[6]
[7]
Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a naturally
occurring phytoalexin present in grapes and other plants. It
has been suggested that its presence in red wine with
concentrations ranging between 0.1 and 15 mgL
lipoprotein (LDL) and liposomes, a potent inhibitor of
[8]
lipoxygenase, and able to protect rat heart fromischaemia
reperfusion injury.[ These facts, coupled with our recent
findings of the antioxidant synergismof vitamin E with green-
±]
À1[1]
is linked
13]
[14]
[15]
to the low incidence of heart diseases in some regions of
France–the so-called ™French paradox∫, that is, that despite a
high fat intake, mortality from coronary heart disease is lower
due to the regular drinking of wine.[ In addition, resveratrol
has been shown to possess cancer chemopreventive activi-
tea polyphenols,[ coumarins and b-carotene, motivated
us to study the antioxidative behaviour of resveratrol and its
analogues, putting emphasis on the structure ± activity rela-
tionship of these compounds. We report herein kinetic and
mechanistic studies on the antioxidation reaction of resvera-
trol and related trans-stilbene analogues, that is 4-hydroxy-
trans-stilbene (4-HS), 3,5-dihydroxy-trans-stilbene (3,5-
DHS), 4,4'-dihydroxy-trans-stilbene (4,4'-DHS), 3,4-dihy-
droxy-trans-stilbene (3,4-DHS), 3,4,5-trihydroxy-trans-stil-
bene (3,4,5-THS) and 3,4,4'-trihydroxy-trans-stilbene (3,4,4'-
THS) on the peroxidation of linoleic acid. The peroxidation
was initiated thermally at physiological temperature by a
water-soluble azo initiator 2,2'-azobis(methylpropionami-
dine) dihydrochloride (AAPH) and conducted in sodium
dodecyl sulfate (SDS) and cetyl trimethylammonium (CTAB)
micelles to mimic the microenvironment of biomembranes.
The interaction of these compounds with a-tocopherol (TOH,
vitamin E) was also investigated.
2]
[
3±4]
ty.
Therefore, the past few years have witnessed intense
research devoted to the biological activity, especially the
antioxidative activity, of this compound,
induced peroxidation of membrane lipids and oxidative
damage of DNA are considered to be associated with a wide
variety of chronic health problems, such as cancer, athero-
[5±±]
since free-radical-
[
a] Prof. Z.-L. Liu, Dr. J.-G. Fang, Dr. M. Lu
Dr. Z.-H. Chen, H.-H. Zhu, Y. Li, Prof. L. Yang, Prof. L.-M. Wu
National Laboratory of Applied Organic Chemistry
Lanzhou University, Lanzhou, Gansu 730000 (China)
Fax : (‡86)±31-8625657
E-mail: liuzl@lzu.edu.cn
Chem. Eur. J. 2002, 8, No. 18
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4191

Z.-L. Liu et al.
FULL PAPER
HO
micelles is shown in Figure 1. It can be seen from the
figure that, upon AAPH initiation, the concentration of
the hydroperoxides increased quickly and linearly with time
in the absence of antioxidants; this demonstrated the fast
HO
HO
OH
trans-resveratrol
4-HS
HO
HO
OH
HO
3,5-DHS
4,4'-DHS
HO
HO
HO
HO
HO
3,4-DHS
3,4,5-THS
CH3
HO
HO
CH3
+
+
-
-
Cl NH2=C-C-N=N-C-C=NH2 Cl
Figure 1. Formation of total hydroperoxides (LOOH) during the perox-
idation of linoleic acid (LH) in SDS (0.1 molL ) micelles at pH 7.5 and
À1
CH3
NH2
OH
H2N
H3C
378C, initiated with AAPH. [LH] ˆ 15.2 mmolLÀ1
,
[AAPH] ˆ
0
0
À1
À1
6
.3 mmolL , [ArOH]
0
ˆ 11.2 mmolL . Uninhibited peroxidation (a) or
3
,4,4'-THS
AAPH
inhibited with b) resveratrol, c) 4-HS, d) 3,5-DHS, e) 4,4'-DHS, f) 3,4-DHS,
g) 3,4,5-THS, h) 3,4,4'-THS.
CH3
HO
H3C
CH3
CH3
CH3
CH3
peroxidation of the substrate. The slope of this line corre-
O
CH3
sponds to the rate of propagation, R . The peroxides×
CH3
p
TOH
formation was remarkably inhibited by the addition of
resveratrol and its analogues during the so-called ™inhibition
period∫ (tinh) or induction period. After the inhibition period,
the rate of hydroperoxide formation increased to close to the
original rate of propagation; this corresponded to the
exhaustion of the antioxidant. During the inhibition period,
the concentration of the hydroperoxides also increased
approximately linearly with time, and the slope of this line
was designated Rinh, which also reflects the antioxidative
potential of the antioxidant. Similar results were obtained in
CTAB micelles (Figure 2), but the kinetic parameters and the
Results and Discussion
Inhibition of linoleic acid peroxidation by resveratrol and its
analogues in micelles: Peroxidation of linoleic acid or its
esters gives different hydroperoxides depending on the
[
16]
reaction conditions.
Hydroperoxide substitution at the
C-± or C-13 positions produces either trans,trans or cis,trans
conjugated dienes, which are the major products in the
absence of antioxidants or in the presence of only small
amount of antioxidants, for example, millimolar concentra-
tions of a-tocopherol.[
16a,b]
It was found recently that these
conjugated dienes were formed from the rapid b-scission of
the primarily formed bis-allylic 11-peroxyl radical,[
16c,d]
and
that the kinetically controlled product, the nonconjugated 11-
substituted hydroperoxide, might become the major product
in the presence of high concentrations of antioxidant, for
example, molar concentrations of a-tocopherol.[
16d]
The
present experiment used very small amounts of antioxidants
micromolar a-tocopherol and/or resveratrol analogues),
(
hence the production of the nonconjugated 11-hydroperoxide
was negligible, and the conjugated hydroperoxides were the
predominant products, which showed characteristic ultravio-
[17]
let absorption at 235 nm
that was used to monitor the
formation of the total hydroperoxides formed during the
peroxidation after separation of the reaction mixture by high-
performance liquid chromatography (HPLC). A set of
representative kinetic curves of the total hydroperoxides
formation during the peroxidation of linoleic acid in SDS
Figure 2. Formation of total hydroperoxides (LOOH) during the perox-
idation of linoleic acid (LH) in CTAB (0.015 molL ) micelles at pH 7.5
À1
À1
and 378C, initiated with AAPH. [LH]
0
ˆ 15.2 mmolL
,
[AAPH]
0
ˆ
À1
À1
6
.3 mmolL , [ArOH]
0
ˆ 11.2 mmolL . Uninhibited peroxidation (a) or
inhibited with b) resveratrol, c) 4-HS, d) 3,5-DHS, e) 4,4'-DHS, f) 3,4-DHS,
g) 3,4,5-THS, h) 3,4,4'-THS.
4192
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Chem. Eur. J. 2002, 8, No. 18

Antioxidant Effects of Resveratrol
41±1±41±8
relative effectiveness of the antioxidants in the two micelles
were appreciably different. The details will be discussed in
following sections.
TOH remarkably prolonged the inhibition period of the latter
and showed a synergistic antioxidation effect, that is, the
inhibition time when the two antioxidants were used in
combination was significantly longer than the sum of the
inhibition times when they were used individually as illus-
trated in Figures 3 and 4. 4-HS and 3,5-DHS could also
prolong the inhibition time of TOH when they were used
together with the latter in both SDS and CTAB micelles, but
the effect was only additive, that is, the inhibition time when
the two antioxidants were used in combination was the sum of
the inhibition times when they were used individually (Figures
not shown). The results are summarized in Table 2, later.
The antioxidant effect of resveratrol and its analogues in the
presence of a-tocopherol: a-Tocopherol (TOH), the most
abundant and active formof vitamin E, is well known and the
principal lipid-soluble chain-breaking antioxidant in plasma
and erythrocytes.[ Its synergistic antioxidative effect with
18]
[1±]
other antioxidants, such as l-ascorbic acid (vitamin C) and
green-tea polyphenols,[ has been well documented. There-
fore, it is interesting to see if TOH can also interact
synergistically with resveratrol and its analogues. In both
SDS and CTAB micelles TOH showed typical antioxidant
13]
Consumption of a-tocopherol: In order to rationalize the
mechanism of the antioxidant synergism of a-tocopherol and
the resveratrol analogues, the decay of a-tocopherol was
studied by HPLC separation of the reaction mixture, followed
by electrochemical determination of a-tocopherol. Represen-
tative results are illustrated in Figure 5. It was found that the
decay of a-tocopherol was approximately linear in the
absence of 3,4,4'-THS in the two micelles (lines a and b in
behaviour against linoleic acid peroxidation (line b in
Figures 3 and 4), as reported previously.[
13±15]
Addition of
resveratrol, 3,4-DHS, 3,4,4'-THS or 3,4,5,-THS together with
Figure 3. Formation of total hydroperoxides (LOOH) during the perox-
À1
idation of linoleic acid (LH) in SDS (0.1 molL ) micelle at pH 7.5 and
À1
3
6
78C, initiated with AAPH. [LH]
0
ˆ 15.2 mmolL
,
[AAPH]
0
ˆ
À1
À1
À1
.3 mmolL , [TOH]
0
ˆ 5 mmolL , [ArOH]
0
ˆ 11.2 mmolL . Uninhibited
peroxidation (a) or inhibited with b) TOH, c) TOH ‡ resveratrol, d) TOH
Figure 5. Consumption of a-tocopherol during the inhibition of linoleic
acid peroxidation in micelles at pH 7.5 and 378C, initiated with AAPH and
‡
3,4,5-THS, e) TOH ‡ 3,4,4'-THS.
À1
inhibited by TOH and/or 3,4,4'-THS (ArOH). [LH]
0
ˆ 15.2 mmolL
,
À1
À1
À1
[
AAPH]
0
ˆ 6.3 mmolL , [ArOH]
0
ˆ 11.2 mmolL , [TOH]
0
ˆ 5 mmolL
.
)
)
À1
À1
a) Decay of TOH in the absence of 3,4,4'-THS in CTAB (0.015 molL
micelle, b) decay of TOH in the absence of 3,4.4'-THS in SDS (0.1 molL
micelle, c) decay of TOH in the presence of 3,4,4'-THS in CTAB micelle,
d) decay of TOH in the presence of 3,4,4'-THS in SDS micelle.
Figure 5), in accordance with the kinetic demand for anti-
oxidation reactions [Eq. (4), vide infra]. When 3,4,4'-THS was
added, however, the decay of a-tocopherol became much
slower before most of 3,4,4'-THS was exhausted (lines c and
d). 3,4-DHS and 3,4,5-THS in both micelles, and resveratrol in
the CTAB micelle showed a similar effect upon the decay of
a-tocopherol, while 4-HS and 3,5-DHS showed no effect
(
3
Figures not shown). These results suggest that resveratrol,
,4-DHS, 3,4,4'-THS and 3,4,5-THS may be able to reduce the
Figure 4. Formation of total hydroperoxides (LOOH) during the perox-
idation of linoleic acid (LH) in CTAB (0.015 molL ) micelle at pH 7.5 and
a-tocopheroxyl radical to regenerate a-tocopherol and,
hence, maintain the concentration of a-tocopherol in the
reaction system. Similar a-tocopherol regeneration reactions
À1
À1
3
6
78C, initiated with AAPH. [LH]
0
ˆ 15.2 mmolL
,
[AAPH]
0
ˆ
À1
À1
À1
.3 mmolL , [TOH]
0
ˆ 5 mmolL , [ArOH]
0
ˆ 11.2 mmolL . Uninhibited
[1±]
by vitamin C
and green-tea polyphenols have been
peroxidation (a) or inhibited with b) TOH, c) TOH ‡ resveratrol, d) TOH
3,4,5-THS, e) TOH ‡ 3,4,4'-THS.
reported previously.[
13]
‡
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Z.-L. Liu et al.
FULL PAPER
Direct determination of the rate of the a-tocopherol regen-
eration reaction: The a-tocopheroxyl radical (TO ) is more
constant versus the concentration of resveratrol gave a
straight line fromwhich the bi mo lecular rate constant
.
.
persistent in micelles than in homogeneous solutions. The rate
between TO and resveratrol [Eq. (±), vide infra] could be
.
.
constants of the bimolecular self-reaction of TO were
obtained. 3,4-DHS reacted with TO much faster than
3
À1 À1
reported to be 3 Â 10 m
s
in benzene/di-tert-butyl perox-
resveratrol (line c in Figure 7). The rate constants for the a-
tocopherol regeneration reaction of resveratrol and 3,4-DHS
[
20]
À1 À1
[15a]
ide and 15m
s
in CTAB micelles,
respectively. They
2
2
À1 À1
correspond to half-lives of 11 seconds and 38 minutes in the
homogeneous solution and the micelle, respectively, taking
were determined to be 0.23 Â 10 and 3.0 Â 10 m
s
respec-
tively in CTAB micelles. These EPR experiments confirm
unambiguously that the antioxidant synergism of a-tocopher-
ol with the resveratrol analogues is due to the a-tocopherol
regeneration reaction by the latter.
.
the initial concentration of TO as 30 mmol. Therefore, the
.
reaction kinetics of TO could be easily determined in micelles
by using stopped-flow electron paramagnetic resonance
(
EPR) spectroscopy[21] at ambient temperature. Figure 6
.
shows the EPR spectrumof TO recorded in CTAB micelles.
Addition of resveratrol through a fast stopped-flow device
remarkably increased the decay of TO , which was found to
be pseudo-first order in the presence of a large excess of
resveratrol (line b in Figure 7). Plotting this first-order rate
Electrochemistry of resveratrol and its analogues: The
electrochemistry of resveratrol and its analogues was studied
by cyclic voltammetry in both SDS and CTAB micelles. It was
found that resveratrol, 4-HS and 3,5-DHS showed irreversible
cyclic voltammograms with higher oxidation potentials, while
[21]
.
3
,4-DHS, 4,4'-DHS, 3,4,5-THS and 3,4,4'-THS showed rever-
sible cyclic voltammograms with lower oxidation potentials.
The oxidation potentials are listed in Table 1.
Kinetics and mechanism: It has been proved that the reaction
kinetics of lipid peroxidation in micelles and biomembranes
follow the same rate law as that in homogenous solutions.[
The kinetics of linoleic acid (LH) peroxidation initiated by
azo-compounds and its inhibition by chain-breaking antiox-
22]
idants (AH) have been discussed in detail in our previous
papers.[
13±14]
The rate of propagation (R ) and the rate of
p
peroxide formation in the inhibition period (Rinh) are given by
Equations (1) and (2), respectively.
)1/2]R
1/2
[LH]
(1)
(2)
d[LOOH]/dt ˆ R
p
ˆ [k
p
/(2k
t
i
R
inh ˆ k
p i
R [LH]/(nkinh [AH])
here k , kt and kinh are rate constants for the chain
p
propagation, chain termination and chain inhibition by
antioxidants, respectively, and R is the apparent rate of chain
i
initiation, which can be obtained by measuring the inhibition
period or decay of the antioxidant (AH), [Eqs. (3) and (4),
respectively].
.
Figure 6. EPR Spectra of the a-tocopheroxyl radical (TO ) recorded in
À1
CTAB (15 mmolL ) micelles at pH 7.4 and room temperature in air. The
TO was generated by oxidizing TOH (1 mmolL ) with PbO
[13]
.
À1
2
. The initial
.
À1
concentration of TO was 28 mmolL
.
R
R
i
i
ˆ n[AH]
0
/tinh
(3)
(4)
ˆ Ànd[AH]/dt
Here n is the stoichiometric factor that designates the
number of peroxyl radicals trapped by each antioxidant
molecule. Since the n value of a-tocopherol is generally
assumed to be 2,[ the R value can be determined from the
22]
i
inhibition period or the decay rate of a-tocopherol.
The kinetic chain length (kcl) defines the number of chain
propagations initiated by each initiating radical and is given
by Equations (5) and (6) for uninhibited and inhibited
peroxidation respectively. The kinetic parameters deduced
fromFigures 1 and 2 are listed in Tables 1 and 2, respectively.
À1
Figure 7. The decay of a-tocopheroxyl radicals in CTAB (15 mmolL
)
micelles at pH 7.4 and room temperature in air. a) intrinsic decay, b) in the
kcl
p
ˆ R
p
/R
i
(5)
(6)
À1
presence of resveratrol (0.78 mmolL ), c) in the presence of 3,4-DHS
À1
kclinh ˆ Rinh/Ri
(
0.13 mmolL ).
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Chem. Eur. J. 2002, 8, No. 18

Antioxidant Effects of Resveratrol
41±1±41±8
Table 1. Inhibition of AAPH-initiated peroxidation of linoleic acid by resveratrol and its analogues in micelles.[a,b]
Micelle
ArOH
R
p
R
inh
À8
t
inh
k
inh
4
n
kcl
p
kclinh
E
pa
[V vs. SCE]
À8
À3 À1
À3 À1
3
3
À1 À1
[10 moldm
s
]
[10 moldm
s
]
[10 s]
[10 dm mol
s
]
SDS
none
resveratrol
8.3
±.0
±.2
7.8
8.0
8.2
8.7
6.8
16.8
16.0
1±.8
15.6
14.8
14.0
16.5
15.5
26.8
2±.1
2±.7
25.1
25.8
26.4
28.1
21.±
20.4
1±.3
23.±
18.8
17.8
16.±
1±.±
18.7
2.8
4.0
3.8
2.6
0.8
1.5
ꢀ 0
3.1
2.1
1.5
3.2
4.±
2.4
7.2
1.3
1.2
1.7
1.4
2.±
3.1
0.8
0.6
0.4
0.±
1.4
0.7
2.0
±.0
12.±
12.2
8.4
0.62
0.64
0.85
0.40
0.34
0.24
0.32
4
3
4
3
3
3
-HS
,5-DHS
,4'-DHS
,4-DHS
,4,5-THS
,4,4'-THS
2.6
4.8
[
c]
[c]
CTAB
none
resveratrol
2.7
3.2
4.2
2.8
1.6
1.6
ꢀ 0
2.7
1.7
2.4
2.6
4.1
2.±
5.1
0.7
1.0
0.5
0.8
0.±
1.3
2.0
1.2
1.8
1.±
3.0
2.1
3.8
3.2
3.8
5.1
3.4
1.±
0.67
0.66
0.7±
0.43
0.36
0.23
0.34
4
3
4
3
3
3
-HS
,5-DHS
,4'-DHS
,4-DHS
,4,5-THS
,4,4'-THS
1.±
[
c]
[c]
[
a] The reaction conditions and the initial concentration of the substrates are the same as described in the legends of Figures 1 and 2 for reactions conducted
in SDS and CTAB micelles respectively. Data are the average of three determinations that were reproducible with a deviation of less than Æ10%. [b] Taking
À1 À1
R
i
as 3.1 and 8.3 nmolL
s
in SDS and CTAB micelles, respectively, see text. [c] Could not be calculated because Rinh is approximately zero.
It can be seen fromFigures 1 and 2 and fromTable 1 that
respectively, see Table 2) and to those of green-tea polyphe-
4
À1 À1
[13b]
resveratrol and its analogues (ArOHs) behave well as chain-
breaking antioxidants against AAPH-induced linoleic acid
peroxidation in both SDS and CTAB micelles. All of them
produced a clear inhibition period in which the rate of
propagation and the kinetic chain length are remarkably
reduced; this demonstrates that they are able to trap the
nols (0.3 ± 3.7 Â 10 m
s
in micelles).
It can also be seen
that the antioxidative activities of 3,4-DHS, 3,4,5-THS and
3,4,4'-THS, that is, the molecules bearing ortho-dihydroxyl
functionality, are appreciably higher than those of resveratrol
and molecules bearing no such functionality. This can be
understood because the ortho-hydroxyl phenoxyl radical, the
oxidation intermediate for these three more active species, is
more stable due to the intramolecular hydrogen bonding
.
propagating linoleic acid peroxyl radicals [LOO , Eq. (7)].
.
kinh
À! LOOH ‡ ArO.
[23]
LOO ‡ ArOH
(7)
interaction, as evidenced recently fromboth experiments
and theoretical calculations.[ The theoretical calculation
24]
The antioxidant potential of these ArOHs can be assessed
by comparing their inhibition rate constant from Equa-
tion (7), kinh, the inhibition period, tinh, or the kinetic chain
length during the inhibition time, kclinh. The kinh of ArOHs is
showed that the hydrogen bond in the ortho-OH phenoxyl
radical is approximately 4 kcalmol stronger than that in the
À1
parent catechol, and that the bond dissociation energy (BDE)
À1
of catechol is ±.1 kcalmol lower than that of phenol and
4
À1 À1
8.8 kcalmol lower than that of resorcinol.[24] In addition, it
À1
about 0.5 ± 3.1 Â 10 m s , comparable to that of a-tocopher-
4
4
À1 À1
ol (3.6 Â 10 and 2.0 Â 10 m
s
in SDS and CTAB micelles,
should be easier to further oxidize the ortho-OH phenoxyl
Table 2. Inhibition of AAPH-initiated peroxidation of linoleic acid by resveratrol and its analogues together with a-tocopherol in micelles.[a,b]
Micelle
ArOH
R
p
R
inh
À8
t
inh
k
inh
4
n'[c]
kcl
p
kclinh
SE
[%]
À8
À3 À1
À3 À1
3
3
À1 À1
[10 moldm
s
]
[10 moldm
s
]
[10 s]
[10 dm mol
s
]
SDS
TOH
esveratrol ‡ TOH
8.1
7.0
7.±
8.3
7.4
6.±
7.7
7.0
16.7
12.3
17.8
13.6
14.3
11.7
13.0
12.8
1.1
0.±
0.8
1.0
0.5
ꢁ 0
0.6
ꢁ 0
2.0
0.1
0.3
0.3
0.2
ꢁ 0
ꢁ 0
ꢁ 0
3.0
6.3
5.3
4.8
6.6
12.6
6.6
13.8
1.2
4.4
2.5
3.0
4.5
7.8
4.±
8.1
3.6
2.0
2.3
2.1
3.1
2.0
1.2
1.0
0.±
1.3
2.4
1.3
2.6
2.0
2.2
1.3
1.5
2.3
4.0
2.5
4.2
26.1
22.6
25.5
26.8
23.±
22.2
24.8
22.6
20.1
14.8
21.4
16.4
17.2
14.1
15.7
15.4
3.6
2.±
2.6
3.2
ꢁ 0
ꢁ 0
ꢁ 0
ꢁ 0
4
3
4
3
3
3
-HS ‡ TOH
,5-DHS ‡ TOH
,4'-DHS ‡ TOH
,4-DHS ‡ TOH
.4.5-THS ‡ TOH
,4,4'-THS ‡ TOH
1.6
[d]
[d]
5±.5
2.±
1.±
22.2
35.3
[d]
[d]
CTAB
TOH
2.0
10.±
6.4
2.4
0.1
0.4
0.4
esveratrol ‡ TOH
12.8
4
3
4
3
3
3
-HS ‡ TOH
ꢁ 0
ꢁ 0
18.4
47.2
1±.5
28.6
,5-DHS ‡ TOH
,4'-DHS ‡ TOH
,4-DHS ‡ TOH
,4,5-THS ‡ TOH
,4,4'-THS ‡ TOH
6.3
7.5
0.2
[
[
[
d]
d]
d]
[d]
[d]
[d]
[
a] The reaction conditions and the initial concentration of the substrates are the same as described in the legends of Figures 3 and 4 for reactions conducted
in SDS and CTAB micelles, respectively. Data are the average of three determinations that were reproducible with a deviation of less than Æ10%. [b] Taking
À3 À1
R
i
as 3.1 and 8.3 nmoldm
s
in SDS and CTAB micelles, respectively, see text. [c] n' ˆ R
i
t
inh/([ArOH]
0
0
‡ [TOH] ). [d] Could not be calculated because Rinh
is approximately zero.
Chem. Eur. J. 2002, 8, No. 18
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Z.-L. Liu et al.
FULL PAPER
radical and/or ortho-semiquinone radical anion to form the
final ortho-quinone[ (Scheme 1). The fact that the stoichio-
metric factor, n, of 3,4-DHS and 3,4,4'-THS is larger than 1
phenolic antioxidants has recently been discussed theoret-
ically.
23]
[24]
It can be seen fromFigures 3 and 4 and Table 2 that
addition of the resveratrol analogues (ArOHs) together with
a-tocopherol (TOH) significantly increases the inhibition
period of the latter. The tinh of TOH and 3,4-DHS when they
were used together in SDS micelles was approximately 60%
longer than the sumof the tinhs when the two antioxidants
were used individually, as expressed by the synergistic
(
Table 1) suggests that the second peroxyl radical is involved
.
HO
HO
LOO
LOOH
HO
.
O
LOO.
[26]
efficiency SE% [Eq. (8)].
+
H
HO
.
-
O
LOOH
t
inhꢂTOH ‡ ArOH† À ‰tinhꢂTOH† ‡ tinhꢂArOH†Š
O
SE% ˆ
 100
(8)
.
t
inhꢂTOH† ‡ tinhꢂArOH†
O
LOO.
ET
As shown in Figures 5 and 7, the antioxidant synergismof
TOH and ArOH can be rationalized by the reduction of the a-
tocopheroxyl radical by the resveratrol analogues [Eq. (±)],
which regenerates a-tocopherol. Since the rate of this TOH-
-
LOO
O
HO
.
O
O
2
À1 À1
regeneration reaction (kreg ꢀ 10 m s ) is approximately two
orders of magnitude slower than that of the antioxidation
+
LOO.
.
O
-
LOO-
O
H
4
À1 À1
reaction [Eq. (7), kinh ꢀ 10 m s ], the antioxidant synergism
can only be observed when the rate of the TOH-regeneration
reaction is high enough to compete with the antioxidation
reaction.
.
.
O
O
ET
Scheme 1. Antioxidative reaction of 3,4-DHS by hydrogen abstraction and
electron transfer.
kreg
.
TO ‡ ArOH
À! TOH ‡ ArO
(±)
in the antioxidation reaction that leads to the formation of the
corresponding ortho-quinones, as shown in the scheme. The
It can also be seen fromTables 1 and 2 that the reaction
medium exerts significant influence on the rate of initiation
and the antioxidant activity of the resveratrol analogues. The
4
'-OH group also enhanced the activity, since the 4'-OH group
can stabilize the semiquinone radical-anion intermediate by
resonance through the trans double bond. It has recently been
R values calculated fromthe inhibition period [Eq. (3)] are
i
À1 À1
3.1 and 8.3 nmolL
s
in SDS and CTAB micelles, respec-
proved that the 4'-OH is more active than the meta-dihydroxyl
Therefore, the antioxidative
activity of 3,4,4'-THS is extremely high. It completely inhibits
tively, which are in good agreement with the values of 3.0 and
groups in resveratrol.[
4, 24, 25]
8.2 nmolL s , respectively, obtained fromthe decay of TOH
À1 À1
À1
[Eq. (4)]. Taking the concentration of AAPH as 6.3 mmolL
peroxidation in both SDS and CTAB micelles and produces a
the Ri value in CTAB micelles corresponds to 1.3 Â
3
À6
À1
longer inhibition period than a-tocopherol (7.2 Â 10 and
10 [AAPH]s ; this is in good agreement with the previously
3
À1
[27]
5
.1 Â 10 s for 11.2 mmolL of 3,4,4'-THS in SDS and CTAB
reported value in liposomal dispersions. However, the Ri
micelles respectively, in comparison with the inhibition period
value of AAPH in SDS micelles is appreciably smaller than
that in CTAB micelles. This can be understood because
AAPH is positively charged, hence it is prone to being
adsorbed onto the surface of SDS micelles; this in turn
reduces the effective initiation, due to the cage effect. On the
other hand, the inhibition rate constant, kinh, of a-tocopherol
and resveratrol analogues is higher in SDS than in CTAB
micelles. This is due to the fact that lipid peroxyl radicals are
polar (dipole moment of ca. 2.6 Debye) and electrophilic.[
Thus, they should move to the surface of micelles and be
subject to intermicellar diffusion[ more quickly in SDS than
in CTAB micelles, so as to react with the antioxidant whose
reactive phenoxyl functional group resides on the surface of
the micelle.[
3
3
À1
of 3.0 Â 10 and 1.2 Â 10 s for 5.0 mmolL of a-tocopherol in
SDS and CTAB, respectively).
It is worth noting that the antioxidant activity of resveratrol
analogues is correlated with the electrochemical behaviour of
the molecule. Molecules with lower oxidation potentials and
reversible cyclic voltammograms, that is, 3,4-DHS, 4,4'-DHS,
3,4,4'-THS and 3,4,5-THS, exhibit higher activity, while
22a]
molecules with higher oxidation potentials and irreversible
cyclic voltammograms, that is, 4-HS, 3,5-DHS and resveratrol,
are less active. This correlation between the activity and the
oxidation potential of the molecules suggests that electron-
transfer antioxidation might take place simultaneously with a
direct hydrogen-abstraction reaction, as exemplified in
Scheme 1. It is well known that phenoxides undergo electron
transfer oxidation more easily to produce relatively stable
phenoxide radical anions in alkaline media. Resveratrol, with
a pKa1 of 6.4,[ partially dissociates under our experimental
conditions (pH 7.4); this makes the electron-transfer reaction
feasible. Cooperation between hydrogen-abstraction and
electron-transfer processes in antioxidation reactions by
28]
18]
In conclusion, this work demonstrates that resveratrol and
its analogues, that is, 4-HS, 3,5-DHS, 4,4'-DHS, 3,4-DHS,
3,4,5-THS and 3,4,4'-THS, are effective antioxidants against
linoleic acid peroxidation in SDS and CTAB micelles. The
antioxidative action involves trapping the propagating per-
25]
.
oxyl radicals (LOO ) on the surface of the micelle and
regenerating a-tocopherol (TOH) by reducing the a-toco-
4196
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Chem. Eur. J. 2002, 8, No. 18

Antioxidant Effects of Resveratrol
41±1±41±8
.
pheroxyl radical (TO ) as depicted in Scheme 2. The obser-
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[
vation that trans-stilbene compounds bearing ortho-dihydrox-
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higher antioxidant activity than the ones bearing no such
functionalities gives us useful information for antioxidant
drug design.
3
8
ROO.
.
R
O2
L.
LH
O2
[
4] L. A. Stivala, M.Savio, P. Carafoli, F. Perucca, L. Bianchi, G. Maga, L.
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LH
LOO
.
ArO.
TOH
ArOH
ArO.
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2
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TO
ArOH
2
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3
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[
[
8
Experimental Section
1
Materials: Resveratrol and its analogues, that is, 4-HS, 3,5-DHS, 4,4'-DHS,
3
,4 -DHS, 3,4,5-THS and 3,4,4'-THS, were prepared according to the
[
2±±30]
available procedures,
and their structures and purity confirmed by MS,
5
4± ± 555.
1
H NMR spectroscopy and HPLC. Linoleic acid (Sigma, Chromatographic
pure), dl-a-tocopherol (Merk, Biochemical reagent, >±±.±%) and 2,2'-
azobis(2-methylpropionamidine) dihydrochloride (AAPH; Aldrich) were
kept under nitrogen in a refrigerator and used as received.
[
[
10] L. J. Marnett, Carcinogenesis 2000, 21, 361 ± 370.
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Medicine, 3rd ed., Oxford University Press, Oxford, 1999.
[
[
12] H. Esterbauer, P. Ramos, Rev. Physiol. Biochem. 1995, 127, 31 ±
Determination of linoleic acid hydroperoxides: Aliquots of the reaction
mixture were taken out of an open vessel at appropriate time intervals and
subjected to high performance liquid chromatography (HPLC) analysis on
a Gilson liquid chromatograph with a ZORBAXODS reversed-phase
column (6 Â 250 mm, Du Pont instruments), then eluted with methanol/
6
4.
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Perkin Trans. 2, 1998, ±11 ± ±15; b) B. Zhou, Z. S. Jia, Z. H. Chen, L.
Yang, L. M. Wu, Z. L. Liu, J. Chem. Soc. Perkin Trans. 2, 2000, 785 ±
7
±1; c) Z. H. Chen, B. Zhou, L. Yang, L. M. Wu, Z. L. Liu, J. Chem.
À1
propan-2-ol (3:1, v/v). The flow rate was set at 1.0 mLmin . A Gilson116
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Yang, Z. L. Liu, Chem. Phys. Lipids 2000, 106, 53 ± 63.
UV detector was used to monitor the total linoleic acid hydroperoxides at
35 nm. Every determination was repeated three times, and the exper-
imental deviations were within Æ10%.
Determination of a-tocopherol: The procedure was the same as described
above for determination of linoleic acid hydroperoxides, except that a
Gilson142 electrochemical detector set at ‡700 mV vs. SCE was used for
monitoring TOH. The column was eluted with methanol/propan-2-ol/
formic acid (80:20:1, v/v/v) containing sodium perchlorate (50 mmolL ) as
supporting electrolyte.
2
[
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±
6± ± ±74; b) Z. Q. Liu, W. Yu, Z. L. Liu, Chem. Phys. Lipids 1999, 103,
125 ± 135.
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Chapter 21, pp. 1±6 ± 20±.
À1
Determination of a-tocopheroxyl radical: EPR measurements were carried
out on a Bruker ER200D spectrometer operated in the X-band with
1
0
00 kHz modulation, a modulation amplitude of 0.25 mT, time constant of
.2 s and microwave power of 25 mW. A flat quartz flow cell (0.4 Â 5.5 Â
0 mm) was used for the stopped-flow determination of the reaction
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15b]
The a-tocopheroxyl radical was
À1
generated by vigorously stirring a-tocopherol (1 mmolL ) and excess
lead oxide with a Vortex mixer for 3 min in CTAB (15 mmolL ) micelles
at pH 7.4 and roomtemperature.
À1
2
8±.
Determination of oxidation potentials: The oxidation potentials of the
ArOHs were determined on a PAR173 potentiostat with a glassy carbon
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as described previously.[
32]
The potential was recorded relative to a
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6
[
Acknowledgement
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23, 1173 ± 1183.
1
We thank the National Natural Science Foundation of China (NSFC) for
financial support (Grant Nos. 2±832040 and 20172025).
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7± ± 8±.
Chem. Eur. J. 2002, 8, No. 18
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0±47-653±/02/0818-41±7 $ 20.00+.50/0
4197

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FULL PAPER
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Received: February 14, 2002 [F3874]
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0±47-653±/02/0818-41±8 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 18