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�-
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-stil-
bene (3,4,4�-THS), against the peroxida-
tion of linoleic acid has been studied in
sodium dodecyl sulfate (SDS) and cetyl-
trimethyl ammonium bromide (CTAB)
micelles. The peroxidation was initiated
thermally by a water-soluble azo initia-
tor 2,2�-azobis(2-methylpropionami-
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 �-
tocopherol (vitamin E) was also studied
by following the decay kinetics of �-
tocopherol and the reaction intermedi-
ate, the �-tocopheroxyl radical. Kinetic
analysis of the antioxidant process dem-
onstrates that these compounds are
effective antioxidants in micelles used
either alone or in combination with �-
tocopherol. The antioxidative action in-
volves trapping the propagating lipid
peroxyl radical and reducing the �-
tocopheroxyl radical to regenerate �-
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.
Keywords: antioxidation ¥ kinetics
¥ lipids ¥ peroxidation ¥ resveratrol
Introduction
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�1[1] is linked
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.[2] In addition, resveratrol
has been shown to possess cancer chemopreventive activi-
ty.[3±4] Therefore, the past few years have witnessed intense
research devoted to the biological activity, especially the
antioxidative activity, of this compound,[5±9] since free-radical-
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-
sclerosis and ageing.[10±12] Resveratrol has been reported to be
a good antioxidant against the peroxidation of low-density
lipoprotein (LDL)[6] and liposomes,[7] a potent inhibitor of
lipoxygenase,[8] and able to protect rat heart from ischaemia
reperfusion injury.[9] These facts, coupled with our recent
findings of the antioxidant synergism of vitamin E with green-
tea polyphenols,[13] coumarins[14] and �-carotene,[15] 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 �-tocopherol (TOH,
vitamin E) was also investigated.
[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)931-8625657
E-mail : liuzl@lzu.edu.cn
FULL PAPER
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FULL PAPER Z.-L. Liu et al.
¹ 2002 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 0947-6539/02/0818-4192 $ 20.00+.50/0 Chem. Eur. J. 2002, 8, No. 184192
HO
HO OH
HO
OH
HO
OH
HO
CH3
CH3
H3C CH3
CH3 CH3 CH3
CH3
HO
HO
HO
HO
HO
HO
HO
HO Cl- NH2=C-C-N=N-C-C=NH2 Cl-
CH3
CH3
CH3
H3C
NH2
H2N
HO
O
4-HS
AAPH
TOH
+ +
trans-resveratrol
3,5-DHS 4,4'-DHS
3,4-DHS 3,4,5-THS
3,4,4'-THS
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
reaction conditions.[16] Hydroperoxide substitution at the
C-9 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 �-tocopherol.[16a,b] It was found recently that these
conjugated dienes were formed from the rapid �-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 �-tocopherol.[16d] The
present experiment used very small amounts of antioxidants
(micromolar �-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-
let absorption at 235 nm[17] 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
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
Figure 1. Formation of total hydroperoxides (LOOH) during the perox-
idation of linoleic acid (LH) in SDS (0.1 molL�1) micelles at pH 7.5 and
37 �C, initiated with AAPH. [LH]0� 15.2 mmolL�1, [AAPH]0�
6.3 mmolL�1, [ArOH]0� 11.2 �molL�1. 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.
peroxidation of the substrate. The slope of this line corre-
sponds to the rate of propagation, Rp. The peroxides×
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
Figure 2. Formation of total hydroperoxides (LOOH) during the perox-
idation of linoleic acid (LH) in CTAB (0.015 molL�1) micelles at pH 7.5
and 37 �C, initiated with AAPH. [LH]0� 15.2 mmolL�1, [AAPH]0�
6.3 mmolL�1, [ArOH]0� 11.2 �molL�1. 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.
Antioxidant Effects of Resveratrol 4191±4198
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relative effectiveness of the antioxidants in the two micelles
were appreciably different. The details will be discussed in
following sections.
The antioxidant effect of resveratrol and its analogues in the
presence of �-tocopherol : �-Tocopherol (TOH), the most
abundant and active form of vitamin E, is well known and the
principal lipid-soluble chain-breaking antioxidant in plasma
and erythrocytes.[18] Its synergistic antioxidative effect with
other antioxidants, such as �-ascorbic acid (vitamin C)[19] and
green-tea polyphenols,[13] 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
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-
idation of linoleic acid (LH) in SDS (0.1 molL�1) micelle at pH 7.5 and
37 �C, initiated with AAPH. [LH]0� 15.2 mmolL�1, [AAPH]0�
6.3 mmolL�1, [TOH]0� 5 �molL�1, [ArOH]0� 11.2 �molL�1. Uninhibited
peroxidation (a) or inhibited with b) TOH, c) TOH � resveratrol, d) TOH
� 3,4,5-THS, e) TOH � 3,4,4�-THS.
Figure 4. Formation of total hydroperoxides (LOOH) during the perox-
idation of linoleic acid (LH) in CTAB (0.015 molL�1) micelle at pH 7.5 and
37 �C, initiated with AAPH. [LH]0� 15.2 mmolL�1, [AAPH]0�
6.3 mmolL�1, [TOH]0� 5 �molL�1, [ArOH]0� 11.2 �molL�1. Uninhibited
peroxidation (a) or inhibited with b) TOH, c) TOH � resveratrol, d) TOH
� 3,4,5-THS, e) TOH � 3,4,4�-THS.
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.
Consumption of �-tocopherol : In order to rationalize the
mechanism of the antioxidant synergism of �-tocopherol and
the resveratrol analogues, the decay of �-tocopherol was
studied by HPLC separation of the reaction mixture, followed
by electrochemical determination of �-tocopherol. Represen-
tative results are illustrated in Figure 5. It was found that the
decay of �-tocopherol was approximately linear in the
absence of 3,4,4�-THS in the two micelles (lines a and b in
Figure 5. Consumption of �-tocopherol during the inhibition of linoleic
acid peroxidation in micelles at pH 7.5 and 37 �C, initiated with AAPH and
inhibited by TOH and/or 3,4,4�-THS (ArOH). [LH]0� 15.2 mmolL�1,
[AAPH]0� 6.3 mmolL�1, [ArOH]0� 11.2 �molL�1, [TOH]0� 5 �molL�1.
a) Decay of TOH in the absence of 3,4,4�-THS in CTAB (0.015 molL�1)
micelle, b) decay of TOH in the absence of 3,4.4�-THS in SDS (0.1 molL�1)
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 �-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
�-tocopherol, while 4-HS and 3,5-DHS showed no effect
(Figures not shown). These results suggest that resveratrol,
3,4-DHS, 3,4,4�-THS and 3,4,5-THS may be able to reduce the
�-tocopheroxyl radical to regenerate �-tocopherol and,
hence, maintain the concentration of �-tocopherol in the
reaction system. Similar �-tocopherol regeneration reactions
by vitamin C[19] and green-tea polyphenols have been
reported previously.[13]
FULL PAPER Z.-L. Liu et al.
¹ 2002 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 0947-6539/02/0818-4194 $ 20.00+.50/0 Chem. Eur. J. 2002, 8, No. 184194
Direct determination of the rate of the �-tocopherol regen-
eration reaction : The �-tocopheroxyl radical (TO.) is more
persistent in micelles than in homogeneous solutions. The rate
constants of the bimolecular self-reaction of TO. were
reported to be 3� 103��1 s�1 in benzene/di-tert-butyl perox-
ide[20] and 15��1 s�1 in CTAB micelles,[15a] respectively. They
correspond to half-lives of 11 seconds and 38 minutes in the
homogeneous solution and the micelle, respectively, taking
the initial concentration of TO. as 30 �mol. 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 spectrum of TO. recorded in CTAB micelles.
Addition of resveratrol through a fast stopped-flow device[21]
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
Figure 6. EPR Spectra of the �-tocopheroxyl radical (TO.) recorded in
CTAB (15 mmolL�1) micelles at pH 7.4 and room temperature in air. The
TO. was generated by oxidizing TOH (1 mmolL�1) with PbO2. The initial
concentration of TO. was 28 �molL�1.
Figure 7. The decay of �-tocopheroxyl radicals in CTAB (15 mmolL�1)
micelles at pH 7.4 and room temperature in air. a) intrinsic decay, b) in the
presence of resveratrol (0.78 mmolL�1), c) in the presence of 3,4-DHS
(0.13 mmolL�1).
constant versus the concentration of resveratrol gave a
straight line from which the bimolecular rate constant
between TO. and resveratrol [Eq. (9), vide infra] could be
obtained. 3,4-DHS reacted with TO. much faster than
resveratrol (line c in Figure 7). The rate constants for the �-
tocopherol regeneration reaction of resveratrol and 3,4-DHS
were determined to be 0.23� 102 and 3.0� 102��1 s�1 respec-
tively in CTAB micelles. These EPR experiments confirm
unambiguously that the antioxidant synergism of �-tocopher-
ol with the resveratrol analogues is due to the �-tocopherol
regeneration reaction by the latter.
Electrochemistry of resveratrol and its analogues : The
electrochemistry of resveratrol and its analogues was studied
by cyclic voltammetry in both SDS and CTABmicelles. It was
found that resveratrol, 4-HS and 3,5-DHS showed irreversible
cyclic voltammograms with higher oxidation potentials, while
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.[22]
The kinetics of linoleic acid (LH) peroxidation initiated by
azo-compounds and its inhibition by chain-breaking antiox-
idants (AH) have been discussed in detail in our previous
papers.[13±14] The rate of propagation (Rp) and the rate of
peroxide formation in the inhibition period (Rinh) are given by
Equations (1) and (2), respectively.
d[LOOH]/dt�Rp� [kp/(2kt)1/2]Ri1/2 [LH] (1)
Rinh�kpRi [LH]/(nkinh [AH]) (2)
here kp, kt and kinh are rate constants for the chain
propagation, chain termination and chain inhibition by
antioxidants, respectively, and Ri is the apparent rate of chain
initiation, which can be obtained by measuring the inhibition
period or decay of the antioxidant (AH), [Eqs. (3) and (4),
respectively].[13]
Ri�n [AH]0/tinh (3)
Ri��nd[AH]/dt (4)
Here n is the stoichiometric factor that designates the
number of peroxyl radicals trapped by each antioxidant
molecule. Since the n value of �-tocopherol is generally
assumed to be 2,[22] the Ri value can be determined from the
inhibition period or the decay rate of �-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
from Figures 1 and 2 are listed in Tables 1 and 2, respectively.
kclp�Rp/Ri (5)
kclinh�Rinh/Ri (6)
Antioxidant Effects of Resveratrol 4191±4198
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It can be seen from Figures 1 and 2 and from Table 1 that
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
propagating linoleic acid peroxyl radicals [LOO. , Eq. (7)].
LOO. � ArOH ��kinh LOOH � ArO. (7)
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
about 0.5 ± 3.1� 104��1 s�1, comparable to that of �-tocopher-
ol (3.6� 104 and 2.0� 104��1 s�1 in SDS and CTAB micelles,
respectively, see Table 2) and to those of green-tea polyphe-
nols (0.3 ± 3.7� 104��1 s�1 in micelles).[13b] 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
interaction, as evidenced recently from both experiments[23]
and theoretical calculations.[24] The theoretical calculation
showed that the hydrogen bond in the ortho-OH phenoxyl
radical is approximately 4 kcalmol�1 stronger than that in the
parent catechol, and that the bond dissociation energy (BDE)
of catechol is 9.1 kcalmol�1 lower than that of phenol and
8.8 kcalmol�1 lower than that of resorcinol.[24] In addition, it
should be easier to further oxidize the ortho-OH phenoxyl
Table 1. Inhibition of AAPH-initiated peroxidation of linoleic acid by resveratrol and its analogues in micelles.[a,b]
Micelle ArOH Rp Rinh tinh kinh n kclp kclinh Epa
[10�8 moldm�3 s�1] [10�8 moldm�3 s�1] [103 s] [104 dm3mol�1 s�1] [V vs. SCE]
SDS none 8.3 26.8
resveratrol 9.0 2.8 3.1 1.3 0.8 29.1 9.0 0.62
4-HS 9.2 4.0 2.1 1.2 0.6 29.7 12.9 0.64
3,5-DHS 7.8 3.8 1.5 1.7 0.4 25.1 12.2 0.85
4,4�-DHS 8.0 2.6 3.2 1.4 0.9 25.8 8.4 0.40
3,4-DHS 8.2 0.8 4.9 2.9 1.4 26.4 2.6 0.34
3,4,5-THS 8.7 1.5 2.4 3.1 0.7 28.1 4.8 0.24
3,4,4�-THS 6.8 � 0 7.2 [c] 2.0 21.9 [c] 0.32
CTAB none 16.8 20.4
resveratrol 16.0 2.7 2.7 0.7 2.0 19.3 3.2 0.67
4-HS 19.8 3.2 1.7 1.0 1.2 23.9 3.8 0.66
3,5-DHS 15.6 4.2 2.4 0.5 1.8 18.8 5.1 0.79
4,4�-DHS 14.8 2.8 2.6 0.8 1.9 17.8 3.4 0.43
3,4-DHS 14.0 1.6 4.1 0.9 3.0 16.9 1.9 0.36
3,4,5-THS 16.5 1.6 2.9 1.3 2.1 19.9 1.9 0.23
3,4,4�-THS 15.5 � 0 5.1 [c] 3.8 18.7 [c] 0.34