Structure-activity relationship studies of resveratrol and its analogues by the reaction kinetics of low density lipoprotein peroxidation

Article abstract of DOI:10.1016/j.bioorg.2006.04.001

Resveratrol (3,5,4′-trans-trihydroxystibene) is a natural phytoalexin present in grapes and red wine, which possesses a variety of biological activities including antioxidative activity. To find more active antioxidants, with resveratrol as the lead compo

Full text of DOI:10.1016/j.bioorg.2006.04.001

Bioorganic Chemistry 34 (2006) 142–157
www.elsevier.com/locate/bioorg
Structure–activity relationship studies of
resveratrol and its analogues by the reaction
kinetics of low density lipoprotein peroxidation
Jin-Chun Cheng, Jian-Guo Fang, Wei-Feng Chen,
*
*
Bo Zhou , Li Yang , Zhong-Li Liu
National Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Gansu 730000, China
Received 2 March 2006
Abstract
0
Resveratrol (3,5,4 -trans-trihydroxystibene) is a natural phytoalexin present in grapes and red
wine, which possesses a variety of biological activities including antioxidative activity. To find more
active antioxidants, with resveratrol as the lead compound, we synthesized resveratrol analogues,
0
0
0
0
0
0
0
i.e., 3,4,3 ,4 -tetrahydroxy-trans-stilbene (3,4,3 ,4 -THS), 3,4,4 -trihydroxy-trans-stilbene (3,4,4 -
0
0
0
THS), 2,4,4 -trihydroxy-trans-stilbene (2,4,4 -THS), 3,3 -dimethoxy-4,4 -dihydroxy-trans-stilbene
0
0
0
0
(
3,3 -DM-4,4 -DHS), 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4 -dihydroxy-trans-stilbene (4,4 -
DHS), 3,5-dihydroxy-trans-stilbene (3,5-DHS) and 2,4-dihydroxy-trans-stilbene (2,4-DHS). Antiox-
idative effects of resveratrol and its analogues against free-radical-induced peroxidation of human
low density lipoprotein (LDL) were studied. The peroxidation was initiated either by a water-soluble
0
2+
initiator 2,2 -azobis(2-amidinopropane hydrochloride) (AAPH), or by cupric ion (Cu ). The reac-
tion kinetics were monitored either by the uptake of oxygen and the depletion of a-tocopherol
(
(
TOH) presented in the native LDL, or by the formation of thiobarbituric acid reactive substances
TBARS). Kinetic analysis of the antioxidation process demonstrates that these trans-stilbene deriv-
2
+
atives are effective antioxidants against both AAPH- and Cu -induced LDL peroxidation with the
0
0
0
0
0
0
activity sequence of 3,4,3 ,4 -THS ꢀ 3,3 -DM-4,4 -DHS > 3,4-DHS ꢀ 3,4,4 -THS > 2,4,4 -THS >
0
0
0
0
0
resveratrol ꢀ 3,5-DHS > 4,4 -DHS ꢀ 2,4-HS, and 3,4,3 ,4 -THS ꢀ 3,4-DHS ꢀ 3,4,4 -THS > 3,3 -
0
0
0
DM-4,4 -DHS > 4,4 -DHS > resveratrol ꢀ 2,4-HS > 2,4,4 -THS ꢀ 3,5-DHS, respectively. Molecules
bearing ortho-dihydroxyl or 4-hydroxy-3-methoxyl groups possess significantly higher antioxidant
activity than those bearing no such functionalities.
*
Corresponding authors. Fax: +86 931 8625657 (B. Zhou).
E-mail address: bozhou@lzu.edu.cn (B. Zhou).
0
045-2068/$ - see front matter ꢀ 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2006.04.001

J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
2006 Elsevier Inc. All rights reserved.
143
ꢀ
Keywords: Resveratrol; Phenolic antioxidants; Low density lipoprotein; Lipid peroxidation; Structure–activity
relationship
1
. Introduction
In the pathogenesis of atherosclerosis, growing evidence suggests that free-radical-in-
duced oxidative modification of low density lipoprotein (LDL) may be involved [1–5].
Therefore, inhibition of LDL peroxidation by addition of antioxidants becomes an attrac-
tive therapeutic strategy to prevent and possibly to treat atherosclerosis and related diseas-
es in human. This leads to a great deal of research devoted to the prevention of lipid
peroxidation of LDL by antioxidants [6–9].
0
Resveratrol (3,5,4 -trihydroxy-trans-stilbene) is a naturally occurring phytoalexin pres-
ent in grapes, nuts and other plants. It is believed that the high level of this compound in
red wine (0.1 ꢀ 15 mg/l) [10] is linked to the low incidence of heart diseases in some
regions of France, the so-called ‘‘French paradox,’’ i.e., despite high fat intake, mortality
from coronary heart disease is lower due to the regular drinking of wine [11,12]. This com-
pound has attracted considerable attention due to its various biological and pharmacolog-
ical activities, including antioxidative [13–16] and anticancer activities [17–20]. As a part of
our ongoing research project on antioxidative effects of natural antioxidants and their
derivatives [21–24] we found recently that some synthetic resveratrol analogues bearing
ortho-dihydroxyl functionality exhibit significantly higher antioxidant and cytotoxic activ-
ity against HL-60 cancer cells than resveratrol and other analogues bearing no such func-
tionality [25–27]. Therefore, it is desirable to see if the same structure–activity relationship
is also valid in LDL peroxidation. We report herein an in vitro study on the antioxidative
effect of resveratrol and related trans-stilbene analogues on free-radical-induced LDL per-
0
0
oxidation. The compounds studied are resveratrol, 3,4,3 ,4 -tetrahydroxy-trans-stilbene
0
0
0
0
0
(
3,4,3 ,4 -THS), 3,4,4 -trihydroxy-trans-stilbene (3,4,4 -THS), 2,4,4 -trihydroxy-trans-stil-
0
0
0
0
0
bene (2,4,4 -THS), 3,3 -dimethoxy-4,4 -dihydroxy-trans-stilbene (3,3 -DM-4,4 -DHS),
0
0
3
,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4 -dihydroxy-trans-stilbene (4,4 -DHS), 3,5-di-
hydroxy-trans-stilbene (3,5-DHS) and 2,4-dihydroxy-trans-stilbene (2,4-DHS) (Fig. 1).
0
The peroxidation was initiated by a water-soluble azo initiator 2,2 -azobis(2-amidinopro-
2
+
pane hydrochloride) (AAPH) and cupric ion (Cu ), and measured by oxygen uptake and
formation of thiobarbituric acid reactive substances (TBARS). It was found that resvera-
trol and its analogues, especially 3,4,3 ,4 -THS, 3,4,4 -THS, 3,4-DHS, and 3,3 -DM-4,4 -
DHS, are good antioxidants for both AAPH- and cupric ion-initiated LDL peroxidation.
The structure–activity relationship is discussed.
0
0
0
0
0
2
. Experimental
2
.1. Materials
0
0
0
Resveratrol, 3,4-DHS, 3,4,4 -THS, 2,4,4 -THS, 3,5-DHS, 4,4 -DHS and 2,4-DHS were
synthesized with reference to the modified Wittig reaction [28,29] using diet-
hylbenzylphosphonate, which was prepared from methoxyl substituted benzyl chloride

144
J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
Fig. 1. Molecular structures of resveratrol and its analogues.
and triethyl phosphate, reacted with methoxyl substituted benzaldehyde and followed by
removing the methyl protecting group with pyridine hydrochloride. This procedure gave
exclusively the trans-isomers and good or moderate yields depending predominantly on
0
0
0
0
the demethylation reaction. 3,4,3 ,4 -THS and 3,3 -DM-4,4 -DHS were synthesized with
reference to the modified McMurry reaction [30,31]. Briefly, zinc powder (2.0 g,
0
.031 mol) was added to a stirred slurry of TiCl (1.5 ml, 0.014 mol) in dry tetrahydrofu-
4
ran (THF) at ꢁ78 ꢁC under an inert atmosphere. After refluxing for 40 min, the black mix-
ture was cooled and a solution of vanillin (4-hydroxy-3-methoxybenzaldehyde, 0.01 mol)
in THF was added. After refluxing for 3–5 h, the mixture was poured into ice-water, and
extracted with ether. The ether layer was washed with brine, dried over anhydrous sodium
sulfate. and concentrated. Removal of the solvent under reduced pressure and purification
0
0
0
of the residue by column chromatography gave the symmetrical stilbene (3,3 -DM-4,4 -
0
DHS). Removing the methyl protecting group by pyridine hydrochloride gave 3,4,3 4 -
THS. Their structures were fully identified with H NMR and EI-MS or FAB-MS and
1

J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
145
the data are consistent with those reported in the literature [28,29]. The purity of the com-
pounds was all checked by HPLC being > 98%.
1
Resveratrol: H NMR (400 MHz, (CD ) CO): d 6.22 (1H, t, J = 2 Hz, H-4), 6.49 (2H,
3
2
0
0
d, J = 2 Hz, H-2 and H-6), 6.75 (2H, d, J = 8.4 Hz, H-3 and H-5 ), 6.77, 6.97 (each 1H, d,
J = 16.3 Hz, ACH@CHA), 7.36 (2H, d, J = 8.4 Hz, H-2 and H-6 ); MS (m/z): 228 [M ],
11, 199, 181, 157, 115.
,4,3 ,4 -THS: H NMR (300 MHz, (CD ) CO): d 6.80 (2H, d, J = 8.1 Hz, H-5 and
3 2
H-5 ), 6.84 (2H, s, ACH@CHA), 6.87 (2H, dd, J = 8.1, 1.8 Hz, H-6 and H-6 ), 7.05
0
0
+
2
0
0
1
3
0
0
0
+
(
2H, d, J = 1.8 Hz, H-2 and H-2 ); MS (m/z): 244 [M ], 221, 207, 165.
0
1
3
,4,4 -THS: H NMR (400 MHz, (CD ) CO): d 6.79 (1H, d, J = 8.1 Hz, H-5), 6.81 (2H,
3 2
0
0
d, J = 8.5 Hz, H-3 and H-5 ), 6.87 (1H, dd, J = 8.1, 2.1 Hz, H-6), 6.79, 6.96 (each 1H, d,
J = 16 Hz, ACH@CHA), 7.05 (1H, d, J = 2.1 Hz, H-2), 7.37 (2H, d, J = 8.5 Hz, H-2 and
0
0
+
H-6 ); MS (m/z): 228 [M ], 211, 199, 181, 157, 115.
0
1
0
0
2
,4,4 -THS: H NMR (400 MHz, (CD ) CO): d 6.73 (2H, d, J = 9 Hz, H-3 and H-5 ),
3 2
0
0
7
.36 (2H, d, J = 9 Hz, H-2 and H-6 ), 6.30 (1H, d, J = 2 Hz, H-3), 6.32 (1H, dd, J = 9,
2
Hz, H-5), 6.77, 6.95 (each 1H, d, J = 16 Hz, ACH@CHA), 7.35 (1H, d, J = 9 Hz,
+
H-6); MS (m/z): 228 [M ], 211, 199, 181, 157, 115.
0
0
1
3
,3 -DM-4,4 -DHS: H NMR (300 MHz, (CD ) CO): d 6.80 (2H, d, J = 8.1 Hz, H-5
3 2
0
0
and H-5 ), 6.98 (2H, s, ACH@CHA), 6.97 (2H, dd, J = 2.1, 8.1 Hz, H-6 and H-6 ), 7.16
0
+
(
2H, d, J = 1.8 Hz, H-2 and H-2 ); MS (m/z): 272 [M ], 259, 218.
1
3
,4-DHS: H NMR (400 MHz, (CD ) CO): d 6.79 (1H, d, J = 8 Hz, H-5), 6.89 (1H,
3 2
dd, J = 8, 2 Hz, H-6), 6.98, 7.01 (each 1H, d, J= 16.4 Hz, ACH@CHA), 7.09 (1H, d,
0
0
J = 2 Hz, H-2), 7.19 (1H, t, J = 7.4 Hz, H-4 ), 7.31 (2H, t, J = 7.4 Hz, H-3 and
0
0
0
+
H-5 ), 7.51 (2H, d, J = 7.4 Hz, H-2 and H-6 ); MS (m/z): 212 [M ], 197, 165, 141,
1
15, 77.
0
1
0
4
,4 -DHS: H NMR (400 MHz, (CD ) CO): d 6.83 (4H, d, J = 8 Hz, H-3, H-5, H-3
3
2
0
0
0
and H-5 ), 6.95 (2H, s, ACH@CHA), 7.40 (4H, d, J = 8 Hz, H-2, H-6, H-2 and H-6 );
MS (m/z): 212 [M ], 197, 165, 141, 115, 77.
+
1
3
,5-DHS: H NMR (400 MHz, (CD ) CO): d 6.27 (1H, t, J = 2.1 Hz, H-4), 6.56 (2H, d,
3
2
J = 2.1 Hz, H-2 and H-6), 7.05, 7.07 (each 1H, d, J = 16.6 Hz, ACH@CHA), 7.22 (1H, t,
0
0
0
0
J = 7.3 Hz, H-4 ), 7.33 (2H, t, J = 7.3 Hz, H-3 and H-5 ), 7.53 (2H, d, J = 7.3 Hz, H-2
0
+
and H-6 ); MS (m/z): 212 [M ], 197, 165, 141, 115, 77.
1
2
,4-DHS: H NMR (400 MHz, (CD ) CO): d 6.30 (1H, d, J = 2 Hz, H-3), 6.32 (1H,
3 2
dd, J = 9, 2 Hz, H-5), 6.97, 7.37 (each 1H, d, J = 17 Hz, ACH@CHA), 7.15 (1H, tt,
0
0
0
J = 8, 1 Hz, H-4 ), 7.28 (2H, d, J = 8 Hz, H-3 and H-5 ), 7.35 (1H, d, J = 9 Hz,
+
H-6), 7.47 (2H, d, J = 8 Hz, H-2 and H-6); MS (m/z): 212[M ], 197, 165, 141, 115,
7
7.
dl-a-Tocopherol (Merck, Biochemical reagent, > 99.9%) and 2,2 -azobis(2-methylpro-
0
pionamidine) dihydrochloride (AAPH, Aldrich) were kept under nitrogen in a refrigerator
and used as received.
The LDL was isolated from human plasma of a healthy donor by the discontinuous
density gradient centrifugation procedure as described in the literature [32] at
4
5,000 rpm (140,000g) for 6 h using a HITACHI 55P-72 ultracentrifuge at 4 ꢁC. The
isolated LDL fraction was then dialyzed with phosphate buffered saline (PBS, com-
posed of 137 mM of NaCl, 2.7 mM of KCl, 8.1 mM of Na HPO and 1.5 mM of
2
4
KH PO in distilled water, pH 7.4) containing 0.1 mM sodium ethylenediaminetetraac-
2
4
etate (EDTA) to prevent oxidation during the isolation. EDTA was removed by dial-

146
J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
ysis with PBS before the oxidation experiments. The concentration of protein was
determined by the method of Lowry [33].
2
.2. Oxygen uptake measurements
The rate of oxygen uptake was measured in a closed glass vessel of ca. 2 ml in vol-
ume, at constant temperature and that was stirred, using a 5946-50 oxygen meter
Cole-Parmer Instruments, USA) which was able to record oxygen concentrations as
(
ꢁ
8
low as 10 M. LDL was suspended in PBS (pH 7.4) to the final concentration of 0.2
mg protein/ml equivalent to approximately 0.4 lM of LDL (mass = 2.5 · 10 g/M and
6
protein content of ca. 20%) [34] under air. Resveratrol and its analogues were dissolved
in DMSO to the concentration of 2 mM as stock solutions. The final concentration of
DMSO in the suspension was less than 0.1% (v/v) of the LDL suspension to avoid dis-
turbance of the system. AAPH was directly dissolved in PBS (pH 7.4) and injected into
the LDL suspension to initiate the peroxidation. Every experiment was repeated three
times and the results were reproducible to within 10% experimental deviation.
2
.3. High performance liquid chromatographic (HPLC) measurements
A Gilson model 702 liquid chromatograph was used to separate a-tocopherol (TOH)
with a Sychropack KPP-100 reversed-phase column (4 · 250 mm) and eluted with metha-
nol–iso-propanol–formic acid (80:20:1 v/v/v) containing 50 mM of sodium perchlorate as
a supporting electrolyte at a flow rate of 1 ml/min. Aliquots of 0.6 ml of reaction mixture
were taken out from three identical reaction vessels of 2 ml in volume at appropriate time
intervals, and TOH was extracted by hexane–ethanol partitioning (hexane : EtOH :
LDL = 12:3:1 v/v/v) which yielded > 97% of TOH [34]. TOH was electrochemically
detected by using a Gilson Model 142 electrochemical detector by setting the oxidation
potential at +700 mV.
2
.4. Malonodialdehyde (MDA) formation measurements
The formation of MDA was determined by thiobarbituric acid assay as a thiobar-
bituacid reactive substances (TBARS) to monitor LDL peroxidation [35]. The LDL
was incubated at 37 ꢁC in 0.1 M potassium phosphate buffer, pH 7.5, and made up
to a final protein concentration of 0.2 mg/ml. The peroxidation was initiated by
1
0 lM of CuSO and inhibited by 2 lM of resveratrol or its analogues which was add-
4
ed as a DMSO solution and the final concentration of DMSO in the suspension was
less than 0.1% v/v that did not show appreciable interference to the reaction as evi-
denced by control experiments. The reaction mixture was gently shaken at 37 ꢁC and
aliquots of the reaction mixture were taken out at specific intervals and a TCA–
TBA–HCl stock solution (15% w/v trichloroacetic acid; 0.375% w/v thiobarbituric acid;
0
.25 N HCl) was added to the reaction mixture, together with 0.02% w/v butylated
hydroxytoluene (BHT). This amount of BHT completely prevented the formation of
any nonspecific TBARS [36]. The solution was then heated in a boiling water bath
for 15 min. After cooling, the precipitate was removed by centrifugation. MDA in
the supernatant was determined at 532 nm using the extinction coefficient of
5
ꢁ1
ꢁ1
1
.56 · 10 M cm [35].

J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
147
3
. Results and discussion
.1. The antioxidant activity of resveratrol and its analogues in AAPH-induced LDL
peroxidation
3
The important event of lipid peroxidation is the uptake of oxygen and formation of lip-
id peroxides [37]. In the present work oxygen uptake was used to measure the reaction
kinetics of the LDL peroxidation. Fig. 2 shows oxygen uptake curves recorded during
the water-soluble radical initiator AAPH-induced LDL peroxidation in the absence and
in the presence of exogenous resveratrol. In the absence of resveratrol the oxygen uptake
did not take place immediately as in the case of lipid peroxidation conducted in model
membranes [21,25,38], but was still inhibited for ca. 20 min (Fig. 2, line a). This demon-
strates the presence of endogenous antioxidants in LDL, such as a-tocopherol (TOH), ubi-
quinol-10 and carotenoids (Eq. (1)), which can trap the propagating radicals to inhibit the
peroxidation. The oxygen uptake rate during the inhibition period is designated as Rinh
.
After the inhibition period the oxygen uptake became faster, indicating depletion of the
endogenous antioxidants. The turning point from the inhibition period to the restoration
of oxygen uptake is the inhibition time, tinh. The slope of the oxygen uptake curve after the
inhibition period represents the intrinsic peroxidation rate, R , of the LDL in the absence
p
of antioxidants. After a short time of the inhibition period, different amounts of resvera-
trol were added. It was found that addition of resveratrol inhibits the LDL peroxidation,
produce a new inhibition period and the inhibition time is proportional to the concentra-
tion of resveratrol (Fig. 2, lines b–d and the inset). This demonstrates that the peroxidation
of LDL is inhibited dose-dependently by resveratrol in the absence of endogenous antiox-
idants. Other resveratrol analogues also show the dose-dependent inhibition for the LDL
peroxidation (Figures not shown).
Fig. 2. Representative oxygen uptake curves recorded during the AAPH-initiated and resveratrol inhibited LDL
peroxidation in PBS (pH 7.4) at 37 ꢁC under atmospheric oxygen. [LDL] = 0.2 mg/ml; [AAPH]
The arrow shows the time of addition of resveratrol. (a) Native LDL containing 2.47 lM of TOH;
b) [resveratrol] = 2 lM; (c) [resveratrol] = 4 lM; (d) [resveratrol] = 8 lM. The inset shows the concentration
dependence of the inhibition time (tinh).
0
= 10.0 mM.
(
0
0
0

148
J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
Fig. 3 shows representative oxygen uptake curves recorded during the AAPH-induced
LDL peroxidation in the presence of the same concentration (4 lM) of resveratrol and
its analogues which were added after the depletion of endogenous antioxidants. It is seen
that after depletion of the endogenous antioxidants, addition of all of these tested
compounds produces a new inhibition period, indicating that LDL peroxidation could
be inhibited by these compounds in the absence of endogenous antioxidants. The
inhibition time, tinh, is significantly different for the different resveratrol analogues and
0
0
0
0
0
0
follows the efficacy sequence of 3,4,3 ,4 -THS ꢀ 3,3 -DM-4,4 -DHS > 3,4-DHS ꢀ 3,4,4 -
0
0
THS > 2,4,4 -THS > resveratrol ꢀ 3,5-DHS > 4,4 -DHS ꢀ 2,4-DHS (the curve for 4,4 -
DHS is not shown for clarity).
When resveratrol or its analogues was added before the AAPH-initiation the intrin-
sic inhibition period of the native LDL peroxidation was remarkably prolonged
(Fig. 4) and the overall inhibition time was approximately equal to the sum of the
intrinsic inhibition time of the native LDL and the inhibition time induced by resvera-
trol or its analogues when it was used after depletion of the endogenous antioxidants
0
0
0
0
(
compare Figs. 3 and 4). The efficacy sequence is 3,4,3 ,4 -THS ꢀ 3,3 -DM-4,4 -
0
0
0
DHS > 3,4-DHS > 3,4,4 -THS > 2,4,4 -THS ꢀ resveratrol > 4,4 -DHS ꢀ 2,4-DHS > 3,5-DHS,
similar to that obtained when these compounds were used after depletion of the antioxidants
in native LDL. This suggests that resveratrol and its analogues serve as chain-breaking anti-
oxidants independently and do not have synergistic interaction with the intrinsic antioxi-
dants, e.g., a-tocopherol (TOH), in the native LDL. It has been reported previously that if
an exogenous antioxidant, such as vitamin C or green tea polyphenols, could react with
TOH in a synergistic fashion in the native LDL, the overall inhibition time would be signif-
icantly longer than the sum of the intrinsic inhibition time of the native LDL and the inhibi-
tion time due to the exogenous antioxidant [8,39].
Fig. 3. Representative oxygen uptake curves recorded during the AAPH-initiated and resveratrol and its
analogues (ROH) inhibited LDL peroxidation in PBS (pH 7.4) at 37 ꢁC under atmospheric oxygen.
[
LDL] = 0.2 mg/ml; [AAPH] = 10 mM; [ROH]
0
= 4 lM. The ROHs were added after the intrinsic inhibition
period. The arrow shows the time of addition of ROH. (a) Native LDL containing 2.47 lM of TOH; (b) 3,5-
0
0
0
0
0
DHS; (c) 2,4-DHS; (d) resveratrol; (e) 2,4,4 -THS; (f) 3,4,4 -THS; (g) 3,4-DHS; (h) 3,4,3 ,4 -THS; (i) 3,3 -DM-
4
0
0
,4 -DHS. Curve for 4,4 -DHS is not shown for clarity.

J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
149
Fig. 4. Representative oxygen uptake curves recorded during the AAPH-initiated and resveratrol and its
analogues (ROH) inhibited LDL peroxidation in PBS (pH 7.4) at 37 ꢁC under atmospheric oxygen.
[
(
4
LDL] = 0.2 mg/ml; [AAPH]
0
= 10 mM; [ROH]
0
= 4 lM. The ROHs were added before the initiation.
0
a) Native LDL containing 2.47 lM of TOH; (b) 2,4-DHS; (c) resveratrol; (d) 2,4,4 -THS; (e) 3,4-DHS; (f) 3,4,
0
0
0
0
0
0
-THS; (g) 3,4,3 ,4 -THS; (h) 3,3 -DM-4,4 -DHS. Curves for 3,5-DHS and 4,4 -DHS are not shown for clarity.
3
.2. Decay kinetics of a-tocopherol during the peroxidation
The decay kinetics of TOH during the antioxidation reaction has been used to deter-
mine the rate of initiation, R , of LDL peroxidation [34] and to study the synergism of
i
TOH with other antioxidants [8,21–25]. In the present work the decay kinetics of TOH
was studied by HPLC separation of the reaction mixture components followed by electro-
chemical determination of TOH. It was found that in the native LDL TOH decayed
approximately linearly with time in the initial two half-lives (Fig. 5, line a), in accordance
with the kinetic demand for antioxidant reactions (Eq. (7), vide infra). The decay rate was
0 0
2
.3 nM/s. Addition of 3,4,3 ,4 -THS decreased significantly the decay rate of TOH, but no
0
0
Fig. 5. Decay of a-tocopherol (TOH) during the AAPH-initiated and 3,4,3 ,4 -THS inhibited peroxidation of
LDL in PBS (pH 7.4) at 37 ꢁC under atmospheric oxygen. [LDL] = 0.2 mg/ml; [AAPH] = 10.0 mM. (a) Native
0
0
0
LDL containing 2.47 lM of TOH; (b) in the presence of 4 lM of 3,3 4,4 -THS.

150
J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
2+
Fig. 6. Inhibition of MDA formtion during the Cu -induced peroxidation of LDL by resveratrol and its
analogues (ROH) at 37 ꢁC. The LDL was suspended in PBS (pH 7.4) at a protein concentration of 0.2 mg/ml.
0
0
0
[
CuSO
4
] = 10 lM; [ROH]
0
= 2 lM. (a) Native LDL; (b) resveratrol; (c) 3,3 -DM-4,4 -DHS; (d) 4,4 -DHS; (e) 3,4-
0
0
0
DHS; (f) 3,4,4 -THS; (g) 3,4,3 ,4 -THS. Curves for other resveratrol analogues are not shown for clarity. Every
experiment was repeated three times.
apparent turning point was observed (Fig. 5, line b). This is in contrast to the case of vita-
min C and green tea polyphenols which exhibited clear turning point in the decay curves of
TOH, demonstrating the antioxidant synergism with TOH [8,22,39]. Therefore, it is
0
0
believed that TOH and 3,4,3 ,4 -THS work independently as chain-breaking antioxidants
in LDL and they do not interact with each other, in accordance with the experimental
results mentioned in the previous section.
2
+
.3. Inhibition of Cu -induced LDL peroxidation
3
2
+
Cu -induced LDL peroxidation is considered to be more relevant to in vivo situation
2
+
than the AAPH-induced peroxidation [1]. Therefore, Cu was also used to induce LDL
peroxidation and the formation of malondialdehyde (MDA), which is the final oxidation
product of lipid peroxidation, was used to monitor LDL peroxidation [35]. Fig. 6 shows
2
+
the MDA formation during the Cu -induced peroxidation of LDL. Similar to the oxygen
uptake experiments mentioned earlier, formation of MDA is inhibited for a short period
of time due to the presence of endogenous antioxidants in the LDL. It is seen that addition
of resveratrol and its analogues significantly suppresses the rate of MDA formation and
increases the inhibition period of the native LDL. The antioxidative activity of resveratrol
and its analogues is assessed by their inhibition period, tinh, and it follows the sequence
0
0
similar to that observed in the oxygen uptake assay mentioned above, i.e., 3,4,3 ,4 -
0
0
0
0
THS ꢀ 3,4-DHS ꢀ 3,4,4 -THS > 3,3 -DM-4,4 -DHS > 4,4 -DHS > resveratrol ꢀ 2,4-
0
DHS > 2,4,4 -THS ꢀ 3,5-DHS (Table 1).
3
.4. Antioxidation kinetics
It has been proven that lipid peroxidation induced by AAPH in model biomembranes
follows the same classical rate law for auto-oxidation as that in homogeneous solutions

Table 1
Inhibition times (tinh) of resveratrol and its analogues (ROH) in Cu -induced peroxidation of human LDL
2+
a
b
0
0
0
0
0
0
0
ROH
TOH
Resveratrol
58 ± 0.6
3,3 ,4,4 -THS
107 ± 4.0
3,4,4 -THS
2,4,4 -THS
3,3 -DM-4,4 -DHS
88 ± 2.6
3,4-DHS
106 ± 3.2
4,4 -DHS
3,5-DHS
43 ± 1.2
2,4-DHS
53 ± 2.0
t
inh (min)
a
30 ± 1.5
105 ± 3.6
43 ± 0.6
77 ± 1.6
4 0
Determined in PBS (pH 7.4) at 37 ꢁC; [LDL] = 0.2 mg/ml; [CuSO ] = 10 lM; [ROH] = 2 lM. Every experiment was repeated three times and the SD are shown
in the table.
b
Intrinsic TOH (2.47 lM) and other antioxidants in native LDL.

152
J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
[
38], and that TOH is the principal lipid-soluble chain-breaking antioxidant in biomem-
branes [37] and in LDL [40]. Although TOH behaves as an antioxidant under normal
experimental conditions in LDL, it is also reported that TOH might become a prooxidant
to accelerate lipid peroxidation in LDL by the so-called tocopherol mediated peroxidation
(
TMP) when the radical flux is low and the concentration of TOH was high [5,34,41]. In
the present study, since the radical flux was high ([AAPH] = 10 mM) and the concentra-
tion of TOH was low ([TOH] = 2.47 lM), TOH behaved as a typical antioxidant as evi-
0
denced by the oxygen uptake kinetics (Figs. 2–4) and its decay kinetics (Fig. 5). Similar
antioxidant behaviour of TOH in LDL has also been reported previously [8,42–44]. There-
fore, the rate of oxygen uptake during the peroxidation of LDL initiated by 10 mM of
AAPH can be expressed as follows:
1=2
1=2
ꢁ
d½O
2
ꢂ=dt ¼ R
p
¼ ðk
p
=ð2k
t
Þ
ÞRi ½LHꢂ;
ð1Þ
where k and k are rate constants for the chain propagation and termination respectively,
p
t
LH represents a lipid molecule with an abstractable hydrogen, i.e., polyunsaturated fatty
acids in LDL, and R is the apparent rate of initiation
i
R
i
¼ 2k
g
ꢃ e½RAN@NARꢂ:
ð2Þ
ꢁ6
Although the radical generation rate of AAPH is known as 1.3 · 10 [AAPH]/s at 37 ꢁC
for protein-containing solutions and liposomal dispersions [1,34], the cage effect parameter
e varies appreciably on the medium and the concentration of the antioxidant and the ini-
tiator [34]. Therefore, the R value is generally determined by the inhibition period and/or
i
by the decay rate of TOH (Eqs. (6) and (7), vide infra).
In the presence of an antioxidant molecule, AH, the peroxyl radical can be trapped and
Å
Å
a new antioxidant radical, A , produced (Eq. (3)). If the A is a stabilized radical (e.g., a-
tocopheroxyl radical or Vitamin C radical anion) which can promote the rate-limiting
hydrogen abstraction reaction (3) and undergo fast termination reaction (Eq. (4)), the per-
oxidation would be inhibited.
kinh
Å
Å
LOO þ AH ! LOOH þ A ;
ð3Þ
ð4Þ
fast
Å
Å
LOO þ A ! LOOA;
where kinh is the rate of inhibition, representing the activity of the antioxidant. During the
inhibition period the rate of peroxyl formation by the initiation equals the rate of peroxyl
trapping, therefore
Å
R
i
¼ Rinh ¼ kinh ꢃ n½AHꢂ ꢃ ½LOO ꢂ;
ð5Þ
where n is the stoichiometric factor that designates the number of peroxyl radicals trapped
by each antioxidant molecule and is given by
n ¼ Ritinh=½AHꢂ0:
ð6Þ
ð7Þ
From Eqs. (3)–(5) we have Eq. (7)
ꢁ
d½AHꢂ=dt ¼ R
i
=n:
The n value of TOH is generally taken as 2 [34,37] according to Eqs. (3) and (4), hence Ri
can be determined from the inhibition period (Eq. (6)) or from the decay rate of TOH (Eq.
(
7)). In the present experiment the R value calculated from the inhibition period is
i

J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
153
4
.2 nM/s. This value is in good agreement with the value of 4.6 nM/s obtained from the
decay of TOH. Similar an R value of 4.5 nM/s has been reported [34] by measuring the
i
decay of TOH with the same concentration of AAPH (10 mM).
The rate of oxygen uptake during the inhibition period is related to k and kinh accord-
p
ing to the steady-state treatment of the above equations as:
ꢁd½O
2
ꢂ=dt ¼ Rinh ¼ k
p
R
i
½LHꢂ=ðnkinh½AHꢂÞ:
ð8Þ
The kinetic chain length defines the number of chain propagations by each initiating rad-
ical and is given by Eqs. (9) and (10) for inhibited and uninhibited peroxidations respec-
tively. The kinetic data obtained from Figs. 2–4 are listed in Table 2.
kclinh ¼ Rinh=R
i
;
ð9Þ
ð10Þ
kcl
p
¼ R
p
=R
i
:
It can be seen from Figs. 2–4 and Table 2 that in the present experimental conditions the
endogenous TOH which contributes > 95% of endogenous antioxidants in native LDL
[
1,43] acts as a chain-breaking antioxidant, in accordance with the previous reports
Table 2
Kinetic parameters for the AAPH-initiated peroxidation of human LDL and its inhibition by resveratrol and its
a
analogues (ROH)
e
ROH
R
inh
ꢁ
R
p
t
inh
kclinh
kcl
p
k
inh
n
8
ꢁ1
ꢁ8
ꢁ1
5
ꢁ1 ꢁ1
(
10 M s
)
(10 M s
)
(min)
(10 M s )
b
f
Native
2.9 ± 0.3
2.5 ± 0.3
1.5 ± 0.4
1.8 ± 0.5
1.8 ± 0.1
1.3 ± 0.2
1.4 ± 0.1
1.8 ± 0.3
4.0 ± 0.2
2.7 ± 0.4
5.9 ± 0.4
4.1 ± 0.5
4.0 ± 0.4
4.5 ± 0.5
4.2 ± 0.2
3.0 ± 0.4
4.6 ± 0.6
4.4 ± 0.2
5.8 ± 0.6
4.5 ± 0.7
19.6 ± 0.8
23.8 ± 1.3
51 ± 2.1
37.2 ± 1.2
27.5 ± 1.6
51 ± 2.3
6.9
5.9
3.5
4.3
4.3
3.2
3.3
4.3
9.5
6.4
14.0
9.8
9.5
10.7
10.0
7.1
11.0
10.5
13.8
10.7
5.7
4.4
3.7
4.2
6.1
4.1
4.9
8.2
3.3
6.2
2
c
Resveratrol
1.5
3.2
2.3
1.7
3.2
2.5
1.2
1.4
1.1
0
0
c
3
3
2
3
3
4
3
2
,4,3 ,4 -THS
0
0
c
,4,4 -THS
,4,4 -THS
,3 -DM-4,4 -DHS
,4-DHS
,4 -DHS
,5-DHS
,4-DHS
c
0
0
c
c
39 ± 2.1
0
c
18.5 ± 0.5
21.7 ± 1.2
17.5 ± 1.9
c
c
d
Resveratrol
2.9 ± 0.5
2.7 ± 0.4
2.0 ± 0.3
2.2 ± 0.3
1.9 ± 0.2
2.7 ± 0.2
3.2 ± 0.2
4.3 ± 0.6
3.2 ± 0.4
4.5 ± 0.4
5.0 ± 0.5
5.0 ± 0.4
5.5 ± 0.5
3.5 ± 0.3
4.9 ± 0.5
5.0 ± 0.4
5.3 ± 0.8
4.6 ± 0.3
45.0 ± 0.6
64.2 ± 2.6
52.5 ± 2.1
43.0 ± 1.1
62.4 ± 2.5
58.0 ± 1.6
41.0 ± 1.1
35.3 ± 1.1
40.0 ± 1.9
6.9
6.5
4.7
5.2
4.5
6.4
7.6
10.2
7.6
10.7
11.9
11.9
13.1
8.3
11.7
11.9
12.6
11.0
2.1
1.8
2.5
2.0
1.7
2.4
2.3
1.6
1.4
1.6
0
0
d
3
3
2
3
3
4
3
2
,4,3 ,4 -THS
1.76
3.11
3.05
2.57
1.82
2.32
1.93
2.46
0
0
d
,4,4 -THS
d
,4,4 -THS
0
0
d
,3 -DM-4,4 -DHS
d
,4-DHS
,4 -DHS
,5-DHS
,4-DHS
a
0
d
d
d
Determined in PBS (pH 7.4) at 37 ꢁC; [LDL] = 0.2 mg/ml; [AAPH] = 10 mM; [ROH]
experiment was repeated three times and the SD are shown in the table. R
0
= 4 lM. Every
i
is taken as 4.2 nM/s by using Eq. (6).
k
inh is calculated by using Eq. (8).
b
Intrinsic TOH (2.47 lM) and other antioxidants in native LDL.
ROH added after the depletion of the intrinsic antioxidants (see Fig. 3).
ROH added before the initiation (see Fig. 4).
c
d
e
f
n = R
i 0 0
tinh/([ROH] + [TOH] ).
Assuming that TOH is the major antioxidants in native LDL and each TOH molecule traps two peroxyl
radicals (see text).

154
J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
[
5
8,42–44]. The inhibition rate constant, k , of a-tocopherol is calculated to be
inh
5
ꢁ1 ꢁ1
ꢁ1 ꢁ1
.7 · 10 M
s
according to Eq. (8), taking the k as 16.6 M
s
in phospholipid
p
membranes [45] and the concentration of polyunsaturated fatty acids, [LH], in LDL as
0
suring the cis, trans/trans, trans product ratios of the cholesteryl linoleate hydroperoxides
5
ꢁ1 ꢁ1
.55 M [1]. This value is close to the value of 5.9 · 10 M
s
reported recently by mea-
4
ꢁ1 ꢁ1
in LDL [44], but it is significantly higher than the value of 8 · 10 M
s
reported pre-
for 1,2-dimyristoylphosphatidyl-choline
DMPC) bilayers [45], due to the very high local concentration of LH in LDL particles.
All these compounds produce clear inhibition periods and have significantly
decreased kinetic chain lengths in the absence of endogenous antioxidants, demon-
strating that they are good antioxidants in LDL. The inhibition rate constant, kinh
of these compounds is comparable to that of TOH and the stoichiometric factor,
4
ꢁ1 ꢁ1
viously for liposomes [38] and 4.7 · 10 M
s
(
,
0
0
0 0
n, of 3,4,3 ,4 -THS is 3.2, implying that each molecule of 3,4,3 4 -THS might be able
to trap about three peroxyl radicals, hence more than one hydroxyl group must be
0
0
involved in the antioxidation process. These facts make 3,4,3 ,4 -THS the most active
antioxidant among these compounds. Based on the inhibition time and/or stoichiom-
etric factor the antioxidant efficacy of resveratrol and its analogues follows the
0
0
0
0
0
0
0
sequence of 3,4,3 ,4 -THS ꢀ 3,3 -DM-4,4 -DHS > 3,4-DHS ꢀ 3,4,4 -THS > 2,4,4 -THS
0
0
>
resveratrol ꢀ 3,5-DHS > 4,4 -DHS ꢀ 2,4-DHS. The similar activity of 3,4,3 ,4 -THS
0
0
0
and 3,3 -DM-4,4 -DHS is most likely due to the higher lipophilicity of 3,3 -DM-
0
4
,4 -DHS that makes it easier partition into LDL particles. We have found previously
that making vitamin C lipophilic could enhance its antioxidant activity in LDL [7].
It is well-known that TOH, the most abundant and active form of vitamin E, is a prin-
cipal lipid-soluble chain-breaking antioxidant in biomembranes [37] and in LDL [40]. Its
synergistic antioxidative effect with other antioxidants, such as L-ascorbic acid (vitamin C)
[
39] and green tea polyphenols [8,22], has been well documented and proven to be due to
Å
the reduction of a-tocopheroxyl radical (TO ) by the co-existent antioxidant (ArOH) to
regenerate TOH [Eq. (11)]. We have proven recently by using stopped-flow electron para-
magnetic resonance (EPR) spectroscopy that resveratrol and 3,4-DHS could reduce a-toc-
opheroxyl radical (TO ) to regenerate TOH (Eq. (11)) with bimolecular rate constants of
0
Å
2
2
ꢁ1 ꢁ1
.23 · 10 and 3.0 · 10 M
s
, respectively, in cetyl trimethylammonium bromide
(
CTAB) micelles [25].
Å
Å
TO þ ArOH ! TOH þ ArO
ð11Þ
It can be seen from Table 2 that the kinh decreases remarkably when resveratrol or
its analogue is added before the initiation, and the overall inhibition time is approx-
imately equal to the sum of the inhibition time of the endogenous antioxidants
(
principally TOH) in native LDL and that of resveratrol or its analogue when it
is added after depletion of the endogenous antioxidants. It is also seen from
Fig. 5 that no appreciable turning point is observed in the decay curve of TOH
0
0
in the presence of 3,4,3 ,4 -THS. These facts suggest that TOH and the exogenous
resveratrol analogue might act independently, i.e., no synergistic antioxidant interac-
tion takes place between the resveratrol analogues and TOH in LDL. This is prob-
ably due to the fact that the reaction rate of resveratrol analogues with peroxyl
5
ꢁ1 ꢁ1
radicals in LDL, kinh, is remarkably faster (10 M
TOH regeneration reaction (10 –10 M
pete with reaction (3).
s
) than the rate of the
1
2
ꢁ1 ꢁ1
s ). Hence the reaction (11) can not com-

J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
155
3
.5. The structure–activity relationship and antioxidant mechanism
The important event of lipid peroxidation is oxygen absorption and the final prod-
uct of the peroxidation is MDA [35]. In the present work the two indexes were used
to evaluate the antioxidative activity of resveratrol and its analogues to find the struc-
tural determinant responsible for the antioxidant activity in vitro. Comparison of the
data of Tables 1 and 2 demonstrates that the antioxidant activity of resveratrol and its
analogues follows a similar pattern, whether the peroxidation is initiated by AAPH or
2
+
by Cu , and in spite of the difference in activity being monitored by oxygen uptake
0
0
or by MDA formation. It is clearly seen that the antioxidative activity of 3,4,3 ,4 -
0
0
0
THS, 3,4-DHS, 3,4,4 -THS and 3,3 -DM-4,4 -DHS is significantly higher than that
of the other compounds. That is, molecules bearing ortho-dihydroxyl or 4-hydroxy-
3
-methoxyl functionality, are appreciably more active than those bearing no such
functionalities. It has been proven that the ortho-methoxyl group can form an intra-
molecular hydrogen bond with the phenolic hydrogen, making the H-atom abstraction
from the ortho-methoxyphenols surprisingly easy [46]. It was also known that the
ortho-hydroxyl substitution on phenol would make the oxidation intermediate, ortho-
hydroxyphenoxyl radical, more stable due to the intramolecular hydrogen bonding
interaction as reported recently from both experiment by spectrophotometric measure-
ments [47] and theoretical calculations [48] (Scheme 1). The theoretical calculation
showed that the intramolecular H-bond in ortho-OH phenoxyl radical is ca. 4 kcal/
mol stronger than that in the parent molecule catechol and the bond dissociation
energy (BDE) of catechol is 9.1 kcal/mol lower than that of phenol [48]. In addition,
ortho-OH phenoxyl radical and/or ortho-semiquinone radical anion will be more easily
further oxidized to form the final product ortho-quinone as exemplified in Scheme 1
0
0
0
[
47]. The fact that the stoichiometric factor, n, of 3,4,3 ,4 -THS, 3,4-DHS, 3,4,4 -
THS is larger than two (Table 2) suggests that the second peroxyl radical must be
involved in the antioxidation reaction leading to the formation of the corresponding
ortho-quinone as shown in Scheme 1. We have recently found the significantly higher
antioxidant activity of molecules bearing ortho-diphenoxyl functionality in flavonols
[
9,21] and curcumin analogues [49,50].
Scheme 1. Mechanism of 3,4-DHS inhibited peroxidation.

156
J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
4
. Conclusion
The present investigation demonstrates that resveratrol and its analogues are effective
2
+
antioxidants against AAPH- and Cu -induced LDL peroxidation. Of particular interest
is the finding that the compounds bearing ortho-dihydroxyl or 4-hydroxy-3-methoxyl
functionality exhibit remarkably higher antioxidative activity than the ones bearing no
such functionalities. It gives us useful information for antioxidant drug design. In addi-
tion, synergistic antioxidant interaction between resveratrol analogues and a-tocopherol
is not observed in LDL.
Acknowledgment
We thank the National Natural Science Foundation of China (Grant Nos. 20502010,
2
0332020 and 20021001) for financial support.
References
[
[
[
[
[
[
[
[
[
1] H. Esterbauer, P. Ramos, Rev. Physiol. Biochem. Pharmacol. 127 (1995) 31–64, and references cited therein.
2] P. Klatt, H. Esterbauer, J. Cardiov. Risk 3 (1996) 346–351.
3] W.A. Pryor, Free Radic. Biol. Med. 28 (2000) 1681–1682.
4] A. Mertens, P. Holvoet, FASEB J. 15 (2001) 2073–2084.
5] J.M. Upston, L. Kritharides, R. Stocker, Progr. Lipid Res. 42 (2003) 405–422.
6] I. Jialal, S.M. Grundy, Circulation 88 (1993) 2780–2786.
7] Z.-Q. Liu, L.-P. Ma, Z.-L. Liu, Chem. Phys. Lipids 95 (1998) 49–57.
8] Z.-Q. Liu, L.-P. Ma, B. Zhou, L. Yang, Z.-L. Liu, Chem. Phys. Lipids 106 (2000) 53–63.
9] L. Hou, B. Zhou, L. Yang, Z.-L. Liu, Chem. Phys. Lipids 129 (2004) 209–219.
[
[
[
10] L. Fremont, Life Sci. 66 (2000) 663–671.
11] G.J. Soleas, E.P. Diamandis, D.M. Goldberg, Clin. Biochem. 30 (1997) 91–113.
12] R. Corder, J.A. Douthwaite, D.M. Lees, N.Q. Khan, A.C.V.D. Santos, E.G. Wood, M.J. Carrier, Nature
414 (2001) 863–864.
[
[
13] M.J. Burkitt, J. Duncan, Arch. Biochem. Biophys. 381 (2000) 253–263.
14] P.S. Ray, G. Maulik, G.A. Cordis, A.A.E. Bertelli, A. Bertelli, D.K. Das, Free Radic. Biol. Med. 27 (1999)
160–169.
[
[
15] R. Amorati, M. Lucarini, V. Mugnaini, G.F. Pedulli, J. Org. Chem. 69 (2004) 7101–7107.
16] M. Murias, W. J a¨ ger, N. Handler, T. Erker, Z. Horvath, T. Szekeres, H. Nohl, L. Gille, Biochem.
Pharmacol. 69 (2005) 903–912.
[
[
17] M.S. Jang, E.N. Cai, G.O. Udeani, K.V. Slowing, C.F. Thomas, C.W.W. Beecher, H.H.S. Fong, N.R.
Farnsworth, A.D. Kinghorn, R.G. Mehta, R.C. Moon, J.M. Pezzuto, Science 275 (1997) 218–220.
18] A. Domianaki, E. Bakogeorgou, M. Kampa, G. Notas, A. Hatzoglou, S. Panagiotou, C. Gemetzi, E.
Kouroumalis, P.M. Martin, E. Castanas, J. Cell. Biochem. 78 (2000) 429–441.
19] M. Jang, J.M. Pezzuto, Drugs Exp. Clin. Res. 25 (1999) 65–77.
[
[
[
[
[
[
[
20] J.K. Kundu, Y.J. Surh, Mutat. Res. 555 (2004) 65–80.
21] B. Zhou, Q. Miao, L. Yang, Z.-L. Liu, Chem. Eur. J. 11 (2005) 680–691.
22] B. Zhou, L.-M. Wu, L. Yang, Z.-L. Liu, Free Radic. Biol. Med. 38 (2005) 78–84.
23] B. Zhou, Z.-L. Liu, Pure Appl. Chem. 11 (2005) 1887–1903.
24] Q.-Y. Wei, B. Zhou, Y.-J. Cai, L. Yang, Z.-L. Liu, Food Chem. 96 (2006) 90–95.
25] J.-G. Fang, M. Lu, Z.-H. Chen, H.-H. Zhu, Y. Li, L. Yang, L.-M. Wu, Z.-L. Liu, Chem. Eur. J. 8 (2002)
4191–4198.
[
[
26] Y.-J. Cai, J.-G. Fang, L.-P. Ma, L. Yang, Z.-L. Liu, Biochim. Biophys. Acta 1637 (2003) 31–38.
27] Y.J. Cai, Q.Y. Wei, J.G. Fang, L. Yang, Z.-L. Liu, J.-H. Wyche, Z. Han, Anticancer Res. 24 (2004) 999–
1002.
[
[
28] K. Thakkar, R.L. Geahlen, M. Cushman, J. Med. Chem. 36 (1993) 2950–2955.
29] F.W. Bachelor, A.A. Loman, I.R. Snowdon, Can. J. Chem. 48 (1970) 1554–1557.

J.-C. Cheng et al. / Bioorganic Chemistry 34 (2006) 142–157
30] J.E. McMurry, Chem. Rev. 89 (1989) 1513–1524.
31] H.A. Ali, K. Kondo, Y. Tsuda, Chem. Pharm. Bull. 40 (1992) 1130–1136.
32] B.H. Chung, T. Wilkinson, I.C. Geer, J.P. Segrest, J. Lipids. Res. 21 (1980) 284–291.
33] O.H. Lowry, N.J. Rosebrough, A.C. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265–275.
34] V.W. Bowry, R. Stocker, J. Am. Chem. Soc. 115 (1993) 6029–6044.
35] J.A. Buege, S.D. Aust, Meth. Enzymol. 52 (1978) 302–310.
36] P. Palozza, N.I. Krinsky, Arch. Biochem. Biophys. 297 (1999) 184–187.
37] G.W. Burton, K.U. Ingold, Acc. Chem. Res. 19 (1986) 194–201.
38] L.R.C. Barclay, Can. J. Chem. 71 (1993) 1–16.
39] E. Niki, T. Saito, A. Kawakami, Y. Kamiya, J. Biol. Chem. 259 (1984) 4177–4182.
40] H. Esterbauer, M. Dieber-Rothenneder, G. Striegl, G. Wang, Am. J. Clin. Nutr. 53 (1991) 314S–321S.
41] V.W. Bowry, K.U. Ingold, Acc. Chem. Res. 32 (1999) 27–34.
42] E. Niki, N. Noguchi, N. Gotoh, Biochim. Soc. Trans. 21 (1993) 313–317.
43] N. Noguchi, N. Gotoh, E. Niki, Biochim. Biophys. Acta 1168 (1993) 348–357.
157
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
44] S.M. Culbertson, F. Antunes, C.M. Havrilla, G.L. Milne, N.A. Porter, Chem. Res. Toxicol. 15 (2002) 870–
76.
8
[
[
[
[
[
[
45] L.R.C. Barclay, M.R. Vinqvist, F. Antunes, R.E. Pinto, J. Am. Chem. Soc. 119 (1997) 5764–5765.
46] M.I. de Heer, P. Mulder, H.G. Korth, K.U. Ingold, J. Lusztyk, J. Am. Chem. Soc. 122 (2000) 2355–2360.
47] M. Foti, G. Ruberto, J. Agric. Food Chem. 49 (2001) 342–348.
48] J.S. Wright, E.R. Johnson, G.A. Dilabio, J. Am. Chem. Soc. 123 (2001) 1173–1183.
49] Q.-Y. Wei, W.-F. Chen, B. Zhou, L. Yang, Z.-L. Liu, Biochim. Biophys. Acta 1760 (2006) 70–77.
50] W.-F. Chen, S.-L. Deng, B. Zhou, L. Yang, Z.-L. Liu, Free Radic. Biol. Med. 40 (2006) 526–535.