Structure-activity relationship and mechanism of the tocopherol- regenerating activity of resveratrol and its analogues

Article abstract of DOI:10.1021/jf802665s

The present study investigated the mechanism of the synergistic antioxidant activity of α-tocopherol with resveratrol (3,5,4′-trihydroxy-trans- stilbene, 3,5,4′-THS) and its synthetic analogues, that is, 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4′-dihydroxy-trans-stilbene (4,4′-DHS), 4-hydroxy-trans-stilbene (4-HS), and 3,5-dihydroxy-trans- stilbene (3,5-DHS). The reaction kinetics of α-tocopheroxyl radical with resveratrol and its analogues were studied in sodium dodecyl sulfate (SDS) and cetyl trimethylammonium bromide (CTAB) micelles using the stopped-flow electron paramagnetic resonance (EPR) technique. It was found that resveratrol and its analogues could regenerate α-tocopherol with rate constants (k _REG) in the order of 3,4-DHS > 4,4′-DHS > resveratrol ≥ 4-HS ≥ 3,5-DHS in SDS and CTAB micelles. It was found that the analogues bearing o-dihydroxyl groups (3,4-DHS) and p-dihydroxyl groups (4,4′-HS) exhibited remarkably higher α-tocopherol-regenerating activity than those bearing no such groups (resveratrol, 4-HS, and 3,5-DHS). In addition, the α-tocopherol-regenerating activity of resveratrol and its analogues was correlated with their electrochemical behaviors, suggesting that electron transfer might play a critical role during the regeneration reaction.

Full text of DOI:10.1021/jf802665s

11458 J. Agric. Food Chem. 2008, 56, 11458–11463
Structure-Activity Relationship and Mechanism of
the Tocopherol-Regenerating Activity of Resveratrol
and Its Analogues
JIAN-GUO FANG AND BO ZHOU*
State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou,
Gansu 730000, China
The present study investigated the mechanism of the synergistic antioxidant activity of R-tocopherol
with resveratrol (3,5,4′-trihydroxy-trans-stilbene, 3,5,4′-THS) and its synthetic analogues, that is, 3,4-
dihydroxy-trans-stilbene (3,4-DHS), 4,4′-dihydroxy-trans-stilbene (4,4′-DHS), 4-hydroxy-trans-stilbene
(4-HS), and 3,5-dihydroxy-trans-stilbene (3,5-DHS). The reaction kinetics of R-tocopheroxyl radical
with resveratrol and its analogues were studied in sodium dodecyl sulfate (SDS) and cetyl
trimethylammonium bromide (CTAB) micelles using the stopped-flow electron paramagnetic resonance
(EPR) technique. It was found that resveratrol and its analogues could regenerate R-tocopherol with
rate constants (kREG) in the order of 3,4-DHS > 4,4′-DHS > resveratrol g 4-HS g 3,5-DHS in SDS
and CTAB micelles. It was found that the analogues bearing o-dihydroxyl groups (3,4-DHS) and
p-dihydroxyl groups (4,4′-HS) exhibited remarkably higher R-tocopherol-regenerating activity than
those bearing no such groups (resveratrol, 4-HS, and 3,5-DHS). In addition, the R-tocopherol-
regenerating activity of resveratrol and its analogues was correlated with their electrochemical
behaviors, suggesting that electron transfer might play a critical role during the regeneration reaction.
KEYWORDS: Reveratrol;r-tocopherol;antioxidant;antioxidantsynergism;lipidperoxidation;structure-activity
relationship
INTRODUCTION
and the most active form of vitamin E, is the major endogenous
lipid-soluble chain-breaking antioxidant in human plasma
and low-density lipoprotein (LDL) (9). It can effectively trap
the lipid peroxyl radical (LOO•) to inhibit the free-radical-
initiated lipid peroxidation within lipid domains by its own
conversion into an oxidized product, R-tocopheroxyl radical
(TO•) (Figure 2). The original form can be regenerated by
another coexisting antioxidant, such as vitamin C, ubiquinol-
10, and green tea polyphenols (10-13). This regeneration
reaction enhances the antioxidant capacity of vitamin E and,
therefore, has led to the clinical investigation of vitamin C used
in concert with vitamin E supplementation (14). We previously
found that resveratrol and its analogues exhibited a synergistic
Resveratrol (3,5,4′-trihydroxy-trans-stilbene) (Figure 1) is a
phytoalexin and a naturally occurring phenolic compound
belonging to a stilbene family found in a wide variety of dietary
sources including grapes, plums, and peanuts (1, 2). Its relatively
high concentration in red wine (0.1-14.3 mg/L) (2) and near
absence in white varietals and grape juice have led to its being
proposed as the agent responsible for the so-called “French
paradox”; that is, mortality from coronary heart disease is lower
in some regions of France than in other western countries due
to the regular drinking of wine despite a high fat intake (3).
Growing evidence suggests that resveratrol plays a role in the
prevention of human diseases, such as heart disease, neurode-
generative disease, and cancer (1, 2). The wide range of
biological effects elicited by resveratrol is believed to be due
to its powerful antioxidant property (1, 4), because free-radical-
mediated peroxidation of membrane lipids and oxidative damage
of DNA are considered to be associated with a wide variety of
chronic health problems, such as atherosclerosis, cancer, and
aging (5, 6). Therefore, research interest in the antioxidative
activity of resveratrol is growing (7, 8).
In the antioxidant family vitamin E plays a central role. It is
well-known that R-tocopherol (TOH), the principal component
* Corresponding author (fax +86-931-8915557; e-mail bozhou@
lzu.edu.cn).
Figure 1. Molecular structures of resveratrol and its analogues.
10.1021/jf802665s CCC: $40.75  2008 American Chemical Society
Published on Web 11/05/2008

Tocopherol-Regenerating Activity of Resveratrol and Its Analogues
J. Agric. Food Chem., Vol. 56, No. 23, 2008 11459
Figure 2. Mechanism for the synergistic antioxidant activity of phenolics (ArOHs) and R-tocopherol (TOH).
resveratrol and its analogues act as antioxidants synergistically
with R-tocopherol and (ii) investigate the reaction kinetics of
R-tocopheroxyl radical with resveratrol and its analogues
(ROHs), that is, 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4′-
dihydroxy-trans-stilbene (4,4′-DHS), 4-hydroxy-trans-stilbene
(4-HS), and 3,5-dihydroxy-trans-stilbene (3,5-DHS) (Figure 1)
in sodium dodecyl sulfate (SDS) and cetyl trimethylammonium
bromide (CTAB) micelles.
MATERIALS AND METHODS
Materials. Resveratrol and its analogues 3,4-DHS, 4,4′-DHS, 4-HS,
and 3,5-DHS were synthesized as previously described (16, 17). This
method produced exclusively the trans-isomer. Their structures were
1
fully identified using H NMR and EI-MS (see below), and the data
were consistent with those reported in the literature (16, 17). The purity
(>98%) of each compound was checked using high-performance liquid
chromatography (HPLC).
Figure 3. Formation of total hydroperoxides (LOOH) during the peroxi-
dation of linoleic acid (LH) in 0.1 M sodium dodecyl sulfate (SDS) at pH
7.4 and 37 °C, initiated with AAPH and inhibited by resveratrol and its
analogues (ROHs) and/or R-tocopherol (TOH): (a) uninhibited peroxidation;
(b) inhibited with TOH; (c) inhibited with resveratrol; (d) inhibited with
3,4-DHS; (e) inhibited with resveratrol and TOH; (f) inhibited with 3,4-
DHS and TOH. [LH]0 ) 15.2 mM, [AAPH]0 ) 6.3 mM, [ROHs] ) 11.2
µM, [TOH] ) 5 µM.
Resveratrol: 1H NMR (300 MHz, (CD3)2CO), δ 6.26 (1H, t, J )
1.8 Hz, H-4), 6.54 (2H, d, J ) 1.8 Hz, H-2 and H-6), 6.83 (2H, d, J )
9.0 Hz, H-3′ and H-5′), 6.89, 6.99 (each 1H, d, J ) 16.5 Hz, H-7 or
H-8), 7.40 (2H, d, J ) 9.0 Hz, H-2′ and H-6′); MS, m/z 228 [M+],
211, 199, 181, 157, 115.
1
3,4-DHS: H NMR (300 MHz, (CD3)2CO), δ 6.81 (1H, d, J ) 8.1
Hz, H-5), 6.91 (1H, dd, J ) 8.4, 1.8 Hz, H-6), 7.00 (1H, s, J ) 7.8 Hz,
H-2), 7.07, 7.12 (each 1H, d, J ) 16.2 Hz, H-7 or H-8), 7.20 (1H, t,
J ) 7.5 Hz, H-4′), 7.32 (2H, t, J ) 7.8 Hz, H-3′ and H-5′), 7.51 (2H,
d, J ) 7.8 Hz, H-2′ and H-6′); MS, m/z 212 [M+], 195, 165, 141, 115,
77.
1
3,5-DHS: H NMR (300 MHz, (CD3)2CO), δ 6.33 (1H, t, J ) 2.4
Hz, H-4), 6.61 (2H, d, J ) 1.5 Hz, H-2 and H-6), 7.10 (2H, s, H-7 and
H-8), 7.24 (1H, t, J ) 7.8 Hz, H-4′), 7.35 (2H, t, J ) 8.1 Hz, H-3′ and
H-5′), 7.55 (2H, d, J ) 7.8 Hz, H-2′ and H-6′); MS, m/z 212 [M+],
195, 165, 141, 115, 77.
4-HS: 1H NMR (300 MHz, (CD3)2CO), δ 6.84 (2H, d, J ) 8.7 Hz,
H-3 and H-5), 7.08, 7.15 (2H, d, J ) 16.5 Hz, H-7 or H-8), 7.23 (1H,
dd, J ) 7.8, 2 Hz, H-4′), 7.34 (2H, t, J ) 8.1 HZ, H-2 and H-6), 7.45
(2H, b, J ) 8.7 HZ, H-3′ and H-5′), 7.54 (2H, d, J ) 7.5 Hz, H-2′ and
H-6′); MS, m/z 196 [M+], 181, 165, 139, 115, 77.
4,4′-DHS, 1H NMR (300 MHz, (CD3)2CO), δ 6.82 (4H, d, J ) 8.7
Hz, H-3, H-5, H-3′, and H-5′), 6.96 (2H, s, H-7 and H-8), 7.39 (4H, d,
J ) 8.7 Hz, H-2, H-6, H-2′, and H-6′); MS, m/z 212 [M+], 197, 165,
141, 115, 77.
Linoleic acid (chromatographic pure, Sigma-Aldrich, St. Louis, MO),
dl-R-tocopherol (biochemical reagent, >99.9%, Merck, Darmstadt,
Germany) and 2,2′-azobis(2-methylpropionamidine) dihydrochloride
(AAPH; Sigma-Aldrich) were kept under nitrogen in a refrigerator. The
surfactants SDS and CTAB were recrystallized from ethyl alcohol and
acetone/water (9:1, v/v), respectively.
Figure 4. Formation of total hydroperoxides (LOOH) during the peroxi-
dation of linoleic acid (LH) in 15 mM cetyl trimethylammonium bromide
(CTAB) at pH 7.4 and 37 °C, initiated with AAPH and inhibited by
resveratrol and its analogues (ROHs) and/or R-tocopherol (TOH): (a)
uninhibited peroxidation; (b) inhibited with TOH; (c) inhibited with
resveratrol; (d) inhibited with 3,4-DHS; (e) inhibited with resveratrol and
TOH; (f) inhibited with 3,4-DHS and TOH. [LH]0 ) 15.2 mM, [AAPH]0 )
6.3 mM, [ROHs] ) 11.2 µM, [TOH] ) 5 µM.
antioxidant activity with R-tocopherol in micelles (15). The
present study was to (i) study the mechanism by which
Determination of Linoleic Acid Hydroperoxides. The linoleic acid
micelles were prepared by mixing appropriate amounts of linoleic acid,

11460 J. Agric. Food Chem., Vol. 56, No. 23, 2008
Fang and Zhou
Table 1. Inhibition of AAPH-Initiated Peroxidation of Linoleic Acid by Resveratrol and Its Analogues (ROHs) and R-Tocopherol (TOH) in Sodium Dodecyl
Sulfate (SDS) and Cetyl Trimethylammonium Bromide (CTAB) Micellesa,b
d
inh/104c M-1 s-1
micelle
ROH
Rp/10-8 M s-1
R
inh/10-8 M s-1
t
inh/103 s
n′
SE (%)
k
SDS
SDS
SDS
SDS
SDS
SDS
SDS
SDS
SDS
SDS
SDS
SDS
none
resveratrol
4-HS
3,5-DHS
4,4′-DHS
3,4-DHS
TOH
resveratrol + TOH
4-HS + TOH
3,5-DHS + TOH
4,4′-DHS + TOH
3,4-DHS + TOH
8.3
9.0
9.2
7.8
8.0
8.2
8.1
7.0
7.9
8.3
7.4
6.9
2.8
4.0
3.8
2.6
0.8
1.1
0.9
0.8
1.0
0.5
∼0
3.1
2.1
1.5
3.2
4.9
3.0
6.3
5.3
4.8
6.6
12.6
1.3
1.2
1.7
1.4
2.9
3.6
2.0
2.3
2.1
3.1
e
0.8
0.6
0.4
0.9
1.4
2.0
1.2
1.0
0.9
1.3
2.4
∼0
∼0
∼0
∼0
59.5
CTAB
CTAB
CTAB
CTAB
CTAB
CTAB
CTAB
CTAB
CTAB
CTAB
CTAB
CTAB
none
16.8
16.0
19.8
15.6
14.8
14.0
16.7
12.3
17.8
13.6
14.3
11.7
resveratrol
4-HS
3,5-DHS
4,4′-DHS
3,4-DHS
TOH
resveratrol + TOH
4-HS + TOH
3,5-DHS + TOH
4,4′-DHS + TOH
3,4-DHS + TOH
2.7
3.2
4.2
2.8
1.6
2.0
0.1
0.3
0.3
0.2
∼0
2.7
1.7
2.4
2.6
4.1
1.2
4.4
2.5
3.0
4.5
7.8
0.7
1.0
0.5
0.8
0.9
2.0
10.9
6.4
6.3
7.5
e
2.0
1.2
1.8
1.9
3.0
2.0
2.2
1.3
1.5
2.3
4.0
12.8
∼0
∼0
18.4
47.2
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, which were reproducible with deviation of <(10%. b Because the n value of R-tocopherol
is generally assumed to be 2 (29), the apparent rate of chain initiation, Ri, can be determined from the inhibition period and/or the decay rate of R-tocopherol. Ri is taken
as 3.1 and 8.3 nM s-1 in SDS and CTAB micelles, respectively. c Calculated from the equation Rinh ) kp Ri[LH]/(nkinh[AH])) by taking kp ) 37 M-1 s-1 (30). d n′ )
Ritinh/([ROH]0 + [TOH]0). e Could not be calculated because Rinh is approximately zero.
Table 2. Rate Constants for the Reactions R-Tocopheroxyl Radical (TO•)
with Resveratrol and Its Analogues (ROHs) in Micellesa,b
SDS
CTAB
kREG 102
(M-1 s-1
Ep
k
REG102
Ep
ROH
)
(V vs SCE)
(M-1 s-1
)
(V vs SCE)
resveratrol
4-HS
3,5-DHS
4,4′-DHS
3,4-DHS
0.19
0.11
0.10
0.61
1.35
0.62
0.64
0.85
0.40
0.34
0.20
0.14
0.19
0.28
2.99
0.67
0.66
0.79
0.43
0.36
a Determined by EPR in sodium dodecyl sulfate (SDS) and 15 mM cetyl
trimethylammonium bromide (CTAB) micelles at pH 7.4 and room temperature
under air. b The values of kREG and Ep are given with errors usually <10%.
phosphate-buffered saline (pH 7.4) and injected into the micelles to
initiate the peroxidation.
The linoleic acid micelles were placed in an open vessel of 2 mL in
volume, thermostated at 37 °C and provided with a magnetic stirrer.
The formation of linoleic acid hydroperoxides (LOOH) was determined
using HPLC as described previously (18). Briefly, the reaction mixture
was sampled at various time intervals and subjected to HPLC analysis
on a Gilson liquid chromatograph equipped with a Zorbax ODS
reversed-phase column (6 × 250 mm, DuPont Instruments), then eluted
with a methanol/water mixture (9:1, v/v). The flow rate was set at 1.0
mL/min. Every determination was repeated three times, and the
experimental deviations were within (10%.
Determination of Oxidation Potentials. The oxidation potentials
of ROHs were determined on a PAR 173 potentiostat with a glassy
carbon electrode in phosphate-buffered micelles (pH 7.4) at room
temperature, as described previously (19). The potential was recorded
relative to a saturated calomel electrode (SCE).
Determination of r-Tocopheroxyl Radical. Electron paramagnetic
resonance (EPR) was carried out on a Bruker ER 200D spectrometer
operated in the X-band with 100 kHz modulation, a modulation
amplitude of 0.25 mT, a time constant of 0.2 s, and a microwave power
of 25 mW. A flat quartz flow cell (0.4 × 5.5 × 60 mm) was used for
Figure 5. Cyclic voltammograms of resveratrol and its analogues (ROHs)
recorded at a glassy carbon electrode in 0.1 M sodium dodecyl sulfate
(SDS) and 30 mM cetyl trimethylammonium bromide (CTAB) micelles at
pH 7.4.
ΤꢀΗ, resveratrol, and its analogues, when required, in an aqueous
solution of 0.1 M SDS or 15 mM CTAB (pH 7.4) followed by vigorous
mixing in a vortex mixer for 2 min. AAPH was directly dissolved in

Tocopherol-Regenerating Activity of Resveratrol and Its Analogues
J. Agric. Food Chem., Vol. 56, No. 23, 2008 11461
were used individually, demonstrating clearly the antioxidant
synergism of 3,4-DHS with TOH. The peroxidation was
completely inhibited by 3,4-DHS and TOH in combination in
SDS micelles. Similar results were also obtained in CTAB
micelles (Figure 4). On the basis of the inhibition period and
synergistic efficiency, the antioxidant activity follows the
sequence 3,4-DHS > 4,4′-DHS > resveratrol ≈ 4-HS > 3,5-
DHS.
SE % ) {tinh(TOH + ArOH)-[tinh(TOH) +
tinh(ArOH)]}/[tinh(TOH) + tinh(ArOH)] × 100 (1)
The kinetic parameters deduced from Figures 3 and 4 are
listed in Table 1. The kinetics of linoleic acid peroxidation
initiated by azo compounds and its inhibition by chain-breaking
antioxidant (AH) have been discussed in detail in our previous
paper (24). In Table 1, kinh is the rate constant for the chain
inhibition by the antioxidant (eq 2), and n is the stoichiometric
factor that designates the number of peroxyl radicals trapped
by each antioxidant molecule.
Figure 6. Decay of R-tocopheroxyl radical (TO•) in 15 mM cetyl
trimethylammonium bromide (CTAB) micelles at pH 7.4 and room
temperature under air: (a) intrinsic decay; (b) in the presence of resveratrol
(0.78 mM); (c) in the presence of 3,4-DHS (0.13 mM). (Inset) EPR spectra
of the TO• recorded in CTAB micelles.
the stopped-flow determination of the reaction kinetics as described
previously (20). R-Tocopheroxyl radical was generated by vigorously
stirring R-tocopherol (1 mM) and excess lead oxide with a vortex mixer
for 3 min in 0.1 M SDS or 15 mM micelles (pH 7.4) at room
temperature.
kinh
LOO• + ROH 8 LOOH + RO•
(2)
Determination of Oxidation Potentials. To rationalize the
structure-activity relationship and mechanism of the antioxidant
synergism of ROHs and ΤꢀΗ, the oxidation potentials were
determined using a cyclic voltammetric method (Figure 5 and
Table 2). It can be seen (Figure 5 and Table 2) that the
analogues bearing o-dihydroxyl or p-dihydroxyl groups (3,4-
DHS and 4,4′-DHS) showed lower oxidation potentials and
reversible cyclic voltammograms, and those bearing no such
groups (resveratrol, 4-HS and 3,5-DHS) showed higher oxida-
tion potentials and irreversible cyclic voltammograms. The
results demonstrate that 3,4-DHS and 4,4′-DHS exhibited higher
activity, whereas resveratrol, 4-HS and 3,5-DHS were less
active.
Direct Determination of the Rate of r-Tocopherol Re-
generation Reaction. TOH, the most biologically and chemi-
cally active form of vitamin E, is considered to be the major
antioxidant in human plasma and low-density lipoprotein (LDL)
(9). The antioxidative activity of TOH can be restored when
the R-tocopheroxyl radical (TO•) is reduced by the coexistent
antioxidant (Figure 2), such as L-ascorbic acid (vitamin C) (10)
and green tea polyphenols (11, 12). To rationalize if resveratrol
and its analogues (ROHs) have similar mechanisms, the decay
kinetics of R-tocopheroxyl radical (TO•) in the presence of the
former were determined by stopped-flow EPR technique as
described previously (20). R-Tocopheroxyl radical (TO•) was
produced by oxidizing TOH with PbO2 in micelles. R-Toco-
pheroxyl radical (TO•) is usually more persistent in micelles
than in homogeneous solutions. The rate constants of the
bimolecular self-reaction of TO• were reported to be 3 × 103
M-1 s-1 in benzene/di-tert-butyl peroxide (25) and 75 and 15
M-1 s-1 in SDS and CTAB micelles (12), respectively.
Therefore, the reaction kinetics of TO• could be easily deter-
mined in micelles at ambient temperature (20). The inset of
Figure 6 shows the EPR spectrum of TO• recorded in CTAB
micelles. Addition of resveratrol 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 6). 3,4-DHS
reacted with TO• more rapidly than resveratrol (line c in Figure
6). Other ROHs showed similar enhanced decay (data not
shown). Plotting this first-order rate constant kobs versus the
concentration of ROHs gave a straight line from which the
second-order rate constant, kREG, between TO• and ROHs, could
RESULTS AND DISCUSSION
Synergistic Inhibition on Linoleic Acid Peroxidation by
Resveratrol, Its Analogues, and r-Tocopherol. Peroxidation
of linoleic acid or its ester as a model of unsaturated lipids gives
different hydroperoxides depending on the reaction conditions
(21). Hydroperoxide substitution at 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 a small amount of antioxidant, for example,
millimolar concentrations of R-tocopherol (21). They showed
characteristic ultraviolet absorption at 235 nm (22) that was used
to monitor the formation of the total hydroperoxides formed
during the peroxidation after separation of the reaction mixture
by HPLC.
A set of representative kinetic curves of the total hydroper-
oxides formation during the peroxidation of linoleic acid in SDS
micelles is shown in Figure 3. It can be seen (line a in Figure
3) that, upon AAPH initiation, concentration of the hydroper-
oxide increased quickly and linearly with time in the absence
of antioxidants, demonstrating the fast linoleic acid peroxidation.
The slope of this line corresponds to the rate of propagation,
Rp. The peroxide formation was remarkably inhibited by the
addition of R-tocopherol (line b) during the so-called “inhibition
period”. After the inhibition period, the rate of hydroperoxide
formation increased to close to the original rate of propagation,
corresponding to the exhaustion of the antioxidant. The turning
point from the inhibition period to the restoration of propagating
rate refers to the inhibition time, tinh. 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 reflects the antioxidative potential of the
antioxidant. Addition of resveratrol or 3,4-DHS showed also a
clear inhibition period, demonstrating that these compounds
acted well as chain-breaking antioxidants in SDS micelles (lines
c and d). Other ROHs (4,4′-DHS, 4-HS, and 3,5-DHS) also
showed similar inhibition periods (data not shown). Addition
of 3,4-DHS together with ΤꢀΗ significantly prolonged the
inhibition period of the latter (line f). The tinh of TOH and 3,4-
DHS when they were used together in SDS micelles was
approximately 60% [(synergistic efficiency SE %) (23), eq 1]
longer than the sum of the tinh values when the two antioxidants

11462 J. Agric. Food Chem., Vol. 56, No. 23, 2008
Fang and Zhou
Scheme 1. Mechanism for the Synergistic Antioxidant Activity of 3,4-DHS and ΤꢀΗ in Micellesa
a The circle denotes the surface of the micelles. LOO•, propagating peroxyl radical; LOOH, linoleic acid hydroperoxides; TO•, R-tocopheroxyl radical; TOH,
R-tocopherol.
be obtained (Table 2). This provides unambiguous evidence
that these comounds could reduce R-tocopheroxyl radical to
regenerate R-tocopherol (eq 3). The rate constant for TOH
regeneration by ROHs, kREG, decreased in the order 3,4-DHS
> 4,4′-DHS > resveratrol g 4-HS g 3,5-DHS in SDS and
CTAB micelles. On the other hand, the Ep values increased in
the order 3,4-DHS < 4,4′-DHS < resveratrol ≈ 4-HS < 3,5-
DHS, as listed in Table 2. The structure-activity relationship
obtained from this experiment is similar to that obtained from
the inhibition of linoleic acid peroxidation by HPLC method
noted above.
Obviously, the relative contribution of these processes depends
on the nature of the reaction medium and the pH, as well as the
oxidation potential and the acid dissociation constant (pKa) of
ROHs. Mukai et al. have proven that the rate constant for TOH
regeneration reaction by green tea polyphenols (11) and
flavonols, such as quercetin and rutin (27), in micellar solution
was pH-dependent and greater with increasing pH value.
The present study demonstrates that analogues bearing
o-dihydroxyl groups (3,4-DHS) or bearing p-dihydroxyl groups
(4,4′-HS) exhibited remarkably higher activity than those bear-
ing no such groups (resveratrol, 4-HS, and 3,5-DHS) in the
tocopherol regenaration reaction. This can be explained because
the o-hydroxyl phenoxyl radical, the oxidation intermediate for
3,4-DHS, is more stable due to the intramolecular hydrogen-
bonding interaction, as evidenced recently from theoretical
calculations (28) (Scheme 1). The theoretical calculation showed
that the hydrogen bond in the o-OH phenoxyl radical was
approximately 4 kcal/mol stronger than that in the parent
catechol and that the bond dissociation energy (BDE) of catechol
was 9.1 kcal/mol lower than that of phenol and 8.8 kcal/mol
lower than that of resorcinol (28). In the case of 4,4′-DHS, the
intermediate formation of p-OH phenoxyl radical and/or p-
semiquinone radical anion can be stabilized by the 4′-OH group
by resonance through the trans double bond.
kREG
TO• + ROH
8 TOH + RO•
(3)
Because the rate of this TOH regeneration reaction (kREG
≈
102 M-1 s-1) is approximately 2 orders of magnitude slower
than that of the antioxidation reaction (eq 2), kinh ≈ 104 M-1
s-1], the antioxidant synergism can only be observed when the
rate of the TOH regeneration reaction is high enough to compete
with the antioxidation reaction.
Structure-Activity Relationship and Mechanism for the
TOH Regeneration Reaction. It is worth noting that the
tocopherol-regenerating activity of ROHs is correlated with
the electrochemical behavior of the molecule (Table 2),
implying that the electron transfer between R-tocopheroxyl
radical (TO•) and ROHs might also contribute to the reaction
as shown in eq 4.
In summary, this work provides unambiguous evidence that
resveratrol and its analogues can effectively reduce R-toco-
pheroxyl radical to regenerate R-tocopherol in the order 3,4-
DHS > 4,4′-DHS > resveratrol g 4-HS g 3,5-DHS in SDS
and CTAB micelles. It was observed that resveratrol analogues
bearing o-dihydroxyl or p-dihydroxyl groups exhibited remark-
ably high synergistic antioxidant activities.
kREG
TO• + RO-
8 TO- + RO•
(4)
It is well-known that the phenoxide (ArO-) more easily
undergoes electron transfer oxidation, producing relatively stable
phenoxyl radical (ArO•) in alkaline solutions. Resveratrol, with
a pKa1 of 6.4 (26), partially dissociates under the present
experimental conditions (pH 7.4); this makes the electron
transfer reaction feasible. Therefore, the electron transfer
regeneration of R-tocopherol by ArO- (eq 4) may take place
simultaneously with the direct hydrogen abstraction (eq 3).
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Received for review August 29, 2008. Revised manuscript received
October 11, 2008. Accepted October 14, 2008. This work was supported
by the National Natural Science Foundation of China (Grants 20502010
and 20621091) and Program for New Century Excellent Talents in
University (NCET-06-0906).
JF802665S