细胞代谢

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 Chem. Eur. J. 2002, 8, No. 18 ¹ 2002 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 0947-6539/02/0818-4191 $ 20.00+.50/0 4191 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 Chem. Eur. J. 2002, 8, No. 18 ¹ 2002 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 0947-6539/02/0818-4193 $ 20.00+.50/0 4193 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 Chem. Eur. J. 2002, 8, No. 18 ¹ 2002 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 0947-6539/02/0818-4195 $ 20.00+.50/0 4195 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