葡萄籽中化学成分的抗氧化作用及对DNA损伤的保护作用
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摘要
葡萄籽中化学成分是一类多酚类化合物，具有明显的抗氧化作用，与其众多的药理作用相关。本论文应用体外脂质过氧化实验方法和单细胞凝胶电泳技术，重点考察了其对氧化应激引起的脂质和DNA损伤的保护作用，以期阐明其抗氧化作用的可能机理。
体外脂质过氧化实验结果表明：葡萄籽中的化学成分白藜芦醇、原花青素、儿茶素及其低聚体和高聚体、以及单体酚、多酚类化合物(1.0，10.0mg／L)，在给药后30、60、90、120、180和240min均可以明显抑制大鼠肝脏脂质过氧化物的生成。其中白藜芦醇、原花青素和儿茶素低聚体的作用强度较高，半数抑制浓度(IC_(50))较小。也可以显著抑制由Fe~(2+)／H_2O_2反应系统产生羟自由基激发的大鼠肝脏脂质过氧化物的生成。
单细胞凝胶电泳实验表明：单独给予白藜芦醇和原花青素(5，50mg／kg，i. p.)对小鼠外周血液淋巴细胞DNA无明显影响。腹腔注射重铬酸钾(10，20，30，40mg／kg)20min，可以造成小鼠外周血液淋巴细胞DNA明显损伤。白藜芦醇和原花青素(5，50mg／kg，i．p．)显著抑制重铬酸钾(20mg／kg，i．p．)引起的外周血液淋巴细胞和肝细胞DNA损伤。急性乙醇(0.5，1.0，2.0，4.0g／kg，i．p．)对DNA有明显的损伤作用。乙醇(2.0g／kg，i．p．)给药后，其DNA损伤作用在20min时最强，180min时基本恢复。给予白藜芦醇和原花青素(5，50mg／kg，i．p．)可以明显减轻乙醇(2.0g／kg，i．p．，20min)对小鼠外周血液淋巴
沈阳药科大学硕士学位论文 中文摘要
细胞和肝细胞的DNA损伤作用。
抗“SARS”方剂＊、Ill、IV（l门临床等效量＊ 床等效量和3倍临床
等效量X 3d，po．）对小鼠外周血液淋巴细胞 DNA无明显影响。而方剂 I、V、
VI＊门临床等效量＊ 床等效量和 3倍临床等效量X3d，po．）对小鼠外周血
液淋巴细胞DNA有明显损伤。给予白蓉芦醇O，100，200 m吵gX3d，po．）
和维生素C＊0，100。200mg／kgX3d，p．o）可以明显减轻方剂1和VI（临床等
效量 X 3人 p．o）造成的损伤，并有良好的剂量依赖性。
综上所述，葡萄籽中化学成分具有良好的抗脂质过氧化活性和对药物致
细胞DNA损伤的抑制作用。
The constituents in grape seeds are mainly polyphenolic compounds with significant antioxidative activities, which are involved in many pharmacological effects. In the present studies, the effects of the constituents in grape seeds on lipid peroxidation, by estimating MDA formation, and on DNA damage induced by oxidative stress, by using single cell gel electrophoresis assay (SCGE), were investigated.
It was shown that the constituents in grape seeds including resveratrol, proanthocyanidins, catechins and its polymers, monophenols and polyphenols (1.0, 10.0 mg/L), could significantly inhibit the formation of LPO in rat liver homogenate m vitro. Resveratrol, proanthocyanidins and the low-polymers of catechin had more significant effects and the lower IC50 than others. Furthermore, all constituents could inhibit the formation of LPO in rat liver homogenate induced by hydroxyl radical in Fenton system (Fe2+/H2O2) in vitro.
The results of SCGE were shown that resverstrol and proanthocyanidins (5, 50 mg/kg, i.p.), given alone, had no significant effect on mouse lymphocyte DNA. Potassium dichromate (10, 20, 30, and 40 mg/kg, i.p.) could induce a siginificant DNA damage in mouse lymphocyte 20 minutes after injection. Resveratrol and proanthocyanidins, at the dose of 5 and 50 mg/kg (i.p.), significantly inhibited the DNA damage induced by potassium dichromate (20 mg/kg, i.p.) in mouse lymphocytes and hepatocytes. Moreover, acute ethanol (2.0 g/kg, i.p.) induced lymphocyte DNA damage, which was maximal at 20 min after administration. The DNA damage almost recovered at 180 min.
Resveratrol and proanthocyanidins (5 and 50 mg/kg, i.p.) showed the obviously inhibiting effects on the DNA damage in mouse lymphocytes and hepatocytes induced by acute ethanol administration (2.0 g/kg, i.p., 20 min).
The anti-SARS prescription II, III, and IV recommended by Chinese Ministry of Health, at the doses of 1/3,1 and 3 times of the clinical dose (3 days, P.O.), had no significant effect on DNA in mouse lymphocytes. However, the prescription I, V, and VI (also 1/3, 1, and 3 tunes of the clinical dose, 3 days, p.o.) showed the obviously harmful effects on mouse lymphocyte DNA. Resveratrol (50, 100, and 200 mg/kg, 3 days, p.o.) and vitamin C (50, 100, and 200 mg/kg, 3 days, p.o.) could dose-dependently restore the DNA damage induced by prescription I and VI (clinical dose, 3 days, p.o.).
In conclusion, these results gave the further evidence that the constituents in grape seed had good activities of anti-LPO formation and firstly demonstrated that these compounds protected cellular DNA damage induced by the noxious substances.
体外脂质过氧化实验结果表明：葡萄籽中的化学成分白藜芦醇、原花青素、儿茶素及其低聚体和高聚体、以及单体酚、多酚类化合物(1.0，10.0mg／L)，在给药后30、60、90、120、180和240min均可以明显抑制大鼠肝脏脂质过氧化物的生成。其中白藜芦醇、原花青素和儿茶素低聚体的作用强度较高，半数抑制浓度(IC_(50))较小。也可以显著抑制由Fe~(2+)／H_2O_2反应系统产生羟自由基激发的大鼠肝脏脂质过氧化物的生成。
单细胞凝胶电泳实验表明：单独给予白藜芦醇和原花青素(5，50mg／kg，i. p.)对小鼠外周血液淋巴细胞DNA无明显影响。腹腔注射重铬酸钾(10，20，30，40mg／kg)20min，可以造成小鼠外周血液淋巴细胞DNA明显损伤。白藜芦醇和原花青素(5，50mg／kg，i．p．)显著抑制重铬酸钾(20mg／kg，i．p．)引起的外周血液淋巴细胞和肝细胞DNA损伤。急性乙醇(0.5，1.0，2.0，4.0g／kg，i．p．)对DNA有明显的损伤作用。乙醇(2.0g／kg，i．p．)给药后，其DNA损伤作用在20min时最强，180min时基本恢复。给予白藜芦醇和原花青素(5，50mg／kg，i．p．)可以明显减轻乙醇(2.0g／kg，i．p．，20min)对小鼠外周血液淋巴
沈阳药科大学硕士学位论文 中文摘要
细胞和肝细胞的DNA损伤作用。
抗“SARS”方剂＊、Ill、IV（l门临床等效量＊ 床等效量和3倍临床
等效量X 3d，po．）对小鼠外周血液淋巴细胞 DNA无明显影响。而方剂 I、V、
VI＊门临床等效量＊ 床等效量和 3倍临床等效量X3d，po．）对小鼠外周血
液淋巴细胞DNA有明显损伤。给予白蓉芦醇O，100，200 m吵gX3d，po．）
和维生素C＊0，100。200mg／kgX3d，p．o）可以明显减轻方剂1和VI（临床等
效量 X 3人 p．o）造成的损伤，并有良好的剂量依赖性。
综上所述，葡萄籽中化学成分具有良好的抗脂质过氧化活性和对药物致
细胞DNA损伤的抑制作用。
The constituents in grape seeds are mainly polyphenolic compounds with significant antioxidative activities, which are involved in many pharmacological effects. In the present studies, the effects of the constituents in grape seeds on lipid peroxidation, by estimating MDA formation, and on DNA damage induced by oxidative stress, by using single cell gel electrophoresis assay (SCGE), were investigated.
It was shown that the constituents in grape seeds including resveratrol, proanthocyanidins, catechins and its polymers, monophenols and polyphenols (1.0, 10.0 mg/L), could significantly inhibit the formation of LPO in rat liver homogenate m vitro. Resveratrol, proanthocyanidins and the low-polymers of catechin had more significant effects and the lower IC50 than others. Furthermore, all constituents could inhibit the formation of LPO in rat liver homogenate induced by hydroxyl radical in Fenton system (Fe2+/H2O2) in vitro.
The results of SCGE were shown that resverstrol and proanthocyanidins (5, 50 mg/kg, i.p.), given alone, had no significant effect on mouse lymphocyte DNA. Potassium dichromate (10, 20, 30, and 40 mg/kg, i.p.) could induce a siginificant DNA damage in mouse lymphocyte 20 minutes after injection. Resveratrol and proanthocyanidins, at the dose of 5 and 50 mg/kg (i.p.), significantly inhibited the DNA damage induced by potassium dichromate (20 mg/kg, i.p.) in mouse lymphocytes and hepatocytes. Moreover, acute ethanol (2.0 g/kg, i.p.) induced lymphocyte DNA damage, which was maximal at 20 min after administration. The DNA damage almost recovered at 180 min.
Resveratrol and proanthocyanidins (5 and 50 mg/kg, i.p.) showed the obviously inhibiting effects on the DNA damage in mouse lymphocytes and hepatocytes induced by acute ethanol administration (2.0 g/kg, i.p., 20 min).
The anti-SARS prescription II, III, and IV recommended by Chinese Ministry of Health, at the doses of 1/3,1 and 3 times of the clinical dose (3 days, P.O.), had no significant effect on DNA in mouse lymphocytes. However, the prescription I, V, and VI (also 1/3, 1, and 3 tunes of the clinical dose, 3 days, p.o.) showed the obviously harmful effects on mouse lymphocyte DNA. Resveratrol (50, 100, and 200 mg/kg, 3 days, p.o.) and vitamin C (50, 100, and 200 mg/kg, 3 days, p.o.) could dose-dependently restore the DNA damage induced by prescription I and VI (clinical dose, 3 days, p.o.).
In conclusion, these results gave the further evidence that the constituents in grape seed had good activities of anti-LPO formation and firstly demonstrated that these compounds protected cellular DNA damage induced by the noxious substances.
引文
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[2] Abuja PM, Albertini R. Methods for monitoring oxidative stress, lipid peroxidation and oxidation resistance of lipoproteins. Clinica Chimica Acta 2001, 306:1-17.
[3] Bagchi D, Bagchi M, Stohs SJ, et al. Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. Toxicology 2000, 148(2-3): 187-197.
[4] Bagchi D, Garg A, Krohn RL, et al. Oxygen free radical scavenging abilities of vitamins C and E, and a grape seed proanthocyanidin extract in vitro. Res Commun Mol Pathol Pharmacol 1997, 95(2): 179-189.
[5] Bagchi D, Ray SD, Patel D, et al. Protection agsinst drug- and chemical-induced multiorgan toxicity by a novel IH 636 grape seed proanthocyanidins extract. Drugs Exp Clin Res 2001, 27(1): 3-15.
[6] Castillo J, Benavente-Garcia O, Lorente J, et al. Antioxidant activity and radioprotective effects against chromosomal damage induced in vivo by X-ray of flavan-3-ols (Procyanidins) from grape seeds (Vitis vinifera): comparative study versus other phenolic and organic compounds. J Agric Food Chem 2000, 48(5): 1738-1745.
[7] Fauconneau B, Waffo-Teguo P, Huguet F, et al. Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vivifera cell cultures using in vitro tests. Life Sci 1997, 61(21): 2103-2110.
[8] Fremont L, Belguendouz L, Delpal S. Antioxidant activity of resveratrol and alcohol-free wine polyphenols related to LDL oxidation and polyunsaturated fatty acids. Life Sci
1999, 64(26): 2511-2521.
[9] Roig R, Cascon E, Arola L, et al. Moderate red wine consumption protects the rat against oxidation in vivo. Life Sci 1999, 64(17): 1517-1524.
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