大青叶提取物对HepG2细胞酒精性损伤的保护作用

Protective Effect of Clerodendrum cyrtophyllum Turcz Leaves Extracts on Alcohol-induced Injury in HepG2 Cells

以大青(Clerodendrum cyrtophyllum Turcz)叶乙醇提取物和石油醚相、二氯甲烷相、乙酸乙酯相、正丁醇相、水相分级萃取物为研究对象,探讨其对肝细胞酒精性损伤的保护作用。采用酒精诱导人肝癌细胞(HepG2)氧化应激建立酒精性肝细胞损伤模型,通过噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测不同浓度大青叶醇提物和五相萃取物预处理对HepG2细胞酒精性损伤后存活率的影响,以2,7-二氢二氯荧光黄双乙酸钠(2,7-dichlorofuorescin diacetate,DCFH-DA)探针法测定细胞内活性氧(reactive oxygen species,ROS)水平,硫代巴比妥酸法测定细胞内丙二醛(malondialdehyde,MDA)含量,二硫代二硝基苯甲酸[5,5'-Dithiobis-(2-nitrobenzoic acid),DTNB]法检测还原型谷胱甘肽(glutathione,GSH)含量,水溶性四唑盐(water-soluble tetrazolium,WST)法检测超氧化物歧化酶(superoxide dismutase,SOD)活力。结果显示,大青叶醇提物和乙酸乙酯相、正丁醇相、水相萃取物可以明显提高HepG2细胞酒精性损伤后的存活率,通过抵御细胞内ROS生成,降低MDA浓度的方式对细胞进行保护,通过提高细胞内抗氧化物GSH含量和抗氧化酶SOD活力来恢复细胞抗氧化能力,石油醚相和二氯甲烷相无明显作用效果。综上,大青叶醇提物能够抵御HepG2细胞的酒精性损伤,具有护肝功能性食品应用研究价值;乙酸乙酯相、正丁醇相、水相是其有效部位,尤其是乙酸乙酯相,可以作为大青叶中天然抗氧化剂的主要研究对象。

The ethanol extracts and petroleum ether,dichloromethane,ethyl acetate,n-butanol and aqueousfractions of Clerodendrum cyrtophyllum Turcz leaves were studied to investigate the protective effects onalcohol-induced injury in hepatocyte. An alcoholic hepatocyte injury model was established by alcohol-inducedhepatocellular carcinoma （HepG2）oxidative stress. The effect of pretreatment with different concentrations ofethanol extract and five fractions extracts on the survival rate of HepG2 cells after alcoholic inducing wasdetected by methyl thizaolyl tetrazolium（MTT）assay. The level of reactive oxygen species （ROS）in HepG2cells was measured by2, 7-dihydrodichlorofluorescein diacetate（DCFH-DA）probe method. The thiobarbituricacid method was used to determine the content of malondialdehyde （MDA） in HepG2 cells. Thedithiodinitrobenzoic acid（DTNB）method was used to detect the content of glutathione（GSH） . Water-solubletetrazolium（WST）method was used to detect the activity of superoxide dismutase（SOD） . The results showedthat ethanol extracts and ethyl acetate,n-butanol and aqueous fractions of C. cyrtophyllum Turcz leaves could significantly increase the survival rate of HepG2 cells after alcohol injury. The HepG2 cells were protected by re原ducing intracellular ROS production and MDA concentrations. The antioxidant capacity of HepG2 cells was re原stored by increasing the intracellular antioxidant GSH content and antioxidant enzyme SOD activity. Thepetroleum ether and dichloromethane fractions did not showa significant effect. In summary,the ethanol extractof C. cyrtophyllum Turcz leaves could resist the alcoholic damage in HepG2 cells,and had a research value inthe application of hepatoprotective functional foods. Ethyl acetate, n-butanol and water fractions were the effective parts,especially the ethyl acetate fraction,which could be used as the main research object of natural antioxidants in C. cyrtophyllum Turcz leaves.