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本文采用柱色谱技术对诸葛菜种子的提取物进行分离纯化,采用波谱技术结合化学方法进行结构鉴定,首次获得新化合物,命名为诸葛菜碱Ⅰ,并通过建立H_2O_2诱导的细胞损伤模型,对诸葛菜碱Ⅰ保护H_2O_2所致Hep G2细胞氧化损伤的作用及其机制进行了研究。于H_2O_2刺激前,加入不同浓度的诸葛菜碱Ⅰ预处理Hep G2细胞12 h,然后以400μmol/L H_2O_2损伤细胞2 h建立模型。MTT法检测细胞存活率,微板法检测细胞培养液中LDH活性和细胞MDA含量及SOD、GSH-PX的活性,Western Blot检测细胞内Nrf 2、HO-1蛋白表达,以评价诸葛菜碱Ⅰ对H_2O_2所致Hep G2细胞氧化损伤的保护作用。结果表明,与模型组相比,诸葛菜碱I给药组(53.5、107、214μmol/L)预处理12 h后,增加了细胞存活率(p<0.01),显著降低LDH向细胞外液的释放(p<0.01),显著降低细胞内MDA含量(p<0.05,p<0.01),显著提高细胞内SOD、GSH-PX的活性(p<0.05,p<0.01),增高了Nrf2蛋白水平。因此,诸葛菜碱Ⅰ对H_2O_2所致Hep G2细胞氧化损伤具有保护作用。
In this paper, the column chromatographic techniques were used to isolate and purify the extracts from the seeds of M. vulgaris. The structures of the compounds were identified by spectroscopic techniques and chemical methods. The new compounds were obtained for the first time and named as the Oryzalcium Ⅰ. Through establishing the cell injury model induced by H_2O_2, The protective effect and mechanism of Ca (OH) 2 on oxidative damage of Hep G2 cells induced by H 2 O 2 were studied. Before H 2 O 2 stimulation, Hep G2 cells were pretreated with different concentrations of taxanes Ⅰ for 12 h, and then induced by 400 μmol / L H 2 O 2 for 2 h. Cell viability was measured by MTT assay. LDH activity, MDA content and SOD, GSH-PX activity in cell culture medium were detected by microplate method. Nrf 2 and HO-1 protein expressions were detected by Western Blot. Protective effect of H_2O_2 on Hep G2 cell oxidative damage. The results showed that compared with the model group, the activity of cell viability increased significantly (p <0.01) after being pretreated with different doses of 53.5,107,214μmol / (P <0.01, p <0.01), significantly decreased intracellular MDA content (p <0.05, p <0.01), significantly increased intracellular SOD, GSH-PX activity (p <0.05, p <0.01), and increased Nrf2 protein level. Therefore, weedin Ⅰ has a protective effect on the oxidative damage of Hep G2 cells induced by H 2 O 2.