目的从尖叶假龙胆不同极性部位中寻找对H_2O_2诱导PC12细胞氧化损伤具有较强保护作用的化合物。方法采用活性追踪分离的方式,通过H_2O_2诱导PC12细胞损伤建立氧化应激损伤模型,利用实时细胞分析技术(real-time cell analysis,RTCA)研究尖叶假龙胆不同极性部位对H_2O_2诱导PC12细胞氧化损伤的保护作用;采用HPLC法测定具有活性的单体化合物的质量分数,并确定其化学结构。结果尖叶假龙胆正丁醇和醋酸乙酯部位对H_2O_2诱导PC12细胞氧化损伤具有保护作用。同时,从尖叶假龙胆正丁醇和醋酸乙酯部位中分离得到的化合物1、2、3、5也对H_2O_2诱导PC12细胞氧化损伤具有保护作用。其中化合物1和2的保护作用在3.125-50.000μmol/L呈一定剂量依赖性,浓度为50.000μmol/L时,对H_2O_2诱导PC12细胞氧化损伤的保护作用最强;而化合物3和5的保护作用在3.125-50.000μmol/L内不具有剂量依赖性,浓度分别为25.000、3.125μmol/L时,对H_2O_2诱导PC12细胞氧化损伤的保护作用最强。经结构鉴定,化合物1、2、3、5分别为雏菊叶龙胆酮、去甲基雏菊叶龙胆酮、当药醇苷及去甲基当药醇苷。结论化合物1、2、3、5对H_2O_2诱导PC12细胞氧化损伤具有保护作用,可能是尖叶假龙胆抗氧化的主要活性成分。
Objective To investigate the compounds of Gentianella acuta with strong protective effect on oxidative damage in PC12 ceils induced by H2O2. Methods Damage model of PC12 cells was established by H2O2 and real-time cell analysis (RTCA) was utilized to evaluate;the protective effect of extracts and compounds. Meanwhile, bioassay-guided method was applied to separating effective components, and HPLC was used for the determination and structure confirmation of compounds. Results Fractions of n-butanol and acetic ether showed protective effect on oxidative damage in PC12 cells induced by H20〉 The anti-oxidant activity of compounds 1 and 2 showed dose-dependent manner in the scope of 3.125--50.000 μmol/L, and the protective effect was the strongest at the concentration of 50.000 -μmol/L. Compounds 3 and 5 showed the best protective effect at the concentration of 25.000 μmol/L and 3.125 μmol/L respectively. Compounds 1, 2, 3, and 5 were identified as bellidifolin, demethylbellidifolin, swertianolin and norswertianolin, respectively. Conclusion Compounds 1, 2, 3, and 5 show protective effect on oxidative damage in PC12 ceils induced by H2O2 maybe the main active components in G acuta. But the mechanism is still uncertain, and further exploration is imperative.