山豆根不同提取部位的毒性作用研究

摘要

Objective To explore the major toxic constituent of Sophorae Tonkinensis.Methods The decoction of Sophorae Tonkinensis were prepared by the usage method of clinic,and then,the alkaloid part and non-alkaloid part (which mainly include flavonoids and saponins) of the decoction were prepared by macroporous resin and polyamide resin.The acute toxicity test of zebrafish was used to evaluate the toxicities of different extractions.In addition,MTT method was adopted to observe the impact of different extractions on growth of HepG2 cells,screening fractions with damage effect and caculadng the half inhibitory concentration (IC50).LDH release assay was used to detect cell membrane injury.Flow cytometry analysis was used to detect apoptosis of HepG2 cells with Annexin V-FITC/PI double dye.Results The results of the toxicity of zebrafish showed that the non-alkaloid part was the main toxic part of Sophorae Tonkinensis.MTT assay also showed that the non-alkaloid site had the strongest inhibitory effect on the viability of HepG2 cells.The non-alkaloid part could induce HepG2 cell membrane injury and the rate of injury reached 67.31％ at the concentration of 2 mg·mL-1.According to flow cytometry analysis,the apoptosis of HepG2 cells induced by non-alkaloid part was higher than that of control (P ＜0.05).Conclusion All the above experiments suggest that the non-alkaloid part of Sophorae Tonkinensis may be the main toxic part.And the non-alkaloid part may affect cell viability by inducing cell membrane damage and apoptosis.%目的 明确山豆根的主要毒性部位.方法 制备山豆根水煎液,过大孔吸附树脂进行分离,得到水洗部位、生物碱部位和非生物碱部位(主要包括黄酮类和皂苷类).斑马鱼急性毒性实验被用于评价山豆根不同提取部位的毒性大小.此外,通过MTT法检测不同提取部位给药24 h对HcpG2细胞活力的影响并计算半数抑制浓度(IC50);LDH释放检测试剂盒检测细胞膜损伤程度;Annexin V-FITC/PI双染,流式细胞术检测细胞凋亡.结果 斑马鱼急性毒性实验结果提示非生物碱部位为山豆根主要毒性部位.MTT法结果也显示非生物碱部位对HepG2细胞活力的抑制作用最强;非生物碱部位可以引起HepG2细胞膜损伤,在2 g·L-1细胞损伤率达到67.31％;非生物碱部位(2g·L-1)给药24 h引起的HepG2细胞凋亡显著高于对照组(P＜0.05).结论 山豆根非生物碱部位可能为山豆根主要毒性部位,非生物碱部位可能通过诱导细胞膜损伤和凋亡从而影响细胞活力.