蟾酥提取工艺优化与提取物体外抗肿瘤活性研究

Extraction from Venenum Bufonis and its antitumor activity in vitro

目的优化蟾酥中脂溶性成分华蟾酥毒基及酯蟾毒配基的提取工艺,并探讨蟾酥提取物对人宫颈癌HeLa细胞和卵巢癌SKOV-3细胞的增殖及细胞周期的影响。方法采用U5(54)均匀设计,考察酒精浓度、酒精用量、提取时间等因素对指标性成分提取的影响;MTT法检测蟾酥提取物对HeLa与SKOV-3细胞增殖的影响;流式细胞技术检测细胞周期变化;倒置显微镜下观察HeLa和SKOV-3细胞形态变化。结果以100倍量95%酒精加热回流提取2次,每次2.5 h,两种指标性成分转移率最大,为(117.20±3.52)%;蟾酥提取物呈时间和剂量依赖性地抑制HeLa和SKOV-3细胞增殖;随着蟾酥提取物剂量的增加,HeLa细胞出现S期和G2/M期阻滞,SKOV-3细胞出现G0/G1期阻滞,两种细胞均出现空泡样变性。结论均匀设计法优选蟾酥有效成分的提取工艺合理可行;蟾酥提取物可显著抑制HeLa和SKOV-3细胞的增殖,使细胞出现空泡样变性,并阻滞细胞周期。

Objective To optimize the extraction technology of liposoluble constituents from Venenum Bufonis and to discuss the effects of extract on proliferation of HeLa and SKOV-3 ceils and their cell cycle. Methods Experiment was arranged by U5 （54 ） table, and the effects of alcohol concentration, volume and extraction time on indicative components were detected; the effects of extraction on proliferation of HeLa and SKOV-3 cells were detected by MTI＇; the cell cycle was measured by flow cytometry ; the morphologic change of HeLa and SKOV-3 cells were observed by inverted microscope. Results By heating reflux method the 95% alcohol was extracted 2 times, 2.5 h per time. The highest extraction efficiency of （117.20 ＋ 3.52） % for the indicator ingredients. The prepared extract significantly restrained the proliferations of HeLa and SKOV-3 cells, which showed the time-and dose-dependent effects. Venenum Bufonis extract arrested cell cycle progression of HeLa in S and G2/M phases while arrested G0/G1 phase for SKOV-3, by flow cytometry. With the increase in drug concentration, vacuolar degeneration was appeared. Conclusion ： The optimized extraction technology was suitable and feasible for Venenum Bufonis. Venenum Bufonis extract showed the excellent antitumor effects on HeLa and SKOV-3 cells in vitro.