蟾酥提取工艺优化与提取物体外抗肿瘤活性研究

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。

Extraction from Venenum Bufonis and its antitumor activity in vitro

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.