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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 process of toad venom toxin and toadin from Toadstool, and to investigate the effects of extract of Toadstool on the proliferation and cell cycle of human cervical cancer HeLa cells and ovarian cancer SKOV-3 cells. Methods Uniform design of U5 (54) was used to investigate the effects of alcohol concentration, alcohol consumption and extraction time on the extraction of index components. The effects of extract of Toadstool on proliferation of HeLa and SKOV-3 cells were detected by MTT assay. Flow cytometry Cell cycle changes; Morphological changes of HeLa and SKOV-3 cells were observed under inverted microscope. The results showed that the extraction rate of HeLa and SKOV was significantly (117.20 ± 3.52)%, with the highest rate of 117.20 ± 3.52%, with the amount of 95% ethanol being 100% -3 cell proliferation. With the increase of dose of toad extract, S phase and G2 / M phase arrest occurred in HeLa cells and G0 / G1 phase arrest occurred in SKOV-3 cells. Both cells showed vacuolar degeneration. Conclusion The uniform design method is preferable for the extraction of the active ingredients from Toadstool. The extract of Toadstool can significantly inhibit the proliferation of HeLa and SKOV-3 cells, make the cells vacuolar degeneration and arrest the cell cycle.