Cinobufacini (huachansu), an aqueous extract from the skin of the toad Bufo bufo gargarizans Cantor, is a traditional Chinese medicinal preparation widely used in clinical cancer therapy in China. Here, we screened and identified active compounds of cinobufacini and investigated their apoptosis-inducing effect on HepG2 cells. Screening was performed using bioassay-guided isolation. The effects of different fractions on the proliferation of HepG2 cells were detected by the MTT assay. The extraction and isolation of active fractions were performed by chloroform extraction, silica column chromatography, preparative thin-layer chromatography and high-performance liquid chromatography. Nuclear magnetic resonance (NMR) imaging and electron ionization-mass spectrometry (EI-MS) were used to identify the structure of the active compounds. The extent of cell apoptosis was detected by Hoechst 33258 staining and flow cytometric analysis. Western blot analysis was used to detect the expression of the apoptosis-related proteins Bax and Bcl-2. Through bioassay-guided isolation, two compounds were isolated from cinobufacini. NMR and EI-MS data revealed these compounds to be resibufogenin and cinobufagin. Cinobufagin was determined to be the more efficient of the two in inhibiting the proliferation of HepG2 cells. Hoechst 33258 staining and flow cytometric analysis indicated that cinobufagin induced marked changes in apoptotic morphology and significantly increased the proportion of apoptotic cells in HepG2 cells. Western blot analysis showed that cinobufagin up-regulated Bax expression and down-regulated Bcl-2 expression. In conclusion, we screened and identified two anti-proliferation compounds of cinibufacini, resibufogenin and cinobufagin. The most effective compound, cinobufagin, inhibited cell proliferation by inducing the apoptosis of HepG2 cells. This was potentially triggered by regulation of the Bax/ Bcl-2 ratio.

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