Abstract
Purpose: To evaluate the effects of ebracteolatain A (EA) and ebracteolatain B (EB) from Euphorbia ebracteolata Hyata (Euphorbiaceae) on the proliferation of HepG2 cells and the possible mechanisms.Methods: EA and EB from E. ebracteolata were obtained by column chromatography. 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays were used to study the cytotoxic and pro-apoptotic activities of EA and EB against HepG2 cells. Western blot assay was used to investigate the possible mechanisms of action.Results: EA and EB were successfully isolated from E. ebracteolata by column chromatography. The results of MTT assay indicate that EA and EB have significant anti-proliferative activities against HepG2 cells in dose- and time-dependent manners with half maximal inhibitory concentration (IC50) of 28.48 and 31.72 μg/mL. respectively. The results of flow cytometry assay suggest that EA and EB significantly (p < 0.01) induced the apoptosis of HepG2 cells at the levels of 47.45 and 42.26 %, respectively. Western blot data indicate that EA and EB significantly (p < 0.05 or 0.01) down-regulated the expression levels of anti-apoptotic proteins (survivin and Bcl-2) and up-regulated the expression levels of proapoptotic proteins (Smac, Bax, c-caspase-3 and c-caspase-9) in mitochondria-mediated apoptotic pathway.Conclusion: EA and EB inhibit the proliferation of HepG2 cells, the probable mechanisms being associated with mitochondria-mediated apoptosis.Keywords: Euphorbia ebracteolata, Phloroglucinol derivatives, Mitochondria-mediated apoptosis, Flow cytometry, Western blot
Highlights
Euphorbia ebracteolata Hyata (Euphorbiaceae) is cultivated in Japan, Korea and China [1] and its dried roots, named “Lang Du”, are usually used to treat ascites, edema, chronic tracheitis, pulmonary tuberculosis and cancer [2]
The results of high performance liquid chromatography (HPLC) area normalization method indicated that the purity levels of ebracteolatain A (EA) and ebracteolatain B (EB) were 93.0 % and 92.8 %, respectively
We studied the effects of EA and EB from E. ebracteolata on the proliferation of HepG2 cells and the possible mechanisms in vitro by MTT, flow cytometry and Western blot assays
Summary
Euphorbia ebracteolata Hyata (Euphorbiaceae) is cultivated in Japan, Korea and China [1] and its dried roots, named “Lang Du”, are usually used to treat ascites, edema, chronic tracheitis, pulmonary tuberculosis and cancer [2]. We used 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), and flow cytometry assays to investigate the effect of EA and EB on the proliferation and apoptosis of HepG2 cells in vitro by determining their inhibition rates and apoptosis rates. The effects of EA and EB on the proliferation of HepG2 cells were evaluated using MTT assay according to the existing literature [13]. The inhibition rates were used to assess the effects of EA and EB on the proliferation of HepG2 cells and calculated using the following equation: Inhibition rate (%) = (OD control – OD treatment)/OD control × 100. HepG2 cells were collected to extract total proteins after treatment with EA or EB at different concentrations of 15, 30 and 45 μg/mL for 48 h. P-value less than 0.05 or 0.01 was taken as statistically significant
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