Determination of three active components in Euphorbia humifusa willd. Using high-performance liquid chromatography with diode-array detection and autophagy and apoptosis analysis of normal rat kidney and hela cells

Abstract

Background: Euphorbia humifusa Willd. (EH) is a kind of Chinese medicinal plant belonging to the family Euphorbiaceae, which is traditionally used for influenza, jaundice, hepatitis B virus, hypotension, inflammation, etc. In a modern study, active ingredients of EH reported to exhibit antioxidant, anticancer, and other properties. However, there are few reports showing that EH and its active compounds have inhibitory effects on cervical cancer through autophagy and apoptosis. Objective: To evaluate the anticervical cancer activity of EH and its main active ingredient Gallic acid (GA) by studying on autophagy and apoptosis of normal rat kidney (NRK) and HeLa cell lines, respectively. Materials and Methods: GA, kaempferol (KA), and quercetin (QU) are the main active compounds of EH. Identification and quantification of three substances from four batch samples obtained from four areas in Xinjiang were analyzed by high-performance liquid chromatography with diode-array detection. The potent effects of EH on anticervical cancer were investigated through autophagy and apoptosis in HeLa and NRK cells with varying concentrations of extracts and active compound GA treatment. The antiproliferation activity against HeLa cells was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Autophagy and apoptosis in NRK and HeLa cells were observed by laser scanning confocal microscope. Quantitative data of apoptosis were estimated by Hoechst staining and Annexin-V binding assay using flow cytometry. The expression levels of autophagy-related protein (LC3 and P62) were subjected to western blot. Moreover, the autophagic vacuoles and other ultrastructures of cells were observed under transmission electron microscopy. Results: The contents of GA, KA, and QU were measured as 2.3342–3.4688, 0.4636–1.5922, and 0.9349–3.1500 mg/g, respectively. We used different concentrations of GA and the extracts of EH to treat with the line of cells, respectively. The best concentration of GA, water, and ethanol extracts inducing autophagy was 25 μg/ml, 10 mg/ml, and 10 mg/ml, respectively. The autophagy mediated with EH induced the accumulation of autophagosome, and even resulted in apoptosis. Conclusion: From our study, these results indicated that EH and GA may induce both autophagy and apoptosis in NRK and HeLa cells. The activity we studied on autophagy and apoptosis of HeLa cells may provide a new foundation for cervical cancer therapy or other related applications. Abbreviations used: EH: Euphorbia humifusa Willd.; HPLC-DAD: High-performance liquid chromatography with diode-array detection; NRK: Normal rat kidney; GA: Gallic acid; FC: Flow cytometry; LSCM: Laser scanning confocal microscope; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; TEM: Transmission electron microscopy; KA: Kaempferol; QU: Quercetin; LC3: The light chain 3; DMEM: Dulbecco's Modified Eagle's medium; FBS: Fetal bovine serum; DMSO: Dimethyl sulfoxide; PBS: Phosphate-buffered saline; PI: Propidium iodide; IC50: Half-maximal inhibitory concentration; BFA: Bafilomycin A1.