Anti-hepatitis B Virus Activity Of Ethanol Extract From Ampelopsis Sinica Root Via P53 Signaling Pathway

Part 1 Preliminary research on anti-hepatitis B virus effects of Ampelopsis Sinica Root in vitroObjective:The present study was performed in order to investigate the anti-HBV activity of the ethanol extract from A. sinica root in vitro.Methods:The stably HBV-transfected HepG2 2.2.15 cells were cellular model. First, the cytotoxic concentration of A. sinica root was detected by CPE and MTT assay. Then, the HepG2 2.2.15 cells were interfered by A. sinica root. The antiviral activity of A. sinica root was examined by detecting the levels of HBsAg, HBeAg and extracellular HBV DNAs in stable HepG2 2.2.15 cells by ELISA and real-time PCR assay respectively.Results:A. sinica root with concentration 200μg/ml and the following had no effect on cell viability (P< 0.05). It effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose-dependent manner, and it also suppressed the amount of extracellular HBV DNA (P< 0.05). But this inhibiting effect had no significant relationship with the action time.Conclusions:A. sinica root had anti-virus activity by inhibiting the secretion of HBsAg, HBeAg and HBV DNA from HepG2 2.2.15 cells. Part 2 Anti-hepatitis B virus mechanisms of extract from Ampelopsis Sinica Root in vitroObjective:To clarify the anti-virus mechanisms of extract from A. Sinica Root in vitro.Methods:First, screening the signaling pathways, the PathDetect Cis-/Trans-Reporting Systems (pNFκB-Luc, pAP-1-Luc, pISRE-Luc, p53-Luc, pFA2-Elkl, pFA2-cJun, pFA2-CHOP, pFA2-CREB, pFR-Luc) were transiently transfected into HepG2 cells, respectively. After 8 h of transfection, cells were treated with 40μg/ml A. Sinica Root for 48 h. The activity of each signaling pathway was determined by measuring the luciferase activities of the reporter in treated and untreated transfectants. After A. Sinica Root treatment, the percentage of apoptotic HepG2 2.2.15 cells were detected by flow cytometric analysis; then, constructing 5 promoter plasmids and 5 truncate plasmid. These plasmids were transiently transfected into HepG2 cells, respectively. After 8 h of transfection, cells were treated with 40μg/ml A. Sinica Root for 48 h. The activity was determined by measuring the luciferase activities of the reporter in treated and untreated transfectants.Results:A. Sinica Root selectively inhibited the activity of p53-associated signaling pathway and the activities of HBV promoters (Cp, S1p and Fp) as well as truncates (t1, t4, t5) significantly(P< 0.05), and could induce apoptosis of HepG2 2.2.15 cells.Conclusions:A. Sinica Root exerts anti-HBV effects via inhibition of HBV transcription and the p53-associated signaling pathway, possibly by inhibiting the p53 pathway to induced apoptosis. Part 3 A optimum technology of extracting bioactive components from Ampelopsis Sinica RootObjective:In order to promote the advanced development of Ampelopsis Sinica Root and provide a scientific evidence for its clinical progress, giving a summary and comparison about the medicine plant extract on a variety of technology of extracting.Methods:Based on water boiling method, reflux method, percolation method, etc, the Ampelopsis Sinica Root was extracted to detect the dry extract and the content of gallic acid, a small-molecule and water-soluble component from extract.Results:In this experiment, the recommended 5# process were used to extract the medicine plant, as a result, the impurities from extract reduced 70%, but the content of gallic acid had no significant difference.Conclusions:The purity of active substance in extract solution or dry extract from Ampelopsis Sinica Root extracted by 5# process was greatly increased. And the extract was made of crud drug with concentration 0.8g/ml, as well as an increased clarity. It could be directly prepared for solution agent. In particular, the extract solution didn't have an element of stimulating mucus, so its cytotoxicity was significantly reduced.