Hongbeiyegen Effective Extraction And In Vitro Parts Of The Mechanism Of Anti-hepatitis B Virus

Hongbeiyegen Chinese herbal medicine commonly used as one of the Lingnan region,Primarily for the treatment of disorders associated with liver. Hongbeiyegen to extract raw materials for solid oral preparations with anti-hepatitis virus and prevent liver fibrosis. But has yet to see on their chemical composition, pharmacological effects reported in the literature area. HepG2.2.15 cell lines genetically modified HBV genome contains, Capable of secreting viral replication and infectious virus particles and HBsAg and HbeAg, As a result,the cells are used generally in screening and therapeutic effect evaluation for antiviral drugs. HepG2.2.15 cells were taken as target cells in this experiment. Firstly, the toxic effect of Hongbeiyegen water extract, ethanol extract, alkaloid containing serum on HepG2.2.15 cells was investigated with MTT colorimetric method. Moreover, The approach of using enzyme-linked immunosorbent assay (ELISA) in cell culture medium was determined HBsAg, HBeAg content, This evaluation Hongbeiyegen extracts the role of anti-HBV.The main observations were reported as following:experimentⅠThe built of the system of HepG2.2.15 cell lineObjective:To make the normal growth of the HepG2.2.15 cells and prepare for the experiment of the drug intervention.Methods:Divide into cell recovery,cell culture,cell passage and cell cryopreservation.Results:After the cell recovery,the growth velocity was slow in three weeks;but the cells grew well after the passage,and could be used in the experiment of drug intervention.ExperimentⅡSecretary curvilinear of HBsAg and HBeAg in the supernatant fluid, Objective:To explore the secretary information of HBsAg and HBeAg thatHepG2.2.15 cells secret into the supernatant fluid.Methods:HepG2.2.15 cells were maintained after serial subcultivation and the supernatant fluid wascollected in day1～6 respectively,kept in-20℃.The contents of HBsAg and HBeAg in supernatant fluid were detected with ELISA method:A450nm was readfrom the enzyme mark instrument after chromogenic reaction and secretary curvilinear was drawn.Results:The contents of HBsAg and HBeAg in the supernatant fluid increased gradually with the prolonging of cultural time after HepG2.2.15 cells got adherent.ExperimentⅢ:Hongbeiyegen Preparation of various extracts and preparation of serum containing.Hongbeiyegen Preparation of various extracts:Hongbeiyegen collected from Huizhou, The Southern Medical University, Department of Chinese medicinal plant identification and Euphorbiaceae plant identified as the red mountains and Stick Alchornea trewioides (Benth.) Muell.-Arg.'s roots.Alkaloids:Hongbeiyegen meal, Soaked in 95% ethanol, after 6h, Extracting 2 times, Every 120 minutes,2 times combined filtrate, then add 1/2 volume,5% hydrochloric acid Isolated impurities and acidic water extract.Recent researches have confirmed that flavonoidsin semen litchi could inhibit the hepatitis B surface antigen(HBsAg),hepatitis Be antigen(HBeAg)and DNA in hepatitis B virus(HBV DNA)significantly.However,whether saponin,as one of the main chemical composition in semen litchi,can inhibit HBV has not been reported yet.Transgenic HepG2.2.15 cells possess the whole genome of HBV so that can undertake viral replication and secrete the infectious viral particle,HBsAg and HBeAg persistently.As a result,the cells are used generally in screening and therapeutic effect evaluation for antiviral drugs.HepG2.2.15 cells were taken as target cells in this experiment. Firstly, the toxic effect of the total saponins in semen litchi on HepG2.2.15 cells was investigated with MTT colorimetric method. Moreover, the contents of HBsAg,HBeAg in supernatant fluid and HBV DNA outside the cells were detected withSA and fluorescent quantitation PCR.In the last,the inhibitory effect of the total saponins in semen litchi on HBV in vitro was estimated.ExperimentⅢToxicity experiment of the total saponins in semen litchion HepG2.2.15 cells.Objective:To investigate the toxicity effects of the total saponins in semen litchi with different concentrations on HepG2.2.15 cells.Methods:The total saponins in semen litchi were prepared for eight concentrations:0.8,0.4,0.2,0.1,0.05,0.025, 0.0125,0.00625g/L while lamivudine was prepared for 0.25g/L.G2.2.15 cells were subcultured in 96-well culture plate for 1×107/L.After the cells had been adherent,media with and without drugs were added.The supernatant fluid was discarded when cells was affected for 6d and MTT(5g/L)20μL was added in each wells,the cells were incubated in 37℃for 4h.Then the supernatant fluid was discarded and DMSO 150μL was added in each wells,the culture plates were quaked for 10min.A490nm was detected in the last.Results:Inhibition ratios of the total saponins in semen litchi(0.8,0.4g/L)on cells were53.7%,39.3%repectively,and TC50 was 0.67g/L.Inhibition ratio of lamivudine (0.25 g/L)on cells was 43.4%.ExperimentⅣDetection of HBsAg and HBeAg in supernatant fluid.Objective:To investigate the effects of the total saponins in semen litchi with different concentrations on HBsAg and HBeAg in supernatant fluid.Methods:The content of HBsAg and HBeAg was detected according to thedircetion of ELIS A kits.Results:Contrast with negative control group,the Avalue decreased in all the total saponins in semen litchi groups and the value decreased more with the increasing of drug concentration and with the prolonging of affect time.