Experiment Of The Total Saponin In Semen Litchi Against Hepatitis B Virus In Vitro

Semen litchi, one of Chinese herbal medicine commonly used, was mainly prescribed in treatment of hepatopathy. Its chemical compositions include saponin, fatty acid and so on. Recent researches have confirmed that flavonoids in semen litchi could inhibit the hepatitis B surface antigen (HBsAg), hepatitis B e 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 with ELISA and fluorescent quantitation PCR. In the last, the inhibitory effect of the total saponins in semen litchi on HBV in vitro was estimated. 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 fluidObjective: To explore the secretary information of HBsAg and HBeAg that HepG2.2.15 cells secret into the supernatant fluid. Methods: HepG2.2.15 cells were maintained after serial subcultivation and the supernatant fluid was collected in day1~6 respectively, kept in -20℃. The contents of HBsAg and HBeAg in supernatant fluid were detected with ELISA method: A450nm was read from 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.ExperimentⅢToxicity experiment of the total saponins in semen litchi on HepG2.2.15 cellsObjective: 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. HepG2.2.15 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 were 53.7%, 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 fluidObjective: 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 the dircetion of ELISA kits. Results: Contrast with negative control group, the A value 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.ExperimentⅤDetection of HBV DNA in supernatant fluidObjective: To investigate the effects the total saponins in semen litchi with different concentrations on HBV DNA in supernatant fluid. Methods: Follow the directions of the Taqman HBV Nucleic acid amplification by fluorescence detection. Primer sequence is P1: 5'-ATC CTG CTG CTA TGC CTC ATC TT-3'; P2: 5'-ACA GTG GGG GAA AGC CCT ACG AA-3'; fluorescent probe sequence: 5'-GGC TAG TTT ACT AGT GCC ATT TG-3'. Every reactons was put into the PCR instrument. Amplification according to the condition: pre-degenerated 2min at 93℃, then 93℃45s to 55℃1min circulate amplification 10 cycles, and 93℃30s to 55℃45s circulate amplification 30 cycles. After the reaction, the computer automatically analyzed the HBV DNA quantity consequences. Results: Contrast with negative control group, the copy numbers of HBV DNA 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.Conclusions:1. HepG2.2.15 cells could secret HBsAg and HBeAg into supernatant fluid and the contents increased with the prolonging of culture time.2. There was little toxicity of the total saponins in semen litchi on HepG2.2.15 cells and the toxic effect increased with increasing of drug concentration.3. The total saponins in semen litchi of different concentrations could inhibit the secret of HBsAg and HBeAg to some extent, the inhibitory effect enhanced with the increasing of concentration and with the prolonging of affect time.4. The total saponins in semen litchi of different concentrations could lower the contents of HBV DNA to some extent, the inhibitory effects enhanced with the increasing of concentration and with the prolonging of affect time.5. The total saponins in semen litchi were effective and harmfulless drug in inhibiting HBV in vitro.