Chemical Composition Of Purple Swertia Content Determination And In Vitro Screening Of Anti-hbv Active Substances

Objective: Adopted in-vitro anti-HBV screening model to screen active components from Swertia punicea Hemsl.The screening results on one hand can direct the next extraction and isolation,on the other hand can help us confirm what are the anti-HBV activity components. Additionally, RP-HPLC was used to determine the content of five main active compounds from Swertia punicea Hemsl.Methods: 2.2.15 cells, a kind of HBV-producing human hepatoblastoma cell line, wasused as the cell model. The samples were dissolved in DMSO and double or triple diluted before used. The cells were grown to confluence in 96-well flat-bottomed tissue culture plates and treated with samples after 24 hours. Seven concentrations and cell control were assayed, each in triplicate culture. Four days following the first addition of samples, the same concentration of the somples were added to the same plate. The living state of cells was observed by microscope at the 8^th day of the addition of samples. Cytopathic effect (CPE) was divided into 5 grades. Cells completely destroyed was regarded as 4, 75% destroyed as 3, 50% destroyed as 2, 25% destroyed as l,and no pathological changes taking place as 0. According to Reed-Muench method, 50% toxicity concentration (TC₅₀) and TCo could be calculated. Cells were seeded into 24-well tissue plates and grown to confluence. The culture medium was changed 24 hours prior to exposure to test samples. During the 8 days of treatment period, the culture medium was removed and test samples were added to the cultures in fresh culture medium at 24-h intervals. Immediately prior to the first dose of test samples (day 0), and after 4 and 8 days of treatment, culture medium was collected and stored at -20 °C for HBsAg and HBeAg analysis by RIA. Cells were lysed at the end of the treatment period and analyzed for intracellular total HBV DNA by Dot blot, and calculated 50% inhibitory concentration (IC50) and selected index ( SI) .Additionally, RP-HPLC was used to determine the content of five main components from Swertia punicea Hemsl at the same time. The five components are swertiamarin, gentipicroside, mangiferin,8-O-β-D- glucopyranosyl-l,5-dihydroxy-3-...