Research On Anti-HBV Ingredients Of Swertia Davidi Franch

Swertia davidi Franch,a precious Chinese Traditional Medica, belongs to Gentianaceae., It has been used to treat HBV, dysentery and pneumonia in the folk for a long time.The extraction, purification, structure, standard, determination of principal Chemical ingredients and anti-HBV activity were studied. The results were as follows:1. The extraction of total xanthonesThe optimal extraction conditions were determined according to uniform design experiments as extraction temperature 50℃, pressure 35MPa, entrainer 100%ethanol(volume fraction) and entrainer flow rate 4.5 mL·min-1. The yield of total xanhtones was 0.425%.Compared with the organic solvents method, the SFE method is more efficient,more rapid,more friendly environ- mently and less cost.2. The extraction of iridoidsThe optimal extraction conditions were determined according to orthogonal experiments as extraction temperature 80℃, solid-liquid ratio was 10, extract 3 times by 3h.The extraction ratio of amarogentin as the index was 0.341%. The method is reliable, stable, accurate and specific.3. Identification of structures of xanhtones and iridoids of S. davidi.A was isolated and prepared from super critical fluid CO2 extracted by silicagel and micrite cellulose column chromatograph; B was isolated and preparedfrom super critical fluid CO2 extract by using silica gel column chromatograph and Pre-TLC; C was isolated and prepared by using silica gel and sephadexLH-20 column chromatograph; D was isolated and prepared by using silica gel column chromatograph. These methods are simple and stable. By employing physical and chemical properties, modern spectrometric analysis techniques such as UV、IR、EI-MS、1H-NMR、13C-NMR、and DEPT, their structures of deme- thylbellidifolin, swerchirin, swertiamarin, amarogentin have been elucidated. These principal components have been chosen as the reference substance for qualitative and quantitative analysis of the crude drug forits speciality and content.4. Quantitative determination of xanhtones and iridoids by HPLC The HPLC method was used to determine the content of these chemical ingredients in S. davidi.Demethylbellidifolin The analysis was carried out on Hypersil C18 column (150mm×4.6mm,5μm).The mobile phase was CH3OH-0.5%H3PO4(56:44).Flow-ratewas 1.0mL·min-1. Temperature was room temperature. Wave-length was 254 nm. The method was simple,fast and had a good liner relationship;Swerchirin The analysis was carried out on Hypersil C18 column (150mm×4.6mm,5μm). The mobile phase was CH3OH-water(76:24). Flow-rate was 1.0 mL·min-1. Wave-length was 254 nm. Temperature was room temperature. The methodwas simple,fast and had a good liner relationship. The content was 0.099～0.116%.Swertiamarin The analysis was carried out on Hypersil C18 column (150mm×4.6mm,5μm).The mobile phase was CH3OH-water(25:75). Flow-rate was 1.0 mL·min-1. Wave-length was 254 nm. Temperature was roomtemperature. The content was 3.62～3.64%.Amarogentin The analysis was carried out on Hyper-sil C18 column (150mm×4.6mm, 5μm).The mobile phase was CH3OH-water(50:50). Flow-rate was 1.0 mL·min-1. Wave-length was 227nm.Temperature was roomtemperature. The content was 0.327～0.341%.5. Activity evaluation in vitroInhibitory effects on the expression of HBsAg and HBeAg on 2.2.15 cells transfected by hepatitis B virus were observed.The results showed that Demethyl-bellidifolin and amarogentin can inhabit expression of HBsAg and HBeAg obvi-ously.