Study On The Screening Of Activite Components From Prismatomeris Connata And Anti-hepatitis B Virus Mechanism Of The Extraction

Objective:The HepG2.2.15 cells line is used as research model,Prismatomeris connata is study object according to its anti-hepatitis B activity.This research mainly consist the chemical constituents from the plant,purification and the structure of identification,and investigate the mechanism of constituents from Prismatomeris connata anti hepatitis B virus.Methods:?The roots of Prismatomeris connata were soaked with 95%ethanol and extracted by sonication extraction.The ethanol extracts were extracted with petroleum ether and ethyl acetate;obtained petroleum extract,ethyl acetate extract and water extract.?To screening the active component,MTT method was used to determine the cytotoxicity of different parts of Prismatomeris connata and the effects of extracts on the secretion of HBeAg and HBsAg were detected by enzyme linked immunosorbent assay?ELISA?kit.?The silica gel column chromatography,Sephadex LH-20column chromatography and recrystallization were used for separation and purification of the active sites,and through FT-IR,HPLC,¹H NMR and ¹³C NMR spectral data were identified by physicochemical properties and structures of these compounds.?According to the screening results about those compounds by MTT and ELISA methods,Rubiadin was chose as drug for further study.?The main experimental groups were divided into control group,different concentrations of the test substance and positive control group?Lamivudine,3TC?.The mechanism of anti-hepatitis B virus was determined by Quantitative Real-time PCR?q PCR?,flow cytometry,Western Blot and Laser confocal microscope.Results:There were 9 compounds isolated from Prismatomeris connata and most of them were anthraquinone compounds.Four of these compounds were identified and named 1,3-Dihydroxy-2-methoxy-9,10-anthraquinone?A?,1,3-Dihydroxy-2-hydroxymethyl-9,10-anthraquinone?D?,1,3-Dihydroxy-2-methyl-9,10-anthraquinone?Rubiadin,F?,anthraquinone bile acid conjugates?H?.Compound H were found for the first from natural plant and the compound D were isolated from Prismatomeris connata for the first time.The IC₅₀0 of compoundsA,D,H,H were 8.03,8.13,17.05,12.41?g/mL and when the concentration was 8?g/mL the inhibition of HBeAg were-20.95%,61.42%,43.79%,69.30%and the inhibition of HBsAg were 9.42%,6.15%,23.34%,43.38%.These experiments show that the compound F?Rubiadin?has best activity in four compounds is chosen as medicine for the mechanism research.Rubiadin would rapidly into the Hep G2.2.15 cells and is maily distributed in the cell membrane,cytoplasm and other organelles.It could significantly decreased HBeAg secretion level,HBV DNA and inhibited HepG2.2.15 cells proliferation by G1 phase.Additionally,HBx protein expression was down-regulated in a dose-dependent manner after Rubiadin treatment.The intracellular calcium concentration was significantly reduced.Conclusion:Prismatomeris connata maily contains anthraquinones compounds.Rubiadin may inhibit the proliferation of HepG2.2.15 cells;reduce the secretion of hepatitis B virus e antigen and the expression of HBx protein or other pathway to complish anti hepatitis B virus effect.It is suggested that Rubiadin is expected become novel anti HBV drug.