Protective Effects On Immunological Liver Injury In Mice And The Compositions Of N-butanol Extraction From Viola Yedoensis Makino.

Objective:To investigate the Anti-Hepatitis B Virus activity and protective effects on immunological liver injury of n-butanol extraction?BE?of Viola yedoensis Makino.,and study on the the component analysis of Viola yedoensis Makino.n-butanol extraction.Methods:1.The pharmacognosy of Viola yedoensis Makino.was studied by morphology identification,microscopic identification,physicaland chemical identification,fluorescence identification.2.The extraction process of Viola yedoensis Makino.was optimized by the L₉?3⁴?orthogonal design,and prepared the n-butanol extract by solvent extraction method.The anti-hepatitis B virus experiments in vitro used Hep G 2.2.15 cell,the four methyl thiazolyl tetrazolium?MTT?method used to observe the toxic effects of Viola yedoensis Makino.n-butanol extraction on HepG 2.2.15 cells,and the inhibitory effects on the HBs Ag and HBe Ag secreted by Hep G 2.2.15 cells detected by ELISA analysis.Real-time fluorescent quantitative polymerase chain reaction was used for the determination of HBV DNA.3.Established Con-A induced fiver injury model,was given different dosages of n-butanol extraction.The collected blood is centrifuged to collect serum preparing for the measure of AST,ALT,observing the change of structure of liver tissue after HE staining.Detect the activity of SOD and GSH-PX in liver tissue and measure the content of MDA,TNF-?,IFN-?in liver tissue.4.The compounds were isolated from the n-butanol extraction using silica gel column chromatography and recrystallization methods.Its structures were confirmed by ¹HNMR,¹³CNMR and MS.Results:1.The morphology identification and microscopic identification of the medicinal materials of Viola yedoensis Makino.all conform to the description of the 2015pharmacopoeia,and contain flavonoids,coumarins,polysaccharides and tannins.The optimum process condition was 60%,1:20,40 min.2.The cell experiment showed that the BE was not toxic to cells in vitro,and could inhibit the secretion of HBsAg and HBs Ag in Hep G 2.2.15cells and the copy of HBV DNA.The inhibition rate was the highest after 6 days of cell culture with 1mg/ml BE which was 26.01%,26.58%and 65.89%.3.The results of animal experiments showed that n-butanol extraction could effectively decrease ALT and AST levels in blood serum and the content of MDA in liver tissue,and significantly increase the activity of SOD,GSH-PX and TNF-?,IFN-?in liver tissues.Compared with each BE groups,the12.00 g/kg dose group was the best on the level of ALT and AST?P<0.01?,and the 6.00 g/kg dose group was the best on the level of SOD,MDA,GSH-PX,TNF-?and IFN-??P<0.001?.Each dosa group liver lesions compared with model group were lighter in mice liver observation.4.The 5 compounds were isolated from the n-butanol extraction using silica gel column chromatography and recrystallization methods.There are Esculetin,Prionanthoside,Cichoriin,Quercetin-3-O-galactoside,Esculin.Conclusion:Studies on pharmacognosy of Viola yedoensis Makino.provides the basis for extraction and indentification.The n-butanol extraction has good effect on anti-HBV and the protective effect on immunological liver injury,the mechanism maybe related to the immune response and oxidative stress in vivo.Four coumarin types and one flavonoid were isolated from the n-butanol components.