| Objective:To study the optimum extraction process of polysaccharide from Ampullaria gigas spix, and explore the inhibitory effect of Ampullaria gigas spix polysaccharide on HBV both vitro and in vivo.Methods:The content of polysaccharide was used to evaluate the factor levels. Four technologica factors of alkali extraction process, concluding alcohol concentration,extraction temperature,pH value of solution and extraction times were optimized by orthogonal test. Then polysaccharide of Ampullaria gigas spix was extracted by the the optimum extraction process.In vitro,HepG 2.2.15 cell transfected with hepatitis B virus gene was used as cell model. cell toxicity of Ampullaria gigas spix polysaccharide on cultured cell line 2.2.15 was determined with MTT methods.In safe concentration, five different concentration of Ampullaria gigas spix polysaccharide and Lamivudine were respectively added into cell culture fluid of 2.2.15 cell strain and the culture fluid was changed every 3 days, the supernatant of culturing fluid was collected on the 9th day. Cell control with non-drug was set at the same time. The effection on HBsAg and HBeAg secreted by HepG2.2.15 cells was evaluated with TRFIA methods; the quantity of HBV-DNA was detected by TaqMan probe fluorescence quantitative PCR. Finally, evaluate the anti-HBV activity in vitro of Ampullaria gigas spix polysaccharide according to all the results above. In vivo, transgenic mice were used as the animal models and were randomly divided into five groups of six mice each including:Ampullaria gigas spix polysaccharide small,middle,large dose groups; Lamivudine group and model group. Each group mouse was intraperitoneal injected with drug for 20 days. The expression of HBV-DNA was detected by PCR test before treatment,at the 5 th,10th,15th and 20th day after treament and the 5th day after drug withdrawal.Results:The ideal extraction process were as follows:dehydrated alcohol was added to the fluid until the alcohol content up to 75%,solution pH value with 9.0,extraction with 50℃,extraction time 8 h. In vitro, Ampullaria gigas spix polysaccharide had no obvious cell toxicity at the concentration of 1mg·mL-1. Ampullaria gigas spix polysaccharide had inhibitory effect on HBsAg and HBeAg in different concentration, and maximum inhibition ratio addig up to 50.57% and 21.12%. The therapeutic index of Ampullaria gigas spix polysaccharide was 12.35 for HBsAg. There was certain inhibition on replication of HBV-DNA(P<0.0 5),but, the inhibition effect was a little weaker than 3TC. In vivo, The levels of HBV-DNA in Ampullaria gigas spix polysaccharide large,middle dose groups at the 15th,20th day after treament, Ampullaria gigas spix polysaccharide small dose group at the 20th day after treament were obviously lower than that before treatment,and there was significant difference (P<0.05,2)P<0.01),but the inhibition rate of every dose group of polysaccharide was lower than 3TC at the same time. The inhibition rate of large dose group of polysaccharide was 15.76% at the 5th day after drug withdrawal ,and compared with the 3TC group was not markedly improved.Conclusions:The optimum extracting procedure was was economical, effective and workable. Ampullaria gigas spix polysaccharide with low- toxicity and large safety margin can obviously inhibit on HBV both vitro and in vivo.Figure [7] table [9] reference [76]... |
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