| Objective To explore the medical influence of shellfish polysaccharide,extracting the polysaccharide from Cipangopaludina chinensis and evaluating the anti-HBV activity in vitro of polysacchafide of Cipangopaludina chinensis.Methods Cipangopahdina chinensis was collected and identified in the locality.Taking the content of polysaccharide as an index,four technological factors of water extraction process,ie, alcohol strength,extraction temperature,water volume used and extraction lime were optimized by orthogonal test.Then polysaccharide of Cipangopaludina chinensis was extracted by the the optimum extraction process.HepG 2.2.15 cell transfected with hepatitis B virus gene was used as cell model in vitro,cell toxicity of polysaccharide of Cipangopaludina chinensis on cultured cell line 2.2.15 was observed by MTT,safe concentration was definite according to the TC50and TC0. Then five concentration-1mg/ml,1×10-1mg/ml,1×10-2mg/ml,1×10-3mg/ml and 1×10-4mg/ml of polysaccharide of Cipangopaludina chinensis and Lamivudine were added into each well,and the culture fluid was changed every 72 hours,the supernatant of culturing fluid was collected and preserved at -20℃on the 9th day.Cell control with non-drug was set at the same lime.And then the inhibition to HBsAg and HBeAg was tested by ELISA;the quantity of HBV-DNA was detected by TaqMan probe fluorescence quantitative PCR.Finally,evaluate the anti-HBV activity in vitro of polysaccharide of Cipangopaludina chinensis according to all the results above.Results The ideal extraction process is as follows:dehydrated alcohol is added to the fluid until the alcohol content up to 75%;extraetion with 60℃;extraction with water for two times; each extraction running 6 hours.1.82g polysaccharide of Cipangopaludina chinensis was extracted. maximum non-toxic conentration(TC0)and half toxic concentration(TC50)of polysaccharide of Cipangopaludina chinensis on cultured cell line 2.2.15 were 1mg/ml and＞10 mg/ml,the cell toxicity was low.Half inhibitory concentration(IC50)of polysaccharide of Cipangopaludina chinensis on HBsAg and HBeAg in cultured cell line 2215 was 0.501mg/ml and 0.401mg/ml, Selectivity index(SI)was＞19.96 and＞24.94;half inhibitory concentration(IC50)of Lamivudine on HBsAg and HBeAg in cultured cell line 2215 was 0.062 mg/ml and 0.238mg/ml,Seleetivity index(SI)was＞161.29 and＞42.02.It showed that polysaccharide of Cipangopaludina chinensis could inhibit the secretion of HBsAg and HBeAg in cultured cell line 2.2.15,and have a stronger effect on HBeAg than on HBsAg,but,the inhibition effect was a tittle weaker than 3TC.The results of FQ-PCR showed that polysaccharide of Cipangopaludina chinensis could inhibit the copy of HBV-DNA in cultured cell line 2.2.15 at concentration-0.1mg/ml and 1mg/ml(P＜0.05),the inhibition effect was weaker than 3TC.Conclusions The optimum extracting procedure was simple,reasonable and reliable.The results indicate that polysaccharide of Cipangopaludina chinensis can suppress the expression of HBsAg and HBeAg and decrease the HBV-DNA level in cultured cell line HepG 2.2.15.The study provides therical and experimental evidence for further research of the shellfish polysaccharide,and also provides a new choice for the effective anti-HBV drug with low- toxicity. |
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