Extraction Of Snail Polysaccharide And Its Anti-HBV Effect In Vitro

Objective To study the optimal condition of extracting snail polysaccharide(SP), and evaluating the anti-HBV effect of SP in vitro.Methods Firstly,the Acusta tourannensis was gathered in open country.After identification,polysaccharide was extracted from Acusta tourannensis by technology of alkaline solution diffusion.And then,the orthogonal test was applied to optimize the extraction conditions including time,temperature,pH value and concentration of alcohol.The crude snail polysaccharide became purified after detachding the protein by Sevag method,decolorization by active carbon and separation hydrone by dislysis. After that,adopt phenol-sulfuric acid chromometry to determine contents of snail polysaccharide.HepG 2.2.15 cell strain was used to study anti-HBV effect of snail polysaccharide in vitro,and Lamivudine was applied as positive control.The safe concentration of drug was calculated after detecting the toxicity of the drug to HepG 2.2.15 by MTT means.And then,different dose(10^-4mg/ml,10^-3mg/ml,10^-2mg/ml, 10^-1mg/ml and 1mg/ml)of snail polysaccharide were added into each cell respectively, and cell control without drug was set at the same time.After nine days,the supernatant of culturing fluid was collected,and ELISA was adopted to detect the level of HBsAg and HBeAg which secreted by HepG 2.2.15 cell strain in supernatant.At the same time, the quantity of HBV DNA was checked by real-time fluorescence quantitative PCR.At last,evaluate the anti-HBV activity of snail polysaccharide in vitro by Selectivity Index which calculated from the results.Results The optimized results by orthogonal test as follows:6 hours,70℃,10.0,and 90%corresponding to extraction time,temperature,pH value and concentration of alcohol.The half toxic concentration(TC₅₀)of SP was over 10mg/ml,and the maximum non-toxic concentration(TC₀)was 1mg/ml which indicated that SP had no toxicity approximately.SP had a striking depressant effect on the HBsAg and HBeAg secreted by HepG 2.2.15 cell strain with maximum inhibition ratio adding up to 42.8% and 52.1%respectively.Lamivudine displayed the same effect with the maximum inhibition ratio reaching to 61.6%and 57.4%respectively.Inhibition on HBeAg surpassed to HBsAg by SP and Lamivudine demonstrated a better depression effect compared to SP.Also,there was certain inhibitionv on replication of HBV DNA given by Lamivudine and SP(P＜0.01),and the former was powerful than the latter.Conclusions It is simple and feasible for the optimized conditions used in alkaline solution diffusion.SP can inhibit the HBsAg and HBeAg secreted by HepG 2.2.15 and the replication of HBV DNA in vitro.Although inhibit effect is inferior to Lamivudine, SP possesses a fine applying prospect for its low toxicity and available.The research provides a experiment base for deeply studying on biologic activity of polysaccharide, and offers a new mentality on screening anti-HBV drug with high effect and low toxicity.