The Study Of Inhibitory Effects Of Liamax Polysaccharide On Hepatitis B Virus In Vitro And In Vivo

Objective To study the optimal condition of extracting Limax polysaccharide and observe the inhibitory effect of Limax polysaccharide on Hepatitis B virus both in vitro and in vivo. It may lead a theoretical foundation on developing of a new kind of anti-virus medicine.Methods Limax was collected and identifid 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 time were optimized by orthogonal test. The crude Limax 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 Limax polysaccharide. HepG2.2.15 cell strain was used to study anti-HBV effect of Limax polysaccharide in vitro, and 3TC was applied as positive control.The different concentration of limax Polysaccharide water solution were respectively added into cell culture fluid of 2.2.15 cell strain. The cell toxicity of Limax polysaccharide was determined with MTT method, and the effect of Limax polysaccharide on HBsAg and HBeAg secreted by HepG2.2.15 cells under the diffenent concentration on the ninth day with ELISA method.Real-time PCR was used for determining secrected HBV-DNA in the cuLture medium. The cell toxicity of limax Polysaccharide was At same time, we took 3TC as a control to evaluate the effect on anti-HBV. Duck model of congenital infection of HBV was used as animal model and were randomly divided into five groups.Each group ducks was given the drug with the doses 25mg/(kg·d) for 10 days. The expression of DHBV-DNA was detected by dot-blot hybridization test before treatment, five days and ten days after treatment..Results The idal extraction process is as follows:dehydrated alcolol is added to the fluid until the alcohol content up to 70%; extraction with 60℃; extraction with water for two times; each extraction running 4 hours. In the cell model, Limax polysaccharide had no obvious cell toxicity at the concentration of 1mg/ml and depressed duplication on HBV-DNA. Limax polysaccharide had obvious restrain effect on HBsAg and HBeAg with maximum inhibition ratio addig up to 56.2% and 58.1%. The therapeutic index of Limax polysaccharide was 19.98% for HBsAg and 22.78% for HBeAg. There was certain inhibition on replication of HBV-DNA(P<0.05). In the animal model,After medicated with Limax polysaccharide for five days and for ten days. The optic density value (A490) of DHBV-DNA in ducklings serum were boviously lower than that before treatment. There was significant difference between them. The inhibition rate of Limax polysaccharide compared with the model group is markedly improved.Conclusion The optimized process is ecomomical, stable and efficient. Limax polysaccharide exerts a certain inhibitory effect on Hepatitis B Virus both in vitro and in vivo. 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.