Comparative Study Of Astragalus Polysaccahride And Its Two Kinds Of Molecular Modifiers On Anti-DHAV Activities

Duck virus hepatitis is caused by duck hepatitis A virus(DHAV),the virus is mainly infected with ducklings.Once the ducklings are infected by the virus,the disease spread rapidly,appear symptoms quickly and cause wide-bound death.So it is one of diseases are defensed in duck breeding industry.This topic aims to study the differences of antiviral activities between Astragalus polysaccharide(APS)and its derivatives and lay the foundation for research of the treatment drugs by using chemical method to obtain phosphorylated(pAPS)and sulfated(sAPS)derivatives,compassion the antiviral activities in vitro,the clinical treatment effect and the immune regulation effect.The research content is divided into the following several parts:Experiment I:Extraction and molecular modification of Astragalus polysaccharides The purpose of this experiment was to extract astragalus polysaccharides and optimize the phosphorylation process for subsequent experiments.The polysaccharide was extracted from Astragalus membranaceus by water detection and the ethanol precipitation method.After being deproteinized by chloroform and urified with the Sevage method and column chromatography with DEAE column,then the sodium trimetaphosphate-sodium tripolyphosphate(STMP-STPP)method and chlorosulfonic acid-pyridine method were applied to obtain the derivatives of phosphorylate and sulfate.The derivatives was determined by FT-IR spectra and the spectragrams were compared with that of Astragalus polysaccharide.The results indicated that the content of purified polysaccharide was 74.86%.The optimal pH of phosphorylation was 8.5,reaction time was 4h and the temperature was 70 ℃.The polysaccharide content of phosphorylated derivatives was 45.28%,phosphate content was 40.31%.Experiment II:Comparison of antiviral activities of Astragalus polysaccharide and its two kinds of derivatives in vitro In order to evaluated the anti-DHAV effect of APS,pAPS and sAPS in vitro,antiviral activities of APS、pAPS and.sAPS were tested by MTT method on duck embryo hepatocyts(DEHs)through different add-drug patterns.Determination of three drugs against virus proliferation in different stages was tested by the RT-PCR method.The results showed that the maximum safe concentration of APS,pAPS and sAPS was 1250、625 and 15.625 μg·mL-1 respectively.In the test of different add-drug patterns,both APS and its derivatives had anti DHAV effect and the antiviral activities of pAPS was the best;RT-PCR results showed that,in the process of virus proliferation,APS and its derivatives mainly inhibited the viral replication and release and the effect of virus inhibition of pAPS was the best.Experiment Ⅲ:Comparison of clinical therapy effect of Astragalus polysaccharide and its two kinds of derivatives This experiment was aim to compare the antiviral effect of APS,pAPS and sAPS in vivo.The ducklings were divided into APS group,pAPS group,sAPS group and VC group after intramuscular injection with DHAV.And the BC group was needed.The survival rate,dynamic survival number and anatomical lesions of each group were caculated and observed.The biochemical indicators of liver injury(ALT、AST、ALP、LDH、TP、ALB and GLO)and oxidative damage indicators were(MDA、NOS、GSH-Px and SOD)determinted by corresponding kits.The RT-PCR method was applied for the testing of DHAV relative expression in blood at the corresponding time point after the attack.The results showed that only the pAPS performed better antiviral activities than APS and sAPS for the significant decrease in the feather of dead ducklings,obvious alleviation of liver injury and the relative expression of DHAV in blood during the later stage.Experiment IV:Comparison on the role of immune regulation of Astragalus polysaccharide and its two kinds of derivatives The purpose of this experiment was to compare the regulating effect on duck immunity of APS,pAPS and sAPS in vitro and in vivo.After determination of the maximum safe concentration of APS,pAPS and sAPS and the optimal concentration of PHA and CyA on peripheral blood lymphocytes of adult ducks,the peripheral blood lymphocytes were isolated by using lymph fluid and being divided into different treatments including single treatment,PHA treatment and CyA treatment respectively.In every treatment,the APS,pAPS and sAPS were added and the activities of lymphocytes were tested by MTT method.The supernatant was collected for the determination of the IL-2 and IFN-y by using ELISA kits.In vivo,the content of IL-2 and IFN-γ in blood plasma after virus inoculation was determinated by ELISA kits.The results exhibited that APS,pAPS and sAPS in three treatments could promote the lymphocytes proliferation and release of IFN-γ.The effect of pAPS was more obvious than APS and sAPS.The results of IFN-y and IL-2 in vivo study were in accord with those in vitro.It can be found that the pAPS possessed the best effect including anti-DHAV activities on DEHs,clinical therapy effect and the function of improving immunity comparing with APS and sAPS.