Effect Of Phosphorylated Gypenosides On Duck Embryonic Hepatocytes Infected By Dhav-1 And Activity Of Duck M?

Gynostemma pentaphyllum,is a famous traditional Chinese medicine,which has the effect of similar ginseng without cold and heat and its side effects,has the reputation of"Ren Jian Xian Cao".With the deepening of the research on Gynostemma in various parts of the world in recent years,it is found that the main active ingredients are saponins,flavonoids and polysaccharides.Gypenosides(GP),one of its major active ingredients,has antiviral,antioxidant and immune enhancing effects and plays an important role in the prevention and treatment of viral hepatitis.Therefore,this study selected GP as a candidate for treatment.In order to enhance or increase the biological activity of traditional Chinese medicine,molecular modification has become an important research field of medicinal chemistry in recent years.The main modification methods include phosphorylation,sulfation and acetylation.In these methods,both phosphorylation and sulfation can significantly enhance the biological activity of the modified components.However,phosphorylation is safer,more convenient and environmentally friendly than sulfation.Therefore,in this study,molecular modification was carried out by phosphorylation method,the optimal modification conditions were obtained by orthogonal test,the hydroxyl groups were replaced by the phosphate groups to form a phosphorylated gypenosides.The activity of gypenosides and phosphorylated gypenosides against DHAV-1 in vitro,antiviral mechanism in vitro,the impact of macrophage immune function were studied,the purpose of this study is to develop a new type of antiviral drug.The test is divided into four parts as follows:Experiment I Gypenosides phosphorylated modifications and its safe concentration on duck embryo hepatocytes The purpose of this study was to investigate the modification conditions of phosphogypsum gypenosides and the structural identification of phosphorylated modifications.Firstly,Using sodium tripolyphosphate and sodium trimetaphosphate phosphate as phosphorylation reagents.The optimal conditions were screened by the orthogonal design of orthogonal test table L9(34),then the structure of pre-modification and modified gypenosides was identified.The experimental results show that when the pH is 9,the reaction time is 4h,the reaction temperature is 65? showed the best technology for the phosphorylation of gypenosides.The product quality according to the best preparation procedure was 159.6 mg,and the content of phosphate was 32.59%and the content of saponin was 67.08%.The maximum safe concentrations of gypenoside and phosphorylated gypenosides on duck embryo hepatocytes measured by MTT assay were 100 ?g/mL and 25 ?g/mL,respectively.Experiment II Study on the effect of phosphorylated gypenosides against DHAV-1 infecting duck embryo hepatocytes In order to observe the anti-DHAV-1 effect,gypenosides(GP)and phosphorylated gypenosides(pGP)were diluted from the maximum safe concentration,and added into cultivating system of duck embryo hepatocytes(DEHs)by three models,pre-,post-adding drug and simultaneous adding drug and DHAV-1.The effects of different concentrations of GP and pGP on DHV infecting DEHs were determined by MTT assay.Real time-PCR method was used to compare the effects of GP and pGP at the most effective concentration on the adsorption,replication and release of DHAV-1 on DEHs.The results showed that both GP and pGP had a good anti-DHAV-1 effect in vitro,and the anti-DHAV-1 effect of pGP was better than that of GP during pre,post-adding drug.During post-adding drug,the two drugs have no obvious effect on the virus adsorption on duck embryo hepatocytes,but can effectively inhibit the replication and release of DHAV-1,and the inhibitory effect of pGP is better than that of GP.This study has shown that phosphorylation modification significantly enhances the direct anti-DHAV-1 effect of gypenosides.Hopefully,phosphorylated gypenoside is developed into a new anti-DHAV-1 drug.Experiment III Effect of phosphorylated gypenoside on apoptosis duck embryo hepatocytes apoptosis induced by DHAV-1 In order to further study the antiviral mechanism of GP and pGP,we used TUNEL and Annexin V/PI flow cytometry to detect the effect of GP and pGP at the optimal anti-DHAV-1 concentrations on apoptosis of duck Embryo Hepatocytes(DEHs)infected by DHAV-1.The results of TUNEL assay showed that compared with the control group,DHAV-1 induced significant apoptosis of DEHs(p<0.05),and the apoptosis rate increased by 24.19%;compared with the virus control group,both GP and pGP significantly inhibited the apoptosis of DEHs induced by DHAV-1,significantly reduced the apoptotic rates(p<0.05),and the apoptotic rate decreased by 17.42%and 20.90%,respectively.The effect of pGP was stronger than that of GP.The results of Annexin V/PI flow cytometry analysis showed that:Compared with the cell control group,DHAV-1 can induce significant apoptosis of DEHs(p<0.05),the rate of apoptosis increased 26.26%;Compared with the virus control group,both GP and pGP significantly inhibited the apoptosis of DEHs induced by DHAV-1,significantly reduced the apoptotic rates(p<0.05),the apoptotic rates decreased by 14.70%and 22.67%,respectively,and the effect of pGP was significantly stronger than GP.The results of Annexin V/PI flow cytometry assay were basically consistent with that of TUNEL assay.This suggests that GP and pGP have the effect of anti-DHAV-1 infection by inhibiting DEHs apoptosis induced by DHAV-1,and the effect of pGP is better than GP.Experiment IV Effect of Phosphorylated Gypenosides on Duck Mcp activity To investigate the effects of GP and pGP on duck peritoneal macrophage activity.Neutrophil red phagocytosis assay,metabolic MTT viability assay and ELISA were used to determine the phagocytic activity of GP and pGP on peritoneal macrophages,metabolic activity of MTT,and secretion of inflammatory cytokines NO,IL-1?,IL-6 and TNF,respectively.The results showed that when macrophages of gypenosides at the concentration of 100 p,g/mL and phosphorylated gypenosides at the concentration of 25 ?g/mL,reached the highest phagocytic capacity and phagocytic index reached the maximum,macrophage metabolism MTT reached the strongest activity;In addition,at this concentration,both GP and pGP could significantly promote the secretion of inflammatory cytokines in macrophages without LPS-stimulated,and at the same time,significantly inhibited the secretion of inflammatory cytokines in macrophages induced by LPS.Therefore,GP and pGP can enhance the immune function of duck macrophages in vitro,and the effection of pGP is stronger than GP.