Study On The Effect Of Several Traditional Chinese Medicine Compositions And Their Modifications On DHAV-1 Proliferation In Vitro

Duck virus hepatitis(DVH),mainly caused by duck hepatitis A virus(DHAV),is an acute and highly fatal disease and it also causes serious economic losses in the duck industry.Currently,there is no effective drug against duck hepatitis A virus,it is extremely important to develop new anti-DHAV drugs.Here,the traditional Chinese medicine compositions with significant anti-DHAV-1 effect,was preliminarily screened out and compared through MTT method.Meanwhile,according to the dynamic changes of the relative DHAV-1 VP1 gene expression,the replication cycle of the virus was analysed.With the replication cycle,the mechanism of the antiviral effect of quercetin and baicalin was also studied.Based on the weakness in property and antiviral activity,geniposide was phosphorylated and quercetin was prepared as quercetin-phospholipid complex,then their anti-DHAV-1 effects were also compared.These researches will enrich the data for DVH control and provide the foundation for future studies on the development of antiviral drugs.Experiment ?:Comparison of several traditional Chinese medicine compositions with anti-DHAV-1 effectThe purpose of this experiment was to preliminarily screen the traditional Chinese medicine compositions with good anti-DHAV-1 effect,and compare their anti-DHAV-1 effect to lay a foundation for subsequent experiments.Here,the safety concentration and anti-DHAV-1 effect of 7 compositions in vitro were measured by MTT method.The result suggested that quercetin and baicalin showed significant anti-DHAV-1 effect,and their highest virus inhibition rates were 185.86%and 146.37%,respectively.Luteolin,galangin and hesperetin showed general antiviral effect their highest virus inhibition rates were 71.06%?69.23%?65.67%,respectively.Besides,with the highest virus inhibition rate was 15.03%,the anti-DHAV-1 effect of naringenin was the worst.Overall,the effect of quercetin,baicalin and geniposide was better.Experiment ?:Effect of quercetin and baicalin on the DHAV-1 replication cycle in vitroThe purpose of this experiment was to explore the mechanism of anti-DHAV-1 effect of quercetin and baicalin in the viral replication cycle,and provide references for the prevention and treatment of DHAV.Here,the dynamic changes of the relative DHAV-1 VP1 gene expression in the replication cycle were detected by real-time PCR,and the morphology of hepatocytes infected with DHAV was evaluated by electron microscope.Based on the replication cycle of DHAV-1,quercetin and baicalin worked in different stages of the replication cycle,and the relative DHAV-1 VP1 gene expressions during adsorption,replication and release were also measured by real-time PCR.The result suggested that the adsorption of DHAV-1 saturated at 90 min post-infection,and the virus particles were observed on the infected cells surface.What's more,the replication lasted around 13 h after the early protein synthesis for about 5 hours,and the release of DHAV-1 was in steady state after 32 h.Quercetin and baicalin in vitro were able to show significant anti-DHAV-1 effect.They could not only inhibit cell lesion caused by virus,could also affect the proliferation of the DHAV-1.Especially,quercetin dramatically reduced the expression of DHAV-1 VP1 gene during replication,so it exerted a strong inhibition to virus replication,and there might be a particular mechanism.While baicalin could reduce the expression of DHAV-1 VP1 gene during replication and release significantly,hence it had wider antiviral range,it had impact on the replication and release of DHAV-1.Experiment?:Preparation and anti-DHAV-1 effect of phosphorylated geniposide and quercetin-phospholipid complexThe purpose of this experiment was to improve the antiviral activity and biological effectiveness of geniposide and quercetin by structural modification,and provide information for the development of anti-duck hepatitis virus drugs.Here,the phosphorylated geniposide was prepared through the method of sodium tripolyphosphate-sodium trimetaphosphate to improve the anti-DHAV-1 effect of geniposide,and the quercetin-phospholipid complex was synthesized by the solvent method to improve the biological effectiveness of quercetin.Meanwhile,the obtained modifications were identified using infrared spectrum,mass spectrometry,etc.In addition,the anti-DHAV-1 effect of these two modifications was also evaluated by MTT method and real-time PCR.The results suggested that the phosphorylated geniposide and quercetin-phospholipid complex were successfully obtained.The best modification condition of phosphorylated geniposide was as follows:the pH was 8.5,the reaction temperature was 70 ? and reaction time was 4 h.After modification,a hydroxyl group of geniposide was replaced by a phosphate group,the highest virus inhibitory rate raised from 52.63%to 73.09%and the expression of DHAV-1 VP1gene reduced to 0.411,significantly.Hence,the anti-DHAV-1 effect of the phosphates was improved.Moreover,the quercetin-phospholipid complex was a new product which was different from quercetin and soybean lecithin.And it had a good virus inhibitory rate and played a role in inhibiting the expression of DHAV-1 VPlgene at low concentration compared to quercetin.Therefore,the complex could not only retain the good anti-DHAV-1 activity of quercetin,but also improve the biological effectiveness.