Inhibitive Effect And Mechanism Of Epimedium Flavone And Its Phosphorylated Derivative On Duck Virus Hepatitis

Epimedium(Epimedium davidii Franch)is Chinese traditional herbal tonic medicine,with "Tonifying Qi,strong bones,waist up,strong effort" effect.With the recent study of its pharmacological function of increasing depth,we found that Epimedium flavones(EF)was the main pharmacological active ingredients for Epimedium.Many clinical and experimental studies have shown that it has anti-viral,anti-oxidant,immune-enhancing effects.However,due to the flavonoids poorly water-soluble,some inconveniences often occured in the clinical application.Therefore,this study focused on improving land Epimedium Flavonoids bioavailability,in order to better play its antiviral effect and serve the clinical application.Aims to study a new type of antiviral drugs,this research is obtained by the orthogonal experiment and confirmed the optimal modification conditions to phosphorylated modification of Epimedium flavones,eventually the hydroxy replaced by the phosphate root to result in phosphorylated epimedium flavonones(pEF).Activity of EF and pEF’s anti duck hepatitis virus 1(DHV-1)on duck embryonic hepatocytes(DEHs),anti-virus mechanisms and lymphocyte proliferation in vitro,and immuno-enhancing and antioxidant activity in vivo were studied and further verified byintervention experiment.The research is divided into the following five sections:Test ⅠPhosphorylation molecular modification and structural identification of Epimedium flavones This experiment was intended to study phosphorylated modification condition of EF and identify the chemistry structural of pEF.The sodium tripolyphosphate-three partial sodium phosphate served as phosphorylation reagent,according to the L9(34)level design orthogonal experiment to screen the optimum modification conditions,confirmed the molecular structure of pEF by FT-IR,HRESIMS and NMR.The results showed that the optimum conditions for the phosphorylation of EF were temperature 70 ℃,reaction time 6 h,the reaction pH=9.The chemical formula of pEF was 8-prenylkaempferol-4’-methylether-3-rhamnosyl-7-(6"’-phosphate)-glycoside.Test Ⅱ Anti-DHV-1 activity of Epimedium:flavone and its phosphorylated derivative in vitro Anti-DHV-1 activities of EF and pEF were compared on DEHs,direct immunofluorescence method and real-time PCR were applied to analyze the antiviral mechanisms of adsorption,replication and release in vitro.The results indicated that EF and pEF effectively resisted DHV-1 against invading DEHs,pEF performed more effectively.EF and pEF performed evenly on DHV-1 replication and release,although pEF restrained virus replication far more effectively.EF and pEF could inhibit DHV-1 adsorption but there was no significant difference between them.Test Ⅲ Roles of the antioxidant properties of Epimedium flavone and its phosphorylated derivative in the protection against DVH This experiment studied antioxidative and hepatoprotective effects of EF and pEF against DVH.180 cherry valley duck were randomly divide four groups:EF group,pEF group,VC group and BC group.Ducklings allocated to the EF,pEF,and VC groups were intramuscularly injected with DHV-1 per duckling.Ducklings allocated to the icariin or p-icariin groups were given aqueous EF or pEF at the dosage of 31.25 mg/Kg of per duckling,SID,for 3 days starting on the same day as DHV-1 injection.Blood samples were taken from five ducklings in each group at 4,8,and 54 h after injecting DHV-1.Determinated index of oxidative damage and liver injury in the blood and analyzed the correlation between them,monitored clinical symptoms and number of dead ducklings.The results showed that DHV-1 induced significant oxidative damage in ducklings.EF and pEF improved the survival and attenuated oxidative stress and liver dysfunction induced by DHV-1.The clinical effects of both EF and pEF were related to their antioxidant activities.Test Ⅳ Roles of the immuno-enhancing of Epimedium flavone and its phosphorylated derivative in the protection against DVH This experiment was designed to study the role of immuno-regulation in anti-DVH activities of EF and pEF.For this purpose,we determined B and T lymphocyte proliferation in vitro,monitored virus content and the levels of serum antibody(Ab),IFN-γ,hepatocyte growth factor(HGF),IL-2,and IL-6 in duck by DHV-1 challenged and studied the correlation between Ab,cytokines and hepatic injury evaluation indexes(ALT、AST、ALP、LDH).The results showed that EF and pEF possessed reliable immuno-enhancing activity.They could promote lymphocyte proliferation,decrease the content of virus in blood,up-regulate the levels of antibody titer,IFN-y,IL-2,IL6 and HGF.Meanwhile,the strong correlation between the immunity indexes and liver injury indexes showed immune enhancement played an important role in the anti-DVH process.Test Ⅴ Oxidative stress and immune injury intervention trials served to verify the anti-DVH mechanism of Epimedium flavone and its phosphorylated derivative This experiment was served as to verify the role of antioxidant and immunomodulatory of EF and pEF of anti-DVH founded in the previous test byintervention trials.Hinokitiol and tacrolimus(FK506),respectively,served as pro-oxidants and immunosuppressants.Death of DHV-1 infected ducks,oxidative damage(GSH-Px,MDA,SOD,catalase)and immune function evaluation(IL-2,IFN-1)changes in vivo were dynamicly observed before and after the intervention..The results showed that EF and pEF suppressesed the pro-oxidant and immunosuppressant role of the Hinokitiol and tacrolimus to increase the survival rate of the duckling challenged by DHV-1 and treated by Hinokitiol and tacrolimus.The results illustrated the antioxidant and immune regulating played an important role for EF and pEF treatment DVH.