Extraction Of Hyperoside From Hawthorn And Its Antiviral Effect On Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea（PED）,a highly contagious,enteric disease of swine characterized by vomiting,diarrhea and dehydration is caused by porcine epidemic diarrhea virus（PEDV）leading to severe economic losses to the pig industry in our country.Vaccination is the primary measure for prevention and control of PEDV.However,with the rapid rate of viral mutation,multiple epidemic strains and lagging vaccine development,PEDV emerges frequently in pigs in our country.It is necessary to carry out antiviral drugs on PEDV.Hyperoside,widely distributed in a variety of plant fruits and herbs,possesses a broad spectrum of biological activities,such as anti-inflammatory,antibacterial,antiviral.Our previous study found that hyperoside inhibited PEDV replication in vitro by antagonizing N protein-induced S-phase arrest,interfering with interaction between N protein and p53.Hawthorn（Crataegus pinnatifida Bunge）,as a medicinal and edible plant,contains abundant hyperoside which can be used as an ideal source for extracting hyperoside.In this study,the optimal conditions for extraction of the natural hyperoside from hawthorn were clarified through single factor test and response surface test,and the antiviral effect on PEDV and mechanism of hyperoside from hawthorn were further confirmed in vitro and in vivo,so as to provide a basis for the development of anti-PEDV drugs.1.Extraction,purification and identification of hyperoside from hawthornIn order to clarify the optimal extraction conditions of hyperoside from hawthorn,single factor and response surface test were used and the optimal extraction conditions of hyperoside from hawthorn by thermal reflux of ethanol were ethanol concentration,70%;extraction time,120 min;ratio of solid to liquid,1:19（g/m L）;and extraction temperature,81℃.Under these conditions,the maximum yield of hyperoside from hawthorn was 0.42 mg/g.The crude hyperoside from hawthorn was purified by the macroporous resins HP-20 and Sephadex-LH20column.The final purity of hyperoside from hawthorn was 98.79%.UV spectrum,infrared spectrum and mass spectrum were used to identify the purified hyperoside from hawthorn.The spectra of UV showed that the hyperoside from hawthorn had strong absorption at 250 nm and360 nm which was consistent with that of the standard hyperoside.The FT-IR spectra showed that the absorption peaks of hyperoside from hawthorn at 3 305,1 656,1 608,1 506,1 365,1259 and 1 091 cm^-1which were consistent with those of standard hyperoside.The MS spectra showed that a high purity molecular ion peak m/z 462 of hyperoside from hawthorn which was consistent with that of the standard hyperoside.2.The anti-PEDV effect of hyperoside extracted from hawthornIn order to verify the anti-PEDV effect of hyperoside from hawthorn,the cytotoxicity assays,50%cytotoxic concentration(CC₅₀)test,Western blot,TCID₅₀test and 50%effective concentration(EC₅₀)test were carried out to analysis effects of the hyperoside from hawthorn on PEDV replication on Vero E6 cells.The results showed that the hyperoside from hawthorn had no cytotoxicity on Vero E6 cells at the concentration of 5.00μg/m L.The CC₅₀of hyperoside from hawthorn was 25.15μg/m L.Compared with PEDV HM2017 infection control group,the5.00μg/m L hyperoside from hawthorn significantly reduced PEDV N protein expression level（p<0.01）.The virus titers of PEDV HM2017 in the 5μg/m L hyperoside from hawthorn group(3.76 log₁₀TCID₅₀/m L)is significantly lower than the virus titer in the PEDV infection control group(4.79 log₁₀TCID₅₀/m L)（p<0.01）.The EC₅₀and SI of hyperoside from hawthorn were2.59μg/m L and 9.70 respectively.For further clarify the anti-PEDV effect of the hyperoside from hawthorn in vivo,the screening of the optimal drug dosage and the viral challenge to suckling piglets were performed to evaluate the anti-infection effect of the hyperoside from hawthorn against PEDV HM2017.The results revealed that hyperoside from hawthorn had no effect on survival rate,body weight,red blood cells,white blood cells and platelets of mice within 14 days when the dosage of hyperoside from hawthorn was less than 500 mg/kg.At 48 h after viral challenge,survival rate of the piglets in the hyperoside from hawthorn group was 75%（3/4）,and the survival rate of the piglets in the PEDV control group was 0%（0/4）.Compared with piglets in PEDV-infected control group,hyperoside from hawthorn reduced the clinical symptoms and intestinal pathological damage of the infected piglets.The viral loads of 12 h and36 h fecal swabs were 10^6.58copies/m L,10^5.41copies/m L,the viral loads of the jejunum and ileum were 10^6.11copies/g,10^5.70copies/g respectively in the hyperoside from hawthorn group,which were significantly lower than those in PEDV infection control group(The viral loads of12 h fecal swabs,36 h fecal swabs,jejunum and ileum were 10^7.48copies/m L,10^7.12copies/m L,10^7.44copies/g and 10^6.67copies/g)and hyperoside from hawthorn inhibited the expression of PEDV N protein in the ileum of the infected piglets（p<0.05）.The results demonstrated that hyperoside from hawthorn had an antiviral effect against PEDV HM2017 in vitro and in vivo.3.Mechanism of hyperoside extracted from hawthorn against PEDVPEDV N protein interacts with p53 to mediate host cell S-phase arrest to enhance PEDV replication.Standard hyperoside could inhibit PEDV replication by antagonizing the interaction between N protein and p53 to relieve S-phase arrest.In order to clarify the mechanism of hyperoside from hawthorn anti-PEDV effect,Co-IP and Western blot were used in vitro and in vivo experiments to analyse the mechanism of hyperoside from hawthorn against PEDV.The results in vitro test showed that 5.00μg/m L hyperoside from hawthorn significantly reduced p53,p21 proteins expression levels（p<0.01）,increased cyclin A protein expression level（p<0.05）and inhibited the interaction between N protein and p53（p<0.05）compared with PEDV infection control group.The results in vivo experiment showed that hyperoside from hawthorn significantly inhibited the expression of p53 protein and inhibited the interaction between PEDV N protein and p53 protein in ileum of challenge piglets（p<0.05）.These results indicated that hyperoside from hawthorn had an antiviral effect on PEDV by inhibiting PEDV N protein interaction with p53 and relieving host cell S phase arrest.In conclusion,hyperoside with high purity was successfully extracted from hawthorn.Hyperoside extracted from hawthorn had an antiviral effect on PEDV in vitro and in vivo by interfering with the interaction of the PEDV N and p53 proteins and intervening virus-mediated host cell cycle arrest.This study lays a theoretical foundation for the development of green,cheap and efficient natural drugs against PEDV.