| Objective:To separate the chemical constituents of antiviral activity in Artemisia rupestris L,obtain the effective constituent or active fraction of antivirus,and prepare the effective constituent or active fraction to varify the antiviral activity of the compounds in Artemisia rupestris L,provide the material basis for the antiviral activity of Artemisia rupestris L.Methods:（1）The separation and purify of the 50%ethanol-eluted fractions of Artemisia rupestris L were performed with polyamide column,ethanol-eluted fractions were obtained;（2）Anti-human cytomegalovirus,anti-respiratory syncytial virus pneumonia,anti-H1N1 influenza virus and anti H3N2 influenza virus activity of different concentrations of ethanol-eluted fractions were screened by the method of in vitro cell culture.Antiviral activity of Artemisia rupestris.L was evaluated by the Cytopathic effect（CPE）;The inhibition of H3N2 influenza virus proteins and RNA in 50%ethanol-eluted fractions were detected by western blotting and Real-time PCR method;Beijing ducklings were used to establish the model of duck Hepatitis B Virus and detected by dot blot method,digoxin kid dot bolt method and digoxin kid southern method,the activity of in vivo anti-HBV was determined by Serum DHBV antigen,Serum DHBV-DNA and Liver DHBV-DNA detection with the method of immunohistochemistry;（3）Antiviral chemical components of Artemisia rupestris L were analyzed by LC-HRMS/MS;（4）The better antiviral activity part were prepared by silica gel column and preparative high performance liquid chromatography（PHPLC）,to determine the structure of the compounds by the method of MS,1H-NMR and 13C-NMR.（5）Anti-human cytomegalovirus,anti-respiratory syncytial virus pneumonia,anti-H1N1 influenza virus and anti-95-359 influenza virus activity of components in Artemisia rupestris L were screened by the method of in vitro cell culture.Antiviral activity of Artemisia rupestris.L was evaluated by the Cytopathic effect（CPE）;The inhibition of H3N2 influenza virus proteins and RNA of components in Artemisia rupestris L were detected by western blotting and Real-time PCR method;Results:（1）Five ethanol-eluted fractions were isolated from Artemisia rupestris L by polyamide column;（2）Through the antiviral experimental in vitro,70%and 95%ethanol-eluted fractions showed some anti-human cytomegalovirus effect,each concentration of ethanol-eluted fractions showed some anti-respiratory syncytial virus pneumonia effect,10%,30%,50%and showed some anti-H1N1 influenza virus effect,50%ethanol-eluted fractions showed some anti H3N2influenza virus effect,50%ethanol-eluted fractions can significantly reduce the expression of M2 protein,NS1 protein and RNA of H3N2 influenza virus.30%and 50%ethanol-eluted fractions have good Anti-hepatitis B virus effect by the in vivo anti-virus method.（3）Through the LC-HRMS/MS method,seven major compounds were speculated from 30%ethanol-eluted fractions as apigenin,herniarin,Linarin,rupestonic acid,artemetin,tianshic acid;five major compounds were speculated from 50%ethanol-eluted fraction as 6-demethoxy-4′-O-methylcapillarisin-7-O-β-D-glucopyranoside,casticin,6-demethoxy-4′-O-methylcapillarisin,artemetin,kaempferol 3,3′,4′-trimethylether.（4）Through the MS and NMR method,three compounds were identified from 50%ethanol-eluted fractions as casticin,6-demethoxy-4′-O-methylcapillarisin,artemetin,trans-spiroketlaenolatherpolyin.（5）Through the antiviral experimental in vitro,compound A2、A9 showed some anti-respiratory syncytial virus pneumonia effects,compound A3、A6、A8、A9 showed some anti-H1N1 influenza virus effects,compound A6、A9 showed some anti A 95-359 influenza virus effect,compound A9 can significantly reduce the expression of M2 protein,NS1 protein and RNA of H3N2 influenza virus.Conclusions:It′s the first time to separate and obtain the better antiviral active ingredient of Artemisia rupestris L by PHPLC.It′s the first time to study antiviral activity of Artemisia rupestris L compounds,To determine the compounds contribution of antiviral,and judge antiviral active substances in Artemisia rupestris L directly,make the material basis of Artemisia rupestris L antiviral activity more clearer. |
