Study On Active Ingredients In Natural Plant SY Targeting Influenza A Virus Nucleoprotein

H1N1 belongs to Orthomyxoviridae,Influenza A Virus,and its antigens are susceptible to mutations.Human beings are usually unable to obtain lasting immunity.Once it outbreaks,it will cause huge human health and social economy loss.Ion channel blockers and neuraminidase inhibitors are currently the main anti-influenza virus drugs,however,some virus has shown drug resistance already.Influenza virus nucleoprotein（NP）plays an important role in all aspects of the viral replication cycle,and the sequence is conserved and the antigen structure is very stable,it is a very ideal drug target.In addition,natural Chinese medicine has a long history and culture in China,its wide source,low price,little side-effect,not easy to produce resistance,has broad prospects for development in the anti-influenza virus drug research and development.In this study,the effects of natural herb SY extractions and separations anti-H1N1influenza virus and its preliminary mechanism were studied at the leval of cell and molecular,and the active ingredients targeting NP protein were isolated and identified.The natural herb SY was extracted with 30%ethanol,60%ethanol and 90%ethanol.Three kinds of crude extracts were obtained by penetration method with three different solvents.The H1N1 influenza virus infected MDCK cells in the presence of the three crude extracts.The results showed that all the extracts had inhibitory effect on the virus,60%of the ethanol extract showed the best inhibiton on the H1N1 virus with,the half inhibitory concentration(IC₅₀)was 1.394±0.11 mg/m L.It’s half toxicity concentration(TC₅₀)of cells was 41.3±3.7 mg/mL.Then,the affinity of the extracts with NP protein was determined by the surface plasmon resonance（SPR）instrument BIA3000.The protein was immobilized on the CM5 chip with bovine serum albumin as the blank control.The results showed that 60%ethanol extract and NP protein had the highest response value,which was consistent with the anti-virus effect at the cell level.The results implied that the antiviral mechanism of 60%ethanol extract was related to NP protein.The active components of 60%ethanol extract on the NP protein were separated and enriched using the protein amino-coupled column.The 1-2 mg of NP protein was incubated with the protein coupling column,then 60%of the alcohol extract was added.The NP-bound components were adsorbed by the column.The elution buffer eluted the column and the combination ingredients were collected in to the EP tube with 1 mL each seperately.The binding affinity of the collection with the NP protein was tested by SPR technique,in which the binding value of G3 and G4 demonstrated significantly increased.G3 and G4 also showed inhibition on influenza virus at the cell level.The ingredients of G3 and G4 were detected by HPLC-MS.The data were alligned with the Chinese Academy of Sciences chemical database and the laboratory of Chinese medicine composition database,and five possible compounds were deduced.Finally,the five compounds were docked with NP protein by DS molecular docking software.The results suggested that these three compounds have a potential inhibitory effect on H1N1 influenza virus,and compound 5 might have the best inhibitory effect with the highest score.The test of compound 5 on H1N1 virus showed a good antiviral activity with IC₅₀ of 190.9±11.05μg/m L.