The Studies Of Several Types Of Natural Products On Anti-H1N1 Virus Activity And Mechanism

Influenza is an acute febrile respiratory infectious disease caused by influenza virus,characterized by strong infectiousness and rapid transmission.According to the estimates of WHO,influenza causes about 200,000 to 500,000 deaths every year,which has a huge impact on global health.Influenza viruses belong to the orthomyxoviridae family,with RNA as the main genetic material.Due to the diversity of influenza virus subtypes and high variability,it is easy to escape the immune clearance of the host by highly mutating through antigenic drift and antigenic conversion,and produce new cross-species infected virus strains,triggering drug resistance and prompting continuo-us updates of corresponding vaccines.The development of a vaccine typically takes six months after an outbreak,and the drugs play an important role before a vaccine can be developed.Therefore,it is necessary to search for new anti-influenza drugs.Using anti-influenza A virus model in vitro,we evaluated anti-influenza A virus activity of 93 crude extract isolated from 18 characteristic medicinal plants such as Sophora flavescens,Vangzan and Rhizoctonia chinensis,and 216 monomeric compou-nds isolated from medicinal plants such as Sophora flavescens,Patchouli and Arachnoides.The structural types of monomer compounds include alkaloids,flavonoi-ds,iridoids,sesquiterpenoids and so on.The results showed that Et OAC phase(EC₅₀=127.25μg/m L,Si>2.00),n-Bu OH phase of Pogostemon cablin(EC₅₀=285.05μg/m L,Si>2.00),Et OAC phase of Elsholtzia densiflora(EC₅₀=50.22μg/m L,SI>2.00),and the total extract of Sophora flavesiens(EC₅₀=76.33μg/m L,Si>2.00),Et OAC phase of Valeriana jatamansi(EC₅₀=78.69μg/m L,Si>2.00),petroleum ether phase of Valeriana officinalis(EC₅₀ was 68.69μg/m L,Si>2.00)and Et OAC phase of honeys-uckle(EC₅₀=128.26μg/m L,Si>2.00),which showed different degrees of anti WSN influenza A virus activitiesn in vitro.And compounds of 132(EC₅₀=134.36μm),135(EC₅₀=19.26μm),140(EC₅₀=85.45μm),189(EC₅₀=138.68μm)and 194(EC₅₀=52.66μm)showed anti WSN influenza A virus activitiesn,and compound 190(EC₅₀=53.68μm)had anti-PR8 influenza A virus activity.Among them,132,135 and 140 are iridoids from Arachnoides fragrans,and 189,190 and 194 are sesquiterpenes from patchouli.The mechanism of iridoid Isovaltrate acetoxyhydrin（135）against WSN influenza A virus in vitro was explored.compound 135 had a significant protective effect on MDCK cells which infected with WSN virus.The mechanism study showed that the anti-influenza virus activity of compound 135 mainly occurred in the early stage of the virus life cycle,cytoplasmic transport and nucleation stage.The interaction of 135 and NP was analyzed using molecular docking.Compound 135 shows that it can bind to aspartic acid（N395）,threonine（T396）,and leucine（L447）in the Nuclear localization signal（NLS3）and NP-NP binding region by hydrogen bond,suggesting that compound135 could inhibit the transport and nucleation process of VRNP by interfering with the function of NP protein.At the same time,135 also had a certain inhibitory effect on NA and inhibited the expression of inflammatory factors in macrophages.The sesquiterpenoid compound（1R,2R,4a R,6R,8a S）-Hexahydro-2,5,5,8a-tetram-ethyl-1,6-methanonaphthalene-1,4a（2H,5H）-diol（190）had a dose-dependent protecti-ve effect on MDCK infected with PR8 virus.By tracking the replication cycle of the virus,we found that the inhibitory effect of compound 190 on PR8 covered most of the life cycle of the virus,and appeared at the stage of inhibiting virus adsorption at the earliest,but the effect was small,which was confirmed by chicken blood erythrocyte agglutination test.When the virus infected the host cells for 8 h,the compounds showed obvious inhibitory effect,suggesting that the compounds played an inhibitory role before the massive replication of the virus,possibly acting on the nucleoviral replication stage.But the specific target remained to be further studied.In conclusion,compound190 might exert anti-influenza virus activity by interfering with the process of virus entry into the nucleus and replication.