The Research On The Mechanism Of Patchouli Alcohol Anti-influenza Activity

Objective:The plant Pogostemon cablin(Blanco)Benth,which is daodi Medicinal Material in Lingnan.It can remove dampness by means of aromatics,stop vomiting by regulating stomach,and dispel summerheat to relieve exterior syndrome.It was recorded for the treatment of cold in ancient Chinese.Its main medicinal ingredient is volatile oil component,and its main components are patchouli alcohol and patchouli ketone.Among them,patchouli alcohol has been reported to have great anti-influenza activity.The aim of this paper is to study the anti-influenza virus effect of patchoulol at the cellular level,RNA level,and protein level.Study for some experiments,such as different periods of administration,Hemagglutinin Inhibition assay,Hemolysis assay,patchouli alcohol in influenza A virus by serial passage experiments and resistant mutation identification,to observe the main stages of its impact on the replication process of influenza virus and related targets.Methods:1.To determine the cell cytotoxicity of patchouli alcohol by cytopathic effect(CPE)assay.To study the virus inhibition effect of patchouli alcohol on influenza virus H1N1 PR8 in MDCK cells and A549 cells by comprehensive administration.To research the inhibition effect of patchouli alcohol on influenza virus by Plaque Reduction Assay and treatment in single administration.Established a model of infected virus cells,to study the anti-influenza activity of patchouli alcohol by Real time RT-PCR and Western blotting assays.To learn the main stage of the effect of patchouli on the replication of influenza virus.2.Adding patchouli alcohol in different time periods(-2h～-1h,-1h～0h,0h～2h,2h～4h,4h～48h),then determine the virus inhibition by CPE assay to find the main stage of the effect of patchouli on the replication of influenza virus.3.The Hagglutination inhibition assay mainly reflects the process of influenza virus adsorption of red blood cells,while the hemolysis assay mainly reflects the process of fusion of influenza virus and host cell membrane.To research the related drug target by Hagglutination inhibition assay,the hemolysis assay and serial passage experiments and resistant mutation identification.Results:1.The median toxic concentration TC50 of patchouli alcohol on MDCK cells was 128.70±6.03 μM;the TC50 on A549 cells was 148.94±3.05 μM.2.On MDCK cells and A549 cells,the median effective concentration IC50 of patchouli alcohol by comprehensive administration was 32.54±2.13 μM and 25.71±2.63 μM.And in the plaque reduction assay,it was observed that patchouli alcohol can effectively inhibit the formation of influenza virus plaques.The median effective concentration IC50 of each single administrat-ion were 41.55±1.83 u M(Prophylactic administration);19.22±2.80 μM(Permixed administration);38.01±2.01 μM(Simultaneous administrati-on);49.04±4.4 μM(Therapeutic administration)3.We were found that patchouli alcohol can effectively reduce viral mRNA of NP transcription and protein of NP expression.4.In the time of Addition Experiments,it was found that there was strongest inhibitory effect at patchouli alcohol was added in the period of Oh～2h,and in the period of 2h～4h had little infibitory effect.5.It was found that patchouli alcohol can inhibitor the hemagglutinin and hemolysis.6.In the serial passage experiments and resistant mutation identi fication,it was found patchouli alcohol caused four sites to change on the HA protein gene segment of the influenza A virus H1N1 PR8.Conclusion:In the study,we found patchouli alcohol can inhibit the influenza virus H1N1 on MDCK cell and A549 cells.Through the Plaque Reduction Assay,treatment in single administration,Real time RT-PCR and Western blotting assays,it was found that the effect of patchouli alcohol on influenza virus occurred in the early stage of virus replication.Through the Hagglutination inhibition assay,the hemolysis assay and serial passage experiments and resistant mutation identification,it was found that the antiviral drug target of patchouli alcohol was determined on the HA protein.