Screening Of Anti-herpes Simplex Virus Type Ⅰ Activity And Molecular Mechanism Of Crude Extract Of Artemisia Argyi.

Objective:To screening the anti-HSV-1 activity of crude extracts（Fr.6 and Fr.8.3）isolated and extracted from Tangyin Artemisia vulgaris L.leaves,and to explore the antiviral mechanism.Methods:The anti-HSV-1 active drugs of the crude extracts of A.vulgaris L.were screened by the CPE cytopathic effect method,and the toxic effects of Fr.6 and Fr.8.3 on different cells were detected by the CCK8 assay.The anti-HSV-1 activity of Fr.6 and Fr.8.3 was detected by plaque reduction test,viral titer determination,viral genomic DNA copy number detection,and western blot etc.On the basis of confirming that Fr.6 has more obvious anti-HSV-1 activity,the anti-HSV-1 mechanism of Fr.6 was clarified by some methods such as direct virus inactivation,viral attachment and viral penetration detection.The main components of Fr.6 were analyzed by LC-MS,and finally,the potential targets of Fr.6 were further identified by biological transmission electron microscope and molecular docking methods in bioinformatics.Results:Fr.6 and Fr.8.3 with anti-HSV-1 activity were screened out by CPE method.Fr.6 and Fr.8.3 were not very toxic to vero cells,with IC₅₀values of 21.47μg/m L and53.26μg/m L,respectively,and the toxic effects of Fr.6 and Fr.8.3 on MA-104 cells were relatively small,at 23.35μg/m L and 54.29μg/m L,respectively.In addition,we also detected the toxicity of Fr.6 to U87-MG and SH-SY5Y two nerve cells with IC₅₀values of47.72μg/m L and 16.42μg/m L,respectively.The results of plaque reduction assay,virus titer determination,viral genome DNA copy number detection and western blot showed that both Fr.6 and Fr.8.3 have anti-HSV-1 activity on vero cells,the results showed that Fr.6 has better anti-HSV-1 activity than Fr.8.3.It was found that Fr.6 and Fr.8.3 also showed good antiviral activity against ACV-resistant strains,the effect of Fr.6 on ACV resistant strains was more significant.Meanwhile,both Fr.6 and Fr.8.3 showed broad antiviral spectrum activity,but Fr.8.3 had better antiviral spectrum activity than Fr.6.In addition,mechanism studies have found that Fr.6 can directly inactivate HSV-1 and has inhibitory effects on both the attachment and penetration of HSV-1.According to LC-MS analysis,there are 12 main components in Fr.6.Among them,DEOXYSAPPANONE B7,3"-DIMETHYL ETHER and 3,7-Dihydroxy-3’,4’-dimethoxyflavone play the main anti-HSV-1 effect.Finally,g B was further predicted as a potential target of Fr.6 by biological transmission electron microscope and molecular docking.Conclusions:Both Fr.6 and Fr.8.3,the crude extracts of Artemisia argyi,showed anti-HSV-1 activity,and the anti-HSV-1 activity of Fr.6 is more obvious.In addition,Fr.6and Fr.8.3 have antiviral activity against ACV-resistant strains.Meanwhile,both Fr.8.3 and Fr.6 exhibited broad antiviral spectrum activity,such as anti-HSV-2 and anti-rotavirus activity,the antiviral spectrum activity of Fr.8.3 was significantly better than that of Fr.6,which means that the specificity of Fr.6 anti-HSV-1 activity was higher than that of Fr.8.3.The anti-HSV-1 activity of Fr.6 was superior to that of its own antiviral spectrum.Mechanism studies show that Fr.6 may exert anti-HSV-1 activity through HSV-1 envelope glycoprotein-g B,and gB may be a potential target of Fr.6.