Study On The Anti-HSV Activity Of Polyphenols From Phyllanthus Emblica L. And Investigation Of Its Possible Mechanisms

Objective:In order to examine the antiviral effect of polyphenols from Phyllanthus emblica L. against herpes simplex virus (HSV) and assess its potential for clinical applications, antiviral activity and mechanism experiments of polyphenols from Phyllanthus emblica L. were carried out in this study.Method:(1) Cytotoxic effects of polyphenols from Phyllanthus emblica L. on Vero cells were determined using MTT (3-{4,5-dimethylthiazol-2-y}-2-5-diphenytetrazolium bromide) method.(2) Acyclovir was used as positive reference drug, anti-HSV-1 and HSV-2 activity of polyphenols from Phyllanthus emblica L. were examined by cytopathic effect (CPE) observation and plaque reduction assay (PRA) in vitro.(3) To investigate the possible anti-HSV-1 mechanisms of polyphenols from Phyllanthus emblica L., virucidal assay, attachment assay, penetration assay and time-of-addition assay were carried out using PRA method.(4) Using immunofluorescence analysis, the effect of polyphenols from Phyllanthus emblica L. on the synthesis of virus protein VP5 of HSV-1 was evaluated by laser scanning confocal microscope.(5) With carbomer 940 as the base excipient, polyphenols from Phyllanthus emblica L. gel was prepared and its preliminary characteristics were evaluated.(6) The guinea pig model with dorsal skin HSV-1 inoculation was used to evaluate whether topical polyphenols from Phyllanthus emblica L.gel had an effect on HSV-1 infection in vivo.Results:(1) Polyphenols from Phyllanthus emblica L. possesses moderate toxicity, with 50% toxic concentration (TC50) value of 353.32μg/ml.(2) Polyphenols from Phyllanthus emblica L. showed anti-HSV-1 and HSV-2 activity in vitro, with 50% inhibiting concentration (IC50) values against HSV-1 and HSV-2 of 7.48μg/ml and 14.31μg/ml, respectively, and with therapeutic index (TI) values more than 47.24 and 24.69, respectively.(3) The results of PRA assay revealed that polyphenols from Phyllanthus emblica L.could inactivate HSV-1 directly and its mechanism may be destructing the structure of the virus envelope and losing ability of infected cells. However, polyphenols from Phyllanthus emblica L. did not interrupt the adsorption and penetration of HSV-1 and HSV-2 to cells. Time addition experiments showed anti-HSV effects of polyphenols from Phyllanthus emblica L. were related to administration time. Treatment with polyphenols from Phyllanthus emblica L.at 3-6h post-infection could effectively inhibit viral replication.(4) Immunofluorescence analysis showed HSV-1 UL19 gene expression and the synthesis of viral capsid protein VP5 was inhibited after treatment of polyphenols from Phyllanthus emblica L.(5) Gel preparetion of polyphenols from Phyllanthus emblica L. had weak acidity, suitable viscosity and stability in characteristics.(6) In vivo experiments showed that treatment of polyphenols from Phyllanthus emblica L. gel could reduce the virus damage to skin and accelerate herpes curst formation in infected guinea pigs.Conclusion:In brief, polyphenols from Phyllanthus emblica L. possesses strong anti-HSV activity in vitro and in vivo. The antiviral mechanisms may be related to inactivating HSV directly and inhibiting the early stage of viral replication cycle.