Experimental Study On The Antiviral Activity And Mechanism Of Ceratostigma Willmattianum Against Herpes Simplex Virus Type 1

Being a kind of little shrub, Ceratostigma willmattianum belongs to ceratostigma genus in the family Plumbaginaceae. There are 23 compounds have been isolated by Tibet Plateau Institute of Biology, of which plumbagin and plumbagic acid are the primary ingredients. It has been reported that Ceratostigma willmattianum has shown strong antibiosis effects, but there is no report with regard to its antiviral activities.In order to examine the antiviral effect of the extract from Ceratostigma willmattianum against herpes simplex virus type-1 (HSV-1) and assess its potential for clinical applications, both in vitro and in vivo antiviral experiments of Ceratostigma willmattianum were carried out in this study.Specifically, Ceratostigma willmattianum in various concentrations was applied to Vero cells infected by HSV-1. Parameters such as fifty percent tissue culture infective dose (TCID₅0), cytopathic effect (CPE), plaque-forming unit (PFU),MTTstaining method were used to characterize the antiviral activity. From MTT staining experiment Ceratostigma willmattianum: gave a fifty percent toxic concentration (TC5l , and the therapeutic index (TI) of 36.56. It confirms that there are no significant differences between the TIs found in Ceratostigma willmattianum and that found in ACV. Also, an evident HSV-1 inhibitory effect of Ceratostigma willmattianum was observed using transmission eled ron microscope at 625 m g *L^' concentration. In conclusion, the extract from the plant exhibits a remarkable inhibitory effect on HSV-1 in vitro.A study designed to investigate the time course effect of the virus infection at different concentrations of Ceratostigma willmattianum revealed that Ceratostigma w. at concentrations 156^<sup>625 mg ? L"1 exhibited the maximum inhibition against HSV-1 infection from 0 to 12 h.In the in vivo experiments, two disease models were made by local-inoculation on the cornea and full-inoculation through tail vein of mice, and then treated with the extract from Ceratostigma willmattianum with different doses. Meanwhile, ACV was employed as a positive control. The focus of the experiments was on the remedial action of Ceratostigma willmattianum to HSV-1 full-infection about variety of viscera pathology and the time of antigen' s appearance and disappearance. They were also aimed at the remedial action to herpes simplex keratitis (HSK) from theacreage variety of cornea pathological changes. Results showed that Ceratostigma willmattianum could decrease the pathological changes in viscera, eliminate the virus antigen notably, and give rise to the effects at a dosage of 0. 3 g/kg/d that is equal to those of positive drug ACV. The data thus seem to indicate Ceratostigma willmattianum has remedial action to HSK.Furthermore, the result obtained at concentration of 625 m g ? L"1 using scanning electron microscope demonstrated that Ceratostigma willmattianum could apparently inhibit HSV-1. The extract of Ceratostigma willmattianum could neither kill HSV-1 directly nor release it from the infected cells, it showed interfering effect on absorption of HSV-1 to Vero cells instead, which might count for the observed inhibitory HSV-1 infectivity.In order to investigate the mechanism on how Ceratostigma willmattianum inhibits the infection of herpes viruses, dot blotting and Northern blotting analysis were used as an estimating index for antiviral activity. Ceratostigma willmattianum could interfere absorption of HSV-1 to Vero cells to prevent HSV-1 infectivity, inhibit HSV-1 gD DNA replication and HSV-1 gD mRNA expression.To further study the mechanisms of Ceratostigma willmattianumagainst HSV-1, HSV-1 DNA synthesis was determined using isopycnic separation of cellular and viral DNA in CsCl gradients centrifugation and 3H-TdR incorporating methods. HSV-1 RNA synthesis was determined using :1H-UTP incorporating method.The results demonstrated that Ceratostigma w. could inhibit HSV-1 DNA synthesis selectively and hence inhibit HSV-1 RNA synthesis.In addtion, SDS-PAGE and Western Blot were employed to determine protein synthesis in the presence of Ceratostigma w. The above concentrations of Ceratostigma w. did not show any inhibitory effects on cellular protein synthesis, but selectively inhibited the viral protein synthesis.In brief, the pharmacological experiments confirmed that Ceratostigma willmattianum indeed showed strong anti-HSV-1 activity. The possible mechanism of action modes might include its effects on: (1) virus absorption; (2) HSV-1 gD gene.replication and transcription; (3) HSV-1 protein translation.