Antiviral Activity Of Glycyrrhizic Acid Against Coxsackieviruse B3 In Vitro

Coxsackievirus is an important pathogen in human, for its inducing serious pancreatitis or myocarditis, which often leads to the death of young individual. In etiology, recent research revealed that Coxsackievirus B was responsible for the acute or chronic viral myocarditis with its end stage scenario梞yocardial failure or dilated myocardiopathy. and by heart biopsy and molecular blot,CVB3 serological form was the most seen form in all CVB infection.CVB3 belongs to the presentive dissolving cell virus, and avoid the immune surveillance in host-dependent manner, the existence of the neutralization antibody remaining the infection in heart tissue, and this persistant infection accompanied with the activation of immune response result in the development of the myocardialpathy. so now still lacks of operative anti-CVB3 drug in clinical. Matsumori with colleagues indicated ribavirin effectively inhibit CVB3 duplication in mouse model, Then, prevent the emergence of myocarditis, but there is certain toxicity; Though CVB3 vaccine can inhibit CVB3B3myocarditis, but it needs mass viral antigen while preparing, so the cost is too high to popularize and use in a large amount. So, screening and developing the new drug resisting CVB3 was paid attention all the time. Glycyrrhizin, widely used in our traditional medicine occurred almost 76% in 50 common complex prescriptions treating viral myocarditits. The components of glycyrrhizin include glycyrrhizic acid(GA), glycyrrhizic chromocor, glycyrrhizic polysaccharide( GPS ) , etc, each has the function of anti inflammation resist virus. Some studies confirmed GPS surpressed several DNA virus, but no CVB3. To verificated the effect of other components of glycyrrhizin in resisting CVB3, our laboratory excerpt glycyrrhizin(supplied by SIGMA Co. Ltd) as the drug. We employ in vitro cell culture system, and the CPE inhibition method, to observe the pathogenicity of CVB3 to the sensitive cell ,and virus titer, then quantitate the ability of glycyrrhizin resisting CVB3. We also use paper chromatography to monitor the change of oligopolyadenylicC 2-5 A) which excreted by the cell in supernatant. 2-5A is the product of 2-5A synthesis enzyme(2-5AS) catalysising ATP, so its content reflects the activity of 2-SAS.From this, we can evaluate the capability of glycyrrhizin resisting CVB3 through IFN pathway. Object and methodIn our research, CVB3 strain is kindly presented by Shanghai ZhongShan hospital, viral myocarditits laboratory of ministry of Health; Glycyrrhizin was bought from SIGMA corporation, The CVB3 permissive cell CV-1 and Vero strain, one passaged from AnHui medical college, department of microbiology, and another was passaged from cell bank of ShangHai biochemistry institute. Inoculating the cell into 96-well plate, when the cells inosculating in monoplayer, series diluting virus and drug liquid, to investigate theB3virus titer and the cytotoxicity of glycyrrhizin; Using the 100TCIDJO virus and the appropriate dosage of glycyrrhizin with no cytotoxicity, to design the resist virus experiment in three groups: the first one, drug treatment at 60 minutes past infection; the second group, adding drug 24 hours before infection; the third group, virus mixtured with glycyrrhizin equivalently, and have water bath 1 hour in37癈,then adding the mixture to the cell. 48 hrs after treatment, recording the CPE phenomenon, to titrated the virus titer and screen the 2-5A content. All data were analysed by t test in SPSS package. Result:l.The cytotoxicity of glycyrrhizin: the maximum no cytotoxicity dosage of glycyrrhizn to CV-1 waslOmg/ml;and to Vero was 1 mg/ml; Within the range of no cytotoxicity dosage, high dose glycyrrhizin could maintenance the cell conformation, elongate the life-span of cell.2. Proliferation inhibition of glycyrrhizin to CVB3:In CV-lcell, the effective concentration was 32ng/ml~10 mg/ml, ED50=104.4u g/ml; In Vero cell, the concentration was8 u g/ml~5 1 2 u g /ml, ED50=288.0 u g/ml;...