Research On Anti HCV Activity Of Flavanoid Compounds

HCV is an enveloped single stranded (+) RNA virus that belongs to genus hepacivirus of the flaviviridae family. It is estimated more than185million people worldwide are infected with HCV, among them55%to85%persons are chronically infected, nearly30%of chronically infected persons will eventually develope to cirrhosis or liver cancer. HCV is a blood borne virus with transmitted routes including transfusion transmission, injecting drug using, sexual contact and unprotected childbirth. During the hepatitis C processing, clinical symptoms of the most infectors are not obvious, when the clinical symptoms appearing, patients have been developed into cirrhosis or liver cancers, which causing serious threaten to human life and health.The commonly clinical treatment of hepatitis C is interferon/ribavirin combination therapy. The effective response to this therapy largely depend on the genotypes of the mutation infected virus. Such as, genotypes1and4are more resistant to interferon treatment, genotype3is associated with a high level of hepatic steatosis and also faster fibrosis progression. Currently using of the RNA dependent RNA polymerase inhibitor sofosbuvir (also known as PSI-7977) largely improved the sustained virological response rate among patients, the occurring of virus genes variations in this gene fragment can also lead to drug resistance, it is urgent to develop new antiviral drugs.The antiviral activity of flavonoid compounds was well documented. However its efficiency of anti-HCV is far unclear. In this study, we introduced HCV cell culture system consisting of J6/JFH1chimeric virus and hepatoma cell line Huh7.5.1as the host cell. Based on this HCV cell culture system (HCVcc), WST was used to detect the compounds cytotoxicity to Huh7.5.1, Real time RT-PCR was used to test HCV viral load for anti-HCV inhibition evaluation. The select index SI value (IC50/EC50) was determined to evaluate the anti-HCV effect of candidate compounds. Here, we have taken pegylated IFN-a2b and RBV as the positive control. It was found that the pegylated IFN-a2b has no significant cytotoxicity with the median effective concentration (EC50) of0.354. IU/ml. In contrast, the median concentration of RBV for cell growth inhibition (IC50) is21.04μg/mL, EC50is2.19μg/mL, the select index SI value is calculated to be9.6. Based on this drug screening system,50kinds of candidate compounds were preliminary screened for anti-HCV activity. Among them,23compounds were suspected to have the obvious anti-HCV activity.To confirm the anti-HCV activity of the compound, indirect immunofluorescence was used to determine the expression of core protein in viaries concentration of compounds as an alternative indicator of HCV RNA detection. In this experiment,1×104cells per well was determined as optimal cell density, multiplicity of infection is1. Using this parameter, the inhibitory effect on HCV of6compounds were evaluated according to the expression of fluorescence. For the positive control drug IFN, it can inhibited HCV effectively in the concentration of1-100IU/ml, green fluorescence was appearing in about60%cell at the concentration of0.1IU/ml. For RBV,90%HCV replication was inhibited at the concentration of100-1000μg/ml, concentration of half HCV replication inhibited was1-10μg/ml. Of the6suspected compounds, no obvious fluorescence can be examined in the maximum concentration of the flavonoid compounds58153a,58158a and6518. Due to their evident cytotoxicity, the anti-HCV activity can’t be reflected. With no obvious cytotoxicity of other remained compounds, the examined fluorescence increased with their concentrations decreasing. Furthermore, the cytotoxicity and anti-HCV effect of FMR-04were examined to be dose-dependent, with the decreasing of its concentration, the cytotoxicity and anti-HCV activity also decrease with the inhibition of the least concentration higher than50%. The compounds WDC-14and WDC-17had little cytotoxicity and can inhibited90%HCV in the maximum concentration. The fluorescence above50%was not detected even in the minimum concentrations.To comfirm the inhibition of the6suspected compounds to the released virus, we detected the virus titer processed by compounds in the concentration which has no obvious cytotoxicity. The titer of virus control group is7×105.25PFU/mL, the virus titer processed by IFN (10IU/mL) is7×101.77PFU/mL, which is far less than the virus control group. The virus titer processed by58153a (10μg/mL) is similar to 58158a (7.5μg/mL), the virus titer is7×104.5PFU/mL and7×104.4PFU/mL respectively. The virus titer for FMR-04(20μg/mL) is7×104.5PFU/mL. The virus titer for both WDC-14(20μg/mL) and WDC-17(20μg/mL) are7×103.67PFU/mL. The inhibition effect of compound6518(10μg/mL) is the best, with the virus titer7×103.67PFU/mL.To clarify the synergistic or antagonism effect between the effective compounds, Macsynergy Ⅱ software was used to analyze the combination effect of4compounds with peg-IFN. In the high concentration, the compound is synergistic, but above all it is antagonism. The obvious antagonism observed in compounds58153a and FMR-04, the compound WDC-14above all is synergistic.On the basis of HCV cell culture system, we used three different drug adding models to analyze the target stage of virus life circle. The compound58153a mainly related to the stage before the virus adsorbed to the cells or the host.6518may mainly affect the stage after the virus absorbed to the cells for example the translation or replication of the virus. FMR-04may affect the commitment after virus absorbed to the cells for strip capside, translation or replication of the virus, but the compound WDC-14is mainly affect the late stage for translation or replication of the virus after they absorbed to the cells.In this study, we use series of screening methods to detect the anti-HCV activity of the flavonoid compounds, the obtained4effective antiviral compounds can ultimately provide important references for anti-HCV flavonoids’design and development of new drugs and clinical therapy.