HCV infection is a serious global health problem necessitating effective treatment. Currently, there is no vaccine available for prevention of HCV infection due to high degree of strain variation. The current treatment of care, Pegylated interferon α in combination with ribavirin is costly, has significant side effects and fails to cure about half of all infections [12
]. Hence, there is a need to develop anti-HCV agents, both from herbal and synthetic chemistry, which are less toxic, more efficacious and cost-effective. Previous studies demonstrated that medicinal plants used for centuries against different diseases including viral diseases and become a focal point to identify, isolate and purify of new compounds to treat diseases such as Hepatitis. Many traditional medicinal plants and herbs were reported to have strong antiviral activity against DNA and RNA viruses by inhibiting virus replication, interfering with virus-to-cell binding and immunomodulation action [14
]. HCV structural proteins (core, E1 and E2) and nonstructural proteins (NS3 protease and NS5B RNA-dependent RNA polymerase) are potent molecular targets of new antiviral compounds.
GL (licorice root extract) has anti-inflammatory and antioxidant activities. GL inhibits CD4+ T-cell and tumor necrosis factor (TNF)-mediated cytotoxicity [16
]. GL has a membrane stabilizing effect [17
] and also stimulates endogenous production of interferon [18
]. 18-β glycyrrhetinic acid, an active constituent of Glycyrrhizic acid shows antiviral activity against a number of DNA and RNA viruses possibly due to activation of NFκB and induction of IL-8 secretion [19
]. GL has been used in Japan for more than 20 years orally and as the intravenous drug Stronger Neo-Minophagen C (SNMC). Oral GL is metabolized in the intestine to a compound called glycyrrhetinic acid (GA) and intravenous GL is metabolized into glycyrrhetinic acid when excreted through the bile into the intestines. GL and glycyrrhetinic acid have both been tested against Hepatitis A, B, C--with some interesting results [20
]. Previous studies report that GL has antiviral activity against HIV by inhibiting virus replication, interfering with virus-to-cell binding and cell-to-cell infection, and inducing IFN activity [23
]. GL has reported antiviral effect against Herpesviridae family viruses (VZV, HSV-1, EBS, CMV) and Flaviviruses by inhibiting the replication of virus [7
]. GL has also antiviral effect against some emerging viruses such as SARS by inhibiting the virus replication and production of NO synthase [26
] The results of our study show that GL has antiviral effect against HCV at non toxic concentrations. Firstly, GL was checked for toxicological analysis in both Huh-7 and CHO cell lines. Our data shows that GL is non toxic at concentrations up to 100 μg (Figure ). The data was further verified by microscopic examination of cells and MTT cell proliferation assay [27
Guha et al. [28
] reported that in vitro cell culture models can at best demonstrate the infectivity of the virus and used in evaluating drugs for antiviral activity or inhibition of HCV infection. Most of the studies all over the world are conducted in Huh-7 derived cell lines and with replicons supporting HCV RNA transcription and protein synthesis. Recently different groups have studied the HCV replication in serum infected liver cell lines for the study of different HCV genotypes which mimics the naturally occurring HCV virions biology and kinetics of HCV infection in humans [29
]. We infected Huh-7 cells with native viral particles from HCV 3a positive serum, the most prevalent type in Pakistan using the same protocol as established [29
]. The results of our data demonstrate that GL has antiviral effect against HCV in a dose-dependent manner (Figure ). The results prove that GL showed 50% reduction of HCV at a concentration of 13 μg. At a concentration of 40 μg, viral inhibition by the GL reached up to 85%.
HCV Core protein modulates gene transcription, cell proliferation, cell death and cell signaling, interferes with metabolic genes and suppresses host immune response [31
] leading to oxidative stress, liver steatosis and eventually hepatocellular carcinoma [32
]. Core protein is also able to up-regulate cyclooxygenase-2 (Cox-2) expression in hepatocytes derived cells, providing a potential mechanism for oxidative stress [33
]. The expression of Cox-2 in HCC was found to correlate with the levels of several key molecules implicated in carcinogenesis such as inducible nitric oxide synthetase (iNOS), activate vascular endothelial growth factor (VEGF) and phosphorylated Akt (p-Akt) [34
]. Our data shows that GL inhibits HCV core gene expression or function in a dose-dependent manner similar to interferon alpha 2a. This may be due to stimulation of interferon pathway by phosphorylation of Stat1 on tyrosine and serine [36
]. GL may show antiviral effect due to its ability to reduce membrane fluidity [37
] and up regulation of Cox2 or related pathway.