Glycyrrhizin as antiviral agent against Hepatitis C Virus

Abstract

Background: Hepatitis C virus is a major cause of chronic liver diseases which can lead to permanent liver damage, hepatocellular carcinoma and death. The presently available treatment with interferon plus ribavirin, has limited benefits due to adverse side effects such as anemia, depression, fatigue, and "flu-like" symptoms. Herbal plants have been used for centuries against different diseases including viral diseases and have become a major source of new compounds to treat bacterial and viral diseases.

Material: The present study was design to study the antiviral effect of Glycyrrhizin (GL) against HCV. For this purpose, HCV infected liver cells were treated with GL at non toxic doses and HCV titer was measured by Quantitative real time RT-PCR.

Results and discussion: Our results demonstrated that GL inhibit HCV titer in a dose dependent manner and resulted in 50% reduction of HCV at a concentration of 14 ± 2 μg. Comparative studies were made with interferon alpha to investigate synergistic effects, if any, between antiviral compound and interferon alpha 2a. Our data showed that GL exhibited synergistic effect when combined with interferon. Moreover, these results were verified by transiently transfecting the liver cells with HCV 3a core plasmid. The results proved that GL dose dependently inhibit the expression of HCV 3a core gene both at mRNA and protein levels while the GAPDH remained constant.

Conclusion: Our results suggest that GL inhibit HCV full length viral particles and HCV core gene expression or function in a dose dependent manner and had synergistic effect with interferon. In future, GL along with interferon will be better option to treat HCV infection.

Figure 1

Toxicological study of GL in Huh-7 and CHO cell: Huh-7 and CHO cells were plated at the density of 4 × 10⁴ in six well plates. After 24 h cells were treated with different concentrations of GL and control consisted of solvent in which compound is disolved. After 24 h incubation period cells were trypsinized and counted with haemocytometer and trypan blue dye explosive method.

Figure 2

Dose dependent inhibition of GL against HCV 3a genotype. Huh 7 cells were infected with 2 × 10⁵ copies of HCV 3a genotype per well in the absence and presence of different concentrations of GL. After 24 h incubation period, total RNA was extracted by Gentra kit, and the levels of HCV RNA remaining were determined by real time Quantitative RT-PCR assay and are shown as percentage of HCV RNA survival in cells. P value > 0.05 vs control was considered as statistically significant.

Figure 3

Synergy in the antiviral activity of GL along with interferon. GL shows synergistic effect with interferon-α (5 IU/well) against HCV in liver cells (Huh-7). Huh-7 cells were incubated for 6 h with GL and interferon alone, or combination of GL and interferon in a 96-well plate. After 6 h cells were infected with 2 × 10⁴ copies of HCV 3a genotype per well and incubated for additional 18 h. At the end of incubation period, total RNA was extracted by Gentra kit, and the levels of HCV RNA remaining were determined, by real time Quantitative RT-PCR assay and are shown as percentage of HCV RNA survival in cells. Results are represented as the average and standard error for three independent experiments. *P value > 0.05 vs control.

Figure 4

Dose dependent inhibition of GL against HCV core gene. Huh-7 cells were transfected with Core in the presence and absence of different concentration of GL. (A) After 24 h incubation period, total RNA was extracted and the levels of HCV core gene were determined by RT-PCR. GAPDH serve as internal control. (B) After 48 h incubation period, protein were isolated and analyzed by western blotting with anti -Core monoclonal antibody and GAPDH served as internal control.