Harnessing Nature’s Gifts: Salix nigra and Its Potential for Combating Hepatitis C Virus (HCV)

[Image: see text] Hepatitis C virus (HCV) causes various liver complications, including fibrosis, cirrhosis, and steatosis, and finally progresses toward hepatocellular carcinoma (HCC). The current study aimed to explore the antiviral activity of the traditional Pakistani medicinal plant Salix nigra (S. nigra) known as black willow against the hepatitis C virus (HCV). The anti-HCV activity of S. nigra was established against stable Hep G2 cell lines expressing the HCV NS3 gene. Various plant-derived compounds with anti-HCV activity were identified, making phytotherapy a promising alternative to conventional treatments due to their cost-effectiveness and milder side effects. The two extraction methods (Maceration and Soxhlet) and four solvents (n-hexane, methanol, ethyl acetate, and water) were used to obtain crude extracts from S. nigra. Cytotoxicity testing showed that methanol (CC(50) 25 μg/mL) and water (CC(50) 30 μg/mL) extracts were highly toxic, while ethyl acetate and n-hexane (CC(50) > 200 μg/mL) extracts were nontoxic at low concentrations (10–50 μg/mL), making them suitable for further anti-HCV investigations. Stable transfection of the NS3 gene was successfully performed in Hep G2 cells, creating a cellular expression system for studying virus–host interaction. The ethyl acetate extract of S. nigra exhibited significant inhibition of NS3 gene expression (mRNA and protein levels). The phytochemical analysis of S. nigra was also performed using the high-performance liquid chromatography (HPLC) technique. The phytochemical analysis identified several polyphenolic substances in the extracts of S. nigra. Our results concluded that the extracts of S. nigra have significantly reduced the expression of the NS3 gene at mRNA and protein levels. These findings contribute to the global efforts to combat hepatitis C by offering plant-based treatment options for HCV management.