In vitro antiviral activity of selected Pakistani medicinal plants against Hepatitis C virus (HCV) and in silico mechanistic investigation of NS5B polymerase inhibition by their major phytoconstituents

Abstract

Pakistan has the second-highest number of HCV patients in the world (10 million). Pakistan has a long history of using hepatoprotective medicinal plants for hepatitis treatment in ethnomedicine, Here, we screened 24 ethnomedicinally chosen Pakistani medicinal plants for in vitro anti-HCV activity against genotypes 3a and 1a, and NS5B polymerase inhibitory mechanism of their major phytoconstituents. Plant extracts were tested for cytotoxicity (MTT assay, CC₅₀) and in vitro anti-HCV activity in HCV 3a- and 1a-infected Huh-7.5 cells. Five of 24 plants (21%) demonstrated significant antiviral activity (>50% inhibition): Solanum nigrum (EC₅₀ = 18.4 μg/mL, SI = 16.9), Acacia nilotica (EC₅₀ = 22.7 μg/mL, SI = 12.7), Syzygium cumini (EC₅₀ = 25.3 μg/mL, SI = 13.5), Momordica charantia (EC₅₀ = 38.6 μg/mL, SI = 7.7), and Fagonia cretica (EC₅₀ = 44.1 μg/mL, SI = 6.3). Time-of-addition assay verified a replication-inhibition mechanism of the five plants. Solanine (ΔG = −8.12 kcal/mol), oleanolic acid (−7.89 kcal/mol) and quercetin (−7.43 kcal/mol) were predicted as superior NS5B inhibitors to sofosbuvir triphosphate (−6.19 kcal/mol) by molecular docking. MD simulation demonstrated stability of the solanine-NS5B complex for 100 ns (RMSD <0.25 nm). EC₅₀ and docking ΔG had a significant Pearson correlation (r = −0.78, p = 0.003). This ethnobotanical, in vitro and in silico study confirms S. nigrum solanine and S. cumini oleanolic acid as mechanistically relevant leads for NS5B polymerase inhibitors that should be considered for further preclinical studies. The high in vitro-in silico correlation offers a principled basis for phytochemical lead discovery for HCV.