Deciphering the Pharmacological Constituents of Cow Urine Distillate Responsible for Antiviral Activity against Chikungunya Virus

This study investigates the antiviral properties of cow urine distillate (CUD) and identifies compounds with therapeutic potential against Chikungunya virus (CHIKV). Crude CUD was evaluated for antiviral activity using in vitro assays, followed by identification of metabolites via GC–MS characterization. Computational docking studies elucidated the binding affinities and critical interactions of these compounds with the CHIKV nsP2 protease (nsP2Pro). Selected compounds were further assessed for inhibition against nsP2Pro using biochemical and cell-based assays. Furthermore, the synergistic effects of CUD combined with natural compounds thymoquinone (TQ) and piperine (PIP) were evaluated against CHIKV replication. Treatment with 2% v/v CUD resulted in ∼90% reduction in CHIKV plaques. Additionally, three of the compounds identified via GC–MS demonstrated substantial inhibition at nontoxic doses, as validated by the FRET assay and the CHIKV plaque-reduction assay. The combination of TQ + PIP + CUD was found to be synergistic, with the combination of 20 μM TQ + 20 μM PIP + 2% CUD exhibiting a 99.85% reduction in CHIKV titer. The present findings establish the antiviral potential of CUD and provide an effective natural remedy against CHIKV infection.