Solanaceae‐Derived Spirostane Saponins Inhibit Dengue Virus Replication and Viral Protein Expression and Exhibit Activity Against Multiple Arboviruses

Mosquito-borne arboviruses, including dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV), represent a growing global health burden, yet no specific antiviral therapies are currently available for most of these infections. This unmet need highlights the importance of identifying novel antiviral compounds from biologically relevant sources such as plant-derived natural products. In this study, a bioactivity-guided approach was applied to methanolic extracts from eight Solanaceae species to identify compounds with antiviral activity against DENV-2 in Vero cells. Five extracts significantly reduced viral infectivity, with Solanum ovalifolium and Cestrum sp. achieving complete inhibition at non-cytotoxic concentrations. Subsequent fractionation and purification led to the isolation of four spirostane-type steroidal saponins, structurally characterized by UPLC-DAD-MS and 1D/2D NMR spectroscopy. Functional assays demonstrated that these compounds inhibit DENV replication, as evidenced by marked reductions in viral RNA levels, NS1 and NS3 protein expression, and infectious viral titers. Stage-specific analyses revealed that their antiviral activity is primarily associated with intracellular phases of the viral life cycle, consistent with inhibition of viral replication and/or protein processing. Molecular docking analyses supported these findings by predicting favorable interactions with key viral enzymes, particularly NS5 RNA-dependent RNA polymerase and NS3 helicase/protease. Importantly, the isolated compounds retained antiviral activity against ZIKV and CHIKV, indicating cross-arboviral efficacy. Together, these findings identify spirostane saponins as promising antiviral scaffolds and support their further development as broad-spectrum antiviral agents.