Abstract

Despite its unique structural characteristics and the limited availability of effective therapeutics options, dengue virus serotype 4 (DENV-4) remains relatively underexplored. This study investigates the antiviral potential of Alpinia purpurata ethanol extract against DENV-4 through an integrated in vitro and in silico approach targeting non-structural proteins (NS1, NS3 helicase, and NS5). The extract was prepared by ethanol maceration, and its antiviral efficacy was evaluated by determining cytopathic effects and half-maximal effective concentration (EC50) using a Viral ToxGloTM assay in DENV-4-infected Vero E6 cells. Cytotoxicity was assessed by MTT assay in Vero E6 cells, and the selectivity index (SI) was determined based on the CC50 and EC50 values. Computational analyses included phytochemical exploration, drug-likeness prediction, molecular docking, molecular dynamics simulations, ADME assessment, and toxicity profiling. At higher concentrations, the extract reduced cytopathic effects from 78.82% to 4.79% in a concentration-dependent manner and exhibited antiviral activity with an EC50 value of 92.38 µg/mL. Cytotoxicity assessment using the MTT assay revealed a CC50 value of 137.77 µg/mL, resulting in a SI of 1.49. In silico analyses identified phenolic compounds, particularly epicatechin, as the most promising candidates, exhibiting strong binding affinities toward NS1 (-4.27 kcal/mol), NS3 helicase (-7.00 kcal/mol), and NS5 (-6.14 kcal/mol). Notably, epicatechin outperformed the reference compound quercetin in its interaction with NS3 helicase. Molecular dynamics simulations confirmed the stability of ligand-protein interactions, particularly within the NS3 helicase complex, which also exhibited favorable binding free-energy profiles. ADME and toxicity predictions indicated favorable pharmacokinetic properties and a generally low toxicity profile. Collectively, these findings identify epicatechin as a promising multitarget inhibitor of key viral replication proteins and demonstrate the moderate yet significant anti-DENV-4 activity of A. purpurata extract. These findings highlight the potential of Indonesian medicinal plants as sources of antiviral agents and provide a foundation for future in vivo investigations and antiviral drug development.