ABSTRACT
Background and Aim: Dengue virus (DENV) infection remains a major public health concern because of its four antigenically distinct serotypes and the limited availability of specific antiviral treatments. Zingiber officinale contains diverse phytochemicals with potential antiviral properties; however, its activity against all four DENV serotypes remains insufficiently investigated. This study evaluated the in vitro antiviral activity and cytotoxicity of Z. officinale ethanolic extract and characterized its metabolites and predicted interactions with DENV NS2B-NS3 proteases.
Materials and Methods: Ginger rhizomes were extracted using 96% ethanol and subjected to liquid chromatography–high-resolution mass spectrometry (LC-HRMS) for metabolite profiling. Putatively annotated metabolites were evaluated using antiviral activity prediction, drug-likeness screening, molecular docking, protein flexibility analysis, and computational pharmacokinetic and toxicity assessment. Extract cytotoxicity was assessed using the MTT assay, whereas antiviral-associated activity against DENV-1–4 was evaluated in Vero E6 cells using a luminescence-based cytopathic effect assay. Experiments were conducted in technical triplicates within a single independent experimental run.
Results: LC-HRMS analysis yielded 45 putatively annotated metabolites. Among the computationally evaluated compounds, 10-deoxymethynolide showed predicted binding affinities of −6.8, −6.5, −6.6, and −7.2 kcal/mol against DENV-1, DENV-2,DENV-3, and DENV-4 NS2B-NS3 proteases, respectively. These predicted interactions require further validation, as themetabolite identity was not confirmed using an authentic analytical standard. The DENV-4 complex exhibited the lowest rootmean square fluctuation (1.789 Å). The extract demonstrated concentration-dependent cytotoxicity, with a 50% cytotoxicconcentration of 359.93 μg/mL. Cytopathic effects (CPE) decreased across all four serotypes, with estimated 50% effectiveconcentrations of 479.03, 452.87, 425.16, and 419.78 μg/mL, respectively. Corresponding selectivity indices ranged from 0.75to 0.85, indicating an unfavorable selectivity profile. Notably, all estimated EC50 values exceeded the highest experimentallytested concentration of 100 μg/mL, indicating that these estimates were extrapolated beyond the tested concentrationrange.
Conclusion: Z. officinale ethanolic extract was associated with measurable changes in dengue-associated CPE across the four serotypes. However, its low selectivity indices and extrapolated EC50 estimates do not support confirmed pan-serotype antiviral efficacy. Computational analysis identified 10-deoxymethynolide as a potential compound for further investigation, but it did not confirm direct protease inhibition. Bioassay-guided fractionation, independent biological replication, purified-compound testing, and direct viral replication assays are required to establish antiviral efficacy and clarify underlying mechanisms.