Hana Md Jelas1*, Mohammad Afiq Aizuddin Rosdi1, Norazlan Mohmad Misnan2, Bazilah Jusoh1,Mohd Ridzuan Mohd Abd Razak1
1. Bioassay Unit, Herbal Medicine Research Centre, Institute for Medical Research, National Institutes of Health, Persiaran Setia Murni, Setia Alam, 40170 Shah Alam, Selangor, Malaysia
2. Phytochemistry Unit, Herbal Medicine Research Centre, Institute for Medical Research, National Institutes of Health, Persiaran Setia Murni, Setia Alam, 40170 Shah Alam, Selangor, Malaysia
*Corresponding author: Nur Hana Md Jelas.
ABSTRACT
INTRODUCTION: Eurycoma longifolia (Tongkat Ali) is a traditional Southeast Asian herb known for its role in enhancing male fertility. Recent studies have demonstrated its potential antiviral properties, with both standardised hot-water extracts and ethanol-isolated quassinoids showing activity against dengue virus. These findings highlight its relevance as a source of natural antiviral agents.
OBJECTIVE(S): This study evaluated the dengue virus serotype 2 (DENV-2) antiviral activity in Huh-7 cells using E. longifolia ethanolic extracts.
MATERIALS & METHODS: Extracts were prepared using water, 20%, 50%, 80%, and 100% ethanol. Huh-7 cells were seeded at 1×10⁴ cells/well and incubated overnight. Cell viability was measured using the Adenosine Triphosphate assay (ATP assay) following 72-hour exposure to ten two-fold dilutions (starting at 100 µg/mL). For antiviral testing, cells were infected with DENV-2 (MOI = 1) for 1 hour prior to treatment with the same extract dilutions. After 72 hours, viral infectivity was analysed by plaque assay, and infected cell count was evaluated by immunofluorescence assay (IFA) targeting the DENV-2 envelope protein. Data analysis was performed using GraphPad Prism.
RESULTS: Cell viability decreased proportionally with ethanol concentration used in extract preparation. Water extract showed no cytotoxicity at concentrations up to 100 µg/mL, whereas 100% ethanol extract yielded a 50% concentration of cytotoxicity (CC₅₀) of 28.05 µg/mL (ATP assay). Antiviral activity in Huh-7 cells demonstrated a strong correlation with the ethanol concentration used for the extraction. Both IFA and plaque assay results confirmed enhanced viral suppression, with half maximal effective concentration (EC₅₀) values decreasing from 28.2 µg/mL (water extract) to 2.04 µg/mL (100% ethanol extract).
CONCLUSION: Increasing ethanol concentration used in E. longifolia extraction resulted in enhanced DENV-2 antiviral activity in Huh-7 cells. This suggests ethanol extraction effectively enriches bioactive compounds—such as quassinoids and alkaloids—that contribute to viral inhibition. These results emphasise the importance of solvent polarity in optimising extract potency for antiviral applications.
KEYWORDS: Dengue Virus, Antiviral, Eurycoma longifolia (Tongkat Ali), Ethanol Extraction
