Bioactivity-Guided Isolation of β-Sitosterol From Terminalia glabrescens as a Potent Anti-Zika Virus Agent

Zika virus (ZIKV) infection remains a global health concern due to its neurological complications and the lack of specific drugs or vaccines. This study investigated the anti-ZIKV potential of an ethanolic extract from Terminalia glabrescens leaves. Dereplication of the extract by UPLC-ESI-MS/MS identified 27 phenolic compounds, while GC-MS analysis of the dichloromethane (DCM) fraction revealed carboxylic acids, esters, an alcohol, a triterpene, and hydrocarbons. The extract (30 µg/mL) reduced viral load by 2.6 log in Vero CCL-81 cells and inhibited viral replication in SH-SY5Y cells (CC₅₀ = 130.2 ± 27.5 µg/mL; EC₅₀ = 20.4 ± 10.2 µg/mL; SI = 6.4). Bioguided fractionation localized the antiviral activity to the DCM fraction, which reduced viral load by 5.1 log in Vero CCL-81 and 4.0 log in SH-SY5Y cells. Chromatographic fractionation of DCM fraction afforded glutinol, β-sitosterol, and a mixture of α/β-amyrin. Among these, β-sitosterol exhibited the most significant activity against ZIKV in SH-SY5Y cells (CC₅₀ > 300 µM; EC₅₀ = 71.3 ± 7.1 µM; SI > 4.2). A 2D similarity analysis based on structural fingerprints revealed that β-sitosterol is structurally distinct from known anti-ZIKV compounds in the ChEMBL database, underscoring its novelty and potential as a lead scaffold for antiviral drug development. These findings identify β-sitosterol as a promising candidate for the development of anti-ZIKV drugs.