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

ABSTRACT 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 (CC 50 = 130.2 ± 27.5 µg/mL; EC 50 = 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 (CC 50 > 300 µM; EC 50 = 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.