From Bee Product to Nutraceutical Lead: Bioactivity-guided Fractionation Reveals Anti-enteroviral Flavonoids in Tunisian Propolis

Propolis is a bee-produced substance derived from plant materials collected from buds, bark, and leaves. Its antiviral properties have been widely reported; however, the bioactive constituents responsible for this activity have not been identified through bioactivity-guided fractionation. The aim of this study is to purify and identify the antiviral constituents of propolis through bio-guided fractionation using TLC and LC-MS. Among the various extracts prepared from propolis, only the ethyl acetate fraction exhibited significant activity against Coxsackievirus B3 (CVB-3), a non-enveloped RNA virus, with a selectivity index of 50.32. In contrast, no activity was observed against herpes simplex virus type 2, differing from previous findings on enveloped viruses. Quantitative RT-qPCR analysis of the active fraction did not reveal a predominant mechanism underlying the antiviral effect. Four bioactive flavonoids (Isorhamnetin, Pinocembrin-5-methyl ether, Pinobanksin-5-methyl ether and Pinobanksin-5-methyl ether 3-O-acetate) were identified within the active fraction by HPLC-DAD-ESI-MS/MS following bio-guided fractionation. An exploratory molecular docking study on five putative cellular and viral targets suggested that the highest predicted binding affinity was associated with the CVB-3 capsid, especially for Isorhamnetin. However, as observed in the in vitro tests, the predicted binding energies across different targets were not sufficiently discriminative to indicate the predominance of one interaction over another, suggesting that complementary approaches and experimental validation remain necessary to fully elucidate the viral inhibition mechanisms of these bioactive constituents from propolis.