Identification of Novel <i>Leishmania donovani</i> Nucleoside Hydrolase Inhibitors from <i>Banisteriopsis laevifolia</i> Using Affinity Selection-Mass Spectrometry

The identification of enzyme inhibitors from natural sources offers a promising pathway for drug discovery. In this study, affinity selection-mass spectrometry (AS-MS) was employed to screen for inhibitors of Leishmania donovani nucleoside hydrolase (<i>Ld</i>NH) from crude extracts of Banisteriopsis laevifolia. The enzyme was immobilized onto magnetic nanoparticles, enabling selective ligand retention and downstream analysis. The leaf extract exhibited significant inhibitory activity, with an IC₅₀ value of 0.73 ± 0.09 μg/mL, prompting further exploration. Analytical-scale fractionation and biochromatogram analysis revealed inhibitory regions, while AS-MS facilitated the annotation of nine flavonoid-based ligands, including procyanidins, glycosylated flavonoids, and rutin. The structures of four ligands (isoquercetin, astragalin, rutin, and orientin) were confirmed using commercial standards. Among these, isoquercetin and astragalin demonstrated potent <i>Ld</i>NH inhibition with IC₅₀ values of 40.2 ± 16.6 and 41.6 ± 8.9 μmol/L, respectively. These findings highlight B. laevifolia as a promising source of bioactive compounds and demonstrate the utility of AS-MS for efficiently identifying enzyme inhibitors in natural libraries.