An innovative strategy coupling cluster fraction with ultrafiltration UPLC-QTOF-MS/MS for screening α-glucosidase...

Affinity ultrafiltration is widely used for rapid, targeted screening of bioactive natural compounds, but high-abundance components in complex matrices often mask low-abundance yet potent inhibitors, reducing sensitivity. Here, barley seedling extracts were preprocessed via HPLC peak-area clustering before affinity ultrafiltration to identify α-glucosidase inhibitors. This "clustering-prior-to-ultrafiltration" strategy enabled the discovery of four additional low-abundance inhibitors overlooked by direct ultrafiltration. These compounds exhibited stronger inhibitory activity (IC₅₀: 217.75-583.35 μg/mL) than the three high-abundance components (IC₅₀: 707.05-1044.95 μg/mL). Enzyme kinetics, spectroscopy, molecular docking, and dynamic simulations revealed their binding mechanisms. This approach enhances screening sensitivity and provides a basis for developing functional foods.