An innovative strategy coupling cluster fraction with ultrafiltration UPLC-QTOF-MS/MS for screening α-glucosidase inhibitors from barley seedlings

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 (IC 50 : 217.75–583.35 μg/mL) than the three high-abundance components (IC 50 : 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. • An innovative strategy for screening α-glucosidase inhibitors was established by cluster fraction with UF-UPLC-QTOF-MS/MS. • The innovative strategy enhanced sensitivity by yielding four additional low abundance inhibitors from barley seedlings. • The α-glucosidase inhibitory activities of four low abundance inhibitors were superior to three high abundance inhibitors. • The low abundance inhibitors bind to α-glucosidase by stable hydrogen bonds and hydrophobic interactions. • The low abundance inhibitors suppress α-glucosidase activity by altering amino-acid microenvironment and secondary structure.