Rapid screening and mechanism analysis of α-amylase inhibitors in apple polyphenols based on immobilized enzyme and HPLC-Q-TOF-MS/MS.

Inhibition of α-amylase (PPA) delays starch digestion and glucose absorption, serving as an effective means to regulate postprandial hyperglycemia. Apple polyphenols (APE) exhibits PPA inhibitory activity, but their core active components and mechanisms of action remain unclear. This study employed immobilized enzyme coupled with HPLC-Q-TOF-MS/MS to screen PPA inhibitors from APE and elucidate their mechanisms of action.Using amino-modified chitosan microspheres as carriers, PPA was covalently immobilized via genipin-mediated Schiff base reaction. The optimal immobilization conditions were optimized (crosslinking agent concentration 0.17%, enzyme concentration 1 mg/mL, crosslinking 6 h, immobilization 14 h), establishing a stable screening system. APE was fractionated into 9 components by solvent precipitation, with component F3 exhibiting the most potent inhibitory activity. Based on the aforementioned techniques, 4 active compounds were identified from F3, and validation confirmed that procyanidin B2 (PC B2) demonstrated the most effective inhibitory effect. In vitro experiments and multi-technology validation demonstrated that PC B2 could delay starch hydrolysis and reduce the production of reducing sugars to regulate postprandial blood glucose, exhibiting reversible competitive inhibition against PPA (IC5=0.25 mg/mL). It could bind to PPA and alter its secondary and tertiary structures, inducing intrinsic protein fluorescence quenching through static quenching. The binding of the two components triggered an exothermic reaction, forming a stable complex.