alpha-Amylase inhibition of chlorogenic acid (CHA) and its component moieties including quinic acid (QA) and caffeic acid (CA) were characterized by IC50, inhibition kinetics, fluorescence quenching, isothermal titration calorimetry, differential scanning calorimetry and molecular docking. QA was found with the highest inhibitory activity in a competitive-mode, and caffeoyl substitution significantly decreased its inhibition but maintained inhibition type. Interestingly, QA hardly quenched alpha-amylase fluorescence, while CA quenched that significantly without inhibitory activity. This resulted from lack of aromatic ring in QA that can form pi-conjugation with alpha-amylase fluorescent residues. Besides, the binding constant of QA with alpha-amylase was higher than CHA. Additionally, QA and CA decreased but CHA remained alpha-amylase thermal stability, indicating that change in alpha-amylase spatial structure was related with enzyme residue sites involved in interactions with inhibitors, instead of with inhibition effect. Conclusively, caffeoyl substitution decreased alpha-amylase inhibition of QA through reducing its binding affinity to the enzyme.
