Applying Isothermal Titration Calorimetry and Saturation Transfer Difference-NMR to Study the Mode of Interaction of Flavan-3-ols with α-Amylase to Understand Their Impact on Starch Hydrolysis

For flavan-3-ols, significant effects to prevent the development of diabetes mellitus are postulated. <i>Inter alia</i>, this is attributed to inhibitory effects on the intestinal α-amylase, in particular for high-molecular-weight procyanidins. In order to gain a deeper insight into the mode of interaction and the resulting α-amylase inhibition, the interaction between the monomers (+)-catechin (CAT) and (-)-epicatechin (EC), the dimers procyanidin (PC) B1 and PC B2, and the trimer PC C1 and their inhibition of porcine pancreatic α-amylase were investigated. Weak interactions were determined by isothermal titration calorimetry (ITC), with no clear difference between monomers and dimers and even no observable interaction with PC C1. Data from saturation transfer difference (STD)-NMR experiments supported these results with respect to reversible interactions. The detailed NMR signal assignments revealed that the formation of rotamers is solvent-dependent, which might explain the differences in the interaction strength between both diastereomers. The results for interaction were in contrast to the accumulating inhibitory strength with an increasing degree of polymerization when monitoring hydrolysis of the natural substrate starch in a novel continuous approach by ITC. By combining the data from the interaction and inhibition studies, we propose that protein aggregation occurs in the presence of flavan-3-ol oligomers, which are responsible for the inhibitory effects. This rather irreversible interaction is not susceptible to detection by ITC and STD-NMR and was also not observable by CD spectroscopy.