Maltoheptaoside hydrolysis with chromatographic detection and starch hydrolysis with reducing sugar analysis: Comparison of assays allows assessment of the roles of direct α-amylase inhibition and starch complexation.

The aim was to determine inhibition of human α-amylase activity by (poly)phenols using maltoheptaoside as substrate with direct chromatographic product quantification, compared to hydrolysis of amylose and amylopectin estimated using 3,5-dinitrosalicylic acid. Acarbose exhibited similar IC50 values (50% inhibition) with maltoheptaoside, amylopectin or amylose as substrates (2.37 ± 0.11, 3.71 ± 0.12 and 2.08 ± 0.01 µM respectively). Epigallocatechin gallate, quercetagetin and punicalagin were weaker inhibitors of hydrolysis of maltoheptaoside (<50% inhibition) than amylose (IC50: epigallocatechin gallate = 20.41 ± 0.25 µM, quercetagetin = 30.15 ± 2.05 µM) or amylopectin. Interference using 3,5-dinitrosalicylic acid was in the order punicalagin > epigallocatechin gallate > quercetagetin, with minimal interference using maltoheptaoside as substrate. The main inhibition mechanism of epigallocatechin gallate and punicalagin was through complexation with starch, especially amylose, whereas only quercetagetin additionally binds to the α-amylase active site. Interference is minimised using maltoheptaoside as substrate with product detection by chromatography, potentially allowing assessment of direct enzyme inhibition by almost any compound.