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

Abstract

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.

Keywords: 3,5-Dinitrosalicylic acid (PubChem CID: 11873); Epigallocatechin gallate (PubChem CID: 65064); Flavonoid; Interference; Maltoheptaose (PubChem CID: 4444637).; Maltopentaose (PubChem CID: 124005); Maltose monohydrate (PubChem CID: 23615261); Maltotetraose (PubChem CID: 439639); Maltotriose (PubChem CID: 439586); Phenolics; Polyphenol; Punicalagin (PubChem CID: 44584733); Quercetagetin (PubChem CID: 5281680); Reducing sugar; Sugar quantification; α-amylase inhibition.