Development of a thin-layer chromatography gel-overlay α-glucosidase inhibition assay

Abstract

Thin-layer chromatography (TLC) coupled with bioassays has proven effective for detecting bioactive compounds in complex samples. In this study, a TLC enzyme-inhibition assay was developed for the detection of α-glucosidase inhibitors. The basic principle of the method involves the enzymatic hydrolysis of the substrate (2-naphthyl-α-d-glucopyranoside), resulting in the formation of β-naphthol that is subsequently reacted with Fast Blue B salt to produce a diazonium dye, resulting in a purple background. Assay development involved the utilization of enzyme gel entrapment, with either agar (over normal-phase plates) or poloxamer (over reversed-phase plates), and the optimization of key parameters including the substrate and enzyme concentrations, derivatization reagent concentrations, buffer type and pH, and plate type. The results showed good linearity within a range of 0.250–2.0 μg for the positive control, acarbose, with a coefficient of determination (r²) of 0.99. Detection and quantification limits were 0.060 μg and 0.199 μg, respectively. To address potential false positive results arising from secondary reactions with the reagents used, a nonenzymatic assay was conducted. In this control assay, the enzyme was replaced by the reaction product (β-naphthol). The developed TLC gel-overlay autographic method was able to detect the α-glucosidase inhibitor chlorogenic acid in a yerba mate extract.