Screening of α-glucosidase inhibitors from natural flavonoids by an in-capillary assay combining PMMA and EMMA
A capillary electrophoresis method combining pressure mediated microanalysis (PMMA) and electrophoretically mediated microanalysis (EMMA) was established and applied to the study of enzyme kinetics and inhibition kinetics of α-glucosidase, and the screening of α-glucosidase inhibitors from natural flavonoids. First, an incubation buffer, enzyme, substrate and incubation buffer were injected by pressure into a capillary as separate plugs for the enzyme kinetics study. While for the inhibition kinetics study and inhibitor screening, plugs of the incubation buffer, enzyme, inhibitor, substrate and incubation buffer were injected in sequence. Subsequently, the injected plugs were mixed and 2 min of incubation time was allowed for the enzymatic reaction. Afterwards, the resulting product p-nitrophenol (pNP) was separated from the reaction mixture by applying a constant voltage of 15 kV and detected at a wavelength of 405 nm. The Michaelis constant (Km) of α-glucosidase was calculated to be 0.83 mM. And the inhibition constant (Ki) and half-maximal inhibitory concentration (IC50) for acarbose were determined to be 200.4 and 320.1 μM, respectively. Finally, the developed method was applied to screen α-glucosidase inhibitors from 9 natural flavonoids. This method enables the injection and mixing of reagents, enzymatic reaction, separation and quantitation of the reaction product in the same capillary, thus simplifying the operation process, resulting in a higher degree of recognition of the product peak, and reducing the screening cost.