On-line high-performance liquid chromatography coupled with biochemical detection method for screening of α-glucosidase inhibitors in green tea.

An on-line high-performance liquid chromatography-biochemical detection (HPLC-BCD) method, in which compounds separated by HPLC were on-line reacted with enzyme and substrate solutions delivered by flow injection and the enzyme inhibition signal was collected by UV detection, was developed to rapidly screen α-glucosidase inhibitors from green tea extracts in this study. The chromatographic fingerprints and enzyme inhibition profiles of the different brands of green tea could be simultaneously detected by the on-line HPLC-BCD method. Enzyme inhibition profiles were detected by the UV detector at 415 nm based on the reaction of α-glucosidase and p-nitrophenyl α-d-glucopyranoside (PNPG). PNPG (1.25 mm), α-glucosidase (0.4 U/mL) and the flow rate 0.07 mL/min were applied as optimized parameters to detect α-glucosidase inhibitors in green tea. Four components in green tea showed α-glucosidase inhibition action and three of them were identified as HHDP-galloyl glucose, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate by HPLC-fourier-transform mass spectrometry (HPLC-FTMS). Two brands of green tea derived from Mengding and Enshi mountainous areas might be superior to the other samples in the prevention and treatment of diabetes owing to their stronger activities of enzyme inhibitors. The proposed on-line HPLC-BCD method could be used to rapidly identify the potential enzyme inhibitors in complex matrixes.