Development of a bioassay-coupled HPLC-SPE-ttNMR platform for identification of α-glucosidase inhibitors in apple peel (Malus ×domestica Borkh.)

► A high-resolution α-glucosidase assay has been combined with a HPLC-SPE-ttNMR system. ► The analytical platform enables efficient identification of α-glucosidase inhibitors. ► Three α-glucosidase inhibitors were identified in extract of ‘Pink Lady’ apple peel. ► IC50[(−)-epicatechin]=1.7mM, IC50[Reynoutrin]≈0.8mM and IC50[Avicularin]≈0.9mM. This work describes an analytical platform based on a high-resolution α-glucosidase inhibition assay in combination with hyphenation of high-performance liquid chromatography, solid-phase extraction, and tube-transfer nuclear magnetic resonance spectroscopy, i.e., HPLC-SPE-ttNMR/high-resolution α-glucosidase assay. The platform enables fast screening for individual α-glucosidase inhibitory analytes in complex matrices, followed by structural identification targeted these α-glucosidase inhibitors, as demonstrated by a proof-of-concept study with extract of ‘Pink Lady’ apple peel. A scout-separation produced a high-resolution biochromatogram and a HPLC chromatogram, which were used for pinpointing HPLC peaks displaying α-glucosidase inhibition. Active analytes were cumulatively trapped on SPE cartridges and the structures identified by 1H NMR experiments obtained in the HPLC-SPE-ttNMR mode. (−)-Epicatechin (1), reynoutrin (3) and avicularin (4) were identified as active compounds. IC50 of the active compounds were determined along with six structurally related compounds. Quercetin was the most potent inhibitor with an IC50 of 8.1±0.4μM. The platform proved to be an efficient method for the identification of α-glucosidase inhibitors.