An improved method for a fast screening of α-glucosidase inhibitors in cherimoya fruit (Annona cherimola Mill.) applying effect-directed analysis via high-performance thin-layer chromatography-bioassay-mass spectrometry.
Laboratory of Advanced Research on Foods and Drugs, Department of Food Science and Technology, Faculty of Pharmacy, University of Concepción, Chile; Center for Biotechnology, University of Concepcion, Chile. Electronic address: [Email]
α-glucosidase inhibitors (AGIs) are very attractive bioactive compounds due to their therapeutic profile that includes beneficial effects over glycemic control in type 2 diabetes mellitus and viral infections. Its detection and identification in plants and fruits has gained growing attention, and certainly requires efficient screening methodologies. The objective of the present work was to develop a fast methodology to detect and identify AGIs in cherimoya fruit (Annona cherimola Mill.) applying effect-directed analysis via high-performance thin layer-chromatography (HPTLC) linked with bioassay and mass spectrometry (MS). Both, HPTLC and bioassay conditions, were optimized accomplishing 50% and 83% reduction on enzyme concentration and incubation time respectively, compared to the original method. Additionally, the contrast between inhibitory bands and purple background was also enhanced by enzyme substrate impregnation on HPTLC plate. The optimized detection conditions established were the following: 5.0 U mL-1 of enzyme solution, 1.0 mg mL-1 of 2-naphthyl-α-D-glucopyranoside substrate, 1.0 mg mL-1 of Fast Blue B salt solution and 10 min as incubation time. Applying this methodology, coupled to HPTLC-MS and ultra-high-performance liquid chromatography (UHPLC)-diode array detector (DAD)-MS/MS, it was possible for the first time to detect and identify three AGIs in cherimoya peel and seeds. Compounds were tentatively assigned as phenolamides (phenylethyl cinnamides): N-trans-feruloyl tyramine (m/z 314 [M+H]+; UV λmax 293 and 316 nm), N-trans-p-coumaroyl tyramine (m/z 284 [M+H]+; UV λmax 296 nm) and N-trans-feruloyl phenethylamine (m/z 298 [M+H]+; UV λmax 288 nm). To the best of our knowledge, the presence of latter compound is reported for the first time in cherimoya.