HPTLC based screening method for the evaluation of α-amylase inhibitory activity in edible flowers

This study introduces a High-Performance Thin-Layer Chromatography (HPTLC)-based method for assessing α-amylase activity and inhibition. The method was designed as an alternative to the conventional 3,5-dinitrosalicylic acid (DNS) assay, which reports only total inhibition and lacks information on specific hydrolysis products. Starch hydrolysis was examined using α-amylase from porcine pancreas, human saliva, and Aspergillus oryzae. Coupling HPTLC with mass spectrometry enabled the identification of six products formed during enzymatic breakdown. For validation, three key markers of activity (maltose, maltotriose, maltotetraose) were quantified after 30 min of incubation, confirming accuracy and reproducibility. The validated approach was applied to twelve edible flowers tested as hydroalcoholic extracts and infusions. Heather, peony, and rose showed the highest inhibitory activity, corresponding to 7.68 ± 0.37, 2.37 ± 0.11, and 1.77 ± 0.07 mg acarbose equivalents per gram of dry weight, respectively. Compared with the DNS assay, the HPTLC method demonstrated greater precision, minimized matrix interference, and, uniquely, allowed direct visualization of how inhibitors altered both total activity and the distribution of hydrolysis products. These advantages highlight the value of HPTLC as a screening tool for complex plant materials and support its potential in food research, nutraceutical development, and quality control.