Bioassay-guided discovery and identification of new potent α-glucosidase inhibitors from Morus alba L. and the interaction mechanism

Morus alba L. (mulberry) is a well-known medicinal species that has been used by herbalist doctors for the treatment of diabetes for a long history, and modern ethnopharmacological studies have demonstrated the ameliorating effects of different mulberry extracts toward diabetes-related symptoms and identified a number of α-glucosidase inhibitors as hypoglycemic ingredients. The present study aims to explore new potent α-glucosidase inhibitors from the root bark of M. alba (known as Sang-Bai-Pi in traditional medicine) based on an in vivo antidiabetic evaluation of its extract fractions and further characterize the preliminary mechanism of the new active constituents. α-Glucosidase inhibitory assay and diabetic mice model were used to locate and evaluate the active fractions from the extract. Diverse separation techniques (e.g. Sephadex LH-20 column chromatograph (CC) and HPLC) and spectroscopic methods (e.g. MS, NMR and ECD) were employed to isolate and structurally characterize the obtained constituents, respectively. Fluorescence quenching, kinetics and molecular docking experiments were conducted to investigate the enzyme inhibitory mechanism of the active compounds. The 80% ethanol eluate from the macroporous resin CC exerted good antidiabetic effects in the tested mice. Fifty-two flavonoids including 22 new ones were then separated and identified, and most of them showed strong inhibition against α-glucosidase with their structure-activity relationship being also discussed. The four new most active ingredients were further characterized to be mixed type of α-glucosidase inhibitors, and their binding modes with the enzyme were also explored. Our current work has demonstrated that the root bark of M. alba is an extremely rich source of flavonoids as potent α-glucosidase inhibitors and potential antidiabetic agents, which makes it a promising candidate species to develop new natural remedies for the prevention and treatment of diabetes.