Enzyme immobilized on polyamidoamine-coated magnetic microspheres for α-glucosidase inhibitors screening from Radix Paeoniae Rubra extracts accompanied with molecular modeling

Abstract

In this study, a method for direct screening and identification of α-glucosidase inhibitors (AGIs) from extracts of natural products was established based on polyamidoamine (PAMAM) coated magnetic microspheres. A facile route to synthesize the magnetic PAMAM was employed and α-glucosidase was successfully covalently attached to its surface through cross linking of glutaraldehyde. Using the enzyme-loaded magnetic microspheres, potential inhibitors were fished out from crude extracts directly, followed by structure confirmation. The inhibitory activities of the screened components were further investigated by the enzyme-loaded magnetic microspheres. The Fe₃O₄ @PAMAM@α-Glu microspheres displayed favorable dispersibility, fast magnetic separation, large enzyme binding amount (42.9 μg•mg^-1) and high enzyme activity. Moreover, the α-glucosidase on the surface of PAMAM coating maintained high storage stability and remarkable reusability. Taking advantage of specific interaction of the α-glucosidase with AGIs, the materials could selectively capture a known AGI (+)-catechin under the interference of an inactive compound salicylic acid, with a binding capacity as high as 15.4%. Additionally, using the Fe₃O₄ @PAMAM@α-Glu microspheres in the inhibition assay, the enzymatic reaction could be stopped by magnetic separation instead of the traditional addition of Na₂CO₃ solution, which not only eliminated the disturbance of termination reagent to the results, but also reused the immobilized α-glucosidase. The screening and inhibitory activity verification of potential ligands in Radix Paeoniae Rubra ("Chi-shao" in Chinese) extracts were achieved by using Fe₃O₄ @PAMAM@α-Glu microspheres, demonstrating practical applicability of our method. Therefore, the magnetic PAMAM-based screening approach could be a feasible and alternative strategy for discovering enzyme inhibitors from natural product extracts.

Keywords: Immobilized enzyme; Magnetic microspheres; Polyamidoamine; Radix Paeoniae Rubra; α-glucosidase inhibitor.