α-Glucosidase Inhibition Assay Protocol

The α-glucosidase inhibition experiment was performed according to the standard protocol, with some modifications [19] (link). RN and UPN were dispersed in deionized water and diluted to different concentrations (15.63, 31.25, 62.50, 125, 250, and 500 μg/mL). After complete ultrasonography, a 0.22 μm filter membrane was filtered for use. In 1 mL of phosphate buffer solution (0.1 M, pH 7.2), 2 μL of α-glucosidase was dissolved. The reaction substrate, 8 mmol/L 4-nitrophenyl-D-glucopyranoside (PNPG) solution, was made by adding 200 mg of powdered PNPG to 125 ml of phosphate buffer. First, 10.6 g of sodium carbonate was dissolved in 100 mL of deionized water. as a stopper. Different concentrations of the nobiletin sample solution (0.1 mL) and the α-glucosidase solution (0.1 mL) were mixed well (2 μL/mL), and each reaction mixture received 200 μL of PNPG solution. The reaction was terminated with Na₂CO₃. The sample absorbance was measured at 405 nm. The procedure's positive control was acarbose, which was used at the same concentration as the sample.