2.5. Dipeptidyl peptidase IV (DPP‐IV) inhibition assay

DPP‐IV inhibition assay was according to the method from Harnedy et al with minor modifications (Harnedy, O'Keeffe, & Fitzgerald, ²⁰¹⁵). The assay was performed in a 96‐well microplate; 25 μl of test sample was preincubated with 25 μl of substrate gly‐pro‐p‐nitroanilide (12 mM) at 37°C for 10 min, after added 50 μl of DPP‐IV (0.02 U/ml of Tris‐HCl buffer, pH 8.0); the mixture was incubated at 37°C for 30 min; and then, 100 μl acetic acid–sodium acetate (1 M) was added to terminate the reaction. The DPP‐IV inhibition rate (%) was calculated as following:

where OD_A is the absorbance of the supernatant in which the tested sample was replaced by the same amount of buffer; OD_S is the absorbance of the supernatant with the tested sample; OD_B is the absorbance of the supernatant in which the tested sample and DPP‐IV solution are replaced by the same amount of buffer; and OD_N is the absorbance of the supernatant in which the DPP‐IV solution is replaced by the same amount of buffer.