3.4. DPP-IV–Inhibitory Activity Assay

DPP-IV activity was analyzed using a DPP-IV inhibitor screening assay kit (Cayman Chemical, Ann Arbor, MI, USA), which provided a fluorescence-based method for screening DPP-IV inhibitors. The assay used the fluorogenic substrate, Gly-Pro-Aminomethylcoumarine (AMC), to measure DPP-IV activity. Cleavage of the peptide bond by DPP released the free AMC group, resulting in fluorescence that could be analyzed using an excitation wavelength of 350–360 nm and an emission wavelength of 450–465 nm.

The tested compounds were initially dissolved in DMSO to produce 50 mM stock solution, and subsequently diluted to the required concentrations using DMSO, and then they were added to a 96-well plate in final volume of 10 μL and a final concentration of 50 μM. The assay procedure is described briefly according to the manufacturer’s protocols as follows: Diluted assay buffer (30 μL) and diluted enzyme solution (10 μL) were added to the 96-well plate containing 10 μL of solvent (blank) or solvent-dissolved test compounds. The reaction was initiated by adding 50 μL of a diluted substrate solution, and the plate was incubated for 30 min at 37 °C. After incubation, fluorescence with an excitation wavelength of 350 nm and an emission wavelength of 450 nm was monitored using a plate reader (TECAN, Männedorf, Switzerland). The percent inhibition was expressed as ([DPP-IV level of vehicle–treated control–DPP-IV level of test samples]/DPP-IV level of vehicle-treated control) × 100.