2.16. DPP-IV Inhibition Assay

The DPP-IV inhibition assay was carried out according to the method described by Al-masri et al. [17] with slight modification. Briefly, 35 μl of the plant extract (50 μg/ml – 100 μg/ml) or positive control (diprotin A; 50 μg/ml) was added to 15 μl of human recombinant DPP-IV enzyme solution (50 μU/μl in Tris buffer) in respective wells of a 96-well plate. After 5 min of incubation at 37°C, 50 μl of 20 mM ρNA substrate (Gly-Pro- ρNA) dissolved in Tris buffer was added to initiate the reaction and further incubated for 30 min at 37°C. After incubation, 25 μl of 25% acetic acid solution was added to stop the reaction and the absorbance was measured at 410 nm. A blank and sample blank were also prepared by adding 35 μl of buffer instead of plant extract and 15 μl of buffer instead of enzyme, respectively. The percentage inhibition was calculated using the following equation: