Prolyl endopeptidase (PEP) inhibitory assay

The inhibitory activity of glucosides to PEP was determined by a modified Yoshimoto T’s method [69]. Firstly, the activity of the purified PEP enzyme was measured using Z-Gly-Pro-4-nitroanilide (GPNA) as a substrate. The reaction mixture contained 1 mM GPNA, 1/10 (v/v) PEP solution (pre-diluted to an appropriate concentration) and 100 mM Tris–HCl buffer (pH 7.0). The reaction was incubated for 10 min at 37℃ and stopped by the addition of 3 volumes of 0.2 M Na₂CO₃ buffer. The reaction without the addition of PEP enzyme was carried out as the control. The formation of product 4-nitroanilide was measured at 410 nm. The activity of the PEP enzyme was calculated as follow:

where n represents total dilution factor, γ represents the extinction coefficient constant of 8800 M⁻¹·cm⁻¹. For determination of PEP inhibitory activity of glucosides, the sample mixture contained 0.005 U/mL PEP, 0.2 mM GPNA, 500 μM glucoside, and 100 mM Tris–HCl buffer (pH 7.0). The control reaction was carried out without the addition of glucoside. All reaction mixtures were incubated at 30 ℃ and the absorption was measured at 410 nm wavelength with an interval of 1 min for a total of 15 min. Percent PEP inhibitory activity was calculated as follow:

where Slop represents absorbance versus time. The IC₅₀ values of the glucosides with inhibition rates greater than 50% were further determined with inhibitor concentrations varying from 10 to 1000 μM.