2.2. sEH Enzymatic Assay

The sEH assay was performed as described previously, with minor modifications [13]. To determine inhibitory activity, 130 μL of the sEH in 25 mM bis-Tris-HCl buffer (pH 7.0) containing 0.1% BSA was added to either 20 μL of inhibitor dissolved in MeOH. Next, 20 μL of PHOME was mixed to each mixture, which was then reacted at 37 °C for sEH hydrolysis. Product formation was monitored fluorometrically at 330 nm excitation and 465 nm emission for approximately 60 min.

sEH inhibition activity was calculated using

where ΔC and ΔI are the difference of the solvent and compound intensities, respectively, after about one hour and

where y₀ is the minimum value along the y-axis, a is the difference between the maximum and minimum values, and b is the x value at 50 percent of the “a” value.