From day 10 to day 14, rats were dosed orally with vehicle or 1,3-Dicyclohexylu at 30 mg/kg twice daily

Cyano(6-methoxy-naphthalen-2-yl)methyl trans-[(3-phenyloxyran-2-yl)methyl] carbonate (CMNPC) is the substrate used for the fluorescent assay. The assay was carried out using a 96 well-plate format. There was a lane for background with PB buffer (100 mM sodium phosphate, pH = 7.4, 200 µL) and two lanes for positive control. Each inhibitor IC50¬ was determined based on triplicate experiments. Each inhibitor (the highest desired concentration, 2 µL, DMSO) was added to the PB buffer (100 mM sodium phosphate, pH = 7.4, 150 µL) at the first set of wells. A series of 2-fold dilution was applied from the first set till to the last set of wells. hSEH solution (25 nM, 100 mM sodium phosphate, pH = 7.4, 20 µL) was added and mixed for 30s. The inhibitor-enzyme mixtures were mixed and incubated at 30 ˚C for 5 min. CMNPC solution (33 µM, 100 mM sodium phosphate, pH = 7.4, 30 µL) was added and mixed. The enzyme activity was measured based on the fluorescence increase.