Urease Assay

The enzyme assay was the modified form of the commonly known Berthelot assay. A total volume of 85 μl assay mixture contained 10 μl of phosphate buffer of pH 7.0 in each well in the 96-well plate followed by the addition of 10 μl of sample solution and 25 μl of enzyme solution (0.1347 units). Contents were pre-incubated at 37°C for 5 minutes. Then, 40 μl of urea stock solution (20 mM) was added to each well and incubation continued at 37°C for further 10 min. After given time, 115 μl phenol hypochlorite reagent was added in each well (freshly prepared by mixing 45 μl phenol reagent with 70 μl of alkali reagent). For color development, incubation was done at 37°C for another 10 min. Absorbance was measured at 625 nm using the 96-well plate reader. The percentage enzyme inhibition was calculated by the following formula: Inhibition (%) = (Abs. of Control- Abs. of Test / Abs. of Control) × 100

IC50 values (concentration at which 50% enzyme catalyzed reaction occurs) of compounds were calculated using EZ-Fit Enzyme Kinetics Software (Perrella Scientific Inc. Amherst, USA) after making suitable dilution of test compound.