The urease inhibitory experiment was performed on the extract fractions of P. cubeba and their respective NPs of P. cubeba. With minimal changes, the previously stated technique was used for this investigation (Weatherburn, 1967 (link)). The experiment was performed in triplicates (n = 3) for each sample. The fractions of the crude extract and their associated NPs were separately added to 96-well plates and incubated for 30 min at 30°C using 5 µL of standard solutions (0.5–0.00625 mM concentrations). The experiment materials (NPs and fractions) were put into reaction mixes that included 55 µL buffer (pH 6.8), jack bean urease (25 µL), and 100 mM urea. Various concentrations of the samples, 0.5 mM (control), 0.625, 1.25, 2.5, and 5 mg of P. cubeba crude extract, and 0.6 mM (control) and 0.05 mg of P. cubeba AgNPs were employed to investigate the kinetics. For that purpose, each well received 70 µL of alkali (0.1% w/v NaOCl and 0.5% w/v NaOH) and 45 µL of phenol reagents (0.005% w/v sodium nitroprusside and 1% w/v phenol). After 1 h, the absorbance was measured at 630 nm. Using the indophenol method and thiourea as a standard, the production of ammonia (NH3) was investigated. Finally, MS-Excel, SoftMax Pro (Molecular Devices, CA, United States), and EZ-FIT software applications were used to analyze the data. The following formula was used to calculate the percentage urease inhibition of each sample (Weatherburn, 1967 (link)):
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