All target thiouracil analogs 3 –9 were further tested for 15-LOX inhibitory activity using Cayman’s Lipoxygenase Inhibitor Screening Assay Kit (Catalog No. 760700, Cayman Chemical, USA.
90 µL of 15-LOX was pipetted into a 96-well plate quickly. The test chemical was then dissolved in DMSO in 10 µL portions at concentrations of 2.5 µM, 5.0 µM, and 10 µM and added to each well. Arachidonic acid, a 110 µL substrate, was added to start the reaction, and the plate was shaken for at least five minutes. In order to interrupt enzyme catalysis and advance the reaction, 100 µL of chromogen was added to each well and made in accordance with the manufacturer’s instructions. In blank wells, 100 µL of assay buffer (0.1 M Tris-HCl, pH 7.4) was utilized. The positive control and 100% beginning activity were Quercetin and DMSO, respectively. The solution’s absorbance was determined at λ 490–500 nm. The percentage inhibition was calculated according to the following equation:
where (IA) is the 100% initial activity and (Ainhibitor sample) is the absorbance of the test sample. A dose-response curve was plotted between % inhibition and the drug concentration. The non-linear dose-response curve was used for calculating drug concentration showing 50% enzyme inhibition.
90 µL of 15-LOX was pipetted into a 96-well plate quickly. The test chemical was then dissolved in DMSO in 10 µL portions at concentrations of 2.5 µM, 5.0 µM, and 10 µM and added to each well. Arachidonic acid, a 110 µL substrate, was added to start the reaction, and the plate was shaken for at least five minutes. In order to interrupt enzyme catalysis and advance the reaction, 100 µL of chromogen was added to each well and made in accordance with the manufacturer’s instructions. In blank wells, 100 µL of assay buffer (0.1 M Tris-HCl, pH 7.4) was utilized. The positive control and 100% beginning activity were Quercetin and DMSO, respectively. The solution’s absorbance was determined at λ 490–500 nm. The percentage inhibition was calculated according to the following equation:
where (IA) is the 100% initial activity and (Ainhibitor sample) is the absorbance of the test sample. A dose-response curve was plotted between % inhibition and the drug concentration. The non-linear dose-response curve was used for calculating drug concentration showing 50% enzyme inhibition.
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