2.8. Lipoxygenase (LOX) Inhibition Assay

The lipoxygenase inhibition assay was performed on a 96-well microplate with linoleic acid as substrate [21]. The test sample was dissolved in DMSO at 8 mg/mL, and diluted solutions were prepared. 3.75 μL of each diluted solution was added to the reaction mixture containing 146.25 μL of an aqueous solution of lipoxygenase (820.51 U/mL). The plate was allowed to incubate for three minutes at room temperature, and the reaction was initiated by adding 150 μL of linoleic acid solution (1.25 mM). An enzyme blank without inhibitor was made of 153.75 μL of borate buffer (0.2 M, pH = 9) and 146.25 μL of an aqueous solution of lipoxygenase (820.51 U/mL). The sample blank (150 μL of borate buffer + 3.75 μL of the test sample and 146.25 μL of enzyme) was also realized. Zileuton was used as a reference compound. Inhibitory activity was measured in triplicate using a microplate spectrophotometer (BioTek Epoch, USA) at 234 nm. The percent of lipoxygenase inhibition was obtained from the following equation:

where A_C is the absorbance of the enzyme—absorbance of enzyme blank—and A_S is the absorbance of the sample—absorbance of the sample blank.