Here we report the inhibitory effects of nine non-steroidal anti-inflammatory drugs (NSAIDs) on soybean 15-lipoxygenase (15-LOX) enzyme (EC by three different methods; UV-absorbance, colorimetric and chemiluminescence methods. Only two drugs, Ibuprofen and Ketoprofen, exhibited enzyme inhibition by UV-absorbance method but none of the drug showed inhibition through colorimetric method. Chemiluminescence method was found highly sensitive for the identification of 15-LOX inhibitors and it was more sensitive and several fold faster than the other methods. All tested drugs showed 15-LOX-inhibition with IC values ranging from 3.52 ± 0.08 to 62.6 ± 2.15 µM by chemiluminescence method. Naproxen was the most active inhibitor (IC 3.52 ± 0.08 µM) followed by Aspirin (IC 4.62 ± 0.11 µM) and Acetaminophen (IC 6.52 ± 0.14 µM). Ketoprofen, Diclofenac and Mefenamic acid showed moderate inhibitory profiles (IC 24.8 ± 0.24 to 39.62 ± 0.27 µM). Piroxicam and Tenoxicam were the least active inhibitors with IC values of 62.6 ± 2.15 µM and 49.5 ± 1.13 µM, respectively. These findings are supported by expression analysis, molecular docking studies and density functional theory calculations. The expression analysis and flow cytometry apoptosis analysis were carried out using mononuclear cells (MNCs) which express both human 15-LOX and 5-LOX. Selected NSAIDs did not affect the cytotoxic activity of MNCs at IC concentrations and the cell death showed dose dependent effect. However, MNCs apoptosis increased only at the higher concentrations, demonstrating that these drugs may not induce loss of immunity in septic and other inflammatory conditions at the acceptable inhibitory concentrations. The data collectively suggests that NSAIDs not only inhibit COX enzymes as reported in the literature but soybean 15-LOX and MNCs LOXs are also inhibited at differential values. A comparison of the metabolomics studies of arachidonic acid pathway after inhibition of either COX or LOX enzymes may reconfirm these findings.
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