Cayman colorimetric cox ovine inhibitor screening assay kit

The extract and its respective fractions were dissolved in 100% DMSO to prepare a stock concentration of 5 mg/ml. The samples were tested in triplicates at serial dilutions of (0.1–100 μg/ml). The inhibitory COX activity was assayed calorimetrically, as previously described method (George et al. 2014 (link)) and using Cayman colorimetric COX (ovine) inhibitor screening assay kit (Cayman Chemical Company, MI, USA), according to the manufacturer’s instructions. Celecoxib was run as the positive control for inhibition of COX-1 and COX-2. A volume of 10 μl each of test samples and vehicle were diluted to 20 μl with 0.1 M Tris–HCl (pH 8.0) and pre-incubated with the enzyme at 37 °C for 15 min before the addition of arachidonic acid. Then, the reaction was initiated by addition of 10 μl 10 mM arachidonic acid after that the reaction mixture was incubated at 37 °C for another 2 min. Reaction was terminated by addition of 50 μl 1 N HCl and saturated stannous chloride. Assays were performed using 100 U of ovine COX-1 and COX-2. An aliquot is removed and the prostanoid produced is quantified spectrophotometrically at 405 nm. The results were expressed in IC₅₀ (μg/mL).

The in vitro ability of test compounds and celecoxib to inhibit the COX-1 and COX-2 isozymes was carried out using Cayman colorimetric COX (ovine) inhibitor screening assay kit supplied by Cayman chemicals, USA. The calculations were performed as per the kit guidelines [27 ].