4.7.2. Cyclooxygenase (COX2) Inhibition In Vitro

The anti-inflammatory activity of EPSR4 was evaluated by examining its inhibitory potency toward the COX2 enzyme at different concentrations (0.98–125-µg/mL in DMSO) compared to Celecoxib (0–31.25 µg/ mL in DMSO). This assay was based on the oxidation of the N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) by arachidonic acid in the presence of COX2 enzyme (EC 1.14.99.1). The inhibitory activity of EPSR4 toward the COX-2 enzyme was assessed following the reported method by Petrovic and Murray and Amessis-Ouchemoukh et al. [55,56]. The inhibitory activity was monitored utilizing a microplate reader (BIOTEK; USA) by evaluating the absorption at 611 nm. Celecoxib was used as the reference drug for the evaluation of COX2 enzymatic activity. The COX2 inhibitory activity was evaluated following the formula:

where, Ax the absorbance in the presence of EPSR4 or Celecoxib; Ay the absorbance of control reaction. The IC₅₀ was determined at 50% inhibition of COX2 enzymatic activity.