Development of a Radiochemical Cyclooxygenase-1 and -2 in Vitro Assay for Identification of Natural Products as Inhibitors of Prostaglandin Biosynthesis

Abstract

A radiochemical enzyme assay for studying cyclooxygenase (COX)-catalyzed prostaglandin biosynthesis in vitro was optimized with respect to both COX-1 and COX-2 activity. The assay can be used to assess the relative selectivity of plant-derived inhibitors on COX-1 and COX-2 Assay conditions were optimized for both enzymes with respect to concentration of cofactors (l-epinephrine, reduced glutathione, and hematin), activation time (enzyme and cofactors), reaction time, and pH. Moreover, the kinetic parameters, Km and Kcat, of both enzymes were estimated. Five COX inhibitors were used to validate the assay, indomethacin, aspirin, naproxen, ibuprofen, and the arylsulfonamide NS-398, all with different COX selectivity and time dependency. Time-dependent inhibition was determined by comparing the inhibition, with and without preincubation of enzyme and inhibitor. Two flavonoids, (+)-catechin and quercitrin, were examined with respect to inhibition of COX-catalyzed prostaglandin biosynthesis. (+)-Catechin showed equal inhibitory effects on the two enzymes. Quercitrin was found to be inactive toward both COX-1- and COX-2-catalyzed prostaglandin biosynthesis. The optimization procedure resulted in a considerable reduction of the amount of enzyme required for adequate prostglandin biosynthesis and a reliable method suited to evaluate natural products on inhibition of COX-2-catalyzed prostaglandin biosynthesis, as well as on COX-1.

Evaluation of the Cyclooxygenase Inhibiting Effects of Six Major Cannabinoids Isolated from Cannabis sativa

“…The IC 50 -values are presented in Table 1. The IC 50 -value of the reference compound indomethacin was within acceptable limits of the value reported previously for this COX-1 assay (1.4 · 10 Ϫ6 M), 26) confirming that the assay was successful. When screened for COX-2 enzyme inhibiting activity D 9 -THCA-A, CBG and CBGA showed more than 30% inhibition.…”

“…Enzyme-Based Inhibition Assay The assay followed the original method described by White and Glassman, 25) with modifications as described by Noreen et al 26) The assay described below was used for both COX-1 and COX-2 enzymes. In short, 20 ml of each sample was dispensed in a 96-well plate.…”

Anti-inflammatory potential of mushroom extracts and isolated metabolites

“…NSAIDs act by inhibiting the intracellular cyclo-oxygenase enzymes which has two isoforms (COX-1 and COX-2). Cyclooxygenase are enzymes involved in the process of inflammation (Noreen, Ringbom, Perera, Danielson, & Bohlin, 1998). They catalyse the rate-limiting step in the biosynthesis of prostaglandins, prostacyclins, and thromboxanes from arachidonic acid (Zhang, Mills, & Nair, 2003;Diyabalanage, Mulabagal, Mills, DeWitt, & Nair, 2008;Stanikunaite, Khan, Trappe, & Ross, 2009;Han, Oh, & Park, 2011;Fangkrathok, Junlatat, & Sripanidkulchai, 2013).…”

“…This is not a very common method for anti-inflammatory activity assessment, but it has been reported in some publications (Noreen, Ringbom, Perera, Danielson, & Bohlin, 1998;Zhang et al, 2003;Yoshikawa et al, 2005;Stanikunaite, Khan, Trappe, & Ross, 2009 …”

Polarity of extracts and fractions of four Combretum (Combretaceae) species used to treat infections and gastrointestinal disorders in southern African traditional medicine has a major effect on different relevant in vitro activities

“…The anti-inflammatory capacities of the extracts were performed using both COX-1 (Jäger et al, 1996) and COX-2 (Noreen et al, 1998) by using radioactively labelled arachidonic acid.…”