A high throughput method for detection of cyclooxygenase-2 enzyme inhibitors by effect-directed analysis applying high performance thin layer chromatography-bioassay-mass spectrometry

Abstract

A high throughput method was developed to detect bioactive molecules with inhibitory activity over cyclooxygenase (COX-2) enzyme applying effect-directed analysis and planar chromatography hyphenated with bioassay and mass spectrometry. The assay was based on the indirect measurement of arachidonic acid transformation into prostaglandin with the colorimetric co-substrate N,N,N’,N’-tetramethyl-p-phenylenediamine. Inhibitory zones were observed as colorless bands over a blue background. Using a central composite design the critical factors like substrate concentration, enzyme: substrate ratio, reaction time, and co-substrate concentration were optimized. Optimal conditions were achieved with 0.03 mg/mL of arachidonic acid, 0.15 U/mL of COX-2, and 8.21 mg/mL of chromogenic reagent. Method usefulness was challenged analyzing fresh Chiloe's giant garlic (Allium ampeloprasum L) ethanol: water (8:2 v/v) extract, finding COX-2 inhibitors that were preliminarily identified as the isomers γ-glutamyl-S-allyl-L-cysteine and γ-glutamyl-S-(trans-1-propenyl)-L- cysteine.