Identification and isolation of the cyclooxygenase-2 inhibitory principle in Isatis tinctoria

Various extracts prepared from the traditional dye and medicinal plant Isat is tinctoria L. were submitted to a broad in vitro screening against 16 ant i-inflammatory targets. Dichloromethane (DCM) extracts from dried leaves sh owed a marked cyclooxygenase (COX) inhibitory activity with a preferential effect on COX-2 catalysed prostaglandin synthesis. A supercritical fluid ex traction (SFE) procedure employing CO2-modifier mixtures was developed by w hich the bioactivity profile and chromatographic fingerprint of the DCM ext ract could be reproduced. High-resolution activity directed on-line identif ication of the COX-2 inhibitory principle, using a combination of LC-DAD-MS with a microtitre-based bioassay, led to the identification of tryptanthri n (1) as the constituent responsible for essentially all COX-2 inhibitory a ctivity in the crude extract. Following on-line identification, 1 was isola ted at preparative scale and its structure confirmed by comparison with syn thetic tryptanthrin. in an assay with lipopolysaccharide stimulated Mono Ma c 6 cells, tryptanthrin (1) was of comparable potency (IC50 = 64 nM) than t he preferential COX-2 inhibitors nimesulide (IC50 = 39 nM) and NS 398 (IC50 = 2 nM). The SFE extract and 1 showed no cytotoxicity in Mono Mac 6 and RA W 264.7 cells when tested at 100 mug/ml and 10 muM, respectively.