Identification and Isolation of the Cyclooxygenase-2 Inhibitory Principle in Isatis tinctoria

Abstract

Various extracts prepared from the traditional dye and medicinal plant Isatis tinctoria L. were submitted to a broad in vitro screening against 16 anti-inflammatory targets. Dichloromethane (DCM) extracts from dried leaves showed a marked cyclooxygenase (COX) inhibitory activity with a preferential effect on COX-2 catalysed prostaglandin synthesis. A supercritical fluid extraction (SFE) procedure employing CO₂-modifier mixtures was developed by which the bioactivity profile and chromatographic fingerprint of the DCM extract could be reproduced. High-resolution activity directed on-line identification of the COX-2 inhibitory principle, using a combination of LC-DAD-MS with a microtitre-based bioassay, led to the identification of tryptanthrin (1) as the constituent responsible for essentially all COX-2 inhibitory activity in the crude extract. Following on-line identification, 1 was isolated at preparative scale and its structure confirmed by comparison with synthetic tryptanthrin. In an assay with lipopolysaccharide stimulated Mono Mac 6 cells, tryptanthrin (1) was of comparable potency (IC₅₀ = 64 nM) than the 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 RAW 264.7 cells when tested at 100 μg/ml and 10 μM, respectively.

Key words

Isatis tinctoria - Brassicaceae - tryptanthrin - anti-inflammatory activity - cyclooxygenase-2 inhibition - on-line spectroscopy - Mono Mac 6 cells

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