Identification and isolation of the cyclooxygenase-2 inhibitory principle in Isatis tinctoria.
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 microg/ml and 10 microM, respectively.
Danz
H
H
Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany.
Stoyanova
S
S
Wippich
P
P
Brattström
A
A
Hamburger
M
M
D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D019607:Brassicaceae / Q000737:chemistry*; D002477:Cells; D004396:Coloring Agents; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015394:Molecular Structure; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology*; D018515:Plant Leaves / Q000737:chemistry; D018517:Plant Roots / Q000737:chemistry; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011799:Quinazolines / Q000138:chemical synthesis / Q000494:pharmacology
D000893:Anti-Inflammatory Agents; D000972:Antineoplastic Agents, Phytogenic; D004396:Coloring Agents; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010936:Plant Extracts; D011799:Quinazolines; C046243:tryptanthrine; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases