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 microg/ml and 10 microM, respectively.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Brassicaceae/chemistry , Cyclooxygenase Inhibitors/isolation & purification , Enzyme Inhibitors/isolation & purification , Isoenzymes/antagonists & inhibitors , Plant Extracts/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Cells , Coloring Agents , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/pharmacology , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Drugs, Chinese Herbal , Enzyme Inhibitors/pharmacology , Isoenzymes/metabolism , Molecular Structure , Plant Extracts/isolation & purification , Plant Leaves/chemistry , Plant Roots/chemistry , Prostaglandin-Endoperoxide Synthases/metabolism , Quinazolines/chemical synthesis , Quinazolines/pharmacologyABSTRACT
Matrix metalloproteinases are secreted from different cells as inactive zymogens. For their activation in vitro organomercurials may be used, the presence of which, however, can falsify activity assays and modulate the effects of the proteases in subsequent investigations. Here, we demonstrate the binding of human matrix metalloproteinase 1 to a thiophilic resin (mercaptoethylquinazolinedione derivatized agarose) and take advantage of this thiophilic interaction for the purification of organomercurial activated matrix metalloproteinase 1 from the supernatant of a thyroid carcinoma cell line in connection with the simultaneous removal of the activator.
Subject(s)
Matrix Metalloproteinase 1/isolation & purification , Matrix Metalloproteinase 1/metabolism , Phenylmercuric Acetate/analogs & derivatives , Sulfhydryl Reagents/pharmacology , Chromatography, Affinity/methods , Culture Media , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Humans , Phenylmercuric Acetate/pharmacology , Substrate Specificity , Thyroid Neoplasms/enzymology , Tumor Cells, CulturedABSTRACT
Several thiophilic adsorbents with mercaptoheterocyclic ligands have been analyzed for their ability to bind human serum proteins in a salt-independent way. In contrast to 2-mercaptopyrimidine, 2-mercaptopyridine derived ligands show a group-selective binding of immunoglobulins and alpha2-macroglobulin, not only in the presence of high concentrations of sodium sulphate but in buffers with low ionic strength. The binding is restricted to thiophilic gels obtained by coupling 2-mercaptopyridine to a vinylsulphone-activated matrix and is not achieved on epichlorohydrin-activated gels. A novel thiophilic ligand based on mercaptonicotinic acid, containing a carboxylic group together with the thiophilic pattern of thioaromatic adsorbents, is demonstrated to be useful as an alternative purification scheme for antibodies.
