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
The suitability of pressurized liquid extraction (PLE) in medicinal plant analysis was investigated. PLE extracts from a selection of representative herbs were compared with extracts obtained according to Pharmacopoeia monographs with respect to yield of relevant plant constituents, extraction time and solvent consumption. In all cases a significant economy in time and solvents was realized, while extraction yields of the analytes were equivalent or higher.
Subject(s)
Plants, Medicinal/chemistry , Carbohydrate Sequence , Chromatography, High Pressure Liquid , Molecular Sequence Data , Solvents , Spectrophotometry, UltravioletABSTRACT
Crystals suitable for X-ray study have been prepared from biochemically active ribosome particles or their complexes with tRNA and polypeptide chains. At ambient temperature the useful lifetime of these crystals under synchrotron irradiation is limited to a few minutes. However, upon cooling to cryogenic temperatures around 85 K, the original resolution limit (up to 4.5 A) can be recorded and radiation damage is virtually eliminated. Hence it has become possible to collect a complete data set from one single crystal. Crystals were cooled as rapidly as possible, either in a cold gas stream, or by immersion in liquid propane. Before cooling crystals were transferred either to an inert hydrocarbon environment, or to solutions similar to the crystallizing ones but with a higher viscosity. In several cases soaking in a cryosolvent was required. Crystallographic data were collected with intense synchrotron radiation. Full data sets have been measured for native and derivatized crystals of 50S ribosomal subunits from H. marismortui as well as from their complexes with tRNA and nascent polypeptide chains, from the wild type and a mutant of 50S subunits from B. stearothermophilus, and from crystals of native and derivatized 30S ribosomal subunits from T. thermophilus.
