A comprehensive metabolite profiling of "Isatis tinctoria" leaf extracts

Woad (Isatis tinctoria L., Brassicaceae) is an ancient indigo dye and anti-inflammatory medicinal plant, which has been used and cultivated in Europe since antiquity. The antiinflammatory potential of lipophilic leaf extracts was recently confirmed in a broad-based pharmacological profiling, in various animal models and in a clinical pilot study. Tryptanthrin, an indolin-2-one derivative, and γ-linoleic acid were identified as pharmacologically active compounds inhibiting cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), the expression of the inducible nitric oxide synthase (iNOS), human neutrophil elastase, and the release of histamine from mast cells. Indirubin inhibits inflammatory reactions in delayed-type hypersensitivity and is a potent inhibitor of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3β (GSK3B). In view of a development of this lipophilic woad extract towards a new anti-inflammatory herbal medicine, analytical tools were developed to address various issues related to quality control. This study was conducted as part of the development process and had the purpose to characterise the pharmacologically active leaf extracts of I. tinctoria and the closely related species I. indigotica Fort. As a first step, a gradient LC-PDA-MS method for the quantitative determination of pharmacologically relevant constituents and the dye indigo was developed and validated. The assay enabled quality assessment in the development of phytomedicinal preparations based on woad extracts. A comprehensive phytochemical profiling of the pharmacologically active dichloromethane extract from I. tinctoria leaves was carried out. The polar constituents of I. tinctoria (MeOH extract) and the taxonomically closely related TCM plant I. indigotica were also profiled for comparative purposes. PDA, ELSD, APCI- and ESIMS, and ESI-TOF-MS detectors were used in parallel to ensure a wide coverage of secondary metabolites with highly diverging structural properties. Off-line microprobe NMR after peak purification by semi-preparative HPLC enabled the structure elucidation of some minor constituents. More than 75 compounds belonging to various structural classes such as alkaloids, flavonoids, fatty acids, porphyrins, lignans, carotenoids, glucosinolates and cyclohexenones were unambiguously identified, and tentative structures were proposed for additional compounds. Numerous compounds were identified for the first time in the genus Isatis, and a new indole alkaloid was discovered. The content of indole glucosinolates in woad, and their possible role as precursors of the alkaloid tryptanthrin was investigated. At first, a robust HPLC-MS method for the quantitative determination of glucosinolates was developed and validated. Direct analysis of glucosinolates was rendered possible by the use of a volatile ion-pairing reagent. Extraction via pressurised liquid extraction was optimized, and purity assessment of reference compounds was carried out with quantitative 1H-NMR. Seasonal fluctuations of glucosinolates in the leaf material of five defined I. tinctoria and one I. indigotica accessions were assessed. Significant qualitative and quantitative differences in the glucosinolate patterns were observed when comparing the two species, while differences among the various I. tinctoria accessions were much smaller. Repeated harvesting during the growth season did not have a major effect on glucosinolate concentrations of newly grown leaves. We investigated the influence of the drying procedure on the glucosinolate content. In contrast to shock frozen samples, none of the glucosinolates could be detected in plant material that had been dried at ambient temperature or at 40 °C. However, none of the known breakdown products of indole glucosinolates could be detected. The glucosinolate patterns in seeds of the five I. tinctoria and two I. indigotica accessions were also investigated. The glucosinolate patterns of the seeds showed characteristic differences compared to leaves. Qualitative and quantitative differences in glucosinolate patterns between both Isatis species were found, enabling a differentiation of the two species at the level of seed material.