Poterat and Hamburge natural products isolation. On-line spectroscopy extract is separated Extract emluent is fra nated in the 96-well fo nat,while the other HPLC chromatogram 2.Bioassay in a smal sovent.typically DMSO.and ith the HPLC chromatog data a out if sequently been implemented in academic research 46 HPLC.Rased profiling in medicinal plant res inhibitory the man'The on the rch for the nt-infan leaf- applied to both and ha in a cell-based assay with variant of bioautography has been n at 8 min The inhibit can he In a d round,the timen 3 n v at higr has bee in particul and maior wa ind hibito COX-2 (IC Mono Ma HPLC-BASED ACTIVITY PROFILING 902 Current Organic Chemistry, 2006, Vol. 10, No. 8 Potterat and Hamburger substrate, and Ellman’s reagent. The enzyme hydrolyses acetylthiocholine resulting in thiocholine which reacts with the Ellman’s reagent. The plate stains yellows and active spots appear as white spots. In an alternative method which has been applied to both actetylcholinesterase and butylcholinesterase, naphthyl acetate is used as a substrate and fast blue salt B as a detection reagent. Inhibitors of cholinesterases produce white spots on the background which is stained purple by the diazonium dye [42]. An interesting variant of bioautography has been developed for visualizing the binding properties of secondary metabolites to biomacromolecules [43, 44]. Binding can be detected via the differential chromatographic mobility of a compound with and without the presence of a target macromolecule. The method has been used in particular to detect interaction with DNA revealed by a significant decrease of the Rf value on the TLC plate. While bioautography is inexpensive and can be set up in almost every laboratory, its applicability remains limited by mainly two factors: The restricted number of relevant biological targets, which can be developed into an assay of this format and the lack of quantitative data. As a further drawback, there is no simple correlation with structural information provided by modern LC-coupled spectroscopic techniques. Dereplication of active compounds is thus not straightforward. HPLC-BASED ACTIVITY PROFILING Over the last decade, the focus in natural product analysis has shifted towards HPLC. The on-line coupling of HPLC with powerful spectroscopic methods provides a wealth of structural information from minute sample amounts without the need for tedious preparative isolation. Preparative HPLC￾MS is nowadays ubiquitous as a purification method in the pharmaceutical industry. The advent of high throughput purification platforms combining UV and MS triggering modes has let to an unprecedented level of automation in natural products isolation. With the development of microtitre based biossays, the great potential of HPLC micro-fractionation became apparent. Bioactivity can be tracked in complex mixtures without isolation of compounds and correlated with spectroscopic information available on-line. The principle of the approach is shown in Fig. 2 [45]: An extract is separated by analytical gradient HPLC. Via a T-split, a portion of the effluent is fractionated in the 96-well format, while the other part serves for the on-line spectroscopic characterization of the eluted peaks. After drying, the fractions are redissolved in a small amount of a suitable solvent, typically DMSO, and assayed for bioactivity. The activity profile is then matched with the HPLC chromatogram and the spectroscopic data. A targeted preparative isolation is carried out if the active principles are deemed of sufficient interest. While the potential of this approach has been first realized in the pharmaceutical industry, similar procedures have subsequently been implemented in academic research [46 - 49]. HPLC-Based Profiling in Medicinal Plant Research The identification of the cyclooxygenase-2 (COX-2) inhibitory principles in Isatis tinctoria L. (Brassicaceae) illustrates the potential of this approach in medicinal plant research [47]. In the search for the anti-inflammatory principles in lipophilic leaf extracts of this traditional dye and medicinal plant, a pronounced COX-2 inhibitory activity had been detected in a cell-based assay with Mono Mac 6 cells. Subsequently, the extract was submitted to activity profiling for rapid identification of the active constituents. The HPLC profiles, fractionation steps and COX-2 inhibition of individual fractions are shown in Fig. 3. In a first step, 11 fractions were taken at 8 min intervals. The inhibition profile revealed that virtually all activity of the extract was located in fraction 4. In a second round, the time window of 24-32 min was assayed at higher resolution. The COX-2 inhibitory principle was concentrated in fraction Tf 25-26 min. Finally, the active compound could be located in the shoulder at 25.0 min preceding the peak at 25.5 min. On the basis of ESI-MS and UV-vis data recorded on-line, the major peak was identified as indirubin (1), whereas the active compound was the indoloquinazoline alkaloid tryptanthrin (2) (Fig. 3). The compound was subsequently characterized as a potent dual inhibitor of COX-2 (IC50 in Mono Mac 6 cells 0.037 µM) Fig. (2). Principle of HPLC-based activity profiling (reprinted from [23] with kind permission of Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart). N H O NH O indirubin (1)