Flavonoid profiling among wild type and related GM wheat varieties

Pleiotropic effects are one of the main concerns regarding genetically modified organisms (GMOs). This includes unintended side effects of the transgene or its genome insertion site on the regulation of other endogenous genes, which could potentially cause the accumulation of different secondary metabolites that may have not only an impact on diet as repeatedly worried by the public but also on the environment. Regarding amount and possible environmental effects, flavonoids represent the most prominent group of secondary metabolites in wheat. Many flavonoids function as signalling or defence molecules. We used a robust and reproducible analytical method to compare the flavonoid content of genetically modified (GM) wheat (Triticum aestivum L., Gramineae) expressing genes that confer increased fungal resistance with their non-GM siblings. The transgenes provide either a broad-spectrum fungal defence (chitinase/glucanase from barley) or bunt-specific resistance by a viral gene (KP4). Significant differences in flavonoid composition were found between different wheat varieties whereas different lines of GM wheat with increased antifungal resistance showed only minor differences in their flavonoid composition relative to their non-GM siblings. In a field test, no significant differences were detectable between infected and non-infected wheat of the same variety regardless of the presence of the transgene. Our results are in agreement with the hypothesis that the transgenes we used to increase wheat defence to fungal pathogens do not interfere with the flavonoid biosynthesis pathway. More significantly, the genetic background resulting from conventional breeding has a direct impact on the biological composition of flavonoids, and thus possibly on the environmen

Determination of trace amounts of ginkgolic acids in Ginkgo biloba L. leaf extracts and phytopharmaceuticals by liquid chromatography-electrospray mass spectrometry

Ginkgolic acids (GAs) are toxic phenolic compounds present in the fruits and leaves of Ginkgo biloba L. (Ginkgoacae). Their maximum level in phytopharmaceuticals containing ginkgo extracts has been recently restricted to 5 mu g/g by the Commission E of the former Federal German Health Authority. In order to detect ginkgolic acids at these low levels, a sensitive and selective analytical method, based on liquid chromatography-electrospray mass spectrometry (LC-ES-MS) has been developed. The three main phenolic acids (1-3) of the chloroform fruit extract were isolated and used as standards for quantification. In the LC-ES-MS negative ion mode, calibration curves with good linearities (r=0.9973, n=6) were obtained in the range of 0.5-10 mu g/g for compounds 1, 2 and between 0.1 and 7.5 mu g/g (r=0.9949, n=6) for ginkgolic acid 3. The detection limits at a SIN ratio of 3 were 0.1 (3) and 0.25 mu g/g (1, 2). Recoveries were around 101% at 5 mu g/g for the substances detected in the leaf extracts. Good precision was achieved with relative standard deviations of less than 4% (n=6). The optimised method was applied to verify whether the amount of gingkolic acids was below 5 mu g/g in a standardised leaf extract which is a constituent of a phytopreparation

Liquid chromatography with ultraviolet absorbance–mass spectrometric detection and with nuclear magnetic resonance spectrometry: a powerful combination for the on-line structural investigation of plant metabolites

In order to discover new bioactive compounds from plant sources which could become new leads or new drugs, extracts should be submitted at the same time to chemical screening and to various biological or pharmacological targets. Metabolite profiling using hyphenated techniques such as LC/UV, LC/MS and more recently LC/NMR, quickly provides plenty of structural information, leading to a partial or a complete on-line de novo structure determination of the natural products of interest. As a complement to this approach, bioassays performed after LC/microfractionation of the extracts allow efficient localisation of the bioactive LC-peaks in the chromatograms. The combination of metabolite profiling and LC/bioassays provides the possibility of distinguishing between already known bioactive compounds (dereplication) and new molecules directly in crude plant extracts. Thus, the tedious isolation of compounds of low interest can be avoided and targeted isolation of new bioactive products or constituents presenting novel or unusual spectroscopic features can be undertaken. Several examples of rapid localisation of bioactive compounds, based on post-chromatographic bioautographic testing of LC/NMR microfractions and subsequent on-line identification will be illustrated. Application of hyphenated techniques for the efficient characterisation of labile constituents or constituents difficult to separate at the preparative scale will also be mentioned. The possibilities and limitations of LC/UV/NMR/MS and LC/bioassay as well as future development expected in this field will be discussed. (C) 2003 Elsevier Science B.V. All rights reserved

The potential of LC-NMR in phytochemical analysis

The coupling of high performance liquid chromatography with nuclear magnetic resonance spectroscopy (LC-NMR) is one of the most powerful methods for the separation and structural elucidation of unknown compounds in mixtures. The recent progress in pulse field gradients and solvent suppression, the improvement in probe technology, and the construction of high held magnets have given a new stimulus to this technique, which has emerged since the mid 1990s as a very efficient method for the on-line identification of organic molecules. LC-NMR thus represents a potentially interesting complementary technique to LC-UV-MS in phytochemical analysis for the detailed on-line structural analysis of natural products. Recent applications have fully demonstrated the usefulness of this technique. A brief review of the applications of LC-NMR in natural product chemistry is presented in this paper, and a summary of the basic principles and modes of operation of LC-NMR is provided. Selected examples of LC-NMR analyses of plant metabolites in crude extracts or in enriched fractions are outlined and used to illustrate the different strategies for employing the technique. The practical possibilities and limitations of LC-MMR in its application to the analysis of crude plant extracts are discussed by means of several examples. Analytical strategies involving LC multi-coupled (hyphenated) techniques for the chemical screening and dereplication of crude plant extracts are presented. An analysis of the future development of the technique with respect to its application in photochemical analysis is also given. Copyright (C) 2001 John Wiley & Sons, Ltd

Liquid Chromatography - Nuclear Magnetic Resonance Spectrometry

The coupling of high-performance liquid chromatography with nuclear magnetic resonance spectroscopy (LCINMR) is one of the most powerful methods for the separation and structural elucidation of unknown compounds in mixtures. The recent progress in pulse field gradients and solvent suppression, the improvement in probe technology, and the construction of high-field magnets have given a new impulse to this technique which has emerged since the mid-1990s as a very efficient method for the online identification of organic molecules. It took nearly 20 years to establish LCINMR and this long period can be mainly attributed to the intrinsic low sensitivity of NMR. LCINMR represents an interesting complementary technique to LCiUV/MS for online identification of LC peaks. Recent applications have demonstrated its usefulness in various fields of analytical chemistry

On-line structure characterization of pyrrolizidine alkaloids in Onosma stellulatum and Emilia coccinea by liquid chromatography-ion-trap mass spectrometry

On-line structure characterization of pyrrolizidine alkaloids in two various plant species (Onosma stellulatum W.K., family Boraginaceae and Emilia coccinea Sims., family Compositae) was performed by a newly elaborated RP-HPLC ion trap MS method with atmospheric pressure chemical ionization (APCI) interface. Different PAs (N-oxides, free bases, otonecine alkaloids) isolated were separated on Waters XTerra C-18 column using a gradient elution. The combination of a CE-SPE with multiple isolation and fragmentation steps for specific masses in ion trap MS detector enabled fast and sensitive analysis of various types of PAs (N-oxides and free bases). In O. stellulatum, spectra 12 various types of structures (13 different alkaloids) have been determined for the first time: leptanthine-N-oxide, lycopsamine-N-oxide, heliospathuline, lycopsamine, trachelanthamine-N-oxide, dihydroechinatine, leptanthine, heliospathuline-N-oxide, 7-acetylintermedine, uplandicine, echimidine and echimidine-N-oxide. In E. coccinea, the following types of PAs were found: platyphylline-N-oxide, platyphylline (three stereoisomers with the same MS' spectrum), ligularidine, neoligularidine, neosenkirkine and also previously reported senkirkine. The method elaborated can be applied in the structural analysis of PAs in newly examined plant materials or food products but further analysis is needed to determine the stereochemistry in details. (C) 2004 Elsevier B.V. All rights reserved

Complete LC/MS analysis of a Tinnevelli senna pod extract and subsequent isolation and identification of two new benzophenone glucosides

The hydroalcoholic extract of Tinnevelli senna is widely used as a laxative phytomedicine. In order to improve the knowledge of the chemical composition of this extract, LC/MS and LC/MSn studies were performed, allowing the on-line identification of most of the known constituents, i.e., flavonoids, anthraquinones and the typical dianthronic sennosides. However, the identity of four compounds could not be ascertained on-line under the given LC/MS conditions. These substances were isolated and their structures elucidated as kaempferol, the naphthalene derivative tinnevellin 8-glucoside and two new carboxylated benzophenone glucosides

Antileishmanial activities associated with plants used in the Malian traditional medicine

Sixty-four extracts issued from twenty-one plants used in the Malian traditional medicine - several of them as antiparasitic drugs - were assayed for their antileishmanial effects against both extracellular and intracellular forms of Leishmania major. Seven extracts from six different plants Sarcocephalus latifolius, Zanthoxylum zanthoxyloides, Entada africana, Bobgunnia madagascarensis, Pseudocedrela kotschyi and Psorosperinum guineense - were found to be significantly active against the intracellular form of the parasite. 4 (c) 2006 Elsevier Ireland Ltd. All rights reserved

The potential of African plants as a source of drugs

African plants have long been the source of important products with nutritional and therapeutical value. Coffee originates from Ethiopia, Strophanthus species are strong arrow poisons and supply cardenolides for use against cardiac insufficiency, the Catharanthus roseus alkaloids are well-known antileukaemic agents - just to mention a few examples. Research is continuing on the vegetable material from this continent in an endeavour to find new compounds of therapeutic interest. An outline is presented here covering the results obtained by the Institute of Pharmacognosy and Phytochemistry of the University of Lausanne during 15 years' work on African plants. The strategy employed for the study of these plants is outlined, covering all aspects from the selection of plant material to the isolation of pure natural products. Different bioactivities have been investigated: the search for new antifungal, molluscicidal and larvicidal agents has been the most important axis. Results are also included for antibacterial, cytotoxicity, anti-inflammatory testin

On-line identification of the bioactive compounds from Blumea gariepina by HPLC-UV-MS and HPLC-UV-NMR, combined with HPLC-micro-fractionation

The dichloromethane extract of the aerial parts of Blumea gariepina (Asteraceae) was shown to be active against the phytopathogenic fungus Cladosporium cucumerinum and to inhibit acetylcholinesterase. In order rapidly to identify the active principles, the crude extract was analysed by on-flow HPLC-H-1-NMR. HPLC-micro-fractionation was performed and all peaks collected were submitted to assays against C. cucumerinum and acetylcholinesterase. By this means, the biological activities could be efficiently associated with selected HPLC peaks. Complementary on-line structural data for all peaks of interest in the crude extract were obtained from HPLC-MS and from HPLC-UV with post-column addition of UV shift reagents. This chemical screening strategy with integrated bioassays permitted the on-line identification of a number of constituents and gave useful information for an efficient isolation procedure. Copyright (C) 2005 John Wiley & Sons, Ltd