Metabolite profiles of essential oils in citrus peels and their taxonomic implications

China is an important center of origin for the genus Citrus L. of the family Rutaceae and is rich in wild Citrus species. The taxonomy of Citrus has been a subject of controversy for more than a half century. We propose that the metabolite profiles of Chinese native Citrus species can be used for classification and understanding of the taxonomic relationships within the Citrus germplasm. In this study, triplicate gas chromatography-mass spectrometry (GC-MS) metabolite profiles of 20 Citrus species/varieties were acquired, including 10 native varieties originating in China. R-(+)-limonene, alpha-pinene, sabinene and alpha-terpinene were found to be major characteristic components of the essential oils analyzed in this study, and these compounds contributed greatly to the metabolic classification. The three basic species of the subgenus Eucitrus (Swingle's system), i.e., C. reticulata Blanco, C. medica L. and C. grandis Osb., were clearly differentiated based upon their metabolite profiles using hierarchical cluster analysis (HCA) and partial least square-discriminant analysis (PLS-DA). All the presumed hybrid genotypes, including sweet orange (C. sinensis Osb.), sour orange (C. aurantium L.), lemon (C. limon Burm.f.), rough lemon (C. jambhiri Lush.), rangpur lime (C. limonia Osb.) and grapefruit (C. paradisi Macf.), were grouped closely together with one of their suggested parent species in the HCA-dendrogram and the PLS-DA score plot. These results clearly demonstrated that the metabolite profiles of Citrus species could be utilized for the taxonomic classification of the genus and are complementary to the existing taxonomic evidence, especially for the identification and differentiation of hybrid species.

Acetone as a greener alternative to acetonitrile in liquid chromatographic fingerprinting

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Methods and strategies to identify the mode of action of phytoactive compounds

Small molecules affecting biological processes in plants are widely used in agricultural practice as herbicides or plant growth regulators and in basic plant sciences as probes to study the physiology of plants. Most of the compounds were identified in large screens by the agrochemical industry, as phytoactive natural products and more recently, novel phytoactive compounds originated from academic research by chemical screens performed to induce specific phenotypes of interest. The aim of the present PhD thesis is to evaluate different approaches used for the identification of the primary mode of action (MoA) of a phytoactive compound. Based on the methodologies used for MoA identification, three approaches are discerned: a phenotyping approach, an approach based on a genetic screen and a biochemical screening approach.rnFour scientific publications resulting from my work are presented as examples of how a phenotyping approach can successfully be applied to describe the plant MoA of different compounds in detail.rnI. A subgroup of cyanoacrylates has been discovered as plant growth inhibitors. A set of bioassays indicated a specific effect on cell division. Cytological investigations of the cell division process in plant cell cultures, studies of microtubule assembly with green fluorescent protein marker lines in vivo and cross resistant studies with Eleusine indica plants harbouring a mutation in alpha-tubulin, led to the description of alpha-tubulin as a target site of cyanoacrylates (Tresch et al., 2005).rnII. The MoA of the herbicide flamprop-m-methyl was not known so far. The studies described in Tresch et al. (2008) indicate a primary effect on cell division. Detailed studies unravelled a specific effect on mitotic microtubule figures, causing a block in cell division. In contrast to other inhibitors of microtubule rearrangement such as dinitroanilines, flamprop-m-methyl did not influence microtubule assembly in vitro. An influence of flamprop-m-methyl on a target within the cytoskeleton signalling network could be proposed (Tresch et al., 2008).rnIII. The herbicide endothall is a protein phosphatase inhibitor structurally related to the natural product cantharidin. Bioassay studies indicated a dominant effect on dark-growing cells that was unrelated to effects observed in the light. Cytological characterisation of the microtubule cytoskeleton in corn tissue and heterotrophic tobacco cells showed a specific effect of endothall on mitotic spindle formation and ultrastructure of the nucleus in combination with a decrease of the proliferation index. The observed effects are similar to those of other protein phosphatase inhibitors such as cantharidin and the structurally different okadaic acid. Additionally, the observed effects show similarities to knock-out lines of the TON1 pathway, a protein phosphatase-regulated signalling pathway. The data presented in Tresch et al. (2011) associate endothall’s known in vitro inhibition of protein phosphatases with in vivo-effects and suggest an interaction between endothall and the TON1 pathway.rnIV. Mefluidide as a plant growth regulator induces growth retardation and a specific phenotype indicating an inhibition of fatty acid biosynthesis. A test of the cuticle functionality suggested a defect in the biosynthesis of very-long-chain fatty acids (VLCFA) or waxes. Metabolic profiling studies showed similarities with different groups of VLCFA synthesis inhibitors. Detailed analyses of VLCFA composition in tissues of duckweed (Lemna paucicostata) indicated a specific inhibition of the known herbicide target 3 ketoacyl-CoA synthase (KCS). Inhibitor studies using a yeast expression system established for plant KCS proteins verified the potency of mefluidide as an inhibitor of plant KCS enzymes. It could be shown that the strength of inhibition varied for different KCS homologues. The Arabidopsis Cer6 protein, which induces a plant growth phenotype similar to mefluidide when knocked out, was one of the most sensitive KCS enzymes (Tresch et al., 2012).rnThe findings of my own work were combined with other publications reporting a successful identification of the MoA and primary target proteins of different compounds or compound classes.rnA revised three-tier approach for the MoA identification of phytoactive compounds is proposed. The approach consists of a 1st level aiming to address compound stability, uniformity of effects in different species, general cytotoxicity and the effect on common processes like transcription and translation. Based on these findings advanced studies can be defined to start the 2nd level of MoA characterisation, either with further phenotypic characterisation, starting a genetic screen or establishing a biochemical screen. At the 3rd level, enzyme assays or protein affinity studies should show the activity of the compound on the hypothesized target and should associate the in vitro effects with the in vivo profile of the compound.

Pancreatic carcinoma, pancreatitis, and healthy controls: metabolite models in a three-class diagnostic dilemma

Metabolomics as one of the most rapidly growing technologies in the "-omics" field denotes the comprehensive analysis of low molecular-weight compounds and their pathways. Cancer-specific alterations of the metabolome can be detected by high-throughput mass-spectrometric metabolite profiling and serve as a considerable source of new markers for the early differentiation of malignant diseases as well as their distinction from benign states. However, a comprehensive framework for the statistical evaluation of marker panels in a multi-class setting has not yet been established. We collected serum samples of 40 pancreatic carcinoma patients, 40 controls, and 23 pancreatitis patients according to standard protocols and generated amino acid profiles by routine mass-spectrometry. In an intrinsic three-class bioinformatic approach we compared these profiles, evaluated their selectivity and computed multi-marker panels combined with the conventional tumor marker CA 19-9. Additionally, we tested for non-inferiority and superiority to determine the diagnostic surplus value of our multi-metabolite marker panels. Compared to CA 19-9 alone, the combined amino acid-based metabolite panel had a superior selectivity for the discrimination of healthy controls, pancreatitis, and pancreatic carcinoma patients [Formula: see text] We combined highly standardized samples, a three-class study design, a high-throughput mass-spectrometric technique, and a comprehensive bioinformatic framework to identify metabolite panels selective for all three groups in a single approach. Our results suggest that metabolomic profiling necessitates appropriate evaluation strategies and-despite all its current limitations-can deliver marker panels with high selectivity even in multi-class settings.

Two new flavonol glycosides and a metabolite profile of Bryophyllum pinnatum, a phytotherapeutic used in obstetrics and gynaecology

Bryophyllum pinnatum is a succulent perennial plant native to Madagascar which is used in anthroposophical medicine to treat psychiatric disorders and as a tocolytic agent to prevent premature labour. We performed a metabolite profiling study in order to obtain a comprehensive picture of the constituents in B. pinnatum leaves and to identify chromatographic markers for quality control and safety assessment of medicinal preparations. Preliminary HPLC-PDA-ESIMS analyses revealed that flavonoid glycosides were the main UV-absorbing constituents in the MeOH extract of B. pinnatum. Two phenolic glucosides, syringic acid β-D-glucopyranosyl ester (1) and 4'-O-β-D-glucopyranosyl-cis-p-coumaric acid (2), as well as nine flavonoids (3-11) including kaempferol, quercetin, myricetin, acacetin, and diosmetin glycosides were unambiguously identified by 1H and 2D NMR analysis after isolation from a MeOH extract. The flavonol glycosides quercetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside 7-O-β-D-glucopyranoside (3) and myricetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (4) were new natural products. With the aid of HPLC-PDA-APCIMS and authentic references isolated from the related species B. daigremontianum, the presence of four bufadienolides, bersaldegenin-1-acetate (12), bryophyllin A (13), bersaldegenin-3-acetate (14), and bersaldegenin-1,3,5-orthoacetate (15) was detected in B. pinnatum.

Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.

Functional genomics of O-glucosyltransferases from Concord grape (Vitis labrusca)

Grape (Vitis spp.) is a culturally and economically important crop plant that has been cultivated for thousands of years, primarily for the production of wine. Grape berries accumulate a myriad of phenylpropanoid secondary metabolites, many of which are glucosylated in plantae More than 90 O-glucosyltransferases have been cloned and biochemically characterized from plants, only two of which have been isolated from Vitis spp. The world-wide economic importance of grapes as a crop plant, the human health benefits associated with increased consumption of grape-derived metabolites, the biological relevance of glucosylation, and the lack of information about Vitis glucosyltransferases has inspired the identification, cloning and biochemical characterization of five novel "family 1" O-glucosyltransferases from Concord grape (Vitis labrusca cv. Concord). Protein purification and associated protein sequencIng led to the molecular cloning of UDP-glucose: resveratrollhydroxycinnamic acid O-glucosyltransferase (VLRSGT) from Vitis labrusca berry mesocarp tissue. In addition to being the first glucosyltransferase which accepts trans-resveratrol as a substrate to be characterized in vitro, the recombinant VLRSGT preferentially produces the glucose esters of hydroxycinnamic acids at pH 6.0, and the glucosides of trans-resveratrol and flavonols at 'pH 9.0; the first demonstration of pH-dependent bifunctional glucosylation for this class of enzymes. Gene expression and metabolite profiling support a role for this enzyme in the bifuncitonal glucosylation ofstilbenes and hydroxycinnamic acids in plantae A homology-based approach to cloning was used to identify three enzymes from the Vitis vinifera TIGR grape gene index which had high levels of protein sequence iii identity to previously characterized UDP-glucose: anthocyanin 5-0-glucosyltransferases. Molecular cloning and biochemical characterization demonstrated that these enzymes (rVLOGTl, rVLOGT2, rVLOGT3) glucosylate the 7-0-position of flavonols and the xenobiotic 2,4,5-trichlorophenol (TCP), but not anthocyanins. Variable gene expression throughout grape berry development and enzyme assays with native grape berry protein are consistent with a role for these enzymes in the glucosylation of flavonols; while the broad substrate specificity, the ability of these enzymes to glucosylate TCP and expression of these genes in tissues which are subject to pathogen attack (berry, flower, bud) is consistent with a role for these genes in the plant defense response. Additionally, the Vitis labrusca UDP-glucose: flavonoid 3-0-glucosyltransferase (VL3GT) was identified, cloned and characterized. VL3GT has 96 % protein sequence identity to the previously characterized Vitis vinifera flavonoid 3-0-glucosyltransferase (VV3GT); and glucosylates the 3-0-position of anthocyanidins and flavonols in vitro. Despite high levels of protein sequence identity, VL3GT has distinct biochemical characteristics (as compared to VV3GT), including a preference for B-ring methylated flavonoids and the inability to use UDP-galactose as a donor substrate. RT-PCR analysis of VL3GT gene expression and enzyme assays with native grape protein is consistent with an in planta role for this enzyme in the glucosylation of anthocyanidins,but not flavonols. These studies reveal the power of combining several biochemistry- and molecular biology-based tools to identify, clone, biochemically characterize and elucidate the in planta function of several biologically relevant O-glucosyltransferases from Vitis spp.

Localization and functional characterization of iridoid biosynthetic genes in Catharanthus roseus /

Catharanthus roseus is the sole biological source of the medicinal compounds vinblastine and vincristine. These chemotherapeutic compounds are produced in the aerial organs of the plant, however they accumulate in small amounts constituting only about 0.0002% of the fresh weight of the leaf. Their limited biological supply and high economical value makes its biosynthesis important to study. Vinblastine and vincristine are dimeric monoterpene indole alkaloids, which consists of two monomers vindoline and catharanthine. The monoterpene indole alkaloids (MIA's) contain a monoterpene moiety which is derived from the iridoid secologanin and an indole moiety tryptamine derived from the amino acid tryptophan. The biosynthesis of the monoterpene indole alkaloids has been localized to at least three cell types namely, the epidermis, the laticifer and the internal phloem assisted parenchyma. Carborundum abrasion (CA) technique was developed to selectively harvest epidermis enriched plant material. This technique can be used to harvest metabolites, protein or RNA. Sequencing of an expressed sequence tagged (EST) library from epidermis enriched mRNA demonstrated that this cell type is active in synthesizing a variety of secondary metabolites namely, flavonoids, lipids, triterpenes and monoterpene indole alkaloids. Virtually all of the known genes involved in monterpene indole alkaloid biosynthesis were sequenced from this library.This EST library is a source for many candidate genes involved in MIA biosynthesis. A contig derived from 12 EST's had high similarity (E'^') to a salicylic acid methyltransferase. Cloning and functional characterization of this gene revealed that it was the carboxyl methyltransferase imethyltransferase (LAMT). In planta characterization of LAMT revealed that it has a 10- fold enrichment in the leaf epidermis as compared to the whole leaf specific activity. Characterization of the recombinant enzyme revealed that vLAMT has a narrow substate specificity as it only accepts loganic acid (100%) and secologanic acid (10%) as substrates. rLAMT has a high Km value for its substrate loganic acid (14.76 mM) and shows strong product inhibition for loganin (Kj 215 |iM). The strong product inhibition and low affinity for its substrate may suggest why the iridoid moiety is the limiting factor in monoterpene indole alkaloid biosynthesis. Metabolite profiling of C. roseus organs shows that secologanin accumulates within these organs and constitutues 0.07- 0.45% of the fresh weight; however loganin does not accumulate within these organs suggesting that the product inhibition of loganin with LAMT is not physiologically relevant. The limiting factor to iridoid and MIA biosynthesis seems to be related to the spatial separation of secologanin and the MIA pathway, although secologanin is synthesized in the epidermis, only 2-5% of the total secologanin is found in the epidermis while the remaining secologanin is found within the leaf body inaccessable to alkaloid biosynthesis. These studies emphasize the biochemical specialization of the epidermis for the production of secondary metabolites. The epidermal cells synthesize metabolites that are sequestered within the plant and metabolites that are secreted to the leaf surface. The secreted metabolites comprise the epidermome, a layer separating the plant from its environment.

Impact of dietary polydextrose fiber on the human gut metabolome

The aim of the present study was to elucidate the impact of polydextrose PDX an soluble fiber, on the human fecal metabolome by high-resolution nuclear magnetic resonance (NMR) spectroscopy-based metabolomics in a dietary intervention study (n = 12). Principal component analysis (PCA) revealed a strong effect of PDX consumption on the fecal metabolome, which could be mainly ascribed to the presence of undigested fiber and oligosaccharides formed from partial degradation of PDX. Our results demonstrate that NMR-based metabolomics is a useful technique for metabolite profiling of feces and for testing compliance to dietary fiber intake in such trials. In addition, novel associations between PDX and the levels of the fecal metabolites acetate and propionate could be identified. The establishment of a correlation between the fecal metabolome and levels of Bifidobacterium (R2 = 0.66) and Bacteroides (R2 = 0.46) demonstrates the potential of NMR-based metabolomics to elucidate metabolic activity of bacteria in the gut.

HPLC-DAD method for metabolic fingerprinting of the phenotyping of sugarcane genotypes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Systematic Evaluation of Extraction Methods for Multiplatform-Based Metabotyping: Application to the Fasciola hepatica Metabolome

Combining data from multiple analytical platforms is essential for comprehensive study of the molecular phenotype (metabotype) of a given biological sample. The metabolite profiles generated are intrinsically dependent on the analytical platforms, each requiring optimization of instrumental parameters, separation conditions, and sample extraction to deliver maximal biological information. An in-depth evaluation of extraction protocols for characterizing the metabolome of the hepatobiliary fluke Fasciola hepatica, using ultra performance liquid chromatography and capillary electrophoresis coupled with mass spectroscopy is presented. The spectrometric methods were characterized by performance, and metrics of merit were established, including precision, mass accuracy, selectivity, sensitivity, and platform stability. Although a core group of molecules was common to all methods, each platform contributed a unique set, whereby 142 metabolites out of 14,724 features were identified. A mixture design revealed that the chloroform:methanol:water proportion of 15:59:26 was globally the best composition for metabolite extraction across UPLC-MS and CE-MS platforms accommodating different columns and ionization modes. Despite the general assumption of the necessity of platform-adapted protocols for achieving effective metabotype characterization, we show that an appropriately designed single extraction procedure is able to fit the requirements of all technologies. This may constitute a paradigm shift in developing efficient protocols for high-throughput metabolite profiling with more-general analytical applicability.

CE-ESI-MS metabolic fingerprinting of Leishmania resistance to antimony treatment

Metabolomics has become an invaluable tool to unveil biology of pathogens, with immediate application to chemotherapy. It is currently accepted that there is not one single technique capable of obtaining the whole metabolic fingerprint of a biological system either due to their different physical-chemical properties or concentrations. In this work, we have explored the capability of capillary electrophoresis mass spectrometry with a sheathless interface with electrospray ionization (CE-ESI-TOF-MS) to separate metabolites in order to be used as a complementary technique to LC. As proof of concept, we have compared the metabolome of Leishmania infantum promastigotes BCN 150 (Sb (III) IC50 = 20.9 mu M) and its variation when treated with 120 mu M of Sb(III) potassium tartrate for 12 h, as well as with its Sb(III) resistant counterpart obtained by growth of the parasites under increasing Sb(III) in a step-wise manner up to 180 mu M. The number of metabolites compared were of 264 for BCN150 Sb(III) treated versus nontreated and of 195 for Sb(III) resistant versus susceptible parasites. After successive data filtering, differences in seven metabolites identified in databases for Leishmania pathways, showed the highest significant differences, corresponding mainly to amino acids or their metabolite surrogates. Most of them were assigned to sulfur containing amino acids and polyamine biosynthetic pathways, of special relevance considering the deterioration of the thiol-dependent redox metabolism in Leishmania by Sb(III). Given the low concentrations typical for most of these metabolites, the assay can be considered a success that should be explored for new biological questions.

The selective determination of sulfates, sulfonates and phosphates in urine by CE-MS

Metabolite identification and metabolite profiling are of major importance in the pharmaceutical and clinical context. However, highly polar and ionic substances are rarely included as analytical tools are missing. In this study, we present a new method for the determination of urinary sulfates, sulfonates, phosphates and other anions of strong acids. The method comprises a CE separation using an acidic BGE (pHmetabolite databases so far. Thus, it is demonstrated that the presented method is complementary to the existing methods for metabolite characterization in urine.

The transporter GAT1 plays an important role in GABA-mediated carbon-nitrogen interactions in Arabidopsis

Glutamate derived γ-aminobutyric acid (GABA) is synthetized in the cytosol prior to delivery to the mitochondria where it is catabolized via the TCA cycle. GABA accumulates under various environmental conditions, but an increasing number of studies show its involvement at the crossroad between C and N metabolism. To assess the role of GABA in modulating cellular metabolism, we exposed seedlings of A. thaliana GABA transporter gat1 mutant to full nutrition medium and media deficient in C and N combined with feeding of different concentrations (0.5 and 1 mM) of exogenous GABA. GC-MS based metabolite profiling showed an expected effect of medium composition on the seedlings metabolism of mutant and wild type alike. That being said, a significant interaction between GAT1 deficiency and medium composition was determined with respect to magnitude of change in relative amino acid levels. The effect of exogenous GABA treatment on metabolism was contingent on both the medium and the genotype, leading for instance to a drop in asparagine under full nutrition and low C conditions and glucose under all tested media, but not to changes in GABA content. We additionally assessed the effect of GAT1 deficiency on the expression of glutamate metabolism related genes and genes involved in abiotic stress responses. These results suggest a role for GAT1 in GABA-mediated metabolic alterations in the context of the C-N equilibrium of plant cells.

Search for Low-Molecular-Weight Biomarkers in Plant Tissues and Seeds Using Metabolomics: Tools, Strategies, and Applications

This chapter summarises the metabolomic strategies currently in force used in plant science and describes the methods used. The metabolite profiling and fingerprinting of plant tissues through MS- and/or NMR-based approaches and the subsequent identification of biomarkers is detailed. Strategies for the microisolation and de novo identification of unknown biomarkers are also discussed. The various approaches are illustrated by a metabolomic study of the maize response to herbivory. A review of recent metabolomic studies performed on seed and crop plant tissues involving various analytical strategies is provided.