The First Comprehensive Chemical Profiling of Vachellia gummifera (Willd.) Kyal. & Boatwr., a Plant with Medicinal Value.

Vachellia gummifera (Willd.) Kyal. & Boatwr. is a medicinal plant endemic to Morocco that has no documented studies on its chemical composition. In this study, the chemical composition of the water/methanol (4 : 1) extracts of air-dried leaf and stem samples of Moroccan V. gummifera was determined using UHPLC-MS and NMR. In total, over 100 metabolites were identified in our study. Pinitol was the major compound in both the leaf and stem extracts, being significantly more abundant in the former. Asparagine and 3-hydroxyheteroendrin were the second most abundant compounds in the stem and leaf extracts, respectively, though both compounds were present in each tissue. The other compounds included flavonoids based on quercetin, and phenolic derivatives. Eucomic acid, only identified in the stems and was the major aromatic compound distinguishing the leaf and stem profiles. Quercetin 3-O-(6''-O-malonyl)-ß-D-glucopyranoside was identified as the major flavonoid in the leaves but was also present in the stems. Other malonylated derivatives that were all flavonol glycosides based on myricetin, kaempferol, and isorhamnetin in addition to quercetin were also identified. This is the first report of eucomic acid and malonylated compounds in Vachellia species. This report provides valuable insights into the chemotaxonomic significance of the Vachellia genus.

γ-Glutamyl-ß-phenylethylamine, a novel α-glucosidase and α-amylase inhibitory compound from Termitomyces robustus, an edible Nigerian mushroom.

Termitomyces species are known edible mushrooms in Nigeria, believed to have exceptional culinary and nutraceutical properties. Methanol extract from fruiting bodies of Termitomyces robustus was evaluated for antidiabetic activity using in vitro α-amylase and α-glucosidase assays. The isolation and structural elucidation of metabolites from the T. robustus extract afforded five compounds including a new natural product γ-glutamyl-ß-phenylethylamine 3 and four known phenyl derivatives: tryptophan 1, 4-hydroxyphenylacetic acid 2, 4-hydroxyphenylpropionic acid 4, and phenyllactic acid 5. Structures were elucidated from analyses of spectroscopic data (1 D and 2 D NMR, HRESIMS) and all isolated compounds were tested for α-amylase and α-glycosidase inhibitory activity. The in vitro assay established crude extract to possess α- amylase and α-glucosidase inhibition with IC50 of 78.05 µg/mL and 86.10 µg/mL, respectively. The isolated compounds compared favourably with the standard drug, acarbose with IC50 ranging from 6.18-15.08 µg/mL and 18.28-44.63 µg/mL for α-amylase and glucosidase, respectively.

Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis.

The co-catabolism of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady-state chemostat system. We demonstrate that Mtb efficiently co-metabolises either cholesterol or glycerol, in combination with two-carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.

A willow UDP-glycosyltransferase involved in salicinoid biosynthesis.

The salicinoids are phenolic glycosides that are characteristic secondary metabolites of the Salicaceae, particularly willows and poplars. Despite the well-known pharmacology of salicin, that led to the development of aspirin >100 years ago, the biosynthetic pathways leading to salicinoids have yet to be defined. Here, we describe the identification, cloning, and biochemical characterization of SpUGT71L2 and SpUGT71L3-isozymic glycosyltransferases from Salix purpurea-that function in the glucosylation of ortho-substituted phenols. The best substrate in vitro was salicyl-7-benzoate. Its product, salicyl-7-benzoate glucoside, was shown to be endogenous in poplar and willow. Together they are inferred to be early intermediates in the biosynthesis of salicortin and related metabolites in planta. The role of this UDP-glycosyltransferase was confirmed via the metabolomic analysis of transgenic plants produced by RNAi knockdown of the poplar orthologue (UGT71L1) in the hybrid clone Populus tremula×P. alba, INRA 717-1B4.

Miyabeacin: A new cyclodimer presents a potential role for willow in cancer therapy.

Willow (Salix spp.) is well known as a source of medicinal compounds, the most famous being salicin, the progenitor of aspirin. Here we describe the isolation, structure determination, and anti-cancer activity of a cyclodimeric salicinoid (miyabeacin) from S. miyabeana and S. dasyclados. We also show that the capability to produce such dimers is a heritable trait and how variation in structures of natural miyabeacin analogues is derived via cross-over Diels-Alder reactions from pools of ortho-quinol precursors. These transient ortho-quinols have a role in the, as yet uncharacterised, biosynthetic pathways around salicortin, the major salicinoid of many willow genotypes.

Comparative Metabolomics and Molecular Phylogenetics of Melon (Cucumis melo, Cucurbitaceae) Biodiversity.

The broad variability of Cucumis melo (melon, Cucurbitaceae) presents a challenge to conventional classification and organization within the species. To shed further light on the infraspecific relationships within C. melo, we compared genotypic and metabolomic similarities among 44 accessions representative of most of the cultivar-groups. Genotyping-by-sequencing (GBS) provided over 20,000 single-nucleotide polymorphisms (SNPs). Metabolomics data of the mature fruit flesh and rind provided over 80,000 metabolomic and elemental features via an orchestra of six complementary metabolomic platforms. These technologies probed polar, semi-polar, and non-polar metabolite fractions as well as a set of mineral elements and included both flavor- and taste-relevant volatile and non-volatile metabolites. Together these results enabled an estimate of "metabolomic/elemental distance" and its correlation with the genetic GBS distance of melon accessions. This study indicates that extensive and non-targeted metabolomics/elemental characterization produced classifications that strongly, but not completely, reflect the current and extensive genetic classification. Certain melon Groups, such as Inodorous, clustered in parallel with the genetic classifications while other genome to metabolome/element associations proved less clear. We suggest that the combined genomic, metabolic, and element data reflect the extensive sexual compatibility among melon accessions and the breeding history that has, for example, targeted metabolic quality traits, such as taste and flavor.

Sulfated flavanones and dihydroflavonols from willow.

Phytochemical profiling of a hybrid species of willow, Salix × alberti L. (S. integra Thunb. × Salix suchowensis W.C. Cheng ex G.Zhu) revealed four sulfated flavonoids, which were then isolated from young stem tissue. The structures of dihydroflavonols (flavanonols) taxifolin-7-sulfate (1) and dihydrokaempferol-7-sulfate (2) and flavanones, eridictyol-7-sulfate (3) and naringenin-7-sulfate (4) were elucidated through NMR spectroscopy and high-resolution mass spectrometry. The identified sulfated flavanones and dihydroflavonols have not been previously seen in plants, but the former have been partially characterised as metabolites in mammalian metabolism of dietary flavonoids. In addition to providing full spectroscopic characterisation of these metabolites for the first time, we also compared the in vitro antioxidant properties, via the DPPH radical scavenging assay, of the parent and sulfated flavanones, which showed that 7-sulfation of taxifolin and eriodictyol attenuates but does not remove anti-oxidant activity.

Phenylalkanoid Glycosides (Non-Salicinoids) from Wood Chips of Salix triandra × dasyclados Hybrid Willow.

Salix triandra (almond leaved willow) is an established crop, grown in coppicing regimes for basket-making materials. It is known as a source of non-salicinoid phenolic glycosides, such as triandrin and salidroside. A spontaneous natural hybrid of S. triandra and S. dasyclados was subjected to metabolite profiling by high resolution LC-MS, and 22 phenolic glycosides, including 18 that are new to the Salicaceae, were identified. Structures were determined by HPLC isolation and NMR methods. The hybridisation process has introduced novel chemistry into the Salix phenolic glycoside palette, in particular, the ability to generate disaccharide conjugates where the glycosyl group is further extended by a range of sugars, including apiose, rhamnose, xylose, and arabinose. Also of note is the appearance of chavicol derivatives, also not previously seen in Salix spp. The work demonstrates the plasticity of the phenolic glycoside biosynthetic pathway, and the potential to improve established crops such as S. triandra and S. dasyclados, via high-value metabolites, for both basketry and bioenergy markets.

The anaplerotic node is essential for the intracellular survival of Mycobacterium tuberculosis.

Enzymes at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate or anaplerotic (ANA) node control the metabolic flux to glycolysis, gluconeogenesis, and anaplerosis. Here we used genetic, biochemical, and 13C isotopomer analysis to characterize the role of the enzymes at the ANA node in intracellular survival of the world's most successful bacterial pathogen, Mycobacterium tuberculosis (Mtb). We show that each of the four ANA enzymes, pyruvate carboxylase (PCA), PEP carboxykinase (PCK), malic enzyme (MEZ), and pyruvate phosphate dikinase (PPDK), performs a unique and essential metabolic function during the intracellular survival of Mtb. We show that in addition to PCK, intracellular Mtb requires PPDK as an alternative gateway into gluconeogenesis. Propionate and cholesterol detoxification was also identified as an essential function of PPDK revealing an unexpected role for the ANA node in the metabolism of these physiologically important intracellular substrates and highlighting this enzyme as a tuberculosis (TB)-specific drug target. We show that anaplerotic fixation of CO2 through the ANA node is essential for intracellular survival of Mtb and that Mtb possesses three enzymes (PCA, PCK, and MEZ) capable of fulfilling this function. In addition to providing a back-up role in anaplerosis we show that MEZ also has a role in lipid biosynthesis. MEZ knockout strains have an altered cell wall and were deficient in the initial entry into macrophages. This work reveals that the ANA node is a focal point for controlling the intracellular replication of Mtb, which goes beyond canonical gluconeogenesis and represents a promising target for designing novel anti-TB drugs.

Salicin-7-sulfate: A new salicinoid from willow and implications for herbal medicine.

Willow (Salix sp.) is a historically well-known herbal medicine that provided the lead compound (salicin) for the discovery of aspirin, one of the most successful plant derived drugs in human medicine. During a metabolomics screen of 86 Salix species contained in the UK National Willow Collection, we have discovered, isolated and fully characterised a new natural salicinoid - salicin-7-sulfate. This molecule may have important human pharmacological actions that need to be considered in determining the efficacy and safety of willow herbal medicines.

Defining genetic and chemical diversity in wheat grain by 1H-NMR spectroscopy of polar metabolites.

SCOPE: The application of high-throughput 1H nuclear magnetic resonance (1H-NMR) of unpurified extracts to determine genetic diversity and the contents of polar components in grain of wheat. METHODS AND RESULTS: Milled whole wheat grain was extracted with 80:20 D2 O:CD3 OD containing 0.05% d4 -trimethylsilylpropionate. 1H-NMR spectra were acquired under automation at 300°K using an Avance Spectrometer operating at 600.0528 MHz. Regions for individual metabolites were identified by comparison to a library of known standards run under identical conditions. The individual 1H-NMR peaks or levels of known metabolites were then compared by Principal Component Analysis using SIMCA-P software. CONCLUSIONS: High-throughput 1H-NMR is an excellent tool to compare the extent of genetic diversity within and between wheat species, and to quantify specific components (including glycine betaine, choline, and asparagine) in individual genotypes. It can also be used to monitor changes in composition related to environmental factors and to support comparisons of the substantial equivalence of transgenic lines.

Metabolite Profiling of Sugarcane Genotypes and Identification of Flavonoid Glycosides and Phenolic Acids.

Sugarcane is an important agricultural crop in the economy of tropical regions, and Brazil has the largest cultivated acreage in the world. Sugarcane accumulates high levels of sucrose in its stalks. Other compounds produced by sugarcane are currently not of economic importance. To explore potential coproducts, we have studied the chemical diversity of sugarcane genotypes, via metabolite profiling of leaves by NMR and LC-DAD-MS. Metabolites were identified via in-house and public databases. From the analysis of 60 HPLC-fractionated extracts, LC-DAD-MS detected 144 metabolites, of which 56 were identified (MS-MS and (1)H NMR), including 19 phenolics and 25 flavones, with a predominance of isomeric flavone C-glycosides. Multivariate analysis of the profiles from genotypes utilized in Brazilian breeding programs revealed clustering according to sugar, phenolic acid, and flavone contents.

Acutifoliside, a novel benzoic acid glycoside from Salix acutifolia.

Ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS) profiling of a polar solvent extract of juvenile stem tissue of Salix acutifolia Willd. identified a range of phenolic metabolites. Salicortin, 1, a well-known salicinoid, was the major compound present and the study identified young stem tissue of this species as a potential source of this compound for future studies. Three further known metabolites (salicin 2, catechin 3 and tremuloidin 4) were also present. The UHPLC-MS analysis also revealed the presence of a further, less polar, unknown compound, which was isolated via HPLC peak collection. The structure was elucidated by high-resolution mass spectroscopic analysis, 1- and 2-D NMR analysis and chemical derivatisation and was shown to be a novel benzoic acid glycoside 5, which we have named as acutifoliside.

(1)H-NMR screening for the high-throughput determination of genotype and environmental effects on the content of asparagine in wheat grain.

Free asparagine in cereals is known to be the precursor of acrylamide, a neurotoxic and carcinogenic product formed during cooking processes. Thus, the development of crops with lower asparagine is of considerable interest to growers and the food industry. In this study, we describe the development and application of a rapid (1)H-NMR-based analysis of cereal flour, that is, suitable for quantifying asparagine levels, and hence acrylamide-forming potential, across large numbers of samples. The screen was applied to flour samples from 150 bread wheats grown at a single site in 2005, providing the largest sample set to date. Additionally, screening of 26 selected cultivars grown for two further years in the same location and in three additional European locations in the third year (2007) provided six widely different environments to allow estimation of the environmental (E) and G x E effects on asparagine levels. Asparagine concentrations in the 150 genotypes ranged from 0.32 to 1.56 mg/g dry matter in wholemeal wheat flours. Asparagine levels were correlated with plant height and therefore, due to recent breeding activities to produce semi-dwarf varieties, a negative relationship with the year of registration of the cultivar was also observed. The multisite study indicated that only 13% of the observed variation in asparagine levels was heritable, whilst the environmental contribution was 36% and the GxE component was 43%. Thus, compared to some other phenotypic traits, breeding for low asparagine wheats presents a difficult challenge.

Characterisation of the willow phenylalanine ammonia-lyase (PAL) gene family reveals expression differences compared with poplar.

Willow is an important biomass crop for the bioenergy industry, and therefore optimal growth with minimal effects of biotic and abiotic stress is essential. The phenylpropanoid pathway is responsible for the biosynthesis of not only lignin but also of flavonoids, condensed tannins, benzenoids and phenolic glycosides which all have a role in protecting the plant against biotic and abiotic stress. All products of the phenylpropanoid pathway are important for the healthy growth of short rotation cropping species such as willow. However, the phenylpropanoid pathway in willow remains largely uncharacterised. In the current study we identified and characterised five willow phenylalanine ammonia-lyase (PAL) genes, which encode enzymes that catalyse the deamination of l-phenylalanine to form trans-cinnamic acid, the entry point into the phenylpropanoid pathway. Willow PAL1, PAL2, PAL3 and PAL4 genes were orthologous to the poplar genes. However no orthologue of PAL5 appears to be present in willow. Moreover, two tandemly repeated PAL2 orthologues were identified in a single contig. Willow PALs show similar sub-cellular localisation to the poplar genes. However, the enzyme kinetics and gene expression of the willow PAL genes differed slightly, with willow PAL2 being more widely expressed than its poplar orthologues implying a wider role for PALs in the production of flavonoids, condensed tannins, benzenoids, and phenolic glycosides, in willow.

An efficient high throughput metabotyping platform for screening of biomass willows.

Future improvement of woody biomass crops such as willow and poplar relies on our ability to select for metabolic traits that sequester more atmospheric carbon into biomass, or into useful products to replace petrochemical streams. We describe the development of metabotyping screens for willow, using combined 1D 1H-NMR-MS. A protocol was developed to overcome 1D 1H-NMR spectral alignment problems caused by variable pH and peak broadening arising from high organic acid levels and metal cations. The outcome was a robust method to allow direct statistical comparison of profiles arising from source (leaf) and sink (stem) tissues allowing data to be normalised to a constant weight of the soluble metabolome. We also describe the analysis of two willow biomass varieties, demonstrating how fingerprints from 1D 1H-NMR-MS vary from the top to the bottom of the plant. Automated extraction of quantitative data of 56 primary and secondary metabolites from 1D 1H-NMR spectra was realised by the construction and application of a Salix metabolite spectral library using the Chenomx software suite. The optimised metabotyping screen in conjunction with automated quantitation will enable high-throughput screening of genetic collections. It also provides genotype and tissue specific data for future modelling of carbon flow in metabolic networks.

Opposite variations in fumarate and malate dominate metabolic phenotypes of Arabidopsis salicylate mutants with abnormal biomass under chilling.

In chilling conditions (5°C), salicylic acid (SA)-deficient mutants (sid2, eds5 and NahG) of Arabidopsis thaliana produced more biomass than wild type (Col-0), whereas the SA overproducer cpr1 was extremely stunted. The hypothesis that these phenotypes were reflected in metabolism was explored using 600 MHz (1) H nuclear magnetic resonance (NMR) analysis of unfractionated polar shoot extracts. Biomass-related metabolic phenotypes were identified as multivariate data models of these NMR 'fingerprints'. These included principal components that correlated with biomass. Also, partial least squares-regression models were found to predict the relative size of plants in previously unseen experiments in different light intensities, or relative size of one genotype from the others. The dominant signal in these models was fumarate, which was high in SA-deficient mutants, intermediate in Col-0 and low in cpr1 at 5°C. Among signals negatively correlated with biomass, malate was prominent. Abundance of transcripts of the FUM2 cytosolic fumarase (At5g50950) showed strong positive correlation with fumarate levels and with biomass, whereas no significant differences were found for the FUM1 mitochondrial fumarase (At2g47510). It was confirmed that the morphological effects of SA under chilling find expression in the metabolome, with a role of fumarate highlighted.

A genome-wide metabolomic resource for tomato fruit from Solanum pennellii.

Tomato and its processed products are one of the most widely consumed fruits. Its domestication, however, has resulted in the loss of some 95% of the genetic and chemical diversity of wild relatives. In order to elucidate this diversity, exploit its potential for plant breeding, as well as understand its biological significance, analytical approaches have been developed, alongside the production of genetic crosses of wild relatives with commercial varieties. In this article, we describe a multi-platform metabolomic analysis, using NMR, mass spectrometry and HPLC, of introgression lines of Solanum pennellii with a domesticated line in order to analyse and quantify alleles (QTL) responsible for metabolic traits. We have identified QTL for health-related antioxidant carotenoids and tocopherols, as well as molecular signatures for some 2000 compounds. Correlation analyses have revealed intricate interactions in isoprenoid formation in the plastid that can be extrapolated to other crop plants.