Hands-on protocol for preparing water-soluble fractions from agri-food samples for NMR-based metabolomics analysis.

Application of multiple chemical and biological approaches for quality assessment of Carthamus tinctorius L. (safflower) by determining both the primary and secondary metabolites

Metabolite variations of sugars, organic acids, fatty acids and amino acids in flower buds of Zingiber mioga Roscoe at different developmental stages

The metabolic products of inorganic carbon taken up through the roots from nutrient solution were studied in willow plants. Willow cuttings (Salix cv. Aquatica gigantea) were supplied with unlabelled or 14C-labelled NaHC03 for 1, 5, 10, and 24 h in light or in darkness. After feeding, the plants were divided into six samples (upper and lower leaves and corresponding stems, cuttings and roots), which were frozen in liquid N2. Freeze-dried ground samples were extracted into water-soluble, chloroform soluble and insoluble fractions. The water-soluble fraction was further separated into basic, acidic, and neutral fractions by ion exchange chromatography. In the light experiment pronase treatment was used to separate the insoluble fraction into proteins and insoluble carbohydrates. After 1 h feeding time, most of the 14C was fixed into organic acids and amino acids both in light and in darkness in all parts of the plants. In the roots a large part of the 14C-carbon was incorporated into the protein and insoluble fractions already during short feeding times, and the amounts incorporated increased with time. In the leaves, after 1 and 5 h the main labelled compounds were the organic acids and amino acids, but after 10 h about half of the total 14C was in protein and in the insoluble fraction. A further analysis of amino acids and organic acids with HPLC showed that C-4 acids were labelled initially and that over time the proportion of different acids changed. These results indicate that the metabolism of carbon in roots might take place via /3-carboxylation of PEP. Part of the fixed 14C is transported from the roots, probably as amino acids and organic acids, to the shoot. In roots the C-4 acids are metabolized further into structural compounds (proteins and insoluble carbohydrates).

Lactic acid bacteria (LAB) were isolated from Korean Meoru (Vitis coigneties) wine and identified as Lactobacillus plantarum meoru0711 (KACC 91436C). The fermentative behavior and metabolic effects of L. plantarum during malolactic fermentation (MLF) were compared with those of the commercial Oenococcus oeni strain through 1H NMR- and GC-based metabolic profiling. Twenty-two primary metabolites of amino acids, carbohydrates, and organic acids, and 55 secondary metabolites of volatile compounds were identified in wines by 1H nuclear magnetic resonance (NMR) spectroscopy and gas chromatography (GC), respectively. Principal component analysis (PCA) revealed that malolactic (ML)-fermented and non-ML-fermented wines, and wines ML-fermented with O. oeni and L. plantarum were clearly differentiated. Both the primary and secondary metabolites were responsible for these differentiations. Compared to non-MLF wines, MLF wines were characterized by increased levels of primary metabolites such as lactic acid, phenylalanine, uracil, ornithine, alanine, threonine, leucine, isoleucine, and valine with decreased levels of monosaccharides, glycerol, malic, and citric acids. In addition, higher levels of secondary metabolites such as butanal, ethyl isobutylate, isobutanol, isoamyl acetate, 2-butanoate ethyl ester, isoamyl alcohol, ethyl hexanoate, glycine, acetic acid, and benzaldehyde characterized the MLF wine. Higher levels of primary metabolites such as tyrosine, monosaccharides, glycerol, alanine, 2,3-butanediol, valine, and leucine, and of secondary metabolites such as propyl acetate, isobutanol, isoamyl acetate, 1-butanol, ethyl hexanoate, prenyl alcohol, glycine, 2-hexen-1-ol, ethyl octanoate, acetic acid, benzaldehyde, and butyric, together with lower levels of lactic acid, were observed in the wines fermented by L. plantarum compared with those by O. oeni. This present study demonstrates that different genera of LAB affect both the primary and second metabolites in wine. Moreover, metabolomics with multivariate statistical analysis provide insight into wine fermentation.

Aim: The purpose of this study was to obtain data concerning growth factor release within liquid and solid platelet-rich fibrin (PRF) matrices and to estimate the amount of potential interindividual variations as a basis for further preclinical and clinical trials. Therefore, we aimed to determine possible differences in the release of growth factors between liquid and solid PRF. Materials and Methods: Blood samples obtained from four subjects were processed to both liquid and solid PRF matrices using a standard centrifugation protocol. Five growth factors (vascular endothelial growth factor, VEGF; epidermal growth factor, EGF; platelet-derived growth factor-BB, PDGF-BB; transforming growth factor-β1, TGF-β1; and matrix metallopeptidase 9, MMP-9) have been evaluated at six time points by ELISA over a total observation period of 10 days (1 h, 7 h, 1 d, 2 d, 7 d, and 10 d). Results: Growth factor release could be measured in all samples at each time point. Comparing liquid and solid PRF matrices, no significant differences were detected (p > 0.05). The mean release of VEGF, TGFβ-1, PDGF-BB, and MMP-9 raised to a peak at time point five (day 7) in both liquid and solid PRF matrices. VEGF release was lower in liquid PRF than in solid PRF, whereas those of PDGF-BB and MMP-9 were higher in liquid PRF than in solid PRF at all time points. EGF had its peak release already at time point two after 7 h in liquid and solid matrices (hour 7 EGF solid: mean = 180 pg/mL, SD = 81; EGF liquid: mean = 218 pg/mL, SD = 64), declined rapidly until day 2, and had a second slight peak on day 7 in both groups (day 7 EGF solid: mean = 182 pg/mL, SD = 189; EGF liquid: mean = 81 pg/mL, SD = 70). Conclusions: This study detected growth factor release within liquid and solid PRF matrices with little variations. Further preclinical trials are needed to precisely analyze the growth factor release in larger samples and to better understand their effects on wound healing in different clinical indications.

Balanites aegyptiaca (L.) Delile (Zygophyllaceae), also known as the desert date, is an edible fruit-producing tree popular for its nutritional and several health benefits. In this study, multi-targeted comparative metabolic profiling and fingerprinting approaches were conducted for the assessment of the nutrient primary and secondary metabolite heterogeneity in different parts, such as leaves, stems, seeds, unripe, and ripe fruits of B. aegyptiaca using nuclear magnetic resonance (NMR), ultra-performance liquid chromatography (UPLC-MS), and gas chromatography mass-spectrometry (GC-MS) based metabolomics coupled to multivariate analyses and in relation to its cytotoxic activities. NMR-based metabolomic study identified and quantified 15 major primary and secondary metabolites belonging to alkaloids, saponins, flavonoids, sugars, and amino and fatty acids. Principal component analysis (PCA) of the NMR dataset revealed α-glucose, sucrose, and isorhamnetin as markers for fruit and stem and unsaturated fatty acids for predominated seeds. Orthogonal projections to latent structure discriminant analysis (OPLS-DA) revealed trigonelline as a major distinctive metabolite in the immature fruit and isorhamnetin as a major distinct marker in the mature fruit. UPLC-MS/MS analysis using feature-based molecular networks revealed diverse chemical classes viz. steroidal saponins, N-containing metabolites, phenolics, fatty acids, and lipids as the constitutive metabolome in Balanites. Gas chromatography-mass spectroscopy (GC-MS) profiling of primary metabolites led to the detection of 135 peaks belonging to sugars, fatty acids/esters, amino acids, nitrogenous, and organic acids. Monosaccharides were detected at much higher levels in ripe fruit and disaccharides in predominate unripe fruits, whereas B. aegyptiaca vegetative parts (leaves and stem) were rich in amino acids and fatty acids. The antidiabetic compounds, viz, nicotinic acid, and trigonelline, were detected in all parts especially unripe fruit in addition to the sugar alcohol d-pinitol for the first time providing novel evidence for B. aegyptiaca use in diabetes. In vitro cytotoxic activity revealed the potential efficacy of immature fruit and seeds as cytotoxic agents against human prostate cancer (PC3) and human colorectal cancer (HCT-116) cell lines. Collectively, such detailed profiling of parts provides novel evidence for B. aegyptiaca medicinal uses.

ABA and GA3 regulate the synthesis of primary and secondary metabolites related to alleviation from biotic and abiotic stresses in grapevine

Liquid matrices were developed and studied for application in the MALDI analysis of polyglycols and poly(dimethylsiloxane). Characteristic positive ion MALDI-TOF mass spectra were obtained from over 20 liquid matrices. These systems are characterized by rapid and simple preparation and exhibited good vacuum stability. The spot-to-spot and point-to-point reproducibility of signal intensity and molecular weight values are indicative of the homogeneous environment created by liquid matrices. The best performance was achieved when the chromophore, 2-cyano-5-phenyl-2,4-pentadienoic acid, was used in the analyte or analyte/mediator systems (mediator = nonabsorbing liquid) at or near saturation concentrations. Internal standards, including silver and low molecular weight polyglycols, have been successfully employed for mass calibration. Comparison of MALDI molecular weight data with those obtained from SIMS and GPC shows some systematic disparities. Possible reasons for these differences are discussed. Excellent agreement is seen between molecular weight values obtained with liquid and solid MALDI matrices. However, conventional solid MALDI matrices generally exhibit better resolution and can be applied for analysis of higher molecular weight materials. This may be related to the higher laser intensity required to produce ions from liquid matrices.

This is the first study presenting a multi-residue method allowing for comprehensive analysis of several chiral pharmacologically active compounds (cPACs) including beta-blockers, antidepressants and amphetamines in wastewater and digested sludge at the enantiomeric level. Analysis of both the liquid and solid matrices within wastewater treatment is crucial to being able to carry out mass balance within these systems. The method developed comprises filtration, microwave assisted extraction and solid phase extraction followed by chiral liquid chromatography coupled with tandem mass spectrometry to analyse the enantiomers of 18 compounds within all three matrices. The method was successfully validated for 10 compounds within all three matrices (amphetamine, methamphetamine, MDMA, MDA, venlafaxine, desmethylvenlafaxine, citalopram, metoprolol, propranolol and sotalol), 7 compounds validated for the liquid matrices only (mirtazapine, salbutamol, fluoxetine, desmethylcitalopram, atenolol, ephedrine and pseudoephedrine) and 1 compound (alprenolol) passing the criteria for solid samples only. The method was then applied to wastewater samples; cPACs were found at concentration ranges in liquid matrices of: 1.7ngL−1 (metoprolol) – 1321ngL−1 (tramadol) in influent, <LOD (desmethylcitalopram and metoprolol) – 506ngL−1 in effluent, and in solid matrix digested sludge: 0.4ngg−1 (metoprolol) – 275ngg−1 (citalopram). Enantiomeric profiling revealed that studied compounds were present in analysed samples in non-racemic composition. Furthermore, enantiomeric composition of studied analytes differed in liquid and solid matrices. This demonstrates that not analysing the solid fraction of wastewater may lead to over-estimation of the removal rates of cPACs as well as possible misrepresentation of the enantiomeric fraction of the compounds as they leave the wastewater treatment plant. Consequently risks from cPACs entering the environment might be higher than anticipated.

This study aimed to elucidate the variations in primary and secondary metabolites during Lycoris radiata flower development using high performance liquid chromatography (HPLC) and gas chromatography time-of-flight mass spectrometry (GC-TOFMS). The result showed that seven carotenoids, seven phenolic acids, three anthocyanins, and galantamine were identified in the L. radiata flowers. Most secondary metabolite levels gradually decreased according to the flower developmental stages. A total of 51 metabolites, including amines, sugars, sugar intermediates, sugar alcohols, amino acids, organic acids, phenolic acids, and tricarboxylic acid (TCA) cycle intermediates, were identified and quantified using GC-TOFMS. Among the hydrophilic compounds, most amino acids increased during flower development; in contrast, TCA cycle intermediates and sugars decreased. In particular, glutamine, asparagine, glutamic acid, and aspartic acid, which represent the main inter- and intracellular nitrogen carriers, were positively correlated with the other amino acids and were negatively correlated with the TCA cycle intermediates. Furthermore, quantitation data of the 51 hydrophilic compounds were subjected to partial least-squares discriminant analyses (PLS-DA) to assess significant differences in the metabolites of L. radiata flowers from stages 1 to 4. Therefore, this study will serve as the foundation for a biochemical approach to understand both primary and secondary metabolism in L. radiata flower development.

Mineral nutrition plays a vital role in plant growth and metabolism. To date, 17 essential nutrients have been identified that are direly needed for plant growth. Nitrogen (N), phosphorus (P), and potassium (K) are among the most important macronutrients, whereas micronutrients include chloride (Cl), copper (Cu), manganese (Mn), iron (Fe), zinc (Zn), cobalt (Co), molybdenum (Mo), and nickel (Ni). Each mineral carries equal importance, and a deficiency of any of them can affect growth and disrupt the plant life cycle. Plants are treasure troves of bioactive metabolites that essentially regulate nearly all fundamental processes like growth, reproduction, and environmental responses. These plant metabolites are divided into two major categories, i.e. primary metabolites (PMs) and secondary metabolites (SMs) according to their functions. There is a strong linkage between plant metabolites and mineral nutrients. Biosynthesis of metabolites involves various metabolic pathways such as absorption, carbon assimilation, photosynthesis, protoplast formation, respiration, transpiration, translocation, and storage. These pathways are regulated by mineral nutrients available in different forms in the soil solution. N is necessary for the biosynthesis of various PMs (i.e. amino acids, chlorophyll, nucleic acids, lipids, proteins, and enzymes) and SMs (i.e. flavonoids and phenolic compounds). P is important for sugar phosphates, phospholipids, DNA, RNA, and nucleotide synthesis as well as actively involved in energy metabolism. K mediates the synthesis of sugars, amino acids, organic acids, and antioxidants and also plays many regulatory roles. Similarly, micronutrients (S, Ca, Mg, Fe, Cu, Mn, B, and Cl) also regulate the synthesis of wide array of PMs and SMs such as proteins, amino acids, sugars, flavonoids, phytohormones, organic acids, vitamins, antioxidants, glucosinolates, and various cofactors that have crucial role in plant growth and development. Availability and composition of mineral nutrients have a profound effect on the type and amount of PMs and SMs produced. So there lies an increased interest in unraveling the regulatory roles of mineral nutrients in the biosynthesis of plant metabolites. Therefore, this chapter specifically aims to improve our understanding regarding the regulatory roles of mineral nutrients in the biosynthesis of plant metabolites.

BackgroundChilling stress is a major factor limiting rice production. Rice genotypes differ greatly in their seedling chilling tolerance (CT), which is known to involve differential expression of large numbers of genes and proteins. To further understand the metabolomic responses of rice to chilling stress, profiles of the 106 primary metabolites of a CT japonica variety, Lijiangxintuanhegu (LTH) and a chilling sensitive indica line, IR29, were investigated under a time-series of chilling stress and non-stress control conditions at the seedling stage.ResultsWe identified 106 primary metabolites that were temporally and genotype-dependently regulated in LTH and IR29 under the time-series chilling stress and subsequent recovery. Three major groups of primary metabolites, amino acids (AAs), organic acids (OAs) and sugars, showed distinct change patterns in both genotypes in response to the chilling stress: a more general accumulation of most AAs, more dramatic decreased levels of most OAs, and greatly reduced levels for most sugars at early time points of stress but increased levels of specific sugars at the later time points of stress. Compared to IR29, LTH had more metabolites showing chilling induced changes, greater levels of these metabolomic changes and a greater ability to recover after stress, implying that LTH used a positive energy-saving strategy against chilling stress. During subsequent recovery, more metabolites were significantly and exclusively up-regulated in LTH, indicating their positive role in chilling tolerance. A comparative analysis of these metabolites data and differentially expressed genes data allowed identification of 7 AAs and related genes that were both chilling responsive and contributed greatly to the CT of LTH.ConclusionsThe metabolomic responses of rice to chilling stress at the seedling stage were dynamic and involved large numbers of the metabolites. The chilling induced changes of three major groups of metabolites, AAs, OAs and sugars, in rice were well coordinated. The high level seedling CT of LTH was apparently attributed to its increased levels of most AAs and reduced energy consumption that resulted in increased glycolysis and strong resilience on recovery. The results of this study extend our understanding of molecular mechanisms of chilling stress tolerance in rice.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-6-23) contains supplementary material, which is available to authorized users.

Honey is a widely consumed natural agricultural product. Honey bees produce honey by collecting nectar from different flowers and then metabolising it to form honey which is stored in the hive. The current focus of research has been on the primary characteristic metabolites of monofloral honey from different plant sources. There is a lack of understanding of the differences in the transformation and composition of overall metabolites between plant nectar and the honey that is processed by bees after nectar feeding. In this study, loquat nectar and mature loquat honey were used for the detection of all non-volatile metabolites in both by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Subsequently, an analysis was conducted on the primary metabolites, including saccharides, amino acids and their derivatives, nucleotides and their derivatives, lipids, and organic acids. In addition, the secondary metabolites, including flavonoids, phenolic acids, and terpenoid, were analysed. The results showed that there was a significant difference in the relative content of non-volatile metabolites between the two. We detected a total of 914 non-volatile metabolites, of which 834 were detected in loquat nectar and 759 in loquat honey. We analyzed the relative content of metabolites based on their classification and found that the relative content of primary and secondary metabolites showed different trends after processing by bees. Among them, the content of nucleotides and their derivatives and sugar metabolites in loquat honey was generally higher than that in loquat honey. This result presents a comprehensive picture of the non-volatile metabolite composition of loquat nectar and loquat honey, and systematically compares the changes in the relative content of the two substances.

Chapter 11 - Bacterial metabolites: an unexplored quarry

Incorporation of photosynthetically fixed 14C was studied at different time intervals of 12, 24, and 36 h in various plant parts—leaf 1 to 4 from apex, roots, and rhizome—into primary metabolites—sugars, amino acids, and organic acids, and secondary metabolites—essential oil and curcumin—in turmeric. The youngest leaves were most active in fixing 14C at 24 h. Fixation capacity into primary metabolites decreased with leaf position and time. The primary metabolite levels in leaves were maximal in sugars and organic acids and lowest in amino acids. Roots as well as rhizome received maximum photoassimilate from leaves at 24 h; this declined with time. The maximum metabolite concentrations in the roots and rhizome were high in sugars and organic acids and least in amino acids. 14C incorporation into oil in leaf and into curcumin in rhizome was maximal at 24 h and declined with time. These studies highlight importance of time-dependent translocation of 14C-primary metabolites from leaves to roots and rhizome and their subsequent biosynthesis into secondary metabolite, curcumin, in rhizome. This might be one of factors regulating the secondary metabolite accumulation and rhizome development.