Nuclear Magnetic Resonance Mass Spectrometry Research Articles

Chemical Constitutes from Mycelia of Laetiporus versisporus (Agaricomycetes).

Edible mushrooms, both wild and cultivated, can be seen as healthy functional food. More and more valuable compounds are obtained from mycelia of macromycetes. However, there was limited report about the medicinal fungus Laetiporus versisporus (Lloyd) Imazeki. Herein, L. versisporus was fermented on rice media and the secondary metabolites of mycelia were investigated. In this study, two-step method was used to obtain fermented products, silica gel column chromatography, recrystallization, medium pressure column chromatography, preparative thin-layer chromatography were applied to separate the chemical constituents. Nine chemical compounds (1-9) including one new triterpenoid acid versisponic acid F were identified by NMR (nuclear magnetic resonance) spectroscopy and MS (mass spectrometry). Seven compounds including monolinoleoyl glycerol, linoleic acid, ergosta-5, 7, 22-triene-3β-ol, β-sitosterol, daucosterol, versisponic acid F were isolated for the first time from L. versisporus.

Comprehensive isotopomer analysis of glutamate and aspartate in small tissue samples

Stable isotopes are powerful tools to assess metabolism. 13C labeling is detected using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS). MS has excellent sensitivity but generally cannot discriminate among different 13C positions (isotopomers), whereas NMR is less sensitive but reports some isotopomers. Here, we develop an MS method that reports all 16 aspartate and 32 glutamate isotopomers while requiring less than 1% of the sample used for NMR. This method discriminates between pathways that result in the same number of 13C labels in aspartate and glutamate, providing enhanced specificity over conventional MS. We demonstrate regional metabolic heterogeneity within human tumors, document the impact of fumarate hydratase (FH) deficiency in human renal cancers, and investigate the contributions of tricarboxylic acid (TCA) cycle turnover and CO2 recycling to isotope labeling invivo. This method can accompany NMR or standard MS to provide outstanding sensitivity in isotope-labeling experiments, particularly invivo.

Isolation and structure elucidation of caryophyllane sesquiterpenoids from leaves of Eremophila spathulata

• This is the first phytochemical investigation of the leaves of Eremophila spathulata . • A combination of HPLC and HPLC-PDA-HRMS-SPE-NMR afforded seven compounds. • The structures were identified as new caryophyllane sesquiterpenoids by HRMS and 2D NMR. Crude extract of Eremophila spathulata leaves was investigated by semi-preparative scale high-performance liquid chromatography (HPLC), analytical scale HPLC, and hyphenated high-performance liquid chromatography-photodiode array-high-resolution mass spectrometry-nuclear magnetic resonance (HPLC-PDA-HRMS-SPE-NMR), which afforded seven previously unreported caryophyllane sesquiterpenoids. Semi-preparative scale separation of the crude extract afforded (1 R* ,4 R *,9 S* , E )-8-formyl-11,11-dimethylbicyclo[7.2.0]undec-7-ene-4-carboxylic acid ( 5 ) and analytical-scale HPLC separation afforded (1 R* ,4 S* ,7 S *,9 S* )-7-hydroxy-11,11-dimethyl-8-methylenebicyclo[7.2.0]undecane-4-carboxylic acid ( 1 ), (1 S* ,6 R* ,9 R*,E )-10,10-dimethylbicyclo[7.2.0]undec-2-ene-2,6-dicarboxylic acid ( 2 ), (1 R* ,4 S* ,9 S* )-11,11-dimethyl-8-oxobicyclo[7.2.0]undecane-4-carboxylic acid ( 3 ), and (1 R* ,4 R* ,9 S* )-11,11-dimethyl-8-oxobicyclo[7.2.0]undecane-4-carboxylic acid ( 4 ). HPLC-PDA-HRMS-SPE-NMR afforded (1 R* ,4 R* ,9 S* )-11,11-dimethyl-8-methylenebicyclo[7.2.0]undecane-4-carboxylic acid ( 6 ) and (1 R* ,4 S* ,9 S* )-11,11-dimethyl-8-methylenebicyclo[7.2.0]undecane-4-carboxylic acid ( 7 ). The structures of all isolated compounds were established based on HRMS as well as extensive 1D and 2D NMR analysis. Relative configurations were determined by correlations in spectra from rotational Overhauser effect spectroscopy.

Inhibitory actions of lupinifolin isolated from Derris reticulata stem against carbohydrate-digesting enzymes

Background: Postprandial hyperglycemia is linked with the development of diabetic complications. Inhibition against the carbohydrate-digesting enzymes, specifically pancreatic α-amylase and intestinal α-glucosidase, delays carbohydrate digestion and subsides postprandial plasma glucose (PPG) levels. Objectives: This study aimed to examine the effects of lupinifolin, purified from Derris reticulata stem, a medicinal plant traditionally used for the treatment of diabetes mellitus, on in vitro α-amylase and α-glucosidase enzyme activities and enzyme kinetics. Subjects and Methods: The identification of the isolated phytochemical was performed using nuclear magnetic resonance–mass spectrometry (NMR-MS) spectrometry.In vitro pancreatic α-amylase and α-glucosidase activities and enzyme kinetics were determined using the enzymatic colorimetric methods. Results: The purified phytochemical was identified as lupinifolin from the NMR-MS spectrometry. The isolated lupinifolin produced a significant α-amylase and α-glucosidase inhibitory actions with the median inhibitory concentrations of 3.43 ± 0.77 and 56.29 ± 11.64 μg/mL, respectively. The study of enzyme kinetics showed that lupinifolin exhibited the mode of non-competitive and mixed enzyme inhibitions against α-amylase and α-glucosidase, respectively. Conclusion: The potent α-amylase and α-glucosidase inhibitory actions of lupinifolin derived from D. reticulata stem evidently suggest its potential use as an alternative for the control of PPG.

Protective Effect and Metabonomics Research of "Biao-Ben Acupoint Combination" Electroacupuncture in Chronic Myocardial Ischemia Model Rats

To observe the effect of electroacupuncture (EA) on ischemic electrocardiogram (ECG), histopathological changes and serum metabolite profile in chronic myocardial ischemia (CMI) rats, so as to reveal its mechanisms underlying protecting ischemic myocardium. A total of 45 male Wistar rats were randomly divided into normal control, CMI model and EA groups, with 15 rats being in each group. The rats in the control group received subcutaneous injection of 0.9% normal saline (5 mg•mg－1•d－1, for 7 days), and those in the model and EA groups received subcutaneous injection of isopropylarterenol hydrochloride (5 mg•mg－1•d－1, for 7 days) to establish CMI model. EA (2 Hz/100 Hz, 1 mA) was applied to bilateral "Zusanli" (ST 36), "Guanyuan" (CV 4) and "Neiguan" (PC 6) for 10 min, once daily for 21 days. The ECG-ST segment of the standard limb lead II was used for evaluating the severity of myocardial ischemia, and the histopathological changes of myocardium were observed under microscope after H．E. staining. The profile of serum metabolites was analyzed by nuclear magnetic resonance mass spectrometry combined with principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) in 21 rats (n＝7 in each group)．. After modeling, the amplitude of ECG-ST was significantly increased in comparison with the normal control group (P<0.01), suggesting a successful establishment of CMI model. H．E. stain showed an apparent injury of the myocardial tissue as interstitial edema, vasodilatation and hemorrhage, infiltration of inflammatory cells, unclear veins, and increased intercellular space, etc. with irregularly arranged myocardial cells being dissolved, ruptured and necrotic, and with obvious edema, blurred shape, uneven staining, karyopyknosis, and glass-like lesions. After EA intervention, the amplitude of ECG-ST in the EA group was evidently lower than that of the model group (P<0.01), and the severity of the myocardial injury was relatively milder relevant to the model group, suggesting an alleviation of the injured myocardium. In the PLS-DA score plot, the normal control group and the EA group outline were close to each other, and Glucose, Lactate, Creatine, Acetate, 3-Hydroybutyrate might be the potential metabolic differential biomarkers (VIP > 1). The PLS-DA analysis revealed deviations in 51 differential biomarkers in serum，among which, Glucose, Lactate, Creatine, Acetate and 3-Hydroybutyrate may contribute to the effect of EA in improving CMI. EA stimulation of acupoints can ameliorate ischemic myocardial injury in CMI rats, which may be related to its effect in regulating serum sugar, lipid metabolism and energy metabolism.

Separation and Identification of Swertiamarin from Enicostema axillare Lam. Raynal by Centrifugal Partition Chromatography and Nuclear Magnetic Resonance-Mass Spectrometry*

Separation and Identification of Swertiamarin from Enicostema axillare Lam.Raynal by Centrifugal Partition Chromatography and Nuclear Magnetic Resonance-Mass Spectrometry* Enicostema axillare, a bitter herb, has been used for the treatment of diabetes in traditional system of medicine in India. Swertiamarin, a bitter and potent hepatoprotective iridoid glucoside (~20 mg) was isolated by Centrifugal Partition Chromatography coupled with Diode Array Detector (DAD) from the crude methanolic extract (1.0 g) of its aerial parts using a biphasic solvent system composed of chloroformmethanol- water (86:74:20, v/v) in ascending mode for the first time. The purity of the compound was found to be >97%. The structure of swertiamarin was determined using spectroscopic data mainly NMR (1H, 13C, DEPT, HSQC and HMBC) and ESI-MS. Compound was also identified by comparing 13C NMR data with those reported and some of the 13C NMR values were revised.

Acaricidal activity of Hypocrella raciborskii Zimm. (Hypocreales: Clavicipitaceae) crude extract and some pure compounds on Tetranychus urticae Koch (Acari: Tetranychidae)

The acaricidal activity of secondary metabolites from an entomopathogenic fungus,Hypocrella raciborskii Zimm. in controlling two-spotted spider mite, Tetranychus urticaeKoch (Acari: Tetranychidae) is explored in this study as a new agent for pest control. The crude extract was prepared in ethyl acetate. Secondary metabolites were elucidated using Nuclear Magnetic Resonance and High-Resolution Mass Spectrometry analysis. The major secondary metabolites of the crude extract were ergosterol, dustanin (15α, 22-dihydroxyhopane) and 3β-acetoxy-15α,22-dihydroxyhopane. The crude extract and pure compounds were used to determine their toxicity, repellency and oviposition deterrence against T. urticae, under the laboratory conditions. Crude extract of 3% w/v showed a strong contact toxicity (97% mortality, one day after treatment) and residual toxicity (80%, three days after treatment). Ergosterol induced 75% mite mortality in a residual toxicity bioassay. Females topically sprayed with 3% crude extract had their egg production reduced by 97%. Direct spraying of 3β-acetoxy-15α, 22-dihydroxyhopane and dustanin reduced egg production of T. urticae by 71 and 70%, respectively. Both crude extract and pure compounds had relatively slight repellency effect on T. urticae. Key words: Fungal metabolites, bioassay, two-spotted spider mite

Multiple Conformers and their Spectroscopic Properties of N-Glycan Predicted using Replica-Exchange Simulations

N-glycans play essential roles in many biological functions such as protein quality control and cell-to-cell communications. Nuclear Magnetic Resonance (NMR) and Mass Spectroscopy (MS) are powerful experimental means to analyze glycan structures, but a precise characterization of various glycan conformers are still challenging. We performed replica-exchange MD (REMD) simulations of N-glycans to identify a family of conformers to interpret experimental data. The results show that N-glycans have distinct multiple conformers and their population sizes change upon the chemical modifications. The experimental NMR data (NOEs, J-couplings) and MS data (collision cross sections) are well reproduced taking the multiple conformers into account. For example, the difference in the major conformer between two isomeric N-glycans leads to the different distribution of collision cross sections observed in the experiment. Further details of the analyses as well as future perspective will be discussed.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Translation of metabolite profiling to infectious diseases

The field of metabonomics involves generation of complex metabolic profiles of tissues and biofluids using analytical platforms such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). Metabolic profiles can reflect changes following an intervention, such as administration of a drug or onset of a disease. Multivariate modeling tools are applied to discriminate profiles and identify biomarkers that differentiate between phenotypes of interest such as control and diseased states. One advantage of metabonomics is that it is a ‘top-down’ approach that reflects an integrated systems response as it takes into account genetic and environmental status. A variety of analytical technologies can be utilized; however NMR spectroscopy and MS represent the most powerful and widely used tools as they can capture information on thousands of metabolites in a biofluid in a single analytical run. The challenge of extracting relevant metabolic information from the complex spectral data sets is tackled using pattern recognition tools that reduce the dimensionality of the data, such as principal components analysis (PCA) and partial least squares regression (PLS) and many novel statistically-driven approaches have been developed in recent years. An example of the application of metabonomics to differentiate cerebrospinal fluid (CSF) samples from clinical bacterial and viral meningitis cases was presented. The application of NMR-based metabolic profiling greatly improved the speed and specificity of sample classification. In addition, patterns of CSF metabolites that altered following bacterial and viral meningitis infection were identifed. Furthermore, gram-stain negative samples of meningococcal meningitis were clearly differentiated from all other cases of bacterial meningitis. A larger study is intended to validate these findings and to also incorporate metabolic profiling of non-invasively collected urine rather than CSF. In terms of diagnostics, metabonomics has many diverse application potentials and could be useful in studying the metabolic signature reflecting host response, in identifying pathogen-derived metabolites, and in differentiating single infection versus co-infection. It may prove a powerful tool for providing systems level insight into mechanisms of pathology associated with infectious diseases, such as Mycobacterium tuberculosis. With respect to therapeutic monitoring, both in vitro and in vivo metabonomic-led approaches could offer insight into the response of both the pathogen and host to intervention, drug resistance, therapeutic efficacy and toxicology.

LC–SPE–NMR–MS: A Total Analysis System for Bioanalysis

Liquid chromatography (LC)-solid-phase extraction (SPE)-nuclear magnetic resonance (NMR)-mass spectrometry (MS) coupling is a key technology for fast and thorough structure elucidation of valuable mass-limited samples. Laborious serial isolation and purification procedures of metabolites, byproducts or impurities from complex biomatrices, natural product extracts or other mixtures of several components can be circumvented by the use of this integrated modular system. This combination of high-end analytical technology significantly accelerates the structure-elucidation process for valuable samples present in minute quantities in mixtures. The information depth is significantly increased by the concurrent availability of NMR and MS data of one chromatographic peak. Thus, this flexible technique is well on its way to becoming the gold standard in analytical chemistry of mixtures. LC-SPE-NMR-MS overcomes the limitations of directly coupled LC-NMR. Full flexibility regarding chromatographic conditions and NMR acquisition is gained by this modular technique. LC-SPE-NMR-MS allows for a rapid structure-elucidation process that would not be possible on the basis of MS or NMR data alone.

Spectroscopic and Statistical Techniques for Information Recovery in Metabonomics and Metabolomics

Methods for generating and interpreting metabolic profiles based on nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and chemometric analysis methods are summarized and the relative strengths and weaknesses of NMR and chromatography-coupled MS approaches are discussed. Given that all data sets measured to date only probe subsets of complex metabolic profiles, we describe recent developments for enhanced information recovery from the resulting complex data sets, including integration of NMR- and MS-based metabonomic results and combination of metabonomic data with data from proteomics, transcriptomics, and genomics. We summarize the breadth of applications, highlight some current activities, discuss the issues relating to metabonomics, and identify future trends.

Opium poppy: blueprint for an alkaloid factory

Opium poppy (Papaver somniferum) produces a large number of benzylisoquinoline alkaloids, including the narcotic analgesics morphine and codeine, and has emerged as one of the most versatile model systems to study alkaloid metabolism in plants. As summarized in this review, we have taken a holistic strategy—involving biochemical, cellular, molecular genetic, genomic, and metabolomic approaches—to draft a blueprint of the fundamental biological platforms required for an opium poppy cell to function as an alkaloid factory. The capacity to synthesize and store alkaloids requires the cooperation of three phloem cell types—companion cells, sieve elements, and laticifers—in the plant, but also occurs in dedifferentiated cell cultures. We have assembled an opium poppy expressed sequence tag (EST) database based on the attempted sequencing of more than 30,000 cDNAs from elicitor-treated cell culture, stem, and root libraries. Approximately 23,000 of the elicitor-induced cell culture and stem ESTs are represented on a DNA microarray, which has been used to examine changes in transcript profile in cultured cells in response to elicitor treatment, and in plants with different alkaloid profiles. Fourier transform-ion cyclotron resonance mass spectrometry and proton nuclear magnetic resonance mass spectroscopy are being used to detect corresponding differences in metabolite profiles. Several new genes involved in the biosynthesis and regulation of alkaloid pathways in opium poppy have been identified using genomic tools. A biological blueprint for alkaloid production coupled with the emergence of reliable transformation protocols has created an unprecedented opportunity to alter the chemical profile of the world’s most valuable medicinal plant.

GacS-Dependent Production of 2R, 3R-Butanediol by Pseudomonas chlororaphis O6 Is a Major Determinant for Eliciting Systemic Resistance Against Erwinia carotovora but not Against Pseudomonas syringae pv. tabaci in Tobacco

Root colonization by a plant-beneficial rhizobacterium, Pseudomonas chlororaphis O6, induces disease resistance in tobacco against leaf pathogens Erwinia carotovora subsp. carotovora SCC1, causing soft-rot, and Pseudomonas syringae pv. tabaci, causing wildfire. In order to identify the bacterial determinants involved in induced systemic resistance against plant diseases, extracellular components produced by the bacterium were fractionated and purified. Factors in the culture filtrate inducing systemic resistance were retained in the aqueous fraction rather than being partitioned into ethyl acetate. Fractionation on high-performance liquid chromatography followed by nuclear magnetic resonance mass spectrometry analysis identified the active compound as 2R, 3R-butanediol. 2R, 3R butanediol induced systemic resistance in tobacco to E. carotovora subsp. carotovora SCC1, but not to P. syringae pv. tabaci. Treatment of tobacco with the volatile 2R, 3R-butanediol enhanced aerial growth, a phenomenon also seen in plants colonized by P. chlororaphis O6. The isomeric form of the butanediol was important because 2S, 3S-butandiol did not affect the plant. The global sensor kinase, GacS, of P. chlororaphis O6 was a key regulator for induced systemic resistance against E. carotovora through regulation of 2R, 3R-butanediol production. This is the first report of the production of these assumed fermentation products by a pseudomonad and the role of the sensor kinase GacS in production of 2R, 3R-butanediol.

On‐line LC‐NMR‐MS characterization of sesame oil extracts and assessment of their antioxidant activity

AbstractSesame lignans were isolated by solvent extraction and subsequently purified by solvent crystallization from crude, unroasted sesame oil, and a sesame oil deodorizer distillate. In addition, an aliquot of the purified sesame oil extract was treated with camphorsulfonic acid to obtain a sesaminol‐enriched extract. The sesame lignan composition of the extracts was characterized by on‐line liquid chromatography nuclear magnetic resonance spectroscopy mass spectrometry coupling (LC‐NMR‐MS). The effect of the sesame oil extracts as well as pure sesame lignans and γ‐tocopherol on the oxidative stability of sunflower oil (lignan‐free) was studied by the Rancimat assay. The Rancimat assay revealed the following oxidative stability order: sesame oil extract &lt; sesame oil deodorizer distillate &lt; sunflower oil (no added sesame oil extracts) &lt; sesamol &lt; sesaminol‐enriched sesame oil extract. In addition, the radical‐scavenging capacity of these extracts was assessed by the Trolox® equivalent antioxidant capacity (TEAC) assay. The TEAC assay revealed a slightly different AOX activity order: sesamin &lt; sesame oil extract &lt; sesaminol‐enriched sesame oil extract &lt; sesamol. In conclusion, the sesaminol‐enriched extract revealed strong antioxidant activity and is therefore suitable to increase the oxidative stability of edible oils high in polyunsaturated fatty acids.

Determination of tacrine metabolites in microsomal incubate by high performance liquid chromatography–nuclear magnetic resonance/mass spectrometry with a column trapping system

A column trapping system has been incorporated into high performance liquid chromatography–nuclear magnetic resonance–mass spectrometry (HPLC–NMR–MS) to reduce data acquisition time of NMR experiments. The system uses a trapping column to capture analytes after the HPLC column and back flush trapped analyte to the flow cell of the NMR probe for detection. A dilution solvent is mixed with eluent from HPLC column to reduce the influence of the organic content in the mobile phase before column trapping. The trapping column is also coupled with a mass spectrometer (MS) to get complementary MS data on the same peak. Studies on 1-hydroxylated 9-amino-1,2,3,4-tetrahydro-acridine (1-OH tacrine), indomethacin and testosterone with the column trapping system showed good recovery of analytes and over 3-fold mean increase in UV–VIS signal intensity. The time saving on NMR experiments with the column trapping system was demonstrated by the analysis of dog microsomal incubate with tacrine.

Application of Directly Coupled High-performance Liquid Chromatography–Nuclear Magnetic Resonance–Mass Spectrometry to the Detection and Characterisation of the Metabolites of 2-Bromo-4-trifluoromethylaniline in Rat Urine

Directly coupled HPLC–NMR–MS was used to identify the major urinary metabolite of 2-bromo-4-trifluoromethylaniline in the rat as the sulfate conjugate of 2-bromo-4-trifluoromethyl-6-hydroxyaniline. This metabolite could not have been fully characterised using HPLC–NMR or HPLC–MS alone as the sulfate group is ‘NMR silent’ and MS would not have enabled the position of substitution on the aromatic ring to have been assigned. The need to carefully select HPLC solvents for directly coupled HPLC–NMR–MS so that NMR and MS data can both be obtained is also shown.

Purity determinations in the bulk drug of the novel indoloquinone antitumor agent EO9.

The pharmaceutical development of the investigational cytotoxic drug EO9 included the structural characterization of the bulk drug by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS) and infrared (IR) spectroscopy, and analytical characterization by high-performance liquid chromatography and ultraviolet/visible spectrophotometry. The presence of impurities in the bulk drug was investigated. The intermediates in the synthesis of EO9 were structurally characterized by NMR spectroscopy and MS, and analytically characterized by HPLC analysis with photodiode array (PDA) detection. All of the intermediates were below their limits of detection in EO9 bulk drug. The amounts of residual organic solvents were determined by gas chromatography. Methanol and ethanol were detected, but the amounts present did not exceed the limits as set in the United States Pharmacopeia XXII.