Comprehensive metabolite profiling in distinct chemotypes of Commiphora wightii

Commiphora wightii (Arn.) Bhandari, known as guggul, produces a medicinally important gum resin which is used extensively by Ayurvedic physicians to treat various ailments. However, most of the studies on C. wightii have been limited to its gum resin. Comprehensive metabolic profiling of leaves, stem and gum resin samples was undertaken to analyse aqueous and non-aqueous metabolites from three distinct chemotypes (NBRI-101, NBRI-102 and NBRI-103) shortlisted from different agro-climatic zones. GC-MS, HPLC and NMR spectroscopy were used for comprehensive metabolomics. Multivariate analysis showed characteristic variation in quinic and citric acids, myo-inositol and glycine (aqueous metabolites) and 2,6-di-tert-butyl-phenol, trans-farnesol and guggulsterones (non-aqueous metabolites) amongst the three chemotypes. Quinic acid, citric acid and myo-ionositol were detected in substantial quantities from leaves and stem samples which provide opportunities for novel nutraceutical and pharmaceutical formulations. Quinic acid, from the leaves, was identified as a marker metabolite for early selection of high guggulsterones-yielding cultivars.     

HR-MAS NMR Applications in Plant Metabolomics

Metabolomics is used to reduce the complexity of plants and to understand the underlying pathways of the plant phenotype. The metabolic profile of plants can be obtained by mass spectrometry or liquid-state NMR. The extraction of metabolites from the sample is necessary for both techniques to obtain the metabolic profile. This extraction step can be eliminated by making use of high-resolution magic angle spinning (HR-MAS) NMR. In this review, an HR-MAS NMR-based workflow is described in more detail, including used pulse sequences in metabolomics. The pre-processing steps of one-dimensional HR-MAS NMR spectra are presented, including spectral alignment, baseline correction, bucketing, normalisation and scaling procedures. We also highlight some of the models which can be used to perform multivariate analysis on the HR-MAS NMR spectra. Finally, applications of HR-MAS NMR in plant metabolomics are described and show that HR-MAS NMR is a powerful tool for plant metabolomics studies.     

Determination of Pentacyclic Triterpenes from Betula utilis by High-Performance Liquid Chromatography and High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

A rapid and reliable method was developed and validated for determining betulin and betulinic acid in bark in Betula utilis by high-resolution magic angle spinning ¹H nuclear magnetic resonance (HR-MAS NMR) spectroscopy. HR-MAS NMR spectroscopy clearly distinguished the resonances of betulin and betulinic acid in the bark of all accessions of B. utilis. The concentrations of betulin and betulinic acid were calculated and added to the spectra. The determination of the targeted metabolites in chloroform extract of bark of each accession of B. utilis was performed by high-performance liquid chromatography (HPLC). Quantitatively, betulin was present at higher concentrations than betulinic acid in all accessions. The HR-MAS NMR and HPLC results showed that betulin and betulinic acid varied significantly among accessions of B. utilis. Principal component analysis of the NMR and HPLC results provided classification into three metabolic groups in which the betulin concentration was high, moderate, or low. The results show that HR-MAS NMR is rapid for fingerprinting of betulin and betulinic acid in the bark of B. utilis, while minimizing the drawbacks associated with solvent extraction.     

The Effect of Blood Contained in the Samples on the Metabolomic Profile of Mouse Brain Tissue: A Study by NMR Spectroscopy

(1) Recently, metabolic profiling of the tissue in the native state or extracts of its metabolites has become increasingly important in the field of metabolomics. An important factor, in this case, is the presence of blood in a tissue sample, which can potentially lead to a change in the concentration of tissue metabolites and, as a result, distortion of experimental data and their interpretation. (2) In this paper, the metabolomic profiling based on NMR spectroscopy was performed to determine the effect of blood contained in the studied samples of brain tissue on their metabolomic profile. We used 13 male laboratory CD-1^® IGS mice for this study. The animals were divided into two groups. The first group of animals (n = 7) was subjected to the perfusion procedure, and the second group of animals (n = 6) was not perfused. The brain tissues of the animals were homogenized, and the metabolite fraction was extracted with a water/methanol/chloroform solution. Samples were studied by high-frequency ¹H-NMR spectroscopy with subsequent statistical data analysis. The group comparison was performed with the use of the Student’s test. We identified 36 metabolites in the brain tissue with the use of NMR spectroscopy. (3) For the major set of studied metabolites, no significant differences were found in the brain tissue metabolite concentrations in the native state and after the blood removal procedure. (4) Thus, it was shown that the presence of blood does not have a significant effect on the metabolomic profile of the brain in animals without pathologies.     

Management of Parthenium hysterophorus (Asteraceae) by Withania somnifera (Solanaceae)

This study was designed to evaluate the herbicidal activity of Withania somnifera (L.) Dunal against the noxious weed parthenium (Parthenium hysterophorus L.). In a laboratory bioassay, the effect of aqueous, methanol and n-hexane shoot and root extracts of 5%, 10%, 15% and 20% w/v concentrations (on a fresh weight basis) of W. somnifera were tested against the germination and seedling growth of parthenium. In general, aqueous and methanol extracts markedly suppressed the germination, root and shoot growth of parthenium. The shoot extracts were more inhibitory than the root extracts. In a foliar spray bioassay, the aqueous and methanol shoot extracts of 10% w/v (on a dry weight basis) concentration were sprayed on 1-week and 2-week-old pot-grown parthenium seedlings. Two subsequent sprays were carried out 5 and 10 days after the first spray. The aqueous and methanol extracts significantly reduced the length and biomass of parthenium shoots. In a soil amendment bioassay, the crushed shoots of W. somnifera were incorporated in the soil at 1-5% w/w. Parthenium seeds were sown one week after the residue incorporation and plants were harvested 40 days after sowing. All the soil amendment treatments significantly reduced seed germination by 43-89%. The highest dosages of 4% and 5% significantly suppressed the root and shoot biomasses of parthenium. This study concludes that foliar spray of aqueous and methanol extracts, and soil amendment with leaf residue of W. somnifera, can control the germination and growth of parthenium, one of the world's worst weeds.     

Improvements in lipid suppression for 1H NMR-based metabolomics: Applications to solution-state and HR-MAS NMR in natural and in vivo samples

Proton nuclear magnetic resonance (NMR) spectra of intact biological samples often show strong contributions from lipids, which overlap with signals of interest from small metabolites. Pioneering work by Diserens et al. demonstrated that the relative differences in diffusivity and relaxation of lipids versus small metabolites could be exploited to suppress lipid signals, in high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. In solution-state NMR, suspended samples can exhibit very broad water signals, which are challenging to suppress. Here, improved water suppression is incorporated into the sequence, and the Carr-Purcell-Meiboom-Gill sequence (CPMG) train is replaced with a low-power adiabatic spinlock that reduces heating and spectral artefacts seen with longer CPMG filters. The result is a robust sequence that works well in both HR-MAS as well as static solution-state samples. Applications are also extended to include in vivo organisms. For solution-state NMR, samples containing significant amount of fats such as milk and hemp hearts seeds are used to demonstrate the technique. For HR-MAS, living earthworms (Eisenia fetida) and freshwater shrimp (Hyalella azteca) are used for in vivo applications. Lipid suppression techniques are essential for non-invasive NMR-based analysis of biological samples with a high-lipid content and adds to the suite of experiments advantageous for in vivo environmental metabolomics.     

The metabolic profile of lemon juice by proton HR-MAS NMR: the case of the PGI Interdonato Lemon of Messina

We have studied by means of High Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) the metabolic profile of the famous Sicilian lemon known as 'Interdonato Lemon of Messina PGI’. The PGI Interdonato Lemon of Messina possesses high organoleptic and healthy properties and is recognised as one of the most nutrient fruits. In particular, some of its constituents are actively studied for their chemo-preventive and therapeutic properties. In this paper, we have determined by means of HR-MAS NMR spectroscopy the molar concentration of the main metabolites constituent the juice of PGI Interdonato Lemon of Messina in comparison with that of the not-PGI Interdonato Lemon of Turkey. Our aim is to develop an analytical technique, in order to determine a metabolic fingerprint able to reveal commercial frauds in national and international markets.     

Metabolic Profiling of Human Colorectal Cancer Using High Resolution 1H Nuclear Magnetic Resonance Spectroscopy

Colorectal cancer (CRC) is the third commonest malignancy cancer worldwide. Clear understandings of global metabolic profiling of the normal mucosa and cancer tissues are vitally important to aid optimizing the clinical management strategy and understanding CRC biology. We studied metabolic characteristics of 20 CRC and 20 distant normal mucosa tissues extracts from 20 patients using high resolution ¹H NMR spectroscopy in conjunction with multivariate analyses, such as principal component analysis (PCA). Compared with distant normal mucosa tissues, lactate, taurine, ornithine and polyamine were present at significantly higher levels in CRC tissue extracts whereas myo‐inositol was present at significantly lower level. Two metabolites ratios such as myo‐inositol/taurine and myo‐inositol/(ornithine+polyamine) appear to be the most valuable biomarkers for the differentiation CRC from normal mucosa tissues. Our data suggested that HR ¹H NMR spectroscopy combined with multivariate analyses is a potentially useful technology for detecting malignant changes in the normal mucosa tissues, the technique may be further exploited for future CRC biomarker research or identification of targets for therapeutic manipulations.     

Identification and quantification of valuable plant substances by IR and Raman spectroscopy

This review presents various infrared and Raman spectroscopic methods applied to the analysis of valuable plant substances or quality parameters in horticultural and agricultural crops. In most cases, vibrational measurements can be performed directly on plant tissues as well as on fractions isolated from the plant material by hydro-distillation or solvent extraction. Generally, both spectroscopy techniques allow to obtain spectra which present some characteristic key bands of individual components. These bands provide information about the chemical composition, including both primary and secondary metabolites, of the investigated samples. Furthermore, based on such markers related to individual plant substances, spectroscopic analyses in principle allow the discrimination of different species, and even chemotypes among the same species. Combination of vibrational spectroscopy and hierarchical cluster analysis provides a fast, easy and reliable method for chemotaxonomy characterization. In most presented cases, determination of main plant components applying the PLS algorithm results in comparatively high R² and low SECV values. The ability to rapidly monitor various plant components makes it possible to efficiently select high-quality single plants from wild populations as well as progenies of crossing experiments. Furthermore, the vibrational spectroscopy methods can also be used by the processing industry in order to perform fast quality checks of incoming raw materials as well as continuous controlling of the production.     

Metabolomics analysis reveals variation in Schisandra chinensis metabolites from different origins

Wu Wei Zi (Schisandra chinensis), an important herbal medicine, is mainly distributed in the northeast of China. Its phytochemical compositions, which depend on geographical origin, climatic conditions and cultural practices, may vary largely among Wu Wei Zi from different areas. In this study, we applied a comprehensive metabolite profiling approach using GC–TOF‐MS, ultra‐performance LC (UPLC) quadrupole TOF (QTOF) MS and inductively coupled plasma MS to systematically investigate the metabolite variations of S. chinensis from four different areas including Heilongjiang, Liaoning, Jilin, and Shanxi of China. A total of 65 primary metabolites, 35 secondary metabolites and 64 inorganic elements were identified. Several primary metabolites, including shikimic acid and tricarboxylic acid cycle intermediates, were abundant in those located in Heilongjiang, Jilin, and Liaoning. Besides, bioactive lignans are also highly abundant in those from northeastern China than those from northwestern China. Inorganic elements varied significantly among the different locations. Our results suggested that the metabolite profiling approach using GC–TOF‐MS, ultra‐performance LC quadrupole TOF MS, and inductively coupled plasma MS is a robust and reliable method that can be effectively used to explore subtle variations among plants from different geographical locations.     

大鼠肝脏纤维化病变的核磁共振研究

利用高分辨魔角旋转核磁共振技术(HR-MAS NMR)以及主成分分析法(PCA)对大鼠肝脏组织纤维化病变前后以及不同程度病变的代谢产物变化进行了研究. 结果表明, 纤维化病变使得肝脏脂肪含量增加; 葡萄糖、糖原、牛磺酸以及肌醇的含量相对减小. 相对磷酸胆碱(PC)而言, 胆碱(Cho)及甘油磷酸胆碱(GPC)降低. 随着正常至纤维化病变程度的加深, 脂肪含量有一个先显著上升, 再缓慢下降的趋势; 而不同程度纤维化病变阶段之间其它代谢产物的变化不明显. 主成分分析证实了上述的结果, 正常与纤维化病变样本中代谢产物的组成存在较大的差异性, 可被明显分为两组; 而各纤维化病变阶段之间的差异不明显.     

Ex-vivo NMR of unprocessed tissue in water: a simplified procedure for studying intracranial neoplasms

Ex-vivo and in-vitro nuclear magnetic resonance (NMR) spectroscopy techniques have been used for studying chemical metabolites in surgically resected specimens of human neoplasms, and may provide complementary information to in-vivo whole-body magnetic-resonance spectroscopy (MRS). We describe an ex-vivo NMR in water method for measurement of water-soluble metabolites in unprocessed normal rat brain tissue and human intracranial neoplasms. The NMR spectra obtained using the method described here were comparable to those obtained using high-resolution magic-angle spinning (HRMAS) NMR methods, with good correlation in metabolite concentrations relative to creatine (r ² = 0.7635). Improved spectral resolution and baseline were noted compared to HRMAS, but macromolecule resonances were not detected. Ex-vivo NMR of unprocessed tissue in water is rapid and technically simple to perform, and has the potential to be used for direct assessment of intracranial neoplasms.     

Determination of Metabolites from Scutellaria baicalensis by GC/MS and 1H NMR

The quality of medicinal herbs and their products is steadily becoming important in tandem with growing interest in complementary alternative medicine for treatment of diseases. Chemical assays with bioactive secondary compounds are usually used for chemical standardization in herbs for quality control purpose. In this study, an analytical platform comprising of a GC-MS technique with an unsupervised multivariate analysis and a complementary one-dimensional ¹H NMR technique was used to obtain the primary metabolite profiling of Scutellaria baicalensis obtained from different medical halls in Singapore. The key primary metabolites such as sucrose, proline, phenylalanine, fructose, and butanedioic acid for the biosynthesis of bioactive secondary metabolites in this medicinal herb were successfully characterized by the combination of the chromatography and spectroscopy techniques. Their results suggested that these compounds could serve as markers for quality control of the herb. The principal component analyses of the GC-MS data reliably discriminated between the various Scutellaria samples indicating that the developed platform was comprehensive and was applicable to assess the quality of other medicinal herbs.     

Gas chromatography/flame ionisation detection mass spectrometry for the detection of endogenous urine metabolites for metabonomic studies and its use as a complementary tool to nuclear magnetic resonance spectroscopy

Metabonomics is a relatively new field of research in which the total pool of metabolites in body fluids or tissues from different patient groups is subjected to comparative analysis. Nuclear magnetic resonance (NMR) spectroscopy is the technology that is currently most widely used for the analysis of these highly complex metabolite mixtures, and hundreds of metabolites can be detected without any upfront separation. We have investigated in this study whether gas chromatography (GC) separation in combination with flame ionisation detection (FID) and mass spectrometry (MS) detection can be used for metabolite profiling from urine. We show that although GC sample preparation is much more involved than for NMR, hundreds of metabolites can reproducibly be detected and analysed by GC. We show that the data quality is sufficiently high--particularly if appropriate baseline correction and time-warping methods are applied--to allow for data comparison by chemometrics methods. A sample set of urines from eleven healthy human volunteers was analysed independently by GC and NMR, and subsequent chemometrics analysis of the two datasets showed some similar features. As judged by NIST database searches of the GC/MS data some of the major metabolites that are detected by NMR are also visible by GC/MS. Since in contrast to NMR every peak in GC corresponds to a single metabolite, the electron ionisation spectra can be used to quickly identify metabolites of interest if their reference spectra are present in a searchable database. In summary, we show that GC is a method that can be used as a complementary tool to NMR for metabolite profiling of urine samples.     

UV-B RADIATION EFFECTS ON PHOTOSYNTHESIS, GROWTH and CANNABINOID PRODUCTION OF TWO Cannabis sativa CHEMOTYPES

The effects of UV-B radiation on photosynthesis, growth and cannabinoid production of two greenhouse-grown C. sativa chemotypes (drug and fiber) were assessed. Terminal meristems of vegetative and reproductive tissues were irradiated for 40 days at a daily dose of 0, 6.7 or 13.4 kJ m^-2 biologically effective UV-B radiation. Infrared gas analysis was used to measure the physiological response of mature leaves, whereas gas-liquid chromatography was used to determine the concentration of cannabinoids in leaf and floral tissue.
There were no significant physiological or morphological differences among UV-B treatments in either drug- or fiber-type plants. The concentration of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), but not of other cannabinoids, in both leaf and floral tissues increased with UV-B dose in drug-type plants. None of the cannabinoids in fiber-type plants were affected by UV-B radiation.
The increased levels of Δ⁹-THC in leaves after irradiation may account for the physiological and morphological tolerance to UV-B radiation in the drug-type plants. However, fiber plants showed no comparable change in the level of cannabidiol (a cannabinoid with UV-B absorptive characteristics similar to Δ⁹ THC). Thus the contribution of cannabinoids as selective UV-B filters in C. sativa is equivocal.     

Use of heteronuclear multiple bond coherence NMR spectroscopy to monitor nitrogen metabolism in a transformed root culture of Datura stramonium

In order to examine the factors that regulate the flux of primary metabolites into tropane alkaloids, Nuclear Magnetic Resonance spectroscopy (NMR) has been used to monitor nitrogen metabolism in transformed root cultures of Datura stramonium fed with (¹⁵NH₄)₂SO₄ and K¹⁵NO₃. This study employs the technique of Heteronuclear Multiple Bond Coherence (HMBC) NMR spectroscopy, which combines the advantages of both bidimensional resolution and ¹H NMR sensitivity. Moreover, the HMBC sequence allows the ¹⁵N bound to labile protons to be observed via ²J and/or ³J couplings, which is very appropriate for the observation of tropane alkaloids. In transformed roots of Datura stramonium, in addition to the amino acids normally observed in monodimensional ¹⁵N NMR, other amino acids are resolved by HMBC. Labelled peaks due to N-acetyl compounds and to uridine are identified. While these primary metabolites were also seen in cell cultures of Nicotiana plumbaginifolia, cross peaks corresponding to secondary metabolites, such as tropine, were also observed. This is the first time that such secondary products have been found by this technique.     

Solid-phase enolate chemistry investigated using HR-MAS NMR spectroscopy.

Supported P4-t-Bu enolate chemistry of phenylacetyloxymethyl polystyrene (PS) resin was investigated using high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. Direct analysis of the crude reaction suspensions through the use of a diffusion filter (DF) allowed a rapid selection of the optimal experimental conditions, but also the characterization of the enolate on the solid phase. Comparison with solution experiments and literature data allowed us to address partially the structure of the enolate. HR-MAS NMR spectra of the enolate revealed also a tight interaction of P4-t-Bu base with the polymer matrix.     

Structure determination of oligomeric alkannin and shikonin derivatives

Monomeric alkannin and shikonin (A/S) are potent pharmaceutical substances with a wide spectrum of biological activity and comprise the active ingredients for several pharmaceutical preparations. Therefore, the determination of the impurities, degradation products or byproducts in alkannin and shikonin samples is of great importance. Oligomeric alkannin and shikonin are formed during biosynthesis of these bioactive secondary metabolites in Boraginaceaous root plants, during tissue culture production of A/S, during alkaline hydrolysis of A/S esters and also thermal treatment of A/S. In the present study, a dimeric alkannin/shikonin compound was isolated by size exclusion chromatography from alkannin and shikonin commercial samples and its structure was determined by one- and two-dimensional NMR spectroscopy. The structure of the most abundant oligomeric species in these samples, a dimeric naphthoquinone, was established for the fi rst time, indicating that coupling of the side chain of one naphthoquinone unit with the aromatic ring of a second naphthoquinone leads to dimer formation. This type of coupling allows further oligomerization by leaving one isohexenyl side chain available at the second monomer unit.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry monitoring of anthocyanins in extracts from Arabidopsis thaliana leaves

Anthocyanins are secondary plant metabolites ubiquitous in the plant kingdom. They have different biological activities, so monitoring their content in plant tissue or in feed/food derived from plants may be an important task in different projects from various fields of molecular biology and biotechnology. Profiling of secondary metabolites with high-performance liquid chromatography/mass spectrometry (HPLC/MS) systems is time-consuming, especially when many samples have to be checked within a defined time frame with a reasonable number of repetitions according to the metabolomic standards. Even application of the advanced ultra-performance liquid chromatography (UPLC)/MS or equivalent systems would require a long time for analysis of numerous samples. We demonstrate the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the assessment of level (concentration) of anthocyanins in leaf tissues of four Arabidopsis thaliana ecotypes grown at normal (20 degrees C/16 degrees C day/night) and decreased (4 degrees C) temperature. The quantitative results were obtained for anthocyanins with MALDI-TOF MS using ferulic acid as a matrix. The amounts of anthocyanins in leaves of A. thaliana varied from 0.3-2.5 microg per gram of leaves for ecotypes Col-0 and C24, respectively, and contents of these markedly increased in plants grown in the cold. The applied analytical method exhibited better repeatability of measurements than obtained with an HPLC/ion trap MS system.