Sun-Hee Hyun

1H-NMR-Based Discrimination of Thermal and Vinegar Treated Ginseng Roots

To investigate the changes in nonvolatile metabolites of thermal and/or vinegar treated ginseng (TVG), samples prepared using various treatment conditions were analyzed using an (1)H-NMR-based metabolomics technique. The processing conditions of the ginseng in this study were 100, 140, and 180 degrees C with and without vinegar and the duration of exposure to each temperature was 10, 30, and 50 min, respectively. There was a clear separation in the score plots among various treatment conditions. Major compounds contributing to the separation of 50% methanol extracts of TVG with various process conditions were valine, lactate, alanine, arginine, glucose, fructose, and sucrose. As temperature increased, valine, arginine, glucose, fructose, and sucrose concentrations decreased, whereas lactate, glucose, and fructose increased in the vinegar-treated samples compared to non-vinegar-treated samples. The present study suggests the usefulness of an (1)H-NMR-based metabolomics approach to discriminate TVG samples, subjected to different processing conditions.

Metabolic Profiling and Predicting the Free Radical Scavenging Activity of Guava (Psidium guajava L.) Leaves According to Harvest Time by 1H-Nuclear Magnetic Resonance Spectroscopy

Guava leaves were classified and the free radical scavenging activity (FRSA) evaluated according to different harvest times by using the 1H-NMR-based metabolomic technique. A principal component analysis (PCA) of 1H-NMR data from the guava leaves provided clear clusters according to the harvesting time. A partial least squares (PLS) analysis indicated a correlation between the metabolic profile and FRSA. FRSA levels of the guava leaves harvested during May and August were high, and those leaves contained higher amounts of 3-hydroxybutyric acid, acetic acid, glutamic acid, asparagine, citric acid, malonic acid, trans-aconitic acid, ascorbic acid, maleic acid, cis-aconitic acid, epicatechin, protocatechuic acid, and xanthine than the leaves harvested during October and December. Epicatechin and protocatechuic acid among those compounds seem to have enhanced FRSA of the guava leaf samples harvested in May and August. A PLS regression model was established to predict guava leaf FRSA at different harvesting times by using a 1H-NMR data set. The predictability of the PLS model was then tested by internal and external validation. The results of this study indicate that 1H-NMR-based metabolomic data could usefully characterize guava leaves according to their time of harvesting.

Rapid sequential separation of essential oil compounds using continuous heart-cut multi-dimensional gas chromatography-mass spectrometry.

A method for separation and identification of peaks in essential oil samples based on rapid repetitive heart-cutting using multidimensional gas chromatography (MDGC)-mass spectrometry (MS) coupled with a cryotrapping interface is described. Lavender essential oil is analyzed by employing repetitive heart-cut intervals of 1.00 and 1.50 min, achieved in a parallel MDGC-MS/GC-FID experiment. The number of peaks that were detected in 1D GC operation above a given response threshold more than tripled when MDGC-MS employing the cryotrapping module method was used. In addition, MDGC-MS enabled detection of peaks that were not individually evident in 1D GC-MS, owing to effective deconvolution in time of previously overlapped peaks in 1D GC. Thus separation using the cryomodulation approach, without recourse to using deconvolution software, was possible. Peaks widths decreased by about 5-7-fold with the described method, peak capacity increased from about 9 per min to 60 per min, and greater sensitivity results. Repeatability of retention times for replicate analyses in the multidimensional mode was better than 0.02% RSD. The present study suggests that the described heart-cutting technique using MDGC-MS can be used for general improvement in separation and identification of volatile compounds.

Biochemical characterization of cultivated Cordyceps bassiana mycelia and fruiting bodies by 1 H nuclear magnetic resonance spectroscopy

In this study, nuclear magnetic resonance techniques coupled with multivariate data analysis were used for the metabolic profiling of mycelia and fruiting bodies of the entomopathogenic fungi, Cordyceps bassiana according to developmental stages. A direct extraction method using two deuterated solvents of D2O and CDCl3 was used to investigate the relative levels of identified metabolites in each extraction condition in the mycelium and fruiting body formation stages. There was a clear separation among mycelia and fruiting bodies with various developmental stages in partial least-squares discriminant analysis (PLS-DA) derived score plots. During the transition from mycelia to fruiting bodies, the major metabolic change observed was the conversion of glucose to mannitol, and beauvericin to phenylalanine and 1-hydroxyisovaleric acid. In the developmental stages of fruiting bodies studied, there was a clear separation between stage 3 and the other stages in PLS-DA derived score plots. Nineteen compounds including 13 amino acids, 2 nucleosides, 3 organic acids, and glucose showed the highest levels in stage 3 fruiting bodies. The flavonoid content in the fruiting bodies showed similar levels during stages 1, 2, and 3, whereas the level at stage 4 was significantly decreased compared to the other stages. Results suggest that the fruiting body of C. bassiana is richer in natural resources at stage 3 compared to the other fruiting body stages due to its high abundance of compounds including total flavonoids. The metabolome information acquired in this study can be useful criteria for the quality control of commercial use of C. bassiana.

Simple preparative gas chromatographic method for isolation of menthol and menthone from peppermint oil, with quantitative GC‐MS and 1H NMR assay

The quantitative performance of a simple home-built preparative gas chromatography (prep-GC) arrangement was tested, incorporating a micro-fluidic Deans switch, with collection of the target compound in a deactivated uncoated capillary tube. Repeat injections of a standard solution and peppermint sample were made into the prep-GC instrument. Individual compounds were eluted from the trapping capillary, and made up to constant volume. Chloronaphthalene internal standard was added in some cases. Recovered samples were quantitatively assayed by using GC-MS. Calibration linearity of GC-MS for menthol standard area response against number of injections (2-20 repeat injections) was excellent, giving R(2) of 0.996. For peppermint, menthol correlation over 2-20 repeated injections was 0.998 for menthol area ratio (versus IS) data. Menthone calibration for peppermint gave an R(2) of 0.972. (1) H NMR spectroscopy was conducted on both menthol and menthone. Good correspondence with reference spectra was obtained. About 80 μg of isolated menthol and menthone solute was collected over a sequence of 80 repeat injections from the peppermint sample, as assayed by 600 MHz (1) H NMR analysis (∼100% recovery for menthol from peppermint). A procedure is proposed for prediction of number of injections required to acquire sufficient material for NMR detection.

NMR and GC-MS Based Metabolic Profiling and Free-Radical Scavenging Activities of Cordyceps pruinosa Mycelia Cultivated under Different Media and Light Conditions

Variation of metabolic profiles in Cordyceps pruinosa mycelia cultivated under various media and light conditions was investigated using 1H nuclear magnetic resonance (NMR) analysis and gas chromatography mass spectrometry (GC-MS) with multivariate statistical analysis. A total of 71 metabolites were identified (5 alcohols, 21 amino acids, 15 organic acids, 4 purines, 3 pyrimidines, 7 sugars, 11 fatty acids, and 5 other metabolites) by NMR and GC-MS analysis. The mycelia grown in nitrogen media and under dark conditions showed the lowest growth and ergosterol levels, essential to a functional fungal cell membrane; these mycelia, however, had the highest levels of putrescine, which is involved in abiotic stress tolerance. In contrast, mycelia cultivated in sabouraud dextrose agar with yeast extract (SDAY) media and under light conditions contained relatively higher levels of fatty acids, including valeric acid, stearic acid, lignoceric acid, myristic acid, oleic acid, palmitoleic acid, hepadecenoic acid, and linoleic acid. These mycelia also had the highest phenolic content and antioxidant activity, and did not exhibit growth retardation due to enhanced asexual development caused by higher levels of linoleic acid. Therefore, we suggested that a light-enriched environment with SDAY media was more optimal than dark condition for cultivation of C. pruinosa mycelia as biopharmaceutical or nutraceutical resources.

Hypoxanthine levels in human urine serve as a screening indicator for the plasma total cholesterol and low-density lipoprotein modulation activities of fermented red pepper paste

Fermented red pepper paste (FRPP) is one of the most well-known traditional foods in Korea. The effects of FRPP in experimental animals and adipocytes have been previously reported. However, the biochemical effects have not yet been validated in humans with various genetic backgrounds and environmental factors. In this study, 28 female volunteers (body mass index, more than 23 kg/m(2)) aged 19 to 60 years were treated with either FRPP or a placebo for 12 weeks. Marked cholesterol modulation was observed in the FRPP-treated group compared with the placebo group. Although the baseline (pretreatment) total cholesterol and low-density lipoprotein levels and body mass index of the volunteers did not differ significantly between the placebo- and FRPP-treated groups, FRPP caused a modulation of cholesterol levels not seen in the placebo group, causing either no variation or a decrease in low-density lipoprotein and total cholesterol levels. Thus, urinary metabolomic profiling of pretreatment samples was carried out in these 2 FRPP-treated groups using (1)H-nuclear magnetic resonance-based metabolomic techniques. These 2 groups, with their opposing cholesterol-modulation tendencies, could be clearly differentiated by orthogonal projections to latent structures-discriminant analysis-derived score plots. In addition, their levels of hypoxanthine differed markedly. We propose that urinary hypoxanthine levels can be used as a screening biomarker to predict the efficacy of the cholesterol-modulating activity of FRPP in human subjects.

Metabolic Profiles and Free Radical Scavenging Activity of Cordyceps bassiana Fruiting Bodies According to Developmental Stage

The metabolic profiles of Cordyceps bassiana according to fruiting body developmental stage were investigated using gas chromatography-mass spectrometry. We were able to detect 62 metabolites, including 48 metabolites from 70% methanol extracts and 14 metabolites from 100% n-hexane extracts. These metabolites were classified as alcohols, amino acids, organic acids, phosphoric acids, purine nucleosides and bases, sugars, saturated fatty acids, unsaturated fatty acids, or fatty amides. Significant changes in metabolite levels were found according to developmental stage. Relative levels of amino acids, purine nucleosides, and sugars were higher in development stage 3 than in the other stages. Among the amino acids, valine, isoleucine, lysine, histidine, glutamine, and aspartic acid, which are associated with ABC transporters and aminoacyl-tRNA biosynthesis, also showed higher levels in stage 3 samples. The free radical scavenging activities, which were significantly higher in stage 3 than in the other stages, showed a positive correlation with purine nucleoside metabolites such as adenosine, guanosine, and inosine. These results not only show metabolic profiles, but also suggest the metabolic pathways associated with fruiting body development stages in cultivated C. bassiana.

Ameliorating effects of Mango (Mangifera indica L.) fruit on plasma ethanol level in a mouse model assessed with 1H-NMR based metabolic profiling

The ameliorating effects of Mango (Mangifera indica L.) flesh and peel samples on plasma ethanol level were investigated using a mouse model. Mango fruit samples remarkably decreased mouse plasma ethanol levels and increased the activities of alcohol dehydrogenase and acetaldehyde dehydrogenase. The 1H-NMR-based metabolomic technique was employed to investigate the differences in metabolic profiles of mango fruits, and mouse plasma samples fed with mango fruit samples. The partial least squares-discriminate analysis of 1H-NMR spectral data of mouse plasma demonstrated that there were clear separations among plasma samples from mice fed with buffer, mango flesh and peel. A loading plot demonstrated that metabolites from mango fruit, such as fructose and aspartate, might stimulate alcohol degradation enzymes. This study suggests that mango flesh and peel could be used as resources for functional foods intended to decrease plasma ethanol level after ethanol uptake.

Physiological and metabolomic analysis of a knockout mutant suggests a critical role of MtP5CS3 gene in osmotic stress tolerance of Medicago truncatula

In the model legume Medicago truncatula, Δ1-pyrroline-5-carboxylate synthetase (P5CS), the rate-limiting enzyme of proline biosynthesis, is encoded by three closely related genes, MtP5CS1, MtP5CS2, and MtP5CS3. While MtP5CS1 is constitutively expressed, MtP5CS2 and MtP5CS3 are induced by adverse environmental conditions, of which MtP5CS3 is prevalently expressed during drought and salinity stresses. Mtp5cs3, a transposon (Tnt1) insertion mutant of MtP5CS3 that cannot synthesize a mature protein, showed decreased proline accumulation and increased sensitivity to salinity, drought, and low water potential stresses, as evidenced by decreased seedling growth and chlorophyll content and increased hydrogen peroxide content. These defective phenotypes were complemented by externally supplied proline or ectopically expressed cDNA to the wild-type gene (MtP5CS3). Gas chromatography–mass spectrometry-based analysis of soluble metabolites revealed that some major metabolites contributing to osmotolerance, including certain amino acids, sugars, and polyols, accumulated more abundantly in the Mtp5cs3 roots than in the wild type, whereas a few other amino acids accumulated less during drought and salinity stresses. While such metabolic reconfiguration apparently fell short of compensating for proline deficiency in Mtp5cs3, overexpression of MtP5CS3 significantly increased tolerance of M. truncatula to salinity and low water potential stress. Thus, MtP5CS3 plays a crucial role in proline accumulation and osmotic stress tolerance of M. truncatula. Manipulation of this predominant proline biosynthetic gene will facilitate the development of environmentally stable legume crops.