Hyung-Kyoon Choi

NMR-based metabolic profiling and differentiation of ginseng roots according to cultivation ages

Ginseng is an important herbal resource worldwide, and the adulteration or falsification of cultivation age has been a serious problem in the commercial market. In this study, ginseng (Panax ginseng) roots, which were cultivated for 2-6 years under GAP standard guidelines, were analyzed by NMR-based metabolomic techniques using two solvents. At first, ginseng root samples were extracted with 50% methanol, and analyzed by NMR with D(2)O as the NMR dissolution solvent. The 2-, 3-, 4-, and 5/6-year-old ginseng root samples were separated in PLS-DA-derived score plots. However, 5- and 6-year-old ginseng roots were not separated by the solvent system. Therefore, various solvents were tested to differentiate the 5- and 6-year-old ginseng root samples, and 100% methanol-d(4) was chosen as the direct extraction and NMR dissolution solvent. In the PLS model using data from the 100% methanol-d(4) solvent, 5- and 6-year-old ginseng roots were clearly separated, and the model was validated using internal and external data sets. The obtained RMSEE and RMSEP values suggested that the PLS model has strong predictability for discriminating the age of 5- and 6-years-old ginseng roots. The present study suggests that the age of ginseng could be successfully predicted using two solvents, and the developed method in this study can be used as a standard protocol for discriminating and predicting the ages of ginseng root samples.

1H-nuclear magnetic resonance spectroscopy-based metabolic assessment in a rat model of obesity induced by a high-fat diet

AbstractObesity, whose prevalence is increasing rapidly worldwide, is recognized as a risk factor for diabetes, cardiovascular disease, liver disease, and renal disease. To investigate metabolic changes in the urine of a rat model of obesity induced by a high-fat diet (HFD), rats were divided into the following four groups based on the diet type and degree of weight gain: normal-diet (ND) low gainers, ND high gainers, HFD low gainers, and HFD high gainers. Biochemical analyses of visceral fat-pad weight, plasma, and liver tissues were performed. The 1H-nuclear magnetic resonance (1H-NMR) spectra of urine were analyzed using multivariate statistical analysis to identify the separation of the groups. It was observed that the metabolic profile of urine obtained by 1H-NMR-spectroscopy-based metabolomic analysis differed between ND low gainers and ND high gainers even though these animals consumed the same normal diet. Several key metabolites in urine, such as betaine, taurine, acetone/acetoacetate, phenylacetylglycine, pyruvate, lactate, and citrate contributed to the classification of these two groups. The metabolic profile of urine also differed between ND low gainers and HFD high gainers, which consumed the different diet and showed a different weight gain. This study has identified features of urine metabolites in various groups and demonstrated the reliability of an NMR-based metabolomics approach to investigate the effects of the diet and the physical constitution on obesity. FigureIntensity of the metabolites (normalized relative to the creatinine intensity) for ND low gainers vs. HFD high gainers. An independent t test was performed to assess the statistical significance between each group. The error bars are expressed as the SEM *P<0.025 vs. each group

Metabolomic Approach for Age Discrimination of Panax ginseng Using UPLC-Q-Tof MS

An ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-Tof MS)-based metabolomic technique was applied for metabolite profiling of 60 Panax ginseng samples aged from 1 to 6 years. Multivariate statistical methods such as principal component analysis and hierarchical clustering analysis were used to compare the derived patterns among the samples. The data set was subsequently applied to various metabolite selection methods for sophisticated classification with the optimal number of metabolites. The results showed variations in accuracy among the classification methods for the samples of different ages, especially for those aged 4, 5, and 6 years. This proposed analytical method coupled with multivariate analysis is fast, accurate, and reliable for discriminating the cultivation ages of P. ginseng samples and is a potential tool to standardize quality control in the P. ginseng industry.

Metabolite profiling of doenjang, fermented soybean paste, during fermentation

BACKGROUND A fermented soybean paste known as doenjang is a traditional fermented food that is widely consumed in Korea. The quality of doenjang varies considerably by its basic ingredients, species of microflora, and fermentation process. The classification of predefined metabolites (e.g. amino acids, organic acids, sugars and sugar derivatives, and fatty acids) in doenjang samples according to fermentation was performed by using GC-FID and GC-MS data sets with the application of a multivariate statistical method. RESULTS The predominantly produced amino acids included alanine, valine, leucine, isoleucine, proline, glutamine, phenylalanine and lysine, showing remarkable increases in amounts during the later stages of fermentation. Carbonic acid, citric acid, lactic acid and pyrogultamic acid were identified as the major organic acids. Significant amounts of erythrose, xylitol, inositol and mannitol were detected during fermentation. Regarding fatty acids, relatively higher amounts of palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid were found in the doenjang at each fermentation time point. Principal component analysis (PCA) successfully demonstrated changes in composition patterns as well as differences in non-volatile metabolites according to fermentation period. CONCLUSION A set of metabolites could be determined representing the quality of doenjang during fermentation, and which might also be correlated with taste ingredients, flavour, nutrition, and physiology activities that are claimed to be dependent on the quality control of commercial doenjang.

Henriciella litoralis sp. nov., isolated from a tidal flat, transfer of Maribaculum marinum Lai et al. 2009 to the genus Henriciella as Henriciella aquimarina nom. nov. and emended description of the genus Henriciella.

A Gram-staining-negative, strictly aerobic bacterium, designated strain SD10(T), was isolated from a tidal flat of the Yellow Sea, South Korea. Cells were non-spore-forming rods that showed catalase- and oxidase-positive reactions. Growth of strain SD10(T) was observed at 15-40 °C (optimum, 25-30 °C), at pH 6.0-9.0 (optimum, pH 6.5-8.5) and in the presence of 1-10 % (w/v) NaCl. Strain SD10(T) contained ubiquinone-10 (Q-10) as a major isoprenoid quinone and C(18 : 1)ω7c (39.3 %), C(16 : 0) (20.2 %), C(17 : 0) (8.9 %) and C(17 : 1)ω6c (8.1 %) as major fatty acids. The cellular polar lipids were identified as phosphatidylglycerol, monoglycosyldiglyceride, glucuronopyranosyldiglyceride and two unidentified glycolipids. The G+C content of the genomic DNA was 55.2 mol%. Based on 16S rRNA gene sequence similarities, the strain was most closely related to Henriciella marina Iso4(T) and Maribaculum marinum P38(T), with similarities of 97.8 and 97.0 %, respectively. The DNA-DNA relatedness between strain SD10(T) and H. marina Iso4(T) was 12.0±3.2 %. A phylogenetic analysis based on 16S rRNA gene sequences showed that M. marinum P38(T) and H. marina Iso4(T) formed a monophyletic cluster and that their 16S rRNA gene sequence similarity was 98.1 %. DNA-DNA hybridization between H. marina Iso4(T) and M. marinum LMG 24711(T) was 22.9±2.7 %, indicating that the two strains belong to separate species. On the basis of chemotaxonomic data and molecular properties, we propose that strain SD10(T) represents a novel species of the genus Henriciella, for which the name Henriciella litoralis sp. nov. is proposed. The type strain is SD10(T) ( = KACC 13700(T)  = DSM 22014(T)). In addition, we propose to transfer Maribaculum marinum Lai et al. 2009 to the genus Henriciella as Henriciella aquimarina nom. nov. (type strain P38(T)  = CCTCC AB 208227(T)  = LMG 24711(T)  = MCCC 1A01086(T)), and we present an emended description of the genus Henriciella.

Effect of osmotic pressure on paclitaxel production in suspension cell cultures of Taxus chinensis

The effect of osmotic pressure on paclitaxel production was investigated in the suspension cell cultures of Taxus chinensis. Paclitaxel production was definitely influenced by the initial sucrose concentration and the highest production yield was achieved at the concentration of 60 g.l(-1) sucrose (300 mOsm.kg(-1)). High osmotic pressure conditions generated by non-metabolic sugar (mannitol and sorbitol) also enhanced paclitaxel production by about two-fold. Kinetic studies revealed that high initial osmotic pressure enhanced paclitaxel production and that high concentration of sucrose was effective for sustaining secondary metabolism after induction of paclitaxel biosynthesis. Stoichiometric analysis with different combinations of sucrose and mannitol confirmed that osmotic pressure was the more important factor for enhancing paclitaxel metabolism. The addition of non-sugar osmotic agent, PEG also enhanced paclitaxel production. In this paper, we showed that high osmotic pressure led to increases in paclitaxel production and proposed that regulation of osmotic pressure may be useful in controlling paclitaxel production.

Metabolite Profiling of Cheonggukjang, a Fermented Soybean Paste, Inoculated with Various Bacillus Strains during Fermentation

Metabolite profiling of Cheonggukjang inoculated with different Bacillus strains including Bacillus amyloliqueciens CH86-1, Bacillus licheniformis 58, and Bacillus licheniformis 67 during fermentation, was performed using gas chromatography-time of flight-mass spectrometry after derivatization, combined with multivariate statistical analysis. A total of 20 amino acids, 10 sugars, five sugar alcohols, and seven organic acids were identified in three Cheonggukjang samples. With fermentation time, most of the amino acids showed increasing amounts. On the other hand, most of the sugars including sucrose, fructose, and glucose decreasing patterns, and the amounts of organic acids varied. In order to observe differences in metabolites with fermentation time and inoculated Bacillus strains, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were carried out, respectively. On PCA plots, some sugars and organic acids including sucrose, fructose, glucose, mannose, succinic acid, and malonic acid, as well as most of the amino acids, contributed mainly to differentiation of the Cheonggukjang samples fermentation time. On the other hand, on PLS-DA, mannose, xylose, glutamic acid, and proline were mainly responsible for differentiating the Cheonggukjang among into various inoculated strains.

Fingerprinting analysis of fresh ginseng roots of different ages using 1H-NMR spectroscopy and principal components analysis

Fingerprinting analysis of fresh ginseng according to root age was performed using1H-NMR spectroscopy and multivariate analysis techniques. Various peaks were detected in the aliphatic (0–3 ppm), sugar (3–6 ppm), and aromatic (6–9 ppm) regions of the1H-NMR spectra of the water extracts of fresh ginseng root. The use of principal components (PCs) analysis (PCA) for metabolomic profiling allowed the large1H-NMR data set obtained for various metabolites to be reduced to PC1, PC2, and PC3. Two dimensional score plots showed clear separations with these three components at different roots ages, and explained 89.6% of the total variance. Canonical discriminant analysis identified the ginseng roots at various ages from the NMR results with over 89.9% discrimination accuracy. These results indicate that the combination of1H-NMR and PCA provides a very promising tool for the authentication and quality control of fresh ginseng roots at different ages.

Mass Production of Paclitaxel by Plant Cell Culture

Samyang Genex succeeded in commercialization of anticancer agent-paclitaxel by plant cell culture technology. The core technology of Samyang Genex relating paclitaxel production includes cell line development, cell line preservation, cell culture, scale-up technology, and purification technology. On the basis of the research, Samyang Genex built the factory operated by CGMP (current good manufacturing practice). The paclitaxel-Genexol-is commercially available in Korea, and it will be launched to world market including USA after approval of US FDA.

Correlation between Antioxidative Activities and Metabolite Changes during Cheonggukjang Fermentation

Liquid chromatography mass spectrometry and multivariate analysis were employed to investigate the correlation between fermentation time-dependent metabolite changes in cheonggukjang, a traditional fermented soybean product, and changes in its antioxidant activity over 72 h. The metabolite patterns were clearly distinguished not by strains but by fermentation time, into patterns I (0–12 h), II (12–24 h), and III (24–72 h), which appeared as distinct clusters on principal component analysis. The compounds that significantly contributed to patterns I, II, and III were soyasaponins, isoflavonoid derivatives, and isoflavonoid aglycons respectively. Partial least square analysis for metabolite to antioxidant effects showed correlations between the ferric reducing/antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay during 24–36 h, and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) test and total phenol content (TPC) during 36–72 h. Compared with the strong negative correlations of glucosylated-isoflavonoids with DPPH, ABTS and TPC during fermentation, the isoflavonoid aglycon displayed strong positive correlations with these compounds during fermentation.