Prediction of the Adult T-Cell Leukemia Inhibitory Activity of Blueberry Leaves/Stems Using Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics.

Although Vaccinium virgatum Aiton leaves and stems inhibit adult T-cell leukemia (ATL) cells, leaves and stems can differ between individual plants and by time and location. In this study, leaf and stem components were profiled in the same individual plant using direct-injection electron ionization-mass spectrometry (DI-EI-MS) metabolomics, with the aims of analyzing the anti-ATL activity, and quantifying proanthocyanidins (PACs). Leaves, stems, and leaf/stem mixtures showed distinct and characteristic spectra. Anti-ATL activity was stronger in stems than leaves, and the PAC content was higher in stems than leaves. These data were subjected to bivariate analysis to identify the factor (m/z) responsible for the inhibitory effect of ATL based on the highest coefficient of determination (R2). The results of this DI-EI-MS metabolomics analysis suggest that among PACs contained in V. virgatum stems and leaves, the fragment ion at m/z 149 contributes significantly to anti-ATL activity.

Use of 13C-NMR Chemical Shifts; Application of Principal Component Analysis for Categorizing Structurally Similar Methoxyflavones and Correlation Analysis between Chemical Shifts and Cytotoxicity.

The 13C-NMR spectral data for the 15-carbon flavonoid skeleton in eleven methoxyflavones isolated from Kaempferia parviflora (Zingiberaceae) were processed by principal component analysis (PCA). Based on the PCA score plots, the methoxyflavones were categorized into three groups according to their structural features. The cytotoxicities of the methoxyflavones toward 3T3-L1 murine preadipocyte cells were evaluated by 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and found to differ according to structure. The relationship between the 13C-NMR chemical shifts of the methoxyflavones and their cytotoxicities was investigated using Pearson's correlation analysis. The 13C-NMR signal at C-10, a quaternary carbon, was correlated with cytotoxicity. Based on these results, a structural design which lowers the 13C-NMR chemical shift at C-10 would be important for the development of cytotoxic compounds. Although quantitative structure-activity and structure-property relationships are well established paradigms for predicting trends among a series of compounds, quantitative property-activity relationships have been relatively unstudied. This approach offers a new strategy for directing structure-activity relationship research.

Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics Method for Analyzing Blueberry Leaf Metabolites That Inhibit Adult T-cell Leukemia Proliferation.

Metabolic profiling is often used to identify possible correlations between a compound's metabolic profile and biological activity. Direct-injection electron ionization-mass spectrometry "fingerprinting" is useful for characterizing biological materials. We demonstrate the utility of direct-injection electron ionization-mass spectrometry for metabolic profiling using 100 different extracts of leaves from 20 blueberry cultivars collected at 5 time points from April to December 2008. A qualitative direct-injection electron ionization-mass spectrometry method was used to profile the major and/or minor constituents in the blueberry leaf extracts. Blueberry leaf extracts could be distinguished by principal component analysis based on the absolute intensity of characteristic fragment ions. Twenty cultivars were categorized into four species, and the most appropriate discriminative marker m/z value for identifying each cultivar was selected statistically. Correlated m/z values indicating the collection month were determined in the same analysis, and air temperature variance factors were extracted from score plots by principal component analysis. We previously reported that blueberry extracts inhibit the proliferation of adult T-cell leukemia cells. Leaves of Vaccinium virgatum collected in December of 2008 exhibited significantly greater inhibition of adult T-cell leukemia cell proliferation than other species. Highly bioactive cultivars or species were identified by direct-injection electron ionization-mass spectrometry metabolomics analysis of blueberry leaf extracts. The components extracted based on our direct-injection electron ionization-mass spectrometry analyses could be used to construct a model to predict anti-adult T-cell leukemia bioactivity. This is the first study to report a relationship between seasonal variation and bioactivity of natural products using a direct-injection electron ionization-mass spectrometry metabolomics method.

Establishment of a Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics Method and Its Application to Lichen Profiling.

Direct-injection electron ionization-mass spectrometry (DI-EI-MS) is a multivariate analysis method useful for characterizing biological materials. We demonstrated the use of DI-EI-MS for metabolic profiling using several closely related lichen species: Cladonia krempelhuberi, C. gracilis, C. pseudogymnopoda, and C. ramulosa. The methodology involves conversion of total ion chromatograms to integrated chromatograms and assessment of reproducibility. The qualitative DI-EI-MS method was used to profile the major and/or minor constituents in extracts of lichen samples. It was possible to distinguish each lichen sample by altering the electron energy in DI-EI-MS and examining the resulting data using one-way analysis of variance. Previously undetectable peaks, which are easy to fragment could be revealed by varying the electron energy. Our results suggest that metabolic profiling using DI-EI-MS would be useful for discriminating between subgroups within the same species. This is the first study to report the use of DI-EI-MS in a metabolomics application.

Application of Mixture Analysis to Crude Materials from Natural Resources (V) [1]: Discrimination of Glycyrrhiza uralensis and G. glabra by El mass spectrometry.

The roots and stolons of some Glycyrrhiza species are used worldwide for traditional folk medicines and commercial pharmaceuticals. Phenolic constituents such as flavonoids and coumarins are medicinal and vary according to species. Therefore, species identification is important for quality analysis. In order to identify Glycyrrhiza species by chemical fingerprinting, methanol extracts of the root bark of Glycyrrhiza uralensis Fischer and G. glabra Linn6 were analyzed using EI-MS. Differences in kinds and quantity of components are reflected in complex EI-MS data and determining characteristic peaks for each species is straightforward.. The chaiacteristic peaks were determined statistically by volcano plot, a multivariate analysis method. EI-MS data of G. uralensis and G. glabra showed differential patterns, and the notable peaks in each pattern were identified. Peaks at m/z 153 and 221 are signature peaks of G. uralensis, and at 11/z 173, 309, and 324 are those of G. glabra. In conclusion, we found species-specific patterns by EI-MS that distinguish G. uralensis and G. glabra. This method based on chemical constituent patterns can be applied to identify other Glycyrrhiza species and similar natural products.

Electron Ionization Mass Spectrometry-based Metabolomics Studies of Sophoraflavescens can Identify the Geographical Origin of Root Samples.

An electron ionization mass spectrometry (EI-MS)-based metabolomic approach was applied to Sophora flavescens to identify the geographical origin of each sample. The score plot from principal component analysis using the EI-MS data showed that Japanese S. flavescens samples tended to cluster away from Chinese S. flavescens samples. Statistical techniques showed that ions arising from kurarinol and kushenol H, which we previously identified as marker molecules for Japanese S. flavescens, were characteristic of Japanese S. flavescens. Therefore, metabolomics based on EI-MS data is a valuable tool for confirming the geographical origins of S. flavescens samples. The results suggest that EI-MS-based metabolomics is suitable for the quality control of traditional medicines containing many components.

Identification of the country of growth of Sophora flavescens using direct analysis in real time mass spectrometry (DART-MS).

In order to identify the country of growth of Sophora flavescens by chemical fingerprinting, extracts of plants grown in China and Japan were analyzed using direct analysis in real time mass spectrometry (DART)-MS. The peaks characteristic of each country of growth were statistically analyzed using a volcano plot to summarize the relationship between the p-values of a statistical test and the magnitude of the difference in the peak intensities of the samples in the groups. Peaks with ap value < 0.05 in the t-test and a ≥ 2 absolute difference were defined as characteristic. Peaks characteristic of Chinese S. flavescens were found at m/z 439 and 440. In contrast, peaks characteristic of Japanese S. flavescens were found at m/z 313, 423, 437 and 441. The intensity of the selected peaks was similar in Japanese samples, whereas the m/z 439 peak had a significantly higher intensity than the other peaks in Chinese samples. Therefore, differences in selected peak patterns may allow identification of the country of growth of S. flavescens.

Extracts and compounds with anti-diabetic complications and anti-cancer activity from Castanea mollissina Blume (Chinese chestnut).

BACKGROUND: Castanea mollissima Blume (Chinese chestnut), as a food product is known for its various nutrients and functional values to the human health. The present study was carried out to analyze the anti-diabetic complications and anti-cancer activities of the bioactive compounds present in C. mollissima. METHODS: The kernels (CK), shells (CS) and involucres (CI) parts of C. Blume were extracted with 90% alcohol. The water suspension of these dried alcohol extracts were extracted using EtOAc and n-BuOH successively. The n-BuOH fraction of CI (CI-B) was isolated by silica gel column, Sephadex LH 20 column and preparative HPLC. The isolated compounds were identified by 1H-NMR, 13C-NMR, HMBC, HMQC and ESI-Q-TOF MS, All the fractions and compounds isolated were evaluated on human recombinant aldose reductase (HR-AR) assay, advanced glycation end products (AGEs) formation assay and human COLO 320 DM colon cancer cells inhibitory assay. RESULTS: CI-B was found to show a significant inhibitory effect in above biological screenings. Six flavonoids and three polyphenolic acids were obtained from CI-B. They were identified as kaempferol (1), kaempferol-3-O-[6''-O-(E)-p-coumaroyl]-ß-D-glucopyranoside (2), kaempferol-3-O-[6''-O-(E)-p-coumaroyl]-ß-D-galactopyranoside (3), kaempferol-3-O-[2''-O-(E)-p-coumaroyl]-ß-D-glucopyranoside (4), kaempferol-3-O-[2", 6"-di-O-(E)-p-coumaroyl]-ß-D-glucopyranoside (5) and kaempferol-3-O-[2", 6"-di-O-(E)-p-coumaroyl]-ß-D-galactopyranoside (6), casuariin (7), casuarinin (8) and castalagin (9). Compounds 2-9 were found to show higher activity than quercetin (positive control) in the AR assay. Compounds 3-6, 8, and 9 showed stronger inhibitory effects than amino guanidine (positive control) on AGEs production. Compounds 4-6, 7, and 8 showed much higher cytotoxic activity than 5-fluorouracil (positive control) against the human COLO 320 DM colon cancer cells. CONCLUSIONS: Our results suggest that flavonoids and polyphenolic acids possesses anti-diabetes complications and anti-cancer properties, and they were presumed to be the bioactive components of Castanea mollissima Blume.

Search of new cytotoxic crude materials against human oral squamous cell carcinoma using 1H NMR-based metabolomics.

BACKGROUND: The 5-year survival rate of the oral cancer patients has remained at approximately the 50% level during the past 30 years, possibly due to the poor tumor-selectivity of conventional anticancer drugs. This prompted us to search new plant extracts that have higher cytotoxicity against cancer cells than normal cells. MATERIALS AND METHODS: Two human oral squamous cell carcinoma cell lines (HSC-2 and HSC-4) and two normal oral cells (gingival and periodontal ligament fibroblasts; HGF and HPLF) were incubated for 48 h with various concentrations of crude plant extract and the viable cell number was determined by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The 50% cytotoxic concentration (CC50) was determined from the dose-response curve. Tumor-specificity (TS) was determined by the following equation: TS=mean CC50 (normal cells)/mean CC50 (cancer cell lines). Metabolic profiling techniques based on (1)H nuclear magnetic resonance (NMR) were applied to gain the chemical structural insight for cytotoxicity induction. RESULTS: Among 24 plant extracts, Camptotheca acuminate leaf, a well-known source for camptothecin, showed the highest TS value (88.3), followed by Vitis s.p.p. (>3.5), Sasa veitchii (>2.3) and Phellodendron amurense (>2.1), whereas other plant extracts showed much lower TS value (<2). These cytotoxic extracts made cluster on principal component analysis (PCA) score plot. CONCLUSION: The TS value determined by the present method seems to reflect the anti-tumor potential of each plant extract, while a part of the cytotoxic compounds present in these extracts may have common chemical structures.

Identification of a xanthine oxidase-inhibitory component from Sophora flavescens using NMR-based metabolomics.

We demonstrate that NMR-based metabolomics studies can be used to identify xanthine oxidase-inhibitory compounds in the diethyl ether soluble fraction prepared from a methanolic extract of Sophora flavescens. Loading plot analysis, accompanied by direct comparison of 1H NMR spectraexhibiting characteristic signals, identified compounds exhibiting inhibitory activity. NMR analysis indicated that these characteristic signals were attributed to flavanones such as sophoraflavanone G and kurarinone. Sophoraflavanone G showed inhibitory activity towards xanthine oxidase in an in vitro assay.

Triterpene glucosides from the leaves of Aralia elata and their cytotoxic activities.

Three new triterpene glucosides, named congmuyenosides C-E (1-3, resp.), along with four known ones, were isolated from an EtOH extract of Aralia elata (Miq.) Seem. leaves. The structures of the new compounds were identified as 3-O-{ß-D-glucopyranosyl-(1→3)-ß-D-glucopyranosyl-(1→3)-[ß-D-glucopyranosyl-(1→2)]-ß-D-glucopyranosyl}caulophyllogenin (1), 3-O-{ß-D-glucopyranosyl-(1→3)-ß-D-glucopyranosyl-(1→3)-[ß-D-glucopyranosyl-(1→2)]-ß-D-glucopyranosyl}hederagenin 28-O-ß-D-glucopyranosyl ester (2), 3-O-{ß-D-glucopyranosyl-(1→3)-ß-D-glucopyranosyl-(1→3)-[ß-D-glucopyranosyl-(1→2)]-ß-D-glucopyranosyl}echinocystic acid 28-O-ß-D-glucopyranosyl ester (3) on the basis of spectral analyses, including MS, (1) H-NMR, (13) C-NMR, DEPT, HSQC, HMBC, NOESY, and HSQC-TOCSY experiments. All isolates obtained were evaluated for their cytotoxic activities against three human tumor cell lines (HepG2, SKOV3, and A549). Compound 3 showed significant cytotoxicity against A549 cell line (IC50 9.9±1.5 µM).

Application of mixture analysis to crude materials from natural resources (IV)[1(a-c)]: identification of Glycyrrhiza species by direct analysis in real time mass spectrometry (II).

In order to identify Glycyrrhiza species by chemical fingerprinting, the bark of the roots and stolons of Glycyrrhiza uralensis Fischer and G. glabra Linné were analyzed using DART (Direct Analysis in Real Time)-MS. The characteristic peaks of each species were determined statistically by volcano plot. This summarizes the relationship between the p-values of a statistical test and the magnitude of the difference in values of the samples in the groups. In this experiment, peaks that had a p value <0.05 in the t test and Z2 absolute difference were defined as characteristic. As a result, characteristic peaks of G. uralensis were found at m/z 299, 315, 341, and 369. In contrast, characteristic peaks of G. glabra were found at m/z 323, 325, 337, 339, and 391. In conclusion, we found several characteristic peaks to distinguish G. uralensis and G. glabra by DART-MS using volcano plot. This method can be applied to identify the Glycyrrhiza species.

Application of mixture analysis to crude materials from natural resources (III): NMR spectral studies to analyze chalcones from Angelica keiskei.

This study presents the application of the NMR-based analyses, DOSY and ROSY, to the chalcones, xanthoangelol (1) and 4-hydroxyderricin (2) from Angelica keiskei. We investigated whether virtual separation and structural information from each compound can be obtained. DOSY displays spectra of (1) and (2) in one dimension and diffusion spectra in the other. And the 1H slice spectra were analyzed in detail by comparison with authentic samples previously isolated from the same material. The resulting ROSY spectrum clearly showed two distinct peaks in the 1H T1 dimension. Each slice of the ROSY spectrum along the 13C dimension contains over-lapped signals, which are difficult to assign at this time.

Classification using NMR-based metabolomics of Sophora flavescens grown in Japan and China.

We demonstrate that NMR-based metabolomics can be used to identify the country of growth (Japan or China) of Sophora flavescens plants. Principle Component Analysis (PCA) conducted on extracts of S. flavescens grown in China provided data distinct from that of extracts of plants grown in Japan. Loading plot analysis showed signals characteristic of Japanese S. flavescens. NMR analyses showed these signals to be due to kurarinol (1) and kushenol H (2). These compounds were confirmed by HPLC analysis to be distinctive markers for Japanese S. flavescens.

Antidiabetic effect of polyphenols from brown alga Ecklonia kurome in genetically diabetic KK-A(y) mice.

CONTEXT: Prevalence of diabetes mellitus type 2 (DM-II) is increasing in Japan. Brown alga Ecklonia kurome Okamura (Laminariaceae) (kurome in Japanese) is rich in phlorotannins, a kind of polyphenol. Phlorotannins have been reported to possess various bioactivities; however, few studies have reported its effect on DM-II. OBJECTIVE: The present study was conducted to investigate the antidiabetic effect of polyphenols from E. kurome (KPP) on KK-A(y) mice, the animal model for human DM-II. MATERIALS AND METHODS: Inhibitory activities of KPP against α-amylase and α-glucosidase in vitro, and effects on oral carbohydrate tolerance test in vivo were investigated. KK-A(y) mice were fed with 0.1% KPP containing water for 5 weeks. A glucose tolerance test was conducted at week 4 of the 5-week period. At the end of experiment, blood biochemical parameters, including blood glucose, insulin, glycoalbumin, and fructosamine were determined. Furthermore, the kidneys and pancreatic islets were histologically examined. RESULTS: KPP showed inhibitory activities on carbohydrate-hydrolyzing enzymes and decreased postprandial blood glucose levels. The body weight gain and blood glucose levels in the KPP group were lower than the control group during the experimental period. KPP improved glucose tolerance and decreased the fasting blood glucose and insulin levels, fructosamine and glycoalbumin levels compared with the control group. Furthermore, KPP contracted the pancreatic islet size and decreased renal mesangial matrix in KK-A(y) mice. DISCUSSION AND CONCLUSION: These results suggest that KPP is effective against DM-II and might provide a source of therapeutic agents for DM-II.

Application to classification of mulberry leaves using multivariate analysis of proton NMR metabolomic data.

Recently, NMR-based metabolomic analysis has been used to acquire information based on differentiation among biological samples. In the present study, we examined whether multivariate analysis was able to be applied to natural products and/or material field. Each extraction of 24 leaf samples, divided into six locations from the tip of the stem in each of four strains, was analyzed by pattern recognition methods, known as Principal Component Analysis (PCA) and Soft Independent Modeling of Class Analogy (SIMCA). Twenty-four extracts from mulberry leaf showed independent spectra by 1H NMR. The separation of leaf extraction data due to the difference at six locations was achieved in the PCA score plot as correlation PC1 (86.1%) and PC3 (4.6%) and showed two loading plots, suggesting classification by leaf position as an independent variable in the loading plot. Moreover, the difference among six locations clarified the seven highest discrimination powers by the SIMCA method. Meanwhile, the PCA score plot obtained classification by the variety of mulberry strains with three loading plots, but the SIMCA method did not give a peak by classification.

Coumarins from Seseli hartvigii.

Linear-type furocoumarins, 5-[(2"E,6"R)-6"-hydroxy-3",7"-dimethylocta-2",7"-dienyloxy]psoralen and 5-[(2"E,6"S)-6"-hydroxy-3",7"-dimethylocta-2",7"-dienyloxy]psoralen (1) were first isolated from whole plants of Seseli hartvigii together with one new natural product 6-(3'-methyl-2'-oxo-3'-butenyl)-7-methoxycoumarin (2), and four known compounds (tamarin, bergaptol, notoptol, and a mixture of beta-sitosterol and stigmasterol). The structure of 1 was elucidated by extensive spectroscopic analysis and chemical conversion. The modified Mosher's method and HPLC were applied to determine its stereochemistry. Both R- and S-configurations exist in 1; after modification by Mosher's reagent, they were effectively separated, and their ratio was deduced to be 59% and 41%, respectively.

Identification of Glycyrrhiza species by direct analysis in real time mass spectrometry.

DART (Direct Analysis in Real Time)-MS is a novel mass spectrometric ion source, and allows the analysis of most compounds at ambient pressure and ground potential by producing [M+H]+ molecular ion species. Using this method, we examined the compounds characteristic of several kinds of licorices. For the analysis of Glycyrrhiza inflata Batalin, the peak at m/z 339 originates mainly from [M+H]+ of licochalcone A (LA), a species-specific compound. This peak was hardly detected in G. glabra Linné and G. uralensis Fischer. These results indicate that G. inflata can be differentiated from the other two species by detection of LA peaks using DART-MS analysis.

Studies of coumarins from the Chinese drug Qianhu, XXVII: structure of a new simple coumarin glycoside from Bai-Hua Qianhu, Peucedanum praeruptorum.

A new simple coumarin glycoside, named praeroside VI (1), along with six known coumarins, were isolated from an aqueous extract of Bai-Hua Qianhu, the root of Peucedanum praeruptorum DUNN. (Umbelliferae). Spectroscopic analysis and chemical studies were carried out to investigate the structure of the new coumarin, which was concluded to be 1. Additionally, two simple glycosidic coumarins and four simple nonglycosidic coumarins were identified as apiosylskimmin (2), hymexelsin (3), umbelliferone (4), scopoletin (5), isofraxidin (6) and 8-carboxy-7-hydroxy coumarin (7), respectively. This is the first reported identification of compound 7 as a constituent of plant materials.