UHPLC/MS and NMR-Based Metabolomic Analysis of Dried Water Extract of Citrus-Type Crude Drugs.

Citrus-type crude drugs (CCDs) are commonly used to formulate decoctions in Kampo formula (traditional Japanese medicine). Our previous study reported metabolomic analyses for differentiation of the methanol extracts of Citrus-type crude drugs (CCDs) using ultra-HPLC (UHPLC)/MS, and 13C- and 1H-NMR. The present study expanded the scope of its application by analyzing four CCD water extracts (Kijitsu, Tohi, Chimpi, and Kippi); these CCDs are usually used as decoction ingredients in the Kampo formula. A principal component analysis score plot of processed UPLC/MS and NMR analysis data indicated that the CCD water extracts could be classified into three groups. The loading plots showed that naringin and neohesperidin were the distinguishing components. Three primary metabolites, α-glucose, ß-glucose, and sucrose were identified as distinguishing compounds by NMR spectroscopy. During the preparation of CCD dry extracts, some compounds volatilized or decomposed. Consequently, fewer compounds were detected than in our previous studies using methanol extract. However, these results suggested that the combined NMR- and LC/MS-based metabolomics can discriminate crude drugs in dried water extracts of CCDs.

Characterization of metabolites in Saposhnikovia divaricata root from Mongolia.

Saposhnikoviae Radix (SR), derived from the dried root and rhizome of Saposhnikovia divaricata, is a popular crude drug used in traditional Chinese and Japanese medicine. To evaluate the metabolites of S. divaricata roots from Mongolia and to investigate their geographical variation, we developed the HPLC method, determined the contents of 9 chromones and 4 coumarins, and conducted multivariate statistical analysis. All Mongolian specimens contained prim-O-glucosylcimifugin (1) and 4'-O-ß-D-glucosyl-5-O-methylvisamminol (3), and their total amount (5.04-25.06 mg/g) exceeded the criterion assigned in the Chinese Pharmacopoeia. Moreover, the content of 1 (3.98-20.79 mg/g) was significantly higher in the Mongolian specimens than in Chinese SR samples. The specimens from Norovlin showed the highest contents of 1 and 3. The total levels of dihydropyranochromones were higher in the specimens from Bayan-Uul. The orthogonal partial least squares-discriminant analysis revealed that the Mongolian specimens tended to be separated into three groups based on growing regions, in which several chromones contributed to each distribution. Furthermore, 1H NMR analysis revealed that Mongolian specimens had less amount of sucrose and a substantial amount of polyacetylenes. Thus, in this study, the chemical characteristics of Mongolian S. divaricata specimens were clarified and it was found that the specimens from the northeast part of Mongolia, including Norovlin, had the superior properties due to higher amounts of major chromones.

Identifying the compounds that can distinguish between Saposhnikovia root and its substitute, Peucedanum ledebourielloides root, using LC-HR/MS metabolomics.

Previously, we established a 1H NMR metabolomics method using reversed-phase solid-phase extraction column (RP-SPEC), and succeeded in distinguishing wild from cultivated samples of Saposhnikoviae radix (SR), and between SR and its substitute, Peucedanum ledebourielloides root (PR). Herein, we performed LC-HR/MS metabolomics using fractions obtained via RP-SPEC to identify characteristic components of SR and PR. One and three characteristic components were respectively found for SR and PR; these components were isolated with their m/z values and retention times as a guide. The characteristic component of SR was identified as 4'-O-ß-D-glucosyl-5-O-methylvisamminol (1), an indicator component used to identify SR in the Japanese Pharmacopoeia. In contrast, the characteristic components of PR were identified as xanthalin (2), 4'-O-ß-D-apiosyl (1 → 6)-ß-D-glucosyl-5-O-methylvisamminol (3), and 3'-O-ß-D-apiosyl (1 → 6)-ß-D-glucosylhamaudol (4) based on spectroscopic data such as 1D- and 2D-NMR, MS, and specific optical rotation. Among them, 4 is a novel compound. For the correlation between the NMR metabolomics results in the present and our previous report, only 1 and 2 were found to correlate with the chemical shifts, and the other compounds had no correlation. As the chemical shifts for compounds 1, 3, and 4 were similar to each other, especially for the aglycone moiety, they could not be distinguished because of the sensitivity and resolution of 1H NMR. Accordingly, combining NMR and LC/MS metabolomics with their different advantages is considered useful for metabolomics of natural products. The series of methods used in our reports could aid in quality evaluations of natural products and surveying of marker components.

1H NMR-based metabolomic analysis coupled with reversed-phase solid-phase extraction for sample preparation of Saposhnikovia roots and related crude drugs.

1H NMR-based metabolomics has been applied in research on food, herbal medicine, and natural products. Although excellent results were reported, samples were directly extracted with a deuterated solvent (e.g., methanol-d4 or D2O) in most reports. As primary metabolites account for most of the results, data for secondary metabolites are partially reflected. Consequently, secondary metabolites tend to be excluded from factor loading analysis, serving as a significant unfavorable feature of 1H NMR-based metabolomics when investigating biologically active or functional components in natural products and health foods. Reversed-phase solid-phase extraction column (RP-SPEC) was applied for sample preparation in 1H NMR-based metabolomics to overcome this feature. The methanol extract from Saposhnikoviae radix (SR), an important crude drug, was fractionated with RP-SPEC into 5% methanol-eluting fractions, and the remaining fraction was collected. Each fraction was subjected to 1H NMR-based metabolomics and compared to results from conventional 1H NMR-based metabolomics. Based on principal component analysis (PCA) and partial least squares projections to latent structures discriminant analysis (PLS-DA), the 5% methanol fraction and conventional method reflected the amount of saccharides such as sucrose on the PC1/PLS1 axes, and wild and cultivated samples were discriminated along those axes. The remaining fraction clearly distinguished SR from Peucedanum ledebourielloides root. The compounds responsible for this discrimination were deemed falcarindiol derivatives and other unidentified secondary metabolites from the s-plot on PLS-DA. The secondary metabolites from original plants were, therefore, presumed to be concentrated in the remaining fraction by RP-SPEC treatment and strongly reflected the species differences. The developed series is considered effective to perform quality evaluation of crude drugs and natural products.

High-Resolution Liquid Chromatography-Mass Spectrometry-Based Metabolomic Discrimination of Citrus-Type Crude Drugs and Comparison with Nuclear Magnetic Resonance Spectroscopy-Based Metabolomics.

Five Citrus-type crude drugs (40 samples) were classified using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. The following six flavonoid derivatives were identified as contributors from the loading plots of multivariate analysis: naringin (1), neohesperidin (2), neoeriocitrin (3), narirutin (9), hesperidin (10), and 3,5,6,7,8,3',4'-heptamethoxyflavone (12). Three coumarin derivatives, namely, meranzin (6), meranzin hydrate (7), and meranzin glucoside (8), were also identified as contributors. Furthermore, compared with our previous studies on proton (1H) and 13C NMR spectroscopy-based metabolomics, the present study revealed that the Citrus-type crude drugs were distinguished with the same pattern; however, the contributors differed between the 1H and 13C NMR spectroscopy-based metabolomics. The high dynamic range of NMR spectroscopy provided broad coverage of the metabolomes including the primary and secondary metabolites. However, LC-MS appeared to be superior in detecting secondary metabolites with high sensitivity, some of which occurred in quantities that were undetectable using NMR spectroscopy.

Botanical origin and chemical constituents of commercial Saposhnikoviae radix and its related crude drugs available in Shaanxi and the surrounding regions.

Saposhnikoviae radix (SR) is described in the Japanese Pharmacopoeia as a crude drug derived from the root of Saposhnikovia divaricata Schischkin (Umbelliferae). According to Flora of China, the root of Peucedanum ledebourielloides K. F. Fu is used as a regional substitute for SR. Therefore, we surveyed the botanical origin of the drug used in China, especially Shaanxi and the surrounding regions, through nucleotide sequence analysis of the internal transcribed spacer region of rDNA. As a result, several samples from Shaanxi () and Shanxi () provinces were identified as Peucedanum ledebourielloides. To prevent this substitute from being distributed as genuine SR, we developed a thin-layer chromatography analysis condition to enable a specific compound of this species to be easily detected. The specific compound was identified as xanthalin, based on 1D- and 2D-NMR and high-resolution mass spectrometry data. The established TLC conditions were as follows-extraction solvent, n-hexane; applied volume, 5 µL; chromatographic support, silica gel; developing solvent, n-hexane:ethyl acetate:acetic acid (20:10:1); developing length, 7 cm; detection, UV (365 nm); R f value, 0.4 (blue fluorescence; xanthalin).

Construction of Prediction Models for the Transient Receptor Potential Vanilloid Subtype 1 (TRPV1)-Stimulating Activity of Ginger and Processed Ginger Based on LC-HRMS Data and PLS Regression Analyses.

To construct a model formula to evaluate the thermogenetic effect of ginger (Zingiber officinale Roscoe) from the ingredient information, we established transient receptor potential vanilloid subtype 1 (TRPV1)-stimulating activity prediction models by using a partial least-squares projections to latent structures (PLS) regression analysis in which the ingredient data from liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and the stimulating activity values for TRPV1 receptor were used as explanatory and objective variables, respectively. By optimizing the peak extraction condition of the LC-HRMS data and the data preprocessing parameters of the PLS regression analysis, we succeeded in the construction of a TRPV1-stimulating activity prediction model with high precision ability. We then searched for the components responsible for the TRPV1-stimulating activity by analyzing the loading plot and s-plot of the model, and we identified [6]-gingerol (1) and hexahydrocurcumin (3) as TRPV1-stimulating activity components.

Cyclopropane-ring formation in the acyl groups of chlorosome glycolipids is crucial for acid resistance of green bacterial antenna systems.

Green photosynthetic bacteria have unique light-harvesting antenna systems called chlorosomes. Chlorobaculum tepidum, a model organism of the bacteria, biosynthesized monogalactosyl- and rhamnosylgalactosyldiacylglycerides possessing a methylene-bridged palmitoleyl group characterized by a cis-substituted cyclopropane ring as the dominant glycolipids of its chlorosome surface. The formation of the cyclopropane ring was chemically inhibited by supplementation of sinefungin, an analog of S-adenosyl-L-methionine, into the bacterial cultivation. The presence of the cyclopropane ring reinforced acid resistance of the light-harvesting chlorosomes and suppressed acidic demetalation (pheophytinization) of bacteriochlorophyll-c pigments constructing the core part of chlorosomes. The ring-formation would represent direct and post-synthetic modifications of chlorosome membrane properties and was tolerant of acidic environments.