UPLC-Q-TOF/MS Based Plasma Metabolomics for Identification of Paeonol's Metabolic Target in Endometriosis.

Endometriosis is a common gynecological illness in women of reproductive age that significantly decreases life quality and fertility. Paeonol has been shown to play an important part in endometriosis treatments. Understanding the mechanism is critical for treating endometriosis. In this study, autologous transplantation combined with a 28 day ice water bath was used to create a rat model of endometriosis with cold clotting and blood stagnation. The levels of estradiol and progesterone in plasma were detected by ELISA, and the pathological changes of ectopic endometrial tissue were examined by H&E staining, which proved the efficacy of paeonol. For metabolomic analysis of plasma samples, UPLC-Q/TOF-MS was combined with multivariate statistical analysis to identify the influence of paeonol on small molecule metabolites relevant to endometriosis. Finally, the key targets were screened using a combination of network pharmacology and molecular docking approaches. The results showed that the pathological indexes of rats were improved and returned to normal levels after treatment with paeonol, which was the basis for confirming the efficacy of paeonol. Metabolomics results identified 13 potential biomarkers, and paeonol callbacks 7 of them, involving six metabolic pathways. Finally, four key genes were found for paeonol therapy of endometriosis, and the results of molecular docking revealed a significant interaction between paeonol and the four key genes. This study was successful in establishing a rat model of endometriosis with cold coagulation and blood stagnation. GCH1, RPL8, PKLR, and MAOA were the key targets of paeonol in the treatment of endometriosis. It is also demonstrated that metabolomic techniques give the potential and environment for comprehensively understanding drug onset processes.

Development and validation of ultra-high performance supercritical fluid chromatography method for quantitative determination of six compounds in Guizhi Fuling capsule and tablet samples.

A fast and simple ultra-high performance supercritical fluid chromatography method has been developed for the determination of six analytes, namely (paeonol, coumarin, cinnamic alcohol, cinnamic acid, paeoniflorin, and amygdalin) in Guizhi Fuling capsule and tablet samples. The influence of the key chromatographic parameters for the separation purposes was evaluated. The optimal column was Trefoil CEL1 column. The optimal mobile phase was a gradient mixture of carbon dioxide and methanol at flow rate of 1.0 mL/min. The back pressure of the system was set to 1.38 × 107  Pa and the temperature to 45°C. The six compounds were separated within 11 min by the proposed ultra-high performance supercritical fluid chromatography method with satisfactory resolution. Method validation confirmed that the procedure is accurate with the recovery rates from 87.04 to 104.30%, intraday precision values less than 4.81% and interday precision less than 5.22%, and linear with R2 higher than 0.9967. Therefore, this work provides a simple and novel method for the simultaneous analysis of six compounds in Guizhi Fuling capsule and tablet samples.

Impact of UV-C treatment and thermal pasteurization of grape must on sensory characteristics and volatiles of must and resulting wines.

UV-C treatment is a commonly known technique to inactivate microorganisms. The objective of this work was to investigate the impact of UV-C treatment of grape must on the sensory characteristics of the resulting wine and on the profile of volatile compounds of grape must and wine. Different UV-C doses were applied to Riesling must and compared with thermal pasteurization. The sensory off-flavor "ATA" and a content of 0.5 µg/L 2-aminoacetophenone were determined in the grape must and in the resulting wine after UV-C treatment with a high dose of 21 kJ/L. Sensory off-flavors did neither occur after thermal pasteurization nor after UV-C treatment with a dose of 2 kJ/L, which is sufficient for the inactivation of microorganisms. Minor changes in the volatiles' profiles of grape must and wine, involving e.g. terpenes and C13-norisoprenoids, occurred in musts treated with thermal pasteurization as well as with a UV-C dose of 2 kJ/L.

Microscopic Characteristic and Chemical Composition Analysis of Three Medicinal Plants and Surface Frosts.

The accumulation of chemical constituents of some medicinal plants, such as Paeonia ostii T. Hong et J. X. Zhang, Houpoëa officinalis (Rehder and E. H. Wilson) N. H. Xia and C. Y. Wu. and Atractylodes lancea (Thunb.) DC, can precipitate on the surface and form frosts after natural or artificial intervention. The characteristics of these three medicinal plants and their frosts were analyzed by light microscope, polarizing microscope, stereomicroscope, and metalloscope. The results of ordinary Raman of P. ostii and H. officinalis showed that the frosts of P. ostii matched paeonol, while that of H. officinalis matched magnolol and honokiol. In P. ostii and its frost, 19 peaks were identified by UPLC-Q/TOF-MS, and the main component was paeonol. Eleven components were identified in H. officinalis and its frosts, and the main components were magnolol and honokiol. A. lancea and its frosts were analyzed by gas chromatography-mass spectrometry (GC-MS), 21 were identified, and its main components were hinesol and ß-eudesmol. These three medicinal plants accumulate compounds and precipitate frosts on the surface. The results show that the components of the frosts provide a basis for quality evaluation and research on similar medicinal plants, and reveals the scientific connotation of "taking the medicinal materials' precipitated frosts as the best" of P. ostii, H. officinalis, and A. lancea, to some extent.

Systematic screening and characterization of multiple constituents in Guizhi Fuling capsule and metabolic profiling of bioactive components in rats using ultra-high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

Guizhi Fuling capsule (GFC), a prestigious traditional Chinese medicinal (TCM) prescription, is efficiently used to treat primary dysmenorrhea in the clinical practice. It's significant to explore the metabolic fate of multiple components in vivo which are responsible for the pharmacological effects but not fully investigated. A rapid and high-throughput method using ultra performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS/MS) was established for systematic investigation on GFC, including GFC chemical compositions, and their absorption and metabolism in rat plasma, urine, uterus and brain after oral administration of GFC. A total of 102 nonvolatile GFC phytochemistry components were identified based on the accurately measured mass value, fragmentation pattern and retention behavior. Compared to the previous GFC study, additional 47 different GFC components were detected. Furthermore 21, 9, 4 and 3 prototype compounds were separately observed in plasma, urine, uterus and brain samples with the support of in vitro GFC study. While 29, 33, 10 and 8 metabolites were also identified with the assistance of the MetaboLynx tool in these biological samples. The result indicated that the developed method was suitable for the components identification even in the complex matrix. The chemical and metabolic profiling of GFC provided an abundant substance foundation for the extensive GFC research, especially for the pharmacodynamic mechanisms research.

Pteleifolols A-E, acetophenone di-C-glycosides and a benzopyran dimer from the leaves of Melicope pteleifolia.

Four new acetophenone di-C-glycosides, pteleifolols A-D (1-4) and a new dimeric benzopyran, pteleifolol E (5), were isolated from the leaves of Melicope pteleifolia. Seven known compounds, including 2,4,6-trihydroxyacetophenone-3,5-di-C-glucopyranoside (6), were also isolated. Structures of the new compounds (1-5) were established by using spectroscopic and spectrometric techniques, including 1D and 2D nuclear magnetic resonance (NMR), UV, and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) data. Pteleifolols A-D (1-4) were E-p-coumaroyl, Z-p-coumaroyl, E-feruloyl, and benzoyl esters of 6, respectively. Pteleifolol E (5) was a dichromene dimerized through a C2 unit.

Application of a quantitative (1)H-NMR method for the determination of paeonol in Moutan cortex, Hachimijiogan and Keishibukuryogan.

Quantitative (1)H-NMR ((1)H-qNMR) was applied to the determination of paeonol concentration in Moutan cortex, Hachimijiogan, and Keishibukuryogan. Paeonol is a major component of Moutan cortex, and its purity was calculated from the ratio of the intensity of the paeonol H-3' signal at δ 6.41 ppm in methanol-d 4 or 6.40 ppm in methanol-d 4 + TFA-d to that of a hexamethyldisilane (HMD) signal at 0 ppm. The concentration of HMD was corrected with SI traceability by using potassium hydrogen phthalate of certified reference material grade. As a result, the paeonol content in two lots of Moutan cortex as determined by (1)H-qNMR was found to be 1.59 % and 1.62 %, respectively, while the paeonol content in Hachimijiogan and Keishibukuryogan was 0.15 % and 0.22 %, respectively. The present study demonstrated that the (1)H-NMR method is useful for the quantitative analysis of crude drugs and Kampo formulas.

Simultaneous determination of seven bioactive components in Guizhi Fuling capsule by microwave-assisted extraction combined with ultra performance liquid chromatography tandem mass spectrometry.

A simple, rapid and reliable microwave-assisted extraction (MAE) combined with ultra performance liquid chromatography tandem mass spectrometry method was developed for simultaneous determination of the seven bioactive constituents in Guizhi Fuling capsule (GFC), namely gallic acid, amygdalin, albiflorin, paeoniflorin, paeonol, cinnamic acid and pachymic acid, respectively. The operation of MAE optimised through orthogonal array design experiment was performed at 80°C for 10 min with methanol-water (70:30, v/v) as the extracting solvent. The method was validated including intra- and inter-day precision, repeatability and stability, with relative standard deviation less than 3.9%, 3.3%, 4.4% and 3.1%, respectively. All analytes showed the good linearity (r >0.999), and their average recoveries varied between 98.2% and 101.2%. The results indicated that this method was simple, effective and suitable for the quality control of GFC.

Ion tree-based structure elucidation of acetophenone dimers (AtA) from Acronychia pedunculata and their identification in extracts by liquid chromatography electrospray ionization LTQ-Orbitrap mass spectrometry.

Acronychia-type acetophenones (AtA) is a chemical group of compounds of important structural and biological interest, abundant in Acronychia species. However, there are no data available for their characterization using mass spectrometry. In the current work, AtA have been investigated by multistage high resolution mass spectrometry and both electrospray ionization and atmospheric pressure chemical ionization, in positive and negative mode, were utilized for their structure elucidation and identification. The analysis of AtA using a linear ion trap-Orbitrap analyzer enabled the structural determination of key fragment ions and cleavages, which can be used for the structural characterization thereof. A systematic nomenclature based on protonated and deprotonated fragment ions under collision-induced dissociation conditions and decision trees for the structural determination of AtA are proposed. Furthermore, taking advantage of the characteristic fragmentation patterns, a selective Ultra High Performance Liquid Chromatography Electrospray Ionization multistage Mass Spectrometry (UHPLC-ESI(-)-MS(n)) method was developed and successfully applied for the dereplication of known AtA and the identification of potentially new ones in Acronychia extracts. Despite the structure similarity and the presence of isomers, accurate characterization of known and unknown AtA derivatives was possible.

Genetic and chemical characterization of white and red peony root derived from Paeonia lactiflora.

Two kinds of peony roots--white peony root (WPR) and red peony root (RPR)--are used for different remedies in traditional Chinese medicine; however, most of them are derived from the same botanical origin, Paeonia lactiflora. The difference between WPR and RPR has been debated for a long time. This study attempted to clarify the genetic and chemical characteristics of WPR and RPR in order to provide a scientific dataset for their identification and effective use. The nucleotide sequence of nrDNA internal transcribed spacer (ITS) and the contents of 8 main bioactive constituents were analyzed from specimens of P. lactiflora, P. veitchii and two related species as well as crude drug samples of WPR, RPR and peony root produced in Japan. Of the samples derived from P. lactiflora, the WPR produced in the southern parts of China and the RPR produced in the northern parts of China were clearly divided into two subgroups within the P. lactiflora group based on similarity of the ITS sequences. The nucleotides at positions 69, 458 and 523 upstream of the ITS sequence served as molecular markers to discriminate between WPR and RPR. Quantitative analysis indicated that the RPR samples obviously contained a higher content of paeoniflorin and paeonol, but a lower content of albiflorin than the WPR produced in the southern parts of China and peony root produced in Japan. The WPR available from Chinese markets was usually processed by sulfur fumigation, which resulted in an extremely low content of paeoniflorin. This study indicated that WPR and RPR were not only geographically isolated, but also genetically and chemically separated. The ITS sequence provided a genetic index for their identification.

Chemical taxonomy of tree peony species from China based on root cortex metabolic fingerprinting.

The section Moutan of the genus Paeonia consists of eight species that are confined to a small area in China. A wide range of metabolites, including monoterpenoid glucosides, flavonoids, tannins, stilbenes, triterpenoids, steroids, paeonols, and phenols, have been found in the species belonging to section Moutan. However, although previous studies have analyzed the metabolites found in these species, the metabolic similarities that can be used for the chemotaxonomic distinction of section Moutan species are not yet clear. In this study, HPLC-DAD-based metabolic fingerprinting was applied to the classification of eight species: Paeoniasuffruticosa, Paeoniaqiui, Paeoniaostii, Paeoniarockii, Paeoniajishanensis, Paeoniadecomposita, Paeoniadelavayi, and Paeonialudlowii. In total, of the 47 peaks that exhibited an occurrence frequency of 75% in all 23 tree peony samples, 43 of these metabolites were identified according to their retention times and UV absorption spectra, together with combined HPLC-QTOF-MS. These data were compared with reference standard compounds. The 43 isolated compounds included 17 monoterpenoid glucosides, 11 galloyl glucoses, 5 flavonoids, 6 paeonols and 4 phenols. Principal component analysis (PCA), and hierarchical cluster analysis (HCA), showed a clear separation between the species based on metabolomics similarities and four groups were identified. The results exhibited good agreement with the classical classification based on the morphological characteristics and geographical distributions of the subsections Vaginatae F.C. Stern and Delavayanae F.C. Stern with the exception of P. decomposita, which was found to be a transition species between these two subsections. According to their metabolic fingerprinting characteristics, P. ostii and P. suffruticosa can be considered one species, and this result is consistent with the viewpoint of medicinal plant scientists but different from that of classical morphological processing. Significantly large variations were obtained in the metabolic profiles of P. delavayi, whereas no significant difference was found between P. delavayi and P. ludlowii. This indicates that these two species have a close genetic relationship. In conclusion, the combination of HPLC-DAD and multivariate analyses has great potential for guiding future chemotaxonomic studies to examine the potential pharmaceutical value of the effective constituents of tree peony species and appears to be able to clarify the confusion and skepticism associated with the reported morphology- and molecular phylogenetics-based taxonomy of tree peonies.

[Study on HPLC chromatograms of different processed Euphorbia ebracteolata products and content change of three chemical components].

To prepare processed products with different methods, in order to study the impact of auxiliary materials and temperature on chemical components of Euphorbia ebracteolata, and establish specific chromatograms of different processed products. Wel-chorm-C18 column (4.6 mm x 250 mm, 5 microm) was used and eluted with a gradient program, with acetonitrile (A)-water(B). The column temperature was 25 degrees C, and the detection wave length was set at 226 nm. The aim was to determine the content of effective components in different processed products--ebracteolata cpd B, ebracteolata cpd C and jolkinolide B and establish respective characteristic fingerprints to compare with similarity. The results showed that the content of ebracteolata cpd B, ebracteolata cpd C first increased and then decreased with the rise in temperature. Different processed products showed significant difference in HPLC spectrograms, with a low similarity. This study showed great impacts of auxiliary materials and temperature on chemical components of E. ebracteolata. As the vinegar processing method had higher attenuation and and synergistic effects than other methods, the auxiliary material vinegar cannot be replaced by chemical reagent acetic acid.

Simultaneous determination of danshensu, salvianolic acid B, and paeonol in ShuangDan oral liquid by HPLC.

An HPLC-photodiode array detector (PDAD) method was developed for simultaneous determination of danshensu, salvianolic acid B, and paeonol in a traditional Chinese medicinal preparation ShuangDan oral liquid, which is widely used to treat coronary heart disease in China. The samples were separated on a SinoChrom ODS-BP C18 column with the column temperature maintained at 30 degrees C. The mobile phase was composed of methanol and 2% (v/v) glacial acetic acid at a flow rate of 1.0 mL/min. The separation was finished within 30 min using linear gradient elution. All calibration curves showed good linearity (r > 0.9997) within the test ranges. The method was simple, reliable, accurate, and specific. The intraday and interday precisions (RSD) were less than 0.85%. The recoveries were between 96.97 and 102.14%. The method was successfully applied to the determination of the three components in three commercial samples of ShuangDan oral liquid. The results indicated that the developed HPLC-PDAD assay could be readily utilized as a comprehensive quality control method for ShuangDan oral liquid.

[Analysis of volatile oil from Gerbera piloselloides by GC-MS].

OBJECTIVE: To identify the chemical components of volatile oil from Gerbera piloselloides. METHODS: Volatile oil was extracted from Gerbera piloselloides by steam distillation, and its chemical components were identified by GC-MS. RESULTS: 22 peaks were detected, among which 17 compounds were identified, accounting for 86.18% of total peak area. CONCLUSION: In the total volatile oil from Gerbera piloselloides, Neryl(s)-2-methylbutanoate (35.99%), 4-hydroxy-3-methylacetophenone (8.74%) and n-Hexadecanoic acid (7.48%) are the main components.

[Study on the influence of the contents of paeonol in the cutting process of fresh Cortex Moutan].

OBJECTIVE: To explore the influence of cutting process on the quality of Cortex Moutan. METHODS: The contents of paeonol in Cortex Moutan collected by different methods were determined by HPLC. RESULTS: The cutting process of fresh Cortex Moutan was as follows: collectd the 4-years-Cortex Moutan, cleaned sliced fresh Cortex Moutan, and then dried them in the sun. CONCLUSION: This study provides a scientific basis for the cutting process of fresh Cortex Moutan.

Phenolic constituents of the Chilean herbal tea Fabiana imbricata R. et P.

"Pichi" or "pichi romero" (Fabiana imbricata R. et. P., Solanaceae) is a Chilean plant used as a tea in the Andean regions of Chile and Argentina. A very simple and direct method was developed for the qualitative analysis of polyphenols in the tea by high-performance liquid chromatography (HPLC) with diode array detection and electrospray ionization tandem mass spectrometry. The phenolic constituents identified in the teas were chlorogenic acid (3-O-caffeoylquinic acid), p-hydroxyacetophenone, scopoletin and quercetin derivatives. The glycosides were mainly glucosides from p-hydroxyacetophenone and scopoletin while di- and tri-glycosides from quercetin were the main flavonoids. The content of the main phenolic compounds in the teas (g/100 g lyophilized infusion) was 0.8-1.9 % for scopoletin, 0.4-6.2 % for p-hydroxyacetophenone and 2.1-4.3 % for rutin, respectively. The health-promoting properties reported for this herbal tea can be associated with the presence of several phenolics with known antioxidant, diuretic and antiinflammatory activity.

Rottlerin and curcumin: a comparative analysis.

Rottlerin and curcumin are natural plant polyphenols with a long tradition in folk medicine. Over the past two decades, curcumin has been extensively investigated, while rottlerin has received much less attention, in part, as a consequence of its reputation as a selective PKCδ inhibitor. A comparative analysis of genomic, proteomic, and cell signaling studies revealed that rottlerin and curcumin share a number of targets and have overlapping effects on many biological processes. Both molecules, indeed, modulate the activity and/or expression of several enzymes (PKCδ, heme oxygenase, DNA methyltransferase, cyclooxygenase, lipoxygenase) and transcription factors (NF-κB, STAT), and prevent aggregation of different amyloid precursors (α-synuclein, amyloid Aß, prion proteins, lysozyme), thereby exhibiting convergent antioxidant, anti-inflammatory, and antiamyloid actions. Like curcumin, rottlerin could be a promising candidate in the fight against a variety of human diseases.

[A novel method for fast determination of components in Guizhi Fuling capsule by near infrared spectroscopy].

OBJECTIVE: To develop a new method to rapidly determine and identify Guizhi Fuling capsule by portable acousto-optic tunable filter-near infrared spectroscopy. METHOD: The qualitative model was set up using principal component analysis. The correlation models between the NIR spectra and the reference values of five major constituents were obtained with partial least squares method. RESULT: The identifying model accurately identified Guizhi Fuling capsule, and quantitative analytical models could precisely predicted the content of ellagic acid, baicalin, benzoylpaenoniflorin, cinnamaldehyde, and paeonol. The correlation coefficients of the calibration models were 0.924 2, 0.938 4, 0.924 2, 0.933 6, 0.934 7, the validation set coefficients of the calibration were 0.924 2, 0.938 4, 0.924 2, 0.933 6, 0.934 7, and the RMSEP were 1.138%, 3.014%, 0.751%, 0.625%, 3.455%, 1.363%, respectively. The results of external validation showed no significant difference between the predictive and the determining values by t-test. CONCLUSION: The method is accurate, rapid and non-destructive, and can be used for determining and identifying Guizhi Fuling capsule.

Electrochemical oxidation and detection of paeonol on modified electrode with acetylene black nanoparticles.

With an aim to construct a sensing platform for the electrochemical detection of paeonol, we modified the glassy carbon electrode with acetylene black nanoparticle (AB). A sensitive oxidation peak of paeonol was observed with remarkably increased peak current on the modified electrode because the electrode has a big surface area due to three dimensional structure of AB nanoparticles. The optimization of detection conditions was performed, including pH value of the buffer, the amount of AB nanoparticles on the electrode surface, the accumulation potential and time of paeonol. Under the optimized conditions, the oxidation peak current of paeonol increased linearly with its concentration over the range from 5×10(-7) to 1×10(-4) M. The detection limit was calculated to be 1×10(-7) M. The modified electrode was successfully applied to detect the content of paeonol in cortex moutan, a common traditional Chinese medicine. The method is new, sensitive, rapid and convenient for the detection of paeonol.

Quality control of modified xiaoyao san through the determination of 22 active components by ultra-performance liquid chromatography.

A simple, sensitive, and reliable ultra-performance liquid chromatography (UPLC) method has been developed for simultaneous determination of 22 major constituents in modified xiaoyao san (MXS), a multiherbal formula. The chromatographic separation was performed on an ACQUITY UPLC BEH C18 column (150 x 2.1 mm, 1.7 microm, particle size), with an aqueous 0.5% acetic acid and acetonitrile mobile phase gradient. The method was validated for linearity (r2 >0.9937), intraday and interday precision (RSD <8.51%), recovery (91.18-107.73%), LOD (0.02-4.17 ng/mL), and LOQ (0.05-12.50 ng/mL). The established method was successfully applied to quantify the 22 marker compounds in MXS, which provided a useful basis of overall evaluation of the quality of MXS.