Ultra-high-performance Liquid Chromatography Coupled To Quadrupole Time-of-flight Mass Spectrometry Research Articles

Chemical constituent characterization and determination of Quisqualis fructus based on UPLC-Q-TOF-MS and HPLC combined with fingerprint and chemometric analysis.

Quisqualis fructus (QF) is a traditional Chinese medicine (TCM) that it has a long history in the therapeutic field of killing parasites, eliminating accumulation, and stopping diarrhea. However, the therapeutic material basis of QF is remaining ambiguous nowadays. The geographical origin differences of QF are also usually ignored in the process of medication. In this study, the alcohol-aqueous soluble constituents in QF from different origins were systematically characterized and accurately measured by ultra-high performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and high-performance liquid chromatography (HPLC) respectively. Chemometric analysis was performed for origin differentiation and screening of potential quality marker (Q-marker). Finally, A total of 106 constituents were tentatively characterized in positive and negative ion modes, including 29 fatty acids, 26 organic acids, 11 amino acids and derivatives, 10 glycosides, 9 alkaloids and derivatives, and 21 other constituents. QF from different origins were effectively distinguished and 16 constituents were selected as the potential Q-markers subsequently. Four representative components (trigonelline, adenosine, ellagic acid, and 3,3'-di-O-methylellagic acid) in QF samples were simultaneously determined. HPLC fingerprint analysis indicated that the similarity between 16 batches of QF was in the range of 0.870-0.999. The above results provide some insights for the research on the pharmacodynamic constituents, quality control, and geographical discrimination of QF.

Jiawei Yanghe Decoction attenuate allergic airway inflammation by suppressing group 2 innate lymphoid cells responses

Ethnopharmacological relevanceJiawei Yanghe Decoction (JWYHD) is modified Yanghe Decoction (YHD). YHD historically utilized as a potent medicinal solution for addressing chronic inflammatory conditions, holds promising therapeutic potential in the treatment of asthma. However, the mechanisms underlying JWYHD's effects on allergic asthma remain unclear. Aim of the studyTo investigate the therapeutic effect as well as the underlying mechanisms of JWYHD on asthmatic mice. Materials and methodsThe ovalbumin (OVA)-induced mouse model was utilized, followed by the administration of JWYHD to allergic asthmatic mice. Subsequently, inflammatory cells in the bronchoalveolar lavage fluid (BALF) and lung tissues were conducted. The levels of various cytokines including interleukin (IL)-4, IL-5, IL-13, IL-33, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in BALF, as well as the total immunoglobulin E (IgE) content in serum, were assessed. Lung function and tissue pathology examinations were performed to assess the protective impacts of JWYHD. The chemical components of JWYHD and its lung prototype compounds (referred to the chemical components present in JWYHD that were observed in the lung) were explored by ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). RNA-seq analysis revealed the regulation mechanisms of JWYHD treating asthma. Furthermore, the effect of JWYHD on type 2 innate lymphoid cells (ILC2s) in asthmatic mice was detected by flow cytometry and Smart-RNA-seq analysis. Then molecular docking analysis was used to show the interaction between identified compounds and key targets. ResultsJWYHD significantly attenuated the airway inflammation of asthmatic mice, reduced the levels of inflammatory cells in BALF, as well the levels of the cytokines IL-4, IL-5, IL-13, IL-33, and TNF-α in BALF and IgE in serum. Airway hyperresponsiveness (AHR) and lung inflammation infiltration were also alleviated by JWYHD. Moreover, RNA-seq analysis revealed that JWYHD attenuated airway inflammation in asthmatic mice via regulating immunity. Flow cytometry confirmed that JWYHD could inhibit ILC2 responses. ILC2 Smart-RNA-seq analysis showed that JWYHD impaired the inflammation reaction-related signaling pathways in ILC2s, and neuropilin-1 (Nrp1), endothelial transcription factor 3 (GATA3) and interleukin 1 receptor like protein 1 (ST2) might be the key targets. The molecular docking analysis investigating the connection between the primary targets and JWYHD's prototype compounds in the lung demonstrated that liquiritin apioside, icariin, glycyrrhizic acid, and uralsaponin B, identified through UPLC-Q-TOF/MS, exhibited significant affinity in binding to the mentioned key targets. ConclusionOur results suggested that the mechanism of JWYHD in treating asthma might be related to limiting ILC2 responses. Our findings provided some pharmacological evidence for the clinical application of JWYHD in the treatment of asthma.

Qualitative and quantitative analysis of triterpenoids in different tissues of Pulsatilla chinensis

Pulsatilla chinensis (P.chinensis) is a traditional Chinese medicine used for the treatment of intestinal amebiasis diseases, vaginal trichomoniasis and bacterial infections. Tritepenoid saponins were important components of P.chinensis. Therefore, we asssessmented expression profiling of triterpenoids in different fresh tissues of P.chinensis by ultra high performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) and ultra high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS). Firstly, we identified 132 triterpenoids, including 119 triterpenoid saponins, 13 triterpenoid acids and forty seven of them were first determined in Pulsatilla genus, including new aglycones and new ways of rhamnose linking to the aglycone. Secondly, we established the analytical method to analysis triterpenoids content of P.chinensis and comprehensively verified the analytical method by linearity, precision, repeatability, stability and recovery. At last, we quantified 119 triterpenoids simultaneously based on UHPLC-QQQ-MS. The results show that the types and contents of triterpenoids had obvious tissue distribution. New components like rhamnose directly linked to the aglycone mainely distributed in aboveground tissues. Additionally, We identified 15 chemical ingredients as differential components between the aboveground and underground tissues of P.chinensis. This study provides an efficient analysis strategy for the qualitative and quantitative analysis of triterpenoids in P.chinensis even in other traditional Chinese medicines. At the same time, it provides important informations to explain the biosynthetic pathway of triterpenoid saponins in P.chinensis.

Metabolomics study of Berberidis Radix in intervening ulcerative colitis based on UPLC-Q-TOF-MS

The effect of Tujia medicine Berberidis Radix on endogenous metabolites in the serum and feces of mice with ulcerative colitis(UC) induced by dextran sulfate sodium(DSS) was analyzed by metabolomics technology to explore the metabolic pathway and underlying mechanism of Berberidis Radix in the intervention of UC. The UC model was induced in mice by DSS. Body weight, disease activity index(DAI), and colon length were recorded. The levels of tumor necrosis factor-α(TNF-α) and interleukin-10(IL-10) in colon tissues were determined by ELISA. The levels of endogenous metabolites in the serum and feces were detected by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS). Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were employed to characterize and screen differential metabolites. The potential metabolic pathways were analyzed by MetaboAnalyst 5.0. The results showed that Berberidis Radix could significantly improve the symptoms of UC mice and increase the level of the anti-inflammatory factor IL-10. A total of 56 and 43 differential metabolites were identified in the serum and feces, respectively, belonging to lipids, amino acids, fatty acids, etc. After the intervention by Berberidis Radix, the metabolic disorder gradually recovered. The involved metabolic pathways included biosynthesis of phenylalanine, tyrosine, and tryptophan, linoleic acid metabolism, phenylalanine metabolism, and glycerophospholipid metabolism. Berberidis Radix can alleviate the symptoms of mice with DSS-induced UC, and the mechanism may be closely related to the re-gulation of lipid metabolism, amino acid metabolism, and energy metabolism.

The “Crosstalk” between Microbiota and Metabolomic Profile of Kefalograviera Cheese after the Innovative Feeding Strategy of Dairy Sheep by Omega-3 Fatty Acids

The purpose of this study was to examine the effects of two different feeding systems, a control or a flaxseed and lupin diet (experimental), for a sheep flock, on the microbiota and metabolome of Kefalograviera cheese samples produced by their milk. In particular, the microbiota present in Kefalograviera cheese samples was analyzed using 16S rRNA gene sequencing, while ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was applied to investigate the chemical profile of the cheeses, considering the different feeding systems applied. The metagenomic profile was found to be altered by the experimental feeding system and significantly correlated to specific cheese metabolites, with Streptococcaceae and Lactobacillaceae establishing positive and negative correlations with the discriminant metabolites. Overall, more than 120 features were annotated and identified with high confidence level across the samples while most of them belonged to specific chemical classes. Characteristic analytes detected in different concentrations in the experimental cheese samples including arabinose, dulcitol, hypoxanthine, itaconic acid, L-arginine, L-glutamine and succinic acid. Therefore, taken together, our results provide an extensive foodomics approach for Kefalograviera cheese samples from different feeding regimes, investigating the metabolomic and metagenomic biomarkers that could be used to foresee, improve, and control cheese ripening outcomes, demonstrating the quality of the experimental Kefalograviera cheese.

Extraction, Isolation and Identification of Low Molecular Weight Peptides in Human Milk

Human milk contains numerous free low molecular weight peptides (LMWPs), which may play an important role in infant health and growth. The bioactivities of LMWPs are determined by their structures, especially the amino acid sequences. In the present study, 81 human milk samples were collected and purified by cation-exchange solid-phase extraction (SPE). Ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used for the separation and detection of free LMWPs in human milk. A total of 56 LMWPs were identified and quantified. These LMWPs were mainly derived from 3 regions of β-casein, which were the amino acid fragments of 16–40, 85–110, and 205–226. The predominant LMWPs were RETIESLSSSEESITEYK, RETIESLSSSEESITEYKQKVEKVK, ETIESLSSSEESITEYK, TQPLAPVHNPIS, and QPLAPVHNPISV with molecular weights of 2247.9573, 2860.2437, 2091.8591, 1372.7666, and 1271.7212, respectively. The results indicated that the technique based on SPE and UPLC-QTOF-MS might greatly facilitate the analysis of LMWPs in human milk.

Organ- and Age-Specific Differences of Dioscorea polystachya Compounds Measured by UPLC-QTOF/MS.

Dioscorea polystachya, named Chinese yam, is widely cultivated as a functional food and natural medicine in China. There is currently little information about the chemical characteristics of Dioscorea polystachya in different organs (tuber cortex and tuber flesh) and at various ages. In this study, an ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was used to profile chemical compounds in Dioscorea polystachya. As a result, thirty-eight compounds were detected in yam tuber cortex and tuber flesh. More compounds were detected in yam tuber cortex than in tuber flesh. Compounds such as dehydroepiandrosterone, allantoin and flavonoids were selected as biomarker candidates. Dehydroepiandrosterone was found more abundant in tuber flesh, while allantoin and flavonoids showed higher levels in tuber cortex. Furthermore, the levels of dioscin, malvalic acid and sucrose differed significantly among age groups and were highest in the tubers at 2 years. While the levels of allantoin, adenosine and glutamine increased with the growing years and were highest at 4 years. Thus, 2-year old Dioscorea polystachya tubers could be harvested to prepare dioscin, malvalic acid and sucrose supplements. The 4-year-old Dioscorea polystachya tubers would be the best choice for obtaining a large amount of allantoin and adenosine in industrial production.

Degradation of okadaic acid in seawater by UV/TiO2 photocatalysis – Proof of concept

The consumption of contaminated shellfish with marine toxins causes adverse socioeconomical, environmental and health impacts. The marine toxin okadaic acid (OA) provokes diarrhetic shellfish poisoning (DSP) syndrome characterized by severe gastrointestinal symptoms. Therefore, there is increasing interest in removing these toxins from the marine environment to protect shellfish harvesting sites. Photocatalysis is proposed as an efficient method to detoxify the marine environment.In this study, Prorocentrum lima was used to produce high purity DSP toxins, in particular OA, for degradation studies. The profiling, characterization and quantification of DSP toxins in the culture of P. lima were achieved by ultrahigh performance liquid chromatography coupled to quadrupole-time of flight mass spectrometry (UPLC-QTOF-MSE) for accurate-mass full spectrum acquisition data. The effectiveness of UV/TiO2 system to degrade OA in seawater was assessed in lab-scale experiments and identification of transformation products was proposed based on the data obtained during analysis by UPLC-QTOF-MSE. The detoxification potential of the UV/TiO2 system was investigated using the phosphatase inhibition assay.Sufficient amount of high-purity OA (25 mg, >90% purity) was produced in-house for use in photocatalysis experiments by simple reversed-phase flash chromatography. Complete degradation of OA was observed in seawater after 30 min and 7.5 min in deionized water. The rate constants fitted with the pseudo-first order kinetic model (R2 > 0.96). High-resolution mass spectrometry analysis of the photocatalyzed OA allowed tentative identification of four transformation products. Detoxification was achieved in parallel with the degradation of OA in deionized water and artificial ocean water (≤20 min) but not for seawater. Overall, results suggest that UV/TiO2 photocatalysis can be an effective approach for degrading OA and their TPs in the marine environment.To the best of our knowledge, this is the first report on the use of photocatalysis to degrade marine toxins and its promising potential to protect shellfish harvesting sites.

Untargeted Metabolomics to Evaluate the Stability of Extra-Virgin Olive Oil with Added Lycium barbarum Carotenoids during Storage.

A carotenoid-rich extract from Lycium barbarum L. was added to extra virgin olive oil (EVOO), obtaining a carotenoid-enriched oil (EVOOCar). The oxidative stability of EVOO and EVOOCar was evaluated during long-term storage of 28 weeks at room temperature, by measuring some classical parameters (acidity and peroxide values, spectrophotometric coefficients, fatty acid composition) and the content of minor compounds (i.e., α-tocopherol and lutein). At the end of the storage, higher content (p < 0.01) of α-tocopherol in EVOOCar in respect to EVOO were observed. Zeaxanthin dipalmitate, the most abundant carotenoid compound of Goji berries, decreased slightly (p < 0.05) in EVOOCar until the end of the storage. In regard to polyphenols, an ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS) using untargeted metabolomics was carried out. This latter approach discriminated the two oil samples during long-term storage, allowing to identify also the phenolic classes most exposed to significant variations during storage (i.e., mainly lignans and flavones). Besides, the addition of Goji carotenoids preserved the stability of tyrosol equivalents in EVOOCar during long-term storage. These results highlighted that the enrichment of EVOO with a carotenoid-rich extract can improve the shelf-life and nutritional value of added-oil, protecting EVOO natural antioxidants during long-term storage.

Saponin-Induced Shifts in the Rumen Microbiome and Metabolome of Young Cattle.

The aim of this study was to explore the effects of saponins on the rumen microbiota and the ruminal metabolome. Alfalfa hay (AH) and soybean hulls (SH) were used as fiber sources for the control diets. The AH and SH diets were supplemented with tea saponins resulting in two additional diets named AHS and SHS, respectively. These 4 diets were fed to 24 young male Holstein cattle (n = 6 per diet). After 28 days of feeding, the rumen fluid from these cattle was collected using an oral stomach tube. Illumina MiSeq sequencing and ultrahigh-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) were used to investigate the changes in the ruminal microbes and their metabolites. The relative abundance of Prevotellaceae_YAB2003 increased, while Ruminococcaceae_NK4A214 and Lachnospiraceae_NK3A20 decreased in SHS and AHS compared to SH and AHS, respectively. Feeding SHS resulted in higher ruminal concentrations of squalene, lanosterol, 3-phenylpropanoic acid, and citrulline compared to SH. The different microbial genes predicted by Tax4Fun were involved in amino sugar and nucleotide sugar metabolism. The pathways of arginine and proline metabolism, purine metabolism, and pyrimidine metabolism were enriched by different metabolites. Moreover, in the SH group, a positive correlation was observed between Prevotella_1 (Bacteroidetes), Prevotellaceae_YAB2003 (Bacteroidetes), and Christensenellaceae_R.7 (Firmicutes), and the metabolites, including citrulline, lanosterol, and squalene. The increased abundances of Prevotella_1, Ruminococcaceae_UCG.002, and Prevotellaceae_YAB2003 might result in increased fiber digestion and nutrient utilization but nutrient digestion was not measured in the current study. In summary, saponins have the ability to modulate the ruminal microbial community and ruminal metabolites and thus affect the rumen environment. However, the response seems to be dependent on the composition of the basal diet. This study provides a comprehensive overview of the microbial and biochemical changes in the rumen of cattle fed saponins.

Metabolites identificaion of two bioactive constituents in Trollius ledebourii in rats using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

Orientin and vitexin, 4'-hydroxyl-2-phenylchromen-4-one, are both major flavones derivatives found in Trollius ledebourii possessing definite pharmacological activities. In this study, in vitro metabolisms investigated on rat liver microsomes (RLMs) and in vivo metabolisms explored on Male Sprague Dawley rats of orientin and vitexin were tested, respectively. A systematic method based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was developed to characterize metabolites by means of electrospray ionization (ESI) mass spectrometry in positive ion mode. An on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to observe probable relevant metabolites. By comparison of chromatographic behaviors with reference substances, exact protonated ions, MS/MS fragment ions and relevant literature, a total of 12 metabolites of orientin and 23 metabolites of vitexin were detected, respectively, which suggested that orientin is more metabolically stable than vitexin. Oxidation, methylation, acetylation, reduction, loss of C6H10O5 and glucuronide conjugation were the major biotransformation routes of both of them in rats. More significant, glutamine conjugation, loss of CO and loss of CH2O were the unique metabolic pathways of vitexin compared with that of orientin for the first time. Besides, most metabolites were observed in rat urine and feces, implying that urine and feces were the active metabolic places for flavones. This is the first study on metabolisms of orientin and vitexin in vitro and in vivo simultaneously and the proposed metabolic pathways of them might provide further understanding of their pharmacological mechanisms and later study on their excretion.

Comparative metabolomics analysis for the compatibility and incompatibility of kansui and licorice with different ratios by UHPLC-QTOF/MS and multivariate data analysis.

Kansui, the root of Euphorbia kansui T.N. Liou ex T.P. Wang (Euphorbiaceae), is a well-known poisonous traditional Chinese medicine (TCM). However, many monographs of TCM indicated that it cannot be co-used with licorice, as kansui-licorice is a typical "eighteen incompatible" medicaments. Our previous studies have indicated that kansui was effective in treating malignant pleural effusion (MPE), and the efficacy could be weakened by the co-use of licorice, even causing serious toxicity at the given ratio. Nevertheless, the actual mechanisms of their dosage-toxicity-efficacy relationship need to be well clarified. The present study aimed to investigate the effect of individual and combined use of kansui and licorice on MPE rats, and explain the underlying mechanisms from a metabolomic perspective. Urine samples were analyzed by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). Partial least-squares discriminate analysis (PLS-DA) models were built to evaluate the interaction between kansui and licorice. Seven potential biomarkers contribute to the separation of model group and control group were tentatively identified. And selenoamino acid metabolism and nicotinate and nicotinamide metabolism with the impact-value 0.31 and 0.24, respectively, were filtered out as the most important metabolic pathways. Kansui and kansui-licorice at a ratio of 4:1 can treat MPE rats by adjusting abnormal metabolic pathways to the normal state, while it may have opposite result with kansui-licorice 1:4. The different influences to the two metabolic pathways may partially explain the dosage-toxicity-efficacy relationship of kansui-licorice with different ratios. The results could offer valuable insights into the compatibility property changes for the two herbs.

Metabolomic approach for Extra virgin olive oil origin discrimination making use of ultra-high performance liquid chromatography – Quadrupole time-of-flight mass spectrometry

The fraudulent miss-description on food product labels regarding origin or composition is a widespread problem. In this work, a metabolomic approach based on the use of ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) has been applied to identify the differentiating chemical markers that allow geographic origin discrimination between different Spanish Extra Virgin Olive Oils (EVOOs). For this purpose, ninety EVOOs from 6 Spanish regions were analyzed. Data processing consisted on peak picking, retention time alignment and response normalization. Partial Least Square Discriminant Analysis (PLS-DA) and orthogonal PLS-DA (OPLS-DA) were applied to identify the most significant markers that allow groups separation. Twelve different compounds were found to correctly separate the EVOOs from their origin and 7 of them could be tentatively identified. The results of our work suggest that UHPLC-QTOF MS-based metabolomic analysis is a suitable approach for biomarker-detection in the food quality/safety field.

UHPLC–MS-based metabolomics analysis on mice bearing neoplasm (H22) for hispidulin

Although some physiological and pathological function parameters of hepatitis and liver cancer have been investigated in relation to hispidulin (5,7,4′-trihydroxy-6-methoxyflavone), the changes of small metabolites in biofluids have been reported rarely. Recent research has shown that metabolic profiling with ultra-high-performance liquid chromatography coupled to quadrupole time of flight mass spectrometry (UHPLC-QTOF/MS) coupled with multivariate statistical analysis provides a good understanding of hispidulin effects on mice vaccinated intraperitoneally with H22 tumor cells. Twenty-five potential biomarkers, up- or down-regulated (P<0.05 or 0.01), were identified, and 17 metabolic pathways were constructed. These potential biomarkers underpin the metabolic pathways, which are disturbed in the mice bearing neoplasm (H22). These pathways include pantothenate and CoA biosynthesis; glycine, serine and threonine metabolism; nicotinate and nicotinamide metabolism; steroid hormone biosynthesis; pyrimidine metabolism; and glyoxylate and dicarboxylate metabolism. Furthermore, 4-phosphopantothenoylcysteine, glycine, niacinamide, cortisol, uracil and 5-thymidylic acid are potential biomarkers that may explain the link between hispidulin and the metabolism of mice bearing neoplasm (H22). Most of the potential biomarkers related to the function of TCA (tricarboxylic acid cycle). The rise of potential biomarkers in the drug groups promoted the up-regulation of TCA cycle compared with the model group.

A metabolomics strategy to explore urinary biomarkers and metabolic pathways for assessment of interaction between Danhong injection and low-dose aspirin during their synergistic treatment.

The drug combination of Danhong injection (DHI) and low-dose aspirin (ASA) was frequently applied for the treatment of cardiovascular and cerebrovascular diseases. Due to the drug interactions, a lot of potential benefits and risks might exist side by side in the course of combination therapy. However, there had been no studies of interaction between DHI and ASA. Metabolomics was a powerful tool to explore endogenous biomarkers and metabolic pathways. In present study, metabolic profiling with ultra-high-performance liquid chromatography coupled to quadrupole time of flight mass spectrometry (UHPLC-QTOF/MS) coupled with multivariate statistical analysis was performed to provide insight into understanding the interaction between DHI and low-dose ASA. Eleven potential biomarkers of three types were identified and seven metabolic pathways were constructed. The results showed that the interaction between DHI and low-dose ASA during synergistic treatment indeed affected some key endogenous biomarkers and metabolic pathways, which could not happen when DHI or low-dose ASA was used alone. The quality and quantity of endogenous metabolite were both influenced by interaction between DHI and low-dose ASA. In details, the amount of flavin mononucleotide, L-2, 4-diaminobutyric acid (DABA) and 4-aminohippuric acid were significantly increased. On the contrary, the amount of 3-methyluridine, 4, 6-dihydroxyquinoline, cortolone-3-glucuronide, and serotonin were significantly decreased. Furthermore, O-phosphotyrosine, 3-methyl-2-butenal, indoxyl sulfate and dolichyl diphosphate were disappeared in urine. As to metabolic pathways, riboflavin metabolism, pentose and glucuronate interconversions, and tryptophan metabolism were all significantly influenced. The emerging alterations of biomarkers and metabolic pathways were associated with a lot of drugs and diseases based on literature researches, which might influence the co-administration of other drugs or the treatments of relevant diseases. Our paper presented some hints to uncover the mechanism of interaction between DHI and low-dose ASA, which would provide some references for application of DHI and low-dose ASA combination.

Urine and plasma metabonomics coupled with UHPLC-QTOF/MS and multivariate data analysis on potential biomarkers in anemia and hematinic effects of herb pair Gui-Hong

The compatibility of Angelicae Sinensis Radix (Danggui) and Carthami Flos (Honghua), a famous herb pair Gui-Hong, can produce synergistic and complementary hematinic effects. Our previous studies have indicated that Gui-Hong has therapeutic potential treatment in hemolytic and aplastic anemia (HAA). The present study aimed to investigate the hematinic effects of Danggui, Honghua and Gui-Hong on HAA rats induced by acetyl phenylhydrazine (APH) and cyclophosphamide (CP) and to explore the underlying hematinic regulation mechanisms. Rats were divided into 5 groups, and drugs were administered by oral gavage one time each day for continuous 7 days from the experiment began. Urine and plasma were analyzed by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). Partial least-squares discriminate analysis (PLS-DA) models were built to evaluate the therapeutic effects of Danggui, Honghua and Gui-Hong. Pearson correlation matrix analysis method was used to discover the correlations between potential biomarkers and biochemical indicators of HAA rats. Seven potential biomarkers contribute to the separation of model group and control group were tentatively identified. The levels of l-kynurenine, phenylalanine, nicotinic acid and sphingosine increased significantly (P<0.05) in HAA rats, while the levels of l-isoleucine, l-tyrosine and serotonin decreased significantly (P<0.05) in comparison with control rats. Those endogenous metabolites were chiefly involved in phenylalanine, tyrosine and tryptophan biosynthesis, valine, leucine and isoleucine biosynthesis, tryptophan metabolism and tyrosine metabolism. The metabolic deviations could be regulated closer to normal level after Danggui, Honghua and Gui-Hong intervention. In term of hematinic effects, Gui-Hong was the most effective as shown by the relative distance in PLS-DA score plots and relative intensity of potential biomarkers. The result reflected the synergic action between Danggui and Honghua. The above results were found to be reasonable in explaining the hematinic effects mechanism of Gui-Hong. The results of routine blood, urinary metabolic pattern and plasma metabolic pattern show the Danggui, Honghua and Gui-Hong groups are moving toward the control group and the HAA was being prevented and alleviated. The effect of Gui-Hong group is more remarkable than Danggui and Honghua groups. Some potential biomarkers like l-kynurenine, phenylalanine, l-isoleucine, l-tyrosine, serotonin, nicotinic acid and sphingosine have been found and identified. The work shows that the metabonomics method is a promising tool in the efficacy and mechanism research of traditional Chinese medicines.

Comparative metabolomics analysis on invigorating blood circulation for herb pair Gui-Hong by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and pattern recognition approach

The compatibility of Angelicae Sinensis Radix (Danggui, DG) and Flos Carthami (Honghua, HH), a famous herb pair Gui-Hong (GH), can produce synergistic and promoting blood effects. Although some physiological and pathological function parameters of the acute blood stasis have been investigated, little information about the changes of small metabolites in biofluids has been reported. In present study, global metabolic profiling with ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) combined with pattern recognition method was performed to discover the underlying blood-activating regulation mechanisms of DG, HH and GH on the acute blood stasis rats induced by subcutaneous injection of adrenaline hydrochloride and ice water bath. The total 14 metabolites (10 in urine and 4 in plasma), up regulated or down regulated (P<0.05 or 0.01), were identified and contributed to the acute blood stasis progress. These promising identified biomarkers underpin the metabolic pathway including phenylalanine metabolism, sphingolipid metabolism, arachidonic acid metabolism and arginine and proline metabolism are disturbed in the acute blood stasis rats, which identified by using pathway analysis with MetPA. The altered metabolites and hemorheological indexes could be regulated closer to normal level after DG, HH and GH intervention. In term of activate blood circulation function, GH was the most effective as shown by the relative distance in PLS-DA score plots and relative intensity of metabolomics trategy, reflecting the synergic action between Danggui and Honghua. The results demonstrated that biofluids metabolomics was a powerful tool in clinical diagnosis and treatment of syndrome of blood stasis for providing information on changes in metabolites pathways.