A method toward constituents with weak response in mass spectra for comprehensively characterizing constituents in traditional Chinese medicine formula, Kangfuxiaoyanshuan as a case.

How to identify the traces with weak response and poor mass spectrometry data is still a barrier to comprehensively characterize chemical constituents of traditional Chinese medicine formula. Thus, we took Kangfuxiaoyanshuan as a carrier to perform a method toward these weak response constituents in mass spectra. Chemical constituents profiling spectra of each herb and formula were firstly obtained by an ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry. Next, the high response constituents in the formula were identified and suspicious constituents with weak response were classified preliminarily according to the reflection of chemical components from each herb. In order to clarify the suspicious, a method increasing detection concentration, optimizing chromatographic separation conditions, and online parameters in mass was established. As a result, a total of 123 chemical components including 43 suspicious components in Kangfuxiaoyanshuan were classified and characterized unambiguously.

Application of characteristic fragment filtering with ultra high performance liquid chromatography coupled with high-resolution mass spectrometry for comprehensive identification of components in Schisandrae chinensis Fructus.

An integrated strategy of characteristic fragment filtering combined with target database screening based on ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry was proposed for comprehensive profiling of components in Schisandrae chinensis Fructus. The strategy consisted of following five steps: (1) Representative standards were analyzed by ultra high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer for characteristic fragments and fragmentation rules of each structure type. (2) The raw data of 70% methanol extract was collected by ultra high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry. (3) The chemical components database that consisted of names, chemical formulas and structures of potential components in Schisandrae chinensis Fructus was established by summarizing previous literature to screen the collected liquid chromatography with mass spectrometry data and obtain matched compounds. (4) Characteristic fragments, literature, and reference standards were used to verify the matches. (5) Characteristic fragment filtering combined with online database querying was used to deduce potential new compounds. As a result, a total of 94 compounds were identified or characterized and 16 of them were potential new compounds. The study provided a reference for comprehensive characterization of ingredients in herbal medicine and formed the foundation for pharmacodynamic study of Schisandrae chinensis Fructus.

[Methods to establishment of serum induced syndrome of liver-depression and spleen-deficiency cell model].

To explore the method of establishing a cell model of traditional Chinese medicine (TCM) syndromes, HepG2 cells were induced by human serum of liver-depression and spleen-deficiency syndrome(LDSDS) to establish a cell model of LDSDS in this research. The concentration of cells, the content of human serum in culture medium and the growth characteristics of model-cell (cell growth curve, the survival rate and apparent morphology were investigated by MTT assay and microscopy. Evaluation of syndrome cell model: metabolomics was used to analyze the human serum of normal individuals and patients with LDSDS, and cell models induced by these serums, respectively. We obtained the difference metabolites from serums and cell models of LDSDS, respectively; then compared the biomarkers from two metabolomics and their metabolic pathways, to verify that the reliability and applicability of the model. Metabolomics data were collected by UPLC-Q-TOF-MS, and then all data were analyzed by multivariate statistical (PCA，OPLS-DA). The results showed that, model cells have the characteristics of normal growth, slow proliferation and stable morphological structure inducted by 10% serum of LDSD in 24-72 h. There were the same 19 difference metabolites which from the human serum of normal individuals and patients with LDSDS, and cell models induced by these serums; including 9 metabolic pathways that play an important role in maintaining normal physiological activities of the human body, such as lipids, amino acids, nucleotides, and energy metabolism etc. It was shown that the established syndrome cell model can reflect the biological basis of LDSDS to some extent. This research provides a reference method for the establishment of TCM syndrome cell model.

Pharmacometabolomics in Endogenous Drugs: A New Approach for Predicting the Individualized Pharmacokinetics of Cholic Acid.

The evaluation of individual variability in endogenous drugs' metabolism and disposition is a very challenging task. We developed and validated a metabotype to pharmacokinetics (PK) matching approach by taking cholic acid as an example to predict the individualized PK of endogenous drugs. The stable isotope-labeled cholic acid was selected as the substitute analyte of cholic acid to ensure the accurate measurement of blood concentration. First, large-scale metabolite profiling studies were performed on the predose urine samples of 28 rats. Then, to examine the individualized PK of deuterium 4-cholic acid (d4-cholic acid) in these rats, we determined its plasma concentrations and calculated the differential AUC values. Subsequently, we conducted a two-stage partial least-squares analysis in which 31 baseline metabolites were screened initially for predicting the individualized AUC values of d4-cholic acid using the data of predose urine metabolites. Finally, network biology analysis was applied to give the biological interpretation of the individual variances in cholic acid metabolism and disposition, and the result further narrowed the selection of baseline metabolites from 31 to 2 (sarcosine and S-adenosyl-l-homocysteine) for such prediction. Collectively, this pharmacometabolomics research provided a new strategy for predicting individualized PK of endogenous drugs.

Characterization of the potential new phthalides in Ligusticum chuanxiong Hort. using ultra-performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry.

The characterization of unknown compounds is still a great challenge currently. A strategy for deduction of potential new phthalides through the characterization of isomers based on ultra-performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry was proposed here to characterize the unknown compounds of Ligusticum chuanxiong Hort. (Chuanxiong). This proposed strategy consisted of four steps: (1) the high resolution MS data was collected, and the peaks were screened preliminarily by UNIFITM platform based on the in-house database; (2) the fragmentation patterns and the characteristic fragments were summarized based on the representative standards; (3) the target compounds were identified based on the fragmentation rules, standards comparison and false positive exclusion; (4) the unknown components were structurally characterized according to the accurate mass and fragmentation patterns analysis. This strategy was successfully applied to the identification and deduction of phthalides in Chuanxiong. A total of 81 phthalides were detected. Fifty-five known phthalides were identified, and 26 potential new phthalides were characterized. This research enriched the material basis of Chuanxiong, and provided a liquid chromatography tandem mass spectrometry-oriented method for the discovery of the potential new compounds.

Applying characteristic fragment filtering for rapid detection and identification of ingredients in rhubarb by HPLC coupled with linear ion trap-Orbitrap mass spectrometry.

Chemical characteristic fragment filtering in MSn chromatograms was proposed to detect and identify the components in rhubarb rapidly using high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry. Characteristic fragments consist of diagnostic ions and neutral loss fragments. Characteristic fragment filtering is a postacquisition data mining method for the targeted screening of groups with specific structures, including three steps: first, in order to comprehensively summarize characteristic fragments for global identification of the ingredients in rhubarb, representative authentic standards of dominant chemical categories contained in rhubarb were chosen, from which fragmentation rules and a characteristic fragments schedule were proposed; second, characteristic fragment filtering was used to rapidly recognize analogous skeletons; finally, combined with retention time, accurate mass, characteristic fragments, and previous literature, the structures of the filtered compounds were identified or tentatively characterized. As a result, a total of 271 compounds were detected and identified in rhubarb, including 34 anthraquinones, 83 anthrones, 46 tannins, 17 stilbenes, 24 phenylbutanones, 26 acylglucosides, 26 chromones, and 15 other compounds, 69 of which are potentially new compounds. The proposed characteristic fragment filtering strategy would be a reference for the large-scale detection and identification of the ingredients of herbal medicines.

[Rapid analysis on phenolic compounds in Rheum palmatum based on UPLC-Q-TOF/MSE combined with diagnostic ions filter].

Diagnostic ions filter method was used to rapidly detect and identify the phenolic compounds in Rheum palmatum based on ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MSE). The representative authentic standards of phenolic compounds, including gallic acid, (+)-catechin, (－)-epicatechin, (－)-epicatechin-3-O-gallate and procyanidin B2, were subjected to analysis by UPLC-Q-TOF/MSE system with negative ion mode. Fragmentation patterns of each standard were summarized based on assigned fragment ions. The prominent product ions were selected as diagnostic ions. Subsequently, diagnostic ions filter was employed to rapidly recognize analogous skeletons. Combined with retention time, accurate mass, characteristic fragments and previous literature data, the structures of the filtered compounds were identified or tentatively characterized. A total 63 phenolic compounds (36 phenolic acid derivatives, 8 flavonoid derivatives and 19 tennis derivatives) in R. palmatum were identified, including 6 potential new compounds. The method of diagnostic ions filter could rapidly detect and identify phenolic compounds in R. palmatum This study provides a method for rapid detection of phenolic compounds in R. palmatum and is expected to complete the material basis of rhubarb.

Integrated Metabolomics and Network Pharmacology Study on the Mechanism of Kangfuxiaoyan Suppository for Treating Chronic Pelvic Inflammatory Disease.

Kangfuxiaoyan suppository (KFXYS) is a commonly used traditional Chinese medicine (TCM) preparation for the treatment of chronic pelvic inflammatory disease (CPID) clinically, and its safety and effectiveness have been well verified. However, the potential mechanism remains unclear. The integrated strategy of metabolomics and network pharmacology was employed in the study to reveal the potential mechanism of KFXYS in the treatment of CPID. Our research consists of five steps. First, the effect of KFXYS in reversing uterine inflammation indexes was verified. Second, based on the comprehensive characterization of 123 chemical ingredients of KFXYS, the ingredients of KFXYS absorbed into blood were identified by UPLC-Q-TOF/MS, then ADME research was carried out on the main ingredients. Third, the differential metabolites with significant correlation to inflammatory indexes were discovered by metabolomics and correlation analysis. Fourth, the potential targets and pathways of KFXYS in treating CPID were predicted by network pharmacology based on the ingredients which had good ADME behavior. Fifth, the proteins in common pathways of metabolomics and network pharmacology were used to screen the key targets from the potential targets of network pharmacology, and the potential mechanism of KFXYS in treating CPID was clarified. As a result, KFXYS significantly reversed the uterine inflammation indexes, including IL-1 and IL-6. The ingredients absorbed into blood including matrine, sophocarpine, aloin, esculetin-O-glucuronide, 7,4'-dihydroxyisoflavone-O-glucuronide, and 4'-methoxyisoflavone-7-O-glucuronide had good ADME behavior in vivo. Among the differential metabolites, Leukotriene A4, 5-Hydroxyindoleacetic acid, Ornithine, Arginine, and PC (20:1 (11Z)/20:4 (8Z,11Z,14Z,17Z)) were significant correlation to inflammation indexes. The integration analysis of metabolomics and network pharmacology shows that KFXYS may regulate the key targets including ARG1, NOS2, NOS3, etc. We speculate that ingredients of KFXYS, such as matrine, sophocarpine, aloin etc. act on the key proteins including ARG1, NOS2, and NOS3, to exert anti-inflammatory effect.

Serum metabolic profiling of traditional Chinese medicine syndromes in patients with diarrhea-predominant irritable bowel syndrome.

OBJECTIVE: The clinical symptoms of diarrhea-predominant irritable bowel syndrome (IBS-D) can be effectively improved by traditional Chinese medicine (TCM) treatment, based on the usage of specific therapies for different TCM syndromes. However, in the stage of diagnosis, the standard criteria for the classification of TCM syndrome were still deficient. Through serum metabolic profiling, this study aimed to explore potential biomarkers in IBS-D patients with different TCM syndromes, which can assist in diagnosis of the disease. METHODS: Serum samples were collected from healthy controls (30 cases), IBS-D patients with Liver-Stagnation and Spleen-Deficiency syndrome (LSSD, 30 cases), Yang Deficiency of Spleen and Kidney syndrome (YDSK, 11 cases) and Damp Abundance due to Spleen-Deficiency syndrome (DASD, 22 cases). Serum metabolic profiling was conducted by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The potential biomarkers were screened by orthogonal partial least square-discriminate analysis, while metabolic pathways undergoing alterations were identified by pathway enrichment analysis in MetaboAnalyst 4.0. RESULTS: Overall, 34 potential biomarkers were identified in LSSD group, 36 in YDSK group and 31 in DASD group. And the 13 metabolites shared by three groups were determined as the potential biomarkers of IBS-D. Glycerophospholipid metabolism was disturbed significantly in IBS-D patients, which may play a role in IBS-D through inflammation. What's more, three TCM syndromes have the specific potential biomarkers in glycerophospholipid metabolism. CONCLUSION: The serum metabolomics revealed that different TCM syndrome types in IBS-D may have different metabolic patterns during disease progression and glycerophospholipid metabolism was one of the pathways, whose metabolism was disturbed differently among three TCM syndromes in IBS-D. Therefore, the specific potential biomarkers in glycerophospholipid metabolism of three TCM syndromes in IBS-D can serve as the objective indicators, which can facilitate the TCM-syndrome objective classification of IBS-D.

Deciphering biochemical basis of Qingkailing injection-induced anaphylaxis in a rat model by time-dependent metabolomic profiling based on metabolite polarity-oriented analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingkailing injection (QKLI) is prepared from eight traditional Chinese medicinal materials or their extracts, which is widely used in clinical practice to treat the upper respiratory inflammation, pneumonia, high fever and viral encephalitis, nonetheless, suffering from serious anaphylaxis. AIM OF STUDY: This study aims to develop an integrative metabolomics approach for deciphering the biochemical basis of QKLI induced anaphylaxis (QKLI-IA). MATERIALS AND METHODS: The accuracy of animal modeling, the coverage of detected metabolites and the timeliness of pathological reaction are three key factors for revealing the biochemical basis of disease with untargeted metabolomics. In this study, firstly, the allergic rats (responders) were first screened by passive cutaneous anaphylaxis experiment and then were utilized for modeling. To cover a wider range of metabolites, a large-scale untargeted metabolomics based on metabolites polarity-oriented analysis was performed using ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Then, to evaluate the timeliness of QKLI-IA, a time-dependent metabolomic profiling including the early, mid and late anaphylaxis stages of QKLI-IA, was performed. RESULTS: Corresponding to early, mid and late anaphylaxis stages of QKLI-IA, 14, 9 and 4 potential biomarkers were identified, respectively. Metabolism pathway analysis revealed that QKLI-IA resulted in dynamic changes in serum amino acid, fatty acid, glycerolipid, and phospholipid metabolisms. Twenty-four metabolites were found with identical fluctuating trends across the three stages of QKLI-IA. The results indicate that the pathogenesis of QKLI-IA is closely related to arachidonic acid metabolism. CONCLUSION: This research provides a methodology reference for revealing the biochemical basis of disease using metabolomic profiling and offers a new insight to understand the pathogenesis of QKLI-IA.

An integrated strategy by using target tissue metabolomics biomarkers as pharmacodynamic surrogate indices to screen antipyretic components of Qingkaikling injection.

Traditional Chinese medicine (TCM) treatment can be valuable therapeutic strategies. However, the active components and action mechanisms that account for its therapeutic effects remain elusive. Based on the hypothesis that the components of a formula which exert effect would be measurable in target tissue, a target tissue metabolomics-based strategy was proposed for screening of antipyretic components in Qingkaikling injection (QKLI). First, we detected the components of QKLI which could reach its target tissue (hypothalamus) by determining the hypothalamus microdialysate and discovered that only baicalin and geniposide could be detected. Then, by conducting hypothalamus metabolomics studies, 14 metabolites were screened as the potential biomarkers that related to the antipyretic mechanisms of QKLI and were used as its pharmacodynamic surrogate indices. Subsequently, the dynamic concentration of baicalin and geniposide in hypothalamus microdialysates and biomarkers in hypothalamus were measured and correlated with each other. The results indicated that only baicalin shown a good correlation with these biomarkers. Finally, a network pharmacology approach was established to validate the antipyretic activity of baicalin and the results elucidated its antipyretic mechanisms as well. The integrated strategy proposed here provided a powerful means for identifying active components and mechanisms contributing to pharmacological effects of TCM.

Using UHPLC Q-Trap/MS as a complementary technique to in-depth mine UPLC Q-TOF/MS data for identifying modified nucleosides in urine.

Modified nucleosides, metabolites of RNA, are potential biomarkers of cancer before the appearance of morphological abnormalities. It is of great significance to comprehensively detect and identify nucleosides in human urine for discovery of cancer biomarkers. However, the lower abundance, the greater polarity and the matrix effects make it difficult to detect urinary nucleosides. In this paper, an integrated method consisted of sample preparation followed by ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC Q-TOF/MS) detection and primary identification, then ultra-high performance liquid chromatography coupled with hybrid triple quadrupole linear ion trap mass spectrometer (UHPLC Q-Trap/MS) further identification and validation were introduced. Firstly, to enrich the nucleosides and eliminate the urine matrix effects, different sorbent materials of solid phase extraction (SPE) and the elution conditions were screened. Secondly, UPLC Q-TOF/MS was used to acquire mass data in MSE mode. The structural formulas of nucleosides in urine sample were primarily identified according to retention time, accurate mass precursor ions and fragment ions from in-house database and online database. Thirdly, the preliminary identified nucleoside structures lacking of characteristic fragment ions were verified by UHPLC Q-Trap/MS in multiple reaction monitoring trigger enhanced product ion scan (MRM-EPI) and neutral loss scan (NL). At last, phenylboronic acid (PBA)-based SPE was utilized due to its higher MS signal and weaker matrix effects under optimized extraction conditions. Fifty-five nucleosides were primarily identified by UPLC Q-TOF/MS, among which 50 nucleosides were confirmed by UHPLC Q-Trap/MS. Five nucleosides, namely 4',5'-didehydro-5'-deoxyadenosine, 4',5'-didehydro-5'-deoxyinosine, isonicotinamide riboside, peroxywybutosine and hydroxywybutosine, were found from urine for the first time. The results will expand the Human Metabolome Database (HMDB).