PDMS-TiO2 composite films combined with LC-MS/MS for determination of phospholipids of urine in non-small cell lung cancer patients with traditional Chinese medicine syndromes.

Lung cancer is one of the most common malignant tumors in China. Most patients are already in the mid to advanced stages during the consultation and the survival rate is less than 23 % with a poor prognosis. Therefore, effective dialectical diagnosis of advanced cancer can guide individualized treatment to improve survival. Phospholipids are the building blocks of cell membranes and abnormal phospholipid metabolism is associated with plentiful diseases. Most studies of disease markers use blood as a sample. However, urine covers extensive metabolites that are produced during the body's metabolic processes. Therefore, the study of markers in urine can be used as a complement to improve the diagnosis rate of marker diseases. Moreover, urine is characterized by high water content, high polarity, and high inorganic salt, therefore the detection of phospholipids in urine is challenging. In this study, an original Polydimethylsiloxane (PDMS)-titanium dioxide (TiO2) composite film for sample pre-treatment coupled with the LC-MS/MS method for the determination of phospholipids in the urine with high selectivity and low matrix effects was prepared and developed. The extraction process was scientifically optimized by the single-factor test. After systematic validation, the established method was successfully applied to the accurate determination of phospholipid substances in the urine of lung cancer patients and healthy subjects. In conclusion, the developed method has great potential for the development of lipid enrichment analysis in urine and can be used as a beneficial tool for cancer diagnosis and Chinese medicine syndrome typing.

A Dual Coverage Monitoring of the Bile Acids Profile in the Liver-Gut Axis throughout the Whole Inflammation-Cancer Transformation Progressive: Reveal Hepatocellular Carcinoma Pathogenesis.

Hepatocellular carcinoma (HCC) is the terminal phase of multiple chronic liver diseases, and evidence supports chronic uncontrollable inflammation being one of the potential mechanisms leading to HCC formation. The dysregulation of bile acid homeostasis in the enterohepatic circulation has become a hot research issue concerning revealing the pathogenesis of the inflammatory-cancerous transformation process. We reproduced the development of HCC through an N-nitrosodiethylamine (DEN)-induced rat model in 20 weeks. We achieved the monitoring of the bile acid profile in the plasma, liver, and intestine during the evolution of "hepatitis-cirrhosis-HCC" by using an ultra-performance liquid chromatography-tandem mass spectrometer for absolute quantification of bile acids. We observed differences in the level of primary and secondary bile acids both in plasma, liver, and intestine when compared to controls, particularly a sustained reduction of intestine taurine-conjugated bile acid level. Moreover, we identified chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, and glycolithocholic acid in plasma as biomarkers for early diagnosis of HCC. We also identified bile acid-CoA:amino acid N-acyltransferase (BAAT) by gene set enrichment analysis, which dominates the final step in the synthesis of conjugated bile acids associated with the inflammatory-cancer transformation process. In conclusion, our study provided comprehensive bile acid metabolic fingerprinting in the liver-gut axis during the inflammation-cancer transformation process, laying the foundation for providing a new perspective for the diagnosis, prevention, and treatment of HCC.

Accurate determination for lipidomics based on LC-tandem-MS parameters modeling, prediction, and database: Monitoring the progression of hepatocellular carcinoma.

The unavailability of adequate lipid reference standards is a major challenge for accurate quantitative analysis of lipidomics. Based on the discovery of regularity and predictability for lipids in chromatography-mass spectrometry behaviors, "target compound-structure correlation-analytical parameter database" protocol and "modeling-prediction" strategy were carried out to calculate the relative coefficients of analytical parameters within each subclass. Then the relevant LC-tandem-MS parameters of unknown lipids were predicted and a quantification parameter database for 4081 lipids was established and validated. Reference standards-independent accurate determination for lipidomics was achieved with the parameter's database and applied to monitor the change of lipid metabolism in the plasma of whole course of health-hepatitis-cirrhosis-hepatocellular carcinoma (HCC). Combined Student's t test, orthogonal partial least squares discrimination analysis (OPLS-DA) and binary logistic regression-ROC analysis, lipid biomarkers for differentiating health from each disease and differentiating different stages of disease were identified and the pathogenesis of HCC was preliminarily clarified. The established methodology would shed light on comprehensive and accurate quantitative lipidomics and exploring the pathomechanism and potential therapeutic targets of HCC.

A convenient and efficient 4-(diethylamino)-butylamine-labeled polarity-response-homodispersed strategy for absolute quantification of carboxyl submetabolome: Monitoring the whole progressive course of hepatocellular carcinoma.

Recent studies have shown that dysregulation of carboxyl submetabolome homeostasis is closely related to the occurrence and development of hepatocellular carcinoma (HCC). However, it is still a challenge to quantify carboxyl metabolites with high efficiency by conventional methods due to their species diversity and nature differences. Moreover, there are few studies on carboxyl submetabolome profiling during the whole progression of HCC. In this study, a convenient and efficient workflow for absolute quantification of carboxyl submetabolome was established based on the 4-(diethylamino)-butylamine (DEABA)-labeled polarity-response-homodispersed strategy. After optimizing derivation conditions with response surface methodology (RSM), the reaction only needed 1 min at room temperature, which radically simplified the labeling process. Compared with nonderivatization, the gaps of polarities and responses of the analytes were narrowed by DEABA labeling, realizing the polarity-response-homodispersion. Then resolution and sensitivity in HPLC-MS/MS were improved significantly. Ultimately, the workflow was applied to monitor carboxyl metabolic profile in human plasma of the whole progressive course of hepatocellular carcinoma. Combined with analysis of variance (ANOVA), orthogonal partial least squares discrimination analysis (OPLS-DA), Bayesian linear discriminant analysis (BLDA) and other data mining methods, the biomarkers of "health-hepatitis-cirrhosis-HCC" were screened out, and the diagnostic model was established. Furthermore, the relevancy between carboxyl submetabolome disorders and the pathogenesis of HCC was investigated. The developed method has the characteristics of high sensitivity, high coverage and high practicability, which is suitable for the study of biomarkers of carboxyl submetabolome in the whole progression of HCC.

Assessment of plasma amino acids, purines, tricarboxylic acid cycle metabolites, and lipids levels in NSCLC patients based on LC-MS/MS quantification.

Non-small cell lung cancer (NSCLC) is the most common type of malignant tumor of the lung with poor prognosis. Currently, there is still no effective strategy for diagnosing lung cancer from the perspective of multiple biomarkers containing both polar and nonpolar molecules. In order to explore the pathological changes of NSCLC at the endogenous molecule levels, and further establish the strategy for identifying and monitoring drug efficacy of NSCLC, targeted metabolomics and lipidomics studies were established with NSCLC patients. Polar metabolites including 21 amino acids, 7 purines, 6 tricarboxylic acid (TCA) cycle metabolites, and nonpolar lipids like phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), sphingomyelin (SM), and ceramide (Cer), diacylglycerol (DG), triacylglycerol (TG), were quantitatively determined based on LC-MS/MS, taking into account their metabolism were significantly concerned with the occurrence of lung cancer in previous study. As a result, 14 polar metabolites and 16 lipids were prominently altered in the plasma of NSCLC patients, among which, after multivariate statistical analysis, LPC 18:0 (sn-2), L-Phenylalanine (Phe), oxaloacetic acid (OAA) and xanthine (XA) were screened out as potential small molecules and lipid biomarkers for NSCLC. Furthermore, a new strategy for formulating equation of NSCLC identification was proposed and clinical utility was successfully evaluated through Kangai injection treatment to NSCLC patients. Taking together, this study investigated the pathological changes of NSCLC from the perspective of endogenous polar and nonpolar molecules, and shed a light on identification of NSCLC.

A Comprehensive Analysis of Microflora and Metabolites in the Development of Ulcerative Colitis into Colorectal Cancer Based on the Lung-Gut Correlation Theory.

The lungs and large intestine can co-regulate inflammation and immunity through the lung-gut axis, in which the transportation of the gut microbiota and metabolites is the most important communication channel. In our previous study, not only did the composition of the gut microbiota and metabolites related to inflammation change significantly during the transition from ulcerative colitis (UC) to colorectal cancer (CRC), but the lung tissues also showed corresponding inflammatory changes, which indicated that gastrointestinal diseases can lead to pulmonary diseases. In order to elucidate the mechanisms of this lung-gut axis, metabolites in bronchoalveolar lavage fluid (BALF) and lung tissues were detected using UHPLC-Q-TOF-MS/MS technology, while microbiome characterization was performed in BALF using 16S rDNA sequencing. The levels of pulmonary metabolites changed greatly during the development of UC to CRC. Among these changes, the concentrations of linoleic acid and 7-hydroxy-3-oxocholic acid gradually increased during the development of UC to CRC. In addition, the composition of the pulmonary microbiota also changed significantly, with an increase in the Proteobacteria and an obvious decrease in the Firmicutes. These changes were consistent with our previous studies of the gut. Collectively, the microbiota and metabolites identified above might be the key markers related to lung and gut diseases, which can be used as an indication of the transition of diseases from the gut to the lung and provide a scientific basis for clinical treatment.

Metabolic Study of Tetra-PEG-Based Hydrogel after Pelvic Implantation in Rats.

In vivo metabolism of polyethylene glycol (PEG) hydrogels has rarely been studied. In this study, we prepared a chemically crosslinked hydrogel formulation using 14C-labeled tetra-armed poly (ethylene glycol) succinimidyl succinate (Tetra-PEG-SS) and 3H-labeled crosslinking agent for implantation into the pelvis of Sprague-Dawley (SD) rats. This radioactive labeling technique was used to investigate the radioactivity excretion rates in of feces and urine, the blood exposure time curve, and the radioactivity recovery rate in each tissue over time. We showed that the primary excretion route of the hydrogel was via urine (3H: about 86.4%, 14C: about 90.0%), with fewer portion through feces (3H: about 6.922%, 14C: about 8.16%). The hydrogel metabolites exhibited the highest distribution in the kidney, followed by the jejunal contents; The 3H and 14C radioactivity exposures in the remaining tissues were low. We also showed that the 3H and 14C radioactivity recovery rates in the blood were usually low (<0.10% g−1 at 12 h after implantation), even though, in theory, the hydrogel could be absorbed into the blood through the adjacent tissues. By using a combination of HPLC-MS/MS and offline radioactivity counting method, we established that the tetra-PEG-based hydrogel was mainly metabolized to lower-order PEG polymers and other low-molecular-weight substances in vivo.

A multidimensional strategy for uncovering comprehensive quality markers of Schisandra chinensis (Turcz.) Baill based on pharmacodynamics and chemical properties.

BACKGROUND: Quality control of Traditional Chinese Medicines (TCMs) has improved greatly, but there is still a lack of a convincing quality evaluation system for TCMs. Developing quality control markers of TCMs based on pharmacodynamics instead of content has been an attractive approach. However, on account of neglecting phytochemistry attributes of TCMs, part of effective markers might be short of specificity and inconvenient for detecting in production manufacture, which is adverse to control the quality of TCMs systematically. PURPOSE: To build a novel and multidimensional quality assessment approach for TCMs based on pharmacodynamics and chemical properties. METHODS: Schisandra chinensis (Turcz.) Baill (S. chinensis) was used as an example and a rat depression model was built by using a chronic unpredictable mild stress procedure. For identifying the antidepressive components of S. chinensis, we elucidated its antidepressant mechanism in first-step by using quantitative RT-PCR and immunoblotting techniques. And accordingly, correlation analysis between ingredients in vivo with target proteins and anti-inflammation experiments in vitro were carried out. On the other hand, HPLC fingerprint combinations with diverse chemometrics methods were applied to analyze 14 preparations of S. chinensis to obtain its characteristic chemical information. Finally, we ascertained the quality control markers of S. chinensis by integrating the efficacious and characteristic constituents. RESULTS: Our research indicated that S. chinensis treated depression by relieving disordered monoaminergic system and ameliorating neuroinflammation. Five effective substances (schisandrol A, schisandrin A, gomisin N, schisandrin B, and schisandrin C) were screened out according to their potential anti-depression efficacy. Besides, 21 common ingredients and 4 representative constituents of S. chinensis were identified by chemical analysis, whereas only 2 characteristic quantitative markers (schisandrol A, schisandrol B) were ultimately ascertained based on previous studies. CONCLUSION: 6 components, schisandrol A, schisandrin A, gomisin N, schisandrin B, schisandrin C, and schisandrol B, possessed efficacy, measurability, and specificity, were selected as the comprehensive markers for quality control of S. chinensis. We proposed a multidimensional strategy for identifying comprehensive quality markers for TCMs in this study.

Salidroside Inhibits Ischemia/Reperfusion-Induced Myocardial Apoptosis by Targeting Mir-378a-3p Via the Igf1r/Pi3k/Akt Signaling Pathway.

BACKGROUND: The present study aimed to investigate the protective effects and mechanism of salidroside (SAL) on hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis and myocardial ischemia/reperfusion (I/R) injury. METHODS: We set up an H/R H9c2 cell model in vitro and an I/R rat model in vivo. Cell viability, apoptosis and histopathologic evaluation were conducted. RESULTS: The cell viability of H/R-induced cardiomyocytes was increased by pretreatment of SAL, whereas the release of lactate dehydrogenase, reactive oxygen species production, and apoptosis were decreased accompanied with reduced Cleaved-caspase-3 and Bax, and increased Bcl-2 expressions. The SAL restored mitochondrial membrane potential both in vitro and in vivo, and improved electrocardiographic abnormality, and attenuated myocardial apoptosis and injury in I/R-induced rats. The transfection of miR-378a-3p inhibitor counteracted the effects of SAL-induced increase of cell viability and decrease of cell apoptosis and mitochondrial membrane potential. SAL reduced the expression of insulin-like growth factor 1 receptor (IGF1R), and increased the expressions of PI3K and Akt, however, these alterations were blocked by miR-378a-3p inhibitor. CONCLUSIONS: miR-378a-3p might participate in the protective effect of SAL in I/R-induced myocardial apoptosis via the IGF1R/PI3K/AKT signaling pathway.

Discrimination of Lonicerae Japonicae Flos according to species, growth mode, processing method, and geographical origin with ultra-high performance liquid chromatography analysis and chemical pattern recognition.

Lonicerae japonicae flos (LJF, Lonicera japonica Thunb.) is often confused and/or adulterated with Lonicerae flos (LF, Lonicera macrantha (D.Don) Spreng.). Ecological conditions and processing methods strongly influenced the safety and efficacy of LJF. For the strict quality control of LJF, a rapid and feasible strategy for identification and classification of LJF by species, growth mode, processing method and geographical origin, based on chromatographic profiles and pattern recognition analysis, in 119 batches of Lonicera samples was systematically established. Firstly, comprehensive analysis of the chemical compositions of LJF was achieved using ultra-high performance liquid chromatography (UHPLC). Next, unsupervised principal component analysis showed that the influence of species, growth mode, processing method and geographical origin displayed a decreasing trend. Subsequently, classification models for authentication of LJF samples were established by linear discriminant analysis (LDA) with good classification abilities. Finally, sweroside and secoxyloganin could be considered as markers associated of cultivated and wild LJF, respectively, while 3-O-caffeoylquinic acid and 3,5-Di-O-caffeoylquinic acid could be regarded as markers for LF. Consequently, the findings suggest that UHPLC profiles combined with pattern recognition analysis is precise and feasible strategy for the discrimination and quality control of LJF.

Nanoconfinement effect based in-fiber extraction and derivatization method for ultrafast analysis of twenty amines in human urine by GC-MS: Application to cancer diagnosis biomarkers' screening.

Amines, including amino acids and biogenic amines are widely recognized as cancer biomarkers for early diagnosis and treatment. But rapid analysis of amines in biological samples with high sensitivity, good durability and low cost is still challenging. Herein, we proposed an ultrafast biological sample preparation method based on in-fiber extraction and derivatization using a homemade needle-tip devise. Under the nanoconfinement effect, the extraction was completed within 2 min in nanofibers, followed by the in-suit derivatization within 3 min under room temperature using pentafluoropropionic anhydride (PFA) as derivatization reagent, then the needle was inserted into the inlet of GC-MS directly for thermal desorption to enable the complete injection. Additionally, the established sample preparation strategy consumed only 20 µL organic solvent and urine sample, and the sensitivity was increased compared with other methods. After systematical optimization and validation, coupled to GC-MS, the established method was successfully applied to quantify twenty amines in urine of healthy people and lung, colorectal and breast cancer patients. The diagnosis biomarkers for each cancer were screened out and accessed by multiple statistical approaches. Then we further constructed the cancer diagnosis model of each cancer through binary logistic regression analysis for clinical use. Taken together, the established nanoconfinement effect based in-fiber extraction and derivatization biological sample preparation strategy in combination with GC-MS was an ultrafast method for biological analysis with high sensitivity, good durability, little organic solvent consumption and low cost. It possesses great potential in clinical cancer screening and treatment.

Induction of P-glycoprotein expression by dandelion in tumor and heart tissues: Impact on the anti-tumor activity and cardiotoxicity of doxorubicin.

BACKGROUND: Previously, we have investigated the anti-tumor activity and mechanism through which dandelion acts against triple-negative breast cancer (TNBC). However, traditional Chinese medicine is mostly accepted as an adjunct therapy during chemotherapy in clinical practice. So far, little is known about the effects of dandelion in conjunction with chemotherapeutic drugs. PURPOSE: To investigate the effects of dandelion on the anti-tumor activity and cardiotoxicity of doxorubicin (DOX), and to further explore the molecular mechanisms through which these effects occur. STUDY DESIGN: At the beginning of this study, dandelion was observed to alleviate DOX-induced cardiotoxicity and reduce the anti-tumor activity of DOX. Subsequently, we investigated whether the resistance to DOX mediated by P-glycoprotein was involved in the above effects. METHODS: The cardioprotective effect of dandelion was investigated on DOX-treated mice by histological analysis, myocardial enzyme assays, and an untargeted metabolomics study based on LC-Q-TOF/MS. TNBC cell lines and 4T1 tumor-bearing mice were employed to investigate the combined anti-tumor activity. Laser scanning confocal microscope and a flow cytometry analysis were employed to measure the intracellular accumulation of DOX. A specific, sensitive, and rapid LC-MS/MS method was developed to detect the efflux of DOX from cells. Expression of P-glycoprotein in mouse tumor and heart tissues was detected via Western blotting analysis. RESULTS: Dandelion was found to significantly alleviate DOX-induced cardiotoxicity, as was evidenced by improved cardiomyocyte morphology, decreased LDH and CK-MB release, and adjusted metabolic biomarker levels. However, in vitro and in vivo studies showed that dandelion could reduce the anti-tumor activity of DOX. This counteraction was achieved by activating of the drug efflux transporter P-glycoprotein, thereby promoting the efflux of DOX from cells and reducing the intracellular accumulation of DOX. Moreover, the activation of P-glycoprotein by dandelion in mouse heart tissue was also observed, thus suggesting that the decrease of cardiac DOX accumulation plays an important role in the cardioprotective effect of dandelion. CONCLUSION: Dandelion can activate the P-glycoprotein in heart and tumor tissues, which ameliorates DOX-induced cardiotoxicity but attenuates DOX cytotoxicity toward TNBC. Our findings have important implications for the correct clinical use of dandelion.

Material Basis Elucidation and Quantification of Dandelion through Spectrum-Effect Relationship Study between UHPLC Fingerprint and Antioxidant Activity via Multivariate Statistical Analysis.

The excessive expression of reactive oxygen species is closely connected to many diseases. Considerable studies have demonstrated dandelion as well as its ingredients exhibited antioxidant activity. However, specific material basis reflecting the antioxidant activity has not been comprehensively investigated. In this study, a spectrum-effect relationship study on dandelion between fingerprinting and antioxidant activity was analyzed in detail, while a UHPLC quantification method developed and completely validated for simultaneous determination of active ingredients in dandelion. With the establishment of dandelion fingerprints of different regions, 24 common peaks were characterized. The classic FRAP method and ABTS methods were then used to detect their antioxidant activity. Partial least squares regression analysis, bivariate correlation analysis and grey correlation method were used to accomplish the spectrum-effect relationship. Eventually, the ingredients with antioxidant activity which could be considered as candidate quality markers of dandelion were discovered through spectrum-effect relationship analysis. The six compounds including caftaric acid, chlorogenic acid, caffeic acid, chicoric acid, isochlorogenic acid A, and isochlorogenic acid C were quantitatively determined. The developed UHPLC assay method was accurate, precise, and reliable. The study has elucidated the antioxidant material basis of dandelion and provided a scientific basis for the quality control of dandelion.

Chemical characteristics of Rhodiola Crenulata and its mechanism in acute mountain sickness using UHPLC-Q-TOF-MS/MS combined with network pharmacology analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhodiola crenulata (Hook.f. & Thomson) H.Ohba has a long history of clinical application for the prevention and treatment of acute mountain sickness (AMS) in traditional Chinese medicine. However, gaps in knowledge still exist in understanding the underlying mechanisms of Rhodiola crenulata against AMS. AIMS: To address this problem, a comprehensive method was established by combining UHPLC-Q-TOF-MS/MS analysis and network pharmacology. MATERIALS AND METHODS: The ingredients of Rhodiola crenulata were comprehensively analyzed using UHPLC-Q-TOF-MS/MS method. On this basis, a network pharmacology method incorporated target prediction, protein-protein interaction network, gene enrichment analysis and components-targets-pathways network was performed. Finally, the possible mechanisms were verified through molecular docking, in vitro and in vivo experiments. RESULTS: A total of 106 constituents of Rhodiola crenulata were charactered via UHPLC-Q-TOF-MS/MS. The 98 potentially active compounds out of 106 were screened and corresponded to 53 anti-AMS targets. Gene enrichment analysis revealed that hypoxia and inflammation related genes may be the central factors for Rhodiola crenulata to modulate AMS. Molecular docking revealed that TNF, VEGFA and HIF-1α had high affinities to Rhodiola crenulata compounds. Subsequently, Rhodiola crenulata extract was indicated to inhibit the protein expression level of TNF in hypoxia induced H9c2 cells. Lastly, Rhodiola crenulata extract was further verified to ameliorate heart injury and decreased the heart levels of TNF, VEGFA and HIF-1α in acute hypoxia-induced rats. CONCLUSIONS: This study used UHPLC-Q-TOF-MS/MS analysis and a network pharmacology to provide an important reference for revealing the potential mechanism of Rhodiola crenulata in the prevention and treatment of AMS.

The therapeutic effect of Xuanbai Chengqi Decoction on chronic obstructive pulmonary disease with excessive heat in the lung and fu-organs based on gut and lung microbiota as well as metabolic profiles.

As a prescription for treating lung inflammation and intestinal diseases, Xuanbai Chengqi Decoction (XBCQD) in clinical practice can effectively treat COPD with excessive heat in the lung and fu-organs, which is characterized by phlegm-heat accumulation in the lung and constipation. This study aims to find the potential biomarkers of COPD with excessive heat in the lung and fu-organs from two aspects of lung and intestine based on metabolomics and microbiota analysis, and to evaluate the efficacy of XBCQD as well as to explore the mechanism of drug function according the regulating effect of drugs on these markers. The HPLC-Q-TOF-MS/MS, 16SrDNA technology and multiple statistical methods were used to trace the process of disease and curative effect with XBCQD. Results showed that the onset and development of disease was associated with the imbalance of 41 differential metabolites in plasma, bronchoalveolar lavage fluid and feces and 82 bacteria at the levels of phylum, class, order, family and genus from lung and intestine, including Escherichia-Shigella. However, after treatment with XBCQD, 30 differential metabolites mainly involving in the metabolism of linoleic acid, taurine and hypotaurine metabolism, arachidonic acid metabolism, biosynthesis of primary bile acids, tryptophan metabolism, arginine and proline metabolism and 65 pulmonary and intestinal bacteria at all levels were reversed in the drug group. In addition, the results of the correlation analysis showed that specific microbiota from lung and intestine and reversed differential metabolites had a significant correlation, and they could affect each other in the course of disease occurrence and treatment. This study preliminarily confirmed that XBCQD can be used to treat COPD with excessive heat in the lung and fu-organs through lung-intestine simultaneous treatment. It also provided new strategies for the treatment of lung diseases or intestinal diseases, and new research ideas for the evaluation of drug efficacy.

Nanoconfined liquid phase nanoextraction combined with in-fiber derivatization for simultaneous quantification of seventy amino-containing metabolites in plasma by LC-MS/MS: Exploration of lung cancer screening model.

Targeted metabolomics with absolute quantification is widely applied in biomarker identification and drug screening. However, due to the complexity of biological matrix and trace amount of metabolites in plasma, simultaneous quantification of highly polar metabolites in plasma with broad coverage in short time is still challenging. Herein, we proposed a nanoconfined liquid phase nanoextraction (NLPNE) combined with in-fiber derivatization (IFD) strategy that enabled simultaneous quantification of seventy amino-containing analytes in plasma, including amines, nucleosides and their metabolites. Methanol-water (2:1, v/v) was selected as nanoconfined solvent (NCS) to quickly extract highly polar analytes based on the nanoconfinement effect, followed by IFD process directly performed by adding the derivatization reagent benzoyl chloride (BzCl) within 5 min. Besides saving time, this combination strategy was environment-friendly with little organic solvent consumption and cost-effective by using reusable carbon nanofibers. Furthermore, the sensitivity was increased up to 4.92-fold compared with protein precipitation (PP) based conventional derivatization method. Key factors that affected derivatization efficiency including the derivatization time, the amount of derivatization reagent, desorption solution and CNFs, were optimized by response surface methodology (RSM). After systematical method validation, this methodology was applied to determine the multi-metabolites index in plasma of lung cancer using an integrated data processing workflow. Then lung cancer diagnosis model was established through binary logistic regression analysis to make a reference for quick lung cancer screening clinically. Taken together, the NLPNE-IFD LC-MS/MS method for targeted metabolomics enables simultaneous quantification of seventy amino-containing analytes with advantages of broad coverage, high sensitivity, time- and solvent-saving, which could be used on cancer diagnosis clinically.

Leonurus japonicus Houtt. (Motherwort): Systematic research through chemical profiling, stability under controlled conditions and pharmacokinetic analysis on screening Q-markers for quality control.

Leonurus japonicus Houtt. (Motherwort) is the fresh or dried aerial part of Leonurus japonicus Houtt. (Labiaceae), which is widely used in clinical practice and daily life, used to treat gynecological diseases. However, the differences between different parts, single index component in Pharmacopoeias and the less stability of active ingredients affect its clinical efficacy. This study aimed to find the multi-active compounds between different parts of Motherwort to ensure its clinical efficacy, which related to stability and had pharmacokinetic behavior. Firstly, HPLC-Q-TOF-MS/MS was used to analyze the components in vitro and in vivo, as well as multivariate statistical analysis and network pharmacology analysis was conducted to find the significant different components related to activity. Secondly, the content determination methods were established to study the stability of effective components during storage in order to establish the content limit for quality control of Motherwort. Thirdly, UFLC-MS/MS was used to analyze the pharmacokinetic behavior of active components in Motherwort. The results showed that a total of 131 chemical constituents were identified in vitro and 21 prototype absorption compounds and 72 metabolites were found in vivo. Meantime, multivariate statistical analysis and network pharmacology analysis was combined to find that leonurine, stachydrine and trigonelline were activity-related substance, which could be used as active components related to pharmacodynamics in different parts. Then the stability variation trend and content limit of three alkaloids were found, which could be used for the quality control of Motherwort. Furthermore, the results showed that three alkaloids had pharmacokinetic behavior in vivo. 3 alkaloids were screened, which could be used as active components most closely related to pharmacodynamics among different parts. The stable stage, assay tolerance and pharmacokinetic characteristics were studied by the active substances, which could provide a basis for quality control and clinical medication of Motherwort.

Isotope labelled in suit derivatization-extraction integrated system for amine/phenol submetabolome analysis based on nanoconfinement effect: Application to lung cancer.

Amine/phenol submetabolome has shown critical role in clinical cancer screening and therapy. In suit derivatization has widely applied in the analysis of amine/phenol submetabolome by LC/MS for simplifying the pretreatment procedures, improving the separation and sensitivity. However, the complexity of biological matrix and trace amount of metabolites in plasma that lead to the limited detection coverage, poor repeatability and low extraction efficiency are still issues for in suit derivatization. Herein, we proposed an isotope labelled in suit derivatization-extraction integrated system for targeted analysis of all the metabolites in amine/phenol submetabolome with high efficiency and repeatability by LC-MS. The processes of in suit derivatization, alkalization and extraction were performed simultaneously in the nanopores highly dispersed between the carbon nanofibers based on the nanoconfinement effect. Isotope labelling derivatization (ILD) reagents benzoyl chloride (BzCl) and BzCl-d5 were used to enhance the accuracy of identification and relative quantification. The detection sensitivity was increased up to 5.91-fold and detection coverage was enhanced more than 25% compared with conventional derivatization method. After systematical validation, the established methodology was applied to profile the amine/phenol submetabolome of human plasma and 1498 metabolites were screened out, among which, 1004 (67.02%) were positively or putatively identified. Furthermore, 106 amine/phenol metabolites exhibited significant difference between lung cancer patients and healthy controls by using multiple data processing methods. Taken together, the isotope labelled in suit derivatization-extraction integrated system was a useful approach for the analysis of amine/phenol submetabolome in plasma with broad metabolome coverage, simple pretreatment steps, high detection sensitivity and accuracy, and could be a potential tool for clinical biomarker discovery of disease.

The Biological Fate of Pharmaceutical Excipient ß-Cyclodextrin: Pharmacokinetics, Tissue Distribution, Excretion, and Metabolism of ß-Cyclodextrin in Rats.

ß-cyclodextrin has a unique annular hollow ultrastructure that allows encapsulation of various poorly water-soluble drugs in the resulting cavity, thereby increasing drug stability. As a bioactive molecule, the metabolism of ß-cyclodextrin is mainly completed by the flora in the colon, which can interact with API. In this study, understanding the in vivo fate of ß-cyclodextrin, a LC-MS/MS method was developed to facilitate simultaneous quantitative analysis of pharmaceutical excipient ß-cyclodextrin and API dextromethorphan hydrobromide. The established method had been effectively used to study the pharmacokinetics, tissue distribution, excretion, and metabolism of ß-cyclodextrin after oral administration in rats. Results showed that ß-cyclodextrin was almost wholly removed from rat plasma within 36 h, and high concentrations of ß-cyclodextrin distributed hastily to organs with increased blood flow velocities such as the spleen, liver, and kidney after administration. The excretion of intact ß-cyclodextrin to urine and feces was lower than the administration dose. It can be speculated that ß-cyclodextrin metabolized to maltodextrin, which was further metabolized, absorbed, and eventually discharged in the form of CO2 and H2O. Results proved that ß-cyclodextrin, with relative low accumulation in the body, had good safety. The results will assist further study of the design and safety evaluation of adjuvant ß-cyclodextrin and promote its clinical development.

Uncovering the mechanisms of dandelion against triple-negative breast cancer using a combined network pharmacology, molecular pharmacology and metabolomics approach.

BACKGROUND: Taraxacum mongolicum, also called dandelion, has been used for thousands of years as a remedy for mammary abscess, mammary gland hyperplasia, and various other diseases afflicting the breast. In modern pharmacological research, dandelion has been proven to be effective against triple-negative breast cancer (TNBC). However, the mechanisms of this anti-tumor effect have not been fully elucidated. PURPOSE: The aim of this investigation was to understand the multi-target mechanisms through which dandelion counteracts TNBC via a network pharmacology strategy as well as to validate its effectiveness by means of molecular pharmacology and metabolomics assessments. METHODS: A liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (LC-Q-TOF/MS) was employed to identify the absorbed components of dandelion in rat plasma. The network pharmacology-based prediction was utilized to uncover the potential mechanisms through which dandelion counteracts TNBC, during which potential targets were identified and pathway enrichment analysis was performed. Subsequently, TNBC cells and 4T1 tumor-bearing mice were used to further verify the molecular mechanisms of dandelion. RESULTS: Twelve active compounds were identified in rat plasma, which were connected with 50 TNBC-related targets. The pathway enrichment showed that dandelion could treat TNBC through regulating a series of biological processes involving cell cycle and metabolism. Experimentally, flow cytometry analysis revealed that dandelion could arrest the G0/G1 and G2/M cell cycles in 4T1 cells. Further western blot analysis evidenced that the protein expression of kinase 6 (CDK6) as well as cyclins B1 and B2 in mice tumor tissue were suppressed by dandelion. In addition, cell metabolomics analysis revealed the changes in the endogenous metabolite levels that result from dandelion treatments, such as the downregulation of arginine and spermine levels. All these findings were consistent with the predicted targets and pathways. CONCLUSION: This study comprehensively demonstrates the multi-target mechanisms of dandelion against TNBC using network pharmacology, molecular pharmacology, and metabolomics approaches. These findings will provide important stepping stones for further mechanism investigations and may lead to the development of highly effective dandelion-based treatments for TNBC.