Exploration of potential active ingredients and mechanism of action of Xihuang pill-medicated serum against glioma based on HPLC-Q-TOF-MS/MS, network pharmacology and experimental verification / 药学学报

Qualitative analysis of the ingredients absorbed into blood and their metabolites of Xihuang pill (XHP) were conducted using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS) technology. Network pharmacology was used to explore the potential anticancer mechanisms of the ingredients against glioma, and their specific mechanisms were validated through molecular docking and experimental verification. SD rats were intragastrically administered with XHP, and rat serum samples were collected. Ingredients absorbed into blood and their metabolites were identified based on the retention time of chromatographic peaks, accurate molecular mass, characteristic fragment ions, and comparisons with reference substances and literature data. PharmMapper and SwissTarget Prediction databases were used to obtain the targets of the XHP-medicated serum, while GeneCards, OMIM, PharmGKB, TTD, and DrugBank databases were used to obtain glioma disease targets. The "component-target" network relationship diagram was constructed using Cytoscape 3.9.1 software. The protein-protein interaction (PPI) network diagram was constructed using the STRING database, and the targets were analyzed using GO and KEGG analyses. Molecular docking was used to verify the binding ability of core targets with their corresponding compounds in XHP-medicated serum. The potential mechanism of the anti-glioma effect of 11-keto-β-boswellic acid (KBA), a representative component of XHP-medicated serum, was verified using CCK-8 and Western blot assays. A total of 40 compounds were identified in the XHP-medicated serum, including 28 prototype components and 12 metabolites. The network pharmacology results showed that elemonic acid, 3-acetyl-β-boswellic acid, KBA, α-boswellic acid, and other 5 compounds might be the active ingredients of XHP-medicated serum in the treatment of glioma. Glutathione reductase (GSR), glucose-6-phosphate dehydrogenase (G6PD), ATP-citrate lyase (ACLY), aldo-keto reductase family 1 member B1 (AKR1B1) and glutaredoxin (GLRX) were identified as key targets, involving pathways such as glutathione metabolism and the pentose phosphate pathway. Further cell experiments showed that KBA significantly inhibited the proliferation of T98G cells with an IC50 of 30.96 μmol·L-1, and KBA (30 μmol·L-1) significantly downregulated the protein expression levels of GSR in T98G cells. In summary, XHP-medicated serum may exert its anti-glioma effect by regulating GSR and G6PD-targeted pathways involved in glutathione metabolism. These results provide valuable evidence for further investigating the mechanism of XHP in treating glioma. The animal welfare and experimental procedures were approved by the Ethical Committee of Laboratory Animals at Nanjing University of Chinese Medicine (approval No. ACU221001).

Mechanism of Didang Decoction in prevention of anti-atherosclerosis and hyperlipidemia by HPLC-Q-TOF-MS/MS and network pharmacology based on theory of "nutrients return to heart and fat accumulates in channels" / 中国中药杂志

Atherosclerosis(AS) is caused by impaired lipid metabolism, which deposits lipids in the intima, causes vascular fibrosis and calcification, and then leads to stiffening of the vascular wall. Hyperlipidemia(HLP) is one of the key risk factors for AS. Based on the theory of "nutrients return to the heart and fat accumulates in the channels", it is believed that the excess fat returning to the heart in the vessels is the key pathogenic factor of AS. The accumulation of fat in the vessels over time and the blood stasis are the pathological mechanisms leading to the development of HLP and AS, and "turbid phlegm and fat" and "blood stasis" are the pathological products of the progression of HLP into AS. Didang Decoction(DDD) is a potent prescription effective in activating blood circulation, removing blood stasis, resolving turbidity, lowering lipids, and dredging blood vessels, with the functions of dispelling stasis to promote regeneration, which has certain effects in the treatment of atherosclerotic diseases. This study employed high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS) to screen the main blood components of DDD, explored the targets and mechanisms of DDD against AS and HLP with network pharmacology, and verified the network pharmacological results by in vitro experiments. A total of 231 blood components of DDD were obtained, including 157 compounds with a composite score >60. There were 903 predicted targets obtained from SwissTargetPrediction and 279 disease targets from GeneCards, OMIM, and DisGeNET, and 79 potential target genes of DDD against AS and HLP were obtained by intersection. Gene Ontology(GO) analysis suggested that DDD presumably exerted regulation through biological processes such as cholesterol metabolism and inflammatory response, and Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis suggested that signaling pathways included lipid and atherosclerosis, insulin resistance, chemo-carcinogenesis-receptor activation, and AGE-RAGE signaling pathways in diabetic complications. In vitro experiments showed that DDD could reduce free fatty acid-induced lipid accumulation and cholesterol ester content in L02 cells and improve cellular activity, which might be related to the up-regulation of the expression of PPARα, LPL, PPARG, VEGFA, CETP, CYP1A1, and CYP3A4, and the down-regulation of the expression of TNF-α and IL-6. DDD may play a role in preventing and treating AS and HLP by improving lipid metabolism and inflammatory response, and inhibiting apoptosis with multi-component, multi-target, and multi-pathway characteristics.

Method establishment of component identification and content determination of 7 components in Mongolian medicine Ixeris chinensis / 中国药房

OBJECTIVE To establish the methods to identify the chemical components of Ixeris chinensis， and determine the contents of 7 components （chlorogenic acid， luteolin， quercetin， rutin， protocatechuic acid， isochlorogenic acid A， luteoloside）. METHODS HPLC-Q-Exactive-MS was used to identify the chemical components of I. chinensis. The contents of 7 components in I. chinensis， including chlorogenic acid， were determined by HPLC-MS/MS. RESULTS A total of 45 components were identified in I. chinensis， including 20 organic acids， 13 flavonoids， 4 fatty acids， 4 amino acids， 3 nucleosides， and 1 coumarin. The linear range of chlorogenic acid， luteolin， quercetin， rutin， protocatechuic acid， isochlorogenic acid A and luteoloside were 503.00- 25 150.00， 42.00-2 100.00， 5.05-252.50， 20.05-1 002.50， 25.10-1 255.00， 750.00-37 500.00， 196.00-9 800.00 ng/mL （r≥0.999 2）， respectively. RSDs of precision， stability and reproducibility tests were all less than 3.00% （n＝6）， and average recovery ranged from 96.72% to 105.84% （all RSD＜4.00%， n＝6）. The contents of 7 components in 3 batches of I. chinensis were 1 145.77- 3 261.25， 23.75-97.90， 0.92-2.12， 1.06-23.18， 9.35-21.85， 833.25-1 045.58， 199.56-1 869.78 μg/g， respectively. CONCLUSIONS The established methods for identification and content determination are rapid and simple， and can be used for the identification of chemical components and the content determination of 7 components in I. chinensis.

Investigation of potential pharmacodynamic substances and mechanism of Qingxin-zishen prescription decoction in treatment of menopause syndrome based on HPLC-Q-TOF-MS/MS and network pharmacology / 中国临床药理学与治疗学

AIM: To analyze the chemical ingredients of Qingxin-zishen prescription decoction (QZPD) and predict its main pharmacodynamic substances and mechanism in the prevention and treatment of menopause syndrome (MPS) with the help of high performance liquid chromatography-quadrupole-time of flight mass spectrometry (HPLC-Q-TOF/MS) combined with network pharmacology. METHODS: The chemical ingredients of QZPD were identified after analyzing the retention time, exact mass, secondary mass spectrometry fragmentation and other information obtained from HPLC-Q-TOF/MS and comparing them with the established chemical ingredients database and the literatures. The targets of ingredients in QZPD were predicted by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and SwissTargetPrediction database. The disease targets of MPS were obtained through Online Mendelian Inheritance in Man (OMIM) and GeneCards Database. Gene ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of potential targets were analyzed with the Metascape database. Cytoscape 3.7.2 software was used to construct the network of active components-key targets-pathways. AutoDockTools 4.2.5 software was applied in the molecular docking verification between the key active components and key targets. RESULTS: A total of 83 components were identified in QZPD and 847 drug targets were predicted. After intersection them with 3 050 disease targets, 395 common targets were obtained. After network topology analysis, 74 key targets were obtained, involving mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt), transforming growth factor-β (TGF-β) and other signaling pathways. Molecular docking analysis results indicated that 23 key active components, such as berberine, epiberberine, coptisine, geissoschizine methyl ether, liensinine, norcoclaurine, palmatine, quercetin, and luteolin, had good binding activity with several of the key targets. CONCLUSION: This study preliminarily identifies the potential effective chemical ingredients of QZPD, predicts its targets in the prevention and treatment of MPS, which provides supporting information for the further study of the pharmacodynamic substances and mechanisms of QZPD.

Identification of Q-markers for Cistanches Herba based on HPLC-Q-TOF-MS/MS and network pharmacology / 中国中药杂志

This study aims to establish a method for analyzing the chemical constituents in Cistanches Herba by high performance liquid chromatography(HPLC) and quadrupole-time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS), and to reveal the pharmacological mechanism based on network pharmacology for mining the quality markers(Q-markers) of Cistanches Herba. The chemical constituents of Cistanche deserticola and C. tubulosa were analyzed via HPLC-Q-TOF-MS/MS. The potential targets and pathways of Cistanches Herba were predicted via SwissTargetPrediction and DAVID. The compound-target-pathway-pharmacological action-efficacy network was constructed via Cytoscape. A total of 47 chemical constituents were identified, involving 95 targets and 56 signaling pathways. We preliminarily elucidated the pharmacological mechanisms of echinacoside, acteoside, isoacteoside, cistanoside F, 2'-acetylacteoside, cistanoside A, campneoside Ⅱ, salidroside, tubuloside B, 6-deoxycatalpol, 8-epi-loganic acid, ajugol, bartsioside, geniposidic acid, and pinoresinol 4-O-β-D-glucopyranoside, and predicted them to be the Q-markers of Cistanches Herba. This study identified the chemical constituents of Cistanches Herba, explained the pharmacological mechanism of the traditional efficacy of Cistanches Herba based on network pharmacology, and introduced the core concept of Q-markers to improve the quality evaluation of Cistanches Herba.

Qualitative and quantitative analysis of chemical compounds in 3 species of wild Veratrum from Inner Mongolia / 中国药房

OBJECTIVE To conduc t qualitative and quantitative analysis for the chemical compounds in 3 species of wild Veratrum（V. nigrum ，V. maackii ，V. dahuricum ）from Inner Mongolia. METHODS HPLC-Q-Exactive-MS/MS technology was used to identify the chemical components of V. nigrum ，V. maackii and V. dahuricum by consulting SciFinder ，ChemSpider database and related literatures and comparing with the reference substance. The contents of polydatin ，oxyresveratrol and resveratrol in 3 species of wild Veratrum were determined by HPLC. RESULTS A total of 31 compounds were identified ，including 13 stilbenes， 11 flavonoids，4 organic acids ，2 glycosides，1 brasilin. Most of the compounds were shared by 2 or 3 species of wild Veratrum， only 2 flavonoids kaempferol and luteolin were owned by V. dahuricum . The total contents of polydatin ，oxyresveratrol and resveratrol in 3 species of wild Veratrum were in the range of 6.618-11.292 mg/g，and the total contents of them in V. nigrum were the highest ，followed by V. maackii and V. dahuricum . The contents of polydatin and resveratrol in V. maackii were the highest ，and the content of oxyresveratrol in V. nigrum was highest. CONCLUSIONS Most of the components of 3 species of wild Veratrum are similar，only kaempferol and luteolin are unique to V. dahuricum . The contents of polydatin ，oxyresveratrol and resveratrol are significantly different among 3 species of wild Veratrum.

Rapid analysis and identification of chemical components of ethanol extract from Mongolian medicine Sanzi san by HPLC-Q-Exactive-MS / 中国药房

OBJECTIVE To systematic ally stu dy the chemic al components of ethanol extract from Sanzi san ，and to provide reference for clarifying the pharmacodynamic material basis of the formulation. METHODS HPLC-Q-Exactive-MS technology was adopted. The determination was performed on Shim-pack GIST-HP C 18 column with mobile phase consisted of acetonitrile- 0.1％ formic acid aqueous solution for gradient elution at the flow rate of 1 mL/min. The column temperature was 40 ℃，and sample size was 10 μL. Mass spectrometry conditions included the electrospray spray ion source was used for detection in positive and negative ion detection modes. Full MS/dd-MS 2 detection mode was adopted ，the resolution of Full MS was 70 000 and the resolution of dd-MS2 was 17 500. The scanning range was m/z 110-1 200. The ion peaks were identified by comparing with the information of control substances ，literature references and self-built database. RESULTS A total of 64 components were identified in the ethanol extract of Mongolian medicine Sanzi san ， including 9 flavonoids，13 iridoids，14 organic acids ，18 tannins，3 triterpenes，3 amino acids and 4 fatty acids. CONCLUSIONS The ethanol extract of Mongolian medicine Sanzi san mainly include iridoids ，tannins and flavonoids ，which might be the pharmacodynamic material basis of Sanzi san.

Analysis of Chemical Constituents in Daishenning by HPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

Objective:High performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry （HPLC-Q-TOF-MS/MS） was used to identify the main chemical constituents of Daishenning. Method:Cosmosil 5 C₁₈-AR-Ⅱ column （4.6 mm×250 mm， 5 μm） was employed for chromatographic separation with mobile phase of acetonitrile （A）-0.5% formic acid aqueous solution （B） for gradient elution （0-10 min， 5%A； 10-20 min， 5%-20%A； 20-30 min， 20%A； 30-55 min， 20%-35%A； 55-65 min， 35%-55%A； 65-75 min， 55%-100%A； 75-80 min， 100%A； 80-85 min， 100%-5%A； 85-90 min， 5%A）， the flow rate was 1 mL·min^-1， column temperature was 40 ℃， and injection volume was 10 μL. Electrospray ionization （ESI）， positive and negative ion detection modes and mass scanning range of <italic>m</italic>/<italic>z</italic> 100-2 000 were selected for mass spectrometry. The main chemical constituents in Daishenning were identified by MassHunter B.06.00 software in combination with PubChem， MassBank， ChemicalBook and other databases， and reference information. Result:A total of 96 components were identified from Daishenning， including 32 flavonoids， 19 organic acids， 6 glycosides， 6 terpenoids， 5 phenylpropanoids， 8 phenols， 14 other components and 6 unknown components. Conclusion:The established method can simultaneously analyze different types of compounds in Daishenning， it is helpful for further research on the extraction and separation of main chemical components and quality control of this preparation. In addition， through the rapid identification of the chemical constituents in Daishenning， it is speculated that the main effective substances of Daishenning may be flavonoids and organic acids.

Qualitative and quantitative analysis of aristolochic acid components in nine Chinese medicinal materials containing toxic aristolochic substances / 药学学报

The objective of this work was to explore the content and composition of aristolochic acid compounds in Chinese medicinal materials containing toxic aristolochic chemicals, so as to ensure the safety of these medicinal materials and their related products. Nine Chinese medicinal materials were selected for study, including the tuber of Aristolochia cinnabarina, the herbs of Asarum forbesii, the stems of Aristolochia manshuriensis., the fruits of Aristolochia debilis, the roots of Aristolochia debilis, the stems and leaf of Aristolochia debilis, the herbs of Aristolochia mollissima, the roots of Aristolochia fangchi, and the roots of Asarum heterotropoides var. mandshuricum. The aristolochic acid components in the nine Chinese medicinal materials were analyzed by high performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC-Q-TOF-MS) combined with high performance liquid chromatography diode-array detection. The separation was performed on an Agilent ZORBAX SB-Aq column (250 mm×4.6 mm, 5 μm) with gradient elution using a mobile phase consisting of acetonitrile and 0.2% acetic acid. ESI positive ion mode MS was used to investigate the ionization pathways of aristolochic acid Ⅰ, Ⅱ, Ⅲa, Ⅳa, Ⅶa, and aristololactam Ⅰ, Ⅱ using seven reference standards, and the structures of the components with UV spectrasimilar to those of the seven reference standards in the selected medicinal materials were qualitatively analyzed by following the investigated ionization pathways. The identified aristolochic acid components were quantified using an external standard method by HPLC-UV with detection at 254 nm. Twenty-two aristolochic acid components including 11 aristolochic acids and 11 aristololactams were identified from the nine selected medicinal materials; 15 aristolochic acids were found in the tuber of Aristolochia cinnabarina and the roots of Aristolochia debilis, followed by 14 aristolochic acids in the fruits of Aristolochia debilis and the stems of Aristolochia manshuriensis. The greatest content of aristolochia components was found in the tuber of Aristolochia cinnabarina and the stems of Aristolochia manshuriensis, ranging from 8.91 mg·g-1 to 13.40 mg·g-1, and the least amount was in the herbs of Asarum forbesii, at less than 0.10 mg·g-1 and containing only two aristolochia components. This study systematically explored the quantity and composition of aristolochic acid components in selected Chinese medicinal materials believed to contain toxic aristolochic compounds, providing a basis for follow-up studies on the toxicity of these substances that can lead to safety standards for their use.

Qualitative and quantitative analysis of alkaloids in Eurycoma longifolia by HPLC-Q-TOF-MS combined with UPLC-QQQ-MS/MS / 中国中药杂志

A sensitive and efficient method was established and validated for qualitative and quantitative analysis of total alkaloids from the extract of Eurycoma longifolia by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(HPLC-Q-TOF-MS) combined with ultra-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry(UPLC-QQQ-MS/MS). The HPLC-Q-TOF-MS conditions are as follows: Welch Ultimate XB-C_(18) column(4.6 mm×250 mm, 5 μm) with acetonitrile(containing 0.1% formic acid)-0.1% formic acid in water as mobile phase for gradient elution. The UPLC-QQQ-MS/MS conditions are as below: Agilent Eclipse Plus C_(18) column(2.1 mm×50 mm, 1.8 μm) with acetonitrile(containing 0.1% formic acid) and 0.1% formic acid in water as mobile phase for gradient elution. MS data were collected by electrospray ionization in positive ion mode. According to the comparison with reference standards and the accurate masses of molecules, a total of 17 alkaloids in E. longifolia extract were identified by HPLC-Q-TOF-MS. The UPLC-QQQ-MS/MS quantitative analysis result of 3 alkaloids showed that the linear ranges of them were good(r≥0.999 7) and the overall recoveries ranged from 108.8%-110.2%, with RSDs of 2.9%-5.3%. The method is accurate, reliable, and efficient, which can comprehensively reflect the constituents and content of alkaloids in E. longifolia. The result can serve as a reference for further elucidating its therapeutic material basis and quality control.

Qualitative analysis of chemical components in Lianhua Qingwen capsule by HPLC-Q Exactive-Orbitrap-MS coupled with GC-MS / 药物分析学报

The Lianhua Qingwen (LHQW) capsule is a popular traditional Chinese medicine for the treatment of viral respiratory diseases.In particular,it has been recently prescribed to treat infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).However,due to its complex composi-tion,little attention has been directed toward the analysis of chemical constituents present in the LHQW capsule.This study presents a reliable and comprehensive approach to characterizing the chemical constituents present in LHQW by high-performance liquid chromatography-Q Exactive-Orbitrap mass spectrometry (HPLC-Q Exactive-Orbitrap-MS) coupled with gas chromatography-mass spectrometry(GC-MS).An automated library alignment method with a high mass accuracy (within 5 ppm) was used for the rapid identification of compounds.A total of 104 compounds,consisting of alkaloids,flavonoids,phenols,phenolic acids,phenylpropanoids,quinones,terpenoids,and other phytochemicals,were suc-cessfully characterized.In addition,the fragmentation pathways and characteristic fragments of some representative compounds were elucidated.GC-MS analysis was conducted to characterize the volatile compounds present in LHQW.In total,17 compounds were putatively characterized by comparing the acquired data with that from the NIST library.The major constituent was menthol,and all the other compounds were terpenoids.This is the first comprehensive report on the identification of the major chemical constituents present in the LHQW capsule by HPLC-Q Exactive-Orbitrap-MS,coupled with GC-MS,and the results of this study can be used for the quality control and standardization of LHQW capsules.

Main chemical constituents of Changyanning Tablets based on HPLC-Q-TOF-MS/MS / 中草药

Objective: To study the chemical constituents of Changyanning Tablets, a high performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC-Q-TOF-MS/MS) was established to recognize and classify the ingredients accurately and rapidly. Methods: Agilent ZORBAX Eclipse XDB-C18 chromatographic column (250 mm × 4.6 mm, 5 μm) was employed and the separation was performed with the mobile phase consisting of methanol-0.05% acetic acid aqueous solution. The information of accurate mass and multistage fragment ions were obtained by the monitored simultaneously for positive and negative ions. The main chemical constituents of Changyanning Tablets were identified by high resolution mass spectrometry data, combining with Pubmed, Hmdb, Massbank network database, reference literature and comparing the reference. Results: Fifty-one chemical components were finally identified in this study, including two phenylpropanoids, eight iridoids, 12 flavonoids, four tannins, 23 organic acids, and two other classes. Conclusion: This study comprehensively studies the material basis in Changyanning Tablets, which provides a basis for improving the quality evaluation system of Changyanning Tablets and lays the foundation for elucidating the active components mechanism.

Rapid Analysis and Identification of Chemical Constituents from Siwei Tumuxiang San by HPLC-Q-Exactive-MS/MS / 中国实验方剂学杂志

Objective::To rapidly characterize and identify the components of Siwei Tumuxiang San by using high performance liquid chromatography tandem quadrupole-electrostatic field orbitrap high resolution mass spectrometry (HPLC-Q-Exactive-MS/MS). Method::Agilent ZORBAX SB-Aq column (4.6 mm×150 mm, 5 μm) was adopted and 5 mmol·L-1 ammonium acetate+ 0.1% formic acid aqueous solution-acetonitrile were used as mobile phase. According to the separation and structure identification results of chemical components based on the methods of ChemSpider and ChemicalBook database retrieval, a local database of molecular formula, molecular weight, structural formula and MS/MS spectrum information of chemical components in Siwei Tumuxiang San was established. An HPLC-Q-Exactive-MS/MS analysis method was established. The complex compounds of Siwei Tumuxiang San were identified rapidly by using Xcalibar 3.0 software, comparison of reference materials and studies on MS/MS fragment pathways. Result::A total of 110 compounds were identified from the Siwei Tumuxiang San by HPLC-Q-Exactive-MS/MS technology, including 3 sesquiterpene compounds from Inulae Radix, 16 alkaloid compunds and 38 isoprene flavonoid compounds from Sophorae Radix flavescentis, 12 triterpenoid saponin compounds, 1 catechin compounds and 4 flavonoid glycoside compounds from Rubus sachalinensis. The fragment pathways of the main types of compounds were summarized. Among them, mass spectrometry information of 31 compounds was reported for the first time. Conclusion::This study can be used to identify the target compounds and non-target compounds in Siwei Tumuxiang San systematically, accurately and quickly, which will lay a foundation for the in vivo analysis and pharmacokinetic study of the formulation.

Research of tissue metabolomics in papillary thyroid carcinoma based on HPLC/Q-TOF-MS / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：用组织代谢组学方法，探讨甲状腺乳头状癌（papillary thyroid carcinoma, PTC）组织及癌旁组织的代谢差异，寻找PTC的潜在生物标志物，探索PTC的发病机制与治疗策略。方法：收集2018年10月至2020年2月期间湖南省人民医院乳甲外科手术切除的40例PTC患者的癌组织及癌旁组织标本。利用HPLC-MS技术平台对PTC组织及癌旁组织样本的差异性代谢物进行多维统计分析，寻找与PTC相关的异常代谢通路。结果：经PCA、PLS-DA、OPLS-DA分析得知癌组织和癌旁组织的代谢轮廓具有显著性差异。经OPLS-Loading plot分析，结合VIP>1、FC>2，且P<0.05，筛选出76个潜在差异性代谢物。其中亮氨酸、2-褪黑素、香草酸等33种代谢物在PTC组织中表达上调；3-葡萄糖苷、甘油磷脂、磷脂酰胆碱、乳糖等43代谢物在PTC癌组织中表达下调。寻找到与差异性代谢物相关的13条异常代谢通路，如半胱氨酸与蛋氨酸代谢、与甘油磷脂代谢、嘧啶代谢、半乳糖代谢以及丙氨酸、天冬氨酸和谷氨酸代谢、柠檬酸循环等，这些代谢通路可能参与PTC代谢的病理生理过程。ROC曲线下面积大于0.9的差异性代谢物有5种，分别是庚二酸、糖醇、辛二酸、乳糖和L-丝氨酸。结论：PTC组织中半乳糖代谢和氨基酸代谢发生改变，PTC组织细胞中存在沃伯格效应(Warburg effect)。庚二酸、糖醇、辛二酸、乳糖、L-丝氨酸五种差异性代谢物可以用来区分PTC患者与正常人。

Qualitative and quantitative analysis of cyclovirobuxine D and related substances by HPLC-CAD in the active pharmaceutical ingredient of Huangyangning tablets / 药学学报

We have developed a new method using HPLC-CAD (charged aerosol detector) for the quantitative analysis of cyclovirobuxine D and related substances in the API of Huangyangning tablets. The related substances were further studied by HPLC-Q-Exactive coupled with hybrid quadrupole-orbitrap mass spectrometry. A HILIC column of XBridge Amide (4.6 mm×250 mm, 5 μm) was used, and the mobile phase was composed of acetonitrile and 100 mmol·L-1 ammonium formate (85∶15), which was adjusted to pH 2.8 with formic acid. Isocratic mode elution was adopted at a flow rate of 1.1 mL·min-1. The column temperature was set at 30 ℃. For CAD, the temperature of atomization and gas pressure were respectively set at 35 ℃ and 62.2 psi. This method detected and quantified five related substances to cyclovirobuxine D. The results showed that the LOD and LOQ of cyclovirobuxine D was 12.588 ng and 28.323 ng, respectively with an average recovery of 95.74% (RSD = 1.79%, n = 6). The content of cyclovirobuxine D in 12 batches of API samples provided by three manufacturers was from 79.94% to 88.49%, with an average value of 82.20%. The total content of the five related substances was from 15.99% to 22.15% with an average value of 20.10%, using an external standard method with cyclovirobuxine D as the reference and according to the CAD uniform response to non-volatile substances. The newly developed HPLC-CAD method has advantages in terms of the comprehensiveness of signals from Buxus alkaloids without UV absorption and with high sensitivity to its trace-related substances; the method yields good separation between the components and is compatible with mass spectrometry. It is applicable for the accurate quantitative analysis of main components and related substances in the API of Huangyangning tablets.

Rapid identification of classical herbal formulae Baoyinjian's chemical substance based by HPLC-Q/TOF-MS / 中草药

Objective: To analyze the chemical constituent cluster of classical herbal formulae Baoyinjian systemically by HPLC-Q/TOF-MS. Methods: The seperation was performed on Diamonsil C18 (250 mm × 4.6 mm, 5 μm) column with gradient elution with acetonitrile-0.1% formic acid. The column temperature was 30 ℃, the flow rate was 1 mL/min, the injection volume was 10 μL, and the mass spectrometry condition was X500R QTOF mass spectrometer, electrospray ion source, positive and negative mode scanning. Results: A total of 52 chemical constituents were identified by reference confirmation, literature comparison, and high mass spectrometry data analysis. The chemical constituent cluster was composed of 17 flavonoids, six phenolics, 12 iridoid glycosides, eight alkaloids, one phenethyl alcohol glycosides, four monoterpene glycoside, two triterpenes and two other compound. Conclusion: This study can identify various chemical constituents of Baoyinjian systematically, accurately, and rapidly, which provides a basis for the determination of the quality attributes of Baoyinjian.

Qualitative Analysis of Multiple Cumarins in Angelicae Sinensis Radix Based on HPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

Objective:To systematically and comprehensively analyze coumarin components in Angelicae Sihensis Radix by an efficient and stable HPLC-Q-TOF-MS/MS method,in order to offer the theoretical basis to develop coumarin,establish the quality control standard and apply in clinic. Method:The separation effect of coumarin components was extracted by adjusting the column,temperature,mobile phase,flow rate,sample concentration and other conditions,and various coumarin components in Angelicae Sihensis Radix were identified by corresponding standards,precise molecular mass,polarity,pyrolysis pattern. Result:In this study,a high-efficiency and stable coumarin separation method was established that can be used to separate complex components,and 14 coumarin components were identified in this study,including phellopterin and osthenol that were rarely reported as effective components in Angelicae Sihensis Radix. Major fragment ions of coumarin components were analyzed. The cleavage in methoxy bond or anisole bond on the parent nucleus was the primary pattern for coumarin components, which was summarized for detecting unknowing coumarins. Conclusion:Abundant coumarins were contained in Angelicae Sihensis Radix. Further qualitative and quantitative analysis of coumarins are conducive to improving the quality standards of Angelicae Sihensis Radix,and providing reference for the development of coumarins and clinical application of Angelicae Sinensis Radix.

Qualitative analysis of metabolites of ethanol extract of Cuscuta chinensis by HPLC-Q/TOF MS/MS / 中草药

Objective To qualitatively analyze the chemical constituents in rats after intragastric administration of estrogenically active ethanol extract of Cuscuta chinensis. Methods The high performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q/TOF MS/MS) was used to identify the prototypes and metabolites in rat urine. Results Twelve chemical constituents were identified in the drug-containing urine, including six prototypes and six metabolites. The prototypes are kaemoferol-3-β-D-glucuronide, 6-O-(E)-p-coumaroyl-β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside, aempferol-7-rhamnoside, chlorogenic acid, and apigenin. The metabolites are p-hydroxycinnamic acid, p-hydroxyphenylpropionic acid, isorhamnetin, kaempferol-3-O-glucoside, acetyl caffeic acid, and quercetin sulfate. Conclusion The method of HPLC-Q/TOF MS/MS is simple and rapid for the analysis of prototype components and metabolites in rats urine after oral administration of ethanol extract of C. chinensis, providing the basis for further clarification of the estrogen material basis of C. chinensis.

Identification and screening of cardiac glycosides in Streptocaulon griffithii using an integrated data mining strategy based on high resolution mass spectrometry / 中国天然药物

The present study was designed to develop a practical strategy to tackle the problem of lacking standard compounds and limited references for identifying structure-related compounds in Streptocaulon griffithii Hook. f., especially those in trace concentrations, with a focus on antitumor activity. The cardiac glycosides (CGs)-enriched part was determined using in vitro bioactive assays in three cancer cell lines and then isolated using macroporous resins. The MS and MS/MS data were acquired using a high performance liquid chromatography coupled with hybrid quadrupole-time of flight (HPLC-Q-TOF-MS) system. To acquire data of trace compound in the extract, a multiple segment program was applied to modify the HPLC-Q-TOF-MS method. A mass defect filter (MDF) approach was employed to make a primary MS data filtration. Utilizing a MATLAB program, the redundant peaks obtained by imprecise MDF template calculated with limited references were excluded by fragment ion classification, which was based on the ion occurrence number in the MDF-filtered total ion chromatograms (TIC). Additionally, the complete cleavage pathways of CG aglycones were proposed to assist the structural identification of 29 common fragment ions (CFIs, ion occurrence number ≥ 5) and diagnostic fragment ions (DFIs, ion occurrence number < 5). As a result, 30 CGs were filtered out from the MDF results, among which 23 were identified. This newly developed strategy may provide a rapid and effective tool for identifying structure-related compounds in herbal medicines.

Identification and screening of cardiac glycosides in Streptocaulon griffithii using an integrated data mining strategy based on high resolution mass spectrometry / 中国天然药物

The present study was designed to develop a practical strategy to tackle the problem of lacking standard compounds and limited references for identifying structure-related compounds in Streptocaulon griffithii Hook. f., especially those in trace concentrations, with a focus on antitumor activity. The cardiac glycosides (CGs)-enriched part was determined using in vitro bioactive assays in three cancer cell lines and then isolated using macroporous resins. The MS and MS/MS data were acquired using a high performance liquid chromatography coupled with hybrid quadrupole-time of flight (HPLC-Q-TOF-MS) system. To acquire data of trace compound in the extract, a multiple segment program was applied to modify the HPLC-Q-TOF-MS method. A mass defect filter (MDF) approach was employed to make a primary MS data filtration. Utilizing a MATLAB program, the redundant peaks obtained by imprecise MDF template calculated with limited references were excluded by fragment ion classification, which was based on the ion occurrence number in the MDF-filtered total ion chromatograms (TIC). Additionally, the complete cleavage pathways of CG aglycones were proposed to assist the structural identification of 29 common fragment ions (CFIs, ion occurrence number ≥ 5) and diagnostic fragment ions (DFIs, ion occurrence number < 5). As a result, 30 CGs were filtered out from the MDF results, among which 23 were identified. This newly developed strategy may provide a rapid and effective tool for identifying structure-related compounds in herbal medicines.