Metabolomic Study of Biochemical Changes related to toxicity induced by bupleurotoxin Using LC-MSCoupled with a Pattern Recognition Approach

The purpose of this study was to detect changes in urine of mice and to clarify the toxicity induced by bupleurotoxin (BETX) using liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). A procedure for urine analysis using pattern recognition was proposed to evaluate the toxicity induced by BETX in male BALB/c mice. BETX at 2.5 mg/kg was administered intraperitoneally (i.p.), and urine samples for the metabolomic study were collected from control and BETX experimental groups. Changes in the concentrations of some urine metabolites were detected exclusively in the experimental group. All results suggested that exposure to BETX might cause a disturbance in fatty acid metabolism and the oxidative stress system. These results may not only clarify the underlying mechanism of diverse intoxication effects of BETX but also provide the guidance in preclinical toxicity screening for new drugs.     

基于血清药物化学的厚藤治疗急性痛风性关节炎质量标志物研究

目的基于中药质量标志物(qualitymarker,Q-Marker)的概念,研究厚藤提取物对急性痛风性关节炎(acutegouty arthritis,AGA)大鼠的治疗作用,并分析厚藤提取物在正常大鼠及AGA大鼠的血中移行成分,为厚藤Q-Marker的确定提供依据。方法采用尿酸钠建立AGA大鼠模型,考察厚藤提取物对AGA大鼠关节肿胀指数、足垫肿胀程度和滑膜组织病理变化的影响,考察厚藤提取物对AGA大鼠血清中白细胞介素-1β(interleukin-1β,IL-1β)、巨噬细胞炎性蛋白-1α(macrophage inflammatory protein-1α,MIP-1α)和MIP-2水平的影响。采用液相色谱-四级杆-飞行时间串联质谱(LC-Q-TOF-MS/MS)技术对厚藤提取物进行定性分析,探寻正常大鼠和AGA大鼠ig厚藤提取物后的入血成分。结果与模型组比较,厚藤提取物组大鼠关节肿胀指数和足垫厚度均显著降低(P0.05、0.01),厚藤提取物高、中剂量组大鼠血清中IL-1β、MIP-1α和MIP-2水平均显著降低(P0.05),厚藤提取物高、中剂量组大鼠滑膜组织炎性细胞浸润减轻。从厚藤提取物中鉴定出树脂糖苷类、黄酮类、有机酸类、香豆素类、核苷类、木脂素类等53个成分,其中有机酸类和黄酮类为其主要成分,并推测了树脂糖苷类、黄酮类、有机酸类、香豆素类部分成分的裂解规律;正常大鼠含药血浆中鉴定出苹果酸、咖啡酸、咖啡酸甲酯和对羟基苯甲酸4个成分,AGA大鼠含药血浆中鉴定出莨菪亭、咖啡酸、咖啡酸甲酯和对羟基苯甲酸4个成分。结论厚藤提取物能够明显改善AGA大鼠关节肿胀,抑制血清中IL-1β、MIP-1α及MIP-2水平,并抑制炎症反应;采用LC-Q-TOF-MS/MS技术可快速鉴定厚藤水提物的主要成分为有机酸类和黄酮类,莨菪亭、咖啡酸、绿原酸及其异构体(新绿原酸、隐绿原酸、异绿原酸B、异绿原酸A、异绿原酸C)和原儿茶酸在AGA大鼠中以原型成分或代谢物形式入血,可作为厚藤治疗AGA的Q-Marker。     

高效液相质谱联用技术鉴定刺五加注射液中苯丙素类化学成分

刺五加药材的化学组成已有报道,而未见刺五加注射液化学物质基础的研究报道。该研究采用液相色谱离子阱质谱（LC-MSⁿ）和液相色谱四极杆飞行时间质谱（LC-Q-TOF-MS）技术对刺五加注射液中苯丙素类化学成分进行定性分析。采用Ultmate^TM XB-C₁₈色谱柱（4.6 mm×250 mm,5 μm）,以0.5%甲酸溶液和乙腈为流动相进行梯度洗脱,流速0.8 mL·min^-1,柱温20℃。根据高分辨和多级质谱数据,并结合对照品及文献数据,在刺五加注射液中共推断出54个苯丙素类成分,包括苯丙酸类成分34个,木脂素类成分16个,香豆素类成分4个,其中28个成分在刺五加中未见报道,通过对照品比对鉴定了14个化合物结构。该研究建立的方法可以简便快速地对刺五加注射液中的苯丙素类化学成分进行鉴定,研究结果为明确刺五加注射液化学物质基础提供了科学数据。     

Metabolic profiling reveals therapeutic biomarkers of processed Aconitum Carmichaeli Debx in treating hydrocortisone induced Kidney-Yang deficiency syndrome rats

Ethnopharmacological relevance

Kidney-Yang Deficiency syndrome (KYDS) is a diagnostic pattern in traditional Chinese medicine (TCM) and clinical data showed that the unbalance in adrenal cortical hormone is the key issue in KYDS patients. The processed Ranunculaceae Aconitum carmichaeli debx (Bai-Fu-Pian in Chinese, BFP) is one of the most commonly used Chinese herbs for treating KYDS. The present study was conducted to explore the therapeutic biomarkers of the BFP in treating hydrocortisone administration induced KYDS rats.

Materials and methods

Thirty male Sprague-Dawley rats were randomly divided into five groups with six in each group. KYDS in rats was induced by i.p. injection of hydrocortisone at the dose of 10 mg/kg per day for 15 days as described previously. The rats with KYDS were administered orally, starting from the day of hydrocortisone administration stopped, with BFP extract at the dose of 0.32 g/kg, 0.64 g/kg and 1.28 g/kg per day respectively for 15 days. The blood samples were collected for the liquid chromatography quadruple time-of-flight mass spectrometry (LC-Q-TOF-MS) test, as well as radioimmunoassay to determine the concentrations of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and adrenocorticotrophic hormone (ACTH). The metabolic responses to BFP administration were investigated by using the principal components analysis (PCA) and orthogonal partial least squares analysis (OPLS). Bioinformatics analyses were performed by using the Ingenuity Pathway Analysis (IPA). Variance analysis and linear regression analysis were used in this study.

Results

The signs and concentrations of cAMP, cGMP and ACTH in the model rats were similar to those previously described about KYDS rats and BFP treatment can reverse the changes. Seventeen significantly changed metabolites among different groups were identified. Thirteen metabolites were identified in the KYDS rats comparing to healthy rats with nine up-regulated and four down-regulated. After BFP treatment at three dosages, five up-regulated metabolites including phosphate, betaine, (4-hydroxyphenyl) acetaldehyde, 5-hydroxyindol-3-acetic acid and 5'-phosphoribosyl-N-formylglycinamide were dose-dependently reversed. The network analysis with IPA showed that four canonical pathways including superpathway of methionine degradation, purine nucleotides De Novo biosynthesis II, tyrosine synthesis and serotonin receptor signaling involved the therapeutic mechanism of BFP in treating the KYDS rats.

Conclusions

Five therapeutic biomarkers (phosphate, betaine, (4-hydroxyphenyl) acetaldehyde, 5-hydroxyindol-3-acetic acid and 5'-phosphoribosyl-N-formylglycinamide) and two corresponding canonical pathways (amino acid metabolism and purine nucleotide metabolism) were identified to be involved in the therapeutic mechanism of BFP treating the KYDS.     

基于UPLC-Q-TOF-MS技术的北青龙衣有效成分动态变化分析

采用UPLC-Q-TOF-MS/MS技术分析不同采集时期北青龙衣有效成分动态变化规律。用Marker View软件对6个采集时期18批样品测定数据进行主成分分析,差异性化合物通过Peakview 2.0/Masterview1.0软件,依据精确质量数和同位素峰度比确定分子式,再通过对照品及数据库的二级谱图比对、裂解规律分析,结合已有文献报道,确定结构式。结果表明不同采集期萘醌类有效成分含量发生了明显的变化,鉴定或推断了北青龙衣中38个萘醌类化合物结构,随着采收时间的变化大部分萘醌类成分含量逐渐下降。该方法快速、准确,为北青龙衣的化学成分鉴定与分析提供一种新的策略,为揭示不同生长期北青龙衣有效成分动态变化规律及确定其药材合理采收期提供基础资料。     

小叶榕干浸膏及咳特灵胶囊的HPLC指纹图谱研究及LC-Q-TOF-MS成分分析

目的:建立小叶榕干浸膏及咳特灵胶囊的HPLC指纹图谱,并对其主要成分进行成分鉴定分析。方法:采用高效液相色谱法建立小叶榕干浸膏及其制剂的指纹图谱并用液相色谱-四极杆-飞行时间质谱(LC-Q-TOF-MS)鉴定指纹图谱主要色谱峰。采用Acquity UPLC HSS T₃C₁₈色谱柱(2.1 mm×100 mm,1.7 μm),流动相甲醇-0.05%甲酸梯度洗脱,流速0.2 mL·min^-1,柱温25℃。对指纹图谱所标识色谱峰进行了部分鉴定。结果:建立了小叶榕干浸膏及咳特灵胶囊的HPLC指纹图谱,通过液相色谱-四极杆-飞行时间质谱对其主要成分进行成分鉴定分析,鉴别出了14个化合物组分。结论:该指纹图谱方法适用于小叶榕干浸膏及咳特灵胶囊,化学成分鉴定准确,完善了高效液相指纹图谱,为小叶榕干浸膏及其制剂的整体质量控制提供了一定的依据。     

Biomarkers in the early period of acute myocardial infarction in rat serum and protective effects of Shexiang Baoxin Pill using a metabolomic method

Ethnopharmacological relevance

To identify the biomarkers in early period of acute myocardial infarction (AMI) in rat serum and reveal the effective mechanism of a Traditional Chinese Medicine (TCM) named Shexiang Baoxin Pill (SBP).

Material and method

A metabolomic approach using reversed-phase liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) was developed.

Results

Fourteen biomarkers in the early period of acute myocardial infarction (AMI) in rat serum were identified. These biomarkers include 5-methylcytosine, cystathionine ketimine, 2-oxoadipic acid, thymidine, epinephrine, homocystine, uric acid, 12(S)-hydroperoxyeicosatetraenoic acid (12s-HPETE), 11-dehydrocorticosterone, 12(S)-hydroxyeicosatetraenoic acid (12s-HETE), deoxycorticosterone, corticosterone, aldosterone and cortisol. Through pathway analysis of these biomarkers, inflammation, hypertrophy and oxidative injury were considered the most relevant pathological changes in early period of AMI.

Conclusion

Identification of AMI biomarkers not only supplied a systematic view of the progression of AMI in the early period but also provided the theoretical basis for the prevention or treatment of AMI. The results demonstrated that SBP pretreatment could offer protective effects for AMI through regulating the pathway of steroid hormone biosynthesis.     

LC-Q-TOF-MS和LC-IT-MS分析桂枝麻黄各半汤中化学成分

目的 对桂枝麻黄各半汤中化学成分进行研究。方法 采用液相色谱-四级杆飞行时间质谱(LC-Q-TOF-MS)和液相色谱-离子阱质谱(LC-IT-MS)对桂枝麻黄各半汤提取物进行分析,色谱柱为Zorbax SB-C₁₈ Rapid Resolution HT,流动相为0.05%甲酸水(A)-乙腈(B),梯度洗脱,流速为0.6 mL·min^-1,柱温30 ℃。结合LC-Q-TOF-MS提供的化合物准确相对分子质量和LC-IT-MS分析获得的化合物多级质谱信息,推测鉴定桂枝麻黄各半汤化学成分结构。结果 共推测鉴定了桂枝麻黄各半汤中119个成分,包括26个黄酮、47个三萜皂苷、11个单萜苷、7个生物碱以及28个其他化合物,其中有25个成分经过与对照品比对确认其结构。结论 基本明确了桂枝麻黄各半汤化学成分,为其质量评价及药效物质研究提供了重要参考。     

LC-Q-TOF-MS和LC-IT-MSn分析当归芍药散中化学成分

目的 对当归芍药散中化学成分进行研究。方法 采用液相色谱-四级杆-飞行时间质谱（LC-Q-TOF-MS）和液相色谱-离子阱-质谱（LC-IT-MSⁿ）对当归芍药散70%乙醇-水提物进行分析,色谱柱为Agilent Zorbax SB-C₁₈、Rapid Resolution HT（50 mm×4.6 mm,1.8 μm）,流动相为0.05%甲酸水（A）-乙腈（B）,梯度洗脱,体积流量为0.6 mL/min。结合LC-Q-TOF-MS提供的化合物准确相对分子质量和LC-IT-MS分析获得的化合物多级质谱信息,对当归芍药散化学成分进行结构推测鉴定,并对化合物的药材来源进行归属。结果 共鉴定了当归芍药散中30个成分,其中14个为首次在当归芍药散中发现,包括以绿原酸（8）和没食子酰蔗糖（5～7）为代表的6个有机酸及其苷类化合物,以16-氧化泽泻醇A 23-乙酸酯（22）为代表的2个原萜烷型三萜类化合物,以环四（异）亮氨酸（14）为代表的4个环肽类化合物以及N-(1-deoxy-D-fructos-1-yl) pyroglutamic acid（1）和腺苷（3）等。结论 研究结果加深了对当归芍药散化学组成的理解,为其药效物质基础研究及质量控制提供了一定的理论依据。     

LC-Q-TOF-MS及LC-IT-MS分析酸枣仁汤的化学成分

采用液相色谱-四级杆-飞行时间质谱（LC-Q-TOF-MS）及液相色谱-离子阱质谱（LC-IT-MS）系统剖析了酸枣仁汤中化学成分。色谱柱为Agilent Zorbax SB-C₁₈,Rapid Resolution HT（4.6 mm×50 mm,1.8 μm）;以乙腈（A）-0.05%甲酸溶液（B）为流动相,梯度洗脱;流速0.6 mL·min^-1;柱温30 ℃。共鉴定了酸枣仁汤中48个化合物,并对化合物的药材来源进行了归属,首次报道了方剂中的31个化合物。LC-Q-TOF-MS结合LC-IT-MS可以简便、快速地对酸枣仁汤中化学成分定性分析,本研究结果为酸枣仁汤质量控制和药效物质基础研究提供了一定的理论依据。