糖肾方对自发性Ⅱ型糖尿病鼠磷脂代谢的影响

利用正相液相色谱飞行时间质谱建立了大鼠血浆磷脂的轮廓谱。通过对磷脂轮廓谱的PLS-DA判别分析及14种化合物的定量,考察了中药糖肾方对自发性II型糖尿病鼠磷脂代谢的影响,并对比了糖肾方与西药蒙诺在糖尿病发展过程中的不同作用。结果表明：与正常对照组相比,糖尿病鼠的血浆磷脂代谢发生异常。糖肾方能够调节糖尿病鼠的磷脂代谢紊乱,蒙诺则加剧了病鼠的磷脂代谢异常。此外,8种磷脂化合物可能成为糖尿病发展过程中的潜在生物标志物。     

复方丹参滴丸抗急性心肌梗死代谢组学研究

采用左冠状动脉前降支结扎的方法,建立SD大鼠急性心肌梗死(Acute myocardial infarction, AMI)模型,使用超高效液相色谱-串联四极杆/飞行时间质谱(UPLC-QTOF/MS)代谢组学研究平台对大鼠血浆样本进行代谢轮廓分析,研究复方丹参滴丸对AMI大鼠心肌保护作用机制.经主成分分析和偏最小二乘法分析,筛选并鉴定出22种与AMI相关的差异代谢物,其中的8种能够被复方丹参滴丸显著调节,分别是硫酸对甲苯、马尿酸、雌马酚葡萄糖苷酸、溶血磷脂胆碱(16∶0)、胆酸、油酸酰胺、棕榈酰胺和鞘脂(d18∶1/16∶0).研究结果表明, 复方丹参滴丸可能是通过调节苯丙氨酸代谢、甘油磷脂代谢、脂肪酸代谢、胆汁酸代谢及鞘脂代谢通路,发挥抗AMI大鼠心肌损伤的作用.     

中药通心络对抑郁-动脉粥样硬化大鼠干预作用的血浆代谢组学研究

采用快速液相色谱串联离子阱飞行时间质谱(UFLC/MS-IT-TOF)技术对通心络干预下的抑郁-动脉粥样硬化大鼠的血浆代谢谱进行研究.结果表明,抑郁-动脉粥样硬化模型大鼠体内的氨基酸代谢、脂肪酸的β-氧化、胆固醇代谢及磷脂代谢发生了异常.中药通心络通过调节模型大鼠体内的色氨酸、苯丙氨酸代谢及某些胆汁酸的代谢,抑制大鼠抑郁-动脉粥样硬化的形成.该研究结果表明代谢组学在中药整体性药效评价、作用机理的阐明等方面具有很好的应用前景.     

基于液相色谱-串联质谱的氨基酸代谢组学方法研究黄芪注射液治疗脑缺血

建立了基于液相色谱-串联质谱技术的氨基酸代谢组学分析方法。选用Diamonsil C18色谱柱,乙腈-0.1%甲酸为流动相,梯度洗脱,三重四级杆质谱检测器,MRM扫描方式,12 min内对血浆中12种氨基酸进行定量分析。应用本方法对脑缺血模型和黄芪注射液的治疗作用做出评价。通过偏最小二乘判别分析统计发现,脑缺血12 h,其血浆中的氨基酸代谢表达存在显著差异,结合载荷图及VIP值确定丝氨酸、天冬氨酸、甘胺酸等7种脑缺血生物标识物;采用黄芪注射液治疗后,从得分图上可以看到氨基酸代谢的轨迹发生了较大变化;与脑缺血模型组比较,丝氨酸、天冬氨酸、甘胺酸等生物标识物均向正常水平趋近。     

基于超高效液相色谱-四极杆飞行时间质谱的缺血性脑卒中的代谢组学研究

采用基于超高效液相色谱-四极杆飞行时间质谱的代谢组学方法,研究缺血性脑卒中患者和健康人群的血浆,分析了缺血性脑卒中的生物标志物.实验收集30例缺血性脑卒中患者和17例健康志愿者的血浆样品,采用主成分分析和正交偏最小二乘判别分析,研究了缺血性脑卒中患者组和健康对照组血浆中的代谢物差异,并进行了代谢通路分析.实验结果表明,患者组血浆中的二氢神经鞘氨醇、植物鞘氨醇等物质含量升高,谷氨酰胺、焦谷氨酸和2-酮丁酸等物质含量降低.结果表明,脑卒中不仅影响了鞘脂类和氨基酸的代谢,还对能量代谢产生了显著的影响.     

基于液相色谱-质谱联用技术的代谢组学方法用于中药通心络和人参对过度疲劳大鼠干预作用的评价

用代谢组学方法评价了中药通心络和人参对过度疲劳大鼠的干预作用.通过构造大鼠过度疲劳模型,并分别用通心络和人参进行干预,采用快速液相色谱-离子阱飞行时间质谱(UFLC-IT-TOF-MS)获取大鼠血浆代谢轮廓,并用正交偏最小二乘法(OPLS)进行多变量统计分析,分别找出用于区分通心络和人参干预组大鼠同正常对照大鼠、过度疲...     

黄芪口服液降低大鼠顺铂毒性作用机制的尿液代谢组学研究

采用基于液相色谱-飞行时间质谱联用(LC-TOF-MS)技术的代谢组学方法,分析大鼠尿液内源性代谢物的变化,研究黄芪口服液(HO)降低大鼠顺铂(CDDP)毒性的作用机制.采用低剂量多次腹腔注射CDDP的方法建立CDDP染毒大鼠模型,并连续给予16天HO.于第18天收集正常对照(Control)组、顺铂模型(CDDP)组和黄芪口服液(HO)组大鼠的24 h尿液, 进行LC-TOF-MS分析,以获取尿液代谢物组数据集,对所得数据进行主成分分析(PCA)和正交偏最小二乘法-判别分析(OPLS-DA)等多元统计分析,以筛选潜在生物标志物.于第20天采集大鼠血清测定肌酐和尿素氮水平.血清指标测定结果表明, HO可以显著降低CDDP染毒大鼠的肌酐和尿素氮水平(p<0.05).PCA得分图显示,3组可分别聚类,HO组位于Control组和CDDP组中间,表明HO可部分改善CDDP所致大鼠尿液代谢产物的异常变化.综合OPLS-DA分析、t检验和倍数变化分析结果,最终共筛选并初步鉴定出35个尿液代谢产物作为HO减毒相关的潜在生物标记物.代谢通路分析结果表明,HO可通过纠正体内氨基酸代谢、能量代谢和核苷酸代谢等通路的紊乱,降低CDDP所致机体毒性.     

基于肝脏代谢组分析研究低聚硒化氨基多糖对黑鲷的免疫调节作用

采用基于液相色谱-飞行时间质谱联用（LC-TOF-MS）技术的代谢组学方法，分析黑鲷肝脏内源性代谢物的变化，研究硒化氨基多糖增强黑鲷的免疫调节机制。采用XCMS^plus软件非靶向分析质谱采集数据，筛选潜在生物标志物，并通过MetaboAnalyst3.0网站分析相关代谢通路。结果表明，饲喂硒化氨基多糖组中的代谢物明显区分于空白组，发现并鉴定了32个有差异的生物标志物。代谢通路分析结果表明，硒化氨基多糖可通过氨基酰基-转运脱氧核糖核苷酸（tRNA）生物合成、氨基酸代谢、核苷酸代谢、氮代谢等代谢通路增强黑鲷自身的免疫机能。该研究为阐明硒化氨基多糖的免疫增强机制提供了科学依据。     

高脂与维生素D缺乏饮食诱导的2型糖尿病小鼠血清和肝脏代谢组学研究

采用高脂与维生素D缺乏(VDD)饮食长期(24周)喂养小鼠,诱导其形成2型糖尿病(T2DM),通过小鼠血清和肝脏代谢组学分析探究了T2DM发生、发展的代谢物和代谢通路变化机制.实验收集小鼠血清和肝脏样品,通过气相色谱-质谱联用技术和硅烷化衍生方法分析得到血清和肝脏代谢轮廓;利用正交偏最小二乘判别分析和非参数检验筛选血清和肝脏代谢组中具有显著性差异的代谢标志物,发现血清样品中乳酸、丙氨酸、甘油、苏氨酸和葡萄糖含量在高脂+VDD小鼠中显著升高,肝脏样品中乳酸、核糖、果糖、葡萄糖、油酸和棕榈酸含量在高脂+VDD小鼠中显著升高.本文还进行了血清和肝脏代谢轮廓整体分析和代谢通路探索,发现高脂+VDD小鼠中三羧酸循环、糖异生、氨基酸以及脂质代谢通量均显著增强,这些代谢路径相互影响共同促进T2DM的发生和发展.本文通过饮食诱导小鼠形成T2DM,得到血清和肝脏代谢物及其代谢通路的变化关系,可为T2DM诊断提供参考信息.     

非靶向尿液代谢组学方法研究五味子治疗精尿病并发症作用机制

采用超高效液相色谱-串联四级杆飞行时间质谱联用技术(Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry,UPLC/Q-TOF-MS),结合主成分分析技术(Principal component analysis,PCA),进行了五味子治疗糖尿病并发症大鼠尿液代谢组学研究,对五味子治疗糖尿病并发症大鼠后尿液生物标志物变化进行了考察.采用UPLC/Q-TOF-MS方法分析了健康组、糖尿病并发症模型组和五味子给药组的大鼠尿液,采用偏最小二乘法-判别分析(Partial least squares discriminant analysis,PLS-DA)和正交偏最小二乘法-判别分析(Orthogonal partial least squares discriminant analysis,OPLS-DA)载荷图筛选,通过分析对各组分离贡献较大(VIP>1,P°0.05)化合物的串联质谱数据,再经Human Metabolome Data-base(HMDB)等数据库检索,进行质谱信息匹配,尿液中共鉴定出28种潜在生物标记物,其中正谱13种,负谱15种.这些生物标记物主要影响戊糖和葡糖醛酸相互转化通路、核黄素代谢、泛酸和CoA合成、精氨酸和脯氨酸代谢、肠内菌代谢、嘌吟代谢、Vc代谢胆酸合成、色氨酸代谢等通路,五味子从能量代谢、氨基酸代谢、肠内菌代谢、脂类代谢等各个角度发挥治疗糖尿病并发症的作用.从各个通路的相关生物功能分析,五味子治疗糖尿病肾病的作用较强,此外还具有降脂、抗氧化的功效.     

Determination of apigenin in rat plasma by high-performance liquid chromatography

Apigenin (4′,5,7-trihydroxyflavone, AP) belongs to a less-toxic and non-mutagenic flavone subclass of flavonoids, the biotransformation and metabolism of which have been little studied until now. Therefore, this study is focussed on the determination of AP in free form. AP was administered to rats via the i.p. route (25 mg kg⁻¹) and then the blood was collected at 10, 15, 30 and 45 min after injection. Methanol was used for rat plasma deproteinization. The HPLC assay (mobile phase, 2% formic acid–acetonitrile–methanol, 40:35:25, v/v; flow-rate, 1 ml min⁻¹; UV detection at 349 nm) for AP determination was validated and used for the quantification of AP in rat plasma. The unknown concentration was calculated from the equation obtained by the least-squares regression analysis (y=0.521x+1.130, r²=0.998). The highest concentration of AP in plasma was found to be 30 min after injection. The concentration profile of AP obtained here may contribute to until known results about AP metabolism. They could be applied to other studies of AP or related flavonoids because of favourable effects on human health.     

Metabolomics analysis of the therapeutic mechanism of Semen Descurainiae Oil on hyperlipidemia rats using 1H‐NMR and LC–MS

Semen descurainiae oil (SDO) is an important traditional Chinese medicine that was recently discovered to have the function of reducing blood lipids. Metabolomics analyses of plasma, liver and kidney in rats were performed using ¹H‐NMR and LC–MS to illuminate the lower blood lipid concentration effect of SDO, and niacin was considered as the active control. The measure of total cholesterol (TC) and low‐density‐lipoprotein cholesterol (LDL‐C) in plasma showed that SDO treatment decreased significantly the content of TC and LDL‐C. An orthogonal partial least squares–discriminant analysis approach was applied to identify the different metabolic profiles of plasma, liver and kidney in rats and to detect related potential biomarkers. The results suggested that the metabolic profiles of the control group and hyperlipidemia group showed significant difference and the SDO and niacin group had effective anti‐hyperlipidemia function. The biomarkers primarily concern lipid metabolism, amino acid metabolism and glycometabolism, and the change in biomarkers indicated that hyperlipidemia could cause the unbalance of these metabolic pathways in vivo. SDO reduced blood lipids by repairing amino acid and lipid metabolism.     

Urinary biomarker and treatment mechanism of Rhizoma Alismatis on hyperlipidemia

Rhizoma Alismatis (RA), a diuretic in Asia and Europe, was found to possess anti‐hyperlipidemic activity. Since the biomarkers and mechanisms of RA in the treatment of hyperlipidemia are inadequate, ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight synapt high‐definition mass spectrometry and multivariate data analysis were employed to investigate the urinary metabolomics of RA on hyperlipidemic rats induced by high‐fat diet. The metabolic profile of RA‐treated hyperlipidemic group located between control and diet‐induced hyperlipidemic groups. Nineteen metabolites with significant fluctuations were identified as potential biomarkers related to the hyperlipidemia and anti‐hyperlipidemia of RA using partial least‐squares‐discriminate analysis, heatmap analysis and correlation coefficient analysis. The fluctuations of these biomarkers represented disturbances in amino acid metabolism, purine metabolism, pyrimidine metabolism and energy metabolism. After RA treatment, these perturbed metabolites were restored to normal or nearly normal levels. RA can alleviate high‐fat diet‐induced dysfunctions in these metabolic pathways.     

Metabolite profiling of four major flavonoids of Herba Epimedii in zebrafish

The zebrafish model organism was applied first in a metabolic study of icariin, baohuoside I, epimedin A and epimedin C, which are flavonoids in Herba Epimedii. Metabolites of these compounds in zebrafish after exposure for 24 h were identified by HPLC-ESI-MS, whereby the separation was performed with a Zorbax C-18 column using a gradient elution of 0.05% formic acid acetonitrile-0.05% formic acid water. The quasi-molecular ions of compounds were detected in simultaneous negative and positive ionization modes. Metabolic products of icariin and epimedin C via cleavage of glucose residue instead of rhamnose residues were found, which coincided with the results using regular metabolic analysis methods. In addition, the zebrafish model was used to predict the metabolism of the trace component epimedin A, whose metabolic mechanisms haven't been clearly elucidated with the current metabolism model. The metabolic pathway of epimedin A in zebrafish was similar to those of its homologue icariin and epimedin C. Our study demonstrated that the zebrafish model can successfully imitate the current models in elucidating metabolic pathways of model flavonoids, which has advantages of lower cost, far less amount of compound needed, easy set up and high performance. This novel model can also be applied in quickly predicting the metabolism of Chinese herb components, especially trace compounds.     

液相色谱-串联质谱法同时测定动物血浆中21种霉菌毒素或其代谢物残留

建立了同时检测动物血浆中黄曲霉毒素B1等21种霉菌毒素或其代谢物残留的液相色谱-串联质谱方法.动物血浆样品中加入0.1％甲酸-乙腈溶液、NaCl和无水MgSO4进行萃取,无水MgSO4和C18,PSA,A-AL对提取液进行脱水净化,经浓缩、复溶和离心后,再进行测定.采用反相C18色谱柱分离,以0.1％甲酸-0.5 mmol/L乙酸铵溶液和0.1％甲酸-甲醇溶液作为流动相进行梯度洗脱,采用电喷雾离子源(ESI)多反应监测离子模式(MRM)进行检测,基质标准曲线外标法进行定量分析,线性范围在0.05 ～ 100 ng/mL之间,方法的定量限为0.05 ～0.5 ng/mL.在高、中、低3个添加浓度水平下,21种霉菌毒素的平均回收率为62.0％ ～ 116.4％,相对标准偏差小于19％.     

非靶向代谢组学用于藜芦妨害人参治疗脾气虚的机制研究

采用超高效液相色谱串联四级杆飞行时间质谱( UPLC/Q-TOF-MS)联用技术,通过非靶向代谢组学方法分析大鼠尿液内源性代谢物的变化,研究藜芦妨害人参发挥药效作用的机制。建立脾气虚大鼠模型,连续给药15天,测定力竭游泳时间及血液中白细胞、红细胞、血红蛋白的含量。结果表明,人参可显著提高脾气虚模型大鼠的力竭游泳时间(p﹤0.01),升高白细胞、红细胞及血红蛋白含量(p﹤0.05,p﹤0.01),藜芦对脾气虚模型大鼠各项指标无明显影响(p>0.05),人参与藜芦配伍后对脾气虚模型大鼠各项指标均无显著影响(p>0.05),表明藜芦妨害了人参发挥药效作用。采用UPLC/Q-TOF-MS技术及非靶向代谢组学的方法分析了空白组、模型组、人参组、藜芦组、参藜组对脾气虚模型大鼠的尿液代谢组差异,其中主成分分析( PCA)得分图显示各组代谢轮廓有显著差别,并通过正交偏最小二乘法-判别分析( OPLS-DA)及数据库检索,鉴定出15种人参干预调节脾气虚模型大鼠的潜在生物标志物,从中找出了7种人参藜芦配伍后减弱人参上述干预作用的潜在生物标志物,并对其涉及的代谢通路进行了系统分析。上述研究结果表明,人参藜芦配伍后妨害了人参对脾气虚模型大鼠的治疗作用,其机理可能是影响人参对体内能量代谢、免疫平衡及氧化还原反应等相关代谢的调节。     

Development of simultaneous analysis of tryptophan metabolites in serum and gastric juice – an investigation towards establishing a biomarker test for gastric cancer diagnosis

To evaluate changes in tryptophan metabolism and discover diagnostic biomarkers for gastric cancer, a quantitative method was developed for tryptophan and its seven metabolites (indole‐3‐lactic acid, anthranilic acid, serotonin, nicotinic acid, kynurenic acid, kynurenine and 3‐indoxyl sulfate) in both human serum and gastric juice using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Serum and gastric juice were prepared with a simple protein precipitation using aqueous 0.1% formic acid and acetonitrile. As a result, it was found that the kynurenine pathway of tryptophan metabolism was activated in gastric cancer and that the metabolic ratio of kynurenine/tryptophan, which reflects the enzyme activity of indoleamine‐2,3‐dioxygenase, was associated with the observed metabolic changes. Finally, the investigation of tryptophan metabolites, especially kynurenic acid, in serum and gastric juice might serve as biomarkers for gastric cancer. The findings in this study provide critical information of tryptophan metabolism which can be applied to a serum‐based diagnostic test for gastric cancer.     

小花玄参化学成分的定性研究及总黄酮含量的测定

采用不同极性溶剂提取小花玄参中的化学成分,应用植物化学成分的系统分析法定性研究,并对其总黄酮含量进行测定.结果表明,小花玄参中含有氨基酸、多肽和蛋白质、糖及苷类、有机酸、鞣质、皂苷、甾体或三萜类、黄酮、酚类、挥发油和油脂、强心苷和生物碱等化学成分,未检出蒽醌及香豆素与萜类内酯;小花玄参中总黄酮的含量为5.689%(56.89mg/g).该结果可为小花玄参化学成分的分离纯化、药理研究以及开发利用等提供基础依据.     

基于气相色谱-质谱联用和图模型分析的脂代谢异常患者血浆代谢组学研究

利用气相色谱-质谱联用技术(GC-MS)和图模型分析方法,寻找脂代谢异常患者可能的血浆代谢标志物群。采用GC-MS技术对37例脂代谢异常患者和10例健康人的血浆样品进行分析,得到血浆代谢物的表达谱。偏最小二乘-判别分析(Partial least squares-discriminant analysis,PLS-DA)得分图可区分脂代谢异常患者与健康人,运用PLS-DA载荷图及t检验发现有9个代谢物在两组间存在显著性差异。经NIST谱库检索,它们分别为缬氨酸、甘氨酸、丙氨酸、焦谷氨酸、葡萄糖醛酸、半乳糖、甘露糖、亚油酸和甘油。在脂代谢异常患者血浆中,除甘油浓度显著高于健康人外,其余8种代谢物浓度均明显低于健康人。图模型分析结果发现这些代谢物与脂代谢异常临床常用诊断指标之间具有很好的相关性。它们可能是脂代谢异常疾病早期诊断和预后新的特异性代谢标志物群。     

Astragalus membranaceus ultrafine powder alleviates hyperuricemia by regulating the gut microbiome and reversing bile acid and adrenal hormone biosynthesis dysregulation

Ethnopharmacological relevanceMetabolic syndrome is closely related to the intestinal microbiota and disturbances in the host metabolome. Hyperuricemia (HUA), a manifestation of metabolic syndrome, can induce various cardiovascular diseases and gout, seriously affecting a patient’s quality of life. Astragalus membranaceus has a long history as a commonly used traditional Chinese medicine to treat kidney disease in China and East Asia.Materials and methodsWe compared the therapeutic effect of benzbromarone and two different doses Astragalus membranaceus ultrafine powder (AMUP) in rats with HUA. Ultra-performance liquid chromatography-mass spectrometer was used to analyze the AMUP metabolism in the plasma, urine, and feces. Further, 16S ribosome RNA sequencing and feces metabolomic were performed to capture the variation of the gut microbiota and metabolites changes before and after drug administration.ResultsAMUP had a notable impact on reducing blood uric acid levels while protecting the liver and kidney. Drug metabolism analysis demonstrated that effective constituent flavonoids are distributed in the blood, whereas saponins remain in the intestine. Gut microbiota analysis showed that low-dose AMUP ameliorated HUA-induced gut dysbiosis by reducing the abundance of harmful bacteria and increasing that of some beneficial bacteria with anti-inflammatory properties, such as Clostridia, Lachnospiraceae, and Muribaculaceae. In addition, HUA-induced changes in metabolite contents in bile acid and adrenal hormone biosynthesis pathways were restored after treatment with AMUP.ConclusionLow-dose AMUP exerts remarkable therapeutic effects on HUA by regulating the gut microbiome and mediating gut metabolism pathways associated with uric acid excretion.