基于血清代谢组学技术探讨桂枝茯苓胶囊及其主要成分治疗原发性痛经的作用机制

目的:采用血清代谢组学技术从内源性代谢物的角度研究桂枝茯苓胶囊及其主要成分(芍药苷、丹皮酚、苦杏仁苷)治疗原发性痛经的作用机制。方法:将36只SD雌性大鼠随机分为正常组、模型组、桂枝茯苓组、芍药苷组、丹皮酚组、苦杏仁苷组,每组6只。注射苯甲酸雌二醇(连续10 d)和缩宫素(d 10)制备大鼠原发性痛经模型,通过扭体反应和子宫药效学指标评价药物的疗效;同时应用UPLC-Q-TOF-MS/MS技术,结合多元变量分析和数据库检索,对不同组别大鼠血清中内源性代谢物进行分析,筛选鉴定出潜在的差异代谢物;并通过Metabo Analyst软件进行代谢通路分析。结果:桂枝茯苓胶囊、芍药苷和丹皮酚均能较好地抑制模型大鼠扭体反应,并能降低模型大鼠子宫中一氧化氮(NO)和丙二醛(MDA)含量。通过主成分分析(PCA)和偏最小二乘法-判别分析(PLS-DA)发现正常大鼠和模型大鼠的代谢轮廓发生明显变化,共鉴定出12种与原发性痛经相关的差异代谢物,涉及的潜在靶标代谢通路(Impact> 0.1)包括亚油酸代谢,苯丙氨酸、酪氨酸和色氨酸的生物合成,苯丙氨酸代谢,花生四烯酸代谢以及色氨酸代谢。经桂枝茯苓胶囊...     

基于序贯代谢模型研究地榆鞣质在大鼠体内的代谢作用

目的：以序贯代谢模型为基础,研究地榆鞣质成分在大鼠体内代谢动态情况。方法：采用超高液相色谱-线性离子阱-串联静电轨道阱高分辨质谱（UHPLC-LTQ-Orbitrap HRMS）技术,以此同时检测多成分变化情况。体内代谢采用在体封闭肠环法研究地榆鞣质口服后多成分经肝肠代谢变化过程。结果：口服地榆鞣质通过肝肠的代谢,经过水解、甲基化及葡萄糖醛酸化后共鉴定出12种代谢产物。结论：采用序贯代谢模型的方法,研究地榆鞣质口服经不同代谢部位的情况进行了定性描述,可为该药物的质量控制、机制阐述及再次开发提供参考依据。     

桂枝茯苓胶囊及其主要成分对原发性痛经模型大鼠肠道菌群的影响

目的:探讨桂枝茯苓胶囊及其主要成分(芍药苷、丹皮酚、苦杏仁苷)对原发性痛经模型大鼠肠道菌群的影响。方法:将雌性SD大鼠随机分为正常组、模型组、胶囊组(桂枝茯苓胶囊内容物,1 000 mg/kg)、芍药苷组(15.0 mg/kg)、丹皮酚组(10.3 mg/kg)和苦杏仁苷组(12.1 mg/kg),每组6只。除正常组外,其余各组大鼠均于背部皮下注射苯甲酸雌二醇和腹腔注射缩宫素建立原发性痛经模型。于皮下注射苯甲酸雌二醇的第4天起,正常组大鼠灌胃等体积生理盐水,模型组大鼠灌胃等体积0.5%羧甲基纤维素钠溶液,各给药组大鼠灌胃相应药物,每天1次,连续7 d。在检测各组大鼠扭体次数和子宫组织中一氧化氮(NO)、丙二醛(MDA)含量的基础上,采用气相色谱法检测其结肠内容物中乙酸、丙酸、丁酸等短链脂肪酸(SCFAs)的含量;以多样性指数为指标,采用细菌基因组重复序列-聚合酶链式反应(Rep-PCR)和肠杆菌基因组间重复序列-聚合酶链式反应(Eric-PCR)技术评价大鼠粪便中肠道菌群的多样性。结果:与正常组比较,模型组大鼠扭体次数显著增多,子宫组织中NO、MDA含量均显著升高,结肠内容物中乙酸、丙...     

黑骨藤主要化学成分在正常和类风湿性关节炎大鼠血浆、尿液、粪便中的代谢产物分析

目的:研究黑骨藤在正常及佐剂性关节炎(AA)大鼠体内的代谢产物,探究类风湿性关节炎(RA)对黑骨藤有效成分在大鼠体内代谢的影响。方法:采用弗氏完全佐剂制备AA大鼠模型;运用UPLC-Q TOF MS^E方法,采用ACQUITY UPLC BEH C₁₈(100 mm×2.1 mm, 1.7μm)色谱柱,以0.01%甲酸水-0.01%甲酸乙腈为流动相梯度洗脱,流速0.25 mL·min^-1,采用电喷雾离子源,在负离子模式下,对正常和AA模型大鼠灌服黑骨藤提取物后的血浆、尿液和粪便进行分析。结果:在正常大鼠体内和AA模型大鼠体内分别检测到2、3个原型成分,32、35个代谢产物,代谢途径主要包括单咖啡酰基奎宁酸还原、甲基化、开环裂解、葡萄糖醛酸化,二咖啡酰基奎宁酸还原、甲基化、乙酰化、异构化、硫酸酯化、葡萄糖醛酸化等。结论:AA模型大鼠血浆和尿液中的代谢产物比正常大鼠更具多样性,RA疾病状态可能会影响黑骨藤有效成分在体内的代谢途径。     

UHPLC-MS/MS测定大鼠尿液中环磷酰胺及其代谢产物浓度

目的 基于超高效液相色谱-串联质谱法（UHPLC-MS/MS）,建立测定给予高剂量环磷酰胺（cyclophosphamide,CTX）后大鼠尿液中CTX及其代谢产物脱氯乙基环磷酰胺（dechloroethylcyclophosphamide,DCCTX）、4-酮基环磷酰胺（4-Ketocyclophosphamide ,4-KetoCTX）、羧基磷酰胺（Carboxyphosphamide ,CEPM）浓度的方法。方法 大鼠尿液样品经10%甲醇水溶液直接稀释,离心后取上清液,采用UHPLC-MS/MS法进行检测分析。色谱柱：Agilent poroshell SB-C18 (2.1 mm × 75 mm,2.7 μm);流动相：甲醇-10 mmol?L-1乙酸铵水溶液,进行梯度洗脱,流速0.25 mL?min-1,柱温25 °C。采用电喷雾离子源,以多重反应监测模式进行正离子检测。用于定量检测分析的离子对分别为：CTX：m/z 261.10→140.10;DCCTX：m/z 199.20→78.00;4-KetoCTX：m/z 275.10→142.00;CEPM：m/z 293.10→221.10;替硝唑（tinidazole,TNZ）：m/z 248.10→121.10。结果 CTX及其代谢产物尿中浓度在40~2000 ng?mL-1范围内线性关系良好（CTX、DCCTX、4-KetoCTX、CEPM的r2分别为：0.9915,0.9910,0.9956,0.9918）,最低定量限均为40 ng?mL-1;日内、日间RSD分别为0.67%~12.76%、1.30%~11.92%;由内标归一化的基质因子计算RSD,结果在0.52%~7.60%之间;该方法的提取回收率为74.00%~102.70% （n=6）,方法回收率为85.89%-110.69% （n=5）;测定成分和内标的稳定性均良好。结论 该方法快捷、准确可靠、重复性好,适用于大鼠高剂量CTX给药后CTX及其代谢产物尿药浓度的测定及排泄研究。     

止得咳颗粒在大鼠体内的代谢产物研究

目的 分析止得咳颗粒在大鼠体内的代谢产物并推测其代谢途径。方法 将雄性SD大鼠随机分为空白组、给药组（止得咳颗粒,9.45 g/kg）,每组灌胃超纯水或相应药液,每天2次,每次灌胃间隔6～8 h,连续3 d。收集各组大鼠血清、粪便、尿液样品,利用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱（UPLC-Q-Exactive-MS）技术鉴定大鼠灌胃止得咳颗粒后上述生物样品中的代谢产物,并推测其代谢途径。结果 大鼠灌胃止得咳颗粒后,在其血清、尿液、粪便样品中共鉴定出16个原型成分（如野鸢尾黄素、黄芩素、绿原酸等）和11个代谢产物（如山柰酚或木犀草素水合产物、绿原酸甲基化产物、黄芩苷羟基化产物）。其中,血清样品中鉴定出8个原型成分和4个代谢产物;尿液样品中鉴定出10个原型成分和7个代谢产物;粪便样品中鉴定出8个原型成分和5个代谢产物。结论 止得咳颗粒在大鼠体内的代谢成分主要包括黄芩苷、野鸢尾黄素、绿原酸,主要涉及甲基化、羟基化、葡萄糖醛酸化等代谢途径。     

UHPLC-Q-Orbitrap HRMS分析吉马酮大鼠体内代谢产物及代谢途径

目的 研究吉马酮在大鼠体内的代谢产物,探讨其可能的代谢途径。方法 采用超高效液相色谱-四级杆/静电场轨道阱高分辨质谱技术（UHPLC-Q-Orbitrap HRMS）对灌胃给予吉马酮（10 mg·kg^-1）后大鼠的生物样品（血浆、尿液和粪便）进行分析,色谱柱采用Waters ACQUITY UPLC^® BEH C₁₈（2.1 mm×50 mm,1.7 μm）,流动相乙腈-0.1%甲酸水溶液,梯度洗脱,Q-Exactive高分辨质谱仪采用HESI离子源,辅助气温度300℃,离子传输管温度320℃,喷雾电压为+3.50 kV或者-2.80 kV,扫描方式Full MS/dd-MS²。分析比对给药组和空白组中高分辨质谱提供的准分子离子峰和碎片离子信息,使用Xcalibur 3.0、Mass Frontier 2.0和Compound Discovery 2.1软件进行数据处理,筛选并鉴定其可能的代谢产物。结果 从大鼠生物样品中共筛选鉴定了包括原型在内的23个代谢产物,推断吉马酮在大鼠体内发生的代谢类型主要有脱水反应、脱饱和反应、氧化反应、葡萄糖胺结合反应、葡萄糖醛酸化反应以及相关的复合反应等。结论 本研究较为详细地阐明了吉马酮在大鼠体内的代谢情况,剖析了其代谢途径,可为其进一步的药效学、药动学及毒理学研究提供理论依据。     

HPLC-ESI-ITMSn法鉴定麻黄碱及其大鼠体内主要代谢产物

目的建立快速灵敏的LC-ESI-ITMSⁿ分析检测麻黄碱及其大鼠体内代谢物的方法。方法以麻黄碱对照品对LC-ESI-ITMS²色谱及质谱条件进行了优化，分析总结其电喷雾质谱的一级电离规律和多级质谱裂解规律，以此作为麻黄碱大鼠体内代谢物分析鉴定的依据。健康大鼠空腹灌胃麻黄碱10 mg·kg^-1，收集0~48 h的尿样，经C₁₈小柱固相萃取分离纯化后，直接采用LC-ESI-ITMSⁿ方法对尿样进行测定。结果根据生物体内药物代谢转化规律及母体药物的色谱-质谱行为规律，在尿样中鉴定出3个第I相代谢产物，未发现第II相代谢产物。结论本方法灵敏、快速、选择性高、专属性好，可用于麻黄碱的代谢产物研究。     

五味子酯甲在大鼠体内代谢产物的鉴定

目的 考察五味子酯甲在大鼠体内的代谢转化。方法 采用超高效液相色谱串联四级杆飞行时间质谱(UPLC-TOF-MS/MS)分析鉴定大鼠灌胃五味子酯甲后,其在尿样中的代谢产物。Phenomenex UPLC C₁₈色谱柱,流动相为乙腈-1‰甲酸水,梯度洗脱,质谱仪离子源为电喷雾离子源(ESI),正离子方式检测。结果 经代谢物软件处理后,根据MS/MS给出质谱碎片信息对五味子酯甲代谢产物进行结构推测,共检测到5种代谢产物。结论 五味子酯甲在大鼠体内的代谢途径主要为氧化反应和还原反应。     

采用UHPLC-MS和质量亏损过滤技术分析二苯乙烯苷在大鼠体内的代谢产物和代谢途径

目的:分析二苯乙烯苷在大鼠体内的代谢产物并推测代谢途径.方法:将雄性SD大鼠随机分为血浆组(n=3)、尿液组(n=3)、胆汁组(n=3)和组织组(n=9),各组大鼠均单次灌胃二苯乙烯苷200 mg/kg,分别收集给药后10、30 min和1、1.5、2、4 h的血浆,给药后0～6 h的尿液,给药后0～4 h的胆汁以及给...     

桂枝茯苓胶囊对实验大鼠血浆内雌二醇、黄体酮、催乳素的影响

目的 :探讨桂枝茯苓胶囊对实验性高雌、孕激素大鼠血浆内雌二醇、黄体酮、催乳素的影响。方法 :取雌性SD大鼠 10 0只给大鼠腹腔注射 ,苯甲酸雌二醇及黄体酮制作高雌二醇、黄体酮大鼠模型 ,将大鼠随机分成 5组 ,空白对照组、模型组 ,他莫昔芬对照组及桂枝茯苓胶囊 2 0 0 ,4 0 0mg·kg- 1组 ,每组2 0只 ,空白对照组和模型组给等体积蒸馏水 ,其余组分别给桂枝茯苓胶囊混悬液 2 0 0 ,4 0 0mg·kg- 1和他莫昔芬 3mg·kg- 1,每日灌胃 ,共 30d。用放射免疫方法测定各组实验性大鼠模型血浆内雌二醇、黄体酮、催乳素的含量。结果 :桂枝茯苓胶囊 2 0 0与4 0 0mg·kg- 1组可明显降低高雌、孕激素大鼠模型血浆雌二醇和黄体酮 ,2组雌二醇分别为 (12 7±s5 2 )ng·L- 1和 (15 3± 4 9)ng·L- 1,黄体酮分别为 (12± 8) μg·L- 1和 (13± 7) μg·L- 1,与模型组雌二醇(2 71± 10 3)ng·L- 1和黄体酮为 (2 1± 15 ) μg·L- 1比较 ,差异均有显著意义 ,P <0 .0 5 ,但对血浆催乳素影响不大。结论 :桂枝茯苓胶囊能够显著降低异常升高的实验性高雌、孕激素大鼠模型的雌二醇、黄体酮的血浓度。     

RP-HPLC法同时测定桂枝茯苓胶囊中3种成分的含量

目的建立RP-HPLC法同时测定桂枝茯苓胶囊中没食子酸、白芍苷和芍药苷含量。方法色谱柱:大连中汇达C18柱(4.6 mm×250 mm,5μm),流动相:乙腈-体积分数为0.1%的磷酸溶液,梯度洗脱,流速:1.0 mL.min-1,检测波长:230 nm,柱温:40℃。结果没食子酸、白芍苷和芍药苷的线性分别为0.69~6.87 mg.L-1(r=0.999 6)、0.88~8.80 mg.L-1(r=0.999 6)和2.00~20.00 mg.L-1(r=0.999 5)。平均回收率:没食子酸为99.7%(RSD=1.0%,n=9)、白芍苷为101.5%(RSD=1.5%,n=9)、芍药苷为100.0%(RSD=0.7%,n=9)。结论此方法可为桂枝茯苓胶囊的质量控制提供方法。     

大鼠灌胃四逆散提取物后血浆、尿液、粪便、胆汁中主要代谢产物的鉴定

采用UPLC-Q-TOF/MS研究四逆散提取物在大鼠体内的代谢产物,利用碰撞能量梯度(MSE)和质量亏损过滤(MDF)技术进行分析,鉴定大鼠灌胃四逆散提取物后血浆、尿液、粪便、胆汁中的代谢产物。四逆散提取物中柚皮苷、柚皮素、橙皮苷、新橙皮苷、甘草苷、甘草素、甘草酸、甘草次酸、柴胡皂苷a、柴胡皂苷d在大鼠不同代谢途径中推测出原形、羟基化、葡糖醛酸化、硫酸化、葡糖醛酸化与硫酸化结合、羟化葡糖醛酸化等共41个代谢产物。     

采用UFLC-IT-TOF/MS鉴定大鼠口服黄芩提取物后胆汁、血浆和尿液中的主要成分(英文)

黄酮类化合物是中国传统中药黄芩的主要活性成分。大鼠口服黄芩提取物后, 采用超快速液相色谱-离子阱飞行时间质谱(UFLC-IT-TOF/MS)对胆汁、血浆和尿液进行分析, 鉴定入血成分和代谢产物。根据对这些成分质谱裂解规律的分析, 共鉴定了36种不同的黄酮类化合物, 其中包括13个新的代谢产物。本实验在体外对黄芩提取物进行分析,鉴定了其中16个黄酮类成分, 大鼠胆汁中鉴定了25个黄酮类化合物, 血浆中15个黄酮类化合物, 尿液中14个黄酮类化合物。结果表明, 黄芩提取物中的黄酮类化合物以葡萄糖醛酸化、硫酸化和甲基化的为主要代谢途径。本实验首次对黄芩提取物的代谢产物进行综合分析。     

Pharmacokinetic parameters of tibolone and metabolites in plasma, urine, feces, and bile from ovariectomized cynomolgus monkeys after a single dose or multiple doses of tibolone.

Levels of nonsulfated and sulfated tibolone metabolites were determined in plasma, urine, and feces from six ovariectomized, mature female cynomolgus monkeys after a single dose and multiple p.o. doses (including bile) of tibolone using validated gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry assays. In plasma, the predominant nonsulfated metabolite after single and multiple dosing was the estrogenic 3alpha-hydroxytibolone; levels of the estrogenic 3beta-hydroxytibolone were 10-fold lower and of progestagenic/androgenic Delta(4)-tibolone, 5-fold lower. Tibolone was undetectable. The predominant sulfated metabolite was 3alphaS,17betaS-tibolone; levels of 3betaS,17betaS-tibolone were about 2-fold lower, and monosulfated 3-hydroxymetabolites were about 10-fold lower. After multiple doses, areas under the curve of nonsulfated metabolites were lower (2-fold), and those of sulfated metabolites were 25% higher. In plasma, >95% metabolites were disulfated. In urine, levels of all the metabolites after single and multiple doses were low. After a single dose, high levels of 3beta-hydroxytibolone and the 3-monosulfated metabolites (3betaS,17betaOH-tibolone and 3alphaS,17betaOH-tibolone) were found in feces. After multiple dosing, 3alpha-hydroxytibolone increased, and the ratio of 3alpha/3beta-hydroxytibolone became about 1. The predominant sulfated metabolite was 3alphaS,17betaS-tibolone. Levels of all the metabolites in feces were higher after multiple doses than after a single dose. Levels of nonsulfated and 3-monosulfated metabolites were higher in feces than in plasma. Bile contained very high metabolite levels, except monosulfates. This may contribute to the metabolite content of the feces after multiple doses. 3beta-Hydroxytibolone and 3alphaS,17betaS-tibolone predominated. In conclusion, tibolone had different metabolite patterns in plasma, urine, feces, and bile in monkeys. The bile contributed to the metabolite pattern in feces after multiple doses. The major excretion route was in feces.     

Identification and quantification of baicalein, wogonin, oroxylin A and their major glucuronide conjugated metabolites in rat plasma after oral administration of Radix scutellariae product

The current study aims to identify and quantify three flavones (baicalein, wogonin and oroxylin A) and their major metabolites (baicalin, wogonoside and oroxylin A-7-O-glucuronide) in rat plasma after oral administration of Radix scutellariae product. A simple HPLC/UV method has been developed to simultaneously determine the three flavones and their major metabolites in rat plasma. The chromatographic separation of the six analytes was achieved by a Thermo C(18) column with linear gradient elution of a mobile phase containing acetonitrile and 20mM sodium dihydrogen phosphate buffer (pH 4.6). All the tested analytes were detected by PDA detector at a wavelength of 320nm. The intra-day and inter-day precision for the current assay of the six analytes was within the range of -2.23% to 15.13% and -10.83% to 6.42%, respectively. All the studied analytes could be efficiently extracted from the rat plasma using HLB cartridge with extraction recoveries above 70% and were stable under different storage conditions. The developed assay method was successfully applied to the pharmacokinetic study of baicalin, wogonoside after oral administration of a commercially available Radix scutellariae containing capsule at a dose of 3.2g/kg to Sprague-Dawley rats. In addition to wogonoside, a new metabolite of wogonin has been identified using LC/MS/MS for the first time.     

Excretion of benzo[a]pyrene and metabolites in urine and feces of rats: influence of route of administration, sex and long-term ethanol treatment

The urinary and fecal excretion of benzo[a]pyrene (B[a]P) and its main metabolites were studied after oral and intraperitoneal administration of B[a]P to male and female ethanol-treated and non-ethanol- treated rats. After oral administration of B[a]P more mutagenic compounds as well as B[a]P metabolites were found in feces than after intraperitoneal administration. The excretion of B[a]P metabolites in urine and feces after oral administration were maximal at days 1 and 2 whereas after intraperitoneal administration excretion was maximal at days 2 and 3. In males, the amounts of excreted phenolic metabolites in urine and feces were generally higher than in females. The amounts of mutagenic products in urine and feces of males were also higher than in females after intraperitoneal and oral administration of B[a]P. In urine of female rats that received B[a]P intraperitoneally, a decreased excretion of phenolic metabolites was found after ethanol treatment. In feces of both male and female rats, a decreased excretion of 3-OH-B[a]P was found after ethanol treatment. In this study, the influence of sex and administration route on the excretion of B[a]P metabolites was more pronounced than the effect of ethanol treatment.