川乌及其配伍的肠内菌代谢及质谱分析方法研究
文献类型：学位论文
| 作者 | 李雪 |
| 学位类别 | 博士 |
| 答辩日期 | 2015-04 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 中国科学院长春应用化学研究所 |
| 导师 | 宋凤瑞 |
| 关键词 | 川乌 中药配伍 肠内菌 质谱技术 DART-MS 微透析 |
| 中文摘要 | 川乌是一种临床常用中药，由于其毒性较大，常与其它药物配合应用，合理的配伍是使川乌减毒增效的关键步骤。口服是中药首选的给药途径，肠道内存在的细菌对口服药物的活性及毒性有重要的影响。本论文以川乌为研究对象，以体外肠内菌代谢为论文研究主线，以超高效液相色谱质谱技术（UPLC-MS）为研究手段，结合多元统计分析方法对川乌及其药对和相关复方的肠内菌代谢进行研究。从川乌配伍前后肠内菌代谢过程中整体化学成分及其代谢产物的变化规律阐明川乌配伍的科学性。 利用UPLC-MS技术结合主成分分析方法研究了制川乌单煎液、制川乌与白芍、制川乌与防己共煎液在大鼠肠内菌中的代谢差异。采用SIMCA-P软件，以肠内菌代谢后乌头类生物碱的相对含量为变量进行PCA分析，根据主成分分析载荷图及独立样本t检验寻找代谢差异标志物。结果表明，制川乌单煎液与制川乌-白芍、制川乌-防己共煎液的肠内菌生物转化存在显著差异。从制川乌-白芍组得到7种差异显著的外源性代谢生物标志物，从制川乌-防己组得到6种代谢生物标志物，其中制川乌-白芍组有4种外源性代谢生物标志物经肠内菌代谢后含量高于制川乌组，而制川乌-防己组有1种化合物含量高于制川乌组，两组中其它外源性代谢生物标志物含量低于制川乌组。推测这些外源性代谢生物标志物是制川乌配伍前后药效差异的物质基础。 采用UPLC-MS技术对乌头汤单煎液、乌头汤与川贝母不同比例配伍（1:2、1:1及1:0.5）共煎液肠内菌代谢前后的化学成分进行了检测，应用PCA对乌头汤配伍前后的肠内菌代谢轨迹进行分析，通过OPLS-DA寻找不同时间点的外源性代谢生物标志物，以这些代谢生物标志物为研究对象，应用PLS-DA对不同比例川贝母在乌头汤肠内菌过程中的影响进行研究。结果表明，乌头汤及其配伍共煎液在肠内菌中培养第1-2天时代谢较快，第9-12时为代谢末期；乌头汤配伍前后的代谢生物标志物主要为乌头生物碱、麻黄生物碱、黄酮、皂苷及皂苷苷元；不同比例的川贝母对乌头汤肠内菌代谢的整体影响一致，对乌头类单酯型、醇胺型生物碱及黄酮类化合物有促进其含量增加的作用，同时还能促进皂苷向苷元转化，其中1:2配伍比例的肠内菌代谢影响作用强于1:1及1:0.5比例。 本文建立了乌头类双酯型生物碱肠内菌生物转化样品的实时直接分析质谱检测方法。首次利用实时直接分析-线性离子阱串联质谱法（DART-IT/MSn）研究了乌头碱（AC）、中乌头碱（MA）和次乌头碱（HA）在大鼠肠内菌中的代谢。代谢样品无需前处理，直接利用实时直接分析质谱离子源进行分析。在本实验中，对实时直接分析质谱离子源的重要实验参数进行优化，并以优化的DART-MSn方法对乌头类双酯型生物碱在大鼠肠内菌作用下的代谢产物进行结构确认及含量分析。每个样品分析时间少于1分钟。该方法检测结果与超高效液相色谱-质谱（UPLC-MS）分析结果具有很好的一致性。结果表明该方法准确、简便、快速，为实现药物代谢的高通量分析提供了一种强大的工具。 应用在线微透析结合超高效液相色谱质谱（MD-UPLC-MS）建立了一种新的动态监测黄芪皂苷I及黄芪皂苷II在复杂肠内微生物系统中生物转化的方法。代谢样品在线检测的实验装置是由微透析取样系统、UPLC-MS检测系统及将二者连接起来的自制六通阀进样系统构成。本研究以黄芪甲苷IV为目标化合物，对微透析实验条件进行优化，结果表明，灌流液中加入2-羟丙基-β-环糊精（20 mg/mL）能够降低肠内菌培养基质中小分子物质的干扰，提高方法的灵敏度。应用此装置成功的监测了黄芪皂苷II在24h内肠内菌代谢情况。与传统培养、取样方法相比，MD-UPLC-MS方法不仅实现了取样、样品前处理及检测连续自动化操作，还降低了基质效应对检测的干扰。 |
| 英文摘要 | Radix Aconiti is a widely used traditional Chinese medicine (TCM) in clinic. Because of its highly toxicity, it is usually combined with other medicinal herbs. Suitable compatibility plays a key role in toxicity reducing and efficacy enhancing for Radix Aconiti. The oral route is the preferred route for TCM administration. The metabolism can render a drug activity or toxicity. In this paper, the metabolism of Radix Aconiti and its compatibility in intestinal bacteria was studied using UPLC-MS to investigate the rationality of its combination with other herb medicines. UPLC-MS was used to investigate the metabolic difference of the decoction of Radix Aconiti Preparata (RAP) and its co-decoctions with Radix Paeoniae Alba (RAP-RPA) or Radix Stephaniae Tetrandrae (RAP-RST) in rat intestinal bacteria. The principal component analysis (PCA) of the relative contents of Aconitum alkaloids after metabolism were performed by SIMCA-P software. The loading plot and independent-samples T test were used to search the metabolic markers of RAP and its co-decoctions (RAP-RPA and RAP-RST). The result indicated that the differences of biotransformation among the groups of PAP, RAP-RPA and RAP-RST were significant. Seven relevant markers with the significant differences were found in the group of RAP-RPA, six relevant markers were obtained in the group of RAP-RST. The relative content of four markers in RAP-RPA was higher than that in RAP, and one marker in RAP-RST was higher than that in RAP. The relative contents of other markers were all lower than that in RAP. These markers may be the effective substance for explaining the different effects of Radix Aconiti Preparata before and after combination with Radix Paeoniae Alba and Radix Stephaniae Tetrandrae. UPLC-MS was used to analyze the components change in Wu-tou-tang (WTT) and its co-decotion with Fritillariae Cirrhosae at in varying proportions (1:2, 1:1, 1:0.5) before and after biotransformation in intestinal bacteria. PCA was used to analyze the metabolic process of WTT and its co-decoctions. The metabolic markers were searched by OPLS-DA. Then the metabolic markers were chosen to investigate the effect of Fritillariae Cirrhosae on the intestinal bacterial metabolism of WTT. The results indicated that the metabolic activity of of WTT in intestinal bacteria is highest in the first two days and the end of the metabolism is in 9 to12 days. These metabolic markers are mono-ester aconitum alkaloids, non-ester aconitum alkaloids, epphedra compounds, flavonoids, saponins and their sapogenin. Fritillariae Cirrhosae can enhance the relative content of mono-ester aconitum alkaloids, non-ester aconitum alkaloids, flavonoids and promote the biotrasformation of saponins to their sapogenin. Between the three percentage of Fritillariae Cirrhosae in the co-decotion, the effect of ratio of 1:2 is more greater than others. In this paper, a direct analysis method of the metabolites of diester-type aconitum alkaloids was established. The study of the metabolites in rat intestinal bacteria was presented for the first time using direct analysis in real time (DART) coupled to multi-stage tandem mass spectrometry. In this study, the samples were directly analyzed by direct analysis in real time (DART) ion source and linear ion trap mass spectrometer. The critical DART parameters were optimized including gas temperature and pressure, source-to-MS distance and autosampler speed, which achieved high sensitivity and enhanced analysis ability. With the optimized method, the metabolites of aconitum alkaloids in intestinal bacteria of rat were analyzed. The tandem mass spectrometry was applied to identify structures of these metabolites.The ananlytical time of analyzing one sample can be less than 10s using this method. The results obtained by DART method were accord with those from the extracts of metabolites using UPLC-MS (ultra-performance liquid chromatography mass spectrometry). Thus, DART-MS method was proved |
| 语种 | 中文 |
| 公开日期 | 2016-05-03 |
| 源URL | [http://ir.ciac.jl.cn/handle/322003/64488]  |
| 专题 | 长春应用化学研究所_长春应用化学研究所知识产出_学位论文 |
| 推荐引用方式 GB/T 7714 | 李雪. 川乌及其配伍的肠内菌代谢及质谱分析方法研究[D]. 中国科学院长春应用化学研究所. 中国科学院研究生院. 2015. |
入库方式： OAI收割
来源：长春应用化学研究所
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