【作者】 赵艳红；

【导师】 阮金秀；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药理学， 2007， 博士

【摘要】 目的以胆汁酸单体、人工牛黄和天然牛黄两种药材及安宫牛黄丸与清开灵注射液两个中成药中的多种胆汁酸作为主要研究对象，建立灵敏、专一、简便快速并能同时检测生物样品中多种胆汁酸的高效液相色谱-质谱法（HPLC-MS法），比较五种受试药中胆汁酸的药代动力学过程，从药代动力学角度分析其药效差异的原因，为进一步优化人工牛黄和方剂配伍提供依据，也为如何研究中药和中药方剂的药代动力学探索思路。方法小鼠血样用微量采血法（全血20μl），乙腈直接沉淀蛋白后用HPLC-MS法检测全血中各种胆汁酸的浓度；组织分布研究取心、肺、肾、脑组织，匀浆离心后取上清液直接沉淀蛋白后检测；建立大鼠原位肠灌流模型，用于分析不同因素对胆汁酸吸收的影响。结果1．建立了能同时检测11种胆汁酸的HPLC-MS检测方法，各成分分离度好，中药成分与内源性成分无干扰，线性范围为4-4000 ng·ml^-1，最低检测浓度为4 ng·ml^-1，日间和日内精密度均小于6.0％，方法可满足药代动力学研究的要求。2．比较胆酸、异去氧胆酸、鹅去氧胆酸和去氧胆酸的药代动力学参数和在组织中的分布，鹅去氧胆酸和去氧胆酸在小鼠体内的AUC和在心脏、肺脏、肾脏、脑组织中的分布明显高于胆酸和异去氧胆酸。其排列顺序与抗肺水肿作用一致。3．比较小鼠腹腔注射清开灵注射液和胆酸／异去氧胆酸单体时胆酸和异去氧胆酸的药代动力学参数及其在主要组织心脏、肺脏、肾脏、脑中的分布，清开灵注射液中的胆酸和异去氧胆酸的AUC、MRT均明显高于胆酸和异去氧胆酸单体（P＜0.05），CL明显低于单体（P＜0.05）。清开灵注射液中胆酸和异去氧胆酸的分布明显优于单体胆酸和异去氧胆酸，胆酸和异去氧胆酸在组织中的浓度：肾脏＞肺脏＞心脏＞脑，肺脏作为靶器官有较高的分布量。4．小鼠口服给予胆汁酸单体、人工牛黄、天然牛黄、清开灵注射液和安宫牛黄丸五种受试药后，结果显示安宫牛黄丸中的胆汁酸在小鼠体内的吸收最好，在肺、脑组织中的浓度最高，其次为清开灵注射液和天然牛黄，第三为人工牛黄，单体胆汁酸在小鼠体内的吸收和分布最差。大鼠原位肠灌流模型结果与整体动物实验结果相类似。5．小鼠口服给予人工牛黄和人工牛黄加胆红素后，比较两组中各种胆汁酸的药代动力学，人工牛黄加胆红素组中胆汁酸的AUC明显高于人工牛黄组中，CL明显低于人工牛黄组；组织分布结果显示胆红素不能促进人工牛黄中胆汁酸在小鼠肺、脑组织中的分布。大鼠原位肠灌流模型结果也显示相似结果，人工牛黄加胆红素组中总胆汁酸的肠吸收明显优于人工牛黄组，其中甘氨胆酸、牛磺胆酸、胆酸、熊去氧胆酸、异去氧胆酸、去氧胆酸在人工牛黄加胆红素组中肠吸收明显优于人工牛黄组。6初步探讨了中药与中药复方药代动力学研究模式。结论1．建立检测各种胆汁酸的HPLC-MS法灵敏度高，特异性好，适用于胆汁酸的体内和体外药代动力学研究。2．胆酸的同系物鹅去氧胆酸、去氧胆酸在小鼠体内的吸收和分布优于胆酸和异去氧胆酸，说明其药理学结果的差异可能是由于其药代动力学差异引起的。3．清开灵注射液中非胆汁酸成分可促进其胆汁酸成分的吸收和分布，胆酸和异去氧胆酸在脑中浓度低说明其不易透过血脑屏障。4．在五种受试药中，单体胆汁酸口服生物利用度低，天然牛黄中的胆汁酸吸收与组织分布优于人工牛黄，而安宫牛黄丸与清开灵注射液又优于单味药材。5．胆红素能促进人工牛黄中胆汁酸的吸收，减慢其在动物体内的消除速率，但不能提高组织中的分布量。更多还原

【Abstract】 OBJIECTIVE: Pharmacokinetics of bile acids in monomer compounds, artificial calculus bovis, natural calculus bovis, qingkailing injection and angongniuhuangwan in biological specimen were studied using a simple, rapid, sensitive and reliable HPLC-MS method. The reasons for difference of pharmacodynamics were analyzed from pharmacokinetics of bile acids in five drugs. It can supply evidence for more optimizing artificial calculus bovis and compatibility of medicines in complex prescription, and search a way to study pharmacyokinetics of traditional Chinese medicines and complex prescription. METHODS: In vivo Drug concentration analytical method and in situ perfused rat intestine model were used. The concentrations of bile acids in mice blood, tissues and perfusate were measured by LC/ESI/MS. RESULTS: 1. The standard curves of bile acids in blood, tissue and perfusate were linear in the range from 4 to 4000 ng? ml^-1 （r>0.999）, the lowest limit of quantitation were 4 ng ? ml^-1. The intra- and inter-day precisions were less than 6.0%. 2. Absorption and distributions in heart, lung, kidney and brain of CDCA and DCA were better than CA and HDCA, and absorption and distributions of HDCA were better than CA. 3. There were significant differences （P<0.05） between qingkailing injection group and CA/HDCA group in the main pharmacokinetic parameters and distributions in heart, lung, kidney and brain. Absorption and distribution of CA and HDCA in qingkailing injection group were better than in CA/HDCA group. The concentratons of CA and HDCA in tissues were C_lung> C_kidney>C_henrt>C_? 4. There were significant differences （P<0. 05） among bile acids in monomer compounds, artificial calculus bovis, natural calculus bovis, qingkailing injection and angongniuhuangwan in absorptions and distributions after oral administration. Absorption and distribution of bile acids in angongniuhuangwan were best, qingkailing injection and natural calculus bovis take second place, artificial calculus bovis third and monomer compounds lowest. The results of in situ perfused rat intestine model were similar. 5. There were significant differences （P<0.05） between absorption of bile acids in artificial calculus bovis group and artificial calculus bovis/bilirubin group. AUC of bile acids in artificial calculus bovis/bilirubin group were better than artificial calculus bovis group. CL were lower than in artificial calculus bovis group. Bilirubin could not advance distribution of bile acids in tissues. CONCLUSION: 1. The method is simple, rapid, sensitive and reliable, and it is applicable to assay bile acids in biological samples. 2. Absorption and distribution of CDCA and DCA are better than CA and HDCA, it explains the pharmacological difference with pharmacokinetic results. 3. The parts of non-cholic acid in qingkailing injection can improve absorption and distribution of CA and HDCA. CA and HDCA permeated blood brain barrier uneasily. 4. Absorption and distribution of Monomer compound in mice is lowest, non-bile acids in angongniuhuangwan and qingkailing injection can advance absorption and distribution of bile acids. Other substances（such as bilirubin, inorganic ions, amino acid） in natural calculus bovis can advance absorption and distribution of bile acids. 5. Bilirubin can promote absorption of bile acids in artificial calculus bovis and slow elimination of bile acids. But bilirubin has no effect on distribution of bile acid in artificial calculus bovis, it shows there are other substances contributing to distribution difference between natural and artificial calculus bovis.更多还原