欧前胡素和异欧前胡素在大鼠体内的药代动力学研究

  本文关键词：欧前胡素和异欧前胡素在大鼠体内的药代动力学研究，由笔耕文化传播整理发布。

        欧前胡素和异欧前胡素属于6,7-呋喃香豆素类化合物，因取代基位置不同互为位置异构体。二者广泛存在于植物界，尤其在伞形科中药中含量丰富，均具有镇痛、抗病毒、抗肿瘤、抗菌和抗HIV等药理作用。此外，欧前胡素和异欧前胡素对药物代谢酶-细胞色素酶P450和谷胱甘肽S-转移酶的活性有一定的影响。中药成分复杂，各成分的体内过程可能存在相互影响，对可影响药物代谢酶活性的成分的药代动力学研究显得尤为重要。目前对于欧前胡素和异欧前胡素的药代动力学研究，多是将其作为药材整体药动学研究中的化合物进行，尚未有二者单体药代动力学的系统研究。本研究以专属、灵敏的高效液相色谱-质谱联用（HPLC-MS）技术为分析手段，分别建立了各种生物样品中欧前胡素和异欧前胡素及其代谢物的定量测定方法，，并用于二者分别灌胃大鼠后的药代动力学研究和比较，为中药整体药动学的阐明奠定了基础，为中药位置异构体药动学规律的寻找提供了借鉴。第一部分欧前胡素的药代动力学研究目的：1优化中空纤维液液微萃取（HF-LPME）条件，用于大鼠血浆、中欧前胡素及其代谢物花椒毒酚测定的样品前处理；2建立血浆中欧前胡素及其代谢物花椒毒酚测定的HPLC-MS方法，研究大鼠灌胃欧前胡素后的药代动力学特征；3建立尿液和胆汁中欧前胡素及其代谢物花椒毒酚和花椒毒素测定的HPLC-MS方法，研究大鼠灌胃欧前胡素后的排泄动力学特征。方法：1以空白大鼠血浆和待测化合物对照品制备模拟生物样本，按照单因素考察的方法依次考察并确定提取溶剂、中空纤维长度、搅拌速率、提取温度、稀释倍数和盐效应等提取条件，确定提取欧前胡素及其代谢物的最佳条件。Electrospray Ionization （ESI）源，源喷射电压：5500V；正离子模式监测；离子源温度（TEM）：650℃；欧前胡素、与花椒毒酚检测离子对和DP电压、CE电压分别为271.1/203.2，25V，15eV和203.2/147.1，20V，32eV。色谱柱为Agilent Zorbax SB-C18，流动相体系为甲醇-1mmol/L NH4Ac，梯度洗脱，流速0.8mL/min；2结合本实验室对欧前胡素体内定性研究的结果进行实验，确定测定化合物。灌胃给予大鼠欧前胡素后采集不同时间点的大鼠血浆，采用已确定的HF-LPME处理血浆样本。采用HPLC-MS方法测定。采用Electrospray Ionization（ESI）源，正离子模式监测。源喷射电压（IS）：5500V；离子源温度：500℃；雾化气（Gas1）：40psi，加热气（Gas2）：50psi，帘气：25psi；以东莨菪内酯为内标，采用Agilent Zorbax SB-C18柱进行分离，流动相体系为甲醇-1mmol/L NH4Ac，梯度洗脱，流速0.8mL/min。36只大鼠灌胃给予欧前胡素后置于代谢笼内，收集72小时内0-4，4-8，8-12，12-24，24-36，36-48，48-60和60-72h的尿液并记录体积，采用与血浆样品相同的HF-LPME预处理方法，另外6只大鼠麻醉后进行胆管插管手术，收集24小时内0-2，2-4，4-8，8-12，12-20和20-24h时间段的胆汁并记录体积，用乙酸乙酯萃取的方式进行预处理。尿液和胆汁样品均在电喷雾离子（ESI）源，正离子多反应监测模式（MRM）下进行检测。欧前胡素、花椒毒酚、花椒毒素和内标的检测离子对和DP电压、CE电压分别为271.1/203.2，25V，15eV、203.2/147.1，20V，32eV、217.2/202.0，10V，25eV和193.1/133.1，49V，25eV，8-甲氧基5-羟基补骨脂素的检测离子对和DP电压、CE电压为193.1/133.1，49V，25eV。色谱分离采用AgilentZorbax SB-C18色谱柱（150mm×4.6mm，5m），柱温：25℃；流动相为甲醇（A）和1mmol/L乙酸铵-水（B），梯度洗脱，洗脱程序如下：0~5min，从50%A线性变化至95%A；5-8.5min，保持95%A；流速：0.8mL/min，进样量10μL。结果：1经过考察确定的提取条件为：以正庚醇为萃取溶剂，中空纤维长度为5cm，磁力搅拌器的转速为675rpm，萃取温度为室温，100μL血浆用2%NaCl稀释至1.5mL，萃取时间为50min；2实验得到欧前胡素和花椒毒酚的药动学参数和药-时曲线。灌胃给予大鼠欧前胡素以后吸收迅速，在1.01h达到最大浓度，T1/2为7.93，K为0.079（1/h），说明欧前胡素消除亦较快。灌胃5min以后即检测到代谢物花椒毒酚，说明欧前胡素被吸收以后迅速被转化为代谢物花椒毒酚；3大鼠灌胃给予欧前胡素后，欧前胡素在尿中45h内基本排泄完全，在胆汁中16h内基本排泄完全。欧前胡素以原型、花椒毒酚和花椒毒素的形式在尿液中的排泄量分别为13.46%，5.296%，和0.1686%；在胆汁中的排泄量分别为0.1214%，0.04282%和0.009042%。表明欧前胡素以原型在大鼠尿液和胆汁中排泄率不高。结论：建立了一种环保、灵敏度高、专属性强的HPLC-MS法测定大鼠血浆、尿液和胆汁中的欧前胡素及其代谢物。欧前胡素在大鼠体内吸收迅速，主要经尿液排泄。第二部分异欧前胡素的药代动力学研究目的：1建立大鼠血浆中异欧前胡素及其代谢物花椒毒酚的HPLC-MS测定方法，并研究大鼠灌胃给予异欧前胡素后的药代动力学特征；2建立大鼠尿液和胆汁中异欧前胡素及其代谢物花椒毒酚和花椒毒素的HPLC-MS测定方法，并研究大鼠灌胃给予异欧前胡素后的排泄动力学特征。方法：1将异欧前胡素混悬在于CMC-Na的灌胃液给予大鼠后，在不同时间点对大鼠进行眼内眦取血，得到血浆生物样本，采用HF-LPME的预处理方法，采用与第一部分血浆测定相同的液相和质谱条件下进行检测；26只SD大鼠灌胃给予异欧前胡素后置于代谢笼内饲养，自由饮水，4h后给予食物，分别收集0-4，4-8，8-12，12-24，24-36，36-48，48-60和60-72h时间段的尿液并记录体积，之后采用与第一部分尿液处理相同的方法进行预处理。另取6只大鼠给药后，以20%乌拉坦麻醉（7mL/kg），进行胆管插管术，收集给药后0-2，2-4，4-8，8-12，12-20和20-24h时间段的胆汁并记录体积，收集的样本使用乙酸乙酯萃取的方法进行预处理，采用第一部分排泄动力学部分的液相和质谱条件下进行检测。结果:1实验结果显示异欧前胡素吸收迅速，计算得到异欧前胡素的T1/2为2.78h，K为0.249（1/h）说明异欧前胡素在大鼠体内消除较快；2在尿液中，72h内异欧前胡素原型在45h内基本排泄完全，代谢物则在56h内基本排泄完全，异欧前胡素分别以原型和代谢物形式排泄量约占总给药量的17.80%和12.34%。而在胆汁中，异欧前胡素以原型形式在12h内基本排泄完全，而代谢物经15h基本排泄完全，两种形式排泄量分别约占总给药量的2.150%和0.0820%。结论：异欧前胡素和欧前胡素在大鼠体内的T1/2、Cmax、Tmax和K无统计学上的差异，二者都吸收迅速，消除较快；在相同给药量的前提下，异欧前胡素有更高的生物利用度；在尿液和胆汁中异欧前胡素以原型排泄的比率更高，说明欧前胡素较异欧前胡素更容易转化为代谢物。

    Imperatorin and isoimperatorin are6,7-furanocoumarins, which widelyexist in medicinal plants, especially in Glehnia littoralis Fr. Schmidt ex Mipand act as the main components of the pharmacological effects. Imperatorinand isoimperatorin possess many pharmacologic activites including anti-viral,anti-tumor, anti-bacterial and anti-HIV. In addition, they have an effect on theactivity of cytochrome P450enzyme and Glutathiones S-transferase, whichwill result in transformation on absorption and metabolism of combinationmedicines. The influences between components have to be solved due to thecomplicacy of traditional Chinese medicine, thus it is of big significance toresearch pharmacokinetics of imperatorin and isoimperatorin. Up to now,there have been some studies on the pharmacokinetics of imperatorin andisoimperatorin in which they were regarded as one or two components whenstudying pharmacokinetics of traditional Chinese medicines, thepharmacokinetic parameters might be changed by the other compounds.Moreover, so far, there is few research to compare the pharmacokinetic of thetwo components of. In this study, the analytes were detected by HPLC-MSafter the plasma, urine and bile samples were pretreated by HF-LPME or ethylacetate. Furthermore, the pharmacokinetics in rat were studied after the ratwas administrated with imperatorin or isoimperatorin.Part one The pharmacokinetics study of imperatorin in ratsObjective:1To develop an environmental friendly and sensitiveHF-LPME-HPLC-MS method qualitatively identify and quantitativelydetermine the imperatorin and its metabolites in rat plasma；2To study thepharmacokinetic of imperatorin in plasma by using the developed HF-LPMEmethod;3To study the excretion of imperatorin in urine and bile.Methods:1The extract conditions were investigated according to single-factor index including extraction solvent, length of the fiber, agitationrate, extraction temperature and time by using spkied samples and the spkiedsamples were prepared by blank plasma and references. A tandem massspectrometric detection with electrospray ion (ESI) source operating in thepositive ionization mode was conducted, the ion spray voltage was set at5500V and the turbo spray temperature was maintained at650℃, the detections, declucstering potential and collision energy of imperatporin, psoralen,isopsoralen, xanthotoxin and xanthotoxol were271.1/203.2,25V,15eV;187.2/131.1,52V,33eV;187.1/131.1,39V,31eV;217.2/202.0,10V,25eVand203.2/147.1,20V,32eV, respectively. The separation was performed onAgilent Zorbax SB-C18(150mm×4.6mm,5m) column with gradient elutionand the mobile phase was consisted of methanol and1mmol/L NH4Ac, theflow rate was0.8mL/min;2On the basis of the qualitation of imperatorin andits metabolites in vivo in our laboratory, the compound to be measured wasdetermined. Plasma samples were collected after administrating imperatorin torats. Afterwards the plasma samples were pretreated with HF-LPME. Atandem mass spectrometric detection was conducted with electrospray ion(ESI) source operating in the positive ionization mode, the ion spray voltagewas set at5500V, Agilent Zorbax SB-C18(150mm×4.6mm，5m） columnwas used to separate the analytes and the mobile phase was consisted ofmethanol and1mmol/L NH4Ac, the flow rate was0.8mL/min and sample sizewas10L.3Urine and bile samples were collected after administratingimperatorin to rats. The analytes were detected by HPLC-MS after the plasma,urine and bile samples were pretreated by HF-LPME or ethyl acetate. Atandem mass spectrometric detection was conducted with electrospray ion(ESI) source operating in the positive ionization mode, the ion spray voltagewas set at5500V and the turbo spray temperature was maintained at650℃.Scopoletin was used as the internal standard. The separation was performed onAgilent Zorbax SB-C18(150mm×4.6mm，5m) column with gradient elutionconsisting of methanol and1mmol/L NH4Ac, the flow rate was0.8mL/min.Results:1The optimum conditions of extraction were:100μL plasma was diluted to1.5mL by2%(w/v) NaCl aqueous, and then the analytes wereextracted by n-heptanol for50min with675rpm at room temperature.2Imperatorin was absorbed quickly after oral administration and reached themaximum concentration at1.01h. The metabolite xanthotoxol had alreadyappeared at5min, which indicated that imperatorin was metabolized toxanthotoxol immediately after it was absorbed. T1/2and K were calculated tobe7.93and0.079h, respectivily, which indicated that imperatorin waseliminated rapidly.3Imperatorin was excreted completely in45h via urineand16h via bile. The excretion rate in urine were13.46%,5.296%and0.1686%act as prototype compound, xanthotoxol and xanthotoxin, while theexcretion rate in bile were0.1214%0.04282%and0.009042%act as activecompound, xanthotoxol and xanthotoxin, respectively. To conclusion, therewas a small amount imperatorin excreted as prototype via urine and bile.Conclusion: An environmental friendly, high sensitive and selectivityHF-LPME-HPLC-MS method was developed to qualitatively andquantitatively determine the imperatorin and its metabolites in rat plasma,urine and bile after the rat was administrated with imperatorin. The resultsshowed that imperatorin was rapidly absorbed and then excreted from rat urinemainly.Part Two The pharmacokinetics study of isoimperatorin in ratsObjective:1To research the pharmacokinetics of isoimperatorin and itsmetabolites in rat plasma;2To research the excretion of isoimperatorin inurine and bile after the rat was administrated with isoimperatorin.Methods:1Plasma, urine and bile samples were collected after the ratwas administrated with isoimperatorin. Plasma samples were pretreated withHF-LPME and bile samples were prepared with ethyl acetate. The analyteswere detected under the same conditions of pharmacokinetics research inplasma in section one. A tandem mass spectrometric detection was conductedwith electrospray ion (ESI) source operating in the positive ionization mode,the ion spray voltage was set at5500V and the turbo spray temperature wasmaintained at650℃.2Urine and bile samples were collected after the rat was administrated with isoimperatorin, the urine samples were preated withHF-LPME while bile samples were pretreated with ethyl acetate. Then theanalytes were detected under the same conditions of excretion study in plasmain section oneResults:1The results showed that isoimperatorin was absorbed rapidlyafter it was administrated to rats and the plasma concentration reached itspeak rapidly. T1/2and K were calculated to be2.78and0.249h, respectivily,which indicated that imperatorin was eliminated rapidly.2Isoimperatorinwas excreted completely in60h via urine, the excretion rate in urine were17.80%and12.34%act as prototytype compound and metabolites,respectively. While isoimperatorin was excreted completely in15h via bile,the excretion rates in bile were2.150%and0.0820%act as prototypecompound and metabolites, respectively.Conclusion: The T1/2, Cmax, Tmaxand K of imperatorin and isoimperatorinwere similar, they were both absorbed and excreted rapidly beingadministrated to rats. Under the same dose, isoimperatorin possessed a higherbioavailability than imperatorin. Isoimperatorin possessed a higher excretionrate of prototype compound than imperatorin, which indicated that imperatorinwas easier to converse to metabolites.

欧前胡素和异欧前胡素在大鼠体内的药代动力学研究

摘要5-9ABSTRACT9-12引言13-14第一部分 欧前胡素的药代动力学研究14-52    前言14-15    一 血浆中欧前胡素及其代谢物中空纤维液液微萃取条件的研究15-20        材料与方法15-17        结果17-20    二 欧前胡素在大鼠血浆中的药动学研究20-24        材料与方法20-23        结果23-24    三 欧前胡素在大鼠尿液和胆汁中的排泄动力学研究24-30        材料与方法24-28        结果28-30    附图30-41    附表41-49    讨论49-50    小结50    参考文献50-52第二部分 异欧前胡素在大鼠体内的药代动力学研究52-82    前言52-53    一 异欧前胡素在大鼠血浆中的药动学研究53-57        材料与方法53-55        结果55-57    二 异欧前胡素在大鼠尿液和胆汁中的排泄动力学研究57-61        材料与方法57-58        结果58-61    附图61-70    附表70-78    讨论78-79    小结79-80    参考文献80-82结论82-83综述 欧前胡素与异欧前胡素的药效学和药代动力学研究进展83-90    参考文献87-90致谢90-91个人简历91-92

  本文关键词：欧前胡素和异欧前胡素在大鼠体内的药代动力学研究，由笔耕文化传播整理发布。