本文關(guān)鍵詞：歐前胡素和異歐前胡素在大鼠體內(nèi)的藥代動力學(xué)研究，由筆耕文化傳播整理發(fā)布。

        歐前胡素和異歐前胡素屬于6,7-呋喃香豆素類化合物，因取代基位置不同互為位置異構(gòu)體。二者廣泛存在于植物界，尤其在傘形科中藥中含量豐富，均具有鎮(zhèn)痛、抗病毒、抗腫瘤、抗菌和抗HIV等藥理作用。此外，歐前胡素和異歐前胡素對藥物代謝酶-細(xì)胞色素酶P450和谷胱甘肽S-轉(zhuǎn)移酶的活性有一定的影響。中藥成分復(fù)雜，各成分的體內(nèi)過程可能存在相互影響，對可影響藥物代謝酶活性的成分的藥代動力學(xué)研究顯得尤為重要。目前對于歐前胡素和異歐前胡素的藥代動力學(xué)研究，多是將其作為藥材整體藥動學(xué)研究中的化合物進行，尚未有二者單體藥代動力學(xué)的系統(tǒng)研究。本研究以專屬、靈敏的高效液相色譜-質(zhì)譜聯(lián)用（HPLC-MS）技術(shù)為分析手段，分別建立了各種生物樣品中歐前胡素和異歐前胡素及其代謝物的定量測定方法，，并用于二者分別灌胃大鼠后的藥代動力學(xué)研究和比較，為中藥整體藥動學(xué)的闡明奠定了基礎(chǔ)，為中藥位置異構(gòu)體藥動學(xué)規(guī)律的尋找提供了借鑒。第一部分歐前胡素的藥代動力學(xué)研究目的：1優(yōu)化中空纖維液液微萃�。℉F-LPME）條件，用于大鼠血漿、中歐前胡素及其代謝物花椒毒酚測定的樣品前處理；2建立血漿中歐前胡素及其代謝物花椒毒酚測定的HPLC-MS方法，研究大鼠灌胃歐前胡素后的藥代動力學(xué)特征；3建立尿液和膽汁中歐前胡素及其代謝物花椒毒酚和花椒毒素測定的HPLC-MS方法，研究大鼠灌胃歐前胡素后的排泄動力學(xué)特征。方法：1以空白大鼠血漿和待測化合物對照品制備模擬生物樣本，按照單因素考察的方法依次考察并確定提取溶劑、中空纖維長度、攪拌速率、提取溫度、稀釋倍數(shù)和鹽效應(yīng)等提取條件，確定提取歐前胡素及其代謝物的最佳條件。Electrospray Ionization （ESI）源，源噴射電壓：5500V；正離子模式監(jiān)測；離子源溫度（TEM）：650℃；歐前胡素、與花椒毒酚檢測離子對和DP電壓、CE電壓分別為271.1/203.2，25V，15eV和203.2/147.1，20V，32eV。色譜柱為Agilent Zorbax SB-C18，流動相體系為甲醇-1mmol/L NH4Ac，梯度洗脫，流速0.8mL/min；2結(jié)合本實驗室對歐前胡素體內(nèi)定性研究的結(jié)果進行實驗，確定測定化合物。灌胃給予大鼠歐前胡素后采集不同時間點的大鼠血漿，采用已確定的HF-LPME處理血漿樣本。采用HPLC-MS方法測定。采用Electrospray Ionization（ESI）源，正離子模式監(jiān)測。源噴射電壓（IS）：5500V；離子源溫度：500℃；霧化氣（Gas1）：40psi，加熱氣（Gas2）：50psi，簾氣：25psi；以東莨菪內(nèi)酯為內(nèi)標(biāo)，采用Agilent Zorbax SB-C18柱進行分離，流動相體系為甲醇-1mmol/L NH4Ac，梯度洗脫，流速0.8mL/min。36只大鼠灌胃給予歐前胡素后置于代謝籠內(nèi)，收集72小時內(nèi)0-4，4-8，8-12，12-24，24-36，36-48，48-60和60-72h的尿液并記錄體積，采用與血漿樣品相同的HF-LPME預(yù)處理方法，另外6只大鼠麻醉后進行膽管插管手術(shù)，收集24小時內(nèi)0-2，2-4，4-8，8-12，12-20和20-24h時間段的膽汁并記錄體積，用乙酸乙酯萃取的方式進行預(yù)處理。尿液和膽汁樣品均在電噴霧離子（ESI）源，正離子多反應(yīng)監(jiān)測模式（MRM）下進行檢測。歐前胡素、花椒毒酚、花椒毒素和內(nèi)標(biāo)的檢測離子對和DP電壓、CE電壓分別為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-羥基補骨脂素的檢測離子對和DP電壓、CE電壓為193.1/133.1，49V，25eV。色譜分離采用AgilentZorbax SB-C18色譜柱（150mm×4.6mm，5m），柱溫：25℃；流動相為甲醇（A）和1mmol/L乙酸銨-水（B），梯度洗脫，洗脫程序如下：0~5min，從50%A線性變化至95%A；5-8.5min，保持95%A；流速：0.8mL/min，進樣量10μL。結(jié)果：1經(jīng)過考察確定的提取條件為：以正庚醇為萃取溶劑，中空纖維長度為5cm，磁力攪拌器的轉(zhuǎn)速為675rpm，萃取溫度為室溫，100μL血漿用2%NaCl稀釋至1.5mL，萃取時間為50min；2實驗得到歐前胡素和花椒毒酚的藥動學(xué)參數(shù)和藥-時曲線。灌胃給予大鼠歐前胡素以后吸收迅速，在1.01h達(dá)到最大濃度，T1/2為7.93，K為0.079（1/h），說明歐前胡素消除亦較快。灌胃5min以后即檢測到代謝物花椒毒酚，說明歐前胡素被吸收以后迅速被轉(zhuǎn)化為代謝物花椒毒酚；3大鼠灌胃給予歐前胡素后，歐前胡素在尿中45h內(nèi)基本排泄完全，在膽汁中16h內(nèi)基本排泄完全。歐前胡素以原型、花椒毒酚和花椒毒素的形式在尿液中的排泄量分別為13.46%，5.296%，和0.1686%；在膽汁中的排泄量分別為0.1214%，0.04282%和0.009042%。表明歐前胡素以原型在大鼠尿液和膽汁中排泄率不高。結(jié)論：建立了一種環(huán)保、靈敏度高、專屬性強的HPLC-MS法測定大鼠血漿、尿液和膽汁中的歐前胡素及其代謝物。歐前胡素在大鼠體內(nèi)吸收迅速，主要經(jīng)尿液排泄。第二部分異歐前胡素的藥代動力學(xué)研究目的：1建立大鼠血漿中異歐前胡素及其代謝物花椒毒酚的HPLC-MS測定方法，并研究大鼠灌胃給予異歐前胡素后的藥代動力學(xué)特征；2建立大鼠尿液和膽汁中異歐前胡素及其代謝物花椒毒酚和花椒毒素的HPLC-MS測定方法，并研究大鼠灌胃給予異歐前胡素后的排泄動力學(xué)特征。方法：1將異歐前胡素混懸在于CMC-Na的灌胃液給予大鼠后，在不同時間點對大鼠進行眼內(nèi)眥取血，得到血漿生物樣本，采用HF-LPME的預(yù)處理方法，采用與第一部分血漿測定相同的液相和質(zhì)譜條件下進行檢測；26只SD大鼠灌胃給予異歐前胡素后置于代謝籠內(nèi)飼養(yǎng)，自由飲水，4h后給予食物，分別收集0-4，4-8，8-12，12-24，24-36，36-48，48-60和60-72h時間段的尿液并記錄體積，之后采用與第一部分尿液處理相同的方法進行預(yù)處理。另取6只大鼠給藥后，以20%烏拉坦麻醉（7mL/kg），進行膽管插管術(shù)，收集給藥后0-2，2-4，4-8，8-12，12-20和20-24h時間段的膽汁并記錄體積，收集的樣本使用乙酸乙酯萃取的方法進行預(yù)處理，采用第一部分排泄動力學(xué)部分的液相和質(zhì)譜條件下進行檢測。結(jié)果:1實驗結(jié)果顯示異歐前胡素吸收迅速，計算得到異歐前胡素的T1/2為2.78h，K為0.249（1/h）說明異歐前胡素在大鼠體內(nèi)消除較快；2在尿液中，72h內(nèi)異歐前胡素原型在45h內(nèi)基本排泄完全，代謝物則在56h內(nèi)基本排泄完全，異歐前胡素分別以原型和代謝物形式排泄量約占總給藥量的17.80%和12.34%。而在膽汁中，異歐前胡素以原型形式在12h內(nèi)基本排泄完全，而代謝物經(jīng)15h基本排泄完全，兩種形式排泄量分別約占總給藥量的2.150%和0.0820%。結(jié)論：異歐前胡素和歐前胡素在大鼠體內(nèi)的T1/2、Cmax、Tmax和K無統(tǒng)計學(xué)上的差異，二者都吸收迅速，消除較快；在相同給藥量的前提下，異歐前胡素有更高的生物利用度；在尿液和膽汁中異歐前胡素以原型排泄的比率更高，說明歐前胡素較異歐前胡素更容易轉(zhuǎn)化為代謝物。

    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.

歐前胡素和異歐前胡素在大鼠體內(nèi)的藥代動力學(xué)研究

摘要5-9ABSTRACT9-12引言13-14第一部分 歐前胡素的藥代動力學(xué)研究14-52    前言14-15    一 血漿中歐前胡素及其代謝物中空纖維液液微萃取條件的研究15-20        材料與方法15-17        結(jié)果17-20    二 歐前胡素在大鼠血漿中的藥動學(xué)研究20-24        材料與方法20-23        結(jié)果23-24    三 歐前胡素在大鼠尿液和膽汁中的排泄動力學(xué)研究24-30        材料與方法24-28        結(jié)果28-30    附圖30-41    附表41-49    討論49-50    小結(jié)50    參考文獻50-52第二部分 異歐前胡素在大鼠體內(nèi)的藥代動力學(xué)研究52-82    前言52-53    一 異歐前胡素在大鼠血漿中的藥動學(xué)研究53-57        材料與方法53-55        結(jié)果55-57    二 異歐前胡素在大鼠尿液和膽汁中的排泄動力學(xué)研究57-61        材料與方法57-58        結(jié)果58-61    附圖61-70    附表70-78    討論78-79    小結(jié)79-80    參考文獻80-82結(jié)論82-83綜述 歐前胡素與異歐前胡素的藥效學(xué)和藥代動力學(xué)研究進展83-90    參考文獻87-90致謝90-91個人簡歷91-92

  本文關(guān)鍵詞：歐前胡素和異歐前胡素在大鼠體內(nèi)的藥代動力學(xué)研究，由筆耕文化傳播整理發(fā)布。