| 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. |
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