臺灣博碩士論文加值系統

漆黃素 (fisetin; 3, 3’, 4’, 7-tetrahydroxyflavone) 是一種具有多重生物活性且廣泛地存在於植物體中的黃酮醇 (flavonol)。在flavonol中，如槲皮素 (quercetin; 3, 3’, 4’, 5, 7-pentahydroxyflavone) 及其glucuronides會被分泌至膽汁進行膽汁排泄，甚至近一步有腸肝循環的特性。化學結構與quercetin相似的fisetin在許多物理、化學及藥理特性上都與quercetin接近。因此，從結構－藥物動力學關係來看，本實驗假設fisetin及其代謝物可能會被分泌至膽汁進行膽汁排泄。為了證明這個假說，實驗中建立了一套高效液相層析結合光二極體陣列式偵測器 (high-performance liquid chromatography method coupled to photo-diode array detector, HPLC-PDA) 之分析方法，以此偵測大鼠血液及膽汁中的fisetin，探討fisetin的藥物動力學及膽汁排泄。選用Sprague-Dawley品種之大鼠進行藥物動力學實驗，透過靜脈注射的路徑將fisetin (30 mg/kg) 進行投予。同時，本實驗也更進一步地探討fisetin之膽汁排泄的可能機轉，如P-醣蛋白的轉運，在給予fisetin (30 mg/kg) 前先投予P-醣蛋白調控劑環孢靈素 (cyclosporine, 20 mg/kg, i.v.)。結果顯示fisetin、其glcururonides及sulfates皆會經過膽汁排泄，而fisetin sulfates不僅是fisetin在靜脈給予下最主要的代謝物，也是膽汁中最多的代謝物。先行投予cyclosporine後，fisetin、其glucuronides及sulfates之血中AUC皆上升，但fisetin與其glucuronides於膽汁中的AUC降低，代表fisetin、其glcururonides及sulfates都是P-醣蛋白的受質，且P-醣蛋白與fisetin、其glcururonides及sulfates被運輸進入膽汁有關。綜合以上結果，本研究首度探討了fisetin、其glcururonides及sulfates的膽汁排泄，並進一步地確認P-醣蛋白參與fisetin、其glcururonides及sulfates進入膽汁排泄的機轉，也發現了除了P-醣蛋白之外，可能還有其他參與fisetin sulfates膽汁排泄之調控機轉。

Fisetin (3, 3’, 4’, 7-tetrahydroxyflavone) is a flavonol possessing multiple bioactivities and widely presents in plants. In the flavonol subgroup, such as quercetin and its glucuronides are well known to go through biliary excretion and enteroheptic circulation. With the resemblance to quercetin (3, 3’, 4’, 5, 7-pentahydroxyflavone) on chemical structure, fisetin shared similar physical, chemical and pharmacological properties with quercetin. In the concern of structure-pharmacokinetic relationships between fisetin and quercetin, the hypothesis is that fisetin and its conjugates would partly being secreted to bile and would undergo biliary excretion. To investigate these hypothesis, a high-performance liquid chromatography method coupled to photo-diode array detector (HPLC-PDA) was developed and applied to assay of fisetin in rat plasma and bile to determine the pharmacokinetics and biliary excretion profile of fisetin. The pharmacokinetic study was conducted on male Spraque-Dawley rats after administration of fisetin (30mg/kg, i.v.). Meanwhile, further investigation of the possible transportation mechanism such as P-glycoprotein (P-gp) which may involve in the biliary excretion of fisetin was conducted. A P-gp inhibitor, cyclosporine (20 mg/kg, i.v.) was pretreated prior to fisetin administration (30 mg/kg, i.v.). The results showed that fisetin, its glucuronides and sulfates all undergo biliary excretion, especially the fisetin sulfates conjugation was the major metabolite. When pretreated with cyclosporine, the area under concentration versus time curves (AUC) of fisetin, its glucuronides and sulfates increased in plasma, but the AUC of fisetin and its glucuronides decreased in bile, which indicates that fisetin, its glucuronides and sulfates maybe the substrates of P-gp and indicates that P-gp is related to the transportation of fisetin, its glucuronides and sulfates into bile. In conclusion, this study first explored the biliary excretion profile of fisetin, its glucuronides and sulfates, and further confirmed the involvement of P-gp in the mechanism of biliary excretion of fisetin, its glucuronides and sulfates. However, the biliary excretion of fisetin sulfates might be co-mediated by other mechanism.

目錄

目錄 i
表目錄 iii
圖目錄 iv
中文摘要 v
Abstract vi
第一章　緒論 1
　第一節　漆黃素 (Fisetin) 1
　　　一、　研究背景 1
　　　二、　相關藥理學研究 2
　　　三、　相關藥物動力學研究 3
　第二節　藥物動力學 4
　　　一、　概論 4
　　　二、　腔室模式 (compartment model) 4
　　　三、　藥物動力學參數－藥物濃度曲線下面積 (Area under concentration versus time curve, AUC) 5
　　　四、　藥物動力學參數－半衰期 (Half-life, t1/2) 6
　　　五、　藥物動力學參數－擬似分布體積 (apparent volume of distribution, Vd) 7
　　　六、　藥物動力學參數－清除率 (Clearance, Cl) 7
　第三節　藥物代謝 8
　　　一、　第一相代謝 (Phase I metabolism) 8
　　　二、　第二相代謝 (Phase II metabolism) 8
　　　三、　第三相膜轉運 (Phase III membrane transportation) 10
　　　四、　Flavonoids的代謝 (Metabolism of flavonoids) 10
　第四節　膽汁排泄 (Biliary excretion) 12
　第五節　P-醣蛋白 (P-glycoprotein, P-gp) 14
　　　一、　簡介 14
　　　二、　P-gp的結構與分布 14
　　　三、　P-gp對藥物動力學的影響 15
　　　四、　P-gp的調控劑 － 環孢靈素 (cyclosporine) 15
第二章　研究動機 16
第三章　實驗材料與方法 17
　第一節　實驗材料與化學試劑 17
　　　一、　試藥與試劑 17
　　　二、　實驗儀器設備與手術器械 18
　　　三、　實驗動物 20
　第二節　Fisetin於大鼠血液及膽汁之定量分析 21
　　　一、　高效液相層析儀結合光二極體偵測器 (HPLC-PDA) 之分析條件 21
　　　二、　血漿及膽汁之檢量線檢品及安定性試驗檢品製備 21
　　　三、　生物檢品之前處理 22
　　　四、　分析方法確效 24
　第三節　Fisetin於大鼠之藥物動力學 27
　　　一、　動物手術程序 27
　　　二、　靜脈給藥與血液、膽汁採集時間 27
　第四節　Fisetin之代謝物鑑定 29
　第五節　應用、數值統計與分析軟體 29
第四章　實驗結果 30
　第一節　Fisetin之分析方法確效 30
　　　一、　分析方法對大鼠血漿及膽汁中fisetin之選擇性 30
　　　二、　分析方法之線性定量範圍 30
　　　三、　生物檢品之萃取回收率 30
　　　四、　分析方法之同日、間日精密度及準確度 31
　　　五、　生物檢品之安定性試驗 31
　第二節　Fisetin於大鼠之藥物動力學 32
　　　一、　Fisetin於大鼠之藥物動力學－腔室模式 32
　　　二、　Fisetin於大鼠之藥物動力學－控制組 32
　　三、 Fisetin於大鼠之藥物動力學－併用Cyclosporin A組 33
　第三節 Fisetin之代謝物鑑定 34
第五章　實驗討論 35
　第一節 Fisetin之分析條件優化 35
　第二節 Fisetin之分析方法確效 37
　第三節 生物檢品前處理 39
　第四節 Fisetin於大鼠血漿及膽汁之藥物動力學 41
　第五節 併用Cyclosporin A對fisetin藥物動力學的影響：P-gp在fisetin之膽汁排泄所扮演的角色 43
　第六節　　Fisetin之代謝物鑑定 45
第六章　結論 46
參考文獻 47


表目錄

Table 1.　 Linearity of fisetin in rat plasma and bile 52
Table 2.　 Recovery of fisetin in rat plasma and bile 53
Table 3.　 Accuracy (bias %) and precision (R.S.D.%) of fisetin in rat plasma and bile 54
Table 4.　 Stability of fisetin in rat plasma and bile 55
Table 5.　 Pharmacokinetic parameters of fisetin (30 mg/kg, i.v.) in rat plasma and bile 56
Table 6.　 Biotransformation rate of fisetin (30 mg/kg, i.v.) in rat 57


圖目錄

Figure 1.　Chemical structure of (a) fisetin (3, 3', 4', 7-tetrahydorxyflavone) and (b) quercetin (3, 3', 4', 5, 7-pentahydorxyflavone) 58
Figure 2.　Hepatic uptake from sinusoid and efflux to bile canaliculi via transporters 13
Figure 3.　Typical chromatograms of (a) drug-free plasma, (b) drug-free plasma spiked standards of fisetin (5 μg/mL) and quercetin (IS, 20 μg/mL), and (c) plasma sampling at 5 min after administration of fisetin (30 mg/kg, i.v.) 59
Figure 4.　Typical chromatograms of (a) drug-free bile, (b) drug-free bile spiked standards of fisetin (5 μg/mL) and quercetin (IS, 20 μg/mL), (c) bile sampling during 0 - 10 min after administration of fisetin (30 mg/kg, i.v.) 60
Figure 5.　Typical chromatograms of (a) plasma sampling at 5 min after administration of fisetin (30 mg/kg, i.v.), (b) plasma sampling at 5 min after administration of fisetin (30 mg/kg, i.v.) and incubated with β-glucuronidase and spiked quercetin (IS; 20 μg/mL), and (c) plasma sampling at 5 min after administration of fisetin (30 mg/kg, i.v.) and incubated with sulfatase and spiked quercetin (IS; 20 μg/mL) 61
Figure 6.　Typical chromatograms of (a) bile sampling at 5 min after administration of fisetin (30 mg/kg, i.v.), (b) bile sampling at 5 min after administration of fisetin (30 mg/kg, i.v.) and incubated with β-glucuronidase and spiked quercetin (IS; 20 μg/mL), and (c) bile sampling at 5 min after administration of fisetin (30 mg/kg, i.v.) and incubated with sulfatase and spiked quercetin (IS; 20 μg/mL) 62
Figure 7.　Plasma concentration-time profile of fisetin (30 mg/kg, i.v.) free form (), treated with β-glucuronidase () and sulfatase () after administration in rat 63
Figure 8.　Bile concentration-time profile of fisetin (30 mg/kg, i.v.) free form (), treated with β-glucuronidase () and sulfatase () after administration in rat 64
Figure 9.　UV-visible spectrum of (a) fisetin (b) unidentified metabolite of fisetin within 190 – 600 nm 65
Figure 10.　(a) Full-scan mass spectrum and (b) MSMS scan spectrum of unidentified metabolite of fisetin 66
Figure 11.　Speculated structures of the unidentified metabolite of fisetin 67
Figure 12.　Proposed metabolic pathway of fisetin, its glucronides, sulfates, possible methyl metabolite and their distribution into plasma and bile 68

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