簡介：秦皮(Cortex fraxini)為常見的傳統中藥材，取自於木犀科白臘樹的莖皮，目前於台灣已有秦皮相關的保健食品、科學中藥以及複方可供使用，多用來治療痛風、關節炎、腹瀉和細菌性痢疾。秦皮苷(fraxin)為秦皮中的重要有效成分之一，於體外試驗證實具有抗發炎、抗氧化及清除自由基等的作用;於體內試驗，在誘導為高尿酸的大鼠實驗中則發現其具有促進尿酸排除以及保護腎臟的效果。口服秦皮苷後，其體內代謝會經由腸胃道的菌叢進行水解切除glycoside後，形成主要代謝物秦皮素(fraxetin)，秦皮素後續經第二相反應(phase II reaction)生成葡萄醣醛酸及硫酸的結合態代謝物fraxetin glucuronide與fraxetin sulfate，再由尿液排除體外；其中，葡萄醣醛酸反應為臨床藥品常見的代謝途徑，重要的參與酵素為尿嘧啶雙磷酸轉移酵素(UGTs)。UGT酵素的代謝是造成許多天然多酚類化合物之生體可用率低下的主因，也是導致食品與藥物交互作用的重要因素。因此同為多酚類化合物的秦皮苷及其經UGT酵素代謝後的相關產物在體內的藥物動力學值得進一步研究。
目的：本研究目的在利用大白鼠動物實驗模式來探討秦皮苷於大白鼠體內動態以及它和臨床使用藥品間的交互作用。為此也將開發靈敏的高效液相層析法，同時定量秦皮苷與其主要代謝物。
方法：在控制組實驗中，分別以靜脈注射與口服投藥方式給予不同劑量的秦皮苷與秦皮素以研究其藥物動態。在交互作用試驗中先給予秦皮素接著給予valproate，採集血液檢品並分析valproic acid、秦皮素與其結合態代謝物，進一步探討其交互作用。
結果：實驗結果已成功開發靈敏且能夠同時分析動物體液中的秦皮苷與其結合態代謝物的液相層析方法且實際應用至大白鼠體內動力學研究。大鼠口服投予秦皮苷或靜脈投予秦皮素後，在血漿檢品層析圖譜上可明顯觀察到疑似代謝產物的層析波峰，猜測可能是fraxetin glucuronide與fraxetin sulfate等相關代謝物。靜脈投予秦皮苷則沒有觀察到相關代謝物生成；口服投予秦皮苷或秦皮素後，皆可觀察到fraxetin glucuronide與fraxetin sulfate等代謝物，秦皮苷與秦皮素具二室的分室模式特性，秦皮苷生體可用率約只有0.8 %，秦皮素約82 %。與valproic acid 交互作用結果顯示，靜脈給藥併用秦皮素與valproate後，兩者血中濃度皆有上升情形且排除速率減緩；口服給藥併用秦皮素與valproate，兩者的血中濃度皆有下降情形，且秦皮素代謝物在給藥後30分鐘內亦觀察到減少現象。
結論：秦皮苷以口服投予後，會在大鼠體內快速且大量代謝成秦皮素、fraxetin glucuronide與fraxetin sulfate，而以靜脈注射投予的話則沒有觀察到相關代謝物的生成，此現象可能是和秦皮苷上的glycoside官能基有關。併用同為UGTs受質的秦皮素與valproic acid在大白鼠體內藥物交互作用之研究中，靜脈給藥的實驗組可以看到valproic acid曲線下面積增加為原本的1.5倍，顯示valproic acid在大鼠體內的排除受到影響，而這樣的交互作用可能會造成valproic acid持續蓄積在體內。口服給藥的實驗組則觀察到valproic acid的吸收顯著受到秦皮素的影響而減少，而valproic acid的排除則沒有改變。

關鍵詞：
秦皮苷，多酚類化合物，第二相反應，葡萄糖醛酸反應，valproic acid

Pharmacokinetics and drug interactions of fraxin in rats
Hui-Chun Hong
Chen-Hsi Chou
Institute of Clinical Pharmacy, Medical College, National Cheng Kung University
Abstract
Background. Cortex fraxini (named Qinpi in Chinese) is the dried bark of plants from the Oleaceae family, including Fraxinus rhynchophylla, F. chinensis, F. szaboana and F. stylosa. Cortex fraxini is commonly used as Chinese herbal drug, proven to be effective in the treatment of diarrhea and dysentery of intense heat type. Fraxin (7-hydroxy-6-methoxycoumarin 8-glucoside), a structurally derivative of coumarin glucoside, is one of the important bioactive components in Cortex fraxini. Fraxin is known to be responsible for the diuretic, anti-inflammatory, anti-oxidant, and anti-hyperuricemic effects. Following oral administration fraxin is extensively metabolized to the aglycone fraxetin (7,8-Dihydroxy-6-methoxycoumarin), which is also an effective constituent of Cortex fraxini and can be extensively metabolized to glucuronides by uridine diphosphate glucuronosyl transferases (UGTs). Glucuronidation mediated by uridine diphosphate glucuronosyl transferases (UGTs) is a common phase II metabolic pathway for various prescribed drugs. In view of these interactions, drugs with narrow therapeutic margins such as valproic acid should be carefully considered with respect to fraxin. Quantification and pharmacokinetics studies on constituents of Traditional Chinese Medicine in plasma are required to offer useful information in clinical application. Although fraxin has been used clinically for many years, its pharmacokinetic property still remains unknown so far due to the lack of quantification methods. Therefore, it is necessary to develop a suitable bioanalytical method for the determination of fraxin in plasma.
Purpose. The aim of this study is to investigate the pharmacokinetics and drug interactions of fraxin in male Sprague-Dawley rats. To characterize the pharmacokinetic properties of fraxin, it is very necessary to develop an accurate and selective bioanalytical method for the determination of fraxin in rat plasma.
Methods. Two sensitive high-performance liquid chromatography (HPLC) methods, one for simultaneous determination of fraxin and its conjugate metabolites using ferulic acid ((2E)-3-(4-hydroxy-3-methoxyphenyl) prop-2-enoic acid) as an internal standard, the other for quantification of fraxetin using piperonyl alcohol (2H-1,3-benzodioxol-5-ylmethanol) as an internal standard in rat plasma were developed. The analytes and internal standards were separated on Thermo Hypurity C18 column (4.6 mm × 250 mm, 5 μm) and protected by an ODS guard colum (10 mm × 4 mm, 5 μm). Fluorescence and ultraviolet detectors were used in this study. Since fraxin has fluorophoric properties, fluorescent detection (FD) was expected to provide an inexpensive, sensitive, and specific detection of fraxin in biological samples. The two methods have been fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery. The Sprague-Dawley rats (8 weeks; body weight: 230~250 g) received fraxin and fraxetin intravenously (iv) and orally at the dose level of 5 and 20 mg per kilogram, respectively. Kinetics of fraxetin following intravenous infusion at a rate of 150 μg per minutes for 20 minutes was also examined. Co-medication of fraxetin and valproate via iv and oral routes were employed to investigate potential drug-drug interaction. Serial blood samples (250 μL) were collected from the carotid intoheparinized 1.5 mL polythene tubes and centrifuged at 13,000 rpm for 10 min and stored frozen at -20 oC until analysis. The plasma concentrations of fraxin, fraxetin and the conjugate metabolites were determined by HPLC methods and the kinetics parameters were estimated by compartmental analysis.
Results. The developed HPLC methods were found to be specific, precise and accurate. Calibration curves for fraxin was constructed over a range of 0.0014 – 27 μM and that of fraxetin was 0.24 – 120 μM. The lower limit of quantitation (LLOQ) for fraxin is 0.0014 μM ng/mL and that of fraxetin is 0.24 μM. The two methods were successfully applied to the pharmacokinetics of fraxin/fraxetin in rats. The disposition kinetics of fraxin in rats displayed two-compartmental characteristics, with a distribution half-life of 20 min and an elimination half-life of 120 minutes. The oral absorption of fraxin/fraxetin was rapid with a peak concentration occurred before 10 minutes. The estimated bioavailability of fraxin was 0.8% and that of fraxetin was 82 %. After iv bolus injection, plasma concentration of fraxetin declined rapidly with a short elimination half-life about 10 minutes, and two major metabolites, with high fluorescence intensity similar to fraxin, were generated. After co-administration with valproate intravenously, the AUC value of valproic acid, fraxetin and its conjugate metabolites were significantly increased. In the oral experiment group, the plasma levels of valproic acid and fraxetin were decreased and the major metabolites of fraxetin were decreased before 30 minutes.
Conclusion. After oral administration of fraxin, it converts to fraxetin rapidly. And, fraxetin undergoes rapid and extensive conjugation metabolism to generate fraxetin glucuronide and fraxetin sulfate. Co-administration of the UGTs substrates fraxetin and valproate by intravenous route in rats resulted in a 1.5-fold increase of the AUC value of valproic acid, indicating that the elimination of valproic acid was affected by fraxetin. In the oral administration group, fraxetin significantly affected the absorption of valproic acid without altered its elimination.

Keywords:
fraxin，polyphenols，phase II reaction，glucuronidation，valproic acid

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