臺灣博碩士論文加值系統

馬兜鈴科植物（Aristolochiaceae Juss.）曾廣泛運用在中醫及世界各地的草藥醫學中，藥理研究指出其主要成分馬兜鈴酸（aristolochic acid, AA）具抗發炎功效，可防止感染、抑制細菌的生長；但馬兜鈴酸已被證實和腎病變、泌尿上皮癌有強烈相關性。一般認為馬兜鈴酸在生物體內會被代謝活化形成Aristolactam，其與DNA形成共價鍵結物，進而致突變與致癌。馬兜鈴酸與DNA的鍵結物在馬兜鈴酸腎病變的病人組織內，二十年後仍可以被檢測到，代表其在體內的持久性。我們預期馬兜鈴酸也會和胺基酸形成共價鍵結物，相較於毒物與DNA的共價鍵結物，毒物與胺基酸的共價鍵結物在生物環境中保持化學穩定、較不受影響，且檢測樣品較易獲得，可提供特定時間的暴露紀錄，是具特殊與專一性且有應用潛力的生物指標。
本研究的目的為合成馬兜鈴酸I（AAI）與胺基酸N-乙醯-L-半胱胺酸（N-acetyl-L-cysteine, NAC）的共價鍵結物（AAI-NAC），大量合成、純化、與鑑定其結構後，預作為馬兜鈴酸暴露的尿中生物指標。合成實驗以AAI的鈉鹽及NAC做為反應物，用鋅粉做為催化劑，在37℃於磷酸鉀緩衝溶液中避光攪拌反應16小時。合成反應最佳化的條件：AAI與NAC的比例為1: 10，磷酸鉀緩衝溶液pH為5.8。大量合成AAI-NAC鍵結物後，使用高效能液相層析儀（High-performance liquid chromatography, HPLC）進行純化，利用半製備級C18層析管柱，以35%移動相A（0.1% formic acid in acetonitrile及65%移動相B (0.1% formic acid in H2O)等梯度沖堤，可順利分離出AAI-NAC鍵結物。以液相層析串聯質譜儀（Liquid Chromatography Tandem Mass Spectrometer, LC-MS/MS）儀器分析和核磁共振光譜儀（Nuclear Magnetic Resonance Spectroscopy, NMR）確認合成產物AAI-NAC鍵結物的結構。目前已證實AAI-NAC鍵結物可在體外環境形成，未來將開發LC-MS/MS分析方法、建立分析方法標準，以發展成為動物或人體暴露馬兜鈴酸或含馬兜鈴酸草藥的生物指標。

Aristolochiaceae plants have been widely used in Traditional Chinese medicine and herbal medicine around the world. Aristolochic acid (AA), a major active component of plants from the Aristolochiaceae family, shows anti-inflammatory properties by pharmacological studies. However, AA has been proven to have a strong correlation with nephropathy and urothelial carcinoma. It is generally believed that AA is metabolically activated to aristolactam that reacts covalently with DNA to form AA-DNA adducts. These DNA adducts were detected two decades later in the tissues of AAN patients resported in Belgium in the early 1990’s, indicating their persistence in vivo. Compared to DNA adducts, amino acid adducts are easily obtained, kept chemically stable in a biological environment, relatively unaffected, and can provide a period of time of exposure to a compound.
The purpose of this study was to synthesize, purify, and characterize the AAI and N-acetyl-L-cysteine (NAC) adducts, AAI-NAC, to be used as a biomarker of AA exposure in the urine. In the synthesis experiments, 2 mmol NAC and 0.2 mmol AAI sodium salt were added in the presence of zinc as a catalyst in 500 mL of pH 5.8 potassium phosphate buffer solution at 37℃ for 16 hours in the dark. Synthetic products of AAI-NAC were analyzed by Liquid Chromatography Tandem Mass Spectrometer (LC-MS/MS). Further purification of AAI-NAC was performed by high performance liquid chromatography (HPLC) using an ODS-3 column and a mobile phase consisting of 35% ACN and 65% H2O in 0.1% FA (isocratic). The yield was about 0.1%. The chemical structure of the purified AAI-NAC was confirmed by Nuclear Magnetic Resonance Spectroscopy (NMR). Our results demonstrated that AAI-NAC adduct can be generated in vitro. In the future, the analytical methods by LC-MS/MS for AAI-NAC will be developed to analyze AAI-NAC in the urine of rodents exposed to AA or Aristolochia plants. The use of AAI-NAC as an exposure biomarker in humans for recent exposure to AA-containing herbs will be explored further.

目 錄
第一章 前　言......1
1.1 研究動機......1
1.2 研究目的......2
第二章 文獻探討......3
第一節 馬兜鈴科植物......3
1. 馬兜鈴科植物......3
2. 馬兜鈴科植物的中藥應用......8
第二節 馬兜鈴酸(Aristolochic acid)......11
1. 馬兜鈴酸的藥理研究 ......12
2. 馬兜鈴酸的代謝與毒理研究......13
3. 馬兜鈴酸的致癌作用......15
3.1 馬兜鈴酸的致癌機轉......15
3.2 馬兜鈴酸與泌尿道上皮癌 ......15
4. 馬兜鈴酸腎病變 ......16
4.1 馬兜鈴酸腎病變的流行病學......17
4.1.1巴爾幹腎病變(Balkan Endemic Nephropathy, BEN)......17
第三節 生物指標......19
1. 暴露生物指標的簡介......19
2. 馬兜鈴酸的DNA鍵結物......21
3. 馬兜鈴酸的胺基酸鍵結物......22
第三章 實驗材料與方法......24
第一節 實驗材料......24
1. 化學試劑與藥品...... 24
2. 儀器設備......25
2.1高效能液相層析儀(High-performance liquid chromatography, HPLC)......25
2.2 真空冷凝離心設備......25
2.3 超純水系統......26
2.4 液相層析串聯質譜儀(Liquid Chromatograph Tandem Mass Spectrometer, LC-MS/MS)......26
2.5 其他......26
3. 實驗器材......27
第二節 實驗方法......28
1. AAI-NAC的合成...... 28
2. 馬兜鈴酸胺基酸鍵結物的純化......29
3. 馬兜鈴酸胺基酸鍵結物的鑑定......32
第四章 結果......33
第一節 AAI-NAC的合成......33
1. AAI-NAC於實驗反應的水層中發現......33
2. 合成反應物AAI與NAC最佳化比例為1: 10......35
3. AAI-NAC合成實驗磷酸鉀緩衝溶液較佳pH為5.8 ......35
第二節 AAI-NAC的純化......37
1. 選擇HPLC層析管柱為半製備級C18 ODS-3管柱......37
2. HPLC純化AAI-NAC的移動相梯度調整......39
第三節 AAI-NAC的鑑定......49
第五章 討論......59
第一節 AAI-NAC的合成與AA- DNA鍵結物做比較......59
第二節 AAI-NAC的純化與結構鑑定......60
第三節 AAI-NAC在體內生成的可能代謝途徑......61
第六章 結論......63
參考文獻......64
英文摘要......77


圖目錄
圖 2. 1馬兜鈴科植物......4
圖 2. 2港口馬兜鈴 ......4
圖 2. 3本草備要馬兜鈴......10
圖 2. 4中藥材馬兜鈴......11
圖 2. 5鐵線蓮狀馬兜鈴......12
圖 2. 6馬兜鈴酸的代謝活化與DNA鍵結物的形成......22
圖 2. 7馬兜鈴酸與胺基酸NAC鍵結物形成的預測......23
圖 3. 1 馬兜鈴酸與胺基酸合成實驗流程圖......28
圖 3. 2 合成馬兜鈴酸胺基酸鍵結物......29
圖 3. 3馬兜鈴酸胺基酸鍵結物預期斷裂碎片......32
圖 4. 1 確認目標產物AAI-NAC位於實驗反應生成物的萃取水層......34
圖 4. 2 AAI-NAC合成反應條件最佳化: 反應物AAI及NAC濃度比例......35
圖 4. 3 AAI-NAC合成反應條件最佳化: 磷酸鉀緩衝溶液pH......36
圖 4. 4 純化AAI-NAC之HPLC層析條件最佳化: 層析管柱、流速與有機相水相比例......38
圖 4. 5純化AAI-NAC之HPLC層析條件最佳化: 移動相梯度......40
圖 4. 6 以LC-MS/MS檢測經HPLC純化的AAI-NAC分液不同滯留時間收集的樣本：移動相梯度 ......42
圖 4. 7 HPLC純化AAI-NAC的實驗水層樣本濃縮......44
圖 4. 8 移動相梯度50% ACN 30:21至30:30 min以LC-MS/MS檢測Full scan圖譜......45
圖 4. 9 移動相梯度50% ACN 30:21至30:30 min樣品質譜圖...... 46
圖 4. 10 移動相梯度50% ACN 30:21至30:30 min樣品內含雜質質譜圖......46
圖 4. 11 純化樣品以LC-MS/MS檢測的質譜圖，在移動相梯度35% ACN時，29:31 min至31:00 min可能有AAI-NAC存在......47
圖 4. 12 純化樣品以LC-MS/MS檢測的質譜圖，在移動相梯度35% ACN時，29:31 min至31:00 min層析峰比較圖......48
圖 4. 13 移動相梯度35% ACN 29:41- 30:50 min的樣品質譜圖......48
圖 4. 14 AAI-NAC樣本進行質譜測試 (MS)....49
圖 4. 15 AAI-NAC樣本進行串聯質譜測試 (MS/MS)......50
圖 4. 16 AAI-NAC樣本進行SRM......51
圖 4. 17 AAI-NAC送測NMR的標號......52
圖 4. 18 純化AAI-NAC的1H NMR 500 MHz圖譜......56
圖 4. 19 純化AAI-NAC的13C NMR 500 MHz圖譜......57
圖 4. 20 純化AAI-NAC的HSQC 500 MHz圖譜......57
圖 4. 21 純化AAI-NAC的COSY 500 MHz圖譜......58
圖 4. 22 合成純化後AAI-NAC的HMBC 500 MHz圖譜......58
圖 5. 1 Glutathione組成及生物體內AAI-NAC形成預測......62

表目錄
表 2. 1 馬兜鈴植物考證舉例簡表......5
表 2. 2 馬兜鈴屬植物於世界各地民俗醫藥應用......6
表 3. 1 質譜游離源參考條件......31
表 4. 1 1H and 13C NMR Interpretation of AAI-NAC......55

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