臺灣博碩士論文加值系統

多甲氧基黃酮 (PMF) 衍生物大多存在於柑橘屬的果皮中，具有抗發炎、抗腫瘤及抗血管動脈粥狀硬化等生物活性，本篇研究首先利用高效能液相層析儀/紫外可見光偵測器 (HPLC/UV-Vis)，分析 11 種台灣柑橘屬果皮中主要的多甲氧基黃酮衍生物，並進一步建立一個簡單、快速及準確之高效能液相層析多次質譜 (LC-MSn) 方法，以正離子之選擇反應偵測 (SRM) 模式，定量分析未經酸處理及酸處理之柑橘屬果皮乙酸乙酯萃取物中 PMFs 及 5-OHPMFs 的組成。
定量方法中，首先分別建立 PMFs 及 5-OHPMFs 標準品之二次或三次質譜，藉由標準品之指紋圖譜以分析柑橘屬果皮萃取物中 PMFs 及 5-OHPMFs 的組成，並建立 12 種標準品之標準曲線，其相關係數 (r2) 皆大於 0.99，偵測極限 (LOD) 與定量極限 (LOQ) 分別介於 0.02 - 0.23 μg/mL 與 0.05 - 0.76 μg/mL，準確度 (RE%) 及精確度 (CV%) 皆於 ± 15% 之內，回收率為 89.5% - 110.9%。
最後並以 Nobiletin 與 Tangeretin 進行抑制酪胺酸酶活性試驗，結果顯示，Nobiletin 與 Tangeretin 抑制酪胺酸酶的能力均優於麴酸 (Kojic acid)，其中以 Nobiletin 抑制酪胺酸酶效果最佳，其 IC50 為 92 μM。因此，本實驗並進一步使用石英晶體微天平儀器 (QCM)，檢測 Nobiletin 與酪胺酸酶分子間的交互作用情形，其解離常數 (Kd )為 0.026 μM，顯示 Nobiletin 與酪胺酸酶分子間具有高結合力。

Polymethoxyflavone derivatives are exclusively existed in Citrus fruit peels. They have shown various biological activities, such as anti-inflammatory, anti-carcinogenic and anti-atherosclerosis. In this study, first a high-performance liquid chromatography equipped with UV-Vis detector was used to analyze the major polymethoxyflavone derivatives in eleven Citrus fruit peels. Furthermore, a simple, quick and reliable LC-MSn method with selective reaction monitoring (SRM) in positive ionization mode was established to determine the contents of polymethoxyflavones (PMFs) and 5-hydroxyl polymethoxyflavones (5-OHPMFs) among the citrus peel extracts treated with or without acid.
The diagnostic MS2 or MS3 spectra were built up from the pure PMF and 5-OHPMF standards. Bases on these fingerprints, the contents of PMFs and 5-OHPMFs in citrus peel ethyl acetate extracts were quantified. All calibration curves were constructed with coefficient of determination (r2) greater than 0.99 among all 12 standards. The limits of detections (LOD) were in the range of 0.02 to 0.23 μg/mL, and the limits of quantifications (LOQ) were in the range of 0.05 to 0.76 μg/mL. Accuracy and precision were within 15%, and the recovery ranges were among 89.5% to 110.9%.
In the final of this study, tyrosinase inhibitory activity of nobiletin and tangeretin was examined. It showed that nobiletin and tangeretin had better tyrosinase inhibitory activity than kojic acid. Nobiletin exhibited the best tyrosinase inhibitory activity with IC50 at 92 μM. The quartz crystal microbalance (QCM) technique was applied to investigate the interaction of nobiletin and tyrosinase. The high affinity between tyrosinase and nobiletin was shown with the dissociation constant (Kd) at 0.026 μM.

中文摘要 .............................................I
英文摘要 .......................................... III
謝誌 ............................................... V
目錄 .............................................. VI
圖次 .............................................. IX
表次 ............................................... X
附圖次 ............................................. XI
附表次 ............................................ XIV
縮寫表 ............................................. XV
壹、前言 ............................................ 1
貳、文獻回顧 ......................................... 2
一、柑橘 ............................................. 2
(一) 柑橘簡介 ....................................... 2
(二) 柑橘類水果果皮之成分 ............................. 5
(三) 柑橘果皮之生理活性 ............................... 9
二、 多甲氧基黃酮 (Polymethoxyflavone, PMF) 衍生物...... 10
(一) PMFs 結構 ..................................... 10
(二) PMFs 生理活性 .................................. 13
(三) PMFs 分析方法 .................................. 15
三、 黑色素 (Melanin) 與美白 .......................... 16
(一) 黑色素之生成 .................................... 16
(二) 酪胺酸酶 ....................................... 16
四、 石英晶體微天平 (Quartz crystal microbalance, QCM).. 19
(一) QCM 原理 ....................................... 19
(二) QCM 應用 ....................................... 20
參、研究目的 .......................................... 21
肆、實驗架構 .......................................... 22
伍、實驗材料與方法 ..................................... 23
一、 實驗材料 ......................................... 23
二、 化學藥品與溶劑 .................................... 23
(一) 試藥 ............................................ 23
(二) 溶劑 ............................................ 25
(三) 其他 ............................................ 25
三、 儀器設備 ......................................... 26
(一) 一般儀器設備 ..................................... 26
(二) 化學分析儀器設備 .................................. 27
(三) 其他 ............................................ 28
四、 實驗方法 ......................................... 29
(一) 柑橘屬果皮粉末樣品製備 ..............................29
(二) HPLC/UV-Vis 分析 11 種柑橘屬果皮 PMFs 及 5-OHPMFs .. 29
(三) 液相層析離子阱式質譜儀 (LC-MSn) 分析條件設定 .......... 31
(四) LC-MSn 分析方法確立 ............................... 33
(五) LC-MSn 分析柑橘屬果皮乙酸乙酯萃取物中 PMFs 及 5-OHPMFs.. 34
(六) 柑橘果皮樣品分離純化 Nobiletin 及 Tangeretin ......... 35
(七) 多甲氧基黃酮抑制酪胺酸酶活性試驗 ....................... 36
(八) 統計分析 .......................................... 38
陸、結果與討論 .......................................... 39
一、 HPLC/UV-Vis 分析 11 種柑橘屬果皮中 PMFs 及 5-OHPMFs... 39
二、 LC-MSn 分析方法設定 ................................. 45
(一) 6 種 PMF 與 6 種 5-OHPMF 標準品指紋圖譜建立 .......... 47
三、 LC/MSn 分析方法確立 ................................. 53
(一) 標準曲線建立 ....................................... 53
(二) 偵測極限 (LOD) 與定量極限 (LOQ)....................... 53
(三) 精確度 (Precision) 與準確度 (Accuracy) .............. 56
(四) 回收率試驗 (Recovery) ............................... 59
四、 未經酸處理及經酸處理之柑橘果皮乙酸乙酯萃取物以 LC/MSn分析 PMFs 及 5-OHPMFs 含量之變化 ..................................... 62
五、 抑制酪胺酸酶活性試驗 .................................. 65
六、 石英晶體微天平 (QCM) 試驗 ........................... 68
柒、結論 ................................................ 71
捌、參考文獻 ............................................. 73
玖、附錄 ................................................ 86

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