臺灣博碩士論文加值系統

蜆、文蛤、牡蠣是台灣人最常食用的貝類，近年來在貝類發現具有
誘導細胞凋亡、抗菌、抗癌及保肝等生理活性的氧化膽固醇。而本研究
目的以 LC-MS/MS 與 GC-MS 為偵測儀器，分析蜆、文蛤、牡蠣等貝類
中的氧化膽固醇。首先將已從花蓮蜆純化的氧化膽固醇 3,5,9-trihydroxycholest-
7-en-6-one, 與 3,7,9-trihydroxy-cholest-5-ene, 以及合成的
cholesterol peroxide 與 ergosterol peroxide 為標準品，以管柱層析、TLC、
HPLC 分析，並從彰化蜆追蹤到此四個標準品。第二部份將蜆、文蛤、
牡蠣三種樣品萃取，以固相萃取法 (SPE) 快速沖提，以 GC-MS, LC-ESIMS/
MS, LC-APCI-MS/MS 分析是否含有氧化膽固醇 ergosterol peroxide,
cholesterol peroxide, 7-hydroxycholesterol, 7-ketocholesterol,
cholesteroltriol, 5,6--epoxycholesterol, 7-hydroxy--sitosterol, �n-sitosterol-
5,6-epoxide。結果發現，以 GC-MS 分析貝類中氧化膽固醇，並未偵測
到八種氧化膽固醇。以 LC-ESI-MS/MS 分析八種氧化膽固醇皆沒有出現
母離子訊號，不能近一步定性，但是皆產生片段 m/z 381.9, 437.9, 354.0
等訊號。而以 LC-APCI-MS/MS 分析貝類中氧化膽固醇，只有發現到微
量的氧化膽固醇。

The freshwater clams, clams, and oysters are popular seafood in Taiwan.
Recently, the bioactive cholesterol oxidation products (COP) have been found
in shellfish, the biological activities of COP including inducing apoptosis,
antifungal, anticancer, and hepatoprotective activities. The aims of this study
were to analyze the COP in the shellfish by LC-MS/MS and GC-MS. The four
COP standards (3,5,9-trihydroxy-cholest-7-en-6-one, 3,7,9-trihydroxycholest-
5-ene, cholesterol peroxide, and ergosterol peroxide) were used to
analyzed Chanhung freshwater clam by thin layer chromatography and high
performance liquid chromatography. To analyze the ergosterol peroxide,
cholesterol peroxide, 7-hydroxycholesterol, 7-ketocholesterol, cholesteroltriol,
5,6-epoxycholesterol, 7-hydroxy--sitosterol, �n-sitosterol-5,6-
epoxide in the shellfishes, COP were enriched by solid phase extraction, and
subsequently analyzed by GC-MS, LC-ESI-MS/MS, and LC-APCI-MS/MS.
Analyses of above 8 COPs in shellfishes by GC-MS were not succeessful,
only cholesterol, -sitosterol, stigmasterol were detcted. On the other hand, no
molecule ions peaks of COP could be detected by LC-ESI-MS/MS, all the of
the COPs provided m/z 381.9, 437.9, 354.0. Nonetheless, whern LC-APCIMS/
MS was use to determine COPs in shellfishes, the results showed that
only μg/g COP in shellfish.

摘要…………………………………………………………………………i
Abstract……………………………………………………………............ii
目錄…………………………………………………………………………I
圖目錄……………………………………………………………………IV
表目錄…………………………………………………………………VII
壹、前言……………………………………………………………………1
貳、文獻整理…………………………………………………………….…3
一、貝類中的固醇………………………….............................................3
二、氧化膽固醇簡介……………………………….................................4
1. 食品中的氧化膽固醇……………………......................……5
2. 海鮮產品中的氧化膽固醇………………….............…………7
3. 氧化膽固醇的吸收……………………..........................……8
4. 體內的氧化膽固醇……………………..........................……8
5. 氧化膽固醇的代謝………………………..........................…9
6. 氧化膽固醇的生理活性………………................…………9
7. Cholesterol peroxide 之生理活性………….............………11
8. Ergosterol peroxide 之生理活性……………...............……12
三、氧化膽固醇的分析…………..........................................…....………16
1. 氧化膽固醇的淨化……………...................................……16
2. 以 GC-MS 分析氧化膽固醇………………....................…17
3. 以 GC 分析 steroidal 5,8-peroxides…….…...........………19
4. 以 GC-MS 分析cholesterol perpxide …….....................…19
5. 以 LC-APCI-MS 分析加工食品中的氧化膽固醇…….....20
6. 以 LC-APCI-MS 分析 ergosterol peroxide………...........22
參、實驗材料與方法……………………………………………………..23
一、材料……………………………….................………………………..23
二、化學藥品……………………………………….……………………..23
三、儀器設備……………………………………….....…………………..24
四、實驗步驟與流程 …………………………………………………...26
標準品合成……………………………………………….....………..26
1. 7-Hydroxycholesterol 以及 7-hydroxy--sitosterol 的合成..26
2. 7-Ketocholesterol 的合成….................................................……29
3. 5,6-Epoxycholesterol 與 -sitosterol-5,6-epoxide 的合成...29
4. Cholestanetriol 的合成.................................................................30
5. Ergosterol peroxide 與 cholesterol peroxide 之合成...................31
6. 3,5,9-Trihydroxy-cholest-7-en-6-one 與 3,7,9-trihydroxy-cholest- 5-
ene.............................................................................................32
第一部份、追蹤蜆中氧化膽固醇……………………….......................33
一、 實驗流程圖…... .………..........…………………………….33
二、 抗癌成分 3,5,9-trihydroxy-cholest-7-en-6-one 之追蹤….34
三、 保肝成分 3,7,9-trihydroxy-cholest-5-ene 之追蹤……......37
四、 Ergosterol peroxide 於彰化蜆中的追蹤分析………….......40
五、 Cholesterol peroxide 於黃金蜆中的追蹤分析………….....43
第二部份、分析貝類中的氧化膽固醇…………………………...............46
一、 實驗流程圖………………………………………………....46
二、 樣品前處理............................................................................47
三、 貝類之固相萃取………………………………................…47
四、 以 GC-MS 分析氧化膽固醇…………………………. .47
五、 LC-ESI-MS/MS 分析氧化膽固醇…………………….….48
六、 LC-APCI-MS/MS 分析氧化膽固醇………………….….49
肆、實驗結果……………………………………………………………...51
伍、結論…………………………………………………………………62
陸、參考文獻……………………………………………………………63

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