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研究生:林曉君
研究生(外文):Hsiao-Chun
論文名稱:各種quercetin代謝產物對Benzo[a]pyrene單獨或合併β-carotene誘發的A549細胞傷害及CytochromeP4501A1/1A2表現之抑制作用
論文名稱(外文):The inhibiting effects of the metabolites of quercetin on the cell damage and the expression of cytochrome P4501A1/1A2 induced by benzo[a]pyrene alone or in combination with Β-carotene in A549 cells
指導教授:葉姝蘭
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:營養學研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:65
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Quercetin 是蔬果當中所富含的一種類黃酮,體外研究顯示quercetin 可能具有抗癌的作用。我們先前的研究指出quercetin會減少β-carotene (BC)在BaP作用的促傷害是透過抑制Cytochrome P450 (CYP) 1A2的表現。然而在人體中quercetin會快速的被phase II 酵素代謝,因此血漿中quercetin 含量相當低(或不存在),而多以接合的代謝產物存在,但這些代謝產物的生理功能還不清楚。因此,本實驗利用人類肺癌細胞株A549細胞探討在BaP的作用下,各種quercetin代謝產物或混合(Q3G:Q3’S: I /2:2:1)與BC之間的相互作用。將A549細胞以quercetin -3-glucuronide (Q3G)、quercetin -3’-sulfate (Q3’S)及isorhamnetin (I)分別以2,5,10 μM單獨或合併20 μM β-胡蘿蔔素(BC)預培養4小時後,再以20 μM benzo[a]pyrene (BaP)刺激後,進行細胞存活分析、DNA斷裂分析、西方墨點法分析cytochrome P450 (CYP) CYP1A1/1A2的表現及測定細胞內ROS分泌量,並與quercetin進行比較。
另外 我們探討quercetin的代謝產物對於在Fe/NTA氧化下BC消耗的影響。而結果顯示,quercetin的代謝產物個別均對BaP或BaP+BC誘發的細胞死亡及DNA傷害有顯著的抑制效果且與quercetin效果相似。
Quercetin的代謝產物也會減少BaP或BaP+BC誘發的CYP1A1/1A2表現其中也以Q3G與Q3’S效果較好,然而只有10 μMQ3’S及Q3G有抑制BaP刺激的ROS生成。而Q3G及quercetin減少在Fe/NTA作用下BC的耗損有相似的效果。更進一步,發現三種代謝產物混合對於在BaP或BaP+BC作用下有抑制細胞傷害或減少CYP1A1/1A2表現。

以上結果證實,Quercetin的代謝產物Q3G、Q3’S、I均會抑制BaP或BaP+BC誘發的細胞傷害及減少CYP1A1/1A2表現且與quercetin相似或更好,而只有Q3G減少在氧化作用下BC的耗損,這表示quercetin的代謝產物與BC有間接或直接的交互影響。
Abstract
Quercetin, a flavonoid, is found ubiquitously in vegetables and fruits. In vitro studies show that quercetin may possess anticancer activity. In our previous study, the results show that quercetin attenuated the harmful effect of β-carotene (BC) induced by benzo[a]pyrene (BaP) through suppressing the expression of cytochrome P450 (CYP) 1A2. However, quercetin aglycone is little (or not) present in the plasma of humans due to its efficient phase II metabolism. Regarding the bioactivities of the conjugated metabolites of quercetin, little has been known. Thus, in this study we used A549 cells, a human lung cell line, to investigate the interaction between each metabolites of quercetin or the mixture (Q3’S: Q3G: I/2:2:1) and BC in the presence of BaP. A549 cells were pre-incubated with 2, 5 or 10 μM quercetin -3-glucuronide (Q3G), quercetin-3’-sulfate (Q3’S) and isorhamnetin (I) alone or in combination with 20 μM BC for 4 hours, followed by incubation with 20 μM benzo[a]pyrene (BaP) for 24 hours. Then, the cell viability, DNA damage, the expression of CYP 1A1/1A2 and the production of intracellular reactive oxygen species (ROS) were examined. We also compared the results with that of quercetin. In addition, we investigate the preventive effect of those quercetin metabolites on the consumption of BC induced by the oxidant, Fe/NTA. The results showed Q3G, Q3’S and I significantly inhibited the cell death and DNA damage induced by BaP or BaP+BC. The efficiencies of those metabolites were similar to that quercetin itself. Those metabolites also decreased the expression of CYP1A1/1A2 induced by BaP or BaP+BC in the order Q3’S, Q3G≧quercetin, I. However, only Q3G and Q3’S at 10 μM decreased the production of ROS induced by BaP. Q3G and quercetin had a similar effect on decreasing the consumption of BC induced by Fe/NTA. Furthermore, we found the mixture of quercetin metabolites tended to additively decrease the cytotoxicity and the expression of CYP1A1/1A2 induced by BaP or BaP+BC.

Taken together, our results showed that Q3G, Q3’S and I could inhibit cell death, DNA damage and the expression of CYP1A1/1A2 induced by BaP or BaP+BC, and the efficiencies were similar to or better than that of quercetin itself. However, only Q3G significantly decreased the consumption of BC induced by the oxidant, implying that those metabolites of quercetin may interact with BC directly or indirectly.
目 錄
縮寫表……………………………………………………………………I
中文摘要………………………………………………………………III
英文摘要…………………………………………………………………V
一、前言……………………………………………………………1
(一)緒論…………………………………………………………………1
(二)文獻探討……………………………………………………………3
1.Quercetin……………………………………………………………3
2. β–胡蘿蔔素…………………………………………………………8
3. Benozo[a]pyrene (BaP)……………………………………………11
4. Cytochrome P450 (CYP)……………………………………………14
5.ROS (Reactive oxygen species, 活性氧)………………………16
6.研究目標……………………………………………………………17
7.實驗架構……………………………………………………………18
二、研究方法………………………………………………………20
(一)材料………………………………………………………………20
1. 儀器…………………………………………………………………20
2. 藥品…………………………………………………………………21
(二)方法………………………………………………………………24
1. 細胞培養……………………………………………………………24
2. 細胞處理……………………………………………………………24
3. 製備quercetin-3-glucuronide……………………………………24
4. 製備quercetin-3''-sulfate(Q3''S)………………………………25
5. 鑑定Q3’S及Q3G………………………………………………….25
6. 細胞存活率分析……………………………………………………26
7. DNA 傷害分析…………………………………………………26
8. 西方墨點法分析……………………………………………………27
9. 測定細胞中reactive oxygen species (ROS)的含量……………30
10. β-胡蘿蔔消耗分析………………………………………………30
11. 統計分析…………………………………………………………31
三、結果與討論……………………………………………………32
1.結果……………………………………………………………32
2.討論……………………………………………………………36
四、結論…………………………………………………………58
五、參考文獻………………………………………………………59














圖表目錄
圖一、BaP的活化代謝過程……………………………………………12
圖二、Quercetin-3-glucuronide純化後HPLC-UV及LC/MS分析之圖譜………………………………………………………………………39
圖三、Quercetin-3’-sulfate 純化後HPLC-UV及LC/MS 分析之圖譜………………………………………………………………………40
圖四、各種quercetin代謝產物對 Benzo[a]pyrene單獨誘發 A549細胞生長的影響…………………………………………………………41
圖五、各種quercetin代謝產物對 Benzo[a]pyrene合併β-carotene (BC)誘發 A549細胞生長的影響………………………………………42
圖六、各種quercetin代謝產物對 Benzo[a]pyrene單獨誘發 A549細胞DNA斷裂的影響……………………………………………………43
圖七、各種quercetin代謝產物對 Benzo[a]pyrene合併β-carotene (BC)誘發 A549細胞DNA斷裂的影響……………………………………44
圖八、各種quercetin代謝產物對 Benzo[a]pyrene單獨誘發 A549細胞CYP1A1的表現……………………………………………………45
圖九、各種quercetin代謝產物對 Benzo[a]pyrene合併β-carotene (BC)誘發 A549細胞CYP1A1的表現……………………………………46
圖十、各種quercetin代謝產物對 Benzo[a]pyrene單獨誘發 A549細胞CYP1A2的表現……………………………………………………47
圖十一、各種quercetin代謝產物對 Benzo[a]pyrene合併β-carotene (BC)誘發 A549細胞CYP1A2的表現…………………………48
圖十二、各種quercetin代謝產物對 Benzo[a]pyrene刺激後對ROS分泌之影響………………………………………………………………49
圖十三、各種quercetin代謝產物對β-carotene耗損之影響………50
圖十四、Quercetin代謝產物混合後對 Benzo[a]pyrene單獨或合併β-carotene (BC) 誘發 A549細胞生長的影響……………………51
圖十五、Quercetin代謝產物混合後對 Benzo[a]pyrene單獨或合併β-carotene (BC) 誘發 A549細胞DNA斷裂的影響………………52
圖十六、quercetin代謝產物混合後對 Benzo[a]pyrene單獨或合併β-carotene (BC)誘發 A549細胞CYP1A1的表現…………………53
圖十七、Quercetin代謝產物混合後對 Benzo[a]pyrene單獨或合併β-carotene (BC)誘發 A549細胞CYP1A2的表現…………………54
圖十八、Quercetin代謝產物混合後對 Benzo[a]pyrene刺激後對ROS分泌之影響……………………………………………………………55
圖十九、Quercetin代謝產物混合對β-carotene耗損之影響………56
表一、quercetin三種代謝產物混合效果的比較……………………57
五、參考文獻
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1. Quercetin代謝產物抑制benzo[a]pyrene單獨或合併β-胡蘿蔔素所誘發的傷害:exvivo及體內試驗
2. 各種quercetin代謝產物對Benzo[a]pyrene單獨或合併β-carotene誘發的A549細胞傷害及CytochromeP4501A1/1A2表現之抑制作用
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