臺灣博碩士論文加值系統

Quercetin是一種廣泛存在於蔬果中的黃酮醇( flavonols)，具有抗
氧化及抗發炎作用，然而卻有研究發現人體食用富含quercetin的食物
後，在其血液中並未能測到或僅能測到很低的quercetin aglycone，反
之血液中卻能測到其代謝產物，亦即其與methyl, sulfate或glucuronic
acid的接合物，因此本研究將分兩部分進行，探討給予gerbil餵食
quercetin後，其血漿或體內的的代謝產物是否能抑制benzo[a]pyrene
( BaP )或BaP＋β-carotene ( 20 μM ) 所造成的體內外傷害。
第一部份
此部份為體外試驗，利用人類肺癌細胞株A549為細胞模式。Quercetin
( 100mg/kgw/week ) 餵食gerbil 之後，收集富含quercetin代謝產物之
血漿( QP )，單獨或合併β-carotene預培養4小時，再經香菸中致癌成
份benzo[a]pyrene ( BaP )刺激24小時，QP中含有多種quercetin代謝產
物，其中包含了quercetin-3-glucuronide, quercetin-3'-sulfate及
methylquercetin，QP是在gerbil管餵quercetin 2小時後進行採血所獲
得，血漿中quercetin代謝產物顯著的增加，總濃度約為5 μM。細胞培
養結果顯示，QP可顯著的降低BaP單獨或合併β-carotene所造成的細
胞死亡、細胞DNA斷裂情形，且其效果與2 μM及5 μM quercetin的功
用相似，QP也能降低細胞經BaP或BaP＋β-carotene誘發的c-Jun的磷酸
化作用。
第二部份
以gerbil為實驗model，單獨或合併管餵β-carotene ( 10 mg/kg bw、3 次
/week )、quercetin ( 100 mg/kg bw、3 次/week )及BaP ( 8 μmole/次、2
次/week ) 6個月，犧牲動物並進行各項分析。收集bronchoalveolar
lavage fluid ( BALF )檢測總細胞數、 macrophages、lymphocytes及
neutrophils數目，利用ELISA檢測BALF中TNF-α ( tumor necrosis
factor-α )及IL-1β ( Interleukin-1β ) 的含量，並利用Western blot分析肺
臟中c-Jun及JNK ( Jun N-terminal kinase )蛋白表現的變化。結果顯示
BaP可顯著的增加BALF免疫細胞的聚集及促發炎激素的含量，另外
血漿中促發炎激素的含量亦會增加，給予β-carotene會促進BaP此一效
果，相反的，管餵quercetin可顯著的降低BaP誘發的免疫細胞的聚集，
及TNF-α及IL-1β的表現，在肺臟中quercetin對於BaP或BaP
+ β-carotene所誘發的c-Jun及JNK磷酸化作用也有明顯的抑制作用，此
表示quercetin經代謝後亦能抑制β-carotene對BaP誘發IL-1β、TNF-α及
JNK、c-Jun磷酸化的促進作用。
縱合以上我們的實驗證實，Quercetin經代謝後能仍對抗BaP或BaP
+ β-carotene所誘發的DNA傷害，顯示quercetin的代謝產物能與
β-carotene產生交互作用，其機制與向下調節JNK及Jun的活化有關。

Quercetin, a flavonoid, is found ubiquitously in the vegetables and
fruits. It possesses antioxidant and anti-inflammatory activity in vitro.
However, the conjugated metabolites rather than quercetin aglycone
present in blood in human after consumption of foods rich in quercetin.
Whether the metabolites of quercetin possess the beneficial effects as
their parent compound remains unclear. Therefore, this thesis work was
divided into two parts to investigate whether the metabolites of quercetin
present in plasma of gerbils, which were fed with quercetin by gavage,
prevent the harmful effects of bezo[a]pyrene ( BaP ) or BaP+β-carotene.
Part 1.
In the ex vivo study, the A549 cells were preincubated with quercetin or
the quercetin-metabolite-enriched plasma ( QP; 10% ), alone or combined
with 20 μM β-carotene for 4 h, and then, the cells were treated with 20
μM of BaP for 24 h. The QP was obtained from gerbils at 2 h after fed
with quercetin at 100mg/kgw/week, while the control plasma was
obtained from gerbils administered vehicle only. Several quercetin
metabolites were observed in QP, including quercetin-3-glucuronide,
quercetin-3'-sulfate and methylquercetin. At 2 h after oral administration
of quercetin, the total quercetin concentration of the plasma in gerbils was
significantly increased and reached to a maximum ( ~ 5 μM ). QP
significantly increased the cell viability and decreased DNA damage in
A549 cells incubated with BaP or BaP+ β-carotene, and the efficiency of
QP was similar to those of quercetin at 2 and 5 μM. QP also significantly
decreased the ratio of phosphor-Jun/total Jun in A549 cells incubated with
BaP or BaP+ β-carotene.
Part 2.
In the in vivo study, we fed gerbil by gavage with β-carotene ( 10 mg/kgw,
3 time/week ), quercetin ( 100 mg/kgw, 3 time/week ) and BaP
( 8 μmole/time, 2 time/week ) alone or combined for 6 months. After
sacrificed, bronchoalveolar lavage fluid ( BALF ) and plasma were
collected to determine cell profile or/and the levels of pro-inflammatory
cytokine, TNF-α ( tumor necrosis factor-α ) and IL-1β ( Interleukin-1β ).
The activation of JNK ( Jun N-terminal kinase ) and c-Jun proteins in
lung was also determined by western blotting. The results showed BaP
significantly increased the numbers of total cell and the macrophage as
well as the levels of pro-inflammatory cytokine in BALF. β-carotene
-administration significantly enhanced such an effect; in contrast,
quercetin- administration significantly decreased the pro-inflammatory
effects of BaP or BaP+ β-carotene. BaP also significantly increased the
ratio of phospho-JNK/total JNK and that of phospho-Jun/total Jun in lung
in gerbils. Similarly, β-carotene-administration significantly enhanced
such an effect while quercetin- administration had a significant
suppressed effect.
The results of these two parts studies demonstrated that the
metabolites of quercetin possess the bioactivity for protect cells against
the damages of BaP or BaP + β-carotene, suggesting that they may
interact with β-carotene. The mechanisms were associated with the
down-regulation of the activation of JNK and Jun.

縮寫表……………………………………………………………..……Ⅰ
摘要………………………………………………………………..……Ⅲ
英文摘要…………………………………………………………..……Ⅴ
一、前言……………………………………….……………………......1
1. 緒論……………………………………………………………..…....1
2. 檞黃酮 ( quercetin )……………………………………...……..…….2
3. β-胡蘿蔔素( β-carotene ) ……………….……………………………6
4. Benzo[a]pyrene ( BaP )…..…………………..………………..……..8
5. Mitogrn-activated-protein kinases ( MAPKs )………..….……..…...10
6. 發炎反應………………………………………………………..…...12
7. 研究目標……………………………………………………….…....14
8. 實驗架構………………………………………………………….…15
二、研究方法
1. 材料
(1) 儀器……………………………………………………………...17
(2) 藥品……………………………………………………………....18
2.方法
2.1 細胞株來源及培養條件...............................................................20
2.2 細胞的處理....................................................................................20
2.3 細胞生長分析...............................................................................21
2.4 DNA 傷害分析..............................................................................22
2.5 Gerbil 血漿中Quercetin 含量分析..............................................23
2.6 西方墨點法...................................................................................24
2.7 Brochoalveolan Lavage…..............................................................27
2.8 cytokines 分析................................................................................28
2.9 統計方法.......................................................................................28
三. 結果與討論
1.第一部份結果.......................................................................................29
2.第一部份討論.......................................................................................32
圖一. gerbil 經管餵quercetin 後，其血漿的組成….............................34
圖二. quercetin 在gerbil體內的濃度隨時間改變的變化量...................35
圖三. quercetin 在gerbil 體內長期累積情形.........................................36
圖四. quercetin 及其代謝產物對BaP 誘發的細胞死亡的影響.............37
圖五. quercetin 及其代謝產物合併β-carotene 對BaP誘發的細胞死亡的
影響................................................................................................38
圖六. quercetin 及其代謝產物對BaP 誘發的細胞DNA 傷害的
影響..............................................................................................39
圖七. quercetin 及其代謝產物合併β-carotene 對BaP 誘發的細胞DNA
傷害的影響....................................................................................40
圖八. quercetin 及其代謝產物單獨或合併β-carotene 對BaP誘發的細胞
Jun 蛋白表現的影響......................................................................41
1.第二部份結果.......................................................................................42
2.第二部份討論.......................................................................................44
圖九. 灌食營養素及BaP 對gerbil 體重之影響…...............................47
表一. Cell profile in bronchalveolar lavage fluid…..............................48
圖十. gerbil 經BaP 刺激細胞激素TNF-α之表現..............................49
圖十一. gerbil 經BaP 刺激細胞激素IL-1β之表現...............................50
圖十二. gerbil 經BaP 刺激肺細胞Jun 蛋白表現的影響......................51
圖十三. gerbil 經BaP 刺激肺細胞JNK 蛋白表現的影響......................52
四. 結論...................................................................................................53
五.參考文獻.............................................................................................54

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