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Cytochrome P4501A1/1A2 表現之抑制作用

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

林曉君(Hsiao-Chun Lin)

指導教授 ： 葉姝蘭

中山醫學大學/健康管理學院/營養學研究所/碩士(2009年)

https://doi.org/10.6834/CSMU.2009.00050

摘要

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。

關鍵字

quercetin ； quercetin -3-glucuronide ； quercetin -3’-sulfate ； isorhamnetin

並列摘要

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.

並列關鍵字

quercetin ； quercetin -3-glucuronide ； quercetin -3’-sulfate ； isorhamnetin

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