Quercetin代謝產物在大鼠、小鼠及沙鼠體內的濃度與分佈及quercetin代謝產物對於A549 細胞增生的影響

Concentration and distribution of quercetin metabolites in rats, mice and gerbils as well as the effects of quercetin metabolites on cell proliferation in A549 cells

林沂怜(Yi-Ling Lin)

指導教授 ： 葉姝蘭 ； 莊正宏

中山醫學大學/健康管理學院/營養學研究所/碩士(2011年)

https://doi.org/10.6834/CSMU.2011.00124

摘要

許多流行病學研究發現，富含類黃酮食物的攝取量與癌症發生率呈逆相關。Quercetin是一種廣泛存在於蔬果中的類黃酮化合物，許多體外實驗顯示，quercetin具有多種生理活性，包括抑制癌細胞增生。然而，人體研究指出，由於quercetin很容易被phase II酵素代謝，因此人體血漿中多可以發現quercetin conjugated metabolites，而aglycone形式quercetin則不一定偵測到。因此，利用適當的體內模式或研究代謝產物的生理活性對瞭解quercetin真正的生理角色而言是必要的。基於上述，本研究將分兩部分進行，第一部份是比較三種動物模式Wistar大鼠、Balb/c小鼠和蒙古沙鼠，其體內quercetin代謝產物含量分布與代謝酵素的活性，以找出與人體最相似的動物模式。第二部分則是研究人體中主要quercetin代謝產物: quercetin-3-glucuronide (Q3G) 及quercetin-3′-sulfate (Q3’S)對A549肺癌細胞株增生的影響。

第一部份
此部分研究，我們比較Wistar大鼠、Balb/c小鼠及蒙古沙鼠三種動物體內quercetin主要代謝產物含量與分布。為了觀察總quercetin濃度在血漿中隨時間變化情形，動物分別經胃管餵低劑量quercetin ( 50 mg/kg body weight)或高劑量quercetin (100 mg/kg body weight)，每週一次，為時5週。接著以相同劑量進行長期管餵研究，管餵次數為每周三次，為時20週後，犧牲動物並分析血漿、肺臟及肝臟中quercetin及代謝產物含量，同時研究小腸及肝臟中phase II代謝酵素活性。在quercetin濃度隨時間變化的研究中，結果顯示三種動物血漿中總quercetin濃度大約在在攝入後quercetin第二小時最高，且蒙古沙鼠總quercetin濃度(12.2 μM)高於另外兩種動物(大鼠6.4 μM及小鼠8.1 μM )，至於三種動物血漿代謝產物分布情形，Q3G及Q3’S濃度高於isorhamnetin (methyl quercetin)及quercetin aglycone，不過，在大鼠與小鼠中，Q3G濃度高於或相似於Q3’S濃度；而沙鼠則是Q3’S濃度大於Q3G濃度。進行管餵20週後，三種動物肺及肝臟中都測得到Q3G、Q3’S、isorhamnetin以及quercetin aglycone。不論血漿或臟器，沙鼠總quercetin濃度均高於另外兩種動物。進一步，我們分析腸黏膜與肝臟中uridine-5’-diphosphate glucuronosyltransferase (UGT) 、phenolsulfotransferase (PST)及catechol-O-methyltransferase (COMT)代謝酵素活性，觀察到三種動物的酵素活性與管餵quercetin量呈劑量效應，並發現UGT活性在小腸黏膜中較高，PST及COMT則是在肝臟中活性較高。就三種動物各種酵素活性進行比較，不論小腸或肝臟，UGT的活性最高的為小鼠，而沙鼠PST活性最高，大鼠則是COMT活性比另外兩種動物高，這與前面三種動物quercetin代謝產物濃度比例趨勢一致。綜合以上，我們結果顯示，在三種動物中沙鼠體內quercetin濃度最高，且管餵兩小時後血漿中Q3G/Q3’S比值近似人類。而長期管餵後，Q3G、Q3’S及isorhamnetin累積在血漿、肺臟及肝臟中百分比與三種動物中小腸及肝臟中代謝酵素活性有關。這些研究結果，顯示沙鼠可能較另外兩種動物適合於研究quercetin體內代謝情形。

第二部份
Q3G與Q3’S為quercetin在人體血漿中主要的代謝產物，然而，此兩種代謝產物的生理活性，目前的研究有限。此部份研究，我們將人類肺癌細胞株A549與Q3G 和Q3’S分別培養，研究quercetin代謝產物對於細胞生長影響及調控peroxisome proliferator-activated receptor-gamma (PPAR-γ)與此影響的相關性。結果顯示，Q3G及Q3’S (0.5、1及5μM)可顯著抑制細胞增生，抑制效果具有時間及劑量效應，5 μΜ的Q3G與Q3’S在48及72小時顯著使細胞週期停滯於G2/M phase (p < 0.05)，比較兩者影響結果，Q3G抑制細胞增生效果較Q3’S好。我們進一步發現，Q3G及Q3’S能增加PPAR-γ和phosphatase and tensin homologue deleted on the chromosome ten蛋白（PTEN；一種抑癌蛋白）的表現量，並減少Akt的磷酸化作用。我們進一步發現，PPAR-γ拮抗劑-GW9662，會降低Q3G及Q3’S對PTEN蛋白質的表現及Akt的磷酸化。綜合以上，這部分結果顯示，Q3G及Q3’S會藉由PPAR-γ蛋白活化途徑，抑制A549細胞增生作用。

關鍵字

槲皮素 ； 槲皮素代謝產物 ； Wistar大鼠 ； Balb/c小鼠 ； 蒙古沙鼠 ； 代謝酵素

並列摘要

Epidemiologic studies have shown an inverse relationship between the dietary intake of flavonoids and cancer risk. Quercetin, a flavonoid, is found ubiquitously in vegetables and fruits. In vitro studies have shown that quercetin possesses various bioactivities including the inhibition of proliferation of cancer cells. However, conjugated metabolites of quercetin rather than quercetin aglycone are regularly present in human plasma because of its efficient phase II metabolism. Thus, it is essential to investigate quercetin using an appropriate in vivo model or to determine the bioactivities of quercetin metabolites in order to understand the real physiological roles of quercetin. Accordingly, this thesis was divided into two parts. First, the distribution of quercetin metabolites and the expression of metabolic enzymes were compared in Wistar rats, Balb/c mice and Mongolian gerbils to find out whether these animal models can be applied to humans. Second, quercetin-3- glucuronide (Q3G) and quercetin-3′-sulfate (Q3’S), two major quercetin conjugated metabolites in humans, were investigated for their effects on the proliferation of human lung cancer A549 cell line.

Part 1.
In this study, we compared the levels and the distribution of major metabolites of quercetin in Wistar rats (rats), Balb/c mice (mice) and Mongolian gerbils (gerbils). Quercetin (50 and 100 mg/kg body weight, once a week) was administrated to the animals by oral gavage for 5 weeks for time course study. Then, quercetin was administrated at the same dose 3 times/week for 20 weeks as a long-term feeding study. We determined the concentration of quercetin and its metabolites in the plasma, lungs or liver. The metabolic activities of phase II enzymes in the animal intestines and livers were also investigated. In the time course study, the results showed that the plasma concentrations of total quercetin (12.2 μM) in the gerbils were higher than in the other animals ( 6.4μM and 8.1 μM in the rats and mice, respectively). The concentrations of Q3G and Q3’S were higher than the levels of isorhamnetin (a methyl quercetin) and quercetin aglycone in all three species of animals. However, the concentrations of Q3G were higher or similar to those of Q3’S in the rats and mice; while the concentration of Q3’S was higher than that of Q3G in the gerbils, which is similar to the situation found in humans. After 20 weeks of feeding, Q3G, Q3’S, isorhamnetin, and quercetin aglycone were found in the lungs and liver of these three species of animals. The concentration of total quercetin in the plasma and tissues of the gerbils still tended to be higher than in the other animals. Although the concentrations of Q3’G were higher than the other compounds in all three species of animals, the concentration of Q3’S in the gerbils tended to be higher than the other animals. The activities of uridine- 5’-diphosphate glucuronosyltransferase (UGT), phenolsulfo
-transferase (PST) and catechol-O- methyltransferase (COMT) in the small intestine and liver in all three species of animals increased after quercetin supplementation in a dose dependent manner. UGT activity was higher in the intestinal mucosa, while PST and COMT activity was higher in the liver in all the animals. Among the three species of animals, UGT activity was highest in mouse tissues; PST activity was highest in gerbil tissues; and COMT activity was highest in rat tissues. These trends were consistent with the distribution of quercetin metabolites in animals. Taken together, our results showed that among the three species of animals, the absorption of quercetin was the highest in the gerbils, and the ratio of Q3G/ Q3’S in the gerbils plasma (after quercetin feeding for 2 h) was similar to that found in humans. The accumulation of Q3G, Q3’S and isorhamnetin in the plasma, lung and liver were associated with the activity of their individual metabolic enzyme in the small intestine and liver in all three species of animals. This study provides some evidence for choosing gerbils as an animal model for quercetin in vivo studies.

Part 2.
Q3G and Q3’S are the two main circulating quercetin metabolites present in human plasma. However, information about the bioactivities of these two metabolites is limited. In this part of our study, human A549 lung cancer cells were incubated individually with Q3G and Q3’S to investigate the effects of these metabolites on the growth of A549 cells and their association with peroxisome proliferator-activated receptor- gamma (PPAR-γ). We found that Q3G and Q3'S (0.5, 1 and 5 μM) inhibited cells growth in a time- and dose-dependent manner. The 5 μM dose of Q3G and Q3’S led to significant G2/M phase cell cycle arrest (p < 0.05) at 48 and 72 h. Q3G seemed to have a greater effect than Q3’S. These two metabolites also significantly increased PPAR-γ expression, which was accompanied by an increase in the expression of a tumor suppressor, phosphatase and tensin homologue deleted on chromosome ten (PTEN) , and a decrease in the phosphorylation of Akt, a protein involved in cellular survival pathways. Furthermore, the antagonist of PPAR-γ, GW9662, diminished the effect of Q3G and Q3’S on the expression of PTEN and phosphorylated Akt. These data suggest that Q3G and Q3’S exert antiproliferative effects in A549 cells at least in part through the activation of the PPAR-γ pathway.

並列關鍵字

quercetin-3’-sulfate ； quercetin-3-glucuronide ； animal model ； catechol-O- methyltransferase ； glucuronosyltransferase

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