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

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.