Quercetin intake from vegetables has been associated with a lower risk of cardiovascular diseases, although the underlying mechanisms are incompletely understood [28
]. Quercetin is rapidly metabolized in the human intestinal mucosa and liver into glucuronide and sulphate conjugates with or without methylation (). After consumption of quercetin-rich foods, quercetin metabolites are largely associated with the albumin-containing fraction of human plasma [29
]. Interestingly, quercetin was detected (after deconjugation) in the aortas of cholesterol-fed rabbits supplemented with quercetin glucosides [30
]. In line with this observation, immunohistochemical studies with monoclonal antibodies raised against quercetin conjugates demonstrated that quercetin glucuronides specifically accumulate in human atherosclerotic lesions, but not in non-lesion aortic areas, and the staining was associated with macrophage-derived foam cells [31
]. These studies suggest that quercetin metabolites can penetrate into the vascular wall and accumulate in atherosclerotic plaque and, hence, may interact with the vascular endothelium.
In this work, we hypothesized that the in vivo effects of dietary quercetin on endothelial cells are modulated by chemical modifications of the quercetin molecule. Therefore, we studied the effects of different O-methylated isomers of quercetin, 3′- and 4′-O-methyl-quercetin, and two additional conjugates with in vivo relevance, quercetin-3-O-glucuronide and quercetin-3′-O-sulphate, on the expression of inflammatory proteins in HAEC. Because quercetin and its metabolites are likely to associate with albumin and other plasma proteins and, furthermore, may undergo enterohepatic circulation, we performed our experiments in high-serum medium (20% FBS) and exposed HAEC to the quercetin metabolites for 18 h prior to the addition of TNFα.
We first studied 3′-O
-methyl-quercetin, commonly known as isorhamnetin, and 4′-O
-methyl-quercetin, to evaluate whether O
-methylation affects the biological activity of quercetin. We found that these methylated quercetin derivatives inhibited TNFα-induced E-selectin and ICAM-1 expression as effectively as the quercetin aglycone. Therefore, it can be speculated that O
-methylation of quercetin by COMT would not affect the potential of dietary quercetin to attenuate vascular endothelial inflammation in vivo
. We have shown previously that the basic A and C-ring flavone structure (see ) is critical for the inhibition of adhesion molecule expression in HAEC, but not the B-ring catechol group [25
], consistent with the results presented here for quercetin and its methyl derivatives. In contrast, the B and C-ring hydroxyl groups were critical for the reducing activity of the molecule [25
], again in agreement with the data presented here. Hence, O
-methyl substitutions in the catechol group of quercetin did not affect its anti-inflammatory activity but lowered its antioxidant capacity.
In contrast to 3′- and 4′-O-methyl-quercetin, quercetin-3-O-glucuronide and quercetin-3′-O-sulphate did not inhibit TNFα-mediated inflammatory responses in HAEC, even when tested at supra-physiological concentrations. Hence, a novel observation of our work is that the biological activity of quercetin with respect to adhesion molecule expression is dependent on the chemical nature of the substituent, not just the position in the molecule that is modified. For example, we found that 3′-O-methylation did not affect the biological activity of quercetin, while 3′-O-sulphation abolished it. Both molecules, however, exhibited similar antioxidant activity, which was lower than the antioxidant activity of quercetin due to lack of the catechol group.
Our data also indicate that HAEC do not have the ability to hydrolyze the metabolites to the active quercetin aglycone, as has been suggested for other cell types [32
]. The introduction of bulky or charged groups and the resulting increased hydrophilicity may impair the ability of quercetin-3-O
-glucuronide and quercetin-3′-O
-sulphate to reach the appropriate intracellular target(s) in HAEC. This notion is supported by the observation that quercetin-3-O
-glucuronide does not seem to be taken up to a significant degree by PC-12 cells [33
]. In contrast, murine macrophages have been shown to accumulate quercetin-3-O
-glucuronide and metabolize it to the much more active aglycone and its methylated form [31
]. In vitro
studies using large unilaminar vesicles have shown that quercetin-3-O
-glucuronide can interact with phospholipid bilayers with low, but significant, affinity, suggesting that an interaction with cell membranes may also be possible [34
While our data indicate that quercetin-3-O
-glucuronide and quercetin-3′-O
-sulphate cannot inhibit inflammatory responses in HAEC, previous work has suggested that these quercetin metabolites may exert relevant biological effects in cells or endothelium. For example, Tribolo et al
] reported that quercetin-3′-O
-glucuronide and 3′-O
-glucuronide inhibited VCAM-1 cell surface expression at concentrations as low as 2 μM. Furthermore, quercetin-3-O
-glucuronide was effective in preventing endothelial dysfunction induced by endothelin-1, and both quercetin-3-O
-glucuronide and quercetin-3′-O
-sulphate inhibited NADPH oxidase-dependent superoxide production in thoracic aortic rings from rats [36
-glucuronide also inhibited angiotensin II-induced hypertrophy in vascular smooth muscle cells, which was attributed in part to its inhibitory effect on the JNK/AP-1 signaling pathway [37
]. The use of different cell types and experimental conditions may account, in part, for these discrepant results.
Our results are, however, in agreement with those reported by Mochizuki et al
] showing that quercetin-3-O
-glucuronide did not inhibit TNFα-induced expression of ICAM in HAEC. Interestingly, these authors also found that inflammation triggered by interleukin-1α resulted in increased cell permeability of quercetin-3-O
-glucuronide, suggesting that it could pass through the endothelium to reach the underlying vascular smooth muscle cells. Taking all our results into account, it can be speculated that the major in vivo
-glucuronide, would be inactive in our model, not because of the biotransformation of the catechol group but because of the glucuronidation in position C3. If intracellular hydrolysis occurred in HAEC in vivo
, the resulting 3′-O
-methyl-quercetin would be an intracellular ‘active’ metabolite. However, non-glucuronidated, O
-methylated quercetin cannot be detected in human plasma, and there is no evidence for hydrolysis of quercetin glucuronides in endothelial cells.
We previously reported that flavonols and flavones were able to inhibit E-selectin and ICAM-1 expression in HAEC, whereas flavanones or flavan-3-ols were not, due to lack of the basic A and C-ring flavone structure [25
]. Accordingly, caffeic acid, which contains a catechol group but lacks a basic flavonoid structure, and–as reported here–phenolic acid metabolites of quercetin were unable to inhibit adhesion molecule expression in HAEC. However, in-vitro
anti-inflammatory effects of phenolic acids have been recently reported. Differences in cell types and inflammatory challenges may account for some of these seemingly discrepant results. For instance, it has been reported that phenolics such as hydrocaffeic, dihydroxyphenyl acetic and hydroferulic acids were able to inhibit interleukin-1β-induced prostaglandin E(2) production by CCD-18 colon fibroblast cells [39
]. The hydroxylated phenolic acids, 3,4-dihydroxyphenylpropionic acid and 3,4-dihydroxyphenylacetic acid, were also able to inhibit lipopolysaccharide-stimulated cytokine release from isolated peripheral blood mononuclear cells [40
], in contrast to our observations in TNFα-exposed HAEC. In general, studies using endothelial cells are scarce. However, Moon et al
] reported that caffeic acid reduced TNFα-induced adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) by inhibiting NF-κB activation. While some of their experimental conditions were similar to ours, e.g
., 18-h pre-incubation with caffeic acid followed by 6-h incubation with TNFα, other conditions differed significantly and may have accounted for the discrepancies in results, e.g.
, use of HUVEC vs
. HAEC and 0.2% vs
. 20% FBS in cell culture media. Our work has consistently found that none of the phenolic acids tested inhibited TNFα-mediated adhesion molecule expression in HAEC, supporting the notion that a basic hydroxyflavone structure is required for activity in this experimental system.
Catechins are widely distributed in the human diet, in particular tea, wine, grapes and cocoa. Considering that tea is the second most consumed beverage in the world after water [42
], catechin consumption by humans can be significant. One catechin, EGCG, is of particular interest because of its numerous biological effects. Although the metabolic transformation of catechins in humans is well understood, relatively little is known about the biological effects of catechin metabolites. It has been reported, for instance, that O
-methylated derivatives of (−)-epicatechin are good inhibitors of peroxynitrite-mediated nitrotyrosine formation [44
]. In intact cells, 3′-O
-methyl-epicatechin inhibited cell death induced by hydrogen peroxide through inhibition of caspases [45
-methyl-epicatechin also was shown to attenuate UVA-induced oxidative damage in human skin fibroblasts [46
]. Interestingly, it was recently reported that HUVEC have the capacity to convert (−)-epicatechin into methyl derivatives, which inhibited NADPH oxidase activity [47
]. In this work, we studied the effect of EGCG–the main catechin in green tea–and two of its in vivo
-methyl-EGCG and 4′,4″-di-O
-methyl-EGCG. EGCG is one of the few dietary flavonoids that have been detected in plasma without further modification, but it is also known that EGCG undergoes extensive biotransformation (see ). Both EGCG and their methylated metabolites are substrates and potent inhibitors of hepatic COMT, suggesting they might have biological activity [48
To investigate EGCG and its metabolites in vitro
, we applied a protocol compatible with EGCG’s in vivo
pharmacokinetic behavior. Because of the much shorter half-life in human plasma of EGCG than quercetin derivatives, HAEC were exposed to EGCG and its metabolites for only 1 h prior to the addition of TNFα. Neither EGCG nor its metabolite 4″-O
-methyl-EGCG inhibited adhesion molecule or MCP-1 expression. In contrast, 4′,4″-di-O
-methyl-EGCG was effective in selectively inhibiting ICAM-1 expression. However, 4′,4″-di-O
-methyl-EGCG was far less effective than quercetin or 3′-O
-methyl-quercetin in our experimental model. Due to the high concentrations required for inhibition of ICAM-1 expression by 4′,4″-di-O
-methyl-EGCG, the physiological relevance of these findings is doubtful, unless high local concentrations accumulate in target cells and tissues, as has been described for quercetin metabolites [31
In addition, further intracellular biotransformation of flavonoid metabolites is possible. Kawai et al. [31
] showed that isolated macrophages can metabolize quercetin glucuronides to quercetin aglycone and methylated quercetin, even in the absence of an inflammatory challenge. Notably, methylated quercetin was the active metabolite, as inhibition of COMT blocked the biological effect of the quercetin glucuronides [31
]. Similarly, Steffen et al. [47
] showed that HUVEC can metabolize (−)-epicatechin to mono-O
-methylated epicatechin, which was the metabolite responsible for NADPH oxidase inhibition. Interestingly, demethylation of flavonoids in human cells also may occur, as has been reported for biochanin A (4′-O
-methyl genistein) in cancer cells [50
]. In this work, we didn’t pursue the identification of intracellular metabolites, but it is possible that the combined effect of selective cellular uptake and intracellular metabolism contributed to our results.
NF-κB activation is the main transcription factor mediating TNFα-induced expression of inflammatory genes [51
]. Our previous results indicated that pharmacological inhibitors of NF-κB completely abolished expression of adhesion molecules in HAEC. However, the active flavonoid metabolites studied here were unable to inhibit NF-κB activation, suggesting that they act through (an) alternative mechanism(s), for example, stimulation of the Nrf2 pathway [52
We have previously shown that the reducing activity of flavonoids is not related to their capacity to inhibit adhesion molecule expression in HAEC [25
]. In this paper, we also measured the ferric reducing activity of the individual compounds and metabolites. As expected, 3′-O
-methyl-quercetin showed lower reducing activity than quercetin, confirming that the catechol group is as an important structural feature. Interestingly, quercetin-3′-O
-sulphate also exhibited good reducing activity, despite its lack of an effect on adhesion molecule expression, confirming that the antioxidant capacity of an individual compound is not relevant for its anti-inflammatory effect. Other authors have also shown that metabolites of quercetin can retain, at least in part, the antioxidant capacity of the quercetin aglycone. For instance, the conjugated metabolite, quercetin-3-O
-glucuronide, effectively prevented peroxynitrite-induced depletion of lipophilic antioxidants in isolated human low-density lipoproteins [53
-glucuronide also inhibited lipid peroxidation of liposome membranes induced by iron, peroxynitrite or lipoxygenase, although the metabolite was less effective than quercetin [34
]. We also compared the reducing activity of EGCG with that of 4′,4″-di-O
-methyl-EGCG and found that the latter was much less active than EGCG, despite being more effective at inhibiting adhesion molecule expression. These results indicate a significant role of the 4′- and 4″-hydroxyl groups for the antioxidant capacity of EGCG, but also highlight that the antioxidant capacity cannot predict the biological effect in this model.
In summary, our work shows that chemical modifications of dietary flavonoids – resulting in formation of different in vivo metabolites – can significantly alter their biological and antioxidant activities. While glucuronidation and sulphation abolished the inhibitory effect of quercetin on adhesion molecule expression, methylation preserved its anti-inflammatory activity. In contrast, 4′,4″-di-O-methyl-EGCG dose-dependently inhibited TNFα-induced expression of ICAM-1 but not other adhesion molecules, while EGCG was ineffective. Thus, the study of the biological responses to physiologically relevant forms and concentrations of circulating flavonoids is pivotal for the proper evaluation of the potential health benefits of dietary flavonoids, considering that the biological activities of the metabolites can be neither predicted nor extrapolated from their dietary forms. In vitro studies of flavonoid glycosides or aglycones are unlikely to be relevant to biological or health effects of flavonoids in humans, with the possible exception of effects in the gastrointestinal tract.