Bioavailability of prenyl quercetin

Prenyl flavonoids are widely distributed in plant foods and have attracted appreciable attention in relation to their potential benefits for human health. Prenylation may enhance the biological functions of flavonoids by introducing hydrophobic properties in their basic structures. Previously, we found that 8-prenyl naringenin exerted a greater preventive effect on muscle atrophy than nonprenylated naringenin in a mouse model. Here, we aimed to estimate the effect of prenylation on the bioavailability of dietary quercetin (Q). The cellular uptake of 8-prenyl quercetin (PQ) and Q in Caco-2 cells and C2C12 myotube cells was examined. Prenylation significantly enhanced the cellular uptake by increasing the lipophilicity in both cell types. In Caco-2 cells, efflux of PQ to the basolateral side was <15% of that of Q, suggesting that prenylation attenuates transport from the intestine to the circulation. After intragastric administration of PQ or Q to mice or rats, the area under the concentration-time curve for PQ in plasma and lymph was 52.5% and 37.5% lower than that of Q, respectively. PQ and its O-methylated form (MePQ) accumulated at much higher amounts than Q and O-methylated Q in the liver (Q: 3400%; MePQ: 7570%) and kidney (Q: 385%; MePQ: 736%) of mice after 18 d of feeding. These data suggest that prenylation enhances the accumulation of Q in tissues during long-term feeding, even though prenylation per se lowers its intestinal absorption from the diet

Mitochondrial Dysfunction Leads to Deconjugation of Quercetin Glucuronides in Inflammatory Macrophages

<div><p>Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-<i>O</i>-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. <i>In vitro</i> experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that β-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of β-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the β-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular β-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides <i>in vivo</i> in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites.</p> </div

Mitochondrial quality control by autophagy is involved in the deconjugation.

<p>(A) Effects of Atg7 knockdown on the β-glucuronidase activity in the medium. (B) Effects of Atg7 knockdown on the intracellular calcium ion levels determined by Fluo4-AM probe. In <i>A</i> and <i>B</i>, RAW264 cells were transfected with siRNA for control or Atg7 and after 2 days, the cells were treated with AMA for 3 h. (C) Confirmation of siRNA knockdown of Atg7 protein by immunoblot analysis of the cell lysates used in <i>A</i> and <i>B</i>. (D-F) The β-glucuronidase activity in the cultured medium of RAW264 cells during treatment with or without chemicals: D, rapamycin (1 μM) for 0-24 h; E, carbonyl cyanide-<i>p</i>-trifluoromethoxyphenylhydrazone (FCCP, 20 μM) for 6 h; F, bafilomycin A₁ (Baf, 200 nM) for 6 h. In D, data points represent duplicate determinations. Data in all bar graphs are presented as the average ± S.D. (n=3).</p

Accumulation and deconjugation of Q3GA in macrophage cells.

<p>(A) HPLC-ECD analysis of quercetin derivatives in various cell lines (upper) and in mouse peritoneal macrophages (lower) treated with Q3GA (20 μM) for 8 h. Q, quercetin; MeQ, 3’- or 4’-<i>O</i>-methylquercetins. Asterisks indicate the peaks corresponding to Q or MeQ. ND, not detected. (B) Effects of inhibitors on accumulation of quercetin and methylquercetins during the treatment of RAW264 cells with Q3GA. <i>Top</i>, no inhibitor; <i>middle</i>, COMT inhibitor 3,5-dinitrocatechol; <i>bottom</i>, β-glucuronidase inhibitor d-saccharic acid 1,4-lactone. Data points represent duplicate determinations. (C) HPLC-ECD analysis of quercetin formed by the deconjugation of Q3GA in the cell-free conditioned medium. The conditioned medium was prepared after culturing each cell line for 24 h, and incubated with Q3GA (50 μM) at 37 °C for 1 h. (D) Relative enzyme activity of β-glucuronidase (left) and sulfatase (right) in the medium during culturing RAW264 cells, determined using 4-methylumbelliferyl substrates. Data in all bar graphs are presented as the average ± S.D. (n=3). Asterisks indicate a significant difference (<i>p</i> < 0.05). NS, not statistically significant.</p

Deconjugation is essential for anti-inflammatory actions of quercetin glucuronide.

<p>(A) Effects of quercetin on the LPS-induced expression of COX-2 protein (upper) and mRNA (lower) in the RAW264 cells after 4 h. The mRNA expression of COX-2 was determined by quantitative real-time RT-PCR (GAPDH as an endogenous control). A hash symbol (#) indicates a significant difference (<i>p</i> < 0.05 vs control). Asterisks (*) indicate a significant difference (<i>p</i> < 0.05 vs the LPS treatment group). Data points represent the average ± S.D. (n=3). (B) Upper, effects of Q3GA on the LPS-induced expression of COX-2 protein (β-actin as an endogenous control) in the RAW264 cells after 4 h. Lower, effects of Q3GA (100 μM) on the LPS-induced COX-2 expression in the presence of β-glucuronidase enzyme (1 μg/ml). (C) Effects of Q3GA (100 μM) on the LPS-induced COX-2 expression in the RAW264 cells in the fresh (FM) or conditioned medium (CM). Conditioned medium was collected from 24-h cultures of the RAW264 cells. (D) Effects of Q3GA on the LPS-induced COX-2 expression in the CM in the presence of β-glucuronidase inhibitor. Experimental condition was same as <i>C</i>. (E) The inhibitory effect of quercetin on the LPS-induced COX-2 expression in the RAW264 cells was enhanced in the presence of the COMT inhibitor. Cells were incubated with LPS and quercetin in the absence or presence of the COMT inhibitor (3,5-dinitrocatechol, 10 μM) for 4 h.</p

Quercetin, but not Q3GA, inhibited LPS-induced JNK activation.

<p>(A and B) Effects of quercetin on the activation of MAP kinases (A) and NFκB pathway (B) in the RAW264 cells treated with LPS for 30 min. (C) Effects of quercetin and Q3GA on the activation of JNK pathway in the RAW264 cells treated with LPS for 30 min. (D) Identification of the signaling pathways responsible for the LPS-induced COX-2 expression in the RAW264 cells. Upper, effects of inhibitors for several signaling pathways on the LPS-induced COX-2 expression in the RAW264 cells. Concentrations of inhibitors are as follows: PD98059 (for MEK/ERK), 10 μM; SP600125 (for JNK), 10 μM; SB203580 (for p38), 10 μM; and wortmannin (for PI3K, 100 nM). Cells were treated with LPS (1 μg/ml) in the presence of inhibitors for 4 h. <i>Lower</i>, overexpression of wild-type JNK1 (WT) significantly induced the COX-2 expression, whereas a dominant negative mutant of JNK1 (DN) did not. (E) Upper, immunoblot analysis of JNK1 and JNK2 (β-actin as control) to confirm the knockdown. Lower, effects of knockdown of JNK1/2 on the inhibitory actions of quercetin (100 μM) on the LPS-induced COX-2 expression in the RAW264 cells. C, control siRNA; JNK1/2, a mixture of JNK1 and JNK2 siRNA. The ratios of COX-2 to β-actin were also presented. ND, not detected. </p

β-Glucuronidase activity in LPS-treated RAW264 cells.

<p>(A) Enhanced deconjugation of Q3GA in the LPS-stimulated RAW264 cells. Cells were pretreated with LPS (1 μg/ml) for 8 h followed by treatment with Q3GA (20 μM) for 1 h. The deconjugated quercetin derivatives, quercetin (Q) and methyquercetins (MeQ), in cells were analyzed by HPLC-ECD. (B) Zymography (left) and immunoblot (<i>right</i>, anti-β-glucuronidase) analysis of the cultured medium of RAW264 cells. Culture medium was collected at each time point after treatment with or without LPS. (C) Immunoblot analysis of the lysates of RAW264 cells treated with or without LPS for 12 h. (D) Acidification is required for the medium β-glucuronidase activity during culturing the RAW264 cells. The β-glucuronidase activity of the cultured medium (for 8 h) at pH 5.0 or intact was determined using Q3GA as a substrate. The arrow head in the HPLC profiles (monitored at 370 nm) shows the peaks for the quercetin aglycone. (E) Acidification of the medium upon LPS treatment. The picture shows the culture plate after treatment of the RAW264 cells with (right) or without (left) treatment with LPS for 24 h. Acidification turns the medium yellow. (F) Medium lactate levels during culturing RAW264 cells in the presence of LPS, antimycin-A (AMA, 5 μg/ml), or 2-deoxyglucose (2DG, 20 mM) for 6 h determined by LC-MS/MS analysis. Blocking of glycolysis by 2DG strongly inhibited the lactate secretion. (G) Effect of lactate supplementation in the medium on the deconjugation of Q3GA. Cells were treated with Q3GA in the absence or presence of lactate (20 mM) for 4 h. Data in all bar graphs are presented as the average ± S.D. (n=3). Asterisks indicate a significant difference (<i>p</i> < 0.05). NS, not statistically significant.</p

Cell-surface binding of Q3GA.

<p>(A) Chemical structure of quercetin-3-<i>O</i>-glucuronide (Q3GA). (B) Time-dependent accumulation of Q3GA in the RAW264 cells. Cells were treated with Q3GA (50 μM) for the indicated time periods. (C) time-dependent dissociation of Q3GA bound to RAW264 cells. Cells were treated with Q3GA (50 μM) for 1 h, washed, and then incubated in the fresh medium for indicated time periods. Data points represent duplicate determinations. (D) Inhibition of Q3GA accumulation in RAW264 cells by various transporter inhibitors. Cells were pretreated with inhibitors for 15 min, followed by Q3GA (50 μM) treatment for 15 min. The concentrations of inhibitors were as follows: DIDS, taurocholic acid (TCA), p-aminohippuric acid (PAH), and estradiol-17-β-glucuronide (E17G), 500 μM; sodium azide, 5 mM. (E) Dose-dependent inhibition of Q3GA accumulation in RAW264 cells by DIDS. Data points represent duplicate determinations. (F) Inhibition of the accumulation of quercetin-3-<i>O</i>-glycosides in RAW264 cells by DIDS. The DIDS-treated cells were incubated with each quercetin-3-<i>O</i>-glycoside (20 μM) for 15 min. Q3G, quercetin-3-<i>O</i>-glucoside; Q3Gal, quercetin-3-<i>O</i>-galactoside. (G) Anti-DIDS immunoreactivity of the lysates of the DIDS-treated RAW264 cells. Cells were treated with DIDS followed by a quick trypsinization for 1 min. β-Actin was detected as a positive control for intracellular proteins. (H) Effects of quick trypsinization of RAW264 cells on the accumulation of Q3GA and quercetin. Q3GA- or quercetin-treated cells were incubated with trypsin for 1 min. (I) Effect of DIDS treatment on the accumulation of quercetin. Data in all bar graphs are presented as the average ± S.D. (n=3). Asterisks indicate a significant difference (<i>p</i> < 0.05). NS, not statistically significant.</p

Deconjugation of quercetin glucuronides <i>in</i><i>vivo</i>.

<p>(A) Immunoblot analysis for β-glucuronidase (GAPDH as control) in the various tissues in ICR mice. (B) Accumulation of the quercetin derivatives determined by HPLC-ECD in the spleen of ICR mice fed with 0.5% quercetin diet for 24 h followed by injection of LPS. <i>Left</i>, total metabolites; middle, aglycones; <i>right</i>, sulfates. (C) Accumulation of quercetin derivatives in the plasma of ICR mice injected with LPS. ND, not detected. Asterisks indicate a significant difference (<i>p</i> < 0.05). NS, not statistically significant. Data points represent the average ± S.D. (n=6). </p

β-Glucuronidase secretion is associated with intracellular calcium ions.

<p>(A) <i>Left</i>, a calcium ionophore A23187 (10 μM, for 2 h) enhanced the β-glucuronidase activity in the medium of RAW264 cells. Reduced β-glucuronidase activity in the medium during culturing the RAW264 cells in the presence of BAPTA-AM (middle) and in Ca²⁺-free medium (right). (B) Effect of AMA treatment (for 3 h) on intracellular calcium ion levels, determined using Fluo4-AM, in the RAW264 cells. (C) Effects of AMA and BAPTA-AM on the β-glucuronidase activity in the medium during culturing the RAW264 cells. Cells were pretreated with BAPTA-AM for 1 h followed by AMA treatment for 3 h. Data in all bar graphs are presented as the average ± S.D. (n=3). Asterisks indicate a significant difference (<i>p</i> < 0.05). NS, not statistically significant. (D) Effects of AMA and Ca²⁺-free medium on the deconjugation of Q3GA. Cells were pretreated with AMA for 3 h (<i>left</i>, DMSO as control) or Ca²⁺-free DMEM medium for 8 h (<i>right</i>, normal DMEM as control) followed by incubating with Q3GA for 1 h. Quercetin derivatives accumulated in the cells were analyzed by HPLC-ECD. </p