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Nutrition Division, Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK;
* School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK; and
Nutrient Bioavailability, Nestlé Research Center, CH-1000 Lausanne 26, Switzerland
3To whom correspondence should be addressed. E-mail: yongping.bao{at}bbsrc.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1 h). However, the expression of COX-2 mRNA in lymphocytes was unchanged by the consumption of high-quercetin onions compared with the low-quercetin group. The results show that a single high dose of the flavonoid quercetin from onions does not change COX-2 mRNA expression in human lymphocytes in vivo even though this change occurred in vitro and ex vivo.
KEY WORDS: • flavonoids • quercetin metabolites • cyclooxgenase-2 • lymphocyte • gene expression
A high consumption of fruits and vegetables protects against different types of cancer (1). However, the mechanisms of this protection are unclear. Phenols, polyphenols, tannins, carotenoids, and vitamin C are dietary "antioxidants" found at high levels in many fruits and vegetables, but the mechanism of action is only partially via direct antioxidant action. Many polyphenols modulate cellular processes by high affinity binding to key receptors, transporters, transcription factors, or by modulation of gene expression, leading to a profound effect on cell growth and defenses (2,3). The type 2 prostaglandin endoperoxide synthase or cyclooxygenase-2 (COX-2)4 gene encodes an inducible enzyme that converts arachidonic acid to prostaglandins, and is upregulated in angiogenesis-related diseases such as rheumatoid arthritis and psoriasis (4–6). COX-2 is also upregulated in many cancer cells (7–9) and is rapidly induced by tumor promoters, growth factors, cytokines, and mitogens (10–13). Nonsteroidal anti-inflammatory drugs have a protective effect in reducing the risk of colorectal and stomach cancers. These effects, in addition to an anti-inflammatory effect, are mediated at least in part through inhibition of COX-2 (14). Antioxidants, such as the dietary flavonoid quercetin and its metabolites possess free radical scavenging properties and inhibit xanthine oxidase and lipoxygenase (15,16). The interaction of these natural antioxidants with reactive oxygen species implicated in inflammation has prompted a number of studies on their effects on the formation of proinflammatory eicosanoids derived from the cyclooxygenase pathway of arachidonic acid metabolism.
Several recent studies have demonstrated that depending on their structures, flavonoids may be potent inhibitors of several enzymes that are involved in regulation of COX-2 expression. Flavonoids and flavonoid-containing foods were investigated as selective inhibitors of COX-2 activity. The 5,7-dihydroxyflavone, galangin, with a 50% inhibitory concentration of 5.5 µmol/L, was the most active cyclooxygenase-inhibiting flavonoid (17). Flavonoids with an ortho-dihydroxy (catechol moiety) in rings A or B were stronger inhibitors of COX-2 than those with a free 3-OH group (17,18). The presence of a C2-C3 double bond appears to be a major determinant of this activity as enzyme inhibitors. More recently, several authors showed that quercetin and other flavonoids, as aglycones, downregulate COX-2 expression in different cell lines (19,20). However, most of these studies were done in vitro and no in vivo data are available at present. Additionally, all studies reported to date used the aglycone form for the in vitro experiments, but this form is not found at significant levels in human plasma (21,22). The major circulating forms of quercetin in the plasma were identified as quercetin 3-glucuronide, 3'-methylquercetin 3-glucuronide, and quercetin 3'-sulfate (21,23). Because the biological activity of the flavonoids is predicted to be highly dependent on the structure, particularly the availability of hydroxyl groups (24), we predict that the results from the in vitro tests warrant reevaluation; the activity also has to be confirmed in vivo. Therefore, in this study, we compared the effect of the human flavonoid plasma metabolites discussed above on the expression of COX-2 mRNA in human lymphocytes ex vivo using TaqMan real-time RT-PCR. In parallel, we measured COX-2 mRNA levels in 8 subjects participating in a 3-way, single-blind, randomized crossover study after consumption of a single meal of white, low-quercetin onions, compared with yellow, high-quercetin onions.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All reagents were obtained from Sigma unless otherwise stated and were of analytical or HPLC grade where applicable. Tissue culture plastics were supplied by Life Technology. Water was purified via a Milli Q plus system (Millipore). Lymphocyte separation medium was obtained from ICN Pharmaceuticals. Quercetin 3-glucuronide was purified from green beans (25). Quercetin 3'-sulfate was kindly provided by Dr. Barron (21). These compounds were confirmed previously by MS, 1H and 13C NMR. All metabolites were checked for purity before use by HPLC and were found to be >98% pure.
Synthesis of 3'-methylquercetin 3-glucuronide (isorhamnetin 3-glucuronide, IR-3-GlcA).
4',7-di-O-Benzyl-3'-O-methylquercetin (26) (0.29 g, 0.58 mmol), methyl 2,3,4-tri-O-acetyl-
-D-glucopyranosyluronate trichloroacetimidate (27) (0.28 g, 0.58 mmol) and 3A molecular sieves (1 g) were suspended with stirring in dichloromethane (5 mL) at -15°C under argon. Boron trifluoride etherate (20 mL) was added to one portion. The mixture was allowed to warm to 10°C over 1.5 h. A further portion of boron trifluoride etherate (20 mL) was added, followed by a final portion (20 mL) after 60 h. After 24 h, the mixture was diluted with 5% methanol:dichloromethane (v:v, 50 mL), and filtered through celite. The filtrate was washed with saturated sodium bicarbonate solution (2 x 50 mL), and water (1 x 50 mL), and dried (anhydrous magnesium sulfate). The solution was evaporated completely. Medium pressure liquid chromatography of the crude product, using 2% methanol:dichloromethane (v:v) as eluant, gave the desired product in protected form. The latter was refluxed with palladium hydroxide (0.25 g) and cyclohexene (10 mL) in ethanol (50 mL) for 15 min. The mixture was filtered through celite, and evaporated. This debenzylated product was suspended and stirred in 50% aqueous methanol (200 mL) under argon and treated at room temperature with 0.5 mol/L sodium bicarbonate solution (6 mL). After 2 h, Dowex resin (50W, H+ form) was added (20 mL). The mixture was stirred until the pH fell to 2.8. The resin was removed by filtration, and the solution evaporated. Preparative HPLC (28) of the residue gave pure isorhamnetin 3-ß-D-glucuronic acid (15 mg, 5%).5
Isolation of lymphocytes, culture of cells, and treatment with quercetin metabolites.
Lymphocytes were immediately isolated from blood samples by centrifugation (800 x g, 20 min) with lymphocyte separation medium following the manufacturer’s instructions. Briefly, cells were washed three times with PBS, resuspended in RPMI-1640 medium, and counted. A portion of the cells were used for total RNA extraction to use it as standard for the construction of the standard curve. The rest of the cells were seeded in 24-well plates at a density of 108 cells/L and allowed to adhere. Cells were then treated with different concentrations of quercetin 3-glucuronide, quercetin 3'-sulfate, and isorhamnetin 3-glucuronide for 6 h. The final concentrations in growth media of these compounds were 4 µmol/L, 1 µmol/L, 100 nmol/L, 10 nmol/L, or 1 nmol/L. For the in vivo study, lymphocytes were isolated from 3 mL of blood using the same method and the total RNA was immediately obtained as described below.
RNA isolation and quantitative RT-PCR of human COX-2 mRNA in lymphocytes.
Total RNA was extracted from cells using a Qiagen RNeasy mini kit according to the protocol described by the manufacturer (Qiagen) after homogenization using a QiAshredder column. RNA was eluted from a binding column with RNase-free water and RNase inhibitor was added immediately (20 U/preparation) and stored at -70°C. The yield of total RNA was determined using Ribogreen RNA Quantitation Kit (Molecular Probes) against a standard curve of ribosomal RNA (16S and 23S rRNA from Escherichia coli).
Target mRNA levels were determined by TaqMan real-time RT-PCR using the ABI prism 7700 Sequence Detection System (Applied Biosystems) using the 
Ct method. Fold of suppression = 2Ct(control) - Ct(treatment). Forward and reverse primers, and the fluorogenic TaqMan probes for the target gene were designed using Primer Express Software (Applied Biosystems). Sequence homology of selected oligomers was checked using a NCBI BLAST search to ensure that sequences were specific to target genes. The TaqMan probe consists of an oligonucleotide with a 5' reporter dye (FAM) and a downstream 3' quencher dye (TAMRA). The forward primer was: GCC CTT CCT CCT GTG CC, the reverse primer was: AAT CAG GAA GCT GCT TTT TAC and the probe was: ATG ATT GCC CGA CTC CCT TGG GT GT. RT-PCR reactions were carried out in a 96-well plate using TaqMan one-step RT-PCR master mix Reagent kit (Applied Biosystems) in a total volume of 25 µL/well consisting of 100 nmol/L probe, 200–300 nmol/L forward and reverse primers. The thermal cycling conditions comprised an initial step at 48°C for 30 min followed by 95°C for 10 min. Subsequent PCR amplifications consisted of 40 cycles of denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min. The standard curve was constructed with serial dilutions of untreated lymphocyte RNA. Glyceraldehyde-3-phoshate dehydrogenase (GAPDH) was used as a reference housekeeping gene for all of the samples. The TaqMan GAPDH control reagent kit was purchased from Applied Biosystems.
Human study.
Human subjects. The study was approved by the University of Surrey Ethics Committee (ACE/2001/64/SBLS) and subjects were recruited from within the School of Biomedical and Life Sciences. Subjects were excluded if they had a BMI >25 kg/m2, a significant current or previous medical history, were receiving regular medication (apart from oral contraceptives) that may have affected gastrointestinal or central nervous systems, consumed >20 U of alcohol (a unit of alcohol is the equivalent of 10 g of alcohol) per week, smoked, or had donated blood within the 4–6 mo preceding the commencement of the study. Eight lean healthy volunteers (5 men, 3 women; age 24–35 y) were admitted to participate. All subjects were interviewed and gave their written consent to participate in this study.
General procedure. The study design was a 3-way, randomized crossover with each subject serving as his or her own control. On 3 separate occasions, subjects were required to come to the clinical investigation unit at 0800 h. The subjects fasted overnight from 2000 h before each study day and were asked to refrain from strenuous exercise and the consumption of alcohol or caffeine the night before. They were also asked to consume a diet high in carbohydrate (>200 g) the day before and a sample menu for this was given. Upon arrival at the clinical investigation unit, an intravenous cannula was inserted into a forearm (antecubital) vein, and a baseline blood sample was taken. Then, the subjects consumed either a control meal (25 g of glucose in 400 mL of water) cooked white onions or cooked yellow onions. Blood samples were taken at frequent intervals for the following 6 h.
Standardization of test meals.
As part of a Plant Research International (Wageningen, The Netherlands) onion-breeding program, intrapopulation crosses were made between yellow and white onion lines. Segregating populations consisting of yellow (high-quercetin) and white (low-quercetin) near-isogenic onion lines were obtained and used for the clinical study. Moisture, ash, calcium, crude protein, and sugars were analyzed as recommended by the AOAC (29). For lipids and crude fiber, the Spanish official methods of analysis were used (30). Each test meal consisted of 368 g of onions that were chopped finely and dry-fried until soft. Maltodextrin (5.5 g) was added to each white onion meal to compensate for the difference in total glucose equivalents between the onion types.
Analysis of quercetin in onion test meals.
Samples were analyzed as described previously (31). Briefly, frozen samples were freeze-dried and milled to fine powders using a food blender. LC/MS quantification of the freeze-dried tissues was performed in triplicate. Ion-pair electrospray ionization LC/MS using selected ion monitoring (SIM) was calibrated by using 25 µmol/L rutin with 0–100 µmol/L quercetin 4'-glucoside and 25 µmol/L rutin with 0–100 µmol/L quercetin 3, 4'-diglucoside. The column was a Phenomenex Luna 5 µm C18 (2) 250 x 4.60 mm with Security-guard precolumn. Solvent A was 0.1% (v:v) trifluoroacetic acid in ultra-pure water, and solvent B was 0.1% (v:v) trifluoroacetic acid in methanol; the flow was 1 mL/min. MS analysis were recorded in the SIM mode at 303 m/z for the quercetin aglycon, 465 for the monoglucoside, 627 for the diglycoside, and 611 for rutin. Quantification of the free quercetin and its glucosides was performed by integrating the SIM peaks with the integration software incorporated into the MassLynx suite of programs.
Extraction and hydrolysis of quercetin metabolites from plasma.
Plasma was immediately prepared from all samples by centrifugation (1500 x g, 10 min at 4°C). The plasma was separated from the RBC and immediately frozen and stored at -20°C. All of the samples were then treated in the same way. Ascorbic acid (final concentration 1 mmol/L) and acetic acid (0.65 mmol/L; 40 µL) were added to plasma to avoid degradation. Plasma samples (0.4 mL) were added to 0.5 mL of 0.1 mol/L phosphate buffer, pH 6.2, with apigenin (180 mmol/L, 100 mL) as internal standard and hydrolyzed with 25 U ß-glucuronidase and 9 U sulfatase at 37°C for 3 h. Methanol (0.5 mL) was added to terminate the reaction. Acetonitrile (2.5 mL) was added to precipitate proteins and extract flavonoids. The samples were mixed on a vortex for 30 s every 2 min over a 10-min period before centrifugation (13,600 x g, 10 min, 4°C). The supernatant was dried, taken up in 0.2 mL water:methanol (1:1, v:v), mixed on a vortex and passed through a 4-mm polyvinylidene-difluoride 0.2-µm syringe filter into vials for HPLC analysis.
HPLC analysis.
A modified version of the previously published analytical HPLC method of Price et al. (25) was adopted. Solvents A, water:tetrahydrofuran:trifluoroacetic acid (98:2:0.1, by vol) and B, acetonitrile, were run at 1 mL/min, using a gradient of 17%B (2 min), increasing to 25% B (5 min), 35% B (8 min), 50% B (5 min), and then to 100% B (5 min). A column clean-up stage was performed at 100% B (5 min) followed by a reequilibration at 17% B (15 min). The column was packed with Prodigy 5 µm ODS (3) reverse phase silica, 250 mm x 4.6 mm i.d. (Phenomenex). The injection volume was 30 µL. Diode array detection monitored the eluent at 270 and 370 nm. Quercetin and isorhamnetin were used as external standards at concentrations ranging from 0.6 to 30 µmol/L. Externals standards were run every 6 h. The identity of the products after the hydrolysis was confirmed in every case by LC-MS using the same conditions as for the quantification of flavonol glycosides in onions.
Direct determination of metabolites in plasma.
Plasma samples (200 µL) containing 1 mmol/L ascorbic acid were added to 500 µL acetonitrile containing apigenin (60 µmol/L, 2 µL) and acetic acid at a final concentration of 0.65 mmol/L. Samples were then mixed on a vortex for 30 s every 2 min for 10 min, centrifuged at 13,000 x g, 10 min, and 150 µL water:methanol (50:50, v:v) added. The mixture was centrifuged (13,000 x g, 3 min), filtered and injected onto the HPLC using the same gradient as for the hydrolyzed metabolites.
Statistical analysis.
Gene expression data were normalized against GAPDH, and the significance of fold-changes (control against treated cells) between control, white-, and yellow onion-treated subjects was analyzed using the 2-factor repeated-measures ANOVA with interaction. Significances of the differences at each time point were analyzed using the Student’s t test using Bonferroni’s correction. The analysis was done using a commercial statistical package (SPSS for Windows version 11.5.1). The quantitative data for quercetin and isorhamnetin in hydrolyzed plasma were examined by noncompartmental pharmacokinetic analysis using PK Solutions 2.0 (Summit Research Services). Differences with P < 0.05 were considered significant. Gene expression and analysis of metabolites are presented as means ± SD or standard error (SEM).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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COX-2 mRNA was detected in lymphocytes and quantified using real-time RT-PCR. None of the treatments produced any visible toxic effects (cell death or loss of cell adhesion) in lymphocytes as assessed microscopically. Figure 1 shows the results as the mean of the results obtained for lymphocytes from three different subjects. Quercetin 3-glucuronide, quercetin 3'-sulfate, and 3'-methylquercetin 3-glucuronide inhibited COX-2 expression in lymphocytes ex vivo in a dose-dependent manner, except for the highest dose of quercetin 3'-sulfate. In that case, the highest concentration of 4 µmol/L was ineffective. The most effective was 3'-methylquercetin 3-glucuronide.
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Volunteers were fed a portion of onions, containing either high or low levels of quercetin. Based on the 368-g cooked portion of onion, the subjects each consumed the following: white onions, 6.1 mg quercetin 3, 4'-diglucoside, 4.2 mg quercetin 4'-glucoside, and 0.9 mg quercetin aglycone; yellow onions, 163.9 mg quercetin 3, 4'-diglucoside, 140.6 mg quercetin 4'-glucoside, and 2.4 mg quercetin aglycone (values expressed as quercetin aglycone equivalents).
Ten minutes before onion administration, plasma quercetin and isorhamnetin concentrations were 0.26 ± 0.07 and 0.038 ± 0.004 µmol/L, respectively. After consumption of the yellow onion test meal, plasma quercetin concentration increased to 3.71 ± 0.40 µmol/L (Fig. 2) and isorhamnetin to 0.36 ± 0.05 µmol/L (mean ± SEM, n = 8). The corresponding maximum concentration (maximum time) [Cmax (Tmax)] values for yellow onions were quercetin 4.38 ± 0.75 µmol/L (2.19 ± 0.35 h) and isorhamnetin 0.36 ± 0.054 µmol/L (1.86 ± 0.30 h).
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Plasma quercetin conjugates.
Quercetin conjugates were measured in plasma by HPLC in 3 of the 8 volunteers after consumption of the high-quercetin onions. The chromatograms were very similar in the three plasma samples. At 30 min (the earliest time point), quercetin sulfates and glucuronides were present. In a typical quercetin metabolic profile (Fig. 3), peaks (4–9) were identified by comparison with standards and by their UV MS and/or MS/MS spectra. The most abundant quercetin metabolites were quercetin 3'-sulfate, quercetin 3-glucuronide, 3'-methylquercetin 3-glucuronide (isorhamnetin 3-glucuronide), and quercetin 3'-glucuronide. Quercetin 7-sulfate and 3'-methylquercetin 4'-glucuronide were minor components. Ion currents for peak 1–3 were too weak for identification although peak 1 had a flavonol-type of UV spectrum. Quercetin 3',4'-diglucoside, quercetin 3-glucoside, and quercetin were not detected in any of the plasma samples as expected. All of the quercetin conjugates identified in Figure 3 were described previously by other authors (21,32). In an examination of the time course of changes in quercetin conjugates (Fig. 4), they reached peak levels at 30 min, whereas two methylquercetin conjugates increased over the period from 30 to 360 min. The major quercetin conjugate, quercetin 3'-sulfate, peaked at 1 h (21 x 10-6 g/L) and decreased gradually to 5 x 10-6 g/L over a 6-h period.
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Lymphocytes were collected before, and at 3 and 6 h after ingestion of the control diet or onion meals. As expected, the control diet did not affect relative COX-2 mRNA expression normalized against the "housekeeping" gene GAPDH. COX-2 gene expression was not affected by the onion diets, even though the levels of quercetin in the plasma should have been sufficient, based on in vitro studies, to downregulate COX-2 transcription.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We first studied the effect of the major quercetin metabolites on COX-2 expression in lymphocytes ex vivo using physiologically achievable concentrations. To determine whether the effect on COX-2 expression could also be observed in vivo, we used lymphocytes from 8 subjects involved in a three-way randomized crossover study to determine the absorption and metabolism of quercetin from onions.
Quercetin was absorbed as expected and demonstrated pharmacokinetics comparable to earlier studies (22,32,37–39). The high dose of quercetin glycosides in the onions led to a high concentration of quercetin conjugates (total of glucuronides and sulfates for both aglycone and methylated aglycone). Based on in vitro data, this concentration of the various quercetin conjugates should have been enough over the 6-h period to suppress COX-2 mRNA to 50%. However, no changes were observed in vivo in these 8 subjects (age 24–35 y). There are reports of elevated production of PGE2 in elderly groups (40) and increased COX-2 mRNA and protein expression in aged mice (41). Therefore, it would be of interest to compare the response of different age groups to a quercetin-rich diet in the future.
Methylation of quercetin enhances its vasorelaxant activity, and this activity is decreased by the removal of endothelium (42). Because it was shown that COX-2 plays an important role in endothelium-dependent vasodilation (43), the effect of the methylation of quercetin on vasorelaxation could be mediated via the COX-2 cascade.
Although the quercetin glucosides in onions were rapidly absorbed and deglucosylated, any subsequent methylation of quercetin occurred relatively slowly. The character of methylated quercetin conjugates in plasma could be explained by the studies of O’Leary et al. (23) and de Santi et al. (44) in vitro in HepG2 cells and human hepatocytes, respectively, and also by Manach et al. (45) in vivo in rats. These studies showed that quercetin glucuronides enter the liver cells and are further metabolized through methylation. In this way, the levels of methylated metabolites would be increased at the expense of nonmethylated metabolites. Some methylation, however, may occur in the gastrointestinal tract (46).
In summary, the results presented here showed that a single high dose of quercetin from onions did not suppress COX-2 mRNA expression in human lymphocytes in vivo although a downregulation by quercetin metabolites was found in vitro and ex vivo. COX-2 is a bifunctional enzyme in immune cells, ensuring the conversion of arachidonic acid to prostaglandin. When giving the flavonoid supplement to humans in a whole-vegetable-based meal, the interactions of dietary components, such as among quercetin metabolites, might be complex. Further studies should focus on the synergistic or antagonistic effects of the factors not only from food but also from endogenous nutrients and metabolites.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Present address: Instituto del Frío (CSIC), Jose Antonio Novais 10, 28040 Madrid, Spain.
4 Abbreviations used: Cmax (Tmax), maximum concentration (maximum time); COX-2, cyclooxygenase-2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SIM, selected ion monitoring.
5 1H NMR (CD3OD): 
7.95 (d, 1 H, J2',6' 2.4 Hz, H-2'), 7.54 (dd, 1 H, J6',5' 8.2 Hz, J6',2' 2.0 Hz, H-6'), 6.86 (d, 1 H, H-5'), 6.37 (d, 1 H, J8,6 2.0 Hz, H-8), 6.17 (d, 1 H, H-6), 5.48 (d, 1 H, J1'',2'' 7.2 Hz, H-1''), 3.93 (s, 3H, OMe), 3.78 (d, 1 H, J5,4 9.2 Hz, H-5''), 3.46–3.62 (m, 3 H, H-2'', H-3'', H-4''). 13C NMR (CD3OD): 158.67(C2); 135.14 (C3); 179.12 (C4); 163.00 (C5); 99.93 (C6); 165.98 (C7); 94.82 (C8); 158.38 (C9); 105.68 (C10); 122.75 (C1'); 114.39 (C2'); 150.85 (C3'); 148.35 (C4'); 116.00 (C5'); 123.52 (C6'); 104.04 (C1''); 75.61 (C2''); 77.46 (C3''); 72.99 (C4''); 77.18 (C5''); 171 64 (C6''). ESMS: m/z 493 [M+H]+; 515 [M+Na]+; 491 [M-H]-.
Manuscript received 29 September 2003. Initial review completed 23 November 2003. Revision accepted 19 December 2003.
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