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Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA United Kingdom
2To whom correspondence should be addressed. E-mail: gary.williamson{at}bbsrc.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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KEY WORDS: • cider • flavonoids • phloretin • polyphenol • quercetin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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–4
). However, very little attention has been given to the absorption and metabolism of low doses of polyphenols (5
). We have, therefore, examined the uptake of polyphenols from a proprietary alcoholic apple cider, which contains a mixture of polyphenol glycosides/esters together with some aglycones at normal dietary levels. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Standards were purchased from Extrasynthese (Genay, France) or from Sigma (Poole, UK). Phloretin-2'-O-xyloglucoside was isolated from apple peel (6
), and B2 (epicatechin-(4ß
8)-epicatechin), 3'-methyl catechin and 3'-methyl epicatechin were gifts from Laurent Rios, INRA (Clermont-Ferrand, France); each compound gave one peak on analytical HPLC. ß-glucuronidase type IX-A from Escherichia coli, and sulfatase type VI from Aerobacter aerogenes were purchased from Sigma. All reagents were of analytical grade or HPLC grade where applicable. Water was purified by a Millex Q-plus system (Millipore, Watford, UK).
Subjects.
Six healthy volunteers (four male and two female) were recruited from the Norwich Research Park by advertisement (age range: 24–42 y; body mass index: 21.1–29.3 kg/m2). All volunteers were assigned a code and personal information kept confidential. Before the study began, written informed consent was obtained from each of the subjects and a fasting blood sample was screened for biochemical suitability (fasting blood glucose, full blood count, urea and electrolytes, and liver function tests) through the Norfolk and Norwich Health Care National Health Service Trust and all were considered normal. Subjects were not taking medication. The study was approved by the Norwich District Research Ethics Committee and was performed under their guidelines.
Study protocol.
Volunteers did not eat any polyphenol-rich foods (no fruits and vegetables, beverages including tea, coffee, red wine; most foods provided: meat, tinned fish, white cheese, white bread, white rice, white pasta, vegetable spread, butter shortbread biscuits; water as the sole beverage) for 48 h before the study. The day before the study, subjects were asked to collect urine for 24 h and to fast from 22:00 h. Immediately before drinking the cider, a cannula was inserted into a vein (anticubical fossa) and a sample of blood (10 mL) was taken as a baseline control. Volunteers were then required to consume 1.1 L of alcoholic cider within a set time frame: the first 550 mL during 10 min, and the second 550 mL during the next 20 min (total 30 min). After completion of drinking the first 550 mL, timing began for the study (t = 0), and blood samples (10 mL) were taken at times 0, 20 min (i.e., after finishing the second 550 mL), 30, 40, 60, and 90 min, and at 2, 4, 6, and 8 h. Subjects returned the following morning for a 24-h blood sample and were asked to continue collection of urine throughout the study day (24 h). Water was offered to drink after 90 min (total 1.5 L up to 8.5 h). A polyphenol-free meal was eaten at t = 4 h in the unit, and after t = 9 h (including more water ad libitum) after commencing the study. Breakfast was served to subjects the next morning after completion of sampling. Plasma was immediately prepared from all samples by centrifugation in Rohre 10-mL lithium-heparin tubes (Sarstedt Ltd, Leicester, UK) at 1500 x g for 10 min. The plasma was separated from the red blood cells and after addition of ascorbic acid (final concentration: 1 mmol/L), samples were frozen on dry ice and stored at -20°C until analysis. These conditions have been previously established (7
). Urine samples were collected over 24 h, frozen immediately and stored at -20°C until analysis.
Extraction of polyphenols in cider and from plasma and urine.
Scrumpy Jack cider (made from apple varieties Dabinett and Chisel Jersey, 330-mL bottles, 6% v/v alcohol; Symonds Cider and English Wine Co Ltd, Stoke Lacy, Hereford, UK) was evaporated to 50 mL under vacuum at 40°C. For analysis of polyphenols, a sample (2 mL) was evaporated just to dryness, taken up in 2-mL 0.1 mol/L phosphate buffer pH 6.2, and 3,4,5 tri-methoxy cinnamate (20 µg) added as internal standard. Plasma samples (2 mL) were added to 1-mL 0.1 mol/L phosphate buffer pH 6.2 with internal standard 3,4,5 tri-methoxy cinnamate (5 µg), and hydrolyzed with 25 U ß-glucuronidase and 0.25 U sulfatase at 37°C for 2 h (7
). Acetonitrile (2.5 vol) was added to precipitate protein and extract flavonoids. The samples were vortexed 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 0.3 mL water: methanol (1:1 v/v), vortexed, microfuged for 4 min at 13,600 x g, and passed through a 4-mm polyvinylidenedifluoride 0.2-µm syringe filter into vials for HPLC analysis (30 µL injection). Urine samples (10 mL) were added to 3-mL 0.1 mol/L phosphate buffer pH 6.2 and 10 µg internal standard, hydrolyzed with 40 U ß-glucuronidase and 0.33 U sulfatase, for 2.5 h at 37°C, and acetonitrile added as above for plasma. Samples were taken up in 0.4 mL for HPLC analysis (30 µL injected). Other flavonoids were also checked during the development of this method (7
).
Identification of compounds by HPLC.
Standards were used to identify (or eliminate) peaks using HPLC retention times and cochromatography in three solvent systems using a modified method described previously (8
). Diode array spectral characteristics were matched to standards and to library spectra. (A) Proportion of B 15%, increasing to 17% after 5 min, held until 15 min, and increasing to 20% at 25 min, 27% at 50 min, 35% at 55 min, 90% at 60 min and held 5 min, before decreasing to 15% at 70 min followed by post-run equilibration for 10 min; (B) B 15%, increasing to 17% after 10 min, held until 15 min, and increasing to 40% at 30 min, 50% at 35 min, 60% at 45 min, 90% at 50 min and held 5 min, before decreasing to 15% at 60 min followed by post-run equilibration; and (C) as Price et al. (8
). The solvent gradient comprised A: water: tetrahydrofuran: trifluoroacetic acid (98:2:0.1) and B: acetonitrile, column Prodigy 5-µm ODS3 reversed phase silica (250 mm x 4.6 mm i.d., guard 30 mm x 4.6 mm i.d.), temperature 30°C, the effluent (1 mL/min) was monitored by diode array detection between 200 and 450 nm. HPLC/fluorescence (9
) was used for analysis of catechins. Identifications from fluorescence or diode array were confirmed by HPLC/MS, using positive and negative ion electrospray HPLC/MS as described previously (7
). Full details of the method and the analysis of cider are available at: http://ifrn.bbsrc.ac.uk/phytochemicals/cider.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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A summary of the HPLC analysis of Scrumpy Jack cider used in the intervention study is shown in Table 1
. Apples and cider contain a complex mixture of polyphenols, some of which are conjugated. Levels of intake of polyphenols were low, and generally lower than in any previous study of this type. For example, only 1.5 mg (5 µmol) of quercetin were given, compared with previous studies, using, for example, doses of 4.8–9.6 mg (16–32 µmol) quercetin from orange juice (10
), 68–89 mg (225–295 µmol) from onions (11
), or 98 mg (325 µmol) from apples (12
).
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Analysis of blood samples revealed no detectable quercetin in any sample, with the exception of one plasma sample that was just above the limit of detection. However, low levels of 3' or 4'-methylquercetin (isorhamnetin and tamarixetin, respectively) were found in some samples before 60 min. There was considerable interindividual variation among subjects (Table 2
), but the mean maximum concentration (3' + 4'-methyl quercetin) was = 0.14 ± 0.19 (SD) µmol/L, range: <0.024 to 0.44 µmol/L (where Cmax = maximum concentration of compound in plasma). Other workers have reported an average plasma concentration of quercetin of 0.7µmol/L (range: 0.08–1.9 µmol/L) (13
), 0.34 µmol/L (range: 0.13–0.84 µmol/L) (14
), 0.6 µmol/L from onions (15
), 0.30 µmol/L from apples and 0.74 µmol/L from onions (12
), but all of these studies used higher doses. The identity of these peaks in all samples was confirmed by coelution after spiking with authentic standards, selected ion monitoring and conventional LC/MS ([M + H]+ = 317) of individual samples and repeat analyses after pooling and concentration of plasma samples from a single volunteer (number 6). LC/UV diode array detection and LC/MS showed possible small amounts of methyl quercetin glucuronide ([M + H]+ = 493) and of methyl quercetin ([M + H]+ = 317) before enzyme hydrolysis; owing to the low levels, this result requires further investigation. However, the samples did not contain quercetin glucuronides or sulfates as found by Day et al. (7
) after consumption of onions by humans, and glucuronides after consumption of quercetin by rats (16
). After consumption of low levels of quercetin, it seems that all of the detectable quercetin (post-enzyme hydrolysis) in plasma is methylated. This is in contrast to feeding higher levels of quercetin to humans: Manach et al. (14
) detected quercetin in all plasma samples but isorhamnetin in only 3 of 10 after an intake of 87 mg quercetin. The intake of methylated quercetin in the cider is only a few micrograms so it is likely that the methyl quercetin in plasma arises from in vivo methylation of quercetin. In agreement with our results, Young et al. (10
) found no quercetin in plasma after low dose intakes of 4.8–9.6 mg. The time of maximum concentration of quercetin in plasma (Cmax) varies dramatically depending on the nature of the consumed quercetin (17
). Pharmacokinetic parameters could not be calculated because the concentrations in plasma were very low and the cider was consumed over a 30-min period, which is longer than the Cmax for quercetin in some studies (15
). Our results suggest that at low levels of quercetin intake, extensive methylation occurs in humans; at higher doses, the pathway becomes saturated readily. Furthermore, the enzyme responsible for methylation is catechol-O-methyl transferase, which has a very low Km for polyphenols, including flavonoids and catechins and known genetic polymorphism in humans (18
–20
). In this study, only some subjects showed detectable levels of methyl quercetin, and so the uptake at low levels shows considerable interindividual variation.
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. The results showed a rapid uptake and metabolism of caffeic acid, but by 90 min the plasma level was below the limit of detection. Neither ferulic acid nor chlorogenic acid was detected in any samples. Chlorogenic acid has generally not been found in plasma (21
), but Olthof et al. (22
) demonstrated uptake in ileostomy patients of 33 ± 17%. Uptake of caffeic acid from esterified compounds found in apples requires de-esterification by feruloyl or other esterases (23
), which are not found in epithelial cells of the intestine but are activities of gut microbial flora. Consequently, some de-esterification must occur in the upper gut, because the appearance of caffeic acid in the circulation was only within 90 min of administration, and absorption in the colon is typically characterized (18
) by concentration peaks of >5 h.
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). Epicatechin and catechin can be readily detected in plasma after consumption of higher levels of these compounds. For example, Donovan et al. (25
) in a study on red wine (intake: 35 mg catechin) found 20% in the form of methylated catechin (0.05–0.176 µmol/L in plasma). In a study on tea intake, Lee et al. (26
) (intake 32 mg epicatechin) commented on individual variability in levels of epicatechin glucuronide metabolites. Intake studies with chocolate (40 g, containing 82 mg epicatechin) showed a maximum plasma concentration at 2 h of 0.383 µmol/L epicatechin (27
); metabolites were not determined in this study. Polyphenol metabolites in urine.
Hippuric acid and phloretin are present in urine as confirmed by HPLC/MS (phloretin [M + H]+ = m/z 275). No substantial amounts of quercetin, methyl quercetin, catechin, epicatechin, methylcatechin or methylepicatechin were found, although a trace of epicatechin was discernable by LC/MS. Wermeille et al. (28
) showed in a human study (feeding 1 g of either catechin or 3'-methylcatechin) that 3'-methylcatechin and 3,3'-dimethylcatechin (both as aglycones and glucuronides) were present in urine, using gas chromatography/mass spectrometry and nuclear magnetic resonance detection. The total amounts of phloretin and of hippuric acid in all subjects are shown in Table 3
. Hippuric acid is a metabolite that results from microbial degradation of polyphenols in the colon followed by hepatic conjugation with glycine, as well as from the metabolism of endogenous catecholamine neurotransmitters (29
). Potential biomarkers for consumption of polyphenols, e.g., quercetin, include the substituted phenyl acetic acids (30
). A consistent biomarker of intake of total polyphenols may be hippuric acid, which is reported as a urinary indicator of polyphenol consumption from tea (31
). Low but measurable levels were detected after 48 h of consuming a polyphenol-free diet, but levels increased 
4-fold after consumption of cider. The range of hippuric acid in urine was only 2-fold among the six subjects. Phloretin (mainly as phloretin glycosides) is found almost exclusively in apples and apples products (6
). Phloretin was not detected in prestudy urine, but increased to 3.8 ± 1.0 µmol (21 ± 5% of dose) in 24-h urine. There was a low (<2-fold) variation among individuals and so 24-h urinary phloretin may be a good biomarker for apple and cider intake. This compound has not previously been reported in human urine, but studies on rats have indicated that many of the metabolites found were microbial degradation products (32
).
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). Additionally, phlorizin is an inhibitor of cellular transporters, such as SGLT1 (34
). However, the effects other dietary components have on flavonoid bioavailability, and vice versa, is an area requiring further study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 17 August 2001. Revision accepted 16 November 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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), 2 (
), 3 (
), 4 (
), 5 (•) and 6 (
). The points are the mean of two to three determinations with an SD of <5% for the method.