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*
State Institute for Quality Control of Agricultural Products (RIKILT), P.O. Box 230, 6700 AE Wageningen, The Netherlands and the
Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK
2To whom correspondence should be addressed. E-mail: a.l.a.sesink{at}rikilt.dlo.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • flavonoids • quercetin glucuronides • intestinal absorption • human • bioavailability
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,2)
. Flavonoids are ubiquitously present
in plant foods and several beverages, such as tea and wine
(3)
, mainly as ß-glycosidic conjugates.
Absorption of flavonoid glycosides is thought to occur in the small
intestine or in the large intestine after bacterial deconjugation. The
sugar moiety of the glycoside seems to be an important determinant for
the site of absorption (4
5
6)
. The precise mechanism of
absorption by the intestinal cells is presently unknown.
Sugar-conjugated flavonoids may be hydrolyzed by the intestinal
microflora (7
,8)
or by hydrolases located at the
intestinal brush border membrane (e.g., lactase phlorizin hydrolase)
(9)
, after which the aglycone may diffuse across the
membrane into the cell. Alternatively, flavonoids may enter the cell as
intact glycosides via the sodium-dependent glucose transporter
(SGLT1)3
(3
,5)
and enter the blood stream as such. However, inside
the enterocyte cytosolic ß-glycosidases may cleave the glycosides
(10
,11)
. Glucuronidation, sulfation and methylation of the
absorbed polyphenols have been shown to occur in humans
(12
,13)
and rats (14
15
16
17)
.
Several reports on the presence of intact glycosides in human plasma
cast doubt on the necessity of hydrolysis of the glycosides before
absorption into the bloodstream (18
19
20
21)
and suggest a
role for SGLT1. There is still debate whether flavonoid-glycosides
can be found circulating in the plasma. It seems that glucosylated and
glucuronidated flavonoids are not easily separated on reversed phase
columns, and detection systems, like ultraviolet spectrophotometry
(diode array) or fluorescence, do not distinguish between these
compounds. The aim of this study was to determine whether flavonoid
glucosides can cross the intestinal barrier as intact molecules. For
this, we analyzed plasma of subjects taken 30 min after oral ingestion
of either quercetin-3-glucoside or quercetin-4'-glucoside
(22)
. Quercetin-glucosides were expected to appear in
the circulation within the initial 30 min postingestion, because
absorption of quercetin took place within this limited period
(22)
. We adapted a HPLC method to distinguish between
glucuronidated and glucosylated quercetin in plasma.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Quercetin, quercetin-3-O-KSO4 and
Isorhamnetin-3-glucoside were obtained from Extrasynthese (Genay,
France), quercetin-3-glucoside was obtained from Apin Chemicals
(Oxfordshire, England) and quercetin-4'-glucoside was obtained from
Carl Roth GmbH (Karlsruhe, Germany). The plasma samples that were used
were collected during a study on the bioavailability of
quercetin-3-glucoside and quercetin-4'-glucoside [design of the study
and data on the bioavailability are reported in (22)
]. In
that study, subjects consumed 
325 µmol of either one of the
glucosides as an oral solution and blood was collected for up to
24 h. For each glucoside, plasma was pooled by mixing equal
volumes of the individual plasma samples, which were collected 30 min
after ingestion of the quercetin glucosides (n = 6
for each group). A mix of quercetin glucuronides used in this study was
kindly provided by Karen O’Leary (IFR, Norwich, UK). The presence of
quercetin glucuronides and the absence of quercetin glucosides in the
standard mixture were confirmed by liquid chromatography-mass
spectrometry.
Extraction of quercetin and metabolites from plasma.
Samples were mixed with two volumes of acetonitrile and thoroughly vortexed. Then, one volume of 20% o-phosphoric acid, containing ascorbic acid (4 g/L) was added. Samples were centrifuged for 10 min at 10,000 x g (4°C), and the supernatant was analyzed by HPLC. Recovery of standards (quercetin-3-O-glucoside, quercetin-4'-O-glucoside, quercetin-3-O-sulfate and the mixture of quercetin glucuronides) added to the plasma ranged from 83% to 115%. All standards and metabolites extracted from the plasma were stable in this solution during storage overnight in the autosampler.
HPLC separation of quercetin and metabolites.
A Merck Hitachi L-6000A pump (Hitachi, Tokyo, Japan), equipped with a Gilson 234 autosampler (fitted with a 100-µL loop) and a coulometric detector (Coularray detector; ESA, Chelmsford, MA), set at 10,250 and 300 mV, was used (Pd as reference). Separation was performed on a Waters Inertsil ODS2 column (150 mm x 4.6 mm i.d., 5µm; Phase Separations, Flintshire, UK), maintained on 30°C with an ESA column heater. The solvents for the gradient elution were 5% acetonitrile (v/v; solvent A) and 40% acetonitrile (v/v; solvent B) in citrate buffer (25 mmol/L; pH = 3.7). The following gradient, at a flow rate of 1 mL/min, was used: 0–3 min, 0% B; 3–20 min, linear gradient to 100% B; 20–27 min, 100% B and 27–32 min, linear gradient to 0% B.
Enzymatic treatment of plasma.
Plasma (25 µL) was incubated with 25 µL ß-glucuronidase from bovine liver (Fluka Chemie AG, Buchs, Switzerland), in 0.1 M acetate buffer, pH = 5.0 (containing 25 mU ß-glucuronidase) for 1 h at 37°C. Samples were extracted and analyzed as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Quercetin glucuronides and quercetin-3-glucoside and
quercetin-4'-glucoside were well separated on our HPLC (Fig. 1
). The glucosides had retention times of 16.0 min (3-derivative) and
17.7 min (4'-derivative). Glucuronides of quercetin appeared at 14.7,
15.4, 16.6 and 17.3 min (the separate glucuronides in the chromatogram
were not identified in this study). The retention time of quercetin
aglycone was 21.1 min (indicated by the arrow, a trace of the aglycone
was present in the glucuronide mixture). Isorhamnetin-3-glucoside had a
retention time that was very similar to the glucuronide, eluting at
17.3 min. However, these compounds could be distinguished due their
different sensitivities to oxidation by the Coularray
[isorhamnetin-3-glucoside could only be detected at voltages above 400
mV (data not shown), and, thus, cannot interfere with the analysis of
the glucuronides and the glucosides].
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Subsequently, we analyzed plasma of subjects who had consumed 325
µmol of pure quercetin-3-glucoside as an oral solution
(22)
. Four peaks appeared in the chromatogram (indicated
by asterisks), which were not present in the plasma before the
ingestion of quercetin-3-glucoside (Fig. 2
). Three of these had retention times similar to quercetin glucuronides
(15.4, 16.6 and 17.3 min). Only a trace amount of quercetin aglycone
was found. There was no indication of the presence of intact
quercetin-3-glucoside in the plasma (position of standard is indicated
with an arrow). A similar chromatogram was obtained when analyzing the
plasma of subjects who had consumed an equal amount of
quercetin-4'-glucoside (data not shown). Again, the 4'-glucoside was
not present in the plasma. In addition, when we analyzed plasma at
higher voltages of the Coularray (450 mV), we did not find the
3'-methoxylated form of quercetin-3-glucoside
(isorhamnetin-3-glucoside) in the plasma from subjects who had consumed
quercetin-3-glucoside (data not shown). To further identify the
circulating conjugates, treatment of the plasma samples with
ß-glucuronidase resulted in a loss of the peaks at 15.4, 16.6 and
17.3 min (corresponding to quercetin glucuronides as indicated above)
and the formation of quercetin (data not shown). The large peak eluting
at 20.6 min was not a glucuronide, because it was refractory to
treatment with ß-glucuronidase. It was not further identified,
because this fell beyond the scope of this study. We then spiked the
plasma (obtained after consumption of quercetin-3-glucoside) with
either the quercetin glucosides or quercetin sulfate. Figure 3
clearly shows that the glucosides had different retention times
compared with the compounds that were present in the plasma originally
(the latter indicated by asterisks). Also,
quercetin-3-O-sulfate did not coelute with one of the plasma
peaks (data not shown). In contrast, when the mixture of the quercetin
glucuronides was added to the plasma, three of four quercetin
glucuronides coeluted with the plasma compounds (retention times of
15.4, 16.6 and 17.3 min). One extra glucuronide peak appeared in the
chromatogram (at 14.7 min), because this compound was not detected in
the plasma. For additional identification of the plasma compounds, we
quantified the ratio of the peak area obtained at 300 mV to the peak
area at 250 mV and compared it with standard quercetin glucuronides.
Table 1
shows that the ratios of the plasma compounds eluting at 15.4, 16.6 and
17.3 min were comparable with the ratios of the
quercetin-glucuronides with identical retention times.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results are in contrast with several studies reporting the presence
of intact quercetin glucosides in plasma. Paganga et al.
(18)
suggested the presence of phlorizin, rutin and
several quercetin glucosides (which were not the 3 and 4'-derivatives)
in the plasma from unsupplemented humans, based on comparison of the
retention times and spectral profiles of plasma compounds with purified
standards. Aziz et al. (19)
reported the presence of
intact quercetin-4'-glucoside and its 3-methoxylated metabolite in the
plasma after consumption of fried onions, based on cochromatography of
added standards. In these studies, no comparison was made with standard
quercetin glucuronides and the plasma compounds were not further
identified. Only Mauri et al. (20)
used mass spectrometry
to identify the metabolites in plasma, confirming the presence of
intact rutin (a rhamnoglucoside of quercetin) in the plasma after the
consumption of tomato puree. In a study by Moon et al.
(13)
, however, it was reported that no quercetin
glucosides were present in plasma from subjects after consuming onions,
because the plasma metabolites (which were further identified) had
different retention times than the glucosides.
Data from experimental studies concerning transport of flavonol
glucosides are limited. Walgren et al. (23
,24)
reported
that intact quercetin-4'-glucoside was poorly absorbed by the SGLT1
transporter from an apical solution into Caco-2 cells, only when it was
present in a very high concentration. However, delivery to the
basolateral side did not occur. In a study with isolated rat small
intestine, quercetin-3-glucoside was completely deconjugated during
transport across the intestinal wall, and only quercetin conjugated
with glucuronic acid and sulfate appeared at the serosal side
(17)
. In rats fed luteolin-7-O-ß-glucoside,
only free and glucuronidated luteolin were detected in the plasma
(14)
. These studies suggest that flavonol glucosides are
not transported across the basolateral side of intestinal epithelial
cells. This is in accordance with our results, showing that during the
trafficking of the quercetin from the intestinal lumen to the
peripheral circulation after passage through the liver, the
ß-glucosidic link between quercetin and the glucose-moiety is
cleaved by hydrolases and the aglycone is then conjugated, for instance
with glucuronic acid.
From our data we cannot determine where the hydrolysis of the quercetin
glucosides and the subsequent conjugation of the aglycone occurred.
Blood samples were taken from the venous circulation, so first-pass
metabolism by the liver may have occurred. Both human intestinal and
liver tissue have been shown to posses ß-glucosidase activity
(11)
and there is direct evidence that quercetin is a
substrate for human liver UDP-glucuronosyltransferase
(25)
. Recently, quercetin and other flavonoids were
reported to be substrates for human intestinal
UDP-glucuronosyltransferases as well (26)
.
In conclusion, our results clearly show that intact quercetin-glucosides are not present in plasma after consumption of either quercetin-3-glucoside or quercetin-4'-glucoside and that quercetin glucuronides are major metabolites.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: SGLT1, sodium-dependent
glucose transporter.
Manuscript received February 14, 2001. Revision accepted April 6, 2001.
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