Characterization of sulfated quercetin and epicatechin metabolites

Article abstract of DOI:10.1021/jf2050203

Different monosulfates of quercetin and epicatechin with metabolic interest were obtained by hemisynthesis and characterized regarding their chromatographic behavior and absorption and mass spectra. Three of these compounds were further isolated, and their structures were elucidated by mass spectrometry and ¹H and ¹³C nuclear magnetic resonance using one- and two-dimensional techniques (heteronuclear single-quantum coherence and heteronuclear multiple-bond correlation). The calculation of the proton and carbon shifts caused by sulfation allowed for the assignment of the position of the sulfate group in the flavonoids, so that the compounds were identified as quercetin-3′-O-sulfate, quercetin 4′-O-sulfate, and epicatechin 4′-O-sulfate. It was found that sulfation at position 3′ induced a large upfield shift in the carbon bearing the sulfate group and downfield displacements of the adjacent carbons, whereas no significant upfield or downfield shifts were observed with respect to the parent flavonoid when sulfation was produced at position 4′.

Full text of DOI:10.1021/jf2050203

Article
pubs.acs.org/JAFC
Characterization of Sulfated Quercetin and Epicatechin Metabolites
Montserrat Duenas, Susana Gonzalez-Manzano, Felipe Surco-Laos, Ana Gonzalez-Paramas,
́
́
̃
and Celestino Santos-Buelga*
Grupo de Investigacion
Farmacia, Campus Miguel de Unamuno, E-37007 Salamanca, Spain
́ ́
en Polifenoles (GIP/USAL), Universidad de Salamanca, Unidad de Nutricion y Bromatología, Facultad de
ABSTRACT: Different monosulfates of quercetin and epicatechin with metabolic interest were obtained by hemisynthesis and
characterized regarding their chromatographic behavior and absorption and mass spectra. Three of these compounds were
1
further isolated, and their structures were elucidated by mass spectrometry and H and 13C nuclear magnetic resonance using
one- and two-dimensional techniques (heteronuclear single-quantum coherence and heteronuclear multiple-bond correlation).
The calculation of the proton and carbon shifts caused by sulfation allowed for the assignment of the position of the sulfate
group in the flavonoids, so that the compounds were identified as quercetin-3′-O-sulfate, quercetin 4′-O-sulfate, and epicatechin
4′-O-sulfate. It was found that sulfation at position 3′ induced a large upfield shift in the carbon bearing the sulfate group and
downfield displacements of the adjacent carbons, whereas no significant upfield or downfield shifts were observed with respect to
the parent flavonoid when sulfation was produced at position 4′.
KEYWORDS: Quercetin, epicatechin, metabolites, sulfates, NMR
is expected to be different of that of the parent flavonoids, as
affected by the nature and position of the conjugating moieties.
Therefore, to better understand the in vivo effects of dietary
flavonoids, it is important to take into account the distribution,
activity, and metabolic fate of the metabolites, as well as to be in
the disposition of analyzing and identifying them in biological
fluids and organs, for which adequate standards are required.
However, most flavonoid metabolites are not commercially
available and must, therefore, be prepared in the laboratory.
Some works have been published dealing with the preparation
of conjugated metabolites of quercetin (glucuronides and
sulfates), using enzymatic or chemical synthesis,18−24 but fewer
studies exist regarding conjugated catechins.18,25 The lack of
suitable procedures is especially important in the case of
sulfates that are more chemically labile than other metabolites,
such as glucuronides or methylethers. In a previous paper of
our group,25 the hemisynthesis of catechin sulfates using an
adaptation of the method described by Jones et al.20 for
quercetin sulfates was reported. In that paper, the identification
of the synthesized compounds was based on their mass spectra
fragmentation patterns that only allowed for establishing their
nature as sulfates and the flavonoid ring on which the sulfation
occurred but not the precise substituted hydroxyl. In view to
confirm their actual structure, in the present work, different
monosulfates of quercetin and epicatechin (Figure 1) were pre-
pared and characterized by nuclear magnetic resonance (NMR).
INTRODUCTION
■
Flavonoids are plant secondary metabolites of phenolic nature
that are widely distributed in the human diet, being present in a
broad range of commonly consumed fruits and vegetables and
derived products, such as cocoa, tea, or wine. In recent years,
there has been a growing interest in the potential health
benefits of dietary flavonoids, because they have been related
to a reduced risk of different chronic diseases and especially
cardiovascular disease.1−3 Flavonols and flavan-3-ols (i.e.,
catechins and proanthocyanidins) are major classes of flavonoids
in the human diet, and among them, quercetin and catechins
have been particularly used in studies of biological activity.
These compounds are generally known to possess antioxidant
and radical-scavenging properties,4,5 as well as the ability to
modulate the activity of several metabolically relevant enzymes
and exert a wide range of biological effects.6 Quercetin acts as
an effective inhibitor of xanthine oxidase and lipoxygenase that
play a key role in processes such as inflammation, atherosclerosis,
cancer, or aging,7 shows vasodilatation and anti-aggregant effects in
vitro, and reduces blood pressure, oxidative status, and end-organ
damage in animal models of hypertension.8,9 Catechins are able to
reduce platelet aggregation and inhibit the growth of human
cancer cell lines,10 as well as to act as powerful inhibitors of low-
density lipoprotein (LDL) oxidation in vitro,11 decrease DNA
damage, and delay tumor promotion in mice.12,13
Health effects of flavonoids depend upon their bioavailability.
Flavonols, such as quercetin, mostly occur in foodstuffs as
glycosides, and in general, the first step in their metabolism is
likely to be deglycosylation before absorption in the small
intestine.14,15 During transfer across the enterocyte and, sub-
sequently, in the liver, quercetin undergoes O-methylation and
other conjugation reactions, namely, glucuronidation and sulfation.
Catechins are not usually glycosylated and can be partially
absorbed as such from the human intestinal tract to be further
transformed into O-methylated derivatives and glucuronide and
sulfate conjugates.16,17 The biological activity of these metabolites
MATERIALS AND METHODS
■
Standards and Reagents. High-performance liquid chromatog-
raphy (HPLC)-grade methanol was purchased from Carlo Erba
(Rodano, Italy). Pyridine and HPLC-grade acetonitrile were obtained
Received: April 15, 2011
Revised: March 8, 2012
Accepted: March 16, 2012
Published: March 16, 2012
© 2012 American Chemical Society
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Figure 1. Structures of quercetin (1) and epicatechin (2). In the case
of the different sulfated metabolites, the corresponding −OH is
substituted by a −O−SO3H group.
from Merck KGaA (Darmstadt, Germany). Trifluoroacetic acid (TFA)
was from Riedel-de-Haen (Seeize, Germany), and analytical-grade
̈
glacial acetic acid was from Panreac (Barcelona, Spain). Quercetin,
epicatechin, sulfur trioxide−N-triethylamine complex, Sephadex
LH-20, and dioxane were purchased from Sigma-Aldrich (Madrid, Spain).
Synthesis of Quercetin and Epicatechin Sulfates. These
metabolites were synthesized by an adaptation of the method
described by Jones et al.20 First water associated with quercetin or
epicatechin (500 mg each) was removed by adding dry pyridine until
dissolved. Pyridine was rotary evaporated, and the dry compound was
dissolved in dioxane (50 mL) and allowed to react in a water bath
(40 °C) for 90 min with a 10-fold molar excess of sulfur trioxide−N-
triethylamine complex under nitrogen to avoid contact with air.
Products of sulfation precipitated out and stuck to the glass. Dioxane
was decanted, and the synthesized mixture redissolved in 10%
methanol in water. The mixtures of quercetin and epicatechin sulfates
were fractioned on a Sephadex LH-20 column (350 × 30 mm),
successively eluted with 10% aqueous ethanol (500 mL) and 20%
aqueous ethanol (500 mL). The fractions containing monosulfates
were collected, concentrated to dryness under vacuum, redissolved in
ultrapure water, and analyzed by high-performance liquid chromatography−
diode array detector−mass spectrometry (HPLC−DAD−MS). Individual
quercetin and epicatechin sulfates were further isolated by semi-preparative
HPLC.
Semi-preparative HPLC. A Waters 600 chromatograph coupled
to an ultraviolet−visible (UV−vis) model 486 detector and an
Ultracarb C18ODS20 5 μm (10 × 250 mm inner diameter) column
from Phenomenex (Supelco Ascentis, Bellefonte, PA) were used.
Solvents and the gradient employed for the separation of quercetin
sulfates were as follows: solvent A, water; solvent B, methanol;
gradient, from 0 to 15% B in 5 min, from 15 to 25% B in 30 min, from
25 to 50% B in 20 min, and isocratic 50% B for 15 min. For the
separation of epicatechin sulfates, the solvents were (A) 5% acetic acid
and (B) methanol. The gradient used was from 0 to 10% B in 40 min
and from 10 to 12% B in 20 min. The flow rate was 3 mL min−1 in
both cases. Detection was carried out at 370 nm (quercetin) and
280 nm (epicatechin). Peaks were automatically collected in a fraction
collector, concentrated under vacuum, and freeze-dried. The identity
of the compounds was checked by HPLC−DAD−electrospray
ionization (ESI)/MS and NMR.
Figure 2. Chromatogram recorded at 370 nm showing the profile of
quercetin monosulfates obtained by hemisynthesis and further
separation on a Sephadex LH-20 column. See Table 1 for peak
identification.
Table 1. Retention Time and UV and Mass Spectral Data
Obtained for Quercetin and Sulfated Metabolites Observed
in the Chromatogram of Figure 2
retention
time
(min)
molecular ion
[M + H]+
(m/z)
peak
λmax
(nm)
compounds number
MS2 (m/z)
quercetin
disulfate
1
2
3
4
5
8.0
10.8
12.2
12.9
14.0
368
351
364
371
366
461
381
381
381
381
381, 301
quercetin 3-
O-sulfate
301
301
301
301
quercetin 5-
O-sulfate
quercetin 7-
O-sulfate
quercetin
4′-O-
sulfate
quercetin
3′-O-
6
14.3
22.1
368
372
381
301
301
sulfate
quercetin
273, 257,
229, 179,
151
HPLC−DAD−MS Analyses. Analyses were carried out in a
Hewlett-Packard 1100 chromatograph (Agilent Technologies, Wald-
bronn, Germany) with a quaternary pump and a DAD coupled to a
HP Chem Station (revision A.05.04) data-processing station.
Separation was achieved on an AQUA (Phenomenex, Torrance,
CA) reversed-phase C18 column (5 μm, 150 × 4.6 mm inner
diameter) thermostatted at 35 °C. For the analysis of quercetin
sulfates, solvents used were (A) 0.1% TFA in water and (B)
acetonitrile and the elution gradient was from 10 to 15% B over 5 min,
from 15 to 25% B over 5 min, from 25 to 35% B over 10 min, from
35 to 50% B over 10 min, isocratic 50% B for 10 min, and re-equilibration
of the column, at a flow rate of 0.5 mL/min. For the analysis of
epicatechin sulfates, a Water Spherisorb S3OD-2 C18 (3 μm, 150 ×
4.6 mm inner diameter) column was used. The solvents were (A) 0.1%
TFA in water and (B) acetonitrile, and the elution gradient was from 0
to 10% B over 5 min, from 10 to 15% B over 20 min, and from 20
to 40% C in B over 5 min, using a flow rate of 0.5 mL min−1.
Double online detection was carried out in the DAD using 280 nm
Figure 3. Chromatogram recorded at 280 nm showing the profile of
epicatechin monosulfates obtained. See Table 4 for peak identification.
(epicatechin) and 370 nm (quercetin) as preferred wavelengths and in
a mass spectrometer connected to the HPLC system via the DAD
cell outlet.
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1
Table 2. H and 13C NMR Data and HMBC Correlations
Table 3. 13C NMR Shifts Induced by 3′- or 4′-Sulfation in
Quercetin
a
Obtained for Quercetin, Quercetin 3′-O-Sulfate, and
Quercetin 4′-O-Sulfate Determined in CD3OD
carbon
quercetin 3′-O-sulfate
quercetin 4′-O-sulfate
a
1
ring position δ H (ppm); m; J (Hz)
Quercetin
δ
13C (ppm)
HMBC
2
+0.9
−0.4
−0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
3
C
2
147.9
137.2
177.3
H-6′, H-2′
4
3
10
1′
2′
3′
4′
5′
6′
4eq
4ax
9
−0.2
−7.9
+4.9
−3.9
−2.0
−5.8
158.2
104.5
162.4
99.2
H-8
10
5
H-8, H-6
H-6
A
B
6
6.16; d; J = 2.0
H-8
a
7
165.5
94.3
H-8, H-6
H-6
Refer to δaglycone − δester
.
8
6.36; d; J = 2.0
7.72; d; J = 2.1
1′
2′
3′
4′
5′
6′
124.1
115.9
146.2
148.7
116.2
121.6
H-5′
Table 4. Retention Time and UV and Mass Spectral Data
Obtained for Epicatechin and Their Monosulfated
Metabolites Observed in the Chromatogram of Figure 3
H-6′
H-5′, H-2′
H-6′, H-2′, H-5′
H-6′
retention
time
(min)
molecular ion
[M − H]−
(m/z)
6.86; d; J = 8.5
peak
λmax
(nm)
compounds number
MS2 (m/z)
7.61; dd; J = 8.4, 2.2
H-2′
Quercetin 4′-O-Sulfate
147.9
epicatechin
1
2
3
4
17.9
18.5
20.4
21.1
27.8
278
277
275
274
279
369
369
369
369
289
217, 245,
247, 289
C
2
H-2′, H-6′
5-O-sulfate
3
137.2
177.3
epicatechin
3′-O-sulfate
201, 231,
245, 289
4eq
4ax
9
epicatechin
7-O-sulfate
217, 245,
247, 289
158.2
104.5
162.5
99.2
H-8
epicatechin
4′-O-sulfate
201, 231,
245, 289
10
5
H-8, H-6
H-6
A
B
epicatechin
245, 203,
179, 151,
137
6
6.16; d; J = 2.0
6.37; d; J = 2.0
7.72; d; J = 2.1
H-8
7
165.5
94.3
H-8, H-6
H-6
8
1′
2′
3′
4′
5′
6′
124.1
115.9
146.2
148.7
116.2
121.6
H-5′
H-6′
voltage was 11 V; and the capillary temperature was 220 °C. Spectra
were recorded in negative-ion mode between m/z 150 and 2000. The
MS detector was programmed to perform a series of two consecutive
scans: a full scan and a MS−MS scan of the most abundant ion in the
first scan, using normalized collision energy of 45%.
H-2′, H-5′
H-6′, H-2′, H-5′
H-6′
6.87; d; J = 8.4
7.62; dd; J = 8.4, 2.2
H-2′
1
NMR Analysis of Quercetin and Epicatechin Sulfates. H NMR
Quercetin 3′-O-Sulfate
(400 MHz) and 13C NMR (100 MHz) spectra of the isolated metab-
olites were measured in CD3OD on a Bruker Avance DRX-400
spectrometer at 298 K. The resonances at 3.30 ppm of the residual
C
2
147.0
137.6
177.4
H-6′, H-2′
3
4eq
4ax
9
methanol in the H spectra and at 39.50 ppm for CD3OD in the 13C
1
spectra were used as internal references. 1H chemical shifts were
158.2
104.5
162.5
99.3
H-8
1
assigned using one-dimensional (1D) and two-dimensional (2D) H
10
5
H-8, H-6
H-6
NMR [correlation spectroscopy (COSY)], while 13C resonances were
assigned using 2D NMR [heteronuclear multiple-bond correlation
(HMBC) and heteronuclear multiple-quantum coherence (HMQC)].
A
B
6
6.16; d; J = 2.0
6.41; d; J = 2.0
8.19; d; J = 2.1
H-8
7
165.7
94.4
H-8, H-6
H-6
8
RESULTS AND DISCUSSION
1′
2′
3′
4′
5′
6′
124.3
123.8
141.3
152.6
118.2
127.4
H-5′
■
H-6′
Preparation of Quercetin and Epicatechin Sulfates.
Chemical hemisynthesis of quercetin and epicatechin sulfates
was performed on the basis of the procedure described by Jones
et al.20 for the preparation of sulfates of quercetin, with some
modifications, as previously reported by our group.25 For both
flavonoids, complex mixtures presenting diversely sulfated
compounds and the aglycones were obtained that were
fractionated on Sephadex LH-20 to separate respective fractions
containing mostly monosulfates (Figures 2 and 3).
H-5′, H-2′
H-6′, H-2′, H-5′
unresolved
H-2′
7.00; d; J = 8.6
8.00; dd; J = 8.6, 2.1
a
s, singlet; d, doublet; dd, double doublets; bs, broad singlet; m,
multiplet.
MS detection was performed in a Finnigan LCQ detector (Thermoquest,
San Jose, CA) equipped with an ESI source and an ion-trap mass
analyzer, which were controlled by the LCQ Xcalibur software. Both
the auxiliary and sheath gases were nitrogen at flow rates of 20 and
80 L/min, respectively. The source voltage was 4.5 kV; the capillary
Identification of Quercetin Sulfates. Table 1 shows
the retention and absorption and mass spectral data of the
peaks corresponding to quercetin sulfates observed in the
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Figure 4. MS2 spectrum of epicatechin 4′-O-sulfate in negative-ion mode.
chromatogram of Figure 2, as well as those of a standard of
quercetin obtained in the same analytical conditions.
peaks observed in the chromatogram of Figure 2 could not be
isolated in a sufficient amount and/or with a sufficient degree
of purity for their identification by NMR.
Tentative assignment of the monosulfates in the chromato-
grams was first made assuming the same elution order in re-
versed phase reported by Jones et al.,20 i.e., quercetin 3-O-
sulfate, quercetin 7-O-sulfate, quercetin 3′-O-sulfate, and
quercetin 4′-O-sulfate. It should be indicated that no formation
of quercetin 5-O-sulfate was reported by those authors. Indeed,
the hydroxyl group at position 5 of quercetin is by far the least
acidic,18 which makes it less likely to be substituted under the
mildly basic conditions employed for the sulfation reaction.
Therefore, the sulfate derivative at position 5 would be expected
to be produced to a lower extent than the other quercetin
monosulfates, so that it could be associated to peak 3 in our
chromatogram.
It has been reported that conjugation of the hydroxyl group
at position 3 of quercetin and other flavonols causes a band I
hypsochromic shift between 13 and 30 nm,22,26−29 whereas
smaller or no shifts in this band occurred when the conjugation
takes place in other positions.22,28 In our case, a large hypso-
chromic shift of λmax in band I (−21 nm) in relation to
quercetin was observed for peak 2, whereas small displacements
of 4−8 nm were found for peaks 3, 5, and 6 and no dis-
placement for peak 4. This observation supports the proposed
identity of peak 2 as quercetin 3-O-sulfate.
The results obtained in the NMR analysis of peaks 5 and 6
are shown in Table 2 compared to a standard of quercetin. Data
obtained for peak 6 agree with those previously published for
quercetin 3′-O-sulfate by other authors.20,21,30 The assignment
of the location of the sulfate group at position 3′ was based on
the characteristic up- and downfield displacements of protons
1
and carbons with respect to quercetin (Table 3). In the H
NMR spectrum, representative downfield shifts of H-2′ (8.19
ppm) and H-6′ (8.00 ppm), which are in positions ortho and
para with respect to the sulfate group, compared to quercetin
(H-2′ and H-6′ at 7.72 and 7.61 ppm, respectively) are
observed. Further confirmation of the presence of the sulfate
group at position 3′ was obtained from the carbon shifts
observed in the 13C NMR spectrum. A large upfield shift of
4.9 ppm was produced in C-3′ in relation to quercetin, while
C-2′/C-4′ and C-6′ (in ortho and para positions with regard to
C-3′, respectively) underwent significant downfield shifts (−7.9,
−3.9, and −5.8 ppm, respectively). These observations are also
in agreement with the expected upfield displacement for carbons
carrying sulfate groups and downfield shifts for the α carbons in
relation to the carbon bearing the sulfate.31
Interestingly, in the case of peak 5, tentatively assigned as
quercetin 4′-O-sulfate based on its elution characteristics,
neither up- nor downfield displacements for protons and
carbons with respect to quercetin were observed in the NMR
spectra (Table 3). The possibility that this result could be due
Separation of the sulfate mixture by semi-preparative HPLC
allowed us to obtain compounds 5 (24 mg, representing a final
yield of 4.8%) and 6 (10 mg, representing a final yield of 2%) in
the pure state to be submitted to NMR analysis. The other
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Table 5. H and 13C NMR Data and HMBC Correlations
Obtained for Epicatechin and Epicatechin 4′-O-Sulfate
Determined in CD3OD
to a loss of the sulfate group during compound isolation or
preparation for NMR analysis was discarded, because further
analysis of the sample by HPLC−DAD−ESI/MS after being
submitted to NMR analysis confirmed that the peak of the
compound appeared at the expected retention time (14 min)
and showed the same absorption spectrum and pseudomo-
lecular ion ([M − H]− at m/z 381) as the original peak.
Therefore, it was concluded that the presence of the sulfate at 4′
position does not modify the magnetic field of the molecule
with respect to the parent quercetin. As far as we know, no
previous NMR data have been reported for quercetin 4′-O-
sulfate, although Barron and Ibrahim30 performed NMR
analysis of quercetin-3,4′-O-disulfate, suggesting some effect
on the shift pattern for the 4′ position. However, those authors
obtained the spectra in dimethyl sulfoxide (DMSO), whereas
our analysis was performed in CD3OD, which may have an
influence on the chemical shifts. On the other hand, the NMR
behavior of the 4′ position of a quercetin disulfate should not
be necessarily reflected in the corresponding monosulfate.
Furthermore, the apparently unexpected behavior observed in
our study for quercetin 4′-O-sulfate could not be totally
unforeseen, because it was in agreement with data predicted by
the “Predict 1H NMR” option of ChemBioDraw Ultra software
based on the tables of chemical shifts for structural determina-
tion of organic compounds.32 Finally, the same behavior was
also observed in the case of epicatechin 4′-O-sulfate, as dis-
cussed below.
Identification of Epicatechin Sulfates. Figure 3 shows
the profile of monosulfates collected after fractionation of the
crude reaction mixture in the Sephadex LH-20 column. Four
compounds were obtained that were tentatively identified as
epicatechin 5-O-sulfate, epicatechin 3′-O-sulfate, epicatechin
7-O-sulfate, and epicatechin 4′-O-sulfate based on their mass
spectra fragmentation patterns and retention characteristics,
as discussed in a previous paper by our group.25 In catechins,
there are no large differences in the acidities of the four
phenolic hydroxyls, which possess pKa values between 9.02 and
9.5833 and are more reactive in the synthesis conditions used
than the non-phenolic hydroxyl at position 3, which explains
that no relevant formation of the corresponding sulfate was
observed.
a
1
ring position δ H (ppm); m; J (Hz)
Epicatechin
δ
13C (ppm)
HMBC
C
2
4.88; s
79.8
67.4
29.2
H-6′, H-2′, H-4
H-2, H-4
H-2
3
4.16; m
4eq
4ax
9
2.72; dd; J = 2.8, 16.8
2.85; dd; J = 4.5, 16.7
157.3
100.8
157.6
95.8
H-4
10
5
H-8, H-6, H-4
H-6, H-4
H-8
A
B
6
5.90; d; J = 2.3
5.93; d; J = 2.3
6.96; d; J = 1.9
7
158.0
96.3
H-8, H-6
H-6
8
1′
2′
3′
4′
5′
6′
132.3
115.3
145.9
145.7
115.8
119.3
H-2, H-5′
H-2, H-6′
H-5′,H-2′
H-6′, H-2′
H-6′
6.74; d; J = 8.1
6.79; dd; J = 8.1, 1.8
H-2′, H-2
Epicatechin 4′-O-Sulfate
79.7
C
2
4.80; s
H-6′, H-2′, H-4
H-4
3
4.16; m
67.3
29.1
4eq
4ax
9
2.72; dd; J = 2.9, 16.8
2.85; dd; J = 4.4, 16.8
unresolved
156.9
100.0
157.6
95.7
unresolved
H-8, H-6, H-4
H-6
10
5
A
B
6
5.90; d; J = 2.2
5.92; d; J = 2.2
6.99; d; J = 1.9
H-8
7
157.9
96.2
H-8, H-6
unresolved
H-5′, H-2
H-6′, H-2
H-5′, H-2′
H-6′, H-2′
H-6′
8
1′
2′
3′
4′
5′
6′
132.2
115.2
145.8
145.6
115.7
119.2
6.69; d; J = 8.1
6.80; dd; J = 8.2, 1.9
H-2′, H-2
a
s, singlet; d, doublet; dd, double doublets; bs, broad singlet; m,
multiplet.
Table 4 shows the chromatographic and UV and mass
spectral data of the four prepared monosulfates and epicatechin.
In this case, modifications in the absorption spectrum are not
much useful for identification purposes, because for the com-
pounds considered, only a small hypsochromic shift (less than
4 nm) in the maximum wavelength (band II) was produced in
relation to epicatechin. Obviously, all monosulfates showed the
same pseudomolecular [M − H]− ion at m/z 369, although
their fragmentation patterns differed depending upon the ring
of substitution on either A or B, as previously reported.25 Thus,
characteristic signals in the MS2 spectrum are observed at m/z
217 (137 + 80; 1,3A− fragment ion bearing a sulfate moiety) and
247 (167 + 80; 1,2A− ion + HSO3) when the sulfate moiety was
located on the A ring, whereas signals at m/z 231 (151 + 80;
1,3B− ion + HSO3) and 201 (121 + 80; 1,2B− ion + HSO3)
appeared when the sulfate moiety was located on the B ring, as
shown in the example of Figure 4.
The results obtained are shown in Table 5 compared to epi-
catechin. All carbons and aromatic protons could be assigned
on the basis of their chemical shifts and HMBC correlations. As
for quercetin 4′-O-sulfate, no significant displacements were
found in protons or carbons with regard to epicatechin, and
further analysis by HPLC−DAD−ESI/MS of the solution
employed in the NMR analysis also confirmed that the com-
pound was not altered and no loss of the sulfate residue was
produced during NMR processing. Overall, this result supports
that the presence of a unique sulfate residue at position 4′ does
not produce upfield displacement in the carbons bearing the
sulfate group nor downfield shifts of the carbons in positions
ortho and para. In our knowledge, this is the first time that
1H and 13C NMR data of an epicatechin sulfate have been
published.
In summary, monosulfate derivatives of quercetin and
epicatechin were prepared by hemisynthesis and characterized
according to their chromatographic behavior, absorption, and
mass spectra. Three compounds could be further isolated and
identified by 1H and 13C NMR as quercetin 3′-O-sulfate, quercetin
4′-O-sulfate, and epicatechin 4′-O-sulfate. It was observed that,
Further fractionation of the monosulfate mixture by semi-
preparative HPLC allowed for the separation of peak 4 (20 mg,
representing a final yield of 4%) in the pure state for its analysis
by 1H and 13C NMR. The rest of the compounds could not be
obtained with the sufficient degree of purity for NMR analysis.
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Journal of Agricultural and Food Chemistry
Article
(12) Agarwal, C.; Veluri, R.; Kaur, M.; Chou, S. C.; Thompson, J. A.;
Agarwal, R. Fractionation of high molecular weight tannins in grape
seed extract and identification of procyanidin B2-3,3′-di-O-gallate as a
major active constituent causing growth inhibition and apoptotic death
of DU145 human prostate carcinoma cells. Carcinogenesis 2007, 28,
1478−1484.
while sulfation at position 3′ induced a large upfield shift in the
carbon bearing the sulfate group and downfield displacements
of the adjacent carbons, no significant up- or downfield shifts
were observed with respect to parent flavonoid when sulfation
was produced at position 4′. All of the observations made
allowed us to assign the peaks corresponding to the different
monosulfate metabolites of quercetin and epicatechin in the
HPLC chromatograms. The obtained results are of interest
because they contribute patterns for the identification of these
metabolites in biological samples and for their preparation in
view to their use as standards with analytical purposes and their
employment in in vitro studies.
(13) Fraga, C. G.; Oteiza, P. I. Dietary flavonoids: Role of
(−)-epicatechin and related procyanidins in cell signaling. Free Radical
Biol. Med. 2011, 51, 813−823.
(14) Day, A. J.; Du Pont, M. S.; Ridley, S.; Rhodes, M.; Rhodes,
M. J. C.; Morgan, M. R. A.; Williamson, G. Deglycosylation of flavonoid
and isoflavonoid glycosides by human small intestine and liver
β-glucosidase activity. FEBS Lett. 1998, 436, 71−75.
(15) Nemeth, K.; Plumb, G. W.; Berrin, J. G.; Juge, N.; Jacob, R.;
Naim, H. Y.; Williamson, G.; Swallow, D. M.; Kroon, P. A.
Deglycosylation by small intestinal epithelial cell β-glucosidases is a
critical step in the absorption and metabolism of dietary flavonoid
glycosides in humans. Eur. J. Nutr. 2003, 42, 29−42.
AUTHOR INFORMATION
Corresponding Author
*E-mail: csb@usal.es.
Funding
GIP/USAL is financially supported by the Spanish Ministerio
́
de Ciencia e Innovacion through the projects AGL2007-66108-
C04-02 and AGL2009-12001 and the Consolider-Ingenio
■
(16) Donovan, J. L.; Luthria, D. L.; Stremple, P.; Waterhouse, A. L.
Analysis of (+)-catechin, (−)-epicatechin and their 3′ and 4′-O-
methylated analogs: A comparison of sensitive methods.
J. Chromatogr., B: Biomed. Sci. Appl. 1999, 726, 277−283.
(17) Baba, S.; Osakabe, N.; Natsume, M.; Terao, J. Absorption and
urinary excretion of procyanidin B2 [epicatechin-(4β-8)-epicatechin]
in rats. Free Radical Biol. Med. 2002, 33, 142−148.
2010 Programme (FUN-c-FOOD, CSD2007-00063). Montserrat
Duenas thanks the Spanish “Ramon
́
y Cajal” Programme for a
̃
contract.
́
(18) Barron, D.; Cren-Olive, C.; Needs, P. W. Chemical synthesis
of flavonoid conjugates. In Methods in Polyphenols Analysis; Santos-
Buelga, C., Williamson, G., Eds.; Royal Society of Chemistry: Cambridge,
U.K., 2003; pp 187−213.
Notes
The authors declare no competing financial interest.
(19) Barron, D. Recent advances in the chemical synthesis and
biological activity of phenolic metabolites. In Recent Advances in
Polyphenol Research; Daayf, F., Lattanzio, V., Eds.; Blackwell
Publishing: Oxford, U.K., 2008; Vol. 1, pp 317−358.
ACKNOWLEDGMENTS
The authors thank Anna Lithgow for her help in NMR analysis
and interpretation.
■
(20) Jones, D. J. L.; Jukes-Jones, R.; Verschoyle, R. D.; Farmer, P. B.;
Gescher, A. A synthetic approach to the generation of quercetin
sulfates and the detection of quercetin 3′-O-sulfate as a urinary
metabolite in the rat. Bioorg. Med. Chem. 2005, 13, 6727−6731.
(21) Needs, P. W.; Kroon, P. A. Convenient syntheses of
metabolically important quercetin glucuronides and sulfates. Tetrahe-
dron 2006, 62, 6862−6868.
(22) Day, A. J.; Bao, Y.; Morgan, M. R. A.; Williamson, G.
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synthesis of quercetin glucosides and glucuronides. In Methods in
Polyphenols Analysis; Santos-Buelga, C., Williamson, G., Eds.; Royal
Society of Chemistry: Cambridge, U.K., 2003; pp 177−185.
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