Enzymatic hemisynthesis of metabolites and conjugates of anthocyanins

Article abstract of DOI:10.1021/jf802844p

This work aims to study the phase II metabolization of anthocyanins that is likely to occur in vivo. Anthocyanins (delphinidin, cyanidin, and malvidin-3-glucosides) were incubated with phase II enzymes in the presence of activated cofactors in order to ob

Full text of DOI:10.1021/jf802844p

J. Agric. Food Chem. 2009, 57, 735–745 735
Enzymatic Hemisynthesis of Metabolites and
Conjugates of Anthocyanins
‡
IVA FERNANDES,† JOANA AZEVEDO,† ANA FARIA,†,‡ CONCEIC¸ AO CALHAU,
VICTOR DE FREITAS,† AND NUNO MATEUS*,†
˜
Chemistry Investigation Centre (CIQ), Department of Chemistry, Faculty of Sciences, University of
Porto, 4169-007 Porto, Portugal, and Department of Biochemistry (U38-FCT), Faculty of Medicine of
the University of Porto, 4200-319 Porto, Portugal
This work aims to study the phase II metabolization of anthocyanins that is likely to occur in vivo.
Anthocyanins (delphinidin, cyanidin, and malvidin-3-glucosides) were incubated with phase II enzymes
in the presence of activated cofactors in order to obtain glutathionyl conjugates, methylated and
glucuronydated compounds. Overall, the three anthocyanins tested were metabolized in vitro. Two
compounds were detected by HPLC after incubation of human liver cytosolic fraction with cyanidin-
3-glucoside and one compound with delphinidin-3-glucoside. These compounds were identified as
monomethylated products. LC-MS analysis yielded mass data that fit with the anthocyanin structures
bearing an additional methyl group in ring B. Several compounds were detected by HPLC after
incubation of human liver microsomes with malvidin, cyanidin, and delphinidin-3-glucosides. These
compounds were identified as monoglucuronides products after HPLC analysis. Conjugation with
glutathione also occurred as proved by the mass data obtained. However, in this case, two anthocyanin
equilibrium forms (flavylium and chalcone or water adducts) conjugated with glutathione were detected.
Overall, the data of the present work shows the feasibility of the in vitro enzymatic hemisynthesis of
metabolites and glutathione conjugates of anthocyanins. This first experimental approach may further
allow the achievement of new purified forms of anthocyanins, some of which do not occur in nature,
and also the determination of whether these compounds are the bioactive forms responsible for some
of the biological activities reported for anthocyanins.
KEYWORDS: Anthocyanins; metabolites; glutathione conjugates; glucuronydation; methylation
INTRODUCTION
Anthocyanins are the most important group of water soluble
plant pigments visible to the human eye. These compounds are
natural food colorants, widely found in plant derived foodstuffs,
such as berries, red grapes, cabbages, and other pigmented foods,
plants, or vegetables (6, 7). Depending on pH and the presence
of chelating metal ions, anthocyanins are intensely colored blue,
violet, or red. The structural differences between individual
anthocyanins are related to the number of hydroxyl and
methoxyl groups, the nature and number of sugars, and the
position of these attachments in the aglycone (Figure 1) (8).
Depending on the nutrition habits, the daily intake of
anthocyanins in humans has been estimated to be from 3 to
15 (9, 10) up to 150 mg/day (11). The moderate consumption
Flavonoids have potential effects in cancer prevention and
other health benefits; however, it has become clear that their
bioactive forms in vivo are not necessarily those which occur
in nature, but conjugates or metabolites arising from them after
absorption (1). In particular, there is now strong evidence for
the extensive phase I deglycosylation and phase II metabolism
of the resulting aglycones to glucuronides and O-methylated
forms during transfer across the small intestine and again in
the liver (2). Thus, these structural modifications may provide
a route for the bioavailability of dietary polyphenols to enable
their beneficial effects. Many studies have been conducted with
some flavonoids, such as (+)-catechin, (-)-epicatechin, (-)-
epigallocatechin-3-gallate, (-)-epicatechin-3-gallate, resveratrol,
and quercetin (3-5); however, very little progress has been
made in establishing the pharmacokinetics of anthocyanins, with
aspects such as absorption, biotransformation, and metabolism
left essentially unstudied.
* Correspondingauthor.Tel:+351.220402562.Fax:+3510.220402659.
E-mail: nbmateus@fc.up.pt.
† Faculty of Sciences, University of Porto.
Figure 1. Structures of Vitis vinifera anthocyanidin monoglucosides
(flavylium form).
‡ Faculty of Medicine of the University of Porto.
10.1021/jf802844p CCC: $40.75  2009 American Chemical Society
Published on Web 12/29/2008

736 J. Agric. Food Chem., Vol. 57, No. 2, 2009
Table 1. LC-MS Analysis of the Anthocyanin Metabolites
Fernandes et al.
reaction
methylation
compound
M+ (m/z) [M + H]+ (m/z)
MS2 (m/z)
MS3 (m/z)
CH3-Dp-3-gluc
CH3-Cy-3-gluc
479
463
317 (-162)
301 (-162)
303 (-14)
287 (-14)
glucuronidation
C6H8O6-Dp-3-gluc
C6H8O6-Cy-3-gluc
C6H8O6-Mv-3-gluc
641
625
669
303 (-338), 479 (-162)
287 (-338), 449 (-176), 463 (-162)
331 (-338), 493 (-176), 507 (-162)
287 (-176)
331 (-176)
glutathione conjugates
SG-Cy-3-gluc
SG-Cy-3-gluc
SG-Dp-3-gluc
SG-Dp-3-gluc
754
772
770
788
463 (-309), 610 (-162), 754 (-18)
479 (-291), 608 (-162)
335 (-144), 353 (-126), 461 (-18)
479 (-309), 608 (-162, -18), 626 (-162) 335 (-144), 353 (-126), 461 (-18)
of such compounds through the intake of products such as red
wine (12) or bilberry extract (13) was described to be associated
with the prevention of diseases related to oxidative stress, such
as coronary heart diseases and cancer (14).
parent glycosides (19-21). It is only recently that researchers
have begun to suggest that anthocyanins are metabolized;
however, the identification of derived metabolites has been
limited as a result of their diversity and low concentrations in
the blood. Anthocyanins are rapidly absorbed from both the
stomach (22) and small intestine (23). They then appear in blood
circulation and urine as intact, methylated, glucurono- and/or
sulfoconjugated forms (24-26).
Studies on the biological activities demonstrated that antho-
cyanins and anthocyanidins are powerful antioxidants in various
test systems (15-17). Furthermore, anti-inflammatory actions
as well as antimicrobial and antitumor activities have been
attributed to anthocyanins (14, 18). So far, most of the research
on the biological activities of anthocyanins with regard to their
potential health effects has been performed in vitro. For that
reason, some research in the past few years have focused on
the bioavailability and biotransformation of anthocyanins, which
could be major determinants of the biological activity of these
compounds in vivo.
It has been previously reported that anthocyanins are poorly
absorbed and circulate in the blood exclusively as unmetabolized
Figure 2. (A) HPLC chromatogram of the methylation products (2 and 3)
of cyanidin-3-glucoside (1) recorded at 520 nm. (B) UV-vis spectra of
the methylated products as recorded by the diode array detector.
Figure 3. (A) HPLC chromatogram of the methylation product (2) of
delphinidin-3-glucoside (1) recorded at 520 nm. (B) UV-vis spectrum of
the methylated product as recorded by the diode array detector.

Enzymatic Hemisynthesis of Metabolites and Conjugates
J. Agric. Food Chem., Vol. 57, No. 2, 2009 737
Figure 4. (A) HPLC chromatogram of cyanidin-3-glucoside (5) and its glucuronidated products (1-4), recorded at 520 nm. (B) UV-vis spectra of the
glucuronidated products as recorded by the diode array detector.
The gut microflora seem to play an important role in the
biotransformation of anthocyanins in the corresponding phenolic
acids derived from the B-ring of the anthocyanin skeleton (27).
As it is fundamental to determine whether these new compounds
are the bioactive forms responsible for some of the biological
activities reported for anthocyanins, the achievement of purified
metabolites of anthocyanins is crucial. Bearing this, the in vitro
phase II enzymatic hemisynthesis of new anthocyanin derived
compounds was attempted. For that purpose, glycosides of cyanidin
(Cy) and delphinidin (Dp), which represent the most abundant
anthocyanins in fruits, and glycosides of malvidin (Mv) were
extracted from grape skins Vitis Vinifera and incubated with rat
liver microsomes/cytosol (catechol-O-methyltransferases (COMT)
and UDP-glucuronyltransferases (UGT)) or comercially available

738 J. Agric. Food Chem., Vol. 57, No. 2, 2009
Fernandes et al.
Figure 5. (A) HPLC chromatogram of delphinidin-3-glucoside (6) and its glucuronidated products (1-5), recorded at 520 nm. (B) UV-vis spectra of the
glucuronidated products as recorded by the diode array detector.
purified by TSK Toyopearl gel column (250 × 16 mm i.d.) chroma-
enzymes (glutathione peroxidase (GP)). The analysis of the reaction
samples was carried out by HPLC-DAD and LC-MS.
tography according to the procedure described previously (28). The
extract was freeze-dried and stored at -18 °C until used.
The extract was analyzed by HPLC (Knauer K-1001) on a reversed-
phase column (250 × 4.6 mm, 5 µm, C18) (Merck, Darmstadt);
detection was carried out at 520 nm using a diode array detector (Knauer
K-2800) as previously reported (28).
MATERIALS AND METHODS
Reagents. Toyopearl gel was purchased from Tosoh (Tokyo, Japan).
Acetonitrile (CH3CN) and dimethyl sulfoxide (DMSO) were purchased
from Fluka (Madrid, Spain). Trichloroacetic acid (CCl3COOH) was
obtained from Merck (Darmstadt, Germany). S-Adenosyl-L-methionine
(SAM), uridine 5′-diphosphoglucuronic acid (UDPGA), glutathione
(GSH), glutathione peroxidase (GP), methanol (MeOH), hydrogen
peroxide (H2O2), and formic acid (HCOOH) were obtained from Sigma-
Aldrich (St. Louis, Missouri).
Anthocyanin Isolation. Grape skin anthocyanins (Vitis Vinifera)
were extracted with an aqueous solution of methanol (1:1) acidified
with HCl, for 2 days at room temperature. The Vitis Vinifera grape
anthocyanin extract was filtered in a 50 µm nylon membrane and then
LC-MS analysis was performed on a liquid chromatograph (Hewlett-
Packard 1100 series) equipped with an AQUATM (Phenomenex,
Torance, CA, USA) reversed-phase column (150 × 4.6 mm, 5 µm,
C18), thermostatted at 35 °C as reported elsewere (28).
HPLC Preparative Conditions. The pigments were purified by
preparative HPLC (Knauer K-1001) on a reversed-phase column
(250 × 25 mm, 10 µm, C18) (Merck, Darmstadt), and detection
was carried out at 520 using a UV-vis detector (Hitachi, L-2420)
(Merck, Darmstadt). The solvents were A, H2O/HCOOH (9:1), and

Enzymatic Hemisynthesis of Metabolites and Conjugates
J. Agric. Food Chem., Vol. 57, No. 2, 2009 739
Figure 6. (A) HPLC chromatogram of malvidin-3-glucoside (5) and its glucuronidated products (1-3), recorded at 520 nm, peonidin-3-glucoside (4). (B)
UV-vis spectra of the glucuronidated products as recorded by the diode array detector.
B, H2O/MeOH/HCOOH (4:5:1). The gradient consisted of 65-15%
A for 70 min at a flow rate of 10 mL min-1. The column was washed
with 100% B for 20 min and then stabilized at the initial conditions
for another 20 min. Each pigment was collected and concentrated
under vacuum. The purity of the isolated compounds was confirmed
by HPLC-DAD-MS. The following anthocyanins were isolated:
delphinidin-3-glucoside (Dp-3-gluc), cyanidin-3-glucoside (Cy-3-
gluc), and malvidin-3-glucoside (Mv-3-gluc), although the last one
was slightly contaminated with peonidin-3-glucoside.
Protein Extraction. Rats were anesthetized with sodium pentobar-
bital (300 mg/kg b.w.), and transcardiac perfusion was performed with
ice-cold NaCl 0.9% (p/v) solution. Rat liver was extracted, weighed,
and homogenized using a Thomas Teflon (DuPont, Wilmington, DE)
homogenizer in solution A (62.5 mM KH2PO4, 50 mM Na2HPO4, and
0.1%Triton X-100, pH 7.4) (2 mL g-1) and kept continuously on ice.
This mixture was centrifuged at 12 000g for 20 min at 4 °C. The
cytosolic supernatant was stored for further protein quantification. The
resultant pellet was mixed with solution B (100 mM Hepes and 100
mM sacarose, pH 7.2) to form microsomes and centrifuged at 100 000g
for 1 h at 4 °C (29). The microsomal pellet was washed in the same
solution and subjected to a second centrifugation at 100 000g for 30
min. Finally, the microsomes were sonicated in solution B.
Rat COMT Catalyzed O-Methylation of Catechol or Pyrogallol-
Containing Anthocyanins. Dp-3-gluc, Cy-3-gluc and Mv-3-gluc were
incubated with rat liver cytosol protein fraction in a final volume of
250 µL of 10 mM Tris-HCl buffer, pH 7.4.
A typical incubation contained 4 mg mL-1 of the cytosol protein,
which was first mixed with 1.2 mM MgCl2, 1 mM DTT, and 2.5 mM
SAM, and finally 400 µM of the substrate dissolved in DMSO (final
concentration 4%) was added and the mixture incubated at 37 °C for
up to 30 min to allow product formation under constant mild shaking.
Negative controls were carried out by omitting SAM.
The reaction was stopped after 30 min by adding methanol (50 µL)
to the reaction mixture to precipitate proteins. After centrifugation (5
min at 7000g), the samples were analyzed using HPLC-DAD (injection
volume 50 µL). The samples were stored at -18 °C until further
analysis. For the identification of the metabolite peaks, HPLC-DAD
and LC-DAD-MS (in positive ion mode) analysis was used.
Rat UGT Catalyzed Glucuronidation of Anthocyanins. Glucu-
ronidation of anthocyanins was carried out as described elsewhere with
slight modifications (27). Dp-3-gluc, Cy-3-gluc, and Mv-3-gluc were
incubated with rat liver microsomes in a final volume of 250 µL of 10
mM sodium phosphate buffer, pH 7.4. A typical incubation contained 4
mg mL-1 of the microsomal protein, which was first mixed with 100 mM
of MgCl2 and 2 mM of UDPGA, and finally 400 µM of substrate dissolved
in DMSO (final concentration 4%) was added and the mixture incubated
at 37 °C for up to 30 min to allow product formation under constant mild
shaking. Negative controls were carried out by omitting UDPGA. The
reaction was stopped as described above for O-methylation.
Protein Determination. The protein content of cytocolic and
microssomal fractions were determined as described by Bradford (30),
with bovine serum albumin as standard. The resulting cytosolic and
microsomal fractions were divided into aliquots and frozen at -80 °C
until use.

740 J. Agric. Food Chem., Vol. 57, No. 2, 2009
Fernandes et al.
The solvents were A, H2O/HCOOH (9:1), and B, H2O/CH3CN/
HCOOH (6:3:1). The gradient consisted of 26-45% B for 50 min,
45-85% B for 25 min, and 85-0% B for 10 min at a flow rate of 1.0
mL min-1. The column was washed with 100% B for 20 min and then
stabilized at the initial conditions for another 20 min. Detected peaks
were scanned between 200 and 600 nm. Compounds were first identified
according to retention time and UV-vis spectra.
The anthocyanin glucuronidated and glutathione conjugated products
were also analyzed by HPLC using the same conditions with a different
gradient: 0-100% B for 70 min at a flow rate of 1.0 mL min-1
.
LC-MS Analysis. HPLC analysis of the anthocyanin methylated
products was performed on a liquid chromatograph (Hewlett-Packard
1100 series) equipped with an AQUATM (Phenomenex, Torance, CA,
USA) reversed-phase column (150 × 4.6 mm, 5 µm, C18), thermo-
statted at 35 °C. Solvents were A, H2O/HCOOH (9.9:0.1), and B, H2O/
CH3CN/HCOOH (6.9:3:0.1). The HPLC gradient used was the same
as that reported above for the HPLC analysis. Double online detection
was done in a photodiode spectrophotometer and by mass spectrometry.
The mass detector was a Finnigan LCQ (Finnigan Corporation, San
Jose, USA) equipped with an API source, using an electrospray
ionization (ESI) interface. Both the auxiliary and the sheath gases were
a mixture of nitrogen and helium. The capillary voltage was 3 V and
the capillary temperature 190 °C. Spectra were recorded in positive
ion mode between m/z 120 and 1500. The mass spectrometer was
programmed to do a series of three scans: a full mass, a zoom scan of
the most intense ion in the first scan, and a MS-MS of the most intense
ion using relative collision energies of 30 and 60.
The anthocyanin glucuronidated and glutathione conjugated products
were also analyzed by LC-MS using the same gradient reported above
for the HPLC analysis.
RESULTS AND DISCUSSION
Delphinidin-3-glucoside (Dp-3-gluc), cyanidin-3-glucoside
(Cy-3-gluc), and malvidin-3-glucoside (Mv-3-gluc) purified
from grape skins (Vitis Vinifera) (Figure 1) were incubated with
rat liver microsomes, cytosol, or glutathione peroxidase as a
source of phase II enzymes in the presence of activated cofactors
in order to obtain glutathionyl conjugates, methylated and
glucuronydated compounds. The metabolites obtained were
analyzed by HPLC-DAD. In order to obtain structural informa-
tion about the metabolites, all solutions were analyzed by LC-
MS. The mass data of each metabolite and their fragments are
summarized in Table 1.
Figure 7. (A) HPLC chromatogram of cyanidin-3-glucoside (2) and its
glutathionyl product (1), recorded at 350 nm. (B) UV-vis spectrum of
the glutathionyl product as recorded by the diode array detector.
Oxidation of Anthocyanins by Horseradish Peroxidase in the
Presence of Glutathione. Oxidation of anthocyanins by horseradish
peroxidase in the presence of glutathione was carried out as described
elsewhere for quercetin with slight modifications (31). Dp-3-gluc, Cy-
3-gluc, and Mv-3-gluc, all added from a 10 mM stock solution in
DMSO, were incubated with glutathione peroxidase in a final volume
of 1000 µL of 50 mM sodium phosphate buffer, pH 7.0. A typical
incubation contained 0.1 µM of glutathione peroxidase, which was first
mixed with 1 mM of GSH and 150 µM substrate dissolved in DMSO
(final concentration 1.5%). The reaction was started by the addition of
200 µM hydrogen peroxide.
The mixture was incubated at 37 °C for up to 30 min to allow product
formation under constant mild shaking. Negative controls were carried
out by omitting GSH.
The following additions were made to the incubation in the course
of time for the generation of sufficient amounts of GSH conjugates of
Dp-3-gluc, Cy-3-gluc, and Mv-3-gluc. Substrate (150 µM) was added
every 5 min during the 30 min incubation period, 0.1 µM HRP was
added at 0, 5, 15, and 20 min of incubation, and glutathione (1 mM)
was added at 0 and 15 min of incubation. The reaction was stopped as
described above for O-methylation.
Methylation. Dp-3-gluc, Cy-3-gluc, and Mv-3-gluc were
incubated with rat liver cytosolic protein fraction as a source
of phase II COMT enzyme in the presence of SAM.
In the case of the incubation of Cy-3-gluc, two new
chromatographic peaks (2 and 3) with close retention times were
detected in the HPLC chromatogram (Figure 2A). Analysis by
LC-MS revealed two peaks at m/z 463, confirming the identity
of these metabolites as monomethylated metabolites of Cy-3-
gluc. Fragmentation of this ion yielded one peak at m/z 301,
corresponding to the methylated aglycone. This observation
indicates that the methylation site was not located on the sugar
moiety. Attending to the enzyme specificity to the ortho-catechol
group, the additional methyl group is supposed to be in ring B.
The analysis of the absorption spectra of the two methylated
products (Figure 2B) allowed the identification of the position
of the attached methyl groups. Metabolite 2 was assigned as
peonidin-3-O-ꢀ-D-glucose from the comparison of the retention
time, mass, and absorption spectra (λma´x. ) 517 nm) of the pure
standard in the conditions of analysis. Metabolite 3 was thus
expected to have a methyl group at the C4′ position corre-
sponding to the structure of isopeonidin-3-O-ꢀ-D-glucose. Cy-
3-gluc has already been found, in vivo, metabolized in both
the 4′ and 3′positions (32).
HPLC-DAD Analysis. HPLC analysis of the anthocyanin methy-
lated products was performed on an Elite Lachrom system (L-2130)
equipped with a 250 × 4.6 mm i.d. reversed phase C18 column (Merck,
Darmstadt); detection was carried out at 520 nm using a diode array
detector (L-2455).

Enzymatic Hemisynthesis of Metabolites and Conjugates
J. Agric. Food Chem., Vol. 57, No. 2, 2009 741
Figure 8. Fragmentation pattern of one Cy-3-gluc glutathionyl product in the form of flavylium (A) and chalcone (B) forms.
However, in the case of Dp-3-gluc, only one chromatographic
peak was detected in the HPLC chromatogram (Figure 3A).
MS fragments of the metabolite agreed with those of O-methyl
delphinidin-3-glucoside (m/z 479) and its aglycone, O-methyl
Dp (m/z 317) (Table 1), indicating that Dp-3-gluc was methy-
lated in its aglycone moiety. However, the HPLC retention time
of the metabolite was completely different from that of
petunidin-3-O-ꢀ-D-glucose (Pt-3-gluc) (3′-O-methyl Dp-3-gluc),
indicating that the site of methylation was likely to be the 4′-
position of ring B. The UV-vis spectrum of the metabolite also
supported the idea that the metabolite was not Pt-3-gluc (λma´x
) 506 nm for O-methyl Dp-3-gluc and 540 nm for Pt-3-gluc)
(Figure 3B). This observation is in accordance with what
happens in vivo. This metabolite has also been identified in
several tissues as a major metabolite, especially in the liver (33),
but no 3′ or 5′-O-methyl Dp-3-gluc were detected. Therefore,
4′-OH methylation is the main path of Dp-3-gluc metabolism
in rats (33). Furthermore, 4-O-methyl gallic acid, which is the
corresponding phenolic acid that probably results from the
cleavage of O-methyl Dp-3-gluc by intestinal microflora, has
been detected in human urine (34, 35).
namely, the hydroxyl positions at C5 and C7 of ring A and the
hydroxyl groups at C3′ and C4′ of ring B.
This information is also important for the interpretation of
the UV-vis spectra of the four monoglucuronide of Cy-3-gluc,
which are shown in Figure 4B. Two of the monoglucuronides
possess only one maximum of absorption, whereas the other
two possess one clear maximum of absorption plus a marked
shoulder peak around 410 nm. According to the literature, the
preferred location for a second sugar would be at position 5
rather than at 7 or 4′ (36). Diglycoside anthocyanins present
absorption maxima toward lower wavelengths than the corre-
sponding monoglycosides, and the presence of a shoulder in
the visible region at about 440 nm in the 3-monoglycosides is
not seen in the 3,5-diglycosides. A shoulder has been observed
in the UV-vis spectra of Cy and Dp derivatives with O-glycosyl
moieties on their B-rings (37, 38). Another work reported that
the UV-vis spectra of anthocyanidins with a sugar moiety in
the 5-position of ring A do not display the shoulder peak around
420-440 nm (39), which could help to explain the observed
results herein. Therefore, it can be proposed that Cy-3-gluc-
monoglucuronides 1 and 3 are glucuronidated at ring A, whereas
Cy-3-gluc-monoglucuronides 2 and 4 are ring B glucuronides.
Concerning the incubation of Dp-3-gluc, five Dp-3-gluc-
monoglucuronides were identified by HPLC-DAD-MS analysis
(Figure 5A). Only one glucuronidated group is present in each
new compound as all displayed the same mass of m/z 641, which
is characteristic of the Dp-3-gluc monoglucuronide. Fragmenta-
tion of this ion by LC-MS yielded two peaks at m/z 303 and
479, corresponding to the loss of glucose and glucuronyl plus
glucose, respectively (Table 1). The fragmentation pattern
indicates that the glucuronidation site was located on ring
A or B.
To clarify the glucuronidation site of the metabolites, UV-vis
spectra of each peak was analyzed and compared with those of
authentic anthocyanin (Figure 5B).
The analysis of the UV-vis spectra of the five glucuronidated
products revealed maximum absorption wavelengths obtained
for peaks 1, 2, 3, 4, and 5 at 521, 522, 506, 526, and 526 nm,
respectively. As seen in Figure 5B, only peak 3 possesses one
clear maximum absorption plus a marked shoulder peak around
415 nm, whereas the other monoglucuronides possessed only
one absorption maximum.
According to the former argument, it was expected that only
two of the detected peaks lacked the presence of a shoulder in
the visible region at about 440 nm. However, in the experimental
conditions used and in result of the minor quantities of
metabolites obtained, as seen in Figure 5B, they could not be
These results are quite interesting because no naturally
occurring food Dp-3-gluc or Cy-3-gluc methylated forms at the
4′ position of ring B have been identified. This work has thus
shown that phase II metabolic modification of ingested antho-
cyanins can give rise to new anthocyanins that cannot be
obtained from food sources. The anthocyanin Mv-3-gluc was
also incubated with rat liver cytosol in the presence of SAM,
but no methylated products were detected.
Glucuronidation. Another reaction in competition with the
intestinal and/or chemical degradation at neutral pH in the gut
is phase II glucuronidation, which could take place in the gut
or, after absorption of the aglycone or the intact glucoside, in
the liver. Therefore, it was assumed that the phase II glucu-
ronidation mainly takes place in the liver and intestinal wall.
The reaction of each anthocyanin with a system consisting of
microsomes and UDPGA was used as an in vitro model for the
glucuronidation reaction.
After incubation of Cy-3-gluc with rat liver microsomes in
the presence of UDPGA, four newly formed compounds were
detected by HPLC (Figure 4A). These compounds were
identified as anthocyanin-monoglucuronides by LC-MS (Table
1). MS2 fragments of the metabolites (m/z 625) were m/z 287
(-338), 449 (-176), and 463 (-162), indicating that Cy-3-
gluc was glucuronidated in its aglycone moiety. The formation
of four different monoglucuronides shows that glucuronidation
takes place at any free hydroxyl position in the Cy molecule,

742 J. Agric. Food Chem., Vol. 57, No. 2, 2009
Fernandes et al.
Figure 9. (A) HPLC chromatogram of delphinidin-3-glucoside (4) and its glutathionyl products (1-3), recorded at 350 nm. (B) UV-vis spectra of the
glutathionyl products as recorded by the diode array detector.
detected. Therefore, it is only possible to assume that peak 3
does not correspond to Dp-3-gluc glucuronidated in ring A.
In the case of Mv-3-gluc, three different glucuronidated
products were detected by HPLC-DAD (Figure 6A) corre-
sponding to the substitution of each of the OH free groups in
the positions C4′, C5, and C7. These peaks were identified as
monoglucuronides by HPLC-MS (Table 1). As previously
reported, a sugar substitution in ring A results in a single peak
in the UV-vis spectra that corresponds to the maximum
wavelength without any shoulder.
It can thus be assumed that the substitution in position C4′
should correspond to metabolite 2, as seen from the respective
UV-vis spectrum (Figure 6B).
Additionally, hypsochromic shifts at maximum wavelengths
observed in the spectra of some conjugates tentatively identified

Enzymatic Hemisynthesis of Metabolites and Conjugates
J. Agric. Food Chem., Vol. 57, No. 2, 2009 743
Figure 10. Fragmentation pattern of one Dp-3-gluc glutathionyl product in the form of flavylium (A) and chalcone (B).
as glucuronidated in ring B were also found for anthocyanidins
glycosylated in the B-ring (38).
Interestingly, the anthocyanidin monoglucosides were all
substrates for UGTs. Curiously, once again no product with
more than one substitution was detected.
Glutathione Conjugates. Previous investigations with chemi-
cal systems have shown that quercetin (40, 41) and other
catechol-containing flavonoids, such as catechin (42), taxifolin,
luteolin (43), fisetin, and 3,3′,4′-trihydroxyflavone (44), react
with GSH to generate mono- and diglutathionyl adducts.
Because the structural similarities between these compounds
and anthocyanins are expected, the same adduct formation
occurs using similar experimental conditions.
and another at m/z 479 (-162 and -129) that may correspond
to the loss of one glucose molecule and a fragment of the
glutathione molecule, probably resulting from fragmentation y3
(Figure 10A). MS2 fragment patterns of peaks 1 and 3 showed
fragments at m/z 754 (-18) corresponding to the loss of one
water, at m/z 610 (-162) corresponding to the loss of one
glucose molecule, and at m/z 479 (-18, -162, and -129) that
may correspond to the two previous losses plus a fragment of
the glutathione molecule, probably resulting from fragmentation
y3 (Figure 10B).
Some quercetin glutathionyl adducts were already described
with a similar fragmentation pattern (45).
The UV-vis spectra of Dp-3-gluc glutathionyl adducts
indicated that peaks 1 and 3 possess one maximum absorption
at 304 and 350 nm, respectively, while peak 2 possesses one
maximum around 490 nm and a shoulder around 380 nm
(Figure 9B). These absorption maxima may indicate that two
anthocyanin equilibrium forms, the flavylium (peak 2) and the
respective chalcone form of anthocyanins (peaks 1 and 3), are
present.
Although peaks 1 and 3 showed the same mass value and
fragmentation pattern, they had different retention times, 17.96
and 22.94 min. The different global polarity and spectroscopic
characteristics of the newly formed compounds thus indicate
that the substitutions may have occurred in different positions
of the anthocyanin. The most probable sites are the C6 and C8
positions of ring A, as the formation of ring A conjugates is
favored by acidic reaction conditions. For quercetin, two ring
A monoglutathione conjugates (8-glutathionyl quercetin and
6-glutathionyl quercetin), at pH 7.0, have recently been observed
(41). In the case of Mv-3-gluc, no glutathionyl adducts were
detected.
The HPLC chromatogram recorded at 350 nm, after the
incubations of Cy-3-gluc with GP/H2O2 in the presence of
glutathione, is shown in Figure 7A.
LC-MS data, have shown a peak at m/z 772 (peak 1), which
fits with the structure of Cy-3-gluc glutathionyl adducts linked
with one water molecule, probably yielding the chalcone form
of the anthocyanin moiety (Table 1). The MS2 fragment pattern
of peak 1 showed fragments at m/z 754 (-18) corresponding
to the loss of one water molecule, at m/z 610 (-162) corre-
sponding to the loss of one glucose molecule, and at m/z 463
(-18, -162, and -129) that may correspond to the two previous
losses plus a fragment of the glutathione molecule, probably
resulting from fragmentation y3 (Figure 8B). The UV-vis
spectrum of this compound 1 showed one maximum absorption
at 350 nm characteristic of the chalcone form of anthocyanins
(Figure 7B).
Traces of the compound detected at m/z 754 probably
corresponding to Cy-3-gluc glutathionyl adduct were also
detected by LC-MS between 25 and 28 min.
The conjugation of glutathione with 3′,4′-dihydroxy fla-
vonoids has been shown to be pH-dependent in cell-free
systems. Effectively, it has been demonstrated that low pH
favored the formation of ring A conjugates, while less acidic
conditions led to the generation of ring B adducts (40). Thus,
in Cy-3-gluc, ring A is likely to be the site of glutathione adduct
formation with anthocyanin.
The HPLC chromatogram recorded at 350 nm, after incuba-
tion of Dp-3-gluc with GP/H2O2 in the presence of glutathione
showed three new chromatographic peaks (1, 2, and 3) with
close retention times (Figure 9A).
More studies are needed for structural identification of the
products detected in order to better understand the molecular
pathways behind glutathionyl conjugate formation.
Conclusions. The present work may provide not only
important purified metabolites for the evaluation of biological
effects of anthocyanin metabolites in comparison with their
precursors but also standards for the following identification of
anthocyanin metabolites in biological samples. The in vitro
hemisynthesis reactions described herein are expected to mimic
the in vivo situations and could help to better understand the
absorption, bioavailability, and metabolism of these compounds.
Overall, where anthocyanins are concerned, a final and
complete evaluation of the biological effects of anthocyanins
will have to take all metabolites and degradation products of
these compounds into account. When considering the possible
bioactive mechanisms of action of anthocyanins and their in
vivo metabolites in cell systems, it is important to consider their
uptake and possible further metabolization by the cells. The
The mass data have shown a peak at m/z 770 (peak 2), which
fits with the structure of the Dp-3-gluc glutathionyl adduct.
Peaks 1 and 3 at m/z 788 possibly match the Dp-3-gluc
glutathionyl adducts bearing one water molecule, corresponding
to the chalcone form of anthocyanin (Table 1).
MS2 fragments of peak 2 originated fragments at m/z 608
(-162) that may correspond to the loss of one glucose molecule

744 J. Agric. Food Chem., Vol. 57, No. 2, 2009
Fernandes et al.
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ABBREVIATIONS USED
Dp-3-gluc, delphinidin-3-glucose; Cy-3-gluc, cyanidin-3-
glucose; Mv-3-gluc, malvidin-3-glucose; HPLC, high-perfor-
mance liquid chromatography; LC-MS, liquid chromato-
graphy-mass spectrometry; COMT, catechol-O-methyltrans-
ferases; UGT, UDP-glucuronyltransferases; GP, glutathione
peroxidase.
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Received for review September 12, 2008. Revised manuscript received
November 17, 2008. Accepted November 19, 2008. This work was
supported by FCT (Fundac¸a˜o para a Cieˆncia e Tecnologia) (POCI,
FEDER, Programa Comunita´rio de Apoio) by two Ph.D. student grants
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