Reaction between hydroxycinnamic acids and anthocyanin-pyruvic acid adducts yielding new portisins

Article abstract of DOI:10.1021/jf070968f

Three new anthocyanin-derived pigments were found to occur in a 2-year-old Port red wine. Their structures were elucidated through LC/DAD-MS and NMR analysis and were found to correspond to a pyranoanthocyanin moiety linked to substituted cinnamyl substit

Full text of DOI:10.1021/jf070968f

J. Agric. Food Chem. 2007, 55, 6349−6356 6349
Reaction between Hydroxycinnamic Acids and
Anthocyanin Pyruvic Acid Adducts Yielding New Portisins
−
†
†
‡
JOANA OLIVEIRA, VICTOR DE FREITAS, ARTUR M. S. SILVA, AND
,
†
NUNO MATEUS*
Centro de Investiga c¸ a˜ o em Qu ´ı mica, Departamento de Qu ´ı mica, Faculdade de Ci eˆ ncias, Universidade
do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal, and Departamento de Qu ´ı mica,
Universidade de Aveiro, 3810-193 Aveiro, Portugal
Three new anthocyanin-derived pigments were found to occur in a 2-year-old Port red wine. Their
structures were elucidated through LC/DAD-MS and NMR analysis and were found to correspond to
a pyranoanthocyanin moiety linked to substituted cinnamyl substituents. The structures of these
compounds are very similar to the one already reported for portisins, with a phenolic moiety replacing
the catechin moiety. The newly formed anthocyanin-derived compounds display a bathochromic shift
of the λmax (∼540 nm) when compared with their anthocyanin-pyruvic acid adduct precursor (λmax
)
5
11 nm), which may be due to the extended conjugation of the π electrons in the structures of those
pigments. Studies performed in model solutions helped to clarify the formation mechanism of these
pigments that can result from the nucleophilic attack of the olefinic double bond of a hydroxycinnamic
acid to the eletrophilic C-10 position of the anthocyanin-pyruvic acid adduct, followed by the loss of
a formic acid molecule and decarboxylation. The chromatic characterization of these malvidin-3-
glucoside-derived compounds revealed a higher resistance to discoloration against the nucleophilic
attack by water and bisulfite when compared to malvidin-3-glucoside that is almost converted into its
colorless hemiacetal form. However, the resistance to discoloration of these new pigments is not as
high as the one reported for catechin-derived portisins. This could be explained by the presence
of a smaller group (hydroxycinnamyl group), which does not protect so efficiently the chromophore
against nucleophilic attack at the C-2 position. The occurrence of these pigments in red wine
highlights new chemical pathways involving anthocyanin-pyruvic acid derivatives as precursors for
the formation of new pigments in subsequent stages of wine aging that may contribute to its color
evolution.
KEYWORDS: Portisins; hydroxycinnamic acids; wine; model solutions; anthocyanin; pyruvic acid
INTRODUCTION
red to red/orange due to several chemical reactions (oxidation,
reduction, and polymerization) in which anthocyanins partici-
pate, being in this way the precursors of new pigments. Figure
Anthocyanins are the most abundant pigments in young red
wines, being responsible for their red/purple color. However,
in solution, the color displayed by anthocyanins is dependent
on the pH of the medium (1, 2). At the wine pH (pH ∼3.5)
anthocyanins are likely to be present mainly in their colorless
hemiacetal form. Nevertheless, the flavylium cation structure
of anthocyanins is stabilized through copigmentation mecha-
nisms, which protect the molecule chromophore against nu-
cleophilic attack by water. These mechanisms may include
molecular association between anthocyanins and other molecules
of anthocyanins (self-association) or complexation with other
molecules, usually noncolored, referred to as copigments (3-
1
summarizes the different families of anthocyanin-derived
pigments that have been detected by HPLC in red wines. The
transformations involved in the formation of anthocyanin-
derived pigments were first thought to arise mainly from the
dimerization between anthocyanins (6) and condensation of
anthocyanins and flavanols in the presence or absence of
acetaldehyde (7-14). However, over the past years new families
of pigments, namely, pyranoanthocyanins, have been identified
and are known to result from the reaction between anthocyanins
and small molecules such as acetaldehyde (15), acetoacetic acid
5
). During maturation and aging, the wine color changes from
(16), pyruvic acid (17), vinylphenol (18), vinylguaiacol (19),
and vinylcatechol (20). This kind of pigment is supposed to
contribute to the orange hues of red wines observed during
maturation. In Port red wine, the anthocyanin-pyruvic acid
adducts (formed from the reaction of anthocyanins with pyruvic
*
Author to whom correspondence should be addressed (e-mail
nbmateus@fc.up.pt; fax +351.226082959; telephone +351.226082862).
†
Centro de Investiga c¸ a˜ o em Qu ´ı mica.
Universidade de Aveiro.
‡
1
0.1021/jf070968f CCC: $37.00
© 2007 American Chemical Society
Published on Web 06/30/2007

6350 J. Agric. Food Chem., Vol. 55, No. 15, 2007
Oliveira et al.
Figure 1. Summary of the main classes of anthocyanin-derived pigments already reported in red wines by HPLC.
acid) were the main pigments detected by HPLC after 1 year
of wine aging (21).
(Madrid, Spain); sinapic acid (g97%), ferulic acid (g98%), and
p-coumaric acid (g98%) were purchased from Fluka (Madrid, Spain).
Hydroxycinnamic acids are phenolic compounds very abun-
dant in grape juice and wine, and the three most common ones
are p-coumaric acid, caffeic acid, and ferulic acid. In grape
berries hydroxycinnamic acids exist as esters of tartaric acid
and are named p-coutaric acid, caftaric acid, and fertaric acid,
respectively. These compounds are present mainly in the pulp
of fruits and thus found in grape juices and wines. Caftaric acid
is by far the predominant hydroxycinnamic acid in grapes and
wines followed by the p-coutaric acid and in smaller amounts
the fertaric acid (22). These naturally occurring esters are
susceptible to hydrolysis by the cinnamyl esterase enzyme (23),
and this occurs in the aqueous acidic solution of wine,
releasing the free hydroxycinnamic acids, which are readily
detected in a few weeks old wine (24). The levels of hydroxy-
cinnamic acids observed in wines are usually 130 mg/L for
whites and 60 mg/L for red ones (25). With respect to red wines,
hydroxycinnamic acids may contribute to their color evolution
because they can participate as copigments through hydrophobic
stacking interactions with anthocyanin chromophores. Haslam
Wine Fractionation. The studied wine was a 2-year-old red Port
wine [pH 3.6, 20% alcohol (v/v), total acidity ) 6.5 g/L, total SO
0 mg/L], made from Touriga Nacional and Touriga Francesa varieties
Vitis Vinifera) grown in the Douro Demarcated Region (northern
2
)
2
(
Portugal). Several fractions were obtained from the Port wine sample
using two types of resins, TSK Toyopearl gel HW-40(S) and polyamide
60-80 mesh, SINOPEC, Hunan, China), and different percentages of
methanol aqueous solutions. The wine fractionation was performed
according to the method of He et al. (29). The fractions obtained were
analyzed by LC/DAD-MS in positive ion mode.
(
Synthesis and Purification of Malvidin-3-glucoside-Pyruvic Acid
Adduct. Grape skin anthocyanins were extracted with an aqueous
solution of ethanol (1:1) acidified with HCl, for 1 day at room
temperature. The anthocyanin extract was purified by TSK Toyopearl
gel column (250 × 16 mm i.d.) chromatography, the malvidin-3-
glucoside fraction being recovered with a solution of 20% (v/v) aqueous
ethanol.
The malvidin-3-glucoside-pyruvic acid adduct was synthesized
through reaction of the previous purified malvidin-3-glucoside with
pyruvic acid and later purified according to the method of Oliveira et
al. (30).
(
26) has also referred to copigmentation as the first step that
led to the formation of a covalent bond between the pigment
anthocyanin) and the copigment (8). In fact, it has already been
Reaction of Malvidin-3-glucoside-Pyruvic Acid Adduct with
Several Hydroxycinnamic Acids (Caffeic, Sinapic, Ferulic, and
p-Coumaric Acids) and Isolation of the Newly Formed Pigments.
Four aqueous solutions of ethanol (30%, v/v) with a 700 µM
concentration of malvidin-3-glucoside-pyruvic acid adduct were
prepared. To each solution was added caffeic acid, sinapic acid, ferulic
acid, or p-coumaric acid to obtain a molar ratio of hydroxycinnamic
acid/malvidin-3-glucoside-pyruvic acid adduct of 50:1. The pH of the
solutions was adjusted to 1.5 with HCl, and the solutions were left to
react at 35 °C in the dark. The formation of new pigments was
monitored by HPLC-DAD. After the maximum formation of the
pigments had been reached, the reaction was stopped, the solvent was
removed, and the major new pigments were purified by Toyopearl gel
column (250 × 16 mm i.d.). The elution was carried out with aqueous
methanol with increasing percentage of methanol up to 60% (v/v). The
(
shown that hydroxycinnamic acids can react directly with
anthocyanins, giving rise to new pigments with different color
hues (20).
More recently, a new class of anthocyanin-derived pigments
with unusual color properties, presenting a blue color in acidic
conditions, were found to occur in a 2-year-old Port wine and
were named portisins (27). Their structural characterization and
studies performed in model solutions revealed that these
pigments arise from the reaction between the anthocyanin-
pyruvic acid adducts and flavanols in the presence of acetal-
dehyde (27, 28). The detection and identification of portisins
point to new chemical reactions that possibly occur in red wines,
in which the major precursors involved are no longer antho-
cyanins but anthocyanin-derived pigments. The present work
deals with the detection in Port wine and characterization of
new portisin-type pigments arising from the reaction between
anthocyanin-pyruvic acid adducts and hydroxycinnamic acids.
5
0% (v/v) aqueous methanol fraction, containing the major compounds,
was then submitted to isolation of the individual pigments by semi-
preparative HPLC. The structure of each compound obtained was
elucidated by LC/DAD-MS and NMR.
HPLC Analysis. All of the extracts were analyzed using a Knauer
K-1001 HPLC on a 250 × 4.6 mm i.d. reversed-phase C18 column
(
5
Merck, Darmstadt, Germany); detection was carried out at 528 and
MATERIALS AND METHODS
40 nm using a Knauer K-2800 diode array detector. The solvents were
(A) H O/HCOOH (9:1) and (B) CH COOH/H O/CH CN (0.5:19.5:80).
The gradient consisted of 20-85% B for 70 min at a flow rate of 1.0
Reagents. TSK Toyopearl gel HW-40(S) was purchased from Tosoh
2
3
2
3
(Tokyo, Japan); caffeic acid (99%) was purchased from Sigma-Aldrich

New Portisins Arising from Hydroxycinnamic Acids
J. Agric. Food Chem., Vol. 55, No. 15, 2007 6351
1
1
Table 1. H Chemical Shifts of Vinylpyranomalvidin-3-O-glucoside
−
Table 2. H Chemical Shifts of Vinylpyranomalvidin-3-O-glucoside
−
OD/TFA (98:2)a
Syringol (Pigment II), Determined in CD
a
Catechol (Pigment I), Determined in CD
3
3
OD/TFA (98:2)
position
δ 1H; J (Hz) δ 13
C
HMBC
HSQC
position
δ 1H; J (Hz) δ 13
C
HMBC
HSQC
pyranomalvidin moiety
pyranomalvidin moiety
2
3
4
160.8
132.8
na
H-2
H-1′′
′
, 6
′
2
3
4
168.9.0
134.4
na
H-2′, 6′
H-1′′
4
5
6
7
8
8
9
1
1
2
3
4
3
a
106.8
152.6
99.4
166.7
101.1
133.2
na
167.2
119.2
107.7
147.5
141.5
55.6
H-6, 8
H-6
4a
5
6
7
8
8a
9
10
109.3
148.9
105.5
169.5
105.6
143.3
109.5
169.3
115.3
105.5
149.9
139.4
56.7
H-6, 8
H-6
7.14; bs
7.30; bs
8.08; s
H-6A
H-8A
6.45; bs
H-6
H-6, 8
H-8
H-6, 8
H-8
7.68; bs
8.09; s
H-8
a
H
H
R
0
′
H
R
, H
â
R
, H
â
H-2
′
, 6
′
1′
2′
3′
4′
3′
H-2
′
, 6
′
′
′
′
′
,6
,5
′
′
7.65; s
3.98; s
H-2
′
B, 6
′
B
,6
,5
′
′
6.89; s
3.88; s
H-2′, 6′
3
CH , H-2
′
, 6′
3
CH , H-2
′, 6′
H-2
′
, 6
′
H-2
′
, 6
′
,5
′
-OMe
CH
3
,5
′
-OMe
CH
3
vinylcatechol group
vinylsyringol group
H
H
R
7.06; d, 15.7
7.87; d, 15.7
115.1
143.9
126.7
126.9
145.8
149.8
114.4
99.6
H
H
R
H
H
R
6.38; d, 15.9
7.59; d, 15.9
114.9
145.8
127.2
105.5
147.1
139.4
56.7
H
H
R
â
â
â
â
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
H
R
, H-6′′′
1
2
3
4
3
′′′
R â
H , H , H-2′′′, 6′′′
7.22; s
H-2′′′
H-5′′′
′′′,6′′′
′′′,5′′′
′′′
6.89; s
3.88; s
H-2′′′, 6′′′
H-2′′′, 5′′′
H-2′′′, 5′′′, 6′′′
H-2′′′, 6′′′
H-2′′′, 6′′′′
6.85; d, 8.1
7.16; d, 8.1
′′′,5′′′-OMe
CH
3
H
â
, H-5′′′
glucose moiety
glucose moiety
1
2
3
4
5
′′
′′
′′
′′
′′
4.80; d, 7.7
3.65; t, 7.7
3.41; *
3.27; *
3.21; *
104.0
74.8
77.0
70.2
77.6
61.5
61.5
H-1′′
H-2′′
H-3′′
H-4′′
H-5′′
H-6a′′, 6b′′
H-6a′′, 6b′′
1′′
2′′
3′′
4′′
5′′
6a
6b
4.75; d, 7.7
3.64;*
3.37;*
3.16;*
3.24;*
103.1
73.9
76.3
77.0
69.8
61.3
61.3
H-1′′
H-2′′
H-3′′
H-4′′
H-5′′
6a′′
6b′′
3.72; *
3.43; *
′′
′′
3.71;*
3.42;*
H-6a′′, 6b′′
H-6a′′, 6b′′
a
*
, unresolved; bs, broad singlet; s, singlet; d, doublet; t, triplet; na, not
a
*, unresolved; bs, broad singlet; s, singlet; d, doublet; na, not assigned.
assigned.
mL/min. The column was washed with 100% B for 20 min and then
stabilized with the initial conditions for another 20 min.
found to be a good source for searching for new polyphenolic
compounds due to its physical-chemical characteristics (21).
Wines are a very complex matrix, which makes it difficult to
screen for new pigments. Therefore, wine samples must be
fractionated to simplify the further interpretation by liquid
chromatography coupled to mass spectrometry (LC/DAD-MS).
For this work, a previously developed procedure was used for
the fractionation of wine samples using two types of resins:
TSK Toyopearl HW-40(S) and polyamide (60-80 mesh) gel
(29). As a result, several fractions were obtained and analyzed
by LC/DAD-MS. The aim was to detect chromatographic peaks
that display a λmax in the visible range different from those of
genuine anthocyanins or other known pigments. Special em-
phasis was given to compounds displaying a higher λmax than
that of anthocyanins. Several chromatographic peaks with these
features were detected in some wine fractions and were assigned
to different known portisins (34). Among these compounds,
in the fraction T50P50 [obtained first from the Toyopearl gel
with a 50% (v/v) methanol aqueous solution and then subfrac-
tionated in a polyamide resin and recovered with a methanol
aqueous solution 50% (v/v)] a chromatographic peak was
LC-MS Analysis. A Finnigan Surveyor series liquid chromatograph,
equipped with a 150 × 4.6 mm i.d., 5 µm LicroCART reversed-phase
C18 column thermostated at 25 °C was used. The mass detection was
carried out by a Finnigan LCQ DECA XP MAX (Finnigan Corp., San
Jose, CA) mass detector with an atmospheric pressure ionization (API)
source of ionization and an electrospray ionization (ESI) interface.
2 3 2 3
Solvents were (A) H O/TFA (99.9:0.1) and (B) CH COOH/H O/CH -
CN (0.5:19.5:80). The HPLC gradient used was the same reported above
for the HPLC analysis. The capillary voltage was 4 V and the capillary
temperature 300 °C. Spectra were recorded in positive ion mode
between m/z 300 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.
NMR. H NMR (500.13 MHz) and 13C NMR (125.77 MHz) spectra
1
were measured in CD
spectrometer at 298 K with TMS as internal standard. H chemical
3
OD/TFA (98:2) on a Bruker-Avance 500
1
1
shifts were assigned using 1D and 2D H NMR (gCOSY and NOESY),
1
3
whereas C resonances were assigned using 2D NMR techniques
gHMBC and gHSQC) (31, 32). The delay for the long-range C/H
(
coupling constant was optimized to 7 Hz. NMR data of pigments I, II,
and III are summarized in Tables 1, 2, and 3, respectively. The data
obtained for pigment IV (vinylpyranomalvidin-3-O-glucoside-phenol)
are already described in the literature (33).
+
detected with a λmax ∼ 540 nm that revealed a [M] molecular
+
ion at m/z 635 with a fragment ion [M - 162] at m/z 473,
possibly corresponding to the loss of the glucosyl moiety. This
molecular ion was attributed to the portisin already described
in the literature, vinylpyranomalvidin-3-O-glucoside-phenol
(33).
RESULTS AND DISCUSSION
Pigment Detection in Port Wine. Different classes of
anthocyanin-derived pigments occurring in red wine such as
pyranoanthocyanins have been described over the past years,
and others are still being researched. Port wine has always been
The same fraction revealed two other [M]+ molecular ions
at m/z 797 and 783. These peaks have a major fragment ion [M
+
- 308] at m/z 489 and 475, respectively, possibly correspond-

6352 J. Agric. Food Chem., Vol. 55, No. 15, 2007
Oliveira et al.
1
Table 3. H Chemical Shifts of Vinylpyranomalvidin-3-O-glucoside
−
Guaiacol (Pigment III), Determined in CD
3
OD/TFA (98:2)a
position
δ 1H; J (Hz) δ 13
C
HMBC
HSQC
pyranomalvidin moiety
2
3
4
162.7 H-2′, 6′
na
na
4
5
6
7
8
8
9
1
1
2
3
4
3
a
108.8 H-6, 8
153.6 H-6
100.8
167.9 H-6, 8
102.6
7.14; bs
7.32; bs
8.08; s
H-6
H-8
a
134.4 H-8
na
0
′
169.4
H
R
, H
â
120.9 H-2
′
, 6
′
′
′
′
′
,6
,5
′
′
7.67; s
3.99; s
109.4
H-2′, 6′
149.5 CH
143.3 H-2
56.8
3
, H-2
′, 6′
′
, 6
′
,5
′
-OMe
CH
3
vinylguaiacol group
H
H
R
6.35; d, 15.8
7.60; d, 15.8
115.7
146.5
H
H
R
â
â
1
2
3
4
5
6
3
′′′
′′′
′′′
′′′
′′′
′′′
′′′-OMe
127.5
7.17; dd, 8.1/1.3 111.2
R â
H , H , 2′′′, 5′′′, 6′′′
H-2′′′
150.0 H-2′′′, 5′′′′
146.5 H-2′′′, 5′′′, 6′′′
116.5
6.81; d, 8.1
H-5′′′
H-6′′′
CH
3
7.07; dd, 8.1/1.3 124.0
3.96; s
56.2
glucose moiety
1′′
2′′
3′′
4′′
5′′
6a
6b
4.74; d, 7.8
3.65; *
3.36; *
3.23; *
3.16; *
104.7
71.2
77.9
71.4
78.8
62.5
62.5
H-1′′
H-2′′
H-3′′
H-4′′
H-5′′
′′
′′
3.70; *
3.41; *
H-6a′′, 6b′′
H-6a′′, 6b′′
a
*, unresolved; bs, broad singlet; s, singlet; d, doublet; dd, double doublets;
na, not assigned.
ing to the loss of a coumaroylglucosyl moiety. The similarity
of the UV-vis spectrum and the fragmentation pattern between
these two compounds with the previously identified vinylpyra-
nomalvidin-3-O-glucoside-phenol suggested that they might
have a closer structure bearing a different phenolic moiety, such
as catechol. The molecular ions at m/z 797 and 783 concur with
the structures of vinylpyranomalvidin-3-O-coumaroylglucoside-
catechol and vinylpyranopetunidin-3-O-coumaroylglucoside-
catechol, respectively.
The compound already reported in the literature, vinylpyra-
nomalvidin-3-O-glucoside-phenol, had been found to arise from
the reaction between malvidin-3-glucoside-pyruvic acid adduct
and vinylphenol (33). The latter precursor could possibly be
formed from the decarboxylation of p-coumaric acid (35). By
similarity, the other two pigments could arise from the reaction
of malvidin-3-coumaroylglucoside and petunidin-3-coumaroyl-
glucoside pyruvic acid adducts with vinylcatechol, formed from
decarboxylation of caffeic acid.
Pigment Formation in Model Solution. To clarify the
structures of these pigments, their formation was thus attempted
in model solutions through the reaction between anthocyanin-
pyruvic acid adducts and hydroxycinnamic acids such as caffeic,
sinapic, ferulic, or p-coumaric acid, which can all be present in
Port wines (18, 20, 35, 36). A malvidin-3-glucoside-pyruvic
acid adduct extract was left to react with the hydroxycinnamic
acids in aqueous ethanol solutions, and the formation of new
pigments was monitored by HPLC/DAD. After 5 days, it was
possible to observe the appearance of new peaks in the HPLC
Figure 2. Chromatograms recorded from the HPLC-DAD at 540 nm of
the reaction of a malvidin-3-glucoside−pyruvic acid adduct (mv-3-glc-py)
extract with different hydroxycinnamic acids after 14 days of reaction:
(A) caffeic acid (pigment I); (B) sinapic acid (pigment II); (C) ferulic acid
(pigment III); (D) p-coumaric acid (pigment IV).
chromatogram (Figure 2). These compounds revealed a λmax
(∼540 nm) bathochromically shifted when compared with that
observed for the malvidin-pyruvic acid adduct precursor (λmax
) 511 nm). The retention time and the λmax are similar to those
of the pigments detected in the wine fraction. After 14 days,
the maximum formation of the pigments was reached, and the

New Portisins Arising from Hydroxycinnamic Acids
J. Agric. Food Chem., Vol. 55, No. 15, 2007 6353
Table 4. Portisins Identified in Port Wine Fractions and Model Solutions by LC/DAD-MS
m/z (M+)
peak identity
m/z
fragment
6
6
6
6
7
7
51*
95*
65*
vinylpyranomalvidin-3-O-glucoside
−
catechol
syringol
guaiacol
489
533
503
473
475
489
vinylpyranomalvidin
vinylpyranomalvidin
vinylpyranomalvidin
vinylpyranomalvidin
−
−
−
−
catechol
syringol
guaiacol
phenol
vinylpyranomalvidin-3-O-glucoside
−
vinylpyranomalvidin-3-O-glucoside-
−
35(26)**
83***
97***
vinylpyranomalvidin-3-O-glucoside
−phenol
vinylpyranopetunidin-3-O-coumaroylglucoside
vinylpyranomalvidin-3-O-coumaroylglucoside
−
catechol
vinylpyranopetunidin
−
catechol
−catechol
vinylpyranomalvidin−catechol
a
*, model solutions; **, model solution and Port wine; ***, Port wine.
Figure 3. Structures of the synthesized pigments: vinylpyranomalvidin-3-O-glucoside−catechol (I); vinylpyranomalvidin-3-O-glucoside−syringol (II);
vinylpyranomalvidin-3-O-glucoside guaiacol (III); vinylpyranomalvidin-3-O-glucoside phenol (IV).
−
−
major compounds were purified for further structural charac-
terization. The purification was performed by TSK Toyopearl
gel HW-40(S) column chromatography, and the main pigments
were isolated by semipreparative HPLC. Their structures were
elucidated by LC/DAD-MS analysis (Table 4) and by 1D and
The formation kinetics of these compounds are different, and
this may be explained by the different substitution patterns of
ring E. The reaction kinetics of the di- and trisubstituted acids
(pigments I-III) with malvidin-3-glucoside-pyruvic acid adduct
seem to be slightly enhanced when compared to the monosub-
stituted one (pigment IV). This feature has already been
described in the literature and is in agreement with the fact that
electron-donating substituents such as hydroxyl and methoxyl
groups stabilize electron-deficient transition states (36).
2D NMR analysis (Tables 1-3). The structural characterization
of the pigments revealed a structure in which a pyranoantho-
cyanin moiety is linked to a hydroxycinnamyl substituent
such as vinylcatechol (pigment I), vinylsyringol (pigment II),
vinylguaiacol (pigment III), or vinylphenol (pigment IV)
Pigment Chromatic Features. Following this, some chro-
matic features of these compounds were also studied, namely,
their color resistance toward discoloration by the nucleophilic
attack by water (color stability at different pH values) and by
bisulfite anion. In acid aqueous buffer conditions (pH 1.0) the
four pigments (I-IV) obtained present a red/violet color. At
these pH conditions, the λmax of the pigments in the visible
region is located at 537, 540, 537, and 533 nm, respectively.
(Figure 3).
The formation mechanism of these pigments can involve a
nucleophilic attack of the olefinic double bond of the hydroxy-
cinnamyl derivative to the C-10 position of the malvidin-3-
glucoside-pyruvic acid adduct that presents eletrophilic char-
acteristics, followed by the loss of a formic acid molecule and
a decarboxylation (Figure 4).

6354 J. Agric. Food Chem., Vol. 55, No. 15, 2007
Oliveira et al.
Figure 4. Proposed mechanism for the synthesis of the hydroxycinnamyl-derived portisins.
Figure 5. UV
−vis spectra of pigment I in model solution at different pH
Figure 6. UV
−
vis spectra of pigment I at pH 2.0 with increasing amounts
values (1.0 11.0).
−
of bisulfite (0
−
200 ppm).
These values are bathochromically shifted to the one observed
This minor resistance may be explained by the presence of a
smaller group (hydroxycinnamyl group), which does not protect
so efficiently the chromophore against the nucleophilic attack
of water at the C-2 position. The bathochromic shift observed
in the λmax for higher pH values (9.0 and 11.0) probably
corresponds to the equilibrium displacement toward the forma-
tion of the quinoidal forms of the pigments. The hydroxycin-
namyl-derived pigments also revealed a great resistance to
discoloration against the nucleophilic attack by bisulfite (Figure
6), especially when compared to the malvidin-3-glucoside
solution that become almost uncolored after the addition of high
concentrations of SO2 (200 ppm) (37). This feature has
previously been shown to occur with the vinylpyranoanthocya-
nin-catechin pigments (37). According to B e´ rk e´ et al., the
nucleophilic addition of anionic bisulfite, in the anthocyanin
structure, can occur at the positively charged C-4 position. The
lower steric hindrance in C-4 makes this position more available
to nucleophilic attack by bisulfite (38). Like vinylpyranoantho-
cyanin-catechin pigments, these new pigments have a substitute
for the malvidin-3-glucoside-pyruvic acid adduct precursor
(511 nm) in similar conditions (data not shown). The substitution
pattern (hydroxylation and/or methoxylation) of ring E affected
slightly the λmax of these pigments. The resistance to discolora-
tion against the nucleophilic attack by water or bisulfite anion
of these four pigments has been revealed to be similar. Thus,
the results obtained for pigment I will be used as an example.
The color displayed by pigment I was shown to change only
slightly with increasing pH of the aqueous solutions (pH 1.0-
4
.0) (Figure 5). At the same pH conditions, malvidin-3-
glucoside is almost converted into its colorless hemiacetal form
37). The higher resistance of the compounds described herein
(
to discoloration can be explained as for the portisins previously
described in the literature (37), by a greater protection of the
chromophore moiety against the nucleophilic attack by water
to give the respective hemiacetal form. However, the resistance
to discoloration of these new pigments is not as high as that
reported for vinylpyranoanthocyanin-catechin pigments (37).

New Portisins Arising from Hydroxycinnamic Acids
J. Agric. Food Chem., Vol. 55, No. 15, 2007 6355
group in C-4 (ring D), which prevents nucleophilic attack of
bisulfite in that position.
Anthocyanin-pyruvic acid adducts and hydroxycinnamic
acids, the precursors of the newly formed pigments reported
herein, are present in appreciable amounts in red wines (12,
(17) Fulcrand, H.; Benabdeljalil, C.; Rigaud, J.; Cheynier, V.;
Moutounet, M. A new class of wine pigments generated by
reaction between pyruvic acid and grape anthocyanins. Phy-
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Cheynier, V.; Favre-Bonvin, J. Structure of new anthocyanin-
derived wine pigments. J. Chem. Soc., Perkin Trans. 1 1996,
15, 17, 21-26). In fact, pigments derived from hydroxycinnamic
acids have already been identified in wines (18, 20). The
characterization of these pigments highlights new chemical
pathways involving anthocyanin-pyruvic acid derivatives as
precursors for the formation of new pigments in subsequent
stages of wine aging that may contribute to its color evolution.
3
5-739.
(
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ester content in mature grapes and during the maturation of white
Riesling grapes. Am. J. Enol. Vitic. 1978, 29, 277-281.
ACKNOWLEDGMENT
We thank Dra. Z e´ lia Azevedo for the LC/DAD-MS analysis.
We also thank Eng. Pedro S a´ from the Sogevinus S.A. Co. for
supplying wine samples.
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Received for review April 3, 2007. Revised manuscript received May
28, 2007. Accepted May 29, 2007. This research was supported by a
Ph.D. grant from FCT (Funda c¸ a˜ o para a Ci eˆ ncia e a Tecnologia -
2
006, 563, 2-9.
Praxis BD/22622/2005), a grant from FCT (PTDC/AGR-ALI/65503/
2006), and a grant from Minist e´ rio da Agricultura (P.O. AGRO 386),
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all from Portugal, and by FEDER funding.
JF070968F