Competition between (+)-catechin and (-)-epicatechin in acetaldehyde- induced polymerization of flavanols

Article abstract of DOI:10.1021/jf980628h

The reactions of (+)-catechin and (-)-epicatechin in the presence of acetaldehyde were studied in model solution systems. When incubated separately with acetaldehyde and at pH values varying from 2.2 to 4.0, reactions were faster with (-)-epicatechin than with (+)-catechin. In mixtures containing both (+)-catechin and (-)-epicatechin with acetaldehyde, new compounds besides the homogeneous bridged derivatives were detected. These compounds were concluded to be heterooligomers consisting of (+)- catechin and (-)-epicatechin linked with an ethyl bridge. In this case, the reaction of (-)-epicatechin was faster than that of (+)-catechin. This was also observed in solutions containing the two flavanols and the (+)-catechin- ethanol intermediate. Under these conditions, the homogeneous (+)-catechin bridged dimers and heterogeneous dimers were obtained by action of the intermediate on (+)-catechin and (-)-epicatechin, respectively. In addition, the homogeneous (-)-epicatechin ethyl-bridged dimers were also detected, showing that ethyl linkages underwent depolymerization and recombination reactions.

Full text of DOI:10.1021/jf980628h

2088
J. Agric. Food Chem. 1999, 47, 2088−2095
Com p etition betw een (+)-Ca tech in a n d (-)-Ep ica tech in in
Aceta ld eh yd e-In d u ced P olym er iza tion of F la va n ols
Nour-Eddine Es-Safi,*,† He´le`ne Fulcrand, Ve´ronique Cheynier, and Michel Moutounet
ISVV-INRA Institut des Produits de la Vigne, Unite´ de Recherche Biopolyme`res et Aroˆmes, 2 place Viala,
34060 Montpellier, France
The reactions of (+)-catechin and (-)-epicatechin in the presence of acetaldehyde were studied in
model solution systems. When incubated separately with acetaldehyde and at pH values varying
from 2.2 to 4.0, reactions were faster with (-)-epicatechin than with (+)-catechin. In mixtures
containing both (+)-catechin and (-)-epicatechin with acetaldehyde, new compounds besides the
homogeneous bridged derivatives were detected. These compounds were concluded to be hetero-
oligomers consisting of (+)-catechin and (-)-epicatechin linked with an ethyl bridge. In this case,
the reaction of (-)-epicatechin was faster than that of (+)-catechin. This was also observed in
solutions containing the two flavanols and the (+)-catechin-ethanol intermediate. Under these
conditions, the homogeneous (+)-catechin bridged dimers and heterogeneous dimers were obtained
by action of the intermediate on (+)-catechin and (-)-epicatechin, respectively. In addition, the
homogeneous (-)-epicatechin ethyl-bridged dimers were also detected, showing that ethyl linkages
underwent depolymerization and recombination reactions.
Keyw or d s: (+)-Catechin; (-)-epicatechin; acetaldehyde; condensation; thiolysis; LC/ DAD;
LC/ MS; competitive action
INTRODUCTION
1992; Santos-Buelga et al., 1995, 1996), and reactions
between them mediated by acetaldehyde (Timberlake
and Bridle, 1976; Baranowski and Nagel, 1983; Roggero
et al., 1987; Bakker et al., 1993; Rivas-Gonzalo et al.,
1995; Dallas et al., 1996a,b; Es-Safi et al., 1996; Escri-
bano-Bailon et al., 1996; Francia-Aricha et al., 1997;
Fulcrand et al., 1996a,b) have been proposed.
Flavanols are polyphenolic compounds found in many
plants, fruits, and beverages such as fruit juices, beer,
and wine. They have attracted much attention in
relation to their potential physiological activities, and
their role has become an important issue in the rela-
tionship between health and human diet. The presence
of these natural compounds in wine has created con-
siderable interest with respect to the potential health
effects associated with moderate consumption.
Polyphenols are also compounds of high reactivity
which react with other phenolic or nonphenolic com-
pounds, giving rise to various new compounds so that
the resulting mixtures become progressively more com-
plex. These transformations affect sensorial properties
such as color, taste, and colloidal stability during storage
and aging of polyphenolic rich foods (Ribereau-Gayon
et al., 1983; Liao et al., 1992; Singleton et al., 1992;
Fulcrand et al., 1996a; Saucier et al., 1996, 1997a).
The color changes produced during storage of red wine
have attracted attention for many years. This has been
attributed to the progressive formation of condensed
pigments resulting from the interaction between free
anthocyanins initially extracted from grapes and other
phenolic compounds, particularly flavanols. Various
mechanisms have been suggested to explain the forma-
tion of these pigments. Processes including copigmen-
tation (Brouillard et al., 1990; Mistry et al., 1991;
Dangles et al., 1992; Figueiredo et al., 1996), direct
condensation between flavanols and anthocyanins (Jurd,
1967; J urd and Somers, 1970; Somers, 1971; Liao et al.,
In addition, changes in taste and astringency also
occur during conservation of red wine. The deastrin-
gency phenomenon has been attributed first as being a
consequence of anthocyanin-flavanol-derived pigment
formation either by direct condensation (Timberlake and
Bridle, 1976; Haslam, 1980; Baranowski and Nagel,
1983; Liao et al., 1992) or through acetaldehyde-medi-
ated reactions (Timberlake and Bridle, 1976; Bara-
nowski and Nagel, 1983; Bakker et al., 1993). However,
a studies conducted on (+)-catechin autopolymerization
induced by acetaldehyde (Fulcrand et al., 1996a; Saucier
et al., 1997a) have proven that this reaction could also
occur and be responsible for flavanol decrease. Occur-
rence of such reaction in grape-derived foods has been
recently proven (Saucier et al., 1997b; Cheynier et al.,
1997) as (+)-catechin ethyl-bridged dimers and trimers
have been detected in red wine samples. This may
explain the partial deastringency observed during aging
of red wine as observed in the case of persimmon fruits
(Tanaka et al., 1994).
The aim of the present work was to study and to
compare the condensation kinetics of flavanols with
acetaldehyde and to establish the effect of pH on these
reactions. The purpose of this work was also to develop
a sensitive thioacidolysis and HPLC analytical method
suitable for identification of the obtained fractions, on
the basis of the released free and thiol units and of
thiolysis kinetics. The major base units of flavanols
* Author to whom correspondence should be addressed.
† Permanent address: EÄ cole Normale Supe´rieure, Labora-
toire de Chimie Organique et d’Etudes Physico-Chimique, B.P.
5118, Takaddoum Rabat, Morocco.
10.1021/jf980628h CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/21/1999

Competition between Flavanols in the Presence of Acetaldehyde
J. Agric. Food Chem., Vol. 47, No. 5, 1999 2089
occurring in wines, that is, (+)-catechin and (-)-epica-
techin, were used in this study.
MATERIALS AND METHODS
Rea gen ts. Deionized water was purified with a Milli-Q
water system (Millipore, Bedford, MA) prior to use. Acetonitrile
was purchased from BDH (Poole, U.K.). Methanol, formic acid,
and acetic acid were obtained from Prolabo (Fontenay S/Bois,
France). (+)-Catechin, (-)-epicatechin and mercaptoethanol
were purchased from Sigma (St. Louis, MO). Acetaldehyde was
obtained from Merck (Darmstadt, Germany).
Rea ction s. An acidic solution was prepared with 17 µL of
acetic acid and 50 µL of ethanol in 373 µL of water, giving a
pH value of 2.2. Six milligrams of (+)-catechin or (-)-
epicatechin and 60 µL of acetaldehyde were then added. The
reactions were monitored by liquid chromatography coupled
with a diode array detector (DAD) and with a mass spectrom-
etry (MS) detector. When (+)-catechin and (-)-epicatechin
were incubated together, 3 mg of each was used and the
reaction was conducted as described above. The various pH
values were obtained by addition of 1 M sodium hydroxide to
the medium described above and were adjusted using a 93313
Bioblock pHmeter.
F igu r e 1. HPLC chromatogram recorded at 280 nm of a
mixture of (-)-epicatechin and acetaldehyde showing residual
flavanol and the newly formed compounds.
An a lytica l HP LC/DAD An a lyses. HPLC/DAD analyses
were performed by means of a Waters system including two
M510 pumps, a U6K manual injector, an automated gradient
controller, and a 990 diode array detector. UV-visible spectra
were recorded from 250 to 600 nm, and peak areas were
measured at 280 nm. The column was a reversed-phase
Lichrospher 100-RP18 (5 µm packing, 250 × 4 mm i.d.)
protected with a guard column of the same material. Elution
conditions were as follows: 1 mL/min flow rate; temperature,
30 °C; solvent A, water/formic acid (98:2, v/v); solvent B,
acetonitrile/water/formic acid (80:18:2, v/v); elution from 5 to
30% B in 40 min, from 30 to 50% B in 20 min, and from 50 to
80% B in 10 min, followed by washing and re-equilibrating
of the column.
Sem ip r ep a r a tive HP LC P u r ifica tion . Semipreparative
HPLC separations were performed by means of a Gilson
system including a 305 master and a 306 slave pump, an 806
manometric module, an 811 dynamic mixer, a 7161 Rheodyne
valve injector, and an 875 UV-visible J asco detector set at
280 nm. The column was a reversed-phase Microsorb C18 (3
µm packing, 250 × 50 mm i.d.). Elution conditions were as
follows: 15 mL/min flow rate; solvent A, water/acetic acid (99:
1, v/v); solvent B, methanol/solvent A (80:20, v/v); elution from
5 to 30% B in 3 min, isocratic 30% B in 2 min, from 30 to 50%
B in 5 min, and from 50 to 80% B in 5 min, followed by washing
and re-equilibrating of the column.
Th iolysis Rea ction s a n d HP LC An a lyses. The thiolysis
reagent was prepared by mixing 1 mL of HCl, 7.5 mL of
mercaptoethanol (HSCH2CH2OH), and 29 mL of water. The
thiolysis reaction was conducted on solutions of pure dimeric
compounds at concentration of 1 mg/mL. After sealing, reac-
tions were carried out at ambient temperature and kinetics
were monitored by HPLC.
HPLC/DAD analyses were performed by means of a Kontron
Instrument systems (Milano, Italy) including a 460 autosam-
pler, a 325 pump system, a 430 detector, and an MSTI 450
data system. UV-visible spectra were recorded from 250 to
600 nm, and peak areas were measured at 280 nm. The column
was a reversed-phase Lichrospher 100-RP18 (5 µm packing,
250 × 4 mm i.d.) protected with a guard column of the same
material. Elution conditions were as follows: 1 mL/min flow
rate; temperaturr, 30 °C; solvent A, water/formic acid (98:2,
v/v); solvent B, acetonitrile/water/formic acid (80:18:2, v/v);
elution begins with isocratic 5% B in 11 min, from 5 to 40% B
in 44 min, isocratic 40% B in 5 min, followed by washing and
re-equilibrating of the column.
F igu r e 2. HPLC chromatogram recorded at 280 nm of a
mixture of (+)-catechin and acetaldehyde showing residual
flavanol and the newly formed compounds.
negative-ion mode. Ion spray voltage was selected at -4 kV
and orifice voltage at -60 V.
HPLC separations were carried out on
a narrow-bore
reversed-phase column with an ABI 140 B solvent delivery
system (Applied Biosystems, Weiterstadt, Germany). The
column was connected with the ES interface via a fused-silica
capillary (length ) 100 cm, 100 µm i.d.). The reaction mixture
was injected with a rotary valve (Rheodyne model 8125) fitted
with a 20 µL sample loop. The separation was achieved on a
Superspher 100-RP18 column (3 µm packing, 125 × 2 mm i.d.,
Merck), using a two-step linear gradient at a flow rate of 200
µL/min. The elution was done with solvents A and B used in
HPLC/DAD analyses and the conditions adapted as follows:
linear gradients from 5 to 30% B in 20 min and from 30 to
50% B in 10 min, followed by washing and reconditioning of
the column. The absorbance at 280 nm was monitored by an
ABI 785A programmable absorbance detector.
RESULTS AND DISCUSSION
To investigate reactions between flavanols and ac-
etaldehyde, (-)-epicatechin and (+)-catechin were in-
dividually incubated with ethanal at various pH values.
The concentrations of the reactants and of the newly
formed compounds were monitored by HPLC with diode
array detection, and their molecular weights were
determined by electrospray mass spectrometry. A gen-
eral decrease in the concentrations of (-)-epicatechin
and (+)-catechin and appearance of numerous products
were observed at all pH values. Figures 1 and 2
represent typical HPLC chromatograms showing, re-
spectively, residual (-)-epicatechin or (+)-catechin and
MS Ap p a r a tu s a n d LC/MS An a lyses. MS measurements
were performed on a Sciex API I Plus simple quadruple mass
spectrometer with mass range of 2400 amu, equipped with an
ion spray ion. The mass spectrometer was operated in the

2090 J. Agric. Food Chem., Vol. 47, No. 5, 1999
Es-Safi et al.
F igu r e 3. General structure of (-)-epicatechin-ethyl dimers.
the newly formed compounds that were eluted later
than their precursors, thus indicating therefore that
they were less polar and/or larger molecules, as observed
in enzymatic oxidation of (+)-catechin (Guyot et al.,
1995).
The UV-visible spectra of the formed compounds
recorded between 250 and 500 nm were similar to that
of (+)-catechin with a maximum absorbance near 280
nm, suggesting that the original flavanol structure was
retained.
LC/MS analysis, conducted in the negative-ion mode,
allowed molecular weight determination of the major
peaks formed through these reactions. The results
obtained indicated that these products are oligomeric
compounds consisting of flavanol units [(-)-epicatechin
or (+)-catechin] bridged by ethyl groups formed accord-
ing to the mechanism postulated by Timberlake and
Bridle (1976) and recently confirmed by Fulcrand et al.
(1996a).
F igu r e 4. Scheme of (-)-epicatechin-ethyl fraction thiolysis.
monomer (Epi; m/z 333), dimer (m/z 921), and trimer
derivatives (m/z 649) were observed.
In the case of (+)-catechin, fractions CD1, CD2, and
CD3 gave a mass signal at m/z 605, which corresponds
to a molecular weight of 606 and thus to a structure in
which two (+)-catechin units are linked by an ethyl
bridge similar to those shown in Figure 3. A recent
study conducted on the condensation of (+)-catechin in
the presence of acetaldehyde has shown the presence
of four dimer derivatives (Saucier et al., 1997a). Fraction
CD2 probably consists of two products that coelute, as
previously observed by Es-Safi et al. (1996), Fulcrand
et al. (1996a,b), and Saucier et al. (1997c).
In addition to these compounds, intermediate adducts
of monomer (Ci; m/z 333), dimer (Cdi; m/z 649), and at
least four trimer derivatives (m/z 921) were also ob-
served (Es-Safi et al., 1996; Fulcrand et al., 1996a,b).
Thus, fractions ED1, ED2, ED3, and ED4, formed
when (-)-epicatechin was incubated with acetaldehyde,
gave a signal at m/z 605, which corresponds to a
molecular weight of 606 and thus to a structure in which
two (-)-epicatechin units are linked by an ethyl bridge
as shown in Figure 3. Recent studies on condensation
of (+)-catechin in the presence of acetaldehyde have
shown the presence of similar derivatives (Es-Safi et al.,
1996; Fulcrand et al., 1996a; Saucier et al., 1997c).
According to the evidence recently contributed (Es-Safi
et al., 1996), flavanols can be linked through C6 or C8,
yielding four products with C6-C6, C8-C8, and C6-
C8 (R and S) bonds, taking into account the presence
of an asymmetric carbon for the C6-C8 isomers (Figure
3). The formation of such bridged products was also
obtained in the case of (+)-catechin, (-)-epicatechin,
procyanidin B2, and malvidin 3-O-glucoside (Es-Safi et
al., 1996; Francia-Aricha et al., 1997; Fulcrand et al.,
1996a,b).
To elucidate the obtained dimeric structures, the four
fractions obtained in the case of (-)-epicatechin (ED1,
ED2, ED3, and ED4) and those formed in the case of
(+)-catechin (CD1, CD2, and CD3) were collected by
HPLC at the semipreparative scale. All of them proved
to be very unstable once isolated, rendering identifica-
tion difficult to achieve by spectral methods (Es-Safi et
al., 1996; Fulcrand et al., 1996b). Nevertheless, degra-
dation by thiolysis (Tanaka et al., 1994) provides es-
sential information on the structures of these com-
pounds. This consists of mixing the obtained flavanol-
ethyl derivatives with thiol reactant in acidic medium.
A free flavanol is thus released in addition to flavanol
ethyl-thiol derivatives according to the considered
thiolyzed isomer. Thus, 6- or 8-flavanol-ethyl thioether
derivatives are released in the case of 6-6 or 8-8 dimer,
respectively, whereas the two thioethers are obtained
starting from a 6-8 dimer (Figure 4). This method has
been used by Tanaka et al. (1994) to elucidate tannin-
acetaldehyde condensed products isolated from persim-
mon fruits.
In addition to these dimers, various oligomeric bridged
compounds up to the tetramer level were also detected.
Among them, intermediate acetaldehyde adducts of

Competition between Flavanols in the Presence of Acetaldehyde
J. Agric. Food Chem., Vol. 47, No. 5, 1999 2091
F igu r e 5. HPLC chromatograms recorded at 280 nm of ED1
and ED2 fraction thiolysis.
F igu r e 7. HPLC chromatograms recorded at 280 nm of CD1,
CD2, and ED3 fraction thiolysis.
Fractions ED1 and ED4, which gave two differents
pairs of thioethers, corresponded to the 8-8 and 6-6
isomers, whereas fractions ED2 and ED3, which gave
all thioethers, corresponded to 6-8 isomers. Kinetics
studies of ED1 and ED4 thiolysis showed that ED1 was
degraded more rapidly than ED4 and should correspond
therefore to an 8-8 isomer (Tanaka et al., 1994),
whereas ED4 corresponds to the 6-6 dimer.
The results obtained in the case of the three (+)-
catechin-ethyl fractions (CD1, CD2, and CD3) are
shown in Figure 7. Examination of the three chromato-
grams obtained after complete thiolysis showed that,
in addition to free (+)-catechin, one, two, or three thiol
derivatives named TEC1, TEC2, and TEC3 are obtained
in the case of CD3, CD1, and CD2, respectively. The
three thiol derivatives are 6- and 8-position isomers with
one position giving two derivatives due to R and S
configurations.
Fractions CD1 and CD3, which gave respectively one
and two different thioethers, probably correspond to 8-8
and 6-6 isomers, whereas compound CD2, which gave
all thioethers, corresponds to the mixture of both 6-8
dimers. Kinetic studies of CD1 and CD3 thiolysis
showed that CD1 was degraded more rapidly than CD3
and should correspond therefore to an 8-8 isomer as
observed in the case of (-)-epigallocatechin 3-gallate
(Tanaka et al., 1994). CD3 corresponds finally to a 6-6
isomer.
F igu r e 6. Mass spectrum of a (-)-epicatechin-ethyl thiol
derivative (TE1).
Examination of the chromatograms obtained after
complete thiolysis of fractions ED1, ED2, ED3, and ED4
showed that, in addition to free (-)-epicatechin, two
differents pairs of thiol derivatives were obtained in the
case of fractions ED1 and ED3, whereas the four thiol
derivatives named TEE1, TEE2, TEE3, and TEE4 were
obtained in the case of fractions ED2 and ED3 (Figure
5). These compounds exhibit spectra similar to that of
(-)-epicatechin with a maximum absorbance near 280
nm. Identical molecular weights of 429 ([M + Cl]-) were
determined by LC/MS analysis conducted in the nega-
tive-ion mode as shown in Figure 6, indicating that an
additional hydroxyethylsulfanyl group is attached to the
ethyl group. This confirmed that the obtained flavanol-
ethyl compounds are sensitive to thiol reagents and that
thiolysis can be used to study their isomerization
(Tanaka et al., 1994). The four thiol derivatives are
evidently 6- and 8-position isomers because the presence
of an asymmetric carbon gives two derivatives for each
position, due to R and S configurations.
To evaluate the effect of pH changes on the composi-
tion of the incubated solutions, the individual condensa-
tions of (-)-epicatechin and (+)-catechin with acetalde-
hyde were monitored by HPLC in the pH range from

2092 J. Agric. Food Chem., Vol. 47, No. 5, 1999
Es-Safi et al.
F igu r e 8. Changes in (-)-epicatechin concentration with time
according to pH.
F igu r e 9. Changes in (+)-epicatechin concentration with time
according to pH.
2.2 to 4.0. Quantification of all products was achieved
on the basis of peak areas at 280 nm, using (-)-
epicatechin and (+)-catechin as standards. The concen-
trations of residual (-)-epicatechin and (+)-catechin at
various pH values are presented in Figures 8 and 9.
(-)-Epicatechin and (+)-catechin concentrations
showed a gradual decrease at all pH values, but the
reaction rate increased with the decrease of pH, owing
to the higher availability of acetaldehyde carbocation
at lower pH values. This phenomenon was also observed
in the case of condensation of malvidin 3-O-glucoside
with (+)-catechin in the presence of acetaldehyde (Gar-
cia-Viguera et al., 1994) and is in agreement with the
mechanism initially postulated by Timberlake and
Bridle (1976) and recently confirmed by us (Es-Safi et
al., 1996; Fulcrand et al., 1996a,b).
The variations in relative amounts of the obtained
dimer fractions during incubation of the reaction media
at various pH values are presented in Figures 10 and
11. Fractions accumulated gradually at the beginning
of the reaction and then decreased, probably as more
polymerized products were formed.
The comparison of the results obtained above, when
the two flavanols were individually incubated with
acetaldehyde, showed that reactions were faster with
F igu r e 10. Changes in relative amounts of fractions ED1,
ED2, ED3, and ED4 according to time and pH.
(-)-epicatechin than with (+)-catechin at all pH values
(Figure 12). The difference was more important as the
pH increased.
To compare the reactivity of the two studied flavanols,
solutions containing (+)-catechin, (-)-epicatechin, and
acetaldehyde were prepared at pH 2.2 and incubated
at room temperature. Changes taking place were moni-
tored by LC/DAD and LC/MS analysis.
Quantification of the two flavanols during the course
of the reaction gave the results shown in Figure 13. The
rate of loss of (-)-epicatechin was higher than that of
(+)-catechin, especially at the beginning of the reaction.
This showed that (-)-epicatechin is more easily incor-

Competition between Flavanols in the Presence of Acetaldehyde
J. Agric. Food Chem., Vol. 47, No. 5, 1999 2093
F igu r e 12. Changes in concentrations of (-)-epicatechin and
(+)-catechin when individually incubated with acetaldehyde
according to time and pH.
F igu r e 11. Changes in relative amounts of fractions CD1,
CD2, and CD3 according to time and pH.
porated in the formed bridged oligomers than (+)-
catechin.
the two flavanols with the intermediate formed by
addition of protonated acetaldehyde onto (+)-catechin.
Thus, after preparation and isolation of the (+)-ca-
techin-ethanol intermediate by semipreparative scale
HPLC, it was incubated with both flavanols at pH 2.2
and at room temperature. The mixture composition was
monitored by HPLC as indicated above. The formation
of the homogeneous (+)-catechin bridged dimers and of
the heterogeneous dimers was first observed. These
products are obviously formed by action of the inter-
mediate on (+)-catechin and (-)-epicatechin, respec-
tively. It was, however, interesting to note the appear-
ance of the (-)-epicatechin homogeneous bridged dimers.
These compounds could not be formed directly by
interaction of the reactants introduced at the beginning
of the reaction. This indicates that the first heteroge-
LC/MS study conducted in the negative ion mode
showed the presence of new dimers (m/z 605) besides
the condensation products obtained when each flavanol
was incubated individually with acetaldehyde at the
same pH. These compounds were concluded to be
heterodimers consisting of (+)-catechin and (-)-epica-
techin units linked with an ethyl bridge. The chromato-
graphic profiles of solutions containing both flavanols
with acetaldehyde and incubated at pH 2.2 were very
complex due to the presence of many new peaks, which
caused peak overlapping.
After comparison of the reactivity of (-)-epicatechin
and (+)-catechin separately and together on acetalde-
hyde, it seemed interesting to study the interaction of

2094 J. Agric. Food Chem., Vol. 47, No. 5, 1999
Es-Safi et al.
The results showed that the first formed products
underwent decomposition and recombination, giving
various bridged oligomers. In both cases, the rate of loss
of (-)-epicatechin was higher than that of (+)-catechin.
LITERATURE CITED
Bakker, J .; Picinelli, A.; Bridle, P. Model wine solutions: colour
and composition changes during ageing. Vitis 1993, 32,
111-118.
Baranowski, E. S.; Nagel, C. W. Kinetics of malvidin-3-
glucoside condensation in wine model systems. J . Food Sci.
1983, 48, 419-429.
Brouillard, R.; Wigand, M. C.; Cheminat, A. Loss of colour, a
prerequisite to plant pigmentation by flavonoids. Phy-
tochemistry 1990, 29, 3457-3460.
Cheynier, V.; Fulcrand, H.; Sarni, P.; Moutounet, M. Applica-
tion des techniques analytiques a` l’e´tude des compose´s
phe´noliques et de leurs re´actions au cours de la vinification.
Analusis 1997, 25, M14-M21.
F igu r e 13. Changes in concentrations of (-)-epicatechin and
(+)-catechin when incubated together with acetaldehyde.
Dallas, C.; Ricardo-da-Silva, J . M.; Laureano, O. Product
formed in model wine solutions involving anthocyanins,
procyanidin B2, and acetaldehyde. J . Agric. Food Chem.
1996a , 44, 2402-2407.
Dallas, C.; Ricardo-da-silva, J . M.; Laureano, O. Interactions
of oligomeric procyanidins in model wine solutions contain-
ing malvidin-3-glucoside and acetaldehyde. J . Sci. Food
Agric. 1996b, 70, 493-500.
Dangles, O.; Wigand, M. C.; Brouillard, R. Polyphenols in plant
pigmentation: The copigmentation case. Bull. Liaison Groupe
Polyphenols 1992, 16, 209-216.
Escribano-Bailon, T.; Dangles, O.; Brouillard, R. Coupling
reactions between flavylium ions and catechin. Phytochem-
istry 1996, 41, 1583-1592.
Es-Safi, N.; Fulcrand, H.; Cheynier, V.; Moutounet, M.;
Hmamouchi, M.; Essassi, E. M. Kinetic studies of acetal-
dehyde-induced condensation of flavan-3-ols and malvidin-
3-glucoside model solutions systems. In Polyphenols Com-
munications 96; Vercauteren, J ., Cheze, C., Dumon, M. C.,
Weber, J . F., Eds.; Groupe Polyphenols: Bordeaux, France,
1996.
Figueiredo, P.; Elhabiri, M.; Toki, K.; Saito, N.; Dangles, O.;
Brouillard, R. New aspects of anthocyanin complexation.
Intramolecular copigmentation as a means for colour loss?
Phytochemistry 1996, 41, 301-308.
Francia-Aricha, E. M.; Guerra, M. T.; Rivas-Gonzalo, J . C.;
Santos-Buelga, C. New anthocyanin pigments formed after
condensation with flavanols. J . Agric. Food Chem. 1997, 45,
2262-2266.
Fulcrand, H.; Docco, T.; Es-Safi, N.; Cheynier, V.; Moutounet,
M. Study of acetaldehyde induced polymerisation of flavan-
3-ols by liquid chromatography-ion spray mass spectrom-
etry. J . Chromatogr. A 1996a , 752, 85-91.
Fulcrand, H.; Es-Safi, N.; Docco, T.; Cheynier, V.; Moutounet,
M. LC-MS study of acetaldehyde induced polymeisation of
flavan-3-ols. In Polyphenols Communications 96; Vercau-
teren, J ., Cheze, C., Dumon, M. C., Weber, J . F., Eds.;
Groupe Polyphenols: Bordeaux, France, 1996b.
Garcia-Viguera, C.; Bridle, P.; Bakker, J . The effect of pH on
the formation of coloured compounds in model solutions
containing anthocyanins, catechin and acetaldehyde. Vitis
1994, 33, 37-40.
Guyot, S.; Cheynier, V.; Souquet, J . M.; Moutounet, M.
Influence of pH on the enzymatic oxidation of (+)-catechin
in model systems. J . Agric. Food Chem. 1995, 43, 2458-
2462.
Haslam, E. In vini veritas: oligomeric procyanidins and the
ageing of red wines. Phytochemistry 1980, 19, 2577-2582.
J urd, L. Anthocyanins and related compounds. XI. Catechin
flavylium salt condensation reactions. Tetrahedron 1967, 23,
1057-1064.
F igu r e 14. Changes in concentrations of (-)-epicatechin and
(+)-catechin when incubated together with the (+)-catechin-
ethanol intermediate.
neous dimers formed underwent decomposition, releas-
ing a mixture of (-)-epicatechin and (+)-catechin inter-
mediates and free (-)-epicatechin and (+)-catechin
units, which by further condensation gave a mixture of
compounds among which were homogeneous (-)-epi-
catechin bridged compounds. This shows the complex
evolution of such mixtures and the interest of working
on model solutions.
The quantification of (+)-catechin and (-)-epicatechin
relative amounts is represented in Figure 14, which
shows that, in this case, the loss of (-)-epicatechin was
also higher than that of (+)-catechin, meaning that it
reacts more readily with the intermediate. The reaction
stopped when the intermediate was depleted.
CONCLUSION
The results obtained in this paper showed that the
acetaldehyde autopolymerizations of (-)-epicatechin
and (+)-catechin are greatly affected by the conditions
used to induce the reaction and that the two studied
flavanols showed different reactivities in such a reac-
tion.
When incubated individually with acetaldehyde, the
reaction was faster with (-)-epicatechin than with (+)-
catechin at all studied pH values. Larger amounts of
bridged compounds and also faster condensation rates
were observed at lower pH values. These phenomena
were related to the availability of the acetaldehyde
cation.
When (-)-epicatechin and (+)-catechin were incu-
bated together with acetaldehyde or with the (+)-
catechin intermediate, heterodimeric ethyl-bridged com-
pounds were obtained in addition to the homo-oligomers.
J urd, L.; Somers, T. C. The formation of xanthylium salts from
proanthocyanidins. Phytochemistry 1970, 9, 419-427.

Competition between Flavanols in the Presence of Acetaldehyde
J. Agric. Food Chem., Vol. 47, No. 5, 1999 2095
Liao, H.; Cai, Y.; Haslam, E. Polyphenols Interaction. Antho-
cyanins: Copigmentation and colour change in Red wines.
J . Sci. Food Agric. 1992, 59, 299-305.
Mistry, T. V.; Cai, Y.; Lilley, T. H.; Haslam, E. Polyphenol
interactions. Part 5. Anthocyanin copigmentation. J . Chem.
Soc., Perkin Trans. 2 1991, 1287-1296.
Ribereau-Gayon, P.; Pontallier, P.; Glories, Y. Some interpre-
tations of colour changes in young red wines during their
conservation. J . Sci. Food Agric. 1983, 34, 505-516.
Rivas-Gonzalo, J . C.; Bravo-Haro, S.; Santos-Buelga, C. Detec-
tion of compounds formed through the reaction of malvidin-
3-monoglucoside and catechin in the presence of acetalde-
hyde. J . Agric. Food Chem. 1995, 43, 1444-1449.
Roggero, J . P.; Coen, S.; Archier, P.; Rocheville-Divorne, C.
Etude par C.L.H.P. de la re´action glucoside de malvidine-
ace´talde´hyde-compose´ phe´nolique. Connaiss. Vigne Vin
1987, 21 (3), 163-168.
Santos-Buelga, C.; Bravo-Haro, S.; Rivas-Gonzalo, J . C. In-
teractions between catechin and malvidin 3-monoglucoside
in model solutions. Z. Lebensm. Unters. Forsch. 1995, 201,
269-274.
Santos-Buelga, C.; Francia-Aricha, E. M.; Rivas-Gonzalo, J .
C. Role of flavan-3-ol structure on direct condensation with
anthocyanins. In Polyphenols Communications 96; Vercau-
teren, J ., Cheze, C., Dumon, M. C., Weber, J . F., Eds.;
Groupe Polyphenols: Bordeaux, France, 1996.
gie 95; Lonvaud, A., Ed.; Lavoisier: Paris, France, 1996; pp
395-400.
Saucier, C.; Bourgeois, G.; Vitry, C.; Roux, D.; Glories, Y.
Characterization of (+)-catechin-acetaldehyde polymers: a
model for colloidal state of wine polyphenols. J . Agric. Food
Chem. 1997a , 45, 1045-4049.
Saucier, C.; Little, D.; Glories, Y. First evidence of acetalde-
hyde-flavanol condensation products in red wine. Am. J .
Enol. Vitic. 1997b, 48 (3), 370-373.
Saucier, C.; Guerra, C.; Pianet, I.; Laguerre, M.; Glories, Y.
(+)-Catechin-acetaldehyde condensation products in relation
to wine-ageing. Phytochemistry 1997c, 46 (2), 229-234.
Singleton, V. L.; Trousdale, E. K. Anthocyanin-tannin interac-
tions explaining differences in polymeric phenols between
white and red wines. Am. J . Enol. Vitic. 1992, 43, 63-70.
Somers, T. C. The polymeric nature of red pigments. Phy-
tochemistry 1971, 10 (9), 2175-2186.
Tanaka, T.; Takahashi, R.; Kouno, I.; Nonaka, K. Chemical
evidence for the de-astringency (insolubilization of tannins)
of persimmon fruit. J . Chem. Soc., Perkin Trans. 1 1994,
3013-3022.
Timberlake, C. F.; Bridle, P. Interactions between anthocya-
nins phenolic compounds and acetaldehyde. Am. J . Enol.
Vitic. 1976, 27 (3), 97-105.
Received for review J une 10, 1998. Revised manuscript
received March 5, 1999. Accepted March 5, 1999.
Saucier, C.; Roux, D.; Glories, Y. Stabilite´ coloidale de poly-
me`res cate´chiques. Influence des polysaccharides. In Œnolo-
J F980628H