Hemisynthesis and structural and chromatic characterization of delphinidin 3- O -glucoside-vescalagin hybrid pigments

Article abstract of DOI:10.1021/jf4033188

During red wine maturation in the presence of oak wood, reactions involving anthocyanins and ellagitannins might affect wine organoleptic properties such as color and astringency. In this work, the condensation reaction between myrtillin (delphinidin 3-O-glucoside) and vescalagin has been performed to determine the behavior of this anthocyanin in this kind of reaction and to assess the possible impact of such a reaction in wine color modulation. Two different hybrid pigments have been hemisynthetized and characterized by HPLC-DAD-MS and NMR spectroscopy. These pigments have been identified as 1-deoxyvescalagin-(1β→8)-myrtillin (major) and 1-deoxyvescalagin- (1β→6)-myrtillin (minor). The minor pigment could be formed both by the condensation reaction and by a regioisomerization process from the major pigment. Moreover, the chromatic properties of these pigments have been studied and compared to those of myrtillin. The hybrid pigments showed an important bathochromic shift (ca. 20 nm) in the maximum absorbance wavelength and lower molar absorption coefficients.

Full text of DOI:10.1021/jf4033188

Article
pubs.acs.org/JAFC
Hemisynthesis and Structural and Chromatic Characterization of
Delphinidin 3‑O‑Glucoside−Vescalagin Hybrid Pigments
Ignacio García-Estevez,† Remi Jacquet,§ Cristina Alcalde-Eon,† Julian C. Rivas-Gonzalo,†
́
́
́
M. Teresa Escribano-Bailon,*,† and Stephane Quideau*,§
́
́
†Grupo de Investigacion
§Universite
de Bordeaux (ISM, CNRS-UMR 5255), Institut Europee
Cedex, France
́
en polifenoles (GIP), Facultad de Farmacia, Universidad de Salamanca, E37007 Salamanca, Spain
n de Chimie et Biologie, 2 rue Robert Escarpit, 33607 Pessac
́
́
ABSTRACT: During red wine maturation in the presence of oak wood, reactions involving anthocyanins and ellagitannins
might affect wine organoleptic properties such as color and astringency. In this work, the condensation reaction between myrtillin
(delphinidin 3-O-glucoside) and vescalagin has been performed to determine the behavior of this anthocyanin in this kind of
reaction and to assess the possible impact of such a reaction in wine color modulation. Two different hybrid pigments have been
hemisynthetized and characterized by HPLC-DAD-MS and NMR spectroscopy. These pigments have been identified as 1-
deoxyvescalagin-(1β→8)-myrtillin (major) and 1-deoxyvescalagin-(1β→6)-myrtillin (minor). The minor pigment could be
formed both by the condensation reaction and by a regioisomerization process from the major pigment. Moreover, the chromatic
properties of these pigments have been studied and compared to those of myrtillin. The hybrid pigments showed an important
bathochromic shift (ca. 20 nm) in the maximum absorbance wavelength and lower molar absorption coefficients.
KEYWORDS: C-glucosidic ellagitannin, oak wood, red wine, myrtillin (delphinidin 3-O-glucoside), vescalagin, anthocyanin reactivity
ellagitannins can take part in some of the anthocyanin
reactions, hence affecting the wine properties.4,5 The most
representative structures of found-in-wine C-glucosidic ellagi-
tannins are the monomeric forms vescalagin and castalagin,
which are the most abundant ellagitannins in oak wood with
contents ranging from 39 to 73% of the total ellagitannin
content.18−21 Lyxose/xylose derivatives (grandinin and roburin
E, respectively) and dimeric forms (roburins A, B, C, and D)
have also been described.20,22−25 In oak heartwood, the C-
glucosidic ellagitannins may represent up to 10% of the dry
wood weight.18 These highly hydrophilic compounds are easily
solubilized by wine, in which they can be involved in different
chemical reactions.
However, there are important differences in the reactivity of
the different C-glucosidic ellagitannins. For example, it has been
proven that castalagin expresses a much lower reactivity than
vescalagin in direct condensation reactions with other wine
components.24,26 In fact, only vescalagin reacts with flavanols or
anthocyanins to form adducts such as flavano-ellagitannins27,28
or anthocyano-ellagitannins.28,29 The reactions of vescalagin
with (epi)catechins yields (epi)acutissimins. These flavano-
ellagitannins feature a C−C linkage between carbon-1 of the
vescalagin moiety and either carbon-8 or carbon-6 of the A ring
of the (epi)catechin unit.26−28 The catechin variants of these
compounds were first isolated and characterized by Ishimaru
and co-workers from the bark of Quercus acutissima,30 and some
studies have also reported their formation during the aging of
wine in contact with oak wood.27,31,32
INTRODUCTION
■
During wine maturation, anthocyanins from red grape
progressively disappear as a result of various chemical reactions
that lead to new derivatives resulting in changes of wine color.
These reactions, which usually involve different other
compounds from grape or from the oak wood made barrels
in which wine is commonly aged, might produce both
qualitative and quantitative changes in wine, affecting its
organoleptic properties such as color and astringency.1−5
Most of the reactions involving wine anthocyanins result in
the formation of more stable derived pigments. Different kinds
of derived pigments such as pyranoanthocyanins and flavanol−
anthocyanin condensation products have been detected in
wines. The first ones result from the cycloaddition of wine
nucleophiles at C4 and O5 of the anthocyanin flavylium
nucleus (followed by aromatization through autoxidation),
which lead to an additional pyrane ring in the pigment
structure. Some nucleophilic compounds present in wines, such
as the enol forms of pyruvic acid or acetaldehyde and
vinylphenols among others, can thus react with anthocyanins
to generate these kinds of pigments.6−11 Anthocyanins have
also been shown to react with flavanols, involving acetaldehyde
or not, to generate either acetaldehyde-derived or direct
flavanol−anthocyanin condensation products.4,12−17 The an-
thocyanin acts as a nucleophile in the formation of
acetaldehyde-derived flavanol−anthocyanin products,4,13
whereas two different mechanisms have been postulated to
explain direct reactions between anthocyanins and flavanols
during which the anthocyanin could act either as an electrophile
or as a nucleophile to form either anthocyanin−flavanol (A-F)
or flavanol−anthocyanin (F-A+) adducts.15
Received: July 30, 2013
Revised: November 6, 2013
Accepted: November 12, 2013
Published: November 12, 2013
Moreover, among the different substances released from oak
wood into the wine solution during aging, the C-glucosidic
© 2013 American Chemical Society
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Figure 1. HPLC-DAD chromatogram (λ = 520 nm) of the reaction between vescalagin and myrtillin: (a) t = 0; (b) t = 24 h. α, vescalagin; β, product
1; γ, product 3; δ, product 2; ε, myrtillin.
The formation of anthocyano-ellagitannin hybrid pigments
has been demonstrated by Quideau and co-workers,28,29 who
were able to achieve the first hemisynthesis of 1-deoxyvesca-
lagin-(1β→8)-malvidin and 1-deoxyvescalagin-(1β→8)-
oenin.28 These authors have also demonstrated the formation
of these hybrid pigments in a standard wine model solution,
confirming the feasibility of these condensation reactions in
such a medium.28 These anthocyano-ellagitannin hybrid
pigments show purple hues in accordance with the bath-
ochromic shift observed in the absorption band of their visible
spectra. Moreover, differences in their kinetic and thermody-
namic parameters as compared to those of the native
anthocyani(di)ns have been reported.29 Therefore, ellagitannins
might also be directly involved in color-modulating reactions in
wine via the formation of this kind of hybrid pigment.
Although the major anthocyanins in grapes and wines are
usually malvidin derivatives, other anthocyanins such as
delphinidin or petunidin derivatives may be, depending on
the grape variety, as quantitatively relevant as malvidin
derivatives.33,34 Moreover, there have been reported differences
in the reactivity of the anthocyanidins upon oxidation
depending on the different substituents of the B-ring,35,36
which may indicate the importance of the substitution pattern
of the B-ring in the reactivity of the different anthocyanins. For
these reasons, it appears interesting to study the behavior of
anthocyanins other than the oenin in this kind of reaction. In
the work described herein, the hemisynthesis of myrtillin
(delphinidin 3-O-glucoside)−vescalagin hybrid pigments has
been carried out, and their chromatic properties have been
examined with the aim of assessing the role of the formation of
such anthocyano-ellagitannins in the modulation of wine color.
MATERIALS AND METHODS
■
Chemicals. Myrtillin (delphinidin 3-O-glucoside) was extracted
from the skins of Vitis vinifera L. cv. Tempranillo grapes. Extraction
was carried out three times using MeOH/12 M HCl 999:1 (v/v), and
the extracts were gathered and evaporated under reduced pressure to
remove the methanol. The residue was redissolved in aqueous HCl
(0.1 M, pH 1) and then loaded on a Sephadex LH-20 (Sigma-Aldrich,
St. Louis, MO, USA) column (30 × 300 mm), which was previously
conditioned using 1 L of aqueous HCl (0.1 M, pH 1). Elution was
carried out using the same aqueous HCl solution, and fractions (25
mL each) were collected. Myrtillin eluted in two fractions after the
elution of the other anthocyanidin-3-O-glucosides. These two fractions
were gathered and then freeze-dried to furnish a reddish purple
powder. Myrtillin purity (>95%) was determined by HPLC-DAD-MS
analysis. (−)-Vescalagin (Vg) was extracted from Quercus robur
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Figure 2. Reaction between vescalagin and myrtillin in an acidic organic solution.
Figure 3. (a) UV−visible spectrum of the major product of the reaction between vescalagin and myrtillin (product 1). (b) Full mass spectrum of
product 1. (c) MS2 fragmentation pattern of the molecular ion of product 1 (m/z 1381). (d) MS3 fragmentation pattern of the main fragment (m/z
1219) obtained in the MS2 analysis.
heartwood and purified as described previously.37 All of the solvents
used were of analytical HPLC grade and were purchased from Sigma-
Aldrich.
Procedures. Reaction. The reaction between vescalagin and
myrtillin was performed according to the procedure described by
Quideau and co-workers for the hemisynthesis of oenin− and
malvidin−vescalagin hybrid pigments.28 Vescalagin (20 mg, 0.021
mmol) and myrtillin chloride (12 mg, 0.024 mmol) were dissolved in
an anhydrous tetrahydrofuran solution containing 1.5 vol % of
trifluoroacetic acid (12 mL). The reaction mixture was stirred at 60 °C
for 24 h, and then the reaction was stopped by evaporation under
reduced pressure. The residue was dissolved in acidic water (H2O/
HCOOH 999:1) and purified by semipreparative HPLC.
Purification and Structural Characterization. Semipreparative
HPLC purification of the products was performed on a Varian
ProStar system using a Dynamax C-18, 5 μm, 21.4 × 250 mm column
(Agilent Technologies, Waldbronn, Germany). This liquid chromato-
graphic purification method used H2O/HCOOH 999:1 as solvent A
and MeOH/HCOOH 999:1 as solvent B in a gradient elution (0−20
min, 0−20% solvent B; 20−35 min, 20−100% solvent B; 35−40 min,
100% solvent B) with a flow rate of 6 mL/min. Column effluent was
monitored by UV detection at 280 nm using a Varian ProStar 320
UV−visible detector controlled by Star Chromatography Workstation
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fragment in MS2 analysis at m/z 1219 ([M+ − 162]),
corresponding to the loss of the glucose moiety of myrtillin,
and a main fragment in MS3 analysis at m/z 917 ([M+ − 162 −
302]), which could correspond to the 1-deoxyvescalagin moiety
formed by the loss of the myrtillin residue. However, this
fragment might also correspond to the 1-deoxyvescalin-
delphinidin moiety formed by the loss of the hexahydrox-
ydiphenoyl (HHDP) residue of the ellagitannin, because it also
represents the loss of 302 units of mass. The MS4 analysis
showed a complicated fragmentation pattern that could
originate from the fragmentation of the 1-deoxyvescalagin
moiety, as well as from the 1-deoxyvescalin-delphinidin moiety.
Both hybrid pigments (products 1 and 2) resulted from an
acid-catalyzed nucleophilic substitution at the C-1 center of
vescalagin with retention of configuration.28 The anthocyanin
molecule has two potential nucleophilic positions (6′-C and 8′-
C),15,41 but due to the higher net negative charge at the ground
state of the anthocyanin 8′-C, this position is more likely to be
involved in the linkage than 6′-C.4 This hypothesis can be
supported by the fact that in most of the anthocyanin
condensation products characterized to this day, the 8′-C of
the anthocyanin is involved in the linkage.13 Nevertheless,
Atanasova and co-workers42 have reported polymeric acetalde-
hyde-mediated self-condensation products of malvidin-3-O-
glucoside in which the 6′-C of the anthocyanin is also involved
in the linkage. Thus, the 6′-C position of the anthocyanin
seems to be also reactive, although to a lesser extent than the
8′-C position. For this reason, and taking into account the rate
at which the compounds were obtained, product 1, the major
one, was identified as 1-deoxyvescalagin-(1β→8)-myrtillin (8′-
C isomer), whereas product 2, obtained in a much lower
proportion, was identified as 1-deoxyvescalagin-(1β→6)-
myrtillin (6′-C isomer). Although some of the 1H NMR
signals of the ellagitannin glucose part of these compounds
could not be fully assigned due to the presence of minor
impurities caused by partial degradation in the NMR solutions,
the signals corresponding to their main protons were attributed
with the help of the analysis of the HMQC and HMBC
correlation data maps of the major product 1 (data not shown)
and comparison with the NMR data of the oenin− and
malvidin−vescalagin adducts.28,29 The main proton signals were
thus assigned as follows.
6.41 software (Agilent Technologies, Waldbronn, Germany). The
obtained fractions were analyzed by HPLC-DAD-ESI+-MS in a
Thermo Finnigan Spectra Systems P1000XR equipped with a Spectra
System UV6000 LP detector (controlled by ChromQuest 4.2 software
Thermoquest, San Jose, CA, USA). The mass analyses were performed
with a Finnigan LCQ ion trap instrument (Thermoquest) equipped
with an electrospray ionization (ESI) interface. HPLC-DAD-MS
analyses were performed using a previously developed method for the
analysis of oenin−vescalagin hybrid pigments.28,29 The fractions
containing each compound of interest were gathered, concentrated
under reduced pressure, and repurified using the same semipreparative
HPLC procedure.
1H NMR analyses were carried out in a Bruker DPX 800NB NMR
spectrometer equipped with a 5 mm direct QNP probed with gradient
capabilities, using MeOH-d4/TFA (99:1) as solvent. Data processing
was performed with Topspin software (Bruker, Billerica, MA, USA)
using a sine-bell multiplication in both dimensions.
Chromatic Characterization. The absorption spectra were
recorded on a Hewlett-Packard UV−visible HP 8453 spectropho-
tometer (Palo Alto, CA, USA), using 10 mm path length quartz cells.
The whole UV−visible spectrum (190−770 nm) was recorded (Δλ =
1 nm) at pH 1 (0.1 M HCl aqueous solution) and at pH 3.2 (model
wine solution: 5 g/L tartaric acid in 12% EtOH/H2O v/v adjusted at
pH 3.2 with 1 M NaOH). The CIELAB parameters (L*, a*, b*, C*ab,
and hab) and color differences (ΔE*ab) were determined by using
CromaLab software.38 The illuminant D65 and CIE 1964 10° standard
observer were used as references in the calculations, following the
recommendations of the Commission International de L’Eclairage.39
RESULTS AND DISCUSSION
■
Hemisynthesis and Structural Characterization. The
reaction mixture between vescalagin and myrtillin initially
showed a dark red color that turned into a deep purple color
over time. The reaction progress was monitored by HPLC-
DAD-MSn to determine the optimum reaction time. After 24 h,
the degradation of the starting materials and the formation of
secondary products were more important than the formation of
direct condensation vescalagin−myrtillin products, and there-
fore this time was set as final time reaction. Nevertheless, high
amounts of the starting vescalagin and myrtillin remained in the
reaction medium, and they could be partially recovered (Figure
1). The reaction (Figure 2) was repeated 10 times. Different
products were obtained (Figure 1), but among them, only two
showed a band of absorption in the visible region of the
spectrum. These two pigments were isolated by semi-
preparative HPLC, hence obtaining 13 mg of product 1
(molar yield of 4.3%) and 1.5 mg of product 2 (molar yield of
0.5%). Their purities (higher than 95 and 90% for products 1
and 2, respectively) were determined by HPLC-DAD-MS
analysis.
1
Product 1 (Figure 2): H NMR (800 MHz, MeOH-d4/TFA
[99:1]) δ 3.55−4.03 (m, 5H, H-2‴, H-3‴, H-4‴, H-5‴, H-6‴),
3.99 (m, 1H, H-6α), 4.63 (s, 1H, H-1), 4.85 (bs, 1H, H-3), 5.21
(m, 1H, H-4), 5.29 (bs, 1H, H-2), 5.33 (d, J = 7.6 Hz, 1H, H-
1‴), 5.61 (d, J = 7.3, 1H, H-5), 5.74 (s, 1H, H-2′(V)), 6.50 (s,
1H, H-2′(III)), 6.77 (s, 1H, H-2′(IV)), 6.79 (s, 1H, H-6′), 7.47
(s, 2H, H-2″, H-6″), 9.14 (s, 1H, H-4′).
The characterization of these hybrid pigments was carried
out by HPLC-DAD-ESI+-MS (full mass and MSn analyses) and
by NMR spectroscopy. The UV−visible spectra of these hybrid
pigments showed two important bands (Figure 3a): one, the
most important, in the UV region with a maximum around 230
nm (characteristic of the ellagitannins) and a shoulder around
280 nm (characteristic of the anthocyanin) and another in the
visible region with a maximum around 540 nm, which is
characteristic of the flavylium form of some purple anthocyanin
derivatives.40
Mass spectrometry analysis allowed the corroboration of the
identity of these products as 1-deoxyvescalagin-myrtillin direct
condensation products. MS analyses of both pigments provided
in full mass analysis a molecular ion at m/z 1381 (Figure 3b)
with the same fragmentation pattern (Figure 3c,d): a main
Product 2 (Figure 2): 1H NMR (800 MHz, MeOH-d4/TFA
[99:1]) δ 3.55−4.03 (m, 5H, H-2‴, H-3‴, H-4‴, H-5‴, H-6‴),
5.29 (s, 1H, H-2), 5.33 (d, J = 7.7 Hz, 1H, H-1‴), 5.61 (d, J =
7.3 Hz, 1H, H-5), 5.74 (s, 1H, H-2′(V)), 6.49 (s, 1H, H-
2′(III)), 6.77 (s, 1H, H-2′(IV)), 6.70 (s, 1H, H-8′), 7.47 (s,
2H, H-2″, H-6″), 9.14 (s, 1H, H-4′).
The reaction between vescalagin and myrtillin showed some
important differences from that between vescalagin and
oenin.28,29 Whereas the reaction between oenin and vescalagin
yielded only one hybrid product, the 8′-C isomer, two isomers
could be isolated from the reaction involving myrtillin and
vescalagin. Moreover, the reaction between myrtillin and
vescalagin generated more side-products than the reaction
between oenin and vescalagin. This is in accordance with
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previous studies carried out in our laboratory about the
formation of pyranoanthocyanins in wine model solutions that
evidenced a higher reactivity for myrtillin than for oenin
(unpublished results). Among the side-products detected, one
of them (product 3) could be isolated with a purity >90%. The
HPLC-DAD-MSn analysis of this side-product confirmed that it
was indeed a product derived from the reaction between
vescalagin and myrtillin. The UV−visible spectrum showed a
band at 230 nm, which is characteristic of the ellagitannins, and
another at 280 nm, which is characteristic of the anthocyanin,
but no band in the visible region could be detected, evidencing
that this product was not colored. The mass spectra showed a
signal at m/z 1397 ([M + H]+), which could correspond to the
pseudomolecular ion of this compound. The fragmentation
pattern confirmed that this product was formed from the
condensation of vescalagin and myrtillin, because the main ion
fragment detected in the MS2 analysis (m/z 1235, [M + H −
162]+) originated from the loss of the glucose moiety of
myrtillin, and the main ion fragment in the MS3 analysis was
detected at m/z 917 ([M + H − 162 − 318]+), which
corresponds to the 1-deoxyvescalagin moiety. Unfortunately,
NMR analyses of this side-product 3 did not provide enough
information to allow its full structural determination.
would cause the regioisomerization of the 1-deoxyvescalagin-
(1β→8)-myrtillin into the 1-deoxyvescalagin-(1β→6)-myrtillin.
To confirm if the 6′-C isomer could thus be formed, the
evolution of an aqueous solution of the 8′-C isomer adjusted to
pH 3 was monitored over time by means of HPLC-DAD-MS.
Figure 5 shows the chromatograms obtained at four different
times (0, 3, 8, and 16 days). At the third day, the percentage of
the area of the peak corresponding to the 8′-C isomer started to
Regioisomerization Process. Two regioisomers thus
resulted from the acid-catalyzed nucleophilic substitution
reaction between vescalagin and myrtillin. In the case of the
malvidin 3-O-glucoside(oenin)−vescalagin and malvidin−ves-
calagin hybrid pigments, Quideau and co-workers detected only
the 8′-C regioisomer,28 although they proposed that the 6′-C
regioisomer could in principle derive from a regioisomerization
process.29 Anthocyanins and related compounds in mildly
acidic aqueous solutions are involved in a series of chemical
reactions leading to mixtures of colored and colorless
species.43,44 Thus, anthocyanin derivatives could undergo a
series of reversible transformations that might lead to a partial
transformation of product 1 into product 2 following the
regioisomerization process shown in Figure 4.29 The hydration
of the major pigment (1-deoxyvescalagin-(1β→8)-myrtillin),
followed by ring-opening, can lead to the E-chalcone, in which
rotation around the C-4′−C-4′a bond is possible. Ring
reclosure and dehydration after rotation around this bond
Figure 4. Putative regioisomerization of 1-deoxyvescalagin-(1β→8)-
myrtillin (product 1) into 1-deoxyvescalagin-(1β→6)-myrtillin (prod-
uct 2) in a mildly acidic aqueous solution (pH 3). Vg = 1-
deoxyvescalagin moiety.
Figure 5. HPLC monitoring of the evolution of a mildly acidic
aqueous solution (pH 3, HCl) of product 1, confirming the
regioisomerization of 1-deoxyvescalagin-(1β→8)-myrtillin (1) into 1-
deoxyvescalagin-(1β→6)-myrtillin (2).
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Figure 6. Visible spectra of myrtillin (4.6 × 10−5 M), 1-deoxyvescalagin-(1β→8)-myrtillin (product 1, 9.2 × 10−5 M), and 1-deoxyvescalagin-(1β→
6)-myrtillin (product 2, 9.2 × 10−5 M) at pH 1: (a) aqueous 1 M HCl solution) and pH 3.2; (b) wine model solution composed of 12% EtOH/H2O
(v/v) and tartaric acid (5g/L) adjusted to pH 3.2 with 1 M NaOH.
decrease concomitantly with an increase of that corresponding
to the 6′-C isomer. At the end of the experience, a clear
formation of the 6′-C isomer was detected (the percentage of
the area of the corresponding peak changed from 2.1% at day 3
to 7.9% at day 16). These changes of the percentages of the
peak areas indicate that the regioisomerization is a slow process,
because no change could be detected in the chromatogram
during the first day (data not shown), and 3 days was necessary
to observe a noticeable increase of the peak corresponding to
the 6′-C isomer. Between days 8 and 16, changes were only
minor, suggesting that equilibrium was reached and that it was
slightly shifted toward a preferred formation of the 8′-C isomer.
The thermodynamic and kinetic properties of the 8′-C isomer45
support that the regiomerization occurs.
Thus, the 6′-C isomer (1-deoxyvescalagin-(1β→6)-myrtillin)
could be formed in two different ways, either by a direct
nucleophilic substitution reaction during which the anthocyanin
attacks the 1-deoxyvescalagin carbocationic intermediate from
its nucleophilic 6′-C center or by an isomerization of the 8′-C
isomer (1-deoxyvescalagin-(1β→8)-myrtillin). During the
reaction, both isomers were detected even in the first hours,
thus suggesting that the direct nucleophilic attack of the
anthocyanin 6′-C center is operational. However, the
chromatographic peak corresponding to the 6′-C isomer was
much less important than that corresponding to the 8′-C
isomer. It is also possible that the 8′-C to 6′-C regioisomeriza-
tion could in part take place during the purification of the
products, because this purification was carried out using a
mildly acidic aqueous mobile phase (H2O/HCOOH 999:1).
Thus, in our opinion, most of the 6′-C isomer isolated for
further characterization might have been formed through this
regioisomerization process.
Chromatic Characterization. The chromatic properties of
these pigments were also determined. The UV−visible spectra
of these compounds (9.2 × 10−5 M) were measured and
compared to the UV−visible spectra of myrtillin (4.6 × 10−5
M) both in aqueous solutions at pH 1 and in wine model
solutions at pH 3.2 (Figure 6). An important bathochromic
shift is observed in the visible band of the hybrid pigment
spectra relative to that of the native myrtillin, which is in
agreement with the violet hue exhibited by these hybrid
compounds. This shift is comparable to that observed in the
acetaldehyde-mediated flavanol−anthocyanin derivative pig-
ments,12,13,16 which show blue hues. The maximum absorption
wavelength is shifted by ca. 20 nm toward higher wavelengths
in the hybrid pigments (Table 1) as compared with myrtillin at
both pH values.
Table 1. Maximum Absorption Wavelength of Myrtillin, 1-
Deoxyvescalagin-(1β→8)-myrtillin (8′-C Isomer) and 1-
Deoxyvescalagin-(1β→6)-myrtillin (6′-C Isomer) at pH 1
and 3.2
λmax (nm)
pH 1
pH 3.2
myrtillin
520
539
539
524
543
545
8′-C isomer
6′-C isomer
The molar absorption coefficients (ε) at pH 1 of myrtillin
and the major pigment (1, 1-deoxyvescalagin-(1β→8)-
myrtillin) have been calculated from the absorbance at the
maximum absorbance wavelength measured at five different
concentrations ranging between 7.6 × 10−6 and 9.2 × 10−5 M.
The value obtained for the hybrid pigment 1 (ε = 9040 cm−1
M−1, λ = 539 nm) was much lower than that corresponding to
the native myrtillin (ε = 21900 cm−1 M−1, λ = 520 nm). The
molar absorption coefficient at pH 1 of the minor pigment (2,
1-deoxyvescalagin-(1β→6)-myrtillin) was estimated from the
absorbance at the maximum absorbance wavelength measured
at two different concentrations (9.2 × 10−5 and 4.6 × 10−5 M).
The value obtained (ε = 8570 cm−1 M−1, λ = 539 nm) was
similar to that determined for product 1. This is in accordance
with the results obtained for the oenin− and malvidin−
vescalagin hybrid pigments, which also showed molar
absorptivities lower than those of their corresponding native
pigments.29 Thus, the presence of the vescalagin moiety linked
to the anthocyanin A-ring through a C−C linkage affects the
molar absorption coefficient of the resulting hybrid pigments.
Moreover, the resulting data perfectly fit with the Lambert−
Beer law, and no effect on the maximum absorbance
wavelength was detected when the pigment concentration
was increased, indicating that this pigment did not form self-
association complexes in the range of concentration studied
(7.6 × 10−6 and 9.2 × 10−5 M). The lack of self-association may
be related to the involvement of the chromophore in
intramolecular copigmentation, as shown for the hybrid
pigment formed with oenin.29
The CIELAB parameters (L*, a*, b*, C*ab, and hab) were
determined from the whole visible spectra of the hybrid
pigments and myrtillin at the same concentration (4.6 × 10−5
M) at two different pH values: pH 1 (0.1 N HCl aqueous
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solution) and pH 3.2 (wine model solution). Table 2 shows the
CIELAB parameters determined, which were employed to
(because the ΔE*ab value is ≥3), but these pigments had
indistinguishable colors at pH 3.2, even when using analytical
methods (ΔE*ab ≤ 1). Thus, at wine pH, these hybrid pigments
showed no difference in their color. Moreover, the color
differences determined for the pigments at different pH values
were higher for the myrtillin−vescalagin pigments than for
myrtillin, the highest color difference being observed in the case
of the 8′-C isomer.
Table 2. Chromatic Parameters (CIELAB Space) of Myrtillin
(4.6 × 10−5 M), 1-Deoxyvescalagin-(1β→8)-myrtillin (8′-C
Isomer, 4.6 × 10−5 M), and 1-Deoxyvescalagin-(1β→6)-
myrtillin (6′-C Isomer, 4.6 × 10−5 M) at pH 1 and 3.2
pH
L*
a*
b*
7.26
C*ab
hab
7.54
Distinctive physicochemical properties of the hybrid pig-
ment45 support the variations of the color parameters upon the
pH change relative to those observed for the native
anthocyanin.
myrtillin
1
73.42
80.30
54.82
41.76
55.30
41.79
3.2
−1.52
−2.08
8′-C isomer
6′-C isomer
1
80.49
91.98
31.99
10.66
−11.32
−4.55
33.93
11.59
−19.49
−23.09
In conclusion, two new regioisomeric anthocyano-ellagitan-
nin hybrid pigments have been obtained by hemisynthesis from
vescalagin and myrtillin: 1-deoxyvescalagin-(1β→8)-myrtillin
(8′-C isomer, major) and 1-deoxyvescalagin-(1β→6)-myrtillin,
(6′-C isomer, minor). This study has also demonstrated that, in
mildly acidic solutions, a partial transformation of the 8′-C
isomer into the 6′-C occurs via a slow regioisomerization
process. These hybrid pigments showed important differences
in their chromatic properties relative to those of myrtillin: a
bathochromic shift (ca. 20 nm) in the maximum absorbance
wavelength at both pH values used, lower molar absorption
coefficients (ε = 9040 cm−1 M−1 for the 8′-C isomer and ε =
8570 cm−1 M−1 for the 6′-C isomer in comparison with ε =
21900 cm−1 M−1 for myrtillin), and differences in their behavior
upon pH changes. Color differences between the two hybrid
pigments were detectable by the human eye only at pH 1, but
these pigments had indistinguishable colors at pH 3.2.
3.2
1
82.12
92.43
28.86
10.02
−9.88
−4.65
30.51
11.05
−18.90
−24.87
3.2
objectively evaluate the color of the hybrid pigments and also to
make comparisons with the myrtillin color. At both pH values,
the lightness (L*) of the hybrid pigments was higher than that
determined for myrtillin, in accordance with the lower molar
absorption coefficient. The hues (hab) determined for the
hybrid pigments were likewise significantly lower than those
determined for myrtillin, in accordance with the bluish hues
exhibited by these pigments.
Moreover, differences in the behavior of the hybrid pigments
and myrtillin with pH changes were evidenced by the variations
in the values of the CIELAB parameters. Whereas lightness
increased around 7 units in the case of myrtillin, the increase in
the L* values of the hybrid pigments with the pH change was
>11 units in both cases. On the contrary, the change in the hue
values was less important in the case of the hybrid pigments
than in the case of myrtillin.
AUTHOR INFORMATION
■
Corresponding Authors
*(M.T.E.-B.) Phone: +34 923294537. Fax: +34 923294515. E-
mail: escriban@usal.es.
To evaluate the importance of these changes and the
differences of the color of the different pigments, color
differences (ΔE*ab) between the different pigments at the
same pH value and for the same pigment at different pH values
were calculated. Table 3 shows the values obtained. The most
important color differences were found between myrtillin and
the hybrid pigments at both pH values, being higher at pH 3.2
than at pH 1. The color differences between the two hybrid
pigments were detectable by the human eye only at pH 1
*(S.Q.) Phone: +33 540003010. Fax: +33 54000221 5. E-mail:
s.quideau@iecb.u-bordeaux.fr.
Funding
We thank the Spanish MICINN and FEDER (Project ref.
AGL2008-05569-C02-01 and AGL2011-30254-C02-01), the
Consolider-Ingenio 2010 Programme (ref FUNC-FOOD,
CSD2007-00063), and the Conseil Interprofessionnel du Vin
de Bordeaux for financial support. We also thank the Spanish
Ministerio de Educacion for an F.P.U. predoctoral scholarship
́
to I.G.-E.
Table 3. Color Differences (ΔE*ab) Obtained in the
Solutions of Myrtillin, 1-Deoxyvescalagin-(1β→8)-myrtillin
(8′-C Isomer), and 1-Deoxyvescalagin-(1β→6)-myrtillin (6′-
C Isomer) at pH 1 and 3.2
Notes
The authors declare no competing financial interest.
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