Thermodynamics and kinetics of cyanidin 3-glucoside and caffeine copigments

Article abstract of DOI:10.1021/jf4006643

The multiequilibrium system of reactions of cyanidin 3-glucoside at acidic and mildly acidic pH values was studied in the presence of caffeine as a copigment. The thermodynamic and kinetic constants were determined using the so-called direct and reverse pH jump experiments that were followed by conventional UV-vis spectroscopy or stopped flow coupled to a UV-vis detector, depending on the rate of the monitored process. Compared with that of free anthocyanin, the copigmentation with caffeine extends the domain of the flavylium cation up to less acidic pH values, while in a moderately acidic medium, the quinoidal base becomes more stabilized. As a consequence, the hydration to give the colorless hemiketal is difficult over the entire range of pH values. At pH 1, two adducts were found for the flavylium cation-caffeine interaction, with stoichiometries of 1:1 and 1:2 and association constants of 161 M^-1 (K₁) and 21 M^-1 (K₂), respectively.

Full text of DOI:10.1021/jf4006643

Article
pubs.acs.org/JAFC
Thermodynamics and Kinetics of Cyanidin 3‑Glucoside and Caffeine
Copigments
Piedad M. Limon,† Raquel Gavara,*,‡ and Fernando Pina*,‡
́
†Department of Physical and Analytical Chemistry, Jaen
́
University, EPS of Linares, Alfonso X El Sabio, n° 28, 23700 Linares, Jaen
́
,
Spain
‡REQUIMTE, Departamento de Química, Faculdade de Cien
̂
cias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte de
Caparica, Portugal
ABSTRACT: The multiequilibrium system of reactions of cyanidin 3-glucoside at acidic and mildly acidic pH values was studied
in the presence of caffeine as a copigment. The thermodynamic and kinetic constants were determined using the so-called direct
and reverse pH jump experiments that were followed by conventional UV−vis spectroscopy or stopped flow coupled to a UV−
vis detector, depending on the rate of the monitored process. Compared with that of free anthocyanin, the copigmentation with
caffeine extends the domain of the flavylium cation up to less acidic pH values, while in a moderately acidic medium, the
quinoidal base becomes more stabilized. As a consequence, the hydration to give the colorless hemiketal is difficult over the
entire range of pH values. At pH 1, two adducts were found for the flavylium cation−caffeine interaction, with stoichiometries of
1:1 and 1:2 and association constants of 161 M−1 (K1) and 21 M−1 (K2), respectively.
KEYWORDS: anthocyanin, cyanidin 3-glucoside, caffeine, copigmentation, color stabilization, hydration
On the other hand, the hemiketal (chromene) reacts via a
tautomeric process that leads to a ring opening with formation
of a cis-chalcone species (Cc). This reaction usually occurs on a
subsecond to second time scale [Figure 1, tautomerization (eq
3)].
INTRODUCTION
■
Anthocyanins are the ubiquitous compounds responsible for
most of the red and blue colors found in flowers and fruits.1
Noncovalent interactions involving the colored species related
to anthocyanins (namely, flavylium cation and quinoidal base)
and other natural compounds are a matter of great importance
with regard to color expression in plants. Noncovalent
interactions have been extensively studied under the category
of copigmentation.2−5
Anthocyanins in acidic to moderately acidic solutions are
involved in a pH-dependent network of reversible reactions
(Figure 1).6−9 The flavylium cation (usually red colored) is the
only stable species under very acidic conditions, and at higher
pH values, the colorless hemiketal is the dominant species
together with quinoidal base (blue color), with chalcones as
minor components. A convenient way to study the network of
chemical reactions involving anthocyanins and related com-
pounds is to conduct pH jumps:10 a direct pH jump is here
defined as a change in pH from equilibrated solutions at very
low pH values (where flavylium cation is stable) to higher pH
values. Conversely, reverse pH jumps11 are defined as the
addition of acid to equilibrated solutions at higher pH values
(typically pH 4−6) to reach a very acidic pH where flavylium is
stable.
The hydration reaction is strongly dependent on the proton
concentration, while the tautomerization is catalyzed at very
acidic and basic pH values.12 In Figure 2, a simulation of the
rate constant of these two kinetic processes is given. Below pH
2, hydration is much faster than tautomerization and occurs on
a millisecond to subsecond time scale. However, at pH >2.5,
hydration is usually much slower than tautomerization.
In conclusion, after a pH jump from a very acidic solution
containing the flavylium cation to a moderately acidic solution,
quinoidal base is formed immediately and constitutes a kinetic
product (because it is usually less stable thermodynamically)
that slowly disappears to reach a pseudoequilibrium involving
all the species except the trans-chalcone.
Finally, the thermodynamic equilibrium is attained from the
cis-chalcone isomerization to give the trans-chalcone (Ct), in a
process that in anthocyanins usually takes a few hours [Figure
1, isomerization (eq 4)].
The three kinetic processes are simulated in Figure 3 for the
case of anthocyanins, and because of their clear separation in
time, they can be studied separately, which simplifies the kinetic
analysis of the system. Moreover, the change of regime in the
case of the second kinetic process is also illustrated in the
figure. Only at lower pH values is the tautomerization reaction
slower than the hydration.
When a direct pH jump is performed, the flavylium cation
(AH+) transfers a proton to water, leading to a zwitterionic
species that rearranges to the quinoidal base A [Figure 1,
deprotonation (eq 1)].
The flavylium cation is also involved in a parallel reaction
that is much slower than the previous one because of the
addition of water at position 2, giving hemiketal species B2
(which will hereafter be termed B) [Figure 1, hydration (eq
Received: February 9, 2013
Revised: May 1, 2013
Accepted: May 3, 2013
a
2)].
© XXXX American Chemical Society
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Figure 1. Network of chemical reactions of cyanidin 3-glucoside in acidic to moderately acidic solutions.
Figure 3. Simulation of the rate constants of the three well-separated
kinetic processes taking place in anthocyanins, first kinetics (),
second kinetics (−−− and −-−), and third kinetics (···): ka = 104 s−1;
k−a = 107.4 s−1 M−1; kh = 0.07 s−1; k−h = 95 s−1 M−1; Ka = 10−3.36; Kt =
0.09; Kh = 7.3 × 10−4 M−1; ki = 7.4 × 10−6 s−1; k−i = 2.5 × 10−5 s−1; kt
+ k−t = 0.7 s−1; kH = 10 s−1 M−1; and kOH = 107 s−1 M−1.
Figure 2. Simulation of the pH-dependent rate constants of hydration
and tautomerization using the values reported in this paper: kh = 0.07
s−1; k−h = 95 s−1 M−1; kt = 0.07 s−1; and k−t = 0.6 s−1. Reliable acid and
basic catalysis constants for the tautomerization reaction were
estimated to be kH = 10 and kOH = 107 s−1 M−1, respectively.
In spite of numerous and well-documented works regarding
the copigmentation with anthocyanins, the effect of copigmen-
tation on the rate and equilibrium constants of the network
described above has been less studied.13,14 For this purpose, the
copigmentation involving cyanidin 3-glucoside and caffeine was
selected (Figure 4). The copigmentation effect of caffeine has
been studied for malvin13,15 and several synthetic flavylium
compounds.16−18 The main driving force for the association
seems to be π−π stacking between the chromophore and the
copigment.15
Figure 4. Chemical structures of cyanidin 3-glucoside and caffeine.
Caffeine and derivatives of cyanidin 3-glucoside are found
together in seeds of cacao19 and in the leaves of a line of red tea
recently developed by natural crossing.20 Additionally, caffeine
is a common food additive, and from this point of view, it is
interesting to know how its presence can affect the reactivity of
anthocyanins, usually employed as food colorants. Recently, we
reported on the effect of self-aggregation on the thermody-
B
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Figure 5. (A) Spectral variations upon addition of caffeine to cyanidin 3-glucoside (2.3 × 10−5 M) at pH 1.0. The spectra correspond to 0, 0.002,
0.006, 0.02, 0.04, 0.06, and 0.08 M caffeine. The spectra for 0 and 0.08 M caffeine are shown as bold lines. (B) Mathematical treatment of the data in
panel A allowed us to obtain the pure spectra corresponding to the 1:1 (−−−) and 1:2 (···) flavylium cation−caffeine adducts. The spectrum of free
anthocyanin is plotted as a solid line. The inset shows the molar fractions of the adducts and free anthocyanin as a function of caffeine concentration.
namics and kinetics of the six most common anthocyanidin 3-
glucosides, including cyanidin 3-glucoside.11 In this work, we
report for the first time an extensive study of the effect of
caffeine copigmentation on the thermodynamics and kinetics of
the network of chemical reactions of cyanidin 3-glucoside in
diluted solutions, where self-aggregation effects can be
neglected.
The 1:1 stoichiometry has been proposed for a number of
flavylium cation−copigment complexes, usually resulting, like
here, in a bathochromic shift and a small decrease in the molar
extinction coefficient.18,23−25 The formation of a second
complex with a 1:2 stoichiometry has been less reported. In
this case, we have to take into account the possibility that the
formation of this complex will vary, depending on the chemical
structure of anthocyanin and copigment as well as the
anthocyanin:copigment molar ratio, among other factors.
Several years ago,18 the formation of the 1:2 complex was
proposed for the 4-methyl-7-hydroxyflavylium−caffeine and 4′-
hydroxyflavylium−caffeine systems at high copigment concen-
trations, but in both cases, the complexation to additional
caffeine induced a greater decrease in the molar extinction
coefficient. Here the effect is different; i.e., the formation of the
second adduct provokes the increase in the molar extinction
coefficient. For some reason (outside the scope of this work),
the interaction with more caffeine increases the value of the
oscillator strength related to the electronic transition (or
transitions) responsible for the absorption band.
Inspection of the inset in Figure 5B shows that
concentrations of caffeine higher than the respective solubility
limit would be necessary to obtain the 1:2 adduct as the only
species. However, for 0.08 M caffeine, practically all the
anthocyanin interacts with the copigment through the 1:1
(36%) and 1:2 (61%) adducts, and we selected this ratio of
concentrations, i.e., 0.08 M caffeine versus 2 × 10−5 M cyanidin
3-glucoside, to analyze the effect of copigmentation on the
thermodynamics and kinetics related to the network of
chemical reactions of the anthocyanin (Table 1).
MATERIALS AND METHODS
■
Cyanidin 3-glucoside chloride (Kuromanin chloride) was purchased
from Extrasynthese; caffeine was purchased from Merck. All the
reagents (≥96%) were used without any further purification. The
solutions were prepared in Millipore water. The pH of the solutions
was adjusted by the addition of HCl, NaOH, and 8.3 × 10−3 M citrate
buffer and was measured on a CRISON pH-Meter BASIC 20+
instrument. The ionic strength of the solutions was kept constant at
0.1 M by the addition of NaCl. UV−vis absorption spectra were
recorded on Varian Cary 100 Bio and Varian Cary 5000
spectrophotometers. The stopped flow experiments were conducted
in an Applied Photophysics SX20 stopped flow spectrometer provided
with a PDA.1/UV photodiode array detector with a minimum scan
time of 0.65 ms and a wavelength range of 200−735 nm.
RESULTS AND DISCUSSION
■
In Figure 5A, the spectral variations observed upon addition of
increasing concentrations of caffeine (up to 0.08 M) to a
diluted solution of cyanidin 3-glucoside (2.3 × 10−5 M) at pH 1
are shown. For lower caffeine concentrations, the absorption
red shifts and slightly decreases and an isosbestic point at 521
nm is observed; for higher caffeine concentrations, the
absorption shifts to higher wavelengths and gives rise to a
small absorption increase and the isosbestic point at 521 nm
disappears, with a new one appearing at 534 nm. At pH 1, the
flavylium cation is the only species present in solution in the
case of the anthocyanin; thus, these spectral changes suggest
the formation of two adducts involving the flavylium cation of
cyanidin 3-glucoside and caffeine, probably with flavylium
cation:caffeine stoichiometries of 1:1 and 1:2. Mathematical
treatment of the system21,22 assuming these stoichiometries
allowed us to obtain the absorption spectra of the two adducts,
as well as the respective association constants, as shown in
Figure 5B.
The pH dependence of the absorption spectra of thermally
equilibrated solutions of cyanidin 3-glucoside 1.7 × 10−5 M in
the presence of caffeine 0.08 M is shown in Figure 6A. Here, K’a
is the global acid−base equilibrium constant that relates the
flavylium cation with the other species in the network (eq 5):26
AH+ + H2O ⇌ A + B Ka′ = Ka + Kh + KhKt
+ Cc + Ct + H3O+
+ KhKtKi
(5)
When these spectra are compared with the absorption spectra
of cyanidin 3-glucoside in the absence of caffeine (Figure 6B),11
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[H+]
1
Table 1. Thermodynamic Constants of 1.7 × 10−5
M
k2nd(hydration control)
=
kh +
k−h[H+]
[H+] + Ka
1 + Kt
Cyanidin 3-Glucoside in the Presence and Absence of 0.08
M Caffeine
(7)
b
caffeine
pKa
Kh (M−1
7.4 × 10−4
3.1 × 10−3
)
Kt
Ki
pKa′
The slower process in Figure 6A corresponds to the
isomerization to give Ct, and its rate depends on the mole
fraction of Cc in the pseudoequilibium involving A, AH+, B, and
Cc (eq 8).9
a
0.08 M
c
3.3(6)
3.8(0)
0.09
0.12
0.3
2.9
2.5
−
−
a
b
Estimated error for all constants except Ki, 15%. The percentage of
c
Ct is ≈2%, and the reported value is a rough estimate. See ref 11.
KhKt
There the reported concentration of anthocyanin was 2 × 10−5 M.
k3rd
=
× ki + k−i
[H+] + Ka + Kh(1 + Kt)
(8)
Because the mole fraction of Ct is very small in anthocyanins
(<5%), the latest process is difficult to observe and quantify
precisely.
it is clear that the copigment stabilizes the flavylium cation,
because its adduct becomes less acidic (pKa′ higher), as well as
the quinoidal base, because a significant amount of this species
is presented for less acidic solutions.
In Figure 7A, the kinetic process with a direct pH jump from
1 to 5.8 for 1.7 × 10−5 M cyanidin 3-glucoside and 0.08 M
caffeine is shown. Immediately after the pH jump, the
characteristic absorption spectrum of the quinoidal base
appears. This process corresponds to the proton transfer
reaction, and its rate that occurs on the microsecond time scale
is too fast to be followed by the common techniques, including
stopped flow6 (eq 6).
In Figure 7B, the absorption spectra of the quinoidal base
(taken immediately after the direct pH jump to pH ≫ pKa) is
compared with that corresponding to cyanidin 3-glucoside in
the absence of caffeine.11 As in the case of the flavylium cation,
the copigmentation with caffeine gives rise to a red shift in the
absorption spectra, indicating that the excited state of the
anthocyanin is more stabilized than the ground state.
The data reported in the inset of Figure 7A allow us to
estimate the equilibrium constant Ka from eq 9,27 where the
initial absorbance is due to the complete conversion of the total
concentration of the anthocyanin (previously in the form of
flavylium cation) to quinoidal base and the final absorbance is
caused by the quinoidal base remaining at the equilibrium; from
the inset of Figure 7A, 35% of quinoidal base remains at
equilibrium (1.7 × 10−5 M cyanidin 3-glucoside and 0.08 M
caffeine). From eq 9 and using a Ka′ value of 10−2.9, a Ka value
of 10−3.36 is obtained.
k1st = ka + k−a[H+]
(6)
The observed decrease in the quinoidal base absorption is
biexponential, the faster process corresponding to the hydration
control (eq 7).9 In other words, because we are in a range of
pH values above 2.5, the hemiketal B equilibrates faster with cis-
chalcone than flavylium cation and hemiketal (faster tautome-
rization) and the term 1/(1 + Kt) corresponds to the mole
fraction of B in equilibrium wtih Cc. Moreover, as reported by
Brouillard and Dubois,6 the quinoidal base does not give
directly the hemiketal at moderately acidic and neutral pH
values; in fact, only the fraction of flavylium cation available
from the initial (faster) equilibrium with quinoidal base leads to
hemiketal. This observation is included in eq 7 by the term
[H+]/([H+] + Ka), corresponding to the mole fraction of the
flavylium cation.
Af
Ai
Ka
Ka′
=
(9)
Several pH jumps like that shown in Figure 7A were performed
at different final pH values. The faster rate constant of the
biexponential process (hydration) is plotted versus pH in
Figure 8 (●) and was fit using eq 7 (−−−) (see below).
The reverse pH jumps give complementary information
about the system. In Figure 9, acid was added to equilibrated
solutions at pH 5.7 to yield a final pH of 1.3, and the spectral
variations were followed by stopped flow (Figure 9A). In this
Figure 6. (A) pH-dependent absorption spectra of thermally equilibrated solutions of 1.7 × 10−5 M cyanidin 3-glucoside and 0.08 M caffeine. (B)
Same in the absence of caffeine [2 × 10−5 M (see ref 11)].
D
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Figure 7. (A) Spectral variations upon a pH jump from 1.0 to 5.8, with 1.7 × 10−5 M cyanidin 3-glucoside and 0.08 M caffeine. (B) Normalized
spectra of the cyanidin 3-glucoside quinoidal base in the presence of caffeine obtained immediately after the pH jump (−−−) and analogous spectra
of cyanidin 3-glucoside in the absence of caffeine for comparison (see ref 11).
and the hydroxyl concentration was very low for detection of
the second.
The amplitudes of the three processes are reported in Figure
9B and give
%Cc
%B
Kt =
(12)
and
Ka
Kh
%A
%B
=
(13)
From the percentages shown in Figure 9B, Kt = 0.09 and Kh =
6.3 × 10−4 M−1. The kinetic constants of the reverse pH jumps
fit with eq 10 are shown in Figure 8 (○).
Knowing Kt and Ka, we can conduct a global fitting of the
kinetic data reported in Figure 8 with eqs 7 and 10, leading to a
kh of 0.07 s−1 and a k−h of 95 M−1 s−1. The ratio between the
hydration and dehydration rate constants gives a Kh of 7.4 ×
10−4 M−1, in good agreement with the value calculated from the
reverse pH jumps (see above). It is worth noting that the
similarity of eq 7 with eq 10 results from the low value of Kt in
this system. Finally, the slower kinetic process in Figure 9B
corresponds to a rate constant k−t of 0.7 s−1, permitting us to
calculate a kt of 0.06 s−1 from the respective equilibrium
constant.
To corroborate the interpretation of the copigmentation
phenomenon, an equilibrated solution of cyanidin 3-glucoside
at pH 5.7 was mixed with caffeine at the same pH value (final
concentrations of 1.7 × 10−5 and 0.08 M, respectively) and the
spectral variations were monitored (Figure 10). An increase in
the level of the caffeine−quinoidal base adduct is clearly
observed. It is worth noting the rate of the kinetic process
shown in Figure 10, which fits with the hydration−dehydration
kinetic process presented in the inset of Figure 8 (□).
In summary, the spectral changes upon addition of caffeine
to cyanidin 3-glucoside show clearly the interaction of the
copigment with the flavylium cation as well as with quinoidal
base. The mole fraction distribution of the species of the
flavylium network in the absence11 and presence of caffeine
0.08 M is represented in Figure 11. The domain of the
flavylium cation is extended by addition of caffeine as a result of
the stabilization of this species, while at moderately acidic pH
●
○
Figure 8. pH dependence of the direct ( ) and reverse ( ) pH jumps
of 1.7 × 10−5 M cyanidin 3-glucoside, in the presence of 0.08 M
caffeine. Fitting was achieved by eq 7 (−−−) and eq 10 (); a point
regarding the kinetics of the complexation of cyanidin 3-glucoside at
□
pH 5.7 is included ( ).
case (low pH value), hydration becomes faster than
tautomerization. The quinoidal base present at pH 5.7 was
immediately converted into flavylium cation, a process that
took place during the mixing time of the stopped flow. This
reaction was followed by the formation of more flavylium
cation through a biexponential process: the first corresponding
to the formation of flavylium cation from the hemiketal present
in the equilibrium at pH 5.7 (eq 10) and the last to the
conversion of Cc into flavylium cation via hemiketal (eq 11). In
this kind of experiment, no reversibility is expected from B to
give Cc.28
[H+]
kobs1(SF)
=
kh + k−h[H+]
Ka + [H+]
(10)
(11)
kobs2(SF) = k−t
In eq 11, neither acidic nor basic catalysis was considered for
the reverse tautomerization reaction,12 because at this pH the
proton concentration was low for the observation of the first
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Figure 9. (A) Reverse pH jump from an equilibrated solution of 1.7 × 10−5 M cyanidin 3-glucoside at pH 5.7 in the presence of 0.08 M caffeine to a
final pH of 1.3, followed by stopped flow. (B) Trace of the kinetics monitored at 535 nm.
Figure 11. Comparison between the mole fraction distribution of
cyanidin 3-glucoside in the absence11 () and presence of 0.08 M
Figure 10. Spectral variations upon addition of caffeine to an
equilibrated solution of cyanidin 3-glucoside at pH 5.7. Final
caffeine (−−−). In this figure, the terms AH+Caf, BCaf, ACaf, and
concentrations of 0.08 M caffeine and 1.7 × 10−5 M cyanidin 3-
CcCaf refer to the molar fractions of AH+, B, A, and Cc, respectively,
glucoside.
in the presence of caffeine.
Table 2. Kinetic Constants of 1.7 × 10−5 M Cyanidin 3-
values, the level of the quinoidal base−caffeine adduct increases
at the expense of the hemiketal.
Glucoside in the Presence and Absence of 0.08 M Caffeine
kh
k−h
kt
k−t
With regard to the kinetic constants, the most interesting
result is the increase in the dehydration rate constant (k−h) for
the anthocyanin−caffeine system (Table 2). Somehow, the
presence of the copigment facilitates the reaction of the
hemiketal to give the flavylium cation. Analogous behavior was
previously observed in the case of the self-aggregation of
anthocyanins (i.e., dehydration is facilitated in more concen-
trated and/or aggregated solutions).11
b
b
caffeine (s−1
)
(M−1 s−1
)
(s−1
)
(s−1
)
ki (s−1
)
k−i (s−1
)
0.08
0.07
95
0.06
0.7
7.4 × 10−6
2.5 × 10−5
a
M
c
−
a
0.11
35
0.07
0.6
−
−
Estimated error for all constants except the isomerization constants,
b
15%. The percentage of Ct is ≈2%, and the reported value is a rough
c
estimate. See ref 11. There the reported concentration of anthocyanin
was 2 × 10−5 M.
In conclusion, copigmentation results in the stabilization of
the colored flavylium cation and quinoidal base species, making
the hydration to give the colorless hemiketal more difficult. The
interaction of the anthocyanin with caffeine mimics in some
way what happens in Nature. The flavylium cation is only stable
at very acidic pH values, and as a consequence, to achieve the
red color inside the vacuoles of the plants (roughly in the pH
interval of 3−6), it is necessary to increase the pH domain of
the flavylium cation. On the other hand, at higher pH values,
the quinoidal base is a very minor species, and thus, interactions
with suitable species are needed to stabilize the quinoidal base
and obtain blue colors.
AUTHOR INFORMATION
■
Corresponding Author
*R.G.: phone, +351212948300, ext. 10946; fax,
+351212948550; e-mail, r.castell@fct.unl.pt. F.P.: phone,
+351212948355; fax, +351212948550; e-mail, fp@fct.unl.pt.
F
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Journal of Agricultural and Food Chemistry
Funding
Article
(16) Wigand, M. C.; Dangles, O.; Brouillard, R. Complexation of a
fluorescent anthocyanin with purines and polyphenols. Phytochemistry
1992, 31, 4317−4324.
(17) El Hajji, H.; Dangles, O.; Figueiredo, P.; Brouillard, R. 3′-(β-D-
Glycopyranosyloxy)flavylium ions: Synthesis and investigation of their
properties in aqueous solution. Hydrogen bonding as a mean of colour
variation. Helv. Chim. Acta 1997, 80, 398−413.
(18) Pina, F. Caffeine interaction with synthetic flavylium salts. A
flash photolysis study for the adduct involving 4′,7′-dihydroxyflavy-
lium. J. Photochem. Photobiol., A 1998, 115, 51−59.
(19) Pereira-Caro, G.; Borges, G.; Nagai, C.; Jackson, M. C.; Yokota,
T.; Crozier, A.; Ashihara, H. Profiles of Phenolic Compounds and
Purine Alkaloids during the Development of Seeds of Theobroma cacao
cv. Trinitario. J. Agric. Food Chem. 2013, 61, 427−434.
(20) Maeda-Yamamoto, M.; Saito, T.; Nesumi, A.; Tokuda, Y.; Ema,
K.; Honma, D.; Ogino, A.; Monobe, M.; Murakami, A.; Murakami, A.;
Tachibana, H. Chemical analysis and acetylcholinesterase inhibitory
effect of anthocyanin-rich red leaf tea (cv. Sunrouge). J. Sci. Food Agric.
2012, 92, 2379−2386.
(21) Antonov, L.; Gergov, G.; Petrov, V.; Kubista, M.; Nygren, J. UV-
Vis spectroscopic and chemometric study on the aggregation of ionic
dyes in water. Talanta 1999, 49, 99−106.
(22) Antonov, L.; Petrov, V. Quantitative analysis of undefined
mixtures: “Fishing net” algorithm. Anal. Bioanal. Chem. 2002, 374,
1312−1317.
̂
This work was supported by Fundaca̧ o para a Ciencia e
̃
Tecnologia through Projects PEst-C/EQB/LA0006/2011 and
PTDC/QUI-QUI/117996/2010. Postdoctoral Grant SFRH/
BPD/44639/2008 (R.G.) is also acknowledged.
Notes
The authors declare no competing financial interest.
ADDITIONAL NOTE
■
a
This nomenclature is due to the fact that in several systems it
is possible to observe the formation of a second hemiketal, from
the addition of water to position 4, which is termed B4.
However, this reaction is negligible for the system being studied
here.
REFERENCES
■
(1) Andersen, Ø. M.; Jordheim, M. The Anthocyanins. In Flavonoids:
Chemistry, Biochemistry and Applications; Andersen, Ø. M., Markham,
K. R., Eds.; CRC Taylor & Francis: Boca Raton, FL, 2006; pp 471−
551.
(2) Asen, S.; Steward, R. N.; Norris, K. H. Copigmentation of
anthocyanins in plant-tissues and its effect on color. Phytochemistry
1972, 11, 1139−1144.
(3) Asen, S.; Steward, R. N.; Norris, K. H. Anthocyanin, flavonol
copigments and pH responsible for larkspur flower color.
Phytochemistry 1975, 14, 2677−2682.
(4) Liao, H.; Cai, Y.; Haslam, E. Polyphenol Interactions. 6.
Anthocyanins copigmentation and color changes in red wines. J. Sci.
Food Agric. 1992, 59, 299−305.
(5) Gonnet, J. F. Colour effects of co-pigmentation of anthocyanins
revisited. 1. A colorimetric definition using the CIELAB scale. Food
Chem. 1998, 63, 409−415.
(6) Brouillard, R.; Dubois, J.-E. Mechanism of structural trans-
formations of anthocyanins in acidic media. J. Am. Chem. Soc. 1977, 99,
1359−1364.
(7) Brouillard, R.; Delaporte, B. Chemistry of anthocyanins pigments.
2. Kinetic and thermodynamic study of proton transfer, hydration and
tautomerization reactions of malvidin-3-glucoside. J. Am. Chem. Soc.
1977, 99, 8461−8468.
(8) Brouillard, R.; Lang, J. The hemiacetal-cis-chalcone equilibrium of
malvin, a natural anthocyanin. Can. J. Chem. 1990, 68, 755−761.
(9) Pina, F.; Melo, M. J.; Laia, C. A. T.; Parola, A. J.; Lima, J. C.
Chemistry and applications of flavylium compounds: A handful of
colours. Chem. Soc. Rev. 2012, 41, 869−908.
(10) Brouillard, R.; Delaporte, B.; Dubois, J.-E. Chemistry of
anthocyanin pigments. 3. Relaxation amplitudes in pH-jumps experi-
ments. J. Am. Chem. Soc. 1978, 100, 6202−6205.
(23) Brouillard, R.; Wigand, M.-C.; Dangles, O.; Cheminat, A. pH
and Solvent Effects on the Copigmentation Reaction of Malvin with
Polyphenols, Purine and Pyrimidine Derivatives. J. Chem. Soc., Perkin
Trans. 2 1991, 1235−1241.
(24) Ferreira da Silva, P.; Lima, J. C.; Freitas, A. A.; Shimizu, K.;
Maca̧ nita, A. L.; Quina, F. H. Charge-Transfer Complexation as a
General Phenomenon in the Copigmentation of Anthocyanins. J. Phys.
Chem. A 2005, 109, 7329−7338.
(25) Franca̧ Rodrigues, R.; Ferreira da Silva, P.; Shimizu, K.; Freitas,
A. A.; Kovalenko, S. A.; Ernsting, N. P. Ultrafast Internal Conversion
in a Model Anthocyanin−Polyphenol Complex: Implications for the
Biological Role of Anthocyanins in Vegetative Tissues of Plants.
Chem.Eur. J. 2009, 15, 1397−1402.
(26) Pina, F.; Petrov, V.; Laia, C. A. T. Photochromism of flavylium
systems. An overview of a versatile multistate system. Dyes Pigm. 2012,
92, 877−889.
(27) Pina, F. Thermodynamics and kinetics of flavylium salts: Malvin
revisited. J. Chem. Soc., Faraday Trans. 1998, 94, 2109−2116 , 3781
(correction in Scheme 1, whereby the Ct and Cc structures should be
exchanged).
(28) Gavara, R.; Yoann Leydet, Y.; Petrov, V.; Pina, F. Photo-
chemistry of 2-(4-hydroxystyryl)-1-naphthopyrylium. Photochem.
Photobiol. Sci. 2012, 11, 1691−1699.
(11) Leydet, Y.; Gavara, R.; Petrov, V.; Diniz, A. M.; Parola, A. J.;
Lima, J. C.; Pina, F. The effect of self-aggregation on the determination
of the kinetic and thermodynamic constants of the network of
chemical reactions in 3-glucoside anthocyanins. Phytochemistry 2012,
83, 125−135.
(12) McClelland, R. A.; Gedge, S. Hydration of the flavylium ion. J.
Am. Chem. Soc. 1980, 102, 5838−5848.
(13) Dangles, O.; Brouillard, R. Polyphenol interactions. The
copigmentation case: Thermodynamic data from temperature
variation and relaxation kinetics. Medium effect. Can. J. Chem. 1992,
70, 2174−2189.
(14) Dangles, O.; Saito, N.; Brouillard, R. Kinetic and thermody-
namic control of flavylium hydration in pelargonidin cinnamic acid
complexation: Origin of the extraordinary flower color diversity of
pharbitis-nil. J. Am. Chem. Soc. 1993, 115, 3125−3132.
(15) Mistry, T. V.; Cai, Y.; Lilley, T. H.; Haslam, E. Polyphenol
Interactions. 5. Anthocyanins copigmentation. J. Chem. Soc., Perkin
Trans. 1991, 1287−1296.
G
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