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Previous and recent advances in pyranoanthocyanins equilibria in aqueous solution
Dyes and Pigments 100 (2014) 190e200
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Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
Review
Previous and recent advances in pyranoanthocyanins equilibria in aqueous solution Joana Oliveira, Nuno Mateus, Victor de Freitas* Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2013 Accepted 10 September 2013 Available online 19 September 2013
Pyranoanthocyanins are anthocyanin-derived compounds found in nature that present a vast palette of colours ranging from yellow to turquoise blue. Studies in aqueous solutions of these pigments by UV eVisible spectroscopy revealed their higher colour stability when compared to their anthocyanin precursors. Through Nuclear Magnetic Resonance (NMR) techniques it was possible to confirm the absence of hydration reactions in pyranoanthocyanins, contrarily to anthocyanins, which helps explaining their higher colour stability with the increase of the pH. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Pyranoanthocyanins Equilibrium forms Aqueous solution Deprotonation NMR UVeVisible spectroscopy
1. Introduction Pyranoanthocyanins are major polyphenolic pigments formed in red wines during their ageing and maturation [1] and are thought to contribute to the orange hues observed in those wines [2]. Their chemical formation pathway involves a cyclic addition onto carbon 4 and the hydroxyl group at the carbon 5 position of the anthocyanin, yielding a fourth ring that is responsible for the higher stability to hydration of these compounds when compared to the original anthocyanins [2e7]. Over the years, several families of pyranoanthocyanins have been described in the literature including A and B-type vitisins [3], methylpyranoanthocyanins [8e10], oxovitisins [11], acetylpyranoanthocyanins [12], pyranoanthocyaninphenolics [4,13,14], pyranoanthocyanin-flavanols [15], A and Btype portisins [16,17], pyranoanthocyanin dimers [18] and pyranoanthocyanin-butadienilydene-phenolics [19] (Fig. 1). In aqueous solution, anthocyanins are present in different forms in equilibrium. The anthocyanins and flavylium-like compounds equilibrium forms in aqueous solution have been vastly described in the literature [20e29]. In very acidic aqueous solution (pH < 1), these pigments are present as red flavylium cations. The increase of pH leads to a reduction of the intensity of the color due to a decrease of the concentration of the flavylium cation that is
* Corresponding author. Tel.: þ351 220402558; fax: þ351 220402658. E-mail address: [email protected] (V. de Freitas). 0143-7208/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dyepig.2013.09.009
converted into its colorless hemiketal form through nucleophilic attack of water. The hemiketal further undergoes a tautomerization reaction to give the pale yellow cis- chalcone (Cc), which isomerizes to trans-chalcone (Ct). At low acidic, neutral and basic pH values, deprotonation of the flavylium cation also occurs, giving rise to the violet/blue quinoidal forms [30] (Fig. 2). Oppositely to anthocyanins, studies of pyranoanthocyanins equilibria in aqueous solutions are scarce in the literature [5e7,31]. Only a few papers have been published related to the physicale chemical properties of pyranoanthocyanins in aqueous solutions. The present review aims to discuss the main features of pyranoanthocyanins in aqueous solutions but also to present new data concerning the physicalechemical features of some recently described pyranoanthocyanins detected in red wines [17,18,32]. Moreover, the ionization constants will be correlated with some structural features that are important to stabilize pyranoanthocyanin in the pyranoflavylium cation form. The diverse colours presented by pyranoanthocyanins pigments and their higher colour stability, namely their colour stability at a wide pH range are important features indicating a putative application of these compounds in food products. 2. Pyranoanthocyanins in aqueous solution Over the years, the equilibrium forms of pyranoanthocyanin pigments have been studied using NMR and UVeVisible spectroscopy [5e7,31,33]. In aqueous solutions pyranoanthocyanins have
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Fig. 1. Anthocyanin-derived pigments found in nature.
shown to co-exist under different equilibrium forms that are pHdependent. NMR spectroscopy gives important information about the type of equilibrium that is occurring in aqueous solution at different pH values. In general, the peaks corresponding to proton exchange equilibrium forms are observed in the aromatic region spectrum of the 1H NMR. No other important 1H NMR peaks at any
pH studied corresponding to the hemiketal form described for anthocyanin compounds were observed [28,36,37] (Fig. 3). Indeed, if the hemiketal form was present, a significant displacement of about 2 ppm in the chemical shift of protons H-9 and H-10 (for vitisin B) to lower ppm values would have been observed. Santos et al. (1993) showed that the chemical shift of proton H-4 in malvin
Fig. 2. Anthocyanin equilibria in aqueous solutions at different pH values.
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Fig. 3. 1H spectra of a pyranoanthocyanin pigments (carboxypyranomalvidin-3-O-glucoside) in D2O at different pD values from 0.67 until 10.99, T ¼ 298 K, total concentration 3.2 mM.
(malvidin-3,5-glucoside) in the hemiketal form is quite different (6.60 ppm) from the one observed for the same proton in the flavylium cation form (8.97 ppm) [36]. The same trend was observed by Jordheim et al. (2006) for the hemiketal and flavylium cation forms in malvidin-3-O-glucoside (6.58 and 9.13 ppm, respectively) [28]. Bearing all this, pyranoanthocyanins do not undergo hydration and only proton transfer reactions occur when the pH change. In fact, a number of studies based on UVeVisible spectroscopy have already established that pyranoanthocyanin pigments are protected from the attack by water when compared with their anthocyanin precursors [2,31,38]. In the pyranoanthocyanins titration through NMR generally, the increase of the pH of the solution until pHw7 leads to a
displacement of the chemical shift of all protons to lower ppm values due to the equilibrium between the pyranoflavylium cation form and the neutral quinoidal form [5,33]. For higher pH values up to pHw12 a similar trend is observed (Fig. 3), corresponding to the faster equilibrium between the neutral quinoidal base form and the anionic quinoidal base. In the UVeVisible spectrum of pyranoanthocyanin commonly, for pH values between 1 and 5 it is possible to observe a batochromic shift in the maximum absorption wavelength that is accompanied by a decrease in the absorbance of the solutions (Fig. 4). The wavelength displacement is attributed to the deprotonation of the pyranoflavylium cation form that leads to the formation of the respective neutral quinoidal base. For higher pH values a similar trend is observed and the equilibrium between
Fig. 4. Absorption spectra (360e830 nm) of aqueous solutions of a pyranoanthocyanin (carboxypyranomalvidin-3-O-glucoside) as a function of pH.
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Table 1 pKa values of different pyranoanthocyanin compounds found in nature determined by UVeVisible and/or NMR spectroscopy. Compound
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Pigment
Pyranomalvidin-3-O-glucoside Pyranomalvidin-3-O-coumaroylglucoside Carboxypyranomalvidin-3-O-glucoside Methylpyranomalvidin-3-O-glucoside Pyranomalvidin-3-O-glucoside-(þ) -catechin Pyranomalvidin-3-O-coumaroylglucoside -(þ)-catechin Pyranomalvidin-3-O-glucoside-() -epicatechin Pyranomalvidin-3-O-coumaroylglucoside -()-epicatechin Pyranomalvidin-3-O-glucoside-catechol Pyranomalvidin-3-O-coumaroylglucoside -catechol Vinylpyranomalvidin-3-O-glucoside-(þ) -catechin Vinylpyranomalvidin-3-O-glucoside -catechol Vinylpyranomalvidin-3-O-glucoside -syringol Pyranomalvidin-3-O-glucoside -butadienylidene-sinapyl Pyranomalvidin-3-O-glucoside methine dímer
UVeVisible
NMR
pKa1
pKa2
<0.63 <0.75 1.18 0.07 4.57 0.07 5.05 0.05
4.34 4.76 4.42 8.23 7.90
5.35 0.08
8.06 0.09
4.80 0.09
pKa1
pKa2
pKa3
References
<0.68 e 1.09 0.09 5.17 0.03 e
4.40 0.08 e 4.93 0.02 8.85 0.08 e
7.45 0.09 e 9.14 0.05 e e
[5] [5] [6] [7]
9.76 0.07
e
e
e
[7]
7.82 0.09
9.49 0.10
e
e
e
[7]
5.24 0.11
8.02 0.10
9.60 0.08
e
e
e
[7]
4.20 0.06 4.31 0.07
7.84 0.05 8.34 0.06
10.28 0.07 10.20 0.06
e e
e e
e e
[7] [7]
4.61 0.03
8.36 0.03
10.11 0.06
e
e
e
4.09 0.03
7.98 0.05
10.18 0.05
e
e
e
3.60 0.02
8.66 0.05
12.16 0.05
e
e
e
3.64 0.01
8.02 0.01
11.19 0.01
e
e
e
4.93 0.04
8.33 0.04
9.10 0.04
e
e
e
the neutral quinoidal base and the anionic quinoidal base is described to occur [5e7,27,33]. Based on the results obtained by both methods, the ionization constants were determined for several pyranoanthocyanins (Table 1). Exceptions were observed for some pyranoanthocyanin pigments and are further discussed. 2.1. B-type vitisins (1) and (2) B-type vitisins (Fig. 5) are the most simple pyranoanthocyanins detected in red wines and they can be formed from the reaction of anthocyanins with acetaldehyde [3] (Fig. 1) present in wines in high amounts [34]. In model solutions these pigments can also be obtained from the reaction of anthocyanins with vinyloxytrimethylsilane [35]. As discussed above, in aqueous solutions and with the increase of the pH vitisin B undergoes deprotonation reactions. However, the positively charged vitisin B was shown to undergo a protonation at very low pH yielding the double-positively charged compound. This idea was supported by NMR and ESI-MS data that revealed the presence of an ion m/2z 259 in the positive ion mode that is compatible with the structure of a double-charged pyranoflavylium form [5]. According to the NMR and UVeVis spectroscopic data, the acide base equilibrium of B-type vitisin pigments in aqueous solution in the pH range between 0 and 8 can be described by the three protonetransfer reactions illustrated in Fig. 6. The three ionization constants (pKa1, pKa2, pKa3) were determined by NMR [36] and UVe Vis [37] (Table 1). The values of the three ionization constants obtained for pyranomalvidin-3-O-glucoside and pyranomalvidin-3-O-coumaroylglucoside (Table 1) are similar, except for the pKa3 value which is smaller in the coumaroyl derivative, indicating that the anionic quinoidal base form of this pigment is formed at lower pH values (6.76). This fact agrees with the results already described in the literature which showed that the pKa of acyl derivatives of anthocyanins are lower than that of the parent anthocyanin resulting in a stabilization of quinonoidal forms, which can be explained by the
pKa3
0.02 0.10 0.01 0.04 0.07
7.34 6.76 7.78 e 9.77
0.03 0.10 0.02 0.06
[19]
intramolecular complexation between the acyl group and the chromophore moiety of vitisin B [38]. 2.2. A-type vitisin (3) A-type vitisins or carboxypyranoanthocyanins (Fig. 5) are the most important anthocyanin-derived compounds detected by HPLC in red wines, being the main anthocyanin derivatives detected in Port wines after only one year of ageing [1]. Their presence in wines arises from the reaction between anthocyanins and pyruvic acid (Fig. 1) which is produced by yeasts during fermentation [2,3,39]. The equilibrium forms of carboxipyranomalvidin-3-O-glucoside in aqueous solutions were firstly studied by Asenstorfer & Jones (2007) using UVeVisible spectroscopy and high-voltage paper electrophoresis [31,40]. In this work, one ionization constant (pKa1 0.95 0.10) was determined by UVeVisible spectroscopy and two hydration constants (pKH1 ¼ 4.51 0.03 and pKH2 ¼ 7.57 0.02) and an additional ionisation constant at high pH (pKa4 ¼ 8.84 0.06) were established by high-voltage paper electrophoresis. However, a recent study confirmed by using NMR techniques (Fig. 3) the absence of hydration reactions in the carboxypyranomalvidin-3-O-glucoside (vitisin A) (3) since no characteristic peaks of the hemiketal form were observed in the NMR spectrum at any pD value studied (pD 0.67e10.99) [33]. With this technique it was also possible to observe that the different protons of vitisin A in the aromatic region are affected differently with the pH increase. By this way it was possible to determine the order by which each proton is removed from the carboxypyranoflavylium cation form. Based on this work, the more acidic proton was found to be the 10COOH group proton and hence the first to be removed (pKa1 ¼ 1.09 0.09) (Table 1), leading to the formation of the carboxypyranoflavylium anion form. With the increase of the pH values, protons 7-OH and 40 -OH are removed from the carboxypyranoflavylium anion form, which leads to the formation of the quinoidal anionic base and the quinoidal di-anionic base, respectively (Fig. 7) (pKa2 ¼ 4.93 0.02 and pKa3 ¼ 9.14 0.05) (Table 1).
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Fig. 5. Structure of the pyranoanthocyanin pigments studied (1e15).
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Fig. 6. Proton transfer equilibria of vitisin B pigments in aqueous solutions at different pH values between 0 and 8.
The ionization constants for vitisin A were also confirmed by UVe Visible spectroscopy (Table 1). 2.3. Methylpyranoanthocyanins (4) Methylpyranoanthocyanins are yellowish pyranoanthocyanins firstly identified by Lu et al. (2000) in black currant seeds [10]. Their
formation was proposed to arise from the reaction of anthocyanins with acetone present during the extraction process [9]. A few years later, He et al. (2006) detected and isolated two new methylpyranoanthocyanins from a 3-year-old Port wine [8]. Their presence in red wines was postulated to be obtained from the reaction of anthocyanins with acetoacetic acid [8] produced by yeasts during fermentation [41] (Fig. 1).
Fig. 7. Proton transfer equilibria of vitisin A in aqueous solutions at different pH values between 1 and 11.
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Fig. 8. Proton transfer equilibrium of methylpyranomalvidin-3-O-glucoside in aqueous solutions at high pH values (pH > 11.5).
The study of methylpyranomalvidin-3-O-glucoside (4) (Fig. 5) equilibrium forms in water at different pH values and its pKa values determination through NMR titration and UVeVisible spectroscopy were reported in the literature by Oliveira et al. (2011) [6]. The chemical behaviour of this compound in aqueous solutions shows a similar trend as the one discussed above. However, at pD 12.40 the 1 H spectra revealed the presence of two additional signals corresponding to two doublets situated at 6.01 and 5.78 ppm with a small coupling constant (J ¼ 2.0 Hz). These signals became more evident for higher pD values and might correspond to the two protons of the methylene group that would be formed upon deprotonation of the methyl group of the methylpyranomalvidin3-O-glucoside (Fig. 8) in strongly alkaline aqueous solution. It was not possible to fully characterize by NMR the structure of the methylene form since that species is relatively unstable and is readily transformed into another compound, the syringetin-3-Oglucoside as reported in the literature [6]. Based on the NMR results it was possible to determine a third ionization constant pKa3 > 11.5 (Table 1) that is due to the deprotonation at the methyl group of the methylpyranomalvidin-3-O-glucoside leading to the formation of a methylene group.
2.4. Pyranoanthocyanin-catechins (5e8) and pyranoanthocyanincatechols (9, 10) Pyranoanthocyanin-catechins and pyranoanthocyanincatechols are orange pigments present in red wines derived from the reaction of anthocyanins with vinyl-catechin or with vinylcatechol [14,15,42]. These compounds are thought to contribute to the orange hues observed in red wines during ageing and maturation. The equilibrium forms of six different compounds (Fig. 3) were studied in aqueous solutions by UVeVisible spectroscopy. No hydration reactions were observed, only electron transfer reactions should take place in these kind of compounds as reported by Cruz et al. (2010) [7] and similarly to what was described for other pyranoanthocyanins [5,6,33]. The hydration process was studied using reverse pH jump experiments (stopped flow). Additionally to the first two deprotonation reactions described above, a third deprotonation was described. A small batochromic shift was observed in maximum absorption wavelength for higher pH values (pH > 10.2) with an increase in the absorbance and these features were postulated to be due to the equilibrium between the anionic quinoidal form and the dianionic quinoidal form that is formed from the deprotonation of one hydroxyl group of the catechol unit in the case of pyranoanthocyanin-catechol pigments or in the phloroglucinol unit in the case of pyranoanthocyanin-catechin compounds. Cruz et al. (2010) also noticed that the pigments presenting a coumaroyl group (esterified in the glucose moiety) in
their structure (6, 8 and 10) showed an absorption band at w360 nm, that is described to be due to the deprotonation of the coumaroyl group of these compounds at high pH values (pKa2 pcoumaric acid ¼ 8.98 [43]) [7]. Based on the titration data and the equilibria postulated for these compounds, the three ionization constants were determined (Table 1) (pKa1, pKa2 and pKa3) and are in agreement with the results described for 7,40 -dihydroxyflavyliums (pKa1 ¼ 4; pKa2 ¼ 8) [44e46] and for other pyranoanthocyanins [5,6]. The pKa3 values obtained for (þ)-catechin and ()-epicatechin-derived pigments are also in agreement with the results described in the literature [47]. The pKa3 values of the catechol-derived compounds were also found to be similar to the ones reported for catechol in aqueous solutions [48].
2.5. A-type portisins (11) and B-type portisins (12, 13) Vinylpyranoanthocyanin-phenolics, commonly known as A and B-type portisins are bluish pyranoanthocyanin pigments detected in two-year-old Port wines [17,32,49]. These compounds were the first bluish compounds at acidic pH detected in wines and their formation can derive from the reaction of carboxypyranoanthocyanins with (þ)-catechin in the presence of acetaldehyde or with hydroxicinnamic acids [17,32,49] (Fig. 1). The equilibrium forms of A and B-type portisins (Fig. 5) were studied in aqueous solutions at different pH values using UVe Visible spectroscopy (Fig. 9). In this case, the study through NMR spectroscopy could not be accomplished since portisins are insoluble in water at the concentrations needed to the NMR titration. The behaviour observed for vinylpyranomalvidin-3-O-glucosidecatechin 11, vinylpyranomalvidin-3-O-glucoside-catechol 12 and vinylpyranomalvidin-3-O-glucoside-syringol 13 in aqueous solutions towards the increase of pH values up to 12.00 was similar for the three compounds. Therefore, the vinylpyranomalvidin-3-Oglucoside-catechin (Portisin A) will be discussed as an example of the equilibrium forms of this kind of pigments in aqueous solution at different pH values. With the increase of pH values from 1.99 to 5.26 it is possible to observe a decrease in the absorbance of the solutions that is accompanied with a shift in the wavelength of maximum absorption for lower values (hypsochromic shift). This tendency was already reported in the literature for other pyranoanthocyanins [7,19] for the equilibrium between the pyranoflavylium form and the neutral quinoidal form. Similarly to what was observed for other pyranoanthocyanins, portisin A did not show evidences of hydration reactions. For higher pH values up to 9.87 the absorbance around 500 nm starts to decrease together with the appearance of a shoulder at w630 nm that becomes more evident with the increase of the pH value. This feature is attributed to the equilibrium between the
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Fig. 9. Absorption spectra (360e830 nm) of aqueous solutions of portisins A as a function of pH (1.99e11.82).
neutral quinoidal base and the respective anionic base. For pH > 9.87 an intensification of the color of the solutions is observed with an important displacement of the maximum absorption wavelength (lmaxw660 nm) that is due to the deprotonation in the hydroxyl group present in carbon C-7 of the (þ)-catechin unit. Based on the data obtained by UVeVisible spectroscopy it was possible to determine the three ionization constants for the three pigments studied shown in Table 1. According to these constants, it seems that the catechin moiety stabilizes more efficiently the pyranoflavylium cation form when compared to the catechol or the syringol units, difficulting the deprotonation reaction. This probably occur because catechin may promote higher electronic delocalization. This trend is observed for the three pKa values when the catechin-derived and the catechol-derived compounds are
compared. However, it looks like that the presence of an electrondonating group such as the methyl group in the syringol moiety will stabilize more readily the neutral quinoidal form of these compounds preventing the second and the third deprotonation reactions. A similar result was observed when comparing the methylpyranomalvidin-3-O-glucoside with the pyranomalvidin-3O-glucoside compound [5,6]. 2.6. Pyranomalvidin-3-O-glucoside-butadienylidene-sinapyl (14) Pyranomalvidin-3-O-glucoside-butadienylidene-sinapyl (14) pigment (Fig. 5) can be synthesised from the reaction of methylpyranomalvidin-3-O-glucoside with synapaldehyde. The structure of this pigment includes a pyranoanthocyanin moiety linked to a
Fig. 10. Absorption spectra (360e830 nm) of aqueous solutions of pyranoanthocyanin dimers as a function of pH (1.80e11.66).
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syringol group through a butadienylidene linkage [19]. This structure is somehow similar to the B-type portisins but in this case there is an additional vinyl group conferring to this family of pyranoanthocyanins a higher electronic delocalization and by this way a more bluish color. The equilibrium forms of the pyranomalvidin-3-O-glucosidebutadienylidene-sinapyl (14) were studied in aqueous solutions at different pH values using UVeVisible spectroscopy [19] and the results obtained are similar to the ones discussed for A and B-type portisins. In aqueous solutions this compound 14 revealed the presence of four equilibrium forms involving three deprotonation reactions at 7-OH, 40 -OH and 400 -OH (Table 1). 2.7. Pyranomalvidin-3-O-glucoside methine dimer (15) Pyranoanthocyanin dimers are a family of turquoise blue color anthocyanin-derived pigments and were first found in a 9-year-old Port wine and in the respective lees [18]. The structure of these compounds includes two pyranomalvidin-3-glucoside moieties linked through a methine linkage (Fig. 5). Studies performed in model solution have shown that these compounds can be formed in red wines from the reaction of a carboxypyranoanthocyanin with the methylpyranoanthocyanin, as described elsewhere [18]. In aqueous solution, according to the structure of this compound 15 (Fig. 5), it was expected for this compound to present four pKa values since there are two 7-OH protons and two 40 -OH protons in its structure. However, based on the results obtained by UVe Visible spectroscopy (Fig. 10) only three deprotonation reactions seem to occur at the pH values studied (1.80e11.66). With the increase of pH for values between 3.15 and 6.55 it is possible to
observe a reduction in the absorbance of the solutions together with a displacement in the wavelength of maximum absorption for lower values and the appearance of a small shoulder at w485 nm (Fig. 10). These features can be attributed to the equilibrium concerning the pyranoflavylium cation dimer and the neutral quinoidal base deprotonated at the first 7-OH (Fig. 11). No hydration reaction seems to occur for these kind of pigments in the conditions studied (pH 1.80e11.66) similarly to what is observed for other pyranoanthocyanins [5e7,33]. For higher pH values (6.55e8.66) an increase in the absorption and the maximum absorption wavelength (from 585 to 610 nm) is observed for all the solutions. A small shoulder at w635 nm starts to appear at pH 8.19. These features can be due to the equilibrium between the neutral quinoidal form and the anionic quinoidal form deprotonated in the 40 OH. With the increase of pH for values >8.66 (till 11.66) a similar trend is observed (Fig. 10) and the equilibrium between the anionic quinoidal form and the di-anionic quinoidal for is expected to occur (Fig. 11). At pH 11.66 when compared to pH 9.59 it is possible to observe a small shift in the wavelength of maximum absorption and a fourth deprotonation could explain this small shift. However, according to the data obtained and with the model adopted it was not possible to determine this ionization constant (it must occur at very high pH values). The ionization constants were determined based on the titration data obtained by UVeVisible spectroscopy and with a specific software pHab 2006 [37]. The pKa values (pKa1 ¼ 4.93 0.04; pKa2 ¼ 8.33 0.04; pKa3 ¼ 9.10 0.04) (Table 1) are in agreement with the ones reported in the literature for other pyranoanthocyanins [5e7]. However, the pKa values for this pyranoanthocyanin dimer are a little higher than the ones observed for
Fig. 11. Proton transfer equilibria of pyranoanthocyanin dimers in aqueous solutions at different pH values between 1 and 12.
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Fig. 12. Color of different pyranoanthocyanin pigments found in nature in water at acidic pH (pHw1.5). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
simpler pyranoanthocyanin pigments such as vitisin B or methylpyranomalvidin-3-O-glucoside (Table 1). This indicates that the presence of two pyranoanthocyanin in the same structure promotes a higher electronic delocalization which stabilizes the pyranoflavylium cation form, preventing the deprotonation reactions that leads to the formation of the quinoidal base forms. 3. Conclusions According to the results discussed above it seems that there is a relation between the structural features of the pyranoanthocyanin compound and the ionization constants observed. In general, the electronic delocalization appears to have an influence in the equilibrium forms of pyranoanthocyanins, especially in the equilibrium between the pyranoflavylium cation form and the respective neutral quinoidal base (pKa1). For instance, vinylpyranoanthocyanincatechin compounds (A-type portisins) that present a higher electronic delocalization when compared to the pyranoanthocyanincatechin compounds showed lower pKa1 values (Table 1). On the other hand, although the presence of coumaroyl groups esterified in the glucose moiety does not contribute to electronic delocalization, it looks like that the presence of this group in the pyranoanthocyanin structure generally increases the stability of the compounds towards deprotonation reactions. This tendency is more evident in the first ionization constant, diminishing its effect for the second and the third deprotonations (Table 1). Catechinderived compounds are also more stable to deprotonation when compared to similar phenolic-derived compounds (comparison between A and B-type portisins). Furthermore, the presence of additional electron-donating groups as observed for the syringol group comparing to the catechol one seems to stabilize the anionic quinoidal base form of the pyranoanthocyanin compounds, reducing its ability to lose the third proton. This was observed for B-type portisins derived from syringol and catechol (pKa3 ¼ 10.18 0.05 and pKa3 ¼ 12.16 0.05, respectively) (Table 1). According to the ionization constants obtained for vitisins A and B and methylpyranomalvidin-3-O-glucoside, the substitution pattern in carbon C-10 does not seem to affect importantly the equilibrium between the pyranoflavylium cation form and the respective neutral quinoidal form as the pKa values are similar for the three pigments (Table 1). However, for the second ionization constant representing the equilibrium between the quinoidal base and the quinoidal anionic forms, the methylpyranomalvidin-3-O-glucoside revealed a
higher value (pKa2 ¼ 8.23 0.04) comparing to the one obtained for pyranomalvidin-3-glucoside (pKa2 ¼ 7.34 0.03). It seems that the methyl group present in the methylpyranoanthocyanin stabilizes the quinoidal base form reducing their ability to lose the second proton. Pyranoanthocyanin pigments found in nature can be present in different colours such as, orange, bluish or turquoise blue colour [15,17,18] (Fig. 12). The physicalechemical properties of these compounds studied over the years revealed their higher stability towards the hydration reactions when compared to their anthocyanin precursors contributing to the colour stability of red wines during the ageing process. These features also bring promising expectation concerning their use in the Food Industry as colorants. Acknowledgements This research was supported by a Post-doctoral grant from FCT (Fundação para a Ciência e a Tecnologia e Praxis BPD/65400/2009) and a grant from FCT (Fundação para a Ciência e a Tecnologia e PTDC/QUI-QUI/117996/2010) all from Portugal and by FEDER funding. References [1] Mateus N, de Freitas V. Evolution and stability of anthocyanin-derived pigments during port wine aging. J Agric Food Chem 2001;49(11):5217e22. [2] Oliveira J, Fernandes V, Miranda C, Santos-Buelga C, Silva A, de Freitas V, et al. Color properties of four cyanidin-pyruvic acid adducts. J Agric Food Chem 2006;54(18):6894e903. [3] Bakker J, Timberlake CF. Isolation, identification, and characterization of new color-stable anthocyanins occurring in some red wines. J Agric Food Chem 1997;45(1):35e43. [4] Fulcrand H, Benabdeljalil C, Rigaud J, Cheynier V, Moutounet M. A new class of wine pigments generated by reaction between pyruvic acid and grape anthocyanins. Phytochemistry 1998;47(7):1401e7. [5] Oliveira J, Mateus N, Silva AMS, de Freitas V. Equilibrium forms of vitisin B pigments in an aqueous system studied by NMR and visible spectroscopy. J Phys Chem B 2009;113(32):11352e8. [6] Oliveira J, Petrov V, Parola AJ, Pina F, Azevedo J, Teixeira N, et al. Chemical behavior of methylpyranomalvidin-3-O-glucoside in aqueous solution studied by NMR and UVeVisible spectroscopy. J Phys Chem B 2011;115(6):1538e45. [7] Cruz L, Petrov V, Teixeira N, Mateus N, Pina F, de Freitas V. Establishment of the chemical equilibria of different types of pyranoanthocyanins in aqueous solutions: evidence for the formation of aggregation in pyranomalvidin-3-Ocoumaroylglucoside-(þ)-catechin. J Phys Chem B 2010;114(41):13232e40. [8] He J, Santos-Buelga C, Silva AMS, Mateus N, De Freitas V. Isolation and structural characterization of new anthocyanin-derived yellow pigments in aged red wines. J Agric Food Chem 2006;54(25):9598e603. [9] Lu Y, Foo LY. Unusual anthocyanin reaction with acetone leading to pyranoanthocyanin formation. Tetrahedron Lett 2001;42(7):1371e3. [10] Lu Y, Sun Y, Foo LY. Novel pyranoanthocyanins from black currant seed. Tetrahedron Lett 2000;41(31):5975e8.
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[11] He J, Oliveira J, Silva AMS, Mateus N, De Freitas V. Oxovitisins: a new class of neutral pyranone-anthocyanin derivatives in red wines. J Agric Food Chem 2010;58(15):8814e9. [12] Gómez-Alonso S, Blanco-Vega D, Gómez MV, Hermosín-Gutiérrez I. Synthesis, isolation, structure elucidation, and color properties of 10-Acetyl-pyranoanthocyanins. J Agric Food Chem 2012;60(49):12210e23. [13] Hayasaka Y, Asenstorfer RE. Screening for potential pigments derived from anthocyanins in red wine using nanoelectrospray tandem mass spectrometry. J Agric Food Chem 2002;50(4):756e61. [14] Schwarz M, Jerz G, Winterhalter P. Isolation and structure of pinotin A, a new anthocyanin derivative from Pinotage wine. Vitis 2003;42(2):105e6. [15] Cruz L, Teixeira N, Silva AMS, Mateus N, Borges J, de Freitas V. Role of vinylcatechin in the formation of pyranomalvidin-3-glucoside-(þ)-Catechin. J Agric Food Chem 2008;56(22):10980e7. [16] Mateus N, Oliveira J, Santos-Buelga C, Silva AMS, de Freitas V. NMR structure characterization of a new vinylpyranoanthocyanin-catechin pigment (a portisin). Tetrahedron Lett 2004;45(17):3455e7. [17] Oliveira J, de Freitas V, Silva AMS, Mateus N. Reaction between hydroxycinnamic acids and anthocyanin-pyruvic acid adducts yielding new portisins. J Agric Food Chem 2007;55(15):6349e56. [18] Oliveira J, Azevedo J, Silva AMS, Teixeira N, Cruz L, Mateus N, et al. Pyranoanthocyanin dimers: a new family of turquoise blue anthocyanin-derived pigments found in port wine. J Agric Food Chem 2010;58(8):5154e9. [19] Oliveira J, Mateus N, de Freitas V. Synthesis of a new bluish pigment from the reaction of a methylpyranoanthocyanin with sinapaldehyde. Tetrahedron Lett 2011;52(16):1996e2000. [20] Dougall DK, Baker DC. Effects of reaction mixture and other components on the determination of the equilibrium and rate constants of the hydration reactions of anthocyanins. Food Chem 2008;107(1):473e82. [21] Freitas AA, Dias LG, Macanita AAL, Quina FH. Substituent effects on the pHdependent multiequilibria of flavylium salt analogs of anthocyanins. J Phys Org Chem 2011;24(12):1201e8. [22] Redus M, Baker DC, Dougall DK. Rate and equilibrium constants for the dehydration and deprotonation reactions of some monoacylated and glycosylated cyanidin derivatives. J Agric Food Chem 1999;47(8):3449e54. [23] Mora-Soumille N, Al Bittar S, Rosa M, Dangles O. Analogs of anthocyanins with a 3 ’,4 ’-dihydroxy substitution: synthesis and investigation of their acid-base, hydration, metal binding and hydrogen-donating properties in aqueous solution. Dyes Pigm 2013;96(1):7e15. [24] Pina F, Benedito L, Melo JM, Parola AJ, Lima JC, Macanita AL. Structural transformations of the synthetic salt 4’,7-dihydroxyflavylium chloride in acid and basic aqueous solutions .1. Ground state. Anales De Quimica 1997;93(2):111e8. [25] Pina F, Benedito L, Melo MJ, Parola AJ, Bernardo A. Photochemistry of 3,4’dimethoxy-7-hydroxyflavylium chloride e photochromism and excited-state proton transfer. J Chem Society-Faraday Trans 1996;92(10):1693e9. [26] Santos H, Turner DL, Lima JC, Figueiredo P, Pina FS, Macanita AL. Elucidation of the multiple equilibria of malvin in aqueous-solution by one-dimensional and 2-dimensional NMR. Phytochemistry 1993;33(5):1227e32. [27] Asenstorfer RE, Iland PG, Tate ME, Jones GP. Charge equilibria and pK(a) of malvidin-3-glucoside by electrophoresis. Anal Biochem 2003;318(2):291e9. [28] Jordheim M, Fossen T, Andersen ØM. Characterization of hemiacetal forms of anthocyanidin 3-O-b-Glycopyranosides. J Agric Food Chem 2006;54(25):9340e6. [29] Brouillard R, Lang J. The hemiacetal-cis-chalcone equilibrium of malvin, a natural anthocyanin. Can J Chemistry-Revue Canadienne De Chim 1990;68(5): 755e61.
[30] Brouillard R. Chemical structure of anthocyanins. In: Markakis P, editor. Anthocyanins as food colors. New York: Academic Press Inc.; 1982. p. 1e38. [31] Asenstorfer RE, Jones GP. Charge equilibria and pK values of 5carboxypyranomalvidin-3-glucoside (vitisin A) by electrophoresis and absorption spectroscopy. Tetrahedron 2007;63(22):4788e92. [32] Oliveira J, Santos-Buelga C, Silva AMS, de Freitas V, Mateus N. Chromatic and structural features of blue anthocyanin-derived pigments present in port wine. Anal Chim Acta 2006;563(1e2):2e9. [33] Oliveira J, Mateus N, de Freitas V. Network of carboxypyranomalvidin-3-Oglucoside (Vitisin A) equilibrium forms in aqueous solution. Tetrahedron Lett 2013 [accepted]. [34] Liu S-Q, Pilone GJ. An overview of formation and roles of acetaldehyde in wine making with emphasis on microbiological implications. Int J Food Sci Technol 2000;35(1):49e61. [35] Oliveira J, de Freitas V, Mateus N. A novel synthetic pathway to vitisin B compounds. Tetrahedron Lett 2009;50(27):3933e5. [36] Frassineti C, Ghelli S, Gans P, Sabatini A, Moruzzi MS, Vacca A. Nuclear Magnetic Resonance as a tool for determining protonation constants of natural polyprotic bases in solution. Anal Biochem 1995; 231(2):374e82. [37] Gans P, Sabatini A, Vacca A. Determination of equilibrium constants from spectrophotometric data obtained from solutions of known pH: the program pHab. Ann Chim 1999;89:45e9. [38] Dangles O, Saito N, Brouillard R. Kinetic and thermodynamic control of flavylium hydration in the pelargonidin-cinnamic acid complexation. Origin of the extraordinary flower color diversity of pharbitis nil. J Am Chem Soc 1993;115(8):3125e32. [39] Bakker J, Bridle P, Honda T, Kuwano H, Saito N, Terahara N, et al. Identification of an anthocyanin occurring in some red wines. Phytochemistry 1997;44(7): 1375e82. [40] Asenstorfer RE, Lee DF, Jones GP. Influence of structure on the ionisation constants of anthocyanin and anthocyanin-like wine pigments. In: Proc 4th symposium on in vino analytica scientia. Montpellier, FRANCE: Elsevier Science Bv; 2005. p. 10e4. [41] Berdanier CD. CRC desk reference for nutrition. Boca Raton, FL: CRC; 1998. [42] Håkansson AE, Pardon K, Hayasaka Y, de Sa M, Herderich M. Structures and colour properties of new red wine pigments. Tetrahedron Lett 2003;44(26): 4887e91. [43] Beltrán JL, Sanli N, Fonrodona G, Barrón D, Özkan G, Barbosa J. Spectrophotometric, potentiometric and chromatographic pKa values of polyphenolic acids in water and acetonitrileewater media. Anal Chim Acta 2003;484(2): 253e64. [44] McClelland RA, McGall GH. Hydration of the flavylium ion. 2. The 40 hydroxyflavylium ion. J Org Chem 1982;47(19):3730e6. [45] Pina F, Melo MJ, Ballardini R, Flamigni L, Maestri M. Flash photolysis of 40 ,7dihydroxyflavylium perchlorate. New J Chem 1997;21:969e76. [46] Pina F, Melo MJ, Parola AJ, Maestri M, Balzani V. pH-controlled photochromism of hydroxyflavylium ions. Chem e A Eur J 1998;4(10):2001e7. [47] Slabbert NP. Ionisation of some flavanols and dihydroflavonols. Tetrahedron 1977;33(7):821e4. [48] Timberlake CF. 997. A potentiometric and polarographic study of coppercatechol complexes. J Chem Soc (Resumed) 1957;0(0):4987e93. [49] Mateus N, Silva AMS, Rivas-Gonzalo JC, Santos-Buelga C, De Freitas V. A new class of blue anthocyanin-derived pigments isolated from red wines. J Agric Food Chem 2003;51(7):1919e23.
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Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
Review
Previous and recent advances in pyranoanthocyanins equilibria in aqueous solution Joana Oliveira, Nuno Mateus, Victor de Freitas* Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2013 Accepted 10 September 2013 Available online 19 September 2013
Pyranoanthocyanins are anthocyanin-derived compounds found in nature that present a vast palette of colours ranging from yellow to turquoise blue. Studies in aqueous solutions of these pigments by UV eVisible spectroscopy revealed their higher colour stability when compared to their anthocyanin precursors. Through Nuclear Magnetic Resonance (NMR) techniques it was possible to confirm the absence of hydration reactions in pyranoanthocyanins, contrarily to anthocyanins, which helps explaining their higher colour stability with the increase of the pH. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Pyranoanthocyanins Equilibrium forms Aqueous solution Deprotonation NMR UVeVisible spectroscopy
1. Introduction Pyranoanthocyanins are major polyphenolic pigments formed in red wines during their ageing and maturation [1] and are thought to contribute to the orange hues observed in those wines [2]. Their chemical formation pathway involves a cyclic addition onto carbon 4 and the hydroxyl group at the carbon 5 position of the anthocyanin, yielding a fourth ring that is responsible for the higher stability to hydration of these compounds when compared to the original anthocyanins [2e7]. Over the years, several families of pyranoanthocyanins have been described in the literature including A and B-type vitisins [3], methylpyranoanthocyanins [8e10], oxovitisins [11], acetylpyranoanthocyanins [12], pyranoanthocyaninphenolics [4,13,14], pyranoanthocyanin-flavanols [15], A and Btype portisins [16,17], pyranoanthocyanin dimers [18] and pyranoanthocyanin-butadienilydene-phenolics [19] (Fig. 1). In aqueous solution, anthocyanins are present in different forms in equilibrium. The anthocyanins and flavylium-like compounds equilibrium forms in aqueous solution have been vastly described in the literature [20e29]. In very acidic aqueous solution (pH < 1), these pigments are present as red flavylium cations. The increase of pH leads to a reduction of the intensity of the color due to a decrease of the concentration of the flavylium cation that is
* Corresponding author. Tel.: þ351 220402558; fax: þ351 220402658. E-mail address: [email protected] (V. de Freitas). 0143-7208/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dyepig.2013.09.009
converted into its colorless hemiketal form through nucleophilic attack of water. The hemiketal further undergoes a tautomerization reaction to give the pale yellow cis- chalcone (Cc), which isomerizes to trans-chalcone (Ct). At low acidic, neutral and basic pH values, deprotonation of the flavylium cation also occurs, giving rise to the violet/blue quinoidal forms [30] (Fig. 2). Oppositely to anthocyanins, studies of pyranoanthocyanins equilibria in aqueous solutions are scarce in the literature [5e7,31]. Only a few papers have been published related to the physicale chemical properties of pyranoanthocyanins in aqueous solutions. The present review aims to discuss the main features of pyranoanthocyanins in aqueous solutions but also to present new data concerning the physicalechemical features of some recently described pyranoanthocyanins detected in red wines [17,18,32]. Moreover, the ionization constants will be correlated with some structural features that are important to stabilize pyranoanthocyanin in the pyranoflavylium cation form. The diverse colours presented by pyranoanthocyanins pigments and their higher colour stability, namely their colour stability at a wide pH range are important features indicating a putative application of these compounds in food products. 2. Pyranoanthocyanins in aqueous solution Over the years, the equilibrium forms of pyranoanthocyanin pigments have been studied using NMR and UVeVisible spectroscopy [5e7,31,33]. In aqueous solutions pyranoanthocyanins have
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191
Fig. 1. Anthocyanin-derived pigments found in nature.
shown to co-exist under different equilibrium forms that are pHdependent. NMR spectroscopy gives important information about the type of equilibrium that is occurring in aqueous solution at different pH values. In general, the peaks corresponding to proton exchange equilibrium forms are observed in the aromatic region spectrum of the 1H NMR. No other important 1H NMR peaks at any
pH studied corresponding to the hemiketal form described for anthocyanin compounds were observed [28,36,37] (Fig. 3). Indeed, if the hemiketal form was present, a significant displacement of about 2 ppm in the chemical shift of protons H-9 and H-10 (for vitisin B) to lower ppm values would have been observed. Santos et al. (1993) showed that the chemical shift of proton H-4 in malvin
Fig. 2. Anthocyanin equilibria in aqueous solutions at different pH values.
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J. Oliveira et al. / Dyes and Pigments 100 (2014) 190e200
Fig. 3. 1H spectra of a pyranoanthocyanin pigments (carboxypyranomalvidin-3-O-glucoside) in D2O at different pD values from 0.67 until 10.99, T ¼ 298 K, total concentration 3.2 mM.
(malvidin-3,5-glucoside) in the hemiketal form is quite different (6.60 ppm) from the one observed for the same proton in the flavylium cation form (8.97 ppm) [36]. The same trend was observed by Jordheim et al. (2006) for the hemiketal and flavylium cation forms in malvidin-3-O-glucoside (6.58 and 9.13 ppm, respectively) [28]. Bearing all this, pyranoanthocyanins do not undergo hydration and only proton transfer reactions occur when the pH change. In fact, a number of studies based on UVeVisible spectroscopy have already established that pyranoanthocyanin pigments are protected from the attack by water when compared with their anthocyanin precursors [2,31,38]. In the pyranoanthocyanins titration through NMR generally, the increase of the pH of the solution until pHw7 leads to a
displacement of the chemical shift of all protons to lower ppm values due to the equilibrium between the pyranoflavylium cation form and the neutral quinoidal form [5,33]. For higher pH values up to pHw12 a similar trend is observed (Fig. 3), corresponding to the faster equilibrium between the neutral quinoidal base form and the anionic quinoidal base. In the UVeVisible spectrum of pyranoanthocyanin commonly, for pH values between 1 and 5 it is possible to observe a batochromic shift in the maximum absorption wavelength that is accompanied by a decrease in the absorbance of the solutions (Fig. 4). The wavelength displacement is attributed to the deprotonation of the pyranoflavylium cation form that leads to the formation of the respective neutral quinoidal base. For higher pH values a similar trend is observed and the equilibrium between
Fig. 4. Absorption spectra (360e830 nm) of aqueous solutions of a pyranoanthocyanin (carboxypyranomalvidin-3-O-glucoside) as a function of pH.
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193
Table 1 pKa values of different pyranoanthocyanin compounds found in nature determined by UVeVisible and/or NMR spectroscopy. Compound
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Pigment
Pyranomalvidin-3-O-glucoside Pyranomalvidin-3-O-coumaroylglucoside Carboxypyranomalvidin-3-O-glucoside Methylpyranomalvidin-3-O-glucoside Pyranomalvidin-3-O-glucoside-(þ) -catechin Pyranomalvidin-3-O-coumaroylglucoside -(þ)-catechin Pyranomalvidin-3-O-glucoside-() -epicatechin Pyranomalvidin-3-O-coumaroylglucoside -()-epicatechin Pyranomalvidin-3-O-glucoside-catechol Pyranomalvidin-3-O-coumaroylglucoside -catechol Vinylpyranomalvidin-3-O-glucoside-(þ) -catechin Vinylpyranomalvidin-3-O-glucoside -catechol Vinylpyranomalvidin-3-O-glucoside -syringol Pyranomalvidin-3-O-glucoside -butadienylidene-sinapyl Pyranomalvidin-3-O-glucoside methine dímer
UVeVisible
NMR
pKa1
pKa2
<0.63 <0.75 1.18 0.07 4.57 0.07 5.05 0.05
4.34 4.76 4.42 8.23 7.90
5.35 0.08
8.06 0.09
4.80 0.09
pKa1
pKa2
pKa3
References
<0.68 e 1.09 0.09 5.17 0.03 e
4.40 0.08 e 4.93 0.02 8.85 0.08 e
7.45 0.09 e 9.14 0.05 e e
[5] [5] [6] [7]
9.76 0.07
e
e
e
[7]
7.82 0.09
9.49 0.10
e
e
e
[7]
5.24 0.11
8.02 0.10
9.60 0.08
e
e
e
[7]
4.20 0.06 4.31 0.07
7.84 0.05 8.34 0.06
10.28 0.07 10.20 0.06
e e
e e
e e
[7] [7]
4.61 0.03
8.36 0.03
10.11 0.06
e
e
e
4.09 0.03
7.98 0.05
10.18 0.05
e
e
e
3.60 0.02
8.66 0.05
12.16 0.05
e
e
e
3.64 0.01
8.02 0.01
11.19 0.01
e
e
e
4.93 0.04
8.33 0.04
9.10 0.04
e
e
e
the neutral quinoidal base and the anionic quinoidal base is described to occur [5e7,27,33]. Based on the results obtained by both methods, the ionization constants were determined for several pyranoanthocyanins (Table 1). Exceptions were observed for some pyranoanthocyanin pigments and are further discussed. 2.1. B-type vitisins (1) and (2) B-type vitisins (Fig. 5) are the most simple pyranoanthocyanins detected in red wines and they can be formed from the reaction of anthocyanins with acetaldehyde [3] (Fig. 1) present in wines in high amounts [34]. In model solutions these pigments can also be obtained from the reaction of anthocyanins with vinyloxytrimethylsilane [35]. As discussed above, in aqueous solutions and with the increase of the pH vitisin B undergoes deprotonation reactions. However, the positively charged vitisin B was shown to undergo a protonation at very low pH yielding the double-positively charged compound. This idea was supported by NMR and ESI-MS data that revealed the presence of an ion m/2z 259 in the positive ion mode that is compatible with the structure of a double-charged pyranoflavylium form [5]. According to the NMR and UVeVis spectroscopic data, the acide base equilibrium of B-type vitisin pigments in aqueous solution in the pH range between 0 and 8 can be described by the three protonetransfer reactions illustrated in Fig. 6. The three ionization constants (pKa1, pKa2, pKa3) were determined by NMR [36] and UVe Vis [37] (Table 1). The values of the three ionization constants obtained for pyranomalvidin-3-O-glucoside and pyranomalvidin-3-O-coumaroylglucoside (Table 1) are similar, except for the pKa3 value which is smaller in the coumaroyl derivative, indicating that the anionic quinoidal base form of this pigment is formed at lower pH values (6.76). This fact agrees with the results already described in the literature which showed that the pKa of acyl derivatives of anthocyanins are lower than that of the parent anthocyanin resulting in a stabilization of quinonoidal forms, which can be explained by the
pKa3
0.02 0.10 0.01 0.04 0.07
7.34 6.76 7.78 e 9.77
0.03 0.10 0.02 0.06
[19]
intramolecular complexation between the acyl group and the chromophore moiety of vitisin B [38]. 2.2. A-type vitisin (3) A-type vitisins or carboxypyranoanthocyanins (Fig. 5) are the most important anthocyanin-derived compounds detected by HPLC in red wines, being the main anthocyanin derivatives detected in Port wines after only one year of ageing [1]. Their presence in wines arises from the reaction between anthocyanins and pyruvic acid (Fig. 1) which is produced by yeasts during fermentation [2,3,39]. The equilibrium forms of carboxipyranomalvidin-3-O-glucoside in aqueous solutions were firstly studied by Asenstorfer & Jones (2007) using UVeVisible spectroscopy and high-voltage paper electrophoresis [31,40]. In this work, one ionization constant (pKa1 0.95 0.10) was determined by UVeVisible spectroscopy and two hydration constants (pKH1 ¼ 4.51 0.03 and pKH2 ¼ 7.57 0.02) and an additional ionisation constant at high pH (pKa4 ¼ 8.84 0.06) were established by high-voltage paper electrophoresis. However, a recent study confirmed by using NMR techniques (Fig. 3) the absence of hydration reactions in the carboxypyranomalvidin-3-O-glucoside (vitisin A) (3) since no characteristic peaks of the hemiketal form were observed in the NMR spectrum at any pD value studied (pD 0.67e10.99) [33]. With this technique it was also possible to observe that the different protons of vitisin A in the aromatic region are affected differently with the pH increase. By this way it was possible to determine the order by which each proton is removed from the carboxypyranoflavylium cation form. Based on this work, the more acidic proton was found to be the 10COOH group proton and hence the first to be removed (pKa1 ¼ 1.09 0.09) (Table 1), leading to the formation of the carboxypyranoflavylium anion form. With the increase of the pH values, protons 7-OH and 40 -OH are removed from the carboxypyranoflavylium anion form, which leads to the formation of the quinoidal anionic base and the quinoidal di-anionic base, respectively (Fig. 7) (pKa2 ¼ 4.93 0.02 and pKa3 ¼ 9.14 0.05) (Table 1).
194 J. Oliveira et al. / Dyes and Pigments 100 (2014) 190e200
Fig. 5. Structure of the pyranoanthocyanin pigments studied (1e15).
J. Oliveira et al. / Dyes and Pigments 100 (2014) 190e200
195
Fig. 6. Proton transfer equilibria of vitisin B pigments in aqueous solutions at different pH values between 0 and 8.
The ionization constants for vitisin A were also confirmed by UVe Visible spectroscopy (Table 1). 2.3. Methylpyranoanthocyanins (4) Methylpyranoanthocyanins are yellowish pyranoanthocyanins firstly identified by Lu et al. (2000) in black currant seeds [10]. Their
formation was proposed to arise from the reaction of anthocyanins with acetone present during the extraction process [9]. A few years later, He et al. (2006) detected and isolated two new methylpyranoanthocyanins from a 3-year-old Port wine [8]. Their presence in red wines was postulated to be obtained from the reaction of anthocyanins with acetoacetic acid [8] produced by yeasts during fermentation [41] (Fig. 1).
Fig. 7. Proton transfer equilibria of vitisin A in aqueous solutions at different pH values between 1 and 11.
196
J. Oliveira et al. / Dyes and Pigments 100 (2014) 190e200
Fig. 8. Proton transfer equilibrium of methylpyranomalvidin-3-O-glucoside in aqueous solutions at high pH values (pH > 11.5).
The study of methylpyranomalvidin-3-O-glucoside (4) (Fig. 5) equilibrium forms in water at different pH values and its pKa values determination through NMR titration and UVeVisible spectroscopy were reported in the literature by Oliveira et al. (2011) [6]. The chemical behaviour of this compound in aqueous solutions shows a similar trend as the one discussed above. However, at pD 12.40 the 1 H spectra revealed the presence of two additional signals corresponding to two doublets situated at 6.01 and 5.78 ppm with a small coupling constant (J ¼ 2.0 Hz). These signals became more evident for higher pD values and might correspond to the two protons of the methylene group that would be formed upon deprotonation of the methyl group of the methylpyranomalvidin3-O-glucoside (Fig. 8) in strongly alkaline aqueous solution. It was not possible to fully characterize by NMR the structure of the methylene form since that species is relatively unstable and is readily transformed into another compound, the syringetin-3-Oglucoside as reported in the literature [6]. Based on the NMR results it was possible to determine a third ionization constant pKa3 > 11.5 (Table 1) that is due to the deprotonation at the methyl group of the methylpyranomalvidin-3-O-glucoside leading to the formation of a methylene group.
2.4. Pyranoanthocyanin-catechins (5e8) and pyranoanthocyanincatechols (9, 10) Pyranoanthocyanin-catechins and pyranoanthocyanincatechols are orange pigments present in red wines derived from the reaction of anthocyanins with vinyl-catechin or with vinylcatechol [14,15,42]. These compounds are thought to contribute to the orange hues observed in red wines during ageing and maturation. The equilibrium forms of six different compounds (Fig. 3) were studied in aqueous solutions by UVeVisible spectroscopy. No hydration reactions were observed, only electron transfer reactions should take place in these kind of compounds as reported by Cruz et al. (2010) [7] and similarly to what was described for other pyranoanthocyanins [5,6,33]. The hydration process was studied using reverse pH jump experiments (stopped flow). Additionally to the first two deprotonation reactions described above, a third deprotonation was described. A small batochromic shift was observed in maximum absorption wavelength for higher pH values (pH > 10.2) with an increase in the absorbance and these features were postulated to be due to the equilibrium between the anionic quinoidal form and the dianionic quinoidal form that is formed from the deprotonation of one hydroxyl group of the catechol unit in the case of pyranoanthocyanin-catechol pigments or in the phloroglucinol unit in the case of pyranoanthocyanin-catechin compounds. Cruz et al. (2010) also noticed that the pigments presenting a coumaroyl group (esterified in the glucose moiety) in
their structure (6, 8 and 10) showed an absorption band at w360 nm, that is described to be due to the deprotonation of the coumaroyl group of these compounds at high pH values (pKa2 pcoumaric acid ¼ 8.98 [43]) [7]. Based on the titration data and the equilibria postulated for these compounds, the three ionization constants were determined (Table 1) (pKa1, pKa2 and pKa3) and are in agreement with the results described for 7,40 -dihydroxyflavyliums (pKa1 ¼ 4; pKa2 ¼ 8) [44e46] and for other pyranoanthocyanins [5,6]. The pKa3 values obtained for (þ)-catechin and ()-epicatechin-derived pigments are also in agreement with the results described in the literature [47]. The pKa3 values of the catechol-derived compounds were also found to be similar to the ones reported for catechol in aqueous solutions [48].
2.5. A-type portisins (11) and B-type portisins (12, 13) Vinylpyranoanthocyanin-phenolics, commonly known as A and B-type portisins are bluish pyranoanthocyanin pigments detected in two-year-old Port wines [17,32,49]. These compounds were the first bluish compounds at acidic pH detected in wines and their formation can derive from the reaction of carboxypyranoanthocyanins with (þ)-catechin in the presence of acetaldehyde or with hydroxicinnamic acids [17,32,49] (Fig. 1). The equilibrium forms of A and B-type portisins (Fig. 5) were studied in aqueous solutions at different pH values using UVe Visible spectroscopy (Fig. 9). In this case, the study through NMR spectroscopy could not be accomplished since portisins are insoluble in water at the concentrations needed to the NMR titration. The behaviour observed for vinylpyranomalvidin-3-O-glucosidecatechin 11, vinylpyranomalvidin-3-O-glucoside-catechol 12 and vinylpyranomalvidin-3-O-glucoside-syringol 13 in aqueous solutions towards the increase of pH values up to 12.00 was similar for the three compounds. Therefore, the vinylpyranomalvidin-3-Oglucoside-catechin (Portisin A) will be discussed as an example of the equilibrium forms of this kind of pigments in aqueous solution at different pH values. With the increase of pH values from 1.99 to 5.26 it is possible to observe a decrease in the absorbance of the solutions that is accompanied with a shift in the wavelength of maximum absorption for lower values (hypsochromic shift). This tendency was already reported in the literature for other pyranoanthocyanins [7,19] for the equilibrium between the pyranoflavylium form and the neutral quinoidal form. Similarly to what was observed for other pyranoanthocyanins, portisin A did not show evidences of hydration reactions. For higher pH values up to 9.87 the absorbance around 500 nm starts to decrease together with the appearance of a shoulder at w630 nm that becomes more evident with the increase of the pH value. This feature is attributed to the equilibrium between the
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Fig. 9. Absorption spectra (360e830 nm) of aqueous solutions of portisins A as a function of pH (1.99e11.82).
neutral quinoidal base and the respective anionic base. For pH > 9.87 an intensification of the color of the solutions is observed with an important displacement of the maximum absorption wavelength (lmaxw660 nm) that is due to the deprotonation in the hydroxyl group present in carbon C-7 of the (þ)-catechin unit. Based on the data obtained by UVeVisible spectroscopy it was possible to determine the three ionization constants for the three pigments studied shown in Table 1. According to these constants, it seems that the catechin moiety stabilizes more efficiently the pyranoflavylium cation form when compared to the catechol or the syringol units, difficulting the deprotonation reaction. This probably occur because catechin may promote higher electronic delocalization. This trend is observed for the three pKa values when the catechin-derived and the catechol-derived compounds are
compared. However, it looks like that the presence of an electrondonating group such as the methyl group in the syringol moiety will stabilize more readily the neutral quinoidal form of these compounds preventing the second and the third deprotonation reactions. A similar result was observed when comparing the methylpyranomalvidin-3-O-glucoside with the pyranomalvidin-3O-glucoside compound [5,6]. 2.6. Pyranomalvidin-3-O-glucoside-butadienylidene-sinapyl (14) Pyranomalvidin-3-O-glucoside-butadienylidene-sinapyl (14) pigment (Fig. 5) can be synthesised from the reaction of methylpyranomalvidin-3-O-glucoside with synapaldehyde. The structure of this pigment includes a pyranoanthocyanin moiety linked to a
Fig. 10. Absorption spectra (360e830 nm) of aqueous solutions of pyranoanthocyanin dimers as a function of pH (1.80e11.66).
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syringol group through a butadienylidene linkage [19]. This structure is somehow similar to the B-type portisins but in this case there is an additional vinyl group conferring to this family of pyranoanthocyanins a higher electronic delocalization and by this way a more bluish color. The equilibrium forms of the pyranomalvidin-3-O-glucosidebutadienylidene-sinapyl (14) were studied in aqueous solutions at different pH values using UVeVisible spectroscopy [19] and the results obtained are similar to the ones discussed for A and B-type portisins. In aqueous solutions this compound 14 revealed the presence of four equilibrium forms involving three deprotonation reactions at 7-OH, 40 -OH and 400 -OH (Table 1). 2.7. Pyranomalvidin-3-O-glucoside methine dimer (15) Pyranoanthocyanin dimers are a family of turquoise blue color anthocyanin-derived pigments and were first found in a 9-year-old Port wine and in the respective lees [18]. The structure of these compounds includes two pyranomalvidin-3-glucoside moieties linked through a methine linkage (Fig. 5). Studies performed in model solution have shown that these compounds can be formed in red wines from the reaction of a carboxypyranoanthocyanin with the methylpyranoanthocyanin, as described elsewhere [18]. In aqueous solution, according to the structure of this compound 15 (Fig. 5), it was expected for this compound to present four pKa values since there are two 7-OH protons and two 40 -OH protons in its structure. However, based on the results obtained by UVe Visible spectroscopy (Fig. 10) only three deprotonation reactions seem to occur at the pH values studied (1.80e11.66). With the increase of pH for values between 3.15 and 6.55 it is possible to
observe a reduction in the absorbance of the solutions together with a displacement in the wavelength of maximum absorption for lower values and the appearance of a small shoulder at w485 nm (Fig. 10). These features can be attributed to the equilibrium concerning the pyranoflavylium cation dimer and the neutral quinoidal base deprotonated at the first 7-OH (Fig. 11). No hydration reaction seems to occur for these kind of pigments in the conditions studied (pH 1.80e11.66) similarly to what is observed for other pyranoanthocyanins [5e7,33]. For higher pH values (6.55e8.66) an increase in the absorption and the maximum absorption wavelength (from 585 to 610 nm) is observed for all the solutions. A small shoulder at w635 nm starts to appear at pH 8.19. These features can be due to the equilibrium between the neutral quinoidal form and the anionic quinoidal form deprotonated in the 40 OH. With the increase of pH for values >8.66 (till 11.66) a similar trend is observed (Fig. 10) and the equilibrium between the anionic quinoidal form and the di-anionic quinoidal for is expected to occur (Fig. 11). At pH 11.66 when compared to pH 9.59 it is possible to observe a small shift in the wavelength of maximum absorption and a fourth deprotonation could explain this small shift. However, according to the data obtained and with the model adopted it was not possible to determine this ionization constant (it must occur at very high pH values). The ionization constants were determined based on the titration data obtained by UVeVisible spectroscopy and with a specific software pHab 2006 [37]. The pKa values (pKa1 ¼ 4.93 0.04; pKa2 ¼ 8.33 0.04; pKa3 ¼ 9.10 0.04) (Table 1) are in agreement with the ones reported in the literature for other pyranoanthocyanins [5e7]. However, the pKa values for this pyranoanthocyanin dimer are a little higher than the ones observed for
Fig. 11. Proton transfer equilibria of pyranoanthocyanin dimers in aqueous solutions at different pH values between 1 and 12.
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Fig. 12. Color of different pyranoanthocyanin pigments found in nature in water at acidic pH (pHw1.5). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
simpler pyranoanthocyanin pigments such as vitisin B or methylpyranomalvidin-3-O-glucoside (Table 1). This indicates that the presence of two pyranoanthocyanin in the same structure promotes a higher electronic delocalization which stabilizes the pyranoflavylium cation form, preventing the deprotonation reactions that leads to the formation of the quinoidal base forms. 3. Conclusions According to the results discussed above it seems that there is a relation between the structural features of the pyranoanthocyanin compound and the ionization constants observed. In general, the electronic delocalization appears to have an influence in the equilibrium forms of pyranoanthocyanins, especially in the equilibrium between the pyranoflavylium cation form and the respective neutral quinoidal base (pKa1). For instance, vinylpyranoanthocyanincatechin compounds (A-type portisins) that present a higher electronic delocalization when compared to the pyranoanthocyanincatechin compounds showed lower pKa1 values (Table 1). On the other hand, although the presence of coumaroyl groups esterified in the glucose moiety does not contribute to electronic delocalization, it looks like that the presence of this group in the pyranoanthocyanin structure generally increases the stability of the compounds towards deprotonation reactions. This tendency is more evident in the first ionization constant, diminishing its effect for the second and the third deprotonations (Table 1). Catechinderived compounds are also more stable to deprotonation when compared to similar phenolic-derived compounds (comparison between A and B-type portisins). Furthermore, the presence of additional electron-donating groups as observed for the syringol group comparing to the catechol one seems to stabilize the anionic quinoidal base form of the pyranoanthocyanin compounds, reducing its ability to lose the third proton. This was observed for B-type portisins derived from syringol and catechol (pKa3 ¼ 10.18 0.05 and pKa3 ¼ 12.16 0.05, respectively) (Table 1). According to the ionization constants obtained for vitisins A and B and methylpyranomalvidin-3-O-glucoside, the substitution pattern in carbon C-10 does not seem to affect importantly the equilibrium between the pyranoflavylium cation form and the respective neutral quinoidal form as the pKa values are similar for the three pigments (Table 1). However, for the second ionization constant representing the equilibrium between the quinoidal base and the quinoidal anionic forms, the methylpyranomalvidin-3-O-glucoside revealed a
higher value (pKa2 ¼ 8.23 0.04) comparing to the one obtained for pyranomalvidin-3-glucoside (pKa2 ¼ 7.34 0.03). It seems that the methyl group present in the methylpyranoanthocyanin stabilizes the quinoidal base form reducing their ability to lose the second proton. Pyranoanthocyanin pigments found in nature can be present in different colours such as, orange, bluish or turquoise blue colour [15,17,18] (Fig. 12). The physicalechemical properties of these compounds studied over the years revealed their higher stability towards the hydration reactions when compared to their anthocyanin precursors contributing to the colour stability of red wines during the ageing process. These features also bring promising expectation concerning their use in the Food Industry as colorants. Acknowledgements This research was supported by a Post-doctoral grant from FCT (Fundação para a Ciência e a Tecnologia e Praxis BPD/65400/2009) and a grant from FCT (Fundação para a Ciência e a Tecnologia e PTDC/QUI-QUI/117996/2010) all from Portugal and by FEDER funding. References [1] Mateus N, de Freitas V. Evolution and stability of anthocyanin-derived pigments during port wine aging. J Agric Food Chem 2001;49(11):5217e22. [2] Oliveira J, Fernandes V, Miranda C, Santos-Buelga C, Silva A, de Freitas V, et al. Color properties of four cyanidin-pyruvic acid adducts. J Agric Food Chem 2006;54(18):6894e903. [3] Bakker J, Timberlake CF. Isolation, identification, and characterization of new color-stable anthocyanins occurring in some red wines. J Agric Food Chem 1997;45(1):35e43. [4] Fulcrand H, Benabdeljalil C, Rigaud J, Cheynier V, Moutounet M. A new class of wine pigments generated by reaction between pyruvic acid and grape anthocyanins. Phytochemistry 1998;47(7):1401e7. [5] Oliveira J, Mateus N, Silva AMS, de Freitas V. Equilibrium forms of vitisin B pigments in an aqueous system studied by NMR and visible spectroscopy. J Phys Chem B 2009;113(32):11352e8. [6] Oliveira J, Petrov V, Parola AJ, Pina F, Azevedo J, Teixeira N, et al. Chemical behavior of methylpyranomalvidin-3-O-glucoside in aqueous solution studied by NMR and UVeVisible spectroscopy. J Phys Chem B 2011;115(6):1538e45. [7] Cruz L, Petrov V, Teixeira N, Mateus N, Pina F, de Freitas V. Establishment of the chemical equilibria of different types of pyranoanthocyanins in aqueous solutions: evidence for the formation of aggregation in pyranomalvidin-3-Ocoumaroylglucoside-(þ)-catechin. J Phys Chem B 2010;114(41):13232e40. [8] He J, Santos-Buelga C, Silva AMS, Mateus N, De Freitas V. Isolation and structural characterization of new anthocyanin-derived yellow pigments in aged red wines. J Agric Food Chem 2006;54(25):9598e603. [9] Lu Y, Foo LY. Unusual anthocyanin reaction with acetone leading to pyranoanthocyanin formation. Tetrahedron Lett 2001;42(7):1371e3. [10] Lu Y, Sun Y, Foo LY. Novel pyranoanthocyanins from black currant seed. Tetrahedron Lett 2000;41(31):5975e8.
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