Food Chemistry 172 (2015) 476–480
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Effect of water content on the acid–base equilibrium of cyanidin-3-glucoside Isabel B. Coutinho a, Adilson Freitas b, António L. Maçanita b, J.C. Lima a,⇑ a b
Requimte, Dep. Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829 516 Caparica, Portugal Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisbon, Portugal
a r t i c l e
i n f o
Article history: Received 8 April 2014 Received in revised form 7 August 2014 Accepted 11 September 2014 Available online 19 September 2014 Keywords: Anthocyanin Acid–base equilibrium Laser Flash Photolysis Water/methanol
a b s t r a c t Laser Flash Photolysis was employed to measure the deprotonation and reprotonation rate constants of cyanidin 3-monoglucoside (kuromanin) in water/methanol mixtures. It was found that the deprotonation rate constant kd decreases with decreasing water content, reflecting the lack of free water molecules around kuromanin, which may accommodate and stabilize the outgoing protons. On the other hand, the reprotonation rate constant, kp, increases with the decrease in water concentration from a value of kp = 2 1010 l mol1 s1 in water up to kp = 6 1010 l mol1 s1 at 5.6 M water concentration in the mixture. The higher value of kp at lower water concentrations reflects the fact that the proton is not freely escaping the solvation shell of the molecule. The deprotonation rate constant decreases with decreasing water content, reflecting the lack of free water molecules around kuromanin that can accommodate the outgoing protons. Overall, the acidity constant of the flavylium cation decreases with the decrease in water concentration from pKa values of 3.8 in water to approximately 4.8 in water-depleted media, thus shifting the equilibrium towards the red-coloured form, AH+, at low water contents. The presence, or lack, of water, will affect the colour shade (red to blue) of kuromanin. This is relevant for its role as an intrinsic food component and as a food pigment additive (E163). Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Cyanidin 3-monoglucoside (kuromanin) belongs to the anthocyanin family of natural polyphenols and is the major pigment present in passion fruit rind (Kidoey et al., 1997). Like all anthocyanins, kuromanin undergoes several equilibria in aqueous media, which affect the colour displayed by the pigment as shown in Scheme 1 (Pina, Melo, Laia, Parola, & Lima, 2012). The equilibrium between the two coloured forms (flavylium cation, AH+, and quinoidal bases, A, see Scheme 1), is responsible for the shading between red (flavylium cation, AH+) and blue (quinoidal base, A) observed in the fruit. Upon light absorption by the pigment, the excited state acid–base reaction provides a pathway for energy dissipation which aids in pigment photostability and photoprotection of the plant cells (Ferreira da Silva et al., 2012). The effect of the solvent medium on the multiequilibria of anthocyanins is of relevance, since in nature the pigment accumulates in vacuoles, where the composition is different from dilute aqueous solutions, and the presence of other organic components
⇑ Corresponding author. http://dx.doi.org/10.1016/j.foodchem.2014.09.060 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
provides microheterogeneous environments of lower dielectric constant than pure water. Several works focusing on the effect of non-aqueous media on the hydration reaction leading to colour loss exist in the literature (Gomes, Parola, Lima, & Pina, 2006; Lima et al., 2002; Pina et al., 2012; Vautier-Giongo et al., 2002). The major purpose of these works has been to find ways to displace the hemiketal (B)-flavylium equilibrium towards AH+, either by stabilization of AH+ in anionic micelles, or through co-pigmentation, thus promoting the existence of the coloured species at higher pH values. However, the effect of the medium (low water concentration, low dielectric constant) on the shading of the colour, i.e., in the equilibrium between the coloured forms AH+ and A, has not been tackled to date. It was shown previously that ultrafast excited state conversion of flavylium cations into their respective bases provides an efficient tool to probe the ground-state interconversion between these species (Maçanita et al., 2002). Because deprotonation of AH+⁄ (where ⁄ stands for excited state) occurs with 99% efficiency (in less than 10 ps) and A⁄ decays to the ground-state in less than 300 ps, absorption of a ns-laser pulse by AH+ generates a transient ground-state concentration of A which is superior to its ground state equilibrium concentration. The recovery of the ground state equilibrium concentrations of AH+ and A can be conveniently
I.B. Coutinho et al. / Food Chemistry 172 (2015) 476–480
followed by transient absorption spectroscopy and follows a first order law with an observed rate constant kobs, which is the sum of the direct and reverse rate constants (Eq. (1)), (Moreira et al., 2003).
kobs ¼ kd þ kp ½Hþ
ð1Þ
2. Materials and methods Kuromanin (cyanidin-3-monoglucoside, PM 484.84 for the chloride salt, e510 = 26,900 M1 cm1 in 0.1 M HCl) was isolated from the rind of purple passion fruit Passiflora edulis using standard separation procedures. Purity was assessed via reversed phase HPLC (Hypersil ODS C18) with detection at 520 nm (>99% peak area) and at 280 nm (>95% peak area) and by mass spectrometry. Samples used in the various experiments were prepared by adding HCl to a kuromanin solution in distilled water. An aliquot of the aqueous solution of known pH was then mixed with methanol (CHROMASOLVÒ, Sigma–Aldrich) to obtain a final volume of 5 ml with the correct H2O:MeOH proportions [volume MeOH:H2O ratios of 0:100 (55.5 M H2O), 50:50 (27.7 M H2O), 70:30 (16.6 M H2O) and 90:10 (5.55 M H2O)]; final anthocyanin concentrations within the sample varied from 1 105 M to 4 105 M. pH values were determined for the samples prepared in water, before the addition of methanol, using a calibrated pH metre (MeterLab pHM240 pH metre from Radiometer Copenhagen or using a Crison BasiC 20 pH metre combined with a Mettler Toledo InLab Micro Ag/AgCl microelectrode). The [H+] values used in the correlations are calculated from the dilution factor and the measured pH in the aqueous phase ½Hþ ¼ ðV H2 O =V mix Þ½Hþ aq . For the measurements of the pKa of the kuromanin in the water:methanol mixtures at 20 °C, the determination of the ss pH (i.e., the log aHþ in solvent mixture) was carried out as described previously (Castells, Ràfols, Rosés, & Bosch, 2003). Flash photolysis experiments were carried out at room temperature using the LK60 Laser Flash Photolysis equipment from
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Applied Photophysics (Pinheiro, Parola, Baptista, & Lima, 2010), with laser excitation at 532 nm (SHG), and monitoring the recovery of the ground-state protonated flavylium absorption band at 510 nm and the decay of the quinoidal base at 570 nm. The observed rate constants (kobs) were obtained through the global fitting of the kinetic traces with single-exponential recovery or decay functions, respectively. The values of kobs used in the linear representations against [H+] are averages of 3 independent measurements, the error in the individual values is always less than 5%. 3. Results and discussion 3.1. Multiequilibria of kuromanin in water Absorption spectra of kuromanin aqueous solutions equilibrated overnight at moderately acidic conditions are shown in Fig. 1. At pH 2 the absorption spectrum of the flavylium cation (AH+) dominates, with an absorption maximum at 512 nm. At pH 5.07, the equilibrium is completely shifted towards the base (A), hemiketal (B) and chalcones (CE and CZ), with absorption maxima at 550, 270 and 330 nm, respectively. The equilibrium concentration of the quinonoidal base is very low, since the overall equilibrium is shifted mainly towards the colourless forms (hemiketal, B and chalcones, CE and CZ, Scheme 1), which absorb below 350 nm. Laser Flash Photolysis of aqueous solutions of kuromanin at pH values below 4 (kexc = 532 nm) results in bleaching of the flavylium cation absorption at 512 nm and the appearance of a positive DOD assigned to the S1 S0 absorption of A, formed during the flash (Fig. 2a). The positive absorption bands formed after the laser flash are due to the formation of the quinoidal base in the ground state with a concentration that is in excess with respect to the ground state equilibrium. The base form thus reacts to yield the flavylium cation, re-establishing the ground state equilibrium concentrations for that particular pH. Both the recovery of the flavylium and
Scheme 1. Multiequilibiria of cyanidin-3-glucoside in moderately acidic aqueous solutions.
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Fig. 1. Absorption spectra of aqueous solutions of kuromanin equilibrated overnight.
disappearance of the base follow first order kinetics (Fig. 2b), with an observed rate constant, kobs, linearly dependent on the [H+] pH (Fig. 2c, Eq. (1)). The rate constants kobs, retrieved from global fits of the decays at 500 and 570 nm with single exponential functions, were measured at several pH values. The values obtained are plotted against [H+] in Fig. 2c. The slope of the fitted line yields the protonation rate constant of the quinoidal base, kp = (2.19 ± 0.02) 1010 l mol1 s1, a value close to the diffusional limit for protonation reactions in water, as found earlier for other anthocyanins and flavylium salts in water (Maçanita et al., 2002; Moreira et al., 2003). The deprotonation rate constant value for the flavylium cation could be estimated from the y-intercept of the same plot, but with considerable error, since its value is comparatively much lower than kp (Maçanita et al., 2002). A more accurate value of kd can, however, be obtained from the combination of kp with the acid–base equilibrium constant Ka = kd/kp (Eq. (2))
kd ¼ kp K a
ð2Þ
The equilibrium constant Ka was determined from Ka = xA ([H+] + K0 a) (Leydet et al., 2012), where xA is the mole fraction of A in solution and K0 a = Ka(1 + Kh + KhKt + KhKtKi) (Vautier-Giongo et al., 2002) is the apparent equilibrium constant, Kh the hydration equilibrium constant, Kt the tautomerization equilibrium constant and Ki the isomerization equilibrium constant (Scheme 1). The apparent equilibrium constant, K0 a, was evaluated from the absorption spectra of thermodynamically equilibrated solutions. Values for xA were obtained by rapidly measuring the absorption spectra of kuromanin as soon as pH was increased to 6 and before significant hydration had occurred, in order to ensure that all the anthocyanin present was in the quinonoidal base form (see Fig. 3, dashed line). Thus, taking a wavelength where only A absorbs, the ratio between the optical density (OD) of A in completely equilibrated solutions and the initial OD at high pH is equal to the mole fraction of A in solution. Fig. 3 shows the absorption spectrum of kuromanin in water at pH 6.03 obtained ca. 5 s after preparation (dashed line) and those obtained after equilibration at several pH values (solid lines). The inset shows the OD of AH+ at 512 nm of these latter solutions plotted as a function of the pH, from which the apparent pK0 a = 2.89 was obtained. As expected, the correct value of kd = 3.31 106 s1 obtained with Eq. (1) is appreciably larger than that obtained from the much less accurate y-intercept of the kobs vs. [H+] plot (kd = 1.3 106 s1).
Fig. 2. (a) Transient absorption spectrum of an aqueous solution of kuromanin (1.5 105 M) at pH = 2.21, 10 ns after laser pulse, (b) time evolution of transients at 500 nm (recovery of depletion of AH+) and 570 nm (decay of A), and (c) plot of the observed decay rate constants kobs as a function of [H+].
Fig. 3. Absorption spectra of thermodynamically equilibrated aqueous solutions of kuromanin in H2O at different pH values (6.03, 4.02, 3.78, 3.17, 2.58 and 1.00) and immediately after preparation at pH 6.03 (dashed line). The inset shows the plot of the OD vs. pH from which the apparent pK0 a = 2.89 was obtained.
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(a)
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(b)
Fig. 4. (a) Plot of the observed rate constants (kobs) as a function of the proton concentration in the mixture for different MeOH:H2O compositions. Series 1 (squares) refers to the assay in water shown in Fig. 2C; (b) plots of kp and kd (obtained from Eq. (1)) as a function of the concentration of water in the mixtures.
Fig. 5. Variation of pKa for the acid–base AH+–A as a function of the dielectric constant/water content of the H2O:MeOH mixtures. The dashed line indicates the average vacuolar pH = 4.8, and the colour bar highlights the dominant coloured species at the different water contents.
3.2. Multiequilibria of kuromanin in water/methanol mixtures The foregoing procedure was applied to evaluate the values of kobs (Fig. 4a), as well as those of pK0 a and pKa in different water:methanol mixtures. The values of kp and kd obtained from the kobs vs. [H+] plots, together with those of kd derived from Eq. (1) can be seen in Table S1 of the supplementary information. Fig. 4b shows plots of kp and kd (from Eq. (1)) for the different mixtures. The interpretation of the plots in Fig. 4a and b is as follows: the deprotonation rate constant decreases with the decrease in water content, reflecting the lower availability of water molecules in the vicinity of the chromophore. The reprotonation rate constant increases with the decrease in water concentration, up to a value well above the diffusion limit in methanol (2 1010 l mol1 s1), which implies an effective concentration of protons in the solvation shell of A higher than in the bulk. This may result from the expected preferential solvation of kuromanin by water molecules (and respective protons) relative to methanol. Both points converge to the conclusion that flavylium is preferentially solvated by water and that the deprotonation/reprotonation reactions apparently involve only water molecules. Fig. 5 shows the effect of the water content on the equilibrium between the flavylium cation and the quinoidal base. The linear relationship between pKa and either the logarithm of the water concentration or the reciprocal of the static dielectric constant of
the medium allows the straightforward prediction of the extent of acid ionization in media of lower dielectric capacity. Solvent mixtures composed of water plus a less polar, watercompatible solvent have been used extensively to deal with weak acids that are sparingly soluble in water, many of which are molecules with pharmaceutical interest (Lakshmi & Barhate, 2011; Sager, Robinson, & Bates, 1964; Sarmini & Kenndler, 1999). For a substantial number of weak acids, it has been found that the pKa increases with increasing proportions of a less polar solvent; plots of pKa against the water content or dielectric data yield robust correlations. In this respect, kuromanin behaves like most other weak acids. This is of relevance for many microheterogeneous media containing anthocyanins. The effect can be exemplified for pH conditions similar to those found in plant vacuoles (colour bars in Fig. 5). For a vacuolar pH = 4.8 (Barkla & Pantoja, 1996; Davies, Hunt, & Sanders, 1994; Kurkdjian & Guern, 1989), at high water contents (vacuolar pH > pKa) the basic blue A form prevails, as is illustrated by the blue bar in Fig. 5a and b. At lower water contents (vacuolar pH < pKa) the acidic red AH+ form is dominant, as shown by the pink bar in Fig. 5a and b. The same reasoning can be extended to other water containing media and different pH values in the mildly acidic region. Thus, a mere alteration in the local water content would affect the AH+/A ratio, with a consequent impact on the colour hue of the anthocyanin containing medium.
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4. Conclusions
References
This work shows that the water content and, arguably, the dielectric constant of the medium, can have significant effects on the equilibrium between the coloured forms AH+ and A, altering the blue-red shade of the colour of anthocyanin-containing tissues such as those found in vegetables, flowers and fruits. In particular, under conditions similar to those found in plant vacuoles (vacuolar pH = 4.8), an alteration in water content would affect the AH+/A ratio with an impact on the colour hue of the plant tissue. The variation of the pKa of a weak acid with the increase of alcohol content in water–alcohol mixtures is well documented, namely in the literature related to pharmacology. On the other hand, kuromanin, along with the other anthocyanins, displays ‘‘reddish’’ colours exclusively in its protonated, cationic flavylium form. In mildly acidic aqueous solution, such colour tends to fade in a short time due to hydration competing reactions. Several studies have focused into the effect of water on the colour stability (hydration equilibrium) of anthocyanins, either in continuous model systems containing mixed solvents or in micro-heterogenous systems. A decrease in water is expected to reduce hydration and contribute to colour stability. In this work, we studied a different part of the anthocyanin multiequilibria: the effect of water on the colour shade (red to blue) of cyanidin-3-glucoside (kuromanin), bearing in mind its role as an intrinsic food component and as a food pigment additive (E163). Via the change in the microscopic deprotonation–reprotonation constants, the pKa value is raised in matrixes with lower water content and the flavylium form is stabilized, with obvious consequences in the appearance and storage of anthocyanin containing materials.
Barkla, B. J., & Pantoja, O. (1996). Physiology of ion transport across the tonoplast of higher plants. Annual Review of Plant Biology, 47, 159–184. Castells, C. B., Ràfols, C., Rosés, M., & Bosch, E. (2003). Effect of temperature on pH measurements and acid–base equilibria in methanol–water mixtures. Journal of Chromatography A, 1002, 41–43. Davies, J. M., Hunt, I., & Sanders, D. (1994). Vacuolar H+-pumping ATPase variable transport coupling ratio controlled by pH. Proceedings of the National academy of Sciences of the United States of America, 91, 8547–8551. Ferreira da Silva, P., Paulo, L., Barbafina, A., Elisei, F., Quina, F. H., & Maçanita, António L. (2012). Photoprotection and the photophysics of acylated anthocyanins. Chemistry – A European Journal, 18, 3736–3744. Gomes, R., Parola, A. J., Lima, J. C., & Pina, F. (2006). Solvent effects on the thermal and photochemical reactions of 4-iodo-8-methoxyflavylium, and their consequences on the colouring phenomenon by anthocyanins in plants. Chemistry – A European Journal, 12, 7906–7912. Kidoey, L., Nygaard, A. M., Andersen, O. M., Pedersen, A. T., Aksnes, D. W., & Kiremire, B. T. (1997). Anthocyanins in fruits of Passiflora edulis and P. suberosa. Journal of Food Composition and Analysis, 10, 49–54. Kurkdjian, A., & Guern, J. (1989). Intracellular pH: Measurement and importance in cell activity. Annual Review of Plant Physiology, 40, 271–303. Lakshmi, N., & Barhate, V. D. (2011). Physico-chemical characterization of some beta blockers and anti-diabetic drugs-potentiometric and spectrophotometric pKa determination in different co-solvents. European Journal of Chemistry, 2, 36–46. Leydet, Y., Gavara, R., Petrov, V., Diniz, A. M., Parola, A. J., Lima, J. C., et al. (2012). 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, 30, 125–135. Lima, J. C., Vautier-Giongo, C., Lopes, A., Melo, E., Quina, F. H., & Maçanita, A. L. (2002). Color stabilization of anthocyanins: Effect of SDS micelles on the acid– base and hydration kinetics of malvidin 3-glucoside (Oenin). Journal of Physical Chemistry A, 106, 5851–5859. Maçanita, A. L., Moreira, P. F., Lima, J. C., Quina, F. H., Yihwa, C., & Vautier-Giongo, C. (2002). Proton transfer in anthocyanins and related flavylium salts. Determination of ground-state rate constants with nanosecond Laser Flash Photolysis. Journal of Physical Chemistry A, 106, 1248–1255. Moreira, P. F., Jr., Giestas, L., Yihwa, C., Vautier-Giongo, C., Quina, F. H., Maçanita, A. L., et al. (2003). Ground and excited proton transfer in anthocyanins: From weak acids to super-photoacids. Journal of Physical Chemistry A, 107, 4203–4210. Pina, F., Melo, M. J., Laia, C. A. T., Parola, A. J., & Lima, J. C. (2012). Chemistry and applications of flavylium compounds: A handful of colours. Chemical Society Reviews, 41, 869–908. Pinheiro, A. V., Parola, A. J., Baptista, P. V., & Lima, J. C. (2010). PH Effect on the photochemistry of 4-methylcoumarin phosphate esters: Caged-phosphate case study. Journal of Physical Chemistry A, 114, 12795–12803. Sager, E. E., Robinson, R. A., & Bates, R. G. (1964). Medium effects on the dissociation of weak acids in methanol–water solvents. Journal of Research of the National Bureau of Standards A, 68, 305–312. Sarmini, K., & Kenndler, E. (1999). Ionization constants of weak acids and bases in organic solvents. Journal of Biochemical and Biophysical Methods, 38, 123–137. Vautier-Giongo, C., Yihwa, C., Moreira, P. F., Jr., Lima, J. C., Freitas, A. A., Alves, M., et al. (2002). Manipulation of the reactivity of a synthetic anthocyanin analogue in aqueous micellar media. Langmuir, 18, 10109–10115.
Acknowledgement The authors want to acknowledge financial support from FCT/ MCTES (project PTDC/QUI/65728/2006). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 09.060.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Effect of water content on the acid–base equilibrium of cyanidin-3-glucoside Isabel B. Coutinho a, Adilson Freitas b, António L. Maçanita b, J.C. Lima a,⇑ a b
Requimte, Dep. Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829 516 Caparica, Portugal Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisbon, Portugal
a r t i c l e
i n f o
Article history: Received 8 April 2014 Received in revised form 7 August 2014 Accepted 11 September 2014 Available online 19 September 2014 Keywords: Anthocyanin Acid–base equilibrium Laser Flash Photolysis Water/methanol
a b s t r a c t Laser Flash Photolysis was employed to measure the deprotonation and reprotonation rate constants of cyanidin 3-monoglucoside (kuromanin) in water/methanol mixtures. It was found that the deprotonation rate constant kd decreases with decreasing water content, reflecting the lack of free water molecules around kuromanin, which may accommodate and stabilize the outgoing protons. On the other hand, the reprotonation rate constant, kp, increases with the decrease in water concentration from a value of kp = 2 1010 l mol1 s1 in water up to kp = 6 1010 l mol1 s1 at 5.6 M water concentration in the mixture. The higher value of kp at lower water concentrations reflects the fact that the proton is not freely escaping the solvation shell of the molecule. The deprotonation rate constant decreases with decreasing water content, reflecting the lack of free water molecules around kuromanin that can accommodate the outgoing protons. Overall, the acidity constant of the flavylium cation decreases with the decrease in water concentration from pKa values of 3.8 in water to approximately 4.8 in water-depleted media, thus shifting the equilibrium towards the red-coloured form, AH+, at low water contents. The presence, or lack, of water, will affect the colour shade (red to blue) of kuromanin. This is relevant for its role as an intrinsic food component and as a food pigment additive (E163). Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Cyanidin 3-monoglucoside (kuromanin) belongs to the anthocyanin family of natural polyphenols and is the major pigment present in passion fruit rind (Kidoey et al., 1997). Like all anthocyanins, kuromanin undergoes several equilibria in aqueous media, which affect the colour displayed by the pigment as shown in Scheme 1 (Pina, Melo, Laia, Parola, & Lima, 2012). The equilibrium between the two coloured forms (flavylium cation, AH+, and quinoidal bases, A, see Scheme 1), is responsible for the shading between red (flavylium cation, AH+) and blue (quinoidal base, A) observed in the fruit. Upon light absorption by the pigment, the excited state acid–base reaction provides a pathway for energy dissipation which aids in pigment photostability and photoprotection of the plant cells (Ferreira da Silva et al., 2012). The effect of the solvent medium on the multiequilibria of anthocyanins is of relevance, since in nature the pigment accumulates in vacuoles, where the composition is different from dilute aqueous solutions, and the presence of other organic components
⇑ Corresponding author. http://dx.doi.org/10.1016/j.foodchem.2014.09.060 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
provides microheterogeneous environments of lower dielectric constant than pure water. Several works focusing on the effect of non-aqueous media on the hydration reaction leading to colour loss exist in the literature (Gomes, Parola, Lima, & Pina, 2006; Lima et al., 2002; Pina et al., 2012; Vautier-Giongo et al., 2002). The major purpose of these works has been to find ways to displace the hemiketal (B)-flavylium equilibrium towards AH+, either by stabilization of AH+ in anionic micelles, or through co-pigmentation, thus promoting the existence of the coloured species at higher pH values. However, the effect of the medium (low water concentration, low dielectric constant) on the shading of the colour, i.e., in the equilibrium between the coloured forms AH+ and A, has not been tackled to date. It was shown previously that ultrafast excited state conversion of flavylium cations into their respective bases provides an efficient tool to probe the ground-state interconversion between these species (Maçanita et al., 2002). Because deprotonation of AH+⁄ (where ⁄ stands for excited state) occurs with 99% efficiency (in less than 10 ps) and A⁄ decays to the ground-state in less than 300 ps, absorption of a ns-laser pulse by AH+ generates a transient ground-state concentration of A which is superior to its ground state equilibrium concentration. The recovery of the ground state equilibrium concentrations of AH+ and A can be conveniently
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followed by transient absorption spectroscopy and follows a first order law with an observed rate constant kobs, which is the sum of the direct and reverse rate constants (Eq. (1)), (Moreira et al., 2003).
kobs ¼ kd þ kp ½Hþ
ð1Þ
2. Materials and methods Kuromanin (cyanidin-3-monoglucoside, PM 484.84 for the chloride salt, e510 = 26,900 M1 cm1 in 0.1 M HCl) was isolated from the rind of purple passion fruit Passiflora edulis using standard separation procedures. Purity was assessed via reversed phase HPLC (Hypersil ODS C18) with detection at 520 nm (>99% peak area) and at 280 nm (>95% peak area) and by mass spectrometry. Samples used in the various experiments were prepared by adding HCl to a kuromanin solution in distilled water. An aliquot of the aqueous solution of known pH was then mixed with methanol (CHROMASOLVÒ, Sigma–Aldrich) to obtain a final volume of 5 ml with the correct H2O:MeOH proportions [volume MeOH:H2O ratios of 0:100 (55.5 M H2O), 50:50 (27.7 M H2O), 70:30 (16.6 M H2O) and 90:10 (5.55 M H2O)]; final anthocyanin concentrations within the sample varied from 1 105 M to 4 105 M. pH values were determined for the samples prepared in water, before the addition of methanol, using a calibrated pH metre (MeterLab pHM240 pH metre from Radiometer Copenhagen or using a Crison BasiC 20 pH metre combined with a Mettler Toledo InLab Micro Ag/AgCl microelectrode). The [H+] values used in the correlations are calculated from the dilution factor and the measured pH in the aqueous phase ½Hþ ¼ ðV H2 O =V mix Þ½Hþ aq . For the measurements of the pKa of the kuromanin in the water:methanol mixtures at 20 °C, the determination of the ss pH (i.e., the log aHþ in solvent mixture) was carried out as described previously (Castells, Ràfols, Rosés, & Bosch, 2003). Flash photolysis experiments were carried out at room temperature using the LK60 Laser Flash Photolysis equipment from
477
Applied Photophysics (Pinheiro, Parola, Baptista, & Lima, 2010), with laser excitation at 532 nm (SHG), and monitoring the recovery of the ground-state protonated flavylium absorption band at 510 nm and the decay of the quinoidal base at 570 nm. The observed rate constants (kobs) were obtained through the global fitting of the kinetic traces with single-exponential recovery or decay functions, respectively. The values of kobs used in the linear representations against [H+] are averages of 3 independent measurements, the error in the individual values is always less than 5%. 3. Results and discussion 3.1. Multiequilibria of kuromanin in water Absorption spectra of kuromanin aqueous solutions equilibrated overnight at moderately acidic conditions are shown in Fig. 1. At pH 2 the absorption spectrum of the flavylium cation (AH+) dominates, with an absorption maximum at 512 nm. At pH 5.07, the equilibrium is completely shifted towards the base (A), hemiketal (B) and chalcones (CE and CZ), with absorption maxima at 550, 270 and 330 nm, respectively. The equilibrium concentration of the quinonoidal base is very low, since the overall equilibrium is shifted mainly towards the colourless forms (hemiketal, B and chalcones, CE and CZ, Scheme 1), which absorb below 350 nm. Laser Flash Photolysis of aqueous solutions of kuromanin at pH values below 4 (kexc = 532 nm) results in bleaching of the flavylium cation absorption at 512 nm and the appearance of a positive DOD assigned to the S1 S0 absorption of A, formed during the flash (Fig. 2a). The positive absorption bands formed after the laser flash are due to the formation of the quinoidal base in the ground state with a concentration that is in excess with respect to the ground state equilibrium. The base form thus reacts to yield the flavylium cation, re-establishing the ground state equilibrium concentrations for that particular pH. Both the recovery of the flavylium and
Scheme 1. Multiequilibiria of cyanidin-3-glucoside in moderately acidic aqueous solutions.
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Fig. 1. Absorption spectra of aqueous solutions of kuromanin equilibrated overnight.
disappearance of the base follow first order kinetics (Fig. 2b), with an observed rate constant, kobs, linearly dependent on the [H+] pH (Fig. 2c, Eq. (1)). The rate constants kobs, retrieved from global fits of the decays at 500 and 570 nm with single exponential functions, were measured at several pH values. The values obtained are plotted against [H+] in Fig. 2c. The slope of the fitted line yields the protonation rate constant of the quinoidal base, kp = (2.19 ± 0.02) 1010 l mol1 s1, a value close to the diffusional limit for protonation reactions in water, as found earlier for other anthocyanins and flavylium salts in water (Maçanita et al., 2002; Moreira et al., 2003). The deprotonation rate constant value for the flavylium cation could be estimated from the y-intercept of the same plot, but with considerable error, since its value is comparatively much lower than kp (Maçanita et al., 2002). A more accurate value of kd can, however, be obtained from the combination of kp with the acid–base equilibrium constant Ka = kd/kp (Eq. (2))
kd ¼ kp K a
ð2Þ
The equilibrium constant Ka was determined from Ka = xA ([H+] + K0 a) (Leydet et al., 2012), where xA is the mole fraction of A in solution and K0 a = Ka(1 + Kh + KhKt + KhKtKi) (Vautier-Giongo et al., 2002) is the apparent equilibrium constant, Kh the hydration equilibrium constant, Kt the tautomerization equilibrium constant and Ki the isomerization equilibrium constant (Scheme 1). The apparent equilibrium constant, K0 a, was evaluated from the absorption spectra of thermodynamically equilibrated solutions. Values for xA were obtained by rapidly measuring the absorption spectra of kuromanin as soon as pH was increased to 6 and before significant hydration had occurred, in order to ensure that all the anthocyanin present was in the quinonoidal base form (see Fig. 3, dashed line). Thus, taking a wavelength where only A absorbs, the ratio between the optical density (OD) of A in completely equilibrated solutions and the initial OD at high pH is equal to the mole fraction of A in solution. Fig. 3 shows the absorption spectrum of kuromanin in water at pH 6.03 obtained ca. 5 s after preparation (dashed line) and those obtained after equilibration at several pH values (solid lines). The inset shows the OD of AH+ at 512 nm of these latter solutions plotted as a function of the pH, from which the apparent pK0 a = 2.89 was obtained. As expected, the correct value of kd = 3.31 106 s1 obtained with Eq. (1) is appreciably larger than that obtained from the much less accurate y-intercept of the kobs vs. [H+] plot (kd = 1.3 106 s1).
Fig. 2. (a) Transient absorption spectrum of an aqueous solution of kuromanin (1.5 105 M) at pH = 2.21, 10 ns after laser pulse, (b) time evolution of transients at 500 nm (recovery of depletion of AH+) and 570 nm (decay of A), and (c) plot of the observed decay rate constants kobs as a function of [H+].
Fig. 3. Absorption spectra of thermodynamically equilibrated aqueous solutions of kuromanin in H2O at different pH values (6.03, 4.02, 3.78, 3.17, 2.58 and 1.00) and immediately after preparation at pH 6.03 (dashed line). The inset shows the plot of the OD vs. pH from which the apparent pK0 a = 2.89 was obtained.
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(a)
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(b)
Fig. 4. (a) Plot of the observed rate constants (kobs) as a function of the proton concentration in the mixture for different MeOH:H2O compositions. Series 1 (squares) refers to the assay in water shown in Fig. 2C; (b) plots of kp and kd (obtained from Eq. (1)) as a function of the concentration of water in the mixtures.
Fig. 5. Variation of pKa for the acid–base AH+–A as a function of the dielectric constant/water content of the H2O:MeOH mixtures. The dashed line indicates the average vacuolar pH = 4.8, and the colour bar highlights the dominant coloured species at the different water contents.
3.2. Multiequilibria of kuromanin in water/methanol mixtures The foregoing procedure was applied to evaluate the values of kobs (Fig. 4a), as well as those of pK0 a and pKa in different water:methanol mixtures. The values of kp and kd obtained from the kobs vs. [H+] plots, together with those of kd derived from Eq. (1) can be seen in Table S1 of the supplementary information. Fig. 4b shows plots of kp and kd (from Eq. (1)) for the different mixtures. The interpretation of the plots in Fig. 4a and b is as follows: the deprotonation rate constant decreases with the decrease in water content, reflecting the lower availability of water molecules in the vicinity of the chromophore. The reprotonation rate constant increases with the decrease in water concentration, up to a value well above the diffusion limit in methanol (2 1010 l mol1 s1), which implies an effective concentration of protons in the solvation shell of A higher than in the bulk. This may result from the expected preferential solvation of kuromanin by water molecules (and respective protons) relative to methanol. Both points converge to the conclusion that flavylium is preferentially solvated by water and that the deprotonation/reprotonation reactions apparently involve only water molecules. Fig. 5 shows the effect of the water content on the equilibrium between the flavylium cation and the quinoidal base. The linear relationship between pKa and either the logarithm of the water concentration or the reciprocal of the static dielectric constant of
the medium allows the straightforward prediction of the extent of acid ionization in media of lower dielectric capacity. Solvent mixtures composed of water plus a less polar, watercompatible solvent have been used extensively to deal with weak acids that are sparingly soluble in water, many of which are molecules with pharmaceutical interest (Lakshmi & Barhate, 2011; Sager, Robinson, & Bates, 1964; Sarmini & Kenndler, 1999). For a substantial number of weak acids, it has been found that the pKa increases with increasing proportions of a less polar solvent; plots of pKa against the water content or dielectric data yield robust correlations. In this respect, kuromanin behaves like most other weak acids. This is of relevance for many microheterogeneous media containing anthocyanins. The effect can be exemplified for pH conditions similar to those found in plant vacuoles (colour bars in Fig. 5). For a vacuolar pH = 4.8 (Barkla & Pantoja, 1996; Davies, Hunt, & Sanders, 1994; Kurkdjian & Guern, 1989), at high water contents (vacuolar pH > pKa) the basic blue A form prevails, as is illustrated by the blue bar in Fig. 5a and b. At lower water contents (vacuolar pH < pKa) the acidic red AH+ form is dominant, as shown by the pink bar in Fig. 5a and b. The same reasoning can be extended to other water containing media and different pH values in the mildly acidic region. Thus, a mere alteration in the local water content would affect the AH+/A ratio, with a consequent impact on the colour hue of the anthocyanin containing medium.
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4. Conclusions
References
This work shows that the water content and, arguably, the dielectric constant of the medium, can have significant effects on the equilibrium between the coloured forms AH+ and A, altering the blue-red shade of the colour of anthocyanin-containing tissues such as those found in vegetables, flowers and fruits. In particular, under conditions similar to those found in plant vacuoles (vacuolar pH = 4.8), an alteration in water content would affect the AH+/A ratio with an impact on the colour hue of the plant tissue. The variation of the pKa of a weak acid with the increase of alcohol content in water–alcohol mixtures is well documented, namely in the literature related to pharmacology. On the other hand, kuromanin, along with the other anthocyanins, displays ‘‘reddish’’ colours exclusively in its protonated, cationic flavylium form. In mildly acidic aqueous solution, such colour tends to fade in a short time due to hydration competing reactions. Several studies have focused into the effect of water on the colour stability (hydration equilibrium) of anthocyanins, either in continuous model systems containing mixed solvents or in micro-heterogenous systems. A decrease in water is expected to reduce hydration and contribute to colour stability. In this work, we studied a different part of the anthocyanin multiequilibria: the effect of water on the colour shade (red to blue) of cyanidin-3-glucoside (kuromanin), bearing in mind its role as an intrinsic food component and as a food pigment additive (E163). Via the change in the microscopic deprotonation–reprotonation constants, the pKa value is raised in matrixes with lower water content and the flavylium form is stabilized, with obvious consequences in the appearance and storage of anthocyanin containing materials.
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Acknowledgement The authors want to acknowledge financial support from FCT/ MCTES (project PTDC/QUI/65728/2006). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 09.060.
