Food Research International 51 (2013) 748755

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Food Research International

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Effect of cyclodextrins on the thermodynamic and kinetic properties of cyanidin-3-O-glucoside

Ana Fernandes, Andr Sousa, Joana Azevedo, Nuno Mateus, Victor de Freitas
Centro de Investigao em Qumica (CIQ), Departamento de Qumica e Bioqumica, Faculdade de Cincias, 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
In this study, the effect of and -cyclodextrin on cyanidin-3-O-glucoside color was investigated by UV visible absorption techniques. The equilibrium and kinetic constants of the network of chemical reactions taking place in cyanidin-3-O-glucoside were also studied in water at 25 C by UVvisible absorption techniques. The results showed that the addition of -cyclodextrin resulted in the fading of anthocyanin solution, and this fading effect was greater at higher pH. This anti-copigmentation effect is caused by the selective inclusion and stabilization of the anthocyanin colorless forms into the -cyclodextrin cavity. Oppositely, no changes were observed in the cyanidin-3-O-glucoside absorption spectra with the addition of -cyclodextrin. Direct pH jump, from thermally equilibrated solutions at pH = 1.0 (avylium cation, AH+), shows three kinetic processes: formation of the base A, hydration reaction to form the hemiketal B and the chalcone cistrans isomerization (CcCt). The results obtained clearly indicated that the equilibrium and kinetic constants of the network of chemical reactions taking place in cyanidin-3-O-glucoside were affected by the presence of -cyclodextrin. Molecular inclusion in the -cyclodextrin cavity resulted in the increase of the isomerization observed rate constant (kobs) at pH 5.3 and in the increase of the hydration equilibrium constant Kh which is in agreement with the fading of the anthocyanin solution. For the macrocycle -cyclodextrin, no signicant changes were observed on the equilibrium and kinetic constants, which suggests that the inclusion of cyanidin-3-glucoside in the -cyclodextrin's cavity is not favored. 2013 Elsevier Ltd. All rights reserved.

Article history: Received 10 October 2012 Accepted 16 January 2013 Keywords: Anthocyanin Cyanidin-3-O-glucoside Cyclodextrin Hydration equilibrium constant Inclusion complex Kinetic constants

1. Introduction Color is one of the most important attributes in food products, as it affects the acceptability of products by consumers (Burin, Rossa, Ferreira-Lima, Hillmann, & Boirdignon-Luiz, 2011). Nowadays, as a consequence of perceived consumer preferences as well as legislative action, there is a worldwide trend towards the development of food colorants from natural sources, and the replacement of synthetic dyes by these natural colorants has been increasing (Idham, Muhamad, & Sarmidi, 2012). In this context, anthocyanins are attractive as food colorants since these natural pigments are responsible for the colors of owers, fruits and vegetables, provide high colorant power and present a low toxicity and water solubility, which permit their incorporation in many food systems (Brouillard, Chassaing, Isorez, Kueny-Stotz, & Figueiredo, 2010; Ersus & Yurdagel, 2007). Moreover, several studies have suggested that these compounds are benecial to human health and have been responsible for several positive therapeutic effects (Bridle & Timberlake, 1997; Clifford, 2000).

Corresponding author. Tel.: +351 220402558; fax: +351 220402659. E-mail address: vfreitas@fc.up.pt (V. de Freitas). 0963-9969/$ see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.01.037

However, the use of these colorants in food products may face some problems due to their instability (Francis & Markakis, 1989). The color and stability of anthocyanins are highly dependent on pH, due to changes in the concentration of the four species present in acidic and neutral aqueous solutions: avylium cation (AH +), quinoidal base (A), hemiketal (B) and chalcone (C) (Fig. 1) (Brouillard, 1982; Brouillard & Delaporte, 1977; Pina, Melo, Laia, Parola, & Lima, 2012). Conversion of one species to another is typically accompanied by dramatic changes in color and stability (Burin et al., 2011; Xiong, Melton, Easteal, & Siew, 2006). To increase the stability of anthocyanins, molecular inclusion and encapsulation with several carbohydrates have been used (Burin et al., 2011; Cai & Corke, 2000; Ersus & Yurdagel, 2007; Idham et al., 2012; Selim, Khalil, Abdel-Bary, & Abdel-Azeim, 2008). Cyclodextrins (CDs) have been used to stabilize anthocyanins (Chandra, Nair, & Iezzoni, 1993; Lewis, Walker, & Lancaster, 1995; Mourtzinos et al., 2008). These compounds are cyclic oligosaccharides composed of -D-glucopyranosyl units linked by -(1 4) bonds. The most common are -, - and -cyclodextrin which have six, seven and eight glucose units, respectively. The glucose monomers are orientated in a cyclic manner, forming a typical conical or truncated cone structure with a hydrophobic hollow cavity of specic diameter and volume and a relatively hydrophilic outer surface (Pegg & Shahidi,

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Fig. 1. Structural transformations of avylium cation in strongly acidic to alkaline aqueous media (Brouillard, 1982; Brouillard & Delaporte, 1977).

2007). The hollow molecular shape gives cyclodextrins their unique ability to form reversible inclusion complexes with a wide variety of organic compounds (often phenolic substances) yielding supramolecules (Hendy & Breslin, 2011). Formation of the hostguest complex can be easily monitored using techniques such as nuclear magnetic resonance (NMR) (Cai et al., 1990; Ishizu, Kintsu, & Yamamoto, 1999; Ishizuka et al., 2002), UVvisible spectroscopy (UVvis) (Divakar, 1993), uorescence spectroscopy (Divakar & Maheswaran, 1997), IR spectroscopy, electrochemical approaches and solubility measurements (Hendy & Breslin, 2011). Concerning anthocyanins, they include in their structure hydrophobic aromatic moieties and hydrophilic polar groups like hydroxyl groups. This amphiphilic character makes anthocyanins a very good candidate for molecular inclusion with cyclodextrins (Dangles, Stoeckel, Wigand, & Brouillard, 1992). Several studies have reported the formation of inclusion complexes of anthocyanins with cyclodextrin macrocycles (Dangles & Brouillard, 1992; Dangles, Stoeckel, et al., 1992; Dangles, Wigand, & Brouillard, 1992; Lewis et al., 1995; Mourtzinos et al., 2008; Nagatomo, 1985; Tamura, Takada, Yamagami, & Shiomi, 1998; Yamada, Komiya, & Akaki, 1980). However, interaction between cyclodextrins and some anthocyanins was found to promote the anthocyanin discoloration and lead to an anti-copigmentation effect (Dangles & Brouillard, 1992; Dangles, Stoeckel, et al., 1992; Lewis et al., 1995; Yamada et al., 1980). The main goal of this study was to promote the use of anthocyanins as food colorants and to overcome the limitations of the application of these natural pigments in food systems, enhancing its stability. In this study, the effect of cyclodextrins ( and ) on the color of cyanidin-3-O-glucoside was evaluated. The effect of these two cyclodextrins on the equilibrium and kinetic constants of the network of chemical reactions taking place in cyanidin-3-O-glucoside was also evaluated by UVvisible absorption techniques.

2. Materials and methods 2.1. Materials -Cyclodextrin (-CD) and -cyclodextrin (-CD) were purchased from Sigma-Aldrich (Madrid, Spain). All aqueous solutions were prepared with distilled water. Cyanidin-3-O-glucoside (cy3glc) was extracted and puried in the laboratory from blackberries (Rubus fruticosus) by semipreparative HPLC with C18 reversed phase column, as described elsewhere (Azevedo et al., 2010). A universal buffer of Theorell Stenhagen was made by dissolving 2.25 mL of phosphoric acid (85% w/w), 7.00 g of monohydrated citric acid, 3.54 g of boric acid and 343 mL of 1 M NaOH solution in water (until 1 L completion) (Kster & Thiel, 1982). 2.2. pH measurements All pH measurements were made in a pH meter WTW pH 320 tted with a Crison electrode. The calibration was made with standard buffers pH 7.00 and pH 4.00 purchased from Crison. 2.3. Effect of cyclodextrins on the color of cyanidin-3-O-glucoside A stock solution of cy3glc (1.50 10 4 M) was prepared using a solution of 0.1 M HCl. The low pH value of the stock solution ensured that most of the cy3glc was in the avylium form and thus prevented pigment degradation. The solution composed of 1.0 mL of cy3glc stock solution, 1.0 mL of NaOH solution (0.1 M) and 0.5 mL of universal buffer solution at pH 1.0 and 2.0 was allowed to equilibrate for 2 h. To these anthocyanin solutions -CD was added at various concentrations (6.00 10 4; 1.80 10 3 and 7.50 10 3 M) and the solutions were left to equilibrate for 2 h. The absorbance (260700 nm) was

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measured in an UVvis spectrophotometer (Thermo Scientic Evolution Array UVvis spectrophotometer) tted with a thermostated 1.0 cm path length cuvette device at 25 C. Similar studies were performed with -CD. 2.4. pH jump All thermodynamic and kinetic constants of the cy3glc solution and of the inclusion complexes cy3glc-CD and cy3glc-CD were determined by a spectrophotometric method, as described elsewhere (Nave et al., 2010; Pina, 1998). A stock solution of cy3glc (1.5010 4 M) was prepared as described above. For the molecular inclusion complex, a -CD and an -CD solution (7.50 10 3 M) were prepared in 0.1 M HCl. Cy3glc was dissolved in this solution at an approximate molar ratio of 1:50 (cy3glc: or -CD, the molar ratio which induced the largest avylium cation absorbance decrease) and left to equilibrate. pH jumps were carried out by adding the necessary amount of NaOH (0.1 M) to neutralize the stock solution of avylium cation (cy3glc) and the necessary amount of a universal buffer solution (at different pH values). The ionic strength was maintained at 0.1 M so that the effect of ion-pair formation could be minimized. The concentration of the buffer (Theorell Stenhagen universal buffer) was maintained low in order to avoid the buffer effects. Throughout the whole experiment the temperature was kept at 25 C. 3. Results and discussion The ability of a macrocycle molecule to form an inclusion complex with a guest molecule depends mainly on two factors: the rst concerns the steric requirements and depends on the relative size of the cyclodextrin and the size of the molecule to include; the second critical requirement corresponds to the thermodynamic interactions between different components of the system (cyclodextrin, molecule to include and solvent) (Bourvellec, 2003). Several forces such as van der Waals forces (hydrophobic interaction, dipoledipole interaction) and hydrogen bonding interaction are involved in the binding of guest molecules to the cyclodextrin cavity. These forces are capable of forming a stable complex, but not permanently as the guest molecule can still be released from the complex to become available for the intended effect of the guest molecule (Hedges, Shieh, & Sikorski, 1995). Among different naturally occurring anthocyanins, cyanidin-3-Oglucoside was selected for this study because of its structural simplicity and reduced steric hindrance, a property needed for a good interaction with cyclodextrin (Dangles, Wigand, et al., 1992). Indeed for sterically hindered pigments such as anthocyanidin diglycosides and anthocyanidin monoglycosides which are heavily substituted on their aromatic rings, the inclusion on the -CD cavity is not favored (Dangles, Stoeckel, et al., 1992). 3.1. Effect of cyclodextrins on the color of cyanidin-3-O-glucoside Addition of -CD to an acidic solution of cy3glc induces a strong decrease in the anthocyanin visible absorption band (hypochromism) (Fig. 2) and this decrease was greater with higher concentrations of -CD added. This effect was lower at pH 1.0, in which cy3glc is practically 100% in its avylium equilibrium form (8% absorbance decrease at pH 1.0 instead of 38% absorbance decrease at pH 2.0, at molar ratio 1:50). This fading effect, also known as anti-copigmentation phenomenon has previously been reported in several works (Dangles, Stoeckel, et al., 1992; Dangles, Wigand, et al., 1992; Lewis et al., 1995; Yamada et al., 1980). Since no change in the shape of the absorption band corresponding to the avylium form was observed, it can be assumed that the avylium cation is not the species interacting with -CD (Dangles, Wigand, et al., 1992). Indeed, anthocyanin avylium cation maximum absorption is very sensitive to solvent effects and the formation of an inclusion complex of the avylium cation

Fig. 2. A, UVvisible spectra of pure cyanidin-3-O-glucoside and in the presence of -CD at different concentrations at pH 2.0 (25 C); Cy3glc: 1.50 10 4 M (0); -CD: 6.00 10 4 M (1); 1.80 10 3 M (2); 7.50 10 3 M (3); B, UVvisible spectra of pure cyanidin-3-O-glucoside and in the presence of -CD at different concentrations at pH 2.0 (25 C); Cy3glc: 1.5010 4 M (0); -CD: 6.00104 M (1); 1.80103 M (2); 7.5010 3 M (3).

with cyclodextrin would induce a large modication in its solvation shell, shifting the visible maximum of absorption of avylium (Dangles, Wigand, et al., 1992). The large loss of color following the addition of -CD to the cy3glc solution points to the preferential inclusion of the colorless forms (B, Cc, Ct) into the -CD cavity, a phenomenon leading to the shifting of the pigment hydration equilibrium towards the formation of more colorless forms (Brouillard et al., 2010; Yamada et al., 1980). A small increase of absorbance at max in the ultraviolet region was observed with the addition of -CD, which agrees with the formation of the anthocyanin colorless forms. These forms are more abundant at pH 2.0 comparing to pH 1.0 which could explain the higher anti-copigmentation effect at the prior pH. Oppositely, addition of -CD to a cy3glc solution at pH 2.0 caused only a small increase at max in the UVvisible spectrum. These results appear to indicate that the inclusion of cy3glc on the -CD's cavity at pH 2.0 is not favored (Fig. 2). -CD differs from -CD only in one glucose unit, having six instead of seven glucose units. This macrocycle has an inside diameter of 5.7 and an outside diameter of 13.7 while -CD presents an inside diameter of 7.8 and an outside diameter of 15.3 (Pegg & Shahidi, 2007). The dimensions of the cyclodextrin's cavity allow some selectivity for the complexation of guest molecules and the smaller cavity of -CD probably could not afford so many interactions between the colorless forms of cy3glc and the cavity walls, thus resulting in weaker binding. On the other hand, by increasing the size of the cyclodextrin, its hydrophobicity is enhanced leading to a signicant increase in its afnity with polyphenols (Tnnesen, Msson, & Loftsson, 2002).

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3.2. pH jumps One useful way to follow the kinetic process occurring in the network of avylium compounds is the use of relaxation techniques, in which the equilibrium of the system is shifted by means of an external inuence, applied very rapidly, forcing the metastable system thus formed to shift to the new state of equilibrium. The most common perturbations used are instantaneous variations of temperature, pressure, concentration or pH, the latter being more advantageous since it does not produce secondary physical effects (Brouillard, Delaporte, & Dubois, 1978). The application of these relaxation methods allows the determination of both equilibrium constants and the reaction rate constants (Brouillard & Dubois, 1977; Maanita et al., 2002). Among the structural transformations that an anthocyanin undergoes in an aqueous solution, the proton transfer equilibrium and the cycle-chain tautomerism are quite instantaneous. By contrast, the hydration reaction and the chalcone isomerization are relatively slow processes occurring on very distinct time scales. For instance, hydration is a process whose equilibrium state is achieved within about 10 s at pH 34 and about a few minutes at a pH value closer to neutrality. The chalcone isomerization is much slower and pH independent and its equilibrium state is fully established within about 2 h. The kinetics of both latter phenomena and the way they are inuenced by the presence of -CD can thus be easily studied using a standard UVvisible spectrophotometer (Dangles, Stoeckel, et al., 1992). In this work, the chemical reactions were studied by performing direct pH jumps from acidic to basic, in thermally equilibrated solutions. As previously reported, in acidic media the following equations account for the thermodynamic and kinetic processes (Brouillard & Delaporte, 1977; Pina, 1998): AH A H AH B H
Kt Kh Ka

K a proton transfer K h hydration

1 2 3 4
Fig. 3. A, Absorption spectra of equilibrated solutions of cy3glc (6.0 10 5 M) at different pH values. Inset: Determination of pKa through the tting of the absorption data taken at 511 nm by means of Eq. (6); B, absorption spectra of equilibrated solutions of the inclusion complex cy3glc:-CD (6.0 10 5 M:3.0 10 3 M) at different pH values. Inset: Determination of pKa through the tting of the absorption data taken at 511 nm by means of Eq. (6).

B Cc K t tautomerization Cc Ct
Ki

K i isomerization :

These can be simplied in one single acidbase equilibrium with constant Ka (Eqs. (5) and (6)) if the A, B, Cc and Ct species are represented together as one generic conjugate base CB (given by the sum of the species A, B, Cc and Ct), which is in equilibrium with the acidic avylium cation AH +: K a Ka Kh KhKt KhKtKi Ka

5 6

CB H ; AH

CB A B Cc Ct :

3.2.1. Thermal equilibrium The constant Ka can be obtained by spectrophotometric measurement (Pina, 1998; Pina, Benedito, Melo, Parola, & Bernardo, 1996). The absorption spectra of the thermally equilibrated solutions of cy3glc and of the inclusion complex cy3glc-CD are displayed in Fig. 3. This gure shows the pH dependence of the absorption spectra of the equilibrated solutions together with the representation of the tting to Eq. (6). The main feature that emerges from these spectra is the progressive decrease, with increasing pH, of the absorption band centered at 511 nm, which can be attributed to the avylium cation (Pina et al., 1996). The data represented in this gure and the tting of Eq. (6) allow us to calculate pKa = 2.8 for cy3glc and pKa = 2.1 for the inclusion complex cy3glc-CD. The difference

observed on the pKa calculated for cy3glc and for cy3glc-CD probably indicates that in the presence of -CD the colorless forms are more favorable. Three main processes with very different timescales can generally be observed for anthocyanins: (i) proton transfer is the fastest and occurs in the micro-second timescale; (ii) hydration (including tautomerization) takes place on a timescale from seconds to several minutes (if pH is not much higher than the pKa); (iii) isomerization occurs on a timescale of seconds or days, depending on the cistrans isomerization barrier. 3.2.2. Proton transfer The rst process that occurs upon a pH jump from pH b 1 to higher values is the transfer of a proton. This reaction is very fast and for this reason, conventional spectrophotometry including stopped-ow analysis is useless (Formosinho & Arnaut, 1993; Pina, 1998). The acidity constant Ka, can be calculated through an alternative method as follows: pH jump from pH b 1 to a value where pH pKa + 2. At this pH the avylium cation is totally converted into a quinoidal base and the hydration process is generally slow enough to be followed by a conventional spectrophotometer. Under these conditions, the

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Fig. 4. A1, Spectral variations versus time of cy3glc (6.0 10 5 M) upon a direct pH jump from pH 1.0 to pH 5.3; A2, variation of the absorbance at 530 nm versus time, pKa = 3.7 determined by means of Eq. (7); B1, spectral variations versus time of the inclusion complex cy3glc:-CD (6.0 10 5 M:3.0 10 3 M) upon a direct pH jump from pH 1.0 to pH 5.3; B2, variation of the absorbance at 530 nm versus time, pKa = 3.2 determined by means of Eq. (7).

Fig. 5. A1, Variation of the absorbance at 530 nm versus time of cy3glc (6.0 10 5 M) upon a direct pH jump from pH 1.0 to pH 5.3; A2, variation of the absorbance at 350 nm versus time of cy3glc (6.0 10 5 M) at the same conditions, showing two kinetic processes; B1, variation of the absorbance at 530 nm versus time of the inclusion complex cy3glc-CD (6.0 10 5 M:3.0 10 3 M) upon a direct pH jump from pH 1.0 to pH 5.3; B2, variation of the absorbance at 350 nm versus time of the inclusion complex cy3glc-CD (6.0 10 5 M:3.0 10 3 M) at the same conditions, showing two kinetic processes.

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absorbance taken immediately after the pH jump, A0, corresponds to the maximum concentration of this species. At the end of the kinetic process (nal equilibrium) the absorbance of the quinoidal base indicates its nal concentration A1. The ratio A1/A0 is equal to the molar fraction distribution of the quinoidal base at the equilibrium (when =1, there is practically no avylium cation) (Eq. (7)). For cy3glc, Ka/Ka =0.12, giving pKa =3.7. For the molecular complex cy3glc-CD, Ka/Ka =0.07, giving pKa =3.1 (Fig. 4). A K a ; C0 K a

A B Cc Ct C0

3.2.3. Hydration and isomerization reactions Direct pH jumps followed in a longer scale of time (using a common spectrophotometer) put in evidence the existence of a much slower process that was assigned to the CcCt isomerization. Three pH jumps to pH 4.5, 5.3 and 6.1 were performed and followed for several hours to study the isomerization process. An example of one of those experiments is displayed in Fig. 5 at pH 5.3, in which the decrease of absorbance at 530 nm together with the increase of absorbance at 350 nm could be an indication of the isomerization process, which led to the formation of colorless forms (Ct). Taking the absorbance values of the consecutive spectra at max = 530 nm and 350 nm and plotting them against time, one can obtain two graphs from Fig. 5(A and B). The tting of the biexponential curve at 350 nm allowed the determination of the observed rate constants (kobs) of the hydration (fastest process absorbance decrease) and the isomerization process (slowest process absorbance increase) (Pina, 1998). It is possible to observe that at pH 5.3, the isomerization occurs with a mean lifetime of 0.96 h for cy3glc and of 0.55 h for the inclusion complex cy3glc-CD (1/kobs). Inclusion of this anthocyanin on the -CD cavity clearly induced acceleration on the isomerization observed rate constant (approximately 1.7 times faster). According to previous works (Houbiers, Lima, Maanita, & Santos, 1998; Nave et al., 2010), for anthocyanins (oenin) the value of Kt is very small and considering only the fastest process (hydration reaction), it is possible to determine the pseudo equilibrium constant K ^a. Bearing this in mind, Eq. (5) could be simplied to give Eq. (8), which allows the calculation of Kh. Nevertheless, this only constitutes an approximation in the measurement of Kh. K ^a K a K h 8

Fig. 6. A, Fitting of the faster observed rate constants as a function of pH for the cy3glc solution (6.0105 M), pKh =2.9, determined by means of Eq. (8); B, tting of the faster observed rate constants as a function of pH for the inclusion complex cy3glc-CD (6.010 5 M:3.010 3 M), pKh =2.5, determined by means of Eq. (8).

As shown in Fig. 6, the tting of the faster observed rate constant (kobs) for cy3glc and for the inclusion complex cy3glc-CD, as a function of pH, allowed the determination of Kh. Hydration of cy3glc is speeded up by the presence of -CD and hydration appears about 1.5 times as fast inside the -CD cavity. From the data presented in Fig. 6, for cy3glc, Kh = 1.8 10 3 M 1 and for the inclusion complex cy3glc-CD, Kh = 2.7 10 3 M 1. The equilibrium and the kinetic constants determined by UVvis absorption, for cy3glc and for the inclusion complex cy3glc-CD are presented in Table 1. The isomerization observed rate constant and hydration constant (Kh) obtained for the molecular inclusion complex cy3glc-CD are in agreement to previous works. Dangles, Stoeckel, et al. (1992) and Dangles, Wigand, et al. (1992) reported the inclusion-induced acceleration for the hydration and isomerization reaction of a synthetic, non-natural anthocyanin, the 3,4-dimethoxy-7-hydroxyavylium. In this work, the evaluation of hydration reaction was made through one pH jump to pH 3.5 and the observed rate constant was about twice as fast inside the -CD cavity. Similarly, this macrocycle catalyzed the chalcone cis trans isomerization, and the isomerization observed rate constant has been estimated to be about ten times faster inside the -CD cavity (Dangles, Stoeckel, et al., 1992).

Similar studies were performed with -CD's smaller homologue, -CD. The interaction of this macrocycle with cy3glc did not alter signicantly the equilibrium and kinetic rate constants of this anthocyanin which clearly indicates that the inclusion of cy3glc on the -CD's cavity is not so favored and thus the impact of this macrocycle on the equilibrium and kinetic constants of cy3glc is not signicant (Table 1).

4. Conclusion The need for safe natural food colors has prompted researchers all over the world to look for new sources of food colors. In order to increase the knowledge about the impact of cyclodextrins on the equilibrium and kinetic constants of the network of chemical reactions taking place in cyanidin-3-O-glucoside a spectroscopic study was performed. Among several cyclodextrins, -CD deserves special attention because it is the most readily available cyclodextrin and is preferred compared to its homologues especially because of its wide use in the food industry and also for its strong interaction with anthocyanins. The spectral properties of cy3glc were changed by the molecular inclusion with -CD. The anti-copigmentation phenomenon observed shows dependence on the pH and on the -CD concentration. The comparison of the values of the equilibrium and kinetic equilibrium constants between cy3glc and the inclusion complex cy3glc-CD showed that the inclusion causes signicant modications in the

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Table 1 Thermodynamic and kinetic constants determined by UVvis absorption for the transformations of cy3glc (6.0 10 5 M) and of inclusion complexes cy3glc-CD (6.0 10 5 M:3.0 10 3 M) and cy3glc-CD (6.0 10 5 M:3.0 10 3 M). Thermodynamic and kinetic constants described for malvidin-3-O-glucoside in the literature (Mv3glc) (Brouillard et al., 1978; Houbiers et al., 1998; Maanita et al., 2002; Nave et al., 2010). Determined values Cy3glc Ka (M 1) Ka (M 1) Kh (M 1) kh (s 1) k h (M 1 s 1) 1.7 10 3 (2.8 0.1) 2.0 10 4 (3.7) 1.8 10 3 (2.9 0.1) 0.087 47.5 Cy3glc-CD 8.4 10 3 (2.1 0.1) 5.9 10 4 (3.2) 2.7 10 3 (2.5 0.1) 0.050 18.0 Cy3glc-CD 2.3 10 3 (2.6 0.1) 2.9 10 4 (3.5) 2.0 10 3 (2.7 0.1) 0.081 39.6 Described values Mv3glc 5.35 10 3 (2.3) 2.74 10 3 (2.54) 1.99 10 4 (3.7) 1.6 10 4 (3.8) 1.6 10 3 (2.8) 4.3 10 3 (2.4) 0.08 19

kinetic and thermodynamic properties. Inclusion of cy3glc in the -CD cavity induced acceleration on the isomerization observed rate constant (kobs) and on the hydration constant (Kh), which is in agreement with the anti-copigmentation phenomenon observed. The results obtained contribute for the elucidation of the impact of cyclodextrins on the color and on the equilibrium and kinetic constants of cyanidin3-O-glucoside, increasing the knowledge regarding the stabilization of anthocyanins. Despite the color diminish molecular inclusion with cyclodextrins may lead to the anthocyanin stabilization, which could promote its use as food colorant.

Acknowledgments The authors thank Professor Fernando Pina for the insights during this research. This research was supported by POPH/FSE and FCT (Fundao para a Cincia e Tecnologia) from Portugal by two PhD scholarships (SFRH/BD/65350/2009 and SFRH/BD/68736/2010). This work was also supported by one project grant (PTDC/QUI-QUI/117996/2010).

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