Intermolecular Copigmentation of Malvidin-3-O-glucoside with Caffeine in Water: The Effect of the Copigment on the pH-Dependent Reversible and Irreversible Processes.

Intermolecular copigmentation of malvidin-3-O-glucoside with caffeine was studied using a holistic procedure that includes the extension to basic pH values. In moderately basic solutions (7.5 < pH < 9.5) and independently of the copigment presence, there is a pH region where degradation of the quinoidal base and anionic quinoidal bases is faster than hydration and OH- nucleophilic addition, preventing the system from reaching the equilibrium. Intermolecular copigmentation with caffeine reduces significantly the degradation rate of quinoidal bases. In a more basic medium, the equilibrium is reached and degradation occurs from the anionic chalcones. In this case, the addition of caffeine also reduces the degradation rate in the interval 10 < pH < 11.5.

Introduction

Anthocyanins are ubiquitous molecules that confer color to the majority of angiosperms.[1] By presenting an attractive color, they favor plant reproduction, attracting pollinators and seed dispersers,[2,3] protect plants from several biotic and abiotic stresses,[4,5] and function as photoprotective agents by absorbing excess visible and UV light.[6,7] The consumption of anthocyanin-rich foods has been associated with numerous beneficial health effects due to their biological properties such as antiproliferative, anti-inflammatory, and antimicrobial properties.[8−12] However, anthocyanins have a severe drawback. They are not stable enough for most of the applications that imply relatively long storage periods.[13−16] Intermolecular copigmentation is used by plants to extend the pH domain of the red flavylium cation and increase the purple color of the quinoidal base.[14] The question is as follows: did intermolecular copigmentation also have some protecting effect on the anthocyanin’s degradation? From this perspective, the tools of the physical chemistry, in particular the studies concerning the pH-dependent thermodynamics and kinetics of anthocyanins, are indispensable to characterize both reversible and irreversible processes occurring in these compounds.[14] This knowledge is critical to design protection strategies to stabilize anthocyanins.

Reversible versus Irreversible Reactions in Anthocyanins

Reversible Equilibrium

Direct pH Jumps

The reversible sequence of chemical reactions that follow the addition of the base to equilibrated solutions of the flavylium cation of malvidin-3-O-glucoside at pH ≤ 1 (direct pH jumps) is illustrated in Scheme .[14,17,18] The first reaction consists of flavylium cation deprotonation to give the respective quinoidal bases with a mole fraction distribution dependent on the final pH. These deprotonation processes are very fast and occur during the mixing time of the stopped flow (first step).[19] Nevertheless, it is possible to calculate the respective acid–base constants (first column of Scheme ) by collecting the absorption spectra using a stopped flow 10 ms after the mixture of the base and representing the absorption versus pH plot at representative wavelengths, as reported in Figure a.

Scheme 1

Sequence of Reversible Reactions upon the Addition of the Base to Equilibrated Solutions of the Flavylium Cation of Malvidin-3-O-glucoside at pH ≤ 1 (Direct pH Jumps)

This scheme is followed by all anthocyanins. Adapted with permission from ref (19). Copyright 2022. American Chemical Society.

Figure 3

(a) Titration curves of malvidin-3-O-glucoside in the presence of caffeine 0.05 M collected by stopped flow 10 ms after a direct pH jump from the flavylium cation at pH = 1 at three representative wavelengths. Fitting was achieved for pKa = 3.5, pKA/A– = 6.7, and pKA–/A2– = 8.3; (b) mole fraction distribution of the malvidin-3-O-glucoside species, flavylium cation, and quinoidal bases in the presence of caffeine taken 10 ms after a direct pH jump; and (c) mole fraction distribution of malvidin-3-O-glucoside in the presence of caffeine at the pseudo-equilibrium after a series of pH jumps monitored by stopped flow and (d) in the absence of caffeine for comparison purposes. Figure c,d, Reprinted with permission from ref (21). Copyright 2019. American Chemical Society.

The quinoidal bases are kinetic products that disappear through the hydration reaction, followed by a faster tautomerization (second step). In the second step, AH and all quinoidal bases (their mole fraction distribution depending on the pH) are in fast equilibrium on one side and B and Cc and the respective anionic forms on the other side. One breakthrough fundamental for the understanding of the multi-equilibria was reported by Brouillard and Dubois,[17] who showed that quinoidal bases do not hydrate and the systems move toward the equilibrium due to the hydration of the flavylium cation. The kinetics of the second step is given by eq , where χAH+ corresponds to the mole fraction of AH in its equilibrium with A and A and χB corresponds to the mole fraction of B in its equilibrium with B, Cc, and Cc.

In eq , the reaction channel occurring in the basic medium due to the OH– nucleophilic addition was added.[14] When the formation of the anionic species was not considered, the experimental data is accounted for by eq .

Inspection of eq shows that the rate of the second step tends to zero with the increase in pH and the rate of OH– nucleophilic addition also tends to zero with the increase in pOH. This is a very relevant result crucial for the rationalization of anthocyanin degradation, see below.

The system reaches the equilibrium through the much slower cis–trans isomerization. In this case, all species except trans-chalcones are in equilibrium, defining the pseudo-equilibrium (identified by the superscript ^). During isomerization, the flavylium cation and all quinoidal bases, hemiketals, and cis-chalcones are in faster equilibrium, allowing for the deduction of the respective observed rate constant, eq .

In eq , ki and k–i are the direct and reverse rate constants for the formation of Ct from Cc, respectively, χCc is the mole fraction of Cc at the pseudo-equilibrium, and χCt is the mole fractions of Ct in its equilibrium with Ct, mutatis mutandis, for the isomerization of Cc/Ct.

The equilibrium of the first row of Scheme is accounted for by eqs –7

This multi-equilibrium can be simplified considering a single acid–base system, eq

According to eq , the complex multi-equilibrium given by eqs –7 is equivalent to the single acid–base equilibrium between the flavylium cation and its conjugated base CB, equal to the sum of A, B, Cc, and Ct, given in eq .

Extending to the neutral species, eqs –14are obtained

A similar simplification can be done to obtain eq with K″a given by eq and [CB–] = [A–] + [B–] + [Cc–] + [Ct–]

In general, in acid to slightly basic solutions, the complex multi-equilibrium of anthocyanins can be reduced to a diprotic acid with constants K′a and K″a.

Considering that the isomerization reaction is by far the slowest kinetic step of the system, a pseudo-equilibrium is attained when the observation time falls in the time range of seconds to a few minutes as in the stopped flow measurements.

In conclusion, in acid medium up to neutral-slightly basic, the equilibrium and the pseudo-equilibrium are equivalent to a diprotic acid and the mole fraction distribution of AH, CB, and CB or the equivalent species, at the pseudo-equilibrium substituting K′a and K″a by K^a and K^̂a, are given by eq .

Reverse pH Jumps

Reverse pH jumps consist in the addition of the acid, generally back to the flavylium cation at pH ≤ 1, to equilibrated solutions or pseudo-equilibrated solutions at higher pH values.[20−22] As shown above in eq , the rate of the hydration reaction increases with the increasing proton concentration. At sufficiently acidic solutions (namely, at pH ≤ 1), the hydration reaction becomes faster than tautomerization, the so-called change of regime.[14,20] The reverse pH jumps rates involving all species except trans-chalcones fall in the domain of seconds, requiring a stopped flow apparatus, while for the trans-chalcones, they occur in hours and can be monitored using a common spectrophotometer. In other words, during stopped flow measurements, the trans-chalcone is “frozen”. In Figure ,the trace of a reverse pH jump of malvidin-3-O-glucoside from pH = 5.5 monitored by stopped flow is shown. Three amplitudes can be observed (Figure a). The first corresponds to the remaining flavylium cation and quinoidal bases that are converted in the flavylium cation during the mixing time of the stopped flow (at pH = 5.5, mainly A). The amplitude of the faster kinetic process corresponds to the conversion of hemiketal in the flavylium cation, and the amplitude of the slower kinetics is the conversion of cis-chalcone in more flavylium cations via hemiketal.[14,20] Normalization of the amplitudes of Figure a gives the respective mole fractions. Fitting of the mole fractions of the pseudo-equilibrium species as a function of pH (see below, Figure c) can be achieved by means of eq .with a0 + b0 + c0 = a1 + b1 + c1 = 1.

Figure 1

(a) Trace of the reverse pH jump of malvidin-3-O-glucoside at pH = 5.5; (b) trace of malvidin-3-O-glucoside obtained using a common spectrophotometer; and (c) mole fraction distribution of the malvidin-3-O-glucoside species at the equilibrium. The yellow band corresponds to the region where equilibrium is not attained. Adapted with permission from ref (21). Copyright 219. American Chemical Society.

Once the coefficients a, b, and c are obtained, the equilibrium constants of the respective species are straightforwardly calculated from the relations of eqs and 23.[21]

For the neutral species

Additionally, for the anionic species

The isomerization rate constants are calculated by means of a reverse pH jump from the equilibrium carried out using a common spectrophotometer (see Figure b). The amplitude of the flavylium at the initial time corresponds to the sum of AH + A + B + Cc and the amplitude of the very slow process (i.e., controlled by the isomerization) permits the calculation of parameters d0 and d1 using eq and consequently Ki and KCt/Ct– using eq . Having obtained all equilibrium constants, the pH-dependent mole fraction distribution of the species at the equilibrium is straightforwardly obtained, as shown in Figure c.[14]

Irreversible Equilibrium

Recently, we reported on an experimental procedure to account for the calculation of the degradation rate in anthocyanins.[19] It is based on a sequence of pH jumps as follows: (i) direct pH jump to an extended pH region including the basic medium; (ii) maintaining the solution at the final pH of the direct pH jump; (iii) taking one aliquot of (ii) after 1 day and performing a reverse pH jump back to pH ≤ 1; and (iv) repeating (iii) for different delay times and calculating the disappearance of the flavylium cation as a function of time. The degradation fraction for each pH value, χdecomposition, is calculated using eq .

A typical result of the flavylium recovery after a certain delay time is shown in Figure a. It is possible to calculate the fraction of the flavylium cation that disappeared for each delay time and obtain the degradation rate for each pH value, as shown in Figure b. Finally, the degradation rate as a function of pH, given in Figure c, can be calculated. The comparison between the reversible processes (hydration and OH– nucleophilic addition) and the degradation rate allows for the detection of a pH range where the equilibrium is not attained. In fact, as shown in Figure d, in the pH region highlighted in red, degradation is faster than any other process toward the equilibrium. The rationalization of the anthocyanin degradation implies the determination of this pH interval.

Figure 2

(a). Recovery of the malvidin-3-O-glucoside flavylium cation after 1 day of delay time, at pH = 11.6, with the red open circle representing the initial flavylium cation absorption; (b) decomposition mole fraction as a function of time, at pH = 11.6, calculated using eq , behaves as a mono-exponential from which the degradation rate constant can be calculated; (c) degradation rate of malvidin-3-O-glucoside as a function of pH; and (d) comparison between the reversible reactions, hydration and OH– nucleophilic addition, and the degradation rate. Adapted with permission from ref (19). Copyright 2022. American Chemical Society.

The question is how to quantify the effect of intermolecular copigmentation[23−25] in anthocyanin degradation. In this work, the copigmentation between malvidin-3-O-glucoside and caffeine was used to illustrate this phenomenon. In particular, caffeine was selected to avoid cinnamic acids, the most common copigments, which have two forms at the working pH range, neutral and negatively charged, interacting differently with the several anthocyanin species, as shown in Scheme .[26] These copigments would introduce an additional level of complexity that would compromise the clarity of the exposition.[27]

Results and Discussion

Reversible Processes

In Figure , the titration of the quinoidal bases of malvidin-3-O-glucoside in the presence of caffeine 0.05 M was monitored by stopped flow 10 ms after a direct pH jump is reported. Fitting was achieved for pKa = 3.5, pKA/A– = 6.7, and pKA–/A2– = 8.3. This allows for calculating the mole fraction distribution of the quinoidal species (first column of Scheme ) in the presence of the copigment, as shown in Figure b. When the acid base constants in the absence of caffeine, pKa = 3.8, pKA/A– = 6.3, and pKA–/A2– = 8.4, are compared with those in its presence, there is an evident decrease of pKa in the presence of caffeine. This indicates that the quinoidal base is more stabilized than the flavylium cation by caffeine and its pH domain is also extended to higher pH values. This result contrasts with the interaction of malvidin-3-O-glucoside with lignosulfonate, where pKa increases to 4.4 due to the stabilization of the flavylium cation by the negatively charged lignosulfonate.[28]

The stabilization of the quinoidal base by the caffeine is evident in the comparison of Figure c with Figure d, where the mole fraction distribution of the species at the pseudo-equilibrium in the absence of caffeine is reported. It is worth noting that in the reverse pH jump experiments shown in Figure c,d, the parameters are as follows: a1 = 1 and b1 = c1 = 0. This was an unexpected result because it suggests that near the neutrality, the only observed species is the quinoidal base. The reason, discussed below in detail, is that in a pH region between neutral and slightly basic, the irreversible processes involving quinoidal bases are the rate-determining step of their relatively slow disappearance.

The pH-dependent rate constants of the second kinetic step (controlled by the hydration reaction in the acidic medium and OH– nucleophilic addition in the basic medium) of malvidin-3-O-glucoside in the presence of caffeine 0.05 M as a function of pH are shown in Figure a.

Figure 4

(a) Hydration reaction of malvidin-3-O-glucoside in the presence of caffeine 0.05 M. Fitting was achieved by means of eq for kh = 0.045 s–1, k–h = 150 M–1s–1, and the values of the protonation constants reported in Figure a; (b) relative energy level of B and A in the absence and presence of caffeine.

Considering that the formation of B– and Cc– does not take place since they fall in the pH range where the degradation reactions of A– and A2– are the rate-determining steps (see below), eq is simplified to eq , and fitting was achieved for kh = 0.045 s–1, k–h = 150 M–1s–1, kOH = 0.03 M–1s–1, and the values of the protonation constants reported in Figure a.

When the hydration and OH– nucleophilic addition rate constants are compared with those obtained in the absence of caffeine, kh = 0.12 s–1, k–h = 35 M–1s–1, and kOH = 0.09 M–1s–1, and it can be concluded that the stabilization by intermolecular copigmentation decreases the rate constant kh and increases k–h, an effect already observed in intermolecular copigmentation of acylated anthocyanins.[19] The OH– nucleophilic addition is also significantly reduced by the intermolecular copigmentation. Figure b shows the relative energy levels of the quinoidal base and hemiketal in the presence and absence of the copigment, calculated as reported in the literature.[14] The copigmentation stabilizes the quinoidal base and destabilizes the hemiketal. Clearly, it is a process to increase the color given by the quinoidal base, as can be visualized when comparing Figure c,d.

Irreversible Processes

The rate constants of the irreversible processes of malvidin-3-O-glucoside in the presence of caffeine 0.05 M were calculated and are represented in Figure a. Fitting was achieved by means of eq , where χ represents the mole fraction distribution of the quinoidal bases calculated in Figure b and kirr. the respective rate constants (degradation constants) with kirr.(A) = 2.5 × 10–6 s–1, kirr.(A−) = 5.5 × 10–6 s–1, and kirr.(A2−) = 6 × 10–7 s–1

Figure 5

(a) Rate constants of the irreversible processes of malvidin-3-O-glucoside in the presence of caffeine 0.05 M. (b) Representation of the hydration, OH– nucleophilic addition, and degradation rates of malvidin-3-O-glucoside in the absence (blue traced lines) and in the presence of caffeine 0.05 M (black full lines), eq (red band) and eq (higher pH values); the red band corresponds to the pH region where the equilibrium is not attained. (c) Contribution for the hydration reaction rate of the term dependent on kh (direct) and the term depending on k–h (reverse) in eq .

In the basic medium, the fitting was performed considering a similar process, given by eq , involving most probably Ct2– and Ct3– with pK = 11.3, kirr.(Ct2−) = 7 × 10–7 s–1, and kirr.(Ct3−) = 3 × 10–6 s–1, but there is a huge uncertainty in these values.

In Figure b, the hydration and OH– nucleophilic addition rates are represented together with the rates of the irreversible processes in the absence and presence of caffeine 0.05 M. Some conclusions can be taken from this figure. The hydration in the presence of the copigment is shifted to higher pH values. Representing the contributions for the hydration reaction rate of the term dependent on kh (direct) and the term depending on k–h (reverse) in eq , as shown in Figure c, it can be concluded that there is an inversion. The hydration rate in the presence of the copigment is shifted to higher pH values, in comparison with the situation in the absence of the copigment, only for pH > 6.5. At these pH values, the influence of the constant k–h is higher. The shift to higher pH values of the hydration curve in the presence of the copigment is explained by considering that the k–h increases from 35 to 150 M–1s–1 in the presence of the copigment.

The OH– nucleophilic addition is shifted to higher pH values, meaning that a higher OH– concentration is necessary for hydroxylation to occur toward the equilibrium. The degradation rates of the trans-chalcones are also significantly decreased in the presence of the copigment caffeine.

Copigmentation at pH = 8.4

In the pH range where degradation is the controlling step, the rate of the anionic quinoidal basis disappearance is slow enough to allow for the determination of the copigmentation constant, as shown in Figure . Caffeine addition to a malvidin-3-O-glucoside solution 8.4 × 10–6 M at pH = 8.4 gives rise to a red shift and a hyperchromic effect, reinforcing the blue hue. Representation of the absorbance at 640 nm as a function of the caffeine concentration can be fitted for a 1:1 copigmentation with an association constant of 1.5 × 103 M–1 (estimated 5% error). For the caffeine concentration of 0.05 M, 99% of the malvidin-3-O-glucoside is involved in the copigmentation process. Despite the fact that degradation is significantly reduced in these conditions, it is still not enough for applications requiring relatively long storage times.

Figure 6

(a) Spectral variations upon the addition of caffeine (pH = 8.4) to a malvidin-3-O-glucoside solution at the same pH and (b) variation of the absorbance at 640 nm vs caffeine concentration. Fitting was achieved for a 1:1 copigmentation with an association constant of 1.5 × 103 M–1 (estimated 5% error).

Experimental Section

Malvidin-3-O-glucoside was purchased from Extrasynthese, and caffeine was obtained from Alfa Aesar Company Ltd. The Teorell and Stenhagen universal buffer[29] was prepared 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 a 1 M NaOH solution in Millipore water until a volume of 1 L.

For the calculation of pK^a, pK′a, and the pKs of the ionized forms, direct pH jumps were carried out by mixing the flavylium stock solution (1 × 10–4 M at pH = 1), NaOH to neutralize the amount of HCl added, buffer at the desired pH (with and without caffeine), and water (or caffeine 0.1 M in water). Reverse pH jumps were performed by adding enough HCl (with or without caffeine) to equilibrated or pseudo-equilibrated solutions to reach pH = 1. pH was recorded on a Radiometer Copenhagen PHM240 pH/ion meter (Brønshøj, Denmark).

UV–vis spectra were recorded on a Varian-Cary 100 Bio or 5000 spectrophotometer (Palo Alto, CA, USA). The reverse pH jumps from the pseudo-equilibrium were observed on an SX20 (Applied Photophysics; Surrey, UK) spectrometer equipped with a PDA.1/UV photodiode array detector. A filter of 385 nm was used to prevent the photochemical reactions that could take place from Cc or B.

Conclusions

Intermolecular copigmentation of malvidin-3-O-glucoside with caffeine affects both reversible and irreversible processes. It destabilizes hemiketal and stabilizes the colored quinoidal base. In the presence of the copigment, there is still a pH range where degradation of quinoidal bases is the faster process. Intermolecular copigmentation with caffeine is able to decrease the malvidin-3-O-glucoside degradation rate by a few folds. While this effect is not enough for applications requiring a relatively long storage time, it constitutes a basic knowledge to find a possible strategy to stabilize the color at neutral to slightly basic pH values. In this pH region, the kinetics of both hydration and OH– nucleophilic addition are very slow. Protection of the quinoidal bases in this pH region could be a way to increase the lifetime of the blue color given by anthocyanins. Studies using cinnamic acids as copigments and degradation of acylated anthocyanins are in progress.