molecules Review The Chemical Reactivity of Anthocyanins and Its Consequences in Food Science and Nutrition Olivier Dangles * ID and Julie-Anne Fenger University of Avignon, INRA, UMR408, 84000 Avignon, France; [email protected] * Correspondence: [email protected]; Tel.: +33-490-144-446 Academic Editors: M. Monica Giusti and Gregory T. Sigurdson Received: 6 July 2018; Accepted: 31 July 2018; Published: 7 August 2018 Abstract: Owing to their specific pyrylium nucleus (C-ring), anthocyanins express a much richer chemical reactivity than the other flavonoid classes. For instance, anthocyanins are weak diacids, hard and soft electrophiles, nucleophiles, prone to developing π-stacking interactions, and bind hard metal ions. They also display the usual chemical properties of polyphenols, such as electron donation and affinity for proteins. In this review, these properties are revisited through a variety of examples and discussed in relation to their consequences in food and in nutrition with an emphasis on the transformations occurring upon storage or thermal treatment and on the catabolism of anthocyanins in humans, which is of critical importance for interpreting their effects on health. Keywords: anthocyanin; flavylium; chemistry; interactions 1. Introduction Anthocyanins are usually represented by their flavylium cation, which is actually the sole chemical species in fairly acidic aqueous solution (pH < 2). Under the pH conditions prevailing in plants, food and in the digestive tract (from pH = 2 to pH = 8), anthocyanins change to a mixture of colored and colorless forms in equilibrium through acid–base, water addition–elimination, and isomerization reactions [1,2]. Each chemical species displays specific characteristics (charge, electronic distribution, planarity, and shape) modulating its reactivity and interactions with plant or food components, such as the other phenolic compounds. This sophisticated chemistry must be understood to interpret the variety of colors expressed by anthocyanins and the color changes observed in time and to minimize the irreversible color loss signaling the chemical degradation of chromophores. The chemical reactivity of anthocyanins is also important to interpret their fate after ingestion and their effects on health, as anthocyanins may be consumed as a complex mixture of native forms, derivatives, and degradation products, which themselves can evolve in the digestive tract [3]. 2. The Basis of Anthocyanin Chemistry 2.1. Anthocyanins Are Weak Diacids Due to conjugation with the electron-withdrawing pyrylium ring, the phenolic OH groups of the flavylium ion at C40, C5, and C7 are fairly acidic [1,2]. In terms of structure–acidity relationships, it is clear that C7-OH is the most acidic group with a pKa1 of ca. 4, i.e., 6 pKa units below the phenol itself. The corresponding neutral quinonoid base (Figure1) can thus be considered to be the prevailing 0 tautomer. At higher pH levels, a second proton loss from C4 -OH (pKa2 ≈ 7 for common anthocyanins) yields the anionic base with maximized electron delocalization over the three rings. Along this deprotonation sequence, the wavelength of maximal visible absorption typically shifts by 20–30 nm Molecules 2018, 23, 1970; doi:10.3390/molecules23081970 www.mdpi.com/journal/molecules Molecules 2018, 23, 1970 2 of 23 Molecules 2018, 23, x 2 of 24 + − (AH20Molecules–30! nmA), 2018 (AH then, 23+ ,→ x by A), 50–60 then nmby 50 (A–60! nmA (A) (Figure → A−) 2(Figure), and the2), and corresponding the corresponding color turns color fromturns2 red offrom 24 to purple-bluered to purple [4-].blue [4]. 20–30 nm (AH+ → A), then by 50–60 nm (A → A−) (Figure 2), and the corresponding color turns from red to purple-blue [4]. FigureFigure 1.1. Flavylium ions are weak diacids. Figure 1. Flavylium ions are weak diacids. FigureFigure 2. 2. (I ()I Absorption) Absorption spectra spectra of of Cat Cat--Mv3Glc:Mv3Glc: pH jump from pHpH == 1.01.0 (100%(100% flavylium) flavylium) to to pH pH 3.00, 3.00, Figure 2. (I) Absorption spectra of Cat-Mv3Glc: pH jump from pH = 1.0 (100% flavylium) to pH 3.00, 3.59,3.59, 4.50, 4.50, 5.70, 5.70, 5.96, 5.96, 6.25, 6.25, and and 7.15, 7.15, respectively. respectively. Spectra recorded 1010 msms after after mixing mixing (negligible (negligible water water 3.59, 4.50, 5.70, 5.96, 6.25, and 7.15, respectively. Spectra recorded 10 ms after mixing (negligible water addition)addition). .( II(II) )Spectra Spectra of of the the componentscomponents obtainedobtained by mathematical decompositiondecomposition. .From From [4] [4] with with addition). (II) Spectra of the components obtained by mathematical decomposition. From [4] with permissionpermission of of the the American American Chemical Chemical SocietySociety.. permission of the American Chemical Society. Molecules 2018, 23, 1970x 3 of 2324 2.2. Anthocyanins Are Hard and Soft Electrophiles 2.2. Anthocyanins Are Hard and Soft Electrophiles By analogy with enones, the C2 and C4 atoms of the pyrylium ring can be regarded as hard and soft electrophilicBy analogy withcenters, enones, respectively. the C2 andHence, C4 they atoms respectively of the pyrylium react with ring hard can (O be-centered) regarded and as hard soft and(S- and soft C electrophilic-centered) nucleophiles, centers, respectively. the first mechanism Hence, they being respectively driven by reactlocal charges with hard and (O-centered) the second andone by soft interaction (S- and C-centered)s between nucleophiles,the frontier molecular the first mechanism orbitals (HOMO being drivenof nucleophile by locals charges and LUMO and the of secondelectrophile one bys). interactions between the frontier molecular orbitals (HOMO of nucleophiles and LUMO of electrophiles). 2.2.1. Nucleophilic Addition at C2 2.2.1. Nucleophilic Addition at C2 Water addition is the ubiquitous process taking place within aqueous anthocyanin solutions Water addition is the ubiquitous process taking place within aqueous anthocyanin solutions [1,2]. [1,2]. It leads to the colorless hemiketal (Figure 3) and can be characterized by the thermodynamic It leads to the colorless hemiketal (Figure3) and can be characterized by the thermodynamic hydration constant Kh, or as an acceptable approximation (chalcones making only a minor hydration constant Kh, or as an acceptable approximation (chalcones making only a minor contribution, contribution, typically less than 20%, of the total pool of colorless forms), by the apparent0 constant typically less than 20%, of the total pool of colorless forms), by the apparent constant K h connecting K′h connecting the flavylium ion and the colorless forms taken collectively. With common0 the flavylium ion and the colorless forms taken collectively. With common anthocyanins, pK h lies anthocyanins, pK′h lies in the range of 2–3, which means that hydration is thermodynamically more in the range of 2–3, which means that hydration is thermodynamically more favorable than proton favorable than0 proton transfer (pK′h < pKa1). Fortunately, it is also much slower, and its pH-dependent transfer (pK h < pKa1). Fortunately, it is also much slower, and its pH-dependent kinetics can be kinetics can be quantified by the apparent rate constant of hydration (kobs) (Equation (1), h = [H+], χAH quantified by the apparent rate constant of hydration (k ) (Equation (1), h = [H+], c = mole fraction = mole fraction of AH+ within the mixture of colored formsobs [2,5]: AH of AH+ within the mixture of colored forms [2,5]: k k h kobs k h= AH k+'h0 h = h + 0 k'h h (1) kobs khcAH k−hh 2 2k−hh.. (1) 11+KKaa11/ h + Kaa11KKa2a/2h/ h 0 kh isis the the absolute absolute rate constant of water addition, kk′−−hh isis the the apparent apparent rate rate constant constant of of water water elimination elimination 0 0 (from thethe mixturemixture of of hemiketal hemiketal and andcis cis-chalcone-chalcone in in fast fast equilibrium), equilibrium) and, andK hK≈′h ≈kh k/hk/k−′−h ((transtrans--chalconechalcone neglected). EquationEquation (1)(1) cancan be be easily easily understood understood by by keeping keeping in in mind mind that that the the flavylium flavylium ion ision the is solethe coloredsole colo formred form that isthat electrophilic is electrophilic enough enough to directly to directly react react with with water. water. Figure 3. Flavylium ions are hard electrophiles reacting at C2 with O-centeredO-centered nucleophiles, such as water (water addition followed by formation of minor concentrationsconcentrations ofof chalcones).chalcones). At a given pH, the initial visible absorbance (A0) (no colorless forms) and the final visible At a given pH, the initial visible absorbance (A0) (no colorless forms) and the final visible absorbance (Af) (hydration equilibrium established) can be easily related through Equation (2): absorbance (Af) (hydration equilibrium established) can be easily related through Equation (2): A 2 f 1 Ka1 / h Ka1Ka2 /2h Af 1 + Ka1/h + Ka1Ka2/h = .2 . (2 (2)) A 1 (K K' 0 ) / h K K 2/ h 0 A0 1 + (aK1 a1 + Khh)/h + Ka1aK1 a2/a2h Molecules 2018, 23, x 4 of 24 MoleculesThus, 2018 the, 23 magnitude, x of color loss can be expressed as (Equation (3)): 4 of 24 A A K' / h Thus, the magnitude of0 colorf loss can be expressed has (Equation (3)): Molecules 2018, 23, 1970 2 . 4 of(3 23) A0 1 (Ka1 K'h ) / h Ka1Ka2 / h A0 Af K'h / h (3) From typical values for the rate and thermodynamic constants 2of. common anthocyanins, Thus, the magnitude of colorA0 loss1 can ( beKa expressed1 K'h ) / ash (Equation Ka1Ka2 (3)):/ h simulations of the pH dependence of the apparent rate constant and percentage of color loss can be 0 constructedFrom typical(Figure values4). The forplotsA 0the− clearly Aratef andshow thermodynamic that theK reversible/h constants color loss of commondue to water anthocyanins, addition to = h (3) simulations of the pH dependence of the apparent rate0 constant and percentage2 of color loss can be the flavylium ion becomes slowerA0 at higher1 + (pHKa 1(less+ K hflavylium)/h + Ka1 Kina 2solution)/h , whereas its magnitude becomesconstructed larger (Figure because 4).