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.

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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+], χ = 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 khχAH 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 Af 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 2 of. 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 0 the− clearly A ratef 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). Theof the plots higher clearly stability show ofthat the the colorless reversible forms. color The loss typical due to time water-dependence addition to of thethe visible Fromflavylium spectrum typical ion becomes values during for slowerwater the addition rateat higher and is pH thermodynamic shown (less inflavylium Figure constants5 in [4] solution). of, commonwhereas its anthocyanins, magnitude simulationsbecomesNear largerneutrality of the because pH water dependence of additionthe higher is of stabilityso the slow apparent (fractionof the colorless rate of flavylium constant forms. andion The < percentage typical0.1%) that time the of-dependence colorcolored loss forms canof be(mixturesthe constructed visible of spectrum neutral (Figure and during4). anionic The water plots bases) clearlyaddition can show, inis shownprinciple that the in ,Figure reversiblepersist 5 for [4] colorhours.. loss However, due to water such additiona reasoning to theignores flavyliumNear the neutrality irreversible ion becomes water mechanisms addition slower at is of higher so color slow pH (fractionloss (less taking flavylium of flavyliumplace near in solution),ion neutrality < 0.1%) whereas that as the the anioniccolored its magnitude formsbase is becomesobviously(mixtures larger of much neutral because more and of sensitiveanionic the higher bases) to stability autoxidation can, in ofprinciple the (noncolorless, persist-enzymatic forms. for hours. The oxidation However, typical by time-dependence such O2 triggered a reasoning by of thetransitionignores visible the metal spectrum irreversible traces) during than mechanisms waterthe flavylium addition of color ion. is shownloss These taking inmechanisms Figure place5 near[4]. will neutrality be addressed as the anionicin Section base 2.3.1. is obviously much more sensitive to autoxidation (non-enzymatic oxidation by O2 triggered by transition metal traces) than the flavylium ion. These mechanisms will be addressed in Section 2.3.1.

Figure 4.4. Simulations of the pH dependence of the apparent rate constant (A) and relative magnitudemagnitude (BB) of color loss. Selected values for parameters: pKa1 = K4, pKa1 = 7,K pK′h = 2.5, Kkh0 = 0.1 s−1, k′−h ≈ kh/K′h−. 1 ( Figure) of color 4. Simulations loss. Selected of the valuespH dependence for parameters: of the apparent p a1 = rate 4, pconstanta1 = 7,(A p) andh =relative 2.5, hmagnitude= 0.1 s , 0 0 k − ≈ k /K . (Bh) of colorh loss.h Selected values for parameters: pKa1 = 4, pKa1 = 7, pK′h = 2.5, kh = 0.1 s−1, k′−h ≈ kh/K′h.

Wavelength (nm) Wavelength (nm)

Wavelength (nm) Wavelength (nm) Figure 5. (I) Spectral changes of Cat-Mv3Glc between 10 ms and 9 s following a pH jump from pH = Figure1Figure to pH 5. =5. (2.45 I()I) Spectral Spectral; half-life changes changes of flavylium of of Cat-Mv3Glc Cat =- Mv3Glc2.4 s. (II between)between pH jump 1010 from msms andand pH 9 =9 s 1s following tofollowing pH = 4.5 a a; pH halfpH jump -jumplife of from from quinonoid pHpH = = 1 to1 pHto pH = 2.45;= 2.45 half-life; half-life of of flavylium flavylium = = 2.4 2.4 s. s. ( II(II)) pH pH jump jump from from pHpH == 1 to pH = 4.5 4.5;; half half-life-life of of quinonoid quinonoid bases = 53.3 s. At pH = 6, the half-life of quinonoid bases ≈ 30 min. From reference [4] with permission of the American Chemical Society.

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Near neutrality water addition is so slow (fraction of flavylium ion < 0.1%) that the colored forms

(mixturesMolecules 2018 of, 23 neutral, x and anionic bases) can, in principle, persist for hours. However, such a reasoning5 of 24 ignores the irreversible mechanisms of color loss taking place near neutrality as the anionic base is obviouslybases = much 53.3 s. more At pH sensitive = 6, the half to autoxidation-life of quinonoid (non-enzymatic bases ≈ 30 min. oxidation From reference by O2 [triggered4] with permission by transition metalof traces) the American than theChemical flavylium Society ion.. These mechanisms will be addressed in Section 2.4.1.

2.2.2. Nucleophilic Addition at C4 BisulfiteBisulfite is an antimicrobial and anti anti-browning-browning agent that that is is frequently frequently used used in in the the food food industry. industry. As a S-centeredS-centered nucleophile, it reversibly reacts with the flavyliumflavylium ion at C4, thus yielding colorless adducts ( (FigureFigure 66))[ [6]6].. No such adducts have been identified identified so far by simply reacting anthocyanins with natural thiols such as cysteine and glutathione (GSH). Unlike bisulfite,bisulfite, which is actually the conjugated base of SO2 (pKa ≈≈ 1.8)1.8) and and can can coexist coexist with with the the fl flavyliumavylium ion ion under acidic conditions, thiolate anions (pKa == 8 8–9)–9) are are usually usually formed formed at at much much higher pH pH levels where the flavyliumflavylium ion is only present as traces.

Figure 6. Flavylium ions are soft electrophiles that react at C4 with S S-- and C-centeredC-centered nucleophiles, such as bisulfitebisulfite and 4-vinylphenols.4-vinylphenols.

A variety of C-centered nucleophiles are also known to add to the flavylium ion, and this A variety of C-centered nucleophiles are also known to add to the flavylium ion, and this chemistry underlies the color changes observed in red wine upon aging [7]. In this context, the most chemistry underlies the color changes observed in red wine upon aging [7]. In this context, the most important C-centered nucleophiles are electron-rich C–C double bonds, such as 4-vinylphenols (4- important C-centered nucleophiles are electron-rich C–C double bonds, such as 4-vinylphenols hydroxystyrenes), formed upon microbial decarboxylation of 4-hydroxycinnamic acids (Figure 6) (4-hydroxystyrenes), formed upon microbial decarboxylation of 4-hydroxycinnamic acids (Figure6) and the enol forms of various aldehydes and ketones such as pyruvic acid and ethanal (acetaldehyde) and the enol forms of various aldehydes and ketones such as pyruvic acid and ethanal [8,9]. In the process, new pigments, called pyranoanthocyanins, are formed, which are resistant to (acetaldehyde) [8,9]. In the process, new pigments, called pyranoanthocyanins, are formed, which are nucleophilic addition at C2 and C4 [10–12]. Their color (shifted to orange-red, compared to the resistant to nucleophilic addition at C2 and C4 [10–12]. Their color (shifted to orange-red, compared to corresponding flavylium ion) is thus more stable. Through their nucleophilic C6- and C8-atoms, the corresponding flavylium ion) is thus more stable. Through their nucleophilic C6- and C8-atoms, flavanols and proanthocyanidins can also add to the electrophilic C4 center of anthocyanins [13]. However, the flavene intermediate thus formed is not accumulated and evolves through two possible routes: (a) under strongly acidic conditions (pH = 2), protonation at C3 allows a second nucleophilic attack by a nearby phenolic OH group of the tannin to yield a colorless product (see Section 2.2. for a similar mechanism); or (b) under moderately acidic conditions (pH = 3–6), dehydration with

Molecules 2018, 23, 1970 6 of 23

flavanols and proanthocyanidins can also add to the electrophilic C4 center of anthocyanins [13]. However, the flavene intermediate thus formed is not accumulated and evolves through two possible routes: (a) under strongly acidic conditions (pH = 2), protonation at C3 allows a second nucleophilic attack by a nearby phenolic OH group of the tannin to yield a colorless product (see Section 2.3 for a similar mechanism); or (b) under moderately acidic conditions (pH = 3–6), dehydration with concomitant formation of an additional pyrane ring is favored and a new pigment bearing a xanthylium chromophore is formed. With its enediol structure, ascorbate (vitamin C) can also react with flavylium ions at C4 but the corresponding adducts have not been reported so far.

2.3. Anthocyanin Hemiketals Are Nucleophiles Basic organic chemistry teaches that electron-donating substituents of benzene rings accelerate aromatic electrophilic substitutions (SEAr) and orient the entering electrophiles to the ortho and para positions. In that perspective, the phloroglucinol (1,3,5-trihydroxybenzene) ring (A-ring) of anthocyanins must be especially favorable to SEAr as the three O-atoms combine their electronic effects to increase the reactivity of C6 and C8. However, the pyrylium ring (C-ring) of the flavylium ion (and, to a lesser degree, the enone moiety of chalcones) is strongly electron-withdrawing, so that only the hemiketal is expected to react by SEAr. Here, again, wine chemistry provides interesting examples of SEAr between anthocyanins and various carbocations derived from other wine components (Figure7)[ 7]. For instance, wine pigments in which anthocyanins and flavanols are linked though an ethylidene bridge between their C6- and/or C8-atoms are formed by double SEAr between A-rings and ethanol [14,15]. The likely intermediates in the reaction are the 6- or 8-vinyl-flavanol and the 6- or 8-vinyl-anthocyanin hemiketals, the protonation of which delivers a benzylic cation that is directly involved in the SEAr reaction. Of course, in addition to the cross reaction products, anthocyanin–ethylidene–anthocyanin and flavanol–ethylidene–flavanol adducts can also form oligomers and mixed oligomers [16]. Even, pyranoanthocyanins stemming from the nucleophilic attack of vinyl-phenols at C4 of anthocyanins can be produced. Flavanol carbocations formed by acidic cleavage of the inter-flavan linkage of proanthocyanidins also react with anthocyanin hemiketals by SEAr [17]. Interestingly, both direct and ethylidene-bridged flavanol–anthocyanin adducts are more purple than the native anthocyanin, but only the latter expresses a color that is stable, i.e., a flavylium nucleus that is less sensitive to water addition [4,18]. A possible explanation is that ethylidene-bridged flavanol–anthocyanin adducts are prone to non-covalent self-association by π-stacking, which provides a less aqueous environment for the flavylium nuclei. Water elimination from the anomeric C-atom of the ellagitannin vescalagin (abundant in oak barrels) also delivers a carbocation for direct coupling with wine anthocyanins [19] and subsequent modest protection against water addition [20]. Finally, the anthocyanin hemiketal can react with the flavylium ion itself, and this pathway provides a route for anthocyanin oligomerization, a poorly documented mechanism as the corresponding oligomers are probably difficult to evidence and quantify. However, an oenin trimer has been found in Port wine, and its structure has been fully elucidated by NMR [21]. The two linkages are of the C4–C8 type. As in the direct flavanol–anthocyanin coupling (see Section 2.2.2), flavene intermediates evolve by C–O coupling and only the lower unit remains colored. Similar oligomers also occur with 3-deoxyanthocyanidins, e.g., in red sorghum, but the detailed structures remain unknown [22]. Anthocyanin hemiketals can also react by Michael addition with o-quinones formed by two-electron oxidation of catechols, such as epicatechin [13] and caffeoyltartaric acid [23]. Molecules 2018, 23, 1970 7 of 23

Molecules 2018, 23, x 7 of 24

Figure 7. Anthocyanin hemiketals are nucleophiles reacting with carbocations (Ar = catechol ring). Figure 7. Anthocyanin hemiketals are nucleophiles reacting with carbocations (Ar = catechol ring). Flavanol carbocations formed by acidic cleavage of the inter-flavan linkage of 2.4. Anthocyaninsproanthocyanidins Are Electron-Donors also react with anthocyanin hemiketals by SEAr [17]. Interestingly, both direct and ethylidene-bridged flavanol–anthocyanin adducts are more purple than the native anthocyanin, but Manyonly polyphenols, the latter express especiallyes a color that those is stable, containing i.e., a flavylium electron-rich nucleus that catechol is less sensitive (1,2-dihydroxybenzene) to water or pyrogalloladdition (1,2,3-trihydroxybenzene) [4,18]. A possible explanationnuclei is that ethylidene are good-bridged electron- flavanol or– H-donors.anthocyanin adducts Electron are transfer is prone to non-covalent self-association by π-stacking, which provides a less aqueous environment for typically faster when the pH is raised, i.e., when the fraction of phenolate anion (a much better the flavylium nuclei. electron-donorWater than elimination the parent from phenol) the anomeric increases. C-atom Electron of the ellagitannin transfer vescalagin from phenols (abundant is involved in oak in their oxidationbarrels) mechanisms also delivers and a alsocarbocation underlies for di therect coupling most common with wine mechanism anthocyanins of[19] antioxidant and subsequent activity, i.e., the reductionmodest of protection reactive against oxygen water species addition (ROS) [20]. Finally, involved the anthocyanin in oxidative hemiketal stress can from react plants with the to humans. flavylium ion itself, and this pathway provides a route for anthocyanin oligomerization, a poorly Anthocyanins are known to be thermally unstable, especially under neutral conditions, and various documented mechanism as the corresponding oligomers are probably difficult to evidence and degradationquantify. products However, have an beenoenin identified.trimer has been Their found antioxidant in Port wine,activity and its structure has been has alsobeen establishedfully in various chemicalelucidated models. by NMR However,[21]. The two detailed linkages knowledge are of the C4 on–C8 the type. mechanisms As in the direct involved flavanol and– on the relative contributionsanthocyanin coupling of the (see different Section colored2.1.2.), flavene and intermediates colorless forms evolve is by still C–O missing. coupling and only the

2.4.1. Oxidation Anthocyanins are among the least thermally stable flavonoids. Anthocyanidins, the corresponding aglycones, are actually only stable under highly acidic conditions and are extensively degraded in less than one hour under physiological conditions (pH = 7.4, 37 ◦C) [24,25]. From the structure of the degradation products, it is clear that a combination of hydrolytic and autoxidative pathways operate, leading to cleavage of the C2–C10, C2–C3 and C3–C4 bonds (Figure8)[ 13,26,27]. A mechanism involving pre-formed hydrogen peroxide actually accounts for the formation of some cleavage products (Figure9). The critical step is the addition of H 2O2 (a hard nucleophile) at C2 of the flavylium ion, Molecules 2018, 23, x 8 of 24

lower unit remains colored. Similar oligomers also occur with 3-deoxyanthocyanidins, e.g., in red sorghum, but the detailed structures remain unknown [22]. Anthocyanin hemiketals can also react by Michael addition with o-quinones formed by two- electron oxidation of catechols, such as epicatechin [13] and caffeoyltartaric acid [23].

2.4. Anthocyanins Are Electron-Donors Many polyphenols, especially those containing electron-rich catechol (1,2-dihydroxybenzene) or pyrogallol (1,2,3-trihydroxybenzene) nuclei are good electron- or H-donors. Electron transfer is typically faster when the pH is raised, i.e., when the fraction of phenolate anion (a much better electron-donor than the parent phenol) increases. Electron transfer from phenols is involved in their oxidation mechanisms and also underlies the most common mechanism of antioxidant activity, i.e., the reduction of reactive oxygen species (ROS) involved in oxidative stress from plants to humans. Anthocyanins are known to be thermally unstable, especially under neutral conditions, and various degradation products have been identified. Their antioxidant activity has been also established in various chemical models. However, detailed knowledge on the mechanisms involved and on the relative contributions of the different colored and colorless forms is still missing.

2.4.1. Oxidation Anthocyanins are among the least thermally stable flavonoids. Anthocyanidins, the corresponding aglycones, are actually only stable under highly acidic conditions and are extensively degraded in less than one hour under physiological conditions (pH = 7.4, 37 °C) [24,25]. From the Moleculesstructure2018, 23 of, 1970 the degradation products, it is clear that a combination of hydrolytic and autoxidative 8 of 23 pathways operate, leading to cleavage of the C2–C1′, C2–C3 and C3–C4 bonds (Figure 8) [13,26,27]. A mechanism involving pre-formed hydrogen peroxide actually accounts for the formation of some 0 followedcleavage by Baeyer–Villiger products (Figure rearrangement, 9). The critical step which is the opens addition routes of H2 forO2 (a cleavage hard nucleophile) of the C2–C1 at C2and of the C2–C3 bondsflavylium [13,26]. ion, However, followed the by preliminaryBaeyer–Villiger formation rearrangement, of H2O which2 remains opens unclear routes for and cleavage must involveof the the directC2 autoxidation–C1′ and C2– ofC3 anthocyanins.bonds [13,26]. However, Thus, an the alternative preliminary mechanism formation beginning of H2O2 remains by electron unclear or and H-atom must involve the direct autoxidation of anthocyanins. Thus, an alternative mechanism beginning by transfer (mediated by unidentified transition metal traces) from the anionic or neutral base to O2 electron or H-atom transfer (mediated by unidentified transition metal traces) from the anionic or would deliver a highly delocalized radical that is susceptible to O2 addition at different centers neutral base to O2 would deliver a highly delocalized radical that is susceptible to O2 addition at (Figure 10). The cleavage of hydroperoxide intermediates thus formed could also yield the degradation different centers (Figure 10). The cleavage of hydroperoxide intermediates thus formed could also productsyield detected.the degradation products detected.

FigureFigure 8. 8.Pathways Pathways of anthocyanin degradation. degradation. Molecules 2018, 23, x 9 of 24

Figure 9. Possible mechanisms of anthocyanin degradation with pre-formed hydrogen peroxide. Figure 9. Possible mechanisms of anthocyanin degradation with pre-formed hydrogen peroxide.

Molecules 2018, 23, 1970 9 of 23 Molecules 2018, 23, x 10 of 24

Figure 10. Possible mechanisms of anthocyanin degradation without pre-formed hydrogen peroxide. Figure 10. Possible mechanisms of anthocyanin degradation without pre-formed hydrogen peroxide.

2.4.2. Antioxidant Activity 2.4.2. Antioxidant Activity Anthocyanins under their native forms can transfer electrons to ROS and could, therefore, provideAnthocyanins protection under to important their native oxidizable forms canbiomolecules, transfer electrons such as polyunsaturatedto ROS and could, fatty therefore, acids provide(PUFAs), protection proteins to, and important DNA. oxidizableThe relevance biomolecules, of such phenomena such as polyunsaturated is probably much fatty higher acids in (PUFAs), food proteins,preservation and DNA. than The in relevance nutrition of and such health, phenomena given is the probably current much knowledge higher in on food antho preservationcyanin thanbioavailability in nutrition and (see health, Section given 3). In the this current section, knowledge we simply on anthocyanin mention that bioavailability anthocyanins (see can Section indeed 3). Ineffectively this section, reduce we simply one-electron mention oxidants that anthocyanins such as the can stable indeed radical effectively DPPH reduce (2,2-diphenyl one-electron-1- oxidantspicrylhydrazyl). such as Structure the stable–activity radical relationships DPPH (2,2-diphenyl-1-picrylhydrazyl).show that hydroxylation at C3′ and C5′ Structure–activity increases the relationshipsH-donating show capacity, that hydroxylationthus suggesting at that C30 theand B C5-ring0 increases is primarily the H-donatinginvolved in capacity,electron donation thus suggesting [28]. thatComparing the B-ring oenin is primarily and the flavanol involved catechin in electron shows donation that the transfer [28]. Comparing of the first oenin(most andlabile) the H flavanol-atom catechinto DPPH shows is roughly that theas fast transfer for both of flavonoids the first (most but that labile) oenin H-atom reduces toat least DPPH twice isroughly as many asradicals fast for boththan flavonoids catechin (Table but that 1) [29] oenin. This reduces advant atage least must twice be rooted as many in the radicals extensive than oxidative catechin degradation (Table1)[ 29 ]. Thisundergone advantage by must oenin be rooted during in the the extensiveDPPH-scavenging oxidative process degradation with undergone the transient by oenin formation during of the DPPH-scavengingintermediates (possibly, process syringic with the acid) transient retaining formation a substantial of intermediates electron-donating (possibly, activity. syringic It is also acid) retainingremarkabl a substantiale that the wine electron-donating pigments combining activity. the It oenin is also and remarkable catechin units that retain the wine a high pigments but combiningcontrasting the DPPH oenin-scavenging and catechin activity units retain[29]: the a high direct but coupling contrasting between DPPH-scavenging the two flavonoid activity units [29 ]: results in a faster first H-atom transfer (higher k1) but markedly lowers the total number of radicals the direct coupling between the two flavonoid units results in a faster first H-atom transfer (higher reduced (ntot), whereas the coupling through an ethylidene bridge apparently leaves each unit free to k1) but markedly lowers the total number of radicals reduced (ntot), whereas the coupling through independently react with DPPH (k1 almost unchanged, approximate additivity in the ntot value), as an ethylidene bridge apparently leaves each unit free to independently react with DPPH (k almost observed with the equimolar oenin–catechin mixture (Table 1). 1 unchanged, approximate additivity in the n value), as observed with the equimolar oenin–catechin tot mixture (Table1).

Molecules 2018, 23, x 11 of 24

Table 1. Antioxidant activity of malvidin 3-O-β-D-glucoside (oenin) and related pigments: reduction of the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical (MeOH, 25 °C , 1 and 2) and inhibition of heme- induced peroxidation of linoleic acid (0.1 mM linoleic acid in acetate buffer + 2 mM Tween-20, 0.1 µM Molecules 2018, 23, 1970 10 of 23 metmyoglobin, pH = 4, 37 °C , 3). From reference [29].

1 −1 2 3 Table 1. AntioxidantAntioxidant activity of malvidin 3-O-β-D-glucosidentot (oenin) andk1 related/s pigments:IC50/μM reduction of ◦ 1 2 the DPPH (2,2-diphenyl-1-picrylhydrazyl)Oenin radical11.26 (MeOH, (±0.08) 25 C, 910and (±70)) and inhibition0.68 of heme- induced peroxidation of linoleic acid (0.1 mM linoleic acid in acetate buffer + 2 mM Tween-20, 0.1 µM metmyoglobin, pH = 4,Catechin 37 ◦C, 3). From reference [4.8629]. (±0.03) 1200 (±110) 0.27

Oenin + Catechin (1:1) 14.041 (±0.10) 1160− (±1 2330) nd 3 Antioxidant ntot k1/s IC50/µM (R)-CatechinOenin-8-CHMe-8-Oenin 11.26 (14.56±0.08) (±0.03) 9101000 (± (±70)320) 0.680.15 Catechin 4.86 (±0.03) 1200 (±110) 0.27 (OeninS)-Catechin + Catechin-8-CHMe (1:1)-8-Oenin 14.04 (14.61±0.10) (±0.18) 1160600 (± (±330)120) 0.41 nd (R)-Catechin-8-CHMe-8-Oenin 14.56 (±0.03) 1000 (±320) 0.15 (S)-Catechin-8-CHMe-8-OeninCatechin-4,8-Oenin 14.61 (±7.160.18) (±0.08) 6005120 (± (±120)1050) 0.410.60 Catechin-4,8-Oenin 7.16 (±0.08) 5120 (±1050) 0.60 1 Antioxidant stoichiometry (number of DPPH radicals reduced per antioxidant molecule). 2 Rate 1 2 constantAntioxidant for stoichiometrythe transfer of (number the first of DPPHH-atom radicals from reduced antioxidant per antioxidant to DPPH molecule).. 3 AntioxidantRate constantconcentration for the transfer of the first H-atom from antioxidant to DPPH. 3 Antioxidant concentration for a doubling of the period of timefor a required doubling for the of accumulation the period of of a time fixed required concentration for of the polyunsaturated accumulation fatty of acid a fixed (PUFA) concentration hydroperoxides of (conjugatedpolyunsaturated dienes). fatty acid (PUFA) hydroperoxides (conjugated dienes).

Oenin, catechincatechin,, and and wine wine pigments pigments were were also compared for their ability to inhibit the peroxidation ofof linoleic linoleic acid acid induced induced by by dietary dietary heme heme iron iron in acidic in acidic micelle micelle solutions, solutions, a chemical a chemical model ofmodel postprandial of postprandial oxidative oxidative stress stress in the in stomach the stomach [29]. [29] As hydrophilic. As hydrophilic antioxidants, antioxidants, polyphenols polyphenols are IV knownare known to act to act at theat the initiation initiation stage stage by by reducing reducing the the hypervalent hypervalent iron iron species species (Fe (FeIV)) involvedinvolved inin the generation of propagating lipid peroxyl radicals (Figure 1111))[ [30]30] which,which, on the other hand hand,, are directly directly reduced by the typical chain-breakingchain-breaking amphiphilic antioxidantantioxidant α--tocopheroltocopherol (vitamin E). The highly hydrophilic oenin was found to be less potent than catechin in the inhibition, inhibition, but coupling both flavonoidsflavonoids via an ethylidene bridge improves theirtheir efficiencyefficiency (Table(Table1 ).1).

FigureFigure 11. 11. PossiblePossible mechanisms mechanisms for forthe theantioxidant antioxidant activity activity of anthocyanins of anthocyanins in food in and food in andthe gastro in the- gastro-intestinalintestinal tract. tract.

Acylation by electron-rich hydroxycinnamic acids, such as sinapic and ferulic acids, potentiates Acylation by electron-rich hydroxycinnamic acids, such as sinapic and ferulic acids, potentiates the the capacity of anthocyanins to inhibit the diazo-initiated autoxidation of styrene in acetonitrile. In capacity of anthocyanins to inhibit the diazo-initiated autoxidation of styrene in acetonitrile. particular, a higher rate constant and stoichiometric factor of radical scavenging were obtained for In particular, a higher rate constant and stoichiometric factor of radical scavenging were obtained acylated (vs. non-acylated) anthocyanins [31]. Curiously, this trend could not be confirmed for the for acylated (vs. non-acylated) anthocyanins [31]. Curiously, this trend could not be confirmed peroxidation of linoleic acid in micelles, as if the intrinsic differences in electron-donating activity for the peroxidation of linoleic acid in micelles, as if the intrinsic differences in electron-donating activity were counterbalanced by differences in the partition of anthocyanins between micelles and the aqueous phase. Molecules 2018, 23, x 12 of 24 were counterbalanced by differences in the partition of anthocyanins between micelles and the aqueous phase.

Molecules 2018, 23, 1970 11 of 23 2.5. Anthocyanin Complexes Phenolic nuclei have an intrinsic ability to develop molecular (non-covalent) interactions as they 2.5. Anthocyanin Complexes combine flat polarizable apolar surfaces (the aromatic nuclei) for strong dispersion interactions and polarPhenolic OH groups nuclei that have are susceptible an intrinsic to ability acting to as develop H-bond molecular donors and (non-covalent) acceptors. interactions as they combine flat polarizable apolar surfaces (the aromatic nuclei) for strong dispersion interactions and 2.5.1.polar Self OH- groupsAssociation that areand susceptible Co-Pigmentation to acting as H-bond donors and acceptors. One of the most remarkable properties of the anthocyanin chromophores is their ability to 2.5.1. Self-Association and Co-Pigmentation develop π-stacking interactions [32–34], mostly driven by dispersion interactions and the concomitantOne of the fav mostorable remarkable release of propertieswater molecules of the anthocyanin from the solvation chromophores shells of is theirthe interacting ability to develop nuclei, knownπ-stacking as interactions the hydrophobic [32–34], mostlyeffect. driven Owing by to dispersion their planar interactions structures and the and concomitant extended favorable electron delocalizationrelease of water over molecules the three from rings, the solvationthe colored shells forms of theare interactingmuch more nuclei, prone knownto π-stacking as the hydrophobic interactions thaneffect. the Owing colorless to theirforms, planar for which structures such andinteractions, extended although electron not delocalization necessarily overabsent, the are three typically rings, neglected.the colored Examples forms are of much π-stacking more prone interactions to π-stacking with interactionsanthocyanins than are theself colorless-association forms, and for binding which betweensuch interactions, anthocyanins although and other not necessarily phenols, a absent,phenomenon are typically called co neglected.-pigmentation. Examples The affinity of π-stacking of co- pigmentsinteractions for with a given anthocyanins anthocyanin are self-association(as measured by and the binding corresponding between thermodynamic anthocyanins and binding other constant)phenols, a decays phenomenon along the called series: co-pigmentation. planar flavonoids The affinity (flavones, of co-pigments flavonols) > fornon a- givenplanar anthocyanin flavonoids (catech(as measuredins), hydroxycinnamic by the corresponding acids > hydroxybenzoic thermodynamic acids binding [32]. As constant) for self- decaysassociation, along it is the stronger series: forplanar the neutral flavonoids base (flavones, than for the flavonols) flavylium > ion non-planar and the anionic flavonoids base, (catechins), as the latter hydroxycinnamic stacks are destabilized acids >by hydroxybenzoic charge repulsion. acids [32]. As for self-association, it is stronger for the neutral base than for the flavyliumThe spectral ion and the consequences anionic base, of as co the-pigmentation latter stacks are are destabilized summarized by in charge Figure repulsion. 12 with malvin (malvidinThe spectral 3,5-di- consequencesO-β-glucoside) of co-pigmentation and a highly water are summarized-soluble rutin in Figure (quercetin 12 with 3 malvin-O-β-rutinoside) (malvidin derivative3,5-di-O-β -glucoside)[35]. In strongly and a highly acidic water-soluble solutions (negligible rutin (quercetin water-to 3--flavyliumO-β-rutinoside) addition), derivative π-stacking [35]. interactionsIn strongly acidic between solutions the two (negligible partners water-to-flavyliumpromote bathochromism addition), as a πconsequence-stacking interactions of co-pigment between-to- pigmentthe two partners charge trapromotensfer. bathochromismChanges in color as intensitya consequence simply of reflectco-pigment-to-pigment differences between charge the transfer. molar absorptionChanges in coefficients color intensity of free simply and reflect bound differences pigments. Underbetween the the mildly molar acidic absorption conditions coefficients typically of encountefree andred bound in natural pigments. media, Under pigment the mildly–co-pigment acidic conditions interactions typically also promote encountered hyperchromism, in natural whichmedia, can pigment–co-pigment be understood as a interactions shift in the alsonow promote established hyperchromism, flavylium–hemiketal which can equilibrium be understood toward as thea shift colored in the form now, establishedwhich is selectively flavylium–hemiketal stabilized equilibriumby its association toward with the colored the co- form,pigment. which This is combinationselectively stabilized of bathochromic by its association and hyperchromic with the co-pigment. shifts makes This co combination-pigmentation of bathochromic one of the most and importanthyperchromic mechanisms shifts makes for color co-pigmentation variation and one stabilization of the most in important plants. It mechanisms can also be noted for color that variation heating usuallyand stabilization attenuates in the plants. hyperchromic It can also shift be noted (Figure that 12) heating as a consequence usually attenuates of the exothermic the hyperchromic character shift of co(Figure-pigmentation 12) as a consequence (∆H0 < 0). of the exothermic character of co-pigmentation (∆H0 < 0).

Figure 12. 12.Co-pigmentation Co-pigmentation of malvin of (malvidin malvin 3,5-diglucoside, (malvidin 3,5 50-diglucoside,µM) by rutin bis(hydrogensuccinate) 50 µM) by rutin bis(hydrogensuccinate)(mixture of 3 regioisomers, (mixture 200 equiv.).of 3 regioisomers, (A) pH = 3.5,200 malvinequiv.). +(A co-pigment) pH = 3.5, malvin at T =15.5 + co- (1),pigment 25.0 (2), at T35.0 = 15.5 (3), (1), 44.2 25.0 (4) (2),◦C, 35.0 malvin (3), 44.2 alone (4) °C at,T mal = 25.3vin alone◦C (5). at T ( B= )25.3 pH °C = (5). 0.9, (B T) =pH 25.0 = 0.9,◦C, T malvin= 25.0 °C alone, malvin (1), alonemalvin (1), + co-pigmentmalvin + co- (2).pigment Adapted (2). fromAdapted reference from reference [35]. [35].

The possibility of developing π-stacking interactions increases with the acylation of anthocyanins on their glycosyl moieties by hydroxycinnamic acid (HCA) residues. Indeed, depending on the location and number of HCA residues, different spatial arrangements can be observed (Figure 13)[34]: Molecules 2018, 23, x 13 of 24

The possibility of developing π-stacking interactions increases with the acylation of MoleculesanthocyaninsMolecules2018 2018, 23, 23,on 1970, x their glycosyl moieties by hydroxycinnamic acid (HCA) residues. 13 Indeed, of12 24 of 23 depending on the location and number of HCA residues, different spatial arrangements can be The possibility of developing π-stacking interactions increases with the acylation of observed (Figure 13) [34]: • anthocyaninsIntramolecular on co-pigmentation: their glycosyl moietiesπ-stacking by interactions hydroxycinnam promoteic acid a conformational (HCA) residues. folding Indeed, of the  dependingpigmentIntramolecular onbringing the co location one-pigmentation: or and more number HCA π-stacking residue(s) of HCA interactions residues, into contact different promote with the spatial a chromophore; conformational arrangements folding can be of • observedEnhancedthe pigment (Figure self-association: bringing 13) [34]: one or the more HCA HCA residues residue(s) can stabilize into contact the chiralwith the stacking chromophore; of chromophores   evidencedEnhancedIntramolecular self by circular-association: co-pigmentation: dichroism. the HCA π-stacking residues interactions can stabilize promote the chiral a conformational stacking of chromophores folding of evidencedthe pigment by circularbringing dichroism. one or more HCA residue(s) into contact with the chromophore;  Enhanced self-association: the HCA residues can stabilize the chiral stacking of chromophores evidenced by circular dichroism.

Figure 13. AcylatedAcylated anthocyanins: anthocyanins: discrimination discrimination of intramolecular of intramolecular co-pigmentation co-pigmentation (type (type 1) and 1) self and - association (types 2 and 3) by circular dichroism (pink or blue CD spectra depending on the chirality self-associationFigure 13. Acylated (types anthocyanins: 2 and 3) by discrimination circular dichroism of intramolecular (pink or blue co-pigmentation CD spectra (type depending 1) and self on- the of the stacks). From [34] with permission of the Royal Society of Chemistry. chiralityassociation of the (types stacks). 2 and From 3) by [34 circular] with dichroism permission (pink of the or Royalblue CD Society spectra of Chemistry depending. on the chirality of the stacks). From [34] with permission of the Royal Society of Chemistry. In such assemblies, the flavylium nucleus has restricted access to the water solvent. In such assemblies, the flavylium nucleus has restricted access to the water solvent. Consequently, Consequently,In such the assemblies, thermodynamics the flavylium of water nucleus addition has are restricted less favorable access0 (increased to the water pK ′h solvent.), and the the thermodynamics of water addition are less favorable (increased pK h), and the percentage of colored percentageConsequently, of colored the thermodynamics forms at equilibrium of water increases addition [5,36 are– 38]less. For favorable instance, (increased at pH = p3,K ca.′h), 90%and theof the forms at equilibrium increases [5,36–38]. For instance, at pH = 3, ca. 90% of the triacylated Morning triacylatedpercentage Morning of colored glory forms pigment at equilibrium is still inincreases colored [5,36 form–38] (mostly. For instance, flavylium) at pH vs. = 3, 15% ca. 90% for of its the non - glory pigment is still in colored form (mostly flavylium) vs. 15% for its non-acylated counterpart acylatedtriacylated counterpart Morning glory (Figure pigment 14). Its is still vulnerability in colored to form water (mostly addition flavylium) prevents vs. 15% the for non its-acylated non- (Figure 14). Its vulnerability to water addition prevents the non-acylated pigment from accumulating pigmentacylated from counterpart accumulating (Figure the 14). neutral Its vulnerability quinonoid base to water at higher addition pH levels, prevents and the the noncorres-acylatedponding the neutral quinonoid base at higher pH levels, and the corresponding solutions are almost colorless. solutionspigment are from almost accumulating colorless. the In neutral contrast, quinonoid 30% of thebase triacylated at higher pH pigment levels, isand present the corres as theponding colored In contrast, 30% of the triacylated pigment is present as the colored neutral base at pH = 5. Moreover, neutralsolutions base are at pHalmost = 5. colorless. Moreover, In thecontrast, π-stacking 30% of interactions the triacylated developed pigment by is thepresent triacylated as the coloredflavylium the π-stacking interactions developed by the triacylated flavylium ion induce a 20 nm bathochromic ionneutral induce base a 20 at nm pH bathochromic = 5. Moreover, shiftthe π of-stacking its λmax interactionscompared to developed its non-acylated by the triacylated counterpart. flavylium shift of its λ compared to its non-acylated counterpart. ionAnthocyanin inducemax a 20 nms with bathochromic an o-dihydroxy shift of its substitution λmax compared on to their its non B-ring-acylated (cyanidin, counterpart. delphinidin , and Anthocyanins with an o-dihydroxy substitution on their B-ring (cyanidin, delphinidin, petunidinAnthocyanin derivatives)s with also an bind o-dihydroxy hard metal substitution ions, such on as their Al3+ Band-ring Fe (cyanidin,3+, in mildly delphinidin acidic to , neutraland 3+ 3+ and petunidin derivatives) also bind hard metal ions, such as Al3+ and3+ Fe , in mildly acidic to neutral solution.petunidin As derivatives)the anthocyanin also bind binds hard as the metal quinonoid ions, such base as withAl and additional Fe , in protonmildly acidicloss from to neutral C3′-OH, solution. As the anthocyanin binds as the quinonoid base with additional proton loss from C30-OH, bathochromismsolution. As the is a observednthocyanin with binds additional as the quinonoid ligand-to base-metal with charge additional transfer proton with loss Fe3+ from (Figure C3′- 15).OH, bathochromismbathochromism is is observed observed with with additionaladditional ligand-to-metalligand-to-metal charge charge transfer transfer with with Fe Fe3+ 3+(Figure(Figure 15). 15 ).

Figure 14. Cont.

Molecules 2018, 23, 1970 13 of 23

Molecules 2018, 23, x 14 of 24

FigureFigure 14.Figure Triacylated 14. 14.Triacylated Triacylated (B) vs. (B )(nonB vs.) vs.- non-acylatedacylated non-acylated (A) Morning ((AA)) MorningMorning glory glory (Pharbitis ((PharbitisPharbitis nil )nil anthocyanins: nil) )anthocyanins: anthocyanins: equilibrium equilibrium equilibrium distributiondistributiondistribution of anthocyanin of of anthocyanin anthocyanin species speciesspecies in aqueous in in aqueous aqueous solution. solution. solution. Red Red solid Red solid line: solid line: flavylium line:flavylium flavylium ion, ion, blue blue ion,solid solid blue line: line: solid neutral base, dotted green line: total colorless forms. Parameters for plots are pK’h = 2.30, pKa1 = 4.21 (A); neutralline: base, neutral dotted base, green dotted line: greentotal colorless line: total forms. colorless Parameters forms. for Parameters plots are p forK’h plots= 2.30, are pK pa1K =’ h4.21= 2.30, pK’h = 4.01, pKa1 = 4.32 (B). From [36,37]. (A); ppKKa1’h = 4 4.21.01, (pAK);a1 p=K 4.32’h = ( 4.01,B). From pKa1 [36,37]= 4.32. (B). From [36,37].

0 0 FigureFigure 15. ( 15.A) 3′(A,4′)- 3Dihydroxy,4 -Dihydroxy-7--7-O-β-OD--glucopyranosyloxyβ-D-glucopyranosyloxyflavyliumflavylium (50 µM) (50 µinM) a pH in a 4 pH buffer 4 buffer (0.1 M (0.1 M acetate),acetate), red spectrum: red spectrum: before before hydration, hydration, blue spectrum: blue spectrum: 10 min 10 after min addition after addition of Al3+ (4 of equiv.); Al3+ (4 ( equiv.);B) equilibrium(B) equilibrium distribution distribution of species of in species aqueous in aqueous solution. solution. Red solid Red line: solid flavylium line: flavylium ion, blue ion,dotted blue line: dotted neutralline: base, neutral dotted base, green dotted line: green total line: colorless total colorlessforms, blue forms, solid blue line: solid Al3+ line:complex. Al3+ Parameterscomplex. Parameters for plots for −4 −4 are pplotsK’h = are3.42, pK p’Kh a1= = 3.42, 4.72, p Ka1M == 2 4.72, × 10K.M From= 2 × [3910]. . From [39].

At least in mildly acidic solution, metal binding is restricted to the colored forms and thus At least in mildly acidic solution, metal binding is restricted to the colored forms and thus efficiently competes with the hydration equilibrium, thereby preventing the formation of the efficiently competes with the hydration equilibrium, thereby preventing the formation of the colorless colorless forms. In the most sophisticated assemblies, metal binding and π-stacking interactions forms. In the most sophisticated assemblies, metal binding and π-stacking interactions combine, combine, thus providing the most common mechanism towards the formation of stable blue colors thus providing the most common mechanism towards the formation of stable blue colors [34,40,41]. [34,40,41]. In the so-called metalloanthocyanins, a fixed metal–pigment–co-pigment stoichiometry of In the so-called metalloanthocyanins, a fixed metal–pigment–co-pigment stoichiometry of 2:6:6 is 2:6:6 is observed: three anthocyanins bind to each metal ion and two equivalent complexes assemble observed: three anthocyanins bind to each metal ion and two equivalent complexes assemble by by left-handed π-stacking interactions between the chromophores. Then, three pairs of flavone or left-handed π-stacking interactions between the chromophores. Then, three pairs of flavone or flavonol co-pigments in left-handed π-stacking intercalate between the pairs of stacked anthocyanins. flavonol co-pigments in left-handed π-stacking intercalate between the pairs of stacked anthocyanins. In this intercalation, right-handed pigment–copigment π-stacking occurs. Large-scale aggregation of In this intercalation, right-handed pigment–copigment π-stacking occurs. Large-scale aggregation of acylated anthocyanins can also result in the formation of highly colored assemblies within the acylated anthocyanins can also result in the formation of highly colored assemblies within the vacuole vacuole (the so-called anthocyanin vacuolar inclusions) [42], the organelle where anthocyanins are (the so-called anthocyanin vacuolar inclusions) [42], the organelle where anthocyanins are stored in stored in plant cells. plant cells.

Molecules 2018, 23, 1970 14 of 23

2.5.2. Binding to Biopolymers Despite the potential significance of such associations in food chemistry and nutrition, the ability of anthocyanins to bind proteins and polysaccharides is still poorly documented at the molecular level. This paragraph focuses on anthocyanins (glycosides), although anthocyanidins are also commonly investigated. Indeed, aglycones are chemically unstable in mildly acidic and neutral conditions and may be substantially degraded over the duration of analysis. Saturation transfer difference (STD)-NMR was used to probe the binding of cyanidin and delphinidin 3-glucosides to pectin from citrus fruits (MM = 111 kDa) [43]. Indeed, magnetization transfer (requiring proton pairs distant by less than 0.5 nm) from irradiated pectin protons to anthocyanin protons provided direct evidence that the two partners are in close contact. STD titrations at pH = 4.0 and pH = 1.5 suggest that the flavylium ion has a higher affinity for pectin than the hemiketal. Assuming the Scatchard model (n identical binding sites having the same binding constant, Kb), pectin was found to bind 180–600 anthocyanin units depending on the selected anthocyanin and 3 −1 pH. The corresponding Kb values are very weak (<10 M ). Thus, the picture emerging from this study is that anthocyanins (as individual species or non-covalent oligomers) provide a coating of the pectin’s surface through the development of very weak interactions (van der Waals contacts, H-bonds). The quenching of intrinsic protein fluorescence by increasing ligand concentrations is a classical method to probe ligand–protein binding and to extract binding parameters. As anthocyanins typically absorb light at the protein’s excitation and emission wavelengths, corrections for these inner-filter effects should be applied [44], which are not systematic [45] and thus lead to discrepancies in Kb values as well as in enthalpy and entropy changes. With human serum albumin (HSA), a globular 5 −1 protein, 1:1 binding is observed with a Kb in the order of 10 M [44,45], meaning a moderate affinity. The influence of the pH (from pH = 4 to pH = 7.4) on the binding strength is very modest [44]. Competition with probes of a known binding site (ibuprofen, warfarin) enables location of the anthocyanin binding site, a hydrophobic pocket lined by positively charged amino-acid residues (Arg, Lys) for possible accommodation of the anionic base [45]. As for the weakly structured salivary proteins, interaction with malvidin-3-glucoside (probed by STD-NMR) was found to be much weaker −1 (Kb ≈ 500 M ) and largely pH-independent (same affinity at pH = 1.0 and pH = 3.4), which suggests that the hemiketal and flavylium ions bind with close affinities [46]. Electrospray ionization MS revealed the formation of soluble aggregates involving 2–6 anthocyanin units and 1–4 peptides (proline-rich proteins or histatin). STD-NMR was also used to investigate the binding of keracyanin (cyanidin 3-rutinoside) to wheat flour gliadins at pH = 2.5 [47]. Protons C20-H, C50-H, C6-H and C8-H appear to be primarily involved in the binding. At this low pH, the corresponding aglycone (cyanidin) is stable and can be also investigated. Its affinity for gliadins appears higher based on the strong shielding of its proton signals when gliadins are added (confirmed by the large retention of cyanidin in the centrifugation pellet: up to 80% vs. only 8% for keracyanin). However, STP-NMR did not point protons specifically involved in the interaction. Cyanidin 3-glucoside expresses a rather high affinity for sodium caseinate (NaCas) [48]. Two binding sites were identified at pH = 2 and 6 −1 pH = 7, one of high affinity (Kb ≈ 1–7 × 10 M depending on pH and T) and a second of lower 5 −1 affinity (Kb ≈ 2–7 × 10 M ). For both sites, the binding was found to be exothermic at pH = 7 but endothermic at pH = 2 and thus is driven by a favorable entropy, which could point to a large contribution of the hydrophobic effect. Interestingly, NaCl addition gradually cancels cyanidin 3-glucoside–NaCas binding at pH = 7 but has no effect at pH = 2. In contrast to the high affinity of cyanidin 3-glucoside for NaCas, malvidin 3-glucoside only weakly binds to α- and β-caseins [49] and 3 −1 to β-lactoglobulin [50] (1:1 binding with Kb < 10 M ). Unlike co-pigmentation, the binding of anthocyanins to biopolymers does not trigger spectacular spectral changes. For instance, in the presence of various polysaccharides [51], no change in the wavelength of maximal visible absorption (λmax) was observed. Interactions of anthocyanins with cellulose, oat bran, and lignin is associated with a weak hypochromic effect, whereas an opposite effect (weak hyperchromism) is observed with highly methylated apple pectins. Sugar beet pectins have Molecules 2018, 23, 1970 15 of 23

Molecules 2018, 23, x 16 of 24 been shown to promote strong bathochromism in solutions of blackcurrant anthocyanins (cyanidin and delphinidin(cyanidinglycosides), and delphinidin but this glycosides) effect is due, but to this endogenous effect is due iron to ions endogenous (bound to iron the polysaccharide)ions (bound to the formingpolysaccharide) blue chelates forming with the blue pigments chelates [52 with]. In the agreement pigments with [52] the. In small agreement spectral with changes the small observed, spectral thechanges binding observed,of anthocyanins the binding to pectin of does anthocyanins not significantly to pectin affect does the thermodynamic not significantly constants affect the of thethermod acid–baseynamic and constants hydration of equilibria the acid– [43base]. In and other hydration words, all equilibria anthocyanin [43]. forms In other (colored words, or all colorless)anthocyanin bind pectin forms with (colored close or affinities. colorless) This bind apparent pectin discrepancy with close affinities. with the STD-NMR This apparent data discrepancy (stronger flavylium–pectinwith the STD- binding)NMR data might (stronger be due flatovylium anthocyanin–pectin self-association, binding) might which be due probably to anthocyanin is significant self- in theassociation concentrated, which solutions probably used is in significant the STD-NMR in the experiments. concentrated In contrast, solutions the used flavylium in the cation STD-NMR of theexperiments. pyranoanthocyanin In contrast, portisin the flavylium is strongly cation stabilized of the by pyranoanthocyanin interactions with anionic portisin wood is strongly lignosulfates stabilized as evidencedby interactions by its with much anionic weaker wood acidity lignosulfates in the presence as evidenced of the polysaccharide by its much (p K weakera1 = 6.6 acidity vs. 4.6 infor the portisinpresence alone) of [the53]. polysaccharide (pKa1 = 6.6 vs. 4.6 for portisin alone) [53].

2.6.2.6. Anthocyanins Anthocyanins in thein the Excited Excited State State AlthoughAlthough their their main main function function is to is absorb to absorb visible visible light light and and express express color, color, anthocyanins anthocyanins are are −3 intrinsicallyintrinsically poorly poorly fluorescent fluorescent with with quantum quantum yields yields typically typically lower lower than than 4 × 104 × 10(meaning−3 (meaning that that less less thanthan one one photon photon out out of of 250 250 absorbed absorbed is is actually actually re-emitted)re-emitted) [[54]54].. Indeed, Indeed, the the fate fate of of anthocyanins anthocyanins after afterabsorption, absorption, i.e. i.e.,, once once in in the the excited excited st state,ate, is a a difficult difficult question question that that must must be be addressed addressed by by sophisticatedsophisticated fast fast techniques, techniques, such such as time-resolvedas time-resolved fluorescence fluorescence and and transient transient absorption-emission absorption-emission spectroscopies.spectroscopies. In theIn the HOMO HOMO→ LUMO→ LUMO transition transition accompanying accompanying the the absorption absorption of visibleof visible light light by by thethe flavylium flavylium ion, ion, electron electron transfer transfer from from the the B-ring B-ring to to the the A-/C-rings A-/C-rings takestakes placeplace (Figure(Figure 1616))[ [55]55].. In In thethe excitedexcited state,state, thethe flavyliumflavylium ionion isis aa strongstrong acidacid (p(pKKaa << 0) that transfers a proton to the solvent on ona picosecond timescale timescale (20 (20 ps ps for for pelargonin pelargonin at at pH pH = = 1) 1) [54,56] [54,56.]. In In the the next next step, step, the the quinonoid quinonoid base baseinin the the excited excited state state is is deactivated by by aa combination combination of of radiative radiative (fluorescence) (fluorescence) and and non-radiative non-radiative (heat)(heat) processes processes and and then then captures captures a proton a proton in thein the ground ground state state to formto form the the ground ground state state flavylium flavylium ion.ion. In In other other words, words, the the quinonoid quinonoid base base is responsible is responsible for for the the (weak) (weak) fluorescence fluorescence observed observed for for anthocyaninsanthocyanins even even in stronglyin strongly acidic acidic solution. solution. In theIn the presence presence of aof co-pigment, a co-pigment, other other mechanisms mechanisms (Figure(Figure 17) 17) supersede supersede the the fast fast flavylium flavylium deprotonation deprotonation observed observed with with free free anthocyanins anthocyanins [57 ][57] in thein the followingfollowing ways: ways: (a) (a) within within the the complex complex in in the the excited excited state,state, throughthrough ultrafastultrafast int internalernal conversion conversion (<1 (<1ps) ps) via via a low-energy low-energy co-pigment-to-pigment co-pigment-to-pigment charge charge transfer transfer state, state, resulting resulting in instatic static fluorescence fluorescence quenching;quenching; and and (b) ( forb) for the the fraction fraction of freeof free anthocyanin, anthocyanin, diffusion-controlled diffusion-controlled electron electron transfer transfer from from thethe co-pigment co-pigment to theto the flavylium flavylium ion ion in thein the excited excited state, state, resulting resulting in dynamicin dynamic fluorescence fluorescence quenching. quenching. TheThe mechanism mechanism of energyof energy dissipation dissipation by by ultrafast ultrafast internal internal conversion conversion has has been been confirmed confirmed for for the the foldedfolded conformation conformation of a of cyanidin a cyanidin glycoside glycoside acylated acylated by pby-coumaric p-coumaric acid acid [58 ].[58] In. addition,In addition, fast fast energy energy transfertransfer to the to the chromophore chromophore following following absorption absorp oftion UV of light UV bylight the by acyl the residue acyl residue operates operates (Figure (Figure 17), thereby17), thereby conferring conferring acylated acylated anthocyanins anthocyanins to have to an have important an important role in plantrole in photoprotection. plant photoprotection.

(A) HOMO LUMO Visible light

Figure 16. Cont.

Molecules 2018, 23, 1970 16 of 23 Molecules 2018, 23, x 17 of 24 Molecules 2018, 23, x 17 of 24

Quinonoid Quinonoid bases bases Excited-state Strong acid Excitedproton-statetransfer Strong acid proton transfer (pKa < 0)   5 - 25 ps (B) (pKa < 0)   5 - 25 ps (B)   100 - 200 ps Flavylium ion   100 - 200 ps Flavylium ion Ground-state Groundproton-state transfer proton transfer

Weak acid (pKa  4) Weak acid (pKa  4)

Figure 16. (A) Frontier MOs of the flavylium ion of cyanidin (from reference [55]) and its most FigureFigure 16. 16. (A(A) ) Frontier Frontier MOs MOs of of the the flavylium flavylium ion ion of of cyanidin cyanidin (from (from reference reference [55] [55)]) and and its its most most representative mesomeric forms in the ground state (left) and first excited state (right). (B) The fate of representativerepresentative mesomeric mesomeric forms forms in in the the ground ground state state (left) (left) and and first first excited excited state state (right). (right). ( (BB)) The The fate fate of of free anthocyanins in the excited state (from references [54,56]). freefree anthocyanins anthocyanins in in the the excited excited state state (from (from references references [54,56] [54,56]).). Flavylium – CP complex Flavylium – CP complex

Heat dissipation Heat dissipation Chromophore Chromophore Low-lying CP → flavylium Low-lying CP → flavylium Glycosyl charge transfer state (ε  0)Glycosyl Energy transfer charge transfer state (ε  0) residue Energy transfer residue Hydroxycinnamoyl Hydroxycinnamoylresidue Absorption of UV light residue Absorption of UV light

(A) (B) (A) (B) Figure 17. The influence of co-pigmentation on the fate of anthocyanins in the excited state. (A) Figure 17. The influence of co-pigmentation on the fate of anthocyanins in the excited state. (A) FigureIntermolecular 17. The influence co-pigmentation of co-pigmentation (from reference on the [57]). fate (B of) Intramolecularanthocyanins in co the-pigmentation excited state. (from Intermolecular co-pigmentation (from reference [57]). (B) Intramolecular co-pigmentation (from (A) Intermolecularreference [58]). co-pigmentation (from reference [57]). (B) Intramolecular co-pigmentation (from referencereference [58] [58]).). 3. The Importance of Anthocyanin Chemistry in Food and Nutrition 3. The Importance of Anthocyanin Chemistry in Food and Nutrition 3. The Importance of Anthocyanin Chemistry in Food and Nutrition 3.1. Formulation of Anthocyanins for Food Applications 3.1.3.1. Formulation Formulation of of Anthocyanins Anthocyanins for for Food Food Applications Applications Anthocyanin degradation typically occurs during thermal processing and storage. The Anthocyanin degradation typically occurs during thermal processing and storage. The knowledgeAnthocyanin on degradation anthocyanin typically–biopolymer occurs interactions during thermal can processing be applied and to storage. devise The formulations knowledge for knowledge on anthocyanin–biopolymer interactions can be applied to devise formulations for on anthocyanin–biopolymerimproved chemical stability. interactions Degradation can be studies applied aimed to devise at demonstrating formulations the for protection improved chemicalafforded by improved chemical stability. Degradation studies aimed at demonstrating the protection afforded by stability.biopolymers Degradation may be studies limited aimed to monitoring at demonstrating the color the loss protection under given afforded conditions by biopolymers of pH, temperature may be , biopolymers may be limited to monitoring the color loss under given conditions of pH, temperature, limitedand to light monitoring exposure. the More color information loss under given is obtained conditions when of samples pH, temperature, are also acidified and light to exposure. pH 1–2 for and light exposure. More information is obtained when samples are also acidified to pH 1–2 for Morequantification information of is obtainedthe residual when flavylium samples ions are also by HPLC acidified or by to pH UV- 1–2visible for quantificationspectroscopy. Withof the this quantification of the residual flavylium ions by HPLC or by UV-visible spectroscopy. With this residualapproach, flavylium color loss ions (directly by HPLC observed or by UV-visible at the monitoring spectroscopy. pH), which With combines this approach, the reversible color loss water approach, color loss (directly observed at the monitoring pH), which combines the reversible water (directlyaddition observed and irreversible at the monitoring phenomena pH), (hydrolysis, which combines autoxidation), the reversible and water true anthocyanin addition and loss addition and irreversible phenomena (hydrolysis, autoxidation), and true anthocyanin loss (irreversible component), can be distinguished. (irreversible component), can be distinguished. In the simplest experiments involving modeling beverages, solutions of anthocyanins and In the simplest experiments involving modeling beverages, solutions of anthocyanins and soluble biopolymers are heated, and their color or residual anthocyanin concentration is monitored soluble biopolymers are heated, and their color or residual anthocyanin concentration is monitored

Molecules 2018, 23, 1970 17 of 23 irreversible phenomena (hydrolysis, autoxidation), and true anthocyanin loss (irreversible component), can be distinguished. In the simplest experiments involving modeling beverages, solutions of anthocyanins and soluble biopolymers are heated, and their color or residual anthocyanin concentration is monitored as a function of time. For instance, yeast mannoproteins (0.5% w/w for both anthocyanins and mannoproteins) increase the half-life of color loss by a factor of 5.4 in experiments conducted at pH = 7 and T = 80 ◦C or 126 ◦C (modeling pasteurization or sterilization) [59]. Similarly, the color loss in solutions of purple carrot anthocyanins at pH = 3.0 and T = 40 ◦C (in light) was shown to be inhibited by the addition of gum arabic (0.05–5.0%) with maximal stability observed at 1.5% (50% color retention after 5 days, vs. 20% in control) [60]. Similar observations were made with pectins or whey proteins (1%), the best result being obtained with heat-denatured whey proteins (70% color retention after 7 days at 40 ◦C, vs. 20% in control) [61]. In these works, fluorescence quenching experiments suggest that color protection involves direct interactions between anthocyanins and proteins (including the glycoprotein of gum arabic). However, the mechanism of protection remains largely unknown. It may be speculated that biopolymers mostly act by providing a more hydrophobic environment to anthocyanins, resulting in slower hydrolysis (despite the weak impact on the hydration equilibrium itself, see Section 2.5.2) and/or by scavenging transition metal traces acting as initiators/catalysts of anthocyanin autoxidation. A more sophisticated approach consists of preparing solid micro- or nanoparticles as delivery systems for anthocyanins. For instance, nanoparticles of whey proteins and beet pectin can be loaded with anthocyanin extracts with a higher efficiency (55%) when anthocyanins are added prior nanoparticle formation [62]. However, when dispersed in pH 4 solution, these nanoparticles do not show improved color stability. Particles of chitosan and carboxymethylchitosan (CMC) loaded with anthocyanins (size ≈ 200 nm, encapsulation efficiencies ranging from 16 to 44% depending on the CMC/chitosan proportions) can be simply prepared by mixing at pH = 5–6 followed by centrifugation [63]. The thermal stability of encapsulated anthocyanins was shown to greatly improve: 12% degradation after 3 days at 40 ◦C, vs. 90% in the control (no particles). Similar protection was observed in samples exposed to white light for 10 days (−20% vs. −80%). Sulfonylated polysaccharides, such as dextran sulfate and carrageenans, can also be used to encapsulate bilberry anthocyanins from acidic solutions (pH ≈ 3) with high efficiency and improved stability [64,65]. The binding of isotherms and HPLC analysis showed that the binding is selective of anthocyanins (the other phenols remaining in solution) and is stronger when the sulfonylation degree is higher. These data strongly suggest that the encapsulation is driven by ionic flavylium–sulfate interactions. Interestingly, the nanoparticles are gradually dissociated under near neutral conditions modeling the small intestine, which is desirable for subsequent intestinal absorption. Combining chitosan and cellulose nanocrystals at pH 2–3 also allows the formation of nanoparticles with high affinity for anthocyanins (up to 94% encapsulation) [66]. When cellulose is replaced by sodium tripolyphosphate, a reticulating agent for the polycationic chitosan chain, gel microcapsules (size ≈ 34 µm, encapsulation yield ≈ 33%) are formed. Finally, large hydrogel particles (size ≈ 2–3 mm) combining alginate and pectin can be used for encapsulation of anthocyanin-rich extracts under acidic conditions (pH = 1–3), and they are released upon dissolution at higher pH [67]. When exposed to white light, the half-life values of anthocyanins in hydrogel, hydrogel particles dispersed in pH 3 solution, and in a control solution (pH = 3) were 630 h, 277 h, and 58 h, respectively. Interestingly, anthocyanin-rich blackcurrant extracts can be incorporated into bread [68]. Replacing wheat flour by a mixture of gluten and starch led to markedly decreased anthocyanin concentrations (especially, for delphinidin glycosides, which are most sensitive to oxidation). This suggests that other flour proteins (e.g., albumins, globulins) and non-starch polysaccharides (e.g., hemicelluloses, β-glucans) may be important to provide chemical stability to anthocyanins in such matrices. Molecules 2018, 23, x 19 of 24 from the low concentrations (generally, <0.1 µM) of native forms (mostly, anthocyanidin glucosides) and anthocyanidin conjugates detected in the general blood circulation [70,71]. These derivatives are formed in the small intestine after enzymatic hydrolysis by membrane-bound lactate phlorizin Molecules 2018, 23, 1970 18 of 23 hydrolase or by cytosolic β-glucosidase, and subsequent conjugation by O-glucuronidation, O- methylation, and/or O-sulfonylation. The detection of native forms in the blood circulation is not equivalent3.2. The Fate to of other Anthocyanins flavonoid in glucosides Humans, Consequences and could be on due the Possible to partial Effects absorption on Health from the stomach. This early absorption has been demonstrated in cell and animal models [72–74] and has been The bioavailability of phenolic compounds has been largely elucidated over the last decades [69]. proposed to involve the organic anion transporter bilitranslocase in the gastric epithelium [72]. This knowledge, which is crucial to the interpretation of the possible effects on health, encompasses the Most importantly, recent investigations, in particular using 13C-labelled compounds [3], have bioaccessibility (the release of phenols from the food matrix during digestion), intestinal absorption, shown that the bulk of the ingested amount of anthocyanins is actually converted into simple metabolism, transport, distribution to tissues, and excretion of dietary phenols and their metabolites. phenolic compounds (Table 2), as a consequence of (a) the chemical instability (under near neutral Anthocyanins have emerged as poorly bioavailable micronutrients as judged from the low conditions) of anthocyanins and, especially, of anthocyanidins [24] and (b) the extensive catabolism concentrations (generally, <0.1 µM) of native forms (mostly, anthocyanidin glucosides) and by the colonic microbiota of the fraction reaching the large intestine. These simple metabolites, which anthocyanidin conjugates detected in the general blood circulation [70,71]. These derivatives are themselves can be further conjugated by intestinal and hepatic enzymes, have been found in the formed in the small intestine after enzymatic hydrolysis by membrane-bound lactate phlorizin blood circulation in much higher concentration than anthocyanidin derivatives [3,75]. hydrolase or by cytosolic β-glucosidase, and subsequent conjugation by O-glucuronidation, O-methylation,Table 2. Serum and/or pharmacokineticO-sulfonylation. profiles of The cyanidin detection 3-glucoside of native (C3G) forms and its in metabolites the blood in circulation humans is not equivalentafter the consumption to other flavonoid of 500 glucosides mg 13C-labelled and could C3G. Frombe due reference to partial [3] absorption (in red is fromthe reference the stomach. This earlycompound absorption and its hasmost been abundant demonstrated metabolites) in. cell and animal models [72–74] and has been proposed to involve the organic anion transporter bilitranslocase in the gastric epithelium [72]. Most importantly,Compound recent investigations,n Cmax/nM in particulartmax/h using 13tC-labelled1/2/h AUC compounds0-48/nM h [3], haveCyanidin shown that-3-glucoside the bulk of (C3G) the ingested 5 amount141 (±70) of anthocyanins1.8 (±0.2) is actually0.4 converted279 (± into170) simple phenolicProtocatechuic compounds acid (Table (PCA)2), as a consequence8 146 (±74) of (a) the3.3 chemical (±0.7) instability9.9 (±3.4) (under1377 near (±760) neutral conditions)Phloroglucinaldehyde of anthocyanins and, especially,4 582 of (± anthocyanidins536) 2.8 (±1.1) [24 ] and (b)nd the extensive7882 (± catabolism7768) by the colonic microbiota of the fraction reaching the large intestine. These simple metabolites, PCA-sulfates 8 157 (±116) 11.4 (±3.8) 31.9 (±19.1) 1180 (±349) which themselves can be further conjugated by intestinal and hepatic enzymes, have been found in the blood circulationVanillic in acid much (VA) higher concentration2 1845 (± than838) anthocyanidin12.5 (±11.5) derivatives6.4 [3,2331975]. (±20650) VA-sulfates 4 430 (±299) 30.1 (±11.4) nd 10689 (±7751) Table 2. SerumFerulic pharmacokinetic acid profiles7 of827 cyanidin (±371) 3-glucoside8.2 (±4.1) (C3G) and21.4 its (± metabolites7.8) 17422 in (± humans11054) after the consumption of 500 mg 13C-labelled C3G. From reference [3] (in red is the reference compound Hippuric acid 8 1962 (±1389) 15.7 (±4.1) 95.6 (±77.8) 46568 (±30311) and its most abundant metabolites).

In agreementCompound with the strongn in vivo C maxcatabolism/nM of anthocyanins,tmax/h in tvitro1/2/h digestion AUC models0-48/nM have h shownCyanidin-3-glucoside that whereas anthocyanins (C3G) 5 are 141 readily (±70) released 1.8 into(±0.2) the acidic 0.4 gastric compartment 279 (±170) and relativelyProtocatechuic stable, acid they (PCA) undergo 8 substantial 146 (± 74) degradation 3.3 (± 0.7) in the near 9.9 (± neutral3.4) upper 1377 (± intestinal760) Phloroglucinaldehyde 4 582 (±536) 2.8 (±1.1) nd 7882 (±7768) compartment, possibly because of autoxidation [76,77]. However, this chemical instability could be PCA-sulfates 8 157 (±116) 11.4 (±3.8) 31.9 (±19.1) 1180 (±349) overestimatedVanillic in acid in (VA)vitro models, 2as the 1845O2 content (±838) is higher 12.5 (± than11.5) under real 6.4 physiological 23319 (conditions.±20650) As a strikingVA-sulfates example, protocatechuic 4 acid 430 (PCA, (±299) 3,4-dihydroxybenzoic 30.1 (±11.4) acid), nd recovered 10689 in (blood±7751) and fecal samples,Ferulic was acid shown to represent 7 827 more (±371) than 70% 8.2 ( of± 4.1) the ingested 21.4 ( ± dose7.8) of 17422 the cyanidin (±11054) O- ± ± ± ± glucosidesHippuric from blood acid orange juice 8 [75] 1962. Interestingly, ( 1389) 15.7 PCA ( 4.1) can be 95.6 formed ( 77.8) by chemical 46568 ( oxidative30311) degradation of anthocyanins and anthocyanidins (Figures 8–10). However, it must be noted that anthocyaninsIn agreement bearing with an the electron strong-richin vivo B-ringcatabolism (e.g., cyanidin of anthocyanins, and delphinidinin vitro glycosides)digestion must models be muchhave shownmore prone that whereasto oxidative anthocyanins degradation are than, readily for instance, released intopelargonidin the acidic derivatives gastric compartment [78], which indeedand relatively could be stable, detected they in higher undergo concentration substantialsdegradation (0.2–0.3 µM) inin the blood near neutral[79]. upper intestinal compartment, possibly because of autoxidation [76,77]. However, this chemical instability could be overestimated in in vitro models, as the O content is higher than under real physiological conditions. 2 As a striking example, protocatechuic acid (PCA, 3,4-dihydroxybenzoic acid), recovered in blood and fecal samples, was shown to represent more than 70% of the ingested dose of the cyanidin O-glucosides Molecules 2018, 23, 1970 19 of 23 from blood orange juice [75]. Interestingly, PCA can be formed by chemical oxidative degradation of anthocyanins and anthocyanidins (Figures8–10). However, it must be noted that anthocyanins bearing an electron-rich B-ring (e.g., cyanidin and delphinidin glycosides) must be much more prone to oxidative degradation than, for instance, pelargonidin derivatives [78], which indeed could be detected in higher concentrations (0.2–0.3 µM) in the blood [79]. In the digestive tract, anthocyanins may also modulate the digestion and uptake of nutrients by interacting with intestinal α-glucosidase [80]. They could, as well, attenuate oxidative stress in the digestive tract, for instance, by inhibiting the peroxidation of dietary lipids induced by heme iron [29,81]. After intestinal absorption, anthocyanin derivatives are probably transported in the blood in moderate association with serum albumin [45] before distribution to tissues, which, again, could involve bilitranslocase, as evidenced in the kidneys of rats [82]. Most importantly, it must be kept in mind that the degradation products of anthocyanins, which are formed in the digestive tract and are generally much more abundant than the residual anthocyanidin derivatives, could mediate most of the potential health effects of anthocyanins [83,84], which remains intriguing given their chemical simplicity [3] (Table2). However, redox-active compounds, such as PCA, could indeed participate in regulating the expression of genes associated with transcription factors susceptible to redox activation. Such mechanisms could, at least partly, underline the induction of antioxidant defense via the Nrf2 pathway and the reduction of inflammation via NF-κB inhibition observed in cells and in rodents with cyanidin derivatives [85] or PCA itself [86].

Author Contributions: O.D. and J.-A.F. analyzed the literature and wrote the paper. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest.

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