16
Anthocyanins

16.1 Introduction

Anthocyanins are the compounds responsible for the color of red and black grapes and red wines; the red and blue colors found in many plants are also attributed to anthocyanins, especially in the flowers and fruits. The perceived red color is due to absorption of green visible light (520 nm), which results from the fully conjugated 10 π‐electron (i.e., aromatic) A–C flavonoid ring system that is also cross‐conjugated into the B ring.¹ If that conjugation is disrupted the color is lost, such as when anthocyanins react with bisulfite, water, or other nucleophiles. Anthocyanin structure and chemistry is further complicated by the ability of anthocyanins to bind both covalently and non‐covalently to common wine constituents. The monomeric anthocyanins react with carbonyl compounds and tannins to produce “stabilized” wine pigments, also called polymeric pigments in some texts. Although monomeric anthocyanins are detectable in small amounts in some wines beyond 5 years, the primary sources of color in most red wines past a few years of age are stabilized pigments.

16.2 Structures and forms

The term for the simple flavonoid ring system is anthocyanidin, but anthocyanidins are not found in grapes or wine except in trace quantities because they are unstable. Anthocyanin refers to glycosylated anthocyanidins. These anthocyanins are also referred to as monomeric pigments to distinguish them from wine pigments formed from their reaction with condensed tannins and other compounds (see Section 16.5). In V. vinifera, the 3‐O‐glucoside is the predominant form of anthocyanins (Figure 16.1). In American species and hybrids (Chapter 31), however, the 3,5‐di‐O‐glucoside is also found and its presence is the basis for identifying the use of non‐vinifera grapes to make wines. The glucose conjugated to anthocyanidins may be further substituted through esterification at the 6‐position of the sugar moiety, either to an acetyl or a coumaroyl group; a small amount of caffeoyl substitution is also observed [1] (Figure 16.1).

Figure 16.1 Malvidin‐3‐glucoside (M3G), the dominant anthocyanin in many grapes, is shown as a general example of the anthocyanin structure. The simple sugar (R = H, malvidin‐3‐glucoside) is most abundant, but varying amounts of acetyl and coumaroyl substituents are found, with traces of caffeoyl, depending on grape variety

There are five major aglycones in red grapes, which differ in their substitution patterns on the B ring (Figure 16.2); the form with only one hydroxyl at the 4′ position, apigenin, is absent in grapes. Considering the B‐ring substitution and acylation noted above, it is possible to observe 10–15 anthocyanins in any particular red grape. Anthocyanin profiles will vary among cultivars, which has led to attempts to use these profiles to authenticate the grapes used to produce young red wines [2]. For instance, Pinot Noir is notable for its lack of acylated glucoside forms, but unfortunately the loss of these grape‐derived monomeric anthocyanins during aging negates the application of this approach to differentiate aged wines (Chapter 28). Malvidin‐3‐glucoside (M3G) and its derivatives dominate the anthocyanin profile in most red grapes and red wines, so most investigations focus on the reactivity and interactions of this anthocyanin alone. However, information from those studies can generally apply to the other anthocyanins.

Figure 16.2 The five versions of the B‐ring found in grape anthocyanins

Anthocyanins present in wine and similar solutions exist as several forms in a pH dependent equilibrium, and the relative proportions strongly affect the color of the solution. The positively charged C‐ring of the flavylium (2‐phenylchromenylium or benzopyrylium) cation is electrophilic, and the C2 and C4 positions can react with nucleophiles present in wine. Common reactions are with water and bisulfite; in both cases, the red color of the flavylium form is lost when the double‐bond conjugation is disrupted. As pH of a solution rises the flavylium cation equilibrates like an acid, reacting with water, to become a colorless neutral species, the carbinol pseudobase, after losing a proton from the nucleophilic water molecule (Figure 16.3). The pK_a of the flavylium–pseudobase equilibrium is 2.7, so at a typical wine pH of 3.7, 90% of the anthocyanin pool is rendered colorless [3]. At low pH, all forms are converted to the flavylium cation, facilitating quantitation of the anthocyanins by light absorbance at 520 nm. In addition, there is a quinone form, which has a violet hue and a pK_a of 4.7, so it is present in small amounts at high wine pH values. One research team has proposed an alternate explanation of anthocyanin color based on the observation that at wine pH, wine pigments appear to have zero charge [4].

Figure 16.3 Equilibrating forms of anthocyanins

16.3 Non‐covalent interactions: co‐pigmentation

Anthocyanins can form non‐covalent interactions with other phenolic compounds in solution to create an effect known as co‐pigmentation. This results in stabilization of the pigmented, aromatic forms of anthocyanins as compared to the non‐aromatic carbinol base. The outcome of co‐pigmentation is that absorbance of anthocyanin‐containing solutions, for example, red wine, are often greater than what is predicted by anthocyanin concentration and the solution pH alone [5]. Co‐factor–anthocyanin interactions can be described by the following relationship:

K_d is the binding constant of the anthocyanin and co‐factor. Typically, the best co‐factors (greatest enhancement of color) are planar aromatic structures, since non‐planar structures will be disfavored by steric effects. For example, the planar quercetin has a binding constant of 2900 M⁻¹, while the non‐planar catechin has a binding constant of 90 M⁻¹. Because the co‐pigmentation reaction is bimolecular, co‐pigmentation complexes will dissipate rapidly as wine is diluted (e.g., a 2‐fold dilution will cause a 4‐fold dilution in complexes). This results in a deviation of wine color from Beer’s Law.

Multiple explanations have been proposed for the nature of chemical bonds in co‐pigmentated complexes. These complexes increase the proportion of anthocyanins that exist in the flavylium form as compared to a solution of anthocyanins alone, in the same manner as decreasing the pH. One possible explanation is charge transfer complexation, which occurs when two aromatic‐ring substances in solution have different electron densities (Figure 16.4) [6]. In wine, the flavylium form of an anthocyanin has a positive charge and is electron poor, and the other phenolic compounds in wine are generally electron rich because the phenol groups are strong electron donors. An alternative explanation is that the co‐pigmentation interactions are primarily hydrophobic – that is, the faces of the planar aromatic molecules arrange in a π–π stacked manner [7]. For complexes found in wine, both charge transfer and hydrophobic interactions may contribute.

Figure 16.4 Formation of a model charge transfer complex with malvidin‐3‐glucoside and phloroglucinol, which has the effect of shifting anthocyanin equilibria towards the red colored form. The red flavylium form is shown with highlighting. Phloroglucinol is used as the electron‐rich partner for simplicity, but it is not found in wine

At wine pH, there is a large pool of the colorless pseudobase (ca. 90%), so the observed co‐pigmentation effect can be very significant – doubling of absorbance is not unusual – and colorimetric analyses of anthocyanin content must be corrected for this effect. However, at low pH, only the flavylium form is present, so under these circumstances, the observed co‐pigmentation contribution to absorbance and bathochromic shift is small. A final complicating issue is that anthocyanins can self‐associate, resulting in strong co‐pigmentation when anthocyanin concentrations are high [8].

16.4 Bisulfite bleaching

Anthocyanins in the flavylium form will react with the bisulfite nucleophile (Chapter 17) on the C‐ring at the C4 position (Figure 16.5), unlike water, which will add at the C2 position to form a pseudobase (cf. Figure 16.3). However, the overall effect is the same as water addition in that pigmentation is lost due to disruption of conjugation. The resulting loss of red color is referred to as “bleaching” [9]. Formation of covalent bonds at the C4 position can block bisulfite addition and decrease this bleaching effect, as described below. Because wine samples often have sulfites present, it is essential to eliminate this bleaching effect when quantifying anthocyanins via spectral analysis [5].

Figure 16.5 Bleaching of malvidin‐3‐glucoside through addition of bisulfite to the flavylium cation at C4

16.5 Wine pigments

During and after fermentation, anthocyanins react with various compounds in the red wine matrix to form modified pigments. Some of the reactions are acid‐catalyzed, while others require an oxidation step. The identity of some specific products is known, but comprehensive characterization of all wine pigments is not yet possible, and the relative significance of any of the pathways described below is not established. Additional details are provided in Chapter 25.

Although the average molar absorptivity of the total anthocyanin pool has decreased as a result of conversion to wine pigments [10], the resulting pigments play an important role in wine color because they are “stabilized,” a descriptor that covers a wide range of phenomena:

• Wine pigments are less prone to degradation during long‐term storage.
• They absorb more strongly at wine pH and demonstrate less pH dependence in their absorbance behavior.
• They are less bleachable by bisulfite.

Modified wine pigments are also referred to in some texts as polymeric pigments (and occasionally other terms), although this term suffers from ambiguity. Initially, “polymeric pigment” was applied to any wine pigment that was not bleachable by bisulfite, as it was believed that the non‐bleachable species were exclusively reaction products of polymeric condensed tannins and anthocyanins [11]. This operational definition is still used in some assays [12]. However, as described below, stable wine pigments like the vitisins (see below), can be formed without the need for reacting with the “polymeric” condensed tannins. Polymeric pigment is also used to describe high molecular weight pigments separated chromatographically, for example, the late eluting hump in an HPLC chromatogram. While this is a more grammatically correct definition, not all of these high molecular weight species demonstrate behavior associated with key wine pigments, for example, resistance to SO₂ bleaching [13] (see below).

The simplest forms of wine pigments are those formed by electrophilic aromatic substitution on the A ring of a flavonoid. In the first case, referred to as the T‐A (i.e., tannin‐anthocyanin) type, the electrophilic cation formed in the course of proanthocyanidin interflavan bond cleavage (Chapter 14) reacts with the A ring of an anthocyanin pseudobase to form a series of pigments from dimer to much larger. Figure 16.6 shows an example of a T‐A type pigment formed from a proanthocyanidin trimer [14]. This occurs since ca. 90% of the anthocyanins in wine are in the neutral pseudobase form (Figure 16.4) and therefore available to act as a nucleophile, attacking the electrophilic C4 carbocation of the flavonoid.

Figure 16.6 Formation of T‐A type wine pigments via condensation of anthocyanins and proanthocyanidins

In the flavylium form, anthocyanins display electrophilic character, as described above, and react directly at the C4 position with the nucleophilic A ring of a proanthocyanidin to create the A‐T (i.e., anthocyanin‐tannin) type pigment (Figure 16.7). This addition disrupts the aromaticity of the flavylium cation, resulting in a colorless flavene product. This could potentially oxidize to generate the aromatic C‐ring and recover the pigmentation, and then react to form a xanthylium cation (Chapter 25), although there is some suggestion that the reaction stops at the flavene, leading to a net loss of color [14].

Figure 16.7 Formation of A‐T type wine pigment resulting in the colorless flavene form of the anthocyanin in the center. Oxidation could potentially return the anthocyanin to its colored flavylium form

Wine oxidation (Chapter 24) and fermentation produce reactive compounds that modify anthocyanins in a number of ways to produce new pigments. The simplest is acetaldehyde, which is produced by yeast sugar metabolism during fermentation or through ethanol oxidation during wine storage. Acetaldehyde is a good electrophile and can act to bridge two flavonoid A rings (Figure 16.8). These can be two flavanols, a flavanol and anthocyanin, or two anthocyanins [15].² These ethylene‐bridged pigments show a bathochromic shift (violet color) and are less susceptible to nucleophilic addition (and color loss) by SO₂ and water. However, this reaction is reversible and could release the anthocyanin. Other aldehydes can initiate related reactions, such as glyceraldehyde formed by oxidation of glycerol (Chapter 3), which can react with anthocyanins and flavanols to form analogous pigments [16].

Figure 16.8 Anthocyanin‐derived wine pigments such as the ethyl‐linked M3G‐flavan‐3‐ol dimer incorporating acetaldehyde, and vitisin A, which results from M3G and pyruvic acid condensation

Electrophiles formed during fermentation or storage are also capable of forming pyranoanthocyanins, which contain an additional pyran ring. A representative structure of vitisin A, formed by reaction of pyruvic acid and malvidin‐3‐glucoside is shown in Figure 16.8 [17]. The pyranoanthocyanin pigments have a blocked C4 position – vitisin A appears to be totally resistant to bleaching up to 250 mg/L of SO₂ and vitisin B (from acetaldehyde addition instead of pyruvic acid) is half bleached at 200 mg/L of SO₂, while malvidin‐3‐glucoside is 80% bleached at 50 mg/L. Both vitisins show only about 10% variation in absorbance intensity between pH 2 and 4, while the anthocyanin shows a 40% change [18]. These pyranoanthocyanins also appear to be more stable during long‐term storage.

Aside from oxidation products, hydroxycinnamates can react directly with anthocyanins to form pinotins [19] in a cyclization with the 5‐OH group of the anthocyanin, forming another pyranoanthocyanin (Figure 16.9). The pinotins are distinguished by the phenolic ring attached to the new pyran ring of the derived pigment. These products are also fairly long lived and certainly contribute to the red pigmentation of aged wine.

Figure 16.9 Pinotin A, arising from reaction of M3G with caffeic acid

The multiple members of the anthocyanin class, along with the varying substitution of the glucoside, makes anthocyanin chemistry complex in wine. However, further details of equilibration with water and bisulfite, plus co‐pigmentation and the multiple reactions to form wine pigments, make it difficult to predict anthocyanin chemistry during aging (Chapter 25).

References

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2. 2. Eder, R., Wendelin, S., Barna, J. (1994) Classification of red wine cultivars by means of anthocyanin analysis. 1st report: application of multivariate statistical methods for differentiation of grape samples. Mitteilungen Klosterneuburg, 44 (6), 201–212.
3. 3. Brouillard, R. and Delaporte, B. (1977) Chemistry of anthocyanin pigments. 2. Kinetic and thermodynamic study of proton‐transfer, hydration, and tautomeric reactions of malvidin 3‐glucoside. Journal of the American Chemical Society, 99 (26), 8461–8468.
4. 4. Asenstorfer, R.E., Iland, P.G., Tate, M.E., Jones, G.P. (2003) Charge equilibria and pK(a) of malvidin‐3‐glucoside by electrophoresis. Analytical Biochemistry, 318 (2), 291–299.
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7. 7. Kunsagi‐Mate, S., May, B., Tschiersch, C., et al. (2011) Transformation of stacked pi‐pi‐stabilized malvidin‐3‐O‐glucoside–catechin complexes towards polymeric structures followed by anisotropy decay study. Food Research International, 44 (1), 23–27.
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10. 10. Zimman, A. and Waterhouse, A.L. (2004) Incorporation of malvidin‐3‐glucoside into high molecular weight polyphenols during fermentation and wine aging. American Journal of Enology and Viticulture, 55 (2), 139–146.
11. 11. Somers, T.C. (1971) The polymeric nature of wine pigments. Phytochemistry, 10 (9), 2175–2186.
12. 12. Harbertson, J.F. and Spayd, S. (2006) Measuring phenolics in the winery. American Journal of Enology and Viticulture, 57 (3), 280–288.
13. 13. Versari, A., Boulton, R.B., Parpinello, G.P. (2007) Analysis of SO₂‐resistant polymeric pigments in red wines by high‐performance liquid chromatography. American Journal of Enology and Viticulture, 58 (4), 523–525.
14. 14. Hayasaka, Y. and Kennedy, J.A. (2003) Mass spectrometric evidence for the formation of pigmented polymers in red wine. Australian Journal of Grape and Wine Research, 9 (3), 210–220.
15. 15. Escribano‐Bailon, T., Alvarez‐Garcia, M., Rivas‐Gonzalo, J.C., et al. (2001) Color and stability of pigments derived from the acetaldehyde‐mediated condensation between malvidin 3‐O‐glucoside and (+)‐catechin. Journal of Agricultural and Food Chemistry, 49 (3), 1213–1217.
16. 16. Laurie, V.F. and Waterhouse, A.L. (2006) Glyceraldehyde bridging between flavanols and malvidin‐3‐glucoside in model solutions. Journal of Agricultural and Food Chemistry, 54 (24), 9105–9111.
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Notes

1 Organic pigment compounds often have high degrees of alternating single and double bonds (“conjugation”). The red and yellow colors of tomatoes, peppers, watermelons, and related members of the nightshade or curcubit family are due to carotenoids (see Chapter 10), composed of 40 carbons arranged in mostly conjugated double bonds. Carotenoids are very non-polar, and a “wine” produced from tomatoes would be nearly colorless.

2 In the US (27 CFR 24.246), addition of acetaldehyde to juice (but not wine) for the purposes of stabilizing color is legal, although not widely practiced.