Flavylium cations

Flavor transport Flavylium cation Flaws Flax... 

As frequently mentioned in the literature, anthocyanins co-exist in equilibrium in four different forms. The pH conditions shift this equilibrium toward a variety of structural forms, with the direct consequences of color changes of these pigments. As a rule, at pH above 4, yellow compounds (chalcone form), blue compounds (quinoid base), or colorless compounds (methanol form) are produced. Anthocyanins have the highest stabilities at a pH between 1 and 2 since the flavylium cation is the most stable predominant form. 

The overall anthocyanin analysis is generally conducted using the Giusti and Wrolstad method based on the differences in absorbance of anthocyanins at pH 1 and pH 4.5. Then the pigment content is determined using the coefficient of molar extinction of the predominant anthocyanin. It should be noted that this technique only allows dosing of anthocyanins with a color difference between the two pH values (due to transition to the flavylium cation form). A more global analysis of total anthocyanin content may be conducted by direct spectrophotometry of the... 

The best cofactors are typically flavonoid derivatives that contain many hydroxyl groups, the most favorable at position 3 of the flavones. The strongest cofactors have electron-rich systems that associate with electron-poor compounds such as the flavylium cation. 

In 1967, Timberlake and Bridle proposed that copigmentation complex formation reactions between cyanidin and quercetin in aqueous buffered solutions took place between the colored forms of the flavylium cation (AH+) cyanidin at pH 3.0 and the quinoidal base (A) at pH 5.0. 

Houbiers, C. et al.. Color stabilization of malvidin 3-glucoside self-aggregation on the of flavylium cation and copigmentation with the Z-chalcone form, J. Phys. Chem. B, 102, 3578, 1998. 

Figure 9.2 Chemical structure of unsubstituted flavylium cation.

Anthocyanins usually give a purple red colour. Anthocyanins are water soluble and amphoteric. There are four major pH dependent forms, the most important being the red flavylium cation and the blue quinodial base. At pHs up to 3.8 commercial anthocyanin colours are ruby red as the pH becomes less acid the colour shifts to blue. The colour also becomes less intense and the anthocyanin becomes less stable. The usual recommendation is that anthocyanins should only be used where the pH of the product is below 4.2. As these colours would be considered for use in fruit flavoured confectionery this is not too much of a problem. Anthocyanins are sufficiently heat resistant that they do not have a problem in confectionery. Colour loss and browning would only be a problem if the product was held at elevated temperatures for a long while. Sulfur dioxide can bleach anthocyanins - the monomeric anthocyanins the most susceptible. Anthocyanins that are polymeric or condensed with other flavonoids are more resistant. The reaction with sulfur dioxide is reversible. 

The anthocyanins exist in solution as various structural forms in equilibrium, depending on the pH and temperature. In order to obtain reproducible results in HPLC, it is essential to control the pH of the mobile phase and to work with thermostatically controlled columns. For the best resolution, anthocyanin equilibria have to be displaced toward their flavylium forms — peak tailing is thus minimized and peak sharpness improved. Flavylium cations are colored and can be selectively detected in the visible region at about 520 nm, avoiding the interference of other phenolics and flavonoids that may be present in the same extracts. Typically, the pH of elution should be lower than 2. A comparison of reversed-phase columns (Ci8, Ci2, and phenyl-bonded) for the separation of 20 wine anthocyanins, including mono-glucosides, diglucosides, and acylated derivatives was made by Berente et al. It was found that the best results were obtained with a C12 4 p,m column, with acetonitrile-phosphate buffer as mobile phase, at pH 1.6 and 50°C. 

Absorption spectra have also been used in the reexamination of pH-dependent color and structural transformations in aqueous solutions of some nonacylated anthocyanins and synthetic flavylium salts." ° In a recent study, the UV-Vis spectra of flower extracts of Hibiscus rosasinensis have been measured between 240 and 748 nm at pH values ranging from 1.1 to 13.0." Deconvolution of these spectra using the parallel factor analysis (PARAFAC) model permitted the study of anthocyanin systems without isolation and purification of the individual species (Figure 2.21). The model allowed identification of seven anthocyanin equilibrium forms, namely the flavylium cation, carbinol, quinoidal base, and E- and Z-chalcone and their ionized forms, as well as their relative concentrations as a function of pH. The spectral profiles recovered were in agreement with previous models of equilibrium forms reported in literature, based on studies of pure pigments. 

Hoshino, T., Self-association of flavylium cations of anthocyanidin 3,5-diglucosides studied by circular dichroism and NMR, Phytochemistry, 31, 647, 1992. 

FIGURE 3.4 Biosynthetic route to proanthocyanidins from leucoanthocyanidins. The product of the ANS is given in the flavylium cation form. Enzyme abbreviations are defined in the text and in Table 3.1. 

The ANR reaction involves a double reduction at the C-2 and C-3 of the anthocyanidin, allowing the inversion of C-3 stereochemistry. Xie et al. postulate four possible reaction mechanisms, proceeding via either flav-3-en-ol or flav-2-en-ol intermediates. The proposed reaction mechanisms are based on anthocyanidins (the flavylium cation forms) as the starting molecules however, as the authors acknowledge, other forms of the anthocyanidin may exist in vivo. In particular, the 3-flaven-2,3-diol pseudobase is thought to be the more likely in vivo product of the ANS. 

Anthocyanins are usually represented as the red flavylium cations (Figure 5.1, left). However, this form is predominant only in very acidic solvents (pH < 2) such as those used for HPLC analysis. In mildly acidic media, the flavylium cations undergo proton transfer and hydration reactions, respectively, generating the quinonoidal base and the hemiketal syn carbinol) form (Figure 5.1, right) that can tautomerize to the chalcone. Thus, at wine pH, malvidin 3-glucoside occurs mostly as the colorless hemiketal (75%), the red flavylium cation, yellow chalcone, and blue quinonoidal base being only minor species. 

Another reaction of the flavylium cation has recently been demonstrated. " It involves concerted addition of compounds possessing a polarizable double bond on the electron-deficient site C-4 and the oxygen of the 5-hydroxyl group of the anthocyanin. The new pigments thus formed, showing a second pyran ring, have been referred to as vitisins, but the term pyranoanthocyanins proposed by Lu and Foo is preferred. 

Nucleophilic addition of anthocyanins and flavanols on electrophiles such as quinones, flavylium cations, protonated aldehydes, and carbocations resulting from acid-catalyzed cleavage of proanthocyanidins. 

Formation of pyranoanthocyanins through reaction of flavylium cations with compounds possessing a polarizable double bond, namely vinylphenol derivatives (including vinylflavanols and hydroxycinnamic acids) and enolizable aldehydes and ketones (e.g., acetaldehyde and pyruvic acid). 

Finally, reactions of flavonoid and nonflavonoid precursors are affected by other parameters like pH, temperature, presence of metal catalysts, etc. In particular, pH values determine the relative nucleophilic and electrophilic characters of both anthocyanins and flavanols. Studies performed in model solutions showed that acetaldehyde-mediated condensation is faster at pH 2.2 than at pH 4 and limited by the rate of aldehyde protonation. The formation of flavanol-anthocyanin adducts was also limited by the rate of proanthocyanidin cleavage, which was shown to take place at pH 3.2, but not at pH 3.8. Nucleophilic addition of anthocyanins was faster at pH 3.4 than at pH 1.7, but still took place at pH values much lower than those encountered in wine, as evidenced by the formation of anthocyanin-caffeoyltartaric acid adducts, methylmethine anthocyanin-flavanol adducts,and flavanol-anthocyanin adducts. The formation of pyranoanthocyanins requiring the flavylium cation was faster under more acidic conditions, as expected, but took place in the whole wine pH range. Thus, the availability of either the flavylium or the hemiketal form does not seem to limit any of the anthocyanin reactions. 

Each anthocyanidin is involved in a series of equilibria giving rise to different forms, which exhibit their own properties including color. One- and two-dimensional NMR have been used to characterize the various forms of malvidin 3,5-diglucoside present in aqueous solution in the pH range 0.3 to 4.5 and to determine their molar fractions as a function of pH. In addition to the flavylium cation, two hemiacetal forms and both the cis and trans forms of chalcone were firmly identified. In a reexamination, the intricate pH-dependent set of chemical reactions involving synthetic flavylium compounds (e.g., 4 -hydroxyflavylium) was confirmed to be basically identical to those of natural anthocyanins (e.g., malvidin 3,5-diglucoside) in... 

Stability is also affected by pH, light, heat, and mechanical stress. Colors change depending on pH and on protonation and hydration reactions during storage. The most stable form, the flavylium cation, predominates at low pH (Torskangerpoll and Andersen, 2005). Stability is... 

The anthocyanins occur in the vacuole as an equilibrium of four molecular species the coloured basic flavylium cation and three... 

Fig. 13 Equilibrium distribution of four anthocyanin forms of malvidin-3-glucoside as a function of pH The red flavylium cation (AH+), the blue quinonoidal base (A), the colorless carbinol pseudobase (B), and the colorless chalcone (C). (From Ref. 138.)...

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