The Flavonoids: Advances in Research since 1980 (1988) pp. 5, 7-8
J. B. Harborne

1.3.4 Zwitterionic anthocyanins

Until recently, acylated anthocyanins were known to be substituted by hydroxycinnamic acids ( ρ-coumaric, caffeic, ferulic or sinapic), by ρ-hydroxybenzoic acid or by acetic acid. However, it is now apparent from extensive electrophoretic surveys (Harborne and Boardley, 1985; Harborne, 1986) and from detailed investigations of individual pigments (e.g. Cornuz et al., 1981) that anthocyanins are also acylated in nature with aliphatic dicarboxylic acids, such as malonic, malic, oxalic and succinic. Such acylation renders the cationic anthocyanin a zwitterion, which means that it is possible to distinguish these pigments from other anthocyanins by paper electrophoresis in a weakly acidic buffer.

Another significant feature of this type of acylation is the instability of the acyl link in vitro, particularly when compared with aromatic acid acylation. If malonated anthocyanins are extracted by standard procedures using methanolic HCl, there is intermediate methyl ester formation at the free carboxyl group, but the main reaction is loss of the malonyl group within a short time. By contrast, hydroxycinnamyl residues are relatively unaffected by such treatment. Successful extraction of zwitterionic anthocyanins depends on the substitution of mineral acid by weaker acids such as acetic, formic or perchloric, and the careful monitoring of the acidity of extracts during the processes of purification. [Exceptionally, a stronger acid (3% aqueous trifluoroacetic) was used by Kondo et al. (1985) to isolate malonated pigments from Monarda didyma.]

Because of this unusual lability, a variety of pigments now known to be zwitterionic were previously reported to be unacylated. In particular, the petal of the blue cornflower, Centaurea cyanis, the classic source of cyanidin 3,5-diglucoside, have now been shown to contain the 3-(6"-succinylglucoside)-5-glucoside using milder methods of extraction and purification (Takeda and Tominaga, 1983; Tamura et al., 1983). Subsequent reinvestigation of other members of the Compositae has shown that most flower pigments are zwitterionic, with malonated anthocyanins occurring in species of Callistephus, Cichorium, Coleostephus, Dahlia and Helenium (Takeda et al., 1986a). Zwitterionic anthocyanins are also distinguished from other anthocyanins by their chromatographic properties and by their high retention times on HPLC (see Table 1.2). FAB-MS and NMR spectroscopy have proved invaluable for their characterization.

Structures of the zwitterionic anthocyanins known to date are listed in Table 1.6. Most are malonic acid derivatives but anthocyanins substituted by malic, oxalic and succinic acids have also been described. Commonly, a single malonic acid is present substituted at the 6-position of the 3-sugar moiety, as in cyanidin 3-(6"-malonylglocoside) from leaves of Cichorium intybus (Bridle et al., 1984). Dimalonates are known, with the second malonic acid substituting at a 5-glucose (as in the 3,5-dimalonylglucosides in Dahlia) or with disubstitution at the same glucose residue (as in the 3-dimalonylglucoside of cyanidin in Coleostephus) (Takeda et al., l986a).

Pigments with both malonic acid and aromatic acid substitution have been encountered. Typical is malonylawobanin (1.5) with p-coumaryl and malonyl residues, which occurs in the blue flowers of Commelina communis (Goto et al., 1983b) and in the bluebell, Hyacinthoides nonscripta (Takeda et al., 1986b). Another example is the pigment of Monarda didyma, monardaein (1.6), which has been the subject of several earlier investigations but is now known to be pelargonidin 3, 5-diglucoside with ρ-coumaric acid acylating the 3-sugar and two malonyl residues at the 5-sugar (Kondo et al., 1985). The most complex structure of this type to date is cinerarin (1.7) from Senecio cruentus (Goto et al., 1984) which is substituted by caffeic and malonic acids. The six pigments, ternatins A-F, from flowers of Clitoria ternatea are also known to have both ρ-coumaric and malonic acid residues linked to delphinidin 3,3',5'-triglucoside, but the precise modes of linkage are still not known (Saito et al., 1985a).

Table 1.7 Families with zwitterionic anthocyanins

Family Species
frequency
DICOTYLEDONS
Caryophyllaceae* 5/12
Compositae* 35/39
Convolvulaceae 1/3
Cruciferae* 5/6
Euphorbiaceae 1/1
Gesneriaceae 1/5
Labiatae* 17/17
Leguminosae* 3/14
Lobeliaceae 1/1
Malvaceae 2/4
Papaveraceae* 1/4
Polemoniaceae 2/3
Ranunculaceae 9/10
Scrophulariaceae 10/14
Solanaceae 1/7
Thymeliaceae 2/2
Verbenaceae* 1/1
MONOCOTYLEDONS
Alliaceae 4/4
Commelinaceae* 1/6
Gramineae* 2/2
Iridaceae 1/17
Lemnaceae* 1/1
Liliaceae* 3/11
Orchidaceae* 3/3

*Pigments identified in members of these families; see Tables 1.7 and 1.9.

Many of the pigments listed in Table 1.6 as being zwitterionic were originally thought to be unacylated. Structural revision of further known anthocyanins will probably be necessitated by the discovery that malonylation is a regular feature of anthocyanin chemistry. Since zwitterionic anthocyanins are relatively widespread, having already been recorded in 24 plant families (Table 1.7) (Harborne, 1986), many more structures of this type remain to be elucidated. Organic acids other than the four so far identified will almost certainly be encountered in future investigations.

Rose Pigments