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Three covalent anthocyanin-flavonol complexes (pigments 1-3) were extracted from the violet-blue flower of Allium 'Blue Perfume' with 5% acetic acid-MeOH solution, in which pigment 1 was the dominant pigment. These three pigments are based on delphinidin 3-glucoside as their deacylanthocyanin and were acylated with malonyl kaempferol 3-sophoroside-7-glucosiduronic acid or malonyl-kaempferol 3-p-coumaroyl-tetraglycoside-7-glucosiduronic acid in addition to acylation with acetic acid. By spectroscopic and chemical methods, the structures of these three pigments 1-3 were determined to be: pigment 1, (6(I)-O-(delphinidin 3-O-(3(I)-O-(acetyl)-β-glucopyranoside(I))))(2(VI)-O-(kaempferol 3-O-(2(II)-O-(3(III)-O-(β-glucopyranosyl(V))-β-glucopyranosyl(III))-4(II)-O-(trans-p-coumaroyl)-6(II)-O-(β-glucopyranosyl(IV))-β-glucopyranoside(II))-7-O-(β-glucosiduronic acid(VI)))) malonate; pigment 2, (6(I)-O-(delphinidin 3-O-(3(I)-O-(acetyl)-β-glucopyranoside(I))))(2(VI)-O-(kaempferol 3-O-(2(II)-O-β-glucopyranosyl(III))-β-glucopyranoside(II))-7-O-(β-glucosiduronic acid(VI)))); and pigment 3, (6(I)-O-(delphinidin 3-O-(3(I)-O-(acetyl)-β-glucopyranoside(I))))(2(VI)-O-(kaempferol 3-O-(2(II)-O-(3(III)-O-(β-glucopyranosyl(V))-β-glucopyranosyl(III))-4(II)-O-(cis-p-coumaroyl)-6(II)-O-(β-glucopyranosyl(IV))-β-glucopyranoside(II))-7-O-(β-glucosiduronic acid(VI)))) malonate. The structure of pigment 2 was analogous to that of a covalent anthocyanin-flavonol complex isolated from Allium schoenoprasum where delphinidin was observed in place of cyanidin. The three covalent anthocyanin-flavonol complexes (pigment 1-3) had a stable violet-blue color with three characteristic absorption maxima at 540, 547 and 618nm in pH 5-6 buffer solution. From circular dichroism measurement of pigment 1 in the pH 6.0 buffer solution, cotton effects were observed at 533 (+), 604 (-) and 638 (-) nm. Based on these results, these covalent anthocyanin-flavonol complexes were presumed to maintain a stable intramolecular association between delphinidin and kaempferol units closely related to that observed between anthocyanin and hydroxycinnamic acid residues in polyacylated anthocyanins. Additionally, an acylated kaempferol glycoside (pigment 4) was isolated from the same flower extract, and its structure was determined to be kaempferol 3-O-sophoroside-7-O-(3-O-(malonyl)-β-glucopyranosiduronic acid)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Six acylated delphinidin glycosides (pigments 1-6) and one acylated kaempferol glycoside (pigment 9) were isolated from the blue flowers of cape stock (Heliophila coronopifolia) in Brassicaceae along with two known acylated cyanidin glycosides (pigments 7 and 8). Pigments 1-8, based on 3-sambubioside-5-glucosides of delphinidin and cyanidin, were acylated with hydroxycinnamic acids at 3-glycosyl residues of anthocyanidins. Using spectroscopic and chemical methods, the structures of pigments 1, 2, 5, and 6 were determined to be: delphinidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(acyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside], in which acyl moieties were, respectively, cis-p-coumaric acid for pigment 1, trans-caffeic acid for pigment 2, trans-p-coumaric acid for pigment 5 (a main pigment) and trans-ferulic acid for pigment 6, respectively. Moreover, the structure of pigments 3 and 4 were elucidated, respectively, as a demalonyl pigment 5 and a demalonyl pigment 6. Two known anthocyanins (pigments 7 and 8) were identified to be cyanidin 3-(6-p-coumaroyl-sambubioside)-5-(6-malonyl-glucoside) for pigment 7 and cyanidin 3-(6-feruloyl-sambubioside)-5-(6-malonyl-glucoside) for pigment 8 as minor anthocyanin pigments. A flavonol pigment (pigment 9) was isolated from its flowers and determined to be kaempferol 3-O-[6-O-(trans-feruloyl)-β-glucopyranoside]-7-O-cellobioside-4'-O-glucopyranoside as the main flavonol pigment. On the visible absorption spectral curve of the fresh blue petals of this plant and its petal pressed juice in the pH 5.0 buffer solution, three characteristic absorption maxima were observed at 546, 583 and 635 nm. However, the absorption curve of pigment 5 (a main anthocyanin in its flower) exhibited only one maximum at 569 nm in the pH 5.0 buffer solution, and violet color. The color of pigment 5 was observed to be very unstable in the pH 5.0 solution and soon decayed. In the pH 5.0 solution, the violet color of pigment 5 was restored as pure blue color by addition of pigment 9 (a main flavonol in this flower) like its fresh flower, and its blue solution exhibited the same three maxima at 546, 583 and 635 nm. On the other hand, the violet color of pigment 5 in the pH 5.0 buffer solution was not restored as pure blue color by addition of deacyl pigment 9 or rutin (a typical flower copigment). It is particularly interesting that, a blue anthocyanin-flavonol complex was extracted from the blue flowers of this plant with H(2)O or 5% HOAc solution as a dark blue powder. This complex exhibited the same absorption maxima at 546, 583 and 635 nm in the pH 5.0 buffer solution. Analysis of FAB mass measurement established that this blue anthocyanin-flavonol complex was composed of one molecule each of pigment 5 and pigment 9, exhibiting a molecular ion [M+1] (+) at 2102 m/z (C(93)H(105)O(55) calc. 2101.542). However, this blue complex is extremely unstable in acid solution. It really dissociates into pigment 5 and pigment 9., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Acylated malvidin 3-glucoside was isolated from the purple flowers of Impatiens textori Miq. as a major anthocyanin component along with malvidin 3-(6''-malonyl-glucoside). Its structure was elucidated to be malvidin 3-O-[6-O-(3-hydroxy-3-methylglutaryl)-beta-glucopyranoside] by chemical and spectroscopic methods.

The structures of 11 acylated cyanidin 3-sophoroside-5-glucosides (pigments 1-11), isolated from the flowers of Iberis umbellata cultivars (Cruciferae), were elucidated by chemical and spectroscopic methods. Pigments 1-11 were acylated with malonic acid, p-coumaric acid, ferulic acid, sinapic acid and/or glucosylhydroxycinnamic acids. Pigments 1-11 were classified into four groups by the substitution patterns of the linear acylated residues at the 3-position of the cyanidin. In the first group, pigments 1-3 were determined to be cyanidin 3-O-[2-O-(2-O-(acyl)-beta-glucopyranosyl)-6-O-(trans-p-coumaroyl)-beta-glucopyranoside]-5-O-[6-O-(malonyl)-beta-glucopyranoside], in which the acyl moiety varied with none for pigment 1, ferulic acid for pigment 2 and sinapic acid for pigment 3. In the second one, pigments 4-6 were cyanidin 3-O-[2-O-(2-O-(acyl)-beta-glucopyranosyl)-6-O-(4-O-(beta-glucopyranosyl)-trans-p-coumaroyl)-beta-glucopyranoside]-5-O-[6-O-(malonyl)-beta-glucopyranoside], in which the acyl moiety varied with none for pigment 4, ferulic acid for pigment 5 and sinapic acid for pigment 6. In the third one, pigments 7-9 were cyanidin 3-O-[2-O-(2-O-(acyl)-beta-glucopyranosyl)-6-O-(4-O-(6-O-(trans-feruloyl)-beta-glucopyranosyl)-trans-p-coumaroyl)-beta-glucopyranoside]-5-O-[6-O-(malonyl)-beta-glucopyranoside], in which the acyl moiety varied with none for pigment 7, ferulic acid for pigment 8, and sinapic acid for pigment 9. In the last one, pigments 10 and 11 were cyanidin 3-O-[2-O-(2-O-(acyl)-beta-glucopyranosyl)-6-O-(4-O-(6-O-(4-O-(beta-glucopyranosyl)-trans-feruloyl)-beta-glucopyranosyl)-trans-p-coumaroyl)-beta-glucopyranoside]-5-O-[6-O-(malonyl)-beta-glucopyranoside], in which acyl moieties were none for pigment 10 and ferulic acid for pigment 11. The distribution of these pigments was examined in the flowers of four cultivars of I. umbellata by HPLC analysis. Pigment 1 acylated with one molecule of p-coumaric acid was dominantly observed in purple-violet cultivars. On the other hand, pigments (9 and 11) acylated with three molecules of hydroxycinnamic acids were observed in lilac (purple-violet) cultivars as major anthocyanins. The bluing effect and stability on these anthocyanin colors were discussed in relation to the molecular number of hydroxycinnamic acids in these anthocyanin molecules.

Anthocyanins were isolated from orange-red flowers of Catharanthus roseus cv 'Equator Deep Apricot', and identified as rosinidin 3-O-[6-O-(alpha-rhamnopyranosyl)-beta-galactopyranoside] (1), and also 7-O-methylcyanidin 3-O-[6-O-(alpha-rhamnopyranosyl)-beta-galactopyranoside] (2) by chemical and spectroscopic methods. Pigment 1 was found to be a major anthocyanin in the flowers of this cultivar. By contrast, the distribution of rosinidin glycosides is very limited in plants, and reported only in the flowers of Primula. Pigment 2 was found in smaller concentrations, but its aglycone, 7-O-methylcyanidin, has been reported only once before, from the fruit of mango.

Three acylated cyanidin 3-(3(X)-glucosylsambubioside)-5-glucosides (1-3) and one non-acylated cyanidin 3-(3(X)-glucosylsambubioside)-5-glucoside (4) were isolated from the purple-violet or violet flowers and purple stems of Malcolmia maritima (L.) R. Br (the Cruciferae), and their structures were determined by chemical and spectroscopic methods. In the flowers of this plant, pigment 1 was determined to be cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-3-O-(beta-D-glucopyranosyl)-beta-D-xylopyranosyl)-6-O-(trans-p-coumaroyl)-beta-D-glucopyranoside]-5-O-[6-O-(malonyl)-(beta-D-glucopyranoside) as a major pigment, and a minor pigment 2 was determined to be the cis-p-coumaroyl isomer of pigment 1. In the stems, pigment 3 was determined to be cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-3-O-(beta-D-glucopyranosyl)-beta-D-xylopyranosyl)-6-O-(trans-p-coumaroyl)-beta-d-glucopyranoside]-5-O-(beta-D-glucopyranoside) as a major anthocyanin, and also a non-acylated anthocyanin, cyanidin 3-O-[2-O-(3-O-(beta-D-glucopyranosyl)-beta-D-xylopyranosyl)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside) was determined to be a minor pigment (pigment 4). In this study, it was established that the acylation-enzymes of malonic acid has important roles for the acylation of 5-glucose residues of these anthocyanins in the flower-tissues of M. maritima; however, the similar enzymatic reactions seemed to be inhibited or lacking in the stem-tissues.

The dicaffeoyl anthocyanin, phacelianin, was isolated from blue petals of Phacelia campanularia. Its structure was determined to be 3-O-(6-O-(4'-O-(6-O-(4'-O-beta-d-glucopyranosyl-(E)-caffeoyl)-beta-d-glucopyranosyl)-(E)-caffeoyl)-beta-d-glucopyranosyl)-5-O-(6-O-malonyl-beta-d-glucopyranosyl)delphinidin. The CD of the blue petals of the phacelia showed a strong negative Cotton effect and that of the suspension of the colored protoplasts was the same, indicating that the chromophores of phacelianin may stack intermolecularly in an anti-clockwise stacking manner in the blue-colored vacuoles. In a weakly acidic aqueous solution, phacelianin displayed the same blue color and negative Cotton effect in CD as those of the petals. However, blue-black colored precipitates gradually formed without metal ions. A very small amount of Al(3+) or Fe(3+) may be required to stabilize the blue solution. Phacelianin may take both an inter- and intramolecular stacking form and shows the blue petal color by molecular association and the co-existence of a small amount of metal ions. We also isolated a major anthocyanin from the blue petals of Evolvulus pilosus and revised the structure identical to phacelianin.

Five acylated peonidin glycosides were isolated from the pale gray-purple flowers of a duskish mutant in the Japanese morning glory (Ipomoea nil or Pharbitis nil) as major pigments, along with a known anthocyanin, Heavenly Blue Anthocyanin (HBA). Three of these were based on peonidin 3-sophoroside and two on peonidin 3-sophoroside-5-glucoside as their deacylanthocyanins; both deacylanthocyanins were acylated with caffeic acid and/or glucosylcaffeic acids. By spectroscopic and chemical methods, the structures of the former three pigments were determined to be 3-O-[2-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-beta-D-glucopyranoside], 3-O-[2-O-(6-O-(3-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-6-O-(4-O-(6-O-(3-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-glucopyranoside], and 3-O-[2-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-6-O-(4-O-(6-O-(3-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranoside] of peonidin. The structures of the latter two pigments were also confirmed as 3-O-[2-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-beta-D-glucopyranoside]-5-O-beta-D-glucopyranoside, and 3-O-[2-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-6-O-(4-O-(6-O-(3-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranoside]-5-O-beta-D-glucopyranoside of peonidin. The mutation affecting glycosylation and acylation in anthocyanin biosynthesis of Japanese morning glory was discussed.

Five polyacylated anthocyanins were isolated from blue-violet flowers of Anemone coronaria 'St. Brigid'. They were identified as delphinidin 3-O-[2-O-(2-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-6-O-(malonyl)-beta-D-galactopyranoside]-7-O-[6-O-(trans-caffeoyl)-beta-D-glucopyranoside]-3'-O-[beta-D-glucuronopyranoside], and its demalonylated form, delphinidin 3-O-[2-O-(2-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-6-O-(2-O-tartaryl)malonyl)-beta-D-galactopyranoside]-7-O-[6-O-(trans-caffeoyl)-beta-D-glucopyranoside]-3'-O-[beta-D-glucuronopyranoside], and its cyanidin analog as well as delphinidin 3-O-[2-O-(2-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-6-O-(2-O-(tartaryl)malonyl)-beta-D-galactopyranoside]-7-O-[6-O-(trans-caffeoyl)-beta-D-glucopyranoside].

The recently isolated pigments from Petunia integrifolia and Triteleia bridgesii present a distinct feature that sheds new light on the understanding of intramolecular copigmentation of anthocyanins. These are among the infrequent anthocyanins that naturally present a coumaric acid substituent in both cis and trans forms. As a consequence, the two isomers demonstrate substantial variations of their thermodynamic and kinetic constants and also colour properties. A possible explanation for these characteristics is presented, making use of molecular modelling and taking into account the three-dimensional structures of the pigments.

Three acylated anthocyanins were isolated from the scarlet flowers of Anemone coronaria 'St. Brigid Red' along with a known pigment, pelargonidin 3-lathyroside. The structures of the acylated pigments were based on a pelargonidin 3-lathyroside skeleton acylated at different positions with malonic acid. The first pigment was identified as pelargonidin 3-O-[2-(beta-D-xylopyranosyl)-6-O-(malonyl)-beta-D-galactopyranoside], the second was pelargonidin 3-O-[2-O-(beta-D-xylopyranosyl)-6-O-(methyl-malonyl)-beta-D-galactopyranoside], and the third was (6''-O-(pelargonidin 3-O-[2''-O-(beta-D-xylopyranosyl)-beta-D-galactopyranosyl]))((4-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-O-tartatryl)malonate.

Four new acylated delphinidin 3,7-glycosides were isolated from the blue-violet flowers of Consolida armeniaca as major anthocyanin pigments. The first pigment was based on delphinidin 3,7-diglucoside and was identified as delphinidin 3-O-[6-O-(malonyl)-beta-D-glucopyranoside]-7-O- [6-O-(4-O-(6-O-(p-hydroxybenzoyl)-beta-D-glucopyranosyl)-p-hydroxybenzoy l)- beta-D-glucopyranoside] by spectral methods. The other three acylated anthocyanins were based on delphinidin 3-glucoside-7-sophoroside. The second pigment was determined to be delphinidin 3-O-[6-O-(malonyl)-beta-D-glucopyranoside]-7-O-[2-O-(beta-D- glucopyranosyl)-6-O-(4-O-(6-O-(p-hydroxybenzoyl)-beta-D-glucopyranosyl)- p- hydroxybenzoyl)-beta-D-glucopyranoside]. The third pigment was elucidated to be delphinidin 3-O-[6-O-(malonyl)-beta-D-glucopyranoside]-7-O-[2-O-(6-O-(p- hydroxybenzoyl)-beta-D-glucopyranosyl)-6-O-(4-O-(6-O-(p-hydroxybenzoyl)- beta-D- glucopyranosyl)-p-hydroxybenzoyl)-beta-D-glucopyranoside], and the fourth as delphinidin 3-O-[6-O-(malonyl)-beta-D-glucopyranoside]-7- O-glucopyranosyl[(2-O-(6-O-(p-hydroxybenzoyl)-beta-D-glucopyranosyl)-6-O - (4-O-(6-O-(p-hydroxybenzoyl)-beta-D-glucopyranosyl)-p-hydroxybenzoyl)-be ta-D- glucopyranoside].

Two series of structurally related anthocyanins, extracted from the blue flowers of Evolvulus pilosus cv. Blue Daze and from the blue-purple flowers of Eichhornia crassipes, exhibit remarkable colour stabilities in aqueous solution at mildly acidic pH values. All the pigments possess the same chromophore (delphinidin), but a different pattern of glycosylation and acylation. Moreover, one of the pigments has an apigenin 7-glucoside molecule (a flavone) attached to the glycosidic chain by two ester bonds with malonic acid, instead of an aromatic acid and is the only known anthocyanin with such a structure. All the molecules studied, except one which has only a 3-gentiobioside (a disaccharide) as substituent, denote an effect of reduction in the hydration constant when compared with the parent delphinidin 3-glucoside or 3,5-diglucoside molecules, which supports the existence of intramolecular hydrophobic interactions between the chromophoric skeleton and the acyl or flavonoid groups. The role played by the sugar units on the hydrophobicity/hydrophilicity of the pigments is also discussed.

A new acylated anthocyanin was isolated from the red-purple flowers of Dendrobium 'Pramot' (phalaenopsis type cv) as a major anthocyanin. The structure of this pigment was determined to be cyanidin-3-O-malonyl)-beta-D-glucopyranoside) 7,3'-di-O-(6-O-(4-O-(beta-D- glucopyranosyl)oxybenzoyl)-beta-D-glucopyranoside) mainly based on the spectroscopic evidence.

A novel acylated delphinidin glycoside was isolated from the blue-purple flowers of Eichhornia crassipes as a major pigment, and was identified as (6'''-O-(delphinidin 3-O-(6''-O-(beta-D-glucopyranosyl)-beta-D-glucopyranosyl))) (6''-O-(apigenin 7-O-(beta-D-glucopyranosyl)))malonate by spectral methods. The three dimensional structure of this pigment was suggested from the observation of the negative Cotton effect at lambda max 535 nm that delphinidin (chromophore) and apigenin (co-pigment) occupy a folding conformation as a binary complex.