Characterization of a Natural, Stable, Reversible and Colourful Anthocyanidin Network from Sphagnum Moss Based Mainly on the Yellow Trans-Chalcone and Red Flavylium Cation Forms.

Anthocyanins with various functions in nature are one of the most important sources of colours in plants. They are based on anthocyanidins or 3-deoxyanthocyanidins having in common a C15-skeleton and are unique in terms of how each anthocyanidin is involved in a network of equilibria between different forms exhibiting their own properties including colour. Sphagnorubin C (1) isolated from the cell wall of peat moss (Sphagnum sp.) was in fairly acidic and neutral dimethyl sulfoxide characterized by nuclear magnetic resonance (NMR) and ultraviolet-visible (UV-vis) absorption techniques. At equilibrium, the network of 1 behaved as a two-component colour system involving the reddish flavylium cationic and the yellow trans-chalcone forms. The additional D- and E-rings connected to the common C15-skeleton extend the π-conjugation within the molecule and provide both bathochromic shifts in the absorption spectra of the various forms as well as a low isomerization barrier between the cis- and trans-chalcone forms. The hemiketal and cis-chalcone forms were thus not observed experimentally by NMR due to their short lives. The stable, reversible network of 1 with good colour contrast between its two components has previously not been reported for other natural anthocyanins and might thus have potential in future photochromic systems. This is the first full structural characterization of any naturally occurring anthocyanin chalcone form.

Nutraceutical Characterization of Anthocyanin-Rich Fruits Produced by "Sun Black" Tomato Line.

Tomato (Solanum lycopersicum L.) is one of the most cultivated vegetable in the world and it represents a large source of bioactive compounds, including carotenoids and polyphenols (phenolic acids and flavonoids). However, the concentration of flavonoids in tomato is considered sub-optimal, particularly because anthocyanins are not generally present. Therefore, this crop has been the object of an intense metabolic engineering in order to obtain anthocyanin-enriched tomatoes by using either breeding or transgenic strategies. Some wild tomato species, such as S. chilense and S. cheesmaniae, biosynthesize anthocyanins in the fruit sub-epidermal tissue, and some alleles from those genotypes have been introgressed into a new developed purple tomato line, called "Sun Black" (SB). It is a tomato line with a purple skin color, both in green and in red fruit stages, due to the biosynthesis of anthocyanins in the peel, and a normal red color pulp, with a taste just like a traditional tomato. SB is the result of a breeding programme and it is not a genetically modified (GM) product. We report the chemical characterization and structure elucidation of the attractive anthocyanins found in the peel of SB tomato, as well as other bioactive compounds (carotenoids, polyphenols, vitamin C) of the whole fruit. Using one- and two-dimensional NMR experiments, the two main anthocyanins were identified to be petunidin 3-O-[6″-O-(4‴-O-E-p-coumaroyl-α-rhamnopyranosyl) -ß-glucopyranoside]-5-O-ß-glucopyranoside (petanin) and malvidin 3-O-[6″-O-(4‴-O-E-p-coumaroyl-α-rhamnopyranosyl)-ß-glucopyranoside]-5-O-ß-glucopyranoside (negretein). The total anthocyanins in the whole ripe fruit was 1.2 mg/g dry weight (DW); 7.1 mg/100 g fresh weight (FW). Chlorogenic acid (the most abundant phenolic acid) was 0.6 mg/g DW; 3.7 mg/100 g FW. The main flavonol, rutin was 0.8 mg/g DW; 5 mg/100 g FW. The total carotenoid content was 211.3 µg/g DW; 1,268 µg/100 g FW. The total phenolic content was 8.6 mg/g DW; 52.2 mg/100 g FW. The vitamin C content was 37.3 mg/100 g FW. The antioxidant activities as measured by the TEAC and ORAC assays were 31.6 and 140.3 µmol TE/g DW, respectively (193 and 855.8 µmol TE/100 g FW, respectively). The results show the unique features of this new tomato genotype with nutraceutical properties.

Auronidins are a previously unreported class of flavonoid pigments that challenges when anthocyanin biosynthesis evolved in plants.

Anthocyanins are key pigments of plants, providing color to flowers, fruit, and foliage and helping to counter the harmful effects of environmental stresses. It is generally assumed that anthocyanin biosynthesis arose during the evolutionary transition of plants from aquatic to land environments. Liverworts, which may be the closest living relatives to the first land plants, have been reported to produce red cell wall-bound riccionidin pigments in response to stresses such as UV-B light, drought, and nutrient deprivation, and these have been proposed to correspond to the first anthocyanidins present in early land plant ancestors. Taking advantage of the liverwort model species Marchantia polymorpha, we show that the red pigments of Marchantia are formed by a phenylpropanoid biosynthetic branch distinct from that leading to anthocyanins. They constitute a previously unreported flavonoid class, for which we propose the name "auronidin," with similar colors as anthocyanin but different chemistry, including strong fluorescence. Auronidins might contribute to the remarkable ability of liverworts to survive in extreme environments on land, and their discovery calls into question the possible pigment status of the first land plants.

Radical Scavenging and Anti-Inflammatory Activities of Representative Anthocyanin Groupings from Pigment-Rich Fruits and Vegetables.

Anthocyanins, the naturally occurring pigments responsible for most red to blue colours of flowers, fruits and vegetables, have also attracted interest because of their potential health effects. With the aim of contributing to major insights into their structure-activity relationship (SAR), we have evaluated the radical scavenging and biological activities of selected purified anthocyanin samples (PASs) from various anthocyanin-rich plant materials: two fruits (mahaleb cherry and blackcurrant) and two vegetables (black carrot and "Sun Black" tomato), differing in anthocyanin content (ranging from 4.9 to 38.5 mg/g DW) and molecular structure of the predominant anthocyanins. PASs from the abovementioned plant materials have been evaluated for their antioxidant capacity using Trolox Equivalent Antioxidant Capacity (TEAC) and Oxygen Radical Absorbance Capacity (ORAC) assays. In human endothelial cells, we analysed the anti-inflammatory activity of different PASs by measuring their effects on the expression of endothelial adhesion molecules VCAM-1 and ICAM-1. We demonstrated that all the different PASs showed biological activity. They exhibited antioxidant capacity of different magnitude, higher for samples containing non-acylated anthocyanins (typical for fruits) compared to samples containing more complex anthocyanins acylated with cinnamic acid derivatives (typical for vegetables), even though this order was slightly reversed when ORAC assay values were expressed on a molar basis. Concordantly, PASs containing non-acylated anthocyanins reduced the expression of endothelial inflammatory antigens more than samples with aromatic acylated anthocyanins, suggesting the potential beneficial effect of structurally diverse anthocyanins in cardiovascular protection.

Aromatic Decoration Determines the Formation of Anthocyanic Vacuolar Inclusions.

Anthocyanins are some of the most widely occurring secondary metabolites in plants, responsible for the orange, red, purple, and blue colors of flowers and fruits and red colors of autumn leaves. These pigments accumulate in vacuoles, and their color is influenced by chemical decorations, vacuolar pH, the presence of copigments, and metal ions. Anthocyanins are usually soluble in the vacuole, but in some plants, they accumulate as discrete sub-vacuolar structures. Studies have distinguished intensely colored intra-vacuolar bodies observed in the cells of highly colored tissues, termed anthocyanic vacuolar inclusions (AVIs), from more globular, membrane-bound anthocyanoplasts. We describe a system in tobacco that adds additional decorations to the basic anthocyanin, cyanidin 3-O-rutinoside, normally formed by this species. Using this system, we have been able to establish which decorations underpin the formation of AVIs, the conditions promoting AVI formation, and, consequently, the mechanism by which they form.

High concentrations of aromatic acylated anthocyanins found in cauline hairs in Plectranthus ciliatus.

Vegetative shoots of a naturalized population of purple-leaved plectranthus (Plectranthus ciliatus, Lamiaceae) were found to contain four main anthocyanins: peonidin 3-(6″-caffeoyl-ß-glucopyranoside)-5-ß-glucopyranoside, peonidin 3-(6″-caffeoyl-ß-glucopyranoside)-5-(6‴-malonyl-ß-glucopyranoside), peonidin 3-(6″-E-p-coumaroyl-ß-glucopyranoside)-5-(6‴-malonyl-ß-glucopyranoside), and peonidin 3-(6″-E-p-coumaroyl-ß-glucopyranoside)-5-ß-glucopyranoside. The first three of these pigments have not been reported previously from any plant. They all follow the typical anthocyanin pattern of Lamiaceae, with universal occurrence of anthocyanidin 3,5-diglucosides and aromatic acylation with p-coumaric and sometimes caffeic acids; however, they differ by being based on peonidin. The four anthocyanins were present in the leaves (22.2 mg g(-1) DW), and in the xylem and interfascicular parenchyma of the stem. They were exceptionally abundant, among the highest reported for any plant organ, in epidermal hairs on some of the stem internodes (101 mg g(-1) DW). Anthocyanin content in these hairs increased more than three-fold from the youngest to the fourth-youngest internodes. In situ absorbances (λmax ≈ 545 nm) were bathochromic in comparison to absorbances of the isolated anthocyanins in their flavylium form in acidified aqueous solutions (λmax = 525 nm), suggesting that the anthocyanins occur both in quinoidal and flavylium forms in constant proportions in the anthocyanic hair cells. The most distinctive observation with respect to relative proportions of individual anthocyanins was found in de-haired internodes, for which anthocyanin caffeoyl-derivatives decreased, and anthocyanin coumaroyl-derivatives increased, from the youngest to the fourth-youngest internode.

Purple anthocyanin colouration on lower (abaxial) leaf surface of Hemigraphis colorata (Acanthaceae).

The functional significance of anthocyanin colouration of lower (abaxial) leaf surfaces is not clear. Two anthocyanins, 5-O-methylcyanidin 3-O-(3″-(ß-glucuronopyranosyl)-ß-glucopyranoside) (1) and 5-O-methylcyanidin 3-O-ß-glucopyranoside (2), were isolated from Hemigraphis colorata (Blume) (Acanthaceae) leaves with strong purple abaxial colouration (2.2 and 0.6mg/gfr.wt., respectively). The glycosyl moiety of 1, the disaccharide 3″-(ß-glucuronopyranosyl)-ß-glucopyranoside), has previously been reported to occur only in a triterpenoid saponin, lindernioside A. The structural assignment of the aglycone of 1 and 2 is the first complete characterisation of a natural 7-hydroxy-5-methoxyanthocyanidin. Compared to nearly all naturally occurring anthocyanidins, the 5-O-methylation of this anthocyanidin limits the type of possible quinoidal forms of 1 and 2 to be those forms with keto-function in only their 7- and 4'-positions.

New anthocyanidin and anthocyanin pigments from blue plumbago.

Phytochemical investigations of blue plumbago ( Plumbago auriculata Poir. syn. Plumbago capensis Thunb.) flowers have led to the isolation of six new anthocyanins based on three new anthocyanidins with 5,7-dimethoxylated A-rings. Their structures were identified by 2D nuclear magnetic resonance and high-resolution mass spectrometry as the 3-O-ß-galactopyranosides (1,2,4) and 3-O-α-rhamnopyranosides (3,5,6) of 5,7-dimethyldelphinidin, 5,7-dimethylpetunidin, and 5,7-dimethylmalvidin. Identification of 1-6 implies new structures for the previously reported anthocyanidins pulchellidin, europinidin, and capensinidin to be 5,7-dimethoxy-3,3',4',5'-tetrahydroxyflavylium, 5,7,3'-trimethoxy-3,4',5'-trihydroxyflavylium, and 5,7,3',5'-tetramethoxy-3,4'-dihydroxyflavylium cations, respectively. The anthocyanins (0.4 mg/g flowers) were accompanied by the dihydroflavonol taxifolin 3'-O-ß-glucopyranoside (1.4 mg/g) and the flavonols 5-methylquercetin 3-O-α-rhamnopyranoside (8.8 mg/g) and 5-methylquercetin (0.4 mg/g). The anthocyanins 1-6 are the first reported natural anthocyanins with no free hydroxyl groups in their 5- and 7-positions on their A-rings. They have thus no possibility of forming the tautomeric quinonoidal bases (anhydrobases), which are related to the free hydroxyl groups in the 5- and 7-positions of previously reported anthocyanins. The genes behind the 5,7-dimethoxylated anthocyanins might be useful for making anthocyanins with special properties (colors, etc.).

Identification of cyanidin 3-O-ß-(6″-(3-hydroxy-3-methylglutaroyl)glucoside) and other anthocyanins from wild and cultivated blackberries.

Anthocyanins from blackberries are natural dietary pigments. The aim of this study was to investigate the occurrence of anthocyanins in fruits of wild Norwegian blackberries and three blackberry ( Rubus fruticosus L.) cultivars and to report the complete identification of cyanidin 3-O-ß-(6″-(3-hydroxy-3-methylglutaroyl)glucopyranoside), 5. This new pigment is most probably the same pigment that has previously been reported to occur in various blackberry samples as cyanidin 3-dioxalylglucoside. All of the examined blackberry samples contained in similar relative proportions the 3-glucoside (1), 3-rutinoside (2), 3-xyloside (3), and 3-O-ß-(6″-malonylglucoside) (4) of cyanidin and 5. The absolute amounts of 1-5 in the wild Norwegian blackberries were 249, 18, 10, 24, and 22 mg of cyanidin 3-glucoside equivalents/100 g of fresh weight, respectively.

Anthocyanins from Fuchsia flowers.

Flowers of Fuchsia arborescens, F. boliviana, F. fulgens var. 'Variegata', F. magellanica (Onagraceae) and twenty-nine F. magellanica cultivars contained some of the thirteen anthocyanidin 3,5-diglucosides (six), 3-monoglycosides (five) and 3-(2"-galloylglucosides) (two), which altogether were identified. Peonidin 3-O-(2"-O-galloyl-beta-glucopyranoside), which has not been reported before, was found in F. magellanica and F. fulgens var. 'Variegata'. The various corollas with purple nuances were correlated with a relatively high content of malvidin 3,5-diglucoside. Flower colors were to a large extent correlated with the number of oxygen substituents on the anthocyanidin B-ring of the major anthocyanins.

Anthocyanins with unusual furanose sugar (apiose) from leaves of Synadenium grantii (Euphorbiaceae).

Four anthocyanins, cyanidin 3-O-(2''-(5'''-(E-p-coumaroyl)-beta-apiofuranosyl)-beta-xylopyranoside)-5-O-beta-glucopyranoside, cyanidin 3-O-(2''-(5'''-(E-p-coumaroyl)-beta-apiofuranosyl)-beta-xylopyranoside), cyanidin 3-O-(2''-(5'''-(E-caffeoyl)-beta-apiofuranosyl)-beta-xylopyranoside) and cyanidin 3-O-(2''-(5'''-(E-feroyl)-beta-apiofuranosyl)-beta-xylopyranoside) were isolated from leaves of African milk bush, (Synadenium grantii Hook, Euphorbiaceae) together with the known cyanidin 3-O-beta-xylopyranoside-5-O-beta-glucopyranoside and cyanidin 3-O-beta-xyloside. The four former pigments are the first reported anthocyanins containing the monosaccharide apiose, and the three 5'''-cinnamoyl derivative-2''-(beta-apiosyl)-beta-xyloside subunits have previously not been reported for any compound.

Flavone C-glycosides from seeds of fenugreek, Trigonella foenum-graecum L.

Fenugreek (Trigonella foenum-graecum L.) is particularly used in Asia, Africa, and Mediterranean countries for its nutritional and medicinal value. The flavone C-glycosides, apigenin 6-C-beta-chinovopyranosyl-8-C-beta-galactopyranoside (6) and apigenin 6-C-beta-xylopyranosyl-8-C-(6'''-O-(3-hydroxy-3-methylglutaroyl)-beta-glucopyranoside) (7), in addition to the known flavone C-glycosides, apigenin 6,8-C-di-beta-galactopyranoside (1), apigenin 6-C-beta-xylopyranosyl-8-C-beta-galactopyranoside (2), apigenin 6-C-beta-arabinopyranosyl-8-C-beta-galactopyranoside (3), luteolin 8-C-beta-glucopyranoside (4), luteolin 6-C-beta-glucopyranoside (5), apigenin 8-C-beta-glucopyranoside (8), apigenin 6-C-beta-glucopyranoside (9), luteolin 8-C-(2''-O-(E)-p-coumaroyl-beta-glucopyranoside) (10), and apigenin 8-C-(2''-O-(E)-p-coumaroyl-beta-glucopyranoside) (11) were isolated from fenugreek seeds. Compounds 1, 5, and 10 were reported for the first time in this species. Signal duplication in the NMR spectra, with exception of spectra of the mono-6-C-substituted compounds, revealed the presence of rotameric conformers, created by rotational hindrance at the C (sp(3))-C (sp(2)) glycosyl-flavone linkage in these flavone C-glycosides.

Structural properties of anthocyanins: rearrangement of C-glycosyl-3-deoxyanthocyanidins in acidic aqueous solutions.

Seven C-glycosyl-3-deoxyanthocyanidins were made from their corresponding C-glycosylflavones. The structures of their rearrangement products, which were formed in acidic aqueous solutions, were elucidated. Rotameric conformers were detected for all of the 8-C-glycosyldeoxyanthocyanidins but were absent for their isomeric 6-C-analogues in acidified methanolic NMR solvent. A correlation method based on HPLC-DAD and NMR integration of similar samples made it possible for the first time to determine accurately the proportions of two isomeric 6-C- and 8-C-glycosylflavonoids occurring in mixtures. Each of the C-glycosyldeoxyanthocyanidins established fixed equilibrium proportions with their corresponding A-ring isomer in aqueous solutions, even under relatively strong acidic conditions (pH approximately 1), whether one started with pure 6-C- or 8-C-glycosyl-3-deoxyanthocyanidin. The nature of the aglycone, C-glycosyl moiety, and temperature were found to affect the equilibrium proportions. Increased water content (to a certain level) and temperatures were shown to increase the isomerization rates. The flavylium cations were the only equilibrium forms present at detectable quantities. The significance of rotation of the A-ring during isomerization was confirmed by lack of rearrangement of both 6-C- and 8-C-glycosyl-3-deoxy-5-carboxypyranoanthocyanidins. The intermediary C-ring open forms of the C-glycosyldeoxyanthocyanidins experience fast ring closure to their cyclic forms, which may reduce irreversible degradation reported for open chalcone forms of the common anthocyanins. The stable C-glycosyl-3-deoxyanthocyanidins may thus attract interest as possible colorants in the food industry, etc.

Acylated anthocyanins in inflorescence of spider flower (Cleome hassleriana).

Five anthocyanins, cyanidin 3-(2''-(6'''-caffeoyl-beta-glucopyranosyl)-6''-(E-p-coumaroyl)-beta-glucopyranoside)-5-beta-glucopyranoside, cyanidin 3-(2''-(6'''-E-sinapoyl-beta-glucopyranosyl)-6''-(E-p-coumaroyl)-beta-glucopyranoside)-5-beta-glucopyranoside, cyanidin 3-(2''-(6'''-feroyl-beta-glucopyranosyl)-6''-(E-p-coumaroyl)-beta-glucopyranoside)-5-beta-glucopyranoside, pelargonidin 3-(2''-(6'''-E-sinapoyl-beta-glucopyranosyl)-6''-(E-p-coumaroyl)-beta-glucopyranoside)-5-beta-glucopyranoside, and pelargonidin 3-(2''-(6'''-E-p-coumaroyl-beta-glucopyranosyl)-6''-(E-p-coumaroyl)-beta-glucopyranoside)-5-beta-glucopyranoside, together with five known anthocyanins have been identified in flowers of Cleome hassleriana Queen line. One monoacylated and four diacylated cyanidin 3-sophoroside-5-glucosides were identified as the main anthocyanins in flowers with mauve colouration, while a homologous glycosidic pattern based on pelargonidin was found in the five main anthocyanins from flowers with pink colouration. The anthocyanins identified in C. hassleriana share the same glycosidic pattern as anthocyanins isolated from the genera Raphanus, Brassica and Iberis in the sister family Brassicaceae.

C-glycosylanthocyanidins synthesized from C-glycosylflavones.

Nine C-glycosyldeoxyanthocyanidins, 6-C-beta-glucopyranosyl-7-O-methylapigeninidin, 6-C-beta-glucopyranosyl-7-O-methylluteolinidin, 6-C-beta-(2''-O-beta-glucopyranosylglucopyranosyl)-7-O-methylapigeninidin, 6-C-beta-(2''-O-beta-glucopyranosylglucopyranosyl)-7,4'-di-O-methylapigeninidin, 8-C-beta-glucopyranosylapigeninidin, 8-C-beta-(2''-O-alpha-rhamnopyranosylglucopyranosyl)apigeninidin, 8-C-beta-(2''-O-alpha-(4'''-O-acetylrhamnopyranosyl)glucopyranosyl)apigeninidin, 6,8-di-C-beta-glucopyranosylapigeninidin (8), 6,8-di-C-beta-glucopyranosyl-4'-O-methylluteolinidin (9), have been synthesized from their respective C-glycosylflavones (yields between 14% and 32%) by the Clemmensen reduction reaction using zinc-amalgam. The various precursors (C-glycosylflavones) of the C-glycosylanthocyanidins were isolated from either flowers of Iris sibirica L., leaves of Hawthorn 'Crataegi Folium Cum Flore', or lemons and oranges. This is the first time C-glycosylanthocyanidins have been synthesized. The structures of all flavonoids including the flavone rotamers were elucidated by 2D NMR techniques and high-resolution electrospray MS. The distribution of the various structural forms of 8 and 9 are different at pH 1.1, 4.5, and 7.0, however, the two pigments undergoes similar structural transformations at the various pH values. Pigments 8 and 9 with C-C linkages between the sugar moieties and the aglycone, were found to be far more stable towards acid hydrolysis than pelargonidin 3-O-glucoside, which has the typical anthocyanidin C-O linkage between the sugar and aglycone. This stability may extend the present use of anthocyanins as nutraceuticals, pharmaceuticals or colorants.

Molar absorptivities and reducing capacity of pyranoanthocyanins and other anthocyanins.

To improve accuracy in the determination of anthocyanin purity and succeeding antioxidant capacity, 1H and 13C nuclear magnetic resonance spectroscopy have been combined with high-performance liquid chromatography (HPLC) equipped with a diode array detector and UV-vis spectroscopy in the analysis of anthocyanidin 3-glycosides and 5-carboxypyranoanthocyanidin 3-glycosides. The molar absoptivity (epsilon) values were found to be relatively similar, in contrast to previously reported literature values, and the average epsilon values for both anthocyanidin 3-monoglycosides and 5-carboxypyranoanthocyanidin 3-glycosides were proposed to be 22,000 and 23,000 in acidified aqueous and methanolic solutions, respectively. To assess the influence of structure on the potential antioxidant capacity of anthocyanins, the 3-glucosides of pelargonidin (1), cyanidin (2), peonidin (3), delphinidin (4), petunidin (5), malvidin (6), 5-carboxypyranopelargonidin (8), 5-carboxypyranocyanidin (9), 5-carboxypyranodelphinidin (11), 5-carboxypyranopetunidin (12), and 5-carboxypyranomalvidin (13) and the 3-galactosides of cyanidin (7) and 5-carboxypyranocyanidin (14) were examined by a ferric ion reducing antioxidant power (FRAP) assay. The reducing capacities of the individual anthocyanins were in the range of 0.9-5.2 micromol of Trolox equivalents/micromol. The two 5-carboxypyranoanthocyanins 11 and 9 and the four common anthocyanins 2, 4, 7, and 14, all possessing pyrogallol or catechol type of B rings, showed the highest antioxidant capacity measured by FRAP. However, the inclusion of the 5-hydroxyl in the D ring and just one oxygen substituent on the B ring in 8 diminished the reducing capacity considerably. Correspondingly, electrochemical behavior of 5-carboxypyranoanthocyanidin 3-glucosides and anthocyanidin 3-glucosides was derived using HPLC coupled to a coulometric array detector set from 100 to 800 mV in increments of 100 mV. The relative order of the reducing capacity of the various 5-carboxypyranoanthocyanidin 3-glucosides and anthocyanidin 3-glucosides were nearly alike, whether determined by coulometric array detection or FRAP.

Reactivity of anthocyanins and pyranoanthocyanins. Studies on aromatic hydrogen-deuterium exchange reactions in methanol.

Reactivity studies involving anthocyanin structures and their equilibrium forms will lead to better understanding of the properties of these antioxidants. Hydrogen-deuterium (H --> D) exchange reactions at various sites of the 3-glucosides of delphinidin (1), petunidin (2), malvidin (3), and the corresponding 3-glucosides of carboxypyranodelphinidin (4), carboxypyranopetunidin (5), carboxypyranomalvidin (6), and the flavonol quercetin 3-O-(6-alpha-rhamnopyranosyl-beta-glucopyranoside)(7) have been examined at room temperature in pure CD 3OD and in CD 3OD acidified with CF 3CO 2D. The H --> D exchange rate constants of H-6 and H-8 of 2 determined from (1)H NMR integration data were found to be independent upon pigment concentration (up to 4 x 10 (-2) M) and trifluoroactic acid concentration (0-15%, v/v), respectively. This suggest that these reactions follow first-order kinetics and unexpectedly to be independent of the acid concentration. H-6 and H-8 of the flavylium cation A-rings of 1- 3, and in the corresponding hydrogens of the hemiketal forms, exchanged with half-lives of approximately 100 h ( 1) and approximately 50 h ( 2 and 3), respectively. The pyranoanthocyanins (4-6) experienced no H --> D exchange for the analogous hydrogens, but H --> D exchange of H-beta (H-4)(t 1/2 approximately 25 h) for these compounds was observed. Only H-8 underwent significant H --> D exchange in 7. It is concluded that a stabilization of the sigma-complexes, assumed to be the intermediates in the reactions, takes place for the common anthocyanins (1-3) contrary to the pyranoanthocyanins (4-6).

Anthocyanins in berries of ribes including gooseberry cultivars with a high content of acylated pigments.

Consumption of berries from various sources including the genus Ribes has been associated with diverse potential health benefits. The 14 examined cultivars of European gooseberry (R. grossularia L.) contained in various proportions the 3-glucoside (3), 3-rutinoside (4), 3-xyloside (7), 3-O-beta-(6' '-E-caffeoylglucopyranoside) (8), and 3-O-beta-(6' '-E-p-coumaroylglucopyranoside) (10) of cyanidin and the 3-rutinoside (6) and 3-glucoside of peonidin (5). Pigments 3, 4, delphinidin 3-rutinoside (2), delphinidin 3-glucoside (1), and minor amounts of 6, 7, and 10 were found in red flowering currant (R. sanguineum Pursh). Golden currant (R. aureum Pursh) contained 3, 4, and trace amounts of 1, 6, and 7, while alpine currant (R. alpinum L.) contained 3, 4, and trace amounts of 10. The major anthocyanins in two cultivars of jostaberries (R. x nidigrolaria Bauer), 1-4, 8, and 10, reflected that this hybrid contained the major anthocyanins of both parents, black currant and gooseberry. This is the first complete identification of 8 and the ring size of the sugar of 10. Pigment 9 was tentatively identified as cyanidin 3-(6' '-Z-p-coumaroylglucoside). This new pigment occurred in minor amounts (<2%) in all R. grosssularia and R. x nidigrolaria cultivars. No commercially available berries have been reported to contain such high proportions of aromatic acylated anthocyanins as found in the gooseberry cultivars "Samsø", "Hinnomäki Red", "Taastrup", "Lofthus", and "Glendal", which are in this context the most obvious candidates for consumption, colorant, and breeding programs.

Anthocyanin 3-galactosides from Cornus alba 'Sibirica' with glucosidation of the B-ring.

The three anthocyanins, delphinidin 3-O-beta-galactopyranoside-3',5'-di-O-beta-glucopyranoside (1), delphinidin 3-O-beta-galactopyranoside-3'-O-beta-glucopyranoside (2) and cyanidin 3-O-beta-galactopyranoside-3'-O-beta-glucopyranoside (3), and the 3-O-beta-galactopyranosides of delphinidin (4) and cyanidin (5) were isolated from the bluish white berries and compound umbel of Siberian dogwood, Cornus alba 'Sibirica'. The ornamental autumn leaves and the characteristic purplish red bark of this variety were found to contain only pigment 5.

Covalent anthocyanin-flavone dimer from leaves of Oxalis triangularis.

The anthocyanin-flavone C-glycoside, (malvidin 3-O-(6(II)-O-alpha-rhamnopyranosyl(AIV)-beta-glucopyranoside(AII))-5-O-beta-glucopyranoside(AIII)) (apigenin 6-C-(2(II)-O-beta-glucopyranosyl(FIII)-beta-glucopyranoside(FII))) malonate(AV) (A(IV)-4-->A(V)-1, F(III)-6-->A(V)-3) (1), has been isolated from leaves of Oxalis triangularis A. St.-Hil. In the 1D (1)H NMR spectrum of 1 dissolved in CD(3)OD-CF(3)CO(2)D (95:5), MTFA, recorded 45 min after sample preparation, this covalently linked dimer occurred mainly as flavylium cation (38%) and two equilibrium forms assigned to be quinonoidal bases (54%), whereas only minor amounts of the hemiacetal forms were present. After five days storage at 300 K, the hemiacetals (39%) and flavylium cation (38%) constituted the main forms of 1. More simple anthocyanins are normally considered to be on the flavylium cation form in acidified deuterated methanol. The cross-peaks observed in NOESY NMR spectra of 1 indicated the presence of vertical 'pi-pi' stacking between the B-ring of the flavone unit and the A-ring of each of the two forms assigned to be quinonoidal bases. It was not possible to discriminate between inter- or intramolecular association mechanisms. The equilibria between the various forms of 1 were studied by two-dimensional NOESY and ROESY NMR spectroscopy. 2D HSQC-TOCSY NMR spectroscopy was among the methods used for characterization of the various forms.