Advancing quantification methods for polyphenols in brown seaweeds-applying a selective qNMR method compared with the TPC assay.

INTRODUCTION: Brown seaweeds are a sustainable biomass with a potential for various industrial applications. Polyphenols are an important contributor to this potential. OBJECTIVE: The aim was total quantification of polyphenols in brown seaweeds from different tidal zones, using a selective 1 H quantitative NMR (qNMR) method, comparing the results with the colorimetric Folin-Ciocalteu total phenolic content (TPC) assay. METHOD: qNMR was performed with integration of selected peaks in the aromatic region (7-5.5 ppm). Deselection of non-polyphenolic 1 H signals was based on information from 2D (1 H-13 C, 1 H-15 N) NMR spectra. 13 C NMR phlorotannin characterisation facilitated the average number of protons expected to be found per aromatic ring used for the 1 H quantification. RESULTS: Selective qNMR and the TPC assay showed similar results for the three sublittoral growing species from the Laminariaceae; lower amounts for Laminaria hyperborea and Laminaria digitata (qNMR: 0.4%-0.6%; TPC: 0.6%-0.8%, phloroglucinol equivalents (PGE), dry weight (DW)) and higher amounts for Saccharina latissima (qNMR: 1.2%; TPC: 1.5%, PGE, DW). For the eulittoral Fucaceae, Fucus vesiculosus (qNMR: 1.1%; TPC: 4.1%; PGE, DW) and Ascophyllum nodosum (qNMR: 0.9%; TPC: 2.0%; PGE, DW), the TPC results were found to be up to three times higher than the qNMR results. The 13 C NMR characterisation showed the highest phlorotannin polymerisation degree for F. vesiculosus. CONCLUSION: The TPC assay provided similar polyphenolic amounts to the selective qNMR method for sublittoral species. For eulittoral growing species, the TPC method showed amounts up to three times higher than the qNMR method-most likely illustrating the lack of selectivity in the TPC assay.

Seasonal variation of phenolic compounds in Zostera marina (Zosteraceae) from the Baltic Sea.

Seasonal variations of phenolic compounds, in leaves of Zostera marina L. from the Baltic Sea near Kiel/Germany were investigated. Dominant compounds were mono- and disulfated flavonoids and phenylpropanoic acids, in particular luteolin 7,3'-O-disulfate and diosmetin 7-O-sulfate as well as rosmarinic acid, a dimeric phenylpropanoid. All detected sulfated flavones showed similar seasonal trends: there were two significant concentration peaks in June and November. Moreover, two geographically distinct flavonoid chemotypes were identified based on their respective main flavonoid; one chemotype was characterized by the prevalence of luteolin 7,3'-O-disulfate (German Baltic Sea), and the other by the prevalence of diosmetin 7-O-sulfate (Norwegian North Sea). Furthermore, an undescribed tetrameric phenylpropanoid, 7'',8''-didehydrosalvianolic acid B, was isolated and its structure was established by extensive NMR, MS, and CD experiments. This compound inhibited activity of Na+/K+-ATPase in the micro-molar range without any cytotoxic effects against human cancer and normal cells.

Variation in Phenolic Chemistry in Zostera marina Seagrass Along Environmental Gradients.

Chemical ecology has been suggested as a less time-consuming and more cost-efficient monitoring tool of seagrass ecosystems than traditional methods. Phenolic chemistry in Zostera marina samples was analyzed against latitude, sea depth, sample position within a seagrass meadow (periphery or center) and wave exposure. Multivariate data analysis showed that rosmarinic acid correlated moderately positively with depth, while the flavonoids had an overall strong negative correlation with increasing depth-possibly reflecting lack of stress-induced conditions with increasing depth, rather than a different response to light conditions. At a molecular level, the flavonoids were separated into two groups; one group is well described by the variables of depth and wave exposure, and the other group that was not well described by these variables-the latter may reflect biosynthetic dependencies or other unrevealed factors. A higher flavonoid/rosmarinic acid ratio was seen in the periphery of a seagrass meadow, while the contrary ratio was seen in the center. This may reflect higher plant stress in the periphery of a meadow, and the flavonoid/rosmarinic acid ratio may provide a possible molecular index of seagrass ecosystem health. Further studies are needed before the full potential of using variation in phenolic chemistry as a seagrass ecosystem monitoring tool is established.

Quantification of Polyphenols in Seaweeds: A Case Study of Ulva intestinalis.

In this case study, we explored quantitative 1H NMR (qNMR), HPLC-DAD, and the Folin-Ciocalteu assay (TPC) as methods of quantifying the total phenolic content of a green macroalga, Ulva intestinalis, after optimized accelerated solvent extraction. Tentative qualitative data was also acquired after multiple steps of purification. The observed polyphenolic profile was complex with low individual concentrations. The qNMR method yielded 5.5% (DW) polyphenols in the crude extract, whereas HPLC-DAD and TPC assay yielded 1.1% (DW) and 0.4% (DW) respectively, using gallic acid as the reference in all methods. Based on the LC-MS observations of extracts and fractions, an average molar mass of 330 g/mol and an average of 4 aromatic hydrogens in each spin system was chosen for optimized qNMR calculations. Compared to the parallel numbers using gallic acid as the standard (170 g/mol, 2 aromatic H), the optimized parameters resulted in a similar qNMR result (5.3%, DW). The different results for the different methods highlight the difficulties with total polyphenolic quantification. All of the methods contain assumptions and uncertainties, and for complex samples with lower concentrations, this will be of special importance. Thus, further optimization of the extraction, identification, and quantification of polyphenols in marine algae must be researched.

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.

Characterization of Polyphenolic Content in the Aquatic Plants Ruppia cirrhosa and Ruppia maritima -A Source of Nutritional Natural Products.

Herein, the polyphenolic content in extracts of Ruppia cirrhosa (Petagna) Grande and Ruppia maritima L.was fully characterized for the first time. High amounts of the main compound chicoric acid (CA) (≤30.2 ± 4.3 mg/g) were found in both Ruppia species. In addition, eight flavonoids, namely the 3-O-glucopyranosides and 3-O-galactopyranosides, as well as malonylated 3-O-glycosides of quercetin and isorhamnetin, were isolated and identified. The antioxidant activity of Ruppia cirrhosa extracts and isolated compounds was investigated spectrophotometrically by a 1,1-diphenyl-2-picrylhydrazyl (DPPH·) radical scavenging assay. IC50 values were 31.8-175.7 µg/mL for Ruppia cirrhosa extracts and 12.1-88.4 µg/mL for isolated flavonoids. Both individual and total phenolic and flavonoid content were quantified in crude extracts using analytical HPLC. The relative high amount of total flavonoids ranged from 5.9 to 14.7 mg/g in both species, with concentrations of individual flavonoids ranging from 0.4 to 2.9 mg/g dry weight. The content of chicoric acid was twofold more in Ruppia maritima than in Ruppia cirrhosa. Seasonal variation of the quantitative content in Ruppia cirrhosa was examined. Total flavonoid content ranged from 8.4 mg/g in October to 14.7 mg/g in August, whereas the highest concentration of chicoric acid was observed in March (29.2 mg/g).

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.

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.

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.

Characterization of hemiacetal forms of anthocyanidin 3-O-beta-glycopyranosides.

The 3-O-beta-glucopyranosides of delphinidin, petunidin, and malvidin (1-3) and cyanidin 3-O-beta-galactopyranoside (4) dissolved in deuterated methanolic solutions without and with acid (5%, CF3COOD) were identified by homo- and heteronuclear NMR techniques. The hemiacetal forms of all the four anthocyanins were characterized as two epimeric 2-hydroxy-hemiacetals on the basis of assignments of both proton and carbon NMR signals together with chemical shift considerations. This is the first report of 13C NMR assignments of two epimeric anthocyanin hemiacetal forms. No 4-hydroxy-hemiacetal form was detected for any of the pigments. For each anthocyanin dissolved in deuterated methanol, the equilibrium between each of the two epimeric hemiacetals and the corresponding flavylium cation was confirmed by the observed positive exchange cross-peaks in the 2D 1H NOESY spectra. The molar proportions of the flavylium cation and the two hemiacetals of 1-4 in deuterated methanol were very similar for all pigments, even during storage for weeks. The majority of the anthocyanins reported to occur in fruits have the same or similar structures as 1-4. These pigments have been proposed to exist predominantly as hemiacetals in slightly acidic to neutral solvents, which is a relevant pH range in plants and in the human gastrointestinal tract.

Preparative isolation and NMR characterization of carboxypyranoanthocyanins.

Three pyranoanthocyanins, the 3-glucosides of 5-carboxypyranodelphinidin (2), 5-carboxypyranopetunidin (4), and 5-carboxypyranomalvidin (6), were produced by nucleophilic addition of pyruvic acid to a purified extract of black beans (Phaseolus vulgaris) containing a mixture of the 3-glucosides of delphinidin (1), petunidin (3), and malvidin (5). A Sephadex LH-20 column was used for preparative separation of both pyranoanthocyanins and anthocyanins. The optimum solvent used for isocratic separation was 20% methanol containing 0.5% trifluoroacetic acid. The chromatographic method applied on the pigment mixture yielded in a one-step separation pigments 1-6, with purities up to 98, 89, 99, 87, 55, and 81%, respectively. The structures of 2 and 4, which previously have been tentatively identified mainly by mass spectrometric data acquired from pigment mixtures in wine samples or modified blueberry extract, were confirmed in the present study by two-dimensional nuclear magnetic resonance spectroscopy.