Quantification of anthocyanins in commercial black currant juices by simple high-performance liquid chromatography. Investigation of their pH stability and antioxidative potency

Quantitative determinations of the four black currant anthocyanins, cyanidin 3-O-beta-glucoside, cyanidin 3-O-beta-rutinoside, delphinidin 3-O-beta-glucoside, and delphinidin 3-O-beta-rutinoside, were achieved in black currant juices by a rapid and sensitive high-performance liquid chromatographic (HPLC) method. The method was validated, and quantification of anthocyanins in 13 commercially available black currant beverages was demonstrated. To optimize the handling of anthocyanin-containing samples, the pH-dependent stability of the anthocyanins was investigated. Four anthocyanins were incubated for 24 h in aqueous solutions at 13 different pH levels between 0.6 and 5.2, after which the samples were analyzed by HPLC. More than 90% of each anthocyanin remained intact up to pH 3.3. At pH 3.8 a local minimum in stability was detected, and at pH >4.5 the stability rapidly decreased. The antioxidant capacity of all 13 black currant juices was investigated by TEAC and FRAP, and the antioxidant potential of both the anthocyanin and the vitamin C contents in the juices was evaluated. This indicated that <70% of the antioxidant capacity of the juices could be attributed to the anthocyanin content or to vitamin C, signifying that other very potent antioxidants are present in commercial black currant juices.     

Preparative-scale isolation of four anthocyanin components of black currant (Ribes nigrum L.) fruits

Four anthocyanin components of black currant, delphinidin 3-O-beta-rutinoside (D3R), cyanidin 3-O-beta-rutinoside (C3R), delphinidin 3-O-beta-glucoside (D3G), and cyanidin 3-O-beta-glucoside (C3G), were successfully isolated as crystalline forms on a preparative scale. In this process, selective hydrolysis of the glucosides (D3G and C3G) and rhamnosides (D3R and C3R) was achieved by treatment with beta-glucosidase and hesperidinase (alpha-rhamnosidase), respectively, to improve resolution of anthocyanin components. Especially, selective conversion of the rutinosides into glucosides made the amounts of D3G and C3G increase about 4- and 7-fold, respectively. D3R, C3R, D3G, and C3G were isolated from enzymatic hydrolysates of black currant anthocyanins through Amberlite XAD-7HP absorption followed by preparative HPLC separation, and their crystals were obtained as the flavylium chloride.     

Anthocyanins from black currants (Ribes nigrum L.)

Fifteen anthocyanin structures are reported from an extract of black currant berries (Ribes nigrum L.). These are the 3-O-glucosides and the 3-O-rutinosides of pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin, cyanidin 3-O-arabinoside, and the 3-O-(6' '-p-coumaroylglucoside)s of cyanidin and delphinidin. The anthocyanins were characterized by means of size exclusion chromatography, high-performance liquid chromatography, UV-visible spectroscopy, and electrospray mass spectrometry. The four main pigments (the 3-O-glucosides and the 3-O-rutinosides of delphinidin and cyanidin) made up >97% of the total anthocyanin content. The minor pigments were enriched from the extract by successive partition against ethyl acetate and by gel fractionation. These chromatographic steps were successfully used to isolate the acylated anthocyanins from the ethyl acetate layer and to separate cyanidin 3-O-arabinoside from the mixture of anthocyanins. The amounts of anthocyanin rutinosides were found to be higher than the amount of the corresponding glucosides for all detected pigments having the same aglycon moiety.     

Anthocyanin-flavanol condensation products from black currant (Ribes nigrum L.)

Putative flavanol-anthocyanin condensation products were detected in a polyphenol-rich concentrate from black currant (Ribes nigrum L.). These compounds had UV-vis spectra similar to those of delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside, but eluted before all previously described anthocyanins on reversed phase HPLC. Mass spectrometric data indicated that they were rutinoside derivatives of novel aglycons 304 amu greater than delphinidin and cyanidin, respectively. The compounds were partly purified by semipreparative HPLC and gave MS and MS2 spectra consistent with anthocyanin rutinosides covalently linked to epigallocatechin or gallocatechin. These compounds are similar in structure to compounds thought to influence color and quality in red wines and strawberry juice products. There was also evidence for the presence of a range of other flavanol-anthocyanin condensation products. The compounds were present at differing levels in juices of 10 black currant varieties, which were roughly correlated to the content of the parent anthocyanins. The flavanol-anthocyanin products were present in polyphenol-enriched concentrates obtained by solid phase extraction, in commercially produced concentrates, and in fresh extracts of black currants. This suggests that the compounds were not artifacts formed during concentration or purification. However, differences in their comparative contents may be related to the lability of the parent anthocyanins during processing. Although present at low levels, the flavanol-anthocyanin products may influence color or quality parameters of black currant juices, and they may confer enhanced stability to the biological activities reported for their anthocyanin parents.     

Absorption and excretion of black currant anthocyanins in humans and watanabe heritable hyperlipidemic rabbits

Anthocyanins are thought to protect against cardiovascular diseases. Watanabe heritable hyperlipidemic (WHHL) rabbits are hypercholesterolemic and used as a model of the development of atherosclerosis. To compare the uptake and excretion of anthocyanins in humans and WHHL rabbits, single-dose black currant anthocyanin studies were performed. Procedures for workup and analyses of urine and plasma samples containing anthocyanins were developed with high recoveries (99 and 81%, respectively) and low limits of quantification (> or =6.6 and > or =1.1 nM, respectively). The excretion and absorption of anthocyanins from black currant juice were found to be within the same order of magnitude in the two species regarding urinary excretion within the first 4 h (rabbits, 0.035%; humans, 0.072%) and t(max) (rabbits, approximately 30 min; humans, approximately 45 min). A food matrix effect was detected in rabbits, resulting in the absorption of a higher proportion of the anthocyanins from black currant juice than from an aqueous citric acid matrix. In humans the absorption and urinary excretion of anthocyanins from black currant juice were found to be proportional with dose and not influenced by the ingestion of a rice cake. In both species a larger proportion of the anthocyanin rutinosides than of the glucosides was absorbed, whereas the structure of the aglycon had no influence on the absorption and excretion. The anthocyanins had no effect in rabbits on the antioxidant capacity of plasma measured as Trolox equivalent antioxidant capacity and ferruc reducing ability of plasma.     

Characterization of anthocyanins in grape juices by ion trap liquid chromatography-mass spectrometry

A reverse phase HPLC and electrospray interface with ion trap mass spectrometer method was developed for the characterization of anthocyanins in Concord, Rubired, and Salvador grape juices. Rubired and Salvador grapes are hybrids from Vitis vinifera and Vitis rupestris. Concord grape is a grape from the native American cultivar Vitis labrusca. Individual anthocyanins in these three varieties were identified on the basis of UV-vis and MS spectra and further elucidated by MS/MS spectra. Anthocyanins in Salvador and Concord grapes were 3-O-glucosides, 3-O-(6' '-O-p-coumaroyl)glucosides, 3-O-(6' '-O-p-acetyl)glucosides, 3,5-O-diglucosides, and 3-O-(6' '-O-p-coumaroyl)-5-O-diglucosides of delphinidin, cyanidin, petunidin, peonidin, and malvidin. Vitisin B was detected in Salvador grape juice. Anthocyanins in Rubired grape juice were primarily anthocyanin diglucosides: peonidin 3,5-O-diglucoside, malvidin 3,5-O-diglucoside, peonidin 3-O-(6' '-O-p-coumaroyl)-5-O-diglucoside, and malvidin 3-O-(6' '-O-p-coumaroyl)-5-O-diglucoside are the four major anthocyanins. The presence of pelargonidin 3-O-glucoside, not previously reported, has been established for the first time in all three juices.     

Biochemical profiling and chemometric analysis of seventeen UK-grown black currant cultivars

Black currant fruits are recognized as being an important dietary source of health-related compounds, such as anthocyanins and ascorbic acid. In the present study, the biochemical composition (viz., nonstructural carbohydrates, individual anthocyanins, total anthocyanins, total phenolics, and organic acids, including ascorbic acid) from 17 UK-grown black currant cultivars was analyzed. Berry composition was significantly affected by genotype. Nonstructural carbohydrates ranged from 85.09 to 179.92 mg g(-1) on a fresh weight (FW) basis, while concentration for organic acids ranged from 36.56 to 73.35 mg g(-1) FW. Relative concentrations of cyanidin 3-glucoside, cyanidin 3-rutinoside, delphinidin 3-glucoside and delphinidin 3-rutinoside were 3.1-7.9%, 35.4-47.0%, 7.6-12.5% and 36.9-50.9%, respectively. Differences in the biochemical profile among cultivars were emphasized by principal component analysis (PCA) and hierarchical cluster analysis (HCA). PCA was able to discriminate between cultivars, especially on the basis of health-related compounds. Initial exploration revealed that individual anthocyanins, total phenolics, and ascorbic acid could be used to characterize and classify different cultivars. HCA showed that the biochemical composition of the different cultivars was related to parentage information.     

Protease-assisted clarification of black currant juice: synergy with other clarifying agents and effects on the phenol content

Conventional clarification with gelatin and silica sol removes a considerable amount of antioxidant phenolics from berry juices. This study examined the clarification and haze-diminishing effects of alternative clarification strategies on black currant juice including centrifugation and addition of acidic protease and pectinolytic enzyme preparations and gallic acid. Centrifugation of freshly pressed juice (10,000 g for 15 min) resulted in a approximately 95% reduction of immediate turbidity and had a decreasing effect on haze development in the juice during cold storage without significantly compromising the total phenols levels. The extent of clarification and haze diminishment varied after individual treatments with five different acidic proteases, but one of the protease preparations, Enzeco, derived from Aspergillus niger, consistently tended to perform best. The individual and interactive effects on juice turbidity, total phenols, and total anthocyanin contents of clarification treatments involving the use of two selected acid proteases (Enzeco and Novozyme 89L), a pectinase (Pectinex BE 3-L), and gallic acid were evaluated in a full factorial 2(4) experimental design. Haze development during cold storage decreased when gallic acid or any of the enzyme preparations were employed individually, but negative interaction effects resulted when the pectinase was employed in combination with any of the proteases. After 28 storage days at 2 degrees C, the lowest levels of haze formation were achieved when the Enzeco protease preparation, added at 0.025 g/L, was added with 0.050 g/L of gallic acid and allowed to react in the juice for 90 min at 50 degrees C. The corresponding anthocyanin reduction was approximately 12% (compared to approximately 30% with gelatin silica sol treatment). The data support the hypothesis that phenol-protein interactions are involved in juice turbidity development during cold storage of berry juices and demonstrate that precentrifugation and protease-assisted clarification show promise as an alternative, phenolics-retaining clarification strategy in black currant juice processing.     

Influence of growing season on phenolic compounds and antioxidant properties of grape berries from vines grown in subtropical climate

The influence of growing season (winter vs summer) on the synthesis and accumulation of phenolic compounds and antioxidant properties was studied in five grape cultivars for three consecutive years. Four phenolic compound parameters (total phenols, flavonoids, flavan-3-ols, and anthocyanins) and three antioxidant property parameters [2,2-diphenyl-1-picrylhydrazyl radical scavenging, 2,2-azinobis(3-ethylbenzothiazolinesulfonic acid) radical scavenging, and ferric reducing antioxidant power] were investigated. Results showed that both phenolic compounds and antioxidant properties in the seed and skin of winter berries were significantly (p < 0.05) higher than those of summer berries for all of the cultivars investigated. The anthocyanin profiles of berry skins appeared to be extremely consistent in different years for the same crop, whereas they varied greatly between the two crops within the same year (winter vs summer). Winter berries contained richer glucosides of delphinidin, cyanidin, peonidin, and malvidin than summer berries. These seasonal variations of phenolic compounds and antioxidant properties on grape berries were largely contributed by climatic factors such as temperature, solar radiation, rainfall, and hydrothermic coefficient between different growing seasons.     

Orally administered delphinidin 3-rutinoside and cyanidin 3-rutinoside are directly absorbed in rats and humans and appear in the blood as the intact forms

Four components of black currant anthocyanins (BCA), delphinidin 3-O-beta-rutinoside (D3R), cyanidin 3-O-beta-rutinoside (C3R), delphinidin 3-O-beta-glucoside (D3G), and cyanidin 3-O-beta-glucoside (C3G), were found to be directly absorbed and distributed to the blood and excreted into urine as the glycosylated forms. In a rat study, following oral administration of purified D3R, C3R, and C3G (800 micromol/kg of body weight), the anthocyanins were detected in the plasma and the C(max) values were 580 +/- 410, 850 +/- 120, and 840 +/- 190 nmol/L, respectively, 0.5-2.0 h after administration. In a human study, when a mixture of BCA [6.24 micromol (3.58 mg) consisting of 2.75 micromol (1.68 mg) of D3R, 2.08 micromol (1.24 mg) of C3R, 1.04 micromol (0.488 mg) of D3G, and 0.37 micromol (0.165 mg) of C3G/kg of body weight)] was orally ingested by eight volunteers, D3R, C3R, D3G, and C3G were detected in the plasma and urine. The plasma C(max) values were 73.4 +/- 35.0, 46.3 +/- 22.5, 22.7 +/- 12.4, and 5.0 +/- 3.7 nmol/L, respectively, 1.25-1.75 h after intake, and the cumulative excretion of the four compounds in urine in the period 0-8 h after intake was 0.11 +/- 0.05% of the dose ingested. These results indicate that 3-O-beta-rutinosyl anthocyanins were directly absorbed and distributed to the blood.     

High-performance liquid chromatography with photodiode array detection (HPLC-DAD)/HPLC-mass spectrometry (MS) profiling of anthocyanins from Andean Mashua Tubers (Tropaeolum tuberosum Ruíz and Pavón) and their contribution to the overall antioxidant activity

Mashua (Tropaeolum tuberosum Ruíz and Pavón), an Andean tuber with high antioxidant activity, has sparked interest because of its traditional medicinal use. In this study, we evaluated the anthocyanin composition for three purple mashua genotypes and their contribution to the overall antioxidant activity of the tuber. Mashua anthocyanins, total phenolics, and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) antioxidant activity ranged from 45.5 to 131.9 mg of cyanidin 3-glucoside equivalents/100 g fresh weight (FW), 174.9 to 275.5 mg of gallic acid equivalents/100 g of FW, and 16.2 to 45.7 micromol of Trolox equivalents/g of FW, respectively. The high-performance liquid chromatography with photodiode array detection (HPLC-DAD) and HPLC-electrospray ionization tandem mass spectrometry (ESI/MS-MS) profiles revealed the presence of 11 different anthocyanins. The two major pigments (56.4-73.0% total area range at 520 nm) were identified as delphinidin 3-glucoside-5-acetylrhamnoside and delphinidin 3-sophoroside-5-acetylrhamnoside. Other pigments were delphinidin 3-glucoside-5-rhamnoside, delphinidin 3-sophoroside-5-rhamnoside, delphinidin 3-glucoside, cyanidin 3-sophoroside, and cyanidin 3-sophoroside-5-rhamnoside. Cyanidin 3-glucoside and cyanidin 3-rutinoside were only found in two genotypes, while pelargonidin 3-sophoroside and pelargonidin 3-sophoroside-5-rhamnoside were only found in the third one. Anthocyanins from mashua were the major contributors to the total ABTS values for only one of the three genotypes, suggesting that other phenolics present are playing a major role in the antioxidant power of mashua tubers. Results from this study provide important information for the Nutraceutical and Functional Food Market for the use of mashua anthocyanins not only as a source of natural colorants but also as a source of phytonutrients.     

Enzyme-assisted processing increases antimicrobial and antioxidant activity of bilberry

The effects of nine cell wall-degrading enzymes on the antimicrobial and antioxidant activities of bilberry were studied. Antimicrobial activity was measured using the human pathogens Salmonella enterica sv. Typhimurium and Staphylococcus aureus as test strains. Enzyme treatments liberated phenolics from the cell wall matrix, which clearly increased the antimicrobial activity of berry juices, press cakes, and berry mashes on the basis of plate counts. Antibacterial effects were stronger against Salmonella than against Staphylococcus bacteria. In general, the increase in activity measured as colony-forming units per milliliter was 3-5 logarithmic units against Salmonella and 1-2 units against Staphylococcus bacteria. Increase in antimicrobial activity was observed only in acidic conditions, which is also the natural environment in various berry products, such as juices. The activity profile of the pectinase preparation affected the chemistry of the phenolics due to the presence of deglycosylating activities in some preparations. The difference in phenolic profiles was reflected in the antimicrobial effects. Bilberry mashes treated with Pectinex Ultra SP-L, Pectinex 3 XL, and Pectinex BE XXL were most efficient against Salmonella bacteria, whereas mashes treated with Pectinex Smash, Pectinex BE 3-L, and Biopectinase CCM showed the strongest antimicrobial activity against Staphylococcus bacteria. Due to the liberation of phenolics from the cell wall matrix the antioxidant activity measured as radical scavenging activity was also increased on average about 30% by the enzymatic treatments. The highest increase in phenolic compounds was about 40%. Highest increases in anthocyanins and in antioxidant activity were observed in berry mash treated with Pectinex Smash XXL enzyme, and the lowest increase was observed after treatment with Pectinex BE 3-L. Enzyme-assisted processing is traditionally used to improve berry and fruit juice yields. However, enzymatic treatments also have an impact on the functional properties of the products. The increased liberation of phenolics from the cell wall matrix can prolong the shelf life of berry products by limiting the growth of contaminants during processing or storage. The increased amount of phenolic compounds may also have a positive effect on gut well-being.     

Increasing antioxidant activity and reducing decay of blueberries by essential oils

Several naturally occurring essential oils including carvacrol, anethole, cinnamaldehyde, cinnamic acid, perillaldehyde, linalool, and p-cymene were evaluated for their effectiveness in reducing decay and increasing antioxidant levels and activities in 'Duke' blueberries ( Vaccinium corymbosum). Carvacrol, anethole, and perillaldehyde showed the capability to promote total anthocyanins and total phenolics and enhance antioxidant activity in fruit tissues expressed as oxygen radical absorbance capacity (ORAC) and hydroxyl radical ( (*)OH) scavenging capacity. All of the essential oils tested in this study were able to inhibit fruit decay development to some degree compared to controls. The most effective compound for mold retardation was p-cymene, followed by linalool, carvacrol, anethole, and perillaldehyde. Cinnamic acid and cinnamaldehyde also suppressed mold growth, but to a lesser extent. Treatment with carvacrol, anethole, or perillaldehyde also significantly increased the levels of fructose, glucose, and citric acid. Individual flavonoids were variably affected by the essential oils. Levels of chlorogenic acid, which was the major phenolic compound in blueberry fruit, were enhanced by all of the essential oils in this study. Increased amounts of quercetin 3-galactoside and quercetin 3-arabinoside were also found in all treated fruit except samples treated with linalool or p-cymene. The major anthocyanin, malvidin 3-galactoside, was enhanced by all essential oils tested except linalool and p-cymene. The levels of other individual anthocyanins including petunidin 3-galactoside, delphinidin 3-galactoside, petunidin 3-glucoside, petunidin 3-arabinoside, delphinidin 3-arabinoside, and cyanidin 3-galactoside were higher in treated fruit compared to controls. Those essential oils that have positive effects on enhancing anthocyanins, phenolic compounds, and antioxidant activity of fruit, but inhibitory effects on microbial growth and decay development, deserve further evaluation.     

Inhibition of protein and lipid oxidation in liposomes by berry phenolics

The antioxidant activity of berry phenolics (at concentrations of 1.4, 4.2, and 8.4 mug of purified extracts/mL of liposome sample) such as anthocyanins, ellagitannins, and proanthocyanidins from raspberry (Rubus idaeus), bilberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), and black currant (Ribes nigrum) was investigated in a lactalbumin-liposome system. The extent of protein oxidation was measured by determining the loss of tryptophan fluorescence and formation of protein carbonyl compounds and that of lipid oxidation by conjugated diene hydroperoxides and hexanal analyses. The antioxidant protection toward lipid oxidation was best provided by lingonberry and bilberry phenolics followed by black currant and raspberry phenolics. Bilberry and raspberry phenolics exhibited the best overall antioxidant activity toward protein oxidation. Proanthocyanidins, especially the dimeric and trimeric forms, in lingonberries were among the most active phenolic constituents toward both lipid and protein oxidation. In bilberries and black currants, anthocyanins contributed the most to the antioxidant effect by inhibiting the formation of both hexanal and protein carbonyls. In raspberries, ellagitannins were responsible for the antioxidant activity. While the antioxidant effect of berry proanthocyanidins and anthocyanins was dose-dependent, ellagitannins appeared to be equally active at all concentrations. In conclusion, berries are rich in monomeric and polymeric phenolic compounds providing protection toward both lipid and protein oxidation.     

Cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside as primary phenolic antioxidants in black raspberry

Anthocyanin constituents in black raspberries (Rubus occidentalis L.) were investigated by HPLC-DAD, and their involvement as potent, significant antioxidants in black raspberries was demonstrated by three common antioxidant assays (FRAP, DPPH, ABTS) in this study. Five anthocyanins were present in black raspberries: cyanidin 3-sambubioside, cyanidin 3-glucoside, cyanidin 3-xylosylrutinoside, cyanidin 3-rutinoside, and pelargonidin 3-rutinoside. Their identities and structures, with particular emphasis on cyanidin 3-xylosylrutinoside, were confirmed by NMR spectroscopy. Two of these anthocyanins, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside, predominated, comprising 24-40 and 49-58%, respectively, of the total anthocyanins in black raspberries. On the basis of both potency and concentration, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside were found to be the significant contributors to the antioxidant systems of black raspberries. These findings indicate that these two anthocyanin compounds may function as the primary phenolic antioxidants in black raspberries. These two compounds exhibit potential biological activities that may be exploited in conjunction with other naturally occurring bioactive compounds in black raspberry fruit-based products used in clinical trials for the treatment of various types of cancer.     

Characterization of anthocyanins and pyranoanthocyanins from blood orange [Citrus sinensis (L.) Osbeck] juice

Anthocyanins from blood orange [Citrus sinensis (L.) Osbeck] juices were isolated and purified by means of high-speed countercurrent chromatography and preparative high-performance liquid chromatography. Structures of the pigments were then elucidated by electrospray ionization multiple mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The major anthocyanins of the juice were characterized as cyanidin 3-glucoside and cyanidin 3-(6"-malonylglucoside). Furthermore, six minor anthocyanins were detected and identified as cyanidin 3,5-diglucoside, delphinidin 3-glucoside, cyanidin 3-sophoroside, delphinidin 3-(6"-malonylglucoside), peonidin 3-(6"-malonylglucoside), and cyanidin 3-(6"-dioxalylglucoside). The occurrence of the latter compound in blood oranges is reported here for the first time, together with full NMR spectroscopic data. Further investigations revealed the presence of four anthocyanin-derived pigments, which are formed through a direct reaction between anthocyanins and hydroxycinnamic acids during prolonged storage of the juice. These novel pyranoanthocyanins were identified as the 4-vinylphenol, 4-vinylcatechol, 4-vinylguaiacol, and 4-vinylsyringol adducts of cyanidin 3-glucoside through comparison of their mass spectrometric and chromatographic properties with those of synthesized reference compounds.     

Influence of vine vigor on grape (Vitis vinifera L. Cv. Pinot Noir) anthocyanins. 2. Anthocyanins and pigmented polymers in wine

The relationships between grapevine (Vitis vinifera) vigor variation and resulting wine anthocyanin concentration and composition and pigmented polymer formation were investigated. The study was conducted in a commercial vineyard consisting of the same clone, rootstock, age, and vineyard management practices. Vine vigor parameters were used to designate vigor zones within two vineyard sites (A and B) to produce research wines (2003 and 2004) and conduct a model extraction experiment (2004 only) to investigate the vine-fruit-wine continuum. Wines and model extracts were analyzed by HPLC and UV-vis spectrophotometry. For the model extractions, there were no differences between sites for pomace weight, whereas juice volume was higher for site A. This was not related to a larger berry size. Site A had a higher anthocyanin concentration (milligrams per liter) in the model extracts than site B specifically for the medium- and low-vigor zones. For anthocyanin composition in the model extraction, site B had a greater proportion of malvidin-3-O-glucoside and less of the remaining anthocyanin glucosides (delphinidin, cyanidin, petunidin, and peonidin) compared to site A. In the wines, there was a vintage effect, with the 2003 wines having a higher anthocyanin concentration (milligrams per liter) than the 2004 wines. This appears to have been primarily due to a greater accumulation of anthocyanins in the fruit. In general, the medium-vigor zone wines had higher anthocyanin concentrations than either the high- or low-vigor zone wines. There was also vintage variation related to anthocyanin composition, with the 2003 wines having a higher proportion of delphinidin and petunidin glucosides and lower malvidin-3-O-glucoside compared to 2004. In both years, there were higher proportions of delphinidin and petunidin glucosides in wines made from low-vigor-zone fruit. Wines made from low-vigor zones showed a greater propensity to form vitisin A as well as pigmented polymers. Low-vigor-zone wines had a approximately 2-fold increase in pigmented polymer concentration (milligrams per liter) over high-vigor-zones wines. There was a strong positive relationship between pigmented polymer concentration, bisulfite bleaching resistant pigments, proanthocyanidin concentration, and color density in wines. Overall, differences found in the wines magnified variation in the fruit.     

Plant phenolics affect oxidation of tryptophan

The effect of berry phenolics such as anthocyanins, ellagitannins, and proanthocyanidins from raspberry (Rubus idaeus), black currant (Ribes nigrum), and cranberry (Vaccinium oxycoccus) and byproducts of deoiling processes rich in phenolics such as rapeseed (Brassica rapa L.), camelina (Camelina sativa), and soy (Glycine max L.) as well as scots pine bark (Pinus sylvestris) was investigated in an H2O2-oxidized tryptophan (Trp) solution. The oxidation of Trp was analyzed with high-performance liquid chromatography using both fluorescence and diode array detection of Trp and its oxidation products. Mechanisms of antioxidative action of the phenolic compounds toward the oxidation of Trp were different as the pattern of Trp oxidation products varied with different phenolic compounds. The antioxidant protection toward oxidation of Trp was best provided with pine bark phenolics, black currant anthocyanins, and camelina meal phenolics as well as cranberry proanthocyanidins.     

Effect of black currant anthocyanins on the activation of endothelial nitric oxide synthase (eNOS) in vitro in human endothelial cells

Polyphenols are known to induce vasodilatory function via activation of the redox-sensitive phosphatidylinositol-3 (PI3)/protein kinase B (Akt) pathway. Black currant fruits have appreciable amounts of polyphenolic compounds including cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, delphinidin-3-O-glucoside, and delphinidin-3-O-rutinoside. It was hypothesized that black currant fruit extracts would cause activation of endothelial nitric oxide synthase (eNOS) through activation of redox-sensitive PI3 kinase/Akt signaling pathway. To test this hypothesis, human umbilical vein endothelial cells (HUVECs) were treated with different concentrations/times of black currant juice concentrates (Ben Gairn and Ben Hope) and the activation of Akt and eNOS was measured using immunoblotting. Vitamin C is also known to activate Akt and eNOS in in vitro models, and black currants are rich in vitamin C. Therefore, the effect of black currant extracts with and without coexisting vitamin C was investigated, using SPE columns to eliminate vitamin C content. The individual (and combined) effects of the major anthocyanins present in black currant juice samples with and without vitamin C were investigated and compared to the effects of the whole extract. Black currant juice samples (1 μL/mL) significantly increased the phosphorylation of Akt (p-Akt) and eNOS (p-eNOS) (P < 0.05). Activation of Akt and eNOS was abolished by incubation with wortmannin, a PI3K inhibitor, supporting the involvement of PI3K/Akt. Vitamin C alone significantly increased the p-Akt and p-eNOS (P < 0.05); however, removal of vitamin C from black currant did not significantly affect p-Akt and p-eNOS compared to black currant with vitamin C. Assessment of individual anthocyanins also showed significant effects on p-Akt and p-eNOS. In summary, in the present study data suggested that black currant concentrates, Ben Gairn and Ben Hope, activated eNOS via Akt/PI3 kinase pathway in vitro in HUVECs and that the effect was not dependent on vitamin C.     

High-performance liquid chromatography (HPLC) analysis of phenolic compounds in berries with diode array and electrospray ionization mass spectrometric (MS) detection: ribes species

High-performance liquid chromatography combined with diode array and electrospray ionization mass spectrometric (MS) detection was used to study phenolic compounds in berries of black, green, red, and white currants (Ribes spp.). UV-visible spectrometry was a valuable tool for the identification of the class of the phenolic compound, whereas MS and MS-MS fragmentation data were useful for further structural characterization. Distinct similarities were found in the relative distribution of conjugated forms of phenolic compounds among the four currants. Phenolic acids were found mainly as hexose esters. Flavonol glycosides and anthocyanin pigments were mainly found as 3-O-rutinosides and second as 3-O-glucosides. However, cyanidin 3-O-sambubioside and quercetin hexoside-malonate were notable phenolic compounds in red currant. Flavonol hexoside-malonates were identified and quantified in the berries of currants for the first time.