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.     

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.     

Enzyme-assisted extraction of antioxidative phenols from black currant juice press residues (Ribes nigrum).

Enzymatic release of phenolic compounds from pomace remaining from black currant (Ribes nigrum) juice production was examined. Treatment with each of the commercial pectinolytic enzyme preparations Grindamyl pectinase, Macer8 FJ, Macer8 R, and Pectinex BE, as well as treatment with Novozym 89 protease, significantly increased plant cell wall breakdown of the pomace. Each of the tested enzyme preparations except Grindamyl pectinase also significantly enhanced the amount of phenols extracted from the pomace. Macer8 FJ and Macer8 R decreased the extraction yields of anthocyanins, whereas Pectinex BE and Novozym 89 protease showed no effect. A decrease in pomace particle sizes from 500-1000 microm to <125 microm increased the phenol yields 1.6-5 times. Black currant pomace devoid of seeds gave significantly higher yields of phenols than pomace with seeds and seedless wine pomace. Four selected black currant pomace extracts all exerted a pronounced antioxidant activity against human LDL oxidation in vitro when tested at equimolar phenol concentrations of 7.5-10 microM.     

Anthocyanins, flavonols, and free radical scavenging activity of Chinese bayberry (Myrica rubra) extracts and their color properties and stability

Characterization of anthocyanins and flavonols and radical scavenging activity assays of extracts from four Chinese bayberry (Myrica rubra) varieties with different fruit colors were carried out. One dominant anthocyanin and three major flavonols were isolated by HPLC, and cyanidin-3-O-glucoside and two of three flavonols, myricetin and quercetin-3-O-rutinoside, were identified by cochromatography with authentic standards. Both DPPH* and ABTS*(+) cation assays indicated that the black varieties (Biji and Hunan) demonstrated much higher radical scavenging activities than the pink (Fenhong) and yellow (Shuijing) varieties, which may be attributed to much higher levels of anthocyanins, flavonoids, and total phenolics in the black varieties. Biji and Hunan had 6.49 and 6.52 mM Trolox equivalent antioxidant capacity (TEAC) per 100 g of fresh weight, whereas the pink (Fenhong) and yellow (Shuijing) bayberries had 1.32 and 1.31 mM TEAC/100 g. Different fruit color was reflected by the surface color and pigment extract color. Color stability of the pigment was dependent on pH, and the pigment was more stable at low pH (pH approximately 1.5). The lightness (L) increased while the chroma (C value) decreased with increase of pH until pH 5, but higher pH caused a small decrease for L and an increase for C.     

Compositional differences of phenolic compounds between black currant (Ribes nigrum L.) cultivars and their response to latitude and weather conditions

Phenolic compounds in black currants of three Finnish cultivars and their response to growth latitude and weather conditions were analyzed over a six-year period. 'Melalahti' had lower contents of total phenolic compounds (31.4% and 29.2% lower than 'Mortti' and 'Ola', respectively), total anthocyanins (32.6% and 30.5%), and total hydroxycinnamic acid derivatives (23.1% and 23.8%) (p < 0.05) and was less affected by growth latitude and weather conditions than 'Mortti' and 'Ola'. However, all the cultivars grown at higher latitude (66°34' N) had lower contents of total flavonols, total anthocyanins, and total phenolic compounds than those grown at lower latitude (60°23' N) (p < 0.05). The content of total hydroxycinnamic acid conjugates did not vary in 'Melalahti' (p > 0.05) but increased as the latitude increased in 'Mortti' and 'Ola' (p < 0.05). Temperature and radiation were the major weather variables influencing the composition of phenolic compounds. Delphinidin-3-O-glucoside, delphinidin-3-O-rutinoside, and myricetin-3-O-glucoside content showed positive correlations with temperature and radiation in all three cultivars. The study gives important guidelines for the selection of raw materials and growth sites as well as for the berry cultivation for commercial exploitation of black currant berries.     

Antioxidants and antioxidant activity of several pigmented rice brans

This study investigated the antioxidant content and activity of phenolic acids, anthocyanins, α-tocopherol and γ-oryzanol in pigmented rice (black and red rice) brans. After methanolic extraction, the DPPH free radical scavenging activity and antioxidant activity were measured. The pigmented rice bran extract had a greater reducing power than a normal rice bran extract from a long grain white rice. All bran extracts were highly effective in inhibiting linoleic acid peroxidation (60-85%). High-performance liquid chromatography (HPLC) analysis of antioxidants in rice bran found that γ-oryzanol (39-63%) and phenolic acids (33-43%) were the major antioxidants in all bran samples, and black rice bran also contained anthocyanins 18-26%. HPLC analysis of anthocyanins showed that pigmented bran was rich in cyanidin-3-glucoside (58-95%). Ferulic acid was the dominant phenolic acid in the rice bran samples. Black rice bran contained gallic, hydroxybenzoic, and protocatechuic acids in higher contents than red rice bran and normal rice bran. Furthermore, the addition of 5% black rice bran to wheat flour used for making bread produced a marked increase in the free radical scavenging and antioxidant activity compared to a control bread.     

Anthocyanin composition and antioxidant activity of the Crowberry (Empetrum nigrum) and other berries

The anthocyanin composition and antioxidant activity of the crowberry ( Empetrum nigrum) were studied. High-performance liquid chromatography (HPLC) combined with a diode array detector and electrospray ionization mass spectrometry were used for identification and quantification of individual anthocyanins. Freeze-dried crowberry powder was extracted with 80% methanol containing 0.5% acetic acid and subjected to HPLC. Thirteen kinds of anthocyanins were identified. The major anthocyanins were cyanidin-3-galactoside and delphinidin-3-galactoside, at 8.04 and 8.62 mg/g extract, respectively. The HPLC profile of crowberry extract was similar to bilberry and blueberry. The total content of anthocyanins in crowberry was 41.8 mg/g extract, higher than the other nine major berry species (2.5-38.8 mg/g extract). The antioxidant activity was also evaluated using the 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azinobis(3-ethybenzothiazoline-6-sulfonic acid) radical quenching assays and the ferric reducing activity power assy. Crowberry extract exerted the strongest antioxidant activity. In conclusion, individual anthocyanins in crowberry were identified and then quantified in this study. Additionally, crowberry is suggested to be associated with a reduction in the risk of developing chronic diseases because of its strong antioxidant activity.     

Screening of free radical scavenging compounds in water extracts of Mentha samples using a postcolumn derivatization method

An on-line high-performance liquid chromatography-1,1-diphenyl-2-picrylhydrazyl (HPLC-DPPH*) method has been improved for the detection of polar and nonpolar radical scavenging compounds in complex plant extracts. Nine water extracts were prepared from different Mentha species, varieties, hybrids, and cultivars. After the components within each extract had been separated by reverse phase chromatography using 10-100% methanol with 2% acetic acid as a mobile phase, analytes within the eluent capable of scavenging a citric acid-sodium citrate-buffered methanol 1,1-diphenyl-2-picrylhydrazyl solution were detected by postcolumn derivatization at 517 nm. The HPLC-DPPH* on-line method was applied to the qualitative and quantitative analysis of Mentha extracts. There was a strong correlation between the scavenging (negative) peak area and the concentration of the radical scavenging reference substances used. The minimum detectable concentration (microg/mL) of the antioxidant compounds was determined. Caffeic acid, eriocitrin (eriodictyol-7-O-rutinoside), luteolin-7-O-glucoside, and rosmarinic acid were identified as the dominant radical scavengers in these extracts by this method.     

Isolation and determination of anthocyanins in seed coats of black soybean (Glycine max (L.) Merr.).

Anthocyanin pigments in seed coats of black soybean (Glycine max (L.) Merr.) were extracted with 1% HCl-CH(3)OH, and the crude anthocyanin extract was purified by Shepadex LH-20 and Lichroprep RP-18 open-column chromatography. Three major anthocyanins were isolated, and their chemical structures were identified by spectroscopic methods (UV-visible, FABMS, (1)H and (13)C NMR, and by TLC). The complete structures of these anthocyanins were elucidated as delphinidin-3-glucoside, cyanidin-3-glucoside, and petunidin-3-glucoside. Among them, petunidin-3-glucoside was identified as a new anthocyanin in black soybeans. On the basis of RP-HPLC with a UV-vis detector, the contents of delphinidin-3-glucoside, cyanidin-3-glucoside, petunidin-3-glucoside, and total anthocyanins in seed coats of 10 black soybeans were found in the ranges of 0-3.71, 0.94-15.98, 0-1.41, and 1.58-20.18 mg/g, respectively. The results obtained in this study imply that the seed coats of black soybean can be used as a good source for cyanidin-3-glucoside and delphinidin-3-glucoside.     

Anthocyanins from bay (Laurus nobilis L.) berries

Anthocyanin composition in the berries of Laurus nobilis L., a perennial tree or shrub typical of the Mediterranean region, was determined for the first time. The pigments were extracted from the berries with 0.1% HCl in methanol, purified on a C-18 solid-phase cartridge, and characterized by means of high-performance liquid chromatography (HPLC)-diode array detection (DAD)-mass spectrometry (MS) analysis. The major anthocyanins were characterized as cyanidin 3-O-glucoside (41%) and cyanidin 3-O-rutinoside (53%). Furthermore, two minor anthocyanins were detected and identified as 3-O-glucoside and 3-O-rutinoside derivatives of peonidin (5%). The two major pigments were also isolated by preparative HPLC and characterized by H1 nuclear magnetic resonance (NMR) spectroscopy. The attractive color and the great abundance of the plant in the south of Italy make Laurus nobilis berries a new and very good source of natural pigments.     

Identification of anthocyanins in the sprouts of buckwheat

The anthocyanin profiles and varieties/breeding line differences of anthocyanin concentrations in common/tartary buckwheat sprouts have been studied. Four anthocyanins, cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, cyanidin 3-O-galactoside, and cyanidin 3-O-galactopyranosyl-rhamnoside, were isolated from the sprouts of common buckwheat, were separated using high-performance liquid chromatography (HPLC), and were identified using reversed-phase liquid chromatography (LC)/electrospray ionization-mass spectrometry (ESI-MS)/MS techniques. In tartary buckwheat sprouts, two anthocyanins (cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside) were identified. Among 19 common/tartary buckwheat varieties/breeding lines, Hokkai T10 contained the highest amounts of anthocyanins. Cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside concentrations in 6-10 days after seeding sprouts of Hokkai T10 ranged from 0.16 to 0.20 mg/g dry wt and from 5.55 to 6.57 mg/g dry wt, respectively. In addition, dark-grown sprouts of Hokkai T10 accumulated 0.091 and 2.77 mg/g dry wt of cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside whereas other varieties/breeding lines accumulated trace amounts of anthocyanins. Given its anthocyanin-rich red cotyledons, Hokkai T10 is a promising line for use as "Moyashi" type sprouts and is strongly recommended as a new functional food, rich in dietary anthocyanins.     

Determination of anthocyanins in Ruscus aculeatus L. berries

Red berries of Ruscus aculeatus L., a wild shrub typical of Mediterranean Europe and Africa, were investigated for the first time in order to determine the profile of anthocyanins. The pigments were extracted from the skins of the berries with 0.1% HCl in methanol, purified using a C-18 solid phase cartridge, and identified by means of high-performance liquid chromatography (HPLC)-diode array detection-mass spectrometry analysis. Information from HPLC profiles, saponification, and acid hydrolysis of the anthocyanins showed that the major anthocyanins were pelargonidin 3-O-rutinoside (64%), pelargonidin 3-O-glucoside (16%), and pelargonidin 3-O-trans-p-coumarylglucoside (13%). The attractive color of R. aculeatus berries and the great abundance of the plant in the south of Italy make these berries a new and promising source of natural colorants.     

Effect of processing of black currant press-residue on polyphenol composition and cell proliferation

The press-residue of black currants provides a good source of phenolic antioxidants. The purpose of this study was to optimize the extraction of phenolic compounds from the press-residue by use of extraction conditions compatible with food use. The effects of temperature, extraction duration, and use of ultrasound-assisted extraction on the juice yield, total phenolics (TP), and anthocyanin content of aqueous extracts were studied. Within the variables and response factors tested, the optimal conditions were a 15 min extraction at 90 °C. No significant effect from ultrasound-assisted extraction was found. The composition of anthocyanins and polyphenols was highly dependent on the extraction temperature. The percentage contribution of delphinidin- and cyanidin-3-rutinoside to TP had a negative linear correlation with temperature, while delphinidin- and cyanidin-3-glucoside had a positive linear correlation with temperature, with a maximum amount obtained at 80 °C and 55 °C, respectively. Furthermore, extracts obtained at higher temperatures showed a stronger inhibition of proliferation of Caco-2, HT-29, and HCT 116 cells than extracts obtained at lower temperatures. This may be due to the decomposition of complex polyphenols at higher temperatures, making them more accessible to the cells.     

Phenolic composition and antioxidant activity in seed coats of 60 Chinese black soybean (Glycine max L. Merr.) varieties

Phenolics in black soybean seed coat (BSSC) are considered to be responsible for the health benefits of black soybean. BSSCs of 60 Chinese varieties were examined for phenolic contents, anthocyanin profiles, and antioxidant activity. Total phenolic and condensed tannin contents ranged from 512.2 to 6057.9 mg gallic acid equivalents/100 g and from 137.2 to 1741.1 mg (+)-catechin equivalents/100 g, respectively. Six anthocyanins (delphinidin-3-glucoside, cyanidin-3-galactoside, cyanidin-3-glucoside, petunidin-3-glucoside, peonidin-3-glucoside, and malvidin-3-glucoside) were detected by HPLC. Total anthocyanin contents (TAC) were from 98.8 to 2132.5 mg/100 g, and cyanidin-3-glucoside was the most abundant anthocyanin in all varieties, with a distribution of 48.8-94.1% of TAC. Antioxidant properties detected by DPPH, FRAP, and ORAC methods all showed wide variations ranging from 4.8 to 65.3 μg/100 mL (expressed as EC(50)), from 17.5 to 105.8 units/g, and from 42.5 to 1834.6 μmol Trolox equivalent/g, respectively. Sixty varieties were classified into four groups by hierarchical clustering analysis, and group 4 consisting of nine varieties had the highest phytochemicals content and antioxidant activity.     

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.     

E-cinnamic acid derivatives and phenolics from Chilean strawberry fruits, Fragaria chiloensis ssp. chiloensis

Three E-cinnamic acid glycosides, tryptophan, and cyanidin-3-O-beta-D-glucopyranoside were isolated from ripe fruits of the Chilean strawberry Fragaria chiloensis ssp. chiloensis. 1-O-E-Cinnamoyl-beta-D-xylopyranoside, 1-O-E-cinnamoyl-beta-D-rhamnopyranoside, and 1-O-E-cinnamoyl-alpha-xylofuranosyl-(1-->6)-beta-D-glucopyranose are reported for the first time. The cinnamic acid glycosides and aromatic compound patterns in F. chiloensis fruits were determined by high-performance liquid chromatography (HPLC). HPLC analyses of extracts showed that cyanidin-3-O-beta-D-glucopyranoside and free ellagic acid are present in achenes while the E-cinnamoyl derivatives and tryptophan were identified only in the thalamus. The free radical scavenging effect of the fruit extract can be associated with the anthocyanin content.     

Influence of vine vigor on grape (Vitis vinifera L. Cv. Pinot Noir) anthocyanins. 1. Anthocyanin concentration and composition in fruit

The relationships between grapevine (Vitis vinifera) vigor variation and resulting fruit anthocyanin accumulation and composition were investigated. The study was conducted in a commercial vineyard consisting of the same clone, rootstock, age, and vineyard management practices. The experimental design involved assigning vigor zones in two vineyard sites based upon differences in vine growth. Fruits and wines were analyzed by HPLC from designated vigor zones in 2003 and 2004. Average berry weight (grams), average dry skin weight (milligrams), degrees Brix, and pH were higher and titratable acidity (grams per liter) was lower in 2003 compared to 2004. In 2003, only the highest and lowest vigor zones had differences in berry weight, whereas there were no differences in 2004. In both years, high vigor zones had lower degrees Brix and higher titratable acidity (milligrams per liter). Accumulation of anthocyanins (milligrams per berry) was greater in 2003 compared to 2004. There was a trend for lower anthocyanin concentration (milligrams per berry) in high vigor zones in both years. In 2004 compared to 2003, there was a higher proportion of malvidin-3-O-glucoside and lower proportions of the other four anthocyanins (delphinidin-, cyanidin-, petunidin-, and peonidin-3-O-glucosides) found in Pinot Noir. In both years, site A had proportionally higher peonidin-3-O-glucoside and lower malvidin-3-O-glucoside than site B. Some of these differences may be related to the higher exposure and temperatures found in site B compared to site A and also in the low vigor zones.     

Composition and thermal stability of anthocyanins from chinese purple corn ( Zea mays L.)

Chinese purple corn extracts ( Zea mays L., Zhuozhou, Hebei, China) (EZPC) were selected among five Chinese purple corn hybrids due to their higher anthocyanin content, and their thermal stability was evaluated. The total anthocyanin content and total phenolic content of EZPC were 304.5 +/- 16.32 mg of cyanidin-3-glucoside equiv/100 g of dry seeds and 489.8 +/- 24.90 mg of gallic acid equiv/100 g of dry seeds, respectively. Moreover, the individual anthocyanins of EZPC were determined by HPLC-DAD/ESI-MS analysis. Seven main compounds were determined, including cyanidin-3-(malonylglucoside), cyanidin-3-O-glucoside-2-malonylglucoside, cyanidin-3-O-glucoside, peonidin-3-O-glucoside, peonidin-3-(malonylglucoside), pelargonidin-3-(6'-malonylglucoside), and peonidin-3-(dimalonylglucoside). The thermal stability of EZPC was studied by differential scanning calorimetry. Thermodynamic analysis showed that the conversion of EZPC followed an Arrhenius relationship, where the delta enthalpy (H) and activation energy (E(a)) were 97.0 J/g and 204 +/- 2.72 kJ/mol, respectively. Furthermore, the relationships between the degree of conversion of EZPC and time or temperature were reported. This study demonstrated that the evaluated Chinese purple corn hybrids are a natural source of anthocyanins and are stable over a wide range of temperatures and times.     

Assessing potential bioavailability of raspberry anthocyanins using an in vitro digestion system

The bioavailability of anthocyanins from raspberry extracts was assessed using an in vitro digestion procedure that mimics the physiochemical and biochemical changes that occur in the upper gastrointestinal tract (GIT). Effectively all of the total phenol content of the raspberry extract survived gastric digestion and partitioned between the IN sample, which represents the serum available material, and the OUT sample, which represents the material that remains in the GIT and passes through to the colon. All of the anthocyanins also survived gastric digestion, but only approximately 5% entered the IN sample and approximately 70% of total anthocyanins were recovered in the IN and OUT samples. Codigestion of the raspberry extract with commonly combined foodstuffs such as bread, breakfast cereal, ice cream, and cooked minced beef gave a different pattern. The total phenol content of the IN samples was slightly reduced by codigestion with ice cream or breakfast cereal but unaffected by codigestion with bread or minced beef. In most cases, the phenol contents of the postgastric and OUT samples were reduced as compared with the expected values. However, the anthocyanin content of the IN samples was unaffected or increased by coincubation with the foodstuffs. This suggests that polyphenols transiently bind to food matrices during digestion, which protects the more labile anthocyanins from degradation, and they are free to diffuse into the IN sample. The anthocyanin composition of the bioavailability samples was monitored by liquid chromatography-mass spectrometry. All eight anthocyanins previously identified in raspberry were detected in the extract and the postgastric samples at similar yields. All eight anthocyanins could be discerned in the IN and OUT samples, but some such as cyanidin-3-O-glucoside were greatly reduced and others such as pelargonidin-3-O-glucoside were apparently increased in abundance. These differences in stability and their importance for the bioavailability of anthocyanins are discussed.