Absorption of black currant anthocyanins by monolayers of human intestinal epithelial Caco-2 cells mounted in ussing type chambers

Anthocyanins (ACNs) have been reported to have multiple biological properties imparting benefits to human health. Their role in human nutrition, however, needs to be related to biokinetic data, such as bioavailability. The purpose of the present study was to focus on the potential absorption of black currant ( Ribes nigrum L.) ACNs. Caco-2 monolayers were used as an in vitro model of the absorptive intestinal epithelium. For absorption studies, Caco-2 cells grown on permeable filters were mounted into Ussing type chambers. The monolayer integrity was monitored by measuring the transepithelial electrical resistance (TEER). Luminal to serosal transport of ACNs was examined by comparing ACN disappearance from the luminal solution of Ussing chambers not containing any inserts (control chambers) with that of Ussing chambers containing inserts. ACNs (C total ACN approximately 180 microM) were not detected in any serosal solution. However, it was shown that ACNs disappeared from the luminal side, not due to ACN degradation processes but rather--at least in part--due to physiological actions of the cells. The luminal net disappearance of ACNs was calculated (max(t20 min) approximately 11% for total ACNs) and labeled as "absorption efficiency". This apical transport might occur to a much larger extent than the further translocation across the basolateral membrane. Thus, cell metabolism and translocation across the basolateral membrane may be the key determinants of ACN absorption and bioavailability.     

Stability and antioxidant activity of black currant anthocyanins in solution and encapsulated in glucan gel

The effects of ferric and ferrous ions, pH, and temperature on the stability and antioxidant activity of black currant anthocyanins (BCA) were studied, and the recovery of BCA from glucan gel [mixed linked (1-->3,1-->4)-beta-D-glucan] after using different encapsulating procedures was determined. The degradation of individual anthocyanins follows first-order kinetics and shows Arrhenius temperature dependence. The activation energies of individual anthocyanins, evaluated over the temperature range 60-100 degrees C, decrease with an increase in pH. While the antioxidant activity of BCA, measured by the ferric reducing antioxidant power assay, decreased with the degradation of anthocyanins, the completely degraded products still exhibited approximately 30% of the initial antioxidant activity. Ferric ions have a detrimental effect on the stability of BCA, especially for delphinidins. Freeze drying of encapsulated BCA gives approximately 20% higher recovery of individual anthocyanins than infrared drying.     

Urinary excretion of black raspberry (Rubus occidentalis) anthocyanins and their metabolites

Anthocyanins are the most abundant phenolic compounds, widely distributed in fruits and vegetables, and exhibit potent antioxidant capacity. Humans ingest a significant amount of anthocyanins in the daily diet. The objective of the current study was to examine human absorption and metabolism of black raspberry anthocyanins when administered at high doses (2.69 +/- 0.085 g/day). Ten healthy men consumed 45 g of freeze-dried black raspberries daily for 1 week. Urine samples were collected over a 12 h period in 4 h intervals at day 1 and day 7. Urinary anthocyanins were analyzed by high-performance liquid chromatography coupled to a photodiode array detector and a tandem mass spectrometer using precursor ion and product ion analyses. Anthocyanins were excreted in intact forms and metabolized into methylated derivatives in human urine. The urinary excretion of anthocyanins reached a maximum concentration (1091.8 +/- 1081.3 pmol/L, n = 10) during the 4-8 h period after black raspberry ingestion. As compared to the anthocyanin distribution in black raspberries, urinary cyanidin 3-xylosylrutinoside was detected at a higher concentration than that of cyanidin-3-rutinoside.     

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.     

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.     

Effect of red wine consumption on low-density lipoprotein oxidation and atherosclerosis in aorta and coronary artery in Watanabe heritable hyperlipidemic rabbits.

Red wine was given to Watanabe heritable hyperlipidemic (WHHL) rabbits (7 mL/kg/day, approximately 23 mg total polyphenol/kg/day) from the age of 4 months for 14 months. With WHHL rabbits given a comparable dose of alcohol used as references, the effect of wine phenolics on the morphology of atherosclerotic lesions in the coronary artery and aorta was investigated in light of the susceptibility of plasma intermediate and low-density protein (ILDL) to oxidation. ILDL of animals consuming red wine showed reduced susceptibility to oxidation, but atherosclerotic lesion progression was not prevented. In WHHL rabbits, a dose of wine phenolics sufficient to inhibit ILDL oxidation might not be adequate to prevent the progression of atherosclerosis morphologically.     

Characterization and fate of black currant and bilberry flavonols in enzyme-aided processing

The fate of black currant ( Ribes nigrum L.) and bilberry ( Vaccinium myrtillus L.) flavonols in enzyme-aided processing was studied. The flavonols were quantified and characterized by high-performance liquid chromatography equipped with a diode array detector and an electrospray ionization mass spectrometer. A tentative identification for 14 black currant and 19 bilberry flavonols is presented representing 11 previously unpublished conjugates. For the first time in any berry, the presence of laricitrin conjugates is reported. The enzyme-aided processing affected the flavonol extractability, elevating the yield in juices and decreasing that in press residues. Importantly, no significant loss of the berry flavonols was observed during the experiments, although some hydrolysis of flavonol conjugates was recorded. To maximize the effect on flavonol extractability, higher enzyme dosages were needed for black currants than for bilberries. The data show that the flavonol extractability and hydrolysis are dependent on the texture of raw material, the glycosylation pattern of the conjugates, and the activity profile of the enzyme preparation.     

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.     

Antioxidant capacity of black currant varies with organ, season, and cultivar

Small berries such as black currant constitute one of the important sources of potential health-promoting phytochemicals because these fruits are rich sources of compounds with high antioxidant properties. In this work, antioxidant capacities of different parts (buds, leaves, fruits) of various black currant cultivars were compared throughout the growing season with the aim to prepare extracts with high antioxidant capacity. Buds (opened, at the end of March) and leaves (in June) had a higher content in phenolics and antioxidants than fully ripened berries (in July) and the best yield (per branch) was obtained with the leaves collected in June due to their higher biomass. The differences observed among the eight cultivars tested were small. Concerning flavonols, quercetin was dominant in all organs and cultivars, myricetin varied widely among the cultivars, and kampferol was very low.     

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.     

Absorption of anthocyanins from blueberries and serum antioxidant status in human subjects

In recent years, numerous studies have shown that the polyphenolics present in fruit and vegetable products exhibit a wide range of biological effects. However, there is little reliable information on the absorption of glycosylated and acylated anthocyanins in humans. In the present study, the absorption of anthocyanins in humans was investigated after the consumption of a high-fat meal with a freeze-dried blueberry powder containing 25 individual anthocyanins including 6 acylated structures. Nineteen of the 25 anthocyanins present in the blueberries were detected in human blood serum. Furthermore, the appearance of total anthocyanins in the serum was directly correlated with an increase in serum antioxidant capacity (ORAC(acetone), P < 0.01). These results show that anthocyanins can be absorbed in their intact glycosylated and possibly acylated forms in human subjects and that consumption of blueberries, a food source with high in vitro antioxidant properties, is associated with a diet-induced increase in ex vivo serum antioxidant status.     

Extraction of polyphenols from processed black currant (Ribes nigrum L.) residues

The total phenol and anthocyanin contents of black currant pomace and black currant press residue (BPR) extracts, extracted with formic acid in methanol or with methanol/water/acetic acid, were studied. Anthocyanins and other phenols were identified by means of reversed phase HPLC, and differences between the two plant materials were monitored. In all BPR extracts, phenol levels, determined by the Folin-Ciocalteu method, were 8-9 times higher than in the pomace extracts. Acid hydrolysis liberated a much higher concentration of phenols from the pomace than from the black currant press residue. HPLC analysis revealed that delphinidin-3-O-glucoside, delphinidin-3-O-rutinoside, cyanidin-3-O-glucoside, and cyanidin-3-O-rutinoside were the major anthocyanins and constituted the main phenol class ( approximately 90%) in both types of black currant tissues tested. However, anthocyanins were present in considerably lower amounts in the pomace than in the BPR. In accordance with the total phenol content, the antioxidant activity determined by scavenging of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation, the ABTS(*)(+) assay, showed that BPR extracts prepared by solvent extraction exhibited significantly higher (7-10 times) radical scavenging activity than the pomace extracts, and BPR anthocyanins contributed significantly (74 and 77%) to the observed high radical scavenging capacity of the corresponding extracts.     

Fatty acid content and juice characteristics in black currant (Ribes nigrum L.) genotypes

The fatty acid compositions of seeds from 29 black currant genotypes were determined using a rapid small-scale procedure. There was interest in alpha-linolenic, stearidonic, and, especially, gamma-linolenic acid (GLA) contents, and most samples showed values between 11.1 and 18.7%, between 2.5 and 4.5%, and between 11.6 and 17.4%, respectively. However, six genotypes exhibited gamma-linolenic contents >18%, and values >20% were recorded in four of these genotypes. The fatty acid contents of the six genotypes were also analyzed by using a conventional procedure, and only slight differences in fatty acid composition were found between the two methods. Although GLA content was not strongly correlated with juice parameters, some genotypes had both high GLA contents and desirable juice characteristics. The results obtained provide evidence that it is possible to select for GLA contents without negatively affecting juice quality, and both aspects can be combined in a single cultivar, thereby increasing the added value of the whole fruit.     

Effects of mulching and cover cropping on soil microbial parameters in the organic growing of black currant

Abstract

Cover cropping and mulching to sustain and improve soil fertility and for weed control are common practices in organic growing systems. In this study, microbial parameters under different kinds of mulches and cover crops were analyzed in a field experiment with organically grown black currant (Ribes nigrum). The experiment comprised a combination of two mulches with bare soil as a control and two cover crops which were compared with bare soil, with and without an extra supply of organic fertilizer. Soil carbon (C) and nitrogen (N) as well as pH were unaffected by any of the treatments. The basal respiration rate was increased by mulching with wood chips throughout the four years of the experiment. During the last two years of the experiment, substrate induced respiration was also measured but was not found to be affected by any of the mulches. The potential ammonium (NH₄ ⁺) oxidation increased significantly after an initial supply of 200 kg N ha^‐1 as solid cattle manure. The increase was significantly lower under wood chips than in bare soil, although an extra 200 kg N ha^‐1 had been supplied under the wood chips. Furthermore, the black currant bushes suffered from a N deficiency in the wood chip treatment. The results showed that there was no substantial lasting build‐up of microbial biomass or organic matter content with wood chips because of lack of N, despite a large initial input of N and easily‐available C. Possible reasons for this deficiency are either increased denitrification under the wood chips or fungal translocation of N to the wood chip layer. Results from this experiment suggests that the evaluation of a few complementary biological soil parameters can be an important tool when developing sustainable growing systems and for indicating environmental stress.     

Extraction of anthocyanins and other phenolics from black currants with sulfured water

Health benefits of fruits, vegetables, and red wine are attributed to anthocyanins and other phytochemicals. In this research, the extraction of phenolics from black currants was optimized using different SO(2) concentrations (28, 300, 700, 1100, and 1372 ppm), temperatures (6, 20, 40, 60, and 74 degrees C), and solvent to solid ratios (S/S) (6, 20, 40, 60, and 74 mL/g). Surface response methodology was used to optimize yields of anthocyanins and total phenolics, as well as their antiradical and antioxidant activities. The extraction of phenolics varied with the SO(2) concentration, S/S, and temperature. Maximum yields of total phenolics and anthocyanins were obtained at an SO(2) concentration of 1000-1200 ppm and 19 L of solvent/kg of milled frozen berries. The increase of extraction temperature increased the rate of extraction and, thus, times to reach equilibrium for the extraction of total phenolics and anthocyanins were reduced. However, for the extraction of anthocyanins it is recommended that temperatures of 30-35 degrees C be used, as higher temperatures will degrade these compounds. Antioxidant activity was affected by all three experimental variables evaluated; however, the main variable affecting it was S/S. The higher the S/S, the lower the antioxidant index.     

Enhanced absorption of anthocyanins after oral administration of phytic acid in rats and humans

Many studies on the bioavailability of polyphenols have been reported. However, the relative urinary excretions of AC are also low, ranging from 0.004% to 0.1%. By contrast, other polyphenols show higher urinary excretion levels. Here, we studied the enhancing effects of phytic acid (IP6) on absorption of blackcurrant anthocyanins (BCAs) in rats and humans. In rats after oral administration of BCAs (as 241 mg of AC/kg body weight) in IP6 (0%, 0.25%, 0.5%, 1%, 2.5%) solution, the ACs recovery in urine was increased dependent on IP6 dose. These results suggest that the IP6 enhances gastrointestinal absorption of ACs. At the further analysis of IP6 enhancement effect in rat, whereas BCAs were normally passed through the stomach and duodenum within 2 h, in IP6 group, after 2-6 h post-administration, stomach and jejunum content's weights were specifically heavy, and large amounts of ACs were also detected in stomach, duodenum, and jejunum. These results suggested that the mixture of BCAs and IP6 reduced the gastrointestinal motility. Prolongation of ACs residue in gastrointestinal tract then caused the enhancing effects of IP6 on absorption of AC. In the human study, each subject was orally administrated a BCA beverage containing BCA concentrate (AC 4 mg/kg body weight), 1% of IP6, and 1% of sodium citrate as a pH stabilizer. Both the plasma level and the urinary excretion of AC were increased as compared to BCA administration without IP6. AC intake with IP6 may increase the bioavailability of AC to the comparative level as other polyphenols. Yet, phytic acid, being a strong chelator of important minerals, contributes to mineral deficiencies. An interference with iron uptake has been reported. Safety tests are therefore necessary before high dose IP6 can be used in foods.     

Influence of thermal treatment on black currant (Ribes nigrum L.) juice aroma

The influence of thermal treatment on black currant juice aroma was investigated in temperature and time ranges relevant for black currant juice concentration processes, namely, 45, 60, 75, and 90 degrees C. Forty-nine aroma compounds were quantified, and the thermal treatment resulted in concentration increases of most terpenes, aldehydes, furans, and phenols, whereas the concentration of esters slightly decreased. Higher temperatures and longer exposure times had larger effects on the aroma compounds. Odor triangle tests showed no sensory difference between pasteurized juice and juice heated at 60 degrees C, whereas juice heated at 90 degrees C differed significantly from pasteurized juice. It is concluded that a 90 degrees C thermal treatment of black currant juice, which is in the temperature range used for conventional evaporation of black currant juice, has an effect on the aroma and sensory properties.     

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.     

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.     

Analysis of anthocyanins in rat intestinal contents--impact of anthocyanin chemical structure on fecal excretion

Absorption of dietary anthocyanins is limited; however, fecal anthocyanin excretion has been rarely studied. We developed a method for extraction and analysis of fecal anthocyanins. Aqueous methanol (60%) maximized extraction efficiency (approximately 88%). Severe anthocyanin degradation (monitored by high-performance liquid chromatography) was observed in feces stored at -18 degrees C; therefore, storage time should be minimized and lower temperatures used. Fecal and cecal content samples were collected from 32 rats receiving either chokeberry, bilberry, grape-enriched (3.85 g monomeric anthocyanin per kg diet), or control diet for 14 weeks. Fecal anthocyanin concentrations were significantly different among groups (0.7/1.8/2.0 g/kg wet feces, chokeberry/bilberry/grape). Anthocyanin profiles of cecal contents and feces were similar. Losses in the intestinal contents were high for anthocyanin glucosides, moderate for galactosides, and negligible for arabinosides or xylosides. Acylation or diglucosylation enhanced anthocyanin stability. High anthocyanin concentration in the fecal content may favor anthocyanin absorption into the colon epithelial cells, resulting in potential health benefits.