Activity and mechanism of the antioxidant properties of cyanidin-3-O-beta-glucopyranoside.

In the present study, the antioxidant activity, the interaction with reactive oxygen species and the redox potential of cyanidin-3-O-beta-glucopyranoside (C-3-G), the main anthocyanin present in juice of pigmented oranges, were evaluated in detail. C-3-G effects on low density lipoproteins (LDL) oxidation induced by 40 microM Cu at a pH of 7.4 were compared with those of resveratrol and ascorbic acid, two other natural antioxidants. All cyanidin-3-O-beta-glucopyranoside concentrations used (1, 2, 5, 10, 20, 50, 100 and 200 microM) inhibited malondialdehyde (MDA) generation (an index of lipid peroxidation), the inhibition being significantly higher than that obtained with equal concentrations of resveratrol and ascorbic acid (IC50 = 6.5 microM for C-3-G, 34 microM for resveratrol and 212 microM for ascorbic acid). Experiments of LDL oxidation performed at a pH of 5.0 or 6.0 showed that C-3-G antioxidant activity is not influenced by pH variations between 5.0 and 7.4. This suggests that metal chelation, exerted by C-3-G through the eventual dissociation of its phenolic groups, plays a minor role in its protective mechanism. The presence of C-3-G produced significantly higher protective effects of pigmented orange juice (obtained from Moro cultivar) with respect to blond orange juice, when tested on copper-induced LDL oxidation. The evaluation of the direct interaction with reactive oxygen species (H2O2, -O2, OH*), demonstrated that C-3-G is quickly oxidized by these compounds and it is, therefore, a highly efficient oxygen free radical scavenger. The powerful C-3-G antioxidant activity is in excellent agreement with the very negative redox potential (-405 mV), determined through direct current cyclic voltammetry measurements. On the basis of these results, C-3-G should be considered as one of the most effective antioxidants that can be assumed with dietary plants; therefore, pigmented oranges represent a very relevant C-3-G source because of the high content of this anthocyanin in their juice.     

Bioactive Compounds of Fruit Parts of Three Eugenia uniflora Biotypes in Four Ripening Stages

Variability of secondary metabolites in edible (peel and pulp) and inedible (seeds) parts of three pitanga varieties, red, red-orange and purple, was investigated during the maturation process. Hydrolysable tannins, anthocyanins, and flavonoids were quantified by HPLC/DAD and carotenoids by absorbance. Peel/pulp showed greater complexity of constituents (carotenoids, anthocyanins, flavonoids, and hydrolysable tannins), while only tannins were identified in seeds, but in quantities of 10 to 100 times greater. The red-orange variety showed the highest levels of phenolic compounds in seeds and peel/pulp, except anthocyanins. The analysis of the principal response curves showed that the pitanga biotype has greater influence on metabolite variation than ripening stages. During peel/pulp maturation, a reduction in the levels of flavonoids and tannins contrasted with an increase in carotenoids and cyanidin-3-O-glucoside in all varieties, whereas in the seeds oenothein B, the major tannin, increased up to 1.32 g/100 g fresh weight. Such marked differences between fruit parts demonstrate that the seeds in stages E3 and E4 are a source of hydrolysable tannins, compounds known for their antitumor activity, while peel/pulp of all varieties in the ripe stage provide natural antioxidants, such as carotenoids and flavonoids. Lastly, the purple biotype can be a rich source of the cyanidin-3-O-glucoside pigment a potent bioactive compound.     

Activity and mechanism of the antioxidant properties of cyanidin-3-O-β-glucopyranoside

In the present study, the antioxidant activity, the interaction with reactive oxygen species and the redox potential of cyanidin-3-O-β-glucopyranoside (C-3-G), the main anthocyanin present in juice of pigmented oranges, were evaluated in detail. C-3-G effects on low density lipoproteins (LDL) oxidation induced by 40 μM Cu²⁺ at a pH of 7.4 were compared with those of resveratrol and ascorbic acid, two other natural antioxidants. All cyanidin-3-O-β-glucopyranoside concentrations used (1, 2, 5, 10, 20, 50, 100 and 200 μM) inhibited malondialdehyde (MDA) generation (an index of lipid peroxidation), the inhibition being significantly higher than that obtained with equal concentrations of resveratrol and ascorbic acid (IC₅₀=6.5 μM for C-3-G, 34 μM for resveratrol and 212 μM for ascorbic acid). Experiments of LDL oxidation performed at a pH of 5.0 or 6.0 showed that C-3-G antioxidant activity is not influenced by pH variations between 5.0 and 7.4. This suggests that metal chelation, exerted by C-3-G through the eventual dissociation of its phenolic groups, plays a minor role in its protective mechanism. The presence of C-3-G produced significantly higher protective effects of pigmented orange juice (obtained from Moro cultivar) with respect to blond orange juice, when tested on copper-induced LDL oxidation. The evaluation of the direct interaction with reactive oxygen species (H₂O₂, ^-O₂, OH^·), demonstrated that C-3-G is quickly oxidized by these compounds and it is, therefore, a highly efficient oxygen free radical scavenger. The powerful C-3-G antioxidant activity is in excellent agreement with the very negative redox potential (405 mV), determined through direct current cyclic voltammetry measurements.

On the basis of these results, C-3-G should be considered as one of the most effective antioxidants that can be assumed with dietary plants; therefore, pigmented oranges represent a very relevant C-3-G source because of the high content of this anthocyanin in their juice.     

Anthocyanin composition of Brassica oleracea cv. Red Danish

Eight anthocyanins were isolated from illuminated red cabbage seedlings. They were identified as: cyanidin-3-sophoroside-5-glucoside, cyanidin-3-malonyl-sophoroside-5-glucoside, cyanidin-3-p-coumaryl-sophoroside-5-glucoside, cyanidin-3-(di-p-coumaryl)sophoroside-5-glucoside, cyanidin-3-ferulyl-sophoroside-5-glucoside, cyanidin-3-(diferulyl)sophoroside-5-glucoside, cyanidin-3-sinapylsophoroside-5-glucoside, and cyanidin-3-(disinapyl)-sophoroside-5-glucoside.     

湖南地区塔罗科血橙花青素苷含量研究初报

为了解湖南地区塔罗科血橙Citrus sinensis ‘Tarocco’花青素苷的合成时期和相对含量,通过调查选择合适的采样点,在开花后100 d起每隔30 d采摘果实,利用pH示差法测定塔罗科血橙果肉的花青素苷含量,并以同果园的纽荷尔脐橙C. sinensis ‘Newhall’为对照。结果表明,纽荷尔脐橙在果实发育阶段中不形成花青素,塔罗科血橙的花青素在开花220 d之后开始积累,至250 d时花青素苷含量为5.98 mg?L-1,280 d时花青素苷含量达12.09 mg?L-1。随着塔罗科血橙果实发育,花青素苷含量逐渐增加。     

Urinary excretion of cyanidin glycosides

Anthocyanins, which are natural plant pigments from the flavonoid family, represent substantial constituents of the human diet. Several fruits (blackcurrant, blue berries, red grape and elderberry) are rich sources of these efficient antioxidant compounds. The present study was designed to determine the potential bioavailability in humans of the anthocyanins of elderberry, mainly cyanidin-3-glucoside and cyanidin-3-sambubioside, and the influence of the simultaneous ingestion of sucrose on the absorption of anthocyanins. Urinary samples from 16 healthy volunteers--8 women and 8 men--were collected before and over a period of 6 h with intervals of 1 h after the ingestion of 11 g elderberry concentrate (containing 1.9 g of anthocyanins equivalent to 235 ml of fresh juice) 1 day diluted with water, the other day with 30 g sucrose. Using high-performance liquid chromatography, it was possible to quantify the two main anthocyanins of elderberry excreted unchanged in the urine (0.003-0.012% of the oral dose). The ingestion of sucrose led to a reduced excretion of anthocyanins.     

Anthocyanins from red cabbage--stability to simulated gastrointestinal digestion

Anthocyanins were the main polyphenol components in extracts of fresh and pickled red cabbage. The composition of anthocyanins in red cabbage was studied using liquid chromatography mass-spectrometry. Eleven major peaks absorbing at 520 nm were discerned, which represented 18 different anthocyanin structures. Another five minor anthocyanin components could be identified by searching at their respective m/z values but only in anthocyanin-enriched concentrates produced by sorption to solid phase extraction matrices. The predominant anthocyanins were constructed of cyanidin-3-diglucoside-5-glucoside "cores" which were non-acylated, mono-acylated or di-acylated with p-coumaric, caffeic, ferulic and sinapic acids. Pelargonidin-3-glucoside and novel forms of cyanidin-3-O-triglucoside-5-O-glucoside di-acylated with hydroxycinnamic acids were also detected in extracts of raw red cabbage, commercially pickled red cabbage and anthocyanin-enriched concentrates. The stability of the anthocyanins to simulated gastrointestinal digestion was assessed. The anthocyanins were effectively stable in the acidic gastric digestion conditions but the total recovery after simulated pancreatic digestion was around 25% compared to around 100% recovery of phenol content. As anthocyanins make up the majority of red cabbage polyphenols, this suggested that anthocyanins broke down to form new phenolic components. The recovery of the individual anthocyanins was monitored by LC-MS(n). All of the anthocyanins were reduced in content after pancreatic digestion but acylated forms were notably more stable than non-acylated forms. There was also a relationship between the type of acylated hydroxycinnamic acid and stability to pancreatic digestion.     

Interaction between flavonoids and the blood-brain barrier: in vitro studies

There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood-brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (Papp) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity.     

FLAVONOID PIGMENTS IN MIMULUS CARDINALIS AND ITS RELATED SPECIES. I. ANTHOCYANINS

Chromatographic and spectrophotometric techniques were used to identify the anthocyanin pigments present in Mimulus cardinalis and its related species of section Erythranthe of the genus Mimulus (Scrophulariaceae). On the basis of rigorous tests, the flowers of M. cardinalis were found to contain pelargonidin-3-glucoside, pelargonidin-3-rhamnoglucoside, the caffeoyl ester of pelargonidin-3-glucoside, cyanidin-3-glucoside, cyanidin-3-rhamnoglucoside, and the caffeoyl ester of cyanidin-3-glucoside. Qualitatively all members of the group contain these six anthocyanins except M. lewisii. All of its populations lack the pelargonidin glycosides and some lack, in addition, the cyanidin-3-rhamnoglucoside. The striking differences in flower color and intensity appear to be due to quantitative differences not here analyzed.     

Changes in citrus leaf flavonoid concentrations resulting from blight-induced zinc-deficiency

Increased flavonoid concentrations were found to correlate with the elevated levels of leaf phenolic compounds occurring in blight-induced zinc-deficient citrus. In orange (Citrus sinensis L.) leaves, the increases occurred primarily in hesperidin and diosmin, whereas in grapefruit (C. paradisi Macf.) the largest increases occurred in naringin and rhoifolin. Zinc-deficiency occurring in the blighted citrus leaves appeared to be the important contributing factor to the increased flavonoid content. Although the leaves from trees with blight were typically smaller than leaves from unaffected trees, the increased flavonoid content was not significantly due to a concentration effect. Large differences occurred in the percent increases in concentrations of certain citrus leaf flavonoids. While large increases occurred for a number of flavanone and flavone glycosides, much smaller percent increases occurred for other minor flavone glycosides, and the polymethoxyflavone aglycones. The parallel increases occurring in the concentrations of certain flavone glycosides and their flavanone analogs provide a further indication that flavanone glycosides are precursors in the biosynthesis of flavone glycosides in citrus.     

The antioxidant activity of regularly consumed fruit and vegetables reflects their phenolic and vitamin C composition

Recent studies are emphasising the importance and putative modes of action of specific flavonoids as bioactive components of the diet in in vivo and in vitro models. Thus, it is important to have a clear idea of the major phenolic families of which fruit and vegetables are comprised and the levels contained therein. Regularly consumed fruit and vegetables of mixed varieties available on the UK market were analysed for the composition of the major individual phenolic components. The total phenolic content (applying the Folin assay) and the vitamin C levels were also determined. The antioxidant capacities of aqueous/methanolic extracts were comparatively assessed using the TEAC (Trolox Equivalent Antioxidant Capacity), the FRAP (Ferric Reducing Ability of Plasma) and ORAC (Oxygen Radical Absorbance Capacity) assays, which comprise contributions from polyphenols, simple phenols and the ascorbate component. The results were calculated in terms of 100 g fresh weight (FW) uncooked portion sizes. Fruit and vegetables rich in anthocyanins (e.g. strawberry, raspberry and red plum) demonstrated the highest antioxidant activities, followed by those rich in flavanones (e.g. orange and grapefruit) and flavonols (e.g. onion, leek, spinach and green cabbage), while the hydroxycinnamate-rich fruit (e.g. apple, tomato, pear and peach) consistently elicited the lower antioxidant activities. The TEAC, FRAP and ORAC values for each extract were relatively similar and well-correlated with the total phenolic and vitamin C contents. The antioxidant activities (TEAC) in terms of 100 g FW uncooked portion size were in the order: strawberry> raspberry = red plum > red cabbage >grapefruit = orange > spinach > broccoli > green grape approximately/= onion > green cabbage > pea > apple > cauliflower tomato approximately/= peach=leek > banana approximately/= lettuce.     

Anthocyanins inhibit peroxyl radical-induced apoptosis in Caco-2 cells

The antioxidant activity of anthocyanins has been well characterized in vitro; many cases has been postulated to provide an important exogenous mediator of oxidative stress in the gastrointestinal tract. The objective of this study was to evaluate the efficacy of anthocyanin protection against peroxyl radical (AAPH)-induced oxidative damage and associated cytotoxicity in Caco-2 colon cancer cells. Crude blackberry extracts were purified by gel filtration column to yield purified anthocyanin extracts that were composed of 371 mg/g total anthocyanin, 90.1% cyanidin-3-glucoside, and 4.9 mmol Trolox equivalent/g (ORAC) value. There were no other detectable phenolic compounds in the purified anthocyanin extract. The anthocyanin extract suppressed AAPH-initiated Caco-2 intracellular oxidation in a concentration-dependent manner, with an IC₅₀ value of 6.5 ± 0.3 μg/ml. Anthocyanins were not toxic to Caco-2 cells, but provided significant (P < 0.05) protection against AAPH-induced cytotoxicity, when assessed using the CellTiter-Glo assay. AAPH-induced cytoxicity in Caco-2 cells was attributed to a significant (P < 0.05) reduction in the G1 phase and increased proportion of cells in the sub G1 phase, indicating apoptosis. Prior exposure of Caco-2 cells to anthocyanins suppressed (P < 0.05) the AAPH-induced apoptosis by decreasing the proportion of cells in the sub-G1 phase, normalized the proportion of cells in other cell cycle phases. Our results show that the antioxidant activity of anthocyanins principally attributed to cyanidin-3-O-glucoside and common to blackberry, are effective at inhibiting peroxyl radical induced apoptosis in cultured Caco-2 cells.     

Comparative analysis of the volatile fraction of fruit juice from different Citrus species

The volatile composition of fruit from four Citrus varieties (Powell Navel orange, Clemenules mandarine, and Fortune mandarine and Chandler pummelo) covering four different species has been studied. Over one hundred compounds were profiled after HS-SPME-GC-MS analysis, including 27 esters, 23 aldehydes, 21 alcohols, 13 monoterpene hydrocarbons, 10 ketones, 5 sesquiterpene hydrocarbons, 4 monoterpene cyclic ethers, 4 furans, and 2 aromatic hydrocarbons, which were all confirmed with standards. The differences in the volatile profile among juices of these varieties were essentially quantitative and only a few compounds were found exclusively in a single variety, mainly in Chandler. The volatile profile however was able to differentiate all four varieties and revealed complex interactions between them including the participation in the same biosynthetic pathway. Some compounds (6 esters, 2 ketones, 1 furan and 2 aromatic hydrocarbons) had never been reported earlier in Citrus juices. This volatile profiling platform for Citrus juice by HS-SPME-GC-MS and the interrelationship detected among the volatiles can be used as a roadmap for future breeding or biotechnological applications.     

Pulsed electric fields-processed orange juice consumption increases plasma vitamin C and decreases F2-isoprostanes in healthy humans

Orange juice, a rich source of vitamin C, accounts for 60% of all fruit juices and juice-based drinks consumed in western Europe. Orange juice preservation is currently accomplished by traditional pasteurization. Pulsed electric fields (PEF) have been studied as a nonthermal food preservation method. Food technology needs in the area of processing are driven by nutrition. Therefore, the objectives of this study were to assess the bioavailability of vitamin C from pulsed electric fields-treated orange juice in comparison with freshly squeezed orange juice and its impact on 8-epiPGF(2alpha) concentrations (biomarker of lipid peroxidation) in a healthy human population. Six subjects consumed 500 mL/day of pulsed electric fields-treated orange juice and six subjects consumed 500 mL/day of freshly squeezed orange juice for 14 days, corresponding to an intake of about 185 mg/day of ascorbic acid. On the first day of the study, subjects drank the juice in one dose, and on days 2-14 they consumed 250 mL in the morning and 250 mL in the afternoon. Blood was collected every hour for 6 hours on the first day and again on days 7 and 14. In the dose-response study, the maximum increase in plasma vitamin C occurred 4 hours postdose. Vitamin C remained significantly higher on days 7 and 14 in both orange juice groups. Plasma 8-epiPGF(2alpha) concentrations was lower at the end of the study (P < 0.001) in both groups. Plasma levels of vitamin C and 8-epiPGF(2alpha) were inversely correlated. Pulsed electric fields-preservation of orange juice retains the vitamin C bioavailability and antioxidant properties of fresh juice with a longer shelf-life.     

Gene characterization, analysis of expression and in vitro synthesis of dihydroflavonol 4-reductase from [Citrus sinensis (L.) Osbeck

Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key "late" step in the biosynthesis of anthocyanins. In this study we showed that a strong reduction in DFR expression occurs in the non-red orange cultivar (Navel and Ovale) compared to that of the red orange (Tarocco) suggesting that the enzyme could be involved in the lack of production of anthocyanins. Therefore, we isolated and compared the cDNAs, the genomic clones, as well as the promoter regions of blood and blond orange dfrs. Our data revealed that the cDNA sequences of pigmented and non-pigmented orange DFRs were 100% homologous and contained a 1017 bp open reading frame which encodes a protein of 338 amino acid residues, corresponding to a molecular mass of 38010.76 Da, with a theoretical pI of 5.96. Moreover, we found that there were no significant differences in non-coding regions (introns and 5' upstream region) of dfr sequences. Southern blot analysis of genomic DNA indicated that dfr was present as a single copy gene in both cultivars. From these findings the low expression level of blond orange dfr, which might play a role in the phenotypic change from blood to blond orange, is thought to be the result of a likely mutation in a regulatory gene controlling the expression of dfr. In addition, here we reported the successful expression of orange DFR cDNAs leading to an active DFR enzyme which converts dihydroquercetin to leucoanthocyanidin, thus confirming the involvement of the isolated genes in the biosynthesis of anthocyanins. Moreover, as far as we know, this is the first report concerning the in vitro expression of DFR from fruit flesh whose biochemical properties might be very different from those of other plant organ DFRs.     

Survey of bioactive components in Western Canadian berries

Berries native to Western Canada were analyzed for total anthocyanins, total phenolics, and trolox equivalent antioxidant activity (TEAC). Values ranged from 1.60 to 9.55 mmol trolox equivalent per 100 g fresh mass. Anthocyanin content ranged from 41.6 (in red twinberries) to 1081 mg cyanidin-3-glucoside equivalents per 100 g fresh mass (in honeysuckle fruits). Honeysuckle fruits contained the highest amount of total polyphenols, 1111 mg gallic acid equivalents per 100 g, among analyzed fruits. Additionally, anthocyanins in the investigated berries were identified and characterized by HPLC - electrospray ionization - tandem mass spectrometric method coupled with diode array detection. The number of anthocyanins varied from 4 in saskatoon berries (Amelanchier alnifolia Nutt.) to 20 in bilberries (Vaccinum myrtilloides Michx.). In all the samples analyzed, 6 common anthocyanidins:, cyanidin, delphinidin, pelargonidin, petunidin, peonidin, and malvidin, were found. Half the analyzed berries contained acylated anthocyanins, but a significant amount was found only in bilberries. The analyzed berry seed oils contained high amounts of unsaturated fatty acids (over 90%), but only the golden currant seed oil contained gamma-linolenic acid.