Effects of polyphenols from grape seeds on oxidative damage to cellular DNA

Grape seed polyphenols have been reported to exhibit a broad spectrum of biological properties. In this study, eleven phenolic phytochemicals from grape seeds were purified by gel chromatography and high performance liquid chromatography (HPLC). The antioxidant activities of five representative compounds with different structure type were assessed by the free radical-scavenging tests and the effects of the more potent phytochemicals on oxidative damage to DNA in mice spleen cells were investigated. Procyanidin B4, catechin, epicatechin and gallic acid reduced ferricyanide ion and scavenged the stable free radical, alpha, alpha-diphenyl-beta-picrylhydrazyl (DPPH) much more effectively than the known antioxidant vitamin ascorbic acid, while epicatechin lactone A, an oxidative derivative of epicatechin, did not reduce ferricyanide ion appreciably at concentrations used and was only about half as effective on free radical-scavenging as epicatechin. Mice spleen cells, when pre-incubated with relatively low concentration of procyanidin B4, catechin or gallic acid, were less susceptible to DNA damage induced by hydrogen peroxide (H2O2), as evaluated by the comet assay. In contrast, noticeable DNA damage was induced in mice spleen cells by incubating with higher concentration (150 microM) of catechin. Collectively, these data suggest that procyanidin B4, catechin, gallic acid were good antioxidants, at low concentration they could prevent oxidative damage to cellular DNA. But at higher concentration, these compounds may induce cellular DNA damage, taking catechin for example, which explained the irregularity of dose-effect relationship.     

Procyanidin B2 has anti- and pro-oxidant effects on metal-mediated DNA damage

Procyanidin B2 (epicatechin-(4beta-8)-epicatechin), which is present in grape seeds, apples, and cacao beans, has antioxidant properties. We investigated the mechanism of preventive action of procyanidin B2 against oxidative DNA damage in human cultured cells and isolated DNA. Procyanidin B2 inhibited the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in the human leukemia cell line HL-60 treated with an H2O2-generating system. In contrast, a high concentration of procyanidin B2 increased the formation of 8-oxodG in HL-60 cells. Experiments with calf thymus DNA also revealed that procyanidin B2 decreased 8-oxodG formation by Fe(II)/H2O2, whereas procyanidin B2 induced DNA damage in the presence of Cu(II), and H2O2 extensively enhanced it. An electron spin resonance spin trapping study utilizing 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) demonstrated that procyanidin B2 decreased the signal of M4PO-OH from H2O2 and Fe(II), whereas procyanidin B2 enhanced the signal from H2O2 and Cu(II). As an antioxidant mechanism, UV-visible spectroscopy showed that procyanidin B2 chelated Fe(II) at equivalent concentrations. As a pro-oxidant property, we examined DNA damage induced by procyanidin B2, using 32P-labeled DNA fragments obtained from genes relevant to human cancer. Our results raise the possibility that procyanidin B2 exerts both antioxidant and pro-oxidant properties by interacting with H2O2 and metal ions.     

Plasma total antioxidant capacity is associated with dietary intake and plasma level of antioxidants in postmenopausal women

Increased plasma total antioxidant capacity (TAC) has been associated with a high consumption of fruits and vegetables. However, limited information is available on whether plasma TAC reflects the dietary intake of antioxidants and the levels of individual antioxidants in plasma. By using three different assays, the study aimed to determine if plasma TAC can effectively predict dietary intake of antioxidants and plasma antioxidant status. Forty overweight and apparently healthy postmenopausal women were recruited. Seven-day food records and 12-h fasting blood samples were collected for dietary and plasma antioxidant assessments. Plasma TAC was determined by vitamin C equivalent antioxidant capacity (VCEAC), ferric-reducing ability of plasma (FRAP) and oxygen radical absorbance capacity (ORAC) assays. TAC values determined by VCEAC were highly correlated with FRAP (r=0.79, P<.01) and moderately correlated with ORAC (r=0.34, P<.05). Pearson correlation analyses showed that plasma TAC values by VCEAC and ORAC had positive correlation with plasma uric acid (r=0.56 for VCEAC; r=0.49 for ORAC) and total phenolics (r=0.63 for VCEAC; r=0.36 for ORAC). However, TAC measured by FRAP was correlated only with uric acid (r=0.69). After multivariate adjustment, plasma TAC determined by VCEAC was positively associated with dietary intakes of γ-tocopherol (P<.001), β-carotene (P<.05), anthocyanidins (P<.05), flavones (P<.05), proanthocyanidins (P<.01) and TAC (P<.05), as well as with plasma total phenolics (P<.05), α-tocopherol (P<.001), β-cryptoxanthin (P<.05) and uric acid (P<.05). The findings indicate that plasma TAC measured by VCEAC reflects both dietary and plasma antioxidants and represents more closely the plasma antioxidant levels than ORAC and FRAP.     

Antioxidant properties of catechins and proanthocyanidins: Effect of polymerisation, galloylation and glycosylation

A range of catechins and oligomeric procyanidins was purified by high performance liquid chromatography (HPLC) from grape seed, apple skin, lentil and almond flesh. Catechins, galloylated epicatechin, glycosylated catechin, procyanidin dimers, galloylated dimers, trimer, and tetramer species were all identified, purified and quantified by HPLC, LC-MS and NMR. The antioxidant properties of these compounds were assessed using two methods: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; (b) scavenging of the radical cation of 2,2'-azinobis(3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). Antioxidant activity in the lipid phase decreased with polymerisation in contrast with antioxidant action in the aqueous phase which increased from monomer to trimer and then decreased from trimer to tetramer. Galloylation of catechin and dimeric procyanidins decreased lipid phase and increased aqueous phase antioxidant activity. Glycosylation of catechin demonstrated decreased activity in both phases.     

Antioxidant properties of catechins and proanthocyanidins: Effect of polymerisation,galloylation and glycosylation

A range of catechins and oligomeric procyanidins was purified by high performance liquid chromatography (HPLC) from grape seed, apple skin, lentil and almond flesh. Catechins, galloylated epicatechin, glycosylated catechin, procyanidin dimers, galloylated dimers, trimer, and tetramer species were all identified, purified and quantified by HPLC, LC-MS and NMR. The antioxidant properties of these compounds were assessed using two methods: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; (b) scavenging of the radical cation of 2,2′-azinobis(3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). Antioxidant activity in the lipid phase decreased with polymerisation in contrast with antioxidant action in the aqueous phase which increased from monomer to trimer and then decreased from trimer to tetramer. Galloylation of catechin and dimeric procyanidins decreased lipid phase and increased aqueous phase antioxidant activity. Glycosylation of catechin demonstrated decreased activity in both phases.     

Relevance of apple polyphenols as antioxidants in human plasma: contrasting in vitro and in vivo effects

Apples are a major source of flavonoids in the Western diet, and flavonoid-rich foods may help protect against chronic diseases by antioxidant mechanisms. In the present study we investigated: (1) the antioxidant capacity of representative apple polyphenols and their contribution to the total antioxidant capacity of apple extracts; (2) the effects of adding apple extract to human plasma in vitro on oxidation of endogenous antioxidants and lipids; and (3) the effects of apple consumption by humans on ex vivo oxidation of plasma antioxidants and lipids. We found that the apple-contained flavonols and flavanols, quercetin, rutin, (-)-epicatechin, and (+)-catechin, had a higher antioxidant capacity than the dihydrochalcones, phloridzin and phloretin, and the hydroxycinnamate, chlorogenic acid. However, together these apple polyphenols contributed less than 20% to the total antioxidant capacity of aqueous apple extracts. When human plasma was exposed to a constant flux of aqueous peroxyl radicals, endogenous ascorbate (70.0 +/- 10.3 microM) was oxidized within 45 min of incubation, while endogenous urate (375 +/- 40 microM) and alpha-tocopherol (24.7 +/- 1.2 microM) were oxidized after ascorbate. Addition of 7.1 or 14.3 micrograms/ml total phenols of apple extract did not protect ascorbate from oxidation, but increased the half-life (t1/2) of urate from 136 +/- 15 to 192 +/- 16 and 208 +/- 23 min, respectively (p < 0.05 each), and t1/2 of alpha-tocopherol from 141 +/- 18 to 164 +/- 8 min (p = ns) and 188 +/- 8 min (p < 0.05). Lipid peroxidation started after ascorbate depletion, and addition of apple extract increased the lag time preceding detectable lipid peroxidation from 36.3 +/- 3.7 to 50.9 +/- 2.7 min (p < 0.05) and 70.4 +/- 4.2 min (p < 0.001). However, when six healthy volunteers ate five apples and plasma was obtained up to 4 h after apple consumption, no significant increases in the resistance to oxidation of endogenous urate, alpha-tocopherol, and lipids were found. Thus, despite the high antioxidant capacity of individual apple polyphenols and apple extracts and the significant antioxidant effects of apple extract added to human plasma in vitro, ingestion of large amounts of apples by humans does not appear to result in equivalent in vivo antioxidant effects of apple polyphenols.     

Procyanidins protect Fao cells against hydrogen peroxide-induced oxidative stress

In this paper, we evaluate the extent to which flavonoids in red wine (catechin, epicatechin, quercetin and procyanidins) protect against hydrogen peroxide-induced oxidative stress in Fao cells. When cells were exposed to H(2)O(2), malondialdehyde (MDA) levels, oxidized glutathione (GSSG) levels and lactate dehydrogenase (LDH) release increased, indicating membrane damage and oxidative stress. All the flavonoids studied, and in particular epicatechin and quercetin, protected the plasma membrane. Only procyanidins lowered MDA levels and LDH leakage, maintained a higher reduced/oxidized glutathione ratio, and increased catalase/superoxide dismutase and glutathione peroxidase/superoxide dismutase ratios, and glutathione reductase and glutathione transferase activities. These results show that the procyanidin mixture has a greater antioxidant effect than the individual flavonoids studied, probably due to its oligomer content and/or the additive/synergistic effect of its compounds. This suggests that the mixture of flavonoids found in wine has a greater effect than individual phenols, which may explain many of the healthy effects attributed to wine.     

The antioxidant properties of zinc: interactions with iron and antioxidants.

Potential mechanisms underlying zinc's capacity to protect membranes from lipid oxidation were examined in liposomes. Using lipid oxidation initiators with different chemical and physical properties (transition metals, lipid- or water-soluble azo compounds, ultraviolet radiation c (UVc), superoxide radical anion (O2*-), and peroxynitrite (ONOO-) we observed that zinc only prevented copper (Cu2+)- and iron (Fe2+)-initiated lipid oxidation. In the presence of Fe2+, the antioxidant action of zinc depended directly on the negative charge density of the membrane bilayer. An inverse correlation (r2: 0.96) was observed between the capacity of zinc to prevent iron binding to the membrane and the inhibitory effect of zinc on Fe2+-initiated lipid oxidation. The interaction of zinc with the bilayer did not affect physical properties of the membrane, including rigidification and lateral phase separation known to increase lipid oxidation rates. The interactions between zinc and the lipid- (alpha-tocopherol) and water- (epicatechin) soluble antioxidants were studied. The inhibition of Fe2+-induced lipid oxidation by either alpha-tocopherol or epicatechin was increased by the simultaneous addition of zinc. The combined actions of alpha-tocopherol (0.01 mol%), epicatechin (0.5 microM) and zinc (5-50 microM) almost completely prevented Fe2+ (25 microM)-initiated lipid oxidation. These results show that zinc can protect membranes from iron-initiated lipid oxidation by occupying negatively charged sites with potential iron binding capacity. In addition, the synergistic actions of zinc with lipid and water-soluble antioxidants to prevent lipid oxidation, suggests that zinc is a pivotal component of the antioxidant defense network that protects membranes from oxidation.     

Vitamin E attenuates cold-induced rat liver oxidative damage reducing H2O2 mitochondrial release

Vitamin E is a major chain-breaking antioxidant which is able to reduce liver oxidative damage without modifying aerobic capacity in T(3)-treated rats. We investigated whether vitamin E has similar effects in hyperthyroid state induced by cold exposure. Cold exposure increased aerobic capacity and O(2) consumption in homogenates and mitochondria and tissue mitochondrial protein content. Vitamin E did not modify aerobic capacity and mitochondrial protein content of cold liver, but increased ADP-stimulated respiration of liver preparations. Succinate-supported H(2)O(2) release rates were increased by cold during basal and stimulated respiration, whereas the pyruvate/malate-supported ones increased only during basal respiration. Vitamin administration to cold-exposed rats decreased H(2)O(2) release rates with both substrates during basal respiration. This effect reduced ROS flow from mitochondria to cytosol, limiting liver oxidative damage. Cold exposure also increased mitochondrial capacity to remove H(2)O(2), which was reduced by vitamin treatment, showing that the antioxidant also lowers H(2)O(2) production rate. The different effects of cold exposure and vitamin treatment on H(2)O(2) generation were also found in the presence of respiration inhibitors. Although this can suggest that the cold and vitamin induce opposite changes in mitochondrial content of autoxidizable electron carriers, it is likely that vitamin effect is due to its capacity to scavenge superoxide radical. Finally, vitamin E reduced mitochondrial oxidative damage and susceptibility to oxidants, and prevented Ca(2+)-induced swelling elicited by cold. In the whole, our results suggest that vitamin E is able to maintain aerobic capacity and attenuate oxidative stress of hepatic tissue in cold-exposed rats modifying mitochondrial population characteristics.     

Abies nephrolepis leaf phenolics prevent the inhibition of gap junction intercellular communication by hydrogen peroxide in rat liver epithelial cells

Recent reports suggest that carcinogenicity of hydrogen peroxide (H2O2) is implicated in inhibition of gap junction intercellular communication (GJIC), which is a cellular event associated with the tumor promotion. The present study investigated the effect of phenolics (KF) from leaves of Abies nephrolepis (Khingan fir) on inhibition of GJIC by H2O2 in WB-F344 rat liver epithelial cells. The phenolics were extracted from fresh leaves by using 80% aqueous methanol, and were analyzed mainly as catechin derivatives including epigallocatechin gallate (EGCG) and catechin itself. KF and EGCG protected the inhibition of GJIC by H2O2, whereas butylated hydroxytoluene, a commercial antioxidant, had no effect. Our results indicate that KF exhibits potential chemopreventive effects against carcinogenesis, which may be attributable to phenolics such as EGCG.     

Antioxidants and antioxidant enzymes protect against pulmonary oxygen toxicity in the rabbit

Two major lines of defense exist against oxidant lung injury: tissue antioxidants and antioxidant enzymes. We studied pretreatment with the antioxidants, vitamin E and butylated hydroxyanisole (BHA), and the antioxidant enzymes, superoxide dismutase (SOD) and catalase, in rabbits exposed to 100% O2 for 48 h. BHA (200 mg/kg ip) or vitamin E (50-100 mg/kg po) were given for 2 or 3 days, respectively, before O2 exposure. Combined therapy with polyethylene glycol- (PEG) conjugated SOD (12 mg/kg) and catalase (200,000 U/kg) was given intraperitoneally 1 h before and 24 h after beginning 100% O2. Hyperoxia significantly increased the pulmonary content of malondialdehyde, indicating enhanced lipid peroxidation. One hundred percent O2 also increased lung weight gain and alveolar-capillary permeability to aerosolized 99mTc-labeled diethylenetriaminepentaacetate (99mTc-DTPA, 500 mol wt) and fluorescein isothiocyanate-labeled dextran (7,000 mol wt). Pretreatment with vitamin E, BHA, or the combination of PEG-SOD and PEG-catalase prevented the increase in malondialdehyde, lung weight gain, and alveolar-capillary permeability caused by hyperoxia. These results indicate that augmenting either tissue antioxidants or antioxidant enzymes can prevent the pulmonary injury caused by 48 h of 100% O2 in rabbits.     

Antioxidant actions of plant foods: Use of oxidative DNA damage as a tool for studying antioxidant efficacy

Plant-food-derived antioxidants and active principles such as flavonoids, hydroxycinnamates (ferulic acid, chlorogenic acids, vanillin etc.), β-carotene and other carotenoids, vitamin E, vitamin C, or rosemary, sage, tea and numerous extracts are increasingly proposed as important dietary antioxidant factors. In this endeavor, assays involving oxidative DNA damage for characterizing the potential antioxidant actions are suggested as in vitro screens of antioxidant efficacy. The critical question is the bioavailability of the plant-derived antioxidants.     

A comparison of antioxidant properties of hypoxen and duroquinone by the method of chemiluminescence

The antioxidant activity of duroquinone and hypoxen was compared with that of alpha-tocopherol and vitamin C in a model system (luminol-peroxidase-H2O2), and their influence on the level of reactive oxygen species in systems containing polymorphonuclear leukocytes of healthy and tumor-bearing animals was studied. It was shown that, in a model chemical system, the concentrations of antioxidants (the inverse of antioxidant activity) necessary to decrease twice the intensity of the chemiluminescence answer (C50%) are arranged in the following order: alpha-tocopherol > duroquinone > hypoxen > ascorbic acid. In this case, the concentrations of the hydrophobic antioxidants (C50% for alpha-tocopherol and duroquinone 10-30 mkM) should be 20-50 times higher than for hydrophilic antioxidants (C50% for vitamin C and hypoxen 0.5-0.6 mkM). It was revealed that the generation of reactive oxygen species by blood phagocytes of tumor-bearing animals is 2-2.5 times higher than by phagocytes of healthy animals. The antioxidant concentration necessary to decrease the chemiluminescence answer in the cellular system should be one order of magnitude higher than in the model chemical system. The distribution of a hydrophobic antioxidant between water/lipid phases promotes an increase in the concentration of the antioxidant necessary to decrease the level of reactive oxygen species twice. Thus, the major factor influencing the antioxidant activity is the constant of distribution of these compounds in a water/lipid system.     

Influence of vitamin C and vitamin E on redox signaling: Implications for exercise adaptations

The exogenous antioxidants vitamin C (ascorbate) and vitamin E (α-tocopherol) often blunt favorable cell signaling responses to exercise, suggesting that redox signaling contributes to exercise adaptations. Current theories posit that this antioxidant paradigm interferes with redox signaling by attenuating exercise-induced reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. The well-documented in vitro antioxidant actions of ascorbate and α-tocopherol and characterization of the type and source of the ROS/RNS produced during exercise theoretically enable identification of redox-dependent mechanisms responsible for the blunting of favorable cell signaling responses to exercise. This review aimed to apply this reasoning to determine how the aforementioned antioxidants might attenuate exercise-induced ROS/RNS production. The principal outcomes of this analysis are (1) neither antioxidant is likely to attenuate nitric oxide signaling either directly (reaction with nitric oxide) or indirectly (reaction with derivatives, e.g., peroxynitrite); (2) neither antioxidant reacts appreciably with hydrogen peroxide, a key effector of redox signaling; (3) ascorbate but not α-tocopherol has the capacity to attenuate exercise-induced superoxide generation; and (4) alternate mechanisms, namely pro-oxidant side reactions and/or reduction of bioactive oxidized macromolecule adducts, are unlikely to interfere with exercise-induced redox signaling. Out of all the possibilities considered, ascorbate-mediated suppression of superoxide generation with attendant implications for hydrogen peroxide signaling is arguably the most cogent explanation for blunting of favorable cell signaling responses to exercise. However, this mechanism is dependent on ascorbate accumulating at sites rich in NADPH oxidases, principal contributors to contraction-mediated superoxide generation, and outcompeting nitric oxide and superoxide dismutase isoforms. The major conclusions of this review are: (1) direct evidence for interference of ascorbate and α-tocopherol with exercise-induced ROS/RNS production is lacking; (2) theoretical analysis reveals that both antioxidants are unlikely to have a major impact on exercise-induced redox signaling; and (3) it is worth considering alternate redox-independent mechanisms.     

Antioxidant combinations are no more beneficial than individual components in combating ram sperm oxidative stress during storage at 5°C

The unfrozen storage of ram semen for 2-4 days is an important goal for the acceptance of artificial insemination in sheep breeding programmes. The objective was to investigate the benefits of antioxidant supplementation on the production of hydrogen peroxide (H(2)O(2)) by ram spermatozoa stored at 5°C over 3 days. Ejaculates from 9 rams were split between two defined diluents, INRA-96 and RSD-1, and cooled slowly to 5°C for storage. Four different additives (vitamin E phosphate 6-100 μmol/L, catalase 500 IU+superoxide dismutase 9-150 μmol/L, and glutathione peroxidase, 20 IU) were investigated both separately and in combination. The amount of H(2)O(2) generated was assessed by use of a 1-step fluorometric micro-plate assay. Sperm viability, acrosome integrity and membrane fluidity were assessed by flow cytometry. H(2)O(2) production in INRA-96- compared with RSD-1-diluted spermatozoa increased approximately 2-3-fold after 24h in storage at 5°C and then declined up to 72 h, while that in RSD-1 showed no change over 72 h; this had no effect on the sperm characteristics. Addition of antioxidants singly reduced H(2)O(2) production in INRA-96, regardless of concentration. Optimal concentrations were vitamin E phosphate 12.5 μmol/L, catalase 500 IU, superoxide dismutase 37 μmol/L, and glutathione peroxidase, 20 IU. In any combination, none was more effective than others. Viability was reduced but acrosomal integrity protected by the antioxidants in INRA-96 but not in RSD-1; membrane fluidity was not affected. Based on this study, there were no combinations more efficient at combating oxidative stress than any one alone.     

Antioxidant actions of plant foods: Use of oxidative DNA damage as a tool for studying antioxidant efficacy

Plant-food-derived antioxidants and active principles such as flavonoids, hydroxycinnamates (ferulic acid, chlorogenic acids, vanillin etc.), β-carotene and other carotenoids, vitamin E, vitamin C, or rosemary, sage, tea and numerous extracts are increasingly proposed as important dietary antioxidant factors. In this endeavor, assays involving oxidative DNA damage for characterizing the potential antioxidant actions are suggested as in vitro screens of antioxidant efficacy. The critical question is the bioavailability of the plant-derived antioxidants.     

Total antioxidant capacity and nuclear DNA damage in keratinocytes after exposure to H2O2

Studies of oxidative stress have classically been performed by analyzing specific, single antioxidants. In this study, susceptibility to oxidative stress in the human keratinocyte cell line NCTC2544 exposed to hydrogen peroxide (H2O2) was measured by the TOSC (total oxyradical scavenging capacity) assay, which discriminates between the antioxidant capacity toward peroxyl radicals and hydroxyl radical. The generation of H2O2-induced DNA damage, total antioxidant capacity and levels of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, glutathione peroxidase) were studied. Exposure to H2O2-induced DNA damage that was gradually restored while a significant reduction in cellular TOSC values was obtained independently of stressor concentrations and the degree of DNA repair. Whereas TOSC values and cell resistance to H2O2 showed a good relationship, the extent of DNA damage is independent from cellular total antioxidant capacity. Indeed, maximum DNA damage and cell mortality were observed in the first 4 h, whereas TOSC remained persistently low until 48 h. Catalase levels were significantly lower in exposed cells after 24 and 48 h. Keratinocytes exposed after 48 h to a second H2O2 treatment exhibited massive cell death. A possible linkage was observed between TOSC values and NCTC2544 resistance to H2O2 challenge. The TOSC assay appears to be a useful tool for evaluating cellular resistance to oxidative stress.     

Attenuation of lipid peroxidation by antioxidants in rat-1 fibroblasts: comparison of the lipid peroxidation reporter molecules cis-parinaric acid and C11-BODIPY(581/591) in a biological setting

Lipid peroxidation is a major factor in the pathogenesis of many disease states. To detect the initial stages of lipid peroxidation or evaluate antioxidant efficacy, cis-parinaric acid (cis-PnA) has been successfully used and thoroughly validated. However, cis-PnA is not very well suited for medium throughput screening of antioxidants in living cells. We recently introduced and validated a lipid peroxidation reporter molecule, C11-BODIPY(581/591). To further explore this probe, we evaluated the protective effect of 12 natural antioxidants in rat-1 fibroblasts subjected to 50 microM cumene-hydroperoxide using both probes. The same pecking order for the individual antioxidant efficacies was obtained: alpha-tocopherol approximately gamma-tocopherol > quercetin approximately lycopene > kaempferol > palm oil > hydroxy-tyrosol > > alpha-carotene = beta-carotene = lutein = tyrosol = chlorogenic acid. This validates the accuracy of the C11-BODIPY(581/591) method and shows that this assay is an accurate and highly flexible method for indexing lipid peroxidation or determining antioxidant efficacy in living cells in a medium throughput scenario. The antioxidant efficacy was compared with their one-electron reduction potential, hydrophobicity and Trolox C equivalent antioxidant capacity. Our results show that although these parameters are valuable for determining structure-function relationships, they have limited predictive value for antioxidant efficacy in vivo.     

The polyphenolic content of fruit and vegetables and their antioxidant activities. What does a serving constitute?

Analysis of the major flavone, flavonol, anthocyanidin and hydroxycinnamic acid constituents (and their glycosides) of onion, tomato, egg plant and apple has been undertaken and the antioxidant activities of the phenolic extracts determined. The major phenolic antioxidant components of egg plant are chlorogenic acid in the flesh and a delphinidin conjugate in the skin. In the case of apple, the major phenolic antioxidants detected are chlorogenic acid, procyanidins/catechin compounds, rutin and phloridzin. Quercetin glycosides are well-known to be the major phenolic components of onion. Assessment of the antioxidant activities of a serving of 100 g fresh weight fruit, vegetable and comparison with previously reported findings for 150 ml beverage (500 ml portion in the case of beer), expressed in μmol Trolox equivalents show that the antioxidant activities of 1 glass (150 ml) red wine ≡ 12 glasses white wine ≡ 2 cups of tea ≡ 4 apples ≡ 5 portions of onion ≡ 5.5 portions egg plant ≡ 3.5 glasses of blackcurrant juice ≡ 3.5 (500 ml) glasses of beer ≡ 7 glasses of orange juice ≡ 20 glasses of apple juice (long life).     

Ascorbic acid 6-palmitate suppresses gap-junctional intercellular communication through phosphorylation of connexin 43 via activation of the MEK–ERK pathway

Although the health benefits of dietary antioxidants have been extensively studied, their potential negative effects remain unclear. L-Ascorbic acid 6-palmitate (AAP), a synthetic derivative of ascorbic acid (AA), is widely used as an antioxidant and preservative in foods, vitamins, drugs, and cosmetics. Previously, we found that AA exerted an antitumor effect by protecting inhibition of gap-junctional intercellular communication (GJIC), which is closely associated with tumor progression. In this study, we examined whether AAP, an amphipathic derivative of AA, has chemopreventive effects using a GJIC model. AAP and AA exhibited dose-dependent free radical-scavenging activities and inhibited hydrogen peroxide (H₂O₂)-induced intracellular reactive oxygen species (ROS) production in normal rat liver epithelial cells. Unexpectedly, however, AAP did not protect against the inhibition of GJIC induced by H₂O₂; instead, it inhibited GJIC synergistically with H₂O₂. AAP inhibited GJIC in a dose-dependent and reversible manner. This inhibitory effect was not due to the conjugated lipid structure of AAP, as treatment with palmitic acid alone failed to inhibit GJIC under the same conditions. The inhibition of GJIC by AAP was restored in the presence of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126, but not in the presence of other signal inhibitors and antioxidant (PKC inhibitors, EGFR inhibitor, NADPH oxidase inhibitor, catalase, vitamin E, or AA), indicating the critical involvement of MEK signaling in the GJIC inhibitory activity of AAP. Phosphorylation of ERK and connexin 43 (Cx43) was observed following AAP treatment, and this was reversed by U0126. These results suggest that the AAP-induced inhibition of GJIC is mediated by the phosphorylation of Cx43 via activation of the MEK–ERK pathway. Taken together, our results indicate that AAP has a potent carcinogenic effect, and that the influence of dietary antioxidants on carcinogenesis may be paradoxical.