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Many oils from plants are important components of our food chain and maintaining their oxidative stability (OS) is economically and nutritionally important. OS is dependent in part on antioxidant capacity (AC) arising from the electron donating ability of endogenous compounds in the oils. Attention has focused on the contribution to AC of phenolic compounds in oils as many have bioactivities in vitro. However, the relevance of such phenolics to healthy nutrition remains unclear. In contrast, many plant-derived oils also contain tocopherol homologs, which as vitamin E are dietary-essential, lipid-soluble antioxidants. We have determined the AC of twelve “off the shelf” edible oils by assessing their ability to quench galvinoxyl, a stable free radical species. The stochiometric reactivity of vitamin E with galvinoxyl indicates that the combined dα-tocopherol and dγ-tocopherol homologs contribute between 20% (olive) and 85% (soya) to AC depending on type of oil. Tocopherols are important contributors to the AC of several vegetable oils. Breeding and photo mixotrophic programmes to enhance content in edible oil crops of this important vitamin may have a positive impact not only on oil stability during storage but also in contributing to provision of optimum dietary intakes for health.

Excessive reactive oxygen species (ROS) can damage proteins, lipids, and DNA, which result in cell damage and death. The outcomes can be acute, as seen in stroke, or more chronic as observed in age-related diseases such as Parkinson’s disease. Here we investigate the antioxidant ability of a novel synthetic flavonoid, Proxison (7-decyl-3-hydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone), using a range ofin vitroandin vivoapproaches. We show that, while it has radical scavenging ability on par with other flavonoids in a cell-free system, Proxison is orders of magnitude more potent than natural flavonoids at protecting neural cells against oxidative stress and is capable of rescuing damaged cells. The unique combination of a lipophilic hydrocarbon tail with a modified polyphenolic head group promotes efficient cellular uptake and mitochondrial localisation of Proxison. Importantly,in vivoadministration of Proxison demonstrated effective and well tolerated neuroprotection against oxidative stress in a zebrafish model of dopaminergic neuronal loss.

Flavonoids are a large group of ubiquitous polyphenolic secondary metabolites in plants with a wide range of properties, including a widely reported anti-cancer effect. The present review focuses on the different known mechanisms partaking in said anti-tumour effects, with particular emphasis on breast cancer. Their structure and reactivity allows flavonoids to work as antioxidant agents and phyto-oestrogens, modulating oestrogen signalling and metabolism to induce an overall anti-proliferative response. Other effects include the ability of flavonoids to modulate the CYP1 (cytochrome P450 1) and ABC (ATP-binding cassette) protein families, involved in carcinogenesis and drug delivery respectively. They can also induce apoptosis and cell cycle arrest and regulate other signalling pathways involved in the development and progression of cancer. In conclusion, there is accumulating evidence on the versatility of flavonoids and the numerous activities contributing to their anti-tumour effect. The complex, yet effective, mechanism of action of flavonoids, together with their interesting pharmacological properties, is the basis for their potential application in breast and other cancers. This rationale has led to the current interest in the application of flavonoids, including clinical trials currently underway and the development of novel flavonoids with improved properties, which hold great promise for tackling breast cancer.

Excessive reactive oxygen species (ROS) can damage proteins, lipids, and DNA, which result in cell damage and death. The outcomes can be acute, as seen in stroke, or more chronic as observed in age-related diseases such as Parkinson’s disease. Here we investigate the antioxidant ability of a novel synthetic flavonoid, Proxison (7-decyl-3-hydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone), using a range of in vitro and in vivo approaches. We show that, while it has radical scavenging ability on par with other flavonoids in a cell-free system, Proxison is orders of magnitude more potent than natural flavonoids at protecting neural cells against oxidative stress and is capable of rescuing damaged cells. The unique combination of a lipophilic hydrocarbon tail with a modified polyphenolic head group promotes efficient cellular uptake and moderate mitochondrial enrichment of Proxison. Importantly, in vivo administration of Proxison demonstrated effective and well tolerated neuroprotection against cell loss in a zebrafish model of dopaminergic neurodegeneration.

We thank SULSA (Scottish Universities Life Science Alliance) for supporting this project through a SULSA BioSkape Industry PhD Studentship and Antoxis Limited for providing additional funding. We also thank the 7th Framework Programme of the European Union (METOXIA project; HEALTH-F2-2009-222741) for support. Background: The natural polyphenol myricetin induces cell cycle arrest and apoptosis in preclinical cancer models. We hypothesised that myricetin-derived flavonoids with enhanced redox properties, improved cell uptake and mitochondrial targeting might have increased potential as antitumour agents. Methods: We studied the effect of a second-generation flavonoid analogue, Oncamex, in a panel of 7 breast cancer cell lines, applying western blotting, gene expression analysis, fluorescence microscopy, and immunohistochemistry to xenograft tissue to investigate its mechanism of action. Results: Proliferation assays showed that Oncamex: treatment for 8 h reduced cell viability and induced cytotoxicity and apoptosis, concomitant with increased caspase activation. Microarray analysis showed that Oncamex was associated with changes in expression of genes controlling cell cycle and apoptosis . Fluorescence microscopy showed the compound’s mitochondrial targeting and ROS-modulating properties, inducing superoxide production at concentrations associated with anti-proliferative effects. A preliminary in vivo study in mice implanted with the MDA-MB-231 breast cancer xenograft showed that Oncamex inhibited tumour growth, reduced tissue viability and Ki-67 proliferation, with no overall systemic toxicity. Conclusion: Oncamex is a novel flavonoid capable of specific mitochondrial delivery and redox modulation. It has shown antitumour activity in preclinical models of breast cancer, supporting the potential of this prototypic candidate for its continued development as an anticancer agent. Publisher PDF

Electron spin resonance (ESR) spectroscopy was applied to humic acids in solid and solution states obtained from a wide range of soil types. The relationships between the relative free-radical concentration and the degree of humification expressed as A 600 / C and log( A 400 / A 600 ) and the aromatic C concentration estimated by cross-polarization magic-angle spinning 13 C nuclear magnetic resonance spectroscopy were analyzed. The hyperfine structure observed in solution ESR spectra was also compared among the humic acids. The free-radical concentration varied up to 147-fold among 42 humic acids, with a sample from a buried Umbric Andosol (Ando soil) having the greatest concentration and that from an upland field soil (Dystric Cambisol) the least. The linewidth of the radical signals ranged between 0.32 and 0.55 mT and showed an inverse correlation with the free-radical concentration, suggesting a decrease in the heterogeneity of free-radicals with their increase. The free-radical concentration was correlated ( P A 600 / C and the aromatic C concentration, whereas it varied in inverse proportion to the log( A 400 / A 600 ) value. Regression analysis suggested that the electron-conjugated systems are more developed in the humic acids with greater aromatic C concentration, which help stabilize free-radicals. Hyperfine splitting observed in the ESR spectra of a range of Ando soil humic acids dissolved in 0.1 M KOH was similar, while the peak strength decreased with increasing degree of humification. Although continuous application of manure to upland field soils scarcely affected the free-radical concentration in their humic acids, their hyperfine structure was weakened. The solution ESR spectra of the green fraction, which contains a chromophore related to 4,9-dihydroxyperylene-3,10-quinone (DHPQ) structure, was composed of basic features with an additional hyperfine structure superimposed on them. Since the basic features were consistent with the hyperfine structure of humic acids from Ando soils and the H layer of a Podzol which do not contain the green fraction, it was considered that the additional hyperfine structure is associated with DHPQ and thus the influence of DHPQ on the ESR spectra of humic acids is minor.

Anthocyanin-rich beverages have shown beneficial effects on coronary heart disease in epidemiological and intervention studies. In the present study, we investigated the effect of black currant anthocyanins on atherosclerosis. Watanabe Heritable Hyperlipidemic rabbits (n = 61) were fed either a purified anthocyanin fraction from black currants, a black currant juice, probucol or control diet for 16 weeks. Purified anthocyanins significantly increased plasma cholesterol and low-density lipoprotein (LDL) cholesterol. Intake of black currant juice had no effect on total plasma cholesterol, but lowered very-low-density lipoprotein (VLDL) cholesterol significantly. There were no significant effects of either purified anthocyanins or black currant juice on aortic cholesterol or development of atherosclerosis after 16 weeks. Probucol had no effect on plasma cholesterol but significantly lowered VLDL-cholesterol and decreased aortic cholesterol accumulation. The erythrocyte antioxidant enzyme glutathione peroxidase was significantly increased by purified anthocyanins and superoxide dismutase was increased by both anthocyanin-containing treatments. Other markers of plasma antioxidant capacity, antioxidant enzymes, protein and lipid oxidation were not affected by any of the anthocyanin treatments. Adverse effects of purified anthocyanins were observed on plasma- and LDL-cholesterol. These effects were not observed with black currant juice, suggesting that black currants may contain components reducing the adverse effects of anthocyanins.

The flavonol myricetin, reacts with oxygen-centred galvinoxyl radicals 28 times faster than d-α-tocopherol (vitamin E), the main lipid-soluble antioxidant in biological membranes. Moreover, each myricetin molecule reduces twice as many such radicals as vitamin E. However, myricetin fails to protect vitamin E-deficient microsomes from lipid peroxidation as assessed by the formation of thiobarbituric acid reactive substances (TBARS). Novel and potentially therapeutic antioxidants have been prepared that combine the radical-scavenging ability of a myricetin-like head group with a lipophilic chain similar to that of vitamin E. C 6 –C 12 alkyl chains are attached to the A-ring of either a 3,3 ′ ,4 ′ ,5 ′ -tetrahydroxyflavone or a 3,2 ′ ,4 ′ ,5 ′ -tetrahydroxyflavone head group to give lipophilic flavonoids ( C log P =4 to 10) that markedly inhibit iron-ADP catalysed oxidation of microsomal preparations. Orientation of the head group as well as total lipophilicity are important determinants of antioxidant efficacy. MM2 models indicate that our best straight chain 7-alkylflavonoids embed to the same depth in the membrane as vitamin E. The flavonoid head groups are prepared by aldol condensation followed by Algar–Flynn–Oyamada (AFO) oxidation or by Baker–Venkataraman rearrangement. The alkyl tails are introduced by Suzuki or Negishi palladium-catalysed cross-coupling or by cross-metathesis catalysed by first generation Grubbs catalyst, which tolerate phenolic hydroxyl and ketone groups.

When TiO2 powder was irradiated with a laser light (>0.8 MW peak pulse power (PPP) at 355 nm) a visible change in its colour from white to dark blue was observed. The initial rate of change of the total colour difference was related to the laser light intensity and the longer the irradiation time the more substantial the colour change. The result of X-ray diffraction (XRD) studies showed that the crystal structure of the TiO2 developed a more rutile form after laser exposure. ESR studies indicated that the colour change was associated with the generation of Ti(III) species in the photocatalyst. Electron microscopic studies showed that more spherical shaped particles of TiO2 were observed after laser treatment although the average particle size remained largely unchanged. No significant changes in the band gap or the surface area of the laser modified TiO2 were observed. The laser modified photocatalyst showed no enhancement in activity for the destruction of methylene blue, rhodamine B and stearic acids, indicating that the rutile/anatase ratio is unimportant in the destruction of the test pollutants used in this work, via TiO2 photocatalysis.

Antioxidant activities of plant extracts from spices, coffee, tea, grape skin, and tomato peel slurry were evaluated using a number of analytical methods including the quantification of principal compounds. Similar rankings in the activities of these extracts were obtained by evaluating their efficiencies as scavengers of stable free radicals: Fremy's salt, galvinoxyl or α,α-diphenyl-β-picrylhydrazyl (DPPH). Similar results were obtained with the lipid oxidation assays based on thermal acceleration (formation of conjugated dienes in methyl linoleate at 40 °C or the Rancimat test at 100 °C with lard). Rankings of the extract activity obtained by scavenging of hydroxyl radicals generated in the Fenton reaction were similar to those obtained by an oxygen consumption assay with linoleic acid as substrate and metmyoglobin as catalyst. However, the results of the latter two assays differed from those of the other assays. In the overall ranking, coffee and rosemary extracts were amongst the most potent extracts whereas the tomato peel slurry showed no activity.

The health benefit of fruit juices have been ascribed, in part, to phenolic antioxidants. The antioxidant potential of a range of fruit juices was assessed by measurement of their ability to reduce a synthetic free radical, potassium nitrosodisulphonate, and also by their ability to reduce Fe(III). Vitamin C was found to account for 65–100% of the antioxidant potential of beverages derived from citrus fruit but less than 5% of apple and pineapple juice. The contribution of carotenoids to antioxidant potential was negligible. Although phenolics appear to be major contributors to the antioxidant potential of the non-citrus juices, their identity and bio-availability requires further investigation.

Health benefits of red wine have been ascribed in part to the antioxidant properties of quercetin and other flavonols. Red wines, however, contain many other polyphenolic compounds with antioxidant potential. The present study has assessed the antioxidant capacity of seven red wines using electron spin resonance spectrometry to measure their ability to donate hydrogen atoms to a stable Fremy's radical. Antioxidant capacity of the wines was strongly related to the total phenol content (r = 0.872, P

In order to determine the dominant properties, which control the interactions of monomeric hydrosoluble phenols with Cu2+ in soil solutions, four different techniques were used. CuSO4 was added to ortho-dihydroxy-phenol (ODP) solutions under controlled temperature, pH, and ionic strength conditions and Cu2+-ODP interactions were studied by alkaline potentiometric titrations, electron spin resonance and UV absorption. Molecular dynamic simulations were also performed for conformational studies. From the titration curves, the shifts in isotropic A and g values, the evolution of the UV absorption band at 400 nm and the calculation of the potential energy of the different complexes, a three-step interaction mechanism was proposed. In the process an inner-sphere complex was formed, Cu(II) is being reduced and an oxydative polymerisation chain reaction initiated. The driving force of the reaction was the energetic stability obtained upon interaction of Cu2+ with the substrate. The ideal conformation was reached at the catechol site and not at carboxylic or hydroxy–methoxy sites. The pKa values of the phenols had little influence on the interaction process but the type and number of interacting sites on the benzene ring influenced the proceeding of progress of the reaction. Increasing the pH of the solution increased the amount of complexed species, but also the number of ligands around the Cu2+ion.

Objective: To assess whether consumption of 100 ml of whisky or red wine by healthy male subjects increased plasma total phenol content and antioxidant capacity. Design: A Latin square arrangement to eliminate ordering effects whereby, after an overnight fast, nine volunteers consumed 100 ml of red wine, malt whisky or unmatured ‘new make’ spirit. Each volunteer participated on three occasions one week apart, consuming one of the beverages each time. Blood samples were obtained from the anticubital vein at intervals up to 4h after consumption of the beverages when a urine sample was also obtained. Results: Within 30 min of consumption of the wine and whisky, there was a similar and significant increase in plasma total phenol content and antioxidant capacity as determined by the ferric reducing capacity of plasma (FRAP). No changes were observed following consumption of ‘new make’ spirit. Conclusions: Consumption of phenolic-containing alcoholic beverages transiently raises total phenol concentration and enhances the antioxidant capacity of plasma. This is compatible with suggestions that moderate alcohol usage and increased antioxidant intake decrease the risk of coronary heart disease. Sponsorship: Funded by the Scottish Office Agriculture, Environment and Fisheries Department (SOAEFD), the ERASMUS exchange program, the EC-Fair program and the Scotch Whisky Research Institute.

Electron spin resonance methodology has been used to assess the hydrogen-donating (antioxidant) ability of catechins and extracts of green and black tea to Fremy's radical and galvinoxyl radical in aqueous and organic solutions, respectively. Catechin flavanols accounted for 77-82% of the total antioxidant activity of green tea but only 47-58% of that of black tea. The radical quenching ability of the green tea was 21-24% more effective than that of the black tea in both aqueous and lipophilic systems. Epigallocatechin gallate was the most effective at reducing the two radical species, whereas epigallocatechin was least effective in the galvinoxyl/ethanol system, and catechin least effective in the Fremy's radical/water system. In a model system comprising a mixture of the flavanols in ethanolic or aqueous solution, in the same concentrations as they were present in the tea extracts, the antioxidant potential was a simple summation of the activity of the individual components indicating no synergistic or antagonistic side reactions. Results demonstrate that flavanol components of teas exhibit potent antioxidant activity in both aqueous and organic media. This will be of physiological relevance if such compounds partition into both the aqueous and lipid compartment of cells.

e14071Background: Clinical trials now underway will assess the use of flavonoids (natural polyphenols) as anticancer agents. Previous work has shown the promising antitumor activity of Oncamex (a n...

The ligands complexing copper in various organic matter fractions isolated from a soil treated with copper-contaminated sewage sludge have been characterized using electron paramagnetic resonance (EPR) spectroscopy. A soil of the Countesswells series was treated with sewage sludge at the rate of 100 t dry matter/ha. Copper in the form of copper carbonate was then applied to give copper loadings of 140 mg/kg and 280 mg/kg in the top 25 cm of the profile, values at the EEC directive limiting values and at twice these values, respectively. After 3 years, the soil was sampled to a depth of 10 cm for analysis. Soil solution obtained by centrifugation of fresh soil contained organically-complexed copper. The EPR spectroscopic parameters (A 11 and g 11 ) for Cu have values consistent with the presence of oxygen coordination at the equatorial position. Organic matter was isolated from soil by successive extractions with 0.1 M sodium pyrophosphate (pH 7) and 0.1 M sodium hydroxide and fractionated by selective adsorption and precipitation procedures. Copper was present in several different complexes. Hyperfine structure from 14 N was observed in pyrophosphate extract unprecipitated by 80% ethanol, the fulvic acid and humic acid fractions, indicating that copper was in coordination with at least two nitrogen atoms in these fractions. The precipitable pyrophosphate-extracted substances only contained oxygen ligands. Soil solutions from three other soil series from sites which had received contaminated sludge treatments 20 years previously were also examined. Their spectra at 100 K were dominated by manganese(II) signals but a copper signal could be resolved which was indistinguishable from that from the Countesswells series soil.

Finnish Landrace sheep with a genetic lesion which results in restricted cysteine transport across the red cell membrane have total glutathione concentrations in their red blood cells that are approximately 40% of those in normal sheep of the same breed. However, dimethyldisulphide-challenged red blood cells from both phenotypes produce an ESR-spin adduct at similar rates. The resistance of the low glutathione phenotype red cells to oxidant challenge is reflected by increases in the activities of antioxidant enzymes. Sheep with a genotypic disorder in cysteine transport may be a suitable model for studying the genetic expression of antioxidant enzymes in response to oxidant loads.

New drugs are required to treat both endocrine resistant and triple negative breast cancers (TNBC). Among new structures being investigated are flavonoids, which are natural polyphenolic compounds with antitumor properties. Clinical trials are underway to assess their application as chemopreventatives and as sensitisers to chemotherapy. Among flavonoids, myricetin has shown particular promise, inducing cell cycle arrest and mitochondrial-dependent apoptosis in cancer cell line models. In this study we evaluated a novel series of myricetin-derived flavonoids with improved antioxidant and mitochondrial targeting properties. We hypothesised that these compounds might have potential in breast cancer management and in particular to treat estrogen receptor positive (ER+) tumors, in which estrogen controls tumor growth at least partially through oxidative stress-related mitochondrial signalling. We first assessed the effect of 8 flavonoids on 4 breast cancer cell lines (MDA-MB-231, BT-549, MCF-7 and HBL-100) and 3 MCF-7-derived cell lines with reduced sensitivity to endocrine therapy (LCC-1/LCC-2/LCC-9). The novel flavonoids were designed to assess the involvement of redox potential and mitochondrial targeting through selective structure-activity changes. Anto-028 was the most potent compound identified with 4 to 140-fold lower IC50 values than myricetin, as shown by sulforhodamine B (SRB) assays applied to assess antiproliferative effects. Although endocrine-resistant cell lines were less sensitive, Anto-028 exerted a strong, ER-independent antiproliferative effect on both ER+ and TNBC cell lines, with IC50 values in the low micromolar range. Treatment for 8 hours exerted dose-dependent reduction in cell viability and induction of cytotoxicity and apoptosis, with a 2 to 5-fold increase in caspase activation, as detected by luminescence-based plate assays. The involvement of reactive oxygen species (ROS) regulation in these effects was demonstrated by plate-based assays and microscopic detection of fluorescent probes for mitochondrial hydrogen peroxide and superoxide. Results indicated that different species of ROS are sequentially generated by treatment with a range of concentrations of Anto-028, suggesting a possible biphasic effect of the drug at different concentrations. Experiments in mice implanted with subcutaneous MDA-MB-231 xenografts showed that Anto-028 can significantly inhibit tumour growth in vivo with associated changes in percentage of viable areas within the xenografts (from 56% in controls to 37% in mice treated with 25mg/kg/day of Anto-028) and reduced Ki67 proliferation index (from 60% in controls to 36% in treated xenografts). Current studies are assessing combinatorial effects with TRAIL and chemotherapy which have produced synergistic effects in ovarian cancer models, with nanomolar concentrations of Anto-028 sensitising resistant cells to induce strong, significant antiproliferative effects. In conclusion, Anto-028 is a novel flavonoid with promising antitumor activity in preclinical models of breast cancer. Citation Format: Carlos Martinez-Perez, Carol Ward, Peter Mullen, Graeme Cook, Donald McPhail, Arran K Turnbull, David J Harrison, Simon P Langdon. Novel flavonoid Anto-028 shows promising antitumor activity in preclinical models of breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-02-08.

To characterize parameters influencing the antioxidant activity at interfaces a novel ESR approach was developed, which facilitates the investigation of the reaction stoichiometry of antioxidants towards stable radicals. To relate the activity of antioxidants towards the location of radicals at interfaces NMR experiments were conducted. Micellar solutions of SDS, Brij and CTAB were used to model interfaces of different chemical nature. The hydrophilic Fremy's radical was found to be solubilized exclusively in the aqueous phase of SDS micellar solution but partitioned partly into the hydrophilic headgroup area of Brij micelles. In contrast the hydrophobic galvinoxyl was exclusively located in the micellar phase with the increasing depth of intercalation in the order SDS < Brij < CTAB. Gallates revealed a higher stoichiometric factor towards galvinoxyl in CTAB systems, which is accounted to a concentration effect of antioxidant and radical being both solubilized in the palisade layer. In contrast, in SDS solutions hardly any reaction between galvinoxyl and gallates was found. SDS acted as a physical barrier between radical (palisade layer) and antioxidant (stern layer). The influence of the hydrophobic properties of the antioxidant was clearly seen in Brij micelles. Elongation of the alkyl chain in gallate molecule resulted in increasing stoichiometric factors in the presence of galvinoxyl being located in the deeper region of the bulky headgroup area. The reverse trend was found in the presence of Fremy's radical being located in the hydrated area of the micelles.

There is current interest in the use of naturally occurring flavonoids as antioxidants for the preservation of foods and the prevention of diseases such as atherosclerosis and cancers. To establish the molecular characteristics required for maximum antioxidant activity, electron spin resonance (ESR) spectroscopy has been used to determine the stoichiometry and kinetics of the hydrogen-donating ability of 15 flavonoids and d-alpha-tocopherol to galvinoxyl, a resonance-stabilized, sterically protected aryloxyl radical. The second-order reaction rates, which will be governed by O-H bond dissociation energies, were myricetin > morin > quercetin > fisetin approximately catechin > kaempferol approximately luteolin > rutin > d-alpha-tocopherol > taxifolin > tamarixetin > myricetin 3',4',5'-trimethyl ether > datiscetin > galangin > hesperitin approximately apigenin. Reactivity is highly dependant on the configuration of OH groups on the flavonoid B and C rings, there being little contribution from the A ring to antioxidant effectiveness. Highest reaction rates and stoichiometries were observed with flavonols capable of being oxidized to orthoquinones or extended paraquinones. However, rates and stoichiometries did not always correlate and the data suggest that kinetic factors may be of greater importance within a biological context.

Objective: To assess whether consumption of 500 ml of blueberry juice or cranberry juice by healthy female subjects increased plasma phenolic content and antioxidant capacity. Design: Latin square arrangement to eliminate ordering effects. After an overnight fast, nine volunteers consumed 500 ml of blueberry juice, cranberry juice or a sucrose solution (control); each volunteer participated on three occasions one week apart, consuming one of the beverages each time. Blood samples were obtained by venipuncture at intervals up to four hours after consumption of the juices. Urine samples were also obtained four hours after consuming the juice. Results: Consumption of cranberry juice resulted in a significant increase in the ability of plasma to reduce potassium nitrosodisulphonate and Fe(III)-2, 4, 6-Tri(2-pyridyl)-s-triazine, these measures of antioxidant capacity attaining a maximum after 60–120 min. This corresponded to a 30% increase in vitamin C and a small but significant increase in total phenols in plasma. Consumption of blueberry juice had no such effects. Conclusion: The increase in plasma antioxidant capacity following consumption of cranberry juice could mainly be accounted for by an increase in vitamin C rather than phenolics. This also accounted for the lack of an effect of the phenolic-rich but vitamin C-low blueberry juice. Sponsorship: Funded by the Scottish Executive Rural Affairs Department and the Danish Government. European Journal of Clinical Nutrition (2000) 54, 405–408

Although many studies have been carried out on copper interactions with humic and fulvic acids, little is known about its interactions with phenolic compounds. In Alpine podzolic systems considerable amounts of phenols are released from the acidophilic vegetation cover (Rh. ferrugineum, Vacc. myrtillus, Calluna vulgaris), together with other primary and secondary metabolites present in leaves (Vaughan and Malcolm 1985). All year-round, and in particular during the period of senescence, these phenols are leached to the soil where they can participate in the humification processes. This study shows that under appropriate pH and redox conditions phenols interact with metallic cations and, therefore, may play a role in their intrapedic transfer. Moreover, the change in redox state as a result of Cu-phenolic interactions may modify the phenol stability itself and provoke the polymerisation of phenolic monomers.

Phytoestrogens (PEs) are diphenolic compounds from plants which can bind to estrogen receptors and have estrogen and antiestrogen effects in man and animals. Like other plant phenolics, PEs may have antioxidant properties through hydrogen/electron donation via hydroxyl groups. They might therefore act as free radical scavengers and inhibit development of coronary heart disease and cancers. The hydrogen-donating ability of a range of phytoestrogens was assessed using electron spin resonance spectroscopy, the ferric-reducing ability of plasma assay, and the Trolox equivalent antioxidant capacity. In addition, the ability of compounds to inhibit lipid peroxidation was examined in vitamin E-deficient liver microsomes. Genistein had the highest activity of the isoflavones; however, the isoflavones were relatively poor hydrogen donors compared with the other estrogenic compounds examined. Coumestrol and equol were more effective antioxidants than genistein but had relatively limited activity in comparison with Trolox. The only estrogenic compound with significant antioxidant activity was kaempferol which is better known as a dietary antioxidant than a phytoestrogen. As the concentrations of PEs used in this study exceed the estimated serum concentrations, their relatively poor antioxidant ability in vitro may indicate little significance as antioxidants in vivo.

Ex vivo studies have been carried out on roe and red deer erythrocytes using electron spin resonance spectroscopy. Free radical formation in cells challenged with the brassica-derived haemolysin, dimethyldisulphide, was measured using spin trapping techniques. Significantly greater amounts of radical were trapped in the roe deer cells which may relate to differences in the antioxidant profile of the two genotypes. Results suggest that roe deer have a greater risk of developing oilseed rape poisoning than red deer.

The antioxidant properties of several tocopherol isomers and of “Palm Vitee” were assessed by measurement of their ability to inhibit NADPH-induced peroxidation of hepatic microsomes prepared from vitamin E-deficient rats. The microsomal concentrations of d γ-, d α- and dl α-tocopherol required to inhibit peroxidation by 50% were 160, 750 and 1850 μg/mg protein respectively; d γ-tocopherol was also the most effective in prolonging the lag phase prior to the initiation of peroxidation. “Palm Vitee” was 3 times more potent than d γ-tocopherol in preventing TBARS formation; this may be due to the tocotrienol components of the preparation. Electron spin resonance (ESR) spectroscopy and spin trapping suggested that the tocopherols were preventing the formation of a carbon-centered radical adduct derived from linoleic acid. Signal height correlated with TBARS formation suggesting that the TBA assay is an accurate measure of lipid peroxidation in this study.

On incubation with the spin trap alpha-(4-pyridyl-l-oxide)-N-tert-butylnitrone (4-POBN), a characteristic electron spin resonance (ESR) signal was produced at a greater rate in hepatic microsomal fractions from malignant hyprthermia susceptible (MHS) pigs compared with resistant (MHR) pigs. This was accompanied by increased formation of thiobarbituric acid reactive substances (TBARS). Supplementation of diets for six weeks with 235 mg alpha-tocopherol acetate/kg significantly increased microsomal vitamin E content of both pigs types. Moreover, the rate of formation of TBARS and ESR signal height of incubated microsomes from supplemented MHS pigs was decreased to that of MHR pigs. Elevated pyruvate kinase activities and TBARS concentrations in plasma of MHS pigs were also moderated by dietary vitamin E. Vitamin E supplementation may decrease the peroxidative events associated with MH.

Microsomes were prepared from livers of malignant hyperthermia susceptible (MHS) or resistant (MHR) pigs. On incubation with the spin trap alpha-(4-pyridyl-l-oxide)-N-tert-butylnitrone (4-POBN), the microsomes from MHS pigs produced a characteristic electron spin resonance (ESR) signal at a greater rate than those from MHR pigs. Increased formation in the incubations of thiobarbituric acid reactive substances (TBARS) by the microsomes of the MHS pigs indicated an enhanced susceptibility to free radical-mediated lipid peroxidation. These results provide further evidence that MHS pigs have an antioxidant abnormality which may contribute to the fatal MH response. However the nature of the abnormality is unclear. The enhanced formation of unstable free radicals and indices of lipid peroxidation was not due to decreased vitamin E concentration or glutathione peroxidase activity in the microsomes. Furthermore, fatty acid profiles were similar in microsomes from MHS and MHR pigs indicating similar amounts of potential substrate for TBARS formation.

Free radicals have been trapped in microsomal fractions from rat livers by the use of alpha-(4-pyridyl-l-oxide)-N-tert-butylnitrone (4-POBN) and detected by ESR spectroscopy. Using this technique we have investigated the effects of low dietary concentrations of vitamin E and selenium (Se) on free radical production. When the microsomal fractions were incubated with Fe, ADP and NADPH significantly greater amounts of radicals were initially trapped when rats were deficient in vitamin E (whether combined with Se deficiency or not) than from Se-deficient animals or Se- and vitamin E-supplemented controls. After prolonged incubation (ca. 30-45 mins) the free radicals trapped from the microsomes from the Se-deficient animals rose to amounts comparable to those seen in the vitamin E-deficient microsomes. Over the same period of incubation approximately half this quantity of radicals were trapped in liver microsomes from Se- and vitamin E-supplemented rats. The effects of 4-POBN on the levels of peroxidation in the various microsomal fractions were assessed by measuring the thiobarbituric acid reactive substances (TBARS). The presence of 4-POBN significantly reduced the amounts of TBARS formed on incubation and there was a clear distinction between the groups on the basis of the vitamin E status of their diets.

Municipal refuse was allowed to decompose in a simulated landfill for 20 months. Three different models were studied in which the refuse, in 40 m 3 lots, was either compacted or mixed 2:1 with sewage sludge, the latter being studied in both uncompacted and compacted states. At 2, 6, 12 and 20 months, humic substances were extracted from samples with 0.1 M Na 2 P 4 O 7 and 0.1 M NaOH, and humic acids isolated by precipitation after acidification with HCl. The humic substances were examined by electron paramagnetic resonance (EPR) to determine the free radical content and the nature of some of the metal complexes present. The principal form of copper was present in either a square planar or a tetragonally-distorted octahedral environment, probably coordinated to two nitrogen and two oxygen atoms. Fe 3+ was present in at least three different environments. In one, it was probably in a complex with rhombic symmetry; another showed Fe 3+ in an axially-symmetric environment, most likely as a ferric porphyrin. No distinctions could be made between the concentrations or forms of metals present in the refuse humic substances as a result of adding sewage sludge, but the additions increased the yield of humic substances, particularly in the uncompacted landfill. Humic substances in the refuse retain metal elements in complexed forms which will restrict their release from the landfill.

The formation of unstable free radical species in homogenates of heart from selenium and/or vitamin E deficient rats has been investigated by electron spin resonance spectroscopy using the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone. There was only a small amount of free radical formation by heart from control or selenium deficient animals. However, the amounts and rates of formation of free radicals were significantly higher in animals with vitamin E deficiency and higher still in those with combined selenium and vitamin E deficiency.

Electron spin resonance spectra are reported for copper(II)–amino-acid complexes [CuL]n+ and [CuL2]m+(for 12 α-amino-acids) and [Cu(HL)L]+(HL = L-histidine). Buffer solutions of aqueous copper(II) and ligand were prepared to give optimum concentrations of these complexes. Isotropic g and A values are in the ranges 2.144–2.157 and 5.5–6.5 mT respectively for [CuL]n+ and 2.118–2.127 and 6.4–7.1 mT respectively for [CuL2]m+. The high-field absorption for [CuL2]m+ complexes showed nitrogen hyperfine structure, which for 63Cu and solvent D2O could be resolved in the second derivative into two overlapping quintet components characteristic of cis(N, N, O–, O–) and trans(N, O–, N, O–) isomers respectively. Spectra have been fitted to this model by computer simulation. From observed nitrogen hyperfine structure, and by comparison with spectra of other amino-acid complexes and that of bis(histamine)copper(II), it is deduced that the histidinate complex [Cu(HL)L]+ has three nitrogen atoms co-ordinated to copper in the planar co-ordination sites, whereas [CuL2] appears to consist of a mixture of structures having three and four nitrogen atoms bound to the copper.

A study has been made of solid and solution electron paramagnetic resonance (EPR) spectra of humic acids from different horizons in a podzolic soil. Hyperfine splitting was observed in the solution spectra of humic acids from all horizons and depended on the strength of alkali and the period of dissolution. The upper organic horizons L, F and O1 contained humic acids with some spectral characteristics in common with lignin. Humic acid from the lower horizons showed different spectra. At least 5 different radical signals were present.

Tissue damage mediated by the formation of chemically reactive free radicals, often derived from oxygen, has been implicated as the cause of many diseases in man and animals. However, in many instances the evidence for the involvement of free radicals in disease processes is indirect. This evidence includes formation of breakdown products from free radical mediated destruction of essential cell constituents and the protective effect of free radical scavengers and antioxidant enzymes. Electron spin resonance (ESR) spectroscopy has been used for the characterisation of free radicals in both chemical and biological systems, either directly in the case of stable radicals or after reaction of unstable radicals with a “spin trap” to form a more long lived radical which can be detected. In this report we describe the application of both direct ESR spectroscopy and ESR spectroscopy after spin trapping to investigate free radicals in heart from Se- and vitamin E-deficient rats.

The relationship among antioxidant activity, based on the electron-spin resonance determination of the reduction of Fremy's radical, vasodilation activity, and phenolic content was investigated in 16 red wines. The wines were selected to provide a range of origins, grape varieties, and vinification methods. Sensitive and selective HPLC methods were used for the analysis of the major phenolics in red wine: free and conjugated myricetin, quercetin, kaempferol, and isorhamnetin; (+)-catechin, (-)-epicatechin, gallic acid, p-coumaric acid, caffeic acid, caftaric acid, trans-resveratrol, cis-resveratrol, and trans-resveratrol glucoside. Total anthocyanins were measured using a colorimetric assay. The total phenolic content of the wines was determined according to the Folin-Ciocalteu colorimetric assay and also by the cumulative measurements obtained by HPLC. The 16 wines exhibited a wide range in the values of all parameters investigated. However, the total phenol contents, measured both by HPLC and colorimetrically, correlated very strongly with the antioxidant activity and vasodilation activity. In addition, the antioxidant activity was associated with gallic acid, total resveratrol, and total catechin. In contrast, only the total anthocyanins were correlated with vasodilation activity. The results demonstrate that the different phenolic profiles of wines can produce varying antioxidant and vasodilatant activities, which opens up the possibility that some red wines may provide enhanced health benefits for the consumer.

The presence of two components, presumably cis and trans isomers, was previously indicated by the 14N superhyperfine structure pattern in the e.s.r. spectra of a number of copper(II)–bis(amino-acid) complexes in aqueous solution. In the present work the g and hyperfine values of the individual species have been obtained from spectra of the 63Cu and 65Cu isotopes. Significantly different values have been observed for the two components and these differences are discussed in terms of possible deviations from the ideal square-planar structure in the cis and trans isomers.

Aspergillus niger, Rhizoctonia solani and Marasmius androsaceous extracts have been studied by e.s.r. spectroscopy as solids and in alkaline solutions. Humic acids (HA) were isolated by extracting the spores of Aspergillus and sclerotia of Rhizoctonia with 0.5 M-alkali and Marasmius stipes with I M-alkali, precipitating at pH 1.0 (HCI) and centrifuging at 2OOOg for 15 min. The sodium humate from Aspergillus was fractionated on Sephadex G-75 at pH 6. Elution with 1% (w/v) NaCl gave three fractions of > 50 000. 2000-5000 and < 500 daltons molecular mass. the latter being similar t o that obtained by cold H 2 0 extraction of the spores. E.s.r. spectra of the solid HAS showed single-peak. free-radical signals and other components from paramagnetic metal ions. The Rhizoctonia HA was dominated by Cu (11) with extensive 14N hfs in t h e g l region. probably arising froin a copper porphyrin. HA from Aspergillus also contained a Cu (11) species but without ligand hfs. Marasrnius produced a complex pattern of peaks which could not be interpreted readily. In solution, all materials showed complex behaviour with the radicals varying with both time and alkali concen

The pH dependence of the formation of complexes between copper(II) and the peptide glycylhistidine has been investigated for metal : ligand ratios of 1 : 1 and 1 : 2 by e.s.r. spectroscopy. Five complex species are seen with the 1 : 2 ratio solutions, 4 of which are also observed with the 1 : 1 ratio solutions. In each case 14N superhyperfine structure is resolved in fluid solution by working with the 63Cu isotope and recording second-derivative spectra, thus producing a direct measurement of the number of nitrogen atoms bound to copper. Superhyperfine structure was also resolved in the frozen-solution spectra of several of the complexes, although this was not always consistent with the conclusions drawn from the fluid-solution spectra. The likely composition and coordination of the various species are discussed with reference to published results from electrochemical and u.v.-visible spectroscopic methods.

E.s.r. spectroscopy has been used to investigate the formation of complexes between oxovanadium(IV) and cysteine (2-amino-3-mercaptopropanoic acid) over a range of pH values. At least six complex species are observed with distinctly different spectral parameters and, although no ligand superhyperfine splitting is resolved, it is possible to produce tentative coordination structures for the various species by reference to previously published correlations for model oxovanadium(IV) complexes.

The pH-dependent formation and stability of a range of oxovanadium(IV) dicarboxylic acid complexes have been studied by e.s.r. spectroscopy. The results from oxalic acid [(CO2H)2] and malonic acid [CH2(CO2H)2] complexes show that there is significant stabilization with respect to oxovanadium(IV) oxidation/hydrolysis, in agreement with published work using electrochemical techniques. This behaviour can be explained by chelate formation, the five-membered chelate ring with oxalate being a slightly more favoured arrangement than the six-membered ring with malonate, where evidence of distortion is seen in the spectral parameters. Addition of further CH2 groups in succinic acid [(CH2)2(CO2H)2] and glutaric acid [(CH2)3(CO2H)2] results in corresponding decreases in complex stability, with the latter giving results similar to those obtained with fumaric acid [trans-(CH)2(CO2H)2], where no chelation is possible. With maleic acid [cis-(CH)2(CO2H)2] a range of complexes is seen, and these have a significantly greater stability range than was observed with succinic acid.