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 < 0.005) positively with the A(600)/C and the aromatic C concentration, whereas it varied in inverse proportion to the log(A400/A600) 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. (c) 2005 Elsevier Ltd. All rights reserved.

The flavonol myricetin, reacts with oxygen-centred galvinoxyl radicals 28 times faster than d-alpha-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.

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.

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.

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

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.

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 < 0.01) but less so to total flavonol content (r = 0.651, P < 0.10). The only flavonols detected in the wines by HPLC with post-column derivatisation were the aglycones and conjugated forms of quercetin and myricetin. These accounted for less than 2% of total phenolic content when expressed as gallic acid equivalents. Although these flavonols showed marked ability to quench Fremy's radical, they contributed less than 1.5% to the total antioxidant capacity of the wines. Consequently, quercetin is not a major antioxidant in red wine. The antioxidant activity of other polyphenols may be of greater importance in contributing to the reputed health benefits of moderate wine consumption. (C) 1999 Society of Chemical industry.

Electron spin resonance (ESR) spectroscopy has been used to assess the antioxidant capacity of eight Scotch whiskeys by measuring the extent by which the original spirits, or pyridine solutions of their residues, reduced Fremy's radical or galvinoxyl radical. All whiskeys displayed antioxidant activity greater than that of a 0.2 mM solution of Trolox in the Fremy's assay and of a 0.1 mM solution of quercetin in the galvinoxyl assay. The relative antioxidant capacities determined according to the two assays were highly correlated and strongly related to the total phenol content as determined by using the Folin-Ciocalteu method. Activity was a consequence of maturation in oak casks with the "newmake" spirit showing no effect. Of 10 aromatic constituents analyzed, activity was most strongly correlated with ellagic acid and gallic acid in both assays. The reductive capacities of four major phenolics were determined, which, in summation, accounted for 31-53% of the total antioxidant activity of the whiskeys. There was no evidence for synergistic interaction between the phenols investigated.

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. (C) 1998 SCI.

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 abilityin vitromay indicate little significance as antioxidantsin vivo.

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 (ESR) solution spectra of soil humic acids (HAs) in alkali show the presence of free radicals. Those giving rise to an unstructured spectrum appear to be very stable, whereas those displaying hyperfine structure are transient. ESR methods have been used to examine the interaction of ascorbate with HA from a peat and a mineral soil. The results demonstrate the ability of HAs to oxidize ascorbate to the ascorbyl radical rapidly as reduction of the HA proceeds. The HA radical species that give rise to structured components are suppressed, while a significant increase occurs in the radical species responsible for the broad, unstructured component, but the two events are not directly related.

Fresh treated sewage sludge from an urban source was applied to a field of granitic sandy loam soil under grass at a rate equivalent to 100 tonnes dry matter ha−1. The field was divided into 32 plots (4 m × 2 m). Treatments with metals (Cd, Cr, Cu, Mo, Ni, Pb, Zn as salts) were applied to blocks of 16 plots. Soil microbial biomass C and basal respiration were measured 3 a after the addition of sludge. After 5 a, plots were sampled, sieved to pass a 10 mm mesh and dried at 40°C. Carbon and N analyses were performed on finely ground soil samples. Total metals (after aqua regia digestion) and 0.1 M CaCl2 extractable metals were measured by ICP. Samples from Cu-treated plots were sequentially extracted with 0.01 N HCl, 0.1 M Na4P2O7 (pH 7) and 0.1 M NaOH and the fractions obtained were examined by Electron Paramagnetic Resonance spectroscopy which showed that some of the Cu had become organically complexed. There was no significant difference in the total soil C contents or microbial parameters as a result of the sludge addition and the presence of the different metals.

SummaryHumic substances contain significant quantities of free radicals which have important functions in many biological processes in soil involving electron transfer. Such radicals may be detected by electron spin resonance spectroscopy in both solid and solution state and are usually reported as giving a single unstructured peak. Humic acids from a range of soil types varying in parent material and pH have been examined by electron spin resonance spectroscopy of their solutions in 0.1 m KOH. Spectra with an effective g‐value typical of an organic free radical were obtained. Observation of hyperfine structure was highly dependent on the instrument settings used. It was present at a modulation amplitude of 0.0142 mT, but not at 0.101 mT. Similarly, there was a reduction in resolution as microwave power was increased from 0.505–20 mW. This may explain the inability of some investigators to observe structure in soil humic acids. Resolution of hyperfine features was greatly enhanced by recording spectra in the second derivative mode. Using appropriate instrument settings all the humic acids showed hyperfine structure, although the intensity of these components varied by a factor of 100, being much greater in humic acid from highly acidic soils.

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 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 (A11 and g11) 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 N-14 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.