Free radical scavenging activity of chitooligosaccharides by electron spin resonance spectrometry

The radical scavenging effects of chitooligosaccharides (COSs) on hydroxyl radical, superoxide radical, alkyl radical, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were investigated using a spin-trapping electron spin resonance (ESR) method and compared with the ESR signal intensity. COSs exhibited strong scavenging activity on hydroxyl radical and superoxide radical and weak scavenging activity on alkyl radical and DPPH radical. The radical scavenging activity of COSs increased with increment of concentration, and it was also dependent on molecular weight. These results suggest that the scavenging activity of COSs is dependent on their molecular weights and tested radicals.     

利用电子自旋共振技术研究美拉德反应自由基变化

为寻求简便而准确地对食品加工和贮藏过程中的美拉德反应进行鉴定的方法,该文研究加热食品中葡萄糖和乳球蛋白建立的美拉德反应体系的自由基变化,主要考察影响美拉德反应的温度和pH值变化导致的自由基改变,从而确定美拉德反应与其自由基变化之间的相互关系。在3种不同温度、有无氧气和3种不同pH值条件下,利用电子自旋共振(electron spin resonance,ESR)光谱检测美拉德反应自由基变化趋势,结果随温度升高,自由基数量增加,反应速度加快;随pH值由酸性向碱性移动,自由基数量增加;自由基数量在无氧条件时多于有氧条件。当自由基积累到一定浓度,由于聚合反应生成高分子聚合物使信号强度下降,最后消失。比较美拉德产物与2种底物产生的自由基发现,无论类型、数量还是生成速率都存在明显差异。研究结果表明,采用自旋捕集剂对不稳定的自由基进行捕集来分析信号能够有效表征加热食品中美拉德反应初中期阶段的自由基变化,从而有效表征和分析美拉德反应过程。与传统研究美拉德反应的分析方法比较,ESR光谱是一种简便灵敏的分析方法,可以作为研究加热食品美拉德反应的重要方法之一。     

Evaluation of free radical scavenging activities of antioxidants with an H(2)O(2)/NaOH/DMSO system by electron spin resonance.

An H(2)O(2)/NaOH/DMSO system has been developed for the formation of three free radicals, and the application of the system was examined with the antioxidants ascorbic acid and tocopherol. Superoxide anion, hydroxyl radical,and methyl radical are simultaneously generated in this system. The scavenging activity of ascorbic acid and tocopherol for these radicals was estimated by 5, 5'-dimethyl-1-pyrroline-N-oxide spin trapping electron spin resonance. Both water-soluble and oil-soluble antioxidants could be evaluated by using this system. Ascorbic acid specifically inhibited the superoxide anion and hydroxyl radical, whereas tocopherol suppressed the methyl radical.     

Characterization of free radicals in soluble coffee by electron paramagnetic resonance spectroscopy

EPR spectra of soluble coffee display single-line free radical signals in both the solid state and aqueous solution, along with signals from the paramagnetic ions Fe(III) and Mn(II). The intensity of the free radical signal in the pure solid was estimated to be ca. 7.5 x 10(16) unpaired electrons/g, and there was no significant change on dissolution in water. In aqueous solutions, however, the free radical signal declined rapidly over ca. 10-15 min in the temperature range 20-65 degrees C, after which only slow changes were observed. This decline, which was essentially independent of atmosphere, was greatest for the lowest temperatures used, and the intensity after 1 h fitted well to an exponential curve with respect to temperature. The free radicals responsible for the single-peak EPR signal did not react with any of the spin traps tested in the present experiments, but unstable free radicals with parameters consistent with adducts of C-centered radicals were detected in coffee solutions in the presence of PBN and 4-POBN spin traps. The presence of oxygen in the solutions increased the initial rate of formation of these free radical adducts. No adducts were detected when DEPMPO was used as spin trap. However, *OH adducts of DEPMPO were shown to be unstable in the presence of coffee, a fact which illustrates the strong free radical scavenging ability of coffee solutions.     

电子自旋共振波谱技术在辐照葡萄检测中的应用

为了应用电子自旋共振（ESR）波谱技术检测辐照葡萄,以葡萄皮、葡萄柄和葡萄籽为试验材料,研究其在0～10.0 kGy剂量范围ESR波谱特征变化以及辐照剂量与信号强度的关系。结果表明： 葡萄皮、葡萄柄和葡萄籽辐照后ESR波谱均有明显的区别,信号强度均随辐照剂量增加而增大。葡萄柄的辐照剂量检出限最低（0.25 kGy）,是鉴定葡萄样品是否经过辐照的理想材料。通过比较葡萄皮、葡萄柄和葡萄籽剂量效应曲线得出葡萄柄拟合曲线最为准确（R2=0.9943）。3种辐照试验材料在贮藏期（15 d）内信号强度均有不同程度的衰减,辐照葡萄皮信号强度衰减最为剧烈（衰减80%）。研究结果为ESR波谱技术在辐照葡萄检测中的应用提供了技术依据。     

Free radical scavenging activities measured by electron spin resonance spectroscopy and B16 cell antiproliferative behaviors of seven plants.

In an effort to discover new antioxidant natural compounds, seven plants that grow in France (most of them in the Limousin countryside) were screened. Among these plants, was the extensively studied Vitis vinifera as reference. For each plant, sequential percolation was realized with five solvents of increasing polarities (hexane, chloroform, ethyl acetate, methanol, and water). Free radical scavenging activities were examined in different systems using electron spin resonance (ESR) spectroscopy. These assays were based on the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), the hydroxyl radicals generated by a Fenton reaction, and the superoxide radicals generated by the X/XO system. Antiproliferative behavior was studied on B16 melanoma cells. ESR results showed that three plants (Castanea sativa, Filipendula ulmaria, and Betula pendula) possessed, for the most polar fractions (presence of phenolic compounds), high antioxidant activities in comparison with the Vitis vinifera reference. Gentiana lutea was the only one that presented a hydroxyl scavenging activity for the ethyl acetate and chloroform fractions. The antiproliferative test results showed that the same three plants are the most effective, but for the apolar fractions (chloroform and hexane).     

Investigation of the presence of OH radicals in electrolyzed NaCl solution by electron spin resonance spectroscopy

In the anode side of a two-chamber electrolyzer, electrolysis of a NaCl solution generates acidic electrolyzed oxidizing (EO) water, which exhibits bactericidal effects against a large number of pathogens. This study was undertaken to investigate whether OH radical species are present in EO water or are formed when EO water reacts with iron ions. Electron spin resonance spectroscopy (ESR) coupled with the spin trapping technique was used for the detection of free radicals. Samples of EO water were collected at 0.5, 1, 2, 3, and 5 min of electrolysis and immediately mixed with the spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The 5,5-dimethyl-2-hydroxypyrrolidine-N-oxyl (DMPO-OH) spin adduct, characteristic of OH radicals, was not observed. Starting with 2-min electrolysis, a seven-line spectrum characteristic of 5,5-dimethyl-2-pyrrolidone-N-oxyl (DMPOX) was formed. The reactions of EO water with Fe3+ and Fe2+ in the presence of DMPO yielded the spin adduct DMPO-OH. However, the addition of OH radical scavengers (ethanol and methanol) did not generate the characteristic DMPO-alkyl spin adducts. This indicated that the DMPO-OH spectrum was due to a nucleophilic addition of water to DMPO and not to trapping of OH radicals.     

Kinetic and stoichiometric assessment of the antioxidant activity of flavonoids by electron spin resonance spectroscopy

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.     

Antioxidant activity in hepatopancreas of the shrimp (Pleoticus muelleri) by electron paramagnetic spin resonance spectrometry

Free radical scavenging properties of hepatopancreas extracts of Pleoticus muelleri were evaluated by electron paramagnetic spin resonance spectrometry methods (EPR) against the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The present study was carried out to characterize different physiological stages of the shrimp under environmental and nutritional stress, evaluating the effect on growth, survival, and functional morphology of the hepatopancreas. Feeding trials were carried out on juveniles (1 g initial weight) held in aquaria. Each diet, with different concentrations of vitamins A and E, was tested in triplicate groups during 25 days. The control groups were fed with fresh squid mantle and with a vitamin-free diet. For all of the diets, the extracts exhibited strong DPPH radical scavenging activity, suggesting that the tissue is a powerful natural antioxidant. Individuals fed with different concentrations of vitamin E showed the strongest effect on the DPPH radicals, reducing the DPPH radicals to 50%, after an incubation period of 3 min. In contrast, the extracts of control animals, fed with squid mantle, had the weakest antioxidant activity (4%). These data indicated that the presence of vitamin E in the diet can provide immediate protection against free radicals.     

Amino acid and protein scavenging of radicals generated by iron/hydroperoxide system: an electron spin resonance spin trapping study

Reduction of free radicals generated by Fe(II)/cumene-hydroperoxide (CumOOH) by amino acids (Gly, Cys, Met, His, and Trp) and proteins (bovine serum albumin (BSA), beta-lactoglobulin, and lactoferrin) was followed by electron spin resonance spectroscopy using alpha-phenyl-N-tert-butylnitrone (PBN), 2-methyl-2-nitrosopropane (MNP), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin traps. The radical species detected were mostly carbon-centered radicals from CumOOH fragmentation (methyl/*H3 and ethyl/*H2CH3), although carbon-centered radicals originated from amino acids could be formed in the presence of Cys, Met, His, or Trp. All proteins and amino acids, except Cys, were effective at inhibiting generation of radicals from the Fe(II)/CumOOH system. Trp was the amino acid with the highest antiradical activity, followed by His > Gly approximately Met. Lactoferrin was the protein showing the most efficient inhibition of radical formation from the Fe(II)/CumOOH system, and BSA and beta-lactoglobulin were not significantly different in their antiradical activities. These results suggest that proteins with higher inhibitory activity on lipid oxidation promoted by transition metal catalytic decomposition of hydroperoxides should be those with elevated metal-chelating and radical-scavenging properties as well as low concentration and accessibility of reducing groups from amino acids capable of activating metals, such as sulfhydryl groups.     

Free radical scavenging activity of grape seed extract and antioxidants by electron spin resonance spectrometry in an H(2)O(2)/NaOH/DMSO system.

The scavenging effects of grape seed extract (GSE) on free radicals formed in an H(2)O(2)/NaOH/DMSO system were examined using a spin-trapping electron spin resonance (ESR) method and compared with other natural antioxidants, ascorbic acid, dl-alpha-tocopherol, and beta-carotene. GSE reduced greatly the ESR signal intensity of superoxide radical-5,5-dimethyl-1-pyrroline-N-oxide (DMPO) adducts. GSE also exhibited weak scavenging activity on hydroxyl radical and a little scavenging activity on methyl radical. Ascorbic acid exhibited strong superoxide and hydroxyl radical scavenging activities, but it increased the amount of methyl radical at high concentration. dl-alpha-Tocopherol reduced the amount of superoxide anion, especially the amount of methyl radical. However, it slightly reduced the amount of hydroxyl radical. beta-Carotene reduced the amount of hydroxyl radical and methyl radical, but it also slightly reduced superoxide anion. In the case of combination use of beta-carotene and dl-alpha-tocopherol, all radical species were suppressed. Combination of GSE and dl-alpha-tocopherol also could reduce all radical species. beta-Carotene and dl-alpha-tocopherol could reduce the methyl radical formation induced by ascorbic acid.     

Dual positional and stereospecificity of lipoxygenase isoenzymes from germinating barley (green malt): biotransformation of free and esterified linoleic acid

The lipoxygenase isoenzymes LOX1 and LOX2 from green malt were separated by isoelectric focusing, and their catalytic properties regarding complex lipids as substrates were characterized. The regio- and stereoisomers of hydroperoxy octadecadienoates (HPODE) resulting from LOX1 and LOX2 enzymatic transformations of linoleic acid, methyl linoleate, linoleic acid glycerol esters monolinolein, dilinolein, and trilinolein, and 1-palmitoyl-2-linoleoyl-glycero-3-phosphocholine (PamLinGroPCho) were determined. In addition, biotransformations of polar and nonpolar lipids extracted from malt were performed with LOX1 and LOX2. The results show that LOX2 catalyzes the oxidation of esterified fatty acids at a higher rate and is more regioselective than LOX1. The dual position specificity of LOX2 (9-HPODE:13-HPODE) with trilinolein as the substrate (6:94) was higher than the resultant ratio (13:87) when free linoleic acid was transformed. A high (S)-enantiomeric excess of 13-HPODE was analyzed with all esterified substrates confirming the formation of 13-HPODE through the LOX2 enzyme; however, 9-HPODE detected after LOX2 biotransformations showed (R)-enantiomeric excesses. PamLinGroPCho was oxygenated by LOX1 with the highest regio- and stereoselectivities among the applied substrates.     

Application of electron spin resonance spectroscopy and spin probes to investigate the effect of ingredients on changes in wheat dough during heating

The change in microviscosity of the aqueous and lipid phases of wheat flour dough, during heating and subsequent cooling, has been measured using novel spin probes based on the isoindolin-yloxyl structure. The spin probes, water and/or lipid soluble, were used with combinations of dough ingredients: diacetyl tartaric acid ester of monoglycerides (DATEM), salt, yeast, and sodium ascorbate. The lipid soluble probe showed that DATEM does not produce a homogeneous phase with endogenous lipids but is found in a separate, less mobile phase. Also, the lipids were shown not to be involved in the baking process, although DATEM may be incorporated into the gelled starch matrix. The water soluble probe enabled starch gelatinization to be investigated in detail and showed that gelatinization produces a reduction of dielectric constant. The technique is appropriate for the detailed examination of the behavior of different ingredients during baking and also potentially to examine interactions between ingredients and flour components in dough.     

Identification of irradiated cashew nut by electron paramagnetic resonance spectroscopy

Cashew nut samples were irradiated at gamma-radiation doses of 0.25, 0.5, 0.75, and 1 kGy, the permissible dose range for insect disinfestation of food commodities. A weak and short-lived triplet (g = 2.004 and hfcc = 30 G) along with an anisotropic signal (g perpendicular = 2.0069 and g parallel = 2.000) were produced immediately after irradiation. These signals were assigned to that of cellulose and CO 2 (-) radicals. However, the irradiated samples showed a dose-dependent increase of the central line (g = 2.0045 +/- 0.0002). The nature of the free radicals formed during conventional processing such as thermal treatment was investigated and showed an increase in intensity of the central line (g = 2.0045) similar to that of irradiation. Characteristics of the free radicals were studied by their relaxation and thermal behaviors. The present work explores the possibility to identify irradiated cashew nuts from nonirradiated ones by the thermal behaviors of the radicals beyond the period, when the characteristic electron paramagnetic resonance spectral lines of the cellulose free radicals have essentially disappeared. In addition, this study for the first time reports that relaxation behavior of the radicals could be a useful tool to distinguish between roasted and irradiated cashew nuts.     

Assessment of the antioxidant potential of scotch whiskeys by electron spin resonance spectroscopy: relationship to hydroxyl-containing aromatic components.

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.     

Pro-oxidant activity of oleuropein determined in vitro by electron spin resonance spin-trapping methodology

In this work, the pro-oxidant behavior of oleuropein (OLP, 1) is characterized in a Fenton-like experiment by means of ESR spectroscopy using the spin trap system DMSO and 4-(pyridyl-1-oxide)-N-tert-butyl nitrone (POBN) in phosphate buffer (PB) solution. Ferrous ions in the absence of hydrogen peroxide cause the formation of the stable nitroxide species 4 and 5 through the intermediate perferryl species. OLP displays its antioxidant activity in vitro blocking the oxidation path that leads to methoxyl radicals hence to the formation of the stable radical species 5. The role of the catechol moiety was proved when the perferryl experiments were repeated in the presence of the dimethylated oleuropein homologue (OLP-Met2, 2). The dual behavior of oleuropein, similar to that ascertained for other catechol and non-catechol natural active species, should provide warnings for its use as nutraceutical or as drug with manifold healing effects.     

Effect of temperature and glassy states on the molecular mobility of solutes in frozen tuna muscle as studied by electron spin resonance spectroscopy with spin probe detection

The mobility of solutes in frozen food systems (tuna muscle, sarcoplasmic protein fraction of tuna muscle, and carbohydrate-water) has been studied using the temperature dependence of the shape of electron spin resonance (ESR) spectra of the spin probe 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL). The spin probe was incorporated into the tuna meat from an aqueous solution of TEMPOL or by contact with a layer of TEMPOL crystals. The melting/freezing of freeze-concentrated solutes in frozen tuna meat was observed to take place over a range of temperatures from -25 to -10 degrees C. Lower temperatures gave ESR powder spectra due to the decreased mobility of the spin probe, and the temperature dependence of the mobility of the spin probe did not show abrupt changes at the glass transition temperatures of the systems. The mobility of nonglass forming solutes is concluded to be decoupled from the glass forming components. Similar behavior was also observed for TEMPOL in frozen, aqueous carbohydrate systems. The temperature dependence of the mobility of TEMPOL in the frozen systems was analyzed using the Arrhenius equation, and the logarithm of the Arrhenius preexponential factor tau(a) was found to be linearly correlated with the activation energy for all of the tuna and carbohydrate samples, indicating a common molecular mechanism for the observed mobility of TEMPOL in all of the systems. The linear correlation also suggests that the observed mobility of TEMPOL in the frozen aqueous systems is dominated by enthalpy-entropy compensation effects, where the mobility of TEMPOL is thermodynamically strongly coupled to the closest surrounding molecules.     

Oxidative reactions during early stages of beer brewing studied by electron spin resonance and spin trapping

An electron spin resonance (ESR)-based method was used for evaluating the levels of radical formation during mashing and in sweet wort. The method included the addition of 5% (v/v) ethanol together with the spin trap alpha-4-pyridyl(1-oxide)- N- tert-butylnitrone (POBN) to wort, followed by monitoring the rate of formation of POBN spin adducts during aerobic heating of the wort. The presence of ethanol makes the spin trapping method more selective and sensitive for the detection of highly reactive radicals such as hydroxyl and alkoxyl radicals. Samples of wort that were collected during the early stages of the mashing process gave higher rates of spin adduct formation than wort samples collected during the later stages. The lower oxidative stability of the early wort samples was confirmed by measuring the rate of oxygen consumption during heating of the wort. The addition of Fe(II) to the wort samples increased the rate of spin adduct formation, whereas the addition of Fe(II) during the mashing had no effect on the oxidative stability of the wort samples. Analysis of the iron content in the sweet wort samples demonstrated that iron added during the mashing had no effect on the iron level in the wort. The moderate temperatures during the early steps of mashing allow the endogenous malt enzymes to be active. The potential antioxidative effects of different redox-active enzymes during mashing were tested by measuring the rate of spin adduct formation in samples of wort. Surprisingly, a high catalase dosage caused a significant, 20% reduction of the initial rate of radical formation, whereas superoxide dismutase had no effect on the oxidation rates. This suggests that hydrogen peroxide and superoxide are not the only intermediates that play a role in the oxidative reactions occurring during aerobic oxidation of sweet wort.     

Measurement of conjugated linoleic acid (CLA) in CLA-rich potato chips by ATR-FTIR spectroscopy

A conjugated linoleic acid (CLA)-rich soy oil has been produced by photoisomerization of soy oil linoleic acid. Nutritional studies have shown that CLA possesses health benefits in terms of reducing certain heart disease and diabetes risk factors. Potato chips are snacks that are readily produced in the CLA-rich soy oil containing CLA levels similar to those of the oil used for frying. The objective of this study was to develop an FTIR method to rapidly determine the CLA content of oil in potato chips. Photoirradiated soy oil samples with ～25% total CLA were mixed with control soy oil, and 100 soy oil samples with total CLA levels ranging from 0.89 to 24.4% were made. Potato chips were fried using each of these 300 g CLA rich soy oil mixtures at 175 °C for approximately 3 min. Duplicate GC-FID fatty acid analyses were conducted on oil extracted from each batch of potato chips. The chip samples were ground and then scanned using ATR-FTIR spectroscopy with the aid of a high-pressure clamp, and duplicate spectra of each sample were averaged to obtain an average spectrum. Calibration models were developed using PLS regression analysis. These correlated the CLA isomer concentrations of potato chips obtained by GC-FID fatty acid analysis with their corresponding FTIR spectral features. The calibration models were fully cross validated and tested using samples that were not used in the calibration sample set. Calibrations for total CLA, trans,trans CLA, trans-10,cis-12 CLA, trans-9,cis-11 CLA, cis-10,trans-12 CLA, and cis-9,trans-11 CLA had coefficients of determinations (R2v) between 0.91 and 0.96 and corresponding root-mean-square error of prediction (RMSEP) ranging from 0.005 to 1.44. The ATR-FTIR technique showed potential as a method for the determination of the CLA levels in unknown potato chip samples.