Free-radical scavenging and mechanism study of flavonoids extracted from the radix ofScutellaria baicalensis Georgi

Free-radical scavenging activities of baicalein, baicalin, wogonin and wogonoside, the four major flavonoids in a traditional Chinese herb medicine, the radix ofScutellaria baicalensis Georgi, were examined by electron paramagnetic resonance (EPR). The results showed that baicalein and baicalin scavenged hydroxyl radical, superoxide anion, 2,2-diphenyl-1-picrylhydrazyl radical and alkyl radical in a dose-dependent manner, while wogonin and wogonoside showed subtle or no effect on these radicals. Baicalein was the most effective free-radical scavenger among the four tested compounds. When 10 mmol/l tested flavonoids dissolved in alkaline solution, only baicalein and baicalin form stable free radicals which can be detected by EPR technique, it was found that the signal came from theiro-di-hydroxyl structure in ring A. To our knowledge, it was the first time demonstrated that flavonoids witho-di-hydroxyl structure in ring A could also form stable semiquinone free radicals. These results demonstrated that flavonoids in radix ofScutellaria baicalensis witho-di-hydroxyl group in ring A such as baicalein and baicalin are good free-radical scavengers and might contribute to some of their pharmaceutical effects.     

Rate constant measurements for initial addition reactions of radicals at the propagation step of photo‐polymerization as studied by pulsed EPR spectroscopy

Pulsed EPR spectroscopy was employed to determine reaction rate constants at an early stage of addition reactions in radical polymerizations triggered by four initiator radicals, which were generated by photodissociation of four parent molecules. Two monomers (tert‐butylacrylate and tert‐butylmethacrylate) were examined as reactant. Stern–Volmer analysis on the measured decay time of electron spin echo intensity of reacting radicals provides rate constants for addition reactions. We focused on rate constants for the second step reaction between monomer and adduct radical that is produced by the first step addition reaction between initiator radical and monomer. The rate constant measured by pulsed EPR was evaluated by theoretical calculations in the light of (1) enthalpy difference between product radical and reactants and (2) charge transfer interaction between reacting radical and monomer. Copyright © 2016 John Wiley & Sons, Ltd.     

电子顺磁共振法(EPR)对银杏叶提取物及其卷烟捕获自由基能力研究

自由基存在于人体和卷烟烟气中,会引起人体病理反应和氧化损伤,种类和形态多样,如：O-·₂,HO·,R·,RO·,ROO·。电子顺磁共振法(EPR)是检测和定量分析自由基的重要方法之一。银杏叶提取物(EGB)是由黄酮、内酯、原花青素类成分组成的植物药,具有较好清除自由基和抗氧化能力。本文以槲皮素、芦丁、原花青素等化合物为对照,运用EPR法结合自旋捕集技术研究EGB和EGB卷烟对DPPH,HO·,O2-·、烟气气相自由基、烟气粒相自由基的抑制水平。结果表明,EGB能够有效抑制化学/生物系统和卷烟烟气产生的自由基。尽管抑制自由基整体水平比黄酮单体和原花青素弱,但EGB提取方法简单容易得到,可作为天然抗氧化剂和自由基清除剂用于食品和烟草等行业。EPR法灵敏度高,稳定性好,是用于自由基研究的可靠方法。捕集剂DEPMPO捕集O2-·更加高效,PBN捕集烟气气相自由基比DMPO更有优势。该研究讨论了EGB清除自由基的机理,丰富的酚羟基结构是其抗氧化作用的主要原因。     

Free radical scavenging activities of white and red wines: An EPR spin trapping study

Free radical scavenging activities of wine samples were investigated by means of electron paramagnetic resonance (EPR) spin trapping. 1,1-Diphenyl-2-picrylhydrazyl served as a source of free radicals, OH radicals were produced by the Fenton system, and in situ thermally generated SO₄⁻ radicals from K₂S₂O₈ and carbon-centered radicals from azo-compounds were also used. 5,5-Dimethylpyrroline-N-oxide (DMPO) was used as a spin trapping agent. Three white and four red wine samples from the Slovak wine regions, along with 12% (v/v) aqueous ethanol solution as a reference sample, were compared. In general, the lowest scavenger activity was found in the reference sample, a higher one in the white, and a considerably higher one in the red wines, except when the Fenton system was used as the radical source. Here, unexpectedly, unlike in the other radical sources, the wine samples increased the yield of DMPO-adducts by about 5 times, compared to the reference sample. This is probably due to an additional activation of the Fenton system by wine components. Higher concentrations of Mn²⁺ ions as determined by EPR spectroscopy corresponded to an increased scavenging activity of wines.     

ESR study on scavenging effect of nicotine on free radicals

Smokers have less risk to catch degenerative diseases such as Alzheimer’s disease and Parkinson’s disease that are both correlated with enhanced oxidative stress. The scavenging effect of nicotine on free radicals was investigated by electron spin resonance techniques. It was found that the scavenging effect of nicotine on hydroxyl radicals and superoxide free radicals was higher than that of vitamin C. It was also found that nicotine could scavenge free radicals in gas-phase cigarette smoke. The data indicate that nicotine may be a potential antioxidant.     

Pulsed EPR measurements on reaction rate constants for addition of photo-generated radicals to double bonds of diethyl fumarate and diethyl maleate

Addition reaction of photo-generated radicals to double bonds of diethyl fumarate (deF) and diethyl maleate (deM), which are geometrical isomers, was studied by means of time-resolved- (TR-) and pulsed-electron paramagnetic resonance (EPR). Analysis of TR-EPR spectra indicates that adduct radicals from deF and deM should have the same structure. The double bonds of these monomers are converted to single ones by addition reaction, which allows hindered internal rotation to give the same structure of adduct radical. The rate constants for addition reaction of photo-generated radicals were determined by Stern–Volmer analysis of the decay time of electron spin-echo intensity of these radicals measured by the pulsed EPR method. Rate constants for deF were found to be larger than those for deM. This relation is in good consistent with efficiency of polymerisation of deF and deM. Experimentally determined rate constants were evaluated by introducing the addition reaction model on the basis of two important factors enthalpy and polar effects.     

The amounts of hydroxyl radicals generated by titanium dioxide and 3.5% hydrogen peroxide under 405-nm diode laser irradiation

A new bleaching agent consisting of a titanium dioxide photocatalyst and 3.5% hydrogen peroxide has recently been developed for in-office bleaching and has proven to react well with irradiated light around a wavelength of 400 nm. This study was conducted to evaluate the efficacy of dental bleaching with a 405-nm diode laser irradiation on titanium dioxide and 3.5% hydrogen peroxide by measuring the generation of hydroxyl radicals. The amount of hydroxyl radicals generated from a titanium dioxide and 3.5% hydrogen peroxide irradiated by a 405-nm diode laser were measured using electron spin resonance spin-trapping techniques. The irradiation conditions of the laser were a spot size of 1 mm, output powers of 100, 200, 400 mW, and 600 mW, and irradiation times of 10, 20, 30, and 60 s. Nonirradiated samples were used as controls. DMPO-OH adduct spectra and DMPO-O ₂ ^? adduct spectra were observed when the bleaching agent was irradiated by laser light. The amount of hydroxyl radicals generated changed in accordance with adjustments in the irradiation power and irradiation time. In the nonirradiated controls, hydroxyl radicals were generated in far smaller amounts. The 405-nm diode laser is effective in inducing the generation of hydroxyl radicals and shows promise as a bleaching light source for use in combination with bleaching agents composed of titanium dioxide and low concentrations of hydrogen peroxide.     

High-power sonication of soy proteins: Hydroxyl radicals and their effects on protein structure

High-power sonication (HPS) is shown to alter protein structure, thus, its functionality, via intermolecular interactions. This study evaluated the effects of HPS on molecular structure of soy proteins in aqueous medium. Free radicals generated during HPS were quantitated using the 5,5-dimethyl-l-pyrrolin N-oxide (DMPO) spin trap method. Electron paramagnetic resonance (EPR) was used to identify them as mostly hydroxyl radicals. The minimum saturation concentration of spin trap solution was determined to be 500 mM of DMPO in water, when exposed to 5 W/cm³ ultrasound power density (PD) for 10 min; subsequently, this concentration was used for quantitating radicals generated in protein samples. Five aqueous soy protein systems, namely, 5% soy protein isolate (SPI), 5% SPI without isoflavonoids (NO-ISO SPI), subunit solutions 1% glycinin (11S) and 1% β conglycinin (7S), and 10% soy flakes (w/v), were sonicated at 2.5 and 5 W/cm³ PDs. Only adducts of hydroxyl radicals (DMPO-OH) were detected in all of these aqueous systems. The highest concentration (3.68 µM) of DMPO-OH adduct was measured in 11S subunit solution at 5 W/cm³, whereas, the lowest (0.67 µM) was in soy flakes solution at 2.5 W/cm³. PD 5 W/cm³ generated higher concentration of radicals in 7S subunit solution, NO-ISO SPI, and soy flakes protein, compared to sonication at PD 2.5 W/cm³. No change in the protein electrophoretic patterns were observed due to HPS. However, some changes due to HPS were observed in the estimated secondary and tertiary structures, and the contents of free sulfhydryl groups and disulfide bonds in proteins.     

Antioxidant Activities of Some New Chromonyl-2,4-Thiazolidinediones and Chromonyl-2,4-Imidazolidinediones Having Chromone Cores

The antioxidant properties of 11 new synthesized chromonyl-2,4-thiazolidinediones and chromonyl-2,4-imidazolidinediones (CBs) were investigated. The antioxidant activities and mechanisms of the CBs interaction with reactive oxygen species (ROS) were clarified using various in vitro antioxidant assay methods including superoxide anion radical ( $ \mathrm{O}\overline{{}_2^{\bullet }} $ ), hydroxyl radical (HO^?), 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH^?) scavenging activity and the iron (II)-ferrozine complex formation. The potassium superoxide/18-crown-6 ether dissolved in dimethylsulfoxide (DMSO) was applied as a source of superoxide anion radical. Hydroxyl radicals were produced in the Fenton-like reaction Fe(II)+H₂O₂. Chemiluminescence, spectrophotometry, and electron paramagnetic resonance (EPR) spectroscopy using 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) as spin trap were applied as the measurement techniques. The CBs examined that exhibited good free radical scavenging activity also showed strong total antioxidant power capacity. Possible mechanisms of antioxidation are proposed to explain the differences in the experimental results between the chromone derivatives with imidazolidine-2,4-dione ring and those with thiazolidine-2,4-dione ring. In conclusion, some of the new CBs are promising to be applied as inhibitors of free radicals.     

Multifrequency CW EPR spectroscopy of extraterrestrial carbonaceous matter

In this work, the carbonaceous matter of Orgueil, Murchison and Tagish Lake carbonaceous meteorites and a reference coal is studied by multifrequency continuous-wave electron paramagnetic resonance (EPR) spectroscopy from 4 to 285 GHz. It is found that the shape of the EPR line of the radicals in meteoritic carbonaceous matter is Lorentzian in all the frequency range, while the line shape of the coal is Lorentzian only below 95 GHz and becomes inhomogeneously broadened at higher frequency, as previously observed for coals by other authors. This points to strong exchange interactions in meteoritic carbonaceous matter, resulting from a pronounced spin clustering that does not occur in biogenic carbonaceous matter (coals). The temperature dependence of the EPR line width has been studied in detail at X- and W-bands for the Orgueil meteorite. It confirmed our previous model of the presence of radicals with thermally accessible triplet states (TATS) in meteorites. These TATS, which were attributed to diradicaloids moieties on the basis of molecular quantum DFT calculations (L. Binet, D. Gourier, S. Derenne, F. Robert, I. Ciofini: Geochim. Cosmochim. Acta 68, 881–891, 2004) do not exist in biogenic carbonaceous matter. This analysis also precised the strength of the clustering effect in meteorites, yielding an estimated local spin concentrationN=5·10²⁰ spin/g, which is two orders of magnitude higher than the average spin concentration in the Orgueil meteorite. It is important to note that such spin clustering has also been observed by other authors in synthetic hydrogenated amorphous carbon. It seems that the clustering of radicals is a common feature of synthetic and extraterrestrial (abiotic) carbonaceous matters, while radicals are homogeneously distributed in biogenic carbonaceous matter.     

葡萄籽中多酚类物质对氧自由基清除作用的ESR研究

用ESR自旋捕集技术研究了从葡萄籽分离的单体、二聚体和三聚体等多酚类物质对羟基自由基和超氧阴离子的精除作用,并与化学发光法的测定结果进行了比较,得到了较一致的结果,证明葡萄籽中多酚类物质对氧自由基的确有较好的清除作用,这很可能是喝少量葡萄酒有利于身体健康的原因.     

Time-resolved EPR study of electron spin polarization and spin exchange in mixed solutions of porphyrin stable free radicals

The time-resolved electron paramagnetic resonance (EPR) spectra are studied in the temperature range of 110–300 K for two mixed solutions of porphyrins, ZnTPP and H₂TPP, in toluene and the stable free radical 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO). The EPR spectra and their kinetic behavior were studied for concentrations of TEMPO varied in the interval from 0.51 to 7.68 mM, while the porphyrin concentration was fixed as 1 mM. The EPR spectra of triplet-state porphyrins and free radicals manifest the chemically induced spin polarization. For the relatively short-lived radical-triplet pairs, with the perturbation theory up to the fourth order, the theoretical expressions are obtained for the triplet and radical spin polarization induced by the enhanced intersystem crossing (ISC) due to the interaction of excited singlet-state porphyrins with free radicals and by the triplet quenching by free radicals. The time-dependent EPR spectra of the triplets are simulated taking into account the spin-lattice relaxation. It is shown that the variation of the triplet EPR spectra shape, when the time of observation increases, arises from the spin-lattice relaxation kinetics. The kinetic behavior of the TEMPO EPR spectrum was simulated on the basis of the kinetic scheme suggested earlier in the literature. The triplet spin-lattice relaxation time, the rate of the ISC and the lifetime of the excited singlet state were estimated by fitting the kinetic curves for the triplet EPR spectra intensity. For the mixed porphyrin-TEMPO solutions, a possible set of the rate constants of important bimolecular processes were determined. For this set of parameters, it turns out that the spin polarization transfer has a smaller rate constant than the rate constant of the diffusion collisions of the triplet and radical. It appears that the rate constant of the ISC catalyzed by radicals is relatively high in the solutions close to the melting point of the solvent and in the soft-glassy state. In the triplet porphyrins the initial spin polarization induced by the spin-selective ISC was found to exceed the equilibrium spin polarization by up to two orders of magnitude.     

EPR Investigation of Radical-Production Cross Sections for Sucrose and L-alanine Irradiated with X-rays and Heavy-ions

Using electron paramagnetic resonance (EPR), we investigated stable radical-production cross sections (σ) of sucrose and L-alanine radicals produced by heavy-ion irradiations with various linear energy transfers (LET). The heavy-ion irradiation results were compared with those of X-ray irradiation at the same dose. The EPR signal areas for the two compounds showed a linear relation with the absorbed dose, as well as a logarithmic correlation with the LET. Further analysis was carried out for the radical-production cross section, which showed that stable radicals of the two compounds were produced through collisions of several particles with a single molecule. The relative σ value of sucrose for C ion irradiation was (1.29 ± 0.64) × 10⁻¹² μm². The σ value of alanine for C ion irradiation was (6.83 ± 0.42) × 10⁻¹³ μm². Considering the structural molecular sizes of sucrose and alanine, the σ values are similar. In addition, a comparison of the EPR results for the C ions and X-rays at 50 Gy dose was made. Sucrose spin concentrations produced by C ions at the LET value of 13.1 keV/μm and X-rays were similar unlike alanine. Thus, the noble EPR results with X-ray and heavy-ion irradiations imply that sucrose can be useful as a radiation indicator.     

Photoinduced decarboxylation of 9‐oxo‐6,9‐dihydro[1,2,5]selenadiazolo[3,4‐f]quinoline‐8‐carboxylic acid

Photoinduced reactions of 9‐oxo‐6,9‐dihydro[1,2,5]selenadiazolo[3,4‐f]quinoline‐8‐carboxylic acid (SeQCA) were investigated in alkaline media (aqueous NaOH solutions) by electron paramagnetic resonance (EPR) spectroscopy, following the in situ formation of paramagnetic species. According to UV–Vis and nuclear magnetic resonance investigations, protonation (pH ≈ 11) and deprotonation (pH ≈ 13) of the imino hydrogen of the 4‐pyridone moiety has to be considered, reflected also in the different EPR spectra observed upon irradiation. Photoinduced generation of radicals was found only for carboxylate substituted SeQCA; other studied selenadiazoloquinolone derivatives, together with those substituted at the C(8) position (R = H, COOCH₂CH₃, COOCH₃, COCH₃ or CN), did not generate paramagnetic species during exposure. Consequently, photodecarboxylation was suggested as the decisive step, accompanied by the decomposition of the selenadiazole ring, resulting in the formation of ortho‐hydroxylate anions. EPR parameters elucidated from experimental EPR spectra obtained at pH ≈ 11 and pH ≈ 13 indicate the formation of oxygen‐centered radicals at the decarboxylated 4‐pyridone ring. EPR spin trapping experiments with nitromethane confirmed a very effective photoinduced electron transfer from all the selenadiazoloquinolones investigated. Copyright © 2011 John Wiley & Sons, Ltd.     

贯众多糖提取及其抗氧化作用

采用水提-醇沉法从贯众中提取多糖,脱脂、去蛋白,经冷冻干燥后得到粗多糖。通过比色法研究其体外清除羟基自由基能力。贯众多糖具有较好的清除.OH的能力,且其清除能力与多糖浓度有明显的量效关系。清除50%羟基自由基所需要的多糖浓度为3.9m g/mL。     

Detection of ketyl radicals using 31P NMR spin trapping

Our recent work has allowed the development of ³¹P NMR spin trapping techniques for the detection and, at times, absolute quantification of many oxygen‐ and carbon‐centered free radical species. These methods are based on the ability of the nitrone phosphorus compound, 5‐diisopropoxy‐phosphoryl‐5‐methyl‐1‐pyrroline‐N‐oxide (DIPPMPO), to react with free radical species and form stable radical adducts, which are suitably detected and accurately quantified using ³¹P NMR. Our continuing efforts have now been focused on the application of this powerful system for the trapping of ketyl radicals, which are very difficult intermediates to be detected and quantified with traditional techniques (i.e., EPR). Ketyl radicals were initially produced using photochemical reactions of acetophenone, whose excited triplet state is able to abstract hydrogen from an H donor. As such, the ³¹P NMR signals for the radical adducts of the DIPPMPO spin trap with the ketyl radicals were assigned. Furthermore, in an effort to confirm the structure of these adducts, their mass spectra and fragmentation patterns were carefully examined under Gas Chromatography–Mass Spectrometry (GC–MS) conditions. Subsequently, the DIPPMPO spin trapping system was applied to the oxidation of 1‐(3,4‐dimethoxyphenyl)ethanol in the presence of horseradish peroxidase (HRP), hydrogen peroxide, and 1‐hydroxybenzotriazole (HBT) as the electron carrier (mediator). Our work confirmed that the mechanism consists of a hydrogen abstraction reaction from the α position, involving the ketyl radical: during the oxidation, the hydroxyl, hydroperoxyl, and ketyl radical intermediates were all detected. These efforts demonstrate the efficacy of our methodology that provides for the first time a facile means for the detection of the otherwise elusive ketyl radical species, with important implications in biology, chemistry, and biochemistry. Copyright © 2009 John Wiley & Sons, Ltd.     

新型饱和烷基类自由基捕捉剂的合成及ESR研究

设计合成了新的饱和烷基类线型硝酮捕捉剂N-（亚乙基）-t-丁胺-N-氧化物(EBN)和N-（亚乙基）-1-二乙氧基磷酰基-1-甲基乙基胺N-氧化物(EPN),并运用ESR、MS、IR、UV等一系列手段对其结构进行了表征,同时对Fenton体系中产生的羟基及不同类型的氧中心、碳中心和硫中心自由基的捕获能力进行了系统的研究. 结果表明,这两种捕捉剂合成方法比较简单,产率较高,对羟基等自由基有比较强的捕捉能力. 期望本文的工作能为自由基捕捉剂的研究提供一个新的思路.     

Types of paramagnetic centers in Cu2+ complexes with model neuromelanins

Electron paramagnetic resonance (EPR) spectroscopy was used to examine free radical properties of model neuromelanins obtained by oxidative polymerization of noradrenaline, adrenaline and dopamine. We compared the lineshape of the experimental spectra, type and concentration of free radicals in the analyzed samples. The effect of different concentrations of Cu²⁺ on free radicals in melanins was studied. The total concentration of free radicals (about 10¹⁸ to 10¹⁹ spin/g) in the studied melanins increases as follows: adrenaline-melanin < dopamine-melanin < noradrenaline-melanin. EPR spectra of dopamine-melanin and adrenaline-melanin were a single EPR line (ΔB _pp, 0.50 and 0.55 mT, respectively). o-Semiquinone free radicals with the characteristicg-value of 2.0040 exist in these melanins. EPR spectra of noradrenaline-melanin were a superposition of two lines (ΔB _pp, 0.45 and 0.81 mT). o-Semiquinone free radicals were responsible for the narrower component. Nitrogen free radicals with ag-factor of 2.0030 were probably responsible for the broader component. Paramagnetic copper ions quenched the EPR signals of melanin free radicals in the studied samples. For melanin-Cu²⁺ complexes, broad EPR lines (ΔB _pp, 10–32 mT) of copper ions with ag-value of about 2.1 appeared. The influence of the microwave power on the EPR spectra of these complexes demonstrated the fast spin-lattice relaxation in the copper system in melanins.     

Formation of Radical Anions by Electrochemical Reduction of Nitroimidazoles in Aprotic Solvents and Mixed Solvent

The EPR spectra of radicals obtained by electrolytical reduction of 2-nitroimidazole and 4-nitroimidazole were measured in mixed solvents. The behavior of the nitrogen coupling constants with the solvent composition is discussed in terms of equilibria between radicals in different solvents. The equilibrium constants of the DMSO/water system were determinated. Molecular orbital calculations in the frame of LCAO-HMO method account correctly for the spin distribution of both compounds investigated.     

Application of EPR Spectroscopy to Examination of Free Radical Formation in Thermally Sterilized Flumetasone

Thermal formation of free radicals in flumetasone sterilized according to the pharmaceutical norms at 160, 170 and 180 °C was examined by electron paramagnetic resonance (EPR) spectroscopy. The microbiological analysis was done. Similar free radical concentrations were measured for flumetasone sterilized at 160 and 170 °C. The concentration was considerably higher than that for flumetasone sterilized at 180 °C. Fast spin–lattice relaxation processes, homogeneously broadened EPR lines, and complex free radicals system characterize the heated flumetasone. Free radicals were not observed 30 days after thermal sterilization. Optimal temperatures of sterilization of flumetasone are 160 and 170 °C.