Reaction of vanadate ion with chlorpromazine,formation of vanadyl ion and chlorpromazine free radical

The red colored product, which was identified as a chlorpromazine (CPZ) free radical, was observed in the reaction of CPZ with the vanadate ion (+5 oxidation state). The product and the mechanism for the reaction were characterized from optical and EPR spectrometries. Optimal conditions for generation of the free radical were determined as reaction time within one minute of pH 6 and free radical stabilizing time of 30 minutes by acidifying with HCl. Under these conditions, the stoichiometry for the reaction was found to be 1:1, indicating the involvement of one electron transfer from CPZ to the vanadate ion to form the free radical and vanadyl ion (+4 oxidation state). A possible reaction scheme was proposed:

The implications of this reaction were discussed with regard to the pharmacological action of the vanadate ion and CPZ.     

The formation of aminopyrine cation radical by the peroxidase activity of prostaglandin H synthase and subsequent reactions of the radical

The oxidation of aminopyrine to an aminopyrine cation radical was investigated using a solubilized microsomal preparation or prostaglandin H synthase purified from ram seminal vesicles. Aminopyrine was oxidized to an aminopyrine cation radical in the presence of arachidonic acid, hydrogen peroxide, t-butyl hydroperoxide or 15-hydroperoxyarachidonic acid. Highly purified prostaglandin H synthase, which processes both cyclo-oxygenase and hydroperoxidase activity, oxidized aminopyrine to the free radical. Purified prostaglandin H synthase reconstituted with Mn2+ protoporphyrin IX, which processes only cyclo-oxygenase activity, did not catalyze the formation of the aminopyrine free radical. Aminopyrine stimulated the reduction of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid to 15-hydroxy-5,8,11-13-eicosatetraenoic acid. Approximately 1 molecule of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid was reduced for every 2 molecules of aminopyrine free radical formed, giving a stoichiometry of 1:2. The decay of the aminopyrine radical obeyed second-order kinetics. These results support the proposed mechanism in which aminopyrine is oxidized by prostaglandin H synthase hydroperoxidase to the aminopyrine free radical, which then disproportionates to the iminium cation. The iminium cation is further hydrolyzed to the demethylated amine and formaldehyde. Glutathione reduced the aminopyrine radical to aminopyrine with the concomitant oxidation of GSH to its thiyl radical as detected by ESR of the glutathione thiyl radical adduct.     

The transition metal-mediated formation of the hydroxyl free radical during the reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase

The NADPH-supported enzymatic reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase was investigated. The ESR spin trapping technique was employed to identify the free radical metabolites of oxygen. The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to trap and identify the oxygen-derived free radicals. [17O]Oxygen was employed to demonstrate that the oxygen-centered radicals arose from molecular oxygen. From the data, the following scheme is proposed: (Formula:see text). The formation of the free hydroxyl radical during the reduction of oxygen was demonstrated with quantitative competition experiments. The hydroxyl radical abstracted hydrogen from ethanol or formate, and the resulting scavenger-derived free radical was trapped with known rate constants. If H2O2 was added to the enzymatic reaction, a stimulation of the production of the hydroxyl radical was obtained. This stimulation was manifested in both the concentration and the rate of formation of the DMPO/hydroxyl radical adduct. Catalase was shown to inhibit formation of the hydroxyl radical adduct, further supporting the formation of hydrogen peroxide as an intermediate during the reduction of oxygen. All three components, ferredoxin, ferredoxin:NADP+ oxidoreductase, and NADPH, were required for reduction. Ferredoxin:NADP+ oxidoreductase reduces ferredoxin, which in turn is responsible for the reduction of oxygen to hydrogen peroxide and ultimately the hydroxyl radical. The effect of transition metal chelators on the DMPO/hydroxyl radical adduct concentration suggests that the reduction of chelated iron by ferredoxin is responsible for the reduction of hydrogen peroxide to the hydroxyl radical via Fenton-type chemistry.     

Free radical scavenging and antioxidant activities of EPS2, an exopolysaccharide produced by a marine filamentous fungus Keissleriella sp. YS 4108

The reactive oxygen species (ROS) and free radical-initiated reactions are ascertained to play multiple roles in degenerative or pathological events such as aging, cancer, heart dysfunction and Alzheimer's disease. EPS2 with a mean molecular weight of 1.3 x 10(5) was characterized as an antioxidant exopolysaccharide from the broth of a marine filamentous fungus Keissleriella sp. YS 4108. Compositionally, it is composed of galactose, glucose, rhamnose, mannose and glucuronic acid in an approximate proportion of 50:8:1:1:0.4. The radical eliminating and antioxidant actions of the glycan was assessed in different in vitro systems showing that EPS2 exhibited profound scavenging activities in superoxide radical. As a reinforcement of the action, similar radical scavenging effects of EPS2 were also discerned with both site-specific and non site-specific hydroxyl radical using the deoxyribose assay method. Moreover, EPS2 effectively blocked as well the non site-specific strand-breaking of DNA induced by the Fenton reaction at concentrations of 0.1 and 1 mg/mL. Further investigation of the effect of EPS2 on human low density lipoprotein (LDL) system demonstrated that it significantly inhibited copper-mediated oxidation of LDL in a dose-dependent manner. These results suggest that EPS2, possessing pronounced free radical scavenging and antioxidant activities, could be of considerable preventive and therapeutic significance to some life-threatening health problems such as cancer, atherogenesis and Alzheimer's disease which pathologically initiated by the presence of free radicals leading to the inevitable peroxidation of important biomolecules.     

Hydralazine-dependent carbon dioxide free radical formation by metabolizing mitochondria

The addition of hydralazine (1-hydrazinophthalazine) to rat liver mitochondria metabolizing malate/glutamate causes formation of a carbon-centered free radical which was spin-trapped with phenyl-t-butylnitrone (PBN) or dimethylpyrrolidine-N-oxide (DMPO). The coupling constants of the spin-trapped free radical were AN = 16.1, AH beta = 4.6 G for PBN and AN = 15.9, AH beta = 18.9 G for DMPO-trapped radical in aqueous solution. The spin-trapped free radical was shown to be the carbon dioxide anion free radical by independent synthesis, high pressure liquid chromatography separation, and electron paramagnetic resonance characterization. The amount of carbon dioxide anion free radical produced was absolutely dependent upon the presence of hydralazine and varied depending on mitochondrial substrate, with by far the highest amount produced by pyruvate. Studies with 13C-labeled pyruvate demonstrated that the carbon dioxide free radical came from C-1 of this compound.     

NaCl、Na2 SO4 和Na2GO3 胁迫对小麦叶片自由基含量及质膜透性的比较研究

 分别用浓度为25mmol／L、50mmol／L、100 mmol／L和200 MMOL／l的Nacl、Na2SO4和Na2CO3的营养液培养小麦4d,较之不含盐的营养液,其自由基含量上升,产生速率增加,叶片质膜透性增加。不同盐的影响也不同,在低浓度时,NaCl的影响大于Na2SO4,高浓度时,NaCl影响小于Na2CO3,Na2CO3的影响最为显著。实验结果也表现出小麦叶片自由基含量和质膜透性呈现较好的相关性。因此可认为,盐胁迫促使自由基含量增加,自由基通过过氧化作用影响质膜透性,从而影响植物的生长。     

Activation of the anticancer drug CCNU into a free radical intermediate via UV irradiation--an ESR study

We studied the formation of a free radical induced by UV irradiation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in benzene. It was determined a stable nitroxide radical by ESR spectroscopy. We confirmed that sterically hindered cyclic amine 2,2,6,6-tetramethyl-4-piperidone (TMPone) was transformed into a corresponding stable free nitroxyl radical during UV irradiation. When CCNU was present, the rate of free radical formation from TMPone increased considerably.     

草菇子实体多肽提取工艺及其抗氧化活性

为了优化草菇子实体多肽的提取工艺和探究其抗氧化活性,以草菇子实体为原料,采用酶解法提取草菇子实体多肽,通过单因素试验得出最佳的酶解工艺,并使用Box-Behnken设计试验组合。结果表明：草菇子实体提取多肽的最佳工艺为料液比1:52 (g/mL)、加酶量7 200 U/g、酶解温度43 ℃,此工艺条件下的多肽得率为67.76%。从1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除能力、铁离子还原能力、超氧阴离子自由基清除能力和羟自由基清除能力4个方面研究其体外抗氧化能力,结果表明,草菇子实体多肽对DPPH自由基清除率为74.11%,超氧阴离子自由基和羟自由基清除率分别在69.64%和91.83%达到稳定,草菇子实体多肽还具有一定的还原力,说明草菇子实体多肽可以作为优质抗氧化肽的良好来源。该研究为草菇多肽的高效制备和抗氧化肽等高附加值产品的研发提供理论依据。     

Generation of free radicals from lipid hydroperoxides by Ni2+ in the presence of oligopeptides.

The generation of free radicals from lipid hydroperoxides by Ni2+ in the presence of several oligopeptides was investigated by electron spin resonance (ESR) utilizing 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. Incubation of Ni2+ with cumene hydroperoxide or t-butyl hydroperoxide did not generate any detectable free radical. In the presence of glycylglycylhistidine (GlyGlyHis), however, Ni2+ generated cumene peroxyl (ROO.) radical from cumene hydroperoxide, with the free radical generation reaching its saturation level within about 3 min. The reaction was first order with respect to both cumene hydroperoxide and Ni2+. Similar results were obtained using t-butyl hydroperoxide, but the yield of t-butyl peroxyl radical generation was about 7-fold lower. Other histidine-containing oligopeptides such as beta-alanyl-L-histidine (carnosine), gamma-aminobutyryl-L-histidine (homocarnosine), and beta-alanyl-3-methyl-L-histidine (anserine) caused the generation of both cumene alkyl (R.) and cumene alkoxyl (RO.) radicals in the reaction of Ni2+ with cumene hydroperoxide. Similar results were obtained using t-butyl hydroperoxide. Glutathione also caused generation of R. and RO. radicals in the reaction of Ni2+ with cumene hydroperoxide but the yield was approximately 25-fold greater than that produced by the histidine-containing peptides, except GlyGlyHis. The ratio of DMPO/R. and DMPO/RO. produced with glutathione and cumene hydroperoxide was approximately 3:1. Essentially the same results were obtained using t-butyl hydroperoxide except that the ratio of DMPO/R. to DMPO/RO. was approximately 1:1. The free radical generation from cumene hydroperoxide reached its saturation level almost instantaneously while in the case of t-butyl hydroperoxide, the saturation level was reached in about 3 min. In the presence of oxidized glutathione, the Ni2+/cumene hydroperoxide system caused DMPO/.OH generation from DMPO without forming free hydroxyl radical. Since glutathione, carnosine, homocarnosine, and anserine are considered to be cellular antioxidants, the present work suggests that instead of protecting against oxidative damage, these oligopeptides may facilitate the Ni(2+)-mediated free radical generation and thus may participate in the mechanism(s) of Ni2+ toxicity and carcinogenicity.     

Detection of lipid radicals by electron paramagnetic resonance spin trapping using intact cells enriched with polyunsaturated fatty acid.

Electron paramagnetic resonance (EPR) spin trapping was used to detect lipid-derived free radicals generated by iron-induced oxidative stress in intact cells. Using the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), carbon-centered radical adducts were detected. These lipid-derived free radicals were formed during incubation of ferrous iron with U937 cells that were enriched with docosahexaenoic acid (22:6n-3). The EPR spectra exhibited apparent hyperfine splittings characteristic of a POBN/alkyl radical, aN = 15.63 +/- 0.06 G and aH = 2.66 +/- 0.03 G, generated as a result of beta-scission of alkoxyl radicals. Spin adduct formation depended on the FeSO4 content of the incubation medium and the number of 22:6-enriched cells present; when the cells were enriched with oleic acid (18:1n-9), spin adducts were not detected. This is the first direct demonstration, using EPR, of a lipid-derived radical formed in intact cells in response to oxidant stress.     

Studies on the antioxidant activity of some thiazolidinedione,imidazolidinedione and rhodanine derivatives having a flavone core

A series of flavonyl‐2,4‐thiazolidinedione, imidazolidinedione and rhodanine derivatives were tested for their antioxidant activity as scavengers of oxygen free radicals. Free radical scavenging activities, including superoxide anion radical , hydroxyl radical (HO^?) and 2,2′‐diphenyl‐1‐picrylhydrazyl free radical have been evaluated using chemiluminescence, electron paramagnetic resonance and spin trapping with 5,5‐dimethyl‐1‐pyrroline‐1‐oxide as a spin trap. Potassium superoxide in dimethylsulfoxide and 18‐crown‐6 ether were used for the production of . Hydroxyl radical was generated using the Fenton reaction. Ten of the eleven examined compounds exhibited decrease in chemiluminescence, but there were large differences in the decrease, ranging from 16% to 89%; also, two of these compounds increased light emission by about 200%. On the contrary, all compounds tested exhibited 30–68% scavenging HO^? and 25–96% scavenging the DPPH^? radical respectively. Possible mechanisms are proposed to explain the results. Copyright © 2014 John Wiley & Sons, Ltd.     

Hydroxyl radical induced cross-linking between phenylalanine and 2-deoxyribose

Hydroxy radicals induce cross-linking between phenylalanine (Phe) and 2-deoxyribose (dR) via formation of corresponding free radical intermediates. The cross-linked products were separated and identified by capillary gas chromatography-mass spectrometry. When phenylalanine and 2-deoxyribose radicals were generated in a 1:1 ratio, the predominant interaction was between Phe and dR radicals while the Phe-Phe and dR-dR cross-links were less abundant. The newly discovered cross-link between 2-deoxyribose and phenylalanine may serve as a model for radiation or free radical induced cross-linking between DNA and proteins and in general between sugar moieties and amino acids.     

芦丁-锗配合物及其自由基清除活性研究

芦丁是存在于多种植物中的天然多羟基黄酮苷,能与多种金属离子形成配合物。本文采用紫外分光度法考察了芦丁与锗离子的配位作用,并研究了芦丁-锗配合物清除超氧自由基和DPPH自由基的作用。结果显示在KH2PO4-NaOH(pH6．70)的缓冲液中,芦丁与锗离子能形成1：1的配合物,其K稳=10^7.46,同时配合物显示有较好的自由基清除活性。     

An Evaluation of EPR Measurements of the Organic Free Radical Content of Individual Seeds in the Non-destructive Testing of Seed Viability

Hepburn, H. A., Goodman, B. A., McPhail, D. B., Matthews, S.and Powell, A. A. 1986. An evaluation of EPR measurements ofthe organic free radical content of individual seeds in thenon-destructive testing of seed viability.—J. exp. Bot.37: 1675–1684. Electron paramagnetic resonance (EPR) spectra of individualintact seeds of a range of legumes and brassicas have been obtainedin order to measure their organic free radical contents. Norelationship was found between free radical content and seedviability or early seedling growth for both legumes and brassicas.The testa had a much greater free radical concentration thaneither the embryo axes or the cotyledons for all cultivars ofseed tested. The general high free radical content of the testasuggests that there is little possibility of EPR being usedto predict the viability of individual seeds. The spectroscopicsplitting factor of g = 2·0045 reported for the intactseeds is consistent with the radicals' arising from melanictype compounds. Key words: Seed viability, free radical content     

Spin-trapping of methyl radical in the oxidative metabolism of 1,2-dimethylhydrazine

A carbon-centered free radical formed during oxidative metabolism of 1,2-dimethylhydrazine has been spin-trapped with alpha-(4-pyridyl-1-oxide)N-tert-butyl nitrone and 2-methyl-2-nitrosopropane. In the horseradish peroxidase/H2O2 catalyzed oxidation, the trapped species was identified as the methyl radical by the characteristic 1:3:3:1 quartet pattern of the 2-methyl-2-nitroso propane adduct. A carbon-centered radical is also formed during microsomal oxidation of 1,2-dimethylhydrazine in the presence of NADPH. However, the alpha-(4-pyridyl-1-oxide)N-tert-butyl nitrone trapped radical has not been unambiguously identified in this latter instance. These results may be of importance in regard to both carcinogenic and antitumor properties of 1,2-disubstituted hydrazine derivatives.     

锌对缺血／再灌注肝脏自由基含量和细胞凋亡的影响

目的：观察补锌对缺血再灌注（HIR）大鼠肝脏自由基含量及细胞凋亡的影响。探讨补锌保护肝损伤的机制。方法：用荧光分光光度法测定血清MDA含量;用电子自旋共振法测定肝脏自由基浓度;用流式细胞术检测肝细胞凋亡。结果：HIR组大鼠血清MDA水平和肝自由基产生均增加,补锌后降低;肝脏缺血再灌注后肝细胞凋亡率达到57.72%,补锌后降低40.85%。结论：减少自由基产生和抑制细胞凋亡是锌保护肝缺血再灌注损伤的重要机制。     

Iron and copper chelation by flavonoids: an electrospray mass spectrometry study

Flavonoids are well known as effective free radical scavengers exhibiting therefore an antioxidant behaviour. Another antioxidant mechanism however may result from the ability they have to chelate metal ions, rendering them inactive to participate in free radical generating reactions. Electrospray mass spectrometry has been used to study metal ion interactions with a set of flavonoids from different classes. Complexes with a range of stoichiometries, of metal: flavonoid, 1:1, 1:2, 2:2, 2:3 have been observed. The stoichiometry 1:2 is in general the preferred one. It is established for flavones and for the flavanone naringenin that the binding metal sites are preferentially at the 5-hydroxyl and 4-oxo groups. Redox reactions are also observed through the change of the oxidation state of the metal, jointly with the oxidation of the flavonoid by loss of hydrogen. Structures of the oxidized species of some flavonoids are proposed.     

Free radicals and their interpretations

Oxygen free radicals can be blamed for evoking gastric mucosal damage, because of the protective effect of some lipid soluble free radical scavengers (vitamin A related compounds, Vitamin E). Direct determination of free oxygen radicals related chemical entities in the gastric tissue during ulcerogenesis yielded controversial results. Aluminum antacid compound together with acid binding property exhibited cytoprotection too, elevating the tissue PGE2 level substantially. Magnesium containing antacid according to our model experiments on red blood cells damage by free radicals, is capable to bind free radicals as well as to counteract with the dangerous intracellular calcium accumulation. It has been concluded that aluminum-magnesium antacid has a cytoprotective effect via: 1. acid binding; 2. prostaglandin generation; 3. free radical scavenging; 4. calcium antagonist activity.     

Synthesis of N-hydroxycinnamoyl amide derivatives and evaluation of their anti-oxidative and anti-tyrosinase activities

Twelve N-hydroxycinnamoyl amino acid amide ethyl esters (CAES) were synthesized by using l-amino acid ethyl ester hydrochloride and corresponding cinnamic acid (ferulic acid, acetylferulic acid and caffeic acid) as raw materials in the presence of a catalytic amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-hydrochloride (EDC) and 1-hydroxybenzotriene (HOBt). The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activities of CAES were evaluated. The anti-tyrosinase activities of N-feruloyl amino acid ethyl esters and the hydroxyl (OH) free radical scavenging activities of N-caffeoyl amino acid ethyl esters were also examined. DPPH free radical scavenging activity was shown in all CAES, of which N-caffeoyl amino acid ethyl esters demonstrated higher radical scavenging activity than N-feruloyl amide derivatives, and (E) -N-(caffeic acid)-l-glycinate ethyl ester (c₅) had the strongest ability to scavenge free radicals with an IC₅₀ value of 18.6 µM. The acetylferuloyl amino acid esters exhibited the highest tyrosinase inhibition activity among the tested amides.