水母胶原蛋白抗氧化活性研究

提取水母胶原蛋白,观察其体内外抗氧化作用。体外采用邻苯三酚自氧化法观察水母胶原蛋白对超氧阴离子(·O2-)的清除作用,采用Fenton反应体系观察对羟自由基(·OH)的清除作用;体内观察水母胶原蛋白对小鼠血清、肝脏、大脑中超氧化物歧比酶(SOD)、过氧化氢酶(CAT),丙二醛(MDA)含量的影响。结果表明:水母胶原蛋白对·O2-、·OH具有良好的清除作用,呈一定的剂量依赖关系,能明显提高小鼠血清、肝脏、大脑中的SOD、CAT含量,能在一定程度上降低血清、肝脏、大脑中的MDA水平。因此,水母胶原蛋白具有显著的抗氧化生物活性。     

Drought acclimation reduces O2*- accumulation and lipid peroxidation in wheat seedlings

Abiotic stresses cause ROS accumulation, which is detrimental to plant growth. It is well known that acclimation of plants under mild or sub-lethal stress condition leads to development of resistance in plants to severe or lethal stress condition. The generation of ROS and subsequent oxidative damage during drought stress is well documented in the crop plants. However, the effect of drought acclimation treatment on ROS accumulation and lipid peroxidation has not been examined so far. In this study, the effect of water stress acclimation treatment on superoxide radical (O(2)(-z.rad;)) accumulation and membrane lipid peroxidation was studied in leaves and roots of wheat (Triticum aestivum) cv. C306. EPR quantification of superoxide radicals revealed that drought acclimation treatment led to 2-fold increase in superoxide radical accumulation in leaf and roots with no apparent membrane damage. However under subsequent severe water stress condition, the leaf and roots of non-acclimated plants accumulated significantly higher amount of superoxide radicals and showed higher membrane damage than that of acclimated plants. Thus, acclimation-induced restriction of superoxide radical accumulation is one of the cellular processes that confers enhanced water stress tolerance to the acclimated wheat seedlings.     

60Co-γ射线对固体透明质酸分子量与抗氧化性的影响

利用60Co-γ射线对崮体透明质酸进行辐射,探讨60Co-γ射线对固体透明质酸分子量和黏度特性及抗氧化性的影响。结果表明：透明质酸的分子量和黏度特性随辐照剂量的增加而降低;对羟自由基（OH·）及超氧阴离子自由基（O2·）清除作用随着剂量的增大逐渐减弱,对DPPH·自由基清除作用和还原能力随着剂量的增大逐渐增强;辐照前后透明质酸外观品质没有明显的变化,流动性增强;辐照对透明质酸的紫外光谱结构没有明显的改变。     

Carboxymethylcellulose–gelatin–superoxidase dismutase electrode for amperometric superoxide radical sensing

A novel, highly sensitive superoxide dismutase biosensor for the direct and simultaneous determination of superoxide radicals was developed by immobilization of superoxide dismutase within carboxymethylcellulose-gelatin on a Pt electrode surface. The parameters affecting the performance of the biosensor were investigated. The response of the CMC-G-SOD biosensor was proportional to O (2) (·-) concentration and the detection limit was 1.25 × 10(-3) mM with a correlation coefficient of 0.9994. The developed biosensor exhibited high analytical performance with wider linear range, high sensitivity and low response time. The biosensor retained 89.8% of its sensitivity after use for 80 days. The support system enhanced the immobilization of superoxide dismutase and promoted the electron transfer of superoxide dismutase minimizing its fouling effect. The biosensor was quite effective not only in detecting O (2) (·-) , but also in determining the antioxidant properties of acetylsalicylic acid-based drugs and the anti-radical activity of healthy and cancerous human brain tissues.     

Inhibition of fibroblast chemotaxis by superoxide dismutase

Superoxide radicals are known to be important mediators in chronic inflammatory and fibrotic processes, in which accumulation of fibroblasts is thought to play a major role in the pathogenetic events. The enzyme superoxide dismutase removes these radicals by a catalytic reaction. Chemotactic response of human fibroblasts and fibrosarcoma-derived cells (HT-1080) to fibroblast conditioned medium, fibronectin and platelet-derived growth factor was inhibited in a dose-dependent manner in the presence of superoxide dismutase, while random migration, cell proliferation, cell viability and synthesis of collagen and non-collagenous proteins was not altered. In contrast, phorbol myristate acetate, an inducer of superoxide generation, stimulated the chemotactic movement of fibroblasts to the attractants. Evidence for the formation of superoxide is provided by the reduction of tetrazolium salt by activated fibroblasts which could be inhibited by superoxide dismutase. Thus, it is concluded that superoxide in small amounts is involved in the mechanism of fibroblast chemotaxis. Superoxide dismutase may, therefore, reduce fibroblast migration into sites of injury or inflammation.     

Reactions of superoxide with the myoglobin tyrosyl radical

The contribution of superoxide-mediated injury to oxidative stress is not fully understood. A potential mechanism is the reaction of superoxide with tyrosyl radicals, which either results in repair of the tyrosine or formation of tyrosine hydroperoxide by addition. Whether these reactions occur with protein tyrosyl radicals is of interest because they could alter protein structure or modulate enzyme activity. Here, we have used a xanthine oxidase/acetaldehyde system to generate tyrosyl radicals on sperm whale myoglobin in the presence of superoxide. Using mass spectrometry we found that superoxide prevented myoglobin dimer formation by repairing the protein tyrosyl radical. An addition product of superoxide at Tyr151 was also identified, and exogenous lysine promoted the formation of this product. In our system, reaction of tyrosyl radicals with superoxide was favored over dimer formation with the ratio of repair to addition being approximately 10:1. Our results demonstrate that reaction of superoxide with protein tyrosyl radicals occurs and may play a role in free radical-mediated protein injury.     

A new approach for extracellular spin trapping of nitroglycerin-induced superoxide radicals both in vitro and in vivo

Anti-ischemic therapy with nitrates is complicated by the induction of tolerance that potentially results from an unwanted coproduction of superoxide radicals. Therefore, we analyzed the localization of in vitro and in vivo, glyceryl trinitrate (GTN)-induced formation of superoxide radicals and the effect of the antioxidant vitamin C and of superoxide dismutase (SOD). Sterically hindered hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidin (PP-H) can be used for in vitro and in vivo quantification of superoxide radical formation. The penetration/incorporation of CP-H or PP-H and of their corresponding nitroxyl radicals was examined by fractionation of the blood and blood cells during a 1-h incubation. For monitoring in vivo, GTN-induced (130 microg/kg) O2*- formation CP-H or PP-H were continuously infused (actual concentration, 800 microM) for 90 to 120 min into rabbits. Formation of superoxide was determined by SOD- or vitamin C-inhibited contents of nitroxide radicals in the blood from A. carotis. The incubation of whole blood with CP-H, PP-H, or corresponding nitroxyl radicals clearly shows that during a 1-h incubation, as much as 8.3% of CP-H but only 0.9% of PP-H is incorporated in cytoplasm. Acute GTN treatment of whole blood and in vivo bolus infusion significantly increased superoxide radical formation as much as 4-fold. Pretreatment with 20 mg/kg vitamin C or 15,000 U/kg superoxide dismutase prevented GTN-induced nitroxide formation. The decrease of trapped radicals after treatment with extracellularly added superoxide dismutase or vitamin C leads to the conclusion that GTN increases the amount of extracellular superoxide radicals both in vitro and in vivo.     

Quantitative determination of superoxide in plant leaves using a modified NBT staining method

Introduction – In plants, the ROS (reactive oxygen species) level is tightly regulated because their accumulation produces irreversible damage leading to cell death. However, ROS accumulation plays a key role in plant signaling under biotic or abiotic stress. Although various methods were reported to evaluate ROS accumulation, they are restricted to model plants or provide only qualitative information. Objective – Develop a simple method to quantify superoxide radicals produced in plant tissues, based on the selective extraction of the formazan produced after nitroblue tetrazolium (NBT) reduction in histochemical staining. Methodology – Plant leaves were stained with a standard NBT method and the formazan precipitated in tissues was selectively extracted using chloroform. The organic phase was dried and formazan residue dissolved in dimethylsulfoxide–potassium hydroxide and quantified by spectrophotometry. The method was tested in strawberry plant leaves under different stressing conditions. Results – Formazan extracted from leaves subjected to stress conditions showed similar absorption spectra to those obtained from standard solutions using pure formazan. Calibration curves showed a linear relationship between absorbance and formazan amounts, within the range 0.5–8 µg. Outcomes suggested that formazan was retained in the solid residue of leaf tissues. This protocol allowed us to quantify superoxide radicals produced under different stress conditions. Conclusions – Chloroform allowed a selective formazan extraction and removal of potential endogenous, exogenous or procedural artefacts that may interfere with the quantitative determination. This protocol can be used to quantify the superoxide produced in plant tissues using any traditional qualitative NBT histochemical staining method.     

藜叶中黄酮类化合物体外抗氧化活性研究

为了探讨藜叶中黄酮类化合物的体外抗氧化活性,采用有机溶剂提取法和色谱柱法对藜叶中化学成分进行提取与分离;以Vc作对照,对分离纯化的芦丁、乙酸乙酯浸膏和正丁醇浸膏进行DPPH·、O2^-·和·OH的清除效果试验。结果表明：三者对DPPH·、O2^-·、·OH均具有清除作用,且与浓度呈量效关系,芦丁对·OH清除效果优于Vc,芦丁具有较强的清除DPPH·能力,其IC50为0．05μg／mL。     

Cellular accumulation and antioxidant activity of acetoxymethoxycarbonyl pyrrolidine nitroxides

Abstract

Nitroxides are widely used in biology as antioxidants, spin labels, functional spin probes for pH, oxygen and thiol levels, and tissue redox status imaging using electron paramagnetic resonance (EPR); however, biological applications of nitroxides is hindered by fast bioreduction to EPR-silent hydroxylamines and rapid clearance. In this work, we have studied pyrrolidine nitroxides with acetoxymethoxycarbonyl groups which can undergo hydrolysis by cellular esterases to hydrophilic carboxylate derivatives resistant to bioreduction. Nitroxides containing acetoxymethoxycarbonyl groups were rapidly absorbed by cells from the media, 3,4-bis-(acetoxymethoxycarbonyl)-proxyl (DCP-AM2) and 3-(2-(bis(2-(acetoxymethoxy)-2-oxoethyl)amino)acetamido)-proxyl (DCAP-AM2) showing the strongest EPR signal of the cellular fraction. Remarkably, the EPR parameters of 3,4-dicarboxy-proxyl (DCP) and its mono- and di-acetoxymethyl esters are different, and consequent intracellular hydrolysis of acetoxymethoxycarbonyl groups in DCP-AM2 can be followed by EPR. To elucidate intracellular location of the resultant DCP, the mitochondrial fraction has been isolated. EPR measurements showed that mitochondria were the main place where DCP was finally accumulated. TEMPO derivatives showed expectedly much faster decay of EPR signal in the cellular fraction, compared to pyrrolidine nitroxides. It was found that supplementation of endothelial cells with 50?nM of DCP-AM2 completely normalised the mitochondrial superoxide level. Moreover, administration of DCP-AM2 to mice (1.4?mg/kg/day) resulted in substantial nitroxide accumulation in the tissues and significantly reduced hypertension. We found that hydroxylamine derivatives of dicarboxyproxyl nitroxide DCP-AM-H can be used for the detection of superoxide in vivo in angiotensin II model of hypertension. Infusion of DCP-AM-H in mice leads to accumulation of persistent EPR signal of nitroxide in the blood and vascular tissue in angiotensin II-infused wild-type but not in SOD2 overexpressing mice. Our data demonstrate that acetoxymethoxycarbonyl group containing nitroxides accumulate in mitochondria and demonstrate site-specific antioxidant activity.     

不同取代度N-邻苯二甲酰壳聚糖抗氧化活性的研究

低聚壳聚糖与邻苯二甲酸酐酰化得到三种取代度不同的N-邻苯二甲酸酐酰低聚壳聚糖NPCOSA、NPCOSB和NPCOSC,取代度分别为0.330、.55和0.65。通过红外光谱对其结构进行表征。并考察了其抗氧化性能。结果表明:COS的抗氧化性能最强;随着取代度的增加,N-邻苯二甲酰低聚壳聚糖对超氧阴离子的清除能力逐渐升高;而对DPPH的清除能力以及还原能力呈逐渐下降趋势;对羟基自由基的清除顺序大小依次为NPCOSB>NPCOSA>NPCOSC,即NPCOSB清除羟基自由基的的能力最佳。     

Superoxide-dependent oxidation of melatonin by myeloperoxidase

Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics.     

Role of superoxide in hemorrhagic shock-induced P-selectin expression

Superoxide has been implicated in the regulation of endothelial cell adhesion molecule expression and the subsequent initiation of leukocyte-endothelial cell adhesion in different experimental models of inflammation. The objective of this study was to assess the contribution of oxygen radicals to P-selectin expression in a murine model of whole body ischemia-reperfusion, i.e., hemorrhage-resuscitation (H/R), with the use of different strategies that interfere with either the production (allopurinol, CD11/CD18-deficient or p47(phox)-/- mice) or accumulation [intravenous superoxide dismutase (SOD), mutant mice that overexpress SOD] of oxygen radicals. P-selectin expression was quantified in different regional vascular beds by use of the dual-radiolabeled monoclonal antibody technique. H/R elicited a significant increase in P-selectin expression in all vascular beds. This response was blunted in SOD transgenic mice and in wild-type mice receiving either intravenous SOD or the xanthine oxidase inhibitor allopurinol. Mice genetically deficient in either a subunit of NADPH oxidase or the leukocyte adhesion molecule CD11/CD18 also exhibited a reduced P-selectin expression. These results implicate superoxide, derived from both xanthine oxidase and NADPH oxidase, as mediators of the increased P-selectin expression observed in different regional vascular beds exposed to hemorrhage and retransfusion.     

Ascorbate reduces superoxide production and improves mitochondrial respiratory chain function in human fibroblasts with electron transport chain deficiencies

The present paper attempts to ascertain the role of ascorbate on the generation of superoxide radicals in skin fibroblasts of patients with deficiency of mitochondrial respiratory chain enzymes. Fibroblast cell lines were grown with or without ascorbate for the last 48 h of their growth period. The amount of superoxide radical production in cells was measured by the reduction of nitroblue tetrazolium and the activities of respiratory chain enzymes were examined in isolated fibroblast mitochondria. The results indicated a significant inverse correlation between the amount of superoxide radicals and the specific activities of complexes I-III and II-III of the respiratory chain. The ascorbate treatment of fibroblasts from control subjects did not show any effect on either superoxide radical production or respiratory chain enzymes' activities. While in patient's fibroblasts, this vitamin significantly decreased the superoxide radicals and increased the specific activities of I-III and II-III complexes but not complex IV. These observations indicate that superoxide radicals are increased in patients with deficient respiratory chain enzymes in their fibroblasts and ascorbate can prevent the loss of these enzymes by acting on the selected sites in the respiratory chain, which are related to the production of free radicals.     

The protective effect of different forms of human ceruloplasmin in copper-induced lysis of red blood cells

The comparison of protective effects of native ceruloplasmin (CP) and of preparation CP1 containing carbohydrate fragment GlcNAc(beta(1,4]GlcNAc which specifically binds on RBC (alpha(1,6)Fuc receptors showed that CP1 exhibits much more powerful protective effect on RBC in copper-induced lysis. It was found, however, that CP2 (native CP devoided of CP1) protected RBC as well as CP despite its inability of binding to RBC membrane. CP and CP1 in a similar way decrease copper concentration in RBC. It was shown that copper accumulation and GSH decrease in RBC are two independent and concurrent processes; the copper and GSH concentrations are not the factors determining RBC resistance to hemolysis. CP inhibits the reaction of superoxide radicals generation as a result of Cu interaction with -SH groups of RBC membrane; the effect is more pronounced than the effect of catalase or superoxide dismutase. CP and CP1 preparations equally inhibit this reaction. Apparently CP reception on RBC leads not only to membrane protection from superoxide and hydroxyl radicals but represents a more complex process.     

Generation of superoxide anion radicals and hydrogen peroxide in the auto-oxidation of caffeic acid

Caffeic acid (5-200 mkM) reduces cytochrome c during autoxidation in potassium phosphate buffer, pH 7-8. The reduction is inhibited by superoxide dismutase, which suggests generation of superoxide anion radicals. The generation rate is 0.028-0.115 mkmoles O2- per min. Superoxide appears to be a side product of the reaction, since the autoxidation of caffeic acid itself (followed by A420) is not inhibited by superoxide dismutase. The autoxidation is accompanied by oxygen of consumption. An addition of catalase results in liberation of some part of consumed oxygen, this being indicative of accumulation of hydrogen peroxide. Caffeic acid is known to be responsible for the resistance of plants to parasites because of its toxicity. This function presumably depends on superoxide or other reactive oxygen species.     

Carbonate anions; effects on the oxidation of luminol, oxidative hemolysis, gamma-irradiation and the reaction of activated oxygen species with enzymes containing various active centres

Presence of carbonate anions increases the oxidation of luminol in different chemical systems. Lysis of human erythrocytes due to the action of dihydroxyfumaric acid or of perborate is also stimulated by carbonate ions. These anions also change considerably the loss of activity of different enzymes treated with superoxide, hydroxyl or formate radicals and can increase or decrease the effect as a function of the nature of the active centre of the enzyme. The relative effects of superoxide, hydroxyl, formate and carbonate radicals for the inactivation of various enzymes (superoxide dismutases, catalase, ribonuclease, glucose oxidase and glutathione peroxidase) have been examined. Three systems were used: gamma-irradiation under different conditions, photoproduction of radicals and sonication. Inactivation of the enzymes is a function not only of the radical used but also of the nature of the active site. Thus glutathione peroxidase is remarkably resistant to hydroxyl radicals while the superoxide dismutases are rapidly inactivated by carbonate radicals. All of the results combine to show that the presence or absence of carbonate anions must be considered in all studies of oxygen containing free radicals whether chemical, biochemical or biological or high energy irradiation.     

Effects of superoxide radicals on transport (Na+K) adenosine triphosphatase and protection by superoxide dismutase

Membrane (Na+K)ATPase isolated from rat brain was preincubated in a medium in which superoxide radicals were generated enzymatically. Exposure to superoxide radicals caused an irreversible inactivation, which could be prevented by further addition of superoxide dismutase. (Na+K)ATPase was also protected by addition of allopurinol, a xanthine oxidase inhibitor, during preincubation. The K-activated nitrophenylphosphatase associated with (Na+K)ATPase was also found to be inactivated by preincubation with superoxide radicals, which could be prevented by superoxide dismutase.     

Reaction of superoxide anions with melanins: electron spin resonance and spin trapping studies

Scavenging of superoxide radicals by melanin is a possible factor in the photoprotection afforded by melanin pigments. The reaction between superoxide anions and melanins has been studied by electron spin resonance and spin trapping methods. It was found that superoxide anions react to produce melanin free radicals in a reaction inhibited by superoxide dismutase but not by catalase. The rate of radical formation depends on the concentration of melanin and superoxide, the pH of the medium and the presence of diamagnetic metal ions. The melanin pigment competes with the enzyme superoxide dismutase for removal of superoxide radicals. It was found that the xanthine-xanthine oxidase system is not suitable for studying the reaction of superoxide with melanin, as the enzymatic activity of xanthine oxidase is considerably inhibited by melanin.