羟自由基对人红细胞磷脂酰乙醇胺脂质体相变性质的影响

研究了过氧化氢与亚铁离子体系产生的羟自由基对人红细胞膜磷脂酸乙醇胺(PE)脂质体相变性质的影响.结果表明,羟自由基导致脂质体不饱和脂肪酸链的含量明显降低和丙二醛(MDA)含量显著升高,同时其膜流动性随之下降.在室温下,羟自由基诱使PF脂质体冰冻断裂面出现脂质颗粒,说明羟自由基通过脂质过氧化作用可促进PE脂质体从脂双层转变为非双层结构.     

植物体内羟自由基的产生及其与脂质过氧化作用启动的关系

本文简要介绍了植物体内羟自由基(OH)的产生及检测方法,并对植物体内羟自由基的产生与启动脂质过氧化的关系进行了讨论。     

甘青铁线莲花水提取物的抗氧化活性研究

本文采用邻二氮菲-Fe2 氧化法,邻苯三酚自氧化法以及卵黄脂蛋白不饱和脂肪酸(PUFA)过氧化体系,对甘青铁线莲花水提取物清除超氧阴离子自由基(O-·2 )和羟自由基(·OH )以及抑制卵黄脂蛋白脂质过氧化(LPO)作用的效果进行了测定.结果表明:甘青铁线莲花水提取物有清除O-2、OH的能力,同时能抑制卵黄脂蛋白脂质过氧化(LPO)作用,在相同干物质浓度下(黄酮含量为2.20 μg),抑制卵黄脂蛋白脂质过氧化(LPO)作用最强、O-2次之、OH最弱,这说明甘青铁线莲花水提取物有抗氧化作用,且水提物抗氧化活性在一定浓度范围内与其黄酮类化合物含量呈正相关.     

掺入胆固醇和阴离子磷脂对阿霉素免疫脂质体性能的影响

本文研究了胆固醇和阴离子磷脂的掺入对阿霉素—磷脂酰乙醇胺免疫脂质体包裹效力及其在PBS缓俄冲液和在50%血清中的稳定性的影响,并对这种影响可能的机理进行了讨论.阴离子磷脂PG和DPG的掺入使ADM免疫脂质体包裹ADM的效力大大增加,使ADM与PE的克分子比从(0.08—0.8)%增加到75%,其中DPG比PG更有效.掺入胆固醇可以明显提高ADM脂质体在缓冲液和在血清中的稳定性,但使脂质体包裹ADM的效力下降.通过脂质的选择和制备条件的摸索,我们成功地用超声法制备了包裹抗癌药(ADM),表面带有抗人胃癌细胞M85单克隆抗体3HII的小单层脂质体(SUV),其起脂质份是PE:PG:Chol:ADM(4:2:2:1),脂质体内的ADM与PE的克分子比为17—25%.经电镜观察,脂质体直径在60—80nm范围内,大以比较均一.这种脂质体在PBS缓冲液中于室温下保存12天,仍然能够保持其包裹药的70%;在50%的血清中,37℃1小时,能够保持其包裹药的80%.     

掺入胆固醇和阴离子磷脂对阿霉素免疫脂质体性能的影响

本文研究了胆固醇和阴离子磷脂的掺入对阿霉素—磷脂酰乙醇胺免疫脂质体包裹效力及其在PBS缓俄冲液和在50%血清中的稳定性的影响,并对这种影响可能的机理进行了讨论.阴离子磷脂PG和DPG的掺入使ADM免疫脂质体包裹ADM的效力大大增加,使ADM与PE的克分子比从(0.08—0.8)%增加到75%,其中DPG比PG更有效.掺入胆固醇可以明显提高ADM脂质体在缓冲液和在血清中的稳定性,但使脂质体包裹ADM的效力下降.通过脂质的选择和制备条件的摸索,我们成功地用超声法制备了包裹抗癌药(ADM),表面带有抗人胃癌细胞M85单克隆抗体3HII的小单层脂质体(SUV),其起脂质份是PE:PG:Chol:ADM(4:2:2:1),脂质体内的ADM与PE的克分子比为17—25%.经电镜观察,脂质体直径在60—80nm范围内,大以比较均一.这种脂质体在PBS缓冲液中于室温下保存12天,仍然能够保持其包裹药的70%;在50%的血清中,37℃1小时,能够保持其包裹药的80%.     

金樱子多糖的抗氧化作用

目的：探讨金樱子多糖(PRL)体外抗氧化作用。方法：邻苯三酚自氧化法测定PRL清除超氧阴离子自由基效果;比色法测定PRL对羟自由基诱导红细胞溶血、脂质过氧化反应的影响。结果：PRL能显著清除超氧阴离子自由基、押制羟自由基对细胞膜的破坏而引起的溶血和脂质过氧化产物的形成。结论：PRL具有显著的抗氧化作用。     

北虫草抗氧自由基和羟自由基作用的研究

基于很多疾病与脂质过氧化有关 ,探讨了利用人工培育的北虫草的抗脂质过氧化作用。结果显示 :人工培育的北虫草子座对 Fenton反应生成的羟自由基具有较强的清除作用 ,且作用明显强于相同剂量的羟自由基特异清除剂甘露醇 (P<0 .0 1 ) ;北虫草对邻苯三酚自氧化体系产生的氧自由基亦具有清除作用 ,与对照组比较 P<0 .0 1 ,但作用弱于相同剂量的抗坏血酸。结果提示 :北虫草具有抗脂质过氧化作用。     

微生物源性抗氧化剂体外抗氧化能力的初步研究

目的:研究微生物源性抗氧化剂的体外抗氧化能力.方法:在体外分别测定微生物源性抗氧化剂、α-生育酚(Vε)、抗坏血酸(VC)、L-硫辛酸、表没食子酸儿茶素的还原能力,羟自由基、超氧阴离子自由基和DPPH自由基清除能力及抗脂质过氧化能力,比较微生物源性抗氧化剂与其他抗氧化剂抗氧化能力.结果:微生物源性抗氧化剂有较强的抗氧化能力,体外清除羟自由基、超氧阴离子自由基、DPPH自由基能力的半数有效量(EC50)分别为184.5μg、48.7μg、66.1 μg.与常见抗氧化剂相比,微生物源性抗氧化剂对氧自由基及氮自由基都有较好的清除自由基作用.结论:微生物源性抗氧化剂体外抗氧化作用明显,有进一步开发的价值.     

脂质体过氧化对DNA的损伤研究

研究了以脂质体为材料的脂质过氧化引起的ＥＮＡ损伤,同时检测了脂质过氧化程度与ＤＮＡ受损情况。结果表明：在脂质体过氧化程度中,ＤＮＡ的增色效应,对核酸酶的敏感程度,ＤＮＡ双链百分含量和ＤＮＡ－溴乙锭复合物的荧光强度随着氧化时间的增加而降低。在四种碱基中,鸟嘌呤损伤最严重。多种自由基清除列实验表明：脂质过氧化所产生的羟基自由基和单线态氧可能是引起ＤＮＡ氧化损伤的重要因素。     

绞股蓝多糖体外抗氧化活性研究

研究绞股蓝多糖的单糖组成及抗氧化活性.采用气相色谱(GC)分析绞股蓝多糖的单糖组成,通过体外抗氧化评价体系研究绞股蓝粗多糖(GPMPP)和精制多糖(GPMP)的总还原力、清除DPPH自由基、羟自由基的活性以及抗脂质过氧化作用.结果显示,绞股蓝多糖由鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖组成,物质的量比为1.39∶3.76∶1.00∶1.64∶4.98∶5.88.绞股蓝多糖具有较好的还原能力,对DPPH自由基和·OH具有较强的清除能力,并且对小鼠肝匀浆自发性脂质过氧化和Fe2+-H2O2诱导的小鼠肝匀浆脂质过氧化具有较好的抑制作用.以上结果表明,绞股蓝多糖具有明显的抗氧化活性.     

Effect of hydroxyl radical on Na(+)-K(+)-ATPase activity of the brain microsomal membranes.

Sphingomyelin liposomes and brain microsomes were oxidized by exposure to hydrogen peroxide and ferrous ion. Lipid peroxidation were measured by the formation of thiobarbituric acid- reactive substances (TBAR). Hydroxyl radical was detected using the spin- trapping technique. Incubation of sphingomyelin liposomes with H2O2-Fe2+ resulted in an increase in the formation of TBAR. Na(+)-K(+)-ATPase activity was markedly inhibited and the SH group content decreased during incubation of microsomes in the presence of H2O2-Fe2+. Sodium ferulate effectively inhibited TBAR formation, protected Na(+)-K(+)-ATPase activity and prevented the oxidative modification of SH groups. Spin-trapping experiments showed that sodium ferulate effectively scavenged the hydroxyl radicals.     

Tyrosyl free radical formation in the small subunit of mouse ribonucleotide reductase

Each R2 subunit of mammalian ribonucleotide reductase contains a pair of high spin ferric ions and a tyrosyl free radical essential for activity. To study the mechanism of tyrosyl radical formation, substoichiometric amounts of Fe(II) were added to recombinant mouse R2 apoprotein under strictly anaerobic conditions and then the solution was exposed to air. Low temperature EPR spectroscopy showed that the signal from the generated tyrosyl free radical correlated well with the quantity of the Fe(II) added with a stoichiometry of 3 Fe(II) needed to produce 1 tyrosyl radical: 3 Fe(II) + P + O2 + Tyr-OH + H+----Fe(III)O2-Fe(III)-P + H2O. + Tyr-O. + Fe(III), where P is an iron-binding site of protein R2 and Tyr-OH is the active tyrosyl residue. The O-O bond of a postulated intermediate O2(2-)-Fe(III)2-P state is cleaved by the extra electron provided by Fe(II) leading to formation of OH., which in turn reacts with Tyr-OH to give Tyr-O.. In the presence of ascorbate, added to reduce the monomeric Fe(III) formed, 80% of the Fe(II) added produced a radical. The results strongly indicate that each dimeric Fe(III) center during its formation can generate a tyrosyl-free radical and that iron binding to R2 apoprotein is highly cooperative.     

铜锌超氧物歧化酶对几种羟自由基产生系统的影响

应用脱氧核糖降解法研究了ＣｕＺｎ－ＳＯＤ对几种·ＯＨ产生系统的作用机理．结果证明：ＳＯＤ对Ｆｅ（３＋）·Ｏ·Ｈ２Ｏ２系统中·ＯＨ的产生有明显的抑制作用,而失活ＳＯＤ或ＢＳＡ对它的抑制作用不大;在Ｆｅ（２＋）·Ｈ２Ｏ２和ＣＵ（２＋）·Ｈ２Ｏ２系统中,ＳＯＤ、失活ＳＯＤ和ＢＡＳ均能抑制·ＯＨ的产生;在Ｆｅ（２＋）·Ｏ系统中,ＳＯＤ对·ＯＨ产生作用不大,而失活ＳＯＤ或ＢＳＡ对它有明显的抑制作用．由此推测ＳＯＤ对·ＯＨ形成可能有三方面的影响：１．对Ｏ的清除作用,阻断Ｈａｂｅｒ－Ｗｅｉｓｓ反应;２．对金属离子的络合作用,降低·ＯＨ的产额;３．促进Ｈ２Ｏ２的积累,加快Ｆｅｎｔｏｎ反应．     

褐蘑菇水溶性多糖抗氧化活性研究

采用水提醇沉以及sevag去蛋白的方法获得褐蘑菇水溶性多糖(WPPA)。通过测定还原力、超氧阴离子清除率、羟基自由基清除率和抑制H2O2诱导红细胞氧化溶血实验评价WPPA抗氧化活性。结果表明:WPPA具有较强的还原力,对O2-.和.OH具有较强的清除作用,IC50分别为527μg/mL、310μg/mL;对H2O2诱导红细胞氧化溶血及MDA生成有很强的抑制作用,IC50分别为700μg/mL和541μg/mL。说明WPPA在一定浓度内具有较强的抗氧化能力。     

The requirement for iron (III) in the initiation of lipid peroxidation by iron (II) and hydrogen peroxide

The initiation of lipid peroxidation by Fe2+ and H2O2 (Fenton's reagent) is often proposed to be mediated by the highly reactive hydroxyl radical. Using Fe2+, H2O2, and phospholipid liposomes as a model system, we have found that lipid peroxidation, as assessed by malondialdehyde formation, is not initiated by the hydroxyl radical, but rather requires Fe3+ and Fe2+. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide and the bleaching of para-nitrosodimethylaniline confirmed the generation of the hydroxyl radical in this system. Accordingly, catalase and the hydroxyl radical scavengers mannitol and benzoate efficiently inhibited the generation and the detection of hydroxyl radical. However, catalase, mannitol, and benzoate could either stimulate or inhibit lipid peroxidation. These unusual effects were found to be consistent with their ability to modulate the extent of Fe2+ oxidation by H2O2 and demonstrated that lipid peroxidation depends on the Fe3+:Fe2+ ratio, maximal initial rates occurring at 1:1. These studies suggest that the initiation of liposomal peroxidation by Fe2+ and H2O2 is mediated by an oxidant which requires both Fe3+ and Fe2+ and that the rate of the reaction is determined by the absolute Fe3+:Fe2+ ratio.     

Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate.

Treatment of the Cu(II)-Fe(III) derivative of pig allantoic fluid acid phosphatase with hydrogen peroxide caused irreversible inactivation of the enzyme and loss of half of the intensity of the visible absorption spectrum. Phosphate, a competitive inhibitor, protected against this inactivation, suggesting that it occurred as a result of a reaction at the active site. The native Fe(II)-Fe(III) enzyme was irreversibly inactivated by H2O2 to a much smaller extent than the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H2O2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H2O2 were increased by addition of ascorbate. These results suggest involvement of a Fenton-type reaction, generating hydroxyl radicals which react with essential active site groups. Experiments carried out on the Fe(II)-Fe(III) enzyme showed that irreversible inactivation by H2O2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of kobs for this reaction against H2O2 concentration (at saturating ascorbate) was hyperbolic, giving kobs(max) = 0.41 +/- 0.025 min-1 and S0.5(H2O2) = 1.16 +/- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H2O2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(III)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.     

Microsomal lipid peroxidation: the role of NADPH--cytochrome P450 reductase and cytochrome P450

The role of NADPH--cytochrome P450 reductase and cytochrome P450 in NADPH- and ADP--Fe3(+)-dependent lipid peroxidation was investigated by using the purified enzymes and liposomes prepared from either total rat-liver phospholipids or a mixture of bovine phosphatidyl choline and phosphatidyl ethanolamine (PC/PE liposomes). The results suggest that NADPH- and ADP--Fe3(+)-dependent lipid peroxidation involves both NADPH--cytochrome P450 reductase and cytochrome P450. Just as in the case of cytochrome P450-linked monooxygenations, the role of these enzymes in lipid peroxidation may be to provide two electrons for O2 reduction. The first electron is used for reduction of ADP--Fe3+ and subsequent addition of O2 to the perferryl radical (ADP--Fe3(+)-O2-), which then extracts an H atom from a polyunsaturated lipid (LH) giving rise to a free radical (LH.) that reacts with O2 yielding a peroxide free radical (LOO.). The second electron is then used to reduce LOO. to the lipid hydroperoxide (LOOH). In the latter capacity, reduced cytochrome P450 can be replaced by EDTA--Fe2+ or by the superoxide radical as generated through redox cycling of a quinone such as menadione.     

Inhibitory effect of eugenol on Cu2+-catalyzed lipid peroxidation in human erythrocyte membranes

1. The effects of eugenol on lipid peroxidation catalyzed by hydrogen peroxide (H2O2) or benzoyl peroxide (BPO) in the presence of copper ions were studied in human erythrocyte membranes. 2. The production of hydroxyl radicals was suggested in the peroxidation system catalyzed by H2O2/Cu2+. 3. H2O2/Cu2+-dependent peroxidation was inhibited by eugenol in a concentration-dependent manner; peroxidation was inhibited 62% by 200 microM eugenol. 4. In the presence of eugenol, the peroxidation catalyzed by BPO/Cu2+ was inhibited in a concentration-dependent manner, and more than 100 microM eugenol completely inhibited peroxidation. 5. The inhibitory effect of eugenol was non-competitive against Cu2+ in H2O2/Cu2+- and BPO/Cu2+-dependent peroxidation. 6. It is suggested that eugenol inhibits formation of hydroxyl radicals.     

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.