天然抗氧化剂与自由基

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,报告人：何山,2006-03-12,天然抗氧化剂与自由基,自由基的发现和进程,自由基：具有未配对电子的原子、分子和离子,研究历程,1900年,Gomberg,发现稳定自由基三苯甲基。,1937年,M.S.Kharasch,发现过氧化物效应，创立自由基化学,40年代苏联物理学家发现电子顺磁共振现象，发明了,ESR,1969,年,Mc Cord,和,Fridovin,发现超氧化物歧化酶,SOD，,是自由基研究发展的里程碑,80年代以后，随着物理、化学检测分析技术的发展，自由基生物学逐步形成多学科交叉特点,90年代以后自由基在生命科学领域中影响进一步扩大，涉及领域也不断扩大。尤其在基础医学各学科及环境公卫和药物保健领域发展最快。多学科交叉的自由基生命科学随之诞生。,对,DNA,、,RNA,、蛋白质和脂类等生物大分子有损伤作用。可引起基因突变、蛋白活性下降和功能丧失，通过脂质过氧化使膜功能发生改变,。损伤最终导致细胞变异、老化、死亡。,自由基具有氧化还原性，可参加机体正常的生理反应，包括有氧代谢、炎症反应等等。有氧代谢中的呼吸链电子的传递都是通过产生自由基中间体来完成，机体炎症反应中，炎症周围聚集大量的中性粒细胞、巨噬细胞，通过释放出大量的自由基，用来消灭入侵的病源微生物。,近来研究表明，细胞一些重要的基因表达和信号通路，都与自由基的调节作用相关。同时，,ROS,本身也扮演第二信使角色。,自由基的主要生物学作用,自由基参与一些重要生命活动,近年来，自由基与生命活动的关系引起人们浓厚兴趣，现主要取得的研究进展是：,1,、发现自由基可以通过影响一些基因表达，来调控一些细胞的生物学功能，包括代谢、记忆、免疫、衰老、疾病发生和发展等。,2,、,NO,是氮中心自由基，除了具有内皮性舒张因子的作用，现发现还有更重要的功能，可以调节线粒体增殖，从而调节能量代谢活动。,3,、发现正常细胞与病理细胞中，胞内氧化和自由基水平不同，提示可采用特异性干预。,4,、越来越多的疾病发生与发展，己证明都与自由基作用有关，在活的病理细胞中巳捕捉到自由基介导的证据。,5,、缺血、缺氧所引起的机体各种损伤，发现都与线粒体膜结构变化和自由基代谢异常密切相关。,Mitochondrial Biogenesis in Mammals: The Role of Endogenous Nitric Oxide,SCIENCE Feb 7 2003;299(5608):869,天然抗氧化剂对自由基损伤的干预,天然抗氧化剂阻断,天然抗氧化剂的分类,内源性：,SOD、,谷胱甘肽过氧化氢酶,维生素：,VE、VC、,胡萝卜素,植物天然产物：多酚类物质、多糖,微量元素：铜、锌、硒,抗氧化剂的作用机制：,1、直接清除自由基：减轻或防止自由基损伤，例如在肝癌病人手术前，采用一些抗氧化剂辅助治疗，以防止感染和并发症。骨关节中注入,SOD,可减轻风湿性关节炎症状。,2、阻断自由基介导：实验证明，某些癌细胞增殖过程中，有自由基介导；如采用一些抗氧化剂，阻止自由基介入，诱导细胞凋亡。,化妆品,SOD,蜜,SOD structure,Cu/Zn-SOD,Mn-SOD,药物葛根素注射液,海藻类,中药,茶,食品加工,保鲜,添加剂,水果,French Paradox,Despite a high fat intake, mortality from coronary heart disease is 40% lower due to the regular drinking of red wine in some regions of France.,Resveratrol, a,polyphenol,present in red wine, has been suggested to be responsible for cardiovascular benefits associated with moderate wine consumption.,植物多酚类物质可以活化延寿基因,麻省理工学院的,Guarante,实验室发现，通过活化单个基因,SIR2,就可以延长大肠杆菌寿命。研究表明，一些多酚类小分子也可以活化人细胞的类似基因,SIR1。,实验还证明,白黎芦醇等天然抗氧化剂可明显延长酵母菌、线虫和果蝇的寿命而不需要限食。而且也不降低动物的生殖能力,（,Nature,430,686，2004),Resveratrol protects rat hearts from ischemia reperfusion injury,Free Radical Biology & Medicine(5.6), 1999, Vol 27, 160-169.,Inhibition of lipid peroxidation,The production of MDA is an index of the occurrence of lipid peroxidation and the development of oxidative stress.,Resveratrol prevents superoxide-dependent inflammatory induced by ischemia/reperfusion,Free Radical Biology & Medicine(5.6), 2003, Vol. 34, 810817.,Effects of resveratrol and superoxide dismutase (SOD) on ischemia/reperfusion (I/R)-induced,leukocyte recruitment,Resveratrol protects myocardial ischemia-reperfusion injury through NO-dependent mechanism,Free Radical Biology & Medicine(5.6), 2004 ,Vol. 36, 774 781.,Cancer Chemopreventive Activity,High expression of cycloxygenase(COX) was observed in many cancer cases.,Prostaglandins can stimulate tumor cell growth and suppress immune surveillance.,COX can activate carcinogens to forms that damage genetic material.,Bioassay-guided fractionation of COX inhibitor from,Cassia quinquangulata,identified resveratrol as active principle.,Science(31.8). 1997. 275. 5297,Cancer Chemopreventive Activity,The process of chemical carcinogenesis can be divided into three general stages: tumor initiation, promotion and progression.,1.Induce phase II drug-metabolizing enzymes, which can detoxifying carcinogens (anti-initiation).,2.Mediated anti-inflammatory effects and inhibited cyclooxygenase and hydroperoxidase functions,(anti-promotion).,3.Inhibited human promyelocytic leukemia cell proliferation (anti-progression).,CD95 Signaling-Dependent Apoptosis in Human Tumor Cells,Blood(9.7), 1998, 92:996-1002.,Analysis of DNA content following resveratrol treatment of HL60 cells indicates an increase in the subdiploid DNA content as shown by the sub-G1 population on cell cycle analysis.,DNA subdiploidy,Externalization of membrane Phosphatidylserine(PS) induced by resveratrol in HL60 cells.,Change in membrane phospholipid asymmetry,Apoptotic cells exhibit increased caspase activity detected by cleavage of caspase-specific substrate PARP that occurs at the onset of apoptosis.,Intracellular caspase activation,resveratrol cytotoxicity is significantly inhibited in the presence of either tetrapeptide inhibitor of caspase-3 (DEVD-CHO) or caspase-1 and caspase-1like proteases (YVAD-CHO), and complete rescue of the cells is obtained when both inhibitors are added together.,Cytotoxicity inhibited by caspase inhibitor,CD95L expression is significantly increased at 24 hours following drug exposure,Inhibition of cell death in the presence of either anti-CD95 or anti-CD95L,Resveratrol does not affect survival of normal human peripheral blood lymphocytes(PBLs),Cytotoxicity of resveratrol oligomers and their acetate derivatives,11,resveratrol oligomers and 6 acetylated derivatives.,7 kinds of tumor cell lines: KB, A549, CAKI-1, MCF-7, 1A9, SaOS-2, HCT-8.,(-)-ampelopsin A showed potent and selective cytotoxic activity with ED50 values of 0.6, 0.7 and 2.0 ug/ml against KB, 1A9 and MCF-7, respectively.,Hopeaphenol and pallidol hexaacetate showed cytotoxicity against KB cells with ED50 values of 1.2 and 1.6ug/mL, respectively.,Acetylation of resveratrol dimers could increase activity and selectivity. The increased lipophilicity of the acetate might play a role in transport and cellular penetration and uptake,.,Bioorganic & Medicinal Chemistry Letters(2.3), 1999, 9, 3057-3060.,Antimutagenic effect of resveratrol against Trp-P-1,YE(methanol extract of,Yucca schidigera,) showed a suppressive effect on umu gene expression of the SOS response induced by S9-activated Trp-P-1 in,Salmonella typhimurium,.,The antimutagenic activity of YE against Trp-P-1 was demonstrated by Ames assay using Salmonella typhimurium TA98.,The SOS suppression and antimutagenicity of resveratrol against Trp-P-1 was determined by umu test and Ames test.,Mutation Research(3.7) 373.1997,.,197200,Estrogenic activity,Resveratrol competed with estradiol,雌二醇,for binding to the estrogen receptor in human breast adenocarcinoma cells.,Resveratrol increased the expression of native-regulated genes and stimulated the proliferation of estrogen-dependent breast cancer cells.,Proc. Natl. Acad. Sci. 1997, 94, 14138-14143,Thioredoxin, a Singlet Oxygen Quencher and Hydroxyl Radical Scavenger,Hydroxyl radicals, Fenton reaction.,Singlet oxygen, photolysis of rose bengal,Biochemical and Biophysical Research Communications(2.9),277, 443447 (2000),singlet oxygen quenching effect,Cysteine is important for the reactive oxygen scavenging properties,Curcumin is a singlet oxygen quencher,Biochemical and Biophysical Research Communications(2.9),295 (2002) 62,66,ESR study on the structure-antioxidant activity relationship of tea catechins and their epimers,Superoxide anion assay,Singlet oxygen assay,Assay for the carbon-centered free radicals from AAPH,DPPH radical assay,Biochimica et Biophysica Acta(3.3) 1427 (1999) 13-23,The gallate group at the 3 position plays the most important role in their free radical-scavenging abilities.,Superoxide anion assay,Singlet oxygen assay,Assay for the carbon-centered free radicals from AAPH,DPPH radical assay,DPPH was dissolved in ethanol to give a 100uM solution,Antioxidant activity of caffeic acid,2-,azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging,DPPH,scavenging,total antioxidant activity by ferric thiocyanate method,total reductive capability using the potassium ferricyanide reduction method,superoxide anion radical scavenging,metal chelating activities,Toxicology(2.7) 217 (2006) 213220,Scavenging effects of tea catechins and their derivatives on DPPH,Free Radical Biology & Medicine(5.6), 1996, 21, 895-902.,Effect of galloyl group,ortho,-dihydroxyls in the B ring is important,Effects of pH and DPPH radical scavenging ability of tea catechins,Protective effect of green tea polyphenols on the SH-SY5Y cells against 6-OHDA induced apoptosis through ROSNO pathway,Assessment of cell viability,Morphological changes,Flow cytometric analysis using Annexin V and PI,Measurement of intracellular ROS,Measurement of mitochondrial membrane potential,Intracellular calcium concentration,Measurement of intracellular NO,Western blot for nNOS and iNOS,Measurement of protein-bound 3-NT,Assay for ROS generation during the autooxidation process of 6-OHDA,ESR assay for p-quinone free radical during the autooxidation process of 6-OHDA,Free Radical Biology & Medicine(5.6) 39 (2005) 682 695,(1),direct inhibition of oxidized 6-OHDA and/or scavenging of ROS; (2) inhibiting the negative effect of 6-OHDA on both PKC and ERK1/2, and EGCG can direct phosphorylative activation of PKC; (3) attenuating NF-,n,B translocation to the nucleus, and inhibiting it activation; (4) modulating the expression of cell death and cell cycle genes; and (5) modulating the intracellular NO level and inhibiting the generation of peroxynitrite,Antioxidant Effects of Resveratrol and its Analogues against the Free-Radical-Induced Peroxidation of Linoleic Acid in Micelles,Chem. Eur. J.(4.5) 2002, 8, No. 18: 4191-4198,Antioxidative reaction of 3,4-DHS by hydrogen abstraction and electron transfer,the antioxidant synergism of a-tocopherol andthe resveratrol analogues,Antioxidative and TOH-regeneration reactions of resveratrol analogues (ArOH) in micelles,