两种天然抗氧化剂对铁离子诱导的羟自由基形成以及细胞毒性的影响
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摘要
天然抗氧化物植酸和秦皮乙素用途广泛。本文以3-羧酸香豆素作为荧光探针研究了植酸、一磷酸腺苷(AMP)、三磷酸腺苷(ATP)、乙烯胺的乙酸衍生物EDTA、NTA和DTPA以及羟自由基清除剂如:二甲基亚砜(DMSO),甲酸(HCOOH)和乙醇(CH_3CH_2OH)对Fenton体系产生羟自由基的影响,同时选取HepG2细胞作为模型,探讨了秦皮乙素对铁参与的生物反应体系的影响,8HQ-Fe与细胞作用30分钟后加入秦皮乙素。
研究结果表明:植酸可促使Fenton体系产生羟自由基(·OH);AMP和ATP对Fenton体系具有同样的促进作用;乙烯胺的乙酸衍生物EDTA、NTA和DTPA也能够促进·OH的产生;DMSO,HCOOH和CH_3CH_2OH可清除植酸-Fenton体系产生的·OH;同样,植酸可促使VO(Ⅱ)-H_2O_2反应(类Fenton体系)产生·OH;0.1 mM的8-羟基喹啉-铁(1:1)复合物对细胞有较大毒性;秦皮乙素本身对细胞不产生毒性,可保护细胞免受铁离子诱导的毒性,二者呈现一定的剂量-效应关系;秦皮乙素类似物4-甲基七叶苷原和秦皮素也具有相似的剂量-效应关系。
其机理与铁的络合作用有关。该结果为进一步认识天然抗氧化物植酸和秦皮乙素提供参考。
Phytic acid and esculetin has been widely used as antioxidant. In this study, coumarin-3-caroxylic acid was used as fluorescent probe to detect hydroxyl radical; meanwhile, the effect of esculetin on 8HQ-Fe-induced cell viability was investigated in HepG2 cells. Esculetin was added to the cells 30 min before the addition of 8HQ-Fe.
The results indicated that: Phytic acid could markedly enhance·OH produced by ferrous iron and hydrogen peroxide; similar enhancing effects were also observed for AMP and ATP;·OH formation could be enhanced by aminoacetic chelating agents such as ethyleneamine ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-penta acetic acid (DTPA) and nitrilotriacetic acid (NTA);·OH produced by Fe/ H_2O_2/phytic acid were markedly inhibited by·OH scanvenging agents such as dimethyl sulfoxide(DMSO), ethanol (CH_3CH_2OH) and formate(HCOOH); similar enhancing effect by phytic acid was observed in the reaction of vanadyl and H_2O_2 (Fenton-like reaction). Esculetin exhibited a dose dependent protection against iron-induced cell toxicity; similar dose-dependent protection was also observed with two other esculetin derivatives such as 6,7-dihydroxy-4-methylcoumarin and 7,8-dihydroxy-6-methoxycoumarin; in the absence of non-toxic esculetin but in the presence of concentrations of 8-hydroxyquinoline-Fe(II) complex (100μM 8-hydroxyquinoline, 100μM ferrous), cell toxicity was observed.
The results are quite related to the mechanism of iron chelation, which help us know more about the property of these two antioxidants
研究结果表明:植酸可促使Fenton体系产生羟自由基(·OH);AMP和ATP对Fenton体系具有同样的促进作用;乙烯胺的乙酸衍生物EDTA、NTA和DTPA也能够促进·OH的产生;DMSO,HCOOH和CH_3CH_2OH可清除植酸-Fenton体系产生的·OH;同样,植酸可促使VO(Ⅱ)-H_2O_2反应(类Fenton体系)产生·OH;0.1 mM的8-羟基喹啉-铁(1:1)复合物对细胞有较大毒性;秦皮乙素本身对细胞不产生毒性,可保护细胞免受铁离子诱导的毒性,二者呈现一定的剂量-效应关系;秦皮乙素类似物4-甲基七叶苷原和秦皮素也具有相似的剂量-效应关系。
其机理与铁的络合作用有关。该结果为进一步认识天然抗氧化物植酸和秦皮乙素提供参考。
Phytic acid and esculetin has been widely used as antioxidant. In this study, coumarin-3-caroxylic acid was used as fluorescent probe to detect hydroxyl radical; meanwhile, the effect of esculetin on 8HQ-Fe-induced cell viability was investigated in HepG2 cells. Esculetin was added to the cells 30 min before the addition of 8HQ-Fe.
The results indicated that: Phytic acid could markedly enhance·OH produced by ferrous iron and hydrogen peroxide; similar enhancing effects were also observed for AMP and ATP;·OH formation could be enhanced by aminoacetic chelating agents such as ethyleneamine ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-penta acetic acid (DTPA) and nitrilotriacetic acid (NTA);·OH produced by Fe/ H_2O_2/phytic acid were markedly inhibited by·OH scanvenging agents such as dimethyl sulfoxide(DMSO), ethanol (CH_3CH_2OH) and formate(HCOOH); similar enhancing effect by phytic acid was observed in the reaction of vanadyl and H_2O_2 (Fenton-like reaction). Esculetin exhibited a dose dependent protection against iron-induced cell toxicity; similar dose-dependent protection was also observed with two other esculetin derivatives such as 6,7-dihydroxy-4-methylcoumarin and 7,8-dihydroxy-6-methoxycoumarin; in the absence of non-toxic esculetin but in the presence of concentrations of 8-hydroxyquinoline-Fe(II) complex (100μM 8-hydroxyquinoline, 100μM ferrous), cell toxicity was observed.
The results are quite related to the mechanism of iron chelation, which help us know more about the property of these two antioxidants
引文
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2.陈红霞.植酸的生物学特性与应用.生物学通报,2006,41(2):14-16
3.陈克然,杨毅军,曹道俊.氧自由基与临床.北京:中国医药出版社,2008
4.从建波,孙存普.自旋捕集短寿命自由基的低温保存.生物化学与生物物理进展,1993,20(4):326-327
5.丛玉隆.流式细胞仪及其临床应用.医学检验与临床,2006,17(4):1-2
6.崔乃杰,崔乃强,傅强.维生素C抗氧化促氧化双向作用的研究.中国中西医结合外科杂志,2004,10(6):419
7.丁克群.SOD应用研究集.北京:原子能出版社,1991
8.方允中,李文杰.自由基与酶--基础理论及其在生物学和医学中的应用.北京:科学出版社,1989
9.方光荣,刘丽明,李玲,刘丽虹,宋功武.羟自由基的流动注射荧光测定.分析科学学报,2004,20(1):63-65
10.黄进,杨国宇,李宏基.抗氧化剂作用机制研究进展.自然杂志,2003,26(2):76
11.霍惠敏,李亚洁.氧自由基对脑损伤的作用和维生素的防护作用.南方护理学报,2001,8(1):18
12.刘向前,刘能保,张敏海.维生素C和维生素E对氢化可的松所致大鼠学习记忆功能损伤的保护作用.华中科技大学学报(医学版),2002,31:238
13.刘立明,宋功武,刘丽虹,方光荣.流动注射荧光法测定羟自由基.分析化学研究简报,2003,6:723-725
14.穆光照.自由基反应.北京:高等教育出版社,1985
15.庞战军,周玫,陈瑗.自由基医学研究方法.北京:人民卫生出版社,2000
16.任凤莲,吴南,吴心传.茜素紫光度法检测H_2O_2/Co~(2+)产生的羟自由基.中南工业大学学报,2003,3l(6):514-517
17.宋军,丁映.植酸的提取方法及其应用.贵州农业科学,1995,4:36
18.王仕良,张曾,黄干强.羟基自由基的产生与测定.造纸科学与技术,2003,22(6):45
19.徐向荣,王文华,李华斌.化学发光法测定Fenton反应中的羟自由基及其应用.环境科学,1983,19(2):51-54
20.杨小峰.生物活性氧的荧光表征及新型螺环型荧光探针的研究.[博士学位论文].厦门:厦门大学图书馆,2002
21.袁倬斌,邹洪.电化学方法测定羟自由基反应速度常数.中国科学技术大学学报,2002,32(3):288-292
22.曾令武.流式细胞术.微生物与感染,2008,3(2):125-126
23.张建中.自旋标记ESR波谱的基本理论和应用.北京:科学出版社,1987
24.张健,秦静芬,曹恩华.DNA损伤的化学发光法测定和茶多酚对它的保护作用.生物物理学报,1996,12(4):691-695
25.张江峰,任凤莲,王磊.水杨基荧光酮荧光法测定羟自由基的探讨.湖南有色金属,2001,17(3):32-34
26.张乃东,郑威,郑永臻.褪色光度法测定芬顿体系中产生的羟自由基.分析化学,2003,31(6):723-725
27.赵士豪,路新利,郭会灿.维生素E保健作用的研究进展.山西食品工业,2005,1:32
28.赵扬帆,郑宝东.植物多酚类物质及其功能学研究进展.福建轻纺,2006,11:106-110
29.Barry H,John G.Free radicals in biology and medicine(Third edition).Oxford science publications.1999
30.Bay B H,Sit K H,Paramanantham R,Chan Y G.Hydroxyl free radicals generated by vanady[Ⅳ]induce cell blebbing in mitotic human Chang liver cells.Bio Metals.,1997,10:119-122
31.Bendich A.The antioxidant role of vitamine C.Adv.Free Radic.Biol.Med.,1986,16(2):419-423
32.Biaglow J E,Kachur A V.The generation of hydroxyl radicals in the reaction of molecular oxygen with polyphosphate complexes of ferrous ion.Radiat Res.,1997,148(2):181-187
33.Blackburn A C,Doe W F,Buffinton G D.Salicylate hydroxylation as an indicator of hydroxyl radical generation in dextran sulfate-induced colitis.Free Radic Biol Med.,1998,25(3):305-313
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