Estimation of free radical formation and deletion of life relation substances

生命相关物质大量参与自由基的产生和清除，与癌症、糖尿病、脑疾病等的关系被指出并从疾病的面对和预防方面进行研究。为了分析超氧阴离子活性氧的作用机制，需要对单个活性氧进行准确的测定。活性氧是多自由基参与的反应体系中的一种。本研究尝试了用高效液相色谱法对自由基进行同时测定，以分离各自由基，从而建立准确的分析方法。此外，高灵敏度分析成为可能，通过使用电化学检测器与HPLC。建立了DMPO与3种自由基的加合物的HPLC/ESR/ECD分析体系，并建立了过氧化氢和DMSO化学生成超氧阴离子、羟基自由基和甲基自由基的方法。同样，由于我们使用PBN作为自旋捕获剂，对于3种在ESR谱中难以区分的PBN加合物，通过所开发的系统实现了对3种PBN加合物的精细基线分离。实验结果表明，抗坏血酸、dl-α-生育酚、β-胡萝卜素作为抗氧化剂，对自由基的清除作用具有剂量依赖性，并结合各抗氧化剂的特点，分析了其清除自由基的作用。此外，该自由基产生系统甚至对于化学药品的自由基缺失活性测量也是有用的，因为即使是水溶性和疏水性的化学品也能够熔化。

The life relation substances are getting involved largely, in the generation and deletion of free radicals that the relations with cancer, diabetes, brain disease etc. which are pointed out and research is going on from the face of the disease and prevention. An individual activity oxygen speceise need to be measured well accuracy, to analyze the action system of the activity oxygens of superoxide. There are many case of activity oxygen speceis that plural free radical contributes to the reaction system that gets involved. We tried the simultaneous determination of free radicals to the separation of each frer radical by using high performance liquid chromatography (HPLC), in order to develop the accrurate analytical methods in this research. Furthermore, high sensitivity analysis became possible by using the electrochemical detector with HPLC. Also, the simultaneous an alysis of DMPO adducts of 3 kinds of free radical became possible by HPLC/ESR/ECD system that we constucted, beside the chemical generation procedure of super oxide anion, hydroxyl radical, and methyl radical by hidrogen peroxide and DMSO are established. Similarly, as a result that we used PBN as the spin trapping agents as for 3 kinds of PBN adducts of which the distinction is difficult in the ESR spectrum, the fine base line separation was achieved regarding 3 kinds of PBN addcuts by the developed system. As a result that we examied the scavenger effect of free radical by the system using the ascorbic acid, dl-α-tocopherol, β-carotin, as the antioxidant the dosage reliance nature is admitted and analyzed the scavenger reaction with the characteristic of each antioxidant. Also, this free radical generation system is conceivable as usefulness even for the free radical deletion activity measurement of the chenmicals, because even the chemical of the water-solubility and also hydrophobic property are able to melt.