羟基自由基和超氧阴离子自由基测定的荧光分析法研究

发布时间：2018-04-25 06:22

本文选题：荧光分析法 + 羟自由基　；参考：《山东农业大学》2009年硕士论文

【摘要】： 生命活动的代谢过程中不断产生各种活性氧自由基(ROS),羟基自由基(·OH),超氧自由基(O_2·~-)是其中具有代表性的两种自由基,它们与生物体的衰老及许多疾病的发生都有重要关系。 ·OH是一种氧化能力很强的自由基,能很容易地氧化各种有机物和无机物,氧化效率高,反应速率快,是造成组织脂质过氧化、核酸断裂、蛋白质和多糖分解的活性氧,与机体的衰老、肿瘤、辐射损伤和细胞吞噬有关。O_2·~-不仅自身具有毒性,而且可以经过一系列反应生成其它活性氧自由基,进一步对生物体产生损伤作用,且停留时间较长。人体内总自由基中约95%以上属于活性氧自由基,因此活性氧自由基的研究对人体有着特殊的意义。通过对活性氧自由基的检测研究,可以帮助我们更好的解释疾病产生的原因,加强有效的预防措施,减少机体的伤害程度。另外在衡量保健食品的抗氧化能力和中草药抗氧化物质的筛选中具有重要的指导意义和理论价值。活性氧自由基反应性强,存在时间短,不易进行直接测定。目前国内外已有较多间接测定·OH和O_2·~-的方法。本研究在全面了解这些方法的基础上,研究了利用荧光分析技术测定活性氧自由基的方法,因为此技术具有灵敏度高、选择性好、操作简便等优点。本文合成了两种新型荧光探针,同时对这两种探针进行了检测选择性的研究,探讨了部分抗氧化剂对活性氧自由基的清除作用。具体研究内容分为以下三点: (1)通过鸟苷与过氧化氢光解反应,形成荧光物质8-羟基鸟苷,分析8-羟基鸟苷在不同条件下存在的形式及不同存在形式与荧光强度的关系。探讨羟基自由基产生及其与鸟苷反应的条件,根据荧光强度与羟基自由基的量效关系,研究羟基自由基与鸟苷作用的分子机理,探讨茶叶提取液和大蒜提取液通过清除羟基自由基对鸟苷的保护作用。 (2)设计合成一种新的荧光探针试剂—2-(2′-吡啶亚胺甲基)苯酚,建立检测O_2·~-和SOD活性的荧光方法,研究测定方法的最佳条件,并用于大蒜、菠菜、木瓜中SOD活性的测定,并与邻苯三酚自氧化法测定出的SOD活性进行了比较。 (3)建立了一种以2-(2′-噻吩基)苯并噻唑啉为新型荧光探针测定O_2·~-和SOD活性的荧光分析方法,探讨了其它物质对体系的干扰影响及荧光探针的检测单一性,研究测定方法的灵敏度,精密度,本法成功应用于大蒜、洋葱等生物样品中SOD活性的测定。本论文首次对羟基自由基与鸟苷作用分子机理的荧光分析做了相关研究,并合成了新型荧光探针,用于O_2·~-和SOD活性的测定,且2-(2′-噻吩基)苯并噻唑啉对O_2·~-的检测具有专一性,得到较为满意的结果,但是也存在一些不足之处,如鸟苷与过氧化氢光解反应体系中测定?OH的含量时灵敏度还不够高,还需进一步探讨。活性氧自由基的测定方法还未能很好的应用于动物体的检测,有待进一步完善。
[Abstract]:Active oxygen free radicals (ROS), hydroxyl radical (/ OH) and superoxide radical (O_2 - -) are two free radicals in the metabolic process of life activities. They are closely related to the aging of organisms and the occurrence of many diseases.
OH is a highly oxidizing free radical, which can easily oxidize various organic and inorganic substances. The oxidation efficiency is high and the reaction rate is fast. It is the active oxygen that causes tissue lipid peroxidation, nucleic acid breakage, protein and polysaccharide decomposition. It is related to body aging, tumor, radiation injury and cell phagocytosis. Not only is it related to its own toxicity, but also related to body aging, tumor, radiation injury and cell phagocytosis. Moreover, a series of reactions can produce other reactive oxygen radicals, which can further damage the organism and stay longer.
More than 95% of the total free radicals in the human body belong to the reactive oxygen free radicals, so the study of reactive oxygen radicals is of special significance to the human body. Through the study of the detection of reactive oxygen radicals, we can help us to better explain the causes of the disease, strengthen effective prevention measures, reduce the injury degree of the body. In addition, it is also a measure of the protection of the body. The antioxidant capacity of healthy foods and the screening of antioxidants in Chinese herbal medicines are of great guiding significance and theoretical value.
Active oxygen radicals have strong reactivity, short existence time and not easy to direct determination. At present, there are many methods of indirect determination of OH and O_2. In this study, based on the comprehensive understanding of these methods, the method for the determination of reactive oxygen radicals by fluorescence analysis technology is studied, because this technique has high sensitivity and good selectivity. Two new fluorescent probes were synthesized in this paper. At the same time, the detection selectivity of these two probes was studied and the scavenging effect of some antioxidants on reactive oxygen radicals was discussed. The specific research contents were divided into three points as follows:
(1) 8- hydroxyguanosine was formed by the photodissociation of guanosine and hydrogen peroxide, and the relationship between the form of 8- hydroxy guanosine under different conditions and the relationship between different forms and fluorescence intensity was analyzed. The production of hydroxyl radicals and the conditions for the reaction with guanosine were discussed. According to the quantitative relationship between the fluorescence intensity and the hydroxyl radical, the hydroxyl group was studied. The molecular mechanism of free radical interaction with guanosine was studied. The protective effects of tea extract and garlic extract on hydroxyl radical scavenging activity of guanosine were investigated.
(2) to design and synthesize a new fluorescent probe reagent, 2- (2 '- pyridimimomethyl) phenol, to establish a fluorescence method for detecting the activity of O_2 - and SOD, to study the optimum conditions for the determination of the method and to determine the activity of SOD in garlic, spinach and papaya, and to compare the activity of SOD with the self oxidation of phthalic three.
(3) a fluorescence analysis method was established by using 2- (2 '- thiophenyl) benzothiazoline as a new fluorescent probe for the determination of O_2. - and SOD activity. The interference effects of other substances on the system and the singularity of the detection of fluorescence probe were discussed. The sensitivity and precision of the method were studied. This method was successfully applied to the biological samples of garlic and onion, such as SOD Determination of activity.
In this paper, the fluorescence analysis of the molecular mechanism of hydroxyl radical and guanosine was studied for the first time. A new fluorescent probe was synthesized and used to determine the activity of O_2 - and SOD. The detection of 2- (2 '- thiophenyl) benzothiazoline has a specificity and satisfactory results for the detection of O_2 - - but there are some shortcomings, such as birds. The sensitivity of the determination of OH in the photodissociation reaction system of glucoside and hydrogen peroxide is not high enough, and further discussion is needed. The determination method of reactive oxygen free radicals is not well applied to the detection of animal body, which needs further improvement.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341

【参考文献】