果蔬籽中还原型谷胱甘肽（GSH）和蛋白巯基（P-SH）含量的测定

发布时间：2018-05-17 10:45

本文选题：果蔬籽 + 巯基化合物　；参考：《延边大学》2017年硕士论文

【摘要】：生物体内含有多种抗氧化剂,其中含半胱氨酸残基的巯基化合物在维持生物体内氧化还原氛围中起到了不可替代的作用。据文献报道;生物体内的巯基化合物含量水平于与众多疾病紧密相连,其含量高低也成为了许多病理条件的一个判断标志。由于生活节奏的日益加速,大多数人时常处在高压力的状态,这也刺激人体内活性氧自由基的过量增加,从而加速机体的衰老,与此同时过量的活性氧自由基也易于诱发基因突变导致癌症等恶性疾病的产生。另据研究表明;人体通过摄入外源性抗氧化剂,像还原型谷胱甘肽(GSH),N-乙酰基-半胱氨酸等含巯基化合物,可明显提高人体血液内的GSH含量水平,这对提高机体抗病能力、保健均具有重要的作用[1]。因此,开发一种准确、灵敏、经济又快速的测定含半胱氨酸残基的巯基化合物含量的分析方法,并以此来筛选富含巯基化合物的各类食材作为外源性抗氧化补充剂,这对防病、治病等方面具有实际意义。本研究利用对巯基具有选择性的、并与其定量衍生化的衍生化试剂5,5'-二硫双-2-硝基苯甲酸(DTNB),用含过量衍生化试剂的提取液,采用细胞破壁、衍生化为一体的前处理方法,分别用HPLC法和光谱法实时测定了果蔬籽中GSH和P-SH含量,并从中筛选出了四种富含GSH和蛋白巯基的果蔬籽。HPLC测定结果发现,柚子籽、南瓜籽、黄瓜子、冬瓜子中含较丰富的GSH和蛋白巯基(P-SH),其中柚子籽中GSH和P-SH巯基含量分别为1.7304 μmol/g(GSH),1.2028μmol/g(P-SH),南瓜籽含量分别为 1.0445 μmol/g(GSH)和 1.8878 μmol/g(P-SH)。黄瓜籽和冬瓜籽中未检测到GSH等小分子巯基化合物,但含相对丰富的蛋白巯基(P-SH),其含量分别为4.0325 μmol/g和4.1623 μmol/g。对所选定的果蔬样品也分别采用文献已报道的荧光分光光度法和紫外-可见分光光度法进行了比较测定,发现荧光光度法测定GSH含量和紫外-可见分光光度法测定结果略微偏离色谱测定结果,这可能是由于样品中的共存组分对GSH的测定有一定的光谱干扰。本研究中采用的HPLC测定方法的回收率在99.92%-101.32%之间,GSH和P-SH测定检测限分别为0.1384 μmol/g和0.2184μmol/g,基本满足准确测定的要求。由于生物样品成分复杂,在果蔬籽中含有大量的不饱和脂肪酸、黄酮类物质、维生素和矿物质等。为了探讨果蔬籽中其他化学成分对实验测定结果的影响,在本研究中将柚子籽和南瓜籽作为样品,采用高效液相色谱法和光谱法分别对黄酮类化合物和不饱和脂肪酸对色谱和光谱测定干扰进行了探究。结果表明;采用光谱法测定样品中巯基化合物含量时受到黄酮类物质的干扰较大,但采用高效液相色谱法测定其含量时并无显著影响;于此同时,样品中的不饱和脂肪酸类物质对样品的测定结果基本不构成干扰或干扰相对较小。综上所述,本研究为进一步从植物中筛选富含巯基化合物的准确分析方法、以及特定果蔬籽中的活性巯基化合物的含量提供了重要的分析数据。
[Abstract]:There are many antioxidants in the organism, and the sulfhydryl compounds containing cysteine residues play an irreplaceable role in maintaining the redox atmosphere in the organism. It is reported in the literature that the levels of sulfhydryl compounds in organisms are closely linked to many diseases, and the content of sulfhydryl compounds has also become one of the many pathological conditions. Judging sign. Because of the increasing pace of life rhythm, most people are often in high stress state, which also stimulates the excessive increase of reactive oxygen radicals in the body, and thus accelerates the aging of the body, while excessive reactive oxygen free radicals can also induce gene mutation to cause cancer and other malignant diseases. Through the intake of exogenous antioxidants, such as GSH, N- acetyl cysteine and other sulfhydryl compounds, the content of GSH in human blood can be significantly increased, which has an important role in improving the body's ability to resist disease and health care. Therefore, a accurate, sensitive, economical and rapid determination of cysteine containing cysteine is made. The analytical method of the content of sulfhydryl compounds in the residues is used to screen all kinds of edible materials rich in sulfhydryl compounds as a exogenous antioxidant supplement, which is of practical significance for the prevention and treatment of the diseases. This study uses the sulfhydryl group, which is selective, and its derivatization reagent 5,5'- two sulfur double -2- Nitrobenzoic Acid. DTNB), the content of GSH and P-SH in fruit and vegetable seeds was measured in real time by HPLC method and spectral method, and the results of four kinds of fruit and vegetable seeds rich in GSH and protein sulfhydryl were detected by HPLC method and spectral method. Rich GSH and protein sulfhydryl (P-SH) were contained in the seeds, in which the content of GSH and P-SH mercapto in grapefruit seed was 1.7304 mu mol/g (GSH), 1.2028 mol/g (P-SH), and the pumpkin seed content was 1.0445 micron (GSH) and 1.8878 mu mol/g (P-SH), and the small molecule mercapto compounds were not detected in cucumber seed and winter melon seed, but the relative rich protein was contained. The content of sulfhydryl group (P-SH) is 4.0325 mu mol/g and 4.1623 mol/g. respectively, and the selected samples of fruits and vegetables are also compared by the reported fluorescence spectrophotometry and UV visible spectrophotometric method. It is found that the determination of GSH content by fluorometric method and the UV visible spectrophotometric method are slightly deviated from the chromatographic test. This may be due to the spectral interference of the coexisting components in the samples for the determination of GSH. The recovery rate of the HPLC method used in this study is between 99.92%-101.32% and GSH and P-SH, respectively. The detection limits are 0.1384 mu mol/g and 0.2184 mu mol/g, respectively. Fruit and vegetable seeds contain a large number of unsaturated fatty acids, flavonoids, vitamins and minerals. In order to explore the effects of other chemical components in fruit and vegetable seeds on the experimental results, pomelo seeds and pumpkin seeds were used as samples in this study. The flavonoids and unsaturated fatty acids were used respectively by high performance liquid chromatography and light spectrum method. The results show that the determination of the content of sulfhydryl compounds in the samples by spectral method is greatly disturbed by the flavonoids, but there is no significant influence on the determination of the content by high performance liquid chromatography. At the same time, the determination results of the unsaturated fatty acids in the samples are basic. No interference or interference is relatively small. In summary, this study provides important analytical data for the accurate analysis of sulfhydryl compounds rich in plants, as well as the content of active sulfhydryl compounds in specific fruit and vegetable seeds.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS209;O657

【参考文献】