Study On On-line Evaluation Method For Antioxidant By Capillary Electrophoresis With Chemiluminescence Detection

The researches of radical and antioxidant are recently regarded with the development of free radical biology and medicine. There are several deficiencies in the traditional evaluation methods of antioxidant activity, which didn't combined with separation methods. The antioxidant activities of polycomponent (mixture) can not be detected and evaluated at one time; and the antioxidant constituents also can not be screened from the complex matrix. In addition, the separation and preparation of constituents from the complex matrix were tedious and time consuming, may cause activity loss and contamination, so it is great important to develop on-line evaluation methods for antioxidant in complex matrix.In this thesis, a novel method of on-line evaluation of antioxidants by capillary electrophoresis (CE) with chemiluminescence (CL) detection was reported. Based on the principle of that a strong and stable background CL signal is generated by luminol-radical reaction, but the radical could be eliminated by the antioxidants, which quenching luminol-radical CL intensity, therefore results in a quantitative decrease in the CL signal, the scavenge ratio for radical and the antioxidative ability of constituents separated by CE were obtained. This method can be applied to screening the antioxidant components in complex matrix.The main research and obtained results are as below:1. A novel method for evaluation antioxidants scavenging hydroxyl radical was established. The hydroxyl radical was produced by Cu2+-H2O2 reaction system, detected by luminol. The production and detection of hydroxyl radical and the evaluation of antioxidant were coupled with CL detection. Optimal CL conditions were obtained, and the antioxidant activities of thiourea and ephedrine were evaluated. The parameter IC50 was introduced to determine the antioxidant of compound. The result was consistent with that of fluorospectrophotometry.2. The method was applied to evaluating antioxidants scavenging singlet oxygen. The singlet oxygen was produced by ClO--H2O2 reaction system, and detected by CL of luminol. The conditions which influenced CL were studied. The antioxidant activities of rutin and quercetin which were flavones were evaluated, and the structure- antioxidant activity relationship was preliminarily discussed.3. The CE-CL instrument was set up and improved. A novel CE system which incorporating an ultra-violet absorption (UV) detector and a CL detector was built in the laboratory. By employing this instrument, the on-line evaluation method was applied to screening and evaluating antioxidants in the extract of Sophora japonica L. The constituents of sample were separated in the optimal CL and CE conditions. The scavenge ratio of singlet oxygen for every constituent were calculated and the antioxidant activities were evaluated.4. The method of evaluation of antioxidants was introduced to the research of traditional chinese medicine–fingerprint (TCM-FP). The antioxidantive biofingerprint of Astraglus membranaceus (Fisch) Bge was studied. The reproducibility, stability of sample were studied, and the similarities of Astragalus from different regions were calculated and the astragaloside was determined.The technology may be further improved and be used to detect and analyze the endogenous antioxidant species which contribute to the studies in life science.