Study On Antioxidants In Herbal Medicines By Capillary Electrophoresis Combined With Chemiluminescence Detection

Reactive oxygen species (ROS) are commonly involved in many oxidative chain reactions in organisms related to the life processes. Researches on the ROS and antioxidants are recently being a focus with the development of free radical biology and preventive medicine. Synthetic antioxidants usually have various side effects, thus, there is a growing tendency to replace synthetic antioxidants by those from natural resources of low toxicity and high efficiency. But there are several deficiencies in the evaluation of antioxidants in hebral medicines by traditional methods, which didn't combine with separation techniques. Capillary electrophoresis (CE) coupled with chemiluminescence (CL) detection was proposed recently for on-line screening and evaluation of antioxidants from complex matrix, which depended on the principal of luminol-ROS reaction. However, this CE-CL method was limited to the analysis of only few standard compounds.In this thesis, the CE-CL method was used for the systemic assessment of various kinds of herbal medicines containing antioxidative components, especially aimed at obtaining some pharmacodynamic and pharmacokinetic parameters of antioxidants in herbal medicines for pharmaceutical exploitation and clinical application. The main research work and obtained results are as follow:1. A CE coupled with CL detection system was designed and established based on a CE-CL interface. The applicability of developed CE-CL system for antioxidants assessment was examined by employing the luminol oxidation assay. Two types of ROS system including singlet oxygen (¹O₂) and hydroxyl radical (·OH) were respectively investigated to achieve constant CL condition. Under optimum CL conditions, the antioxidative activity of matrine and oxymatrine against ¹O₂ and·OH were evaluated, the IC50 was introduced to determine the ROS scavenging capacity of compounds. Furthermore, fluorospectrophotometry as an auxiliary measurement was applied to examine the reliability of results obtained by CE-CL system.2. The proposed CE-CL system was used to screen the antioxidants in five representative herbal medicines with alkaloids as major components, including Herba Ephedrae, Radix Sophorae Flavescentis, Rhizoma Coptidis, Cortex Phellodendri Chinensis, and Bulbus Fritillariae Cirrhosae, under the optimum conditions by employing the ¹O₂ system. The distribution and activity differences of antioxidants in herbal medicines were revealed by the calculation of relative ROS scavenging ratios. Furthermore, plasma protein binding effects of extracts from herbal medicines was studied by employing equilibrium dialysis.3. The CE-CL method was used to establish novel antioxidative biological fingerprints. The repeatability and reproducibility of Radix Astragalus from 8 different regions were investigated, and the similarities in scavenging·OH of samples were calculated. Based on the curvilinear equation between concentrations and scavenging ratios for·OH of astragaloside, the content of astragaloside in Radix Astragalus was determined. Furthermore, the antioxidants in Radix Astragalus injection were examined for quality control in pharmaceutical engineering.4. Microemulsion was introduced to realize the combination of microemulsion electrokinetic chromatography (MEEKC) with CL detection, as to improve the on-line assessment of antioxidants, especially those of lipid-solubility. Four types of microemulsion constructed with different surfactants including SDS, Tween-80, Tx-100, and Np-40 were separately investigated on their application in CE-CL. The extracts from Radix et Rhizoma Salviae Miltiorrhizae were analyed under optimum conditions, and the log Pow values of some antioxidants in Radix et Rhizoma Salviae Miltiorrhizae were caculated.The methodology may be further developed by improving CE-CL interface and employing some enzyme reactions to genarate ROS for the evaluation of more antioxidative herbal medicines.