Purification Of SOD From Spinach And The Determination Of Salidroside From Rhodiola Eoccinea With HPLC

Based on much more high performance, high sensitivity and high speed, high performance liquid chromatography(HPLC)has already become a powerful tool for separation and purification of natural and active components from plant, animal, and so on. Especially, the separartion and analysis of herbal medicines, proteins, enzymes from complex samples is changing towards more quickly, environmental protection and lower cost. Therefore, in this thesis, the salidroside from rhodiola eoccinea was determined with HPLC, and superoxidedismutase from spinach was also separated and purified with HPLC.The dissertation consists of four sections:1. Literature review:The mechanism and applicaion of HPLC, such as reversed-phase liquid chromatography (RPLC), ion exchange chromatograpgy (IEC), hydrophobic interaction chromatography(HIC), affinity chromatography(AFC)and size exclusion chromatoraphy(SEC) were introduced, respectively. The advance and application of on-line or off-line two dimensional liquid chromatography on the separation and analysis of complex samples were also reviewed.2. Isolation and determination of salidroside from rhodiola eoccinea by HP-RPLC: The isolation and determination of salidroside from rhodiola eoccinea with RPLC was studied in this part. The chromatographic conditions, standard curve, repeatability, stability, standard addition test and content determination were investigated in detail. When using water and methanol to extract salidroside from rhodiola eoccinea, the extaction efficiency of salidroside were 9.25 mg/g and 9.27 mg/g. It indicates that water can be used as the solvent instead of methanol so that the extraction method become green and environmental protection.3. Coarse separation of SOD from spinach:Firstly, The extraction conditions of SOD from spinach, such as the solid to liquid ratio (m:v, RMV), pH, temperature and the volume of acetone as extraction solvent was optimized, and the activity determination method was also screened. The coarse separation of SOD from spinach was studied in detail. As a result, the activity ratio of SOD can reach to be 72.38 U/mg, and purified multiple to be 4.2 folds under the optimal experimental conditions. With the identificaiton of SOD from spinach by isozymes electrophoresis, the result shows that SOD from spinach is Cu,Zn-SOD, which molecular weight is 31.8 kDa with two subunits, and 19.8 kDa for its subunit detemined with MALDI-TOF MS and SDS-PAGE, respectively. 4. Separation and purification coarse superoxidedismutase from spinach by HPLC: Separation and purification coarse SOD from spinach by HIC, SEC and HIC-SEC, respectively. The activity of purified SOD was determined to be 119.82 U/mg by HIC,159.92 U/mg by IEC, and 284.65 U/mg by HIC-SEC, respectively. To compare the pury and activity of SOD, the result indicated that the two-dimensional chromatography(HIC-SEC)is the best choice to separate and purify SOD from spinach.