Study On Characteristic Chromatogram Of Glycosides Of Apostichopus Japonicus By HPLC/ELSD And Its Application

Sea cucumber (Holothuroidea) is a class of marine animals (Phylum Echinodermata). It is used extensively as food and medicine in China. Many substances found in sea cucumber have bioactivity character. The presence of triterpene glycosides is characteristic for most of sea cucumbers. Sea cucumber glycosides are lanostane derivatives. Antifungal, hemolyzation, hemolyzation, cell growth suppression, antitumor and immunoregulation bioactivities of these glycosides have been found.This thesis is aim to study the glycosides of sea cucumber used as food and in northern China. An HPLC-ELSD method has been established to analyze the glycosides of Apostichopus japonicus. In this thesis, the composition of mobile phase and the condition of ELSD was established and optimized. The different of glycosides from same species of sea cucumber dealing with different methods and from different districts were studied.1. Extraction of triterpene glycosides of Apostichopus japonicas. The sea cucumbers were cut into pieces and extracted with refluxing 65 % ethanol in 80℃water bath for 2h×3 times. The combined extracts were concentrated, and the extracted residue was dissolved in water. The solution was extracted by petroleum ether and n-BuOH saturated with water consecutively. The n-BuOH extracts were concentrated to semi-solid. Dissolve the extracts with 60 % MeOH, centrifugate at 12000r/min, sample the upper solution.2. An optimized analysis condition was obtained by adjusting mobile phase and the parameters of ELSD. A Waters HPLC system equipped with1525 HPLC pump, a 2996 photodiode array detector, an ELSD detector, an on-line degasser and a Waters Symmetry C18 (150mm×4.6mm, 5μm) column setting at a temperature of 30℃was used in this study. The mobile phase was composed of Solvent A (water, 0.2 % HAc, v/v) and solvent B (acetonitrile) for gradient elution to get an optimum separation. The flow rate is 1.0 ml/min. The temperature of drift tube of ELS detector is 70℃, and the pressure of carrier gas (compressed air) was set at 25 psi.3. The validation of the method has done, and the results were good. Sample all of the samples. The results show that all the chromatogram have No. 6 common peak. It is an evidence of the presence of sea cucumber glycosides of Apostichopus japonicus.4. The analysis results of five products of Apostichopus japonicas and 2 plants (genseng and mongolian milkvetch root) containing glycosides show that the specificity of the method for glycosides of Apostichopus japonicas test is good.