Studying On Isolation,Purification And Activity Of Cerebrosides Hydrolysate From Asteroidea Dissertation Submitted To Dalian Ocean University

In this study, visceral organs of Asteroidea were used as raw material, immersed in organic solvent(petroleum ether and ethanol) so that cerebrosides could be extracted. Crude hydrolysate of cerebrosides were produced after a series of steps including saponification and hydrolysis. Pure products were obtained by purification methods such as column chromatography(silica gel, Sephadex LH-20 as packing loaded stock), prep-TLC, prep-HPLC, etc. Structural identification and related activities were detective on purified hydrolysate of cerebrosides. The main results were as follows:1. Crude cerebrosides were extracted by petroleum ether and 95% ethanol in room temperature from powder of visceral organs in Asteroidea, hydrolysate of cerebrosides were obtained through steps including saponification, hydrolysis, purification, etc.2. FTIR, MSD were devoted to analysis structure of cerebrosides along with its hydrolysate. The structure of cerebrosides were as follows: C-H, C=O,C=C, C-O,-CH2- could be found in cerebrosides, and the possible molecular weight were 790.6439, 804.6601 and 818.6752, molecular formula were C44H87O10 N, C45H89O10 N and C46H91O10 N, accordingly. In the hydrolysate of cerebrosides, F1 sample contained C-H, C=O, C=C, C-O, and CH2. It was similar except the N-H structure included in sample F2. By high resolution mass spectrometry analysis, the possible relative molecular mass of sample F1 was 384, corresponding formula C22H40O5, while sample F2 was 412, corresponding formula C21H36O6N2.3. In antioxidant activity experiment, hydrolysate of cerebrosides showed a higher reactivity than cerebrosides in aspect of the remove DPPH radicals and hydroxyl radicals, same results were concluded in reducing activity experiment.4. Bacteriostatic experiments were conducted to make a comparison between cerebrosides and its hydrolysate. Conclusion was made that cerebrosides and its hydrolysate both had the ability of antibacterial, and hydrolysate contained better activity than cerebrosides. The antibacterial activity of these two samples had a selection on five kinds of strain. Cerebrosides were the most effective on clostridium perfringens while hydrolysate on staphylococcus aureus.5. Cerebrosides and its hydrolysate were also proceeded on ACE inhibitory activity experiment. It turned out that both samples showed low activity on a high level of concentration. Therefore the samples didn`t have the lower blood activity.6. Result from in vitro Anti-tumor experiment showed that cerebrosides and its hydrolysate showed cytotoxicity. In MTT experiment, tumor cells including MCF-7 cell, Hela cell, Hep G-2 cell and SW480 cell could be inhibited by cerebrosides in concentration range 10~200μg/ml with time and dose dependent, in which hydrolysate went better activity.7. In cell cycle and Annexin V/PI staining experiment, cell cycle of MCF-7 cell was blocked in G2 phase under the treatment of cerebrosides, while hydrolysate made MCF-7 cell block in S phase and directly caused cell death. In the mean time, late apoptosis and cell necrosis of MCF-7 cell were inducted by higher concentration of hydrolysate in Annexin V/PI staining experiment.