Isolatntion, Purification And Activity Analysis Of Glycoprotein And Polysaccharide From Corbicula Fluminea

Corbicula fluminea, a kind of freshwater bivalve mollusk, is to assist in the treatment of some diseases such as hepatitis, measles and fever in Chinese traditional medicine. However, few studies on its components and biological functional were carried out. It was proved by our laboratory that proteins and polysaccharides were its important bioactive components. That provides a major interest to study the active components ofCorbicula Jluminea for better utilization. The main results are as follow:In this study, an active protein, named CFp-a, was isolated and purified from the fresh Corbicula fluminea with the methods of ammonium sulfate precipitation, Blue Sepharose 6 Fast Flow, SP Sephorose Fast Flow column chromatography and HPLC. With the affinity chromatography, CFp-a was isolated and concentrated. HPLC analysis proved the purity of the protein. The purified CFp-a was a monomeric protein with 24.0 kDa in relative molecular mass, estimated by SDS-PAGE. It was a glycoprotein containing 2.3% sugar by phenol-sulfuric acid reaction. Analyzed with amino acid composition, Phe had the highest content followed by Cys and Tyr, but there was a lack of Pro.The antitumor activity in vitro of CFp-a was determined by using BEL7404 human hepatoma cell line. BEL7404 cell were treated with 10,20,40,80,160 ug mL-1 CFp-a respectively for 48h and 72h.The growth inhibition rates of tumor cell were measured by MTT method. BEL7404 cell were treated with 10 ug mL-1, 20 ug mL-1 and 40ug mL-1 CFP-a for 48h. Cell apoptosis was observed by flow cytometry (FCM).The results showed that CFp-a display strong growth inhibitory effect in a dose and time dependant manner against BEL7404 cells with estimated IC50 valves of 46 ug mL-1 after 72h of treatment with CFp-a. The morphological change of apoptosis cell was also found by electronic microscopy. After cells weretreated by CFp-a for 48h, many microvilli falling off, nuclear chromatin concentrated, fragmentation and augophagosome formation in the cytoplasm were observed; the nuclei shrunk, and the chromatin condensed to form high density clumps, which located along the nuclear envelope. Cell-cycle kinetics of BEL7404 cells was analyzed by flow cytometry. An accumulation of the cells in G0-G1 phase was caused with different dose of CFp-a for 48h. A typical subdiploid peak sub G1 appeared on DNA histogram in FCM. The effect of CFp-a on the growth inhibition and the induction of apoptosis of BEL7404 cancer cells depended on the concentration. Above results revealed that the CFp-a inhibited the proliferation and induced the apoptosis of BEL7404 cells in vitro.Up to now, the polysaccharides from the shellfish had been little studied. In order to investigate the chemical structures and biological activities of polysaccharides in the shellfish, the isolation, purification, preparatory structural elucidation and pharmacological tests were performed on the polysaccharides from Corbicula fluminea. Two polysaccharides COGI and COGII were obtained from Corbicula fluminea and purified by using DEAE-cellulose-52 and SephacrylS-200.The inhibitory effect of polysaccharides on TMV was examined by half leaf method, results indicated that COGII had the active of anti-TMV. COGI,howver, had no effect on TMV. The COGII smear test was performed at different interval(0.5h- 12h).The inhibitory rate was about 81% for 0.5h and 63% for 12h.. The result demostrated that COGII probaly may compete with the invasion site of the virus, thus decreasing the affinity of TMV and its host. The mixture of TMV and CFp-a was investigated by electronic microscopy, not finding the morphological change of the virus compared with the reference group. However when the COGII contacted with the surface of TMV, the part of the virus collapsed. The result indicated that the isolated glycoprotein could interact with TMV anddestruct the structure of the virus. The system assay proved that the inhibitory effect of the glycoprotein COGII on TMV in advance 24 hours was better than that of in advance 3 hours.In addition, the scanvage...