Study On The Preliminary Purification And Bioactivity Of Chlorella Growth Factor (CGF) Active Substance

This study mainly focuses on three parts: the extraction method of chlorellagrowth factor, the research of CGF activity and the preliminary separation andpurification of CGF.Firstly, the compare of the hot water extraction method, ultrasonic method,enzymatic method, shiqun Han method by immune and antioxidant activity, the resultshow that the yield of hot water extraction method lower than enzymatic method, butit can be showed better effect on promoting Ana-1, RAW264.7cells. It can promotethe Ana-1phagocytic, rate was210.5%.It also significantly cleared OH-, rate was80%. Ultimately consider the best extraction method for the hot water extract.Secondly, CGF was cultured with Ana-1for24hours, at the concentration of100μg/mL, not only can significantly promote phagocytosis of neutral red, rate was213.5%, also can inhibited NO for LPS-induced cells to release. Mice peritonealmacrophages Ana-1proliferated52.3%when cultured with CGF (75μg/mL) for24hours, HEK-293cells can proliferated310%when cuLtured with CGF (200μg/mL)for48hours. The CGF treatment inhibited Hela and HepG2cell growth intime-dependent manner. When the concentration of400μg/mL, CGF was cuLturedwith Hela, HepG2for72hours to24hours, cells proliferative activity decreased by50%,66.9%. CGF can also promote E. coli, Lactobacillus cell growth in adoes-dependent manner.Thirdly, CGF was separated by acetone and ethanol, get ethanol precipitationethanol supernatant, acetone precipitation, acetone supernatant. The Ana-1activitywith precipitation was better than supermatant, the effects for ethanol precipitationwas higher than acetone precipitation, the best proliferation was59.2%. CGF wasfurther separated into crude protein, crude polysaccharide, which the better effects onpromoting Ana-1cell growth for crude protein than the other components,proliferative rate was55.7%. The Ana-1cells activity was significantly reduced, whenCGF and crude protein by Trypsin digestion. It can consider that protein is the mainactive substances of CGF. Finally, the crude protein was found to promote the growth of RAW264.7,HEK-293cells significantly, proliferation activity was66.9%,401.6%. Highconcentrations of crude protein will cause Hela and HepG2cells shrinkage, off thewall, cracking, mortality. It can also cause DNA damage of Hela cells as measured byComet assay, the extent of damage in a dose-and time-dependent manner.