Study On The Preparation Of Genipin By Polyurethane Foam Immobilized Cellulase And The Anti Tumor Activity Of Genipin

Genipin is an iridoid compound,it is also a kind of low toxicity biological crosslinking agent.Geniposide can not only the preparation of biological materials,but also can protect the liver,lowering blood sugar,blood pressure and other pharmacological effects.Geniposide is genipin main raw materials for preparation,geniposide sources widely and in higher levels of eucommia bark and gardenia plants,geniposide also has many kinds of pharmacological activity,antitumor activity of which has been repeatedly reported.Genipin is the aglycone of Geniposide,Genipin has same mother nucleus structure with Geniposide.So,in this text we use the new polyurethane foam iumobilized cellulose prepare the genipin,and detect the half death rate of purified geniposide on three kinds of tumor cells in vitro.his experiment was used for anti tumor effect of genipin provides a certain basis.Polyurethane foam is a kind of material which is easy to get,the preparation method is simple,and the use effect is good.In the classification,soft polyurethane foam and half hard foam,because of its character of light quality,soft texture,Porous structure,is a kind of perfect material to supported catalyst,such as enzyme.In the natural world,cellulase is rich in resources,but the utilization rate is poor,and the waste is serious.Application of the appropriate polyurethane foam material load of cellulase,can be repeated use several times,in the reaction of gardenia blue.Meanwhile,the use efficiency of cellulase was greatly improved.1.In this article,We determined the optimum conditions for the preparation of polyurethane foam carriers through the Central Composite Design design optimization method.And the physical and chemical properties,repeated use,enzyme kinetic parameters and so on were detected.Their main results are as follows:1.Through the center point of the design optimization method to prepare and optimize the best raw material consumption is 1.62%deionized water,polyether modified silicone oil 0.35%,DBTDL 0.35%,PEG400/MDI=1.25.Under the optimum conditions of prepared with polyurethane foam carriers per unit volume of water absorbing capacity of the average value of 3.8247 g/cm3.2.Under the optimum conditions,the polyurethane foam carrier could be loaded with cellulase 27.12%.Physical and chemical properties of the test results show that cellulase was successfully loaded in polyurethane foam.Compared with the polyurethane foam,the immobilized cellulase has little change in average pore size distribution,but the pore size distribution is increased,cross sectional appearance has also changed.TG and DSC test results showed that the immobilized cellulase and its corresponding polyurethane foam carriers had no fixed melting point.2.The optimum reaction conditions Immobilized cellulose,kinetic parameter and its cycle use:Immobilized cellulase optimum reaction temperature of 45?,Optimum pH=5.5,reaction time should be greater than or equal to 75 min;free cellulase optimum reaction temperature is 45?,Optimum pH=4.5,reaction time should be greater than or equal to 65 min.Under the above conditions,the enzyme activity was 6274.78+294.91U/g.After the cyclic enzymatic reaction was carried out for 7 times.The main purpose of this paper is to detect free cellulase and immobilized cellulase enzyme kinetic parameters by Michaelis Menten equation and double reciprocal plot method.The results showed that the free enzyme Y1=0.0557X1+0.0843,R2=0.9374,Km1=0.6613,Vmax1=11.8624;immobilized cellulase Y2=0.1991X2+0.1429,R2=0.9153,Km2=1.3933,Vmax2=6.9979.3.0n the basis of the optimum conditions of Immobilized Cellulase,We detected the immobilized cellulase enzyme promoting a geniposide generating the time course of genipin reaction,kinetic parameters,and immobilized cellulase in the reaction cycle use.The result is The buffer pH=5.5 conditions,the reaction temperature is 45?,the reaction time of genipin reaction should be greater than 75 min.The kinetic parameters of enzyme immobilized cellulase in the reaction obtained by Michaelis equation and double reciprocal plot.The result is that Lineweaver-Burk fitting curve for Y=2.4660x-0.7660,R2=0.9337,the reaction of Km=3.2193t Vmax=1.3054.Immobilized cellulase in genipin reaction in repeated use,in the process of the seventh reaction,the transformation rate of geniposide was 40.6033%.Finally,on the reaction of genipin were purified purification results:through purification purity could be obtained for the 98.34%of high purity genipin.4.With the hepatocellular carcinoma cell line HepG2 and breast cancer cell MCF-7 and lung cancer cells A549 and MTT assay method of Geniposide,we do the text of genipin antitumor activity test,The results showed that the geniposide and genipin of HepG2 cells,MCF-7 cells and A549 cells were significantly inhibited and the geniposide half of the three kinds of cell death rate respectively:400 ?M,5 ?M,155.51 ?M;genipin half of the three cell death rate respectively:800 ?M,25 ?M,274.06 ?M.