The Optimization Of The Extraction Process Of Kelp Polysaccharide And The Study Of Its Interaction With Lipid Membrane

Laminaria japonica polysaccharides(LJP)are important components which have complex physiological functions such as anti-atherosclerosis,immunomodulatory,antithrombotic activity,resisting oxidation and so on.The extraction of Laminaria japonica polysaccharides is the basis and prerequisite for the study of its structure and activity.Present studies show that hot water extraction is time-consuming,enzymatic hydrolysis is high-cost the acid extraction is easy to destroy the structure and the polysaccharides extracted by alkaline extraction are not dissolved in hot water,which indicates that there is not a method with high extraction efficiency and the extraction process needs to be improved.Meanwhile,it is not clear how does LJP work on cells owing to its bioactivity.Therefore,the study on extraction process and interaction with biological molecules of Laminaria japonica polysaccharide is of great significance in its clinical application.In this paper,Laminaria japonica polysaccharides were extracted from Laminaria japonica by ultrasonic assisted extraction.The influence of liquid-solid ratio(mL/g),extraction time(min),ultrasonic power(W)and temperature(?)on the extraction yield was investigated by single factor experiments,then designed the response surface experiment and obtained the best extraction conditions.Moreover,we investigated the effect of LJP on cationic 1,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP)and anionic 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-1-glycerol)](DPPG)monolayers at the air-water interface by the pressure-area isotherms(?-A),adsorption curves(?-t)and morphology measurements with atomic force microscopy(AFM)technique.The specific experimental results are as follows:(1)First,we optimized the ultrasonic assisted extraction of polysaccharides from Laminaria japonica.The optimum conditions obtained from the experiments of single factor were the liquid-solid ratio of 50mL/g,the extraction time of 35min,the ultrasonic power of 500W and the temperature of 70?.According to the results of single factor experiments,the factor of temperature with the least influence on yield was eliminated,and three factors including liquid-solid ratio,ultrasonic power and extraction time were selected to design response surface experiments.We analyzed the results and obtained the two regression equation of polysaccharides yield.The optimum conditions were the liquid-solid ratio of 57.35mL/g,ultrasonic power of 563.37W and ultrasonic time of 35.35min,by which the yield of polysaccharides was 7.60%.(2)Next,We studied the interaction between Laminaria japonica polysaccharide and monolayers according to the ?-A curve,the molecular limit area and the compression modulus of DOTAP and DPPG monolayers,we obtained the conclusion that as the polysaccharides added into the subphase,the curves of DOTAP and DPPG shifted to larger mean molecular area,which implied that the permeation of LJP into lipids expanded the monolayers.There was also electrostatic interaction between lipid molecules and polysaccharides.The polysaccharides molecules could stabilize the heads with the positive charge due to electrostatic attraction and resulted in that the collapse pressures of monolayers with LJP had no significant decrease compared with pure lipid.The electrostatic repulsion between polysaccharides molecules and DPPG lipid molecules led to a significant decrease of the collapse pressures when LJP permeated into the monolayers.In addition,compression modulus showed the effect of LJP decreased the elasticity of monolayers for both DOTAP and DPPG.The result of adsorption curves showed the increase of surface pressure for both DOTAP and DPPG at the surface pressure of lOmN/m,meanwhile,the change of DOTAP was bigger than DPPG,which indicated the interaction between DOTAP and LJP was greater.Nevertheless,DOTAP and DPPG monolayers exhibited different behavior at 20mN/m.Compared with the curves at lOmN/m,the value of surface pressures at 20mN/m increased faster and reached a steady state quickly for DOTAP while DPPG monolayers with LJP showed slight decrease.The different phenomena might result from the following:The structure of DPPG became denser at higher pressure,so when the polysaccharide molecules permeated into the film,the electrostatic repulsion made some of the lipid molecules squeeze out,which led to the loss in surface pressure.In addition,the kinetic equation of the adsorption curve was a good explanation of this behavior.The results of AFM showed that the adsorption of LJP and monolayer films increased with the increase of LJP concentration at pressure of 10mN/m,which was consistent with the previous conclusions.