Separation,Structure Identification Of Paramylon And Study On Its Bioactivities

Paramylon is a kind of functional food certified by FDA.It is found that paramylon has the functions of anti-oxidation,anti-radiation,reducing blood lipid,anti-inflammatory,immune regulation and anti-cancer.Paramylon and its derivatives(such as sulfated paramylon)also have antiviral activity.At present,the extraction process of paramylon is relatively complex,and there are less studies on purity and yield of the extraction.Moreover,the characterization of its structure,especially its molecular weight,is sometimes contradictory in diffirent literatures.However,the purity and structure of the paramylon have an important influence on its bioactivity,so it is urgent to carry out relevant research.In this paper,the Euglena gracilis power was used to study on separation,structure identification of paramylon and its bioactivities.At first,congo red staining method was used to establish a quantitative method for the determination of paramylon content.Then,the extraction process of paramylon was optimized by response surface design.The optimal conditions of ultrasonic-assisted extraction are as follows:SDS concentration 8.50g/L,liquid-material ratio 20.00mL/g,ultrasonic time 18.50min.Under this condition,the extraction rate of paramylon is 95.26%,its purity is 95.69%.After that,the structure of paramylon was elucidated.By UV spectrum analysis,the extracted paramylon doesn't contain protein and nucleic acid.The molecular weight of paramylon is about 1.9×105 Da by Gel Permeation Chromatograph(GPC).Through the analysis of infrared spectrum,1H NMR and 13C NMR,it can be seen that there is only one monosaccharide of glucose in the polysaccharide The bond between the monosaccharide residues of the polysaccharides is 1-3 bond.The residue type of monosaccharide is ?-(1?3)-D-glucose.By scanning electron microscopy,paramylon is ellipsoidal.The X-ray single crystal diffraction showed that paramylon has high crystallinity.Then we made the raw material of 1%paramylon and 1%Euglena gracilis powder into the feed respectively,carried out the feeding experiment of mice,and observed the effect of paramylon and Euglena powder on gouty arthritis.It was found that paramylon and Euglena powder could not improve the pain threshold,but Euglena powder had some alleviating effect on the swelling degree of paw.We found that paramylon and Euglena powder reduced the inflammation of mouse foot with HE staining.In order to identify the type of apoptotic effect,we used JC-1 staining to observe the change of mitochondrial membrane potential.The results showed that the breakdown of mitochondrial membrane potential could be caused by the paramylon.In addition,the results of western blot showed that the change of cytochrome C content in the cytoplasm was an important process of apoptosis signal transduction pathway mediated by mitochondria.Therefore,it was finally determined that the apoptosis of HepG2 cells was induced by paramylon through mitochondrial pathway.In addition,the expression levels of Bcl-2 protein family and caspase-3/-9 protein were detected.The results showed that the expression of Bcl2 decreased and Bax increased.The expression of caspase-3/-9 protein was further activated and induced,leading to apoptosis.The results of this experiment provide the experimental basis and theoretical basis for the clinical research of paramylon.