Study On The Protective Effect Of Polysaccharides Phellinus Vaninii Ljub On Myocardial Cell Injury And Development Of Phellinus Vaninii Ljub Products

As an important local characteristic fungus in Jilin Province,Phellinus vaninii Ljub has been gradually concerned by many scholars because of its rich nutritional value,anti-tumor,immunity enhancement,anti arrhythmia and other biological activities.Cardiovascular disease is one of the most serious fatal diseases in the world.Timely reperfusion therapy to restore blood supply of ischemic myocardium is considered to be the most effective treatment to prevent myocardial necrosis.However,reperfusion therapy itself may aggravate the degree of myocardial injury,and even seriously affect the life of patients.This paper mainly studied the extraction and separation of polysaccharide,structure composition,anti myocardial damage activity and food development of Phellinus vaninii Ljub.The main research contents and results were as follows:1.Extraction,isolation and structure analysis of Polysaccharides from Phellinus vaninii LjubThe dried mulberry fruit was crushed,and the polysaccharide was extracted and separated by water extraction alcohol precipitation.After deproteinization and depigmentation,30%to90%of the crude polysaccharides(F30-F90)were obtained,and the yield was 1.58%,0.92%,0.08%,0.132%,0.18%,0.022%and 0.34%.According to the screening results of H9c2myocardial cell toxicity and myocardial cell injury protective activity,F80 polysaccharide has obvious protective effect on cardiac myocytes.Therefore,the polysaccharide polysaccharide Sephadex-100 gel column was used to separate and purify the crude polysaccharide extract,and the purity of each polysaccharide was analyzed by the evaporative light detector.Three single polysaccharides,namely F80-1,F80-2 and F80-3,were extracted.The extraction rates were0.022%,0.018%,0.013%;the total sugar content after purification was 90.15%,94.21%,88.04%;the protein content was 0.24%,0.14%,0.02%;the content of glycoaldolic acid was14.18%,13.06%,16.42%;the molecular weight was 5.49×10⁴Da,5.47×10⁴Da and 5.38×10⁴ Da respectively.By GC analysis,the three single polysaccharides were mainly composed of D-galactose and D-mannose;the molar ratio of d-arabinose,D-glucose,D-galactose and D-mannose in F80-1 polysaccharide was 1.29:0.52:19.04:1.14;the molar ratio of L-rhamnose,D-galactose and D-mannose in F80-2 polysaccharide was 21.33:1.10:25.09;the molar ratio of D-mannose,D-glucose and D-galactose in F80-3 polysaccharide was 31.23:0.09 99:1.55.2.Protective effect of polysaccharides on H₂O₂ induced cardiomyocyte injuryThe oxidative stress injury model of H9c2 rat cardiomyocytes induced by H₂O₂ was used to determine the antioxidant activity of Polysaccharides from Phellinus vaninii Ljub.The proliferation of cardiomyocytes was determined by CCK-8 method.The results showed that F80polysaccharide had more obvious protective effect on cardiomyocytes.The concentration of F80-1 was 0.2?g/m L and 0.4ng/m L,and the concentration of F80-2 was 0.2?g/m L and 0.4ng/m L.LDH,MDA,SOD,GSH-Px and CAT kits were used to analyze the damage degree of cardiomyocytes,and the production of ROS was observed by fluorescence microscope.The results showed that the activities of SOD,CAT and GSH-PX in cardiomyocytes were significantly increased,and the release of LDH and the expression of MDA in cardiomyocytes were decreased after treated with the polysaccharides of F80-1 and F80-2.The production of ROS in H9c2 cardiomyocytes induced by H₂O₂ was significantly reduced.Western blot was used to detect the expression of H₂O₂ model related proteins.After H₂O₂ treatment,the expression level of Bcl-2 protein in H9c2 cardiomyocytes was significantly lower than that in the normal group,and the expression levels of Bax and Cleaved caspase-3 protein were significantly higher than those in the normal group.These results indicate that the polysaccharide of Phellinus vaninii Ljub can prevent cell apoptosis.3.Protective effect of polysaccharides on cardiomyocyte injury induced by hypoxia reoxygenationThe myocardial ischemia-reperfusion injury model of H9c2 rat was induced by hypoxia reoxygenation(H/R),and the anti ischemia-reperfusion activity of Polysaccharides from Phellinus vaninii Ljub was determined.CCK-8 method was used to determine the proliferation ability of cardiomyocytes.Three concentrations of F80 single polysaccharide were selected.F80-3:2?g/m L,0.02?g/m L,0.002?g/m L.Cell apoptosis was detected by fluorescence microscopy and flow cytometry.The results showed that different doses of F80-3 pretreatment showed excellent anti apoptotic effect.Western blot was used to detect the expression of PI3K and AKT protein in H/R model.The phosphorylation expression of PI3K and AKT protein increased gradually after treated with Phellinus vaninii Ljub polysaccharide.4.Food development of Phellinus vaninii LjubWith Phellinus vaninii Ljub as the main raw material,one kind of substitute tea and one kind of solid beverage of Phellinus vaninii Ljub were developed.