Extraction,Purification,Structural Analysis And Application Of Bioactive Polysaccharide In Grifola Frondosa

The main active substance in macrofungi is polysaccharide,which has attracted extensive attention because of its biological activities such as antioxidant,antibacterial,antitumor and immune regulation.As a new type of traditional Chinese medicine in China,Grifola frondosa has been widely concerned for its anti-inflammatory,lipid-lowering,liver protection and enhancing immune function.So far,there are a litter of reports on the extraction method,process optimization and bioactive mechanism of Grifola frondosa polysaccharide.In this thesis,Grifola frondosa polysaccharide was systematically studied from the aspects of extraction method and extraction process optimization,separation and purification of crude polysaccharide,physical and chemical properties,structural characterization,antioxidant,antibacterial,antitumor,lung protection and immune regulation of pure polysaccharide.The methods of fractional extraction and supercritical CO₂ extraction were developed to extract polysaccharides from Grifola frondosa.The processes of fractional extraction and supercritical CO₂ extraction were optimized by response surface method,and the best extraction conditions were found.The optimum technological parameters of ultrasonic assisted fractional extraction of Grifola frondosa polysaccharide were extraction temperature 35.6℃,liquid material ratio33.8m L/g,extraction time 47 min and ultrasonic power 104.6 W.under these conditions,the yield of Grifola frondosa polysaccharide was 6.91±0.11%（n=5）.The technological parameters of extracting polysaccharide from Grifola frondosa by supercritical CO₂ technology are as follows:the extraction temperature is 36.7℃,the extraction pressure is 34.5MPa,the extraction time is 116.3min,and the amount of entrainer is 286m L.The yield of Grifola frondosa polysaccharide is 4.63±0.13%（n=5）.Compared with the traditional extraction methods of Grifola frondosa polysaccharide,fractional extraction and supercritical CO₂ extraction not only have higher yield,but also are friendly to polysaccharide and will not damage its own structure.The polysaccharide extracted by supercritical CO₂ was purified by DE-52 and glucose agar G-300 chromatography,and a main purified component was obtained,named GFP;The polysaccharide from Grifola frondosa was extracted by ultrasonic fractionation.The polysaccharide was purified by DE-52 and glucose agar G-300chromatography.Two main purified components were obtained,named GFP-1-a and GFP-2-a respectively.The physicochemical properties and structure of the above three polysaccharides were analyzed.The general analysis showed that GFP,GFP-1-a and GFP-2-a were acidic polysaccharides containing sulfate groups;The relative molecular mass of GFP,GFP-1-a and GFP-2-a was by high performance gel permeation chromatography（HPGPC）.1.80×10⁶Da,1.22×10⁶Da and 1.71×10⁶Da;GFP,GFP-1-a and GFP-2-a were measured by GC-MS.They were composed of monosaccharides such as glucose,xylose,galactose,rhamnose,fucose,arabinose and mannose in different proportions;Congo red test and transmission electron microscope analysis showed that the three polysaccharides had three helical conformations.The results showed that the three polysaccharides may have good biological activities.The ability to scavenge DPPH radical,ABTS,superoxide anion and iron ion chelation by polysaccharide,The antioxidant capacity of Grifola frondosa polysaccharide in vitro was evaluated;The antioxidant capacity and liver protective effect of Grifola frondosa polysaccharide in vivo were evaluated by constructing a mouse model of polar alcohol liver injury.The results showed that Grifola frondosa polysaccharide had good antioxidant activity in vitro.Grifola frondosa polysaccharide can effectively reduce ALT and AST in serum of mice caused by alcohol,and make SOD in liver of mice caused by alcohol The decrease of GSH-Px and CAT activities and the decrease of MDA content can effectively inhibit TNF-αand IL-1βlevel.Grifola frondosa polysaccharide may inhibit IL-1βand TNF-αand other proinflammatory factors,improve the activity of antioxidant enzymes in vivo,chelate metal ions and scavenge free radicals in vivo.The antibacterial activity of Grifola frondosa polysaccharide in vitro was comprehensively evaluated by its antibacterial effect and antibacterial ability against Escherichia coli,Staphylococcus aureus and Pseudomonas aeruginosa;The antibacterial activity of Grifola frondosa polysaccharide in vivo was evaluated by constructing a mouse model infected with Staphylococcus aureus and Pseudomonas aeruginosa.The results showed that Grifola frondosa polysaccharide had good antibacterial activity in vitro.Grifola frondosa polysaccharide can effectively reduce the mortality,liver coefficient,kidney coefficient,white blood cell（WBC）and neutrophil rate（NEUT%）of mice infected with Staphylococcus aureus and Pseudomonas aeruginosa.At the same time,it can also restore the body cells damaged by infection.Grifola frondosa polysaccharide can better play the role of antibacterial in vivo.A mouse model of lung injury caused by fine particulate matter(PM_2.5)was established.The effects of Grifola frondosa polysaccharide on PM_2.5 were analyzed by molecular biology,cytology and biochemical analysis The protective effect of on lung injury in mice.Grifola frondosa polysaccharide can effectively reduce PM_2.5 in mice.Reduce the degree of pulmonary edema in lung injury,alleviate the inflammatory reaction caused by lung injury,reduce neutrophil aggregation and reduce PM_2.5 toxic effect on macrophages,protect the integrity of capillary membrane in alveoli,and reduce the TP,CRP,ALB,LDH and The leakage of MPO,AKP and ASM inhibited IL-1β、TNF-αAnd the release of IL-6 alleviated PM_2.5 in mice oxidative stress response to lung injury,increase the activities of GSH-Px,CAT and SOD in lung tissue,and reduce the contents of PCG,MDA and 8-OHd G.It shows that Grifola frondosa polysaccharide plays its role in mice due to PM_2.5 through the joint action of antioxidant and antibacterial activities protective effect on lung injury.The inhibitory effect of Grifola frondosa polysaccharide on cervical cancer cells（Hela cells）and gastric cancer cells in vitro was evaluated,and the effect of polysaccharide on the growth of normal liver cells in vitro was observed.At the same time,Lewis lung cancer mouse model was constructed to observe the effect of polysaccharide on lung cancer mice.Through experiments,it is found that polysaccharide can inhibit the proliferation of tumor cells in vivo,effectively restore the body weight,thymus coefficient and spleen coefficient of lung cancer mice,activate macrophages in the abdominal cavity of lung cancer mice,stimulate the proliferation of lymphocytes in the spleen,and effectively improve cytokines and TNF-αAnd the expression level of IL-12.It shows that Grifola frondosa polysaccharide can exert its antitumor activity in vivo through immune regulation.The protective effect of Grifola frondosa polysaccharide on immunosuppression in mice was evaluated by biochemical analysis,molecular biology and cytology.The results showed that Grifola frondosa polysaccharide could restore the organ coefficients of heart,spleen,liver,thymus and kidney of immunosuppressive mice,improve the phagocytosis of macrophages in immunosuppressive mice,enhance the release of no from macrophages,and increase the proliferation of lymphoid T cells and lymphoid B cells,Activate the toxic effects of NK cells and CTL cell,and increase the cytokines IL-2 and IL-2 in IFN-γ.The levels of Ig G,Ig M and Ig A enhance the T-AOC,GSH-Px,CAT and SOD activities,and reduce the content of MDA at the same time.Grifola frondosa polysaccharide can effectively improve the immune function and exert its antioxidant activity,antibacterial and antitumor activity.There were 81 figures,32 tables,and 391 references in this thesis.