Phellinus Igniarius Active Polysaccharide Separation, Purification, Structure Identification And In Vivo Biological Activity Research

Phellinus igniarius is one of precious medicinal fungus,the polysaccharides obtained from this fungus has been reported to possess antioxidant,anti-tumor and other biological activity.However,due to some factors such as climate and temperature,the wild fruiting of Phellinus igniarius is difficult to form in nature.The artificial cultivation technology at present is not yet mature,as a result,obtain the wild fruiting of Phellinus igniarius and its products by liquid fermentation technology is very important.In this study,Phellinus sp.mycelia were cultured by liquid fermentation technology.After isolation and purification from the mycelia Phellinus sp.,two kinds of purified polysaccharides were obtained,and their physicochemical properties,structure characteristics and biological activity were discovered by experiment.Two kinds of phellinus sp.extracellular polysaccharide were obtained in our laboratory,and structure characteristics and antioxidant activity in vivo were studied by experiment.This study can play an important role for further for the further exploitation of this rare medicinal fungus Phellinus sp.The main results are as follows:1.Studies on the new extraction process to obtain mycelia polysaccharide of Phellinus sp.with high yield and high activity were perfomed.comparison of hot water extraction,ultrasonic crushing method,low temperature low pressure method.The results showed that low temperature low pressure method is the optimal extraction method.The extraction conditions were optimized by response surface optimization,and the obatined optimization of extraction conditions is extraction time 1.8 h,pressure 0.059 Mpa,solid-liquid ratio 52.5:1.The yield of polysaccharide reached 4.41%(relative wet mycelium),the total antioxidant activity reached 6.33 mM.2.After extraction of mycelia polysaccharide of Phellinus sp.at low temperature and low pressure,the obtained crude polysaccharides were sequentently subjeted to remove for protein,pigment and inorganic salt.Then DEAE cellulose column and gel column were perfomed to further purify the polysaccharides and which was determined for the purity of polysaccharide,confirmed as pure polysaccharide.The obtained two polysaccharides were named SHJS1 and SHJS4,respectively.3.Studies on physicochemical properties of polysaccharide were perfomed.SHJS1 was composed of mannose,glucose,galactose,arabinose,fucose,with a molar ratio of 3.92:1.96:4.03:0.88:2.03;the SHJS4 was composed of glucose,galactose,fucose,with a molar ratio of 1.00:0.61:0.83_The average molecular weights were determinedto bel.65×105 and 7.14×105,respectively.Infrared spectroscopy analysis showed that SHJS1 has beta glycosidic bond,and these two kinds of polysaccharides were pyranose configuration.4.Methylation and NMR analysis(1H,13C,1H-1HCOSY,TOCSY,HMQC,HMBC,NOESY spectrum)showed that the backbone of SHJS1 was(1?4)-a-D-Fucp,(1?3,6)-a-D-Arap and(1?2,4)-?-D-Galp,the side chain is composed of a-D-Manp and a-D-Glcp.Polysaccharide SHJS4,has not yet been speculated the structure possibly due to its high viscosity.5.Study on the immunological activity in vivo for intracellular polysaccharide SHJS1 and SHJS4 was perfomed.This result indicated that polysaccharide SHJS1 could obviously increase the content of serum IL-4,IEN-?,IFN-?.SHJS4 can significantly increase the content of serum IL-2,IFN-? and IFN-?.6.The structure of the extracellular polysaccharides SH1-3 and SH4-1 in our laboratory was analyzed.Based on the analysis of the methylation reaction and NMR,the backbone of polysaccharides SH1-3 was(1?2,4)-a-D-Glcp and(1?2)-?-D-Manp,the side chain is formed by a-D-Glcp.The backbone of polysaccharide SH4-1 is(1?3)??-D-Araf,(1?4)-?-D-Manp,(1?2,6)-a-D-Glcp and(1?6)-a-D-Galp,the side chain is composed of a-D-Glcp and a-D-Manp.7.Study on the anti-aging activity in vivo for individual SH1-3 and SH4-1 was perfomed.the results indicated that two kinds of extracellular polysaccharide can increase the activity of superoxide dismutase(SOD),catalase(CAT)and antioxidant capacity(TEAC)in serum,and reduce the Lipofuscin level in mice liver,the mouse liver tissue slice observation.These results suggest that that the polysaccharide can reduce liver cell damage resulted from D-galactose.