Study On The Preparation And Structure-Activity Relationships Of Carboxymethyl-Pachymaran

Mushroom-derived?-glucan has a wide range of biological activities,including immune regulation,anti-tumor,anti-inflammation,anti-oxidation,anti-virus,metabolic regulation and so on.At present,the structure-activity relationship of?-glucan is not clearly understood,which is an obstacle to guide the application of?-glucan in the field of medicine and health care.Poria is the dried sclerotia of Poria cocos(Schw.)Wolf which is a fungus of the family Polyporaceae.?-Glucan accounts for 70%?90%of the dry weight of sclerotia.After carboxymethylation,its biological activities will be enhanced.In this study,carboxymethyl-pachymaran(CMP)with different degrees of substitution and different molecular weights were prepared to explore the relationships between the structure(the degree of substitution and molecular weight)and the immunomodulatory,anti-inflammatory,anti-tumor and antioxidant activities.The main experimental contents and results studied in this dissertation were as follows:(1)Preparation of CMP by the process in aqueous solution and its activity verification:The crude CMP was prepared in aqueous solution with polysaccharides content of 80.3%.and degree of carboxymethyl substitution of 0.46.The inhibition rate of CMP on S180tumor in mice was 27%.In addition,CMP can enhance the phagocytosis of monocytes,delayed-type hypersensin,hemolysin formation,splenic lymphocyte proliferation and NK cell killing activity in immunosuppressed mice.(2)Preparation and structure identification of CMP with different degrees of substitution and different molecular weights:By adjusting the preparation parameters of CMP,CMP with different degrees of substitution(0.13,0.32,0.95)and different molecular weights Mw(5.93×10~4Da,11.58×10~4Da,26.02×10~4Da)were isolated.The polysaccharides content of CMP were more than 92%,and none of them contained protein or nucleic acid.Infrared spectrum showed that CMP were?-pyranose.NMR results proved that the CMP were composed of a monosaccharide.Besides,the degree of introduction of carboxymethyl groups was C-6,C-4 and C-2,respectively.Congo red test showed that CMP with medium degree of substitution and large Mw was beneficial to maintain the triple-helix structure.(3)Regularity of different degrees of substitution and different molecular weights of CMP on immunomodulatory and anti-inflammatory activities:RAW264.7 macrophage model was used to evaluate the effects of CMP with different structures on the release of NO and TNF-?.The immune response of RAW264.7 macrophages could be stimulated by different molecular weights of CMP.The CMP of moderate molecular weight could enhance the immune activity,while the CMP of low molecular weight was the lowest.The high degree of CMP promoted NO synthesis effectively,while the medium degree of CMP promoted TNF-?synthesis best.In the model of inflammation induced by LPS in RAW264.7,CMP with moderate degree of substitution was the most beneficial to inhibit NO,while CMP with low degree of substitution was the best to inhibit TNF-?.The higher the Mw,the stronger the inhibition of CMP on the synthesis of NO and TNF-?.Receptor identification tests showed that Dectin-1 was one of the immune receptors of CMP.(4)Effects of CMP with different degrees of substitution and different molecular weights on the inhibition of tumor cell proliferation and antioxidant activity:MTT assay showed that CMP with moderate degree of substitution and high molecular weight were beneficial to Hep G-2 tumor cell inhibition.DPPH method showed that moderate substitution degree of CMP was better at scavenging DPPH free radicals,while the low Mw was beneficial to scavenge DPPH free radicals.In this dissertation,the content of structure-activity relationship of?-glucan was improved by studying the relationship between the activity of CMP with different degrees of substitution and different molecular weights,which provided experimental basis for the application of?-glucan.