Study On Extraction Of Pine Cone Scale Polysaccharide And Its Immunological Activity

Pinecone is the seed of Pinaceae.Pinecone is rich in plant resources in China,widely distributed in Northeast,East China,North China,northwest,Sichuan and Yunnan.In Northeast China,pinecone of pinus koraiensis is the main one.As far as medicinal value is concerned,pinecone scale can relieve and treat diffuse acute bronchitis,dry eye and dry cough.In the food industry,we have developed and designed pinecone melaleuca crisp,pinecone wine and other products,which have good health care effect on human body.In recent years,studies have shown that pinecone scale extract has a good immune enhancing effect,in which polysaccharide is the main active component.But at present,the relevant research on pinecone scale is still in the primary stage of extracting mixture,and the physiological effects of specific components are rare,especially the scientific research on which functional components in pinecone can enhance immunity.Therefore,this study was based on the optimization of the extraction process of Polysaccharides from pinecone scales,and the obtained polysaccharides were separated and purified.On this basis,the basic structure of polysaccharide was elucidated.And further select pinecone scale polysaccharide to study the function of immune regulation,to explore its mechanism of action.This study has a positive practical significance for the comprehensive utilization of pinecone scales and the promotion in the field of food.The specific contents of this study are as follows:1.In this study,water extraction-alcohol precipitation were used to extract polysaccharides from pinecone scales.Single factor combined with response surface methodology was used to optimize the extraction process,including solid-liquid ratio,extraction temperature and extraction time.The optimum conditions were as follows:the ratio of solid to liquid was 1:30,the extraction temperature was 90?,and the extraction time was 2 h.Under these conditions,the yield of polysaccharide was 5.56%.The crude polysaccharide was deproteinized by sevag method,and the deproteinization rate was 73.5%;The decolorization rate of crude polysaccharide by activated carbon method was 80.14%;The DEAE-Sephacel cellulose column was combined with Sephadex G-200 dextran gel column to further separate and purify the polysaccharide.The results showed that a single polysaccharide was obtained by column chromatography and named PSP-I.the purity of PSP-I was 92.03%.2.the structure of single polysaccharide was analyzed by a series of chromatographic methods such as UV,IR,X-ray diffraction,HPLC and high effective gel.The results of UV spectrum analysis showed that PSP-I had no characteristic absorption peaks at 260 nm and 280nm,indicating that there was no nucleic acid or protein in PSP-I;The results of infrared spectrum analysis showed that PSP-I showed a strong band at 3398.42 cm^-1,which was O-H stretching vibration of hydrogen bond,C-H stretching vibration of methylene group at 2925.88cm-1,and carboxyl stretching vibration at 1487.05 cm^-1;The above three types are typical polysaccharide absorption peaks,so it can be judged that PSP-I has polysaccharide structure;The band at 1400-1000 cm^-1was caused by C-O-C vibration.The band at 745.92 cm^-1 was a typical glucopyranose.The band at 843.51 cm^-1 indicated the presence of?-glucuronic acid in the polysaccharide,indicating that PSP-I was an acidic polysaccharide;X-ray diffraction analysis shows that PSP-I is amorphous;The molecular weight of the gel was determined to be12 KDa by high performance gel chromatography.The monosaccharide composition was analyzed by Pre-column Derivatization High Performance Liquid Chromatography(PMP-HPLC).The results showed that the monosaccharide composition was as follows:mannose,glucuronic acid,galacturonic acid,glucose,galactose,xylose,arabinose,and the molar ratio was 1.60:1.81:39.52:40.79:12.27:0.67:3.34.3.In this study,RAW264.7 macrophages were used to investigate the immunoregulatory effect of PSP-I in vitro.The test was divided into blank group and positive group,PSP-I was divided into four dose groups(50,100,200,400?g/m L).The results showed that PSP-I in the range of 50?400?g/m L could significantly promote the proliferation of RAW264.7macrophages,increase the secretion of IL-1?,IL-6 and TNF-?cytokines and NO secretion,and enhance the phagocytosis of RAW264.7 macrophages.4.In this study,the immunosuppressive mouse model was established with cyclophosphamide to explore the immunomodulatory effect of PSP-I in vivo.The animals were divided into blank group,model group,positive group and low,medium and high PSP-I groups(5,10 and 20 mg/kg).After administration,the indexes of spleen and thymus,the morphology of spleen,the proliferation of splenic lymphocytes,the secretion levels of cytokines(IL-4,IL-2,IFN-?)and the content of serum hemolysin were investigated.The results showed that low,medium and high doses of PSP-I could significantly increase the spleen and thymus index of immunosuppressive mice induced by cyclophosphamide,alleviate the morphological abnormality of spleen tissue induced by cyclophosphamide,promote the secretion of IL-4,IL-2and IFN-?cytokines in serum of immunosuppressive mice,the proliferation of T and B cells,and the production of serum hemolysin.These results indicate that PSP-I can give full play to the reasonable immunoregulation of humoral and cellular immunity.The results show that PSP-I can enhance the immunity by regulating humoral immunity and cellular immunity.