Isolation,purification And Immunological Activity Of Polysaccharides From Tibetan Medicine Sibiraea Angustata

Sibiraea laexigata(L.)Maxim(SLM)is also named Tibetan sibiraea angustata,which belongs to Rosaceae(Genus Xianbei).Its young leaves and branches are often used as tea drinks by tibetan folk,and it is a precious medicinal and food homology plant resource.Sibiraea angustata polysaccharides(SAPs)is the main bioactive substance,which has the biological activities including clearing heat,helping digestion,regulating lipid metabolism,anti-oxidation,anti-tumor and enhancing immune activity.At present,the physicochemical properties and molecular spatial structure characterization of SAPs have not been found,and the relationship between biological activity and structure-activity,and mechanism of SAPs is still unclear.In this study,we extracted polysaccharide from Sibiraea angustata(SAPs)by polyethylene glycol ultrasonic-assisted enzymatic method,the effects of different parameters on the extraction efficiency of SLMPs were optimized by RSM.Then,the preliminarily structure of SLMPs was characterized by HPLC,FT-IR,and SEM.Moreover,the immune activity and regulatory mechanism of SAPs were analyzed by a mouse model of immunosuppression,which could provide a scientific basis for the comprehensive development and utilization of SLM.The main research methods and results are as follows:(1)The basic nutritional components of SLM were determined,and it was found that the crude protein content of fresh SLM was 4.03±0.02 g/100 g,the water content was 60.44%,the fat content was 1.71%,and the ash content was 4.76±0.12 g/100 g.The reducing sugar content was 21.81±0.17 g /100 g,and it was rich in trace elements such as Mg,K,Zn,Mn and essential amino acids such as Asp,Glu,Leu and Lys.(2)PEG-ultrasonic enzyme-assisted extraction of SAPs,and response surface method with Box-Behnken design(BBD)was used to optimize the extraction process of SAPs.The extraction rate of SLMs was used to as evaluation index,the effects of PEG molecular weight,PEG concentration,enzyme addition,p H value,ultrasonic power,ultrasonic time and ultrasonic temperature on SAPs extraction rate were studied.On the basis of single factor experiment,BBD response surface methodology with four factors and three levels was used to optimize the extraction process of SAPs,and the optimal extraction parameters were determined as follows: The maximum extraction yield of SAPs was 10.95±0.36% and the polysaccharide content was21.82±0.13% under the conditions of 2% enzyme content,p H 4.0,2.0 h ultrasonic time and 80 ? ultrasonic temperature.(3)The crude SAPs were separated and purified by protein removal decoloration,ultrafiltration and gel chromatography.The results showed that 0.1 g/L crude SAPs solution was treated with Sevage reagent(n-butanol: chloroform=1:5)After decolorization of protein and 0.06% activated carbon,the purified SAPs were successively separated by ultrafiltration in different molecular weight segments(Mw >10 k Da,3.5 k Da < Mw < 10 k Da,Mw < 3.5 k Da),and then collected by evaporation and concentration of filtrate solution and alcohol precipitation and freeze-drying.Cellulose was separated by DEAE-52 column chromatography and the two fractions were divided into SAPs-1 and SAPs-2.After concentration,distilled water dialysis,freeze-drying and further separation and purification by Sephadex G-100,SAPs-1-1and SAPS-2-1 were obtained.(4)The structures of SAPs and its components SAPs-1-1 and SAPs-2-1 were analyzed by UVS,HPGPC,HPLC-PMP,FT-IR,NMR,SEM detection and Congo red test.The results showed that: The UVS of sample SAPs and its components SAPs-1-1and SAPs-2-1 showed that the three samples contained no nucleic acid and protein and had high purity.The average molecular weight was 1.06 KDa,1.03 KDa and 1.02 KDa,respectively.By HPLC-PMP analysis,the monosaccharide composition of SAPs and its components mainly consisted of rhamnose,glucose,galactose,fructose and arabinose,but the contents were slightly different.FT-IR spectra showed that the sample had similar absorption peaks and was a pyranoid polysaccharide containing?-glycosidic bond.The results showed that ?-type pyranose and ?-type pyranose were found in SAPs and its components SAPS-1-1 and SAPS-2-1.The results of SEM show that all of them have obvious amorphous structure.It has a three-strand helical conformation and is metastable in the concentration range of 0.1-0.6mol/L Na OH.(5)The mice were injected subcutaneously with cyclophosphamide(Cyclophosphamide,CTX)to prepare a mouse model of immunosuppression.Effects of immune activity in mice.The results showed that compared with the PS control group,the weight growth rate,immune organ and organ index,carbon particle clearance index and blood routine index levels,IL-2,IFN-?,TNF-? in plasma of the mice in the CTX model group were significantly increased,Ig A content were significantly decreased(P < 0.05,P < 0.01);SAPs-M dose group increased body weight,immune organ and organ index,carbon particle clearance index and blood routine index levels,plasma IL-2,IFN-?,TNF-? and Ig A were significantly higher than those in CTX model group(P < 0.01),SAPs-L and SAPs-H groups(P < 0.05).Among them,SAPs have a certain regulatory effect on the damage of mouse immune organs(spleen and thymus),and have a significant effect on the expression levels of TLR4 and NF-?B in mouse spleen tissue.