Chemical Studies On The Acid Hydrolysate Of The Polysaccharide Adjuvant PCP-I From Poria Cocos

ObjectionThe purpose of this paper is at first to extract the crude polysaccharides from the Poria cocos,the crude polysaccharides are isolated to obtain homogeneous polysaccharide components and the physicochemical properties of the isolated products are determined.Then,the optimal condition for the partial acid hydrolysis of the polysaccharide components are selscted to prepare a main oligosaccharide fragment.Furthermore,the physicochemical properties of the main oligosaccharide fragment would be determined and its chemical structural characteristics would be studied,which laid a solid experimental foundation for the further studies of the polysaccharide adjuvant from P.cocos.MethodThe crude polysaccharides of P.cocos are prepared by extraction with warm water,precipitation with ethanol,dialysis after redissolving in water and freeze-drying.The crude polysaccharides are separated by DEAE-Cellulose to gain homogeneous polysaccharides components.Firstly,the acid hydrolysates of polysaccharide components are splitted by different concentrations of TFA(0.025-0.5 mol/L),temperatures(40℃,60℃,80℃,100 ℃ and 120 ℃)and reaction times(1 h,2 h,4 h,6 h and 8 h).Analysis is conducted using thin layer chromatography(TLC)and high performance gel permeation chromatography(HPGPC),Secondly,a L9(34)orthogonal design of three factors and three levels is used to study acid hydrolysis of polysaccharide components before an optimal hydrolysis condition is determined.The optimal hydrolysis condition is used to enrich main oligosaccharide fragment.The physicochemical properties of the main oligosaccharide fragment are determined by measuring its monosaccharide composition and molecular weight.The structural characteristics of the main oligosaccharide fragment are elucidated by periodate oxidation,Smith degradation,methylation reaction,infrared spectroscopy and nuclear magnetic resonance spectroscopy.Results1.The crude polysaccharide(PCP)is obtained from P.cocos powder by water bath extraction at 55℃,graded ethanol precipitation,redissolving,dialysis and freeze-drying.The yield of PCP is 0.15%.PCP has three forms of molecular weight,among which the peak molecular weight(Mp)is 1.71 ×104 Da,weight-average molecular weight(Mw)is 1.94×104 Da and number-average molecular weight(Mn)is 1.40×104 Da.The monosaccharide molar ratio of PCP is Glc:Fuc:Gal:Man=0.65:1.00:6.00:1.86.2.The crude polysaccharide(PCP)is separated into two fractions(PCP-I and PCP-II)by DEAE-cellulose column and their yields are 32.0%and 39.2%respectively.PCP-I mainly contains one chromatographic peak in high performance gel permeation chromatography,whose Mp is 1.69×104 Da,Mw is 1.92×104 Da and Mn is 1.38×104 Da.The monosaccharide molar ratio of PCP-I is Glc:Fuc:Gal:Man=0.07:1.00:4.33:1.16.PCP-II mainly contains one chromatographic peak,whose Mp is 1.75×104Da,Mw is 1.98×104Da and Mn is 1.43×104Da.The monosaccharide molar ratio of PCP-II is Glc:Fuc:Gal:Man=0.11:1.00:3.88:1.11.3.The hydrolysates of PCP-I under different concentrations of TFA,temperatures and reaction times are analyzed by TLC and HPGPC.As is shown by the results of single factor experiments,(1)under 80 ℃ and after two hours of hydrolysis,0.05-0.1 mol/L of TFA is suitable;(2)under 0.05 mol/L TFA and after two hours of hydrolysis,80-100℃ is suitable;(3)under 100℃and 0.1 mol/L TFA for hydrolysis,1-4 h is desirable.The results of orthogonal design show that the condition of 0.1 mol/L TFA and 2 h under 100℃ is ideal for acid hydrolysis of PCP-I which is easy to operate and prepare a main oligosaccharide fragment.4.The acid hydrolysates of PCP-1 under optimal hydrolysis condition are separated and purified by Sephadex G-25 Medium column chromatography to gain a homogeneous oligosaccharide(PCP-I-hy-1)with a weight-average molecular weight of 1726 Da.Its monosaccharide molar ratio is Glc:Fuc:Gal:Man=0.49:0.62:24.08:1.00.The structural characteristics of PCP-I-hy-1 are analyzed by methods of periodate oxidation,Smith degradation,methylation reaction,IR,NMR.PCP-I-hy-1 might be galactan with a anomeric carbon and βanomeric carbon.The linking residues of galactan are(1→)、(1→6)and(1→2,6).The possible structure is guessed as follow:(?)ConclutionCrude polysaccharide(PCP)is extracted from the P.cocos,and two polysaccharide components(PCP-Ⅰ and PCP-Ⅱ)are obtained by chemical separation.Then,the influences of different concentrations of TFA,temperatures and reaction times on the acid hydrolysates of PCP-I are analyzed to determine an optimal hydrolysis condition.The main oligosaccharide fragment is prepared under the optimal hydrolysis condition,and its physicochemical properties and structural characterization are investigated.