Extraction, Separation, Purification And Preliminary Structural Analysis Of Lycium Barbarum Polysaccharide

Objective:To study the extraction,separation,antitumor activity and chemical structure of Lycium barbarum polysaccharides against human hepatoma HepG2 cells.There are few studies on biological activity and related structure,so on the basis of extraction and separation,this paper studies the effects of different fractions of Lycium barbarum polysaccharides on human liver cancer HepG2 cells,and characterizes the structure of the most significant fractions LBP4-1.Methods:Crude Lycium barbarum polysaccharides（LBP）,was extracted by water extraction and alcohol precipitation.Crude Lycium barbarum polysaccharides（LBP）was isolated and purified by DEAE-Sepharose.Then the physicochemical properties,monosaccharide composition and relative content,relative molecular weight and molecular conformation of different fractions of Lycium barbarum polysaccharides were studied by phenol-sulfuric acid method,m-hydroxybiphenyl method and coomassie brilliant blue method.HPAEC-PAD,HPGPC-RI and MALLS viscometer,and then the anti-hepatoma activity of different fractions was preliminarily evaluated.Considering the yield,total sugar content and activity of each fractions of polysaccharide,the best fractions LBP4 was determined.Finally,the homogeneous polysaccharide LBP4-1,was further isolated by gel chromatography（Superdex200）.The structure of LBP4-1 was characterized by UV,IR,HPAEC-PAD,HPGPC-RI,methylation binding GC-MS and scanning electron microscope（SEM）observation.Results:1.The extraction rate of crude polysaccharide was 8.9%.LBP was separated and purified by DEAE,and five fractions were named LBP 1,LBP2,LBP3,LBP4 和 LBP5.The yield was 10.12%,8.46%,7.69%,9.02%and 7.89%,respectively.2.The total sugar,acid sugar and protein content of LBP,LBP1,LBP2,LBP3,LBP4 和 LBP5 was 33.34%,39.56%,38.77%,30.12%,38.22 and 31.66%;36.37%,37.89%,22.34%,19.75%,20.76 and 39.21%;4.53%,4.13%,4.04%,1.02%,1.16 and 0.98%,respectively.The protein content of LBP 1～LBP5 decreased after DEAE cellulose column chromatography,but there were still a small amount of protein in LBP 1 and LBP2,which may be bound with polysaccharide.3.The monosaccharide composition of LBP1,LBP2,LBP3,LBP4 and LBP5 are fucose,arabinose,glucosamine,galactose,glucose,xylose,mannose,fructose,ribose,galacturonic acid and glucuronic acid.The corresponding molar ratio of LBP1 is 0.65:4.08:5.24:14.08:29.61:10.16:7.98:2.75:0.10:0.72:0.21,that of LBP2 is 0.60:2.65:0.22:16.31:23.08:3.94:0.78:1.46:1.04:0.72:0.21,that of LBP3 is 0.67:3.73:2.66:34.40:28.51:6.87:3.50:1.64:0.12:5.06:0.74,that of LBP4 is 0.70:4.77:3.46:45.88:31.07:13.19:4.41:3.10:0.62:5.79:0.16,and that of LBP5 is 0.65:3.48:0.36:20.22:17.19:7.21:0.29:0.08:0.13:8.01:0.04.LBP is mainly composed of arabinose,glucosamine,galactose,glucose,xylose,mannose,fructose,ribose,galacturonic acid and glueuronic acid.The molar ratio of each monosaccharide is 1.26:0.18:1.74:74.18:2.46:10.91:18.85:0.12:0.21:1.16,and the content of glucose and fructose is the highest.The average molar ratio of each monosaccharide of LBP1-LBP5 is 0.65:3.74:2.39:26.18:25.89:8.27:3.39:1.81:0.40:4.06:0.27,the general trend of monosaccharide composition is the same as that of LBP,but there are some differences.For example,the highest content of LBP1-LBP5 is glucose and galactose,and the lower content of fructose.The above results show the complexity of monosaccharide composition in Lycium barbarum polysaccharide.4.The results of GPC after universal correction and MALLS of LBP1-1,LBP1-2,LBP2-1,LBP2-2,LBP3-1,LBP4-1,LBP5-1 are 4.136 × 10⁵ and 1.348×10⁶,7.820×10⁴ and 3.435 ×10⁵,1.974×10⁶ and 1.232×10⁶,9.911×10⁴ and 1.166×10⁵,3.367×10⁵ and 3.901×10⁵,1.828×10⁵ and 2.521×10⁵,2.383×10⁵ and 2.784×10⁵ Da,respectively.Finally,GPC after universal correction is used to determine the molecular weight,and the method is verified by MALLS.The polydispersity of LBP 1-1,LBP1-2,LBP2-1,LBP2-2,LBP3-1,LBP4-1 and LBP5-1 are 1.54,1.00,2.54,1.07,1.21,2.37 and 4.15,respectively.LBP1-2 is uniformly distributed,LBP1-1,LBP2-2 and LBP3-1 are narrow distribution,LBP2-1,LBP4-1 and LBP51 are wide distribution.The hydrodynamic radii rh of LBP1-1,LBP1-2,LBP2-1,LBP2-2,LBP3-1,LBP4-1 and LBP5-1 are 51.3,202.3,121.0,81.0,64.0,74.4 and 204.4nm,respectively.Radii rn are 46.2,64.7,32.5,36.1,72.2,22.7 and 107.2 nm,respectively.The α values of LBP1,LBP2,LBP3,LBP4 and LBP5 are 0.7,0.5,0.8,0.4 and 0.3,respectively.The molecular conformations of LBP 1 and LBP3 are linear molecules,and the molecular conformations of LBP2,LBP4 and LBP5 are linear irregular coils.5.The MTT results showed that there was a significant difference between all concentration groups of LBP4 and the control group（**P<0.01）.The inhibition rate was concentration dependent.With the increase of treatment time,the inhibition rate increased first and then decreased in the treatment groups of 200,400 and 1000 μg/mL.The inhibition rates of all concentration groups of LBP4 increased when treatment time was between 48～72 h.And the inhibition rate of the treatment group with 1000 μg/mL was the highest（62.81%）.At the concentrations of 200,400,800 and 1000 μg/mL,the highest inhibition rates all appeared at 72 h and were（13.41 ± 1.28）%,（28.62 ± 3.85）%,（45.33 ± 2.84）%and（62.81 ± 6.94）%,respectively.The inhibition rates increased with the increase of the concentration,which was concentration dependent.Considering the total sugar content and yield of each fractions,LBP4 was next to LBP1 The total sugar content and yield were（38.22 ± 0.06）%and（9.02± 0.12）%respectively.Therefore,LBP4 was selected as the target of further structural study,which laid the foundation for further exploring the mechanism and structure-activity relationship of polysaccharide against hepatoma cells,and expected to provide certain experimental basis and reference value.6.LBP4-1 has a single absorption peak of polysaccharide at 196 nm,and there is no characteristic absorption peak of carotenoid in the wavelength range of 350 nm～550 nm.There is no obvious absorption peak at 260 nm,indicating that the polysaccharide of this fractions does not contain nucleic acid or micro nucleic acid,but there is an absorption peak at 260 nm280 nm,indicating that LBP4-1 is a kind of glycoprotein complex with uniform fractions.LBP4-1 has the typical characteristic absorption peaks 3424,2918,1629,1420 cm^-1 of polysaccharide,and contains the ring skeleton structure of pyranosyl ring.LBP4-1 was homogeneous polysaccharide.The relative molecular weight is calculated by substituting the retention time into the standard curve fitted by GPC,and the relative molecular weight of LBP4-1 is 2.212×10⁵ Da.LBP4-1 was mainly composed of fucose,arabinose,glucosamine,galactose,glucose,xylose,mannose,fructose,ribose,galacturonic acid and glucuronic acid.The molar ratio of each monosaccharide was 0.92:5.01:0.09:54.21:19.74:10.14:1.70:1.19:2.34:6.32:3.95,and the content of each monosaccharide was 0.60,3.01,0.05,38.99,14.21,6.08,1.23,0.86,1.41,4.90 and 3.00 μg/mg,respectively.There are seven kinds of glycosidic bonds in LBP4-1,which are T-Glcp,1,4-Glcp,1,3,6-Manp,2,6-Galp,2,3,6-Manp,2,3,5Araf and 2,4,6-Galp,with a molar ratio of 14.78:6.09:21.90:1.22:3.90:5.44:46.66,LBP4-1 contains both linear polysaccharides and branched chain polysaccharides.Branched chain polysaccharides are about 2.5 times as much as linear polysaccharides.The（DB）value of the branching degree of LBP4-1 is calculated to be 92.69%.LBP4-1 had a rough rod like structure.Conclusion:In this study,we preliminarily established a HPAEC-PAD method for the determination of the monosaccharide composition and content of Lycium barbarum polysaccharides without sugar derivation,good separation effect and high sensitivity,and a method for the determination of molecular weight by universal calibration HPGPC-RI method and MALLS verification.In addition,the structure of LBP4-1 was analyzed preliminarily,which provides experimental basis for further study on the biological activity and molecular structure of Lycium barbarum polysaccharides.