Research Of Effective Parts Of Ginseng Polysaccharides Based On The Immunological Activities

Systematical studied the extraction and purification process of the polysaccharides from Panax ginseng,screened out the immune active site,carried on the preliminary structure analysis on those active site,and established quality standards(protocol),to lay a foundation for depth research and drug's development of ginseng polysaccharide.1.The research on extraction and purification of ginseng polysaccharidesWith the yield and molecular weight of polysaccharide for index,compared the results of water extraction,acid extraction and alkaline extraction,and the optimal extraction conditions were obtained by orthogonal test:15 times solvent volume,extract temperature 100?,extract 3 times,3 hours per time.This method was simple and stability.With the removing rate of protein,the retaining rate of polysaccharides and molecular weight for indicators,compared the results of two removing protein methods:Sevag method and TCA method,and the optimal removing protein conditions were obtained by orthogonal test:Sevag reagent volume was equal to 1/3 times of 5%ginseng polysaccharides solution,the volume ratio of CHCl3 and normal butanol was 4:1,removing 5 times.This method was stability.GP was separated by Q-Sepharose FF column,eluted with H2O and 0.3 mol/L NaCl,collecting every fractions,two ploysaccharides fractions were got:GPN and GPA.GPA was separated by Q-Sepharose FF column,eluted with H2O,0.1,0.2 and 0.3 mol/L NaCl,collecting every fractions,four ploysaccharides fractions were got:GPA-1,GPA-2,GPA-3 and GPA-4.GPN and GPA-1 didn't have uronic acid,GPA-2,GPA-3 and GPA-4 were acidic fractions.Different polysaccharide acid were separated from GP with Q-Sepharose FF.GPN,GPA-1,GPA-2,GPA-3 and GPA-4 were separated by Sepharose CL-6B column,eluted with H2O,collecting every fractions,eleven fractions were got:GPN-1,GPN-2,GPA-1-a,GPA-1-b,GPA-2-a,GPA-2-b,GPA-3-a,GPA-3-b,GPA-3-c,GPA-4-a and GPA-4-b.2.The research on immune active of ginseng polysaccharidesThe in vivo experimental results showed that GP can promoted nonspecific immunity,GPN-2 can markedly promoted nonspecific immunity and specific immunity;GPN-2,GPA-2-a,GPA-2-b,GPA-3-b,GPA-3-c,GPA-4-a and GPA-4-b can markedly increased the spleen index and promote the macrophages' phagocytosis.From those results we can see that the immune active site and the unactive site got separated.The immunological fractions that separated from ginseng polysaccharides were GPN-2,GPA-2-a,GPA-2-b,GPA-3-b,GPA-3-c,GPA-4-a and GPA-4-b.3.Preliminary study on the structure of immunological fractionsGPN-2:contained polysaccharides was 87%,didn't contain uronic acid and protein,the molecular weight was 4.8×104,composed by glucose,rhamnose,arabinose and galactose,and their mole ratio was:1:0.04:0.49:0.74.The IR spectra showed that it connected by a-pyranoid glycosidic bond.GPA-2-a:contained polysaccharides was 85%,contained uronic acid was 6%,didn't contain starch and protein,the molecular weight was 1.3×105,composed by glucose,rhamnose,arabinose,galactose,galacturonic acid and glucuronic acid,and their mole ratio was:1:0.14:0.505:0.71:1.28:0.13.The IR spectra showed that it connected by a-pyranoid glycosidic bond.GPA-2-b:contained polysaccharides was 75%,contained uronic acid was 4%,didn't contain starch and protein,the molecular weight was 7.3×103,composed by glucose,rhamnose,arabinose,galactose,galacturonic acid and glucuronic acid,and their mole ratio was:1:0.09:0.25:0.43:1.06:0.14.The IR spectra showed that it connected by a-pyranoid glycosidic bond.GPA-3-b:contained polysaccharides was 40%,contained uronic acid was 42%,didn't contain starch and protein,the molecular weight was 5.8×104,composed by glucose,rhamnose,arabinose,galactose,galacturonic acid and glucuronic acid,and their mole ratio was:1:3.05:12.00:10.87:16.72:10.80.GPA-3-c:contained polysaccharides was 54%,contained uronic acid was 64%,didn't contain starch and protein,the molecular weight was 1.7×104,composed by glucose,rhamnose,arabinose,galactose,galacturonic acid and glucuronic acid,and their mole ratio was:1:0.43:1.54:1.10:9.90:4.31.GPA-4-a:contained polysaccharides was 46%,contained uronic acid was 91%,didn't contain starch and protein,the molecular weight was 1.1 ×105,composed by glucose,rhannose,arabinose,galactose,galacturonic acid and glucuronic acid,and their mole ratio was:1:2.14:2.78:2.21:20.76:1.80.GPA-4-b:contained polysaccharides was 39%,contained uronic acid was 72%,didn't contain starch and protein,the molecular weight was 1.3×104,composed by glucose,rhamnose,arabinose,galactose,galacturonic acid and glucuronic acid,and their mole ratio was:1:1.28:8.39:6.67:14.05:1.59.4.The establishment for quality standard of ginseng polysaccharidesunder the condition of the identify physical and chemical property,quality and immune action,the quality control of GPN-2(protocol)was establied which contained the characteristics,identification,limit check,loss on drying,heavy metal;residue on ignition and protein of polysaccharide.the contains of polysaccharide should no less than 80.0%.The molecular weight distribution of GPN-2 was 3.0×104?6.0×104.