| In this project, we extracted and purified the polysaccharide from dried Polygonatum cyrtonema Hua, calculated its molecular weight and made primary analysis on the pharmacological effect of the crude polysaccharide.Anthrone sulfuric-UV spectrophotometry was chosen as the standard method for polysaccharides content determination. Three extraction methods:water decoction, water dipping, microwave-water extraction retrieved21.6%,6.5%,17.9%of the total polysaccharide respectively, and we chose water decoction in this study for its best extraction rate. For higher extraction rate, we tested three major factors in the water decoction technique that matter most in a L9(3)4orthogonal test, that is, extraction times,extraction time and water consumption. Solid-liquid ratio and alcohol concentration of the alcohol precipitation step were also optimized. The final optimized conditions were:use10volume of water for decoction,2h each time for3times; then concentrate to a solid-liquid ratio of1:1; add alcohol to a concentration of75%for precipitation. In such a way,28.5%of the polysaccharide can be rerieved.For the deproteinization method, we chose Sevage out of two optional methods for its optimized protein removal and polysaccharide retention; and in a6-times repeated experiment, an average of72.7%of the protein was removed and84.6%of the polysaccharide was retained. After the deproteinization,decoloration and dialysis, the polysaccharide went through DEAE-52cellulose column to get PCPd-1PCPd-2, PCPd-3components, each of which then went through Sephadex G-150for further purification and resulted in PCPs-1,PCPs-2PCPs-3respectively. All three components were analyzed by HPGPC and showed single symmetrical peak, indicating high purity.Using the TCL method, we found that PCPs-1and PCPs-2contain glucose and galactose while PCPs-3was mainly composed of galactose. In UV absorption spectra, all three components showed no absorption at260nm or280nm indicating no nucleic acids or protein contained; and in IR absorption spectra, they all showed absorption properties of glycosidic bond with a β configuration. And HPGPC-ELSD method was used for determination of the molecular weight distribution of Polygonatum polysaccharide; the chromatographic conditions were Shodex OHpak SB-804HQ (8.0×300mm),0.1mol/L ammonium acetate as the mobile phase, flow rate set at0.8mL/min, column temperature30℃. The number average molecular weight of PCPs-1, PCPs-2, PCPs-3were determined as5640,4760,4380Dalton respectively; the weight average molecular weight13000,13900,12200Dalton; and the distribution coefficient were2.31,2.93,2.80.Crude polysaccharide from polygonatum cyrtonema Hua was extracted by water decoction and alcohol precipitation, then further divided by proper solvent into two parts:CPPC-1and CPPC-2; and the two parts were subjected to primary in vitro immune enhancement experiment. The effect of CPPC-1and CPPC-2on lymphocyte transformation and proliferation were assessed by MTT assay, and their effect on NO generation of RAW264.7was assessed by Griess test. Our results showed that crude polysaccharide CPPC-1and CPPC-2had no synergistic effect on lymphocyte transformation, but they can moderately promote splenic lymphocytes proliferation; CPPC-1achieve its best promoting rate,20.3%, at50ug/mL while the CPPC-2achieve the best promoting rate,13.3%, at500ug/mL. Neither CPPC-1nor CPPC-2had obvious effect on NO generation of RAW264.7. These results indicated that rude polygonatum polysaccharide may have some immune-enhancing effect. |
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