Study On The Extraction, Isolation And Purification Of Polysaccharide In Chrysanthemum Morifolium Ramat

Chrysanthemum Morifolium Ramat, which belongs to the head flower of chrysanthemum family, is usually used as Chinese traditional herb medicine and has lots of important functions. Chrysanthemum polysaccharide, one of main bioactivity components in C. Morifolium R., has many pharmacological effects, such as enhancing immunity and preventing aging and so on.The extraction, isolation and purification as well as molecular weight determination of polysaccharide from C. Morifolium Rama Huang Shan were studied in this paper.Taking glucose as control, the phenol-sulfuric acid method examining content of chrysanthemum polysaccharide was established. The results showed that the revised method was stable and had good repeatability and high accuracy when the volume ratio of 5% phenol to sulfuric acid was 5:1, the volume ratio of sulfuric acid to total volume of solution was 5:8 and reaction solution was cooled for 30 min after bathing 15min in boiling water.Factors that had influence on the yield of chrysanthemum polysaccharide were mainly considered, including extraction temperature, extraction time, the solvent-solid ratio and ethanol terminal concentration. On the basis of single factor experiments, orthogonal design was adopted to search the optimum extracting parameter of chrysanthemum coarse polysaccharides. The result showed that optimum extracting parameter as follows: 95℃of extraction temperature, 3h of extraction time, 25:1 of the solvent-solid ratio and 80% of ethanol terminal concentration. Moreover, one extracting time and pH7.0 were suitable. The yield of chrysanthemum coarse polysaccharides was 18.54%. The extracting efficiency of polysaccharide between microwave-assisted extraction and water extraction was compared. The result indicated the yield of rude polysaccharides with microwave-assisted extraction increased by 13.3% compared with that of water extraction.For purification of polysaccharide, the three methods to remove protein, including Sevag, trichloroacetic and ethanol precipitation plus Sevag, were compared. The result showed that the protein-removed rate by the methods of Sevag, trichloroacetic and ethanol precipitation plus Sevag were 40%, 66% and 39%, respectively. The loss rate of polysaccharide with the methods of Sevag, trichloroacetic and ethanol precipitation plus Sevag was 30%, 46% and 61%, respectively. Considering the loss ratio of polysaccharide and protein removing rate, Sevag method was adopted to remove protein.The crude polysaccharide was separated by negative ion exchange DEAE-Cellurose 52. Two polysaccharide components CPS- I and CPS- II were obtained. The further purification was finished by column chromatograph of Sephadex G-75 and single symmetric peak of both CPS-1 and CPS-II was gained. Both CPS- I and CPS-II had characteristic absorption below wavelength 200 nm. The purity and molecular weight of CPS-1 and CPS-2 were identified by HPLC. The results showed that purification of CPS-I and CPS-II both amounted to 99% and that their molecular weights were 13427 and 5268, respectively.