The Purification And The Structure Representation Of Polysaccharides From Anoectochilu Roxburghii (Wall) Lindl And The Anti-tumor Activities Research

In this paper, polysaccharide from Anoectochilus roxburghii (ARPS) was obtained with the ultrasound extraction, the content determination of them were studied. The High-speed counter current chromatography(HSCCC)was successlly applied to separate and purify ARPS. The structural identification of ARPS was confirmed. HPLC method was described for the quantitative analysis and molar ratio of component monosaccharides of ARPS. The anticancer effects of ARPS on five tumor cells and morphology effect of K562 which had the strongest reaction with ARPS were investigated. The antitumor activity on K562 and its mechanism of ARPS was approached initially.This thesis includes four parts as follows:PartⅠ: Optimization of Phenol-Sulfuric Acid Determination Conditions of Polysaccharide in Anoectochilu roxburghii(wall) Lindl by Orthogonal TestPolysaccharide was obtained with the ultrasound extraction and alcohol precipitation method. Taking phenol-sulfuric acid method to the content determination, and glucose as parameters. The absorbance was measured at 490 nm. L9(34) Orthogonal Design optimum determined conditions. When the sample solution used 1.0 mL, 0.6 mL of 5% solution of phenol, sulfuric acid 6.0 mL, 100℃water bath for 15 min reaction conditions determination, the results is best. The content of crude polysaccharide was 12.09% in A. roxburghii.PartⅡ: High Speed Countercurrent chromatographic purification of polysaccharides from Anoectochilu roxburghii (wall) Lindl with an aqueous two-phase system and the structure representation of ARPSPolysaccharides were purified from A. roxburghii by HSCCC. The two phase system consisted of an aqueous polymer two-phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000, 10% (w/w) potassium dihydrogen phosphate, and 10% (w/w) dipotassium hydrogen phosphate buffer at pH 6.8. An orthogonal design was used for optimizing the revolution speed of the separation column, flow rate of the mobile phase and separation temperature, which were 850 r/min, 1.0 mL/min and 25℃, respectively. UV Spectrum and Sephadex G-100 analysis of the fractions collected by preparative HSCCC showed that ARPS was homogeneous fractions related to molecular weight. The preparative separation of A. roxburghii (100 g) was successfully performed, yielding 2 g of polysaccharides at high purity. The structural identification of ARPS was confirmed by FT-IR and 1HNMR. The average molecular weight of ARPS was 18197 which determined by gel chromatography. HPLC method was described for the quantitative analysis of component monosaccharides of ARPS which was carried out on a RP-C18 column using precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP), and the result showed that ARPS was a typical heteropolysaccharide and was composed of rhamnose, xylose, galactose, fucose and arabinose. As to their peak area, the corresponding molar ratio was 4.43: 14.04: 1.00: 2.28: 6.58, respectively.PartⅢ: Anticancer activities of the polysaccharide from A.roxburghii.We investigated the anticancer effects of ARPS on human chronic leukemia K562 cells, liver carcinoma HepG-2 cells, gastric carcinoma SGC-7901 cells, MGC-803 cells and colon carcinoma HCT-116 cells. Results showed that the IC50 on the anterior three cells were 19.28,34.45 and 53.93 nmol/mL, respectively. The strongest effect on the mentioned cells is K562. The antitumor activity on K562 and its mechanism of ARPS was approached initially.PartⅣ: A review of the research about the extract method and technology of polysaccharide from Chinese herbal medicine was given.