Study On Separation And Purification,Structure Characterization,Antitumor Effect In Vitro And Mechanism Of Polysaccharide From Artificial Cultivated Anoectochilus Roxburghii

In this paper,Anoectochilus Roxburghii polysaccharide?ARPS?were prepared by water extraction-alcohol precipitation method.The most suitable standard monosaccharide was chosen by comparing the UV spectra of different monosaccharides and ARPS and to compare the content of ARPS in the plants with different producing areas and culture conditions.The two solvent system was optimized to separate and purify ARPS and the structure of it was characterized.MTT method was used to investigate ARPS which was from artificial cultivated plants in the inhibitory effects of different tumor cells include time-effect and dosage-effect.Fluorescence analysis and flow cytometry were used to explore the mechanism of antitumor activities of ARPS.These investigations would provide the theoretic basis for the further study on the applications of artificial cultivated Anoectochilus Roxburghii in clinical reserch.This thesis consists of four chapters:1 Study on the analysis of polysaccharide content in Anoectochilus Roxburghii from different producing areas and culture conditionsIt is the first time to the systematic study on the selection of standard monosaccharide.As its UV spectrum shows most similarity with ARPS,glucose was selected to be the standard monosaccharide by comparing the UV spectrum of mannose,rhamnose,glucose,galactose and fucose after phenol-sulfuric acid method.The conversion factor f was calculated by refined ARPS and it was used to the content analysis of ARPS from different producing areas and culture conditions plants.The result shows ARPS content in the plants ranges from 2.01% to 17.49%.The conclusion is that the content of ARPS in the plants with different producing areas and culture conditions was very different.2 Separation and purification of ARPS with HSCCCHSCCC was used to the separation and purification of ARPS.By comparing the partition coefficient value?K?of ARPS in the two-phase system consists of PEG600,800,1000 and different proportions of salt,the most suitable two-phase solvent system was selected for the separation and purification of ARPS and the content of the purified ARPS was detected.The result shows that 12.5% PEG1000-20% phosphate system isbest suited for the purification of ARPS and the content of purified ARPS was 95.01%.It can successfully realize the separation and purification of ARPS.3 Structure characterization of ARPSThe structure of ARPS was characterized by UV,HPGPC,HPLC,LC-MS,IR,NMR,AFM and Congo red test.The result shows that the average molecular weight of ARPS was 26090 Da and it consists of mannose,ribose,glucose,galactose and arabinose.The molar ratio of monosaccharides was mannose:ribose: glucose: galactose:arabinose =1.00:8.47:47.30:1.17:1.19.The glycosidic bond was ? configuration and there is no triple helical conformation.The AFM microscopic observation showed that the moleculars aggregate closely together as globular and the diameter of it is about 120 380 nm.4 The antitumor effect of ARPS and the preliminary study of its mechanism in vitroThe antitumor effect of ARPS obtained from artificial cultivated Anoectochilus Roxburghii was tested by MTT method on lung cancer A549,osteosarcoma 143 B,the rat adrenal pheochromocytoma PC 12,breast cancer MCF-7,acute leukemia HL 60,chronic leukemia K562,colon cancer SW620,esophagus cancer OE 19,liver cancer HepG2 and neuroglioma U251,and analyzed by statistical method.The result shows that ARPS has the best inhibitory effect on esophagus cancer OE 19 with 48 h and the IC₅0 was 5.67?moL/L.Fluorescence analysis and flow cytometry were used to explore the mechanism of antitumor activities of ARPS.The result shows that ARPS induce esophagus cancer OE 19 cells to apoptosis and it was retardant in G₂/M phase.