Study On Isolation,Purification,Structural Token And Activity Of Polysaccharide From Pinus Massoniana Pollen

The extraction process of polysaccharides from pinus massoniana pollen was studied in this thesis.The DEAE-sephadex A-25 chromatography and Sephadex G-75 gel chromatography were used to isolate and purify the crude pollen polysaccharides; Infrared spectroscopy,UV scanning,gas chromatography(GC) and GC-MS were used to analyze the structures of pollen polysaccharides;The simulation experiments in vitro and MTT assay were used to determine the effects of different types of pinus pollen polysaccharides on the effects on scavenging active oxygen free radicals and the growth of three tumor cell lines Sp2/0,HepG2 and Hela.The main findings are as follows:1.The extraction process of pollen polysaccharides in pinus massonianaThe influence of methods of pollen-wall broken,temperature,the ratio of solid to liquid,extraction time,and extraction times for extracting pinus massoniana pollen polysaccharides were tested.On the basis of single-factor experiments,the optimum extracting technology was obtained through orthogonal desingn.That was pollen breaking by superfine comminution,extraction for 2 hrs,extracting temperature 85℃, the ratio of solid to liquid 1:15,extracting for 4 times.The highest yield of pollen polysaccharides reached 1.70 percent.2.Isolation and purification of pinus massoniana pollen polysaccharidesDEAE-SephadexA-25 chromatography was used for a preliminary separation on crude pollen polysaccharides,and Sephadex G-75 gel column chromatography was selected for a further classification.The crude pollen polysaccharide passed through DEAE-Sephadex A-25 chromatography column after precipitated by 95%ethanol and four components-MTP1,MTP2,MTP3 and MTP4 were produced.After dialysis, enrichment,freeze-dried,light yellow powders were gotten,with the yield of 27.18%, 7.03%,9.22%,19.60%.MTP1-A and MTP4-A were purified from MTP-1 and MTP-4 through the Sephadex G-75 gel chromatography column,with the yield of 33.0 percent and 26.0 percent respectively.3.Preliminary analysis of the structures of MTP1-A,MTP4-AThe preliminary analysis of structure of the pollen polysaccharide was finished through infrared spectroscopy,UV scanning analysis,gas chromatography and GC-MS analysis.The results showed that:MTP1-A is composed of mannose,glucose and arabinose,and the molar ratio of monosaccharide composition is 1:0.43:0.33; MTP4-A is composed of galactose,arabinose,rhamnose and mannose,and the molar ratio is 1:0.30:0.14:0.11 respectively.GC-MS analysis showed that there are furan rings and pyrans in two pure polysaccharides.UV scanning indicated there is no sugary protein in pollen polysaccharides.I₂-KI reaction showed that there are longer side chains and more branches in polysaccharide andβ-elimination showed that there are O-glucosides.4.Antioxidant and anti-tumor activity of pinus massoniana pollen polysacchari-des in vitroThrough simulation experiments in vitro,we researched the effects on scavenging active oxygen radical of different types of pinus massoniana pollen polysaccharides.The influence of pollen polysaccharides on the growth of three tumor cell lines Sp2/0,HepG2 and Hela by MTT method was tested.The different types of pollen polysaccharides showed certain dose-effect relationships to O₂^-and·OH,but it was not obvious.The scavenging tests on DPPH radical showed a good activity and the clearance rate of crude polysaccharide MTP,purifier polysaccharide MTP1-A and MTP4-A showed good dose-effects.MTP1,MTP2,MTP3,MTP4 inhibited the growth of the tumour cells such as Sp2/0,HepG2 and Hela,and indicated dose-effect and time effect in the range of their concentrations.