Study On Extraction, Purification And Anti-Oxidant Activity Of The Polysaccharide Isolated From Trollious Chinensis

Trollius chinensis, dried flowers of Trollius chinensis Bunge, has been used as an important traditional Chinese medicine in folk for a long history, which widely grows in the southwest, northwest, northeast. Bencao Gangmu Shiyi said that it was "bitter in taste, cold-natured, asepsis" and could be used for the treatment of aphtha, larynxboss, light fever atrophy of the gum, ear ache, ophthalmalgia, and it had the effects on eyesight improvement and anti-miasma. Modern pharmacological studies show that it possesses antimicrobial and antiviral functions and has been used widely to treat cold, fever, chronic tonsillitis, acute tympan-itis, urinary tract infection and other inflammations. Currently the studies on Trollious chinensis are only limited to small molecules. And the study on active polysaccharide has not been reported yet. In this project, Trollious chinensis from Zhangjiakou Bashang were used as experimental materials. Firstly, the optimum extraction techniques of polysaccharide from Trollious chinensis was estabilished. Secondly, one kind of optimum decoloration and deprotein mefhod for Trollious chinensis polysaccharide was presented. Thirdly, isolation and purification, molecular weight and monosaccharide were studied systematically. Finally, the work focused on antioxidant activity assay. This study provides basical information for development and utilization of Trollious chinensis resource efficiently.1The extraction process of polysaccharide from Trollious chinensisThree different methods of reflux extraction, ultrasonic extraction and supercritical CO2fluid extraction were respectively adapted to extract polysaccharide from Trollius chinensis. The best extraction technology was selected by taking the extraction ratio of polysaccharide as index. 2Decolorization and deproteinization technology of polysaccharide from Trollius chinensisFive kinds of resin-AB-8、D-101、DM-301、717and732were evaluated, with decoloration rate, protein removal rate, total sugar retention rate and compositive weighted score as index. Then, one kind of resin was confirmed to carry out single-factor experiments. The orthogonal tests L9(33) were carried out based on single factor test to choose the optimal condition of decolorization and deproteinization of polysaccharide.3Isolation, purification and structural analysis of polysaccharide from Trollius chinensisA DEAE-52cellulose ion exchange column a Sephadex G-150gels filter column were successively used to further separate crude polysaccharides, and then determining the product purity and molecular weight with high performance gels filtration chromagraphy(HPGFC). High performance liquid Chromatography(HPLC) was applied to study the momosaccharide composition of Trollius chinensis polysaccharide.4Study on the antioxidation activity of polysaccharide from Trollius chinensisAntioxidative capability of polysaccharide was determined by salicylic-Fe2+-H2O2system,1,1-Diphenyl-2-picrylhydrazyl radical2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl(DPPH) system, and means of pyrogallol oxidation.The research results were as follows:1the yield of reflux extraction was prefer to those of ultrasonic extraction and supercritical CO2fluid extraction. The yield were in turn1.21%,0.24%and0.00187%. Then the optimal reflux extraction conditions was determined with single-factor method and orthogonal method. The results indicated as following:the ratio of solution to material20, time3h, temperature95℃, and the rate of extracting is1.45%. 2AB-8resin can be used as a high effective one to romove pigment and protein of Trollius chinensis polysaccharide. The optimal technological parameters were as follows:the ratio of resin and crude polysaccharides solution (the concentration of polysaccharides solution was5mg·mL-1) was0.06g·mL-1, temperature40℃, time1.5h. The decoloration rate was75.4%, the protein removal rate was88.2%, total sugar retention rate was85.7%.3Four fractions of TCBP1, TCBP2, TCBP3and TCBP4were obtained after fractionating through DEAE-52cellulose and Sephadex G-150column. TCBP1, with a relative molecular weight of3283220Da, consisted of Rha, Gal, Ara, with the molar ratio of0.84:1:1.16. TCBP2, with a relative molecular weight of156819Da, consisted of Man, Rha, GlcUA, GalUA, Gal, Ara, with the molar ratio of1.36:0.45:1:0.29:5.68:1.46. TCBP3, with a relative molecular weight of10949Da, consisted of Rha, GlcUA, GalUA, Glc, Gal, Ara, with the molar ratio of0.86:0.15:1:0.12:3.54:2.17. TCBP4, with a relative molecular weight of1068Da, consisted of Rha, GlcUA, GalUA, Glc, Gal, Ara, with the molar ratio of0.96:1.23:1:0.42:4.10:3.46.4TCBP1, TCBP2, TCBP3and TCBP4all exhibited strong scavenging capability to hydroxyl radical. The sequence from strong to weak of this effects is:TCBP2>TCBP3>TCBP1>TCBP4. Clearance ratio of TCBP2and TCBP3rise to the maximum, by99.3and86.9per cent, at0.30mg·ml-1of solution concentration. With regard to Inhibiting DPPH, TCBP2and TCBP3had certain effect. TCBP1and TCBP4had no effect on cleaning DPPH. For scavenging·O2, They all had no effects on scavenging superoxide anion free radicals. Instead, The rate of pyrogallor autoxidation had an ascending tread with the increasing of polysaccharide concentration.