金莲花多糖的分离纯化及其抗氧化活性研究

Purification and Anti-oxidant Activity of the Polysaccharide Isolated from Trollius Chinensis

目的:对金莲花粗多糖进行分离纯化,研究纯化多糖的体外抗氧化作用,为进一步开发金莲花中药资源提供实验依据。方法:常规回流提取、乙醇沉淀得到金莲花粗多糖,经AB-8大孔吸附树脂脱色、脱蛋白后得精制的金莲花多糖(Trollius chinensis Bunge polysaccharide,TCBP),以蒸馏水、不同浓度的Nacl和NaOH溶液为洗脱剂,应用DEAE-52纤维素离子交换柱层析,Sephadex G-150凝胶过滤柱层析对得到的主要组分多糖进一步分离纯化,采用高效凝胶过滤色谱(high performance gels filtration chromagraphy,HPGPC)鉴定所得组分纯度并测定其相对分子质量,通过高效液相色谱(high performance liquid chromatography,HPLC)分析多糖的单糖组成。通过水杨酸-Fe^(2+)-H_2O_2体系(·OH)、二苯代苦味肼基自由基(1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl,DPPH)体系和邻苯三酚自氧化(·0_2^-)法对分离纯化的金莲花多糖组分的抗氧化能力进行研究。结果:得到TCBP1,TCBP2,TCBP3,TCBP4四个多糖组分均为杂多糖,其中TCBP1分子量为3 283 220 Da、TCBP2分子量为156 819 Da、TCBP3分子量为10 949 Da、TCBP4分子量为1 068 Da;TCBP1,TCBP2,TCBP3,TCBP4均具有清除·OH的能力,其强弱顺序为:TCBP2>TCBP3>TCBP1>TCBP4,TCBP2和TCBP3对DPPH具有一定清除作用,TCBP1,TCBP2,TCBP3,TCBP4均没有清除·O_2^-的能力。相反,还能诱导·O_2^-的产生,且随着它们浓度的增大,邻苯三酚自氧化程度也增强。结论:金莲花多糖为复杂的杂聚多糖,且具有一定的抗氧化活性。

Objective： To study purification and anti-oxidant activity of the polysaccharide Isolated from Trollius chinensis. This study provides basic information to develop and utilize Trollius chinensis resource efficiently. Methods：The methods of reflux extraction were adapted to extract polysaccharide from Trollius chinensis.,AB-8 resin can be used as a high effective one to romove pigment and protein of Trollius chinensis Bunge polysaccharide （TCBP）. DEAE-52 cellulose ion exchange column,a Sephadex G-150 gels filter column were successively used to further separate crude polysaccharides,and then determining the product purity and molecular weight with high performance gels filtration chromatography （HPGPC）. High performance liquid chromatography（HPLC）was applied to study the momosaccharide composition of TCBP. Antioxidative capability of polysaccharide was determined by salicylicFe2＋-H2O2 system （-OH）, I, 1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1- （2,4, 6-trinitrophenyl） hydrazyl （DPPH）system, and means of pyrogallol oxidation （ · O2-）. Results： Our results showed that four polysaccharides were complex polysaccharide with a variety of monosaccharides. Molecular weight of TCBP1 is 3 283 220 Da,TCBP2,156 819 Da,TCBP3, 10 949 Da and TCBP4,1 068 Da. Four polysaccharides had significant effects on scavenging hydroxyl radical,The sequence from strong to weak of this effects is ：TCBP2〉TCBP3〉 TCBPI〉TCBP4. TCBP2 and TCBP3 had certain effect on 1,1-diphenyl -2- picrylhydrazyl radical. They all induced superoxide radical instead of scavenging. Conelusion：TCBP1, TCBP2, TCBP3 and TCBP4 are complex polysaccharide with a variety of monosaccharides, they have the certain antioxidant activity.