凝集素亲和层析技术分离金花茶多糖的研究

Study on the Lectin Affinity Chromatography of Polysaccharides in Camellia chrysantha(Hu) Tuyama

以蓖麻凝集素(RCA)为亲和配体制备亲和层析树脂,在比较2种固定载体制备亲和树脂对金花茶多糖分离效果的基础上,探讨了多糖上样量、NaCl浓度以及洗涤量对RCA-Sepharose-2B亲和树脂结合率或再生效果的影响,并采用高效液相色谱(HPLC)对亲和多糖进行分离效果分析,为将亲和层析技术应用于金花茶活性多糖的分离纯化提供科学依据。研究结果显示,相比AF-Amino-650 m树脂,RCA-Sepharose 2B亲和树脂的分离效果比较好,而且可以进行反复使用多次。多糖上样量显著影响多糖与树脂的亲和效果,多糖结合率随上样量增加呈倒U型变化,当多糖上样量为10.4 mg/g亲和层析树脂时,其亲合率最高,达到57%左右;NaCl浓度对树脂的再生效果也有显著影响,用含0.2 mol/L NaCl的磷酸缓冲液洗涤树脂可以有效恢复树脂对多糖的亲和效果。HPLC分析结果显示分离获得的亲和活性多糖是由3种不同分子量的多糖组分组成。

Using ricinus agglutinin （RCA） as ligand, two kinds of lectin affinity chromatography resins RCA-Amino and RCA-Sepharose 2B were made, based on the comparison of the two resins＇ separation effect on polysaccharide, the effect of the loading amount of polysaccharide, the concentration of sodium chloride （NaCl） and the dosage of washing liquid for regeneration on binding rate or regeneration effect of the RCA-Sepharose 2B were studied, and separation effect of Camellia chrysantha polysaccharide was studied by high performance liquid chromatography （HPLC） , so as to provide scientific theory bases for the separation of Camellia chrysantha polysaccharide by lectin affinity chromatography technology. The results showed that the separation effect of lectin affinity chromatography resin RCA-Sepharose 2B was better, and could be used multiple times. The loading amount of polysaccharide significantly affected the binding rate of polysaccharide with affinity chromatography resin; the binding rate curve was inverted U-shaped as the increase of loading amount of polysaccharide; when the loading amount was 10.4 mg/g resin, the binding rate of Camellia chrysantha polysaccharide with RCA-Sepharose 2B was biggest （57%） ; the regeneration effect of lectin affinity chromatography resin was significantly affected by NaCl concentration, affinity effect of lectin affinity chromatography resin with polysaccharide could be regenerated by 0.2 M NaCl solution. HPLC analysis showed that the isolated polysaccharide was composed of three kinds ofpolysaccharide with different molecular weight.