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目的建立板蓝根多糖的系统分离纯化方法,并对分离得到的均一多糖进行组成测定。方法以抗病毒活性较好的80%醇沉板蓝根粗多糖为研究对象,对其进行DEAE-Sepharose Fast Flow柱色谱、Sephacryl-200葡聚糖凝胶柱色谱以及高效凝胶色谱系统分离纯化。分别采用1-苯基-3-甲基-5-吡唑啉酮(PMP)和邻苯二甲醛(OPA)衍生化HPLC法对分离得到的板蓝根均一多糖进行单糖组成和氨基酸组成测定。结果利用系统分离纯化策略,从80%醇沉板蓝根粗多糖中分离得到2种均一板蓝根多糖(IRPS1A和IRPS1B)和2种均一板蓝根糖蛋白(IRPS2A和IRPS3A)。其中,IRPS1A与IRPS1B的单糖组成均为甘露糖、葡萄糖、半乳糖和阿拉伯糖;IRPS2A的单糖组成为半乳糖醛酸、葡萄糖、半乳糖、阿拉伯糖;IRPS3A的单糖组成为甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖。糖蛋白IRPS2A与IRPS3A均含有天门冬氨酸、谷氨酸、丝氨酸、组氨酸、甘氨酸、苏氨酸、精氨酸、丙氨酸、胱氨酸、缬氨酸、苯丙氨酸、异亮氨酸、亮氨酸、赖氨酸,共14种氨基酸残基。同时,IRPS2A还含有酪氨酸残基。结论该系统分离纯化的策略可应用于板蓝根均一多糖(糖蛋白)的获取,为板蓝根多糖的结构分析及药理活性研究奠定基础。
OBJECTIVE To establish a systematic method for the isolation and purification of polysaccharides from Radix Isatidis, and to determine the composition of the isolated polysaccharides. Methods 80% ethanolic crude Radix Isatidis with high antiviral activity was used as the research object, which was separated and purified by DEAE-Sepharose Fast Flow column chromatography, Sephacryl-200 dextran gel column chromatography and high performance gel permeation chromatography. The monosaccharide composition and amino acid composition of the isolated polysaccharides were determined by 1-phenyl-3-methyl-5-pyrazolone (PMP) and phthalaldehyde (OPA) Results Two kinds of homo-IRP (IRPS1A and IRPS1B) and two kinds of uniform IRP (IRPS2A and IRPS3A) were isolated from 80% ethanolic Radix Isatidis by means of systematic isolation and purification. The monosaccharide compositions of IRPS1A and IRPS1B were mannose, glucose, galactose and arabinose. The monosaccharide composition of IRPS2A was galacturonic acid, glucose, galactose and arabinose. The monosaccharide composition of IRPS3A was mannose, Rhamnose, galacturonic acid, glucose, galactose and arabinose. Both glycoproteins IRPS2A and IRPS3A contain aspartate, glutamate, serine, histidine, glycine, threonine, arginine, alanine, cystine, valine, phenylalanine, Leucine, leucine, lysine, a total of 14 kinds of amino acid residues. Meanwhile, IRPS2A also contains tyrosine residues. Conclusion The isolation and purification strategy of this system can be applied to the acquisition of uniform polysaccharide (glycoprotein) from Radix isatidis, which lays a foundation for the structural analysis and pharmacological activity of Radix Isatidis.