板蓝根多糖的系统分离纯化与组成分析

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。

System isolation and purification of Isatidis Radix polysaccharides and determination of their compositions

Objective To isolate and purify the polysaccharides (glycoproteins) from Isatidis Radix (Banlangen) systematically and to study the composition of them. Methods Crude polysaccharides precipitated by 80% ethanol from the water extract of Isatidis Radix, which has the antiviral activity, were fractionated by DEAE-Sepharose Fast Flow, Sephacryl-200, and high gel chromatography system sequentially. The composition of monosaccharides and amino acids of polysaccharides (glycoproteins) was then determined by HPLC with pre-column derivatization using 1-phenyl-3-methyl-5-pyrazolone (PMP) and o-phthalaldehyde (OPA), respectively. Results Two of homogeneous polysaccharides named IRPS1A and IRPS1B and two of homogeneous glycoproteins named IRPS2A and IRPS3A were obtained from Isatidis Radix by systematical separation and purification. The monosaccharide composition of IRPS1A and IRPS1B was of arabinose, mannose, galactose, and glucose. IRPS2A contained galacturonic acid, glucose, galactose, and arabinose. While IRPS3A contained mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose. The amino acid compositions of IRPS2A and IRPS3A were 14 kinds of amino acid residues including Asp, Glu, Ser, His, Gly, Thr, Arg, Ala, Cys, Val, Phe, Iso, Leu, and Lys. Besides all, IRPS2A also contained Tyr. Conclusion This strategy can be used for the isolation and purification of homogeneous polysaccharides/glycoproteins from Isatidis Radix which provides a possible support for the elucidation of the structure and the pharmacologic action of Isatidis Radix polysaccharides (glycoproteins).