目的 从枳壳粗多糖中分离纯化得到精制枳壳多糖(CALB-1),并对其结构和免疫调节活性进行研究。方法 采用热水提取法提取获得枳壳粗多糖(CALB),通过阴阳离子交换树脂、离子交换琼脂糖凝胶等方法对CALB进行分离纯化,得到CALB-1。采用高效液相色谱法(HPLC)测定CALB-1的相对分子质量。综合运用红外(IR)光谱、高碘酸氧化、Smith氧化降解、甲基化、扫描式电子显微镜(SEM)和原子力显微镜(AFM)等方法考察CALB-1的结构及分子形态。采用体外脾细胞增殖实验及在体对小鼠单核细胞吞噬作用的影响实验测定CALB-1的免疫调节活性。结果 经HPLC测定CALB-1为均一多糖,其相对分子质量为3.28×10⁷。综合化学手段和IR光谱分析得到CALB-1为多支结构的酸性多糖,其主链主要由阿拉伯糖(Ara)、甘露糖(Man)和半乳糖(Gal)构成,且主要以1→、1→4键型存在。分子形态考察结果显示CALB-1为非晶态固体。CALB-1具有促进小鼠脾细胞增殖的作用,并可提高免疫低下模型小鼠的廓清指数(K)值和吞噬指数(α)。结论 CALB-1为多支结构的均一相对分子质量的酸性多糖,且具有较好的免疫调节作用,为枳壳多糖进一步的深入研究奠定了理论基础。

Objective To separate and purify the crude polysaccharide from Aurantii Fructus to obtain CALB-1 and to study the structure of CALB-1 and immunoregulatory activity. Methods A refined CALB-1 was obtained from F. aurantii by hot water extraction, then separated and purified by ion exchange resin and ion exchange agarose gel. The molecular weight of CALB-1 was measured by HPLC. The chemical structure and molecular morphology of CALB-1 were determined by IR, periodate oxidation, methylation analysis, scanning electron microscopy (SEM), and atomic force microscope (AFM). The immunoregulatory activity of CALB-1 was evaluated by splenocyte proliferation and mononuclear-macrophage phagocytic function in hypo-immunologic mice. Results CALB-1 was a homogeneous polysaccharide measured by HPLC and the molecular weight of CALB-1 was estimated to be 3.28 × 10⁷. Chemical and spectroscopic analyses illustrated that CALB-1 was highly branched acidic polysaccharides, contained Ara, Man, and Gal as monosaccharide constitutes, and it consisted of backbone chain of 1→ and 1→4 linkages. Through SEM and AFM observations, we indicated that the molecular morphology of CALB-1 was amorphous solid. Besides, CALB-1 significantly stimulated the splenocyte proliferation in vitro and improved the K value of expurgation index and α value of phagocytic index in comhypo-immunologic mice. Conclusion CALB-1 is highly branched acidic polysaccharides and uniform relative molecular mass. Moreover, CALB-1 could present the certain immune regulation in vivo and in vitro. Our study provides a theoretical basis for the development and utilization of CALB-1.

国家重点基础研究发展计划"973"项目(2013CB531800);国家自然科学基金资助项目(81473351);黑龙江教育厅科学技术研究项目(12521516);黑龙江中医药大学优秀创新人才支撑计划