金蝉花多糖的含量组成分析及其对LPS诱导的THP-1细胞NF-κB活性的研究

Polysaccharide Content and Composition Analysis of Golden Cicada Flowers and Effect on NF-κB Activity of LPS Inducted THP-1 Cells

目的:阐明金蝉花多糖的糖含量及单糖组成,研究其对脂多糖(Lipopolysaccharide,以下简称LPS)诱导的THP-1细胞NF-κB的活性。方法:以金蝉花为原料药材,经脱脂干燥、水提、浓缩、透析、乙醇沉淀和真空干燥后,得到金蝉花水提粗多糖。然后经二乙氨乙基纤维素(DEAE-纤维素)阴离子交换柱纯化获得4种纯化多糖。应用紫外分光光度计、高效凝胶渗透色谱(HPGPC)和气相色谱(GC)方法对其中性糖含量、糖醛酸含量,相对分子质量和单糖组成进行测量分析,同时对4种纯化金蝉花多糖进行了体外细胞免疫活性测定。结果:金蝉花粗多糖的中性糖含量为48.36%,糖醛酸含量为12.39%,总多糖含量为60.75%,相对分子质量为9.3×103Da,单糖组成物质的量比分别为阿拉伯糖(Ara)∶木糖(Xyl)∶甘露糖(Man)∶半乳糖(Gal)∶葡萄糖(Glc)=0.42∶0.29∶4.14∶6.38∶6.8。4种金蝉花纯化多糖中,只有0.2 mol·L^(-1)氯化钠(NaCl)溶液洗脱的金蝉花纯化多糖(CSTA2)在高浓度(1000μg·mL^(-1))时具有较强的免疫抑制作用,其对NF-κB的活化率仅为77.2%,不仅远低于阳性对照LPS组,而且比空白对照还要低。高浓度(1000μg·mL^(-1))和低浓度(100μg·mL^(-1))的蒸馏水洗脱金蝉花纯化多糖(CSTA0)均具有较强的免疫抑制作用,且显示良好的浓度依赖性,浓度越低抑制作用越强。结论:金蝉花粗多糖主要由葡萄糖(Glc)、半乳糖(Gal)、甘露糖(Man)和少量的阿拉伯糖(Ara)和木糖(Xyl)组成。金蝉花纯化多糖对LPS诱导THP-1细胞产生的NF-κB的抑制作用随着质量浓度的升高和(或)洗脱剂种类和浓度的不同会发生明显变化,高浓度(1 000μg·mL^(-1))的0.2 mol·L^(-1) NaCl溶液洗脱金蝉花纯化多糖(CSTA2)和低浓度(100μg·mL^(-1))的蒸馏水洗脱金蝉花纯化多糖(CSTA0)具有较强的免疫抑制作用,具有较高的肿瘤抑制活性,具有深入研究的价值。

Objective： To clarify golden cicada flower polysaccharide content and monosaccharide composition of polysaccharides, and study the nf-kappa B activity of THP1 cells induced by LPS. Methods： Polysaccharide from golden cicada flower was obtained by water extraction, dialysis purification, ethanol precipitation and vacuum drying, and further purified by DEAE cellulose-anion exchange column. 4 kinds of purified polysaccharides were obtained. HPGPC and GC were applied to determine uronic acid content, the relative molecular mass and the monosaccharide composition. 4 kinds of polysaceharides were tested for LPS induced the nf-kappa B signals of THP1 cells. Results： The neutral polysaccharide content of crude Golden cicada flower was 48.36%, the polysaccharide uronie acid content was 12. 39%. The total polysaccharide content of crude Golden cicada flower was 60. 75%. The relative molecular mass was 9. 3×10^3 Da, molar ratio of monosaccharide composition was Xyl-Man-Gal-Glc （0. 42：0. 29：4. 14：6. 38：6. 8）. Polysaccharide purified by O. 2 mol. L-1 NaCl solution elution （CSTA2） at high concentration （1000μg·mL^-1） showed the strong immune inhibitory effect, the activation rate reached only 77.2%, which was not only far lower than the positive control LPS group, and even lower than ck. High concentration （ 1000 μg·mL^-1 ） and distilled water of low concentration （ 100μg·mL^-1 ） elution golden cicada flower purified polysaccharide （CSTA0） showed lower activation rate, and a good concentration dependence. Conclusion： Golden cicada polysaccharide is mainly composed of glucose （Glc）, galactose （Gal）, mannose （Man） and a small amount of arabinose （Ara） and xylose （Xyl）. The purified polysaccharides shows THP 1 cells induced by LPS nf-kappa B inhibition. The purified polysaccharide CSTA2 and CSTA0 shows stronger immune inhibition, and deserve for further study.