补骨脂多糖的分离纯化、结构鉴定及其对RAW264.7巨噬细胞活力的影响

Purification, Structural Identification and Effect on Cell Viability of RAW264.7 Macrophages of Polysaccharides from Psoralea corylifolia L. Fruits

对补骨脂多糖(Psoralea corylifolia polysaccharides,PPs)进行分离纯化,并对多糖的一般理化性质、单糖组成、摩尔质量、红外光谱、核磁共振(nuclear magnetic resonance,NMR)谱(1H-NMR和13C-NMR)、微观结构及其对RAW264.7活力的影响进行研究。利用热水提取乙醇沉淀,除蛋白,经DEAE-52纤维素色谱柱(0.05 mol/LNaCl溶液洗脱)和Sephadex G-100柱色谱分离纯化得到2 种多糖(PPs-1-1和PPs-2-1),均为白色絮状粉末,能溶于水,不溶于乙醇、正丁醇、丙酮、氯仿、石油醚等有机溶剂;考马斯亮蓝染色、斐林试剂反应、三氯化铁反应、碘-碘化钾反应均为阴性,总糖质量分数分别为(94.35±0.23)%和(95.27±0.42)%。PPs-1-1和PPs-2-1均由鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖以不同物质的量比组成,平均摩尔质量分别为3.98×106 g/mol和4.47×106 g/mol;PPs-1-1具有表面光滑且致密的三维层流结构,具有α-和β-构型的呋喃糖苷;PPs-2-1具有表面疏松多孔的层状结构,具有β-构型的吡喃糖苷;PPs-1-1和PPs-2-1质量浓度分别低于0.16 μg/mL和0.01 μg/mL时,可明显激活RAW264.7巨噬细胞的活力。

In this study, purified polysaccharides from the fruits of Psoralea corylifolia L. (PPs) were investigated for theirgeneral physicochemical properties, monosaccharide composition, molecular mass, infrared spectroscopic characteristics,nuclear magnetic resonance (1H-NMR and 13C-NMR) characteristics and microstructure together with their effects on the cellviability of RAW264.7 macrophages. The crude polysaccharides, obtained by hot water extraction and ethanol precipitation,were deproteinized by Sevag’s method and then fractionated into two polysaccharides (PPs-1-1 and PPs-2-1) by DEAE-52with 0.05 mol/L NaCl as the eluent and Sephadex G-100 column chromatography. Both PPs-1-1 and PPs-2-1 were dried intoa white flocculent powder, soluble in water but insoluble in ethanol, n-butanol, acetone, chloroform, petroleum ether andother organic solvents. They were negative to Coomassie brilliant blue staining, and reaction with Fehling reagent, ferricchloride and iodine-potassium iodide, and their total sugar contents were (94.35 ± 0.23)% and (95.27 ± 0.42)%, respectively.Both polysaccharides were composed of rhamnose, arabinose, xylose, mannose, glucose and galactose with different molarratios, and their average molecular masses were 3.98 × 106 and 4.47 × 106 g/mol, respectively. PPs-1-1, showing a threedimensionallaminar structure with a smooth and dense surface, contained α-/β-furanosides. PPs-2-1, having β-pyranosides, exhibited a multilayer lamellar structure with loose and porous surface morphology. PPs-1-1 and PPs-2-1 at concentrationslower than 0.16 and 0.01 μg/mL, respectively could significantly activate RAW264.7 macrophages.