高速逆流色谱分离纯化枸杞多糖在神经炎症中的作用

郝高峰; 曹妤; 赵宁; 刘建飞; 邸多隆

doi:10.13386/j.issn1002-0306.2023110273

高速逆流色谱分离纯化枸杞多糖在神经炎症中的作用

Separation and Purification of Lycium barbarum Polysaccharides in Neuroinflammation by High-speed Countercurrent Chromatography

摘要

摘要: 目的：构建高速逆流色谱技术分离纯化枸杞多糖的新方法，并对分离得到的枸杞多糖进行结构表征，探究其对神经炎症的影响。方法：通过分配系数K值和固定相保留率S_f，筛选并优化双水相体系偶联高速逆流色谱分离纯化枸杞多糖的条件，利用高效分子排阻色谱联用激光光散射仪和示差检测器串联检测法、1-苯基-3-甲基-5-吡唑啉酮柱前衍生结合高效液相色谱法、傅立叶变换红外光谱仪、热重分析仪、扫描电子显微镜对枸杞多糖进行初步结构表征，利用脂多糖诱导的小胶质细胞炎症模型研究枸杞多糖对神经炎症的作用。结果：确定最佳双水相体系为乙醇、饱和硫酸铵、水、异丙醇的质量比为1.4:1.0:2.58:0.3，高速逆流色谱最佳分离纯化条件为转速900 r/min，流动相流速0.8 mL/min。在上述条件下，分离得到3个枸杞多糖组分LBPs-1、LBPs-2和LBPs-3，多糖含量分别为78.35%±1.52%，69.03%±1.71%和62.11%±2.31%，分子量分别为7.40×10⁴ Da，2.73×10⁴ Da和1.47×10⁴ Da，单糖组成分别为甘露糖:鼠李糖:半乳糖醛酸:葡萄糖醛酸:葡萄糖:半乳糖:阿拉伯糖，其中摩尔比分别为1.9:4.9:1.3:8.1:7.6:32.6:41.8、2.1:4.3:1.5:8.3:5.1:28.6:37.9和5.9:4.6:1.1:0.7:13.8:66.8:5.3，同时热稳定性和微观形貌也有明显差异。生物活性研究结果表明，不同分子量的枸杞多糖可通过抑制炎症因子分泌，缓解脂多糖诱导的小胶质细胞炎症。结论：本研究构建了双水相体系偶联高速逆流色谱分离纯化枸杞多糖的新方法，为植物多糖高效分离纯化提供了技术支撑，为枸杞多糖在抑制神经炎症相关疾病中的应用提供了科学依据。

Abstract: Objective: This study constructed a novel method for the separation and purification of polysaccharides from Lycium barbarum polysaccharide (LBPs) by high-speed countercurrent chromatography with aqueoust two-phase system, and investigated its effect on neuroinflammation. Methods: Through the partition coefficient (K) value and stationary phase retention rate S_f, the conditions for separating and purifying LBPs using aqueous two-phase system coupled with high-speed countercurrent chromatography (HSCCC) were screened and optimized. Preliminary structural characterisation of LBPs was performed using high performance size exclusion column-multiangle laser light scatter-refractive index detector (HPSEC-MALLS-RID), 1-phenyl-3-methyl-5-pyrazolone precolumn derivatization coupled to high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analyzer (TG) and scanning electron microscopy (SEM). Moreover, the anti-neuroinflammation effect was investigated on LPS induced BV2 cell model. Results: The optimal biphasic system consisted of ethanol, ammonium sulphate, water and isopropanol had a ratio of 1.4:1.0:2.58:0.3. Under the separation conditions (900 r/min and 0.8 mL/min mobile phase flow rate), three polysaccharide fractions, named LBPs-1, LBPs-2 and LBPs-3, was isolated. Polysaccharide contents were 78.35%±1.52%, 69.03%±1.71% and 62.11%±2.31%. Molecular weights were 7.40×10⁴ Da, 2.73×10⁴ Da and 1.47×10⁴ Da. The monosaccharide composition was as follows: Man:Rha:GalA:GlcA:Glu:Gal:Ara, with the molar ratios of 1.9:4.9:1.3:8.1:7.6:32.6:41.8, 2.1:4.3:1.5:8.3:5.1:28.6:37.9 and 5.9:4.6:1.1:0.7:13.8:66.8:5.3, respectively. There was also a significant difference in terms of thermal stability and micro-morphology. The LBPs exhibited varying degrees of protective activity on LPS-induced BV2 cells. Conclusions: This study would provide technical support for the efficient separation and purification of plant polysaccharides, and scientific basis for the application of LBPs in the inhibition of neuroinflammation related diseases.