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

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 Sf, 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.

Keywords

• high speed countercurrent chromatography (hsccc)
• aqueous two-phase system
• lycium barbarum polysaccharide
• separation and purification
• structure analysis
• neurological inflammation