远志多糖除蛋白工艺优化、单糖组成分析及神经保护研究

Optimization of Protein Removal Process, Monosaccharide Composition Analysis and Neuroprotective Activity of Polygala Tenuifolia Polysaccharide

关键词:  远志多糖  除蛋白  单糖组成  神经保护

Key words:  Polygala tenuifolia polysaccharide  deproteinization  monosaccharide composition  neuroprotection

摘要:

目的 研究远志多糖最佳除蛋白工艺以及单糖组成，对远志多糖的神经保护活性进行初步探讨。方法 采用热水浸提法提取远志总多糖，从酶法、Sevag法、酶-Sevag法以及三氯乙酸（trichloroacetic acid，TCA）-正丁醇法4种除蛋白方法中筛选出远志总多糖除蛋白的最佳方法，并用响应面法优化该方法得到远志总多糖除蛋白最佳工艺。将除蛋白后的远志总多糖超滤纯化得到远志多糖。将远志多糖完全酸水解后，经1-苯基-3-甲基-5-吡唑啉酮（PMP）衍生后，用HPLC研究远志多糖的单糖组成及比例。用H₂O₂诱导损伤的PC12神经细胞研究远志多糖神经的保护作用。结果 TCA-正丁醇法是远志总多糖最佳除蛋白方法，经响应面优化，远志总多糖TCA-正丁醇法除蛋白的最佳工艺为TCA：正丁醇=1：7.5，远志总多糖溶液：TCA-正丁醇溶液=1：4.0，振摇时间为33.0 min。远志多糖由半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖4种单糖组成，其摩尔比为2.37：4.79：1：2.97。远志多糖对PC12细胞增殖没有明显的毒性，且对H₂O₂诱导损伤的PC12细胞有保护作用。结论 TCA-正丁醇法能有效除去远志总多糖的蛋白质，远志多糖由半乳糖醛酸、半乳糖、葡萄糖和阿拉伯糖组成，且有神经保护作用。

Abstract:

OBJECTIVE To study the best protein removal process and monosaccharide composition of Polygala tenuifolia polysaccharide, and to preliminarily explore the neuroprotective activity of Polygala tenuifolia polysaccharide. METHODS The total polysaccharide of Polygala tenuifolia was extracted by hot water extraction. The best method for protein removal of total polysaccharide of Polygala tenuifolia was selected from four methods:enzyme method, Sevag method, enzyme-Sevag method and trichloroacetic acid(TCA)-n-butanol method. The method was optimized by response surface methodology to obtain the best process for protein removal of total polysaccharide of Polygala tenuifolia. Polygala tenuifolia polysaccharide was purified by ultrafiltration after protein removal. After complete acid hydrolysis of Polygala tenuifolia polysaccharide and derivatization by 1-phenyl-3-methyl-5-pyrazolone(PMP), the monosaccharide composition and proportion of Polygala tenuifolia polysaccharide were studied by HPLC. The protective effect of Polygala tenuifolia polysaccharide on PC12 nerve cells injured by H₂O₂ was studied. RESULTS TCA-n-butanol method was the best method for protein removal of Polygala tenuifolia polysaccharide. Through response surface optimization, the best process for protein removal of total Polygala tenuifolia polysaccharide by TCA-n-butanol method was TCA:n-butanol=1:7.5, Polygala tenuifolia polysaccharide solution:TCA-n-butanol solution=1:4.0, shaking time was 33.0 min. Polygala tenuifolia polysaccharide was composed of galacturonic acid, glucose, galactose and arabinose, and its molar ratio was 2.37:4.79:1:2.97. Polygala tenuifolia polysaccharide had no obvious toxicity to the proliferation of PC12 cells, and had protective effect on PC12 cells damaged by H₂O₂.CONCLUSION TCA-n-butanol method can effectively remove the protein of Polygala tenuifolia polysaccharide, which is composed of galacturonic acid, galactose, glucose and arabinose, and has neuroprotective effect.