巴戟天糖聚物的分离纯化、结构鉴定及抗骨质疏松活性研究

发布时间：2018-09-01 16:06

【摘要】：本文对巴戟天(Morinda officinalis How)中的糖聚物进行提取分离,经柱层析纯化得到均一糖聚物,并对其结构进行研究。同时对巴戟天糖聚物的体内、体外抗骨质疏松活性进行研究。首先采用热水/碱液将巴戟天中的水溶性糖聚物和碱溶性糖聚物进行提取,采用分级醇沉法得到四个部位糖聚物MO50、MO70、MO90、MOB,苯酚硫酸法测得四个部位的糖含量分别为:68.8%,71.3%,54.3%,32.1%。采用Sevage法对其中溶解性较好MO70和MO90分别进行脱蛋白处理,然后经DEAE-Cellulose52阴离子交换柱层析分离,再经Sephacryl S-100凝胶柱层析进一步纯化,得到MOW70-1、MOP70-2、MOW90-1三个糖聚物。采用高效凝胶渗透色谱法对其纯度进行验证,结果明MOW70-1、MOP70-2、MOW90-1为均一糖聚物,相对分子量分别是1248 Da、3109 Da、2172 Da。综合高效离子色谱,红外光谱,甲基化气质联用分析,核磁共振分析等方法得到3个均一糖聚物的一级结构特征,结果表明MOW70-1为果寡糖,MOP70-2、MOW90-1为果聚糖,它们的主链为(2→1)连接β-D-果糖,末端为葡萄糖残基。紫外光谱扫描表明各糖聚物均不含有蛋白质和核酸。采用扫描电镜、圆二色谱对高级结构进行初步研究,扫描电镜结果显示3个均一糖聚物微观形态各不相同、圆二色谱结果表明它们均不含有三股螺旋结构。其中两个均一糖聚物MOP70-2和MOW90-1的结构均未见报道。体内抗骨质疏松活性研究采用双侧卵巢摘除法建立原发性骨质疏松模型,通过检测巴戟天糖聚物对大鼠骨密度、骨量、骨生物力学性能、尿液及血清生化指标、骨微观结构及参数的影响来评价其抗骨疏松活性,结果表明各部位巴戟天糖聚物均能显著增加去卵巢大鼠股骨、腰椎骨的骨密度和骨量,尤其是MO50组对于全股骨和远端股骨骨密度的提升作用、MO50和MO90组对腰椎骨骨量的提升作用明显强于强于阳性组E2;提高股骨、腰椎骨的生物力学性能,增强股骨和椎骨承受外力的能力,其中MO50和MO90组对大鼠生物力学性能的提升效果更强;改善尿液及血清中骨转换指标,提升骨转换之间的平衡;修复损坏的骨微观结构,增加骨小梁数量、厚度,其中MO50、MO90、MOB组修复效果更为突出;改善股骨干骺端相关物理参数,逆转骨质疏松大鼠骨结构退变。体外促进骨形成活性研究表明3个巴戟天糖聚物都能促进MC3T3-E1细胞增殖,其中MOP70-2效果更为显著。选择MOP70-2继续测定其对成骨细胞分化、矿化及成骨相关基因量表达量的影响,结果显示MOP70-2能够显著提高MC3T3-E1细胞的ALP活性和矿化能力并表现出浓度依赖性,明显上调成骨相关基因的表达量。大鼠体内抗骨质疏松实验和体外促成骨细胞活性实验显示,巴戟天糖聚物通过改善骨吸收和骨形成之间的平衡来发挥治疗大鼠骨质疏松症的作用,其中MO50和MO90的体内活性最为显著,MOP70-2的体外活性更强。
[Abstract]:In this paper, the polysaccharides from Morinda officinalis How were extracted and separated, purified by column chromatography, and their structures were studied. At the same time, the anti-osteoporosis activities of Morinda officinalis polysaccharides in vivo and in vitro were studied. Firstly, the water-soluble and Alkali-soluble Polysaccharides from Morinda officinalis How were prepared by hot water/alkali solution. The polysaccharides of MO50, MO70, MO90, MOB and phenol-sulfuric acid were obtained by fractional alcohol precipitation. The contents of sugar in the four parts were 68.8%, 71.3%, 54.3% and 32.1% respectively. The MO70 and MO90 with better solubility were separated by DEAE-Cellulose 52 anion exchange column chromatography. The purity of MOW70-1, MOP70-2 and MOW90-1 was verified by high performance gel permeation chromatography. The results showed that MOW70-1, MOP70-2 and MOW90-1 were homogeneous polymers with relative molecular weights of 1248 Da, 3109 Da and 2172 Da, respectively. The results showed that MOW70-1 was fructooligosaccharide, MOP70-2 and MOW90-1 were fructo-oligosaccharides. Their main chains were (2_1) linked with beta-D-fructose and their ends were glucose residues. Scanning electron microscopy (SEM) and circular dichroism (CD) were used to study the high-level structure of the three monosaccharides. The results of scanning electron microscopy (SEM) and circular dichroism (CD) showed that the three monosaccharides did not contain triple helix structure. The structures of two monosaccharides, MOP70-2 and MOW90-1, were not reported. The effects of Morinda officinalis polysaccharides on bone mineral density, bone mass, bone biomechanical properties, urine and serum biochemical indices, bone microstructure and parameters were examined to evaluate their anti-osteoporosis activity. The results showed that Morinda officinalis polysaccharides in different parts of the ovariectomized rats could significantly increase the femur of the ovariectomized rats. Bone mineral density and bone mass of bone and lumbar vertebrae, especially of MO50 and MO90, were significantly higher than those of E2. The biomechanical properties of femur and lumbar vertebrae were improved, and the ability of femur and vertebrae to bear external force was enhanced in MO50 and MO90 groups. The biomechanical properties were improved more effectively; the bone turnover indexes in urine and serum were improved to balance the bone turnover; the damaged bone microstructure was repaired and the number and thickness of bone trabeculae were increased, especially in the MO50, MO90 and MOB groups; the related physical parameters of femoral metaphysis were improved to reverse the bone structure degeneration of osteoporotic rats. Three Morinda officinalis polysaccharides could promote the proliferation of MC3T3-E1 cells in vitro, especially MOP70-2. The effects of MOP70-2 on osteoblast differentiation, mineralization and expression of osteogenesis-related genes were determined. The results showed that MOP70-2 could significantly increase the ALP activity and mineralization of MC3T3-E1 cells. Anti-osteoporosis test in vivo and osteoblast-promoting activity test in vitro showed that Morinda officinalis polysaccharides played an important role in the treatment of osteoporosis in rats by improving the balance between bone resorption and bone formation, including the in vivo activities of MO50 and MO90. The most significant activity of MOP70-2 was stronger in vitro.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914;R96

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