巴戟天糖聚物的分離純化、結(jié)構(gòu)鑒定及抗骨質(zhì)疏松活性研究
發(fā)布時(shí)間：2018-09-01 16:06
【摘要】：本文對(duì)巴戟天(Morinda officinalis How)中的糖聚物進(jìn)行提取分離,經(jīng)柱層析純化得到均一糖聚物,并對(duì)其結(jié)構(gòu)進(jìn)行研究。同時(shí)對(duì)巴戟天糖聚物的體內(nèi)、體外抗骨質(zhì)疏松活性進(jìn)行研究。首先采用熱水/堿液將巴戟天中的水溶性糖聚物和堿溶性糖聚物進(jìn)行提取,采用分級(jí)醇沉法得到四個(gè)部位糖聚物MO50、MO70、MO90、MOB,苯酚硫酸法測(cè)得四個(gè)部位的糖含量分別為:68.8%,71.3%,54.3%,32.1%。采用Sevage法對(duì)其中溶解性較好MO70和MO90分別進(jìn)行脫蛋白處理,然后經(jīng)DEAE-Cellulose52陰離子交換柱層析分離,再經(jīng)Sephacryl S-100凝膠柱層析進(jìn)一步純化,得到MOW70-1、MOP70-2、MOW90-1三個(gè)糖聚物。采用高效凝膠滲透色譜法對(duì)其純度進(jìn)行驗(yàn)證,結(jié)果明MOW70-1、MOP70-2、MOW90-1為均一糖聚物,相對(duì)分子量分別是1248 Da、3109 Da、2172 Da。綜合高效離子色譜,紅外光譜,甲基化氣質(zhì)聯(lián)用分析,核磁共振分析等方法得到3個(gè)均一糖聚物的一級(jí)結(jié)構(gòu)特征,結(jié)果表明MOW70-1為果寡糖,MOP70-2、MOW90-1為果聚糖,它們的主鏈為(2→1)連接β-D-果糖,末端為葡萄糖殘基。紫外光譜掃描表明各糖聚物均不含有蛋白質(zhì)和核酸。采用掃描電鏡、圓二色譜對(duì)高級(jí)結(jié)構(gòu)進(jìn)行初步研究,掃描電鏡結(jié)果顯示3個(gè)均一糖聚物微觀形態(tài)各不相同、圓二色譜結(jié)果表明它們均不含有三股螺旋結(jié)構(gòu)。其中兩個(gè)均一糖聚物MOP70-2和MOW90-1的結(jié)構(gòu)均未見報(bào)道。體內(nèi)抗骨質(zhì)疏松活性研究采用雙側(cè)卵巢摘除法建立原發(fā)性骨質(zhì)疏松模型,通過(guò)檢測(cè)巴戟天糖聚物對(duì)大鼠骨密度、骨量、骨生物力學(xué)性能、尿液及血清生化指標(biāo)、骨微觀結(jié)構(gòu)及參數(shù)的影響來(lái)評(píng)價(jià)其抗骨疏松活性,結(jié)果表明各部位巴戟天糖聚物均能顯著增加去卵巢大鼠股骨、腰椎骨的骨密度和骨量,尤其是MO50組對(duì)于全股骨和遠(yuǎn)端股骨骨密度的提升作用、MO50和MO90組對(duì)腰椎骨骨量的提升作用明顯強(qiáng)于強(qiáng)于陽(yáng)性組E2;提高股骨、腰椎骨的生物力學(xué)性能,增強(qiáng)股骨和椎骨承受外力的能力,其中MO50和MO90組對(duì)大鼠生物力學(xué)性能的提升效果更強(qiáng);改善尿液及血清中骨轉(zhuǎn)換指標(biāo),提升骨轉(zhuǎn)換之間的平衡;修復(fù)損壞的骨微觀結(jié)構(gòu),增加骨小梁數(shù)量、厚度,其中MO50、MO90、MOB組修復(fù)效果更為突出;改善股骨干骺端相關(guān)物理參數(shù),逆轉(zhuǎn)骨質(zhì)疏松大鼠骨結(jié)構(gòu)退變。體外促進(jìn)骨形成活性研究表明3個(gè)巴戟天糖聚物都能促進(jìn)MC3T3-E1細(xì)胞增殖,其中MOP70-2效果更為顯著。選擇MOP70-2繼續(xù)測(cè)定其對(duì)成骨細(xì)胞分化、礦化及成骨相關(guān)基因量表達(dá)量的影響,結(jié)果顯示MOP70-2能夠顯著提高M(jìn)C3T3-E1細(xì)胞的ALP活性和礦化能力并表現(xiàn)出濃度依賴性,明顯上調(diào)成骨相關(guān)基因的表達(dá)量。大鼠體內(nèi)抗骨質(zhì)疏松實(shí)驗(yàn)和體外促成骨細(xì)胞活性實(shí)驗(yàn)顯示,巴戟天糖聚物通過(guò)改善骨吸收和骨形成之間的平衡來(lái)發(fā)揮治療大鼠骨質(zhì)疏松癥的作用,其中MO50和MO90的體內(nèi)活性最為顯著,MOP70-2的體外活性更強(qiáng)。
[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.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R914;R96
本文編號(hào)：2217690
[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.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R914;R96
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