草叶马尾藻多糖的制备及其抗草酸钙结石作用研究
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摘要
泌尿系统结石是一种世界范围的常见病,其中大多数为草酸钙结石,为了治疗泌尿系统结石和降低其复发率,从海洋中寻找新的活性物质来满足临床需要意义重大。草叶马尾藻(Sargassum graminifolium)是广泛分布在我国东海及南海的一种褐藻,本课题主要研究了其多糖(polysaccharides from Sargassum graminifolium, SGP)的制备及其理化性质、体外抗氧化活性与抑制草酸钙结晶活性,并从抗氧化角度、减少草酸钙结晶沉积和保护线粒体功能等角度探讨了其体内治疗高草酸结石模型大鼠的功效和作用机制。具体阐述如下：
首先对于SGP的制备,基于制药和保健应用的考虑,本文采用单因素和正交试验方法,确定了SGP的超声辅助提取工艺和脱蛋白脱色工艺。对于SGP的理化性质,运用了紫外,红外光谱,质谱和薄层色谱以及电泳等多种分析手段,测定了其硫酸基、糖醛酸、蛋白质的含量(硫酸基含量为13.2%,糖醛酸含量为10.11%,蛋白质为1.03%)和SGP的分子量(11887Da),初步推测SGP可能是由葡萄糖,半乳糖和甘露糖醛酸组成的硫酸多糖。
其次,为了分析SGP的体外抗氧化活性,分别测定了SGP的还原能力,清除超氧阴离子自由基能力和对DPPH自由基的抑制能力。结果表明SGP的浓度与以上3种抗氧化能力存在明显的量效关系,SGP对超氧阴离子清除活性的ICso为1.9mg/mL,对DPPH自由基的清除活性的IC50为0.56mg/mL。其中清除DPPH自由基活性显著优于已有研究的马尾藻多糖。
再次,研究了SGP体外抑制草酸钙结晶的活性。考察了影响草酸钙成核、生长和聚集过程中的关键指标：最大诱导时间,成核抑制率和聚集抑制率,并对草酸钙结晶形貌进行了观测。此外,对添加不同浓度的SGP后的草酸钙结晶混悬液进行了Zeta电位测定,以发现SGP浓度与草酸钙微粒表面带的负电荷之间的关系。结果表明与柠檬酸三钠相比,SGP抑制草酸钙结晶的效果较佳,SGP对草酸钙的成核、聚集抑制率分别为69.2%、76.8%。
最后,为研究SGP治疗大鼠草酸钙结石的功效和作用机制,本文建立了高草酸导致的大鼠肾损伤的草酸钙结石模型,使用SGP分别以高、中、低剂量对患有结石的大鼠进行治疗。测定了各组大鼠的尿液生理指标及血清生化指标；运用组织切片方法观察了肾组织中草酸钙结晶沉积的情况,并使用各类相关酶活力测试盒和丙二醛(MDA)测试盒,分别对各组大鼠肾组织线粒体的ATP酶活力、过氧化氢酶活力、谷胱甘肽过氧化酶活力、超氧化物歧化酶活力、琥珀酸脱氢酶活力、线粒体肿胀度测定和MDA的水平进行了测定。在此基础上阐明了SGP通过抗氧化、减少草酸钙结晶沉积和保护线粒体功能等多途径作用发挥对高草酸尿症大鼠肾脏的保护作用,并揭示了SGP抗草酸钙结石活性的作用机制,因其具有显著的抗氧化活性并能调节大鼠肾脏的线粒体功能障碍从而能有效治疗草酸钙结石。
Urinary stones affect a large proportion of the population, most of urinary stones are calcium oxalate stones. Therefore, it is important to search for new bioactive compounds to meet the clinical needs that prevent calcium oxalate stones and reduce their recurrence rate. Sargassum graminifolium, a brown seaweed extensively distributed along the coasts of the South and the East China Sea. This thesis researched on preparation of polysaccharides from Sargassum graminifolium (SGP), and its physical and chemical properties, antioxidant effects and also the inhibition of calcium oxalate crystals activity in vitro, furthermore the treatment efficacy and mechanism of SGP on renal injury induced by hyperoxaluria in vivo. The main view is based on antioxidant effects, reduction of calcium oxalate crystal deposition and protection mitochondrial function. The summary as follows:
Firstly for SGP preparation technology, considering pharmaceutical and health care applications, the processes of ultrasonic assisted extraction, deproteinization and decolorization were used, with the methods of single factor and orthogonal array design. In order to identify SGP's physical and chemical properties, the methods include electrophoresis, ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry and thin-layer chromatography were used, got the content of sulfate, glucuronic acid and protein (sulfate13.2%, glucuronic acid10.11%, protein1.03%), and also molecular weight of SGP was obtained(11887Da). We can conclude that SGP is a kind of sulfated polysaccharide composed of glucose, galactose and mannuronic acid.
Secondly, for analyzing the antioxidant activity of SGP in vitro, the reducing power, the ability of scavenge superoxide radicals, and the activity of scavenge DPPH were determinted respectively. The results showed there is obvious dose-effect relationship between concentrations of SGP and the three antioxidant activities, increasing concentrations of SGP resulted in increased scavenging of superoxide anions and DPPH radicals (IC50=1.9and0.56mg/mL, respectively).
Thirdly this thesis researched on the effects of SGP on calcium oxalate crystallization in vivo. We studied the maximum time, the rates of nucleation and aggregation that characterize the crystallization process and also investigated the morphology of calcium oxalate crystals. Furthermore, the Zeta potential measurement of calcium oxalate crystals liquids was tested after adding different concentrations of SGP in order to find the relationship between concentrations of SGP and the negative charge of calcium oxalate particle surface. The results show that SGP'rates of nucleation and aggregation were69.2%and76.8%, and it's better than with trisodium citrate.
At last, this thesis researched the effects of SGP on hyperoxaluria rats and its action mechanisms. For the rats with calcium oxalate stones, they were taken high, middle and low dose of SGP. On following aspects the rats were measured:the urine physiological indicators, the biochemical serum markers, the kidney mitochondria indicators (the activity of ATP enzyme, GSH-PX enzyme, SDH enzyme and SOD enzyme and mitochondrial swelling, MDA levels), and also renal morphology was investigated using tissue slice method. Base on these experiments, this thesis researched SGP protects the kidney cells of hyperoxaluria rats from damaging through antioxidant, reducing calcium oxalate crystal deposition, and protection mitochondria. Then it discovered the vital mechanism of SGP on the inhabition activity of calcium oxalate stones, for it has obvious antioxidant ability, and can regulate the mitochondrial dysfunction of rat kidney.
首先对于SGP的制备,基于制药和保健应用的考虑,本文采用单因素和正交试验方法,确定了SGP的超声辅助提取工艺和脱蛋白脱色工艺。对于SGP的理化性质,运用了紫外,红外光谱,质谱和薄层色谱以及电泳等多种分析手段,测定了其硫酸基、糖醛酸、蛋白质的含量(硫酸基含量为13.2%,糖醛酸含量为10.11%,蛋白质为1.03%)和SGP的分子量(11887Da),初步推测SGP可能是由葡萄糖,半乳糖和甘露糖醛酸组成的硫酸多糖。
其次,为了分析SGP的体外抗氧化活性,分别测定了SGP的还原能力,清除超氧阴离子自由基能力和对DPPH自由基的抑制能力。结果表明SGP的浓度与以上3种抗氧化能力存在明显的量效关系,SGP对超氧阴离子清除活性的ICso为1.9mg/mL,对DPPH自由基的清除活性的IC50为0.56mg/mL。其中清除DPPH自由基活性显著优于已有研究的马尾藻多糖。
再次,研究了SGP体外抑制草酸钙结晶的活性。考察了影响草酸钙成核、生长和聚集过程中的关键指标：最大诱导时间,成核抑制率和聚集抑制率,并对草酸钙结晶形貌进行了观测。此外,对添加不同浓度的SGP后的草酸钙结晶混悬液进行了Zeta电位测定,以发现SGP浓度与草酸钙微粒表面带的负电荷之间的关系。结果表明与柠檬酸三钠相比,SGP抑制草酸钙结晶的效果较佳,SGP对草酸钙的成核、聚集抑制率分别为69.2%、76.8%。
最后,为研究SGP治疗大鼠草酸钙结石的功效和作用机制,本文建立了高草酸导致的大鼠肾损伤的草酸钙结石模型,使用SGP分别以高、中、低剂量对患有结石的大鼠进行治疗。测定了各组大鼠的尿液生理指标及血清生化指标；运用组织切片方法观察了肾组织中草酸钙结晶沉积的情况,并使用各类相关酶活力测试盒和丙二醛(MDA)测试盒,分别对各组大鼠肾组织线粒体的ATP酶活力、过氧化氢酶活力、谷胱甘肽过氧化酶活力、超氧化物歧化酶活力、琥珀酸脱氢酶活力、线粒体肿胀度测定和MDA的水平进行了测定。在此基础上阐明了SGP通过抗氧化、减少草酸钙结晶沉积和保护线粒体功能等多途径作用发挥对高草酸尿症大鼠肾脏的保护作用,并揭示了SGP抗草酸钙结石活性的作用机制,因其具有显著的抗氧化活性并能调节大鼠肾脏的线粒体功能障碍从而能有效治疗草酸钙结石。
Urinary stones affect a large proportion of the population, most of urinary stones are calcium oxalate stones. Therefore, it is important to search for new bioactive compounds to meet the clinical needs that prevent calcium oxalate stones and reduce their recurrence rate. Sargassum graminifolium, a brown seaweed extensively distributed along the coasts of the South and the East China Sea. This thesis researched on preparation of polysaccharides from Sargassum graminifolium (SGP), and its physical and chemical properties, antioxidant effects and also the inhibition of calcium oxalate crystals activity in vitro, furthermore the treatment efficacy and mechanism of SGP on renal injury induced by hyperoxaluria in vivo. The main view is based on antioxidant effects, reduction of calcium oxalate crystal deposition and protection mitochondrial function. The summary as follows:
Firstly for SGP preparation technology, considering pharmaceutical and health care applications, the processes of ultrasonic assisted extraction, deproteinization and decolorization were used, with the methods of single factor and orthogonal array design. In order to identify SGP's physical and chemical properties, the methods include electrophoresis, ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry and thin-layer chromatography were used, got the content of sulfate, glucuronic acid and protein (sulfate13.2%, glucuronic acid10.11%, protein1.03%), and also molecular weight of SGP was obtained(11887Da). We can conclude that SGP is a kind of sulfated polysaccharide composed of glucose, galactose and mannuronic acid.
Secondly, for analyzing the antioxidant activity of SGP in vitro, the reducing power, the ability of scavenge superoxide radicals, and the activity of scavenge DPPH were determinted respectively. The results showed there is obvious dose-effect relationship between concentrations of SGP and the three antioxidant activities, increasing concentrations of SGP resulted in increased scavenging of superoxide anions and DPPH radicals (IC50=1.9and0.56mg/mL, respectively).
Thirdly this thesis researched on the effects of SGP on calcium oxalate crystallization in vivo. We studied the maximum time, the rates of nucleation and aggregation that characterize the crystallization process and also investigated the morphology of calcium oxalate crystals. Furthermore, the Zeta potential measurement of calcium oxalate crystals liquids was tested after adding different concentrations of SGP in order to find the relationship between concentrations of SGP and the negative charge of calcium oxalate particle surface. The results show that SGP'rates of nucleation and aggregation were69.2%and76.8%, and it's better than with trisodium citrate.
At last, this thesis researched the effects of SGP on hyperoxaluria rats and its action mechanisms. For the rats with calcium oxalate stones, they were taken high, middle and low dose of SGP. On following aspects the rats were measured:the urine physiological indicators, the biochemical serum markers, the kidney mitochondria indicators (the activity of ATP enzyme, GSH-PX enzyme, SDH enzyme and SOD enzyme and mitochondrial swelling, MDA levels), and also renal morphology was investigated using tissue slice method. Base on these experiments, this thesis researched SGP protects the kidney cells of hyperoxaluria rats from damaging through antioxidant, reducing calcium oxalate crystal deposition, and protection mitochondria. Then it discovered the vital mechanism of SGP on the inhabition activity of calcium oxalate stones, for it has obvious antioxidant ability, and can regulate the mitochondrial dysfunction of rat kidney.
引文
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