连钱草多糖分离纯化、结构分析及生物活性初探
发布时间：2018-09-11 15:54
【摘要】：连钱草[Glechoma longituba(Nakai)Kupr.]为唇形科活血丹属草本植物,具有溶石、抗菌、消炎等作用。本文对连钱草多糖进行研究,优化了连钱草多糖的脱蛋白脱色素工艺,分离纯化出分子量均一分布的多糖并对其理化性质、结构组成及生物活性进行了研究,为连钱草多糖的功能性开发奠定了基础。1.通过水提醇沉法获取连钱草粗多糖(GP),以多糖保留率、蛋白质清除率和脱色率为指标,在单因素试验的基础上采用正交试验分别考察了活性炭、大孔树脂D301G对GP溶液的脱蛋白脱色素效果,确定最优脱蛋白脱色素工艺。结果表明,活性炭对GP的最佳脱蛋白脱色素条件为:活性炭添加量1.0%(g/mL),振荡时间1.5 h,多糖浓度6 mg/mL,样液pH 3.0,此条件下多糖保留率、蛋白质清除率、脱色率分别为(95.32±1.66)%、(94.59±0.34)%、(86.49±0.81)%;D301G对GP溶液的最佳脱蛋白脱色素条件为:大孔树脂添加量15%(g/mL),振荡时间0.5 h,多糖浓度2 mg/mL,样液pH 7.0,此条件下多糖保留率、蛋白质清除率、脱色率分别为(93.41±0.87)%、(95.82±0.19)%、(96.97±0.05)%;综合比较,D301G对GP的脱蛋白脱色素效果优于活性炭。脱蛋白脱色素后的粗多糖GPC依次经DEAE Sephadex A-25离子交换层析和Sephacryl S-200凝胶过滤层析,得到三个组分:GRa-1、GRb-1、GRb-2。2.考察了GRa-1、GRb-1、GRb-2的理化性质和纯度,并分析了GRa-1的单糖组成及结构特点。结果表明,GRa-1、GRb-1为中性多糖,GRb-2为碱性多糖,三者均不含蛋白质;HPLC分析显示GRa-1为分子量均一分布的多糖,相对分子质量为3 584 Da,而GRb-1和GRb-2则含多种多糖组分,有待进一步纯化;GRa-1的红外图谱显示该多糖含有D型甘露呋喃糖;GC-MS分析GRa-1的单糖组成为阿拉伯糖、甘露糖、葡萄糖、半乳糖四种,摩尔比为0.005:0.014:0.168:0.813。3.研究连钱草多糖分离纯化中各组分的体外抗氧化活性和分布。结果表明,粗多糖GP和GPC在各方面都显示出较强的体外抗氧化活性;GRa-1在Fe2+螯合及O-2·、OH·、DPPH+·清除上表现出较强的活性;GRb-1对O-2·和OH·表现出较强的清除能力;GRb-2除Fe2+螯合和O-2·、OH·、DPPH+·清除力较强外还具有强的总还原力。4.通过腹腔注射葡萄糖耐量试验(Intraperitoneal Glucose Tolerance Test,IPGTT)、口服脂肪耐量试验(Oral Fat Tolerance Test,OFTT)、二甲苯致小鼠耳肿胀试验、老年小鼠抗氧化试验,检测了GRa-1的降血糖、降血脂、抗炎和体内抗氧化活性,结果表明,GRa-1能显著提高小鼠对葡萄糖的利用能力;加速小鼠体内甘油三酯的代谢;显著抑制二甲苯致小鼠耳肿胀;增强老年小鼠血清和肝脏中部分抗氧化酶活性,降低脂质过氧化产物(MDA)含量。
[Abstract]:Glechoma longituba (Nakai) Kupr. It is a herbaceous plant of Huoxue Dan genus in Lip family. It has the functions of litholysis, antibacterial, anti-inflammatory and so on. In this paper, we studied the polysaccharides of Polygonum chinensis, optimized the deproteinization and depigmentation process, isolated and purified the polysaccharides with uniform molecular weight distribution, and studied their physicochemical properties, structural composition and biological activities. It lays a foundation for the functional development of polysaccharides. 1. The crude polysaccharide (GP), obtained by water extraction and alcohol precipitation was characterized by polysaccharide retention rate, protein clearance rate and decolorization rate. On the basis of single factor experiment, the activated carbon was investigated by orthogonal test. The effect of macroporous resin D301G on the depigmentation of GP solution was studied. The results showed that the optimum conditions for deproteinization of GP by activated carbon were as follows: the dosage of activated carbon was 1.0% (g/mL), the oscillating time was 1.5 h, and the concentration of polysaccharide was 6 mg/mL, sample solution pH 3.0. Under this condition, the polysaccharide retention rate and protein clearance rate were obtained. The decolorization rates were (95.32 卤1.66), (94.59 卤0.34), (86.49 卤0.81) and (86.49 卤0.81), respectively. The optimum conditions for decolorization of GP solution were as follows: the amount of macroporous resin was 15% (g/mL), the oscillating time was 0.5 h, and the concentration of polysaccharide was 2 mg/mL, like solution pH 7.0. The decolorization rates were (93.41 卤0.87), (95.82 卤0.19), (96.97 卤0.05), respectively. The crude polysaccharide GPC was separated by DEAE Sephadex A-25 ion exchange chromatography and Sephacryl S-200 gel filtration chromatography. The physicochemical properties and purity of GRa-1,GRb-1,GRb-2 were investigated, and the monosaccharide composition and structure of GRa-1 were analyzed. The results showed that GRa-1 GRb-1 was a neutral polysaccharide and GRb-2 was a basic polysaccharide. HPLC analysis showed that GRa-1 was a homogeneous polysaccharide with a molecular weight of 3 584 Da, while GRb-1 and GRb-2 contained a variety of polysaccharides. The IR spectra of the polysaccharides to be further purified showed that the polysaccharides contained D-type mannofuran sugar. GC-MS showed that the monosaccharides of GRa-1 were composed of arabinose, mannose, glucose and galactose, and the molar ratio was 0.005: 0.014: 0.168: 0.813.3. To study the antioxidant activity and distribution of each component in the separation and purification of polysaccharides. The results show that Crude polysaccharides (GP and GPC) showed strong antioxidant activity in vitro. GRa-1 showed strong activity in Fe2 chelation and O-2 hydroxyl radical scavenging. GRb-1 showed stronger scavenging ability to O-2 and OH. GRb-2 chelates with Fe2 and O-2 with hydroxyl hydroxide with higher scavenging ability. Strong external also has a strong total reduction force. 4. By intraperitoneal injection of glucose tolerance test (Intraperitoneal Glucose Tolerance Test,IPGTT), oral fat tolerance test (Oral Fat Tolerance Test,OFTT), ear swelling test induced by xylene in mice and antioxidation test in aged mice, the antihyperglycemic, antilipids, anti-inflammatory and anti-oxidation activities of GRa-1 were measured. The results showed that GRa-1 could significantly increase the utilization of glucose in mice, accelerate the metabolism of triglycerides in mice, inhibit ear swelling induced by xylene, and enhance the activities of some antioxidant enzymes in serum and liver of aged mice. The content of (MDA) was decreased.
【学位授予单位】：湖北民族学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ464.1
[Abstract]:Glechoma longituba (Nakai) Kupr. It is a herbaceous plant of Huoxue Dan genus in Lip family. It has the functions of litholysis, antibacterial, anti-inflammatory and so on. In this paper, we studied the polysaccharides of Polygonum chinensis, optimized the deproteinization and depigmentation process, isolated and purified the polysaccharides with uniform molecular weight distribution, and studied their physicochemical properties, structural composition and biological activities. It lays a foundation for the functional development of polysaccharides. 1. The crude polysaccharide (GP), obtained by water extraction and alcohol precipitation was characterized by polysaccharide retention rate, protein clearance rate and decolorization rate. On the basis of single factor experiment, the activated carbon was investigated by orthogonal test. The effect of macroporous resin D301G on the depigmentation of GP solution was studied. The results showed that the optimum conditions for deproteinization of GP by activated carbon were as follows: the dosage of activated carbon was 1.0% (g/mL), the oscillating time was 1.5 h, and the concentration of polysaccharide was 6 mg/mL, sample solution pH 3.0. Under this condition, the polysaccharide retention rate and protein clearance rate were obtained. The decolorization rates were (95.32 卤1.66), (94.59 卤0.34), (86.49 卤0.81) and (86.49 卤0.81), respectively. The optimum conditions for decolorization of GP solution were as follows: the amount of macroporous resin was 15% (g/mL), the oscillating time was 0.5 h, and the concentration of polysaccharide was 2 mg/mL, like solution pH 7.0. The decolorization rates were (93.41 卤0.87), (95.82 卤0.19), (96.97 卤0.05), respectively. The crude polysaccharide GPC was separated by DEAE Sephadex A-25 ion exchange chromatography and Sephacryl S-200 gel filtration chromatography. The physicochemical properties and purity of GRa-1,GRb-1,GRb-2 were investigated, and the monosaccharide composition and structure of GRa-1 were analyzed. The results showed that GRa-1 GRb-1 was a neutral polysaccharide and GRb-2 was a basic polysaccharide. HPLC analysis showed that GRa-1 was a homogeneous polysaccharide with a molecular weight of 3 584 Da, while GRb-1 and GRb-2 contained a variety of polysaccharides. The IR spectra of the polysaccharides to be further purified showed that the polysaccharides contained D-type mannofuran sugar. GC-MS showed that the monosaccharides of GRa-1 were composed of arabinose, mannose, glucose and galactose, and the molar ratio was 0.005: 0.014: 0.168: 0.813.3. To study the antioxidant activity and distribution of each component in the separation and purification of polysaccharides. The results show that Crude polysaccharides (GP and GPC) showed strong antioxidant activity in vitro. GRa-1 showed strong activity in Fe2 chelation and O-2 hydroxyl radical scavenging. GRb-1 showed stronger scavenging ability to O-2 and OH. GRb-2 chelates with Fe2 and O-2 with hydroxyl hydroxide with higher scavenging ability. Strong external also has a strong total reduction force. 4. By intraperitoneal injection of glucose tolerance test (Intraperitoneal Glucose Tolerance Test,IPGTT), oral fat tolerance test (Oral Fat Tolerance Test,OFTT), ear swelling test induced by xylene in mice and antioxidation test in aged mice, the antihyperglycemic, antilipids, anti-inflammatory and anti-oxidation activities of GRa-1 were measured. The results showed that GRa-1 could significantly increase the utilization of glucose in mice, accelerate the metabolism of triglycerides in mice, inhibit ear swelling induced by xylene, and enhance the activities of some antioxidant enzymes in serum and liver of aged mice. The content of (MDA) was decreased.
【学位授予单位】：湖北民族学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ464.1
【参考文献】
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1 张逸;王旺;蔡寅;王e，
本文编号：2237145
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