【作者】 孙明礼；

【导师】 张静；

【作者基本信息】 陕西师范大学 ， 食品科学， 2008， 硕士

【摘要】 半枝莲为唇形科植物半枝莲Scutellaria barbata D.Don的干燥全草,分布在我国大部分地区。半枝莲作为传统的中药具有抗肿瘤、抑菌、解热等活性,其主要化学成分为黄酮类化合物、生物碱、挥发油及多糖等,多糖是半枝莲中的主要化学成分之一,有多种生物活性,但目前对半枝莲多糖的分离纯化及结构性质的研究报道甚少。本文研究了半枝莲多糖的提取、分离、纯化及硫酸化修饰,并且对半枝莲多糖SBP、SBP₁、SBP₂、SBPⅠ、SBP-L、SBP-L₁和SBP-L₂的体外抗氧化活性及其化学结构进行了研究,为今后半枝莲多糖更深入的研究与开发奠定了基础。研究结果如下:1.在单因素实验基础上,设计正交实验优化了半枝莲粗多糖的提取工艺,得到热水提取半枝莲粗多糖的最佳工艺参数为:提取温度60℃,料液比1:20,提取时间2h,提取次数4次,半枝莲粗多糖的提取率为2.82%,并采用苯酚.硫酸法对多糖含量进行了测定。2.进行了多糖脱色工艺的研究,对活性炭、中性氧化铝、大孔吸附树脂AB-8、LSA-21和NKA-9五种脱色剂的脱色性能进行了比较,从中筛选出了最佳的脱色剂为大孔吸附树脂AB-8。通过单因素实验确定了脱色的最佳条件是pH为7,温度为40℃,溶液中多糖含量为1046.5 mg/L。采用最佳条件脱色验证,测定多糖的保留率为88.46%,脱色率为90.65%。3.脱色后的多糖（SBP）采用季铵盐沉淀法（Hexadecyltrimethylammoniumbromide,CTAB）进行分离,得到半枝莲多糖亚组分SBP₁和SBP₂。采用DE-52纤维素离子交换柱层析对多糖SBP进行了分级分离,得到水洗组分SBPⅠ用0.1 moL/LNaCl溶液洗脱得到SBPⅡ。利用Sephdex-200柱层析、高效液相色谱、紫外光谱和冻融法对SBPⅠ进行纯度鉴定,证明了SBPⅠ为组成均一的多糖组分。4.对多糖进行了硫酸化修饰。采用氯磺酸-吡啶法对半枝莲多糖SBP进行硫酸酯化修饰,得到多糖SBP-L,其水溶性较原多糖明显提高,通过紫外分光光度计扫描在262 nm处有吸收峰,测得的SO₄^2-浓度为5.73 mg/mL,取代度为0.35。红外光谱显示在1258 cm^-1附近出现硫酸基(-O-SO^3-)的特征吸收峰。5.采用气相色谱法对半枝莲多糖的单糖组分进行了分析。比较了硅烷衍生化、糖腈衍生化法和糖醇衍生化三种衍生化方法,确定了糖腈乙酰化法为最佳的衍生化方法;通过单因素实验确定了硫酸水解半枝莲多糖的最佳条件为:硫酸浓度为0.5 mol/L、体积2 mL、水解时间12 h、温度100℃:气相色谱测定半枝莲多糖SBP由鼠李糖阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖组成,摩尔比为3.26:0.37:4.85:5.16:4.10:9.47,SBP-L由阿拉伯糖、甘露糖、葡萄糖和半乳糖组成,摩尔比为0.84:6.67:3.62:5.53,SBP1,SBP2,SBP-L1,SBP-L2和SBPⅠ都由鼠李糖木糖、甘露糖、葡萄糖和半乳糖组成,摩尔比依次为4.51:5.50:4.86:4.90:8.22,3.25:6.20:5.85:4.05:11.30,3.86:4.39:4.96:4.95:7.73,3.04:6.87:5.51:4.37:10.90,3.53:4.62:7.22:4.08:8.37。6.利用高效液相色谱测定多糖的分子量,得到半枝莲多糖SBP1、SBP2、SBPⅠ、SBP-L₁和SBP-L₂的分子量依次为:290000Da、340000Da、300000Da、360000Da和390000Da;采用部分酸水解、高碘酸氧化、Smith降解结合气相色谱和红外光谱对半枝莲多糖进行结构分析,确定了多糖的结构:SBP₁和SBP₂糖链连接方式都是由β-（1→2）—吡喃葡聚糖及半乳糖构成主链,其中SBP₁由（1→3,4,6）—鼠李糖、阿拉伯糖、木糖、甘露糖和葡萄糖残基组成侧链分支,多糖中（1→6）键、（1→3）键、（1→2或1→4）键摩尔百分比约为9%:22%:69%,SBP₂由（1→3,4,6）—鼠李糖、阿拉伯糖、甘露糖和葡萄糖残基组成侧链分支,多糖中（1→6）键、（1→3）键、（1→2或1→4）键摩尔百分比约为12%:26%:62%:SBPⅠ以β—（1→2,3）—吡喃葡聚糖及半乳糖构成主链,由（1→4,6）—鼠李糖、阿拉伯糖、甘露糖和葡萄糖残基组成侧链分支,多糖中（1→6）键、（1→3）键、（1→2或1→4）键摩尔百分比约为10.6%:47%:42.4%:SBP-L₁以β—（1→2）—吡喃葡聚糖及半乳糖构成主链,由（1→3,4,6）—鼠李糖、阿拉伯糖、木糖、甘露糖和葡萄糖残基组成侧链分支,并存在少量α—糖苷键型吡喃糖基,多糖中（1→6）键、（1→3）键、（1→2或1→4）键摩尔百分比约为9.6%:38.6%:51.8%;SBP-L₂以β—（1→2）—吡喃葡聚糖及半乳糖、阿拉伯糖构成主链,由（1→3,4,6）—鼠李糖、甘露糖和葡萄糖残基组成侧链分支,多糖中（1→6）键、（1→3）键、（1→2或1→4）键摩尔百分比约为10.6%:24.2%:65.2%。7.对七种半枝莲多糖（SBP、SBP1、SBP₂、SBPⅠ、SBP-L、SBP-L₁和SBP-L₂）的体外抗氧化活性进行了研究:采用邻苯三酚自氧化方法,测定半枝莲多糖对O^2-自由基的抑制作用;采用邻二氮菲-亚铁离子—H₂O₂体系测定了半枝莲多糖对羟基自由基的抑制作用;体外模拟胃液pH值条件下测定了半枝莲多糖对NO₂^-清除能力。结果表明,七种多糖均具有不同的抗氧化活性,呈剂量依赖关系。半枝莲多糖SBP的抗氧化性要强于分离后的多糖SBP₁和SBP₂,硫酸化修饰后的多糖SBP-L和SBP-L₂的抗氧化性较原多糖SBP和SBP₂的抗氧化性增强,多糖SBP-L₁较多糖SBP₁对羟基自由基和NO^2-的清除效果有所降低,对O^2-自由基清除效果有所提高。本文研究表明,几种半枝莲多糖都具有不同百分比的β—（1→3）葡聚糖苷键,因此,七种多糖出现不同程度的抗氧化性。更多还原

【Abstract】 Scutellaria barbata D.Don,a herb belonging to the Lamiaceae family,is widely distributed in china.This herb is known in traditional Chinesse Medicine Ban-Zhi-Lian and has been used as an antitumor,anti-inflammatory,antifebrile agent.The main chemical compositions are flavonoids,alkaloid,essential oils and polysaccharide.Polysaccharide which is the one of major component of Scutellaria barbata D.Don have many kinds of bioactivities.But the study were seldom carried out on isolation,purification,structure and property of polysaccharides from Scutellaria barbata D.Don.This paper studied the extraction,separation,purification and sulfation of Scutellaria barbata D.Don polysaccharide,the antioxidant activity of SBP,SBP₁,SBP₂,SBPⅠ、SBP-L、SBP-L₁ and SBP-L₂ and their chemical structure,which gave a foundation for the further research..The main results and innovations were as follows:1.Based on the single factor experiment,designing orthogonal experiments to optimize the extraction process of crude Scutellaria barbata polysaccharide,the optimal parameters of extracting Scutellaria barbata polysaccharide by hot water were:the extraction temperature was 60℃, solid-liquid ratio was 1:20,extraction time was 2 h and extraction times were 4,and the rate of polysaccharides extraction was 2.82%.The amount of carbohydrates was measured with phenol-sulfuric acid method.2.This paper studied on de-coloration of Scutellaria barbata polysaccharide.which compared the de-coloration capacity of five different kinds of de-coloration agents:active carbon,neutral alumina,macroporous adsorption resins AB-8,LSA-21 and NKA-9.The better de-coloration agents was AB-8.Decoloring experiments were carried out by single factor experiment.The best conditions for de-coloration were determined as follows:the pH is 7,the temperature is 40℃,the content of polysaccharides is 1046.5 mg/L.Adopt the best condition,holding rate of polysaccharides is 88.46%,de-coloration rate is 90.65%.3.Polysaccharides（SBP） were treated after de-coloring by quaternary ammonium salt precipitation method.SBP₁ and SBP₂ were obtained.SBP was isolated using DE-52 cellulose chromatography in series processes.SBPⅠand SBPⅡwas obtained respectively eluting the DE-52 cellulose chromatography with distilled water and the solution of 0.1 moL/L NaCI.SBPⅠwas identified as homogeneous polysaccharide component by SephadexG-200 gel chromatography,HPLC,UV spectrum and freeze-thawing. 4.Chemical modification of sulfation reaction about SBP,Scutellaria barbata polysaccharide Sulfate（SBP-L） was obtained and its water-sohibility was increased.By scanning it with UV spectrum,the results showed that it had absorption peaks at 262 nm.The content of sulphate groups was 5.73 mg/mL,the proportion of sulfate substitution was 2.89.IR showed that it has a-O-SO^3- absorption peaks at 1258 cm^-1.5.The monosaccharide components of Scutellaria barbata polysaccharide were measured by GC.Three methods of TMS,acety.lated aldononitriles and sugar alcohol acetates were compared to confirm acetylated aldononitriles which is one of the best methods.Hydrolyzing experiment were carried out by single factor experiment.The best conditions were determined as follows:the concentration of sulphuric acid was 0.5mol/L,the volume was 2 mL,the time was 12 h and the temperature was 100℃.GC result showed that SBP was mainly composed of rhamnose,arabinose, xylose,mannose,glucose and galactose,and the mole proportion of monosaccharide was 3.26:0.37:4.85:5.16:4.10:9.47.SBP-L was mainly composed of arabinose,mannose,glucose and galactose,and the mole proportion of monosaccharide was 0.84:6.67:3.62:5.53.SBP₁,SBP₂,SBP-L₁, SBP-L₂ and SBP I were mainly composed of rhamnosexylose,mannose,glucose,galactose and the mole proportion of monosaccharide respectively were 4.51:5.50:4.86:4.90:8.22,3.25:6.20:5.85: 4.05:11.30,3.86:4.39:4.96:4.95:7.73,3.04:6.87:5.51:4.37:10.90,3.53:4.62:7.22:4.08:8.37.6.The average molecular weights of SBP₁,SBP₂,SBPⅠ,SBP-L₁ and SBP-L₂ were approximately 290000,340000,300000,360000 and 390000Da.,determined respectively by HPLC. Partial hydrolysis with sulphuric acid,periodate oxidation,Smith degradation,GC and IR was used to determine their structure.The results showed that:The main chain of SBP₁ and SBP₂ was made up ofβ—（1→2）—pyranose glucosan and galactose,and the side chain of SBP₁ was composed of（1→3, 4,6）—rhamnose,arabinose,xylose,mannose and glucose.The mole proportion of bond（1→6）: （1→3）:（1→2 or 1→4）was 9%:22%:69%.The side chain of SBP₂ was composed of（1→3, 4,6）—rhamnose,arabinose,mannose and glucose.The mole proportion of bond（1→6）:（1→3）: （1→2 or 1→4） was 12%:26%:62%.The main chain of SBPⅠwas made up ofβ—（1→2,3）—pyranose glucosan and galactose,and the side chain of SBPⅠwas composed of（1→4,6）—rhamnose,arabinose,mannose and glucose.The mole proportion of bond（1→6）:（1→3）:（1→2 or 1→4）was 10.6%:47%:42.4%.The main chain of SBP-L₁ was made up ofβ—（1→2）—pyranose glucosan and galactose,and the side chain of SBP-L₁ was composed of（1→3,4,6）—rhamnose, arabinose,xylose,mannose and glucose and had a fewα—pyranose glucesidal bond.The mole proportion of bond（1→6）:（1→3）:（1→2 or 1→4） was 9.6%:38.6%:51.8%.The main chain of SBP-L₂ was made up ofβ—（1→2）—pyranose glucosan,galactose and arabinose,and the side chain of SBP-L₂ was composed of（1→3,4,6）—rhamnose,mannose and glucose.The mole proportion of bond（1→6）:（1→3）:（1→2 or 1→4） was10.6%:24.2%:65.2%.7.Antioxidative activities of seven kinds of Scutellaria barbata polysaccharides（SBP,SBP₁, SBP₂,SBPⅠ,SBP-L,SBP-L₁ and SBP-L₂） in vitro were studied:the capability of the polysaccharides to eliminate O^2- was determined through pyrogallol auto oxidation;the ability of the polysaccharides to scavenge hydroxyl radicals was determined by the system of ferrosin-iron-H₂O₂ and the ability of the polysaccharides to scavenge NO₂^- in the condition of pH for simulation gastric juice in vitro.The result showed that the seven kinds of Scutellaria barbata polysaccharides had different antioxidation activities in vitro,and their activities depended on their amounts.The antioxidation activities of SBP was better than separate polysaccharides SBP₁ and SBP₂.The antioxidant activities of sulfation of Scutellaria barbata polysaccharide SBP-L and SBP-L₂ were increased than SBP and SBP₂,through comparing with SBP₁ and SBP-L₁ the ability of the SBP₁ to scavenge hydroxyl radicals and NO₂^- is better,but the capability of the polysaccharides to eliminate O^2- was reverse.This paper studied that seven kinds of Scutellaria barbata polysaccharides had different antioxidation activities in vitro because all kinds of them had different percent ofβ-（1→3） glucesidal bond.更多还原