【作者】 高晓旭；

【导师】 孟宪军；

【作者基本信息】 沈阳农业大学 ， 食品科学与工程， 2009， 博士

【摘要】 五味子（Schisandrachinensis（Turcz）Baill）是国家中药现代化科技产业基地的五大品种之一，特别是北五味子，是五味子中的极品，它含有多糖等活性成分，既可食用也可药用，具有广泛的营养保健作用及药理活性。是优势产业链发展的理想资源，开发前景广阔。本文以北五味子果实为原料，研究北五味子多糖的提取工艺、分离纯化技术、理化性质、结构及活性。以期为北五味子的产业化开发奠定基础。主要结论如下：1．采用水提醇沉法提取北五味子多糖，通过正交试验确定最佳工艺条件为：料水比1：30；浸提温度100℃；浸提时间3h,在此条件下北五味子多糖得率为10．62％，以提取2次较为合适；若经超声波预处理40min，可使多糖得率从10．62％提高到13．26％；提取液醇沉的优化条件为：5倍体积95％乙醇，醇沉6h。2．探讨了五种方法对北五味子多糖中蛋白质的脱除效果。结果表明：酶法和三氯乙酸-正丁醇法结合使用效果较好，即酶2％，pH5．0，60℃酶解2h，再用等体积的三氯乙酸-正丁醇溶液处理5次，此时蛋白质含量为0．71mg/g，多糖损失率为17．03％。3．对比了活性炭和过氧化氢脱色效果，结果表明活性炭对北五味子多糖的脱色效果更好。活性炭脱色的最佳条件为：60℃、pH4．0、添加1．5％的活性炭、搅拌50min，脱色率达91．7％．4．北五味子多糖过DEAE-52纤维素柱后出现四个洗脱峰，依次为SCP-AⅠ、SCP-BⅠ、SCP-BⅡ和SCP-BⅢ，含量分别占0．42％、4．83％、4．03％和1．25％。将SCP-BⅠ、SCP-BⅡ和SCP-BⅢ分别过Sephadex S-300葡聚糖凝胶柱进一步纯化，冻干后，经醋酸纤维薄膜电泳和Sephadex S-300凝胶过滤等方法鉴定，证明SCP-BⅠ、SCP-BⅡ和SCP-BⅢ均为均一多糖。5．北五味子多糖理化实验结果表明：三种北五味子多糖比旋光度分别为：+28．0°、+45．0°、+30.0°0。；经X-射线衍射试验结果证明三种北五味子多糖都不是晶体；溶解性实验表明北五味子多糖低温下不易于溶解，溶解性受酸碱度影响较小；粘度特性实验表明北五味子多糖溶液的粘度随溶液浓度增大而增大，随着温度的升高而降低，但温度对其影响具有可逆性；溶液粘度随剪切速率的增加而降低；通过透射电镜和扫描电镜的观察，北五味子多糖分子在极稀的水溶液中呈一定形状的聚集态，其单分子呈线性短棒状，由三股单链螺旋在一起。6．北五味子多糖的含量分析试验结果表明：SCP-BⅠ、SCP-BⅡ、SCP-BⅢ糖醛酸含量的含量分别为11．03％、10．40％和9．18％，粗多糖糖醛酸含量2．59％-2．96％；SCP-BⅠ、SCP-BⅡ、SCP-BⅢ总糖含量分别为91．3％、90．6％、89．6％、；北五味子多糖元素分析表明，三种多糖是由C、H元素组成，都不含N元素；SCP-BⅠ、SCP-BⅡ、SCP-BⅢ的分子质量为：27Kda、23Kda、15Kda。7．通过气相色谱（GC）分析结果表明SCP-BⅠ是一种含有GalA酸性杂多糖，其单糖组成主要为阿拉伯糖（Ara），半乳糖（Gal），葡萄糖（Glc）和半乳糖醛酸（GalA），其中摩尔比依次为2．6：3．1：2．1：1．0。8．SCP-BⅠ经红外光谱分析、核磁共振分析、部分酸水解、高碘酸氧化、Smith降解、甲基化、气相色谱分析、GC-MS分析等方法，确定其分子结构为：SCP-BⅠ主链主要由Ara（1→5）和Gal（1→6）交替连接组成，其间有痕量的GalA（1→3）；支链由Gal（1→3）,Gal（1→4）,Glc（1→4）构成；分支点由Glc（1→4，6），Gal（1→3，6）构成；末端残基为Gal,Glc.SCP-BⅠ存在吡喃糖苷，半乳糖醛酸以内酯形式存在，并且SCP-BⅠ中存在α、β两种构型。9．北五味子多糖对油脂的抗氧化性能试验结果证明：北五味子多糖对油脂具有一定的抗氧化作用；抗坏血酸与北五味子多糖，对油脂具有较好的抗氧化协同增效作用。10．通过SCP-BⅠ对·OH和O^2-·的清除作用实验，得出SCP-BⅠ对·OH和O^2-·都有明显的清除作用，且随着多糖浓度的升高均呈现良好的量效关系。在清除·OH时，当多糖浓度达到35mg／ml时增加幅度减缓，曲线趋于平缓；而在清除O^2-·的过程中，当多糖浓度达到43．636mg／ml时增加幅度减缓，SCP-BⅠ对·OH的清除作用优于其对O^2-·的清除作用。11．对D-半乳糖（100mg／Kg.d）致衰老模型小鼠，用400mg／Kg.d、200mg／Kg.d、100mg／Kg.d等剂量的北五味子多糖生理盐水溶液灌胃30d。结果表明，北五味子多糖能显著提高小鼠血清、肝组织、脑组织中SOD、GSH-P_x的活性，减少MDA含量。表明北五味子多糖在抗氧化、防衰老方面具有重要作用12．北五味子多糖减肥降脂试验结果表明：北五味子多糖能明显降低肥胖小鼠体重、脂肪指数、总胆固醇、甘油三酯、低密度脂蛋白、血糖含量，对肥胖小鼠血清中高密度脂蛋白有明显的升高作用，证明北五味子活性多糖具有明显减肥降脂作用。13．北五味子多糖降血糖试验表明SCP能降低正常小鼠血糖（P＜0．05）以及四氧嘧啶诱导的糖尿病小鼠血糖（P＜0．01）。SCP还能缓解糖尿病小鼠的症状，恢复小鼠体重，减少饮水量，但对小鼠饮食量影响不显著。14．建立小鼠游泳训练模型，研究北五味子多糖的抗运动疲劳的作用．结果表明：服用一定剂量北五味子多糖能提高肌糖原和肝糖原含量，减少力竭运动后血乳酸的堆积，维持血糖恒定，使血清酶呈现不同程度的良性变化，延长小鼠力竭时间，并能极显著延长小鼠在缺氧条件下的存活时间，与对照组比较，延长率在14．4％～36．8％之间．本实验充分证明北五味子多糖具有抗运动疲劳的作用。更多还原

【Abstract】 Schisandra chinensis（ Turcz）Baill is One of five varieties of national modernization oftraditional Chinese medicine industry base, Especially schisandrae chinensis is nonsuch inSchisandra chinensis （Turcz）Baill, It contains polysaccharide, etc, Both edible but alsomedicinal,and Widespread nutritional health care function and pharmacological activities. It isthe ideal resources of Industrial development, and Developping prospect is broad.Schisandrachinensis fruit is used as raw materials in this article, Schisandra chinensis polysaccharideextracting technology, purification technology, physical and chemical properties, structure andactivity was studied, It will lay the foundation developping the industrialization ofschisandrae chinensis .Main conclusions are as follows:1. Polysaccharides are extracted by the methods of water extracting, precipitation withalcohol, and the optimum extraction technology is determined by orthogonal experiments.The optimum parameters to extract are as follows: the ratio of water to material 1:30,temperature 100℃, extracting time 3h and extracting times 2. In this condition, the extractionpercent of polysaccharide is 10.62%. With ultrasound -assisted extraction for over 40 minutesThe rate of extracting Polysaccharide would increased to 13.6% from 10.62% The bestcondition that the extraction and alcohol separated : with 5times volume of 95% ethanol for 6hours.2. Five methods are used to remove protein from SCP. The results show that the methodof combining enzyme and Trichloroaceticacid-normal-Butanol is better than others. By using2% protease, the polysaccharide solution is enzymic hydrolysisfor 2 hours in 40℃water andpH5.0, added Trichloroaceticacid-normal-Butanol volume as much as polysaccharide solutionfor 5 times, In this condition ,the content of protein is 0.71mg/g, and the rate of lost thePolysaccharide is 17.03%.3. The decoloration of active carbon and H₂O₂ were studied,the results showed that activecarbon was more effective to decolor SCP than H₂O₂,the optimum conditions were as follows:the decoloration temperature,30℃; pH4.0; the dosage of active carbon, 1%; the decolorationduration, 30 min; The rate of discoloration reach 91.7%4. SCP without protein is separated into four fractions on DEAE-52 celluloseChromatography. They are SCP-AⅠ、SCP-BⅠ、SCP-BⅡand SCP-BⅢ.The content of them is0.42%、4.83%、4.03% and 1.25%. Then they are purified by SephadexS-300 gel chromatography into SCP-BⅠ、SCP-BⅡand SCP-BⅢ, and After freeze-dried they arehomogeneous tested by cellulose acetate film eletrophoresis and gel filtration on SephadexS-300 column chromatography. The result showed that they was pure component.5. The result of phisical chemical testing is showed that specific rotation of SCP-BⅠ、SCP-BⅡand SCP-BⅢis +28.0°、+45.0°、+30.0°respectively; The result of X-ray diffractionis showed that they are not crystal, the solubility testing of SCP is showed that they are noteasily dissolved at low temperature and Solubility are less effect by PH. Viscosity propertiesof SCP experiments showed that the viscosity solution of polysaccharides solutionconcentration increases with thickening, Solution viscosity is rreduced with the rise oftemperature, but reversible, Solution viscosity is reduced with the rise of shearing rate ; Byobserving with transmission electron microscope and scanning electron microscopy, SCPpresent Certain shapes state of aggregation in extremely dilute aqueous solution, singlemolecule is linear short clavite, three single chain is spiral together.6. The content of SCP analysis experiment results showed that The contents of uronicacid are 11.03%、10.40% and 9.18% respectively, and The contents of uronic acid of crudeSCP is 2.59%-2.96%; The content of total sugar of them,is 91.3%、90.6% and 89.6%respectively; The element analysis of SCP showed that three kinds of polysaccharides isconstituted by C and H elements, and they don’t contain N elements; The molecular weight ofSCP-BⅠ、SCP-BⅡand SCP-BⅢare estimated to be 27Kda、23Kda、15Kda respectively.7. Gas chromatography analysis results showed that SCP-BI is an acidheteropolysaccharide consisting of Ara, Gal, Glc and GalA in ratio of 2.6:3.1:2.1:1.8. SCP-BI was Studied by the infrared spectrum analysis, nuclear magnetic resonance（NMR） analysis, part acid hydrolysis, periodate oxidation, Smithdegradation, methylation,GC and GC-MS analysis method, its molecular structure is:The backbone of SCP-BI wascomposed of （1→5）-linked Ara, （1→6）-linked Gal with 3 branches attached to O-3 of（1→6）-linked Gal and terminated with either Gal （1→3）or Gal（1→4）and Glc（1→4）,of Glc and the majority of Gala are distributed in branches. SCP-BI is made ofβ-D-Galactopyranoside; Galaurnic acid and Galaurnic acid exist in lactone and there are twokinds form which isαandβ-configuration.9. The antioxidant properties test result showed that SCP has strong anti-oxidative effectsfor oil.The SCP and Vc on lard and gingili have synergic antioxidative 10. The experiment which studied on the effect of SCP-BI on scavenging·OH^- andO^2-·showed the conclusion that the effect of SCP-BI was obviously on scavenging·OH^- andO^2-·and with the more percent of Polysaccharide was added in, there was good relationbetween the number and the effect. When the percent of Polysaccharide was 35mg/ml, itwould not rise sharply as before and the curve was becoming on level .It has the samefunction to O^2-·,but the effect is not as good as to·OH^-.It has the same function when theamount must be reached to 43.636mg/ml .So the effect of SCP-BI on scavenging·OH^- isbetter than O^2-·.11. Aging model mice induced by D-galactose（100mg/Kg.d） are respectively igrrigatedwith three doses （400mg/Kg.d, 200mg/Kg.d, 100mg/Kg.d） crude polysaccharide dissolved innormal saline for 30 days. The results show that SCP can increase SOD and GSH-P_X activitiesof serum, liver and brain organization, decrease the contents of their MDA. So the resultsindicate that SCPplays an important role in anti-oxidation and anti-decrepit function.12. The results of test reducing weight and fat with SCP showed that:the SCP canobviously reduce the adiposity mice’s weight,fat index number, TC, TG, LDL and GLU, andimprove their HDL in blood serum too, So the SCP possesses great functions in reducingweight and fat.13. Pharmaceutical experiments showed SCP administered orally in alloxan-induceddiabetic mice can significantly reduce blood glucose levels （P<0.01）and water intake, andincrease the body weight of diabetic mice compared with alloxan-induced diabetic controlgroup. But no significant influence to of the mice’s appetite.14.Taking male mice as experimental objects, the strength increasing swimming modelswere built up. To Study the Functions of SCP Against exercise Fatigue. The results showedthat the Active SCP can increase the contents of muscle glycogen and liver glycogen, reducethe accumulation of blood lactic acid after exhaustion, maintain the blood sugar constant,raisethe activities of superoxide dismutase and glutathione peroxide in serum, reduce theactivities of creatine kinase and lactatedehydrogenase in serum, and prolong the exhaustingtime of mice and the survival time under hypoxic condition. Compared with controls,prolong the rate at 14.4%～36.8%. It was proved that the Active SCP can alleviate exercisefatigue.更多还原