【作者】 汪艳群；

【导师】 孟宪军；

【作者基本信息】 沈阳农业大学 ， 食品科学， 2012， 博士

【摘要】 五味子（Schisandra chinensis (Turcz.) Baill），木兰科五味子属植物，主要分布于辽宁、吉林、黑龙江等地，是一种药食两用的资源。早期国内外对五味子研究多集中在木脂素、三萜、挥发油等脂溶性成分上，生产中提取过脂溶性成分的五味子常作为残渣被废弃，而近年来发现五味子水溶性成分中含有大量多糖，引起研究者的关注。本文以提取过脂溶性成分的五味子残渣为原料，研究五味子多糖的提取工艺、分离纯化技术，并对得到的五味子多糖进行了理化性质、结构、抗氧化作用及免疫调节活性研究，以期为五味子的产业化开发奠定基础。主要结论如下：1、利用Design Expert7.0和Minitab15两款专业软件通过响应面法（RSM）优化传统热水浸提、超声波辅助提取及酶解辅助提取五味子多糖的工艺。（1）热水浸提法的最佳工艺条件为：料液比1:40，提取温度90.3℃，提取时间5h，五味子粗多糖的提取率为（10.99±0.22）%；（2）超声波辅助提取法的最佳预处理条件为：料液比1:40，超声波功率为300W，处理时间为1h，处理温度为30℃，预处理后进行热水浸提，五味子粗多糖的提取率为（16.73±0.25）%；（3）酶解辅助提取法的最佳预处理条件为：料液比1:40，果胶酶用量为1.19%，酶解时间为4h，温度为30℃，酶解pH值为4.21，酶解后迅速升温至100℃，15min灭酶，调整pH为中性，然后进行热水浸提，五味子粗多糖的提取率为（20.82±0.25）%。2、利用溶剂法、酶法、柱层析法对五味子粗多糖进行纯化，结果表明：最佳脱蛋白方法为酶法与三氯乙酸-正丁醇法结合，酶解条件为：蛋白酶用量为1%、酶解温度为50℃、酶解时间为140min、pH值为5，酶解之后再用三氯乙酸-正丁醇法处理4次，可除去蛋白质，多糖保留率为68.3%。脱蛋白后用聚酰胺柱层析进行脱色和分离，得到三个组分SCP-A、SCP-B和SCP-C，分别占多糖总量的67.3%、2.42%、1.35%；将SCP-A经Sephacryl S-300HR凝胶柱层析后分离得到一个组分SCP-0，将SCP-0经紫外光谱、Sephacryl S-300HR凝胶柱层析法、醋酸纤维薄膜电泳法及凝胶色谱法进行纯度鉴定，表明其为均一度较高的多糖，纯度达94.6%。将SCP-0经HPLC进一步纯化，得到SCP-01，经高效液相凝胶色谱法鉴定为单一尖锐对称峰形，其分子量分布宽度Mw/Mn为1.22，表明其为高纯度均一多糖。3、五味子多糖的理化性质研究表明：（1）五味子多糖溶解性受温度影响较大，低温下不易于溶解，60℃为其适宜溶解温度；在食品加工中常见的盐浓度和酸碱度下都有较好溶解性，受盐浓度和pH值的影响较小，碱性条件下溶解更快。（2）五味子多糖溶液的粘度受浓度和温度影响较大，随着浓度增大，粘度显著提高；随着温度升高，粘度下降。五味子多糖溶液的粘度受NaCl溶液浓度、pH值和热处理的影响较小，具有较好的加工稳定性。（3）五味子多糖SCP-0总糖含量为88.7%，糖醛酸含量为12.7%，重均分子量为394kDa。（4）超声波处理会使五味子多糖降解，平均分子量下降。4、利用高效液相色谱、气相色谱、红外光谱、原子力显微镜等结合多糖的经典化学分析方法，对五味子多糖SCP-01的化学组成及结构进行了初步研究，结果表明：（1）五味子多糖含有Man、Rha、Glc、Ara；各单糖摩尔比为Man：Rha：Glc：Ara=3.6：2.25：9.08：1。（2）含有α-D-吡喃糖苷及α-D-Man，无β构型或含量很低。（3）分子中1→、1→6键型的糖残基约占31.68%；1→2、1→2，6、1→4、1→4，6键型的糖残基约占28.64%；1→3、1→2，3、1→2，4、1→3，4、1→3，6、1→2，3，4的糖残基约占39.68%；每3.2个糖残基有一个非还原末端1→或1→6糖苷键。（4）分子主链由Glc、Man、Rha和Ara以1→、1→6和1→4、1→4，6键构成，并有少量Man和部分Glc以1→3、1→2，3、1→2，4、1→3，4、1→3，6、1→2，3，4键型间隔存在其中；支链由Glc、Man和少量GlcA以1→3、1→2，3、1→2，4、1→3，4、1→3，6、1→2，3，4键构成。（5）五味子多糖分子为带支链的多分枝结构，分子链高度为0.35~0.7nm；分子间存在螺旋(有序)和线圈(无序)结构，在水溶液中，依聚集程度不同，聚集体的形态可为多分枝团簇状、螺旋状、凝胶网络状、颗粒状等。5、体外抗氧化试验表明：传统浸提和超声波辅助提取的五味子多糖对羟自由基（·OH）和DPPH自由基（DPPH·）有较强的清除作用，对DPPH·的清除能力要强于对·OH的清除能力；且清除作用随多糖浓度增加而提高；但对清除超氧阴离子自由基（·O2-）几乎没有作用。超声波辅助提取的五味子多糖其清除自由基能力强于传统浸提的五味子多糖。6、体外细胞试验表明：（1）传统浸提和超声波辅助提取的五味子多糖都具有不同程度的促进小鼠脾淋巴细胞体外增殖的作用，其中以传统浸提的SCP-0的作用最强，其次为超声波辅助提取的SCP-3。（2）SCP-0、SCP-3对人PBMC的体外增殖具有较好刺激作用：在一定浓度范围内，单独作用即可刺激PBMC极显著地增殖；对PHA和LPS诱导的增殖作用也有极显著的促进作用；其余各样品，对人PBMC的体外增殖无明显作用。（3）SCP-0具有刺激细胞因子分泌的作用。在一定浓度范围内，单独作用于PBMC时，可显著刺激细胞因子IFN-γ、IL-4和TNF-α的分泌；并可以显著促进PHA刺激细胞因子IFN-γ、IL-4和TNF-α的分泌；但对LPS刺激PBMC分泌细胞因子IFN-γ、IL-4和TNF-α的作用无显著性影响。（4）在一定浓度范围内，SCP-0单独使用或与LPS联合使用，均可极显著地刺激PBMC产生抗体IgM和IgG；与LPS联合使用，该刺激作用更显著。7、动物试验表明，五味子多糖SCP-0可逆转环磷酰胺对小鼠非特异性和特异性免疫功能的抑制作用，对免疫低下动物模型的免疫功能有良好的调节作用：（1）可显著增强免疫抑制小鼠的碳廓清能力、血清溶血素水平、抗体形成细胞水平，促进ConA和LPS诱导的脾淋巴细胞增殖、提高脾脏NK细胞的杀伤活性；（2）可显著提高免疫抑制小鼠血清INF-γ、TNF-α和IL-4水平；（3）可显著提高免疫抑制小鼠肝组织中SOD、CAT和GSH-PX活性，降低肝组织中MDA水平。更多还原

【Abstract】 Schisandra chinensis (Turcz.) Baill（Magnoliaceae, schisandra）is an edible Chinese herb mainlydistributed in Liaoning, Jilin, Heilongjiang provinces of China. Early researches about Schisandrachinensis were focused on lipid-soluble composition such as lignan, triterpenoid, and essential oil, etc. Andthe material is discarded after lipid-soluble components extraction. In recent years, researches found thatwater-soluble ingredients contain a lot of polysaccharides, which causes attentions of researchers. Theresidue of Schisandra chinensis from lipid-soluble components extraction is used as raw materials in thisarticle, the extraction, purification, physical and chemical properties, structure, antioxidant andimmunodulatory activities of polysaccharide were studied. And hope to lay the foundation for theSchisandra chinensis industrialized development. And the main results are as follows.1. Professional softwares Design Expert7.0and Minitab15were used to optimize the polysaccharidesextraction by hot water and extraction associated with ultrasonic and enzyme pretreatment by responsesurface method (RSM). The optimum conditions of extraction by hot water are ratio of material to water for1:40, temperature at90.3℃, time for5hours, and the yield of crude polysaccharide is (10.99±0.22)%. Theoptimum ultrasonic pretreatment conditions are ratio of material to water for1:40, power of ultrasonic at300W, temperature at30℃, and time for1hour. Afterultrasonic pretreatment, traditional extraction is doneand the yield of crude polysaccharide is (16.73±0.25)%. The optimum enzyme pretreatment conditions areratio of material to water for1:40, pectinase dosage of1.19%, temperature at30℃, time for4hours, andpH value at4.21. After the pretreatment, traditional extraction is done and the yield of crude polysaccharideis (20.82±0.25)%.2. Crude polysaccharides were purified by organic solvents, enzymatic treatments and columnchromatography and results show as follows: method of acetocaustin-n-butyl alcohol combining enzymepretreatment is the best way to get rid of protein. After enzymic hydrolysis pretreatment of adding1%protease to the polysaccharide solution at50℃with pH value of5for140min, acetocaustin-n-butyl alcoholis added with the same volume of polysaccharide solution. Treatment of acetocaustin-n-butyl alcohol isrepeated for four times to get rid of protein and the retention rate of polysaccharides is68.3%.Polysaccharides were decolored and separated by polyamide column chromatography after deproteinization,and three fractions SCP-A, SCP-B and SCP-C were got with accounts for67.3%,2.42%and1.35%of totalpolysaccharides respectively. SCP-0seprated from SCP-A through Sephacryl S-300HR gel column was identified as a homogeneous component by ultraviolet spectrum, Sephacryl S-300HR gel columnchromatography, cellulose acetate membrane electrophoresis method and gel chromatography, and thepurity of SCP-0is94.6%. SCP-0was further purified by HPLC then SCP-01was obtained. Gelchromatogram of SCP-01showed a single sharp symmetrical peak form indicating a high purity withmolecular weight distribution width of1.22.3. Results of physical and chemical properties researches are as follows.(1) The solubility ofpolysaccharide is affected by temperature intensively and dissolving slowly at low temperature, theappropriate temperature is60℃. The polysaccharidehas good solubility at different pH value and goodsolubility in salt solution with concentration commonly in food processing. The solubility is affected by saltsolution and pH value less and the polysaccharides dissolve faster in alkaline conditions.(2) The viscosityof polysaccharide solution is affected by concentration and temperature more. With the increasingconcentration, the viscosity increased significantly, as the temperature increases, viscosity falls. NaClsolution, pH value and heat treatment affect the viscosity of polysaccharide solution less and thepolysaccharide has good stability in processing.(3) The total sugar content of SCP-0is88.7%, the uronicacid content is12.7%, and the average molecular weight is394kDa.(4) Ultrasonic treatment will causepolysaccharide degradation and average molecular weight down.4. Chemical composition and structure of polysaccharide SCP-01was studied by high performanceliquid chromatography, gas chromatography, infrared spectrum, atomic force microscope and such as thecombination of the classical chemical analysis method. And the results show as follows.(1)Monosaccharide composition of SCP-01contains Man, Rha, Glc and Ara with molar ratio of3.6:2.25:9.08:1.(2) SCP-01has α-D-pyranoside and α-D-Man, no or few β configuration.(3)Monosaccharide residues linked by1→,1→6bond types accounts for31.68%of total residues, residueslinked by1→2,1→2,6,1→4,1→4,6bond types accounts for28.64%and residues linked by1→3,1→2,3,1→2,4,1→3,4,1→3,6,1→2,3,4bond types accounts for39.68%. There is a nonreducing end group1→or a1→6glycosidic bond per3.2residues.(4) Main chain of SCP-01is formed of Man, Rha, Glc and Aralinked by1→,1→6,1→4and1→4,6bond types, and a few Man and some Glc linked by1→3,1→2,3,1→2,4,1→3,4,1→3,6,1→2,3,4bond types exist in the main chain at intervals. SCP-01has manybranches which are formed of Glc, Man and few GlcA linked by1→3,1→2,3,1→2,4,1→3,4,1→3,6,1→2,3,4bond types.(5) The height of polysaccharide molecular chain is about0.35~0.7nm and spiral and coil structures are found in molecules. Polysaccharide aggregates in water are observed as clusters, spirals,gel network and particles.5. Antioxidant tests in vitro show that SCP extracted by hot water and associated with ultrasonic havestrong activities of·OH and DPPH· scavenging, the effect of scavenging DPPH· is more remarkable thanthat of·OH and scavenging rate increases with the concentration of polysaccharide. However,·O2-scavenging activity of SCP-01is hardly observed. Polysaccharides extracted associated with ultrasonichave stronger scavenging activity than that extracted by hot water.6. The results of cell culture in vitro are as follows.(1) SCP extracted by hot water and associated withultrasonic can promote mice splenic lymphocytes proliferation and the best is SCP-0, then the SCP-3.(2)Samples SCP-0and SCP-3can promote PBMC proliferation in some concentration range and also canenhance the proliferation induced by PHA and LPS. However, the promotion effects of other SCP sampleson PBMC are hardly observed.(3) SCP-0has effect on stimulation of cell factors secretion and canstimulate the secretion of IFN-γ, IL-4and TNF-α, promote the secretion of IFN-γ, IL-4and TNF-α inducedby PHA and has no effect on the secretion induced by LPS.(4) SCP-0can stimulate PBMC producing IgMand IgG alone or in combination with LPS and the stimulation of combination is more effective.7. Animal experiments indicate that SCP-0has good regulating action on immunity of the low immunemice confronting the inhibitory effect of cytoxan on nonspecific and specific immune function of mice.(1)SCP-0can enhance the carbon clearance index, the serum hemolysin level, the antibody-forming cell level,the splenic lymphocyte proliferation induced by ConA and LPS and the NK cell activity.(2) SCP-0canincrease the secretion of IFN-γ, IL-4and TNF-α in serum.(3) SCP-0can increase the activities of SOD,CAT and GSH-PX in hepatic tissue of mice meanwhile reduces the MDA production.更多还原