【作者】 朱建飞；

【导师】 吴谋成；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2008， 博士

【摘要】 菜籽粕是菜籽榨油后的副产物。菜籽多糖的研究与开发有利于菜籽粕的综合利用。对于菜籽多糖的结构国外已有一些研究,而对于其功能活性则未见有文献报道。本文以华杂4号“双低”菜籽粕为原料,利用傅里叶变换红外光谱学（FT-IR）、气相色谱（GC）、高效液相色谱（HPLC）、气相色谱质谱联用（GC-MS）、核磁共振（NMR）对分离纯化得到的菜籽多糖进行组成和结构表征,并采用化学发光分析评价其体外清除自由基能力,MTT法、逆转录聚合酶链反应（RT-PCR）等细胞生物学和分子生物学方法研究了菜籽多糖对脾淋巴细胞、腹腔巨噬细胞的免疫调节作用。主要研究结果如下:1菜籽多糖的制备及表征。菜籽多糖的制备及表征国外已有较多研究。本文重点研究了华杂4号菜籽粕中多糖的提取优化条件、绿色环保的纯化工艺以及表征方法。1.1经过前处理的脱脂菜籽粕,按照中心组合实验（CCD）得到的水溶性多糖提取优化工艺条件,即以水按液料比为28:1（mL/g）在94℃浸提2.9 h,一次提取菜籽多糖得率为2.18%,连续提取三次将水溶性多糖提取殆尽。剩余的残渣料再以5%NaOH溶液作提取剂,按料液比1:20,60℃提取2 h,得到的提取液以4 M HCl溶液中和至pH=5。得到的水溶性多糖和碱溶性多糖两部分提取液分别经筛选出的特1号大孔吸附树脂柱层析、分步醇沉、DE-52纤维素离子交换柱层析等方法纯化,分别得到各自的主要级分WPS-1和APS-2。WPS-1和APS-2再通过葡聚糖凝胶G-200柱层析和凝胶渗透色谱（GPC）进行纯度鉴定,结果表明WPS-1和APS-2都是相对均一多糖。1.2 WPS-1和APS-2均为白色絮状的非淀粉多糖;中性糖含量分别为83.2%和66.8%;糖醛酸含量分别为6.1%和9.3%;蛋白质含量分别为3.78%和8.65%。WPS-1和APS-2中蛋白质部分都由天门冬氨酸、谷氨酸、丝氨酸、甘氨酸、精氨酸、苏氨酸、丙氨酸、酪氨酸、缬氨酸、胱氨酸、异亮氨酸、亮氨酸、苯丙氨酸等9种氨基酸组成。GPC法测定WPS-1和APS-2的重均分子量（M_w）分别为7.20×10⁵和1.61×10⁵,数均分子量（M_n）分别为2.45×10⁵和6.79×10⁵。FT-IR、GC、HPLC初步分析WPS-1和APS-2的官能团特征及单糖组成,确定WPS-1单糖主要由阿拉伯糖（Ara）、半乳糖（Gal）、葡萄糖（Glc）、葡萄糖醛酸（GlcA）组成,其摩尔百分含量分别为66.6%、18.3%、12.6%、2.51%;APS-2单糖组成包括Ara、Gal、Glc、甘露糖（Man）、半乳糖醛酸（GalA）、GlcA、Rha摩尔百分含量分别为52.8%、21.7%、10.6%、5.0%、4.8%、3.6%、1.4%。通过甲基化分析以及一维NMR(包括¹³C NMR和¹H NMR)对WPS-1的结构表征进行了研究,结合前人的相关研究成果,得出WPS-1的主体部分是主要是由（1→5）键和（1→2）键连接的Ara残基组成的多支链α-阿拉伯聚糖。2菜籽多糖的体外活性。菜籽多糖的生物活性本实验室曾作初步研究,国内外未见有菜籽多糖生物活性的报道。本文重点从细胞、分子水平上研究了菜籽多糖的生理活性。2.1分别采用邻苯三酚-鲁米诺、硫酸铜-邻菲哕啉-抗坏血酸-双氧水、鲁米诺-双氧水三种化学发光体系,通过微弱发光测量仪测定了WPS-1和APS-2对超氧阴离子(O₂^·-)、羟基自由基（HO^·）和双氧水（H₂O₂）三种ROS的体外清除作用。结果表明WPS-1和APS-2对各ROS都有良好的体外清除作用,且都呈剂量-效果关系。WPS-1和APS-2在实验最高浓度2000μg/mL时,对O₂^·-的极大抑制率分别为90.4%和89.6%。WPS-1和APS-2对O₂^·-的半数抑制浓度(IC₅₀)分别为400±44μg/mL和450±64μg/mL。WPS-1比APS-2具有更好的清除O₂^·-能力。WPS-1和APS-2在实验最高浓度1000μg/mL时,其对HO^·的极大抑制率分别为93.8%和86.7%。WPS-1和APS-2对HO^·的IC₅₀分别为240±18μg/mL和293±24μg/mL。WPS-1比APS-2具有更好的清除HO^·的能力。WPS-1和APS-2在实验最高浓度50μg/mL时,其对H₂O₂的极大抑制率分别为84.2%和85.2%。WPS-1和APS-2对H₂O₂的IC₅₀分别为10.0±0.8μg/mL和6.1±0.5μg/mL。APS-2清除H₂O₂的能力略强于WPS-1。比较IC₅₀值,WPS-1和APS-2对各自由基的清除能力都表现为:H₂O₂＞HO^·＞O₂^·-。2.2采用尼龙毛柱法从脾淋巴细胞分离T、B淋巴细胞。通过MTT法检测,菜籽多糖对总脾淋巴细胞和T淋巴细胞具有明显的增殖活性,并表现出明显的剂量-效果关系,增殖的最佳浓度为40μg/mL,并且WPS-1比APS-2作用效果更好。采用半定量RT-PCR进一步研究菜籽多糖WPS-1对T淋巴细胞分泌的细胞因子白细胞介素2（IL-2）、IL-4、IL-6、干扰素γ（IFN-γ）mRNA表达的影响。首先研究了各细胞因子mRNA随WPS-1作用时间的变化,WPS-1促进IL-2、IL-4、IL-6 mRNA表达最佳作用时间都为24 h,而促进IFN-γmRNA表达的最佳作用时间为12 h。采用各细胞因子mRNA表达的最佳作用时间,进一步考察WPS-1浓度的影响,得到其对促进IL-2、IL-6、IFN-γmRNA表达的最佳浓度都为40μg/mL,而对IL-4则为20μg/mL。菜籽多糖可能对淋巴细胞的免疫功能具有调节作用。2.3采用MTT法检测细胞的活性,菜籽多糖能促进巨噬细胞增殖,并表现出明显的剂量-效果关系,菜籽多糖促进巨噬细胞增殖的最佳浓度为40μg/mL,WPS-1比APS-2对巨噬细胞增殖活性的促进作用略大。菜籽多糖对巨噬细胞的影响与对T淋巴细胞的检测结果相类似,。采用半定量RT-PCR进一步研究不同作用时间下40μg/mL菜籽多糖WPS-1对腹腔巨噬细胞IL-6、肿瘤坏死因子α（TNF-α）这两种细胞因子以及诱生型一氧化氮合酶（iNOS）mRNA表达的影响。WPS-1对腹腔巨噬细胞IL-6、TNF-α和iNOS mRNA的表达量最大的时间为24 h。菜籽多糖可能对巨噬细胞的免疫功能具有调节作用。更多还原

【Abstract】 Rapeseed meal is the byproduct of rapeseed after oil extraction.The study and exploitation of rapeseed polysaccharides is beneficial to the comprehensive utilization of rapeseed meal.There have been several research results on the structure of rapeseed polysaccharides,but their bioactivity has not been reported up to now.In this paper,the components stuctural characterization of rapeseed polysaccharides isolated and purified from Huaza No.4 rapeseed meal was identified by Fourier transform infrared spectrometry（FT-IR）,gas chromatography（GC）,Gas chromatography-mass spectrometry（GC-MS）,high performance liquid chromatography（HPLC）,and nuclear magnetic resonance（NMR）.The chemiluminescence（CL） method was used to investigate the free radical scavenging activity of rapeseed polysaccharides.The immunomodulatory activity was investigated by MTT colorimetric assay and semi-quantitative reverse transcription-polymerase chain reaction（RT-PCR）.The major content and results are as follows:1 Preparation and characterization of rapeseed polysaccharides.There are some reports on pareparation and characterization of rapeseed polysaccharide abroad.The emphasis of this research is on optimization of extraction technology,environmentally friendly purification process,and characterization methods of polysaccharides from Huaza No.4 rapeseed Meal.1.1 The optimized extraction technology,of water-soluble polysaccharides derived from the pretreated rapeseed meal was performed by the central combinational design（CCD）. the extraction technology of water-soluble polysaccharides composed of 94℃,2.9 h,and 28:1（mL/g） ratio of water to rapeseed meal.After extraction of three times,The insoluble residue on the filter paper was then resuspended and further treated（1 g/20 mL） with 5%NaOH and heated at 60℃for 2 h.Alkali-insoluble material was removed by filtration,and the filtrate was acidified to pH=5 with 4 mol/L HCl to yield an alkali-soluble polysaccharide fraction.Two major polysaccharide fractions,WPS-1 and APS-2,were isolated from water-soluble and alkali-soluble extracts of Huaza No.4 rapeseed meal with a stepwise procedure of Special No.1 macroporous adsorption resin column chromatography,ethanol precipitation and DE-52 cellulose column chromatography.WPS-1 and APS-2 were both relative homogeneous fractions,which were identified by Sephadex G-200 chromatography and gel permeation chromatography （GPC）.1.2 Both WPS-1 and APS-2 were non-starch polysaccharides with white and flocculent appearance.UV,FT-IR,GC and HPLC were used to analyze the components of WPS-1 and APS-2.It was found that the weight-average molecular weight（Mw） of WPS-1 and APS-2 were 7.20×10⁵ and 1.61×10⁵,respectively.The number-average molecular weight（Mn） of WPS-1 and APS-2 were 2.45×10⁵ and 6.79×10⁵,respectively. It was found that WPS-1 and APS-2 were 2 kinds of polysaccharides containing proteins and uronic acids.The contents of neutral sugar,uronic acid,and protein of them were 83.2%and 66.8%;6.1%and 9.3%;3.8%and 8.7%,respectively.The associated protein portions in both polysaccharide fractions consisted of 13 different amino acids,including aspartic acid,glutamic acid,serine,glycine,arginine,threonine,alanine,tyrosine,valine, cystine,isoleucine,leucine and phenylalanine.To their monosaccharide composition, WPS-1 consisted primarily of Ara（66.6 mol%） and Gal（18.3 mol%） accompanied by some Glc（12.6 mol%） and GlcA（2.5 mol%）;APS-2 consisted primarily of Ara（52.9 mol%） and Gal（21.7 mol%） accompanied by some Glc（10.6 mol%）,Man（5.0 mol%）, GalA（4.8 mol%）,GlcA（3.6 mol%）,and Rha（1.4 mol%）.The structural characterization of WPS-1 was further investigated by methylation linkage analysis and 1-dimensional NMR(including ¹³C NMR and ¹H NMR).On the basis of these study results and considering other relevant studies,it was concluded that WPS-1 consisted manily of arabinan fragment,which was mainly（1→5） and（1→2） linked.2 In vitro activity of rapeseed polysaccharides.The bioactivity of rapeseed polysaccharides has been investigated preliminary by our lab.However,research on the bioactivity of rapeseed polysaccharides has received little attention up to now.The emphasis of this research is on the in vitro activity of polysaccharides at the cellular and molecular levels2.1 The chemiluminescence（CL） method was used to investigate the free radical scavenging activity of rapeseed polysaccharides.The scavenging ability of WPS-1 and APS-2 for O₂^·-,HO^·,and H₂O₂ were determined by a pyrogallol-luminol system, CuSO₄-phenanthroline-ascorbate-H₂O₂ system,and luminol-H₂O₂ system,respectively, on a BPCL Ultra-weak luminescence analyzer.The results indicated both WPS-1 and APS-2 have prominent ability of scavenging free radical in a concentration-dependent manner.The maximum inhibition rates of WPS-1 and APS-2 were 90.4%and 89.6%at the 2000μg/mL concentration,respectively.Their half-maximal inhibitory concentration (IC₅₀) values were 400±44μg/mL for WPS-1 and 450±64μg/mL for APS-2.WPS-1 was slightly more effective than APS-2 at scavenging O₂^·-.Both WPS-1 and APS-2 could scavenge HO^·and the maximum inhibition of CL by WPS-1 and APS-2 were 93.8%and 86.7%,respectively.The IC₅₀ value of WPS-1 was 240±18μg/mL,while that of APS-2 was 293±24μg/mL.WPS-1 was more effective than APS-2 at scavenging HO^·.The maximum inhibition by WPS-1 was 84.2%and by APS-2 was 85.2%.The IC₅₀ value of WPS-1 was 10.0±0.8μg/mL,while that of APS-2 was 6.1±0.5μg/mL.It was concluded that APS-2 was more effective than WPS-1 at scavenging H₂O₂.Compared with APS-2,WPS-1 was more effective at scavenging superoxide radical(O₂^·-) and hydroxyl radical（HO^·）,but less effective at scavenging hydrogen peroxide（H₂O₂）.In decreasing order,free radical scavenging activity of WPS-1 and APS-2 towards reactive oxygen species（ROS） was:H2O2＞HO^·＞O₂^·-.2.2 A nylon wool column was used to separate of spleen T and B lymphocytes. Through the MTT assay,rapeseed polysaccharides were found to significantly enhance proliferation of totol spleen lymphocytes and T lymphocytes.The optimal concentration of both WPS-1 and APS-2 to promote proliferation were 40μg/mL,but WPS-1 was more effective than APS-2 at promoting cell proliferation.Semi-quantitative RT-PCR was performed to determine changes in cytokine mRNA expression.The influence of WPS-1 to cytokine interleukin-2（IL-2）,IL-4,IL-6,and interferonγ（IFN-γ） mRNA expression in T lymphocytes were investigated.First,the relation between time of T lymphocytes cells incubated with WPS-1 and the cytokine mRNA expression was studied.The best time of WPS-1 to promote IL-2,IL-4,IL-6 mRNA of expression were all 24 h.The best time to that of IFN-γwas 12 h.The best time of the cytokines mRNA expression were adopted,to further investigate the effect of the concentration of WPS-1 to cytokine mRNA expression.The results showed the best concentration of WPS-1 to promote IL-2,IL-6, and IFN-γmRNA expression were all 40μg/mL,to that of IL-4 was 20μg/mL.The results indicates that rapeseed polysaccharide can enhance the immune regulation of spleen T lymphocyte.2.3 Rapeseed polysaccharides promote proliferation macrophage cells,in a dose-dependent manner.The optimal concentration of both WPS-1 and APS-2 to promote proliferation were 40μg/mL,but WPS-1 was more effective than APS-2 at promoting cell proliferation,which similar to lymphocytes proliferation.Semi-quantitative RT-PCR was performed to determine changes in cytokine mRNA expression.The influence of WPS-1 to IL-6,tumor necrosis factor-α（TNF-α）,and inducible nitric oxide synthase（iNOS） mRNA expression were all 40μg/mL.The results indicates that rapeseed polysaccharide can enhance the immune regulation of peritoneal macrophages.更多还原