【作者】 李景恩；

【导师】 谢明勇； 聂少平；

【作者基本信息】 南昌大学 ， 食品科学与工程， 2014， 博士

【摘要】 江香薷主要分布于江西省，是江西的特产资源，属于唇形科，在民间是一种药食两用的植物。目前已有部分关于其挥发油、黄酮类化合物的报道，但有关江香薷多糖的结构和活性目前国内外还处于空白。本文系统地研究了江香薷中多糖的提取、分离纯化、理化性质、一级结构、抗氧化、免疫调节作用等生物活性。研究结果可为江香薷的药理作用研究提供理论基础，并为江香薷的开发和利用提供实验数据和理论依据。论文第二部分主要研究了阿拉伯胶（Acaica senegal）和赛伊尔相思树胶（Acacia seyal）经过7小时部分水解产物的一级结构（单糖组成、糖残基连接方式及顺序），并通过美拉德反应改善其乳化性，从而提高它们的商业价值，拓展其应用空间。本文主要研究内容与结论如下：1.在单因素实验基础上，采用响应曲面法（RSM）对影响江香薷粗多糖提取率的三个主要因素：提取温度、提取时间和液固比进行优化。结果表明：提取温度86.9℃、提取时间4.10h、液固比17.7：1（mL/g），江香薷粗多糖提取率的预测值为3.38%，与实测值3.41%基本相符。粗多糖经过Sevag法脱除蛋白，得到江香薷精制多糖（MP）。MP先后经过DEAE-52纤维素色谱柱和SephacrylTMS-400HR凝胶层析柱分离得到一个中性纯多糖MP-1。当MP使用Q SepharoseTMFast Flow阴离子交换色谱进行分离，用1M NaCl进行洗脱，将其中得率较高的一个组分采用乙醇分级沉淀法进行分离，当乙醇浓度为40%（v/v）时，得到一种酸性纯多糖MP-A40。2.江香薷粗多糖CMP，精制多糖MP，中性纯多糖MP-1和酸性纯多糖MP-A40的中性糖含量分别为37.84±0.04%，33.96±0.31%，71.86±0.22%，10.30±0.04%；糖醛酸含量分别为5.18±0.16%，11.00±0.24%，0%，68.63±0.06%；蛋白质含量分别为6.14±0.01%，9.05±0.04%，1.96±0.08%，0.17±0.00%。气相色谱法分析CMP的单糖组成分别为鼠李糖，核糖，岩藻糖，阿拉伯糖，木糖，甘露糖，葡萄糖和半乳糖，摩尔比为3.72：2.45：0.92：6.00：2.76：5.09：13.53：9.58；MP的单糖组成分别为鼠李糖，阿拉伯糖，木糖，甘露糖，葡萄糖，半乳糖，摩尔比分别为5.364：12.260：3.448：12.260：32.567：30.651；MP-1单糖组成为葡萄糖、半乳糖、阿拉伯糖、甘露糖、鼠李糖，摩尔比为16.02：10.01：4.92：4.62：2.23，以及痕量岩藻糖。MP-A40的单糖组成使用离子色谱法检测，其中性糖包括阿拉伯糖（4.94%），半乳糖（3.07%），鼠李糖（2.13%），甘露糖（1.62%）及葡萄糖（1.29%），以及68.63%的半乳糖醛酸。MP包含四个组分，分子量分别为4522723Da，254918Da，127350Da和18664Da，而MP-1和MP-A40的分子量分别为587530Da和32600Da。经过HPSEC法鉴定，MP-1和MP-A40均为均一纯多糖。3. MP-1经过甲基化分析确定其糖残基间的连接方式包括→4-Glcp1→（31.76%），→2-Rhap1→（17.11%），t-Galp1→（11.02%），→2,4-Rhap1→（6.47%），→3,4-Galp1→（5.05%），t-Rhap1→（4.31%），t-Glcp1→（4.04%），t-Araf1→（4.01%），→4-Manp1→（3.61%），→5-Araf1→（3.10%），→3-Galp1→（3.05%），→2,4-Manp1→（2.55%），t-Fucp1→（2.39%）和→4,6-Glcp1→（1.53%）。根据糖残基比例判断其主链由→4-Glcp1→，→2-Rhap1→，→2,4-Rhap1→构成，末端主要为t-Galp1→。4.通过甲基化和1D/2D NMR分析，MP-A40的一级结构重复单元为→4)-[α-GalpA6Me-(1→]m-[4-α-GalpA-(1→]n。红外光谱法分析MP-A40酯化度约为32%。5.本章研究了中性纯多糖MP-1和酸性纯多糖MP-A40的体外抗氧化作用。通过DPPH自由基清除实验，铁离子还原能力，还原力实验和胡萝卜素-亚油酸体系四个抗氧化体系，证明江香薷多糖具有一定的抗氧化性，而且MP-A40的抗氧化性优于MP-1。6.使用化疗药物环磷酰胺（CY）诱导构建小鼠免疫抑制模型，以江香薷精制多糖灌胃小鼠，考察小鼠肝脏、心脏和肾脏中各种抗氧化酶活性的恢复情况。小鼠肝脏、心脏和肾脏分别用0.1g/mL的冰生理盐水研磨成匀浆，以3000rpm的速度，4℃下离心10min，取上清液分别检测总抗氧化（T-AOC），丙二醛（MAD），过氧化氢酶（CAT），超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-PX）等参数。结果证明MP不仅提高了脾脏指数和胸腺指数，而且显著提高了T-AOC，CAT，SOD和GSH-PX的酶活，降低了MDA的水平。因此，精制多糖（MP）可以降低环磷酰胺引起的的小鼠免疫抑制作用，在免疫系统防止抗氧化损伤过程中扮演重要的角色。7.粗多糖CMP刺激原代巨噬细胞吞噬中性红的实验结果表明，随着多糖浓度的增加，巨噬细胞对中性红的吞噬作用也增强。CMP刺激原代小鼠腹腔巨噬细胞产生NO，也随多糖浓度的增加而增加，并呈剂量依赖关系。脾淋巴细胞增殖实验表明MP-1可以显著促进T淋巴细胞和B淋巴细胞的增殖，而且对T淋巴细胞增殖更有效。当MP-A40浓度为500μg/mL时，K562的抑制率为31.32%。MP-A40浓度为10μg/mL时，NO的产量是阴性组的15倍。当多糖浓度从200μg/mL增至500μg/mL，NO产量从32.58μM显著增加至35.02μM。结果证明MP-A40对促进RAW264.7产生NO具有显著作用。8.将2g阿拉伯胶Acacia seyal和Acacia senegal分别溶于200mL0.1MTFA，于100℃水解7h，比较两种阿拉伯胶部分酸水解产物的一级结构信息。通过甲基化分析，得出seyal-7中各糖基的连接方式及比例分别为t-Galp（13.76%），→3-Galp1→（9.70%），→4-GalpA1→（13.70%），→6-Galp1→（30.98%），→4,6-Galp1→（1.63%），→3,6-Galp1→（27.54%），→3,4,6-Galp1→（1.40%），→2,3,6-Galp1→（1.27%）；senegal-7中各糖基的连接方式及比例分别为t-Galp（11.65%），→3-Galp1→（12.41%），→6-Galp1→（33.85%），→3,4-Galp1→（1.34%）→4,6-Galp1→（1.75%），→3,6-Galp1→（34.61%），→2,6-Galp1→（1.14%），→2,3,6-Galp1→（3.24%）。再经过1D/2D NMR（1H，13C，COSY，TOCSY，HSQC，HMBC，NOESY）对seyal-7糖残基的连接顺序进行分析，得出重复单元的结构信息，最终判断其主链结构为→1,6-Galp1→，并且在O-2，O-3，O-4三个位置有连接位点。9.将两种阿拉伯胶Acacia seyal和Acacia senegal置于60℃，湿度为75%（饱和盐水提供）的密闭环境中，样品发生美拉德反应。结果表明，反应时间为7天时，乳化液的（10%菜籽油，v/v）粒径最小，稳定性最好。更多还原

【Abstract】 Mosla chinensis Maxim. cv. jiangxiangru is mainly distributed in Jiangxiprovince. Also as the unique resource in Jiangxi, it belongs to the member of Labiataefamily, and is usually used for both food and medicine. There were already manyreports about its essential oil and flavonoids compounds, however, the studies aboutthe structure and bioactivities of polysaccharides were still in blank both in domesticand abroad. Polysaccharides were isolated and purified from this plant, and theirprimary structures, antioxidant activities and immunomodulatory effects wereinvestigated systematically in this study. The results could provide theoretical basis ofpharmacologic action of Mosla chinensis Maxim. cv. jiangxiangru, and theexperimental data could also help promote the development and utilization of thisplant.The second part of this research was about the primary structure information(monosaccharide composition, the linkage patterns and sequence of sugar residues) ofhydrolysates from Acacia seyal and Acacia senegal after7hours hydrolysis by0.1MTFA at100℃. Their emulsifying properties of these two Arbic gums were bothimproved by Maillard reaction. This research helped improve the commercial valueand expand its application space.The main research contents and conclusions were summarized as follows,1. On the basis of single factor experiments, response surface methodology(RSM) was used to optimize the extraction conditions of crude polysaccharides fromMosla chinensis Maxim. cv. jiangxiangru (CMP). Three independent variables,extraction temperature, extraction time and water to solid ratio were investigated.Based on the RSM analysis, the optimal extraction conditions were determined asextraction temperature of86.9℃, extraction time of4.1h and water to solid ratio of17.7:1(mL/g). The predictive value of optimal extraction yield was3.38%, which wasconsistent with the real value of3.41%. Protein of CMP was removed by Sevagmethod, fine polysaccharide (MP) was obtained. A neutral polysaccharide MP-1wasisolated and purified by DEAE-52cellulose chromatography column and SephacrylTMS-400HR column. If MP was purified by loading on a Q SepharoseTMFast Flow ionexchange column eluted with1M NaCl and gradient precipitation with ethanol, awater-soluble pectic polysaccharide MP-A40was obtained. 2. The neutral sugar contents of crude polysaccharide (CMP), finepolysaccharide (MP), neutral polysaccharide (MP-1) and acidic polysacchairde(MP-A40) from Mosla chinensis Maxim. cv. jiangxiangru were37.84±0.04%,33.96±0.31%,71.86±0.22%and10.30±0.04%, respectively. Uronic acid content of themwere5.18±0.16%,11.00±0.24%,0%,68.63±0.06%. Potein contents were6.14±0.01%,9.05±0.04%,1.96±0.08%,0.17±0.00%. Monosaccahride compositions ofCMP determined by gas chromatograph were Rha, Rib, Fuc, Ara, Xyl, Man, Glc andGal with a molar ratio of3.72:2.45:0.92:6.00:2.76:5.09:13.53:9.58.Monosaccahride compositions of MP were Rha, Ara, Xyl, Man, Glc and Gal with amolar ratio of5.364:12.260:3.448:12.260:32.567:30.651. Monosaccahridecompositions of MP-1were Glc, Gal, Ara, Man and Rha with a molar ratio of16.02:10.01:4.92:4.62:2.23, and trace amounts of Fuc. MP-A40was composed with4.94%Ara,3.07%Gal,2.13%Rha,1.62%Man and1.29%Glc and68.63%galacturnic acid. The results from HPLC indicated that MP was consisted with at leastfour fractions, and their average molecular weights were4522723Da,254918Da,127350Da and18664Da, repectively. The average molecular weights of MP-1andMP-A40determined by HPSEC were587530Da and32600Da. MP-1and MP-A40were uniform pure polysaccharides determniend by HPSEC method.3. The linkage patterns of MP-1determined by methylation analysis included→4-Glcp1→(31.76%),→2-Rhap1→(17.11%), t-Galp1→(11.02%),→2,4-Rhap1→(6.47%),→3,4-Galp1→(5.05%), t-Rhap1→(4.31%),t-Glcp1→(4.04%), t-Araf1→(4.01%),→4-Manp1→(3.61%),→5-Araf1→(3.10%),→3-Galp1→(3.05%),→2,4-Manp1→(2.55%), t-Fucp1→(2.39%) and→4,6-Glcp1→(1.53%). The backbone was made of→4-Glcp1→，→2-Rhap1→，→2,4-Rhap1→, and the main terminal residue was t-Galp1→.4. The primary structure of repeat unit of MP-A40was→4)-[α-GalpA6Me-(1→]m-[4-α-GalpA-(1→]n, and the degree of esterification (DE)of MP-A40was32%determined by FTIR method.5. The antioxidant activities in vitro of neutral polysaccharide MP-1and acidicpolysaccharide MP-A40were investited, including DPPH free radical scavengingactivity, ferric ion reducing antixodant power, reducing power and β-carotene-linoleicacid system. The result indicated that MP-1and MP-A40possessed certainantioxidant activities. What’s more, MP-A40had better antoxidant activities.6. The immunosuppressive mice model was built by injecting cyclophosphamide (CY), a common used chemotherapy drug. The restorations of various kinds of relatedenzymes were investigated after feeding the mice by gavag method for a period. Thetissue homogenate was made by granding liver, heart or kidney with0.1g/mL icenormal saline, respectively. And then the homogenate was centrifuged under3000rpm at4°C for10min. The supernatant was used to detect total antioxidant (T-AOC),malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD) andglutathione peroxidase (GSH-PX), and other biochemical indicators. The resultsindicated that the spleen and thymus indices and the levels of T-AOC, CAT, SOD, andGSH-PX were raised by MP significantly. However, the MDA level was decreasedunder the same condition. The results proved that MP was able to overcome theimmunosuppression incuced by cyclophosphamide. It indicated that MP might play avery important role during the anti-oxidative damage process in the immun system.7. The capability of the original generation of macrophage’s phagocytosis wasdetected by using neutral red method. As the concentrations of crude polysaccharideCMP increased, the phagocytosis of macrophage was also enhanced. CMP couldstimulate the original generation of peritoneal macrophages to produce NO. As theconcentrations increased, the NO production was increased in a dose-dependentmanner. Spleen lymphocyte proliferation assay indicated that MP-1promoted theproliferation of both T lymphcytes and B lymphcytes. What’s more, MP-1was morelikely to promote T lymphcytes proliferation. The inhibition rate was31.32%whenthe concentration of MP-A40was500μg/mL. The NO production was15times ofthat of the negative control, when MP-A40was of10μg/mL. The NO was from32.58μM to35.02μM when MP-A40was of200μg/mL to500μg/mL. The result indicatedthat MP-A40could increase NO production from RAW264.7.8.2g Acacia seyal or Acacia senegal was dissolved and hydrolyzed in200mL0.1M TFA for7h at100°C. The primary structure informations of hydrolysates wereinvestigated. Methylation analysis indicated that the linkage patterns and their ratiosof Acacia seyal were t-Galp (13.76%),3-Galp (9.70%),4-GalpA (13.70%),6-Galp(30.98%),4,6-Galp (1.63%),3,6-Galp (27.54%),3,4,6-Galp (1.40%) and2,3,6-Galp9(1.27%), and for Acacia Senegal, they were t-Galp (11.65%),→3-Galp1→(12.41%)，→6-Galp1→(33.85%),→3,4-Galp1→(1.34%),→4,6-Galp1→(1.75%),→3,6-Galp1→(34.61%),→2,6-Galp1→(1.14%),→2,3,6-Galp1→(3.24%). Based on1D/2D NMR (1H,13C, COSY, TOCSY, HSQC, HMBC, NOESY) analysis, thesequence of sugar residues and the structure information of repeating unit were obtained. The backbone was determined to be1,6-Galp with branch linked at O-2，O-3and O-4positions.9. The emulsifying properties of Acacia seyal and Acacia senegal were improvedby Maillard reaction. The reaction was occurred in a closed environment withtemperature of60°C and humidity of75%providing by saturated salt water. Theresult indicated that when the reaction time was7days, the particle size of theemulsion (10%canola oil, v/v) was minimal, and the stability of emulsion was thebest.更多还原