【作者】 叶红；

【导师】 曾晓雄；

【作者基本信息】 南京农业大学 ， 生物工程， 2008， 博士

【摘要】 马尾藻（Sargassum pallidum）属褐藻门马尾藻科植物,盛产于我国黄海、渤海沿岸各地,资源十分丰富,由于不能直接食用,以致目前马尾藻的加工利用范围比较窄,只有小部分被用作饲料、藻胶和医药工业的原料,大部分尚未得到全面的开发利用。褐藻糖胶（褐藻多糖）是褐藻中的一种杂多糖,具有多样的生理功能,是褐藻中重要的生物活性物质。目前国内外对马尾藻中的多糖（S.pallidum Polysaccharides,SPP）研究很少,且不够深入。鉴于此,本文对采自山东荣成的马尾藻中多糖的体外抗肿瘤活性与体外抗氧化活性,以及多糖的结构进行了研究,以期了解它们的生理活性与结构。本论文共有四个部分,分别对马尾藻多糖的提取、分离纯化和理化性质、体外抗肿瘤活性与抗氧化活性、结构鉴定进行了研究。主要结果如下:1马尾藻多糖的提取利用超临界二氧化碳（SC-CO₂）脱除马尾藻粉中的脂类物质,研究了超临界CO₂萃取压力、萃取温度、萃取时间对马尾藻脱脂过程的影响。研究结果表明CO₂的流量为20 L/h时,超临界CO₂脱脂的最佳工艺条件为:萃取压力45 MPa,萃取温度55℃,时间6 h,萃取率为8.53‰。将响应面分析法用于马尾藻多糖的提取工艺,对影响马尾藻多糖提取的主要因素超声波破碎时间、提取温度、提取时间的最佳水平范围及其交互作用进行了研究和探讨,依据回归分析结果,最佳工艺条件是:超声波破碎时间为469.54 s,提取温度为90.94℃,提取时间为4.96 h,多糖得率的理论值为2.48%。对三氯乙酸（TCA）脱蛋白条件进行了优化,选择TCA终浓度为2%左右,TCA沉淀蛋白的时间为2h或稍长。在压力0.5 MPa,温度20℃下,对脱蛋白后的提取液进行膜分离,提取液依次用孔径0.1μm的微滤膜、截留分子量为100 KDa、50 KDa、10 KDa和3 KDa的超滤膜进行膜分离。研究结果表明多糖组分主要包含在分子量大于50 KDa的截流液中,由于大于孔径0.1μm的截流溶液中所含多糖量较多,成分较复杂,将其单独收集分析。分子量小于50 KDa的溶液多糖含量较少,将其合并。在以后的实验中将SPP提取液分成三部分SPP-1（＞0.1μm）、SPP-2（＜0.1μm且＞50 KDa）和SPP-3（＜50 KDa）。研究了马尾藻料液浓度对膜分离的影响,在料液比分别为1:20（m/V）、1:30、1:100、1:150,压力为0.5 MPa,温度为20℃时,将脱蛋白后的提取液进行膜分离,结果表明随着料液比的增加,膜分离后的多糖损失越少。综合考虑,在膜分离前将待处理的溶液按1:150稀释。在温度20℃时,压力分别在0.1、0.3、0.5 MPa下进行膜分离,结果表明操作压力对多糖损失并无显著影响,在膜分离设备允许压力范围内,0.1-0.5 MPa都是适合的。2马尾藻多糖的纯化多糖组分SPP-1、SPP-2和SPP-3占粗多糖百分比分别为72.15%、16.69%和11.16%,其中的多糖含量依次为58.13%、18.98%和16.38%,蛋白含量依次为1.09%、1.75%和1.73%,硫酸基含量依次为15.68%、15.26%和22.62%,糖醛酸含量依次为14.22%、14.30%和5.20%。利用DEAE Cellulose-52层析柱分别对SPP-1、SPP-2和SPP-3三个多糖组分进行分离纯化,其中由SPP-1得到两个主要的组分SPP-1-1和SPP-1-2,得率分别为32.53%和8.67%;由SPP-2得到SPP-2-1、SPP-2-2和SPP-2-3三个组分,得率分别为10.53%、9.20%和36.33%;由SPP-3得到SPP-3-1和SPP-3-2两个组分,得率分别19.52%和57.27%。在纯化过程中多糖有部分损失,同时粗多糖中的蛋白基本被除去。利用Sephadex G-100层析柱分别对SPP-1-1、SPP-1-2、SPP-2-1、SPP-2-2、SPP-2-3、SPP-3-1和SPP-3-2七个组分进行分离纯化,以用于进一步的结构分析。3马尾藻多糖的体外抗肿瘤活性与抗氧化活性以MTT比色法探讨了经DEAE Cellulose-52纯化得到的七个多糖组分对人肺癌细胞株A549、人肝癌细胞株HepG2和人胃癌细胞株MGC-803生长的体外抑制作用。组分SPP-3-1和SPP-3-2对人肝癌细胞株HepG2、人肺癌细胞株A549和人胃癌细胞株MGC-803的生长具有明显的体外抑制作用,且抑制能力具有剂量依赖性。组分SPP-2-3只对肝癌HepG2细胞表现出强抑制率,并呈剂量依赖性。组分SPP-3-1和SPP-3-2具有较高的体外抗肿瘤活性可能与它们的低分子量（＜50 KDa）及高硫酸基含量（22.62%）有关。在浓度为2 mg/mL时,粗多糖组分SPP-1、SPP-2和SPP-3对DPPH自由基的清除率分别为23.84%、13.23%和12.83%;总抗氧化能力分别为0.087、0.083和0.088（μmolFeSO₄/mg）;还原力(以700 nm处的吸光度A₇₀₀表示)分别为0.785、0.446和0.541;对·OH的清除率分别为68.39%、13.22%和27.59%。研究了乙醇浸提温度对马尾藻中总多酚含量的影响,在30℃马尾藻中的多酚类物质不能完全浸提出来;在70℃因浸出时间短不易控制,所以选择50℃作为乙醇浸提温度。马尾藻经50℃、70%乙醇浸提后,依次用不同有机溶剂萃取后得到乙酸乙酯萃取物、正丁醇萃取物及萃取剩余物,其占总萃取物百分比依次为15.76%、5.28%、78.96%,采用Folin-Ciocalteu比色法测定其中的总多酚含量,以绿原酸（CHA）作为标准物质,依次为3.20 mgCHA/g干重、12.12 mgCHA/g干重和52.08 mgCHA/g干重,总萃取物得率为8.07%。乙酸乙酯萃取物和正丁醇萃取物在浓度为2 mg/mL时对DPPH自由基清除率分别为30.59%和29.36%。乙酸乙酯萃取物和正丁醇萃取物的总抗氧化能力较高,分别为0.520和0.345（μmolFeSO₄/mg）。乙酸乙酯萃取物、正丁醇萃取物和萃取剩余物的还原力较高,由高到低为萃取剩余物＞正丁醇萃取物＞乙酸乙酯萃取物。乙酸乙酯萃取物、正丁醇萃取物和萃取剩余物在浓度为2 mg/mL时对金属离子的螯合率分别为48.25%、51.34%和17.21%。4马尾藻活性多糖的结构分析利用高效凝胶过滤色谱法（HPGFC）测定多糖组分SPP-2-3、SPP-3-1、SPP-3-2的分子量和检测其纯度,其平均分子量（M_w）依次为132.999 KDa、5.874 KDa、7.249KDa。气相色谱法分析多糖的单糖组成,SPP-2-3单糖组成及摩尔比为鼠李糖:木糖:岩藻糖:甘露糖:葡萄糖:半乳糖=0.15:2.74:0.64:1.00:0.49:1.11,SPP-3-1单糖组成及摩尔比为甘露糖:葡萄糖:半乳糖=1.00:11.18:0.96,SPP-3-2单糖组成及摩尔比为鼠李糖:木糖:岩藻糖:甘露糖:葡萄糖:半乳糖=0.17:2.53:0.61:1.00:0.46:0.92。通过红外吸收光谱（IR）分析得知SPP-2-3和SPP-3-2中存在（?）糖苷键。高碘酸氧化、Smith降解以及甲基化分析结果显示SPP-2-3中的甘露糖的连接方式以（1→3）糖苷键为主,葡萄糖和半乳糖除了存在（1→3）键型外,还存在（1→6）键连接方式;木糖的连接方式以（1→2）键为主;岩藻糖除了存在（1→3）键型外,还存在（1→4）键型。SPP-3-2中的甘露糖和半乳糖的连接方式以（1→3）键为主;葡萄糖是以（1→6）或者（1→2）键型连接;木糖和岩藻糖以（1→2）或（1→4）键型连接为主。更多还原

【Abstract】 Sargassum pallidum belongs to Phaeophyta Sargassaceae and are extensively distributed in China Yellow Sea and Bohai Sea.Due to not being edible,S.pallidum is used in relatively narrow scope at present,and only a small part is used as feed,algin and raw materials of pharmaceutical industry,and most of them has not yet been fully utilized.Alga glycine is a kind of miscellaneous polysaccharide.It has a variety of physiological functions,and is an important bioactive substance in brown alga.However,little information about S.pallidum polysaccharides（SPP） and biological activity is available compared with those of the other Sargassum genus at present.In this paper S.pallidum collected from Rongcheng in Shandong Province was studied,including the antitumor activity and antioxidant activity in vitro of SPP,and identification,of polysaccharides structure.This paper is composed of four parts:extraction of polysaccharides from S.pallidum, purification and identification of physical-chemical characters,antitumor activity and antioxidant activity of SPP in vitro,and identification of polysaccharides structure.Main results are listed as follows:1.Extraction of S.pallidum polysaccharidesSupercritical CO₂（SC-CO₂） extraction was used to degrease the lipophylic substances in S.Pallidum.The effect of extraction pressure,extraction temperature and extraction time were investigated.As results,the optimum operating conditions for treatment were as follows:temperature at 55℃,pressure at 45 MPa,a flow rate of CO₂ at 20 L/h,and extraction time for 6 h,and the yield was 8.53‰.Extraction of S.pallidum polysaccharide was optimized by the method of reSPPonse surface analysis（RSA）.The effects of the main variables namely:ultrasonic time,extraction temperature and extraction time as well as the mutual interaction between variables were studied.The maximum yield of polysaccharides was up to 2.48%under the conditions as follows:ultrasonic time 469.54 s;extraction temperature 90.94℃;extraction time 4.96 h. The conditions of deproteinization by trichloroaeetie acid（TCA） were optimized.TCA final concentration was about 2%,and precipitation time was 2 h or longer.Membrane separation was performed at 0.5 MPa and at 20℃.The deproteinized polysaccharides solution was pumped to membrane surface（tangential flow） with different nominal molecular weight cut-offs（0.1μm,100 KDa,50 KDa,10 KDa,3 KDa）,in turn. The results showed that S.pallidum polysaccharides were mainly included in the fractions with molecular weight more than 50 KDa.Due to the fraction with pore size more than 0.1μm contained much polysaccharides,having complex composition,it was collected and analyzed singly.Fractions with molecular weight less than 50 KDa containing less polysaccharide were combined.In later experiments S.pallidum polysaccharides（SPP） were divided into 3 main crude polysaccharides,SPP-1（＞0.1μm）,SPP-2（＜0.1μm and＞50 KDa） and SPP-3（＜50 KDa）.Effect of the ratio（material to water） on membrane separation was studied.At the ratio（material to water） of 1:20（m/V）,1:30,1:100 and 1:150,pressure at 0.5 MPa, temperature at 20℃,the deproteinized polysaccharides solution were treated by membrane separation.The results showed that a higher ratio（material to water） led to a less loss of polysaccharides in membrane separation.It was considered to be suitable to dilute the solution at the ratio of 1:150 before membrane separation.At 20℃,membrane separation was performed at the pressure of 0.1,0.3 and 0.5 MPa,respectively.The results showed that the operating pressure had no significant effect on polysaccharides loss.So the pressure between 0.1 and 0.5 MPa are suitable in the scope allowed by membrane separation equipment.2.Purification of S.pallidum polysaccharidesThe yield of SPP-1,SPP-2 and SPP-3 accounted for 72.15%,16.69%and 11.16%, respectively of total crude polysaccharides.The polysaccharides contents were 58.13%, 18.98%and 16.38%,respectively and the protein contents were 1.09%,1.75%and 1.73%, respectively.The sulfate contents were 15.68%,15.26%and 22.62%,respectively and the uronic acid contents were 14.22%,14.30%and 5.20%,respectively.A lyophilized fraction of polysaccharides SPP-1 was chromatographed on a DEAE Cellulose-52 anion-exchange column to yield two peaks,SPP-1-1 and SPP-1-2.In a similar manner,SPP-2-1,SPP-2-2 and SPP-2-3,SPP-3-1 and SPP-3-2 were obtained from SPP-2 and SPP-3,respectively.The recovery of SPP-1-1 and SPP-1-2 were 32.53%and 8.67%, respectively.The recovery of SPP-2-1,SPP-2-2 and SPP-2-3 were 10.53%,9.20%and 36.33%,respectively.The recovery of SPP-3-1 and SPP-3-2 were 19.52%and 57.27%, respectively.During the purification of crude polysaccharides a little part of polysaccharides were losed,while the protein in crude polysaccharide was almost removed. Fractions of polysaccharides SPP-1-1,SPP-1-2,SPP-2-1,SPP-2-2,SPP-2-3,SPP-3-1 and SPP-3-2,respectively were chromatographed on a Sephadex G-100 chromatography column.So they could be used for further structural analysis.3.Antitumor activity and antioxidant activity in vitro of S.pallidum polysaccharidesThe inhibition effects of 7 fractions purified by DEAE Cellulose-52 on the growth of HepG2 cells,A549 cells and MGC-803 cells were evaluated in vitro by MTT assay. Fractions of polysaccharides SPP-3-1 and SPP-3-2 presented significantly higher antitumor activity against the HepG2 cells,A549 cells and MGC-803 cells in vitro than blank control groups,and the inhibition ability was dose-dependent.SPP-2-3 only presented significantly higher antitumor activity against the HepG2 cells in vitro than a blank control, dose-dependently.It seems that SPP-3-1 and SPP-3-2 from SPP-3 with relatively lower molecular weight（＜50KDa） and higher sulfate contents（22.62%） showed significantly higher antitumor activity against the HepG2 cells,A549 cells and MGC-803 cells in vitro.At the concentration of 2mg/mL,scavenging of DPPH radical of crude polysaccharide SPP-1 and SPP-2 and SPP-3 were 23.838%,13.232%and 12.828%,respectively.The total antioxidant capacity of SPP-1 and SPP-2 and SPP-3 were 0.087,0.083 and 0.088, respectively（μmolFeSO4/mg）.The reducing power（expressed as absorbanee at 700 nm） and OH radical scavenging of SPP-1,SPP-2 and SPP-3 increased with the increasing concentration in experimental scope.Their reducing power were 0.785,0.446 and 0.541, respectively at 2 mg/mL.Their OH radical scavenging were 68.391%,13.218%and 27.586%,respectively at 2 mg/mL.Effect of ethanol extraction temperature on total polyphenol content extracted from S. pallidum were studied.At 30℃total polyphenol could not been fully extracted from S. pallidum,while at 70℃the extraction time was too short to be controlled,so 50℃was selected as alcohol extraction temperature.S.pallidum was extracted by 70%ethanol at 50℃,and then extracted by chloroform,ethyl acetate and n-butanol in turn.The yeied of total extract was 8.07%.Ethyl acetate extract,n-butanol extract and extract residue accounted for 15.76%,5.28%and 78.96%,respectively of total extract,and their total polyphenol contents were 3.20 mgCHA /gDW,12.12 mgCHA/gDW and 52.08 mgCHA/gDW, respectively determined by Folin-Ciocalteu method.DPPH radical scavenging of ethyl acetate and n-butanol extract were 30.59%and 29.36%,respectively at 2 mg/mL.Their total antioxidant capacity were 0.520 and 0.345 （μmolFeSO4/mg）,respectively.Reducing power of ethyl acetate extracts,n-butanol extract and extract residue were high.The descending sequence was extract residue,n-butanol extract and ethyl acetate extracts.Metal ions scavenging of ethyl acetate extracts,n-butanol extracts and extract residue were 48.25%,51.34%and 17.21%at 2 mg/mL.4.Structural analysis of S.pallidum polysaccharidesThe average molecular weight and purity of polysaccharides fractions SPP-2-3, SPP-3-1 and SPP-3-2 were defined by high performance of gel filtration chromatography （HPGFC）.They were 132.999KDa,5.874KDa and 7.249KDa,respectively. Monosaccharide composition and molar ratio of SPP-2-3,SPP-3-1 and SPP-3-2 were analyzed by Gas chromatography（GC）.They were rhamnose:xylose:fucose:mannose: glucose:galactose= 0.15:2.74:0.64:1.00:0.49:1.11,mannose:glucose:galactose= 1.00:11.18:0.96,and rhamnose:xylose:fucose:mannose:glucose:galactose=0.17: 2.53:0.61:1.00:0.46:0.92,respectively.The infrared absorption spectroscopy（IR） of SPP-2-3 and SPP-3-2 showed a character of口-glycosidic bond.Periodate oxidation,Smith degradation and methylation analysis of SPP-2-3 showed that mannose were linked mainly by（1→3） glycosidic bond.In addition to（1→3） glycosidic bond in glucose and galactose,there was also（1→6） glycosidic bond.Xylose was linked mainly by（1→2） glycosidic bond.In addition to（1→3） glycosidic bond in fucose,there was also（1→4） glycosidic bond.For SPP-3-2,mannose and galactose were linked mainly by（1→3） glycosidic bond.Glucose was linked by（1→6） or（1→2） glycosidic bond.Fucose and L-xylose was linked by（1→2） or（1→4） glycosidic bond.更多还原