【作者】 蒋长兴；

【导师】 曾晓雄；

【作者基本信息】 南京农业大学 ， 食品科学， 2011， 博士

【摘要】 青蛤俗名蛤蜊,属软体动物门,双壳纲,青蛤属,为我国主要的水产贝类,在海洋水产业中具有重要地位。青蛤肉质鲜美,营养丰富,富含蛋白质、氨基酸、脂类、糖类、矿物质元素。传统医学证实,青蛤软体组织水浸提物对发炎、哮喘、口腔溃疡等病症疗效较佳。青蛤水提物的生理活性可能与其富含多糖类物质有关。研究表明,一些源于海洋贝类诸如文蛤、菲律宾蛤仔、翡翠贻贝的多糖具有抗肿瘤、消炎、免疫调节等方面的生理活性,并且关于贝类多糖分离纯化、理化性质、单糖组成、结构等方面研究均有报道。而关于青蛤多糖的研究报道甚少。本实验以青蛤为原料,对青蛤多糖进行提取制备、分离纯化,并对多糖的化学组成、结构特征、化学修饰、生理活性等方面进行分析与研究。研究内容及研究结果具体如下：(一)青蛤多糖提取条件优化采用单因素实验研究提取工艺参数(提取温度、提取时间、提取次数、水料比)对青蛤多糖产率的影响,结果表明,提取温度、提取时间、提取次数对青蛤多糖产率有显著影响。随着提取温度升高,青蛤多糖产率基本呈上升趋势；提取次数2次以上时,随着提取次数增多,多糖产率变化不明显。在单因素研究的基础上,采用二次正交旋转组合实验设计对提取工艺参数进行优化。通过Design expert 7.0统计软件对实验数据进行处理,得到青蛤多糖提取最佳工艺条件(提取温度90℃,提取时间250 min,水料比29,提取次数2次)。在此工艺条件下青蛤多糖的实际产率为15.52%士1.26%,与模型预测结果(15.62%)基本相符。(二)青蛤多糖分离纯化、理化性质及结构分析采用DEAE-纤维素柱层析法对青蛤粗多糖(Crude CSPS)进行初步分离,获得三个组分(F1、F2、F3)。采用Sephdex G-100凝胶柱层析进一步分离纯化,获得三个纯化组分(CSPS-1、CSPS-2、CSPS-3)。采用HPLC法对CSPS-1、CSPS-2、CSPS-3的纯度进行鉴定,结果表明,CSPS-1、CSPS-2、CSPS-3为均一的多糖组分。采用苯酚-硫酸法、硫酸-间羟联苯法、考马斯亮蓝法、氯化钡-明胶法分别对Crude CSPS、CSPS-1、CSPS-2、CSPS-3中总糖、糖醛酸、蛋白质、硫酸基含量进行分析。结果表明,Crude CSPS、CSPS-1、CSPS-2、CSPS-3,总糖含量分别为83.81%、98.75%、95.58%、84.71%；糖醛酸含量分别为1.58%、0.16%、0.96%、2.13%；硫酸基含量分别为0.92%、1.22%、2.08%、3.58%：Crude CSPS、CSPS-3蛋白质分别为3.08%、6.34%,CSPS-1、CSPS-2均未能检出；GC分析表明,Crude CSPS由鼠李糖、岩藻糖、葡萄糖、半乳糖组成,摩尔百分比分别为：10.88%、12.45%、42.78%、33.89%；CSPS-1由木糖、葡萄糖组成,摩尔百分比分别为：4.92%、95.08%。CSPS-2由葡萄糖组成。CSPS-3由鼠李糖、岩藻糖、甘露糖、葡萄糖、半乳糖组成,其摩尔百分比分别为：11.48%、17.15%、12.44%、21.57%、37.36%。HPLC分析结果表明,CSPS-1、CSPS-2、CSPS-3的平均分子量分别为68.6 kDa、80.6 kDa、100.6 kDa。采用FT-IR、NMR、高碘酸氧化、Smith降解、甲基化反应、GC-MS对CSPS-1、CSPS-2、CSPS-3结构进行分析。FT-IR分析表明,CSPS-1、CSPS-2、CSPS-3具有多糖的特征吸收,存在α型构型；NMR进一步证实,CSPS-1、CSPS-2、CSPS-3均为α型吡喃糖。高碘酸氧化、Smith降解、甲基化反应、GC-MS分析表明,CSPS-1主链由(1→4)-葡萄糖构成,支链为(1→6)-葡萄糖。CSPS-2主链由(1→3)-葡萄糖、(1→4)-葡萄糖构成,支链为(1→2)-葡萄糖、(1→6)-葡萄糖。CSPS-3主链为(1→3)-葡萄糖、(1→3)-半乳糖,支链为(1→6)-葡萄糖。(三)青蛤多糖硫酸酯化及抗肿瘤活性采用氯磺酸吡啶法对青蛤多糖纯化组分CSPS-1进行硫酸酯化。通过三因素三水平正交实验研究酯化条件(氯磺酸吡啶比例、酯化温度、酯化时间)对硫酸基取代度(DS)、总糖含量的影响。在正交实验条件制备了DS、总糖含量不同的CSPS-1硫酸酯化样品。CSPS-1硫酸酯化正交实验结果表明,酯化条件对DS的影响由弱到强的顺序为酯化时间<酯化温度<氯磺酸吡啶比例。酯化条件对总糖含量的影响由弱到强的顺序为酯化时间<氯磺酸吡啶比例<酯化温度。采用MTT法研究不同酯化条件下制备的硫酸酯化多糖对人胃癌细胞BGC-823的增殖抑制效果,结果表明,不同DS或总糖含量的多糖样品对人胃癌细胞BGC-823的增殖具有不同的抑制效果。硫酸酯化多糖样品的DS、总糖含量越高,其对人胃癌细胞BGC-823的增殖抑制能力越强。氯磺酸吡啶比例为1：4、酯化温度为65℃、酯化时间为2h时,硫酸酯化多糖的DS、总糖含量均达到最大值(0.102、72.19%),并且对人胃癌细胞BGC-823的增殖抑制率最高达89.44%(400μg/ml,72 h)。(四)青蛤多糖抗癌活性采用MTT法研究Crude CSPS、CSPS-1、CSPS-2、CSPS-3对人胃癌细胞BGC-823的增殖抑制效果。结果表明,在50-400μg/ml的多糖浓度范围内,Crude CSPS.CSPS-1、CSPS-2、CSPS-3对人胃癌细胞BGC-823的增殖抑制率呈现一定的量效关系。随着多糖浓度提高,Crude CSPS、CSPS-1、CSPS-2、CSPS-3对人胃癌细胞BGC-823的增殖抑制率呈上升趋势。当多糖浓度为400谬g/ml, Crude CSPS、CSPS-1、CSPS-2、CSPS-372 h时对人胃癌细胞BGC-823的增殖抑制率分别为35.16%、58.98%、42.91%、79.89%。在Crude CSPS、CSPS-1、CSPS-2、CSPS-3中,CSPS-3对人胃癌细胞BGC-823的增殖抑制效果最好,抗肿瘤活性最高。CSPS-3较强的抗肿瘤活性可能与其较高的蛋白质、糖醛酸、硫酸基含量及相对复杂的单糖组成、结构有关。(五)青蛤多糖抗氧化活性采用化学方法对Crude CSPS、CSPS-1、CSPS-2、CSPS-3进行体外抗氧化活性测定。结果表明,Crude CSPS、CSPS-1、CSPS-2、CSPS-3均具有不同的超氧自由基清除能力、羟自由基清除能力、还原力、脂质过氧化抑制活性、金属离子螯合能力。Crude CSPS、CSPS-1、CSPS-2、CSPS-3具有一定的体外抗氧化活性。在0.4～4.0 mg/ml的范围内,随着多糖浓度提高,Crude CSPS、CSPS-1、CSPS-2、CSPS-3的体外抗氧化活性呈上升趋势。青蛤多糖的超氧自由基清除活性由小到大的顺序为CSPS-2< CSPS-1< Crude CSPS< CSPS-3,羟自由基清除活性为CSPS-1< CSPS-2< CSPS-3< Crude CSPS,还原力为Crude CSPS< CSPS-1< CSPS-2< CSPS-3,脂质过氧化抑制活性为CSPS-2< CSPS-1< CSPS-3< Crude CSP,金属离子螯合能力为CSPS-1< CSPS-2 < CSPS-3< Crude CSPS。CSPS-1、CSPS-2、CSPS-3抗氧化活性方面的差异可能是由于多糖的化学组成、分子量、聚合度、硫酸基取代位置、主链特征等方面的差异所致。青蛤粗多糖体内抗氧化活性采用CCl4诱导肝损伤模型小鼠进行评价。蛋白质含量、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性、丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)等生理生化指标均采用商业试剂盒进行测定。青蛤多糖体内抗氧化实验结果表明,与正常对照组相比,模型对照组处理能够显著提高小鼠血清ALT、AST活性(P<0.05),降低肝SOD、GSH-Px活性(P<0.05),说明本实验的小鼠CCl4诱导肝损伤模型被成功建立。与模型对照组相比,多糖组能够显著提高小鼠肝SOD、GSH-Px活性(P<0.05),降低小鼠肝MDA水平(P<0.05),表明青蛤多糖对CC14诱导的肝损伤小鼠具有保护作用。青蛤多糖的护肝效果可能与青蛤多糖提高小鼠自身抗氧化能力以及抑制脂质过氧化有关。更多还原

【Abstract】 Cyclina sinensis, commonly called clam, a well-known bivalve mollusk in the family of Veneridae, is widely distributed along the coastal waters of China. It is one of the most important bivalve species in Chinese aquaculture. It has been reported that Cyclina sinensis can be used for the treatment of inflammation, asthma and dental ulcer in traditional Chinese medicine. In addition, it has been demonstrated that it is rich in protein, amino acid, lipid, minerals and polysaccharides that may contribute to the biological functions, such as anti-tumor, anti-inflammation and immune-regulation. However, little attention has been devoted to the isolation, purification, chemical and structural characterization, sulfated modification and biological activities of polysaccharides from Cyclina sinensis (CSPS) compared with those of some other bivalvia species such as Meretrix meretrix Linnaeus, Ruditapes philippinarum and Perna viridis. Therefore, we report here optimization of extraction parameters, isolation and purification, chemical characterization, structures, sulfation, antioxidant and anticancer activities of CSPS. Main results are listed as follows:1. Optimization for extraction of CSPSThe effects of extraction temperature, extraction time, ratio of water to raw material and extraction times on the extraction yields of CSPS were investigated by using single-factor experiments. The results showed that all these variables markedly affected the polysaccharides yield. The yield of CSPS was increased with the increase of extraction temperature. The yield of CSPS stopped growing with increased extraction times when the extraction times were more than 2 times. Based on the single-factor experiments, response surface methodology (RSM) was applied for the parameter optimization for CSPS production. By using the software of Design Expert version 7.0, the optimum values of the tested variables for the extraction of CSPS were obtained as follows:extraction temperature 90℃, extraction time 250 min and ratio of water to raw material 29. Using the optimal conditions, the maximum predicted extraction yield of CSPS was 15.62%, which corresponded fairly well to that of real extraction (15.52%±1.26%).2. Isolation, purification, chemical characterization, structures of CSPSThe crude CSPS was firstly separated through acolumn of DEAE-cellulose (Whatman DE 52). As a result, three independent elution peaks (F1, F2 and F3) were obtained. The three fractions were loaded onto a column of Sephadex G-100, affording CSPS-1, CSPS-2 and CSPS-3, respectively. Purity of CSPS-1, CSPS-2 and CSPS-3 was further confirmed by using HPLC, and results showed that CSPS-1, CSPS-2 and CSPS-3 were homogeneous polysaccharides respectively.The chemical characterizations of CSPS were investigated by various methods. As to crude CSPS, CSPS-1, CSPS-2 and CSPS-3, polysaccharides content, measured by employing the method of sulfuric acid-phenol coloration, was 83.81%,98.75%,95.58% and 84.71%, respectively. Uronic acid content, determined by using the method of sulfate-3-Phenylphenylol assay, was 1.58%,0.16%,0.96% and 2.13%, respectively. Sulfuric radical content, determined by employing the method of gelatin-barium chloride assay, was 0.92%,1.22%,2.08% and 3.58%, respectively. Protein content, measured by applying the method of coomassie brilliant blue coloration, was 3.08%, not detected, not detected and 6.34% respectively. The relative molecular weight of CSPS-1, CSPS-2 and CSPS-3, determined by HPLC, was 68.6 kDa,80.6 kDa and 100.6 kDa, respectively. According to GC analysis, CSPS-1 was composed of xylose and glucose in a molar percent of 4.92 and 95.08, respectively, and CSPS-2 was only composed of glucose. CSPS-3 was composed of rhamnose, fucose, mannose, glucose and galactose with a molar percent of 11.48,17.15,12.44,21.57 and 37.36, respectively.The structural characterizations of CSPS were investigated by FT-IR, periodte oxidation, Smith degradation, methylation, GC-MS and NMR analysis. In FTIR spectrum of CSPS, characteristic absorptions of polysaccharides, carboxyl group and pyranose ring were observed. NMR spectroscopy confirmed that there were pyranose rings in CSPS-1, CSPS-2 and CSPS-3. Results of periodate oxidation, Smith degradation, methylation and GC-MS analysis suggested that CSPS-1 was a glucan with a backbone ofα-D-(1→4) Glc, branched withα-D-(1→6) Glc. CSPS-2 was a glucan with a backbone ofα-D-(1→3) Glc andα-D-(1→4) Glc branched withα-D-(1→2) andα-D-(1→6) Glc. CSPS-3 had a backbone ofα-D-(1→3) Glc andα-D-(1→3) gala, branched withα-D-(1→6) Glc.3. Sulfated modification of CSPS-1 and its anticancer activitiesNine modification conditions were designed to sulfate CSPS-1 by chlorosulfonic acid-pyridine (CSA-Pyr) method according to the orthogonal test and focusing on three affecting factors such as ratio of CSA to Pyr, reaction temperature and reaction time. And nine sulfated derivatives with various degrees of substitution (DS) and carbohydrate content were obtained. The results indicated that the extent of the impact of variables on DS followed the order:reaction time< reaction temperature< ratio of CSA to Pyr, and the extent of the impact of variables on carbohydrate content followed the order:reaction time < ratio of CSA to Pyr< reaction temperature. The anticancer activities of the derivatives in vitro were evaluated by determining their inhibitory rates against human gastric cancer BGC-823 cells. The results indicated that the derivaitves with different DS or carbohydrate content showed different anticancer activities. It seemed that polysaccharides with relatively higher DS and carbohydrate content exhibit stronger antitumor activity in vitro. According to results of the orthogonal test, the optimum sulfated conditions of CSPS-1 should be the ratio of CSA to Pyr of 1:4, reaction time of 2 h and reaction temperature of 65℃. Using the optimal conditions, DS and carbohydrate content of products were 0.102 and 72.19%, respectively. And the inhibitory rates against human gastric cancer BGC-823 cells reached 89.44% at 72 h at a concentration of 400μg/ml.4. Anticancer activities of CSPSThe anticancer activities in vitro of crude CSPS, CSPS-1, CSPS-2 and CSPS-3 were evaluated by determining their inhibitory rates against human gastric cancer BGC-823 cells. The results showed that all polysaccharides exhibited a dose-dependent activity within the concentration range of 50-400μg/ml, and the inhibitory effects of crude CSPS, CSPS-1, CSPS-2 and CSPS-3 increased with the increase of sample concentration. At a concentration of 400μg/ml, the inhibitory effects of crude CSPS, CSPS-1, CSPS-2 and CSPS-3 at 72 h were 35.16%,58.98%,42.91% and 79.89%, respectively. CSPS-3 showed strong inhibitory effect on the growth of BGC-823 cells. The higher activity of CSPS-3 might be attributed to its high contents of protein, uronic acid and sulfuric radical as well as relative more complicated monosaccharide composition and structure.5. Antioxidant activities of CSPSAntioxidant activities in vitro of CSPS, CSPS-1, CSPS-2 and CSPS-3 were measured by using the chemical methods. Results showed that crude CSPS, CSPS-1, CSPS-2 and CSPS-3 had different superoxide radical scavenging activity, hydroxyl radical scavenging activity, reducing power, lipid peroxidation inhibition effect and ferrous ion chelating activity. Crude CSPS, CSPS-1, CSPS-2 and CSPS-3 had some antioxidant activities in vitro. Antioxidant activities of crude CSPS, CSPS-1, CSPS-2 and CSPS-3 were increased with the increase of concentrations ranged from 0.4 mg/ml to 4.0 mg/ml. For superoxide radical, the scavenging activity increased in the order of CSPS-2< CSPS-1< Crude CSPS< CSPS-3. Hydroxyl radical scavenging activity increased in the order of CSPS-1< CSPS-2 < CSPS-3< Crude CSPS. Reducing power increased in the order of Crude CSPS< CSPS-1 < CSPS-2< CSPS-3. Lipid peroxidation inhibition effect increased in the order of CSPS-2 < CSPS-1< CSPS-3< Crude CSPS. Ferrous ion chelating activity increased in the order of CSPS-1< CSPS-2< CSPS-3< Crude CSPS. The difference in antioxidant activities were related to the difference in chemical components, molecular weights, polymerization degree, the position of sulfate groups and polymer backbone of polysaccharides.Antioxidant activities in vivo of CSPS were evaluated by its attenuation of carbon tetrachloride-induced hepatotoxicity in mice. Protein content, activities of SOD and GSH-Px as well as level of MDA, ALT and AST were determined by using commercially available kits. The results showed that the activities of antioxidant enzymes (SOD and GSH-Px) in liver homogenate of the CCl4-induced mice were significantly decreased and MDA and serum enzymes (ALT and AST) levels were significantly increased compared to that of the normal control (P< 0.05), suggesting that the hepatotoxicity mice model was established successfully. The results also showed that oral administration of Crude CSPS significantly reduced the elevated serum levels of ALT and AST, the level of MDA in the liver that were induced by CCl4 in mice. Moreover, the Crude CSPS treatment was also found to significantly increase the activities of SOD and GSH-Px in the liver. The results suggested that Crude CSPS exhibited potent hepatoprotective effects on CCl4-induced liver damage in mice, likely due to both the increase of antioxidant-defense system activity and the inhibition of lipid peroxidation.更多还原