【作者】 廖宁波；

【导师】 刘东红；

【作者基本信息】 浙江大学 ， 食品科学， 2014， 博士

【摘要】 河蚬(Corbicula fluminea)是双壳类软体动物,又称黄蚬,在我国的东部沿海地区多有分布。本草纲目记载河蚬作为中药的药材,具有开胃、通乳、明目、利尿、去湿毒、治疗肝病、麻疹、退热、止咳化痰、解酒等功效。目前对河蚬功能成份的研究主要集中在蛋白和脂类物质方面,对多糖的研究还较少,而且不够深入。本论文对河蚬多糖进行系统的研究,包括提取条件优化、分离纯化、结构特性、抗氧化活性、抗肿瘤活性以及对人体肠道菌群微生态的影响等方面。主要结果如下：1河蚬多糖超声辅助酶法提取条件的优化选择提取温度、提取时间、液料比及超声强度4个因素分别进行单因素实验,确定了各因素在RSM实验中的取值中心点。在单因素实验基础上,运用doehlert matrix (DM)设计对提取温度、提取时间、液料比及超声强度4个参数的实验组合进行优化。运用Matlab软件对实验数据进行处理分析,得出最优提取条件为提取温度60℃、提取时间30min、液料比35ml/g、超声强度300W。在此条件下,河蚬得率值为35.4%。2河蚬多糖的分离纯化及理化性质运用阴离子交换柱和凝胶层析柱对河蚬粗多糖进行分离纯化,得到CFPS-1和CFPS-2。采用HPSEC对CFPS-1和CFPS-2的纯度和分子量作近一步鉴定,其相对分子质量分别是283和22kDa。运用化学、物理的方法分别对粗CFPS、CFPS-N、 CFPS-1和CFPS-2的化学特征进行分析。其中CFPS-1的蛋白含量(10.8%)、糖醛酸含量(14.34%)相对较高,而CFPS-2的总糖含量(94.4%)及硫酸基含量(8.1%)相对较高。单糖分析表明粗CFPS、 CFPS-N. CFPS-1主要由葡萄糖组成,而CFPS-2主要由葡萄糖、半乳糖、岩藻糖、氨基半乳糖、氨基葡萄糖组成,各单糖比例分别是0.7：0.3：0.9：0.2：0.2。3河蚬多糖的结构解析运用原子力显微镜(AFM)对河蚬多糖进行观察,发现CFPS-N分子相互盘绕呈球形。CFPS-1和CFPS-2中可以看到单独游离于溶液中的糖链,糖链直径是在10-20nm之间,长度为1500-2000nm之间。结合多糖甲基化、GC-MS及核磁共振技术分析的结果,推测CFPS-1可能的结构是：CFPS-2可能的结构是：4河蚬多糖的抗氧化、抗肿瘤活性体外抗氧化活性研究发现,与河蚬粗糖(CFPS和CFPS-N)相比,纯化产物CFPS-1和CFPS-2有较强的抗氧化活性。体外抗肿瘤活性筛选结果表明,CFPS-1对人乳腺癌细胞(MDA-MB-231)的抗增殖效果最为显著。末端转移酶标记技术(TUNNEL)显示随着CFPS-1浓度的增加可以很好的诱导MDA-MB-231细胞的凋亡。因此,我们近一步采用蛋白质印记技术研究细胞周期相关蛋白的表达情况,结果表明其机制很可能是通过对ASK1蛋白的活化,进一步激活p38/p53途径。p53蛋白的增加一方面抑制Cyclin D1CDK4和PCNA蛋白的表达,最终诱导细胞周期阻滞,抑制癌症细胞过度增殖。同时,还可以使线粒体中Caspase-3酶原裂解成为活性蛋白Caspase-3酶,最终激活凋亡特异性核酸内切酶诱导肿瘤细胞发生凋亡。5河蚬多糖对人体肠道菌群微生态的影响我们发现在没有微生物参与的情况下,胃-肠道消化液对河蚬多糖CFPS-N的降解作用很有限。近一步研究肠道菌群与CFPS-N的代谢关系中发现,CFPS-N能够被肠道微生物所降解,并且发现代谢产物中乙酸、丙酸和丁酸的含量较高。DGGE的结果发现代谢多糖后的菌群结构主要由厚壁菌门、变形菌门、放线菌门和拟杆菌门组成。qPCR检测结果表明,参与多糖代谢的很可能包括双歧杆菌、肠球菌、梭菌、乳酸菌和拟杆菌。在对河蚬多糖降解菌的分离过程中,我们分离到的能够降解河蚬多糖的肠道微生物主要分布在双歧杆菌、乳酸菌和梭菌属中。其中双歧杆菌中的Bifidobacterium longum strain L-25和乳酸菌中的Lactobacillus fermentum GM-4降解效果最好。更多还原

【Abstract】 Asian clam(Corbicula fluminea), commonly called Hunagxian, is currently one of the most economically important aquatic species in the south part of China. The extracts of C. fluminea have been reported to possess various activities including hepato-protective, antioxidant, anticancer, antihypertension, and hypocholesterolemic effects. However, few reports have focused on the active constituents of C. fluminea in a purified component form. In this paper, the purified polysaccharides of C. fluminea were studied systemically, including optimization of extraction parameters, isolation and purification, physicochemical characterization, antioxidant and antitumor activities, and the effect of crude polysaccharide fraction (CFPS) on human intestinal microflora. Main results are listed as follows:1Optimization of extraction parameters of crude C. fluminea polysaccharideThe optimal parameters for extraction of crude C. fluminea polysaccharide were obtained by using a doehlert matrix (DM) design as follows:ratio of water to raw material35ml/g; extraction time of30min; ultrasound power of300W, and extraction temperature of60℃. In these extraction conditions, the maximum polysaccharide yield of35.4%was achieved.2Isolation, purification, and characterization of C. fluminea polysaccharidesCFPS-1and CFPS-2were purified from Corbicula fluminea by anion-exchange and gel-permeation chromatography. They were homogenous with a molecular weight of283kDa and22kDa by HPSEC, respectively. Basic characterization of CFPS-1and CFPS-2were determined by using the methods of chemistry and physics. The protein content (10.8%) and uronic acid content (14.34%) of CFPS-1were higher than that of CFPS-2. However, CFPS-2had the highest content of sulfuric radical (8.1%) and sugar (94.4%). Monosaccharide composition showed that CFPS-1was mainly composed of glucose. While, CFPS-2was composed of glucose, galactose, fucose, galactosamine, glucosamine with a molar ratio of0.7:0.3:0.9:0.2:0.2.3Structural features of C. fluminea polysaccharideThe molecular structure of Corbicula fluminea polysaccharides were observed by atomic force microscopy (AFM). CFPS-1and CFPS-2showed their single molecule stretching behavior in the solution. The diameter were about10-20nm, and the height were about1500-2000nm. According to the results of GC-MS and nuclear magnetic resonance (NMR) experiments, the structure of CFPS-1and CFPS-2were established: CFPS-14Antioxidant and antitumor activities of C. fluminea polysaccharidesAntioxidant activities in vitro showed that CFPS-1and CFPS-2exhibited much higher antioxidant activities than that of CFPS and CFPS-N. Methylene blue, TUNEL and western blot assays were performed to determine whether C. fluminea polysaccharides had antitumor activity and the mechanism of action. The results suggested that CFPS-1had slightly inhibitory effect on MDA-BD-231cells. Further analysis showed that the inhibitory effect was associated with an up-regulation of ASK1, p53, p21, Bax and caspase-3expression, and down-regulation of Cyclin D1, CDK4, PCNA and Bcl-2expression. Taken together, our studies indicate that CFPS-1is capable of inhibiting MDA-BD-231cell proliferation and inducing apoptosis in these cells.5Effect of crude polysaccharide (CFPS-N) on human intestinal microfloraCFPS-N could not be degraded without intestinal microflora, suggesting that intestinal microflora may be responsive to CFPS-N digestion. The effect of CFPS-N on human fecal microflora in a batch culture system was investigated. CFPS-N can be fermented to short chain fatty acids (SCFAs) by human intestinal microflora. A significant amount of acetate, propionate and butyrate was generated in the CFPS-containing culture (p<0.05). Denaturing gradient gel electrophoresis (DGGE) and qPCR analysis revealed that the bacterial community structures were mainly affiliated with Bacteriodes prevotella, Bifidobacterium, Clostridium, Enterbacteriaceae, and Lactobacillus groups. Various bacteria isolated from human feces were screened for their capacity to utilize CFPS-N by measuring the Mw of polysaccharide. Most strains of Escherichia coil, Lactococcus lactis, Enterococcus feacalis, and Streptococcus thermophiles could not degrade CFPS-N, while only Bifidobacterium longum strain L-25,and Lactobacillus fermentum GM-4could efficiently utilize the polysaccharide.更多还原