The Structures And Bioactivities Of Corbicula Fluminea Polysaccharides And Their Effect On Human Intestinal Microflora

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.