Isolation, Structural Elucidation, Sulfated Modification And Biological Activities Of Polysaccharides From Cyclina Sinensis

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-(1â†'4) Glc, branched withα-D-(1â†'6) Glc. CSPS-2 was a glucan with a backbone ofα-D-(1â†'3) Glc andα-D-(1â†'4) Glc branched withα-D-(1â†'2) andα-D-(1â†'6) Glc. CSPS-3 had a backbone ofα-D-(1â†'3) Glc andα-D-(1â†'3) gala, branched withα-D-(1â†'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.