| The researches of this paper was supported by the R&D Special Fund for Public Welfare Industry from State Ocean Administration P.R. China (No:201305007), and the National Nature Science Foundation(No:30900293). Crude polysaccharide was extracted from Mactra veneriformis by water extraction and alcohol precipitation, and we got the Mactra Veneriformis polysaccharide the percentage composition of which was 90%, we studied its oxidation resistance, and further we got the acid hydrolyzate of Mactra Veneriformis polysaccharide. First we optimized the acid solution process and got the best one, then analysed the composition of the product and studied the antioxidant and hepatoprotective activity of the acid hydrolyzate of Mactra Veneriformis polysaccharide. Following parts were mainly included:1. In vitro antioxidant activity studies on Mactra Veneriformis polysaccharideTCA method was used for removal of protein and further we used dialysis impurity, and its component analysis showed that it contains 90% of the total sugar and a small amount of protein; Determined the hydroxyl radical scavenging ability and DPPH of Mactra Veneriformis polysaccharide by colorimetry, and determined the reducing power by using Prussian Blue reaction; As a result, with the increasing of the concention of the Mactra Veneriformis polysaccharide solution, hydroxyl radical and DPPH scavenging ability increased, and also the reducing power.2. The Mactra Veneriformis polysaccharide acid hydroltsis process research and product analysys(1) To screening the acid to produce the acid hydrolyzate of Mactra Veneriformis polysaccharide. This experiment researched the type the acid, included hydrochloric acid, aulfric acid and trifluoroacdtic acid, the concentrations of the three acids were all lmol/L, hydrolysis time was 2h, and deceted the hydroxyl radical and DPPH scavenging ability and reducing power of the three hydrolyzates, to determine the type of the acid. Results showed that the hydroxyl radical and DPPH radical scavenging ability of hydrochloric acid and trifluoroacetic acid products were equal, and were better than the sulfuric acid.(2) Analysis of the hydrolyzate, this experiment detected the composition and the relative cintents of the hydrolysis by HPLC-ELSD, and determined the constitution of the product by LC-MS. According to the results of HPLC, hydrochloric acid and trifluoroacetic acid acidolysis product components and each component relative content were similar, and were better than the sulfuric acid. Sulfuric acid degradation ability were stronger than the former two, and we got little oligosaccharides. According to the results of LC-MS, the hydrolysis is mainly composed of glucose, dextran, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide and so on.(3) Studied the single factor of the hydrolysis, this experiment researched the acid hydrolysis progress, from the following three main factors, include time, concentration of the acid and the temperature, and detected the hydroxyl radical scavenging ability, and we got the optimum progress, which was at 80℃,1 mol/L hydrochloric for 2 hours.3. The protective effect of the acid hydrolyzate of Mactra Veneriformis polysaccharide at the cellular level for the liver(1) Determination of cell viability by MTS, found that in human normal liver cells activity the acid hydrolyzate of Mactra veneriformis polysaccharides showed no inhibition between 0-5000 μg/mL for 24 hours, but with the increase of concentration, began to show the promoting effect on cells. So we selected the concentration which had no effect on cell proliferation, that was 20-320 μg/mL for the later experiment.(2) To investigate the protective effect of the acid hydrolyzate of Mactra veneriformis polysaccharides on hydrogen peroxide and ethanol caused injury of human liver cell. Human liver cells L-02 was after different doses and different time exposed to hydrogen peroxide and ethanol, to determine the hydrogen peroxide and ethanol on liver cell damage sensitive exposure time and doses. With the acid hydrolyzate of Mactra veneriformis polysaccharide by different exposure time of exposure, to determine the role of sample in advance. Detected the ALT, AST, SOD and MDA activity in the cell culture supernatant, to study the the liver protective effect of the acid hydrolyzate of Mactra veneriformis polysaccharide. Results showed that the exposure of the hydrogen peroxide concentration was 2 mmol/L, and exposure time was 8 h; preact time of Mactra veneriformis polysaccharide hydrolyzate was 1 h; the antioxidant capacity of hydrolyzate increased linearly with concentration.4. The liver protective effect of the acid hydrolyzate of Mactra Veneriformis polysaccharide on the overall level of animalStudied the protective effect of the acid hydrolyzate of Mactra Veneriformis polysaccharide on the acute liver injury in mice caused by carbon tetrachloride and ethanol. Using carbon tetrachloride and ethanol induced acute liver injury in mice, the mice were euthanized by cervical dislocation to get serum, then monitored the ALT and AST activity, got the liver and calculated the liver index, and then determined the SOD activity and MDA content, did a statistical breakdown of the data and observed the mice liver biopsy. As a result, moddle and high dose could observably brought down the content of MDA, the liver index, enzyme activity of ALT and AST, and improved the enzyme activity of SOD. Conclusion:the product had obvious protective effect on acute liver injury in mice induced by CCl4 and ethanol. |
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