Isolation And Purification Of Fucoidan From Kelp And Its Activities Study

Kelp polysaccharide were isolated from kelp by hot water extract method. Statistics-based response surface methodology was used to optimize the extraction process of kelp polysaccharide. Single factor experiments were undertaken for determining the optimum range of each of four factors (pH, temperature, time and ratio of water to kelp) and these factors were further optimized using the response surface methodology with CCD design. The optimum conditions were found to be:extraction pH3.42, extraction temperature83℃, extraction time3.95h, ratio of water to kelp1:23. Under these conditions, the polysaccharide yield was up to1.259%, which was agreed closely with the predicted yield value.In this paper, during the purification of crude kelp polysaccharide, two kinds of methods, trichloroacetic acid and Sevage method, of removing protein from crude kelp polysaccharide were studied. And the relationship between the effect of removing protein and the loss of kelp polysaccharide was compared. The results show that the Sevage method is better than trichloroacetic acid. The mechanism making the protein precipitate might be the main reason that caused polysaccharide losses while removing protein. So, we choose the way of Sevage method to remove protein.During the purification, we choose the high-speed countercurrent chromatography and DEAE-Sepharose Fast Flow to purificate the kelp polysaccharide. High-speed countercurrent chromatography (HSCCC) has been applied for the separation of crude polysaccharide. The HSCCC run was carried out with a two-phase solvent system composed of PEG1000-K2HPO4-KH2PO4-H2O (0.5:1.25:1.25:7.0, w/w) by eluting the lower aqueous phase at2.0ml/min at600r/min. Two of polysaccharide, KPS-1and KPS-2, were isolated by HSCCC. Then, the KPS-2was purified by DEAE-Sepharose F.F to afford3fractions, KPS-2-1, KPS-2-2, KPS-2-3, respectively. High performance liquid chromatography (HPLC) analysis results show that KPS-2-1is the purified fucoidan.On the basis of getting high purity fucoidan, we studied its physical and chemical properties and its antioxidative activity. The results show that fucoidan is difficult to dissolve in cold water and easy to dissolve in hot water, dilute acid and dilute alkali. Not soluble in chloroform, ethanol, acetone, ethylether, butanol, and other organic solvents. The pH of1%of fucoidan solution is3.48. After dialysis, the polysaccharide does not contain reducing sugar, no starch, non-phenolic or tannin.The scavenging percentages of·OH,·O2-and DPPH using purified fucoidan were up to99.4%,99.26%and23.83%, respectively, implying that it could be a potential natural antioxidant for future use.We studied the effects of the concentrations of various fucoidan on the oxidation of L-tyrosine, by tyrosinase. The tyrosinase was inhibited by varying concentrations of fucoidan (from0.2to1.0mg/mL). With increasing concentrations of fucoidan, the activity of tyrosinase markedly decreased. When the concentration of inhibitor reached1.0mg/mL, enzyme activity was inhibited by62.21%. The IC50value of fucoidan was determined to be0.82mg/mL. The plots of the remaining enzyme activity versus the concentrations of enzyme in the presence of different concentrations of fucoidan gave a family of straight lines, which all passed through the origin. Increasing the inhibitor concentration resulted in a descending of the slope of the line, indicating that the inhibition by fucoidan was reversible. The presence of fucoidan did not bring down the amount of the efficient enzyme, but just resulted in the inhibition of enzyme activity.Then, the inhibitory kinetics of tyrosinase by fucoidan were studied. Under the experimental conditions employed, the oxidation reaction of L-tyrosinase follows Michaelis-Menten kinetics. Double-reciprocal plots yielded a family of straight lines intersected at the3rd quadrant. The values both of Km and Vm decreased with increasing inhibitor concentration. Thus, fucoidan was a competitive type inhibitor. Ki was obtained from the double-reciprocal plots of the slope versus the concentration of fucoidan respectively. The value of Ki was determined to be0.9907mg/mL...