Fractionation Of Propylene Glycol Alginate Sodium Sulfate (PSS) And The Study On Its Biology Activities

Polysaccharides and their derivatives have attracted considerable attention in biomedicine and have been used as a variety of biomaterials. Currently, the study of polysaccharides is mainly focused on their structure-activity relationships. And more and more studies have been concentrated on these polysaccharides, which molecular weight (Mw) are several thousand Daltons and have the outstanding bioactivities.Sulfated alginate derivatives is a kind of heterogeneous and highly dispersed sulfated polysaccharide, and it had various bioactivities, e.g. anticoagulation, anti-oxidation, anti-HIV, heparanase inhibition, anti-inflammation and preventing calcium phosphate crystals formation in urine tract. However, little is known about the structure-activity relationships of sulfated alginate derivatives.Propylene Glycol Alginate Sodium Sulfate (PSS), which has been used as a marine derived drug for lowering blood lipid in China for nearly 20 years, is one of the most important sulfated alginate derivatives, it is necessary to make an intensive study of structure-activity relationships of PSS. In this study, the relationship between molecular mass of PSS and its several activities was investigated. The structural characteristics and activities of PSS and its fractions were studied in detail.The main results and conclusions of the research were as follows:1,PSS was fractionated by low-pressure gel permeation chromatography and ultrafiltration. Four column fractions (Z-PSS1-4) and five ultrafiltration fractions (C-PSS1-5) with different weight-averaged molecular mass (Z-PSS1-4: 51.95, 25.62, 11.76 and 5.41 kDa, C-PSS1-5: 21.97, 20.25, 18.27, 11.35, 4.93 kDa, respectively) were obtained, and their structural characteristics were determined and compared by Fourier transform infrared and nuclear magnetic resonance spectroscopes. The degrees of sulfate substitutions of most fractions were at 1.2-1.3 analyzed by oxygen flask method. The FT-IR data provided the information of backbone and sulfation of PSS and its fractions. That meant that the backbones of PSS and its fractions was made up ofα-1, 4-guluronic acids andβ-1, 4-mannuronic acids, respectively, and the sulfation was really occurred. The NMR data indicated that the propylene glycol group was linked to the C-6 position of the hexauronic acid residues of alginate and sulfate group was located at C-2 and C-3 positions of PSS and its fractions.The results of chemical analysis showed that PSS and PSS1-4 have similar structure characteristics except for the molecular mass.2,The comparative study of bioactivities of PSS and its fractions can be summarized as below:2.1,The anticoagulant activity of the column fractions was studied by evaluating their influence on the prothrombin time, activated partial thromboplastin time and thrombin time. The results showed that the highest activity of PSS and its column fractions was found in the TT, followed by that in the APTT, the moderate activity was found in the PT. And that the anticoagulant increased with the ascending molecular weight of its fractions. That proved that the anticoagulant of PSS was molecular weight dependence.2.2,The results of anticoagulant experiments of the ultrafiltration fractions were same as the results of the column fraction anticoagulant experiments. That showed that PSS and its fractions had the highest activity of TT, followed by that in the APTT, the moderate PT activity. At the same time, the molecular weight dependence effect of anticoagulant of PSS was also observed.2.3,Based on the above results of anticoagulant 3 items, their anticoagulant activities were studied by evaluating their influence on anti-thrombin and anti-FXa activities mediated by antithrombin III and heparin cofactor II furthermore. The results demonstrated that Z-PSS1 and Z-PSS2 strongly inhibited the activity of thrombin mediated by heparin cofactor II and antithrombin III, whereas PSS4 only showed very weak anti-thrombin activity. Z-PSS1-3 weakly inhibited the activity of coagulation factor Xa mediated only by antithrombin III while Z-PSS4 lacked the anti-Xa activity.2.4,The results of the protection of PSS on mastoparan-induced apoptosis of cultured cerebellar granule neurons showed that PSS can protect the mastoparan- induced apoptosis of cultured cerebral granule neurons. Measurements of intracellular free calcium concentration ([Ca2+]i) with Fluo-3/AM proved that PSS and Z-PSS1-3 debased the elevated [Ca2+]i induced by mastoparan. That clued on the protection of PSS on apoptosis neuron was through the inhibition on elevated [Ca2+]i induced by mastoparan. Z-PSS1-3 also showed the inhibition effect on apoptosis of cerebellar granule neurons, but Z-PSS4 was not. This meant the protection effect had the limit of molecular weight. But the molecular weight dependence was not observed as the effect did not increase with the ascending molecular weight of fractions.2.5,The immunoregulation activity of PSS and its fractions was investigated using immunocyte cultivation technique in vitro. The structure-activity relationship was analyzed on the basis of the structure studies of PSS'fractions. The experimental results showed that PSS could improve spleen cell proliferation, increase macrophage phagocytic function and inhibit the proliferation of T-cell induced by Concanavalin A (Con A) and B-cell induced by lipopolysaccharide (LPS). That proved that PSS possesses the significant immunoregulation effect, whilst the immunocompetence comparison of PSS'column fractions proved that the different immunocyte had different requirement for saccharides length. For spleen cell proliferation, the best molecular weight was about 10-20 kDa. For macrophage phagocytic function, the bigger Mw fraction of PSS had the stronger effect.2.6,In-vitro antibacterial activities of PSS and all fractions were compared and analyzed in the studies of the inhibition effect on Gram-Negative Bacteria (G+) (S. Aureus) and Gram-Positive Bacteria (G-) (E. Coli). The results showed that PSS, Z-PSS:1-3, C-PSS1-4 possessed the favorable antibacterial activity. Z-PSS2-3 and C-PSS4 showed the stronger effect than that of Z-PSS1 and C-PSS1-3, whilst Z-PSS4 and C-PSS lose the effect. This meant that the smaller molecular weight had the stronger antibacterial activities. To some extent, PSS had the molecular weight dependence of antibacterial activity.2.7,Based on the screen assay of PSS'promotion on insulin secretion in vitro, hypoglycemic effect of C-PSS4 was studied. C-PSS4 showed the possibility of decreasing blood glucose, but the statistical significant difference was not displayed (p>0.05). The effect needs the further investigation.