Guangxi Seaweed Polysaccharides Lo1 Purification And Mechanism Of The Antithrombotic Effect

Polysaccharide of Laminaria is an active composition of Laminaria japonica Aresch. The study had shown that it has a variety of pharmacology functions. But its compositions are complicated, there are many differences in its molecular weight, the length of the sugar chain, the constitute of the single sugar and connect form from the different sea area, withdrawing part, extracting and separating method. So the different structure will produce different pharmacology function. Furthermore, only a small quantity of Polysaccharide of Laminaria was extracted by the traditional method, which limited the pharmacology studies in the further scope. As a result, it is attractive and challenge to acquire the higher and more active composition of Polysaccharide of Laminaria from different sea area with different methods, and carry on a series of scientific research of its pharmacology functions. A new method was used to purify and get a fragment Polysaccharide (L01) from Guangxi Laminaria and the function and mechanism of antithrombus were studied in this research, which will lay the foundation for the further research of the ocean and antithombus medicine. Methods:1. A raw product of Polysaccharide of Laminaria was extracted by the technique of enzymolysis, alkali digesting and alcohol deposition from Guangxi Laminaria.Then the sample was decolored and refined, and the effective fragment (L01) with higher content was isolated by ion exchange chromatography on DEAE-cellulose. Furthermore, the purity degree of L01 was identified by paper chromatography, PAGE, and dextran liquid chromatography. The molecular weight was measured by HPGPC. The sulfate radical content was determined by glutin turbidimetry. The Polysaccharide content was determined by sulfuric acid-phenol methods. The protein was detected by ultraviolet spectrometer. And the L01 spectrum was analysed by infrared spectrometer.2. Various animal models of thrombus formation were established, such as by carotid-stimulating electrically, by artery-vein loop, and by ting up lower cava, to investigate the effect of L01 on thrombus formation.3. The influence of L01 on blood coagulation was observed through the experiments of anticoagulant and AT-IIa compound formation in vivo and in vitro to explore the mechanism of its anticoagulant.4. A rat model of endothelial injured was established via injecting adrenaline and the damaged degree of vascular endothelial was evaluated by aortic immunity histochemistry. The platelet adhesion rate and the platelet aggregation rate were evaluated by filtration method, collodion-glass plate method and turbidimetry respectively, to observe the influence of L01 on platelet activation in vascular endothelial injured rats.5. To observe the influence of L01 on plasma level of TXB2, 6-Keto- PGF₁ α and GMP-140 in vascular endothelial injured rats, the contents of plasma TXB₂ and6-Keto-PGF₁ α were measured by radioimmunoassay, and the concentration of plasma GMP-140 was measured by ELISA.6. A rat model of endothelial injured was established and HUVEC was cultured in vitro. The content of vWF in plasma or in supernatant was measured by ELISA in order to observe the influence of L01 on endothelial injured rats and HUVEC stimulated by adrenaline. The influence of L01 on vWF expression of lung tissue section was observed via the immunohistochemistry method. The content of plasma vWF before and after platelet aggregation induced by ristocetin in vitro was measured by ELISA and the vWF stain in the platelet aggregation deposition was evaluated by immunohistochemitry method in order to observe the inhibition function of L01 on platelet aggregation mediated by vWF. Results:1. A new fragment of Polysaccharide of Laminaria (L01) was got from Guangxi Laminaria. Its molecular weight was 49546, the content of sulfate radial was 83.28mg/g, and the content of Polysaccharide was 71.0%. It was proved to be a single component by several analytic methods. No peak of protein absorption was showed by ultraviolet spectrometer scanning, and general characteristic absorption peaks of Polysaccharide and special absorption peaks of L01 were seen through the infrared spectrometer analysis.2. The occlusion times (min) of carotid artery thrombus of L01 high-dose group (50mg/kg), low-dose group (10mg/kg) and NS group were 20.57 ± 3.64, 18.52± 3.10, and 13.67±2.75 respectively. The wet weights of artery-vein loop thrombuswere 0.1162±0.0248, 0.1443±0.0263, 0.2303 ± 0.0740 respectively, and the wet weights of vein thrombus were 0.2195±0.0902, 0.2839±0.0395, and 0.4154± 0.1148 respectively. Comparing with the control group, both high-dose L01 and low-dose L01 prolonged the occlusion time of thrombus mediated by carotid-stimulating electrically, and decreased the wet weight of artery-vein loop and vein thrombus (P<0.05, P<0.01), showing a dose-dependent relation.3. The anticoagulation effect of L01 was showed in vivo and in vitro. When the eventual concentrations of L01 were 0.2, 2.0, and 20.0mg/mL and the infection doses were 50mg/kg and 10mg/kg, the CT, PT, and APTT were prolonged in vitro or in vivo respectively, with statistic significant difference (P<0.05) and dose-dependent relation. The experiment of AT- II a compound formation indicated that in all the reactive liquid comprising with thrombin, antithrombin showed the electrophoresis bend of AT-IIa compound when L01(1ug/mL and 10ug/mL) or heprin was added into the reactive liquid, indicating the anticoagulant function of L01 was correlative with enhancing the activities of antithrombin.4. The vascular endothelial cell of rats could be injured by repeat infection of adrenaline. During the 4th and 5th day of the model made, the morphology observation of the aorta tissue section demonstrated that the endothelial trauma was severely injured in model group. The intact endodermis lengths (μm) stained by immunohistochemistry were 208.45±42.46, 184.94±25.31, and 135.88 ± 49.06, respectively, for the L01 high-dose group (50mg/kg), low-dose group(10mg/kg) and model group on the 4th day; and 210.61+28.94, 202.44+29.26, and 121.16 ± 60.135, respectively, on the 5th day. The lengths of two L01 groups were obviously longer than the model group (P<0.05), indicating that L01 could protect the endothelial cell. On the 4th day, the platelet adhesion rates(%) of L01 high-dose group, low-dose group and model group were 16.778±7.466, 15.605±6.272, 25.782±5.781 respectively and 13.81±7.68, 14.86±12.89 and 30.51±5.37 on the 5th day, respectively. The maximal platelet aggregation rates (%) induced by ristocetin were 32.68 + 10.60, 37.70+11.94 and 52.22+10.74 respectively. Comparing with the model group, various parameters of LOl group were significantly different (P<0.05). The study of collodion-glass plate method showed both L01 groups decreased aggregation rate on the 4th and 5th day. Comparing with the model group, regardless of the total aggregation rate or the aggregation rate of both small and large groups were obviously lower (P<0.05). The results indicated that L01 could decrease platelet adhesion and aggregation functions in vascular endothelial cell injured rats mediated by adrenaline. 5. On the 5th day of adrenaline injection, the plasma TXB₂ contents (pg/ml) of L01 high-dose group (50mg/kg), L01 low-dose group (10mg/kg) and model group were 64.49+12.79, 74.39+10.20 and 82.81 + 15.15 respectively. There was a significant difference between high-dose group and model group. At the same time, plasma 6-Keto-PGF₁ contents (pg/ml) of high-dose group, low-dose group and model group were 874.36 + 134.10, 806.81 + 163.90 and 910.65 + 146.59 respectively. Comparing with the model group, the contents of 6-Keto-PGF1 in both L01 groups were obviously lower (P<0.05). On the 4th and 5th day, the plasmaGMP-140 contents (ng/ml )of high-dose group, low-dose group and model group were 0.5212±0.1305, 0.5498±0.1271, 0.8880±0.3555 and 0.5164±0.2247, 0.5927±0.1468, 0.8991±0.2810 respectively. There were significant differences between both L01 groups and model group, indicating that L01 could decrease plasma TXB2 and GMP-140 level and increased 6-Keto-PGF₁ level in vascular endothelial cell injured rats mediated by adrenaline.6. On the 4th and 5th days of adrenaline injection, the plasma vWF contents (%) of L01 high-dose group (50mg/kg), L01 low-dose group (10mg/kg) and model group were 51.766±8.269 , 60.801±17.910 , 72.410±12.242 and 61.588±6.604 , 62.440±7.966,75.509±8.880 respectively. On the 4th day, there was a significantly different between high-dose group and the model group (P<0.05). On the 4th and 5 day, there were significant differences between high-dose group and model group or low-dose group and model group (P<0.05). In the study of cultured HUVEC, on the 24h, L01 groups (0.01mg/mL and 0.lmg/mL) decreased the supernatant vWF level, and on the 48h, high-dose group (0.lmg/mL) also decreased the supernatant vWF level, with significant difference compared with adrenaline group (10ug/mL, P<0.05). The supernatant vWF contents in these three groups were 29.003±7.233, 29.354±6.347, and 39.145±9.562 on 24h, and 32.588±7.404, 30.684±6.710, and 37.166±8.996 on 48h, respectively. The immunohistochemistry analysis of lung tissue slices showed the average grey values (%) of vWF stain of lung tissues and vascular endothelial in L01 high-dose group, low-dose group and model group on the 4th day of adrenaline injection were195.53 + 11.10, 191.27 + 15.52, and 172.06 + 7.33. On the 5th day, it showed 197.47 + 8.91, 192.07+13.14, and 172.32+5.77. The average grey values in both L01 high-dose group and low-dose group were higher than the model group. All results showed that L01 decreased the vWF expression not only in the vascular endothelial injured rats but also in cultured HUVEC stimulated by adrenaline. The study of platelet aggregation by ristocetin showed that L01 decreased the platelet aggregation rate. The maximal platelet aggregation rates (%) in three L01 dose groups (0.04mg/mL, 0.2mg/mL, and lmg/mL) were 24.96+4.55,20.72+4.95 and 1.91 + 1.79, respectively, with significant differences (P<0.001) comparing with PBS group (60.81+5.27). The differences of plasma vWF content before and after aggregation in these three L01 dose groups were 12.313+4.988,9.815+5.328 and 2.511 +2.109, with significant differences (P<0.05, P<0.01, P<0.001 )comparing with PBS group (23.648+7.286). L01 also decreased immunochemistry products of vWF in the platelet aggregation deposition. The effect of L01 on platelet aggregation was negative correlation with the difference of plasma vWF content before and after platelet aggregation, and with the vWF content in the platelet aggregation deposition, which indicated that L01 perhaps inhibited the site where vWF and platelet combine. Conclusion:L01 was a new composition of Polysaccharide of Laminaria. It was proved to have the effect of antithrombosis. Its function mechanism involved anticoagulation and the inhibition of platelet activation. On anticoagulation aspect, LOl enhanced the...