Degradation Enhances The Antiplatelet Aggregation And Anticoagulant Activities Of Polysaccharide From Lycium Barbarum L.leaves

Excessive platelet aggregation can cause hromboembolic disease,so the study of antiplatelet aggregation and anticoagulant activity is of great significance for the prevention and treatment of thromboembolic diseases..In the present paper,five polysaccharides were extracted from Lycium barbarum L.leaves by hot water,purified by DEAE-52 cellulose column,eluted by different concentrations of Na Cl?0,0.05,0.1,0.2,0.4 M?.After freeze-drying,five polysaccharides were obtained.Their antiplatelet aggregation activity was determined.It was found that the polysaccharides eluted by 0.2M Na Cl had the strongest inhibitory activity.The purified polysaccharide LP was further purified by Sephadex G-100 column.The LP was degraded by hydrogen peroxide and ascorbic acid with concentration of 5,10,15,20,30 and 50m M,respectively named LP1,LP2,LP3,LP4,LP5 and LP6?collectively referred to as CP?.The effects of hydrogen peroxide degradation of LP on platelet aggregation and anticoagulant activity were studied,and the relationship between the characteristics and activity of degraded polysaccharides was analyzed.The main results were as follows:?1?Purification and characteristic structure analysis of polysaccharide from Lycium barbarum L.leaves:The crude polysaccharide was obtained by water extraction and alcohol precipitation,sevage to remove protein.After elution by DEAE-52 cellulose column,it was found that the polysaccharide fraction eluted by 0.2 M Na Cl solution showed the strongest platelet aggregation inhibitory activity?36.5%,0.5 mg/ml?.This component was further purified by Sephadex G-100 column to obtain Lycium barbarum L.leaves polysaccharide?LP?,and its physical and chemical properties and structure were determined.The content of polysaccharide was 88.0±7.3%,the contents of glucuronic acid and protein were was 29.0±4.1%,2.2±0.3%,respectively,which indicated that LP was a highly purified polysaccharide.LP is a homogenous polysaccharide composed of seven monosaccharides:arabinose,rhamnose,ribose,xylose,mannose,galactose and glucose.The mole ratio was4.4:2.0:7.0:1.0:3.1:6.8:13.4.Its molecular weight?Mw?was 46.3 k Da,and its dispersion coefficient?Mw/Mn?was 1.709.?2?Preparation and physicochemical characteristics of degradation polysaccharide from Lycium barbarum L.leaves:When the concentration of hydrogen peroxide and ascorbic acid increased from 0to 50 mm,the Mw of polysaccharides before and after degradation decreased from 4.630×10⁴ to 3.450×10⁴ Da,Mw/Mn increased from 1.709 to 2.160,indicating that degradation could reduce the molecular weight of LP and enlarge the range of molecular weight distribution,and the DD of polysaccharide increased from 3.5%of LP1 to 25.5%of LP6,the zeta potential of polysaccharide increased from-8.01±0.58 m V?LP?to 5.35±0.19 m V?LP6?The results showed that the high concentration of hydrogen peroxide and ascorbic acid can cause the destruction of polysaccharide chain.The contents of total sugar?82.9-91.1%?and glucuronic acid?26.9-34.6%?of the six degradation products were almost the same as those of LP,indicating that the degradation did not significantly affect the contents of total sugar and glucuronic acid.The infrared spectrum analysis showed that the wavelength from 4000 to 400 cm^-1,the main characteristic peak of CP was almost the same as that of LP.In addition,the carbon and hydrogen spectrum of CP was very similar to that of LP,which indicated that there was no major functional group transition during the degradation of LP.?3?Effect of polysaccharide degradation on antiplatelet aggregation and anticoagulant activities:In vitro experiments showed that degradation can significantly improve the antiplatelet aggregation activity of LP?p<0.05?.The inhibitory effect of polysaccharide with the highest degradation degree on platelet aggregation induced by arachidonic acid and thrombin was higher than that of aspirin at 0.5 mg/ml?88.67%vs 80.62%;69.67%vs 65.73%?,and anticoagulation experiments showed that LP and CP could prolong APTT,but had little effect on PT,indicating that LP and CP could inhibit endogenous coagulation.Further analysis confirmed that after degradation,polysaccharide changed from a compact spherical structure to a random spiral structure in aqueous solution,which promoted the interaction between polysaccharide and platelet..LP and LP6 significantly inhibited COX₁ activity and TXA₂production.Compared with LP,LP6 had a stronger inhibitory effect?P<0.05?,which indicated that LP and LP6 play a strong role in antiplatelet aggregation through cyclooxygenase pathway,and the degradation of polysaccharide enhanced this activity.After reduction of glucuronic acid,the content of glucuronic acid decreased by 60-70%,and the antiplatelet activity of LP and CP decreased to 40-60%,and APTT also decreased significantly,indicating that glucuronic acid is very important for antithrombotic activity.To sum up,polysaccharide from Lycium barbarum L.leaves can effectively enhance the antiplatelet aggregation and anticoagulant activity after degradation by hydrogen peroxide and ascorbic acid,and uronic acid is an important active group.In this study,the structure and function of polysaccharide from Lycium barbarum L.leaves were analyzed,which provided theoretical basis for the study of safe and natural antiplatelet components.