Extraction And Structural Characterization Of Auricularia Auricula Anticoagulant Polysaccharide And Its Inhibition Mechanism On Thrombosis

There are complex and perfect hemostasis,coagulation,anticoagulation and fibrinolytic systems as well as fine balance regulation mechanisms in the body.Under normal physiological conditions,blood flowing in the blood vessels will neither bleed nor clot to form thrombus.Increased labor intensity of static work,irregular work and rest schedules,unhealthy living habits such as smoking and improper diet,and extremely cold living environment can all increase the risk of abnormal coagulation of blood in the body.Abnormal coagulation of blood is the main cause of inducing cardiovascular and cerebrovascular diseases in the body,and the WHO predicts that in the recent decade,cardiovascular and cerebrovascular diseases will be the highest cause of death in human beings.At present,several mainstream anticoagulants on the market have their own disadvantages,such as the limitation of the way of administration,hemolysis and other serious side effects.Therefore,the development of natural drugs with low toxic side effect and appropriate anticoagulant effect can reduce the occurrence and recurrence of cardiovascular diseases,from the treatment and post-treatment maintenance.In this study,the wild Auricularia auricula（A.auricula）in northeast China was taken as the research object,and natural polysaccharides were extracted from A.auricula by alkali-soluble alcohol precipitation with UAE.The extraction process of A.auricula polysaccharides（AAP）was optimized by response surface design method,and the deproteinization process of A.auricula polysaccharides was optimized by orthogonal experiments.A.auricula polysaccharide anticoagulant component aAAPⅠ-b2 with strongest anticoagulant activity was prepared by using cetyl trimethyl ammonium bromide（CTAB）coupled with diethylamine（DEAE）ethyl cellulose ion exchange chromatography screening and gel chromatography purification,and structure characterization was carried out.In addition,this study systematically explored the anticoagulant function of aAAPⅠ-b2 in vivo and its inhibitory effect on thrombus formation,as well as the different antithrombotic pathways of aAAPⅠ-b2 through establishing a mouse thrombus model.Box-Behnken response surface optimization method was used to determine the optimal extraction parameters of AAP:extraction temperature of 70℃,extraction time of 25 min,liquid/material ratio of 100 m L/g,ultrasonic power of 225 W,polysaccharide yield of 14.07±0.21 mg/g.The optimum conditions for Papain-Sevag deproteinization were determined by orthogonal experiments:the crude polysaccharide solution was degraded by papain under the conditions of enzymatic hydrolysis temperature 55℃,enzymatic hydrolysis time 2.5 h and enzyme dosage 0.2%.The enzymatic solution was shaken up and down for 15 min according to the volume ratio of 3:1 with Sevag reagent,in which the volume ratio of chloroform to butanol was 4.The operation was repeated 3 times.The protein removal rate was 81.43%,and the retention rate of AAP was 80.23%.The component aAAPⅠ-b2 with the strongest anticoagulant activities in vitro was isolated through CTAB precipitation and DEAE Sepharose Fast Flow ion exchange chromatography,and further purification with Superdex-200 glucan gel chromatography,getting 7.6%of recovery rate.aAAPⅠ-b2 has the relative molecular weight of 9772 Da,consisting of glucuronic acid,mannose,glucose and xylose at a molar ratio of 89.25:30.5:4.25:1.aAAPⅠ-b2 molecular structure was determined by FT-IR scanning,partial acid hydrolysis,periodate oxidation,Smith degradation,methylation,GC-MS and NMR,and its main repeat unit was aboutα-D-Manp-（1→3）-α-D-Manp-（1→2,3）-α-D-Manp-（1→4）-β-D-Glc Ap-（1→,where Man and Glc A are main chain,and with→4）-Glcp-(1→on the side chain of connections of Man-2-C.On the basis of establishing the model of tail thrombus induced by carrageenin in mice,and taking aspirin as positive control,through APTT,PT,TT and FIB experiments,it was confirmed that aAAPⅠ-b2 also had strong anti-endogenous and anti-exogenous clotting function in mice.The comparison of black tail inhibition rate,bleeding time and tail thrombus H.E.staining in each group also showed that aAAPⅠ-b2 also had significant anticoagulant activity in vivo and inhibition of thrombosis.On this basis,the expression levels of the platelet surface receptors PGI₂,TXB₂,e NOs and ET-1 in each group were further studied,and it was determined that aAAPⅠ-b2 could inhibit the formation of thrombus by inhibiting platelet activation.Also,through compared AT-Ⅲand PC of mice liver tissue from different groups,it was found that the aAAPⅠ-b2 can also inhibit the activity of thrombin and factorⅤandⅧ,through adjusting the activity of antithrombinⅢand PC expression level,namely inhibiting the formation of blood clots through anticoagulant way.In the same way,AT-Ⅲand PC of mice liver tissue from different groups were compared,and the results indicated that the aAAPⅠ-b2 can also inhibit the activity of thrombin and factorⅤandⅧ,through adjusting the activity of AT-Ⅲand PC expression level,namely through anticoagulant way inhibit the formation of blood clots.AAAPⅠ-b2 cannot increase potential fibrinolytic activity by PLG,thus suppressing the effect of thrombosis,but it can improve the expression of HMWK,increase by a factor ofⅫfactor activation.It was found that AAP showed strong anticoagulant activity in vitro and in vivo,and could inhibit the formation of thrombus by oral administration.It was also found that the anticoagulant AAP mainly realized the antithrombotic purpose by inhibiting platelet activity and anticoagulation.This study can provide a certain theoretical basis for the development and utilization of anticoagulant and antithrombotic natural polysaccharides,as well as new ideas for the further development of related health food and drugs.