Isolation And Extraction Of Hypoglycemic And Anticoagulant Components Of Cyclocarya Paliurus

Cyclocarya paliurus（C.paliurus）is a deciduous tree belonging to the family Juglandaceae in the order of Fagales.It has various biological activities such as hypoglycemic,hypolipidemic,anticoagulant,and anti-inflammatory effects.C.paliurus is commonly used in traditional Chinese medicine to treat diabetes and blood stasis-related disorders.In order to further explore the material basis of its hypoglycemic and anticoagulant activities,this study investigated the different extracts of C.paliurus.The specific results are as follows:1、The leaves of C.paliurus were powdered and extracted with 70%ethanol to obtain the ethanol extract（yield:24.68%）.The residue was then extracted with deionized water to obtain the water extract.After alcohol precipitation,the alcohol sink（yield:4.29%）and alcohol solution（yield:2.12%）were obtained.The hypoglycemic and anticoagulant activities of different C.paliurus extracts were evaluated using STZ-induced diabetic mice and acute blood stasis rats induced by epinephrine and ice water.The results showed that the ethanol extract of C.paliurus had significant hypoglycemic and anticoagulant effects（P<0.01）,and the alcohol sink of C.paliurus had significant hypoglycemic activity（P<0.01）.2、After DEAE-52 cellulose chromatography of the alcohol sink of C.paliurus,two fractions were obtained:the water-eluted fraction B-1 and the salt-eluted fraction B-2.The fraction B-1 had significant hypoglycemic activity in STZ-induced diabetic mice（P<0.01）.B-1 was further purified by HW-55F and Sephacryl S 400 gel to obtain two monomeric compounds,CPP-1 and CPP-2.Among them,CPP-1 significantly improved the symptoms of polydipsia and polyphagia in diabetic mice,reduced fasting blood glucose（FBG）and hemoglobin A1c（Hb A1c）levels,and increased oral glucose tolerance（P<0.01）.Ultraviolet spectrum analysis of CPP-1 showed that it was a pure polysaccharide without nucleic acid or protein components.The monosaccharide composition analysis showed that CPP-1 was mainly composed of glucose and glucuronic acid.The molecular weight determination results showed that the weight-average molecular weight and number-average molecular weight of CPP-1 were12.59 k Da and 9.61 k Da,respectively,with a polydispersity index of 1.31.Nuclear magnetic resonance（NMR）spectroscopy analysis showed that the backbone of CPP-1 was composed of（1→2）-β-glucose,withβ-glucuronic acid ethyl ester side chains attached to the C₃ position of the main chain.3、In the study of the anticoagulant activity of different extracts of C.paliurus,it was found that the alcohol extract had significant anticoagulant activity,so this fraction was selected for further separation.The ethanol extract of C.paliurus was separated by macroporous resin AB-8 to obtain three compounds.Based on mouse model evaluation,the 75%ethanol elution fraction from the large pore resin separation exhibited anticoagulant activity and was further separated using an MCI column to yield four components.Among them,the 30%and 60%ethanol elution fractions significantly reduced coagulation time（P<0.01）and prolonged tail bleeding time in mice（P<0.01）.The two active fractions were further purified using an ODS-A column to yield two monomeric compounds named CPT-1 and CPT-2.NMR analysis revealed that CPT-1 was 16-acetylchy kirenol,and CPT-2 was kirenol.The anticoagulant mechanisms of CPT-1 and CPT-2 were investigated using a mouse tail thrombosis model induced by carrageenan.It was found that CPT-1 inhibited the activity of thrombin by increasing the expression level of protein C,thereby suppressing the amplification of coagulation and inhibiting the conversion of fibrinogen to fibrin.CPT-2,on the other hand,inhibited platelet aggregation by reducing the number of platelets on the surface of blood vessels.It also increased the expression of TFPI to inhibit the catalytic activity of the FVIIa-TF complex,which suppressed the activation of prothrombin and inhibited the initiation of the extrinsic coagulation pathway and platelet activation mediated by thrombin.