Quality Evaluation And Antithrombotic Effects Comparison Of Several Representative Astragalus Plants Of Yunnan

Objective:With A.mongholicus（Genuine Astragalus）as the control,the level of main active ingredients and similarity of chromatographic fingerprint about several Astragalus,which are the representative Astragalus in Yunnan were compared.The antithrombotic effects of A.yunnanensis,A.ernestii,and A.mongholicus were compared in vitro and in vivo.The research results of this paper will provide data support for the development and utilization of Astragalus in Yunnan.Method:1.Determination of main active ingredients.（1）Determination of the content of calycosin-7-O-β-D-glucoside.The content of calycosin-7-O-β-D-glucoside in Astragalus was determined by standard curve method.The method was appllied with Aglient 1260 HPLC,DAD detector,Aglient SB-C₁₈ column（4.6 mm×150 mm,3.5μm）.Water（A）–acetonitrile（B）was used as a mobile phase with gradient elution.In addition,the detecting wavelength was set at 254 nm.（2）Determination of total saponin.The content of total saponin in Astragalus was determined by Vanillin-perchloric acid colorimetry methods.The method use Astragaloside IV as the control substance.The absorbance was measured at 540 nm.2.Comparison of chromatographic fingerprint similarity.The chromatographic fingerprints of crude extracts and different organic reagents soluble portion of crude extracts of several Astragalus were constructed by HPLC.Water（A）–acetonitrile（B）was used as a mobile phase with gradient elution,then,the similarity was calculated by the Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine（Version2012）.The Comparison of chromatographic fingerprint similarity included three parts.（1）The similarity of the underground parts between Astragalus of Yunnan and A.mongholicus.（2）The similarity of underground parts of Astragalus of Yunnan.（3）The similarity between the aboveground and underground parts of Astragalus of Yunnan.3.Comparison of the antithrombotic effects.（1）Experiment in vitro.Blood from rat abdominal aorta was naturally coagulated at 37℃to form thrombus in vitro.Subsequently,extracts of high,medium,and low concentrations of A.mongholicuss,A.yunnanensis,and A.ernestii were added separately,and incubated with thrombus for 24 hours.After that,the remained blood clots were weighed,and the thrombolysis rate was calculated to compare the anticoagulant effects of the three Astragalus.（2）Experiments in vivo.After continuous gavaged of high and low doses of A.mongholicuss,A.yunnanensis,and A.ernestii extracts to rats for 7 days,a thrombus model in vivo was established by wraping filter paper soaked Fe Cl₃ around the arteries of rats.Then,the blood sample was taken after 20 minutes and the level of TXB₂ and 6-K-PGF_1αin plasma was determined by Elisa.After that,the thrombus in blood vessel was weighed to compare the anticoagulant effects of the three Astragalus in vivo.4.Comparison of the bleeding risks.After continuous gavaged of high and low doses of A.mongholicuss,A.yunnanensis,and A.ernestii extracts to rats for 4 days,the tail bleeding time was measured to evaluate the bleeding risks of the three Astragalus.Results:1.The contents of main ingredients.（1）Calycosin-7-O-β-D-glucoside.The content of calycosin-7-O-β-D-glucoside in A.mongholicus was 0.035%.Among the Astragalus in Yunnan,only A.ernestii contain calycosin-7-O-β-D-glucoside,whose content was 0.017%.（2）The total saponins.The total saponin content in A.mongholicus was 3.84%.The total saponin content in Astragalus of Yunnan,from the sequence of high to low was A.polycladus（6.51%）,A.ernestii（4.38%）,A.forrestii（4.30%）,A.acaulis（2.87%）,A.pullus（2.79%）,A.skythropos（2.50%）,A.bhotanensis（2.25%）,A.yunnanensis（1.84%）,A.englerianus（1.82%）.2.Similarity of chromatographic fingerprint.（1）The similarity between the underground parts of Astragalus in Yunnan and A.mongholicus.The similarity of the crude extract ranged from 0.103 to 0.906.A.pullus and A.mongholicus had the highest similarity.The similarity of the PE soluble portion of the crude extract ranged from 0.041 to 0.810.A.ernestii and A.mongholicus had the highest similarity.The similarity of the Et OAc soluble portion of the crude extract ranged from 0.002 to 0.302.A.yunnanensis and A.mongholicus had the highest similarity.The similarity of the n-Bu OH soluble portion of the crude extract ranged from0.025 to 0.193.A.polycladus and A.mongholicus had the highest similarity.The similarity of the water soluble portion of the crude extract ranged from 0.565 to 0.984.A.englerianus and A.mongholicus had the highest similarity.（2）The similarity between the underground parts of Astragalus in Yunnan.The similarity between the crude extracts of these Astragalus ranged from 0.059 to 0.888,with an average similarity of 0.404,the A.englerianus and A.pullus had the highest similarity.The similarity between the PE soluble portion ranged from0.046 to 0.962,with an average similarity of 0.469,the A.englerianus and A.bhotanensis had the highest similarity.The similarity between the Et OAc soluble portion ranged from 0 to0.506,with an average similarity of 0.071.The A.skythropos and A.pullus had the highest similarity.The similarity between the n-Bu OH soluble portion ranged from 0.010 to 0.417,with an average similarity of 0.176.The A.bhotanensis and A.englerianus had the highest similarity.The similarity between the water soluble portion ranged from 0.092 to 0.980,with an average similarity of 0.666.The A.bhotanensis and A.polycladus had the highest similarity.（3）The similarity between the aboveground and underground parts of Astragalus in Yunnan.The aboveground and underground parts of each Astragalus plants in Yunnan had a certain degree of similarity,ranging from 0.143 to 0.703.The aboveground and underground parts of A.polycladus had the highest similarity.3.Comparison of antithrombotic activity.（1）Experiments in vitro.At high concentrations（30 mg/m L）,the antithrombotic ability of these three Astragalus in vitro was A.yunnanensis>A.ernestii>A.mongholicus.At medium concentrations（15 mg/m L）,the antithrombotic ability of these three Astragalus was A.ernestii>A.yunnanensis>A.mongholicus.At low concentrations（7.5 mg/m L）,all these three types of Astragalus showed no significant ability to dissolve thrombus in vitro.（2）Experiments in vivo.At high doses（300 mg/kg）,the ability of these three types of Astragalus to dissolve thrombus in vivo was A.ernestii>A.mongholicus>A.yunnanensis.At low doses（150 mg/kg）,the ability of these three types of Astragalus to dissolve thrombus in vivo was A.ernestii>A.yunnanensis>A.mongholicus.Compared with the sham surgery group,when the ratio of TXB₂/6-K-PGF_1αin the thrombotic model group increased,it could be reduced by the three types of Astragalus,and the reduction ability was A.yunnanensis>A.ernestii>A.mongholicus.4.Comparison of bleeding risk.A.mongholicus at low dose（300 mg/kg）,A.ernestii at high dose（600 mg/kg）or low dose（300 mg/kg）could significantly prolong the tail bleeding time of rats.The bleeding risk of the three types of Astragalus ranged from large to small as A.ernestii,A.mongholicus,A.yunnanensis.Conclusion:1.The results of the active ingredients contents indicated that the calycosin-7-O-β-D-glucoside,which is the main active ingredient of A.mongholicus,was only presented in A.ernestii.All Astragalus in Yunnan tested contained saponins.The total saponins of A.ernestii,A.forrestii and A.polycladus were higher than those in A.mongholicus.Thus,A.ernestii was likely to be used as a substitute for A.mongholicus.The similarity of chromatographic fingerprint indicated that the chemical composition of Astragalus in Yunnan was significant different from A.mongholicus.Thus,these Astragalus in Yunnan were less likely to be a substitute of A.mongholicus.There were significant differences in chemical composition among different Astragalus from Yunnan.Thus,There was little chance of substitution for each other.The similarity of chemical composition between the aboveground and underground parts of A.polycladus was the highest,Thus,the aboveground part is more likely to replace the underground part.2.A.ernestii inhibited thrombosis both in vitro and in vivo,but it had a certain bleeding risk.Although the antithrombotic effect of A.yunnanensis was weaker than A.ernestii,its bleeding risk was lower.Compared to A.mongholicus,A.ernestii and A.yunnanensis had more potential for the development of antithrombotic drugs.