| Background:Scolopendra,which comes from Scolopendra subspinipes mutilans L.Koch,is one of traditional Chinese medicinal materials with a long history of treating many kinds of diseases,such as furunculosis,infantile convulsion,various pain,hemiplegia,traumatic injury and so on.Modern study shows that it has lots of effects on analgesia,anti-inflammatory,anti-tumor,anti-convulsant,anticoagulation thrombolytic and so on.Tcm dispensing granule is a kind of granule which is processed from Chineses medicinal sliceses,prepared with extraction,low temperature concentration and rapid drying technologies and provides clinical or commercial use.Thromboembolic is one of the most serious fatal disease which is initiated by thrombosis and thromboembolism in modern society.Disseminated intravascular coagulation(DIC)is a serious clinical syndrome with high mortality rate,which is characted by thrombus,embolization,widely bleeding,shock and hemolysis.There ara some of the research showing that Scolopendra materials have some special effects on anticoagulation and thrombolysis in recent years.However,the traditional decoction has disadvantage of big dosage,unstable quality,unconvenient decoction,take and carry,relatively difficult storage and transport,therefore makes it to be Scolopendra dispensing granule which is stable and reliable in quality,accurate in dosage,safe and effective,convenient to take,easy to carry,store and transport.It not only makes sure the clinical use convenient,safe,cffevtive but also expands its use crowd.Objective:mainly through researching the quality control methods of Scolopendra materials,preparation craft optimization and anticoagulation thrombolytic effect of its dispensing granule to provide the scientific basis to make sure the raw material and end produet’s stable and controllable,the clinical use security and effective,meanwhile to provide a model for the correlation researeh.Methods:In the rwa materials’quality study aspcet,this topic did some comparison research to four different batch numbers but same habitats Scolopendra raw materials in the collection,including origin,charaeter,water content,ash and extract by the methods in Chinese Pharmacopoeia while identified the free arginine(Arg)and serine(Ser)in scolopendra by TCL,screened the samples preparation process and developing solvent systems,determined the anti-coagulant activity by method of titration with thrombin and screened the pretreatment methods.The results of titration with thrombin were signed with measurement data(X±S).Those with homogeneous variance received t test,while those with non-homogeneous variance received t’ test.Statistical significance was obtained when P<0.05.In the preparation of the Scolopendra dispensing granule aspect,water were used to extract the Scolopendra materials and enzymatic hydrolysis was used to treat the herb residue.To study the preparation craft of the Scolopendra Dispensing Granule,the suitable conditions of water extraction and enzymatic hydrolysis were confirmed by single factor method and the best preparation were selected by orthogonal design.All of the results were signed with measurement data(X±S).The results of single factor method were analyzed with one-way ANOVA,while the results of orthogonal design were analyzed with Univariat method in General Linear Model.When doing the multiple comparison,those with homogeneous variance received LSD test,while those with non-homogeneous variance received Dunnett’s T3 test.Statistical significance was obtained when P<0.05.To study the anticoagulation thrombolytic effect,this topic compared the effect of Scolopendra raw materials,its water extraction and dispensing granule on thrombolysis in vitro with streptokinase(SK)used as positive drugs and the thrombus wet weight of different group were measured at different time points(W0,W2,W18)as main index.All the results were signed with measurement data(X±S)and analyzed with repeated Measurement method in General Linear Model.Mauchly’s test of sphericity should be used to judge whether there were relations among the repeatedly measured data.If any(P<0.05),Greenhouse-Geisser corrected results should be taken.Mouse model of DIC induced by lipopolysaccharide(LPS)were used to study their anticoagulation thrombolytic effect in vivo.Heparin Sodium and aspirin(ASP)were used as positive drugs and the mouse coagulation time(CT),platelet(PLT)and plasma protamine paracoagulation(3P)test were measured after the DIC model was achieved.The results of CT and PLT were signed with measurement data(X±S)and analyzed with one-way ANOVA.When doing the multiple comparison between different groups,those with homogeneous variance received LSD test,while those with non-homogeneous variance received Dunnett’s T3 test,statistical significance was obtained when P<0.05.3P test results were signed with ranked data and analyzed with Kruskal-Wallis H test.Mann-Whitney U test could be used to do the multiple comparison and Bonferroni method should be used to correct the results.Results:in the rwa material study aspect,all batches of Scolopendra raw materials accord the regulation except the batch 20101001,the water content of which is higher than the Chinese Pharmacopoeia standard.When medicinal materials are extracted by formic acid and 95%ethanol(1:1)with ultrasonic method and developed by n-butanol-acetic acid-water(12:5:4),the spots of Arg and Ser can be well separated.Comparing with the water extraction method,ultrasonic method is more suitable for preparating the anti-coagulant components in scolopendra materials when its anti-coagulant activity is obviously high than the water extraction(P<0.001),and determining it by method of titration with thrombin could get a well reproducibility.The whole activity of anti-thrombin among four different batch numbers’ rwa materials are 14.00±1.53U/g、13.00±0.58U/g、17.00±1.15U/g、15.67±1.53U/g respectively,range from 12.42～18.15U/g.In the study of the preparation craft of the Scolopendra dispensing granule respect,the best processing are:the Scolopendra materials were extracted by 12 times 0.02mol/L Na3PO4 buffer solution in 65℃ for 75 min,and the herb residue were comfirmed by trypsin enzymatic hydrolysis in artificial intestinal juice,when the substrate concentrations was 1/10,the enzyme concentration was 6%and the enzymatic hydrolysis time was 5h.The anti-coagulant activity of the Scolopendra dispensing granule was 69.00U/g.In the study of the vitro thrombolysis effect respect,the activity of thrombolysis in vitro of the raw materials,its water extraction and dispensing granule would increased signtificantly with time increasing(P<0.001).Comparing with the raw materials and its water extraction,the thrombolysis activity of dispensing granule in vitro doesn’t show a signtificant difference at each dose(P>0.05)in a short time,while it shows a signtificant improvement at the middle and high doses(P<0.05)in a long time.The effect of vitro thrombolysis on Scolopendra doesn’t change obviously when extracted by water.All the three kinds of drugs show a dose-effect relationship.In the respect of the effect on the DIC mouse,all drugs in the experiment can shorten the CT signtificantly(P<0.05)at the 45th min after the DIC model finished and the order from low to high is the Scolopendra raw materials<ASP<Heparin Sodium<the water extraction<the dispensing granule.At the 5th hour after the DIC model achieved,the Scolopendra raw materials,water extraction and the dispensing granule all can obviously inhibit the platelet aggregation and increase the PLT level of the DIC mouse(P<0.05),but only the dispensing granule can inhibit the hyperfibrinolysis.The inhibition of the Scolopendra dispensing granule is significantly greater than the raw materials and the water extraction(P<0.05).Conclusion:the qualities of Scolopendra materials collected in this topic accord the Chinese Pharmacopoeia regulation.The methods of TLC identification and anti-coagulant activity quantitatively could be used as a basis for improving the quality standard of scolopendra materials.The preparation craft optimized is suit for the Scolopendra dispensing granule preparation.The vitro thrombolysis activity and the vivo anticoagulation of the Scolopendra dispensing granule is greater than the raw materials and the water extraction. |
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