| The traditional Chinese medicine aspongopus is a dried whole insect of aspongopus genus Aspongopus chinensis Dallas of Pentatomidae,It is mainly produced in Guizhou,Sichuan,Yunnan and other places.Jiangsu,Anhui,Zhejiang,Hunan,Hubei,Guangdong,Guangxi and other places are also produced.Aspongopus is endemic to China.Its medicinal materials are expensive,and there are many kinds of adulterants in the market.The quality of medicinal materials varies greatly.Morphological identification is a commonly used identification method in aspongopus.However,aspongopus and its adulterants have similar morphological characteristics and need strong taxonomic expertise and subjectivity.HPLC method can reflect the types and contents of chemical components in traditional Chinese medicine,and is an effective method for identification of authenticity and quality control of traditional Chinese medicine.This study intends to explore the feasibility of identifying the authenticity of aspongopus medicinal materials through the chemical components contained in it,so as to provide a more objective chemical identification method for the authenticity identification of aspongopus medicinal materials.Traditional Chinese medicine believe that aspongopus has the effects of regulating qi and relieving pain,warming the middle and helping yang,Modern medical clinical studies show that aspongopus has better anticoagulantand antitumor effects,but its related pharmacological studies are few,the effective active parts and pharmacological mechanism are still unclear,and the active ingredients have not been reported.This study hopes to explore the mechanism of anticoagulant activity of aspongopus through animal in vivo experiments and screen out effective active sites,so as to provide basis for clinical application and research of aspongopusObjective: To identify the authenticity of aspongopus,confirm the anticoagulant activity of aspongopus extract,explore the mechanism and location of its anticoagulant activity.Methods:1.Collect 25 batches of aspongopus medicinal products from Yunnan,Guizhou,Sichuan and Guangxi,establish HPLC fingerprints,and use principal component analysis and cluster analysis methods in Stoichiometry to identify genuine and fake aspongopus medicinal products.2.Anticoagulant activities of aspongopus water-decocted extract,water-extracted alcohol precipitate upper layer solution,water-extracted alcohol precipitate and ethanol extract were studied by measuring activated partial thromboplastin time(APTT)and prothrombin time(PT).On this basis,the blood coagulation time(CT)value of mice was determined by capillary method,and the tail-end bleeding time(BT)value of mice was determined by tail-end method to verify the anticoagulant effect of aspongopus in animals.The mechanism of anticoagulation in aspongopus was preliminarily explored through the experiments of coagulation function in rats and platelet aggregation in rabbits in vitro.3.Furthermore,the aspongopus extract with the strongest anticoagulant activity is used for extraction and separation,and is subjected to D101 macroporous adsorption resin column chromatography separation to obtain elution parts with different ethanol concentrations,and the anticoagulant activity is respectively measured to find out the parts with the strongest activity.Recovery purified the most active part to obtain aspongopus anticoagulant active monomer.Results: 1.Identification of aspongopus: 25 batches of aspongopus medicinal materia were identified as genuine 8 batches,counterfeit 14batches(containing Cyclopelta perva Distana ? Erthesina fullo Thunberg ?Megymenum inerme H.-S ? Aspongopus nigriventris Westwood ? Essaratoma papillosa Druryand other varieties)and adulterated 3 batches.The HPLC method was used to analyze 25 batches of medicinal products.Among them,there was no common peak between Erthesina fullo Thunberg and aspongopus genuine.Only No.3 and No.4 were common peaks between aspongopus nymph and aspongopus genuine,but the contents were quite different and the pattern shapes were different.HPLC method can directly identify Erthesina fullo Thunberg and nymph.The remaining 22 batches of medicinal materials were established HPLC fingerprints to generate control chromatogram R,containing 6common peaks.The similarity between HPLC chromatogram and control chromatogram R between different batches is between 0.643 and 0.964,and the content of chemical components between authentic and adulterated products is quite different.Principal component analysis was used to analyze the peak areas of common peaks in 22 batches of samples.Among them,the cumulative contribution rate of the first,second and third principal components reached91.55%,which was highly representative.The results showed that there was a big difference between Cyclopelta perva Distana and genuine products from aspongopus.On the basis of principal component analysis,taking the three principal components after dimension reduction as variables,the systematic clustering method adopts the linking method between groups and the squared Euclidean distance method.The results show that the genuine products in aspongopus are grouped into one category,the Cyclopelta perva Distana is grouped into one category,and the rest samples are divided into two categories.Only one batch of adulterated products are classified into the genuine products in aspongopus,which may be due to the fact that aspongopus accounts for more ofthe adulterated products and the chemical components are mainly represented by the genuine products.The results of cluster analysis and principal component analysis are basically the same.2.Anticoagulant Activity Research:The aspongopus water decoction extract and the water extraction and alcohol precipitation upper layer solution have obvious anticoagulant effect,and anticoagulant effective substances should be concentrated in the water extraction and alcohol precipitation upper layer solution.Aspongopus water extract and its upper layer solution after further water extraction and alcohol precipitation can obviously prolong the coagulation time CT value and tail tip bleeding time BT value of mice,indicating that aspongopus water extraction and alcohol precipitation extract has obvious anticoagulant effect in animals.Through the coagulation function test of rats,it is proved that the anticoagulant process in aspongopus acts on coagulation factors existing in intrinsic pathway through APTT pathway.The rabbit platelet aggregation experiment in vitro shows that aspongopus water extract and alcohol precipitate extract can affect the normal physiological function of platelets and have significant inhibitory effect on platelet aggregation.3.Screening of anticoagulant active sites and components in aspongopus:After the water extract and alcohol precipitate from aspongopus are separated by D101 macroporous adsorption resin,the 50%,70% and 90% ethanol elution sites have the strongest activity and are anticoagulant active sites.The above active sites were further isolated and purified,and four monomer compounds were obtained.However,only one monomer compound has been identified as aspongester A so far.Conclusion: Aspongopus medicinal products can be objectively and accurately identified by HPLC fingerprint and chemometrics.Among the three extraction methods of boiling water decoction,ethanol cold extraction and waterextraction and alcohol precipitation,the best extraction method for anticoagulant activity is water extraction and alcohol precipitation.Aspongopus water extract and alcohol precipitation extract exert anticoagulant effect in rats through intrinsic pathway and can inhibit platelet aggregation.In addition,after the aspongopus water extraction and alcohol precipitation extract is separated by D101 macroporous adsorption resin,the effective active sites are concentrated in the high concentration(50%,70% and 90%)ethanol elution sites,and a lipid compound aspongester A is obtained by separation and purification.Based on the above experimental results,it can be seen that the anticoagulant active ingredients of aspongopus are various ingredients that can be extracted by decocting with water.Boiling water extraction can remove a large number of impurities such as fatty acids,but can retain alcohol soluble active ingredients at the same time,which can be further refined by water extraction and alcohol precipitation.At the same time,it shows that the anticoagulant activity may be the result of the joint action of many components,not a single chemical component.This study provides a more objective chemical identification method for the authenticity identification of aspongopus medicinal materials.Animal in vivo experiments have verified the anticoagulant activity of aspongopus extract,and explored its anticoagulant activity mechanism and effective active site,providing a certain basis for further development and utilization of aspongopus. |
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