Analysis Of Chemical Components Of Thistle And Evaluation Of Its Efficacy In Cooling Blood, Stopping Bleeding And Protecting Live

Objective:Cirsium tianmushanicum Shih,a plant of genus Cirsium Mill.in Asteraceae,is a indigenous folk medicin used in Liyang and Huchen areas of Ninghai county,Zhejiang province in China for hundreds of years,which has the effects of removing jaundice,lowering blood pressure and protecting liver.As a folk medicinal plant,it has enomous economic potential in the local area.However,at present,the research on quality control,effective components and pharmacological effects of C.tianmushanicum is still rare,and there is basically no scientific evidence for the clinical uses of the herb.Based on these,the presented experiments of pharmacognostical identification,chemical composition characterization,pharmacological evaluation and mechanism research are carried out aiming at providing scientific basis for its future development and utilization.Methods:1 Pharmacognosy identification of Cirsium tianmushanicum Shih:Using the research method of pharmacognosy identification,high-definition digital camera,optical microscope and digital imaging technology,referring to the microscopic identification method(2001)of China Pharmacopoeia(2020 edition),the appearance and morphology of Cirsium tianmushanicum Shih were studied,and the surface photos of the medicinal materials were taken and their microscopic characteristics were recorded.2 Chemical composition analysis of C.tianmushanicum:A Waters Acquity UPLC BEHC18 column(2.1 m×50 mm,1.7 μm)was used,and acetonitrile(A)-0.1%formic acid aqueous solution(B)was used as mobile phase for gradient elution.In ESI negative ion mode,the extracts of Cirsium japonicum Fisch.ex DC.,Cirsium setosum(Willd)MB.and Cirsium tianmushanicum Shih were analyzed by one-stage and multi-stage full scanning mass spectrometry to collect mass spectrometry data.At the same time,using the global natural product molecular network GNPS molecular network technology and Cytoscape3.6.1 software,the molecular clusters with similar structures were screened according to the similarity of MS/MS fragment patterns and the relative contents were compared.3 Study on the pharmacological effects of C.tianmushanicum on cooling blood and hemostasis:36 healthy C57/BL6 mice(SPF grade)were prepared and fed adaptively for one week,then they were randomly divided into normal group,model group,YB group,CSL group(125 mg·kg-1),CSM group(250 mg·kg-1)and CSH group(500 mg·kg-1).The dosage of the drug group was calculated according to the crude drug dose.The normal group and the model group were given the same volume of normal saline and 0.5%CMC-Na solution respectively,and the YB group was given 0.5%CMC-Na suspension of 0.025 g·mL-1 Yunnan Baiyao(0.5g·kg-1)C.tianmushanicum administration group intragastric administration of the corresponding dose of extract.The mice were fed once a day for seven consecutive days.After intragastric administration for 6 days,except the normal group,the intraperitoneal injection dose of LPS was 10 EU·kg-1.The injection volume of each group was 10 mL·kg-1,and 20%dry yeast suspension(10 mL·kg-1)was injected subcutaneously on the back for 12 hours,and the mice were sacrified to establish a blood-heat bleeding model in accordance with the theoretical characteristics of traditional Chinese medicine.In the experiment,the anal temperature,clotting and hemostatic time,four indexes of the coagulation function,whole blood high,medium and low shear viscosity,platelet aggregation rate and maximum aggregation time were recorded in each group of the mice.At the same time,the effects of C.tianmushanicum on fibrinolytic system and thyroid system of the model mice were studied.The research indexes included triiodothyronine T3,thyroxine T4,thyrotropin TSH and the ratio of t-PA,PAI-1 content to t-PA/PAI-1.Finally,the pulmonary hemorrhage was observed by the pathological section.4 Study on the pharmacological effects of C.tianmushanicum on alcoholic liver injury:C57/BL6 mice were randomly divided into normal group,model group,BIF group,0.8 mg·kg-1 group,CSL group,125 mg·kg-1 group,CSM group 250 mg·kg-1 and CSH group 500 mg·kg-1 group.After adaptive feeding,50%ethanol was given to each group except the normal group,and the normal group was given the same volume of normal saline.One hour later,the drug was given to the drug group,the normal group and the model group were given the same volume of normal saline,the positive drug group was given 0.8 mg·kg-1 biphenyl diester,and the C.tianmushanicum group was given intragastric administration according to the above-mentioned low,middle and high doses for 30 days,once a day.Drugs are prepared with normal saline.The mice in each group were sacrificed after fasting for 16 hours.The study indexes included calculating liver index and detecting the level of TG,ALT,AST,GSH and MDA.At the same time,the pathological structure,lipid deposition and apoptosis of liver and small intestine of mice in each group were observed under microscope,and the regulatory effect of C.tianmushanicum extract on apoptosis pathway Caspase3/Bax/Bcl-2 was further studied.Results:1 In this study,the original plant characters,cross sections of the plant stems and leaves and microscopic characteristics of powder were described systematically,and the basis of pharmacognostical identification was summarized,the plant was identified as Cirsium tianmushanicum Shih,belonging to the genus Cirsium of Asteraceae.The obtained results and data deliver a scientific foundation for the identification and quality control of Cirsium Mill.2 Fifty-three compounds in C.tianmushanicum were quickly characterized by LC-MS,including 25 organic acids,19 flavonoids,1 quinone,2 volatile oils,1 triterpenoid and 5 other compounds.At the same time,the LC-MS data of Cirsium japonicum Fisch.ex DC.and C.japonicum Fisch.ex DC.of the same genus were compared.36 compounds were common components of the three species,and the common number of compounds of C.tianmushanicum and C.japonicum,C.setosum were 39 and 38,respectively.The relative contents of effective components of three kinds of plants were evaluated by molecular network technique.the results showed that the relative contents of compounds such as 3-0feruloylquinicacid and diosmin were higher in C.tianmushanicum.The content of anthraquinones is higher in C.japonicum.3 The results of pharmacological experiment on the cooling blood and hemostasis of C.tianmushanicum showed that compared with the model group,the bleeding time,clotting time,TT,APTT,FIB content,platelet aggregation time and t-PA/PAI-1 ratio decreased significantly,while the maximum platelet aggregation rate and t-PA content increased significantly.At the same time,all the dose groups of C.tianmushanicum could reduce the low shear viscosity of the whole blood of the model mice,but there was no significant change in the high shear viscosity,but only the CSM and CSH groups significantly decreased the middle shear viscosity,and the anal temperature was used as an important sign index of the model of blood heat hemorrhage.The results show that C.tianmushanicum can lower the body temperature by regulating thyroid hormones.Finally,the pathological results showed that all the dose groups could significantly reduce the pulmonary hemorrhage and inflammatory cell infiltration in the model group,and the high dose group had more obvious alleviating effect on the symptoms of pulmonary hemorrhage.4 In the pharmacological study on the treatment of chronic alcoholic liver injury,the results showed that compared with the model group,CSL,CSM and CSH groups significantly decreased the liver index,serum ALT,AST,MDA and TG activities in liver tissue,and increased the level of GSH in liver tissue.The pathological examination showed that the degree of liver tissue necrosis and inflammatory cell infiltration were significantly improved in the treatment group compared with the model group,and the CSM and CSH groups had a better protective effect on the intestinal wall and villus integrity of the mice.The results of Tunel and WB showed that the apoptosis in liver tissue of each treatment group was significantly decreased compared with the model group,while the expression of Bax,Cyp-D,Cyt-C,Cleaved Caspase-3 protein decreased,the expression of Bcl-2 protein increased,and the expression of Caspase-3 protein showed no difference.Conclusion:C.tianmushanicum,as a plant of the genus Cirsium,exhibit obvious plant characters and microscopic characteristics of the original plant,and the study fully provides a basis for the identification of C.tianmushanicum in the future.at the same time,a method for rapid screening and analysis of C.tianmushanicum compounds was established.It is proved that C.tianmushanicum has high clinical research value and medicinal potential at the component level.Finally,pharmacological experiments show that C.shanicum has a good effect on cooling blood and effect on hemostasis,and effect on chronic alcoholic liver injury,and its mechanism of liver protection may be related to antioxidation,regulation of lipid metabolism and regulation of Caspase3/Bax/Bcl-2 pathway to inhibit apoptosis.To sum up,for the first time,this study conducted a systematic basic research on C.tianmushanicum from the aspects of pharmacognosy identification,component analysis and the efficacy evaluation of cooling blood and hemostasis and protecting liver,so as to provide a scientific basis for the follow-up development and research of it.