| Objective1.To establish a UV spectrophotometric method for the determination of total polysaccharides,total flavonoids and total saponins in Mutouhui.At the same time,gas chromatography（GC）and high performance liquid chromatography（HPLC）methods for the determination of volatile and non-volatile components in Toutouhui were established.2.Evaluate the hemostatic effect of the water extract from the tomb head.3.Screen the possible mechanism of hemostasis through network pharmacology.Methods:1.Research on the quality control of the tomb back:1.1 Determination of total components in Mutouhui:A method for the determination of total polysaccharides was established by phenol concentrated sulfuric acid colorimetry with D-glucose as control.The color conditions of phenol concentration,color developing temperature,color developing time and stable time were optimized by single factor experiment and orthogonal experiment,and the methodological investigation was completed;The content determination method of total flavonoids was established by using rutin reference substance and Na NO2-Al（NO3）3-Na OH colorimetry.The addition amount of Al（NO3）3solution,standing time and color developing temperature were optimized by single factor experiment and orthogonal experiment,and the methodological investigation was completed;The content determination method of total saponins was established by using 1%vanillin acetic acid perchloric acid colorimetry with oleanolic acid as reference substance.The addition amount of 1%vanillin perchloric acid solution,the addition amount of glacial acetic acid solution,the color developing temperature and heating time were optimized by single factor experiment combined with orthogonal experiment,and the methodological investigation was completed.1.2 Determination of the content of volatile components in the tomb headUsing GC method.The Agilent DB-5 capillary column（30m×0.25mm×0.25μm）was used as the analytical column,and the flame ionization detector（FID）was used as the detector.The detector temperature is 250°C,the inlet temperature is 250°C,the temperature is programmed,the carrier gas is high-purity nitrogen,and the flow rate is 1.0m L/min.1.3 Determination of non-volatile components in Toutouhui:HPLC method.Diamonsil C18（250mm×4.6mm,5μm）was used as the chromatographic column,the mobile phase was acetonitrile-0.2%phosphoric acid aqueous solution（80:20）,the flow rate was 1.0m L/min,the column temperature was 25℃,the injection volume was 10μL,and the detection wavelength was 205nm.2.Study on the effect of hemostasis in Toutouhui2.Study on the effect of hemostasis in Toutouhui2.1 Hemostatic effect of Mutouhui on hemorrhagic hemorrhage model rats:dry yeast and absolute ethanol were subcutaneously injected to establish hemorrhagic hemorrhage rat model.The SD rats were randomly divided into blank group,model group,Yunnan Baiyao positive group,Toutouhui low-dose group,Toutouhui medium-dose group,and Toutouhui high-dose group.The detection indicators include four indicators of rat rectal temperature,body weight,food consumption,water consumption,and coagulation function,namely plasma thrombin time（TT）,activated partial thromboplastin time（APTT）,prothrombin time（PT）and fibrin Original（FIB）,platelet and red blood cell indicators,including:（1）Platelet indicators:platelet count（PLT）,mean platelet volume（MPV）,platelet distribution width（PDW）,platelet pressure（PCT）;（2）red blood cell indicators:red blood cell count（RBC）,hemoglobin volume（HGB）,hematocrit（HCT）,mean corpuscular volume（MCV）,mean corpuscular hemoglobin content（MCH）and mean corpuscular hemoglobin concentration（MCHC）.2.2 Study on the effect of Mutouhui on bleeding time and clotting time in normal mice:50 mice were subjected to bleeding time experiment and clotting time experiment respectively.The mice were randomly divided into blank group,Yunnan Baiyao positive group,and Mutohouhui low-dose group There are 5 groups,10 rats in each group,middle-dose group and high-dose group.Tail-cut method was used to determine bleeding time,and capillary method was used to determine clotting time.3.Screen the possible mechanism of hemostasis in Toutou by network pharmacologyTCMSP,Pub Chem and other databases were used to screen the active ingredients and action targets.The target screening of hemorrhagic diseases was based on DRUGBANK,Dis Ge NET and other databases.After the intersection of the two,the compositional target network was constructed by using Spring online platform and Cytoscape software,and the core target genes were enriched by the David database for GO analysis and KEGG pathway analysis.Molecular docking technology was used to dock the previous correlation between the five proteins with the highest binding energy and the core component quercetin.Results:1.Research on the quality control of the tomb back1.1 Determination of total components in TouhuiDetermination of total polysaccharide content in Toutouhui:The optimal color development conditions are 9%phenol concentration,water bath temperature 70℃,and heating time 10 min.The results of methodological investigation are regression equation A=0.1431C-0.1454,r=0.9976,D-anhydrous glucose solution shows good linearity in the concentration range of 0.00513～0.1026mg/m L;RSD values of precision,stability and repeatability are all less than 3.0%;the average recovery rate of the sample is 99.75%.Determination of total flavonoids in Toutouhui:The optimal color development conditions are 1.5m L of Al（NO3）3solution,standing time 25 min,and color development temperature 30℃.The methodological investigation result is that the regression equation A=0.8573C+0.1583,r=0.9954,the rutin solution shows a good linear relationship within the concentration range of 0.062～0.62mg/m L;the precision,stability,and repeatability RSD values are all less than 3.0%;The average recovery rate of the sample is 100.91%.Determination of total saponins in the head of tomb:the optimum color developing condition was 80℃,reaction time 20 minutes,1%vanillin acetic acid-perchloric acid solution 0.5m L.The results of the methodology study were as follows:linear regression equation A=1.6162C+0.2431,r=0.9997,The oleanolic acid reference solution showed a good linear relationship in the concentration range of 0.104～1.04mg/m L;precision,stability,repeatability RSD values are less than 3.0%;the average recovery rate of the sample is 99.67%.1.2 Determination of the content of volatile components in the tomb headA method for the determination ofβ-caryophyllene and oxycaryophyllene in Toutouhui was established by gas chromatography.The mass concentration ofβ-caryophyllene showed a good linear relationship between 6.13～61.31μg/m L（r=0.9990）,and the precision,stability,and repeatability RSD values were all less than 3.0%.The average sample recovery rate was 100.81%,and the RSD was 0.81%.The mass concentration of caryophyllene oxide shows a good linear relationship in the range of4.55～45.50μg/m L（r=0.9995）.The precision,stability,and repeatability RSD values are all less than 3.0%.The average sample recovery rate was 99.52%,and the RSD was 0.70%.1.3 Determination of the content of non-volatile components in the tomb head backA method for the determination of oleanolic acid and ursolic acid in Mutouhui was established by high performance liquid chromatography.Oleanolic acid and ursolic acid showed a good linear relationship in the range of 0.1042～1.042mg/m L（r=0.9999）and0.1024～1.024mg/m L（r=0.9996）,respectively.The RSD values of precision,repeatability and stability are all less than 3.0%.The average recovery rate of oleanolic acid was100.27%and the RSD was 1.39%.The average recovery rate of ursolic acid was 98.73%and the RSD was 1.07%.2.Study on the effect of hemostasis in Toutouhui2.1 Study on the hemostatic effect of Mutouhui on hemorrhagic hemorrhage model ratsAs for the comparison between the model group and the blank group:the rectal temperature of the rats increased after 2 hours,reaching 38℃and lasting for 6 hours,indicating that the heat syndrome model was successfully replicated.Weight decreased significantly（P<0.01）,water intake increased（P<0.01）,and diet decreased,but the difference was not statistically significant.PT,APTT and TT of the model group were significantly prolonged（P<0.01）,FIB was extremely significantly increased（P<0.01）;PLT and PCT in the model group were significantly reduced（P<0.05,P<0.01）,MPV,PDW Significant increase（P<0.01）.RBC and HCT increased significantly（P<0.01）,and HGB and MCHC increased significantly（P<0.05）.The difference between MCV and MCH was not statistically significant.In terms of the comparison between each administration group and the model group:the body weight of the low and medium dose groups increased,and the weight of the high dose group decreased.The dietary intake of rats in the positive group and Mutouhui each dose group decreased,but the difference was not statistically significant.The amount of drinking water of rats in each dosage group in Toutouhui also increased.The PT,APTT,TT and FIB of rats in the positive group and each dose of Toutou water extract group were significantly reduced（P<0.05,P<0.01）.Except that the PDW of the rats in the low-dose group was reduced but not statistically significant,the PLT and PCT of the rats in the other treatment groups were significantly increased（P<0.01）,and both MPV and PDW were significantly reduced（P<0.01）.PLT and PCT in the positive group and Mutouhui each dose group increased significantly（P<0.01）,and HCT decreased significantly（P<0.01）.In addition to the low-dose Toutouhui group,RBC and MCHC were significantly reduced in the positive group,the middle-dose Toutouhui group and the high-dose group（P<0.05）,and MPV and PDW were significantly reduced（P<0.01）.The middle-dose group can significantly reduce HGB（P<0.05）,and the high-dose group can significantly reduce HGB（P<0.01）.2.2 Research on the effect of tomb head back on bleeding time and clotting time in normal miceIn terms of bleeding time,the positive group could significantly shorten the bleeding time compared with the blank group（P<0.01）,indicating that the results of this bleeding experiment were reliable.The bleeding time of Cucumis cephalus low-dose group and Cucumis cephalus medium-dose group was also significantly shortened（P<0.05）,but there was no significant difference in the bleeding time of Cucumis cephalus high-dose group.Compared with the positive group,the bleeding time of each dose of cephalocervical gyrus group was significantly prolonged.The bleeding time of each dose group was prolonged in each dose group.The higher the dose was,the shorter the bleeding time was,which indicated that the effect of shortening the bleeding time and coagulation time was related to the dose.In terms of coagulation time,compared with blank group,the coagulation time of positive group and low,medium and high dose gravetarii groups was significantly shortened（P<0.01）;It is suggested that the results of this coagulation experiment are reliable.Compared with the positive group,the clotting time of each dose group was shortened.The higher the dose was,the shorter the coagulation time was.The higher the dose was,the shorter the coagulation time was.The high dose group could shorten the coagulation time more than the low and medium dose groups.The results showed that the effect of the drug on shortening the coagulation time in mice was related to the dose.3.Screening of possible mechanism of hemostasisSeven active components and 76 action targets were screened out for hemostasis of the first gyri of the tomb.By GO enrichment analysis and KEGG enrichment analysis,259enrichment items that may be related to hemostasis mechanism of the first gyri of the tomb and 21 enrichment items that may be related to hemostasis pathway of the first gyri of the tomb were obtained,respectively.The results showed that the possible mechanism of occipitric gyrus hemostasis was through the key targets of CCR5,JAK2,MMP9,MPO and STAT3,and the involved pathways included neuroactive ligand-receptor interaction signaling pathway,calcium signaling pathway,arachidonic acid metabolism signaling pathway,HIF-1 signaling pathway,etc.The above hemostasis mechanism was verified by molecular docking,and the results showed that the 7 active components had strong affinity with the 5 core targets,especially quercetin with the lowest binding energy and the strongest affinity.Graphical docking of quercetin with five core targets was carried out.Conclusion1.The content of total polysaccharides,total flavonoids and total saponins in Toutouhui were determined by ultraviolet spectrophotometry,and the triterpenoid components oleanolic acid and ursolic acid were volatile by high performance liquid chromatography and gas chromatography.The content of the sexual componentsβ-caryophyllene and oxycaryophyllene were determined.The precision,stability,repeatability and sample recovery rate of the established method were all less than 3%.The content of 10 batches of tomb heads from different origins was determined.The method is simple,easy to implement,accurate and reliable,and provides a scientific basis for the quality control of medicinal materials from the tomb head.2.The experiment passed the research on the hemostatic effect of Toutouhui,and the rats were selected for the determination of the blood-heat hemorrhage model.The results showed that Yunnan Baiyao positive group and each dose group of Toutouhui could shorten the PT and APTT of the model group to varying degrees.And TT time,reduce FIB content,and can significantly increase PLT and PCT,and significantly reduce HCT.In addition,the bleeding time and clotting time of mice were measured to evaluate the hemostatic effect of Toutouhui.Compared with the Toutouhui group of each dose,the bleeding and clotting time were shortened to a certain extent.The higher the dose,the shorter the bleeding and clotting time.Obviously,it shows that the effect of shortening the bleeding and clotting time of mice has a certain dose-effect relationship with the dosage.3.The experiment screened 76 targets for hemostasis in Toutouhui through network pharmacology,259 were obtained through GO enrichment analysis,and 21 pathways that might be related to the hemostatic effect of Toutouhui were obtained through KEGG enrichment analysis,and selected The HIF-1 signal channel serves as the hemostasis mechanism for the next study.Through the docking of 7 components with 5 core target molecules,the results show that quercetin has the lowest binding energy and the strongest affinity to the 5 key core targets. |
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