Screening Of Effective Parts And Separation Analysis Of Chemical Composition Based On Blood Nourishing,Blood Circulation And Laxative Of Angelica Sinensis

Taking Angelica sinensis as the study object,based on traditional efficacy,completed three aspects of research:to select the effective parts of blood nourishing,relieving constipation and blood stasis,clear angelica blood circulation,laxative effect of the material basis,qualitative and quantitative analysis of main active components in Angelica Sinensis.The main methods and results are as follows:1.Based on the extraction and preparation methods of various Angelica preparations on the market,seven different extracts of Radix Angelicae Sinensis were prepared.Compound diphenoxylate mouse constipation model was established,The effect of laxative in 7 different extraction sites was compared with the first time of excretion,the amount of defecation in 6 h,the weight of stool,the rate of small intestine propulsion and the water of intestine.The mice model of blood deficiency was established by the combination of cyclophosphamide（CTX）and acetophenone hydrazine（APH）.Thymus index（TI）,spleen index（RBC）,white blood cell count（WBC）,platelet count PLT),hemoglobin（HCT）and hematocrit（HGB）were observed to compare the effect of blood enrichment in seven different extraction sites.Acute blood stasis model was established by subcutaneous injection of epinephrine hydrochloride combined with ice water bath.Plasma viscosity（PV）,whole blood viscosity（WBV）,prothrombin time（PT）,thrombin time（TT）APTT and fibrinogen（FIB）were used as the observation indexes to compare the effect of activating blood circulation in seven different extraction sites.The data were first processed by SPSS 20.0 statistical software,expressed as`x±S,and then the fuzzy matter-element model based on the coefficient of variation coefficient was used for comprehensive evaluation.2.Using column chromatography,preparative liquid method to separate and purify the chemical components of the selected active site,the structure of the compound was analyzed by physicochemical properties and spectroscopic data(¹H-NMR,¹³C-NMR,DEPT,etc.).The fingerprint of the active site was established by UPLC-MS.Some of the main chemical components were identified by the control samples,based on the molecular weight of the known compounds other compounds.At the same time,the UHPLC multi-component was used to establish the quantitative analysis method of multi-component（ferulic acid,senkyunolide I,senkyunolide H,ferulic acid coniferyl ester,Z-n-butyl phthalide,ligustilide,Z-butenylphthalide,and angelica lactone A）in effective part.Results1.laxative cathartic experiment:Compared with the normal control group,the model group for the first row of black stool time extension,6 h reduced the number of stool,fecal weight,carbon reduction rate decreased,intestinal weight was significantly reduced,the difference was statistically Significance（p<0.05）.Compared with the model group,part 1,part 2,part 3,part 5 and part 6 significantly shortened the first discharge time,increased the number of stool and stool weight,the difference was statistically significant（p<0.05）;part 1,Part 2,part 5 and part 6 could significantly promote the small intestine movement,enhance the propulsive peristalsis of the small intestine,increase the length of the carbon end and increase the carbon advancing rate,the difference was statistically significant（P<0.05）;part 1,part 2,Part 3,part 5 and part 6 increased significantly（P<0.05）.The maximum value of patch progress calculated by fuzzy matter-element model was 0.70（part 2,70%ethanol impregnation part）.2.Blood enrichment experiments:Compared with the normal control group,the thymus index of the model group was significantly lower（p<0.01）,the spleen index was significantly increased（p<0.01）,the RBC,WBC,PLT,HCT and HGB were significantly decreased（P<0.001）,Compared with the model group,the positive group（p<0.01）,part 1（p<0.05）increased thymus index in mice;positive group and parts 1-7 can significantly reduce the mouse spleen index（P<0.01）.The RBC,WBC,PLT,HCT and HGB in the positive group and the Angelica sinensis group were all increased to some extent,especially in the Angelica sinensis water group and the immersion group（P<0.01）Of the 7 sites,the maximum value of the paste progress calculated by the fuzzy matter-element model was 0.50（part 2,70%ethanol-impregnated part）.3.Blood circulation experiments:Compared with normal control group,the WBV and PV in the model group were significantly increased（P<0.01）,the PT,APTT and TT time shortened,and the differences of APTT and TT were statistically significant（P<0.01）,and the FIB was significantly higher（P<0.05）.Compared with the model group,the TT of the part 1and the part 2 were significantly increased（p<0.05）,and the difference was statistically significant（p<0.01）.The maximum value of patch placement calculated by the fuzzy matter-element model was 0.527（part 2 and 70%ethanol-impregnated part）.4.By isolated and purified,a total of 20 compounds were obtained,and the structures of16 compounds were finally confirmed.They were 3（R）,8（S）-falcarindiol（1）,angelica lactone A（2）,7-hydroxy-6-methoxycoumarin（3）,ethyl caffeate（4）,ferulic acid（5）,4β,10a-Aromadendranediol（6）,a-spinasterol（7）,4-hydroxy-acetophenone（8）,Linoleic acid（9）,linoleic acid triglyceride（10）,linoleic acid methyl ester（11）,β-sitosterol（12）,daucosterol（13）,vanillin（14）,2-n-Propylphenol（15）,Tokinolide B（16）.5.The identification of the compounds in the active site by UPLC-MS was based on the comparison of the molecular weight of the reference substance with other known compounds and related references to confirm the basis of the pharmacodynamic substance represented by the 12 compounds.Under the optimized chromatographic conditions,ferulic acid,senkyunolide I,senkyunolide H,ferulic acid coniferyl ester,Z-n-butyl phthalide,ligustilide,Z-butenylphthalide,and angelica lactone A showed good linearity in the range of0.00174⁰.0348,0.00958⁰.1916,0.0016⁰.032,0.00646⁰.1292,0.00326⁰.0652,0.00842⁰.1648,0.01036⁰.2072,0.00766⁰.1532 mg/m L,respectively RSD of repeatability and stability were less than 3%.Through the above experiment,we conclude that Angelica blood,promoting blood circulation,laxative effective part is 70%ethanol impregnateded.The structures of 16compounds were identified from the active sites,among which 4β,10a-Aromadendranediol（6）,4-hydroxyacetophenone（8）,2-propylphenol For the first time isolated from the Angelica.The established qualitative and quantitative analysis method is accurate,high sensitivity.It can provide experimental basis for more comprehensive control of the quality of Angelica sinensis.