| Cistanche deserticola mainly used to nourish kidney Yang,enrich blood and moistening intestines and bowel movement.The research group has confirmed that cistanche deserticola total glycosides;in the early(to gauge acteoside content is 62.5%)is the valid target of play estrogenic activity.Based on the previous work basis,the subject intends to establish a model of "intrinsic component-estrogenic activity" research model of spectrum-effect relationship,and reveal the material basis of cistanche deserticola total glycosides of play estrogenic effect.Through the establishment of cistanche deserticola total glycosides serum fingerprints and pharmacodynamic study of drug containing serum,build "intrinsic components-body composition-estrogenic activity" research model of spectral efficiency in vivo,reveals cistanche deserticola total glycosides play estrogenic effect of direct acting substance.Combination of internal and external spectrum-effect relationship results,reveals cistanche deserticola total glycosides play estrogenic effect of material basis,and on the basis of drug effect material for pharmacokinetic studies,reveals the play a role of quasi estrogen efficacy material base of process in the body.The main research contents and results are as follows:(1)Optimization of experimental conditions of MTT by orthogonal design,The selected cells were human breast cancer cells MCF-7,by L9(33)orthogonal experimental design,respectively investigate the number of cells plated(A)?dosage of serum containing medicine(B)and serum reaction time(C),optimization of the optimum experimental conditions.The experimental results show that the impact of various factors on the proliferation of MCF-7 cells in turn the role of serum reaction time>dosage of serum containing medicine>number of cells plated.Comprehensive intuitionistic analysis and variance analysis of the results,to determine the best combination of factors of MCF-7 cell proliferation is A2B3C2,the number of cells were plated for 1.5 x 104/hole,the dosage of serum containing medicine was 5%,the action time of the drug containing serum is 72 hours and the verification experiment was carried out.The test results show that the process is stable and feasible and has good repeatability.(2)The early stage of the project group analysis of the chemical constituents of the cistanche deserticola total glycosides by UPLC/Q-TOF-MS,43 compounds are demarcated in the total ion flow chart.Based on the previous work of the research group,by MCF-7 cell proliferation experiment and mouse uterine weight experiment.Inspected estrogenic effect of cistanche deserticola total glycosides,and establish the relationship between the 10 different areas of cistanche deserticola total glycosides each characteristic peak,correlation analysis of the spectrum-effect relationship of the cistanche deserticola total glycosides,through the bivariate correlation analysis and grey correlation analysis indicated that there are 10 components are related to the "mouse womb weight gain" experiment,20 components are associated with the "MCF-7 cell proliferation" experiment.(3)Analysis of components in the cistanche deserticola total glycosides by UPLC/Q-TOF-MS,Analysis by mass spectrometry and comparison with previous work and literature report,85 compounds are demarcated in the total ion flow chart,It includes 14 archetypal components and 71 metabolic components.(4)The fingerprints of 10 different blood sampling time points were established and 42 common peaks were calibrated.The relative retention time and relative peak area RSD of precision,repeatability and stability are less than 1%.The correlation between the common peaks of cistanche deserticola total glycosides and the proliferation of MCF-7 cells was analyzed at 10 different time points.Study on the correlation of the spectrum-effect relationship of the cistanche deserticola total glycosides.through the bivariate correlation analysis and grey correlation analysis indicated:A total of 38 chemical constituents of cistanche deserticola total glycosides play a direct role of estrogen in vivo substances.Among them,19 components were associated with in vitro metabolism,respectively:8-epiloganic acid,desmethyl(2E,6Z)-8-O-?-D-glucopyranosyloxy-2,6-dimethyl-2,6-octadienoic acid,methyl-O-salidroside,methyl-O-geniposide,methyl-O-decaffeoyl acteoside,syringalide sulfate conjugate,syringalide glucuronic acid conjugate,acetylation dehydrodiconiferyl alcohol-4-O-?-D-glucoside,dehydrodiconiferyl alcohol-4-O-?-D-glucoside,dehydrodiconiferyl alcohol-4-O-?-D-glucoside glucuronic acid conjugate,3,4-dihydroxy phenetidine,hydroxylated ononin,methyl-O-crenatoside,dehydroxylated crenatoside,demethylated crenatoside,hydroxylated crenatoside,methylated tubuloside B,bartsioside sulfate conjugate,acteoside.(5)To determine the content of syringalide,acteoside,salidroside and echinacoside in rats after oral cistanche deserticola total glycosides,and the metabolism kinetics was studied.The results showed that the method had good specificity and high sensitivity,and the precision,stability,recovery and matrix effect were all in accordance with the requirements of biological samples.Pharmacokinetic results indicated that syringalide,acteoside,salidroside and echinacoside were quickly absorbed in the body.The average peak time in plasma was 1.5 h,1.0 h,0.5 h and 1.0 h,the average peak concentrations were 123.79,96.28,99.30 and 110.52 ng/mL,and the AUC was 625.67.351.02,406.43 and 646.23 ng/mL*h,and the half-lives were 1.88 h,4.17 h,5.11 h and 2.24 h.It can be seen that they metabolize faster in the body.In summary,this study reveals cistanche deserticola total glycosides efficacy material base of play the role of estrogen,and reveal the effectiveness of material base for pharmacokinetic studies,to reveal the internal process of giving play to the substance basis of estrogenic effect,to provide a scientific and reliable theoretical basis for the development of the cistanche deserticola total glycosides.At the same time,it lays the foundation for the deep development of herba cistanche deserticola as the 5 new drugs of phytoestrogens. |
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