| Chinese medicine is China's cultural treasures.There is a long history of the use of traditional Chinese medicine for the treatment of various diseases.However,with the continuous development of modern technology and the introduction of Western medicine,the diversity ingredients hindered Chinese medicine's process of internationalization.Chinese medicine has complex diversity of ingredients which are still not clear of the principle of operation.In order to implement the intermationalization of Chinese medicine,we need to build on the basis of its quality control standards desiderately.While in the process of the establishment of Chinese medicine quality control standards,the research of Pharmacodynamic substance basis is indispensable which can help reveal the specific efficacy ingredients of traditional Chinese medicine.Cistanche deserticola is a classic tonic Chinese herbs,with a reputation of "Desert Ginseng",which found to have the phytoestrogen effect in recent study.Phytoestrogen may adjust the balance level of estrogen in the female body.This study focuses on the estrogenic effect of C.deserticola.During the study,first we screen the active site of the quasi estrogen activity of C.deserticola through the combination of whole animal experiments and in vitro experiments,by using the mouse uterine weight gain test and MCF-7 cell proliferation experiment.Followed by high performance liquid chromatography tandem mass spectrometry?HPLC-MS?techniques,we analysis the ingredients in the serum,urine,feces of rats and the serum of rats in different physiological state which gavaged with the quasi estrogen active site of C.deserticola.Thus,the results can help to reveal the material basis for the quasi estrogen activity of C.deserticola.Here are the main contents and results:In the first part,we screened the different extracts and different doses of C.deserticola,combined with the mouse uterine weight test and MTT cell proliferation assay.The results show that 95%ethanol extracts of C.deserticola,dose of 30g·kg-1·d-1 group,can significantly improve the immature mouse uterine factor?P<0.01?,and the drug serum also can significantly promote the proliferation of MCF-7 cells?P<0.01?.Thus we conclude that 95%ethanol extracts,30g·kg-1·d-1?crude medicine material?as quasi estrogen active site.This results lay the foundation for further study.In the second part of the study,high performance liquid chromatography tandem mass spectrometry?HPLC-MS?was used to analysis the drug-contained serum of normal female rats after gavaged with the liquid of quasi estrogen effect sites of C.deserticola.We marked 13 chromatogiphic peaks in the serum,and finally idetified 13 chemical constituents based on the MS information and parts of the standard substance information,including 6 prototype components:Geniposidic acid,Salidroside,Cistanoside A,Echinacoside,Acteoside,Cistanoside D;7 metabolites components:3,4-Dihydroxyphenylethanol glycosides,methylation acteoside,acetylation Cistanoside G,3,4-Dihydroxyphenylethanol,acetyl tubuloside B,Cistanoside B glucuronide metabolite and methylation-3,4 Dihydroxyphenylethanol.In the third part,We compared and analyzed the total ion mass spectrometry of drug-contained serum of rats in different physiological conditions after gavaged with the liquid of quasi estrogen effect sites of C.deserticola.The differences between the three groups?normal group,estrogen deficiency group,estrogen excess group?,obtained from MS analysis,containing 4 common peaks:Cistanoside A,Echinacoside,Acteoside,Cistanoside B glucuronic acid conjugates;in estrogen deficiency group,the unique composition may be:methyl-O-salidroside,hydroxylation echinacoside,tubuloside A,methylation 8-epiloganic acid.This part of the research can provided the material basis for the two-way adjustment of C.deserticola,s phytoestrogen effect.In the fourth part of the study,high performance liquid chromatography tandem mass spectrometry?HPLC-MS?was used to analysis the drug-contained urine of normal female rats after gavaged with the liquid of quasi estrogen effect sites of C.deserticola.We mark 15 chromatogrphic peaks in the urine sample,and finally idetified 15 chemical constituents based on the MS information and parts of the standard substance information,including 7 prototype components:Geniposidic acid,8-epiloganic acid,salidroside,syringin A3,-?-L-rhamnopyranoside,echinacoside,acteoside,2'-acetyl acteoside;8 metabolites components:Caffeic acid glucuronide conjugation,Homovanillic acid glucuronide conjugation,Homovanillic alcohol glucuronide conjugation,Homovanillic alcohol sulfate conjugation,Salidroside glucoside glycine metabolites,Hydroxytyrosol glucuronide conjugation,Geniposidic acid hydrolyzation,acetylation caffeic acid.In the fifth part of the study,high performance liquid chromatography tandem mass spectrometry?HPLC-MS?was used to analysis the drug-contained feces of normal female rats after gavaged with the liquid of quasi estrogen effect sites of C.deserticola.We marked 14 chromatogrphic peaks in the feces samples,and finally idetified 14 chemical constituents based on the MS infornation and parts of the standard substance information,including 5 prototype components:Decaffeoylacteoside,Echinacoside,Acteoside,Cistanoside D,Cistanoside G;9 metabolites components:Acetylation Cistanoside F,Caffeic acid,Salidroside sulfate conjugation,lugrandoside,Methyl syringin A3'-?-rhannopyranoside,Methyl caffeic acid sulfate conjugation,3,4-Dihydroxyphenylethanol,Cistanoside B sulfate conjugation,Methyl 3,4-dihydroxyphenethyl glycoside.In the research above,we determine the estrogenic effect active site of C.desericola,combined with liquid chromatography-mass spectrometry techniques to analyze and identify the ingredients in the drug-contained serum,urine,feces of rats.There is important scientific value to direct the clinical use of C.deserticola,at the same time,the results is of great significance to the medicinal material basic research. |
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