Research On Pharmacodynamic Material Basis Of Danggui Shaoyao San In The Treatment Of Primary Dysmenorrhea

Primary Dysmenorrhea（PD）was a common gynecological disease accompanied by spastic pain in the lower abdomen,nausea,vomiting,dizziness,diarrhea and other symptoms etc.Although western medicine was effective in treating PD,it was easy to cause adverse reactions and drug resistance,so more effective treatment drugs are urgently needed.Traditional Chinese medicine（TCM）had unique theoretical and experience in the treatment of dysmenorrhea-related diseases,and had become an important direction in drug development in recent years.Danggui Shaoyao San（DSS）from Synopsis of Golden Chamber written by Zhang Zhongjing,was a classic Chinese medicine prescription for dysmenorrhea,with definite efficacy and no adverse reactions.Classic formulas was used to explore the pharmacodynamic material basis of TCM,and develop new drugs,which had become a hot spot.In this study,based on the primary dysmenorrhea-related rat model induced by estradiol benzoate and oxytocin,the biological characteristics of the model and the effectiveness of DSS were evaluated from the perspectives of rat behavior,histopathology,clinical biochemical indexes and metabolomics,and the potential pharmacodynamic material basis of DSS for the treatment of PD were discovered with the help of Chinmedomics,in order to lay an experimental and theoretical foundation for the development of new drugs for the treatment of PD.Objective:The metabolic markers and metabolic pathways of PD were revealed,the pathogenesis of PD and the effectiveness of DSS in the treatment of PD were elucidated from the metabolic level,and the potential pharmacodynamic material basis of DSS in the treatment of PD were discovered,laying a foundation for the discovery of new drugs for the treatment of PD.Methods:1.The PD rat model was replicated by estradiol benzoate combined with oxytocin,and the model was evaluated by weight,twisting reaction and twisting latency,clinical biochemical indexes and histopathology.The UPLC-Q-TOF-MS technology was used to establish a metabolomic analysis method for PD rat model,analyze the urine and blood metabolic profiles of model rats,identify differential biomarkers and related pathways,and finally clarify its pathogenesis.2.After administration,the changes in body weight,behavior,hormone levels and tissue morphology of PD model rat were determined.The effects of DSS on metabolic profiles,metabolic markers and pathways were analyzed by metabolomics technology.3.Based on the method of Chinmedomics,the components of DSS were characterized by UPLC-Q-TOF-MS technology,and the blood components from DSS were analyzed and identified with the help of pattern recognition method.The potential pharmacodynamic material basis of DSS for the treatment of PD was verified by associating the active substances in DSS with biomarkers.Results:1.Replication and evaluation of PD model ratsThe number of twists in PD rats was significantly increased,the incubation period of twisting was significantly shortened,weight gain was slow,severe exfoliation and edema of the endometrium appeared,the content of pain factor E₂,ET,PGF_2aand PGE₂ was significantly increased,and the content of Prog andb-EP was significantly reduced.A metabolomic analysis method for PD model rats based on UPLC-Q-TOF-MS was established.41 urine biomarkers related to PD were identified,mainly including kynurenic acid,hippuric acid,18-hydroxycorticosterone,12-hydroxydodecanoic acid,dodecanedioic acid,azelaic acid,sebacic acid,etc.,mainly involved in phenylalanine metabolism,arachidonic acid metabolism,steroid hormone biosynthesis,pentose and glucuronate interconversions.Meanwhile,29 serum biomarkers including citric acid,docosapentaenoic acid,2-methylhippuric acid,L-tyrosine,cholesterol,etc,were identified,mainly involving in citrate cycle,primary bile acid biosynthesis,glycerophospholipid metabolism,phenylalanine,tyrosine and tryptophan biosynthesis pathways.2.The evaluation of the effectiveness of DSSAfter DSS intervention,the number of twisting times in rats was significantly reduced,the incubation period of twisting was prolonged,the weight was reversed,the degree of endometrial peeling and epithelial edema was significantly reduced,the content of pain factors E₂,ET,PGF_2aand PGE₂ was significantly reduced,and the content of Prog andb-EP was significantly increased.After DSS intervention,36 urine biomarkers and 22 blood biomarkers were recalled by affecting arachidonic acid metabolism,primary bile acid biosynthesis,phenylalanine and tyrosine metabolism,glycerophospholipid metabolism,and steroid hormone biosynthesis pathways,and adjusting the metabolic trajectories of markers such as hippuric acid,phenylacetaldehyde,prostaglandin G2,6-hydroxy-5-methoxyindole glucuronide,L-tyrosine,glycocholic acid,citric acid,cholesterol,etc.3.Study on pharmacodynamic material basis of DSSA component analysis method for DSS based on UPLC-Q-TOF-MS technology was established,and the mass spectra of in vitro samples and drug-containing serum and control serum of DSS were acquired.Referring to the identification results of the external chemical components of DSS,a total of 22 blood components were characterized,namely cianidanol,jasminoside B,levistilide A,poricoic acid A,isovalerophenone,griffonilide,alisol C,23-acetate and alisol B,respectively acetate,atractylenolide III,Z-ligustilide,sedanolide,paeoniflorin,senkyunolide I,alisol F,ferulic Acid,gentisic acid5-O-glucoside,4-hydroxy-3-（3-methylbut-2-enyl）benzoic acid,ethyl gallate,dehydrotumulosic acid,chlorogenic acid,albiflorin,alisol A.Only when the number of active components of DSS in vivo extremely correlated with disease biomarkers reached more than 5,can we consider it as the potentia pharmacodynamic material basis.Z-ligustilide,levistilide A,chlorogenic acid,paeoniflorin,poricoic acid A,sedanolide,senkyunolide I,cianidanol,isovalerophenone,griffonilide,alisol B acetate,alisol F and jasminoside B can be considered as the pharmacodynamic basis of DSS for the treatment of PD.Conclusions:In this study,DSS can effectively reduce the metabolic disorder of rats with PD.The main target of its intervention was to regulate hippuric acid,phenylacetaldehyde,prostaglandin G2,6-hydroxy-5-methoxyindole glucuronide,lyso PC,glycocholic acid,cholic acid,tyrosol glucuronide,which affected key pathways such as primary bile acid biosynthesis,steroid hormone biosynthesis,phenylalanine and tyrosine metabolism pathways.On the basis of the effectiveness of DSS in the treatment of PD,the 22 blood components of DSS were characterized by UPLC-Q-TOF-MS,it was found that Z-ligustilide,Levistilide A,chlorogenic acid,paeoniflorin,poricoic acid A,sedanolide,senkyunolide I,cianidanol,isovalerophenone,griffonilide,alisol B acetate,alisol F and jasminoside B could be used as the potential pharmacodynamic material basis of Danggui Shaoyao San.