| Hyperplasia of Mammary Gland(HMG)is a common clinical gynecological disease.The main symptoms are breast pain,nodules or masses.The prescription of Shuangjin Sanjie Granules(SJSJG)comes from the clinical experience of the chief physician of the famous old Chinese medicine doctor Qin Yuehao.It is mainly composed of 12 traditional Chinese medicines such as radix bupleuri,radix paeoniae alba,radix angelica sinensis,radix curcμmae,membrane of chicken gizzard,bulbus fritillariae thunbergll and herba taraxaci.The composition is based on the principle of "soothing the liver and regulating qi,activating blood and freeing the network vessels,transforming phlegm and dissipating binds",and has a good therapeutic effect on HMG with "liver depression and qi stagnation,phlegm and static blood binding together".In order to prepare it into a granule formulation more suitable for clinical application,this topic combined with pharmacodynamic research to screen the extraction process route of SJSJG,and carried out research on its preparation process.At the same time,the use of network pharmacology research to predict the mechanism and material basis of SJSJG in the treatment of HMG,combined with UHPLC-LTQ-Orbitrap MS and pharmacodynamic experiments of HMG SD rats to conduct preliminary verification of the prediction results,with a view provide experimental basis and reference for the research and development and clinical application of TCM and SJSJG.Objective:Taking SD rats with HMG as the efficacy evaluation model,the extraction process of SJSJG was screened,and then the extraction,purification and molding process parameters were optimized to determine the scientific and reasonable preparation process.Based on network pharmacology research combined with UHPLC-LTQ-Orbitrap MS technology and HMG SD rats pharmacodynamic experiments,the material basis and mechanism of SJSJG in treating HMG were initially explored.Method:(1)Study on preparation technology of SJSJG:Exogenous intramuscular injection of estradiol benzoate and progesterone injection to construct a rat model of hyperplasia of the breast.Using the breast tissue pathological sections as evaluation indicators,the extraction process of SJSJG was screened.Taking the transfer rate of paeoniflorin as the inspection index,the orthogonal extraction design method was used to optimize the extraction process parameters of the prescription;further,the purification,concentration and drying process parameters were optimized;the optimum molding process is determined based on the particle molding rate,solubility and other indicators,such as the concentration of wetting agent and the type and ratio of auxiliary materials.(2)Network pharmacological study of SJSJG:The BATMAN-TCM database was used to collect the chemical components in SJSJG and the target proteins that may interact with them.The disease targets related to HMG were collected through the GeneCards database and the OMIM database.The clusterProfiler package was used for DO enrichment analysis with the targets related to HMG and JUN CHEN ZUO SHI.And the KOBAS web server was used to perform GO biological processes and KEGG pathway enrichment on the direct targets of SJSJG for HMG analysis.(3)Material basis research of SJSJG:UHPLC-LTQ-Orbitrap MS technology was used to detect the main chemical constituents of SJSJG,and the basis of their pharmacodynamic substances was analyzed by combining with previous experimental data.(4)Validation of SJSJG in treating rats with HMG:Based on the network pharmacology research,using the samples of the drug efficacy test in the process screening,the content of estradiol(E2),progesterone(P)and prolactin(PRL)in serum,and the estrogen receptor a(ERa),androgen receptor(AR),progesterone receptor(PR)and tumor necrosis factor-a receptor(TNF-α)protein expression levels in breast tissues were detected,preliminary verification of network pharmacology prediction results.Result:(1)Study on preparation technology of SJSJG:The results of animal model evaluation experiments showed that after treatment of the rats with HMG,the abnormal hyperplasia of mammary gland tissues in all administration groups was weakened.The number is the smallest(3/8),and the number of cases of breast acinar becoming smaller or atrophy is the largest(4/8),so that the prescription extraction process is determined by full water extraction.And the best extraction process for pharmaceutical research was the whole prescription were extracted with 12 folds of water for 3 times,1 hour for each time.Subsequently,it was concentrated to a relative density of 1.1(60℃)by means of separate concentration,and vacuum dried at 80℃ to a dry paste.The dry paste powder and dextrin were mixed uniformly at a ratio of 1:1 to 1:1.25,and 90%ethanol was used as a wetting agent to prepare granules by wet granulation.(2)Network pharmacological study of SJSJG:Network pharmacology research collected 215 prescription-related chemical components,corresponding to 697 targets,and 459 protein targets related to HMG,including 50 direct treatment targets.The results of KEGG enrichment analysis showed that there were 20 enrichment results that played an important role in the occurrence and development of breast hyperplasia.DO enrichment analysis results shown the results that the common DO entries of the HMG and JUN CHEN ZUO SHI targets are clustered into 4 categories,which are Endocrine disorders related diseases;Abnormal hyperplasia related diseases;Qi stagnation syndrome and Blood stasis syndrome.GO biological process and KEGG pathway analysis results of direct treatment targets indicated that SJSJG may affect cell proliferation and apoptosis by regulating the endocrine system and inflammatory response in the body,and have a therapeutic effect on HMG.(3)Material basis research of SJSJG:The results of basic material research showed that 85 chemical components were identified from SJSJG,of which 16 were alkaloids,7 were flavonoids,15 were terpenes,and 9 were phenolic acid compounds,3 coumarin compounds,10 esters and lactone compounds,7 fatty acids compounds,4 amino acids compounds,and 14 other types of ingredients.Verticinone and peimine were detected in the positive ion mode,Saikosaponin A,D and paeoniflorin were detected in the negative ion mode.It was speculated that Verticinone and Peimine were from bulbus fritillariae thunbergl,Saikosaponin A,D were from radix bupleuri and paeoniflorin were from radix paeoniae alba.These ingredients are closely related to the efficacy of SJSJG.(4)Validation of SJSJG in treating rats with HMG:The results showed that compared with the normal group,serum E2 significantly increased(P<0.01);ERα,AR,PR,and TNF-α increased expression;compared with the model group,serum E2 was decreased significantly(P<0.01);ERα,AR,PR,and TNF-α protein expressions were significant decreased(P<0.01)).Conclusions:Based on the research of pharmacodynamics,this subject screened out the extraction process route of SJSJG,and further established the extraction,purification and molding process parameters of SJSJG.The determined process is reasonable and feasible,which lays the foundation for the subsequent development of new Chinese medicines.The combination of network pharmacology research and UHPLC-LTQ-Orbitrap MS and pharmacodynamic experiment of HMG SD rats found that the treatment of HMG with SJSJG is a process of multiple targets and multiple pathways.The mechanism is mostly related to hormones and inflammatory pathways in the body,and it is speculated that the alkaloids and terpenes may be closely related to the effect of the drug,which may provide a reference for the clinical application of SJSJG. |
PDF Full Text Request