Study On Metabolism And Prediction And Analysis Of Network Pharmacology Of Berberis Amurensis

The research on the effective substances basis and mechanism of Traditional Chinese Medicines?TCMs?has always been a challenge for researchers due to its complexity.It is well-known that TCMs are often administered orally in the form of decoction,and oral drug usually need to across the gastrointestinal barrier before they can enter the blood circulation system.In a series of processes,not all ingredients of TCMs can enter the circulation system and exert therapeutic action.Therefore,the ingredients of TCMs that eventually enter the blood circulation are the potential substances for the pharmacological effects.In recent years,due to the development of analytical techniques,UPLC-Q-TOF-MS/MS has become an important technical means for analyzing multiple components and their metabolites in complex systems of traditional Chinese medicine because of its high-efficiency,high-sensitivity and rapid separation.With the development of bioinformatics,network pharmacology has become an important technical method studying the mechanism of TCMs.Berberis amurensis is a shrub of the Berberis genus in the Berberidaceae family.It has ornamental value and a wide range of pharmacological effects.Folks often use their roots and stems as medicines for the treatment of hypertension,inflammation,dysentery,enteritis,jaundice and other diseases.Modern pharmacological studies have shown that it could inhibit the growth of S180 sarcoma.However,its chemical composition and molecular mechanism are not yet clear.In this study,Berberis amurensis was used as the research subject,its pharmacological substance basis was initially clarified by studying its metabolism in vivo and in vitro,and network pharmacology analysis.The research content of this paper is mainly divided into the following three parts:1.UPLC-Q-TOF-MS/MS technology was used to analyze the chemical constituents of the Berberis amurensis extract and constituents migrating to blood.?1?UPLC-Q-TOF-MS/MS technology combined with Peakview/Masterview data processing method was used to analyze the chemical constituents of the Berberis amurensis extracts.A total of 33 compounds were isolated from extract,and 28 compounds were identified for the mass spectrum information of the database compounds as well as the reference substance,including 17 alkaloids,3 organic acids,2 iridoids,2 lignans,1 coumarin,1 glycolipid,1 nucleoside,1 glycoside compounds.?2?On the basis of identifying the chemical constituents of the Berberis amurensis,its constituents migrating to blood were analyzed in SD rats.We collected the rats'plasma samples after gavage of Berberis amurensis extract.Chromatographic column Synergi^TMFusion-RP100A?2.0 mm×100 mm,2.5?m?was used for gradient elution with 0.05%formic acid acetonitrile?A phase?—1 mmol/L ammonium acetate and 0.05%formic acid water?B phase?as mobile phase in positive ion mode.Peakview/Masterview software was employed to compare the mass spectrometric data of Berberis amurensis extract,blank plasma,and plasma after administration.Nine prototype compounds and ten metabolic compounds were identified from plasma after administration.The prototype compounds include Berberine,Jatrorrhizine,Adenosine,Palmatine,Eleutheroside E,Magnoflorine,Tetrandrine,Berberamine and Berberrubine.2.UPLC-Q-TOF-MS/MS technology was used to analyze the metabolism of the main components of Berberis amurensis in vitro and in vivo.?1?We established a pseudo-germ-free rat model using antibiotic cocktails at first.The difference of metabolites of major components between normal rats and pseudo-germ-free rats in urine,feces and bile were compared to elucidate the effect of intestinal flora on the metabolism of Berberis amurensis.As a result,46 metabolites were found in normal rats,while only 29 metabolites were detected in pseudo-germ-free rats,and there were no dehydrogenated metabolites in pseudo-sterile rats.The results of the study showed that the metabolism of Berberis amurensis in pseudo-germ-free rats with inhibited intestinal bacterial activity was changed.?2?The fecal incubation method was used to co-cultivate Berberis amurensis extract and normal rat's feces in an anaerobic environment at 37°C in vitro.The metabolites of the main components of Berberis amurensis after 24 hours incubation by intestinal flora were analyzed.As a result,7 metabolites were detected from the intestinal flora incubation solution.It shows that the intestinal flora could cause the biotransformation of the main components of Berberis amurensis.3.Using network pharmacology to explain the mechanism of anti-sarcoma of Berberis amurensis.Using the Chinese Medicine System Pharmacology Database?TCMSP?and the Swiss Target Prediction database to predict the core target of the blood constituents of Berberis amurensis,while collecting the targets of sarcoma-related diseases,based on the PPI protein interaction database for Network construction of compound-disease common targets,and then use the DAVID database to collect the channels related to the core target,and analyze the channel enrichment.The results of the study showed that there are 31 core target proteins for preventing and treating sarcoma,mainly involving TP53,GAPDH,CCND1,ESR1,FOS,RELA,AR,SMAD3,HSP90AA1,H1F1A,etc.These target pathways have cancer-related pathways?Such as pancreatic cancer,prostate cancer,small cell lung cancer,etc.?,cell growth and differentiation pathways?such as PI3K-AKt signaling pathway,cell cycle?,inflammation-related signaling pathways?such as hepatitis B,HTLV-I infection,measles,etc.?.In summary,on the basis of clarifying the chemical substances of Berberis amurensis,this study explained the intestinal flora's metabolic effect on Berberis amurensis through in vivo and in vitro metabolism experiments,and constructed the network of"blood component-target-pathway"to explain its mechanism of the anti-sarcoma action.It lays the foundation for the future pharmacological research and clinical use of Berberis amurensis.