Screening Of The Anti-tumor,Anti-oxidation And Hypolipidemic Synergistic Compounds From Paeoniae Radix Rubra Based On Multi-level Interaction Network

Paeoniae Radix Rubra is a dried root of the plant Paeoniae lactiflora Pallas and Paeoniae veitchii Lynch,which exerts a variety of biological and pharmacological activities,such as anti-tumor,anti-oxidation and hypolipidemic.However,its major pharmacodynamic compounds and the related mechanisms of action have been still unclear.Since research reports on the interaction between various pharmacological mechanisms have been lacking at present,this study constructed a multi-level interaction network of Paeoniae Radix Rubra compounds-targets?biomolecules?-three diseases?tumor-related diseases,oxidation-related diseases and blood lipid-related diseases?using a network pharmacology approach.This network was applied to identify the potential compounds,the combination of which could reveal synergistic therapeutic effects in terms of anti-tumor,anti-oxidation and hypolipidemic.Subsequently,these representative compounds were assessed to verify the synergistic activities.Here in this dissertation contained five chapters to demonstrate the construct of the multi-level interaction network and related verification experiments.Part?.ReviewThe aim of this chapter is to present an overview of the history and research progress of Paeoniae Radix Rubra,and to give an overview of the various approaches to predict targets and the network pharmacology research.First,this review compared the pharmacological effects of the compounds that have been uncovered in Paeoniae Radix Rubra to pave the way for the identification of active compounds.Then the prediction approaches that were developed based on the structures of compounds and targets were collected and discussed.Lastly,the review presented an overview of progress and problems in network pharmacology in terms of data sources,network construction and network analysis.To sum up,this chapter showed promising techniques for identify the therapeutic targets and bioactive compounds in Paeoniae Radix Rubra.It would provide theoretical,data and technical basis for this research and provide a reference for the smooth progress of this research.Part?.Data collection and organizationIn this chapter,the comprehensive biomedical data of the Paeoniae Radix Rubra were partly extracted from the established databases,and some of the data were collected from relevant text mining.Finally,109 compounds that were isolated from the Paeoniae Radix Rubra were collected,and these compounds covered a variety of disease targets and drug-target interactions,including 51 compounds with targets,390compound targets,946 pairs of compound-target interactions,168 tumor-related diseases,459 biomolecules?targets?related to tumor-related diseases,657 pairs of tumor-related disease-biomolecule?target?interactions,36 oxidation-related diseases,90 biomolecules?targets?of oxidation-related diseases,135 pairs of oxidation-related disease-biomolecule?target?interactions,40 blood lipid-related diseases,80biomolecules?targets?of blood lipid-related diseases,102 pairs of blood lipid-related disease-biomolecule?target?interactions and 60079 pairs of protein?target?-protein?target?interactions.The data collection provides the data base for the study.Part?.Extended prediction of Paeoniae Radix Rubra interaction dataThe established network was used to predict the interactions of the Paeoniae Radix Rubra compound-target and three diseases?tumor-related diseases,oxidation-related diseases and blood lipid-related diseases?-biomolecules?target?.On the basis of simple prediction,a dataset was obtained,including 60882 pairs of new compound-target interactions,12339 new compound targets,39399 pairs of new tumor-related disease-biomolecule?target?interactions,10706 new tumor-related disease biomolecules?targets?,5739 pairs of new oxidation-related disease-biomolecule?target?interactions,2479 new related to oxidation-related disease biomolecules?targets?,1259 pairs of new blood lipid-related disease-biomolecule?target?interactions,891 new related blood lipid-related disease biomolecules?targets?.Subsequently,923 targets corresponding to 42 Paeoniae Radix Rubra compound were predicted through the multi-compound-multi-target interaction prediction model,and 2060 pairs of new compound-target interactions were obtained.Through predictive expansion,the integrity of the Paeoniae Radix Rubra network was improved.Part?.Construction and Analysis of the Multi-level Interaction Network of Paeoniae Radix RubraA multi-level interaction network based on known Paeoniae Radix Rubra biomedical data and extended predicted data was constructed.In comparison with the basic network,the expanded network was much larger,showing more nodes and edges,and more perfect interactions.This result indicated that our expanded network can potentially boost the prediction accuracy and provide new insights into drug-target interactions.The expanded network was analyzed by the MCODE algorithm using Cytoscape software,and 15 functional clusters with scores greater than 3 were obtained in which 9 clusters related with Paeoniae Radix Rubra compounds and three types of diseases were identified.Subsequently,the relevant pathways involved in the targets of the compounds and every compound in the identified 9 clusters were further analyzed by KEGG Mapper.Taking into account the pathways which simultaneously regulated three diseases and which could be treated more types of diseases,and the number of diseases pathways related to a certain type of disease related pathways,the bioactive compounds were gallic acid,propyl gallate and naringenin,showing the potential effects on the treatment of three diseases?tumor-related diseases,oxidation-related diseases and blood lipid-related diseases?.Part?.Experimental validation of the predictionsIn this study,the anti-tumor,anti-oxidation and hypolipidemic pharmacological activities of gallic acid,propyl gallate and naringenin were verified experimentally.The high-performance liquid chromatography?HPLC?method of gallic acid,propyl gallate and naringenin was established and methodological investigation was did.Results proved that the HPLC method for the simultaneous measurement of gallic acid,propyl gallate and naringenin,was linear over a concentration range of of 5-250?g/mLwith less than 2%precision and accuracy levels.Using this HPLC method,the concentrations of three compounds per gram of Paeoniae Radix Rubra were 1.867 mg of gallic acid,0.561 mg of propyl gallate and 0.093 mg of naringenin,respectively.Taking galanthenic acid,propyl gallate,and naringenin as reference,in vitro anti-tumor results demonstrated that either single compound,or a combination of three compounds,or extract from Paeoniae Radix Rubra could exert somewhat cytotoxicity on cancer cells.Compared to that of Paeoniae Radix Rubra extract,a combination of three compounds had a lower IC₅₀0 value of 89.38?M,showing a better anti-tumor activity.In addition,cellular results revealed the differentiated anti-tumor abilities of single compound,that is,IC₅₀0 levels were 85.10?M for naringenin,275.29?M for gallic acid and 394.55?M for propyl gallate,respectively.The anti-oxidation results also demonstrated that all samples,including single compound,a combination and Paeoniae Radix Rubra extract,were effective to remove oxidants.As a result,a combination of three compounds exerted the best anti-oxidation activity,showing that 50%of oxidant DPPH,was cleared at a low concentration of 0.6?g/mL.However,single compound should increase its concentration to achieve the same clearance rate,confirming the synergistic anti-oxidation effect of three-compound combination.In addition,50%clearance rate were 4.7?g/mL for naringenin,1.7?g/mL for gallic acid,2.0?g/mL for propyl gallate and 16.8?g/mL for Paeoniae Radix Rubra extract,respectively.Additionally,hypolipidemic effects of these samples were evaluated.All of these groups appeared to have a blood lipid lowering effect.However,no significant difference was observed between the three-compound combination group and positive control group.It could be seen that gallic acid,propyl gallate,and naringenin have synergistic anti-tumor,anti-oxidation,and hypolipidemic effects.In this study,three representative compounds were obtained by screening of the anti-tumor,anti-oxidation and hypolipidemic synergistic compounds from Paeoniae Radix Rubra based on multi-level interaction network and verified its effectiveness.The results showed that gallic acid,propyl gallate,and naringenin have synergistic anti-tumor,anti-oxidation and hypolipidemic pharmacological effects.The results of this study will provide a reference for the study of Chinese Materia Medica"one drug multi-effect"and quality control.