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The Study On The Chemical Constituents Of Polygala Japonica Houtt. And Exploration In Targets Action Of Cancer Based On Network Pharmacology Method

Posted on:2021-03-29Degree:MasterType:Thesis
Country:ChinaCandidate:J MinFull Text:PDF
GTID:2404330611495988Subject:Pharmaceutical
Abstract/Summary:PDF Full Text Request
The Polygala japonica Houtt.is a material of the tradional chinese medicine,widely used in the folk.According to the Chinese Materia Medica,it is used to dispel phlegm and cough,promote blood circulation and reduce swelling.Anti-inflammatory,anti-tumor,anti-depression are reported by modern medical research.In the research of the medicine already prepared by a pharmacy,we found that most of the unqualified medicinal materials of the Polygala japonica Houtt.were still widely applied in the market,and the users responded that the effect of pharmacology was very good.Therefore,it is of great significance for the development and utilization of the Polygala japonica Houtt.to make a thorough and comprehensive study on its basic source,composition and efficacy.Objective:To establish the HPLC and UPLC-Q-TOF-MS for the multi-component analysis of the Polygala japonica Houtt.Based on the analysis and comparison of several batches of materials,the antitumor targets of its were predicted according to different chemical components,and then the components of the Polygala japonica Houtt.were separated and purified.Method:(1)According to the current quality standard of the Polygala japonica Houtt.,12 batches medicines purchased from the market were tested.At the same time,in order to analyze more components of the Polygala japonica Houtt.,a HPLC method for simultaneous determination of three saponins(polygalasaponin V,polygalasaponin F,polygalasaponin XXXI)was established by selecting all kinds of preparation methods and chromatographic conditions of the test solution.The material was prepared by the ultrasonic extractde in the methanol,with C18 bonded silica gel as the stationary phase and acetonitrile:methanol(90:10)as mobile phase A;0.3% Formic acid as mobile phase B,gradient elution and ELSD as detector.The method was used to test 12 batches of the Polygala japonica Houtt.The principal component analysis(PCA)of cluster analysis was used to compare the content distribution of three saponins in different parts of medicinal materials.(2)UPLC-Q-TOF-MS was used to analyze the chemical structures and recognized its chemicals of the two kinds of medicinal materials,which were the representative batch numbers of 170110 and 180306.The stationary phase was C18 column,mobile phase a was acetonitrile methanol(90:10),mobile phase B was acetonitrile methanol(90:10)0.3%Formic acid,gradient elution,mass spectrometry condition is electrospray ion source(ESI),positive and negative ion mode monitoring,data acquisition range m/z is 300-2000.(3)Chemraw and Schrodinger 2018 software were used to describe the molecular information of medicinal ingredients,and the pharmacodynamic groups of the compounds were predicted by comparing with Lipinski rule,systematic pharmacology and the required principles of drug-making in the references.At the same time,the protein molecules in the process of tumor genesis and formation were searched.PPI network analysis,go and KEGG enrichment analysis were used to predict the interaction sites,functions and pathways of the compounds.(4)A method for the separation of components of two kinds of medicinal materials,the Polygala japonica Houtt.,was established.50%ethanol was used for ultrasonic extraction,75% ethanol and 90% ethanol were used for hierarchical extracts,and then the multi-solvent washing(petroleum ether,dichloromethane,ethyl acetate,water saturated n-butanol)was carried out in turn.The ethyl acetate extraction liquid and water saturated n-butanol extraction liquid were used to separate the chemical components of the herbs by macroporous resin column chromatography.At the same time,the components of the products in the process of extraction,purification and separation were analyzed by TLC and HPLC in method 1.Result:(1)According to the current quality standard of the Polygala japonica Houtt.,only 4 of 12 batches of the content polygalasaponin F were qualified.At the same time,according to the different concentration of retention time in the liquid chromatography,the herbs were divided into two categories: the first category was the main components with retention time of 5-35 min,and the second category was the ones with retention time of 40-68 min.The new HPLC method was verified to be accurate and feasible by methodology.Among the three saponins determined by this method,the content of polygalasaponin F is consistent with that determined by the current quality standard method of the Polygala japonica Houtt.The results showed that the content and distribution of three saponins in different parts of medicinal materials were the contents of polygalasaponin F.and polygalasaponin V as follows leaf > stem > root,and pplygalasaponin XXXI as follows root > leaf > stem.(2)The results of liquid analysis of two kinds of medicinal materials are as follows: the first kind of medicinal materials mainly contains 31 compounds,including 18 saponins(polygalasaponin V,polygalasaponin F,polygalasaponin XXXI,polygalasaponin II,astragalin,tenuifolin,etc.),6flavones(polygalin C,polygalin B,etc.),4 glycolipids and 3 xanthones.The second kind of medicinal materials mainly contain 30 compounds,including 10 saponins(polygalasaponin XXXI,polygalasaponin E,polygalasaponin J,polygalasaponin XXI),10 glycolipids(tenuifoliose F,tenuifoliose A,tenuifoliose L),6 flavonoids(polygalin C,polygalin B,etc.),3 xanthones and 1 phenylpropanoid.At the same time,we found that the retention time of saponins was generally shorter than that of flavonoids.In reversed-phase chromatography,saponins have higher polarity and lower affinity with stationary phase.Mobile phase can quickly elute these components,so the retention time is shorter.However,the retention time of flavonoids is longer because the polarity of mother nucleus is smaller and the affinity with stationary phase is better. (3)The anti-tumor targets of the two kinds of medicinal materials are predicted as follows: the first kind of medicinal materials have 19anti-tumor targets,such as MMP2,IGF1,kit,etc.19 target proteins affect186 Gene Ontology(go)biological processes,such as biological regulation,metabolism and stress,which are mainly enriched in chemokines,PI3 K Akt metabolic pathway,nod like receptor signaling pathway,etc.The second kind of drugs has 12 anti-tumor targets,such as kit,JAK3 and Src.12 target proteins affect the biological processes of 150 gene ontology go,such as biological regulation,cell signal transduction,mainly enriched in estrogen signal,cancer metabolic pathway,endocrine resistance receptor signal pathway,etc.The difference of the target protein and the activity of the two kinds of drugs are different.(4)Through the extraction,purification and separation of two kinds of medicinal materials,two purification products with more components were obtained.The two purified products are respectively 40%-80%ethanol eluate purified by macroporous resin from 180306 batches of water saturated n-butanol extract,mainly saponins;60%-80% ethanol extract purified by macroporous resin from 170110 batches of ethyl acetate.Mainly flavonoids.Conclusion:In this study,through the establishment of analytical methods,the analysis of ingredients,the prediction of anti-tumor targets and the preliminary purification and separation of ingredients,we can get a more comprehensive understanding of the chemical information of herbs.According to the different concentration of components in the retention time of the components detected in the liquid phase,the medicinal materials are divided into two categories.There are 21 components in the two kinds of medicinal materials,including polygalasaponin II,polygalin C,and 17 different components,including polygalasaponin X,polygalasaponin XXI and polygalasaponin E.Among the two kinds of medicinal materials,the structure of polygalasaponin XXI,polygalasaponin II,polygalin C and polygalin B are similar,which may have similar activity.At the same time,three kinds of saponins in different medicinal materials were determined.It was found that there were more polygalasaponin in the leaves,which might be related to different medicinal parts.To sum up,in the research of the Polygala japonica Houtt.,many components in the medicinal materials have pharmacological activities and can act on multiple targets.On the one hand,it reflects the anti-tumor characteristics of multi-component,multi activity and multi-target of cucurbit seed gold.This characteristic provides a new thought and method for the comprehensive study of the physicochemical characteristics of the Polygala japonica Houtt.On the other hand,this study shows that it is not rigorous to use polygalasaponin F as the content determination index for the quality control of medicinal materials.In this study,a more reasonable analysis method of medicinal materials components is proposed to provide reference for improving the quality standard.
Keywords/Search Tags:Polygala japonica Houtt., Chemical Compound, NetworkPharmacology, Therapeutic Targets
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