Study On The Active Components And Anti-Type ? Diabetes Function Of Arctium Lappa L.(Burdock)

Arctium lappa L.?Burdock?is rich in resources and widely distributed in the world.The burdock is not only a healthy and nutritive dietary supplement,it is also a folk herbal medicine with heat-clearing and detoxifying effect.Treatment of“Xiaoke”in traditional Chinese medicine is very similar to diabetes,and it will give us the new treatment to treat the diabetes.The study was based on the traditional medical theory combined with modern biotechnology.In this thesis,the research on analysis and isolation of active components,evaluation of antidiabetic function,prediction of targets and pathways,and metabolism of active ingredients in the body were applied.The purpose of the study is to improve diabetes through diet,and provide new references for the development of new specialty foods.The main results are indicated as follows:?1?The analysis results under different mobile phase,different wavelengths,different kinds of the columns and different extracts from burdock were investigated respectively.The results showed that the part of ethyl acetate is analogous to the part of ethanol,which will be used to further multicomponent separation in next step.Furthermore,Acetonitrile is better than methanol as a mobile phase,and analysis time can be shortened by 40 minutes.The optimal HPLC analysis conditions are acetonitrile-water?0.1%formic acid?system at 280 nm using a Gemini-NX C₁₈ column for analysis.?2?A new method based on DPPH-HPLC-DAD-Q-TOF-MS was used for the rapid screening and identification of free radical scavengers in burdock extract has been developed.The sample was divided into two parts after the first HPLC,one flowed to the second HPLC with the DPPH solution for on-line screening the antioxidants,the other flowed to Q-TOF-MS for on-line rapid identification.Nineteen kinds of caffeic acid and its derivatives were identified in negative ion mode,combined with accurate molecular weight,UV absorption,relevant literature and standard samples.This method is a reliable and powerful tool for rapid screening and identification of free radical scavenging compounds from complicated systems such as food and herbal extracts.?3?A combinative method of HSCCC and Pre-HPLC was developed for the caffeoylquinic acid derivatives from the burdock.The ethyl acetate extract of burdock was fractionated three fractions?Fr.1-3?by MCI chromatography from the elution of 40%methanol.Then,these three fractions were separately subjected to HSCCC for purification with the solvent system composed of petroleum ether-ethyl acetate-methanol-water at different volume ratios,and the mixtures were further purified by Pre-HPLC.As a result,a total of 13 known caffeoylquinic acid derivatives including caffeic acid,caffeic acid methyl ester,3-O-caffeoylquinic acid,3-O-Caffeoylquinic acid methyl ester,1,3-O-dicaffeoylquinic acid,1,5-O-dicaffeoylquinic acid,3,5-O-dicaffeoyl-quinic acid,3,5-O-dicaffeoyl-methyl ester quinic acid,4,5-O-dicaffeoyl-quinic acid,1,5-O-dicaffeoyl-3-O-?4-maloyl?-quinic acid,1,5-O-dicaffeoyl-3-O-?4-maloyl methyl ester?-quinic acid,1,5-O-dicaffeoyl-3-O-succinylquinic acid,1,5-O-dicaffeoyl-4-O-succinylquinic acid,and two new compounds were obtained.The new compounds were 1,4-O-dicaffeoyl-3-succinyl methyl ester quinic acid and 1,5-O-dicaffeoyl-3-O-succinyl methyl ester quinic acid.The research indicated that the combination of HSCCC and Pre-HPLC was a highly efficient approach for preparative separation of the instability and bioactive caffeoylquinic acid derivatives from natural products.?4?Hep G2 cells were used as a insulin resistance screening model to screen each compound for anti-diabetic activity.The results showed that the extracts and monomer components have the effect of increasing the glucose consumption in insulin-resistant model.The 1,3-O-dicaffeoylquinic acid has the best effect and its function was close to metformin.The burdock is a new drug resource which has latent value for exploitation.?5?The Anti-diabetic active components-target-pathway through the network pharmacology research method.The results showed that the caffeoylquinic acid components of burdock can exert anti-diabetic activity through various biological pathways.Their pathways were far more than that of pentacyclic triterpenoid such as oleanolic acid and ursolic acid.The target corresponding to caffeic acid coveres 15 potential targets associated with type?diabetes.The main active components of burdock mainly exert anti-diabetic activity by regulating the nutrients,and affecting bile secretion and insulin resistance.The prediction method can provide scientific basis and reference for the research on the anti-diabetic mechanism of function of burdock.?6?The metabolites of caffeoylquinic acid compounds in the ethyl acetate fraction of burdock were established using HPLC-Q-TOF-MS.A total of 13 caffeoylquinic acid metabolites in the plasma were identified by accurate molecular weight information provided by Q-TOF-MS after 45 minutes.From the information of metabolites,it can be deduced that the caffeoylquinic acid compounds degrade into chlorogenic acid,caffeic acid and quinic acid,caffeic acid degrades into dihydro caffeic acid and 3-hydroxypropionic acid,quinic acid degrades into benzoic acid and hippuric acid.Metabolic pathways of related degradation products include hydrogenation,methylation,dimethylation,acetylation,sulfation,glucuronidation and so on.The method established in this study is simple and reliable,and can provide a theoretical basis for exploring the active material of burdock.?7?Simultaneous detection of oleanolic acid and ursolic acid in rat blood by in vivo microdialysis can provide important pharmacokinetics information.Microwave-assisted derivatization coupled with magnetic dispersive solid phase extraction was established for the determination of oleanolic acid and ursolic acid by liquid chromatography tandem mass spectrometry.2?-carbonyl-piperazine rhodamine B was firstly designed and synthesized as the derivatization reagent,which was easily adsorbed onto the surface of Fe₃O₄/graphene oxide.Simultaneous derivatization and extraction of oleanolic acid and ursolic acid were performed on Fe₃O₄/graphene oxide.The permanent positive charge of the derivatization reagent significantly improved the ionization efficiencies.The limits of detection were 0.025 and0.020 ng/m L for oleanolic acid and ursolic acid,respectively.The validated method was shown to be promising for sensitive,accurate and simultaneous determination of oleanolic acid and ursolic acid.It was used for their pharmacokinetics study in rat blood after oral administration of the burdock extract.