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Screening,isolation And Bioactivity Of Effective Components From Gastrodia Elata And Pinellia Ternata

Posted on:2024-02-03Degree:MasterType:Thesis
Country:ChinaCandidate:Y J LiFull Text:PDF
GTID:2531307139978159Subject:Chemistry
Abstract/Summary:PDF Full Text Request
Gastrodiae Rhizoma(Tianma)is the dried tuber of Gastrodiae Rhizoma Bl.plant(Orchidaceae).It is neutral in nature and sweet in taste.It has the effect of calming endogenous wind and stop spasm,calming the liver and suppressing Yang,and dispelling wind and dredging collateral.It is a traditional and valuable Chinese herbal medicine in our country.Pinellia tuber is derived from the dried tuber of Pinellia tuber Breitenbach(Araceae).It is pungent flavor and warm nature.It has the effect of drying dampness and resolving phlegm,reducing adverse reaction and anti-emesis,eliminating epiphysis and dispersing nodules,as well as anti-tumor.Therefore,in this paper,the active components in the above two natural medicines were systematically extracted,separated and the in vitro activity was studied to provide a certain theoretical basis for the exploration of Gastrodia Rhizoma and Pinellia tuber activity as well as drug research and development.Firstly,the extraction rate of active ingredients in Gastrodia Rhizoma was used as the evaluation index,and liquid-to-material ratio,ethanol concentration,extraction times and extraction time were selected for single factor experiments,and the results were functionally fitted.Matlab genetic algorithm was further used to globally optimize the extraction conditions of active ingredients from Gastrodia Rhizoma.The results showed that the optimum extraction process of Gastrodiae Rhizoma was as follows:ethanol concentration of 63.18%,liquid-to-material ratio of 1:20.89,extraction times of 3.13 times,ultrasonic extraction time of 2.64 h.The extraction rate of active ingredient in Gastrodia Rhizoma was 1.09 mg/g.Xanthine oxidase was selected as target protein to screen potential enzyme inhibitors in Gastrodia Rhizoma by ultrafiltration-mass spectrometry.The affinity ultrafiltration results were analyzed by calculating the binding strength,and the binding of active ingredients to target proteins was studied by molecular docking and molecular dynamics simulation techniques,and the results of affinity ultrafiltration experiments were verified to further elucidate the binding mechanism.Three potential xanthine oxidase inhibitors were screened experimentally,including Gastrodin,P-hydroxybenzyl alcohol and Parishin A in Gastrodia Rhizoma.The results of molecular docking and molecular dynamics simulation showed that the main binding forces between the three active ingredients and the target protein were van der Waals forces,and the binding energies were-13.70,-10.90,-9.30 kcal/mol,respectively.Under the guidance of activity screening,the active ingredients in Gastrodia Rhizoma were separated and purified by semi-preparative high performance liquid chromatography combined with high-speed countercurrent chromatography,and the purity of the obtained monomers was analyzed by high-performance liquid chromatography;the structure of the isolated active ingredient monomers was identified by UPLC-Q-Exacive techniques.A total of three chemical constituents were isolated from Gastrodia Rhizoma,which were Gastrodin,P-hydroxybenzyl alcohol and Parishin A,respectively.Capillary electrophoresis was used to study the inhibitory effect of active ingredients in Gastrodia Rhizoma on xanthine oxidase.The inhibition ability was investigated using the peak area of the product as a quantitative basis and allopurinol as a positive control.The results showed that all three active components had good inhibitory ability on xanthine oxidase,and the IC50 values of Gastrodin,P-hydroxybenzyl alcohol and Parishin A were 44.44,16.48 and 7.63μg/m L,respectively.Secondly,the extraction rate of active ingredients in Pinellia tuber was used as the evaluation index,and liquid-to-material ratio,ethanol concentration,extraction times and extraction time were selected for single factor experiments,and the results were functionally fitted.Matlab genetic algorithm was further used to globally optimize the extraction conditions of active ingredients from Pinellia tuber.The results showed that the optimum extraction process of Pinellia tuber was as follows:ethanol concentration of 66.28%,liquid-material ratio of 1:24.39,extraction times of 2.13times,ultrasonic extraction time of 2.03 h.The extraction rate of active ingredient in Pinellia tuber was 1.09 mg/g.Xanthine oxidase was selected as target protein to screen potential enzyme inhibitors in Pinellia tuber by ultrafiltration-mass spectrometry.The affinity ultrafiltration results were analyzed by calculating the binding strength,and the binding of active ingredients to target proteins was studied by molecular docking and molecular dynamics simulation techniques,and the results of affinity ultrafiltration experiments were verified to further elucidate the binding mechanism.Five potential xanthine oxidase inhibitors were screened experimentally,including Uridine,Guanosine,Adenosine,Liquiritin and Liquiritigenin in Pinellia tuber.The results of molecular docking and molecular dynamics simulation showed that the main binding forces between the three active ingredients and the target protein were van der Waals forces,and the binding energies were-8.0、-9.6、-7.7、-6.5、-9.2 kcal/mol,respectively.Under the guidance of activity screening,the active ingredients in Pinellia tuber were separated and purified by semi-preparative high performance liquid chromatography combined with high-speed countercurrent chromatography,and the purity of the obtained monomers was analyzed by high-performance liquid chromatography;the structure of the isolated active ingredient monomers was identified by UPLC-Q-Exacive.A total of five chemical constituents were isolated from Pinellia tuber,which were Uridine,Guanosine,Adenosine,Liquiritin and Liquiritigenin,respectively.High performance liquid chromatography(HPLC)was used to study the inhibitory effect of active ingredients on xanthine oxidase in Pinellia tuber.The inhibition ability was investigated using the peak area of the product as a basis for quantification.The results showed that all five active ingredients had good inhibitory ability on xanthine oxidase,and the IC50 values of Uridine,Guanosine,Adenosine,Liquiritin and Liquiritigenin were 49.17,33.24,42.83,40.84 and 61.36μg/m L,respectively.Finally,the molecular biological mechanism of active ingredients in Gastrodia Rhizoma and Pinellia tuber in the treatment of gout was studied based on network pharmacology.To construct the target of correlation between active ingredients in Gastrodia Rhizoma and Pinellia tuber,GO functional enrichment analysis KEGG signaling pathway enrichment analysis was used to elucidate the molecular mechanism of active ingredients in Gastrodia Rhizoma and Pinellia tuber in the treatment of gout.The results showed that the three active components in Gastrodia Rhizoma involved a total of 156 drug targets,and there were 1684 strongly correlated targets for gout,of which 63 formed common targets with the active components in Gastrodia Rhizoma.These 63 common targets were used as potential targets of active components in the treatment of gout to construct PPI networks and perform topological analysis and cluster analysis,in order to select the core targets.Meanwhile,GO and KEGG enrichment analysis were performed for these 63 common targets.According to the results of pathway enrichment analysis,the main metabolic pathways involved cell proliferation pathway,protein autophosphorylation pathway,negative regulation pathway of apoptosis process,prostate cancer metabolic pathway and proteoglycan pathway in cancer,and it was speculated that potential xanthine oxidase inhibitors in Gastrodia Rhizoma may play a good therapeutic effect on gout through the above metabolic pathways.There were 198 related targets of the five active components and1726 related targets of gout in Pinellia tuber.Eighty-five common targets of active components and gout were screened by intersection of the two targets,and the target protein interaction network was constructed.The results showed that the core targets included ALB,EGFR,VEGFA,SRC,HSP90AA1.According to the results of pathway enrichment study,the main metabolic pathways involved lipid and atherosclerosis pathways,cancer pathways,proteoglycan pathway in cancer,PI3K-Akt signaling pathway and Rap1 signaling pathway,and it was speculated that potential xanthine oxidase inhibitors in Pinellia tuber may have a therapeutic effect on gout through the above metabolic pathways.In this paper,the extraction conditions,ultrafiltration activity screening,separation and purification of monomer,structural analysis and identification of monomer,inhibition ability evaluation and mechanism of action of active ingredients in Gastrodia Rhizoma and Pinellia tuber were systematically studied,providing the basis for the research and development and comprehensive utilization of anti-gout drugs in Gastrodia Rhizoma and Pinellia tuber.
Keywords/Search Tags:Gastrodiae Rhizoma, Pinellia tuber, Gout, Active component screening, Isolation and purification, Mechanism of action
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