| Purpose:Based on the microfluidic chip technology,the effective components of Hairyvein agrimony were screened out,and the extraction and purification process of the effective components of Hairyvein agrimony was studied.At the same time,it's related quality standards were established.On this basis,the in vitro anti-hepatoma effect and mechanism of the effective components of Hairyvein agrimony were studied,which could provide some experimental reference for the development of new anti-lung cancer drugs.Method:1.Based on microfluidic chip technology,Hoechst 33342/PI fluorescence double-staining method was used to screen the effective components of Hairyvein agrimony with the index of cell apoptosis and necrosis rate.The extraction conditions of total flavonoids were optimized with the factors of ethanol volume fraction,material-liquid ratio,extraction time.The purification process conditions of total flavonoids were optimized with the factors of resin type,sample concentration,pH value of sample solution,solvent quantity of elution and dissolution.2.The fingerprints of nine Hairyvein agrimony'total flavonoids from different habitats were established by using high performance liquid chromatography combined with multi-wavelength fusion technology.The characterization of total flavonoids of Hairyvein agrimony was carried out by using UPLC-Q-TOF-MS technology through comparing the mass spectrum information with reference substances,related literature and related databases.To establish a comprehensive,comprehensive and effective quality evaluation system for the total flavonoids of Hairyvein agrimony.3.Microfluidic chip technology and flow cytometry were used to study the effects of total flavonoids of Hairyvein agrimony on cell cycle,cell apoptosis,necrosis,migration and invasion of hepatocellular carcinoma cell line HepG2,and to evaluate its anti-hepatoma efficacy comprehensively.4.Using real-time fluorescence quantitative PCR and Western Blot technology,the preliminary anti-hepatoma mechanism of the total flavonoids of Hairyvein agrimony was explored at the level of gene and protein,respectively.Results:1.Two kinds of pharmacodynamics research chips have been constructed by combining soft lithography,injection molding and plasma bonding.One is a multi-functional high-throughput drug screening chip,which can realize on-line screening of multiple pharmacodynamics of homologous cells.The other is a model chip for tumor cell migration and invasion,which can realize the migration and invasion of tumor cells.It provides a model for anti-cancer drug efficacy research.The new technology platform also lays a solid foundation for the further experiment.2.There are some differences among the flavonoid components of Hairyvein agrimony from nine different habitats.Ten compounds were identified:quercetin,kaempferol,quercitrin,citrin,isoquercetin,astragaloside,catechin,apigenin,scutellarin and ellagic acid.At the same time,the content of flavonoids in Hairyvein agrimony from different habitats was different.Among them,the highest content of Isoquercitrin was found in Changzhou,Jiangsu Province,at 0.5386 mg·g-1;the highest content of scutellarin was found in Bozhou,Anhui Province,at0.8316 mg·g-1;and the highest content of quercitrin was found in Nanning,Guangxi Province,at 0.4152 mg·g-1.3.The total flavonoids of Hairyvein agrimony can significantly promote the apoptosis and necrosis of human hepatocellular carcinoma HepG2 cells.The cell apoptosis and necrosis rate of 0.75 mg·mL-11 reaches?67.97±1.9?%.The total flavonoids of Hairyvein agrimony have significant G2/M blocking effect on the cell cycle of HepG2 cells.Meanwhile,the total flavonoids of Hairyvein agrimony can significantly inhibit the migration and invasion of HepG2 cells based on the tumor cell migration and invasion model chip.At the same time,with the increase of dosage,the drug effect gradually increased,showing a strong anti-hepatoma effect.4.The results of PCR showed that total flavonoids of Herba Agrimoniae could significantly up-regulate the expression of PTEN,Caspase-3 and Caspase-9 genes,significantly down-regulate the expression of PI3K,AKT,CyclinB1,CDC2,COX-2 and VEGF genes.Western Blot showed that total flavonoids of Herba Agrimoniae significantly reduced the expression of PI3K,AKT,VEGF,COX-2 and CyclinB1 proteins,and significantly increased the expression of PTEN and Caspase-9 proteins.Integrating the data of genomics and proteomics,it was concluded that total flavonoids of Herba Agrimoniae exerted anti-hepatoma effects by regulating PTEN-PI3K/AKT signaling pathway.Conclusion:1.Two kinds of pharmacodynamics research chips have been constructed by combining soft lithography,injection molding and plasma bonding.One is a multi-functional high-throughput drug screening chip,which can realize on-line screening of multiple pharmacodynamics of homologous cells.The other is a model chip for tumor cell migration and invasion,which can realize the migration and invasion of tumor cells.It provides a model for anti-cancer drug efficacy research.The new technology platform also lays a solid foundation for the further experiment.2.In this study,the extraction and purification method of total flavonoids of Hairyvein agrimony was established.The process was simple and easy to operate,which laid a foundation for the follow-up study of the pharmacodynamics of total flavonoids of Hairyvein agrimony.3.From the point of view of fingerprint,component analysis and quantitative analysis of multi-index components of total flavonoids of Hairyvein agrimony,a comprehensive,effective quality evaluation system of total flavonoids of Herba Agrimoniae was established by combining HPLC with UPLC-Q-TOF-MS technology,which laid an experimental foundation for its development,utilization and pharmacodynamic research.4.The total flavonoids of Hairyvein agrimony can inhibit the proliferation of tumor cells,promote the apoptosis of tumor cells,block the cell cycle,and inhibit the migration and invasion of tumor cells.It has significant anti-hepatoma effect.5.The total flavonoids of Herba Agrimoniae exert anti-hepatoma effects by regulating PTEN-PI3K/AKT signaling pathway. |
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