| Objective:Sanguisorba officinalis L.was first published in the book of shennong materia medica,it is the dry root of Sanguisorba officinalis L.or Sanguisorba officinalis L.var.longifolia?Bert.?Yu et Li.It has the effect of cooling blood to stop bleeding and detoxifying ambuncles.Based on the definite pharmacological and pharmacodynamic effects of Sanguisorba officinalis L.against liver tumor,in view of the unclear basis and mechanism of the antihepatic tumor pharmacodynamic substances of Sanguisorba officinalis L.,macroporous adsorption resin method and microfluidic-controlled chip combined with flow cell technology were used to study the pharmacodynamic material basis and preliminary mechanism of action of Sanguisorba officinalis L.against liver cancer,which provides experimental basis for the development and clinical application of new anti-liver tumor drugs of Sanguisorba officinalis L..Method:1.Taking total flavonoids and total tannin content as evaluation indexes,single factor method was used to determine the best extraction method of effective components from Sanguisorba officinalis L..Taking ethanol concentration,extraction time,solvent dosage and extraction times as factors,the comprehensive score of content and paste yield as the index,L9?34?orthogonal test design was used to optimize the extraction process of effective components of Sanguisorba officinalis L..Taking the purity of total flavonoids and total tannins as the index,the optimum purification process of effective components of Sanguisorba officinalis L.was optimized by static and dynamic adsorption and desorption experiments.2.High performance liquid chromatography?HPLC?was used to investigate the methodology and establish the fingerprint of the active components of Sanguisorba officinalis L.from different places,and the similarity of traditional Chinese medicine chromatogram fingerprint was calculated by using similarity evaluation software.The chemical composition characterization of effective components of Sanguisorba officinalis L.was studied by using liquid-mass coupling technique.Through literature review and comparison of reference materials,the monomer components of effective components were analyzed and identified,based on which the quality control of multi-effective components of Sanguisorba officinalis L.was studied.3.CorelDraw software was used to design an in vitro anti-liver tumor efficacy screening chip.On this basis,Hoechst 33342/propidine iodide?PI?double staining technique was used to carry out the in vitro pharmacodynamics study on the effective components and monomer components of Sanguisorba officinalis L.from different areas on HepG2 human liver cancer cells. 4.Flow cytometry and Annexin v-fitc/PI double staining and PI single staining were used to investigate the effect of effective components of Sanguisorba officinalis L.on the cell cycle division and apoptosis of HepG2 cells in human liver cancer.Real-time fluorescence quantitative PCR was used to detect the relative expression of related genes after the action of effective components in Sanguisorba officinalis L.based on the apoptosis of liver cancer cells and migration pathway,and to explore the initial mechanism of action of Sanguisorba officinalis L.against liver tumor.Results:1.In this study,the optimal extraction process of flavonoids from Sanguisorba officinalis L.was optimized as follows:70%ethanol,15:1 liquid-material ratio,reflux extraction for 3 times,each time extraction for 1.5h,extraction with this extraction process,the content of flavonoids in Sanguisorba officinalis L.was 14.43%,RSD was 1.08%.The optimal purification process was as follows:HPD-300 resin was used,the concentration was0.1g/mL of the pharmaceutical solution,and the sample was loaded according to the proportion of 0.5g/mL?crude drug dosage/resin volume?,5BV 70%ethanol elution,the eluent was collected.Purified by this purification process,the purity of flavonoids was88.60%,the RSD was 0.98%,the yield was 80.76%,and the RSD was 0.68%.2.The optimal extraction process of tannins from Sanguisorba officinalis L.was optimized as follows:60%acetone,15:1 liquid-material ratio,ultrasonic extraction for 3times,each time extraction for 1h,extraction with this extraction process,the content of tannins in Sanguisorba officinalis L.was 15.08%,RSD was 1.04%.The optimal purification process was as follows:HPD-400 resin was used,the liquid concentration was 3.94mg/mL,4times the volume of the column resin,and was eluted with 70%ethanol solution of 6 times the volume of the column resin.After the first eluent was collected and dried,the upper column was used for the second time.The purity of tannin and RSD was 74.72%and 0.91%,respectively.3.High performance liquid chromatography?HPLC?was used to establish the fingerprint of the effective components of Sanguisorba officinalis L.from 6 different places,among which 12 common peaks of flavonoids were determined,and the similarity of 6 batches of samples was greater than 0.88.Among them,15 common peaks of tannin components were determined,and the similarity of 8 batches of samples was greater than 0.94.Five chemical components of effective components of Sanguisorba officinalis L.were determined,the flavonoids were kaempferin and quercetin,and the tannins were gallic acid,catechin and ferulic acid.The content of Sanguisorba officinalis L.from 6 different places was detected.The highest content of kaolin was 0.0368mg/g in anhui province,quercetin was0.0693mg/g in heilongjiang province,gallic acid was 0.5977mg/g in henan province,catechin was 0.7549mg/g and ferulic acid was 0.5137mg/g in gansu province.4.This project designed and produced a two-layer PDMS microfluidic chip for simultaneous online screening the effect of anti-liver cancer.16pneumatic microvalves?A-H?were placed on the first PDMS film layer,and two pairs were paired on control channels,eight liquid valves symmetrically arranged to realize different kinds of different concentrations of liquid medicine in different channels.The second PDMS film layer is provided with 16 elliptical cell culture chambers and fluid channels.The chip can simultaneously screen different components and monomer components in anti HepG2 cells.5.In this study,the in vitro efficacy of effective components and monomer components and effective components from different habitats were carried out.The results showed that total flavonoids,total tannin and monomer components?quercetin and gallic acid?have inhibitory effects of HepG2 cells.The rate of apoptosis and necrosis of total flavonoids is15.89%-26.38%,and the rate of apoptosis and necrosis of total enamels is 62.34%-90.52%.The effect of total enamels is better than that of total flavonoids.The flavonoids from Heilongjiang have the best efficacy in the production of cancer cells,and the enamel from Shandong the best efficacy in the production of cancer cells.6.In this study,different doses of enamel and different doses of flavonoids could induce apoptosis and block cycle of HepG2 cells.The rate of apoptotic after stimulation for36 hours in the high dose group of enamels was?86.0±0.2?%,in which the rate of apoptotic after 36 h in high dose group of flavonoids was?24.3±0.3?%.Real-time quantitative PCR analysis showed that total flavonoids and total enamel could increase the expression of Bax and Caspase-3 mRNA and decrease the expression of Bcl-2 mRNA in HepG2 cells,and the effect of total enamel was more significant.Conclusion:1.The optimized extraction and purification process of flavonoids and tannins from Sanguisorba officinalis L.is stable and feasible,and the purity of flavonoids and tannins produced by this process is high.The optimized process can provide a material basis for studying the pharmacodynamics and mechanism of effective components from Sanguisorba officinalis L.2.In this study,the fingerprints of flavonoids from six different origins and the fingerprints of tannins from eight different origins were establish using HPLC.The components in the fingerprints were characterized by HPLC-QTOF-MS.Bsed on the components characterized above,quantitative control of multi-index components were analyzed.The results provide an experimental basis for quality evaluation and quality control of Sanguisorba officinalis L.3.This study designed and produced a pharmacodynamic screening chip for screening the anti-liver cancer components of Sanguisorba officinalis L.At the same time,the chip was applied to detect pharmacodynamic effect of the effective components,monomer components and effective components of different habitats.The results show that total flavonoids and total enamels of Sanguisorba officinalis L.have anti-liver cancer effects in vitro.The apoptosis rate of gallic acid cells at different concentrations was not significantly different from that of total enamels cells,suggesting that gallic acid may play a major role in anti-liver cancer.There are some differences in anti-liver cancer effect of effective components from different regions due to the different content of chemical composition.This part provides an important preliminary basis for deeply study the mechanism of Sanguisorba officinalis L.in anti-liver cancer.4.This study demonstrates that flavonoids and tannins both can induce apoptosis of HepG2 cells,but their effects are different.The total enamels can inhibit the synthesis of DNA and prolong the proportion of cells in S phase,hence inhibit proliferation of liver cancer cells.It has a dependence on the inhibition of S phase of liver cancer cells with the increase of the dose.The total flavonoids can inhibit the proliferation of liver cancer cells by prolonging the proportion of cells in S phase and G2/M phase,inhibiting the synthesis of DNA and cell division.The effect may be related to regulate the expression of Bcl-2 and Bax in upstream signals,and activate caspase 3.This study clarified the preliminary mechanism of anti-liver cancer in vitro,and provided experimental and theoretical basis for the study of Sanguisorba officinalis L.. |
PDF Full Text Request