| Purpose:In this paper,the anti-gastric cancer effect of ramie root in vitro was researched on the basis of the research on the optimal extraction and purification technology of the effective substance of ramie root anti-gastric cancer.Ramie root anti-gastric cancer drug components were characterized to clarify the composition of its material system through HPLC-Q-TOF-MS.The mechanism of ramie root anti-gastric cancer in vitro was elucidated through flow cytometry,cell scratch assay and Transwell chamber assay.Whether the effective components of ramie root can regulate the immune cells and provide a basis for the further anti-tumor effect of ramie root through immune regulation in the future.The pharmacodynamic mechanism of effective substance components of ramie root against gastric cancer was studied from the angle of related genes,and the multi-target and multi-pathway characteristics of Chinese medicine was also discussed.It provides a theoretical basis for the further application in new drugs for anti-gastric cancer of ramie root medicine in the future.Material and method:1.Orthogonal design was used to screen the best extraction method of effective components of ramie root anti-gastric cancer by the amount of solvent,time and times of extraction of effective components of ramie root against gastric cancer and the content was determined by UV spectrophotometry.The effects of sample concentration,eluent concentration,sample acidity and alkalinity,elution rate on the purification process of effective components of ramie root were further investigated by macroporous adsorption resin method,and then the best purification method of effective components of ramie root anti-gastric cancer was screened out.2.MTT assay was used to detect the proliferation inhibition effect of ramie root active components on human gastric cancer hgc-27 cells,so that clarify the anti-gastric cancer effect of ramie root active components in vitro.3.The effective component fingerprint of ramie root was established by high performance liquid chromatography(HPLC),and the content of the ingredients was detected.The ramie root effective component fingerprint was established in 10 different areas,and the components of ramie root effective component were characterized by liquid mass coupling.On this basis,the quantitative analysis of its multi-index components can provide some reference value for the quality control of ramie root medicine.4.Flow cytometry was used to investigate the effect of effective components of ramie root on the induction of hgc-27 cycle division and apoptosis and necrosis in human gastric cancer cells through the Annexin V-FITC/PI double staining and PI single staining method.The effect of ramie root active components on the migration and invasion of human gastric cancer cells hgc-27 was investigated by cell scratch assay and Transwe11 chamber assay.The effect of the effective components on immune cells was studied by using mouse macrophages as the research object.To detect the effect of ramie root active components on the relative expression of genes related to cell cycle apoptosis,migration and invasion in human gastric cancer cell line hgc-27 by QPCR related technology.And then the mechanism of ramie root anti-gastric cancer was elucidatedResults:1.Flavonoids were extracted from ramie root twice with 15 times the concentration of 50%ethanol for 1 hour each time using the extraction method of heating reflux was the best extraction method for the effective components of ramie root anti-gastric cancer.The optimal purification conditions of effective substance components of ramie root anti-gastric cancer were obtained as follows: Hpd-100 macroporous adsorption tree was used to extract ramie root at a concentration of 0.225 g/m L(medicinal materials/wet resin).Impurity was removed with 2 BV water,and eluted with 6BV 70% ethanol.Eluent was collected and dried in a water bath.The optimized purification process made the effective substance purity of ramie root anti-gastric cancer reach about 83%.2.Ramie root active material components can significantly inhibit the proliferation of human gastric cancer cells hgc-27,and showed a good time-dose-effect relationship,suggesting that ramie root active material components have a better anti-gastric cancer efficacy in vitro.3.The chemical constituents of ramie root were analyzed through HPLC-Q-TOF-MS and identifying seven chemical components.The three-wavelength fusion fingerprint of effective components of ramie root in 10 different areas was established.The similarity evaluation and analysis showed that the similarity of medicinal materials in different areas was different.The content of 4 components in the effective components of ramie root were measured simultaneously.4.The 26 chemical constituents in the petroleum ether of ramet root were analyzed by gc-ms,including sterols(37.228%),esters(21.445%),acids(21.315%),phenols(1.245%)and anthraquinones(0.882%).The total contents of sitosterols and leptosterols in the petroleum ether of ramie root were determined to be 33.2% and 6.5% respectively.The total contents of sitosterol and stigmasterol in the petroleum ether of ramie root were determined to be 33.2%and palmitic acid 6.5% respectively.5.The results of apoptosis detection by flow cytometry showed that the effective component of ramie root could induce the apoptosis of human gastric cancer hgc-27 cells,and the apoptosis ratio increased with the increase of drug concentration.The results of cell cycle detection by flow cytometry showed that the effective components of ramie root acted on hgc-27 cells of human gastric cancer and the cells were mainly blocked in S phase,thus affecting the normal progress of cell cycle and inhibiting cell proliferation.6.Cell scratch assay was used to detect the effect of effective components of ramie root on migration ability of human gastric cancer HGC-27 cells,and the results showed that the effective components of ramie root could significantly inhibit the migration of human gastric cancer HGC-27 cells compared with the blank control group.Transwell chamber method was used to detect the effect of ramie root active components on the invasion ability of human gastric cancer HGC-27 cells and the results showed that ramie root effective constituents in the gastric cancer HGC-27 cells invasion ability has significant inhibitory effect,differences statistically significant(F = 58.7,P < 0.05).Ramie root components showed a dose-dependent increasing ability to inhibit invasion and migration of gastric cancer cells with the increase of dosage concentration.7.The effect of effective components of Ramie root on immune cells was studied in this study.The results showed that the effective components of Ramie root could promote the secretion of NO by promoting cell proliferation and activation.It plays a key role in the immune process and in inhibiting the killing of tumor cells.It can also reduce the risk of invading foreign viruses by engulfing foreign microbes or viruses.It is suggested that the effective components of Ramie root can play an immunomodulatory role and provide some basis for the research and development of Ramie root in immunomodulation and anti-tumor.8.QPCR results showed that the effective components of ramie root at different concentrations all affected the m RNA expression of Bax,bcl-2,caspase-3,cyclin-e,cdk-2,PTEN,MMP9,VEGF,AKT and PI3 K in hgc-27 cells that regulate cell cycle,apoptosis,migration and invasion.Among them,the relative expressions of tumor suppressor gene PTEN and pro-apoptotic gene Bax increased,while the relative expressions of anti-apoptotic genes bcl-2,caspase-3,cyclin-e,cdk-2,PTEN,MMP9,VEGF,AKT and PI3 K decreased.Conclusion:1.Orthogonal experimental design method and macroporous adsorption resin method were the optimal extraction and purification method of effective substance components of ramie root anti-gastric cancer.It provides a reference for the basic research on the effective components of ramie root anti-gastric cancer and the preliminary mechanism of action.2.Antitumor studies showed that the effective components of ramie root had a good antitumor effect on gastric cancer in vitro.3.In this study,the overall characteristic fingerprint of effective components of ramie root was obtained by HPLC and similarity evaluation software.On top of that,a quantitative method for multi-index components of effective components of ramie root was established based on full time three-wavelength fusion fingerprint at the same time.Thus it can reflect the internal quality of ramie root medicine.The method is stable and reliable,which provides reference value for further quality control and evaluation system of ramie root.4.Flow cytometry was used to study the effect of effective components of ramie root on apoptosis and cell cycle of human gastric cancer hgc-27 cells.The effects of effective components of ramie root on the migration and invasion of human gastric cancer cells hgc-27 were investigated by cell scratch assay and Transwell chamber assay.The mechanism of action of ramie root anti-gastric cancer was discussed.5.In recent years,traditional Chinese medicine affects the occurrence and development of tumor cells through immunomodulation.Therefore,in this experiment,mouse macrophages were selected as the research object,and the effective components of Ramie root were found to have a certain regulatory effect on immune cells.It provides a basis for further anti-tumor effects through immunomodulation in the future.6.The anti-gastric cancer active components of ramie root of traditional Chinese medicine can significantly regulate the PI3K-AKT signaling pathway,regulate cell cycle apoptosis,migration and invasion related gene expression,tumor suppressor gene PTEN and the relative expression of pro-apoptotic gene Bax increased,anti-apoptotic genes Bcl-2,caspase-3,cyclin-e,CDK-2,PTEN,MMP9,VEGF,AKT and PI3 K relative expression decreased.It can effectively control the proliferation and growth of tumor cells,and provide some experimental basis for clarifying the mechanism of action of anti-cancer drugs. |
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