Study On The Extraction And Isolation Of Ginsenoside From White Ginseng And Its Inhibitory Effect On Glioma Cells

Objective: In this study,HPLC-MS was used to establish a rapid qualitative and quantitative method of ginsenoside,to study the antitumor effects of ginsenoside Rc,Rd,Re on human glioma SHG-44,U251 MG.Methods: In the qualitative and quantitative analysis of ginsenosides,a rapid qualitative and quantitative method was established by using the combination of ultra-high performance liquid chromatography(thermo ultimate 3000)and triple quadrupole mass spectrometer(thermo TSQ Endura);Different concentrations of ginsenoside Rc,Rd and Re were used to treat human glioma cells SHG-44 and U251 MG respectively.CCK-8 method was used to detect the proliferation activity of SHG-44 and U251 MG cells.Cell morphology was observed under normal light microscope,cell cycle was detected by flow cytometry,annexin was detected V/FITC double staining was used to detect the apoptosis of cells and to study the mechanism of ginsenoside Rc,Rd,Re on human glioma cells SHG-44,U251 MG.Results: In the qualitative and quantitative analysis of ginsenosides,13 kinds of ginsenosides in the extract of raw ginseng can be analyzed by HPLC-MS;At the same time,quantitative analysis of ginsenoside Rc,Rd and Re showed that the linear relationship of ginsenoside Rc and Re was good in the range of 2-20 ?g/mL the correlation coefficients were 0.9994 and 0.9991,respectively,and the linear relationship of ginsenoside Rd was good in the range of 0.2-2 ?g/mL,the correlation coefficients were 0.9951.The content of ginsenoside Rc,Rd and Re in the extract of raw sun exposure ginseng was 2.59±0.03 mg/g?0.22±0.01 mg/g?4.19±0.04 mg/g.The activity of ginsenoside Rc,Rd and Re on SHG-44 and U251 MG cells showed that ginsenoside Rc(1-100 ?g/mL)had weak inhibitory effect on SHG-44 cells,and ginsenoside Rc(12.5-200 ?g/mL)had weak inhibitory effect on U251 MG cells;The inhibition rate of cell proliferation was 97.28±0.94% after ginsenoside Rd(100 ?g/mL)treated SHG-44 cells for 72 hours,94.51±1.86%,93.86±0.40%,95.32±0.68% after ginsenoside Rd(200 ?g/mL)treated U251 MG cells for 24,48 and 72 hours,and 51.96±2.36% after ginsenoside Re(200 ?g/mL)treated U251 MG cells for 72 hours.The results of cell cycle showed that the SHG-44 cycle could be blocked in S phase by ginsenoside Rd(100 ?g/mL)for 72 hours,and U251 MG cell cycle was blocked in S phase by ginsenoside Rd(200 ?g/m L)for 24 hours.The results of apoptosis showed that the ratio of late apoptosis and total apoptosis of SHG-44 cells increased significantly(P<0.05)after 72 h of ginsenoside Rd(100 ?g/mL),and the ratio of early apoptosis and late apoptosis of U251 MG cells increased after 24 h of ginsenoside Rd(200 ?g/mL).Under the normal light of microscope,it can be seen that the number of cells in the treated group decreased,the number of cells shrunk,the volume became smaller,and the number of suspended dead cells increased gradually.Conclusion: In this study,HPLC-MS method was used to establish a rapid qualitative and quantitative analysis method to detect the extract of shengshai ginseng.Ginsenoside Rd has a significant inhibitory effect on the proliferation of human glioma cells SHG-44 and U251 MG.Its mechanism is to induce the apoptosis of SHG-44 and U251 MG cells by regulating the cell cycle of SHG-44 and U251 MG cells.This experiment provides reference for the establishment of the experimental method flow of ginsenoside extraction,separation,analysis and biological activity evaluation,and excavates the application of Ginsenoside in anti-tumor.