Study On The Material Basis Of The Anti-A549Cells Of Non-Small Cell Lung Cancer By Buxus Microphylla

Human non-small cell lung cancer（non-small cell lμng cancer,NSCLC）is the most common type of lung cancer,accounting for about 80%of the total number of lung cancer.Currently,clinical treatment modalities include surgery,drug therapy and radiotherapy,and drug therapy is still the main treatment.The existing clinical non-small cell lung cancer drugs have many adverse reactions,and the development of safer,more effective,stable and controllable drugs is of great significance for the treatment of NSCLC.Buxus microphylla is a plant of the genus Buxus in the family Buxaceae.In this thesis,the dried leaves of Buxus microphylla were used to optimize the extraction process,isolate and purify the active ingredients,confirm the structure and inhibit the activity of non-small cell lung cancer A549 cells in vitro,etc.The results are as follows:1.The best extraction process of Buxus microphylla was screenedThe CCK-8 method was used to determine the active site of the anti-small cell lung cancer A549 effect of Buxus microphyll was the dried leaves;the best extraction conditions of Buxus microphyll were screened by single-factor and orthogonal tests:75%ethanol was mixed at a material-to-liquid ratio of 1:10,and the extract was refluxed three times for 2 h each.2.Completed the isolation and purification of the effective components of Buxus microphylla leavesThe active fractions of dried Buxus microphylla leaves were identified as petroleum ether phase and trichloromethane phase by solvent extraction and CCK-8 method;then the active components were separated by silica gel column chromatography,thin-layer chromatography and recrystallization,and a total of four active monomer compounds were obtained,and the IC₅₀ values of compounds 1-4 in vitro inhibited non-small cell lung cancer A549 cells were 24.48±0.75μg/ml,53.01±1.73μg/ml,19.07±0.46μg/ml,28.55±0.81μg/ml,respectively.3.Clarified the structure of the active ingredients in Buxus microphylla leavesThe four effective monomer compounds were confirmed by wave spectral analysis as:lupinol,betulinol,hydroxycycloprotobuxine,16-benzenecarboxamide-23-ethyl ester-cycloprotobuxine,of which hydroxycycloprotobuxine,16-benzenecarboxamide-23-ethyl ester-cycloprotobuxine are new derivatives.4.A preliminary study of the proliferation inhibitory activity of hydroxycycloprotobuxine on A549 cells was carried outAfter treatment of non-small cell lung cancer A549 cells with hydroxycycloprotobuxine for 24 h,the morphology of some cells was changed,and the cell volume was reduced and rounded,and the number of cells with changed morphology increased with the increase of the administered concentration,accompanied by the production of cell debris,with an IC₅₀ value of 20.06±1.87μg/ml.The results of the scratch test,transwell migration and invasion assays showed that hydroxycycloprotobuxine inhibited the migration and invasion of A549 cells in a dose-dependent manner in the concentration range of 12.5μg/ml to 50μg/ml,with significant differences compared to the Control group.The results of cell cycle,apoptosis and caspase 3 activity assay showed that the expression of apoptosis and caspase 3 increased in A549 cells with the increase of drug concentration,among which the increase of early apoptosis was more obvious in the concentration range of 12.5μg/ml to 50μg/ml in a dose-dependent manner,and its mechanism of action may be related to the blockage of cell cycle in G0/G1 phase and upregulation of The mechanism of action may be related to blocking the cell cycle in the G0/G1 phase,upregulating the expression of apoptotic protein caspase 3 in A549 cells,and thus inducing apoptosis.It was suggested that hydroxycycloprotobuxine could inhibit the proliferation,migration and invasion of non-small cell lung cancer A549 cells and induce their apoptosis,which provided some experimental basis for the anti-lung cancer effects of boxwood alkaloids.