| Purpose:to optimize the extraction process of Stellera chamaejasme,and to study the effect and mechanism of Stellera chamaejasme extract on human lung adenocarcinoma A549 cells in vitro.Material and method:with the coumarin compound of Stellera chamaejasme as the index,the best extraction technology was studied by HPLC Combined with response surface analysis(box Behnken).The best concentration of Stellera chamaejasme extract(SCE)on human lung adenocarcinoma A549 cells was determined by MTT,the inhibition of SCE on human lung adenocarcinoma A549 cells was observed by inverted microscope,the inhibition of JC-1staining and hoechst33342 staining were observed by laser confocal microscopy and fluorescence microscopy respectively The expression of p53 and MDM2 was detected by Western blot.Results:1.The best extraction process parameters of Stellera chamaejasme extract are: ultrasonic extraction,extraction time 50 min,ethanol concentration 90%,material liquid ratio7.5:1;quadratic regression model equation: y=185.96+17.17a-9.58b+0.70c+15.25ab+3.91ac-8.76 bc +6.79a2-29.84b2-23.63c2,theoretical extraction rate of daphnoretin 189.6 ? g/g.Under the optimal conditions,three batches were extracted for validation,and the average extraction rate was 188.55 ? g / g.2.The results of MTT test showed that the inhibition rate of cell growth increased with the increase of drug concentration in the range of 1ug /ml-20 ug / ml after the effect of SCE on human lung adenocarcinoma A549 cells,showing a good dose-response relationship.The IC50 was 37.5 ? g / M and 22.16 ? g / ml at 24 h and 48 h,respectively.The best concentration of cell action was 10 ?g /ml according to the relationship between drug solubility and dose-response.3.Cell scratch observation showed that SCE inhibited the growth of human lung adenocarcinoma A549 cells.24 h cell migration rate: the control group(24.39 ± 1.21)washigher than the drug group(19.51 ± 1.23)(P < 0.01);48 h cell migration rate: the control group(58.54 ± 2.10)was higher than the drug group(45.78 ± 1.67)(P < 0.01).4.JC-1 staining results showed that SCE in different dose groups could promote mitochondrial apoptosis in varying degrees.Compared with the normal group and the low-dose group,the mitochondrial membrane potential of the middle,high-dose group and CCCP positive group changed significantly.5.Hoechst33342 cell apoptosis test showed that human lung adenocarcinoma A549 cells showed light blue after normal staining.After SCE effect,the blue of low dose group to high dose group increased to gradually bright white.After apoptosis,the nucleus was dense and fragmented or condensed.6.Western blot results showed that compared with the control group,p53 expression increased in the low,medium and high dose groups(P <0.05),and MDM2 expression in the medium and high dose groups decreased significantly(P <0.01)Conclusion:1.HPLC Combined with response surface optimization analysis showed that the content of cyhaloxine was reliable,the chromatographic conditions and detection methods were stable,operable,reproducible and recovery were good,and the predicted values of regression model fit well with the expected values of theoretical values.The SCE obtained by this extraction process could be used as a sample for in vitro activity study.2.The results of MTT experiments showed that the extract of Stellera chamaejasme Poison(SCE)could inhibit human lung adenocarcinoma A549 cells in a certain concentration range,and the concentration was dependent.3.The cell scratch test further verified the inhibitory effect of Stellera chamaejasme poison extract(SCE)on human lung adenocarcinoma A549 cells.4.Mitochondrial apoptosis and Hoechst33342 cell apoptosis results indicate that Stellera chamaejasme Poison Extract(SCE)may cause apoptosis of human lung adenocarcinoma A549 cells by acting on mitochondria to cause changes in membrane potential.5.The effect of Stellera chamaejasme Venom Extract(SCE)on human lung adenocarcinoma A549 cells in vitro may play a role in inhibiting the proliferation of A549 cells by acting onthe p53-MDM2 negative feedback pathway. |
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