The Study Of Selection And Action Mechanism On Anti-Tumor Effect Extracts Of Ferula Sinkiangensis K.M.Shen

Objective:Using the methods of of DPPH antioxidant,in vitro experiments,We researched anti-tumor,antioxidant effect of Ferula sinkiangensis K.M.Shen different polar parts,We selected Ferula anti-tumor parts active parts;We made clear anti-tumor activiey of Ferula sinkiangensis K.M on the basis of serum pharmacology,scratch experiment,Transwell Small parts experiments;We observe the tumor inhibiting effect of Ferula active parts in mice S-180 ascites tumor and mice S-180 solid tumor,And to further explore the molecular mechanisms of anti-tumor active parts.We provided experimental basis for the development and effective utilization of the Xinjiang Uighur local herbs.Methods:1.Through sulfonyl rhodamine B colorimetric method?SRB?,We detected proliferation inhibition effects of Ferula sinkiangensis K.M.Shen the polarity parts?petroleum ether part,ethyl acetate part,n-butanol part,methanol part?on human colon cancer cell HCT116,colon cancer cells Caco-2,HepG2 liver cancer cell;By flow cytometry methods,We observed promoting apoptosis effects of Ferula diffent polarity parts on HCT116,Caco-2,HepG2 cells,Using cell proliferation inhibition rate,total apoptosis rate as index,We screened Ferula anti-tumor active parts.2.Through method of the TLC-DPPH and DPPH free radicals experiment,We Evaluated antioxidant ability effects of Ferula sinkiangensis K.M.Shen polarity parts?petroleum ether part,ethyl acetate part,n-butanol part,methanol part?.3.By the method of serum pharmacology,We detected proliferation inhibiting effects of Ferula anti-tumor active parts drug-containing serum on HCT116?Caco-2?HepG2 cells.4.Using scratch experiment,Transwell Small chamber experiments,With migration inhibition rate,invasion inhibition rate for evaluation,We studied the inhibiting migration and invasion ability of anti-tumor active parts on HCT116,Caco-2,HepG2 cells.5.To establish mice S-180 ascites tumor models:120 mice,randomly divided into 6groups,Normal control group,and model group,high dose group,middle dose group,low-dose group,positive control group,Ten days after intragastric infusion with ethyl acetate parts,We observed the survival time of mice,spleen index and thymus index and weight.6.To establish mouse S-180 solid tumor model:60 mice,randomly divided into 6groups,Normal control group,model group,high dose group,middle dose group,low-dose group,positive,control group.Ten days after intragastric infusion with ethyl acetate parts,We observed tumor inhibition rate,spleen index and thymus index and weight,Check tumor in mice with HE staining.7.Using flow cytometry methods,We studied the effect on human colon cancer cell HCT116 cell cycle,Using Hoechst33258 staining methods,We studied influence on HCT116 cells apoptosis morphology,With the methods pf transmission electron microscope,We observed apoptosis cells ultrastructure of HCT116 cells.8.Using the real time quantiative PCR method,We detected the influence of gene expression of EGFR,PTEN,mTOR,Akt1,Bcl-xl,p53,Pl3Kp85 on PI3K-Akt pathway.9.With the application of Western Blot method,We detected the influence of protein expression of PTEN,mTOR,Bcl-xl,p53 on PI3K-Akt pathway.Results:1.Ferula sinkiangensis K.M.Shen were separated 4 parts?petroleum ether parts,ethyl acetate parts,n-butanol parts,methanol parts?.With petroleum ether parts and ethyl acetate parts accounted for the highest yield rate of 0.12 g·g^-11 and 0.15 g·g^-1;The Ferula polarity parts could inhibit 3 kinds of tumor cell proliferation,The ethyl acetate part is best of all,We Optimized IC₅₀0 of HCT116,Caco-2,HepG2 cells were 43.7?15.0?28.7?g·mL^-1;The Ferula polarity parts could promoting 3 kinds of tumor cells apoptosis.The ethyl acetate part effect is best of all.We optimized apoptosis rate were 46.6%,64.0%and80.8%respectively of HCT116,Caco-2,HepG2 cells.DPPH free radical method indicated that in addition to the petroleum ether part,the other polarity parts had clear effect on DPPH free radicals,IC₅₀0 of the ethyl acetate,n-butanol were 34.1 mg·L^-11 and 33.8 mg·L^-1;TLC-DPPH method showed that ethyl acetate,n-butyl alcohol had good antioxidant activity.2.The results drug-containing serum showed that proliferation inhibition rate was25.3%,27.6%and 12.2%,respectively of 50%of Ferula ethyl acetate parts medicated serum on HCT116,Caco-2,HepG2 cells.3.Scratch experiment showed that 48 hours cell migration inhibitory rate of Ferula ethyl acetate parts were 35.8%,24.4%and 35.8%respectively on HCT116,Caco-2,HepG2 cells;Transwell Chambers migration experiment showed 48 hours cell migration inhibitory rate of Ferula ethyl acetate parts was 70.7%on HCT116 cells;Transwell small chamber attack experiment showed that 72 hours of cell inhibiting invasion rate of Ferula ethyl acetate parts were 55.1%,89.5%,75.0%on HCT116,Caco-2,HepG2 cells.4.Establishing mice S-180 ascites tumor model,administered Ferula ethyl acetate extract for 10 days,It showed that survival time was longer and spleen index were significantly increased in Ferula low,medium and high dose groups.Compared with the control group,the difference was statistically significant?P<0.05?,No obvious change in the thymus index and weight.Establishing mice S-180 tumor model,Intragastric administrated Ferula ethyl acetate extract for 10 days,It showed that tumor growth inhibition rates were 40.4%,50.9%,58.2%and spleen index were significantly increased in Ferula low,medium and high dose groups.Compared with the control group,the difference was statistically significant?P<0.05?,No obvious change in the thymus index and weight.5.Flow cytometry showed that Ferula ethyl acetate part role in HCT116 cell cycle,The cell cycle was blocked in G0/G1 and S period;Hoechst33258 staining and transmission electron microscopy showed HCT116 cells nuclear chromatin became condensation and marginalized,apoptotic body could be seen.Using application of real time quantiative PCR method,It showed that the EGFR,PI3Kp85 relative gene expression level bad on change,Compared wity the control group,the difference was not statistically significant;the PTEN,p53 gene expression significantly increased,Akt1,mTOR,Bcl-xl gene expression significantly decreased,Compared with the blank control group,difference was statistically significant?P<0.05?.Using Western Blot method,It showed that PTEN,p53 protein expression was significantly increased,mTOR Bcl-xl protein expression was significantly decreased,Compared with the control group,the difference was statistically significant?P<0.05?.Conclusion:1.Through cell in vitro,we concluded that oxidation resistance,inhibiting tumor cell proliferation,promoting tumor cell apoptosis effect of Ferula ethyl acetate parts is best,While the extract yield is highest,It showed that Ferula ethyl acetate parts has certain value,Therefore identified as Ferula anti-tumor active part.2.Ferula ethyl acetate parts medicated serum could inhibit HCT116 cell proliferation strongly,Ferula ethyl acetate parts could inhibit HCT116 cell migration,invasion,It may play a role by influencing the PI3K-Akt signaling pathways.3.Ferula ethyl acetate parts could prolong survival time of mice S-180 ascites tumor models,The tumor growth inhibition rate was greater than or equal 40%in mice S-180solid tumor models?P<0.05?,We could concluded that Xinjiang Ferula had anti-tumor effects.