Optimization Of Extraction Process And Anti-tumor Effect Of Total Flavonoids From Nigellaglandulifera Preynet Sint

Objective:This study intends to use the response surface method to optimize the extraction process of total flavonoids from Rhizoma Parvofolia.To test the anti-tumor cell proliferation ability and the anti-tumor cell migration ability of the total flavonoids of Nigellaglandulifera Preynet Sint,and to study the anti-tumor activity and the autophagy-related gene detection of the total flavonoids of Nigellaglandulifera Preynet Sint,and explore the possible mechanism of action.Methods : Response surface methodology(RSM)was used to optimize the conventional extraction process of total flavonoids from nigellaglandulifera preynet sint.By choosing methanol volume fraction,extraction time and extraction temperature as the evaluation indexes.The anti-proliferation ability of total flavonoids from nigellaglandulifera preynet sint human colon cancer cell line SW480? human colon cancer cell line Caco-2? human breast cancer cell line MCF-7 and human glioma cell line U251 was detected by MTT method.The morphological changes of human colon cancer cell line SW480,human colon cancer cell line Caco-2,human breast cancer cell line MCF-7 and human glioma cell line U251 were observed by inverted microscope.The effect on the migration ability of tumor cells was detected by scratch test.The expression levels of autophagy related genes BECN1 and MAPILC3 B were detected by qPCR.Results :1.Response surface methodology(RSM)was used to optimize the extraction process of total flavonoids from nigellaglandulifera preynet sint.: methanol volume fraction 63%,extraction time 1.5 h,extraction temperature62 ?,solid-liquid ratio 1:25 g/mL.the average yield of total flavonoids of from nigellaglandulifera preynet sint was 2.2314 mg/g?2.Detection of the anti-proliferation ability of total flavonoids from nigellaglandulifera preynet sint in vitro: The total flavonoids of nigellaglandulifera preynet sint had inhibitory effects on proliferation ofhuman colon cancer cell line Caco-2,human colon cancer cell line SW480,human glioma cell line U251 and human breast cancer cell line MCF-7.The total flavonoids of nigellaglandulifera preynet sint began to work on human colon cancer cell line Caco-2and human colon cancer cell line SW480 at a drug concentration of 15 ?g/mL(p <0.05),and then increased with the concentration of the drug.The inhibitory rate of total flavonoids from nigellaglandulifera preynet sint on human colon cancer cell line SW480 was also increasing,showing a good dose-effect dependence;The total flavonoids of nigellaglandulifera preynet sint began to take effect on human glioma cell line U251 and human breast cancer cell line MCF-7 at a drug concentration of 10 ?g/mL(P<0.05),followed by drug concentration.The inhibitory rate of the total flavonoids of the nigellaglandulifera preynet sint of the genus Hemp sinensis on the human glioma cell line U251 is also increasing,showing a good dose-effect dependence.3.Detection of anti-tumor cell migration ability of total flavonoids from nigellaglandulifera preynet sint:After 24 h of drug intervention,the cells of human colon cancer cell line Caco-2,human colon cancer cell line SW480,human glioma cell line U251,and human breast cancer cell line MCF-7 control group tended to confluence.There was little change in the scratches of each drug intervention group.The migration distance of tumor cells in each drug intervention group was smaller than that in the blank control group,and the difference was statistically significant(P<0.05).4.The results of real-time PCR analysis of autophagy-related genes showed that the relative expression levels of autophagy-related gene BECN1 in human colon cancer cell line SW480 and human breast cancer cell line MCF-7 were different compared with the blank control group.The relative expression of autophagy-related gene BECN1 in human colon cancer cell line Caco-2 and human glioma cell line U251 decreased in different degrees;the difference was statistically significant(P<0.05).Compared with the blank control group,the relative expression of autophagy-related gene MAPILC3 B in human colon cancer cell line SW480,human colon cancer cell line Caco-2,and human breast cancer cell line MCF-7 was higher than that of the blank control group.The relative expression of autophagy-related gene MAPILC3 B in human glioma cell line U251 was significantly lower than that in the blank control group;the difference was statistically significant(P<0.05).Conclusion: In summary,the response surface method was optimized for the extraction of total flavonoids from nigellaglandulifera preynet sint,and the response Inhibition of in vitro proliferation of human colon cancer cell line Caco-2,human colon cancer cell line SW480,human glioma cell line U251 and human breast cancer cell line MCF-7 was observed.The totalflavonoids of nigellaglandulifera preynet sint have inhibited the migration of tumor cells in vitro against human colon cancer cell line Caco-2,human colon cancer cell line SW480,human glioma cell line U251 and human breast cancer cell line MCF-7.The results of real-time PCR showed that the induction of autophagic death in tumor cells may be one of the mechanisms of anti-tumor effect of total flavonoids from nigellaglandulifera preynet sint.