Studies On The Extraction And Separation Process And In Vitro Activity Of Total Flavonoids From Ampelopsis Grossedentata

In recent years,the pharmacological effects of rubaquinone compounds such as anti-tumor,liver protection,hypoglycemia,anti-inflammatory and antibacterial have gradually been paid attention to by scholars at home and abroad,and the demand has also increased year by year.At present,the method for obtaining quinone compounds is mainly through ethanol extraction,but the extraction rate of rubia quinone compounds is low,and its main anti-cancer active components are not yet clear,resulting in the lack of effective application of some medicinal values of rubia quinone.The study of compound extraction process and its anticancer mechanism is of great significance.In this study,ultrasonic-assisted ethanol extraction was used to extract Rubia quinones,the optimal extraction process parameters were optimized by response surface methodology,and UPLC-Q-TOF-MS/MS technology was used to qualitatively analyze the total components of Rubia rubia ethanol extract.MTT method,scratch test,Western Blot and other experiments were used to further explore the effect of the main active components of rubia alcohol extract on the proliferation,migration and apoptosis of human breast cancer MCF-7 cells.The specific research results are as follows:1.The optimal process parameters for the extraction of rubia quinones by ultrasonic extraction:extraction time,ethanol concentration,and liquid-solid ratio were 31.78 min,82.09%,10.36:1 mL/g,and the yield was 29.88%.2.Using ultra-performance liquid chromatography-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）technology,the chemical constituents of rubia rubia ethanol extracts were qualitatively analyzed in combination with the chemical constituents database of rubia spp.and reference substance database.Thirty-four compounds were identified,including 30 positive ions,4 negative ions,and 29 quinone compounds.3.The results of MTT showed that the median inhibitory concentration（IC50）of rubia rubia extract（125-2000 μg/mL）on breast cancer MCF-7 was 211 μg/mL,its main active ingredient,mollugin（10-160 μg/mL）,has a median inhibitory concentration（IC50）of 56.18μg/mL on human breast cancer MCF-7 cells,which inhibits cell proliferation in a concentration-dependent manner.4.The scratch test results showed that a certain dose of mollugin（0,30,60,120 μg/mL）significantly inhibited the migration of breast cancer MCF-7 cells within 12,24,and 48 h.5.Western Blot results showed that mollugin could significantly up-regulate the protein expression levels of P53,caspase 9,caspase 3 and Bax,down-regulate the protein expression level of Bcl-2,up-regulate the ratio of Bax/Bcl-2,and promote the apoptosis of breast cancer MCF-7 cells.In this experiment,response surface methodology was used to optimize the extraction process of Rubia quinones,and the results showed that the optimized ultrasonic-assisted extraction method had a yield of 29.88%.Mechanistic studies showed that rubidin could inhibit the proliferation and migration of breast cancer MCF-7 cells;up-regulate the expression levels of P53,Bax,caspase 3,and caspase 9 proteins,down-regulate the expression levels of Bcl-2 and other proteins,and induce breast cancer MCF-7 cells apoptosis.Finally,it was found that mollugin in rubia ethanol extract was the main active component of rubia rubia ethanol extract against breast cancer,which provided the basis for the development of anti-cancer drugs of rubia.