Inhibition Of Total Flavonoids From Cudrania Tricuspidata On Proliferation Of Lung Cancer Cell And Its Mechanism

Lung cancer is the most common cancer in our country,its morbidity and mortality are all in the forefront of many tumors.At present,most of the chemotherapeutic drugs used to treat cancer are components of western medicine,and their serious side effects restrict the use of western medicine.Therefore,the research on multi-component and multi-targets of traditional Chinese medicine has drawn increasing attention.Cudrania tricuspidata is a new anti-tumor medicine,the main chemical components of flavonoids,alkaloids and polysaccharides.At present,Cudrania tricuspidata is widely accepted by researchers because of its anti-tumor pharmacological effects,and its pharmacological research is also expanding.The existing research has made it clear that Cudrania tricuspidata has certain therapeutic and remission effects on gastric cancer,intestinal cancer,breast cancer and liver cancer.Although many researches show that total flavonids of Cudrania tricuspidata can inhibit the growth of tumor cells,the research about the effect of total flavonids from Cudrania tricuspidata on lung cancer cells has not been reported yet.In this paper,the best extraction process of total flavonoids from Cudrania tricuspidata was optimized;the method of D101 macroporous adsorption resin was used to explore its process and optimize the enrichment of total flavonids from Cudrania tricuspidata;LLC cells were used as research object to investigate the effect of total flavonids from Cudrania tricuspidata on lung cancer mice.The anti-tumor activity of the preliminary study of its mechanism of action for clinical research to provide a theoretical basis.Response Surface Method(RSM)was applied to optimize the extraction process conditions of total flavonoids from Cudrania tricuspidata.According to the principle of Box-behnken response surface method,four influencing factors viz.,ethanol concentration,solid-liquid ratio,extraction time and extracting frequency were selected to investigate flavonoids yield based on single factor test.To establish quadratic regression model,and to determine optimum technological conditions for extracting flavonoids.The optimum extraction process conditions were shown as follows:the concentration of ethanol was 90%,solid-liquid ratio was 16:1,the extraction time was 116min,extracting frequency was 3 times.The extraction rate of flavonoids under this condition was 3.33%,with small theoretical prediction relative error of 2.63%(<5%).D101 macroporous resin was found to have good the adsorption and elution effects,the best conditions for separation and purification were pH value of 4.50,the sample mass concentration was 1.88mg/mL,the loaded amount was 6mg/mL,on the sample volume flow was 1Bv/h,and then the sample was washed with 2BV water,2BV 20 percent ethanol and 5BV 75 percent ethanol in turn.The purity of total flavonoids increased to 66.24 percent after the purification with D101 resins.The D101 macroporous resin is best used for enriching total flavonids of Cudrania tricuspidata,and it is ideal separation and purification medium.In vivo studies have investigated the anti-tumor activity of total flavonoids of Eucalyptus urophylla on LLC lung cancer mice.The results showed that total flavonoids of eucalyptus could improve the body weight of mice and protect immune organs such as spleen and thymus.The high dose group significantly inhibited the growth of tumors,and the tumor inhibition rate was as high as 67.04%.Pathological sections showed that the nuclei became smaller,the nuclear pyknosis,and the shape of the tumor cells were significantly damaged.In the tumor tissues,a large area of necrosis and a large number of nuclear fragmentation were common,with inflammatory cell infiltration around them.Finally,the serum levels of TNF-α,IL-2,IL-6 and IL-12 in mice were detected by ELISA method.Compared with the model group,the total flavonoids in the treated group could increase its content,suggesting that the total flavonoids of eucalyptus It may be able to achieve anti-tumor effects by regulating immune function.In order to further explore the antitumor mechanism of total flavonoids from Alnus japonicus,the components of total flavonoids from Alnus crestis were identified by UPLC.The flavonoids were initially identified.Compound 1 was Taxifolin,Compound 2 was naringenin,Compound 3 For kaempferol.Finally,MTT assay of three monomer components on the inhibition of LLC cells,measured OD values to determine the proliferation of LLC lung cancer cells to explore the role of anti-tumor mechanism.Taxifolin inhibited LLC cell proliferation in a concentration range of 0.156-5mM in a concentration-dependent manner with an IC50 value of about 0.45mM.Naringenin inhibited the proliferation of LLC cells in a concentration range of 12.5-200μM in a concentration-dependent manner,while 12.5μM and 25μM had no significant effect on cell proliferation with an IC50 value of about 0.43 mM.The kaempferol inhibits the proliferation of LLC cells in a concentration range of 12.5-200μM in a concentration-dependent manner,while 12.5μM has no significant effect on cell proliferation with an IC50 of about 60μM.The effects of kaempferol on P62 and LC3 in LLC cells were detected by Western Blot.Autophagy was detected by GFP-LC3 and MDC fluorescence intensity was quantified by flow cytometry.All the results showed that kaempferol could promote autophagy in LLC lung cancer cells.