| Oxytropis falcata is a treasure of Chinese medicinal herbs,which is also known as ’the King of Herbal Medicines’ for its good anti-inflammatory and analgesic effects.Modern research shows that chronic inflammation is closely related to the occurrence and development of cancer.Persistent inflammation increases the probability of transformation of surrounding tissue cells to cancer cells.A large number of studies in the early period have shown that the O.falcata is rich in flavonoids and has good anti-inflammatory,anti-oxidation,antibacterial and anti-tumor activities.Therefore,the anti-tumor effect of the O.falcata is widely concerned.However,the active compounds of O.falcata and their anti-tumor mechanisms are not clear,and the relationship between the anti-inflammatory effects and its anticancer effects of O.falcata is really worthy of study.This study began with serum pharmacology,and the influences of flavonoids extracted from O.falcata on the viability,apoptosis and cell cycle arrest of human lung adenocarcinoma A549 cell line were studied in this paper.The effects of the medicated serum on the growth cycle of human lung adenocarcinoma A549 cells were studied.Secondly,the flavonoids of O.falcata were isolated and prepared,and the contents of two flavonoids were determined simultaneously.Finally,the anti-tumor effect and mechanism of flavonoids on A549 cells were studied in vitro.The influence of flavonoids on TLR4/NF-κB pathway in A549 cells were detected by molecular biological techniques.This research could provide theoretical basis for the anti-lung cancer mechanism of O.falcata.1.Effect of serum containing flavonoid of O.falcata(OFF)on viability of human lung adenocarcinoma A549 cells in vitroO.falcata were extracted with 95%ethanol at room temperature,and separated to obtain flavonoid extract(OFF);SD rats were administered medicated serum of OFF at low,medium and high doses,and treated for 4 days.A549 cells of human lung adenocarcinoma were treated with different doses of OFF-containing serum.CCK-8 method was used to detect the antitumor activity of OFF-containing serum and the cell morphological changes were observed.Flow cytometry was used to detect the apoptosis induction and cycle arrest of OFF-containing serum on A549 cells.The results showed that the OFF-containing serum could inhibit the activity of A549 cells,and the cell morphology showed obvious shrinkage and necrosis,with a dose-dependent manner.Compared with blank group,after 24 h of treatment,,the apoptotic rate of A549 cells increased significantly(P<0.001),and the proportion of cells in the G0/G1 growth cycle was significantly increased(P<0.05 or<0.01),with a dose-dependent manner.The results showed that OFF could inhibit the viability of A549 cells,and the action mechanism may be related to the induction of apoptosis and cell cycle arrest in the G0/G1 phase.2.Separation of flavonoids from O.falcata by polyamide chromatography combined with medium pressure preparative liquid chromatographyPolyamide chromatography has a good effect on the enrichment and purification of various flavonoids.The method of medium pressure preparative liquid chromatography is suitable for large-scale extraction and separation of compounds because of its large sample loading and simple procedure.In this study,polyamide was used to fill the chromatographic column,and the flavonoids from O.falcata were preliminarily enriched and separated by dry sampling and ethanol-water gradient elution.Then after the separation of medium pressure preparative chromatography,two flavonoids were isolated and purified by gradient elution of petroleum ether-acetone on the medium pressure preparative chromatography column.The the two flavonoids were identified by 1H NMR,13C NMR and MS to be 7-hydroxy dihydroflavanone(7-HF)and 2’,4’-dihydroxy(2’,4’-DDC)dihydrochalcone.The purities of the two flavonoids were both more than 98%by HPLC analysis.3.Determination of 7-hydroxy dihydroflavone(7-HF)and 2’,4’-dihydroxy dihydrochalcone(2’,4’-DDC)in the extract of O.falcata by HPLCThe contents of 7-HF and 2’,4’-dihydroxy dihydrochalcone were determined by HPLC.The calibration curve of 7-HF was Y=8921.6 X+5.2181,R2=0.9991.It shows that 7-HF has a good linear relationship in the range of 19.80 μg to 198.00 μg.The calibration curve of 2’,4’-DDC was Y=29505 X-1.9793,R2=0.9997.It shows that the 2’,4’-DDC also has a good linear relationship in the range of 20.20 μg to 202.00 μg.The results showed that the contents of 7-HF and 2’,4’-DDC were 37.62 mg·g-1 and 5.51 mg·g-1,respectively.4.Anti-lung cancer mechanism of 2’,4’-DDCCCK-8 assay was used to detect the inhibitory effect of 2’,4’-DDC on A549 cells.Flow cytometry was used to detect its effect on apoptosis of A549 cells.Western Blot and ELISA were used to detect expression levels of TLR4 and downstream proteins and inflammatory factors in A549 cells stimulated by lipopolysaccharide(LPS).The results showed that compared with the blank control group,2’,4’-DDC could inhibit the viability of A549 cells and significantly increase the apoptosis rate of A549 cells,with a dose-dependent trend(P<0.05 or P<0.01).After LPS treatment,the expression levels of TLR4,MyD88,TRAF6,NF-κB proteins and TNF-α,IL-6,IL-1β,VEGF and MMP-2 in A549 cells were all significantly up-regulated(P<0.05 or P<0.01).After 2’,4’-DDC treatment for 24h,the expression levels of these proteins and factors in A549 cells were significantly reduced in this pathway(P<0.05 or P<0.01),suggesting that the anti-lung cancer mechanism of 7-HF was related to blocking of TLR4/NF-κB pathway. |
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