| Objective'Tong-guan-teng':the dry stems of Marsdenia tenacissima(Roxb.)Wight et Am.,is a traditional anti-tumor medicine in Dai medicine,and it has been widely used in the treatment of cancer in Yunnan and Guizhou province.Xiaoaiping,an extract from Marsdenia tenacissima,has been clinically proved to be effective for esophageal cancer,stomach cancer,liver cancer,lung cancer,colorectal cancer,cervical cancer,leukemia,etc.It has good efficacy,high safety and low adverse reactions.It can also cooperate with the adjuvant therapy of radiotherapy and chemotherapy,which has the effect of reducing toxicity,improving the immunity of patients and extending the survival period.It is a natural anticancer drug with good development prospect,but its anti-tumor material base and mechanism are not clear.Lung cancer is a serious threat to human health and survival quality,and has become one of the leading cause of death in human cancer.Its incidence and mortality are in the first place in all kinds of malignant tumor.Therefore,based on the theory of Dai medicine,combined with modern research techniques,we plan to research the effective constituents to anti-neoplasma from Tong-guan-teng and the mechanism of C21 steroid saponins separated from Marsdenia tenacissima to anti-A549 cells on proliferation and metastasis in vitro.What we do will provide a reference for the development of therapeutic drugs to treat lung cancer.Method1.Based on single factor test,using ethanol concentration,liquid ratio and extraction temperature as response factors,the extraction ratio of total saponins as response value,Box-Behnken response surface design was adopted to optimize the extraction process.The macroporous adsorption resin was used to purify total saponins with ethanol elution conentration as influence factor and elution rate as index.2.Marsdenia tenacissima was extracted by ethyl acetate.The extract was isolated and purified by various chromatographic techiques such as silica gel column,sephadex LH-20 column,ODS column and semi-preparative HPLC.The structures of isolated compounds were elucidated on the basis of 'UV,ESIMS,'H-NMR,13C-NMR,1H-1H COSY,HMBC,HMQC,and NOESY analysis.3.CCK-8 method was used to screen the activity of different parts and compounds isolated from Marsdenia tenacissima to inhibit the proliferation of A549 cells.4.The effects of four C21 steroids isolated from Marsdenia tenacissima with good anti-A549 cell proliferation activity screened by CCK-8 method on the migration and invasion of A549 cells were detected by scratch test and transwell test.5.PI/RNase,Annexin V-FITC/PI,JC-1 and DCFH-DA staining and flow cytometry detection were used to detect the effect of four active monomers of C21 steroid on A549 cell cycle phase distribution,cell apoptosis,mitochondrial membrane potential and intracellular reactive oxygen species level.6.The protein expression associated with migration,invasion and apoptosis of A549 cells treated with four active monomers were analyzed by western blot method.Results1.The optimal extraction process was as follows as ethanol concentration of 85%,solvent-solid ratio of 18.5:1(mL/g),extraction temperature of 80 ?.In three verification tests,the average extraction rate of total saponins was 4.80%(RSD=1.06%,n=3),it was close to predicted value 4.89%,and the deviation was 1.84%.D101 macroporous absorption resin had the best purified efficiency when 45%ethanol to remove the impurity and 90%ethanol to purify.In three verification tests,the average total saponins content of total saponins was 62.45%(RSD=0.88%,n=3).2.Totally twenty compounds including fifteen C21 steroids were isolated from Marsdenia tenacissima.Three compounds(15,16 and 18)are novel compounds and cimigenol(14)is isolated from the plant for the first time.3.The inhibitory effect of the different extraction sites and total saponins in vitro against A549 cells was as follows:purified total saponins>ethyl acetate extraction>n-butanol extraction>water extraction>petroleum ether extraction.Seven steroids had certain inhibition effect on the growth of A549 cells in vitro,and six of them(7,8,9,10,13 and 15)showed good activity inhibition effect.4.The relative width of A549 cells treated 28?M TGT-7,44?M TGT-9,29?M TGT-13 and 47?M TGT-15 were significantly higher than that of the blank control group,and the scratch healing rate was lower than that of the blank control group.The number of invasion of A549 cells treated with TGT-7,9 and 13 were decreased by 35.4,39.2 and 6.2 compared with the control group.5.The cell percentage of G0/G1 phase treated with TGT-7,9,13 and 15 was significantly increased from(65.31 ±3.79)%to(75.58±0.44)%,(71.63±2.02)%,(80.27±2.13)%and(69.17± 1.05)%.The apoptosis rate of A549 cells was significantly higher than that of the control group,and was positively correlated with the drug concentration.After JC-1 staining,the red and green fluorescence ratio was decreased from 8.08±1.31 to 1.07±0.92,1.94±0.35,1.58±0.74,3.28±0.57.Compared with the control group,there was a statistical difference(n=3,P<0.001).After DCFH-DA staining,the intensity of green fluorescence treated with TGT-7,9,13,and 15 was 1251±78,1379±94,792±86,and 534±21.Compared with the blank control group 464±28,there was a statistical difference(n=3,P<0.05).6.The expression of MMP-2,MMP-9 and Bcl-2 in A549 cells decreased and the expression of Bax and Cytochrome C increased significantly after the treatment of TGT-7,9,13 and 15.Conclusion1.Follow the optimal condition,the total saponins with high content(>60%)can be prepared,and the method is simple and feasible,and is applicable for large-scale industrialized production.2.Through systematic research of chemical constituents of Marsdenia tenacissima,the chemical composition of C21 steroids were supplemented,and the chemical composition type was enriched.3.C21 steroids compounds had a strong inhibition effect on A549 cells.4.The possible mechanism of TGT-7,9,13 and 15 against A549 cells were as follows:? They all induced G0/G1 phase arrest.?Whey inhibited the migaration and invasion of A549 cells through down-regulating MMP-2 and MMP-9 protein expression.?They finduced apoptosis of A549 cells through decreasing mitochondrial membrance potential and increasing reactive oxygen species.?They could up-regulate the expression Bax and down-regulate bcl-2,release Cytochrome C to induce apoptosis of A549 cells through mitochondrial pathway. |
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