The Proliferation Inhibition Effection Of Ingredients Extracted From Yiliuning On Tumor Cells In Vitro

Background and ObjectiveThe malignant tumor is one of those disease that threatening the health and life of human beings seriously. The mortality of it is first in all the diseases. Now, the treatment on it is the synthetic application of surgery, radiotherapy, chemotherapy and biotherapy. Modern chemotherapeutics play an important role in treating malignant tumor. But, following the profounder research, we found that chemotherapeutics have not only definite effects, but some limitations in the clinical application such as severe toxicity, primary or secondary drug resistance, being unable to radical cure and so on. However, traditional Chinese medicines, especially compound prescriptions, are based on an overall analysis of the illness and othe patients' condition, treat tumors in a systemic way, and have their merits such as low toxicity, persistent and stable effect. Clinical observations show some abstractions from herbs not only can anti-tumor directly, but can decrease the toxicity and increase antitumor efficiency of radiotherapy and chemotherapy, and prevent precancerous lesion developing. Effective abstractions or ingredients are basic of herbs' function. More herbs have being applied to explore and develope to new clinical medicine following the development of modern techonology. Thus they are becoming the inevitably tendency in the researche of new antitumor drugs.Yiliuning is a traditional Chinese medicine compound prescription composed of Rab, Aca, Sol and Duc. It has definite curative effect on treating digestive system tumors, and has little adverse reactions. According to our previous experimental results, the four compositions' crude extracts can obviously inhibit the growth of tumor cells of digestive system, which has great research value and application prospect.However, the researchs on abstractions or ingredients from herbs are staying on single ingredient level now, and their clinical curative effect is unsatisfied because of overdose, although they have showed definite antitumour effect in vivo and vitro. There is no report about several ingredients cooperation from different herbs. So this study extracted Oridonin from Rab, ASS(Acanthopanax Senticosus) from Aca, Solanine from Sol, Ducheside from Duc, four ingredients single or compound, use A, B, C, D represent Oridonin, ASS , Solanine, Ducheside separately. We selected human breast cancer MDA-MB-231 cell, lung cancer A549 cell, lung cancer H520 cell and uterine cervix cancer Hela cell be used in the experiment. We took A as the essential ingredient, then compounded with other one, two or three ingredients into experimental groups. We picked out the best grouping and dosing which has the maximum cytotoxicity and the minimum dosis.Methods1. Grouping. We set to set control group and 11 experimental groups, they are: A,B,C,D,A+B,A+C,A+D,A+B+C,A+B+D,A+C+D,A+B+C+D.2. The growth inhibitory rate of each group was measured by MTT assay. The cells in the logarithmic phase were inoculated in the plate with 96 wells, and inoculated for 24h , each group of different concentrations was set in 6 wells and reapeated 3 times, treating on MDA-MB-231,A549,NCI-H520,Hela cells for 48h , then test the growth inhibition ratioes.3. After each group treating on MDA-MB-231 cells for 48h, the variations of morphology would be observed in culture medium by inverted microscope.4. The cell cycle and apoptosis of MDA-MB-231 cells were detected by flow cytometry (FCM).5. Statistical analysis SPSS11.0 software was used to carry out statistical treatment. T-test and x²-test were employed to analysis statistics of one group. and q-test was employed to analysis statistics of parameters between different groups, The significance level of test was "α= 0.05". Results1. The proliferate inhibition analysis of MDA-MB-231,A549,NCI-H520,Hela cells after four ingredients single or compound treating on them for 48h:1.1 MDA-MB-231 cells: Among single ingredient groups, the inhibition function of A is the best (IC₅₀=4.78μg/ml) ,C is the second (IC₅₀=1.15mg/ml), B is the worst(IC₅₀=2.15mg/ml). Among compound groups, four ingredients compound group (A 2μg/ml + B 0.6 mg/ml + C 0.4 mg/ml + D 0.4 mg/ml) is the best group, having the maximum cytotoxicity and the minimum dosis.1.2 A549 cells: Among single ingredient groups, the inhibition function of A is the best (IC5o=16.26ng/ml), C is the second (IC₅₀=0.51 mg/ml), B is the worst(IC₅₀=1.92mg/ml) . Among compound groups, four ingredients compound group (A 4μg/ml + B 0.2 mg/ml + C 0.1 mg/ml + D 0.4 mg/ml) is the best group, having the maximum cytotoxicity and the minimum dosis.1.3 NCI-H520 cells: Among single ingredient groups, the inhibition function of A is the best (IC₅₈₀=31.56μg/ml), D is the second (IC₅₀=0.71 mg/ml), B is the worst(IC₅₀=2.43mg/ml)o Among compound groups, four ingredients compound group (A 10μg/ml + B 0.6 mg/m + C 0.1 mg/ml + D 0.2 mg/ml) is the best group, having the maximum cytotoxicity and the minimum dosis.1.4 Hela cells: Among single ingredient groups, the inhibition function of A is the best (IC₅₀=23.44μg/ml), C is the second (IC₅₀=1.73mg/ml), D is the worst(IC₅₀=2.26mg/ml). Among compound groups, four ingredients compound group (A 10μg/ml + B 0.6 mg/ml + C 0.4 mg/ml + D 0.4 mg/ml) is the best group, having the maximum cytotoxicity and the minimum dosis.2. After each group treating on MDA-MB-231 cells for 48h, the variations of morphological observation with inverted microscope:2.1 Control group: MDA-MB-231 cells are adhering to the bottom of culture capsule, long and thin like shuttles and contacting with each other, all cells are transparent, the state of growth is very well.2.2 A group : We can observe that some MDA-MB-231 cells have changed in 48h, detached from neighbours, rounded up, shrinked, some cells are not transparent like control group, the state of growth is very well.2.2 Compound group of "A+B+C+D": We can observe that many MDA-MB-231 cells have changed in 48h, cells detached from neighbours, rounded up, shrinked, most of cells are not transparent, in which often contained intact organells and intact plasma membranes, the nucleus shrinked and the chromatin condensed.3. Testing the cell cycle and apoptosis ratios of MDA-MB-231 cells by means of flow cytometry.After A group and compound group of "A+B+C+D" treating on cells for 48h, the s-period cells' percentage of each experimental group was obviously increased and the cells' percentage from G₀-period to G₁-period was obviously decreased, and each experimental group presented the sub-G₁-summit which was the sign of apoptosis, the apoptosis ratio was increased. Compared with blank groups, the difference had statistical significance (P<0.05) .Conclusion1. Among four ingredients' single function on four tumor cells: the inhibition function of A is the best, in which its function on MDA-MB-231,A549 cells is better than NCI-H520,Hela cells relatively.2. Four ingredients have great cooperation effect, they can increase inhibition effection cooperatively with each other. "A+B+C+D" group has the maximum cytotoxicity and the minimum dosis comparing with single action.3. The best composition of Yiliuning's effective antitumor ingredients is "A+B+C+D". Its inhibition function on MDA-MB-231 cell is the best, on A549 cell is the second and Hela cell is the worst relatively.4.Either single action or compound action of four ingredients from Yiliuning have great inhibition function. S-period blockage and induction apoptosis of tumor cells are the mechanisms of anti-tumor action. These results provide scientific basis for the further study and clinical application of this prescription.