The Study On The Extrasomatic Proliferate Inhibition Of Yiliuning In Treating Alimentary System Tumor Cell

ObjectiveMalignant tumor is a common disease that can severely threaten our health and life, it is also a hang-up of the global medical science. Now, the treatment to it is the synthetic application of surgery, radiotherapy, chemotherapy and biotherapy. The medication has an influential position in the treatment of the patients who are in the advanced stage.Chemotherapeutics have definite efficacy, but they also have some problems for their severe side effects, high price and multidrug resistance,these can lead to the aggravation of patients' conditions and the degression of quality of life, even lead the patients to give up.Traditional Chinese medicines, especially compound prescriptions, treat tumors in a systemic way, have their merits such as widespread medicinal herbs resource,low price and little side effects.The chemistry of traditional Chinese medicine look for active components through extraction, abstraction, depuration and assessment of simplex and complex prescriptions.This is becoming an important way to obtain new anticancer drugs, thus it is becoming the hot spot in the researches of antitumor drugs.Yiliuning is a traditional Chinese compound prescription composed of Rab, Aca, Sol and Duc. It has definite curative effect on the treatment of tumours of digestive system, and has little side effects. According to our experimental result of its four compositions' crude extracts, Rab compound with Duc and Rab compound with Aca, Sol and Duc both can obviously inhibit the growth of tumour cells of digestive system, Rab compound with Aca, Sol and Duc can arrest the Eca-109 cells during S-phase and induce cell apoptosis. But it hasn't been made the pharmacodynamics study of the four compositions' effective antitumor ingredients, the composition and dose of recipe have yet to be selected through the experiments. We selected human esophageal cancer Eca-109, gastric cancer cell line BGC-823, liver cancer cell Bel-7402 and colon cancer cell line Ls-174t to carried out this extrosomatic antitumor experiment in vitro. By consulting the reference literatures,we chose ingredient A(Oridonin) from Rab, ingredient B (ASS) from Aca, ingredient C (Solanine) from Sol and ingredient D (Ducheside) from Duc,and 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 which has the maximum cytotoxicity and the minimum dosage.Methods:1. The growth inhibitory rates in each group were measured by MTT assay. The cells in the logarithmic phase were inoculated in the board with 96 holes, and were taken incubation after 24 hours, to set control group and experimental group.Every concentration was set in 6 holes and reapeated 3 times, after acted on Eca-109,BGC-823,Bel-7402,Ls-174t cells for 48h, then test the growth inhibition rates.2. After the medicine acted on Eca-109 cells for 48h, the variations of morphology were observed in culture medium by inverted microscope.3. The cell cycle and apoptosis of Eca-109 cells were detected by flow cytometry (FCM).4. With SPSS10.0 software package,one-way ANOVA and Chi-Square test were used to analyze all experimental data, size of the test was P<0.05.Results:1. The proliferate inhibition on Eca-109,BGC-823,Bel-7402,Ls-174t cells. 1.1 The proliferate inhibition on Eca-109 cells. When the compositions were " A (10μg/mL) + B (1mg/mL)" , "A (15μg/mL) + B (1mg/mL) +C (0.4mg/mL)", "A(10μg/mL)+B(1mg/mL)+D(3.2mg/mL)"or "A(10μg/mL)+B(0.5mg/mL)+C (0.4mg/mL) +D (1.6mg/mL)" ,the growth inhibitory rate could reach the highest, it was 100%.1.2 The proliferate inhibition on BGC-823 cells. When the compositions were "A(20μg/mL)+B(1mg/mL)" , "A(20μg/mL)+B(1mg/mL)+C(0.8mg/mL)" , "A(30μg/mL) +B(1mg/mL)+D(3.2mg/mL)" , "A(15μg/mL)+B (0.75mg/mL)+C(0.4mg/mL)+D(3.2mg/mL) " , the growth inhibitory rate could reach the highest, it was 100%.1.3 The proliferate inhibition on Bel-7402 cells. Two ingredients: the highest growth inhibitory rate was 90.80% when the composition was "A (15μg/mL) + B(2mg/mL) " .Three ingredients : the highest growth inhibitory rate was 76.31 % when the composition was"A(30μg/mL)+ B( 1mg/mL)+C( 1.6mg/mL)". Four ingredients: the highest growth inhibitory rate was 94.23% when the composition was "A(20μg/mL) +B (1mg/mL) +C (0.4mg/mL) +D (3.2mg/mL)" .1.4 The proliferate inhibition on Ls-174t cells. Two ingredients: the highest growth inhibitory rate was 31.46% when the composition was "A (30μg/mL) + B(1mg/mL)" .Three ingredients:the highest growth inhibitory rate was 28.76% when the composition was"A(20μg/mL)+ B(1mg/mL)+C(0.8mg/mL)" . Four ingredients: the highest growth inhibitory rate was 36.54% when the composition was "A(20μg/mL) +B (1mg/mL) +C (0.8mg/mL) +D (1.6mg/mL)" .2. By means of flow cytometry to test the cell cycle and apoptosis rates of Eca-109 cells.After acting on cells for 48 hours, the S phase cells percentage in every experimental group was obviously increased and the G₀/G₁ phase cells percentage was obviously decreased. Each experimental group presented the sub-G₁-summit which was the sign of apoptosis, the apoptosis rate was increased.Compared with blank group,the difference had statistical significance (P<0.05) . Conclusion:1. The compositions "A + B"or"A + B+ C+ D"have a markedly extrasomatic proliferate inhibition on Eca-109,BGC-823,Bel-7402,Ls-174t cells.2. The compositions "A + B"and "A + B+ C+ D" can arrest the Eca-109 cell in S phase and induce its apoptosis.3. The best compositions of Yiliuning's effective antitumor ingredients are "A + B"and "A + B+ C+ D", and they provide scientific basis for the further study and clinical application.