Preliminary Investigation On The Antitumor Effect And Mechanism Of New Chinese Medicine Compound Preparation With Low Concentration Ethanol Extraction(LTCM)

Objective:1,To screen out the new Chinese medicine compound preparation of RX1-1^RX9-1 and RX1-4^RX9-4 series of traditional Chinese medicine compound preparation,which posses the strongest antitumor effect.2,To investigate the antitumor effect of the new Chinese medicine compound preparation furtherly through animal experiments.3,To investigate the mechanism of antitumor effect of the new compound preparation.Methods:1,50%ethanol is used to prepare RX1-1^RX9-1 series of traditional Chinese medicine compound preparation and 40%ethanol is used to prepare RX1-4^RX9-4 series of traditional Chinese medicine compound preparation.Inhibitory effect of the new Chinese medicine compound preparation on L02,Bel-7402,SMMC-7721,HepG2,A549,NCI-H460,Calu-1,MCF-7 and T47D is detected with MTT assay,in order to filter out the new Chinese medicine compound preparation which posses the strongest antitumor effect and the corresponding tumor cell.2,Subcutaneous tumor model is made with tumor cell in athymic mice,after the model is prepared successfully,gavage with different doses of traditional Chinese medicine compound preparation selected.Every 2 days after administration,the tumor volume,the amount of diet and the weight of athymic mice are observed.At the end of the experiment,the weight of tumor,liver and kidney function are detected,and the pathological sections are stained with HE.3,The cell cycle and apoptosis are detected by Flow Cytometry（FCM）.4,Data statistic is analyzed by variance and rank sum test,and P<0.05 is considered statistically significant.Results:1,RX_1-1-1 of RX_1-1^RX_9-1-1 series of new Chinese medicine compound preparation posses the strongest inhibitory effect on Bel-7402 of liver cancer cells(IC₅₀=0.202 mg/ml)and the less toxicity on L02 of normal liver cells(IC₅₀=0.583 mg/ml).RX_1-4-4 of RX_1-4^RX_9-4-4 series of new Chinese medicine compound preparation posses the strongest inhibitory effect on T47D of breast cancer cells(IC₅₀=0.223 mg/ml).RX_1-1-1 of RX_1-1^RX_9-1-1 and RX_1-4^RX_9-4-4 series of new Chinese medicine compound preparation posses the strongest inhibitory effect on Bel-7402 of liver cancer cells(IC₅₀=0.202 mg/ml)and the less toxicity on L02 of normal liver cells(IC₅₀=0.583 mg/ml).2,The eight days after administered,the difference of tumor volume and body weight of each group is not statistically significant（P>0.05）,and after eight days the difference of tumor volume and body weight of each group is statistically significant（P<0.05）.The inhibitory rate on tumor of the daily administered group is more than the next day administered group at the same dose,and the 300 mg/kg daily administered group posses the strongest inhibitory rate（93.2%）.The diet of different dosage groups and control group is not dose and time dependent.The nephrotoxicity in athymic mice of each dosage group and control group is almost little.The hepatotoxicity of the daily administered group is less than the next day administered group at the same dose.Compared with the control group,the hepatotoxicity of the 300 mg/kg dosage group decreases,and the hepatotoxicity of the 200 mg/kg dosage group and the 100 mg/kg dosage group enhances.The pathological changes of the next day administered group are more obvious than that of the daily administered group.The liver,lung and kidney tissues show no tumor like pathological changes,however,the liver tissue is obviously necrotic.3,Different concentration of RX_1-1-1 induces apoptosis of L02,Bel-7402 and SMMC-7721.The apoptosis rate is in a concentration-dependent manner,with the increase of concentration,the apoptosis rate increases,but the apoptosis rate is weak.At the same concentration,the apoptosis rate of SMMC-7721 is the highest,Bel-7402 Secondly and L02 lowest.RX_1-1-1 blocks the cell cycle of L02in G₀/G₁ phase,the cell cycle of Bel-7402 in G₀/G₁ phase and the cell cycle of SMMC-7721 in S phase,however,the effect is weak and not concentration-dependent.Conclusion:1,RX_1-1-1 posses the strongest inhibitory effect on Bel-7402 of liver cancer cells and the less toxicity on L02 of normal liver cells.2,The antitumor effect of RX1-1 is significant in vivo,however,there is no dose-dependence,moreover,300 mg/kg shows the strongest anti-tumor effect.Increased with the time of administration,the gastrointestinal injury increases gradually.The renal toxicity is little and the toxicity on liver function is obvious,however,the toxicity on liver function of 300 mg/kg dose is the least.3,RX_1-1-1 induces apoptosis and cell cycle arrest of normal liver cells and liver cancer cells.The effect of apoptosis and cell cycle arrest induced by RX_1-1-1 is weak,therefore,it is speculated that RX_1-1-1 exerts its anti-tumor effect not mainly realizing on cell apoptosis and cell cycle arrest.