| Objective:Primary liver cancer is one of the malignant tumors,and nearly 600 thousand people die each year.China is a high incidence area of liver cancer,and its incidence is increasing.The onset of primary liver cancer is occult.Most of the cases are in the late stage of diagnosis,and have lost the best time for radical operation,and the recurrence rate of the disease is high.Radiotherapy,chemotherapy and targeted therapy can control the development of liver cancer to some extents,but at the same time,that kind of treatments can cause secondary injury to patients.Therefore,it is of great practical significance to explore safe and effective anti hepatoma drugs.Turmeric,Astragalus and gynostemma pentaphyllum are commonly used traditional Chinese medicinal herbs.The study shows that all of them have anti-tumor effects,The inhibition of tumor cell proliferation has its limitations.Therefore,this study follows the characteristics of compatibility of Chinese medicine.Turmeric,astragalus,Gynostemma pentaphyllum and other six kinds of Chinese medicine were formulated according to different proportions,in order to interact with each other and achieve synergistic effect.Useing 95% and 75% ethanol to extract the effective components,and study the antitumor activities,apoptosis andintracellular distribution.Methods:1.Setting up several Chinese herbal prescription with turmeric as the main component,using 95% and 75% ethanol to extract the effective components,respectively.2.In this study,hepatoma cell lines SMMC-7721,SK-HEP-1,HepG2 were used as experimental subjects.We used the methyl thiazolyl tetrazolium(MTT)and cell morphology observation to evaluate the inhibition of Chinese medicine extract on hepatoma cell lines at 48 h,72 h and 96 h,respectively.Comparing the effect of Chinese medicine extract that extracted by two different volumes of ethanol,and screen out the most sensitive cell line,the best effect of Chinese medicine extract.3.The distribution of R1 and R7 in the cancer cells was detected by confocal laser scanning microscope.4.The apoptosis of SMMC-7721 cell dosed of R1 and R7 respectively was inspected with flow cytometry.Results:1.The extract of Chinese herbal prescription with turmeric as the main component were extracted successfully.2.MTT experiment showed that the extracts R1,R2,R3,R4,R5,R6,R7 extracted by 95% ethanol to 3 cancer cell lines had a manner of time and dose-dependent,the inhibitory effects of extracts R3,R4 and R5 on the proliferation of 3 hepatoma cell lines were significantly lower than that of otherfour drugs.At 72 h,the IC50 of R3 to SMMC-7721、SK-HEP-1 and HepG2 were 203 μg/ml,211 μg/ml and 220 μg/ml,the IC50 of R4 and R5 has not been detected.At 48 h,the inhibition of R1,R2,R6,R7 to the 3 hepatoma cell lines were in the order of R1 > R7 > R6 > R2;At 72 h and 96 h,the inhibition of R1,R2,R6,R7 to SMMC-7721 and HepG2 were in the order of R1 > R7 > R6 >R2,to SK-HEP-1 was in the order of R1 > R6 > R7 > R2.The extracts R1’,R2’,R6’,R7’extracted by 75% ethanol to 3 cancer cell lines at 48 h,72 h and 96 h,respectively,only the IC50 of the extract R1’ can be detected,extract R1’ was sensitive to SMMC7721 cell line,the IC50 of R2’,R6’and R7’has not been detected.Comparing the effect of Chinese medicine extract that extracted by two different volumes of ethanol,the effect of 95% ethanol extract is stronger than that of 75% ethanol extract.At 72 h and 96 h,the IC50 value of R1 against SMMC-7721 cell was 1 times lower than that of R1’.Derivatives R1 and R2 had good tumor cell proliferation inhibitory activities.The most sensitive hepatoma cell line to extracts action was SMMC-7721.The toxicities of R1,R2,R6,R7 to normal liver cell L02 are low.3.In the drug group,the nucleus of SMMC-7721,SK-HEP-1,HepG2 cells were not only dyed blue by PI,at the same time,the cell nucleus had the green fluorescence of the drugs,but in the blank group,the nucleus of cancer cells only had blue fluorescence of PI.This shows that R1 and R7,a mainly entered into the nucleus of cancer cells for playing a antitumor role.4.At different concentrations(20 μg/ml,15 μg/ml,10 μg/ml),the SMMC-7721 cell apoptosis of R1 and R7 were 19.89%,12.76%,8.86% and15.78%,11.80%,8.73% respectively at 48 h.It showed that R1 and R7 both could promote SMMC-7721 cell apoptosis.Conclusions:1.R1 and R7 have good tumor cell proliferation inhibitory activities,their toxicities to normal liver cells are low.The most sensitive hepatoma cell line to extracts action was SMMC-7721.2.Comparing the effect of Chinese medicine extract that extracted by two different volumes of ethanol,the effect of 95% ethanol extract is stronger than that of 75% ethanol extract.3.R1 and R7 mainly entered into the nucleus of cancer cells for playing a antitumor role.4.R1 and R7 can inhibit the proliferation of hepatoma cells by promoting apoptosis mechanism. |
