| Objective:To establish a rapid and simple way to identify the species of Glycyrrhiza Inflata, and to prepare target compounds by modifying the structure of isoliquiritigenin which is lead compounds,and study on the anticancer activity of human cervircal cancer. Methods:(1) With the special flavonoid licochalcone A as reference, the species of Glycyrrhiza inflata by using TLC were identified. (2) 3-methoxy isoliquiritigenin (3-MO-ILG) was synthesized by three methods of acid catalysis, the hydroxyl-protecting and microwave solid-phase synthesis, and then we selected a high yield synthetic method, which was the microwave solid-phase synthesis. The rest of the target compounds were synthesized with microwave solid-phase synthesis. (3)With the SiHa cells and HeLa cells of human cervircal cancer in vitro model and clinical commonly cisplatin as positive control, the target compounds were intervening for SiHa cells and HeLa cells in 24 h,48 h, and 72 h. The proliferation inhibition rates of target compounds for SiHa cells and HeLa cells were determined by MTT assay in order to the candidate compounds, which have strong biological activity. The apoptosis of SiHa cells and HeLa cells were measured by flow cytometry. Finally, The structure-activity relationship of candidate compounds was analysised. Results:(1) According to the results of TLC identification, a bright yellow fluorescent spots of licochalcone A could be observed at ethanol extract of Glycyrrhiza inflata from different areas. By preparative TLC, separation, purification and recrystallization to get the bright yellow needles, which was licochalcone A with the physical and chemical and spectral identification. (2) The yields of 3-methoxy isoliquiritigenin was respectively 8.9%、9.27% and 57.60% by using three methods of acid catalysis, the hydroxyl-protecting and microwave solid-phase synthesis. With a higher yield synthetic method-microwave solid-phase synthesis, the yields of Isoliquiritigenin, 3,4’-Dihydroxy-4-methoxy-chalcone,3-hydroxyl-isoliquiritigenin and 4,4’-dihydroxy-chalcone was 58.9%、52.9%、48.89%、38.75%.(3)According to the experimental results of proliferation inhibition of SiHa cells and HeLa cells by MTT assay, when the concentration was at 12.5μg/ml,25μg/ml,50μg/ml,100μg/ml,150μg/ml and 200 βg/ml, the target compounds were intervening for SiHa cells and HeLa cells in 24 h,48 h, and 72 h. The inhibition rates were respectively about 10%～50%、20%～70% and 40%～80%.The target compounds have the time-dependent manner. (4) The target compound screened-Isoliquiritigenin,’3-methoxy-isoliquiritigenin,3-hydroxyl-isoliqui-ritigenin and 3,4’-Dihydroxy-4-methoxy-chalcone have strong biological activity. Then, the apoptosis of SiHa cells and HeLa cells is measured by flow cytometry with Isoliquiritigenin,3-methoxy-isoliquiritigenin,3-hydroxyl-isoliqui-ritigenin and 3,4’-Dihy-droxy-4-methoxy-chalcone. The experimental results show that its apoptosis of SiHa cells and HeLa cells was> 30% at the concentration of 100μg/ml. It means that they have a significant biological effect on SiHa cells and HeLa cells. Conclusion:(1)TLC is a effective method for the rapid and simple identification of the species of Glycyrrhiza inflate. It has a certain reference value for the rapid identification of Glycyrrhiza inflate, research resources of licorice and quality control of its products. (2) With the study of acid catalysis, hydroxyl protection and microwave solid-phase synthesis methods, we have screened out a green, low energy consumption, no pollution, easy operation, low cost and high yield method-microwave solid phase synthesis to finish structural modify and synthesis of isoliquiritigenin derivatives and target compounds, which have significant inhibition rates and apoptosis rates of target compounds for SiHa cells and HeLa cells by MTT assay and by flow cytometry. It is to lay an important material foundation for effective candidate drugs with cervical cancer from licorice chalcone isoliquiritigenin derivatives. |
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