Preparation And Anti-cervical Cancer Activity Of Glycyrrhiza Inflata Chalcones And Its Derivatives

Objective:?1?Extraction and purification of total flavonoids from Xinjiang Glycyrrhiza inflata and isolation of natural active ingredients which named Licochalcone A and Licochalcone B.?2?Chalcone derivatives were designed and synthesized by using licochalcone B as a lead compound,then the structure was characterized.?3?To study the inhibitory activity of each compound on human cervical cancer cells SiHa,HeLa and normal cell CHO,and to screen candidate drugs with strong anti-cervical cancer activity and low toxicity.?4?To study the pro-apoptotic effects of cervical cancer cells and their effects on the cell cycle.?5?Predict of the binding strength and mode of drug candidates to cervical cancer-related proteins Cyclin-D1,SIRT1,CDK4 and MDM2,and determine the effect of drugs on the content of these proteins.Methods:The crude extract of Glycyrrhiza inflata was extracted by ultrasonic-assisted ethanol extraction,and the total flavones were purified by polyamide and AB-8 macroporous resin,and the Licochalcone A and Licochalcone B were separated by preparative thin layer chromatography combined with silica gel column chromatography.?2?The chalcone derivatives were synthesized by Claisen-Schmidt reaction using substituted acetophenone and substituted benzaldehyde,which were separated and purified by water recrystallization and silica gel column chromatography,its structural characteristics characterized by ¹H-NMR and¹³C-NMR.?3?Cisplatin was used as a positive control,determine the inhibitory activity of each compound on the proliferation of human cervical cancer SiHa and HeLa cells and the toxicity to Chinese hamster normal ovarian cells CHO.The IC50 was calculated using SPSS21.0 and the drug candidates were determined based on the anti-cancer activity and toxicity data.?4?The effects of drugs candidates on the pro-apoptotic effects and cell cycle of SiHa and HeLa cells were determined by flow cytometry.?5?Predict the binding mode of drugs and Cyclin-D1,SIRT1,CDK4 and MDM2 by the computer-aided molecular docking method,and the content of these protein were determine by ELISA.Results:?1?The extract yield of Glycyrrhiza inflata was 21.4%,the flavonoid content in the extract was 17.98%,and the total flavonoid content after purification is 57.96%,which was increased by 40%.?2?Twenty-four chalcone derivatives were synthesized and identified by TLC and structure identification as the target compounds with high purity,and the yield was 10.3⁹1.7%.?3?The MTT results of SiHa?HeLa and CHO cells showed that derivatives had different degrees of inhibition on the three types of cells in a concentration range of 0¹00?g/mL,and the inhibition rate was related to the dose and intervention time positive correlation.The inhibitory activity of most of the derivatives on SiHa and HeLa is higher than lead compound lico B,especially compound 21?2,3,4-trimethoxy-3?,4?-dimethoxychalcone?,it's IC₅₀ of SiHa and HeLa for 24 h was 9.66and 5.64?g/mL,while the IC₅₀ for CHO was 74.36?g/mL.?4?Flow cytometry results showed that Lico A,compound 17 and 21 all had significant pro-apoptotic effects on SiHa and HeLa cells,the apoptosis rate was compound 21>compound 17>Lico A.The cycle results showed that the drug candidate blocked the cell cycle at G₂/M.?5?Molecular docking and ELISA results show that LicoB,LicoA,compound 17 and compound 21have significant binding ability with Cyclin-D1,SIRT1,CDK4 and MDM2,and up-regulate this in the cervical cancer cell signaling pathway to varying degrees.Conclusion:The methoxylated modification of Lico B significantly improved the compound's anti-cervical cancer activity,and chalcone derivatives mainly exerted anti-cervical cancer effects by promoting cervical cancer cell apoptosis and blocking the cell cycle in the G₂/M phase.At the same time,chalcone compounds can also down-regulate the contents of Cyclin-D1,SIRT1,CDK4,and MDM2 in SiHa cells,and exerting anti-cervical cancer effects by inhibiting the degradation of tumor suppressor genes p53 and pRb.