Preparation,Anti-cancer Activity And Molecule Mechanism Of Saponins From Polygonatum Sibiricum

Polygonatum sibiricum is a homologous resource of medicine and food recognized by the Ministry of Health of China,and was broadly applicated in natural medicine and functional food industry.Saponins are the main active substances in Polygonatum sibiricum.Due to their various structures,their antibacterial and anti-tumor mechanisms are also different.Research on these mechanisms will provide theoretical basis and technical support for the comprehensive utilization of Polygonatum sibiricum resources.In this thesis,high-purity dioscin and methyl protodioscin were prepared from Polygonatum sibiricum.The anti-tumor activity and related molecular mechanisms were studied.The main research contents and results are shown as follows:(1)Separation and purification of saponins from Polygonatum sibiricum.The optimal process conditions for ultrasound-assisted extraction of saponins in Polygonatum sibiricum were determined by single factor and response surface optimization experiments: ethanol concentration of 78%,solid-liquid ratio of 1:20 g/mL,ultrasonic temperature at 58 °C and ultrasonic time of 75 min.In this condition,the yield of saponin was 2.72%.On this basis,dioscin and methyl protodioscin with purity of 98.05% and 95.13% were obtained by silica gel column chromatography and Sephadex column chromatography and were identified by UPLC-TOF-MS/MS and NMR analysis.(2)Inhibition and molecular mechanism of diosgenin on human hepatocellular carcinoma cell line HepG2.Cell viability and cycle analysis showed that the semi-inhibitory concentration was 8.34 μM,and it can arrest the cell cycle in G2/M phase.After treated with 9 μM dioscin,the rate of early apoptotic cells could reach 34.0%.The changes in expression levels of cell cycle-related genes and proteins including CHK2,p53,p21,CDK1 and Cyclin B1 were analyzed by qPCR and Western blot,and indicated that dioscin can block cell cycle in G2/M phase;and changes in the expression levels of Bak,Bcl-xl,FasL,TNF-R1,and Caspase-3,8,9 and other genes and proteins indicate that dioscin can induce apoptosis of HepG2 cells through the mitochondrial pathway and the death receptor pathway.Other genes,such as PI3 K / Akt / mTOR signaling pathway-related genes,Smac and JNK,are also involved in the differentiation of dioscin-induced apoptosis,and the accumulation of intracellular reactive oxygen species may also be one of the causes of the apoptosis.(3)Inhibition and molecular mechanism of methyl protodioscin on human cervical cancer cell line HeLa.Cell viability and cycle analysis showed that the semi-inhibitory concentration was 18.31 μM,and it can arrest the cell cycle in G2/M phase.After treated with 18 μM dioscin,the rate of apoptotic cells reached up to 46.58%.By detecting the expression levels of Bak,Bcl-xl,Fas and Caspase-3,8,9 and other genes and proteins,as well as the expression levels of related proteins after Caspase-8 inhibitor Z-IETD-FMK and Caspase-9 inhibitor Z-LEHD-FMK pretreatment,we proved that methyl protodioscin can induce apoptosis of Hela cells through mitochondrial and death receptor pathway.In addition,the increase of intracellular reactive oxygen species and the changes of mRNA levels of Smac,IAPs,NF-κB and JNK may also be associated with methyl protodioscin-induced apoptosis of Hela cells.