Preparation Of Gypenoside L And Gypenoside LI And Their Screening And Mechanism Of Action On Different Cancer Cells

ObjectiveGypenoside L and gypenoside LI are saponins of Gynostemma pentaphyllum.The aim of the paper was to prepare gypenoside L and gypenoside LI from heat-processed G.pentaphyllum,screen their activities against human cancer cells and analyze their anti-cacer mechanism.Methods1.Gypenoside L and gypenoside LI were isolated by liquid-liquid extraction,silica gel column chromatography,rapid preparation chromatography and semi-preparative HPLC from heat-processed G.pentaphyllum.The chemical structures were identified with UV,ESI-MS and NMR data.2.The activities of gypenoside L and gypenoside LI against A549 cells,HepG2 cells,EC-109 cells,and PC-3 cells were investigated using CCK 8 assay.The activity was expressed as the IC50(concentration in?g/mL required to inhibit cancer cells by 50%).The differences of their activities against the cancer cells were compared.3.CCK 8 assay was used to test the relationship between cytotoxicity and time or dosage of gypenoside L and gypenoside LI.The activities against A549 cells of them were compared with the positive control ginsenoside Rg3.4.The A549 cell cycle and apoptosis of gypenoside L and gypenoside LI were detected by PI/Rnase staining,Annexin V-FITC/PI,JC-1,DCFH-DA staining and flow cytometry.5.The effects of gypenoside L and gypenoside LI on the migration and invasion of A549 cells were detected by scratch test and transwell test.6.The protein expression associated with apoptosis,migration and invasion of A549 cells treated with gypenoside L and gypenoside LI was detected by western blot method.Results1.Gypenoside L(2.2 g)and gypenoside LI(0.9 g)were isolated from heat-processed G.pentaphyllum 80%ethanol total extract and the purity was over 98%.2.Gypenoside L and gypenoside LI showed cytoxic activities against A549 cells,HepG2 cells,EC-109 cells,and PC-3 cells in a dose-dependent manner.When treated for 24 h,the IC50 of gypenoside L were 21.09 ± 3.60,28.31 ±1.48,78.70 ± 3.06 and 96.16 ± 2.43 ?g/mL,respectively and the IC50 of gypenoside LI were 17.09 ± 0.63,17.70 ±1.15,19.05 ± 2.82 and 46.03 ± 6.54 ?g/mL,respectively.3.The activities of gypenoside L and gypenoside LI against A549 cells showed in a dose-and time-dependent manners.They showed stronger anticancer activities than Rg3 at 30 ?g/mL for 24 h.4.When treated with 24 ?gg/mL gypenoside L,the percentage of G0/G1 phase A549 cells was increased significantly from(66.42 ?0.15)%to(76.23 ± 1.41)%.The apoptotic rate of A549 cells was increased from(0.57 ± 0.18)%to(24.62 ± 0.70)%.The ratio of red/green fluorescence was decreased from(15.44 ± 2.29)%to(4.09 ± 0.23)%,indicated that mitochondrial membrane potential depolarization was obvious.The production of reactive oxygen was obviously increased and the mean fluorescence intensity was increased from 14.3 ± 0.14 to 142.5± 4.03.When treated with 17 ?g/mL gypenoside LI,the percentage of G2/M phase A549 cells was increased significantly from(6.9 ± 0.32)%to(25.83 ± 1.63)%.The apoptotic rate of A549 cells was increased from(0.57 ± 0.18)%to(22.44 ± 0.71)%.The ratio of red/green fluorescence was decreased from(15.44 ± 2.29)%to(4.43 ± 0.02)%,indicated that mitochondrial membrane potential depolarization was obvious.The production of reactive oxygen was obviously increased and the mean fluorescence intensity was increased from 14.3 ± 0.14 to 152.0 ± 5.59.5.The scratch healing rate of A549 cells treated with 20 ?g/mL gypenoside L and 15 ?g/mL gypenoside LI was 30.45%and 19.87%lower than that of the control group,and the cell invasion rate was 39.03%and 51.22%lower than that of the control group.6.The expression of IL-24 protein in A549 cells was increased and the expression of MMP-2 and MMP-9 proteins were decreased significantly treated with low-,middle-and high-dose of gypenoside L and gypenoside LI.Conclusion1.20(S)-gypenoside L and 20(R)-gypenoside LI can be isolated from the heat-processed G.pentaphyllum.2.Gypenoside L and gypenoside LI showed inhibitory activities against A549,HepG2,EC-109 and PC-3 cells by CCK 8 assay,especially they showed strong inhibitory effects on A549 and HepG2 cells,while weak inhibitory effect on PC-3 cells.Gypenoside LI showed stronger inhibitory activity than that of gypenoside L on EC-109 cells.3.The possible mechanism of gypenoside L and gypenoside LI against A549 cells were as follows:? Gypenoside L induced G0/G1 phase arrest and gypenoside LI induced G2/M phase arrest in A549 cells.? Both of them induced apoptosis of A549 cells mitochondria-mediated intrinsic pathway through the loss of mitochondrial membrane potential and the generation of reactive oxygen species.? They inhibited migration and invasion of A549 cells by decreasing scratch hilling rate,invasion rate,MMP-2 protein and MMP-9 protein.? Gypenoside L and gypenoside LI up-regulated the expression of IL-24,which indicated both of them may inhibit the proliferation,migration and invasion of A549 cells and induce apoptosis of A549 cells through IL-24 related pathways.