| ObjectiveThe aims of the study were to isolate and identify novel saponins to enrich the chemical structures of gypenosides from“Gocaekmbaw",Gynostemma pentaphyllum(Thunb.)Makino;to study the structure-cytotoxic activity relationship(SAR)of the isolated saponins to provide scientific references for clinical drug use;to elucidate the mechanism of G.pentaphyllum via cytotoxicity,apoptosis,cell cycle and cell migration of gypenosides.Methods1.The possibility of novel saponins was discovered by literature-oriented and mass spectrometry information comparison.Saponins from the raw herbs Gynostemma pentaphyllum were isolated by synthetically using positive and negative separation packing and normal,medium and high pressure chromatographic column system.Structure elucidation was determined through UV,IR,MS,and NMR spectra and comparison with known literature.Monosaccharide type was identified through acidic hydrolysis and Rfvalue in TLC.2.CCK 8 assay was used to investigate the gypenoside cytotoxicity in NSCLC A549 cells.SAR was derived by comparing chemical structures and IC50 values.3.Inhibitory effect of damulin B on MRC-5,A549 and H1299 cells was preliminarily investigated by using CCK 8 assay.Several related Kits and corresponding antibodies were used to investigate the inhibitory effect of damulin B from apoptosis,cell cycle and migration.4.Inhibitory effect of stereoisomers,Gyp L and Gyp LI,on A549 cells was preliminarily investigated by using CCK 8 assay.Several related Kits and corresponding antibodies were used to investigate the different inhibitory effect of Gyp L and Gyp LI on A549 cells from apoptosis,cell cycle and migration.5.The inhibitory activity of novel saponins in five human cancer cells was investigated by using CCK 8 assay.H2O2-induced cytotoxicity in neuroblastoma cell line SH-SY5Y was regarded as a cultured cell model of oxidative damage to evaluate neuroprotection.Results1.Nineteen saponins were isolated from the raw herbs of"Gocaekmbaw".They were identified as gypenoside LXXVII(GPF-1),2a,3β,12β,20(S)-tetrahydroxydammar-24-ene-3-O-β-D-glucopyranoside-20-O-β-D-glucopyranoside(GPF-2),gypenoside Ⅸ(GPF-3),gypenosideⅧ(ginsenoside Rd,GPF-4),gypenoside LXⅧ(GPF-5),2α,3β,12β,20-tetrahydroxy-25-hydroperoxy-dammar-23-ene-3-O-β-D-glucopyranos yl-(1→2)-β-D-glucopyranosyl-20-O-β-D-xylopyranosyl-(1→6)-β-D-gluco pyranoside(GPF-6),2a,3β,12β,20,24(S)-pentahydroxydammar-25-ene-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-20-O-β-D-glucopy-ranoside(GPF-7),gypenoside LVII(GPF-8),3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-2a,3β,12β,20,24(S)-tetrahydroxy-dammar-25-ene-20-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside(GPF-9),2a,3β,12β,20,25-pentahydroxydammar-23-ene-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-20-O-β-D-glucopyranoside(GPF-10),3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-2a,3β,12β,20,24(R)-tetrahydroxy-dammar-25-ene-20-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside(GPF-11),2a,3β,12β,20-tetrahydroxy-dammar-25-en-24-one-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-20-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside(GPF-12),gypenoside GD5(GPF-13),gypenoside LVI(GPF-14),gypenoside XLVI(GPF-15),gypenoside L(GPF-16),gypenoside LI(GPF-17),damulin B(GPF-18)and damulin A(GPF-19).Among them,there were six novel saponins,which were respectively named as gypenoside J4(GPF-6),gypenoside J1(GPF-7),gypenoside J5(GPF-9),gypenoside J2(GPF-10),gypenoside J6(GPF-11),gypenoside J3(GPF-12).2.Gypenoside LXXVII(GPF-1),gypenoside L(GPF-16),gypenoside LI(GPF-17),damulin B(GPF-18)and damulin A(GPF-19)showed strong inhibitory effect on A549 cells in a dose-dependent manner and their IC50 values were as follows:53.6±2.6μM、30.6±1.4μM、22.7±1.3μM、21.9±1.3μM、22.5±0.9μM,while other gypenosides exhibited weak inhibitory effect with IC50 values more than 80μM.3.Inhibitory effect of damulin B on human lung cancer cellsCytotoxicity:Damulin B showed stronger cytotoxic activities(7.52-30.71%higher inhibition)against A549 and H1299 cells in a dose-dependent manner than that of MRC-5 cells.A dose-dependent manner was more clearly noted treated with damulin B than positive control taxol.Compared with MRC-5 cells,A549 and H1299.cells were more susceptible to damulin B with more obvious cavitation,and they showed a significant increase in detached cells in culture medium,with a round and shrunken shape.Cell apoptosis:Both NSCLC cells treated with damulin B showed green TUNEL-positive cells in a dose-dependent manner and greener and browner fluorescence positive-staining with Annexin V/PI double staining assay.Both apoptotic cell ratio increased in a dose-dependent manner from(5.2±2.3)%to(42.4±3.3)%(A549)and from(7.8±0.5)%to(22.5 ± 4.1)%(H1299).Both cancer cells treated with different concentrations of damulin B for 24 h emitted an apparent green fluorescence,whereas control cells produced an orange-red fluorescence with a slight green fluorescence.The ratio of red fluorescence to green fluorescence decreased from 3.4±0.7 to 0.4±0.1(A549),from 2.0±0.5 to 0.3±0.2.Both cancer cells treated by damulin B emitted a clearer green fluorescence than that of control groups.Flow cytometry analysis was used to investigate production of ROS induced by damulin B.The mean fluorescence intensity increased in a dose-dependent manner from 14.3±0.1 to 143.3±3.6(A549)and from 9.1±0.2 to 76.4±7.6.In both NSCLC cell groups treated by damulin B,extrinsic protein procaspase-8 and intrinsic protein procaspase-9 were both activated in both NSCLC cells.Anti-apoptotic protein Bcl-2 was suppressed compared with that in control groups.Cleaved caspase-8,pro-apoptotic proteins Bax,Bid and tBid were up-regulated in both treatment groups.The release of cytochrome c in the cytoplasm was more than that in the control groups.Cell cycle:Damulin B induced a drastic dose-dependent reduction in both number and size of colonies formed,and colony formation ratio reduced in a dose-dependent manner from(63.5± 2.1)%to(4.0±1.4)%(A549),from(66.0± 2.8)%to(4.8 ± 1.4)%(H1299).In particular,the colony formation rate was less than 5%at concentration of 24 μM.Cell cycle phase distribution induced by damulin B was analyzed using flow cytometry.The percentage of cells at G0/G1 phase increased in a dose-dependent manner from(54.3 ± 2.1)%to(72.9 ± 0.7)%(A549),(66.8± 2.8)%to(82.4± 1.2)%(H1299);cells at S phase decreased from(27.9±2.5)%to(19.0±0.7)%(A549),(21.5±0.3)%to(7.5±1.1)%(H1299);cells at G2/M phase decreased from(17.9±0.4)%to(8.1 ±0.1)%,(16.7±4.0)%to(9.8±0.3)%.The G0/G1 arrest-related proteins,p53,CDK2,CDK4,CDK6,Cyclin D1 and Cyclin E1,were investigated by western blotting.After treated by damulin B,the expression of p53 was up-regulated;the expression of CDK4,CDK6 and Cyclin D1 were suppressed;but those of CDK2 and Cyclin E1 were not downregulated.Cell migration:Transwell assay was performed to detect the damulin B effect on migration.Damulin B inhibited both cancer cell migration from the upper to the lower chamber through the membrane in a dose-dependent manner with migration inhibition rate(42.5±1.8)%,(62.1±3.7)%for A549 cells and(40.7±2.5)%,(59.4±1.6)%for H1299 cells when treatment concentration was 20 μM and 24μM,respectively.The migration-related proteins MMP-2 and MMP-9 were investigated,and their expression was inhibited.Meanwhile,the expression of IL-24 protein was upregelated.4.Similarities and differences of Gyp L and Gyp LI against A549 cellsCytotoxicity:Gyp LI(R configuration in C-20)showed stronger inhibition in A549 cells than Gyp L(S configuration in C-20).Compared with control group,A549 cells treated with Gyp L and Gyp LI for 24 h showed a significant increase in detached cells in culture medium and displayed obvious cavitation.Cells treated by Gyp L showed a thin shape,while treated by Gyp LI showed a round shape.Cell apoptosis:One Step TUNEL Apoptosis Assay found that A549 cells treated with Gyp L and Gyp LI for 24 h showed much stronger green fluorescence than that of control cells.Annexin V-FITC/PI double-staining assay was further used to determine the apoptotic cells.A549 cells treated with Gyp L and Gyp LI for 24 h showed more green fluorescence in low concentration group and more orange or red fluorescence staining in medium or high concentration than that of control cells.The apoptotic cell increased from(7.3 ± 0.2)%to(72.3±1.5)%in Gyp L group,(7.3±0.2)%to(87.4±1.2)%in Gyp LI group.Both gypenosides could increase the release of cytochrome c release into cytoplasm and cause less expression of procaspase 8.Cell cycle:Gyp L and Gyp LI both induced a drastic dose-dependent reduction in both number and size of the colonies formed.Colony formation ratio were respectively 59.3±10.4,39.8 ± 8.2,4.4±0.5 and 57.5±10.6,43.7±1.9,2.5±1.5 after low,medium,high concentration Gyp L and Gyp LI of treatment for 24 h.In particular,the colony formation rate was less than 5%at high concentration of Gyp L and Gyp LI.Gyp L and Gyp LI could induce aberrant distribution at different phases of the A549 cells.Gyp L could increase G0/G1 phase cells with(70.9±0.4)%、(77.7±0.6)%、(83.8±0.6)%at low,medium and high concentration,while the ratio in control group was(66.0±0.8)%.Gyp L could decrease cells in S phase from(25.2±1.6)%to(10.8±0.5)%and cells in G2/M phase from(8.7±2.2)%to(5.4 ± 0.1)%.Gyp LI could increase G2/M phase cells with(24.7±1.0)%、(27.3±0.4)%、(29.8±6.2)%at low,medium and high concentration,while the ratio in control group was(9.6± 3.5)%.Gyp LI could decrease cells in G0/G1 phase from(65.4±1.6)%to(54.3±1.6)%and cells in S phase from(24.9± 2.1)%to(15.9± 4.6)%.The data indicated that Gyp L arrested A549 cells in G0/G1 phase,whereas Gyp LI arrested A549 cells in G2/M phase.For cell cycle-related proteins,Gyp L could obviously down-regulate the expression of CDK2 and CDK4 proteins,whereas the expression of CDK1 protein was close to control group,while Gyp LI could obviously suppressed the expression of CDK1 protein rather than CDK2 and CDK4 proteins,which were even somehow upregulated.Cell migration:Transwell assay indicated that Gyp L and Gyp LI inhibited A549 cell migration from the upper to the lower chamber through the membrane.The inhibition rate of A549 cell migration was(40.3± 7.6)%,(51.5±7.1)%and(65.2±0.1)%treated by Gyp L;(29.5±3.4)%,(42.6±1.3)%and(64.9±0.4)%treated by Gyp LI.The inhibition was in a dose-dependent manner.Both Gyp L and Gyp LI could inhibit MMP2 and MMP9 expression in a dose-dependent manner.5.Novel gypenoside monomers showed weak anti-cancer activity in five human cancer cells(lung cancer A549 and H1299 cells,liver cancer HepG2 cells,gastric cancer SGC7901,prostate cancer PC-3 cells,and neuroblastoma SH-SY5Y cells)with less than 15%inhibition at 100μM.Compared to positive control vitamin C,novel gypenoside monomers had considerable protective effect with 2-15%higher cell viability than H2O2-induced model group.ConclusionNineteen saponins were isolated and elucidated from the raw herbs"Gocaekmbaw".Among them,six gypenosides were new saponins,which has somewhat enriched chemical structures of gypenosides.Summarization about the chemical structures of gypenoside monomers and their inhibitory effect on A549 cells has provided reference for scientific,rational and efficient drug use in clinic.Inhibitory effect of representative saponins on NSCLC cells was investigated.NSCLC cells were more susceptible to damulin B than human normal fibroblasts.damulin B not only induced both intrinsic and extrinsic apoptosis,arrested cell cycle at early G0/G1 phase,but inhibited migration in lung cancer cell lines.Multi-pathways were involved in the process of damulin B inhibiting NSCLC cells.The stereoisomers,Gyp L and Gyp LI both could induce A549 cell apoptosis through extrinsic pathway and intrinsic pathway.Also,they inhibited A549 cell migration through regulating MMP-2 and MMP-9 expression.However,Gyp LI(R configuration at C-20)showed stronger inhibitory effect on A549 cells than Gyp L(S configuration at C-20).Gyp L could block A549 cell cycle at G0/G1 phase,while Gyp LI induced G2/M arrest.To some extent,the material basis of the anti-NSCLC effect of"Gocaekmbaw"was elucidated.The anti-cancer activity of novel gypenosides was much weak,but all showed neuroprotection,which provided drug candidates for neuroprotection in clinic. |
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