Studies On The Chemical Constituents,Anti-Tumor Activity And Relationships Of Dioscorea Plants

Dioscorea species are important medicinal plants widely distributed in China,among which comprehensive chemical and pharmacological studies are still needed.Moreover,a considerable inconsistency still exists in the taxonomy of this genus.In this study,the chemical constituents of D.nipponica subsp.rosthornii and D.althaeoides from sect.Stenophora were systematically isolated and identified.The anti-lung cancer mechanisms of main constituents were also evaluated.Additionally,the metabolites of 21 Dioscorea species were comprehensively analyzed by nuclear magnetic resonance(NMR)and liquid chromatography-mass spectrometry(LC-MS)technology.Their genetic relationships were further investigated based on the partial least squares-discriminant analysis(PLS-DA)or orthogonal projections to latent structuresdiscriminant analysis(OPLS-DA)and hierarchical cluster analysis(HCA)results of untargeted and targeted metabolomics in combination with the correlations among phylogenetic relationships,chemical constituents and modern pharmacological activities.Through systematic phytochemical separation,17 compounds including two new compounds 2,2’,7,7’-tetramethoxy-[1,1’-biphenanthrene]-4,4’,6,6’-tetrol(n1)and diorosthornoumarin(n2),were all firstly identified from the rhizomes of D.nipponica subsp.rosthornii.Compound n11 were firstly obtained from both genus Dioscorea and family Dioscoreaceae.For D.althaeoides,15 compounds were identified from its rhizomes,including a new spirostanol saponin dioscin-6’-O-acetate(DA,a1).Of these,compounds a14-a15 were firstly obtained from genus Dioscorea,while a3-a8 and a10-a15 were firstly isolated from this plant.The above results enriched the variety and quantity of the constituents of Dioscorea.Cell experiments in vitro were utilized to detect the anti-lung cancer effects of main compounds.The results revealed that DA exhibited potent anti-tumor activity against 4 kinds of human lung cancer cell lines.It dramatically inhibited cell proliferation and migration,induced cell apoptosis and cell cycle arrest,and triggered ROS generation and loss of mitochondrial membrane potential.Further in-depth mechanism studies showed that DA could significantly increase the levels of cleaved caspase 3,cleaved caspase 8,cleaved caspase 9,cytochrome C(intracellular),cleaved PARP,Bax,p21,p53,phosphorylated JNK and p38 MAPK,and markedly decreased the expression of Bcl-2,p-ERK,p-PI3 K,p-AKT,NF-κB,MMP-2 and MMP-9,demonstrating that DA exerted strong anti-lung cancer activity through ROS-mediated PI3K/AKT,MAPK and NF-κB signaling pathways.NMR-based metabolomics was firstly used to investigate the metabolites,taxonomic relationships,differential metabolites and related metabolic pathways of 21 Dioscorea species.A total of 63 constituents including mainly primary metabolites were identified by NMR spectra.OPLS-DA and HCA of NMR-based untargeted metabolomics were performed to study the genetic relationships of these plants.15 and 17 differential metabolites(mainly amino acids and steroids)were found among species of different groups within Dioscorea genera and within sect.Stenophora,respectively.The HCA revealed that sect.Stenophora was a strongly supported monophyletic group.The classification status of D.bulbifera and D.zingiberensis was also verified.D.bulbifera was closely related to three Sect.Lasiophyton species(D.esquirolii,D.melanophyma and D.hispida).D.banzhuana should be reclassified into sect.Stenophora,D.nipponica subsp.rosthornii and D.collettii var.hypoglauca were suggested to be separated as independent species from their original subspecies/varieties.LC-MS-based metabolomics was used to investigate the secondary metabolites,genetic relationships,differential constituents and related metabolic pathways of 12 Dioscorea species rich in steroidal saponins.A total of 49 metabolites including mainly steroidal saponins were identified by UHPLC-Q-TOF/MS analysis.Ten major steroidal saponins were further quantified simultaneously by LC-MS method.The(+)-ESI-MS/MS fragmentation patterns of new steroidal saponin DA was firstly reported.Genetic relationships of these species were discussed according to the PLS-DA and HCA results of LC-MS-based untargeted and targeted metabolomics,and 36 differential metabolites were identified.D.bulbifera was distinguished with species from sect.Stenophora for three unique diterpenoid lactones and pennogenin steroidal saponin PNG.D.zingiberensis was far related with other species of sect.Stenophora due to four special steroidal saponins.The classification status of D.zingiberensis and D.parviflora were strongly supported.D.banzhuana with abundant steroidal saponins should be reclassified into sect.Stenophora.D.nipponica subsp.rosthornii and D.collettii var.hypoglauca,far related with their original subspecies/varieties,should be separated as individual species.Finally,regarding the theory of plant pharmacophylogeny,the genetic relationships of 21 Dioscorea species were systematically discussed based on HCA results of phytochemical constituents and modern pharmacological activities.This study enriched the basic data of medicinal plants of Dioscorea,and provided theoretical basis for the conservation and utilization of resources of this genus.