Background: Biosynthetic pathways to structurally complex plant medicinals are incomplete or unknown. Results: Next generation sequencing/bioinformatics and metabolomics analysis of Podophyllum tissues gave putative unknown genes in podophyllotoxin biosynthesis. Conclusion: Regio-specific methylenedioxy bridge-forming CyP450s were identified catalyzing pluviatolide formation. Significance: Database of several medicinal plant transcriptome assemblies and metabolic profiling are made available for scientific community.

Podophyllum hexandrum and, to a much lesser extent P. peltatum, are sources of podophyllotoxin, extensively used as a chemical scaffold for various anti-cancer drugs. In this study, integrated omics technologies (including advanced mass spectrometry/metabolomics, transcriptome sequencing/gene assemblies, and bioinformatics) gave unequivocal evidence that both plant species possess a hitherto unknown aporphine alkaloid metabolic pathway. Specifically, RNA-seq transcriptome sequencing and bioinformatics guided gene assemblies/analyses in silico suggested presence of transcripts homologous to genes encoding all known steps in aporphine alkaloid biosynthesis. A comprehensive metabolomics analysis, including UPLC-TOF-MS and MALDI-MS imaging in situ, then enabled detection, identification, localization and quantification of the aporphine alkaloids, magnoflorine, corytuberine and muricinine, in the underground and aerial tissues. Interestingly, the purported presence of alkaloids in Podophyllum species has been enigmatic since the 19th century, remaining unresolved until now. The evolutionary and phylogenetic ramifications of this discovery are discussed.

Chromatographic fractionation of a dichloromethane extract from the leaves of Piper scutifolium yielded two new isobutyl amides, scutifoliamide A ( 1) and scutifoliamide B ( 2), together with the known compounds piperolactam C ( 3), piperovatine ( 4), piperlonguminine ( 5), corcovadine ( 6), isopiperlonguminine ( 7), and isocorcovadine ( 8). From the dichloromethane extract from the leaves of P. hoffmannseggianum two new isobutyl amides, hoffmannseggiamide A ( 9) and hoffmannseggiamide B ( 10), were obtained together with the known compounds isopiperlonguminine ( 7) and isocorcovadine ( 8), sitosterol, and stigmasterol. The structures of the new compounds were established on the basis of spectroscopic data analysis. The inhibitory activity of compounds 1-10 against the growth of the fungi Cladosporium sphaerospermum and C. cladosporioides was determined by bioautography.