Accurate mass-time tag library for LC/MS-based metabolite profiling of medicinal plants.

We report the development and testing of an accurate mass-time (AMT) tag approach for the LC/MS-based identification of plant natural products (PNPs) in complex extracts. An AMT tag library was developed for approximately 500 PNPs with diverse chemical structures, detected in electrospray and atmospheric pressure chemical ionization modes (both positive and negative polarities). In addition, to enable peak annotations with high confidence, MS/MS spectra were acquired with three different fragmentation energies. The LC/MS and MS/MS data sets were integrated into online spectral search tools and repositories (Spektraris and MassBank), thus allowing users to interrogate their own data sets for the potential presence of PNPs. The utility of the AMT tag library approach is demonstrated by the detection and annotation of active principles in 27 different medicinal plant species with diverse chemical constituents.

Next generation sequencing in predicting gene function in podophyllotoxin biosynthesis.

Podophyllum species are sources of (-)-podophyllotoxin, an aryltetralin lignan used for semi-synthesis of various powerful and extensively employed cancer-treating drugs. Its biosynthetic pathway, however, remains largely unknown, with the last unequivocally demonstrated intermediate being (-)-matairesinol. Herein, massively parallel sequencing of Podophyllum hexandrum and Podophyllum peltatum transcriptomes and subsequent bioinformatics analyses of the corresponding assemblies were carried out. Validation of the assembly process was first achieved through confirmation of assembled sequences with those of various genes previously established as involved in podophyllotoxin biosynthesis as well as other candidate biosynthetic pathway genes. This contribution describes characterization of two of the latter, namely the cytochrome P450s, CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum. Both enzymes were capable of converting (-)-matairesinol into (-)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates tested. Interestingly, the enzymes described herein were highly similar to methylenedioxy bridge-forming enzymes from alkaloid biosynthesis, whereas candidates more similar to lignan biosynthetic enzymes were catalytically inactive with the substrates employed. This overall strategy has thus enabled facile further identification of enzymes putatively involved in (-)-podophyllotoxin biosynthesis and underscores the deductive power of next generation sequencing and bioinformatics to probe and deduce medicinal plant biosynthetic pathways.

Antifungal amides from Piper scutifolium and Piper hoffmanseggianum.

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.