Molecular Network-Guided Alkaloid Profiling of Aerial Parts of Papaver nudicaule L. Using LC-HRMS.

Papaver nudicaule L. (Iceland poppy) is widely used for ornamental purposes. A previous study demonstrated the alleviation of lipopolysaccharide-induced inflammation mediated by P. nudicaule extract through nuclear factor-kappa B and signal transducer and activator of transcription 3 inactivation. As isoquinoline alkaloids are chemical markers and bioactive constituents of Papaver species, the present study investigated the alkaloid profile of aerial parts of five P. nudicaule cultivars with different flower colors and a P. rhoeas cropped for two years. A combination of liquid chromatography high-resolution mass spectrometry and molecular networking was used to cluster isoquinoline alkaloids in the species and highlight the possible metabolites. Aside from the 12 compounds, including rotundine, muramine, and allocryptopine, identified from Global Natural Products Social library and reported information, 46 structurally related metabolites were quantitatively investigated. Forty-two and 16 compounds were proposed for chemical profiles of P. nudicaule and P. rhoeas, respectively. Some species-specific metabolites showed similar fragmentation patterns. The alkaloid abundance of P. nudicaule differed depending on the flower color, and the possible chemical markers were proposed. These results show that molecular networking-guided dereplication allows investigation of unidentified metabolites. The derived chemical profile may facilitate evaluation of P. nudicaule quality for pharmacological applications.

Papaver nudicaule (Iceland poppy) alleviates lipopolysaccharide-induced inflammation through inactivating NF-κB and STAT3.

BACKGROUND: Papaver nudicaule belongs to the Papaveraceae family, which is planted as an annual herbaceous species generally for ornamental purpose. Papaver rhoeas in the same family has been reported to have various pharmacological activities such as antioxidant and analgesic effects. In contrast, little is known about the pharmacological activity of Papaver nudicaule. In this study, the anti-inflammatory activity of Papaver nudicaule extracts and the action mechanisms were investigated in RAW264.7 macrophage cells. METHODS: To investigate the anti-inflammatory activity of five cultivars of Papaver nudicaule with different flower color, samples were collected from their aerial parts at two growth stages (60 and 90 days) and their ethanol extracts were evaluated in the lipopolysaccharide (LPS)-treated RAW264.7 cells by measuring nitric oxide (NO) and prostaglandin E2 (PGE2) levels. Interleukin 1-beta (IL-1ß), Interleukin-6 (IL-6) and Tumor necrosis factor alpha (TNF-α) production were also analyzed by RT-PCR and multiplex assays. Nuclear Factor-kappa-light-chain-enhancer of activated B cells (NF-κB) and Signal transducer and activator of transcription 3 (STAT3) signaling pathways were examined using western blotting and luciferase reporter assays to reveal the action mechanism of Papaver nudicaule extracts in their anti-inflammatory activity. RESULTS: All of the Papaver nudicaule extracts were effective in reducing the LPS-induced NO, which is an important inflammatory mediator, and the extract of Papaver nudicaule with white flower collected at 90 days (NW90) was selected for further experiments because of the best effect on reducing the LPS-induced NO as well as no toxicity. NW90 lowered the LPS-induced PGE2 level and decreased the LPS-induced Nitric oxide synthase 2 (NOS2) and Cyclooxygenase 2 (COX2). In addition, NW90 reduced the LPS-induced inflammatory cytokines, IL-1ß and IL-6. Furthermore, NW90 inhibited the LPS-induced activation of NF-κB and STAT3. CONCLUSIONS: These results indicate that NW90 may restrain inflammation by inhibiting NF-κB and STAT3, suggesting the potential therapeutic properties of Papaver nudicaule against inflammatory disease.

Transcriptome Profiling of Two Ornamental and Medicinal Papaver Herbs.

The Papaver spp. (Papaver rhoeas (Corn poppy) and Papaver nudicaule (Iceland poppy)) genera are ornamental and medicinal plants that are used for the isolation of alkaloid drugs. In this study, we generated 700 Mb of transcriptome sequences with the PacBio platform. They were assembled into 120,926 contigs, and 1185 (82.2%) of the benchmarking universal single-copy orthologs (BUSCO) core genes were completely present in our assembled transcriptome. Furthermore, using 128 Gb of Illumina sequences, the transcript expression was assessed at three stages of Papaver plant development (30, 60, and 90 days), from which we identified 137 differentially expressed transcripts. Furthermore, three co-occurrence heat maps are generated from 51 different plant genomes along with the Papaver transcriptome, i.e., secondary metabolite biosynthesis, isoquinoline alkaloid biosynthesis (BIA) pathway, and cytochrome. Sixty-nine transcripts in the BIA pathway along with 22 different alkaloids (quantified with LC-QTOF-MS/MS) were mapped into the BIA KEGG map (map00950). Finally, we identified 39 full-length cytochrome transcripts and compared them with other genomes. Collectively, this transcriptome data, along with the expression and quantitative metabolite profiles, provides an initial recording of secondary metabolites and their expression related to Papaver plant development. Moreover, these profiles could help to further detail the functional characterization of the various secondary metabolite biosynthesis and Papaver plant development associated problems.

Assembly of the Mitochondrial Genome in the Campanulaceae Family Using Illumina Low-Coverage Sequencing.

Platycodongrandiflorus (balloon flower) and Codonopsislanceolata (bonnet bellflower) are important herbs used in Asian traditional medicine, and both belong to the botanical family Campanulaceae. In this study, we designed and implemented a de novo DNA sequencing and assembly strategy to map the complete mitochondrial genomes of the first two members of the Campanulaceae using low-coverage Illumina DNA sequencing data. We produced a total of 28.9 Gb of paired-end sequencing data from the genomic DNA of P.grandiflorus (20.9 Gb) and C.lanceolata (8.0 Gb). The assembled mitochondrial genome of P.grandiflorus was found to consist of two circular chromosomes; the master circle contains 56 genes, and the minor circle contains 42 genes. The C.lanceolata mitochondrial genome consists of a single circle harboring 54 genes. Using a comparative genome structure and a pattern of repeated sequences, we show that the P.grandiflorus minor circle resulted from a recombination event involving the direct repeats of the master circle. Our dataset will be useful for comparative genomics and for evolutionary studies, and will facilitate further biological and phylogenetic characterization of species in the Campanulaceae.

Identification and metabolite profiling of alkaloids in aerial parts of Papaver rhoeas by liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Papaver plants can produce diverse bioactive alkaloids. Papaver rhoeas Linnaeus (common poppy or corn poppy) is an annual flowering medicinal plant used for treating cough, sleep disorder, and as a sedative, pain reliever, and food. It contains various powerful alkaloids like rhoeadine, benzylisoquinoline, and proaporphine. To investigate and identify alkaloids in the aerial parts of P. rhoeas, samples were collected at different growth stages and analyzed using liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. A liquid chromatography with mass spectrometry method was developed for the identification and metabolite profiling of alkaloids for P. rhoeas by comparing with Papaver somniferum. Eighteen alkaloids involved in benzylisoquinoline alkaloid biosynthesis were used to optimize the liquid chromatography gradient and mass spectrometry conditions. Fifty-five alkaloids, including protoberberine, benzylisoquinoline, aporphine, benzophenanthridine, and rhoeadine-type alkaloids, were identified authentically or tentatively by liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry in samples taken during various growth stages. Rhoeadine alkaloids were observed only in P. rhoeas samples, and codeine and morphine were tentatively identified in P. somniferum. The liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry method can be a powerful tool for the identification of diverse metabolites in the genus Papaver. These results may help understand the biosynthesis of alkaloids in P. rhoeas and evaluate the quality of this plant for possible medicinal applications.

The multipartite mitochondrial genome of Cynanchum wilfordii (Gentianales: Apocynaceae).

Cynanchum wilfordii is a traditional herbal medicine and belongs to the family Apocynaceae. The C. wilfordii mitochondrial genome consists of three circular chromosomes (named chromosomes I-III), the lengths of which are 379,060, 352,767 and 111,332 nucleotides. The mitochondrial genome encodes 58 genes, including 38 protein-coding genes, 17 transfer RNA genes and three ribosomal RNA genes. Of these 58 genes, 37 are located in chromosome I, 35 in chromosome II and 15 in chromosome III. Phylogenetic analysis suggests that among the 14 reported species of Asterids, C. wilfordii is most closely related to Asclepias syriaca.