Lycopodium Alkaloids from Phlegmariurus carinatus with Cytotoxic and Neuroprotective Effects.

One new fawcettimine-type alkaloid, phlecarinadine A (1), and twelve known ones (2-13) were isolated from the whole plant of Phlegmariurus carinatus. Their chemical structures were unambiguously established by extensive spectroscopic analyses, including nuclear magnetic resonance (NMR) spectroscopic and high resolution electrospray ionization mass spectrometry (HR-ESI-MS). The absolute configuration of 1 was elucidated by the electronic circular dichroism (ECD) technique. These compounds were tested for their cytotoxic and neuroprotective activities. None of these compounds revealed cytotoxic activity against five tumor cells. Phlegmariurine B (2) exhibited potential neuroprotective effects against hemin-induced HT22 cell damage, with a 17.76 % increase in cell survival at 10 µM. In further study, 2 can ameliorate hemin-induced neuronal cell death via an anti-apoptotic pathway. These findings suggest that 2 might be a valuable lead compound with neuroprotective activity.

Two new serratene triterpenes from club moss cultivars.

Two new serratene triterpenes, 14α,21ß-dihydroxyserrat-3ß-yl acetate and 3α,21ß-dihydroxyserrat-14-en-23-oic acid, together with eight known compounds were isolated from two club moss cultivars, Phlegmariurus carinatus (Desv.) Ching and Phlegmariurus nummulariifolius (Blume) Ching. 14α,21ß-Ddihydroxyserrat-3ß-yl acetate (1) was isolated from P. carinatus, and 3α,21ß-dihydroxyserrat-14-en-23-oic acid (2), an undescribed carboxylic group at C-23 position of the serratene triterpenoids, was isolated from P. nummulariifolius. The structures of these new compounds were elucidated by using HR-ESIMS, UV, IR, 1D (1H and 13C NMR spectra), 2D NMR spectra, experimental ECD spectrometry and the single-crystal X-ray analysis. Biological evaluation of 14α,21ß-dihydroxyserrat-3ß-yl acetate (1) and lycoclavanol (8) revealed moderate cytotoxic activity on three tumor cell lines (HepG2, A549 and HuCCA-1) whereas 3α,21ß-dihydroxyserrat-14-en-23-oic acid (2) showed strong inhibitory effect on HuCCA-1 cells with the IC50 of 4.72 µM.

Phlegcarines A-C, three Lycopodium alkaloids from Phlegmariurus carinatus (Desv. ex Poir.) Ching.

Three undescribed Lycopodium alkaloids, phlegcarines A-C, along with nine known alkaloids, were isolated from the aerial parts of a gardening clubmoss Phlegmariurus carinatus (Desv. ex Poir.) Ching. Phlegcarine A is an undescribed Lycopodium alkaloid possessing an unprecedented 5/9/6/6 fused-tetracyclic ring system consisting of an oxa-cyclononanone, a piperidine, a methylcyclohaxane and an oxazolidine. Phlegcarine B is the first N-chloromethyl piperidinium Lycopodium alkaloid of (+)-lycoflexine. The semi-synthesis of phlegcarine B was investigated from (+)-fawcettimine. Phlegcarine C, an undescribed epimer of 12-epilycodoline, is a rare lycopodine-type alkaloid with ß-oriented H-4. Transformation of phlegcarine C and lycodoline to 12-epilycopodine N-oxide via keto-enol tautomerization was investigated using m-CPBA. The structural assignments were established through comprehensive spectroscopic techniques and chemical correlations. Phlegcarines A-C were assayed for their anti-acetylcholinesterase activity, but none of them exhibited biological activity more potent than that of huperzine A.

Abietane diterpenoids with neuroprotective activities from Phlegmariurus carinatus.

Two new abietane diterpenoids, phlecarinatone A (1) and phlecarinatone B (2), along with two known analogues (3 and 4), were isolated from Phlegmariurus carinatus. The structures of 1 - 4 were unambiguously elucidated by comprehensive spectroscopic analyses. Abietane diterpenoids were isolated from the plant for the first time. All isolates were tested for their neuroprotective activities against H2O2-induced SH-SY5Y cells injury, and compound 2 showed moderate effect at the concentrations ranging from 5 ∼ 20 µM in vitro assay.

Abietane diterpenoids from Phlegmariurus carinatus and their biological activities.

Five undescribed abietane diterpenoids, along with eight known analogs, were isolated from Phlegmariurus carinatus. Their structures were unambiguously elucidated by extensive analysis of spectroscopic data and comparison between the literature. The absolute configuration of phlecarinatone C was determined by evaluating ECD spectra. Four undescribed abietane diterpenoids and eight known analogs were tested for their neuroprotective and cytotoxic activities, separately. Teuvincenone C showed potential neuroprotective effect against Hemin-induced HT22 cell damage. Importantly, phlecarinatone C showed pronounced cytotoxic effect against U251 cells in vitro assays. The biological evaluation revealed that phlecarinatone C could inhibit proliferation, migration, and invasion in a concentration-dependent manner of U251 cells. Meanwhile, phlecarinatone C effectively reversed epithelial-to-mesenchymal transition (EMT) and promoted U251 cells apoptosis via a mitochondrial apoptotic pathway. Taken together, phlecarinatone C might be a valuable candidate for anti-metastatic agents against glioblastoma treatment.

The complete chloroplast genome sequence of Phlegmariurus carinatus.

The first complete chloroplast genome (cpDNA) sequence of Phlegmariurus carinatus was determined from Illumina HiSeq pair-end sequencing data in this study. The cpDNA is 150,349 bp in length, contains a large single-copy region (LSC) of 100,582 bp and a small single-copy region (SSC) of 19,455 bp, which were separated by a pair of inverted repeat (IR) regions of 15,156 bp. The genome contains 120 genes, including 79 protein-coding genes, 8 ribosomal RNA genes, and 33 transfer RNA genes. The overall GC content of the whole genome is 34.0%, and the corresponding values of the LSC, SSC, and IR regions are 31.6%, 30.4%, and 44.2%, respectively. Further phylogenomic analysis showed that P. carinatus and Huperzia serrata clustered in a clade in family Lycopodiaceae.

Carinatines A and B, Lycopodium Alkaloids from Phlegmariurus carinatus.

Carinatine A (1), a C16N2-type Lycopodium alkaloid possessing a 5/6/6/6 ring system formed by a new C-4/C-12 bond, and carinatine B (2), the first derivative of lycojaponicumin C, along 16 known compounds, were isolated from the whole plant of Phlegmariurus carinatus. Their structures were elucidated based on the spectroscopic data. The two new isolates were no inhibitory activity for the acetylcholinesterase (AChE).

Cloning and sequence analysis of PcFPS1 gene in Phlegmariurus carinatus / 中草药

Objective: To clone and analyze the coding region of Farnesyl diphosphate synthase (FPS) gene PcFPS1 from Phlegmariurus carinatus. Methods: According to the acquired transcriptome dataset of P. carinatus, one transcript coding FPS was obtained. The coding region sequence was obtained using RT-PCR method, and the physicochemical properties, protein secondary structure, and three-dimensional structure of PcFPS1 protein were predictively analyzed. Results: The cloned PcFPS1 gene contained a 1 119-bp coding region for encoding a predicted protein of 372 amino acids with high homology (70%) to FPS gene in Picea abies. PcFPS1 contained almost no transmembrane region and had the conserved domain of terpenoid cylases, without signal peptide. Conclusion: This study clones and analyzes the FPS gene from P. carinatus which is obtained for the first time. The results will provide a foundation for exploring the function of PcFPS1 in terpene and sterol biosynthesis of plants in Huperziaceae.