Plastome phylogenomics of Cephalotaxus (Cephalotaxaceae) and allied genera.

BACKGROUND AND AIMS: Cephalotaxus is a paleo-endemic genus in East Asia that consists of about 7-9 conifer species. Despite its great economic and ecological importance, the relationships between Cephalotaxus and related genera, as well as the interspecific relationships within Cephalotaxus, have long been controversial, resulting in contrasting taxonomic proposals in delimitation of Cephalotaxaceae and Taxaceae. Based on plastome data, this study aims to reconstruct a robust phylogeny to infer the systematic placement and the evolutionary history of Cephalotaxus. METHODS: A total of 11 plastomes, representing all species currently recognized in Cephalotaxus and two Torreya species, were sequenced and assembled. Combining these with previously published plastomes, we reconstructed a phylogeny of Cephalotaxaceae and Taxaceae with nearly full taxonomic sampling. Under a phylogenetic framework and molecular dating, the diversification history of Cephalotaxus and allied genera was explored. KEY RESULTS: Phylogenetic analyses of 81 plastid protein-coding genes recovered robust relationships between Cephalotaxus and related genera, as well as providing a well-supported resolution of interspecific relationships within Cephalotaxus, Taxus, Torreya and Amentotaxus. Divergence time estimation indicated that most extant species of these genera are relatively young, although fossil and other molecular evidence consistently show that these genera are ancient plant lineages. CONCLUSIONS: Our results justify the taxonomic proposal that recognizes Cephalotaxaceae as a monotypic family, and contribute to a clear-cut delineation between Cephalotaxaceae and Taxaceae. Given that extant species of Cephalotaxus are derived from recent divergence events associated with the establishment of monsoonal climates in East Asia and Pleistocene climatic fluctuations, they are not evolutionary relics.

Cytotoxic secondary metabolites from the vulnerable conifer Cephalotaxus oliveri and its associated endophytic fungus Alternaria alternate Y-4-2.

Rare and endangered plants (REPs) and their associated endophytes survived in unique habitats are promising sources for natural product-derived drug discovery. In this study, six new (cephaloverines A-F, 1-6, resp.) and 16 known (11-26) cephalotaxine-type alkaloids, together with three new (oliverbiflavones A-C, 7-9, resp.) and 11 known (27-37) biflavonoids were isolated and characterized from the twigs and leaves of Cephalotaxus oliveri, an endangered plant endemic to China. Meanwhile, a preliminary investigation on the secondary metabolites from a selected fungal endophyte (i.e., Alternaria alternate Y-4-2) associated with the title plant led to the isolation of 21 structurally distinct polyketides including one new dimeric xanthone (10). The new structures (1-10) with the absolute configurations were determined by detailed spectroscopic analyses, electronic circular dichroism (ECD) or Na2MoO4-induced ECD, the modified Mosher's method, and some chemical transformations. Compounds 1-4 are the first representatives of naturally occurring N-oxides of cephalotaxine esters, while compounds 7-9 have a special structural feature of having a C-methylated biflavonoid skeleton. The Cephalotaxus alkaloids with ester side-chains at C-3 (1-6, 13-22, and 26) and four biflavonoids (27-29 and 34) were found to show pronounced cytotoxicities against a small panel of human cancer cell lines (A549, NCI-H460, HL60, NCI-H929, and RPMI-8226), with IC50 values mainly ranging from 0.003 to 9.34 µM. The most potent compound, deoxyharringtonine (16), generally exhibited IC50 values less than 10 nM. The structure-activity relationship (SAR) of the aforementioned Cephalotaxus alkaloids was briefly discussed.

Cephaliverins A-D, four new lignans from Cephalotaxus oliveri Mast.

Four new lignans named cephaliverins A-D (1-4), along with seven known analogues (5-11), were isolated from Cephalotaxus oliveri Mast. Their structures were elucidated on the basis of HR-ESI-MS and NMR analyses, and their absolute configurations were determined by ECD comparison. Cephaliverin A (1), herpetotriol (5) and hedyotol A (6) exhibited moderate antitumor activity against HepG2 and A549 cell lines.

Diterpenolignans and cephalotane diterpenoids from Cephalotaxus oliveri mast. with antitumor activity.

Four undescribed naturally diterpenolignans, and two cephalotane diterpenoids, along with seven known compounds, including two pairs of enantiomers, were isolated from the twigs and leaves of Cephalotaxus oliveri Mast. Their structures were elucidated via spectroscopic data interpretation, chiral-phase HPLC analysis, NMR calculations, and electronic circular dichroism analysis. All the isolated compounds were evaluated for their cytotoxic activities against three kinds of human tumor cell lines. Among them, compound 8 exhibited the most potent activities against MCF-7, HepG2 and A549 cell lines with IC50 values of 2.83, 4.75 and 2.77 µM, respectively.

Highly modified cephalotane-type diterpenoids from Cephalotaxus fortunei var. alpina and C. sinensis.

Cephalotanes are a rare class of diterpenoids occurring exclusively in Cephalotaxus plants. The intriguing structures and promising biological activities for this unique compound class prompt us to investigate C. fortunei var. alpina and C. sinensis, leading to the isolation of six undescribed cephalotane-type diterpenoids and/or norditerpenoids, ceforloids A-F (1-6). Their structures were elucidated by comprehensive analysis of spectroscopic data, including ECD and single-crystal X-ray diffraction studies, as well as quantum chemical calculations. Compound 1 possesses an unprecedented norditerpenoid skeleton featuring an unusual acetophenone moiety, and originated putatively from a disparate biogenetic pathway. Compounds 4 and 5 incorporate a unique 12,13-p-hydroxybenzylidene acetal motif. Compound 6 is a rare cephalotane-type diterpenoid glycoside. Immunosuppressive assays showed that compounds 2 and 6 exhibited mild suppressive activity against the activated T and B lymphocytes proliferation. These findings not only expanded the structural diversity of this small group of diterpenoids, but also explored their potential as novel structures for the development of immunosuppressive agents.

An integrative view on the systematic position of the cupressophyte Cephalotaxus.

We made an in-depth review of historical studies of the cupressophyte conifer genus Cephalotaxus Siebold & Zucc. with an emphasis on its systematic position. We suggest that the systematic position of the genus is better understood using an integrative approach, so the evolution of phenetic characters is discussed within the context of recent phylogenomics. We propose that the genus should be classified as a separate family Cephalotaxaceae belonging to the clade consisting of Cupressaceae, Cephalotaxaceae, and Taxaceae; the family Cephalotaxaceae is sister to the Taxaceae but not nested within the Taxaceae and is characterized by a unique set of characters including morphology, anatomy, embryology, and chemistry. The family Cephalotaxaceae shows transitional characters between the Cupressaceae and the Taxaceae; the family possesses female cones with a primary cone axis bearing 5-8 pairs of decussate bracts, which is similar to the typical female cones of the Cupressaceae, on the one hand, and may have given rise to the reduced female cone of the Taxaceae with one terminal ovule partially or completely enclosed in a fleshy aril. In parallel, the compound male cone of the Cephalotaxaceae evolved into the seemingly "simple" male cones of the Taxaceae by means of reduction, elimination, and fusion.

Taxonomic status and nomenclature of Cephalotaxuslanceolata (Cephalotaxaceae).

The nomenclature of Cephalotaxuslanceolata is controversial. After a thorough literature investigation, the nomenclatural problems have been resolved. This name was published in W. C. Cheng et al. (1975) and, although ascribed to "K. M. Feng", there is no suggestion that the descriptive material in the protologue was provided by K. M. Feng. Under Art. 46.5 of the ICN, this name should be attributed to K. M. Feng ex W. C. Cheng et al. but not to K. M. Feng alone. It has been claimed that the name is an illegitimate later homonym of one published by Beissner in 1901, but Beissner never accepted this name in any of his publications and so, under Art. 36.1, he did not validly publish an earlier homonym. Cephalotaxuslanceolatus was first validly published in W. C. Cheng et al. (1975).

Enantiomeric Cephalotaxus alkaloids from seeds of Cephalotaxus oliveri.

Five undescribed alkaloids were isolated from the seeds of Cephalotaxus oliveri along with 27 known ones. The new structures were elucidated based on spectroscopic data including 1D and 2D NMR, MS and calculated ECD spectra. Among them, (+)-acetylcephalofortine C was an enantiomeric Cephalotaxine alkaloids. The performed bioassay revealed that those alkaloids were not cytotoxic against cancer cells and had no neuroprotective properties in the HEI-OC-1 cells model.

Cephalotaxine homologous alkaloids from seeds of Cephalotaxus oliveri Mast.

Six undescribed isoquinoline alkaloids, named as cephaloliverines A-F, were isolated from the seeds of Cephalotaxus oliveri. They were identified by NMR and MS spectroscopic data analyses, combined with the time-dependent density functional theory ECD calculation for cephaloliverines A and B and also by X-ray crystal diffraction for cephaloliverine E. Biosynthetic considerations suggest that cephaloliverines A-D are homologous of cephalotaxine-, homoerythrina- and Erythrina-type alkaloids. The performed bioassay revealed no cytotoxic activity against cancer cells and no neuroprotective properties on HEI-OC-1 cells model.

Abietane diterpenes from the twigs and leaves of Cephalotaxus oliveri Mast. with antitumor activity.

Twenty one abietane diterpenes were isolated from Cephalotaxus oliveri Mast. The structures of 7 undescribed diterpenoids, named cephaloliverins A-G, were elucidated via spectroscopic data interpretation and electronic circular dichroism (ECD) analysis. The isolated diterpenoids were evaluated for their cytotoxicity against three kinds of human tumor cell lines (MCF-7, HepG2, and A549). Metaglyptin A was the most active with IC50 values of 16.34, 15.63 and 21.33 µM and was further investigated for colony formation and apoptosis in HepG2 cells.

Flavonolignans and biflavonoids from Cephalotaxus oliveri exert neuroprotective effect via Nrf2/ARE pathway.

Plants of the Cephalotaxus genus are rich in structurally diverse and naturally bioactive components, while limited studies have been reported for Cephalotaxus oliveri. Two undescribed flavonolignans and four undescribed biflavonoids, as well as thirteen known compounds, were isolated from the twigs and leaves of C. oliveri. Their structures were characterized by spectroscopic data analysis, and the absolute configurations were determined by electronic circular dichroism (ECD) calculations. All the isolated compounds were assayed for their neuroprotective activity against hydrogen peroxide (H2O2)-induced SH-SY5Y cell injury. All six undescribed compounds were effective to some degree, and umcephabiflovin B, apigenin 5-O-α-L-rhamnopyranosyl-(1 â†’ 2)-6″-acetyl-ß-D-glucopyranoside, and apigenin 7-O-ß-D-glucoside exhibited good neuroprotective activity. Umcephabiflovin B protected SH-SY5Y cells against H2O2-induced neurotoxicity by repressing oxidative stress and apoptosis and by activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant-response element (ARE) pathway.

Progress in structure, synthesis and biological activity of natural cephalotane diterpenoids.

The Cephalotaxus genus is well-known owing to the numerous complex, biologically relevant natural products that can be obtained from its constituent species. The successful identification of various Cephalotaxus alkaloids and natural, structurally diverse cephalotane diterpenoids that exhibit antitumor activities and excellent pharmacological properties has encouraged the discovery of previously undescribed compounds from this genus. The present review summarizes the different strategies for the total synthesis of cephalotane diterpenoids as well as their diverse chemical structures, antitumor activities, structure-activity relationships (SARs), and biosynthetic pathways.

Cephalotaxine-type alkaloids from the seeds of Cephalotaxus fortunei and their cytotoxic activities.

Six new Cephalotaxus alkaloids, including five cephalotaxine-type alkaloids, and one homoerythrina-type alkaloid, along with six known analogues, were isolated from the seeds of Cephalotaxus fortunei. Their structures were elucidated by combination of spectroscopic data analyses, time-dependent density functional theory (TDDFT) ECD calculation, and single-crystal X-ray diffraction. Cephalofortine B represents the first example of C-5 epi-cephalotaxine-type alkaloid. All isolated compounds were tested for cytotoxicities against HCT-116, A375, and SK-Mel-28 cell lines. Cephalofortine E showed moderate activity against HCT-116 cell line, with an IC50 value of 7.46 ± 0.77 µM.

Bioactive norditerpenoids from Cephalotaxus fortunei var. alpina and C. lanceolata.

Twenty-eight naturally occurring Cephalotaxus tropone analogues, including 19 previously undescribed ones, were identified from Cephalotaxus fortunei Hook. var. alpina H. L. Li and C. lanceolata K. M. Feng. The presence of the C20 cephinoids A-E revealed that these tropones were assigned to the norditerpenoids and were perhaps derived from labdane-type diterpenoids. These norditerpenoids showed excellent cytotoxicity against human cancer cells (IC50, 20-0.1 µM) in vitro. The SAR (structure-activity relationship) analysis disclosed that the tropone moiety and the lactone ring were crucial structural features for the observed activities. Further SAR analyses led to a new candidate, cephinoid H, which demonstrated an inhibition of 49.0% by administration to zebrafish at a dose of 60.0 ng/mL, compared to cisplatin (DDP, 22.4%) at 15.0 µg/mL. These compounds might affect the NF-κB signaling pathway rather than binding to microtubules. Additionally, the isolated norditerpenoids showed almost equal anti-inflammatory activities compared to the positive control, MG132.

Development and characterization of 15 microsatellite markers for Cephalotaxus fortunei (Cephalotaxaceae).

PREMISE OF THE STUDY: To survey population variation and the adaptive evolution of Cephalotaxus fortunei (Cephalotaxaceae), an endemic and endangered conifer in China, microsatellite markers were developed and characterized for this species. METHODS AND RESULTS: Based on the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, 15 microsatellite markers were developed for C. fortunei, 13 of which were polymorphic within a sample of 75 individuals representing five natural populations. The number of alleles per locus ranged from one to seven. The expected and observed heterozygosities were 0.108-0.738 and 0.000-1.000, respectively. Ten polymorphic loci were also successfully amplified in C. oliveri. CONCLUSIONS: These polymorphic loci provide a valuable tool for population genetic analysis of C. fortunei, which will contribute to its management and conservation.