Total Synthesis of the Cephalotaxus Norditerpenoids (±)-Cephanolides A-D.

Concise syntheses of the Cephalotaxus norditerpenoids cephanolides A-D (8-14 steps from commercial material) using a common late-stage synthetic intermediate are described. The success of our approach rested on an early decision to apply chemical network analysis to identify the strategic bonds that needed to be forged, as well as the efficient construction of the carbon framework through iterative Csp2-Csp3 cross-coupling, followed by an intramolecular inverse-demand Diels-Alder cycloaddition. Strategic late-stage oxidations facilitated access to all congeners of the benzenoid cephanolides isolated to date.

Diterpenoids and Flavonoids from the Twigs of Cephalotaxus fortunei var. alpina.

Three new diterpenoids (a cephalotane, an abietane and a 9(10→20)-abeo-abietane) and one new flavonoid, together with 11 known compounds, were isolated from the twigs of Cephalotaxus fortunei var. alpina. The new compounds were identified by comprehensive spectroscopic (including 1D and 2D-NMR and HR-ESI-MS) analysis. Anti-inflammatory, immunosuppressive and cytotoxic activities of three new compounds were evaluated. 3ß,20-epoxyabieta-8,11,13-triene-3α,12-diol showed weak cytotoxicity against tumor cell lines NCI-H1975, HepG2, MCF-7, while fortalpinoid R and 3-acetonyl-3,5,7,4'-tetrahydroxy-2-methoxyflavanone were not active at 80 µM. None of these compounds showed anti-inflammatory and immunosuppressive activities.

Insights from the pollination drop proteome and the ovule transcriptome of Cephalotaxus at the time of pollination drop production.

BACKGROUND AND AIMS: Many gymnosperms produce an ovular secretion, the pollination drop, during reproduction. The drops serve as a landing site for pollen, but also contain a suite of ions and organic compounds, including proteins, that suggests diverse roles for the drop during pollination. Proteins in the drops of species of Chamaecyparis, Juniperus, Taxus, Pseudotsuga, Ephedra and Welwitschia are thought to function in the conversion of sugars, defence against pathogens, and pollen growth and development. To better understand gymnosperm pollination biology, the pollination drop proteomes of pollination drops from two species of Cephalotaxus have been characterized and an ovular transcriptome for C. sinensis has been assembled. METHODS: Mass spectrometry was used to identify proteins in the pollination drops of Cephalotaxus sinensis and C. koreana RNA-sequencing (RNA-Seq) was employed to assemble a transcriptome and identify transcripts present in the ovules of C. sinensis at the time of pollination drop production. KEY RESULTS: About 30 proteins were detected in the pollination drops of both species. Many of these have been detected in the drops of other gymnosperms and probably function in defence, polysaccharide metabolism and pollen tube growth. Other proteins appear to be unique to Cephalotaxus, and their putative functions include starch and callose degradation, among others. Together, the proteins appear either to have been secreted into the drop or to occur there due to breakdown of ovular cells during drop production. Ovular transcripts represent a wide range of gene ontology categories, and some may be involved in drop formation, ovule development and pollen-ovule interactions. CONCLUSIONS: The proteome of Cephalotaxus pollination drops shares a number of components with those of other conifers and gnetophytes, including proteins for defence such as chitinases and for carbohydrate modification such as ß-galactosidase. Proteins likely to be of intracellular origin, however, form a larger component of drops from Cephalotaxus than expected from studies of other conifers. This is consistent with the observation of nucellar breakdown during drop formation in Cephalotaxus The transcriptome data provide a framework for understanding multiple metabolic processes that occur within the ovule and the pollination drop just before fertilization. They reveal the deep conservation of WUSCHEL expression in ovules and raise questions about whether any of the S-locus transcripts in Cephalotaxus ovules might be involved in pollen-ovule recognition.

Effects and action mechanisms of sodium fluoride (NaF) on the growth and cephalotaxine production of Cephalotaxus mannii suspension cells.

To explore the effects of NaF on the growth and cephalotaxine production of Cephalotaxus mannii suspension cells, NaF was added into the C mannii cell suspension cultures at day 15 of culture time. It is documented that NaF suppressed cell growth but enhanced cephalotaxine production. The largest yield obtained of cephalotaxine reached to 9.57mg/L when the cultures were treated with an appropriate dosage of 15mg/L NaF, which was 3.7 times that of the control cultures (2.58mg/L). Additionally, NaF markedly enhanced the activity of glucose 6-phosphate dehydrogenase (G6PDH) and reduced the level of hydrogen peroxide (H2O2) of cells. It was also found that NaF weakened the oxidative damage of cell membrane and led to lower content of malonyl dialdehyde (MDA) in NaF-treated cells compared with the control cells. The MDA content of NaF-treated cells decreased 91% compared to the controls. Although lower membrane lipid peroxidation in NaF-treated cells, its membrane permeability was higher than the control cells and showed a high product secret rate. What is more, NaF boosted the activity of phenylalanine ammonium-lyse (PAL), but did not burst a peak of PAL activity in the time curve of PAL activity. These results indicated NaF acted as an inhibitor of the Enbden Parnas (EMP) pathway, not as an elicitor to promote cephalotaxine production.

Development of microsatellite loci for Cephalotaxus oliveri (Cephalotaxaceae) and cross-amplification in Cephalotaxus.

PREMISE OF THE STUDY: Microsatellite loci were developed for Cephalotaxus oliveri, an endemic and endangered conifer in China, which will allow assessment of the levels of genetic diversity and a means to understand the genetic consequences of habitat fragmentation. METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences COntaining (FIASCO) Repeats protocol, 19 microsatellite loci were identified in C. oliveri, 13 of which were polymorphic within a sample of 52 individuals representing five natural populations. The actual number of alleles per locus ranged from one to five. Twelve polymorphic loci were also successfully amplified in C. fortunei. CONCLUSIONS: These microsatellite loci will provide a useful tool for further investigating genetic variation in natural populations of C. oliveri, which will inform future conservation and management strategies. Additionally, cross-amplification in C. fortunei suggested the potential utility of these loci in this and other congeneric species.

[Biotransformation of artemisinic acid by cell suspension cultures of Cephalotaxus fortunei and Artemisia annua].

OBJECTIVE: To investigate the biotransformation of artemisinic acid by cell suspension cultures of Cephalotaxus fortunei and Artemisia annua. METHODS: Artemisinic acid was added into to the media of the suspension cells of Cephalotaxus fortunei and Artemisia annua in their logarithmic growth phase. The biotransfromed product was detected with HPLC and isolated by silica gel column, Sephadex LH20 and ODS chromatography methods. The chemical structure of biotransformed product was elucidated on the basis of physical-chemical properties and spectroscopic data. Otherwise, the influence of co-cultured time on conversion ratio was investigated with HPLC. RESULTS: One biotransformed product, 3-alpha-hydroxyartemisinic acid, was obtained after two days of artemisinic acid administration to the suspension cells of Cephalotaxus fortunei and Artemisia annua. The optimal co-cultured time in suspension cells of Cephalotaxus fortunei was 2 days with the highest biotransformation rate of 8.42%, and in the case of Artemisia annua, it was 3 days and 3.95% respectively. CONCLUSION: It was the first time for the biotransformation of artemisinic acid to 3-alpha-hydroxyartemisinic acid by using cell suspension cultures of Cephalotaxus fortunei and Artemisia annua.