Galanthamine production by Leucojum aestivum L. shoot culture in a modified bubble column bioreactor with internal sections

Shoot culture of summer snowflake (Leucojum aestivum L.) was successfully cultivated in an advanced modified glass‐column bioreactor with internal sections for production of Amaryllidaceae alkaloids. The highest amounts of dry biomass (20.8 g/L) and galanthamine (1.7 mg/L) were achieved when shoots were cultured at 22°C and 18 L/(L·h) flow rate of inlet air. At these conditions, the L. aestivum shoot culture possessed mixotrophic‐type nutrition, synthesizing the highest amounts of chlorophyll (0.24 mg/g DW (dry weight) chlorophyll A and 0.13 mg/g DW chlorophyll B). The alkaloids extract of shoot biomass showed high acetylcholinesterase inhibitory activity (IC₅₀ = 4.6 mg). The gas chromatography–mass spectrometry (GC/MS) profiling of biosynthesized alkaloids revealed that galanthamine and related compounds were presented in higher extracellular proportions while lycorine and hemanthamine‐type compounds had higher intracellular proportions. The developed modified bubble‐column bioreactor with internal sections provided conditions ensuring the growth and galanthamine production by L. aestivum shoot culture.     

An improved technique for obtaining well-spread metaphases from plants with numerous large chromosomes

Preparations that contain well-spread metaphase chromosomes are critical for plant cytogenetic analyses including chromosome counts, banding procedures, in situ hybridization, karyotyping and construction of ideograms. Chromosome spreading is difficult for plants with large and numerous chromosomes. We report here a technique for obtaining cytoplasm-free, well-spread metaphases from two Amaryllidaceae species: Sprekelia formosissima (2n = 120) and Hymenocallis howardii (2n = 96). The technique has three main steps: 1) pretreatment to cause chromosome condensation, 2) dripping onto tilted slides coated with a thin layer of pure acetic acid and 3) application of steam and acetic acid to produce cytoplasmic hydrolysis, which spreads the chromosomes.     

Floral and macroecological evolution within Cyrtanthus (Amaryllidaceae): Inferences from combined analyses of plastid ndhF and nrDNA ITS sequences

One of the most diverse members of Amaryllidaceae is Cyrtanthus Aiton, a large, sub-Saharan Africa genus of approximately 55 species found mostly in South Africa. To investigate phylogenetic and biogeographic relationships within Cyrtanthus, sequence data from the plastid ndhF gene and the ITS nrDNA region for 41 species were analyzed with parsimony, maximum likelihood, and Bayesian-inference approaches. Various recombination detection algorithms were used to test for interspecific hybridization in the ITS alignment. The genus resolved as monophyletic, comprising three poorly to well-supported major lineages: a predominantly Afrotemperate lineage, largely restricted to seasonally moist sites in summer rainfall southern Africa, a subtropical lineage found mostly in nonseasonal rainfall regions, often in dry habitats, and a Cape Floristic Region-centered lineage in which most species are concentrated in the summer-dry to nonseasonal rainfall southwest. The ITS sequence alignment shows no evidence for reticulation between any of the species. Relationships inferred by the molecular data disagree with those derived from morphological data, but agree with previously published groupings based on karyotype morphology. Fitch optimization of selected floral characters on the combined gene tree reveals recurrent patterns of convergence. Ornithophilous floral forms occur in parallel among the three primary clades, putatively sphingophilous species are concentrated in the Afrotemperate lineage in seasonally moist upland grasslands; the brush-type Aeropetes tulbaghia butterfly and inferred long-proboscid fly pollination syndromes are unique in the Cape lineage. Macroecological factors inferred to have influenced the evolution of Cyrtanthus are changes in rainfall seasonality, the advent of fire, and the availability of new habitats at high and low altitudes and in rock-free soils or rock crevices. This study gives greater clarity on relationships within the genus and enables its division into three informal infrageneric groups.     

Using a phylogenetic approach to selection of target plants in drug discovery of acetylcholinesterase inhibiting alkaloids in Amaryllidaceae tribe Galantheae

We present phylogenetic analyses of 32 taxa of Amaryllidaceae tribe Galantheae, 6 taxa of other Eurasian genera of Amaryllidaceae and Phaedranassa dubia as outgroup in order to provide a phylogenetic framework for selection of candidate plants for lead discovery in relation to Alzheimer’s disease. We used DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS) and the plastid matK and trnL-F regions. Phylogenetic analyses using maximum parsimony and Bayesian inference strongly support a monophyletic tribe Galantheae in a narrow sense, including only Acis, Galanthus and Leucojum. Infrageneric relationships of Galanthus only partly support previous classifications. Alkaloid profiles and inhibition of acetylcholinesterase (AChE) were investigated for 18 taxa using gas chromatography–mass spectrometry (GC–MS) and an assay measuring inhibition of AChE activity. AChE inhibitory activity was found in all investigated clades and was correlated with alkaloid profiles of the plants. Lowest IC₅₀ values were expressed by extracts containing either galanthamine or lycorine type compounds. Evaluation of available chemistry and activity data in a phylogenetic framework could be used to select target species for further investigation.     

长筒石蒜鳞茎中的生物碱类成分(英文)

从长筒石蒜鳞茎的乙醇提取物中分离得到7个生物碱类化合物,经理化方法和波谱分析,分别鉴定为galanthamine(1),lycoramlne(2),6β-hydroxycrinamine(3a),6α-hydroxycrinamine(3b),(-)-amarbellisine(4),tazettine(5),macowine(6).其中化合物3a～6为首次从该植物中分离得到.     

Synthesis and biological evaluation of unnatural derivatives of narciclasine: 7-aza-nornarciclasine and its N-oxide

Several unnatural derivatives of narciclasine were prepared in which the C-7 carbon was replaced with nitrogen. The 7-aza derivative and its N-oxide were prepared by the coupling of iodopicolinic acid with a conduramine unit derived chemoenzymatically from bromobenzene. Intramolecular Heck reaction was used to construct the isocarbostyryl ring system. The compounds were submitted to biological screening against cancer cell lines. Full experimental and spectra data are provided for all new compounds.     

Plants of the ‘Libellus de Medicinalibus Indorum Herbis’ from Mexico, 1552. Zephyranthes fosteri (Amaryllidaceae) Alkaloids

The Libellus de Medicinalibus Indorum Herbis (Booklet of Indian Medicinal Plants) is the first book of medicinal plants written in the American continent. It was first published in 1939 as ‘An Aztec Herbal’. One of the depicted plants is Huetzcanixochitl (laughing flower) interpreted as Zephyranthes fosteri (Amaryllidaceae). No chemical or pharmacological studies are reported for this species; so, we decide to investigate it. The GC/MS of the bulbs and aerial parts extracts indicated that they contain Amaryllidaceae alkaloids, among them: lycorine, 3-O-acetylpowelline, and norlycoramine. An unknown major alkaloid was isolated and identified by ¹H, ¹³C-NMR and MS, as 3′-demethoxy-6-epimesembranol ( 1 ). The methanolic extract, the alkaloid fraction, and compound 1 inhibited acetylcholinesterase in vitro. Mesembrine alkaloids are found in Sceletium species (Aizoaceae). Several are known as serotonin recapture inhibitors and have been proposed as potential antidepressant drugs. The presence of 1 suggests that Z. fosteri was probably used in pre-Columbian times in Mexico as a ‘stimulant and euphoriant’, alike Sceletium tortuosum by several ethnic groups in South Africa.     

Stimulating effect of both 4’‐O‐methylnorbelladine feeding and temporary immersion conditions on galanthamine and lycorine production by Leucojum aestivum L. bulblets

Bulb cultures of Leucojum aestivum and L. aestivum ‘Gravety Giant’ were subcultured in medium containing the precursor 4’‐O‐methylnorbelladine (MN) at various concentrations [0 (control), 0.15 and 0.3 g/L]. The cultures were conducted in bioreactor RITA® and lasted for 15, 30, 40 and 50 days. The growth rate and the alkaloid accumulation in bulblets were studied. For this latter purpose, a purification method was developed. It comprised a highly selective solid phase extraction using on the one hand, UPTI‐CLEAN SI and SCX cartridges for plant extracts and on the other hand, 2H cartridges for culture media. Pure alkaloidal fractions were, thus, analyzed by LC‐ESI‐MS allowing the quantitative evaluation of galanthamine and lycorine from culture extracts. Precursor feeding along with temporary immersion conditions was found to significantly improve the accumulation of both galanthamine and lycorine. The maximal concentrations of galanthamine (0.81 mg/g DW) and lycorine (0.54 mg/g DW) in L. aestivum bulblets were reached, respectively, after 40 days of culture with 0.15 g/L of precursor and after 30 days of culture with 0.3 g/L of precursor. In L. aestivum ‘Gravety Giant’ bulb cultures, 0.3 g/L of precursor was the best condition for both galanthamine (0.6 mg/g DW after 50 days) and lycorine (1.13 mg/g DW after 30 days).     

The corona of the daffodil Narcissus bulbocodium shares stamen‐like identity and is distinct from the orthodox floral whorls

The structural homology of the daffodil corona has remained a source of debate throughout the history of botany. Over the years it has been separately referred to as a modified petal stipule, stamen and tepal. Here we provide insights from anatomy and molecular studies to clarify the early developmental stages and position of corona initiation in Narcissus bulbocodium. We demonstrate that the corona initiates as six separate anlagen from hypanthial tissue between the stamens and perianth. Scanning electron microscope images and serial sections demonstrate that corona initiation occurs late in development, after the other floral whorls are fully developed. To define more precisely the identity of the floral structures, daffodil orthologues of the ABC floral organ identity genes were isolated and expression patterns were examined in perianth, stamens, carpel, hypanthial tube and corona tissue. Coupled with in situ hybridisation experiments, these analyses showed that the expression pattern of the C‐class gene NbAGAMOUS in the corona is more similar to that of the stamens than that of the tepals. In combination, our results demonstrate that the corona of the daffodil N. bulbocodium exhibits stamen‐like identity, develops independently from the orthodox floral whorls and is best interpreted as a late elaboration of the region between the petals and stamens associated with epigyny and the hypanthium.     

Patterns of fruit and seed set within inflorescences of Pancratium maritimum (Amaryllidaceae): nonuniform pollination,resource limitation,or architectural effects?

We investigated patterns of fruit and seed production on inflorescences of a population of Pancratium maritimum in northwest Spain over a 2-yr period. Initial findings showed that the earliest opening flowers on an inflorescence are more likely to set fruit and produce more seeds than later opening flowers and that this pattern is maintained throughout the flowering season. Supplementary pollination and flower-removal experiments were performed to investigate whether the observed pattern is attributable (a) to variation in pollen receipt, (b) to sequestration of resources by the earliest flowers on an inflorescence, and/or (c) to "architectural" limitations on the fruit/seed production of later flowers. Supplementary pollination did not improve fruit or seed production by late flowers in either of the 2 yr of study. In flower-removal experiments, the remaining flowers showed improved fruit set and mean number of seeds per flower, by comparison with flowers in the same position on control inflorescences. When all flowers except the latest third were removed, these showed fruit set and seed production similar to those of early flowers on control inflorescences. These results strongly suggest that the observed within-inflorescence patterns of fruit and seed production in P. maritimum are mainly attributable to competition for resources (i.e., explanation b), though other adaptive explanations cannot be ruled out.