Leucojum Aestivum Research Articles

4′‐O‐Methylnorbelladine feeding enhances galanthamine and lycorine production by Leucojum aestivum L. shoot cultures

Galanthamine and lycorine are Amaryllidaceae alkaloids that exhibit a wide range of pharmacological activities. Organic synthesis and plant extraction are the two main commercial sources of galanthamine. Since total organic synthesis is low yielding and complex, and the harvesting of plant biomass is causing wild plant depletion, in vitro cultures offer an alternative approach. Amaryllidaceae alkaloids can be regarded as derivatives of the common precursor 4′‐O‐methylnorbelladine via intramolecular oxidative phenol coupling. The aim of this work was to enhance the biosynthetic pathway of these alkaloids using in vitro techniques. The precursor 4′‐O‐methylnorbelladine was incorporated into the liquid medium of Leucojum aestivum shoot cultures at different concentrations (0, 0.05, 0.1, or 0.2 g/L). The cultures were conducted for various periods of time (15, 30, and 40 days). After alkaloid extraction and according to LC‐ESI‐MS analysis, the precursor highly stimulated the biosynthesis of both galanthamine (0.5 mg/g dry weight [DW], V/S 0.01 mg/g DW in the control sample, i.e. without precursor) and lycorine (0.2 mg/g DW, V/S 0.04 mg/g DW in the control sample) after its biotransformation by the shoot cultures. The optimal culture conditions for the production of both alkaloids were 0.1 g/L of precursor and 15 days of culture.

Morphological and anatomical features of Leucojum aestivum L. (lower reaches of the Dnieper, Ukraine)

Morphological and anatomical features of Leucojum aestivum L. (lower reaches of the Dnieper, Ukraine)

THE INFLUENCE OF ENVIRONMENTAL CONDITIONS ON SEASONAL CYCLES OF DEVELOPMENT OF THE LEUCOJUM AESTIVUM, GALANTHUS WORONOWII AND PANCRATIUM MARITIMUM

Ecological conditions of habitats essentially influence on phenological phases terms at Leucojum aestivum, Galanthus woronowii and Pancratium maritimum on the Black Sea coast ofRussia. The general duration of vegetation of Leucojum aestivum and Galanthus woronowii populations on the shaded habitats exceeds that for the opened habitats on 3 and 2 weeks respectively; Pancratium maritimum, on the contrary, differs continuous vegetation on open habitats. Despite earlier beginning of flowering in the shaded conditions, fruits maturing of Leucojum aestivum and Galanthus woronowii is noted for a week later, than on the shined habitats; flowering of Pancratium maritimum in the shaded conditions can not be observed.

Galanthamine biosynthesis in plant in vitro systems

The Amaryllidaceae alkaloid galanthamine (Gal) is a long‐acting, selective, reversible, and competitive acetylcholinesterase inhibitor used for the treatment of early‐ to mid‐stage Alzheimer's disease, poliomyelitis, and other neurological diseases. Currently, Gal is produced by extraction from plants such as daffodils (Narcissus cultivar Carlthon), snowflake (Leucojum aestivum), “red‐tubed lily” (Lycoris radiata), and Ungernia victoria, and alternatively by chemical synthesis. Due to the increased demand by the generic pharmaceutical companies and the limited availability of plant sources, the biosynthesis of Gal by plant in vitro systems has been considered as an alternative approach for its sustainable production. The present article reviews the state of the art of in vitro Gal biosynthesis including growth regulators, medium components, culture conditions, elicitation, and bioreactor systems. It may be used as a starting point for further studies in this area leading to a progress in biotechnological production of this valuable alkaloid.

Leucojum aestivum L. in vitro bulbs induction and acclimatization

Abstract The effects of growth retardants (paclobutrazol and ancymidol), sucrose, GA3 (gibberellic acid) and physical state of the medium (solid and liquid — Rita® temporary immersion system) on in vitro induction of Leucojum aestivum bulbs and their acclimatization were studied. Paclobutrazol, regardless of the physical state of the medium, stimulated the formation of bulbs (99.3%). Under the influence 90 g L−1 of sucrose or paclobutrazol the bulbs with the highest fresh weight (FW) were formed (250 mg and 208.8 mg, respectively). However, the addition of ancymidol to the liquid medium led to obtaining the bulbs showing the highest number of leaves and roots (63.2% and 91.7%, respectively). The scanning microscopy study proved that plants obtained in the medium containing GA3 produced the stomata which most closely resembled to the one observed in the mother plant. Cytometric analysis of all regenerants revealed absence of changes in the nuclear DNA content. The maximum survival rate (100%) was observed for plants derived from liquid medium containing 90 g L−1 of sucrose. Somewhat fewer plants were acclimatized after their cultivations in liquid medium enriched with paclobutrazol or ancymidol. The temporary immersion system led to perform successful ex vitro adaptation of Leucojum aestivum plants.

Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus.

Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer’s disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4′-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4′-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.

Medical Resource Value Appraisal for Leucojum aestivum in Turkey.

In this study, we investigate the medical and economical value of Leucojum aestivum. Leucojum aestivum contains the alkaloid galanthamine, which is one of the main active substances in Reminyl, a commonly used drug for the treatment of Alzheimer's disease (AD). In this analysis, we estimate that there are 13 million mild to moderate patients with Alzheimer's who use Reminyl. Our results suggest that the market change value of L aestivum required for 1 unit of Reminyl is US$62. The value of total change of galanthamine and L aestivumis estimated to be US$18.6 billion per year. Alzheimer's drugs that use L aestivum are relatively expensive, ranging in cost from US$183 to more than US$400 per month. The minimum annual cost of this is US$2196 per person for "patients with mild and moderate stage AD." The using value of 6 million units of L aestivum bulbs, which is the amount exported from Turkey, is estimated to be US$27 million per year. The determined value for L aestivum is equivalent to the value of a rare flower that can be used to treat the serious illness of AD.

Elicitation of galanthamine biosynthesis by Leucojum aestivum liquid shoot cultures

The effects of methyl jasmonate and jasmonic acid on galanthamine production, phenolic acid content and growth of Leucojum aestivum L. shoot culture, cultivated in submerged conditions were investigated. The best time-point for addition of elicitors was during the exponential phase of the culture growth. The maximal contents of galanthamine and lycorine (226.9μg/flask and 491.4μg/flask, 1.36 and 1.67-fold higher compared to the control, respectively) were achieved after elicitation with jasmonic acid, whereas the elicitation with methyl jasmonte resulted in maximal accumulation of phenolic acids. It was demonstrated that the boosting effect of jasmonic acid on Amaryllidacea alkaloid biosynthesis was due to induction of the activity of tyrosine decarboxylase, whereas methyl jasmonate stimulates the biosynthesis of phenolic acids by inducing mainly the activity of phenylalanine ammonia-lyase.

Late-discovered petaloid monocotyledons: separating the native and alien flora

The criteria that might help in assessing native or alien status are set out and any recent developments noted. The major change has been a better understanding of north-western European distribution and alien plant occurrences, mainly through the production of dot-map floras. Six petaloid monocotyledons are examined — all well-known and long-grown garden plants, and all with not only a late discovery, but also with a subsequent almost immediate clumping of records from a number of counties. This is contrasted with the discovery and traits of species of similar broad habitats in the same counties, which were almost entirely found much earlier. Two probably alien species, Leucojum aestivum and Muscari neglectum, are examined in depth, and the history of their discovery contrasted with a certainly native third, Allium schoenoprasum. The advantages and disadvantages of using just the first date of discovery as a criterion for native or alien status are noted, as well as the perceived lack of any change in public attitudes towards alien plants. A significant improvement in the numbers of species covered in popular, keyed, floras, since 1990 has led, in turn, to a rapid increase in the number of recorders dealing with them and an equally rapid increase in records. This has complicated our understanding of the perceived threat to the native flora from alien species. Progress in Europe is noted and it is questioned whether there is the same concern there with the assignment of species to native and alien categories.

Influence of auxins on somatic embryogenesis and alkaloid accumulation in Leucojum aestivum callus

Abstract In vitro cultures of Leucojum aestivum are considered as an alternative for the production of galanthamine, which is used for the symptomatic treatment of Alzheimer’s disease. We studied the effects of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), 3,6-dichloro-o-anisic acid (dicamba) at concentrations of 25 and 50 µM on the induction of embryogenic callus and its capacity to induce somatic embryogenesis and alkaloid accumulation. The embryogenic response of the explants was from 30% for 25 µM of dicamba to 100% for picloram (for both 25 and 50 µM). 2,4-D (50 µM) stimulated greater callus proliferation and somatic embryo induction as compared to the other auxins. Polyethylene glycol (PEG) stimulated somatic embryo maturation. Callus grown on media containing 50 µM of auxins produced fewer phenolic compounds as compared with callus grown on media containing 25 µM of auxins. GC-MS analyses showed seven alkaloids in the in vivo bulbs and two to four in callus culture. Galanthamine was detected in callus cultivated with 2,4-D (25, 50 µM), picloram (25 µM), and dicamba (50 µM). Other alkaloids, trisphaeridine, tazettine, and 11-hydroxyvittatine were accumulated only in callus growing on medium with picloram (50 µM).

Elicitation of galanthamine production by leucojum aestivum shoots grown in temporary immersion system

The influence of different elicitors (copper sulfate, silver nitrate, salicylic acid and methyl jasmonate), on both the growth and alkaloid production of Leucojum aestivum shoots grown in a temporary immersion system was studied. Seven Amaryllidaceae alkaloids and three protoalkaloids were quantitatively determined by GC-MS analysis in leaves and bulblets, separately. Methyl jasmonate was found to significantly improve the production of galanthamine (GAL) in both leaves and bulblets. The content of GAL released to the liquid nutrient medium was also measured. The release of GAL into the liquid medium took place mainly in the first 2 weeks determined by harvesting the liquid nutrient medium after 2 weeks and measuring the GAL content (1st subculturing step).

A Zákányi-dombok égeres mocsárerdei [Angelico sylvestri-Alnetum glutinosae Borhidi in Borhidi & Kevey 1996

We present in this paper the phytosociological analysis of 50 samples of peat deficient alder swamps (Angelico sylvestri-Alnetum glutinosae) found in the Zákány Hills along the Drava river in southwestern Hungary. Most of these swamps are found on nutrient-rich alluvial soil along the Visszafolyó stream. The species composition of them does not show signs of sub-Mediterranean and Illyrian influences, which generally are characteristic of the Zákány Hills. Instead, it is characterized by the occurrence of a number of regional rarities, such as Alnus incana, Cardamine amara, Carex elongata, C. pseudocyperus, C. rostrata, C. strigosa, Doronicum austriacum, Dryopteris carthusiana, D. dilatata, D. expansa, Equisetum hyemale, Geranium palustre, Leucojum aestivum, Peucedanum palustre, Ranunculus lingua, Ribes rubrum, Rubus idaeus, Scilla drunensis, Thelypteris palustris, Veratrum album. The stands are somewhat related to alder gallery forests (Carici pendulae-Alnetum glutinosae), but are more closely related to typical alder swamps (Carici elongatae-Alnetum glutinosae). As an associastion, it is classified in the suballiance Scirpo sylvaticiAlnenion glutinosae Kevey 2008a.

Burkholderia Gladioli and Pseudomonas Marginalis Pathogens of Leucojum Aestivum

ABSTRACTLeucojum aestivum (summer snowflake) is a perennial, wild plant species of medical importance. L. aestivum is in danger of extinction and has been thus put under a regulated regime for conservation. However, its natural fields in Bulgaria continue to deteriorate, one of the reasons being contamination of rivers with industrial waste. The plant has been long known not to suffer diseases. In the period 2006–2011, however, diseased bulbs with symptoms of bacteriosis were observed. This paper presents the third of a series of investigations of L. aestivum in Bulgaria with symptoms of bacterial rot.After the pathogenicity tests and on the basis of physiological characteristics, metabolic fingerprints, and PCR with specific primers, bacterial strains—causal agents of bacterial rot of L. aestivum were identified as Burkholderia gladioli and Pseudomonas marginalis. Additional PCR using the RAPD technique differentiated Burkholderia gladioli pv. alliicola. It could be speculated that contamination of rivers may have been the reason for the spread of pathogens which normally infect other bulbous plants and can survive in soil and water, thus contributing to deterioration of the natural L. aestivum fields.

Leucojum aestivum plants propagated in in vitro bioreactor culture and on solid media containing cytokinins

Cytokinins are growth regulators that stimulate cell division and control morphogenesis in plants, however their role in regulating secondary metabolism is not well studied. The influence of various cytokinins (benzyladenine, zeatin, kinetin, meta‐topolin, thidiazuron) and culture systems (solid and temporary immersion RITA® system) on the quality Leucojum aestivum plant regenerated from somatic embryos was investigated. The largest number of regenerated plants (181.6 and 168.8) was obtained from the embryos cultivated on media enriched with meta‐topolin and benzyladenine. Thidiazuron and meta‐topolin led to the highest number of normally developed plants (94.8 and 90.6). The random amplified polymorphic DNA analysis of in vitro and in vivo plants showed four clusters of similarity. The highest biomass (growth index: 2.49) was obtained with the temporary immersion RITA® system. Alkaloid extracts were analyzed by LC‐MS, leading to the quantification of galanthamine and lycorine both in plant materials and in liquid media. The highest contents of galanthamine (0.05% dry weight) were observed in plants cultivated in the presence of thidiazuron in bioreactor system. Galanthamine was accumulated (highest content 0.05% dry weight) in plants cultivated in the presence of thidiazuron in bioreactor system whereas lycorine was synthetized mainly in plants cultivated on solid media.

The geographic isolation of Leucojum aestivum populations leads to divergation of alkaloid biosynthesis

Leucojum aestivum, an industrial source of the acetylcholinesterase inhibitor galanthamine, shows a great chemodiversity in its alkaloid synthesis. Samples from various geographically distinct populations from Bulgaria and the Balearic Islands were studied by GC–MS. The alkaloid pattern of the plants of L. aestivum (subsp. pulchellum) from the Balearic Islands were dominated by crinine type compounds. Populations of homolycorine chemotype were distributed along the Danube river in the north part of Bulgaria, which is separated from the south part by the Balkan mountains. Populations with high accumulation of lycorine were found in East Bulgaria near the sea coast, while the south populations were dominated by galanthamine type synthesis. The average of the galanthamine content was found to vary from 0.003 to 0.08% (referred to dry weight) in the north, and up to 0.42% in the southern Bulgarian populations. Some individuals showed up to 0.65% galanthamine. The galanthamine content of the plants from the Balearic island was 0.1% of DW. The galanthamine percentage in the total alkaloid mixture ranged from 0.2 to 95% of the total alkaloids. Our study demonstrated that the geographic isolation of the populations of L. aestivum has led to divergation in the alkaloid biosynthesis and consequently to the occurrence of different chemotypes. This chemodiversity in both alkaloid patterns and galanthamine content provides an opportunity for further selection work toward a galanthamine-rich crop, on the one hand, and makes the species an excellent biological system for molecular studies leading to further improvement of the galanthamine production, which is a valuable alkaloid used in medicine for the treatment of Alzheimer's disease.

Ultrastructural studies on the sporogenous tissue and anther wall of Leucojum aestivum (amaryllidaceae) in different developmental stages

In this study, ultrastructures of anther wall and sporogenous tissue of Leucojum aestivum were investigated during different developmental stages. Cytomictic channels were seen between pollen mother cells during prophase I. Polar distribution was described in the organelle content of pollen mother cells and microspores in early phases of microsporogenesis and also in pollen mitosis. Active secretion was observed in tapetal cells. Previous reports about developmental stages of male gametophyte were compared with the results of this study.

Some histochemical features of anther wall of Leucojum aestivum (Amaryllidaceae) during pollen development

In this study, polysaccharide and RNA contents of anthers were investigated on different phases of sporogenesis by using light microscopy techniques from histological and cytological point of view in Leucojum aestivum. Paraffin and semi-thin sections of anthers were stained with toluidine blue and PAS. Anthers were tetrasporangiate. The wall of the anther consists of an epidermis, endothecium, middle layer and glandular tapetum. During one nucleated microspore and mature pollen phase microspores and tapetum cells began to degenerate and they were become very rich of RNA in L. aestivum. And also RNA content was increased in endothecium and middle layer cells except the epidermis cells of anther wall. An increase in RNA content indicates cell activation. Polysaccharides were not seen in young anther wall but they were seen in older ones. They were generally condensed in the cell walls and especially in the cell walls of vascular bundles of connective tissue. This could be thought that insoluble polysaccharides were used in metabolic events in early developmental stages. Appearance of polysaccharides in late phases was indicated that polysaccharides were used in the formation of cuticule and differentiation of endothelium cell walls.

GC‐MS of amaryllidaceous galanthamine‐type alkaloids

Galanthamine-type alkaloids produced by plants of the Amaryllidaceae family are potent acetylcholinesterase inhibitors. One of them, galanthamine, has been marketed as a hydrobromide salt for the treatment of Alzheimer's disease. In the present work, gas chromatography with electron impact mass spectrometry (GC-EIMS) fragmentation of 12 reference compounds isolated from various amaryllidaceous plants and identified by spectroscopic methods (1D and 2D nuclear magnetic resonance, circular dichroism, high-resolution MS (HRMS) and EIMS) was studied by tandem mass spectrometry (GC-MS/MS) and accurate mass measurements (GC-HRMS). The studied compounds showed good peak shape and efficient GC separation with a GC-MS fragmentation pattern similar to that obtained by direct insertion probe. With the exception of galanthamine-N-oxide and N-formylnorgalanthamine, the galanthamine-type compounds showed abundant [M](+.) and [M-H](+) ions. A typical fragmentation pattern was also observed, depending on the substituents of the skeleton. Based on the fragmentation pathways of reference compounds, three other galanthamine-type alkaloids, including 3-O-(2'-butenoyl)sanguinine, which possesses a previously unelucidated structure, were identified in Leucojum aestivum ssp. pulchelum, a species endemic to the Balearic islands. GC-MS can be successfully applied to Amaryllidaceae plant samples in the routine screening for potentially new or known bioactive molecules, chemotaxonomy, biodiversity and identification of impurities in pharmaceutical substances.

Galanthamine production by Leucojum aestivumL. 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.

Production of Galanthamine by Leucojum aestivum Shoots Grown in Different Bioreactor Systems

The production of galanthamine by shoots of Leucojum aestivum grown in different bioreactor systems (shaking and nonshaking batch culture, temporary immersion system, bubble bioreactor, continuous and discontinuous gassing bioreactor) under different culture conditions was studied. The influence of the nutrient medium, weight of inoculum, and size of bioreactor on both growth and galanthamine production was studied. The maximal yield of galanthamine (19.416 mg) was achieved by cultivating the L. aestivum shoots (10 g of fresh inoculum) in a temporary immersion system in a 1-L bioreactor vessel which was used as an airlift culture vessel, gassing 12 times per day (5 min).