Leucojum Aestivum Research Articles

Plants of the Amaryllidaceae family grown and introduced in Georgia: A source of galanthamine

A series of plants of the Amaryllidaceae family (Galanthus woronowii A. Los., Galanthus krasnowii Hohrjakov, Galanthus caucasicus Bacer A. Grossheim, Galanthus latifolius Wor, Leucojum aestivum L., Stenbergia colchiciflora Waldst et Kit, Pancratium maritimum L., Crinum giganteum L., Narcissus tazetta L.), grown and introduced in Georgia, has been studied for their alkaloid composition. Alkaloids galanthamine, lycorine, tazettine, galanthine, galanthusine, demethylhomolycorine, and gemanthamine have been isolated and chemically characterized. The dynamics of galanthamine accumulation during vegetation has been studied. It is established that the content of galanthamine reaches maximum in bulbs at the end of the vegetation stage (except for L. aestivum, in which the maximum is reached at the beginning of vegetation). The bulbs should be harvested in the indicated vegetation periods. The plants of G. woronowii, G. krasnowii, N. tazetta, and L. aestivum species are recommended as raw materials for obtaining galanthamine hydrobromide.

Results with the establishment of in vitro culture of Leucojum aestivum

Leucojum aestivum is a native, protected ornamental and medicinal plant in Hungary and in Ukraine too. The aim of our work was to establish in vitro cultures of this bulbous plant. Prior to surface sterilisation the old leaves and roots were dissected from the bulbs and they were stored in a refrigerator (2-3°C) for different periods (1 week for the first starting experiment and 5 weeks for the second one). After sterilisation, bulbs, bulb scales and leaves of the bulbs were placed on Murashige and Skoog's (1962) medium with 1 mg/1 benzyl-adenine (BA) and 0,1 mg/1 naphthalene acetic acid (NAA). At the first starting experiment 81,3%, and at the second one 92,3% of the explants turned to be sterile. Bulblets and roots were developed on the explants in the case of using bulb plates together with bulb scales and leaves as inoculua. The best result was achieved after 5 weeks chilling and it was possible to gain little bulbs from the bulb leaves too.

Hairy root and tissue cultures of Leucojum aestivum L.—relationships to galanthamine content

Galanthamine, an isoquinoline alkaloid acetylcholinesterase inhibitor, is an important agent used all around the world for the symptomatic treatment of senile dementia of the Alzheimer’s type. The production of this metabolite and the availability of the plant are limited and prompted the search for an alternative way to obtain this valuable metabolite using in vitro cultures of Leucojum aestivum L. It is known that cell differentiation level shows a major influence upon the accumulation of alkaloids. For this reason, tissue cultures of L. aestivum showing different stages of morphogenesis controlled by exogenous growth regulators were established. Agrobacterium rhizogenes strain LBA 9402 has been tested for its capacity to induce hairy roots of this monocotyledonae plant.

Galanthamine production by Leucojum aestivum in vitro systems

The callus induction from young fruits of Leucojum aestivum was performed on Murashige–Skoog nutrient medium supplemented with 4 mg/L 2,4 dichlorphenoxyacetic and 2 mg/L 6-benzylamynopurine. Further, by planting the obtained calluses on the same nutrient medium supplemented with 1.15 mg/L α-naphtylacetic acid and 2.0 mg/L 6-benzylamynopurine shoot cultures were established. The growth and galanthamine accumulation of obtained L. aestivum in vitro systems were studied. It was established that the amount of accumulated galanthamine strongly depended on the level of the differentiation. The maximum yield of biomass (17.8 g/L) and the maximum amount of accumulated galanthamine (2.5 mg/L) were achieved after day 35 of submerged cultivation of L. aestivum 80 shoot culture, performed under illumination. Data concerning the time courses of the utilization of the main nutrient components of the medium during cultivation of L. aestivum shoot culture are presented as well.

Galanthamine Content of Bulbs and in Vitro Cultures of Leucojum Aestivum L

In vitro cultures, at different stages of morphogenesis, were established from leaves of Leucojum aestivum (Amaryllidaceae) for determination of their galanthamine content, an alkaloid that possesses cholinesterase inhibitory activity and which is used for the treatment of Alzheimer's disease. A suitable HPLC method for qualitative and quantitative determination of galanthamine in both in vitro and in vivo extracts has been developed. Confirmation of the identity of galanthamine in sample extracts was achieved using LC-MS-MS. A correlation was observed between the state of differentiation and the galanthamine content of the tissue cultures. No galanthamine was detected in the roots grown in vitro, while all bulblets grown in vitro showed the presence of this alkaloid, with dramatic variations in concentration levels, according to the growth substance balance. The best result (6.79 × 10−3 % of D.W.) was obtained with bulblets initiated with NAA (10 μM) combined with BA (0.5 μM).

A study on nitrate reductase activity (NRA) of geophytes from Mediterranean environment

Nitrate reductase activity (NRA) in different compartments of 14 Mediterranean geophytes (bulbous, tuberous and rhizomatous) and actual mineral nitrogen (NO 3 − and NH 4 +) in their soils were investigated. The nitrate reduction capacities of each species were determined as NRA per total plant material. Differences among compartments for NRA were significant in all species. The highest NRA was found in leaves of tuberous species ( Anemone coronaria, Cyclamen coum) and of most bulbous species ( Allium flavum, Allium guttatum, Bellevelia sarmatica, Galanthus plicatus, Leucojum aestivum, Ornithogalum nutans, Tulipa sylvestris). Therefore, in this group of species the contribution of the leaves to total plant NRA was the highest. The other bulbous species ( Allium scorodoprasum, Crocus chrysanthus, Fritillaria bithynica, Muscari neglectum) and one rhizomatous taxon ( Iris suaveolens) have a different NRA distribution within the plants. In these species the highest values of NRA were found in different organs. For example, in Allium scorodoprasum the highest NRA was in tunics, and in flowers in M. neglectum. Although leaves are the main compartments reducing nitrate in most of the studied geophytes, other compartments also contribute to total plant nitrate reduction. Our results show that the nitrate reduction capacity is different among geophyte species. Even if it roughly reflects the nitrogen supply in a habitat, differences in nitrate reduction capacities of different species collected from same sites indicate that the nitrate reducing capacity is species-specific.

Effects of sucrose and silver thiosulphate pulsing on stem-base cracking and vase-life in Leucojum aestivum flowers

SummaryCut Leucojum aestivum L. (cv. ‘Summer Snowflake’) flowers were treated with distilled water (control), sucrose (5–10%), silver thiosulphate (STS; 1–2 mM), or combinations of sucrose plus STS. Pulse treatments were applied for 2 h in a controlled environment room maintained at 20°–21°C. A vase-life of 4.8 d was obtained for flowers treated with distilled water (control). The longest vase-life of 7.8 d was obtained for flowers treated with 1 mM STS + 10% sucrose.This treatment also significantly reduced stem cracking. Accordingly, it may serve as a practical treatment for preserving cut Leucojum flowers.

Determination of Phenolic Acids in Amaryllidaceae Species by High Performance Liquid Chromatography

Five Bulgarian Amaryllidaceae species; Pancratium maritimum. L., Sternbergia colchiciflora. W. K., Galanthus nivalis. L., Galanthus elwesii. Hook., and Leucojum aestivum. L., were analyzed for their high-performance liquid chromatography (HPLC) profiles of phenolic acids. An improved HPLC method was developed for the simultaneous determination of seven phenolic acids. Protocatechuic, 4-hydroxybenzoic, vanillic, caffeic, syringic, p.-coumaric, and ferulic acids were identified in the studied species. Protocatechuic, 4-hydroxybenzoic, vanillic, and syringic acids are reported for the first time in Amaryllidaceae. Also, this is the first report on phenolic acid composition in the genus Pancratium., as well as in Galanthus elwesii, Sternbergia colchiciflora., and Pancratium maritimum..

CGC-MS of alkaloids inLeucojum aestivum plants and theirin vitro cultures

Underivatised alkaloid mixtures extracted from intact plants and in vitro cultures of Leucojum aestivum (Amaryllidaceae) were investigated by capillary GC-MS. Excellent peak resolution for the alkaloids was exhibited and isomers of galanthamine and N-formylnorgalanthamine were well separated. Fourteen alkaloids of galanthamine, lycorine and crinane types were identified, 11 in the intact plants and eight in the in vitro cultures.

Bulb Yield and Some Plant Characters of Summer Snowflake (Leucojum aestivum L.) Under Shading as Affected by GA3 and NAA at Different Concentrations

Bulb Yield and Some Plant Characters of Summer Snowflake (Leucojum aestivum L.) Under Shading as Affected by GA3 and NAA at Different Concentrations

The Effects of Different N Doses and Harvesting Times on Bulb Yield and Some Plant Characters of Summer Snowflake (Leucojum aestivum L.)

The Effects of Different N Doses and Harvesting Times on Bulb Yield and Some Plant Characters of Summer Snowflake (Leucojum aestivum L.)

Leaf and tepal anatomy, plastid ultrastructure and chlorophyll content in Galanthus nivalis L. and Leucojum aestivum L.

The leaf structure of Galanthus nivalis L. (snowdrop) and Leucojum aestivum L. (snowflake) is characterized by a homogeneous mesophyll tissue. The dominant characters of the leaves are cavities with mucose substance. There is a striking difference between these plants tepal anatomy. A central cavity occurs only in snowdrop tepals. Plastids from white parts of the tepals have a poorly developed membrane system. Leaves and green parts of tepals of both species possess amoeboid chloroplasts and contain chlorophyll a and b. The chlorophyll content in tepals is lower than in leaves, but the chlorophyll a:b ratio is always 2:1. Both, snowdrop and snowflake are from the family Amaryllidaceae, but their ecology is different. This paper presents common features related to systematic relatedness and differences induced by ecological factors.

Antimalarial activity screening of some alkaloids and the plant extracts from Amaryllidaceae.

Four groups of Amaryllidaceae alkaloids, namely lycorine-, crinine-, tazettine-, and galanthamine-type, as well as plant extracts of the Amaryllidaceae plants (Pancratium maritimum, Leucojum aestivum, and Narcissus tazetta ssp. tazetta) growing in Turkey were evaluated in vitro for their ability to inhibit Plasmodium falciparum growth by a high-throughput screening method with a 96-well microtiter plate. All four groups of alkaloids exhibited antimalarial activity at different potencies. 6-Hydroxyhaemanthamine, haemanthamine and lycorine were found to be the most potent alkaloids against P. falciparum (T9.96) and galanthamine and tazettine had the least potent activity against P. falciparum (K1).

Bioactivity-Directed Fractionation of Alkaloids from Some Amaryllidaceae Plants and Their Anticholinesterase Activity

Chloroform:methanol extracts of the bulbs of five Amaryllidaceae plants, namely Galanthus elwesii Hooker fil., G. Ikariae L., Narcissus tazetta subsp. tazetta L., Leucojum aestivum L., and Pancratium maritimum L. growing in Turkey, were evaluated for their anticholinesterase activity by the Ellman method in comparison with galanthamine as the standard drug. Bioactivity-directed fractionation and isolation studies carried out on G. ikariae and N. tazetta subsp. tazetta extracts afforded eight Amaryllidaceae-type alkaloids in total. We found that the activity of both plant extracts was due to the synergistic interaction of the alkaloids isolated.

Tracer studies with 13C-labeled carbohydrates in cultured plant cells. Retrobiosynthetic analysis of chelidonic acid biosynthesis

The biosynthesis of chelidonic acid was studied in cell suspension cultures of Leucojum aestivum. Cell cultures were supplied with [U- 13C 6]glucose, [1- 13C]glucose or [U- 13C 5]ribose/ribulose in standard medium containing unlabeled glucose. 13C labeling patterns of amino acids obtained by hydrolysis of biomass were determined by NMR spectroscopy and compared to the labeling pattern of chelidonic acid. The data document the incorporation of a contiguous 4-carbon fragment derived from the pentose phosphate pool into chelidonic acid. This suggests a biosynthetic pathway involving the condensation of phosphoenolpyruvate with a pentose phosphate followed by dehydration, dehydrogenation, ring closure and decarboxylation conducive to the loss of C-5 of the pentose precursor.

Antiviral Activities in Extracts of Turkish Medicinal Plants

A total of 16 ethanol extracts of Turkish medicinal plants were evaluated for antiviral activities against herpes simplex virus (HSV) and Sindbis virus (SINV). Extracts of Galanthus elwesii and Rheum ribes showed the most potent anti-HSV activities, while six other extracts had weaker activities. Galanthus elwesii and Leucojum aestivum were the most potent anti-SINV extracts with four others showing weaker activities. In total, five extracts were active against both viruses, three were selective for HSV and one was selective for SINV. Evidence for an antiviral photosensitizer was obtained in two anti-HSV extracts, in which activity was either completely dependent on light, or was con-siderably enhanced by light. Thus, several Turkish medicinal plants appear to be promising sources of antiviral activities.

Antiviral Activities in Extracts of Turkish Medicinal Plants

A total of 16 ethanol extracts of Turkish medicinal plants were evaluated for antiviral activities against herpes simplex virus (HSV) and Sindbis virus (SINV). Extracts of Galanthus elwesii and Rheum ribes showed the most potent anti-HSV activities, while six other extracts had weaker activities. Galanthus elwesii and Leucojum aestivum were the most potent anti-SINV extracts with four others showing weaker activities. In total, five extracts were active against both viruses, three were selective for HSV and one was selective for SINV. Evidence for an antiviral photosensitizer was obtained in two anti-HSV extracts, in which activity was either completely dependent on light, or was con-siderably enhanced by light. Thus, several Turkish medicinal plants appear to be promising sources of antiviral activities.

Biosynthesis of the amaryllidaceae alkaloid Galanthamine

Application of radioactively as well as 13C-labelled 4′- O-methylnorbelladine to organs of field grown Leucojum aestivum plants resulted in an optimal incorporation into galanthamine (27%), and N-demethylgalanthamine (31%), respectively. In contrast to expectations based on results from the literature, the N-methylated 4′- O-methylnorbelladine was metabolized into a minor extent in L.aestivum and was incorporated into galanthamine as well as into N-demethylgalanthamine to only about 1\\3 of the rate of 4′- O-methylnorbelladine. Furthermore, it was shown that N-demethylgalanthamine is N-methylated to galanthamine in the final step of biosynthesis. A revised scheme for the biosynthesis of galanthamine is presented involving the phenol oxidative coupling of 4′- O-methylnorbelladine to a postulated dienone which undergoes spontaneous closure of the ether bridge to yield N-demethylnarwedine which upon stereoselective reduction and N-methylation yields the cholineesterase inhibitor galanthamine.

Characteristics of natural growing areas forLeucojum aestivumL. in Bulgaria based on chemical composition of plants

The characterization Bulgarian natural growing areas of Leucojum aestivum L. was on the basis of the chemical composition of the plants and some soil characteristics which give an opportunity to choose those conditions suitable for growing and obtaining enlargement of the source of plant material with high galanthamine content. This information should benefit the improvement of natural growing areas for Leucojum aestivum L. as well as for the creation of new industrial plantations.

Morphogenetic potential and in vitro micropropagation of endangered plant species Leucojum aestivum L. and Lilium rhodopaeum Delip.

The present work deals with the in vitro methods for rapid propagation, and morphogenetic potential of the rare and endangered bulb species Leucojum aestivum L., Amaryllidaceae, and Lilium rhodopaeum Delip., Liliaceae. The morphogenetic potential of different plant organs (bulb, stem, leaves and ovaries) was studied. Leaves of Leucojum aestivum L. and basal parts of the bulb in Lilium rhodopaeum Delip. possess the highest regeneration activity. Murashige and Skoog (MS) medium + 1 mg/l 6-benzylaminopurine (BAP) + 1 mg/l kinetin and Linsmaier and Skoog (LS) medium + 0.5 mg/l 1-naphthaleneacetic acid (NAA) + 0.1 mg/l kinetin were favourable for direct organogenisis from these explants. A stimulating effect of alow gamma-irradiation dose (5 Gy) upon the quantity and growth intensity of the bulbs formed by the explants in in vitro conditions is observed.