Integrating Narcissus-derived galanthamine production into traditional upland farming systems.

Alzheimer's disease (AD) is a disorder associated with progressive degeneration of memory and cognitive function. Galantamine is a licenced treatment for AD but supplies of the plant alkaloid that it is produced from, galanthamine, are limited. This three-year system study tested the potential to combine Narcissus-derived galanthamine production with grassland-based ruminant production. Replicate plots of permanent pasture were prepared with and without bulbs of Narcissus pseudonarcissus sown as lines into the sward. Two different fertiliser regimes were imposed. The above-ground green biomass of N. pseudonarcissus was harvested in early spring and the galanthamine yield determined. In the second harvest year a split-plot design was implemented with lines of N. pseudonarcissus cut annually and biennially. All plots were subsequently grazed by ewes and lambs and animal performance recorded. Incorporation of N. pseudonarcissus into grazed permanent pasture had no detrimental effects on the health or performance of the sheep which subsequently grazed the pasture. There was no consistency to the effects of fertiliser rates on galanthamine yields. There was no difference in overall galanthamine yield if N. pseudonarcissus was cut biennially (1.64 vs. 1.75 kg galanthamine/ha for annual combined vs biennial cuts respectively; s.e.d = 0.117 kg galanthamine/ha; ns). This study verified the feasibility of a dual cropping approach to producing plant-derived galanthamine.

Relative expression of putative genes involved in galanthamine and other Amaryllidaceae alkaloids biosynthesis in Narcissus field and in vitro tissues.

The Narcissus pseudonarcissus cv. Carlton contains Amaryllidaceae alkaloids namely galanthamine, lycorine, homolycorine, narciclasine, which are noted for their pharmaceutical properties such as for the treatment of early to mid-stage Alzheimer's diseases, cancer, tumor etc. Alkaloid biosynthesis using plant in vitro systems has been considered as a tool for drug discovery and the pathways are starting to be understood but still far from complete. Therefore, the study was emphasized to observe the relative expressions of putative genes involved in the biosynthetic pathway leading to the Amaryllidaceae alkaloids in field grown bulbs and developing cell culture systems in Narcissus. MS media fortified with growth regulators were used for the development of tissue culture from Carlton twin-scale explants. MS medium with high auxin, 20 mg/l NAA was the best medium for callus growth and maintenance while media with low auxin, 4 mg/l NAA and MS basal media gave the maximum bulblets. Field tissues showed a higher amount of galanthamine content; i.e. basal plate (1050-1310 µg Gal/g FW) and bulb (980-1150 µg Gal/g FW) than the culture derived samples; callus (1.0-7.0 µg Gal/g FW) and bulblets (12-215 µg Gal/g FW) on a fresh weight (FW) basis. GC-MS chromatograms of samples under study also showed the presence of other important alkaloids i.e. lycorine, homolycorine, lycorenine, haemanthamine, crinamine, lycoramine and tazettine. RNA extracted from in vitro callus, bulblets and field grown bulb, basal plate were used for PCR to detect the relative expression of putative genes; P450, PAL, TYDC and NpO4OMT normalized to actin. The selected transcripts for P450s and TYDC were expressed in both field and in vitro tissues. Higher expressions of PAL were observed in calli than field samples. The expression of NpN4OMT was notably higher in field samples than in vitro tissues. Therefore, in vitro tissues could be a good source for the reproducible and easy extraction of alkaloids from plants.

Functional characterization of phenylalanine ammonia-lyase- and cinnamate 4-hydroxylase-encoding genes from Lycoris radiata, a galanthamine-producing plant.

Galanthamine (GAL), the well-known Amaryllidaceae alkaloid, is a clinically used drug for the treatment of Alzheimer's disease. L-Phenylalanine (Phe) and trans-cinnamic acid (CA) were enzymatically transformed into the catechol portion of GAL. Herein, a Phe ammonia-lyase-encoding gene LrPAL3 and a cinnamate 4-hydroxylase-encoding gene LrC4H were cloned from Lycoris radiata, a GAL-producing plant. LrPAL3 was overexpressed in Escherichia coli and purified to homogeneity. LrPAL3 catalyzes the forward deamination conversion of L-Phe into trans-CA. The 3-chloro- and 4-fluoro-L-Phe were deaminated to generate the corresponding 3-chloro- and 4-fluoro-trans-CA by LrPAL3. LrPAL3-catalyzed reverse hydroamination was confirmed by the conversion of trans-CA into L-Phe with exceptional regio- and stereo-selectivity. LrC4H was overexpressed in E. coli with tCamCPR, a cytochrome P450 reductase-encoding gene. LrC4H catalyzes the regioselective para-hydroxylation on trans-CA to form p-coumaric acid. The transcriptional levels of both LrPAL3 and LrC4H were positively associated with the GAL contents within the leaves and flowers of L. radiata, which suggested that their expression and function are co-regulated and involved in the biosynthesis of GAL. The present investigations on the biosynthetic genes of GAL will promote the development of synthetic biology platforms for this kind of important drug via metabolic engineering.

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 (226.9 µg/flask and 491.4 µg/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.

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).

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).

Alkaloid patterns in Leucojum aestivum shoot culture cultivated at temporary immersion conditions.

The alkaloid patterns in Leucojum aestivum L. shoot culture cultivated at temporary immersion conditions were investigated using gas chromatography-mass spectrometry. 18 alkaloids were identified, and galanthamine, hamayne and lycorine were dominant. The L. aestivum 80 shoot culture, cultivated at temporary immersion conditions, is a prospective biological matrix for obtaining wide range Amaryllidaceae alkaloids, showing valuable biological and pharmacological activities. The temperature of cultivation influenced enzyme activities, catalyzing phenol oxidative coupling of 4'-O-methylnorbelladine and formation of the different groups Amaryllidaceae alkaloids. Decreasing the temperature of cultivation of L. aestivum 80 shoot culture led to activation of para-ortho' phenol oxidative coupling (formation of galanthamine type alkaloids) and inhibited ortho-para' and para-para' phenol oxidative coupling (formation of lycorine and haemanthamine types alkaloids).

An overview on natural cholinesterase inhibitors--a multi-targeted drug class--and their mass production.

Cholinesterase enzyme family consisting of acetylcholinesterase (AChE) and butrylcholinesterase (BChE) is important in pathogenesis of Alzheimer's disease (AD), explained by "cholinergic hypothesis". Accordingly, deficiency of the neuromediator called "acetylcholine" excessive amount of BChE has been well-described in the brains of AD patients. Consequently, cholinesterase inhibition has become one of the most-prescribed treatment strategies for AD. In fact, cholinesterase inhibitors have been also reported for their effectiveness in some other diseases including glaucoma, myasthenia gravies, as well as Down syndrome, lately. They play a role in the action of mechanism of insecticidal drugs such as carbamate derivatives as well as nerve gases such as malathion and parathion. All these utilizations can make them a multi-targeted drug class putting a special emphasis on AD therapy in the first place. Several inhibitors of cholinesterases with synthetic and natural origins are available in drug market; however, the reasons including side effects, relatively low bioavailability, etc. limit their uses in medicine and there is still a great demand to discover new cholinesterase inhibitors. Galanthamine, an alkaloid derivative isolated from snowdrop (Galanthus nivalis L.), is the latest anticholinesterase drug used against AD. Huperzine A, isolated from Huperzia serrata (Thunb.) Trev. is the most-promising drug candidate with potent anticholinesterase effect and it is a licensed anti-AD drug in China. In this review, a short introduction will be given on known cholinesterase inhibitors and, then, galanthamine and huperzine A will be covered in regard with their cholinesterase inhibitory potentials and mass productions by organic synthesis and in vitro culture techniques.

Galanthamine and related alkaloids production by Leucojum aestivum L. shoot culture using a temporary immersion technology.

The process of galanthamine and related alkaloids production by Leucojum aestivum shoot culture in a temporary immersion system was studied. It was established that temporary immersion approach is prospective for development of a biosynthetic process for obtaining valuable Amaryllidaceae alkaloids. Both immersion frequency and temperature had significant effect on biomass accumulation and the yields of galanthamine and related alkaloids. The maximal yield of galanthamine was achieved at the cultivation of L. aestivum shoot culture in temporary immersion RITA(®) system at immersion frequency 15 min flooding and 8 h stand-by periods, at 26 °C. Data on the relationships in the biological system "Nutrient medium-L. aestivum shoot culture-galanthamine" are presented as well.

Galanthamine production by Leucojum aestivum cultures in vitro.

The results described in these studies proved that the successful in vitro bioproduction of galanthamine from L. aestivum shoot-clumps required mainly the selection of in vitro clones with a genetically determined high ability to produce the desired alkaloids, although the expression of this ability could be additionally influenced by diverse exterior factors, such as some components of the nutrient medium, or the cultivation conditions of the ambience. Tissue differentiation was also of great importance for the biosynthetic capacity of the cultures. The most suitable inocula for in vitro biosynthesis of galanthamine in liquid medium were the directly regenerated shoot-clumps, ensuring high alkaloid concentrations between 1 and 2 mg/g DW for the selected clones. We observed astonishing clone-specific dynamics of the biosynthetic activity of all of the studied in vitro clones. The dynamics were obviously related to the strong biological clock of the species, persisting even in several-year old cultures. These dynamics did not coincide with those usual for the plants growing in situ and under controlled field conditions. In our opinion, the clone specificity of the biosynthetic dynamics could be due to the disturbance of the plant regulation mechanism under the equal conditions of the ambience in the culture room. The sharp decrease of the alkaloid concentrations were transient, followed by an increase, so that cultures were retaining their biosynthetic capacity. The biosynthesis of the main alkaloids, galanthamine and lycorine, was influenced by diverse stimulants such as substances causing stress (JA), feeding with alkaloid precursors (the amino acids phenylalanine and tyrosine, and CH), and physical treatment (acoustic waves). However, the course of the biosynthetic dynamics during the period of the treatments was always the most important factor for the success of secondary metabolism stimulation. As far as scaling-up of the in vitro biosynthesis of valuable compounds, a stable and predictable yield is required, and additional investigations aimed at the annulment of the effect plant biological clock on alkaloid biosynthesis are needed. The elucidation of the relative influences of the diverse factors modulating alkaloid biosynthesis was of great importance. The high galanthamine concentrations of the selected in vitro clones are a promising basis for future studies.

Optimized nutrient medium for galanthamine production in Leucojum aestivum L. in vitro shoot system.

The common effect of NH4+, NO3-, KH2PO4 and sucrose on the biosynthesis of galanthamine by a Leucojum aestivum shoot culture was studied. Polynominal regression models were elaborated for the description of the galanthamine biosynthesis as a consequence of variation of the investigated variables (NH4+ between 0.20 and 0.54 g/L; NO3- between 1.44 and 3.44 g/L; KH2PO4 between 0.10 and 0.24 g/L, and sucrose between 30.00 and 60.00 g/L). Optimization procedures allowed us to establish the optimal concentrations of the investigated variables and to propose the modified MS nutrient medium, with 4.50 g/L KNO3, 0.89 g/L NH4NO3, 1.25 g/L (NH4)2SO4, 0.10 g/L KH2PO4 and 60 g/L sucrose, for the galanthamine production by a Leucojum aestivum shoot culture. The proposed modified MS medium provided considerable increase of both the production yield and the relative content of the target alkaloid in the alkaloid mixture.

Improved production of galanthamine and related alkaloids by methyl jasmonate in Narcissus confusus shoot-clumps.

Liquid-shake cultured shoot-clumps of Narcisus confussus were treated with the commonly used biotic elicitors methyl jasmonate, arachidonic acid, chitosan and salicylic acid. The effects of these compounds on the growth of the explants, as well as on the amount of the alkaloids released to the liquid culture medium and accumulated in the tissues at the end of the experiment were studied. The obtained results showed that, in general, high doses of these compounds had a negative effect on the growth of the explants, particularly the salicylic acid. On the contrary, the addition of methyl jasmonate, mainly at 25 microM, promoted the release of galanthamine and other related alkaloids to the liquid medium in proportions of up to 300% in relation to the control explants, and also their accumulation in tissues. The other elicitors studied did not have any interesting effects on the production of these Amaryllidaceae-type alkaloids.

Galanthamine pattern in Narcissus confusus plants.

Galanthamine is an Amaryllidaceae-type alkaloid with acetylcholinesterase inhibitory activity which is used in the treatment of Alzheimer's disease. The distribution of galanthamine and four other alkaloids in different organs of Narcissus confusus plants, as well as the variations occurring during the ontogenic cycle of this plant species, was studied. The five alkaloids were found to be present in all the organs at every stage, with the exception of haemanthamine in senescent flowers. The highest amount of alkaloids occurred in the bulb at the emerging stage, galanthamine being the most abundant, reaching a concentration of up to 2.5 % referred to dry weight.

Evaluation of four Narcissus cultivars as potential sources for galanthamine production.

Galanthamine, an alkaloid present in the Amaryllidaceae is currently undergoing clinical trials for the treatment of Alzheimer's. Common daffodils, Narcissus spp., contain galanthamine and other alkaloids. Four commercial Narcissus cultivars were evaluated as potential sources of galanthamine. Planting depths, planting densities, bulb size or flower bud removal did not affect galanthamine content.