Final Report Summary - SUPROGAL (Sustainable Production of Galanthamine by both in vitro and agricultural crops of highly galanthamine-containing plants)
Executive summary:
An international consortium integrated by four SMEs and three RTD performers was constituted to develop the research project entitled "Sustainable production of galanthamine by both in vitro culture and agricultural crops of highly galanthamine-containing plants" (SUPROGAL) within the 7th Framework Programme of the European Union. Galanthamine is an alkaloid produced exclusively by plants of the family Amaryllidaceae, mainly belonging to the genus Galanthus, Leucojum and Narcissus. Owing to its acetylcholinesterase inhibitory activity, galanthamine is used and marketed for the treatment of the Alzheimer's disease, among others. Although it is obtained by organic synthesis, galanthamine is also being extracted from natural sources. Both Galanthus and Leucojum plants are very widespread in the Eastern European countries, whereas Narcissus plants are mainly distributed in the Mediterranean region. For industrial purposes, Leucojum aestivum plants are gathered from wild populations in their natural habitat, which causes increasing problems regarding quality of the plant material as well as depletion pressure on the natural populations. By development of an in vitro production method, both problems will be overcome. Narcissus confusus, an endemic species growing in Spain, was found to produce around two-fold higher amount of galanthamine than L. aestivum.
Most of the objectives and technical goals for the project were achieved:
i) plant populations of high galanthamine-producing plants of L. aestivum and N. confusus were detected,
ii) selected genotypes were propagated by in vitro techniques;
iii) in vitro plant material was produced and supplied to other partners for further experiments;
iv) liquid medium culture was started for mass propagation;
v) cultivation processes of the plant material in airlift bioreactors and temporary immersion systems were developed;
vi) both species were transferred and successfully maintained in soil conditions;
vii) a new protocol for the extraction and purification of galanthamine at pilot plant level was developed, and
viii) an overview of the current status of the galanthamine market was performed.
Project Context and Objectives:
PROJECT CONTEXT AND MAIN OBJECTIVES
Galanthamine is an alkaloid produced exclusively by plants of the family Amaryllidaceae, mainly belonging to the genus Galanthus, Leucojum and Narcissus. Owing to its acetylcholinesterase inhibitory activity, galanthamine is used and marketed for the treatment of the Alzheimer's disease, among others. Although it is obtained by organic synthesis, galanthamine is also being extracted from natural sources. Both Galanthus and Leucojum plants are very widespread in the Eastern European countries, whereas Narcissus plants are mainly distributed in the Mediterranean region. For industrial purposes, Leucojum aestivum plants are gathered from wild populations in their natural habitat, which causes increasing problems regarding quality of the plant material as well as depletion pressure on the natural populations. By development of an in vitro production method, both problems will be overcome. Narcissus confusus, an endemic species growing in Spain, was found to produce around two-fold higher amount of galanthamine than L. aestivum.
So, the main goals of the project can be summarized as follows:
i) to ensure a regular supply of plant raw material under conditions that can be certified by GAP (Good Agricultural Practices) standards for the extraction of galanthamine;
ii) to increase the quality of the natural galanthamine through the adoption of GMP (Good Manufacturing Practices) methods;
iii) to reduce the overall production cost of galanthamine, and
iv) to adopt a marketing stance ensuring the promotion of galanthamine produced by SUPROGAL and set and policed in Europe.
WORK PERFORMED AND MAIN RESULTS ACHIEVED
The project started with plants obtained in former years and also with plants collected from nature. Different wild populations of plants were identified and registered using GPS data. Leucojum aestivum and Narcissus confusus plants from different sites were maintained in ex vitro conditions in Ihtiman, near Sofia (Bulgaria), and Barcelona (Spain), respectively. The plants were checked for their galanthamine content, adapted to soil conditions in a different environment (Netherlands) and they were also introduced and well acclimated to the in vitro culture for mass propagation. Although no serious problems of contamination appeared throughout the cultivation under these conditions (in vitro), cultures of N. confusus evidenced some technical problems in their propagation, which were solved further.
EXPECTED FINAL RESULTS AND THEIR POTENTIAL IMPACTS AND USE
Most of the objectives and technical goals for the project were achieved:
i) plant populations of high galanthamine-producing plants of L. aestivum and N. confusus were detected,
ii) selected genotypes were propagated by in vitro techniques;
iii) in vitro plant material was produced and supplied to other partners for further experiments;
iv) liquid medium culture was started for mass propagation;
v) cultivation processes of the plant material in airlift bioreactors and temporary immersion systems were developed;
vi) both species were transferred and successfully maintained in soil conditions;
vii) a new protocol for the extraction and purification of galanthamine at pilot plant level was developed, and
viii) an overview of the current status of the galanthamine market was performed.
Project Results:
Main S & T Results/foregrounds
The research program was broken down to seven workpackages, each of them divided into different tasks. The work carried out within the project is here described separately for each workpackage.
Workpackage 1) Bioprospecting and conservation of genetic resources
Collection of plant material from different natural populations (ABI & UB)
Summer snowflake, Leucojum aestivum L. (Amaryllidaceae), is a very important plant species in Bulgaria, which is commonly used for commercial production of the alkaloid galanthamine. One of the tasks of AGROBIOINSTITUTE within the project was to collect snowflake plants from as much wild populations as possible.
The expedition was organized at the beginning of spring of 2008 (April-May) during the flowering stage of the plants. Most of the natural populations are under control of the Bulgarian Ministry of Environmental and Water, and there is a low for their protection. Independently of this circumstance, the state of some populations was not satisfactory. A general evaluation of the natural populations for their potential capacity as sources of galanthamine will be performed at the end of the project. In the case of N. confusus, a few populations are growing in Spain. The pants had been previously collected and maintained in the greenhouse at the Faculty of Pharmacy of the University of Barcelona, and they were also screened for alkaloids.
Creation and maintenance of ex situ collections of Leucojum aestivum (ABI)
The plant materials (bulbs and leaves) collected from each population of L. aestivum were separated and prepared accordingly to the requirements of the aims and intended analysis. Apart from this, 10 bulbs from each population of snowflake were used for creation of an ex situ collection in Bulgaria. The bulbs were placed in soil in a parcel located in the Ihtiman region, at about 50 km far from Sofia, where the plants are being cultivated under field conditions without weeds and with sufficient light and humidity.
Preparation and extraction of the plant material for further analyses (ABI and UB)
Leaf and bulb samples from plants of Leucojum aestivum collected individually from each wild population were dried at 60 0C until constant weight and then sent to UNIVERSITY OF BARCELONA for further chemical analysis of both alkaloid profile and galanthamine content (TLC, GC-MS). Plant material of Narcissus confusus was prepared by UNIVERSITY DE BARCELONA in the routine conditions used by the team. On the other hand, young leaves of twenty individuals from each population of L. aestivum were separated and storage at -20 0C, and then used for DNA analysis. Since the beginning of the project, this task has been continuously performed for the further analyses of the samples which are obtained as a result of the different tasks within the workpackages.
Characterization and assessment of genetic resources of Leucojum aestivum (ABI)
Two sets of naturally accessions were analyzed. A set of Leucojum aestivum L. individuals was collected from 31 populations in Bulgaria and the other set of Narcissus confusus were from four different sites in Spain. Three different DNA marker systems were used for the molecular assessment of genetic diversity and population differentiation (RAPD, AFLP and ISSR). During the second period of the project 10 referent individuals with proven chemotype of both L. aestivum and N. confusus from each population were selected for molecular analysis. The genomic DNA was extracted from bulbs and leaves by using commercial innuPREP Plant DNA Kit (analytikjena biosolutions).
RAPD Polymorphism in summer snowflake (Leucojum aestivum L.)
For genotyping and genetic diversity assessment of L. aestivum, reference individuals with proven chemotype by RAPD markers were applied two kind of profiling: 1) RAPD profiling of individual accessions; 2) RAPD profiling of mixed gDNA samples. Screening of 22 RAPD primers allowed selection of two primers (OPA17 and OPB19) which revealed a higher level of polymorphism. The Genetic Similarity (GS) among Bulgarian accessions evaluated from RAPD data ranged from 0.38 to 1.00 thus indicating that a high level of gene diversity is present in the selected genotypes. For the nine accessions supplied by LUDWIG, GS values ranged from 0.43 to 0.96. RAPD analysis proved to be efficient for discrimination of L. aestivum individuals with similar morphological characters and origin.
RAPD Polymorphism in Narcissus confusus L.
For genotyping and genetic diversity assessment of N. confusus, 19 accessions were selected from four different sites in Spain: Barco de Ávila, Peguerinos, Candelario and Santa Maria. RAPD analysis of 19 narcissus accessions was performed using set of five previously selected decamer oligonucleotides with arbitrary sequence and 60-70 % (G+C) content. RAPD patterns obtained are characterized with high reproducibility under the established PCR conditions. A total of 35 fragments, ranging from 190 to 1400 base pairs (bp) were recorded, 57 % of which were polymorphic. The five primers (OPA-01, OPA-02, OPA-03, OPA-17 and OPB-19) used in RAPD analysis generated reproducible polymorphic fragments and could be successfully used for discrimination of the Spanish accessions. There is only one unique fragment of 520 bp in NPC44 amplified by OPB-19. Data analysis showed a middle level of polymorphism ranged from 0.55 to 1.0.
AFLP polymorphism in Leucojum aestivum and Narcissus confusus
The assessment of genetic diversity of L. aestivum was initiated by analysis of four referents accessions collected from four different Bulgarian sites and characterizing by type of galanthamine synthesis (galanthamine, homolycoryne, lycorine and mix: galanthamine+lycorine types). AFLP analysis was performed by using 18 selective primer combinations. A total of 402 fragments, ranging from 70 to 500 bp were recorded, 15 % of which were polymorphic. The eighteen selective primer combinations using in AFLP analysis generated reproducible polymorphic fragments and could be successfully used for discrimination of accessions according to their locations as well as by their chemotype. Analysis of bulked samples from each population also has been done. Data analysis showed variation of genetic distances represented as genetic similarity coefficients (GS) ranged from 0.72 between Archar and Elhovo to 1.0 between Goritsa and Kosharitsa.
Internal Simple Sequence Repeats (ISSR) Population analysis
Internal Simple Sequence Repeats PCR was used to search for polymorphism in the profile of 24 Leucojum populations for further marker development for galanthamine content. Primers for CA-type of microsatellite repeats were used. Inter-microsatellite analysis is a viable alternative to RAPD analysis. On the other hand it has some limitations especially when compared to AFLP. Our results suggest that ISSR-PCR with primers recognizing CA-based repeats or related sequences (CT/CTC, GTG/TG) did not produce sufficient polymorphism to discriminate individual genotypes. This might be due to narrow genetic base of the examined populations. ISSR was further used for identification of chemotype-based differences. Three chemotypes - homolycorine, mixed and galantamine were analyzed with five ISSR primers. Several polymorphic bands were observed in the lines of different chemotypes. Some differences were observed in band intensity obtained with primer 844 for homolycorine and mixed chemotypes. Primer 844 produced single polymorphic band for galanthamine chemotype (844-G650).
Comparison between applied DNA marker systems (RAPD, AFLP and ISSR)
Two natural species, Leucojum aestivum L. and Narcissus confusus L., characterizing as a richest sources of galanthamine, were genotyped by using different genomic DNA fingerprinting techniques, known as dominant DNA marker systems: Random Amplified Polymorphism DNA (RAPD), Internal Simple Sequence Repeats (ISSR) and high resolution - Amplified Fragment Length Polymorphism (AFLP). As regard to generated level of polymorphism and comparing between different methods, RAPD and AFLP were suggested as more informative. A number of chemotype specific DNA markers were found mainly by RAPD method which clearly discriminated three basic chemotypes: homolycorine, galanthamine and lycorine.
Workpackage 2) Chemical and biological analysis
Selection of the best wild plant material on the basis of its galanthamine content (UB and ABI)
Two types of samples were analyzed: samples from individuals, and averaged leaf or bulb samples (from around 20-30 individuals). The first type of samples gives information on the chemical variability within the populations, while the second one characterizes the population as a whole. For the selection of the best plant materials, two chemical parameters were monitored: the galanthamine content, expressed as percentage referred to dry weight (DW), and its proportion in the alkaloid mixture, expressed as % of galanthamine with respect to the total alkaloids.
Screening for alkaloids (galanthamine)
For a fast screening of galanthamine content in leaf samples, a simple thin-layer chromatography (TLC) method was developed. For quantification purposes, the QUANTISCAN software from Biosoft (UK) was used. The method showed RSD values below 10 % thus allowing a comparative analysis of the galanthamine content and alkaloid profile of the samples. By using this fast method, it was easy to select the best plants on the basis of its content of galanthamine before sending the bulbs to LUDWIG and VITROFLORA for further assays. As an average, leaves of fifty plants of L. aestivum per population were screened for alkaloids, what means that a global figure of around 1500 individuals of snowflake were tested with this purpose.
Chemical analysis of plant samples
The TLC analysis revealed a remarkable difference in the alkaloid profile of L. aestivum and N. confusus plants. In the first species, the alkaloid profiles consisted mainly of galanthamine, while in N. confusus it was a mixture of galanthamine and two other main accompanying compounds. After quantitative determination of the alkaloids using the QUANTISCAN program, the analysis of galanthamine in plants of L. aestivum revealed great differences in its content within the same population, ranging from 0.2 to 0.7 % DW.
Alkaloid variability
TLC and GC-MS analyses of N. confusus plants from several individuals within a population revealed altered biosynthesis of their main alkaloids. Thus, for instance, plants of one site were characterised by a high percentage of homolycorine with respect to the total alkaloids of their leaves. Nonetheless, other plants showed no homolycorine but high levels of its precursor, 8-O-demethylhomolycorine, whilst another individual contained neither homolycorine nor 8-O-demethylhomolycorine. What is most interesting is that these plants retained their characteristic spectra when growing in the greenhouse. These biochemical variants showed high levels of galanthamine in their alkaloid mixtures and are of interest for further micropropagation purposes.
Screening of different genetic resources of amaryllidaceous plants for both galanthamine content and acetylcholinesterase inhibitory activity (UB)
Apart from the plant samples mentioned above, other amaryllidaceous plants were studied also for their alkaloid composition searching for new potential sources of galanthamine. Thus, LUDWIG supplied UNIVERSITY OF BARCELONA with different plant materials belonging to the genus Leucojum, and also with bulbs of around one hundred ornamental varieties belonging to the genus Narcissus. These materials were grown in the greenhouse of the Faculty of Pharmacy. Samples were collected in spring of 2010 and they were studied for their alkaloid content and composition, and also for their acetylcholinesterase inhibitory activity.
Comparative study of plant populations
GC-MS analysis of averaged samples (about 50 individuals pooled) from 30 Bulgarian populations of L. aestivum showed a remarkable variability of galanthamine content. The proportion of galanthamine in the alkaloid mixture varied from 0.2 to 95 %. Three geographically distributed chemotypes were revealed whih accumulated mainly homolycorine type compounds, mainly galanthamine type alkaloids, or both lycorine and galanthamine type compounds.
Quantitative analysis of galanthamine and related alkaloids in the in vitro obtained plant material and ex-situ collections and cultivated plants (UB)
A method for the quantitative determination of galanthamine was previously validated for quality control of different plant materials (in vitro and intact plants). Bulbs and leaves of N. confusus, bulbs of N. pseudonarcissus cv. Carlton, leaves of L. aestivum and in vitro cultures of L. aestivum, representing different matrixes, were used for the validation of the method. The extraction process was optimized with respect to pH and time. Derivatization and chromatographic conditions were also optimized. The limits of detection and quantification were determined by analysing samples with a known amount of analyte. Precision and accuracy tests showed acceptable variation according to the Horwitz equation.
Evaluation of the quality of galanthamine substance (UB)
Both the endo- and exo- configurations of the methyl group at the nitrogen atom of some Amaryllidaceae alkaloids have been described in the literature. Also the substituent at the N atom has strong effect on the AChE inhibitory activity of the galanthamine type compounds. For that reason, galanthamine substances from both natural (from Bulgarian Leucojum aestivum plants, supplied by BULPHYTOOILS) and syntheic (supplied by Sopharma, a Bulgarian company) origines were subjected to X-ray analysis. The results indicated that both the natural and synthetic galanthamine substances have identical three-dimensional configuration.
Workpackage 3) Material supplying by in vitro propagation
Introduction of the first material to the in vitro culture (VITROFLORA)
Leucojum aestivum
Initially, VITROFLORA received material of Leucojum aestivum from ABI as in vitro tissues culture material, which was not tested for its galanthamine content. The purpose of working on this non-tested supplied material was the establishment of a micropropagation protocol. The establishment of an in vitro gene bank of L. aestivum was done later starting from other galanthamine reach material.
Narcissus confusus
Bulbs of N. confusus received from UNIVERSITY OF BARCELONA were introduced to in vitro conditions. At the beginning of the work, and similarly to L. aestivum, the first step was gathering knowledge on this new crop. The material was very hard in multiplication. After transferring it to basic culture medium, keeping same proportions of material on the same level, and doing trials with different kinds of multiplication media, it was not succeed. After treating the material in many different ways and grown on different culture media, it was not able to react properly under new conditions, and it finally had to be discarded.
Optimization of micropropagation protocols (VITROFLORA)
Leucojum aestivum
During several months of research work, the material was a basis for different experiments with nutrients (different types and concentrations of growth regulators, macro- and microelements, vitamins), photoperiod, way of cutting, subculture duration, temperature, light conditions, etc. First sample was rooted (48 plants) and sent to LUDWIG for agronomical trials in April 2009. The samples were small (that was the beginning of work with L. aestivum) but allowed LUDWIG to start first trials on early stage of the project.
Narcissus confusus
Similarly to Leucojum plants, the material introduced to in vitro conditions was a basis for propagation experiments. Those explants showing the best multiplication capability in the different combinations of media and culture conditions were chosen for further work. During the work with N. confusus, from July 2009 to January 2010, ca. 80 different media and ca. 40 variants of growing conditions were tested in several experiments. In the second year of project, experiments with different growth conditions were continued.
Multiplication and rooting (VITROFLORA)
Leucojum aestivum
VITROFLORA multiplied material for other partners of the SUPROGAL consortium. Thus, in April 2009, first 20 shoot-clumps were transferred to BIOPLANTA for bioreactor experiments. The same month, first 11 shoot-clumps were sent to UNIVERSITY OF BARCELONA for testing their galanthamine content. In July 2009, a second batch of 100 shoot-clumps was prepared and transferred to BIOPLANTA for bioreactor propagation. Also a second batch of 10 shoot-clumps was sent to UNIVERSITY OF BARCELONA to check if the galanthamine content was stable during the multiplication process. Material was still multiplied to build up stock for supplying to BIOPLANTA. In November 2009, ca. 500 shoot-clumps were delivered to BIOPLANTA for bioreactor experiments. After having established the multiplication protocol, most of the plant material was used for rooting trials. A second batch of rooted plants was delivered to LUDWIG in August 2009.
Narcissus confusus
The first material, 20 shoot-clumps originated from 40 bulbs received from UNIVERSITAT DE BARCELONA was transferred to BIOPLANTA in February 2010 for bioreactor experiments. Likewise, about 100 shoot-clumps from the same starting material were shipped also to BIOPLANTA in the second year or project (August, 2010). Starting from March 2010, the first experiments for rooting trials were set up at VITROFLORA for the optimization of the micropropagation protocol, for the maintenance of the gene bank and for rooting for LUDWIG. In addition, a gene bank from new clones coming from galanthamine rich material was created.
Workpackage 4) Agronomical assays
Establishment of the optimal conditions for the growth (LUDWIG)
Leucojum aestivum
Bulbs of Leucojum aestivum were received from Bulgaria in June 2008. The general impression was that they were in good condition. After arrival, the bulbs were unpacked, weighted and stored to keep the bulbs healthy. One month later, the bulbs got a warm water treatment and then they were dried and placed in a cold storage room. In the first two weeks of October 2009, after preparation of the land, the bulbs were disinfected and planted outside on the field. In this phase different tests couldn't be performed because the bulbs were harvested during flowering. In spring the amount of leaves was little and they didn't bloom.
Narcissus confusus
First bulbs of Narcissus confusus were received on May 25, 2008, from UNIVERSITY OF BARCELONA. The bulbs were packed one by one in a bag, each with their own number. The bulbs were each weighted and then placed into an empty plastic pot until August 4, 2008. After that the bulbs were treated with hot water in order to become more resistant to diseases like narcissus bulb fly and stagonospora, and to develop more rapidly. On October 1st, 2008, the bulbs were taken out of the cold storage room, disinfected and planted.
Scaling of the bulbs for multiplication (LUDWIG)
Leucojum aestivum
Besides the bulbs received from Bulgaria, in summer 2008 a lot of extra bulbs of snowflake were purchased to a supplier from Turkey to do more trials. After a warm water treatment, the bulbs were scaled. After cutting, the segments were disinfected and all packed in peat, then covered and placed dry in the heat chamber. Two different methods to determine how many young bulbs can be obtained from one big bulb were tested. We wanted to make as much young bulbs as possible to get a large stock of Leucojum aestivum in a short period. The result of the test also showed what the minimum size of a part must be to form into a young bulb. Finally, these data are important to calculate the cost price.
Narcissus confusus
On July 20th 2009 a few bulbs were scaled to find out how fast these bulbs can be multiplied to make a calculation on the amount of bulbs that can be produced in what time. The scales, which were checked regularly, were packed separately whereby the whole growing process can be followed per bulb. Meanwhile, in the first week of September 2009 we had noticed that the development of new scales went very well. The temperature of the growing chamber was fixed at 23 °C. In the first week of September 2009 the development of new scales went very well.
Plant breeding (LUDWIG)
Leucojum aestivum
Plants of L. aestivum from several populations of Bulgaria, Turkey and Netherlands were planted. The first selection came from Bulgaria, but did not flower because the bulbs were harvested in 2008 during flowering, and the bulbs could not get the right treatment. The second selection came originally from Turkey and had two different bulb sizes (12 and 10 cm). The results led to the conclusion that bulb size affects the flowering development. The bulbs from the third selection, large bulbs from Holland which are in the soil at our nursery for a few years, were all flowering. The bulbs from Bulgaria were harvested, sorted and weighted separately. After the warm water treatment, the bulbs were packed separately and put in different storage rooms to determine if the different treatments resulted in different alkaloid content. The bulbs from Turkey were cultivated after harvesting and they were prepared for planting for the next season. The bulbs from the Dutch wholesaler were also purchased and a few bulbs bloomed. The bulbs made seeds by themselves, which were collected.
Narcissus confusus
The first plants of Narcissus confusus did not flower. After planting the bulbs in autumn of 2008, the plants quickly developed. The bulbs received a natural dormancy treatment and were planted outside in September 2008. In mid February 2009 the leaves (around 20 cm long) were further developed. Due to the small bulb size, the amount of leaves of the crop had always been quite limited. On April 15, 2009, the first leaves were harvested to be tested for galanthamine. In order to avoid rot problems, the plant material was dried immediately before sending to UNIVERSITY OF BARCELONA. From mid April to July 2009, leaf samples of N. confusus were regularly (every two weeks) sent to UNIVERSITY OF BARCELONA to be further analyzed for alkaloids which allowed seeing the process of the compounds. It started with the first samples when the first buds started to bloom, and it ended when the leaves were fully dried.
Storage during dormancy (LUDWIG)
Leucojum aestivum
Many storage tests were performed to determine the optimal growing conditions to get the highest percentage of galanthamine combined with a high production of plant material. The bulbs were stored at different temperatures during the summer. The highest temperature was 40 °C. After 7 days it was clear that the bulbs could not bear this temperature and the bulbs were fossilized. The bulbs of all the other storage treatments (lower storage temperatures) did very well. It was concluded that storage of the bulbs during the dormancy period at a low temperature is better than a warm one. For cost price reasons, however, cold temperatures are more expensive and give the same results as warm temperatures.
Narcissus confusus
After harvesting and drying the bulbs in 2009, the bulbs of N. confusus were packed in different bags and labelled, and they got different storage treatments. The objective of the different tests was to test the following:
i) influencing the amount of leaves per bulb;
ii) influencing the percentage of galanthamine, and
iii) optimize the growth of the bulbs (if this process can be influenced, it is possible to get a higher profit per ha).
In vitro plants (LUDWIG)
Leucojum aestivum
At the end of April 2009, tissue culture plants of L. aestivum from VITROFLORA (Poland) were supplied to LUDWIG. This material, without any roots, was planted in a crate. The first plants made roots and the first leaves started to grow slowly. The second supply of tissue material was received on November 1st of the same year and it was directly planted in the greenhouse. After planting the in vitro plants, they developed immediately and by April 1st, 2010, all the bulbs produced roots and leaves. The development of the growth of the tissue culture was similar to the growth of the young bulbs coming from the scaling bulbs. It could be concluded that the growing conditions of the plant material (for example growing and storage temperature) are more important than the origin of the material (scaled bulbs versus tissue culture).
Galanthamine content (UB)
Leucojum aestivum
Samples from all different tests and materials from the different selections (Bulgaria, Turkey and Netherlands) were harvested and processed prior to send them to UNIVERSITY OF BARCELONA for further analysis of the galanthamine content. In the leaves of the Bulgarian snowflakes, the highest content of galanthamine took place during the flowering season. After flowering, the content was decreasing again until the starting point of dormancy of the leaves. The Dutch material was found to contain a very low amount of galanthamine, and the leaves of Turkey didn't contain galanthamine at all. The content of galanthamine per bulb was also quite different. As a conclusion, it can be stated that the content of galanthamine in leaves of L. aestivum is the highest during flowering time and that it is depending on the population. So, it is important to use plants of the population with the highest percentage of galanthamine as starting material for further works.
Narcissus confusus
After the results of the tests with the available bulb material of N. confusus, different soil treatment tests were performed with another variety of narcissus, named Carlton. Narcissus cv Carlton is the cultivar which is used for the production of galanthamine at this moment. The control group didn't receive any treatment. In the other treatments the soil was added with different additives like borax, zinc sulphate, magnesium sulphate, zinc, copper sulphate and bonzie. It was concluded that the content of galanthamine in the bulbs is high, and that it is influenced in a different way compared with the control group, according to the compound added in the treatment.
Workpackage 5) In vitro large scale production
Adaptation of in vitro cultures to cultivation in liquid nutrient media (BIOPLANTA)
In 2009 and 2010, in vitro plantlets of Leucojum aestivum and Narcissus confusus as well as Leucojum clones with higher galanthamine content were successfully transferred from VITROFLORA to BIOPLANTA laboratory. In vitro cultures provided by VITROFLORA were tested for the adaptation of the in vitro cultures to cultivation in liquid nutrient media. The composition of the liquid nutrient medium was optimised. Therefore, the influence of plant growth regulators, different amounts of carbohydrates as well as the influence of micro and macro elements and nitrogen contents was investigated.
Leucojum aestivum
In 2009 in vitro plantlets of L. aestivum were successfully transferred to the BIOPLANTA laboratory by VITROFLORA in three different dates. According to protocols from VITROFLORA (medium composition, cutting method) in vitro shoots of L. aestivum were micropropagated by regularly subculture on fresh medium to get enough plant material for experiments.
In a first experiment, in vitro plantlets of L. aestivum were transferred to standard liquid medium (propagation medium without gelling agent). Simultaneously, the effect of macro and micro nutrients, phytohormons, nitrogen (NH4+) and sucrose concentration on the growth was tested in liquid medium. It was shown that shoots of L. aestivum can be successfully transferred to standard liquid medium. In comparison to solid standard medium, in vitro plantlets of L. aestivum showed a considerable growth and less callus formation. The propagation rate of biomass increased significantly in liquid culture medium. The variation of medium composition had no phenotypic effect on the growth of in vitro bulbs compared to the control. Applying other phytohormone composition than the prescribed 1-naphthalene acetic acid (NAA) and kinetin did not show any positive effect on the growth of the in vitro plantlets of L. aestivum compared to the control.
Narcissus confusus
Due to the fact that culture establishment of N. confusus was found to be difficult at VITROFLORA laboratory, only three in vitro shoots of N. confusus were supplied to BIOPLANTA in November, 2009. According to the micropropagation protocol used for L. aestivum, in vitro bulbs of N. confusus were successfully transferred to liquid culture medium (propagation medium without gelling agent). In comparison to solid medium, in vitro plantlets of N. confusus showed a considerable growth.
The effects of reduced micro and macronutrients and doubled sucrose concentration were simultaneously tested in liquid medium. The variation of medium composition had no phenotypic effect on the growth of in vitro bulbs compared to the control. There were only slight deviations in the propagation rate of biomass; no statistical differences could be found. At the same time, culture medium with another phytohormone composition (NAA and 6-benzylaminopurine) was tested, and other changes in the medium composition (reduced nitrogen content and increased sucrose concentration) were also examined again.
Optimization of the airlift bioreactor system for the growth of in vitro cultures and the galanthamine biosynthesis (BIOPLANTA)
Leucojum aestivum and Narcissus confusus were investigated for the production of biomass and the biosynthesis of galanthamine in airlift bioreactor systems. Different levels of gassing rate, dissolved oxygen, illumination regimes and temperature were analysed. Furthermore, the influence of different inoculum types, ages (cut versus uncut shoots) and amounts of inoculated shoots were also tested. This was to lead to the optimization of the airlift bioreactor system in order to maximise the yields of galanthamine.
Leucojum aestivum
In a first study, shaking liquid cultures of in vitro bulbs of L. aestivum were compared with plantlets grown in standard culture vessels without shaking. After four weeks of initial culture, bulbs of both variants showed growth, but the bulbs growing in the liquid-shaked culture showed visual a likely greater growth than plantlets in standard culture vessels without shaking. Comparing the propagation rates of the biomass, no significant difference between both parameters was observed due to the high standard deviation. Furthermore, after analysing the galanthamine content there were no differences between both variants.
Leucojum aestivum clones with higher content of galanthamine
In August 2010, BIOPLANTA received four different clones of L. aestivum with a high content of galanthamine. First investigations were performed with 1 L airlift bioreactors. By analysing the results, optimized parameters for the L. aestivum clones could be found. The propagation rate of cut shoots was clearly higher than of uncut shoots varying dependent on the L. aestivum clones. This was due to the inoculation process. Cut shoots showed a better growth than uncut shoot clusters. Also, some clones exhibited a higher galanthamine content when cut during the inoculation process. Only in one clone a higher galanthamine content was determined in uncut shoot clusters. These results were used for a further scale up experiment to bioreactors of five litres in volume. Similar results in comparison to the experiment with 1 L bioreactors were determined. The transfer from the small scale bioreactor to a scale up bioreactor (volume of five litres) was successfully carried out.
Scale up of Leucojum aestivum biomass production
Different scale up experiments were performed. When an experiment was finished, sterile in vitro shoots were sometimes used for inoculating a scale up experiment. The shoots were transferred without any cutting to a 5 L bioreactor with temporary gassing for eight weeks. After the transfer process the shoots showed growth of biomass, but the growth rate did not reach any of the values of the small scale bioreactor experiments. The galanthamine content in shoots, leaves and bulblets in the 5 L bioreactors was marginally lower than in 1 L bioreactors with continuous gassing.
Narcissus confusus
After the time delay in receiving in vitro plantlets from VITROFLORA, experiments in different airlift bioreactors were performed under very specific well chosen conditions.
In the first experiment, different gassing regimes were tested. In vitro bulbs of N. confusus were inoculated to a bioreactor without any gassing. Other bioreactor vessels were shaken continuously. The growth of the in vitro plantlets exhibited large pale leaves with only small amounts of green. In agreement with that, grown in vitro plantlets were cut, but not divided into leaves and bulblets.
In comparison to that, 1 L airlift bioreactor with temporary gassing and 1 L airlift bioreactor with continuous gassing were tested for both biomass growth and galanthamine biosynthesis. With temporary gassing shoots of N. confusus looked very well, leaves of those shoots were dark green. Shoots of the third variant (continuous gassing) were well grown, but in comparison with temporary gassing the green of the leaves was less dark.
Optimization of the Temporary Immersion bioreactor system for the growth of in vitro cultures and the galanthamine biosynthesis (BIOPLANTA)
Different immersion frequencies were analyzed in order to determine the optimal conditions for the growth of the in vitro cultures and to reduce hyperhydricity. Different elicitors and the point of time of application were also investigated. Different nutrient medium compositions were tested for optimization of the biomass growth as well as the galanthamine biosynthesis.
Leucojum aestivum
In a first study, the influence of different immersion frequencies was investigated. In vitro bulbs of L. aestivum were inoculated in TIS (Temporary Immersion System) bioreactors of one litre in volume with two different immersion regimes. The shoots grown in TIS bioreactors with intermittent immersion in the nutrient medium showed little growth and strong callus formation, independently of the immersion frequency. After 131 days of culture a biomass production of factor 16 was determined at an immersion frequency of six times per day. In comparison to that, in airlift bioreactors with temporary or continuous gassing, a lower factor of biomass production was calculated in a shorter culture period. Nonetheless, only traces of galanthamine could be determined in those samples. Concerning the growth rate, both variants were not statistically different.
Narcissus confusus
Different frequencies of immersion were tested in TIS bioreactors with in vitro bulbs of N. confusus. Biomass production and galanthamine content were analysed. There was no correlation between biomass production and immersion frequency. In contrast to that, with decreasing frequency of immersion the averaged maximum length of shoots increased. Likewise, the fresh weight of shoots also increased with lowered frequency of immersions. Moreover, it seems that the galanthamine content in bulblets increased with a decreased amount of immersions. But the value showed a high variability, so that it was not possible to make a clear conclusion about the optimized parameter for biomass production as well as for galanthamine biosynthesis. More repeats and other conditions need to be tested to evaluate the best culture parameters for temporary immersion bioreactor systems.
Release of galanthamine to the liquid culture medium
The possible release of galanthamine to the liquid culture medium was investigated in the experiments involving Leucojum aestivum shoot-clumps growing in bioreactors. To check it, samples of liquid medium were freeze-dried by lyophilisation before alkaloid analysis. Interestingly, the samples were found to contain galanthamine. Progress of the alkaloid release was in accordance to the determined content of galanthamine in the bulbs what means that the galanthamine content in the culture media followed the distribution of galanthamine in the in vitro cultured bulbs. On this basis, it was investigated if there was any dependency of release during the whole culture period. For this, samples of liquid medium were taken when replacing the nutrient medium (every two weeks).
Some questions involving alkaloid release remain still unsolved; so further investigations need to be done in this regard.
Workpackage 6) Scale up extraction
Owing to the scarce material of L. aestivum and N. confusus obtained within the project (bulbous plants need several years to reach the adult size), BULPHYTOOILS has successfully developed a new technology for extracting galanthamine from an ornamental daffodil, Narcissus pseudonarcissus cv. Carlton, with a similar alkaloid pattern to that of N. confusus and with a reasonable amount of galanthamine. Bulbs of Narcissus cv. "Carlton" were selected as a natural alternative source for obtaining galanthamine hydrobromide because it is economically advantageous for both laboratory and practical purposes, as compared to the leaves of Leucojum aestivum. An approach for the purification of technical galanthamine HBr to a compound with a purity of 99.5 % was developed. A laboratory Standard Operation Procedure for production of galanthamine HBr with 99.5 % purity was also developed.
Development of technology for the extraction of galanthamine from different genetic resources (BULPHYTOOILS)
The methodology developed by BULPHYTOOILS includes three phases, which are here described.
Phase 1
During this phase a complete literature search related to both the isolation and purification of galanthamine from plant sources and the synthesis and chemical transformations of galanthamine derivatives was made. This literature search was necessary to answer the following questions:
i) what is the international excellence for the production of galanthamine from natural sources and synthesis?;
ii) what are the existing technologies?;
iii) is our idea competitive to these technologies?, and
iv) are there other alternatives for galanthamine production?
Using Internet and different literature sources in the Bulgarian National Library and other libraries in Sofia, including that of the Bulgarian Academy of Sciences, the existing scientific literature on galanthamine was examined. Exhaustive information about scientific and technical achievement in the isolation and purification of galanthamine from plant sources and its chemical synthesis was obtained. On the other hand, in order to comply with the existing patent rights, it was necessary to make a detailed study of the existing patents on galanthamine and its salts. The databases at the Patent Office of Bulgaria, and the websites of both the U.S. Patent Office and European Patent Office were assessed for existing patents. Detailed information on the existing and expiring patents was acquired.
Phase 2
During this phase, plant material from four cultivars of Narcissus and material of Leucojum aestivum was purchased to serve for comparison. Various extracting agents and different operating conditions such as temperature, pH of the medium, duration and dynamics of extraction, and particle size of the raw material were tested. The effect of these factors on each phase of the extraction process, mainly isolation and purification of alkaloids and in particular the major alkaloid galanthamine, was studied. The processes were monitored mainly by qualitative reactions for alkaloids (using the reagents of Dragendorff and Meyer) and by thin layer chromatography (TLC) and UV. After an adequate preliminary test and discussion of the obtained results, a method to test different (and available at the time) plant samples for their galanthamine content was chosen. In laboratory conditions, the extracts were prepared from plant materials in order to quantify and evaluate the alkaloid composition. The following cultivars of narcissus were used: "Carlton", "Actea III", "White Marvel" and "Tete a tete". Leucojum aestivum L. plants were used as reference material to compare the results of the Narcissus cultivars.
Phase 3
The main objective during this phase was to find suitable laboratory methods and conditions to obtain galanthamine hydrobromide with technical purity, which can serve as a source of end product with a purity of 99.5 %. On the basis of the results from the previous phase, it was concluded that Narcissus cv Carlton was the most prospective source of galanthamine. Therefore, new laboratory experiments were scaled up and performed with 100 kg of bulbs. The galanthamine was extracted from the bulbs as a base and as a salt. As a base it was extracted on the basis of previous experience with Leucojum aestivum, which gives good results and it is economically effective. Various extracting agents and different operating conditions such as temperature, pH of the medium, duration and dynamics of extraction, and particle sizes of the extracted material were tested. Different conditions of extraction were also experimented when extracting the galanthamine as a salt.
Comment on the HPLC analysis of samples
HPLC analysis of the obtained alkaloids showed that the extraction of galanthamine as a base from the plant material resulted in a technical product with a purity of about 89 %. Three other accompanying alkaloids, representing from 2.2 to 2.9 %, were observed, while the remaining impurities were generally below 1%. This analysis gave a basis to look for an opportunity to obtain the desired purity of the product after a proper purification process.
Conclusion
The laboratory experiments allowed confirming the obtaining of technical galanthamine hydrobromide with a sufficiently high purity by using appropriate reagents, to serve as a basis for a product with high purity of around 99.5 %. The results of the scale experiments allowed concluding that the cultivar Narcissus "Carlton" is prospective and economically advantageous for both the laboratory and practical purposes. So, it can serve as an alternative source for isolation of galanthamine. As a result of the conducted experiments, 13.89 g of galanthamine hydrobromide were isolated in total, which can be used for development of a method for purifying technical galanthamine hydrobromide with purity of 99.5 %.
Laboratory isolation of higher amounts of galanthamine with technical purity (BULPHYTOOILS)
Laboratory isolation of higher amount of galanthamine HBr with technical purity 85-90 %
The main objective in this phase was to obtain technical product galanthamine HBr with sufficient purity to serve as a source of the final product with a desired purity of 99.5 %. Experiments on alkaloid extraction from plant raw materials were based on two methods: extraction of galanthamine in the form of base or salt. Different operating conditions such as temperature, pH of the medium, duration, dynamics of extraction, and fineness of the extraction material were tested. The impact of these parameters on each phase of extraction, isolation and purification, and particularly on the major alkaloid galanthamine, was checked. All these processes were monitored by qualitative reactions for alkaloids (reaction of Mayer and Dragendorff) and by thin layer chromatography (TLC) and UV light.
Development of a method for purification of galanthamine with technical purity 85-90% up to galanthamine with purity of 99.5%
The main objective during this phase was to develop a method for the purification of technical galanthamine, purity 89 % (obtained during the previous stage) up to galanthamine with purity 99.5 %. During the purification process, technical galanthamine isolated from Narcissus pseudonarcissus variety "Carlton" and another technical galanthamine HBr isolated from Leucojum aestivum were used. The purity of the galanthamine extracted as a base, analyzed by HPLC, was about 85 to 89 %. Three major impurities were found, ranging from 2.2 to 2.9 %, while the remaining impurities were generally below 1 %. This technical product allows further purification to obtain galanthamine HBr at a sufficiently high purity. The purity of galanthamine isolated as a salt was 82-89 %. In this case two major impurities of about 10 % and 1.8 % were found, while the remaining impurities were generally below 1 %. This technical product also allows further purification to obtain galanthamine HBr at a sufficiently high purity. Most of the technical galanthamine HBr used for purification was found to contain 86.15 % of galanthamine according to HPLC analysis.
Conclusion
All the research activities and laboratory experiments of the project have been completed. The obtained results give a good reason to conclude that the objectives have been achieved. The results can be summarized as follows:
-Bulbs of Narcissus pseudonarcissus cv "Carlton" have been selected for the extraction of galanthamine HBr as an alternative source to Leucojum aestivum. This plant material is economically advantageous for both the laboratory and practical purposes.
-Development of an approach for the purification of technical galanthamine HBr to a purity of 99.5 %.
-Development of a laboratory regulation for the production of galanthamine HBr with a purity of 99.5 %.
Workpackage 7) Marketing
The market for Alzheimer treatment drugs is still young in comparison to more established areas, but given the projected age demography it is not surprising that there is a substantial amount of interest in developing drugs for this market. Axonyx, Pfizer, Johnson and Johnson, Novartis and Forest Labs are pharmaceutical companies which fight for the market. About a dozen candidate drugs are in different phases/clinical trials but still there is no success. Each year new drugs are tested and cancelled.
Potential Impact:
The cooperation between SMEs and RTD performers within the SUPROGAL consortium has lead the SMEs to obtain some interesting results which are expected will improve their capability:
i) to propagate high galanthamine-containing plants by both in vitro tissue culture (VITROFLORA, Poland) and conventional technology under Good Agricultural Practices (LUDWIG, Netherlands),
ii) to produce high purity galanthamine at a lower cost (BULPHYTOOILS, Bulgaria), and
iii) to put it into the market at a competitive price (HOLLAND BIODIVERSITY, Netherlands).
Thus, once the project has been finished, VITROFLORA has achieved to develop in vitro tissue culture protocols for a rapid propagation of both Leucojum aestivum and Narcissus confusus plants, LUDWIG has adapted these two plant species to grow in soil conditions, BULPHYTOOILS has developed new technology for the extraction, separation and purification of galanthamine, and HOLLAND BIODIVERSITY has now a better knowledge of the galanthamine market.
It has to be noted, however, that the rhythm of growth of the bulbous plants is quite slow, so that they need several years to reach the adult size (required for vegetative propagation). At present, the material of L. aestivum and N. confusus obtained throughout the project has been adapted to soil conditions and the plants will go on growing until they can be used for extraction purposes. Full sized, flowering plants cultivated under agronomic conditions are estimated to be obtained in 2-3 years for N. confusus and 3-4 years for L. aestivum after completion of the project. Therefore, the first field cultivated galanthamine-rich plant material for industrial extraction is expected three years after the project end. In the meanwhile, BULPHYTOOILS has successfully developed the technology for the extraction and purification of galanthamine not with leaves of L. aestivum (used by them until now) but with bulbs of Narcissus pseudonarcissus cv. Carlton, an ornamental variety closely related to N. confusus. The company will extract and purify the galanthamine api substance, a process estimated to start in the third year. The full capacity for production of galanthamine-rich biomass by the Consortium is estimated to be achieved 4-5 years after the project's end - considering the time necessary for plantations to be fully developed for industrial exploitation.
In vitro production of snowflake biomass will markedly reduce depletion pressure and thus help to maintain biological diversity. Narcissus confusus will be propagated and introduced to conventional cultivation to be further exploited in a sustainable fashion as an industrial crop owing to its high content of galanthamine. Thus, although the prime aim of the SUPROGAL project is to concentrate on the production of "natural", high quality galanthamine, it will additionally contribute to agricultural species diversification in Europe by establishing two very interesting crops. It will also contribute to preserve plant biodiversity in Europe by avoiding further destruction of the natural populations of L. aestivum and by ensuring the future of those of N. confusus. Producing these bulbs in large scale, commercial, agronomic conditions guarantees both the sustainable production of galanthamine and the optimal standard quality of extracted alkaloids.
Dissemination activities
At the beginning of the project, the Coordinator, on behalf of the consortium, sent a communication of the SUPROGAL project to the University of Barcelona for dissemination purposes. As a consequence, several journals and some radio station made him interviews which were further published in both internet and national press (Eccus, Correo Farmacéutico, Diario Médico, La Vanguardia, El Médico Interactivo). A website on the SUPROGAL project (see http://www.suprogal.eu online) was also created at the beginning of the project to explain the objectives, research activities, partners involved, etc. to the large public. The webpage also includes a special area restricted to the partners of the consortium (password required) for the exchange of any kind of information regarding the project (announcements of meetings, results, protocols, confidential documents, reports, minutes, etc.).
LIST OF TENTATIVE PUBLICATIONS
1. GEOGRAPHIC DIVERSITY IN ALKALOID PATTERNS OF LEUCOJUM AESTIVUM
Samples of Leucojum aestivum from various geographicaly distinct populations from Bulgaria and the Balearic islands were studied by gas chromatography coupled to mass espectrometry (GC-MS). The results and conclusions on the chemical diversity and alkaloid content will be discussed.
2.ASSESSMENT OF GENETIC DIVERSITY IN LEUCOJUM AESTIVUM L. BY RAPD MARKERS
The potential use of the Randomly Amplified Polymorphic DNA (RAPD) technique for characterization and assessment of genetic relationships was investigated in 18 Leucojum aestivum L. genotypes differentiated by type of alkaloid biosynthesis and different ecological habitats. It has been found that RAPD markers can detect enough polymorphism among Leucojum genotypes, even among closely related genotypes. Two of the RAPD primers have generated chemotype specific banding patterns mainly for homolycorine and galanthamine genotypes. Cluster analysis showed good discrimination between genotypes and grouping into two main subclusters. Grouping of accessions originating from different ecological habitats was observed.
3.MOLECULAR CHARACTERIZATION OF DIVERSITY IN LEUCOJUM AESTIVUM L. ASSESSED BY AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP)
The assessment of genetic diversity was initiated by analysis of four referents accessions collected from different Bulgarian sites. AFLP analysis was performed by using 18 selective primer combinations. A total of 402 fragments, ranging from 70 to 500 bp were recorded, 15% of which were polymorphic. The 18 selective primer combinations usesd in AFLP analysis generated reproducible polymorphic fragments and could be successfully used for discrimination of some groups with common pedigree but characterizing with different morphological characters. Chemotype specific DNA banding patterns was suggested as candidate DNA markers.
4.COMPARATIVE STUDY OF GENETIC DIVERSITY IN LEUCOJUM AESTIVUM L. AND NARCISSUS CONFUSUS L. ASSESSED BY RAPD AND AFLP MARKERS
The genetic relationships and diversity assessment in Leucojum aestivum and Narcissus confuses plants have been carried out by successfully application of two multilocus DNA markers systems (RAPD and AFLP). Comparison according to their level of polymorphism was done and best primer combinations were selected. Generally two dominant marker systems (RAPD and AFLP) could be used successfully for discrimination and genetic diversity assessment in L. aestivum and N. confuses. A set of chemotype specific DNA markers has been suggested as candidate markers. Grouping of accessions from different ecological habitats was observed.
5.GALANTHAMINE CONTENT, ALKALOID PROFILE AND ACETYLCHOLINESTERASE INHIBITORY ACTIVITY IN NARCISSUS CULTIVARS
One hundred ornamental daffodils have been extracted for their alkaloids. The level of galanthamine in both bulbs and leaves has been quantitatively determined by gas chromatography coupled to mass espectrometry (GC-MS). The alkaloid profile has been also determined by GC-MS in order to identify as much alkaloids as possible. The acetylcholinesterase inhibitory activity has also been evaluated in order to correlate such an activity with the occurrence of galanthamine and other related alkaloids in daffodils.
6. QUALITY CONTROL OF GALANTHAMINE SUBSTANCE
Results from GC-MS, X-ray and GC-isotopic ratio analyses of galanthamine substances and galanthamine containing extracts will be presented. Possibilities for quality control with respect to the impurities (quantitative and qualitative determination) and origine of the galanthamine substance will be discussed.
7.OPTIMIZATION OF GALANTHAMINE PRODUCTION IN BIOREACTORS
Effects of different parameters on the production of biomass and galanthamine in in vitro micropropagating Leucojum aestivum L. will be demonstrated. Therefore, different medium compositions and the influence of different bioreactor types were investigated. The influence of shaking as well as the influence of continuously, of temporary and also of discontinuously gassing was studied. Furthermore, the amount of inoculated shoots was changed to examine any depending effect on biomass and galanthamine production. Finally, results of scale up experiments with the optimized culture parameters will be shown.
8. ELICITATION OF GALANTHAMINE BIOSYNTHESIS IN LEUCOJUM AESTIVUM IN VITRO CULTURES. A METABOLOMIC STUDY
Effect of elicitation on both galanthamine production and the metabolic profile of Leucojum aestivum in vitro shoots growing in an airlift bioreactor with discontinuously gassing will be demonstrated. In the planned publication different types of elicitors (abiotic and biotic) as well as the influence of different concentrations and time point of application will be described. Furthermore, besides the data of biomass production the content of galanthamine and related alkaloids will be shown. Additionally, the metabolic profile of the different samples will be discussed.
On the other hand, project participants from AGROBIOINSTITUTE are intending to arrange a meeting with representatives of the Bulgarian Ministry of Agriculture in the coming months in order to make them acquainted with the impressions on the state of the natural habitats of snowdrop (Leucojum aestivum L.) in Bulgaria, and also with the results obtained at ABI. This contact will help to a better maintainance of the natural snowdrop natural habitats and their potential use.
Exploitation of the results
The SUPROGAL project deals with the initiation of two new crops of high galanthamine-producing plants, Leucojum aestivum and Narcissus confusus, which have been previously selected and further propagated by in vitro culture techniques and also by conventional methods. So, only small plantlets have been obtained at the end of the project, although the methodology for their multiplication has been successfully developed. Since the length of the project was only two years (the maximum admitted), some extra time (several years) will be required for the supply of big amounts of raw material for the industrial extraction of galanthamine from these plant species. This is the main reason why the SME performers have preferred not to establish specific plans for the use and dissemination of the foreground. So, there are no existing or anticipated business agreements of commitments, or other restrictions, which may impose limitations on the subsequent exploitation of technical information or inventions generated as a result of the research. The SMEs involved in the project have decided to exploit the results individually by establishing bilateral agreements among them depending on the situation.
A "Plan for the Use and Dissemination of the Foreground", which was discussed and agreed among all the partners, includes:
a) Distribution of the project results and IPR among the SMEs
The SMEs agreed to share the IPR on the project results and knowledge as indicated in Table 2 of Part A of the proposal on the basis of their particular participation in the production pipeline and business interests.
b) Patent applications
A patent application will be possible for the registration of both Leucojum aestivum abd Narcissus confusus at the COMMUNITY PLANT VARIETY OFFICE (France) as new crops to be commercially marketed.
c) Scientific publications
List of Websites:
http://www.suprogal.eu
An international consortium integrated by four SMEs and three RTD performers was constituted to develop the research project entitled "Sustainable production of galanthamine by both in vitro culture and agricultural crops of highly galanthamine-containing plants" (SUPROGAL) within the 7th Framework Programme of the European Union. Galanthamine is an alkaloid produced exclusively by plants of the family Amaryllidaceae, mainly belonging to the genus Galanthus, Leucojum and Narcissus. Owing to its acetylcholinesterase inhibitory activity, galanthamine is used and marketed for the treatment of the Alzheimer's disease, among others. Although it is obtained by organic synthesis, galanthamine is also being extracted from natural sources. Both Galanthus and Leucojum plants are very widespread in the Eastern European countries, whereas Narcissus plants are mainly distributed in the Mediterranean region. For industrial purposes, Leucojum aestivum plants are gathered from wild populations in their natural habitat, which causes increasing problems regarding quality of the plant material as well as depletion pressure on the natural populations. By development of an in vitro production method, both problems will be overcome. Narcissus confusus, an endemic species growing in Spain, was found to produce around two-fold higher amount of galanthamine than L. aestivum.
Most of the objectives and technical goals for the project were achieved:
i) plant populations of high galanthamine-producing plants of L. aestivum and N. confusus were detected,
ii) selected genotypes were propagated by in vitro techniques;
iii) in vitro plant material was produced and supplied to other partners for further experiments;
iv) liquid medium culture was started for mass propagation;
v) cultivation processes of the plant material in airlift bioreactors and temporary immersion systems were developed;
vi) both species were transferred and successfully maintained in soil conditions;
vii) a new protocol for the extraction and purification of galanthamine at pilot plant level was developed, and
viii) an overview of the current status of the galanthamine market was performed.
Project Context and Objectives:
PROJECT CONTEXT AND MAIN OBJECTIVES
Galanthamine is an alkaloid produced exclusively by plants of the family Amaryllidaceae, mainly belonging to the genus Galanthus, Leucojum and Narcissus. Owing to its acetylcholinesterase inhibitory activity, galanthamine is used and marketed for the treatment of the Alzheimer's disease, among others. Although it is obtained by organic synthesis, galanthamine is also being extracted from natural sources. Both Galanthus and Leucojum plants are very widespread in the Eastern European countries, whereas Narcissus plants are mainly distributed in the Mediterranean region. For industrial purposes, Leucojum aestivum plants are gathered from wild populations in their natural habitat, which causes increasing problems regarding quality of the plant material as well as depletion pressure on the natural populations. By development of an in vitro production method, both problems will be overcome. Narcissus confusus, an endemic species growing in Spain, was found to produce around two-fold higher amount of galanthamine than L. aestivum.
So, the main goals of the project can be summarized as follows:
i) to ensure a regular supply of plant raw material under conditions that can be certified by GAP (Good Agricultural Practices) standards for the extraction of galanthamine;
ii) to increase the quality of the natural galanthamine through the adoption of GMP (Good Manufacturing Practices) methods;
iii) to reduce the overall production cost of galanthamine, and
iv) to adopt a marketing stance ensuring the promotion of galanthamine produced by SUPROGAL and set and policed in Europe.
WORK PERFORMED AND MAIN RESULTS ACHIEVED
The project started with plants obtained in former years and also with plants collected from nature. Different wild populations of plants were identified and registered using GPS data. Leucojum aestivum and Narcissus confusus plants from different sites were maintained in ex vitro conditions in Ihtiman, near Sofia (Bulgaria), and Barcelona (Spain), respectively. The plants were checked for their galanthamine content, adapted to soil conditions in a different environment (Netherlands) and they were also introduced and well acclimated to the in vitro culture for mass propagation. Although no serious problems of contamination appeared throughout the cultivation under these conditions (in vitro), cultures of N. confusus evidenced some technical problems in their propagation, which were solved further.
EXPECTED FINAL RESULTS AND THEIR POTENTIAL IMPACTS AND USE
Most of the objectives and technical goals for the project were achieved:
i) plant populations of high galanthamine-producing plants of L. aestivum and N. confusus were detected,
ii) selected genotypes were propagated by in vitro techniques;
iii) in vitro plant material was produced and supplied to other partners for further experiments;
iv) liquid medium culture was started for mass propagation;
v) cultivation processes of the plant material in airlift bioreactors and temporary immersion systems were developed;
vi) both species were transferred and successfully maintained in soil conditions;
vii) a new protocol for the extraction and purification of galanthamine at pilot plant level was developed, and
viii) an overview of the current status of the galanthamine market was performed.
Project Results:
Main S & T Results/foregrounds
The research program was broken down to seven workpackages, each of them divided into different tasks. The work carried out within the project is here described separately for each workpackage.
Workpackage 1) Bioprospecting and conservation of genetic resources
Collection of plant material from different natural populations (ABI & UB)
Summer snowflake, Leucojum aestivum L. (Amaryllidaceae), is a very important plant species in Bulgaria, which is commonly used for commercial production of the alkaloid galanthamine. One of the tasks of AGROBIOINSTITUTE within the project was to collect snowflake plants from as much wild populations as possible.
The expedition was organized at the beginning of spring of 2008 (April-May) during the flowering stage of the plants. Most of the natural populations are under control of the Bulgarian Ministry of Environmental and Water, and there is a low for their protection. Independently of this circumstance, the state of some populations was not satisfactory. A general evaluation of the natural populations for their potential capacity as sources of galanthamine will be performed at the end of the project. In the case of N. confusus, a few populations are growing in Spain. The pants had been previously collected and maintained in the greenhouse at the Faculty of Pharmacy of the University of Barcelona, and they were also screened for alkaloids.
Creation and maintenance of ex situ collections of Leucojum aestivum (ABI)
The plant materials (bulbs and leaves) collected from each population of L. aestivum were separated and prepared accordingly to the requirements of the aims and intended analysis. Apart from this, 10 bulbs from each population of snowflake were used for creation of an ex situ collection in Bulgaria. The bulbs were placed in soil in a parcel located in the Ihtiman region, at about 50 km far from Sofia, where the plants are being cultivated under field conditions without weeds and with sufficient light and humidity.
Preparation and extraction of the plant material for further analyses (ABI and UB)
Leaf and bulb samples from plants of Leucojum aestivum collected individually from each wild population were dried at 60 0C until constant weight and then sent to UNIVERSITY OF BARCELONA for further chemical analysis of both alkaloid profile and galanthamine content (TLC, GC-MS). Plant material of Narcissus confusus was prepared by UNIVERSITY DE BARCELONA in the routine conditions used by the team. On the other hand, young leaves of twenty individuals from each population of L. aestivum were separated and storage at -20 0C, and then used for DNA analysis. Since the beginning of the project, this task has been continuously performed for the further analyses of the samples which are obtained as a result of the different tasks within the workpackages.
Characterization and assessment of genetic resources of Leucojum aestivum (ABI)
Two sets of naturally accessions were analyzed. A set of Leucojum aestivum L. individuals was collected from 31 populations in Bulgaria and the other set of Narcissus confusus were from four different sites in Spain. Three different DNA marker systems were used for the molecular assessment of genetic diversity and population differentiation (RAPD, AFLP and ISSR). During the second period of the project 10 referent individuals with proven chemotype of both L. aestivum and N. confusus from each population were selected for molecular analysis. The genomic DNA was extracted from bulbs and leaves by using commercial innuPREP Plant DNA Kit (analytikjena biosolutions).
RAPD Polymorphism in summer snowflake (Leucojum aestivum L.)
For genotyping and genetic diversity assessment of L. aestivum, reference individuals with proven chemotype by RAPD markers were applied two kind of profiling: 1) RAPD profiling of individual accessions; 2) RAPD profiling of mixed gDNA samples. Screening of 22 RAPD primers allowed selection of two primers (OPA17 and OPB19) which revealed a higher level of polymorphism. The Genetic Similarity (GS) among Bulgarian accessions evaluated from RAPD data ranged from 0.38 to 1.00 thus indicating that a high level of gene diversity is present in the selected genotypes. For the nine accessions supplied by LUDWIG, GS values ranged from 0.43 to 0.96. RAPD analysis proved to be efficient for discrimination of L. aestivum individuals with similar morphological characters and origin.
RAPD Polymorphism in Narcissus confusus L.
For genotyping and genetic diversity assessment of N. confusus, 19 accessions were selected from four different sites in Spain: Barco de Ávila, Peguerinos, Candelario and Santa Maria. RAPD analysis of 19 narcissus accessions was performed using set of five previously selected decamer oligonucleotides with arbitrary sequence and 60-70 % (G+C) content. RAPD patterns obtained are characterized with high reproducibility under the established PCR conditions. A total of 35 fragments, ranging from 190 to 1400 base pairs (bp) were recorded, 57 % of which were polymorphic. The five primers (OPA-01, OPA-02, OPA-03, OPA-17 and OPB-19) used in RAPD analysis generated reproducible polymorphic fragments and could be successfully used for discrimination of the Spanish accessions. There is only one unique fragment of 520 bp in NPC44 amplified by OPB-19. Data analysis showed a middle level of polymorphism ranged from 0.55 to 1.0.
AFLP polymorphism in Leucojum aestivum and Narcissus confusus
The assessment of genetic diversity of L. aestivum was initiated by analysis of four referents accessions collected from four different Bulgarian sites and characterizing by type of galanthamine synthesis (galanthamine, homolycoryne, lycorine and mix: galanthamine+lycorine types). AFLP analysis was performed by using 18 selective primer combinations. A total of 402 fragments, ranging from 70 to 500 bp were recorded, 15 % of which were polymorphic. The eighteen selective primer combinations using in AFLP analysis generated reproducible polymorphic fragments and could be successfully used for discrimination of accessions according to their locations as well as by their chemotype. Analysis of bulked samples from each population also has been done. Data analysis showed variation of genetic distances represented as genetic similarity coefficients (GS) ranged from 0.72 between Archar and Elhovo to 1.0 between Goritsa and Kosharitsa.
Internal Simple Sequence Repeats (ISSR) Population analysis
Internal Simple Sequence Repeats PCR was used to search for polymorphism in the profile of 24 Leucojum populations for further marker development for galanthamine content. Primers for CA-type of microsatellite repeats were used. Inter-microsatellite analysis is a viable alternative to RAPD analysis. On the other hand it has some limitations especially when compared to AFLP. Our results suggest that ISSR-PCR with primers recognizing CA-based repeats or related sequences (CT/CTC, GTG/TG) did not produce sufficient polymorphism to discriminate individual genotypes. This might be due to narrow genetic base of the examined populations. ISSR was further used for identification of chemotype-based differences. Three chemotypes - homolycorine, mixed and galantamine were analyzed with five ISSR primers. Several polymorphic bands were observed in the lines of different chemotypes. Some differences were observed in band intensity obtained with primer 844 for homolycorine and mixed chemotypes. Primer 844 produced single polymorphic band for galanthamine chemotype (844-G650).
Comparison between applied DNA marker systems (RAPD, AFLP and ISSR)
Two natural species, Leucojum aestivum L. and Narcissus confusus L., characterizing as a richest sources of galanthamine, were genotyped by using different genomic DNA fingerprinting techniques, known as dominant DNA marker systems: Random Amplified Polymorphism DNA (RAPD), Internal Simple Sequence Repeats (ISSR) and high resolution - Amplified Fragment Length Polymorphism (AFLP). As regard to generated level of polymorphism and comparing between different methods, RAPD and AFLP were suggested as more informative. A number of chemotype specific DNA markers were found mainly by RAPD method which clearly discriminated three basic chemotypes: homolycorine, galanthamine and lycorine.
Workpackage 2) Chemical and biological analysis
Selection of the best wild plant material on the basis of its galanthamine content (UB and ABI)
Two types of samples were analyzed: samples from individuals, and averaged leaf or bulb samples (from around 20-30 individuals). The first type of samples gives information on the chemical variability within the populations, while the second one characterizes the population as a whole. For the selection of the best plant materials, two chemical parameters were monitored: the galanthamine content, expressed as percentage referred to dry weight (DW), and its proportion in the alkaloid mixture, expressed as % of galanthamine with respect to the total alkaloids.
Screening for alkaloids (galanthamine)
For a fast screening of galanthamine content in leaf samples, a simple thin-layer chromatography (TLC) method was developed. For quantification purposes, the QUANTISCAN software from Biosoft (UK) was used. The method showed RSD values below 10 % thus allowing a comparative analysis of the galanthamine content and alkaloid profile of the samples. By using this fast method, it was easy to select the best plants on the basis of its content of galanthamine before sending the bulbs to LUDWIG and VITROFLORA for further assays. As an average, leaves of fifty plants of L. aestivum per population were screened for alkaloids, what means that a global figure of around 1500 individuals of snowflake were tested with this purpose.
Chemical analysis of plant samples
The TLC analysis revealed a remarkable difference in the alkaloid profile of L. aestivum and N. confusus plants. In the first species, the alkaloid profiles consisted mainly of galanthamine, while in N. confusus it was a mixture of galanthamine and two other main accompanying compounds. After quantitative determination of the alkaloids using the QUANTISCAN program, the analysis of galanthamine in plants of L. aestivum revealed great differences in its content within the same population, ranging from 0.2 to 0.7 % DW.
Alkaloid variability
TLC and GC-MS analyses of N. confusus plants from several individuals within a population revealed altered biosynthesis of their main alkaloids. Thus, for instance, plants of one site were characterised by a high percentage of homolycorine with respect to the total alkaloids of their leaves. Nonetheless, other plants showed no homolycorine but high levels of its precursor, 8-O-demethylhomolycorine, whilst another individual contained neither homolycorine nor 8-O-demethylhomolycorine. What is most interesting is that these plants retained their characteristic spectra when growing in the greenhouse. These biochemical variants showed high levels of galanthamine in their alkaloid mixtures and are of interest for further micropropagation purposes.
Screening of different genetic resources of amaryllidaceous plants for both galanthamine content and acetylcholinesterase inhibitory activity (UB)
Apart from the plant samples mentioned above, other amaryllidaceous plants were studied also for their alkaloid composition searching for new potential sources of galanthamine. Thus, LUDWIG supplied UNIVERSITY OF BARCELONA with different plant materials belonging to the genus Leucojum, and also with bulbs of around one hundred ornamental varieties belonging to the genus Narcissus. These materials were grown in the greenhouse of the Faculty of Pharmacy. Samples were collected in spring of 2010 and they were studied for their alkaloid content and composition, and also for their acetylcholinesterase inhibitory activity.
Comparative study of plant populations
GC-MS analysis of averaged samples (about 50 individuals pooled) from 30 Bulgarian populations of L. aestivum showed a remarkable variability of galanthamine content. The proportion of galanthamine in the alkaloid mixture varied from 0.2 to 95 %. Three geographically distributed chemotypes were revealed whih accumulated mainly homolycorine type compounds, mainly galanthamine type alkaloids, or both lycorine and galanthamine type compounds.
Quantitative analysis of galanthamine and related alkaloids in the in vitro obtained plant material and ex-situ collections and cultivated plants (UB)
A method for the quantitative determination of galanthamine was previously validated for quality control of different plant materials (in vitro and intact plants). Bulbs and leaves of N. confusus, bulbs of N. pseudonarcissus cv. Carlton, leaves of L. aestivum and in vitro cultures of L. aestivum, representing different matrixes, were used for the validation of the method. The extraction process was optimized with respect to pH and time. Derivatization and chromatographic conditions were also optimized. The limits of detection and quantification were determined by analysing samples with a known amount of analyte. Precision and accuracy tests showed acceptable variation according to the Horwitz equation.
Evaluation of the quality of galanthamine substance (UB)
Both the endo- and exo- configurations of the methyl group at the nitrogen atom of some Amaryllidaceae alkaloids have been described in the literature. Also the substituent at the N atom has strong effect on the AChE inhibitory activity of the galanthamine type compounds. For that reason, galanthamine substances from both natural (from Bulgarian Leucojum aestivum plants, supplied by BULPHYTOOILS) and syntheic (supplied by Sopharma, a Bulgarian company) origines were subjected to X-ray analysis. The results indicated that both the natural and synthetic galanthamine substances have identical three-dimensional configuration.
Workpackage 3) Material supplying by in vitro propagation
Introduction of the first material to the in vitro culture (VITROFLORA)
Leucojum aestivum
Initially, VITROFLORA received material of Leucojum aestivum from ABI as in vitro tissues culture material, which was not tested for its galanthamine content. The purpose of working on this non-tested supplied material was the establishment of a micropropagation protocol. The establishment of an in vitro gene bank of L. aestivum was done later starting from other galanthamine reach material.
Narcissus confusus
Bulbs of N. confusus received from UNIVERSITY OF BARCELONA were introduced to in vitro conditions. At the beginning of the work, and similarly to L. aestivum, the first step was gathering knowledge on this new crop. The material was very hard in multiplication. After transferring it to basic culture medium, keeping same proportions of material on the same level, and doing trials with different kinds of multiplication media, it was not succeed. After treating the material in many different ways and grown on different culture media, it was not able to react properly under new conditions, and it finally had to be discarded.
Optimization of micropropagation protocols (VITROFLORA)
Leucojum aestivum
During several months of research work, the material was a basis for different experiments with nutrients (different types and concentrations of growth regulators, macro- and microelements, vitamins), photoperiod, way of cutting, subculture duration, temperature, light conditions, etc. First sample was rooted (48 plants) and sent to LUDWIG for agronomical trials in April 2009. The samples were small (that was the beginning of work with L. aestivum) but allowed LUDWIG to start first trials on early stage of the project.
Narcissus confusus
Similarly to Leucojum plants, the material introduced to in vitro conditions was a basis for propagation experiments. Those explants showing the best multiplication capability in the different combinations of media and culture conditions were chosen for further work. During the work with N. confusus, from July 2009 to January 2010, ca. 80 different media and ca. 40 variants of growing conditions were tested in several experiments. In the second year of project, experiments with different growth conditions were continued.
Multiplication and rooting (VITROFLORA)
Leucojum aestivum
VITROFLORA multiplied material for other partners of the SUPROGAL consortium. Thus, in April 2009, first 20 shoot-clumps were transferred to BIOPLANTA for bioreactor experiments. The same month, first 11 shoot-clumps were sent to UNIVERSITY OF BARCELONA for testing their galanthamine content. In July 2009, a second batch of 100 shoot-clumps was prepared and transferred to BIOPLANTA for bioreactor propagation. Also a second batch of 10 shoot-clumps was sent to UNIVERSITY OF BARCELONA to check if the galanthamine content was stable during the multiplication process. Material was still multiplied to build up stock for supplying to BIOPLANTA. In November 2009, ca. 500 shoot-clumps were delivered to BIOPLANTA for bioreactor experiments. After having established the multiplication protocol, most of the plant material was used for rooting trials. A second batch of rooted plants was delivered to LUDWIG in August 2009.
Narcissus confusus
The first material, 20 shoot-clumps originated from 40 bulbs received from UNIVERSITAT DE BARCELONA was transferred to BIOPLANTA in February 2010 for bioreactor experiments. Likewise, about 100 shoot-clumps from the same starting material were shipped also to BIOPLANTA in the second year or project (August, 2010). Starting from March 2010, the first experiments for rooting trials were set up at VITROFLORA for the optimization of the micropropagation protocol, for the maintenance of the gene bank and for rooting for LUDWIG. In addition, a gene bank from new clones coming from galanthamine rich material was created.
Workpackage 4) Agronomical assays
Establishment of the optimal conditions for the growth (LUDWIG)
Leucojum aestivum
Bulbs of Leucojum aestivum were received from Bulgaria in June 2008. The general impression was that they were in good condition. After arrival, the bulbs were unpacked, weighted and stored to keep the bulbs healthy. One month later, the bulbs got a warm water treatment and then they were dried and placed in a cold storage room. In the first two weeks of October 2009, after preparation of the land, the bulbs were disinfected and planted outside on the field. In this phase different tests couldn't be performed because the bulbs were harvested during flowering. In spring the amount of leaves was little and they didn't bloom.
Narcissus confusus
First bulbs of Narcissus confusus were received on May 25, 2008, from UNIVERSITY OF BARCELONA. The bulbs were packed one by one in a bag, each with their own number. The bulbs were each weighted and then placed into an empty plastic pot until August 4, 2008. After that the bulbs were treated with hot water in order to become more resistant to diseases like narcissus bulb fly and stagonospora, and to develop more rapidly. On October 1st, 2008, the bulbs were taken out of the cold storage room, disinfected and planted.
Scaling of the bulbs for multiplication (LUDWIG)
Leucojum aestivum
Besides the bulbs received from Bulgaria, in summer 2008 a lot of extra bulbs of snowflake were purchased to a supplier from Turkey to do more trials. After a warm water treatment, the bulbs were scaled. After cutting, the segments were disinfected and all packed in peat, then covered and placed dry in the heat chamber. Two different methods to determine how many young bulbs can be obtained from one big bulb were tested. We wanted to make as much young bulbs as possible to get a large stock of Leucojum aestivum in a short period. The result of the test also showed what the minimum size of a part must be to form into a young bulb. Finally, these data are important to calculate the cost price.
Narcissus confusus
On July 20th 2009 a few bulbs were scaled to find out how fast these bulbs can be multiplied to make a calculation on the amount of bulbs that can be produced in what time. The scales, which were checked regularly, were packed separately whereby the whole growing process can be followed per bulb. Meanwhile, in the first week of September 2009 we had noticed that the development of new scales went very well. The temperature of the growing chamber was fixed at 23 °C. In the first week of September 2009 the development of new scales went very well.
Plant breeding (LUDWIG)
Leucojum aestivum
Plants of L. aestivum from several populations of Bulgaria, Turkey and Netherlands were planted. The first selection came from Bulgaria, but did not flower because the bulbs were harvested in 2008 during flowering, and the bulbs could not get the right treatment. The second selection came originally from Turkey and had two different bulb sizes (12 and 10 cm). The results led to the conclusion that bulb size affects the flowering development. The bulbs from the third selection, large bulbs from Holland which are in the soil at our nursery for a few years, were all flowering. The bulbs from Bulgaria were harvested, sorted and weighted separately. After the warm water treatment, the bulbs were packed separately and put in different storage rooms to determine if the different treatments resulted in different alkaloid content. The bulbs from Turkey were cultivated after harvesting and they were prepared for planting for the next season. The bulbs from the Dutch wholesaler were also purchased and a few bulbs bloomed. The bulbs made seeds by themselves, which were collected.
Narcissus confusus
The first plants of Narcissus confusus did not flower. After planting the bulbs in autumn of 2008, the plants quickly developed. The bulbs received a natural dormancy treatment and were planted outside in September 2008. In mid February 2009 the leaves (around 20 cm long) were further developed. Due to the small bulb size, the amount of leaves of the crop had always been quite limited. On April 15, 2009, the first leaves were harvested to be tested for galanthamine. In order to avoid rot problems, the plant material was dried immediately before sending to UNIVERSITY OF BARCELONA. From mid April to July 2009, leaf samples of N. confusus were regularly (every two weeks) sent to UNIVERSITY OF BARCELONA to be further analyzed for alkaloids which allowed seeing the process of the compounds. It started with the first samples when the first buds started to bloom, and it ended when the leaves were fully dried.
Storage during dormancy (LUDWIG)
Leucojum aestivum
Many storage tests were performed to determine the optimal growing conditions to get the highest percentage of galanthamine combined with a high production of plant material. The bulbs were stored at different temperatures during the summer. The highest temperature was 40 °C. After 7 days it was clear that the bulbs could not bear this temperature and the bulbs were fossilized. The bulbs of all the other storage treatments (lower storage temperatures) did very well. It was concluded that storage of the bulbs during the dormancy period at a low temperature is better than a warm one. For cost price reasons, however, cold temperatures are more expensive and give the same results as warm temperatures.
Narcissus confusus
After harvesting and drying the bulbs in 2009, the bulbs of N. confusus were packed in different bags and labelled, and they got different storage treatments. The objective of the different tests was to test the following:
i) influencing the amount of leaves per bulb;
ii) influencing the percentage of galanthamine, and
iii) optimize the growth of the bulbs (if this process can be influenced, it is possible to get a higher profit per ha).
In vitro plants (LUDWIG)
Leucojum aestivum
At the end of April 2009, tissue culture plants of L. aestivum from VITROFLORA (Poland) were supplied to LUDWIG. This material, without any roots, was planted in a crate. The first plants made roots and the first leaves started to grow slowly. The second supply of tissue material was received on November 1st of the same year and it was directly planted in the greenhouse. After planting the in vitro plants, they developed immediately and by April 1st, 2010, all the bulbs produced roots and leaves. The development of the growth of the tissue culture was similar to the growth of the young bulbs coming from the scaling bulbs. It could be concluded that the growing conditions of the plant material (for example growing and storage temperature) are more important than the origin of the material (scaled bulbs versus tissue culture).
Galanthamine content (UB)
Leucojum aestivum
Samples from all different tests and materials from the different selections (Bulgaria, Turkey and Netherlands) were harvested and processed prior to send them to UNIVERSITY OF BARCELONA for further analysis of the galanthamine content. In the leaves of the Bulgarian snowflakes, the highest content of galanthamine took place during the flowering season. After flowering, the content was decreasing again until the starting point of dormancy of the leaves. The Dutch material was found to contain a very low amount of galanthamine, and the leaves of Turkey didn't contain galanthamine at all. The content of galanthamine per bulb was also quite different. As a conclusion, it can be stated that the content of galanthamine in leaves of L. aestivum is the highest during flowering time and that it is depending on the population. So, it is important to use plants of the population with the highest percentage of galanthamine as starting material for further works.
Narcissus confusus
After the results of the tests with the available bulb material of N. confusus, different soil treatment tests were performed with another variety of narcissus, named Carlton. Narcissus cv Carlton is the cultivar which is used for the production of galanthamine at this moment. The control group didn't receive any treatment. In the other treatments the soil was added with different additives like borax, zinc sulphate, magnesium sulphate, zinc, copper sulphate and bonzie. It was concluded that the content of galanthamine in the bulbs is high, and that it is influenced in a different way compared with the control group, according to the compound added in the treatment.
Workpackage 5) In vitro large scale production
Adaptation of in vitro cultures to cultivation in liquid nutrient media (BIOPLANTA)
In 2009 and 2010, in vitro plantlets of Leucojum aestivum and Narcissus confusus as well as Leucojum clones with higher galanthamine content were successfully transferred from VITROFLORA to BIOPLANTA laboratory. In vitro cultures provided by VITROFLORA were tested for the adaptation of the in vitro cultures to cultivation in liquid nutrient media. The composition of the liquid nutrient medium was optimised. Therefore, the influence of plant growth regulators, different amounts of carbohydrates as well as the influence of micro and macro elements and nitrogen contents was investigated.
Leucojum aestivum
In 2009 in vitro plantlets of L. aestivum were successfully transferred to the BIOPLANTA laboratory by VITROFLORA in three different dates. According to protocols from VITROFLORA (medium composition, cutting method) in vitro shoots of L. aestivum were micropropagated by regularly subculture on fresh medium to get enough plant material for experiments.
In a first experiment, in vitro plantlets of L. aestivum were transferred to standard liquid medium (propagation medium without gelling agent). Simultaneously, the effect of macro and micro nutrients, phytohormons, nitrogen (NH4+) and sucrose concentration on the growth was tested in liquid medium. It was shown that shoots of L. aestivum can be successfully transferred to standard liquid medium. In comparison to solid standard medium, in vitro plantlets of L. aestivum showed a considerable growth and less callus formation. The propagation rate of biomass increased significantly in liquid culture medium. The variation of medium composition had no phenotypic effect on the growth of in vitro bulbs compared to the control. Applying other phytohormone composition than the prescribed 1-naphthalene acetic acid (NAA) and kinetin did not show any positive effect on the growth of the in vitro plantlets of L. aestivum compared to the control.
Narcissus confusus
Due to the fact that culture establishment of N. confusus was found to be difficult at VITROFLORA laboratory, only three in vitro shoots of N. confusus were supplied to BIOPLANTA in November, 2009. According to the micropropagation protocol used for L. aestivum, in vitro bulbs of N. confusus were successfully transferred to liquid culture medium (propagation medium without gelling agent). In comparison to solid medium, in vitro plantlets of N. confusus showed a considerable growth.
The effects of reduced micro and macronutrients and doubled sucrose concentration were simultaneously tested in liquid medium. The variation of medium composition had no phenotypic effect on the growth of in vitro bulbs compared to the control. There were only slight deviations in the propagation rate of biomass; no statistical differences could be found. At the same time, culture medium with another phytohormone composition (NAA and 6-benzylaminopurine) was tested, and other changes in the medium composition (reduced nitrogen content and increased sucrose concentration) were also examined again.
Optimization of the airlift bioreactor system for the growth of in vitro cultures and the galanthamine biosynthesis (BIOPLANTA)
Leucojum aestivum and Narcissus confusus were investigated for the production of biomass and the biosynthesis of galanthamine in airlift bioreactor systems. Different levels of gassing rate, dissolved oxygen, illumination regimes and temperature were analysed. Furthermore, the influence of different inoculum types, ages (cut versus uncut shoots) and amounts of inoculated shoots were also tested. This was to lead to the optimization of the airlift bioreactor system in order to maximise the yields of galanthamine.
Leucojum aestivum
In a first study, shaking liquid cultures of in vitro bulbs of L. aestivum were compared with plantlets grown in standard culture vessels without shaking. After four weeks of initial culture, bulbs of both variants showed growth, but the bulbs growing in the liquid-shaked culture showed visual a likely greater growth than plantlets in standard culture vessels without shaking. Comparing the propagation rates of the biomass, no significant difference between both parameters was observed due to the high standard deviation. Furthermore, after analysing the galanthamine content there were no differences between both variants.
Leucojum aestivum clones with higher content of galanthamine
In August 2010, BIOPLANTA received four different clones of L. aestivum with a high content of galanthamine. First investigations were performed with 1 L airlift bioreactors. By analysing the results, optimized parameters for the L. aestivum clones could be found. The propagation rate of cut shoots was clearly higher than of uncut shoots varying dependent on the L. aestivum clones. This was due to the inoculation process. Cut shoots showed a better growth than uncut shoot clusters. Also, some clones exhibited a higher galanthamine content when cut during the inoculation process. Only in one clone a higher galanthamine content was determined in uncut shoot clusters. These results were used for a further scale up experiment to bioreactors of five litres in volume. Similar results in comparison to the experiment with 1 L bioreactors were determined. The transfer from the small scale bioreactor to a scale up bioreactor (volume of five litres) was successfully carried out.
Scale up of Leucojum aestivum biomass production
Different scale up experiments were performed. When an experiment was finished, sterile in vitro shoots were sometimes used for inoculating a scale up experiment. The shoots were transferred without any cutting to a 5 L bioreactor with temporary gassing for eight weeks. After the transfer process the shoots showed growth of biomass, but the growth rate did not reach any of the values of the small scale bioreactor experiments. The galanthamine content in shoots, leaves and bulblets in the 5 L bioreactors was marginally lower than in 1 L bioreactors with continuous gassing.
Narcissus confusus
After the time delay in receiving in vitro plantlets from VITROFLORA, experiments in different airlift bioreactors were performed under very specific well chosen conditions.
In the first experiment, different gassing regimes were tested. In vitro bulbs of N. confusus were inoculated to a bioreactor without any gassing. Other bioreactor vessels were shaken continuously. The growth of the in vitro plantlets exhibited large pale leaves with only small amounts of green. In agreement with that, grown in vitro plantlets were cut, but not divided into leaves and bulblets.
In comparison to that, 1 L airlift bioreactor with temporary gassing and 1 L airlift bioreactor with continuous gassing were tested for both biomass growth and galanthamine biosynthesis. With temporary gassing shoots of N. confusus looked very well, leaves of those shoots were dark green. Shoots of the third variant (continuous gassing) were well grown, but in comparison with temporary gassing the green of the leaves was less dark.
Optimization of the Temporary Immersion bioreactor system for the growth of in vitro cultures and the galanthamine biosynthesis (BIOPLANTA)
Different immersion frequencies were analyzed in order to determine the optimal conditions for the growth of the in vitro cultures and to reduce hyperhydricity. Different elicitors and the point of time of application were also investigated. Different nutrient medium compositions were tested for optimization of the biomass growth as well as the galanthamine biosynthesis.
Leucojum aestivum
In a first study, the influence of different immersion frequencies was investigated. In vitro bulbs of L. aestivum were inoculated in TIS (Temporary Immersion System) bioreactors of one litre in volume with two different immersion regimes. The shoots grown in TIS bioreactors with intermittent immersion in the nutrient medium showed little growth and strong callus formation, independently of the immersion frequency. After 131 days of culture a biomass production of factor 16 was determined at an immersion frequency of six times per day. In comparison to that, in airlift bioreactors with temporary or continuous gassing, a lower factor of biomass production was calculated in a shorter culture period. Nonetheless, only traces of galanthamine could be determined in those samples. Concerning the growth rate, both variants were not statistically different.
Narcissus confusus
Different frequencies of immersion were tested in TIS bioreactors with in vitro bulbs of N. confusus. Biomass production and galanthamine content were analysed. There was no correlation between biomass production and immersion frequency. In contrast to that, with decreasing frequency of immersion the averaged maximum length of shoots increased. Likewise, the fresh weight of shoots also increased with lowered frequency of immersions. Moreover, it seems that the galanthamine content in bulblets increased with a decreased amount of immersions. But the value showed a high variability, so that it was not possible to make a clear conclusion about the optimized parameter for biomass production as well as for galanthamine biosynthesis. More repeats and other conditions need to be tested to evaluate the best culture parameters for temporary immersion bioreactor systems.
Release of galanthamine to the liquid culture medium
The possible release of galanthamine to the liquid culture medium was investigated in the experiments involving Leucojum aestivum shoot-clumps growing in bioreactors. To check it, samples of liquid medium were freeze-dried by lyophilisation before alkaloid analysis. Interestingly, the samples were found to contain galanthamine. Progress of the alkaloid release was in accordance to the determined content of galanthamine in the bulbs what means that the galanthamine content in the culture media followed the distribution of galanthamine in the in vitro cultured bulbs. On this basis, it was investigated if there was any dependency of release during the whole culture period. For this, samples of liquid medium were taken when replacing the nutrient medium (every two weeks).
Some questions involving alkaloid release remain still unsolved; so further investigations need to be done in this regard.
Workpackage 6) Scale up extraction
Owing to the scarce material of L. aestivum and N. confusus obtained within the project (bulbous plants need several years to reach the adult size), BULPHYTOOILS has successfully developed a new technology for extracting galanthamine from an ornamental daffodil, Narcissus pseudonarcissus cv. Carlton, with a similar alkaloid pattern to that of N. confusus and with a reasonable amount of galanthamine. Bulbs of Narcissus cv. "Carlton" were selected as a natural alternative source for obtaining galanthamine hydrobromide because it is economically advantageous for both laboratory and practical purposes, as compared to the leaves of Leucojum aestivum. An approach for the purification of technical galanthamine HBr to a compound with a purity of 99.5 % was developed. A laboratory Standard Operation Procedure for production of galanthamine HBr with 99.5 % purity was also developed.
Development of technology for the extraction of galanthamine from different genetic resources (BULPHYTOOILS)
The methodology developed by BULPHYTOOILS includes three phases, which are here described.
Phase 1
During this phase a complete literature search related to both the isolation and purification of galanthamine from plant sources and the synthesis and chemical transformations of galanthamine derivatives was made. This literature search was necessary to answer the following questions:
i) what is the international excellence for the production of galanthamine from natural sources and synthesis?;
ii) what are the existing technologies?;
iii) is our idea competitive to these technologies?, and
iv) are there other alternatives for galanthamine production?
Using Internet and different literature sources in the Bulgarian National Library and other libraries in Sofia, including that of the Bulgarian Academy of Sciences, the existing scientific literature on galanthamine was examined. Exhaustive information about scientific and technical achievement in the isolation and purification of galanthamine from plant sources and its chemical synthesis was obtained. On the other hand, in order to comply with the existing patent rights, it was necessary to make a detailed study of the existing patents on galanthamine and its salts. The databases at the Patent Office of Bulgaria, and the websites of both the U.S. Patent Office and European Patent Office were assessed for existing patents. Detailed information on the existing and expiring patents was acquired.
Phase 2
During this phase, plant material from four cultivars of Narcissus and material of Leucojum aestivum was purchased to serve for comparison. Various extracting agents and different operating conditions such as temperature, pH of the medium, duration and dynamics of extraction, and particle size of the raw material were tested. The effect of these factors on each phase of the extraction process, mainly isolation and purification of alkaloids and in particular the major alkaloid galanthamine, was studied. The processes were monitored mainly by qualitative reactions for alkaloids (using the reagents of Dragendorff and Meyer) and by thin layer chromatography (TLC) and UV. After an adequate preliminary test and discussion of the obtained results, a method to test different (and available at the time) plant samples for their galanthamine content was chosen. In laboratory conditions, the extracts were prepared from plant materials in order to quantify and evaluate the alkaloid composition. The following cultivars of narcissus were used: "Carlton", "Actea III", "White Marvel" and "Tete a tete". Leucojum aestivum L. plants were used as reference material to compare the results of the Narcissus cultivars.
Phase 3
The main objective during this phase was to find suitable laboratory methods and conditions to obtain galanthamine hydrobromide with technical purity, which can serve as a source of end product with a purity of 99.5 %. On the basis of the results from the previous phase, it was concluded that Narcissus cv Carlton was the most prospective source of galanthamine. Therefore, new laboratory experiments were scaled up and performed with 100 kg of bulbs. The galanthamine was extracted from the bulbs as a base and as a salt. As a base it was extracted on the basis of previous experience with Leucojum aestivum, which gives good results and it is economically effective. Various extracting agents and different operating conditions such as temperature, pH of the medium, duration and dynamics of extraction, and particle sizes of the extracted material were tested. Different conditions of extraction were also experimented when extracting the galanthamine as a salt.
Comment on the HPLC analysis of samples
HPLC analysis of the obtained alkaloids showed that the extraction of galanthamine as a base from the plant material resulted in a technical product with a purity of about 89 %. Three other accompanying alkaloids, representing from 2.2 to 2.9 %, were observed, while the remaining impurities were generally below 1%. This analysis gave a basis to look for an opportunity to obtain the desired purity of the product after a proper purification process.
Conclusion
The laboratory experiments allowed confirming the obtaining of technical galanthamine hydrobromide with a sufficiently high purity by using appropriate reagents, to serve as a basis for a product with high purity of around 99.5 %. The results of the scale experiments allowed concluding that the cultivar Narcissus "Carlton" is prospective and economically advantageous for both the laboratory and practical purposes. So, it can serve as an alternative source for isolation of galanthamine. As a result of the conducted experiments, 13.89 g of galanthamine hydrobromide were isolated in total, which can be used for development of a method for purifying technical galanthamine hydrobromide with purity of 99.5 %.
Laboratory isolation of higher amounts of galanthamine with technical purity (BULPHYTOOILS)
Laboratory isolation of higher amount of galanthamine HBr with technical purity 85-90 %
The main objective in this phase was to obtain technical product galanthamine HBr with sufficient purity to serve as a source of the final product with a desired purity of 99.5 %. Experiments on alkaloid extraction from plant raw materials were based on two methods: extraction of galanthamine in the form of base or salt. Different operating conditions such as temperature, pH of the medium, duration, dynamics of extraction, and fineness of the extraction material were tested. The impact of these parameters on each phase of extraction, isolation and purification, and particularly on the major alkaloid galanthamine, was checked. All these processes were monitored by qualitative reactions for alkaloids (reaction of Mayer and Dragendorff) and by thin layer chromatography (TLC) and UV light.
Development of a method for purification of galanthamine with technical purity 85-90% up to galanthamine with purity of 99.5%
The main objective during this phase was to develop a method for the purification of technical galanthamine, purity 89 % (obtained during the previous stage) up to galanthamine with purity 99.5 %. During the purification process, technical galanthamine isolated from Narcissus pseudonarcissus variety "Carlton" and another technical galanthamine HBr isolated from Leucojum aestivum were used. The purity of the galanthamine extracted as a base, analyzed by HPLC, was about 85 to 89 %. Three major impurities were found, ranging from 2.2 to 2.9 %, while the remaining impurities were generally below 1 %. This technical product allows further purification to obtain galanthamine HBr at a sufficiently high purity. The purity of galanthamine isolated as a salt was 82-89 %. In this case two major impurities of about 10 % and 1.8 % were found, while the remaining impurities were generally below 1 %. This technical product also allows further purification to obtain galanthamine HBr at a sufficiently high purity. Most of the technical galanthamine HBr used for purification was found to contain 86.15 % of galanthamine according to HPLC analysis.
Conclusion
All the research activities and laboratory experiments of the project have been completed. The obtained results give a good reason to conclude that the objectives have been achieved. The results can be summarized as follows:
-Bulbs of Narcissus pseudonarcissus cv "Carlton" have been selected for the extraction of galanthamine HBr as an alternative source to Leucojum aestivum. This plant material is economically advantageous for both the laboratory and practical purposes.
-Development of an approach for the purification of technical galanthamine HBr to a purity of 99.5 %.
-Development of a laboratory regulation for the production of galanthamine HBr with a purity of 99.5 %.
Workpackage 7) Marketing
The market for Alzheimer treatment drugs is still young in comparison to more established areas, but given the projected age demography it is not surprising that there is a substantial amount of interest in developing drugs for this market. Axonyx, Pfizer, Johnson and Johnson, Novartis and Forest Labs are pharmaceutical companies which fight for the market. About a dozen candidate drugs are in different phases/clinical trials but still there is no success. Each year new drugs are tested and cancelled.
Potential Impact:
The cooperation between SMEs and RTD performers within the SUPROGAL consortium has lead the SMEs to obtain some interesting results which are expected will improve their capability:
i) to propagate high galanthamine-containing plants by both in vitro tissue culture (VITROFLORA, Poland) and conventional technology under Good Agricultural Practices (LUDWIG, Netherlands),
ii) to produce high purity galanthamine at a lower cost (BULPHYTOOILS, Bulgaria), and
iii) to put it into the market at a competitive price (HOLLAND BIODIVERSITY, Netherlands).
Thus, once the project has been finished, VITROFLORA has achieved to develop in vitro tissue culture protocols for a rapid propagation of both Leucojum aestivum and Narcissus confusus plants, LUDWIG has adapted these two plant species to grow in soil conditions, BULPHYTOOILS has developed new technology for the extraction, separation and purification of galanthamine, and HOLLAND BIODIVERSITY has now a better knowledge of the galanthamine market.
It has to be noted, however, that the rhythm of growth of the bulbous plants is quite slow, so that they need several years to reach the adult size (required for vegetative propagation). At present, the material of L. aestivum and N. confusus obtained throughout the project has been adapted to soil conditions and the plants will go on growing until they can be used for extraction purposes. Full sized, flowering plants cultivated under agronomic conditions are estimated to be obtained in 2-3 years for N. confusus and 3-4 years for L. aestivum after completion of the project. Therefore, the first field cultivated galanthamine-rich plant material for industrial extraction is expected three years after the project end. In the meanwhile, BULPHYTOOILS has successfully developed the technology for the extraction and purification of galanthamine not with leaves of L. aestivum (used by them until now) but with bulbs of Narcissus pseudonarcissus cv. Carlton, an ornamental variety closely related to N. confusus. The company will extract and purify the galanthamine api substance, a process estimated to start in the third year. The full capacity for production of galanthamine-rich biomass by the Consortium is estimated to be achieved 4-5 years after the project's end - considering the time necessary for plantations to be fully developed for industrial exploitation.
In vitro production of snowflake biomass will markedly reduce depletion pressure and thus help to maintain biological diversity. Narcissus confusus will be propagated and introduced to conventional cultivation to be further exploited in a sustainable fashion as an industrial crop owing to its high content of galanthamine. Thus, although the prime aim of the SUPROGAL project is to concentrate on the production of "natural", high quality galanthamine, it will additionally contribute to agricultural species diversification in Europe by establishing two very interesting crops. It will also contribute to preserve plant biodiversity in Europe by avoiding further destruction of the natural populations of L. aestivum and by ensuring the future of those of N. confusus. Producing these bulbs in large scale, commercial, agronomic conditions guarantees both the sustainable production of galanthamine and the optimal standard quality of extracted alkaloids.
Dissemination activities
At the beginning of the project, the Coordinator, on behalf of the consortium, sent a communication of the SUPROGAL project to the University of Barcelona for dissemination purposes. As a consequence, several journals and some radio station made him interviews which were further published in both internet and national press (Eccus, Correo Farmacéutico, Diario Médico, La Vanguardia, El Médico Interactivo). A website on the SUPROGAL project (see http://www.suprogal.eu online) was also created at the beginning of the project to explain the objectives, research activities, partners involved, etc. to the large public. The webpage also includes a special area restricted to the partners of the consortium (password required) for the exchange of any kind of information regarding the project (announcements of meetings, results, protocols, confidential documents, reports, minutes, etc.).
LIST OF TENTATIVE PUBLICATIONS
1. GEOGRAPHIC DIVERSITY IN ALKALOID PATTERNS OF LEUCOJUM AESTIVUM
Samples of Leucojum aestivum from various geographicaly distinct populations from Bulgaria and the Balearic islands were studied by gas chromatography coupled to mass espectrometry (GC-MS). The results and conclusions on the chemical diversity and alkaloid content will be discussed.
2.ASSESSMENT OF GENETIC DIVERSITY IN LEUCOJUM AESTIVUM L. BY RAPD MARKERS
The potential use of the Randomly Amplified Polymorphic DNA (RAPD) technique for characterization and assessment of genetic relationships was investigated in 18 Leucojum aestivum L. genotypes differentiated by type of alkaloid biosynthesis and different ecological habitats. It has been found that RAPD markers can detect enough polymorphism among Leucojum genotypes, even among closely related genotypes. Two of the RAPD primers have generated chemotype specific banding patterns mainly for homolycorine and galanthamine genotypes. Cluster analysis showed good discrimination between genotypes and grouping into two main subclusters. Grouping of accessions originating from different ecological habitats was observed.
3.MOLECULAR CHARACTERIZATION OF DIVERSITY IN LEUCOJUM AESTIVUM L. ASSESSED BY AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP)
The assessment of genetic diversity was initiated by analysis of four referents accessions collected from different Bulgarian sites. AFLP analysis was performed by using 18 selective primer combinations. A total of 402 fragments, ranging from 70 to 500 bp were recorded, 15% of which were polymorphic. The 18 selective primer combinations usesd in AFLP analysis generated reproducible polymorphic fragments and could be successfully used for discrimination of some groups with common pedigree but characterizing with different morphological characters. Chemotype specific DNA banding patterns was suggested as candidate DNA markers.
4.COMPARATIVE STUDY OF GENETIC DIVERSITY IN LEUCOJUM AESTIVUM L. AND NARCISSUS CONFUSUS L. ASSESSED BY RAPD AND AFLP MARKERS
The genetic relationships and diversity assessment in Leucojum aestivum and Narcissus confuses plants have been carried out by successfully application of two multilocus DNA markers systems (RAPD and AFLP). Comparison according to their level of polymorphism was done and best primer combinations were selected. Generally two dominant marker systems (RAPD and AFLP) could be used successfully for discrimination and genetic diversity assessment in L. aestivum and N. confuses. A set of chemotype specific DNA markers has been suggested as candidate markers. Grouping of accessions from different ecological habitats was observed.
5.GALANTHAMINE CONTENT, ALKALOID PROFILE AND ACETYLCHOLINESTERASE INHIBITORY ACTIVITY IN NARCISSUS CULTIVARS
One hundred ornamental daffodils have been extracted for their alkaloids. The level of galanthamine in both bulbs and leaves has been quantitatively determined by gas chromatography coupled to mass espectrometry (GC-MS). The alkaloid profile has been also determined by GC-MS in order to identify as much alkaloids as possible. The acetylcholinesterase inhibitory activity has also been evaluated in order to correlate such an activity with the occurrence of galanthamine and other related alkaloids in daffodils.
6. QUALITY CONTROL OF GALANTHAMINE SUBSTANCE
Results from GC-MS, X-ray and GC-isotopic ratio analyses of galanthamine substances and galanthamine containing extracts will be presented. Possibilities for quality control with respect to the impurities (quantitative and qualitative determination) and origine of the galanthamine substance will be discussed.
7.OPTIMIZATION OF GALANTHAMINE PRODUCTION IN BIOREACTORS
Effects of different parameters on the production of biomass and galanthamine in in vitro micropropagating Leucojum aestivum L. will be demonstrated. Therefore, different medium compositions and the influence of different bioreactor types were investigated. The influence of shaking as well as the influence of continuously, of temporary and also of discontinuously gassing was studied. Furthermore, the amount of inoculated shoots was changed to examine any depending effect on biomass and galanthamine production. Finally, results of scale up experiments with the optimized culture parameters will be shown.
8. ELICITATION OF GALANTHAMINE BIOSYNTHESIS IN LEUCOJUM AESTIVUM IN VITRO CULTURES. A METABOLOMIC STUDY
Effect of elicitation on both galanthamine production and the metabolic profile of Leucojum aestivum in vitro shoots growing in an airlift bioreactor with discontinuously gassing will be demonstrated. In the planned publication different types of elicitors (abiotic and biotic) as well as the influence of different concentrations and time point of application will be described. Furthermore, besides the data of biomass production the content of galanthamine and related alkaloids will be shown. Additionally, the metabolic profile of the different samples will be discussed.
On the other hand, project participants from AGROBIOINSTITUTE are intending to arrange a meeting with representatives of the Bulgarian Ministry of Agriculture in the coming months in order to make them acquainted with the impressions on the state of the natural habitats of snowdrop (Leucojum aestivum L.) in Bulgaria, and also with the results obtained at ABI. This contact will help to a better maintainance of the natural snowdrop natural habitats and their potential use.
Exploitation of the results
The SUPROGAL project deals with the initiation of two new crops of high galanthamine-producing plants, Leucojum aestivum and Narcissus confusus, which have been previously selected and further propagated by in vitro culture techniques and also by conventional methods. So, only small plantlets have been obtained at the end of the project, although the methodology for their multiplication has been successfully developed. Since the length of the project was only two years (the maximum admitted), some extra time (several years) will be required for the supply of big amounts of raw material for the industrial extraction of galanthamine from these plant species. This is the main reason why the SME performers have preferred not to establish specific plans for the use and dissemination of the foreground. So, there are no existing or anticipated business agreements of commitments, or other restrictions, which may impose limitations on the subsequent exploitation of technical information or inventions generated as a result of the research. The SMEs involved in the project have decided to exploit the results individually by establishing bilateral agreements among them depending on the situation.
A "Plan for the Use and Dissemination of the Foreground", which was discussed and agreed among all the partners, includes:
a) Distribution of the project results and IPR among the SMEs
The SMEs agreed to share the IPR on the project results and knowledge as indicated in Table 2 of Part A of the proposal on the basis of their particular participation in the production pipeline and business interests.
b) Patent applications
A patent application will be possible for the registration of both Leucojum aestivum abd Narcissus confusus at the COMMUNITY PLANT VARIETY OFFICE (France) as new crops to be commercially marketed.
c) Scientific publications
List of Websites:
http://www.suprogal.eu