galanthamine biosynthesis in plant in vitro systems

www.els-journal.com Engineering in Life Sciences

Received: 17-May-2014; Revised: 18-Jul-2014; Accepted: 23-Sep-2014.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting,

typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of

Record. Please cite this article as doi: 10.1002/elsc.201300159.

This article is protected by copyright. All rights reserved.

Review

Galanthamine biosynthesis in plant in vitro systems

Strahil Berkov

Institute of Biodiversity and Ecosystem Research, 23 Acad. G Bonchev Str, 1113 Sofia,

Bulgaria

Ivan Ivanov

Department of Organic Chemistry, University of Food Technology, 26 Maritza Boulevard,

Plovdiv 4002, Bulgaria

Vasil Georgiev

Laboratory of Applied Biotechnology, The Stefan Angeloff Institute of Microbiology,

Bulgarian Academy of Sciences, 139 Ruski Boulevard, Plovdiv4000, Bulgaria

Center for Viticulture and Small Fruit Research, Florida A&M University, 6505, Mahan

Drive, Tallahassee 32317, USA

Carles Codina

Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmacia,

Universitat de Barcelona, Avda. Diagonal 643, 08028 Barcelona, Spain

Atanas Pavlov

Department of Analytical Chemistry, University of Food Technology, 26 Maritza Boulevard,

Plovdiv 4002, Bulgaria

Laboratory of Applied Biotechnology, The Stefan Angeloff Institute of Microbiology,

Bulgarian Academy of Sciences, 139 Ruski Boulevard, Plovdiv 4000, Bulgaria

Keywords: Acetylcholinesterase inhibitor, Biosynthesis, Bioreactors, Galanthamine, Plant in

vitro systems



Abbreviations

AChE - acetylcholinesterase

DW – dry weight

FDA - U.S. Food and Drag Administration

Gal - galanthamine

GC-MS – gas chromatography–mass spectrometry

L-phe - L-phenylalanine

L-tyr - L-tyrosine

PAL - phenylalanine ammonia lyase

RITA -Récipient à Immersion Temporaire Automatique

TIS – temporarry immersion system

Abstract

The Amaryllidaceae alkaloid galanthamine is a long-acting, selective, reversible and

competitive acetylcholinesterase (AChE) inhibitor used for the treatment of early- to mid-

stage Alzheimer’s disease, poliomyelitis and other neurological diseases. Currently,

galanthamine is produced by extraction from plants such as daffodils (Narcissus cultivar

Carlthon), snowflake (Leucojum aestivum), ‘red-tubed lily’ (Lycoris radiata), and Ungernia

victoria, and alternatively by chemical synthesis. Due to the increased demand by the generic

pharmaceutical companies and the limited availability of plant sources, the biosynthesis of

galanthamine by plant in vitro systems has been considered as an alternative approach for its

sustainable production. The present article reviews the state-of-the-art of in vitro

galanthamine biosynthesis including growth regulators, medium components, culture

conditions, elicitation and bioreactor systems. It may be used as a starting point for further

studies in this area leading to a progress in biotechnological production of this valuable

alkaloid.



1 Introduction

Galanthamine is an Amaryllidaceae alkaloid which is a long-acting, selective,

reversible and competitive acetylcholinesterase (AChE) inhibitor [1] crossing the blood-

brain, and marketed as a hydrobromide salt under the name of Razadine® (formerly

Reminyl®) and Nivalin® for the treatment of Alzheimer’s disease [2]. AChE is responsible

for the degradation of acetylcholine at the neuromuscular junction, in peripheral and central

cholinergic synapses and in parasympathetic target organs. AChE inhibitors compensate the

diminished cholinergic function and are widely used for symptomatic treatment of dementia.

Four AChE inhibitors have received approval for clinical use in early- to mid-stage

Alzheimer’s disease: tacrine, donepezil, rivastigmine and galanthamine. Tacrine, approved by

FDA in 1993, has been largely withdrawn due to adverse side effects, including possible liver

damage. Galanthamine hydrobromide have shown superior pharmacological profiles and

increased tolerance compared to the original AChE inhibitors, physostigmine or tacrine [3].

This natural compound stimulates pre- and postsynaptic nicotinic receptors and amplifies the

nerve-muscle transfer. Its pharmacological profile also includes causation of respiration and

reduction of the intraocular pressure. Besides this, it has shown a mild analeptic, analgesic,

hypotensive and a weak antimalarial activities [4].

Galanthamine was discovered in Galanthus woronowii by Proskurina and Yakovleva

in 1952 [5]. It has been first extracted by Sopharma (Bulgaria) in the early 1960s from G.

nivalis and later from considerably bigger plant Leucojum aestivum [6]. Galanthamine has

been found also in plants from the genera Amaryllis, Hippeastrum, Lycoris, Ungernia,

Narcissus, Zephyranthes, Hymenocallis, and Haemanthus [7] but it is currently being

extracted from daffodils (Narcissus cultivar Carlthon) in central and west Europe, snowflake

(Leucojum aestivum) in East Europe, ‘red-tubed lily’ (Lycoris radiata) in China, and

Ungernia victoria in Uzbekistan and Kazakhstan. Galanthamine content in plants used for

industrial extraction, reffered as percent from the dry weight, varies from traces to ca. 0.5%

(generally ca. 0.1-0.3%) in L. aestivum, from 0.10% to 0.15% in Narcissus cultivars, and up

to 0.52% in U. victoria [6].

The biosynthesis of galanthamine starts with enzymatic conversion of L-

phenylalanine (L-phe), including the enzyme phenylalanine ammonia lyase (PAL), and L-

tyrosine (L-tyr), including the enzyme tyrosine decarboxylase, to protocatechuic aldehyde

and tyramine, respectively. The junction of the tyramine and the protocatechuic aldehyde

results in a Schiff’s base, which is converted to norbelladine. Norbelladine or related

compounds could undergo oxidative coupling in Amaryllidaceae plants. 4’-O-

methylnorbelladine is considered as a key intermediate. Experiments with application of 13

C-

labelled O-methylnorbelladine to field grown Leucojum aestivum have indicated that the

biosynthesis of galanthamine involves the phenol oxidative coupling of O-

methylnorbelladine to a postulated dienone, which undergoes spontaneous conversion to N-

demethylnarwedine, giving norgalanthamine after stereoselective reduction. Norgalanthamine

is N-methylated to galanthamine in the final step of biosynthesis [8] (Figure 1). Later studies

indicated that deuterated-4'-O-methylnorbelladine is incorporated into three different groups

of Amaryllidaceae alkaloids that are biosynthesized by three modes of intramolecular

oxidative phenol coupling [9, 10].



Plants remain the main source of galanthamine although its chemical synthesis has

been achieved [11]. Due to the increased demand by the generic pharmaceutical companies

and the limited availability of plant sources, the biosynthesis of galanthamine by plant in

vitro systems has been considered as an alternative approach for sustainable production of

this valuable natural product. The first articles on the biosynthesis of galanthamine by

Narcissus confusus and L. aestivum in vitro shoot cultures were published in the early 1990s

[12-18].

The undifferentiated cell cultures derived from N. confusus and L. aestivum produce

low levels of alkaloids – about 30 μg/g DW and 12 μg/g DW, respectively [17, 19, 20]. The

small amounts of alkaloids indicate that undifferentiated cell cultures are unsuitable for

development of a biotechnological process [17]. Differentiation results in an increased

amount of biosynthesized galanthamine and its related alkaloids [17, 20]. The biosynthesis of

galanthamine and the Amaryllidaceae alkaloids by shoot cultures derived from different

explants—bulbs [15, 16, 18, 20], young fruit, and seeds—has been investigated. They

accumulate 25 to 454 μg Gal/g DW [16]. There are also reports in scientific literature on the

successful transformation of L. aestivum with Agrobacterium rhizogenes, where the authors

obtained a transformed root culture with stable morphological characteristics, which,

however, did not produce galanthamine [21].

Callus re-differentiation in the shoot culture correlates with higher levels of alkaloids,

including galanthamine [19]. Cellular differentiation affects the biosynthetic pathway of

alkaloids, in particular it increases the para-ortho’ oxidative phenol coupling, yielding

galanthamine precursors [22]. The following studies demonstrate that shoot cultures are a

good experimental system for galanthamine and its accompanying alkaloids [15, 17].

2 Effect of growth regulators on galanthamine biosynthesis

Growth regulators significantly affect galanthamine biosynthesis because of their

specific connection with cellular differentiation. There are studies on the effect of auxins,

cytokinins, and ethylene on the yield of galanthamine. The maximum galanthamine yield of

1354 μg/g of fresh biomass in N. confusus shoot cultures was reported for 3 mg/L

benzyladenine, where the amount of biosynthesized galanthamine correlates with the

benzyladenine concentration [22]. Diop et al. [15] have discussed the variations in the

accumulation of galanthamine in L. aestivum in vitro cultures, grown in media with various

combinations of growth regulators. In the absence of growth regulators the amount of

galanthamine is 1.14x10-3

% of dry biomass. When cultivated in media with 10μM α-

naphthyl acetate and 0.5 μM benzyladenine, the highest yield was obtained – 6.79x10-3

% of

dry biomass. The influence of cytokinins (benzo adenine, zeatin, kinetin meta-topolin and

thidiazuron) on galanthamine biosynthesis by L. aestivum shoot cultures cultivated in RITA

systems with periodic agitation has been studied and it was found that the maximum yield of

galanthamine (0.05% DW) was achieved when the cytokinin thidiazuron was used [23].

The same authors have proved that ethylene exerts inhibitory action on the

biosynthesis of galanthamine and lycorine in various L. aestivum in vitro [24, 25]. The

ethylene precursor 1-aminocyclopropane-1-carboxylic acid on the other hand caused a six-

fold increase in the amount of accumulated galanthamine and it reached up to 2% of the dry



biomass [25]. Tahchy et al. [26], studying the effect of growth regulators and sucrose

concentrations in tissue cultures of L. aestivum, Galanthus elwesii and N. pseudonarcissus

found a species specific response on AChE inhibitory activity and galanthamine content.

These results indicate that growth regulators affect secondary metabolism differently in

different in vitro cultures derived from the same plant. Their influence is specific and should

be studied separately for each individual culture. An overview of galanthamine content in

different in vitro systems is presented in Table 1.

3 Effect of medium components and cultivation conditions on galanthamine

biosynthesis

The optimization of the medium composition is an important step towards creating a

biotechnological process for biosynthesis of secondary metabolites [27]. Most often, the

composition of nutrient media is optimized with respect to the concentrations of the key

nutrient components (carbon, nitrogen and phosphorus sources) and/or the C/N ratio [28].

The links in the biological system “nutrient medium - in vitro culture - product(s)” are

examined by a multifactorial analysis. Then, depending on the results obtained, the nutrient

medium may be optimized to obtain maximum yields of the target product, biomass or both

[27].

The primary carbon source employed in in vitro systems biosynthesizing

galanthamine is sucrose [18, 27]. The study of the influence of sucrose amounts on

galanthamine biosynthesis in Narcissus confusus in vitro shoot cultures has found that the

highest amount of galanthamine (0.457 mg per culture) is accumulated at sucrose

concentration of 180 g/L in the nutrient medium [18]. At the same time, the best culture

growth was observed in a medium containing 90 g/L sucrose. The alkaloids haemanthamine,

tazetin and N-formilnorgalantamin accompanying galanthamine have maximum levels at low

sucrose concentrations. The sucrose concentration affects not only the biosynthesis of

alkaloids, but also the morphology of the test culture, and the activity of the photosynthetic

systems. For example, shoot cultures grown in sucrose concentration less than 9% are dark

green and consist of segments with foliage length of 5-6 cm. If grown in media with a higher

sucrose concentration, they are pale green with vitrification tendency, can form necrotic spots

and demonstrate late elongation. High sucrose levels are a stress factor affecting shoot

cultures also by changing the osmotic pressure [18]. On this basis, the optimization of the

nutrient media should be carried out not only with regard to the maximum yield of the target

product, but also should take into account the growth characteristics of the culture.

The combined influence has been studied of four independent variables in the culture

medium (sucrose, ammonium, nitrate and phosphate ions) on the production of biomass and

on galanthamine biosynthesis by a L. aestivum shoot culture by a multifactorial statistical

analysis [27]. The maximum values of the independent variables for a maximum

galanthamine yield were determined using a statistical analysis. Based on the results

obtained, Georgiev et al. [27] have proposed a modified variant of an MS growth medium for

maximum galanthamine biosynthesis by L. aestivum shoot cultures.



The nitrogen source in the nutrient media, used for cultivation, is a combination of

ammonium and nitrate ions. It has been found that in the optimal medium at NO3-/NH4

+ ratio

of (8.6/1) the biosynthesized galanthamine has a maximum level, while in the standard

medium the ratio is 6.5/1. This phenomenon is accompanied by the fact that nitrate ions are

absorbed more slowly (compared to ammonia), and nitrogen is needed mainly in the form of

nitrate ions in the secondary metabolism of cells [27]. Another important factor in the

optimization of nutrient media is the C/N ratio. Maximum amounts of galanthamine are

biosynthesized at higher concentrations of the carbon source (C/N – 21/1) compared to the

standard medium (15/1) [29]. The elevated carbon and nitrogen concentrations expectedly

have a positive impact due to the relationship between the carbon source and the biosynthesis

of secondary metabolites, on the one hand, and the presence of a nitrogen atom in the

galanthamine molecule, on the other [27]. In the optimal medium the alkaloid profile in the

shoot culture also changes. GC-MS analyses revealed that by cultivating the in vitro culture

in the modified medium, an increase occurred in the relative concentrations of intracellular

galanthamine (1.14 times) compared to the total ion current [27].

4 Light as a factor influencing galanthamine biosynthesis

Light has a positive effect on the growth of in vitro cultures and on the production of

secondary metabolites, in particular galanthamine [13, 17]. Light affects the plant cell with

differentiated shoot cultures by changing its metabolism from heterotrophic to mixotrophic.

Light also enhances the biosynthesis of phenol-containing amino acids (phenylalanine and

tyrosine), which are alkaloid direct precursors, in particular galanthamine, by inducing the

shikimate pathway enzymes [30].

The N. confusus shoot cultures have a better growth index when cultivated under light

conditions compared to those grown in dark conditions [13]. In vitro systems cultivated under

light conditions have green leaves while those grown in the dark are with pale leaves. The

fresh to dry biomass ratio is higher when cultivation is conducted in the dark, due to

differences in metabolism. Cultures grown under light conditions have photomixotrophic

metabolism, and those grown in the dark have heterotrophic metabolism. Maximum

galanthamine production was achieved in cultures grown under light conditions [13].

The growth characteristics of the L. aestivum in vitro shoot culture cultivated under

light and dark conditions in flasks gave a clear picture of the relationship between

illumination, culture development and galanthamine biosynthesis [17].

The results of the comparative study of 10 L. aestivum shoot lines cultivated in dark

and light conditions show that there are no significant differences in the alkaloid profile of

the cultures, i.e. light synchronously activates the biosynthesis of all types of alkaloids

identified in the L. aestivum in vitro cultures [19]. Tyramine protoalkaloids decrease in the

light, which indicates that light activates the enzymes in the initial stages of the synthesis of

alkaloids as they are precursors in the biosynthetic pathway of the Amaryllidaceae alkaloids.

On the other hand, galanthamine in the cultures is more than twice as high as those cultivated

under light – 73.8 μg/g dry biomass, unlike those cultivated under dark conditions – 38.5 μg/

dry biomass [19].



5 Elicitation

The main functions of secondary metabolites are associated with the protection of

plant organisms against various pathogens, as well as with their resistance to various biotic

and abiotic stress factors, whereas their biosynthesis is stress-induced by various elicitors and

signalling molecules [31]. On this basis a variety of strategies have been developed to

improve the yield of secondary metabolites by treating plant cells with elicitors or abiotic

stress [32]. Elicitors are chemical molecules or biofactors isolated from different sources that

can induce a physiological or morphological response by biosynthesizing phytoalexins [31].

Elicitation is a current commonly used method for optimization of the biosynthesis of various

secondary metabolites by plant in vitro systems.

Elicitation with biotic and abiotic elicitors is a method allowing a manifold increase in

the production of secondary metabolites by plant in vitro systems [33]. The effects of various

concentrations of four elicitors (methyl jasmonate, chitosan, arachidonic acid, and salicylic

acid) on galanthamine biosynthesis by Narcissus confusus shoot cultures have been studied

[34]. It has been found that the addition of methyl jasmonate at the start of the cultivation

process results in a twofold increase in the concentration of biosynthesized galanthamine.

Methyl jasmonate also increases the concentrations of the alkaloids accompanying

galanthamine: N-formyl-norgalanthamine, haemanthamine, homolycorine and pretazetine –

where the total amount of alkaloids is three times higher than the control. Schumann et al.

[29] studied the effect of copper sulphate, silver nitrate, salicylic acid and methyl jasmonate

on the growth and biosynthesis of galanthamine by L. aestivum shoot cultures. In that study

again it was found that methyl jasmonate increased galanthamine biosynthesis most strongly.

Recent studies in our laboratory [35] have indicated that methyl jasmonate increase mainly

the transcription of the enzyme phenylalanine ammonia lyase, and to a lesser degree that of

tyrosine decarboxylase. On the other hand, jasmonic acid advantageously increases the

transcription of tyrosine decarboxylase, and to a lesser extent the transcription of

phenylalanine ammonia lyase. Due to the different induction levels of both enzymes in the

initial stages of the biosynthetic pathway of Amaryllidaceae alkaloids by methyl jasmonate

and jasmonic acid, the biosynthesized galanthamine amounts are different. Galanthamine

biosynthesis was induced to the highest degree by jasmonic acid – 1.36 times more than the

control. This increase in galanthamine yield is due mainly to the enhanced expression of the

tyrosine decarboxylase gene, which in turn leads to an increase in the amounts of

biosynthesized tyramine – a direct precursor of the Amaryllidaceae alkaloids [35]. These

results indicate that the mechanisms of eliciting the biosynthesis of Amaryllidaceae alkaloids

are unclear and further in-depth studies are required on the interactions of jasmonic acid and

methyl jasmonate with the signal system that switches on in response to stress conditions in

the plant cell - calcium/calmodulin and protein phosphorylation/dephosphorylation, and the

induction and expression of the tyrosine decarboxylase and phenylalanine ammonia lyase

genes, as well as the other genes involved in the biosynthetic pathway of the Amaryllidaceae

alkaloids. The clarification of these relationships will have a significant impact on the

structuring of a cost-effective technology for the production of galanthamine from in vitro

systems of the Amaryllidaceae plants.



6 Bioreactor systems for galanthamine biosynthesis

The cultivation of differentiated organ cultures in bioreactors is associated with a

number of difficulties, mainly due to their morphology. Culture growth requires bioreactors

of special design in terms of the mass transfer of nutrients and gas phase. Despite these

difficulties, various bioreactors have been developed for cultivation of shoot organ cultures

[36]. Bioreactors in plant biotechnology are used for micropropagation of various species of

decorative plants and industrial crops, as well as for the biosynthesis of various secondary

metabolites [36]. Cultivation in a bioreactor occurs in depth, which may result in limitation of

the gas exchange between the culture fluid and the in vitro culture, causing vitrification and

hyperhydration of the plant tissue [37]. To overcome the problems encountered in deep

cultivation in liquid media, different cultivation systems have been developed such as

bioreactors with liquid phase diffuser and culture systems with periodic agitation [38, 39].

Regardless of the ever-increasing interest in plant in vitro technologies for producing

biologically active substances (including galanthamine), up till now a large-scale technology

based on plant organ cultures has not been implemented. One of the main reasons for the

limited industrial application of plant shoot cultures are the difficulties associated with their

cultivation in large volumes. The various bioreactor systems used for the cultivation of plant

organ cultures all too often are with a complicated design and expensive. Another thing is

that the problem concerning the inoculation of the bioreactor systems with large workloads

has not been solved [1].

For the successful development and introduction of new biotechnology for

galanthamine production by plant organ cultures, it is essential to establish the optimal

conditions for the accumulation of the product and to choose the most productive culture

system (bioreactor system). The interest is primarily directed towards the systems with

periodic agitation, considering the biological matrix producer of galanthamine (organ

culture). These systems are characterized by a low level of mechanical stress and a low

degree of hyperhydration.

The main parameters (agitation period and cultivation temperature) affecting the

growth and synthetic characteristics of line L. aestivum G 80 in RITA culture system were

optimized by statistical analysis [40]. The maximum galanthamine yield, 265 μg/RITA (1.17

mg/L), was obtained under optimal conditions (15 min agitation period and 8 hours resting

period, cultivation temperature 26°C). The optimal conditions for galanthamine accumulation

coincide with the optimal growth conditions (GI – 2.98) and maximum absorption of the

nutrients [40]. Ivanov et al. [41] studied the effect of the agitation period and cultivation

temperature on the alkaloid profile of line L. aestivum G 80 cultivated in RITA® system. By

GC-MS analysis, it was found that the cultivation temperature had a significant influence on

the biosynthesis of alkaloids. The highest relative amount of galanthamine was synthesized at

22°C, twice as much as the amount synthesized at 26°C [41].

Schumann et al. [29] investigated the growth and biosynthetic characteristics of L.

aestivum shoot cultures cultivated in different bioreactor systems - TIS, bubble and air-lift



column bioreactors. The agitation period and the amount of the culture were optimized. It

was found that the TIS system – 1L air-lift column bioreactor with agitation of 12 times daily

for 5 min was the most productive. In that cultivation system, productivity reached 214.7 μg

Gal/L per day, the in vitro culture biosynthesized 1.49 mg Gal/g DW. Good results were also

obtained in the cultivation of L. aestivum shoot cultures in the 1, 5 and 10 L bubble column

bioreactors (0.52, 0.72 and 0.68 mg Gal/g DW, respectively). It was found that maximum

galanthamine yield was obtained in deep cultivation conditions [29]. The authors did not

explore the influence of the cultivation conditions and type of cultivation system on the

alkaloid profile of the intracellular and extracellular alkaloid mixtures.

The course of bioreactor investigations has logically led to the further development of

a new type of cultivation system by Georgiev et al. [42]. The column bioreactor with internal

sections allows the distribution of the culture in separate sections in the whole reactor (Figure

2). This impedes the flotation of the culture and improves the mass transfer. That bioreactor

system entirely satisfies the requirements pointed out by Schumann et al. [29] for maximum

galanthamine yield. This system ensures good growth of the L. aestivum shoot cultures. A

maximum biomass yield of 20.8 g/L was achieved, which was about twice as high as the

yield obtained in the RITA cultivation system. The period of doubling the biomass was

shortened (from 282 h in the RITA system to 175 h in the modified column bioreactor); the

growth index increased. A statistical optimization method was used to obtain the optimal

values for the independent variables (cultivation temperature and air intake volume), that

influence galanthamine biosynthesis. A maximum galanthamine yield of line L. aestivum G

80 (1.7 mg/L) was reached at a cultivation temperature of 22°C and incoming air flow

velocity of 18 L/L.h. The yield achieved was 1.45 higher compared to the yield achieved in

the RITA cultivation system [42].

The authors investigated the effect of the growth conditions (intake airflow rate and

cultivation temperature) in a column bioreactor with inner sections on the alkaloid profile of

synthesized Amaryllidaceae alkaloids. Seventeen alkaloids of galanthamine, lycorine,

homolycorine and haemanthamine type were identified by a GC-MS analysis. Galanthamine

and lycorine were dominant, both in the intracellular and extracellular alkaloid mixtures (50-

70% of the total ion current for lycorine, and 19-60% of the total ion current for

galanthamine) [42]. The cultivation temperature had a significant influence on the alkaloid

profile in the cultivation of line L. aestivum G 80 in the column bioreactor with internal

sections. The relative concentration of the intracellular and extracellular galanthamine was

the highest at 22°C; similar results were also reported in the cultivation of line L. aestivum G

80 in the RITA® culture system [41, 42]. Under optimal conditions for galanthamine

biosynthesis of 22°C and intake airflow rate of 18 L/L.h, over 40% of the biosynthesized

galanthamine was extracellular with 45% purity of the total ion current. All these results

indicate that the column bioreactor with inner sections is a suitable cultivation system for

galanthamine production by shoot cultures of L. aestivum. The design of the bioreactor

allows a subsequent increase in the cultivation volume.

On this basis, a successful two-phase technology process can be developed for in situ

extraction of galanthamine biosynthesized in a column bioreactor with inner sections by

means of adsorption resins with low ion exchange capacity Amberlite XAD. This would

increase the yield of galanthamine, and would also increase its relative concentration, i.e.



biosynthesis of galanthamine at the expense of the accompanying Amaryllidaceae alkaloids.

The reported galanthamine yields, though high, are not satisfactory for a large-scale in vitro

technology for galanthamine biosynthesis. It is necessary to breed new highly productive L.

aestivum shoot lines with new improved genetic characteristics that can ensure steady growth

on the one hand, and high yields of galanthamine, eliminating the accompanying alkaloids,

on the other.

7 Concluding remarks

Plant in vitro systems are an alternative for galanthamine bioproduction. However, the

obtained yields are still too low and not attractive for the pharmaceutical industry. To

overcome this disadvantage, an integrated approach for process optimization should be

applied. A critical point in such an approach is the development of a new suitable design of

bioreactors for shoot cultivation.

The authors have declared no conflicts of interest.



References [1] Thomsen, T., Bickel, U., Fischer, J., Kewitz, H., Stereoselectivity of cholinesterase

inhibition by galanthamine and tolerance in humans, European Journal of Clinical

Pharmacology, 1998, 39, 603-605.

[2] Heinrich, M., Teoh, H.L., Galanthamine from snowdrop-the development of a modern

drug against Alzheimer’s disease from local Caucasian knowledge, Journal of

Ethnopharmacology, 2004, 92, 147–162.

[3] Alfirevic, A., Mills, T., Carr, D., Barratt, B.J. et al., Tacrine-induced liver damage: an

analysis of 19 candidate genes. Pharmacogenet Genomics, 2007, 17, 1091-1100.

[4] Bastida, J., Lavilla, R., Viladomat, F., Chemical and biological aspects of Narcissus

alkaloids, in: Cordell, G.A. (Ed.), The Alkaloids, Vol. 63, Elsevier Scientific,

Amsterdam, The Netherlands 2006, pp. 87–179.

[5] Proskurnina, N.F., Yakovleva, A.P., Alkaloids of Galanthus woronowi. II Isolation of

a new alkaloid, Zhurnal Obshchei Khimii, 1952, 22, 1899–1902.

[6] Berkov, S., Georgieva, L., Kondakova, V., Atanassov, A., et al., Plant sources of

galanthamine: phytochemical and biotechnological aspects, Biotechnol & Biotechnol

Eq., 2009, 23, 1170-1176.

[7] Cherkasov, O., Plant sources of galanthamine, Khim-Farm. Zhur., 1977, 11, 84-87.

[8] Eichhorn, J., Takada, T., Kita, Y., Zenk, M., Biosynthesis of the Amaryllidaceae

alkaloid galanthamine. Phytochemistry, 1998, 49, 1037–1047.

[9] El Tahchy, A., Boisbrun, M., Ptak, A., Dupire, F., et al., New method for the study of

Amaryllidaceae alkaloid biosynthesis using biotransformation of deuterium-labeled

precursor in tissue cultures. Acta Biochimica Polonica, 2010, 57, 75-82.

[10] El Tahchy, A., Ptak, A., Boisbrun, M., Dupire, F., et al., Kinetic Study of Deuterium-

labelled 4’-O-methylnorbelladine Rearrangement in Leucojum aestivum shoot cultures

by mass spectroscopy. Influence of precursor feeding on Amaryllidaceae alkaloid.

Journal of Natural Products, 2011, 74, 2356-2361.

[11] Satcharoen, V., McLean, N. J., Kemp, S. C., Camp, N. P., et al., Stereocontrolled

synthesis of (-)-galanthamine, Org. Lett., 2007, 9, 1867-1869.

[12] Bergoñón, S., Codina, C., Bastida, J., Viladomat, F., et al., The shake liquid culture as

an alternative way to the multiplication of Narcissus plants, Acta Horticulture, 1992,

325, 447-452.

[13] Bergoñón, S., Codina, C., Bastida, J., Viladomat, F., et al., Galanthamine production

in shoot-clumps culture of Narcissus confusus in liquid-shake medium, Plant Call,

Tissue and Organ Culture, 1996, 45, 191-199.

[14] Berkov, S., Pavlov, A., Iliava, M., Burrus, M., et al., CGC/MS of alkaloids in

Leucojum aestivum plants and there in vitro cultures, Phytochemical Analysis, 2005,

16, 98-103.

[15] Diop, M. F., Ptak, A., Chrétien, F., Henry, M., et al., Galanthamine content of bulbs

and in vitro cultures of Leucojum aestivum L, Natural Product Communication, 2006,

1(6), 475-479.

[16] Georgieva, L., Berkov, S., Kondakova, V., Bastida, J., et al., Alkaloid variability in

Leucojum aestivum from wild populations, Zeitschrift für Naturforschung, 2007, 62c,

627-635.

[17] Pavlov, A., Berkov, S., Courot, E., Gocheva, T., et al., Galanthamine production by

Leucojum aestivum in vito systems, Process Biochemistry, 2007, 42, 734-739.

[18] Sellés, M., Bergoñón, S., Viladomat, F., Bastida, J., et al., Effact of sucrose on growth

and galanthamine production in shoot-clump culture of Narcissus confuses in liquid-

shake medium, Plant Cell, Tissue and Organ Culture, 1997, 49, 129-136.

http://www.ncbi.nlm.nih.gov/pubmed/18004213

http://www.ncbi.nlm.nih.gov/pubmed/18004213

http://www.sciencedirect.com/science/article/pii/S0031942297010248




http://www.sciencedirect.com/science/journal/00319422



[19] Berkov, S., Pavlov, A., Georgiev, V., Bastida, J., et al., Alkaloid synthesis and

accumulation in Leucojum aestivum in vitro cultures, Natural Product

Communications, 2009, 4(0), 1-6.

[20] Sellés, M., Viladomat, F., Bastida, J. Codina, C., Callus induction, somatic

embryogenesis and organogenesis in Narcissus confusus: correlation and related

alkaloids, Plant Cell Reports, 1999, 18, 646-651.

[21] Diop, M. F., Hehn, A., Ptak, A., Chretien, F., et al., Hairy root and tissue culture of

Leucojum aestivum L. – relationships to galanthamine content, Phytochemistry

Reviews, 2007, 6, 137-141.

[22] Codina, C., Production of galanthamine by Narcissus tissue in vitro, in: Hanks, G. R.

(Ed.), Narcissus and Daffodil. The genus Narcissus, Taylor and Francis, London and

New York 2002, 215-241.

[23] Ptak, A., Simlat, M., Kwiecień, M., Laurain-Mattar, D., Leucojum aestivum plants

propagated in in vitro bioreactor culture and on solid media containing cytokinins,

Engineering in Life Sciences, 2013, 13(3), 261–270.

[24] Ptak, A., Tahchy, A. E., Dupire, F., Boisbrun, M., et al., LCMS and GCMS for the

screening of alkaloids in natural and in vitro extraction of Leucojum aestivum, Journal

of Natural Products, 2009, 72, 142-147.

[25] Ptak, A., Tahchy, A. E., Wyzgolik, G., Henry, M., et al., Effects of ethylene on

somatic embryogenesis and galanthamine content in Leucojum aestivum L. cultures,

Plant Cell, Tissue and Organ Culture, 2010, 102, 61-67.

[26] El Tahchy, A., Bordage, S., Ptak, A., Dupire, F., et al., Effect of sucrose and plant

growth regulators on acetylcholinesterase inhibitory activity of alkaloids accumulated

in shoot cultures of Amaryllidaceae, Plant Cell Tissue and Organ Cultures, 2011,

106, 381-390.

[27] Georgiev, V., Berkov, S., Georgiev, M., Burrus, M., et al., Optimized nutrient

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

Zeitschrift für Naturforschung, 2009, 64c, 219-224.

[28] Wilhelmson, A., Hakkinen, S. T., Kallio, P. T., Oksman-Caldentey, K. M., et al.,

Heterologous expression of vitreoscilla haemoglobin (VHB) and cultivation

conditions affect the alkaloid profile of Hyoscyamus muticus hairy roots,

Biotechnology Progress, 2006, 22(2), 350-358.

[29] Schumann, A., Berkov, S., Claus, D., Gerth, A., et al., Production of galanthamine by

Leucojum aestivum shoot grown in different bioreactor systems. Appl. Biochem.

Biotechnol., 2012, 167, 1907-1920.

[30] Beaudoin-Eagan, L. D., Thorpe, T. A., Shikimate pathway activity during shoot

initiation in tobacco callus cultures, Plant Physiology, 1983, 73, 228-232.

[31] Zhao, J., Davis, L. C., Verpoorte, R., Elicitor signal transduction leading to

production of plant secondary metabolites, Biotechnology Advances, 2005, 23(4),

283-333.

[32] Zhao, J., Zhu, W. H., Hu, Q., He, X. W., Improved indole alkaloid production in

Cataranthus roseus suspension cell culture by various chemicals, Biotechnology

Letters, 2000, 22, 1221-1226.

[33] Radman, R., Saez, T., Bucke, C., Keshavarz, T., Elicitation of plants and microbial

cell systems, Biotechnology and Applied Biochemistry, 2003, 37, 91-102.

[34] Colque, R., Viladomat, F., Bastida, J., Codina, C., Improved production of

galanthamine and related alkaloids by methyl jasmonate in Narcissus confuses shoot-

clumps, Planta Medica, 2004, 70, 1180-1188.



[35] Ivanov, I., Georgiev, V., Pavlov, A., Elicitation of galanthamine biosynthesis by

Leucojum aestivum liquid shoot culture, Journal of Plant Physiology, 2013, 170(12),

1122-1129.

[36] Steingroewer, J., Bley, T., Georgiev, V., Ivanov, I., et al., Bioprocessing of

differentiated plant in vitro systems, Engineering in Life Sciences, 2013, 13(1), 36-38.

[37] Debergh, P., Maene, L., Pathological and physiological problems related to the in

vitro culture of plant, Parasitica, 1984, 40, 69-75.

[38] Afreen, F., Temporary immersion bioreactor. Engineering consideration and

applications in plant micropropagation, in: Dutta Gupta, S. and Ibaraki, Y. (Eds.),

Plant Tissue Culture Engineering. Springer, The Netherlands 2006, pp. 187-201.

[39] Ducos, J.P., Terrier, B., Courtois, D., Disposable bioreactors for plant

micropropagation and mass plant cell culture, in Scheper, T., (Ed.), Advances in

Biochemical Engineering/Biotechnology vol 115, Disposable Bioreactors, Eibl, R.,

Eibl, D., (Eds)., Springer-Verlag, Heidelberg, Germany 2009, pp. 89-115.

[40] Ivanov, I., Georgiev, V., Georgiev, M., Ilieva, M., et al., Galanthamine and related

alkaloids production by Leucojum aestivum L. shoot culture using a temporary

immersion technology. Applied Biochemistry and Biotechnology, 2011, 163(2), 268-

277.

[41] Ivanov, I., Georgiev, V., Berkov, S., Pavlov, A., Alkaloid patterns in Leucojum

aestivum shoot culture cultivated at temporary immersion conditions, Journal of Plant

Physiology, 2012, 169, 206-211.

[42] Georgiev, V., Ivanov, I., Berkov, S., Ilieva, M., et al., Galanthamine production by

Leucojum aestivum L. shoot culture in a modified bubble column bioreactor with

internal sections, Engineering in Life Sciences, 2012, 12(5), 534-543.



Table 1. Galanthamine biosynthesis in different plant in vitro cultures

Plant In vitro culture Cultivation

system

Galanthamine

References

Narcissus

confussus

Callus

Shoot

Flask 30-60 μg/g DW

110-130 μg/g

DW

[11]

Leucojum

aestivum

Callus

Shoot

Flask 12 μg/g DW

100-200 μg/g

DW

[19, 41]

Shoot Flask 25-454 μg/g DW [26]

In vitro bulblets Flask 1.14-6.79x10-3

%

DW

[15, 21]

Shoot RITA 1.17 mg/L [16]

Shoot TIS systems 2.40 mg/g DW [25]

Shoot Bubble bioreactor 0.72 mg/g DW

Shoot Bubble column

bioreactor with

internal section

1.7 mg/L [9]

Shoot RITA 0.05% DW [35]



Figure 1. Biosynthesis of galanthamine



Figure 2. (A) Construction and (B) schematic diagram of modified bubble-column bioreactor

with internal sections: 1, bubble column with internal sections; 2, fluorescent lamps; 3, filters;

4, sampling. (C) Twenty-eight day of cultivation of L. aestivum line 80 shoot culture in

modified bubble-column bioreactor with internal sections at 22◦C and flow rate of inlet air of

18 L/(L·h) [18]

galanthamine biosynthesis in plant in vitro systems

Documents