Journal of Plant Physiology 169 (2012) 206–211
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Journal of Plant Physiology
journal homepage: www.elsevier.de/jplph
Alkaloid patterns in Leucojum aestivum shoot culture cultivated at temporary
immersion conditions
Ivan Ivanov a , Vasil Georgiev a,∗ , Strahil Berkov b , Atanas Pavlov a,c
a
b
c
Laboratory of Applied Biotechnologies, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 139 Ruski Blvd, 4000 Plovdiv, Bulgaria
AgroBioInstitute, 8 Dragan Tzankov Blvd., 1164 Sofia, Bulgaria
University of Food Technologies, Department of Organic Chemistry and Microbiology, 26 Maritza Blvd, 4002 Plovdiv, Bulgaria
a r t i c l e
i n f o
Article history:
Received 18 March 2011
Received in revised form
12 September 2011
Accepted 12 September 2011
Keywords:
Amaryllidaceae alkaloids
Culture conditions
Gas chromatography–mass spectrometry
Leucojum aestivum L.
Shoot culture
Temporary immersion system
a b s t r a c t
The alkaloid patterns in Leucojum aestivum L. shoot culture cultivated at temporary immersion conditions were investigated using gas chromatography–mass spectrometry. 18 alkaloids were identified, and
galanthamine, hamayne and lycorine were dominant. The L. aestivum 80 shoot culture, cultivated at temporary immersion conditions, is a prospective biological matrix for obtaining wide range Amaryllidaceae
alkaloids, showing valuable biological and pharmacological activities. The temperature of cultivation
influenced enzyme activities, catalyzing phenol oxidative coupling of 4′ -O-methylnorbelladine and formation of the different groups Amaryllidaceae alkaloids. Decreasing the temperature of cultivation of L.
aestivum 80 shoot culture led to activation of para-ortho’ phenol oxidative coupling (formation of galanthamine type alkaloids) and inhibited ortho-para’ and para-para’ phenol oxidative coupling (formation
of lycorine and haemanthamine types alkaloids).
© 2011 Elsevier GmbH. All rights reserved.
Introduction
Summer snowflake (Leucojum aestivum L.), a EuroMediterranean region plant, belongs to the Amaryllidaceae
family, is a well known producer of pharmaceutically important
alkaloids. Among the range of biosynthesized alkaloids, galanthamine was found to be the major bioactive compound. It is
well known as a long-acting, selective, reversible and competitive
acetylcholineesterase inhibitor (Diop et al., 2006). Nowadays,
galanthamine is widely used for treatment of neurological
disorders, mainly Alzheimer’s disease. This dibenzofuran nucleuscontaining alkaloid is obtained by para-ortho’ phenol oxidative
coupling of the common precursor of all Amaryllidaceae alkaloids
– 4′ -O-methylnorbelladine [19] (Bastida et al., 2006). Although
chemical synthesis of galanthamine is performed at a commercial
scale (Magnus et al., 2009), the preferred source for pharmacy
still remains the processing of harvested plant biomass. Intensive gathering from natural habitats during the last decade has
resulted in serious depletions of wild L. aestivum L. populations.
Abbreviations: BAP, 6-benzylaminopurine; GC/MS, gas chromatography–mass
spectrometry; MS, Murashige and Skoog nutrient medium; NAA, 1-naphthylacetic
acid; TIC, total ion current.
∗ Corresponding author. Tel.: +359 32 642 430; fax: +359 32 642 430.
E-mail address: vasgeorgiev@gmail.com (V. Georgiev).
0176-1617/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2011.09.010
This widening problem challenges scientists to intensify their
investigations, searching for new, alternative approaches for
galanthamine production and/or related alkaloids. As a result,
several reports concerning cultivation of L. aestivum L. shoot
cultures at submerged conditions of cultivation and in temporary
immersion systems have been reported (Pavlov et al., 2007;
Georgiev et al., 2009; Ivanov et al., 2011). The application of the
latter for secondary metabolite production by differentiated plant
in vitro systems represents an innovative, inexpensive approach,
with further possibilities for effective scale-up (Ivanov et al., 2011).
Despite the incontrovertible advantages of temporary immersion
systems, currently there are not available data concerning the
relationships between the cultivation conditions on the one
hand, and the changes in alkaloid profiles and the bioactivity
of the produced alkaloid mixtures during the cultivation of L.
aestivum L. shoot cultures in them, on the other. Focusing scientific
attention on the development of a biotechnological process for
galanthamine production by L. aestivum L. shoots, and production
and related concomitant alkaloids are poorly investigated. To
date, over 300 Amaryllidaceae alkaloids have been isolated and
described (Bastida et al., 2006). Most of them belong to one of the
following groups, defined on the basis of the principal molecule
skeleton types: galanthamine, lycorine, haemanthamine, tazettine,
montanine, homolycorine narciclasine, crinine and norbelladine.
Recent data revealed a wide range of pharmacological properties,
possessed by some of these structures, including anticancer,
28.0 ± 2.5
0.2b
0.7 ± 0.3
0.8b
0.8b
0.2 ± 0.1
–
0.6 ± 0.3
1.5 ± 0.8
–
tr
0.1b
tr
2.0 ± 0.5
0.2b
64.7 ± 4.8
–
tr
32.8 ± 6.1
1.3b
0.3 ± 0.1
0.9 ± 0.2
0.8 ± 0.2
0.4b
0.8b
tr
tr
–
0.1b
tr
–
3.9 ± 1.2
–
58.6 ± 4.8
tr
tr
27.8 ± 1.1
0.4 ± 0.3
0.5b
0.7 ± 0.1
0.7 ± 0.1
0.2b
–
0.5 ± 0.2
1.1 ± 0.1
tr
tr
tr
tr
1.9 ± 0.3
0.2b
65.7 ± 2.1
tr
0.1b
34.1 ± 4.6
1.2 ± 1.1
0.2 ± 0.1
0.6b
0.8 ± 0.1
0.4 ± 0.1
0.9 ± 0.2
tr
tr
–
0.1b
tr
–
5.2 ± 2.1
–
56.2 ± 1.0
tr
0.1b
27.6 ± 1.4
0.2b
0.5b
0.7b
0.6b
0.2 ± 0.1
–
1.1 ± 0.7
1.3 ± 0.8
tr
tr
tr
–
1.7 ± 0.6
0.5b
65.5 ± 0.2
tr
tr
25.2 ± 3.7
0.3 ± 0.2
tr
0.5 ± 0.2
0.3 ± 0.3
0.4 ± 0.3
0.7 ± 0.7
tr
tr
–
0.1b
0.1b
–
2.5 ± 1.5
–
69.70 ± 6.1
tr
tr
24.3 ± 2.2
0.2 ± 0.1
0.6 ± 0.2
0.8b
1.2 ± 0.3
0.4 ± 0.2
–
1.2 ± 0.1
3.2 ± 1.1
tr
tr
0.1b
tr
1.1 ± 0.4
0.2b
66.5 ± 4.6
tr
0.1b
26.2 ± 7.1
1.1 ± 0.7
tr
0.4b
0.7 ± 0.2
0.8 ± 0.5
0.4 ± 0.2
tr
0.1b
–
0.1b
0.1b
–
3.1 ± 1.3
–
66.8 ± 6.6
–
0.1b
32.9 ± 3.1
tra
0.6 ± 0.2
0.9 ± 0.2
0.8b
0.2b
–
0.5 ± 0.2
0.4 ± 0.2
tr
tr
0.1b
–
1.5 ± 0.1
0.4b
61.6 ± 2.8
tr
tr
Traces < 0.1% of TIC.
The standard deviations were lower than 0.05.
a
12 h/15 min
Exo
Endo
10 h/15 min
Exo
8 h/15 min
Exo
Endo
Exo
6 h/15 min
20.9 ± 8.4
0.7 ± 0.2
0.1 ± 0.1
0.5 ± 0.2
0.5 ± 0.3
0.1b
0.3 ± 0.2
–
tr
–
tr
0.1b
–
4.6 ± 2.5
–
72.0 ± 11.8
tr
tr
287
273
271
285
251
331
273
315
313
287
287
249
301
287
331
287
301
315
b
The GC–MS analyses were performed with a Hewlett
Packard 6890+/MSD 5975 (Hewlett Packard, Palo Alto, CA, USA)
20.49
21.12
21.48
21.66
21.91
21.93
22.06
22.26
22.32
22.98
23.14
23.48
23.30
24.94
25.07
25.53
26.51
26.55
Gas chromatography–mass spectrometry (GC/MS) analyses of
alkaloids
Galanthamine [1]
N-Demethylgalanthamine [2]
Vittatine [3]
Narwedine [4]
Anhydrolycorine [5]
6-Methoxylycorenine [6]
8-O-Demethylmaritidine [7]
Norpluviineacetate [8]
Acetylcaranine [9]
Pluviine [10]
Pancratinine C [11]
11,12-Dehydroanhydrolycorine [12]
Haemanthamine [13]
Hamayne [14]
Sternbergine [15]
Lycorine [16]
N-Formynorlgalanthamine [17]
8-O-Demethylhomolycorine [18]
Extracellular alkaloids
Fifty milliliters of culture liquids were evaporated to dryness and
dissolved in 10 mL methanol. After centrifugation and separation
of pellets, 8 mL of supernatants were evaporated to dryness and
residuals were dissolved in 2× 2 mL of 3% H2 SO4 and then processed
as described above.
4 h/15 min
Intracellular alkaloids
Dry biomass (0.2–0.3 g) was extracted three times with 5 mL of
methanol in an ultrasonic bath for 15 min. The combined extracts
were concentrated under vacuum and dissolved in 2× 2 mL of 3%
sulfuric acid. The neutral compounds were removed by extraction
(three times) with diethyl ether. The alkaloids were fractionated
after basification of the extracts with 1 mL of 25% ammonia and
extraction with chloroform (3× 3 mL). The chloroform extracts
were then dried over anhydrous sodium sulfate and evaporated
to dryness.
Endo
Extraction of alkaloids
M+
The L. aestivum L. line 80 shoot culture was cultivated for 35 days
in RITA® apparatus (CIRAD Ltd., France) with 200 mL optimized MS
medium (Georgiev et al., 2009). Each RITA was inoculated with 12 g
fresh shoots. The cultivation was performed on a thermostat chamber, under illumination (16 h light/8 h dark per day) and a flow rate
of the inlet air of 60 L h−1 for each RITA apparatus. For experiments,
different immersion frequencies (15 min flooding and 4, 6, 8, 10,
and 12 h stand-by periods) and different temperatures (18 ◦ C, 22 ◦ C
and 26 ◦ C) were investigated. In agreement with the experimental
design, all experiments on optimization of immersion frequency
were performed at a temperature of 26 ◦ C. The next optimization of
temperature was carried out by using an optimal immersion regime
of 15 min flooding and 8 h stand-by period.
Rt
Conditions of the temporary immersion cultivation
Alkaloid
The L. aestivum L. line 80 shoot culture was established and
selected after the planting of the previously obtained calli (Pavlov
et al., 2007) on Murashige and Skoog (MS) nutrient medium, supplemented with 30 g L−1 sucrose, 1.15 mg L−1 1-naphthylacetic acid
(NAA, Duchefa, The Netherlands), 2.0 mg L−1 6-benzylaminopurine
(BAP, Duchefa, The Netherlands), and 5.5 g L−1 “Plant agar”
(Duchefa, The Netherlands). The culture was maintained for more
than 7 years at 26 ◦ C under illumination (16 h light/8 h dark per
day). The subcultivation period was 28 days.
Table 1
Influence of immersion frequency on the percentage contribution of the alkaloids mixtures of the L. aestivum L. line 80 shoot culture, expressed as percentage of TIC.
Leucojum aestivum L. shoot culture
Endo
Material and methods
Endo
Exo
References
antiacetylcholinesterase, antiviral, antimalarial, antiprotozoal
antidepressant and anticonvulsant activities (Evidente et al., 2004;
Kornienko and Evidente, 2008; Lamoral-Theys et al., 2010; Osorio
et al., 2010). The production of such compounds could be applied
for an alternative to galanthamine in disease treatment.
In this study, for the first time, we demonstrated that controlling
the cultivation conditions, we can manipulate the alkaloid profile to
produce one or other desired compounds of the alkaloid mixtures,
biosynthesized by L. aestivum L. shoot culture, during its cultivation
in temporary immersion systems.
207
Berkov et al. (2005)
Berkov et al. (2005)
Berkov et al. (2008)
Berkov et al. (2005)
Berkov et al. (2008)
Kreh et al. (1995)
Berkov et al. (2005)
Berkov et al. (2009a)
Berkov et al. (2005)
Berkov et al. (2005)
Cedrón et al. (2009)
Berkov et al. (2009a)
Berkov et al. (2008)
Berkov et al. (2008)
Evidente et al. (1984)
Berkov et al. (2008)
Berkov et al. (2005)
Berkov et al. (2008)
I. Ivanov et al. / Journal of Plant Physiology 169 (2012) 206–211
208
I. Ivanov et al. / Journal of Plant Physiology 169 (2012) 206–211
Table 2
Influence of the temperature of cultivation on the percentage contribution of the alkaloids mixtures of the L. aestivum L. line 80 shoot culture, expressed as percentage of TIC.
18 ◦ C
Alkaloid
Galanthamine [1]
N-Demethylgalanthamine [2]
Vittatine [3]
Narwedine [4]
Anhydrolycorine [5]
6-Methoxylycorenine [6]
8-O-Demethylmaritidine [7]
Norpluviineacetate [8]
Acetylcaranine [9]
Pluviine [10]
Pancratinine C [11]
11,12-Dehydroanhydrolycorine [12]
Haemanthamine [13]
Hamayne [14]
Sternbergine [15]
Lycorine [16]
N-Formynorlgalanthamine [17]
8-O-Demethylhomolycorine [18]
a
b
22 ◦ C
26 ◦ C
Endo
Exo
Endo
Exo
Endo
Exo
44.5 ± 7.4
1.4 ± 0.1
tra
0.5 ± 0.1
1.3 ± 0.4
0.9 ± 0.1
0.2 ± 0.1
tr
tr
–
0.2 ± 0.2
0.5b
–
2.0 ± 1.9
–
48.4 ± 6.4
tr
tr
55.6 ± 11.0
0.1 ± 0.1
tr
0.6 ± 0.1
1.0 ± 0.6
0.3 ± 0.1
–
0.2b
0.3b
tr
tr
0.5 ± 0.2
tr
0.6 ± 0.1
tr
40.6 ± 10.2
tr
tr
59.8 ± 11.0
1.9 ± 0.5
tr
0.8 ± 0.1
1.4 ± 1.0
0.9 ± 0.6
0.3b
tr
0.7b
–
0.1b
1.0 ± 0.3
–
1.2 ± 0.9
–
31.6 ± 12.4
0.1b
tr
66.3 ± 11.1
0.5 ± 0.2
0.1b
1.2 ± 0.5
2.1 ± 0.5
0.4 ± 0.1
–
0.3b
0.3 ± 0.2
tr
tr
1.1 ± 0.2
0.3b
0.4 ± 0.1
0.2b
26.7 ± 12.4
0.1b
tr
25.2 ± 3.7
0.3 ± 0.2
tr
0.5 ± 0.2
0.3 ± 0.3
0.4 ± 0.3
0.7 ± 0.7
tr
tr
–
0.1b
0.1b
–
2.5 ± 1.5
–
69.70 ± 6.1
tr
tr
27.6 ± 1.4
0.2b
0.5b
0.7b
0.6b
0.2 ± 0.1
–
1.1 ± 0.7
1.3 ± 0.8
tr
tr
tr
–
1.7 ± 0.6
0.5b
65.5 ± 0.2
tr
tr
Traces < 0.1% of TIC.
The standard deviations were lower than 0.05.
instrument operating in EI mode at 70 eV. A HP-5 MS column
(30 m × 0.25 mm × 0.25 m) was used. The temperature program
was: 100–180 ◦ C at 15 ◦ C min−1 , 180–300 at 5 ◦ C min−1 and 10 min
hold at 300 ◦ C. The injector temperature was 250 ◦ C. The flow rate
of carrier gas (helium) was 0.8 mL min−1 . Split ratio was 1:20. One
microliter of the solution was injected. The spectra of co-eluting
chromatographic peaks were examined and deconvoluted by using
AMDIS 2.6 (NIST, Gaithersburg, MD) software before area integration. The contributions of each compound in the extracts are shown
in Table 1 as a percentage of the total ion current (TIC). The area of
the GC–MS peaks depends not only on the concentration of the
corresponding compounds, but also on the intensity of their mass
spectral fragmentation, so data given in the table do not express
absolute values (do not represent a true quantification) but can be
used for comparison of the samples, which was the objective of this
work.
The data presented are the averages from two independent
experiments, which were repeated twice, and expressed as the
means with standard deviations (±S.D.).
Results
Seven years ago, we developed a protocol for shoot cultures of
L. aestivum L. obtaining via callus (Pavlov et al., 2007). The shoot
line 80, showed stable growth and biosynthetic characteristics, was
selected as prospective for the further experiments. Recently, the
nutrient medium for maximal galanthamine yields was optimized
during its submerged cultivation (Georgiev et al., 2009). The profiles
of the both extra- and intracellular alkaloid fractions of L. aestivum
L. line 80 shoot culture have been investigated as well (Berkov et al.,
2005, 2009b). The results showed that, in addition to major alkaloids (galanthamine, lycorine and N-demethylgalanthamine), the
shoots synthesized many alkaloids in minor concentrations that
could be of interest because they are possible new and unknown
carriers of bioactivity. Our last investigations showed that the temporary immersion approach is prospective for development of a
biosynthetic process for obtaining alkaloids as both immersion
frequency and temperature had significant effect on biomass accumulation and the yields of galanthamine (Ivanov et al., 2011). The
possibilities for manipulation of the biosynthetic process of alkaloids through environmental conditions provoked our interest to
investigate the influence of the immersion frequency and the temperature during the cultivation of L. aestivum L. line 80 shoot culture
in a temporary immersion RITA system, on the profiles of the synthesized valuable Amaryllidaceae alkaloids.
Gas chromatography–mass spectrometry is a powerful tool for
fast and accurate identification of alkaloid mixtures (Berkov et al.,
2011) and for multimetabolite analyses as well (Georgiev et al.,
2010). The immersion frequency and the temperature of cultivation
are both parameters that could be varied during the cultivation of plant in vitro systems at temporary immersion conditions
(Debnath, 2009). The results obtained concerning their influences
on the patterns of alkaloids biosynthesized by L. aestivum L. line
80 shoot culture are presented in Tables 1 and 2. 18 alkaloids were
detectable in shoot cultures cultivated at all tested regimes. Three of
them were dominant: galanthamine [1], hamayne [14] and lycorine
[16].
Discussion
Effect of immersion frequency on the alkaloid pattern
Lycorine [16] had the highest percentage contribution in alkaloid patterns – between 60% and 70% of the TIC. This was
Table 3
Influence of the temperature on the percent distribution of the different Amaryllidaceae alkaloid groups in mixtures of the L. aestivum L. line 80 shoot culture, expressed as
sum of percentages of TIC.
Alkaloid groups
18 ◦ C
22 ◦ C
Endo
Lycorine type
Homolycorine type
Haemanthamine type
Galanthamine type
50.4
0.9
2.2
46.4
Exo
±
±
±
±
7.0
0.1
2.0
7.6
42.6
0.3
0.6
56.3
26 ◦ C
Endo
±
±
±
±
11.0
0.1
0.1
11.2
34.8
0.9
1.5
62.6
Exo
±
±
±
±
13.7
0.6
0.8
11.6
30.6
0.4
0.8
68.1
Endo
±
±
±
±
13.3
0.1
0.1
11.8
70.2
0.4
3.2
26.0
Exo
±
±
±
±
6.4
0.3
2.2
4.1
69.0
0.2
2.2
28.5
±
±
±
±
1.7
0.1
0.6
1.4
I. Ivanov et al. / Journal of Plant Physiology 169 (2012) 206–211
209
OH
MeO
NH
HO
4'-O-methylnorbelladine
19
ortho - para'
para - ortho'
para - para'
R1
R2
R2
O
R1
OR1
R2
H
OR4
OR4
OR4
H
H
OR3
N
OR3
N
N
R3
Lycorine type
Galanthamine type
Haemanthamine type
8 R1=Ac; R2=H; R3=H; R4=Me
9 R1=Ac; R2=H; R3 + R4=CH2
10 R1=H; R2=H; R3=Me; R4=Me
15 R1=Ac; R2=OH; R3=Me; R4=Me
16 R1=H; R2=OH; R3 + R4=CH2
1
2
4
17
3 R1=OH; R2=H; R3 + R4=CH2
7 R1=OH; R2=H; R3=H; R4=Me
13 R1=OMe; R2=OH; R3 + R4=CH2
14 R1=OH; R2=OH; R3 + R4=CH2
R1=OH; R2=H; R3 =Me; R4=Me
R1=OH; R2=H; R3=H; R4=Me
R1 + R2=O; R3=Me; R4=Me
R1=OH; R2=H; R3=CHO; R4=Me
Homolycorine type
Me
HO
O
O
N
N
O
OR4
H
N H
O
5
11
O
OR3
O
R2
R1
6 R1=OMe; R2=H; R3=Me; R4=Me
18 R1 + R2=O; R3=H; R4=Me
N
O
12
Fig. 1. Biosynthetic relationships of the identified alkaloids in L. aestivum L. line 80 shoot culture during its cultivation in a temporary immersion RITA system after Berkov
et al. (2009b).
an important result because this pyrrolophenanthridine alkaloid
attracts huge interest because of its strong biological and pharmacological activities (Yui et al., 1998; Evidente et al., 2004; Li et al.,
2007; Kornienko and Evidente, 2008; Liu et al., 2009; LamoralTheys et al., 2010). The galanthamine [1], well known with its
anti-cholinesterase activity, was 25–30% of TIC. The content of
galanthamine [1] in extracellular and intracellular alkaloid fractions was comparable (Tables 1 and 2). It should be underlined
that immersion frequency did not influence the percentage contribution of galanthamine [1] and lycorine [16] in the alkaloid
patterns of L. aestivum L. line 80 shoot culture during its cultivation in temporary immersion system (Table 1). In contrast to
210
I. Ivanov et al. / Journal of Plant Physiology 169 (2012) 206–211
galanthamie [1] and lycorine [16], hamayne [14] was presented
in intracellular fractions between 3% and 5% of TIC, while in
extracellular fractions, it was about 2-fold lower. This alkaloid is
also of interest because of its cytostatic and anticancer activities
(Campbell et al., 2000). N-Demethylgalanthamine [2], the precursor of galanthamine [1], was presented in higher percentage in
intracellular alkaloid fractions, while narwendine [4], a product
of galanthamine [1] was in higher percentages in extracellular
alkaloid fractions (Table 1). Other alkaloids identified in minor
percentages of TIC were: 8-O-demethylmaritidine [7], identified
only in intracellular alkaloid mixtures and possessing antibacterial
activity (Evidente et al., 2004); pluviine [10], norpluviineacetate
[8], acetylcaranine [9], haemanthamine [13] and sternbergine [15].
Recently, it was reported that alkaloids [8], [10], [13] and [15] possess strong cytostatic and anticancer activities (Campbell et al.,
2000; Lamoral-Theys et al., 2009, 2010). Alkaloids vittatine [3]
and anhydrolycorine [5] were identified both in the intracellular
and in the extracellular alkaloid mixtures. These alkaloids possess
antibacterial activity (Evidente et al., 2004). In addition, the vittatine [3] improve the analgesic effect of morphine (Lamoral-Theys
et al., 2010), and anhydrolycorine [5] possesses anticancer activity
(Lamoral-Theys et al., 2009). Presented results clearly underlined
that the L. aestivum L. line 80 shoot culture, cultivated at temporary
immersion conditions, was a good biological matrix both for galanthamine production (Ivanov et al., 2011) and for obtaining related
Amaryllidaceae alkaloids, possessing valuable biological and pharmacological activities.
Effect of temperature of cultivation on the alkaloid pattern
Three different temperatures (18 ◦ C, 22 ◦ C and 26 ◦ C) were
tested. It was established that the temperature of cultivation had
significant effect on the biosynthesis of alkaloids by L. aestivum
L. line 80 shoot culture (Table 2). A decrease in the cultivation temperature from 26 ◦ C to 22 ◦ C led to a 2-fold increase of
the presence of galanthamine (1). It became the dominant alkaloid: intracellular galanthamine [1] increased from 25.2 ± 3.7%
of TIC (at 26 ◦ C) to 59.8 ± 11.0% of TIC (at 22 ◦ C) and extracellular from 27.6 ± 1.4% of TIC (at 26 ◦ C) to 66.3 ± 11.1% of TIC
(at 22 ◦ C), respectively (Table 2). The rest of the galanthamine
type alkaloids (N-demethylgalanthamine [2], narwedine [4] and
N-formynorlgalanthamine [17]) increased their percentages in
both intracellular and extracellular alkaloid mixtures. In contrast, lycorine [16] decreased its presence with the decrease of
the temperature. The percents of lycorine [16] were reduced
2.2-fold in the intracellular alkaloids [from 69.7 ± 6.1% of TIC
(at 26 ◦ C) to 31.6 ± 12.4% of TIC (at 22 ◦ C)] and 2.4-fold in
the extracellular alkaloids [from 65.5 ± 0.2% of TIC (at 26 ◦ C) to
26.7 ± 12.4% of TIC (at 22 ◦ C)], respectively (Table 2). Other alkaloids whose biosynthesis was affected by changes in cultivation
temperature were sternbergine [15], anhydrolycorine [5] and
11,12-dehydroanhydrolycorine [12] (Table 2). Further decrease of
the temperature (from 22 ◦ C to 18 ◦ C) had no significant influence
on galanthamine [1] and lycorine [16] percentage in the alkaloid
mixture (Table 2).
Conclusion
The temperature of cultivation had a significant effect on
the alkaloid biosynthesis by L. aestivum L. line 80 shoot culture
cultivated at temporary immersion conditions. The optimal temperature for galanthamine [1] biosynthesis was 18–22 ◦ C, while
that for lycorine [16] biosynthesis was at 26 ◦ C. At 22 ◦ C galanthamine type alkaloids increased their presence in the alkaloid
mixtures and they became the dominant group (Table 3). These
alkaloids each had a para-ortho’ phenol oxidative structure (Fig. 1).
On the other hand, the presence of lycorine type alkaloids (with
ortho-para’ phenol oxidative structure, Fig. 1) decreased 2-fold in
comparison to the cultivation at 26 ◦ C. Haemanthamine type alkaloids (with para-para’ phenol oxidative structure, Fig. 1) increased
in the alkaloid mixtures with increasing the temperature of cultivation (Table 3). These results clearly show that temperature of
cultivation influenced enzyme activities, catalyzing phenol oxidative coupling of 4′ -O-methylnorbelladine [19] and formation of the
different groups Amaryllidaceae alkaloids (Fig. 1). Decreasing the
temperature of cultivation of L. aestivum L. line 80 shoot culture
led to activation of para-ortho’ phenol oxidative coupling (formation of galanthamine type alkaloids) and inhibited ortho-para’ and
para-para’ phenol oxidative coupling (formation of lycorine and
haemanthamine types alkaloids).
Acknowledgement
This research was supported by the Bulgarian Science
Foundation, Bulgarian Ministry of Education and Science (DO02/105-2009).
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