Journal of Plant Physiology 169 (2012) 206–211 Contents lists available at SciVerse ScienceDirect 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). References Bastida J, Lavilla R, Viladomat F. Chemical and biological aspects of Narcissus alkaloids. In: Cordell G, editor. The alkaloids: chemistry and biology, vol. 63. Oxford: Academic Press; 2006. p. 87–179. Berkov S, Pavlov A, Ilieva M, Burrus M, Popov S, Stanilova M. CGC–MS of alkaloids in Leucojum aestivum plants and their in vitro cultures. Phytochem Anal 2005;16:98–103. Berkov S, Bastida J, Sidjimova B, Viladomat F, Codina C. Phytochemical differentiation of Galanthus nivalis and Galanthus elwesii (Amaryllidaceae). 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