Dynamics of Growth and Galanthamine Biosynthesis in Hippeastrum papilio (Ravena) Van Sheepen Hydroponic Culture

Abstract

Hippeastrum papilio (Ravena) van Sheepen is a bulbous evergreen species and considered a potential new source of galanthamine. This natural compound approved by the FDA is used for the cognitive treatment of Alzheimer’s disease. To optimize the galanthamine yield from this species, it is necessary to study the effects of plant age and fertilization on the alkaloid content, as well as alkaloid and biomass accumulation dynamics in plant organs. H. papilio plants of different ages, which were ex vitro acclimatized (age 0) and previously grown for one (age 1) and two (age 2) vegetation seasons, were cultivated in a flood and drain hydroponic system with different fertilizer solutions for six months. Samples from the roots, bulbs, and leaves were gathered at the end of the vegetation, and the fresh and dry biomasses were measured and then analyzed by GC–MS to establish their alkaloid content. Depending on the age and fertilizer, the galanthamine content varied from 4.5 ± 1.8 to 11.2 ± 2.8 mg/g DW in the roots, from 3.4 ± 0.5 to 5.8 ± 1.3 mg/g DW in the bulbs, and from 3.2 ± 0.3 to 5.7 ± 0.6 mg/g DW in the leaves. The main part (53–61%) of galanthamine was accumulated in the bulbs, while the leaves and roots stored 25–30% and 13–19%, respectively. Higher amounts of N, K, and Ca in the fertilizer did not positively influence the alkaloid yield in plants of ages 1 and 2. Despite the lower biomass accumulation per individual, the plants grown for two seasons (age 1) showed a comparable galanthamine yield (per square meter) at the end of vegetation to those grown for three seasons (age 2) due to their higher density of cultivation. The dynamics of alkaloid and biomass accumulation, studied in plants from age 1 during the vegetation season, showed that the highest galanthamine content in the plant organs is at the beginning of vegetation. Still, the end of vegetation is the best time to harvest the plant biomass for galanthamine extraction. Hydroponic cultivation of H. papilio is an interesting alternative for the production of galanthamine.

1. Introduction

Hippeastrum papilio (Ravena) van Sheepen (subfamily Amaryllidoideae) is a bulbous evergreen species endemic to South America and cultivated as an ornamental plant in Europe. It is a potential new source of the alkaloid galanthamine [1] used as an AChE inhibitor for the cognitive treatment of Alzheimer’s disease. Even though its chemical synthesis has been achieved [2], the main source for industrial production of galanthamine (Figure 1) remains the plants. This alkaloid is currently produced from the bulbs of Narcissus pseudonarcissus cv. Carlton (containing about 2.2–3.3 mg/g DW) [2,3,4] in central and west Europe from the leaves of a snowflake (Leucojum aestivum, galanthamine content ranges from traces to about 5.0 mg/g DW) [5] in east Europe and from the bulbs of Lycoris species (galanthamine content from 0.2 to 5.0 mg/g DW) [6] in China. The use of L. aestivum, however, is very limited because it is categorized as endangered in the Red Data Book of Bulgaria for which there is no agrotechnology for cultivation and its leaf biomass is gathered from natural populations under regulation [7,8]. On the other hand, H. papilio is a considerably bigger plant as compared to the other galanthamine sources, and its galanthamine content in the leaves of some genotypes was found to be 0.54% (5.4 mg/g DW). Moreover, galanthamine comprises 87% of all alkaloids in the n-hexane fraction from bulbs and 54%, 62%, and 88% in chloroform fractions from leaves, bulbs, and roots, respectively, reaching ca. 98% in selected genotypes [1]. Another valuable alkaloid, produced in H. papilio at relatively high levels, is haemanthamine (Figure 1) [9], which has shown potent antitumor activities inducing apoptosis in various tumor cells at micromolar concentrations [10].

The main challenge for the introduction of H. papilio as a new galanthamine source is the development of an efficient agrotechnology ensuring high biomass and alkaloid yields. Factors affecting the alkaloid yield such as plant age, time of harvesting, fertilization, and the dynamics of biomass and alkaloids in the plant organs have to be studied.

The content of galanthamine in H. papilio is expected to vary during the vegetation season like in other alkaloid-producing plants [11,12,13]. Its biosynthesis and accumulation were found to be an organ-specific process, indicating the bulbs as the main reservoir for galanthamine [9]. The harvest time may vary depending on the plant organ used for galanthamine extraction. The leaves of L. aestivum are gathered during flowering (April–May), while the bulbs of Narcissus cv. Carlton during the leaf dieback [13] when the plant transferred the metabolites from the leaves into the bulbs. Chang [6] and Akram et al. [14] have found that the accumulation of alkaloids in Amaryllidoideae plants is also an age-dependent process.

Alkaloid accumulation and plant growth may be influenced by fertilizers. Since alkaloids contain N, it has been generally believed that increased fertilization with this element would lead to a higher accumulation of alkaloids in plants, which was the case with several alkaloid-producing plants, including periwinkle (Catharanthus roseus) and datura (Datura innoxia) [15,16,17]. On the other hand, several recent studies on alkaloid-producing plants from genera Narcissus, Duboisia, and Mytragyna have shown that while increased N fertilization positively affects biomass accumulation, it is negatively related or does not affect the alkaloid biosynthesis [3,18,19]. The type of N fertilization (NO³⁻ and/or NH⁴⁺) also has an impact on the growth of Hippeastrum plants [20,21]. Additional application of K in pot experiments enhanced both the biomass and galanthamine accumulation in Lycoris radiata, but they are significantly decreased by levels above the optimal one [6].

Hydroponic cultivation is a suitable approach to study the effects of fertilization on alkaloid biosynthesis that could provide control on plant nutrition and environmental parameters [22,23,24]. The potential for the production of secondary metabolites and alkaloids, in particular, from hydroponic cultures is poorly studied.

As a part of our ongoing studies on H. papilio as a potential industrial source of bioactive compounds, the aim of the present study was to obtain preliminary information that could encompass the way to optimize the galanthamine yield, answering the following questions: (1) what is the best age of the plant to harvest? (2) what is the best time during the vegetation season to harvest? (3) can fertilization (macronutrients) enhance biomass and galanthamine biosynthesis? and (4) can hydroponic cultures of H. papilio be applied for galanthamine production?

2. Materials and Methods

2.1. Plant Material

The experiments were performed with H. papilio plants of three different ages abbreviated in the text as age 0 for 4-months plants after ex vitro acclimatization in perlite, age 1 for plants grown in soil for one vegetation season after twin-scaling, and age 2 for plants grown in soil for two vegetation seasons after twin scaling. Plants of age 0 were obtained in vitro and acclimatized ex vitro as described in Berkov et al. [1]. Bulbs of plants from ages 1 and 2 as well as the plant material used for induction of in vitro cultures were kindly provided by Ludwig and Co BV, Budel, the Netherlands.

2.2. Cultivation and Experimental Design

The plants were grown on a flood and drain hydroponic system in plastic crates (40 cm × 60 cm) with perlite for plants of age 0 and clay pebbles for plants of ages 1 and 2 for 6 months (from the beginning of May 2022 until the end of October 2022) at the greenhouse of the Institute of Biodiversity and Ecosystem Research at the Bulgarian Academy of Sciences (Figure 2). During the first three months, the plants were grown with a basic nutrient solution (Fertilizer I) described in the recipe formulations proposed by Kroggel and Kubota [25] for optimized greenhouse production of tomatoes (

Table 1. Solution recipes for standard soluble fertilizers according to Kroggel and Kubota, [25]. All amounts are for a 100× concentrated stock solution.

	Fertilizer I	Fertilizer II	Fertilizer III
Macronutrient	g/L	g/L	g/L
KNO3	0.0	22.2	50.1
KH2PO4	20.7	20.7	20.7
MgSO4-7H2O	61.2	61.2	61.2
K2SO4	20.7	49.6	24.1
Ca(NO3)2	57.9	57.9	78.9
CaCl2	9.4	13.9	13.9
Micronutrient	mg/L	mg/L	mg/L
Borax	165.9	165.9	165.9
MnSO4-H2O	177.4	177.4	177.4
CuSO4-5H2O	20.0	20.0	20.0
Na2MoO4-2H2O	12.6	12.6	12.6
ZnSO4-H2O	145.1	145.1	145.1
Fe chelate (DTPA)	2.0	2.0	2.0

). During the next three months, the plants were grown with three different fertilizers (Fertilizer I, Fertilizer II, and Fertilizer III, Table 1). The temperature inside the greenhouse ranged between 14–25 °C (night) and 18–37 °C (day) during the cultivation period. The pH of the nutrient solution was maintained below 6.5 while its electroconductivity was maintained at 1500 ± 300 mS m⁻¹. The nutrient solution was circulated for 15 min at intervals of 4 h. The plant density was as follows: for age 0–75 plants per crate (313 plants per m²), for age 1–25 plants per crate (104 plants per m²), and for age 2–12 plants per crate (50 plants per m²). For each variant of the experiment (age and fertilizer), 3 crates with plants were used (in total for age 0–675 plants, 225 per fertilizer; for age 1–225 plants, 75 per fertilizer and for age 2–108 plants, 36 per fertilizer). Every month, 5 plants from each age and fertilizer were collected randomly from different crates. Every plant was subsequently divided into roots, bulbs, and leaves. The fresh weight (FW) and dry weight (DW) of each part of the individuals was recorded.

2.3. Phytochemical Analysis

2.3.1. Sample Preparation

Fresh roots, bulbs, and leaves were cut, dried at 60 °C separately for each individual, and then powdered. The sample preparation was carried out as described previously [1]: 100 mg of dried plant material was macerated in screw-top Eppendorf tubes (2 mL of volume) with 1 mL of methanol adjusted to pH 8 with 25% of ammonia and containing 50 µL of codeine as an internal standard. After 24 h of extraction at room temperature, the samples were centrifugated at 6000 rpm for 1 min. Then, 500 µL aliquots were transferred to other Eppendorf tubes and dried overnight. Afterward, 500 µL of 2% sulfuric acid in distilled water was added and the neutral compounds were eliminated by triplicate extraction (vortexing) with 500 µL chloroform. The purified mixtures were then basified with 60 µL 25% ammonia and the alkaloids were extracted in triplicate with 500 µL chloroform and collected in a glass vial. The organic solvent was evaporated and the dry extract was derivatized with 50 μL of N,O-bis-(trimethylsilyl)trifluoro-acetamide (BSTFA) in 50 μL of pyridine for 2 h at 50 °C. After cooling, 300 µL of chloroform was added to the derivatized fractions and analyzed by gas chromatography–mass spectrometry (GC–MS).

2.3.2. Gas Chromatography–Mass Spectrometry

The GC–MS chromatograms and spectra were recorded on a Thermo Scientific Focus GC coupled with a Thermo Scientific DSQ II mass detector (Thermo Fisher Scientific Inc., Waltham, MA, USA) operating in EI mode at 70 eV. A DB-5MS column (30 m × 0.25 mm × 0.25 mm) was used. The temperature program was 100–180 °C at 15 °C × min⁻¹, 180–300 °C at 5 °C × min⁻¹, and 10 min hold at 300 °C. The injector temperature was 250 °C. The flow rate of the carrier gas (Helium) was 0.8 mL × min⁻¹. In total, 1 mL of the derivatized samples was injected at a split ratio of 1:10.

2.3.3. Quantification of Galanthamine and Haemanthamine

The quantification of galanthamine was carried out after a previously validated GC–MS method [26]. A calibration curve for galanthamine quantification was prepared using eight concentrations (20, 50, 100, 200, 400, 600, 800, and 1200 µg/mL) of galanthamine and 50 µg of codeine as an internal standard (IS), which were analyzed as TMS derivatives. The ratios of the peak areas of selected (base) ions in the selected ion monitoring (SIM) mode of galanthamine TMS (m/z at 216, RT 38.81) versus those of codeine (m/z at 178, RT 39.32) were plotted against the corresponding concentration of galanthamine to obtain the calibration curve (Supplementary Materials, Table S2, Figures S1 and S2). Galanthamine content was expressed as a milligram per gram of the sample’s dry weight (DW). Haemanthamine content was calculated as a galanthamine equivalent in the samples using the base ion at m/z 181 (RT 40.28) for haemanthamine TMS-derivative [1,9].

2.4. Statistical Analysis

The statistical analyses were performed using Minitab Statistical Software v. 19 (Minitab LLC, State College, PA, USA) (free trial). The one-way ANOVA method was used when the effect of one factor was investigated. The multiple linear regression method was used when the effect of more than one factor was analyzed. Both methods were followed by Tukey’s test at a significant level of p < 0.05. The data values are expressed as mean ± standard deviation (n = 5).

3. Results

3.1. The Effects of Age and Fertilizers on Galanthamine and Biomass Accumulation

The results on the alkaloid and biomass accumulation in H. papilio plants at the end of vegetation (October) from three different ages and treated with three different fertilizers are shown in Figure 3 and Supplementary Materials, Figures S3–S9.

In general, the roots showed higher alkaloid content as compared to the bulbs and leaves (Figure 3A). The effect of age on the galanthamine content in the plant organs was studied by comparing plants grown with the same fertilizer (Supplementary Materials, Figures S4–S7). The results showed that the roots of plants from age 1 tend to have higher galanthamine content, where the differences for fertilizer I and II (9.4 ± 1.5 and 11.2 ± 2.8 mg/g DW) were significant as compared to those of plants from ages 0 (6.2 ± 1.9 and 4.5± 1.9 mg/g DW) and 2 (7.6 ± 0.8 and 8.2 ± 1.4 mg/g DW). Similarly, the bulbs of plants from age 1 tend to accumulate more galanthamine where the differences for fertilizer II (5.3 ± 0.8 mg/g DW) are significant as compared to those of plants from age 2 (3.4 ± 0.5 mg/g DW, Supplementary Materials, Figure S6). The haemanthamine content and haemantamine/galanthamine ratio in the bulbs and leaves from plants of age 0 are considerably higher than those in the bulbs and leaves from plants of age 1 and 2 (Table S1, Supplementary Materials).

The effect of the different fertilizers on the galanthamine content was studied by comparing plants of the same age (Figure 3A). The galanthamine content in the bulbs (5.5 ± 0.6 mg/g DW) and leaves (4.6 ± 0.6 mg/g DW) of plants from age 2 grown with the fertilizer I was significantly higher as compared to the other fertilizers (Supplementary Materials, Figures S8 and S9).

The biomass accumulation of the roots, bulbs, and leaves of H. papilio was not influenced significantly by the fertilizers (Figure 3B). The values of the root dry weight per plant varied around 0.1 ± 0.0 g for age 0, between 0.6 ± 0.2 and 1.1 ± 0.6 g for age 1 and between 1.8 ± 0.5 and 2.3 ± 0.6 g for age 2. The values of the bulb dry weight were between 0.4 ± 0.2 and 0.7 ± 0.5 g for age 0, 5.0 ± 1.0 and 6.3 ± 2.0 g for age 1, and 11.8 ± 2.8 and 14.2 ± 2.1 g for age 2. The values of the leaf dry weight were between 0.3 ± 0.1 and 0.7 ± 0.4 g for age 0, 3.9 ± 1.4 and 4.7 ± 2.5 g for age 1, and 6.5 ± 1.8 and 7.8 ± 1.1 g for age 2.

The highest biomass yield from roots (116 ± 28 g/m²) was found in plants from age 2 grown with the fertilizer I (

Table 2. Yield of biomass (g DW/m²) (A) and galanthamine (mg/m²) (B) in H. papilio hydroponic cultures, shown as mean ± standard deviation (n = 5).

(A)	Age 0			Age 1			Age 2
Fertilizer	I	II	III	I	II	III	I	II	III
Roots	25 ± 8	17 ± 8	21 ± 11	110 ± 58	64 ± 18	83 ± 23	116 ± 28	114 ± 47	90 ± 27
Bulbs	142 ± 49	125 ± 52	214 ± 149	624 ± 266	520 ± 99	655 ± 207	677 ± 132	712 ± 105	592 ± 142
Leaves	109 ± 36	125 ± 54	218 ± 134	492 ± 91	417 ± 91	403 ± 143	343 ± 76	392 ± 54	324 ± 92
Total	275 ± 92	267 ± 114	453 ± 292	1226 ± 580	1000 ± 196	1141 ± 368	1136 ± 227	1217 ± 136	1006 ± 235
(B)	Age 0			Age 1			Age 2
Fertilizer	I	II	III	I	II	III	I	II	III
Roots	167 ± 96	82 ± 75	113 ± 40	983 ± 405	665 ± 136	722 ± 240	874 ± 313	869 ± 254	622 ± 55
Bulbs	623 ± 214	566 ± 286	1043 ± 708	3650 ± 2213	2704 ± 670	3510 ± 1160	3816 ± 988 *	2386 ± 270	2452 ± 462
Leaves	598 ± 162	470 ± 387	759 ± 554	1558 ± 759	1469 ± 235	1567 ± 647	1559 ± 248	1268 ± 354	1125 ± 215
Total	1388 ± 440	1118 ± 691	1915 ± 1269	6191 ± 3275	4838 ± 724	5799 ± 1884	6249 ± 1487	4523 ± 559	4198 ± 631

* represents a significant difference between the different fertilizers tested among the same organ and age at a significant level of p < 0.05.

A). The highest biomass yield from leaves (492 ± 91 g/m²) was found in plants of age 1, treated with fertilizer I. The highest biomass yield from bulbs (712 ± 105 g/m²) was found in plants of age 2 grown with fertilizer II. The highest biomass yield from whole plants was found for plants from age 1 grown with fertilizer I (1226 ± 580 g/m²) and it was comparable with those of plants of age 2 grown in fertilizer II (1217 ± 136 g/m²). The biomass distribution of the roots, bulbs, and leaves was 6.3%, 48.4%, and 45.4% for age 0, 7.6%, 53.4%, and 38.9% for age 1, and 9.5%, 59.0%, and 31.5% for age 2, respectively.

The yield of galanthamine per square meter at the end of vegetation was calculated based on plant density and the dry weight of the plant organs and their galanthamine content (Table 2B). The highest value for the galanthamine yield from roots (983 ± 405 mg/m²) was found in plants from age 1 grown with the fertilizer I. The highest galanthamine content in leaves (1567 ± 647 mg/m²) was found in plants of age 1, treated with fertilizer III. The highest galanthamine yield from bulbs (3816 ± 988 mg/m², significant difference) and whole plants (6249 ± 1487 mg/m²) were found in plants of age 2 grown with fertilizer I. The galanthamine yield from the whole plants of age 1 grown in fertilizer I is 6191 ± 3275 mg/m².

3.2. Dynamics of Galanthamine and Biomass Accumulation

The dynamics of galanthamine and biomass accumulation in H. papilio plants of age 1 from the beginning of May until the end of October are presented in Figure 4.

The results showed that the dormant bulbs have the lowest galanthamine content (4.3 ± 0.4 mg/g DW). A month after the beginning of growth, the galanthamine content in the bulbs reached the highest values (5.9 ± 1.0 mg/g DW) and then gradually decreased until September. At the end of vegetation in October, the galanthamine content in the bulbs was slightly increased to 5.3 ± 0.8 mg/g DW (Figure 4A).

The highest galanthamine content (6.2 ± 1.0 mg/g DW) in the leaves was found two months after the beginning of cultivation when it gradually decreased until the end of vegetation season, reaching 3.7 ± 0.5 mg/g DW. The dynamics of galanthamine content in the roots followed that of the bulbs, reaching the highest level at the end of the vegetation season (11.2 ± 2.8 mg/g DW).

The biomass accumulation of bulbs showed a trend similar to that of galanthamine accumulation (Figure 4B). During the first months of cultivation, the bulbs’ dry biomass increased from a mean of 2.0 ± 1.0 g to 4.8 ± 0.3 g, reaching 5.0 ± 1.0 g at the end of the vegetation season, which is an increase of 2.5 times. For comparison, the bulbs of age 2 increased their biomass 2.2 times (from 6.6 ± 2.0 g to 14.2 ± 2.1 g).

The biomass of the leaves of plants from age 1 increased during the vegetation season to a mean of 4.0 ± 1.0 g of DW per plant, showing the most intensive growth in August-September. For comparison, the mean leaf dry biomass of plants from ages 0 and 2 reached 0.4 ± 0.2 g and 7.8 ± 1.1 g, respectively.

Like the bulbs, the roots grew intensively during the first months of cultivation, and their biomass remained relatively constant until the end of the vegetation season.

4. Discussion

The plants of age 0 started their growth almost immediately after their transfer at the greenhouse, while the bulbs from age 1 and 2 came out of dormancy two weeks after the planting when the root growth started. The leaf growth of the plants of ages 1 and 2 started about a month after the planting. In general, the development of the plants at age 2 was slower than that of age 1.

From a practical point of view, plants of ages 1 and 2 should be considered as raw material for galanthamine extraction due to their bigger size (Figure 3B). The galanthamine content has been found to vary in the different parts of the plant organs of H. papilio (e.g., inner, middle, and outer parts of the bulb) [9]. In the present study, the values of the galanthamine content are representative for the whole plant organs and indicate that H. papilio is the richest natural source of galanthamine with 5.3 ± 0.8 mg/g DW in the bulbs and 3.7 ± 0.5 mg/g DW in the leaves at the end of vegetation season in certain conditions.

The results showed that the effect of age on the galanthamine content depends on the fertilizers. The bulbs from plants of age 1 grown with fertilizers II and III accumulated 55.9% (significant difference) and 34.1% more galanthamine as compared with the respective bulbs of age 2 (Figure 3A, Supplementary file, Figure S6). Similarly, the roots of plants from age 1 grown with fertilizers I and II have significantly higher galanthamine content as compared to those of age 2. These results are in line with the study of Akram et al. [14] who found that the age of the bulbs of N. pseudonarcissus cv. Carlton is an important factor affecting the industrial production of galanthamine. The authors have found that two-year-old bulbs have higher galanthamine content as compared with one- and three-year-old ones, which in certain conditions may lead to an increase in the yield of 10–15%. Likewise, Chang [6] reported that the highest galanthamine content in bulbs of L. radiata is after the third year of cultivation.

The galanthamine content in H. papilio was influenced by the fertilizers. The fertilizers used in this study differ in the content of nitrogen (N), potassium (K), and calcium (Ca). Fertilizer I was used as the basic nutrient solution while fertilizer II was formulated with 33% more N, 143% more K, and 11% more Ca. Fertilizer III had 111% more N, 143% more K, and 39% more Ca as compared to fertilizer I [25]. The difference between fertilizers II and III is in the content of N and Ca. The fertilizers did not show significant effects on the galanthamine content in plants of age 1, while fertilizer I was found to be optimal for the bulbs and leaves of the plants of age 2 (Figure 3A, Supplementary file, Figures S8 and S9) where the application of fertilizers with higher concentrations of N, K, and Ca showed a tendency to decrease the galanthamine content in the plant organs. The tendency for negative effects of fertilizers II and III is clearer considering the galanthamine yield (Table 2B), where fertilizer I showed the best results with an exception for the leaves of plants grown with fertilizer III. Similarly, Ullrich et al. [18] reported decreased levels of scopolamine in Duboisia species when a higher supply of N was applied. Luanratana and Griffin [27] also found decreased alkaloid yields by applying fertilizers with higher N content in a hydroponic culture of Duboisia hybrid. Lubbe et al. [3] observed a higher level of amino acids than alkaloids in bulbs from N. pseudonarcissus cv. Carlton, which is cultivated with double N fertilization, indicating that primary metabolism is dominant.

In contrast to the plants of ages 1 and 2, the plants of age 0 showed a tendency of having increased galanthamine yield when the plants were grown with fertilizer III (Table 2B), which may be explained by the different physiological status of the young plants.

The application of higher amounts of K, N, and Ca did not significantly influence the dry biomass per plant while the age logically affected it (Figure 3B). The bulbs from plants of age 2 have about two times more biomass as compared to the bulbs from age 1. Similarly, the leaves of plants from age 2 have considerably more biomass as compared to the leaves from plants of age 1.

Considering the yield of galanthamine from H. papilio per square meter of horizontal hydroponic culture (Table 2), the plants of age 1 and those of age 2 showed comparable results, indicating that the biomass production of the smaller plants from age 1 is compensated by the increased plant density. This also indicates that despite the higher biomass of the plants from age 2, the plants from age 1 are preferable for biomass production in hydroponic cultures because of the reduced time and costs for cultivation. From the tested fertilizers, fertilizer I was found to be optimal for the hydroponic cultivation of H. papilio for galanthamine production. Depending on the fertilizer and age, the bulbs are the plant organ, where between 53% and 61% of the galanthamine was stored. The leaves, however, accumulated between 25% and 30% of the whole galanthamine amount, indicating that they are also attractive for galanthamine extraction. Roots also stored considerable amounts of galanthamine, between 13% and 19%. Further increases in biomass and galanthamine yield can be achieved by vertical hydroponic systems and manipulation of alkaloid biosynthesis. Hydroponic cultivation is an interesting alternative to field cultivation because it can provide some advantages characteristic of bioreactors such as control of plant nutrition and environmental parameters (e.g., temperature, light, humidity, etc.) allowing for the manipulation of plant growth and metabolism [22,23,24]. Other advantages of hydroponic cultures, especially those in a controlled environment, are associated with reduced consumption of water (70–90%) and biocides (for pest control), year-round production of biomass, etc. [22,24].

In difference to the other well-studied sources of galanthamine, such as the European N. pseudonarcissus cv. Carlton and L. aestivum, H. papilio is an ever-green tropical plant species and it is of interest to study the seasonal variation in its biomass and galanthamine accumulation to determine the best time-point for harvesting. The fact that the plant is tropical, however, limits its field cultivation in Europe. In the present study, the dynamics of alkaloid and biomass accumulation were studied in plants of age 1 grown with the fertilizer I due to the similar yields to those of the plants of age 2 and the advantage of a shorter total cultivation period. The galanthamine content in the bulbs from age 1 of H. papilio rapidly increased in the first 2 months to the highest level, which can be explained by the start of the alkaloid biosynthesis most probably in the inner part of the bulbs comprising young leaves [9]. The galanthamine content in the leaves of H. papilio was highest at the beginning of growth and declined with the augmentation of their biomass during the vegetation season. The increase in the galanthamine content and biomass of the bulbs at the end of the vegetation season coincides with the stunted leaf growth and rapidly decreasing leaf galanthamine content indicating the transfer of alkaloids and nutrient compounds from the aerial parts to the bulbs. Similar alkaloid dynamics have been observed for the bulbs of N. pseudonarcissus cv. Carlton, which showed a maximum of galanthamine content before flowering in April, which decreased after flowering yet had a slight increase to about 2.5 mg/g DW at the end of the vegetation season. The galanthamine content in the leaves of this daffodil was stable (less than 2 mg/g DW) during the vegetation and dropped in senescent leaves in June at the end of the vegetation season [13].

5. Conclusions

The main conclusions of this study are that H. papilio accumulates superior quantities of galanthamine in its bulbs and leaves than the other actual plant sources of this valuable alkaloid (e.g., N. pseudonarcissus cv. Carton). The highest galanthamine content in the plant organs was found at the beginning of vegetation, but the end of vegetation season is the best time for harvesting the plant biomass for galanthamine extraction. The main part (53–61%) of galanthamine is stored in the bulbs. The plant is evergreen (in contrast to N. pseudonarcissus cv. Carton) and the leaves and roots can be also used for galanthamine extraction as they store considerable amounts of this alkaloid, specifically 25–30% and 13–19%, respectively. Fertilization and age affect galanthamine biosynthesis. Higher amounts of N, K, and Ca in the nutrient solution do not positively influence the alkaloid biosynthesis and biomass. Despite the lower biomass accumulation, the plants grown for two seasons (age 1) yield comparable amounts of galanthamine per square meter as those grown for three seasons (age 2) due to their higher density of cultivation. Further field experiments are necessary to determine the most suitable plant age and density for cultivation that could provide optimal galanthamine yield. Hydroponic cultivation of H. papilio is an interesting alternative for the production of galanthamine, which can be optimized in further research on plant density (vertical farming), nutrient solution formulation and electroconductivity, lightening (intensity and quality of light), humidity, etc. Studies on the expression of genes involved in galanthamine biosynthesis could provide an explanation of the driving mechanisms to optimize its production.