In vitro micropropagation and alkaloids analysis by GC–MS of Chilean Amaryllidaceae plants:

Rhodophiala pratensis

Abstract
Introduction: Plants from Amaryllidaceae family are of interest since they produce a particular type of
alkaloid useful for the treatment of neurodegenerative diseases of the central nervous system, such as
Galanthamine. Given the low content of these secondary metabolites in the plant, it is necessary to study
mechanisms to increase the productivity of them.
Objective: To obtain fast qualitative and quantitative analysis of the alkaloids and extend the understanding
of biosynthesis and metabolism in these kinds of plants. Furthermore, establish a reliable, simple and fast
analytical method for the in vitro callus culture of vegetative organs for Rhodophiala pratensis species.
Methods: The alkaloids composition of the callus culture of R. pratensis were analysed by gas
chromatography coupled with mass spectrometry (GC–MS).
Results: A methodology for the qualitative and quantitative analysis of the alkaloids present in fresh callus
culture of this wild plant species was established. The analysis showed alternation in the alkaloids type ratio
and number of compounds between wild bulbs, in vitro bulbs and callus. It was possible to identify 24
alkaloids from a pool of 60 signals whose fragmentation pattern corresponds to the alkaloids of
Amaryllidaceae plants. Together with the aforementioned, the amount and type of alkaloid present in the
plant material obtained by in vitro culture of R. pratensis was determined in the same way. The results show
the high biosynthetic potential of in vitro grown bulbs and callus tissue that are able to produce significant
amounts of pharmacologically relevant alkaloids from R. pratensis in various proportions that depend on
the culture conditions such as supplementation with growth substances. The in vitro grown bulbs produce
an alkaloidal extract that contain a 52.6% w/w of alkaloids.
Conclusion: This study allowed the alkaloid content in callus culture of R. pratensis to be found by means
of GC–MS. These results allowed a relationship between the type of growth regulator and the type of
alkaloids found to be established. Finally, we can say that the results achieved to state that the production
of alkaloids using different combinations of growth regulators could be directed during in vitro
micropropagation from provided plant material.

1 | INTRODUCTION
Plants from Amaryllidaceae family include about 1600 species in 73 genus distributed largely over
tropical and subtropical regions. Particularly in Chile 35 to 45, native and introduced species have been
described. This family has produced a large number of structurally diverse alkaloids with a wide range of
biological activities such as antitumor, antiviral, acetylcholinesterase inhibitors, antimalarial and
hypotensive activities. Some of these alkaloids are of particular interest because of their potential use in
clinical therapy, particularly in Alzheimer's disease.
Knowledge about the chemical composition of endemic species of Chilean Amarillidaceae is
scarce. One of the native species studied corresponds to Rhodophiala ananuca (Phil.) Traub & Uphof (=
Hippeastrum ananuca). The alkaloids lycorine, 17‐epi‐homolicorin, homolycorine, 11‐hydroxy‐1,2‐
dihydromaritidine, maritidine, hippeastidine and haemantamine were isolated from individuals of this
species and identified by spectroscopy and X‐ray diffraction techniques.
Recently, Tallini et al., reported the identification of 37 alkaloids from five species of the
Rhodophiala genus that grow in Chile as in other passages of South America.
However, it is known that the content of alkaloids in these plants is very low, which hinders the
biological study of these class of compounds. In order to improve the production of Amarillidaceae
alkaloids for therapeutically relevant applications, two approaches have been proposed. The first one, total
chemical synthesis, is not very effective and very complex. The second one uses plants or in vitro culture
of some Amarillidaceae species. Literature data have reported that contents of these alkaloids in these
plants, particularly galanthamine, oscillate between 0.01 and 2.0% dry weight (DW). To the earlier
mentioned is added the fact that the multiplication of bulbous plants from seeds will show wide variability
in morphological features, whereas separation of bulbs is a very slow process for obtaining a large number
of plants in a short time. Normally, a plant produces two to three bulbs in a year of growth. A more
acceptable method for the rapid propagation of the bulbous plants with stable morphological features would
be micropropagation. Cell cultures provide a viable system for the production of these compounds; the
performance can be modulated by using different combinations of auxins and cytokinins. The use of illicit
drugs should also be considered, which according to the literature could stimulate the production of specific
secondary metabolites, which are concentrated in the biomass produced. In this context, we hypothesise
that in vitro culture of R. pratensis offer stable production of alkaloids with higher productivities than plant
in nature. In order to test this hypothesis, the aims of the present study were: (1) to obtain fast qualitative
and quantitative analysis of the alkaloids and extend the understanding of biosynthesis and metabolism in
these kinds of plants; (2) to quantify alkaloids in callus culture of R. pratensis by gas chromatography
coupled with mass spectrometry (GC–MS) procedure; (3) to quantify alkaloids in cell that had been
subcultures in vitro with different proportions of vegetable hormone; (4) to quantify alkaloids in plants
produced by microprogation; (5) compare the productivity to plants in nature with those presented
by in vitro systems.

2 | EXPERIMENTAL
2.1 | Material, chemicals and reagents
 All the chemicals and solvents were obtained from Merck (Darmstadt, Germany). Thin layer
chromatography (TLC) was carried out on precoated Silica Gel 60 GF254 plates (Merck). Spots on
chromatogram were visualised under ultraviolet (UV) light and by spraying with 5% sulphuric acid
(H2SO4) in methanol (MeOH), and then heating at 110°C for 5 min. 2,4‐Dichlorophenoxyacetic acid (2,4‐
D), indole acetic acid (IAA), 6‐benzylaminopurine (BAP), Agar and Murashige and Skoog basal medium
were purchased from Sigma (St Louis, MO, USA).
2.2 | Plant material
Rhodophiala pratensis bulbs about 2.5 cm diameter and 2.0 g of weight were collected during the
month September of 2017 from Hualpen city (36°47′S–73°10′W), VIII Region del Bio‐Bio, Chile. The
different parts of the plant were separated, washed with tap water, cut into small pieces of approximately 3
cm and lyophilised for later extraction of alkaloids and analysis by GC–MS. However, bulbs were cut into
eight parts after removal of roots, leaves, and dry outer scales. Segments were then separated, which were
washed under running tap water, then they were immersed in sodium hypochlorite solution at 4% for 10
min, then transferred to ethanol solution at 70% for 10 s, and then were washed three times with sterile
water.
2.3 | In vitro plant material establishment
Bulbs were cut longitudinally in eight parts, obtaining double scale explants 0.4–0.6 cm wide and
0.7–1.3 cm long joined by a thin segment of the basal plate obtaining 16 explants from each bulb. All
explants were cultured in petri dishes with 15 mL of Murashige and Skoog culture medium supplemented
with 3% sucrose, containing IAA and BAP as regulators of growth. Five combinations of IAA (10,
15, 10, 10, 20 μM) were tested with BAP (5, 5, 10, 15, 10 μM), seeding 16 explants in each experiment.
The pH of the medium was adjusted to 5.5 with 1 M sodium hydroxide (NaOH) or 1 M hydrochloric
acid (HCl), solidifying with 9.0% agar and autoclaved at 120.0°C, 117.679 kPa for 20 min. All explants
were incubated at a temperature of 25 ± 1°C with a photoperiod of 12 h and a relative humidity of 80%.
The explants obtained in the induction of calluses (Figure 1) were sub‐cultured individually in
culture flasks (100 mL) with 30 mL of Murashige and Skoog culture medium supplemented with 3%
sucrose, with 2,4‐D and BAP in different concentrations. Five combinations were tested: 2,4‐D (9, 12, 14,
16, 18 μM) and BAP (10 μM), culturing eight explants for each experiment (Table 1).
2.4 | In vitro regeneration of the plant
The stabilisation of the explants obtained (Figure 2) was carried out considering the optimal
concentrations of the growth regulators used in the formation and multiplication of the explants. Three
combinations were selected: control culture C0 (10 μM IAA/10 μM BAP), culture C1 (16 μM 2,4‐D/10 μM
BAP) and culture C2 (16 μM 2,4‐D/10 μM IAA/10 μM BAP). In each experiment, six explants were
cultured for 8 weeks. At the end of the cultivation period, the metabolites were lyophilised and extracted,
evaluating the isoquinolic alkaloid content by GC–MS (Table 1).
2.5 | Extractions of alkaloids
Bulbs, leaves, roots and callus were extracted separately with MeOH. The combined methanolic
extracts were evaporated, dissolved in 10 ml of 2% H2SO4 and filtered. The acidic solution was defatted
with diethyl‐ether (3 × 10 mL) and the basified with 25% ammonium hydroxide (NH4OH). The alkaloids
were extracted with chloroform (3 × 50 mL) and the solvent evaporated under a stream of nitrogen. The
dried alkaloids mixture was re‐dissolved in MeOH (5 mg of alkaloids extract/250 μL of MeOH) for further
analysed using GC–MS analysis.
2.6 | Gas chromatography–mass spectrometry (GC–MS) analysis
The analysis by GC–MS was developed in an Agilent 7890 A GC (Agilent, Palo Alto, CA, USA)
with multimodal injector, Agilent triple Quad 7000 GC/MS detector (SCAN analysis by a quadrupole). An
HP‐5 MS capillary column (30 m × 0.250 mm × 0.25 μm, Agilent) was used. The temperature programme
was: 100–180°C at 15°C/min, 180–300°C at 5°C/min and 10 min hot at 300°C. The injection was carried
out at 250°C. The flow of carrier gas (helium) was 0.8 mL/min. Furthermore, 1 μL of alkaloid extract was
injected in to the GC–MS directly. Codeine was used as internal standard.
Kovats retention index (RI) of the compounds was recorded with a standard calibration n‐
hydrocarbon mixture (C8–C32). The percentage of the total ion current (TIC) for each compound is given
in
Table 2. The compounds were identified by comparing their mass
spectral fragmentation with standard reference spectral using mass library (NIST 2.0). Moreover, data
obtained from the literature were used for the identification of the compounds. The proportion of each
compound in the tested extracts was connoted as the percentage of the total alkaloid content. The area of
the GC–MS peaks depends not only on the concentration of the related compounds but also
on the intensity of their mass spectral fragmentation. Hence, percentages shown in Table 2 are semi‐
quantitative, but can be used for a relative comparison of the alkaloids between different
treatments.

3 | RESULTS AND DISCUSSION

3.1 | In vitro plant material
Twin scale of bulbs as indicated in the methodology were cultivated in Murashige and Skoog
medium, containing five combinations of auxin and cytokinins, observing the callus formation in the BAP
(10 μM)/ IAA (10 μM) combination. The callus obtained were grown in Murashige and Skoog medium
containing a mixture of 2,4‐D (16 μM) and BAP (5 μM), achieving with this combination the formation of
bulbs and their growth. A third hormonal combination formed by IAA (10 μM), BAP (5 μM) and 2,4‐D
(16 μM), allowed to increase the number of bulbs formed, achieving an average of six bulbs per
explants (Figures 1 and 2).

FIGURE 1 In vitro culture of Rhodophiala pratensis. (A) Induction of callus; (B) outbreak formation; (C)
regeneration of the plant; (D) multiplication of the plant [Colour figure can be viewed at
wileyonlinelibrary.com]

Alkaloid in the alkaloidal fraction. Note: IAA, indole acetic acid; BAP, 6‐benzylaminopurine; 2,4‐D, 2,4‐
dichlorophenoxyacetic acid.

FIGURE 2 Effect of different combinations of growth inducers in the production of alkaloids. C0, control
culture (10 μM IAA/10 μM BAP); C1, culture with 16 μM 2,4‐D/5 μM BAP; C2, culture with 16 μM 2,4‐
D/10 μM IAA/5 μM BAP (IAA, indole acetic acid; BAP, 6‐benzylaminopurine; 2,4‐D, 2,4‐
dichlorophenoxyacetic) [Colour figure can be viewed at wileyonlinelibrary.com]

3.2 | Alkaloid profiles

Following the methodology described, the alkaloid extract of leaves, stem, bulbs and roots from
wild plants were prepared, and their alkaloid content was determined by GC–MS (Figure 3) for each of the
parts under study (Table 2). In the same way, we worked with bulbs obtained by in vitro culture. The
chromatographic analysis allowed to investigate the presence of 34 alkaloids, identifying 24 of them
(Scheme 1). The roots and bulbs have a greater number of alkaloids, followed by stems, and a smaller
amount in the leaves. GC–MS was used for the identification and determination of the different groups of
isoquinolinic alkaloids of Amaryllidaceae. In our study, quantification was done relative to the standard of
codeine, since standards of these kinds of alkaloids were not available in our laboratory. Galanthamine is
routinely used as standard, however in our samples its concentrations was very low. However, other
alkaloids such as haemanthamine, that appears in the samples studied in this work, present serious problems
of thermal‐decomposition in GC, so its quantification is not reliable. Therefore, our work followed the
conditions described previously, which used the areas of different alkaloids and standardised with the area
of codeine (internal standard).
Previous studies in species of the Rhodophiala genus have reported the presence of 37 alkaloids
characteristic of the family Amaryllidaceae and 40 compounds corresponding to alkaloids, which according
to their fragmentation patterns would present a skeleton of the lycorine type, haemanthamine, tazettine,
homolycorine, galanthamine, montanine and narciclasine. Our study contributes to the knowledge of the
alkaloids present in the species R. pratensis, which had not been included in the previously reported studies.
When comparing the amount and type of alkaloids obtained from both wild bulbs and those
obtained by in vitro culture, it can be observed that wild plants have 34 alkaloids whereas in vitro bulbs
show only 12. Of the total alkaloids detected, these are distributed differently in the different parts of the
plant, observing a large number of alkaloids in the bulbs (Table 1). These alkaloids are derived from the
following groups: lycorine, haemanthamine, homolycorine and tazettine, most of which belongs to the O‐
methyllycorine group.
Nevertheless, the bulbs obtained in vitro from the culture of callus supplemented with appropriate
concentrations and the combination

FIGURE 3 GC–MS of the alkaloid fraction obtained from (A) wild bulbs and in vitro culture, (B) IAA/BAP,
(C) IAA/BAP/2,4‐D and (D) BAP/2,4‐D of Rhodophiala pratensis. The numbers of the peaks correspond
to the compound numbering shown in
Table 2. Internal standard (IS): codeine; IAA, indole acetic acid; BAP, 6‐benzylaminopurine; 2,4‐D, 2,4‐
dichlorophenoxyacetic

SCHEME 1 Biosynthetic relationships between the different alkaloid types identified in Rhodophiala
pratensis of growth regulators resulted in a lower number of alkaloids (Table 1).
Thus, the alkaloid extract obtained from calluses grown with the combination IAA (10 μM)/BAP
(10 μM) contains only 12 alkaloids with a mass of 52.66%, w/w. The combination IAA (10 μM)/BAP (10
μM)/ 2,4‐D (16 μM) produces an alkaloid extract containing 10 alkaloids with a mass of 44.13% w/w.
However, the combination 2,4‐D (16 μM)/BAP (10 μM) contains 11 alkaloids, but with a total
mass of 23.44% w/w. Unlike what is observed in the wild bulbs, alkaloids obtained from bulbs produced in
vitro are formed by an arrangement ortho‐para′ (o‐p′) and p‐p of the precursor O‐methylnorbelladine.
Therefore, we find alkaloids of lycorine, homolycorine, haemanthamine, tazettine, and narciclasine type.
Galanthamine‐type alkaloids are not present, which is explained because they are formed by arrangement
p‐o′ of precursor Omethylnorbelladine (Scheme 2). This may be due to the effect of plant growth regulators
(PGRs), such as auxin and cytokinins used in this report. It is known that PGRs have effects not only on
cell differentiation and proliferation but also on the pathway of cell biosynthesis.

For instance, 2,4‐Dinduces callus formation when it is used in combination with auxins because it
promotes DNA synthesis and mitosis. The research shows that the method used allows a good number
of bulbs to be obtained quickly and inclusion of different combinations of growth regulators may
significantly influence the alkaloid biosynthesis which makes it a suitable route for alkaloid production.
The results found for R. pratensis correlate with the results of micropropagation in vitro and the production
and identification of alkaloids in explants by GC–MS for the species of Amaryllidaceae Leucojum aestivum
and Galanthus transcaucasicus.
ACKNOWLEDGEMENTS
This work was jointly supported by the CONICYT‐FONDECYT N°1130463, CONICYT
FONDECYT N° 1161157 and CONICYT FONDECYT 1150948 and CONICYT FONDEQUIP 150025
grants.
The authors greatly appreciate the internal grants from the Dirección de Investigación, Universidad
del Bio Bio, Chillan, Chile: DIUBB # 083009‐2R, # 122509 and # 132209 GI/C grants. These funders
played no roles in the study design, data collection and analysis, and decision to publish.