OBTAINING AND SELECTION OF PANCRATIUM MARITIMUM L.

IN VITRO
CULTURES WITH ACETYLCHOLINESTERASE INHIBITORY ACTION

V. Georgiev, I. Ivanov and A. Pavlov

The Stephan Angeloff Institute of Microbiology, Department of Applied Microbiology – Laboratory in Plovdiv,
Bulgarian Academy of Sciences, Plovdiv, Bulgaria
Correspondence to Vasil Georgiev
E-mail: vasgeorgiev@gmail.com

ABSTRACT
Two types of plant in vitro systems - calli and shoot cultures were obtained from ovaries of sea daffodil (Pancratium
maritimum L.) – a Bulgarian threatened plant belonging to the Amaryllidaceae family. Using TLC method, ten different
alkaloids were separated in extracts from in vitro cultures, as well as their abilities to inhibit the acetylcholinesterase (EC.
3.1.1.7) were evaluated by the same techniques. All investigated lines from both type in vitro systems exhibited high levels of
somaclonal variability concerning the number and the levels of alkaloids produced. However, the callus cultures produced
alkaloids in significantly lower levels compared to the shoot cultures. Two shoot lines (Н6L7 and Н6L6) producing four
alkaloids with high acetylcholinesterase inhibitory action (with Rf values of 0.47, 0.43, 0.25 and 0.12) they were selected as
prospective source for further screening of new acetylcholinesterase inhibitors.

Keywords: Amaryllidaceae alkaloids, Acetylcholinesterase
inhibitors, Pancratium maritimum L., TLC
Introduction
The sea daffodil (Pancratium maritimum L.) is a perennial
Mediterranean dune plant belonging to the Amaryllidaceae
family. As consequence of the steadily increasing urban
expansion and other human activities, the species intensively
lost their natural habitats and was characterized as
endangered and protected in Bulgaria (3) and Greece (8), and
recently, it was classified as threatened in France, Spain, Italy
and Turkey (7) and vulnerable in Lebanon (9). Moreover, the
recent studies showed that the P. maritimum L. populations
management and restorations were embarrassed because of
the suggestion that the species is self-pollinated (9) as well as
by the higher seedling mortality (only 3% of the seeds
resulted in established seedlings) (1). This revealed the
necessity for developing protocols for in vitro propagation.
Sea daffodil attracts great interest by researchers because
of the produced alkaloids, some of which possessing valuable
pharmaceutical properties (3, 4).
It is well known that alkaloids synthesized by plants from
Amaryllidaceae family are important acetylcholinesterase
inhibitors (2). The development of in vitro techniques for
screening and producing of these alkaloids is important
technological step by the mean to prevent the natural
populations lost as well as to ensure the continuous supply of
these substances to pharmacy. Up to now, there are scanty
data concerning the alkaloid production by P. maritimum L.
in vitro cultures (4, 5) and such data for in vitro production of
compounds with acetylcholinesterase inhibitory action by P.
maritimum L. were not found in accessible scientific
literature.
In the present study we report the effective protocol for
obtaining of P. maritimum L. callus and shoot cultures
producing amarillidaceae alkaloids. The fast procedure for
screening and evaluation of acetylcholinesterase inhibitory
action of produced alkaloids, based on TLC assay was
developed and applied to select lines with higher biosynthetic
potential of acetylcholinesterase inhibitors.

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Materials and Methods TABLE 1
Combinations of growth regulators used for initiation of
Plant material different in vitro cultures by Pancratium maritimum L. plant
Young epigeous parts of Pancratium maritimun L. plants
were collected from their natural habitat located near to the Combination Cytokinins, mg/L Auxins, mg/L
camping site Kavatsite, Silistar Beach on the Bulgarian Black number Kinetin BAP NAA 2,4 D
See coast. For the experiments, the steams and the young 1 0.5 0.0 0.0 0.5
fruit from one cluster were cut and surface sterilized with 2 0.5 0.0 0.0 1.0
70% ethanol for 20 sec followed by sterilization in 20% 3 0.5 0.0 0.0 2.0
Domestos bleach solution (www.unilever.com) for 20 min. 4 0.5 0.0 0.0 4.0
The sterilized explants were rinsed by sterile distilled water 5 1.0 0.0 0.0 0.5
(triplicate), dried on sterile filter paper and after removing of 6 1.0 0.0 0.0 1.0
death parts were cut in pieces and used for the next initiation 7 1.0 0.0 0.0 2.0
of in vitro cultures. 8 1.0 0.0 0.0 4.0
9 2.0 0.0 0.0 0.5
Obtaining of in vitro cultures
10 2.0 0.0 0.0 1.0
The calli and the shoots formation was initiated by
11 2.0 0.0 0.0 2.0
transferring of the sterile explants on MS media, supplied
12 2.0 0.0 0.0 4.0
with 3% sucrose (Duchefa, The Netherlands), 5.5 g/L “Plant
agar” (Duchefa, The Netherlands) and different combinations 13 0.0 0.5 0.0 0.5
of auxins and cytokinins, presented in Table 1. The 14 0.0 0.5 0.0 1.0
cultivation was carried out at 26°C, in darkness or under 15 0.0 0.5 0.0 2.0
illumination program including 16 hours exposure on light 16 0.0 0.5 0.0 4.0
and 8 hours on darkness. The subcultivation period of the 17 0.0 1.0 0.0 0.5
explants, as well as of the obtained in vitro cultures was 28 18 0.0 1.0 0.0 1.0
days at the same conditions. 19 0.0 1.0 0.0 2.0
20 0.0 1.0 0.0 4.0
Extraction of alkaloids
21 0.0 2.0 0.0 0.5
The alkaloids, produced by investigated in vitro lines were
22 0.0 2.0 0.0 1.0
analyzed after their extraction from 100-200 mg or 200-500
23 0.0 2.0 0.0 2.0
mg lyophilized biomass for shoot and callus cultures,
24 0.0 2.0 0.0 4.0
respectively. The extraction procedure includes one step of
25 0.0 1.0 0.0 0.2
16 hour extraction in 5 ml methanol, followed by two times
26 0.0 2.0 0.0 0.2
extractions in 5 ml methanol for 30 min. Combined methanol
27 0.0 3.0 0.0 0.2
extracts were evaporated to dryness on 55°C and the residue
28 0.0 2.0 1.15 0.0
was dissolved in 2x2 ml of 3% Sulphuric acid (pH of the
solution is around 2). The neutral compounds were removed 29 0.0 5.6 0.2 0.0
by extraction (tree times) with 5 ml of diethyl ether. The 30 2.0 0.0 0.2 0.0
alkaloids were fractionated after basification of the extracts 31 3.0 0.0 1.0 0.0
with 1.0 ml of 25% ammonia (pH of the solution is around 32 2.0 0.0 0.0 0.2
11) and extracted (3 x 3 ml) with chloroform. The chloroform 33 3.0 0.0 0.0 1.0
extracts were dried over anhydrous sodium sulphate and 34 0.0 1.0 0.1 0.0
evaporated to dryness. 35 0.0 1.0 1.0 0.0
36 0.0 2.0 1.0 0.0

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 The alkaloids from the dry residues were transferred spots of alkaloids with acetylcholinesterase inhibitory actions
quantitatively with 1.2 ml methanol in Eppendorf tubes and remain uncoloured and their areas were proportional to their
were dried for 8 hours on 60°C. The obtained dry alkaloids inhibitory activities.
mixtures were used for the next analyzes.

TLC alkaloids assay Statistic analyzes

The dried alkaloid mixtures were dissolved in 100 μL All data were collected from two independent analyzes and
methanol and 5 μL or 20 μL of extracts (for shoots or calli, the results are presented as mean values. Data was analyzed
respectively) were dropped on Silicagel G60 TLC plate by unpaired t-test with a level of significance of P≤0.05 by
(ALUGRAM® SIL G Silica gel 60, Macherey-Nagel, SigmaPlot v.7.0 software (Systat Software, Inc., USA).
Germany). The galanthamine standard (Galantamine
hydrobromide, Sigma-Aldrich, Germany) was spotted in
concentrations of 2, 5, 10 and 20 μg on the same plate as Results and Discussion
well. The chromatogram was developed by using Chloroform One of the biggest problems, concerning plant in vitro
: Methanol : 26% Ammonia (in proportion of 12 : 1 : 0.1) as cultures iniciation from dune plants is the sterilization of the
mobile phase, as well as Dragendorff reagent for spots plants. Growing deeply in the low content of organic matter
visualization. The alkaloid contents were quantified and nutritional substances sand, Sea daffodil was in close
densitometry using QuantiScan® software (BioSoft, relations with many symbiotic micro-organisms, which poses
Cambridge, UK). The amounts of the detected alkaloids were great difficulties with sterilization and increase the risk of
expressed as GAL equivalents, using galanthamine standards explants contamination (7; 8). To avoid this risk we used
to build the calibration curve. steams and the young fruits for initiation of in vitro systems
by P. maritimum L. These organs were located above the soil
and easily survived during sterilization procedure, giving
TLC acetylcholine esterase inhibitory actions assay
large number of aseptic explants. These explants were
The assay was performed according tio the method discribed
transferred on the MS media with combinations of auxins and
by Marston et al. (6), modified as follow: The dried alkaloid
cytokinins presented in Table 1. The experiments were
mixtures were dissolved in 100 μL methanol and 2 μL of
carried out with equivalent number of explants cultivated in
them were spotted on previously washed with acetone and
darkness and under illumination (16h light/8h darkness) as
dried Silicagel G60 TLC plate (ALUGRAM® SIL G Silica
well. The first calli appeared on day 35 of the cultivation in
gel 60, Macherey-Nagel, Germany). The galanthamine
darkness on MS media supplied with 2.0 mg/L BAP and 4.0
standard (Galantamine hydrobromide, Sigma-Aldrich,
mg/L 2,4D (Combination number 24, Table 1) or 2.0 mg/L
Germany) was spotted in concentrations of 2, 4 and 5 μg on
BAP and 1.0 mg/L 2,4D (Combination number 22, Table 1).
the same plate as well. The plate was developed by mobile
The calli were white-yellow colored, with friable structure
phase of Chloroform : Methanol : 26% Ammonia (12 : 1 :
and showed stable growth characteristic.
0.1) for alkaloids separation and after drying, it was
immediately sprayed with enzyme solution First shoot appeared on day 48 of the cultivation under
(Acetylcholinesterase EC. 3.1.1.7, Sigma-Aldrich, Germany illumination on MS medium supplied with 1.15 mg/L NAA
– 500U in 150 ml of 0.05 M Tris-HCl buffer with pH=7.8, and 2.0 mg/L BAP (Combination number 28, Table 1). The
supplied with 150 mg bovine serum albumin - Sigma- separated shoots were stable and possess fast growth and
Aldrich, Germany) and dried on could air. The dried plate event formed small in vitro plants after several
was incubated at 37°C, under high humidity, for 20 min and subcultivations.
then was sprayed with freshly prepared mixture of one part 1- It should be noted, that the both calli and shoot cultures
naphthyl acetate (250 mg in 100 mL ethanol, Sigma-Aldrich, raised from explants of young fruits, as well as that the steam
Germany) and four parts Fast Blue B Salt (250 ng in 100mL explants did not form any in vitro cultures on all tested media
dH2O, Fluka, Switzerland), coloring the plate in purple. The combinations.
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TABLE 1
TLC of alkaloid fractions of P. maritimum L. in vitro systems and evaluation of their acetylcholinesterase inhibitory
actions
Culture Line Number on Alkaloid Rf Amount, Acetylcholinesterase
type plate (Position, according to value g GAL eq/g inhibitory action**
Fig. 1A) DW*
shoot Н7L4 А1 6 0,31 Trace -
shoot Н7L4 А1 8 0,23 24,25 -
shoot Н7L7 А2 6 0,31 Trace +
shoot Н7L7 А2 8 0,23 Trace -
callus Н7 А3 - - - -
callus Н6 А4 4 0,43 Trace +
shoot Н6L7 А5 3 0,47 272,83 +++
shoot Н6L7 А5 4 0,43 118,60 +++
shoot Н6L7 А5 5 0,32 Trace +
shoot Н6L7 А5 8 0,23 Trace ++
shoot Н6L8 А6 3 0,47 38,40 +++
shoot Н6L8 А6 4 0,43 10,19 ++
shoot Н6L8 А6 5 0,32 Trace +
shoot Н6L8 А6 8 0,23 Trace +
shoot Н6L6 А7 1 0,8 27,06 -
shoot Н6L6 А7 3 0,47 568,72 +++
shoot Н6L6 А7 4 0,43 153,68 +++
shoot Н6L6 А7 5 0,32 43,83 +
shoot Н6L6 А7 7 0,25 3,25 +++
shoot Н6L6 А7 8 0,23 Trace +
shoot Н6L6 А7 9 0,17 Trace -
shoot Н6L6 А7 10 0,12 Trace +++
shoot Н7L13 А8 3 0,47 Trace ++
shoot Н7L13 А8 5 0,32 Trace +
shoot Н7L13 А8 7 0,25 59,07 +
shoot Н7L13 А8 10 0,12 Trace ++
shoot Н7L14 А9 3 0,47 Trace ++
shoot Н7L14 А9 5 0,32 Trace +
shoot Н7L14 А9 7 0,25 16,96 -
shoot Н7L14 А9 10 0,12 Trace +
* - The amount of the alkaloids was calculated as μg galanthamine equivalents per g dry biomass;
** - Acetylcholinesterase inhibitory action was evaluated using the following scale:
(-) - lack of activity;
(+) - some activity;
(++) - high activity;
(+++) - strong activity.

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 The obtained in vitro lines were screened by their ability
to produce alkaloids, on the one hand, as well as by the
evaluation of the acetylcholinesterase inhibitory activities of
produced alkaloids, on the other. The screening was made by
using TLC technique (Fig. 1) and the results were presented
in Table 2.
The analyze of the results showed that the shoot type in
vitro systems produced more alkaloids in comparison to the
calli (Fig. 1A and Table 2). Neither shoot cultures, nor calli
produced galanthamine, but shoot lines Н6L7 and Н6L6 were
found to produced high levels of alkaloids with Rf values of
0.47 and 0.43 (Positions 3 and 4, according to Fig. 1A). It
should be noted that even initiated from the same explants
type on the same medium combination, the different shoot
lines demonstrated great variability concerning the number
and the levels of the produced alkaloids (Fig. 1A). This is
evidence for existance of high levels of somaclonal
variability exhibited by this type plant in vitro systems.
Fig. 1. TLC separaton of produced alkaloids (A) by P. maritimum L. in vitro
systems and assay of their acetylcholinesterase inhibitory action (B): A1 -
shoot line Н7L4; A2 - shoot line Н7L7; A3 - callus line Н7; A4 - callus line
Н6; A5 - shoot line Н6L7; A6 - shoot line Н6L8; A7 - shoot line Н6L6; A8 -
shoot line Н7L13; A9 - shoot line Н7L14; C2, C4, C5, C10 and С20 –
Galanthamine standards
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To evaluate the acetylcholinesterase inhibitory activities
of the produced by P. maritimum L. in vitro cultures
alkaloids, the fast TLC assay was applied (Fig. 1B).
Alkaloids with Rf values of 0.47, 0.43, 0.25 and 0.12
(Positions 3, 4, 7 and 10, according to Fig. 1B) demonstrated
higher potential to inhibit the acetylcholinesterase enzyme.
Surprisingly, some minor alkaloids, presented in low
concentrations in calli, were not detected by Dragendorff
visualization but successfully inhibit the acetylcholinesterase
enzyme (Positions 3, 7 and 10 in samples A2 and A3,
according to Fig. 1B). This is an evidence for the higher
sensitivity and accuracy of the TLC acetylcholinesterase
inhibitory assay compared to the TLC alkaloid visualization
by Dragendorff reagent.
As a result of the screening implemented, two shoot lines
(Н6L7 and Н6L6) were selected as prospective producers of
new acetylcholinesterase inhibitors. These lines produced
four alkaloids (Rf values of 0.47, 0.43, 0.25 and 0.12) with
high acetylcholinesterase inhibitory activities.
Conclusions
In conclusion, the effective protocol for obtaining of P.
maritimum L. in vitro systems producing amarillidaceae
alkaloids was developed. Based on the fast TLC assays, two
shoot lines (Н6L7 and Н6L6) were selected as prospective
source for further screening of new acetylcholinesterase
inhibitors. The results obtained by this study could be useful
for the future screening and isolation of new
acetylcholinesterase inhibitors as well as for the development
of protocols for in vitro techniques focused on preservation of
the wild populations of Pancratimum maritimum L. plants.
Acknowledgment
This research was supported by the Bulgarian Science
Foundation, Bulgarian Ministry of Education and Science
(project TK – Б – 1605/2006).
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