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Antioxidant and antimicrobial actions of the clubmoss
Lycopodium clavatum L.
Ilkay Orhan ÆBerrin O
¨zc¸elik ÆSinem Aslan Æ
Murat Kartal ÆTaner Karaoglu ÆBilge S¸ener Æ
Salih Terzioglu ÆM. Iqbal Choudhary
Received: 23 May 2006 / Accepted: 1 December 2006 / Published online: 3 March 2007
Springer Science+Business Media B.V. 2007
Abstract Purpose of the present study was to
evaluate antioxidant, antibacterial, antifungal,
and antiviral activities of the petroleum ether,
chloroform, ethyl acetate and methanol extracts
as well as the alkaloid fraction of Lycopodium
clavatum L. (LC) from Lycopodiaceae growing in
Turkey. Antioxidant activity of the LC extracts
was evaluated by 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical-scavenging method at 0.2 mg/ml
using microplate-reader assay. Antiviral assess-
ment of LC extracts was evaluated towards the
DNA virus Herpes simplex (HSV) and the RNA
virus Parainfluenza (PI-3) using Madin-Darby
Bovine Kidney (MDBK) and Vero cell lines.
Antibacterial and antifungal activities of the
extracts were tested against standard and iso-
lated strains of the following bacteria; Escherichia
coli,Pseudomonas aeruginosa,Proteus mirabilis,
Acinobacter baumannii,Klebsiella pneumoniae,
Staphylococcus aureus,Bacillus subtilis as well as
the fungi; Candida albicans and C. parapsilosis.
All of the extracts possessed noteworthy activity
against ATCC strain of S. aureus (4 lg/ml), while
the LC extracts showed reasonable antifungal
effect. On the other hand, we found that only the
chloroform extract was active against HSV
(16–8 lg/ml), while petroleum ether and alka-
loid extracts inhibited potently PI-3 (16–4 lg/ml
and 32–4 lg/ml, respectively). However, all of the
extracts had insignificant antiradical effect on
DPPH. In addition, we also analyzed the con-
tent of the alkaloid fraction of the plant by
capillary gas chromatography-mass spectrometry
(GC-MS) and identified lycopodine as the major
alkaloid.
Keywords Lycopodium clavatum
Lycopodiaceae Antioxidant Antimicrobial
Alkaloid
I. Orhan (&)B. S¸ener
Department of Pharmacognosy, Faculty of Pharmacy,
Gazi University, Ankara, Turkey
e-mail: iorhan@gazi.edu.tr
B. O
¨zc¸ elik
Department of Pharmaceutical Microbiology, Faculty
of Pharmacy, Gazi University, Ankara, Turkey
S. Aslan M. Kartal
Department of Pharmacognosy, Faculty of Pharmacy,
Ankara University, Ankara, Turkey
T. Karaoglu
Department of Virology, Faculty of Veterinary,
Ankara University, Ankara, Turkey
S. Terzioglu
Department of Forest Botanic, Faculty of Forestry,
Karadeniz Technical University, Trabzon, Turkey
M. I. Choudhary
H.E.J. Research Institute of Chemistry, International
Center for Chemical Sciences, University of Karachi,
Karachi, Pakistan
123
Phytochem Rev (2007) 6:189–196
DOI 10.1007/s11101-006-9053-x
Introduction
From ancient to modern history, traditional plant-
based medicines have played an important role in
health care. Many countries in Africa, Asia and
Latin America still rely on traditionally used
herbal medicines for primary health care needs.
Traditional medicine has maintained its popular-
ity in developing countries and has been becoming
fashionable in industrialized countries as well.
Lycopodium serratum Thunb. (syn. Huperzia
serrata (Thunb.) Trev. has been used in traditional
Chinese medicine since ages for treatment of
amnesia, contusion, strain, haematuria, schizo-
phrenia, and swelling (Bai 1993; Zhu et al. 1996).
The anticholinesterase alkaloids, huperzine A and
B, were firstly discovered in this plant (Liu et al.
1986). More to the point, huperzine A was
reported to have in vivo antioxidant activity in
several studies (Xiao et al. 1999,2000; Zhang
and Tang 2000). Another Lycopodium species,
L. varium growing in New Zealand, was shown to
possess insecticidal activity towards the flies An-
threnocerus australis,Lucilia cuprina, and Tineda
bisselliella, which led to isolation of huperzine A
as the active component (Ainge et al. 2002).
The Lycopodium genus in Turkey is repre-
sented by five species, namely L. alpinum L.,
L. annotinum L., L. clavatum L., L. complanatum
ssp. chamaecyparissus (A. Br.) Do
¨ll, and L. selago
L. (Davis and Cullen 1984). Among them,
L. clavatum (LC), the most common species in
Anatolia, has been reported to have a healing
effect on wounds and dermatological diseases
including rush in babies in Anatolia and, there-
fore, called ‘‘belly powder’’ (Baytop 1999).
On this purpose, the aim of this work was to
examine antioxidant, antibacterial, antifungal, and
antiviral properties of petroleum ether, chloro-
form, ethyl acetate and methanol extracts as well
as the alkaloid fraction of Lycopodium clavatum
L. (LC) from Lycopodiaceae growing in Turkey.
Materials and methods
Plant material
The plant material was collected from two
different localities in northern Anatolia, firstly
from the vicinity of Oymalitepe village, Yomra
town, Trabzon at 600 m altitude (coded as LC-T)
and secondly from Bagirankaya plataeu, Ikizdere
town, Rize at 2000 m altitude (coded as LC-R) in
2001. The identification of the plant samples was
carried out by Dr. Salih Terzioglu from the
Department of Forest Botany, Faculty of For-
estry, Karadeniz Technical University, Trabzon,
Turkey. The voucher specimen (GUE 2216) has
been deposited at the Herbarium of the Faculty
of Pharmacy of Gazi University, Ankara, Turkey.
Preparation of the extracts
The plant materials of LC-T and LC-R were
independently dried under shade, ground
mechanically to fine powders in a grinder and
weighed accurately as 374.91 and 382.88 g,
respectively. Same procedure for solvent extrac-
tion was applied to both materials. Initially, the
petroleum ether extract was prepared by macer-
ating with 2 l of petroleum ether for each sample
at room temperature, filtrating through the fil-
ter paper and concentrating to dryness under
reduced pressure, which was sequentially fol-
lowed by chloroform (1 l ·3), ethyl acetate
(1 l ·2) and methanol (1 l ·0.3). In order to
prepare the alkaloid fraction, acid–base extrac-
tion of 100 g of the plant material was performed
with chloroform (0.5 l ·3). pH of the chloroform
extract was finally adjusted to pH 12 using 25%
NH
4
OH (Merck) and a special indicator paper
scaled between pH 8–12 (Merck). All of the crude
extracts prepared from LC-T and LC-R samples
were comparatively checked on TLC (Kieselgel
HF
254 + 366
, Merck), using the solvent systems
chloroform:methanol (10:1) for the petroleum
ether and chloroform extracts along with the
alkaloid fraction and chloroform:methanol (5:1)
for the ethyl acetate and methanol extracts. TLC
spots of the extracts were also observed under the
ultraviolet light at 254 and 366 nm and also
revealed by both Dragendorff revelator and 5%
H
2
SO
4
, independently. The TLC results of the
extracts of LC-T and LC-R proved that both LC
samples contained completely the same phyto-
chemical compounds, showing no differences at
all independent on locality difference. Therefore,
the dual extracts belonging to LC-T and LC-R
190 Phytochem Rev (2007) 6:189–196
123
were combined. Their yield percentages (w/w) as
well as codes were also given as follows: LC-PE
(petroleum ether extract, 5.30%), LC-CHCl
3
(chloroform extract, 15.79%), LC-CHCl
3
-alk
(alkaloid fraction, 22.56%), LC-EtOAc (ethyl
acetate extract, 20.34%), LC-MeOH (methanol
extract, 51.34%).
DPPH free radical-scavenging assay
Antiradical activity of the plant extracts and the
reference were assessed on the basis of the radical
scavenging effect of the stable 1,1-diphenyl-2-
picrylhydrazyl radical (DPPH) free radical (Lee
et al. 1998). The concentration of DPPH was kept
as 300 lM. The extracts and reference were
dissolved in DMSO, while the DPPH solution
was prepared in ethanol. 10 ll of each extract and
reference was allowed to react with 200 llof
stable free radical DPPH at 37C for 30 min in a
96-well microtiter plate. After incubation, de-
crease in absorption for each solution was mea-
sured at 515 nm using ELISA microplate reader
(Spectra MAX-340 Molecular Devices, USA).
The corresponding blank readings were also taken
and the remaining DPPH was calculated. Percent
radical scavenging activity by samples was deter-
mined in comparison with a DMSO treated
control group. BHA was used as reference.
Inhibition of free radical DPPH in percent (I%)
was calculated in following way: I%¼ð1
Asample=Ablank Þ100;where A
blank
is the absor-
bance of the control reaction (containing all
reagents except the test sample), and A
sample
is
the absorbance of the extracts/reference.
Microbiological studies
All of the LC extracts were dissolved in etha-
nol:hexane (1:1) by using 1% tween 80 solution at
a final concentration of 1024 lg/ml and sterilized
by filtration using 0.22 lm Millipore (MA 01730,
USA) and used as the stock solutions. Standard
antibacterial powders of ampicillin (AMP, Fako),
ofloxacin (OFX, Hoechst Marion Roussel), and
also standard antifungal powders of ketoconazole
(KET, Bilim) and fluconazole (FLU, Pfizer), were
obtained from their respective manufacturers and
dissolved in phosphate buffer solution (AMP, pH:
8.0, 0.1 mol/l), DMSO (KET), in water (FLU and
OFX). The stock solutions of the agents were
prepared in medium according to the NCCLS
recommendations (National Committee 1996).
Microorganisms
Standard and the isolated strains of the following
bacteria, namely Escherichia coli (ATCC 35218),
Pseudomonas aeruginosa (ATCC 10145), Proteus
mirabilis (ATCC 7002), Klebsiella pneumoniae
(RSKK 574), Acinetobacter baumannii (RSKK
02026), Staphylococcus aureus (ATCC 25923),
and Enterococcus faecalis (ATCC 29212) for
determination of antibacterial activity, along with
standard strains of Candida albicans (ATCC
10231) and C. parapsilosis (ATCC 22019) were
used for determination of antifungal activity.
Inoculum preparation
Mueller-Hinton Broth (Difco) and Mueller-
Hinton Agar (Oxoid) were applied for growing
and diluting of the bacteria. Sabouraud liquid
medium (Oxoid) and Sabouraud dextrose agar
(SDA) (Oxoid) were applied for growing and
diluting of the fungi. The medium RPMI-1640
with L-glutamine was buffered pH: 7 with
3-[N-morpholino]-propansulfonic acid (MOPS).
Prior to the tests, strains of bacteria and fungi
were cultured on media and passaged at least
twice to ensure purity and viability at 35C for
24–48 h. Culture suspensions were prepared
according to the NCCLS M27-A (O
¨zc¸elik et al.
2005). The bacterial suspensions used for inocu-
lation were prepared at 10
5
cfu/ml by diluting
fresh cultures at McFarland 0.5 density (10
8
cfu/
ml). The fungi suspension was prepared by the
spectrophotometric method of inoculum prepara-
tion at a final culture suspension of 2.5 ·10
3
cfu/
ml (National Committee 2002).
Antibacterial and antifungal tests
The microdilution method was employed for
antibacterial and antifungal activity tests. Media
were placed into each well of the 96 well-
microplate. Extract solutions at 1024 lg/ml were
added into first raw of microplates and two fold
Phytochem Rev (2007) 6:189–196 191
123
dilutions of the compounds (512–0.25 lg/ml)
were made by dispensing the solutions to the
remaining wells. 10 ll culture suspensions were
inoculated into all the wells. The sealed micro-
plates were incubated at 35C for 24 and 48 h in
humid chamber. The lowest concentration of the
extracts that completely inhibit macroscopic
growth was determined and minimum inhibitory
concentrations (MICs) were reported. Antimicro-
bial activity of the oils was tested against two
Gram-positive, five Gram-negative bacteria and
two yeast-like fungi, using AMP, OFX, KET, and
FLU as the standards (National Committee 2002;
O
¨zc¸elik et al. 2004a,b).
Cytotoxicity and antiviral tests
Cell line and growth condition
African green monkey kidney (Vero cell line) and
Madin-Darby bovine kidney (MDBK) used in
this study were obtained from Department of
Virology, Faculty of Veterinary, Ankara University
(Turkey). The culture of the cells was grown
in Eagle’s Minimal Essential Medium (EMEM)
enriched with 10% fetal calf serum (FCS) (Biochrom,
Germany), 100 mg/ml of streptomycin and 100 IU/
ml of penicillin in a humidified atmosphere of 5%
CO
2
at 37C. The cells were harvested using trypsin
solution (Bipco Life Technologies, UK).
Test viruses
In order to establish the antiviral activity, Herpes
simplex virus (HSV) and Para-influenza-3 virus
(PI-3) were used. The test viruses were obtained
from Department of Virology, Faculty of Veter-
inary, Ankara University (Turkey).
Antiviral Activity
Media (EMEM) were placed into each 96 wells of
the microplates (Greiner
R
, Germany). Stock solu-
tions of the extracts were added into first raw of
microplates and twofold dilutions of the extracts
(512–0.25 lg/ml) were made by dispensing the
solutions to the remaining wells. Twofold dilutions
of each material were obtained according to Log
2
on the microplates. Acyclovir (Biofarma) and
oseltamivir (Roche) were used as the references.
Strains of HSV and PIV titers were calculated by
the Frey and Liess method as TCID
50
(Frey and
Liess 1971). They were inoculated into all the
wells. The sealed microplates were incubated in
5% CO
2
at 37C for 2 h to detect the possible
antiviral activities of the samples. After incuba-
tion, 50 ll of the cell suspension of 300,000 cells/
ml which were prepared in EMEM + 5% fetal
bovine serum was put in each well and the plates
were incubated in 5% CO
2
at 37C for 48 h. After
the end of this time, the cells were evaluated using
cell culture microscope (·400), comparing with
treated-untreated control cultures and with acy-
clovir and oseltamivir. Consequently, maximum
cytopathogenic effect (CPE) concentrations as the
indicator of antiviral activities of the extracts were
determined (O
¨zc¸elik et al. 2005).
Cytotoxicity
The maximum non-toxic concentration (MNTC)
of each sample was determined according to the
method described elsewhere based on cellular
morphologic alteration (O
¨zc¸elik et al. 2005).
Several concentrations of each sample were
placed in contact with confluent cell monolayer
and incubated in 5% CO
2
at 37C for 48 h.
MNTC values were determined by comparing
treated and controlling untreated cultures.
Conditions of gas chromatography-mass
spectrometry (GC-MS) analysis
Chromatographic analysis was carried out on
Agilent 6890N Network GC system combined
with Agilent 5973 Network Mass Selective Detec-
tor (GC-MS). The capillary column used was an
Agilent 19091N-136 (HP Innowax Capillary;
60.0 m ·0.25 mm ·0.25 lm). Helium was used
as carrier gas at a flow rate of 2.4 ml/min for MS
and 2.3 ml/min for FID columns with 1 ll injec-
tion volume. Samples were analyzed with the
column started initially 150C, then increased to
250C with 5C/min heating ramp and kept at
250C for 15 min. The injection was performed
in split mode (20:1). Detector and injector
192 Phytochem Rev (2007) 6:189–196
123
temperatures were 250 and 250C, respectively.
Run time was 35 min. MS interface temperature
was 280C. MS scan range was (m/z): 15–450
atomic mass units (AMU) under electron impact
(EI) ionization (70 eV).
Identification of the alkaloidal components
Identification of the peaks was done using Wiley
and Nist Library, comparison of retention time
(Rt) and comparison of standard compounds with
mass spectrums. Relative contents of % alkaloids
were determined with area under peaks using
Agilent software. The results were expressed as
an average of three determinations in all cases.
Statistical analysis of data
Data obtained from in vitro experiments with
antiradical activity against DPPH were expressed
as mean standard error (±SEM). Statistical dif-
ferences between the treatments and the control
were evaluated by ANOVA test. P< 0.05 was
considered to be significant [*P< 0.05;
**P< 0.01; ***P< 0.001].
Results and discussion
Results of the antibacterial and antifungal tests
are given in Table 1. According to data we
obtained from the antibacterial assay, all of the
extracts belonging to LC displayed inhibition
against each bacterium used at the same MIC
values ranging between 4 and 64 lg/ml, whereas to
they were observed to be more susceptible against
isolated bacterium strains. The extracts seemed
to the most active towards the ATCC strain of
S. aureus (4 lg/ml), while they showed inhibition
against the standard strains of P. mirabilis,
K. pneumoniae,A. baumannii, and E. faecalis,as
well as the isolated strain of S. aureus (16 lg/ml).
As for the antifungal tests, LC-CHCl
3
-Alk, LC-
EtOAc, and LC-MeOH inhibited C. albicans and C.
parapsilosis (16 lg/ml) moderately but better than
LC-PE and LC-CHCl
3
(32 lg/ml). On the other
hand, LC-CHCl
3
extract exerted good antiviral
effect towards the DNA virus HSV (16–8 lg/ml)
with the MNTC of 16 lg/ml, similar to that of
Table 1 Antibacterial and antifungal activities of the LC extracts as mnimum inhibitory concentrations (MICs) (lg/ml)
Microorganisms E. coli P. aeruginosa P. mirabilis K. pneumoinae A. baumannii S. aureus E. faecalis C. albicans C. parapsilosis
ATCC Isol. ATCC Isol. ATCC Isol. ATCC Isol. ATCC Isol. ATCC Isol. ATCC Isol.
Extracts
LC-PE 32 64 32 64 16 32 16 32 16 64 4 16 16 32 32 32
LC-CHCl
3
32 64 32 64 16 32 16 32 16 64 4 16 16 32 32 32
LC-CHCl
3
-Alk 32 64 32 64 16 32 16 32 16 64 4 16 16 32 16 16
LC-EtOAc 32 64 32 64 16 32 16 32 16 64 4 16 16 32 16 16
LC-MeOH 32 64 32 64 16 32 16 32 16 64 4 16 16 32 16 16
AMP
a
264––2 4 2 4 2 4 <0.12 8 0.5 1 ––
OFX
b
0.12 1 1 4 <0.12 1 <0.12 1 0.12 2 0.5 4 1 2 ––
LVX
c
<0.12 0.25 1 2 <0.12 1 <0.12 1 0.12 2 0.5 4 0.5 2 ––
KET
d
––– –––– –– –– –––11
FLU
e
––– –––– –– –– –––44
a
AMP: Ampicilline,
b
OFX: Ofloxacine,
c
LVX: Levofloxacine,
d
KET: Ketoconazole,
e
FLU: Fluconazole, –: No activity observed
Phytochem Rev (2007) 6:189–196 193
123
acyclovir (16 to <0.25 lg/ml), except for its thera-
peutic range of LC-CHCl
3
was narrower (Table 2).
As to PI-3, LC-PE and LC-CHCl
3
-Alk exhibited
some inhibition (16–4 and 32–4 lg/ml, respectively).
In particular, the alkaloid fraction of LC showed
quite similar anti-PI-3 effect and MNTC value to
that of oseltamivir (32 to <0.25 lg/ml). Additionally,
LC-PE and LC-CHCl
3
were more cytotoxic (32 and
16 lg/ml) as compared to the rest of the extracts.
However, LC-CHCl
3
and LC-EtOAc were rather
cytotoxiconVerocells(8lg/ml).
Nevertheless, all of the extracts revealed to
exhibit of no consequence antioxidant property
against DPPH radical at 0.2 mg/ml with the
highest inhibition of 44.1% from LC-CHCl
3
-
Alk, when compared to BHA, whose inhibition
was 92.7% (Table 3).
The antioxidant activity of Lycopodium ex-
tracts has not been reported previously, however,
huperzine A, the potent anticholiesterase alkaloid
isolated from L. serratum (Huperzia serrata), was
found to have in vivo antioxidant capacity (Xiao
et al. 1999,2000; Zhang et al. 2000). This prop-
erty is also important for huperzine A, which may
show its neuroprotective effect through reducing
amyloid-bplaque formation in the brains of
Alzheimer’s disease (AD) patients. Because, a
common theory for AD pathogenesis has been
suggested to be cell death due to oxidative stress
mediated by free radicals (Markesbery and Car-
ney 1999). In our recent study on the same subject
with L. complanatum subsp. chamaecyparissus
(LCC) of Turkish origin, we gathered the similar
results in antioxidant activity against DPPH
(Orhan I et al. submitted). In fact, in one our
previous studies, we performed a qualitative
analyze on the extracts of both L. clavatum and
L. complanatum subsp. chamaecyparissus by
liquid chromatography-mass spectrometry (LC-
MS) in order to detect huperzine A, and were not
able to find this alkaloid in Turkish species of
Lycopodium (Orhan et al. 2002).
On the other hand, L. clavatum was formerly
reported to contain various phenolic acids such as
dihydrocaffeic, vanillic, p-hydroxy-benzoic, syrin-
gic, p-coumaric, and ferulic acids (Towers and
Maass 1965) and phenolic acids are known to
display antimicrobial activity against a variety of
microorganisms (Herald and Davidson 1983; Stead
1993). For instance; ferulic acid has been shown to
have antimicrobial activity by several researchers
against S. aureus,B. subtilis,P. aeruginosa, and
C. albicans as well as Listeria monocytogenes
(Fernandez et al. 1996; Kwon et al. 1997; Panizzi
et al. 2002; Wen et al. 2003). In Fernandez et al.’s
Table 2 Antiviral
assessment of the LC
extracts against MDBK
and Vero cell lines
a
MNTC: Maximum non-
toxic concentration,
b
CPE: Cytopathogenic
effect, –: No activity
observed
MDBK
a
cells (lg/ml) Vero
b
cells (lg/ml)
Extracts MNTC (lg/
ml)
CPE inhibitory
concentration
MNTC (lg/
ml)
CPE inhibitory
concentration
HSV PI-3
Maximum Minimum Maximum Minimum
LC-PE 32 ––16 16 4
LC-CHCl
3
16 16 8 8 ––
LC-CHCl
3
-
Alk
64 ––32 32 4
LC-EtOAc 64 –– 8––
LC-MeOH 64 ––64 ––
Acyclovir 16 16 <0.25 –––
Oseltamivir –––32 32 <0.25
Table 3 Antioxidant activity of the LC extracts against
DPPH radical at 0.2 mg/ml
Extracts Inhibition%
LC-PE 11.3 ± 1.64***
LC-CHCl
3
–
LC-CHCl
3
-Alk 44.1 ± 5.94***
LC-EtOAc 26.6 ± 0.70***
LC-MeOH 30.3 ± 0.15***
BHA 92.7 ± 0.21
–: No activity observed
194 Phytochem Rev (2007) 6:189–196
123
study (1996), syringic, caffeic, isovanillic, ferulic,
and p-hydroxycinnamic acids were also stated to
possess antimicrobial activity. For that reason, the
presence of these compounds in this plant may
explain their antibacterial activity.
In the past, plants have afforded a number of
anti-infective agents including emetine, quinine,
and berberin (Iwu et al. 1999). The Lycopodium
genus is also known to be rich in alkaloids with
high toxicity (Ayer 1991). This may also contribute
to the antimicrobial activity of the LC extracts as
Ainge et al.’s study (2002) declared that huperzine
A was also responsible or the insecticidal activity
of L. varium extract.
In continuation of this study, we aimed to
investigate the LC-CHCl
3
-Alk extract by GC-MS
in order to elucidate its main alkaloidal constitu-
ents of Turkish species of L. clavatum. Our GC-
MS analysis demonstrated that LC-CHCl
3
-Alk
consisted of lycopodine (m/z 247, 89.7 ± 0.02%),
as the major alkaloid, along with dihydrolycopo-
dine (m/z 249, 7.6 ± 0.08%), and lycodine (m/z
242, 2.7 ± 0.09%). Existence of all the three
alkaloids were previously reported from L. clav-
atum of different origins, which is in consistence
with our data (Luan and Xu 1986).
Conclusions
Plant extracts are known to have a quite complex
mixture comprised of structurally diverse compo-
nents and therefore, high probability of compet-
ing or synergistic interactions within the same
extract may exist for any biological activity, which
leads to a starting point for an activity-guided
search for active metabolites. In this case, LC-
CHCl
3
-Alk extract might be considered worthy of
advance phytochemical investigation to find out
the active component(s) for its antiviral activity,
although lycopodine, the major alkaloid, seems to
be possibly responsible, which needs to be con-
firmed. To best of our knowledge, for the first
time, we herein report antioxidant, antibacterial,
antifungal, and antiviral activities of L. clavatum
L. and also explaining the alkaloidal content of
the Turkish counterpart of the plant.
Acknowledgement This work was financially supported
by a research grant provided by the Foundation for
Scientific Research Projects of Gazi University (Project
code no: 02/2006–30).
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