Projekat ministarstva br. 143026B i 143041

Soković, Marina

Vinterhalter, Branka

Vinterhalter, D.

Ćirić, Ana

Šavikin-Fodulović, Katarina

English

 Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)

Celandine (Chelidonium majus L.) (Papaveraceae) is an impor-
tant medical herb used in traditional and folk medicine through-
out the world. In China it is used as a remedy for whooping 
cough, chronic bronchitis, asthma, jaundice, gallstones and 
gallbladder pains (C h a n g  and C h a n g , 1986). In folk medi-
cine of the Balkan countries, it is widely used for its choleric, 
spasmolytic, and sedative properties. Extracts from celandine 
are supposed to have antibacterial, antiviral, antifungal and anti-
inflammatory effects. Fresh latex is used to remove warts, which 
are a visible manifestation of papilloma viruses (C o l o m b o 
and To m e , 1995; R o g e l j  et al., 1998). The main active 
constituents of celandine are the alkaloids chelidonine, chelery-
thrine, sanguinarine, isochelidonine, and isoquinoline alkaloids 
with protopine (F r a n z  and F r i t z , 1979; � e R o s a and � i    
V i n c e n z o , 199�). The alkaloid fraction is generally assumed       
to contribute to the antimicrobial activity of plant extracts 
(H a h n  and N a h r s t e d t , 1991).
Celandine is a very common weed growing around human 
habitats, usually along walkways and footpaths, in crevices 
of walls and buildings, and in neglected spots and places. 
Containing the yellow latex rich with alkaloids, the aerial parts 
of celandine perish with the first frost, making the plant unavail-
able during winter until the early spring.
Celandine leaves, petioles, and seeds used for in vitro 
culturing were collected from plants growing in Kalemegdan 
Park, Belgrade, Serbia. Shoots and leaves of plants grown in 
vitro were collected from shoots produced by recurrent somatic 
embryogenesis on hormone-free medium as elaborated by 
V i n t e r h a l t e r  and V i n t e r h a l t e r  (�00�). 
In vitro plant cultures often produce secondary metabolites 
in quantities equal to those produced by plants growing in 
nature. Thus, R o s i ć  et al. (�000) demonstrated high anthra-
quinone production by in vitro cultures of Rhamnus fallax, and 
Š a v i k i n  et al. (1999) described production of saponins in 
callus cultures of Dioscorea balcanica. In celandine, alkaloids 
produced by shoot cultures which leeched into the medium 
were visible as media discoloration (V i n t e r h a l t e r  and 
V i n t e r h a l t e r , �00�). 
The main purpose of this study was to examine total alka-
loid content and antimicrobial activity of methanol extracts 
derivates of tissues from plants growing in nature and under 
conditions of in vitro culture. 
HPLC analysis of total alkaloids was expressed as chelido-
nine (Table 1). �etection of chelidonine was done according to 
European Pharmacopoeia IV (H e n n i n g  et al., �003). 
Antibacterial and antifungal tests were done with 96% 
methanol extract derivates (S o k o v i ć  et al., �000) from leaves 
and petioles grown in nature and from shoots and somatic 
embryos cultured in vitro.
The following Gram (+) bacteria were used: Bacillus subtilis 
ATCC 10707,  Micrococcus luteus ATCC 9341, Sarcinia lutea 
ATCC 9391, and Staphylococcus aureus ATCC 6538, in addition 
to the Gram (-) bacteria Agrobacterium rhizogenes A4M70GUS, 
A. tumefacians A�81, Escherichia coli ATCC 35�18, Proteus 
mirabilis (clinical isolates), and Salmonella enteritidis ATCC 
13076; and the yeast Candida albicans (clinical isolates). The 
molds were from the collection of the IBISS Mycological 
Laboratory. 
Test microorganisms were cultivated overnight at 37°C in 
TSB medium (tryptic soy broth - Biolife). Suspensions contain-
ing ~ 109 cells/ml were used. 
Minimum inhibitory concentration (MIC) determinations 
were performed by a serial dilution technique using 96-well 
microtiter plates. The compounds investigated were dissolved 
in nutrient broth (TSB) with microorganism inocula. The plates 
containing bacteria were incubated for �4 h at 37°C, that of C. 
albicans for 48 h at �8°C. The following day, 50 µL of 0.� mg/mL 
solution of INT (p-iodonitrotetrazolium violet, Sigma) was 
added to each row and the plate was returned to the incubator 
for at least half an hour to ensure adequate color development. 
Inhibition of growth was indicated by a clear solution or a 
definite decrease in the color reaction (E l o o f , 1998). This value 
was taken as the minimum inhibitory concentration (MIC) of 
the compounds. The experiments for each sample were con-
ducted in duplicate. Bifonazole was used as a positive control for 
C. albicans and streptomycin for bacterial species. This method 
was chosen because of its simplicity, sensitivity, and relatively 
low cost, being rapid and uncomplicated at the same time.
CHEMICAL ANALYSIS AND ANTIMICROBIAL ACTIVITY OF METHANOL EXTRACTS OF 
CELANDINE (CHELIDONIUM MAJUS L.) PLANTS GROWING IN NATURE AND CULTURED 
IN VITRO. Ana Ćirić1, Branka Vinterhalter1, Katarina Šavikin-Fodulović2, Marina Soković1, and D. 
Vinterhalter1.  1Siniša Stanković Institute for Biological Research, 11060 Belgrade, Serbia; �Dr. Josif Pančić 
Institute of Medicinal Plant Research, 11000 Belgrade, Serbia
U�C  58�.675.5:581.1
7P
Arch. Biol. Sci., Belgrade, 60 (1), 7P-8P, �008		 	 	 	 	 																	              �OI:10.��98/ABS080107PC
Keywords: Chelidonium majus, chelidonine, antimicrobial activity
Chemical analysis (Table 1) showed that the total alkaloid 
content in shoots and embryos of in vitro cultured plants was 
higher than in leaves and petioles of plants growing in nature. 
The results of testing antimicrobial activity are presented 
in Table �. Methanol extracts of all samples showed significant 
activity against Gram (+) and Gram (-) bacteria and C. albicans. 
The extracts were not effective only against P. mirabilis. Extracts 
from various explants had higher antimicrobial activity against 
the tested microorganisms than that recorded for extracts from 
leaves and petioles of plants growing outdoors. When the results 
were compared with the antimicrobial activities of positive 
controls (streptomycin or bifonazole), some extracts showed 
equal antimicrobial activity against E. coli,  S. enteritidis, and 
C. albicans.
The obtained results justify the use of Chelidonium majus 
in traditional folk medicine. Also, during the winter season, 
when we cannot collect C. majus, plants cultured in vitro can 
completely replace plants in nature.
Acknowledgments - The presented research was funded by 
the Serbian Ministry of Science and Environmental Protection 
through projects 1430�6B and 143041B.
References - Chang, H. M., and P. P. H. Chang (1986). Pharmacology 
and Applications of Chinese Material Medica, 1, 390. Singapore. 
- Colombo, L. M., and F. Tome (1995). Chelidonium majus L. 
(greater celandine): in vitro culture and production of sanguina-
rine, coptisine, and other isoquinoline alkaloids, In: Biotechnology 
in Agriculture and Forestry (Ed. Y. P. S. Bajaj) 33, 157-159. 
Berlin. - �e Rosa, S., and G. �i Vincenzo (199�).         Phytochem. 
3, 1058. - Eloff, �. N. (1998).    Planta Med. 64, 711. - Franz, C., 
and �. Fritz (1979). Planta Med. 36, �46. - Hahn, R., and A. 
Nahrstedt (1991). Planta Med. 57, 119. - Henning, H. G., and A. 
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8P
Total alkaloids 
(%)
collected	from	nature in vitro cultured
leaves petioles embryos shoots
chelidonine	 1.40	±	0.2 1.17	±	0.2 1.58	±	0.3 1.53	±	0.2
Table 1. Total alkaloids calculated as chelidonine contained in various explants of Celandine tissues. Values are means ± SE (n = 16)..
Table 2. Antimicrobial activity (MIC - mg/ml) of methanol extracts from various Celandine explants as determined by the microdilution method..
Microorganisms
Plants	grown
	in	nature
Plants	cultured
in vitro control
leaves petioles embryos shoots
Agrobacterium rhizogenes	 80 80 40 20 10a
Agrobacterium tumefacians  80 80 80 40 10a
Bacillus subtilis  80 80 80 40 10a
Candida albicans - - - 80 80b
Escherichia coli  80 80 80 80 80a
Micrococcus luteus  40 80 80 80 10a
Proteus mirabilis - - - - -
Salmonella enteritidis 80 - 80 80 80a
Sarcinia lutea 80 80 80 80 10a
Staphylococcus aureus 80 80 80 80 20a
–:	no	activity.
	a	Streptomycin;	b	bifonazole.


Chemical analysis and antimicrobial activity of methanol extracts of celandine (Chelidonium majus L.) plants growing in nature and cultured in vitro

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Chemical analysis and antimicrobial activity of methanol extracts of celandine (Chelidonium majus L.) plants growing in nature and cultured in vitro

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