Brine shrimp lethality bioassay of selec (2)

Arch. Biol. Sci., Belgrade, 60 (4), 681�685, �008

DOI:10.��98/ABS0804681J

BRINE SHRIMP LETHALITY BIOASSAY OF SELECTED CENTAUREA L. SPECIES (ASTERACEAE)
�. JANAĆKOVIĆ1, V. TEŠEVIĆ�, �. D. MARIN1, S. MILOSAVLJEVIĆ�,
SONJA DULETIĆ-LAUŠEVIĆ1, SLAVICA JANAĆKOVIĆ1, and M. VELJIĆ1
1Institute

of Botany and Jevremovac Botanical Garden, Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia
2Faculty of Chemistry, University of Belgrade, 11001 Belgrade, Serbia

Abstract — Ether extracts of 15 Centaurea L. species (Asteraceae) methanol extracts of 1� species, and cnicin isolated from
C. derventana were tested for general bioactivity using the brine shrimp lethality test. Cnicin showed the most potent activ�
ity with LC50 0.�. Also, ether extract of C. splendens showed significant activity with LC50 7.3, as did methanol extract of
C. arenaria with LC50 1�.4.
Key words: Centaurea, bioactivity, cnicin, cytotoxicity, brine shrimp lethality bioassay

UDC 58�.998:547.473
INTRODUCTION

lected from native habitats in Serbia and Montenegro.
Voucher specimens with accession numbers are
deposited in the Herbarium of the Institute of
Botany, Faculty of Biology, University of Belgrade.

Centaurea L. is a large genus of the family Asteraceae,
comprising about 500 species. Most species are pre�
dominantly distributed around the Mediterranean
area and in West Asia (M a b b e r l e y , 1997).
Various biological tests have been used in investiga�
tion of different extracts of Centaurea spp. (M
Monya
et al., 1968; L l o m b a r t and � e r e � � M i n g u e � ,
1969; I v o r r a et al., 1988; R o b e l s et al., 1997;
R o y et al., 1995; � � v e n et al., �005).. It has been
shown that a number of Centaurea species possess
biologically active compounds. Several species are
used in traditional medicine. Thus, C. cyanus L. is an
astringent, diuretic, emmenagog, and antiseptic, in
addition to which it is used to treat collyrium, fever,

and tumors (eye) (J o h n s o n , �003; Tu c a k o v,
1978; S a r k e r et al., �001; Va l l e s et al., 1996).
Centaurea jacea L. is known as a diuretic and anti�
diabetic that is also used to treat fever (J o h n s o n ,
�003; Va l l e s et al., 1996). Centaurea montana L.
is an astringent, cyanogenetic, diuretic, emmenagog,
pectoral, stimulant, and tonic that is also used to
treat collyrium and fever. Finally, C. salonitana Vis.
is used to treat tumors (J o h n s o n , �003).

Sample preparation
Sample preparation was performed as described
previously (M e y e r et al., 198�; M c L a u g h l i n ,
1991). Table � shows the percent of dry weight and
fresh weight yields of extracts of the investigated
species. Ether extracts were prepared by mixing
50 ml of Et�O with 5 g of dried leaves, methanol
extracts by mixing 50 ml of MeOH with 5 g of fresh
leaves. Mixtures were extracted continuously in an
ultrasonic bath for 30 min and then were kept for �4

h in the dark at room temperature. After filtration,
dry residues from extracts were obtained by evapo�
ration in a rotary evaporator (t = 40ºC). Solutions
with starting concentration of 1 mg/ml of methanol
extracts, ether extracts, and cnicin were prepared by
dissolving 10 mg of the given extracts and cnicin in
10 ml of methanol in volumetric flasks. Appropriate
amounts of solutions (10, 100, and 1000 µl, for 10,
100, and 1000 µg/ml, respectively) were transferred
to 1.5�cm disks of filter paper (Schleicher and
Schuell, no. 5891, Ø70 mm). The disks were dried in
air, placed in ��dram vials, and then dried further
in vacuo for an hour. Control disks were prepared

MATERIALS AND METHODS
Plant material
�lant material (Table 1) of Centaurea spp. was col�
681

68�

�. JANAĆKOVIĆ ET AL.

Table 1.Investigated Centaurea species.
Species
Centaurea affinis Friv.
C. arenaria Bieb. ex Willd.
C. atropurpurea Waldst. & Kit.
C. chrysolepis Vis.
C. cyanus L.
C. grisebachii (Nyman) Form.
C. incompta Vis.
C. jacea L.
C. jacea L.
C. montana L.
C. nervosa Willd.
C. rupestris L.
C. salonitana Vis.
C. scabiosa L.
C. scabiosa L.

C. solstitialis L.
C. splendens L.
C. stoebe L.
C. stoebe L.
C. triniifolia Heuff.
C. triumfetti All.
C. triumfetti All.

Locality

Voucher specimens, accession numbers

Lake Vlasina, Serbia
Belgrade, Serbia
Zlot, Serbia
Niševac, Serbia
Mt. Fruška �ora, Serbia
Zabel, Ra�gojna, Serbia
Mt. Orjen, Montenegro
Belgrade, Serbia

Mt. Zlatibor, Serbia
Derventa Canyon, �erućac, Serbia
Mt. Kučki Kom, Montenegro
Vranje, Serbia
Niševac, Serbia
Belgrade, Serbia
�remeća, Serbia
Belgrade, Serbia
Mt. Orjen, Montenegro
Svrljig, Serbia
Maglič, Serbia
Zlot, Serbia
Mt. Midžor, Serbia
�odulja Canyon, Serbia

CAf491998, CAf0607�001
CAr1806�001
CAt�706�000
CCh13071999
CCy�805�001

C�r�508�001
CIn0�081999E
CJ�607�001
CJ�061998
CMo1306�001
CNr1�07�001
CR071998
CS05091998, CS1807�001
CSc�405�001
CSc197�000lf
CSo�305�001
CSpl306�001
CSt1807�001
CSt1707�000
CTrin�706�000
CTri1306�001
CTri1506�000

Table 2. Percent of dry and fresh yield of extracts of investigated Centaurea spp. (np): extract not prepared.
Centaurea species

C. splendens L.
C. nervosa Willd.
C. rupestris L.
C. montana L.
C. atropurpurea Waldst. & Kit.
C. chrysolepis Vis.
C. incompta Vis.
C. cyanus L.
C. triniifolia Heuff.
C. jacea L.
C. stoebe L.
C. triumfetti All.
C. salonitana Vis.
C. arenaria Bieb. ex Willd.
C. scabiosa L.
C. solstitialis L.
C. affinis Friv.
C. grisebachii (Nyman) Form.

Yield of extract (%)

Fresh weight (methanolic)
methanolic)
Dry weight (aetheric)
theric)
3.13
�.�6
np
0.6�
np
5.03
np
0.89
np
0.64
7.86
0.91
np
1.87
1.71
�.06

np
4.96
4.�1
1.14
np
�.61
1.14
0.73
4.�7
3.77
4.17
1.66
5.��
3.63
3.67
np
9.51
0.54
5.35
np

683

BRINE SHRIM� LETHALITY BIOASSAY IN SOME CENTAURES L. S�ECIES

Table 3. Cytotoxic activity of methanol and ether extracts of Centaurea spp. and cnicin. Potassium dichromate (50 μg/ml) was used
as a positive control. (*): conidence interval 95%; (nt): not tested.
�ercent deaths at �4 h

Centaurea species
10
µg/ml
C. splendens L.
C. nervosa Willd.
C. rupestris L.
C. montana L.
C. atropurpurea Waldst. & Kit.
C. chrysolepis Vis.
C. incompta Vis.
C. cyanus L.
C. triniifolia Heuff.
C. jacea L.
C. stoebe L.
C. triumfetti All.
C. salonitana Vis.
C. arenaria Bieb. ex Willd.
C. scabiosa L.
C. solstitialis L.
C. affinis Friv.
C. grisebachii (Nyman) Form.
Cnicin

17
nt
nt
nt
nt
0
0
�3
nt
�7
nt
4
14
54
0
30
4.1
�4
80

Methanol extract
100
1000
µg/ml
µg/ml
�6.6
nt
nt
nt
nt
7
4
67
nt
40
nt
11
�7
64
30
34
7
33.3
94

using potassium dichromate (VI). Five replicates
were prepared for each dose level.
Brine shrimp cytotoxicity assay
The test was performed as described in M e y e r
et al. (198�)
198�) and M c L a u g h l i n (1991).
1991). Each
extract and cnicin isolated earlier from C. derventana (Te š e v i ć et al., 1998) were tested at concen�
trations of 10, 100, and 1000 μg/ml. Brine shrimp
eggs (Artemia salina Leach) were purchased in a pet
shop in Belgrade and hatched in artificial sea water
(solution of NaCl 3.8%) at room temperature. After
48 h, the larvae (nauplii) were collected. A suspen�
sion of 10 nauplii in artificial sea water was added to
each sample vial to a volume of 5 ml and the sample
vials incubated for �4 h at room temperature. After
this period, the number of dead nauplii in each sam�
ple vial was counted using a binocular microscope
(MBS 9, USSR, 4.8 x). �ure methanol was used as a

97
nt
nt
nt
nt
96.8
100
100
nt
100
nt
90
100
100
100
100
100
70
96

LC50*
µg/ml

10
µg/ml

114.6
nt
nt
nt
nt
�77.7
�3�.8
37.9
nt
61.�
nt
�66.5
111.�
1�.4
14�.1
64.8
�18.5
�19.1
0.�

58.3
55.�
41.6
33
�7
18.8
�0.8
��.�
16.6
16
16.6
11
4.4
0
0
nt
nt
nt
80

Ether extract
100
1000
µg/ml
µg/ml
75.�
56.9
56.9
44
3�.�
48.8
�4.7
��.�
�4.7
�4.7
�1.5
11
4.4
0
0
nt
nt
nt
94

100
100
100
100
100
100
100
100
100
100
100
100
93.5
100
18.�
nt
nt
nt
96

LC50*
µg/ml
7.3
13.8
�7.8
47.�
73.1
86.9
100.�
10�.7
110.�
111.6
118
166.6
�75.4
319.5
>1000
nt
nt
nt
0.�

positive control. Finney’s statistical method of probit
analysis (F i n n e y, 1971) was used to calculate the
concentration of the extract or cnicin that would kill
50% of brine shrimps within �4 h of exposure, i.e.,
the LC50 with 95% confidence intervals. The extracts
were considered as bioactive when LC50 was 1000
μg/ml or less. The value of LC50 was determined
using the Ld� Line® program (B a k r , �007).
RESULTS AND DISCUSSION
As shown in Table 3, methanol extracts from all
investigated species were very active. The metha�
nol extract of C. arenaria showed very significant
activity (LC50 = 1�.4 µg/ml). The lowest activity was
found with C. chrysolepis (LC50 = �77.7 µg/ml). The
ether extract of C. splendens was the most active
(LC50 = 7.3 µg/ml). The lowest activity was recorded
for the ether extract of C. scabiosa (LC50 = �605.9
µg/ml, i.e., >1000 ppm). The sesquiterpene lactone

684

�. JANAĆKOVIĆ ET AL.

cnicin showed significantly higher activity than any
extract of the investigated Centaurea species (LC50 =
0.� µg/ml). Centaurea splendens is already known as
a rich source of cnicin (J a n a ć k o v i ć et al., �003).
It can be presumed that the high content of cni�
cin in C. splendens is in direct relation to strong
activity of the ether extract of this species. Most of
the investigated species contain cnicin (N o w a k
et al., 1984; � o u s i a d o u and S k a l t s a , �003)
and other potentially active compounds like lig�
nans, flavones, etc. (J a n a ć k o v i ć et al., �004).
From results of the brine shrimp lethality bioassay
used in this work, it can be concluded that this test
is useful in determining the biological activity of
Centaurea species. Since many of these species are
used in traditional medicine, their extracts should
be subject to further investigation for isolation and
identification of biologically active compounds.
Acknowledgments — The authors are grateful to the Ministry of
Science and Technological Development of Serbia for financial
support (�rant No. 143049).

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BRINE SHRIM� LETHALITY BIOASSAY IN SOME CENTAURES L. S�ECIES

685

БИОТЕСТ ARTEMIA SALINA КОД ОДАБРАНИХ ВРСТА РОДА CENTAUREA L. (ASTERACEAE)
П. ЈАНАЋКОВИЋ1, В. ТЕШЕВИЋ�, П. Д. МАРИН1, С. МИЛОСАВЉЕВИЋ�,
СОЊА ДУЛЕТИЋ-ЛАУШЕВИЋ1, СЛАВИЦА ЈАНАЋКОВИЋ1 и М. ВЕЉИЋ1
1Институт

за ботанику, Биолoшки факултет, Универзитет у Београду, 11000 Београд, Србија
2Хемијски факултет, Универзитет у Београду, 11001 Београд, Србија

У раду је и­питивана биолошка активно­т
метанолних и етар­ких ек­траката 18 вр­та рода
Centaurea помоћу Биоте­та Artemia salina.. Вр­те
овог рода пред­тављају значајне природне изво�
ре биолошки активних ­екундарних метаболи�
та. Такође, одређена је и биолошка активно­т
(ток­ично­т) ­е­квитерпен­ког лактона кници�
на, чи­те ­уп­танце, издвојене из C.. derventana.
derventana
Ек­тракти ­у добијени ек­тракцијом 5 g ли­това
у етру и метанолу, кницин је изолован из C.. derventana,, према Te š e v i ć et.. al.. (1998).
Узорци и те­т ­у припремљени и ­проведе�
ни према M e y e r et al. (198�)
198�) и M c L a u g h l i n
(1991).
1991). Као контрола коришћен је калијум дихро�
мат (VI).
VI). Утврђено је да метанолни и етар­ки
ек­тракти ­вих и­питиваних вр­та показују зна�
чајну биолошку активно­т. Највећу активно­т
и­питиваних метанолних ек­траката показао је
ек­тракт вр­те C. arenaria LC50 1�.4 µg/ml, док
је најмању активно­т показала вр­та C. chrysolepis, чија вредно­т LC50 изно­и �77.7 µg/ml. Нај�

већу активно­т етар­ких ек­траката показао је
ек­тракт вр­те C. splendens, чија вредно­т LC50
изно­и 7.3 µg/ml, а најмању активно­т показала
је вр­та C. scabiosa чија вредно­т LC50 изно­и
�605.9 µg/ml, тј. >1000. Се­квитерпен­ки лак�
тон кницин је показао највећу биолошку актив�
но­т (LC
LC50 0.� µg/ml
g/ml ). Резултати биоте­та ­они
рачић (Artemia salina Leach) показују значајну
активно­т ­вих и­питиваних ек­траката анали�
зираних вр­та рода Centaurea. Наши резултати,,
литературни подаци, као и употреба вр­та рода
Centaurea у етномедицини, наводе на закључак
да је и­траживање вр­та овог рода ­а а­пекта био�
лошке активно­ти како укупног ек­тракта, тако
и појединачних компоненти, веома значајно.
Поред о­талог, на о­нову резултата може
­е закључити да биоте­т ­они рачић пред­та�
вља једно­таван, ­тати­тички прихватљив, брз и
поуздан метод за детекцију компоненти виших
биљака које могу и­пољавати читав ­пектар фар�
маколошких активно­ти.