Cytotoxic and Antibacterial Activities of Centaurea cadmea Boiss.

Turk J Pharm Sci 11(1), 101-106, 2014
Original article
Cytotoxic and Antibacterial Activities of Centaurea
cadmea Boiss.
Kaveh Alizadeh ASTARİ 1 , Şura BAYKAN EREL 1 , Fadime AYDIN KÖSE 2 ,
KÖKSAL 3 , Canan KARAALP 1 *
£inel
'Ege University, Faculty of Pharmacy, Department of Pharmaceutical Botany, 35100 Bornova,
İzmir, TURKEY, 2Ege University, Faculty of Pharmacy, Department of Biochemistry, 35100
Bornova, İzmir, TURKEY, 3Ege University, Faculty of Science, Department of Biology, 35100
Bornova, İzmir, TURKEY
The cytotoxic activity of the extracts obtained from roots and the aerial parts of Centaurea cadmea Boiss.
(Asteraceae) were analyzed by cell proliferation assay using WST-1 reagent against three human cancer
cell lines; HeLa, A549 and U20S and one non-cancer cell line; 293HEK. The chloroform extract of the
aerial parts of the plant exhibited inhibitory activities against all cell lines (IC50: 14.24-43.10 μg/mL). The
antibacterial activity of the extracts were tested against four gram negative (Escherichia coli ATCC 23999,
Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium CCM 5445 and Klebsiella pneumoniae
CCM 2318 and four gram positive (Staphylococcus aureus ATCC 6538/P, S. epidermidis ATCC 12228,
Enterococcus faecalis ATCC 29212 and Bacillus cereus ATCC 7064) bacteria strains by broth dilution
method. The chloroform extract of the aerial parts of the plant showed strong activity on E. faecalis (8 μg/
mL) and B. cereus (16 μg/mL).
Key words: Centaurea cadmea, Antibacterial activity, Cytotoxic activity.
Centaurea cadmea Boiss.’in Sitotoksik ve Antibakteriyal Aktiviteleri
Centaurea cadmea Boiss. (Asteraceae) kök ve topraküstü kısımlarından elde edilen ekstrelerin sitotoksik
aktiviteleri, WST-1 reaktif kullanılarak üç insan kanser hücre hattı; HeLa, A549 ve U20S ve bir kanser
olmayan hücre hattı; 293HEK üzerinde, hücre proliferasyon testi ile analiz edilmiştir. Topraküstü kısımların
kloroform ekstresi tüm hücre hatlarında aktif bulunmuştur (IC50: 14.24-43.10 μg/mL). Ekstrelerin
antibakteriyel aktivitesi dört gram negatif (Escherichia coli ATCC 23999, Pseudomonas aeruginosa ATCC
27853, Salmonella typhimurium CCM 5445 ve Klebsiella pneumoniae CCM 2318) ve dört gram pozitif
(Staphylococcus aureus ATCC 6538/P, S. epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212
ve Bacillus cereus ATCC 7064) bakteri üzerinde broth dilüsyon metodu ile test edilmiştir. Toprak üstü
kısımların kloroform ekstresi, E. faecalis (8 μg/mL) ve B. cereus (16 μg/mL) üzerinde güçlü etki göstermiştir.
Anahtar kelimeler: Centaurea cadmea, Antibakteriyal aktivite, Sitotoksik aktivite.
*Correspondence: E-mail: [email protected], Tel: +90 232 3114084
101
Kaveh Alizadeh ASTARİ, §ura BAYKAN EREL, Fadime AYDIN KÖSE, Qinel KÖKSAL, Canan Karaalp
INTRODUCTION
EXPERIMENTAL
The genus Centaurea L. (Asteraceae) is Plant material
Centaurea cadmea Boiss. was collected from
represented by 199 taxons in Turkish fora and
o
61.1% of them are endemic (1-10). Various Denizli, Evrantepe, 1512 m, in June 2004 (37
o
species of Centaurea are used as herbal 41’ 18.6’’N; 29 00’ 07’E) and identifed by Prof.
remedies for their digestive, tonic, expectorant, Dr. Ozcan Secmen, from Section of Botany,
antipyretic and antidiarrheal effects in Department of Biology, Faculty of Science,
traditional medicine (11). Pharmacological Ege University, Izmir, Turkey and a voucher
studies on some Centaurea species have specimens were deposited in the Herbarium
reported anti-infammatory, antimicrobial, of Ege University, Faculty of Pharmacy, Izmir,
antipyretic, cytotoxic and immunological Turkey (IZEF 5670).
activities (12).
Extraction and isolation
Centaurea cadmea Boiss. belonging to
Dried and powdered roots (200 g) were
section Phalolepis (Cass.) DC. (Asteraceae) extracted sequentially with chloroform and
with purple forets is an endemic taxon for methanol and the aerial parts (600 g) were
Anatolia, growing wild in N, W & SW of extracted also sequentially with n-hexane,
Turkey (1). Phytochemical studies revealed chloroform and methanol (3x10 mL/g, for
the presence of a sesquiterpene lactone, each), sonicated at room temperature for 24h,
ivalin, together with eupatorin, 5-hydroxy- and then fltered. All solvents were analytical
3’,4’,6,7-tetramethoxyfavone, β-sitosterol and grade and obtained from Sigma Aldrich. The
β-sitosterol-3-O-β-D-glucopyranoside from the combined extracts were evaporated under
CHCl3 and MeOH extracts of aerial parts of C. reduced pressure to dryness at 40 0 C.
cadmea (13). Essential oil analysis was also
Cytotoxic activity
reported for the plant (14).
The cytotoxic activity was analyzed by
In vitro anti-infammatory, antioxidant,
cell
proliferation assay. U2OS (human
antiprotozoal and antifungal activities of the
osteocarcinoma),
A549
(human
lung
aerial parts of C. cadmea extracts have been
adenocarcinoma), HeLa (human cervical
previously studied (15, 16). Formerly, we have
carcinoma) and non-tumoral 293HEK (human
reported the cytotoxic and antibacterial efects
embryonic kidney) cell lines were cultured in
of the roots of C. cadmea as a poster presentation DMEM supplemented with L-glutamine (2
(17). As a continuation of this study, aerial parts mmol/L), 100 U/mL penicillin, 100 μg/mL
of the plant was planned to be searched for streptomycin and 10% fetal bovine serum. In
the same activities. So, the present study aims order to perform cytotoxicity assay, 5.000 cells
to investigate the cytotoxic and antibacterial were seed in 96 well dishes and 24 hours later
activities of extracts obtained from roots and samples were added in various concentrations
the aerial parts of C. cadmea. Cytotoxic activity (500, 250, 100, 50 mg/mL). Forty-eight hours
was performed by cell proliferation assay using after drug exposure, cell viability was measured
WST-1 reagent against three human cancer using WST-1 cell proliferation reagent (Roche)
cell lines; U2OS (human osteocarcinoma), according to the manufacturer instructions (18).
(adenocarcinoma), HeLa (human cervical All measurements were performed in triplets.
carcinoma) and one non-cancer cell line;
293HEK (human embryonic kidney). T e Antibacterial activity
Antibacterial acitivity tests of C. cadmea
antibacterial activity of the extracts were
tested against four gram negative (Escherichia extracts were evaluated by using Minimum
coli ATCC 23999, Pseudomonas aeruginosa Inhibitory Concentration (MIC) measurements
ATCC 27853, Salmonella typhimurium CCM (19, 20). The MIC values were determined
5445 and Klebsiella pneumoniae CCM 2318) for eight bacterial strains [Escherichia coli
and four gram positive (Staphylococcus aureus (ATCC 23999), Pseudomonas aeruginosa
(ATCC 27853), Salmonella thyphimurium
ATCC 6538/P, S. epidermidis ATCC 12228,
(CCM 5445), Klebsiella pneumoniae (CCM
Enterococcus faecalis ATCC 29212 and Bacillus
2318), Staphylococcus aureus (ATCC 6538cereus ATCC 7064) bacteria strains by broth
P), Staphylococcus epidermidis (ATCC
dilution method.
12228), Bacillus cereus (ATCC 7064), and
Enterococcus faecalis (ATCC 29212)].
102
Turk J Pharm Sci 11(1), 101-106, 2014
Those strains were inoculated on MuellerHinton broth (Difco) and incubated at 37
± 0.1oC for 24 h. The inoculated strains
were prepared from 24 h broth cultures and
suspensions were adjusted to 0.5 McFarland
standard turbidity and diluted 1:100 (v/v) in
Mueller-Hinton broth. Dilution series of the
compounds were prepared in test tubes and
transferred to the broth in 96-well micro titer
plates. Final concentrations were 256 to 0.25
µg/mL in the medium. The last well contained
100 mL of nutrient broth without compounds
and 10 mL of the inoculums on each strip was
used as a negative control and Gentamycin
(Sigma Aldrich) was used as a positive control.
All plates were covered with a sterile plate
sealer and incubated at 37oC for 24 h.
After incubation, MIC values were detected
Those strains were inoculated on Muellerby adding 50 mL of 0.5% triphenyl tetrazolium
Hinton broth (Difco) and incubated at 37 ±
chlooride (TTC, Fluca) aqueous solution and
0.1 Cwere
for defned
24 h. The
inoculated
strainstration
were
they
as the
lowest conce
prepared
from
24 growth
h brothas cultures
that
inhibited
visible
indicated and
by
suspensions
were
adjusted
to
0.5
the TTC reduction. In the presence ofMcFarland
bacterial
strongest activity was observed on HeLa (IC50:
14.24 µg/mL) by chloroform extract of aerial
parts. But it is also active against non-cancerous
cell line 293 HEK, which is used for detecting
selectivity. Hexane extract had weak activity
on U2OS and showed moderate effect on the
other cell lines (43.05 to 79.03 µg/mL). MeOH
extract of aerial parts and the extracts of roots
were not exhibited cytotoxic activity.
In a previous report we have evaluated in
vitro cytotoxic properties of extracts of fve
Centaurea species and the strongest effect has
been determined on chloroform extract of C.
polyclada on KB (human epidermal carcinoma,
oral) and BT-549 (breast ductal carcinoma) cell
lines (33 and 30 µg/mL, respectively) (21).
Most of the studies involved the activities of
pure compounds isolated from various extracts
concentration that inhibited visible growth as
of C aurea species. Especial y isolated
indicated by the TTC reduction. In the
sesquiterpenes and favonoids were found to be
presence of
bacterial
growth
by(22-26).
reduction
responsible
for
cytotoxic
p perties
reactions,
TTC
changed
the
color
In our earlier study, we hav isolated of
a
microorganisms
from ivalin,
colorless
red.
This
sesquite pe e lactone,
fromtothe
CHCl
3
provided
clearly
defined
easily(13).
readable
gsrtoawndtharbdy turrebdiudcityionanrdeadcitliuotnesd, T1 :T1C00 c (hva/nvg)e idn extract
of aeri
l parts
of C.and
cadmea
The
thMeuceolleor-Ho finmtoincroborrogthan.iDsmilsutfiroonm sceroileosrleosfs t thoe aecntidvpitoyinotfs.chAl lolr of othrmeaesxstaryasctw oefreaepreiarflopr amr tesd o ifn
recdo.m Tphoiusndpsrowviedreed pcrlepaarlryeddeinfntedst a tnudbeesasainlyd thtreipplliacnatem.a y b e d u e t o t h e p r e s e n c e o f i v a l i n
retraadnasbfelerreedndtopotihnetsb.roAtlhl inof96th-weeallssmayicsrow tei trer that is known as a potent cytotoxic compound
ppelraftoersm.eFdin ianl trciopnl icceantetr.ations w e r e 2 5 6 t o 0 . 2 5on several tumor cell lines (27).
µg/mL in the medium. The last well contained RAEntSimUiLcrTobSiaAlNacDtivDitIieSsC wUeSreS ItOesNted against
RESU TS AND DISCUSSION
8 bacteria strains by using NCCLS method.
100 µL of nutrient broth without compounds ReYsuiletlsdasr e o sfhonw-hne ixnanTea,bleC 3H .CTl h3e acnhdlorMofoeOrmH
aYndiel1d0s µLofof nt-hhee ixnaonceu,lumCHsCo nl3 eanchd stMripeOwHaseexxtrtaracctt osf othbetaianeerdial fproamrts roofothse apnldanth sehoawe rei dal
strong
on E. f ecali
(8 mg/mL)
nd B.
euxstreadctassobatanineegdatfirvoem c ronottrso alnadn tdheG aernitalm pyacrtisn
parts activity
of C. cadmea
is shown
in Table
1.
cereus
(16
mg/mL)
with
concen
rations
more
o(fS Cig.mcadmAeladrisic shh)owwn a isn Tuasbelde 1 a.sExatracptosstiht ievne
Extracts then were tested for their potential
equal toand
the antibacterial
standart antibiotic
gentamycin.
wceornetrotels.tAedll fpol ratethse wi rerpeotceonvteiarledcywtoitthoxaicstearnidleorcytotoxic
activities.
apnltaibteacsteearliearl ancdtivinitcieusb.ated a t 3 7 o C f o r 2 4 h .
MeOH extract also had a strong effect on these
TAhfetecry itnoctouxbiactioanc,tivMi tIyC rveasulultess awrereprdeesetenct teeddstrains (16 mg/mL, both). Hexane extract of the
inby Taabdledin2g. T5h0e nµ-hLexaonfe a0n.5d% chltorripohfoernmy l aerial parts and MeOH extract of the roots have
etxettracztos
l oiufm
t h e c ahel roiraild
pear(tTs
fC t,heF
plluacnat)
exahqiubeitoeuds
weak activity against all tested microorganisms
inhibit ry
activities
against
alloTcell
lines.
The
solution and they were defined as the lowest (64-256 mg/mL).
Table 1. Yields of various extracts of C. cadmea.
Obtained extracts (g)
Yield of extracts
(% of dry weight)
Root CHCl3
1.5
0.75
Root MeOH
5.36
2.68
Aerial parts n-hexane
7.8
1.3
Aerial parts CHCl3
23.58
3.93
Aerial parts MeOH
53.51
8.91
The cytotoxic activity results are presented
in Table 2. The n-hexane and chloroform
extracts of the aerial parts of the plant
exhibited inhibitory activities against all cell
lines. The strongest activity was observed on
HeLa (IC50: 14.24 µg/mL) by chloroform
extract of aerial parts. But it is also active
been determined on chloroform extract of C.
polyclada on KB (human epidermal
carcinoma, oral) and BT-549 (breast ductal
103
carcinoma) cell lines (33 and 30 µg/mL,
respectively) (21). Most of the studies
involved the activities of pure compounds
isolated from various extracts of Centaurea
Antimicrobial activities were tested against
more or
the standart
antibiotic
Kaveh Alizad e h ASTARl, §ura BAYKAN EREL, Fa dime AYDIN ROSE, Qinel KOKS AL, Canan Karaalp
Antimicrobial activities were tested against
8 bacteria strains by using NCCLS method,
Results are shown in Table 3. The chloroform
extract of the aerial part s of the plant showed
strong activity on E. faecalis (8 mg/mL) and B.
cereus ( 16 mg /mL) with conce ntrations more
Table 2. Cytotoxic activities of C. cadmea extracts (IC 50 , |lg/mL).
U20S
Root CHCl 3
Root CHCl 3
Root MeOH
Root MeOH
Aerial parts nhAeexraianle parts nhexane
Aerial parts
CAHerCiall3 parts
CHCl3
A e r i a l parts
MA eerOi aHl p a r t s
NT: Not tested, NA: Not
A549
HeLa
293HEK
NA
NA
43.05
23.50
>100
NA
>100
79.03
HeLa
NT
NT
NT
NT
50.25
43.10
35.00
14.24
NA
NA
NA
NA
NA
NA
active at 500 µg/mL concentration.
o r Ae qnut iamlitcorot hbeia slt ancdtiavrittiaenstiwb ieor teictegsetnetdamagyaciins. t mThoerere oa r e esqeuvaelraltorepthoertss t o a n d a n r t imainctriobbioiatilc
M 8e AOb naHtcit me xriictar aoscbtrialisn oas c htbiavydituai e ssitn r wgo neNgr e Ce ftCfe eLsctSeta do
cgmtm
eionvrtieatn ai me
egthtahei
soy ons
rco des
ifn .t
e.dq i Muf faelr OeHnt ot eCxtehtnreatactus traelnas do asphr tea cd ia eans t isfbtriomntigc
s tR8raebisnausclt(se1 r6ai ra me sgsth/r maoiwLn sn,b ibonyth Tu)a.s bHi lneg x 3aN. nTCe h TCeexL
eguferfkneet
tcSrhalcomt
cayt m reotfh
. oy otVcnohraimde
nr it.oh
. MueseO e sHxttr raeixcntsra c(ot1 f 6alsµoi x g h/maCdLena,tasbut orrotehna)g .
a eRxreitasr ulaclpttasroat fsre at hns edhoaMwe reni Oa il Hn p Tae rxatbtsr l aeoc f t3 to. hfTe t h pe tl acr
aHexfnohtol
eof exnotssrho
castne(o
hof awovremed
Cen.xt rtphasceetsu eod fo sstchraeib naisoesria(a1 l 6sp uabµr stgsp/.ma nLgdl,eMcbheonOtihiH,) .
wseetxratokr na agc cttaoi cvfti itvyh i eat y ga aeoirniastEl a.plalfarttessc toaefld i s tmh(ei8 c p rCeH.
µol aognr/
xetsxrpaicn
tmg asLhntae)oio swma
taef, nesd
x tthrCea. c rtog olaft s t thifeaov laieae rw, i aeCla .kpasaractlstoi vnaintydanaMag,aeiOCn Hs.
(6Bs t4.r -o2cn5eg6reamucsgti/vm(i1tLy6 ).oµng E/m.Lfa)ecwailtihs (8coµncge/mntLra)tbaeiaonds
lxlstart eamscitetaod f a m nt hdi ceCr or.oboregt sha enhnias)vm heasw d(6eba4ek-e2 na5 ce6tviµvailgtu/yam taeLgd)a .fi
B. cereus (16 µg/mL) with concentrations
all tested microorganisms (64-256 µg/mL).
Antibacterial activities of
extracts (µg/mL).
Table 3. Antibacterial activities of C. cadmea extracts (ug/mL).
Aerial
Microorganisms
pAaretrsianlAerial
Aerial
Root
Root
phaerxtas nneMicroorganisms
Gentamycin
parts
parts
CHC13
MeOH
hexane
MeOH
CHCI3
128
AEsTcCheCri2c3h9ia99coli
128
> 256
128
128
128
SAtTapChCyl2o3c9o9cc9us aureus
1.0
256
AStTapChCyl6o5c3o8cc/Pus aureus
> 256
256
256
128
128
SA.T eCpiCde6r5m3i8d/iPs
1.0
128
AS.T eCpiCde1r2m2i2d8is
256
128
128
> 256
128
SAaTlmCoCn 1e2ll2a2 t8yphimurium
1.0
128
CSaClmMo5n4e4ll5a typhimurium
64
128
128
> 256
128
BCaCcMillu5s44ce5reus
1.0
64
ABaTcCilClus70c6e4reus
128
16
16
> 256
64
KAlTeCbsCie 7ll0a6 p4neumoniae
4.0
128
CKCleMbsi2e3ll1a8 pneumoniae
128
128
128
128
>256
ECnCtMero 2c3o1c8cus faecalis
4.0
64
AEnTtCerCoc2o9c2c1u2s faecalis
128
8
16
> 256
64
PAsTeCudCo2m9o2n1a2s aeruginosa
16.0
128
APsTeCudCo2m7o8n5a3s aeruginosa
64
128
128
> 256
128
ATCC 27853
2.0
hsteaplxei
ncoaontisad
thaecTitrih v eai rtniete i sma roi ecfrdos eibfvifaelraealn cttr ieCvpietoni rettasu broyen a dasi nspcteicmdieifcs
urosf riboinma
l ,(e Cs.C ph.saedgu l dasoshtsoi cfwoa lbni ia o, s toaC s.uh bavsapel.o gsnliegt acn
e fbsfpaei
stat ma
tag Ca ai.n sdtgl Ca .s Bt bi rf eao hnl iehana,) m hCeal.ld a sbaeleconan tieat avr nrahal ua, alitse
mTa ecut hri kvoeidtyi. e . sE Vtohaf a r ndioifulf se a rnednx tterCat hceytnsltaaoucfretasitxes p eCxceit
enrlcastac
fturi,aroemfa
S bt aaplhs ay mloictaocacnuds a Cu.r ebues h aend) Hhaedlicboebeanctevraplyulaotreid
CT.urgkleayst.ifoVliaar iohuasv exstrhaocwtsedof stsrioxngCeancttaivuirteya
on Staphylococcus epidermidis and Proteus (29).
mirabilis when compared with ciprofoxacin
E. faecalis can cause life threatening
(28). In another report, water extract of C. gastrointestinal infections in humans which
104
Turk J Pharm Sci 11(1), 101-106, 2014
has high levels of antibiotic resistance and
B. cereus is responsible for a minority of
foodborne illnesses, causing severe nausea,
vomiting and diarrhea (30). C. cadmea may
be a potential natural source for discovering
new anti bacterial agents due to its remarkable
activity on these pathogens.
ACKNOWLEDGEMENT
Authors are appreciated to U. KarabayYavasoğlu, Ph.D. and P. Ballar, Ph.D. for their
scientifc contribution.
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Received: 28.03.2013
Accepted: 02.05.2013