Cytotoxic and Antibacterial Activities of Centaurea cadmea Boiss.
Transkript
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). 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