Material and methodsDried leaf material collected at natural habitats (Katalinić brig i Sustipan in Split,Croatia) was used for the preparation of the extracts and the isolation procedure.Isolation of compounds from C. ragusina was carried out using liquidchromatography coupled to mass spectrometry and nuclear magnetic resonance.Antibacterial activities of isolated compounds against Acinetobacter baumanniiDURN [2] and Staphylococcus aureus ATCC25923 were tested according to Lee et al.[3]. Antioxidant activity of isolated compounds was determined by DPPH [4] andABTS [5] assays. Cytotoxic activity was measured by the crystal violet bioassay oncell lines SCCVII, FsaR and B16-F10 [6]. Interactions of isolated compounds withdouble stranded polynucleotides (poly A – poly U and ctDNA) were evaluated by CDspectroscopy [7].

BIOLOGICAL ACTIVITY OF ISOLATED COMPOUNDS FROM

CENTAUREA RAGUSINA L.

Valerija VUJČIĆ1, Sandra RADIĆ BRKANAC1, Marijana RADIĆ STOJKOVIĆ2, Irena ŽILIĆ3, Sonja TOLIĆ3, Adela KRIVOHLAVEK3, Siniša

IVANKOVIĆ4, Dušica IVANKOVIĆ4, Ranko STOJKOVIĆ4, Jasna HRENOVIĆ5, Mirko RUŠČIĆ6, Ulrike GRIENKE 7, and Judith Maria ROLLINGER7

Faculty of Science, University of Zagreb, Department of Biology, Division of Botany1, Ruđer Bošković Institute, Division of Organic Chemistry and Biochemistry2, Institute of Public Health ‘‘Dr. Andrija

Štampar’’, Department of Ecology3, Ruđer Bošković Institute, Division of Molecular Medicine4, Faculty of Science, University of Zagreb, Department of Biology, Division of Microbiology5, 10000 Zagreb,

Croatia, University of Split, Department of Biology6, Split, Croatia, University of Vienna, Department of Pharmacognosy7,Vienna, Austrija

Introduction Centaurea genus represents an attractive source of secondary metabolites, predominantly sesquiterpenelactones (SL) and flavonoids which were shown to possess a wide variety of promising pharmacological andbiological activities [1]. Antibacterial, antioxidant and cytotoxic activity of three flavonoids (chrysin, oroxylin Aand hispidulin) and sesquiterpene lactones (hemistepsin A, deacylcynaropicrin and (3aR,4S,6aR,8S,9aR,9bR)-[Dodecahydro-8-dihydroxy-3,6,9-tris(methylene)-2oxo-2(3H)-azuleno[4,5-b]furanyl]-3-methyl-butanoate)isolated from ethanolic (96%) extract of leaves of Croatian endemic plant species Centaurea ragusina L. werestudied. The interactions of isolated compounds with double stranded polynucleotides ctDNA and poly A – polyU (possible biological targets) were studied using circular dichroism spectroscopy (CD spectroscopy) to providea better understanding of the biological activity of tested compounds.

ConclusionsTwo sesquiterpene lactones (3aR,4S,6aR,8S,9aR,9bR)-[Dodecahydro-8-dihydroxy-3,6,9-tris(methylene)-2oxo-2(3H)-azuleno[4,5-b]furanyl]-3-methyl-butanoate) andhemistepsin A showed significant antibacterial activity against S. aureus (MIC 31,3 mg mL-1) and weak antibacterial activity against A. baumannii. Flavonoid hispidulinshowed moderate antioxidant activity evaluated by ABTS method while other isolated compounds from ethanolic extract showed weak antioxidant activity. Among isolatedcompounds, only SL1 exerted selective and prominent tumor cell-growth inhibitory activity towards SCCVII cell line (IC50 = 2.55µM). Simultaneously, SL1 showed weakinteraction with DNA indicating that some other target/mechanism is responsible for its prominent cytotoxic activity towards SCCVII cell line.

Acknowledgments This study has been co-financed by the European

Union: the European Social Fund as part of the human Resources Development 2007 - 2013, as part of Project "HR.3.2.01-0290 Biological and phytochemical activity

of Centaurea ragusina L. (BioFitoCen)“. http://www.biofitocen.biol.pmf.hr

CHRYSIN (CRE03_13) 254,24 g mol−1

C15H10O4

OROXYLIN A (CRE04_07) 284,26 g mol−1

C16H12O5

HISPIDULIN (CRE06_07) (CAS-No. 1447-88-7, MW 300)

300,27 g mol−1

C16H12O6

HEMISTEPSIN A (CRE15_02) (CAS-No. 208449-03-0, MW 346,37)

346,37 g mol−1

C19H22O6

DEACYLCYNAROPICRIN (CRE17_01) (CAS-No. 31565-50-1, MW 262,31

262,31 g mol−1

C15H18O4

CRE27_02 (= ((3aR,4S,6aR,8S,9aR,9bR)-[Dodecahydro-8-dihydroxy-3,6,9-

tris(methylene)-2oxo-2(3H)-azuleno[4,5-b]furanyl]-3-methyl-butanoate)

CAS-No. 120826-46-2)346,42 g mol−1

C20H26O5

References:

1.Khammar A, Djeddi S. Eur J Sci Res 2012; 84: 398-416.2.Hrenović J, Durn G, Goić-Barisić I, Kovačić A. Applied and environmental microbiology 2014; 80(9): 2860-2866.3.Lee DD, Lee EY, Jeong SH, Chang CL. Korean J Clin Microbiol 2007; 10(1): 49-53.4.Germano MP, Pasquale RD, D’Angelo V, Catania S, Silvari V, Costa C. J Agric Food Chem 2002; 50: 1168–11715.Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Free Radic Biol Med 1999; 26: 1231–1237.6.Ivanković S, Stojković R, Galić Z, Galić B, Ostojić J, Marasović M, Miloš M. J Enzyme Inhib Med Chem 2015; 30: 354-359.7.Tumir L-M, Radić Stojković M, Piantanida I. Beilstein J Org Chem 2014; 10: 2930-2954.

Sample DPPH ABTSCRE03_13 – chrysin 0,091c 0,232d

CRE04_07 – oroxylin A nd 15,027b

CRE06_07 – hispidulin 4,020b 43,972a

CRE15_02 - hemistepsin A (SL2) nd ndCRE17_01 – deacylcynaropicrin (SL3) nd nd

CRE27_02 (SL1) 5,778a 2,293c

GA 95,374 99,421Values represent mean of 3 replicates. Different letters indicate significant difference at p < 0.05. SD < 5%, nd – not detected

Table 2. Antioxidant activity of isolated compoundsestimated by DPPH (DPPH; % inhibition) and ABTS (ABTS; %inhibition)

Bacterial strain A. baumannii S. aureus

Sample MIC (mg mL-1 ) MIC (mg mL-1)

CRE03_13 – chrysin >62,5 62,5

CRE04_07 - oroxylin A >62,5 62,5

CRE06_07 - hispidulin >62,5 62,5

CRE15_02 - hemistepsin A(SL2) >62,5 31,3

CRE17_01 – deacylcynaropicrin (SL3) >62,5 >62,5CRE27_02 (SL1) >62,5 31,3

Values represent mean of 3 replicates. SD < 5%

Table 1. Minimum inhibitory concentrations (MIC) of isolatedcompounds against Acinetobacter baumannii DURN and Staphylococcusaureus ATCC25923 cde

abc

ab

e

h

f

bcdab

de cde

g

e

0

50

100

Control CRE03_13 CRE04_07 CRE06_07 CRE17_01 CRE27_02 CRE15_02

SCCVII 1 1/2ab

c

ab ab ab

bc

d d

abcabc ab

bc

d

c

0

50

100

Control CRE03_13 CRE04_07 CRE06_07 CRE17_01 CRE27_02 CRE15_02

FsaR

bcde

abcabc

bcde cde

ede

cde

bcde bcdebcde

decde

0

50

100

Control CRE03_13 CRE04_07 CRE06_07 CRE17_01 CRE27_02 CRE15_02

B16-F10

Figure 2. Percentage of cell survival of SCCVII, FsaR and B16-F10 cells after exposure to isolated compounds at higher (5.1µM) and lower concentration (2.55 µM).

Figure 1. CD titration of ctDNA (c = 3.0 10-5 M) withCRE27_02 (pH 7.0, buffer sodium cacodylate, I = 50mM).

Sesquiterpene lactone - SL1

Sesquiterpene lactone - SL2

Sesquiterpene lactone - SL3

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