biological activity of isolated compounds from€¦ · isolation of compounds from c. ragusina was...
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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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