Chemical constituents， cytotoxic， antifungal and antimicrobial properties of Centaurea diluta Ait. subsp. algeriensis （Coss. & Dur.） Maire

Hanne Zater， Jo?lle Huet， Véronique Fontaine， Samir Benayache， Caroline Stévigny，Pierre Duez，6*， Fadila Benayache*Unité de recherche : Valorisation des Ressources Naturelles， Molécules Bioactives et Analyses Physicochimiques et Biologiques （VARENBIOMOL）， Faculté des Sciences Exactes， Université Frères Mentouri Constantine ， 2000 Constantine， Algérie

2Université Ziane Achour， Cité du 5 Juillet， Route Moudjbara BP : 3117， 17000 Djelfa， Algérie

3Laboratoire de Pharmacognosie， de Bromatologie et de Nutrition Humaine， Université Libre de Bruxelles （ULB）， 1050 Bruxelles， Belgique

4Laboratoire de Biopolymère et nanomatériaux supramoléculaire， Université Libre de Bruxelles （ULB）， 1050 Bruxelles， Belgique

5Unité de Microbiologie Pharmaceutique et Hygiène， Faculté de Pharmacie， Université Libre de Bruxelles （ULB）， 1050 Bruxelles， Belgique

6Service de Chimie Thérapeutique et de Pharmacognosie， Université de Mons （UMONS）， 7000 Mons， Belgique

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Chemical constituents， cytotoxic， antifungal and antimicrobial properties of Centaurea diluta Ait. subsp. algeriensis （Coss. & Dur.） Maire

2Université Ziane Achour， Cité du 5 Juillet， Route Moudjbara BP : 3117， 17000 Djelfa， Algérie

3Laboratoire de Pharmacognosie， de Bromatologie et de Nutrition Humaine， Université Libre de Bruxelles （ULB）， 1050 Bruxelles， Belgique

4Laboratoire de Biopolymère et nanomatériaux supramoléculaire， Université Libre de Bruxelles （ULB）， 1050 Bruxelles， Belgique

5Unité de Microbiologie Pharmaceutique et Hygiène， Faculté de Pharmacie， Université Libre de Bruxelles （ULB）， 1050 Bruxelles， Belgique

6Service de Chimie Thérapeutique et de Pharmacognosie， Université de Mons （UMONS）， 7000 Mons， Belgique

ARTICLE INFO ABSTRACT

Article history:

in revised form 16 March 2016 Accepted 15 April 2016

Available online 20 June 2016

Flavonoids

Lignans

Centaurea diluta

Asteraceae

Cytotoxic activity

Direct and indirect antimicrobial activity

MRSA

Objective: To investigate the chemical composition of a moderately polar extract （CHCl3soluble part of the MeOH-H2O extract） obtained from the aerial parts （leaves and flowers） of Centaurea diluta Ait. subsp. algeriensis （Coss. & Dur.） Maire， a species endemic to Algeria and Morocco on which no reports are available to date. To evaluate in vitro the cytotoxic， antifungal and antimicrobial activities of this extract and the cytotoxic and antimicrobial activities of its isolated secondary metabolites. Methods: The cytotoxic effects of the extract were investigated on 3 human cancer cell lines i.e. the A549 non-small-cell lung carcinoma （NSCLC）， the MCF7 breast adenocarcinoma and the U373 glioblastoma using a MTT colorimetric assay. Biological data allowed to guide the fractionation of the extract by separation and purification on silica gel 60 （CC and TLC）. The isolated compounds which were characterized by spectral analysis， mainly HR-ESIMS， HR-EIMS，UV and NMR experiments （1H，13C， COSY， ROESY， HSQC and HMBC） and comparison of their spectroscopic data with those reported in the literature， were evaluated for cytotoxic activities on six cancer cell lines （A549， MCF7， U373， Hs683 human glioma， PC3 human prostate and B16-F10 murine melanoma）. The direct and indirect antibacterial and antifungal activities were determined using microdilution methods for the raw extract and TLC-bioautography and microdilution methods against standard and clinical strains for the isolated compounds. Results: The raw extract reduced cell viability with IC50s of 27， 25 and 21 μg/mL on A549， MCF7 and U373， respectively. Five secondary metabolites: two phenolic compounds （vanillin 1， paridol 3）， a lignan ［（-）-arctigenin 2］and two flavonoid aglycones （eupatilin 4 and jaceosidin 5）， were then isolated from this extract. Moderate cytotoxic effects were observed for （-）-arctigenin 2 （IC50s: 28 and 33 μM on Hs683 and B16-F10， respectively）， eupatilin 4 （IC50s: 33 and 47 μM on B16-F10 and PC3， respectively） and jaceosidin 5 （IC50s: 32 and 40 μM on PC3 and B16-F10， respectively）. Conclusions: All the isolated compounds were described for the first time from this species. Although inactive against 7 tested microorganisms （fungi， bacteria and yeast， human or plant pathogens）， the raw extract was able to potentiate the effect of beta-lactam antibiotics on methicillin-resistant Staphylococcus aureus （MRSA），reducing the minimal inhibitory concentrations （MICs） by a factor of 2-32-fold. No synergy was found between the extract and streptomycin. From the five isolated compounds only jaseosidin 5 showed a moderate antimicrobial activity.

1. Introduction

The genus Centaurea （tribe Cynareae， family Asteraceae） is one of the most widely distributed plant genera in the world. Centaurea includes more than 500 species， 45 of which grow spontaneouslyin Algeria， with 7 species localized in the Sahara［1， 2］. Although， to our best knowledge， no traditional uses or pharmacological studies are reported so far for the species Centaurea diluta （C. diluta）， many other Centaurea species are well known in traditherapy. For example，in Turkey， dried flowers of Centaurea cyanus are used in infusion to relieve diarrhea， gain energy， increase appetite， and to relieve chest tightness； Centaurea calcitrapa is used （infusion） as a febrifuge；Centaurea jacea is used to reduce fever， to start menstruation， to relieve constipation and increase appetite［3， 4］. In Tunisia， Centaurea furfuracea， an endemic species from the desert regions of the North of Africa［5］， is used as astringent and diuretic［6］， while， in Algeria，the roots of Centaurea incana are used in the area of Aurès for the treatment of liver diseases［7］ and Centaurea pullata is used in the preparation of a local traditional dish called “El Hammama”［8］. Various studies have shown medicinal properties of Centaurea species， mainly as analgesic［9］， cytotoxic［10］， antibacterial［11］ and antifungal［12］.

Centaurea typically present high structural diversity in major bioactive compounds， including triterpenes， flavonoids， lignans and sesquiterpene lactones［13-21］. In specimens of C. diluta， cultivated in the botanical garden of the Technical University of Braunschweig，Germany， polyacetylenic compounds have been reported［22-24］. In the essential oil of C. diluta Aiton aerial parts， collected from Sicily， Italy［25］， the most abundant compounds were fatty acids and derivatives， notably hexadecanoic acid （21.3%） and （Z，Z）-9，12-octadecadienoic acid methyl ester （12.2%）， followed by hydrocarbons （15.3%）， terpenoids being present in low amounts（2.8 %）.

Given the interest of Centaurea pharmacology and phytochemistry，the present paper concentrates on a relatively unknown subspecies，C. diluta Ait. subsp. algeriensis （Coss. & Durieu） Maire［26］， endemic to Algeria and Morocco［2］.

2. Material and methods

2.1. Chemicals， reagents and general

Solvents were analytical grade. Trypsin 0.5% in EDTA， RPMI1640 red phenol and fetal bovine serum （FBS） were purchased from Gibco? Invitrogen （Merelbeke， Belgium）. 3- （4，5-dimethylthiazol-2-yl） -2，5-diphenyl tetrazolium bromide （MTT） was obtained from Sigma Aldrich?（Bornem， Belgium）. Dimethyl sulfoxide（DMSO） was obtained from Merck?（Overijse， Belgium）. RNAsefree water was from Braun?（Machelen， Belgium）. The Penicillin V was purchased from Certa SA ACA Pharma NV， the ampicillin，amoxicillin and oxacillin were purchased from Sigma-Aldrich.

The absorbance of the reaction mixture of MTT test was measured by spectrophotometer microplate reader Model 680XR， Bio-Rad?，Nazareth Eke （Belgium）. The cells were counted by Cells Culture Counter， Beckman （Analis?， Suarlée， Belgium）. The following apparatus were also used: optical microscope PCM-type Axiovert S100 （Zeiss， Nederlands） and laminar flow hood class II （IKS?，Leerdam， Nederlands）.

Melting points were determined on a SMP10 Büchi B-540 Stuart Biocote apparatus and are uncorrected. Plant material powdering: Mill: Culatti， CZ13 model， Reference DCFH48. TLC: pre-coated aluminium foil silica gel 60F254& TLC silica gel 60F254Plastic roll 500×20 cm （Merck KGaA， Germany）， visualized using UV lamp（CAMAG 254 nm & 366 nm） and by detection with a spraying reagent （vanillin-sulfuric at 10% and/or anisaldehyde） followed by heating at 100 ℃ for 3-5 minutes. Column chromatography （CC）: silica gel 60 （Merck KGaA， Germany， 230-400 mesh ASTM）. Routine Preparative thin-layer chromatography （PLC）: silica gel plates （20×20 cm Silica gel 60 PF254， Merck）， Optical rotation: Perkin-Elmer 241 polarimeter atλNa589 nm.

UV spectra were recorded using a Thermo Electron Corporation evolution 300 spectrophotometer.1H NMR and13C NMR spectra were recorded on Bruker Avance 300， 400 MHz and Varian 600 MHz； 2D-NMR experiments （COSY， HSQC， HMBC， NOESY and ROESY） were performed on Bruker Avance 400 MHz or Varian 600 MHz spectrometers. Spectra of compounds 1， 2 and 3 were recorded in CDCl3， compound 4 in DMSO-d6and compound 5 in CD3OD. A Shigemi tube was used for compound 2.

High resolution mass spectra in positive mode were recorded by direct infusion using a 6520 series quadrupole time-of-flight（Q-TOF） mass spectrometer （Agilent， Palo Alto， CA， USA） fitted with an electrospray ionization （ESI） source in positive mode. The error between the observed and calculated masses is expressed in ppm； below 5 ppm， the compounds were considered to correspond to predicted formula.

2.2. Plant material

The aerial parts of C. diluta Ait. subsp. algeriensis （Coss. & Dur.）were collected in the flowering stage in the area of Djelfa （1 038 m， 34o53′39.6′′N， 3o3′56.3′′E） in June 2012. The plant was authenticated by Professor Mohamed Kaabache， specialist in the identification of Algerian Centaurea species （Ferhat Abbas University，Setif， Algeria）. A voucher specimen has been deposited in the National Herbarium of Belgium （National Botanical Garden of Meise） under the number BR0000013666187.

2.3. Extraction and isolation

Air-dried aerial parts （leaves and flowers， 1.5 kg） of C. diluta Ait. subsp. algeriensis （Coss. & Dur.） were powdered （slight grinding with controlled temperature， up to 35 ℃） and macerated at room temperature with MeOH-H2O （77:23， v/v） （25 L） for 48 h， four times. The filtrates were combined， concentrated under reduced pressure， diluted in H2O （600 mL） under magnetic stirring and maintained at 4 ℃for one night to precipitate a maximum of chlorophylls. After filtration， the resulting solution was successively extracted with solvents with increasing polarities （petroleum ether，chloroform， ethyl acetate and n-butanol） ［27，28］. The present study focused on the chloroform soluble part which was dried with anhydrous Na2SO4， filtered and concentrated under vacuum at room temperature to yield the CHCl3extract （4.0 g， yield: 0.27%， w/w）. The chloroform extract was fractionated by column chromatography（120 g of silica gel； CH2Cl2/EtOAc/MeOH step gradients） to yield 23 fractions （F1-F23）， combined according to their TLC profiles.

Fraction F3 （26.2 mg） （CH2Cl2/EtOAc 98:2） was subjected to preparative TLC on silica gel； eluting with petroleum ether/EtOAc /acetone （6:3:1） yielded vanillin 1 as white crystals （3.5 mg） ［29，30］. Fractions F4 （12.2 mg） （CH2Cl2/EtOAc 98:2）， F5 （13.0 mg）（CH2Cl2/EtOAc 98:2） and F6 （28.5 mg） （CH2Cl2/EtOAc 95:5） were combined and rechromatographed by CC （600 mg of silica gel；cyclohexane/EtOAc/acetone step gradients） to yield 21 subfractions（F’1-F’21） according to TLC profiles. Subtraction F’3 （cyclohexane/ EtOAc/acetone 6:6:2） yielded （-）-arctigenin 2 （3.2 mg） ［17， 31］ and Subtraction F’4 （cyclohexane/EtOAc/acetone 5:2.5:2.5） gave paridol 3 （4.5 mg） ［32， 33］. Fraction F8 （CH2Cl2/EtOAc 95:5） （44.4 mg） yielded up on concentration a yellowish compound which was washed with MeOH to obtain eupatilin 4 （7.5 mg） as needles ［11， 34］. Fraction F10 （CH2Cl2/EtOAc 87.5:12.5） （19.60 mg） was chromatographed on preparative plates of silica gel eluted with CH2Cl2/EtOAc （4:1） to give jaceosidin 5 as a yellowish powder （5.0 mg） ［35， 36］.

2.4. Cell cultures

The human cancer cell lines included the A549 （Deutsche Sammlung von Mikroorganismen und Zellkulturen， DSMZ code ACC107）， NSCLC carcinoma， the U373 （European Collection of Cell Culture， ECACC 08061901） glioblastoma， the PC3 prostate carcinoma （DSMZ code ACC465）， the Hs683 glioma （American Type Culture Collection， ATCC code HTB-138） and the MCF7（DSMZ code ACC115） breast adenocarcinoma. The murine tumor cell line included the B16-F10 （American Type Culture Collection ATCC code CRL-6475） melanoma.

2.4.1. Viability assay

The cytotoxic properties of the raw chloroform extract and isolated compounds were assessed， using a 3-（4，5-dimethylthiazol-2-yl）-2，5-diphenyltetrazolium bromide （MTT） assay ［37，38］. Briefly， this test is based on the capability of living cells to reduce the yellow MTT to a blue formazan compound， a reaction mediated by the mitochondrial succinate dehydrogenase. Cells were seeded （cells per well， A549: 1 500； B16F10: 1 000； Hs683:1 500； MCF7:2 800； PC3:3 000；U373:1 800） and allowed to adhere for 24 h before adding test compounds （100 μL； final concentrations from 10-4M to 10-8M）. In the same condition， for the chloroform extract， cells were seeded（cells per well， A549: 1 200； MCF7: 2 500； U373: 1 800）； 100 μL；final concentrations from 100 μg/mL to 10-2μg/mL）. The cells in medium alone without drug were considered as a negative control. After 72 h contact， the culture medium was replaced by a 0.5 mg/mL MTT solution in RPMI medium without phenol red （100 μL/well）. After 3 h incubation， the formazan crystals were centrifuged and dissolved in 100 μL/well of DMSO. The absorbance of each well was then measured at 570 nm and 690 nm （reference） wavelength. The IC50values were calculated as follows:

IC50= ［（X2 - X1） x （50 - Y1） / （ Y2 - Y1）］+ X1， where

X1 and X2: are the higher and lower concentrations that border the concentration that reduces the global cell growth by the value closest to 50 %.

Y1 and Y2: are the mean percentages of viable cells at the X1 and X2 concentrations.

2.5. Antimicrobial and antifungal assays

2.5.1. Microorganisms

The microorganisms used in the antimicrobial tests were: （1）Gram-positive bacteria: Staphylococcus aureus ATCC 6538 （S. aureus ATCC6538 ）， Staphylococcus aureus C98506 （S. aureus C98506）， Staphylococcus aureus C100459 （S. aureus C100459）and Staphylococcus aureus ATCC 33591（S. aureus ATCC 33591）；（2） Gram-negative bacteria: Escherichia coli ATCC 25922 （E. coli ATCC 25922） and a plant pathogen， Pseudomonas syringae DC 3000； （3） plant pathogen fungi: Fusarium oxysporum， Fusarium oxysporum sporulent， Cladosporium cucumerinum， Botrytis cinerea，Colletotrichum lagenarium and Pythium aphanidermatum； and （4）a plant pathogen yeast: Rhodotorula aurantiaca. The ATCC strains were obtained from the American Type Culture Collection； strains C98506 and C100459 were clinical isolates， a generous gift from the Centre Hospitalier Universitaire of Charleroi， Belgium （Mr. Lerson）. Strains C98506， C100459 and ATCC 33591 are methicillin-resistant S. aureus （MRSA）. The different plant pathogens were provided by the Centre Wallon de Biologie Industrielle， Bio-Industrie Unité Gembloux Agro-Bio Tech， Université de Liège， 5030 Gembloux，Belgique （Dr. Ongena）.

2.5.2. Direct and indirect antimicrobial effects

Direct and indirect antibacterial effects were evaluated by a broth microdilution method［39］. The raw extract and isolated compounds，dissolved in DMSO， were further diluted in Mueller Hinton broth（MHB）， the final DMSO concentration being maximum 4%. These solutions were transferred into 96-wells plates and serially diluted using MHB. The bacterial inoculum prepared from an overnight culture， diluted in 0.85 % NaCl to achieve 0.5 Mc Farland （108cells/mL）， was further diluted 1/100 to be inoculated in the 96-wells plates （100 μL/well）. The plates were incubated at 37 ℃for 24 h， added with an aqueous solution of MTT （0.8 mg/mL）and reincubated for 4 h. The minimum inhibitory concentrations（MIC） were the lowest concentrations that completely inhibited the growth of microorganisms， detected by unaided eyes using the MTT staining.

2.5.3. Direct and indirect antibacterial bioautography

TLC was performed for the extract and the purified compounds on precoated silica gel 60 F254glass plates （Merck， Darmstadt，Germany）. Plates were thoroughly dried at room temperature. One mL of 0.5 Mc Farland microorganism suspension was added to 9 mL MH agar （107CFU/mL） at 37 ℃ and poured on the TLC plates. After solidification， the plates were incubated overnight at 37 ℃. The bioautography was subsequently visualized by spraying MTT（0.8 mg/mL） followed by an additional incubation at 37 ℃ for 4 h［40］.

To study indirect antibacterial activity against MRSA， a subinhibitory concentration of penicillin V （1 μg/mL） was incorporated in the mixture of MHB and agar； products with no direct antibacterial activity were selected， chromatographed， and bioautographed with this medium as described above.

3. Results

3.1. Structural elucidation of compounds 1-5

The structures of the isolated compounds were established by spectral analysis， mainly UV-Vis， HRESI-MS，1H-，13C-， and 2DNMR （COSY， ROESY， HSQC and HMBC） as well as by comparing their spectroscopic data with those reported in the literature.

Vanillin 1: White crystals； MP = 82 ℃；UV （MeOH）λmax（nm）: 230，279， 309； HRESI-QTOF -MS （positive mode） m/z: 153.0545 ［M+H］+（calculated for C8H9O3: 153.0546）， 175. 0372 ［M+Na］+（calculated for C8H8O3Na: 175.0366）， 191.0216 ［M+K］+（calculated for C8H8O3K: 191.0105）， measured exact mass: 152.0471 （calculated for C8H8O3: 152.0473）， molecular formula C8H8O3；1H NMR （300 MHz， CDCl3）δ（ppm， J /Hz）: 9.83 （1H， s， H-7） ，7.44 （1H， d， J = 1.8 Hz， H-2），7.41 （1H， dd， J = 9.0， 1.8 Hz， H-6）， 7.03， 1H， d， J = 9.0 Hz， H-5），6.62 （1H， brs， 4-OH） ， 3.98 （3H， s， OCH3-3）；13C NMR （75 MHz，CDCl3）δ （ppm）: 190.86 （C， C-7）， 151.62 （C， C-4）， 147.05 （C， C-3），130.33 （C， C-1）， 127.45 （CH， C-6）， 114.04 （CH， C-5）， 108.75 （CH，C-2）， 56.40 （CH3， OCH3-3）.

（-）-Arctigenin 2: White powder； MP = 103 ℃； ［α］20D= -17.27o（EtOH， c， 0.145）； HRESI-QTOF-MS （positive mode） m/z: 373.1655 ［M+H］+（calculated for C21H25O6: 373.1646）， 395.1478［M+Na］+（calculated for C21H24O6Na: 395.1467）， measured exact mass:372.1569 （calculated for C21H24O6: 372.1577）. These data led to the molecular formula C21H24O6； HRESI-QTOF-MS/MS: m/z: 355.1556 ［M+H-H2O］+（C21H23O5） which confirm the presence of a hydroxyl group；1HNMR （600 MHz， CDCl3）δ（ppm， J /Hz）: 6.80（1H， d， J = 7.9 Hz， H-5’）， 6.72 （1H， d， J = 8.1 Hz， H-5）， 6.62 （1H，d， J= 1.7 Hz， H-2’）， 6.59 （1H， dd， J = 7.9， 1.7 Hz， H-6’）， 6.53 （1H，dd， J = 8.1， 1.7 Hz， H-6）， 6.44 （1H， d， J = 1.7 Hz， H-2）， 5.50 （1H，brs， 4’-OH）， 4.12 （1H， dd， J=9.0， 7.4 Hz， H-9α）， 3.87 （1H， dd， J = 9.0， 7.6 Hz， H-9β）， 3.83 （3H， s， OCH3-3’）， 3.80 （3H， s， OCH3-4），3.79 （3H， s， OCH3-3）， 2.92 （1H， dd， J = 14.1， 5.3 Hz， H-7’a）， 2.89（1H， dd， J = 14.1， 7.1 Hz， H-7’b）， 2.61（1H， dd， J = 14.7， 7.4 Hz，H-7a）， 2.54 （1H， m， H-8’）， 2.52 （1H， m*， H-7b）， 2.47 （1H， m， H-8），*: partially overlapped by the signal of H-8’；13C NMR （150 MHz，CDCl3） δ （ppm）: 178.96 （C， C-9’）， 149.24 （C， C-3）， 148.05 （C， C-3’），146.91 （C， C-4）， 144.76 （C， C-4’）， 130.65 （C， C-1）， 129.72 （C， C-1’），122.32 （CH， C-6’）， 120.80 （CH， C-6）， 114.31 （CH， C-5’）， 111.97（CH， C-2’）， 111.71 （CH， C-2）， 111.48 （CH， C-5）， 71.53 （CH2， C-9），56.12 （CH3， OCH3-4）， 56.07 （CH3， OCH3-3’）， 56.02 （CH3， OCH3-3），46.82 （CH， C-8’）， 41.14 （CH， C-8）， 38.42 （CH2， C-7）， 34.74 （CH2，C-7’）. Our results which were confirmed by the analysis of the ROESY spectrum experiment complete the spectroscopic data previously reported for this molecule ［31， 41］.

Paridol 3: White powder； MP = 128 ℃； HRESI-QTOF-MS（positive mode） m/z: 153.0544 ［M+H］+（calculated for C8H9O3: 153.0546）， 175.0369 ［M+Na］+（calculated for C8H8O3Na: 175.0369），343.0543 ［2M+K］+（calculated for C16H16O6K: 343.0546）， measured exact mass: 152.0472， （calculated for C8H8O3: 152.0473）， molecular formula C8H8O3.

HRESI-QTOF-MS/MS of ［M+H］+: 153.0547 ［M+H］+， 135.0239［M+H-H2O］+， 121.0289 ［M+H- CH3OH］+， these two last ions confirmed the presence of the hydroxyl and methoxyl groups in the molecule；1H NMR （300 MHz， CDCl3）δ（ppm， J /Hz）: 7.95 （2H， d，J = 8.9 Hz， H-2 & H-6）， 6.86 （2H， d， J= 8.9 Hz， H-3 & H-5）， 5.98（1H， brs， 4-OH）， 3.88 （3H， s， OCH3-7） ；13C NMR （75 MHz， CDCl3） δ167.08 （C， C-7）， 159.90 （C， C-4）， 132.06 （CH， C-2 & C-6），122.93 （C， C-1）， 115.33 （CH， C-3 & C-5）， 52.08 （CH3， OCH3-7）. Eupatilin 4: Yellow crystals； MP = 236 ℃； UV （MeOH）λmax（nm）:276， 340； +NaOH: 276， 320， 360 （with hypochromic effect）；+AlCl3: 282， 368； + AlCl3+ HCl: 283， 361； + NaOAc: 276， 366；+NaOAc +H3BO3: 276， 357； HRESI-QTOF-MS （positive mode）m/z:345.0968 ［M+H］+（calculated for C18H17O7: 345.0969）， 367.0788［M+Na］+（calculated for C18H16O7Na: 367. 0788）， 383.0537［M+K］+（calculated for C18H16O7K: 383.0528）， 689.1736 ［2M+H］+（calculated for C36H33O14: 689.1865）， 712.1717 ［2M+Na］+（calculated for C36H32O14Na: 712.1718）， 727.1077 ［2M+K］+（calculated for C36H32O14K: 712.1424）， measured exact mass: 344.0888 （calculated for C18H16O7: 344.0896）， molecular formula C18H16O7；1H NMR （400 MHz， DMSO-d6）δ（ppm， J/Hz）: 13.04 （1H， s， OH-5）， 7.68 （1H，dd， J = 8.5， 2.0 Hz， H-6’）， 7.56 （1H， d， J = 2.0 Hz， H-2’）， 7.13 （1H，d， J = 8.5 Hz， H-5’）， 6.97 （1H， s， H-3）， 6.64， （1H， s， H-8）， 3.88（3H，s， 3’-OCH3）， 3.85， （3H， s， 4’-OCH3） 3.75， （3H， s， 6-OCH3） ；13C NMR （100 MHz， DMSO-d6）； δ （ppm）: 182.01 （C， C-4）， 163.26 （C，C-2），157.12 （C， C-7）， 152.83 （C， C-5）， 151.96 （C， C-9）， 149.06 （C，C-4’）， 148.84 （C， C-3’）， 131.11 （C， C-6）， 122.83 （C， C-1’）， 120.10（CH， C-6’）， 111.55 （CH， C-5’）， 109.26 （CH， C-2’）， 104.22 （C，C-10）， 103.36 （CH， C-3）， 94.33 （CH， C-8）， 59.97 （CH3， OCH3-6），55.88 （CH3， OCH3-4’）， 55.76 （CH3， OCH3-3’）.

Jaceosidin 5: Yellowish powder； MP = 237 C， UV （MeOH）λmax:276，346； + NaOH: 276， 314， 360 （with hyperchromic effect）； + AlCl3: 282， 368； + AlCl3+ HCl: 283， 361； + NaOAc: 278， 366； + NaOAc + H3BO3: 276， 357； HRESI-QTOF-MS （positive mode） m/z: 331.0811（calculated for C17H15O7: 331.0812）， 353.0625 ［M+Na］+（calculated for C17H14O7Na: 353.0632）， 661.1542 ［2M+H］+（calculated for C34H29O14: 661.1552）， 683.1485 ［2M+Na］+（calculated for C34H28O14Na: 683.1371），701.0942 ［2M+K］+（calculated for C34H28O14K: 701.1137）， 991.2454 ［3M+H］+（calculated for C51H43O21: 991.2291）， measured exact mass: 330.0744， （calculated for C17H14O7: 330.0740）， molecular formula C17H14O7；1H NMR （400 MHz， CH3OH-d4）δ（ppm， J /Hz）: 7.52 （1H， dd， J = 8.5， 2.0 Hz，H-6’）， 7.50 （1H， d， J = 1.9 Hz， H-2’）， 7.48 （1H， d， J = 8.5 Hz， H-5’），6.94 （1H， s， H-8）， 6.64 （1H， s， H-3）， 3.96 （3H， s， OCH3-3’）， 3.88（3H， s， OCH3-6）.13C NMR （75 MHz， CH3OH-d4）δ（ppm）: 184.21（C， C-4）， 166.36 （C， C-2）， 158.92 （C， C-7）， 154.83 （C， C-5）， 154.70（C， C-9）， 151.70 （C， C-4’）， 149.44 （C， C-3’）， 132.92 （C， C- 6），123.90 （C， C-1’）， 121.64 （C， C-6’）， 116.91 （CH， C-5’）， 110.34 （CH，C-2’）， 105.58 （C， C-10）， 103.59 （CH， C-3）， 95.65 （CH， C- 8）， 61.04（CH3， OCH3-6）， 56.50 （CH3， OCH3-3’）.

3.2. Biological activities

3.2.1. Cytotoxic effects

The CHCl3extract showed cell growth inhibitory activity against all 3 tested cell lines in the μg/mL range （Figure 1）. These results are in agreement with previous data from an Algerian Centaurea species；the raw chloroformic extract of Centaurea musimomum （musimonum）Maire showed on KB cells， cytotoxic activity with growth inhibition of 89% at 10 μg/mL and 26% at 1 μg/mL［10］.

The evaluation of the isolated compounds 1 to 5 indicated moderate growth inhibitory/cytotoxic activities for eupatilin 4 （33 - 85 μM），jaceosidin 5 （32 - 49 μM）， and （-）-arctigenin 2 （28 - 82 μM） （Figure 1）.

Figure 1. Cytotoxic effects （IC50） of the chloroform extract and the isolated compounds on different tumor cell lines.

Our results showed that the chloroformic extract displayed more significant cytotoxic effects on cancer cells A549， MCF7 and U373 than the isolated pure compounds. This could be attributed to the synergetic interactions， more especially as this extract contains flavonoids for which it is thought that they may have a role to play in increasing the biological activity of other compounds by synergistic or other mechanisms［42］.

3.2.2. Antifungal and antimicrobial activities

Although inactive against 7 tested microorganisms （fungi，bacteria and yeast， human or plant pathogens， Table 1）， the raw extract was able to potentiate the effect of beta-lactam antibiotics on methicillin-resistant S. aureus （MRSA）， reducing the minimal inhibitory concentrations （MICs） by a factor of 2-32-fold （Table 2）. In a direct antibacterial TLC-bioautography assay， compound 5（jaceosidin）， showed the highest activity （Tables 3 and 4）. This was further investigated in a direct antibacterial assay， but the activity was relatively quite low on Gram positive and negative bacteria（MIC of 200 μg/mL on MRSA C98506， MRSA C100459， MRSA ATCC33591， MSSA ATCC6538， E. coli ATCC25922）.

Table 1MIC of the chloroform extract （μg/mL）.

Table 2Impact of the chloroform extract （200 μg/mL） on the susceptibility of the MRSA towards various beta-lactam antibiotics.

Table 3Antibacterial activity of the purified compounds （1-5） measured by a direct TLC-bioautography.

Table 4Antibacterial activity of eupatilin 4 and jaceosidin 5， measured by a direct TLC- bioautography with different amounts spotted.

4. Discussion

4.1. Phytochemical investigation

We report in this work the isolation， purification and structural elucidation of chemical components of the chloroform soluble part of the MeOH-H2O （77%） extract obtained from the aerial parts（leaves and flowers） of C. diluta Ait. subsp. algeriensis （Coss. & Durieu） Maire （Asteraceae）. No report is available so far on the phytochemistry of this species endemic to Algeria and Morocco. The present phytochemical investigation allowed the isolation of a lignan［（-）-arctigenin］， flavonoids （eupatilin and jaceosidin） and phenols（vanillin and paridol）. These results are in agreement with major studies reported on different Centaurea species［14，43-48］.

4.2. Biological activities

4.2.1. Cytotoxic effects

The raw extract and the isolated compounds were evaluated for cytotoxic activity. Moderate cytotoxic effects were observed for three compounds， （-）-arctigenin 2， eupatilin 4 and jaceosidin 5， with IC50s in the range 25-50 μg/mL. These data are in agreement with previous studies. Indeed， arctigenin （unspecified stereoisomer） as tumor specific agent that showed cytotoxicity to lung cancer （A549），liver cancer （HepG2） and stomach cancer （KATO III） cells， but not cytotoxic to several normal cell lines［49］. Arctigenin specifically inhibited the proliferation of cancer cells， which might consequently lead to the induction of apoptosis and is cytotoxic for human hepatocellular carcinoma cell lines， the IC50values after 12 h， 24 h and 48 h of treatment were respectively 38.29， 1.99 and 0.24 μM［50］， the highest activity was demonstrated with IC50values of 0.73 μM （HeLa）， 3.47 μM （MCF7） and 4.47 μM （A431）［46］. Eupatilin reduces aortic smooth muscle cell proliferation and migration by inhibiting PI3K， MKK3/6， and MKK4 activities （IC50， in Hec1A and KLE cells was 82.2 and 85.5 μM） ［51， 52］ and jaceosidin can induce G2/M cell cycle arrest by inactivating cdc25C-cdc2 via ATMChk1/2 activation［53］.

4.2.2. Antifungal and antimicrobial activities

The raw extract and the isolated secondary metabolites were evaluated for antimicrobial activity. Although the raw extract didn’t show any antimicrobial effect on various bacteria or fungi， it could potentiate the effect of beta-lactam antibiotics on methicillinresistant S. aureus （MRSA）， reducing the minimal inhibitory concentrations （MICs） by a factor of 2-32- fold. Jaceosidin 5 showed a moderate antimicrobial activity （MIC of 200 μg/mL on MRSA C98506， MRSA C100459， MRSA ATCC33591， MSSA ATCC6538，E. coli ATCC25922）. This is in agreement with previous results ［54］. Jaceosidin 5 had the greatest potency （MICs 16-32 μg/mL） against most S. aureus isolates ［55］.

The identification of five compounds， vanillin， （-）-arctigenin，paridol， eupatilin and jaceosidin， from the aerial parts （leaves and flowers） of C. diluta Ait. subsp. algeriensis （Coss. & Dur.） M.（Asteraceae） emphasized the possible relevance of this plant for Algerian traditional medicine and it is surprising that no report has been published so far on eventual ethnomedical uses of this species. This may be due to a low distribution of this species or to an eventual toxicity that could have discouraged its use in traditherapy；this warrants investigation. A promising effect on bacterial resistance needs to be further investigated to identify the compound（s） able to reverse bacterial betalactam resistance.

Declare of interest statement

We declare that we have no conflict of interest.

Acknowledgements

We thank Algerian government for financial support， HCDS Djelfa for helping us in the process of exploration and the harvest of plant material， professor M. Kaabeche for the identification of the plant material and C. Delporte （Laboratoire de Chimie Pharmaceutique Organique， Faculté de Pharmacie， Université Libre de Bruxelles（ULB） for the mass spectrometry measurement.

We thank Professor V. Mathieu （Laboratoire de Cancérologie et Toxicologie， Université Libre de Bruxelles） for access to her laboratory and help in performing the cytotoxicity experiments，Dr. Ongena Marc for access to her laboratory （Centre Wallon de Biologie Industrielle， Bio-Industrie Unité Gembloux Agro-Bio Tech，Université de Liège， 5030 Gembloux， Belgique） and Dr. E. Gicquel and J. Vancautenberg for the measurement of the optical rotation（Institut Meurice - Service de Chimie Organique Haute Ecole Lucia de Brouckère Avenue Emile Gryzon， 11070 Bruxelles）.

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Document heading 10.1016/j.apjtm.2016.04.016

15 February 2016

*Corresponding author: Corresponding authors: Pierre Duez， Laboratoire de Pharmacognosie， de Bromatologie et de Nutrition Humaine， Université Libre de Bruxelles （ULB）， 1050 Bruxelles， Belgique.

E-mail : pierre.duez@umons.ac.be

Fadila Benayache， Unité de recherche : Valorisation des Ressources Naturelles， Molécules Bioactives et Analyses Physicochimiques et Biologiques（VARENBIOMOL）， Faculté des Sciences Exactes， Université Frères Mentouri Constantine 1， 25000 Constantine， Algérie.

Tel. /Fax: +213 31 81 11 03

E-mail: fbenayache@yahoo.fr