Industrial Crops and Products 46 (2013) 317 323

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Chemic of tNigella on

A. Pirasa eri c

a Dipartimento taria db Dipartimento nserrac ITI M. Giua,d Department oe Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara, Turkey

a r t i c l e i n f o

Article history:Received 16 OReceived in re17 December 2Accepted 1 Feb

Keywords:Nigella sativaEssential oilFixed oilIn vitro biologiAntibacterialAntifungalAntituberculosSupercritical e

a b s t r a c t

1. Introdu

Nigella sannual herbgrowing inslightly bittlar, of genethey are use

CorresponE-mail add

0926-6690/$ http://dx.doi.october 2012vised form012ruary 2013

cal activity

isxtraction

Nigella sativa L. (Ranunculaceae), commonly known as black cumin, is an erect herbaceous annual plant.N. sativa seeds have traditionally been used in folk medicine as a natural remedy for various diseases aswell as a spice. The seeds contain both xed and essential oils, proteins, alkaloids and saponins. Much ofthe biological activity of the seeds has been shown to be due to thymoquinone, the major component ofthe essential oil. The xed oil is composed mainly of unsaturated fatty acids, including the unusual C20:2eicosadienoic acid.

Isolation of volatile and xed oils from N. sativa seed of Turkey and Egypt has been obtained by super-critical fractioned extraction with carbon dioxide. Extraction experiments were carried out at pressuresof 90 and 300 bar and temperature of 40 C. The extraction step performed at 90 bar produced a volatilefraction mainly formed by tymoquinone (79 86%) and o-cymene (5 11%). The oil yield relative to thisstep of the process was 0.1 0.3% by weight of the charge. The last extraction step at 300 bar produced axed oil. The yield of this step was 2126% by weight. The most represented fatty acids of xed oil fromN. sativa were 18:2 n 6 (54 55%), 18:1 n 9 (22 23%), 16:0 (12 13%), 18:0 (3%), and 20:2 (2 3%).

The volatile and xed oils obtained from N. sativa were evaluated for the antibacterial activity byemploying standard strains of Escherichia coli, Pseudomonas aeroginosa, Acinetobacer baumannii, Staphylo-coccus aureus, Enterococcus faecalis. In vitro antifungal activity of the derivatives against Candida albicans, C.tropicalis, and C. krusei were screened by using ketoconazole, and uconazole as control agents. The anti-mycobacterium activity breakpoint concentration (g mL1) was determined against standard strainsof Mycobacterium tuberculosis H37Rv and M. avium (ATCC 15769). The volatile and xed oils displayedantimicrobial activity toward all of the standard (ATCC, RSKK) strains of the tested bacteria at MIC valuesof 864 g mL1 and were revealed to be ineffective against isolated strains (MIC; >256 g mL1). Thevolatile and xed oils emerged as effective against the bacteria of M. avium with MIC values of 8 g mL1.Moreover, all the extracts exhibited antifungal activity against C. albicans, C. tropicalis, and C. krusei at MICvalues of 1664 g mL1.

2013 Elsevier B.V. All rights reserved.

ction

ativa L., commonly known as black cumin seed, is anaceous plant belonging to the Ranunculaceae family

countries bordering the Mediterranean Sea. It tasteser and peppery with a crunchy texture. Seeds are angu-rally small size (15 mg), dark gray or black color andd for edible and medicinal purposes in many countries

ding author. Tel.: +39 070 675 4124; fax: +39 070 675 4032.ress: anrosa@unica.it (A. Rosa).

(Cheikh-Rouhou et al., 2007). The composition and properties ofthis species have been fairly well investigated.

The seeds contain a yellowish volatile oil, a xed oil, proteins,amino acids, reducing sugars, mucilage, alkaloids, organic acids,tannins, resins, toxic glucoside, metarbin, bitter principles, glycosi-dal saponins, crude ber, minerals, and vitamins (Ramadan, 2007).

Several authors have investigated the volatile oil of nigella seedsand isolated and identied active constituents that have benecialclinical effects. Egyptians believe that nigella seeds increase humanimmunity. The volatile oil has been produced by pressing the rawor roasted seeds (Atta, 2003).

Nigella seed xed oil is considered as one among newer sourcesof edible oils, thanks to its important role in human nutrition and

see front matter 2013 Elsevier B.V. All rights reserved.rg/10.1016/j.indcrop.2013.02.013al composition and in vitro bioactivity sativa L. extracted by supercritical carb

, A. Rosab,, B. Marongiua, S. Porceddaa, D. Falconi di Scienze Chimiche e Geologiche, Universit degli Studi di Cagliari, Cittadella Universi

di Scienze Biomediche, Universit degli Studi di Cagliari, Cittadella Universitaria di Mo via Montecassino, Cagliari, Italyf Microbiology, Faculty of Pharmacy, Gazi University, Ankara, Turkey/ locate / indcrop

he volatile and xed oils of dioxide

, M.A. Dessb, B. Ozcelikd, U. Kocae

i Monserrato, Cagliari, Italyto, Cagliari, Italy

318 A. Piras et al. / Industrial Crops and Products 46 (2013) 317 323

health. Black cumin crude xed seed oil is a valuable source ofessential fatty acids, glycolipids, phospholipids, and bioactive phy-tosterols (Ramadan, 2007; Ramadan et al., 2012a). This oil hasbeen reported to possess antitumor activity, antioxidant activity,anti-inameffect on thbeen focusehas increasusually proand even atextraction calteration ofunctional, et al., 2007to obtain bl

Fats andimportant ivegetable ovitamins animportant tas a source

The objeof supercritand to invesbial activityand fractionout at low t(SFE) offersniques, likethat it uses vent (Piras 2003).

2. Materia

2.1. Chemic

Triolein,methyl estpurchased fthe highestmethanoliccis,trans-13cis,trans-9-hobtained frothe other ch

2.2. Plant m

Cultivatecities of Tzli). Only tplants, whideposited anumbered tially. The Minardi (Bawas groundoverheatingm.

2.3. SFE ext

Supercriapparatus, earator vesse

Extraction was carried out in a semi batch mode: batch charging ofvegetable matter and continuous ow solvent. The N. sativa volatileoil was obtained working at 90 bar and 40 C in the extraction ves-sel, at 90 bar and 10 C in the rst separator and at 20 bar and

n thee sa

obta by uract.

and

lyzeraphS). At, M

dett HP5

0.25 soft

at 3pera:10; MS odelr, aAgil

lm tl-meions /minr waL/m

Theilention e, qua04

rams dilut

of th sys

al Lib by con ins, 20

relatintercentak are

xed o

Prepaaratit al.,f EtOf a w

KOtemp

L o phane to

andiabcid hmatory activity, antibacterial activity and a stimulatorye immune system. Actually, a great deal of attention hasd on black cumin seed oils, and thus their consumptioned, especially in Middle East countries. The oil has beenduced by a hot solvent extraction method at 4060 C

70 C, using the Soxhlet extractor. This hot method ofould affect the oil properties and may induce partialf the majority of minor constituents that have manyantioxidative and pro-oxidative effects (Cheikh-Rouhou). Recently, the cold-pressing procedure has been usedack cumin seed oil (Ramadan et al., 2012a).

oils play an important role in the food industry and aren human nutrition for a variety of reasons. Particularly,ils are becoming increasingly important as suppliers ofd essential unsaturated fatty acids. Therefore, it is veryo investigate the possibility of using some raw materialsof vegetable oils and vitamins.ctive of this work is to explore the potential applicationical CO2 to the extraction of oils from N. sativa seedstigate their chemical composition and their antimicro-. Supercritical CO2 is a promising solvent for extractionation of edible oils, since the extraction can be carriedemperature. Besides, the supercritical uid extraction

many other favorable features over the traditional tech- steam distillation and solvent extraction, due to the facta clean, inexpensive, non-ammable and non-toxic sol-et al., 2009, 2012; Porcedda et al., 2009; Marongiu et al.,

ls and methods

als

trilinolein, standards of fatty acids and fatty aciders, and desferal (deferoxamine mesylate salt), wererom SigmaAldrich (Milan, Italy). All solvents used, of

available purity, were also from SigmaAldrich. The HCl (3 N) was purchased from Supelco (Bellefonte, PA).-Hydroperoxyoctadecadienoic acid (c,t-13-HPODE) andydroperoxyoctadecadienoic acid (c,t-9-HPODE) werem Cascade (Cascade Biochem. Ltd., London, UK). All ofemicals used in this study were of analytical grade.

aterials

d N. sativa seeds were obtained from the cultivationurkey namely T1 (Antalya), T2 (Aydn), T3 (Deni-he seeds were obtained from the cultured N. sativach were authenticated by us, voucher specimens weret the Gazi University, Department of Pharmacognosy,as UK2010NST1, UK2010NST2, UK2010NST3 sequen-seeds from Egyptian N. sativa, E1, were supplied bygnacavallo-Ravenna, Italy). Before utilization, matter

with a Malavasi mill (Bologna, Italy) taking care to avoid and the particles sizes were in the range (250425)

raction

tical CO2 extractions were performed in a laboratoryquipped with a 320 cm3 extraction vessel and two sep-ls of 300 and 200 cm3 respectively connected in series.

15 C ithe samoil wassel andthe ext

2.4. GC

Anamatog(GCM(AgilenizationAgilen30 m Stationraisingtor temratio 1

GClent, Minjectoferent i.d., phenyconditat 3 Cinjectoat 1.0 m(1:10).MS, Agionizat200 Crange 3matogwith aparisonGCMSSpectrrmedretenti(Adamminedlinear

PerGC pea

2.5. Fi

2.5.1. Sep

(Rosa e5 mL o1 mL oof 10 Nroom and 7 mhexanen-hexapH 34(saponfatty a second one. The extraction of the xed oil was run onmples of N. sativa previously treated at 90 bar; the xedined working at 300 bar and 40 C in the extraction ves-sing only one separator (at 20 bar and 15 C) to recover

GCMS analysis of essential oil

s of the volatile extracts were carried out by gas chro-y (GC) and by gas chromatographymass spectrometrynalytical GC was carried out in a gas chromatographodel 7890A, Palo Alto, CA), equipped with a ame ion-ector (FID), an autosampler (Agilent, Model 7683B),

fused silica column (5% phenyl-methylpolysiloxane), mm i.d., lm thickness 0.25 m, and a Agilent Chem-ware system. Oven temperature was settled at 60 C,C/min to 250 C and then held 20 min at 250 C; injec-ture: 250 C; carrier gas: helium at 1.0 mL/min; splittingdetector temperature: 300 C.analyses were carried out in a gas chromatograph (Agi-

6890N, Palo Alto, CA) equipped with a splitsplitlessn autosampler Agilent model 7683 and two dif-ent fused silica capillary columns (30 m 0.25 mmhickness 0.25 m) of different polarities (HP-5, 5%thylpolysiloxane; DB-WAXetr, polyethylene glycol). GCused were: programmed heating from 60 to 250 C

followed by 20 min under isothermal conditions. Thes maintained at 250 C. Helium was the carrier gasin; the sample (1 L) was injected in the split mode

GC was tted with a quadrupole mass spectrometer, model 5973 detector. MS conditions were as follows:nergy 70 eV, electronic impact ion source temperaturedrupole temperature 150 C, scan rate 3.2 scan/s, mass80 u. Software adopted to handle mass spectra and chro-

was a ChemStation. Samples were run in chloroformion ratio of 1:100. Compounds were identied by com-eir mass spectra with those of NIST02 library data of the

tem and Adams libraries spectra (NIST/EPA/NIH Massrary, 2002; Adams, 2007). The results were further con-omparison with the compounds elution order with theirdices on semi-polar phases reported in the literature07). Retention indices of the components were deter-ive to the retention times of a series of n-alkanes withpolation.ge of individual components was calculated based onas without FID response factor correction.

il

ration of fatty acidson of fatty acids was obtained by mild saponication

2011) as follows: 3 mg of the xed oil were dissolved inH and 100 L of desferal solution (25 mg/mL of H2O),ater solution of ascorbic acid (25% w/v), and 0.5 mL

H were added. The mixtures were left in the dark aterature for 14 h. After addition of 10 mL of n-hexanef H2O, samples were centrifuged for 1 h at 900 g. These was collected, and after addition of further 10 mL of

the mixtures, samples were acidied with 37% HCl to then centrifuged for 1 h at 900 g. The hexane phasele fraction) with free fatty acids and conjugated dieneydroperoxides (HP) was collected and the solvent was

A. Piras et al. / Industrial Crops and Products 46 (2013) 317 323 319

evaporated. A portion of the dried residue was dissolved in 500 Lof CH3CN with 0.14% CH3COOH (v/v) and aliquots of the sampleswere injected into the HPLC system.

An aliquot of dried fatty acids was methylated with 1 mL ofmethanolicat room temof H2O, samane phase wwas evaporand aliquotrecovery ofan externaland trilinoleevaporation

2.5.2. HPLCAnalyses

were carriematograph diode arrayacids and Hcarried out ticle size) (AEclipse (12.lent Techno(75/25/0.12The tempercation of fand the secoerated withcurves of aland were fo

2.5.3. GC anFatty ac

matograph Alto, USA)with a cya(30 m 0.32as carrier gwas set at 1the detectoesters werestandard cocalculated ausing the H

2.6. Microb

2.6.1. Test mExtracts

(70%) at a ltration usthe stock sazid, ethamstandard anrial agents MO, USA) apH: 6.0; 0.1(meropenemsolutions ofClinical and(CLSI, 2008powder walter-sterili

2.6.2. Microorganisms and inoculum preparationAntibacterial activity and antifungal activity tests were car-

ried out against standard (ATCC; American type culture collection,RSKK; Culture collection of Rek Saydam Central Hygiene Institute;

Natiol isoicinestand), Pse

(RSK (ATC

(ATctivi

), andfungaeller

Oxosionfferelturet al.

weg frens o

exte baccfu mu mLtome

Antib micgal acroplst ro560rema

intoun oat 35icro

westscopiory c

(z

Anti- effeti tu

M. ) wa

H37ce senstgar (menmenl anin 0.justnd 1iters d 10rile .06 te. O HCl (3 N) (Christie, 1993; Rosa et al., 2011) for 30 minperature. After addition of 4 mL of n-hexane and 2 mLples were centrifuged for 20 min at 900 g. The hex-ith fatty acid methyl esters was collected, the solvent

ated, the residue was dissolved in 250 L of n-hexanes of the samples were injected into the GC system. The

fatty acids during saponication was calculated using standard mixture prepared dissolving 1 mg of trioleinin in 5 mL of EtOH and processed as samples. All solvent

was performed under vacuum.

analyses of unsaturated fatty acids, and oxidative productsd out with an Agilent Technologies 1100 liquid chro-(Agilent Technologies, Palo Alto, CA) equipped with a

detector (HPLC-DAD). Analyses of unsaturated fattyP, detected at 200 and 234 nm, respectively, were

with a XDB-C18 Eclipse (150 mm 4.6 mm, 3.5 m par-gilent Technologies) equipped with a Zorbax XDB-C18

5 mm 4.6 mm, 5 m particle size) guard column (Agi-logies), with a mobile phase of CH3CN/H2O/CH3COOH, v/v/v), at a ow rate of 2.3 mL/min (Rosa et al., 2011).ature of the column was maintained at 37 C. The identi-atty acids and HP was made using standard compoundsnd derivative, as well as conventional UV spectra, gen-

the Agilent Chemstation A.10.02 software. Calibrationl of the compounds were constructed using standardsund to be linear with correlation coefcients >0.995.

alysisid methyl esters were measured on a gas chro-Hewlett-Packard HP-6890 (Hewlett-Packard, Palo

with a ame ionization detector and equippednopropyl methyl-polysiloxane HP-23 FAME column

mm 0.25 m) (Hewlett-Packard). Nitrogen was usedas at a ow rate of 2 mL/min. The oven temperature75 C; the injector temperature was set at 250 C; andr temperature was set at 300 C. The fatty acid methyl

identied by comparing the retention times to those ofmpounds. The composition of individual fatty acid wass a percentage of the total amount of fatty acids (g%),ewlett-Packard A.05.02 software.

iological studies

aterials were dissolved in dimethylsulfoxide (30%) and H2Onal concentration of 512 g mL1 and sterilized bying 0.22 (m Millipore (MA 01730, USA), and used asolutions. Ampicillin, gentamycin, levooxacin, isoni-butol, ketoconazole, and uconazole were used as thetibacterial and antifungal drugs. Reference antibacte-were purchased from Sigma Chemical Co. (St. Louis,nd dissolved in phosphate buffer solution (ampicillin;

mol/mL), dimethylsulfoxide (ketoconazole), or in water, gentamycin, levooxacin, uconazole). The stock

the agents were prepared in medium according to the Laboratory Standards Institute (CLSI formerly; NCCLS)). A stock solution of the resazurin sodium salt (Sigma)s prepared at 0.01% in sterile distilled water. It waszed and kept at 4 C.

NCPF; (clinicaof Med

As 35218manniiaureussubtilisterial a13803of anti

Mu(MHA;suspenwas buand cu(Koca eulationdilutinpensiodilutedria. That 105

(108 cftropho

2.6.3. The

antifunthe miinto rples (2to the ulatedeach rbated pure mThe lomacroinhibitviously

2.6.4. The

for ansis and(REMAculosisreferenon Low7H11 asupplesuppleglyceropared that ad1:20 amicrolerol, anof a ste(2560the planal Collection of Pathogenic Fungi) and isolated strainslate obtained from Department of Microbiology, Faculty, Gazi University).ards; gram negative strains of Escherichia coli (ATCCudomonas aeroginosa (ATCC 10145), Acinetobacer bau-K 02026), and as gram positive strains of StaphylococcusC 25923), and Enterococcus faecalis (ATCC 29212), B.

CC 6633) were used for the determination of antibac-ty. Candida albicans (ATCC 10231), C. tropicalis (ATCC

C. krusei (ATCC 6258) were used for the determinationl activity.Hinton Broth (MHB; Difco) and Mueller Hinton Agarid) were applied for growing and diluting of the bacterias. The synthetic medium RPMI-1640 with l-glutamined to pH 7 with 3-[N-morpholino]-propansulfonic acid

suspensions were prepared as described previously, 2010). The microorganism suspensions used for inoc-re prepared at 105 cfu (colony forming unit/mL) bysh cultures at McFarland 0.5 density (108 cfu mL1). Sus-f bacteria and fungi were added in each well of theracts, density of 105 cfu mL1 for fungi, and for bacte-terial suspensions used for inoculation were preparedL1 by diluting fresh cultures at McFarland 0.5 density

1). The fungi suspension was prepared by the spec-tric method of inoculums (zc elik et al., 2012).

acterial and antifungal testsrodilution method was employed for antibacterial andctivity tests. Media were placed into each 96 wells ofates. Volatile and xed oils at 512 g mL1 were addedws of microplates and two fold dilutions of the sam-.125 g mL1) were made by dispensing the solutionsining wells. The culture suspensions (10 L) were inoc-

all the wells. All organisms were tested in triplicate inf the experiments. The sealed microplates were incu-C for 24 h and 48 h in humid chamber. DMSO, H2O,

organisms and pure media were used as control wells. concentration of the samples that completely inhibitc growth of the cultures was determined and minimumoncentrations (MICs) were reported as described pre-c elik et al., 2012).

mycobacterium activityct of the volatile and xed oils of N. sativa was searchedberculosis activity against Mycobacterium tuberculo-avium, which Resazurin microplate assay procedures carried out (Orhan et al., 2012). The strains M. tuber-Rv (ATCC 27294; American Type Culture Collection)train and M. avium (ATCC 15769) were maintainedeinJensen medium and subcultured on MiddlebrookBecton Dickinson) resuspended in 7H9-S broth mediumted with 10% [OADC; 0.1% casitone, 0.5% glycerol,ted oleic acid, albumin, dextrose, and catalase], 0.2%d 0.1% Bacto casitone (Difco). Suspensions were pre-04% (v/v) Tween 800.2% + bovine serum albumin soed to McFarland tube number 1. This was diluted to00 L aliquot was used as inoculum. One hundredof Middlebrook 7H9 broth (0.1% casitone, 0.5% glyc-% OADC; Becton-Dickinson) was dispensed in each wellat-bottom 96-well plate, and serial twofold dilutionsg mL1) of each compound were prepared directly inne hundred microliters of inoculum was added to each

320 A. Piras et al. / Industrial Crops and Products 46 (2013) 317 323

Table 1Chemical composition of Nigella sativa volatile oil obtained by SFE.

IR (HP-5) IR (DB-WAXetr) T1 T2 T3 E1 Compound Class

930 1032 0.4 -Thujene MH1027 10311062 1179 1198 1252 12931303 1402 1418

well. A grofor each isoavoid evapoand incubaof incubatiowell, and thfrom blue (of bacteria, the sample

3. Results

3.1. Volatile

The volacollected in

All the von two coltied compboth HP-5 aidenticatiove oxygencarbon) whoil consisteaccompaniebons (5.4 hydrocarbo

Among tmajor consby lower coacetate andThe monotecymene (5.4were presethat N. sativchemotype

Our resvolatile oilmental andbeen descroil was fonents and d2003); otheria (Benkacfound p-cywith 33.0% monoterpe(Moretti et

-cym for Nits anentie (2undsp-cym

xed o

the er) an

yieldli-quse th

HPssed

oilstion ic aci, 23%-vac

polynd eicid pllabotechressit of ed b200 aortedeternt ox1271 7.6 5.4 5.41200 1247 0.6 0.6 1595 0.51664 0.6 2989 77.2 78.1 86.21647 1.5 2201 5.8 7.9 2.91556 2.4 2.0 2.01586 0.6

Total identied 95.2 95.1 97.0% MH 6.0 8.2 5.4% MO 86.6 84.5 89.6% SH 2.6 2.4 2.0

wth control and a sterile control were also includedlate. Sterile water was added to all perimeter wells toration during the incubation. The plate was covered,

ted at 37 C under a normal atmosphere. After 7 daysn, 10 g mL1 of resazurin solution was added to eache plate was reincubated overnight. A change in color

oxidized state) to pink (reduced) indicated the growthand the MIC was dened as the lowest concentration ofthat prevented this change.

and discussion

oil

tile oils extracted by SFE from the seeds of N. sativa, four diverse locations, ranged from 0.1% to 0.3% (w/w).olatile fractions were analyzed by GC and by GCMSumns of different polarity. Table 1 reports the iden-onents, their percentages, their retention indices onnd DB-WAXetr columns. Analysis of the oils led to then of 11 components (four monoterpenes hydrocarbon,ated monoterpenes, and two sesquiterpenes hydro-ich represented 93.1 97.0% of the total amount. Thed chiey of oxygenated monoterpenes (79.5 89.6%)d by noticeable contents of monoterpenes hydrocar-

11.7%) and much smaller amounts of sesquiterpenesns (1.9 2.6%).he oxygenated monoterpenes, thymoquinone was thetituent in all four samples (77.2 86.2%), accompaniedntents of terpinen-4-ol, methyl chavicol, trans-sabynil

carvacrol not detected at all in all four oils (see Table 1).rpene hydrocarbons were distinctly dominated by o-

11.0%), while -thujene, limonene and -terpinenent at much lower amounts. The analytical data showed

60.2% p(2008)

Burthe essquinoncomposativa

3.2. Fi

On (300 bawith a

QuacompoGC and(expreAll thecentrapalmittively)and cis58% ofn 6 afatty aand coferent cold-pcontendetectn 9, as repHPLC ddifferea from Turkey and Egypt belongs to the thymoquinone, with a high level of thymoquinone (77.2 86.2%).ults reinforce previous data on the variability seeds, depending on the origin of the samples, environ-

climatic conditions. A variety of chemotypes haveibed in the literature. An Iranian N. sativa essentialund to be dominated by phenylpropanoid compo-isplayed a trans-anethole chemotype (Nickavar et al.,r N. sativa from Iran (Hajhashemi et al., 2004), Alge-i-Ali et al., 2007) and India (Singh et al., 2005) wasmene/thymoquinone chemotype. While a chemotypep-cymene and 26.8% thymol and the preponderance ofnes was reported for N. sativa essential oil from Moroccoal., 2004; DAntuono et al., 2002) and a chemotype with

3.79 0.20,measured f

Extracte18:2 n 6 oleic acid (ously publiN. sativa oitonseed oilby a high rsents a southat cannothuman devacid compolipid-solubl11.0 o-Cymene MH0.7 Limonene MH

-Terpinene MH Terpinen-4-ol MO Methyl chavicol MO

79.5 Tymoquinone MO trans-Sabynil acetate MO Carvacrol MO1.9 Longifolene SH (E)-Caryophyllene SH

93.111.779.51.9

ene and 12.9% -terpinene was reported by Wajs et al.. sativa from Poland.d Bucar (2000) reported the chemical composition of

al oils from N. sativa from Austria. They found thymo-7.2 57.0%) and p-cymene (7.0 15.7%) as the major. Recently, Bourgou et al. (2010) found in Tunisian N.ene as main component (60.5%).

il

xhausted matrix a further extraction at higher pressured 40 C was performed for the extraction of xed oil,

of 21.0 26.0%.antitative information on the individual fatty acids thate lipid classes of N. sativa xed oils was obtained by

LC analyses. Table 2 shows the fatty acid compositionas % of total fatty acids, g/100 g) of N. sativa oils by GC.

showed a similar fatty acid composition, with a con-of approximately 16% of saturated fatty acids (mainlyd 16:0, and stearic acid 18:0, 1213% and 23%, respec-

of monounsaturated (mainly oleic acid 18:1 n 9cenic acid 18:1 n 7, 22% and 0.5%, respectively), andunsaturated, mainly constituted by linoleic acid 18:2cosadienoic acid 20:2, 5455 and 2%, respectively. Thisrole was comparable to those reported by Ramadamrators for black cumin seed oil obtained by two dif-niques, Soxhlet extraction (Ramadan et al., 2003) andng procedure (Ramadan et al., 2012a). Furthermore, thethe main unsaturated fatty acids in the oils was alsoy HPLC, as follows: approximately 500 mg/g of 18:1nd 3 mg/g of 18:2 n 6 and 18:3 n 3, respectively,

in Table 3. The oil oxidative status was evaluated bymination of the HP level. The extracted oils showed aidative status and an average HP content of 6.48 0.52, 13.50 0.43, and 2.81 0.16 mol/g of xed oil wasor T1, T2, T3, and E1, respectively.d N. sativa oils contained mostly the essential fatty acid(55%), but also exhibited a signicant high content of22%) and these results are in agreement with previ-shed data (Ramadan et al., 2003, 2012a; Atta, 2003).l, like wheat germ oil (Piras et al., 2009), corn oil, cot-, and soybean oil (Ong and Goh, 2002), is characterizedatio of unsaturated to saturated fatty acids, and repre-rce of the essential fatty acid 18:2 n 6, a compound

be synthesized de novo by humans, indispensable forelopment and health (Hornstra, 2000). The special fattysition as well as the presence of valuable amounts ofe bioactive compounds make the N. sativa oil a special

A. Piras et al. / Industrial Crops and Products 46 (2013) 317 323 321

Table 2Fatty acid composition (% of total fatty acids, g/100 g) by GC of Nigella sativa xed oils obtained by SFE at 300 bar and 40 C.

Fatty acid T1 T2 T3 E1

12:0 0.07 0.03 0.11 0.04 0.09 0.03 0.05 0.0114:0 0.29 0.01 0.27 0.02 0.33 0.03 0.30 0.0316:0 12.30 0.52 12.27 0.14 13.31 0.33 13.51 0.5516:1 0.29 0.06 0.26 0.01 0.31 0.05 0.26 0.0218:0 2.79 0.06 2.64 0.08 2.69 0.04 2.83 0.2618:1 n 7 0.59 0.04 0.51 0.22 0.49 0.04 0.48 0.0818:1 n 9 22.75 0.50 22.55 0.43 22.29 0.13 22.53 0.5118:2 n 6 55.20 1.31 54.20 0.63 55.51 0.36 55.31 0.6718:3 n 3 0.30 0.03 0.25 0.00 0.25 0.00 0.25 0.0220:0 0.16 0.04 0.12 0.00 0.14 0.02 0.14 0.0120:1 n 9 0.26 0.02 0.24 0.01 0.23 0.03 0.24 0.0220:2 2.75 0.07 2.81 0.42 2.305 0.19 2.39 0.18SFA 15.60 0.50 15.41 0.16 16.57 0.32 16.86 0.68MUFA 23.89 0.59 23.57 0.54 23.31 0.11 23.51 0.49PUFA 58.90 1.25 58.11 0.46 58.29 0.32 58.15 0.59

SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Data are mean values (4 samples) with standard deviations (sd).

Table 3Unsaturated fatty acid (UFA) composition (mg/g weight) by HPLC of Nigella sativa xed oils obtained by SFE at 300 bar and 40 C.

UFA T1 T2 T3 E1

18:1 18:2 n 6 18:3 n 3

Data are mean

componentand Wahda

3.3. Antimi

The xedples were eAntibacteriof E. coli, PB. subtilis vactivity of C. krusei wconazole asthe breakpostandard st15769) by (REMA).

Antimicris reported ineffective effective Mbacteria we

s E. y agae anded oei at azolet C. atratint foy val

gramd theL1

xedolatid ac

Table 4Screening for

Extracts

T1 T2 T3 E1 AMPLVX GN KET FLU

AMP, ampicillisulfamethoxaz231.22 9.10 228.38 13.60 525.26 16.89 519.83 27.74

3.00 0.09 3.13 0.17 values (4 samples) with standard deviations (sd).

for nutritional application (Ramadan, 2013; Ramadann, 2012b).

crobial activity

and volatile oils extracted from different N. sativa sam-valuated for their antimicrobial activities (Tables 47).al activity was evaluated by employing standard strains. aeroginosa, A. baumannii, S. aureus, E. faecalis, andia broth microdilution method; in vitro antifungalthe samples against C. albicans, C. tropicalis, andere evaluated by comparing ketoconazole, and u-

control agents. As for anti-mycobacterium activity,int concentration (g mL1) was determined againstrains of M. tuberculosis H37Rv and M. avium (ATCCmeans of the colorimetric resazurin microtiter assay

such aactivitvolatiland xC. krusuconagainsconcendiffereactivit

Forshowe64 g mtested ples (velevateobial activity toward of standard and isolated strainsin Tables 47. All tested samples were determined asagainst isolated strains (MIC; >256 g mL1). The mostIC values of 16 and 32 g mL1 against gram negativere seen with volatile and xed oil of E1, respectively,

All the volatuberculosisless active wof 16 g mLmycin) shoMIC values

xed oil antimicrobial activity against gram-negative bacteria and fungi (MIC in g mL1

Gram-negative bacteria

E. coli P. aeruginosa A. baumanni

ATCC 35218 Isolated strain ATCC 10145 Isolated strain RSKK 02026

>256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256

32 >256 32 >256 64 128 0.5 >128 0.12 0.25 128 0.5 32 0.5 2

ne; LVX, levooxacin; GM, gentamicine; KET, ketaconazole; FLU, uconazole; E. coli isolaole, tazobactam), A. baumannii isolates (trimethoprim-sulfamethoxazole resist).222.43 12.85 201.13 4.72523.85 26.59 479.41 23.63

2.95 0.08 2.60 0.16

coli and P. aeruginosa. Similarly E1 showed moderateinst A. baumannii at MIC values of 32 and 64 g mL1 for

xed oil, respectively. On the other hand, all the volatileils (T1, T2, T3, E1) exhibited antifungal effect against64 g mL1, which is close to the effect of the control

(Table 4). Less effective concentration was obtainedlbicans and C. tropicalis at 16 and 32 g mL1 inhibitionon for volatile and xed oil, respectively, which was notr both strains. In addition, the controls showed differentues in the range 264 g mL1.

positive bacteria (S. aureus, E. faecalis, B. subtilis), E1 best activity against all standard strains at 32 and, for volatile and xed oil respectively, the rest of the

oils were ineffective against all of the strains. All sam-le and xed oils T1, T2, T3, and E1) demonstrated antivity against M. avium with MIC values of 8 g mL1.

tile oil samples had shown similar effect against M.

(Table 7), on the other hand, all xed oil samples wereere less active against M. tuberculosis with MIC values

1, which the controls (isoniazid, ethambutol, strepto-wed inhibitory activity between the 0.1252 g mL1

(Table 5).

).

Fungi

i C. albicans/C. tropicalis C. krusei

Isolated strain ATCC 10231/ATCC 13803 ATCC 6258

>256 32/32 64>256 32/32 64>256 32/32 64>256 32/32 64

0.5 32/32 64

0.5/2 42/4 64

tes; (+ESLs enzyme), P. aeruginosa isolates (resist to trimethoprim-

322 A. Piras et al. / Industrial Crops and Products 46 (2013) 317 323

Table 5Screening for xed oil antimicrobial activity against gram-positive bacteria and antitubercular (MIC in g mL1).

Gram-positive bacteria Mycobacterium

. subti

TCC 6

T1 256 T2 256 T3 256 E1 64AMP 0.1LVX INH EMB SM

AMP, ampicill d stra(cephalosporin

Table 6Screening for v g mL

Extracts

T1volT2volT3volE1volAMP LVX GN KET FLU

AMP, ampicillsulfamethoxaz

Table 7Screening for v

Extracts

T1volT2volT3volE1volAMP LVX INH EMB SM

AMP, ampicill(cephalosporin

As a reseffective agTropicalis, afour more studies on tthat volatilis in accordtion or diffeantimicrobiHasan et al.

4. Conclus

Chemicaby SFE froS. aureus E. faecalis B

ATCC 25923 Isolated strain ATCC 29212 Isolated strain A

>256 >256 >256 >256 >>256 >256 >256 >256 >>256 >256 >256 >256 >

64 >256 64 >256128 0.5 >128 0.25 128 0.5 32

ine; LVX, levooxacin; INH, Isoniazid; EMB, ethambutol; SM, streptomycin; isolate resist), isolated strain of B. subtilis (ceftriaxon resist).

olatile oil antimicrobial activity against gram-negative bacteria and fungi (MIC in Gram-negative bacteria

E. coli P. aeruginosa A. baumanni

ATCC 35218 Isolated strain ATCC 10145 Isolated strain RSKK 02026

128 >256 128 >256 128 128 >256 128 >256 128 128 >256 128 >256 128 16 >256 16 >256 32 128 0.5 >128 0.12 0.25 128 0.5 32 0.5 2

ine; LVX, levooxacin; GM, gentamicine; KET, ketaconazole; FLU, uconazole; E. coli isolaole, tazobactam), A. baumannii isolates (trimethoprim-sulfamethoxazole resist).

olatile oil antimicrobial activity against gram-positive bacteria and antitubercular (MIC

Gram-positive bacteria

S. aureus E. faecalis B. sub

ATCC 25923 Isolated strain ATCC 29212 Isolated strain ATCC

64 >256 64 >256 64 64 >256 64 >256 64 64 >256 64 >256 64 32 >256 32 >256 32 128 0.5 >128 0.120.25 128 0.5 32

ine; LVX, levooxacin, INH, isoniazid, EMB, ethambutol; SM, streptomycin; isolated stra resist), isolated strain of B. subtilis (ceftriaxon resist).

ult, all the volatile oil samples were two fold moreainst gram negative bacteria, fungi C. albicans and C.nd M. tuberculosis, additionally, volatile oil samples arefold effective against gram positive bacteria. Previoushe antimicrobial activity of N. sativa seeds had showne oil is much more effective than the xed oil, whichance with our results; moreover, the phenolic frac-rent solvent extracts from N. sativa seeds demonstratedal activity (Toppozada et al., 1965; Agarwal et al., 1979;, 1989; Hanafy et al., 1991).

ions

l composition of essential and xed oils extractedm the seeds of N. sativa, collected in four diverse

locations, hthymoquinmajor consfour sampleberculosis aand xed oies will be chemical co

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Chemical composition and in vitro bioactivity of the volatile and fixed oils of Nigella sativa L. extracted by supercritic...1 Introduction2 Materials and methods2.1 Chemicals2.2 Plant materials2.3 SFE extraction2.4 GC and GCMS analysis of essential oil2.5 Fixed oil2.5.1 Preparation of fatty acids2.5.2 HPLC analyses2.5.3 GC analysis

2.6 Microbiological studies2.6.1 Test materials2.6.2 Microorganisms and inoculum preparation2.6.3 Antibacterial and antifungal tests2.6.4 Anti-mycobacterium activity

3 Results and discussion3.1 Volatile oil3.2 Fixed oil3.3 Antimicrobial activity

4 ConclusionsReferences