Industrial Crops and Products 46 (2013) 317323

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Industrial Crops and Products
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Chemical composition and in vitro bioactivity of the volatile and xed oils of
Nigella sativa L. extracted by supercritical carbon dioxide
A. Piras
a
, A. Rosa
b,
, B. Marongiu
a
, S. Porcedda
a
, D. Falconieri
c
, M.A. Dess
b
, B. Ozcelik
d
, U. Koca
e
a
Dipartimento di Scienze Chimiche e Geologiche, Universit degli Studi di Cagliari, Cittadella Universitaria di Monserrato, Cagliari, Italy
b
Dipartimento di Scienze Biomediche, Universit degli Studi di Cagliari, Cittadella Universitaria di Monserrato, Cagliari, Italy
c
ITI M. Giua, via Montecassino, Cagliari, Italy
d
Department of Microbiology, Faculty of Pharmacy, Gazi University, Ankara, Turkey
e
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 October 2012
Received in revised form
17 December 2012
Accepted 1 February 2013
Keywords:
Nigella sativa
Essential oil
Fixed oil
In vitro biological activity
Antibacterial
Antifungal
Antituberculosis
Supercritical extraction
a b s t r a c t
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 as
well as a spice. The seeds contain both xed and essential oils, proteins, alkaloids and saponins. Much of
the biological activity of the seeds has been shown to be due to thymoquinone, the major component of
the essential oil. The xed oil is composed mainly of unsaturated fatty acids, including the unusual C20:2
eicosadienoic 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 pressures
of 90 and 300 bar and temperature of 40

C. The extraction step performed at 90bar produced a volatile

fraction mainly formed by tymoquinone (79 86%) and o-cymene (5 11%). The oil yield relative to this
step of the process was 0.1 0.3% by weight of the charge. The last extraction step at 300 bar produced a
xed oil. The yield of this step was 2126% by weight. The most represented fatty acids of xed oil from
N. 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 by
employing 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 mL
1
) was determined against standard strains
of Mycobacterium tuberculosis H37Rv and M. avium (ATCC 15769). The volatile and xed oils displayed
antimicrobial activity toward all of the standard (ATCC, RSKK) strains of the tested bacteria at MIC values
of 864 g mL
1
and were revealed to be ineffective against isolated strains (MIC; >256 g mL
1
). The
volatile and xed oils emerged as effective against the bacteria of M. avium with MIC values of 8 g mL
1
.
Moreover, all the extracts exhibited antifungal activity against C. albicans, C. tropicalis, and C. krusei at MIC
values of 1664 g mL
1
.
2013 Elsevier B.V. All rights reserved.
1. Introduction
Nigella sativa L., commonly known as black cumin seed, is an
annual herbaceous plant belonging to the Ranunculaceae family
growing in countries bordering the Mediterranean Sea. It tastes
slightly bitter and peppery with a crunchy texture. Seeds are angu-
lar, of generally small size (15mg), dark gray or black color and
they are used for edible and medicinal purposes in many countries

Corresponding author. Tel.: +39 070 675 4124; fax: +39 070 675 4032.
E-mail address: anrosa@unica.it (A. Rosa).
(Cheikh-Rouhou et al., 2007). The composition and properties of
this 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 investigatedthe volatile oil of nigella seeds
and isolated and identied active constituents that have benecial
clinical effects. Egyptians believe that nigella seeds increase human
immunity. The volatile oil has been produced by pressing the raw
or roasted seeds (Atta, 2003).
Nigella seed xed oil is considered as one among newer sources
of edible oils, thanks to its important role in human nutrition and
0926-6690/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2013.02.013
318 A. Piras et al. / Industrial Crops and Products 46 (2013) 317323
health. Black cumin crude xed seed oil is a valuable source of
essential fatty acids, glycolipids, phospholipids, and bioactive phy-
tosterols (Ramadan, 2007; Ramadan et al., 2012a). This oil has
been reported to possess antitumor activity, antioxidant activity,
anti-inammatory activity, antibacterial activity and a stimulatory
effect on the immune system. Actually, a great deal of attention has
been focused on black cumin seed oils, and thus their consumption
has increased, especially in Middle East countries. The oil has been
usually produced by a hot solvent extraction method at 4060

C
and even at 70

C, using the Soxhlet extractor. This hot method of

extraction could affect the oil properties and may induce partial
alteration of the majority of minor constituents that have many
functional, antioxidative andpro-oxidative effects (Cheikh-Rouhou
et al., 2007). Recently, the cold-pressing procedure has been used
to obtain black cumin seed oil (Ramadan et al., 2012a).
Fats and oils play an important role in the food industry and are
important in human nutrition for a variety of reasons. Particularly,
vegetable oils are becoming increasingly important as suppliers of
vitamins and essential unsaturated fatty acids. Therefore, it is very
important toinvestigate the possibilityof usingsome rawmaterials
as a source of vegetable oils and vitamins.
The objective of this work is to explore the potential application
of supercritical CO
2
to the extraction of oils from N. sativa seeds
and to investigate their chemical composition and their antimicro-
bial activity. Supercritical CO
2
is a promising solvent for extraction
and fractionation of edible oils, since the extraction can be carried
out at low temperature. Besides, the supercritical uid extraction
(SFE) offers many other favorable features over the traditional tech-
niques, like steamdistillationandsolvent extraction, due to the fact
that it uses a clean, inexpensive, non-ammable and non-toxic sol-
vent (Piras et al., 2009, 2012; Porcedda et al., 2009; Marongiu et al.,
2003).
2. Materials and methods
2.1. Chemicals
Triolein, trilinolein, standards of fatty acids and fatty acid
methyl esters, and desferal (deferoxamine mesylate salt), were
purchased from SigmaAldrich (Milan, Italy). All solvents used, of
the highest available purity, were also from SigmaAldrich. The
methanolic HCl (3N) was purchased fromSupelco (Bellefonte, PA).
cis,trans-13-Hydroperoxyoctadecadienoic acid (c,t-13-HPODE) and
cis,trans-9-hydroperoxyoctadecadienoic acid (c,t-9-HPODE) were
obtained fromCascade (Cascade Biochem. Ltd., London, UK). All of
the other chemicals used in this study were of analytical grade.
2.2. Plant materials
Cultivated N. sativa seeds were obtained from the cultivation
cities of Turkey namely T1 (Antalya), T2 (Aydn), T3 (Deni-
zli). Only the seeds were obtained from the cultured N. sativa
plants, which were authenticated by us, voucher specimens were
deposited at the Gazi University, Department of Pharmacognosy,
numbered as UK2010NST1, UK2010NST2, UK2010NST3 sequen-
tially. The seeds from Egyptian N. sativa, E1, were supplied by
Minardi (Bagnacavallo-Ravenna, Italy). Before utilization, matter
was groundwithaMalavasi mill (Bologna, Italy) takingcaretoavoid
overheating and the particles sizes were in the range (250425)
m.
2.3. SFE extraction
Supercritical CO
2
extractions were performed in a laboratory
apparatus, equipped with a 320cm
3
extraction vessel and two sep-
arator vessels of 300 and 200cm
3
respectively connected in series.
Extraction was carried out in a semi batch mode: batch charging of
vegetable matter and continuous owsolvent. The N. sativa volatile
oil was obtained working at 90bar and 40

C in the extraction ves-

sel, at 90bar and 10

C in the rst separator and at 20bar and

15

C in the second one. The extraction of the xed oil was run on
the same samples of N. sativa previously treated at 90bar; the xed
oil was obtained working at 300bar and 40

C in the extraction ves-

sel and by using only one separator (at 20bar and 15

C) to recover
the extract.
2.4. GC and GCMS analysis of essential oil
Analyzes of the volatile extracts were carried out by gas chro-
matography (GC) and by gas chromatographymass spectrometry
(GCMS). Analytical GC was carried out in a gas chromatograph
(Agilent, Model 7890A, Palo Alto, CA), equipped with a ame ion-
ization detector (FID), an autosampler (Agilent, Model 7683B),
Agilent HP5 fused silica column (5% phenyl-methylpolysiloxane),
30m0.25mm i.d., lm thickness 0.25m, and a Agilent Chem-
Station software system. Oven temperature was settled at 60

C,
raising at 3

C/min to 250

C and then held 20min at 250

C; injec-
tor temperature: 250

C; carrier gas: heliumat 1.0mL/min; splitting

ratio 1:10; detector temperature: 300

C.
GCMS analyses were carried out in a gas chromatograph (Agi-
lent, Model 6890N, Palo Alto, CA) equipped with a splitsplitless
injector, an autosampler Agilent model 7683 and two dif-
ferent Agilent fused silica capillary columns (30m0.25mm
i.d., lm thickness 0.25m) of different polarities (HP-5, 5%
phenyl-methylpolysiloxane; DB-WAXetr, polyethylene glycol). GC
conditions used were: programmed heating from 60 to 250

C
at 3

C/min followed by 20min under isothermal conditions. The

injector was maintained at 250

C. Helium was the carrier gas

at 1.0mL/min; the sample (1L) was injected in the split mode
(1:10). The GC was tted with a quadrupole mass spectrometer,
MS, Agilent model 5973 detector. MS conditions were as follows:
ionization energy 70eV, electronic impact ion source temperature
200

C, quadrupole temperature 150

C, scan rate 3.2 scan/s, mass

range 30480u. Software adoptedtohandle mass spectra andchro-
matograms was a ChemStation. Samples were run in chloroform
with a dilution ratio of 1:100. Compounds were identied by com-
parisonof their mass spectrawiththoseof NIST02librarydataof the
GCMS system and Adams libraries spectra (NIST/EPA/NIH Mass
Spectral Library, 2002; Adams, 2007). The results were further con-
rmedby comparisonwiththe compounds elutionorder withtheir
retention indices on semi-polar phases reported in the literature
(Adams, 2007). Retention indices of the components were deter-
mined relative to the retention times of a series of n-alkanes with
linear interpolation.
Percentage of individual components was calculated based on
GC peak areas without FID response factor correction.
2.5. Fixed oil
2.5.1. Preparation of fatty acids
Separation of fatty acids was obtained by mild saponication
(Rosa et al., 2011) as follows: 3mg of the xed oil were dissolved in
5mL of EtOH and 100L of desferal solution (25mg/mL of H
2
O),
1mL of a water solution of ascorbic acid (25% w/v), and 0.5mL
of 10N KOH were added. The mixtures were left in the dark at
room temperature for 14h. After addition of 10mL of n-hexane
and 7mL of H
2
O, samples were centrifuged for 1h at 900g. The
hexane phase was collected, and after addition of further 10mL of
n-hexane to the mixtures, samples were acidied with 37% HCl to
pH 34 and then centrifuged for 1h at 900g. The hexane phase
(saponiable fraction) with free fatty acids and conjugated diene
fatty acid hydroperoxides (HP) was collected and the solvent was
A. Piras et al. / Industrial Crops and Products 46 (2013) 317323 319
evaporated. A portion of the dried residue was dissolved in 500L
of CH
3
CN with 0.14% CH
3
COOH (v/v) and aliquots of the samples
were injected into the HPLC system.
An aliquot of dried fatty acids was methylated with 1mL of
methanolic HCl (3N) (Christie, 1993; Rosa et al., 2011) for 30min
at roomtemperature. After addition of 4mL of n-hexane and 2mL
of H
2
O, samples were centrifuged for 20min at 900g. The hex-
ane phase with fatty acid methyl esters was collected, the solvent
was evaporated, the residue was dissolved in 250L of n-hexane
and aliquots of the samples were injected into the GC system. The
recovery of fatty acids during saponication was calculated using
an external standard mixture prepared dissolving 1mg of triolein
andtrilinoleinin5mL of EtOHandprocessedas samples. All solvent
evaporation was performed under vacuum.
2.5.2. HPLC analyses
Analyses of unsaturated fatty acids, and oxidative products
were carried out with an Agilent Technologies 1100 liquid chro-
matograph (Agilent Technologies, Palo Alto, CA) equipped with a
diode array detector (HPLC-DAD). Analyses of unsaturated fatty
acids and HP, detected at 200 and 234nm, respectively, were
carried out with a XDB-C
18
Eclipse (150mm4.6mm, 3.5mpar-
ticle size) (Agilent Technologies) equipped with a Zorbax XDB-C
18
Eclipse (12.5mm4.6mm, 5mparticle size) guard column (Agi-
lent Technologies), with a mobile phase of CH
3
CN/H
2
O/CH
3
COOH
(75/25/0.12, v/v/v), at a ow rate of 2.3mL/min (Rosa et al., 2011).
The temperature of the columnwas maintainedat 37

C. The identi-
cation of fatty acids and HP was made using standard compounds
and the second derivative, as well as conventional UV spectra, gen-
erated with the Agilent Chemstation A.10.02 software. Calibration
curves of all of the compounds were constructed using standards
and were found to be linear with correlation coefcients >0.995.
2.5.3. GC analysis
Fatty acid methyl esters were measured on a gas chro-
matograph Hewlett-Packard HP-6890 (Hewlett-Packard, Palo
Alto, USA) with a ame ionization detector and equipped
with a cyanopropyl methyl-polysiloxane HP-23 FAME column
(30m0.32mm0.25m) (Hewlett-Packard). Nitrogenwas used
as carrier gas at a ow rate of 2mL/min. The oven temperature
was set at 175

C; the injector temperature was set at 250

C; and
the detector temperature was set at 300

C. The fatty acid methyl

esters were identied by comparing the retention times to those of
standard compounds. The composition of individual fatty acid was
calculated as a percentage of the total amount of fatty acids (g%),
using the Hewlett-Packard A.05.02 software.
2.6. Microbiological studies
2.6.1. Test materials
Extracts were dissolved in dimethylsulfoxide (30%) and H
2
O
(70%) at a nal concentration of 512gmL
1
and sterilized by
ltration using 0.22(m Millipore (MA 01730, USA), and used as
the stock solutions. Ampicillin, gentamycin, levooxacin, isoni-
azid, ethambutol, ketoconazole, and uconazole were used as the
standard antibacterial and antifungal drugs. Reference antibacte-
rial agents were purchased from Sigma Chemical Co. (St. Louis,
MO, USA) and dissolved in phosphate buffer solution (ampicillin;
pH: 6.0; 0.1mol/mL), dimethylsulfoxide (ketoconazole), or inwater
(meropenem, gentamycin, levooxacin, uconazole). The stock
solutions of the agents were prepared in mediumaccording to the
Clinical and Laboratory Standards Institute (CLSI formerly; NCCLS)
(CLSI, 2008). A stock solution of the resazurin sodiumsalt (Sigma)
powder was prepared at 0.01% in sterile distilled water. It was
lter-sterilized and kept at 4

C.
2.6.2. Microorganisms and inoculumpreparation
Antibacterial activity and antifungal activity tests were car-
ried out against standard (ATCC; American type culture collection,
RSKK; Culture collectionof Rek SaydamCentral Hygiene Institute;
NCPF; National Collection of Pathogenic Fungi) and isolated strains
(clinical isolate obtainedfromDepartment of Microbiology, Faculty
of Medicine, Gazi University).
As standards; gram negative strains of Escherichia coli (ATCC
35218), Pseudomonas aeroginosa (ATCC 10145), Acinetobacer bau-
mannii (RSKK02026), andas grampositive strains of Staphylococcus
aureus (ATCC 25923), and Enterococcus faecalis (ATCC 29212), B.
subtilis (ATCC 6633) were used for the determination of antibac-
terial activity. Candida albicans (ATCC 10231), C. tropicalis (ATCC
13803), and C. krusei (ATCC 6258) were used for the determination
of antifungal activity.
Mueller Hinton Broth (MHB; Difco) and Mueller Hinton Agar
(MHA; Oxoid) were applied for growing and diluting of the bacteria
suspensions. The synthetic medium RPMI-1640 with l-glutamine
was buffered to pH 7 with 3-[N-morpholino]-propansulfonic acid
and culture suspensions were prepared as described previously
(Koca et al., 2010). The microorganism suspensions used for inoc-
ulation were prepared at 10
5
cfu (colony forming unit/mL) by
diluting freshcultures at McFarland0.5density(10
8
cfumL
1
). Sus-
pensions of bacteria and fungi were added in each well of the
diluted extracts, density of 10
5
cfumL
1
for fungi, and for bacte-
ria. The bacterial suspensions used for inoculation were prepared
at 10
5
cfumL
1
by diluting fresh cultures at McFarland 0.5 density
(10
8
cfumL
1
). The fungi suspension was prepared by the spec-
trophotometric method of inoculums (zc elik et al., 2012).
2.6.3. Antibacterial and antifungal tests
The microdilution method was employed for antibacterial and
antifungal activity tests. Media were placed into each 96 wells of
the microplates. Volatile and xed oils at 512gmL
1
were added
into rst rows of microplates and two fold dilutions of the sam-
ples (2560.125gmL
1
) were made by dispensing the solutions
to the remaining wells. The culture suspensions (10L) were inoc-
ulated into all the wells. All organisms were tested in triplicate in
each run of the experiments. The sealed microplates were incu-
bated at 35

C for 24h and 48h in humid chamber. DMSO, H

2
O,
pure microorganisms and pure media were used as control wells.
The lowest concentration of the samples that completely inhibit
macroscopic growth of the cultures was determined and minimum
inhibitory concentrations (MICs) were reported as described pre-
viously (zc elik et al., 2012).
2.6.4. Anti-mycobacteriumactivity
The effect of the volatile and xed oils of N. sativa was searched
for anti tuberculosis activity against Mycobacterium tuberculo-
sis and M. avium, which Resazurin microplate assay procedure
(REMA) was carried out (Orhan et al., 2012). The strains M. tuber-
culosis H37Rv (ATCC 27294; American Type Culture Collection)
reference strain and M. avium (ATCC 15769) were maintained
on LowensteinJensen medium and subcultured on Middlebrook
7H11agar (BectonDickinson) resuspendedin7H9-S brothmedium
supplemented with 10% [OADC; 0.1% casitone, 0.5% glycerol,
supplemented oleic acid, albumin, dextrose, and catalase], 0.2%
glycerol and 0.1% Bacto casitone (Difco). Suspensions were pre-
pared in 0.04% (v/v) Tween 800.2%+bovine serum albumin so
that adjusted to McFarland tube number 1. This was diluted to
1:20 and 100L aliquot was used as inoculum. One hundred
microliters of Middlebrook 7H9 broth (0.1% casitone, 0.5% glyc-
erol, and 10% OADC; Becton-Dickinson) was dispensed in each well
of a sterile at-bottom 96-well plate, and serial twofold dilutions
(2560.06gmL
1
) of each compound were prepared directly in
the plate. One hundred microliters of inoculumwas added to each
320 A. Piras et al. / Industrial Crops and Products 46 (2013) 317323
Table 1
Chemical 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 MH
1027 1271 7.6 5.4 5.4 11.0 o-Cymene MH
1031 1200 0.7 Limonene MH
1062 1247 0.6 0.6 -Terpinene MH
1179 1595 0.5 Terpinen-4-ol MO
1198 1664 0.6 Methyl chavicol MO
1252 2989 77.2 78.1 86.2 79.5 Tymoquinone MO
1293 1647 1.5 trans-Sabynil acetate MO
1303 2201 5.8 7.9 2.9 Carvacrol MO
1402 1556 2.4 2.0 2.0 1.9 Longifolene SH
1418 1586 0.6 (E)-Caryophyllene SH
Total identied 95.2 95.1 97.0 93.1
% MH 6.0 8.2 5.4 11.7
% MO 86.6 84.5 89.6 79.5
% SH 2.6 2.4 2.0 1.9
well. A growth control and a sterile control were also included
for each isolate. Sterile water was added to all perimeter wells to
avoid evaporation during the incubation. The plate was covered,
and incubated at 37

C under a normal atmosphere. After 7 days

of incubation, 10gmL
1
of resazurin solution was added to each
well, and the plate was reincubated overnight. A change in color
from blue (oxidized state) to pink (reduced) indicated the growth
of bacteria, and the MIC was dened as the lowest concentration of
the sample that prevented this change.
3. Results and discussion
3.1. Volatile oil
The volatile oils extracted by SFE from the seeds of N. sativa,
collected in four diverse locations, ranged from0.1% to 0.3% (w/w).
All the volatile fractions were analyzed by GC and by GCMS
on two columns of different polarity. Table 1 reports the iden-
tied components, their percentages, their retention indices on
both HP-5 and DB-WAXetr columns. Analysis of the oils led to the
identication of 11 components (four monoterpenes hydrocarbon,
ve oxygenated monoterpenes, and two sesquiterpenes hydro-
carbon) which represented 93.197.0% of the total amount. The
oil consisted chiey of oxygenated monoterpenes (79.589.6%)
accompanied by noticeable contents of monoterpenes hydrocar-
bons (5.411.7%) and much smaller amounts of sesquiterpenes
hydrocarbons (1.92.6%).
Among the oxygenated monoterpenes, thymoquinone was the
major constituent in all four samples (77.286.2%), accompanied
by lower contents of terpinen-4-ol, methyl chavicol, trans-sabynil
acetate and carvacrol not detected at all in all four oils (see Table 1).
The monoterpene hydrocarbons were distinctly dominated by o-
cymene (5.411.0%), while -thujene, limonene and -terpinene
were present at much lower amounts. The analytical data showed
that N. sativa fromTurkey and Egypt belongs to the thymoquinone
chemotype, with a high level of thymoquinone (77.286.2%).
Our results reinforce previous data on the variability seed
volatile oils, depending on the origin of the samples, environ-
mental and climatic conditions. A variety of chemotypes have
been described in the literature. An Iranian N. sativa essential
oil was found to be dominated by phenylpropanoid compo-
nents and displayed a trans-anethole chemotype (Nickavar et al.,
2003); other N. sativa from Iran (Hajhashemi et al., 2004), Alge-
ria (Benkaci-Ali et al., 2007) and India (Singh et al., 2005) was
found p-cymene/thymoquinone chemotype. While a chemotype
with 33.0% p-cymene and 26.8% thymol and the preponderance of
monoterpenes was reportedfor N. sativa essential oil fromMorocco
(Moretti et al., 2004; DAntuono et al., 2002) and a chemotype with
60.2% p-cymene and 12.9% -terpinene was reported by Wajs et al.
(2008) for N. sativa fromPoland.
Burits and Bucar (2000) reported the chemical composition of
the essential oils from N. sativa from Austria. They found thymo-
quinone (27.257.0%) and p-cymene (7.015.7%) as the major
compounds. Recently, Bourgou et al. (2010) found in Tunisian N.
sativa p-cymene as main component (60.5%).
3.2. Fixed oil
On the exhausted matrix a further extraction at higher pressure
(300bar) and 40

C was performed for the extraction of xed oil,

with a yield of 21.026.0%.
Quali-quantitative informationonthe individual fatty acids that
compose the lipid classes of N. sativa xed oils was obtained by
GC and HPLC analyses. Table 2 shows the fatty acid composition
(expressed as % of total fatty acids, g/100g) of N. sativa oils by GC.
All the oils showed a similar fatty acid composition, with a con-
centration of approximately 16% of saturated fatty acids (mainly
palmitic acid 16:0, and stearic acid 18:0, 1213% and 23%, respec-
tively), 23% of monounsaturated (mainly oleic acid 18:1 n9
and cis-vaccenic acid 18:1 n7, 22% and 0.5%, respectively), and
58% of polyunsaturated, mainly constituted by linoleic acid 18:2
n6 and eicosadienoic acid 20:2, 5455 and 2%, respectively. This
fatty acid prole was comparable to those reported by Ramadam
and collaborators for black cumin seed oil obtained by two dif-
ferent techniques, Soxhlet extraction (Ramadan et al., 2003) and
cold-pressing procedure (Ramadan et al., 2012a). Furthermore, the
content of the main unsaturated fatty acids in the oils was also
detected by HPLC, as follows: approximately 500mg/g of 18:1
n9, 200 and 3mg/g of 18:2 n6 and 18:3 n3, respectively,
as reported in Table 3. The oil oxidative status was evaluated by
HPLC determination of the HP level. The extracted oils showed a
different oxidative status and an average HP content of 6.480.52,
3.790.20, 13.500.43, and 2.810.16mol/g of xed oil was
measured for T1, T2, T3, and E1, respectively.
Extracted N. sativa oils contained mostly the essential fatty acid
18:2 n6 (55%), but also exhibited a signicant high content of
oleic acid (22%) and these results are in agreement with previ-
ously published data (Ramadan et al., 2003, 2012a; Atta, 2003).
N. sativa oil, like wheat germ oil (Piras et al., 2009), corn oil, cot-
tonseed oil, and soybean oil (Ong and Goh, 2002), is characterized
by a high ratio of unsaturated to saturated fatty acids, and repre-
sents a source of the essential fatty acid 18:2 n6, a compound
that cannot be synthesized de novo by humans, indispensable for
human development and health (Hornstra, 2000). The special fatty
acid composition as well as the presence of valuable amounts of
lipid-soluble bioactive compounds make the N. sativa oil a special
A. Piras et al. / Industrial Crops and Products 46 (2013) 317323 321
Table 2
Fatty acid composition (% of total fatty acids, g/100g) by GC of Nigella sativa xed oils obtained by SFE at 300bar 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 0.01
14:0 0.29 0.01 0.27 0.02 0.33 0.03 0.30 0.03
16:0 12.30 0.52 12.27 0.14 13.31 0.33 13.51 0.55
16:1 0.29 0.06 0.26 0.01 0.31 0.05 0.26 0.02
18:0 2.79 0.06 2.64 0.08 2.69 0.04 2.83 0.26
18:1 n7 0.59 0.04 0.51 0.22 0.49 0.04 0.48 0.08
18:1 n9 22.75 0.50 22.55 0.43 22.29 0.13 22.53 0.51
18:2 n6 55.20 1.31 54.20 0.63 55.51 0.36 55.31 0.67
18:3 n3 0.30 0.03 0.25 0.00 0.25 0.00 0.25 0.02
20:0 0.16 0.04 0.12 0.00 0.14 0.02 0.14 0.01
20:1 n9 0.26 0.02 0.24 0.01 0.23 0.03 0.24 0.02
20:2 2.75 0.07 2.81 0.42 2.305 0.19 2.39 0.18
SFA 15.60 0.50 15.41 0.16 16.57 0.32 16.86 0.68
MUFA 23.89 0.59 23.57 0.54 23.31 0.11 23.51 0.49
PUFA 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 3
Unsaturated fatty acid (UFA) composition (mg/g weight) by HPLC of Nigella sativa xed oils obtained by SFE at 300bar and 40

C.
UFA T1 T2 T3 E1
18:1 231.22 9.10 228.38 13.60 222.43 12.85 201.13 4.72
18:2 n6 525.26 16.89 519.83 27.74 523.85 26.59 479.41 23.63
18:3 n3 3.00 0.09 3.13 0.17 2.95 0.08 2.60 0.16
Data are mean values (4 samples) with standard deviations (sd).
component for nutritional application (Ramadan, 2013; Ramadan
and Wahdan, 2012b).
3.3. Antimicrobial activity
The xedandvolatile oils extractedfromdifferent N. sativa sam-
ples were evaluated for their antimicrobial activities (Tables 47).
Antibacterial activity was evaluated by employing standard strains
of E. coli, P. aeroginosa, A. baumannii, S. aureus, E. faecalis, and
B. subtilis via broth microdilution method; in vitro antifungal
activity of the samples against C. albicans, C. tropicalis, and
C. krusei were evaluated by comparing ketoconazole, and u-
conazole as control agents. As for anti-mycobacterium activity,
the breakpoint concentration (gmL
1
) was determined against
standard strains of M. tuberculosis H37Rv and M. avium (ATCC
15769) by means of the colorimetric resazurin microtiter assay
(REMA).
Antimicrobial activity toward of standard and isolated strains
is reported in Tables 47. All tested samples were determined as
ineffective against isolated strains (MIC; >256gmL
1
). The most
effective MIC values of 16 and 32gmL
1
against gram negative
bacteria were seen with volatile and xed oil of E1, respectively,
such as E. coli and P. aeruginosa. Similarly E1 showed moderate
activity against A. baumannii at MIC values of 32 and 64gmL
1
for
volatile andxedoil, respectively. Onthe other hand, all the volatile
and xed oils (T1, T2, T3, E1) exhibited antifungal effect against
C. krusei at 64gmL
1
, which is close to the effect of the control
uconazole (Table 4). Less effective concentration was obtained
against C. albicans and C. tropicalis at 16 and 32gmL
1
inhibition
concentration for volatile and xed oil, respectively, which was not
different for both strains. In addition, the controls showed different
activity values in the range 264gmL
1
.
For gram positive bacteria (S. aureus, E. faecalis, B. subtilis), E1
showed the best activity against all standard strains at 32 and
64gmL
1
, for volatile and xed oil respectively, the rest of the
tested xed oils were ineffective against all of the strains. All sam-
ples (volatile and xed oils T1, T2, T3, and E1) demonstrated an
elevated activity against M. avium with MIC values of 8gmL
1
.
All the volatile oil samples had shown similar effect against M.
tuberculosis (Table 7), on the other hand, all xed oil samples were
less active were less active against M. tuberculosis with MIC values
of 16gmL
1
, which the controls (isoniazid, ethambutol, strepto-
mycin) showed inhibitory activity between the 0.1252gmL
1
MIC values (Table 5).
Table 4
Screening for xed oil antimicrobial activity against gram-negative bacteria and fungi (MIC in gmL
1
).
Extracts Gram-negative bacteria Fungi
E. coli P. aeruginosa A. baumannii C. albicans/C. tropicalis C. krusei
ATCC 35218 Isolated strain ATCC 10145 Isolated strain RSKK 02026 Isolated strain ATCC 10231/ATCC 13803 ATCC 6258
T1 >256 >256 >256 >256 >256 >256 32/32 64
T2 >256 >256 >256 >256 >256 >256 32/32 64
T3 >256 >256 >256 >256 >256 >256 32/32 64
E1 32 >256 32 >256 64 >256 32/32 64
AMP <0.12 >128 0.5 >128 0.12 0.5 32/32 64
LVX 0.25 128 0.5 32
GN 0.5 2
KET 0.5/2 4
FLU 2/4 64
AMP, ampicilline; LVX, levooxacin; GM, gentamicine; KET, ketaconazole; FLU, uconazole; E. coli isolates; (+ESLs enzyme), P. aeruginosa isolates (resist to trimethoprim-
sulfamethoxazole, tazobactam), A. baumannii isolates (trimethoprim-sulfamethoxazole resist).
322 A. Piras et al. / Industrial Crops and Products 46 (2013) 317323
Table 5
Screening for xed oil antimicrobial activity against gram-positive bacteria and antitubercular (MIC in gmL
1
).
Gram-positive bacteria Mycobacterium
S. aureus E. faecalis B. subtilis M. tuberculosis M. avium
ATCC 25923 Isolated strain ATCC 29212 Isolated strain ATCC 6633 Isolated strain ATCC 27294 ATCC 15769
T1 >256 >256 >256 >256 >256 >256 16 8
T2 >256 >256 >256 >256 >256 >256 16 8
T3 >256 >256 >256 >256 >256 >256 16 8
E1 64 >256 64 >256 64 >256 16 8
AMP <0.12 >128 0.5 >128 0.12 0.5
LVX 0.25 128 0.5 32
INH 0.125 0.125
EMB 2 2
SM 1 2
AMP, ampicilline; LVX, levooxacin; INH, Isoniazid; EMB, ethambutol; SM, streptomycin; isolated strain of S. aureus (methicillin resist; MRSA), isolated strain of E. faecalis
(cephalosporin resist), isolated strain of B. subtilis (ceftriaxon resist).
Table 6
Screening for volatile oil antimicrobial activity against gram-negative bacteria and fungi (MIC in gmL
1
).
Extracts Gram-negative bacteria Fungi
E. coli P. aeruginosa A. baumannii C. albicans/C. krusei C. tropicalis
ATCC 35218 Isolated strain ATCC 10145 Isolated strain RSKK 02026 Isolated strain ATCC 10231/ATCC 13803 ATCC 6258
T1
vol
128 >256 128 >256 128 >256 32/16 64
T2
vol
128 >256 128 >256 128 >256 32/16 64
T3
vol
128 >256 128 >256 128 >256 32/16 64
E1
vol
16 >256 16 >256 32 >256 32/16 64
AMP <0.12 >128 0.5 >128 0.12 0.5
LVX 0.25 128 0.5 32
GN 0.5 2
KET 0.5/2 4
FLU 2/4 64
AMP, ampicilline; LVX, levooxacin; GM, gentamicine; KET, ketaconazole; FLU, uconazole; E. coli isolates; (+ESLs enzyme), P. aeruginosa isolates (resist to trimethoprim-
sulfamethoxazole, tazobactam), A. baumannii isolates (trimethoprim-sulfamethoxazole resist).
Table 7
Screening for volatile oil antimicrobial activity against gram-positive bacteria and antitubercular (MIC in gmL
1
).
Extracts Gram-positive bacteria Mycobacterium
S. aureus E. faecalis B. subtilis M. tuberculosis M. avium
ATCC 25923 Isolated strain ATCC 29212 Isolated strain ATCC 6633 Isolated strain ATCC 27294 ATCC 15769
T1
vol
64 >256 64 >256 64 >256 8 8
T2
vol
64 >256 64 >256 64 >256 8 8
T3
vol
64 >256 64 >256 64 >256 8 8
E1
vol
32 >256 32 >256 32 >256 8 8
AMP <0.12 >128 0.5 >128 0.12 0.5
LVX 0.25 128 0.5 32
INH 0.125 0.125
EMB 2 2
SM 1 2
AMP, ampicilline; LVX, levooxacin, INH, isoniazid, EMB, ethambutol; SM, streptomycin; isolated strain of S. aureus (methicillin resist; MRSA), isolated strain of E. faecalis
(cephalosporin resist), isolated strain of B. subtilis (ceftriaxon resist).
As a result, all the volatile oil samples were two fold more
effective against gram negative bacteria, fungi C. albicans and C.
Tropicalis, and M. tuberculosis, additionally, volatile oil samples are
four more fold effective against gram positive bacteria. Previous
studies on the antimicrobial activity of N. sativa seeds had shown
that volatile oil is much more effective than the xed oil, which
is in accordance with our results; moreover, the phenolic frac-
tion or different solvent extracts fromN. sativa seeds demonstrated
antimicrobial activity (Toppozada et al., 1965; Agarwal et al., 1979;
Hasan et al., 1989; Hanafy et al., 1991).
4. Conclusions
Chemical composition of essential and xed oils extracted
by SFE from the seeds of N. sativa, collected in four diverse
locations, had been investigated. The oxygenated monoterpene
thymoquinone and the essential fatty acid 18:2 n6 were the
major constituents of essential and xed oils, respectively, in all
four samples. All volatile andxedoils displayedinteresting antitu-
berculosis and antifungal activities. Egyptian Nigella sativa volatile
and xed oils showed the best antimicrobial activity. Further stud-
ies will be performed to better clarify the relationship between
chemical composition and observed biological activity.
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