Food and Chemical Toxicology 48 (2010) 2186–2192

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Food and Chemical Toxicology

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Chemical composition and biological activities of Tunisian Cuminum cyminum L.

essential oil: A high effectiveness against Vibrio spp. strains
Hafedh Hajlaoui a,*, Hedi Mighri b, Emira Noumi a, Mejdi Snoussi a,c,1, Najla Trabelsi d, Riadh Ksouri d,
Amina Bakhrouf a
a
Laboratoire d’Analyse, Traitement et Valorisation des Polluants de l’Environnement et des Produits, Département de Microbiologie, Faculté de Pharmacie, Rue Avicenne,
Monastir 5000, Tunisia
b
Laboratoire d’Ecologie Pastorale, Institut des Régions Arides, Km 22.5, Route du Djorf, 4119 Médenine, Tunisia
c
Laboratoire de Traitement et de Recyclage des Eaux, Centre de Recherches et des Technologies des Eaux (CRTE), Technopole de Borj-Cédria, BP 273, Soliman 8020, Tunisia
d
Laboratoire des Plantes Extrêmophiles, Centre de Biotechnologie, Technopole de Borj-Cédria (CBBC), BP 901, 2050 Hammam-Lif, Tunisia

a r t i c l e i n f o a b s t r a c t

Article history: Essential oil extracted by hydrodistillation from Tunisian variety of Cuminum cyminum was characterized
Received 11 January 2010 by means of GC and GC–MS. Twenty-one components were identified and C. cyminum contained cuminl-
Accepted 12 May 2010 aldehyde (39.48%), gamma-terpinene (15.21%), O-cymene (11.82%), beta-pinene (11.13%), 2-caren-10-al
(7.93%), trans-carveol (4.49%) and myrtenal (3.5%) as a major components. Moreover, C. cyminum oil
exhibited higher antibacterial and antifungal activities with a high effectiveness against Vibrio spp.
Keywords: strains with a diameter of inhibition zones growth ranging from 11 to 23 mm and MIC and MBC values
Cuminum cyminum
ranging from (0.078–0.31 mg/ml) to (0.31–1.25 mg/ml), respectively.
Essential oil
GC–MS
On the other hand, the cumin oil was investigated for its antioxidant activities using four different tests
Antimicrobial activity then compared with BHT. Results showed that cumin oil exhibit a higher activity in each antioxidant sys-
Vibrio spp. tem with a special attention for b-carotene bleaching test (IC50: 20 lg/ml) and reducing power (EC50:
Antioxidant activity 11 lg/ml).
In the light of these findings, we suggested that C. cyminum essential oil may be considered as an inter-
esting source of antibacterial, antifungal and antioxidants components used as potent agents in food
preservation and for therapeutic or nutraceutical industries.
Ó 2010 Published by Elsevier Ltd.

1. Introduction distinctive aroma. Cumin is popular in Indian, Pakistan, North Afri-

Cuminum cyminum is a small annual and herbaceous plant
belonging to Umbelliferae. It is one of the popular spices regularly
used as a flavouring agent. It is cultivated in Arabia, India, China
and in the countries bordering the Mediterranean Sea (Thippesw-
amy and Naidu, 2005). Cumin seeds resemble caraway seeds, being
oblong in shape, thicker in the middle, compressed laterally with
nine fine ridges about 5-in long and yellow–brown in colour, like
other members of the Umbelliferae family such as caraway, parsley
and dill (Hashim and E1-Kiey, 1962). It is an herbaceous annual
plant, with a slender branched stem 20–30 cm tall. The leaves
are 5–10 cm long, pinnate or bipinnate, thread-like leaflets. The
flowers are small, white or pink, and borne in umbels. The fruit
is a lateral fusiform or ovoid achene 4–5 mm long, containing a sin-
gle seed. Today, cumin is the second most popular spice in the
world after black pepper. Cumin seeds are used as a spice for their
* Corresponding author. Tel.: +216 73 461000; fax: +216 73 461830.
E-mail address: hajlaoui_htn@yahoo.fr (H. Hajlaoui).
1
Tel.: +216 79 325 199; fax: +216 79 325 802.
can, Middle Eastern, Sri Lankan, Cuban, Northern Mexican cuisines
and the Western Chinese cuisines of Sichuan and Xinjiang (Daniel
and Maria, 2000, http://www.foodreference.com/html/fcu-
min.html). Cumin can be found in some Dutch cheeses like Leyden
cheese, and in some traditional bread from France. It is also com-
monly used in traditional Brazilian cuisine. Cumin can be an ingre-
dient in (often Texan or Mexican-style) chili powder, and is found
in achiote blends, adobos, sofrito, garam masala, curry powder, and
bahaarat.
Cumin can be used to season many dishes, either ground or as
whole seeds, as it draws out their natural sweetnesses. It is tradi-
tionally added to curries, enchiladas, tacos, other Middle Eastern,
Indian, Cuban and Mexican-style foods. Cumin has also been used
on meat in addition to other common seasonings. The spice is a
familiar taste in Tex–Mex dishes and is extensively used in the cui-
sines of the Indian subcontinent. Cumin was also used heavily in
ancient Roman cuisine. Cultivation of cumin requires a long and
a hot summer of 3–4 months, with daytime temperatures around
30 °C; it is drought tolerant, and is mostly grown in Mediterranean
climates. It is grown from seed, sown in spring, and needs fertile

0278-6915/$ - see front matter Ó 2010 Published by Elsevier Ltd.

doi:10.1016/j.fct.2010.05.044
 H. Hajlaoui et al. / Food and Chemical Toxicology 48 (2010) 2186–2192
with well-drained soil. All the cumin varieties are used in tradi-
tional and veterinary medicine as a stimulant, a carminative, an
astringent, and as remedy against indigestion, flatulence and diar-
rhea (Norman, 1990). Cuminaldehyde, cymene and terpenoids are
the major constituents of volatile oils of cumin (El-Hamidi and
Ahmed, 1966). Cumin’s distinctive flavour and strong, warm aroma
is due to its essential oil content. Its main constituent and impor-
tant aroma compound is cuminaldehyde (4-isopropylbenzalde-
hyde). Important aroma compounds of toasted cumin are the
substituted pyrazines, 2-ethoxy-3-isopropylpyrazine, 2-methoxy-
3-s-butylpyrazine, and 2-methoxy-3-methylpyrazine.
Recently, spices are gaining importance as bionutrients, both as
functional food ingredients and nutritional supplements. The use
of spices as food additives has been widely practised since ancient
times. Spices have a definite role to play in enhancing the taste and
flavour of any food. Apart from this, spices are believed to have
medicinal value. They have been used in a large number of medic-
inal preparations for the treatment of several disorders, particu-
larly of the digestive system (Muthamma et al., 2008).
In traditional medicine, the seeds have been used for treatment
of toothache, dyspepsia, diarrhea, epilepsy and jaundice (Eikani
et al., 1999; Nostro et al., 2005). It also used due to its diuretic, car-
minative, emmanogogic and antispasmodic properties (Janahmadi
et al., 2006; Singh et al., 2002). China is known as the home of C.
cyminum and is an important exporter of this commodity. Besides
its use in traditional medicine, C. cyminum is widely used in food.
The proximate composition and the physicochemical properties
of the essential oil have been reported (Li and Jiang, 2004). The
spice is well known appetizers and is considered essential in culi-
nary art all over the world. In traditional usage, C. cyminum seeds
are generally ground to a powder, but this may bring about disad-
vantages such as bad taste and rapid loss of flavour. Cumin is lar-
gely used in Tunisian kitchen and is locally known as
‘‘kammoun”. This spice is an ingredient of most curry powders
and many savoury spice mixtures, and is mainly used to prepare
fish dishes in mixture with salt and olive oil. In Sfax city, this con-
diment is largely used to prepare many dishes like ‘‘Kammounia”
and fish plates.
The aim of this work was to study the chemical composition of a
Tunisian variety of C. cyminum seeds commonly used in the Tuni-
sian kitchens and to evaluate their antioxidant and antimicrobial
effects against several pathogenic microorganisms isolated from
seawater and fish food and associated with human infection due
to consumption of raw or undercooked sea products.
2. Materials and methods
2.1. Plant material and extraction of essential oil
C. cyminum plants were freshly collected in May 2008 from Swassi Tunisian
locality. The specie was identified according to the flora of Tunisia (Pottier-Alape-
tite, 1961). The seeds were dried at room temperature. The 100 g of seeds were sub-
jected to hydrodistillation for 3 h with 500 ml distilled water using a Clevenger-
type apparatus according to the European Pharmacopoeia (1975). The oil obtained
was collected and dried over anhydrous sodium sulfate and stored in sealed glass
vials in a refrigerator at 4 °C prior to analysis. Yield based on dried weight of the
sample was calculated.
2.2. Analyses of the essential oil
2.2.1. Gas chromatograph
The gas chromatography analysis of the volatile oil was performed using a HP
5890-series II equipped with Flame ionization detectors (FID), HP-5 (BP-1) (5% phe-
nyl + 95% dimethylpolysiloxane) 30 m  0.25 mm ID, 0.25 lm film thickness fused
capillary column. The carrier gas was nitrogen (1.2 m/mn). The oven temperature
program was 1 min isothermal at 50 °C, then 50–280 °C (BP-1) and 50–220 °C
(BP-20) at rate of 5 °C/min and held isothermal for 1 min. The injection port tem-
perature was 250 °C, detector 280 °C. Volume injected: 1 ll of 1% solution (diluted
in hexane). Percentages of the constituents were calculated by electronic integra-
tion of FID peak areas.
2187
2.2.2. Gas chromatograph–mass spectrometry
The analyses of the volatile constituents were run on a Hewlett–Packard GC–
MS system (GC: 5890-series II; MSD 5972). The fused-silica HP-5 MS capillary
column (30 m  0.25 mm ID, film thickness of 0.25 lm) was directly coupled
to the MS. The carrier gas was helium, with a flow rate of 1.2 ml/mn. Oven tem-
perature was programmed (50 °C for 1 min, then 50–280 °C at 5 °C/min) and sub-
sequently, held isothermal for 2 min. Injector port: 250 °C, detector: 280 °C, split
ratio 1:50. Volume injected: 1 ll of 1% solution (diluted in hexane): HP 5972
recording at 70 eV; scan time 1.5 s; mass range 40–300 amu. Software adopted
to handle mass spectra and chromatograms was a Chem Station. The compo-
nents of the oil was identified by comparison of their mass spectra with those
in the Wiley 275 GC–MS library and those in the literature (Adams, 1995), as
well as by comparison of their retention indices with literature data (Adams,
1995; Sibanda et al., 2004). Retention indices of the components were deter-
mined relative to the retention times of a series of n-alkanes (relative to C9–
C28 on the HP5 and HP-20M columns).
2.3. Antioxidant activity
2.3.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion radical-scavenging
activity
The effect of the tested essential oil on DPPH degradation was estimated
according to the method described by Hanato et al. (1988) and Espin et al. (2000)
with small modifications. The essential oil was diluted in pure methanol at different
concentrations, and then 2 ml were added to 0.5 ml of a 0.2 mmol/l DPPH metha-
nolic solution. The mixture was shaken vigorously and left standing at room tem-
perature for 30 min. The absorbance of the resulting solution was then measured
at 517 nm measured after 30 min. The antiradical activity (three replicates per
treatment) was expressed as IC50 (lg/ml), the antiradical dose required to cause a
50% inhibition. A lower IC50 value corresponds to a higher antioxidant activity of
essential oil (Patro et al., 2005). The ability to scavenge the DPPH radical was cal-
culated using the following equation:
DPPH scavenging effect ð%Þ ¼ ½ðA0  A1 Þ  100=A0 : ð1Þ
where A0 is the absorbance of the control at 30 min, and A1 is the absorbance of the
sample at 30 min.
Superoxide anion scavenging activity was assessed using the method described
by Duh et al. (1999). The reaction mixture contained 0.2 ml of essential oil has dif-
ferent concentration, 0.2 ml of 60 mM PMS stock solution, 0.2 ml of 677 mM NADH,
and 0.2 ml of 144 mM NBT, all in phosphate buffer (0.1 mol/l, pH 7.4). After incuba-
tion at ambient temperature for 5 min, the absorbance was read at 560 nm against a
blank. Evaluating the antioxidant activity was based on IC50. The IC50 index value
was defined as the amount of antioxidant necessary to reduce the generation of
superoxide radical anions by 50%. The IC50 values (three replicates per treatment)
were expressed as lg/ml. As for DPPH, a lower IC50 value corresponds to a higher
antioxidant activity of plant extract. The inhibition percentage of superoxide anion
generation was calculated using the following formula:
Superoxide quenching ð%Þ ¼ ½ðA0  A1 Þ  100=A0
where A0 and A1 have the same meaning as in Eq. (1).
2.3.2. Reducing power
The ability of the extracts to reduce Fe3+ was assayed by the method of Oyaizu
(1986). Briefly, 1 ml of C. cyminum essential oil were mixed with 2.5 ml of phos-
phate buffer (0.2 M, pH 6.6) and 2.5 ml of 1% K3Fe(CN)6. After incubation at 50 °C
for 25 min, 2.5 ml of 10% trichloroacetic acid was added and the mixture was cen-
trifuged at 650g for 10 min. Finally, 2.5 ml of the upper layer was mixed with 2.5 ml
of distilled water and 0.5 ml of 0.1% aqueous FeCl3. The absorbance was measured
at 700 nm. The mean of absorbance values were plotted against concentration and a
linear regression analysis was carried out. Increased absorbance of the reaction
mixture indicated increased reducing power. EC50 value (mg/ml) is the effective
concentration at which the absorbance was 0.5 for reducing power. Ascorbic acid
was used as positive control.
2.3.3. b-Carotene-linoleic acid model system (-CLAMS)
The b-CLAMS method by the peroxides generated during the oxidation of lino-
leic acid at elevated temperature (Koleva et al., 2002). In this study the b-CLAMS
was modified for the 96-well micro-plate reader. In brief, the b-carotene was dis-
solved in 2 ml of CHCl3, to which 20 mg of linoleic acid and 200 mg of tween 40
were added. CHCl3 was removed using rotary evaporator. Oxygenated water
(100 ml) was added, and the flask was shaken vigorously until all material dis-
solved. This test mixture was prepared fresh and using immediately. To each well,
250 ll of the reagent mixture and 35 ll sample or standard solution were added.
The plate was incubated at 45 °C. Readings were taken at 490 nm using visible/
UV micro-plate kinetics reader (EL  808, Bio-Tek instruments).
Readings of all samples were performed immediately (t = 0 min) and after
120 min of incubation. The antioxidant activity (AA) of the extracts was evaluated
in term of b-carotene blanching using the following formula:
2188 H. Hajlaoui et al. / Food and Chemical Toxicology 48 (2010) 2186–2192
AA ð%Þ ¼ ½ðA0  A1 Þ=A0   100
where A0 is the absorbance of the control at 0 min, and A1 is the absorbance of the
sample at 120 min. The results are expressed as IC50 values (lg/ml). All samples
were prepared and analyzed in triplicate.
2.4. Antimicrobial activity
2.4.1. Microorganisms
The test microorganisms included the following Gram-positive bacteria: Staph-
ylococcus aureus ATCC 25923, Staphylococcus epidermidis CIP 106510, Micrococcus
luteus NCIMB 8166, Bacillus cereus ATCC 11778, B. cereus ATCC 14579AND Gram-
negative bacteria: Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC
27853, Salmonella typhimurium LT2 DT104, Listeria monocytogenes ATCC 19115,
Enterococcus feacalis ATCC 29212. The antibacterial effect was also tested against
11 species (14 strains) belonging to Vibrio genus, four Candida species (albicans, par-
apsilosis, glabrata and krusei) and one Saccharomyces cerivisae strain. These microor-
ganisms were kindly provided by Prof. Stefania Zanetti from the Department of
Biomedical Sciences (University of Sassary, Sardegna, Italy).
2.4.2. Disc-diffusion assay
Antimicrobial activity testing was done according to the protocol described by
Vuddhakul et al. (2007) and modified by Snoussi et al. (2008a) for Vibrio spp.
strains. For the experiments, a loopful of the microorganisms working stocks were
enriched on a tube containing 9 ml of Mueller–Hinton broth (for bacteria) and Sab-
ouraud Chloramphenical broth (for Candida), then incubated at 37 °C for 18–24 h.
The overnight cultures were used for the antimicrobial activity of the essential oil
used in this study and the optical density was adjusted at 0.5 McFarland turbidity
standards with a DENSIMAT (Biomérieux). The inoculums of the respective bacteria
and fungus were streaked onto MH or SB agar plates using a sterile swab. For Vibrio
strains, the MH medium was supplemented with 1% NaCl.
A sterile filter discs (diameter 6 mm, Whatman Paper No. 3) were impregnated
with 10 ll of essential oil placed on the appropriate agar mediums (SB, MH and
MH + 1% NaCl). Gentamycin (10 lg/disc) and amphetorecin B (20 lg/disc) were
used as positive reference standards to determine the sensitivity of one strain/iso-
late in each microbial species tested.
Tetracycline (30 lg/disc) was used in this study as positive controls for Vibrio
spp. strains. The antibiotic susceptibility was determined by using the Kirby–Bauer
method and Mueller–Hinton agar plates supplemented with 1% NaCl as described
by Ottaviani et al. (2001). After incubation at 37 °C for 18–24 h, the diameter of
the inhibition zone was measured with 1 mm flat rule and the diameters were
interpreted according to the Comité de la Société Française de l’Antibiogramme
(Cavallo et al., 2006).
The dishes were incubated at 37 °C for 18–24 h for microbial strains. The diam-
eter of the zones of inhibition around each of the discs was taken as measure of the
antimicrobial activity. Each experiment was carried out in triplicate and the mean
diameter of the inhibition zone was recorded.
2.4.3. Micro-well determination of MIC and MBC
The minimal inhibition concentration (MIC) and the minimal bactericidal
concentration (MBC) values were determined for all bacterial strains used in this
study as described by Gulluce et al. (2007) and Snoussi et al. (2008b) for Vibrio
strains. The inoculums of the bacterial strains were prepared from 12 h broth
cultures and suspensions were adjusted to 0.5 McFarland standard turbidity.
The essential oil of C. cyminum dissolved in 10% dimethylsulfoxide (DMSO), were
first diluted to the highest concentration (20 mg/ml) to be tested, and then serial
twofold dilutions were made in a concentration range from 0.0488 to 50 mg/ml
in 5 ml sterile test tubes containing nutrient broth. The 96-well plates were pre-
pared by dispensing into each well 95 ll of nutrient broth and 5 ll of the inoc-
ulum. A 100 ll aliquot from the stock solutions of each essential oil was added
into the first wells. Then, 100 ll from the serial dilutions were transferred into
11 consecutive wells. The last well containing 195 ll of nutrient broth without
essential oil and 5 ll of the inoculum on each strip was used as the negative
control. The final volume in each well was 200 ll. The plates were incubated
at 37 °C for 18–24 h. The essential oil tested in this study was screened two
times against each organism. The MIC was defined as the lowest concentration
of the compounds to inhibit the growth of the microorganisms. The BMC values
were interpreted as the highest dilution (lowest concentration) of the sample,
which showed clear fluid with no development of turbidity and without visible
growth. All tests were performed in triplicate.
3. Results and discussion
3.1. Essential oil composition
Twenty-one compounds were identified in the essential oils
used in this study. The qualitative and quantitative compositions
of the oils are given in Table 1, where the components were listed
according to their elution on the apolar column. The oil yield of the
Tunisian variety of cumin was 1.22%. The extraction yield of Ira-
nian variety of cumin was 1.45% (Mehdi et al., 2007). The essential
oil yield of the cumin seeds from the local market of India was
2.33% (Sowbhagya et al., 2008). The different constituents of the
samples were identified and quantified by GC and GC–MS. The
oil of C. cyminum is particularly rich in beta-pinene (11.13%), O-
cymene (11.82%), gamma-terpinene (15.21%), trans-carveol
(4.49%), cuminlaldehyde (39.48%), 2-caren-10-al (7.93%) and myrt-
enal (3.5%). Thirty-two compounds were identified in the essential
oil of C. cyminum from Iran (Gachkar et al., 2007) and the main
components were: a-pinene (29.1%), limonene (21.5%), 1,8 cineole
(17.9%), linalol (10.4%), a-terpineole (3.17%) and linalyl acetate
(4.8%). Our literature survey could not find a report on the chemi-
cal composition of Tunisian C. cyminum oil.
In fact, the composition of the essential oil of C. cyminum de-
pends on many factors, such as plant part, harvest-time, extrac-
tion-method, type of cultivar, geographic origin and storage
conditions. In general, cumin aldehyde, menthane derivatives, c-
terpinene, p-cymene and b-pinene are major components of many
essential cumin oils and are mainly responsible for the aroma and
biological effects (Nestorova et al., 1977; Lis-Balchin et al., 1998;
Hiller, 1999). It has been shown that the components of essential
oil (aldehydes, ketones, and alcohols) showed the strongest antimi-
crobial activity (Arora and Kaur 1999; Marino et al., 1999). Differ-
ent antimicrobial effects were obtained with cumin (Gachkar et al.,
2007; Iacobellis et al., 2005; Leopold et al., 2005; Özcan and Erk-
men, 2001; Viuda-Martos et al., 2008).
3.2. Antioxidant activities
Several antioxidant methods have been proposed to evaluate
antioxidant characteristics and to explain antioxidants mecha-
nisms and actions. Of these, free synthetic radical scavenging like
DPPH or ABTS+, superoxide anion scavenging, reducing power
and antioxidant assay using b-carotene linoleate system are most
commonly used for the evaluation of the total antioxidant behav-
iour of extracts and essential oils (Amarowicz et al., 2000; Chang
et al., 2002).
DPPH is a free radical that accepts an electron or hydrogen rad-
ical to become a stable molecule. DPPH radical scavenging is one of
the important methods to evaluate antioxidant activity of essential
oils and phenolic extracts. Table 2 illustrates scavenging of the
DPPH radical by C. cyminum essential oil. The scavenging effect of
essential oil and standard (BHT) on the DPPH radical expressed
as IC50 values was 31 lg/ml for C. cyminum oil and 11.5 lg/ml for
BHT. In fact, Fakoor and Rasooli (2008) reported that C. cyminum
and Rosmarinus officinalis essential oils notably reduced the con-
centration of DPPH free radical, with an efficacy lower than that
of reference oil Thymus x-porlock (69.29% inhibition) and slightly
lower than that of Trolox. The performance of the rosemary oil
was better than that of C. cyminum.
In fact, oils with higher monoterpenic abundance were reported
to be almost ineffective. This result is in agreement with the poor
performance given by the oils with similar patterns and by single
monoterpenic hydrocarbons (Ruberto and Baratta, 2000).
The superoxide anion is the most common free radical gener-
ated in vivo. Under oxidative stress, the concentration of this spe-
cies can increase dramatically in all cells, inducing several
pathophysiological processes, due to its transformation into more
reactive species (Gülçin et al., 2007). In the PMS-NADH-NBT sys-
tem, superoxide anion, derived from dissolved oxygen from the
coupling reaction of PMS-NADH, reduces NBT. The decrease in
absorbance at 560 nm with antioxidants indicates the consump-
tion of superoxide anion in the reaction mixture. Table 4 showed
that the results of the inhibiting capacities of superoxide were very
 H. Hajlaoui et al. / Food and Chemical Toxicology 48 (2010) 2186–2192 2189

Table 1 food industry in order to find possible alternatives to synthetic pre-

Percentage composition of the essential oils from seeds of a Tunisian variety of cumin servatives (namely BHT). In this context, C. cyminum essential oil
(C. cyminum).
gave interesting results, being one of the promising performed ex-
No. Compounds Retention indices Percentage Identification tracts in terms of ability to neutralize free radicals and prevent
identified (RI) HP-5 unsaturated fatty acid oxidation. The results presented here may
1 Alpha-thujene 928 0.20 MS, RI also contribute to knowledge of the antioxidative and antimicro-
2 Alpha-pinene 935 0.40 MS, RI bial potentials of these species reported elsewhere.
3 Sabinene 975 0.56 MS, RI
4 Beta-pinene 979 11.13 MS, RI
5 Beta-myrcene 990 0.76 MS, RI
6 Alpha-phellendrene 1006 0.18 MS, RI 3.3. Antimicrobial activity
7 Delta-3-carene 1011 0.42 MS, RI
8 O-cymene 1027 11.82 MS, RI
The antimicrobial activities of C. cyminum L. essential oil against
9 Limonene 1030 1.42 MS, RI
10 Gamma-terpinene 1061 15.21 MS, RI microorganisms examined in the present study and its potency
11 Linalool 1089 0.08 MS, RI were qualitatively and quantitatively assessed by the presence or
12 cis-Sabinene hydrate 1099 0.34 MS, RI the absence of inhibition zone diameter; MIC and MBC values.
13 Verbenol 1141 0.18 MS, RI The results were given in Table 3. The results showed that the
14 trans-Pinocarveol 1168 0.26 MS, RI
essential oil of Tunisian variety of C. cyminum had substantial anti-
15 Terpinene-4-ol 1179 0.30 MS, RI
16 trans-Carveol 1196 4.49 MS, RI microbial activity against 24 bacteria and five yeasts species tested.
17 Cuminlaldehyde 1248 39.48 MS, RI In fact, the data obtained of zones of growth inhibition (mm)
18 Phellandral 1278 0.39 MS, RI scored in Mueller–Hinton agar demonstrated that Gram-positive
19 2-Caren-10-al 1289 7.93 MS, RI
bacteria exhibited the highest diameters of growth inhibition (be-
20 Myrtenal 1294 3.50 MS, RI
21 Beta caryophyllene 1425 0.33 MS, RI tween 10 and 35 mm). Cumin essential oil was particularly effec-
tive against M. luteus NCIMB 8166 with a diameter of inhibition
Total identified 99.37
Yield (g/100 g dry weight) 1.22 about 35 mm. In the other hand, Gram-negative bacteria were less
Monoterpene hydrocarbons 42.42 sensitive to cumin essential oil with a diameter of growth inhibi-
Oxygenated monoterpenes 56.61 tion ranging from 10 (E. feacalis ATCC 29212) to 12 mm (E. coli
Sesquiterpene hydrocarbons 0.33 ATCC 35218).
Oxygenated sesquiterpenes –
Concerning Vibrio spp. strains, the diameters of growth inhibi-
tion were ranging from 11 mm (Vibrio alginolyticus ATCC 33787)
to 23 mm (Vibrio cholerae ATCC 9459). C. cyminum essential oil
Table 2 was also effective against all yeast tested with a diameter of
Antioxidant activity of essential oil extract from Tunisian variety of cumin seeds (C. growth inhibition ranging from 17 to 22.67 mm. Iacobellis et al.
cyminum): scavenging activity (expressed as IC50 values: lg/ml), DPPH, superoxide (2005) have demonstrated that the essential oil of C. cyminum pos-
radicals and b-carotene bleaching test. Reducing power was expressed as EC50 values
(lg/ml).
sessed a high antibacterial activity against the genera Clavibacter,
Curtobacterium, Rhodococus, Erwinia, Xanthomonas, Ralstonia and
Essential oil of Tunisian cumin BHT Agrobacterium. The lowest activity was observed with Pseudomonas
DPPH IC50 (lg ml1) 31 11.5 genus.
1
O
2 IC50 (lg ml ) 16 1.5 Viuda-Martos et al. (2008) have tested the in vitro antibacterial
P R EC50 (lg ml1) 11 75
activities of six essential oils (thyme, sage, cumin, rosemary, clove
b-Carotenes IC50 (lg ml1) 20 75
and oregano) against six microorganisms. These authors reported
that cumin essential oil was the most effective essential oil after
the oregano one, which showed inhibition zones between
interesting for the C. cyminum essential oil, but is inferior as com- 31.23 mm on Lactobacillus sakei and 38.17 mm.
pared to BHT. The values of IC50 obtained are, respectively, 16 and MIC and MBC values indicate that the essential oil of cumin was
1.5 lg/ml. efficient against all tested bacteria with MIC about 0.078 mg/ml for
Another reaction pathway in electron donation is the reduction Gram-positive bacteria, from 0.078 to 0.15 mg/ml for Gram-nega-
of an oxidized antioxidant molecule to regenerate the ‘‘active” re- tive bacteria. For Vibrio spp. Strains, MIC values were ranging from
duced antioxidant. As showed in Table 4, the reducing power of C. 0.078 to 0.31 mg/ml. The MBC values were also important and low
cyminum essential oil, expressed as CE50, was clearly more impor- concentration of cumin essential oil were sufficient to eliminate
tant than that of positive control BHT (11 and 75 lg/ml, the growth of M. luteus (MBC: 0.15 mg/ml) and E. feacalis and
respectively). E. coli (MBC: 0.625 mg/ml). It has shown also that 1.25 mg/ml of
In this model, b-carotene undergoes rapid discoloration in the essential oil was sufficient to stop the growth of several pathogenic
absence of an antioxidant. The presence of an antioxidant such as Vibrio species including: V. cholerae, Vibrio parahaemolyticus and
phenolics can hinder the extent of b-carotene destruction by ‘‘neu- Vibrio vulnificus ones.
tralizing’’ the linoleate free radical and any other free radicals The MIC and MBC values obtained with cumin oil for V. parahae-
formed within the system (Kamath and Rajini, 2007). Table 3 de- molyticus, V. alginolyticus, V. vulnificus and Vibrio fluvialus strains
picts the inhibition of b-caroten bleaching by the C. cuminum were similar to those previously obtained with thyme oil (Snoussi
essential oil and by BHT. The values of IC50 are, respectively, 20 et al., 2008d). Özcan and Erkmen (2001) have studied the antibac-
and 75 lg/ml. Gachkar et al. (2007) compared the lipid peroxida- terial activities of nine Turkish plant species including C. cuminum.
tion inhibitory activities of the cumin and rosemary essential oils The results obtained showed that the essential oils tested varied in
were assessed by the b-carotene bleaching test with the Thymus, their antimicrobial activity. Cumin oil at 1% inhibited E. coli KUEN
Trolox and BHA showed that the percentage of activity of the C. 1504; at 10% Yersinia enterocolitica, S. aureus and E. feacalis. While,
cyminum oil is more than 60%. Salmonella typhimurium and B. cereus were inhibited by cumin oil
These antioxidants properties (DPPH radical-scavenging activ- at 15%. The same authors showed that mold species required high-
ity, superoxide anion radical-scavenging activity, reducing power, er concentrations of cumin essential oil (10%) to inactivate popula-
b-carotene linoleate system) of essential oil are well needed for tions than did bacteria (1%) while it was ineffective on nolds.
2190 H. Hajlaoui et al. / Food and Chemical Toxicology 48 (2010) 2186–2192

Table 3
Antibacterial and antifungal activity of essential oil of Tunisian C. cyminum against human pathogenic bacteria and yeast strains using agar disc diffusion method and
determination of MIC (mg/ml) and MBC (mg/ml) values.

Microorganisms IZ (mm ± SD) Antibiotics MIC MBC

Bacterial strains
Gram-positive bacteria Gen
Staphylococcus epidermidis CIP 106510 10.67 ± 0.58 21.33 ± 0.58 0.078 0.31
Staphylococcus aureus ATCC 25923 10 ± 0 32.67 ± 0.58 0.078 0.625
Micrococcus luteus NCIMB 8166 35 ± 0 27.67 ± 1.53 0.039 0.15
Bacillus cereus ATCC 11778 21.67 ± 0.58 26 ± 1 0.078 0.625
Bacillus cereus ATCC 14579 22.67 ± 0.58 28 ± 1 0.078 0.625
Gram-negative bacteria
Escherichia coli ATCC 35218 12 ± 1 27.33 ± 0.58 0.078 0.625
Listeria monocytogenes ATCC 19115 12 ± 0 37.67 ± 0.58 ND ND
Enterococcus feacalis ATCC 29212 10 ± 0 26 ± 1 0.078 0.625
Pseudomonas aeruginosa ATCC 27853 11.67 ± 0.58 30.33 ± 0.58 ND ND
Salmonella typhimirium LT2 DT104 11 ± 0 21 ± 1 0.15 1.25
Vibrio spp. strains Tet
V. cholerae ATCC 9459 23 ± 1 25 ± 1 0.078 1.25
V. parahaemolyticus ATCC 17802 15 ± 0 21 ± 0 0.15 1.25
V. parahaemolyticus ATCC 43996 13.33 ± 0.58 20 ± 0 0.078 0.625
V. alginolyticus ATCC 33787 11 ± 0 20 ± 0 0.31 0.625
V. alginolyticus ATCC 17749 20.33 ± 0.58 7±0 0.15 0.625
V. vulnificus ATCC 27562 12 ± 0 13.33 ± 0.57 0.15 1.25
V. harveyi ATCC 18293 15.33 ± 0.58 18.33 ± 0.58 0.15 0.625
V. proteolyticus ATCC 15338 15 ± 1 20 ± 1 0.078 1.25
V. furnisii ATCC 35016 18.67 ± 1.15 20.33 ± 0.57 0.15 1.25
V. mimicus ATCC 33653 13.67 ± 0.58 20 ± 0 0.15 1.25
V. furnisii ATCC 33813 12 ± 0 19 ± 0 0.078 0.625
V. natrigens ATCC 14048 14 ± 0 21 ± 0 ND ND
V. carhiaccae ATCC 35084 14 ± 0 18.33 ± 0.58 0.078 0.31
V. fluvialis ATCC 33809 14.67 ± 0.58 18.33 ± 0.57 0.31 1.25
Yeast strains AmB
Candida albicans ATCC 90028 17 ± 1 11 ± 0 0.039 0.31
Candida glabrata ATCC 90030 22.67 ± 0.58 14.33 ± 0.57 0.009 0.019
Candida parapsilosis ATCC 22019 17 ± 0 10.33 ± 0.57 0.078 0.31
Candida krusei ATCC 6258 23 ± 1 12 ± 0 0.004 0.039
Saccharomyces cerevisae 17.33 ± 0.58 18 ± 0 0.009 0.019

IZ: Inhibition zone in diameter (mm ± SD) around the discs impregnated with 10 ll of essential oil. SD: standard deviation; Gen: gentamycin (10 lg/disc); Tet: tetracycline
(30 lg/disc); AmB: amphotericin B (10 lg/ml); ND: not determined.

Table 4
Comparison between the zones of growth inhibition (mm), MIC (mg/ml) and MBC (mg/ml) values obtained with clove, thyme and cumin oils against some Vibrio spp. strain.

Strains E. caryophyllata T. vulgaris C. cyminum

Z (mm ± SD) MIC MBC Z (mm ± SD) MIC MBC Z (mm ± SD) MIC MBC
V. alginolyticus
ATCC 33787 11.33 ± 0.57 0.15 1.25 13.33 ± 0.57 0.078 0.625 11 ± 0 0.312 0.625
ATCC 17749 10.66 ± 0.57 0.31 2.5 14 ± 1 0.156 1.25 20.33 ± 0.58 0.156 0.625
V. parahaemolyticus
ATCC 17802 13.66 ± 0.57 0.156 >1.25 14.66 ± 0.57 0.156 0.312 15 ± 0 0.156 1.25
ATCC 43996 12.33 ± 0.57 0.156 0.625 22.33 ± 0.57 0.156 0.312 13.33 ± 0.58 0.078 0.625
V. vulnificus
ATCC 27562 11.66 ± 0.57 0.156 1.25 12.66 ± 0.57 0.156 1.25 12 ± 0 0.156 1.25
V. fluvialis
ATCC 33809 10 ± 0 0.156 1.25 13 ± 1 0.156 0.625 14.67 ± 0.58 0.312 1.25

Cumin at 1% was effective to inhibit the growth of Saccharomyces
cerevisiae.
Iacobellis et al. (2005) reported that in the case of C. cyminum,
results from the disc diffusion method, and determination of min-
imal inhibitory and bactericidal concentrations (MIC and MBC),
indicate that E. coli is the most sensitive microorganism with the
lowest MBC value (1 ll/ml). Other sensitive microorganisms are
S. aureus and L. monocytogenes with the MIC values of 1 and 2 ll/
ml, respectively. Although L. monocytogenes required higher oil
concentration (2 ll/ml) for complete elimination, it showed great-
er zone of inhibition (17.67 mm) on disc diffusion plates. Complete
death time on exposure to C. cyminum and R. officinalis oils were
(20 and 25 min), (180 and 240 min) and (90 and 120 min) for
E. coli, S. aureus and L. monocytogenes, respectively. It can be con-
cluded that E. coli is the most vulnerable and S. aureus is the least
vulnerable microorganisms to the oils under study. These values
suggest the duration of time required for complete bactericidal ef-
fects of the oils.
Recently, Gachkar et al. (2007) have demonstrated that the
essential oil of Iranian C. cyminum exhibited good to moderate
antimicrobial activities against all microorganisms tested. In fact,
E. coli was the most sensitive microorganism with the lowest
MBC value (1 ll/ml) and L. monocytogenes required higher oil con-
centration (2 ll/ml) for complete elimination with a greater zone
 H. Hajlaoui et al. / Food and Chemical Toxicology 48 (2010) 2186–2192
of inhibition (17.67 mm). These authors have calculated the dura-
tion of time required for complete bactericidal effect of both Ros-
marinus and Cumin essential oils. They founded that complete
death times after exposure to the MBC level of the essential oil of
C. cyminum were 20, 180 and 90 min for E. coli, S. aureus and L.
monocytogenes, respectively. It can be concluded that E. coli is most
vulnerable and S. aureus is the lowest vulnerable microorganisms.
In a previous study, Snoussi et al. (2008c) have tested the anti-
Vibrio spp. activity of five Tunisian essential oils extracted from
Mentha longifolia, Mentha pulegium, R. officinalis, Eugenia caryophyl-
lata and Thymus vulgaris (Table 4). These authors reported that
thyme and clove oils exhibited a high degree of anti-Vibrio spp.
activity, especially against V. parahaemolyticus strains with a diam-
eter of zone growth inhibition ranging from 14.66 to 28 mm. these
authors reported that MIC and MBC value were interestingly low
for thyme oil (MIC 0.078–0.156 mg/ml) and (MBC > 0.31–
1.25 mg/ml).
Most studies investigating the action of whole essential oils
against food spoilage organisms and food-borne pathogens agree
that, in general, essential oils are slightly more active against Gram
positive than Gram-negative bacteria (Cosentino et al., 1999;
Cimanga et al., 2002; Hajlaoui et al., 2008; Harpaz et al., 2003; Kar-
aman et al., 2003; Ruberto et al., 2000). However, our results were
in discordance with those reported by Viuda-Martos et al. (2008)
and Sokmen et al. (2004). These authors showed that spice essen-
tial oil did not possess any selective antimicrobial activity on the
basis of the cell wall differences of bacteria.
To our knowledge, this work represents the first attempt to
study the chemical composition and the biological activities of
Tunisian variety of C. cyminum, and especially to study his antibac-
terial effect against several Vibrio spp. strains. In this context, C.
cyminum essential oils gave interesting results in terms of both
antimicrobial activity and ability to neutralize free radicals and
prevent unsaturated fatty acid oxidation. As a whole, these findings
may confirm the interesting potential of this spice as a valuable
source of natural bioactive molecules and highlights its important
role to prevent contamination due to Vibrio spp. strains after con-
sumption of seawater products.
Conflict of Interest
The authors declare that there are no conflicts of interest.
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