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Inhibition of Aflatoxin Production and Growth of Aspergillus parasiticus by

Cuminum cyminum , Ziziphora clinopodioides , and Nigella sativa Essential Oils

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DOI: 10.1089/fpd.2011.0929 · Source: PubMed

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FOODBORNE PATHOGENS AND DISEASE
Volume 8, Number 12, 2011
ª Mary Ann Liebert, Inc.
DOI: 10.1089/fpd.2011.0929

Inhibition of Aflatoxin Production and Growth

of Aspergillus parasiticus by Cuminum cyminum,
Ziziphora clinopodioides, and Nigella sativa Essential Oils

Ali Reza Khosravi,1 Hojjatollah Shokri,2 and Mohammadhassan Minooeianhaghighi 3

Abstract
Aflatoxins are highly toxic and carcinogenic metabolites produced by Aspergillus parasiticus on food and agri-
cultural commodities. Natural products may control the production of aflatoxins. The aims of this study were to
evaluate the effects of the essential oils (EOs) of Cuminum cyminum, Ziziphora clinopodioides, and Nigella sativa on
growth and aflatoxins production by A. parasiticus. Minimal inhibitory concentrations (MICs) and minimal
fungicidal concentrations (MFCs) of the EOs were determined and compared with each other. Determination of
aflatoxins (AFB1, AFB2, AFG1, and AFG2) was performed by immunoaffinity column extraction using reverse
phase-high performance liquid chromatography. The major oil components were a-pinene (30%) in C. cyminum,
pulegone (37%) in Z. clinopodioides, and trans-anthol (38.9%) in N. sativa oils. In broth microdilution method,
C. cyminum oil exhibited the strongest activity (MIC90: 1.6; MFC: 3.5 mg/mL), followed by Z. clinopodioides
(MIC90: 2.1; MFC: 5.5 mg/mL) and N. sativa (MIC90: 2.75; MFC: 6.25 mg/mL) oils against A. parasiticus ( p < 0.05).
Aflatoxin production was inhibited at 0.25 mg/mL of C. cyminum and Z. clinopodioides oils, of which that of
C. cyminum was a stronger inhibitor. C. cyminum EO caused significant reductions in values of 94.2% for AFB1,
100% for AFB2, 98.9% for AFG1, 100% for AFG2, and 97.5% for total aflatoxin. It is concluded that the EOs of
C. cyminum, Z. clinopodioides, and N. sativa could be used as natural inhibitors in foods at low concentrations to
protect from fungal and toxin contaminations by A. parasiticus.

Introduction Studies on controlling aflatoxin contamination of plants

and susceptible crops are mainly focused on fungal processes

F ungi are important spoilage microorganisms of food

and feedstuffs during the storage, thereby rendering them
unfit for human consumption by retarding their nutritive
value and sometimes by producing mycotoxins (Kumar et al.,
2007). Mycotoxins are toxic to humans and animals, cause
significant reductions in crop yield, and result in economic
losses (Iheshiulor et al., 2011). Their occurrence in various
countries has been well documented (Bathnagar and Garcia,
2001). Aflatoxins are secondary metabolites synthesized
by several Aspergillus spp. (A. parasiticus, A. flavus, and
A. nomius), as they grow on economically important crops
such as corn, wheat, peanuts, tree nuts, dried fruits, vegeta-
bles, and medicinal plants (Vardon, 2003; Georgianna and
Payne, 2009). The food and agriculture organization of the
United Nations estimated that at least 25% of the cereal grains
in the world are contaminated by mycotoxins including af-
latoxins (Bhatnagar and Cleveland, 2004).
needed for plant invasion and mycotoxin production as well
as developing of genetically modified crops, which are resis-
tant to fungal invasion (Cleveland et al., 2003). Besides the
chemical agents (Razzaghi-Abyaneh et al., 2006), some plants
and microbes or their active metabolites have been introduced
as inhibitors of aflatoxin biosynthesis (Sakuda et al., 2000;
Rasooli and Razzaghi-Abyaneh, 2004; Yoshinari et al., 2007;
Yahyaraeyat and Khosravi, 2010; Roze et al., 2011). Within the
wide range of natural sources such as microorganisms, plants,
and insects, essential oils (EOs) from plants have received
major consideration with regard to their relatively safe status
and enrichment by a wide range of structurally different
useful constituents (Bruneton, 1995; Reddy et al., 2010). Many
EOs have also been reported as effective inhibitors of fungal
growth and aflatoxin production (Yoshinari et al., 2007).
Plants from Iranian biomes, such as Cuminum cyminum
(Apiaceae; known as Ziree), Ziziphora clinopodioides (Labiatae;

1
Faculty of Veterinary Medicine, Mycology Research Center, University of Tehran, Tehran, Iran.
2
Faculty of Veterinary Medicine, University of Mazandaran, Amol, Iran.
3
Department of Basic Sciences, Faculty of Medicine, Gonabad University of Medical Sciences, Gonabad, Iran.

1275
1276
known as Avishan), and Nigella sativa (Ranuculaceae; known
as black seed) have been used as natural medicines by local
populations in the treatment of several diseases (Avicenna,
980-1037AD; Tadjbakhsh, 2003). Further, a report from our
laboratory showed that the above-mentioned plants exhibit
antifungal activities (Khosravi et al., 2011). Up to now, no data
have been reported about the effects of these interesting plants
on aflatoxin biosynthesis by A. parasiticus. The present study
was undertaken to investigate the effect of C. cyminum, Z.
clinopodioides, and N. sativa EOs on the growth and aflatoxin
production of A. parasiticus when the fungus was grown on
specific fungal media.
Materials and Methods
Organism
A. parasiticus (ATCC 56775) was obtained from Department
of Mycology, Faculty of Veterinary Medicine, University of
Tehran, Iran. The strain was cultured on potato dextrose agar
(Merck) slope for 10–14 days at 26C – 1C. Conidia were
harvested by adding 10 mL of 0.05% Tween 80 solution
(Merck) to culture and gently scraping the mycelia with a
sterile inoculating loop to free spores. Spore suspensions were
prepared and diluted in 2% yeast extract sucrose (2% YES)
broth to a concentration of *106 spores per mL. Spore popu-
lation was counted by using a hemocytometer. YES broth also
served as aflatoxin production medium (Khosravi et al., 2011).
Oil extraction
The plants (C. cyminum, Z. clinopodioides, and N. sativa)
were collected from different regions of Khorasan Razavi
Province (Eastern part of Iran) in 2009. The plant materials
were steam distilled for 90 min in a full glass apparatus. The
oils were extracted using a Clevenger-type apparatus. The
extraction was carried out after 4-h maceration in 500 mL of
water. The EOs were stored in dark glass bottles in a freezer
at -12C until they were used (Baydar et al., 2004).
EOs analysis
Gas chromatography (GC) analyses were performed using a
Shimadzu-9A (Shimadzu Co.) gas chromatograph equipped
with a flame ionization detector, and quantitation was carried
out on Euro Chrom 2000 software from Knauer by the area
normalization method neglecting response factors (Davies,
1998). The analysis was carried out using a DB-5 fused-silica
column (30 m · 0.25 mm, film thickness 0.25 lm; J & W Scien-
tific Inc.). The operating conditions were as follows: injector
and detector temperatures were 250C and 265C, respectively;
carrier gas was Helium. Oven temperature program was 40C–
250C at the rate of 4C/min. The gas chromatography/mass
spectrometry (GC/MS) unit consisted of a Varian Model 3400
gas chromatograph coupled to a Saturn II ion trap detector was
used. The column was the same as in GC, and the GC condi-
tions were as just mentioned. Mass spectrometer conditions
were as follows: ionization potential 70 eV; electron multiplier
energy 2000 V. The identities of the oil components were
established from their GC retention indices, relative to C7-C25
n-alkanes, by comparison of their MS spectra with those re-
ported in the literature, and by computer matching with the
Wiley 5 mass spectra library (Wiley), whenever possible, by
coinjection with standards available in the laboratories.
KHOSRAVI ET AL.
Antifungal analysis
The minimal inhibitory concentration (MIC)90 and minimal
fungicidal concentration (MFC) values of C. cyminum, Z.
clinopodioides, and N. sativa EOs were determined by broth
macro- and microdilution methods, according to the protocol
in M38-A for filamentous fungi with some modifications
(CLSI, 2002). For the broth macrodilution method, 900 lL of
the final conidia suspensions were mixed with 100 lL of the
test EO in 12 · 75 mm test tubes and incubated at 28C for 48 h.
The positive control tube contained 900 lL of conidial sus-
pension plus 100 lL of Roswell Park Memorial Institute
medium (RPMI) 1640, and the negative one contained 1 mL of
RPMI 1640 only. The lowest oil concentration inhibiting fun-
gal growth by 90% was identified as the MIC90. In addition,
flat-bottom microdilution plates containing 96 wells were
employed for the broth microdilution method. One-hundred
microliters of final conidia suspension was added to each well
containing 100 lL of the oil. Positive control was the well
containing 100 lL of the inoculum suspension and 100 lL of
the RPMI only, and the negative control was a well containing
200 lL of RPMI 1640. The MFCs were determined by sub-
culturing 10 lL aliquot from all MIC90 wells showing no vis-
ible growth on to Sabouraud glucose agar plates. Antifungal
analysis was replicated four times, and the results were ex-
pressed as the average of four repetitions.
Aflatoxin production assay
For evaluation of aflatoxin formation, EOs at concentra-
tions lower than MIC90 values (0.25 mg/mL for C. cyminum,
Z. clinopodioides, and 1.5 mg/mL for N. sativa) were used. Fifty
microliters of spore suspension (106 spores per mL) was ad-
ded to 25 mL of 2% YES broth containing different concen-
trations of EOs in 100 mL Erlenmeyer flask and incubated for
10 days at 26C – 1C in the darkness. After the incubation
period, cultures were autoclaved at 121C for 30 sec, to inac-
tivate mycelia and conidia, and filtered through Whatman
No. 1 filter paper. The mycelia were dried to a constant weight
at 80C, and the weight of dried mat was estimated (Patkar
et al., 1993). Determination of aflatoxins (AFB1, AFB2, AFG1,
and AFG2) was performed by immunoaffinity column ex-
traction using reverse phase-high performance liquid chro-
matography (RP-HPLC) according to AOAC (2000). Briefly,
the filtrated content of each flask was mixed with 150 mL
MeOH:H2O (80:20) and 2.5 g NaCl, followed by vortexing for
3 min. Sixty-five microliter of phosphate buffer solution (PBS)
was added to 10 mL of this mixture, vigorously shaken, and
passed through a glass fiber filter. Seventy milliliter of solu-
tion was transferred onto an immunoaffinity column (Puri-
Fast-AFLA IAC) in a flow rate of 3 mL/min. The column was
then washed with 15 mL PBS, dried by passing air gently
through it, and aflatoxins were eluted with adding 500 and
750 lL methanol with 1 min interval. The elution diluted with
1750 lL H2O and the aliquot of 200 lL was injected into HPLC
system equipped with a separator module (2695; Waters), a
Nova-Pak LC-18 column, and a fluorescence detector (474;
Waters). Aflatoxins were derivatized by KB Cell post column
derivatization system (Libios) in a H2O–MeCN–MeOH mo-
bile phase containing HNO3 and KBr at a flow rate of 1 mL/min
and detected at an excitation wavelength of 365 nm and an
emission wavelength of 435 nm. Quantization of aflatoxins
was performed using the peak height by Millenium 32 v 4.0
AFLATOXIN INHIBITION BY ESSENCES 1277

Table 1. The Compositions of Cuminum cyminum, Ziziphora clinopodioides, and Nigella sativa Essential
Oils Identified by Gas Chromatography and Gas Chromatography–Mass Spectrometry

No. Cuminum cyminum Ziziphora clinopodioides Nigella sativa

1 a-Pinene 30% Pulegone 37% Trans anthol 38.9%

2 Limonene 21% q-Peritone 19.6% q-Cymene 17%
3 1,8 cineol 18.5% Carvacrol 5.4% Limonene 4.8%
4 Linalool 10% Neomenthol 5% Carvone 4.4%
5 Linalil acetate 4% 1,8 cineol 3.5% a-Thujene 2.8%
6 a-Terpineol 3% Menthol 3% Anisaldehyde 1.9%
7 a-Terpineol acetate 1.5% Verinone 2.3% N-Nonane 1.9%
8 Geraniol 1.5% Thymol 2.2% Carvacrol 1.8%
9 Methyl eugenol 1.2% c-Terpinene 1.8% Miristicine 1.7%
10 Sabinene 0.5% q-Pritenone 1.5% Sabinene 1.7%
11 Terpinene-4 ol 0.5% Gemacrine-D 1.1% a-Pinene 1.5%
12 Terpinolene 0.5% Carvone 0.5% Phencen 1.4%
13 c-Terpinene 0.5% Eugenol 0.5% Apiol 1.3%
14 q-Cymene 0.3% Linalool 0.5% Terpinene-4 ol 0.8%
15 a-Thujene 0.2% Iso menthyl acetate 0.3% Thymokinone 0.7%
16 Myrcene 0.1% b-Pinene 0.2% 1-Methyl-3-propyl benzene 0.5%
17 c-Terpineol 0.08% — — N-Decane 0.4%
Total — 93.27% — 84.4% — 83.5%

software (Waters). Aflatoxin standards were purchased from Results

Sigma. Sensitivity of the HPLC method was tested by exam-
ining the limit of detection (LOD) and limit of quantification
(LOQ). The LOD was calculated based on the concentration of
the analyte that produced a peak, whose height was thrice the
height of the noise from a blank sample. The LOQ is the
lowest concentration of analyte in a sample that can be de-
termined with acceptable precision and accuracy; it was cal-
culated by taking three replications of the lowest calibration
standard. The percent inhibition of aflatoxin production was
calculated by the following equation:
Ac  As
Inhibition of aflatoxin production (%) ¼ · 100
Ac
where Ac is the amount of aflatoxin in control sample, As is the
amount of aflatoxin in treated sample.
Statistical analyses
The quantitative data of fungal growth and RP-HPLC an-
alyses were subjected to Mann-Whitney and Kruskal-Wallis
tests using SPSS software version 12.0 (SPSS Inc.). The dif-
ferences with p < 0.05 were considered significant.
Chemical composition of EOs
Chemical analysis of the EOs led to identification of 17, 16,
and 17 components in C. cyminum, Z. clinopodioides, and
N. sativa, respectively (Table 1). The major components
were a-pinene (30%) in C. cyminum, pulegone (37%) in
Z. clinopodioides, and trans-anthol (38.9%) in N. sativa oils.
Antifungal activities of EOs on A. parasiticus
MIC and MFC techniques were employed to assess fungi-
static and fungicidal properties of the EOs. As shown in
Table 2, fungal growth inhibitions were found to be correlated
dose-dependently with anti-mould activities. Based on broth
macrodilution method, C. cyminum and Z. clinopodioides oils
showed the MIC90 of 0.37 mg/mL; whereas the MIC90 value
for N. sativa oil was 1.75 mg/mL against toxigenic A. para-
siticus. In broth microdilution method, C. cyminum oil ex-
hibited the strongest activity, with MIC90 value of 1.6 mg/mL
against test organism. The MIC90 value for Z. clinopodioides
was 2.1 mg/mL for A. parasiticus, whereas the oil from N.
sativa exhibited relatively moderate activity with an MIC90 of
2.75 mg/mL for the fungus ( p < 0.05). These results generally

Table 2. Anti-Aspergillus parasiticus Susceptibility of Cuminum cyminum, Ziziphora clinopodioides,

and Nigella sativa in Broth Macro- and Microdilution Methods (mg/mL)

Broth macrodilution Broth microdilution

Aspergillus parasiticus A. parasiticus

MIC90 (mean – SD) MFC (mean – SD) MIC90 (mean – SD) MFC (mean – SD)

Plant essential oil

C. cyminum 0.37 – 0.1 1 – 0.4 1.6 – 0.2 3.5 – 0.5
Z. clinopodioides 0.37 – 0.1 1.2 – 0.5 2.1 – 0.5 5.5 – 2.8
N. sativa 1.75 – 0.3 2.5 – 0.4 2.75 – 0.9 6.25 – 2.3

The values in the table are an average of four experiments.

SD, standard deviation.
1278 KHOSRAVI ET AL.

Table 3. Comparison of the Effect of Cuminum cyminum, Ziziphora clinopodioides,

and Nigella sativa Oils on Mycelia and Aflatoxin Productions by Aspergillus parasiticus

Aflatoxin (mg/kg)

Dried weight of mycelia B1 B2 G1 G2 Total

Sample (mg) (mean – SD) (mean – SD) (mean – SD) (mean – SD) (mean – SD) (mean – SD)

C. cyminum (0.25 mg/mL) 1079.4 – 19.6 2.6 – 0.02 0.004 – 0.0002 1.2 – 0.4 0.003 – 0.0001 4 – 0.05
Z. clinopodioides (0.25 mg/mL) 855.3 – 17 2.6 – 0.02 0.003 – 0.0001 1.8 – 0.06 2 – 0.03 6.4 – 0.11
N. sativa (1.5 mg/mL) 1109.4 – 12.8 4.6 – 0.07 0.001 – 0.0005 1.7 – 0.02 0.4 – 0.03 6.4 – 0.04
Positive control 3406.1 – 25.5 9.3 – 0.02 2.9 – 0.4 110 – 0.04 3.4 – 0.05 165 – 0.04

confirmed those obtained in the broth macrodilution assay.
Subcultures of these treated inoculums were negative, thus
confirming fungicidal effects against A. parasiticus at concen-
trations mentioned in Table 2. The MFC value for Z. clin-
opodioides was 5.5 mg/mL for A. parasiticus, representing a
significant difference with others ( p < 0.05).
Effect of different concentrations
of EOs on mycelial growth
EOs of C. cyminum (0.25 mg/mL), Z. clinopodioides
(0.25 mg/mL), and N. sativa (1.5 mg/mL) exhibited a growth
inhibition percent of mycelia production by A. parasiticus in
values of 68.3%, 74.9%, and 67.4%, respectively (Table 3).
Statistical analysis showed significant differences among the
EOs ( p < 0.05).
Inhibitory effects on aflatoxin production
When the exact concentrations of C. cyminum, Z. clin-
opodioides, and N. sativa EOs were added to the cultures, sig-
nificant reductions in aflatoxin synthesis were observed
(Table 3). C. cyminum EO caused significant reductions in
values of 94.2% for AFB1, 100% for AFB2, 98.9% for AFG1,
100% for AFG2, and 97.5% for total aflatoxin ( p < 0.05). With
regard to aflatoxins (B1, B2, G1, G2, and total), inhibitory ef-
fects of Z. clinopodioides EO were 90.7%, 100%, 98.3%, 38.8%,
and 94.9%, respectively. These cases were 91.4%, 100%, 98.5%,
87.3%, and 96.2% for N. sativa, respectively. The LOD and
LOQ were found to be 0.04 and 0.16 lg/kg for AFB1, 0.0001
and 0.0004 lg/kg for AFB2, 0.03 and 0.12 lg/kg for AFG1,
0.0005 and 0.002 lg/kg for AFG2, and 0.005 and 0.02 lg/kg
for total aflatoxin, respectively.
Discussion
The increasing worldwide concern about food safety has
enhanced interest in fungal infection and subsequent pro-
duction of mycotoxins, especially aflatoxins, in food products
(Williams et al., 2004). Due to the increasing public awareness
of the pollutive, residual, carcinogenic, and phytotoxic effects
of many synthetic fungicides, the importance of natural
products to control phytopathogenic fungi is gaining popu-
larity (Bankole, 1997). Among medicinal plants, species be-
longing to the genera C. cyminum, Z. clinopodioides, and N.
sativa gained increasing interest, because they are composed
of different bioactive chemicals (Khosravi et al., 2011).
In the current study, we showed a new biological activity for
C. cyminum, Z. clinopodioides, and N. sativa EOs as inhibitors of
AFB1, AFB2, AFG1, and AFG2 production by A. parasiticus, in
addition to the ability for strong fungal growth inhibition. As a
result of GC/MS analyses, C. cyminum, Z. clinopodioides, and N.
sativa contained a-pinene (30%), pulgone (37%), and trans-
anthol (38.9%), respectively, as the major compounds. Our
results were consistent with other investigators, representing
a-pinene (29.1%) in C. cyminum by Gachkar et al. (2007), pule-
gone (31.8%) in Z. clinopodioides by Ozturk and Ercisli (2007),
and trans-anthol (38.3%) in N. sativa by Nickavar et al. (2003).
The differences in chemical compositions of oils could be at-
tributed to the climatic effects on the plants (Marzoug et al.,
2011; Polat et al., 2011).
MIC90 and MFC assays were employed to determine fun-
gistatic and fungicidal properties of the EOs. The EO of C.
cyminum had the highest inhibitory effect, followed by Z.
clinopodioides and N. sativa on fungal development of A.
parasiticus. Inhibitory effect of C. cyminum is associated with a
group of terpenes such as a-pinene and 1,8 cineole (Davidson
and Naidu, 2000; Hammer et al., 2003). Antibacterial activities
of these EOs have been reported in previous studies (Iaco-
bellis et al., 2005; Aghajani et al., 2008; Salman et al., 2008). In
addition, antifungal activities of the EOs of C. cyminum
(Bansod and Rai, 2008), Z. clinopodioides (Behravan et al., 2007),
and N. sativa (Naeini et al., 2009) were tested against different
pathogenic and saprophytic fungi. Khosravi et al. (2011)
showed that C. cyminum and, to a lesser extent, Z. clin-
opodioides EOs exhibited the strongest activity against A. fu-
migatus and A. flavus with MIC90 ranging from 0.25 to 1.5 mg/mL;
whereas the oil from N. sativa exhibited relatively moderate
activity against the two fungi just mentioned with MIC90
ranging from 1.5 to 2 mg/mL. This study showed that the
extent of inhibition of fungal growth and aflatoxins produc-
tion was dependent on the concentration of the EOs used. In
addition, EO-related inhibition in mycelial growth was ob-
served to be associated with decreased levels of aflatoxin
production, which was in agreement with other studies (Ra-
sooli and Razzaghi-Abyaneh, 2004; Razzaghi-Abyaneh et al.,
2008). In this study, aflatoxin production was significantly
inhibited at lower than fungistatic concentrations of the EOs
tested. Interestingly, aflatoxin B2 was more affected than AFB1
by the EOs. There is a 10-kb gene cluster that controls the ac-
tivity of the aflatoxins biosynthesis pathway. Regarding the
different aflatoxins, it seems that some of the genes have more
activity than the others. The correlations between some gene
expressions and production of each kind of aflatoxins have
been noted. There is speculation that EOs may be affected by
some special aflatoxin B2 gene expressions (Yahyaraeyat and
Khosravi, 2010). Further studies are needed to approve this
matter. One of the characteristics of aflatoxin deactivation
processes is that they should destroy the mycelia and spores
AFLATOXIN INHIBITION BY ESSENCES
of the toxic fungi, which may proliferate under favorable con-
ditions (Namazi et al., 2002; Khosravi et al., 2011). The results of
this study comply with the findings just specified. These in-
hibitory effects are interesting in connection with the preven-
tion of aflatoxin contamination in many foods. C. cyminum oil
exerted higher antifungal as well as antitoxic effects than those
of Z. clinopodioides and N. sativa. These differences in antifungal
and aflatoxin inhibition efficacy of the EOs may be attributable
to the oil compositions. It has been established that the com-
positions of the EOs depend on the plant species, the chemo-
types, and the climatic conditions; therefore, their antimicrobial
activities could vary (Shu and Lawrence, 1997).
Conclusion
These results indicated the potential of the EOs of C. cym-
inum, Z. clinopodioides, and N. sativa as natural inhibitors in
foods against A. parasiticus, the well-known causal agents of
foodborne diseases and food poisonings.
Acknowledgments
This work was supported by the Research Council of
University of Tehran.
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Ali Reza Khosravi, D.V.M., Ph.D.
Faculty of Veterinary Medicine
Mycology Research Center
University of Tehran
Azadi St.
Tehran 345261789
Iran
E-mail: khosravi@ut.ac.ir