Inhibition of Aflatoxin Production and Growth of Aspergillus parasiticus by Cuminum cyminum,Ziziphora clinopodioides,and Nigella sativa Essential Oils

Abstract

Aflatoxins are highly toxic and carcinogenic metabolites produced by Aspergillus parasiticus on food and agricultural 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 α-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

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, vegetables, 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 aflatoxins (Bhatnagar and Cleveland, 2004).

Studies on controlling aflatoxin contamination of plants and susceptible crops are mainly focused on fungal processes needed for plant invasion and mycotoxin production as well as developing of genetically modified crops, which are resistant 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; 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 26°C±1°C. 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 population 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 −12°C 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 μm; J & W Scientific Inc.). The operating conditions were as follows: injector and detector temperatures were 250°C and 265°C, respectively; carrier gas was Helium. Oven temperature program was 40°C–250°C at the rate of 4°C/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 conditions 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 reported 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.

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 μL of the final conidia suspensions were mixed with 100 μL of the test EO in 12×75 mm test tubes and incubated at 28°C for 48 h. The positive control tube contained 900 μL of conidial suspension plus 100 μL of Roswell Park Memorial Institute medium (RPMI) 1640, and the negative one contained 1 mL of RPMI 1640 only. The lowest oil concentration inhibiting fungal 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 μL of the oil. Positive control was the well containing 100 μL of the inoculum suspension and 100 μL of the RPMI only, and the negative control was a well containing 200 μL of RPMI 1640. The MFCs were determined by subculturing 10 μL aliquot from all MIC₉₀ wells showing no visible growth on to Sabouraud glucose agar plates. Antifungal analysis was replicated four times, and the results were expressed as the average of four repetitions.

Aflatoxin production assay

For evaluation of aflatoxin formation, EOs at concentrations 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 added to 25 mL of 2% YES broth containing different concentrations of EOs in 100 mL Erlenmeyer flask and incubated for 10 days at 26°C±1°C in the darkness. After the incubation period, cultures were autoclaved at 121°C for 30 sec, to inactivate mycelia and conidia, and filtered through Whatman No. 1 filter paper. The mycelia were dried to a constant weight at 80°C, 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 extraction using reverse phase-high performance liquid chromatography (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 solution 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 μL methanol with 1 min interval. The elution diluted with 1750 μL H₂O and the aliquot of 200 μL 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 mobile 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 software (Waters). Aflatoxin standards were purchased from Sigma. Sensitivity of the HPLC method was tested by examining 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 determined with acceptable precision and accuracy; it was calculated by taking three replications of the lowest calibration standard. The percent inhibition of aflatoxin production was calculated by the following equation: \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland, xspace} \usepackage{amsmath, amsxtra} \pagestyle{empty} \DeclareMathSizes {10} {9} {7} {6} \begin{document} \begin{align*} \hbox {\rm Inhibition of aflatoxin production} \ ( \% ) = \frac { A_c - A_s } { A_c } \times 100 \end{align*} \end{document}

where A _c is the amount of aflatoxin in control sample, A _s is the amount of aflatoxin in treated sample.

Statistical analyses

The quantitative data of fungal growth and RP-HPLC analyses were subjected to Mann-Whitney and Kruskal-Wallis tests using SPSS software version 12.0 (SPSS Inc.). The differences with p<0.05 were considered significant.

Results

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 α-pinene (30%) in C. cyminum, pulegone (37%) in Z. clinopodioides, and trans-anthol (38.9%) in N. sativa oils.

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	α-Pinene	30%	Pulegone	37%	Trans anthol	38.9%
2	Limonene	21%	ρ-Peritone	19.6%	ρ-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%	α-Thujene	2.8%
6	α-Terpineol	3%	Menthol	3%	Anisaldehyde	1.9%
7	α-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%	γ-Terpinene	1.8%	Miristicine	1.7%
10	Sabinene	0.5%	ρ-Pritenone	1.5%	Sabinene	1.7%
11	Terpinene-4 ol	0.5%	Gemacrine-D	1.1%	α-Pinene	1.5%
12	Terpinolene	0.5%	Carvone	0.5%	Phencen	1.4%
13	γ-Terpinene	0.5%	Eugenol	0.5%	Apiol	1.3%
14	ρ-Cymene	0.3%	Linalool	0.5%	Terpinene-4 ol	0.8%
15	α-Thujene	0.2%	Iso menthyl acetate	0.3%	Thymokinone	0.7%
16	Myrcene	0.1%	β-Pinene	0.2%	1-Methyl-3-propyl benzene	0.5%
17	γ-Terpineol	0.08%	—	—	N-Decane	0.4%
Total	—	93.27%	—	84.4%	—	83.5%

Antifungal activities of EOs on A. parasiticus

MIC and MFC techniques were employed to assess fungistatic 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. parasiticus. In broth microdilution method, C. cyminum oil exhibited 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 confirmed those obtained in the broth macrodilution assay. Subcultures of these treated inoculums were negative, thus confirming fungicidal effects against A. parasiticus at concentrations mentioned in Table 2. The MFC value for Z. clinopodioides was 5.5 mg/mL for A. parasiticus, representing a significant difference with others (p<0.05).

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.

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).

Table 3.

Comparison of the Effect of Cuminum cyminum, Ziziphora clinopodioides, and Nigella sativa Oils on Mycelia and Aflatoxin Productions by Aspergillus parasiticus

		Aflatoxin (μg/kg)
Sample	Dried weight of mycelia (mg) (mean±SD)	B₁ (mean±SD)	B₂ (mean±SD)	G₁ (mean±SD)	G₂ (mean±SD)	Total (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

Inhibitory effects on aflatoxin production

When the exact concentrations of C. cyminum, Z. clinopodioides, and N. sativa EOs were added to the cultures, significant 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 effects 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 μg/kg for AFB₁, 0.0001 and 0.0004 μg/kg for AFB₂, 0.03 and 0.12 μg/kg for AFG₁, 0.0005 and 0.002 μg/kg for AFG₂, and 0.005 and 0.02 μg/kg for total aflatoxin, respectively.

Discussion

The increasing worldwide concern about food safety has enhanced interest in fungal infection and subsequent production 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 popularity (Bankole, 1997). Among medicinal plants, species belonging 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 α-pinene (30%), pulgone (37%), and trans-anthol (38.9%), respectively, as the major compounds. Our results were consistent with other investigators, representing α-pinene (29.1%) in C. cyminum by Gachkar et al. (2007), pulegone (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 attributed to the climatic effects on the plants (Marzoug et al., 2011; Polat et al., 2011).

MIC₉₀ and MFC assays were employed to determine fungistatic 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 α-pinene and 1,8 cineole (Davidson and Naidu, 2000; Hammer et al., 2003). Antibacterial activities of these EOs have been reported in previous studies (Iacobellis 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. clinopodioides EOs exhibited the strongest activity against A. fumigatus 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 production was dependent on the concentration of the EOs used. In addition, EO-related inhibition in mycelial growth was observed to be associated with decreased levels of aflatoxin production, which was in agreement with other studies (Rasooli 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 activity 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 of the toxic fungi, which may proliferate under favorable conditions (Namazi et al., 2002; Khosravi et al., 2011). The results of this study comply with the findings just specified. These inhibitory effects are interesting in connection with the prevention 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 compositions of the EOs depend on the plant species, the chemotypes, 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. cyminum, Z. clinopodioides, and N. sativa as natural inhibitors in foods against A. parasiticus, the well-known causal agents of foodborne diseases and food poisonings.

Footnotes

Acknowledgments

This work was supported by the Research Council of University of Tehran.

Disclosure Statement

No competing financial interests exist.

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