Inhibitory effects of three monoterpenes from ginger essential oil on growth and aflatoxin production of Aspergillus flavus and their gene regulation in aflatoxin biosynthesis

Abstract

Ginger (Zingiber officinale) essential oil (ZOE) possesses strong antibacterial and antifungal activities. In this study, the antifungal activity of ZOE against Aspergillus flavus was investigated, and a chemical analysis was carried out to identify compounds that control fungal growth. A total of 37 compounds were identified by gas chromatographic analysis with a mass detector, and the antifungal and antiaflatoxigenic properties of three constituents, γ-terpinene, isoborneol, and citral, against A. flavus were tested. All compounds exhibited strong antifungal activity at 1000 μg/mL, and the antifungal activity of γ-terpinene and citral remained until treatment with tenfold diluted solution. The decrease in aflatoxin production by the three compounds was observed until treatment with 10 μg/mL. To evaluate their antiaflatoxigenic activity, RT-qPCR was used to compare the expression of 11 genes involved in aflatoxin biosynthesis by A. flavus. Among the three compounds, γ-terpinene and citral markedly reduced the expression of most of the tested genes but a different pattern of downregulation of the expression was observed. γ-Terpinene did not downregulate aflR, aflS, and yap, whereas citral did not alter the expression of aflC and aflG. Therefore, γ-terpinene and citral may have the potential to control A. flavus growth and aflatoxin production in agricultural products, including at the storage stage.

Introduction

The phytochemicals in ginger (Zingiber officinale) can control viruses, including Feline calicivirus, and bacteria [1,2,3]. The major constituents of fresh ginger are phenol derivatives such as gingerols, which are converted into shogaols during heat treatment [4]. These natural compounds exhibit strong pharmacological activities, such as lowering of lipid and glucose content in the blood [5], antiplatelet activity by decreasing production of platelet thromboxane-B2 [6], and anti-inflammatory effects [7].

In a previous study, Zingiber officinale essential oil (ZOE) was extracted by steam distillation and its constituents were identified by gas chromatography with a mass detector (GC–MS) [8]. ZOE showed antibacterial activity against eight pathogenic bacteria (125–500 μg/mL) and antifungal activities against two fungal strains (250 μg/mL) [9]. They identified sabinene, (E)-1-(3′,4′-dimethoxyphenyl)buta-1,3-diene, terpinen-4-ol, γ-terpinene, and β-phellandrene as the major constituents of ZOE among the 49 compounds determined [9]. In addition, ZOE exhibited antibacterial activity against bacteria isolated from fish and shellfish [10]. ZOE also inhibited reproduction of the cattle tick Rhipicephuls microplus [11].

Aspergillus flavus produces cancer-stimulating compounds, such as aflatoxins. Birds are particularly susceptible to these natural compounds [12, 13]. Selenium and zinc can neutralize aflatoxin toxicity in poultry through dietary supplementation [13]. Interestingly, many essential oils have been used as reducing agents against aflatoxin contamination by A. flavus or A. parasiticus in agricultural products [14, 15]. The direct inhibition of aflatoxin biosynthesis in A. flavus or indirect inhibition pathways that reduce the respiration ability in aflatoxin-producing fungi are responsible for the antiaflatoxigenic properties [16].

Recently, we identified natural compounds containing a methylenedioxy moiety that exhibit antifungal and antiaflatoxigenic activities against A. flavus by direct inhibition of aflatoxin production through the suppression of certain major genes involved in the biosynthesis [17, 18]. 4-Hydroxy-7-methyl-3-phenylcoumarin also possessed similar inhibitory properties against A. flavus by strongly suppressing aflK-expressing versicolorin B synthase [19].

In the present study, the antifungal activity of ZOE against A. flavus was investigated, and a chemical analysis was performed using GC–MS. The antifungal and antiaflatoxigenic activities of the three compounds, which are easily available in the market, against A. flavus were evaluated. Finally, the modes of antiaflatoxin action of the three compounds were evaluated and confirmed by RT-qPCR by comparing the gene expression after treatment.

Materials and methods

Chemicals and microorganisms

ZOE was purchased from Lhasa Karnak Herb Company (Berkeley, CA, USA). Tested γ-terpinene, isoborneol, and citral were also purchased from Sigma-Aldrich Co. (St. Louise, MO, USA). Aspergillus flavus ATCC 22546 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Preparation of spore solution

For the preparation of the isolate, A. flavus was grown on malt extract agar (MEA: Difco Laboratories, Sparks, MD, USA) and this isolate generated aflatoxin B1 and B2, but not G1 and G2 [19]. It was grown on MEA medium at 30 °C for 5 days until fungal spores formed. After spore formation, they were collected from slants by shaking with 0.05% (v/v) of Tween 80 and stored at − 70 °C in 20% glycerol solution (v/v).

Chemical analysis of ZEO using GC–MS

Chemical analysis for ZOE was undertaken using a HP-6890 (Agilent Technologies, Wilmington, DE, USA) gas chromatograph equipped with a DB-5 fused silica capillary column (60 m × 0.25 mm, 0.25 μm film thickness) interfaced with a 59,731 V (Agilent) mass detector. The GC–MS was connected to a computer equipped with Wiley 7 spectra library. The condition of GC–MS was column temperature ranged from 60 to 250 °C with an elevation of 5 °C. Injector temperature was set at 250 °C, and injection volume was 1.0 μl with a split ratio of 1:50. The determined mass range was 50–600 amu. Compounds were identified by comparing their mass spectra with those in Wiley 7 mass spectral database for GC–MS (Table S1).

Aflatoxin analysis using HPLC-fluorescence detector

Fungal spore suspension adjusted to 10⁶ density was inoculated into the liquid culture media comprising potato dextrose broth (25 mL) (Difco Laboratories). All the test compounds were spiked into the corresponding liquid media serially, and the culture was incubated at 25 °C for 5 days under shaking conditions. All experiments were performed in triplicate for each concentration of the tested compounds.

Following cultivation in liquid medium for 5 days, the fungal growth was measured using a filter paper to weigh the mycelial and sclerotial residues with overnight drying in an oven. Separately, the mycelia from each treatment were subjected to the extraction procedure using an ultrasonic cleaner, and aflatoxin B (AFB) and G were analyzed using an HPLC-fluorescence detector [19]. The average of the three replicates was calculated for each experiment. One-way ANOVA was used for comparisons between two groups, and statistical significance was set at p < 0.05 [20].

Real-time qPCR after isolation of total RNA

To understand the inhibition of fungal growth and aflatoxin production, real-time qPCR was employed. Fungal mycelia in liquid media were carefully collected and total RNA was extracted using the QIAzol Lysis reagent, supplied by QIAGEN Inc. (Dusseldorf, Germany), after grinding to a fine powder under addition of liquid nitrogen. Total RNA extracted from the treated fungi were quantified with μDrop™ Plate (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the extracted RNAs were quantified using 1% of agarose gel with ethidium bromide. Complementary DNA (cDNA) for extracted RNAs (2 μg) was synthesized using Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).

RT-qPCR was performed using Rotor-Gene SYBR Green PCR Kit (QIAGEN Inc.) with an appropriate amount of cDNA (100 ng). Primers for genes, such as yap, aflR, aflS, aflK, aflD, aflQ, and 18S rRNA, were synthesized by Genotech (Daejeon, Korea) (Table S2) and used to understand the relationship between aflatoxin biosynthesis and the active compound [18]. Forty cycles of thermal cycling were performed for amplification (denaturation at 95 °C for 30 s, annealing at 60 °C for 20 s, and elongation at 72 °C for 30 s, followed by an additional step at 95 °C for 5 min). RT-qPCR was performed in triplicate for each treatment. Significant differences in gene expression were calculated using delta/delta Ct methods [21]. Data were standardized with 18S rRNA, and the gene expression between the treatment group and controls was compared.

Results

The components of ZEO were identified by GC–MS analysis (Table S1). After the analysis, γ-terpinene, isoborneol, and citral were chosen to investigate their inhibitory effects on the growth of A. flavus and aflatoxin production with the assessment of their molecular modes of inhibitory action.

The three major components of ZEO showed a strong inhibitory effect against A. flavus growth at 1000 μg/mL. This inhibition by isoborneol decreased approximately twofold when the fungi were treated with 250 μg/mL of ZEO and disappeared at 100 μg/mL. γ-Terpinene exhibited 50% inhibitory effect on the fungal growth at 100 μg/mL, but this effect was not observed at 10 μg/mL. Citral exhibited a similar inhibitory pattern as the other two monoterpenes (Table 1).

Table 1 Effects of monoterpenes present in ginger essential oil on Aspergillus flavus growth

Aflatoxin production after treatment with the three compounds was determined (Table 2), and the three compounds completely inhibited aflatoxin biosynthesis at 100 μg/mL. At 10 μg/mL, γ-terpinene and citral showed some antiaflatoxigenic activities.

Table 2 Effects of monoterpenes derived from ginger essential oil on aflatoxin production

To understand the inhibitory effect of γ-terpinene on aflatoxin production, the expression of 11 genes, which are involved in aflatoxin biosynthesis, was analyzed by RT-qPCR (Fig. 1). The expression of aflC, aflD, aflE, aflK, aflO, and aflQ genes was inhibited by approximately 80% compared with that of the control at 100 μg/mL. However, aflR, aflS, and yap were not affected by the treatment (Fig. 1). At tenfold dilution, γ-terpinene downregulated only five genes by approximately 50% and upregulated aflG gene approximately threefold (Fig. 1).

Fig. 1

Expression of genes involved in aflatoxin biosynthesis in Aspergillus flavus after the treatment of γ-terpinene. (A), aflC; (B), aflD; (C), aflE; (D), aflG; (E), aflK; (F), aflL; (G), aflO; (H), aflQ; (I), aflR; (J), aflS; (K), yap. 18S rRNA was used for the standardization of gene expression. Different letters indicate significantly different from the control group (p < 0.05)

Citral downregulated the expression of most of the tested genes, except aflC and aflG, at 100 μg/mL (Fig. 2). However, the inhibition was higher at 10 μg/mL than at 100 μg/mL. In addition, aflR was the only gene to be more strongly inhibited by citral at 100 μg/mL.

Fig. 2

Expression of genes involved in aflatoxin biosynthesis in Aspergillus flavus after the treatment of citral. (A), aflC; (B), aflD; (C), aflE; (D), aflG; (E), aflK; (F), aflL; (G), aflO; (H), aflQ; (I), aflR; (J), aflS; (K), yap. 18S rRNA was used for the standardization of gene expression. Different letters indicate significantly different from the control group (p < 0.05)

Discussion

In a previous study, ZEO exhibited antifungal activity against A. flavus and antiaflatoxigenic activity [22]; the mycelial growth of A. flavus was completely inhibited at 150 μg/mL. The major components in ZEO are monoterpenes such as β-phellandrene (0.95%), camphene (0.61%), and β-pinene (0.61%) as well as sesquiterpenes such as zingiberene (29.54%), β-sesquiphellandrene (18.42%), and germacrene D (3.58%) (Onyenekwe and Hashimoto, 1999). However, higher amounts of monoterpenes such as camphene (3.0%), β-phellanfrene (1.4%), 1,8-cineole (1.9%), borneol (2.1%), neral (7.4%), and geranial (25.9%) and lower amounts of sesquiterpenes such as zingiberene (9.5%), β-sesquiphellandrene (5.1%), and germacrene B (0.3%) in ZEO have also been reported (Singh et al., 2008). In our study, γ-terpinene (1.9%), campene (17.2%), shisool (19.86%), citral (3.41%), γ-curcumene (3.03%), and ar-curcumene (6.16%) were the main volatile components extracted from ZEO (Table S1). ZEO contained 0.22% zingiberene, and its proportion was much lower than that reported earlier [23, 24].

Compositional difference in essential oils extracted from the same plant sources may be due to the extraction methods for essential oils [25]. They used two different extraction methods, hydrodistillation and supercritical fluid extraction (SCFE), to obtain essential oils from cumin. Then, the authors analyzed these two essential oils by GC–MS [25]. Cumin essential oil obtained by hydrodistillation had a higher proportion of cuminaldehyde (52.6%) than that in the essential oil (37.3%) obtained by SCFE extraction method. In the case of cuminic alcohol, the essential oil obtained by SCFE contained higher portions than that by hydrodistillation [25]. In addition, the essential oil composition may differ with weather condition during cultivation, e.g., guava cultivated during the dry season primarily comprises nerolidol [26]. Finally, one of primary parameters for the difference in composition may be related to the plant chemo-type [27]. Therefore, essential oils, including ZEO, need to be standardized before use in the agricultural or other industries [28, 29].

The antifungal activity and antiaflatoxigenic activities of three monoterpenes, γ-terpinene, isoborneol, and citral, against A. flavus were evaluated in this study. γ-Terpinene is a major component of several essential oils that are known to inhibit Aspergillus spp. growth and aflatoxin production [22, 30, 31]. However, its antifungal and antiaflatoxigenic activities against A. flavus have not been evaluated yet. Isoborneol has not been reported to exhibit antifungal and antiaflatoxigenic activities against A. flavus. In our study, γ-terpinene and isoborneol strongly inhibited A. flavus growth at 1000 μg/mL and aflatoxin production at 100 μg/mL. However, their mode of action in terms of the downregulation pattern of aflatoxin biosynthesis genes was different. γ-Terpinene downregulated 7 genes at 100 μg/mL, while isoborneol did not downregulate genes at the same concentration. γ-Terpinene showed a similar pattern of gene downregulation as eugenol and cinnamaldehyde, which inhibited aflatoxin production by modifying the major genes for aflatoxin biosynthesis in A. flavus [32]. Another aflatoxin-producing fungi, A. parasiticus, has been used to study the inhibitory effect of eugenol on its growth and aflatoxin production [33]. Eugenol downregulated aflatoxin biosynthetic genes ver-1, nor-1, pks A, omt A, and aflR at 62.5–125 μg/mL [33]. ver-1 is equivalent to aflM in this study, nor-1 to aflD, pksA to aflC, and omtA to aflP [34]. In our study, the expression of aflC was only 20% in the presence of 100 μg/mL of γ-terpinene. aflD was downregulated approximately twofold and fivefold in the presence of 10 and 100 μg/mL of γ-terpinene, respectively. However, the expression of aflR was not affected. Therefore, the inhibition of major genes involved in the biosynthesis of aflatoxins in A. flavus contributes toward the inhibitory action of γ-terpinene.

In the case of isoborneol, aflK, aflL, and aflQ were downregulated approximately twofold at 10 μg/mL. It is likely that isoborneol possesses a different mode of inhibitory action on aflatoxin production, suggested by the penetration of H₂O₂ generated after treatment with isoborneol [35].

In a previous study, citral showed antifungal and antiaflatoxigenic activities against A. flavus growth and aflatoxin biosynthesis, respectively [32]. They demonstrated that at 2.80 mmol/L, citral completely inhibited A. flavus growth and aflatoxin production [32]. It also downregulated aflT completely and inhibited the expression of aflM, aflP, aflL, aflR, and aflD 257-, 29-, 3.5-, and 2.5-fold compared with the control group. In our study, citral treatment downregulated aflC, aflD, aflE, aflG, aflK, aflL, aflO, aflQ, aflR, aflS, and Yap in A. flavus. All the tested genes were affected by the addition of 10 μg/mL citral (Fig. 3). We did not use aflT in this study. However, our results were similar to those reported [32] that aflL, aflR, and aflD were downregulated following treatment with citral. This indicates that citral inhibits aflatoxin production in A. flavus by interfering with the expression of aflatoxin biosynthesis genes. In addition to the downregulation of the expression of aflatoxin-producing genes, another inhibitory mechanism has been suggested—the secretion of H₂O₂ after treatment with citral [35]. With these monoterpenoids, other major constituents such as turmerone from turmeric essential oil may also need to determine their antifungal and antiaflatoxigenic activities to A. flavus to develop ecofriendly used fungicides in agricultural fields [36].

Fig. 3

Expression of genes involved in aflatoxin biosynthesis in Aspergillus flavus after the treatment of isoborneol. (A), aflC; (B), aflD; (C), aflE; (D), aflG; (E), aflK; (F), aflL; (G), aflO; (H), aflQ; (I), aflR; (J), aflS; (K), yap. 18S rRNA was used for the standardization of gene expression. Different letters indicate significantly different from the control group (p < 0.05)

Conclusively, three natural compounds (isoborneol, γ-terpinene, and citral) in ZOE showed antifungal and antiaflatoxigenic activities against A. flavus. Aflatoxin production by A. flauvs was strongly inhibited at 100 μg/mL of γ-terpinene and citral. These two compounds downregulated the primary genes involved in aflatoxin biosynthesis in A. flavus at 10 μg/mL. However, their inhibitory action on gene expression was different, and γ-terpinene suppressed aflC, aflD, aflE, aflK, aflO, and aflQ genes, which express polyketide synthase, reductase, NOR-reductase, VERB synthase, O-methyltransferase B, and oxidoreductase, respectively. Citral downregulated nine genes among the tested genes, except aflC and aflG, which express polyketide synthase and P450 monooxygenase, respectively. Isoborneol did not suppress any of the tested genes. The different inhibitory patterns of gene expression in A. flavus warrant further studies.