Search for novel antifungals from 49 indigenous medicinal plants: Foeniculum vulgare and Platycladus orientalis as strong inhibitors of aflatoxin production by Aspergillus parasiticus

Abstract

In a search for novel antifungals from natural sources, essential oils (EOs) and extracts of 49 medicinal plants were studied against an aflatoxin (AF)-producing Aspergillus parasiticus using a microbioassay technique. AF levels were measured in culture broth by high performance liquid chromatography. The EOs were analyzed by gas chromatography/mass spectrometry (GC/MS). Based on the results obtained, Achillea millefolium subsp. elborsensis, Ferula gummosa, Mentha spicata, Heracleum pubescens and Thymus fedtschenkoi markedly inhibited A. parasiticus growth by IC₅₀ values of 35 to 1,815 μg/ml without affecting AF production by the fungus. The EOs of flowers and roots of Foeniculum vulgare significantly inhibited both fungal growth (∼70.0%) and production of AFs B₁ and G₁ (∼99.0%). The ethyl acetate extract of Platycladus orientalis leaves suppressed AFB₁ (∼90.0%) but not fungal growth and AFG₁ production. This work provides evidence for the first time that F. vulgare and P. orientalis are strong inhibitors of aflatoxin biosynthesis in A. parasiticus. The antifungal activities of the bioactive plants introduced in the present study could make an important contribution to explaining the use of these plants as effective antimicrobial candidates to protect foods and feeds from toxigenic fungus growth and subsequent AF contamination.

Introduction

The members of Aspergillus section Flavi comprise an important group of human pathogens, mycotoxin producers and food contaminants all over the world (Samson et al. 2000; Hedayati et al. 2007). Two species of this section, i.e., Aspergillus flavus and Aspergillus parasiticus have received major attention, not only for their ability to contaminate a wide array of substrates including peanuts, corn, pistachio nuts and oil seeds, but also for their production of carcinogenic aflatoxins (AFs) (Payne 1998). AFs are a real public health hazard due to their carcinogenic, mutagenic, teratogenic and immunosuppressive effects on various biological systems (Hedayati et al. 2007). On the other hand, economic losses from AF contamination of food and feed are a worldwide problem, costing millions of US dollars each year.

Despite the existence of a lot of useful information on AF inhibitors from natural sources (Holmes et al. 2008; Razzaghi-Abyaneh, et al. 2008, 2009, 2010), fungal invasion and subsequent AF contamination of food and feed is not yet under adequate control. Chemicals are used widely to control the detrimental effects of AF-producing fungi. However, chemical treatments suffer from severe limitations, including adverse reactions on biological systems, development of resistance by fungal pathogens, and undesirable effects on non-target organisms sharing the ecosystem. Thus, there is a clear tendency towards optimization of environmentally friendly fungicides that cause minimal damage to human health and the surrounding ecosystem (Ghisalberti 2000).

In recent years, researchers have focused on finding novel antimicrobials from natural sources including higher plants, microorganisms, insects, nematodes and vertebrates. Plants are rich sources of beneficial secondary metabolites. Their essential oils (EOs) and extracts have a wide array of biological activities, especially antimicrobial effects on different groups of pathogenic organisms (Shams-Ghahfarokhi et al. 2006; Bakkali et al. 2008; Webster et al. 2008; Tolouee et al. 2010). Nowadays, an expanding list of plant EOs has been classified as generally recognized as safe (GRAS) by the Unites States Food and Drug Administration (FDA) as approved flavors or food additives (Tripathi and Dubey 2004).

Interestingly, production of an extremely wide array of bioactive compounds by medicinal plants is completely dependent on parameters such as plant characteristics (i.e., species, variety and growth cycle), geographic condition, soil composition, etc. Thus, screening of a large number of plants from different geographic locations may increase the chance of finding novel bioactive compounds inhibitory to AF-producing fungi.

With respect to seasonal variations, geographic conditions and unique ecosystems, a wide array of medicinal plants are grown in Iran—some being quite specific at the genus or species level. As a continuation of our ongoing research on natural antifungals, EOs and extracts of 49 indigenous medicinal plants belonging to 21 major families were evaluated in relation to their ability to inhibit A. parasiticus growth and AF (AFB₁ and AFG₁) production. Inhibitory components of the bioactive plants determined by GC/MS were given special consideration.

Materials and methods

Fungal strain and growth media

Aspergillus parasiticus NRRL 2999, a known producer of AFs of the B and G series, was used throughout the study. The fungus was cultured on potato dextrose agar (PDA; Merck, Darmstadt, Germany) slants for 7 days at 28°C. Spore suspension was prepared by gently scraping the culture surface using a sterile glass rod in the presence of a 0.1% aqueous solution of Tween 80. Potato dextrose broth (PDB; Scharlau Chemie, Barcelona, Spain) was the medium used for submerged cultures of A. parasiticus for AF production.

Plant materials, EOs and extract preparation

As indicated in Table 1, a total of 49 plant species belonging to 21 different families was studied. The plants were collected during April–June 2009. Vouchers were stored in the herbarium of the Research Institute of Forest and Rangelands. Plant materials (leaves, seeds, aerial parts, roots, flowers) were steam distilled for 90 min in a fully glass apparatus. EOs were prepared by hydro-distillation of sterilized plant parts using a Clevenger-type apparatus during a 4-h time period (Bradley 1993). The extraction was carried out for 120 min in 500 ml water. The EO yields were in the range of 0.25–1.2% of total weight, and were kept at 4°C until use. To prepare extracts, plant materials were air-dried and then powdered using a homogenizer. Amounts of 10 g of each air-dried plant material were extracted separately with 100 ml ethyl acetate (EtOAc) and n-hexane in Erlenmeyer flasks for 24 h. The extracts were filtered through Whatman No.1 filter papers and evaporated to near dryness by a rotary evaporator. Extracts were kept at 4°C until use.

Table 1 General features and preliminary data of antifungal and antiaflatoxigenic potential of the essential oils and extracts of 49 medicinal plants belonging to 21 different families. AFB ₁ Aflatoxin B₁, AFG ₁ aflatoxin G₁

GC/MS analyses of EOs

GC/MS analyses were performed using a Varian 3400 GC/MS apparatus coupled to a Saturn II ion trap detector (http://varianinc.com/). Quantitation was performed using Euro Chrom 2000 software (http://www.knauer.net) by the area normalization method neglecting response factors. GC analysis was carried out using a DB-5 fused silica capillary column (60 m × 0.25 mm × film thickness 0.25 μm; J & W Scientific, Rancho Cordova, CA). The operating conditions were as follows: injector and detector temperature, 250°C and 265°C, respectively; helium as carrier gas. Oven temperature programme was 40°C–250°C at the rate of 4°C/min. Mass spectrometry conditions were: ionization potential of 70 eV and electron multiplier energy equal to 2,000 V. The identities of EOs components were established from their GC retention indices relative to C7-C25 n-alkenes, by comparison of their MS spectra with those reported in the literature, and by computer matching with the Wiley 5 mass spectra library (http://eu.wiley.com/WileyCDA) and whenever possible, co-injection with a standard available in the laboratory (Davies 1998).

Microbioassay

Aspergillus parasiticus NRRL 2999 was cultured on PDB in 6-well flat-bottom microplates (Greiner bio-one, http://www.greinerbioone.com; well diameter 36.0 mm) in the presence of plants EOs, EtOAc and/or n-hexane extracts using a microbioassay technique (Razzaghi-Abyaneh et al. 2007). Culture medium (5 ml/well) was added to the microplates, which were inoculated with fungal spore suspension (5 × 10⁶ spores/well) prepared in an aqueous solution of 0.1% Tween 80. Serial two-fold dilutions of the EOs and/or extracts (from 15.62 to 2,000 μg/ml) prepared in methanol (final concentration 1.0%) were added separately to the test wells. The control wells were treated in the same manner except that they did not contain plant EOs and extracts. Triplicate microplates were incubated for 96 h at 28°C under static conditions in two separate experiments.

Fungal dry weight determination

The total contents of each well including culture medium and fungal biomass were filtered through a thin layer of cheese cloth and then thoroughly washed with distilled water. A known weight of mycelium was placed in a stainless steel container and allowed to dry at 80°C to constant weight. The net dry weight of mycelium was then determined.

HPLC assay of AFs

AFs were first qualitatively detected by observing the thin layer chromatography (TLC) pattern of cultures spotted on Silica gel 60 F₂₅₄ plates under UV light (365 nm). The AF content of cultures was measured using HPLC (Knauer D-14163 UV-VIS system, Berlin, Germany) according to Razzaghi-Abyaneh et al. (2007) with some modifications. A 50-μl aliquot of each sample (culture filtrate) was injected into the HPLC column (TSKgel ODS-80TS; 4.6 mm ID × 15.0 cm, Tosoh Bioscience, Japan) and eluted at a flow rate of 1 ml/min. using water/acetonitrile/methanol (60:25:15, v/v/v) as mobile phase. The amounts of AFB₁ and AFG₁ were measured at a wavelength of 365 nm by comparison of the area under the curve (AUC) of unknown samples with authentic standards treated in the same manner. The retention times of AFB₁ and AFG₁ were 11.3 and 9.2 min, respectively.

Statistical analysis

Data on fungal growth and AF content were subjected to analysis of variance (one-way ANOVA) in Tukey range using a SPSS Version 10.0 Programme for Windows (http://www.spss.com/). Differences with P < 0.05 were considered significant.

Results

Plant characteristics and chemical composition of EOs

The general features of plants used in the present study are summarized in Table 1. A total of 49 medicinal plants belonging to 21 different families was evaluated in relation to their antifungal activities. The main EO constituents of the bioactive plants, i.e., Foeniculum vulgare, A. millefolium subsp. elborsensis, Ferula gummosa and M. spicata identified by GC-MS are summarized in Table 2 according to their retention indices (RI) and percentage composition. A total of 23 compounds were identified in the flower EOs of A. millefolium subsp. elborsensis, of which chamazulene (48.9%) was the main substance, followed by isoborneol (10.2%) and camphor (9.5%). Twelve compounds were identified in the leaves EO of F. gummosa with β-pinene (54.4%), guaiyl acetate (11.6%) and guaiol (9.1%) as the main constituents. Dillapiol (90.1%) was the main component of F. vulgare root EO, while trans-anethole (68.4%) was the principle substance of the plant flower EO. Among the 18 compounds identified in the leaf EO of M. spicata, piperitenone oxide (34.7%) was the main constituent, followed by cis-carveol (21.7%) and 1,8-cineole + limonene (11.3%).

Table 2 Chemical composition of Ferula gummosa, Achillea millefolium subsp. elborsensis, Foeniculum vulgare and Mentha spicata essential oils

Effect of plant EOs and extracts on A. parasiticus growth

Figure 1 shows the inhibitory effects of bioactive plant species on A. parasiticus growth without affecting AF production by the fungus. All the plant EOs and extracts inhibited fungal growth in a dose-dependent manner in the range of 4.21% to 100%. The maximum growth inhibition observed was at a final concentration of 2,000 μg/ml for all plants in the order of M. spicata (100%), T. fedtschenkoi n-hexane extract (99.58%), H. pubescens (99.10%), A. millefolium subsp. elborsensis (93.03%), T. fedtschenkoi EtOAc extract (92.04%), and F. gummosa (53.45%). The IC₅₀ values for these plants were reported as 35, 125, 370, 580, 720 and 1815 μg/ml, respectively. For all the plants except A. millefolium subsp. elborsensis, growth inhibitory activity was significant at concentrations higher than 31.25 μg/ml compared to appropriate controls (ANOVA, P < 0.05).

Fig. 1

Inhibitory effects of essential oils (EOs) and extracts from bioactive plants on Aspergillus parasiticus NRRL 2999 growth in microbioassay. Results are the mean ± SD obtained from two separate experiments in triplicate. * Statistically significant differences with a control (ANOVA, P < 0.05)

Inhibition of AFs B₁ and G₁ by F. vulgare EO

As shown in Table 3, EOs from the flowers, roots and stems of F. vulgare suppressed AF production by the fungus. The root EO inhibited both AFB₁ and AFG₁ production in parallel with a marked retardation in fungal growth. The maximum inhibition rates of fungal growth, and of AFB₁ and AFG₁ production were 65.66%, 99.48% and 99.62%, respectively. The growth inhibitory activity was significant at all concentrations except 15.62 μg/ml in comparison with controls (ANOVA, P < 0.05). Besides plant roots, a concentration of 2000 μg/ml of the plant flower and seed EOs was also examined. As indicated in Table 3, flower EO significantly inhibited fungal growth (68.58%) and production of AFB₁ (98.95%) and AFG₁ (99.81%) in comparison with controls (ANOVA, P < 0.05). The stem EO inhibited AFG₁ production significantly while not affecting fungal growth and AFB₁ production (Table 3).

Table 3 Inhibition of fungal growth and production of AFs B₁ and G₁ in A. parasiticus NRRL 2999 exposed to the EOs prepared from Foeniculum vulgare roots, flowers and stems

Inhibition of AFB₁ production by the EtOAc extract of P. orientalis leaves

Table 4 presents the biological activity of the EtOAc extract of P. orientalis leaves on A. parasiticus growth and AF production. The plant extract markedly suppressed AFB₁ production, while it did not inhibit fungal growth and AFG₁ production (data not shown), even at the highest concentration of 2,000 μg/ml. The inhibition of AFB₁ production was significant for all plant concentrations except 15.62 μg/ml, with a maximum of 89.48% at 2,000 μg/ml (ANOVA, P < 0.05). The IC₅₀ value for AFB₁ inhibition was calculated as 55.0 μg/ml. Surprisingly, fungal growth was enhanced by the plant extract at concentrations greater than 125 μg/ml and reached a significant level at 2,000 μg/ml, despite the potent inhibition of AFB₁ production by the fungus (Table 4).

Table 4 Inhibition of AFB₁ production in A. parasiticus NRRL 2999 by the ethyl acetate (EtOAc) extract of Platycladus orientalis leaves. Fungal growth was stimulated by the plant extract to a significant degree at the highest concentration of 2,000 μg/ml

Discussion

In the present study, a total of 49 medicinal plants belonging to 21 different families were evaluated for their antifungal activities. Antifungal activity of the EOs from A. millefolium subsp. elborsensis, Ferula gummosa, Foeniculum vulgare, and M. spicata, EtOAc extract of H. pubescens, and EtOAc and n-hexane extracts of T. fedtschenkoi against aflatoxigenic A. parasiticus was shown. The plants F. vulgare and P. orientalis were identified as potent inhibitors of fungal AF biosynthesis. All the bioactive plants except P. orientalis inhibited A. parasiticus growth in a dose-dependent manner to different extents. Based on the IC₅₀ values of the plant preparations, M. spicata was the most effective fungal growth inhibitor followed by T. fedtschenkoi, H. pubescens, A. millefolium subsp. elborsensis, F. vulgare and F. gummosa. To the best of our knowledge, this is the first report of antifungal activity of T. fedtschenkoi and A. millefolium subsp. elborsensis against aflatoxigenic A. parasiticus.

Plant EOs are composed of a wide array of chemicals that are characterized by two or three major components at high concentrations (20–70%) and other components present in trace amounts. Generally, the major components determine the biological properties of EOs (Bakkali et al. 2008). In the present study, β-pinene, guaiyl acetate and guaiol were identified as major components of F. gummosa EO; chamazulene, isoborneol and camphor as components of A. millefolium subsp. elborsensis EO; piperitenone oxide, cis-carveol and 1,8-cineole + limonene as components of M. spicata EO, and dillapiol (roots) and trans-anethole (flowers) as components of EO of F. vulgare. The majority of these compounds are monoterpenes (cineole, camphor, β-pinene, cis-carveol, piperitenone oxide, isoborneol) and the others belong to phynelpropanoids (trans-anethole, dillapiol), terpenoids (chamazulene) or cyclic terpenes (limonene). All the monoterpenes, trans-anethole and limonene are known for their inhibitory effects on the growth of various fungal species to different extents (Bakkali et al. 2008). Thus, they might be responsible for the antifungal activity of the corresponding plants against A. parasiticus demonstrated in the present study.

The results of the AF assay in the presence of plant EOs and extracts revealed more interesting data than that of fungal growth. Two species, i.e., F. vulgare and P. orientalis were found to be potent inhibitors of AF production by A. parasiticus. The EOs from F. vulgare roots and flowers inhibited both AFB₁ and AFG₁ production dose dependently, consistent with their inhibitory effects on fungal growth. F. vulgare Mill. (Fennel) is a perennial herb belonging to the Apiaceae family that is distributed widely throughout the world both as wild and cultivated species. Despite the existence of interesting data on the antifungal activity of different plant preparations (Kwon, et al. 2002; Mimica-Dukic et al. 2003; Singh et al. 2006; Napoli et al. 2010), no data is available about their effects on AF production by the producing fungi. Our results demonstrate for the first time a strong inhibitory activity of the EOs of both roots and flowers on production of AFs B₁ and G₁ by A. parasiticus in parallel with a marked suppression of fungal growth. The antifungal activity of F. vulgare toward A. parasiticus demonstrated in the present work may be accounted for by the presence of 1,8-cineol, limonene, trans-anethole and fenchone—the major constituents of F. vulgare roots and flowers, which have been reported previously to have similar activities. The main component of F. vulgare roots identified in the present study, i.e., dillapiol, is a phenylpropanoid responsible for pathway-specific inhibition of AFG₁ as previously reported by Razzaghi-Abyaneh et al. (2007). Limonene—a major constituent of F. vulgare flower EO—was previously identified as an inhibitor of AF production by A. parasiticus (Greene-McDowelle et al. 1999). However, it was not identified as a constituent of F. vulgare root EO in the present work. Hence, the active component(s) of root EOs of the plant causing AFB₁ inhibition require further characterization.

The potent inhibition of AF production by the EtOAc extract of P. orientalis leaves was interesting in relation to the fact that it significantly suppressed AFB₁ production with no effect on AFG₁ synthesis, and had a remarkable stimulatory effect on fungal growth. Platycladus orientalis is an evergreen coniferous tree in the cypress family Cupressaceae that is distributed widely from the West to the North of Iran. Leaves of this plant are commonly used in oriental traditional medicine. Although a wide array of biological activities have been reported for extracts prepared from different parts of the plant, little has been documented about plant antimicrobial activities (Lu et al. 2006; Chen et al. 2007; Wang et al. 2008). It has been shown that the flavonoid constituents, such as pinusolide, rutin, quercitrin, quercetin, myricetin, aromadendrin, amentoflavone and hinokiflakone, of P. orientalis leaves are responsible for its biological activities (Lu et al. 2006). The majority of these compounds are known as potent antioxidants in the sense that they are active toward free radicals such as reactive oxygen species. Thus, they may responsible for the plant-induced AFB₁ inhibition observed in the present study by a mechanism of suppressing the oxidative stress response to the toxigenic fungus. The growth stimulatory effect of P. orientalis for toxigenic A. parasiticus in parallel with the marked suppression of AFB₁ but not AFG₁ production reported here is a very promising result. This finding further substantiates the complex nature of the relationship between fungal growth as an index of primary metabolism and AF production as a hallmark of secondary metabolism in toxigenic fungi.

Overall, the results of the present study show clearly that bioactive plants, especially F. vulgare and P. orientalis, with strong inhibitory activity toward A. parasiticus growth and/or AF production, are potential targets for use as natural preservatives to control toxigenic fungal growth and subsequent AF contamination of foods, feeds and agricultural commodities. Further identification of the inhibitory compounds of these bioactive plants and a comprehensive ecological study to evaluate their effectiveness in the field are recommended.