Antifungal Activity and Detoxification of Aflatoxins by Plant Extracts : Potential for Food Applications

Aflatoxins are secondary metabolites produced by fungi of the genus Aspergillus, which occur naturally in cereals like corn, beans and rice. Aflatoxin B1 causes an extensive number of toxic effects in animals and humans. This mycotoxin is a stable term and can act in low concentrations due to their higher toxicity. Management to prevent commodities aflatoxin contamination is essential during the production, mainly in preand post-harvest steps. A number of essential oils and aqueous plant extracts have been reported to be fungal growth inhibitors and may provide an attractive alternative to prevent aflatoxin contamination in foods. Thus, the aim of this review is to highlight recent data on the in vitro antifungal activity of essential oils and aqueous extracts from plants and discuss the perspectives of their use in food products.


INTRODUCTION
Aflatoxins are highly toxic secondary metabolites produced by fungi of the genus Aspergillus, mainly species A. flavus, A. parasiticus and A. nomius, which can develop naturally in food products such as beans, corn, rice and wheat, among others [1].There are currently more than 20 different compounds known as aflatoxin, although the most important types from a public health perspective are the aflatoxins B 1 (AFB 1 ), B 2 (AFB 2 ), G 1 (AFG 1 ) and G 2 (AFG 2 ) [2].AFB 1 is the main fraction produced by the fungi and has the highest toxicity, mainly targeting the liver, while also exhibits teratogenic, mutagenic and carcinogenic effects in several animal models [3].In 1987, the International Agency for Research on Cancer classified AFB 1 in Group 1 -human carcinogen [4].
The aflatoxins are chemically stable and heat-resistant, and may remain in the foods even after fungi have been removed by regular food treatment and preservation processes [5].Considering aflatoxins thermic stability and high toxicity, management to prevent commodities contamination are essential throughout the production.In this context, plant extracts and essential oils have been studied as fungal growth inhibitors and regarded as safe alternatives in the prevention of mycotoxins in foods [6 -10].Some aqueous plant extracts have chemically active compounds that inhibit the biosynthesis of aflatoxins [11], which have increased the scientific attention on these issues.A number of recent publications have shown the efficiency of plant extracts against Aspergillus and/or aflatoxin production.The objective of this review is to highlight recent data on in vitro antifungal activity by essential oils and aqueous extracts from plants and discuss the perspectives for using these compounds in food products.

ANTIFUNGAL ACTIVITY OF PLANT EXTRACTS CONTAINING ESSENTIAL OILS
Throughout evolution, plants have developed adaptation mechanisms as a response to stress caused by abiotic or biotic factors [12].Several essential plant oils are involved in the mechanisms of plant protection involving a range of antimicrobial effects in vitro [13 -15] and in vivo [16,17].Essential oils have low risk in the development of antimicrobial resistance and are nontoxic, being classified as GRAS (generally recognized as safe) by the United States Food and Drug Administration [18].Essential oils damage the enzymatic system of fungi cells by reducing the synthesis of proteins and structural compounds.The mechanism action is denaturing enzymes responsible for spore germination and interfering with amino acids involved in germination [19].Some compounds, such as monoterpenes and limonene, have been identified as potential inhibitors of pectin methylsterase, which is responsible for building the main components of the cell wall in fungi [20].The greatest difficulty of studies on the antifungal activity of essential oils depends on the sufficient production of the raw materials.In this context, different processes have been used for extraction, such as classical methods including hydro-distillation [7], effleurage [21], organic solvent extraction [8] and maceration [11].Laboratory-scale techniques like Clevenger distillation and innovative methods such as microwave and supercritical fluid also are being studied [22].
Studies attempting to understand the mechanisms of action and toxicological safety of the use of plants are still scarce, preventing more effective use of these compounds in agriculture, livestock production and the industry.Essential oils antimicrobial actions involve several chemical compounds found in plants, and this activity cannot be attributed to a single cell mechanism, but a set of them [23].Vilela et al. [24] tested Eucalyptus globulus (common eucalyptus) essential oil and its main component alone (1,8-cineol) against A. flavus and A. parasiticus.They observed that 1,8-cineol has lower antifungal activity than the essential oil.These findings suggest that other components found in lower levels in the oil may be critical for promoting synergism and enhancing the effects [25].
Essential oils have two different modes of action: reduced fungal growth or aflatoxin biosynthesis and secretion.Therefore, inhibition of AFB 1 production may not be completely attributed to reduced fungal growth.Tian et al. [26] demonstrated that 5 µL/mL and 4 µL/mL of essential oils from Cicuta virosa L. var.latisecta were sufficient for antifungal and anti-aflatoxigenic activities of A. flavus, respectively.However, another study [27] reported that the oil extract from Carum carvi L. was only effective in reducing aflatoxin inhibition, with no effect on A. parasiticus growth.Essential oils prepared from flowers, herbs, seeds, leaves and roots have different inhibitory activities against the growth of toxigenic fungi (A.flavus and A. parasitucus), and aflatoxin production.Table 1 presents an overview of plants containing essential oils from the most commonly studied plant species that have antifungal effects in vitro.Inhibition reported in those studies varied from 48 to 100%, with a huge variation among the extract's concentrations used in the assays (0.25 to 3,000 µg/mL).The variation in the results may be partially attributable to different processes used for extraction in each experiment, leading to different concentrations of essential oils in the extracts.However, strong antifungal activities at lower extract concentrations (0.25 to 5.0 µL/mL) were reported for extracts from Ageratum conyzoides [28], Carum carvi [54], Origanum vulgare [17] and Rosmarinus officinalis [17];
A. conyzoides (baume, chick weed) is a popular medicinal plant in Brazil.Therapeutic properties have been attributed to volatile, terpenes and coumarins, and non-volatile phenolic acids and flavonoids.Nogueira et al. [28] found nearly 48% inhibition of A. flavus growth in Sabouraud agar with 5.0 µL in a 6mm diameter of filter paper disk of the A. conyzoides extract.The component found in the highest concentration in the extract was precocene II (46.3%).Studies using Rosmarinus officinalis (rosemary) [29], which also has a large amount of precocene II, demonstrated important changes in the mitochondria and endomembrane system of fungal cells.The antifungal action of these plants is attributed to the presence of this substance, as well as its synergistic action with other compounds (e.g., carvacrol, in the case of rosemary).C. carvi (caraway) is an important member of the Apiaceae family, which includes carrot and fennel.Studies demonstrated that C. carvi lacks inhibitory activity against A. parasiticus growth, although it can affect AFB 1 and AFG 1 production [27].The main components of this plant are myristicin and dillapiole (two phenylpropanoids) which are responsible for the inhibition of aflatoxin production [29].
Plants in the family Lamiaceae have been drawing huge attention from the scientific community [30,31].They have two types of phenols, carvacrol and thymol.Studies have demonstrated that these substances are able to inhibit the growth of a wide range of microorganisms, including fungi and bacteria [15,32].These two classes of compounds affect the integrity of the cell membrane in microorganisms, thus changing the pH homeostasis and the inorganic ion balance [33].Carvacrol is found in the highest percentage in Origanum spp., ranging from 50 to 86% depending on the species [34].This plant has been widely employed for a long time as a therapeutic agent and as an antioxidant in the food industry.However, its antimicrobial and anti-aflatoxin characteristics are still issues that should be studied due to conflicting results described in different studies [14].
There is evidence that other plant extracts also inhibit Aspergillus spp.growth, although at much higher concentrations.Chenopodium ambrosioides (Mexican tea, wormseed) is used in the treatment of respiratory and digestive problems in Central and South America.It originated in Mexico and belongs to the Amaranthaceae family.Kumar et al. [35] demonstrated that the essential oil from its leaves (100 µg/mL) caused the lysis of mycelia and spores, thereby reducing aflatoxin production of two aflatoxigenic strains of A. flavus, Navjot 4NSt and Saktiman 3NSt (procured from Mycotoxin Laboratory, University of Bhagalpur, India).Foeniculum vulgare (fennel) has been widely known and used as a medicinal plant for many years.The roots of this plant contain approximately 90% dillapiol, which may be responsible for the inhibitory action of AFG 1 production [36].However, it has been reported that high concentrations (2,000-3,000 µg/mL) of F. vulgare extracts are required for growth inhibition of A. flavus and A. parasiticus [13].Alinezhad et al. [37] reported 99% inhibition of AFB 1 and AFG 1 production.The authors attributed the effects to the most prevalent components in the oil, 1,8-cineole, limonene, trans-anethole, and fenchone, whose antifungal activities are well described in the literature [20,24,36].The essential oil from Ocimum basilicum (basil) at a concentration of 3,000 µg/mL had fungistatic and fungicidal activity against A. parasiticus and A. flavus strains [13].These effects are attributed to the two substances found in the highest concentration in the oil, estragole (50%) and ocimene (11.2%), which are known for their antifungal activity [13].Soković et al. [15] carried out studies with the flowers, stems, leaves, roots and seeds of Daucus carota (wild carrot) and their potential inhibition effect on fungal and bacterial growth.Results showed that fungi are more sensitive than bacteria to the essential oils of this species of plant.The analysis of the main components of the oil showed two important terpenes in high concentrations (α-pinene and sabinene), to which the antifungal activity of the oil may be attributed.

EFFECT OF PLANT EXTRACTS CONTAINING ESSENTIAL OILS ON AFLATOXIN BIOSYNTHESIS
The results from several in vitro studies on plant extracts containing essential oils with inhibitory activity against aflatoxin production by Aspergillus species are presented in Table 2. Similar to the antifungal activity of plant extracts, the concentrations of the extract reported for total inhibitory effect against aflatoxin production varied markedly among different studies, from 0.1 to 2.5 µL/mL, or 100 to 50,000 µg/mL.[45] 1 Culture medium, temperature and time of incubation used in each assay. 2 Values refer to the plant extract concentrations that resulted in total (100%) inhibition of aflatoxin production. 3Values expressed in µL/mL.
A. conyzoides extracts showed the greatest inhibition capacity on aflatoxin production by A. flavus, at concentrations above 0.1 µg/mL [28].The essential oils from Lippia alba, a species belonging to Verbenaceae family, caused complete inhibition in vitro of the AFB 1 production at the concentration tested, 0.6 to 1.0 µg/mL [38].Cinnamomum tamala was effective against aflatoxin production by A. flavus at very low concentration, 0.75 µg/mL [39].In this study, the aflatoxigenic activity was attributed to the compound with the highest concentration in the plant, eugenol (45.6%).The leaves from C. zeylanicum, another plant also belonging to the family Lauraceae, and widely used as condiment and flavoring of cooked foods, had similar antifungal activity [40].The authors [40] also noted that high levels of eugenol were present in the leaf extracts (87.3%) and that 2.0 µL of the extract resulted in 100% inhibition of the fungal activity.
Cuminum cyminum (cumin) is one of the most known spices in the world, commonly found in oriental food preparations.Kedia et al. [32] investigated the applicability of these essential oils as an antifungal agent in foods and found that 0.5 µg/mL inhibited AFB 1 production.The same research group also demonstrated the potential of Mentha spicata (spearmint), a native species from Africa belonging to the family Lamiaceae.Although 13 compounds were identified in the essential oil composition of spearmint, the major components were carvone (59%) and the limonene (26%), and the minimum concentration of the extract to inhibit AFB 1 production was 0.9 µg/mL [41].
Curcuma longa, also known as turmeric, is a plant from the southeast of Asia.Food industries use this spice as a seasoning and coloring agent.It is also an essential ingredient of industrialized products, such as mustard.The synergistic and cumulative action of its main component (turmerone) promotes antimicrobial, antifungal, antiinflammatory, and antioxidant activity [42,43].Ferreira et al. [44] confirmed the complete inhibitory activity of plant extracts from C. longa on production AFB 1 and AFB 2 adding 0.5% (v/v) of the oil on YES medium was 96% and 98.6%, respectively.It has been assumed that the inhibition mechanism is based on cell lipid oxygenation and peroxidation caused by phenolic compounds.Investigations of the essential oil extracted from the fruits of Trachyspermum ammi (ajowan), an important member of the Apiaceae family, suggested a strong inhibition effect at 0.75 µL/mL on AFB 1 production, and it had no mammalian toxicity when tested in vivo in mice [45].These data highlight the potential of essential oil from T. ammi as a food preservative with anti-aflatoxigenic activity.
Studies carried out by Reddy et al. [11] have shown that aqueous extracts also have chemically active properties against aflatoxin production.These authors elucidated the effect of some dried plant extracts mixed with rice seeds on the production of AFB 1 , and found that extracts from Allium sativum (garlic), Ocimum sanctum (basil), Curcuma longa (turmeric), and Syzigium aromaticum (clove) at 5.0 g/kg concentration effectively inhibited aflatoxin production by 75%, 85.7%, 72.2% and 100%, respectively.The use of aqueous extracts is an attractive alternative compared with essential oils which need specific equipment to be extracted.In summary, the potential application of plants extracts has been attracting greater attention in the scientific community because they are biodegradable, environmental friendly, biologically safe, renewable and potentially low-cost [46].

PERSPECTIVES FOR USING ESSENTIAL OILS AND PLANT AQUEOUS EXTRACTS IN FOOD PRODUCTS
In the last 25 years, several studies have attempted to evaluate the efficacy of natural compounds from different species of plants, regional or not, against agricultural pests.Food products such as corn, rice, nuts, beans, wheat, peanuts and chickpeas are the most important agricultural commodities that are affected by fungal contamination and oxidative deterioration during processing, transportation and storage [12].There is scant information on methods for application of the plant extracts to effectively prevent the fungal development in foods.However, fumigation is one of the best methods to protect commodities against contamination during storage.Plant extracts fumigation has already been considered an eco-friendly alternative to prevent fungal growth and the production of mycotoxins [47].The vapor phases of purified essential oils or plant extracts preserve their bioactivity, hence making them highly effective fumigants for protecting stored products [12].
The application of essential oils as antimicrobial and antioxidant agents in food and food packaging has no safety restrictions because they are considered Generally Recognized as Safe (GRAS) by the Food and Drug Administration [48].Although the majority of studies evaluating the antifungal activity of plant extracts have been conducted in vitro or in vivo under laboratory conditions, a few experiments described the efficacy on some grains, fruits and vegetables.Reddy et al. [11] observed that fungal development and aflatoxin production were completely inhibited after the application of aqueous extracts from S. aromaticum on healthy seeds of a rice cultivar for five days.In another study, wheat and chickpea were stored for one year at 10-46°C and relative humidity of 30-90%.The samples were inoculated with A. flavus and then fumigated with essential oils from cumin seed.The percent protection from fungal development was 65.8% for wheat and 75.0% for chickpea [32].Chickpea was also used in an in vivo experiment to evaluate the efficacy of fumigant essential oils from Cananga odorata, Hedychium spicatum, O. majorana, Coriandrum sativum and Commiphora myrrha [16].The authors observed that incubation for six months at 28°C and 70% relative humidity resulted in 77.4%, 72.0%, 67.9%, 65.5% and 55.4% protection values, respectively.Tian et al. [49] evaluated the essential oil from Anethum graveolens (dill) as a potential food preservative.The study analyzed wound-inoculated cherry tomatoes with A. flavus and healthy tomatoes as in vivo models.The essential oil was sprayed and the samples were incubated for 9 days.As a result, fungal development was completely inhibited at 120 µg/mL of essential oil for the contaminated tomatoes and 100 µg/mL for healthy tomatoes.Feng and Zeng [50] demonstrated the efficacy of the Cassia fistula (golden shower) essential oil in vivo experiments.As an alternative to synthetic chemicals to control the Alternaria alternata postharvest contamination, this essential oil demonstrated results with concentrations of 300-500 µg/mL.Anžlovar et al. [51] showed another example of using essential oil with fumigation technique.Essential oil of thyme was tested for the protection of wheat grain from fungal colonizers (Alternaria alternata, Alternaria infectoria, Aspergillus flavus, Epicoccum nigrum and Fusarium poae).The fungitoxicity potential was good against all the strains tested.The highest tolerance for the essential oil was A. flavus.These results differ from those with the fumigation of Boswellia carterii essential oil on Piper nigrum fruits (black pepper) [47].The authors found an increased protection against A. flavus during a 6-month period of storage [47].
According to write of authors [52], the major obstacle to the use of essential oils in foodstuffs is the reproducibility of their activity caused by the variations in the bioactive components and their strong aroma which may restrict their applications.Despite this, some studies have demonstrated the efficacy of using plants incorporated without relevant negative effects in foods, as alternative additives with antioxidant and antimicrobial activities [53].Azizkhani and Tooryan [54] tested rosemary and mint extracts incorporated in beef sausage during storage at 4°C, and observed that both extracts were efficient against major spoiling microorganisms including yeasts and molds.Vilela et al. [55] demonstrated that application of essential oils from R. officinalis and Laurus nobilis (laurel) in the packaging of "Maronesa" beef was efficient in reducing spoilage caused by bacteria and fungi.The same study concluded that the addition of this oil also contributed to the fresh beef color, an important point for consumers.These results suggest the potential for further development of methods using essential oils and aqueous plant extracts as antifungal food preservatives.

CONCLUSION
The development of new reliable techniques to eliminate the aflatoxin contamination of foods and commodities is a very important task for the food industry, as well as for food production.In this context, considerable experimental research has demonstrated that essential oils and aqueous plant extracts inhibit the fungal development and/or the biosynthesis of aflatoxins, hence demonstrating a potential for their use in food products.However, there is limited information available on the use of essential oils or aqueous plant extracts directly on food commodities to prevent Aspergillus growth or aflatoxin production.Therefore, more studies are necessary to evaluate the potential application of plant extracts under field conditions, particularly on stored cereals and their manufactured products.As a first step, the procedures for preparation of plant extracts and/or essential oils need standardization.Additionally, further studies are necessary to identify the main active compounds of plant extracts and understand their mechanisms of action, as well to determine the safety levels for their use by the food or feed industry.Some practical aspects of using essential oils and aqueous plant extracts in food products also need to be investigated, especially their potential effects on sensory characteristics of foods, and their shelf life for the maintenance of antifungal properties under different environmental conditions.