Control of Aflatoxin Production Using Herbal Plant Extract

The aflatoxins are a group of chemically similar poisonous, carcinogenic fungal second - ary metabolites produced by Aspergillus flavus , A. parasiticus , and A. nomius , which are abundant in warm and humid regions of the world. They are probably the most inten sively researched toxins in the world due to their carcinogenic and mutagenic effects. Aflatoxins have also been identified as a potential biological weapon for food and water contamination. The four major aflatoxins commonly isolated from different foods and feed stuffs are AFB1, AF B2, AFG1, and AFG2. Aflatoxin contamination of food and feed has gained global significance as a result of its deleterious effects on human as well as livestock health including gastrointestinal dysfunction, reduced feed utilization, anemia, jaundice, liver damage and immunity suppression. The profitability and marketing of various agricultural products are adversely affected by either contamination of aflatox - ins or aflatoxin‐producing fungi. The foods at highest risk of aflatoxin contamination are maize, chilies, peanuts, and cotton seeds. There are various physical, chemical, and natural methods investigated to prevent aflatoxin production and the growth of afla toxin‐producing fungus in various agricultural products. Here, we describe various natu - ral plant extracts that would be potential source of controlling aflatoxin production in agricultural products.

The aflatoxin was a combination of three words: first letter "A" from genus Aspergillus, next three letters "FLA" from species flavus, and the noun "TOXIN" [16]. Aflatoxins are quite stable and found resistant to degradation. Among the 18 different groups, aflatoxins B1, B2, G1, G2, M1, and M2 are the major classes and derivative of bifuranocoumarins. The aflatoxins B1 and B2 give blue color, while G1 and G2 give a yellowish green color under UV light. Aflatoxins M are hydroxylated derivatives of aflatoxins B and first isolated from milk. A. flavus produces only AFB1 and AFB2, but it is also able to synthesize cyclopiazonic acid. However, A. parasiticus produces AFBI, AFB2, AFG1, and AFG2 [17,18].
The International Agency for Research on Cancer (IARC) classified AFB1 as class I human carcinogens [19] and have a positive association between dietary aflatoxins and liver cell cancer (LCC). This was the third leading reason of cancer death around the world [20]. The cytochrome p450 metabolized AFB1 in their epoxide form. Depurination occurs, when epoxide reacts with DNA or RNA. That will obstruct DNA and protein synthesis in active tissues of bone marrow, intestine, and liver. The order of toxicity of aflatoxins is AFB1 > AFB2 > AFG1 > AFG2 [21], and the critical point, which determined the biological activity of this group of mycotoxins, is terminal furan moiety of aflatoxin [22]. In cereal and their derivatives, maximum residual limits (MRLs) of aflatoxins are 2 μg kg 1 for AFB1 and 4 μg kg⁻ 1 for the sum of four aflatoxins. In processed cereal-based foods and baby foods for infants and young children, the level of AFB1 is 0.1 μg kg -1 . These values were recommended by the European Union Commission Regulation (EC) [21]. According to the Food and Drug Administration (FDA), the safe limit of aflatoxins is 20 ppb (Figure 1) [23].
In developing countries, about 4.5 billion people are chronically exposed to uncontrolled amounts of aflatoxins [24]. Consumption of contaminated products causes aflatoxicosis in humans and animals. Aflatoxicosis may be acute and chronic. Acute condition caused death, while chronic condition results in immune suppression and cancer. In human, it is characterized by vomiting, abdominal pain, pulmonary edema, convulsions, coma, and death with cerebral edema and fatty involvement of the liver, kidneys, and heart [25]. Due to aflatoxicosis, in Kenya about 215 people died in 2004 [26][27][28]. In animal, aflatoxicosis is characterized by gastrointestinal dysfunction, reduced feed utilization, anemia, jaundice, liver damage, decreased milk and egg production, and immunity suppression [29]. In plants, AFs retarded seed germination, seedling growth, and root elongation. It also inhibits chlorophyll, carotenoid, and some enzymes synthesis [30].
Although A. parasiticus and A. flavus are related fungi, they are different from each other on the basis of their color and length of conidiophore. Sterigmata were the main characteristics, which differentiate the two Aspergillus species. The sterigmata of A. flavus were biseriate as compared to A. parasiticus, which has uniseriate sterigmata [31]. In 2006, Cary and Ehrlich reported that about 12 A. flavus groups are 96% similar to A. parasiticus. Another character that distinguishes the two fungi species is their adapted environment. The A. flavus acclimated to aerial and foliar environment, mostly prominent in tree nuts, corn, and cottonseed, while the A. parasiticus adapted to soil environment and dominated in peanuts [32]. A. flavus exists in two forms: one is the S type, while the other is the L type on the basis of morphological, physiological, and genetic characteristics [33]. On average, S-strain isolates produce much more aflatoxins than L-strain isolates [34]. S strain synthesized frequently small sclerotia that are less than 400 μm and processes lesser conidia as compared to L-strain isolates whose sclerotia sizes are greater than 400 μm [34,35].The members of genus Aspergillus mostly contaminate agriculture commodities in tropic and sub-tropic region. Contamination may occur at different stages such as in pre-harvesting stage, harvesting stage, post-harvesting stage, or in storage and transportation stage. In pre-harvesting, the field fungi attack on growing crop because of different reasons. It may be the environmental stress (hot and dry condition and soil moisture), mechanical damage (by arthropods, birds, rodents, and nematodes), or delayed harvesting. While in post-harvesting, contamination occurred due to improper drying, storage in polythene bags, damage during shelling, or storage in poorly ventilated warm environment.
Contamination rate of aflatoxin depends upon humidity, temperature, storage, and soil conditions [36]. Optimum condition for fungal growth in cereal is moisture content about 18% (equal to 85% relative humidity) and temperature about 12-42°C with an optimum at 27-30°C in tropical and sub-tropical areas [37]. An important point to be considered was the time of incubation that effects the production of toxin by Aspergillus species [38].
Optimum duration for the production of aflatoxins was 14 days of incubation at 30°C. When the length of incubation time increased, there will be reduction in aflatoxin level because of re-adsorption or degradation by fungus [39]. The fungal growth is effected by 20% CO 2 and 10% O 2 level [40]. The metals such as manganese and zinc are crucial for aflatoxin production. But the mixture of cadmium and iron mixture reduces the mold growth and aflatoxin synthesis [41].
The infectious cycle of Aspergillus species is mostly dependent upon host species. Overwinter fungus developed either mycelium or sclerotia (resistant structure) that have the ability to grow on soil surface [42,43]. Under favorable condition (high temperature and moisture level) in summer, it either produced hyphae or conidia (asexual spores). Through air or insects, conidia spread in soil and on silk and kernels and contaminate agriculture commodities (Figure 2) [44].

Aflatoxin-Control, Analysis, Detection and Health Risks
Aflatoxin contamination is inescapable due to health hazards in human and animal, crops deterioration, and economical loses. In the past, many strategies (physical, chemical, and biological) are used to avoid aflatoxin contamination. Physical strategies usually used are rodent-proof room, cold storage of feeds with less than 100-g/kg moisture level, use rapid drying and gamma radiation, and so on. In chemical strategies, propionic acid, acetic acid, benzoic acid, citric acid, hydrogen peroxide, copper sulfate, and ammonium hydroxide are used to inhibit the growth of fungi and aflatoxin production. But the formation of toxic residue by chemical treatment was the main concern that causes potent health problems. As compared to chemical, physical practice is a healthier option but it is slow processes. Other strategies used were the biological control in which different microorganisms such as bacteria, yeast, and non-toxic stain of A. flavus and A. parasiticus were used to detoxify aflatoxins by microbial binding and biotransformation [45][46][47][48]. This is a laborious and costly process. Therefore, to avoid potential risk, the use of safe, renewable, and biodegradable natural plant extracts to remove aflatoxin contamination [49] is required.

Effect of active ingredients of medicinal plants on aflatoxins producing fungus
Modern research found that phytophenols as plant secondary metabolite existed above 8000 structures. These structures resemble with tannin and phenolic acid [50]. Phytophenols showed antiallergenic, antioxidant, anti-inflammatory, antimicrobial, antiorthrogenic, and antithrombotic activity [51]. These plant compounds exhibited key biological activity in the degradation of many microorganisms [52]. Plants, herbs, essential oils, and spices in powder or extracts form are used to detoxify microbes due to the presence of flavonoids, betalain, phenolics, phytoalexins, and thiosulfonates. But mostly antimicrobial and antioxidant activities of plant extracts were due to their phenolic alignments [53].
A recent study exposed the antifungal and antiaflatoxigenic nature of phenolic components of plant extracts [54][55][56]. The syringaldehyde, sinapic acid, and acetosyringone were the plant phenolic compounds that inhibited the production of aflatoxin B1 [57]. However, salicylic acid, thymol, vanillyl acetone, cinnamic acid, and vanillin were phenolic compounds that ceased A. flavus growth by targeting oxidative mitochondrial stress as defense system [58].  [61]. The above study found that ethanolic extracts of some medicinal plant showed the inhibition of aflatoxins producing fungus.
The highest activity was showed by betel vine, a traditional Thai medicine, followed by false coriander, Indian mulberry, Chaa Phluu, Chinese radish, and clove. The leaf of betel vine is used topically for urticaria, contains eugenol and chavicol, and mostly chewed by mouth as antiflatulent, antimicrobial, and antipruritic [62].
Crude ethanolic extract of olive callus in different ratios was used to inhibit the aflatoxins synthesis [63] by the addition of appropriate amounts of extracts onto potato dextrose agar (PDA) to obtain the final concentration of 0.5 and 1%, and Aspergillus was then point-inoculated into PDA.
The results showed that ethanolic extract of olive callus had no inhibitory effect on fungal growth but it reduced 90% of aflatoxin synthesis. The main compounds in olive callus are reported as caffeic acid, coumarin; o-, p-, or m-coumaric acid and catechin which facilitate the reduction of aflatoxin. Only o-coumaric acid and caffeic acid showed antifungal and antibacterial activity.
Various concentrations (0, 2, 4, 6, 8, and 10% (w/v)) of clove, garlic, and carrot's crude aqueous extracts were tested for their possible inhibitory effect on Aspergillus growth and aflatoxin production in 50 g of rice. The results showed that garlic and clove at 10% (w/v) and carrot at 2% inhibited the Aspergillus growth and also reduced the level of aflatoxin production in rice [64]. Crude extracts of garlic, eugenol, and onion were used to reduce A. flavus growth as well as aflatoxin synthesis in maize and SKMY liquid medium [65]. The study showed that garlic extract inhibited 61.94% fungus growth. However, onion extract ceased about 60.44% aflatoxin synthesis. While on maize grain, eugenol extract reduced 60.35% aflatoxins synthesis. Hussain and Ali [48] compared the antifungal activity of some herbal spices, chemicals, and plants to inhibit the growth of aflatoxins producing fungus like A. flavus and A. parasiticus. They found that benzoic and propionic acid showed complete inhibition of A. flavus at (0.1-0.5%) and A. parasiticus at (0.2-0.5%), while clove (0.5%), garlic (0.5%), and onion (0.5%) showed complete inhibition of both Aspergillus.
The aqueous and phenolic extracts of several other natural and medicinal plants have been tested against Aspergillus [66]. Aqueous extracts of Lupinus albus (Leguminosae), Ammi visnaga (Umbelliferae), and Xanthium pungens (Compositae) were found to cease the growth of A. flavus and also the production of aflatoxin [67]. It was also found that the inhibitory effect was proportional to the applied concentration.

Role of essential oils on the inhibition of aflatoxins producing fungus and its production
The search for naturally occurring compounds or metabolites having bioactivity against aflatoxins producing fungi has been the target of interest in the search for ecologically friendly products [68]. There are many essential oils produced by medicinal plants that have been tested for their inhibiting ability of aflatoxin production [69,70].
Essential oils were extracted from 16  Different ratios (50, 25, 12.5, 6.25%) of each essential oil were placed onto a cylinder cup (6 mm dia) on agar plate streaked with A. flavus. It was observed that the essential oil extracted from white wood showed the highest inhibition followed by the essential oils of cinnamon and lavender, respectively. Sindhu et al. [72] used Curcuma longa leaves essential oil of 0.01, 0.05, 0.1, 0.5, 0.75, 1, and 1.5% concentration in YES broth that was inoculated with A. flavus spores. C. longa oil of 1 and 1.5% concentration reduced 95.3 and 100% aflatoxin (AFB1, AFG1) synthesis, respectively. They analyzed α-phellandrene, terpinolene, and p-cymene as an active compound in turmeric leave oil extract by gas chromatography-mass spectrometry (GC-MS).
Mahmoud [73] also used 0.01% of five essential oils namely geraniol, nerol and citronellol (aliphatic oils), cinnamaldehyde (aromatic aldehyde), and thymol (phenolic ketone) to suppress the Aspergillus growth. The result showed the complete inhibition of A. flavus growth.

Conclusions
Despite all efforts, it has been very difficult to control the exposure of man and animals to aflatoxins, because of their natural occurrence in the environment. Although the prevention of aflatoxin contamination by inhibiting the fungal growth in food and feeds is the best practice, other measures are also necessary. The advantage of using active compound based on natural plant is that they are safer, ecologically friendly than any chemical compounds, and synthetically produced antimicrobial agents. Other procedures such as the removal or decomposition of aflatoxins are also necessary as the prevention of contamination alone may not always be successful.