**1. Introduction**

Mycotoxins, i.e., aflatoxins, are a type of fungal polyketide secondary metabolite that are produced mostly by *Aspergillus*, including *Aspergillus flavus* [1,2]. Currently, there are 18 types of aflatoxin produced by *Aspergillus* spp., of which the four principal kinds are Aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2) [3]. AFB1 is the most common mycotoxin in nature and is functionally carcinogenic in animal models as well as mammals if the toxicity level exceeds a certain threshold [4]. Aflatoxigenic fungi can cause damage loss in seeds growth, preservation, or viability [5]. Aflatoxin contamination primarily affects dried fruits (such as nuts and peanuts), cereal grains (maizes, etc.), some spices, and oils [6,7]. The primary sources of the world's exposure to aflatoxins are maize and peanuts because of their high consumption [8]. Consequently, solutions for the control of aflatoxigenic *A. flavus* in maize grains and food during storage are in demand worldwide [9]. Chemical treatments can effectively control aflatoxins, but they cannot be used on grains, cereals, or other food materials due to hazardous residues, teratogenicity, carcinogenicity, spermatotoxicity, and hormonal imbalances, as well as the development of resistance microbes against antimicrobial agents [10–12]. Currently, the use of plant-based natural antifungal agents is considered a beneficial and healthy practice in this regard [13,14]. Plant extracts could be employed as antimicrobial agents or for improved food storage and preservation due to their high activity, the simplicity of their production and utilization, their reliability, and their biocompatibility [15,16].

The release of large quantities of agro-by-product wastes such as peels and seed husks is one of the biggest problems facing society, as they are grave threats to the environment [17,18]. As a result, researchers continue to assess into the possibility of reusing these wastes. Such wastes encompass a wide range of compositions, including high levels of proteins, carbohydrates, and minerals. It was reported that many fruit peels offer a range of biological and medicinal properties and are known to contain them [19,20]. Pomegranate peels, lemon peels, and green walnut husks have been reported to be effective natural antimicrobials in various investigations [21–23]. Pomegranate peel extracts are high in functional molecules, such as flavones, phenylpropanoids, and alkaloids, which feature potent antioxidant properties [24,25]. Many investigators have reported the significant antifungal activity of pomegranate and eggplant peel extracts against many phytopathogens [26–29]. Linoleic acid is known to feature antifungal activity in larger plants as a substrate for producing a series of trihydroxy oxylipins [30]. The growth and biomass production of *Rhizoctonia solani* was reduced by 74% and *Pythium ultimum* by 65% when 1000 μM linolenic acid and allylphenol were applied together [31]. Several studies have reported that such natural compounds, including essential oils or extracts such as monoterpenoids and sesquiterpenes could suppress *A. flavus* growth and AFB1 formation by downregulating the transcription of genes involved in AFB1 synthesis [32,33].

Generally, AFB1 is produced from a complicated biosynthetic pathway, including at least 28 enzymatic steps. The structural genes encoding these enzymes are grouped in one gene cluster while two cluster-specific regulators, aflR and aflS, mainly regulate their expression [34,35]. It was reported that the decrease in the transcription levels of aflatoxin genes was associated with a reduction in AFB1 production. The ability of many plant-derived substances to stop AFB1 production or inhibit its expression has been observed [36–38]. Several investigations have shown that various doses of different plant extracts suppress the expression of 25 of the 27 studied genes in the AFB1 biosynthesis pathway [39,40]. The purpose of this study was to evaluate the effectiveness of different extracts of pomegranate, sugar apple, and eggplant peels to inhibit *A. flavus* growth; to test the ability of the peel extracts to suppress the expression of AFB1 biosynthesis genes in maize grains compared with Topsin fungicide; and to identify the different bioactive compounds of the best extracts using the GC-MS analysis technique.

### **2. Materials and Methods**

### *2.1. Fungus Isolation and Identification*

Aspergillus isolate was isolated from local maize grains, purified, characterized morphologically, and assessed for its ability to produce AFB1. The aflatoxigenic isolate was identified by sequencing the amplified ITS region [41,42].

### *2.2. Peel Extracts Preparation*

The pomegranate (*Punica granatum* L.), sugar apple (*Annona squamosa* L.), and eggplant (*Solanum melongena* L.) edible fruits were purchased from local markets in Alexandria Governorate, Egypt. All the fruits were washed and surface sterilized; the peels obtained, air-dried, and pulverized to a fine powder [43]. Twenty grams of the fine powder for each fruit peel was mixed with 100 mL of each of the four solvents: ethanol, diethyl ether, methanol, and acetone, with three concentrations, of 25%, 50%, and 75% (solvent/water, *v*/*v*). The preparations were left overnight on an orbital shaker (Heidolph, Schwabach, Germany) at 200 rpm. All the mixtures were filtered using Whatman No. 1 and stored in a refrigerator (at 5 ◦C) until further use.

### *2.3. Total Phenolics Content*

The Folin–Ciocalteau reagen<sup>t</sup> (FCR) assay (Sigma-Aldrich, Taufkirchen, Germany) [44], was used to determine the total polyphenols content (TPC), with slight modifications. A total of 0.1 mg/mL of extract was dissolved in distilled water. Next, 0.5 mL FCR (1 mol/L) and 1.5 mL of sodium carbonate (10% *w*/*v*) were added to 0.5 mL of each extract. The final mixture was kept for 30 min in the dark, and the absorbance values at a wavelength (λ) = 725 nm were measured. The TPC was calculated according to a standard curve using gallic acid prepared in methanol with 12.5, 25, 50, 75, and 100 μg/mL concentrations. The concentrations of TPC were expressed in milligrams of gallic acid equivalents per gram of dry extract weight (mg GAEs/g DW) [45].

### *2.4. DPPH Radical Scavenging Ability*

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (Sigma-Aldrich, Taufkirchen, Germany) was used to test antioxidant activity, and the capacities of the extracts to scavenge free radicals were determined as described by Asnaashari et al. [46]. The calculation equation was: (DPPH) % = [(Ab − Abs)/Ab] × 100 where Ab is the blank absorbance value and Abs is the sample absorbance value.

### *2.5. Gas Chromatography-Mass Spectroscopy Analysis*

Thermo Scientific ISQ Quadrupole GC-MS with Trace GC Ultra (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a capillary column TG–5MS (30 m × 0.25 mm × 0.25 μm film thickness) was used as previously described [47,48]. The separation conditions were performed as outlined by Okla et al. [49]. For the identification of the different compounds in the fruit peel extract samples, the retention times and mass spectra databases were compared to those of authentic standards.

### *2.6. Effect of Fruit Peel Extracts on Fungal Biomass and Aflatoxin Production* 2.6.1.FungalBiomassDetermination

Fifteen mL of potato dextrose agar (PDA) media was poured into a petri dish, and after the solidification, a 5 mm disc of the aflatoxigenic fungus was placed in the center of the PDA petri dish and incubated for 7 days at 30 ◦C. In total, 1 mL of each fruit peel extract, previously prepared as described in Section 2.2, was added to 50 mL yeas<sup>t</sup> extract sucrose (YES) broth in a conical flask. Next, a fungus disc was placed in each conical flask and set for 15 days at 30 ◦C. Each treatment's fungal mat was oven-dried. The wet and dry weights (g) of the fungal mat were recorded in all the treatments, and the filtrates were

maintained at 4 ◦C for later aflatoxin B1 analysis. The following equation was used to calculate the aflatoxin inhibition (AI) percentage ratio [50]:

$$(\text{AI})\% = \left[\frac{\text{AFB1 control} - \text{AFB1 treatment}}{\text{AFB1 control}}\right] \times 100\%$$

### 2.6.2. Maize Storage Experiment

Fifty grams of maize grains were treated with the fruit peel extracts, yielding the most significant results, as in the previous section. The treated grains were placed in sterilized-glass bottles. The fungicide treatment (2.5 mg/mL) was treated with Topsin (Thiophanate methyl, 70% wettable powder, United Phosphorus, Inc., King of Prussia, PA, USA), which was used as a control. Subsequently, each bottle was inoculated with a 5 mm disc of the aflatoxigenic fungus and kept for 30 days at 30 ◦C. The shape and odor of the maize grains were assessed after the storage period, using the scale developed by Youssef et al. [15]. All of the analyzed grains were crushed and refrigerated at 4 ◦C until they were used for further aflatoxin studies.

### 2.6.3. Aflatoxin B1 Extraction

Aflatoxin B1 (AFB1) was extracted by mixing 2 mL of fungal filtrate YES broth medium with chloroform (1:1 *v*/*v*). The mixture was centrifuged at 10,000 rpm for 5 min; a total of 2 mL of the bottom layer was transferred to a fresh glass vial. After evaporating under a moderate air stream, the dried chloroform extracts were re-dissolved with 1 mL methanol [51]. To extract the AFB1 from the contaminated maize grains, about 20 g of crushed grains was mixed with 100 mL of methanol and 12 mL of 4% potassium chloride (*w*/*v*), according to Hoeltz et al. [52], with some adjustments. The samples were filtered after a spin for 2 min at 10,000 rpm. The filtrate was then added to 100 mL of 10% ( *w*/*v*) CuSO4, mixed, and filtered. To extract the AFB1, 15 mL of an equal volume of chloroform and distilled water (1:1 *v*/*v*) was mixed with the filtrate in the separating funnel; this process was repeated twice. The solvent extracts were collected and evaporated. Before high-performance liquid chromatography (HPLC) analysis, all the samples were filtered into HPLC vials using a 0.2 m syringe filter (Thermo Fisher Scientific, Waltham, MA, USA).

#### 2.6.4. Preparation of AFB1 Standard and HPLC Conditions

To prepare the AFB1 standard (Merck, MO, USA), 1 mg was dissolved in 100 mL of toluene: acetonitrile (9:1, *v*/*v*) to obtain a final concentration of 10 μg/L. A working standard solution was prepared with a sample diluent (7% methanol + 92% 0.01 phosphate-buffered saline + 1% dimethylformamide) at concentrations of 5, 2, 1, 0.5, 0.2, and 0 μg/L [53]. The limit of detection and quantification for AFB1 as detected by the UV detector were 0.01 μg/L and 1 μg/L, respectively. Agilent HPLC (Santa Clara, CA, USA) was used to analyze the AFB1 using a Zorbax Eclipse Plus C18 column (4.6 mm 150 mm, 3.5 m) and a UV 365 nm detector. The mobile phase ratios were water, methanol, and acetonitrile (50:40:10, *v*/*v*/*v*). The flow rate was 0.8 mL/min, at ambient temperature, and the injection volume was 10 μL, with a concentration of 0.044 mg/mL [51].

### 2.6.5. RNA Extraction, cDNA Synthesis, and qRT-PCR Assay

The guanidium isothiocyanate technique was used to isolate whole-plant RNA, with certain modifications [54]. As previously described, the reverse transcription procedure was carried out [55,56]. The real-time PCRs (Qiagen Rotor-Gene Q2, Qiagen, Hilden, Germany) were carried out with designated primers targeting the aflatoxin biosynthesis pathway (Table 1). For normalization, the β-tubulin gene was served as an internal reference. As previously indicated, 20 μL SYBR Green qPCR reactions were performed [57,58]. The 2−ΔΔCT method was used to calculate the relative gene expression levels from the threshold cycle [59].


**Table 1.** Primer sequences were used in this study.
