3.3.1. AFB1 Production

Table 3 demonstrates that 50% diethyl ether eggplant peel extract was the most effective treatment, with the most negligible value of AFB1 (20.72 μg/L) and an AI of 96.11%, followed by 75% ethanol sugar apple peel extract with an AI of 94.85% (27.39 μg/L). Compared to the other treatments, the value of AFB1 in the 25% diethyl ether pomegranate peel extract was 112.64 μg/L. These findings are similar to those of Oliveira and Furlong [20], who found that the presence of 30μg phenol/mL agar eggplant bulb extract can reduce *A. flavus* AFB1 production by 87.80%.

**Table 3.** AFB1 production (μg/L) and aflatoxin inhibition ratios (AI) after the storage period correspond to peel extract treatments.


3.3.2. Grain Shape and Odor as Affected by Applied Fruit Peel Extract Treatments

Table 4 demonstrates that the tested fruit peel extracts and Topsin fungicide dramatically modified the grains' appearance compared to the control. The eggplant peel extract, followed by the sugar apple and the pomegranate peel extracts, demonstrated outstanding antifungal efficacy and a high-grain appearance compared to the control. The Topsin fungicide treatment of inoculated grains at authorized quantities resulted in rotting, deformation, foul odors, and unapproved grains. Our findings are consistent with those of Gemeda et al. [66] and El-Aziz et al. [67]. They reported similar reductions in *Aspergillus* fungal dry weight after using essential oils. Doum, banana, and licorice peel extracts exhibited similar decreases [15]. The findings were similarly consistent with those of Yazdani et al. [68] and Oliveira and Furlong [29]. They found that some phenolic compounds could reduce aflatoxin production AFB1.

**Table 4.** Grains' shape and odor correspond to peel extract treatments.


*3.4. Transcriptional Levels of AFB1 Biosynthesis Genes*

The biosynthesis of aflatoxin compounds, particularly AFB1, is a complicated enzymatic pathway [69]. AFB1 is generated in *A. flavus* from acetyl CoA by a 75 kb cluster of genes that encodes more than 18 enzymatic steps [70,71]. Such pathways are organized by different structural and regulatory genes [72]. These regulatory genes include many genes such as aflR and aflS, while structural genes contain more than 20 genes, such as aflD, aflG, aflH, aflI, aflK, aflM, aflO, aflP, and aflQ [73,74]. In the present study, the influence of the three fruit peel extracts, as well as Topsin fungicide, on the relative expression of two regulatory genes (aflR and aflS) and three structural genes (aflD, aflP, and aflQ) was investigated (Figure 1). The aflD enzyme is required to convert norsolorinic acid to averantin in the early stages of AFB1 biosynthesis. In the late stages of the AFB1 pathway, the aflP and aflQ genes encode enzymes that convert sterigmatocystin to o-methylsterigmatocystin and AFB1, respectively [75,76].

**Figure 1.** The expression levels of regulatory genes (aflR and aflS) and structural genes (aflD, aflP, and aflQ) of the AFB1 biosynthesis pathway. Columns with the same letters among each gene are not significantly different at *p* ≤ 0.05.

The untreated maize grains (infected control) demonstrated the highest relative transcription level of aflD (5.69-fold), followed by the Topsin-treated grains, with a relative expression level 2.29-fold higher than the control (Figure 1). In addition, the acetone 25% pomegranate, diethyl ether 75% sugar apple, and eggplant peel extract treatments showed decreasing transcriptional of aflD, with relative expression levels of 1.98-, 1.58-, and 1.48-fold, respectively (Figure 1). Similarly, the infected control treatment presented the highest relative expressions levels of aflP and aflQ, which were 4.91- and 7.81-fold, respectively, higher than the control (Figure 1). Interestingly, the two-aflP and aflQ genes exhibited the lowest relative expression levels, of 1.51-fold, with acetone 25% pomegranate and diethyl ether 75% eggplant peel extract treatments, respectively (Figure 1). The findings are consistent with those of Mayer et al. [77], who reported that the relative expression level of aflD in wheat experimentally infected with an aflatoxin-producing *A. flavus* was linked with AFB1 production and fungus growth kinetics. As a result, the expression of the aflD, aflP, and aflQ genes can support valuable distinguishing between aflatoxigenic and nonaflatoxigenic *A. flavus* strains [78,79]. Generally, the aflR and aflS are essential regulatory genes for AFB1 synthesis. Many reports assed significant correlations between the transcriptional levels of the two regulatory genes and AFB1 production [15,80]. The results showed the downregulation of two genes, aflR, and aflS, for all treatments compared to infected (untreated) controls. The diethyl ether 75% eggplant peel extract treatment exhibited the lowest aflR and alfS relative expression levels of 2.01- and 1.56-fold, respectively (Figure 1). The untreated infected control exhibited relative expression levels of 8.28- and 5.26-fold for aflR and alfS, respectively (Figure 1). Similar to the structural genes, it was demonstrated that the expression levels of aflS and aflD could be used to distinguish AFB1-producing *Aspergillus* strains from non-producing *Aspergillus* strains [81]. The results indicated that all the tested extracts exert an inhibitory effect on growth and aflatoxin production and could be used as antimycotoxigenic agents against AFB1-producing *Aspergillus* strains.

#### *3.5. Total Phenolics Content of the Fruit Peel Extracts*

The TPC was calculated as mg GAEs/g DW for each of the plant materials evaluated. The fruit peel extracts tested presented TPC levels ranging from 30.26 to 116.88 mg of GAEs/g DW (Table 5). The 25% diethyl ether pomegranate peel extract (116.88 mg GAEs/g

DW) featured the greatest TPC. The TPC of the 75% ethanol sugar apple peel extract was 35.09 mg GAEs/g DW, and 50% diethyl ether eggplant peel extract featured the lowest TPC value (30.26 mg GAEs/g DW). It was shown that TPC values in various 70% ethanol pomegranate peel fractions ranged from 78.10 to 200.90 mg GAE/g. Meanwhile, the ethyl acetate fraction featured the highest TPC, whereas the petrol-ether fraction presented the lowest. In the ethyl acetate fraction, gallic acid and ellagic acid were found in the highest quantities. However, in the water fraction, punicalin and punicalagin were found in the highest concentrations [82]. Manochai et al. [83] found that TPC ranged from 33.80 to 140.40 mg GAEs/g in ten Thailand sugar apple peel ethanol extracts, while Petch Pakchong cultivar featured the lowest TPC, of 33.80 mg GAEs/g. In another study, Ji et al. [84] found that the total phenolic content of eggplant peel water extract was more than four times higher than that of eggplant pulp. Malviya et al. [85] reported that the highest value of TPC was detected in a 100% aqueous pomegranate extract. At the same time, the lowest TPC was found in the 70% ethanol extracts. Phenolic contents are the most common secondary metabolites in plants. Their high antioxidant activities and significant impact in preventing oxidative stress-related disorders have drawn increasing attention [86].

**Table 5.** Total phenolics (TPC) and total antioxidant activity (TAA) of the fruit peel extracts.


### *3.6. DPPH Scavenging Ability*

DPPH is a method for evaluating the antioxidant potential of extracts and testing the ability of substances to serve as free radical scavengers or hydrogen donors [87]. For DPPH scavenging IC50 values, total antioxidant activities (TAA) were evaluated in all the investigated fruit peels. The results revealed that most of the extracted peels examined featured reasonably strong antioxidative activity values (Table 5). The highest TAA was found in the sugar apple peel extract (94.02 μg/mL), followed by 86.76 and 52.94 μg/mL in pomegranate and eggplant peels, respectively. The findings were similar to those of Ji et al. [84], who observed a higher amount of ascorbic acid in eggplant peel (51.88 mg/100 g). Meanwhile, Jayaprakasha and Rao [88]'s results suggested that methanol pomegranate peel extract offered the highest antioxidant activity among all the tested extracts in scavenging or preventive capacity against superoxide anion, hydroxyl, and peroxyl radicals [89]. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was found to be the highest for the methanol pomegranate peel extract and the 70% aqueous ethanol extract (79.50; 94.60), respectively [85]. Ten Thailand cultivars of sugar apple peel extracts featured antioxidant activity ranging between 0.42 and 3.06 mg/mL, and the "Nhur Thong" cultivar peel ethanol extract featured the highest antioxidant capacity [83].

### *3.7. Bioactive Compounds Identified in Fruit Peel Extracts*

The GC-MS analysis of the pomegranate, sugar apple, and eggplant peel extracts revealed many bioactive components in each extract. Table 6 presents the main extract compounds with peak area percentages (%) at different retention times (RT). The pomegranate peel extract included a total of 29 components. High relative abundance concentrations were observed in p-allylphenol (20.78%), 3,5 dihydroxy phenol (9.37%), linoleic acid (7.35%), xanthinin (7.05%), sorbitol (5.66%), ethylnorbornane (4.93%), levoglucosenone (4.76%), Dmannose (4.70%), α-himachalene (4.33%), and octadecanoic acid (3.65%). The sugar apple peel extract included a total of 32 components, with a high relative abundance concentration in α-fenchene (11.03%), octadecanoic acid (10.34%), alpha-kaurene (6.33%), hexestrol (5.87%), longipinene (5.83%), methyl isopimarate (5.67%), rhodopin (5.61%), valproic acid (5.04%), (S)-(-)-citronellic acid (4.76%), 4-methylcatechol (4.75%), and 1,16-hexadecanedioic acid (4.20%). As stated previously, the polyphenol content of the pomegranate peel extract

included ellagic and punicalagin compounds, or their derivatives [90]. The eggplant peel extract included a total of 43 components, with a high relative abundance concentration of alpha-kaurene (25.67%), p-allylphenol (8.50%), methyl isopimarate (6.01%), linoleic acid (5.22%), α-fenchene (4.67%), phyllocladene (4.29%), rhodopin (4.19%), octadecanoic acid (3.98%), dimrthoxydurene (3.76%), and (+)-beyerne (3.03%). The most relative abundance of octadecanoic acid, the ester of linoleic acid, which operates as a signaling molecule, was 10.34%, at an RT of 15.70 min.


**Table 6.** GC-MS phytochemical analysis of pomegranate, sugar apple, and eggplant peels extracts.


It was reported that linolenic acid and p-allylphenol (syn. chavicol) possess antifungal properties [91,92]. They may help to manage plant infections, such as *Botrytis cinerea*, which increases fungal oxygen consumption by 19.58% at 5 ppm and features fungicidal properties against various taxa, including *Alternaria* and *Sclerotinia* species [91,92]. Hydroxychavicol derivative found in betel leaf chloroform extract inhibited *Aspergillus* species with minimum fungicidal concentration (MFC) ranging from 125 to 500 μg/mL employed with broth microdilution method [93]. Similarly, several linoleic acid derivatives have been shown to inhibit the production of AFB1 by various *Aspergillus* species [68].

The second abundant phenolic compound found in the pomegranate peel extract was 3,5-dihydroxy phenol (syn., phloroglucinol). Acylated phloroglucinol is employed as an antifungal agen<sup>t</sup> against *A. flavus* and *A. niger* at a low minimum inhibitory concentration (MIC), 1.0 μg/mL [94]. Flavones, catechins, tannins, and quinines are phenolic compounds that interact with proteins and inactivate them by modifying their structure. Different phenolic compounds, such as dihydrochalcone and chavibetol, were identified from *Piper betle* extract, as reported by Ali et al. [93] and Yazdani et al. [68]. Polyphenolic compounds could halt the *A. flavus* production pathway of AFB1 by reducing norsolorinic acid accumulation, according to Hua et al. [95]. Pomegranate peel extract also contains xanthinin, which acts as a plant growth regulator and features phytotoxicity. It was purified from *Xanthium macrocarpum* fruit and evaluated against *A. fumigates*; it exhibited antifungal activity with MIC > 250 μg/mL. A possible metabolization leading to an unsaturated carbonyl group by eliminating an acetate ion could thus explain its antifungal activity [96]. Furthermore, the pomegranate and eggplant peel extracts contain the energy source linoleic acid, which is crucial for maintaining the membrane fluidity of the epidermis' transdermal water barrier. The biological inhibitory effect of hexestrol was investigated in a study by Inamori et al. [97], who found potent antifungal activity on the growth of plant pathogenic fungi, *Fusarium oxysporum f*. sp. *lycopersici* and *Botrytinia fuckeliana* with MIC 5 and 10 μg/mL, respectively.

Terpenes are the most diverse category of bioactive molecules identified in many plant extracts, with significant antibacterial activities that can be boosted synergistically through the interplay of multiple compounds (from the plant's crude extracts). The biochemical composition of these extracts varies based on the plant species and the plant part used [98]. The compound α-fenchene, also known as (-)-7,7-dimethyl-2-methylene bi-cycle [2.2.1]

heptane, was the most organic compound in sugar apple peel extract considered bicyclic monoterpenoids. The α-kaurene (ent-kaurene) was the most diterpene chemical compound detected in the eggplant peel extract. Low antimicrobial properties of ent-kaurene against *E. coli*, *P. aeruginosa*, *S. aureus*, *C. albicans T. mentagrophytes*, and *T. rubrum* were observed; in addition, no activity was observed against *A. niger* at 30 μg/mL [99,100]. Since monoterpenes, diterpenes, and sesquiterpenes were detected in the three studied fruit peel extracts, they could be effective alone or synergistically at killing fungi or preventing aflatoxin production in media or stored grains. Similarly, Bisht et al. [101] found that *Origanum vulgare* hydro-distilled oil (the main constituent is the oxygenated monoterpene *p*-cymene) strongly inhibited both fungi *A. flavus* and *A. niger*, with the highest inhibition zone of 30 mm. Linalool and citral terpenes are effective against *C. albicans*, and when combined with fluconazole, they produce tremendous synergistic action against a fluconazole-resistant *C. albicans* [102].

Even though eggplant peel extract includes a high amount of polyphenols, terpenoids, and fatty acids, it suppresses *A. flavus* AFB1 production by up to 95%. By contrast, a combined treatment (low dosages of pomegranate peel extract and the azole fungicide prochloraz) resulted in the total prevention of toxin synthesis over 72 h. A qRT-PCR analysis revealed the downregulation of most aflatoxin biosynthetic cluster genes [61]. Youssef et al. [48] reported that phytochemical compounds, such as fatty acids or their esters (octadecanoic acid, n-hexadecanoic acid, and hexadecanoic acid methyl ester), as identified in beetroot extracts, offered potential activity against the mycotoxin produced by *Alternaria alternata*. Overall, these findings sugges<sup>t</sup> that fruit peel extracts could be promising as efficient and sustainable green sources of antioxidants, inhibit aflatoxin production, and potentially become protective grain storage saver applications instead of the chemicals currently used.
