2.6. Phenolic Acid Composition
The compounds that present in the PGO are characterized by utilizing the well-defined peaks with maximum absorbance at 284 nm for phenolic acids. The inspection of chromatographic separations enabled us to identify 20 compounds (
Table 2). The quantitative determination of each compound was recorded. Gallic acid was the main component found in the PGO (784.13 ± 0.25 mg/100g), followed by Catechin (396.63 ± 0.45 mg/100g sample). To the best of our knowledge, this is the first time in which twenty phenolic compounds analytically were identified in the PGO oleoresin.
Different ranges of phenolic fractions, including chlorogenic, rutin, quercetin, protocatechuic, kaempferol, lutein, 4-hydroxybenzoic, vanillic, and vanillin were detected, while sinapic acid was not detected in the PGO. Nearly, most of these compounds investigated for their biological and pharmacological properties and literature data had described a range of interesting activities: antibacterial, antiviral, antioxidant, anticancer, anti-inflammatory, anti-aging, and hepato-protective. In recent decades, an increasing number of publications on phenolic compound bioactivity support these data. This finding demonstrates the importance given to understanding the biological efficacy behind these compounds, for instance, regarding their antioxidant, anticancer, antitumor, and cytoprotective activities, and analyzing the possible benefits to derive from their dietary ingestion, as well as, their pharmacological and therapeutic potential.
2.7. Fatty Acid Composition
The fatty acid compositions were determined where the results are shown in
Table 3. The PGO was rich in polyunsaturated fatty acids (PUFA) (87.51%). The PUFAs were essential fatty acids, namely, linoleic acid 18:2 (omega 6), linolenic acid C18:3 (omega 3), as well as a bioactive conjugated punicic acid (CLnA, omega 5), and eicosapentaenoic (20:5 omega 3) and were 5.24, 0.18, 81.29 and 0.8, respectively. Punicic acid was determined by comparing with the standard of the GC retention time and mass spectra. It was 81.29% of total fatty acids in pomegranate seed oils. This was following the previous reports on punicic acid in pomegranate seed oils [
13,
19].
The PGO contained an extreme amount of CLnA than other well-known CLnA-rich seeds, which recommended the PGO to act a reduced impact against cancer, tumors, and obesity [
20]. The total saturated fatty acids (SFA) of the present PGO recorded at 5.78% of total fatty acids. A total of 95% SFA in the PGO were palmitic acid (2.57%) and stearic acid (2.07%), which were higher than cultivars grown in Turkey and lower than Georgian cultivars [
13,
19]. Palmitoleic, oleic, vaccenic, gondoic, erucic, and nervonic acid were the monounsaturated fatty acid (MUFA) detected in the PGO and accounted for 6.71% of total fatty acids (
Table 3). The MUFA content in the PGO was comparable to that in Georgian and Turkish cultivars [
13,
19]. However, the PGO contained much less oleic acid than red raspberry seeds (12.4%), blueberry (22.9%), onion (24.8–26%), parsley (80.9–81%), cardamom (49.2%), and pumpkin (36.3%) [
21,
22].
2.11. Cytotoxicity and Protection Effect of the PGO
The cytotoxic effect of the PGO was investigated in vitro on an isolated murine hepatocytes cell line using the MTT assay. The powerful EC100 value indicates the high safety of the PGO. This result shows that no significant difference observed between the EC100 of the PGO, and these doses were higher. The assay results indicated that PGO failed to show any cytotoxic effect on the isolated murine hepatocytes cell line at the recommended dosage level, and the EC100 value was recorded at 0.0288 ± 0.002 µg/mL.
Moreover, the protective effect of the PGO against aflatoxin-induced hepatotoxicity showed in
Figure 3. The result illustrates that PGO possessed a higher significant protection effect (>63%) against aflatoxin-induced hepatotoxicity. The hepatotoxicity percentage in aflatoxin-exposed cells decreased by 63% when the AFB
1 was mixed with the PGO oleoresin before injected into the cell line media. In
Figure 3A, concentrations of the PGO, PGO+AFB
1, and AFB
1 lonely were applied in the cell line media. The AFB
1 is known to occur carcinogenicity and cell death, which appeared in the figure. The results have expressed the amelioration that happened to the cell viability by adding PGO oleoresin to cell line media. However, the application of just PGO oleoresin reflects a few impacts on the cell line viability. This result has confirmed in
Figure 3B, where the anti-hepatotoxic dose recorded as 0.028 and 0.184 µg/mL for the PGO and PGO–AFB
1 applied in cell line media, respectively. These results reflect the bioactive role of the PGO generally and against mycotoxins in a specific.
Based on the fact that plant extracts (polar or non-polar) possessed bioactive molecules in various amounts, which previously were reported by a capability to reduce mycotoxin excretion [
27,
28]. The obtained results will be discussed in this section to prove the synergized impact of PGO oleoresin in minimizing the mycotoxin formation. The minimizing action encouraged using the PGO, which occurred by the insertion of PGO in the fungal growth of liquid media. The mycotoxin decreases compared to the control reflect how the PGO components were synergized. This change in the condition of the growth media did not encourage mycotoxin secretion. The AFs are groups of health-hazard compounds, and also are classified as polyketide-derived [
29]. Their production relies on more than twenty genes clustered together in the DNA-sequence region. The genes encode a plurality of enzymes participatory in toxin synthesis as transcription agents [
30]. A nearer strategy of synthesis process was reported for the ZEA toxins, where it is also having a lacto-coumarin ring connected to their toxicity. Subsequently, the synthesis of enzymes related to these genes could be affected by the existence of PGO active molecules.
The impact of plant phytochemicals on the aflatoxin gene cluster of toxigenic fungi and the biosynthetic pathway of aflatoxin was reported in several previous investigations. For instance, the expression of aflatoxin genes was reported downregulating in a presence of essential oils [
31]. In addition, the phytochemicals, such as phenolic acids, are known to suppress gene expression in the aflatoxin pathway [
32,
33]. Moreover, the plant phytochemicals represent pivotal components that command the gene regulation in toxigenic fungi [
34].
In specific, oxidizing agents can induce AF biosynthesis [
35]. By the incidence of oxidative stress, the fungal molecules and enzymes are generated concomitantly to the AF biosynthetic gene cluster [
36]. Therefore, it was also proposed that toxin production could also be acting to blend oxygen atoms and defend cells from oxidative damage through fungal life [
37]. Otherwise, the plant phytochemicals of antioxidant molecules act as antifungals and affect the biosynthetic pathway of the aflatoxin gene cluster. It could serve out the limitation of aflatoxin biosynthesis by scavenging the factors that lead the fungi to feel oxidative stress, which may redirect the biological activity of fungi [
38,
39].
Indubitably, several plant types of enzymes had a capacity diminishing aflatoxin secretion due to the genetic impact on the AF cluster gene [
40,
41]. The effect of plant molecules on aflatoxin secretion varied according to the plant-active molecule. The AFB
1 altered in diverse behaviors, such as double-bond eradication from a furan ring plus the readjustment for the lactone-ring structure to reproduce less toxic compounds [
42].
Mycotoxins mainly destroy the cell system through their protein adducted or DNA-adducted action. The active form that reportedly attacks the cell-system is the 8–9 epoxide derivative [
29,
43]. This derivative is distinguished by the unsaturated bonds, which are classified as a principle step for cell mutations occurred. The fungal cells betake through several transactions during their life cycle. Pivotal stages are related to the fungal suffering of the stresses such as oxidative tension [
6]. This stress could procure fungal mutations leading to changes in hormone and enzyme creation of the cell life-cycle [
44,
45]. In addition, oxidative stress is considered a significant factor of aflatoxin synthesis by fungi; it mainly deems the principle reason for aflatoxin biosynthesis by the excitation of the aflatoxin gene cluster. Oxidative stress of fungal cells is the playmaker that causes the cell conversion to produce aflatoxin [
39,
46]. As well, the AFs’ creation by fungal cells requires specific enzymes, which are approximately 18 enzyme types that are significant to complete the AF creation steps [
38,
47]. Most of these creation steps depend on the monooxygenation reactions. The presence of bioactive components possessed an antioxidant function leads for suppressing the aflatoxin creation [
24,
39,
40]. This action was achieved by scavenging the free radicals, due to the PGO oleoresin bioactivity. The interaction between bioactive molecules presented by the PGO in growth-media and fungal metabolic reactions that lead to an inhibition of the mycotoxin production reflected as mycotoxin amount decreases. This action also may illustrate through the suppression of enzymes’ secretion of mycotoxin creation [
41].
Several physiological proceedings in fungi cells are organized by oxidative changes, including the secretion of secondary metabolism components. In particular, the presence of oxidants and free radicals in the growth media are capable encourages the biosynthetic of AFs molecules [
25]. In this regard, the abundant tocol derivatives (mainly gamma fractions) of oil can synergize antioxidant phenolics that also were existed in oil to change the condition media of fungal growth. These changes offer a favorable condition to fungi, which affected the fungal metabolism and converting to limit the mycotoxin formation.
In light of the model created by Kenne et al. [
47], the aflatoxin biosynthesis by the fungal cell was to protect their cells against the reactive oxygen species accumulation, since the mono-oxygenase reactions are the prime response that regulates the conversion into aflatoxin in fungal cells [
48,
49]. The occurrence of oxidative stress and its related factors deems the kickoff point that forced the fungi for the mycotoxin formation. In this case, the mycotoxin creation reaction will help the fungal cell to scavenge its content of the free radicals and also to mitigate the oxidative stress influences [
47,
50]. This point could explain the cell transformation between primary and secondary metabolism production, where the latter occurs under the condition of oxidative stress [
37,
51,
52]. The existence of the free-radicals in the fungal-media conducts the fungal strain to mitigate the unsuitable conditions and convert to excrete secondary metabolites [
47,
51].
It was pointed out that AF production could be a format to mingle atoms of oxygen to protect living-cell against oxidative damage [
23]. In this regard, if there is a source of other molecules that are available in fungal medium and assist the scavenging free-radicals from it [
37,
39,
53]. This action will be limited to the fungal oxidative stress, and the strain may be discontinued secretion of mycotoxins. The active molecules, which possess that action, was expressed by phytochemical antioxidants that are comprehensive the tocotromanols, phenolic acids, and carotenoids. These molecules are stimulated concomitantly and interact with the AF gene cluster throughout the AF biosynthesis process by the fungal cells [
22,
54]. This influence extended to the fungal cells for stopping secondary metabolites production or redirection to the vegetative growth [
24,
25,
55].
Technically, by the PGO additive to the fungal growth media of filamentous fungi, the contents of PGO oleoresin interacts and fuses into the fungal media content and offers wide varieties of bioactive molecules [
4,
16]. The phenolic antioxidants, tocopherols, and tocotrienols represent the principle scavengers, which when present in fungal media, could mitigate the oxidative stress action on the fungal growth [
3,
24,
39]. Moreover, it looks like PGO components represent a part of media micro-nutrients for the fungi, which affects the biochemical processes inside the fungal strain [
13]. The mechanism, where mycotoxin has secreted, was influenced by the PGO presence and is related to the interaction between the PGO antioxidant potency and the free radical in fungal media. The antioxidant activity resulted from antioxidant phenolics and tocols components that account for the mitigation of fungal oxidative stress, which is finally reflected as a decrement in mycotoxin production of AFs or ZEA compounds.