*2.1. Antioxidant Activity*

Reactive oxygen species (ROS), formed in normal cellular metabolic processes or generated from exposure to ionizing or xenobiotic radiation, are held concausal factors in a large amount of chronic diseases. The toxicity of ROS is attributable to the ability of damaging essential biological substrates, such as DNA, RNA, proteins, and membrane lipids. ROS comprise superoxide radicals, lipoperoxide oxides, hydrogen peroxide, and hydroxyl free radicals [8]. An antioxidant is generally defined as a natural (fruit and vegetables) or artificial substance that can neutralize or protect a biological system from free radicals, such as oxygen, nitrogen, and lipid radicals [9,10]. These antioxidant properties make fruit and vegetables elements with good health properties, avoiding or decreasing the risk of su ffering from determined degenerative diseases [11–18].

Anthocyanins, phenols [19], and vitamins as A (14), C (15), and E (16) [20] confer the high antioxidant power to pomegranate as reported by several authors in both in vitro and in vivo models [21].

Some authors [22] state that the antioxidant capacity of phenolic compounds is a consequence of their ability to capture free radicals and their chelating ability of metal cations.

Gil et al. [23] reported that the antioxidant e ffect possessed by pomegranate juice is 3 times higher than that of red wine or green tea and 2, 6, and 8 times superior than that found in red berries, grapefruit, and orange juice, respectively.

In 2013, a parallelism of total phenolic content and antioxidant properties between several extraction solvents of pomegranate seed (PS) and pomegranate defatted seed (PDS) was carried out. Data revealed this trend, in decreasing order, for the used solvents regarding the radical scavenging activity methanol > water > acetone > butanol > ethyl acetate > hexane (EC50 antiradical potential amounting to 0.14 for PS and 0.19 μg/g for PDS). Similarly, the reducing activity test, decreed that methanol extract of PS an PDS possessed the greater reducing strength [24].

Derakhshan et al. in 2018 investigated the antioxidant activity and the total phenolic content of pomegranate peels, juice, and seeds from three regions of Natanz, Shahreza, and Doorak using as solvent ethanol. The best antioxidant activity was obtained by Doorak's seed and peel, as well as for the higher total phenolic content [25].

More recently, the analysis of five pomegranate juices genotypes (*Mollar*, *Kingdom*, *Dente di Cavallo*, and two old populations *Francofonte* and *Santa Tecla*) assessed that the total phenolic content ranged between 741.9 ± 55.8 and 424.2 ± 47.5 mg GAE/100 mL and the *Francoforte* genotype exhibited the higher amount. Furthermore, twenty-three phenolic compounds were detected. In particular, cyanidin-3,5-*O*-diglucoside and pelargonidin-3,5-*O*-diglucoside were the kind of anthocyanins present in all genotypes; the *Santa Tecla* population possessed the richest amount of these anthocyanins with values of 97.64 and 40.29 mgL−1, respectively, whereas in the *Francoforte* population, ferulic acid hexoside was the most abundant compound (391.18 mgL−1). The antioxidant activity values ranged between 221.5 and 36.73 μmol Trolox equivalents/100mL of juice and the higher one was observed for the *Santa Tecla* pomegranate population [26].

Preliminary studies performed by Bernabucci et al. explored the possible beneficial properties of pomegranate peel extracts (PPE) on bovine mammary epithelial cells BME-UV1. Their outcomes evidenced the ability of peel extract to decrease ROS production and malondialdehyde induced by the presence of hydrogen peroxide or lipopolysaccharide. Furthermore, PPE lowered pro-inflammatory cytokines expressions, exhibiting an anti-inflammatory effect on BME-UV1 treated with lipopolysaccharide [27].

A new and interesting study conducted by Hanani et al. addressed this issue from another perspective. Indeed, pomegranate peel powder was integrated into fish gelatin film-forming solution (FFS) with the aim to create an active packaging film. They found that the addition of 1% of pomegranate peel powder significantly increased the DPPH radical-scavenging activity to 59.74%, whereas the addition of 5% led to a percentage of 71.82%, and the fish gelatin film without pomegranate peel powder used as control achieved only the 53% [28].

Similarly, Bertolo et al. aimed to incorporate PPE at different doses to chitosan/gelatin gels. As a consequence, the antioxidant activity of the generated mixture was significantly improved. Moreover, concerning the rheological properties, PPE extended linear viscoelastic range and enabled the samples to easily flow under the applied shear rate boosting, in this way, the functional characteristics of chitosan/gelatin-based materials were improved [29]. In agreemen<sup>t</sup> with all these studies are the findings reported by Jalal et al., whose intention was to compare antioxidant activity of pomegranate peel and seed powder extracts through both DPPH and ABTS methods. The two assays evidenced the higher antioxidant capacity in peel powder samples with respect to seed powder samples. Furthermore, the methanolic extracts achieved the best results compared to water extract [30].

Surek et al. analyzed and compared the antioxidant activities of co-products from industrial pasteurized pomegranate nectar (PN), processing the peel (PP), press cake (PC), and precipitate after clarification (PAC) in comparison with raw material (arils) and final products (CON and PN) using DPPH, CUPRAC (Cupric ion reducing antioxidant capacity), FRAP (Ferric Antioxidant Power), and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) methods. PP has shown to possess the greater antioxidant capacity in all the assays. This effect could be due to the presence of a rich amount of phenols, flavonoids, and tannins, compared with the other tested extracts. Consequently, it is plausible to think that discarding the peel would cause a decrease of antioxidant potential [31].

In 2019, deeper analyses were conducted on the principal antioxidants enzymes (peroxisomal catalase and superoxide dismutase (SOD) isozymes) and the NADPH-regenerating system of pomegranate and their possibility to vary for different cultivar and genotypes. In the reported study, there were analyzed seeds and juices from two pomegranate varieties (*Valenciana* and *Mollar*) grown in two diverse Spanish locations. The evaluations of the isoenzymatic superoxide dismutase (SOD) activity pattern showed one Mn-SOD and five CuZn-SODs (I–V) whose richness depended on the variety while immunoblot assays exhibited at least one additional Fe-SOD with a subunit size of about 23 kDa in both varieties. Moreover, a strong metabolism of ROS could be due to the presence of the H2O2-scavenging peroxisomal catalase in seeds and juice [32].

The research works mentioned in this section have proven that the major factor responsible for pomegranate antioxidant activity is the presence of polyphenols and anthocyanins. In particular, it has been demonstrated that peel extract is the richest of these elements, which makes it suitable as a natural source of anti-free radicals.
