*2.6. Determination of Antioxidant Activities in Pomegranate Peel*

The antioxidant activity against DPPH radical (2,2-diphenyl-1-picryl-hydrazyl-hydrate) was determined with the method reported in Muscolo et al. [25]. The DPPH concentration in the cuvette was chosen to give absorbance values of ∼1.0. Absorbance changes in the violet solution were recorded at 517 nm after 30 min of incubation at 37 ◦C. The inhibition I (%) of radical-scavenging activity was calculated as:

$$\text{I (\%)}=\text{[(A0 -- AS)/A0]} \times 100\tag{1}$$

where A0 is the absorbance of the control and AS is the absorbance of the sample after 30 min of incubation. Results were expressed as μmol Trolox/g DW.

The 2,2 -azino-bis-3-ethylbenzothiazoline-6-sulfonic acid assay (ABTS) was carried out according to Muscolo et al. [25] using a solution of 7 mM of ABTS in phosphate buffered saline (PBS). Aliquots of ethanol extracts (25, 50, and 100 μL) were added to 0.5 mL of ABTS+• solution and brought to a final volume of 600 μL with PBS. After 6 min of incubation in the dark at room temperature the absorbance of the samples was measured at 734 nm. Results were expressed as μmol Trolox/g DW.

The total antioxidant capacity (TAC) was performed according to Muscolo et al. [25]. Sample absorbance was measured at 695 nm using UV-Vis spectrophotometer. Methanol (0.3 mL) in place of the extract was used as blank. The antioxidant activity was expressed as μg of α-tocopherol g−<sup>1</sup> DW on a calibration curve.

#### *2.7. RP-DAD-HPLC Identification of Phenolic and Flavonoid Components*

Pomegranate peel was finely ground for analysis. By-product samples were subjected to solvent extraction before HPLC analysis for determination of the single phenolic and flavonoid compounds. Each sample was extracted in two different ways—0.1 g of previously lyophilised pomegranate peels was dissolved in 10 mL of 1% of HCl in methanol and 0.1 g of sample was dissolved in 10 mL of acetone solution: 1% of HCl in methanol (1:1). Each sample was analyzed in six independent replicates [25]. Reverse-phase–diode array detector–high-performance liquid chromatography (RP-DAD-HPLC) analyses of samples was carried out with a Shimadzu system (Kyoto, Japan), consisting of an LC-10AD pump system, a vacuum degasser, a quaternary solvent mixer, an SPD-M10A diode array detector, and a Rheodyne 7725i injector (Merck KGaA, Darmstadt, Germany). Separation of each compound was done on a 250 × 4.6 mm i.d. 5 μm Discovery C18 column supplied by Supelco Park (Bellefonte, PA, USA) and equipped with a 4.0 × 20 mm guard column. The column was placed in a column oven set at 25 ◦C. The injection loop was 20 μL and the flow rate was 1.0 mL/min. The mobile phase consisted of a linear gradient of solvent A (acetonitrile) in 2% acidified water (acetic acid:H2O, 2:98) as follows: 0–80% (0–55 min), 90% (55–70 min), 95% (70–80 min), 100% (80–90 min), and 0% (90–110 min). UV-Vis spectra were measured between 200 and 600 nm and simultaneous detection using a diode array at 278 and 325 nm. Compounds were measured using their retention time and UV spectra (Dueñas and Estrella, 2002), through comparison with purified standards (Sigma Chemical Co., Saint Louis, MO, USA).

#### *2.8. Statistical Analysis*

Analysis of variance was carried out for all the data sets. One-way ANOVA with Tukey's Honestly Significant Difference tests were carried out to analyze the effects of treatment/cultivar on each of the various parameters measured. ANOVA and a *T*-test were carried out using SPSS software (IBM Corp. 2012, New York, NY, USA). Effects were significant at *p* ≤ 0.05. To explore relationships among different treatments/cultivars and chemical parameters, datasets were analyzed using principal component analysis (PCA).

#### **3. Results and Discussion**

Results evidenced that total carbohydrates were contained in a higher quantity in peels of other cultivars than in peels of Wonderful. The lowest carbohydrate content was found in spray-dried Wonderful peel (PSD) (Figure 1). Total proteins had an opposite trend, with the lowest in the peels of the other cultivars and the highest in PSD (Figure 1A). Total phenols and total flavonoids were present in the highest quantity in the Calabrian Wonderful spraydried peels and in the lowest amount in the other cultivar peels. Total phenols were higher than total flavonoids in all the samples analyzed (Figure 1B). These data evidenced that the Wonderful cultivar had the majority of total phenols and flavonoids. These data highlighted that the geographic conditions, in which a determined type of cultivar grows, can drive the synthesis of bio-compounds, shifting the metabolism from primary to secondary. Data from Ramakrishna and Ravishankar [29] showed how drought conditions increased, in different plants and in different part of the plants, the amount of total flavonoids and phenolic acids that were used as antioxidants to overcome stress conditions. Vaneková et al. [30] showed how environmental factors such as altitude, habitat type, and sunlight exposure influenced the synthesis of total phenols and flavonoids in seven different cultivars of berries. The distribution of drylands is quite accentuated in southern and Mediterranean countries, and Calabria in particular is dominated by climate conditions mainly characterized by dry summers and mild wet winters that, as demonstrated by Fialho et al. [31], in pomegranates increased secondary metabolites with nutraceutical properties. The data of this research are in line with literature findings and highlight that the major quantity of total phenols and flavonoids contained in Wonderful peel cultivated in Calabria could be the result of the microclimatic conditions, which in turn affect metabolism, increasing antioxidants as well as antioxidant activities, and the soluble protein amount, which can have a double function of working as osmolytes or as antioxidative enzymes, as demonstrated by Kosová et al. [32]. In the spray-dried peel, the greatest amount of these compounds was found, evidencing that the innovative methodology used to dry the peels did not denature the bio-compounds but rather concentrated them. Vitamins were contained in greater amounts in the Wonderful cultivar than in the other cultivars, and were more concentrated in PSD and CFD. Vitamin E was the most abundant in all the pomegranate peel samples, except for the peel of the Indian cultivars. Vitamins have a great role as antioxidants and have important health benefits when consumed with the diet. The PSD contained a huge amount of vitamins (Figure 2). It was demonstrated that vitamins are enzymatic cofactors and act as antioxidants. Vitamin C increases under stress conditions to protect plants from oxidative stress by acting to detoxify reactive oxygen species (ROS) by direct scavenging or by acting as cofactors in the enzymatic reactions that involved ascorbate peroxidase and glutathione reductase enzymes [33]. Vitamin E, which is the most abundant vitamin, is a major single oxygen scavenger that provides protection against lipid peroxidation [34]. In support of the above findings, the activities of the antioxidant enzymes were greater in PSD than in the other samples. All the activities (DPPH, ABTS, and TAC) were expressed more in PSD and CFD than in the peels of the other samples (Figure 3). These data evidence that the innovative dry process did not affect the biological compounds and the enzymes.

Single phenolic acids were higher in Wonderful peels than in the peels of the other cultivars. In PSD, the greatest amount of single phenolic acids was detected (Table 1). Ellagic acid was the most abundant, followed in ranking by 2-5 dihydroxy-benzoic, gallic, protocatechuic, *p*-coumaric, chlorogenic, and ferulic acids. It has been widely demonstrated that ellagic acid (EA) is a potent antioxidant with antimicrobial, anti-inflammatory, neuroprotective, antihepatotoxic, anticholestatic, antifibrogenic, anticarcinogenic, cytotoxic, and antiviral effects [35]. Recently, Reis Jordão et al. [36] evidenced that ellagic acid can be a promising alternative treatment for hypertension and cardiovascular disease, and Pei et al. [37] demonstrated that EA can be used to prevent diabetic cardiac dysfunction. The doses of EA generally tested in the prevention health treatments were 30 mg/kg. PSD peel contained a great amount of EA (240 mg/g), suggesting its possible use as a nutraceutical supplement for the prevention of numerous diseases. Additionally, gallic, 2-5 dihydroxy-benzoic, protocatechuic, and ferulic acids have been found in a number of phytomedicines with diverse biological and pharmacological activities, including radical scavenging, apoptosis of cancer cells, antihyperglycemic, antioxidant effects, and cardioprotective activity [38–41]. Among the single flavonoids (Table 2), procyanidin 2, punicalagin, procyanidin 1, and pelargonidin were, in this order, the most abundant compounds in PSD. Conversely, in the freeze-dried Calabrian and South African Wonderful peel, a lesser amount of single flavonoids than PSD was found, but in a greater quantity than the Indian cultivars. CFD had a greater amount of punicalagin (65 mg/g), procyanidin 1 (1.6 mg/g), procyanidin 2 (1.6 mg/g), and delphinidin than SAFD. IC contained a great amount of procyanidin 2 only in respect to CFD and SAFD.

**Figure 1.** Soluble proteins and total carbohydrates (**A**) and total phenols and total flavonoids (**B**) in peels of different pomegranate cultivars dried differently. PSD (spray-dried Wonderful peel, experimental data); CFD (Calabrian Wonderful peel freeze-dried, experimental data); SAFD (South African Wonderful peel freeze-dried, literature data); IC (Indian cultivar peel freeze-dried, literature data). The experimental data are the mean of six replicates. Soluble protein (mg BSE g−<sup>1</sup> DW), carbohydrates (mg g−<sup>1</sup> DW), total phenols (μg TAE g−<sup>1</sup> DW), total flavonoids (μg quercetin g−<sup>1</sup> DW).

**Figure 2.** Vitamin A (μg retinol g−<sup>1</sup> DW), C (μg ascorbate g−<sup>1</sup> DW), and E (μg α-tocopherol g−<sup>1</sup> DW) in peels of different pomegranate cultivars dried differently. PSD (spray-dried Wonderful peel, experimental data); CFD (Calabrian Wonderful peel freeze-dried, experimental data); SAFD (South African Wonderful peel freeze-dried, literature data); IC (Indian cultivar peel freeze-dried, literature data). The experimental data are the mean of six replicates.

**Figure 3.** Antioxidant activities expressed as 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), total antioxidant capacity (TAC), and 2,2 -azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) in peels of different pomegranate cultivars dried differently. PSD (spray-dried Wonderful peel, experimental data); CFD (Calabrian Wonderful peel freeze-dried, experimental data); SAFD (South African Wonderful peel freeze-dried, literature data); IC (Indian cultivar peel freeze-dried, literature data). The experimental data are the mean of six replicates. Different letters indicate significant differences *p* ≤ 0.05. Lowercase (DPPH), capital (ABTS), and italic (TAC).

**Table 1.** Single phenolic acids contained in the peels of different pomegranate cultivars dried differently. PSD (spray-dried Wonderful peel, experimental data); CFD (Calabrian Wonderful peel freeze-dried, experimental data); SAFD (South African Wonderful peel freeze-dried, literature data); IC (Indian cultivar peel freeze-dried, literature data). The experimental data are the mean of six replicates.


Different letters in the same row indicate significant differences *p* ≤ 0.05.

**Table 2.** Single flavonoids in peels of different pomegranate cultivars dried differently. PSD (spraydried Wonderful peel, experimental data); CFD (Calabrian Wonderful peel freeze-dried, experimental data); SAFD (South African Wonderful peel freeze-dried, literature data); IC (Indian cultivar peel freeze-dried, literature data). The experimental data are the mean of six replicates.


Different letters in the same row indicate significant differences *p* ≤ 0.05.

Pearson's correlation was used to determine the degree of correlation between selected reference data and variables (Table 3). As expected, TP was positively correlated with all the variables except for CARB. Vitamins and proteins were also positively correlated with all the variables except for carbohydrates. Total flavonoids did not correlate with the antioxidant activities and CARB. In addition, strong positive correlations between the antioxidant assays were reported at r = 0.998, *p* = 0.05, between DPPH and ABTS; at r = 0.996, *p* = 0.05, between DPPH and TAC; and at r = 0.988, *p* = 0.05, between ABTS and TAC (Table 3). This agrees with the results in Figure 1B suggesting that the high phenolic content in peel extracts determines the strong antioxidant activity.

**Table 3.** Pearson's correlations (r) between total phenols (TP), total flavonoids (TF), vitamin A (VIT A), vitamin C (VIT C), vitamin E (VIT E), 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), total antioxidant capacity (TAC), 2,2 -azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), total protein (PRO), and total carbohydrates (CARB). Values in bold are different from 0 with a significance level alpha = 0.05.


PCA analysis confirmed this assertion, and evidenced that TP, TF, VIT C, and VIT A were mainly correlated with PSD (Figure 4). No correlation between SAFD and IC was evidenced. Single phenolic acids correlated only with PSD (Figure 5), whereas single flavonoids were mainly correlated with PSD and in part with CFD. Rutin, luteolin, delphinidin, and apigenin were the single flavonoids in the highest quantities and correlated with CFD (Figure 6).

Procyanidins B1 and B2 were discovered to inhibit human colorectal adenocarcinoma and to improve the survival of chronic disease patients by reducing the complications of cardiovascular disease and metabolic syndrome, improving the overall quality of life [42]. Additionally, punicalagin is a flavonoid with proven antioxidant, hepatoprotective, antiatherosclerotic, and antitumoral activity [43]. An ethical study was performed in 50 subjects (25 treated with supplements and 25 with placebo) to identify clinical features induced by 25 mg dried pomegranate (*Punica granatum*) fruit extract (which in turn contained 3.75 mg procyanidins) and 8.75 mg punicalagin–ellagic acid. Results evidenced after 60 days of treatment that the values for systemic oxidative stress, plasmatic antioxidant capacity, and skin antioxidant power increased significantly [44]. Other authors evidenced that the daily intake of pomegranate juice, rich in flavonoids and phenols, decreased the susceptibility of low-density lipoproteins (LDLs) to aggregate, and in cultured human coronary artery endothelial cells exposed to high shear stress, it down-regulated the expression of redoxsensitive genes and increased the functioning of blood endothelial cells [7]. Considering that peels contain many more phenols and flavonoids than juice, as already demonstrated by the previous study of Derakhshana et al. [45] and Russo et al. [46] carried out on different cultivars, it is possible to conclude that pomegranate peels represent a resource comparable to the fruit, if not better, that can be used as a source of bio-compounds with high added

value in the nutraceutical field to formulate new supplements with beneficial effects on human health.

**Figure 4.** Total phenols (TP), total flavonoids (TF), proteins (PRO), vitamin C (VIT C), vitamin A (VIT A), vitamin E (VIT E), total carbohydrates (CARB), soluble proteins, 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH), total antioxidant capacity (TAC), and 2,2 -azino-bis-3-ethylbenzothiazoline-6 sulfonic acid (ABTS) contained in peels of different pomegranate cultivars dried differently. PSD (spray-dried Wonderful peel, experimental data); CFD (Calabrian Wonderful peel freeze-dried, experimental data); SAFD (South African Wonderful peel freeze-dried, literature data); IC (Indian cultivar peel freeze-dried, literature data).

**Figure 5.** PCA (principal component analysis) diagram of single phenolic acids contained in the peels of different pomegranate cultivars dried differently. PSD (spray-dried Wonderful peel, experimental data); CFD (Calabrian Wonderful peel freeze-dried, experimental data); SAFD (South African Wonderful peel freeze-dried, literature data); IC (Indian cultivar peel freeze-dried, literature data).

**Figure 6.** PCA (principal component analysis) diagram of single flavonoids contained in the peels of different pomegranate cultivars dried differently. PSD (spray-dried Wonderful peel, experimental data); CFD (Calabrian Wonderful peel freeze-dried, experimental data); SAFD (South African Wonderful peel freeze-dried, literature data); IC (Indian cultivar peel freeze-dried, literature data).
