*Myrtus Communis* **Liquor Byproduct as a Source of Bioactive Compounds**

**Fabio Correddu 1,**†**, Mariateresa Maldini 2,**†**, Roberta Addis 2, Giacomo Luigi Petretto 2, Michele Palomba 2, Gianni Battacone 1, Giuseppe Pulina 1, Anna Nudda 1,\* and Giorgio Pintore <sup>2</sup>**


Received: 27 May 2019; Accepted: 27 June 2019; Published: 30 June 2019

**Abstract:** The fatty acid (FA), polyphenol content and evaluation of the antioxidant capacity of exhausted *Myrtus communis* berries (EMB) resulting from the production of myrtle liqueur were assessed. All parts of the exhausted berries exhibited high concentrations of carbohydrates, proteins, lipids and phenolic compounds. The lipid fraction contained a high amount of poly unsaturated fatty acids (PUFA), mainly represented by linoleic acid (>70%). Of the phenolic acids evaluated by liquid chromatography/mass spectrometry, ellagic acid was the most predominant (>50%), followed by gallic and quinic acids. Quercetin and quercetin3-O-rhamnoside were the most abundant flavonoids. The seed extracts showed a higher antioxidant potential than the pericarp extracts; the same trend was observed for total phenolic compounds evaluated by spectrophotometric assay. The overall high content of bioactive compounds and the high antioxidant potential of this byproduct sustain its suitability for a number of industrial applications, such as a food ingredient in novel foods, an additive in cosmetic formulations or a component of animal feed formulations.

**Keywords:** LC-MS/MS; fatty acids; polyphenols; antioxidant activity

#### **1. Introduction**

Over recent years, interest in the recovery of high added-value products from waste plant material has grown worldwide, as the re-use and valorization of these byproducts have become important economic issues in a number of industrial sectors [1].

Food processing waste often consists of organic material, the disposal of which presents a serious pollution risk. However, the appropriate management and disposal of such materials entails additional cost. Attempting to extract extra value out of agricultural waste is thus a major step towards alleviating this problem. Many byproducts arising from the processing of fruit and vegetables are rich in phytochemicals that may still retain valuable chemical and biological properties [2]. For example, it has been repeatedly demonstrated that such byproducts can possess high amounts of phenolic compounds—a large group of secondary metabolites that includes: phenolic acids, flavonoids, anthocyanins, and proanthocyanidins [3]. These metabolites have received a great deal of attention because of their numerous biological properties, such as their anti-mutagenic, cardioprotective, anti-inflammatory, anti-carcinogenic, anti-allergic, antiviral and antioxidant activities [4]. Indeed, myrtle liqueur itself has been reported to exhibit strong antioxidant activity [5]. Furthermore, several epidemiological studies suggest that a diet rich in antioxidants may have a positive impact by increasing the reactive antioxidant potential of an organism and reducing the risk of certain degenerative diseases that originate from deleterious free radical reactions [6].

Polyphenol-rich byproducts could be used as functional antioxidant ingredients in the food industry; this possibility is particularly interesting because the currently available and widely used synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), have been suspected to cause negative health effects [7]. Polyphenol-rich byproducts could also provide attractive solutions for the pharmaceutical and cosmetic industries [8,9].

The utilization of byproducts rich in polyphenols as feedstuff has also been explored since recent studies into ruminants showed beneficial effects on health conditions [10], protein and lipid ruminal metabolism [11,12] and the immune system [13], as well as an enhancement of milk production and quality [14,15]. In addition, their use as feed in animal diets may help avoid expensive byproduct treatments, which often lead to further waste production [1].

To the best of our knowledge, no studies have been performed on the byproducts derived from myrtle liqueur production. The berries of *Myrtus communis* are used to produce a sweet myrtle liqueur by their hydroalcoholic infusion (> 40 ◦C) lasting at least 15 days [16]. More than three million bottles of myrtle liqueur are currently produced in Sardinia per year, and it is fast becoming one of the most popular Sardinian exports [17]. Of consequence, Sardinia produces a considerable amount of exhausted berries of *Myrtus communis* as a waste product, estimated to be approximately 200,000 tons/year.

The present study aimed to characterize the chemical composition and the phenolic profile of exhausted berries of *Myrtus communis* resulting from myrtle liqueur production and to test their antioxidant activity in order to evaluate their potential for further exploitation.

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

#### *2.1. Reagents and Standards*

The solvents used for extraction (methanol, acetonitrile and formic acid) were purchased from Sigma-Aldrich Chemical Company (St Louis, MO, USA). A Milli-Q purification system (Millipore, Bedford, MA, USA) was used to prepare high-performance liquid chromatography (HPLC) grade water. Standards of gallic acid, caffeic acid, p-coumaric acid, ellagic acid, ferulic acid, sinapic acid, quinic acid, syringic acid, chlorogenic acid, phloridzin, kaempferol, luteolin, quercetin, isorhamnetin, myricetin, apigenin, epicatechin and catechin were purchased from Sigma-Aldrich. Standards of quercetin rhamnoside, isoquercetin, rutin, robinin, isorhamnetin rutinoside, neohesperidin, quercetin galactoside, myricitrin, myricetin galactoside, epigallocatechin, epigallocatechin gallate, procyanidin B1, procyanidin B2, cyanidin-3-O-glucoside, cyanidin-3-O-arabinoside, cyanin, delphinidin-3-O-glucoside, malvidin-3-O-glucoside, and pelargonidin-3-O-glucoside chloride were purchased from Extrasynthese (Genay, France).

The reference standard mixture of 37 FAME (FAME mix 37) was acquired from Supelco (Sigma-Aldrich, Bellefonte, PA, USA); other reference standards were purchased from Matreya Inc. (Pleasant Gap, PA, USA): PUFA-2, a nonconjugated 18:2 isomer mixture of individual PUFA, eicosapentaenoic acid, (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA), C18:3 cis-6,9,12, and C18:3 cis-9,12,15.

#### *2.2. Samples Collection and Extracts Preparation*

The exhausted myrtle berries (EMB) analyzed were obtained from a local distillery. The EMB were dried at ambient temperature for a week, successively, in an air oven at 40 ◦C until complete drying (24 h) and stored at 0–5 ◦C for later uses. For analysis, seeds were manually separated from pericarps by screening after air-drying, and samples of whole EMB, seeds and pericarps were finely ground.

For polyphenolic analysis and antioxidant assays, the following extraction procedure was employed: the samples were sonicated for 60 min in a solution of 70:30 ethanol:water (*v*/*v*) with a sample to solvent ratio of 13:25 (*w*/*v*) and kept in the dark overnight. After filtration, a rotary evaporator was used to remove completely the solvent. Ultrapure water at the same volume of extraction was used to dissolve the dried samples that were then filtered using 0.20-μm syringe PVDF filters (Whatmann International Ltd., Maidstone, UK).

#### *2.3. Chemical Composition*

The dry matter (DM) content of the samples was determined by oven-drying at 105 ◦C for 24 h. The fiber fractions content (neutral detergent fiber, NDF; acid detergent fiber, ADF; and acid detergent lignin, ADL) was determined following the sequential procedure described by Van Soest, Robertson and Lewis [18], using the filter bag equipment of Ankom (Ankom Technology Corp., Fairport, NY, USA). Ash, protein (CP) and ether extract (EE) contents were determined following the analytical procedures (methods 942.05, 988.05 and 920.39, respectively) reported by AOAC [19,20]. Organic matter (% DM) was calculated as follow: 100 – ash. NFC (non-fiber carbohydrate) was calculated as follows: NFC (% DM) = 100 − (NDF + CP + ash + EE). Hemicelluloses and cellulose were calculated as NDF – ADF and ADF – ADL, respectively. Carbohydrates and gross energy were calculated according to Guimarães, Barros, Carvalho and Ferreira [21] as follow: carbohydrates (% DM) = 100 − (CP + ash + EE); and energy (kcal/100 g DM) = 4 × (CP + carbohydrate) + 9 × (EE). These parameters (except for energy) were expressed as percentage of DM. Analyses were carried out in triplicate, and results were reported as mean ± SD.

#### *2.4. Fatty Acid Profile*

The FA profiles of seeds, pericarps and whole EMB were determined following the method of Kramer, Fellner, Dugan, Sauer, Mossoba and Yurawecz [22] with some modifications. The powder was processed with 2 mL of 0.5 M methanolic sodium methoxide (Sigma-Aldrich, Spain) kept in a water bath at 50 ◦C for 10 min and then cooled at room temperature. The samples were then processed (in a water/ice bath) with 3 mL of HCl/methanol (3M) prepared, freshly, with acetyl chloride and methanol. The samples were heated again in a water bath at 50 ◦C for 10 min and cooled to room temperature. After adding 1 mL of solution containing methyl nonadecanoate (C19:0) as internal standard (Sigma Chemical Co., St. Louis, MO, USA) and 7.5 mL of K2CO3 (0.43 M) the samples were shaken and centrifuged (1500× *g*, room temperature, 5 min), and supernatant was kept in an amber vial for GC analysis. The Fatty acid methyl esters (FAME) determination was carried out using a 7890A GC System (Agilent Technologies, Santa Clara, CA, USA), provided with an autosampler (7693, Agilent Technologies, Santa Clara, CA, USA), a split/splitless injection port (split mode, 1:80), and a flame ionization detector (FID). FAME separation was carried out on a capillary column (CP-Sil 88, 100 m × 0.250 μm i.d., 0.25 μm film thickness, Agilent Technologies, Santa Clara, CA, USA). The oven temperature was maintained at 45 ◦C for 4 min, increased by 13 ◦C/min to 175 ◦C, and held for 27 min; finally, it was increased by 4 ◦C/min until 215 ◦C, and held for 35 min. Carrier gas (Helium) was used at a flow rate of 1 mL/min and with a pressure of 28 psi. Sample volume injection was 1 μL. Both injector and detector temperatures were 250 ◦C. Peak detection was operated using OpenLAB CDS GC ChemStation Upgrade software data system (Revision C.01.04, Agilent Technologies Inc., Santa Clara, CA, USA). Identification of individual FAME was carried out by the comparison of their retention times with those of standards methyl ester, and isomeric profiles found in the literature [23]. Analysis were carried out in triplicate, and results were expressed as mean ± SD.

#### *2.5. Antioxidant Capacity*

The antioxidant capacity in seeds and pericarp extracts was evaluated by two colorimetric assays measuring the activity of the samples to scavenge the two radicals DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid).

#### *2.6. DPPH Radical Scavenging Activity*

The DPPH radical scavenging assay was performed according to the method proposed by Brand-Williams, Cuvelier and Berset [24] with some modifications as previously reported by Maldini et al. [25]. The DPPH (2.4 mg) was dissolved in 10 mL of ethanol 70% and stored, in the dark, at −20 ◦C. An aliquot of 1 mL of this solution was added to 45 mL ethanol 70%, to prepare a work solution having an absorbance of 1.2 ± 0.02, at λ of 517 nm. The ethanolic extracts (100 μL) at different concentrations (from 0.1 to 100 μg) were added to the work solution, to reach 1 mL of final volume. The solutions were then mixed thoroughly and kept in the dark at 25 ◦C. The extent of DPPH radical reduction was measured by reading the solution's absorbance at 517 nm at zero and after 30 min. A Trolox calibration curve in the range 0.25–7.5 μg/mL was used as positive reference. The spectrophotometer used for the assay was an Ultrospec 4300 pro UV–vis (Amersham Biosciences, Piscataway, NJ, USA), equipped with a temperature controller. Solutions were read in 1 cm quartz cuvette.

The following equation was used to calculate the scavenging activity of the DPPH radical:

$$\text{1\% scavenging of DPPH radical} = \text{[(Ab}-\text{As)/Ab]} \times 100\tag{1}$$

where Ab is the absorbance of the control reaction (blank); and As is the absorbance of the hydroalcoholic extracts (sample). Analyses were carried out in triplicate, and results were expressed as mean ± SD.

#### *2.7. ABTS Radical Scavenging Assay*

The ABTS radical scavenging assay was performed following the method detailed by Petretto et al. [26]. The assay is based on the properties of an antioxidant compound to reduce the radical cation ABTS· <sup>+</sup> (chromophore blue/green) to ABTS. The extent of the reduction and the timescale depend on the concentration and the antioxidant power of the considered compound and on the duration of the reaction. The first step was the production of the radical cation obtained by the reaction between ABTS and potassium persulfate (2.45 mM) to reach a final concentration of 7 mM. The solution was kept in the dark at 25 ◦C for 12–16 h. Before use, the ABTS· <sup>+</sup> solution was diluted with ethanol 70% to obtain a work solution with an absorbance of 0.7 ± 0.02 at λ of 734 nm. The ethanolic extracts (100 μL at different concentrations (from 0.1 to 100 μg) were added to the work solution, to reach 1 mL of final volume. The reduction of ABTS· <sup>+</sup> radical cation was recorded (each concentration in triplicate) at zero and after 50 min. Antioxidant capacity of each sample was reported as percent of inhibition. In addition, the IC50 value (reported as mean ± SD) was calculated from regression analysis.

#### *2.8. Determination of Total Phenols*

Total phenols were measured by a colorimetric assay based on procedures described by Lizcano, Bakkali, Ruiz-Larrea and Ruiz-Sanz [27] with some modifications as previously described [25]. Results were expressed as μg of gallic acid equivalent (GAE) per mg of each EMB part.

#### *2.9. ESI-MS and ESI-MS*/*MS Analysis*

MS analysis was carried out using an ABSciex (Foster City, CA, USA) API4000 Q-Trap spectrometer. Depending on the investigated compound, the spectrometer was set in the both negative/positive ion mode. To optimize the experimental conditions, a solution of each standard (1 μg/mL in methanol:water 50:50) was infused at 10 μL/min into the source.

#### *2.10. HPLC–ESI-MS and HPLC–ESI-MS*/*MS Analysis*

An UHPLC system was used to perform quantitative on-line UHPLC-ESI-MS/MS analyses; the system was interfaced to an ABSciex (Foster City, CA, USA) API4000 Q-Trap instrument in Multiple Reaction Monitoring (MRM) mode, with the mass spectrometer operating as a triple quadrupole analyzer.

Liquid chromatography analysis was conducted using a Flexar UHPLC AS system (Perkin-Elmer, USA). The system was equipped with: autosampler, degasser, pump (Flexar FX-10), and column oven (PE 200). Injection volume of each sample was 5 μL and polyphenolic compounds were separated on a

X Select CSH C18 column (Waters, Milford, MA) (100 mm × 2.1 mm i.d., 2.5 μm d). The temperature was kept at 47 ◦C and 2 mobile phases were used: A (formic acid 0.1% in H2O) and B (formic acid 0.1% in acetonitrile). For anthocyanin compounds, a XSelect HSS T3 column (Waters, Milford, MA , USA) (100 mm × 2.1 mm i.d., 2.5 μm d) was selected and elution was carried out at 41 ◦C. The flow and the solvent gradient used for elution of phenolic compounds and anthocyanins were different and were previously reported in the work of Maldini et al. [28].

For each compound, selected transitions and the optimized parameters were listed in supplementary material (Table S1). Analyst software 1.6.2. was used for the data acquisition and processing.

#### *2.11. Calibration and Quantification of Phenolic Compounds*

A stock solution for each standard was prepared at 1mg/mL in methanol:water (50:50). To calculate the calibration curves for each compound, five work solutions at the concentrations of 0.01, 0.05, 0.1, 1, 5 and 10 μg/mL of standards were prepared by diluting the stock solution with methanol. (each work solution was analyzed in 3 replicates).

#### *2.12. Method Validation*

Validation of LC–MS/MS method was performed following the guidelines of the European Medicines Agency (EMEA), concerning the analytical methods validation [29].

The determination of the limit of detection (LOD) and the limit of quantification (LOQ) for each standard compound were by the serial dilution of a stock solution until the signal:noise (S/N) ratios were 3:1 and 10:1, respectively. The LOD and LOQ values for each compound are reported in the Supplementary Materials (Table S2).

To evaluate the precision of the method, variations of intraday and interday analysis were assessed as follows: for each sample, 3 aliquots within the same day, and other 3 aliquots during three consecutive days (one per day) were analyzed. The precision of the method was expressed as percentage relative standard deviation (RSD) (Supplementary Materials, Table S2).

The efficiency of extraction and the analytical method were evaluated by performing recovery tests (in triplicate, with the optimized parameters). LC–MS/MS analysis were carried out on samples after the addition of standard solutions (at different concentration). The recovery (%) ranged from 94.6% to 106.7% within the same day.

#### *2.13. Statistical Analysis*

The one-way analysis of variance (ANOVA) was used to determine significance differences between seeds, pericarps and whole-EMB. Means were separated using Tukey's test (*p* < 0.05). Differences in total phenols contents over concentrations between seeds and pericarps were assessed using linear regression in which the slope variations were compared with a global test of coincidence using an online statistical calculator (http://www.danielsoper.com/statcalc3/calc.aspx?id=103 [30]). When the data were normally distributed, the association between variables was evaluated by the Pearson product moment correlation coefficient.

#### **3. Results and Discussions**

The average proportions of seeds and pericarps in the whole EMB after drying at 40 ◦C were 58.60 ± 4.8 and 41.40 ± 4.8 (mean ± SD), respectively.

#### *3.1. Chemical Composition*

The results of the chemical analyses performed on whole EMB and the separated seeds and pericarps are reported in Table 1. The seeds presented slightly higher values for DM and organic matter than for pericarps (*p* < 0.01), whereas the ash content was higher for the pericarps than for seeds (*p* < 0.01). Regarding the fiber content, NDF was higher in seeds than in pericarps (*p* < 0.01), whereas ADF and lignin contents were both higher in pericarps than in seeds (*p* < 0.01). The differences in fiber content and composition for the two EMB fractions was further highlighted by the higher hemicellulose (27.75 vs. 21.24) and cellulose (25.21 vs. 8.62) contents in seeds than in pericarps (*p* < 0.01). Non-fiber carbohydrates (NFC) were more abundant in pericarps than in seeds (*p* < 0.01). The crude protein and fat contents were higher in seeds than in pericarps (*p* < 0.01); in particular, the values for crude protein and fat were about 2-fold and almost 10-fold higher in seeds compared with pericarps, respectively. These differences result in a significantly higher energy value for seeds compared with pericarps (445 vs. 384 kcal/100 g DM, *p* < 0.01). Overall, the chemical composition of whole EMB showed interesting value from a nutritional point of view, suggesting a possible use as feedstuff. This is evidenced by the value of gross energy (425 kcal/100 g of DM), which is comparable to that of typical feeds used in ruminant nutrition, as soybean meal (350–450 kcal/100 g of DM). Recently, the EMB was used as supplement in two nutritional trials in sheep [31,32], evidencing contrasting results in term of milk production (no effect or reduction of milk yield) and milk composition (no effect or reduction of protein and fat content, and reduction or no effect of milk urea content), but both studies agreed on the suitability of this by product as feed in sheep.

**Table 1.** Chemical composition of seeds, pericarps and whole exhausted myrtle berries.


Means in the same row with different superscripts differ (*p* < 0.05). Values are means with standard deviation (*n* = 3). \*\* *p* < 0.01; \*\*\* *p* < 0.001. <sup>1</sup> NDF, neutral detergent fiber; ADF, acid detergent fiber; NFC, non-fiber carbohydrates; ADL, acid detergent lignin.

#### *3.2. Fatty Acid Composition*

The fatty acid (FA) profiles of pericarps, seeds and whole EMB are presented in Table 2. The FA profiles for EMB and seeds were very similar due to the low contribution of pericarps to the lipid content of whole EMB (see Table 1). The FA profile of pericarps showed a composition similar to that of seeds, but with a different proportion of each FA. Linoleic acid (C18:2 n-6, LA) was the most abundant FA in seeds and in whole EMB, accounting for 75% and 71% of total FA, respectively. The other most representative FAs in seeds and whole EMB were oleic acid (C18:1 cis-9, OA; 9.25% and 9.41%, respectively), palmitic acid (C16:0, PA; 8.30% and 9.34%, respectively), and stearic acid (C18:0, SA; 3.99% and 4.26%, respectively). In pericarps, the most abundant FA was PA, accounting for about 25%, followed by LA, OA, SA, arachidic acid (C20:0, AA) and LNA (17.31%, 11.69%, 8.12%, 5.20% and 4.24%, respectively). Interestingly, pericarps showed a higher proportion of LNA and saturated and unsaturated long chain FAs when compared with seeds (*p* < 0.01). In general, these results are in line with the FA composition of seeds and pericarps of fresh myrtle berries as reported in previous studies [33,34]. However, a different FA profile was reported by Cakir [35], who found the OA content of seeds and mesocarps to be 64% and 72%, respectively, with LA accounting for only 12.7% and 1.7%, respectively. This discordance could be ascribed to a difference in the maturation stage of the berries. In fact, when the variations in FA composition of myrtle berries were studied at different

time points during fruit maturation [36], PA and OA were shown to be the most abundant FA in the first stage of ripening (37.03% and 21.89%, respectively), whereas their proportions decreased progressively throughout all stages of ripening (until 13.58 and 6.49%, respectively). On the other hand, the proportion of LA only accounted for 12.21% at 30 days after flowering and increased progressively to 71.34% in fully ripe fruit (180 days post flowering), thus reaching comparable values to those observed in our study.


**Table 2.** Fatty acid profile (mean of FAME ± SD) of seeds, pericarps and whole exhausted barriers of myrtle resulted from liquor production.

Means in the same row with different superscripts differ (*p* < 0.05). NS *p* > 0.05; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001. <sup>1</sup> FAME, fatty acid methyl ester; PA, palmitic acid; SA, stearic acid; OA, oleic acid; LA, linoleic acid; LNA, α-linolenic acid; EPA, eicosapentaenoic acid; SFA, total saturated fatty acids; MUFA, total monounsaturated fatty acids; PUFA, total polyunsaturated fatty acids.

The high amount of PUFA makes the lipid content of EMB potentially useful because of the beneficial biological and nutritional properties of these compounds. Indeed, LA could be included in cosmetic formulations since it exhibits important skin protection properties [37]. Moreover, the inclusion of lipid sources (with a high proportion of PUFA) in ruminant diets represents a useful strategy to increase the proportion of beneficial FA in meat and milk and their derived products [38]. Values of LA that exceed 50% of total FA are typical of plant oils, such as soybean, sunflower and grape seed oils [39]. In particular, the FA profile reported in our study for the myrtle seeds is very close to

that of the grape seed byproduct [40], which was found to enhance the concentration of beneficial FA in sheep milk when added to the animals' diet [14].

#### *3.3. Polyphenolic Compounds*

A preliminary screening of polyphenol total content was performed using the Folin–Ciocalteau method and data were expressed as μg GAE/mg of dry extract; the results are in line with those reported by Wannes and Marzouk [41] relating to fresh berry parts. As evidenced by the results (Table 3), the total polyphenol content was higher in seed extracts than pericarps (*p* < 0.01). In two recent trials on sheep nutrition, the presence of polyphenols in EMB has been associated to the reduction in blood and milk urea concentration [31] and in ammonia accumulation in rumen [42]. It seems correlated to the ability of polyphenols to bind dietary proteins and to reduce their ruminal degradation. In addition, EMB was found to be effective in reducing the proteolytic bacteria in rumen [42]. These findings also point out that *Myrtus* byproduct could be used to increase feed efficiency in animals, in terms of better protein utilization.

**Table 3.** Determination of total phenols by the Folin–Ciocalteau method in extracted of seeds and pericarps of EMB. Data are expressed as mean ± SD of 3 independent experiments. Each result showed a positive correlation (*p* < 0.001) with DPPH and ABTS results.


<sup>1</sup> GAE, gallic acid equivalent.

All secondary metabolites detected in EMB samples were identified by comparing their chromatographic behaviors and their MS and MS/MS spectra with those of standard reference compounds, when available.

The MS conditions were optimized using reference standards to achieve optimal MS sensitivity for detection and to obtain abundant fragment ions for structural elucidation. Molecules that were identified in negative ion mode belonged to the flavonoid and phenolic acid compound classes. On the other hand, due to the presence of a positive charge in the chemical structure of anthocyanin, good signal sensitivity could also be obtained in positive ion mode.

All compounds were finally confirmed by monitoring their characteristic transitions in MRM mode and comparing their retention times with those of the corresponding authentic standards.

The analytes listed in Table S1 were monitored for their occurrence and 31 compounds were identified in the investigated samples (Table 4).

The precursor/product transitions selected to develop the MRM method are described in Table S1. Quantitative results are reported in Table 4. Each of the three samples was analyzed in triplicate, and the results obtained are expressed as average values of the three analyses.

As shown, ellagic acid was found as the most representative compound in all samples with the highest content in seeds (345 mg/100 g FW), followed by whole EMB (281 mg/100 g FW) and pericarps (244 mg/100 g FW). The other most abundant acids were gallic and quinic acids, ranging 63–123 mg/100 g FW and 77–121 mg/100 g FW, respectively.

With regard to flavonoids, quercetin and quercetin 3-O-rhamnoside were the most abundant (the greatest levels being found in seeds [21 mg/100 g FW and 24 mg/100 g FW, respectively]) followed by isorhamnetin, with values in the range 8–15 mg/100 g FW. Myricetin 3-O-galactoside content was higher in pericarps (10 mg/100 g FW) than in seeds or whole EMB. Overall, the seeds contained the highest level of total polyphenols, at 566 mg/100 g FW. No anthocyanin compounds were found in our samples; this is probably because these compounds are exhaustively extracted during the hydroalcoholic infusion of the myrtle berries in liqueur production. In addition, the low stability of these compounds, which are easily degraded by light, high temperature and air, is widely reported in the literature [43].


**Table 4.** Polyphenolic contents (mg/100 g DW ± standard deviation) and percentages (%) of different part of exhausted berries of *Myrtus communis*.

Means in the same row with different superscripts differ (*p* < 0.05). NS *p* > 0.05; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001. ND, not determined (below LOD).

Only few studies have assessed and quantified the polyphenolic composition of myrtle berries: three were focused on whole fresh berries [5,44,45]; one on pericarps [46]; and one specifically looked at the various myrtle berry parts [41]. Thus, a real comparison of our data with other published results is difficult. Nevertheless, the majority of secondary metabolites identified in our samples have previously been reported as present in fresh myrtle fruit; with the exception of caffeic acid, p-coumaric acid, ferulic acid, sinapic acid, quinic acid, syringic acid, chlorogenic acid, isorhamnetin, robinin, isorhamnetin 3-O-rutinoside, neohesperidin, phloridzin, apigenin, luteolin and epicatechin, which were not investigated in the cited papers.

The liquor preparation by hydroalcoholic infusion of berries, extract some of the polyphenolic compounds. Consequently, as expected, the detected levels of the main bulk of polar compounds in EMB were lower than those reported in the literature for fresh myrtle fruit, apart from ellagic acid that was more abundant in our samples. Ellagic acid is a naturally occurring phenolic compound found at high concentrations in many berries; in plants, it forms structural components in the plant cell wall and cell membrane in the form of hydrolysable tannins (ellagitannins), where it is esterified with glucose. Several papers have investigated the biological properties of ellagic acid, which include antioxidant, antimicrobial, anti-inflammatory and antimutagenic activities, as reviewed in [47].

#### *3.4. Antioxidant Activity*

The free radical-scavenging properties of the exhausted myrtle berry byproduct are presented in Figure 1, where a lower IC50 value (μg/mL) implicates higher antioxidant activity. The ability of DPPH radical scavenging was significantly higher in seeds (*p* < 0.01) than in pericarps, with a three-fold higher antioxidant activity at both time points investigated (0 and 30 min). Our results are in line with those reported by Wannes and Marzouk [41] for the separate myrtle fruit parts, where seeds showed the highest antioxidant activity. This result could be explained by considering the higher content of phenolic acids and flavonols in seeds than in pericarps, as the antioxidant activity of fruit is mainly obtained from phenolic compounds [41].

**Figure 1.** Scavenging of 50% of DPPH and ABTS radical by Trolox and ethanolic extracts from different fruit parts (seeds and pericarps) of exhausted myrtle berries (EMB) at different time points (0 and 30 min). Data were expressed as means ± SD of three independent experiments. Different letters (a,b) indicate significant differences (*p* < 0.01) between seeds and pericarps of EMB at each time point.

The ABTS· <sup>+</sup> assay showed that antioxidant activity was also significantly higher in seeds (*p* < 0.01) than in pericarps, with values two-fold higher at both time points (Figure 1). A highly significant positive correlation was found by comparing the results obtained using the Folin–Ciocalteau method with the DPPH and ABTS results, respectively (Table 3), confirming the well documented [48] role of phenols in antioxidant activity.

#### **4. Conclusions**

Our results demonstrate that exhausted myrtle berries, left over following hydroalcoholic infusion, can still provide a rich source of commercially viable phytochemicals with high antioxidant capacity, carbohydrates, proteins, lipids and polyphenols. These features and the high antioxidant activity of the byproduct support the notion that EMB, in particular the seeds, could be further processed to provide a source of bioactive compounds of bioactive compounds. The possibility of using this byproduct, in its whole form, in feed formulations should also not be excluded.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/8/7/237/s1, Table S1: LC–MS/MS conditions for quantification of detected compounds by negative/positive ion MRM, Table S2: Accuracy, precision, linearity, LOQ and LOD of LC-ESI-QqQ-MS/MS MRM method for the analysis of standard compounds.

**Author Contributions:** Formal analysis, writing—original draft preparation, F.C. and M.M.; Formal analysis R.A. and G.L.P.; review and editing, M.P.; data curation supervision, G.B. and G.P. (Giuseppe Pulina); supervision, project administration, A.N.; and review and editing G.P. (Giorgio Pintore).

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Characterization of Polyphenolic Compounds in Cantaloupe Melon By-Products**

#### **Filomena Monica Vella 1, Domenico Cautela <sup>2</sup> and Bruna Laratta 1,\***


Received: 2 April 2019; Accepted: 1 June 2019; Published: 6 June 2019

**Abstract:** The Muskmelon (*Cucumis melo* L.), which includes several crops of great economic importance worldwide, belongs to the Cucurbitaceae family, and it is well recognized for culinary and medicinal purposes. The high fruit consumption produces a large quantity of waste materials, such as peels and seeds that are still rich in molecules like polyphenols, carotenoids, and other biologically active components that possess a positive influence on human health and wellness. A sustainable development in agro-food and agro-industry sectors could come through the reutilization and valorization of these wastes, which in turn, could result in reducing their environmental impact. The current study provides a biochemical characterization of cantaloupe by-products, peels and seeds, through evaluating total polyphenols, *ortho*-diphenols, flavonoids, and tannins content. Furthermore, the antioxidant activity was assessed in order to understand potential benefits as natural antioxidants. Overall, the peel extract revealed the highest radical's scavenging and reducing activities, moreover, it showed higher polyphenolic content than seed extract as revealed by both cromatographic and spectrophotometric analyses. The results of the present study indicate that the melon residues are a good source of natural phytochemicals useful for many purposes, such as ingredients for nutraceutic, cosmetic, or pharmaceutical industries, development of functional ingredients and new foods, and production of fertilizers and animal feed.

**Keywords:** *Cucumis melo*; polyphenols; flavonoids; antioxidants; by-products; waste valorization

#### **1. Introduction**

The Cucurbitaceae family covers several species of great economic importance, including muskmelon (*Cucumis melo* L.), which is largely cultivated and consumed in Europe. Muskmelon encircles a wealth of varietal types, such as smooth-skinned varieties like Honeydew, Crenshaw, and Casaba (*C. melo* var. *inodorous*), rough-skinned varieties like Cantaloupe, Persian melon, and Santa Claus or Christmas melon (*C. melo* var. *reticulatus*), and varieties used when they are immature as vegetables like Barattiere, Carosello, and Armenian Cucumber (*C. melo* var. *flexuosus*). The Cantaloupe melon is well recognized by its net-like slightly ribbed, gray-to-green or light brown skin. It is one of the most consumed melons worldwide thanks to its sweetness, juicy taste, pleasing flavor, and nutritional value [1,2]. In 2016, about 1.9 million tons of melon were harvested in the Mediterranean area, with Spain, Italy, and France representing the main European producers, accounting for 35%, 34%, and 13% overall yield, respectively [3]. In Italy, Cantaloupe is the most cultivated variety. Its name is supposed to derive from Italian "Cantalupo in Sabina", which was formerly a papal county seat near Rome [4].

Cantaloupe is an excellent source of vitamin A, vitamin C, and microelements such as potassium and magnesium [1,4,5]. In recent years, it has been shown to possess useful medicinal properties such as analgesic, anti-inflammatory, antioxidant, antiulcer, anticancer, antimicrobial, diuretic, and antidiabetic properties [2,6,7]. Furthermore, it showed a hepato-protective effect, activity against hypothyroidism and immune-modulator action [6].

Ever-increasing demand for healthy food has stimulated the manufacturing sectors to search for new natural sources of nutritional and healthful components to be employed as food additives or supplements, with high nutritional value [8,9]. As a consequence, the European Union has encouraged the exploitation of fruit by-products for their use as a source of nutritionally and therapeutically functional ingredients to utilize for dietary intake, and as active ingredients in pharmaceuticals and cosmetic industries [10,11].

During fresh consumption and industrial processing of melons (juices, compotes, and salads), large quantities of peels and seeds are produced, and are considered waste. The complete utilization of these by-products could minimize the litter volume, so reducing the environmental impact and the economic costs associated to their disposal. Peels and seeds, in fact, are potential sources of phytochemicals, such as polyphenols, carotenoids, flavonoids, and other bioactive compounds with potential health-promoting effects [12,13]. Among them, polyphenol compounds show antioxidant activity, delaying or inhibiting the oxidation of lipids and other molecules, thus playing an important role in defending cells against free radical damage, a very important way of preventing diseases like cancer and cardiovascular disorders [12–15].

Studies related to melon peels and seeds are scarce; data concerning their whole biochemical characterization are insufficient. Recently, Mallek-Ayadi et al. [5] studied only the phenolic composition and functional properties of peels, concluding that this by-product could be considered as a rich source of carbohydrates, proteins, calcium, potassium, and polyphenols. Fundo et al. [1] characterized the edible and the waste parts of cantaloupe melon, only considering the bioactive compounds showing antioxidant activity. These studies suggested that the valorization of cantaloupe by-products should be encouraged because they are important sources of healthy compounds for food, cosmetics, and nutraceutical products.

On this basis, this research aims to examine the peels and seeds from cantaloupe melon, evaluating total polyphenol, *ortho*-diphenol, flavonoid, and tannin contents. At the best of our knowledge, this is the first time that such a comprehensive investigation, by means of spectrophotometric, chromatographic and in vitro assays, is achieved on both extracts from seeds and peels, in order to explore their potential attitude as natural sources of antioxidants.

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

#### *2.1. Reagents and Standards*

Sodium carbonate, Folin–Ciocalteu reagent, sodium nitrite, sodium molybdate, aluminum chloride 6-hydrate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), sodium acetate, iron(III) chloride 6-hydrate, 2,4,6-tripyridyl-s-triazine (TPTZ), bovin serum albumin (BSA), standards phenolic acids (gallic, caffeic, chlorogenic, syringic, ferulic, and ellagic acids), flavonoids (rutin, quercetin, kaempferol and isorhamnetin) and the HPLC-grade solvents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Sodium hydroxide, hydrochloric acid and ethanol were obtained from Carlo Erba Reagents (Milan, Italy).

#### *2.2. Extraction of Bioactive Compounds*

Three "Prescott" varieties of cantaloupe melons (*Cucumis melo* L. var. *reticulatus*) were purchased in a local market at commercial ripening stage. Peels were manually removed with a knife and seeds were separated too. Peels and seeds were oven-dried at 37 ◦C, until constant weight. Randomly chosen samples were utilized for analyses. Both samples were milled with a food mixer (Moulinex, Italy) and kept at −20 ◦C until extractions were performed. For bioactive compounds recovery, 200 mg of fine powder of cantaloupe peels and seeds were extracted with 10 mL of 95% ethanol (ratio 1:50 w/v) for 6 h at 50 ◦C in a closed vessel using an ultrasonic bath. The extracts were recovered by centrifugation at 13,000× *g* for 10 min at 4 ◦C, and dried using a rotary evaporator (IKA RV8, IKA-Werke GmbH & Co, Staufen, Germany).

#### *2.3. Total Polyphenols Content*

Total polyphenols were spectrophotometrically determined according to the Folin–Ciocalteu method [16]. In brief, 150 μL of extracts were mixed with 750 μL of Folin–Ciocalteu reagent and 600 μL of 7.5% (*w*/*v*) Na2CO3. After incubation, the absorbance was read at 765 nm (UV-Vis spectrophotometer, model DMS-200, Varian, Leini, Italy). Total phenolic amount was calculated by a six point calibration curve obtained with different quantities of gallic acid standard solution ranging from 1.5 to 10 μg. (y = 0.0768 x; R2 = 0.9909) and the results were expressed as mg of gallic acid equivalents (GAE) per g of extract.

#### *2.4. Ortho-Diphenols Content*

*Ortho*-diphenols content was evaluated by Arnow assay [17]. Briefly, 400 μL of extracts were mixed with 400 μL of 0.5 M HCl, 400 μL of 1.45 M NaNO2—0.4 M Na2MoO4 and 400 μL of 1 M NaOH. The absorbance was recorded at 500 nm and *ortho*-diphenols were determined by a calibration curve obtained using caffeic acid as standard. The *ortho*-diphenolic content was determined by a calibration curve obtained using a caffeic acid standard solution ranging from 5 to 50 μg (y = 0.0152 x; R<sup>2</sup> = 0.9921), and the results were expressed as mg of caffeic acid equivalents (CAE) per g of extract.

#### *2.5. Flavonoid Content*

Flavonoid content in the extracts was determined according to the colorimetric method based on the formation of flavonoid-aluminum compounds [18]. In the assay, extracts were mixed with distilled water and NaNO2. After 5 min, AlCl3 × 6H2O were added and the reaction was stopped by adding 1 M NaOH and distilled water. The absorbance was read at 510 nm and (+)-catechin was used to create the standard curve. (+)-Catechin, from 5 to 100 μg was used to create the calibration curve (y = 0.009 x; R2 = 0.9940) and the results were expressed as mg of catechin equivalents (CE) per g of extract.

#### *2.6. Tannins Content*

Total tannins were assessed as reported by Vella et al. [19] incubating extracts with BSA at 30 ◦C for 1 h. The supernatant, representing the non-tannin fraction, was collected by centrifugation at 13,000× *g* for 10 min at 4 ◦C and was analyzed using the Folin-Ciocalteu method. Tannins were determined by difference from the amounts of the polyphenols determined before and after BSA precipitation. Tannins were expressed as mg of gallic acid equivalents (GAE) per g of extract.

#### *2.7. In Vitro Antioxidant Activity*

The antioxidant activity of cantaloupe peels and seed extracts were evaluated by means of two in vitro biochemical assays: The Ferric Reducing Antioxidant Power (FRAP) and the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity.

As reported by Benzie and Strain [20], freshly prepared FRAP reagent were added to the extracts. The absorbance was recorded after 4 min at 593 nm. The antioxidant activity of samples was calculated from a calibration curve with L-ascorbic acid ranging from 0.5 to 5 μg (y = 0.1662 x; R<sup>2</sup> = 0.9918) and the results were expressed as mg of ascorbic acid equivalents (AAE) per g of extract.

The free radical scavenging activity (RSA) of the extracts was assessed according to the procedure of Blois [21]. In brief, different concentrations of peels and seeds extracts were mixed with DPPH methanolic solution. The absorbance reduction at 517 nm of the DPPH was determined continuously. The RSA was calculated as a percentage of DPPH discoloration, using the following equation:

$$\% \, RSA = \left[ \frac{[A\_{DPPH} - A\_s]}{A\_{DPPH}} \right] \ast 100,\tag{1}$$

where AS is the absorbance of the solution when the extract was added and ADPPH is the absorbance of the DPPH solution. The EC50 value was obtained from the graph of %RSA against the extract concentrations in mg/mL.

#### *2.8. Cromatographic Analyses*

High performance liquid chromatography-photodiode-array-mass spectrometry (HPLC-PDA-ESI-MS/MS) analyses were performed using a Surveyor LC pump, a Surveyor autosampler, coupled with a photodiode array detector (PDA) Surveyor and a LCQ Advantage ion trap mass spectrometer (Thermo Finnigan, Waltham, MA, USA) equipped with Xcalibur 3.1 software (Thermo Fisher Scientific, Waltham, MA, USA).

A volume of 5 μL was employed for the analysis on a Supelco Spherisorb® ODS2 HPLC Column (250 × 4.6 mm), and the column was thermostatically controlled at 35 ◦C. The elution was conducted, as already reported [22], by employing 0.3% acetic acid solution (solvent A) and acetonitrile (solvent B). A gradient elution was performed as following: the initial solvent was 90% A and 10% B; the gradient elution was changed from 10% to 20% B in a linear mode for 15 min; this composition was maintained at isocratic flow for 10 min; the solvent B reached 50% in 10 min and from 50 to 90% B in 10 min.

Elution was performed at a flow rate of 0.5 mL/min with a splitting system of 2:8 to the MS detector (100 μL/min) and PDA detector (400 μL/min). Analyses were performed with an electrospray ionization (ESI) interface in the negative mode. The optimization of the instrumental parameters for bioactive compounds was performed by continuous infusion (FIA)-ESI MS/MS analyses. Parameters for analysis were set using negative and positive ion modes, with spectra acquired over a mass range from *m*/*z* 50 to 1100. The ionization conditions were optimized, and the parameters used were as follows: capillary temperature, 210 ◦C; capillary voltage, −10.0 V; tube lens offset, −50.0 V; sheath gas flow rate, 60.00 arbitrary units; auxiliary gas flow rate, 20.00 arbitrary units; spray voltage, 4.50 kV; and scan range of *m*/*z* 150–1200. In the MS/MS experiments, normalized collision energy of 35.0% was applied.

PDA data were recorded with 200–600 nm range, and HPLC/UV chromatograms were acquired at three different wavelengths (226, 284 and 369 nm) according to the absorption maxima of analyzed compounds. Figure 3 shows the 284 nm absorption data for all compounds. For the quantitative analysis of phenolic compounds, a calibration curve was obtained by the injection of different concentrations of each standard. Peak identification of phenolic compounds was performed according to their retention time, UV-Vis and mass spectra.

For the quantification of phenolic compounds by HPLC-UV, a calibration curve was obtained through injection of different concentrations of standard mixture, blending aliquots of different stock individual standards into a 10 mL glass volumetric flask. The standard solutions were prepared in methanol in the range of 0.0025–0.045 mg/mL. The reproducibility of the detector response at each concentration level was evaluated by a triplicate injection of standard mix and expressed as percentage of relative standard deviations (RSD%). The RSDs were expected to be less than 2%. The limits of detection (LOD) were established at a signal to noise ratio (S/N) of 3. The limits of quantification (LOQ) were established at a signal to noise ratio (S/N) of 10. LOD and LOQ were experimentally verified by the nine injections of reference compounds in LOQ concentrations.

#### *2.9. Statistical Analysis*

All samples were analyzed in triplicates and the results were expressed as mean ± standard deviation (SD). Means, SD, calibration curves and linear regression analyses (R2) were determined using Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA).

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

The phenolic composition and functional activity of melon residues, peels and seeds, were studied in order to explore their beneficial properties in sight of potential industrial applications. In this contribution, a biochemical characterization was obtained through the evaluation of total polyphenols, *ortho*-diphenols, flavonoids, tannins, and antioxidants by means of photometric assays and by HPLC profiling.

In Figures 1 and 2 polyphenol, *ortho*-diphenol, flavonoid, and tannin contents in cantaloupe peels and seeds, respectively, are reported.

**Figure 1.** Polyphenol, *ortho*-diphenol, flavonoid, and tannin contents in cantaloupe peels.

**Figure 2.** Polyphenol, *ortho*-diphenol, flavonoid, and tannin contents in cantaloupe seeds.

In peels, the polyphenol content was 25.48 ± 1.44 mg GAE/g, which is 6- and 8-fold higher than that reported by Isamil et al. [2] and Mallek-Ayadi et al. [5], respectively. This gap could be attributed to several factors, including cultivar, degree of ripening, and environmental elements, such as climatic conditions and geographical origin [4,19,22]. Conversely, cantaloupe seeds content of polyphenols was 1.50 ± 0.02 mg GAE/g, and this result matches with the range reported in literature data [1,2]. Polyphenols are commonly found in both edible and non-edible plant parts, being essential compounds for their growth and reproduction pathways. They also play an important role in modulating the

defense response against insects, pathogens, and microorganisms. Moreover, they take an active part in determining color, flavor, taste, and appearance of fruits. Among phenolics, *ortho*-diphenols are recognized as the most important in relation to their antioxidant activity, since they are able to improve radical stability by forming an intra-molecular hydrogen bond between the hydrogen and phenoxyl radicals. As reported in Figures 1 and 2, *ortho*-diphenols content was 17.86 ± 1.43 and 0.92 ± 0.04 mg CAE/ g in peels and seeds, respectively. To the best of our knowledge, data on *ortho*-diphenols have never been reported in cantaloupe until now.

Considering flavonoids, they are the most common and widely distributed group of plant phenolics, being very effective antioxidants [23]. Cantaloupe peels showed the highest flavonoid content of 15.19 ± 1.88 mg CE/g, meanwhile seeds had 0.74 ± 0.03 mg CE/g, as shown in Figures 1 and 2, respectively. In addition, these components scored higher content values, as well for the total phenolic compounds, than those reported in the literature [2,5]. Moreover, similarly to polyphenols, flavonoid content has many sources of variation such as genotype, fruit ripening, plant phenotypic state and pedoclimatic conditions [4,19,22].

Tannins have been considered health-promoting components of plants, since possessing anti-carcinogenic and anti-mutagenic potentials, as well as antimicrobial, antioxidant and antiradical properties [24–27]. In the present study, tannins content was higher in peels than in seeds, displaying 11.83 ± 1.44 and 0.92 ± 0.03 mg GAE/g, respectively. The literature reports many studies comparing polyphenolic and flavonoid contents of different parts of cantaloupe [1,2,5] but tannins were never assessed.

As the phenolic compounds are known to protect cellular components against free radicals, the antioxidant properties of cantaloupe peels and seed extracts were estimated by means of the FRAP assay and by the DPPH radical scavenging activity, whose results are shown in Table 1. As reported by Benzie and Strain [20], the FRAP assay measures the reduction of a ferric 2,4,6-tripyridyl-s-triazine complex (Fe3+-TPTZ) to the ferrous form (Fe2+-TPTZ) in the presence of an antioxidant compound. The antioxidant ability, measured by the FRAP assay, indicated a higher value in peels as compared to seeds, with values of 12.27 ± 1.22 mg AAE/g and 0.31 ± 0.02 mg AAE/g, respectively.


**Table 1.** Antioxidants content and activity in cantaloupe peels and seeds.

\* The antioxidant power was measured by FRAP assay; \*\* EC50 was calculated by DPPH assay.

The scavenging activity was studied by means of the DPPH assay, based on the evaluation of the reduction of the DPPH radical to hydrazine as a consequence of the antiradical activity of the extracts. Similarly to the results of the FRAP assay, scavenging activity in the peels extracts was stronger since the EC50 after 15 min was 6.65 mg/mL, while seed extracts showed a value of 55.03 mg/mL, thus indicating a lower antioxidant activity of this latter cantaloupe by-product. These results were in agreeance with literature data: in particular, the cantaloupe peels extract in our experiment proved to be 1.4 fold more active when compared to the results published by Isamil et al. [2]. Conversely, seed extract showed an activity that was half than that observed and reported by Isamil et al. [2]. The results of the DPPH assay suggest that extracts are capable of scavenging free radicals via electron or hydrogen-donating mechanisms. Moreover, DPPH activity of these cantaloupe by-products showed similar behavior with the polyphenols, *ortho*-diphenols, flavonoids, and tannins content, thus indicating that radical scavenging activity of cantaloupe peels and seeds extracts is related to the amount of phenolic compounds.

Nowadays, synthetic antioxidants are widely used as additives in food, pharmaceuticals, and cosmetics, but their uses have been questioned because of their possible toxic or carcinogenic activities due to some components formed during their degradation occurring in industrial processing [28]. Therefore, the application of natural plant-based substances may be a suitable alternatives to replace artificial molecules, not only because of their safety, but also since they protect food, feed, and derivatives from the deleterious effects of natural oxidation.

The two extracts from melon peels and seed were also analyzed by HPLC to evaluate the real composition of the phenolic profiles (Figure 3).

**Figure 3.** HPLC chromatograms of melon peel and seed extracts, monitored at 284 nm. (**A**) Standard mixture of bioactive compounds. (**B**) Melon peels ethanolic extract. (**C**) Melon seeds ethanolic extract. Gallic acid (Gal), Chlorogenic acid (Clo), Caffeic acid (Caf), Syringic acid (Sir), Rutin (Rut), Ferulic acid (Fer), Ellagic acid (Ell), Quercetin (Que), Kaempferol (Kae), Isorhamnetin (Iso).

Phenolic acids and flavonoids were quantified according to HPLC-PDA data recorded at 284 nm, where gallic acid (2.45 ± 0.08 mg/g), ellagic acid (0.57 ± 0.01 mg/g), and kaempferol (0.32 ± 0.03 mg/g) were the main bioactive compounds found in the peels extract (Table 2; Figure 3B). In seed extract, the richest phytochemicals were ferulic acid (1.51 ± 0.02 mg/g), followed by kaempferol (0.54 ± 0.02 mg/g), and gallic acid (0.07 ± 0.02 mg/g), as reported in Table 2 and in Figure 3C. Peak identification of phenolic compounds was performed according to their retention time, UV-Vis and mass spectra (Table 2).


**Table 2.** Bio-active contents in melon peel and seed extracts (UV-Vis, HPLC and MS data).

LOD = limit of detection S/N: 3 (*n* = 9) LOD = 0.02 μg/mL.

Gallic acid, ellagic acid and kaempferol have been reported to have antiviral, anti-mutagenic, anticancer, antioxidant and cytotoxic effects [29–31]. Ferulic acid is a ubiquitous natural phenolic compound in seeds; it exhibits a wide variety of biological activities such as antioxidant, anti-inflammatory, antimicrobial, antiallergic, hepatoprotective, anticarcinogenic, antithrombotic, antiviral and vasodilatory actions, increase sperm viability, metal chelation, modulation of enzyme activity, activation of transcriptional factor [31]. Peel and seed HPLC profiles showed some differences from what was reported by Mallek-Ayadi et al. [5] and Zeb [32]. We suppose that these differences might be due to variation relating to different cultivar, environmental conditions during plant growth and fruiting, plant phenotypic state, and possibly due to extraction conditions too [4,19,22].

Altogether, HPLC analysis confirms a higher level of phenolic acids and flavonoids in melon peel than in seed extract. The diverse tissue distribution of bioactive compounds may be attributed to different metabolic roles and networks active in peels and seeds due to their different roles in plant architecture and physiology. Furthermore, it is important to underline that in this study ethanol was used for chemical extraction of peels and seeds and, since it is a GRAS (Generally Recognized As Safe) solvent, it can be utilized safely for bioactive compound recovery, to be used in the food industry.

#### **4. Conclusions**

Normally, the non-edible parts of the melon (seeds and peels) are discarded during production processes, reaching approximately 8 to 20 million tons of waste per year worldwide [33]. The extracts of melon exhibit valuable functional and nutraceutical properties, in the light of all the data, spectrophotometry, HPLC profiling and biological activity, obtained in the course of the present study.

With the aim of developing new nutraceuticals, such as supplements, dietary and nutritional products, these cantaloupe by-products seem to be very promising, opening up new perspectives for their use, mainly due to the solubility in water and the stability of their extracts. Therefore, the melon extracts could be used in the production of functional waters, greatly demanded by markets and consumers all over the world, or in food and cosmetic products. Indeed, it has been observed that these by-products act against the oxidation process, thus suggesting their possible future uses as natural colorants and antioxidants in yogurt, biscuits, cupcakes, jellies, sweets and bread, and in anti-wrinkle creams, soaps and bathroom foams, as reported in the literature [33]. Moreover, in this study the reduction to a minimum of the waste volumes and the possibility of developing new products, with the recovery of biomolecules with high added value, may contribute to the sustainable management of waste biomasses that otherwise imply environmental and economic costs.

**Author Contributions:** Conceptualization, B.L.; formal analysis, F.M.V., D.C. and B.L.; investigation, F.M.V. and D.C.; writing—original draft, F.M.V. and B.L.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
