**3. Results and Discussion**

This study involved the screening and characterization of phenolic compounds with antioxidant potential from twenty different fruit peel samples. An untargeted polyphenol identification and characterization were achieved by the LC-ESI-QTOF-MS/MS, an advanced analytical technique which can provide comprehensive phytochemical screening and MS/MS characterization. For the quantification of phenolic compounds, the twenty most abundant phenolic compounds including (10) phenolic acids and (10) flavonoids present in different fruit peels were targeted and quantified by the HPLC-PDA. A strong correlation between phenolic compound levels and antioxidant activities was observed in all selected fruit peel samples.

#### *3.1. Phenolic Estimation (TPC, TFC and TTC)*

Fruit peels contain high concentrations of phenolic compounds including flavonoids, phenolic acids, and tannins. The phenolic contents in different fruit peel samples were determined with TPC, TFC, and TTC assays.

Table 1 summarizes the polyphenol concentrations and antioxidant potentials of twenty selected fruit peel samples. The TPC values of these fruit peel samples varied widely, with mango, grapefruit, and lime peel samples exhibiting the highest TPC values (27.51 ± 0.63, 27.22 ± 1.00 and 23.32 ± 2.07 mg GAE/g, respectively), followed by orange and avocado peel samples. The lowest phenolic contents were detected in dragon fruit, nectarine, and passion fruit peels. Comparing all the peel samples, the mango peel sample had significantly higher phenolic contents (*p* < 0.05) than any other fruit peels. Previously, Nguyen, et al. [23] reported significantly higher phenolic contents in mango peels as compared to other tropical fruits, including passion fruit and dragon fruit, which is consistent with our results. In our study, total phenolic content was measured using the Folin-Ciocalteu reagent that has the ability to react with both phenolics and non-phenolic compounds such as ascorbic acid and other reducing substances [24]. Grape and lime peel were reported to be rich in ascorbic acid, which may be one of the contributors to their high total polyphenol content [25]. However, grapefruit and lime peel were previously found to be abundant in polymethoxylated flavones, phenolic acids, and flavanones including naringin and neohesperidin [26]. Previously, similar trends but with higher TPC values were detected in different fruit juices, including grapefruit (657.65 ± 69.20 mg GAE/g), lime (579.41 ± 91.14 mg GAE/g) and orange (523.44 ± 87.20 mg GAE/g) [27]. Nurliyana, et al. [28] also found that dragon peel has a high phenolic content, most likely due to the abundance of betacyanins (pigments) rather than polyphenols, which increased the TPC values [29]. Considering these facts, polyphenol characterization through advanced analytical techniques including LC-MS/MS can provide more reliable and useful information for their applications in different food, feed, nutraceutical, and pharmaceutical industries.


**Table 1.**The polyphenol concentrations and antioxidant potentials of twenty different selected fruit peels.

All values are expressed as mg/g mean ± standard deviation (*n* = 3). Alphabetic letters indicate the significant difference (*p* < 0.05) in a row using a one-way analysis of variance (ANOVA) and Tukey's test. TPC, Total phenolic content; TFC, total flavonoid content; TTC, total tannins content; FRAP, ferric reducing antioxidant power assay; DPPH, 2,2′-diphenyl-1-picrylhydrazyl assay; ABTS, 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid assay; TAC, total antioxidant capacity; GAE, gallic acid equivalents; CE, catechin equivalents; QE, quercetin equivalents; AAE, ascorbic acid equivalents.

*Foods* **2020**, *9*, 1206

Flavonoids are the predominant class of phenolic substances found in almost all plants, which was determined via the aluminium chloride colorimetric method in this study. Aluminium chloride reacts with carbonyl group present in flavonoids, forming a stable complex [30]. The highest amount of flavonoid was found in the mango peel (1.75 ± 0.08 mg QE/g), followed by pineapple and banana peels (1.47 ± 0.07 and 1.32 ± 0.12 mg QE/g, respectively). Marina and Noriham [31] also reported higher flavonoid contents in mango peel than other tropical fruit peels such as papaya and guava peels, which is consistent with our study. Previously, Morais, et al. [32] determined the TFC in different parts of the avocado (*Persea americana*), and found that avocado peels had more flavonoids than seeds and pulp. Ayala-Zavala, et al. [33] also reported that the peels of tropical exotic fruits like avocado, pineapple, banana, papaya, passion fruit, and melon contain more phenolic acids and flavonoids than pulp. Overall, TFC values of our twenty different fruit peels were slightly higher than previously reported values, which may be due to the difference in the growing area, climatic conditions, varietal differences, and extraction. Fruits growing under different climatic regions have different flavonoid content in their peels, the peels being the outer part of fruit bodies exposed to more to sunlight as compared to pulp, leading to the synthesis of the abundant and diverse nature of flavonoids. Nogata, et al. [34] reported that the flavonoid contents in the outer layer of citrus fruits are higher than the inner layers and pulps. The flavonoid profile differs among species and cultivars of the same fruits grown in different regions under different climatic conditions, soil characteristics, and cultivation techniques [35]. Moreover, the efficiency of the extraction of flavonoids also varies under different extraction conditions, such as the type of solvents, solvent concentration, extraction time and temperature, solvent-to-solid ratio, etc. [36,37].

Tannins are also one of the important groups of phenolic compounds which can be classified into hydrolysable tannins and condensed tannins. Avocado peels exhibited the highest TTC values of 9.01 ± 0.20 mg CE/g, followed by mango (8.99 ± 0.13 mg CE/g), lime, and custard apple peels (8.42 ± 0.63 and 8.32 ± 0.56 mg CE/g, respectively), while few tannins were detected in dragon fruit, melon, nectarine, passion fruit, peach and pear peels. Overall, most of our TTC values are in accordance with previously published work, while we also had high values of tannins in mango peel as compared to the previously published literature. Previously, the mango fruit peel has already been reported to be a rich source of hydrolysable tannins, while hydrolysable tannins can decrease significantly during the ripening process [38]. One of the possible reasons might be the difference in sample preparations, storage conditions, and extraction techniques. In our study, all the fruit peels were freeze-dried prior to the extraction of polyphenols; it has been reported that freeze-drying facilitates the overall polyphenol extraction. Freeze-drying can also preserve the highest percentage of condensed tannins as compared to other conventional drying methods. Freeze-drying also helps to accelerate the release of bounded phenolic compound [39], deactivating oxidative and hydrolytic enzymes, improving the extraction and protecting the phenolic compounds [40].

#### *3.2. Antioxidant Potential (DPPH, ABTS, FRAP and TAC)*

To further investigate the antioxidant potential of the twenty different fruit peels, different antioxidant assays based on different mechanisms were applied in this study. Antioxidant assays including DPPH and ABTS were used to measure the radical scavenging ability, while FRAP and TAC assays were used to determine the reducing power of samples. The results shown in Table 1 were reported in mg ascorbic acid equivalents (AAE) per g of samples (mg AAE/g).

The DPPH assay is widely used to determine the free radical scavenging activity, which is mainly attributed to polyphenols [15]. Grapefruit, mango, and avocado peels exhibit higher DPPH radical scavenging ability (9.17 ± 0.19, 8.67 ± 0.49 and 8.67 ± 0.44 mg AAE/g, respectively). Previously, different varieties of mango peel extracts have shown concentration-dependent DPPH free radical scavenging activity [41]. Most of our DDPH values are in accordance with the previously published literature. Moreover, the DPPH assay showed significantly higher levels of antioxidant capacity in freeze-dried fruit peels as compared to fresh fruit peels. The freeze-drying process generates

redox-active metabolites that can scavenge and neutralize free radicals [42]. The DPPH assay is one of the non-specific free radical scavenging assays since it measures scavenged free radicals from both phenolic and non-phenolic compounds, including ascorbic acid. Therefore, the antioxidant potential of plant polyphenols cannot be properly assessed only through DPPH assays. For this reason, a set of different in vitro reagent-based assays can be applied to estimate antioxidant potential, while the confirmation of these antioxidant compounds can be achieved through the LC-MS characterization.

The ABTS assay is another widely used method for determining the antiradical scavenging abilities based on the hydrogen atom donating tendency of phenolic compounds. The scavenged ABTS free radicals were measured using a colorimetric assay where antioxidants in samples reduce ABTS<sup>+</sup> and form a stable free radical [15]. The ABTS assay exhibits high similarity with that of the DPPH assay with the highest ABTS value from the grapefruit peel with 10.79 ± 0.56 mg AAE/g, followed by the mango peel (9.32 ± 0.24 mg AAE/g), kiwi fruit peel (8.95 ± 0.18 mg AAE/g), and avocado peel (7.19 ± 0.72 mg AAE/g) samples. In comparison, banana, dragon fruit, melon, nectarine, passion fruit, peach, pear, and plum peels exhibited relatively low ABTS radical scavenging ability. Previously, a similar ABTS·+scavenging tendency was found in white and pink freeze-dried grapefruit peel extracts [42]. Pal, et al. [43] also found the ABTS radical scavenging ability in kiwi fruit at different ripening stages. Tremocoldi, et al. [44] reported slightly higher ABTS radical scavenging activities in different avocado varieties, including Hass and Fuerte peel samples, as compared to our results. Ortega-Arellano, et al. [45] also reported the ABTS antioxidant activity for both Hass and Reed peels, which is consistent with our results.

The FRAP assay evaluates the ability of samples to donate electrons to reduce a Fe+<sup>3</sup> -TPTZ complex to a blue Fe+<sup>2</sup> -TPTZ complex. Grapefruit peel exhibited the highest FRAP reducing power with 9.22 ± 0.25 mg AAE/g, followed by mango peel (6.19 ± 0.26 mg AAE/g), avocado peel (3.65 ± 0.07 mg AAE/g) and apple peel samples (3.20 ± 0.04 mg AAE/g), while the FRAP reducing power from dragon fruit, melon, passion fruit, pear, and plum peels were relatively low as compared to other fruit peels. Previously, Oboh and Ademosun [46] also reported high FRAP activity in orange and apple peels that was attributed to their bound phenolics compounds and flavonoids. Furthermore, FRAP activities previously reported in other fruit peels including kiwifruit, lime, pineapple, banana, and mango was also in accordance with our study [47].

The total antioxidant capacity (TAC) assay is based on an electron transfer mechanism. This assay is very similar to FRAP, where molybdenum (VI) will be reduced to molybdenum (V) through antioxidant compounds or phenolic compounds. Similar to the results of FRAP assay, the highest TAC values were reported in the grapefruit peel (8.77 ± 0.34 mg AAE/g), followed by mango, avocado, and apple peels (6.19 ± 0.23, 4.50 ± 0.16 and 2.97 ± 0.16 mg AAE/g, respectively). In comparison, dragon fruit, kiwi fruit, melon, nectarine, papaya, peach, pear, and plum peels had relatively low TAC values. The strong antioxidant activities including DPPH, ABTS, and FRAP of different citrus fruits have already been reported, while grapefruit had the strongest antioxidant potential [48]. Antioxidant assays involved multiple reactions and mechanisms to estimate the antioxidant potential of any plant material, and unfortunately, there is no single method that can accurately reflect the overall antioxidant potential due to the complex nature of phytochemicals. For this reason, the MS/MS characterization is one of the key areas in phytochemical research to used compute overall phenolic compounds and their antioxidant potential.

In general, grapefruit, mango, and avocado peels exhibit distinctive antioxidant activity in four different types of antioxidant assays. Our polyphenolic and antioxidant results indicated that further research is needed to determine the actual contribution of polyphenols toward the antioxidant potential by minimizing other distracting factors of in vitro reagent-based assays, including the contribution of non-phenolic compounds toward the antioxidant potential.
