3.3.2. Flavonoids

Flavonoids (in total 86) are the most abundant class with antioxidant potential found in the fruit peels. Flavonoids were divided into eight subclasses, including flavanols (11), flavones (12), flavanones (8), flavonols (19), dihydrochalcones (3), dihydroflavonols (2), anthocyanins (12) and Isoflavonoids (19).

A total of eight flavanones was discovered in the peels. Quercetin 3'*-O-*glucuronide (Compound **82**) and myricetin 3*-O-*arabinoside (Compound **83**) were found in both modes and tentatively identified by the precursor ions [M − H]<sup>−</sup> *m*/*z* at 477.067 and [M − H]<sup>−</sup> *m*/*z* at 449.0716. The product ion at *m*/*z* 301 in the MS<sup>2</sup> spectrum of quercetin 3'*-O-*glucuronide was produced by the loss of glucuronide (176 Da) from the precursor [56], and the peaks at *m*/*z* 317 (loss of pentose moiety, 132 Da) confirmed the identity of myricetin 3*-O-*arabinoside [57].

#### 3.3.3. Other Polyphenols

A total of 25 other polyphenols were identified from the peels, which were further divided into hydroxycoumarins (5), hydroxybenzaldehydes (2), hydroxybenzoketones (2), hydroxyphenylpropenes (1), curcuminoids (3), furanocoumarins (1), phenolic terpenes (2), tyrosols (5) and other polyphenols (4).

Coumarin (Compound **138**) and scopoletin (Compound **139**) were found in both negative and positive modes and tentatively identified according to the precursors [M + H]<sup>+</sup> at *m*/*z* 147.0448 and [M − H]<sup>−</sup> at *m*/*z* 191.0345. In the MS<sup>2</sup> experiment of 147.0448, peaks at *m*/*z* 103 [M + H − CO2] and *m*/*z* 91 [M + H − 2CO] achieved the identification of coumarin, and in the MS/MS spectra of *m*/*z* 191.0345, peaks at 176 [M − H − 15, loss of CH3] are characteristic for scopoletin [58,59].

#### 3.3.4. Lignans

A total of eleven lignans were identified in most of the fruit peels. Compounds **161** and **163** presenting in the positive mode were identified as enterolactone and schisandrin C according to the *m*/*z* 299.1283 and *m*/*z* 385.1652, respectively. The MS/MS experiment achieved the identification of these lignans. Enterolactone exhibited the fragment ions at *m*/*z* 281, 187, and 165, representing the loss of H2O, C6H8O<sup>2</sup> and C9H8O2, respectively [60]. The presence of schisantherin C was verified by the product ions at *m*/*z* 370 (loss of CH3, 15 Da), *m*/*z* 315 (loss of C5H10, 70 Da) and *m*/*z* 300 (loss of CH<sup>3</sup> and C5H10, 85 Da) [61].

#### 3.3.5. Stilbenes

A total of five stilbenes were identified in different fruit peel samples. Resveratrol (Compound **173,** [M − H]<sup>−</sup> *m*/*z* at 227.0709 presenting in custard apple and avocado peels) and resveratrol 5-*O*-glucoside (Compound **174**, [M − H]<sup>−</sup> *m*/*z* at 389.1245 appearing in passion fruit, pomegranate, and kiwi fruit peels) were detected in both ionization modes. In the MS<sup>2</sup> spectra, Resveratrol showed the characteristic *m*/*z* at 212 (loss of CH3), 185 (loss of CHCOH), 157 (loss of CHCOH and CO), and 143 (loss of CHCOH and C2H2O) [62]. The excepted loss of glucoside (162 Da) was observed in the MS<sup>2</sup> fragmentation of resveratrol 5-*O*-glucoside, which allowed the identification of this compound [63].

The LC-MS/MS characterizations of phenolic compounds presented in different fruit peels have remarkable antioxidant capacities. Most of the hydroxycinnamic hydroxybenzoic acids and their derivatives and flavonoid and their derivatives have strong free radical scavenging ability. The presence of these phenolics in different fruit peel samples indicates that these food wastes could be valuable sources of natural antioxidant compounds. In short, these fruit peels could be utilized in different food, feed, nutraceutical, and pharmaceutical industries.

#### *3.4. Distribution of Phenolic Compounds—Venn Diagram*

To further investigate the distribution of phenolic compounds in different fruit peels, Venn diagrams were generated among fruits grown in different climate zones including tropical, sub-tropical, and temperate (Figure 3). Although the aim of this study was not to explore the relationship between

growing regions and phenolic contents in different fruit peel samples, we tentatively characterized their phenolic profiling through Venn diagrams. This preliminary analysis indicates that it is worth further exploring the relationship between growing regions and phenolic contents in different fruit peel samples. The comparison showed that there are differences in the phenolic compositions of fruits grown in different climate zones, and so it may be possible to optimize phenolic levels in these fruits and their peels through the targeted selection of the growing location.

**Figure 3.** Venn diagram of phenolic compounds presented in different fruit peel samples grown in different regions. (**A**) shows the relations of total phenolic compounds present in different fruit peel samples grown in three different zones. (**B**) shows the relations of phenolic acids present in different fruit peel samples. (**C**) shows the relations of flavonoids present in different fruit peel samples. (**D**) shows the relations of other phenolic compounds present in different fruit peel samples.

Fruit peel samples were divided into three groups according to their growing regions, which were tropical (banana, custard apple, dragon fruit, mango, papaya, and pineapple peels), sub-tropical (pomegranate, passion fruit, orange, grapefruit, avocado, lime peels), and temperate (apple, apricot, kiwi fruit, peach, pear, melon, plum, nectarine peels).

From Figure 3A, a total of 375 phenolics were tentatively identified in all twenty selected fruit peels. Among "*total phenolic compounds*", 69.9% of them were commonly identified in all three zones, including tropical, sub-tropical, and temperate regions. From Figure 3B,C, a total of 72.8% of the "*phenolic acids*" and 68.3% of the "*flavonoids*" were commonly identified in all three zones. The proportions of common phenolic acids and flavonoids shared by all the fruit peels were almost similar to that of total phenolic compounds, which indicated the compositions of these compounds were similar in tropical, sub-tropical, and temperate fruits, despite different growing regions. However, Figure 3D shows that "*other phenolic compounds*" had only 56.5% of commonly identified compounds in the three groups, the proportions of which were much lower than those in the total phenolic compounds. The lower proportion of shared compounds of *"other phenolic compound"* indicated that they might be the main contributors responsible for the differences in overall phenolic concentrations and antioxidant activities of different fruit peels collected from three different climatic zones. Additionally, tropical and sub-tropical fruits were more similar in the compositions of other phenolic compounds, while temperate fruits had a quite different composition. The difference may be explained by a previous study, which indicated that tropical fruits often had richer phenolic contents and stronger antioxidant capabilities than temperate fruits due to the presence of some phenolic compounds in tropical fruits that functioned as lipid peroxidation inhibitors and decreased deleterious effects in plants caused by the strong ultraviolet radiation in tropical regions [64]. For example, stilbenes possess an antioxidant ability that can decrease oxidative stress caused by UV irradiation, as well as for the defense system of plants against fungi and bacteria [65]. Also, other phenolic compounds with anti-insect functions might exist exclusively in tropical fruits, as a warmer climate usually favors pest threats [66,67].

In this work we found that there is a strong relationship between growing regions and phenolic contents in different fruit peel samples, and we elucidate the differences in the compositions of phenolic compounds, particularly "*other phenolic compounds*". Further work is required to explore the impacts of individual phenolics.

#### *3.5. HPLC-PDA Quantitative Analysis*

HPLC has been widely used as an effective tool for the identification and quantification of phenolic compounds in different fruit and vegetable samples. The twenty most abundant phenolic compounds present in the different fruit peels, including 10 phenolic acids and 10 flavonoids, were selected for quantification. Tables 2 and 3 show the quantified phenolic acids and flavonoids by comparing retention time with reference standards, and results were calculated using standard curves.

#### 3.5.1. Phenolic Acids

In our study, ten targeted phenolic acids were quantified in the twenty fruit peels. Table 2 showed that the mango peel was most abundant in terms of the overall phenolic acids (72.2 ± 4.5 mg/g) and most of the individual phenolic acid, while melon peels significantly had the lowest overall phenolic acid content. Mango peels significantly had the highest phenolic content for seven out of ten targeted phenolic acids, including gallic acid (14.5 ± 0.4 mg/g), chlorogenic acid (13.8 ± 0.9 mg/g), caffeic acid (4.5 ± 0.4 mg/g), *p*-hydroxybenzoic acid (10.5 ± 0.4 mg/g), syringic acid (11.5 ± 0.7 mg/g), ferulic acid (6.3 ± 0.4 mg/g), and coumaric acid (5.1 ± 0.2 mg/g), respectively. Previously, Palafox-Carlos, et al. [68] detected gallic acid, chlorogenic acid, and protocatechuic acids in different mango varieties, including in both pulp and peels. Chlorogenic acid was the most abundant in their study, while gallic acid was the most abundant in our study. However, Kim, et al. [69] reported that gallic acid was the predominant phenolic acid in mango peels, which is in agreement with our results. In another study, Hu, et al. [70], reported a gallic acid concentration of 0.08–0.59 mg/g among mango peel samples, which is much

lower than our results. These variations can be explained by the variability of phenolic content with cultivar type and maturity, growing regions, and climatic conditions.

Another study of Marina and Noriham [31] indicated that mango peels had higher phenolic content than papaya peels, which also agrees with our quantification results for the ten targeted phenolic acids. Moreover, Gorinstein, et al. [71] also reported a similar trend that mango had significantly higher phenolic contents than avocado peel samples. However, they also reported a higher phenolic content in kiwifruit than in mango, which is in contrast with our results. The difference might be caused by the difference in varieties and growing conditions as they, used grown fruits from Singapore, while our study was conducted on grown fruits from Australia. Apart from mango, other tropical fruits, including banana, custard apple, dragon fruit, papaya, passion fruit, and pineapple peels, did not show significantly higher phenolic contents than other temperate or subtropical fruits, although some of these fruits, such as the banana, were reported to have high phenolic contents and antioxidant ability [72].

Pomegranate was another fruit other than mango which had a significantly higher content for most of the phenolic acids. Previously, Li, et al. [73] detected 249.4 ± 17.2 mg/g phenolic contents in pomegranate peels, which indicated that this fruit was an excellent source of phenolics. Moreover, the study of Marina and Noriham [31] also indicated that pomegranates possessed high phenolic contents. From our results, similar conclusions can be postulated, as pomegranate has a significantly higher phenolic acid content compared with other fruit peels. Additionally, Pal, et al. [74] reported approximately a three-fold higher phenolic content in the pomegranate peel than in the orange peel, which is consistent with our study. Apart from pomegranate, grapefruit and lime peels were also quantified in our study and significantly showed the highest contents for several phenolic acids. Previously, Sir Elkhatim, et al. [75] compared the phenolic contents between peels of citrus fruits including orange, grapefruit, and lime, and reported that grapefruit peels had the highest phenolic content, followed by lime and orange peels, which showed a similar pattern with our results for the targeted phenolic acids. Li, et al. [76] also reported similar results that the grapefruit peel had the highest phenolic contents compared with other citrus fruit peels.

As for temperate fruits, apple peels had significantly higher contents of protocatechuic acid (7.4 ± 0.4 mg/g) than all other fruits. While the apple peel did not have higher overall phenolic acid contents among all the 20 fruits, it is one of the most widely consumed fruits known for its antioxidant ability [77], and importantly, the peel is often consumed. Previously, Russell, et al. [78] reported a higher content of phenolic acids, including gallic acid, protocatechuic acid, *p*-hydroxybenzoic acid, syringic acid, and sinapinic acid, in apple peels than in the peel of pears of Scottish varieties, which is consistent with our results. The study of Mihailovi´c, et al. [79] indicated that chlorogenic acid was the most dominant phenolic acid presented in the apple peel, which is in agreement with our study, which detected the highest chlorogenic acid content of 11.2 ± 0.1 mg/g for apple peels. Moreover, Veberic, et al. [80] also reported that chlorogenic acid was the most abundant phenolic acid in the apple peel with the content range of 4.1–79.5 mg/100 g. The variation can be attributed to a difference in apple varieties. Previous studies have suggested that most tropical fruits have higher phenolic contents than temperate fruits, as phenolic compounds are essential for inhibiting lipid peroxidation and deleterious effects in plant tissues caused by strong ultraviolet radiation in tropical areas [64]. It can also be concluded from previous studies that, although some temperate fruits were already potential phenolic sources, topical fruits had richer phenolic contents, which makes them better sources of phenolic acids [64]. In our study, the tropical fruit mango showed significantly higher phenolic acid content in the peel than all the sub-tropical and temperate fruits, which is consistent with previous studies.

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**Table 2.**Phenolic acids quantified in different fruit peel samples using HPLC-PDA.

All values are expressed as "mg/g", mean ± standard deviation (*n* = 3). Alphabetic letters indicate significant difference (*p* < 0.05) in a row using a one-way analysis of variance (ANOVA) and Tukey's test. Fruit peel samples were mentioned in abbreviations. Apple peel "APL-P", Apricot peel "APR-P", Avocado peel "AVO-P", Banana peel "BNA-P", Custard apple peel "CTA-P", Dragon fruit peel "DGF-P", Grapefruit peel "GRF-P", Kiwifruit peel "KWF-P", Lime peel "LMN-P", Mango peel "MNG-P", Melon peel "MEL-P", Nectarine peel "NEC-P", Orange peel "ORN-P", Papaya peel "PAP-P", Passionfruit peel "PSN-P", Peach peel "PEC-P", Pear peel "PER-P", Pineapple peel "PIN-P", Plum peel "PLM-P", and Pomegranate peel "POM-P".

#### 3.5.2. Flavonoids

Flavonoids are the largest group of phenolics and are present in most of the fruits. Among the fruit peels investigated, the mango peel has the highest content for overall flavonoids (57.1 ± 2.4 mg/g), while passion fruit had the lowest (10.4 ± 1.4 mg/g) listed in Table 3.

Mango peels showed similarly high contents for flavonoids as for phenolic acids, significantly with the highest contents of epicatechin gallate (3.2 ± 0.9 mg/g), quercetin-3-galactoside (10.9 ± 0.1 mg/g), quercetin-3-glucuronide (11.5 ± 0.7 mg/g), quercetin (11.9 ± 0.4 mg/g), and kaempferol (9.8 ± 0.7 mg/g). Previously, catechin and quercetin-3-galactoside were quantified by López-Cobo, et al. [81] in different mango peel samples. Compared with other fruits, Marina and Noriham [31] reported higher catechin and epicatechin contents in mango peels than other tropical fruit peels, such as papaya peel and guava peel, which is consistent with our study. However, a few studies reported lower flavonoids in mango pulp as compared to kiwifruit and avocado pulp, which did not agree with our fruit peel extracts [71]. The contradictory results might be explained by previous literature indicating that peels contained more flavonoids as compared to pulp [68].

Apart from mango peel, dragon fruit peel was also found to be abundant with flavonoids while catechin was dominantly detected in it with a concentration of 7.5 ± 0.9 mg/g. Previously, flavonoids including kaempferol and quercetin derivatives were detected and quantified in dragon fruit peels [82]. The pineapple peel sample had a relatively low flavonoid content among all the twenty fruits which showed a different pattern from mango and dragon fruit peels, but these results agree with the previous study of Silva, et al. [83], who reported significantly higher flavonoid contents in mango, papaya, and passion fruit than in pineapple, in which only a few flavonoids were detected in the pineapple peel sample. The pomegranate peel sample also had higher flavonoids (35.7 ± 4.7 mg/g) similar to phenolic contents. For individual flavonoids, pomegranate peel had the highest epicatechin content (4.1 ± 0.3 mg/g). Previously, Li, Guo, Yang, Wei, Xu and Cheng [73] reported higher flavonoids in pomegranate peel than our results, which may be because we only quantified the ten most abundant flavonoids across the fruit samples observed, and there is a chance that individual fruits may have high concentrations of a flavonoid outside this group. Another study showed that flavonoid contents in pomegranate and mango juices were significantly lower than the phenolic acid contents, which is consistent with our results [68]. Our results indicated that both mango and pomegranate peels are excellent sources of phenolic compounds.

For citrus fruit peels, the kaempferol-3-glucoside content was highest in lime peel (3.7 ± 0.4 mg/g), which is higher than those in orange, grapefruit, and pomegranate. Previously, Singh and Immanuel [84] reported similar results that lime peel had a higher total flavonoid content compared with other citrus species, such as orange. However, a more recent study of Sir Elkhatim, Elagib, and Hassan [75] showed that orange peel contained higher amounts of flavonoids than lime and grapefruit peels, which is in contrast with our results. The variation can be attributed to the difference in fruit varieties and extraction methods. In temperate fruits, quercetin-3-glucoside was the most abundant in apple peel, with a concentration of 4.5 ± 0.9 mg/g. Previously, Schieber, et al. [85] also reported quercetin-3-glucoside was present in apple pomace at a low concentration. Another study of Mihailovi´c, Mihailovic, Kreft, Ciric, Joksovi´c and Djurdjevic [79] reported flavonoids including catechin (0.187 ± 0.007 mg/g) and quercitrin (0.256 ± 0.002 mg/g) from peels of wild apple varieties, which were also detected in our study. In summary, all twenty fruit peel samples have a considerable quantity of phenolic compounds, including both phenolic acids & flavonoids, and these fruit peels are potential commercial sources of these phenolics.


**Table 3.**Flavonoids quantified in different fruit peel samples using HPLC-PDA.

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 ANOVA and Tukey's test. Fruit peel samples were mentioned in abbreviations. Apple peel "APL-P", Apricot peel "APR-P", Avocado peel "AVO-P", Banana peel "BNA-P", Custard apple peel "CTA-P", Dragon fruit peel "DGF-P", Grapefruit peel "GRF-P", Kiwifruit peel "KWF-P", Lime peel "LMN-P", Mango peel "MNG-P", Melon peel "MEL-P", Nectarine peel "NEC-P", Orange peel "ORN-P", Papaya peel "PAP-P", Passionfruit peel "PSN-P", Peach peel "PEC-P", Pear peel "PER-P", Pineapple peel "PIN-P", Plum peel "PLM-P" and Pomegranate peel "POM-P".
