*2.8. Statistical Analysis*

In order to evaluate if the total phenolic contents (TPC) contribute to the antioxidant activity evaluated with the DPPH and ORAC methodologies, a correlation analysis was carried out between the TPC values and the DPPH and ORAC results. Also, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was applied to the TPC, DPPH, and ORAC values, and differences were considered significant at *p* < 0.05.

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

#### *3.1. Phenolic Yield and Total Phenolic Contents*

The extraction process described in the Materials and Methods section allowed the phenolic-enriched extracts to be obtained, which are expressed as g of phenolic enriched extract/100 g of dry material and are summarized in Table 1. Keitt cultivar skin presented the highest yield (2.77 g extract/100 g dry material) whereas Tommy Atkins flesh showed the lowest value (0.57 g extract/100 g dry material). In both cultivars, skin extract yields were higher than flesh extracts.


**Table 1.** Extraction yield and total phenolic content.

1 g of dry material/100 g of fresh weight 2 g of phenolic enriched extract/100 g of dry material 3 mg of gallic acid equivalents (GAE)/g extract. 4 Values are expressed as mean ± standard deviation (S.D.) 5 Different superscript letters in the column indicate differences are significant at *p* < 0.05.

The total phenolic contents (TPC) summarized in Table 1 show results ranging between 162.7 and 698.7 gallic acid equivalents (GAE)/g dry extract. The one-way analysis of variance (ANOVA), with a Tukey post hoc as the statistical test, showed a significant di fference (*p* < 0.05) between results for the skin and flesh of *M. indica* samples, with a much higher average for skins corresponding to 671.4 GAE/g dry extract compared to a three times lower average value of 226.9 GAE/g dry extract for flesh. Total phenolic contents (TPC) previous reports for the flesh of T. Atkins cultivars from Mexico and Spain range between 15.3 and 21.77 mg GAE/100 g FW [8,33], whereas our finding of 16.3 mg GAE/100 g FW (value calculated using TPC and lyophilization yields from Table 1) fits within that range. Meanwhile, values reported for the skins of T. Atkins (43.17 mg GAE/100 g FW) and Pica (72.01 mg GAE/100 g FW) cultivars from Chile [34] are lower than our result of 380.9 mg GAE/100 g FW (value calculated using TPC and lyophilization yields from Table 1). In respect to Keitt flesh, results from the literature show variability, reporting values ranging between 17.99 and 59.43 mg GAE/100 g FW for Keitt and other cultivars from Italy and China [21,33] whereas our result of 33.9 mg GAE/100 g FW (value calculated using TPC and lyophilization yields from Table 1) fits in that range. Finally, regarding Keitt skin, previous results for this and other cultivars from China range between 368.52 and 641.9 mg GAE/100 g FW [21]. Our finding of 402.5 mg GAE/100 g FW (value calculated using TPC and lyophilization yields from Table 1) fit within that range and are higher than values found for the Irwin cultivar (26.9 mg GAE/g extract) from Korea [35]. TPC content variations are linked to polyphenols present in extracts [36,37] and the influence of these metabolites in extracts' biological properties, such as the antioxidant capacity [38,39] and cytotoxic activity [14,22], as discussed in the following sections.

#### *3.2. Profile by UPLC-DAD-ESI-TQ-MS Analysis*

The UPLC-DAD-ESI-MS/MS analysis described in the Materials and Methods section allowed identification of 71 compounds, including 32 gallates and gallotannins, six xanthonoids, eight hydroxybenzophenones, eight flavonoids, and 11 phenolic acids and derivatives, in Costa Rican Keiit and T. Atkins commercial cultivars. Figures 1 and 2 show the chromatograms of the 71 di fferent compounds and Table 2 summarizes the results of the identification analysis.

**Figure 1.** High Performance Liquid Chromatography (HPLC) chromatogram of *Mangifera indica* Tommy Atkins cultivar skin extract, in a Hypersil Gold AQ RP-C18 column (200 mm × 2.1 mm × 1.9 μm) using an LTQ Orbitrap XL Mass spectrometer (Thermo Scientific™, Walthman, MA, USA) in a mass range from 100 to 2000 amu.

**Figure 2.** High Performance Liquid Chromatography (HPLC) chromatogram of *Mangifera indica* Keitt flesh extract in a Hypersil Gold AQ RP-C18 column (200 mm × 2.1 mm × 1.9 μm) using an LTQ Orbitrap XL mass spectrometer (Thermo Scientific™, Walthman, MA, USA) in a mass range from 100 to 2000 amu.


**Table 2.** Profile of phenolic compounds identified by UPLC-DAD-ESI-TQ-MS analysis for mangoes Keitt and T. Atkins samples.


**Table 2.** *Cont.*

#### *Foods* **2019**, *8*, 384






**Table 2.** *Cont.*

#### 3.2.1. Benzoic and Hydroxycinnamic Acids

Peaks 6 and 9 had an [M−H]<sup>−</sup> ion at *m*/*z* 299.0780 (C13H15O8) and a main MS2 fragment at *m*/*z* 137 [M−H−162]<sup>−</sup> corresponding to a loss of hexose (Glc). Thus, those peaks were identified as isomers of hydroxybenzoic acid hexoside [40], as shown in Figure 3.

**Figure 3.** Structure and fragments of acid derivatives (6) and (9).

As represented in Figure 4, peak 12 was identified as 5-hydroxyferuloyl hexoside, with [M−H]<sup>−</sup> = 371.0993 (C16H19O10), with fragments at *m*/*z* 233 due to the fragmentation of the aromatic moiety, and at *m*/*z* 209 [M−H−162]<sup>−</sup> due to loss of an hexoside. [41] Peak 17 had an [M−H]<sup>−</sup> ion at 193.0515 (C10H9O4) that agreed with ferulic acid, with fragments at *m*/*z* 178, 149, and 134, due to a loss of methyl groups [M−H−15]<sup>−</sup>, carbon dioxide from the carboxylic acid [M−H−44]<sup>−</sup>, and cleavage through the double bond [M−H−59]<sup>−</sup> [42]. Peak 24 was assigned to sinapic acid due to its [M−H]<sup>−</sup> ion at 223.0618 (C11H11O5), which had main fragments at *m*/*z* 208 [M−H−15]<sup>−</sup>, 179 [M−H−44]<sup>−</sup>, and 164 [M−H−59]<sup>−</sup>. Peaks 27 ([M−H]<sup>−</sup> = 517.2304, C24H37O12) and 28 ([M−H]<sup>−</sup> = 519.2462, C24H39O12) were identified respectively as sinapic acid *O*-pentosyl-hexoside and dihydrosinapic acid *O*-pentosyl-hexoside, which showed an MS2 fragment at [M−H−132]<sup>−</sup> due to a loss of pentoside, and an MS3 fragment at [M−H−132−162]<sup>−</sup> due to a subsequent loss of hexoside. [20].

**Figure 4.** Structure and fragments of acid derivatives (12), (17), (24), (27), and (28).

Peaks 35, 37, and 38 (Figure 5) were identified as isomers of a derivate of syringic acid hexoside, which has an [M−H]<sup>−</sup> ion at 403.1621 (C18H27O10) and successive fragments at *m*/*z* 241 (loss of hexoside) and 197 (aglycone of syringic acid) [43]. Peak 47 had [M−H]<sup>−</sup> ion at 300.9995 (C14H5O8) that is coincident with ellagic acid, whose fragments at *m*/*z* 257 and 229 were previously reported [19,44].

**Figure 5.** Structure and fragments of acid derivatives (35), (37) and (38).

## 3.2.2. Other acids

As shown in Figure 6, peak 1, [M−H]<sup>−</sup> = 191.0568, whose molecular formula was C7H11O6, agreed with quinic acid. Peaks 16 and 33 had an [M−H]<sup>−</sup> ion at 443.1934 (C21H31O10), and fragments at *m*/*z* 425 (loss of water), 281 (loss of hexoside), 237 (α-cleavage to carbonyl group), and 219 (cleavage of double bond); so they were identified as isomers of dihydrophseic acid hexoside [45].

**Figure 6.** Structure and fragments of acid derivatives (1), (16), and (33).
