Polyphenolic QTOF-ESI MS Characterization and the Antioxidant and Cytotoxic Activities of Prunus domestica Commercial Cultivars from Costa Rica

There is an increased interest in plum research because of their metabolites’ potential bioactivities. In this study, the phenolic profiles of Prunus domestica commercial cultivars (Methley, Pisardii and Satsuma) in Costa Rica were determined by Ultra Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry using a quadrupole-time-of-flight analyzer (UPLC-ESI-QTOF MS) on enriched phenolic extracts obtained through Pressurized Liquid Extraction (PLE) under acidic and neutral extraction conditions. In total, 41 different phenolic compounds were identified in the skin and flesh extracts, comprising 11 flavan-3-ols, 14 flavonoids and 16 hydroxycinnamic acids and derivatives. Neutral extractions for the skins and flesh from all of the cultivars yielded a larger number of compounds, and were particularly rich in the number of procyanidin trimers and tetramers when compared to the acid extractions. The total phenolic content (TPC) and antioxidant potential using the DPPH and ORAC methods exhibited better results for neutral extracts with Satsuma skins and Methley flesh, which showed the best values (685.0 and 801.6 mg GAE/g extract; IC50 = 4.85 and 4.39 µg/mL; and 12.55 and 12.22 mmol TE/g extract, respectively). A Two-Way ANOVA for cytotoxicity towards AGS gastric adenocarcinoma and SW620 colon adenocarcinoma indicated a significant difference (p < 0.05) for PLE conditions, with better results for neutral extractions, with Satsuma skin delivering the best results (IC50 = 60.7 and 46.7 µg/mL respectively) along with Methley flesh (IC50 = 76.3 and 60.9 µg/mL, respectively). In addition, a significant positive correlation was found between TPC and ORAC (r = 0.929, p < 0.05), as well as a significant negative correlation (p < 0.05) between TPC and cytotoxicity towards AGS and SW620 cell lines (r = −0.776, and −0.751, respectively). A particularly high, significant, negative correlation (p < 0.05) was found between the number of procyanidins and cytotoxicity against the AGS (r = −0.868) and SW620 (r = −0.855) cell lines. Finally, the PCA clearly corroborated that neutral extracts are a more homogenous group exhibiting higher antioxidant and cytotoxic results regardless of the part or cultivar; therefore, our findings suggest that PLE extracts under neutral conditions would be of interest for further studies on their potential health benefits.


Introduction
The bioactive properties of polyphenols have generated much interest in recent years, increasing the consumption of drinks and foods rich in these compounds, and generating greater attention for their research. One of the major interests of these compounds relates to their antioxidant activity, i.e., the prevention of oxidative stress measured by radical activity and its related health benefits [1].
There are reports on the beneficial effects of polyphenols in long-term health protection based on data collected in population studies and clinical trials [2]. For instance, polyphenols offer protection against reactive oxygen and nitrogen radical species, UV light, and pathogens, but also reduce the risk of chronic and degenerative diseases, cardiovascular problems, and cancer [3]. These outstanding properties are still in intense study due to new evidence suggesting that their radical scavenging activities are involved in their metabolic modulation properties in vivo [4].
Plums are fruits rich in polyphenols such as proanthocyanidins, flavonoids, hydroxycinnamic acids and coumarins, and in some countries these fruits-which exhibit a great diversity in shape, size, appearance and taste-are used as medicinal products [5,6]. The global annual production of plums is approximately 11,000,000 tons, and they are marketed worldwide, with the highest demand in Europe, North America and Japan [7].
Although attention has increased towards the ways in which polyphenolic extraction methods and conditions may influence the final composition of samples [8], the reports for plums mainly focus on one type of polyphenols, such as acids or flavonoids. In addition, the reports are often performed for the fruit as a whole, or in a selected fruit part, for instance the flesh or skins [5] [7]. Based on preliminary findings showing the potential of polyphenolic contents in the main Costa Rican commercial plum cultivar [9], we aimed to evaluate the skins and flesh of different plum commercial cultivars under different extraction conditions in order to study their polyphenols and their biological activities.
Therefore, the aim of the present work was to obtain enriched polyphenolic extracts of P. domestica commercial cultivars in Costa Rica, and to characterize them through Ultra Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry (UPLC-ESI-MS). Furthermore, the evaluation of the total polyphenolic contents and antioxidant activity using 2,2-diphenyl-1-picrylhidrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) methods, as well as the cytotoxic activity (MTT) on adenocarcinoma AGS gastric cell lines and adenocarcinoma SW620 colon cell lines, was also carried out with the different extracts.

Phenolic Yield and Total Phenolic Contents
The total phenolic content (TPC) was evaluated separately for the skin and flesh from the three main Costa Rican plum cultivars, namely Methley, Satsuma and Pisardii, using Pressurized Liquid Extraction (PLE) under neutral and acidic aqueous methanolic conditions, as described in the Materials and Methods section. The results for the TPC are summarized in Table 1 for the twelve extracts. The results indicated that neutral PLE extractions showed higher TPC content than PLE acid extractions for all of the samples. When comparing among the cultivars, Methley flesh and Satsuma skins showed the highest TPC value (801.6 mg gallic acid equivalents (GAE)/g extract and 685 mg GAE/g extract respectively) using PLE under neutral conditions, while Methley cultivar skin and flesh extracts exhibited the highest values in acidic PLE conditions (539.3 and 567.7 mg GAE/g extract respectively). In turn, Pisardii flesh and skin extracts under acid conditions showed the lowest values (319 and 438.5 mg GAE/g extract, respectively) among all twelve extracts.  2 Values are expressed as the mean ± standard deviation (S.D.). 3 Different superscript letters in the same column or different superscript signs in the same row indicate that the differences are significant at p < 0.05 using one-way analysis of variance (ANOVA) with a Tukey post hoc.
In order to assess the influence of the fruit part and type of extraction on the TPC, a two-way Analysis of Variance (ANOVA) was carried out. The results showed no significant differences for the fruit part; however, there is significant difference (p < 0.05) regarding the extraction method, with neutral PLE extraction yielding better TPC values for both fruit parts. Previous studies have indicated a dependence on the nature of the polyphenols present in the matrix, e.g., with neutral conditions being more favorable because of the structural stability of flavan-3-ols [1], while acidic conditions give better results for anthocyanin in acidic conditions [8]. On the other hand, previous literature reports on the total phenolic contents for plums indicated values for the whole fruits varying between 18.82 to 25.58 mg GAE/g DW for French cultivars [10], and from 1.38 to 7.61 mg GAE/g FW [11,12] for Japanese and European cultivars. Other studies showed TPC values ranging between 85.2 and 773 mg GAE/100 g FW [13][14][15] for the skins of Spanish and Romanian plum cultivars, while our results indicate values between 161 and 631 mg GAE/100 g FW, which are within the range when comparing them with these studies. In respect to the flesh, previous reports have shown TPC values varying from 36.4 to 180.8 mg GAE/100 g FW for European cultivars [13,14], while Costa Rican plum values are higher, ranging from 91.1 to 323 mg GAE/100 g FW. Finally, a report for plum leaf extracts from Romanian cultivars showed values from 82.84-139.67 mg GAE/g extract [16], which is lower than our results for the skin and flesh of Costa Rican plum fruits extracted under neutral PLE conditions ranging between 319.9 and 801.6 mg GAE/g extract.

Profile by UPLC-ESI-MS/MS Analysis
The UPLC-ESI-QTOF MS analysis described in the Materials and Methods section allowed us to identify 41 phenolic compounds in the skin and flesh extracts from the main Costa Rican commercial cultivars, including nine procyanidin oligomers (dimers, trimers and tetramers), the two flavan-3-ol monomers, 14 flavonoids (kaempferol and quercetin derivatives), and 16 hydroxycinnamic acids and related compounds (HCA). Figure 1 shows the chromatograms of the different compounds, and Table 2 summarizes the results of the identification analysis.   In the first group of compounds, a series of 4-hydroxiccinamic acid derivatives were found ( Figure 2). For instance, peaks 24 (Rt = 16.99 min) and 29 (Rt = 20.55 min), which correspond to methyl-coumaroylquinic acid at m/z 351. 10 [5], with a main fragment at m/z 173 that corresponds to the quinic acid fragment with the loss of water.

Flavan-3-ols Derivatives
The third group of compounds are flavan-3-ols, corresponding to monomers and procyanidin oligomers, including dimers, trimers and one tetramer.  On the other hand, several procyanidin oligomers were identified, which can follow different fragmentation pathways-including Retro Diels-Alder (RDA), Heterocyclic Ring Fission (HRF) with the loss of phluoroglucinol (126 Da), and the Quinone-methide (QM) pathway with fragmentation between (epi)catechin units-to yield one of two different QM ions. Figure 5 illustrates this for procyanidin A-type dimers, of which the (epi)catechin units are linked through C2-O-C7 and C4-C8 bonds [26].   [26].
Regarding the total number of polyphenols in P. domestica commercial cultivars from Costa Rica, 306 compounds were found (Table S1), including 84 procyanidin oligomers, 102 flavonoids, and 120 hydroxycinnamic acids and related derivatives (HCA). The Methley and Satsuma cultivars showed the greatest numbers for both parts and extraction methods, with 110 and 102 compounds, respectively, while the Pisardii cultivar exhibited the lowest number, with 94 compounds.
Concerning the skin extracts, the Methley and Satsuma cultivars presented a similar number of compounds, which were higher than the Pisardii cultivar in both acid and neutral extractions, with flavonoids and HCA as the more abundant groups. In sum, the acidic and neutral skin extractions yielded 175 compounds, comprising 67 HCA, 65 flavonoids, and 43 flavan-3-ol monomers and procyanidin oligomers. For the flesh samples, the results showed a greater number of compounds in the neutral PLE conditions with respect to acidic PLE conditions for all of the cultivars. The neutral extraction of Satsuma showed the greatest number of procyanidin oligomers, while Pisardii flesh showed a greater number of HCA derivatives, especially in the acid extraction. In sum, the acid and neutral flesh extractions yielded 131 compounds, including 53 HCA, 37 flavonoids, and 41 flavan-3-ol monomers and procyanidin oligomers.
Finally, comparing the extractions, neutral PLE conditions showed more compounds than acidic PLE ones, with 172 compounds under neutral conditions versus a total of 134 compounds in acidic extractions. Neutral extractions for both skin and flesh are par-ticularly abundant in procyanidins when compared to acid extractions, and are further particularly rich in the number of procyanidin trimer and tetramer oligomers (Table S1).
When comparing with literature reports, our results show a larger number of compounds than those reported for whole plum fruits from Egypt, USA, Serbia and Romania [16,[28][29][30] in flavan-3-ols and HCA, as well as similar results in flavonoids to those found for Egyptian cultivars [29]. Within studies focused on plum skins, our results show a greater number of compounds than cultivars from the USA and Germany [7,31]. In the case of plum flesh, our findings indicate a similar number of compounds to those reported in German cultivars [5], especially for HCA, and higher than those reported for US cultivars [31].

Antioxidant Activity
The DPPH radical scavenging activity and Oxygen Radical Absorbance Capacity (ORAC) results for the twelve extracts from Costa Rican plum commercial cultivars are summarized in Table 3. Among the neutral PLE extractions for the DPPH assay, Methley flesh (IC50 = 4.39 µg/mL) and Satsuma cultivar skins (IC50 = 4.85 µg/mL respectively) showed the highest antioxidant capacity. In PLE acid extractions, Satsuma skin yielded the highest value (IC 50 = 6.73 µg/mL), followed by Methley flesh (IC50 = 6.81 µg/mL). In turn, the lowest value of the three cultivars was found for Pisardii skin and flesh (IC 50 = 12.72 µg/mL and IC 50 12.44 µg/mL) for acid extractions. A two-way ANOVA analysis was performed to evaluate the influence of the two factors, i.e., the fruit part and PLE extraction conditions. In respect to DPPH, the results showed no significant difference for the fruit part or for the extraction conditions. In turn, the ORAC results showed a significant difference (p < 0.05) for the extraction method, with neutral PLE conditions rendering better results when compared to the acidic PLE extraction. Finally, the ORAC values indicated no significant difference for the fruit part. As shown in Figure 7, a correlation analysis was carried out between the TPC (Table 1), DPPH and ORAC values.  The findings indicated a significant positive correlation (p < 0.05) between the TPC and ORAC antioxidant results (r = 0.929), as well as a significant negative correlation (p < 0.05) between the TPC and DPPH values (r = −0.860). Therefore, our results align with previous studies reporting a correlation between the total polyphenolic contents and antioxidant results [34,35]. Finally, our findings indicate a negative correlation (p < 0.05) between the DPPH and ORAC values (R = −0.797), which were in agreement with previous studies [36].
A correlation analysis was also performed between the antioxidant values (Table 3) and the presence of polyphenolic groups of the compounds in the twelve extracts. The results indicated no significant correlation (p < 0.05) for flavonoids or HCA, which would explain the fact that although HCA constituted the largest number of skin acidic extracts, it seemed to have a low influence in the antioxidant activity.
In turn, a significant negative correlation (p < 0.05) was found between procyanidins and DPPH radical scavenging (r = −0.790), as well as a significant positive correlation between procyanidins and ORAC antioxidant capacity (r = 0.828), indicating that samples with a predominance of procyanidins, such as skin and flesh extracts under neutral conditions, would yield a higher antioxidant capacity (Table 4). This is in agreement with previous reports on procyanidins [37]; furthermore, the influence of a higher number of oligomers such as procyanidin trimers and tetramers would account for greater antioxidant values, as published earlier on procyanidin oligomers' influence in antioxidant activities [38]. Table 4 summarizes the IC50 values for the cytotoxic effect of P. domestica extracts on three different cell lines, namely AGS gastric adenocarcinoma cell lines, SW620 colon adenocarcinoma cells and Vero normal epithelial kidney cells. In addition, Figure 8 shows the cell viability dose-response curves for each extract.  A two-way ANOVA analysis of the IC50 values presented in Table 4 was performed to evaluate the influence of two factors: the fruit part and the extraction method. The findings indicate there is not a specific part of the fruit with a statistically improved cytotoxic activity; in some cultivars, the flesh showed better cytotoxicity, and in others it was the skin. On the other hand, the two-way ANOVA of the cytotoxic effect also reaffirmed a significant difference (p < 0.05) for extraction conditions, as was previously discussed in this paper for other parameters, such as the total phenolic content and antioxidant activities. The samples extracted in neutral PLE conditions yielded the best cytotoxicity, with values between 46.7 and 186 µg/mL, while the samples extracted in acidic PLE conditions displayed a less cytotoxic effect, with a range from 161 µg/mL to more than 500 µg/mL. As stated earlier, neutral extractions being more favorable could be related to the higher stability of the characterized polyphenols in these conditions [1].

Cytotoxicity
The dose-response curves for each extract displayed in Figure 8 confirm the neutral extraction methodology as the best approach to obtain bioactive compounds. In fact, the four plots demonstrate a marked slope in the dose-response curves for the neutral extractions compared to the acid extractions. This pattern is independent of the cell line evaluated and the fruit part used for the polyphenol extracts. The comparison between the cytotoxic curves demonstrates that the Satsuma cultivar, for both the skin and flesh neutral extracts, as well as Methley flesh and Pisardii skin, present some of the best slopes in the dose-response curves in both tumoral cell lines tested.
Few previous publications reported the cytotoxic effect of P. domestica extracts in tumoral cell lines. A study that compared P. domestica cultivars displayed IC 50 values ranging from 71.1 µg/mL to 144.5 µg/mL for a colorectal carcinoma cell line (HCT116); among them was a Methley cultivar grown in India, for which an IC50 of 85.9 µg/mL was reported [39]. In our assessment, Satsuma skins and Methley flesh extracts display IC 50 values of 46.7 and 60.9 µg/mL, respectively, incubated with a colon adenocarcinoma cell line (SW-620), indicating a higher cytotoxic activity.
A significant negative correlation (p < 0.05) was observed between the TPC and IC50 cytotoxic values on AGS adenocarcinoma (r = −0.776), as well as TPC and IC50 cytotoxic values on SW620 adenocarcinoma (r = −0.751). These correlations suggest a role of the total content of polyphenolic compounds in the cytotoxic effect in tumoral cells. In addition, a specific correlation analysis was performed between the IC50 cytotoxic activity (Table 4) and the number of compounds of each type of polyphenol group identified (Tables 2 and 3). The results showed no significant correlation with HCA or flavonoids for either the AGS or SW620 cell lines. However, the cytotoxic activity exhibited an important, significant negative correlation (p < 0.05) with the number of procyanidins for both cell lines, AGS (r = −0.868) and SW620 (r = −0.855). These specific r coefficient values are higher than the TPC r coefficients, suggesting procyanidins as the strong determinants of the anti-tumor activity. This suggestion is also supported by the fact that neutral PLE extractions have better cytotoxicity than acidic extracts, as discussed before, and according to the ESI-QTOF MS profile, the neutral extracts are particularly abundant in procyanidins compared to acid extractions (Tables 2 and 3).
Procyanidins association with cytotoxicity against tumoral cell lines have been described previously for human breast adenocarcinomas [40], oral squamous cells [41], prostate carcinomas [42], hepatocellular carcinomas [43,44], and mostly for colorectal carcinomas [45][46][47][48][49][50]. The publications that assessed the cytotoxicity of tumoral cells linked to procyanidins mainly used extracts from sources such as grape seeds and cocoa [41,46,51]. In the case of grape seed procyanidins, extracts incubated with colorectal carcinomas showed a maximum level of cytotoxicity of 33% in HT-29, and 56% in SW-480 cells treated for 48 h with 100 µg/mL [48]. In our study, for a Satsuma skin neutral extract, a 50% cytotoxic activity was observed in SW-620 cells treated with 46.7 µg/mL, and for Methley flesh neutral extract with 60.9 µg/mL, thus exhibiting better results than those grape skin extracts. Another report on a procyanidin extract from a different species, Bactris guineensis, showed better cytotoxicity (IC 50 16.6 µg/mL) towards SW-620 cells [50] while exhibit-ing a lower cytotoxicity towards AGS gastric adenocarcinoma cell lines with an IC 50 of 113.6 µg/mL. Our study shows a better cytotoxicity towards these AGS gastric cancer tumor lines for neutral extracts from Satsuma skins and flesh, as well as from Methley flesh with IC 50 values of 60.7, 75.0 and 76.3 µg/mL, respectively. In addition to this, studies show the relationship between procyanidins and higher cytotoxicity on human cancer cells [46,51]; these flavan-3-ol sources may display different in vitro bioactivities for various cancer cells, as the data is influenced by their structure and interactions at the biological level.
In respect to selectivity towards the tumoral cell lines (SW-620, and AGS) when compared to the non-tumor cells (Vero), for all acidic and neutral extracts, a larger cytotoxicity effect was observed. In order to quantify this specificity against the cancer cells, Table 4 shows the values of the selectivity index, which is defined as the ratio of the IC50 values of non-tumor cells to cancer cells. Particularly in the neutral extracts, the highest selectivity index corresponds to SW-620 cells, with SI values from 3.8 to 8.2. In the case of AGS cells, the SI values were between 2.7 and 6.6. This selectivity reported for Costa Rican plum cultivars is better than the SI of 4.1 reported for a plum extract which was used to treat breast cancer cells (MDA-MB-435) compared to breast epithelial cells [52]. Higher values of the selectivity index are desirable, and suggest a possible therapeutic potential. Extracts with an SI greater than 3 are considered to have a high selectivity towards cancer cells [53,54]. This selectivity could be associated with an apoptotic activity [54]. The apoptotic effect has been previously described for a plum extract used to treat human hepatoma HepG2 cells [55], and in the literature this effect has also been associated with the presence of procyanidins [50,56].
These findings merit further studies on other cancer cell types, as plums are edible sources of these bioactive polyphenols, and the results suggest these plum extracts-and particularly Satsuma skins and Methley flesh-as strong candidates for future preventive and therapeutic approaches.

Principal Component Analysis for Polyphenolic Extracts of P. domestica
In order to summarize the findings, a statistical Principal Component Analysis (PCA) was performed (n = 12) considering TPC, DPPH, and ORAC antioxidant values, as well as the cytotoxicity results on gastric adenocarcinoma AGS and colon adenocarcinoma SW620 of polyphenolic extracts. Two components (PC1 and PC2) were obtained (loadings > 0.41), as shown in Figure 9. The first component (PC1) represented 85.83% of the total variance and showed a positive correlation with ORAC, as well as a negative correlation with the DPPH, AGS and SW620 IC 50 values. The second component (PC2) accounted for 8.36% of the total variance, and was positively correlated to the TP values.
As illustrated in the plane represented by the two components (Figure 9), the skin and flesh extracts are distributed along PC1, and show a less important distribution along PC2, indicating a high variability for their ORAC and DPPH values and cytotoxicity IC 50 results on AGS and SW620 cancer cell lines. However, a clear trend can be observed for all of the samples obtained under neutral extraction conditions with better values than the ones obtained under acidic extraction conditions. Some samples have particularly low values in PC1-for instance, Pisardii cultivar skins and flesh extracts under PLE acid conditions-exhibiting poorer antioxidant and anticancer results. Meanwhile, Methley flesh and Satsuma skin neutral extracts show the highest PC1, which agrees with the results discussed in the previous sections, accounting for their especially high antioxidant and cytotoxic activity among all twelve extracts. Furthermore, our findings showed that all of the acid extracts are grouped with low scores in PC1 and, in contrast, neutral samples are more homogenously grouped in the higher score in PC1, with a greater antioxidant capacity and cytotoxic activity towards AGS and SW620 tumor cells, independently of the part or cultivar involved. In sum, as discussed previously, the results show that P. domestica extracts obtained under Neutral PLE conditions, particularly from the flesh of the Methley cultivar and from the skins of the Satsuma cultivar, are promising substrates for further studies to assess their actual benefits on human health.

Materials, Reagents and Solvents
P. domestica fruits of the Methley, Pisardii and Satsuma cultivars were acquired in a ripe state from Frutalcoop local producers in Los Santos, Costa Rica. The cultivars were confirmed with the support of the Costa Rican National Herbarium, and vouchers were deposited there. The reagents, such as fluorescein, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), 2,2-diphenyl-1-picrylhidrazyl (DPPH), Trolox, gallic acid, Amberlite XAD-7 resin, fetal bovine serum, glutamine, penicillin, streptomycin, amphotericin B, and trypsin-EDTA, were provided by Sigma-Aldrich (St. Louis, MO, USA). The human gastric adenocarcinoma cell line AGS, human colorectal adenocarcinoma SW 620 and monkey normal epithelial kidney cells Vero were obtained from the American Type Culture Collection (ATCC, Rockville, MD), while the solvents-such as acetone, chloroform and methanol-were purchased from Baker (Center Valley, PA, USA), and the DMSO was acquired from Sigma-Aldrich (St. Louis, MO, USA).

Phenolic Extracts from Prunus domestica Fruits
The P. domestica fruits were rinsed in water, peeled, and both the skin and flesh material were freeze-dried in a Free Zone −105 • C, 4.5 L, Cascade Benchtop Freeze Dry System (Labconco, Kansas, MO, USA). The freeze-dried material was preserved at −20 • C until extraction. The freeze-dried samples were extracted under Pressurized Liquid Extraction (PLE) conditions in a Dionex™ ASE™ 150 Accelerated Solvent Extractor (Thermo Scientific™, Walthman, MA, USA) using methanol:water (70:30) as the solvent in a 34 mL cell, at 40 • C, under neutral conditions or using 0.1% formic acid. Next, the extract was evaporated under vacuum to eliminate the methanol, and the aqueous phase was washed with ethyl acetate and chloroform to remove the less-polar compounds. Afterwards, the aqueous extract was evaporated under vacuum to eliminate organic solvent residues, and was eluted (2 mL/min) in an Amberlite XAD7 column (150 mm × 20 mm), starting with 300 mL of water to remove the sugars, and then with 200 mL each of methanol and water (80:20) and pure methanol to obtain the polyphenols. Finally, the enriched extract was obtained after evaporating to dryness using a Buchi™ 215 (Flawil, Switzerland) rotavapor.

Total Phenolic Content
The polyphenolic content was determined as previously reported [57] by a modification of the Folin-Ciocalteu (FC) method [58], for which the reagent is composed of a mixture of phosphotungstic and phosphomolybdic acids. Each sample was dissolved in MeOH (0.1% HCl) and combined with 0.5 mL FC reagent. Afterwards, 10 mL Na 2 CO 3 (7.5%) was added and the volume was completed to 25 mL with water. The blanks were prepared in a similar way, but using 0.5 mL MeOH (0.1% HCl) instead of the sample. The mixture was left standing in the dark for 1 h, and then the absorbance was measured at 750 nm. The values obtained were extrapolated in a gallic acid calibration curve. The total phenolic content was expressed as mg gallic acid equivalents (GAE)/g sample. The analyses were performed in triplicate.

UPLC-ESI-MS Analysis
The UPLC-MS system used to analyze the composition of the P. domestica extracts consisted of an Xevo G2-XS QTOF (Waters, UK) coupled with an AQUITY H Class UPLC system with a quaternary pump. The ESI source parameters were set to a capillary voltage of 2 kV, a sampling cone of 20 eV, a source temperature of 150 • C, and a source offset of 10 • C. The desolvation temperature was set to 450 • C, the cone gas flow was set to 0 L/h, and the desolvation gas flow was set to 900 L/h.
The measurement was performed in MSe negative mode using an acquisition mass range from 100 m/z to 2000 m/z and a scan rate of 0.5 s, where the fragmentation was carried out using Independent Data Acquisition for all of the eluting compounds, with a collision energy ramp from 20 V to 30 V for the high-energy function. Instrument calibration was applied in the mass range of the measurement with sodium formate. Lock mass correction was applied directly to the measurement using a leucine enkephalin infusion measured every 30 s during the run.
The separation was carried out on a Luna RP-C18 column (150 mm × 4.6 mm i.d. × 4 µm, Phenomenex, Torrance, CA) with a pre-column filter (Phenomenex, Torrance, CA, USA). In total, 1 uL of the sample was injected at a flow rate of 0.3 mL/min using a gradient of Mobile phase A and B consisting of a combination of 0.1% formic acid in water, v/v, and 0.1% formic acid in acetonitrile, v/v, respectively. The gradient was from 4% to 20% B (v/v) at 20 min, to 35% B at 35 min, and to 100% B at 40 min, and was held at 100% B to 45 min.

DPPH Radical-Scavenging Activity
The DPPH evaluation was performed as previously reported [59], and was expressed as IC 50 (µg/mL), which is the amount of sample required to reach the 50% radicalscavenging activity. Briefly, a solution of 2,2-diphenyl-1-picrylhidrazyl (DPPH) (0.25 mM) was prepared using methanol as the solvent. Next, 0.5 mL of this solution was mixed with 1 mL of the extract at different concentrations, and incubated at 25 • C in the dark for 30 min. The DPPH absorbance was measured at 517 nm. Blanks were prepared for each concentration. The percentage of the radical-scavenging activity of the sample was plotted against its concentration to calculate IC 50 (µg/mL). The samples were analyzed in three independent assays. Trolox was used as the reference antioxidant agent. A methanolic solution at different concentrations was used instead of the samples along the described procedure. Trolox IC 50 (µg/mL) was converted to mmol/mL using the Trolox molecular weight (250.29 mg/mmol), and was then divided by the IC 50 of each sample in order to express the DPPH results as mmol of Trolox equivalents (TE)/g extract.

ORAC Antioxidant Activity
The ORAC (Oxygen Radical Absorbance Capacity) antioxidant activity was determined following a method previously described [60,61] using fluorescein as a fluorescence probe. The reaction was performed in 75 mM phosphate buffer (pH 7.4) at 37 • C. The final assay mixture consisted of AAPH (12 mM), fluorescein (70 nM), and either Trolox (1-8 µM) or the extract at different concentrations. The fluorescence was recorded every minute for 98 min in black 96-well untreated microplates (Nunc, Denmark), using a Polarstar Galaxy plate reader (BMG Labtechnologies GmbH, Offenburg, Germany) with 485-P excitation and 520-P emission filters. Fluostar Galaxy software version 4.11-0 (BMG Labtechnologies GmbH, Offenburg, Germany) was used to measure the fluorescence. Fluorescein was diluted from a stock solution (1.17 mM) in 75 mM phosphate buffer (pH 7.4), while the AAPH and Trolox solutions were freshly prepared. All of the reaction mixtures were prepared in duplicate, and three independent runs were completed for each extract. The fluorescence measurements were normalized to the curve of the blank (no antioxidant). From the normalized curves, the area under the fluorescence decay curve (AUC) was calculated as: where f 0 is the initial fluorescence reading at 0 min, and f i is the fluorescence reading at time i. The net AUC corresponding to a sample was calculated as follows: The regression equation between the net AUC and antioxidant concentration was calculated. The ORAC value was estimated by dividing the slope of the latter equation by the slope of the Trolox line obtained for the same assay. The final ORAC values were expressed as the mmol of Trolox equivalents (TE)/g of the phenolic extract. The human gastric adenocarcinoma cell line AGS, the human colorectal adenocarcinoma SW 620, and monkey normal epithelial kidney cells Vero were grown in minimum essential Eagle's medium (MEM) containing 10% fetal bovine serum (FBS) in the presence of 2 mmol/L glutamine, 100 IUmL −1 penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin B. The cells were grown in a humidified atmosphere containing 5% CO 2 at 37 • C, and were sub-cultured by detaching them with trypsin-EDTA solution at about 70-80% confluence. For the experiments, 100 µL of a cell suspension of 1.5 × 10 5 cells/mL was seeded overnight into 96-well plates. The cells were further exposed for 48 h to various concentrations of extracts (50 µL), dissolved in DMSO, and diluted with cell culture medium to final concentrations between 15-500 µg/mL. The DMSO concentrations used in the experiments were below 0.1% (v/v), and the control cultures were prepared with the addition of DMSO (the vehicle control).

Assessment of the Cytotoxicity by the MTT Assay
After incubation for 48 h, MTT assays were performed to evaluate the cytotoxicity. Briefly, the medium was eliminated, the cells were washed twice with 100 µL PBS and incubated with 100 µL MTT solution (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, 5 mg/mL in a cell culture medium) for 2 h at 37 • C. The formazan crystals formed were dissolved in 100 µL 95% ethanol, and the absorbance was read at 570 nm in a microplate reader. Dose-response curves were established for each extract, and the concentration which was enough to reduce the cell viability by 50% (IC 50 ) was calculated.
In order to evaluate whether the cytotoxicity activity was specific against the cancer cells, a selectivity index (SI) was determined. This index is defined as the ratio of the IC 50 values of normal epithelial kidney cells (Vero) to cancer cells (AGS or SW620).

Statistical Analysis
One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was applied to the TPC, DPPH, ORAC and cytotoxicity results, and the differences were considered significant at p < 0.05. Two-way analyses of variance (ANOVA) were applied to evaluate both factors, i.e., the part of the fruit and the extraction conditions used. In order to evaluate whether the total phenolic contents (TPC) contribute to the antioxidant activity evaluated with the DPPH and ORAC methodologies, a correlation analysis was carried out as well as the cytotoxicity assays. Finally, a Principal Component Analysis (PCA) was performed using R (version x64 4.1.1) as the statistical program.

Conclusions
This paper reports valuable information on the profile of the phenolic-enriched extracts of the main commercial cultivars of P. domestica in Costa Rica, using UPLC-ESI-QTOF MS techniques, showing a total of 306 compounds characterized in the twelve extracts obtained under acidic and neutral PLE conditions for the skins and flesh of the Methley, Pisardii and Satsuma cultivars. Extracts obtained under Neutral PLE conditions exhibited a higher number of polyphenols for the skins and flesh in all three cultivars, and indicated a higher and more diverse number than the cultivars from other countries reported in the literature. The PCA results align with the higher values obtained under neutral PLE conditions for TPC, DPPH and ORAC antioxidant activities, and with the cytotoxicity towards AGS and SW 620 adenocarcinoma cell lines. Furthermore, these cytotoxicity values show a high significant negative correlation with procyanidins (r= −0.868 and r= −0.855 respectively). As discussed, the relationships of these metabolites and their antioxidant capacity with health benefits [62], including anti-inflammatory effects and cancer chemo-preventive properties, suggest their potential application as functional ingredients [63]. In sum, the overall results-and particularly those obtained for Methley flesh and Satsuma skins-suggest the potential of these Neutral PLE extracts to carry further research, for instance, using other cancer cell lines and antioxidant models [64], towards a comprehensive approach to their potential application in the nutraceutical industry.