HRMS Characterization, Antioxidant and Cytotoxic Activities of Polyphenols in Malus domestica Cultivars from Costa Rica

There is increasing interest in research into fruits as sources of secondary metabolites because of their potential bioactivities. In this study, the phenolic profiles of Malus domestica Anna and Jonagold cultivars from Costa Rica were determined by Ultra Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry (HRMS) using a quadrupole-time-of-flight analyzer (UPLC-QTOF-ESI MS), on enriched-phenolic extracts from skins and flesh, obtained through Pressurized Liquid Extraction (PLE). In total, 48 different phenolic compounds were identified in the skin and flesh extracts, comprising 17 flavan-3-ols, 12 flavonoids, 4 chalcones, 1 glycosylated isoprenoid and 14 hydroxycinnamic acids and derivatives. Among extracts, the flesh of Jonagold exhibits a larger number of polyphenols and is especially rich in procyanidin trimers, tetramers and pentamers. Evaluating total phenolic content (TPC) and antioxidant activities using ORAC and DPPH procedures yields higher values for this extract (608.8 mg GAE/g extract; 14.80 mmol TE/g extract and IC50 = 3.96 µg/mL, respectively). In addition, cytotoxicity evaluated against SW620 colon cancer cell lines and AGS gastric cancer cell lines also delivered better effects for Jonagold flesh (IC50 = 62.4 and 60.0 µg/mL, respectively). In addition, a significant negative correlation (p < 0.05) was found between TPC and cytotoxicity values against SW620 and AGS adenocarcinoma (r = −0.908, and −0.902, respectively). Furthermore, a significant negative correlation (p < 0.05) was also found between the number of procyanidins and both antioxidant activities and cytotoxicity towards SW620 (r = −0.978) and AGS (r = −0.894) cell lines. These results align with Jonagold flesh exhibiting the highest abundance in procyanidin oligomers and yielding better cytotoxic and antioxidant results. In sum, our findings suggest the need for further studies on these Costa Rican apple extracts—and particularly on the extracts from Jonagold flesh—to increase the knowledge on their potential benefits for health.


Introduction
The increasing popularity and acceptability of herbal medicine is based on natural products being safe and readily available [1]. This awareness is well justified, since evidence of the past decades demonstrates the medicinal properties and functionalities of dietary derived natural compounds and their several health implications. Thus, people Reports from the literature indicate variability among findings in different apple cultivars with total phenolic contents (TPC) values ranging between 5.2-18.0 mg GAE/g DW for skin and 1.3-3.6 mg GAE/g DW for flesh [23,24] for cultivars from Denmark and Germany. Other studies indicate values between 78.2-201.2 mg GAE/100 g FW for skin and 15.9-109.5 mg GAE/100 g FW for flesh [25,26] for cultivars from Canada and China. Comparing with these findings, our results for skin (7.8-8.7 mg/g DW and 173.6-178.5 mg/100 g FW) are within those ranges while they are higher in the case of flesh (3.8-8.6 mg/g DW and 60.3-121.1 mg/100 g FW).

Profile by UPLC-QTOF-ESI MS Analysis
The UPLC-QTOF-ESI MS analysis described in the Materials and Methods section enabled us to identify 48 different compounds, including 4 chalcones, 15 procyanidin oligomers and the 2 flavan-3-ol monomers, 12 flavonols and glycosylated flavonols, 1 glycosylated isoprenoid derivative and 14 hydroxycinnamic acids and related derivatives (HCA), present in Anna and Jonagold Costa Rican apple cultivars. Figures 1 and 2 show the chromatograms of the four samples and Table 2 summarizes the analysis results for the 48 compounds.       is tentatively assigned to the aglycone phloretin, which shows a characteristic main fragment at m/z 167 due to the loss of the benzylic moiety, as previously described [28,29]. Chalcones constitute one group of compounds found in these fruit samples. For instance, peaks 33 (Rt = 26.14 min) and 34 (Rt = 26.95 min) with [M-H] − at m/z 567.1725 (C26H31O14) were identified as diglycoside derivatives of phloretin. They exhibit a main fragment at m/z 273 due to the loss of glycosides to yield the phloretin ion aglycone, thus these peaks are tentatively assigned to phloretin-pentosylhexoside isomers ( Figure 3). In turn, peak 41 (Rt = 30.00 min) with [M-H] − at m/z 435.1312 (C21H23O10) is tentatively identified as phloridzin (phloretin 2′-O-glucose) with main fragments at 273 due to the cleavage of the glycoside [27] and at m/z 167 due to loss of the benzylic group. Finally, peak 48 (Rt = 40.98 min) with [M-H] − at m/z 273.0757 (C15H13O5) is tentatively assigned to the aglycone phloretin, which shows a characteristic main fragment at m/z 167 due to the loss of the benzylic moiety, as previously described [28,29].   (Figure 4). The resulting ion is coincident with vomifoliol, thus allowing the peak to be assigned to a vomifoliol-pentosylhexoside isomer [30]. A relevant and abundant group of compounds found in Costa Rican Anna and Jonagold apples is constituted by flavan-3-ols, corresponding to monomers and procyanidin oligomers, including dimers, trimers, tetramers and several pentamers. Firstly, monomers catechin and epicatechin which were present in peaks 8 (Rt = 9.01 min) and 13 (Rt = 12.14 min). As shown in Figure 5 Figure 4). The resulting ion is coincident with vomifoliol, thus allowing the peak to be assigned to a vomifoliol-pentosylhexoside isomer [30]. Chalcones constitute one group of compounds found in these fruit samples. For instance, peaks 33 (Rt = 26.14 min) and 34 (Rt = 26.95 min) with [M-H] − at m/z 567.1725 (C26H31O14) were identified as diglycoside derivatives of phloretin. They exhibit a main fragment at m/z 273 due to the loss of glycosides to yield the phloretin ion aglycone, thus these peaks are tentatively assigned to phloretin-pentosylhexoside isomers ( Figure 3). In turn, peak 41 (Rt = 30.00 min) with [M-H] − at m/z 435.1312 (C21H23O10) is tentatively identified as phloridzin (phloretin 2′-O-glucose) with main fragments at 273 due to the cleavage of the glycoside [27] and at m/z 167 due to loss of the benzylic group. Finally, peak 48 (Rt = 40.98 min) with [M-H] − at m/z 273.0757 (C15H13O5) is tentatively assigned to the aglycone phloretin, which shows a characteristic main fragment at m/z 167 due to the loss of the benzylic moiety, as previously described [28,29].   (Figure 4). The resulting ion is coincident with vomifoliol, thus allowing the peak to be assigned to a vomifoliol-pentosylhexoside isomer [30]. A relevant and abundant group of compounds found in Costa Rican Anna and Jonagold apples is constituted by flavan-3-ols, corresponding to monomers and procyanidin oligomers, including dimers, trimers, tetramers and several pentamers. Firstly, monomers catechin and epicatechin which were present in peaks 8 (Rt = 9.01 min) and 13 (Rt = 12.14 min). As shown in Figure 5  A relevant and abundant group of compounds found in Costa Rican Anna and Jonagold apples is constituted by flavan-3-ols, corresponding to monomers and procyanidin oligomers, including dimers, trimers, tetramers and several pentamers. Firstly, monomers catechin and epicatechin which were present in peaks 8 (Rt = 9.01 min) and 13 (Rt = 12.14 min). As shown in Figure 5             For these compounds, main fragment ions were observed from QM cleavage as multiples of the monomer: m/z 289, 577 and 865 for tetramers, and additionally m/z 1153 for pentamers [32].    For these compounds, main fragment ions were observed from QM cleavage as multiples of the monomer: m/z 289, 577 and 865 for tetramers, and additionally m/z 1153 for pentamers [32].
Flavonoids constitute another group of compounds found on these apple extracts.      Phenolic acids and derivatives constitute another group of compounds found in these samples. The smallest acid found was peak 18 (Rt = 15.06 min) corresponding to Phenolic acids and derivatives constitute another group of compounds found in these samples. The smallest acid found was peak 18 (Rt = 15.06 min) corresponding to shikimic acid ( Figure 13). Main fragments at m/z 111 and 93 were generated from RDA fission and from subsequent loss of water, respectively.  A series of 4-hydroxycinnamic acid derivatives were identified, as summarized in Table 2. For instance, as shown in Figure 14 A series of 4-hydroxycinnamic acid derivatives were identified, as summarized in Table 2. For instance, as shown in Figure 14  A series of 4-hydroxycinnamic acid derivatives were identified, as summarized in Table 2. For instance, as shown in Figure 14, peak 17 (Rt = 14.55 min) with [M-H] − at m/z 337.0912 (C16H17O8) was assigned to a coumaroylquinic acid with a main fragment at m/z 173 due to the loss of water of the quinic acid ion [34].   173 due to the loss of water of the quinic acid ion [34].

43, 45
OH H H  When comparing data in the literature regarding compound characterization from apple skins, our results for Anna and Jonagold cultivars are similar to the total number of compounds and diversity in Golden Delicious and Braeburn cultivars from Slovenia [28]. In addition, both Costa Rican cultivars show a greater number and diversity in respect to When comparing data in the literature regarding compound characterization from apple skins, our results for Anna and Jonagold cultivars are similar to the total number of compounds and diversity in Golden Delicious and Braeburn cultivars from Slovenia [28]. In addition, both Costa Rican cultivars show a greater number and diversity in respect to other cultivars from Brazil and Canada [25,42,43]. In respect to flesh, the Jonagold cultivar is far superior to Anna and other cultivars from the literature, especially regarding proanthocyanidins both in total occurrence and in greater polymerization degree [24,25,44], for instance in procyanidin trimers, tetramers and pentamers found in Costa Rican Jonagold flesh.
In the case of glycosylated flavonoids, our findings show a similar number of compounds to European cultivars [24,28,44] and they indicate more diversity in quercetin derivatives. In respect to the occurrence of hydroxycinnamic acid derivatives, our results are within the range reported for cultivars from South Korea [45] and Europe [46][47][48]. Finally, for the chalcones group, our findings are similar to results reported for cultivars from Canada and China [25,49].
In sum, as within recent studies on other fruits [50], the profiling of polyphenols reveals high diversity in Costa Rican cultivars, which is in agreement with evidence showing that apples' secondary metabolites profile is greatly influenced by location [44,51] as well as with findings from studies on other species indicating that tropical forests have a greater diversity of secondary metabolites [52]. Thus, the present work can be of interest for further research and future studies should take into consideration the parameters from the cultivars themselves, such as origin, location, soil composition and their relationship with chemical metabolites and bioactivities.

Antioxidant Activity
The DPPH and ORAC values obtained are summarized in Table 3 [53] and 710 µg/mL for flesh [48] in cultivars from India and Portugal, respectively, thus extracts from Anna and Jonagold cultivars show better results for both skins and flesh, as shown in Table 3. Another study on cultivars from Austria reported DPPH values ranging between 2.29 and 7.44 mmol TE/100 g DM [54] with our results for Anna and Jonagold cultivars showing values (2.12-8.05 mmol TE/100 g DM) within that range.  [24] and Norway [47]. On the other hand, other studies report values varying between 0.45-10.62 mmol TE/100 g FW for skins and between 0.19 and 2.61 mmol TE/100 g FW for flesh in cultivars from Chile [56] and Italy [55]. Our results for skins (15.53-18.43 mmol TE/100 g DM and 3.10-4.23 mmol TE/100 g FW) and for flesh (6.37-20.99 TE/100 g DM and 1.01-2.94 mmol TE/100 g FW) from Anna and Jonagold cultivars fall within those ranges.
In addition, a correlation analysis was performed among the total phenolic contents (TPC, Table 1) and the antioxidant activity results from ORAC and DPPH methods. A significant negative correlation (p < 0.05) was found between TPC and DPPH results (R = −0.983) as wells as a significant positive correlation (p < 0.05) between TPC and ORAC values (R = 0.980). Therefore, these results align with previous findings reporting a correlation between total polyphenolic contents and different types of antioxidant activities [57].
Finally, the results for Jonagold flesh are of particular importance since there are few reports on the flesh being richer in polyphenols and having higher antioxidant activity than the skin, which points to the interest for further research on biological models. For instance, some studies have described the antioxidant mechanisms associated with proanthocyanidins with an increase in the Nuclear factor E2-related factor 2 (Nrf2) translocation to the nucleus [58], which activates the transcription of genes responsible for maintaining cellular redox homeostasis and protect cells from oxidative damage [59]. Table 4 summarizes the IC 50 values for the cytotoxic effect of M. domestica extracts on different human carcinoma cells related to the digestive tract, namely AGS (gastric adenocarcinoma) and SW-620 (colorectal adenocarcinoma) cell lines, while the dose-response curves are displayed in Figure 18. The development of digestive tract cancers has been associated with lower consumption of vegetables and fruits [60]; in particular, 60% of stomach cancer and 43% of colon cancer are attributed to deficient consumption of vegetables [61]. In Costa Rica, colon cancer is the second most common cancer and gastric cancer has the third and fourth incidence rate in men and women, respectively [62]. Proanthocyanidins found in apples have been associated with exert antitumoral effects reaching and interacting directly with the gastrointestinal cells [63,64]. Thus, it is of interest to evaluate these extracts' cytotoxicity using as targets these tumoral cancer cell lines.  As observed in Table 4, the best cytotoxic effects of Costa Rican M. domestica against AGS and SW-620 cells were observed for Jonagold cultivar, with the flesh sample (IC 50 values of 60.0 ± 1.7 and 62.4 ± 5.2 µg/mL, respectively). Anna cultivar skin sample showed a moderate cytotoxic effect against AGS cells (IC 50 of 167 ± 10 µg/mL).

Cytotoxicity
The dose-response curves for each extract displayed in Figure 18 confirm Jonagold flesh as the best extract with the highest cytotoxic effect on both AGS and SW620 adenocarcinoma cell lines. In fact, both plots demonstrate a marked slope in the dose-response curves for this extract compared to the other samples. Anna and Jonagold skins show a more moderate cytotoxicity to obtain bioactive compounds, while Anna flesh represents the sample with the lowest cytotoxic effect in both tumoral cell lines tested.
Some studies have evaluated the cytotoxic effect of apples (Malus domestica) in tumoral cell lines and variations were observed for samples of the same species cultivated in different locations. Studies using French apples evaluated the cytotoxic effect against colorectal adenocarcinoma cells (SW-620) and esophageal adenocarcinoma (OE-33) showing 50% of cytotoxicity with similar concentrations of our study (45-60 µg/mL); however, for both studies, the extracts were enriched with a specific type of polyphenol [65,66]. Other studies showed moderate cytotoxic effect with acetone extracts from whole apple extracts from Lithuanian cultivars against the human colon adenocarcinoma cell line (HT-29) and human glioblastoma cell line (U-87), reporting an IC 50 of 113.3 µg/mL and 119.7 µg/mL, respectively [67]. A lower cytotoxic effect was reported for acetone and alcoholic extracts from apple (M. domestica) pomace cultivated in India. These Indian apples achieved 50% cytotoxicity only in oral carcinoma (KB) with concentrations of 100 µg/mL, but for cervical squamous cells carcinoma (SiHa) and colorectal adenocarcinoma (HT-29), 50% was not reached even with treatment of 400 µg/mL [68]. On the other hand, studies of Indian M. domestica apples achieved an improved cytotoxic effect using innovative delivering strategies such as silver nanoparticles. This approach permits an IC 50 of 10 µg/mL [69] and 33.8 µg/mL [70] to be achieved against breast cancer cells (MFC-7).
The cytotoxic effect in tumor cell lines has been reported for other Malus species, also demonstrating very fluctuating results. Malus sieversii acetone extracts, grown in China, were assessed on breast cancer cell lines (MCF-7 and MAD-MB-231) and showed a very low cytotoxic effect (IC 50 of 33.44 mg/mL and 20.94 mg/mL) [71]. Similarly, a methanolic extract of Chinese apples, Malus pumila, were evaluated against cancer colon cells (SW-480), stomach cancer cells (BCG 803) and esophageal cancer cells (CaEs-12) and a weak cytotoxic activity was reported with IC 50 varying between 3.5-4.3 mg/mL in all cell lines [72]. In the opposite side, the cytotoxicity of Chinese apples, M. pumila, was evaluated against liver hepatocellular carcinoma (HepG2) and a strong inhibitory grown rate of 50% was achieved with concentration of less than 4 µg/mL for pulp extracts and less than 20 µg/mL for skin extracts [73]. Finally, ornamental crabapple Malus sp. ("red splendor") has also been studied, and the cytotoxic activity showed values of 48.3 µg/mL, 64.5 µg/mL and 78.9 µg/mL, for SW-480, BCG 803 and CaEs-17, respectively [72].
In addition to the IC 50 values used to quantify the cytotoxic effect, Table 4 shows the selectivity index, which is defined as the ratio of IC 50 values of non-tumor cells to cancer cells. The highest selectivity index values in this study correspond to the Jonagold flesh sample (5.1 for AGS cells and 4.9 for SW-620 cells) and the Anna skin sample (3.0 for AGS cells). According to previous reports, extracts with SI greater than three are considered to have high selectivity towards cancer cells and suggest a possible therapeutic potential [74,75]. For M. domestica extracts, a comparison of IC 50 values of non-tumor cells to cancer cells has been reported previously for breast cancer cell lines. The selectivity ratio in this Indian apple was 2.2, which is a lower value compared to our study, even though the Indian apple extracts were applied to the cells using nanoparticle delivery systems [70].
In addition, correlation analysis was performed between the cytotoxicity results obtained and total phenolic contents. Significant negative correlation (p < 0.05) was found between IC 50 cytotoxic values on SW620 cancer cells and TPC (r = −0.908) and between IC 50 cytotoxic values on AGS cancer cells and TPC (r = −0.902). Furthermore, correlation analysis performed between the IC 50 cytotoxic activity in both adenocarcinoma cell lines (Table 4) and the number of compounds identified for each polyphenol group (Table 2) showed no significant correlation (p < 0.05) with HCA, chalcones or flavonoids for either SW620 or AGS cell lines. In contrast, a significant negative correlation (p < 0.05) with the number of procyanidins was found for cytotoxicity results on both SW620 (r = −0.978) and AGS (r = −0.894) cell lines. These r coefficients represent similar and higher values, respectively, than the ones for TPC, suggesting procyanidin's major contribution to the cytotoxic activity against both tumor cells.
The predominant role of proanthocyanidins in the cytotoxic effect against tumoral cells has been widely documented for grape seeds extracts [76][77][78][79]. However, not many reports are available for other natural sources; some of the few reports include exotic fruits such as Japanese Quince [80] and Bactris guineensis [81] and other widespread consumed fruits, such as berries [82].
The association of proanthocyanidins and cytotoxic effect in tumoral cells has been linked to the degree of polymerization of these polyphenols. For grapes, grape seeds and pine bark assays in colon cancer cells (HCT116, SW-480, SW-620, HT-29, Caco-2, RKO and LoVo), the anti-proliferative effect positively correlated with an increase in the degree of polymerization [83]. Another report [65] compared two polyphenol-enriched fractions from M. domestica, reporting a 50% inhibition of colorectal carcinoma (SW-620) cell growth with 45 µg/mL of the fraction rich in polymers and no effect in the monomer fraction even at a concentration of 100 µg/mL. Other studies in esophageal gastric adenocarcinoma demonstrated that oligomer procyanidins showed more potent antiproliferative activities that the monomeric and dimeric procyanidins [66]. These reports are consistent with the pattern shown in the present study. The strongest cytotoxic activity, an IC 50 of 60 and 63 µg/mL in AGS and SW-620, respectively, was assessed for flesh samples of the Jonagold cultivar (Table 4) which is the one showing an enriched profile of proanthocyanidins oligomers (Table 2), specifically trimers, tetramers and pentamers B-type procyanidins.
The antitumor effect of proanthocyanidins has been associated with an apoptotic induction and a regulation of inflammatory pathways that ends in an inhibition of the tumor cell proliferation [81,84,85]. Some reports from grape seeds and apple procyanidins (M. pumila) have described an induction of cell cycle arrest by down-regulation of cyclin D1, CDK4 and survivin. In addition, these reports describe an induction of apoptosis through an increase in mitochondrial membrane permeability, a cytochrome c release and enhance of caspase 3 and caspase 9 expression and activation, which represents a hallmark of apoptosis [76,85,86]. However, despite these preliminary reports, the specific mechanism has yet to be elucidated. Reports on the bioavailability of procyanidins indicate that these molecules reach the colon almost intact and would interact there with colorectal cancer cells [64] similar to the ones evaluated in this work. In sum, the promising results obtained for Jonagold flesh suggest that there is a need for further studies, for instance in other cancer cell lines, to determine the prospective of these Costa Rican apples as source of enriched proanthocyanidins extracts and their related bioactivities.

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

Phenolic Extracts from Malus Domestica Fruits
M. domestica fruits were rinsed in water, peeled, and both skin and flesh material were frozen at −20 • C and then lyophilized in a Free Zone Cascade Benchtop Freeze Dry System 720401000 (Labconco, Kansas, MO, USA), with Ice Holding Capacity of 4.5 L and Collector Temperature of −105 • C and system vacuum level < 133 × 10 −3 mbar. The lyophilized material was preserved at −20 • C until extraction. 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 solvent in a 34 mL cell, at 40 • C. Next, the extract was evaporated under vacuum to eliminate the methanol and the aqueous phase was washed with ethyl acetate and chloroform to remove less polar compounds. Afterwards, the aqueous extract was evaporated under vacuum to eliminate organic solvent residues and was eluted (2 mL/min) in Amberlite XAD7 column (150 mm × 20 mm), starting with 300 mL of water to remove sugars, and then with 200 mL each of methanol:water (80:20) and pure methanol to obtain the polyphenols. Finally, the enriched extract was obtained after evaporating to dryness at 40 • C using a Buchi™ 215 (Flawil, Switzerland) rotavapor.

Total Phenolic Content
The polyphenolic content was determined as previously reported [87] by a modification of the Folin-Ciocalteu (FC) method [88], whose 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 of FC reagent. Afterwards, 10 mL of Na 2 CO 3 (7.5%) were added and the volume was completed to 25 mL with water. Blanks were prepared in a similar way, but using 0.5 mL of 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. Values obtained were extrapolated in a gallic acid calibration curve. Total phenolic content was expressed as mg gallic acid equivalents (GAE)/g sample. Analyses were performed in triplicate.

UPLC-ESI-MS Analysis
The UPLC-MS system used to analyze the composition of M. domestica extracts consisted of a Xevo G2-XS QTOF (Waters, UK) coupled with an AQUITY H Class UPLC system with quaternary pump. ESI source parameters were set to a capillary voltage of 2 kV, sampling cone of 20 eV, source temperature of 150 • C, and source offset of 10 • C. The desolvation temperature was set at 450 • C, the cone gas flow at 0 L/h and the desolvation gas flow at 900 L/h. Measurement was performed in MS e high resolution negative mode using an acquisition mass range from 100 m/z to 2000 m/z and a scan rate of 0.5 s, where fragmentation was carried out using Independent Data Acquisition for all eluting compounds with collision energy ramp from 20 V to 30 V storing at 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 leucine enkephalin infusion measured each 30 s during the run. The data was analyzed using MassLynx V4.2 software from Waters.
Separation was carried out on a Luna RP-C18 column (150 mm × 4.6 mm i.d. × 4 µm, Phenomenex, Torrance, CA, USA) with a pre-column filter (Phenomenex, Torrance, CA, USA). Solvents used in the mobile phase were water with 0.1% formic acid (A), methanol with 0,1% formic acid (B) and acetonitrile with 0.1% formic acid (C). Then, 5 µL of sample was injected with a flow rate of 0.5 mL/min at 40 • C. The chromatographic gradient started at 83% A, 12% B and 7% C, changing to 79.2% A, 12% B and 8.8% C at 4.8 min, then to 74% A, 15% B, and 11% C at 14.8 min, then to 0% A, 85% B and 15% C at 48 min, holding it for 10 min. Then, the column was equilibrated for 5 min to initial conditions.

DPPH Radical-Scavenging Activity
DPPH evaluation was performed as previously reported [89] and was expressed as IC 50 (µg/mL), which is the amount of sample required to reach the 50% radical-scavenging activity, and also as mmol of Trolox equivalents (TE)/g extract. Briefly, a solution of 2,2-diphenyl-1-picrylhidrazyl (DPPH) (0.25 mM) was prepared using methanol as solvent. Next, 0.5 mL of this solution was mixed with 1 mL of extract or Trolox at different concentrations, and incubated at 25 • C in the dark for 30 min. DPPH absorbance was measured at 517 nm. Blanks were prepared for each concentration. The percentage of the radical-scavenging activity of the sample or Trolox was plotted against its concentration to calculate IC 50 (µg/mL). The samples were analyzed in three independent assays. In order to express the DPPH results as mmol TE/g extract, the IC 50 (µg/mL) of Trolox was converted to mmol/mL using Trolox molecular weight (250.29 mg/mmol) and then dividing by the IC 50 of each sample.

ORAC Antioxidant Activity
The Oxygen Radical Absorbance Capacity (ORAC) antioxidant activity was determined following a method previously described [90] 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. 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 fluorescence. Fluorescein was diluted from a stock solution (1.17 mM) in 75 mM phosphate buffer (pH 7.4), while AAPH and Trolox solutions were freshly prepared. All reaction mixtures were prepared in duplicate and three independent runs were completed for each extract. 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: Net AUC = AUC antioxidant − AUC blank (2) The regression equation between 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. Final ORAC values were expressed as mmol of Trolox equivalents (TE)/g of phenolic extract.

Cell Culture
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 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 were 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 of 0.1% (v/v) and control cultures were prepared with the addition of DMSO (vehicle control).

Assessment of Cytotoxicity by MTT Assay
After incubation for 48 h, MTT assays were performed to evaluate the cell viability. The decrease in the viability correlates with the cytotoxic activity of the extract. Briefly, the medium was eliminated, cells were washed twice with 100 µL of PBS and incubated with 100 µL MTT solution (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, 5 mg/mL in cell culture medium) for 2 h at 37 • C. The formazan crystals formed were dissolved in 100 µL of ethanol 95% and the absorbance was read at 570 nm in a microplate reader. Dose-response curves were established for each extract and the concentration, which is 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 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 TPC, DPPH, ORAC and cytotoxicity results, and differences were considered significant at p < 0.05. In order to evaluate whether the total phenolic contents (TPC) contributes to the antioxidant activity evaluated with DPPH and ORAC methodologies, a correlation analysis was carried out as well as cytotoxicity assays. R (version x64 4.1.1) was used as the statistical program.

Conclusions
The UPLC-HRMS analysis using the QTOF-ESI MS technique allowed 106 compounds to be characterized in phenolic enriched extracts of skins and flesh of Anna and Jonagold apple cultivars in Costa Rica. Among them, the flesh of the Jonagold cultivar displayed the most abundant number of polyphenols and also exhibited higher and more diversified procyanidin oligomers than cultivars from other countries reported in the literature. Furthermore, this extract also showed the best results for TPC, ORAC and DPPH antioxidant activities as well as for cytotoxicity IC 50 values against SW620 and AGS cancer cell lines. In addition, the abundance of procyanidins showed a significant positive correlation (p < 0.05) with the ORAC results and a significant negative correlation (p < 0.05) with DPPH and cytotoxicity towards AGS and SW620 tumor. These findings align with the fact that procyanidin oligomers were more abundant and presented a higher degree of polymerization, including tetramers and pentamers, in the flesh of Jonagold extract, displaying better bioactivity effects. The overall results from this study and particularly the ones obtained for the flesh of Jonagold cultivar, support findings suggesting the importance of considering fruit varieties [91]. As mentioned, the higher degree of polymerization in procyanidins has been linked with anti-inflammatory and anticancer activities [92], therefore additional research would contribute to determining the potential health benefits of these extracts.