**1. Introduction**

Several studies have linked vegetable consumption, especially fruits, with a reduced risk for cardiovascular disease and cancer, thus the importance of metabolites' characterization. Mango (*Mangifera indica* L.) is a commercial fruit cultivated worldwide that holds the fifth position in total production amongs<sup>t</sup> the main fruit crops, with 5.4 million hectares in approximately 100 countries, especially in areas with subtropical and tropical climates [1]. Out of the large number of cultivars reported, Keitt and Tommy Atkins are the most important commercialized mango cultivars in Costa Rica.

The bioactive e ffects reported for *M. indica* include antioxidant activity, anti-inflammatory, antipyretic, antibacterial, antiviral, antimicrobial, and anticancer, as well as hepatoprotective and gastroprotective properties, in addition to immunomodulatory and lipid-lowering drug e ffects [2–5]. Particularly, it has been reported that mango exhibits antiproliferative activity in MDA-MB-231 adenocarcinoma breast cell lines, HepG2 liver, and HL-60 leukemia cancer cells [6], as well as antitumoral e ffects on MCF-7 breast carcinoma cells [7], Molt-4 leukemia, A-549 lung, LnCap prostate, and SW-480 colon cancer cells [8]. These studies report di fferent results depending on mango cultivar and on cancer cell lines; however, the e ffects have mainly been attributed to the fruits' polyphenolic contents.

In fact, several studies have reported polyphenolics benefits on health based on findings from the above bioactivities [9] and have established their role in reducing the risk of degenerative and chronic diseases, therefore contributing to long-term health protection [10]. For instance, their contents in fruits have been associated to a lower risk for cardiovascular diseases and cancer, hence the increase in interest in fruit consumption and the importance of scientific research for polyphenols' characterization and their associated valuable e ffects on health.

Previous studies of *M. indica* polyphenols have focused, for instance, on properties of xanthonoid compounds, mainly mangiferin isomers [11,12] and gallotannins [13], which have been studied for their anticarcinogenic e ffects [6,14]. Other studies have involved gallotannins and hydroxybenzophenones [15], xanthonoids and flavonoids [16,17], or phenolic acids and gallotannins [18]. Few reports have studied all five types of compounds [19,20] and their biological activities [21].

Polyphenols' antioxidant properties have been linked, among others, with their anti-inflammatory and anticancer activities, which have been reported to increase with gallotannins' degree of polymerization of specific structures, which has been found to enhance such properties [22], thus further knowledge on phenolic structures' characterization in mango fruits would contribute to a better understanding of their implications in the fruits' quality as a source of dietary compounds with potential biological properties.

Therefore, the objective of the present work was to obtain enriched polyphenolic extracts of fruits from *M. indica* commercial cultivars in Costa Rica and to characterize them through ultra performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-DAD-ESI-MS), with an emphasis on the five types of compounds previously reported. An 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 the adenocarcinoma AGS gastric cell line, adenocarcinoma HepG2 hepatic cell line, and adenocarcinoma SW620 colon cell line was also carried out in the di fferent extracts.

#### **2. Materials and Methods**

#### *2.1. Materials, Reagents and Solvents*

*Mangifera indica* fruits were acquired in the ripe state from a local producer from Marichal Orotina Orotina (Keitt cultivar) and Fabio Baudrit Station (Alajuela). 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, Amberlite XAD-7 resin, fetal bovine serum, glutamine, penicillin, streptomycin, amphotericin B, and trypsin–ethylenediaminetetraacetic acid (EDTA), were provided by Sigma-Aldrich (St. Louis, MO, USA). Human gastric adenocarcinoma cell line AGS, human colorectal adenocarcinoma SW 620, and human hepatocellular carcinoma Hep-G2 were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). In order to evaluate the specificity of the cytotoxic activity towards these cancer cells with respect to normal cells, a selectivity index (SI) was determined by also measuring the cytotoxicity on normal non-cancer cells, according to previous publications [23–25]. Di fferent cell lines are used in the literature, such as normal mouse subcutaneous fibroblast L929 in studies evaluating cytotoxicity on HeLa and SiHa cervical cancer cells [26]; normal human dermal fibroblast TelCOFSO2MA used in comparative cytotoxicity studies with Caco-2 colon and OE19 esophageal adenocarcinoma cell lines [27] and normal monkey epithelial kidney Vero cells used as non-tumoral control cells in studies evaluating cytotoxicity towards MCF-7 breast and HeLa cervix cancer cells [24]; Caco-2 colon and A549 lung cancer cells [23]; AGS gastric and SW620 adenocarcinoma cells [28]; and malignant HepG2 hepatoma cells [25]. These Vero cell lines were selected for this study due to previous reports and accessibility (American Type Culture Collection, Rockville, MD, USA). Finally, 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).

#### *2.2. Phenolic Extracts from Mangifera. Indica Fruits*

*M. indica* fruits were rinsed in water, peeled, and both the skin and flesh material were freeze-dried in a Free Zone at −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. Freeze-dried samples were extracted in a Dionex ™ ASE ™ 150 Accelerated Solvent Extractor (Thermo Scientific ™, Walthman, MA, USA) using methanol:water (70:30) as solvent for 7.5 g of sample 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 an 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 evaporation to dryness using a Buchi ™ 215 (Flawil, Switzerland) rotavapor.

#### *2.3. Total Phenolic Content*

The polyphenolic content was determined by modification of the Folin–Ciocalteu (FC) method [29], which is based on the oxidation of the hydroxyl groups of phenols by the mixture of phosphotungstic and phosphomolybdic acids. Briefly, each polyphenolic enriched extract was dissolved in MeOH (0.1% HCl) to obtain a 500 ppm solution and 2 mL were combined with 0.5 mL of FC reagent. Afterwards 10 mL of Na2CO3 (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 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.

## *2.4. UPLC-DAD-ESI-TQ-MS Analysis*

The UPLC-MS system used to analyze the composition of *M. indica* fruit extracts consisted of an LTQ Orbitrap XL mass spectrometer with an Accela 1250 binary Pump, PAL HTC Accela TMO autosampler, PDA detector (Thermo Fisher Scientific, San Jose, CA, USA), and G1316A column compartment (Agilent, Palo Alto, CA, USA). Separation was carried out by a modification of a method

previously described [30]. Briefly, a Hypersil Gold AQ RP-C18 UHPLC column (200 mm × 2.1 mm i.d., 1.9 μm, Thermo Fisher Scientific) with an UltraShield pre-column filter (Analytical Scientific Instruments, Richmond, CA, USA) were used at a flow rate of 0.3 mL/min. Mobile phases A and B consist of a combination of 0.1% formic acid in water, *v*/*v* and 0.1% formic acid in acetonitrile, *<sup>v</sup>*/*<sup>v</sup>*, respectively. The linear gradient is from 4% to 20% B (*v*/*v*) at 20 min, to 35% B at 30 min and to 100% B at 31 min, and held at 100% B to 35 min. The UV/Vis spectra were acquired from 200 to 700 nm. The mass spectrometer was calibrated using Pierce ™ LTQ ESI Negative Ion Calibration Solution, and the conditions for the negative electrospray ionization mode used were set as follows: Sheath gas, 70 (arbitrary units); aux and sweep gas, 15 (arbitrary units); spray voltage, 4.8 kV; capillary temperature, 300 ◦C; capillary voltage, 15 V; and tube lens, 70 V. The mass range was from 100 to 2000 amu with a resolution of 30,000, FTMS AGC target at 2e5, FT-MS/MS AGC target at 1 × 105, isolation width of 1.5 *amu*, and max ion injection time of 500 ms. A clean chromatographic separation was obtained, and the most intense ion was selected for the data-dependent scan to o ffer MS<sup>2</sup> to MS<sup>5</sup> product ions, respectively, with a normalization collision energy at 35%.

#### *2.5. DPPH Radical-Scavenging Activity*

DPPH evaluation was performed by a modification of the original method [31], based on antioxidant determinations using a stable free radical. 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 were mixed with 1 mL of each polyphenolic-enriched extract at di fferent concentrations ranging between 4 and 40 ppm, and incubated at 25 ◦C in the dark for 30 min. The mixture 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 IC50 (μg/mL). The samples were analyzed in three independent assays. Results were expressed as IC50 (μg/mL), which is the amount of sample required to reach 50% radical-scavenging activity.

#### *2.6. ORAC Antioxidant Activity*

The ORAC (oxygen radical absorbance capacity) antioxidant activity was determined by modification of a method using fluorescein as a fluorescence probe [32]. Briefly, the reaction was performed in 75 mM phosphate bu ffer (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 di fferent 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, O ffenburg, Germany) with 485-P excitation and 520-P emission filters. Fluostar Galaxy software version 4.11−0 (BMG Labtechnologies GmbH, O ffenburg, Germany) was used to measure fluorescence. Fluorescein was diluted from a stock solution (1.17 mM) in 75 mM phosphate bu ffer (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:

$$A \text{lIC} = 1 + \sum\_{i=1}^{i=98} \int\_i \Big/ \int\_0^{\cdot} \tag{1}$$

where 0 is the initial fluorescence reading at 0 min and *i* is the fluorescence reading at time *i*. The net AUC corresponding to a sample was calculated as follows:

$$\text{Net AUC} = \text{AUC}\_{\text{antioxidation}} - \text{AUC}\_{\text{blank}} \,\text{s} \,\tag{2}$$

The regression equation between the net AUC and the 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 are expressed as mmol of Trolox equivalents (TE)/g of phenolic extract.

#### *2.7. Evaluation of Cytotoxicity of Extracts*

## 2.7.1. Cell Culture

The human gastric adenocarcinoma cell line AGS, human colorectal adenocarcinoma SW 620, human hepatocellular carcinoma Hep-G2, 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 IU mL−<sup>1</sup> penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. The cells were grown in a humidified atmosphere containing 5% CO2 at 37 ◦C and sub-cultured by detaching with trypsin–EDTA solution at about 70% to 80% confluence. For the experiments, 100 μL of a cell suspension of 1.5 × 10<sup>5</sup> 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 and 500 μg/mL. The DMSO concentrations used in the experiments were below 0.1% (*v*/*v*) and control cultures were prepared with the addition of DMSO (vehicle control).

#### 2.7.2. Assessment of Cytotoxicity by MTT Assay

After incubation for 48 h, MTT assays were performed to evaluate cytotoxicity. 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 that is enough to reduce the cell viability by 50% (IC50) was calculated.

In order to evaluate if the cytotoxicity activity was specific against the cancer cells, a selectivity index (SI) was determined. This index is defined as the ratio of IC50 values of normal epithelial kidney cells (Vero) to cancer cells (AGS, HepG2, or SW620).
