Polyphenolic Characterization and Antioxidant Activity of Malus domestica and Prunus domestica Cultivars from Costa Rica

The phenolic composition of skin and flesh from Malus domestica apples (Anna cultivar) and Prunus domestica plums (satsuma cultivar) commercial cultivars in Costa Rica, was studied using Ultra Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry (UPLC-DAD-ESI-MS) on enriched-phenolic extracts, with particular emphasis in proanthocyanidin and flavonoids characterization. A total of 52 compounds were identified, including 21 proanthocyanidins ([(+)-catechin and (−)-epicatechin]) flavan-3-ols monomers, five procyanidin B-type dimers and two procyanidin A-type dimers, five procyanidin B-type trimers and two procyanidin A-type trimers, as well as one procyanidin B-type tetramer, two procyanidin B-type pentamers, and two flavan-3-ol gallates); 15 flavonoids (kaempferol, quercetin and naringenin derivatives); nine phenolic acids (protochatechuic, caffeoylquinic, and hydroxycinnamic acid derivatives); five hydroxychalcones (phloretin and 3-hydroxyphloretin derivatives); and two isoprenoid glycosides (vomifoliol derivatives). These findings constitute the first report of such a high number and diversity of compounds in skins of one single plum cultivar and of the presence of proanthocyanidin pentamers in apple skins. Also, it is the first time that such a large number of glycosylated flavonoids and proanthocyanidins are reported in skins and flesh of a single plum cultivar. In addition, total phenolic content (TPC) was measured with high values observed for all samples, especially for fruits skins with a TPC of 619.6 and 640.3 mg gallic acid equivalents/g extract respectively for apple and plum. Antioxidant potential using 2,2-diphenyl-1-picrylhidrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) methods were evaluated, with results showing also high values for all samples, especially again for fruit skins with IC50 of 4.54 and 5.19 µg/mL (DPPH) and 16.8 and 14.6 mmol TE/g (ORAC) respectively for apple and plum, indicating the potential value of these extracts. Significant negative correlation was found for both apple and plum samples between TPC and DPPH antioxidant values, especially for plum fruits (R = −0.981, p < 0.05) as well as significant positive correlation between TPC and ORAC, also especially for plum fruits (R = 0.993, p < 0.05) and between both, DPPH and ORAC antioxidant methods (R = 0.994, p < 0.05).


Introduction
Polyphenols effects on health are based on results obtained from bioactivity studies, which in turn, has increased the interest in the consumption of foods and beverages rich in polyphenols as well as the importance of related scientific research. Such bioactive effects include antioxidant properties, prevention of oxidative stress associated diseases like cardiovascular, neurodegenerative diseases and cancer [1] and their role in long-term health protection by reducing the risk of chronic and degenerative diseases [2].
Several studies have linked vegetable consumption, specially fruits with a reduced risk for cancer and cardiovascular disease, thus characterization of polyphenols is essential to increase the knowledge on fruits contents and their related beneficial effects. Malus domestica (apple) and Prunus domestica (plum) are trees from the Rosaceae family, both native from southern Europe and western Asia that were introduced in Costa Rica as an initiative of local producers to diversify their crops, constitute fruits of high consumption in the country.
Studies on M. domestica have found these fruits to have a potent antioxidant activity and to inhibit the growth of cancer cells in vitro [3,4]. Similar properties have been described for plum [5,6] and some of these effects were attributed at least partially to its polyphenolic contents.
Previous studies on polyphenols of M. domestica have shown mainly flavonoids, phenolic acids, and chalcones, while proanthocyanidins included only catechin and epicatechin monomers and procyanidin dimers [7][8][9] and other reporting also trimers mainly in skin [10,11]. In the case of P. domestica, studies have focused mostly in a specific type of compounds, such as caffeolylquinic acid derivatives [12,13], flavonoids such as quercetin derivatives [14], and both type of compounds [15]. Few reports have studied also extensively proanthocyanidins, findings indicating mainly monomers and procyanidin dimers [16,17].
However other findings [18] would suggest proanthocyanidins with higher polymerization degree, without having reported their particular characterization. Despite the increasing number of studies on phenolics, the characterization of proanthocyanidins remains a complex task because of the need for high-end techniques such as high-resolution mass spectroscopy (HRMS). Proanthocyanidins, which are condensed flavan-3-ols, constitute an important group of polyphenols because of their bioactivities, for instance, mainly to their antioxidant capacity, which in turn is linked, among others, with their ant-inflammatory and anti-cancer activities [19].
Antioxidant properties have been reported to increase with proanthocyanidins degree of polymerization and the presence of specific structures such as procyanidin tetramers and pentamers has been found to enhance such properties [20,21], thus further knowledge on these phenolic structures characterization in apple and plum 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. domestica and P. domestica 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 particular emphasis in flavonoids and proanthocyanidins. Evaluation of the total polyphenolic contents and antioxidant activity using 2,2-diphenyl-1-picrylhidrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) methods, was also carried out in the different extracts.

Phenolic Yield and Total Phenolic Contents
The extraction process described in the Materials and Methods section, allowed to obtain phenolic enriched extracts, as summarized in Table 1. P. domestica (satsuma cultivar) skin presented the highest yield (2.74%) whereas M. domestica (Anna cultivar) flesh showed the lowest value (0.51%). In both fruits, skin extract yields were higher than flesh extracts. The total phenolic contents (TPC) shown also in Table 1, resulted in high values for all samples with both skins exhibiting slightly higher results, ranging from 619.6-640.3 gallic acid equivalents (GAE)/g dry extract. Results from literature show variability among reports from other apple cultivars with total phenolic contents (TPC) values ranging between 0.3-25.9 mg GAE/g DW for skin and 1.6-14.8 mg GAE/g DW for flesh [22][23][24][25]. Our results for Anna cultivar skin and flesh 7.4 and 3 mg GAE/g DW respectively (values calculated using TPC and extract yields from Table 1) are within that range. A similar situation occurs in the case of plum, with determination of total phenolic contents in the literature revealing variability, with values ranging between 18.4-495 mg GAE/100 g fresh weight (FW) [14,26,27], whereas our findings of 109-179 mg GAE/100 g FW (values calculated using TPC, extract and lyophilization yields from Table 1) are in agreement with the published results.

Profile by UPLC-DAD-ESI-TQ-MS Analysis
The UPLC-DAD-ESI-MS/MS analysis described in the Materials and Methods section, allowed to identify 52 compounds, including 21 proanthocyanidins and flavan-3-ol monomers, 15 glycosylated flavonols and nine acids and derivatives in Costa Rican apple (Anna cultivar) and plum (Satsuma cultivar) only commercial cultivars; as well as five hydroxy chalcones and two glycosylated isoprenoids characteristic of apple fruits. Figures 1 and 2 show the chromatograms of the samples and Table 2 summarizes the analysis results for the 52 compounds.      The first common group of compounds, proanthocyanidins, corresponded to oligomers of flavan-3-ols catechin and epicatechin. The monomeric units of these proanthocyanidins are linked through a C4-C8 or C4-C6 bond (B-type), which coexist with an additional C2-O-C7 linkage (A-type) [28].  The first common group of compounds, proanthocyanidins, corresponded to oligomers of flavan-3-ols catechin and epicatechin. The monomeric units of these proanthocyanidins are linked through a C4-C8 or C4-C6 bond (B-type), which coexist with an additional C2-O-C7 linkage (A-type). [28].  The first common group of compounds, proanthocyanidins, corresponded to oligomers of flavan-3-ols catechin and epicatechin. The monomeric units of these proanthocyanidins are linked through a C4-C8 or C4-C6 bond (B-type), which coexist with an additional C2-O-C7 linkage (A-type). [28].       [33].
The second group of common compounds, glycosylated flavonol derivatives were elucidated based in the fragmentation pattern from the aglycone due to the loss of glycosides ( Figure 6). showed its main fragment at m/z 271, corresponding to naringenin-hexoside [35].      On the other hand, a series of p-coumaric acid derivatives was identified, as shown in Figure 8.  On the other hand, a series of p-coumaric acid derivatives was identified, as shown in Figure 8.  [11,39].
The fifth group of compounds, glycosylated isoprenoid derivatives, shown in Figure 10 to the loss of a pentoside and a pentosylhexoside respectively. The resulting ion is coincident with vomifoliol, allowing the peaks to be assigned to vomifoliol-pentosylhexoside isomers [40].  The fourth group of compounds, chalcones, shown in Figure 9, were found only in apples.  [11,39].
The fifth group of compounds, glycosylated isoprenoid derivatives, shown in Figure  Polyphenol profiling reveals great similarities between skins and flesh, with a high number of compounds and high diversity for both fruits. When comparing the reports for apple cultivars in the literature, our results for Anna cultivar from Costa Rica show greater number and diversity of polyphenols than the findings on sixteen cultivars from Norway, Italy, Canada and United States [7][8][9][10], and similar to only two cultivars, namely Golden Delicious and Braeburn from Slovenia [11]. Further, findings on proanthocyanidins indicate better results both in total occurrence and in greater polymerization degree, for instance procyanidin tetramers and pentamers in the skins of Costa Rican Anna apple cultivar.
In the case of plums, when comparing the reports of twenty-four cultivars from United States, Germany and Portugal [15][16][17][18], greater number and diversity of polyphenols is observed in the skins of Satsuma cultivar from Costa Rica. Likewise, results for Satsuma flesh are also superior to twenty-three of the cultivars, being similar to President cultivar from Germany. Of special interest is the presence of a greater number of procyanidins oligomers, such as procyanidin trimers and one tetramer as well as of glycosylated flavonols, showing different quercetin derivatives not reported in previous characterizations of plum skins.
It is important to highlight the characterization of proanthocyanidin oligomers present in both fruits, which is of special interest due to procyanidins antioxidant results having been reported to increase with higher degree of polymerization, for instance trimers, tetramers and pentamers   Polyphenol profiling reveals great similarities between skins and flesh, with a high number of compounds and high diversity for both fruits. When comparing the reports for apple cultivars in the literature, our results for Anna cultivar from Costa Rica show greater number and diversity of polyphenols than the findings on sixteen cultivars from Norway, Italy, Canada and United States [7][8][9][10], and similar to only two cultivars, namely Golden Delicious and Braeburn from Slovenia [11]. Further, findings on proanthocyanidins indicate better results both in total occurrence and in greater polymerization degree, for instance procyanidin tetramers and pentamers in the skins of Costa Rican Anna apple cultivar.
In the case of plums, when comparing the reports of twenty-four cultivars from United States, Germany and Portugal [15][16][17][18], greater number and diversity of polyphenols is observed in the skins of Satsuma cultivar from Costa Rica. Likewise, results for Satsuma flesh are also superior to twenty-three of the cultivars, being similar to President cultivar from Germany. Of special interest is the presence of a greater number of procyanidins oligomers, such as procyanidin trimers and one tetramer as well as of glycosylated flavonols, showing different quercetin derivatives not reported in previous characterizations of plum skins.
It is important to highlight the characterization of proanthocyanidin oligomers present in both fruits, which is of special interest due to procyanidins antioxidant results having been reported to increase with higher degree of polymerization, for instance trimers, tetramers and pentamers Polyphenol profiling reveals great similarities between skins and flesh, with a high number of compounds and high diversity for both fruits. When comparing the reports for apple cultivars in the literature, our results for Anna cultivar from Costa Rica show greater number and diversity of polyphenols than the findings on sixteen cultivars from Norway, Italy, Canada and United States [7][8][9][10], and similar to only two cultivars, namely Golden Delicious and Braeburn from Slovenia [11]. Further, findings on proanthocyanidins indicate better results both in total occurrence and in greater polymerization degree, for instance procyanidin tetramers and pentamers in the skins of Costa Rican Anna apple cultivar.
In the case of plums, when comparing the reports of twenty-four cultivars from United States, Germany and Portugal [15][16][17][18], greater number and diversity of polyphenols is observed in the skins of Satsuma cultivar from Costa Rica. Likewise, results for Satsuma flesh are also superior to twenty-three of the cultivars, being similar to President cultivar from Germany. Of special interest is the presence of a greater number of procyanidins oligomers, such as procyanidin trimers and one tetramer as well as of glycosylated flavonols, showing different quercetin derivatives not reported in previous characterizations of plum skins.
It is important to highlight the characterization of proanthocyanidin oligomers present in both fruits, which is of special interest due to procyanidins antioxidant results having been reported to increase with higher degree of polymerization, for instance trimers, tetramers and pentamers showing better results than dimers or flavan-3-ol monomers [20,21]. The detailed characterization of these fruit cultivars provides an important contribution expanding the knowledge for their exploitation as available sources of such diversity of polyphenols, which in turn can be of interest for further research due to their potential biological activities.

Antioxidant Activity
The DPPH and ORAC values obtained are summarized in Table 3 [41], thus Anna cultivar extracts showing better results. DPPH values expressed as mmol TE/g extract were obtained as described in the experimental section (in respect to Trolox IC 50 = 5.62 µg/mL ) and allow to compare our results with those reported in the literature for different extracts, for instance our values fit in the range between enju and grape seed extracts (0.76-1.35 mmol TE/g extract) used as antioxidant food additives [42,43]. On the other hand, for ORAC, a study of ethanolic extracts of Pelingo cultivar apples [10], reported values of 44.07 µmol Trolox eq./g DW for skin and 23.19 µmol Trolox eq./g DW for flesh, while findings for aqueous extracts showed values of 42.97 µmol Trolox eq./g DW and 31.99 µmol Trolox eq./g DW for skin and flesh respectively, therefore ORAC of extracts from Anna cultivar apples are superior for both skin and flesh since our findings indicate values of 57.33 µmol Trolox eq./g DW for flesh and 202.03 µmol Trolox eq./g DW (values calculated using ORAC from Table 3 and extract yields from  Table 1).
The difference in antioxidant values among extracts could be attributed to the differences in their phenolic content and distribution. Thus, in order to investigate if the total phenolic contents (TPC, Table 1) contributes to the antioxidant activity, a correlation analysis was carried out between these TPC values with DPPH and ORAC results. Significant positive correlation (p < 0.05) was found for both apple and plum samples between TPC values and ORAC with R = 0.827 and R = 0.993 respectively, as well as significant negative correlation (p < 0.05) between TPC results and DPPH with R = −0.833 and R = −0.981 respectively. Therefore, our results are in agreement with previous studies reporting correlation between total polyphenolic contents and ORAC antioxidant activity [44]. Finally, our findings indicate positive correlation (p < 0.05) between both DPPH and ORAC antioxidant values (R = 0.994), thus in agreement with previous studies [43].

Materials, Reagents and Solvents
Malus domestica and Prunus domestica fruits were acquired in ripe state from FRUTALCOOP, a local producer cooperative located in Los Santos in 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 were provided by Sigma-Aldrich (St. Louis, MO, USA), while solvents such as acetone, chloroform and methanol were purchased from Baker (Center Valley, PA, USA).

Phenolic Extracts from M. domestica and P. domestica Fruits
M. domestica and P. domestica fruits were rinsed in water, peeled, and both, 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), and 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 acetone:water (70:30) as solvent in a 34 mL cell, at 40 • C. Next, the extract was evaporated under vacuum to eliminate the acetone 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 using a Buchi™ 215 (Flawil, Switzerland) rotavapor.

Total Phenolic Content
The polyphenolic content was determined by a modification of the Folin-Ciocalteu (FC) method [45], 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 sample. The mixture was let 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.

UPLC-DAD-ESI-TQ-MS Analysis
The UPLC-MS system used to analyze the composition of M. domestica and P. domestica fruit extracts consisted of an LTQ Orbitrap XL mass spectrometer with an Accela 1250 binary Pump, a PAL HTC Accela TMO autosampler, a PDA detector (Thermo Fisher Scientific, San Jose, CA, USA), and a G1316A column compartment (Agilent, Palo Alto, CA, USA). Separation was carried out on 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) 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, v/v, 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 200-700 nm.
Negative electrospray ionization mode was used and the conditions 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; tube lens, 70 V. The mass range was from 100 to 2000 amu with a resolution of 30,000, FTMS AGC target at 2 × 10 5 , FT-MS/MS AGC target at 1 × 10 5 , isolation width of 1.5 amu, and max ion injection time of 500 ms. The most intense ion was selected for the data-dependent scan to offer their MS 2 to MS 5 product ions, respectively, with a normalization collision energy at 35%.

DPPH Radical-Scavenging Activity
DPPH evaluation was performed as previously reported [46] 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 were 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 ORAC (Oxygen Radical Absorbance Capacity) antioxidant activity was determined following a method previously described [47] 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: i 0 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 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.

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

Conclusions
The qualitative analysis of phenolic-enriched extracts of the only commercial cultivars of M. domestica (Anna cultivar) and P. domestica (Satsuma cultivar) in Costa Rica, using UPLC-DAD-ESI-MS techniques, shows 52 compounds characterized, distributed as 21 proanthocyanidins, including procyanidin A-type and B-type dimers and trimers, B-type tetramer and pentamers, flavan-3-ol monomers and gallates; 15 flavonoids, including kaempferol, quercetin and naringenin derivatives, and eight phenolic acid derivatives in both fruits; as well as chalcones and isoprenoid glycosides in Anna apples. These findings constitute the first report of such a high number and diversity of compounds in skins of one single plum cultivar and of the presence of proanthocyanidin pentamers in apple skins. Also, it is the first time that such a large number of glycosylated flavonoids and proanthocyanidins are reported in skins and flesh of a single plum cultivar. Further, significant negative correlation was found for both apple and plum samples between TPC and DPPH antioxidant values, especially for plum fruits (R = −0.981, p < 0.05) as well as significant positive correlation between TPC and ORAC, also especially for plum fruits (R = 0.993, p < 0.05). DPPH and ORAC methods show high values for all samples, especially for fruits skins, thus indicating the potential value of these extracts. The presence of procyanidin tetramers and pentamers in apple skin could be responsible for the higher antioxidant potential, in agreement with reports indicating higher antioxidant values related to the presence of this type of procyanidin oligomers [20,21]. Further purification or fractioning of these extracts would be important to evaluate their structure-bioactivity relationship and, for instance, specific proanthocyanidin structures effect on epithelial gastrointestinal cancer cells, of particular relevance due to promising related results [3,48] and the fact that proanthocyanidins low absorption make gut epithelial cells [49] likely one of the main tissues where these compounds can actually exert their biological effects.