Evaluation of Phenolic Profile and Antioxidant Activity of Eleven Pistachio Cultivars (Pistacia vera L.) Cultivated in Andalusia

Pistachio (Pistacia vera L.) is a nut with a good adaptability to the Mediterranean conditions of cultivation, specifically in the Andalusian region, becoming an emerging crop. Moreover, it has been getting attention in the past years for the great content of bioactive compounds such as polyphenols. Although some studies have reported the polyphenolic profile of pistachios, most of them have analyzed the hull part, considered as a residue, and not the kernel which is the edible part. Therefore, characterization of eleven varieties of pistachios kernels cultivated in Andalusia and harvested in 2019 and 2020 was carried out by UHPLC-MS (ultra-high-performance liquid chromatography high-resolution mass spectrometry). The identification and quantification of 56 polyphenolic compounds was performed, being the hydroxybenzoic acids group the most abundant with a 71–86% of the total amount followed by flavan-3-ols group that accounted for 8–24%. Moreover, 3,4-dihydroxybenzoic acid was the main compound in most of the varieties, followed by vanillic acid hexoside. Larnaka, Avdat, Aegina, and Mateur presented the highest amount of total polyphenols, while Kalehghouchi, Joley, Lost Hills, Kerman, and Golden Hills were the varieties with the lowest content. Regarding the harvest season, no significant differences (p < 0.01) were found in the total amount of polyphenols between 2019 and 2020. In addition, the antioxidant activity was measured by DPPH (1,1-diphenyl-2-picryl-hydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), and ORAC (oxygen radical absorbance capacity) assays, showing a similar trend as that of the polyphenols.


Introduction
The pistachio (Pistacia vera L.) is a nut widely consumed throughout the world, originated from arid regions of Central and West Asia. This crop belongs to the Anacardiaceae family, and its health benefits and nutritional characteristics have been recently recognized by some studies [1,2].
The pistachio kernel or edible seed is commonly consumed as a natural or roasted salted snack food. Moreover, pistachio kernels are used in the industry as a food ingredient to elaborate pastry, ice cream, and confectionary production [1]. The edible kernel of pistachio has a thin layer which is a purplish skin covered by a hard shell, which is also covered by another shell known as hull (exocarp) characterized by turning the color from green to yellow-red depending on the ripeness [3].

Materials and Sample Preparation
Pistachios (Pistacia vera L.) were kindly provided from an experimental field of pistachio varieties of IFAPA in Guadix (Granada, Spain) which were planted in 2012. Eleven varieties were collected during two consecutive seasons (2019 and 2020): Aegina, Avdat, Golden Hills, Joley, Kalehghouchi, Kastel, Kerman, Larnaka, Lost Hills, Mateur and Sirora. The experimental design consisted in four blocks of random repetitions per variety. Fresh pistachios were peeled and ground using a homogenizer (SAMMIC, Madrid, Spain) and stored at −80 • C until analysis.

Extraction of Polyphenols
The extraction of polyphenols from pistachio samples was adapted from Ordóñez-Díaz et al. [33] with some modifications. One gram of sample was homogenized in an Ultra-Turrax (Stauten, Germany) with 12 mL of a methanol/acidified water mixture (80:20, v/v) with 0.1 % formic acid. The samples were centrifuged at 5000 rpm for 15 min at 4 • C, and supernatants were collected. The pellet was re-extracted following the same indications abovementioned. All the supernatants were pooled and made up to a final volume of 25 mL and stored at −80 • C until analysis.

ABTS Assay
Free radical scavenging activity was measured using the ABTS (2,2 -azino-bis(3ethylbenzothiazoline-6-sulfonic acid) decolorization method [34] with some modifications [35]). Briefly, the ABTS radical cation (ABTS ·+ ) was produced by reacting ABTS ·+ with a 2.45 mM of potassium persulfate solution stirring in dark at room temperature for 12-16 h before use. The ABTS ·+ solution was diluted with ethanol to get a final absorbance of 0.8 ± 0.02 at 730 nm. Fresh ABTS ·+ solution was prepared for each assay and day. Samples were resuspended in 500 µL of 7% methylated β-cyclodextrin (RMCD) in acetone/water (1:1, v/v) solution and 25 µL was added to 190 µL of ABTS ·+ solution in a 96-well microplate measuring the absorbance every 20 s at 30 • C for over 6 min in a Synergy HTX Multi-Mode Microplate Reader (Biotek Instruments, Winooski, VT, USA). The antioxidant activity was expressed as mmol of Trolox equivalents per 100 g of sample (mmol TE/100 g). Each value is the average of three determinations.

DPPH Assay
Free radical DPPH (1,1-diphenyl-2-picryl-hydrazyl) scavenging capacity was determined using the previously described methods by Sánchez-Moreno et al. [36]. The antioxidant activity was expressed as mmol of Trolox equivalents per 100 g of sample (mmol TE/100 g). Each value is the average of three measurements.

ORAC Assay
Oxygen radical absorbance capacity (ORAC) assay was measured according to the method previously published by Huang et al. [37] and modified by Pereira-Caro et al. [35]. Briefly, samples were resuspended in 500 µL of 7% methylated β-cyclodextrin (RMCD) in acetone/water (1:1, v/v) solution. Then, 25 µL of either Trolox or sample extract or 75 mM phosphate buffer as a blank was added to a 96-well microplate followed by the addition of 150 µL of fluorescein work solution (8.5 × 10 −5 mM) prepared in 75 mM phosphate buffer (pH 7.4). The microplate reader (Synergy HTX Multi-Mode Microplate Reader (Biotek Instruments, Winooski, VT, USA)) was programmed to record every two minutes for 120 min at 485 and 528 nm excitation and emission wavelengths, respectively, the fluorescence after the addition of 30 µL of AAPH (153 mM) as peroxyl radical generator, prepared in 75 mM phosphate buffer (pH 7.4). ORAC values are expressed as mmol Trolox equivalents per 100 g of sample (mmol TE/100 g).

Total Phenolic Content
Total phenolic content (TPC) was determined by the Folin-Ciocalteu assay following the methodology of Slinkard and Singleton [38], with modifications of Hervalejo et al. [39] Total phenolic content was expressed as mmol of gallic acid per 100 g of sample (mmol GAE/100 g).

UHPLC-HRMS Polyphenol Analysis
Identification and quantification of polyphenols in the pistachio extracted samples were carried out by using UHPLC-HRMS mass spectrometer system (Thermo Scientific, San José, CA, USA) comprising a UHPLC pump, a PDA detector scanning from 200 to 600 nm, and an autosampler operating at 4 • C (ThermoFisher Scientific, San Jose, CA, USA).
Separation of phenolic compounds was performed on a Zorbax SB-C18 RRHD column (100 × 2.1 mm i.d., 1.8 µm (Agilent, Santa Clara, CA, USA) preceded by a guard precolumn of the same stationary phase and maintained at 40 • C. The flow rate was set to 0.2 mL/min with a 26 min gradient of phase A: deionized water with 0.1% formic acid and B: acetonitrile with 0.1% formic acid. The gradient started at 3% B, was maintained for 2 min, then rose to 65% B in 18 min, before rising to 80% B in 1 min and being maintained for 6 min with a 26 min gradient. After that, the column was equilibrated to the previous conditions within 10 min. After passing through the flow cell of the PDA detector part of the column, the eluate (0.2 mL/min) went into an Exactive Orbitrap mass spectrometer (Thermo Scientific, San José, CA, USA) fitted with a heated electrospray ionization probe (HESI) operating in negative ionization mode for the determination of polyphenols. Full scans were recorder in m/z range from 100 to 1200 with a resolution of 50,000 Hz and with a full AGC target of 100,000 charges, using 2 microscans. Analyses were also based on scans with in-source collision-induced dissociation (CID) at 25.0 eV. The capillary temperature of the MS experiment with HESI in negative ionization mode was at 320 • C, the sheath gas was 35 units, the heater temperature was 150 • C, the auxiliary gas was 10 units, and the spray voltage was 4.0 kV. Data acquisition and processing were carried out using Xcalibur 3.0 software (Thermo Scientific, San José, CA, USA).
Targeted identification of phenolic compounds was achieved comparing the exact mass and the retention time with available standards. In the absence of standards, compounds were tentatively identified by comparing the theoretical exact mass of the molecular ion with the measured accurate mass of the molecular ion and searched against metabolite databases including Metlin, Phenol Explorer and more general chemical databases such as PubChem and ChemSpider. Compounds having molecular masses within the pre-specified tolerance (≤10 ppm) of the query masses are retrieved from these databases. Quantification of phenolic compounds was carried out by selecting the theoretical exact mass of the molecular ion with reference to standard curves. In absence of reference compounds, they were quantified by reference to the calibration curve of a closely related parent compound.

Statistical Analysis
Two-way ANOVA and Tukey post-hoc tests were applied to identify the differences among samples using R software (v. 3.6.3, R Core Team, Vienna, Austria). A principal component analysis (PCA) was carried out for data exploration.

Antioxidant Activity
Total antioxidant activity was evaluated for the eleven varieties of pistachios following three scavenging methods: ABTS, DPPH, and ORAC assays. Results are displayed in Table 1 and significant statistical differences (p < 0.01) were observed among varieties. Larnaka, Mateur, Avdat, and Aegina were the varieties with the highest antioxidant capacity measured by ABTS assay. Regarding DPPH methodology, Larnaka presented the highest antioxidant activity, followed by Mateur, Avdat, and Aegina, while Golden Hills and Kalehghouchi varieties showing the lowest values. In the same way, Larnaka also presented higher values of antioxidant capacity by the ORAC assay, followed by Mateur, Avdat, and Aegina, while Joley, Kalehghouchi, and Golden Hills showed the lowest ones. The measurement of the total phenolic content (TPC) also showed Larnaka as the variety with the highest amount, followed by Aegina, Mateur, and Advat. In general, Larnaka, Mateur, Avdat, and Aegina showed the highest antioxidant activity values for the three scavenging methods and the total phenolic content. This finding agreed with Ojeda-Amador et al. [40] who reported Larnaka as the variety with the highest values for the antioxidant capacity evaluated by DPPH and ORAC assays followed by Aegina and Avdat. In the same manner, Noguera-Artiaga et al. [30] found similar results for Larnaka measured by ABTS radical but, in contrast with our findings in that study, Mateur variety had the lowest values. Significant differences (p < 0.01) in the antioxidant activity values measured by ABTS and DPPH, as well as in the TPC were found for the harvesting year, showing higher values for the samples harvested during 2020. This fact could be explained due to the different climatic conditions between years. No significant differences were shown for data measured by the ORAC method. To sum up, Larnaka variety presented the highest antioxidant activity in two of the three methods analyzed.

Polyphenols by UHPLC
A total of 56 polyphenolic compounds were identified and quantified by UHPLC-HRMS in the eleven varieties of pistachios from two seasons of harvest (2019 and 2020). Details of the characteristics for the identification of compounds, including the retention time (Rt), the experimental accurate mass, and the error (ppm) between the exact accurate mass and the mass found of the detected compounds are described in Table S1 (Supplementary Information). Polyphenolic compounds identified and quantified include thirteen hydroxybenzoic acids, ten galloyl derivatives, five hydroxycinnamic acids, four flavones, nine flavonols, nine flavan-3-ols, four flavanones, one flavanonol, and one stilbene. Table 2 provides information about the total amount of the 56 polyphenolic compounds identified in the eleven varieties of pistachio grouped by families, for both 2019 and 2020 harvesting seasons. Regarding the qualitative profile among varieties, all of them showed a similar polyphenolic profile with the presence of all groups of polyphenols identified.
Significant differences (p < 0.01) among the eleven varieties were observed for the total content of polyphenols (Table 2). Larnaka, Avdat, Aegina, and Mateur presented the highest amount of the total polyphenols, which agrees with the antioxidant activity results. Based on that, we can conclude that the antioxidant activity is closely linked to the total polyphenols content. Most of the studies published on pistachios have characterized the hull part of the Turkey varieties [16,17,27,41,42] as well as the hulls of Bronte [18,21,22,26] and Kerman [2,43]. In agreement with our results, Noguera-Artiaga et al. [30] and Mannino et al. [22] have reported the Larnaka variety as one of the varieties with highest concentrations of polyphenolic compounds in pistachio kernels and seed skin respectively. However, in contrast, in the same study of Noguera-Artiaga et al. [30], found that Aegina and Avdat were the varieties with the lowest values for polyphenols. Furthermore, Kerman kernels has been widely studied and highly appreciated by the industry worldwide as a snack due to its quality [25,31,44].
In general, the group of the hydroxybenzoic acids was the most abundant for the eleven varieties under study, accounting for 71-86% of the total polyphenol content. Flavan-3-ols was the second more abundant group of polyphenols for all the varieties contributing from 8 to 24% of the total compounds. The rest of the seven groups of polyphenols were found as minority, representing less than 3% of the total content.
Significant differences (p < 0.01) among varieties for each group of polyphenols, (V) were observed, except for the flavanonols. It could be highlighted that varieties with the lower values for the hydroxybenzoic acid group (Aegina Avdat, Kerman, Larnaka and Mateur) presented the highest values for the flavan-3-ols group, which may be due to the genetic synthesis mechanisms of polyphenols.
Regarding the harvesting season (S), in terms of the total polyphenols content ( Table 2), no significant differences were found between the 2019 and 2020. Nevertheless, some significant differences were found for the individual polyphenol groups, but for the galloyl derivatives group. Those differences between years are compensated for the total amount Antioxidants 2022, 11, 609 7 of 16 of polyphenols. It was observed that the concentration of hydroxybenzoic and hydroxycinnamic acid groups, as well as flavanonols, tend to decrease significantly (p < 0.01) from 2019 to 2020, whereas for the rest of the groups increased.
Considering that hydroxybenzoic acids group is the main contributor to the total polyphenols content, Table 3 shows detailed information for the thirteen identified and quantified compounds of this category in the eleven varieties of pistachios. Significant differences (p < 0.01) were observed for all the compounds for the varieties except for the benzoic acid derivative III. In this way, we can conclude that Larnaka, Avdat, Aegina, and Mateur presented three-fold concentration than the rest of varieties. The main compound was the 3,4-dihydroxybenzoic acid for most of the studied varieties, ranging from 43 to 67% of the total content of this group, followed by vanillic acid hexoside (28-51%) except for Kalehghouchi and Kerman for which the major one was the vanillic acid hexoside and 3,4-dihydroxybenzoic acid respectively. The rest of the compounds were found in minor amounts. Results are in agreement with the literature, where 3,4-dihydroxybenzoic acid was described as one of the principal compounds detected, for instance, in the shells of Bronte variety [45] and in unknown varieties from Turkey (name is not mentioned) [42]. In contrast with our results, Tomaino et al. [21], found quercetin-3-O-rutinoside as the most abundant polyphenol in the kernels from the pistachio studied variety (Bronte) while eriodictyol-7-O-glucoside and gallic acid were described as the main compounds in the skins.
As mentioned before, the total amount of hydroxybenzoic acids decreased significantly (p < 0.01) between the two harvest years (2019 and 2020) because of 3,4-dihydroxybenzoic acid which is the major compound, since the tendency of most compounds is to increase. Nonetheless, no significant differences were found for theogallin, galloylshikimic acid, digallic acid, and benzoic acid derivative based on the harvesting year factor. Moreover, the nine compounds of the flavan-3-ols group (second more abundant group) are presented in Table 4, showing that Larnaka, Avdat, Aegina, and Mateur presented between six and eight-fold higher concentration than the rest of the varieties, for which no significant differences were observed for total of flavan-3-ols content. Epigallocatechin gallate was the most abundant compound (ranging from 39 to 83% of the total of this group), followed by catechin (7-22%), and gallocatechin (5-8%). Proanthocyanidins have been previously reported in pistachios hulls [18,43], in the skin of the seed [22,23], and in the oil obtained from pistachios [46]. Regarding pistachio kernels, Liu et al. [25] and Tomaino et al. [21] identified and quantified catechin and epicatechin by HPLC-DAD in Bronte and Kerman varieties. However, to our best knowledge, epiafzelechin 3-gallate, epicatechin gallate, and three afzelechin derivatives have been also detected for the first time in pistachio kernels.
In contrast with the hydroxybenzoic group, a significant (p < 0.01) increase was observed between 2019 and 2020 in flavan-3-ols. The trend in most of the compounds was the same, but for Afzelechin I, for which no significant differences were found.

Multivariate Analysis of Data (PCA)
To determine the main variation sources in the data from the groups of polyphenols in pistachios, a PCA was constructed, and the subspace spanned by the first two principal components (PCs), which explained the 71% of the total variance, was plotted ( Figure 1). naka, Avdat, Aegina, and Mateur varieties which are the most abundant in polyphenols were clearly separated from the ones with less content of compounds (Joley, Lost Hills, Kerman, Golden Hills, and Kalehghouchi). The main families of polyphenols involved in such differences were hydroxybenzoic acids, flavan3-ols, stilbenes, flavonols, and flavanones which could be primarily responsible for the high antioxidant activity measured by ABTS, DPPH, and ORAC assays, being present in the same square ( Figure 2). Additionally, the PC2 explained the 14% of the total variance, showing the difference between Kastel and the rest of the varieties (Figure 2, bottom part), mainly due to the contribution of hydroxycinnamic acids (Table 2). Kastel variety presented the highest content of hydroxycinnamic acids on account of the great amount found in ferulic acid derivative (Table S3).  Different clusters related to the different families of polyphenols in pistachios were observed. In the direction of PC1, which explained around 57% of the total variance, Larnaka, Avdat, Aegina, and Mateur varieties which are the most abundant in polyphenols were clearly separated from the ones with less content of compounds (Joley, Lost Hills, Kerman, Golden Hills, and Kalehghouchi). The main families of polyphenols involved in such differences were hydroxybenzoic acids, flavan3-ols, stilbenes, flavonols, and flavanones which could be primarily responsible for the high antioxidant activity measured by ABTS, DPPH, and ORAC assays, being present in the same square ( Figure 2). Additionally, the PC2 explained the 14% of the total variance, showing the difference between Kastel and the rest of the varieties (Figure 2, bottom part), mainly due to the contribution of hydroxycinnamic acids ( Table 2). Kastel variety presented the highest content of hydroxycinnamic acids on account of the great amount found in ferulic acid derivative (Table S3). Moreover, for more detailed information related to the distribution of the 56 polyphenols, a hierarchical cluster analysis (HCA) was performed ( Figure 3). This hierarchical chemotype clustering information reflects the classification of two groups of varieties, showing the cluster with highest concentrations of polyphenols found in Larnaka, Avdat, Aegina, and Mateur separated from the rest of the varieties with lower values. The analysis also emphasized that 3,4-dihydroxybenzoic acid (3,4-DHBA) is the major compound found in Larnaka, Avdat, Aegina, and Mateur varieties in agreement with the results previously discussed in Table 3. Likewise, another cluster is differentiated by vanillic acid hexoside and epigallocatechin gallate (EGCG), which follow the same trend, presenting the highest content in the four major varieties. Moreover, for more detailed information related to the distribution of the 56 polyphenols, a hierarchical cluster analysis (HCA) was performed ( Figure 3). This hierarchical chemotype clustering information reflects the classification of two groups of varieties, showing the cluster with highest concentrations of polyphenols found in Larnaka, Avdat, Aegina, and Mateur separated from the rest of the varieties with lower values. The analysis also emphasized that 3,4-dihydroxybenzoic acid (3,4-DHBA) is the major compound found in Larnaka, Avdat, Aegina, and Mateur varieties in agreement with the results previously discussed in Table 3. Likewise, another cluster is differentiated by vanillic acid hexoside and epigallocatechin gallate (EGCG), which follow the same trend, presenting the highest content in the four major varieties.

Conclusions
Results of this study report an exhaustive characterization of 56 polyphenolic compounds present in pistachio kernels from eleven varieties cultivated in Andalusia, describing hydroxybenzoic acids and flavan-3-ols as the most abundant groups. Likewise, the main polyphenolic compound was 3,4-dihydroxybenzoic acid for most of the varieties, followed by vanillic acid hexoside. Larnaka, Avdat, Aegina, and Mateur presented the highest amount of the total polyphenols, while Kalehghouchi, Joley, Lost Hills, Kerman, and Golden Hills were the varieties with the lowest content. Regarding the harvest season, no significant differences (p < 0.01) were found between 2019 and 2020 in the total amount of polyphenols. In addition, antioxidant activity was measured, showing Larnaka as the

Conclusions
Results of this study report an exhaustive characterization of 56 polyphenolic compounds present in pistachio kernels from eleven varieties cultivated in Andalusia, describing hydroxybenzoic acids and flavan-3-ols as the most abundant groups. Likewise, the main polyphenolic compound was 3,4-dihydroxybenzoic acid for most of the varieties, followed by vanillic acid hexoside. Larnaka, Avdat, Aegina, and Mateur presented the highest amount of the total polyphenols, while Kalehghouchi, Joley, Lost Hills, Kerman, and Golden Hills were the varieties with the lowest content. Regarding the harvest season, no significant differences (p < 0.01) were found between 2019 and 2020 in the total amount of polyphenols. In addition, antioxidant activity was measured, showing Larnaka as the highest variety in two of the three methods analyzed without significant differences in the remaining varieties. Results suggest that antioxidant activity is mainly attributable to the content of the family of hydroxybenzoic acids. Moreover, the different results obtained among the antioxidant methods may be linked to the contribution of other antioxidant compounds in the hydrophobic fraction which react with the radicals. This study was carried out to characterize the polyphenols presented in the different varieties of pistachios and the variability of the compounds, since it is the basis for subsequent digestibility analyses, which are necessary to evaluate the bioaccessibility and bioavailability of these compounds.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11040609/s1. Table S1: UHPLC-HRMS characteristics of the polyphenols identified in pistachios varieties. Table S2: Influence of the cultivar and harvesting season on the galloyl derivatives group. Table S3: Influence of the cultivar and harvesting season on the hydroxycinnamic acids group. Table S4: Influence of the cultivar and harvesting season on the flavones group. Table S5: Influence of the cultivar and harvesting season on the flavonols group. Table S6: Influence of the cultivar and harvesting season on the flavanones group. Table S7: Influence of the cultivar and harvesting season on the flavanonols group. Table S8: Influence of the cultivar and harvesting season on the stilbenes group.