Phenolic Profile and Antioxidant Activity of Italian Monovarietal Extra Virgin Olive Oils

In the last years, the interest in Italian monovarietal oils has increased due to their specific organoleptic qualities. Extra virgin olive oils (EVOOs) are rich in phenolic compounds, secondary metabolites well known and studied for their nutraceutical properties. However, among EVOOs, there is great variability in phenolic composition due to the origin, the production technique, and mainly, the genotype. The aim of this work was to evaluate the different phenolic profiles and the antioxidant activities of monovarietal oils. The results confirm this variability. In fact, the overall content of oleuropein varies up to four times between the different genotypes (from 33.80 to 152.32 mg/kg oil), while the oleocanthal content is significant only in two oils. The antioxidant activity, determined with 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) assays, is correlated with the content of total phenolic substances, with half maximal inhibitory concentration (IC50) values for the DPPH test ranging from 160 to 91 mg of oil, while the ORAC test shows values between 5.45 and 8.03 μmol Trolox equivalent (TE)/g oil.


Introduction
Extra virgin olive oil (EVOO) is the unique oil obtained by mechanical cold processes from the fruits of the olive. EVOO mainly consists of triglycerides (about 98%), and among them, oleic acid represents the main component (about 50 to 80%). Linoleic acid (from 3.5% up to 20%) and small amounts of linolenic acid (about 1%) are usually found in EVOO too. In the remaining 2%, there are several compounds such as phytosterols, vitamin E, triterpenes, and aliphatic alcohols, as well as a series of phenolic compounds that characterize the quality of the EVOO [1].
The phenolic compounds typically present in EVOO are mainly derivatives of hydroxytyrosol and tyrosol and are classified as secoiridoids. There are also small quantities of lignans, pinoresinol and acetoxypinoresinol being the most common. Among phenolic acids, vanillic, coumaric, and protocatechuic acid can be found in EVOO. Finally, there are some flavonoids, among which luteolin and apigenin are the most important [2][3][4][5][6][7][8].

Plant Material and Oil Production
Oils from eight genotypes/cultivars (Table 1) were extracted from olives of trees grown in an identical climate/environment area: the province of Lecce, Apulia, Italy (40 • 21'17.32" N, 18 • 10'20.78" E). To limit the variation in phenolic content due to the ripening stage, the olives were harvested when they reached a ripening index (Jaén index) equal to 3 [25,28] and then processed with a mini-laboratory crusher. The oil was stored in amber-colored bottles. Immediately after oil production, the bottles were closed and kept in an environment protected from sunlight. Oil analyses were completed within a month after olive oil production. The oils were analyzed and classified as EVOO according to EU laws (data not shown).

Phenolic Compounds: Extraction, Identification, and Quantification
The phenolic compounds were extracted by placing 5 g of oil into a 50 ml tube. For a better oil fluidification, 2 ml of hexane were added and subsequently, 5 ml of methanol: water 80:20 v/v. The solution was vortexed for 10 min. The emulsion was subjected to centrifugation for 20 min at 5500× g at 4 • C to separate the two phases. The alcoholic extract was recovered, and this procedure was repeated three times. Finally, the alcoholic extract was evaporated in cold and reduced pressure conditions. The dried extract was resuspended in 1 ml of 80% methanol.
The total phenolic content was determined using the spectrophotometric Folin-Ciocalteu method [29]. Data were expressed as mg of gallic acid equivalent (GAE) per kg of EVOO.
The phenolic identification was performed using the Agilent 1200 Liquid Chromatography System (Agilent Technologies, Palo Alto, CA, USA) equipped with a standard autosampler and Agilent column Zorbax extended C 18 50 × 2.1 mm, 1.8 µ. The separation was carried out at 30 • C with a gradient elution program at a flow rate of 0.4 mL/min. The mobile phases consisted of water plus 0.1% formic acid (A) and acetonitrile (B). The following multistep linear gradient was applied: 0 min, 10% B; 10 min, 25% B; 14 min, 50% B; 20 min, 80% B; 20 min 90% B. The injection volume in the HPLC system was 5 µL. The HPLC system was coupled to an Agilent diode array detector (DAD) (λ detection was 280 and 330 nm) and Agilent 6320 Time-of-Flight (TOF) mass spectrometer equipped with a dual electro spray interface (ESI) (Agilent Technologies) operating in negative ion mode. Detection was carried out within a mass range of 50-1700 m/z. Accurate mass measurements of each peak from the total ion chromatograms (TICs) were obtained by means of an Isocratic Pump (Agilent G1310B, company, Santa Clara, CA, USA) using a dual nebulizer ESI source that introduces a low flow (20 µL/min) of a calibration solution that contains the internal reference masses at m/z 112.9856, 301.9981, 601.9790, 1033.9881, in negative ion mode. The accurate mass data of the molecular ions were processed through the software Mass Hunter (Agilent Technologies, Santa Clara, CA, USA). The quantification of phenolic compounds was achieved using calibration curves of authentic chemical standards: hydroxytyrosol, oleuropein, pinoresinol, luteolin, and apigenin.

Antioxidant Activity
Antioxidant activity was evaluated using a 2,2-Diphenyl-1-picrylhydrazyl (DPPH) test as reported by Bondet et al. [30] and determined as half maximal inhibitory concentration, IC 50 per mg of oil, and an oxygen radical absorbance capacity (ORAC) test, as reported by Ou et al. [31] and expressed as µmol Trolox Equivalent (TE)/g oil.

Statistical Analysis
All data were reported as the mean ± standard deviation (SD), with at least three replications for each olive oil. Statistical evaluation was conducted by Duncan's test to discriminate among the mean values. To evaluate the correlation between antioxidant activity and total phenolic content (TPC) and between antioxidant activity and the single compounds, the Pearson correlation was calculated. Moreover, to provide a concise but comprehensive presentation of chemical compounds in relation to the different EVOOs, a heatmap was drawn [32].

Results
The total phenolic content (TPC) of eight monovarietal oils was expressed as gallic acid equivalents ( Table 2). TPC ranged between 138 and 278 mg GAE/ kg, in the Spina and Ogliarola di Lecce cultivars, respectively. This result highlighted a great difference in the oil phenolic compounds content, suggesting that the genotype may be responsible for about 50% of this parameter. As far as the identification and characterization of phenolic compounds in EVOO extracts is concerned, 30 compounds, already reported in other EVOO extracts [23][24][25][26][27], were identified, although these were not always present in all examined oils. Figure 1 displays a representative chromatogram of the phenolic extract obtained from the monovarietal oil of the Oliva Grossa cultivar (the chromatograms related to EVOO extracts from the other cultivars are essentially similar), while Table 3 shows the results of the qualitative analysis obtained by high-performance liquid chromatography time-of-flight mass spectrometry (HPLC/MS-TOF). The antioxidant activity results are reported in Table 5. Relative to the DPPH test, the radical scavenging IC50 ranged between a best value of 91 mg (Oliva Grossa) and the worst of 160 mg (Spina). The mean IC50 was 102 mg. The antioxidant activity values measured by ORAC assay ranged between 5.45 µ mol Trolox equivalent (TE)/g oil (Spina) and 8.03 µ mol TE/g oil (Oliva Grossa). The mean value was 7.14 µ mol TE/g oil.
The antioxidant activity values were, as expected, closely related to the TPC oil va lues; Figure 2 shows the correlations between TPC and antioxidant activities. The two antioxidant assays showed a similar correlation coefficient (0.7788 for the DPPH assay and 0.8095 for the ORAC assay).  Table 3.  Table 3.
The main compound in the Colozzese and Barone di Monteprofico oils was oleuropein aglycone isomer 3, with 93.7 and 63.6 mg/kg, respectively, which is also present in a high concentration in Oliva Grossa EVOO. In Cellina di Nardò and Oliva Grossa, the dominant compound was decarboxymethyl oleuropein aglycone, with 73.6 and 124.4 mg/kg oil, respectively. Lastly, in Cornola, Ogliarola di Lecce, Oriella, and Spina, hydroxyoleuropein aglycone was the most represented compound. Among the other compounds, the content of decarboxymethyl ligstroside aglycone (oleocanthal) in the Colozzese and Oliva Grossa EVOOs, is especially meaningful, with values of 75.4 and 103.4 mg/kg, respectively. The amount of hydroxytyrosol was very variable. In particular, Colozzese contained about 16.1 mg/kg oil, while the other genotypes had lower values that ranged between 0.2 and 8.8 mg/kg oil. The luteolin and apigenin flavonoids were present in all EVOOs, with values ranging between 2.2 and 16.7 mg/kg (luteolin), respectively, in the Cornola and Oliva Grossa oils, and between 0.2 and 9.2 mg/kg (apigenin) in the Ogliarola di Lecce, Cellina di Nardò, and Cornola oils. High contents of lignans were observed in Barone di Monteprofico and Oliva Grossa EVOOs; the amounts of pinoresinol were 18.6 and 21.9 mg/kg oil, respectively. In contrast, Cellina di Nardò showed low levels of pinoresinol (1.4 mg/kg oil), whereas the mean value across all oils was 3.2 ± 2.1 mg/kg oil.
The antioxidant activity results are reported in Table 5. Relative to the DPPH test, the radical scavenging IC 50 ranged between a best value of 91 mg (Oliva Grossa) and the worst of 160 mg (Spina). The mean IC 50 was 102 mg. The antioxidant activity values measured by ORAC assay ranged between 5.45 µmol Trolox equivalent (TE)/g oil (Spina) and 8.03 µmol TE/g oil (Oliva Grossa). The mean value was 7.14 µmol TE/g oil.
Elenoic acid is.    Figure 2 shows the correlations between TPC and antioxidant activities. The two antioxidant assays showed a similar correlation coefficient (0.7788 for the DPPH assay and 0.8095 for the ORAC assay).   The measured Pearson correlation coefficient between ORAC and DPPH tests was -0.84, showing a negative correlation between the two tests. This negative value was expected due to the opposite representation of the result (µ mol Trolox equivalent/g versus IC50 mg oil).
To evaluate the possible correlation between a single compound and antioxidant activity, the Pearson correlation was measured and is reported in Table 6. Decarboxymethyl oleuropein aglycone and decarboxymethyl ligstroside aglycone are the two compounds with a higher correlation to antioxidant activities. In addition, quinic acid shows a high correlation with antioxidant activity, even if the concentration is very low in the analyzed olive oils.  The measured Pearson correlation coefficient between ORAC and DPPH tests was −0.84, showing a negative correlation between the two tests. This negative value was expected due to the opposite representation of the result (µmol Trolox equivalent/g versus IC 50 mg oil).
To evaluate the possible correlation between a single compound and antioxidant activity, the Pearson correlation was measured and is reported in Table 6. Decarboxymethyl oleuropein aglycone and decarboxymethyl ligstroside aglycone are the two compounds with a higher correlation to antioxidant activities. In addition, quinic acid shows a high correlation with antioxidant activity, even if the concentration is very low in the analyzed olive oils.
Furthermore, for a better graphical representation of the EVOO chemical compound distribution among genotypes, a heatmap [32] was drawn (Figure 3). The relative unsupervised clustering highlighted two main clusters: a first group including Oliva Grossa, Ogliarola, and Cellina di Nardò genotypes, and a second cluster including Cornola, Orniella, Spina, Colozzese, and Barone di Monteprofico. Among the chemical compounds, decarboxymethyl oleuropein aglycone and decarboxymethyl ligstroside aglycone cluster together. Furthermore, for a better graphical representation of the EVOO chemical compound distribution among genotypes, a heatmap [32] was drawn ( Figure 3). The relative unsupervised clustering highlighted two main clusters: a first group including Oliva Grossa, Ogliarola, and Cellina di Nardò genotypes,

Discussion
The TPC detected in the eight monovarietal EVOOs ranged between 138 and 278 mg GAE/kg oil, and they agree with the values reported by Baiano et al. [33] (133-322 mg GAE/kg oil) for olive

Discussion
The TPC detected in the eight monovarietal EVOOs ranged between 138 and 278 mg GAE/kg oil, and they agree with the values reported by Baiano et al. [33] (133-322 mg GAE/kg oil) for olive orchards located in the north of the Apulia region. Similarly, oils from environments like that of the Province of Lecce (Apulia, Italy) showed a TPC of approximately 300 mg GAE/kg oil [10], whereas Ninfali et al. [7] reported values between 50 and 236 mg/kg for plants cultivated in the center of Italy. Ricciutelli et al. [23] analyzed commercial oil and reported values ranging from 136 and 437 mg GAE/kg oil. In Chile [34], Spain [35], Greece [36], and Tunisia [25] the phenolic compounds quantified in olive oil were even higher (680 mg GAE/kg oil). Therefore, the TPC in the eight EVOOs analyzed is in agreement with the average values observed in other extra virgin olive oils [7,10,23]. However, the TPC of EVOOs is strictly related to the olive harvesting time, oil extraction techniques, and also to quantification methodologies [37,38].
Among the many phenolic compounds identified in the eight EVOOs, decarboxymethyl ligstroside aglycone (oleocanthal) deserves special attention because of its several nutraceutical activities highlighted by many studies and reviews [16,20,39,40]. In the analyzed EVOOs, we found high oleocanthal concentrations in Colozzese and Oliva Grossa oils (75.4 and 103.4 mg/kg oil, respectively). The oleocanthal in olive oils showed wide concentration ranges, as reported by many authors [24,26,34,35,41]. In Spanish olive oils, the mean value was 3.90 mg/kg [35], whereas the Sicilian oils had 25 mg/kg, and the Tunisian oils had about 40 mg/kg [41]. Maximum values were reported by Fuentes et al. [34]: 77 mg/kg oil. Considering the central role of this molecule in the nutraceutical properties of EVOO, Colozzese and Oliva Grossa genotypes can represent an excellent source of this compound, and further studies are therefore needed to evaluate the nutraceutical activities of EVOOs produced from the above-mentioned cultivars.
Another interesting molecule present in EVOOs is hydroxytyrosol, which is worthy of investigation for its nutraceutical properties [42]. In the Colozzese cultivar, we found 16 mg of hydroxytyrosol/kg oil, whereas other authors reported much lower values in monovarietal oils, ranging between 0.36 and 2.85 mg/kg oil [33]. In contrast, oils produced from olives in the of south Italy showed a high hydroxytyrosol concentration (41 mg/kg oil) [10], and Ricciutelli et al. [23] have identified a mean value of 9.9 mg/kg oil for high-quality commercial oils. Oil from Spain, Morocco, and Tunisia contain, respectively, 14.34, 6.65, and 22.57 mg/kg oil [24,25,27]. Since EVOO from the Colozzese cultivar also showed a high hydroxytyrosol concentration in comparison with other oils, this cultivar is worthy of interest to obtain EVOO blends with increased nutraceutical properties.
Moreover, oleuropein aglycone, which normally decreases from drupes to malaxation paste and to the final oil [36], ranges from 33.8 to 152.3 mg/kg in the Orniella and Oliva Grossa samples, respectively, and falls within the range of data reported in the literature. In fact, similar values were reported in oils from southern Italy and in oils from Tunisia [10,25,41], whereas lower values were reported in Spanish oils [35]. In contrast, higher values were observed in oils obtained in Morocco [24].
In this report, we performed the analysis of antioxidant activity using two different assays: DPPH and ORAC. Since the solvents of DPPH and ORAC assays are methanol and water, respectively, differences in the reported antioxidant activities are expected. Nevertheless, the tests showed a correlation, as reported by other authors [34,43].
Hydroxytyrosol and oleuropein are known as scavengers of hydrophilic peroxyradicals and are usually associated with the antioxidant activities of olive products [44]. However, the data collected from these eight EVOOs did not show a tight correlation between antioxidant activity and the concentration of hydroxytyrosol or oleuropein. As reported by Presti and colleagues [45], hydroxytyrosol is not always correlated to the measured antioxidant activity, whereas the antioxidant activity of oleuropein seems to be linked to the presence of the carboxy-methyl group [6].
The comparison of antioxidant activity between the eight EVOOs and other oils reveals similar trends: Lavelli [46], as well as other authors [10,47], reported DPPH antioxidant activity (IC 50 values) ranging from 27 to 156 mg. For the ORAC assay, the values ranged from 1.7 and 8.2 µmol TE/g oil in Italian oils [7,48] and from 1.4 and 4.9 µmol TE/g oil in Spanish oils [43]. In the olive oils analyzed in this paper, the ORAC values ranged between 5.4 and 8.0 µmol TE/g oil, high values, on average, for Italian oils. Antioxidant activity is a good index for representing oil quality because it is positively correlated to phenolic compounds ( Figure 2 and Table 6).
In particular, we observed that quinic acid, decarboxymethyl oleuropein aglycone, and decarboxymethyl ligstroside aglycone positively correlate to antioxidant activity. However, quinic acid showed low concentrations and probably does not represent the main determinant of the antioxidant properties of these oils. In contrast, decarboxymethyl oleuropein aglycone and decarboxymethyl ligstroside aglycone are known to possess antioxidant activities [6,47,49].
In fact, compounds with o-dihydroxyl functionalities, such as decarboxymethyl oleuropein aglycone, are able to form intramolecular hydrogen bonds during the reaction with free radicals [6,47].
The heatmap plot also highlighted that some compounds are able to characterize some genotypes. For example, elenoic acid, deoxyelenoic acid, and hydroxytyrosol are more concentrated in olive oils from the Colozzese cultivar. In contrast, apigenin, acetoxypinoresinol, and ligstroside is.1 are more concentrated in the Cornola cultivar.

Conclusions
The results of the present work support the literature in the conclusion that extra virgin oils show a very high variability regarding the content of phenolic compounds. Therefore, a detailed characterization of EVOOs is important to better understand and attest to the antioxidant values and nutraceutical proprieties of this key food of the Mediterranean diet. In addition, the wide variation in the presence of different polyphenols in the tested monovarietal EVOOs demonstrates the remarkable biodiversity typical of Italian olive oils and confirms their nutraceutical and economic value. In particular, Colozzese and Oliva Grossa EVOOs exhibited high amounts of oleocanthal and hydroxytyrosol and good antioxidant activities. Further studies will evaluate nutraceutical features, such as the anti-inflammatory and anti-proliferative activities of such EVOOs, with the aim of suggesting the best use and therefore maximize their health beneficial effects.