The evidence suggesting many beneficial health effects associated with the dietary consumption of extra virgin olive oils (EVOO-s) has steadily accumulated for decades. More recent studies and reviews, for example, by Oliveras-Lopez et al. (2014), Garcia-Martinez et al. (2014a, 2014b), Estruch et al. (2013), Sofi et al. (2010) and Fung et al. (2009) fit the same general picture presented by Keys (1970) and Martinez-Gonzales et al. (2002) and López-Miranda et al. (2010) or Lipworth et al. (1997). The phenolic compounds, naturally present in EVOO-s are deemed to be of central importance for these beneficial cardiovascular, according to Estruch et al. (2013), Fung et al. (2009), Valls et al. (2015), antiatherogenic/antiinflammatory, Scoditti et al. (2014), Lu et al. (2013), Cabrerizo et al. (2013), anticancer, Hashim et al. (2008), Lamy et al. (2014), Hashim et al. (2014), antioxidant and antimicrobial effects Cicerale et al. (2012), Karaosmanoglu et al. (2010), Paiva-Martins et al. (2013), Giordano et al. (2014).

Olive cultivation and olive oil consumption are traditional in southern Croatia. In contrast to the millenary tradition on the Croatian coast and islands of the Adriatic, olive oil consumption is substantially lower in the northern continental part of Croatia. Although people in northern Croatia traditionally use sunflower oil more frequently than olive oil, a lack of enforced standards for EVOO in the consumer market also may play a role. As Clodoveo et al. (2014) noted, consumers place a high value on the accurate and defensible labeling of EVOO-s, also involving voluntary information such as the olive variety used. In order to extend the consumption of EVOO-s in northern Croatia, an incentive has been considered to provide labeled extra virgin olive oils as a food supplement in pharmacies, where a number of food supplements associated with some health effect expectations, such as blackberry wine, for example, had already been well marketed. This should involve an innovative labeling that includes, among others, the contents of essential phenolics such as hydroxytyrosol, tyrosol and oleuropein as well as total phenolics and possibly some important minor phenolics. In addition, accordinng to prevailing practices and popular traditions in the country, extra virgin olive oils from autochthonous olive cultivars are preferred. Here, the results of the analysis of the phenolic profile of the extra virgin olive oils obtained from the three autochthonous Croatian olive cultivars, Buža, Plominka and Žižolera, and the domesticated cultivar Bjelica, being a part of the above mentioned incentive, are reported for the first time. Furthermore, the determination also involves two more autochthonous cultivars i.e. Oblica and Lastovka and the Italian cultivar Leccino. The investigation includes the biomedicinally most significant simple phenolic compounds, hydroxytyrosol and tyrosol, along with nine other simple phenolics and the complex phenolic compound oleuropein in the oils from two succesive crop years and different agroclimatic areas.

The selected data obtained during the investigation of the phenolic fraction of the EVOO-s from the autochthonous Croatian olive cultivars Buža, Plominka, Žižolera, Oblica and Lastovka, the domesticated (since 1929.) cultivar Bjelica and the Italian cultivar Leccino (cultivated at the Istra peninsula) are given in Tables 1 and 2. The phenolic profiles of the EVOO-s from the autochthonous cultivars Buža, Plominka, Žižolera and the domesticated cultivar Bjelica were examined for the first time. All the EVOO-s examined were obtained from conventional cultivation. In Table 1 the data obtained for the EVOO-s of Bjelica, Buža and Leccino cultivars for the two olive-oil years are presented. The data enable a comparison of the phenolic contents of the EVOO-s of Bjelica, Buža and Leccino cultivars with regard to one rainy and a succesive drought olive-oil year, also involving two significantly different agroclimatic locations. The locations (Istra peninsula) differ in their height above sea-level and their distance from the sea i.e. the southern location (Pula/Barbariga and Vodnjan), is about 10 m above sea-level and close to the sea coast and the northern location (Sovinjak), is 300 m above sea-level and 25 km from the sea coast. In Table 2, the results from one crop year (corresponding to the second year denoted in Table 1) for the autochthonous cultivars Plominka, Žižolera, Oblica and Lastovka are presented.

The data presented in the Tables reveal the commonly observed difference in the contents of hydroxytyrosol and tyrosol, and other simple phenolics; whereas concentrations of hydroxytyrosol are in the range 1–12 mg·kg⁻¹ and tyrosol between 3 and 140 mg·kg⁻¹ (the latter value in the case of two samples), “minor” phenolics appeared in most cases in concentrations less than 1mg·kg⁻¹ of the oil. The concentrations of hydroxytyrosol in the EVOO-s of Bjelica and Buža are in a commonly observed range, found for instance by Tura et al. (2007), Šarolić et al. (2014), Ramos-Escudero et al. (2015), Bajoub et al. (2015), and Ballus et al. (2015). Hydroxytyrosol and tyrosol in the EVOO of Leccino are present in concentrations similar to that obtained by Šarolić et al. (2014), for the same cultivar from the city of Zadar (about 200 km to the south-east of the Istra peninsula). The total phenolic content is effectively the same in that case (Leccino, Zadar 246 mg·kg⁻¹) as for Leccino in the rainy crop year in this study (254 mg·kg⁻¹). The concentrations of the complex phenolic compound oleuropein are in the range of 1.5 and 4 mg·kg⁻¹ and could be similar, for example, to those reported by O. Garcia-Martinez et al. (2014a), for the Sicilian varieties Biancolilla, Passulunara, Tonda iblea etc., or Ilyasoglu et al. (2010), for the Turkish variety Ayvalik.

It could be seen that phenolic profile is characteristic of a particular olive cultivar. This can also be seen from the correlation test and PC analysis (see below). Thus, although the ripening index was significantly lower in the case of the cultivar Bjelica than that for Buža and Leccino, both hydroxytyrosol and tyrosol and total phenolic content of Bjelica are greater than those for Buža and Leccino and this holds for the rainy and drought crop year as well. Furthermore, while the observed content of hydroxytyrosol and total phenolics for cultivars Bjelica and Leccino were much greater in the drought than in the rainy year, the opposite could be seen for tyrosol and there is a difference in the content of hydroxytyrosol with regard to the Buža cultivar. Following an investigation comprising four successive crop seasons, Romero et al. (2003) concluded that phenolic profiles were mainly influenced by cumulative rainfall. Similarly, Ilyasoglu et al. (2010) found that the total phenolic contents of some Aegean VOO-s are greater in the drought than in the rainy crop year, but their observed range of variation is much smaller than that observed in this study. It seems that the total phenolic content could depend less on the plantation area but is much more characteristic of the cultivar and strongly depends on the climate characteristics of the year (see also the above mentioned case of Leccino). In this context some findings of Tura et al. (2009) may be of interest; they suggested that the cultivation sites did not affect oil composition and profile of EVOO of the same cultivar in the same crop year. However, Table 1 shows the concentrations of a majority of individual phenols in the EVOO-s to be greater in oils from the southern location. This seems not to be unexpected since environments where olive trees grow may likely influence plant physiology as well as the olive ripening process and subsequently the phenolic profiles. Here, it might be noted that, for example, the finding of a total phenolic content of 1589 mg·kg⁻¹ of EVOO in the Bjelica cultivar from Pula (Barbariga) is consistent with the historically well known great appreciation that the olive oil from the Istra peninsula received from the ages of the Roman Empire. To emphasize this issue, as well as, for example, the unusually high concentration of tyrosol in the EVOO (see below) could be additional line within the intended framework for a more innovative labeling of EVOO aimed at being presented as a food supplement.

The examined EVOO-s from the cultivar Bjelica have very high concentrations of tyrosol in some cases. While the observed concentrations range from 3 mg·kg⁻¹ to 140 mg·kg⁻¹ of tyrosol, the reported average value of the tyrosol content in VOO-s from the 23 samples from Spain, Italy, Greece and Tunisia (Hrncirik et al., 2004) is tenfold or even lower than our upper value for this phenol. This upper value presents one of the highest observed contents of tyrosol in an EVOO and values well below 13 mg·kg⁻¹ of tyrosol were often reported (Ramos-Escudero et al., 2015, Bajoub et al., 2015, Ballus et al., 2015). Given the great biomedicinal potential of tyrosol suggested which, among others involves antioxidants, Cicerale et al. (2012), neuroprotective, Vauzour et al. (2010), cardiovascular Nakbi et al. (2011), antimicrobial, Karaosmanoglu et al. (2010), anti-inflammatory, Lu et al. (2013), Cárdeno et al. (2014), antigenotoxic, Anter et al. (2014), to mention only a few, future selection of the olives based on the presented results could be of use to obtain oils which are more abundant in particular simple phenol compounds.

The presence of biomedicinally important “minor” phenolic compounds such as p-coumaric, caffeic acid, homovanillyl alcohol, vanilic acid and others in the examined EVOO-s (see Table 1 and 2) should be of particular interest. Because of, for instance, beneficial anticancer effects, Nichenametla et al. (2006), anti-inflammatory potential, Pragasam et al. (2013) potential beneficial effects in treating metabolic disorders, Yoon et al. (2013), antibacterial effects, Lou et al. (2012) and a possible preventive effect on myocardial infarct size, Roy et al. (2013) of p-coumaric acid, or the beneficial effects of caffeic acid, El Seedi et al. (2012), Vauzour et al. (2010), da Cunha et al. (2004), these and other “minor” phenolics should be an inevitable part in the new labeling framework.

Here, a feasibility of using a back-label on EVOO bottles to additionally quote scientific references (perhaps including citation of short statements from such references) is under consideration. Namely, in the case of olive oil, European Regulation 432/2012 states that the health effect can be claimed only if the oil contains more than 5mg of hydroxytyrosol and its derivatives (e.g., oleuropein complex and tyrosol) in 20 g of oil, but the quotation of scientific references is not explicitly forbidden.

The phenolic profiles of EVOO-s of the autochthonous cultivars Plominka, Žižolera, Oblica and Lastovka (Table 2) are not very different from those of Buža and Bjelica. It could be noted that Oblica is the most represented olive variety in Croatia. Table 2 shows fairly high concentrations of tyrosol, hydroxytyrosol and p-coumaric acid both in EVOO-s of Oblica and Lastovka. The case of Žižolera could be of some interest because oil from three hundred year olives was examined. Namely, there are at the island of Pag (Lun, about 50 km to the south-east of the Istra peninsula) about 1000 very old olive trees (mainly of the autochthonous Croatian varieties Mastrinka and Oblica); the majority of these olives comprise trees older than 700 years, and there is also a sizable group of trees more than 1000 years old. Their location is of special interest within the framework of the incentive mentioned at the beginning. It should be noted that the European Community (EC) Council of Regulation established compulsory standards for olive oil production with regard to labeling (European Commission Regulation 182/2009, cf. in references); the regulation has compelling virgin and extra-virgin olive oil producers to indicate the geographical location of olive harvesting and oil production on the label, but the age of the olive trees is not involved. There are thousands of very old autochthonous olive orchards across the Croatian Adriatic coast and islands.

A correlation test involving data obtained from the analysis of the phenolic profile of EVOO-s from the cultivars Bjelica, Buža and Leccino (Table 1, 3, 4, 5) shows significant characteristic correlation profiles pertaining to a particular cultivar. It could be speculated that the relationships among phenolic compounds reflect more complex relationships between genetic and biosynthetic patterns, thus describing the “identity” of a particular olive variety.

Recently, Tura et al. (2007) and El Riachy et al. (2012a) reported the correlation between tyrosol and vanillic acid, and El Riachy et al. (2012b) the correlation between vanilic acid and p-coumaric acid, ascribing the findings to the same biosynthetic pathway of these compounds.

A principal component analysis (PCA) also was carried out in an attempt to explore some underlying pattern that may be taken as a source of variation in EVOO phenolic compound composition.

The data set for different cultivars, involving two different agroclimatic locations and obtained over two harvest years that differ significantly in the amount of rainfall, regarding the variables of phenolics was subjected to PCA to investigate possible grouping of samples on the basis of cultivar, harvest year and agroclimatic location.

PCA showed that 68% of the total variance was associated with the first three principal components. The first component accounted for 33.5% of the total variance, with a high positive correlation with 3,4dihydroxybenzoic acid, p-hydroxybenzoic acid, homovanillyl alcohol and syringic acid; the second component accounted for 21.5% of the total variance and was positively correlated to tyrosol, hydroxytyrosol, p-coumaric and ferulic acid and the third component accounted for 13.4% of the total variance with a high positive correlation with vanillic and caffeic acid.

The biplot on PC1 and PC2 (Figure 1), explaining 55% of the total variance, allows for differentiation of the two groups according to harvest year mainly on the basis of the first principal component values. Almost all EVOO samples from the rainy harvest year have positive scores for the first component, while almost all drought year EVOO samples have negative scores. There is also obvious differentiation taking into regard cultivar, mainly on the basis of the second principal component values. All EVOO samples corresponding to the Bjelica cultivar have positive scores for the second component, while Buža and Leccino samples have negative scores. Furtherly, there is differentiation between Buža and Leccino samples, with Buža samples found between Bjelica and Leccino. The biplot on PC1 and PC3 (Figure 2), explaining 47% of the total variance, also shows good separation of samples regarding harvest year, while in the biplot on PC2 and PC3 (Figure 3), explaining 35% of the total variance, the grouping is less obvious. Regarding cultivar, the biplot on PC2 and PC3 shows good grouping of the Bjelica cultivar and some overlap between Leccino and Buža; while in the biplot on PC1 and PC3 there is no obvious grouping of different cultivars. Interestingly, and consistent with some of the mentioned considerations (see above) regarding agroclimatic location, there is no significant differentiation in the score plots.

Phenolic profile analyses of the extra virgin olive oils obtained from the three autochthonous Croatian olive cultivars, Buža, Plominka and Žižolera, and the domesticated cultivar Bjelica, are reported for the first time. The analyses have been performed within the framework of an incentive that has been considered to provide labeled extra virgin olive oils as a food supplement in pharmacies, in order to extend the consumption of EVOO-s in northern Croatia. The obtained data, also involving a correlation test and the insight from PCA reveal that the cultivars are highly individualized in character with regard to relationships among phenolic compounds. This finding as well as the observation of very significant differences in the content of phenolic compounds between the rainy and successive drought crop years will be of importance for targeted acquisition and perhaps the price of the oils. The observation of an extremely high concentration of tyrosol in some cases can be useful not only for future selection of the olives to obtain oils which are more abundant in a particular simple phenol compound. While the information on the most important phenolic compounds and the total phenolic content should be a part of the main label, on the back-label of EVOO bottles appropriate biomedicinally important scientific references (perhaps including citation of short statements from such references) with regard to selected and especially abundant phenolic compound(s) in the oil could be additionally quoted. The information on the variety used, the age of very old olive trees, the location of orchard and the harvest year should follow a tendency toward an innovative labeling aimed at better presenting the authenticity, quality, excellence and uniqueness of the EVOO-s. Finally, the issue reported also seems to be consistent with the The Framework Programme for Research and Innovation - 2014-2020 of the European Commission, promoted as a research program aimed at preserving the image of olive oil and to guarantee its quality and authenticity. A very important issue related to the uniqueness, authenticity and quality of EVOO-s is the question of the aromatic profile of oils (Reboredo-Rodríguez et al., 2012, 2013a, b, c, 2014a, b); although not elaborated in this study, it should be an unavoidable part of subsequent future work in the matter.