**1. Introduction**

The olive cultivar "Cellina di Nardò" (CdN), also known as "Leccese", "Saracena", "Visciola", "Asciulo", or "Muredda", is an Italian variety that is widespread in Salento (Apulia, South Italy), especially in the Province of Lecce, although it is also grown in the territories of Taranto and Brindisi, covering a total of about 60,000 hectares. However, since 2013, Salento has been su ffering from a devastating olive disease (the olive quick decline syndrome) caused by the bacteria *Xylella fastidiosa* [1,2] that has progressively destroyed hundreds of thousands of olive trees. Unfortunately, the CdN cultivar is sensitive to *Xylella fastidiosa*, and in the coming years is likely to disappear completely from the Salento area [3,4]. For this reason, a complete characterization of such traditional cultivars is required, as many plants are still cultivated in South Italy.

The CdN tree is vigorous; the branches at the top are erect, while the lateral ones almost pendulous, and the tree can reach a height of 20 m. The leaves have an elliptical elongated shape; the upper side of the leaf is dark green while the lower one is silvery gray. The blooming stage is quite early, and the inflorescence results in 15–20 flowers. The fruit is an elliptical drupe, slightly asymmetrical, with a color ranging from green to black; when ripening is completed, it has a reduced size, with a weight ranging from 1.5 to 2.0 g, and a low oil yield (15–17%).

The drupes have a high resistance to detachment and are not suitable for mechanical harvesting. On the contrary, this cultivar is valued by local farmers due to the slow vegetative growth, good fructification in adverse conditions, and good tolerance to cold and various pests. Despite the low oil yield and di fficulties in harvesting, the CdN is cultivated mainly for oil production, which is characterized by an intense fruity flavor. The oil obtained from CdN and Ogliarola olives (another traditional cultivar widespread in Salento) is guaranteed by the brand "Terra d'Otranto" as a "Protected Designation of Origin", and presents some specific characteristics as a consequence of the geographical influence, pedoclimatic conditions, agronomic techniques, and oil processing.

The olive fruit consists of water (about 50%) and fats (20%), and the remaining part is made up of nitrogenous compounds, cellulose, sugars, and secondary metabolites [5]. The secondary metabolites of fresh fruit and fermented olives can vary greatly. In fact, the di fferent processes of extraction and purification can modify the chemical structure of the molecules due to exposure to oxygen or solvents or even pH changes, situations that can commonly occur in phenolic metabolism [6]. The proportion of phenolic compounds within the edible part of the olive is considerable and can reach concentrations ranging from 1% to 3% of the fresh weight of the pulp [7]. There is a complex mixture of phenolic compounds in olives, some of which are present at very low concentrations and as a consequence are di fficult to identify [8]. Moreover, phenolic and secondary metabolites are not uniformly present in diverse parts of the fruit: most of them are present in the pulp (about 85–90%), followed by the peel (about 8–12%), and then by the seed (1–2%).

Among phenolic compounds, oleuropein is generally the most represented among the various olive cultivars, reaching concentrations up to 140 mg/g fresh weight (FW) [9]. Oleuropein belongs to the secoiridoid family, as well as other compounds usually found in olives like dimethyl oleuropein, verbascoside, ligstroside, and nüzhenide [10]. In particular, oleuropein, dimethyl oleuropein, and verbascoside have been found in the all parts of the olive fruit (pulp, skin, and seed); conversely, the presence of nüzhenide was reported only in the seed [11]. In the olive fruit there are also flavones such as luteolin-7-glucoside, flavonols such as quercetin 3-rutinoside [12], anthocyanins like cyanidin-3-rutinoside and cyanidin-3-glucoside, and phenolic acids such as hydroxybenzoic, gallic, ferulic, ca ffeic, vanillic, and syringic acid [13].

All these secondary metabolites are of grea<sup>t</sup> interest for human health because of their antioxidant activity and properties with respect to cancer prevention, inflammatory disorders, and cardiovascular diseases [14,15].

The health properties of CdN when used as table olive have not ye<sup>t</sup> been reported, and the *Xylella fastidiosa* threat suggests their urgen<sup>t</sup> investigation due to the extinction risk caused by the pathogen. Therefore, in this paper a characterization of the secondary metabolites during ripening stages was carried out. Moreover, six di fferent table olive cultivars were compared to CdN cultivars to establish the best food in terms of antioxidant e ffects and phenolic compounds.

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

#### *2.1. Plant Material and Samples Preparation*

To evaluate the antioxidant activity and phenolic content during ripening, the olives of CdN cultivar were collected from four of the eight Maturity Index (MI) classification groups (0–7) of olives described by Guzman et al. [16] (Figure 1).


Drupes were collected from three different orchards in Salento.

**Figure 1.** Cellina di Nardò olives. The images represent four of the eight Maturity Index (MI) classification groups (0–7) of olives described by Guzman et al. [16]. Stage 0: green olives; Stage 2: Olive peel partially pigmented and green pulp; Stage 4: dark/black peel and yellow pulp; Stage 7: dark/black peel and pulp.

To obtain CdN table olives, fully-ripened olives are traditionally first washed for two days (replacing the water several times) and then deposited in barrels of about 300 kg, covered with saturated brine, and left to ferment naturally (without the addition of chemical products for debittering) until they reach a pH below 4.0; the process takes an average of six months. CdN table olives were analyzed and compared with commercial table olives (Leccino, Blanqueta, Ogliarola, Empeltre, Hojiblanca, Kalamata) purchased on the market. Leccino and Ogliarola table olives were locally fermented essentially as described for CdN olives, whereas table olives of the other cultivars (much more common on the market) were harvested green and darkened by oxidation.

To extract the phenols from edible part of olives, the pulp of 60 olives was homogenized and 5 g were sampled. Then, 50 mL of a cold solution of methanol/water (80/20 *v*/*v*) acidified with HCl (pH 2.5) were added. Methanol and pulp were mixed by continuous agitation for 30 min. The homogenate was then filtered. The pulp was recovered from the filter and mixed again with 50 mL of the same methanol/water solution. A second vacuum filtration was then performed, and the filtrate was added to the previous one. Since the eluates could contain lipid traces, two treatments with an equal volume of hexane (≥97%, HPLC grade) were carried out to remove fatty acids. The extracts were finally concentrated by evaporation under low pressure and cold, avoiding any residues of methanol and obtaining, therefore, aqueous extracts (approximately 10 mL).
