CHARACTERISTICS of DRUPES, PHENOLIC CONTENT and ANTIOXIDANT CAPACITY of ITALIAN OLIVE FRUITS ABSTRACT Drupe Characteristics

CHARACTERISTICS OF DRUPES, PHENOLIC CONTENT AND ANTIOXIDANT CAPACITY OF ITALIAN OLIVE FRUITS

ANTONIETTA BAIANO1,2,3, GIUSEPPE GAMBACORTA1,2, CARMELA TERRACONE1, MARIA ASSUNTA PREVITALI1 and ENNIO LA NOTTE1,2

1Department of Food Science

2Istituto per la Ricerca e le Applicazioni Biotecnologiche per la Sicurezza e la Valorizzazione dei Prodotti Tipici e di Qualità University of Foggia Via Napoli 25 – 71100 Foggia, Italy

Submitted for Publication January 6, 2009 Revised Received and Accepted March 5, 2009

ABSTRACT

Drupe characteristics, phenolic content and antioxidant capacity of olive fruits were studied for 12 Italian oil cultivars grown in two areas (Cerignola and Torremaggiore) of Daunia district. The effects of plantation place were higher than those exerted by cultivars on drupe characteristics such as weight, pulp/stone ratio, oil content and maturation index. Olive fruits from Cerignola, which is located at a lower altitude than Torremaggiore, showed the highest phenolic content. The highest phenolic content (14 g gallic acid/kg dry olive) was detected on Peranzana and Cellina di Nardò grown in Cerignola groves. The lowest values were registered for FS17 and Cima di Melﬁ (about 7 g/kg). 1-Acetoxypinoresinol, 3,4-dihydroxyphenylethyl 4-formyl-3- formylmethyl-4-hexenoate, the dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA), hydroxytyrosol and luteolin, were the phenolic compounds detected in a greater amount and in most of the cultivars. Independently on the assay applied, the highest antioxidant activity was detected for Cellina di Nardò (Cerignola). The lowest antioxidant activity values were different depending on the method applied. According to the 2,2Ј-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) assay, the lowest value was detected for Moraiolo from Torremaggiore, whereas Nociara showed the lowest contribution to the inhibition of lipid oxidation (b-carotene beaching assay). A strong positive linear correlation (R = 0.82) existed between

3 Corresponding author. TEL: (+39) 881-589117; FAX: (+39) 881-589308; EMAIL: a.baiano@

unifg.it_ 209..226

Journal of Food Lipids 16 (2009) 209–226. All Rights Reserved. © 2009, Wiley Periodicals, Inc. 209 210 A. BAIANO ET AL. total phenolic content and antioxidant activity measured according to the ABTS method, whereas a low linear correlation coefﬁcient (R = 0.36) was obtained when the b-carotene bleaching assay was used.

PRACTICAL APPLICATIONS

The experimental results obtained with this research could help farmers to increase their knowledge of the minor components of some major and minor Italian olive cultivars. In fact, there are very few works on the characterization of olive fruits destined for oil production. The analytical ﬁndings of this study can also provide useful information to the oil industries interested in process- ing olives having high phenolic content and high antioxidant capacity. Fur- thermore, the diffusion of these results could help consumers to choose, among monovarietal extra virgin oils available in the market, those derived from the cultivars rich in antioxidant compounds.

INTRODUCTION

Olive (Olea europaea L.) is one of the oldest cultivated fruits, largely found in the Mediterranean countries. The Italian olive germplasm includes hundreds of cultivars, most of them propagated at the farm level since ancient times including minor local cultivars widespread in different olive-growing areas (Cantini et al. 1999). The study of minor cultivars is important because they may have traits such as vigor, resistance to low temperature, salinity tolerance, as well as acidic and phenolic composition important for olive oil quality. Only a few cultivars are grown to any signiﬁcant extent. Some important cultivars in Italian olive oils include Frantoio, Leccino, Coratina, Peranzana, Pendolino and Moraiolo. The growing interest in the determination of phenolic compounds in vegetables and fruits is due to the natural antioxidant activity of these compounds. Phenolic compounds are secondary metabolites produced in plants as a mechanism of protection against microorganisms, pathogens and strong ultraviolet (UV) radiation. The increasing popularity of olives (O. europaea) and olive oil is mainly attributed to their content in bioactive compounds including carotenoids, tocopherols and other phenolic compounds that are natural alternatives to synthetic antioxidants as they possess similar or even higher antioxidant activity. The antioxidant activity of phenolic compounds is due to their ability in quenching free radicals and metal chelation (Saija et al. 1998). Some biophenols present PHENOLIC ANTIOXIDANTS OF ITALIAN OLIVE FRUITS 211 in olive drupes seem to be involved in several activities such as prevention of coronary artery disease and atherosclerosis because of their ability to inhibit platelet aggregation (Carluccio et al. 2003), modulation of the arachidonic acid metabolism (Kohyama et al. 1997) and inhibition of low-density lipopro- tein peroxidation (Visioli et al. 2002). Phenolic compounds of the Oleaceae family are characterized by the presence of a number of coumarin-like compounds known as secoiridoids. Oleuropein, demethyloleuropein and ligstroside represent the predominant phenolic oleosides (Soler-Rivas et al. 2000), whereas verbascoside (Ryan et al. 1999) is the main hydroxycinnamic acid derivative of the olive fruit. In particular, oleuropein, which is a secoiridoid glycoside with hydroxyaromatic functionality deriving from the shikimate and phenylpropanoid metabolism, represents the major biophenol in olive leaf and fruit (Benavante-Garcia et al. 2000) and may reach concentrations of up to 140 mg/g on a dry matter basis in young olives (Amiot et al. 1986). Hydroxytyrosol and oleuropein act as potent radical scavengers (Saija et al. 1998; Benavante-Garcia et al. 2000; Briante et al. 2001; Gordon et al. 2001; Saija and Uccella 2001; Paiva-Martins et al. 2003). Some of the phenolic compounds in olive show antimicrobial activity by inhibiting the growth of a wide variety of bacteria, fungi (Aziz et al. 1998) and viruses (Fredrickson 2000). Furthermore, phenolic compounds substantially affect both the sensory properties of olive fruits (oleuropein and ligstroside are responsible for the bitter and spicy taste) and the shelf life of olives. An important aspect of the study of olive biophenols has been the mea- surement of antioxidant activity according to available tests such as Rancimat, scavenging of the 2,2′-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) radical cation, thiobarbituric acid reactive substances and diene formation. Bianco and Uccella (2000) investigated the concentrations of different biophenolic compounds in olives in order to develop appropriate procedures for determination of these compounds in fresh and processed table olives and in the olive drupes for olive oil production. Despite the large number of studies on olive phenolic composition, not all these compounds have been identiﬁed, and, generally, the antioxidant activity is measured on methanolic extracts fractionated on a polarity basis (McDonald et al. 2001). The aim of this study was to investigate the phenolic content and antioxidant activity of olive pulp from 12 Italian cultivars grown in the Daunia district. The antioxidant activity was measured according to the ABTS and the b-carotene bleaching methods. 212 A. BAIANO ET AL.

MATERIALS AND METHODS

Olives Healthy olive fruits (O. europaea L.) were manually picked in two olive groves located in the countryside near Cerignola and Torremaggiore (Apulia, Italy), respectively. Cerignola is located in the valley of the Ofanto river at 120 m on the sea level and has a continental climate, characterized by a large temperature range. The coolest month is January, with temperatures of 4–11C, whereas the hottest one is August (19–31C). Rarely does the temperature fall below 0C. Rain is scarce (600 mm per year) and concen- trated in the period between September and February. Summer is usually droughty. It is subjected to both winds hot and cold. Torremaggiore is located on a hill at 169 m on the sea level. Climate is similar to that described for Cerignola. Samples of Cellina di Nardò, FS17, Grignano, Moraiolo, Nociara, Ogliarola barese, Peranzana and Picholine were with- drawn in the Cerignola groves, whereas samples of Cima di Melfi, Coratina, Frantoio, Leccino, Moraiolo, Nociara and Peranzana were collected in the Torremaggiore groves. Olives were collected in the period that coincided with the time when olives are usually harvested for oil production. Olive trees were planted on a 6m¥ 6 m spacing layout and drip irrigated. The same agronomic techniques were applied. The following agronomical parameters were determined for samples from each cultivar (about 100 olives randomly chosen among 4 kg of fruits were used for each analysis): mean drupe weight, pulp/stone ratio, percent- age of drupes damaged by Bactrocera oleae, moisture content (Ünal and Nergiz 2003), oil extraction yield (expressed as L oil/100 kg olives) and maturation index (MI). The MI was determined according to a simple technique that is based on the assessment of the color of the olive skins (International Olive Oil Council 1984). One hundred olive fruits were randomly drawn from 1 kg of each sample and divided into the following categories: DGS = the skin is a deep or dark green color; OV = the skin is a yellow, yellowish, reddish, light violet color or the skin is black and the flesh is a green or violet color almost to the stone; BS = the skin is black and the flesh is completely dark. The MI was calculated as:

MI=×()1 DGS +× 2 OV +× 3 BS 100.

According to this formula, the MI values were between 1 (100% green olives) and 3 (100% black olives). After they were sampled, fruits were rapidly frozen and stored at -20C for a maximum of 24 h prior to analysis. PHENOLIC ANTIOXIDANTS OF ITALIAN OLIVE FRUITS 213

Extraction of Phenolic Compounds Phenolic compounds were extracted from frozen olive pulp, according to Ryan et al. (2001) with some adjustments. Approximately 2 g of ground olive pulp were measured into a beaker, mixed with 20 mL of 50% (v/v) aqueous methanol containing 400 mg/L sodium metabisulfite, and then homogenized for 20 s using a laboratory blender (Waring, Torrington, CT). The mixtures were kept in the dark for 30 min to increase the extraction yield and then centrifuged (3,000 g, 20 min, 4C) to remove the solid phase. The liquid phase was diluted with 50% (v/v) aqueous methanol solution up to 36 g of weight and filtered through nylon filters (0.45 mm, DISMIC-13NP, Advantec, Toyo Roshi Kaisha, Tokyo, Japan). Extractions were performed in duplicate.

Determination of the Total Phenolic Content Total phenolic compounds were determined according to the Folin– Ciocalteau method as adapted by Di Stefano et al. (1989). In a test tube, 100 mL of phenolic extract or phenolic standard were mixed with the Folin– Ciocalteau reagent (2 M, 100 mL) and, after 4 min, with an aqueous solution of Na2CO3 (5%, 800 mL). The mixture was heated in a 40C water bath for 20 min and the total phenol content was determined colorimetrically at 750 nm. Stan- dard curve was prepared using gallic acid in methanol/water (50:30, v/v). The total phenolic content was conventionally expressed as grams of gallic acid equivalents per kilogram of dry pulp (determined by oven drying at 105C for 16 h).

High-Performance Liquid Chromatography (HPLC) Analysis The HPLC analysis of the phenolic extracts was carried out according to Gambacorta et al. (2006), using an HPLC binary system (Model G1311A, Agilent, Santa Clara, CA) equipped with a 7725 Rheodyne injector, a 20 mL sample loop, a diode array detector (Model G1315Bm, Agilent) and a Chem- Station integrator (Agilent) for data acquisition. The stationary phase was a Nova-Pack C18 analytical column (150 ¥ 3.9 mm i.d.) with a particle size of 4 mm (Waters, Milford, MA). The mobile phases for chromatographic analysis were (1) water/acetic acid (98:2, v/v) and (2) methanol/acetonitrile (1:1, v/v) at constant ﬂow rate of 1 mL/min. The gradient program of solvent was 0–30 min, 100% A; 30–45 min, 70% A; 45–55 min, 50% A; 55–65 min, 40% A; and 65–75 min, 0% A. The identiﬁcation of some phenolic components was carried out by comparing the peak retention times with those obtained by injection of the pure standards 3,4-dihydroxyphenylethanol or hydroxytyrosol (3,4-DHPEA) and p-4-hydroxyphenylethanol or tyrosol (p-HPEA), purchased from 214 A. BAIANO ET AL.

Extrasynthese, Genay Cedex, France, and vanillin, ferulic acid, coumaric acid, luteolin purchased from Sigma-Aldrich, Milan, Italy, by analyzing the obtained spectra and by employing LC-MS. The identiﬁcation of compounds such as pinoresinol, 1-acetoxypinoresinol, 3,4-dihydroxyphenylethyl 4-formyl-3-formylmethyl-4-hexenoate, the dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA), 3,4- dihydroxyphenylethanol-elenolic acid, an isomer of oleuropein aglycone (3,4-DHPEA-EA), 4-(acetoxyethyl)-1,2-dihydroxybenzene (3,4-DHPEA- AC), p-4-hydroxyphenylethanol-elenolic acid, an isomer of ligstroside aglycone (p-HPEA-EA) and dialdehydic form of the decarboxymethyl elenolic acid linked to tyrosol (p-HPEA-EDA) was made on the basis of literature publications (Servili et al. 1999; Brenes et al. 2000; Capannesi et al. 2000; Sivakumar et al. 2005). Quantiﬁcation of phenolic compounds was performed according to the method of the internal standard (gallic acid, Extrasynthese, Genay Cedex) and on the basis of the response factors. The response factors were determined, taking into account the recovery percentages of the phenolic compounds and the internal standard. The recovery percentages were 76.85% p-HPEA-EDA, 76.98% pinoresinol, 77.35% 3,4-DHPEA-EDA, 77.71% hydroxytyrosol, 79.19% tyrosol and 81.24% gallic acid.

Antioxidant Capacity The antioxidant capacity of the pulp phenolic extracts was evaluated according to two tests. These were the b-carotene-linoleate model system (Lee et al. 2005), in which the antioxidant activity was measured by the ability of a compound to minimize the loss of b-carotene in an emulsified aqueous solution in the presence of oxygen at high temperatures (50C), and the ABTS method (Miller et al. 1993), based on the abilities of the antioxidants present in the extracts to scavenge the ABTS radical cation in comparison with that of a standard antioxidant (Trolox). The b-carotene-linoleate test is based on the thermal (50C) autoxidation of linoleic acid and subsequent formation of peroxyl radical that is able to scavenge hydrogen atoms from the b-carotene molecule, determining its bleaching. The b-carotene bleaching is detected by the absorbance decrease that is greater when the antioxidant content is low. Five milligrams of b-carotene (Sigma, St. Louis, MO) was dissolved in 50 mL of chloroform (J.T. Baker, Mallinckrodt Baker, Milano, Italy). Three milliliters of this solution were pipetted into a round-sized flask containing 40 mg of linoleic acid (Sigma) and 400 mg of Tween 40 (Sigma). After evaporation of chloroform to dryness under vacuum at 40C, 100 mL of distilled water enriched with oxygen were added to the flask and the mixture was shaken to form an emulsion. PHENOLIC ANTIOXIDANTS OF ITALIAN OLIVE FRUITS 215

Aliquots of 1.5 mL of this solution were pipetted into test tubes containing 20 mL of phenolic extracts immediately put into a water bath at 50C. The absorbance of samples and of a control containing an aqueous solution of methanol (50%, v/v) at 470 nm were monitored every 15 min on a Varian Cary 50 Scan UV-Visible spectrophotometer (Palo Alto, CA) until the complete b-carotene bleaching (after about 2 h). The antioxidant capacity was expressed as the antioxidant activity coefﬁcient (AAC):

⎛ 100−− Abs of extract 0 min Abs of extract 120 min⎞ AAC = ⎜ ⎟ ×100 ⎝ Abs of conntrol 0 min− Abs of control 120 min ⎠

The antioxidant capacity was also measured through the ability of antioxidants to scavenge the ABTS radical cation, thus reducing its absorption at 734 nm. The ABTS•+ is the oxidizable substrate. Metmyoglobin (MetMb) supplies the ferric ions needed for the formation of hydroxyl radicals following the Fenton reaction (Winterbourn 1995). H2O2 is the iniziator. A solution of ABTS (246 mL, 610 mM), phosphate buffered saline (PBS, 524 mL, pH 7.4) and puriﬁed MetMb in PBS (410 mL, 6.1 mM) were prepared. Fresh ABTS solutions were prepared each day. Three hundred microliters of hydrogen peroxide were added to generate the ABTS•+. Twenty microliters of the phenolic extracts were added to the mixture in order to evaluate their antioxidant properties. A 2.5-mM Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid) (Aldrich, Steinhein, Germany) was prepared in aqueous methanol (50%, v/v) as a stock standard. The antioxidant activity of this solution and of aqueous methanol (50%, v/v) was also measured. The antioxidant activities of Trolox and the methanolic solution were considered as equal to 100 and 0% inhibition of the formation of the radical ABTS•+, respectively. Obviously, the phenolic extracts showed intermediate values.

Statistical Analysis Means, standard deviations and R values were determined. Analysis of variance, the Holm test and the pair comparison test at a conﬁdence level of 95% (P < 0.05) were performed by means of the Kaleidagraph Statistical Software (ver. 3.6.2; Synergy Software, Reading, PA).

RESULTS AND DISCUSSION

Characteristics of Drupes Table 1 summarizes information about olive drupes tested. Both the highest (Picholine) and the lowest (Cellina di Nardò, Ogliarola barese) mean 216 A. BAIANO ET AL.

TABLE 1. CHARACTERISTICS OF DRUPES FROM OLIVE TREES GROWN IN CERIGNOLA AND TORREMAGGIORE AREAS

Cultivar Mean Pulp/Stone Olives damaged Moisture Oil MI weight ratio by Bactrocera content content (g) oleae (%) (%) (%)

Cellina di Nardò 1.8 4.1 0 76.3 9.2 2.95 FS 17 3.5 8.9 0 70.7 15.7 1.56 Grignano 2.9 4.5 0 67.6 12.8 2.77 Moraiolo 2.6 3.7 0 68.1 13.1 2.70 Nociara 3.7 5.3 0 67.9 12.1 2.70 Ogliarola barese 1.8 4.9 0 66.9 19.6 2.68

From Cerignola Peranzana 4.5 5.8 0 67.5 11.9 2.34 Picholine 6.6 9.2 0 71.8 7.7 2.20 Cima di Melﬁ 3.0 3.8 0 69.2 12.4 1.53 Coratina 3.0 3.8 0 67.4 14.1 1.30 Frantoio 3.1 4.1 1 81.5 12.0 1.73 Leccino 3.0 4.1 0 71.5 15.2 3.00 Moraiolo 3.1 3.9 0 67.1 16.5 1.53 Nociara 2.6 4.7 0 68.0 13.9 2.04

From Torremaggiore Peranzana 2.9 4.3 2 77.2 12.7 1.20

MI, maturation index. weight were measured on drupes picked in groves of the Cerignola countryside, whereas those picked in the Torremaggiore countryside showed intermediate weights and a very small weight difference (0.5 g) between the heaviest (Moraiolo) and the lightest (Nociara). Concerning the olives from Torremaggiore, an inverse correlation existed between weight and pulp/stone ratio (R = 0.845). Olives were in a satisfactory health state as demonstrated by the absence of damaged fruits for almost all the cultivars and the low percent- age of drupes affected by B. oleae detected on Frantoio and Peranzana. The moisture content of the olives from Cerignola was included in the range 67–76%, whereas those picked from Torremaggiore fields ranged between 67 and 81%. The oil content appeared inversely correlated with the moisture showed, but the low values of the correlation coefficient (R = 0.580 and 0.638 for olives from Cerignola and Torremaggiore, respectively) probably depended on factors such as cultivar and maturity index. Both the lowest (Peranzana) and the highest (Leccino) MI were noted on drupes picked in groves of the Torremaggiore countryside. An interesting finding was represented by the effects that the plantation place showed on the characteristics of the drupes of Moraiolo, Nociara and Peranzana grown both in Cerignola and Torremaggiore groves. In fact, these cultivars showed noticeable differences depending on the plantation place, especially for parameters such as weight, pulp/stone ratio, oil content and MI. PHENOLIC ANTIOXIDANTS OF ITALIAN OLIVE FRUITS 217

Total Phenolic Content of Olives Figure 1 shows the total phenolic contents of pulp samples. The signifi- cantly highest phenolic content was detected on the extracts of Peranzana from Cerignola (over 14 g gallic acid/kg dry pulp). The lowest phenolic content (about 7 g gallic acid/kg dry olive) was noted on drupes of FS17 from Ceri- gnola and Cima di Melfi from Torremaggiore. Two interesting results were represented by the absence of any correlation between phenolic content and maturity index and the presence of an inverse correlation (R = 0.750) between phenolic and oil contents of the three cultivars (Moraiolo, Nociara, Peranzana) grown both in Cerignola and Torremaggiore fields. Caponio et al. (2001), Morelló et al. (2004) and Rotondi et al. (2004) found a reduction in the phenolic content and antioxidant activity with the ripening progress of the drupes. The inverse correlation among phenolic and oil content can be easily explained. In fact, oil extraction is more effective with olives of lower water content (Di Giovacchino et al. 1994). Furthermore, phenolic compounds are water soluble, and so high values of moisture effectively reduce the phenolic extraction. In order to evaluate the effect of cultivar and plantation place, a pair comparison test was applied to the phenolic content of the three cultivars (Moraiolo, Nociara, Peranzana) grown both in Cerignola and Torremaggiore

18,00 C,D 16,00 D C,D C,D 14,00 B,D C C 12,00 C B,C B B 10,00 B A B 8,00 A

6,00

4,00

2,00

0,00 Phenolic content (g gallic acid / kg dry matter) e- C o- T

FS17- C holine- C Nociara- C Leccino- T Nociara-anzana- T T Grignano-Moraiolo- C C Pic Coratina-Frantoio- T T Moraiol Peranzana- C Per Cima di Melfi- T Cellina di Nardò- C Ogliarola Bares

FIG. 1. TOTAL PHENOLIC CONTENT OF PULP SAMPLES Different letters indicate signiﬁcant differences (P < 0.05). 218 A. BAIANO ET AL.

TABLE 2. TOTAL PHENOLIC CONTENT OF THE THREE VARIETIES (MORAIOLO, NOCIARA, PERANZANA) CULTIVATED BOTH IN CERIGNOLA AND TORREMAGGIORE FIELDS

Sample Total phenolic content (g gallic acid/kg dry matter)

Moraiolo – T 8.24 Ϯ 0.27a Moraiolo – C 9.38 Ϯ 0.59b Nociara – T 8.48 Ϯ 0.75a′ Nociara – C 11.15 Ϯ 0.48b′ Peranzana – T 11.69 Ϯ 0.49a″ Peranzana – C 14.86 Ϯ 0.25b″

Different letters indicate signiﬁcant differences (P < 0.05) within each couple. C, Cerignola; T, Torremaggiore. groves. The results shown in Table 2 highlighted the greater inﬂuence of the plantation place with respect to the cultivar. In fact, the highest phenolic contents were detected in olives picked in Cerignola groves that are located at lower altitude than Torremaggiore. These results agree with those described by Mousa et al. (1996) for oils derived from mastoid olives and by Paz Aguilera et al. (2005) for the phenolic content of Frantoio and Leccino extra-virgin olive oils.

Phenolic Profiles of Olives As an example, the phenolic profile of Picholine olives is reported in Fig. 2. The phenolic composition of drupes is reported in Tables 3 and 4 (Cerignola and Torremaggiore groves, respectively). Among the 13 phenolic compounds identified, 1-acetoxypinoresinol, 3,4-DHPEA-EDA, hydroxytyrosol and luteolin were those detected in a greater amount and in most of the cultivars. 3,4-DHPEA-EDA was the most abundant phenolic compound in Cellina di Nardò, Nociara and Ogliarola barese, whereas 1-acetoxypinoresinol was mainly detected in Grignano, Moraiolo, Peranzana, Picholine, Cima di Melfi, Coratina and Peranzana. 1-Acetoxypinoresinol belongs to the class of lignans, is present in a great number of monovarietal olive oil and can be used in authentication of Picual oil, as it is present in very low concentrations of this compound (Brenes et al. 2002). The content in oleosidic forms of 3,4- dihydroxyphenolethanol and the dialdehydic form of elenolic acid linked to 3,4-DHPEA are known to be correlated with the oxidative stability of the corresponding oil (Baldioli et al. 1996). Hydroxytyrosol, after gallic acid, is believed to be one of the most powerful antioxidants. PHENOLIC ANTIOXIDANTS OF ITALIAN OLIVE FRUITS 219

FIG. 2. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY CHROMATOGRAMS OF PHENOLIC EXTRACTS OF PICHOLINE FROM CERIGNOLA GROVES This injection was made without the internal standard. Peak numbers are identiﬁed as: (1) 3,4-(dihydroxyphenyl)ethanol (hydroxytyrosol, 3,4-DHPEA); (2) p-(hydroxyphenyl)ethanol (tyrosol, p-HPEA); (3) vanillin; (4) p-coumaric acid; (5) 4-(acetoxyethyl)-1,2-dihydroxybenzene (3,4-DHPEA-AC); (6) ferulic acid; (7) 3,4-DHPEA-EDA (3,4-dihydroxyphenylethyl 4-formyl-3-formylmethyl-4-hexenoate); (8) p-HPEA-EDA (dialdehydic form of the decarboxymethyl elenolic acid, an isomer of oleuropein aglycone); (9) 1-acetoxypinoresinol; (10) pinoresinol; (11) 3,4-DHPEA-EA (where EA is elenolic acid, oleuropein aglycone); (12) luteolin; and (13) p-HPEA-EA (p-4-hydroxyphenylethanol-elenolic acid, an isomer of ligstroside aglycone).

Concerning Moraiolo, Nociara and Peranzana, the drupes picked in Cerignola and Torremaggiore groves showed similar phenolic profiles for the major compounds, pointing to a strong influence of the cultivar. Analogous results were also obtained by Vinha et al. (2005) in an investigation concerning Portuguese olive fruits. The chromatographic profiles showing the lowest number of peaks were those referred to Frantoio (presence of peaks referred to 1-acetoxypinoresinol, 3,4-DHPEA-EA and luteolin) and FS17 (hydroxytyrosol, p-HPEA-EDA, 1-acetoxypinoresinol, luteolin, p-HPEA-EA), which were also included among the cultivars having the lower phenolic contents.

Antioxidant Capacity of Olives In this work, two tests were applied: the ABTS and the b-carotene bleaching assays. As can be inferred from the data shown in Table 5, the application of more than one method for the evaluation of the antioxidant activity gave rise to different results. Thus, the relationships among data obtained according to the considered assays were evaluated and a low correlation coefﬁcient (R = 0.540) was found. This behavior can be explained by the fact that while ABTS assays for radical scavenging ability in an aqueous phase 2 .BAIANO A. 220

TABLE 3. PHENOLIC COMPOSITION OF OLIVE FRUITS FROM CERIGNOLA GROVES

Phenolic compounds Cellina di FS17 Grignano Moraiolo Nociara Ogliarola Peranzana Picholine identiﬁed Nardò barese

Hydroxytyrosol 137.5 Ϯ 14.9 58.2 Ϯ 0.9 118.3 Ϯ 2.1 97.3 Ϯ 28.9 160.7 Ϯ 40.6 169.8 Ϯ 0.0 247.8 Ϯ 0.3 228.0 Ϯ 4.8 Tyrosol 59.5 Ϯ 4.0 0.0 Ϯ 0.0 32.6 Ϯ 4.9 60.2 Ϯ 31.1 81.0 Ϯ 59.7 104.6 Ϯ 0.0 56.9 Ϯ 2.0 34.7 Ϯ 3.8 Vanillin 0.0 Ϯ 0.0 0.0 Ϯ 0.0 28.6 Ϯ 0.4 20.0 Ϯ 0.5 64.2 Ϯ 39.5 34.9 Ϯ 0.0 40.9 Ϯ 3.5 61.4 Ϯ 3.9 p-Coumaric acid 291.5 Ϯ 9.6 0.0 Ϯ 0.0 49.4 Ϯ 12.2 30.5 Ϯ 4.8 47.3 Ϯ 8.4 55.0 Ϯ 0.0 39.1 Ϯ 0.9 18.7 Ϯ 2.6 3,4-DHPEA-AC 349.8 Ϯ 193.8 0.0 Ϯ 0.0 29.7 Ϯ 8.8 22.4 Ϯ 2.6 22.3 Ϯ 1.0 10.2 Ϯ 0.0 24.4 Ϯ 1.5 31.1 Ϯ 3.7 Ferulic acid 110.7 Ϯ 53.4 0.0 Ϯ 0.0 33.4 Ϯ 5.7 35.5 Ϯ 1.7 11.2 Ϯ 4.8 0.0 Ϯ 0.0 36.9 Ϯ 6.4 39.4 Ϯ 27.5 3,4-DHPEA-EDA 11,754.6 Ϯ 160.8 0.0 Ϯ 0.0 247.5 Ϯ 2.0 89.7 Ϯ 6.9 379.8 Ϯ 88.5 798.7 Ϯ 0.0 218.1 Ϯ 7.8 97.6 Ϯ 43.4 p-HPEA-EDA 1,075.5 Ϯ 18.7 31.2 Ϯ 1.0 85.1 Ϯ 1.0 127.0 Ϯ 52.1 102.3 Ϯ 76.7 41.9 Ϯ 0.0 174.8 Ϯ 42.0 116.3 Ϯ 38.6 AL. ET 1-Acetoxypinoresinol 905.6 Ϯ 12.4 84.2 Ϯ 2.2 915.3 Ϯ 17.5 480.2 Ϯ 130.8 258.5 Ϯ 93.0 408.6 Ϯ 0.0 1,955.9 Ϯ 19.0 628.1 Ϯ 317.6 Pinoresinol 65.9 Ϯ 13.9 0.0 Ϯ 0.0 58.2 Ϯ 9.8 83.5 Ϯ 20.0 64.1 Ϯ 6.6 35.0 Ϯ .0 48.8 Ϯ 21.4 34.3 Ϯ 6.5 3,4-DHPEA-EA 46.3 Ϯ 25.3 0.0 Ϯ 0.0 61.1 Ϯ 3.5 76.9 Ϯ 45.0 65.0 Ϯ 25.1 73.6 Ϯ 0.0 138.8 Ϯ 22.6 55.2 Ϯ 3.6 Luteolin 80.1 Ϯ 5.6 72.2 Ϯ 0.8 45.6 Ϯ 1.4 116.8 Ϯ 74.8 107.2 Ϯ 161.1 19.8 Ϯ 0.0 430.2 Ϯ 34.2 77.9 Ϯ 3.4 p-HPEA-EA 135.6 Ϯ 3.1 55.8 Ϯ 1.3 33.9 Ϯ 2.2 102.1 Ϯ 7.4 262.4 Ϯ 22.4 328.6 Ϯ 0.0 100.4 Ϯ 19.3 105.6 Ϯ 23.4

Results are expressed as mg/kg dry matter. 3,4-DHPEA-AC, 4-(acetoxyethyl)-1,2-dihydroxybenzene; 3,4-DHPEA-EDA, 3,4-dihydroxyphenylethyl 4-formyl-3-formylmethyl-4-hexenoate, the dialdehydic form of elenolic acid linked to hydroxytyrosol; p-HPEA-EDA, dialdehydic form of the decarboxymethyl elenolic acid linked to tyrosol; 3,4- DHPEA-EA, 3,4-dihydroxyphenylethanol-elenolic acid, an isomer of oleuropein aglycone; p-HPEA-EA, p-4-hydroxyphenylethanol-elenolic acid, an isomer of ligstroside aglycone. TABLE 4.

PHENOLIC COMPOSITION OF OLIVE FRUITS FROM TORREMAGGIORE GROVES FRUITS OLIVE ITALIAN OF ANTIOXIDANTS PHENOLIC

Phenolic compounds Cima di Melﬁ Coratina Frantoio Leccino Moraiolo Nociara Peranzana identiﬁed

Hydroxytyrosol 49.8 Ϯ 9.0 56.9 Ϯ 17.7 178.9 Ϯ 0.9 116.1 Ϯ 17.3 84.0 Ϯ 3.3 144.9 Ϯ 1.2 236.6 Ϯ 47.0 Tyrosol 0.0 Ϯ 0.0 36.9 Ϯ 14.6 15.0 Ϯ 3.5 17.9 Ϯ 2.6 0.0 Ϯ 0.0 64.3 Ϯ 5.4 0.0 Ϯ 0.0 Vanillin 14.6 Ϯ 3.9 27.3 Ϯ 5.8 18.4 Ϯ 0.0 23.4 Ϯ 2.9 0.0 Ϯ 0.0 39.9 Ϯ 4.5 44.1 Ϯ 7.0 p-Coumaric acid 69.1 Ϯ 14.0 44.0 Ϯ 9.1 63.9 Ϯ 4.8 44.9 Ϯ 2.9 26.3 Ϯ 0.4 54.1 Ϯ 1.7 61.4 Ϯ 16.2 3,4-DHPEA-AC 12.8 Ϯ 3.7 14.4 Ϯ 0.2 33.0 Ϯ 2.5 37.1 Ϯ 2.1 13.6 Ϯ 0.4 17.5 Ϯ 0.6 28.8 Ϯ 4.2 Ferulic acid 0.0 Ϯ 0.0 35.0 Ϯ 6.2 19.4 Ϯ 0.4 40.4 Ϯ 0.3 0.0 Ϯ 0.0 12.9 Ϯ 0.9 55.2 Ϯ 9.3 3,4-DHPEA-EDA 51.7 Ϯ 16.0 169.1 Ϯ 26.2 59.0 Ϯ 20.9 151.1 Ϯ 15.4 31.6 Ϯ 3.2 148.5 Ϯ 5.1 308.4 Ϯ 82.7 p-HPEA-EDA 35.1 Ϯ 4.7 163.9 Ϯ 27.0 125.7 Ϯ 10.5 48.5 Ϯ 9.7 45.9 Ϯ 5.1 88.2 Ϯ 29.2 134.9 Ϯ 58.5 1-Acetoxypinoresinol 363.4 Ϯ 186.5 1,612.2 Ϯ 268.9 618.8 Ϯ 4.7 440.9 Ϯ 38.0 270.9 Ϯ 76.2 45.0 Ϯ 9.1 1,698.0 Ϯ 328.4 Pinoresinol 0.0 Ϯ 0.0 67.2 Ϯ 20.4 78.7 Ϯ 27.0 73.3 Ϯ 8.1 0.0 Ϯ 0.0 35.3 Ϯ 10.5 0.0 Ϯ 0.0 3,4-DHPEA-EA 31.1 Ϯ 4.3 0.0 Ϯ 0.0 261.4 Ϯ 15.6 27.4 Ϯ 18.9 81.8 Ϯ 6.0 44.3 Ϯ 2.7 132.9 Ϯ 0.4 Luteolin 33.6 Ϯ 9.1 629.6 Ϯ 384.3 215.3 Ϯ 17.1 39.6 Ϯ 13.3 51.6 Ϯ 26.6 61.7 Ϯ 0.7 219.5 Ϯ 115.6 p-HPEA-EA 55.0 Ϯ 8.8 269.3 Ϯ 108.4 44.7 Ϯ 3.0 35.5 Ϯ 7.3 0.0 Ϯ 0.0 54.7 Ϯ 9.5 123.0 Ϯ 78.0

TABLE 5. ANTIOXIDANT CAPACITY OF OLIVE EXTRACTS

Olive samples % Antioxidant activity AAC (ABTS assay) (b-carotene beaching assay)

Cellina di Nardò-C >100g 55.90 Ϯ 4.99d FS17-C 45.08 Ϯ 3.23b 49.96 Ϯ 4.60d Grignano-C 83.26 Ϯ 2.47f 37.34 Ϯ 4.57c Moraiolo-C 52.33 Ϯ 2.14c 37.86 Ϯ 6.81c Nociara-C 56.78 Ϯ 6.41c 10.48 Ϯ 7.14a Ogliarola barese-C 70.44 Ϯ 1.96e 41.57 Ϯ 4.50c c,d,e c From Cerignola Peranzana-C 66.42 Ϯ 17.85 38.25 Ϯ 2.20 Picholine-C 75.82 Ϯ 2.92e 41.03 Ϯ 3.25c Cima di Melﬁ-T 38.55 Ϯ 9.45a,b 11.06 Ϯ 6.28a Coratina-T 64.90 Ϯ 4.96d 41.83 Ϯ 9.63c,d Frantoio-T 52.23 Ϯ 3.90c 35.18 Ϯ 3.27c Leccino-T 55.16 Ϯ 2.16c 25.89 Ϯ 1.62b Moraiolo-T 37.15 Ϯ 3.56a 23.90 Ϯ 4.08b Nociara-T 52.91 Ϯ 4.64c 12.27 Ϯ 3.24a Ϯ d Ϯ a,b From Torremaggiore Peranzana-T 65.49 1.25 14.83 6.79

Different letters indicate significant differences (P < 0.05). AAC, antioxidant activity coefficient; ABTS, 2,2′-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid. and is usually used for the measure of the total antioxidant capacity, b-carotene tests the ability to inhibit lipid oxidation (Schwarz et al. 2001). According to the ABTS assay, the highest antioxidant capacity was detected for Cellina di Nardò grown in the Cerignola countryside, whereas the lowest value was measured on Moraiolo olives picked in Torremaggiore groves. When the b-carotene assay was applied, Cellina di Nardò from Cerignola showed the highest antioxidant capacity, whereas Nociara deriving from the same countryside showed the lowest AAC values. A strong positive linear correlation was found between total phenolic content and antioxidant capacity measured by the ABTS method (R = 0.82), indicating that phenolic compounds made an important contribution to the capacity of scavenging free radicals in olive extracts. A weak linear correlation (R = 0.36) was found between total phenolics and antioxidant capacity measured by the b-carotene bleaching assay. Thus, phenolic compounds do not appear to make a major contribution to the inhibition of lipid oxidation. Furthermore, the antioxidant activity depends not only on the phenolic con- centration but also on the specific chemical structure of each phenolic compound, as already demonstrated by McDonald et al. (2001), which indicated a hierarchy for antioxidant activity and reduction potential of phenols. These authors tested the antioxidant activity of various phenolic compounds on the oxidation of linoleic acid and found the following order: caffeic acid > PHENOLIC ANTIOXIDANTS OF ITALIAN OLIVE FRUITS 223 tyrosol, naringin, chlorogenic acid > vanillic acid > 4-hydroxybenzoic acid, p-coumaric acid and gallic acid > naringenin > 2,4-dihydroxybenzoic acid and oleuropein. When the antioxidant activity was measured in an aqueous phase, the order was: 2,4-dihydroxybenzoic acid, gallic acid, vanillic acid, and oleuropein > 4-hydroxybenzoic acid, caffeic acid, cinnamic acid, tyrosol, naringin and naringenin.

CONCLUSIONS

The plantation place affected parameters such as weight, pulp/stone ratio, oil content, MI, phenolic content and antioxidant activity in a higher extent than cultivar. Olive fruits from Cerignola, which is located at a lower altitude than Torremaggiore, showed the highest phenolic content and antioxidant activity. The phenolic content was inversely correlated with the oil content of the drupes. Concerning Moraiolo, Nociara and Peranzana, the drupes picked in Cerignola and Torremaggiore groves showed similar phenolic profiles for the major compounds, pointing to a strong influence of the cultivar. A good correlation was found between total phenolic content and antioxidant capacity measured according to the ABTS assay but not between total phenolic content and b-carotene data. This finding indicates that phenolic compounds greatly contributed to the total antioxidant activity of olives (due to their ability in scavenging free radicals) but did not make a great contribution to the inhibition of lipid oxidation.

REFERENCES

AMIOT, M.J., FLEURIET, A. and MACHEIX, J.J. 1986. Importance and evolution of phenolic compounds in olive during growth and maturation. J. Agric. Food Chem. 34, 823–826. AZIZ, N.H., FARAG, S.E., MOUSA, L.A. and ABO-ZAID, M.A. 1998. Comparative antibacterial and antifungal effects of some phenolic compounds. Microbios 93, 43–54. BALDIOLI, M., SERVILI, M., PERRETTI, G. and MONTEDORO, G.F. 1996. Antioxidant activity of tocopherols and phenolic compounds of virgin olive oil. J. Am. Oil Chem. Soc. 73, 1589–1593. BENAVANTE-GARCIA, O., CASTELLO, J., MORENTE, J., ORTUÑO, A. and DEL RIO, J.A. 2000. Antioxidant activity of phenolics extracted from Olea europea L. leaves. Food Chem. 68, 457–462. BIANCO, A. and UCCELLA, N. 2000. Biophenolic components of olives. Food Res. Int. 33, 475–485. 224 A. BAIANO ET AL.

BRENES, M., GARCÍA, A., GARCÍA, P. and GARRIDO, A. 2000. Rapid and complete extraction of phenols from olive oil and determination by means of a coulometric electrode array system. J. Agric. Food Chem. 48, 5178– 5183. BRENES, M., GARCÍA, A., GARCÍA, P., RIOS, J.J. and GARRIDO, A. 2002. Acetoxypinoresinol to authenticate Picual olive oils. Int. J. Food Sci. Technol. 37, 615–625. BRIANTE, R., LA CARA, F., TONZIELLO, M.P., FEBBRAIO, F. and NUCCI, R. 2001. Antioxidant activity of the main bioactive derivates from oleuropein hydrolysis by hyperthermophilic b-glycosidase. J. Agric. Food Chem. 49, 3198–3203. CANTINI, C., CIMATO, A. and SANI, G. 1999. Morphological evaluation of olive germplasm present in Tuscany region. Euphytica 109, 173–181. CAPANNESI, C., PALCHETTI, I., MASCINI, M. and PARENTI, A. 2000. Electrochemical sensor and biosensor for polyphenols detection in olive oils. Food Chem. 71, 553–562. CAPONIO, F., GOMES, T. and PASQUALONE, A. 2001. Phenolic compounds in virgin olive oils: Inﬂuence of the degree of olive ripeness on organoleptic characteristics and shelf-life. Eur. Food Res. Technol. 212, 329–333. CARLUCCIO, M.A., SICULELLA, L., ANCORA, M.A., MASSARO, M., SCODITTI, E., STORELLI, C., VISIOLI, F., DISTANTE, A. and DE CATERINA, R. 2003. Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: Antiatherogenic properties of Mediterra- nean diet phytochemicals. Arterioscler. Thromb. Vasc. Biol. 23, 622–629. DI GIOVACCHINO, L., SOLINAS, M. and MICCOLI, M. 1994. Effect of extraction systems on the quality of virgin olive oil. J. Am. Oil Chem. Soc. 71, 1189–1194. DI STEFANO, R., CROVARO, M.C. and GENILIZZI, N. 1989. Metodi per lo studio dei polifenoli nei vini. L’Enotecnico 5, 83–89. FREDRICKSON, W.R. 2000. Method and composition for antiviral therapy. US Patent 6117844 (Appl. No. 668324). GAMBACORTA, G., PREVITALI, M.A., PATI, S., BAIANO, A. and LA NOTTE, E. 2006. Characterization of the phenolic proﬁles of some monovarietal extravirgin olive oils of Southern Italy. XXIII International Conference on Polyphenols.Winnipeg, MA, Canada,August 22–25, 2006. GORDON, M.H., PAIVA-MARTINS, F. and ALMEIDA, M. 2001. Antioxi- dant activity of hydroxytyrosol acetate compared with that of other olive oil polyphenols. J. Agric. Food. Chem. 49, 2480–2485. INTERNATIONAL OLIVE OIL COUNCIL (IOOC). 1984. Document no. 6. IOOC, Madrid, Spain. PHENOLIC ANTIOXIDANTS OF ITALIAN OLIVE FRUITS 225

KOHYAMA, N., NAGATA, T., FUJIMOTO, S. and SEKIYA, K. 1997. Inhibition of arachidonate lipoxygenase activities by 2-(3,4- dihydroxyphenyl)ethanol, a phenolic compound from olives. Biosci. Bio- technol. Biochem. 61, 347–350. LEE, C.H., YANG, L., XU, J.Z., YEUNG, S.Y.V., HUANG, Y. and CHEN, Z.Y. 2005. Realtive antioxidant activity of soybean isoﬂavones and their glycosides. Food Chem. 90, 735–741. MCDONALD, S., PRENZLER, P.D., ANTOLOVICH, M. and ROBARDS, K. 2001. Phenolic content and antioxidant activity of olive extracts. Food Chem. 73, 73–84. MILLER, N.J., RICE-EVANS, C., DAVIES, M.J., GOPINATHAN, V. and MILNER, A. 1993. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin. Sci. 84, 407–412. MORELLÓ, J.R., ROMERO, M.P. and MOTIVA, M.J. 2004. Effect of the maturation process of the olive fruit on the phenolic fraction of drupes and oils from Arbequina, Farga, and Morrut cultivars. J. Agric. Food Chem. 52, 6002–6009. MOUSA, Y.M., GERASOPOULOS, D., METZIDAKIS, I. and KIRITSAKIS, A. 1996. Effect of altitude on fruit and oil quality characteristics of mastoids olives. J. Sci. Food Agric. 71, 345–349. PAIVA-MARTINS, F., GORDON, M.H. and GAMEIRO, P. 2003. Activity and location of olive oil phenolic antioxidants in liposomes. Chem. Phys. Lipids 124, 23–36. PAZ AGUILERA, M., BELTRAN, G., ORTEGA, D., FERNANDEZ, A., JIMENEZ, A. and UCEDA, M. 2005. Characterisation of virgin olive oil of Italian olive cultivars: “Frantoio” and “Leccino,” grown in Andalusia. Food Chem. 89, 387–391. ROTONDI, A., BENDINI, A., CERRETANI, L., MARI, M., LERCKER, G. and TOSCHI, T.G. 2004. Effect of olive ripening degree on the oxidative stability and organoleptic properties of cv. Nostrana di Brisighella extra virgin olive oil. J. Agric. Food Chem. 52, 3649–3654. RYAN, D., ROBARDS, K., PRENZLER, P., JARDINE, D., HERLT, T. and ANTOLOVICH, M. 1999. Liquid chromatography electrospray ionisa- tion mass spectrometry detection of phenolic compounds from Olea europea. J. Chromatogr. A 855, 537–599. RYAN, D., LAWRENCE, H., PRENZLER, P.D., ANTOLOVICH, M. and ROBARDS, K. 2001. Recovery of phenolic compounds from Olea europaea. Anal. Chim. Acta 445, 67–77. SAIJA, A. and UCCELLA, N. 2001. Olive biophenols: Functional effects on human wellbeing. Trends Food Sci. Technol. 11, 357–363. 226 A. BAIANO ET AL.

SAIJA, A., TROMBETTA, D., TOMAINO, A., LO CASCIO, R., PRINCI, P., UCCELLA, N., BONINA, F. and CASTELLI, F. 1998. In vitro evaluation of the antioxidant activity and biomembrane interaction of the plant phenols oleuropein and hydroxytyrosol. Int. J. Pharmacol. 166, 123–133. SCHWARZ, K., BERTELSEN, G., NISSEN, L.R., GARDNER, P.T., HEINONEN, M.I., HOPIA, A., HUYNH-BA, T., LAMBELET, P., MCPHAIL, D., SKIBSTED, L.H. et al. 2001. Investigation of plant extracts for the protection of processed foods against lipid oxidation. Eur. Food Res. Technol. 212, 319–328. SERVILI, M., BALDIOLI, M., SELVAGGINA, R., MINIATI, E., MACCHIONI, A. and MONTEDORO, G. 1999. High-performance liquid chromatography evaluation of phenols in olive fruit, virgin olive oil, vegetation waters, and pomace and 1D- and 2D-nuclear magnetic resonance characterization. J. Am. Oil Chem. Soc. 76, 873–882. SIVAKUMAR, G., BRICCOLI BATI, C. and UCCELLA, N. 2005. HPLC-MS screening of the antioxidant profile of Italian olive cultivars. Chem. Nat. Comp. 41, 588–591. SOLER-RIVAS, C., ESPIN, J.C. and WICHERS, H.J. 2000. Oleuropein and related compounds. J. Sci. Food Agric. 80, 1013–1023. ÜNAL, K. and NERGIZ, C. 2003. The effect of table olive preparing methods and storage on the composition and nutritive value of olives. Grasas Aceites 54, 71–76. VINHA, A.F., FERREREI, F., SILVA, B.M., VALENTÃO, P., GONÇALVES, A., PEREIRA, J.A., OLIVEIRA, M.B., SEABRA, R.M. and ANDRADE, P.B. 2005. Phenolic profiles of Portuguese olive fruits (Olea europaea L.): Influences of cultivar and geographical origin. Food Chem. 89, 561–568. VISIOLI, F., POLI, A. and GALLI, C. 2002. Antioxidant and other biological activities of phenols from olives and olive oil. Med. Res. Rev. 22, 65–75. WINTERBOURN, C.C. 1995. Toxicity of iron and hydrogen peroxide: The Fenton reaction. Toxicol. Lett. 82/83, 969–974.