**8.1 Extraction and quantification**

The analytical method used to quantify phenolics present in EVOO has an influence on the reported concentration and therefore is an important consideration when interpreting and comparing the data of such investigations (Carrasco-Pancorbo et al., 2005). Regarding the

Oleocanthal: A Naturally Occurring Anti-Inflammatory Agent in Virgin Olive Oil 367

(Gomez-Rico et al., 2006) also demonstrated the negative effect of irrigation on oleocanthal concentration. Rain-fed olive trees produced EVOO containing higher concentrations of oleocanthal (229.0 ± 48.0 to 498 ± 47.0 mg/kg) compared to those that underwent the highest amount of irrigation (206 mm water per year), (119.0 ± 36.0 to 336.0 ± 81.0 mg/kg). Two additional studies (Romero et al., 2002, Servili et al., 2007a) are also in agreement with this data in that, they both observed a 37-38% decrease in oleocanthal concentration amongst the

Maturation of the olive fruit at harvest is an important predictor of the phenolic composition and concentration in EVOO. In regards to oleocanthal, one study found that with extended picking date and increased olive fruit ripeness, the concentration of oleocanthal in EVOO decreased by 43% (148.0 mg/kg to 84.0 mg/kg) over a short two month period (Morello et al., 2004). The researchers, Gomez-Rico et al. (Gomez-Rico et al., 2006) also observed a similar decrease of 20% and 54% in oleocanthal with increasing maturity index using two

In general, the processing of olive fruit to oil has a substantial effect on the concentrations of phenolic compounds in EVOO (Kalua et al., 2006b, Cerretani et al., 2005, Vierhuis et al., 2001, Romero et al., 2004, Gimeno et al., 2002). EVOO produced by the traditional processing method (whereby the entire olive fruit is crushed, including the stone), was found to contain lower quantities of oleocanthal (43.8 ± 3.1 mg/kg) compared to EVOO produced by the stoning method in which the olive stone is removed before crushing (54.8 ± 3.1 mg/kg). The researchers from this study hypothesized that the differences may be due to the increased peroxidase (POD) activity that tends to accompany the crushed olive stone, which has an

EVOO produced under nitrogen (N2) flushing and with use of enzymatic treatment (which aids cell wall degradation and thus improves phenolic extraction) (Vierhuis et al., 2001), was found to contain oleocanthal concentrations of 31.4 ± 1.0 mg/kg. EVOO produced with no added enzymes and without nitrogen (N2) flushing (therefore allowing oxygen (O2) to be present) was found to contain a lower amount of oleocanthal (24.8 ± 1.9 mg/kg). EVOOs produced with use of N2 flushing alone and enzymatic treatment alone, contained 28.4 ± 1.4

EVOOs produced using two-phase centrifugation which uses no added water in the processing method, was found to contain a higher phenolic concentration compared to EVOOs obtained from three-phase centrifugation which utilizes a considerable amount of water (approximately 400 L/h) (De Stefano et al., 1999). De Stefano and et al. (De Stefano et al., 1999) found oleocanthal concentration in EVOO obtained from the two-phase centrifuge to be higher (78.2 0.5 mg/kg) than that produced from the three-phase method (67.3 ± 2.6 mg/kg). The addition of water in the three-phase centrifugation method, may have a reducing effect on the more water-soluble phenolics from the oil phase during processing, thus reducing the concentration of oleocanthal in the resultant

EVOOs from the highly irrigated olive trees compared to those least irrigated.

oxidizing effect on oleocanthal concentration (Servili et al., 2007b).

mg/kg and 29.4 ± 0.8 mg/kg oleocanthal respectively (Vierhuis et al., 2001).

**8.5 Olive maturation** 

olive cohorts.

**8.6 Processing** 

EVOO (Cicerale et al., 2009c).

analysis of oleocanthal, Beauchamp and colleagues (Beauchamp et al., 2005) in collaboration with Impellizzeri et al. (Impellizzeri and Lin, 2006), developed an extraction and quantification method specific for this compound. Utilizing this method, Impellizeri et al. (Impellizzeri and Lin, 2006) and Franconi et al. (Franconi et al., 2006) found the concentration of oleocanthal ranged between 8.3 ± 4.0 and 189.9 ± 2.7 mg/kg. An adaption of the Beauchamp and co-workers' (Beauchamp et al., 2005) methodology, was also utilized in studies by Cicerale and colleagues (Cicerale et al., 2009a, Cicerale et al., 2009b, Cicerale et al., 2011b, Cicerale et al., 2011a). The resultant oleocanthal concentrations in these investigations were similar to those found by Franconi et al. (Franconi et al., 2006) and Impellizzeri et al. (Impellizzeri and Lin, 2006) (53.9 ± 7.7 to 152.2 ± 10.5 mg/kg). However, a number of methods not specific for oleocanthal have also been used to quantify this compound and may account partially for the large variation in oleocanthal concentration observed (5.0 ± 0.3 – 498.0 ± 47.0 mg/kg) (Vierhuis et al., 2001, Servili et al., 2007b, Servili et al., 2007a, Romero et al., 2002, Morello et al., 2004, Tovar et al., 2001, De Stefano et al., 1999, Montedoro et al., 1992, Gomez-Alonso et al., 2003, Gomez-Rico et al., 2006, Allouche et al., 2007)

#### **8.2 Geographic region**

Geographic region in which olives are grown has been shown to be an important factor in regards to phenolic composition and concentration in general (Vinha et al., 2005, Cerretani et al., 2005). Beauchamp et al. (Beauchamp et al., 2005) demonstrated that EVOOs produced in differing countries had variable oleocanthal concentrations. For instance, EVOO produced in the U.S.A., contained a low concentration of oleocanthal (22.6 ± 0.6 mg/kg), however EVOOs produced in Italy contained some of the highest quantities of this compound (up to 191.8 ± 2.7 mg/kg).

#### **8.3 Cultivar**

Several studies have demonstrated differences between olive cultivar and oleocanthal concentration in the oil produced. In one study, the Coratina cultivar EVOO contained 78.2 ± 0.5 mg/kg oleocanthal, whereas the Oliarola cultivar EVOO contained 21.0 ± 0.8 mg/kg oleocanthal, a 3-fold difference (De Stefano et al., 1999). In another study, EVOO produced from the Frantoio cultivar had an oleocanthal concentration of 43.8 ± 3.1 mg/kg, whilst EVOO obtained from the Coratina cultivar, contained a 2-fold higher oleocanthal content at 92.8 ± 7.8 mg/kg (Servili et al., 2007b). A study by Franconi and colleagues (Franconi et al., 2006) also showed significant differences in oleocanthal concentration amongst differing olive cultivars. For instance, an oleocanthal concentration of 8.3 ± 4.0 mg/kg and 53.0 ± 12.0 mg/kg in EVOOs produced from the Taggiasca and Seggianese cultivars respectively, was noted.

#### **8.4 Agricultural methods**

The concentration of phenolic compounds in EVOO is greatly affected by agricultural techniques used in the cultivation of olive fruit (Gomez-Rico et al., 2006, Ayton et al., 2007). Tovar and co-workers (Tovar et al., 2001) demonstrated that with increased irrigation applied to the olive tree, oleocanthal concentration decreased. For instance, in the EVOO obtained from the least irrigated olive trees (46 mm water per year) oleocanthal concentration was determined to be 50.9 ± 6.5 mg/kg. For the EVOO produced from highly irrigated olive trees (259 mm water per year), oleocanthal concentration was 23.1 ± 1.3 mg/kg. Gomez-Rico et al. (Gomez-Rico et al., 2006) also demonstrated the negative effect of irrigation on oleocanthal concentration. Rain-fed olive trees produced EVOO containing higher concentrations of oleocanthal (229.0 ± 48.0 to 498 ± 47.0 mg/kg) compared to those that underwent the highest amount of irrigation (206 mm water per year), (119.0 ± 36.0 to 336.0 ± 81.0 mg/kg). Two additional studies (Romero et al., 2002, Servili et al., 2007a) are also in agreement with this data in that, they both observed a 37-38% decrease in oleocanthal concentration amongst the EVOOs from the highly irrigated olive trees compared to those least irrigated.
