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- Materials and Methods
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ABSTRACT: Phenolic composition and antioxidant activity of extra-virgin olive oils extracted from several Italian varieties were studied at production and during storage. The antioxidant activity was measured according to the following tests: in the aqueous phase, by radical scavenging of the 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical cation; and in the lipid phase, using the β-carotene bleaching method. The phenolic content was not correlated to the oxidation indices (peroxide value and spectrophotometric constants). The phenolic contents and profiles of the various cultivars showed remarkable differences. The phenolic content was strongly correlated with the antioxidant activity measured according to the β-carotene test and weakly correlated with the radical scavenging ability.
- Top of page
- Materials and Methods
- Results and Discussion
The Mediterranean diet, which is largely vegetarian in nature, includes the consumption of noticeable amounts of extra-virgin olive oil. To be an extra-virgin olive oil, it must be obtained from the fruit of the olive tree solely by mechanical or other physical means under conditions that do not lead to alteration in the oil and without any treatment other than washing, decantation, centrifugation, or filtration (EC Reg. 1513/2001).
The health-promoting properties of extra-virgin olive oil concern the ability to prevent diseases that may be related to oxidative damages such as coronary heart diseases, stroke, and certain types of cancers (Fito and others 2000; Covas and others 2001; Aguilera and others 2002; Warhrburg and others 2002; Bendini and others 2007). The protective role of virgin olive oil is the result of its specific composition including high proportion of monounsaturated fatty acids (oleic acid), a balanced presence of polyunsaturated fatty acids, and minor components such as phenolic compounds (Owen and others 2000) tocopherols, and carotenoids, known to act as antioxidants against reactive species (Boskou 1996) at different levels in the oxidative sequence involving lipid molecules.
A virgin olive oil contains at least 30 phenolic compounds (Bendini and others 2007). Phenolic total amount and composition of olive oil varies from 50 to 1000 mg/kg (Montedoro and others 1992a), depending on cultivars, place of origin, agronomic techniques, olive ripening, possible infestation by the olives fly Bactrocera oleae (Gómez-Caravaca and others 2008), extraction methods, and storage conditions but results obtained by different researchers are hardly comparable because of the variety of methods proposed for their determination. In fact, the widely employed Folin–Ciocalteau reagent is not specific for phenols and the HPLC analysis is limited by the complexity of the phenolic fraction.
The polar fraction of virgin olive oil is rich in simple and complex phenolic antioxidants with the latter being the most abundant (Nissiotis and Tasioula-Margari 2002). Phenolic compounds identified in virgin olive oil include 3,4-dihydroxyphenylethanol (3,4-DHPEA, or hydroxytyrosol) and p-4-hydroxyphenylethanol (p-HPEA, or tyrosol) (Montedoro and others 1992b, 1993; Angerosa and others 1995), gallic, caffeic, vanillic, p-coumaric, syringic, ferulic, homovanillic, phydroxybenzoic, protocatecuic acids (Montedoro and others 1992b; Mannino and others 1993), and lignans (Brenes and others 1999; Owen and others 2000). Derivates of 3,4-DHPEA, in particular the dialdehydic form of elenolic acid linked to 3,4-DHPEA (3,4-DHPEA-EDA), an isomer of oleuropein aglycon (3,4-DHPEA-EA), and the dialdehydic form of elenolic acid linked to p-HPEA (p-HPEA-EDA) have been identified as the major secoiridoid compounds of virgin olive oil (Montedoro and others 1993; Baldioli and others 1996; Brenes and others 1999). More than 45 phenolic compounds were characterized in olive oil through the application of capillary electrophoresis and HPLC-TOF-MS (Carrasco-Pancorbo and others 2007). The type as well as the level of these compounds is an important parameter in evaluating the quality and nutritive value of virgin olive oil.
Lavelli and others (2006) studied the extent of degradation of phenolic derived from olive secoiridoids and the antioxidant activity of several monovarietal extra-virgin olive oils during storage. They found that degradation occurred in a similar pattern in all the considered oils and that oleuropein derivatives were less stable than the corresponding ligstroside derivatives. Based on their results, despite the antioxidant depletion, the oils with high phenolic content maintained their beneficial properties during all the commercial life. Gómez-Alonso and others (2007) investigated the evolution of phenolics during 21 mo storage at room temperature founding that the reduction of total phenolic compounds ranged from 43% to 73%, and was higher in samples whose initial phenol contents were greater. Hydroxytyrosol increased linearly in most samples, whereas its complex forms decreased considerably.
Storage affects the phenolic profile through the oxidative stress and the consequent formation of oxidized phenols (Armaforte and others 2007). In particular, 3,4-DHPEA and p-HEA increased during storage. The authors concluded that it may be feasible to use the ratio fresh phenols/oxidized phenols as an interesting means of determining the freshness/aging ratio of the oil.
Natural antioxidants exert their antioxidant activity through various mechanisms: preventing first chain initiation by scavenging initiating radicals, metal chelating, decreasing localized oxygen concentration, and decomposing peroxides (Aruoma 1996). The antioxidant properties of o-diphenols can be related to hydrogen donation, that is, their ability to improve radical stability by forming an intramolecular hydrogen bond between the free hydrogens of their hydroxyl group and their phenoxyl radicals. Although investigations on the structure–activity relationship of olive oil phenols are yet to be carried out, similar studies have been performed on flavonoids and have indicated that, in general, the degree of antioxidant activity is correlated with the number of hydroxyl substitutions (Rice-Evans and others 1996; Cao and others 1997).
According to Chimi and others (1991), among the phenolic compounds contained in olive oil, the antioxidant effect was, in a decreasing order: hydroxytyrosol > oleuropein > tyrosol (Chimi and others 1991). A synergistic effect between oleuropein, which is a derivative of hydroxytyrosol, and α-tocopherol was reported (Baldioli and others 1996). A more recent study by Carrasco-Pancorbo and others (2005) classified hydroxytyrosol, deacetoxy oleuropein aglycon, and oleuropein aglycon as the strongest antioxidant in virgin olive oils.
An important aspect of the study of phenolic compounds is the assessment of their antioxidant activity. Various methods have been introduced to test the antioxidant activity of olive oil. They can be divided into 2 groups: (1) assay of the radical scavenging ability and (2) assay of the ability to inhibit the oxidation of a lipidic substrate (Schwarz and others 2001). The radical scavenging tests measure either the reduction of stable radicals or radicals generated by radiolysis, photolysis, or other reactions. The elements involved in an oxidation reaction are a substrate, an oxidant, an initiator, intermediates, and final products and the measurement of one of these can be used to assess antioxidant activity (Antolovich and others 2002). An important limitation of these tests is that the reducing capacity does not necessarily reflect antioxidant activity (Katalinic and others 2006; Wong and others 2006).
In this study, methanolic extracts of extra-virgin olive oil from 15 Italian cultivars grown in the Daunia district were analyzed for their phenolic content and antioxidant activity and the change of these indices during storage was also considered. The antioxidant activity was measured according to the following tests: in the aqueous phase, by radical scavenging of the 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical cation; and in the lipid phase, using the β-carotene bleaching method.