PHENOLIC COMPOUNDS IN OLIVE OIL AND OLIVES

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1 Current Topics in Nutraceutical Research Vol. 3, No. 2, pp , 2005 ISSN print, Copyright 2005 by New Century Health Publishers, LLC All rights of reproduction in any form reserved PHENOLIC COMPOUNDS IN OLIVE OIL AND OLIVES D. Boskou, G. Blekas, and M. Tsimidou Aristotle University of Thessaloniki, School of Chemistry, Lab. Food Chemistry and Technology, Thessaloniki, Greece [Received December 15, 2004 ; Accepted March 23, 2005] ABSTRACT: Over the past three decades accumulated laboratory and epidemiological studies have lead to the consensus that the consumption of virgin olive oil helps prevent chronic diseases and that many of the health benefits can be attributed to the presence of polar phenolic compounds. The latter are mainly tyrosol and hydroxytyrosol and their derivatives (aglycones of oleuropein and ligstroside, deacetoxy and dialdehydic forms of the aglycones), hydroxytyrosol acetate, the lignans pinoresinol and 1-acetoxypinoresinol, luteolin, apigenin and phenolic acids. In table olives free hydroxytyrosol and tyrosol prevail. The levels of total phenols and individual phenols profiles in the raw olives and the extracted oil depend on agronomic factors, maturity of olives, processing, packaging and storing. Health benefits attributed to olive oil phenols have been linked to their antioxidant properties and their potential to scavenge radicals and reactive species. Attempts to understand better the biological role of these biophenols focus on mechanisms related to improvement of in vivo antioxidant defences, biochemical markers for the assessment of oxidant stress and metabolism in the body. KEY WORDS: Biophenols; Nutritional significance, Olive oil; Technology Corresponding Author: Dr. Maria Tsimidou, Aristotle University of Thessaloniki, School of Chemistry, Lab. Food Chemistry and Technology, Thessaloniki, Greece; Fax: ; tsimidou@chem.auth.gr INTRODUCTION Virgin olive oil has a unique place among other vegetable oils because of its minor constituents, and much has been written in the past two decades about their importance. Both completed and ongoing studies associate these constituents with the beneficial role of olive oil in human health. Phenolic compounds present in olive oil were in the past conventionally characterised as polyphenols, though not all of them are polyhydroxy aromatic compounds. They are part of the polar fraction usually obtained from the oil by extraction with methanol-water. They are important minor constituents linked both to the flavour of virgin olive oil and to its keeping ability. Many publications deal not only with the nutritional effects of polyphenols, but also with the agronomic factors that influence their presence in olives and in olive oil, the mechanisms that contribute to a long shelf life and the importance of the processing conditions. Traditional olive farming in the major olive oil producing countries of the Mediterranean basin varies widely in terms of climate, agricultural, technological and varietal factors. The phenolic content of the fruits is strongly affected by cultivar and stage of maturation. Genetic characteristics and alternate bearing of the tree influence the content and pattern of phenolic compounds of the drupe and, consequently, of the oil or table olives (Ryan and Robards, 1998; Romani et al., 1999; Cortesi et al., 2000; Ucella, 2001; Ryan et al., 2001; Rovellini and Cortesi, 2003). Pedoclimatic conditions and agronomic practices are equally important parameters to enhance natural variation (Tovar et al., 2001; Botia et al., 2001; Romero et al., 2002; Patumi et al., 2002; Morello et al., 2003; Beltrán et al., 2005). As regards Good Agricultural and Manufacturing Practices emphasis is paid to the effect of the degree of olive infestation. Affected olives yield oils with lower amounts of total phenols and, consequently, of lower stability (Delrio et al., 1995; Zunin et al., 1995). COMPOSITION AND PROPERTIES Phenolic composition of olive fruits As analytical options increase, the list of olive phenolics is continuously updated. The predominant compounds in raw olives are certain secoiridoids derived from oleosides, a combination of elenolic acid and glucose residue. Oleuropein, an heterosidic ester of elenolic acid with 3,4-dihydroxyphen ethylalcohol (hydroxytyrosol), demethyloleuropein, the acid derivative of oleuropein and ligstroside, an heterosidic ester of elenolic acid with 4-hydroxyphenethylalcohol (tyrosol) are repeatedly reported as the main secoiridoids of the fruit (Panizzi et al., 1960; Ragazzi et al., 1973; Gariboldi et al., 1986). Olive fruit contains also some other oleuropein derivatives, e.g. oleuropein aglycon, hydroxytyrosilelenolate,

2 126 Olive Oil Phenolics enololeuropeindiale (Bianco and Uccella, 2000), hydroxytyrosol and tyrosol glucosides, such as hydroxytyrosol- 1-O-β-glucoside, tyrosol-1-o-β-glucoside, hydrotyrosol-3 - O-β-glucoside and hydrotyrosol-4 -O-β-glucoside (Bianco and Uccela, 2000; Bastoni et al., 2001) and verbascoside, which is the caffeoylrhamnosylglucoside of hydroxytyrosol (Andary et al., 1982) Benzoic, cinnamic, phenylacetic and phenylpropionic acid hydroxyderivatives, such as p-hydroxybenzoic, protocatechuic, vanillic, syringic, o- and p-coumaric, caffeic, chlorogenic, ferulic, sinapic, p-phenylacetic, 3,4-dihydroxyphenylacetic, homovanillic and dihydrocaffeic acids, are also present in olive at levels depending on fruit variety (Bianco and Uccela, 2000). Flavonoids present in olive fruit are flavones, mainly luteolin, flavone and flavonol glucosides, mainly rutin and luteolin 7- glucoside, and anthocyanins, mainly cyanidin 3- glycosides (Vlahov, 1992; Romani et al., 1999). The concentration of luteolin 7-glucoside and rutin depends on fruit maturation (Amiot at al, 1986; Vlahov, 1992; Esti et al., 1998; Servilli et al., 1999; Bouaziz et al., 2004). Tyrosol and hydroxytyrosol oleosides and the respective free alcohols have been also identified at considerable amounts (Soler-Rivas et al., 2000; Bianco and Ucella, 2000). Servili et al., (1999) characterised the phenolic fraction of peel, pulp and seed of three Italian cultivars used for oil production (Coratina, Leccino, Moraiolo). Oleuropein, demethyloleuropein and verbascoside were found in all parts of the fruit, luteolin-7-glucoside and rutin only in the peel whereas nuzhenide was reported for the first time exclusively in seeds. Biotransformation of phenolic compounds and metabolic relationships between phenolic content and various parts (leaf, fruit, stone, seed) were recently discussed by Ryan et al., (2002). 3,4-Dihydroxyphenylglycol, a metabolite of norepinephrine, has also been found in olive fruit at levels higher than that of hydroxytyrosol (Bianchi and Pozzi, 1994). Phenolic compounds in virgin olive oil Compounds which often appear in lists of olive oil polyphenols are (in alphabetical order): 4-acetoxy-ethyl-1,2- dihydroxybenzene, 1-acetoxy-pinoresinol, apigenin, caffeic acid, cinnamic acid (not a phenol), o- and p-coumaric acids, elenolic acid (not a phenol), ferulic acid, gallic acid, homovanillic acid, p-hydroxybenzoic acid, hydroxytyrosol, luteolin, oleuropein, pinoresinol, protocatechuic acid, sinapic acid, syringic acid, tyrosol, vanillic acid, vanillin (Morales and Tsimidou, 2000, García et al., 2001, Mateos et al., 2001, Owen et al., 2000, Boskou, 2002). Tyrosol and hydroxytyrosol in their various forms are reported to be the major constituents. The more polar part of the methanol-water extract contains free phenols and phenolic acids (Figure 1). The less polar part contains aglycones of oleuropein and ligstroside (the hydroxytyrosol and tyrosol glycosides), deacetoxy and dialdehydic forms of the aglycones (Fig. 2) flavonoids (luteolin, apigenin, (Fig. 3), the lignans 1-acetoxypinoresinol and pinoresinol (Fig. 4), elenolic acid and cinnamic acid (Fig. 5). Litridou et al (1997) reported the presence of an ester of tyrosol with a dicarboxylic acid. The same investigators demonstrated that the otal content of phenols and o-diphenols was higher in the less polar part of the methanol-water extracts. Glycosides were found to be present only in trace amounts. In a recent report García and his co-workers (2001) determined the dialdehydic forms of elenolic acid linked to hydroxytyrosol and tyrosol, 1-acetoxy-ethyl-1,2-dihydroxtbenzene (hydroxytyrosol acetate), 1-acetoxypinoresinol, pinoresinol, oleuropein aglycone, luteoli, and ligstroside aglycone as phenols with the higher concentration in Italian oils. The polyphenol content difers from oil to oil. Wide ranges have been reported ( mg/kg) but values are usually between 100 and 300 mg/kg. The cultivar, the system of extraction, and the conditions of processing are critical factors for the content of polyphenols. Polyphenols are important for the flavour and the stability of the oil. When their content exceeds 300 mg/kg the oil may have a bitter taste. Formation of 4-vinylphenol from p- coumaric acid by decarboxylation or the presence of esters of cinnamic cid may also contribute to the flavour in a negative way. However, a high polyphenol content appears to be beneficial for the shelf life of the oil and there is a good correlation of stability and total phenol content (Tsimidou et al., 1992, Monteleone, et al., 1998). Among the various phenolic compounds tested for their contribution to the antioxidant effect, hydroxytyrosol was found to be the most potent and more effective than butylated hydroxytoluene (BHT) (Boskou, 1996). Gutierrez-Rosales and Arnaud (2001) found that the concentration of hydroxytyrosol, dialdehydic form of elenolic acid linked to hydroxytyrosol and oleuropein aglycone are highly correlated to the oil stability. The effect of maturation on olive oil quality In a thorough review on olive phenolics Ryan and Robards (1998) commended on the difficulties associated with maturation studies and the various ways researchers design the respective experiments (sampling at distinct ripening stages or at technological ripening stage). There are defined criteria to follow growth such as the appearance or disappearance of certain compounds, various characteristic ratios between certain compounds and the evolution of enzymatic activity resulting in biosynthesis or degradation of phenolic compounds. Amiot et al (1986) hypothesised a biochemical relationship between oleuropein and verbascoside, since the second is not found in young fruits. Moreover, the same authors pointed out that small-fruit varieties are characterised by high oleuropein and low verbascoside contents while large fruit varieties are characterised by low oleuropein and high verbascoside content. During maturation oleuropein content is constantly reduced and is at a minimum in overripe drupes. At the same time, demethyl-oleuropein replaces oleuropein in about the same amounts. According to Amiot et al. (1989) the chemical relationship between oleuropein, elenolic acid glycoside and demethyloleuropein and their respective levels during fruit

3 Olive Oil Phenolics 127 maturation imply that these compounds may be related biosynthetically. The investigators suggested that possibly the last two components are formed from oleuropein by the action of esterases. Another extrapolation is that elenolic acid glycoside and demethyloleuropein may be intermediates in the biosynthesis of oleuropein and may accumulate progressively during maturation when biosynthesis slows down. The fruit of O. europea appears to accumulate only glycosylated derivatives of oleuropein, which are probably less toxic than aglycones. Hydroxytyrosol is also related with ripe fruits. It seems probable that oleuropein can be converted by the action of glycosidases but the intermediate compounds are probably immediately re-metabolised in the fruit. Briante et al. (2002) paid attention to the esterases activity not only during fruit maturation but even during processing, as activation during crushing and malaxation is most probable. Oxidation of phenols by phenol oxidases and polymerization of free phenols are also expected to occur (Ryan et al., 2002). Delaying or anticipating the harvest time may, consequently, be crucial in maintaining oleuropein derivatives in olive oil (because of differences in their distribution between the oil and aqueous phases) and balancing bitter to pungent taste in the oil (Esti et al., 1998; Caponio et al., 2001; Skevin et al., 2003 ). Gimeno et al., (2002) studied comparatively the fate of olive phenolics and other endogenous antioxidants (beta-carotene, alphatocopherol) during ripening and found a reverse relationship in the accumulation between polar phenols and tocopherols. The literature concerning the effect of maturation on the fate of phenolics in specific cultivars is considerable for Italian and Spanish cultivars. Less or no information is available for cultivars from other regions. The aim of such studies varies as investigators focus on the identification of biomarkers for oil authentication or characterisation as PDO (Protected Denomination of Origin) and also on quality aspects of olives in relation to production. Brenes et al. (1999) studied the phenolic pattern of a great number of Spanish oils from 13 different cultivars with various degrees of ripeness, obtained under identical processing conditions (Abencor analyzer). In all the samples examined, hydroxytyrosol, tyrosol, vanillic acid, p-coumaric acid and ferulic acid were present whereas caffeic, syringic and homovanillic acids were not detected. Instead, vanillin was identified (by means of retention time and spectrum characteristics) for the first time in all samples. The presence of luteolin and apigenin was verified in most of the samples as well as the presence of oleuropein aglycone and ligstroside aglycone, and the dialdehydic forms of hydroxytyrosol or tyrosol. The authors also reported the presence of a new compound, 4-(acetoxyethyl)-1,2-dihydroxybenzene, in most samples and at considerable levels in Arbequina, Empeltre and Hojiblanca oils. The presence of peaks, later identified as lignans (Brenes et al., 2000) was also reported for certain cultivars. Two tendencies were reported in simple phenols as regards maturation: the constant presence and level (~2-4ppm) of vanillic acid, vanillin, p-coumaric acid, and ferulic acid and the increase in tyrosol and hydroxytyrosol content. The concentration of tyrosol was always higher than that of Figure 1. Free phenols and phenolic acids

4 128 Olive Oil Phenolics Figure 2. Glycosides and aglycones Figure 4. Lignans Figure 5. Closely related non-phenolic compounds extracted with phenols Figure 3. Flavonoids hydroxytyrosol. Though luteolin concentration increased with maturation (~10 ppm in some samples) apigenin did not show a definite tendency with fruit ripening. The reduction in the levels of complex forms of phenols coincides with the decrease in the content of glycosides in fruits during maturation but is also influenced by enzymic reactions during oil extraction. Morrello et al.. (2004) studied the effect of maturation on the phenolic fraction of drupes and oils from cultivars from Ebro Valley in Spain (Arbequina, Ferga, Morrut). Bouaziz et al., (2004) in their work on olive cultivar Chemlali from Tynisia reported that the antioxidant activity of methanol extracts increased during maturation though some uncertainty is expressed concerning the specificity of the Folin-Ciocalteu method. Similar observations were recently reported for Portuguese olive fruits by Vinha et al., (2005), who emphasised the effect of cultivar in relation to origin. The interest in phenol evolution during ripening and the effect of maturation stage on the stability and overall organoleptic quality of the oil is continuous. Linked to the latter is expected to be the effect of storage of olives prior to milling. Holding of olives has as a result a considerable loss of antioxidants due to both degradation of the cell structure and growth of lipolytic moulds (Brenes et al., 1992; Mariani et al., 1991; García et al., 1996; Agar et al., 1998).

5 Olive Oil Phenolics 129 Milling, level of phenols and oxidative stability Many studies are known for the conditions of milling and the content of tocopherols and polar phenols. Salvador et al (2003) reported the results of a study with samples from 5 crop seasons. The three main extraction systems, pressure, dual-phase and triple-phase were examined. Total phenols and o-diphenols were found to be present at higher levels in the oil obtained by the two-phase decanters. The oil obtained by these decanters was superior in oxidative stability and overall quality. These properties were followed by a slightly higher index of bitterness. The lower phenol content of the oil extracted in three phase centrifuges is due to the addition of water, which reduces the concentration of the polar phenolic compounds. In the two phase system this drawback is lessened because the wastewater is recycled as soon as it is produced and used instead of the added water for the dilution of the paste. The above results are in accordance with data presented in other studies (Di Giovacchino et al, 2001, Cert et al, 1996). In addition to the system of extraction, the mode of fruit crushing seems to be also important. To upgrade oil quality, olives very rich in phenols can be crushed using a stone mill. In this way, the level of phenols is reduced and as a result bitterness and pungency are eliminated. On the contrary, to increase phenol content hammer crushers are recommended (Caponio et al, 1999). When hammer crushers are used, even the rotation rate may be critical, as indicated by Fogliano and his co-workers (1999). A change from 2200 rpm to 2900 rpm resulted in about 40% increase in the total antioxidant power of the polar fraction. This was attributed to a better fragmentation of olive tissues and a release of 3,4-DHPEA- EDA (the dialdehydic form of elenolic acid linked with hydroxyltyrosol), due to activation of hydrolytic enzymes during the subsequent paste mixing. It has to be stressed also that the conditions of kneading (temperature, time) are also critical for the level of phenols (Garcia et al., 2001, Angerosa, 2001). According to Garcia and his co-workers the malaxation stage may reduce the concentration of ortho-diphenols ca 50-70%. Other phenols seem to be more stable. Angerosa observed in the first 15 minutes of malaxation losses ranging from due to change in the temperature from 25 to 35 C.When at laboratory scale the paste was malaxed under nitrogen, losses of phenols were avoided (Garcia, 2001). This indicates the importance of chemical and enzymatic reactions taking place because of the presence of oxidoreductases. Glycosidases present in the olive fruit and consequently in the paste result in the formation of aglycone forms of secoiridoids. The oxidizing enzymes oxidize the latter. Servili and his coinvestigators (2003) suggested that the time of exposure of olive paste to air during the malaxation is a processing parameter that can be used to control endogenous oxidoreductases such as phenol oxidase, peroxidase and lipoxygenase. Finally it has to be mentioned that the addition of cell wall degrading enzymes during the extraction process increases the level of phenols in the product, especially the levels of the dialdehydic form of elenolic acid linked to hydroxytyrosol (Ranalli, 1997, Vierhuis, 2001). Bitter taste and polyphenols The bitterness of virgin olive oil has been related to the concentration of total polyphenols (Angerosa et al., 2000) and total oleuropein derivatives (Garcia et al., 2001). In a more recent report Gutierrez-Rosales et al., (2003) attempted to correlate bitter intensity of a large number of virgin olive oils with the concentration of individual phenols. The oil samples were evaluated for bitterness by a panel using a 1-5 scale (imperceptible, slight, moderate, great, extreme), while solid phase extraction, preparative HPLC, analytical HPLC and on-line liquid chromatography-electrospray ionization massspectrometry were used to separate, identify and quantify individual phenols for sensorial analysis. The results of their study indicated that the dialdehydic and aldehydic forms of decarboxymethyl oleuropein aglycone and the dialdehydic form of decarboxymethyl-ligstroside aglycone are the compounds mainly responsible for the bitter taste. Andrewes et al. (2003) carried out a very systematic study to establish relations between sensory properties and individual phenols. They tentatively identified the dialdehydic forms of deacetoxy-oleuropein and diacetoxy-ligstroside aglycon, derivatives and isomers of ligstroside and oleuropein aglycons and many other compounds, which were all described as bitter and astringent. The fraction containing deacetoxy-ligstroside aglycone was found to produce a strong burning pungent sensation at the back of the throat, while the deacetoxyoleuropein aglycon was slightly burning and the sensation was perceived mainly on the tongue. Tyrosol was astringent but not bitter. Hydroxytyrosol was not tested due to the difficulty in the preparation in sufficient pure form. It is concluded from the above that pungent virgin olive oils have higher levels of the deacetoxy-ligstroside aglycon. Phenolic compounds in table olives Raw olives, with a few exceptions, are not edible because their flesh is astringent and bitter. Thus appropriate processing is necessary to obtain edible and good tasting table olives. The processing methods are related to the chosen maturation stage of the olive fruit. The most commonly prepared final products are Spanish (or Sevillian)-style green olives in brine, Californian-style black olives in brine and Greek-style naturally black olives in brine (Garrido-Fernández et al., 1997). The processing for preparing Spanish-style green olives includes lye treatment, washing, and lactic fermentation in brine. In the Californian method the olives are processed by successive treatments using lye. At the end of each lye treatment, the olives are washed and aerated (darkening process). After fixing the black color in an aqueous solution of ferrous gluconate or lactate, the olives are canned in brine and the process is completed by heat-sterilization. In the case of the Greek-style process, olive fruits are fermented after washing into the brine for at least six months. Responsible for the fermentation are yeasts. Table olives have a different qualitative and quantitative phenolic composition than the raw olive fruits from which they are prepared. The reason is the diffusion of phenols

6 130 Olive Oil Phenolics and other water soluble constituents from the olive fruit to the surrounding medium (water, brine or lye) and vice versa, and, in the case of lye treated products, the reactions in olive flesh between sodium hydroxide and constituents that have carboxylic and hydroxyl functional groups yielding hydrophilic derivatives that are washed away. During the lye treatment, oleuropein and verbascoside are hydrolyzed (Brenes-Balbuena et al., 1992; Brenes et al., 1995; Marsilio et al., 2001). The glycosides formed (oleoside 11-methylester, hydroxytyrosol-1-o-rhamnosylglucoside) are hydrolyzed to hydroxytyrosol during lactic fermentation of the washed olives in brine by preparing Spanish-style green olives (Brenes et al., 1995; Brenes and de Castro, 1998). Acid hydrolysis of hydroxytyrosol and tyrosol glucosides, anthocyanins, luteolin 7-glucoside, oleuropein, verbascoside, ligstroside and some of the derived products (caffeoylrhamnoglucoside, oleuropein derivatives, ligstroside aglycone) takes also place during the fermentation in brine when Greek-style naturally black olives are prepared (Brenes et al., 1992; Romero et al., 2004a). Anthocyanins are also polymerized during fermentation (Romero et al., 2004b). Hydroxytyrosol and caffeic acid are decreased markedly during the darkening process by preparing Californian-type black olives. The diminution of these orthodiphenols in flesh is directly related to the olive fruit browning development (Brenes-Balbuena et al., 1992; Marsilio et al., 2001). Iron salts, used for color fixation, catalyze the oxidation of hydroxytyrosol, which disappears (Marsilio et al., 2001). Commercially available Greek table olive samples, analyzed for individual phenols by RP-HPLC, were found to contain hydroxytyrosol as the prevailing phenolic compound (Blekas et al., 2002). Its levels (in flesh) were found to be higher than 100 and 170 mg/kg in Greek-style naturally black olives and Spanish-style green olives in brine, respectively. However, in Kalamata olives, a special type of Greek-style naturally black olives, this phenol was found at levels ranging from 250 to 760 mg/kg. Remarkable levels of luteolin were determined only in Greek-style naturally black olive samples (25-75 mg/ kg), whereas tyrosol was found in both types of table olives analyzed at levels ranging from 20 to 170 mg/kg. Individual phenols were also determined by HPLC in the juice and oil of packed table olive samples (Romero et al., 2004b). The samples were Spanish-style green olives, Californian-style black olives, Greek-style naturally black olives, and turning color olives in brine that are prepared using fruits with yellowpurple color according to the Greek-style method. The oil and juice phases had a different phenolic composition. Tyrosol and hydroxytyrosol were the main phenols in olive juice. The same phenols, their acetates and two lignans (pinoresinol and 1-acetoxypinoresinol) were the main phenolic compounds in oil. The juice of Greek-style naturally black olive and turning color olive samples was found to contain also tyrosol-1-o-βglucoside at remarkable levels, and verbascoside. The oil phase in all samples was found to contain also catechol, vanillic acid and vanillin. Taking into account the oil and moisture content of the olive flesh, the calculated levels of the main above-mentioned phenolic compounds varied between (a) 117 and 980 mg/kg for hydroxytyrosol, (b) 36 and 134 mg/kg for tyrosol, (c) 0.5 and 24 mg/kg for hydroxytyrosol acetate, (d) 4.5 and 11.5 mg/kg for tyrosol acetate, (e) 0.2 and 12.3 mg/kg for 1-acetoxypinoresinol, and (f) 0.5 and 6.8 mg/kg for pinoresinol. Catechol, vanillin and vanillic acid levels were lower than 5, 1.5 and 1 mg/kg, respectively. Tyrosol-1- O-glucoside levels were 92 and 87 mg/kg in the samples of Greek-style naturally black olives and 57 mg/kg in the turning color olive sample. The same samples contained verbascoside at levels lower than 0.5 mg/kg. It can be concluded from the above that table olives are excellent sources of antioxidants, baring in mind that 1-2 olives may provide approx 10 mg of polyphenols. This quantity corresponds to approximately g of olive oil. BIOLOGICAL PROPERTIES In the last decade the number of publications related to the biological role of olive oil and its minor constituents has increased significantly. The collected data suggest that the phenolic compounds present in olive oil may contribute to the health benefits attributed to Mediterranean diet. The latter is linked to a lower incidence of coronary heart disease, prostate and colon cancer (Assmann and Wahrburg, 1999, Keys, 1995, Wahrburg, et al., 2002). The work published focused mainly on the antioxidant properties of olive oil phenols and their capability to break peroxidative chain reactions. Biological properties of olive oil phenols have been reviewed by Visioli and Galli (1998), Soler- Rivas et al., (2000), Boskou and Visioli, (2002), Tuck and Hayball, (2002) and Visioli et al., (2002). Low-density lipoprotein oxidation The in vivo lipoprotein oxidation is considered a dominant risk factor for the development of atherosclerosis (Assman and Wahrburg, 1999, Salonen, 2000, Aviram, 2002). Phenolic compounds present in olive oil are potent in vitro inhibitors of low-density lipoproteins (LDLs) oxidation. This was demonstrated in a series of papers (Visioli and Galli, 1994 a,b, Visioli et al., 1995, Visioli and Galli, 1998, Aruoma et al., 1998, Caruso et al., 1999). Visioli s work was concentrated mainly on hydroxyltyrosol and its derivatives, but the antioxidant/atherosclerosis theory stimulated experimental studies with other olive oil bioactive phenols. Nardini and his co-investigators (1995) studied the activity of hydroxycinnamic acids on in vitro LDL oxidation with Cu++. Caffeic acid was found to completely protect LDL. Another phenolic acid, protocatechuic acid was studied by Masella and others (2004), who used macrophages - like cells in the LDL model oxidation. Pilot studies were also conducted in vitro for the evaluation of changes induced by oxidised LDL in human colon adenocarcinoma cell line CaCo-2. Tyrosol, a major constituent of the polar fraction of olive oil was found to have a protective effect and reduce cytostatic and cytotoxic effects caused by oxidized LDL (Giovannini et al., 1999). Coni et al. (2000) conducted an in vivo study with laboratory

7 Olive Oil Phenolics 131 rabbits fed special diets containing olive oil and oleuropein. The biochemical parameters measured in the rabbit plasma and the isolated LDL verified the antioxidant efficiency of virgin olive oil phenols and in particular of oleuropein. Masella et al. (2001) studied the effect of dietary intake of extra virgin olive oil on the oxidative susceptibility of LDL, isolated from the plasma of hyperlipaemic patients. The intake of extra virgin olive oil did not affect LDL fatty acid composition but significantly reduced the formation of LDL oxidation products induced by CuSO 4. These results are attributed to the presence of phenolic antioxidants in olive oil. Isoprostane formation The isoprostanes are a series of prostaglandin-like compounds formed in vivo from peroxidation of long-chain PUFA. They are used as biochemical markers for the assessment of oxidant stress and lipid peroxidation (Salomen, 2000). Visioli and his co-workers (2000) attempted to evaluate in humans the antioxidant activity of catecholic structure phenols in olive oil. Human volunteers were administrated olive oil with varying levels of o-diphenols. Isoprostane 8-iso-PGF 2a was determined in the urine by mass-spectrometry after incubation with β-glucuronidase. From their study the authors concluded that the administration of hydroxytyrosol and oleuropein aglycones results in a dose-depended reduction of the urinary formation of isoprostanes. It is interesting to note also that a statistically negative correlation was observed between F2- isoprostane excretion and formation of homovanilyl alcohol, hydroxytyrosol s major metabolite. Scavenging of radical and other reactive species Olive oil phenols, especially oleuropein and hydroxytyrosol, have been studied with respect to their potential to scavenge synthetic radicals, peroxyl radicals, superoxide radicals and hypochlorous acid and to reduce damages induced by hydrogen peroxide and peroxinitrate ion. Free radical scavenging activity of hydroxytrosol and its derivatives using 1,7-diphenyl-2-picrylhydrazyl radical (DPPH.)was measured by Visioli, et al. (1998a, b), Saija et al. (1998), Gordon et al. (2001), Tuck et al. (2002), Lavelli, V., (2002). Tuck et al. (2002) studied the scavenging activity not only of hydroxytyrosol but also of its metabolites in rats (homovanillic acid, homovanillic alcohol, glucuronide conjugate, and sulphate conjugate). The glucuronide was found to be more potent antioxidant compared to hydroxytyrosol itself. Saija et al. (1998), in addition to DPPH test conducted additional measurements to obtain more information for the scavenging activity of hydroxytyrosol and oleuropein against peroxyl radicals near the membrane surface and within the membranes, using a model system consisting of dipalmitoy lphosphatidylcholine/linoleic acid unilamellar vesicles and a water soluble azo compounds and free radical generator. The radical scavenging capacity of the major phenols present in olive oil was also measured by Briante et al. (2003), who used the stable red radical cation DMPD.+. The same authors attempted to differentiate phenols not only by their activity to scavenge radicals but also by their ability to chelate metal ions. A metal-chelate mechanism for antioxidant activity of olive oil phenols was also suggested by Visioli and Galli (1998). In order to evaluate olive oil as a source of antioxidants, many researchers attempted to measure ORAC (oxygen radical absorbance capacity) values, which indicate a capacity to protect against oxidation by peroxyl radicals (Ninfalli et al., 2001). Pellegrini et al. (2002), determined the TEAC (Trolox equivalent antioxidant capacity). FRAP (Ferric-reducing antioxidant capacity) and TRAP (total-radical-trapping antioxidant parameter) in plant food beverages and various edible oils including olive oil in an attempt to obtain additional information, necessary to investigate the relation between antioxidant intake and oxidative stress induced diseases. Scavenging of reactive nitrogen species Deiana and others (1999) found that hydroxytyrosol was very protective against peroxynitrite dependent nitration of tyrosine and DNA damage by peroxynitrite in vitro. Scavenging activities of the major olive oil phenols against reactive nitrogen species (nitric oxide, peroxinitrite) were studied by De la Puerta and his co-workers (2001). Caffeic acid, oleuropein and hydroxytyrosol reduced the amount of nitric oxide formed by sodium nitroprusside and were also found to have the ability to reduce chemically generated peroxinitrite. However, as Visioli and his co-workers demonstrated (1998b) oleuropein seems to enhance NO production, from LPSchallenged mouse macrophages. De la Puerta concluded that this glycoside has both the ability to scavenge nitric oxide but also to cause an increase in the inducible nitric oxide synthase (INOS) expression in the cell. CONCLUDING REMARKS Accumulated data suggest that the polar phenolic compounds present in olive oil may improve in vivo antioxidant defences and that their role in disease prevention is greater than previously thought. Hydroxytyrosol in its various forms seems to be one of the key bioactive phenolic compounds. That confers many of the health benefits attributed to olive oil and the Mediterranean diet. The production of high quality virgin olive oil with a sufficient level of biophenols can be guaranteed by present day chemical knowledge and the combination of tradition and technology. Table olives are also good sources of biophenols and they should be evaluated not only for their colour and appearance but their hydroxytyrosol content as well. CONFLICT OF INTEREST DISCLOSURE: None disclosed REFERENCES Agar, I. T., Hess-Pierce, B., Sourour, M. M. and Karer, A. A. (1998) Quality of fruit and oil of black-ripe olives is influenced by cultivar and storage period. Journal of Agriculture and Food

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