Review article	UDC 633.852.73:615.01 (497.4 Istra)
Received: 2006-09-06
OLIVE TREE - THE SOURCE OF PHARMACODYNAMICALLY
ACTIVE SUBSTANCES
Metoda LIPNIK-ŠTANGELJ
University of Ljubljana, Faculty of Medicine, Department of Pharmacology and Experimental Toxicology, SI-1000 Ljubljana, Korytkova 2
and
University of Primorska, Science and Research Center Koper, SI-6000 Koper, Garibaldijeva 1 E-mail: metoda.lipnik-stangelj@mf.uni-lj.si
Milena BUČAR-MIKLAVČIČ
University of Primorska, Science and Research Center Koper, SI-6000 Koper, Garibaldijeva 1
and
LABS, LLC - Institute for Ecology, Olive Oil and Control, SI-6310 Izola, Zelena ulica 8
Bojan BUTINAR
University of Primorska, Science and Research Center Koper, SI-6000 Koper, Garibaldijeva 1
ABSTRACT
Olive and its products have been recognized as an important component of a healthy diet. Increase olive oil consumption is implicated in reduction of cardiovascular diseases, rheumatoid arthritis and a variety of cancers. In the prevention of these diseases, antioxidant activity of olive oil seems to play the key role. In addition, some compounds from olive oil, particularly polyphenols, show potent effects on cell signalling via modulation of enzymes activity and protein expression, therefore their use became interesting not only in prevention, but also in the treatment of certain diseases.
Key words: olive oil, polyphenols, antioxidants, healthy diet
OLIVO - FONTE Dl SOSTANZE FARMACODINAMICAMENTE ATTIVE
SINTESI
L'olivo ed i suoi prodotti vengono riconosciuti come un'importante componente di una dieta salutare. L'aumento del consumo dell'olio d'oliva e implicato nella riduzione delle malattie cardiovascolari, dell'artrite reumatica e di varie forme di cancro. Nella prevenzione di tali malattie, l'attivita antiossidante dell'olio d'oliva ha una funzione chiave. Alcuni composti dell'olio d'oliva, in particolare i polifenoli, hanno un potente effetto sulla segnalazione cel-lulare tramite modulazione dell'attivita enzimatica e dell'espressione proteica, pertanto il loro utilizzo e diventato interessante non solo nella prevenzione ma anche nella cura di alcune malattie.
Parole chiave: olio d'oliva, polifenoli, antiossidanti, dieta salutare
INTRODUCTION
Olive and its products have been recognized as an important component of a healthy diet. The number of reports, describing the beneficial properties of olive oil, has dramatically increased in the last couple of years, and recent data have suggested that olive oil has more health benefits that previously thought. The growing popularity of the Mediterranean diet is due to several epidemiological studies that show the lowest incidence of coronary heart disease and certain cancers, e.g., breast and colon cancers in the Mediterranean basin. It has been suggested that this is largely due to the relatively safe and even protective dietary habits of this area, where olive oil represents the principal source of fat in a diet (Hertog et al., 1995; Keys, 1995; Visioli et al.,
2005).
Olive oil's vital components, monounsaturated fatty acids and antioxidant and anti-thrombotic substances are attributed with its protective effects against, among others, atherosclerosis and other cardiovascular diseases (Manna et al., 2004; Dell'Agli et al., 2006; Perona et al.,
2006),	diabetes (Al-Azzawie et al., 2006), certain cancers (Owen et al., 2004; Visioli et al., 2004; Hashim et al., 2005), inflammation diseases (Pattison et al., 2004; Puel et al., 2004; Beauchamp et al., 2005) and age related cognitive decline (Wahle et al., 2004).
This review is focuses on biologically active constituents of olive and particular olive oil and their importance for human health. The molecular mechanisms involved in pharmacologically effects of certain compounds are highlighted.
THE COMPOSITION OF OLIVE OIL
Olive oil is obtained from the drupes of olive tree that is best grown between the 30° and the 450 parallel. Accordingly, the Mediterranean countries supply more than 95% of the world olive oil production that is around 2.000.000 tons/year (Visioli et al., 2002). Depending on its chemical properties, organoleptic characteristics and its degree of acidity, olive oil is classified into different grades (EEC Council regulations, 1991). From this classification, the most valuable is the extravirgin oil, obtained from intact olives that are quickly processed and cold-pressed. In this way, activation of cellular lipases and degradation of the triglycerides is minimized (Visioli et al., 2002).
The composition of olive oil is primarily saponifiable glyceridic compounds as triglycerides (Montedoro, 1972), where the oleic acid, a monounsaturated acid
(18:1n-9) represents 56 to 84% of total fatty acid, while linoleic acid (18:2n-6) is present in 3 to 21% (Boskou, 2000; Butinar et al., 2004).
The biological effects of monounsaturated fatty acids in olive oil on circulating lipids and lipoproteins in human body are somewhat controversial (Visioli at al., 2002): while the major effects of high monounsaturated fatty acids intakes on serum cholesterol are generally attributed to the associated replacement of saturated fatty acids (Hegsted et al., 1993; Gardner et al., 1995), some studies attributed a direct, although modest cholesterol-lowering effect to monounsaturated fatty acids alone, when they equicalorically replace carbohydrates. Furthermore, monounsaturated fatty acids increase the levels of the protective high-density lipoprotein (HDL) more than polyunsaturated when these two classes of fatty acids replace carbohydrates in the diet (Mensink et al., 1992).
Unsaponifiable compounds represent 0.5 to 1.0% of constituents of minor fraction in olive oil. Among minor constituents of virgin olive oil, there are vitamins such as a- and y-tocopherols (around 200 ppm) and P-carotene, phytosterols, pigments, terpenoids, flavonoids such as luteolin and quercetin, squalene, and more than 30 different phenolic compounds (Montedoro, 1972; Butinar et al., 1999; Boskou, 2000), some of them with potent antioxidant activity, which is important in the prevention of cardiovascular and cancer diseases and inflammation (Boskou, 1996) (Tab. 1).
The amount of phenolic compounds in olive oil depends on several factors, including cultivar, degree of maturation, possible infestation by the olive fly Dacus olea, and climate (Boskou, 2000; Butinar et al., 2000a), and usually decreases with over-maturation of olives (Visioli et al., 2002).
The three phenolic compounds in highest concentration in olive oil are the glycoside oleuropein, hy-droxytyrosol (3,4-dihydroxyphenyl ethanol) and tyrosol. These three compounds are related structurally. Hy-droxytyrosol and tyrosol are structurally identical except that hydroxytyrosol possesses an extra hydroxy group in the meta position (Tuck et al., 2002). Oleuropein is an ester composed of hydroxytyrosol and elenolic acid. Oleuropein is the major phenolic compound in olive drupes, whereas hydroxytyrosol is the major phenolic component in olive oil (Amiot et al., 1996). As the olive drupe matures, the concentration of oleuropein decreases, while hydroxytyrosol, a hydrolysis product of oleuropein, increases (Cimato et al., 1990; Ryan et al., 1999).
Tab. 1: The chemical composition of olive oil. Tab. 1: Kemična sestava oljčnega olja.
Olive oil	Subfraction	Component
Major fraction (98-99%, saponifiable)	triglycerides	oleic acid, linoleic acid
Minor fraction (1-2%, unsaponifiable)	hydrocarbons	squalene, P-carotene, polycyclic aromatic hydrocarbon
	sterols	P-sitosterol, campesterol, A7-stigmasterol, brassicasterol
	terpenic dialcohols	erythroidol, uvaol
	tocopherols	a-tocopherol, P-tocopherol, y-tocopherol, A-tocopherol
	phenolic compounds	tyrosol, hydroxytyrosol, caffeic acid, oleuropein
	others	flavour components
PHARMACODYNAMICALLY EFFECTS OF OLIVE OIL CONSTITUENTS
Recent studies showed that certain olive oil constituents exert strong pharmaco-dynamic effects in human body. They have potent modulatory effect on cell signalling and became interesting not only in prevention, but also in the treatment of certain diseases. So far, several biologic activities of olive oil compounds have been demonstrated, like: scavering of superoxide and other reactive oxygen substances (ROS) (Le Toutour et a!., 1992; Aeschbach et a!., 1994; Manna et a!., 1997) inhibition of low-density lipoprotein (LDL) oxidation (Scac-cini et a!., 1992; Visioli et a!., 1994), inhibition of apoprotein derivatization (Visioli et a!., 1995), reduced thromboxan B2 and leukotriene B4 production by activated leukocytes, inhibition of platelet aggregation and thromboxane generation (Petroni et a!., 1995), inhibition of peroxynitrite-induced DNA damage and inhibition of peroxynitrite-induced tyrosine nitration (Deiana et a!., 1999), inhibition of bacterial growth and activity, and decreased isoprostane excretion in humans and in side-stream smoke-exposed rats (Tuck et a!., 2002), and others, like scavering of hypochlorus acid, increased nitric oxide production by mouse macrophages, cytostasis, hypotensive action, and increased plasma antioxidant capacity (for review see Visioli et a!., 2002).
Antioxidant activity of olive oil constituents
Antioxidant activity of olive oil constituents, particularly polyphenols, seems to play a key role in the beneficial effect in several diseases, like cardiovascular diseases, cancer and inflammation. The main mechanism by which the components of olive oil express their antioxidant activity is inhibition and/or scavenging of ROS, which can activate different signalling pathways that lead to progression of disease. In the excess of ROS, they can also react with different cellular constituents and cause cell damage. ROS are produced during normal metabolism or after oxidative processes and include superoxide anion (O2~) and hydrogen peroxide (H2O2)
(Voetsch et a!., 2004). In the increased production of O2~ and H2O2, they react rapidly with nitric oxyde (NO) to form peroxynitrite (OONO), thus inactivating NO and leading to different physiological dysfunctions (Perona et a!., 2006) (Fig. 1).
The formation of ROS is balanced by a range of antioxidant defences, but the excess can overwhelm these systems and leads to oxidative stress, which importantly contributes to the development of certain diseases. Several constituents in olive oil potently modulate the ROS production. Oleic acid and P-sitosterol may reduce intracellular ROS by creating a less-oxidant environment through inhibition of intracellular ROS production. P-Sitosterol may also enhance superoxyde dysmutase activity, hence decreasing O2~ levels. This reduction has also been observed for the terpenoid oleanolic acid, although the mechanism is not presently known. Tocopherols and phenolic compounds are potent antioxidants that may help reduce lipid peroxidation and scavenge intracellular ROS and free NO, reducing the formation of OONO . ROS can activate the nuclear factor kB (NFkB), which is then translocated into the nucleus, where it binds to recognition sequences in DNA to induce gene expression. This mobilization of NFkB is blocked by a-tocopheryl succinate but not by a-tocopherol. In contrast, phenolic compounds have been proposed to act blocking the formation of NFkB/DNA binding complexes. NFkB modulates the expression of cytokines, enzymes lipoxygenase (LOX) and cycloxy-genase (COX), thereby affecting the levels of adhesion molecules and eicosanoids. However, some of the minor compounds of olive oil may act directly on these enzymes and cytokines. LOX and COX activities are inhibited at different points by phenolics and triterpenoids, whereas interleukin-1 P (IL-1P) expression is inhibited by phenolics and tocopherols, contributing to protect the endothelium against vasoconstriction, platelet aggregation and monocyte adhesion. Vasodilatation is also suggested to be enhanced by oleuropein and oleanolic acid through an increase in the production of NO (Tab. 2) (for review see Perona et a!., 2006).
Fig. 1: Different biochemical pathways of reactive oxygen substances (ROS) production in the cell (SODsuperoxyde dysmutase).
Sl. 1: Različne biokemične poti nastanka reaktivnih kisikovih spojin (ROS) v celici (SOD-superoksid-dismutaza).
Tab. 2: Influence of olive oil constituents on biochemical processes in the cell. Tab. 2: Vpliv sestavin oljčnega olja na biokemične procese v celici.
Olive oil constituent	Mechanism of action
oleic acid	Inhibition of ROS production
polyphenols	Reduction of lipid peroxidation Scavenging intracellular ROS Reduction of OONO- formation Reduction of NFkB/DNA binding complexes formation Inhibition of LOX and COX Inhibition of IL-P expression Increasing NO production
tocopherols	Scavenging intracellular ROS Reduction of OONO- formation Block of NFkB mobilisation to the nucleus Inhibition of IL-P expression
ß-sitosterol	Inhibition of ROS production Activation of superoxyde dismutase
terpenoids	Inhibition of LOX and COX
The effects of olive oil constituents in cardiovascular diseases
Olive oil constituents show complex benefit effects in prevention of cardiovascular diseases. Cardiovascular diseases risk factors, like hypercholesterolemia (Ohara et al., 1993; Stokes et al., 2002), diabetes mellitus (Guzik et al., 2002) and hypertension (Just, 1997; Kerr et al., 1999), are all strongly related to increased production of ROS, inactivation of NO and endothelial dysfunction. ROS can also react with polyunsaturated fatty acids
contained in lipoproteins in the vessel wall, initiating lipid peroxidation. The hydroperoxides formed in this process can in turn react with NO to form OONO, inactivating NO, and directly decrease the endothelial synthesis of NO (Chin et al., 1992). Monounsaturated fatty acids in olive oil increase the levels of the protective HDL and improve therefore lipoprotein profile in the body. Beside this, they reduce the thrombogenic-atherogenic process by various actions on arterial thrombus formation, such as decreased monocyte adhesion, increased fibrinolysis and decreased arte-
rial pressure. Additionally, the antioxidant substances found in olive oil (i.e. tocopherols, polyphenols) could influence atherogenesis by inhibition of LDL-cholesterol oxidation, protection against free radicals and their toxic effects, inhibition of platelet aggregation and thromboxane generation, stimulation of anti-inflammatory agents and increased nitric oxide production (Perona et al., 2006).
The effects of olive oil constituents in inflammation
Certain compounds from olive oil have been shown to modulate immune function that might be interesting in the treatment of inflammatory processes, associated with the immune system like rheumatoid arthritis (Patti-son et al., 2004; Puel et al., 2004; Beauchamp et al., 2005).
In the inhibition of inflammatory processes by olive oil constituents, the reduction of intracellular ROS production is important, since ROS enhances transcriptional activity via activation of NFkB that modulates the expression of certain pro-inflammatory cytokines like IL-1P, IL-6 and tumour necrosis factor a. Beside this, reduction of ROS production inhibits LOX and COX transcription and activity. Consequently, the cyclooxygenase and lipoxigenase pathway of arachidonic acid metabolism, and the production of prostaglandins and other inflammatory mediators are diminished. Some of the minor compounds of olive oil may act directly on selected enzymes and cytokines, involved in inflammation; LOX and COX activities are inhibited at different points by phenolics and triterpenoids, whereas IL-ip expression is inhibited by phenolics and tocopherols (Beauchamp et al., 2005; Perona et al., 2006).
The effects of olive oil constituents in cancer
In the ethiopathogenesis of cancer disease, there are several factors contributing to the development and progress of the disease. Among them, oncogenic substances, oxidative stress and angiogenesis play an important role.
Several epidemiological studies show beneficial effects of olive oil constituents in prevention of cancer
diseases (Owen et al., 2004; Visioli et al., 2004; Hashim et al., 2005). Olive oil constituents inhibit production of ROS and have protective role in conditions, where the excess production of ROS leads to oxidative stress and causes oxidative cell damage. Certain compounds from olive oil also inhibit the expression of pro-oncogenic substances (Nelson, 2005). Recent studies showed inhibitory effect of olive oil polyphenols on proliferation of human promyelocytic leukemia cell by inducing apop-tosis and differentiation (Fabiani et al., 2006). Beside this, it was found strong anti-angiogenic effect of oleu-ropein, which causes irreversible changes in cancer cells, preventing their replication, motility and invasiveness (Hamdi et al., 2005).
These effects may explain the cancer-protective effects of the olive-rich Mediterranean diet and may also have important therapeutic implications in the treatment of cancer disease.
CONCLUSION
Epidemiological studies show that populations consuming a predominantly plant-based Mediterranean-style diet exhibit lower incidences of chronic diseases than those consuming a northern European or North American diet. Although total fat intake in Mediterranean populations can be higher than in other regions, the greater proportion is derived from olive oil and not animals. Increased olive oil consumption is implicated in a reduction in cardiovascular diseases, rheumatoid arthritis and, to a lesser extent, a variety of cancers. Olive oil intake also has been shown to modulate immune function, particularly the inflammatory processes associated with the immune system (Wahle et al., 2004).
Typical of olive oil is combination of high oleic acid content and content of a variety of plant antioxidants, particularly oleuropein, hydroxytyrosol, and tyrosol. In the prevention of diseases, strong antioxidant activity of olive oil seems to play the key role. In addition, some compounds from olive oil, particularly polyphenols, showed potent effects on cell signalling via modulation of enzymes activity and protein expression, therefore their use became interesting not only in prevention, but also in the treatment of certain diseases.
OLJKA - VIR FARMAKODINAMIČNO UČINKOVITIH SNOVI
Metoda LIPNIK-ŠTANGELJ
Univerza v Ljubljani, Medicinska fakulteta, Inštitut za farmakologijo in eksperimentalno toksikologijo, SI-1000 Ljubljana, Korytkova 2
in
Univerza na Primorskem, Znanstveno-raziskovalno središče Koper, SI-6000 Koper, Garibaldijeva 1 E-mail: metoda.lipnik-stangelj@mf.uni-lj.si
Milena BUČAR-MIKLAVČIČ
Univerza na Primorskem, Znanstveno-raziskovalno središče Koper, SI-6000 Koper, Garibaldijeva 1
in
LABS, LLC - Inštitut za ekologijo, oljčno olje in kontrolo, SI-6310 Izola, Zelena ulica 8
Bojan BUTINAR
Univerza na Primorskem, Znanstveno-raziskovalno središče Koper, SI-6000 Koper, Garibaldijeva 1
POVZETEK
Oljka in izdelki iz oljke so že dolgo znani kot pomembna sestavina zdrave prehrane. Dokazani so bili številni koristni učinki sestavin oljčnega olja, plodov in listov oljke. S količinskega vidika so glavna komponenta oljčnega olja umiljivi triacilgliceroli, medtem ko manjši del sestavljajo neumiljive snovi, kot so steroli, terpenski alkoholi, to-koferoli, polifenoli in druge spojine. Večina sestavin (oleinska kislina, polifenoli, tokoferoli, /3-sitosterol, terpenoidi) ima močan modulatoren učinek na celično signaliziranje, zato so postale zanimive za preprečevanje in zdravljenje nekaterih bolezni.
Najbolj preučevan učinek je zaščitna vloga oljčnega olja pri boleznih srca in ožilja, kjer imata ključno vlogo moteno delovanje žilnega endotelija in razvoj ateromatoznih plakov. Glavni mehanizem delovanja, po katerem sestavine oljčnega olja vplivajo na delovanje žilnega endotelija, je zaviranje delovanja reaktivnih kisikovih spojin in lovljenje prostih radikalov. To je posledica zaviranja nastanka reaktivnih kisikovih spojin pa tudi aktivacije encima superoksid-dismutaze. Poleg tega oleuropein in oleanolna kislina s povečanjem nastanka NO povzročata tudi razširitev žil.
Polifenoli so učinkoviti tudi pri zaviranju aktivnosti nekaterih pro-vnetnih citokinov, kot je npr. IL-1//, ter encimov ciklo-oksigenaze in lipo-oksigenaze. Ti procesi so posredovani najmanj po dveh poteh. Prvo pot predstavlja zaviranje mobilizacije NFkB, ki je pomembna za izražanje genov številnih pro-vnetnih citokinov in encimov. Poleg tega delujejo polifenoli in tokoferoli tudi kot lovilci prostih radikalov in s tem zavirajo nastanek vnetnih mediatorjev iz arahidonske kisline. Ker polifenoli tako neposredno vplivajo na nastanek vnetnega in/ali imunskega odgovora, so postali zanimivi za zdravljenje bolezni, kot je npr. revmatoidni artritis, ter degenerativnih boleznih živčnega sistema, kakršna je npr. Alzheimerjeva bolezen. Nekatere sestavine oljke, zlasti tokoferoli, skvaleni in polifenoli, pa so zaradi antioksidativnega in protitumorskega učinka zanimive tudi za preprečevanje nastanka raka.
Ključne besede: oljčno olje, polifenoli, antioksidanti, zdrava prehrana
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