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Introduction. Traditional Mediterranean diet, characterized by high intake of olive oil, fruit, nuts, vegetables, cereals and a moderate intake of fish and wine, is widely recognized and approved as beneficial for human health. Olive oil has been the subject of numerous studies, as valuable source of bioactive phenols and possible use in prevention/treatment of cardiovascular and neurodegenerative diseases, cancer, diabetes mellitus, metabolic syndrome. The composition and the level of the phenolic compounds in the olive oil are affected by a number of factors as cultivar, climate and other agro-ecological factors. Olive fruit, even richer in phenolic compounds, presents an interesting and possibly not enough exploited source of these bioactive molecules. The aim of this study was to evaluate and to compare the content of phenolic compounds in the olive oil and the olive fruit, particularly those characteristic for olive species, with prominent biological activity (hydroxytyrosol, HOTyr ; tyrosol, Tyr ; oleuropein, Ole ; decarboxymethyl-oleuropein aglycon, 3, 4-DHPEA-EDA). Materials and methods. Fresh and health olive fruits of Oblica cultivar (Ugljan island, Croatia) have been picked carefully. One part of the fruits was kept frozen until analysis, while the other one was used for the extraction of olive oil, using the centrifugal line OLIOMIO 350. Extraction of phenolic compounds from extra virgin olive oil (EVOO) and de-stoned olive fruits (dsOF) was carried out using the ultrasound-assisted liquid-liquid extraction method according to the literature, with some modifications. The total phenol (TP) content was determined by the Folin-Ciocalteu method. The concentration of o-diphenols was determined with the sodium molybdate assay. The phenolic compounds content (OHTyr, Tyr, Ole and 3, 4- DHPEA-EDA) has been determined following the HPLC-DAD analysis performed in essence by applying the previously used procedure. Results. The determined TPs in EVOO and dsOF in cultivar Oblica (Ugljan) were 507±63 mg gallic acid equivalent (GAE)/kg EVOO and 3102±608 mg GAE/kg dsOF, respectively. The o-diphenols were 241±58 mg GAE/kg EVOO and 2190±593 mg GAE/kg dsOF. Following the HPLC-DAD analysis, the content of OHTyr, Tyr, Ole and 3, 4-DHPEA-EDA in EVOO and dsOF, as well as the relationship between the content in EVOO and dsOF were determined. The dsOF showed roughly tenfold greater content of HOTyr and Tyr, while the EVOO showed higher content of 3, 4-DHPEA-EDA. Conclusion. The content of the total phenols, o-diphenols, HOTyr, Tyr, Ole is roughly tenfold greater in the olive fruit than in the olive oil. The determined relationship between the content in the olive oil and the olive fruit could be useful in recognition of the “fruits” which should give the oil with the best content of certain phenolic compounds. Furtherly, olive fruit itself could be used as a potential source of biologically active phenolic compounds.

Introduction. Mediterranean populations experience reduced incidence of cardiovascular disease, atherosclerosis, diabetes mellitus, metabolic syndrome, neurodegenerative diseases, certain types of cancer and higher life expectancy. These health benefits could be at least partially attributed to the olive oil, and more specifically to the phenolic compounds naturally present in olive oil. Phenolic compounds can interact synergistically with each another, resulting in higher biological activity of an extract than the sum of activities of its individual components. The aim of the present work is to determine the total phenol (TP) content and antioxidant activity of the phenolic compounds in extra virgin olive oils (EVOOs) from Croatian cultivar Buža and Žižolera (Istra). Cytotoxic activity of the phenolic compounds in EVOO and their interaction effects with anticancer drug cisplatin have been evaluated on human cervical carcinoma (HeLa) cells. Materials and methods. Extraction of phenolic compounds from EVOOs was carried out using the ultrasound-assisted liquid-liquid extraction method. The TP content in the EVOOs was determined by the Folin- Ciocalteu (FC) colorimetric assay. The antioxidant activity of phenolic compounds from EVOOs was performed using 2, 2-diphenyl-1- picrylhydrazyl radical scavenging assay. The biological activity of phenolic compounds included in EVOOs and their interaction effects with anticancer drug cisplatin (cDDP) were evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide colorimetric assay in HeLa cells upon 72 h incubation. Results. Analysed phenolic compounds in EVOOs from Croatian cultivar Buža and Žižolera showed concentration of TP 270±8 mg gallic acid equivalent (GAE)/kg EVOO (Buža) and 391±34 mg GAE/kg EVOO (Žižolera), respectively. The EC50 values were 190 µg TP GAE/ml of EVOO phenolic extract (Buža) and 198 µg TP GAE/ml of EVOO phenolic extract (Žižolera), respectively. The effect of EVOOs on HeLa cells metabolic activity was similar for both EVOOs and dose- dependent. The combination of both EVOOs with cDDP showed increased metabolic activity of HeLa cells compared to cDDP alone implying their protection effect. Conclusion. Despite the fact that the concentration of phenolic compounds in both EVOOs (Buža and Žižolera) is different their antioxidant activity is similar. The both results are comparable with those found in the literature. The investigated phenolic extracts from two EVOOs showed similar effect on a decrease in metabolic activity of HeLa cells in a dose-dependent manner. The increased metabolic activity detected upon combination treatment of both EVOOs with cDDP suggests theirs chemo protective effect and the need for careful use of olive oils during chemotherapy.

Introduction. Extra virgin olive oil (EVOO) has nutritional and sensory characteristics that make it unique among vegetable oils and as such, it is a basic component of the Mediterranean diet. The importance of EVOO is attributed both to its high content of oleic acid and richness in phenolic compounds, which act as natural antioxidants and may contribute to the prevention of several human diseases. Apart from the interest in composition of the polar fraction of EVOO, the determination of these compounds also helps to understand their health benefits including reduction of risk factors of coronary heart disease, prevention of several varieties of cancer and modification of immune and inflammatory responses. The polar phenolic compounds of EVOO belong to different classes: simple phenols, phenolic acids and their derivatives, flavonoids, lignans and secoiridoids. Some of phenolic acids such as caffeic, vanillic, syringic, p-coumaric, o- coumaric, protocatechuic, sinapic and p- hydroxybenzoic acids were the first group of polyphenols observed in EVOO. Materials and methods. Three samples of EVOO from Istra (Bjelica, Buža i Žižolera) and a sample from Ugljan island were analysed in this study. Extraction of phenols from EVOO was carried out using the ultrasound-assisted liquid-liquid extraction (US-LLE) method. The HPLC-DAD analysis was performed. Results. Results show that phenolic acids and their derivatives (p- hydroxybenzoic acid, homovanilyl acohol, vanillin, vanillic, syringic, p-coumarinic, benzoic and ferulic acids) are present in each analysed EVOO. Their concentration is below 1mg/kg olive oil, as it is usually reported for EVOO in literature. Conclusion. Analysed EVOO derived from Croatia (Istra and Ugljan island) are rich in important minor phenolic acids and their derivatives which provide good properties of oil in nutrition and disease prevention.

Introduction. Free radicals/reactive oxygen species (ROS) are generated from both endogenous and exogenous sources. Most of the body macromolecules, such as lipids, proteins, DNA and carbohydrates are susceptible to damage by ROS. Extra virgin olive oil (EVOO) could be considered as example of a functional food, with a variety of components that may contribute to its overall therapeutic characteristics. EVOO contains at least 30 phenolic compounds. The major phenolic compounds are oleuropein derivatives, based on hydroxytyrosol which are strong antioxidants and radical scavengers. The most widely employed methods for evaluating the total phenolic content (TP) of extra virgin olive oil extract (EVOOE) are the Folin–Ciocalteu (FC) colorimetric assay. Another colorimetric assay that could be used is the method with sodium molybdate, specific for the quantification of o- diphenols. The antioxidant activity of these extract can be estimated by reaction with the stable radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH). Materials and methods. Three different EVOO from Istra (Bjelica, Buža and Žižolera) and from Ugljan island have been analysed in this study. The TP content was determined by the FC method according to the analytical protocol. The concentration of o-diphenols in the EVOOE was determined with sodium molybdate assay. The antioxidant activity of EVOOE was performed using the stable radical DPPH according to the procedure with some modifications. Results. The concentration of TP and o- diphenols in extracts was expressed as mg gallic acid equivalent (GAE) per kg of EVOO (Fig. 1.). Gallic acid served as a standard for the preparation of the calibration curve. The antioxidant activity expressed as EC50 value denotes the concentration leading to 50% reduction of the initial DPPH concentration. Conclusion. Analysed EVOO derived from Croatia (Istra and Ugljan island) have concentration of TP, o-diphenols and EC50 values comparable to those in literature which provide good properties of oil in disease prevention

Istražen je ucinak djelomicno hidrofobnih molekula (organskog otapala i kvarterne amonijeve soli) na protonom spregnuti prijelaz elektrona (PCET) u reakciji askorbata s heksacijanoferat(III) ionom u vodenoj sredini, te je opažen prijelaz iz aktivacijskog režima u režim tuneliranja, dodatkom organskih otapala i kvarternih iona. Eksperimentalna opažanja koja ukljucuju povecane vrijednosti kinetičkog izotopnog učinka (KIE) u odnosu na reakciju u vodi, izotopne omjere AH/AD Arrheniusovih predeksponencijalnih faktora znacajno manje od semiklasično predviđenih, kao i izotopne razlike aktivacijskih energija procesa veće od semiklasičnih, objašnjena su u okviru markusianskog modela tuneliranja koji uzima u obzir dinamičke aspekte sustava i okoliša. Prijelaz protona dinamički je povezan s femtosekundnom dinamikom OH skupine vode. Dodatak djelomično hidrofobnih molekula u vodenu sredinu dovodi do promjena dinamike OH vode zbog interakcije s hidrofobnim dijelom molekule i ta promjena intermolekularnim rezonantnim vibracijskim mehanizmom uključuje molekulu vode u prijelaznoj konfiguraciji i promotivne vibracije povezane s reakcijskom koordinatom protonsko-elektronskog prijelaza u procesu. The influences of molecular hydrophobic interfaces, involving organic solvents and quarternary ammonium ions, on the proton-coupled electron transfer (PCET) reaction of ascorbate monoanion with hexacyanoferrate(III) ion in water has been investigated. The kinetic isotope effect (KIE) in the reaction and the isotopic differences in Arrhenius activation energies increased and the isotopic ratios of the corresponding pre-factors AH/AD decreased significantly, suggesting a change from the apparent over-the-barrier process in neat water to a tunnelling regime on involving hydrophobic interfaces in the water solvent system. The observed phenomena are ascribed, invoking a Marcus-like tunnelling model that includes dynamical aspects of system and surroundings, to hydrogen tunnelling in reaction dynamically connected with femtosecond motions of water OH, where presence of hydrophobic interfaces causes changes in water dynamics coupled with reaction coordinate of the PCET process through intermolecular vibrational coupling mechanism of water.

Olive oil phenolic compounds, particularly tyrosol (Tyr), hydroxytyrosol (HO-Tyr), oleuropein (Ole), although being minor components, are of great importance, taking into account a broad spectra of their biomedicinal activity antimicrobial, antioxidant and anti-inflammatory features. We determined Tyr, HO-Tyr, Ole in extra virgin olive oils (EVOO) obtained from three native Croatian olive cultivar, Bjelica, Buža and Črnica.The study comprises the comparision of the Croatian olive oil phenolic compound content with content in literature, in olive oils from Italy, Spain, Turkey, Greece and Tunisia. The oil was obtained using a small extracting system. The phenolic compounds extraction and HPLC analysis were performed applying in essence the procedure of Montedoro et al.. The content of Tyr, HO-Tyr and Ole was reported here as mean value from all analyzed samples, and also the highest value in the individual sample. In the course of this preliminary study, we found that Croatian EVOOs from three investigated cultivar are particularly rich in Tyr, in comparison with EVOOs from other countries, Italy, Spain, Greece, Turkey and Tunisia. Among the Croatian cultivar, the olive oil from cultivar Bjelica is most abundant with Tyr, with mean value 35.3 mg/kg, and the highest value 140 mg/kg. According to the literature data, the Tyr content in the EVOO is always below 65 mg/kg. The olive oil from cultivar Bjelica is also the richest in HO-Tyr, with mean value 5.24 mg/kg, and the highest value 14.8 mg/kg. The olive oil from cultivar Buža is the richest in Ole, with the highest value of 21.0 mg/kg. The content of HO-Tyr and oleuropein determined in Croatian EVOOs are generally similar or even smaller in comparison with the literature data. This result could be promising for the use pharmaceutical quality olive oil, especially rich in Tyr, taking into regard the well known importance of this natural phenolic compound.

Vitamin C, more precisly ascorbate monoanion, is well known antioxidant and cofactor in large number of enzyme reactions in human body. Some ascorbate redox reactions, for example reactions with N-containing toxins, nitrosobenzene and TEMPO radical ; and metal complexes, hexacyannoferrate(III) ion and ferricinium ion, are PCET reactions, reactions based on concerted electron and proton transfer, that avoided highly reactive intermediates, which are present in case of stepwise electron/proton transfer reactions. Some other important antioxidants, as tocopherol, ubiqinon, uses PCET mechanism, as well. Generally, PCET reactions are of essential importance in many biological systems, and enzyme reactions [1, 2]. Figure 1. A graphical presentation of a PCET (Proton Coupled Electron Transfer) reaction of ascorbate and a reactant R (reactants R are listed in a Table 1). The evidence for PCET reaction mechanism usually comes from thermochemical analysis and kinetic isotope effects, determined to be 3.10, 24.2, 4.60, 1.91 for the reaction between ascorbate and nitrosobenzene, TEMPO radical, hexacyannoferrate(III) ion and ferricinium ion, respectively [3]. In these ascorbate reactions, an important role has quantum-mechanical phenomenon of hydrogen tunnelling, as seen from KIE and isotopic activation parameters, H/D ratio of Arrhenius preexponential factors AH/AD and ΔEa(D-H). AH/AD determined in the reaction between ascorbate and hexacyannoferrate(III) ion are 0.07 and 2.30 in 0.25:0.75 dioxane-water solvent mixture and in 0.5 M aqueous NaCl solution, respectively ; and in the reaction between ascorbate and TEMPO radical, in MeCN-water 0.50:0.50 solvent mixture, in presence of 0.5 M tetraethylammonium chloride AH/AD 0.22. The observed values are typical evidence indicating H tunnelling. The results are explained within quantum-mechanical model for H tunnelling (Marcus like tunnelling model) [4], which concerns dynamical role of activated complex and it`s environment in the process. The obtained results are of importance for better understanding the reactivity of ascorbate, and the role of ascorbate in biological and biochemical systems. Table 1. Experimental data for the reactions of ascorbate with reactant R [3, 5-7] ; a - in water ; b - in 50:50 v/v MeCN-water solvent mixture, in presence of 0.5 M tetraethylammonium chloride ; c - 25:75 v/v dioxane-water solvent mixture ; d - 0.5 M aqueous NaCl solution References: 1. Weinberg, D. R. ; Gagliardi, C. J. ; Hull, J. F. ; Fecenko Murphy, C. ; Kent, C. A. ; Westlake, B. C. ; Paul, A. ; Ess, D. H. ; Granville McCafferty D. ; Meyer, T. J. Proton-Coupled Electron Transfer, Chem. Rev., 2012, 112, 4016−4093. 2. Reece, S. Y. ; Nocera, D. G. Proton-coupled electron transfer in biology: results from synergistic studies in natural and model systems, Annu. Rev. Biochem., 2009, 78, 673-99. 3. Sajenko, I. ; Pilepić, V. ; Jakobušić Brala, C. ; Uršić, S. Solvent Dependence of the Kinetic Isotope Effect in the Reaction of Ascorbate with the 2, 2, 6, 6-Tetramethylpiperidine-1-oxyl Radical: Tunnelling in a Small Molecule Reaction. J. Phys. Chem. A, 2010, 114, 3423-3430. 4. Knapp, M. J. ; Rickert, K. ; Klinman J.P. Temperature-dependent isotope effects in soybean lipoxygenase-1: Correlating hydrogen tunneling with protein dynamics, J. Am. Chem. Soc., 2002, 124, 3865-3874. 5. Vuina, D. ; Pilepić, V. ; Ljubas, D. ; Sanković, K. ; Sajenko, I. ; Uršić, S. PCET in the oxidation of ascorbate. Dramatic change of the kinetic isotope effect on the change in solvent polarity, Tetrahedron Lett., 2007, 48, 3633–3637. 6. Jakobušić Brala, C. ; Pilepić, V. ; Sajenko, I. ; Karković, A. ; Uršić, S. Ions Can Move a Proton-Coupled Electron-Transfer Reaction into Tunnelling Regime, Helv. Chim. Acta, 2011, 94, 1718 – 1731. 7. Jakobušić Brala, C. ; Karković, A. ; Klepac, K. ; . Vučinović, A. M. ; Pilepić, V. ; Uršić, S. Small Molecule Tunnelling Systems. Variation of Isotope Effects, Z. Phys. Chem., 2011, 225, 821-841.

Cilj ovog rada bio je određivanje biomedicinski važnih fenolnih spojeva u maslinovom ulju i plodu masline kultivara Oblica. Nakon ekstrakcije, provedena je HPLC DAD analiza uzoraka na valnoj duljini od 278 nm te su utvrđene koncentracije fenolnih spojeva u ulju masline: 20,6 mg/kg ulja hidroksitirozola, 8,36 mg/kg ulja tirozola i 304 mg/kg ulja oleaceina, U plodu masline određene su koncentracije 215 mg/kg ploda hidroksitirozola, 61 mg/kg ploda tirozola i 272 mg/kg ploda oleuropeina. Prisutnost ovih polifenola u ulju i plodu masline ukazuje na prednosti mediteranske prehrane za ljudsko zdravlje i nudi potencijalna farmakološka rješenja u smjeru antioksidativnog, protuupalnog i antitumorskog učinka The aim of this work was quantitative analysis of biomedically import ant phenolic compounds in olive oil and fruit in Croatian cultivar Oblica. Aft er extraction, HPLC DAD analysis was carried out with the apsorption of eluates detected on 278 nm with following results: 20,6 mg hydroxytyrosol, 8,36 mg tyrosol an d 304 oleacein per kg of oil and 215 mg hydroxytyrosol, 60,9 mg tyrosol and 272 mg of oleuropein per kg of fruit. Presence of these polyphenols with various biological properties emphasizes the advantages of Mediterranean diet. In addition, it offers new pharmacological possibilities due to its antioxidative, anti inflammatory and anticancer effects.