Effects of enrichment of refined olive oil with phenolic compounds from olive leaves

The possibility of preparing olive oil, with the same nutritional value and stability characteristics found in virgin olive oil, by the enrichment of refined olive oil with olive leaf polyphenols was studied. To obtain antioxidant phenols similar to those found in virgin olive oil, these components were extracted from the leaves of several olive cultivars from the Northern region of Portugal, namely, Carrasca, Ripa, Negruche, Cordovil, Verdeal, Madural, and Bical cultivars, under several conditions. The concentration of a leaf extract required for addition to refined olive oil to obtain the same stability as virgin olive oil was determined. The extract from 1 kg of leaves was sufficient to fortify 50-320 L of refined olive oil to a similar stability as a virgin olive oil sample depending on the metal concentration of the oil, cultivar, and time of the year when the leaves were picked.

Emerging technologies for recovery of value-added components from olive leaves and their applications in food/feed industries

Olive leaves are the most abundant agricultural waste source rich in polyphenolics. Due to the numerous health benefits associated with these compounds, the interest in recovering polyphenols from olive leaves has increased in the scientific community over the last decade. Recent studies have focused on improved extraction techniques and processing methods that are most suited for agro-biological industries involved in the development of nutraceutical and functional products. The major problems in olive leaves processing include bitter taste and the low stability of various phenolic compounds. Oleuropein and hydroxytyrosol are the most important phenolic compounds extracted from olive leaves. The present review highlights the importance of olive leaves, their composition, preparation methods, major phenolic compounds, and commercial applications. This review article focuses on integrating studies on olive leaf extract (OLE) pertinent to nutrition, health, and beauty. The different board categories of delivery systems available for the encapsulation of OLE are given. These novel delivery systems could improve fortification, supplementation, and dietary diversification in food and pharmaceutical products.

Olive leaf phenolics and cardiovascular risk reduction: Physiological effects and mechanisms of action

Olive oil, an important component of the Mediterranean diet, is rich in polyphenols and is known to possess positive health effects relative to other dietary fats. In addition, the leaves of the olive plant (Olea europaea) contain similar phenolics (oleuropein, luteolin-7-glucoside, apigenin-7-glucoside, verbascoside and hydroxytyrosol) to those of olives and olive oil, although at higher concentrations. For example, the most abundant is the secoiridoid, oleuropein, representing 1–14% of olive leaf weight vs. 0.005–0.12% in olive oil. Although currently considered a waste product of the olive oil industry, recent research has suggested beneficial effects of phenolic-rich olive leaf extracts (OLE) in modifying cardiovascular risk biomarkers such as blood pressure, hyperglycaemia, oxidative stress and inflammation, as well as improving vascular function and lipid profiles. Despite this, data regarding the biological actions of OLE has mostly derived from animal, in vitro and ex vivo studies, with limited evidence deriving from human trials. Although the absorption and metabolism of olive oil phenolics has been investigated, less is known about the bioavailability of phenolics from OLE, limiting the interpretation of existing in vitro and ex vivo data. The current review will begin by describing the phenolic composition of olive leaves in comparison with that of the better studied olive oil. It will then review the effects of OLE on cardiovascular risk factors, covering both animal and human studies and will end by considering potential mechanisms of action

Effects of land management on different forms of soil carbon in olive groves in Mediterranean areas

This study analyses soil organic carbon (SOC) and hot-water extractable carbon (HWC), both measures of soil quality, under different land management: (1) conventional tillage (CT); (2) CT plus the addition of oil mill waste alperujo (A); (3) CT plus the addition of oil mill waste olive leaves (L); (4) no tillage with chipped pruned branches (NT1); and (5) no tillage with chipped pruned branches and weeds (NT2); in a typical Mediterranean agricultural area; the olive groves of Andalucía, southern Spain. SOC values in CT, A, NT1 and NT2 decreased with depth, but in NT2 the surface horizon (0-5 cm) had higher values than the other treatments, 47% more than the average values in the other three soils. In L, SOC also decreased with depth, although there was an increase of 88.5% from the first (0-10 cm) to the second horizon (10-16 cm). Total SOC stock values were very similar under A (101.9 Mg ha−1), CT (101.7 Mg ha−1), NT1 (105.8 Mg ha−1) and NT2 (111.3 Mg ha−1, if we consider the same depth of the others). However, SOC under L was significantly higher (p < 0.05) at 250.2 Mg ha−1. HWC decreased with depth in A, CT and NT1. NT2 and L followed the same pattern as the other management types but with a higher value in the surface horizon (2.3 and 4.9 mg g−1 respectively). Overall, our results indicate that application of oil mill waste olive leaves under CT (L) is a good management practice to improve SOC and reduce waste.

Soil quality assessment based on carbon stratification index in different olive grove management practices in Mediterranean areas

In Mediterranean areas, conventional tillage increases soil organic matter losses, reduces soil quality, and contributes to climate change due to increased CO2 emissions. CO2 sequestration rates in soil may be enhanced by appropriate agricultural soil management and increasing soil organic matter content. This study analyzes the stratification ratio (SR) index of soil organic carbon (SOC), nitrogen (N) and C:N ratio under different management practices in an olive grove (OG) in Mediterranean areas (Andalusia, southern Spain). Management practices considered in this study are conventional tillage (CT) and no tillage (NT). In the first case, CT treatments included addition of alperujo (A) and olive leaves (L). A control plot with no addition of olive mill waste was considered (CP). In the second case, NT treatments included addition of chipped pruned branches (NT1) and chipped pruned branches and weeds (NT2). The SRs of SOC increased with depth for all treatments. The SR of SOC was always higher in NT compared to CT treatments, with the highest SR of SOC observed under NT2. The SR of N increased with depth in all cases, ranging between 0.89 (L-SR1) and 39.11 (L-SR3 and L-SR4).The SR of C:N ratio was characterized by low values, ranging from 0.08 (L-SR3) to 1.58 (NT1-SR2) and generally showing higher values in SR1 and SR2 compared to those obtained in SR3 and SR4. This study has evaluated several limitations to the SR index such as the fact that it is descriptive but does not analyze the behavior of the variable over time. In addition, basing the assessment of soil quality on a single variable could lead to an oversimplification of the assessment. Some of these limitations were experienced in the assessment of L, where SR1 of SOC was the lowest of the studied soils. In this case, the higher content in the second depth interval compared to the first was caused by the intrinsic characteristics of this soil's formation process rather than by degradation. Despite the limitations obtained SRs demonstrate that NT with the addition of organic material improves soil quality.

Atividade antioxidante in vitro de extrato de folhas de oliveira (Olea europaea L.) e seu efeito protetor sobre danos oxidativos em eritrócitos humanos

O objetivo do presente estudo foi avaliar a atividade antioxidante de extrato de folhas de oliveira (EFO) (Olea europaea L.) por diferentes metodologias analíticas in vitro e in situ, para verificação de efeito em sistemas biológicos. O extrato foi obtido a partir de folhas secas de oliveira, previamente micronizadas, em metanol/água (80/20%) na proporção 1:20 (m/v), após remoção de compostos solúveis em n-hexano. Após liofilização, no EFO foi avaliado o poder redutor por Folin-Ciocalteau, conteúdo de flavonoides totais, teor de oleuropeina, poder de redução do íon férrico (FRAP) e atividade antioxidante sobre DPPHo, ABTSo+, ânion superóxido (O2o-), ácido hipocloroso (HOCl) e óxido nítrico (NOo). O extrato foi também avaliado quanto ao efeito protetor sobre danos oxidativos em eritrócitos humanos. O ácido ascórbico foi utilizado como referência. O experimento foi repetido seis vezes (n = 6) e os ensaios realizados em duplicata. O poder redutor do extrato e o conteúdo de flavonoides totais e oleuropeína foram 131,7 ± 9,4 mg equivalente de ácido gálico/g extrato seco (ms), 19,4 ± 1,3 mg equivalente de quercetina/g ms e 25,5 ± 5,2 mg oleuropeína/g ms, respectivamente. O ensaio de FRAP apresentou 281,8 ± 22,8 mg equivalente de trolox/g ms. O EFO foi efetivo na inibição dos radicais DPPHo e ABTSo+, dependente da concentração de extrato, com valores de IC50 de 13,8 ± 0,8 e 16,1 ± 1,2 µg/mL, respectivamente. Com relação à atividade antioxidante sobre espécies reativas de importância biológica, o EFO apresentou forte capacidade de inibição de O2o- (IC50 = 52,6 ± 2,1 µg/mL) e NOo (IC50 = 48,4 ± 6,8 µg/mL), quando comparado ao ácido ascórbico. Porém, a inibição de HOCl não foi tão eficiente (IC50 = 714,1 ± 31,4 µg/mL). O EFO inibiu a hemólise induzida em eritrócitos de maneira dependente da concentração (IC50 = 7,8 ± 1,1 µg/mL), assim como a peroxidação lipídica e a formação de meta-hemoglobina, com valores de IC50 de 38,0 ± 11,7 e 186,3 ± 29,7 µg/mL, respectivamente. Os resultados obtidos neste estudo sugerem que extrato de folhas de oliveira possui efetiva atividade antioxidante em sistemas biológicos, pelo efeito sequestrador de determinadas espécies reativas que participam dos processos bioquímicos, e pela prevenção de danos oxidativos em eritrócitos humanos. Portanto, sua ingestão pode estar relacionada com a prevenção de estresse oxidativo in vivo, com consequentes benefícios à saúde.

Extração de oleuropeína de folhas de oliva com solvente hidroalcoólico e efeito dos extratos sobre a estabilidade oxidativa de óleos vegetais

A oleuropeína é o composto fenólico mais abundante presente nas folhas da oliveira, sendo que muitos estudos vêm demonstrando que este composto apresenta importantes propriedades antimicrobiana, antioxidante, anti-inflamatória, entre outras, surgindo o interesse em estudos de métodos para sua extração e aplicação em produtos na área alimentícia, cosmética e farmacêutica. O objetivo deste estudo foi a extração da oleuropeína à partir de folhas de oliva, utilizando solvente não tóxico, para posterior aplicação dos extratos em óleos vegetais a fim de se verificar seu efeito sobre a estabilidade oxidativa dos mesmos. O solvente selecionado para o estudo foi uma mistura de etanol e água (70:30, em massa, condição obtida através de um trabalho prévio), na presença de 1 % de ácido acético. Em uma primeira etapa, foram realizados experimentos de extração utilizando-se as técnicas de maceração (tipo I) e ultrassom (tipo II), em diferentes condições de temperatura (20, 30, 40, 50 e 60°C). Em uma segunda etapa, através de experimentos com maceração à temperatura ambiente, estudou-se o efeito da razão folhas:solvente (1:8, 1:6 e 1:3) e a influência da presença de ácido acético sobre o processo de extração (tipo III). Por fim, realizando-se a maceração na presença de ácido acético, temperatura ambiente e proporção folhas: solvente igual a 1:3, realizaram-se extrações sequenciadas a partir de uma mesma matéria-prima (tipo IV). Os resultados desses experimentos foram expressos em rendimento de oleuropeína (RO), teor de oleuropeína nos extratos (TO) e rendimento global (RG). Analisando-se os experimentos I e II, verificou-se que a temperatura não exerceu influência significativa sobre as respostas RO, TO e RG. Além disso, verificou-se que os valores das respostas para os experimentos com a maceração foram um pouco maiores do que os valores obtidos para as extrações com o auxílio do ultrassom. Nos experimentos tipo III, em linhas gerais, observou-se a influência positiva da presença do ácido acético sobre as respostas estudadas. Verificou-se também que, na presença de ácido, o aumento da quantidade de solvente na extração conduz ao aumento de RO e RG, e à diminuição de TO. Através do experimento tipo IV, constatou-se que mesmo após quatro extrações sequenciadas, ainda não foi possível esgotar a oleuropeína da matéria-prima. Após a obtenção de todos os extratos hidroalcoólicos, selecionou-se um contendo aproximadamente 19 % de oleuropeína para o estudo da estabilidade oxidativa em óleos vegetais (oliva e girassol) utilizando o método Rancimat. A presença de extrato aumentou em 3 horas o tempo de indução do azeite de oliva extra-virgem, e em 2 horas o tempo de indução do azeite de oliva comum. Os óleos de girassol bruto e refinado não apresentaram melhora na estabilidade oxidativa quando adicionados dos extratos. Foram realizados também testes de estabilidade oxidativa através da adição direta de folhas de oliva em pó nos azeites de oliva extra-virgem e comum. Para o azeite extra-virgem, a adição das folhas não proporcionou melhora da estabilidade oxidativa, porém para o azeite comum, houve um aumento de mais de 2 horas no tempo de indução.Os resultados apresentados neste trabalho demonstraram que é possível obter extratos contendo teores significativos de oleuropeína utilizando-se um solvente renovável. Além disso, constatou-se que os mesmos podem ser utilizados como um antioxidante natural em azeite de oliva, melhorando sua estabilidade oxidativa.

Tutankhamun's Embalming Cache (KV 54), excavated artifact, Floral Collar

Valley of the Kings (burial site), Egypt; 1 ft. 5 21/64 in.x 49 ft. 4 33/64 in.; papyrus, olive leaves, cornflowers, linen

Tutankhamun's Embalming Cache (KV 54), excavated artifact, Floral Collar: detail

Valley of the Kings (burial site), Egypt; papyrus, olive leaves, cornflowers, linen

Tutankhamun's Embalming Cache (KV 54), excavated artifact, Floral Collar: detail

Valley of the Kings (burial site), Egypt; papyrus, olive leaves, cornflowers, linen

Tutankhamun's Embalming Cache (KV 54), excavated artifact, Floral Collar: back

Valley of the Kings (burial site), Egypt; 1 ft. 5 21/64 in.x 49 ft. 4 33/64 in.; papyrus, olive leaves, cornflowers, linen

Simulation of oleuropein structural conformation in vacuum, water and triolein-water systems using molecular dynamics

Oleuropein, the main phenolic compound of olive leaves, exhibits a unique blend of biological activities and has been shown to locate itself at the oil-water (O/W) interface. This behavior could influence the physico-chemical properties of dispersed systems such as emulsions. In this work, we study the effect of the microenvironment (vacuum, water, and triolein-water) on the conformational preferences of oleuropein using molecular dynamics (MD) simulations at 300K for at least 30ns. The seven torsions that describe the flexible skeleton of oleuropein were monitored together with the distance between the glucose (Glu) and hydroxytyrosol (Hyd) moieties (dglu-hyd) of the molecule. The obtained trajectories demonstrated that oleuropein adopts different conformations that depend on the environment. The preferential conformers in each system were analyzed for their molecular geometry and internal energy. In vacuum, the oleuropein preferential conformation is tight with the glucose moiety in close proximity with the hydroxytyrosol moiety. In water, oleuropein preferential conformers presented large differences in their structural properties, varying from a close like U form, and a semi-opened form, to an opened form characterized by high fluctuations in dglu-hyd values. In a triolein-water system, oleuropein tends to adopt a more open form where the glucose moiety could be approximately aligned with the hydroxytyrosol and elenolic acid moieties. Based on a calculation at the HF/6-31G* level, these flexibilities of oleuropein required energy of 19.14kcal/mol in order to adopt the conformation between water and triolein-water system. A radial distribution function (RDF) analysis showed that specific hydroxyl groups of Hyd and Glu interact with water molecules, enabling us to understand the amphiphilic character of oleuropein at the triolein-water interface. MD calculations together with interfacial tension measurements revealed that the oleuropein binding at O/W interface is an enthalpy driven mechanism.

SHORT-TERM EFFECTS OF SALINITY ON SOME PHYSIOLOGICAL PARAMETERS OF YOUNG OLIVE TREES OF ARBEQUINA, COBRANÇOSA AND GALEGA VARIETIES

SHORT-TERM EFFECTS OF SALINITY ON SOME PHYSIOLOGICAL PARAMETERS OF YOUNG OLIVE TREES OF ARBEQUINA, COBRANÇOSA AND GALEGA VARIETIES Ana Elisa Rato1,4, Renato Coelho1, Margarida Vaz1, Teresa Carola2, Dália Barbosa2, Nádia Silva1, José dos Santos2, Lourenço Machado2, João Godinho2, Luzia Ruas2, Margarida Barradas2, Hernani Pereira2, Sara Porfírio4 1 ICAAM, Universidade de Évora, Apartado 94, 7002-554 Évora, Portugal 2 Master students, Universidade de Évora, Apartado 94, 7002-554 Évora, Portugal 3 Ph.D. student, Universidade de Évora, Apartado 94, 7002-554 Évora, Portugal 4 aerato@uevora.pt Due to the desertification in some regions, the interest in plant’s tolerance to salinity has been increasing, as this response is determining for plant survival in stress conditions. This work reports the investigation of tolerance to salt in two year-old olive trees (Olea europaea L.) of three varieties, Arbequina, Cobrançosa and Galega vulgar. Plants were grown in 10 L plastic pots containing approximately 9 Kg of a sandy granitic soil, on a greenhouse. For 3 months (from the beginning of February to the end of April 2012), they were subjected to three levels of salinity in the irrigation water, 0 mM, 80 mM and 200 mM NaCl (6 plants per salinity level in a total of 18 plants of each variety),. Stomatal conductance (gs) and relative leaf chlorophyll content were assessed on each plant in February, March and April. Mid-day leaf water potential () and soil salinity were measured at the end of the experiment (April). On average, concerning all treatments and dates of determination, stomatal conductance of Arbequina and Galega vulgar was quite similar, around 40 mmol m-2 s-1, but Cobrançosa had a value of gs 36% higher, almost 50% higher (61 mmol m-2 s-1) when compared with the controls (0 mM salt) of the other two varieties. In percentage of controls, there was little difference in gs between varieties and between salinities during February and March. In contrast, in April, after about 90 days of exposure to salt, there was a clear decrease in gs with salt irrigation, proportional to salt concentration. Compared with controls, plants irrigated with 200 mM salt showed around 80% (Arbequina) or 85% (Cobrançosa and Galega vulgar) decrease in gs. Chlorophyll content of leaves showed less than 5% difference between varieties on the average of all treatments and dates of determination. During the course of this experiment, the salinity levels used did not show any relevant effect on chlorophyll content. Overall, at the end of the experimental period (April), leaf water potential () at midday was significantly higher in Cobrançosa (-1,4 MPa) than in Galega vulgar (-1,7 MPa) or Arbequina (-1,8 MPa), and salt decreased  of control plants (-1,25 MPa) by an average 30% (with 80 mM) and 65% (with 200 mM). At the end of the experiment, salinity in the soil irrigated with 0 mM, 80 mM or 200 mM NaCl was, on average of all varieties, 0,2 mS, 1,0 mS or 2,0 mS, respectively. Soil salinity was quite similar in Arbequina and Galega vulgar but about 35% lower in the pots of Cobrançosa, on average of all salt-irrigation levels. Plants of Cobrançosa had higher stomatal conductance, however they showed higher water potential and lower salinity in the soil. These apparently contradictory results seem to suggest that Cobrançosa responds to salt differently from the other two varieties. This issue needs further investigation.

Salinity-induced effects on transpiration rate, stomatal conductance and leaf area of three olive (Olea europaea L.) varieties

Transpiration of two year-old olive trees of three different varieties, Arbequina, Cobrançosa and Galega (18 trees per variety), irrigated with three levels of salt (0, 80 or 200 mM NaCl) for about 90 days, was measured by a gravimetric method. To determine leaf area, each tree was photographed from the side against a white background and the total area of each projected image was determined with ImageJ software. To calibrate these area determinations, one tree of each variety was subsequently stripped of all its leaves and its total leaf area was accurately measured. A correlation was then obtained between the area on the photograph of this particular tree and the total area of the detached leaves of the same tree. Using the leaf area determined by this procedure, transpiration rates of the trees could be calculated. Knowing leaf and air temperatures and RH, it was possible to determine the difference in molar fraction of water between the leaf and the air. Using this and the values of the transpiration rate, stomatal conductance could be calculated (gs calc) and compared with the conductance measured on the same trees with a porometer (gs). Actual leaf area of a plant was 1,40 (Arbequina), 1,42 (Cobrançosa) or 1,24 (Galega) times the area measured with ImageJ on the photograph of the same plant. Leaf area of the trees, on average of all salt irrigations, was significantly higher on Arbequina (0,187 m2) then on the other two varieties (0,138 m2 or 0,148 m2, for Cobrançosa or Galega, respectively), but did not differ significantly in percentage of controls (0 salt). On average of all three varieties, leaf area was also higher on plants irrigated without salt (0,181 m2) than on plants exposed to 80 or 200 mM NaCl (0,152 m2 or 0,140 m2, respectively), which did not differ between them. The same significant difference was observed when leaf area was expressed as percentage of controls. Transpiration rate was significantly higher on Cobrançosa (1,17 mmol m-2 s-1), on average of all treatments, but there were no significant differences between Arbequina (1,08 mmol m-2 s-1) and Galega (0,82 mmol m-2 s-1). In percentage of controls, there were no significant differences between varieties. Salt reduced significantly the transpiration rate in all varieties, both the actual and percentual values, to about 50% or 30% of controls when exposed to 80 mM or 200 mM NaCl, respectively. Stomatal conductance (gs), assessed by porometry, was significantly higher in control plants, mainly in Cobrançosa (102 mmol m-2 s-1), then in Arbequina (77 mmol m-2 s-1) and the lower values were found in Galega (51 mmol m-2 s-1). Salt reduced gs, on average of the three varieties to 30% or 10% of controls on exposure to 80 mM or 200 mM NaCl, respectively. Calculated (gs calc) and measured (gs) values of stomatal conductance showed a close relation between them (0,967, R2 = 0,837) which indicates this non-destructive method to determine whole-plant leaf area to be reasonably accurate.