Estudio de la estabilidad oxidativa de aceites funcionalizados con derivados de hidroxitirosol

Las materias grasas sufren procesos de autooxidación que conducen a la formación de productos volátiles que les confieren olor y sabor desagradable, haciéndolas inaceptables para el consumo humano. Para evitarlo, se utilizan aditivos que retrasan este proceso, con la consiguiente prolongación de la vida útil del producto. Sin embargo la gama de productos con carácter de aditivo antioxidante para alimentos es reducida pues, aparte de los extractos vegetales, la mayoría son productos fenólicos sintéticos, excepto los tocoferoles y el palmitato de ascorbilo que son los que presentan una menor capacidad de protección. Por este motivo la industria alimentaria está requiriendo antioxidantes más potentes, a ser posible de origen natural, que no tengan sabor y que además confieran al alimento propiedades funcionales. El hidroxitirosol (HTy) y una serie de derivados son orto-difenoles con fuerte carácter antioxidante que se encuentran en el aceite de oliva y que poseen numerosas propiedades farmacológicas y nutricionales que dan lugar a que el aceite de oliva virgen sea reconocido como cardiosaludable. Los derivados mayoritarios del HTy en el aceite de oliva tienen marcado sabor amargo y el HTy libre es un producto siruposo de difícil disolución en grasas por lo que no se han utilizado en los alimentos grasos. Sin embargo, ciertos derivados del hidroxilo primario, tales como éteres y ésteres de hidroxitirosilo, han demostrado ser insípidos y tener buena solubilidad en grasas manteniendo un fuerte carácter antioxidante. En la presente Tesis se profundiza en la investigación de las propiedades antioxidantes de los alquil éteres (AEHTy), ésteres grasos (EGHTy) y acetato (AcHTy) de hidroxitirosilo y sus interacciones con el α-tocoferol (α-T), antioxidante presente en la mayoría de los aceites vegetales, para que puedan ser utilizados como aditivos liposolubles en alimentos grasos. Así mismo, se estudia un procedimiento de obtención de EGHTy y se optimizan métodos analíticos para la determinación de todos estos derivados en matrices oleosas. Los AEHTy son sustancias obtenidas por reacción de yoduros de alquilo con el HTy con los OH fenólicos protegidos, y que han mostrado tener interesantes propiedades farmacológicas y nutricionales ya que después de la ingesta no se hidrolizan en el tracto intestinal, manteniendo su carácter lipofílico hasta que se forman los glucurónidos o sulfatos. Mediante la oxidación acelerada Rancimat® se ha investigado la actividad antioxidante de los AEHTy en matrices oleosas de diferente grado de insaturación, así como el efecto de la presencia de α-T. Utilizando el sistema de oxidación a baja temperatura open cup, se ha investigado también el efecto de la insaturación de la matriz, siguiéndose la evolución de los parámetros de calidad y de las concentraciones de α-T y AEHTy. Las capacidades antioxidantes fueron ligeramente mayores que las de los ésteres homólogos. El método más conveniente para la obtención de EGHTy es por transesterificación de ésteres alquílicos de ácidos grasos con HTy libre. Debido a que para disponer de los AEHTy y EGHTy es necesario proceder a una síntesis seguida de una purificación del producto de la reacción, en esta Tesis se ha investigado la síntesis de los EGHTy por reacción del HTy libre con los triacilgliceroles que componen los aceites, en un proceso simple y económico que puede ser aplicado fácilmente por la industria refinadora de aceites y que no requiere el aislamiento y la purificación del producto. Como producto de la reacción se obtiene un aceite que contiene una alta concentración de una mezcla de EGHTy, y que puede ser usado directamente como aditivo antioxidante para alimentos grasos. La reacción se ha desarrollado en aceites con muy diversa composición en ácidos grasos y se han estudiado las variables que intervienen en el proceso, tales como relación HTy/aceite, cantidad de enzima, soporte sólido para la dispersión del HTy, temperatura y tiempo de reacción. Los aceites resultantes se han caracterizado por la composición en ácidos grasos, y los perfiles glicerídico y de EGHTy, así como por el contenido total en HTy solubilizado y ésteres. Los EGHTy están presentes en pequeña cantidad en el aceite de oliva virgen y tienen un marcado carácter lipofílico que se pierde en parte tras la ingesta ya que en el tracto intestinal se hidrolizan a HTy libre. Para el estudio de la capacidad antioxidante de los ésteres se han aplicado procesos de oxidación open cup a temperatura ambiente así como la oxidación acelerada a alta temperatura en equipo Rancimat®, utilizándose el acetato de hidroxitirosilo (AcHTy) como representante de esta familia de ésteres. Se ha estudiado su actividad en comparación con la del α-T y la de las mezclas de ambos, para conocer la influencia de este compuesto sobre la capacidad de los ésteres. Como resultado se ha observado que los ésteres tienen una alta capacidad antioxidante y que ejercen un efecto protector sobre el tocoferol al ralentizar la disminución de su concentración con el tiempo. Por otra parte el α-T ejerce un efecto aditivo positivo para bajas concentraciones de AcHTy mientras que el efecto es sustractivo para altas. Finalmente, utilizando aceites con una alta concentración de EGHTy obtenidos in situ se han enriquecido aceites de diverso grado de insaturación a diversas concentraciones, se ha determinado la estabilidad Rancimat y se ha estimado la vida útil de los aceites a temperatura ambiente, mediante la extrapolación de los periodos de inducción Rancimat a temperaturas elevadas. Como conclusión final, se afirma que tanto los éteres como los ésteres de hidroxitirosilo tienen alta capacidad antioxidante, en ventaja con el α-T y los antioxidantes fenólicos sintéticos de uso habitual, por lo que pueden ser usados como aditivos antioxidantes de las materias grasas, aportando carácter funcional al alimento y presentando una actividad que depende de la composición en ácidos grasos de la matriz lipídica y de su contenido de tocoferoles

Antioxidant activity of alkyl hydroxytyrosyl ethers in unsaturated lipids

The antioxidant activity of ethyl and octyl hydroxytyrosyl ethers toward lipids was determined using the Rancimat and open cup methods at high temperatures and 50 °C, respectively. The effect of the un- saturation of the matrix was evaluated using sunflower, soya, and fish refined oils. The antioxidant activities of alkyl hydroxytyrosyl ethers (HTy ethers), hydroxytyrosyl esters, and free hydroxytyrosol are similar, and are much higher than that of !-tocopherol at the same millimolar concentration. The relationship between the induction period and the concentration of the HTy ethers is a sigmoidal curve; an accurate concentration of HTy ethers is necessary to achieve maximum activity, as it increases with the level of matrix unsaturation. The presence of tocopherols in commercial oils affects the antioxidant effect of HTy ethers. Thus, the addition of a low concentration of HTy ethers results in a positive effect, whereas the effect of the addition of high amounts of ethers is slightly less than that of the phenol alone. The addition of HTy ethers to commercial refined oils increases the stability of the oils and preserves tocopherols and polyunsaturated fatty acids from oxidation, enabling the oils to maintain their nutritional properties for longer periods of time.This work was supported by Grants AGL2007-66373 from Ministerio de Educación y Ciencia and P09-AGR-5098 from Junta de Andalucía (Spain).Peer Reviewe

Determination of saturated aliphatic hydrocarbons in vegetable oils

9 Páginas.-- 3 Figuras.-- 3 TablasThe aim of this work is to inform about the development of a simple and reliable off-line method for the determination of saturated hydrocarbons (SH) in vegetable oils. SH can be used as markers for fuel or for mineral oil contamination in edible oils and fats. The method consists of the isolation of the fraction by LC on deactivated silver-silica gel and subsequent on-column GC-FID analysis. This stationary phase was prepared avoiding any kind of activation. The method was developed and validated through the participation in both a proficiency test organized by the Joint Research Centre of the European Commission, and a collaborative trial carried out with trained Spanish laboratories according to the standard ISO 5725. Results showed acceptable repeatability and reproducibility values, and Horrat index, being this protocol in use with satisfactory results ever since. The method’s LOQ is 15 mg·kg–1 and its LOD 5 mg·kg–1, which make it suitable to quantify the 50 mg·kg–1 limit established by the EU, and to detect mineral oil content within the 10–500 mg·kg–1 range. Although other procedures with lower LOD have been developed throughout the years, the use of just regular laboratory equipment such as GC-FID makes the proposed method appropriate for application on a routine basis.The Spanish Ministry of Science and Innovation funded this work (ref. AGL2009-07618). The authors would like to thank Ms. Rosario González Cordones for her assistance in the laboratory.Peer reviewe

Hydroxytyrosyl acetate contributes to the protective effects against oxidative stress of virgin olive oil

The effects of virgin olive oil phenols, hydroxytyrosyl acetate (HTy-Ac) and hydroxytyrosol (HTy), on cell integrity and steady-state values of cellular redox status were assessed in HepG2 cells, as well as their potential protective effects against oxidative stress induced by tert-butyl hydroperoxide (t-BOOH). Direct treatment for 20 h with 0.5-10 μM HTy or HTy-Ac reduced ROS generation and increased glutathione peroxidase activity at the higher concentrations. Furthermore, after t-BOOH exposure, pretreatment with HTy-Ac and HTy for 2 or 20 h counteracted cell viability damage from 1 μM, counterbalanced reduced glutathione levels from 0.5 μM, protected against lipid peroxidation from 0.5 μM, decreased ROS generation from 1 μM as well as antioxidant enzyme activities from 1 μM. All these changes were statistically significant. HTy-Ac presents antioxidative stress protective effects at physiological concentrations similar to or even slightly higher than that of HTy, thus contributing to the protective role of virgin olive oil. © 2011 Elsevier Ltd. All rights reserved.This work was supported by projects AGL2007-64042, AGL2007-66373-C03/ALI and CSD2007-00063 from the Consolider-Ingenio Program of the Spanish Ministry of Science and Innovation (CICYT), a grant (RTA2007-000036-00-00) from the Research National Institute of Food, Agriculture and Technology (INIA), a contract (110105090014) with CSIC-IFAPA.Peer Reviewe

Nutraceutical composition, antioxidant activity and hypocholesterolemic effect of a water-soluble enzymatic extract from rice bran

This study was designed to characterize the nutraceutical composition of a water-soluble enzymatic extract from rice bran (EERB) and to evaluate its antioxidant and hypocholesterolemic activities. The EERB contains broad functional components in water soluble form such as sterols, tocopherols, tocotrienols, γ-oryzanol and peptides. The antioxidant behaviour of EERB specifically against lipid oxidation was measured in an emulsion prepared with fish oil containing fatty acid very susceptible to oxidation (EPA; DHA 30%); the emulsion shows a stable antioxidant activity, specifically preserving the lipid from peroxidation phenomena and inhibiting the generation of thiobarbituric acid-reactive substances (TBARS) and lipid peroxides. Moreover, the hypocholesterolemic activity was evaluated in male Wistar rats through two different hypercholesterolemic diet models: a reduction in total cholesterol levels and an increase in HDL-cholesterol were found in both models. EERB is a new product from rice bran that can control lipid oxidation in emulsions and induce a physiological hypocholesterolemic effect. These findings may facilitate the development and use of new products derived from rice within the functional food field.This research was supported by an MEC (Spanish Ministry for Education and Science) grant CTM2007-60210/TECNO.Peer reviewe

Effect of rice parboiling on the functional properties of an enzymatic extract from rice bran

5 Tablas.-- 1 FiguraWe have studied the influence of the parboiling process on enzymatic extract obtained from rice bran. Two rice bran enzymatic extracts have been obtained; one from non-parboiled (RBEE), the other from parboiled rice (RBEEp), and their chemical composition and antioxidant capacities have been compared. These extracts differ in their main chemical composition; RBEEp has less carbohydrates and more fat content than RBEE. No differences in protein content were found. With regard to bioactive compounds, both extracts are rich in phytosterols, tocopherols, tocotrienols and γ-oryzanol, being γ-oryzanol similar in both extracts. However RBEEp is richer in total tocols and phytosterols and RBEE has the highest content in hydrophilic phenols. Functional properties such as total antioxidant activity revealed that RBEE has higher capacity to scavenger peroxyl radicals than RBEEp. Accordingly, specific antioxidant studies showed that RBEE has greater protective capacity against lipid and protein oxidation than RBEEp. Therefore, we can conclude that RBEE has better bioactive properties than RBEEp. In conclusion, these findings suggest that the parboiling pretreatment of rice modifies the bioactive composition of derived products such as rice bran enzymatic extract, that have been shown to exert bioactive properties with application in the nutraceutical and cosmetic fields.This work was supported by gs1:Ministerio de Ciencia e Innovación, CTM2011-29930-01, and Junta de Andalucía, Proyecto de Excelencia P11-RNM-7887

Acetylation of hydroxytyrosol enhances its transport across differentiated Caco-2 cell monolayers

Hydroxytyrosol and hydroxytyrosyl acetate are two well-known phenolic compounds with antioxidant properties that are present in virgin olive oil. Since the in vivo biological activity of polyphenols is dependent on their intestinal absorption and metabolism, the absorption of hydroxytyrosol and hydroxytyrosyl acetate and the extent to which they are conjugated and metabolised during transfer across intestinal Caco-2/TC7 cell monolayers, was investigated. LC-DAD and LC-MS were used for the quantification and identification of metabolites. Further evidence was obtained by observing metabolite susceptibility to β-glucuronidase treatment and by comparison of products of in vitro conjugation reactions of authentic phenolics with those produced by the CaCo-2 cells. Homovanillyl alcohol was the only conjugate detected as a result of hydroxytyrosol metabolism, and accounted for 20% of the total metabolites detected in the basolateral compartment after 2h of incubation. Hydroxytyrosyl acetate was largely converted into free hydroxytyrosol (38.4%) and subsequently metabolised into homovanillyl alcohol (6.7%). In addition, hydroxytyrosyl acetate glucuronide (17.4%) together with non-metabolised hydroxytyrosyl acetate (37.5%) were also detected. Both hydroxytyrosyl acetate and hydroxytyrosol were transferred across human Caco-2/TC7 cell monolayers, but the acetylated compound exhibited an apparent permeability (PappAP→BL/Papp BL→AP) 2.1-fold higher than free hydroxytyrosol. For both compounds, all conjugates were preferentially transported to the basolateral side. These results show that the acetylation of hydroxytyrosol significantly increases its transport across the small intestinal epithelial cell barrier, and supports further research into hydroxytyrosyl acetate as a hydroxytyrosol prodrug offering enhanced bioavailability. © 2010 Elsevier Ltd.This work was supported by projects AGL2007-64042, AGL2007-66373-C04/ALI and CSD2007-00063 as part of the Consolider-Ingenio Program from the Spanish Ministry of Science and Innovation (CICYT), Grant RTA2007-000036-00-00 from the Research National Institute of Food, Agriculture and Technology (INIA), a contract (110105090014) with CSIC-IFAPA, and a core grant from the Biotechnology and Biological Sciences Research Council (to PAK and SS). G.P.-C. is a predoctoral fellow of the National Research Institute of Food, Agriculture and Technology (INIA).Peer Reviewe

Synthesis and antioxidant evaluation of isochroman-derivatives of hydroxytyrosol: Structure-activity relationship

© 2014 Elsevier Ltd. All rights reserved. Isochroman-derivatives of the natural olive oil phenol hydroxytyrosol (HT) have been synthesised via Oxa-Pictet-Spengler reaction in high yields. Lipophilicity and antioxidant activity were determined to establish the structure-activity relationship of isochromans compared to HT, BHT and α-tocopherol. Antioxidant capacity was tested in two different media: bulk oils, using the Rancimat test, and brain homogenates, by measuring malondialdehyde (MDA) levels as a lipoperoxidation biomarker. In addition, other antioxidant assays (FRAP, ABTS and ORAC) were carried out. Rancimat and MDA results show that antioxidant activity was related with lipophilicity, directly in brain homogenates and inversely in the oils, in agreement with the polar paradox. Free o-diphenolic groups positively determined the activity in the oils, whereas reducing and radical-scavenging activities were related to the number of free hydroxyl moieties. BHT and α-tocopherol showed lower antioxidant activity than isochromans and HT. We conclude that HT-isochromans present significant potential as bioactive compounds.This research was supported by Grant P09-AGR-5098 from Junta de Andalucía (Spain) and AGL2010-18269 and Consolider-Ingenio CSD2007-00063 from Spanish Ministry of Science and innovation.Peer Reviewe

Lipophilic hydroxytyrosyl esters. Antioxidant activity in lipid matrices and biological systems

Antioxidant activities of lipophilic hydroxytyrosyl acetate, palmitate, oleate, and linoleate were compared with those of hydroxytyrosol, α-tocopherol, and butylhydroxytoluene (BHT) in both glyceridic matrix and biological systems. Aliquots of a glyceridic matrix spiked with various concentrations of antioxidant were subjected to accelerated oxidation in a Rancimat apparatus operated at 90 °C. The relationships between induction time (IT) and antioxidant concentration (mmol/kg) presented by hydroxytyrosol and hydroxytyrosyl acetate, palmitate, oleate, and linoleate were similar. Hydroxytyrosol and its esters showed greater antioxidant activity than α-tocopherol or BHT. We also evaluated the capacity of hydroxytyrosyl esters to protect proteins and lipids against oxidation caused by peroxyl radicals, using a brain homogenate as an ex vivo model. All tested compounds showed a protective effect in these systems, which was greater in preventing the generation of carbonyl groups in protein than of malondialdehyde in lipid. Inclusion of a lipophilic chain in the hydroxytyrosol molecule enhanced its antioxidant capacities in this biological model.We thank the Ministerio de Educacion y Ciencia of Spain for financial support (Grant Nos. AGL2004-07935-C03 and REN2002-02101).Peer reviewe