WO2012175771A1 - Phenol extract from heat-treated olive subproducts - Google Patents

Phenol extract from heat-treated olive subproducts Download PDF

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Publication number
WO2012175771A1
WO2012175771A1 PCT/ES2012/070441 ES2012070441W WO2012175771A1 WO 2012175771 A1 WO2012175771 A1 WO 2012175771A1 ES 2012070441 W ES2012070441 W ES 2012070441W WO 2012175771 A1 WO2012175771 A1 WO 2012175771A1
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Prior art keywords
phenolic
extract
extraction
phenolic extract
fraction
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PCT/ES2012/070441
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Spanish (es)
French (fr)
Inventor
Juan FERNÁNDEZ-BOLAÑO GUZMÁN
Guillermo RODRÍGUEZ GUTIÉRREZ
Antonio LAMA MUÑOZ
Fátima SENENT RUBIO
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Consejo Superior De Investigaciones Científicas (Csic)
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Publication of WO2012175771A1 publication Critical patent/WO2012175771A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3472—Compounds of undetermined constitution obtained from animals or plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/63—Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • A61K8/922—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of vegetable origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/522—Antioxidants; Radical scavengers

Definitions

  • the present application is addressed to the food, pharmaceutical, cosmetics and agriculture sector.
  • Olives have a characteristic phenolic composition.
  • the existence and quantity of specific phenols in the olive depend on the variety and state of maturity, climatic, seasonal and geographical conditions. Only a small amount (1-2%) become part of the oil phase during the extraction of olive oil while the rest remains in the residue or alperujo (Rodis, PS, Karathanos, VT and Mantzavinou, A. (2002 ), J. Agrie. Food Chem., 50, 596-601). So far the alperujo has not been properly exploited due in some way to the difficulty involved in its manipulation. Therefore, the extraction of these phenols as high value-added products could be considered as an interesting alternative to make mill waste profitable.
  • the object of the present invention is a new extract from residues or by-products generated from the extraction of olive oil or table olives, once they have undergone a heat treatment.
  • the new extract has a phenolic content different from the extracts that can be obtained from the same by-products without heat treatment, and is characterized by significantly increasing certain phenols and also by the presence of compounds that are formed during said treatment and that contribute to greatly to the improvement of its properties (antioxidants, anti-inflammatory, free radical sequestrant, etc.).
  • the novelty of the application of heat treatments lies in obtaining a phenolic extract with a greater amount of simple and new phenols, favored by heat treatment, while facilitating phase separation and thereby simplifying extraction. From the phenolic extract different fractions rich in different types of phenols can be obtained that provide a wide variety of properties, or even isolate certain compounds of high added value. Detailed description of the invention
  • the application of a thermal treatment substantially favors the subsequent extraction of phenolic compounds and of interest present in them.
  • the treatment not only the extraction is facilitated but also the formation of new and active compounds while hydrolyzing phenols in their most active form.
  • the final result is an extract enriched in bioactive compounds and with a greater antioxidant activity than an extract obtained from a byproduct of olive oil without heat treatment.
  • the by-product mass is treated in a temperature range between 50 ° C and 250 ° C, more preferably between 70 ° C and 200 ° C, by direct or indirect heat input, either through a steam inlet or other inert gas, or through a heating jacket or electrical resistance that is in contact with the material to be treated.
  • the reaction time can vary greatly depending on the type of heating, temperature and design of the thermal reactor, but it can range from two minutes to longer treatments of up to 3 or 5 hours.
  • a solid-liquid separation can be carried out in order to obtain the extract of each phase separately, or the extract of both phases can be obtained without previously separating them.
  • the separation facilitates the extraction in the case of this being carried out with organic solvents and is the way to obtain the concentrated extract of the liquid fraction as the final product rich in phenols.
  • the extraction is carried out at a temperature between 50 ° C and 100 ° C.
  • the process may comprise an additional step that may consist of microencapsulation or nanoencapsulation of the phenolic extract; the absorption or adsorption of the phenolic extract in any type of support; or the formation of an emulsion of the phenolic extract.
  • a food, cosmetic and / or pharmaceutical formulation characterized in that it comprises a phenolic extract as described has been the subject of the invention.
  • phenolic extract as an antioxidant for the preparation of both lipophilic and hydrophilic preparations, which may have food, cosmetic and / or pharmaceutical use.
  • a sample of 20 kg of fresh alperujo was subjected to several heat treatment processes. The conditions they were in this case 160 ° C for 30, 60 and 90 minutes by direct heating with water vapor.
  • the untreated alperujo is subjected to an extraction with 80% ethanol (EtOH) in water. It starts from 2,690 kg of the fresh non-heat treated lupine, which has been previously defatted with hexane.
  • the defatted lupine is brought into contact under gentle agitation with 80% EtOH at room temperature for 30 minutes, using 15 ml of 80% EtOH for every 10 g of lupine, the extraction is repeated twice more, using 10 ml of 80% EtOH for every 10 g of alperujo.
  • the fractions are filtered through a paper filter in Buchner, combined and the EtOH is evaporated until an aqueous extract is obtained.
  • the phenolic compounds are extracted with ethyl acetate, for this purpose a hot and countercurrent extraction with ethyl acetate is carried out for 8 hours, 500 ml of ethyl acetate are used for every 200 mL of sample.
  • EFNT dry untreated phenolic extract
  • Table 1 The amount of phenolic extract obtained from alperu or treated and not heat treated is shown in Table 1, where a significant increase can be seen with the treatment.
  • Three of the extracts treated plus the untreated control have been characterized in terms of their total composition in Table 2.
  • the percentage e in moisture is higher in the case of heat treated extracts, while the content of total and lipid phenols does not seem to vary significantly. Thanks to the heat treatment and subsequent extraction, a new component appears consisting of a mixture of phenolic polymers (FFP), among others, which can have important bioactive properties.
  • FFP phenolic polymers
  • the amount of protein increases with treatments while the amount of sugar decreases.
  • Table 3a and Table 3b a more detailed characterization of the content of phenolic compounds was carried out as detailed in Table 3a and Table 3b.
  • the notable differences in composition as well as the appearance of new phenolic and other sugar degradation compounds make an important difference with the phenolic extracts that can be obtained from untreated by-products. These differences translate into greater activity due to a greater presence of more active and new phenols with important health properties, among others.
  • the new features of the heat treated extracts are detailed below.
  • Phenolic and sugar degradation components present in the different EF extracts at different temperatures and for the EFNT expressed in mg of compound per kilogram of alperuj or fresh.
  • Vanillic acid 3 362 n. d 5, 013 2,089 Phenolic acids
  • Phenolic alcohols 0, 961 26, 406 13, 058 12, 351 total
  • the amount of extract obtained increases with the heat treatment and in the case of the present example increases with the treatment time;
  • 1-Phenyl-6, 7-dihydroisocromone belongs to the hydroxy-isochroman family, which are part of the phenolic nature components from olive oil (Bianco, A., et al., (2001), Food Chem. 77, pp. 405-411, ) . However, they have not been previously described in the alperujo;
  • the EF is rich in a great variety of compounds that can present different activities, all contributing in sum to the final activity of the extract.
  • the fractionation of said extract may be beneficial to enhance certain specific activities of the fractionated components. Therefore, a study has also been carried out on the activity of the heat treated EF fractions. As an example, the fractions in which the extract is divided 160 ° C / 60min are presented and the antioxidant activity tests are carried out on each of them.
  • the composition of the different fractions obtained is shown by way of example in Table 4.
  • Table 5 is an example of the in vitro activity of the antioxidant and free radical sequestrant activity of the new extract and its different fractions, the following tests whose results are compared with the untreated phenolic extract as a control (EFNT), Vitamin E (VE), hydroxytyrosol (HT) and 3,4-dihydroxyphenyl glycol (DHFG), the latter being two of the most active single phenols present in the olive.
  • fractions 1, 2B, 3C, 3D, 3E, 3F, 4C, 4D, 4E, 4F and 4G are those fractions that have a greater capacity to capture DPPH radicals, exceeding the heat treated EF itself. These fractions are rich in DHFG, hydroxytyrosol, l-phenyl-6,7-dihydroisocromone, vanillic acid, 1-acetoxypyrosinol and comsegolide. It should be noted that the 4E fraction is also active, which is the one formed by FFP. The results of antiradical activity are comparable to the results obtained with vitamin E.
  • fractions 2B, 4F and 4G have an ability to capture ABTS radicals of the same order as observed in the case of HT and DHFG, and that together with 2C, 3C, 3E and 4C improve extract activity .
  • Fraction 2B is characterized by having a high percentage of HT.
  • the 4F fraction in addition to hydroxytyrosol contains other species such as vanillic acid or verbascoside that are likely to act synergistically.
  • the 4G fraction is characterized by having a high percentage of comsegoloside.
  • fractions 2C, 3C, 3E, 4C, 4D and 4E have an ABTS radical scavenging capacity similar to that observed by vitamin E, due to the presence of compounds such as protocateic acid, acid vanillic, 1-acetoxyproinoresinol, pinoresinol, verbascoside, comsegolide and FFP.
  • fraction 4C the capacity for inhibition of primary oxidation is even above the values obtained for vitamin E.
  • This fraction is rich in 1-acetoxyproinoresinol.
  • Fractions 1, 2B, 3C, 3D, 3E, 3F, 4D, 4E, 4F and 4G also have an ability to inhibit primary oxidation above the heat treated extract. In fraction 1, 2B and 3C this effect may be due to the high concentrations of HT and DHFG. In the other cases the activity may be due to the same as in the cases prior to the presence of l-phenyl-6,7-dihydroisocromone, protocateic acid, vanillic acid, 1-acetoxypinotesinol, verbascoside, comsegolide and FFP.
  • the high percentage of inhibition shows the new phenolic extract as a possible agent for the prevention of cardiovascular diseases.

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Abstract

The invention relates to a phenol extract obtained from subproducts derived from the extraction of olive oil and/or from the olive preparation industry, which is characterized in that it includes an average composition of total phenol alcohols over 5% in dry weight and an average composition in total phenol acids over 0.3% by dry weight. Likewise, the invention relates to the method for obtaining said extract and to the use thereof as an antioxidant in the preparation of food, cosmetic and/or pharmaceutical preparations, as an agent inhibiting the action of the enzyme L-tyrosinase, or as an agent inhibiting platelet aggregation in the prevention of coronary diseases. Finally, the invention relates to the partially or totally isolated fraction or fractions that can be obtained from said phenol extract.

Description

Extracto fenólico de los subproductos de la aceituna tratados Phenolic extract of the olive by-products treated
térmicamente  thermally
Sector de la técnica  Technical sector
La presente solicitud está dirigida al sector de la alimentación, farmacéutico, de la cosmética y de la agricultura .  The present application is addressed to the food, pharmaceutical, cosmetics and agriculture sector.
Estado de la técnica State of the art
Recientes estudios demuestran que los fenoles son poderosos antioxidantes y poseen otras actividades biológicas que podrían explicar en parte los efectos saludables observados en la dieta Mediterránea. Estos compuestos también juegan un importante papel en la estabilidad, inhibiendo la peroxidación lipídica, y en las propiedades químicas y organolépticas de los productos de las aceitunas (aceite de oliva y aceituna de mesa), y tienen significativos efectos nutricionales , fisiológicos y farmacéuticos sobre la salud humana. Estudios epidemiológicos han asociado la baja incidencia de enfermedades coronarias, ateroesclerosis , y algunos tipos de cáncer (pecho y colon) con el consumo de aceite de oliva en la dieta Mediterránea (López-Miranda, J. et al., (2010), Nutrition, Metabolism & Cardiovascular Diseases, 20, 284-294) . La ingesta habitual de aceite de oliva proporciona un continuo aporte de antioxidantes, que pueden reducir el estrés oxidativo en el cuerpo humano. Numerosos estudios relacionan los efectos beneficiosos del aceite de oliva, con una gran variedad de antioxidantes fenólicos (fenoles simples, secoroideos, flavonoides, lignanos), y su contenido fenólico. Todas estas sustancias son potentes inhibidores del ataque de especies de oxígeno reactivas y en la actualidad hay evidencias que ponen de manifiesto que las especies oxidantes (oxígeno activo, radicales libres, etc.) están implicadas en la etiología de enfermedades tales como el cáncer (Menéndez, J.A. et al., (2007), BMC Cáncer, 7, 80) . Además, incrementan los niveles de disponibilidad de óxido nítrico, suprimen la agregación plaquetaria, disminuyen la presencia de moléculas de adhesión y promueven el contenido fenólico total de las LDL retrasando así su oxidación y, por tanto, la formación y el crecimiento de la placa de ateroma. Recent studies show that phenols are powerful antioxidants and have other biological activities that could partly explain the healthy effects observed in the Mediterranean diet. These compounds also play an important role in stability, inhibiting lipid peroxidation, and in the chemical and organoleptic properties of olive products (olive oil and table olives), and have significant nutritional, physiological and pharmaceutical effects on the Human health. Epidemiological studies have associated the low incidence of coronary heart disease, atherosclerosis, and some types of cancer (breast and colon) with the consumption of olive oil in the Mediterranean diet (López-Miranda, J. et al., (2010), Nutrition , Metabolism & Cardiovascular Diseases, 20, 284-294). The usual intake of olive oil provides a continuous supply of antioxidants, which can reduce oxidative stress in the human body. Numerous studies relate the beneficial effects of olive oil, with a wide variety of phenolic antioxidants (simple phenols, secoroids, flavonoids, lignans), and their phenolic content. All these substances are potent inhibitors of the attack of reactive oxygen species and at present there are evidences that show that the oxidizing species (active oxygen, free radicals, etc.) are involved in the etiology of diseases such as cancer (Menéndez , JA et al., (2007), BMC Cancer, 7, 80). In addition, they increase the levels of nitric oxide availability, suppress platelet aggregation, decrease the presence of adhesion molecules and promote the total phenolic content of LDL thus delaying their oxidation and, therefore, the formation and growth of the plaque. atheroma
Las aceitunas poseen una composición fenólica característica. La existencia y cantidad de fenoles específicos en la aceituna dependen de la variedad y estado de madurez, condiciones climáticas, estacionales y geográficas. Sólo una pequeña cantidad (1-2%) entran a formar parte de la fase oleosa durante la extracción del aceite de oliva mientras que el resto permanece en el residuo o alperujo (Rodis, P.S., Karathanos, V.T. y Mantzavinou, A. (2002), J. Agrie. Food Chem. , 50, 596-601). Hasta el momento el alperujo no ha sido adecuadamente explotado debido en cierta manera a la dificultad que entraña su manipulación. Por lo tanto, la extracción de estos fenoles como productos de alto valor añadido podría ser considerada como una interesante alternativa para hacer provechosos los residuos de almazaras. Se han desarrollado y patentado diversos métodos y sistemas encaminados a la obtención de extractos que contienen compuestos fenólicos desde la aceituna y sus subproductos, así como la purificación de algunos de ellos (hidroxitirosol , tirosol y oleuropeína fundamentalmente) .  Olives have a characteristic phenolic composition. The existence and quantity of specific phenols in the olive depend on the variety and state of maturity, climatic, seasonal and geographical conditions. Only a small amount (1-2%) become part of the oil phase during the extraction of olive oil while the rest remains in the residue or alperujo (Rodis, PS, Karathanos, VT and Mantzavinou, A. (2002 ), J. Agrie. Food Chem., 50, 596-601). So far the alperujo has not been properly exploited due in some way to the difficulty involved in its manipulation. Therefore, the extraction of these phenols as high value-added products could be considered as an interesting alternative to make mill waste profitable. Various methods and systems aimed at obtaining extracts containing phenolic compounds from the olive and its by-products have been developed and patented, as well as the purification of some of them (hydroxytyrosol, tyrosol and oleuropein mainly).
En muchos casos se utilizan técnicas de extracción con solvente o procedimientos de separación y concentración selectiva por ultrafiltración, osmosis inversa, evaporación, separación cromatográfica y extracción de fluidos supercríticos , a partir tanto de las aguas de vegetación separadas del alperujo, como de las aguas de procesado de la aceituna de mesa. Estas técnicas pueden ser usadas individualmente o de modo integrado. Así, después de efectuar una ultrafiltración de las aguas generadas en el procesado de la aceituna de mesa se procedía a efectuar un proceso de adsorción en resina no iónica (tipo XAD) para obtener un extracto rico en hidroxitirosol (ES 2186467) . También se han descrito técnicas de separación por membrana de microfiltración, ultrafiltración, nanofiltración y osmosis inversa, seguido de un proceso de purificación en columna (US 2006/0070953) . In many cases solvent extraction techniques or procedures for separation and selective concentration by ultrafiltration, reverse osmosis, evaporation, chromatographic separation and extraction of supercritical fluids are used, both from the vegetation waters separated from the alperujo, and from the waters of Table olive processing. These techniques can be used individually or in an integrated way. Thus, after ultrafiltration of the water generated in the processing of the table olive, a process of adsorption on non-ionic resin (type XAD) to obtain an extract rich in hydroxytyrosol (ES 2186467). Microfiltration membrane separation, ultrafiltration, nanofiltration and reverse osmosis techniques have also been described, followed by a column purification process (US 2006/0070953).
Otros procedimientos emplean una extracción sólido- liquido sometiendo directamente el alperu o a una extracción con una mezcla de etanol-agua, para posteriormente la fase orgánica ser sometida a una extracción en fluido supercritico (WO 2007/013032) . Por otro lado también se obtienen extractos de hidroxitirosol a partir de las aguas de vegetación de la aceituna mediante fluidos supercriticos , tal como el dióxido de carbono (US 2002/0198415) .  Other procedures employ a solid-liquid extraction by directly subjecting the alperu or to an extraction with an ethanol-water mixture, and then the organic phase is subjected to a supercritical fluid extraction (WO 2007/013032). On the other hand, hydroxytyrosol extracts are also obtained from the vegetation waters of the olive by means of supercritical fluids, such as carbon dioxide (US 2002/0198415).
Uno de los procedimientos más simple, práctico y económico de los descritos es el que permite obtener un hidroxitirosol con alto grado de pureza a partir de cualquier fuente acuosa (alpechín, fase acuosa del proceso hidrotérmico del alperujo, agua de lavado de la elaboración de las aceitunas de mesa, etc.) mediante una cromatografía en dos pasos, una primera con una resina de intercambio iónico y una segunda con una resina adsorbente del tipo XAD (US 2004/0102657) .  One of the most simple, practical and economical procedures described is that which allows a hydroxytyrosol with a high degree of purity to be obtained from any aqueous source (alpechin, aqueous phase of the hydrothermal process of the alperujo, wash water from the preparation of table olives, etc.) by two-step chromatography, a first with an ion exchange resin and a second with an XAD type adsorbent resin (US 2004/0102657).
También se ha descrito la extracción del aglicón de la oleuropeína a partir del alpechín mediante una extracción con hexano : acetona (50/50, v:v) (US 2005/01037111), así como otros procedimientos que permiten obtener extractos polifenólicos mediante extracciones líquido-líquido a partir de productos derivados de la aceituna (concentrado de alpechín o fase acuosa de las aceitunas), utilizando acetato de etilo (ES 2051238) o mezcla de acetato de etilo y etanol o acetato de etilo, etanol y agua (ES 2311401) para posteriormente llevar a cabo tratamientos de la fase orgánica con una cromatografía de alta eficacia en fase reversa en el caso de la primera invención o en una resina del tipo XAD para la segunda, que permiten obtener unos extractos con una alta concentración de hidroxitirosol . It has also been described the extraction of oleuropein aglycon from alpechin by an extraction with hexane: acetone (50/50, v: v) (US 2005/01037111), as well as other procedures that allow obtaining polyphenolic extracts by liquid extractions - liquid from products derived from the olive (concentrate of alpechin or aqueous phase of the olives), using ethyl acetate (ES 2051238) or mixture of ethyl acetate and ethanol or ethyl acetate, ethanol and water (ES 2311401) to subsequently carry out treatments of the organic phase with a high efficiency chromatography in the reverse phase in the case of the first invention or in a resin of the XAD type for the second, they allow to obtain extracts with a high concentration of hydroxytyrosol.
Descripción de la invención Description of the invention
El objeto de la presente invención es un nuevo extracto procedente de los residuos o subproductos generados a partir de la extracción del aceite de oliva o de la aceituna de mesa, una vez éstos han sido sometidos a un tratamiento térmico .  The object of the present invention is a new extract from residues or by-products generated from the extraction of olive oil or table olives, once they have undergone a heat treatment.
El nuevo extracto posee un contenido fenólico distinto a los extractos que se puedan obtener a partir de los mismos subproductos sin tratar térmicamente, y se caracteriza por aumentar significativamente ciertos fenoles y además por la presencia de compuestos que se forman durante dicho tratamiento y que contribuyen en gran medida a la mejora de sus propiedades (antioxidantes, anti-inflamatorias , secuestrante de radicales libres, etc.).  The new extract has a phenolic content different from the extracts that can be obtained from the same by-products without heat treatment, and is characterized by significantly increasing certain phenols and also by the presence of compounds that are formed during said treatment and that contribute to greatly to the improvement of its properties (antioxidants, anti-inflammatory, free radical sequestrant, etc.).
La aplicación de determinados procesos, como es el caso de tratamientos térmicos, ayuda a la solubilización de los fenoles y con ello facilita su posterior extracción. Se han estudiado procedimientos en un alto rango de temperaturas que facilitan sustancialmente no sólo la liberación de los fenoles y otros compuestos interesantes, sino que además, favorecen la hidrólisis en fenoles más simples y la aparición de otros nuevos con un alto valor añadido.  The application of certain processes, such as thermal treatments, helps the solubilization of phenols and thus facilitates their subsequent extraction. Procedures in a high temperature range have been studied that substantially facilitate not only the release of phenols and other interesting compounds, but also favor hydrolysis in simpler phenols and the appearance of new ones with a high added value.
La novedad de la aplicación de tratamientos térmicos radica en la obtención de un extracto fenólico con una mayor cantidad de fenoles simples y nuevos, favorecido por el tratamiento térmico, al mismo tiempo que facilita la separación de fases y con ello se simplifica la extracción. A partir del extracto fenólico se pueden llegar a obtener distintas fracciones ricas en diferentes tipos de fenoles que proporcionan una amplia variedad de propiedades, o incluso aislar determinados compuestos de alto valor añadido. Descripción detallada de la invención The novelty of the application of heat treatments lies in obtaining a phenolic extract with a greater amount of simple and new phenols, favored by heat treatment, while facilitating phase separation and thereby simplifying extraction. From the phenolic extract different fractions rich in different types of phenols can be obtained that provide a wide variety of properties, or even isolate certain compounds of high added value. Detailed description of the invention
Partiendo de cualquier tipo de subproducto del aceite de oliva o de la industria de aderezo, ya sea el caso del alperujo, alpechín, orujo, mezcla de ellos, o salmueras, la aplicación de un tratamiento térmico favorece sustancialmente a la posterior extracción de compuestos fenólicos y de interés presentes en los mismos. Durante el tratamiento no sólo se facilita la extracción sino también la formación de nuevos y activos compuestos al mismo tiempo que se hidrolizan fenoles en su forma más activa. El resultado final es un extracto enriquecido en compuestos bioactivos y con una mayor actividad antioxidante que un extracto obtenido a partir de un subproducto del aceite de oliva sin tratar térmicamente.  Starting from any type of by-product of olive oil or from the dressing industry, be it the case of alperujo, alpechín, orujo, mixture of them, or brines, the application of a thermal treatment substantially favors the subsequent extraction of phenolic compounds and of interest present in them. During the treatment not only the extraction is facilitated but also the formation of new and active compounds while hydrolyzing phenols in their most active form. The final result is an extract enriched in bioactive compounds and with a greater antioxidant activity than an extract obtained from a byproduct of olive oil without heat treatment.
La masa de subproducto se trata en un rango de temperatura de entre 50°C y 250°C, más preferentemente entre 70°C y 200°C, por aporte directo o indirecto de calor, ya sea a través de una entrada de vapor u otro gas inerte, o bien a través de una camisa calefactora o resistencia eléctrica que esté en contacto con el material a tratar. El tiempo de reacción puede ser muy variable dependiendo del tipo de calentamiento, la temperatura y diseño del reactor térmico, pero puede oscilar entre los dos minutos hasta tratamientos más largos de hasta 3 ó 5 horas.  The by-product mass is treated in a temperature range between 50 ° C and 250 ° C, more preferably between 70 ° C and 200 ° C, by direct or indirect heat input, either through a steam inlet or other inert gas, or through a heating jacket or electrical resistance that is in contact with the material to be treated. The reaction time can vary greatly depending on the type of heating, temperature and design of the thermal reactor, but it can range from two minutes to longer treatments of up to 3 or 5 hours.
Tras el tratamiento térmico puede procederse a una separación sólido-líquido con el fin de obtener el extracto de cada fase por separado, o bien se puede proceder a la obtención del extracto de ambas fases sin separarlas previamente. La separación facilita la extracción en el caso de llevarse ésta a cabo con disolventes orgánicos y es la vía para obtener el extracto concentrado de la fracción líquida como producto final rico en fenoles.  After the heat treatment, a solid-liquid separation can be carried out in order to obtain the extract of each phase separately, or the extract of both phases can be obtained without previously separating them. The separation facilitates the extraction in the case of this being carried out with organic solvents and is the way to obtain the concentrated extract of the liquid fraction as the final product rich in phenols.
De manera preferente, la extracción se lleva a cabo a una temperatura comprendida entre 50°C y 100°C.  Preferably, the extraction is carried out at a temperature between 50 ° C and 100 ° C.
Por lo tanto se puede partir de cualquier subproducto de la aceituna, sólido o líquido. En una realización preferente de la invención, el proceso puede comprender una etapa adicional que puede consistir en la microencapsulación o nanoencapsulación del extracto fenólico; la absorción o adsorción del extracto fenólico en cualquier tipo de soporte; o la formación de una emulsión del extracto fenólico. Therefore, it can be derived from any by-product of the olive, solid or liquid. In a preferred embodiment of the invention, the process may comprise an additional step that may consist of microencapsulation or nanoencapsulation of the phenolic extract; the absorption or adsorption of the phenolic extract in any type of support; or the formation of an emulsion of the phenolic extract.
Adicionalmente , es objeto de la invención una formulación alimentaria, cosmética y/o farmaceútica caracterizada por que comprende un extracto fenólico según ha sido descrito.  Additionally, a food, cosmetic and / or pharmaceutical formulation characterized in that it comprises a phenolic extract as described has been the subject of the invention.
Finalmente, es objeto de la invención el uso del extracto fenólico como antioxidante para la preparación de preparados tanto lipófilos como hidrófilos, los cuales pueden presentar uso alimentario, cosmético y/o farmaceútico . Breve descripción de las figuras  Finally, it is an object of the invention to use the phenolic extract as an antioxidant for the preparation of both lipophilic and hydrophilic preparations, which may have food, cosmetic and / or pharmaceutical use. Brief description of the figures
La Figura 1 muestra la inhibición de la agregación plaquetaria de un extracto de alperujo (EA) usando colágeno (a) y TRAP (b) como agentes agonistas o estimulantes de la agregación. La inhibición plaquetaria está expresada en % de disminución de las áreas bajo la curva control de agregación plaquetaria. El número de muestras n=12-13 incubaciones por test. Todos los valores muestran una alta inhibición estadísticamente significativa frente al control (p<0.05) . (-) Valor promedio.  Figure 1 shows the inhibition of platelet aggregation of an extract of alperujo (EA) using collagen (a) and TRAP (b) as agonist or aggregation stimulating agents. Platelet inhibition is expressed in% decrease in areas under the control curve of platelet aggregation. The number of samples n = 12-13 incubations per test. All values show a statistically significant high inhibition against control (p <0.05). (-) Average value.
Ejemplos de la invención Examples of the invention
A continuación se muestran a modo de ejemplo ilustrativo y no limitativo tratamientos térmicos aplicados a una muestra de alperujo como subproducto del aceite de oliva y la obtención a partir de los mismos del extracto fenólico.  The following are illustrative and non-limiting examples of heat treatments applied to a sample of alperujo as a byproduct of olive oil and the obtaining thereof of the phenolic extract.
Ej emplo Example
Una muestra de 20 kg de alperujo fresco fue sometida a varios procesos de tratamientos térmicos . Las condiciones fueron en este caso de 160°C durante 30, 60 y 90 minutos mediante un calentamiento directo con vapor de agua. A sample of 20 kg of fresh alperujo was subjected to several heat treatment processes. The conditions they were in this case 160 ° C for 30, 60 and 90 minutes by direct heating with water vapor.
Tras dicho proceso se obtiene una fracción sólida y otra liquida que son separadas mediante centrifugación, obteniéndose 51 litros de fracción liquida. Se toma una alícuota de 10 litros de la fracción líquida y se concentra a 1 litro. Se extraen los compuestos apolares (grasa) con hexano (2x100 mi por cada 200 mi de fracción soluble) .  After this process, a solid fraction and a liquid fraction are obtained, which are separated by centrifugation, obtaining 51 liters of liquid fraction. A 10-liter aliquot of the liquid fraction is taken and concentrated to 1 liter. The apolar compounds (fat) are extracted with hexane (2x100 ml for every 200 ml of soluble fraction).
A continuación se procede a la extracción de los compuestos fenólicos, para ello se realiza una extracción en caliente y a contracorriente con acetato de etilo durante 8 horas, empleándose 500 mi de acetato de etilo por cada 200 mi de muestra. De la fase orgánica se obtienen las cantidades señaladas en la tabla 1 de extracto fenólico seco (EF) .  Then the phenolic compounds are extracted, for this purpose a hot and countercurrent extraction with ethyl acetate is carried out for 8 hours, using 500 ml of ethyl acetate for every 200 ml of sample. The amounts indicated in Table 1 of dry phenolic extract (EF) are obtained from the organic phase.
La misma extracción se realiza directamente sobre la muestra original de alperujo sin tratar térmicamente (EFNT) para que sirva como control y poder establecer los efectos del tratamiento térmico:  The same extraction is performed directly on the original sample of non-heat treated lupine (EFNT) to serve as a control and to establish the effects of heat treatment:
Para ello se somete el alperujo sin tratar a una extracción con etanol (EtOH) al 80% (v/v) en agua. Se parte de 2.690 kg del alperujo fresco no tratado térmicamente, que ha sido previamente desgrasado con hexano. El alperujo desgrasado se pone en contacto bajo agitación suave con EtOH 80% a temperatura ambiente durante 30 minutos, empleando 15 mi de EtOH 80% por cada 10 g de alperujo, la extracción es repetida dos veces más, utilizando 10 mi de EtOH 80% por cada 10 g de alperujo. Las fracciones son filtradas a través de un filtro de papel en Buchner, reunidas y el EtOH es evaporado hasta obtener un extracto acuoso. Al concentrado obtenido se le extraen los compuestos fenólicos con acetato de etilo, para ello se realiza una extracción en caliente y a contracorriente con acetato de etilo durante 8 horas, se emplean 500 mi de acetato de etilo por cada 200 mL de muestra. De la fase orgánica se obtienen 15.56 g de extracto fenólico seco no tratado (EFNT) . La cantidad de extracto fenólico obtenido a partir del alperu o tratado y no tratado térmicamente se muestra en la tabla 1, en donde se aprecia un significativo aumento con el tratamiento. Tres de los extractos tratados más el control sin tratar han sido caracterizados en cuanto a su composición total en la tabla 2. To do this, the untreated alperujo is subjected to an extraction with 80% ethanol (EtOH) in water. It starts from 2,690 kg of the fresh non-heat treated lupine, which has been previously defatted with hexane. The defatted lupine is brought into contact under gentle agitation with 80% EtOH at room temperature for 30 minutes, using 15 ml of 80% EtOH for every 10 g of lupine, the extraction is repeated twice more, using 10 ml of 80% EtOH for every 10 g of alperujo. The fractions are filtered through a paper filter in Buchner, combined and the EtOH is evaporated until an aqueous extract is obtained. To the concentrate obtained, the phenolic compounds are extracted with ethyl acetate, for this purpose a hot and countercurrent extraction with ethyl acetate is carried out for 8 hours, 500 ml of ethyl acetate are used for every 200 mL of sample. From the organic phase 15.56 g of dry untreated phenolic extract (EFNT) are obtained. The amount of phenolic extract obtained from alperu or treated and not heat treated is shown in Table 1, where a significant increase can be seen with the treatment. Three of the extracts treated plus the untreated control have been characterized in terms of their total composition in Table 2.
Tabla 1. Gramos de extracto obtenido por kilogramo de alperujo fresco tratado o no térmicamente (AFNT) .
Figure imgf000010_0001
Table 1. Grams of extract obtained per kilogram of fresh or non-thermally treated lupine (AFNT).
Figure imgf000010_0001
Tabla 2. Composición de los extractos fenólicos obtenidos a partir de un alperujo sin tratar (EFNT) y tratado a 160°C para tres tiempos (n.d. , no detectado) . Fracción fenólica polimérica (FFP) . Table 2. Composition of phenolic extracts obtained from an untreated alperujo (EFNT) and treated at 160 ° C for three times (n.d., not detected). Phenolic polymeric fraction (FFP).
Sin tratar Tratamiento térmico a 160°C Untreated Heat treatment at 160 ° C
Componentes (%) AFNT 30 (min) 60 (min) 90 (min)" Components (%) AFNT 30 (min) 60 (min) 90 (min) "
Humedad 8, 62 12, 36 15, 60 13, 02Humidity 8, 62 12, 36 15, 60 13, 02
Fenoles 25,70 48,40 19, 93 22, 68Phenols 25.70 48.40 19, 93 22, 68
FFP n.d. 7, 32 1, 09 14,21FFP n.d. 7, 32 1, 09 14.21
Proteínas 1,41 1, 81 3, 09 3, 90Proteins 1.41 1, 81 3, 09 3, 90
Cenizas 0,01 0, 02 0, 04 0, 03Ashes 0,01 0, 02 0, 04 0, 03
Azúcares 7,30 5,75 5,54 3, 52Sugars 7.30 5.75 5.54 3, 52
Lípidos 52, 64 20,25 42, 64 35, 08Lipids 52, 64 20.25 42, 64 35, 08
Ácidos urónicos n.d. n.d. n.d. n.d.Uronic acids n.d. n.d. n.d. n.d.
Total 95, 68 95, 91 87, 93 92, 44 Total 95, 68 95, 91 87, 93 92, 44
El porcenta e en humedad es mayor en el caso de los extractos tratados térmicamente, mientras que el contenido en fenoles totales y lípidos no parece variar significativamente. Gracias al tratamiento térmico y posterior extracción aparece un nuevo componente que consiste en una mezcla de polímeros fenólicos (FFP), entre otros, que puede presentar importantes propiedades bioactivas . La cantidad de proteínas aumenta con los tratamientos mientras que la de azúcares disminuye. Para estos mismos extractos se llevó a cabo una caracterización más detallada sobre el contenido de compuestos fenólicos como se detalla en la tabla 3a y en la tabla 3b. Las notables diferencias de composición asi como la aparición de nuevos compuestos fenólicos y otros de degradación de azúcares marcan una importante diferencia con los extractos fenólicos que se pueden llegar a obtener a partir de los subproductos sin tratar. Estas diferencias se traducen en una mayor actividad por una mayor presencia de fenoles más activos y otros nuevos con importantes propiedades para la salud, entre otras. A continuación se detallan más a fondo las nuevas características de los extractos tratados térmicamente. The percentage e in moisture is higher in the case of heat treated extracts, while the content of total and lipid phenols does not seem to vary significantly. Thanks to the heat treatment and subsequent extraction, a new component appears consisting of a mixture of phenolic polymers (FFP), among others, which can have important bioactive properties. The amount of protein increases with treatments while the amount of sugar decreases. For these same extracts, a more detailed characterization of the content of phenolic compounds was carried out as detailed in Table 3a and Table 3b. The notable differences in composition as well as the appearance of new phenolic and other sugar degradation compounds make an important difference with the phenolic extracts that can be obtained from untreated by-products. These differences translate into greater activity due to a greater presence of more active and new phenols with important health properties, among others. The new features of the heat treated extracts are detailed below.
Tabla 3a. Componentes fenólicos y de degradación de azúcares presente en los diferentes extractos EF a distintas temperaturas y para el EFNT expresados en mg de compuesto por kilogramo de alperuj o fresco. Table 3a. Phenolic and sugar degradation components present in the different EF extracts at different temperatures and for the EFNT expressed in mg of compound per kilogram of alperuj or fresh.
mg/Kg alperujo fresco  mg / kg fresh lupine
Cantidades totales EFNT 160°C/30 160°C/60 160°C/90 Total quantities EFNT 160 ° C / 30 160 ° C / 60 160 ° C / 90
3, 4-Dihidroxi-3, 4-Dihydroxy
24, 907 123, 433 70,18 71, 64 fenilglicol 24, 907 123, 433 70.18 71, 64 phenyl glycol
Hidroxitirosol 15, 733 1624, 825 776,548 1127, 53 Hydroxytyrosol 15, 733 1624, 825 776,548 1127, 53
Tirosol 14, 970 108, 072 80, 648 168, 038Tyrosol 14, 970 108, 072 80, 648 168, 038
Vainillina n . d . 0, 095 n . d 11,122Vanillin n. d. 0.095 n. d 11,122
4-metilcatecol n . d . 1, 921 n . d n . d4-methylcatechol n. d. 1, 921 n. d n. d
Alcoholes fenólicos Phenolic alcohols
55, 610 1858,347 927, 376 1378, 329 totales  55, 610 1858,347 927, 376 1378, 329 total
Hidroximetil- n . d . 1, 325 4, 68 21,29 furfural  Hydroxymethyl- n. d. 1, 325 4, 68 21.29 furfural
Ácido 3,4 Dihidroxi- n . d . 15, 563 n . d 19,59 fenilacetico  Acid 3,4 Dihydroxy. d. 15, 563 n. d 19.59 phenylacetic
Ácido protocateico 9, 174 24, 464 18, 462 2,74 Protocateic acid 9, 174 24, 464 18, 462 2.74
Ácido cafeico n . d . 7, 115 2, 658 0, 957Caffeic Acid n. d. 7, 115 2, 658 0, 957
Ácido p-Ohbenzoico 0,4 1, 657 1, 432 1, 067P-Ohbenzoic acid 0.4 1, 657 1, 432 1, 067
Derivados del ácido Acid derivatives
n . d . 5, 137 n . d n . d protocateico  n. d. 5, 137 n. d n. protocatetic d
4-metilcatecol n . d . 1, 921 n . d n . d 4-methylcatechol n. d. 1, 921 n. d n. d
Ácido p-cumárico 2, 929 n . d 1,496 n . dP-cumaric acid 2, 929 n. d 1,496 n. d
Der ac p-cumárico n . d . n . d 0,049 n . dDer ac p-cumárico n. d. n. d 0.049 n. d
Ácido clorogénico n . d . n . d n . d 19,572Chlorogenic Acid n. d. n. d n. d 19,572
Ácido siríngico n . d . n . d n . d 1,786Syringic acid n. d. n. d n. d 1,786
Ácido vainíllico 3, 362 n . d 5, 013 2,089 Ácidos fenólicos Vanillic acid 3, 362 n. d 5, 013 2,089 Phenolic acids
15, 865 55, 857 29,11 47, 802 totales  15, 865 55, 857 29.11 47, 802 total
Hemiacetal de la  Hemiacetal of the
aglicona de la n . d n . d 0, 019 n . d oleuropeina aglycone of n. d n. d 0, 019 n. d oleuropein
Derivados de la  Derivatives of the
n . d 8,382 1,39 n . d oleuropeina 4C  n. d 8,382 1.39 n. d oleuropein 4C
Oleuropeina 2,743 0, 922 0, 461 n . d Oleuropein 2,743 0, 922 0, 461 n. d
Desmetiloleuropeina n . d n . d n . d 14, 535Demethyloleuropein n. d n. d n. d 14, 535
Secologanosido n . d 7, 355 n . d n . dSecologanoside n. d 7, 355 n. d n. d
Oleuropeina aglicón Oleuropein Aglycon
n . d n . d 0, 196 n . d derivado  n. d n. d 0, 196 n. d derivative
10-Hidroxi- n . d 4,277 n . d n . d metiloleuropeina  10-Hydroxy. d 4,277 n. d n. d methylleuropein
Derivados de la  Derivatives of the
2,743 20, 937 0, 676 14, 535 oleuropeina, totales  2,743 20, 937 0, 676 14, 535 oleuropein, total
Derivado del ácido  Acid derivative
1339, 749 664, 381 191, 299 527, 404 elenoico A  1339, 749 664, 381 191, 299 527, 404 Elenoic A
Derivado del ácido  Acid derivative
n . d 294, 459 123, 562 n . d elenoico C  n. d 294, 459 123, 562 n. d elenoic C
Derivado del ácido  Acid derivative
n . d 26,48 0, 577 9,39 elenoico B  n. d 26.48 0.577 9.39 elenoic B
Derivados del ácido  Acid derivatives
1339, 749 985, 32 315, 438 536, 794 elenoico totales  1339, 749 985, 32 315, 438 536, 794 total elenoic
Ligstrosido n . d n . d 0, 042 n . d Ligstroside n. d n. d 0, 042 n. d
Desmetilligstrosido n . d 18,716 2, 207 111, 395Demethylltrostroside n. d 18,716 2, 207 111, 395
Derivados del Derivatives of
n . d 18,716 2,25 111, 395 ligstrosido, totales  n. d 18,716 2.25 111, 395 ligstroside, total
Luteolin-7-Ο- n . d 1,34 0, 072 0, 125 Rutinosido  Luteolin-7-Ο- n. d 1.34 0.072 0.012 Rutinoside
Apigenin-7-Ο- n . d n . d 0, 179 n . d Rutinosido  Apigenin-7-Ο- n. d n. d 0, 179 n. d Rutinoside
Luteolin-7-Ο- Luteolin-7-Ο-
3, 697 n . d 0,58 n . d Glucosido 3, 697 n. d 0.58 n. d Glucoside
Cianidina-3- n . d 0, 055 n . d n . d Oruriosido  Cyanidine-3- n. d 0, 055 n. d n. d Orurioside
Cfeoil-6 ' -0- n . d 8, 752 n . d 0, 809 Secologanosido  Cfeoil-6 '-0- n. d 8, 752 n. d 0, 809 Secologanoside
Flavonoides totales 3, 697 10, 147 0, 831 0, 934 Total flavonoids 3, 697 10, 147 0, 831 0, 934
1-Acetoxipinoresinol n . d 154, 671 59, 831 174, 9221-Acetoxyproinoresinol n. d 154, 671 59, 831 174, 922
Pinoresinol n . d 25, 021 5, 939 n . dPinoresinol n. d 25, 021 5, 939 n. d
Lignanos totales n . d 179, 692 65, 77 174, 922 l-Fenil-6, 7- n . d 5, 097 36, 926 21,056 dihidroisocromona Total Lignans n. d 179, 692 65, 77 174, 922 l-Phenyl-6, 7- n. d 5, 097 36, 926 21.056 dihydroisochromone
Verbascósido n . d 2, 025 8, 432 n . d Verbascoside n. d 2, 025 8, 432 n. d
6' -0- [ (2E) -2, 6-6 '-0- [(2E) -2, 6-
Dimetil-8 -hidroxi- n . d 2, 141 n . d n . dDimethyl-8-hydroxy. d 2, 141 n. d n. d
2octonoiloxi] secologanosido 2octonoyloxy] secologanoside
Comsegolosido 184, 427 58, 93 22, 684 5, 121 Comsegolide 184, 427 58, 93 22, 684 5, 121
Nuzherina n . d 2, 432 n .d n . dNuzherina n. d 2, 432 n. d n. d
Ácido 3-Acid 3-
Hidroximetil-2 , 3- dihidroxi-5- n . α 207, 18 n . α n . dHydroxymethyl-2, 3- dihydroxy-5- n. α 207, 18 n. α n. d
(metoxicarbonil ) -2- metil-2H-piran-4- acetico metil éster (methoxycarbonyl) -2- methyl-2H-pyran-4- acetic methyl ester
Varios, totales 184, 427 277, 805 68, 042 26,177 Miscellaneous, total 184, 427 277, 805 68, 042 26,177
FFP n . d 515, 823 77, 658 1286, 409 FFP n. d 515, 823 77, 658 1286, 409
Tabla 4b . Componentes fenólicos y de degradación de azúcares presente en los diferentes extractos EF a distintas temperaturas y para el EFNT expresados porcentaje (gramos de componente por lOOg de extracto fenólico) . Table 4b Phenolic and sugar degradation components present in the different EF extracts at different temperatures and for the EFNT expressed percentage (grams of component per lOOg of phenolic extract).
% en el extracto  % in the extract
Cantidades totales EFNT 160°C/30 160°C/60 160°C/90 Total quantities EFNT 160 ° C / 30 160 ° C / 60 160 ° C / 90
3, 4-Dihidroxi- 0, 431 1,754 0, 988 0, 792 fenilglicol 3, 4-Dihydroxy-, 431 1,754 0, 988 0, 792 phenylglycol
Hidroxitirosol 0,272 23, 088 10, 935 9, 58 Hydroxytyrosol 0.272 23, 088 10, 935 9, 58
Tirosol 0,259 1, 536 1, 136 1, 857Tyrosol 0.259 1, 536 1, 136 1, 857
Vainillina n . d . 0, 001 n . d 0, 123Vanillin n. d. 0.001 n. d 0, 123
4-metilcatecol n . d . 0, 027 n . d n . d4-methylcatechol n. d. 0.027 n. d n. d
Alcoholes fenólicos 0, 961 26, 406 13, 058 12, 351 totales Phenolic alcohols 0, 961 26, 406 13, 058 12, 351 total
Hidroximetil- n . d . 0, 019 0, 066 0, 235 furfural  Hydroxymethyl- n. d. 0, 019 0, 066 0, 235 furfural
Ácido 3,4 Dihidroxi- fenilacetico  3,4-Dihydroxyphenylacetic acid
Ácido protocateico n . d 0,221 n . d 0, 217 Protocateic acid n. d 0.221 n. d 0, 217
Ácido cafeico 0, 159 0, 073 0,26 0, 03Caffeic Acid 0, 159 0, 073 0.26 0.03
Ácido p-Ohbenzoico n . d 0, 101 0, 037 0, 011P-Ohbenzoic acid n. d 0, 101 0, 037 0, 011
Derivados del ácido 0, 007 0, 024 0, 02 0, 012 protocateico Derivatives of 0.007 0.024 0 0.02 0.012 protocateic acid
4-metilcatecol n . d 0,348 n . d n . d 4-methylcatechol n. d 0,348 n. d n. d
Ácido p-cumárico n . d n . d n . d n . dP-cumaric acid n. d n. d n. d n. d
Der ac p-cumárico 0, 051 n . d 0, 021 n . dDer ac p-cumaric 0.051 n. d 0, 021 n. d
Ácido clorogénico n . d n . d 0, 001 n . dChlorogenic Acid n. d n. d. 001 n. d
Ácido siringico n . d n . d n . d 0,216Syringic acid n. d n. d n. d 0.216
Ácido vainillico n . d n . d n . d 0, 023Vanillic acid n. d n. d n. d 0, 023
Ácidos fenólicos 0, 058 n . d 0, 071 0, 02 totales Phenolic acids 0.058 n. d 0, 071 0, 02 total
Hemiacetal de la 0,274 0,766 0, 41 0, 528 aglicona de la  Hemiacetal of 0.274 0.766 0, 41 0, 528 aglycone of the
oleuropeina  oleuropein
Derivados de la n . d n . d n . d n . d oleuropeina 4C Oleuropeina n . d 0, 119 0, 02 n . dDerivatives of the n. dn. dn. dn. d oleuropein 4C Oleuropein n. d 0, 119 0, 02 n. d
Desmetiloleuropeina 0, 047 0, 013 0, 006 n . dDesmethylouropein 0.047 0.013 0.06 n. d
Secologanosido n . d n . d n . d 0, 161Secologanoside n. d n. d n. d 0, 161
Oleuropeina aglicón n . d 0, 105 n . d n . d derivado Oleuropein aglycone n. d 0, 105 n. d n. d derivative
10-Hidroxi- n . d n . d 0, 003 n . d metiloleuropeina  10-Hydroxy. d n. d 0.003 n. d methylleuropein
Derivados de la n . d 0, 061 n . d n . d oleuropeina, totales  Derivatives of the n. d 0, 061 n. d n. d oleuropein, total
Derivado del ácido 0, 047 0, 297 0, 029 0, 161 elenoico A  Acid derivative 0.047 0, 297 0.029 0, 161 elenoic A
Derivado del ácido 23, 161 9, 441 2, 694 5,829 elenoico C  Derivative of acid 23, 161 9, 441 2, 694 5,829 elenoic C
Derivado del ácido n . d 4, 184 1,74 n . d elenoico B  Acid derivative n. d 4, 184 1.74 n. d elenoic B
Derivados del ácido n . d 0, 376 0, 008 0, 104 elenoico totales  Acid derivatives n. d 0, 376 0.008 0, 104 total elenoic
Ligstrosido 23, 161 14, 001 4,442 5, 933 Ligstrosido 23, 161 14, 001 4,442 5, 933
Desmetilligstrosido n . d n . d 0, 001 n . dDemethylltrostroside n. d n. d. 001 n. d
Derivados del n . d 0,266 0, 031 1,231 ligstrosido, totales Derivatives of n. d 0.266 0.031 1.231 ligstroside, total
Luteolin-7-Ο- n . d 0,266 0, 032 1,231 Luteolin-7-Ο- n. d 0.266 0.032 1.231
Rutinosido Rutinoside
Apigenin-7-Ο- n . d 0, 019 0, 001 0, 001 Apigenin-7-Ο- n. d 0, 019 0, 001 0.001
Rutinosido Rutinoside
Luteolin-7-Ο- n . d n . d 0, 003 n . d Luteolin-7-Ο- n. d n. d 0.003 n. d
Glucosido Glycoside
Cianidina-3- 0, 064 n . d 0, 008 n . d Cyanidine-3, 064 n. d. 008 n. d
Oruriosido Orurioside
Cfeoil-6 ' -0- n . d 0, 001 n . d n . d Cfeoil-6 '-0- n. d. 001 n. d n. d
Secologanosido Secologanoside
Flavonoides totales n . d 0, 124 n . d 0, 009 Total flavonoids n. d 0, 124 n. d 0.009
1-Acetoxipinoresinol 0, 064 0, 144 0, 012 0, 011-Acetoxyproinoresinol 0, 064 0, 144 0, 012 0.01
Pinoresinol n . d 2, 198 0, 842 1, 933Pinoresinol n. d 2, 198 0, 842 1, 933
Lignanos totales n . d 0, 356 0, 084 n . d l-Fenil-6, 7- n . d 2, 553 0, 926 1, 933 dihidroisocromona Total Lignans n. d 0, 356 0, 084 n. d l-Phenyl-6, 7- n. d 2, 553 0, 926 1, 933 dihydroisochromone
Verbascósido n . d 0, 072 0, 52 0, 233 Verbascoside n. d 0, 072 0, 52 0, 233
6' -0- [ (2E) -2, 6- n . d 0, 029 0, 119 n . d6 '-0- [(2E) -2, 6- n. d 0, 029 0, 119 n. d
Dimetil-8 -hidroxi-Dimethyl-8-hydroxy-
2octonoiloxi] 2octonoyloxy]
secologanosido secologanoside
Comsegolosido n . d 0, 03 n . d n . d Comsegolide n. d 0, 03 n. d n. d
Nuzherina 1, 198 0, 837 0,319 0, 057Nuzherina 1, 198 0, 837 0.319 0, 057
Ácido 3- n . d 0, 035 n . d n . dAcid 3- n. d 0, 035 n. d n. d
Hidroximetil-2 , 3- dihidroxi-5-Hydroxymethyl-2, 3- dihydroxy-5-
(metoxicarbonil ) -2- metil-2H-piran-4- acetico metil éster (methoxycarbonyl) -2- methyl-2H-pyran-4- acetic acid methyl ester
Varios, totales n . d 2, 944 n . d n . dSeveral, total n. d 2, 944 n. d n. d
FFP 1, 198 3, 947 0, 958 0, 2892342 FFP 1, 198 3, 947 0, 958 0, 2892342
Principales ventajas del nuevo extracto Main advantages of the new extract
De la caracterización se pueden destacar los siguientes puntos o ventajas del tratamiento térmico que diferencian al nuevo extracto obtenido:  The following points or advantages of heat treatment that distinguish the new extract obtained can be highlighted from the characterization:
La cantidad de extracto obtenido aumenta con el tratamiento térmico y en el caso del presente ejemplo aumenta con el tiempo de tratamiento;  The amount of extract obtained increases with the heat treatment and in the case of the present example increases with the treatment time;
En el EF obtenido tras el tratamiento térmico se produce un incremento de la concentración de los alcoholes fenólicos. El 3 , -dihidroxifenilglicol aumenta su concentración de 3 a 5 veces en el extracto. En el caso del hidroxitirosol la concentración aumenta hasta 100 veces con respecto a la concentración inicial. La concentración de tirosol se incrementa hasta más de 10 veces con respecto al EFNT. Por lo tanto, se observa que el extracto fenólico obtenido tras el tratamiento térmico se encuentra altamente enriquecido en estos antioxidantes naturales;  In the EF obtained after heat treatment there is an increase in the concentration of phenolic alcohols. 3, -dihydroxyphenyl glycol increases its concentration 3 to 5 times in the extract. In the case of hydroxytyrosol the concentration increases up to 100 times with respect to the initial concentration. The concentration of tyrosol is increased up to more than 10 times compared to the EFNT. Therefore, it is observed that the phenolic extract obtained after the heat treatment is highly enriched in these natural antioxidants;
De forma global se experimenta un incremento de las concentraciones de los fenoles ácidos presentes en EF con respecto al EFNT, aumentando su porcentaje hasta 3.5 veces en el extracto. Hay que destacar que tras el tratamiento térmico se observa la presencia de ácido cafeico, el cual antes del tratamiento no había sido detectado. Aunque se encuentra presente en otras muestras su concentración aumenta notablemente tras el tratamiento térmico. Esta molécula se encuentra formando parte de estructuras más complejas como el comsegolosido, el cual puede sufrir una ruptura en el reactor liberando la especie. El ácido protocateico experimenta un aumento de la concentración y porcentaje de más del doble en el extracto; En el EF también destaca la nueva presencia de compuestos como el pinoresinol y 1 -acetoxipinoresinol (hasta un 0.36 y 2.2% respecti amente) que no han sido identificados en el EFNT. En el alperujo el pinoresinol y 1-acetoxipinoresinol solo han sido descritos por primera vez en un articulo reciente (Suárez, M. et al., (2009), J Agrie Food Chem. , 57, 1463-72.), aunque las concentraciones a las que se encuentran son mucho menores que las presentes en el extracto fenólico aquí tratado. Es importante señalar que prácticamente ningún autor identifica a estas especies en el alperujo, probablemente debido a las bajas concentraciones en las que se encuentran; Overall, there is an increase in the concentrations of the acidic phenols present in EF with respect to the EFNT, increasing its percentage up to 3.5 times in the extract. It should be noted that after the heat treatment the presence of caffeic acid is observed, which had not been detected before the treatment. Although it is present in other samples, its concentration increases markedly after heat treatment. This molecule is part of more complex structures such as comsegolide, which can suffer a break in the reactor releasing the species. Protocateic acid experiences an increase in the concentration and percentage of more than double in the extract; In the EF also highlights the new presence of compounds such as pinoresinol and 1-acetoxyproinoresinol (up to 0.36 and 2.2% respectively) that have not been identified in the EFNT. In the alperujo, pinoresinol and 1-acetoxyproinoresinol have only been described for the first time in a recent article (Suárez, M. et al., (2009), J Agrie Food Chem., 57, 1463-72.), Although concentrations those found are much smaller than those present in the phenolic extract treated here. It is important to note that practically no author identifies these species in the alperujo, probably due to the low concentrations in which they are found;
Se observa la presencia de nuevos compuestos en el EF obtenido tras el tratamiento térmico que no se observan en EFNT, tales como el hidroximetilfurfural y el l-fenil-6,7- dihidroisocromona, que alcanzan porcentajes de hasta un 0.24 y 0.52% en el EF respectivamente. El hidroximentil furfural no ha sido descrito como componente del alperujo. Su presencia se debe a la degradación de hexosas que tienen lugar en el reactor como consecuencia del calentamiento;  The presence of new compounds is observed in the EF obtained after the heat treatment that are not observed in EFNT, such as hydroxymethylfurfural and l-phenyl-6,7-dihydroisocromone, which reach percentages of up to 0.24 and 0.52% in the EF respectively. Hydroxymethyl furfural has not been described as a component of the alperujo. Its presence is due to the degradation of hexoses that take place in the reactor as a result of heating;
El 1-Fenil- 6, 7-dihidroisocromona pertenece a la familia de los hidroxi-isocromanos , los cuales forman parte de los componentes de naturaleza fenólica procedente del aceite de oliva (Bianco, A., et al., (2001), Food Chem. 77, pp . 405- 411,). Sin embargo no han sido descritos con anterioridad en el alperujo; 1-Phenyl-6, 7-dihydroisocromone belongs to the hydroxy-isochroman family, which are part of the phenolic nature components from olive oil (Bianco, A., et al., (2001), Food Chem. 77, pp. 405-411, ) . However, they have not been previously described in the alperujo;
Compuestos como el comsegolosido, verbascósido, los derivados de la Oleuropeina y del Ligstrosido sufren un descenso en su concentración. Este hecho se justifica debido a que el tratamiento térmico provoca la ruptura de estas moléculas complejas, provocando la liberación de otras de menor tamaño tales como el hidroxitirosol o el tirosol, que forman parte de su estructura, aumentando la concentración de éstas como ya se ha visto con anterioridad; El tratamiento térmico seguido por una extracción fenólica favorece la aparición de una nueva fracción fenólica polimérica (FFP) , hasta un 14% en el extracto y con interesantes propiedades para la salud. Compounds such as comsegolide, verbascoside, derivatives of Oleuropein and Ligstroside suffer a decrease in their concentration. This fact is justified because the heat treatment causes the breakdown of these complex molecules, causing the release of other smaller ones such as hydroxytyrosol or tyrosol, which are part of its structure, increasing the concentration of these as it has already been seen before; The heat treatment followed by a phenolic extraction favors the appearance of a new polymeric phenolic fraction (FFP), up to 14% in the extract and with interesting health properties.
El incremento tan importante que ocurre para los compuestos fenólicos con tan importantes propiedades bioactivas asi como la aparición de nuevos componentes hacen que el nuevo extracto presente nuevas y mejores propiedades beneficiosas para la salud. Éstas quedan claramente reflejadas en los ensayos de actividad que a continuación se muestran .  The very important increase that occurs for phenolic compounds with such important bioactive properties as well as the appearance of new components make the new extract present new and better health-beneficial properties. These are clearly reflected in the activity tests shown below.
Ensayos de Actividad  Activity Essays
Actividad de los nuevos extractos fenólicos tratados térmicamente (EF) y de sus fracciones.  Activity of the new heat-treated phenolic extracts (EF) and their fractions.
El EF es rico en una gran variedad de compuestos que pueden presentar distintas actividades, contribuyendo todas ellas en suma a la actividad final del extracto. El fraccionamiento de dicho extracto puede resultar beneficioso para potenciar ciertas actividades concretas de los componentes fraccionados. Por ello, se ha realizado también un estudio sobre la actividad de las fracciones del EF tratado térmicamente. A modo de ejemplo se presentan las fracciones en las cuales se dividen el extracto 160°C/60min y se le realizan los ensayos de actividad antioxidante a cada una de ellas. La composición de las distintas fracciones obtenidas se muestra a modo de ejemplo en la tabla 4.  The EF is rich in a great variety of compounds that can present different activities, all contributing in sum to the final activity of the extract. The fractionation of said extract may be beneficial to enhance certain specific activities of the fractionated components. Therefore, a study has also been carried out on the activity of the heat treated EF fractions. As an example, the fractions in which the extract is divided 160 ° C / 60min are presented and the antioxidant activity tests are carried out on each of them. The composition of the different fractions obtained is shown by way of example in Table 4.
Se expone en la tabla 5 como ejemplo de la actividad in vitro de la actividad antioxidante y secuestrante de radicales libres del nuevo extracto y de sus distintas fracciones los siguientes ensayos cuyos resultados obtenidos son comparados con el extracto fenólico no tratado como control (EFNT) , la vitamina E (VE) , el hidroxitirosol (HT) y el 3, 4-dihidroxifenilglicol (DHFG) , siendo éstos últimos dos de los fenoles simples más activos presentes en la aceituna. Tabla 4: Composición de cada una de las fracciones en las cuales se divide el extracto fenólico obtenido tras tratar el alperujo a 160°C durante 60 minutos . Se especifica las especies de naturaleza fenólica presentes en cada fracción y la concentración de las mismas en ng/ul. (Fr.= Fracción) Table 5 is an example of the in vitro activity of the antioxidant and free radical sequestrant activity of the new extract and its different fractions, the following tests whose results are compared with the untreated phenolic extract as a control (EFNT), Vitamin E (VE), hydroxytyrosol (HT) and 3,4-dihydroxyphenyl glycol (DHFG), the latter being two of the most active single phenols present in the olive. Table 4: Composition of each of the fractions into which the phenolic extract obtained after treating the alperujo at 160 ° C for 60 minutes is divided. The species of phenolic nature present in each fraction and their concentration in ng / ul are specified. (Fr. = Fraction)
Fr . Compuestos [ng/μΐ] Fr . Compuestos [ng/pL]Fr. Compounds [ng / μΐ] Fr. Compounds [ng / pL]
1 DHFG 2457, 377 3F Ácido protocateico 150, 142 1 DHFG 2457, 377 3F Protocateic acid 150, 142
Ácido p- Acid p-
2A DHFG 74, 445 10, 663 hidroxibenzoico 2A DHFG 74, 445 10, 663 hydroxybenzoic
Hidroximetilfu  Hydroxymethylfu
93, 015 3G Ácido Cafeico 106, 863 rfural  93, 015 3G Caffeic Acid 106, 863 rfural
Hidroxi- Hydroxy
99, 883 4A Glicol 5, 445 tirosol 99, 883 4A Glycol 5, 445 tyrosol
Tirosol 664, 213 Hidroxitirosol 12, 276 Tyrosol 664, 213 Hydroxytyrosol 12, 276
2B DHFG 284, 163 Tirosol 2,7752B DHFG 284, 163 Tyrosol 2,775
Hidroximetilfr Derivado Ácido Hydroxymethylfr Acid Derivative
53, 505 211,814 fural Elenoico A  53, 505 211,814 fural Elenoic A
Hidroxi- 29890, 67  Hydroxy- 29890, 67
4B Hidroxitirosol 78, 815 tirosol 4  4B Hydroxytyrosol 78, 815 tyrosol 4
Derivado Ácido 4967, 17 Acid Derivative 4967, 17
Tirosol 861, 222 Tyrosol 861, 222
Elenoico C 5 Elenoic C 5
Ácido Derivado de Acid Derived from
2C 534, 619 56, 105 2C 534, 619 56, 105
Protocateico Oleuropeina Protocateic Oleuropein
Hidroximetilfu  Hydroxymethylfu
3A 38, 753 4C Hidroxitirosol 78, 820 rfural  3A 38, 753 4C Hydroxytyrosol 78, 820 rfural
Hidroximetilfu 1- 2260, 06 Hydroxymethylfu 1- 2260, 06
3B 2, 953 3B 2, 953
rfural Acetoxipinoresinol 6 rfural Acetoxypinoresinol 6
Hidroxi-Hydroxy
9, 547 Oleuropeina 18,548 tirosol 9, 547 Oleuropein 18,548 tyrosol
Derivado Ácido  Acid Derivative
3639, 062 Ligstrosido 1, 705 Elenoico A  3639, 062 Ligstroside 1, 705 Elenoic A
Hemiacetal de  Hemiacetal of
la Oleuropeina  Oleuropein
0, 760 7, 870 aglicona de la aglicon derivado  0,760 7,870 aglycone derived aglycone
oleuropeina  oleuropein
Hidroxi- Hydroxy
3C 888,204 4D Hidroxitirosol 64, 102 tirosol 3C 888,204 4D Hydroxytyrosol 64, 102 tyrosol
Derivado Ácido Derivado Ácido  Acid Derivative Acid Derivative
582, 152 23, 194 Elenoico A Elenoico B  582, 152 23, 194 Elenoic A Elenoic B
1- one-
Tirosol 1489, 292 153, 009 Tyrosol 1489, 292 153, 009
Acetoxipinoresinol  Acetoxypinoresinol
Pinoresinol 238, 763 4E FFP  Pinoresinol 238, 763 4E FFP
Hidroxi- Hydroxy
3D 51, 951 4F Hidroxitirosol 42, 644 tirosol 3D 51, 951 4F Hydroxytyrosol 42, 644 tyrosol
Derivado del  Derived from
Ácido p- 1, 964 Ácido Vainillico 107, 208 cumárico Apigenin-7-0 Acid p- 1, 964 Vanillic Acid 107, 208 Cumaric Apigenin-7-0
Tirosol 224,514 7, 191  Tyrosol 224,514 7, 191
Rutinosido  Rutinoside
Derivado Ácido  Acid Derivative
2631, 371 Verbascósido 293, 091 Elenoico A  2631, 371 Verbascoside 293, 091 Elenoic A
l-fenil-6, 7- dihidro- 1484,426 4G Ácido p-Cumárico 60, 168 isocromona  l-phenyl-6, 7- dihydro- 1484,426 4G p-Cumaric acid 60, 168 isochromone
Luteolin-7-0  Luteolin-7-0
Verbascósido 45, 877 23, 298  Verbascoside 45, 877 23, 298
Glucósido  Glycoside
Ácido  Acid
3E 53, 506 Comsegolosido 911, 887 protocateico  3E 53, 506 Comsegolide 911, 887 protocatech
Ácido p- hidroxi- 46, 905  P-hydroxy acid 46, 905
benzoico  benzoic
Derivado ác.  Derivative ac.
625, 750  625, 750
Elenoico A  Elenoic A
Luteolin-7-0  Luteolin-7-0
2, 908  2, 908
Rutinosido  Rutinoside
Ác . Vainillico 94, 309  Ac. Vanilla 94, 309
Tabla 5. Ensayos de actividad biológica de los extractos obtenidos tras distintos tratamiento térmicos y sus fracciones comparados con el extracto sin tratamiento y compuestos como el HT, el DHFG y la vitamina E. (1= Poder Reductor; 2= Actividad antirradical DPPH; 3= Captación de radicales libres ABTS; 4= Inhibición a la Oxidación Ia; 5= Inhibición a la Oxidación 2a; 6= Inhibición a la actividad tirosinasa) Table 5. Biological activity tests of the extracts obtained after different thermal treatments and their fractions compared with the extract without treatment and compounds such as HT, DHFG and vitamin E. (1 = Reducing Power; 2 = DPPH anti-radical activity; 3 = ABTS free radical uptake; 4 = Oxidation inhibition I a ; 5 = Oxidation inhibition 2 a ; 6 = Tyrosinase activity inhibition)
1 2 3 4 5 6 mg/ml  1 2 3 4 5 6 mg / ml
EC50 TEAC EC 50 EC50 Abs trolox  EC50 TEAC EC 50 EC50 Abs trolox
EFNT 0, 15 5,59 0,22 1, 80 0,79 0, 84 EFNT 0.15 5.59 0.22 1, 80 0.79 0.84
EF (160/15) 0,45 1, 64 0, 53 1,46 0, 97 0, 48EF (160/15) 0.45 1, 64 0, 53 1.46 0, 97 0.48
EF (160/30) 0,34 1,50 0,46 1,31 1, 14 0,51EF (160/30) 0.34 1.50 0.46 1.31 1, 14 0.51
EF (160/45) 0,48 1, 11 0,51 1, 06 0, 94 0, 43EF (160/45) 0.48 1, 11 0.51 1, 06 0, 94 0, 43
EF (160/60) 0,49 1,23 0,54 0, 96 1, 13 0, 44EF (160/60) 0.49 1.23 0.54 0.96 1, 13 0, 44
EF (160/75) 0,42 1, 06 0, 42 1,38 1, 02 0, 35EF (160/75) 0.42 1, 06 0, 42 1.38 1, 02 0.35
EF (160/90) 0,50 1,72 0,51 1, 64 1, 05 0,51EF (160/90) 0.50 1.72 0.51 1, 64 1, 05 0.51
Fracción 1 0, 32 1, 08 0, 40 1, 08 0, 60 -Fraction 1 0, 32 1, 08 0, 40 1, 08 0, 60 -
Fracción 2A 0, 17 6, 03 0,20 6, 03 1, 06 -Fraction 2A 0, 17 6, 03 0.20 6, 03 1, 06 -
Fracción 2B 1,29 0, 81 1,38 0, 80 0, 14 -Fraction 2B 1.29 0, 81 1.38 0, 80 0, 14 -
Fracción 2C 1,79 2,79 0,70 2,79 0, 08 -Fraction 2C 1.79 2.79 0.70 2.79 0, 08 -
Fracción 3A 0,06 1,75 0, 17 1,76 1, 66 -Fraction 3A 0.06 1.75 0, 17 1.76 1, 66 -
Fracción 3B 0,09 3,22 0, 17 3,22 1, 88 -Fraction 3B 0.09 3.22 0, 17 3.22 1, 88 -
Fracción 3C 0,78 0, 81 0,71 0, 81 0, 35 -3C fraction 0.78 0.81 0.71 0, 81 0.35 -
Fracción 3D 0, 97 0, 94 0,28 0, 94 0,29 -3D fraction 0, 97 0, 94 0.28 0, 94 0.29 -
Fracción 3E 0,84 0, 67 0, 67 0, 67 0, 16 -3E fraction 0.84 0, 67 0, 67 0, 67 0, 16 -
Fracción 3F 0, 96 0, 60 0,29 0,59 0, 35 - Fracción 3G 0,43 7,24 0,28 7,24 0,34 -Fraction 3F 0.96 0.60 0.29 0.59 0.35 - 3G fraction 0.43 7.24 0.28 7.24 0.34 -
Fracción 4A 0, 03 8,58 0, 06 8, 57 5, 65 -Fraction 4A 0, 03 8.58 0, 06 8, 57 5, 65 -
Fracción 4B 0,24 3,29 0,24 3,29 0, 84 -Fraction 4B 0.24 3.29 0.24 3.29 0.84 -
Fracción 4C 0,83 0, 32 0, 69 0, 32 0, 10 -Fraction 4C 0.83 0.32 0, 69 0, 32 0, 10 -
Fracción 4D 0, 66 1, 01 0, 47 1, 01 0, 11 -4D fraction 0, 66 1, 01 0, 47 1, 01 0, 11 -
Fracción 4E 0,54 0, 57 0, 45 0, 57 0,24 -4E fraction 0.54 0, 57 0, 45 0, 57 0.24 -
Fracción 4F 0,80 0, 44 1,29 0, 44 0, 33 -4F fraction 0.80 0.44 1.29 0, 44 0.33 -
Fracción 4G 0, 52 0,86 0, 95 0,86 0, 41 -4G fraction 0.52 0.86 0.95 0.86 0.41 -
HT 1, 60 0, 11 1, 53 0, 68 0, 07 -HT 1, 60 0, 11 1, 53 0, 68 0, 07 -
DHFG 1, 96 0, 17 0, 99 0, 83 0, 03 -DHFG 1, 96 0, 17 0, 99 0, 83 0, 03 -
Vit E - 0, 83 0,50 0, 52 0,23 - Vit E - 0.83 0.50 0.52 0.23 -
1. - Poder reductor : El poder reductor de los extractos estudiados se expresa como equivalentes en mg/ml de Trolox que se requieren para llevar a cabo una inhibición de la oxidación de igual magnitud a la que se produce con una concentración de 1 mg/ml de compuesto/extracto. Se observa (Tabla 5) que los extractos fenólicos obtenidos a partir de alperu o tratado térmicamente (EF) presentan una actividad muy superior al del obtenido a partir del alperujo no tratado térmicamente, e inferior en este caso a la actividad mostrada por el HT y el DHFG como compuestos control. 1. - Reducing power: The reducing power of the studied extracts is expressed as equivalents in mg / ml of Trolox that are required to carry out an oxidation inhibition of equal magnitude to that produced with a concentration of 1 mg / ml of compound / extract. It is observed (Table 5) that the phenolic extracts obtained from alperu or heat treated (EF) have a much higher activity than that obtained from the non-thermally treated alperujo, and in this case inferior to the activity shown by HT and DHFG as control compounds.
En cuanto a la actividad de las distintas fracciones se puede comprobar algunas como la 2B y 2C cuyo valor reductor se encuentra por encima del Trolox, siendo la 2B la más rica en hidroxitirosol , y la 2C está constituida exclusivamente por ácido protocateico . La mayoría de las fracciones poseen un valor superior al del propio EF tratado térmicamente, por lo que se verifica que en casos como éste un posterior fraccionamiento ayuda a aumentar las propiedades del extracto.  Regarding the activity of the different fractions, some such as 2B and 2C, whose reducing value is above Trolox, can be verified, with 2B being the richest in hydroxytyrosol, and 2C consists exclusively of protocateic acid. Most of the fractions have a higher value than the thermally treated EF itself, so it is verified that in cases like this a subsequent fractionation helps to increase the properties of the extract.
2. - Captación de radicales DPPH: Los resultados se expresan como EC50, de forma que se indica la concentración de los extractos/compuestos necesarias para inhibir la oxidación en un 50%. Cuanta más capacidad antirradical posea la especie a estudio menos cantidad de la misma se requiere para disminuir la oxidación en un 50%. Como puede comprobarse en la tabla 5, los extractos obtenidos tras el tratamiento poseen mayor capacidad de captación que el extracto control sin tratar y a su vez los valores son próximos a los valores que proporciona la vitamina E. 2. - DPPH radical uptake: The results are expressed as EC 50 , so that the concentration of the extracts / compounds necessary to inhibit oxidation by 50% is indicated. The more antiradical capacity the species under study has, the less amount of it is required to reduce oxidation by 50%. As can be seen in Table 5, the extracts obtained after treatment They have a higher uptake capacity than the untreated control extract and in turn the values are close to the values provided by vitamin E.
Los resultados muestran que las fracciones 1, 2B, 3C, 3D, 3E, 3F, 4C, 4D, 4E, 4F y 4G son aquellas fracciones que presentan una mayor capacidad de captación de radicales DPPH, superando al propio EF tratado térmicamente. Estas fracciones son ricas en DHFG, hidroxitirosol , l-fenil-6,7- dihidroisocromona, ácido vainillico, 1-acetoxipinoresinol y comsegolosido . Hay que destacar que también presenta actividad la fracción 4E que es la que está formada por FFP . Los resultados de actividad antirradical son comparables a los resultados obtenidos con la vitamina E.  The results show that fractions 1, 2B, 3C, 3D, 3E, 3F, 4C, 4D, 4E, 4F and 4G are those fractions that have a greater capacity to capture DPPH radicals, exceeding the heat treated EF itself. These fractions are rich in DHFG, hydroxytyrosol, l-phenyl-6,7-dihydroisocromone, vanillic acid, 1-acetoxypyrosinol and comsegolide. It should be noted that the 4E fraction is also active, which is the one formed by FFP. The results of antiradical activity are comparable to the results obtained with vitamin E.
3.- La captación de radicales ABTS+ es comparada con la capacidad del Trolox bajo las mismas condiciones. Los resultados se expresan como "Capacidad antioxidante en equivalentes de Trolox" (TEAC) . Como se puede comprobar en la tabla 5 la capacidad captadora de los EF tratados superan ampliamente al no tratado y presentan una actividad similar a la vitamina E. Por otro lado la actividad mostrada por los extractos tratados no solo de debe a la presencia de HT . 3.- The uptake of ABTS + radicals is compared with the capacity of Trolox under the same conditions. The results are expressed as "Antioxidant capacity in Trolox equivalents" (TEAC). As can be seen in Table 5, the capacity of the treated EFs is far superior to the untreated and they have an activity similar to vitamin E. On the other hand, the activity shown by the treated extracts is not only due to the presence of HT.
Los resultados muestran que las fracciones 2B, 4F y 4G poseen una capacidad de captación de radicales ABTS del mismo orden que el observado en el caso del HT y DHFG, y que junto con el 2C, 3C, 3E y 4C mejoran la actividad del extracto. La fracción 2B se caracteriza por poseer un elevado porcentaje de HT . La fracción 4F además de hidroxitirosol contiene otras especies como el ácido vainillico o el verbascósido que probablemente actúen de forma sinérgica. La fracción 4G se caracteriza por poseer un elevado porcentaje de comsegolosido. Por otro lado, las fracciones 2C, 3C, 3E, 4C, 4D y 4E poseen una capacidad de captación de radicales ABTS similar a la observada por la vitamina E, debido a la presencia de compuestos como el ácido protocateico, el ácido vainillíco, el 1-acetoxipinoresinol , el pinoresinol, el verbascósido, el comsegolosido y la FFP. The results show that fractions 2B, 4F and 4G have an ability to capture ABTS radicals of the same order as observed in the case of HT and DHFG, and that together with 2C, 3C, 3E and 4C improve extract activity . Fraction 2B is characterized by having a high percentage of HT. The 4F fraction in addition to hydroxytyrosol contains other species such as vanillic acid or verbascoside that are likely to act synergistically. The 4G fraction is characterized by having a high percentage of comsegoloside. On the other hand, fractions 2C, 3C, 3E, 4C, 4D and 4E have an ABTS radical scavenging capacity similar to that observed by vitamin E, due to the presence of compounds such as protocateic acid, acid vanillic, 1-acetoxyproinoresinol, pinoresinol, verbascoside, comsegolide and FFP.
4. - Inhibición de la oxidación primaria: Se determina la inhibición a la oxidación primaria del ácido linoleico. Los resultados se expresan como EC50. En la Tabla 5 se muestra que los valores obtenidos para todos los EF tratados térmicamente superan al extracto no tratado y se encuentran próximos a los obtenidos con HT y DHFG. De nuevo se verifica que la actividad que presentan los extractos tratados no se puede justificar sólo por la presencia de H .  4. - Inhibition of primary oxidation: The inhibition of primary oxidation of linoleic acid is determined. The results are expressed as EC50. Table 5 shows that the values obtained for all thermally treated EFs exceed the untreated extract and are close to those obtained with HT and DHFG. Again, it is verified that the activity presented by the treated extracts cannot be justified only by the presence of H.
En la fracción 4C la capacidad de inhibición de la oxidación primaria se encuentra incluso por encima de los valores obtenidos para la vitamina E. Esta fracción es rica en 1-acetoxipinoresinol. Las fracciones 1, 2B, 3C, 3D, 3E, 3F, 4D, 4E, 4F y 4G también poseen una capacidad de inhibición de la oxidación primaria por encima del extracto tratado térmicamente. En la fracción 1, 2B y 3C este efecto se puede deber a las elevadas concentraciones de HT y DHFG. En los demás casos la actividad puede deberse al igual que en los casos previos a la presencia de l-fenil-6,7- dihidroisocromona, al ácido protocateico, ácido vainillico, 1-acetoxipinotesinol , verbascósido, comsegolosido y FFP.  In fraction 4C the capacity for inhibition of primary oxidation is even above the values obtained for vitamin E. This fraction is rich in 1-acetoxyproinoresinol. Fractions 1, 2B, 3C, 3D, 3E, 3F, 4D, 4E, 4F and 4G also have an ability to inhibit primary oxidation above the heat treated extract. In fraction 1, 2B and 3C this effect may be due to the high concentrations of HT and DHFG. In the other cases the activity may be due to the same as in the cases prior to the presence of l-phenyl-6,7-dihydroisocromone, protocateic acid, vanillic acid, 1-acetoxypinotesinol, verbascoside, comsegolide and FFP.
5. Inhibición de la oxidación secundaria: Se basa en la medida de la cantidad de malondialdehido (MDA) , un subproducto formado en sistemas de lipoperoxidación . En la tabla 5 se muestran los resultados obtenidos de EC50. Los resultados ponen de manifiesto que los valores obtenidos de los EF se encuentran por debajo de los compuestos de referencia y también del EFNT. A pesar de ello tras el fraccionamiento una gran cantidad de fracciones presentan valores de EC50 muy por debajo del extracto sin tratar térmicamente, mejorando bastante las propiedades en la prevención a la oxidación en medios lipidíeos. 5. Inhibition of secondary oxidation: It is based on the measurement of the amount of malondialdehyde (MDA), a byproduct formed in lipoperoxidation systems. Table 5 shows the results obtained from EC 50 . The results show that the values obtained from the EFs are below the reference compounds and also from the EFNT. Despite this, after fractionation, a large number of fractions have EC 50 values well below the extract without heat treatment, greatly improving the oxidation prevention properties in lipid media.
6. - Inhibición de la actividad de la tirosinasa de hongos in vitro: Esta enzima cataliza las primeras dos reacciones de la síntesis de la melanina, la hidroxilación de la L- tirosinasa para dar 3, 4-dihidroxifenilalanina (L-DOPA) y la oxidación de la L-DOPA a dopaquinona. Esta quinona es altamente reactiva y puede polimerizar espontáneamente a melanina. Los efectos de los extractos sobre las actividades de monofenolasa, utilizando L-tirosinasa como sustrato y de difenolasa sobre L-DOPA, se siguieron mediante la inhibición de la formación de dopacromo mediante medidas espectrofotométricas . Se muestran los resultados de absorbancia mostrados a 300 segundos, como tiempo final, para concentraciones de 0.75mg/ml de los extractos en la tabla 5. Cuanto menor sea la absorbancia desarrollada mayor es la capacidad de inhibición. Todos los extractos tratados térmicamente se encuentran dentro del mismo rango y poseen mayor capacidad de inhibición que el extracto fenólico no tratado térmicamente. 6. - Inhibition of fungal tyrosinase activity in vitro: This enzyme catalyzes the first two reactions of the synthesis of melanin, the hydroxylation of L-tyrosinase to give 3, 4-dihydroxyphenylalanine (L-DOPA) and the oxidation of L-DOPA to dopaquinone. This quinone is highly reactive and can spontaneously polymerize melanin. The effects of the extracts on monophenolase activities, using L-tyrosinase as a substrate and diphenolase on L-DOPA, were followed by inhibiting the formation of dopachrome by spectrophotometric measurements. The absorbance results shown at 300 seconds are shown, as the final time, for concentrations of 0.75mg / ml of the extracts in Table 5. The lower the absorbance developed, the greater the inhibitory capacity. All heat treated extracts are within the same range and have a greater capacity for inhibition than the heat-treated phenolic extract.
7. Inhibición de la agregación plaquetaria: Se estudió un rango de concentraciones de entre 50-2000 mg/1 del extracto EF, usando colágeno y TRAP (péptido agonista del receptor de la trombina) como agonistas o estimulantes de la agregación. Los porcentajes de inhibición observados (Figura 1) son bastante altos, incrementándose éstos con la concentración usada de EF, los cuales llegan hasta un 90% en el caso de usar colágeno o de un 70% para el TRAP, o de un 10% en el caso de concentraciones de EA a las que sus componentes lo están a nivel fisiológico (100-200 mg de EA/1) .  7. Inhibition of platelet aggregation: A range of concentrations between 50-2000 mg / 1 of the EF extract was studied, using collagen and TRAP (thrombin receptor agonist peptide) as agonists or aggregation stimulants. The percentages of inhibition observed (Figure 1) are quite high, increasing with the concentration of EF used, which reach up to 90% in the case of using collagen or 70% for TRAP, or 10% in the case of concentrations of EA at which its components are at the physiological level (100-200 mg of EA / 1).
El alto porcentaje de inhibición muestra al nuevo extracto fenólico como un posible agente para la prevención de enfermedades cardiovasculares.  The high percentage of inhibition shows the new phenolic extract as a possible agent for the prevention of cardiovascular diseases.

Claims

Reivindicaciones Claims
1. Extracto fenólico obtenido a partir de subproductos derivados de la extracción del aceite de oliva y/o de la industria de aderezo de la aceituna, caracterizado por que comprende una composición media en alcoholes fenólicos totales por encima del 5% en peso seco y una composición media en ácidos fenólicos totales por encima del 0.3% en peso seco . 1. Phenolic extract obtained from by-products derived from the extraction of olive oil and / or from the olive dressing industry, characterized in that it comprises an average composition in total phenolic alcohols above 5% by dry weight and a average composition in total phenolic acids above 0.3% dry weight.
2. Extracto fenólico, de acuerdo a la reivindicación 1, caracterizado por que comprende al menos un compuesto fenólico seleccionado entre pinoresinol, 1- acetoxipinoresinol , hidroximetil furfural , l-fenil-6,7- dihidroi socromona o una fracción fenólica polimérica FFP, asi como cualquiera de sus combinaciones. 2. Phenolic extract according to claim 1, characterized in that it comprises at least one phenolic compound selected from pinoresinol, 1- acetoxyproinoresinol, hydroxymethyl furfural, l-phenyl-6,7-dihydroi socromone or a polymeric phenolic fraction FFP, thus Like any of your combinations.
3. Proceso de obtención de un extracto fenólico a una cualquiera de las reivindicaciones 1 a 2, caracterizado por que comprende una etapa previa de tratamiento térmico entre 50°C y 250°C del subproducto de aceituna, seguido de una etapa de extracción del extracto fenólico. 3. Process for obtaining a phenolic extract according to any one of claims 1 to 2, characterized in that it comprises a previous stage of heat treatment between 50 ° C and 250 ° C of the olive by-product, followed by an extraction stage of the extract phenolic
4. Proceso de acuerdo a la reivindicación 3, caracterizado por que comprende una etapa adicional de separación sólido- liquido, posterior al tratamiento térmico, dando lugar a una fase sólida y a una fase liquida, seguida de una etapa de extracción por separado del extracto fenólico de la fase liquida y de la fase sólida. 4. Process according to claim 3, characterized in that it comprises an additional solid-liquid separation stage, after the heat treatment, giving rise to a solid phase and a liquid phase, followed by a separate extraction stage of the phenolic extract of the liquid phase and the solid phase.
5. Proceso de acuerdo a la reivindicación 3 ó 4, donde dicha extracción se lleva a cabo mediante el empleo de al menos un disolvente orgánico. 5. Process according to claim 3 or 4, wherein said extraction is carried out by using at least one organic solvent.
6. Proceso, de acuerdo a una cualquiera de las reivindicaciones 3 a 5, donde durante la extracción se lleva a cabo la aplicación de temperatura entre 50°C y 100°C. 6. Process according to any one of claims 3 to 5, wherein during the extraction the temperature application between 50 ° C and 100 ° C is carried out.
7. Proceso, de acuerdo a una cualquiera de las reivindicaciones 3 a 6, caracterizado por que comprende una etapa adicional de concentración total o parcial del extracto fenólico. 7. Process according to any one of claims 3 to 6, characterized in that it comprises an additional stage of total or partial concentration of the phenolic extract.
8. Proceso, de acuerdo a una cualquiera de las reivindicaciones 3 a 7, caracterizado por que comprende una etapa adicional de microencapsulación o nanoencapsulacion del extracto fenólico; de absorción o adsorción del extracto fenólico en cualquier tipo de soporte; o de formación de una emulsión del extracto fenólico. 8. Process according to any one of claims 3 to 7, characterized in that it comprises an additional step of microencapsulation or nanoencapsulation of the phenolic extract; of absorption or adsorption of the phenolic extract in any type of support; or for the formation of an emulsion of the phenolic extract.
9. Extracto fenólico obtenible a partir de un proceso de acuerdo a una cualquiera de las reivindicaciones 3 a 8. 9. Phenolic extract obtainable from a process according to any one of claims 3 to 8.
10. Formulación alimentaria, cosmética y/o farmaceútica caracterizada por que comprende un extracto fenólico de acuerdo a las reivindicaciones 1, 2 ó 9. 10. Food, cosmetic and / or pharmaceutical formulation characterized in that it comprises a phenolic extract according to claims 1, 2 or 9.
11. Uso de un extracto fenólico según una cualquiera de las reivindicaciones 1, 2 ó 9 como antioxidante para la preparación de preparados tanto lipófilos como hidrófilos, con uso alimentario, cosmético y/o farmaceútico . 11. Use of a phenolic extract according to any one of claims 1, 2 or 9 as an antioxidant for the preparation of both lipophilic and hydrophilic preparations, with food, cosmetic and / or pharmaceutical use.
12. Uso de un extracto fenólico según una cualquiera de las reivindicaciones 1, 2 ó 9 como agente inhibitorio de la acción de la enzima L-tirosinasa, implicada en procesos de pardeamiento . 12. Use of a phenolic extract according to any one of claims 1, 2 or 9 as an inhibitory agent for the action of the enzyme L-tyrosinase, involved in browning processes.
13. Uso de un extracto fenólico según una cualquiera de las reivindicaciones 1, 2 ó 9 como agente inhibitorio de la agregación plaquetaria en la prevención de enfermedades coronarias . 13. Use of a phenolic extract according to any one of claims 1, 2 or 9 as an inhibitor of platelet aggregation in the prevention of coronary heart disease.
14. Fracción o fracciones parcial o totalmente aisladas obtenibles a partir de un extracto fenólico según una cualquiera de las reivindicaciones 1, 2 14. Partial or fully isolated fraction or fractions obtainable from a phenolic extract according to any one of claims 1, 2
PCT/ES2012/070441 2011-06-21 2012-06-14 Phenol extract from heat-treated olive subproducts WO2012175771A1 (en)

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