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WO2018134460A1 - Egg preparation with anti-inflammatory and anti-oxidant properties - Google Patents

Egg preparation with anti-inflammatory and anti-oxidant properties Download PDF

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Publication number
WO2018134460A1
WO2018134460A1 PCT/ES2018/070032 ES2018070032W WO2018134460A1 WO 2018134460 A1 WO2018134460 A1 WO 2018134460A1 ES 2018070032 W ES2018070032 W ES 2018070032W WO 2018134460 A1 WO2018134460 A1 WO 2018134460A1
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Prior art keywords
egg
preparation
temperature
amount
pressure
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PCT/ES2018/070032
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Spanish (es)
French (fr)
Inventor
Crisanto Palacios Gavilán
Christian Palacios Gazules
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Liofilizados Girona, Sl
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Publication of WO2018134460A1 publication Critical patent/WO2018134460A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/57—Birds; Materials from birds, e.g. eggs, feathers, egg white, egg yolk or endothelium corneum gigeriae galli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C50/00—Quinones
    • C07C50/02—Quinones with monocyclic quinoid structure
    • C07C50/04—Benzoquinones, i.e. C6H4O2

Definitions

  • the present invention relates to a fertilized egg preparation comprising coenzyme Q10 and one or more antioxidants, its preparation process, and its pharmaceutical, nutraceutical or cosmetic compositions. Additionally, it also refers to the use of said pharmaceutical, nutraceutical or cosmetic egg compositions for use as an anti-inflammatory, analgesic or cosmetic agent.
  • CFS chronic fatigue syndrome
  • Fibromyalgia is a syndrome characterized by widespread chronic pain and the presence of between 1 and 18 painful points of connection between muscle and tendon, which are known as "trigger points.”
  • the current therapeutic alternatives offer a wide range of treatments, without a largely significant response, which include a rheumatologic, neurological, psychiatric medical approach, treatment with anti-inflammatories or analgesics.
  • a syndrome of difficult diagnosis and treatment for which there is no clear etiology to date and there are enormous difficulties in the treatment of associated pain, which is often accompanied by depression and other psychiatric disorders derived from loss of mobility, chronic pain, and even social isolation in the most serious cases.
  • Inflammasomes which are macromolecules of the protein type, comprise NOD type receptors, an apoptosis-associated protein (ASC) and procaspase-1; such inflamasomes can be activated by the variation of different factors, including ionic concentration, intracellular or extracellular ATP, destabilization of phagolysosome, or for example, mechanisms of oxoreduction.
  • ASC apoptosis-associated protein
  • procaspase-1 procaspase-1
  • IL-1 ⁇ interleukin 1 ⁇
  • IL-18 interleukin 18
  • Zhou et al (2009) have also suggested the link between the inhibition of mitochondrial respiratory chain complexes I and III and the triggering of musculoskeletal syndromes, particularly fibromyalgia.
  • a lack of coenzyme Q10 was also observed (Villalba et al, 2000; Modi et al, 2006), as well as an increase in oxidative stress.
  • ROS reactive oxygen species
  • Coenzyme Q10 or CoQ10 is a 1,4-benzoquinone that can be found in three oxidation states known as ubiquinone (oxidized form), ubiquinol (reduced form) and semiquinone or ubisemiquinone (partially reduced form).
  • Coenzyme Q10 It is present in most eukaryotic cells, mainly in mitochondria, where it plays a key role in the oxidative phosphorylation chain responsible for the production of ATP (Mancini et al, 2011; Litarru et al, 2007).
  • CoQ10 can act as a carrier of 1 or 2 electrons, playing a central role in the electron transport chain, because it has iron-sulfur clusters that They can only accept one electron at a time, and because it acts as an antioxidant capable of capturing free radicals.
  • CoQ10 is currently classified as a lipophilic antioxidant, particularly in its reduced form, which represents more than 80% of the total CoQ10 in human plasma, and protects biological membranes and lipoproteins.
  • CoQ10 can also participate, in addition, in several aspects of redox control of cell signaling pathways, such as in the self-oxidation of semiquinone, which represents a primary source of hydrogen peroxide.
  • eggs represent one of the great pillars of human food, being a valuable source of protein;
  • egg preparations provide safe, non-toxic compositions, which in various studies have demonstrated their effectiveness in use as an anti-inflammatory and as an analgesic.
  • EP0904090 describes an anti-inflammatory composition obtained by separating a water soluble fraction from a whole egg, the white or the yolk, of an animal in a superimmunized state, from which an extract less than 3000 dalton is obtained. of average molecular weight with anti-inflammatory properties.
  • EP2765869 describes an egg preparation comprising a mixture of yolk and white with a ratio of yolk to white between 98%: 2% and 60%: 40%, respectively, both extracted from a fertilized egg incubated during a period between 18 and 36 hours.
  • the compositions described herein show certain expression patterns of growth factors suitable for the use of said compositions as analgesic, anti-inflammatory and regenerative agents.
  • CA2197050 describes the use of compositions derived from incubated fertilized eggs, comprising physiologically tolerable carriers and excipients, for the treatment or prevention of cancer.
  • An objective of the present invention is to provide an egg preparation comprising an egg product extracted from an incubated fertilized egg, coenzyme Q10, and one or more antioxidants.
  • stimulation phase is understood the stage of embryonic development that happens to the formation of the blastula, and which results in the formation of the three fundamental layers of the embryo, i.e. ectoderm, mesoderm and endoderm.
  • Neurostimulation phase means the stage of embryonic development that occurs in the final stages of gastrulation, and which includes the formation of the central nervous system.
  • Supercritical fluid extraction or SFE (ie supercritical fluid extractior ⁇ ) is known in the state of the art as the process of separating a component (ie extractant) with respect to another (ie matrix) using supercritical fluids as extracting solvents, such as carbon dioxide.
  • supercritical fluid refers to a gas subjected to high compression, under certain critical conditions of temperature and pressure, so that it has combined properties of gases and liquids.
  • supercritical carbon dioxide supercritical extraction conditions assume a critical temperature of 31 ° C and a critical pressure of 74 bar.
  • bitterry product or “nutraceutical composition” refers to products or compositions derived from foods intended to provide additional health benefits compared to the basic nutritional value of such foods. Usually, these products are highly regulated by the authorities, and are usually pharmaceutical grade nutrients.
  • acceptable pharmaceutical excipients or carriers refers to suitable excipients or carriers, from a medical point of view, for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or any other problem or complication, commensurable with a reasonable benefit / risk ratio.
  • bitteretically acceptable excipients or carriers refers to suitable excipients or carriers, from the food point of view, for use in nutraceutical products or compositions.
  • cosmetically acceptable excipients or carriers refers to excipients or carriers suitable for use in contact with human or animal skin without excessive toxicity, incompatibility, instability, allergic response, among others.
  • the term "effective amount” refers to an amount sufficient to obtain an expected effect.
  • the present invention comprises an egg preparation comprising: a) an egg product that has been previously extracted from an incubated fertilized egg,
  • said egg product has been previously extracted from a fertilized egg incubated for a period of time between 0 and 17 hours, which makes it possible to control that the egg remains in the gastrulation phase, without the neurulation phase having yet begun. , thus ensuring that said egg preparation contains pluripotent cells.
  • 0 hours refers to the moment in which fertilization occurs and therefore the beginning of embryonic development.
  • the egg product has been previously extracted from a fertilized bird egg.
  • said bird is selected from hen, goose, duck, quail, turkey, ostrich, pheasant and dove; In an even more preferred embodiment, said bird is a chicken.
  • the egg preparation of the present invention comprises an amount of coenzyme Q10 comprised between 0.1% and 4.5% by weight with respect to the total weight of said preparation, more preferably an amount of coenzyme Q10 comprised between 0.2% and 2.2% by weight with respect to the total weight of said preparation, and even more preferably, an amount of coenzyme Q10 comprised between 0.4% and 0.7% by weight with respect to the total weight of said preparation.
  • the egg preparation of the invention comprises an amount of one or more antioxidants comprised between 2.2% and 6.7% by weight with respect to the total weight of said preparation; preferably, the egg preparation comprises an amount of one or more antioxidants comprised between 1.7% and 5.6% by weight with respect to the total weight of said preparation.
  • Said one or more antioxidants are selected from the group consisting of, but not limited to superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), ascorbic acid (ie vitamin C), ⁇ -tocopherol (ie vitamin E), retinol, ⁇ -carotene (ie vitamin A precursor), metallothionein, melatonin, taurine, selenium, resveratrol, methionine, polyphenols, isoflavonones, S-adenosyl-methionine, and any of its mixtures.
  • SOD superoxide dismutase
  • SOD comprises a cofactor, which is selected from the group consisting of copper, zinc, manganese and iron.
  • Another aspect of the present invention comprises a process for the preparation of the egg preparation described above, comprising: a) incubating a fertilized egg,
  • a fertilized egg is preferably incubated for a period of time between 0 and 17 hours, in order to control that the incubated fertilized egg is kept in gastrulation phase, without The neurulation phase has still begun, thus ensuring that the egg preparation obtained contains pluripotent cells. More preferably, the fertilized egg is incubated for a period of time between 0 and 17 hours, at a temperature between 34 ° C and 41 ° C, and a relative humidity between 30% and 75%; even more preferably, the fertilized egg is incubated for a period of time between 0 and 17 hours at a temperature of 38 ° C and a relative humidity between 55% and 60%.
  • Stage c) corresponds to a sterilization process to suppress bacteria, molds and other microorganisms potentially harmful to health, which is based on the optimization of a process known as high pressure processing or HPP ("high-pressure processing”), widely used in the food sector.
  • the pressure of step c) of the process described above is 6000 bar.
  • the temperature of step c) is 9 ° C; further, preferably, the duration of step c) is 5 minutes. More preferably, step c) is carried out at a pressure of 6000 bar and a temperature of 9 ° C, for 5 minutes.
  • stage d) The selection of the temperature of stage d) is important for the maintenance of the biological conditions suitable for the good preservation of the egg product and its properties.
  • the temperature when the drying of the egg product is carried out at atmospheric pressure, the temperature must be below 60 ° C, preferably, a temperature between 5 ° C and 50 ° C, to avoid the acceleration of embryonic development; more preferably, the drying of the egg product of step d) is carried out at a temperature between 25 ° C and 40 ° C.
  • the temperature may be equal to or greater than 60 ° C. It will be obvious to the person skilled in the art that when working under pressure conditions corresponding to vacuum pressure, it will be possible to work with a temperature equal to or greater than 60 ° C, although it will be necessary to adjust these conditions to preserve the properties of the egg product .
  • Said step d) of drying can be carried out by numerous methods known within the state of the art, such as lyophilization, spray-drying, microwave drying, spray drying, and mixer drying under vacuum conditions.
  • a mixer under vacuum conditions preferably a temperature of 30 ° C will be used.
  • Step f) of adding an amount of coenzyme Q10 and an amount of one or more antioxidants is preferably carried out in a mixer.
  • steps d) -f) can be carried out in a band mixer at a rotation speed equal to or greater than 5 turns per minute. The use of this type of mixer allows to carry out, simultaneously, the drying of the product, and a homogeneous micronization and mixing.
  • the egg product obtained through the present process of the invention is in the form of nanocapsules.
  • Said nanocapsules can be obtained by means of any of the methods of obtaining known in the state of the art, which will be obvious to the person skilled in the art.
  • This final egg product in the form of nanocapsules has an analgesic, anti-inflammatory or cosmetic activity 10 times higher than that obtained with the non-encapsulated egg product.
  • the method of obtaining the egg preparation described above further comprises a step of extracting cholesterol by means of supercritical fluid extraction (SFE) technology, which can be carried out after stage d), and prior to stage e).
  • Said additional extraction stage will be carried out at a pressure between 150 and 450 bar, and a temperature between 15 ° C and 70 ° C.
  • said additional stage of supercritical fluid extraction of growth factors and cholesterol can be carried out at a pressure of 275 bar and a temperature of 40 ° C.
  • the supercritical fluid extraction technique is a clean and safe, completely safe option, which allows components to be separated efficiently, while preserving their characteristics, which has led to its rapid implementation in the food sector.
  • examples of supercritical fluid applicable in step e) include carbon dioxide or SC-C0 2 , water, ethane, propane, methanol and ethanol, all in supercritical form, that is, handled under temperature conditions and Critical pressure suitable for each of them, as will be apparent to a person skilled in the art.
  • the method of obtaining the egg preparation described above comprises an additional step comprising the extraction of saturated and unsaturated fats and oils from said egg preparation by extraction with supercritical fluid; said additional stage can be carried out according to the conditions described above, and can be carried out after stage d) and prior to stage e).
  • SFC extraction it is possible to separate the saturated and unsaturated fats and oils, so that the subsequent grinding (stage e) and addition of a quantity of coenzyme Q10 and an amount of one or more antioxidants (stage f) and takes conducted only on an egg protein concentrate free of fats and oils.
  • This alternative embodiment also comprises a second additional stage, subsequent to step f), of re-incorporating saturated and unsaturated fats and oils in a whisk, in which the corresponding final emulsion is obtained. That is, according to this alternative embodiment, the method of obtaining the egg preparation described above comprises the following steps:
  • step d2) extract saturated and unsaturated fats and oils from the product obtained in step d) by extracting with supercritical fluid
  • step d2) grind the egg protein concentrate free of saturated and unsaturated fats and oils resulting from step d2).
  • step f) add an amount of coenzyme Q10 and an amount of one or more antioxidants, f2) reinstate the saturated and unsaturated fats and oils obtained in step d2), on the mixture obtained in step f.
  • the egg preparation of the present invention is administered to mammals, more preferably, to humans.
  • this third aspect of the invention relates to pharmaceutical, nutraceutical or cosmetic compositions comprising an effective amount of egg preparation described above, and one or more suitable pharmaceutical, nutraceutical or cosmetic excipients or carriers, respectively.
  • the present invention provides a new solution that in addition to providing the natural nutrients of the egg itself, provides antioxidants, with the consequent reduction of oxidative stress and coenzyme Q10, which in combination with said antioxidants, has demonstrated a significant effect. in the control of oxidative stress, and in the improvement of the efficiency of the mitochondrial respiratory chain.
  • Said pharmaceutical, nutraceutical or cosmetic compositions are preferably characterized by a dosage form selected from: a) a solid form selected from powder, tablet, coated tablet, capsule, coated capsule, granule, envelope, encapsulated nanoparticle, and
  • a liquid form selected from solution, spray, cream, emulsion, gel, paste, shampoo and serum.
  • these pharmaceutical, nutraceutical or cosmetic compositions are described which comprise an effective amount of egg preparation for use as an anti-inflammatory agent for the treatment of anti-inflammatory conditions.
  • this composition may be administered in combination with one or more known anti-inflammatory agents.
  • these pharmaceutical, nutraceutical or cosmetic compositions which comprise an effective amount of egg preparation for use as an analgesic agent for the treatment of acute or chronic pain under pain-related conditions.
  • this composition may be administered in combination with one or more known analgesic agents.
  • Conditions related to acute or chronic pain include, but are not limited to, chronic fatigue syndrome, fibromyalgia or adrenal fatigue.
  • Figure 1 shows the percentage of healthy cells (fibroblasts) after 17, 23 and 44 hours of treatment.
  • Figure 1 shows the percentage of tumor cells (HepG2) after 17, 23 and 44 hours of treatment.
  • Figure 3 Percentage of healthy cells (fibroblasts) at the time OH, 24 and 48 hours of treatment.
  • Figure 4 shows the protein study of the effect of egg extract treatment.
  • FIG. 5 shows the weight control in mice at the beginning and end of the different treatments.
  • Figure 6 shows the pain test as an effect of the different treatments.
  • Figure 7 shows the depression trial (forced swimming test for depression) as an effect of the different treatments.
  • Figure 8 shows the levels of the inflammatory marker IL-1 beta.
  • Figure 9 shows the weight control at the beginning and end of the different treatments.
  • Figure 10 shows the pain test as an effect of the different treatments.
  • Figure 11 shows the depression test as an effect of the different treatments.
  • Figure 12 shows the levels of the inflammatory marker IL-1 beta.
  • Figure 13 shows the protein study of the effect of treatment with egg extract and CoQ10.
  • Figure 14A shows the levels of ATP; 14B: shows the levels of lipid peroxidation determined in muscle and liver 14C.
  • Figure 15 shows the levels of MnSOD determined in muscle. Detailed description of the invention
  • the present invention relates to a fertilized egg preparation comprising coenzyme Q10 and one or more antioxidants, its preparation process and pharmaceutical, nutraceutical or cosmetic compositions comprising said egg preparation and the use thereof for use as an anti-inflammatory agent. , analgesic or cosmetic.
  • Example 1 In vitro study of the effects of egg extract.
  • Example 2 In vivo study of the effects of egg extract.
  • mice were subjected to different treatments, 5 to the treatment of para-minoabenzoic acid (PABA), an inhibitor of the Te CoQ10 synthesis that reproduces the symptoms of fibromyalgia, 5 to PABA plus Egg of 17 hours and 5 without Treatment as a negative control.
  • PABA para-minoabenzoic acid
  • FIG. 6 shows the results of the hot plate test in which the mice, subjected to a temperature of 56 degrees, assess the time it takes to react to pain and therefore the pain threshold. A lower threshold was observed in the PABA group, as well as a significant increase in the egg group which demonstrates the increase in analgesia.
  • Example 3 In vivo study of the effects of egg extract compared to CoQ10.
  • mice were subjected to different treatments, 5 to the treatment of para-minoabenzoic acid (PABA), a coQ10 synthesis inhibitor that reproduces the symptoms of fibromyalgia and that our group has patented, to 5 PABA plus Egg of h hours, 5 PABA plus CoQ10 30mg, 5 the combination of PABA, egg 0 hours and CoQ10 and 5 without treatment as a negative control.
  • PABA para-minoabenzoic acid
  • egg treatment induced high levels of ATP production, thus stimulating bioenergetic cells against the PABA-induced deficit.
  • fertilized egg extract induces changes both at the cellular level and in animal models that lead us to think of a beneficial effect from the point of view of oxidative stress and inflammation. It induces cell growth and moderates the effect of apoptosis in addition to stimulating the production of ATP. In addition, it shows beneficial effects in terms of analgesia and antidepressant effect in our model. patented mouse. It shows no toxicity effects in the parameters evaluated by us or in the metabolism.
  • the combination with the well-known antioxidant CoQ10 shows greater efficacy in depression, but not in pain where CoQ10 is most effective.

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Abstract

The present invention relates to a fertilised egg preparation comprising co-enzyme Q10 and one or more anti-oxidants, to the method for preparing same and to pharmaceutical, nutraceutical or cosmetic compositions containing same. In addition, the invention also relates to the use of these pharmaceutical, nutraceutical or cosmetic egg compositions as anti-inflammatory, analgesic or cosmetic products.

Description

Preparado de huevo con propiedades antiinflamatorias y antioxidantes  Egg preparation with anti-inflammatory and antioxidant properties
DESCRIPCIÓN DESCRIPTION
La presente invención se refiere a un preparado de huevo fertilizado que comprende coenzima Q10 y uno o más antioxidantes, su procedimiento de preparación, y sus composiciones farmacéuticas, nutracéuticas o cosméticas. Adicionalmente, también se refiere al uso de dichas composiciones farmacéuticas, nutracéuticas o cosméticas de huevo para su uso como agente antiinflamatorio, analgésico o cosmético. The present invention relates to a fertilized egg preparation comprising coenzyme Q10 and one or more antioxidants, its preparation process, and its pharmaceutical, nutraceutical or cosmetic compositions. Additionally, it also refers to the use of said pharmaceutical, nutraceutical or cosmetic egg compositions for use as an anti-inflammatory, analgesic or cosmetic agent.
Antecedentes de la invención Background of the invention
Existen numerosas enfermedades, tales como la fibromialgia y el síndrome de fatiga crónica (SFC), caracterizadas por dolor músculo-esquelético generalizado e hipersensibilidad, pero en las que no se evidencian causas orgánicas demostrables. There are numerous diseases, such as fibromyalgia and chronic fatigue syndrome (CFS), characterized by widespread musculoskeletal pain and hypersensitivity, but in which no demonstrable organic causes are evident.
La fibromialgia es un síndrome caracterizado por dolor crónico generalizado y la presencia de entre 1 1 y 18 puntos dolorosos de conexión entre músculo y tendón, que se conocen como "puntos gatillo". Las alternativas terapéuticas actuales ofrecen un amplio abanico de tratamientos, sin una respuesta ampliamente significativa en su gran mayoría, que incluyen un abordaje médico desde el punto de vista reumatológico, neurológico, psiquiátrico, el tratamiento con antiinflamatorios o con analgésicos. Además, ya que se trata de un síndrome de difícil diagnóstico y tratamiento, para el que no existe hasta la fecha una etiología clara y existen enormes dificultades en el tratamiento del dolor asociado, que a menudo se acompaña de depresión y otros trastornos psiquiátricos derivados de la pérdida de movilidad, dolor crónico, e incluso aislamiento social en los casos más graves. Según la revisión realizada por Busse et al (2013), la tasa de prevalencia de fibromialgia en España en 2013 era de un 2,4%, y por ejemplo, un 2% de la población total de Estados Unidos. Esto se traduce también en un aumento de los costes sanitarios y del absentismo laboral, con un impacto económico significativo. Fibromyalgia is a syndrome characterized by widespread chronic pain and the presence of between 1 and 18 painful points of connection between muscle and tendon, which are known as "trigger points." The current therapeutic alternatives offer a wide range of treatments, without a largely significant response, which include a rheumatologic, neurological, psychiatric medical approach, treatment with anti-inflammatories or analgesics. In addition, since it is a syndrome of difficult diagnosis and treatment, for which there is no clear etiology to date and there are enormous difficulties in the treatment of associated pain, which is often accompanied by depression and other psychiatric disorders derived from loss of mobility, chronic pain, and even social isolation in the most serious cases. According to the review by Busse et al (2013), the prevalence rate of fibromyalgia in Spain in 2013 was 2.4%, and for example, 2% of the total population of the United States. This also translates into an increase in health costs and absenteeism, with a significant economic impact.
A lo largo de los años se han planteado diferentes hipótesis acerca de este tipo de síndromes de afectación músculo-esquelética, y entre ellas, merecen una especial mención los estudios realizados por Salem et al (2003), Wang et al (2008), Poazzichi et al (2007), l Nakamura et al (2010) y Togo et al (2009). Dichos estudios comprobaron que uno de los mecanismos de activación de estos síndromes, especialmente la fibromialgia, están relacionados con la presencia de inflamación, disfunción mitocondrial y estrés oxidativo. Over the years, different hypotheses have been raised about this type of musculoskeletal involvement syndromes, and among them, studies by Salem et al (2003), Wang et al (2008), Poazzichi deserve special mention et al (2007), l Nakamura et al (2010) and Togo et al (2009). These studies found that one of the mechanisms of activation of these syndromes, especially fibromyalgia, are related to the presence of inflammation, mitochondrial dysfunction and oxidative stress.
Se conoce que la inflamación es una respuesta biológica del sistema inmune dirigida a eliminar estímulos capaces de producir daño, y a iniciar la curación y la reparación. Los inflamasomas, que son macromoléculas de tipo proteico, comprenden unos receptores de tipo NOD, una proteína asociada a apoptosis (ASC) y la procaspasa-1 ; dichos inflamasomas pueden ser activados por la variación de diferentes factores, entre ellos la concentración iónica, el ATP intracelular o extracelular, la desestabilización del fagolisosoma, o por ejemplo, mecanismos de oxoreducción. También sustancias que puedan formarse durante una infección, un daño en un tejido e incluso un desequilibro metabólico podrían desencadenar la formación de la caspasa-1 , que de forma proteolítica activa las citoquinas proinflamatorias interleucina 1 β (IL-1 β) y la interleucina 18 (IL-18). Además, la activación de los inflamasomas puede llegar a causar incluso una forma rápida proinflamatoria de muerte celular conocida como piroptosis (Latz et al, 2013). It is known that inflammation is a biological response of the immune system aimed at eliminating stimuli capable of causing damage, and initiating healing and repair. Inflammasomes, which are macromolecules of the protein type, comprise NOD type receptors, an apoptosis-associated protein (ASC) and procaspase-1; such inflamasomes can be activated by the variation of different factors, including ionic concentration, intracellular or extracellular ATP, destabilization of phagolysosome, or for example, mechanisms of oxoreduction. Also substances that can be formed during an infection, a tissue damage and even a metabolic imbalance could trigger the formation of caspase-1, which in a proteolytic way activates the proinflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 (IL-18). In addition, the activation of inflamasomes can even cause a rapid proinflammatory form of cell death known as piroptosis (Latz et al, 2013).
Adicionalmente, Zhou et al (2009) también han sugerido el vínculo existente entre la inhibición de los complejos I y III de la cadena respiratoria mitocondrial y el desencadenamiento de los síndromes músculo-esqueléticos, particularmente fibromialgia. La afectación en los complejos de la cadena respiratoria mitocondrial, especialmente los complejos I, III y IV, provoca una disminución significativa en el ATP resultante, y en consecuencia, un deterioro de la respiración celular. En muchos casos de afectación de los complejos mitocondriales, se observó además una carencia de coenzima Q10 (Villalba et al, 2000; Modi et al, 2006), así como un aumento del estrés oxidativo. Este último conduce a la rápida y excesiva formación de especies reactivas de oxígeno (ROS o reactive oxygen species) debido a una disfunción en el metabolismo oxidativo mitocondrial, que afectarán a la capacidad de respuesta antioxidante de las células, conduciendo a un daño macromolecular, y numerosas enfermedades tales como arterieesclerosis, diabetes, cáncer, neurodegeneración o envejecimiento (Tsuji et al, 2012). Additionally, Zhou et al (2009) have also suggested the link between the inhibition of mitochondrial respiratory chain complexes I and III and the triggering of musculoskeletal syndromes, particularly fibromyalgia. The involvement in mitochondrial respiratory chain complexes, especially complexes I, III and IV, causes a significant decrease in the resulting ATP, and consequently, a deterioration of cellular respiration. In many cases of mitochondrial complex involvement, a lack of coenzyme Q10 was also observed (Villalba et al, 2000; Modi et al, 2006), as well as an increase in oxidative stress. The latter leads to the rapid and excessive formation of reactive oxygen species (ROS or reactive oxygen species) due to a dysfunction in mitochondrial oxidative metabolism, which will affect the antioxidant response capacity of cells, leading to macromolecular damage, and numerous diseases such as arteriosclerosis, diabetes, cancer, neurodegeneration or aging (Tsuji et al, 2012).
La coenzima Q10 o CoQ10 es una 1 ,4-benzoquinona que se puede encontrar en tres estados de oxidación conocidos como ubiquinona (forma oxidada), ubiquinol (forma reducida) y semiquinona o ubisemiquinona (forma parcialmente reducida). La coenzima Q10 está presente en la mayoría de células eucariotas, fundamentalmente en las mitocondrias, en donde juega un papel clave en la cadena de fosforilación oxidativa encargada de la producción de ATP (Mancini et al, 2011 ; Litarru et al, 2007). En función de su estado de oxidación y situación del equilibrio de oxidación-reducción, la CoQ10 puede actuar como portadora de 1 o 2 electrones, desempeñando un papel central en la cadena transportadora de electrones, debido a que esta presenta clústeres de hierro-azufre que únicamente pueden aceptar un electrón cada vez, y porque actúa como un antioxidante capaz de capturar radicales libres. En este sentido, la CoQ10 se clasifica actualmente como un antioxidante lipofílico, en particular en su forma reducida, que representa más del 80% del total de CoQ10 en el plasma humano, y protege membranas biológicas y lipoproteínas. El CoQ10 también puede participar, además, en varios aspectos del control redox de las vías de señalización celular, como por ejemplo en la autooxidación de la semiquinona, que representa una fuente primaria de peróxido de hidrógeno. Coenzyme Q10 or CoQ10 is a 1,4-benzoquinone that can be found in three oxidation states known as ubiquinone (oxidized form), ubiquinol (reduced form) and semiquinone or ubisemiquinone (partially reduced form). Coenzyme Q10 It is present in most eukaryotic cells, mainly in mitochondria, where it plays a key role in the oxidative phosphorylation chain responsible for the production of ATP (Mancini et al, 2011; Litarru et al, 2007). Depending on its oxidation state and oxidation-reduction equilibrium situation, CoQ10 can act as a carrier of 1 or 2 electrons, playing a central role in the electron transport chain, because it has iron-sulfur clusters that They can only accept one electron at a time, and because it acts as an antioxidant capable of capturing free radicals. In this sense, CoQ10 is currently classified as a lipophilic antioxidant, particularly in its reduced form, which represents more than 80% of the total CoQ10 in human plasma, and protects biological membranes and lipoproteins. CoQ10 can also participate, in addition, in several aspects of redox control of cell signaling pathways, such as in the self-oxidation of semiquinone, which represents a primary source of hydrogen peroxide.
De todo lo anterior se desprende la necesidad de desarrollar nuevas composiciones que permitan controlar el estrés oxidativo y por lo tanto también la formación de especies reactivas de oxígeno, con el objetivo final de ofrecer una alternativa viable para el tratamiento de enfermedades asociadas a dolor crónico o agudo, tales como la fibromialgia o el síndrome de la fatiga crónica, asociadas a condiciones inflamatorias a través del vínculo observado entre los inflamasomas y el desencadenamiento de dichos procesos inflamatorios. Además, resultaría especialmente ventajoso el desarrollo de nuevas composiciones que evitasen el uso habitual de dosis significativas de antiinflamatorios y analgésicos de potencia elevada, para evitar efectos secundarios derivados de su uso prolongado, algo habitual en las condiciones arriba mencionadas. From all of the above, it is clear the need to develop new compositions that allow the control of oxidative stress and therefore also the formation of reactive oxygen species, with the final objective of offering a viable alternative for the treatment of diseases associated with chronic pain or acute, such as fibromyalgia or chronic fatigue syndrome, associated with inflammatory conditions through the observed link between inflamasomes and the triggering of such inflammatory processes. In addition, it would be especially advantageous to develop new compositions that avoid the usual use of significant doses of high-potency anti-inflammatories and analgesics, to avoid side effects derived from their prolonged use, which is common in the above-mentioned conditions.
Se conoce ampliamente en el estado de la técnica que los huevos representan uno de los grandes pilares de la alimentación humana, siendo una valiosa fuente de proteínas; además, tras una correcta esterilización, los preparados de huevo proporcionan composiciones seguras, no tóxicas, que en diversos estudios han demostrado su eficacia en el uso como antiinflamatorio y como analgésico. It is widely known in the state of the art that eggs represent one of the great pillars of human food, being a valuable source of protein; In addition, after proper sterilization, egg preparations provide safe, non-toxic compositions, which in various studies have demonstrated their effectiveness in use as an anti-inflammatory and as an analgesic.
El documento EP0904090 describe una composición antiinflamatoria obtenida por separación de una fracción soluble en agua de un huevo completo, la clara o la yema, de un animal en estado superimmunizado, de la cual se obtiene un extracto inferior a 3000 dalton de peso molecular promedio con propiedades antiinflamatorias. EP0904090 describes an anti-inflammatory composition obtained by separating a water soluble fraction from a whole egg, the white or the yolk, of an animal in a superimmunized state, from which an extract less than 3000 dalton is obtained. of average molecular weight with anti-inflammatory properties.
Por otro lado, EP2765869 describe un preparado de huevo que comprende una mezcla de yema y clara con una proporción de yema respecto a clara comprendida entre 98%:2% y 60%:40%, respectivamente, ambas extraídas de un huevo fertilizado incubado durante un período comprendido entre 18 y 36 horas. Las composiciones aquí descritas muestran ciertos patrones de expresión de factores de crecimiento aptos para el uso de dichas composiciones como agentes analgésicos, antiinflamatorios y regeneradores. On the other hand, EP2765869 describes an egg preparation comprising a mixture of yolk and white with a ratio of yolk to white between 98%: 2% and 60%: 40%, respectively, both extracted from a fertilized egg incubated during a period between 18 and 36 hours. The compositions described herein show certain expression patterns of growth factors suitable for the use of said compositions as analgesic, anti-inflammatory and regenerative agents.
En tercer lugar, CA2197050 describe el uso de composiciones procedentes de huevos fertilizados incubados, que comprenden portadores y excipientes fisiológicamente tolerables, para el tratamiento o prevención de cáncer. Thirdly, CA2197050 describes the use of compositions derived from incubated fertilized eggs, comprising physiologically tolerable carriers and excipients, for the treatment or prevention of cancer.
Sin embargo, aunque estos documentos del estado de la técnica muestran diferentes aproximaciones para el tratamiento de enfermedades antiinflamatorias, ninguno de ellos parece sugerir un enfoque específicamente dirigido hacia el control del estrés oxidativo y la corrección de la deficiencia de CoQ10, habitualmente asociada a la existencia de dicho estrés y al exceso de especies reactivas de oxígeno. However, although these state-of-the-art documents show different approaches for the treatment of anti-inflammatory diseases, none of them seems to suggest an approach specifically directed towards the control of oxidative stress and the correction of CoQ10 deficiency, usually associated with the existence of said stress and the excess of reactive oxygen species.
Descripción de la invención Description of the invention
Un objetivo de la presente invención consiste en proporcionar un preparado de huevo que comprende un producto de huevo extraído de un huevo fertilizado incubado, coenzima Q10, y uno o más antioxidantes. An objective of the present invention is to provide an egg preparation comprising an egg product extracted from an incubated fertilized egg, coenzyme Q10, and one or more antioxidants.
Por "fase de gastrulacion" se entiende la etapa de desarrollo embrionario que sucede a la formación de la blástula, y que tiene como resultado la formación de las tres capas fundamentales del embrión, i.e. ectodermo, mesodermo y endodermo. By "gastrulation phase" is understood the stage of embryonic development that happens to the formation of the blastula, and which results in the formation of the three fundamental layers of the embryo, i.e. ectoderm, mesoderm and endoderm.
Por "fase de neurulación" se entiende la etapa de desarrollo embrionaria que se produce en las etapas finales de la gastrulacion, y que comprende la formación del sistema nervioso central. "Neurulation phase" means the stage of embryonic development that occurs in the final stages of gastrulation, and which includes the formation of the central nervous system.
La "extracción con fluidos supercríticos" o SFE (i.e. supercritical fluid extractiorí) se conoce en el estado de la técnica como el proceso de separación de un componente (i.e. extractante) respecto a otro (i.e. matriz) utilizando fluidos supercríticos como disolventes extractores, como por ejemplo dióxido de carbono. "Supercritical fluid extraction" or SFE (ie supercritical fluid extractiorí) is known in the state of the art as the process of separating a component (ie extractant) with respect to another (ie matrix) using supercritical fluids as extracting solvents, such as carbon dioxide.
El término "fluido supercrítico" se refiere a un gas sometido a una elevada compresión, bajo ciertas condiciones críticas de temperatura y presión, de modo que presenta propiedades combinadas de gases y líquidos. Por ejemplo, en el caso del dióxido de carbono supercrítico, las condiciones de extracción supercrítica suponen una temperatura crítica de 31 °C y una presión crítica de 74 bar. The term "supercritical fluid" refers to a gas subjected to high compression, under certain critical conditions of temperature and pressure, so that it has combined properties of gases and liquids. For example, in the case of supercritical carbon dioxide, supercritical extraction conditions assume a critical temperature of 31 ° C and a critical pressure of 74 bar.
El término "producto nutracéutico" o "composición nutracéutica" se refiere a productos o composiciones derivados de alimentos destinados a proporcionar beneficios adicionales de salud en comparación con el valor nutricional básico de dichos alimentos. Habitualmente, se trata de productos altamente regulados por las autoridades, y suelen ser nutrientes de grado farmacéutico. The term "nutraceutical product" or "nutraceutical composition" refers to products or compositions derived from foods intended to provide additional health benefits compared to the basic nutritional value of such foods. Usually, these products are highly regulated by the authorities, and are usually pharmaceutical grade nutrients.
La expresión "excipientes o portadores farmacéuticos aceptables" hace referencia a excipientes o portadores adecuados, desde el punto de vista médico, para su uso en contacto con tejidos de humanos y animales sin una excesiva toxicidad, irritación, respuesta alérgica, o cualquier otro problema o complicación, conmensurable con una relación beneficio/riesgo razonable. The term "acceptable pharmaceutical excipients or carriers" refers to suitable excipients or carriers, from a medical point of view, for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or any other problem or complication, commensurable with a reasonable benefit / risk ratio.
La expresión "excipientes o portadores nutracéuticamente aceptables" hace referencia a excipientes o portadores adecuados, desde el punto de vista alimentario, para su uso en productos o composiciones nutracéuticas. The term "nutrachetically acceptable excipients or carriers" refers to suitable excipients or carriers, from the food point of view, for use in nutraceutical products or compositions.
La expresión "excipientes o portadores cosméticamente aceptables" se refiere a excipientes o portadores adecuados para su uso en contacto con la piel humana o animal sin excesiva toxicidad, incompatibilidad, inestabilidad, respuesta alérgica, entre otros. The term "cosmetically acceptable excipients or carriers" refers to excipients or carriers suitable for use in contact with human or animal skin without excessive toxicity, incompatibility, instability, allergic response, among others.
La expresión "cantidad efectiva" se refiere a una cantidad suficiente para obtener un efecto esperado. The term "effective amount" refers to an amount sufficient to obtain an expected effect.
La presente invención comprende un preparado de huevo que comprende: a) un producto de huevo que ha sido previamente extraído de un huevo fertilizado incubado, The present invention comprises an egg preparation comprising: a) an egg product that has been previously extracted from an incubated fertilized egg,
b) una cantidad de coenzima Q10, y  b) an amount of coenzyme Q10, and
c) una cantidad de uno o más antioxidantes.  c) an amount of one or more antioxidants.
Preferiblemente, dicho producto de huevo ha sido previamente extraído de un huevo fertilizado incubado durante un período de tiempo comprendido entre 0 y 17 horas, lo que permite controlar que el huevo se mantenga en fase de gastrulación, sin que haya comenzado todavía la fase de neurulación, asegurando así que dicho preparado de huevo contenga células pluripotentes. Preferably, said egg product has been previously extracted from a fertilized egg incubated for a period of time between 0 and 17 hours, which makes it possible to control that the egg remains in the gastrulation phase, without the neurulation phase having yet begun. , thus ensuring that said egg preparation contains pluripotent cells.
En la presente invención por "0 horas" se refiere al momento en el que se produce la fecundación y por lo tanto el inicio del desarrollo embrionario. In the present invention, "0 hours" refers to the moment in which fertilization occurs and therefore the beginning of embryonic development.
Más preferiblemente, el producto de huevo ha sido previamente extraído de un huevo fertilizado de ave. Todavía más preferiblemente, dicha ave se selecciona entre gallina, ganso, pato, codorniz, pavo, avestruz, faisán y paloma; en una realización todavía más preferida, dicha ave es una gallina. More preferably, the egg product has been previously extracted from a fertilized bird egg. Even more preferably, said bird is selected from hen, goose, duck, quail, turkey, ostrich, pheasant and dove; In an even more preferred embodiment, said bird is a chicken.
El preparado de huevo de la presente invención comprende una cantidad de coenzima Q10 comprendida entre 0, 1 % y 4,5% en peso con respecto al peso total de dicho preparado, más preferiblemente una cantidad de coenzima Q10 comprendida entre 0,2% y 2,2% en peso con respecto al peso total de dicho preparado, y todavía más preferiblemente, una cantidad de coenzima Q10 comprendida entre 0,4% y 0,7% en peso con respecto al peso total de dicho preparado. The egg preparation of the present invention comprises an amount of coenzyme Q10 comprised between 0.1% and 4.5% by weight with respect to the total weight of said preparation, more preferably an amount of coenzyme Q10 comprised between 0.2% and 2.2% by weight with respect to the total weight of said preparation, and even more preferably, an amount of coenzyme Q10 comprised between 0.4% and 0.7% by weight with respect to the total weight of said preparation.
Por otro lado, el preparado de huevo de la invención comprende una cantidad de uno o más antioxidantes comprendida entre 2,2% y 6,7% en peso respecto al peso total de dicho preparado; preferiblemente, el preparado de huevo comprende una cantidad de uno o más antioxidantes comprendida entre 1 ,7% y 5,6% en peso respecto al peso total de dicho preparado. On the other hand, the egg preparation of the invention comprises an amount of one or more antioxidants comprised between 2.2% and 6.7% by weight with respect to the total weight of said preparation; preferably, the egg preparation comprises an amount of one or more antioxidants comprised between 1.7% and 5.6% by weight with respect to the total weight of said preparation.
Dichos uno o más antioxidantes se seleccionan entre el grupo que consiste en, pero no limitado a superóxido dismutasa (SOD), catalasa (CAT), glutatión (GSH), ácido ascórbico (i.e. vitamina C), α-tocoferol (i.e. vitamina E), retinol, β-caroteno (i.e. precursor de vitamina A), metalotioneína, melatonina, taurina, selenio, resveratrol, metionina, polifenoles, isoflavononas, S-adenosil-metionina, y cualquiera de sus mezclas. Particularmente, la superóxido dismutasa (SOD) comprende un cofactor, que se selecciona entre el grupo que consiste en cobre, zinc, manganeso y hierro. Said one or more antioxidants are selected from the group consisting of, but not limited to superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), ascorbic acid (ie vitamin C), α-tocopherol (ie vitamin E), retinol, β-carotene (ie vitamin A precursor), metallothionein, melatonin, taurine, selenium, resveratrol, methionine, polyphenols, isoflavonones, S-adenosyl-methionine, and any of its mixtures. Particularly, superoxide dismutase (SOD) comprises a cofactor, which is selected from the group consisting of copper, zinc, manganese and iron.
Otro aspecto de la presente invención comprende un procedimiento para la preparación del preparado de huevo arriba descrito, que comprende: a) incubar un huevo fertilizado, Another aspect of the present invention comprises a process for the preparation of the egg preparation described above, comprising: a) incubating a fertilized egg,
b) romper el huevo y obtener el producto de huevo correspondiente,  b) break the egg and obtain the corresponding egg product,
c) someter el producto de huevo a una presión comprendida entre 1000 y 9000 bar, durante un tiempo comprendido entre 1 y 20 minutos, a una temperatura comprendida entre 6°C y 20°C,  c) subjecting the egg product to a pressure between 1000 and 9000 bar, for a time between 1 and 20 minutes, at a temperature between 6 ° C and 20 ° C,
d) secar el producto de huevo a una temperatura determinada, con la condición de que:  d) drying the egg product at a certain temperature, with the proviso that:
i. si la presión es atmosférica, dicha temperatura es inferior a 60°C, y ¡i. si la presión es presión de vacío, dicha temperatura es igual o superior a 60°C,  i. if the pressure is atmospheric, said temperature is below 60 ° C, and ¡i. if the pressure is vacuum pressure, said temperature is equal to or greater than 60 ° C,
e) moler la mezcla obtenida en la etapa d), y,  e) grind the mixture obtained in step d), and,
f) añadir una cantidad de coenzima Q10 y una cantidad de uno o más antioxidantes.  f) add an amount of coenzyme Q10 and an amount of one or more antioxidants.
Respecto a las condiciones de la etapa a) del presente procedimiento, preferiblemente se incuba un huevo fertilizado durante un período de tiempo comprendido entre 0 y 17 horas, con el fin de controlar que el huevo fertilizado incubado se mantenga en fase de gastrulación, sin que haya comenzado todavía la fase de neurulación, asegurando así que el preparado de huevo obtenido contenga células pluripotentes. Más preferiblemente, se incuba el huevo fertilizado durante un período de tiempo comprendido entre 0 y 17 horas, a una temperatura comprendida entre 34°C y 41 °C, y una humedad relativa comprendida entre 30% y 75%; todavía más preferiblemente, se incuba el huevo fertilizado durante un período de tiempo comprendido entre 0 y 17 horas a una temperatura de 38°C y una humedad relativa comprendida entre 55% y 60%. La etapa c) corresponde a un proceso de esterilización para suprimir bacterias, mohos y otros microorganismos potencialmente nocivos para la salud, que se basa en la optimización de un proceso conocido como procesado a altas presiones o HPP ("high-pressure processing"), ampliamente utilizado en el sector alimentario. With respect to the conditions of step a) of the present process, a fertilized egg is preferably incubated for a period of time between 0 and 17 hours, in order to control that the incubated fertilized egg is kept in gastrulation phase, without The neurulation phase has still begun, thus ensuring that the egg preparation obtained contains pluripotent cells. More preferably, the fertilized egg is incubated for a period of time between 0 and 17 hours, at a temperature between 34 ° C and 41 ° C, and a relative humidity between 30% and 75%; even more preferably, the fertilized egg is incubated for a period of time between 0 and 17 hours at a temperature of 38 ° C and a relative humidity between 55% and 60%. Stage c) corresponds to a sterilization process to suppress bacteria, molds and other microorganisms potentially harmful to health, which is based on the optimization of a process known as high pressure processing or HPP ("high-pressure processing"), widely used in the food sector.
En una realización preferida, la presión de la etapa c) del procedimiento arriba descrito es de 6000 bar. En otra realización, la temperatura de la etapa c) es de 9°C; además, preferiblemente, la duración de la etapa c) es de 5 minutos. Más preferiblemente, la etapa c) se lleva a cabo a una presión de 6000 bar y una temperatura de 9°C, durante 5 minutos. In a preferred embodiment, the pressure of step c) of the process described above is 6000 bar. In another embodiment, the temperature of step c) is 9 ° C; further, preferably, the duration of step c) is 5 minutes. More preferably, step c) is carried out at a pressure of 6000 bar and a temperature of 9 ° C, for 5 minutes.
La selección de la temperatura de la etapa d) es importante para el mantenimiento de las condiciones biológicas adecuadas para la buena conservación del producto de huevo y sus propiedades. Así pues, cuando el secado del producto de huevo se realice a presión atmosférica, la temperatura debe ser inferior a 60°C, preferiblemente, una temperatura comprendida entre 5°C y 50°C, para evitar la aceleración del desarrollo embrionario; más preferiblemente, el secado del producto de huevo de la etapa d) se lleva a cabo a una temperatura comprendida entre 25°C y 40°C. The selection of the temperature of stage d) is important for the maintenance of the biological conditions suitable for the good preservation of the egg product and its properties. Thus, when the drying of the egg product is carried out at atmospheric pressure, the temperature must be below 60 ° C, preferably, a temperature between 5 ° C and 50 ° C, to avoid the acceleration of embryonic development; more preferably, the drying of the egg product of step d) is carried out at a temperature between 25 ° C and 40 ° C.
Por otro lado, si dicha etapa d) se realiza en condiciones de presión de vacío, entonces la temperatura podrá ser igual o superior a 60°C. Resultará obvio para el experto en la materia que cuando se trabaje en condiciones de presión correspondientes a presión de vacío, será posible trabajar con una temperatura igual o superior a 60°C, aunque será necesario ajustar dichas condiciones para preservar las propiedades del producto de huevo. On the other hand, if said step d) is performed under vacuum pressure conditions, then the temperature may be equal to or greater than 60 ° C. It will be obvious to the person skilled in the art that when working under pressure conditions corresponding to vacuum pressure, it will be possible to work with a temperature equal to or greater than 60 ° C, although it will be necessary to adjust these conditions to preserve the properties of the egg product .
Dicha etapa d) de secado se puede llevar a cabo mediante numerosos métodos conocidos dentro del estado de la técnica, tales como liofilización, spray-drying, secado por microondas, secado por atomización, y secado con mezclador en condiciones de vacío. Cuando el secado se realiza con un mezclador en condiciones de vacío, preferiblemente se utilizará una temperatura de 30°C. Said step d) of drying can be carried out by numerous methods known within the state of the art, such as lyophilization, spray-drying, microwave drying, spray drying, and mixer drying under vacuum conditions. When drying is carried out with a mixer under vacuum conditions, preferably a temperature of 30 ° C will be used.
La etapa f) de adición de una cantidad de coenzima Q10 y una cantidad de uno o más antioxidantes se lleva a cabo, preferiblemente, en un mezclador. Particularmente, las etapas d)-f) se pueden llevar a cabo en un mezclador de bandas a una velocidad de rotación igual o superior a 5 vueltas por minuto. La utilización de este tipo de mezclador permite llevar a cabo, de forma simultánea, el secado del producto, y una micronización y mezclado homogéneos. Step f) of adding an amount of coenzyme Q10 and an amount of one or more antioxidants is preferably carried out in a mixer. In particular, steps d) -f) can be carried out in a band mixer at a rotation speed equal to or greater than 5 turns per minute. The use of this type of mixer allows to carry out, simultaneously, the drying of the product, and a homogeneous micronization and mixing.
En una realización adicional de la presente invención, el producto de huevo obtenido a través del presente proceso de la invención está en forma de nanocápsulas. Dichas nanocápsulas se pueden obtener por medio de cualquiera de los métodos de obtención conocidos en el estado de la técnica, que resultarán obvios para el experto en la materia. Este producto de huevo final en forma de nanocápsulas presenta una actividad analgésica, antiinflamatoria o cosmética 10 veces superior a la obtenida con el producto de huevo en forma no encapsulada. In a further embodiment of the present invention, the egg product obtained through the present process of the invention is in the form of nanocapsules. Said nanocapsules can be obtained by means of any of the methods of obtaining known in the state of the art, which will be obvious to the person skilled in the art. This final egg product in the form of nanocapsules has an analgesic, anti-inflammatory or cosmetic activity 10 times higher than that obtained with the non-encapsulated egg product.
En otra realización de la presente invención, con el fin de mejorar las condiciones cosméticas del huevo, el procedimiento de obtención del preparado de huevo arriba descrito comprende, además, una etapa de extracción del colesterol mediante tecnología de extracción con fluido supercrítico (SFE), que se puede llevar a cabo tras la etapa d), y previamente a la etapa e). Dicha etapa adicional de extracción se realizará a una presión comprendida entre 150 y 450 bar, y una temperatura comprendida entre 15°C y 70°C. Preferiblemente, dicha etapa adicional de extracción con fluido supercrítico de factores de crecimiento y colesterol se puede llevar a cabo a una presión de 275 bar y una temperatura de 40°C. In another embodiment of the present invention, in order to improve the cosmetic conditions of the egg, the method of obtaining the egg preparation described above further comprises a step of extracting cholesterol by means of supercritical fluid extraction (SFE) technology, which can be carried out after stage d), and prior to stage e). Said additional extraction stage will be carried out at a pressure between 150 and 450 bar, and a temperature between 15 ° C and 70 ° C. Preferably, said additional stage of supercritical fluid extraction of growth factors and cholesterol can be carried out at a pressure of 275 bar and a temperature of 40 ° C.
La técnica de extracción con fluido supercrítico constituye una opción limpia y segura, totalmente inocua, que permite separar componentes de manera eficiente, a la vez que preserva sus características, lo cual ha conducido a su rápida implantación en el sector de la alimentación. A modo de ilustración, ejemplos de fluido supercrítico aplicables en la etapa e) incluyen dióxido de carbono o SC-C02, agua, etano, propano, metanol y etanol, todos ellos en forma supercrítica, es decir, manipulados bajo condiciones de temperatura y presión críticas adecuadas para cada uno de ellos, tal como resultará evidente para un experto en la materia. The supercritical fluid extraction technique is a clean and safe, completely safe option, which allows components to be separated efficiently, while preserving their characteristics, which has led to its rapid implementation in the food sector. By way of illustration, examples of supercritical fluid applicable in step e) include carbon dioxide or SC-C0 2 , water, ethane, propane, methanol and ethanol, all in supercritical form, that is, handled under temperature conditions and Critical pressure suitable for each of them, as will be apparent to a person skilled in the art.
De acuerdo con esto, en una realización alternativa, el procedimiento de obtención del preparado de huevo arriba descrito comprende una etapa adicional que comprende la extracción de las grasas y aceites saturados e insaturados de dicho preparado de huevo mediante extracción con fluido supercrítico; dicha etapa adicional se puede realizar de acuerdo con las condiciones arriba descritas, y se puede llevar a cabo tras la etapa d) y previamente a la etapa e). A través de esta extracción SFC se consigue separar las grasas y aceites saturados e insaturados, de modo que la posterior molienda (etapa e) y adición de una cantidad de coenzima Q10 y una cantidad de uno o más antioxidantes (etapa f) y se lleva a cabo únicamente sobre un concentrado de proteína de huevo libre de grasas y aceites. Esta realización alternativa también comprende una segunda etapa adicional, posterior a la etapa f), de reincorporación de las grasas y aceites saturados e insaturados en un batidor, en el que se obtiene la emulsión final correspondiente. Es decir, de acuerdo con esta realización alternativa, el procedimiento de obtención del preparado de huevo arriba descrito comprende las siguientes etapas: Accordingly, in an alternative embodiment, the method of obtaining the egg preparation described above comprises an additional step comprising the extraction of saturated and unsaturated fats and oils from said egg preparation by extraction with supercritical fluid; said additional stage can be carried out according to the conditions described above, and can be carried out after stage d) and prior to stage e). Through this SFC extraction it is possible to separate the saturated and unsaturated fats and oils, so that the subsequent grinding (stage e) and addition of a quantity of coenzyme Q10 and an amount of one or more antioxidants (stage f) and takes conducted only on an egg protein concentrate free of fats and oils. This alternative embodiment also comprises a second additional stage, subsequent to step f), of re-incorporating saturated and unsaturated fats and oils in a whisk, in which the corresponding final emulsion is obtained. That is, according to this alternative embodiment, the method of obtaining the egg preparation described above comprises the following steps:
a) incubar un huevo fertilizado,  a) incubate a fertilized egg,
b) romper el huevo y obtener el producto de huevo correspondiente,  b) break the egg and obtain the corresponding egg product,
c) someter el producto de huevo a una presión comprendida entre 1000 y 9000 bar, durante un tiempo comprendido entre 1 y 20 minutos, a una temperatura comprendida entre 6°C y 20°C,  c) subjecting the egg product to a pressure between 1000 and 9000 bar, for a time between 1 and 20 minutes, at a temperature between 6 ° C and 20 ° C,
d) secar el producto de huevo a una temperatura determinada, con la condición de que:  d) drying the egg product at a certain temperature, with the proviso that:
i. si la presión es presión atmosférica, dicha temperatura es inferior a 60°C, y ¡i. si la presión es presión de vacío, dicha temperatura es igual o superior a 60°C,  i. if the pressure is atmospheric pressure, said temperature is below 60 ° C, and ¡i. if the pressure is vacuum pressure, said temperature is equal to or greater than 60 ° C,
d2) extraer las grasas y aceites saturados e insaturados del producto obtenido en la etapa d) mediante extracción con fluido supercrítico,  d2) extract saturated and unsaturated fats and oils from the product obtained in step d) by extracting with supercritical fluid,
e) moler el concentrado de proteína de huevo libre de grasas y aceites saturados e insaturados resultante de la etapa d2).  e) grind the egg protein concentrate free of saturated and unsaturated fats and oils resulting from step d2).
f) añadir una cantidad de coenzima Q10 y una cantidad de uno o más antioxidantes, f2) reincorporar las grasas y aceites saturados e insaturados obtenidos en la etapa d2), sobre la mezcla obtenida en la etapa f.  f) add an amount of coenzyme Q10 and an amount of one or more antioxidants, f2) reinstate the saturated and unsaturated fats and oils obtained in step d2), on the mixture obtained in step f.
Esta realización alternativa permite obtener el preparado de huevo en forma de emulsión, algo que resultará especialmente útil para las aplicaciones cosméticas de dicho producto final. En otro aspecto de la invención, el preparado de huevo de la presente invención se administra a mamíferos, más preferiblemente, a seres humanos. This alternative embodiment allows to obtain the egg preparation in the form of an emulsion, something that will be especially useful for the cosmetic applications of said final product. In another aspect of the invention, the egg preparation of the present invention is administered to mammals, more preferably, to humans.
Particularmente, este tercer aspecto de la invención se refiere a composiciones farmacéuticas, nutracéuticas o cosméticas que comprenden una cantidad efectiva de preparado de huevo arriba descrito, y uno o más excipientes o portadores farmacéuticos, nutracéuticos o cosméticos adecuados, respectivamente. Particularly, this third aspect of the invention relates to pharmaceutical, nutraceutical or cosmetic compositions comprising an effective amount of egg preparation described above, and one or more suitable pharmaceutical, nutraceutical or cosmetic excipients or carriers, respectively.
A través de estas composiciones, la presente invención proporciona una nueva solución que además de aportar los nutrientes naturales propios del huevo, proporciona antioxidantes, con la consiguiente reducción de estrés oxidativo y coenzima Q10, que en combinación con dichos antioxidantes, ha demostrado un efecto significado en el control del estrés oxidativo, y en la mejora de la eficiencia de la cadena respiratoria mitocondrial. Through these compositions, the present invention provides a new solution that in addition to providing the natural nutrients of the egg itself, provides antioxidants, with the consequent reduction of oxidative stress and coenzyme Q10, which in combination with said antioxidants, has demonstrated a significant effect. in the control of oxidative stress, and in the improvement of the efficiency of the mitochondrial respiratory chain.
Dichas composiciones farmacéuticas, nutracéuticas o cosméticas están caracterizadas, preferiblemente, por una forma de dosificación seleccionada entre: a) una forma sólida seleccionada entre polvo, pastilla, pastilla recubierta, cápsula, cápsula recubierta, gránulo, sobre, nanopartícula encapsulada, y Said pharmaceutical, nutraceutical or cosmetic compositions are preferably characterized by a dosage form selected from: a) a solid form selected from powder, tablet, coated tablet, capsule, coated capsule, granule, envelope, encapsulated nanoparticle, and
b) una forma líquida seleccionada entre solución, aerosol, crema, emulsión, gel, pasta, champú y suero.  b) a liquid form selected from solution, spray, cream, emulsion, gel, paste, shampoo and serum.
En un cuarto aspecto de la invención, se describen estas composiciones farmacéuticas, nutracéuticas o cosméticas que comprenden una cantidad efectiva de preparado de huevo para su uso como agente antiinflamatorio para el tratamiento de condiciones antiinflamatorias. Opcionalmente, esta composición se podrá administrar en combinación con uno o más agentes antiinflamatorios conocidos. In a fourth aspect of the invention, these pharmaceutical, nutraceutical or cosmetic compositions are described which comprise an effective amount of egg preparation for use as an anti-inflammatory agent for the treatment of anti-inflammatory conditions. Optionally, this composition may be administered in combination with one or more known anti-inflammatory agents.
En un quinto aspecto de la invención, se describen estas composiciones farmacéuticas, nutracéuticas o cosméticas que comprenden una cantidad efectiva de preparado de huevo para su uso como agente analgésico para el tratamiento del dolor agudo o crónico en condiciones relacionadas con el dolor. Opcionalmente, esta composición podrá administrarse en combinación con uno o más agentes analgésicos conocidos. Ejemplos de condiciones relacionadas con el dolor agudo o crónico incluyen, pero no se limitan a, síndrome de fatiga crónica, fibromialgia o fatiga adrenal. In a fifth aspect of the invention, these pharmaceutical, nutraceutical or cosmetic compositions are described which comprise an effective amount of egg preparation for use as an analgesic agent for the treatment of acute or chronic pain under pain-related conditions. Optionally, this composition may be administered in combination with one or more known analgesic agents. Examples of Conditions related to acute or chronic pain include, but are not limited to, chronic fatigue syndrome, fibromyalgia or adrenal fatigue.
A lo largo de la descripción y de las reivindicaciones, la palabra "comprende" y sus variaciones, tales como la palabra "incluye", no pretenden excluir otras características técnicas, aditivos, componentes o etapas. Los objetos, ventajas y características adicionales de la invención resultarán evidentes para los expertos en la materia tras el análisis de la descripción. Adicionalmente, la presente invención cubre todas las posibles combinaciones de las formas de realización particulares y preferidas arriba descritas. Throughout the description and the claims, the word "comprises" and its variations, such as the word "includes", are not intended to exclude other technical characteristics, additives, components or steps. The objects, advantages and additional features of the invention will be apparent to those skilled in the art after analysis of the description. Additionally, the present invention covers all possible combinations of the particular and preferred embodiments described above.
Descripción de las figuras Description of the figures
La figura 1 muestra el porcentaje de células sanas (fibroblastos) tras 17, 23 y 44 horas de tratamiento. Figure 1 shows the percentage of healthy cells (fibroblasts) after 17, 23 and 44 hours of treatment.
La figura 1 muestra el porcentaje de células tumorales (HepG2) tras 17, 23 y 44 horas de tratamiento.  Figure 1 shows the percentage of tumor cells (HepG2) after 17, 23 and 44 hours of treatment.
La figura 3. Porcentaje de células sanas (fibroblastos) en el momento OH, 24 y 48 horas de tratamiento.  Figure 3. Percentage of healthy cells (fibroblasts) at the time OH, 24 and 48 hours of treatment.
La figura 4 muestra el estudio proteico del efecto del tratamiento con extracto de huevos. Figure 4 shows the protein study of the effect of egg extract treatment.
La figura 5 muestra el control del peso en ratones al inicio y final de los diferentes tratamientos. Figure 5 shows the weight control in mice at the beginning and end of the different treatments.
La figura 6 muestra el ensayo de dolor como efecto de los diferentes tratamientos.  Figure 6 shows the pain test as an effect of the different treatments.
La figura 7 muestra el ensayo de depresión (prueba de natación forzada para depresión) como efecto de los diferentes tratamientos.  Figure 7 shows the depression trial (forced swimming test for depression) as an effect of the different treatments.
La figura 8 muestra los niveles del marcador inflamatorio IL-1 beta.  Figure 8 shows the levels of the inflammatory marker IL-1 beta.
La figura 9 muestra el control del peso al inicio y final de los diferentes tratamientos.  Figure 9 shows the weight control at the beginning and end of the different treatments.
La figura 10 muestra el ensayo de dolor como efecto de los diferentes tratamientos.  Figure 10 shows the pain test as an effect of the different treatments.
La figura 11 muestra el ensayo de depresión como efecto de los diferentes tratamientos. Figure 11 shows the depression test as an effect of the different treatments.
La figura 12 muestra los niveles del marcador inflamatorio IL-1 beta. Figure 12 shows the levels of the inflammatory marker IL-1 beta.
La figura 13 muestra el estudio proteico del efecto del tratamiento con extracto de huevos y CoQ10. La figura 14A, muestra los niveles de ATP; 14B: muestra los niveles de peroxidación lipídica determinados en músculo e hígado 14C. Figure 13 shows the protein study of the effect of treatment with egg extract and CoQ10. Figure 14A shows the levels of ATP; 14B: shows the levels of lipid peroxidation determined in muscle and liver 14C.
La figura 15 muestra los niveles de MnSOD determinados en músculo. Descripción detallada de la invención  Figure 15 shows the levels of MnSOD determined in muscle. Detailed description of the invention
La presente invención se refiere a un preparado de huevo fertilizado que comprende coenzima Q10 y uno o más antioxidantes, su procedimiento de preparación y composiciones farmacéuticas, nutracéuticas o cosméticas que comprenden dicho preparado de huevo y el uso de las mismas para su uso como agente antiinflamatorio, analgésico o cosmético. The present invention relates to a fertilized egg preparation comprising coenzyme Q10 and one or more antioxidants, its preparation process and pharmaceutical, nutraceutical or cosmetic compositions comprising said egg preparation and the use thereof for use as an anti-inflammatory agent. , analgesic or cosmetic.
A continuación, se presentan algunos ejemplos que muestran la invención y proporcionan ilustraciones adicionales de la misma. Below are some examples that show the invention and provide additional illustrations thereof.
Ejemplo 1 : Estudio in vitro de los efectos del extracto de huevo. Example 1: In vitro study of the effects of egg extract.
Para este objetivo, evaluamos tres extractos de huevo, aislados a las 17, 23 y 44 horas a 0.5% de concentración. Se añadieron diferentes cantidades de cada extracto y se ensayó su efecto en el crecimiento celular como principal marcador de toxicidad. Los extractos fueron sonicados para su mejor disolución. For this purpose, we evaluate three egg extracts, isolated at 17, 23 and 44 hours at 0.5% concentration. Different amounts of each extract were added and its effect on cell growth was tested as the main toxicity marker. The extracts were sonicated for better dissolution.
Este ensayo nos mostró un claro crecimiento inducido por el tratamiento con el huevo de 17 horas en los fibroblastos en el tratamiento de 5μί (figura 1), y no en las células tumorales (HepG2), (figura 2). This trial showed a clear growth induced by the 17-hour egg treatment in the fibroblasts in the 5μί treatment (figure 1), and not in the tumor cells (HepG2), (figure 2).
Dado que el tratamiento más efectivo fue el del extracto de huevo aislado a menos horas, se realizaron ensayos con extractos en horas más tempranas, incluyendo 0 horas (inmediatamente después de producirse la fecundación). En el siguiente experimento se evaluó la eficacia en la inducción del crecimiento celular de 0 horas, 2 horas, 8 horas e incluimos el tiempo más eficiente el experimento anterior, 17 horas. En este ensayo se observó un incremento del crecimiento celular progresivo en el tratamiento de 0 horas, nuevamente a la misma concentración de 5μί. Desde el punto de vista proteico, se evaluó e que finalmente fue considerado el tratamiento más efectivo, 0 horas, en dos dosis diferentes en comparación con células sin tratamientos y con otro tratamiento de eficacia menor, 2 horas. El resultado mostró que el tratamiento con huevo de 0 horas induce una activación del sistema antioxidante celular mediante el incremento de la Manganeso Superóxido Dismutasa (MnSOD) y la catalasa. Esta inducción suponemos que es debida a un incremento moderado del estrés oxidativo (OGG-1). Además, el tratamiento con 0 horas no indujo activación de la muerte celular (apoptosis) dado que no observamos incremento de la forma activa de Caspasa 3 ni modifico los niveles de BCL-2, una proteína anti- apoptótica (figura 4). Since the most effective treatment was that of isolated egg extract at less hours, tests were carried out with extracts at earlier hours, including 0 hours (immediately after fertilization occurred). In the following experiment the efficacy in the induction of cell growth of 0 hours, 2 hours, 8 hours was evaluated and we included the most efficient time the previous experiment, 17 hours. In this trial, an increase in progressive cell growth was observed in the treatment of 0 hours, again at the same concentration of 5μί. From a protein point of view, it was evaluated that it was finally considered the most effective treatment, 0 hours, in two different doses compared to cells without treatments and with another treatment of less efficacy, 2 hours. The result showed that the 0-hour egg treatment induces an activation of the cellular antioxidant system by increasing the Manganese Superoxide Dismutase (MnSOD) and catalase. This induction assumes that it is due to a moderate increase in oxidative stress (OGG-1). In addition, treatment with 0 hours did not induce activation of cell death (apoptosis) since we did not observe an increase in the active form of Caspase 3 nor did I modify the levels of BCL-2, an anti-apoptotic protein (Figure 4).
Ejemplo 2: Estudio in vivo de los efectos del extracto de huevo. Example 2: In vivo study of the effects of egg extract.
Para este objetivo, se sometieron 15 ratones a diferentes tratamientos, 5 al tratamiento de ácido para-minoabenzoico (PABA), un inhibidor de la síntesis te CoQ10 que reproduce los síntomas de la fibromialgia, 5 a PABA mas Huevo de 17 horas y 5 sin tratamiento como control negativo. For this purpose, 15 mice were subjected to different treatments, 5 to the treatment of para-minoabenzoic acid (PABA), an inhibitor of the Te CoQ10 synthesis that reproduces the symptoms of fibromyalgia, 5 to PABA plus Egg of 17 hours and 5 without Treatment as a negative control.
En este ensayo pudimos comprobar que el tratamiento con huevo evitó un incremente del peso de los ratones, aunque este resultado hay evaluarlo con precaución dado que se observaron resultados similares en el grupo PABA. Por otro lado, si se observó una reducción o protección de los síntomas de dolor y depresión que indujo el tratamiento con PABA. Estos son los dos principales síntomas de la fibromialgia. En la figura 6 se observan los resultados de la prueba de placa caliente en la que los ratones, sometidos a una temperatura de 56 grados se evalúa el tiempo que tardan en reaccionar al dolor y por tanto el umbral de dolor. Se observó un menor umbral en el grupo PABA, así como un importante incremento en el grupo de huevo lo que demuestra el aumento de analgesia. Por otro lado, e la figura 7 se evalúa la depresión mediante la prueba de natación forzada en la que se ve tiempo que permanecen inmóbiles los animales en un baño de agua a 37°C. El grupo PABA muestra más permanencia inmóvil, síntoma de mayor grado de depresión el cual es recuperado en el grupo de huevo. In this trial we could verify that the egg treatment prevented an increase in the weight of the mice, although this result should be evaluated with caution since similar results were observed in the PABA group. On the other hand, if there was a reduction or protection of the symptoms of pain and depression that induced treatment with PABA. These are the two main symptoms of fibromyalgia. Figure 6 shows the results of the hot plate test in which the mice, subjected to a temperature of 56 degrees, assess the time it takes to react to pain and therefore the pain threshold. A lower threshold was observed in the PABA group, as well as a significant increase in the egg group which demonstrates the increase in analgesia. On the other hand, in Figure 7 depression is evaluated by the forced swimming test in which it is seen time that the animals remain immobile in a 37 ° C water bath. The PABA group shows more immobile permanence, a symptom of a greater degree of depression which is recovered in the egg group.
Por otro lado, no se observó daño tisular por el huevo en parámetros de daño hepático (GOT y GPT), muscular (Creatina Kinasa) ni alteraciones de glucosa ni perfil lipídico. Triglicéridos Glucosa Colesterol GPT GOT Creatina On the other hand, no tissue damage was observed by the egg in parameters of liver damage (GOT and GPT), muscle damage (Creatine Kinase) or glucose alterations or lipid profile. Triglycerides Glucose Cholesterol GPT GOT Creatine
kinasa  kinase
WT 0, 1634 25,912 1 ,406 507,488 520,906 878,084  WT 0, 1634 25,912 1, 406 507,488 520,906 878,084
PABA 0, 1604 29,516 1 ,292 962,79 1429,51 1588,2  PABA 0, 1604 29,516 1, 292 962.79 1429.51 1588.2
PABA 0, 16375 24,9 0,837 482,0425 407,8725 659,9  PABA 0, 16375 24.9 0.837 482.0425 407.8725 659.9
+ Huevo  + Egg
Tabla 1. Valoración de daño metabólico y tisular  Table 1. Assessment of metabolic and tissue damage
Por otro lado, el tratamiento con huevo indujo una protección frente a la inflamación inducida por PABA, tal y como muestran los niveles de IL-1 beta, una citoquina pro- inflamatoria relacionada con el inflamasoma. On the other hand, egg treatment induced protection against PABA-induced inflammation, as shown by levels of IL-1 beta, a pro-inflammatory cytokine related to inflammation.
Ejemplo 3: Estudio in vivo de los efectos del extracto de huevo en comparación con CoQ10. Example 3: In vivo study of the effects of egg extract compared to CoQ10.
Para este objetivo, se sometieron 25 ratones a diferentes tratamientos, 5 al tratamiento de ácido para-minoabenzoico (PABA), un inhibidor de la síntesis te CoQ10 que reproduce los síntomas de la fibromialgia y que nuestro grupo tiene patentado, a 5 PABA más Huevo de h horas, 5 PABA mas CoQ10 30mg, 5 la combinación de PABA, huevo 0 horas y CoQ10 y 5 sin tratamiento como control negativo. For this purpose, 25 mice were subjected to different treatments, 5 to the treatment of para-minoabenzoic acid (PABA), a coQ10 synthesis inhibitor that reproduces the symptoms of fibromyalgia and that our group has patented, to 5 PABA plus Egg of h hours, 5 PABA plus CoQ10 30mg, 5 the combination of PABA, egg 0 hours and CoQ10 and 5 without treatment as a negative control.
En este ensayo, los datos mostraron que el peso de los ratones es mantenido y no incrementado por los tratamientos, con ligero incremento en el caso de los tratamientos en los que aparece el CoQ10. Por otro lado, el dolor era reducido por todos los tratamientos, huevo, CoQ10 y la combinación, siendo más significativo en el CoQ10. En cuanto a la depresión, la combinación fue considera más efectiva que el huevo solo y el CoQ10 solo. In this trial, the data showed that the weight of the mice is maintained and not increased by the treatments, with a slight increase in the case of the treatments in which CoQ10 appears. On the other hand, pain was reduced by all treatments, egg, CoQ10 and the combination, being more significant in CoQ10. As for depression, the combination was considered more effective than egg alone and CoQ10 alone.
Figure imgf000017_0001
Figure imgf000017_0001
Tabla 2. Valoración de daño metabólico y tisular Igualmente, no se observó daño tisular por el huevo en parámetros de daño hepático (GOT), ni alteraciones de glucosa ni perfil lipídico. Table 2. Assessment of metabolic and tissue damage Similarly, no tissue damage was observed by the egg in liver damage parameters (GOT), glucose alterations or lipid profile.
Por otro lado, el tratamiento con huevo y la combinación huevo más CoQ10 indujo una protección frente a la inflamación inducida por PABA, tal y como muestran los niveles de IL- 1 beta, una citoquina pro-inflamatoria relacionada con el inflamasoma. On the other hand, treatment with egg and the combination of egg plus CoQ10 induced protection against PABA-induced inflammation, as shown by levels of IL-1 beta, a pro-inflammatory cytokine related to inflammation.
Dado los efectos analgésicos observados y la fisiopatología muscular de nuestro modelo de fibromialgia se analizaron los tejidos musculares. Tal y como muestra a figura 13, se observa un incremento del daño oxidativo en el grupo de PABA que conlleva la activación de los antioxidantes (MnSOD y catalasa). Se observó una reducción en los grupos tratados con huevo, CoQ10 y combinación intuyéndose más significativa en la combinación lo que demuestra que la combinación es efectiva desde el punto de vista oxidativo. También se observó una reducción de la inflamación mediada por IL-1 beta y de la muerte celular por apoptosis mediada por la proteína pro-apoptótica BAX. Paralelamente, se observa un incremento de la autofagia que muestra un mayor reciclaje celular. Given the observed analgesic effects and muscular pathophysiology of our fibromyalgia model, muscle tissues were analyzed. As shown in Figure 13, an increase in oxidative damage is observed in the PABA group that involves the activation of antioxidants (MnSOD and catalase). A reduction was observed in the groups treated with egg, CoQ10 and combination, intuiting more significant in the combination which demonstrates that the combination is oxidatively effective. A reduction in IL-1 beta-mediated inflammation and cell death due to apoptosis mediated by pro-apoptotic protein BAX was also observed. In parallel, there is an increase in autophagy that shows greater cell recycling.
Por otro lado, el tratamiento con huevo indujo altos niveles de producción de ATP, estimulando así la bioenergética células frente al déficit inducido por PABA. On the other hand, egg treatment induced high levels of ATP production, thus stimulating bioenergetic cells against the PABA-induced deficit.
Finalmente, y dado que el músculo es el principal tejido afectado en patologías de dolor como la fibromialgia, evaluamos el potencial antioxidante de los tratamientos en el tejido muscular mediante la determinación de la actividad enzimática de la MnSOD, una enzima antioxidante altamente relacionada con la actividad mitocondrial. El tratamiento con nanopartículas cargadas con CoQ10 mejoró considerablemente la respuesta antioxidante, lo cual concuerda con la bibliografía actual sobre el efecto del CoQ10. Interesantemente, el tratamiento con CoQ10 en nanopartículas muestra una tendencia a mejorar al CoQ10 solo, sin nanopartículas. Finally, and since muscle is the main tissue affected in pain pathologies such as fibromyalgia, we evaluate the antioxidant potential of treatments in muscle tissue by determining the enzymatic activity of MnSOD, an antioxidant enzyme highly related to activity mitochondrial Treatment with nanoparticles loaded with CoQ10 significantly improved the antioxidant response, which is consistent with the current literature on the effect of CoQ10. Interestingly, treatment with CoQ10 in nanoparticles shows a tendency to improve CoQ10 alone, without nanoparticles.
Los ensayos demostraron que el extracto de huevo fertilizado induce cambios a nivel tanto celular como en modelos animales que nos llevan a pensar en un efecto beneficioso desde el punto de vista del estrés oxidativo y la inflamación. Induce el crecimiento celular y modera el efecto de la apoptosis además de estimular la producción de ATP. Además, muestra efectos beneficiosos en cuanto a analgesia y efecto antidepresivo en nuestro modelo patentado de ratón. No muestra efectos de toxicidad en los parámetros evaluados por nosotros ni en el metabolismo. The trials showed that fertilized egg extract induces changes both at the cellular level and in animal models that lead us to think of a beneficial effect from the point of view of oxidative stress and inflammation. It induces cell growth and moderates the effect of apoptosis in addition to stimulating the production of ATP. In addition, it shows beneficial effects in terms of analgesia and antidepressant effect in our model. patented mouse. It shows no toxicity effects in the parameters evaluated by us or in the metabolism.
Por otro lado, la combinación con el conocido antioxidante CoQ10 muestra una mayor eficacia en la depresión, no así en el dolor donde el CoQ10 es más efectivo. Sin embargo, evaluando el conjunto de beneficios por parte de la combinación para un tratamiento idóneo con potencial antioxidante, antiinflamatorio, anti-apoptótico que no induce toxicidad en los parámetros evaluados por nosotros. On the other hand, the combination with the well-known antioxidant CoQ10 shows greater efficacy in depression, but not in pain where CoQ10 is most effective. However, evaluating the combination of benefits by the combination for an ideal treatment with antioxidant, anti-inflammatory, anti-apoptotic potential that does not induce toxicity in the parameters evaluated by us.
También podemos concluir que existe un alto grado de efectividad de las nanopartículas cargadas tanto con CoQ10 como la combinación de CoQ10 y huevo a 0 horas. En ambos casos es significativamente mejorado el dolor y síntoma depresivo. Además, en condiciones patológicas como es el caso del modelo animal de fibromialgia, ofrecen un potencial antioxidante y antiinflamatorio con pocos signos de toxicidad. We can also conclude that there is a high degree of effectiveness of nanoparticles loaded with both CoQ10 and the combination of CoQ10 and egg at 0 hours. In both cases the pain and depressive symptom is significantly improved. In addition, in pathological conditions such as the animal model of fibromyalgia, they offer an antioxidant and anti-inflammatory potential with few signs of toxicity.

Claims

REIVINDICACIONES
1. Preparado de huevo caracterizado porque comprende: a) un producto de huevo que ha sido previamente extraído de un huevo fertilizado incubado,  1. Egg preparation characterized in that it comprises: a) an egg product that has been previously extracted from an incubated fertilized egg,
b) una cantidad de coenzima Q10, y  b) an amount of coenzyme Q10, and
c) una cantidad de uno o más antioxidantes.  c) an amount of one or more antioxidants.
2. Preparado de huevo de acuerdo con la reivindicación 1 , caracterizado porque la incubación de dicho huevo fertilizado se lleva a cabo durante un período de tiempo comprendido entre 0 y 17 horas. 2. Egg preparation according to claim 1, characterized in that the incubation of said fertilized egg is carried out for a period of time between 0 and 17 hours.
3. Preparado de huevo de acuerdo con la reivindicación 1 o 2, caracterizado porque el producto de huevo ha sido previamente extraído de un huevo fertilizado de ave en donde dicha ave se selecciona entre gallina, ganso, pato, codorniz, pavo, avestruz, faisán y paloma. 3. Egg preparation according to claim 1 or 2, characterized in that the egg product has been previously extracted from a fertilized bird egg wherein said bird is selected from hen, goose, duck, quail, turkey, ostrich, pheasant and dove.
4. Preparado de huevo de acuerdo con la reivindicación 3, caracterizado porque el ave es una gallina. 4. Egg preparation according to claim 3, characterized in that the bird is a chicken.
5. Preparado de huevo de acuerdo con las reivindicaciones 1-4, caracterizado porque la cantidad de coenzima Q10 está comprendida entre 0, 1 % y 4,5% en peso con respecto al peso total de dicho preparado. 5. Egg preparation according to claims 1-4, characterized in that the amount of coenzyme Q10 is between 0.1% and 4.5% by weight with respect to the total weight of said preparation.
6. Preparado de huevo de acuerdo con las reivindicaciones 1-5, caracterizado porque la cantidad de coenzima Q10 está comprendida entre 0,2% y 2,2% en peso con respecto al peso total de dicho preparado. 6. Egg preparation according to claims 1-5, characterized in that the amount of coenzyme Q10 is between 0.2% and 2.2% by weight with respect to the total weight of said preparation.
7. Preparado de huevo de acuerdo con cualquiera de las reivindicaciones 1-6, caracterizado porque la cantidad de coenzima Q10 está comprendida entre 0,4% y 0,7% en peso con respecto al peso total de dicho preparado. 7. Egg preparation according to any of claims 1-6, characterized in that the amount of coenzyme Q10 is between 0.4% and 0.7% by weight with respect to the total weight of said preparation.
8. Preparado de huevo de acuerdo con cualquiera de las reivindicaciones 1-7, caracterizado porque la cantidad de uno o más antioxidantes está comprendida entre 2,2% y 6,7% en peso respecto al peso total de dicho preparado. 8. Egg preparation according to any of claims 1-7, characterized in that the amount of one or more antioxidants is comprised between 2.2% and 6.7% by weight with respect to the total weight of said preparation.
9. Preparado de huevo de acuerdo con cualquiera de las reivindicaciones 1-8, caracterizado porque la cantidad de uno o más antioxidantes está comprendida entre 1 ,7% y 5,6% en peso respecto al peso total de dicho preparado.  9. Egg preparation according to any of claims 1-8, characterized in that the amount of one or more antioxidants is comprised between 1.7% and 5.6% by weight with respect to the total weight of said preparation.
10. Preparado de huevo de acuerdo con cualquiera de las reivindicaciones 1-9, caracterizado porque dichos uno o más antioxidantes se seleccionan entre el grupo que consiste en superóxido dismutasa, catalasa, glutatión, ácido ascórbico, a-tocoferol, retinol, β-caroteno, metalotioneína, melatonina, taurina, selenio, resveratrol, metionina, polifenoles, isoflavononas, S-adenosil-metionina, y cualquiera de sus mezclas. 10. Egg preparation according to any of claims 1-9, characterized in that said one or more antioxidants are selected from the group consisting of superoxide dismutase, catalase, glutathione, ascorbic acid, a-tocopherol, retinol, β-carotene , metallothionein, melatonin, taurine, selenium, resveratrol, methionine, polyphenols, isoflavonones, S-adenosyl-methionine, and any of their mixtures.
11. Preparado de huevo de acuerdo con la reivindicación 10, caracterizado porque dichos uno o más antioxidantes consisten en superóxido dismutasa, y porque dicha superóxido dismutasa comprende un cofactor seleccionado entre el grupo que consiste en cobre, zinc, manganeso y hierro. 11. Egg preparation according to claim 10, characterized in that said one or more antioxidants consist of superoxide dismutase, and that said superoxide dismutase comprises a cofactor selected from the group consisting of copper, zinc, manganese and iron.
12. Procedimiento para la preparación del preparado de huevo de las reivindicaciones 1-11 , caracterizado porque comprende:  12. Method for preparing the egg preparation of claims 1-11, characterized in that it comprises:
a) incubar un huevo fertilizado,  a) incubate a fertilized egg,
b) romper el huevo y obtener el producto de huevo correspondiente,  b) break the egg and obtain the corresponding egg product,
c) someter el producto de huevo a una presión comprendida entre 1000 y 9000 bar, durante un tiempo comprendido entre 1 y 20 minutos, a una temperatura comprendida entre 6°C y 20°C,  c) subjecting the egg product to a pressure between 1000 and 9000 bar, for a time between 1 and 20 minutes, at a temperature between 6 ° C and 20 ° C,
d) secar el producto de huevo a una temperatura determinada, con la condición de que:  d) drying the egg product at a certain temperature, with the proviso that:
i. si la presión es presión atmosférica, dicha temperatura es inferior a 60°C, y ¡i. si la presión es presión de vacío, dicha temperatura es igual o superior a 60°C,  i. if the pressure is atmospheric pressure, said temperature is below 60 ° C, and ¡i. if the pressure is vacuum pressure, said temperature is equal to or greater than 60 ° C,
e) moler la mezcla obtenida en la etapa d), y  e) grind the mixture obtained in step d), and
f) añadir una cantidad de coenzima Q10 y una cantidad de uno o más antioxidantes. f) add an amount of coenzyme Q10 and an amount of one or more antioxidants.
13. Procedimiento de acuerdo con la reivindicación 12, caracterizado porque la etapa a) se lleva a cabo a una temperatura comprendida entre 34°C y 41 °C, y una humedad relativa comprendida entre 30% y 75%. 13. Method according to claim 12, characterized in that step a) is carried out at a temperature between 34 ° C and 41 ° C, and a relative humidity between 30% and 75%.
14. Procedimiento de acuerdo con la reivindicación 12 o 13, caracterizado porque la etapa a) se lleva a cabo a una temperatura de 38°C, y una humedad relativa comprendida entre 55% y 60%. 14. Method according to claim 12 or 13, characterized in that step a) is carried out at a temperature of 38 ° C, and a relative humidity comprised between 55% and 60%.
15. Procedimiento de acuerdo con cualquiera de las reivindicaciones 12-14, caracterizado porque la presión en la etapa c) es de 6000 bar. 15. Method according to any of claims 12-14, characterized in that the pressure in step c) is 6000 bar.
16. Procedimiento de acuerdo con cualquiera de las reivindicaciones 12-15, caracterizado porque la temperatura en la etapa c) es de 9°C. 16. Method according to any of claims 12-15, characterized in that the temperature in step c) is 9 ° C.
17. Procedimiento de acuerdo con cualquiera de las reivindicaciones 12-16, caracterizado porque la duración de la etapa c) es de 5 minutos. 17. Method according to any of claims 12-16, characterized in that the duration of step c) is 5 minutes.
18. Procedimiento de acuerdo con cualquiera de las reivindicaciones 12-17, caracterizado porque cuando la presión es presión atmosférica en la etapa g), se seca el producto de huevo a una temperatura comprendida entre 5°C y 50°C. 18. Method according to any of claims 12-17, characterized in that when the pressure is atmospheric pressure in step g), the egg product is dried at a temperature between 5 ° C and 50 ° C.
19. Procedimiento de acuerdo con cualquiera de las reivindicaciones 12-18, caracterizado porque las etapas d) y e) se llevan a cabo en un mezclador de bandas a una velocidad de rotación igual o superior a 5 vueltas por minuto. 19. Method according to any of claims 12-18, characterized in that steps d) and e) are carried out in a band mixer at a rotation speed equal to or greater than 5 turns per minute.
20. Procedimiento de acuerdo con cualquiera de las reivindicaciones 12-19, caracterizado porque comprende una etapa adicional tras la etapa d), y previamente a la etapa e), que comprende una extracción con fluido supercrítico de colesterol a una presión comprendida entre 150 y 450 bar y una temperatura comprendida entre 15°C y 70°C. 20. Method according to any of claims 12-19, characterized in that it comprises an additional stage after stage d), and prior to stage e), which comprises an extraction with supercritical cholesterol fluid at a pressure between 150 and 450 bar and a temperature between 15 ° C and 70 ° C.
21. Procedimiento de acuerdo con cualquiera de las reivindicaciones 12-18, caracterizado porque comprende: una etapa adicional d2), posterior a la etapa d), que comprende la extracción de las grasas y aceites saturados e insaturados del preparado de huevo, y 21. Method according to any of claims 12-18, characterized in that it comprises: an additional stage d2), after stage d), which includes the extraction of saturated and unsaturated fats and oils from the egg preparation, and
una etapa adicional f2), posterior a la etapa f), que comprende la reincorporación de las grasas y aceites saturados e insaturados extraídos en la etapa d2) a la mezcla obtenida en la etapa f).  an additional stage f2), subsequent to stage f), comprising the reinstatement of saturated and unsaturated fats and oils extracted in step d2) to the mixture obtained in stage f).
22. Composición farmacéutica, nutracéutica o cosmética caracterizada porque comprende una cantidad efectiva de un preparado de huevo tal como se define en cualquiera de las reivindicaciones 1 -1 1 , y uno o más excipientes o portadores farmacéuticos, nutracéuticos o cosméticos aceptables, respectivamente. 22. Pharmaceutical, nutraceutical or cosmetic composition characterized in that it comprises an effective amount of an egg preparation as defined in any of claims 1 -1 1, and one or more pharmaceutical, nutraceutical or acceptable cosmetic excipients or carriers, respectively.
23. Composición farmacéutica, nutracéutica o cosmética de acuerdo con la reivindicación 22, caracterizada por una forma de dosificación seleccionada entre: c) una forma sólida seleccionada entre polvo, pastilla, pastilla recubierta, cápsula, cápsula recubierta, gránulo, sobre, nanopartícula encapsulada, y 23. Pharmaceutical, nutraceutical or cosmetic composition according to claim 22, characterized by a dosage form selected from: c) a solid form selected from powder, tablet, coated tablet, capsule, coated capsule, granule, envelope, encapsulated nanoparticle, Y
d) una forma líquida seleccionada entre solución, aerosol, crema, emulsión, gel, pasta, champú y suero.  d) a liquid form selected from solution, spray, cream, emulsion, gel, paste, shampoo and serum.
24. Composición farmacéutica o nutracéutica o cosmética de acuerdo con la reivindicación 22 o 23, para uso como agente antiinflamatorio para el tratamiento de condiciones inflamatorias. 24. Pharmaceutical or nutraceutical or cosmetic composition according to claim 22 or 23, for use as an anti-inflammatory agent for the treatment of inflammatory conditions.
25. Composición farmacéutica, nutracéutica o cosmética de acuerdo con la reivindicación 22 o 23, para uso como agente analgésico para el tratamiento del dolor agudo o crónico en condiciones relacionadas con el dolor. 25. Pharmaceutical, nutraceutical or cosmetic composition according to claim 22 or 23, for use as an analgesic agent for the treatment of acute or chronic pain under pain-related conditions.
PCT/ES2018/070032 2017-01-19 2018-01-17 Egg preparation with anti-inflammatory and anti-oxidant properties WO2018134460A1 (en)

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