WO2007026038A2 - Use of a mycosporin-type amino acid (shinorine) as an antioxidant - Google Patents

Use of a mycosporin-type amino acid (shinorine) as an antioxidant Download PDF

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WO2007026038A2
WO2007026038A2 PCT/ES2006/000492 ES2006000492W WO2007026038A2 WO 2007026038 A2 WO2007026038 A2 WO 2007026038A2 ES 2006000492 W ES2006000492 W ES 2006000492W WO 2007026038 A2 WO2007026038 A2 WO 2007026038A2
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shinorine
amino acid
type amino
mycosporine
red algae
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PCT/ES2006/000492
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Spanish (es)
French (fr)
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WO2007026038A3 (en
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Francisca De La Coba Luque
José AGUILERA ARJONA
Félix LÓPEZ FIGUEROA
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Universidad De Málaga
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A61K36/04—Rhodophycota or rhodophyta (red algae), e.g. Porphyra
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
    • A61K8/442—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof substituted by amido group(s)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9706—Algae
    • A61K8/9717—Rhodophycota or Rhodophyta [red algae], e.g. Porphyra
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9733—Lichens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/522—Antioxidants; Radical scavengers

Definitions

  • the present invention is part of the biotechnology sector and describes the potential use as antioxidants of certain secondary metabolites called mycosporin-type amino acids (MAAs) isolated from red algae and marine lichens in addition to their possible application in pharmaceutical preparations, nutraceuticals, functional foods for oxidative stress prevention
  • UV radiation is one of biological factors that limit the survival, physiology and growth of many organisms. Some of the multiple harmful effects of UV radiation include the alteration of DNA and protein molecules, inactivation of enzymes and the formation of free radicals, which attack cell membranes and other target molecules altering their functionality. All aerobic organisms have a wide variety of both enzymatic and non-enzymatic antioxidant defense systems that cooperatively coordinate and protect the body from the risks of oxidative stress.
  • ROS oxygen molecule
  • Free radicals give rise to important alterations in molecules such as DNA, lipids and proteins, seriously altering the cycle and cellular functionality.
  • the DNA can suffer loss of bases as well as the rupture of one or both strands of the genetic material, alterations that can result in irreversible mutations.
  • Many proteins are capable of absorbing a large amount of oxidation without apparently affecting their function.
  • the damages produced by OH • and 1 O 2 are irreversible and in general terms mark proteins for degradation.
  • Cell membranes can also be seriously damaged in oxidative stress because phospholipids that have fatty acids with several double bonds are very susceptible to oxidation due to the loss of a hydrogen (allyl).
  • allyl Once the carbon radical has been generated in a fatty acid, it reacts with molecular oxygen forming a peroxyl radical.
  • the peroxyl radical can take an allylic hydrogen to another methylene whereby the reaction is propagated.
  • Hydroperoxides which are stable compounds, if they come into contact with transition metal ions will produce more free radicals that will initiate and propagate other chain reactions. Thus, the membranes are seriously damaged and therefore their functionality is altered.
  • Free radicals are associated with a wide range of pathologies and diseases such as Alzheimer's or Parkinson's and conditions related to sun exposure such as the appearance of cataracts, photoaging, inflammatory episodes and neoplasms. They are also responsible for the oxidation of food fats, which is the most important form of deterioration after alterations caused by microorganisms. With oxidation, stale odors and flavors appear, the color and texture are altered, and the nutritional value decreases when some vitamins and polyunsaturated fatty acids are lost. In addition, products formed in oxidation can become harmful to health.
  • Mycosporin-type amino acids consist of a cyclohexenone or cyclohexenimine ring, conjugated with a nitrogenous substituent of an amino acid or its amino alcohol that acts as a chromophore allowing the absorption of certain shortwave radiation.
  • the metabolites that are isolated in fungi have an absorption range between 310 and 320 nm and have cyclohexenone rings exclusively, known by the name of Mycosporins in reference to their origin. On the contrary, metabolites that are isolated from marine organisms and algae contain cyclohexenimine rings, with maximum absorptions between 310 and 360 nm and are known as mycosporine-type amino acids or MAAs.
  • mycosporine-glycine and mycosporine taurine are aminocyclohexenones isolated from marine organisms.
  • mycosporins described in fungi and 23 MAAs in marine organisms. They are small molecules, with molecular weights around 330 Da and have high photostability. They behave like amphoteric molecules, similar to amino acids, so that they have positive and negative charges on the same molecule. They show physicochemical characteristics of ionic compounds, for example, high effusion point and high water solubility.
  • An antioxidant is defined as a substance that in low concentrations compared to an oxidizable substrate, delays or prevents its oxidation.
  • the present invention describes the potentiality of shinorine MAA isolated from Gymnogongrus devoniensis as a radical scavenger and lipid peroxidation inhibitor. In the tests carried out, the new antioxidant is compared with another known antioxidant, ⁇ -tocopherol.
  • the described compound could be used in therapeutic applications, and in non-medical applications for the stabilization of compounds susceptible to oxidative deterioration, in the preservation of food or related products, and in nutritional, nutraceutical, functional or parapharmacy supplements for their antioxidant properties for prevent oxidative stress.
  • the present invention presents an isolated compound of Gymnogongrus devoniensis with the following structure and useful as an antioxidant and free radical sequestrant.
  • the purification was performed in three consecutive steps in which chromatographic absorption techniques are combined by the application of active carbon, precipitation of polysaccharides by adding 100% methanol to the sample and final separation by ion exchange chromatography. Finally, aqueous solutions of MAA were obtained in a high degree of purity at concentrations of the order of mM.
  • the ABTS peroxidase method allows to determine the total antioxidant activity (TAA) of a sample understood as a parameter that allows quantifying the capacity of a sample, natural or processed, of sequestering free radicals present in an aqueous solution.
  • TAA total antioxidant activity
  • Shinorine isolated from Gymnogongrus devoniensis does not show significant antioxidant activity at any pH tested as an inhibitor of water-soluble free radical production (ABTS +) .
  • Shinorine isolated from Gymnogongrus devoniensis was studied as an inhibitor of lipid peroxidation in vitro using the ⁇ -carotene bleaching technique.
  • the method of decolorization of ⁇ -carotene is widely used to determine the antioxidant capacity of various substances in lipophilic medium, most of them extracted from fruits, vegetables and other products intended for food consumption in order to determine their greater or lesser degree of self-preservation in a natural state.
  • ⁇ -tocopherol ( ⁇ -TOC) was used as a positive control.
  • Shinorine isolated from Gymnogongrus devoniensis shows moderate antioxidant activity at the level of lipid peroxidation inhibition. It is thus constituted as an antioxidant of moderate activity in vitro.
  • this compound in extracts or preparations containing it, could be used in pharmaceutical preparations or formulations for the prevention and therapeutic treatment of diseases or conditions related to free radicals, in parapharmacy products, in functional foods, nutritional supplements and nutraceutical preparations, and in the food industry as an antioxidant potential (additive).
  • Figure 1 Area (%) of eluted peaks and concentrations expressed in mg g "1 PS of different MAAs present in methanolic extracts of algae Porphyra leucosticta,
  • Gymnogongrus devoniensis Gelidium sesquipedale and lichen Lichina pygmaea.
  • the presence of a type of majority MAA in each organism > 66%) together with others
  • FIG. 1 Chromatogram of an aqueous extract of shinorine isolated from Gymnogongrus devoniensis eluted from the column loaded with DOWEX resin.
  • Figure 3. Table. Dose ( ⁇ M) - antioxidant activity response (%) of shinorine isolated from Gymnogongrus devoniensis with respect to 10 ⁇ M of ⁇ -tocopherol by the method of decolorization of ⁇ -carotene. The values represent the mean values and standard deviation of 3 experiments. Shinorine isolated from Gymnogongrus devoniensis is a good antioxidant at a concentration of 100-200 ⁇ M.
  • Figure 4. Sequestration capacity of superoxide radicals generated by the shinorine pyrogallol method isolated from Gymnogongrus devoniensis. The mean and standard deviation of three experiments are represented.
  • the mobile phase used was 2.5% methanol (v / v, HPLC quality) plus 0.1% acetic acid (v / v) isocratically pumped at a flow rate of 0.5 ml min. "1 A UV detector was used. visible (photodiode detector 996), which measured the absorbency for each sample between 290 and 400 nm.
  • Figure 1 shows the percentages in area of the chromatographed peaks of different algal extracts, some identified as MAAs and others unknown. Purification objective was to isolate the major MAA in Gymnogongrus devoniensis in the aqueous phase, in addition to eliminating traces and other types of unidentified compounds.
  • Preparative scale extraction was performed by dissolving 60-80 g (PF) of biological material in 1 liter of methanol to 20% v / v and incubated in a thermostatic bath at 45 0 C for 2 hours. Extract at 14,000 rpm for 15 min and then centrifuged at rotary evaporation 45 was 0 C to remove part of the methanol in the sample.
  • the purification is carried out in three consecutive steps in which chromatographic absorption techniques are combined by the application of active carbon, precipitation of polysaccharides by adding 100% methanol to the sample and final separation by ion exchange chromatography (Dowex 50 W x 8 resin -100).
  • chromatographic absorption techniques are combined by the application of active carbon, precipitation of polysaccharides by adding 100% methanol to the sample and final separation by ion exchange chromatography (Dowex 50 W x 8 resin -100).
  • ion exchange chromatography Dowex 50 W x 8 resin -100
  • elution of the amino acid type mycosporine shinorine double-distilled water was used as eluent, with a slightly alkaline pH (7.2).
  • aqueous solutions of MAA were obtained in a high degree of purity at concentrations of the order of mM.
  • the ABTS peroxidase method allows to determine the total antioxidant activity (TAA) of a sample understood as a parameter that allows quantifying the capacity of a sample, natural or processed, of sequestering free radicals present in an aqueous solution.
  • TAA total antioxidant activity
  • This parameter is aimed at giving information on the antioxidant activity that a specific sample can present, regardless of the partial activities that each of its components may present or the synergism effects that could be established.
  • ABTS 2,2'-Azino-bis- (3- ethyl-benzotizoline-6- sulfonic acid) or ABTS is a compound that has great chemical stability, high water solubility and maximum absorption in the UVA band at 342 nm .
  • This compound in the presence of H 2 O 2 and peroxidases enzymes derives to a metastable radical (ABTS + ) with a characteristic absorption spectrum and different from ABTS, presenting maximum absorption in the UV spectral region and visible at 413, 645, 727 and 811 nm.
  • ABTS is a product that has great stability over a wide pH range, showing the same absorbance spectrum at pH 4 and pH 8.5.
  • the formation of the ABTS + radical is also carried out in that pH range but the enzymatic activity of the peroxidase itself is dependent on the pH of the reaction medium so that when the activity is alkalized the activity decreases, thus increasing the period of delay or "lag time".
  • the activity of our enzyme could be adjusted to an exponential curve so that it is maximum at pH 4.5 and ceases to be active at pH greater than 10.
  • Our assays will run at pH 6-8.5 so that we ensure the activity of the enzyme.
  • the quantification of the free radical sequestration capacity of a sample is carried out by decolorization tests in which the formation of ABTS + results in a characteristic coloration that will decrease proportionally to the amount of substances capable of trapping these radicals that are added to the reaction volume.
  • This loss of color can be measured by kinetic monitoring of loss of absorbency at 413 nm (wavelength that does not interfere with other molecules) over a minute using HRP as peroxidase and ascorbic acid (L-ASC) as a negative control.
  • the reaction medium comprises 50 mM phosphate buffer pH 6, 7.5, 8, 2 mM H 2 O 2, 2 mM ABTS, 0.25 uM HRP enzyme and increasing concentrations shown.
  • TAA The calculation of TAA is established according to the relationship between the slopes (Abs / min) of enzymatic tests in which the course of the reaction is estimated in the absence of antioxidants (positive control), and in the presence of different concentrations of substances with possible activity antioxidant
  • the slope of the control kinetics would correspond to a TAA of zero percent, based on this the percentage of inhibition of the other curves.
  • Lipid peroxidation is a well-established mechanism of cell damage in plants and animals, as well as food spoilage (thickening). This process leads to the production of lipidium peroxides and degradation aldehydes that leads to loss of cell membrane function and integrity. Shinorine isolated from Gymnogongrus devoniensis was studied as an inhibitor of lipid peroxidation in vitro using the ⁇ -carotene bleaching technique.
  • the method of decolorization of ⁇ -carotene is widely used to determine the antioxidant capacity of various substances in lipophilic medium, most of them extracted from fruits, vegetables and other products intended for food consumption in order to determine their greater or lesser degree of self-preservation in a natural state. It is a spectrophotometric method that measures the inhibition caused by an antioxidant on the discoloration of ⁇ -carotene in an aqueous system emulsified with Tween 20 and linoleic acid.
  • Linoleic acid self-oxidizes at a high rate in the presence of specially activated hydrogen atoms.
  • ⁇ -carotene a precursor to vitamin A, is also known as a lipophilic antioxidant that prevents lipid peroxidation in membranes by sequestering singlet oxygen molecules and peroxyl lipidium radicals.
  • the ⁇ -carotene when it is in the presence of linoleic acid, yields electrons delaying the initiation stage of the linoleic acid self-oxidation process as well as limiting the propagation phase of the damage by simultaneously eliminating formed peroxidic radicals.
  • ⁇ -carotene has a maximum absorption at 470 nm. This maximum varies when the molecule oxidizes since it loses double bonds and the structure of the molecule's chromophore is It is altered, thus losing its characteristic orange color and can be detected spectrophotometrically.
  • the absorbance of the reaction medium will remain unchanged over time in the presence of antioxidant substances, with a drop in the absorbance of the sample when measured in the absence of antioxidants.
  • the measurement of the antioxidant capacity of a substance will be inversely proportional to the slope drop of the curve that describes the oxidation of ⁇ -carotene (measured at a wavelength of 470 nm).
  • ⁇ -tocopherol ( ⁇ -TOC) was used as a positive control.
  • P refers to the slopes of the obtained fading curves (Abs / time).
  • the superoxides (O 2 " ) radicals are mediators of autooxidation reactions of some compounds. Most of the time these oxidized compounds are characterized by having a characteristic and quantifiable absorption spectrum by spectrophotometry. Pyrogallol (1,2,3-benzenotriol ) is a substance that rapidly oxidizes in the presence of oxygen, especially in alkaline solutions, at pH 7.9 the SOD inhibits 99% of the reaction, indicating a practically total participation of the superoxide anion O 2 " in the reaction.
  • the oxidized pyrogallol has a maximum absorption at 420 nm so that the ability of MAAs to sequester superoxide radicals was measured as loss of absorbance of kinetic tests monitored spectrophotometrically (Shimadzu UV 1603) during one minute of reaction.
  • the protocol that was carried out was based on Marklund & Marklund (1974, Eur. J. Biochem., 47: 469-474) with some modifications.
  • the reaction mixture contained 0.4 mM pyrogallol and MAA at different concentrations in 50 mM phosphate buffer at pH 8.2, containing 1 mM diethylenetriaminepentaacetic acid in a final ImI incubation volume. The temperature was stable at 20 ⁇ 1 0 C.
  • the positive control was the kinetic curve of generation of oxidized pyrogallol radicals in the absence of antioxidants to compare them with different concentrations of SOD as a known antioxidant.
  • Dose-response relationships for the MAAs under study were determined at Different concentrations.
  • the sequestration capacity of superoxide radicals of the purified extracts was evaluated according to the following formula:
  • P refers to the slopes of the kinetic oxidation curves of pyrogallol (Abs / time).

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Abstract

The invention relates to a mycosporin-type amino acid (shinorine) which can be used as an antioxidant and which is suitable for use in the biotechnological industry. The invention outlines the potential use of a mycosporin-type amino acid (MAA) as an antioxidant substance, specifically shinorine isolated from red alga Gymnogongrus devoniensis, and to the possible use thereof in pharmaceutical, nutraceutical or functional food preparations, among others, for the prevention of oxidative stress.

Description

AMINOÁCIDO TIPO MCOSPORINA (SHINORINE) COMO ANTIOXIDANTE MCOSPORINE TYPE AMINO ACID (SHINORINE) AS AN ANTIOXIDANT

SECTOR TÉCNICOTECHNICAL SECTOR

La presente invención se encuadra en el sector biotecnológico y describe el potencial uso como sustancias antioxidantes de determinados metabolitos secundarios denominados aminoácidos tipo micosporina (MAAs) aislados de algas rojas y liqúenes marinos además de su posible aplicación en preparados farmacéuticos, nutraceúticos, alimentos funcionales para la prevención de estrés oxidativo.The present invention is part of the biotechnology sector and describes the potential use as antioxidants of certain secondary metabolites called mycosporin-type amino acids (MAAs) isolated from red algae and marine lichens in addition to their possible application in pharmaceutical preparations, nutraceuticals, functional foods for oxidative stress prevention

TÉCNICA ANTERIORPREVIOUS TECHNIQUE

La radiación ultravioleta es uno de factores biológicos que limitan la supervivencia, fisiología y crecimiento de muchos organismos. Algunos de los múltiples efectos dañinos de la radiación UV incluye la alteración de moléculas de ADN y proteínas, inactivación de enzimas y a la formación de radicales libres, los cuales atacan a membranas celulares y otras moléculas diana alterando su funcionalidad. Todos los organismos aerobios disponen de una gran variedad de sistemas de defensa antioxidante tanto enzimáticos como no enzimáticos que se coordinan cooperativamente y protegen al organismo de los riesgos que conlleva el estrés oxidativo. Entre ellos destacan las actividades enzimáticas de la superóxido dismutasa (SOD), glutatión peroxidasa (GPX) y catalasa (CAT); además del ácido ascórbico (vitamina C), α- tocoferol (vitamina E), glutatión (GSH), β- caroteno, vitamina A, flavonoides y ácidos fenólicos entre otros. Se entiende por radical libre a cualquier especie química que contiene uno o más electrones desapareados en sus orbitales externos de manera que un compuesto puede convertirse en radical libre captando o perdiendo un electrón. Aunque existen radicales libres de muy distinta naturaleza, son las especies que derivan de la molécula de oxígeno (ROS) las más abundantes en los organismos aerobios destacando productos de la ruptura o la excitación del O2 como el oxígeno singlete 1O2 y especies de oxígeno que están parcialmente reducidas como el radical hidroxilo (OH»), aniones superóxido (O2 ") y peróxido de hidrógeno (H2O2). Estas moléculas inestables recorren el organismo tomando electrones con lo que recuperan su estabilidad electroquímica, esto las hace muy peligrosas porque para conseguirlo atacan moléculas estables. Una vez que el radical libre ha conseguido tomar el electrón que necesita para emparejar su electrón libre, la otra molécula se convierte a su vez en un radical libre, iniciándose así un ciclo destructivo para nuestras células.Ultraviolet radiation is one of biological factors that limit the survival, physiology and growth of many organisms. Some of the multiple harmful effects of UV radiation include the alteration of DNA and protein molecules, inactivation of enzymes and the formation of free radicals, which attack cell membranes and other target molecules altering their functionality. All aerobic organisms have a wide variety of both enzymatic and non-enzymatic antioxidant defense systems that cooperatively coordinate and protect the body from the risks of oxidative stress. Among them, the enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) stand out; in addition to ascorbic acid (vitamin C), α-tocopherol (vitamin E), glutathione (GSH), β-carotene, vitamin A, flavonoids and phenolic acids among others. Free radical means any chemical species that contains one or more missing electrons in its external orbitals so that a compound can become a free radical by capturing or losing an electron. Although free radicals of a very different nature exist, it is the species that derive from the oxygen molecule (ROS) the most abundant in aerobic organisms, highlighting products of the rupture or excitation of O 2 such as singlet oxygen 1 O 2 and species of oxygen that are partially reduced such as the hydroxyl radical (OH » ), superoxide anions (O 2 " ) and hydrogen peroxide (H 2 O 2 ). These unstable molecules travel through the body taking electrons, thus recovering their electrochemical stability, this it makes them very dangerous because to achieve this they attack stable molecules. Once the free radical has managed to take the electron it needs to match its free electron, the other molecule in turn becomes a free radical, thus initiating a destructive cycle for our cells.

Los radicales libres dan lugar a alteraciones importantes en moléculas como ADN, lípidos y proteínas, alterando gravemente el ciclo y la funcionalidad celular. El ADN puede sufrir pérdida de bases así como la ruptura de una o de ambas hebras del material genético, alteraciones que pueden traducirse en mutaciones irreversibles. Muchas proteínas son capaces de absorber una gran cantidad de oxidaciones sin que aparentemente se vea afectada su función. Sin embargo, es indudable que las consecuencias de las alteraciones en algunas funciones, por ejemplo, la recepción y transmisión de señales, el transporte de iones, la duplicación y reparación del ADN, las respuestas a condiciones de tensión y el metabolismo energético, la transcripción y traducción pueden ser críticas para la célula. Los daños producidos por el OH • y el 1O2 son irreversibles y en términos generales marcan las proteínas para su degradación. Las membranas celulares también pueden resultar seriamente dañadas en situación de estrés oxidativo ya que fosfolípidos que tienen ácidos grasos con varios dobles enlaces son muy susceptibles a la oxidación por pérdida de un hidrógeno (alílico).Una vez generado el radical carbono en un ácido graso, éste reacciona con el oxígeno molecular formando un radical peroxilo. El radical peroxilo puede tomar un hidrógeno alílico a otro metileno con lo cual se propaga la reacción. Los hidroperóxidos, que son compuestos estables, si entran en contacto con iones metálicos de transición producirán más radicales libres que iniciarán y propagarán otras reacciones en cadena. Así, las membranas resultan seriamente dañadas y por tanto su funcionalidad se ve alterada.Free radicals give rise to important alterations in molecules such as DNA, lipids and proteins, seriously altering the cycle and cellular functionality. The DNA can suffer loss of bases as well as the rupture of one or both strands of the genetic material, alterations that can result in irreversible mutations. Many proteins are capable of absorbing a large amount of oxidation without apparently affecting their function. However, there is no doubt that the consequences of alterations in some functions, for example, the reception and transmission of signals, the transport of ions, the duplication and repair of DNA, the responses to stress conditions and energy metabolism, transcription and translation can be critical for the cell. The damages produced by OH • and 1 O 2 are irreversible and in general terms mark proteins for degradation. Cell membranes can also be seriously damaged in oxidative stress because phospholipids that have fatty acids with several double bonds are very susceptible to oxidation due to the loss of a hydrogen (allyl). Once the carbon radical has been generated in a fatty acid, it reacts with molecular oxygen forming a peroxyl radical. The peroxyl radical can take an allylic hydrogen to another methylene whereby the reaction is propagated. Hydroperoxides, which are stable compounds, if they come into contact with transition metal ions will produce more free radicals that will initiate and propagate other chain reactions. Thus, the membranes are seriously damaged and therefore their functionality is altered.

Los radicales libres se asocian con un amplio rango de patologías y enfermedades como el Alzheimer o el Parkinson y afecciones relacionadas con la exposición solar como la aparición de cataratas, fotoenvejecimiento, episodios inflamatorios y neoplasias. También son los responsables de la oxidación de las grasas de los alimentos, que es la forma de deterioro más importante después de las alteraciones producidas por microorganismos. Con la oxidación, aparecen olores y sabores a rancio, se altera el color y la textura, y desciende el valor nutritivo al perderse algunas vitaminas y ácidos grasos poliinsaturados. Además, los productos formados en la oxidación pueden llegar a ser nocivos para la salud. Los aminoácidos tipo micosporina están constituidos por un anillo de ciclohexenona o de ciclohexenimina, conjugado con un sustituyente nitrogenado de un aminoácido o su aminoalcohol que actúa como cromóforo permitiendo la absorción de determinada radiación de onda corta. Los metabolitos que se aislan en hongos presentan un rango de absorción entre 310 y 320 nm y poseen anillos de ciclohexenona exclusivamente, conociéndoseles por el nombre de micosporinas en referencia a su origen. Por el contrario, los metabolitos que se aislan de organismos marinos y algas contienen anillos de ciclohexenimina, con absorciones máximas entre 310 y 360 nm y se les conoce con el nombre de aminoácidos tipo micosporina ó MAAs. Aún así, la mycosporine-glycine y la mycosporine taurine son aminociclohexenonas aisladas de organismos marinos. En la actualidad hay descritas 13 micosporinas distintas en hongos y 23 MAAs en organismos marinos. Son moléculas pequeñas, con pesos moleculares que rondan los 330 Da y presentan una alta fotoestabilidad. Se comportan como moléculas anfóteras, similares a los aminoácidos, de manera que presentan cargas positivas y negativas en la misma molécula. Muestran características físico-químicas propias de compuestos iónicos por ejemplo alto punto de efusión y alta solubilidad en agua.Free radicals are associated with a wide range of pathologies and diseases such as Alzheimer's or Parkinson's and conditions related to sun exposure such as the appearance of cataracts, photoaging, inflammatory episodes and neoplasms. They are also responsible for the oxidation of food fats, which is the most important form of deterioration after alterations caused by microorganisms. With oxidation, stale odors and flavors appear, the color and texture are altered, and the nutritional value decreases when some vitamins and polyunsaturated fatty acids are lost. In addition, products formed in oxidation can become harmful to health. Mycosporin-type amino acids consist of a cyclohexenone or cyclohexenimine ring, conjugated with a nitrogenous substituent of an amino acid or its amino alcohol that acts as a chromophore allowing the absorption of certain shortwave radiation. The metabolites that are isolated in fungi have an absorption range between 310 and 320 nm and have cyclohexenone rings exclusively, known by the name of Mycosporins in reference to their origin. On the contrary, metabolites that are isolated from marine organisms and algae contain cyclohexenimine rings, with maximum absorptions between 310 and 360 nm and are known as mycosporine-type amino acids or MAAs. Still, mycosporine-glycine and mycosporine taurine are aminocyclohexenones isolated from marine organisms. There are currently 13 different mycosporins described in fungi and 23 MAAs in marine organisms. They are small molecules, with molecular weights around 330 Da and have high photostability. They behave like amphoteric molecules, similar to amino acids, so that they have positive and negative charges on the same molecule. They show physicochemical characteristics of ionic compounds, for example, high effusion point and high water solubility.

Son muchas las funciones que se les ha atribuido a estas moléculas en el organismo: desde osmolito orgánico en comunidades cianobacterianas Chlorogloeopsis, pigmentos accesorios fotosintéticos o precursores de estos, a moléculas determinantes en procesos reproductivos de algunas especies de peces, sin embargo es el papel fotoprotector frente a la radiación UV el más aceptado y documentado ya que al parecer actúan protegiendo parcialmente a los componentes celulares y procesos fisiológicos. Un número de trabajos han evaluado este tipo de moléculas por sus actividad como fotoprotectores de uso tópico vehiculizando extractos naturales con un alto porcentaje de MAAs y viendo su FPS y su potencial fotoprotector en células vegetales, queratinocitos humanos, etc. (Patente US 6787147; Patente WO 02/39974; Patente WO 03/020236). También se han publicado trabajos que hacen referencia a las propiedades antioxidativas de extractos obtenidos de algas y corales.There are many functions that have been attributed to these molecules in the organism: from organic osmolyte in Chlorogloeopsis cyanobacterial communities, photosynthetic accessory pigments or precursors of these, to determining molecules in reproductive processes of some species of fish, however it is the photoprotective role against UV radiation the most accepted and documented since apparently they act partially protecting cellular components and physiological processes. A number of studies have evaluated these types of molecules for their activity as topical photoprotectors, vehiculating natural extracts with a high percentage of MAAs and seeing their SPF and their photoprotective potential in plant cells, human keratinocytes, etc. (US Patent 6787147; WO 02/39974; WO 03/020236). Works have also been published that refer to the antioxidant properties of extracts obtained from algae and corals.

Dunlap y Yamamoto en 1995 (Comp. Biochem. Physiol. 112: 105-114) apuntaron a una posible actividad antioxidante de mycosporine-glycine mediante ensayos in vitro de peroxidación lipídica (método de la fosfatidilcolina) a partir de extractos de organismos marinos que contenían MAAs, mientras que otras iminoMAAs como porphyra 334, shinorine, palythine, asterine 330 y palythinol se mostraban oxidativamente robustas y no participaban en reacciones de oxidación-reducción. No obstante, como se ha indicado, dichos ensayos fueron realizados con extractos algales que conteman además de MAAs un alto porcentaje de otros componentes celulares como polisacáridos, enzimas, etc., por lo que no es posible afirmar firmemente la actividad antioxidante de mycosporine-glycine (o M-gly) en base a dicho trabajo.Dunlap and Yamamoto in 1995 (Comp. Biochem. Physiol. 112: 105-114) pointed to a possible antioxidant activity of mycosporine-glycine by in vitro assays of lipid peroxidation (phosphatidylcholine method) from extracts of marine organisms containing MAAs, while other iminoMAAs such as porphyra 334, shinorine, palythine, asterine 330 and palythinol were oxidatively robust and did not participate in oxidation-reduction reactions. However, as indicated, these tests were carried out with algal extracts that contain in addition to MAAs a high percentage of other cellular components such as polysaccharides, enzymes, etc., so it is not possible to firmly affirm the antioxidant activity of mycosporine-glycine (or M-gly) based on that work.

Nakayama y colaboradores en 1999 (J. Am. OiI Chem. Soc, 76: 649-653) aislaron un nuevo aminoácido tipo micosporina del alga roja Porphyra yezoensis llamado usujilene, ya identificado en Palmaria palmata pero no en ninguna especie de Porphyra. Sus resultados indicaban que tal MAA mostraba actividad antioxidante frente a la autoxidación del ácido linoleico (Métodos del ácido tiobarbitúrico y tiocianato férrico), donando determinados hidrógenos a radicales lipidíeos LOO- y dando lugar a moléculas de MAAs estabilizadas por resonancia al igual que el α- tocoferol.Nakayama et al. In 1999 (J. Am. OiI Chem. Soc, 76: 649-653) isolated a new mycosporin-like amino acid from the red seaweed Porphyra yezoensis called usujilene, already identified in Palmaria palmata but not in any species of Porphyra. Their results indicated that such MAA showed antioxidant activity against acid autoxidation. linoleic acid (Methods of thiobarbituric acid and ferric thiocyanate), donating certain hydrogens to LOO- lipid radicals and giving rise to resonance stabilized MAA molecules like α-tocopherol.

Suh y colaboradores en 2003 (Photochem. Photobiol. 78: 109-113) sugieren también la función antioxidante de la M-gly, pero probablemente actuando junto con otros MAAs activos. La M-gly, entre otros, podría jugar un importante papel participando en la eliminación de O2 " generado por sistemas fotosintetizadores endógenos.Suh et al. In 2003 (Photochem. Photobiol. 78: 109-113) also suggest the antioxidant function of M-gly, but probably acting together with other active MAAs. M-gly, among others, could play an important role by participating in the elimination of O 2 " generated by endogenous photosynthesizer systems.

Yakovleva y colaboradores en 2004 (Comp. Biochem. PhysioL, 139: 721-739 examinaron la importancia de la M-gly como antioxidante funcional frente a estrés térmico en dos corales, Platygyra ryukyuensis y Stylophora pistillata, en base a la correlación entre el grado de susceptibilidad y la concentración endógena de M-gly.Yakovleva et al. In 2004 (Comp. Biochem. PhysioL, 139: 721-739 examined the importance of M-gly as a functional antioxidant against thermal stress in two corals, Platygyra ryukyuensis and Stylophora pistillata, based on the correlation between grade of susceptibility and endogenous concentration of M-gly.

También la patente US2004228875 hace referencia a las propiedades antioxidativas de extractos obtenidos a partir de algas del género Porphyra, aunque sin concluir sobre el posible papel jugado por MAAs. Publicaciones más recientes analizan las propiedades antioxidativas de extractos obtenidos a partir de algas pero sin concretar la participación de MAAs (Yuan y colaboradores, 2005, Food Chem. ToxicoL, 43: 1073-1081; Kuda y colaboradores, 2005, J. Food Compos. Anal, 18: 625-633).Also the patent US2004228875 refers to the antioxidant properties of extracts obtained from algae of the genus Porphyra, although without concluding on the possible role played by MAAs. More recent publications analyze the antioxidant properties of extracts obtained from algae but without specifying the participation of MAAs (Yuan et al., 2005, Food Chem. ToxicoL, 43: 1073-1081; Kuda et al., 2005, J. Food Compos. Anal, 18: 625-633).

Se puede concluir, por tanto, que el papel antioxidante de los iminoMAAs como tales, es decir, purificados o aislados en un alto grado de pureza, no se conoce bien, como tampoco su comportamiento a nivel de secuestro de radicales libres hidrosolubles.It can be concluded, therefore, that the antioxidant role of iminoMAAs as such, that is, purified or isolated in a high degree of purity, is not well known, nor is their behavior at the level of water-soluble free radical sequestration.

Un antioxidante se define como una sustancia que en bajas concentraciones comparado con un substrato oxidable, retrasa o previene su oxidación. La presente invención describe la potencialidad del MAA shinorine aislado de Gymnogongrus devoniensis como secuestrador de radicales e inhibidor de la peroxidación lipídica. En los ensayos realizados, el nuevo antioxidante es comparado con otro antioxidante ya conocido, el α-tocoferol. El compuesto descrito podría utilizarse en aplicaciones terapéuticas, y en aplicaciones no médicas para la estabilización de compuestos susceptibles del deterioro oxidativo, en la preservación de alimentos o productos relacionados, y en complementos nutricionales, nutracéuticos, alimentos funcionales o de parafarmacia por sus propiedades antioxidantes para prevenir el estrés oxidativo.An antioxidant is defined as a substance that in low concentrations compared to an oxidizable substrate, delays or prevents its oxidation. The present invention describes the potentiality of shinorine MAA isolated from Gymnogongrus devoniensis as a radical scavenger and lipid peroxidation inhibitor. In the tests carried out, the new antioxidant is compared with another known antioxidant, α-tocopherol. The described compound could be used in therapeutic applications, and in non-medical applications for the stabilization of compounds susceptible to oxidative deterioration, in the preservation of food or related products, and in nutritional, nutraceutical, functional or parapharmacy supplements for their antioxidant properties for prevent oxidative stress.

DIVULGACIÓN DE LA INVENCIÓN La presente invención presenta un compuesto aislado de Gymnogongrus devoniensis con la siguiente estructura y de utilidad como antioxidante y secuestrador de radicales libres.DISCLOSURE OF THE INVENTION The present invention presents an isolated compound of Gymnogongrus devoniensis with the following structure and useful as an antioxidant and free radical sequestrant.

Figure imgf000006_0001
Figure imgf000006_0001

Se han purificado compuestos del tipo aminoácidos tipo micosporina en fase acuosa partiendo de Gymnogongrus devoniensis. Los compuestos se han detectado y caracterizado por HPLC. Se empleó un detector UV-visible (detector de fotodiodos 996), que medía la absorbancia para cada muestra entre los 290 y 400 nm. Una vez extraído el cromatograma a 330 nm, se identificaron los picos por co-cromatografía según sus espectros y tiempos de retención, comparándose con estándares de MAAs. La extracción a escala preparativa se realizó disolviendo 60-80 g (PF) de material biológico en 1 litro de metanol al 20% v/v e incubándose en un baño termostático a 45 0C durante 2 horas. Posteriormente se centrifuga el extracto a 14000rpm durante 15 min y rotavaporación a 45 0C para eliminar parte del metanol de la muestra.Compounds of the mycosporin type amino acid type have been purified in the aqueous phase starting from Gymnogongrus devoniensis. The compounds have been detected and characterized by HPLC. A UV-visible detector (photodiode detector 996) was used, which measured the absorbance for each sample between 290 and 400 nm. Once the chromatogram was extracted at 330 nm, the peaks were identified by co-chromatography according to their spectra and retention times, compared to MAAs standards. Preparative scale extraction was performed by dissolving 60-80 g (PF) of biological material in 1 liter of methanol to 20% v / v and incubated in a thermostatic bath at 45 0 C for 2 hours. The extract centrifuged 14000rpm subsequently for 15 min and rotary evaporation at 45 0 C to remove part of the methanol in the sample.

La purificación se realizó en tres pasos consecutivos en los que se combinan técnicas cromatográficas de absorción mediante la aplicación de carbono activo, precipitación de polisacáridos al añadir a la muestra metanol 100% y separación final mediante cromatografía de intercambio iónico. Finalmente se obtuvieron soluciones acuosas de MAA en alto grado de pureza en concentraciones del orden de mM.The purification was performed in three consecutive steps in which chromatographic absorption techniques are combined by the application of active carbon, precipitation of polysaccharides by adding 100% methanol to the sample and final separation by ion exchange chromatography. Finally, aqueous solutions of MAA were obtained in a high degree of purity at concentrations of the order of mM.

Capacidad antioxidante de los aminoácidos tipo micosporinaAntioxidant capacity of mycosporin-like amino acids

Para medir la actividad como secuestrador de radicales hidrosolubles se ha utilizado el método de la ABTS peroxidasa, el cual permite determinar la actividad antioxidante total (TAA) de una muestra entendida como un parámetro que permite cuantificar la capacidad de una muestra, natural o procesada, de secuestrar radicales libres presentes en una solución acuosa. Shinorine aislado de Gymnogongrus devoniensis no presenta actividad antioxidante signifcativa a ningún pH ensayado como inhibidor de la producción de radicales libres hidrosolubles (ABTS+).To measure the activity as a water-soluble radical sequestrant, the ABTS peroxidase method has been used, which allows to determine the total antioxidant activity (TAA) of a sample understood as a parameter that allows quantifying the capacity of a sample, natural or processed, of sequestering free radicals present in an aqueous solution. Shinorine isolated from Gymnogongrus devoniensis does not show significant antioxidant activity at any pH tested as an inhibitor of water-soluble free radical production (ABTS +) .

Capacidad como inhibidor de la peroxidación lipídicaCapacity as a lipid peroxidation inhibitor

Shinorine aislado de Gymnogongrus devoniensis se estudió como inhibidor de la peroxidación lipídica in vitro mediante la técnica de decoloración del β-caroteno. El método de decoloración del β-caroteno es ampliamente utilizado para la determinación de la capacidad antioxidante de diversas sustancias en medio lipofílico, la mayoría de ellas extraídas de frutas, vegetales y demás productos destinados a consumo alimentario para poder determinar su mayor o menor grado de autoconservación en estado natural. En este ensayo, como control positivo se utilizó el α-tocoferol (α-TOC).Shinorine isolated from Gymnogongrus devoniensis was studied as an inhibitor of lipid peroxidation in vitro using the β-carotene bleaching technique. The method of decolorization of β-carotene is widely used to determine the antioxidant capacity of various substances in lipophilic medium, most of them extracted from fruits, vegetables and other products intended for food consumption in order to determine their greater or lesser degree of self-preservation in a natural state. In this test, α-tocopherol (α-TOC) was used as a positive control.

Shinorine aislado de Gymnogongrus devoniensis muestra actividad antioxidante moderada a nivel de inhibición de la peroxidación lipídica. Se constituye, pues, como un antioxidante de actividad moderada in vitro.Shinorine isolated from Gymnogongrus devoniensis shows moderate antioxidant activity at the level of lipid peroxidation inhibition. It is thus constituted as an antioxidant of moderate activity in vitro.

Secuestro de radicales superóxidoAbduction of superoxide radicals

El protocolo que se llevó a cabo fue basado en Marklund & Marklund (1974, Eur. J.The protocol that was carried out was based on Marklund & Marklund (1974, Eur. J.

Biochem., 47: 469-474) con algunas modificaciones. Relaciones de dosis-respuesta para las MAAs objeto de estudio se determinaron a diferentes concentraciones.Biochem., 47: 469-474) with some modifications. Dose-response relationships for the MAAs under study were determined at different concentrations.

Shinorine aislado de Gymnogongrus devoniensis a concentraciones de 1 mM inhibe al 50% la cinética de oxidación del pirogalol. Al actuar como antioxidante y secuestrador de radicales libres, este compuesto, en extractos o preparados que lo contengan, podría utilizarse en preparados o formulaciones farmacéuticas para la prevención y el tratamiento terapéutico de enfermedades o afecciones relacionadas con los radicales libres, en productos de parafarmacia, en alimentos funcionales, complementos nutricionales y preparados nutracéuticos, y en la industria alimentaria como potencial antioxidante (aditivo).Shinorine isolated from Gymnogongrus devoniensis at concentrations of 1 mM 50% inhibits the oxidation kinetics of pyrogallol. By acting as an antioxidant and free radical scavenger, this compound, in extracts or preparations containing it, could be used in pharmaceutical preparations or formulations for the prevention and therapeutic treatment of diseases or conditions related to free radicals, in parapharmacy products, in functional foods, nutritional supplements and nutraceutical preparations, and in the food industry as an antioxidant potential (additive).

BREVE DESCRIPCIÓN DE LOS DIBUJOSBRIEF DESCRIPTION OF THE DRAWINGS

Figura 1. Área (%) de picos eluídos y concentraciones expresadas en mg g"1 PS de diferentes MAAs presentes en extractos metanólicos de las algas Porphyra leucosticta,Figure 1. Area (%) of eluted peaks and concentrations expressed in mg g "1 PS of different MAAs present in methanolic extracts of algae Porphyra leucosticta,

Gymnogongrus devoniensis, Gelidium sesquipedale y del liquen Lichina pygmaea. Se observa la presencia de un tipo de MAA mayoritario en cada organismo ( > 66%) junto con otrasGymnogongrus devoniensis, Gelidium sesquipedale and lichen Lichina pygmaea. The presence of a type of majority MAA in each organism (> 66%) together with others

MAAs minoritarias y trazas de sustancias no identificadas.Minority MAAs and traces of unidentified substances.

Figura 2. Cromatograma de un extracto acuoso de shinorine aislado de Gymnogongrus devoniensis eluído de la columna cargada con resina DOWEX. Figura 3. Tabla . Dosis (μM) - respuesta de la actividad antioxidante (%) de shinorine aislado de Gymnogongrus devoniensis con respecto a 10 μM de α-tocoferol por el método de decoloración del β-caroteno. Los valores representan los valores medios y desviación estándar de 3 experimentos. Shinorine aislado de Gymnogongrus devoniensis resulta un buen antioxidante a concentración de 100-200 μM. Figura 4. Capacidad de secuestro de radicales superóxido generados por el método del pirogalol de shinorine aislado de Gymnogongrus devoniensis. Se representa la media y desviación estándar de tres experimentos.Figure 2. Chromatogram of an aqueous extract of shinorine isolated from Gymnogongrus devoniensis eluted from the column loaded with DOWEX resin. Figure 3. Table. Dose (μM) - antioxidant activity response (%) of shinorine isolated from Gymnogongrus devoniensis with respect to 10 μM of α-tocopherol by the method of decolorization of β-carotene. The values represent the mean values and standard deviation of 3 experiments. Shinorine isolated from Gymnogongrus devoniensis is a good antioxidant at a concentration of 100-200 μM. Figure 4. Sequestration capacity of superoxide radicals generated by the shinorine pyrogallol method isolated from Gymnogongrus devoniensis. The mean and standard deviation of three experiments are represented.

MANERA(S) DE REALIZAR LA INVENCIÓNWAY (S) OF CARRYING OUT THE INVENTION

Purificación a escala preparativa de shinorine a partir del alga roja Gymnogongrus devoniensisPreparatory scale purification of shinorine from Gymnogongrus devoniensis red algae

Se ha purificado compuestos del tipo aminoácidos tipo micosporina en fase acuosa partiendo de Gymnogongrus devoniensis. Los compuestos se han detectado y caracterizado por HPLC (Waters 600). La columna empleada para la separación de los MAAs en el HPLC fue una C8 (Sphereclone ™, Phenomenex, Aschaffenburg, Alemania), empaquetada con micropartículas porosas de sílica de 5 mm de diámetro con superficie derivatizada con una cadena alifática de 8 átomos de carbono (octadecil silano). Su tamaño era de 250 x 4.6 mm. Se empleó una precolumna (Phenomenex, Aschaffenburg, Alemania) afín a la columna empleada. La fase móvil que se empleó fue metanol al 2.5% (v/v, calidad HPLC) más 0.1 % de ácido acético (v/v) bombeada isocráticamente a una velocidad de flujo de 0.5 mi min"1. Se empleó un detector UV- visible (detector de fotodiodos 996), que medía la absorbencia para cada muestra entre los 290 y 400 nm. En la figura 1 vienen recogidos los porcentajes en área de los picos cromatografiados de distintos extractos algales, algunos identificados como MAAs y otros desconocidos. El objetivo de la purificación fue aislar en fase acuosa el MAA mayoritario en Gymnogongrus devoniensis además de eliminar trazas y otro tipos de compuestos no identificados.Compounds of the mycosporin type amino acid type have been purified in the aqueous phase starting from Gymnogongrus devoniensis. The compounds have been detected and characterized by HPLC (Waters 600). The column used for separation of MAAs in the HPLC was one C 8 (Sphereclone ™, Phenomenex, Aschaffenburg, Germany), packed with porous microparticles of silica of 5 mm in diameter with surface derivatized with an aliphatic chain of 8 carbon atoms (octadecyl silane). Its size was 250 x 4.6 mm. A pre-column (Phenomenex, Aschaffenburg, Germany) related to the column used was used. The mobile phase used was 2.5% methanol (v / v, HPLC quality) plus 0.1% acetic acid (v / v) isocratically pumped at a flow rate of 0.5 ml min. "1 A UV detector was used. visible (photodiode detector 996), which measured the absorbency for each sample between 290 and 400 nm. Figure 1 shows the percentages in area of the chromatographed peaks of different algal extracts, some identified as MAAs and others unknown. Purification objective was to isolate the major MAA in Gymnogongrus devoniensis in the aqueous phase, in addition to eliminating traces and other types of unidentified compounds.

Una vez extraído el cromatograma a 330 nm, se identificaron los picos por co- cromatografía según sus espectros y tiempos de retención, comparándose con estándares de MAAs, proporcionados por el profesor Dr. UIf Karsten (Universidad de Rostock, Alemania) extraídos de distintos organismos marinos: Mastocarpus stellatus (shinorine), Porphyra yezoensis (porphyra-334), Bostrychia scorpioides (palythine), los ojos de la trucha del coral Plectropomus leopardus (asterine 330) y el liquen Lichina pygmaea recolectado en Francia (M- glycine). El cromatograma de los extractos recogidos después del paso por la columna de intercambio iónico DOWEX50 se muestran en la figura 2. Shinorine apareció en un alto grado de pureza, observándose la ausencia de compuestos desconocidos con picos de absorción entre los 320 - 330 nm.Once the chromatogram was extracted at 330 nm, the peaks were identified by chromatography according to their spectra and retention times, compared with MAA standards, provided by Professor Dr. UIf Karsten (University of Rostock, Germany) extracted from different organisms Marine: Mastocarpus stellatus (shinorine), Porphyra yezoensis (porphyra-334), Bostrychia scorpioides (palythine), the eyes of the coral trout Plectropomus leopardus (asterine 330) and the lichen Lichina pygmaea collected in France (M-glycine). The chromatogram of the extracts collected after passing through the DOWEX50 ion exchange column are shown in Figure 2. Shinorine appeared to a high degree. of purity, observing the absence of unknown compounds with absorption peaks between 320-330 nm.

La extracción a escala preparativa se realizó disolviendo 60-80 g (PF) de material biológico en 1 litro de metanol al 20% v/v e incubándose en un baño termostático a 45 0C durante 2 horas. Posteriormente se centrifuga el extracto a 14000 rpm durante 15 min y rotavaporación a 45 0C para eliminar parte del metanol de la muestra.Preparative scale extraction was performed by dissolving 60-80 g (PF) of biological material in 1 liter of methanol to 20% v / v and incubated in a thermostatic bath at 45 0 C for 2 hours. Extract at 14,000 rpm for 15 min and then centrifuged at rotary evaporation 45 was 0 C to remove part of the methanol in the sample.

La purificación se realiza en tres pasos consecutivos en los que se combinan técnicas cromatográficas de absorción mediante la aplicación de carbono activo, precipitación de polisacáridos al añadir a la muestra metanol 100% y separación final mediante cromatografía de intercambio iónico (resina Dowex 50 W x 8-100). Para la elución del aminoácido tipo micosporina shinorine se utilizó como eluyente agua bidestilada, con un pH ligeramente alcalino (7.2 ). Finalmente se obtuvieron soluciones acuosas de MAA en alto grado de pureza en concentraciones del orden de mM.The purification is carried out in three consecutive steps in which chromatographic absorption techniques are combined by the application of active carbon, precipitation of polysaccharides by adding 100% methanol to the sample and final separation by ion exchange chromatography (Dowex 50 W x 8 resin -100). For the elution of the amino acid type mycosporine shinorine, double-distilled water was used as eluent, with a slightly alkaline pH (7.2). Finally, aqueous solutions of MAA were obtained in a high degree of purity at concentrations of the order of mM.

Capacidad antioxidante a nivel de secuestro de radicales hidrosolubles ABTSAntioxidant capacity at the level of ABTS water-soluble radical sequestration

Para medir la actividad como secuestradores de radicales hidrosolubles se ha utilizado el método de la ABTS peroxidasa, el cual permite determinar la actividad antioxidante total (TAA) de una muestra entendida como un parámetro que permite cuantificar la capacidad de una muestra, natural o procesada, de secuestrar radicales libres presentes en una solución acuosa. Este parámetro está orientado a dar información de la actividad antioxidante que puede presentar una muestra concreta con independencia de las actividades parciales que puedan presentar cada uno de sus componentes o los efectos de sinergismo que pudiesen establecerse.To measure the activity as water-soluble radical sequestrants, the ABTS peroxidase method has been used, which allows to determine the total antioxidant activity (TAA) of a sample understood as a parameter that allows quantifying the capacity of a sample, natural or processed, of sequestering free radicals present in an aqueous solution. This parameter is aimed at giving information on the antioxidant activity that a specific sample can present, regardless of the partial activities that each of its components may present or the synergism effects that could be established.

El 2,2'- Azino —bis- ( 3- etil-benzotizolina-6- ácido sulfónico) o ABTS es un compuesto que presenta gran estabilidad química, alta solubilidad en agua y un máximo de absorción en la banda del UVA a 342 nm. Este compuesto en presencia de H2O2 y enzimas peroxidasas deriva a un radical metaestable (ABTS+) con un espectro de absorción característico y diferente al ABTS, presentando máximos de absorción en la región espectral del UV y visible a 413, 645, 727 y 811 nm. El ABTS es un producto que presenta gran estabilidad en un amplio rango de pH, mostrando el mismo espectro de absorbancia a pH 4 y pH 8.5. Así mismo, la formación del radical ABTS+ también se lleva a cabo en ese rango de pH pero la actividad enzimática de la peroxidasa sí que es dependiente del pH del medio de reacción de manera que al alcalinizarse éste la actividad disminuye, aumentando así el periodo de retardo o "lag time". La actividad de nuestra enzima se podría ajustar a una curva exponencial de manera que es máxima a pH 4.5 y deja de ser activa a pH superiores a 10. Nuestros ensayos discurrirán a pH 6-8.5 de manera que aseguramos la actividad de la enzima.2,2'-Azino-bis- (3- ethyl-benzotizoline-6- sulfonic acid) or ABTS is a compound that has great chemical stability, high water solubility and maximum absorption in the UVA band at 342 nm . This compound in the presence of H 2 O 2 and peroxidases enzymes derives to a metastable radical (ABTS + ) with a characteristic absorption spectrum and different from ABTS, presenting maximum absorption in the UV spectral region and visible at 413, 645, 727 and 811 nm. ABTS is a product that has great stability over a wide pH range, showing the same absorbance spectrum at pH 4 and pH 8.5. Likewise, the formation of the ABTS + radical is also carried out in that pH range but the enzymatic activity of the peroxidase itself is dependent on the pH of the reaction medium so that when the activity is alkalized the activity decreases, thus increasing the period of delay or "lag time". The activity of our enzyme could be adjusted to an exponential curve so that it is maximum at pH 4.5 and ceases to be active at pH greater than 10. Our assays will run at pH 6-8.5 so that we ensure the activity of the enzyme.

La cuantificación de la capacidad de secuestro de radicales libres de una muestra se llevan a cabo mediante ensayos de decoloración en los cuales la formación de ABTS+ da lugar a una coloración característica que disminuirá de manera proporcional a la cantidad de sustancias con capacidad de atrapar estos radicales que se le añadan al volumen de reacción. Esta pérdida de color puede medirse mediante seguimientos cinéticos de pérdida de absorbencia a 413 nm (longitud de onda que no interfiere con otras moléculas) a lo largo de un minuto utilizando como peroxidasa la HRP y como control negativo el ácido ascórbico (L-ASC). El medio de reacción se compone de tampón fosfato 50 mM pH 6, 7.5, 8, H2O2 2mM, ABTS 2 mM, enzima HRP 0.25 μM y muestra a concentraciones crecientes.The quantification of the free radical sequestration capacity of a sample is carried out by decolorization tests in which the formation of ABTS + results in a characteristic coloration that will decrease proportionally to the amount of substances capable of trapping these radicals that are added to the reaction volume. This loss of color can be measured by kinetic monitoring of loss of absorbency at 413 nm (wavelength that does not interfere with other molecules) over a minute using HRP as peroxidase and ascorbic acid (L-ASC) as a negative control. . The reaction medium comprises 50 mM phosphate buffer pH 6, 7.5, 8, 2 mM H 2 O 2, 2 mM ABTS, 0.25 uM HRP enzyme and increasing concentrations shown.

El cálculo de TAA se establece según la relación entre las pendientes (Abs/min) de ensayos enzimáticos en los cuales el curso de la reacción es estimado en ausencia de antioxidantes (control positivo), y en presencia de diferentes concentraciones de sustancias con posible actividad antioxidante. De este modo, la pendiente de la cinética control correspondería a una TAA del cero por ciento, calculándose en base a ésta los porcentajes de inhibición de las demás curvas.The calculation of TAA is established according to the relationship between the slopes (Abs / min) of enzymatic tests in which the course of the reaction is estimated in the absence of antioxidants (positive control), and in the presence of different concentrations of substances with possible activity antioxidant Thus, the slope of the control kinetics would correspond to a TAA of zero percent, based on this the percentage of inhibition of the other curves.

Capacidad como inhibidor de la peroxidación lipídicaCapacity as a lipid peroxidation inhibitor

La peroxidación lipídica es un mecanismo bien establecido de daño celular en plantas y animales, así como de deterioro de alimentos (enranciamiento). Este proceso conduce a la producción de peróxidos lipidióos y aldehidos de degradación que conlleva pérdida de la función de la membrana celular y de su integridad. Shinorine aislado de Gymnogongrus devoniensis se estudió como inhibidor de la peroxidación lipídica in vitro mediante la técnica de decoloración del β-caroteno.Lipid peroxidation is a well-established mechanism of cell damage in plants and animals, as well as food spoilage (thickening). This process leads to the production of lipidium peroxides and degradation aldehydes that leads to loss of cell membrane function and integrity. Shinorine isolated from Gymnogongrus devoniensis was studied as an inhibitor of lipid peroxidation in vitro using the β-carotene bleaching technique.

El método de decoloración del β-caroteno es ampliamente utilizado para la determinación de la capacidad antioxidante de diversas sustancias en medio lipofílico, la mayoría de ellas extraídas de frutas, vegetales y demás productos destinados a consumo alimentario para poder determinar su mayor o menor grado de autoconservación en estado natural. Se trata de un método espectrofotométrico que mide la inhibición que causa un antioxidante sobre la decoloración del β-caroteno en un sistema acuoso emulsifϊcado con Tween 20 y ácido linoleico.The method of decolorization of β-carotene is widely used to determine the antioxidant capacity of various substances in lipophilic medium, most of them extracted from fruits, vegetables and other products intended for food consumption in order to determine their greater or lesser degree of self-preservation in a natural state. It is a spectrophotometric method that measures the inhibition caused by an antioxidant on the discoloration of β-carotene in an aqueous system emulsified with Tween 20 and linoleic acid.

El ácido linoleico se autoxida a una alta velocidad ante la presencia de átomos de hidrógeno especialmente activados. El β-caroteno, precursor de la vitamina A, también es conocido como antioxidante lipofílico que previene de la peroxidación lipídica en membranas secuestrando moléculas de oxígeno singlete y radicales lipidióos peroxilos. El β-caroteno, cuando se encuentra en presencia de ácido linoleico, cede electrones retardando la etapa de iniciación del proceso de autooxidación del ácido linoleico así como limitando la fase de propagación del daño al eliminar simultáneamente radicales peróxidos formados. Si añadimos una nueva sustancia con posible capacidad antioxidante al medio de reacción que contiene ácido linoleico y β-caroteno, ésta nueva sustancia tenderá a oxidarse ella preferentemente al β- caroteno, compitiendo con este por el secuestro de estos radicales.Linoleic acid self-oxidizes at a high rate in the presence of specially activated hydrogen atoms. Β-carotene, a precursor to vitamin A, is also known as a lipophilic antioxidant that prevents lipid peroxidation in membranes by sequestering singlet oxygen molecules and peroxyl lipidium radicals. The β-carotene, when it is in the presence of linoleic acid, yields electrons delaying the initiation stage of the linoleic acid self-oxidation process as well as limiting the propagation phase of the damage by simultaneously eliminating formed peroxidic radicals. If we add a new substance with possible antioxidant capacity to the reaction medium containing linoleic acid and β-carotene, this new substance will tend to oxidize it preferentially to β-carotene, competing with it for the sequestration of these radicals.

El β-caroteno presenta un máximo de absorción a 470 nm. Este máximo varía cuando la molécula se oxida ya que pierde dobles enlaces y la estructura del cromóforo de la molécula se ve alterada, perdiendo así su característico color naranja y pudiendo ser detectado espectrofotométricamente. La absorbancia del medio de reacción permanecerá invariable a lo largo del tiempo en presencia de sustancias antioxidantes, advirtiéndose una caída en la absorbancia de la muestra cuando se mida en ausencia de antioxidantes. Así pues, la medida de la capacidad antioxidante de una sustancia será inversamente proporcional a la caída de pendiente de la curva que describe la oxidación del β-caroteno (medida a longitud de onda de 470 nm).Β-carotene has a maximum absorption at 470 nm. This maximum varies when the molecule oxidizes since it loses double bonds and the structure of the molecule's chromophore is It is altered, thus losing its characteristic orange color and can be detected spectrophotometrically. The absorbance of the reaction medium will remain unchanged over time in the presence of antioxidant substances, with a drop in the absorbance of the sample when measured in the absence of antioxidants. Thus, the measurement of the antioxidant capacity of a substance will be inversely proportional to the slope drop of the curve that describes the oxidation of β-carotene (measured at a wavelength of 470 nm).

En este ensayo, como control positivo se utilizó el α-tocoferol (α-TOC).In this test, α-tocopherol (α-TOC) was used as a positive control.

La actividad de la solución se evaluó según el grado de decoloración del β-caroteno, aplicando la fórmula propuesta por Hidalgo y colaboradores (1994, Phytochemistry, 37: 1585- 1587) con algunas modificaciones:The activity of the solution was evaluated according to the degree of discoloration of β-carotene, applying the formula proposed by Hidalgo et al. (1994, Phytochemistry, 37: 1585-1587) with some modifications:

AA = [ P muestra - P control / P Patrón- P control ] • 100AA = [P sample - P control / P Pattern- P control] • 100

P hace referencia a las pendientes de las curvas de decoloración obtenidas (Abs/ tiempo).P refers to the slopes of the obtained fading curves (Abs / time).

Para ello ajustamos mediante regresión lineal la parte de la curva cinética que describe un comportamiento lineal. Los coeficientes de correlación para cada réplica de cada muestra eran todos superiores a 0.98.To do this, we adjust the part of the kinetic curve using linear regression that describes a linear behavior. The correlation coefficients for each replica of each sample were all greater than 0.98.

Secuestro de radicales superóxidoAbduction of superoxide radicals

Los radicales superóxidos (O2 ") son mediadores de reacciones de autooxidación de algunos compuestos. La mayoría de las veces estos compuestos oxidados se caracterizan por poseer un espectro de absorción característico y cuantificable por espectrofotometría. El pirogalol (1,2,3-benzenotriol) es una sustancia que se autooxida rápidamente en presencia de oxígeno especialmente en soluciones alcalinas. A pH 7.9 la SOD inhibe el 99% de la reacción indicando una participación prácticamente total del anión superóxido O2 " en la reacción. El pirogalol oxidado presenta un máximo de absorción a 420 nm de manera que la capacidad de las MAAs para secuestrar radicales superóxido fue medida como pérdida de absorbancia de ensayos cinéticos monitorizados espectrofotométricamente (Shimadzu UV 1603) durante un minuto de reacción. El protocolo que se llevó a cabo fue basado en Marklund & Marklund (1974, Eur. J. Biochem., 47: 469-474) con algunas modificaciones. La mezcla reacción contenía 0.4 mM de pirogalol y el MAA a diferentes concentraciones en 50 mM de tampón fosfato a pH 8.2, conteniendo 1 mM de ácido dietilenotriaminopentaacético en un volumen final de incubación de ImI. La temperatura se mantuvo estable a 20 ± 1 0C. El control positivo fue la curva cinética de generación de radicales de pirogalol oxidado en ausencia de antioxidantes para compararlos con distintas concentraciones de SOD como antioxidante conocido. Relaciones de dosis-respuesta para las MAAs objeto de estudio se determinaron a diferentes concentraciones. La capacidad de secuestro de radicales superóxido de los extractos purificados se evaluó siguiendo la siguiente fórmula:The superoxides (O 2 " ) radicals are mediators of autooxidation reactions of some compounds. Most of the time these oxidized compounds are characterized by having a characteristic and quantifiable absorption spectrum by spectrophotometry. Pyrogallol (1,2,3-benzenotriol ) is a substance that rapidly oxidizes in the presence of oxygen, especially in alkaline solutions, at pH 7.9 the SOD inhibits 99% of the reaction, indicating a practically total participation of the superoxide anion O 2 " in the reaction. The oxidized pyrogallol has a maximum absorption at 420 nm so that the ability of MAAs to sequester superoxide radicals was measured as loss of absorbance of kinetic tests monitored spectrophotometrically (Shimadzu UV 1603) during one minute of reaction. The protocol that was carried out was based on Marklund & Marklund (1974, Eur. J. Biochem., 47: 469-474) with some modifications. The reaction mixture contained 0.4 mM pyrogallol and MAA at different concentrations in 50 mM phosphate buffer at pH 8.2, containing 1 mM diethylenetriaminepentaacetic acid in a final ImI incubation volume. The temperature was stable at 20 ± 1 0 C. The positive control was the kinetic curve of generation of oxidized pyrogallol radicals in the absence of antioxidants to compare them with different concentrations of SOD as a known antioxidant. Dose-response relationships for the MAAs under study were determined at Different concentrations The sequestration capacity of superoxide radicals of the purified extracts was evaluated according to the following formula:

AA= 100 - [ P muestra • 100/ P control]AA = 100 - [P sample • 100 / P control]

P hace referencia a las pendientes de las curvas cinéticas de oxidación del pirogalol (Abs / tiempo). P refers to the slopes of the kinetic oxidation curves of pyrogallol (Abs / time).

Claims

REIVINDICACIONES 1. Extracto purificado de aminoácido tipo micosporina shinorine extraído del alga roja Gymnogongrus devoniensis caracterizado por no presentar actividad antioxidante significativa como inhibidor de la producción de radicales libres hidrosolubles en el rango de pH 6 - 8,5, por presentar actividad antioxidante moderada a nivel de inhibición de la peroxidación lipídica, presentando a una concentración de 10 μM una actividad próxima al 5 % de la que corresponde a una concentración similar de α-tocoferol; y por presentar actividad antioxidante significativa a nivel de secuestro de radicales superóxido a concentración igual o superior a 100 μM, inhibiendo al 45 % la cinética de oxidación del pirogalol cuando la concentración del extracto purificado es de 1 mM,1. Purified extract of mycosporine shinorine-type amino acid extracted from the Gymnogongrus devoniensis red algae characterized by not presenting significant antioxidant activity as an inhibitor of the production of water-soluble free radicals in the pH range 6 - 8.5, for presenting moderate antioxidant activity at the level of inhibition of lipid peroxidation, presenting at an concentration of 10 μM an activity close to 5% of which corresponds to a similar concentration of α-tocopherol; and for presenting significant antioxidant activity at the level of sequestration of superoxide radicals at a concentration equal to or greater than 100 μM, inhibiting 45% the oxidation kinetics of pyrogallol when the concentration of the purified extract is 1 mM, 2. Uso del extracto de aminoácido tipo micosporina shinorine extraído del alga roja Gymnogongrus devoniensis de acuerdo con la reivindicación 1 para la preparación de un producto para la prevención y el tratamiento terapéutico de enfermedades coronarias y aterosclerosis.2. Use of the mycosporin shinorine-type amino acid extract extracted from the Gymnogongrus devoniensis red algae according to claim 1 for the preparation of a product for the prevention and therapeutic treatment of coronary heart disease and atherosclerosis. 3. Uso del extracto de aminoácido tipo micosporina shinorine extraído del alga roja Gymnogongrus devoniensis de acuerdo con la reivindicación 1 para la preparación de un producto para la prevención de procesos cancerígenos.3. Use of the mycosporine shinorine-type amino acid extract extracted from the Gymnogongrus devoniensis red algae according to claim 1 for the preparation of a product for the prevention of carcinogenic processes. 4. Uso del extracto de aminoácido tipo micosporina shinorine extraído del alga roja Gymnogongrus devoniensis de acuerdo con la reivindicación 1 para la preparación de un producto para la prevención y el tratamiento terapéutico del Parkinson y Alzheimer.4. Use of the mycosporine shinorine-type amino acid extract extracted from the Gymnogongrus devoniensis red algae according to claim 1 for the preparation of a product for the prevention and therapeutic treatment of Parkinson's and Alzheimer's. 5. Uso del extracto de aminoácido tipo micosporina shinorine extraído del alga roja Gymnogongrus devoniensis de acuerdo con la reivindicación 1 para la preparación de un producto para la prevención de cataratas.5. Use of the mycosporine shinorine-type amino acid extract extracted from the Gymnogongrus devoniensis red algae according to claim 1 for the preparation of a product for the prevention of cataracts. 6. Uso del extracto de aminoácido tipo micosporina shinorine extraído del alga roja Gymnogongrus devoniensis de acuerdo con la reivindicación 1 para la mejora en el rendimiento de prácticas deportivas de montaña y trabajos realizados en altura en condiciones de hipoxia. 6. Use of the mycosporine shinorine-type amino acid extract extracted from the Gymnogongrus devoniensis red algae according to claim 1 for the improvement in the performance of mountain sports practices and work performed at height in hypoxic conditions. 7. Uso del extracto de aminoácido tipo micosporina shinorine extraído del alga roja7. Use of shinorine mycosporine-type amino acid extract extracted from red algae Gymnogongi'us devoniensis de acuerdo con la reivindicación 1 para la preparación de un producto para la prevención y el tratamiento de estados anímicos depresivos.Gymnogongi'us devoniensis according to claim 1 for the preparation of a product for the prevention and treatment of depressive moods. 8. Uso del extracto de aminoácido tipo micosporina shinorine extraído del alga roja8. Use of the amino acid extract type mycosporin shinorine extracted from red algae Gymnogongrus devoniensis de acuerdo con la reivindicación 1 para la preparación de un producto para la prevención y el tratamiento terapéutico de eritema actínico, fotocarcinogénesis y fotoenvejecimiento.Gymnogongrus devoniensis according to claim 1 for the preparation of a product for the prevention and therapeutic treatment of actinic erythema, photocarcinogenesis and photoaging. 9. Uso del extracto de aminoácido tipo micosporina shinorine extraído del alga roja9. Use of the mycosporine shinorine type amino acid extract extracted from red algae Gymnogongrus devoniensis de acuerdo con la reivindicación 1 como potencial antioxidante o aditivo en la preparación de productos de la industria alimentaria tales como preparados nutracéuticos o alimentos funcionales.Gymnogongrus devoniensis according to claim 1 as an antioxidant or additive potential in the preparation of food industry products such as nutraceutical preparations or functional foods. 10. Uso del extracto de aminoácido tipo micosporina shinorine extraído del alga roja10. Use of shinorine mycosporine-type amino acid extract extracted from red algae Gymnogongrus devoniensis de acuerdo con la reivindicación 1 para la prevención de la oxidación (deterioro) en productos cosméticos y farmacéuticos.Gymnogongrus devoniensis according to claim 1 for the prevention of oxidation (deterioration) in cosmetic and pharmaceutical products. 11. Uso del extracto de aminoácido tipo micosporina shinorine extraído del alga roja Gymnogongrus devoniensis de acuerdo con la reivindicación 1 para la preparación de un productos de parafarmacia, productos farmacéuticos, productos cosméticos, preparados nutracéuticos o en alimentos funcionales para el tratamiento terapéutico de enfermedades y afecciones relacionadas con los radicales libres enumeradas en la reivindicaciones 2,3, 4, 5, 6, 7,8.11. Use of the mycosporine shinorine-type amino acid extract extracted from the Gymnogongrus devoniensis red algae according to claim 1 for the preparation of a parapharmacy products, pharmaceutical products, cosmetic products, nutraceutical preparations or in functional foods for the therapeutic treatment of diseases and conditions related to free radicals listed in claims 2,3, 4, 5, 6, 7,8. 12. Uso del extracto de aminoácido tipo micosporina shinorine extraído del alga roja Gymnogongrus devoniensis de acuerdo con la reivindicación anterior para la preparación de productos de parafarmacia, productos farmacéuticos o productos cosméticos de aplicación tópica para la prevención de enfermedades y afecciones relacionadas con los radicales libres enumeradas en la reivindicación 8. 12. Use of the mycosporine shinorine-type amino acid extract extracted from the Gymnogongrus devoniensis red algae according to the preceding claim for the preparation of parapharmacy products, pharmaceutical products or cosmetic products of topical application for the prevention of diseases and conditions related to free radicals listed in claim 8.
PCT/ES2006/000492 2005-08-31 2006-08-28 Use of a mycosporin-type amino acid (shinorine) as an antioxidant WO2007026038A2 (en)

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ES2284344B1 (en) 2008-11-01
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ES2301425A1 (en) 2008-06-16
ES2301428A1 (en) 2008-06-16
ES2284344A1 (en) 2007-11-01
ES2301426A1 (en) 2008-06-16
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ES2301425B1 (en) 2009-05-01
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