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  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
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  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
  • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
• • A—HUMAN NECESSITIES
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  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • 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
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
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  • A61P27/12—Ophthalmic agents for cataracts
• • A—HUMAN NECESSITIES
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  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/08—Anti-ageing preparations
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/12—Carboxylic acids; Salts or anhydrides thereof

Definitions

• the present invention relates to the use of an acid enriched in docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) or DHA-derived EPA for manufacturing a drug for the treatment of processes that involve associated oxidative damage.
• DHA docosahexaenoic acid
• EPA eicosapentaenoic acid
• DHA-derived EPA DHA-derived EPA
• omega-3 fatty acids are necessary for maintaining cellular functional integrity, and are necessary in general for human health.
• Docosahexaenoic acid (22:6 n-3, DHA) an important omega-3 component of fish oil and of marine algae, is concentrated in the brain, in the photoreceptors and in the synapses of the retina.
• DHA-enriched diets are initially metabolised by the liver and afterwards distributed via the lipoproteins in the blood in order to meet the needs of the various organs.
• the administration of DHA leads to an increase of its concentration at tissue level, inducing also an increase in the concentration of omega-3 eicosapentaenoic acid (EPA) which in linked metabolically, whereas the administration of EPA only increases its concentration decreasing that of DHA at cell level.
• EPA omega-3 eicosapentaenoic acid
• the DHA is incorporated into the phospholipids of the cell membrane, which have effects on its composition and functionality, on the production of reactive oxygen species (ROS), on membrane lipid oxidation, on transcription regulation, on the biosynthesis of eicosanoids and on intracellular signal transduction. Furthermore, in the central nervous system, the DHA is involved in the development of the learning capacity related to the memory, in the excitable functions of the membrane, in biogenesis of the photoreceptor cells and in transducing the signal dependent upon quinase protein. A potential dietary therapy would be based on correcting the optimum levels of omega-3 fatty acids to prevent certain pathologies from originating or progressing, such as inflammatory pathologies, tumoral processes, cardiovascular diseases, depression and neurological disorders.
• ROS reactive oxygen species
• a potential dietary therapy would be based on correcting the optimum levels of omega-3 fatty acids to prevent certain pathologies from originating or progressing, such as inflammatory pathologies, tumoral processes, cardiovascular diseases, depression and neurological disorders.
• DHA In the central nervous system, both the brain and the retina show an unusual capacity for retaining DHA, even under situations of very prolonged dietary deficiencies of omega-3 fatty acids.
• Several studies have described the protective effect of DHA on neurones, in which it is present in very high levels. For example, it is involved in protecting the neuronal cells from death by apoptosis.
• DHA found in reduced amounts in the hippocampus of rats of advanced age, is capable of protecting primary cultures of said cells against the cytotoxicity induced by glutamate.
• DHA has also been shown to modulate the levels of the pro- and anti- apoptotic proteins of the Bcl-2 family.
• the external segments of the retinal photoreceptor contain rodopsin, as well as a higher DHA content than any other type of cell.
• the DHA is concentrated in the phospholipids of the photoreceptor segment disc's outer membranes. Retinal dysfunctions have been observed under conditions of reduction of optimal DHA concentration.
• the retina pigmentary epithelial cell (RPE) plays a very active role in DHA take-up, conservation and transport.
• the high DHA content in the photoreceptor and in the RPE cells is mainly linked to domains in the membrane with physical characteristics that contribute to the modulation of receptors, ionic channels, carriers, etc., while it also appears to regulate the concentration of phosphatidilserine .
• the PLA 2 releases the DHA from the membrane phospholipids and the LOX converts it into its metabolically active derivatives.
• the reactive oxygen species are produced during normal cellular functioning.
• the ROS include the superoxide anion, hydrogen peroxide and the oxydril radical. Their high chemical reactivity leads to the oxidation of proteins, of DNA or of lipids.
• the superoxide dismutase (SOD) , the catalase (CAT) and the glutation peroxidase (GPx) are the primary antioxidant enzymes that protect against the molecular and cellular damage caused by the presence of ROS.
• SOD superoxide dismutase
• CAT catalase
• GPx glutation peroxidase
• the oxidative stress activates many metabolic channels; some are cytoprotective, while others lead to death of the cell. Recent studies indicate that an imbalance between ROS production and breakdown is a significant risk factor in the pathogenesis of many illnesses, in some cases related to a deterioration of the antioxidant system.
• the DHA is presented as a target of the ROS that produces damage to the cell of the photoreceptor and to the RPE.
• the retinal degeneration induced by light promotes loss of DHA in the photoreceptors.
• photoreceptor function deteriorates because the RPE cells are essential for its survival.
• death of the RPE cell under the effect of oxidative stress leads to a deterioration of eyesight, particularly when the cells of the macula are affected, since it is responsible for eyesight acuity.
• the pathophysiology of many retinal degenerations e.g., macular degenerations related to age and to Stargardt disease) involves oxidative stress that leads to RPE cell apoptosis.
• RPE cell apoptosis appears to be the dominant factor in the macular degeneration observed with age.
• Such studies suggest that said cells have developed highly effective antioxidant mechanisms to protect themselves from their high DHA content and show notable adaptive capacity.
• the relationship between the free radicals and ageing is perfectly well accepted, based on the evidence that free radicals produced during aerobic respiration cause oxidative damage that accumulates and leads to a gradual loss of the homeostatic mechanisms, interference in gene expression patterns and a loss of the cell's functional capacity, leading to ageing and death.
• An interrelation exists between the generation of oxidants, antioxidant protection and repair of the oxidative damage. Many studies have been carried out to determine whether antioxidant defences decline with age.
• erythema or burns can be produced, while chronic over-exposure increases the risk of skin cancer and ageing.
• the cutaneous cells can respond to acute or chronic oxidative stress by increasing expression of a variety of proteins, such as the enzymes involved in maintaining cell integrity and resistance to oxidative damage.
• telomeres are non-coding DNA regions located at the ends of eukaryotic chromosomes. These are constituted by highly conserved DNA sequences, repeated in tandem (TTAGG) n and associated proteins, and have a special structure which hinders the ligation to the ends of other chromosomes, preventing the telomeric fusion. They have an essential role in the preservation of the chromosomic integrity, protecting the coding DNA from the enzymatic action and its degradation, contributing to the mainteinance of the chromosomic stability.
• telomeres In contrast with coding sequences which have a semiconservative replication, the telomeres undergo a progressive loss of its repetitive sequences during the successive cell division.
• a minimum telomeric length is required in order to keep the telomere function and when these reach a critical size they have difficulties for the division in the mitosis, generating telomeric association (TAS) and chromosomic instability.
• TAS telomeric association
• chromosomic instability would be associated with an increase in the probability of producing errors capable of generating significant genetic changes .
• omega-3 fatty acids are considered to be molecular targets for generation and propagation of free radicals during the oxidative stress processes related to generation of lipidic peroxides. Contradictory results have been obtained, however, in various studies of susceptibility to oxidative stress owing to dietary supplements of omega-3 fatty acids. Some studies in humans have shown increased oxidation of the LDL, while others have found no such effect. In studies with animals, treatment with omega-3 fatty acids has been found to lead to increased or reduced susceptibility to oxidation of the LDL. On the other hand, an over-expression of the genes involved in the antioxidant defence system has been found in the livers of mice fed on a fish-oil-enriched diet for three months.
• Free radicals also affect DNA and acute physical exercise increases damage in DNA, as evidenced by the increase of 8-0xodG. Exhausting physical effort (running a marathon) causes damage in DNA which is evident for some days after the trial and also causes damage in immunocompetent cells (which can be associated with the immune decrease shown in sportsmen after such a trial) .
• n-3 fatty acids related to sports performance were focused on the antiinflammatory effect and, indeed, first assays tried to find the possible action of these nutrients improving the alveolar- capillary absorption by diminishing the intensive physical exercise-induced broncoconstriction .
• Mickleborough proved that after administering 3.2 g EPA and 2.2 g DHA regime proinflammatory cytokines were attenuated by diminishing the presence of TNF- ⁇ and IL-l ⁇ in an elite athlete, along with a decrease in the broncoconstriction.
• Walser related n-3 fatty acids vascular effects to positive effects in people showing intolerence to physical exercise.
• van Houten et al studied that a n-3 fatty acid high ingestion was associated with a better recovery in patients carrying out a cardiac rehabilitation after a coronary syndrome.
• Exercise increases glucose uptake, capillary perfusion, glycogen synthesis rate and insulin sensitivity.
• muscular contraction changes are produced in temperature, intracellular pH, ATP/ADP ratio, as well as Ca ++ intracellular concentration and other metabolites which could act as messengers in the cellular functioning regulation with exercise.
• Ca ++ regulates a great amount of intracellular proteins, including calmodulin kinase, protein kinase C (PKC) and calcineurin which are important intermediates in the signals of intracellular transduction.
• AMPK AMP kinase
• AMPK activation effects probably include stimulation of GLUT4 and hexokinase expression, as well as mitochondria enzymes.
• AMPK activation is not the unique way (independent of insulin) wherein the exercise increases the response to glucose in skeletal muscle. See Mora and Pessin, J. Biol. Chem. 2000; 275 (21) : 16323-16328, showed that an increase in the glucose response in the muscle, indeed, there are several transcription factors such as MEF2A and MEF2D activating GLUT4 and those factors are activated by exercise.
• the present invention concerns the unexpected discovery that the administration of docosahexaenoic acid (herein also referred to as DHA) or eicosapentaenoic acid (EPA) or DHA-derived EPA, whether in free form or incorporated into a triglyceride, among others, acts as a cellular antioxidant.
• DHA docosahexaenoic acid
• EPA eicosapentaenoic acid
• DHA-derived EPA acts as a cellular antioxidant.
• An object of the present invention is therefore the use of docosahexaenoic acid for the manufacturing of a pharmaceutical composition for the treatment of cellular oxidative damage.
• Another object of the present invention is the use of docosahexaenoic acid (DHA) at a specific position of the glycerol backbone, the two remaining positions of the glyceride being also specified in their composition for the treatment of cellular oxidative damage.
• DHA docosahexaenoic acid
• a further object of the present invention is the use of docosahexaenoic acid (DHA) for manufacturing a composition for the treatment of the cellular oxidative damage at DNA level.
• DHA docosahexaenoic acid
• the use of docosahexaenoic acid has the application as a protective agent in the natural process of telomere shortening and as an inhibitory agent of premature senescence in a treatment of cellular oxidative damage.
• docosahexaenoic acid for manufacturing a composition for the treatment of cellular ageing and hereditary pathologies associated with disorders in the mitochondrial respiratory chain, as well as a composition for treating Down's Syndrome.
• a further object of the present invention is the use of docosahexaenoic acid (DHA) for manufacturing a composition for the treatment of the cellular oxidative damage associated with physical exercise.
• DHA docosahexaenoic acid
• the use of docosahexaenoic acid has application as an enhancer agent in the sports performance and as a regulating agent of blood glucose levels during physical effort.
• docosahexaenoic acid for manufacturing a composition for enhancing sports performance, as well as a composition for maintaining blood glucose levels after physical exercise by means of, mainly, the administration of a food, a dairy product or any suitable administration form typically used by people when doing physical exercise .
• the expression "cellular oxidative damage” means any process that involves an imbalance between the generation and degradation of cellular oxidant species of endogenous or exogenous origin .
• DHA is capable of inhibiting the production of reactive oxygen species (ROS) , whether related to a dependent induction of peroxides or superoxides. More specifically, it reduces the production of superoxide anion and therewith of all the derived species produced in the oxidative cascade, such as for example a highly significant reduction of lipidic peroxidation.
• ROS reactive oxygen species
• an increase in antioxidant enzyme activity was found, which suggests an adaptation of the cell by inducing the expression of antioxidant agents, basically enzymes, and by repressing the expression of pro-oxidant agents such as the A 2 phospholipase .
• said docosahexaenoic acid is incorporated into a monoglyceride, diglyceride, triglyceride, phospholipid, ethyl ester or free fatty acid.
• said docosahexaenoic acid is incorporated into a triglyceride.
• docosahexaenoic acid incorporated into a glyceride is taken to mean a monoglyceride, diglyceride, triglyceride, phospholipid, with at least one of the three positions esterified with a docosahexaenoic acid and, optionally, at least one of the remaining esterified positions further with one acid selected from a short-, mid- or long-chain fatty acid and a phosphoric acid.
• said glycerol is a triglyceride .
• triglyceride as chemical structure of the DHA is based on data taken from a study which compared the bioavailability of four omega-3 acid concentrates in the form of ethyl esters, phospholipids, free fatty acids and triglycerides following oral administration, which demonstrated that the re-esterified triglycerides presented a higher bioavailability than the other preparations.
• said docosahexaenoic acid is found in a percentage by weight of between 20 and 100% in relation to the total fatty acids, preferably between 40 and 100% in relation to total fatty acids, and more preferably said docosahexaenoic acid is in a percentage by weight between 66 and 100% in relation to total fatty acids.
• said docosahexaenoic acid is incorporated into at least one specific position of a glycerol via an ester bound, a structured lipid, for manufacturing a pharmaceutical composition for the treatment of cellular oxidative damage .
• Such a glycerol may further comprise at least one fatty acid and/or one phosphoric acid so that said docosahexaenoic acid being incorporated into a position selected from sn-1, sn-2 and sn-3, may further comprise, optionally, at least one acid selected from a short- and/or mid-chain fatty acid and a phosphoric acid, and when incorporated into the sn-2 position may further comprise, optionally, at least one acid selected from a fatty acid and a phosphoric acid.
• said docosahexaenoic acid incorporated into a position selected from sn-1, sn-2 and sn-3 may or not further comprise at least one acid selected from a short- and/or mid-chain fatty acid and a phosphoric acid, or otherwise that said docosahexaenoic acid incorporated into the sn-2 position may or not further comprise at least an acid selected from a long-chain fatty acid and a phosphoric acid.
• the inventors of the present invention have found that the use of structured glycerols wherein the position of the docosahexaenoic acid has been selected and the composition of the rest of the compound bound to the glycerol, leads to an unexpected increase, at least twice or even thrice, the therapeutic efficiency of the use of docosahexaenoic acid for manufacturing a pharmaceutical composition for the treatment of cellular oxidative damage.
• the common definition relates to fats containing fatty acids located in specific positions in the glycerol backbone.
• the long-chain polyunsaturated fatty acids are located preferably in the sn-2 position of the glycerol and taking into account the intestinal absorption process, triglycerides are hydrolized by lipases to free fatty acids, di- and monoglycerides, from which the free fatty acids and sn-2 monoglycerides are absorbed directly by intestinal epithelial cells, named enterocytes .
• docosahexaenoic acid incorporated into a specific position of the glycerol backbone, via an ester bound provides an increased bioactivity, an increased antioxidant protection at the same molar percentage in respect with the whole amount of fatty acids present and a diminished dependency on the administration dosage in respect with the antioxidant effect of the docosahexaenoic acid in the glyceride.
• the inventors of the present invention have found that the use of docosahexaenoic acid incorporated into a position of the glycerol selected from sn-1, sn-2 and sn-3, and optionally said glycerol further comprising at least one acid selected from a short- and/or mid-chain fatty acid and a phosphoric acid, provides an increased bioactivity, an increased antioxidant protection at the same molar percentage in respect with the whole amount of fatty acids present and a diminished dependency on the administration dosage in respect with the antioxidant effect of the docosahexaenoic acid in the glycerol.
• the inventors of the present invention have found that the use of docosahexaenoic acid incorporated into a sn-2 position of a glycerol and optionally said glycerol further comprising at least one acid selected from a long-chain fatty acid and a phosphoric acid, provides also an increased bioactivity, an increased antioxidant protection at the same molar percentage in respect with the whole amount of fatty acids present and a diminished dependency on the administration dosage in respect with the antioxidant effect of the docosahexaenoic acid in the glycerol.
• acids also present in a glycerol with the docosahexaenoic acid will be short-chain fatty acids (C1-C8) or mid-chain fatty acids (C9-C14) or a phosphoric acid, since these have no functional activity, but only energetic activity and, therefore, will not compete with the docosahexaenoic acid.
• the present inventions relates to the use of docosahexaenoic acid incorporated into a glycerol wherein one of the positions sn-1 and sn-3 are free or occupied by a mid-chain fatty acid (C9-C14) or short-chain fatty acid (C1-C8) or a phosphoric acid and in which sn-2 position is occupied by functional DHA.
• a still higher increase of DHA is achieved since it is more efficiently absorbed in the intestinal cells.
• the synthesis of structured glycerides wherein the docosahexaenoic acid has been incorporated into any position of the glycerol when it does not compete with other fatty acids and wherein the DHA has been incorporated into the sn-2 position of the glyceride when it competes with at least one fatty acid shows improvements related to its antioxidant effect and, therefore, it is a preferred way for manufacturing a composition for the treatment of the oxidative cellular damage .
• a cell enriched with a composition with DHA in accordance with the invention, is better prepared to face up to a new situation of oxidative stress and thus to minimise the adverse effects that can derive therefrom. That is, the presence of the DHA in the biomembranes induces a cellular adaptive response to the oxidative stress.
• Adaptive response is a cellular phenomenon by which exposure to a toxic agent (in sub-lethal concentrations) provokes a cellular response which will subsequently protect the cell against the deleterious effects of that same toxic agent at lethal concentrations, or, put another way, it is a beneficial effect unleashed by a low level of exposure to an agent that is harmful at high levels.
• DHA has the following substantial advantages: a) Increased cellular antioxidant activity; b) Absence of cellular cytotoxicity at the dosages administered; c) Absence of significant alterations to cellular oxidant status at the dosages administered; d) Adaptive cellular antioxidant activity.
• the present invention relates to the use of docosahexaenoic acid for manufacturing a pharmaceutical composition for treating a pathology associated with cellular oxidative damage, said pathology being a neurodegenerative pathology, preferably selected from the group that comprises: multiple sclerosis, Alzheimer's disease, Parkinson's disease, amiotrophic lateral sclerosis and muscular dystrophy, among others.
• the pathology associated with the oxidative damage is an ocular pathology, preferably one selected from the group that comprises pigmentary retinosis, macular degeneration and cataracts, among others.
• the pathology associated with the oxidative damage is an ischaemic pathology, particularly a myocardial infarct, cerebral infarct, etc.
• the pathology associated with the oxidative damage is an inflammatory process, preferably selected from the group comprising arthritis, vasculitis, glomerulonephritis and eritomatose lupus, among others.
• the pathology associated with the oxidative damage is atherosclerosis.
• Another aspect of the present invention is the use of DHA as a protective agent in the natural process of telomere shortening and as an inhibitory agent of premature senescence.
• telomeric associations TAS
• telomerase is very active in foetal cells, but has not much activity in adult tissue cells. TAS have seldom found in normal cells, but they have been observed in infected cells by virus or tumour cells.
• telomere shortening occurs naturally with the cellular replication, a premature senescence and breakages of telomeres when inducing oxidative damage in the DNA have been observed.
• the telomeres are more sensitive to oxidative damage and their breakages are less efficiently repaired than other parts of the genome. This leads to an accumulation of telomeric damage which produces a faster shortening during the DNA replication reducing the cellular replicative life expectancy.
• the reactive oxygen species (ROS) particularly superoxide anions, hydrogen peroxide and oxidril radicals, can accelerate the losses in the telomeres during the replication of some cellular types, even though they also induce premature senescence regardless of the telomere shortening .
• the present inventors have found a reverse correlation between the shortening rate of telomeres and the cellular antioxidant capacity in more than 20 fibroblasts human strains. Most of the cellular parameters of these prematurely aged fibroblasts are the same as the normal ageing of these cells (morphology, accumulation of lipofuscin and changes in the genie expression) .
• the fibroblasts with a lower antioxidant defence shorten their telomeres faster and vice versa.
• the shortening rate of the telomere is higher in cells with a lower antioxidant defence.
• free radical scavengers reduce the shortening rate of the telomere.
• telomere shortening rate of telomeres in human fibroblasts.
• the length of telomeres is determined mainly by the relation between the oxidative stress and the cellular antioxidant defence capacity.
• the length of age- dependent telomeres is an accumulative measurement of the history of the oxidative damage that a cell has undergone along its life.
• a correlation between oxidative stress and shortening rate of telomeres has been shown for hereditary pathologies associated with disorders in the mitochondrial respiratory chain and for Down's Syndrome.
• the pharmaceutical composition comprising DHA can be found in the form of an oil or an emulsion, which can be administered by oral, sublingual, intravenous, intramuscular, topical, subcutaneous or rectal routes, or even by merely bringing the active ingredient of the microemulsion of the invention in liquid or vapour form into contact with the olfactory organs situated at the entrance of the respiratory tracts.
• the administration can be carried out by spraying, misting or atomisation of the microemulsions or by inhalation.
• said pharmaceutical composition further comprises a second active ingredient.
• the pharmaceutical composition comprising DHA can be used in the food industry for the purpose of enriching food products (e.g. lactic products such as yoghurts, milk, etc) with a natural antioxidant agent such as DHA.
• lactic products such as yoghurts, milk, etc
• DHA natural antioxidant agent
• said pharmaceutical composition is administered to a patient who is already receiving a treatment against a pathology associated with oxidative damage.
• Another object of the present invention is the use of DHA as an enhancer agent in the sports performance and as a regulating agent of blood glucose levels during physical effort.
• the present inventors have shown that a statistically significant increase of the oxygen consumption (VO2) , both absolute (p ⁇ 0.019) and relative (p ⁇ 0.036) values to the weight in the ventilatory threshold 2 when comparing basal triangular effort trials with those carried out after four months of treatment with DHA.
• VO2 oxygen consumption
• p ⁇ 0.019 absolute
• p ⁇ 0.036 relative
• Another parameter related to an increase in the sports performance is the increase in the cardiac frequency wherein the UV2 of the effort trial is set, since in case the cardiac frequency increases in the anaerobic threshold, the sportsmen are considered to be capable of slightly increasing its ability of keeping the aerobic metabolism in higher intensities.
• MDA Malonyldialdehyde
• 8-oxodG is a oxidative stress biomarker. There is an increase of oxidative damage to the DNA while carrying out rectangular effort trials (p ⁇ 0.011). This oxidative damage diminishes after administering DHA for 3 weeks
• hypoglycemia Since beverage with carbohydrates were not ingested, an hypoglycemia was expected. This hypoglycemia of second extraction (twenty minutes after the end of the trial in respect to the starting sample obtained twenty minutes before the start) , is shown in the first effort trial, as it was expected. However, data obtained after the DHA administration for four months show a statistically significant glycemia maintainement, which was not observed previously and it represents a surprising finding in the realized research.
• Another object of the present invention is the use of docosahexaenoic acid for manufacturing a composition for enhancing sports performance and maintaining blood glucose levels after physical exercise administered by any suitable means.
• At least 75% of caloric energy must derive from carbohydrates with a high glycemic charge (glucose, maltodextrine, sucrose)
• Vitamine Bl 0,2 mg/100 mg carbohydrates
• the inclusion of carbohydrates is aimed to maintain the glycemia in order to avoid the fast consumption of muscular and hepatic glycogen. It should be considered the drawbacks of gastric emptying diminished due to the increase of osmolarity generating the presence of concentrations of carbohydrates, associated with the feeling of gastric fullness undesirably for a lot of sportsmen. Por consiguiente, preparing a beverage with a loer concentration of carbohydrates by adding DHA could be an ergogenic advantage of undoubted interest in the sports performance .
• another aspect of the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising DHA which can be used in the in the food industry for the purpose of enriching food products (e.g. dairy products such as yoghurts, milk, etc) with a natural antioxidant agent such as DHA, or further, incorporated into a suitable administration form selected from the group comprising a beverage in all its characteristics for before, during and after physical exercise; energy-giving bar; ergogenical bars; solids and preparations for provisioning; dietetic supplement and polivitaminic preparation (in the form of, for example, capsules, tablets, pills, lyophilised form, or any suitable mean of administration); ergogenical aids; textiles with nanocapsules for skin absorption and any other suitable mean of administration.
• dairy products e.g. dairy products such as yoghurts, milk, etc
• a natural antioxidant agent such as DHA
• a suitable administration form selected from the group comprising a beverage in all its characteristics for before, during and after physical exercise; energy
• FIG. 1 Effect of DHA concentration in the Foreskin cells culture medium on the intracellular generation of ROS.
• the cells were cultured in the presence of a triglyceride with 70% by weight of DHA in relation to the total fatty acids for three days prior to the experiment.
• FIG. 1 Comparative effect of the proportion of DHA of a triglyceride in the Foreskin cells culture medium on the intracellular generation of ROS.
• A The cells were cultured in the presence of each triglyceride for three days prior to the experiment. The concentration on the x- axis is the equivalent that would be obtained with a triglyceride having a DHA content of 70% by weight. The detection of ROS was carried out with DHR 123 on cells treated with 40 mM AAPH for 180 min. The data represent the mean of three independent experiments.
• B Representation of the antioxidant protection in relation to DHA concentration in the oil of 20, 50 and 70%.
• FIG. 3 Effect of DHA concentration on the production of TBARS in Foreskin cells.
• the cells were cultured in the presence of a triglyceride with 70% by weight of DHA in relation to the total fatty acids for three days prior to the experiment at the concentration indicated.
• the oxidative stress was induced with 40 mM AAPH for 6 h and 24 h of latency.
• the data represent the mean of three independent experiments.
• Figure 4 Effect of DHA concentration in the Foreskin cells culture medium on the generation of superoxide anions.
• the cells were cultured in the presence of a triglyceride with 70% by weight of DHA in relation to the total fatty acids for three days prior to the experiment.
• the detection of superoxide anions was carried out by chemiluminiscence immediately following oxidative induction of the cells with 40 mM AAPH and in some experiments in the presence of 10 mM Tyron or of 0.1875 UA/ ⁇ l of exogenous SOD. The data are representative of three independent experiments.
• FIG. 5A Effect of DHA concentration in the Foreskin cells culture medium on SOD activity.
• the cells were cultured in the presence of a triglyceride with 70% by weight of DHA in relation to the total fatty acids for three days prior to the experiment at DHA concentrations of 0.5 (A), 5 (B) and 50 ⁇ M (C).
• the SOD activity was analysed indirectly by analysing the decrease in the chemiluminiscence generated by the luminol as a consequence of the endogenous SOD activity.
• Oxidative induction was carried out with the 0.1 mM xanthine / 0.005 U/ml xanthine oxidase system that immediately generates superoxide anions. The data are representative of three independent experiments.
• FIG. 5B Effect of DHA concentration in the Foreskin cells culture medium on SOD activity.
• the cells were cultured in the presence of a triglyceride with 70% by weight of DHA in relation to the total fatty acids for three days prior to the experiment.
• the SOD activity was evaluated on the non-induced cellular system or the system induced with 40 mM AAPH.
• the data are representative of three independent experiments.
• Figure 6 Effect of DHA concentration in the Foreskin cells culture medium on GPx activity.
• the cells were cultured in the presence of a triglyceride with 70% by weight of DHA in relation to the total fatty acids for three days prior to the experiment.
• GPx activity was evaluated on the non-induced cellular system or the system induced with 40 mM AAPH. The data are representative of three independent experiments.
• FIG. 7 Effect of DHA concentration in culture medium of ARPE-19 cells on the intracellular generation of ROS.
• the cells were cultured in the presence of a triglyceride with 70% by weight of DHA in relation to the total fatty acids for three days prior to the experiment.
• FIG. 8 Comparative effect of DHA concentration of a triglyceride in the culture medium of ARPE-19 cells on the intracellular generation of ROS.
• the cells were cultured in the presence of each triglyceride for three days prior to the experiment.
• the concentration on the x- axis is the equivalent that would be obtained with triglyceride having a DHA proportion of 70% by weight.
• the detection of ROS was carried out with DHR 123 on cells treated with 40 mM de AAPH for 180 min.
• the data represent the mean of three independent experiments.
• (B) Representation of the antioxidant protection in relation to DHA concentration in the oil of 20, 50 and 70%.
• Figure 9 Effect of DHA concentration on the production of TBARS in ARPE-19 cells.
• the cells were cultured in the presence of a triglyceride with 70% by weight of DHA in relation to the total fatty acids for three days prior to the experiment at the indicated concentration.
• the oxidative stress was induced with 40 mM AAPH for 6 h and 24 h of latency.
• the data represent the mean of three independent experiments.
• Figure 10 Effect of DHA concentration in the ARPE-19 cells culture medium on the generation of superoxide anions.
• the cells were cultured in the presence of a triglyceride with 70% by weight of DHA in relation to the total fatty acids for three days prior to the experiment.
• the detection of superoxide anions was carried out by chemiluminiscence immediately following oxidative induction of the cells with AAPH 40 mM.
• the data are representative of three independent experiments.
• Figure 11 Effect of DHA concentration in the ARPE-19 cells culture medium on GPx activity.
• the cells were cultured in the presence of a triglyceride with 70% by weight of DHA in relation to the total fatty acids for three days prior to the experiment.
• GPx activity was evaluated on the non-induced cellular system or the cellular system induced with 40 mM AAPH.
• the data are representative of three independent experiments.
• FIG. 12 Effect of DHA concentration in the ARPE-19 cells culture medium on SOD activity.
• the cells were cultured in the presence of a triglyceride with 70% by weight of DHA in relation to the total fatty acids for three days prior to the experiment.
• SOD activity was evaluated on the non-induced cellular system or the cellular system induced with 40 mM AAPH. The data are representative of three independent experiments.
• Figure 13 Effect of DHA concentration obtained by chemical synthesis (A and C) or enzymatic synthesis (B and D) on the percentage of cellular protection versus oxidative stress in ARPE-19 cells (A and B) or Foreskin cells (C and D) .
• Figure 14. Influence of purification degree of the oil obtained by chemical synthesis on the percentage of cellular protection versus oxidative stress induced by DHA in ARPE-19 cells.
• Figure 15. Influence of chemical structure on the percentage of cellular protection versus oxidative stress induced by DHA in ARPE-19 cells.
• FIG. 1 Effect of DHA concentration on intracellular concentration of glutation in Foreskin cells. Influence of the presence of BSO.
• Figure 20 Effect of EPA concentration on the percentage of cellular protection versus oxidative stress in Foreskin cells. Comparative study with DHA.
• Figure 21 Effect of EPA concentration on intracellular concentration of glutation in Foreskin cells. Influence of the presence of BSO.
• Figure 22 is a comparative bar graphic showing the effect of the DHA percentage in a structured and non- structured triglyceride at different dosages in respect with the percentage of cell protection.
• Said figure 22 shows the surprising results of the object of the present addition when comparing a non- structured glyceride chemical structure (triglyceride) with the same structure wherein sn-1 and sn-3 positions have been replaced with caprylic acid (structured) , both from an enzymatic source with two starting levels in content of DHA of 20 and 70%.
• the percentage of protection of the docosahexaenoic acid incorporated into the sn-2 position of a glyceride (structured) shows an efficiency which is approximately 3 times higher than that of a glyceride containing non- structured DHA.
• the protection percentage indicates the relationship between the difference in the intracellular concentration of reactive oxygen species of control cells and those treated with DHA in respect with the control cells, both subjected to the same oxidative stress expressed in percentage.
• the existence of a protection percentage indicates in the treated cells a significant statistically less intracellular generation of reactive oxygen species in respect with the control.
• Figure 23 is a comparative graphic showing the average length of the telomere in human fibroblasts cultured under oxidative stress with or without DHA incorporated vs. the pass number of cellular populations.
• Said figure 23 shows the surprising results of the object of the present addition at observing that in presence of DHA under oxidative stress conditions, the telomere shortening index is lower in respect to the control or without DHA.
• Figure 24 is a graphic representing the absolute oxygen consumption in the "ventilatory threshold 2" (UV2) for competing, non-competing and all cyclists at basal level and after 4 months taking DHA.
• UV2 ventilation threshold 2
• Figure 25 is a graphic representing the cardiac frequency in UV2 for competing, non-competing and all cyclists at basal level and after 4 months taking DHA.
• Figure 26 is a graphic representing the time needed to reach the UV2 for competing, non-competing and all cyclists at basal level and after 4 months taking DHA.
• Figure 27 is a graphic representing the cardiac frequency during the consumption of 2000 ml/min O 2 in the ventilatory threshold for competing, non-competing and all cyclists at basal level and after 4 months taking DHA.
• Figure 28 is a graphic representing the plasma total antioxidant capacity for competing, non-competing and all sportsmen at basal level and after 3 weeks taking
• Figure 29 is a graphic representing the oxidative damage to plasmatic lipids according to MDA concentration for competing, non-competing and all sportsmen at basal level and after 3 weeks taking DHA. In each case, there is shown the oxidative damage before (left bar) and the oxidative damage after (right bar) the effort trial.
• Figure 30 is a graphic representing the oxidative damage to DNA using the oxidative stress biomarker 8-oxodG for competing, non-competing and all sportsmen at basal level and after 3 weeks taking DHA. In each case, there is shown the oxidative damage before (left bar) and the oxidative damage after (right bar) the effort trial.
• Figure 31 is a graphic representing the glycemia in competing sportsmen during a physical effort who did not take DHA or did it for 3 weeks or 4 months.
• Figure 32 is a graphic representing the glycemia in non-competing sportsmen during a physical effort who did not take DHA or did it for 3 weeks or 4 months.
• Figure 33 is a graphic representing the glycemia in competing and non-competing sportsmen during a physical effort who did not take DHA or did it for 3 weeks or 4 months .
• the following examples are included by way of illustrative and non-limitative examples of the invention.
• the cellular models used were Foreskin cells
• ARPE-19 cells retina pigmentary epithelial cells, CRL-
• the cell cultures were kept in suitable growth conditions of temperature (37°C), CO2 concentration (5%) and humidity (95%) in an incubator specially designed for this purpose.
• the ARPE-19 cells were maintained in growth up to confluence of 0.3xl0 4 cells/cm 2 in culture flasks with DMEM-F12 medium (Biological Industries) supplemented with 10% bovine foetal serum, penicillin antibiotics (100 U/mL) , streptomycin (100 ⁇ g/mL) and glutamine (Biological Industries).
• the CRL-2076 fibroblasts were kept growing in culture flasks in Iscove's modified Dulbecco's medium (Biological Industries) supplemented with 10% bovine foetal serum, penicillin antibiotics (100 U/mL), streptomycin (100 ⁇ g/mL) and glutamine (Biological Industries) .
• the cells were transferred for adherence to the substrate 24 h at 37°C from the 75 ml flasks to 6, 12 or 96-well plates in order to be able to carry out the experiment (10 6 cells/mL) .
• DHA-TG was added at various concentrations (0.5-50 ⁇ M) starting with the DHA-TG enriched with 20, 50 and 70% (oil density 0.92 g/mL) , made by dissolving the oil in ethanol for the stock solution (1:100) and preparing the working solutions in a culture medium prepared with serum.
• the cells were cultured with supplemented DHA-TG medium for 3 days at 37°C.
• xanthine/xanthine oxidase system 0.8 mM/10 ⁇ 2 U/mL that catalyses the oxidation of hypoxanthine and xanthine to uric acid, with reduction of O 2 to O- "2 and H 2 O 2 .
• AAPH 2, 2' -azobis- (2-amidinopropane) dihydrochloride 1-100 mM widely used as a hydrophilic initiator of free radicals by inducing lipidic and protein peroxidation.
• the AAPH oxidises the DNA, the proteins and the lipids through the action of the formed peroxil radicals. It further acts on the endogenous defence system, since it deactivates the key enzyme, the SOD, thereby losing the protective capacity of the CAT and the GPx.
• ROS reactive oxygen species
• the ROS level was measured in primary cultures of human skin CRL-2076 fibroblasts and in ARPE-19 retinal epithelial cells by employing the fluorimetric technique using dihydrorodamine 123 (DHR123, Molecular Probes) and 2, 7-dichlorofluorescein diacetate (H 2 DCFDA, Molecular Probes) as fluorescent probes in a continuous system measuring every 30 min until 180 minutes. In both cases, this is an unspecific measurement of ROS generation.
• the fluorescent probes were added to the cells (IxIO 6 cells/ mL) at a final concentration of 10 ⁇ M.
• the fluorescence of the oxidised probes (2, 7-dichlorofluorescein and rodamine 123) was measured in a Mithras fluorescence reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm in function of the time.
• the fluorescence obtained is modulated with the cellular viability determinations by the MTT spectrophotometric technique outlined below.
• This method consists of adding the MTT reagent (3- (4, 5- dimethylthiazol-2-yl) -2, 5-diphenyltetrazoyl bromide, Sigma) , soluble in aqueous medium, to the incubation medium.
• the viable cells metabolise this compound and it is converted into formazan salt.
• This salt is a colorimetric compound insoluble in aqueous medium, soluble in DMSO and usable for measuring cellular viability.
• the method consists of adding 20 ⁇ l per well of a 7.5 mg/ml
• the study of the efficiency of the product is carried out after exposing the cells to the product for 72 h in a sufficiently wide range of concentrations to find the value of IC 5 O.
• the experimental results are adjusted to the Hill equation using the Sigma Plot 8.0 to determine the IC 50 , defined as the DHA concentration necessary to reduce the viability of the culture to 50% in relation to the control.
• the determination is based on colorimetric detection and total quantification of the proteins with an optimised dizinconinic acid formulation that allows proteins to be measured in diluted samples in a concentration range of 0.5-20 ⁇ g/ml .
• the method uses a detector for Cu +1 , which is reduced by the proteins in alkaline medium to Cu +2 .
• the purple reaction product is formed by chelation of two molecules of BCA with the cuprous ion.
• the water-soluble complex absorbs at 562 nm.
• the commercial kit used is the MicroBCA from Pierce (No. 23235) .
• MDA malonildialdehyde
• HAE 4- hydroxyalkenals
• the condensation of one molecule of MDA with two molecules of the chromogenic reagent gives a stable chromophore with maximum absorbance at 586 nm, with the detection limit being 0.1 ⁇ M.
• the induction was carried out for 6 h with 40 mM AAPH and 24 hours of latency.
• the cells (10 7 cells/mL) were lysed by means of cycles of freezing and thawing in liquid N2.
• the samples were fractionated in order to measure MDA and protein. The results were expressed in ⁇ M of MDA/mg of protein.
• the capacity for inhibiting the production of superoxide anion was also investigated using a specific superoxide anion sequestering agent, Tyron (4, 5-dihydroxy-l, 3-benzene disulphonic acid, Sigma) frequently used for in vitro blocking assays on ROS production, being permeable to the cell membrane and superoxide dismutase (SOD, Sigma) was used as an enzyme blocker, constituting a first-line enzyme in the endogenous antioxidant defence.
• SOD superoxide dismutase
• the chemiluminiscence measurement in the cells submitted to the AAPH oxidative stress inducing treatment was analysed every 60 seconds for a total time of 4100 seconds, at a frequency of 120 sec/cycle. The results were expressed in UA of chemiluminiscence / mg protein.
• GPx catalyses the reduction of hydroperoxides to reduced glutation, the function being to protect the cell from oxidative damage. It uses glutation as last electron donor to regenerate the reduced form of selenocysteine .
• the indirect measurement of GPx is obtained by coupled reaction with glutation reductase.
• the oxidated glutation (GSSG) produced by the reaction with the hydroperoxides by action of the GPx is recycled to its reduced state by the glutation reductase using NADPH as coenzyme.
• Oxidation from NADPH to NADP + is accompanied by reduction of its absorbance at 340 nm.
• the rate of reduction of the absorbance at 340 nm is directly proportional to the GPx activity of the sample.
• the ELISA microplate spectrophotometric kit from Cayman (No. 703102) was used for detecting the GPx in cell lysates of primary cultures.
• the cells were cultured by adherence to the substrate for 24 h at 37°C.
• the cell lysate was obtained by sonication in Tris 50 mM pH 7.5, EDTA 5 mM and DTT 1 mM.
• the activity of the GPx is obtained by determining the change of A 340 nm /min ( ⁇ A340) , expressed as nanomoles NADPH/min/mg of protein from the sample.
• SOD superoxide dismutase activity
• the superoxide dismutase activity (SOD) in cellular lysates by means of the reaction using tetrazolium salts for detecting superoxide radicals generated by xanthine oxidase/hypoxanthine system has been also determined.
• An spectrophotometric method is used on a microplate for measuring the 3 types of SOD (Cu-Zn-SOD; Mn-SOD and Fe-SOD) , that is cytosolic and mitochondrial) .
• One unit of SOD is defined as the quantity of enzyme required for dismuting 50% of the generated superoxide anion.
• a Cayman kit N. 706002 was used following the protocol optimized by the manufaturer.
• the dynamic range of the assay is 0.025-0.25 SOD units/ml.
• Direct kinetic assay for measuring reduced glutation (GSH) in cellular lysates Glutation can be found inside the cells mainly in the reduced form (90-95% of total glutation) , being the main antioxidant in tissues. Its role is detoxifying xenobiotics and removing hydroperoxides so as to keep the cellular redox state.
• the technique employed measures the total glutation (GSSG + GSH) in a biological sample (cellular lysate) previously deproteinized with sulphosalicylic acid (Sigma-Aldrich CS0260 kit).
• GSH causes a continuous reduction from 5,5'- dithiobis (2-nitrobenzoic) acid (DTNB) to 5-thio (2- nitrobenzoic acid (TNB) and the GSSG formed is recycled by glutation reductase and NADPH.
• TNB is spectrophometrically measured at 412 nm.
• Buthionine sulfoximine (BSO) specifically inhibiting gamma- glutamylcysteine synthetase was used as a synthesis inhibitor .
• Foreskin cells concentrations of less than 1000 ⁇ M of DHA did not affect cellular viability in studies at 3 days. Neither was cellular viability affected for the studies of oxidative stress with the xanthine/xanthine oxidase system or with AAPH. It has also been shown that the incorporation of DHA up to a concentration of 50 ⁇ M in a culture of Foreskin cells for 3 days does not significantly increase the cellular oxidative level measured as cellular fluorescence associated with two probes, dihydrorodamine (DHR 123) and 2,7- dichlorofluorescein (H 2 DCFDA) , more specific for superoxide anion and for the detection of hydroperoxides, respectively.
• DHR 123 dihydrorodamine
• H 2 DCFDA 2,7- dichlorofluorescein
• the general antioxidant capacity of the DHA incorporated into the membrane of the Foreskin cells was evaluated against oxidative stress induced by xanthine/xanthine oxidase or by AAPH.
• the DHA shows an inhibiting effect on the generation of the reactive oxygen species, both at the concentration of 0.5 ⁇ M (59% protection) and at 5 ⁇ M (33% protection) , showing a lower effect at 10 ⁇ M (26% protection) or no effect at 50 ⁇ M of DHA (Fig. IA) .
• the DHA shows a protective effect against the generation of ROS, both at 0.5 ⁇ M concentration (40% protection) and 5 ⁇ M (29% protection), but losing it at higher concentrations of DHA (Fig. IA) .
• Fig. IB we observe that they exercise similar protection kinetics (with DHA inhibiting cellular oxidation by 33.46% and vitamin E by 30%) .
• the protection kinetic response of the DHA always presents a maximum antioxidant effect between 60-120 minutes after carrying out the induction, thus denoting a saturation in the hydroperoxides and superoxide anion sequestering capacity of the DHA.
• the antioxidant behaviour is critically dose-dependent, since increasing the concentration thereof leads to a loss of ROS sequestering capacity, with the 0.5 ⁇ M concentration having the most effective antioxidant capacity.
• another critical parameter in terms of optimising the efficiency of the system is the proportion of DHA in relation to total fatty acids.
• the DHA presents an antioxidant activity protecting the fibroblasts from generating membrane hydrolipidic peroxides, its action being of the inverse concentration-dependent type.
• the protection with DHA was 87% for 0.5 ⁇ M DHA, 85% for 5 ⁇ M and 48% for 50 ⁇ M DHA-TG (Fig. 3) .
• Foreskin cells submitted to an oxidative stress with 40 mM AAPH generated a superoxide anion production 2.5 times greater than the non-induced cells, which maintained a constant superoxide anion level (Fig. 4) .
• the cells with integrated DHA do not show a higher level of intercellular superoxide anion in relation to control (Fig. 4) .
• the DHA inhibits generation of the superoxide anion by 16.5% at a concentration of 0.5 ⁇ M, by 10% at a concentration of 5 ⁇ M and by 9% at a concentration of 50 ⁇ M.
• the DHA as antioxidant may prevent deactivation of the SOD entrusted with dismutation of the superoxide anion, maintaining in the cell the endogenous antioxidant defence of the catalase and the glutation peroxidase.
• SOD activity is shown not to be increased in basal state with DHA being present (-10/-15%), but loss of SOD activity inherent to the oxidative stress process is inhibited with DHA being present keeping or even increasing SOD activity (10/20%).
• GPx activity Fig.
• the cellular model was based on ARPE-19 cells (pigmentary retinal epithelial cells, ATCC CRL-2302), being a suitable cellular type due to their good in vitro response to various oxidant inducers, as well as being a primary culture with normal nutritional requirements and culture conditions. It also constitutes a good ocular model, since it keeps the biological and functional properties of the retinal pigmentary epithelial cells.
• the DHA On inducing a moderate oxidative stress with 40 mM AAPH and using DHR123 as ROS detector, the DHA shows an inhibiting effect on the generation of the reactive oxygen species, at the concentrations of 0.5 ⁇ M (43% protection) and 5 ⁇ M (32% protection) , but with a lower effect at 50 ⁇ M (4% protection) of DHA (Fig. 7A) .
• the DHA shows a protective effect against ROS generation, at the 0.5 ⁇ M concentration (13% protection) and lower at higher concentrations of DHA (Fig. 7A).
• the protection kinetics of the DHA also always presents a maximum antioxidant effect 60-120 minutes after carrying out the induction, denoting a saturation in the DHA' s hydroperoxides and superoxide anion sequestering capacity.
• the antioxidant capacity is critically dose-dependent, since when DHA concentration is increased there is a loss of ROS sequestering capacity, with the 0.5 ⁇ M concentration being the most effective in its antioxidant capacity (Fig. 7A and 7B) .
• another critical parameter in terms of optimising the efficiency of the system is the ratio of DHA to total fatty acids.
• lipidic peroxides Fig. 9
• superoxide anions Fig. 10
• the results obtained are very similar to those obtained with the Foreskin cells.
• the cells treated with AAPH generate a higher concentration of substances reactive to thiobarbituric acid (TBARS) and of superoxide anions in relation to the non-induced cells.
• DHA DHA-derived lipidic peroxidation
• the DHA presents a cellular antioxidant activity inhibiting them from generating membrane lipidic hydroperoxides in an inverse ratio to their concentration.
• the protection with DHA was 64% for 0.5 ⁇ M DHA, 58% for 5 ⁇ M and 42% for 50 ⁇ M DHA (Fig. 9) .
• Generation of the superoxide anion was then analysed. In the absence of oxidative induction, the cells with integrated DHA do not present a higher level of intracellular superoxide anion in relation to the control (Fig. 10A) .
• An oxidative stress with 40 mM AAPH generates a superoxide anion production that is partially inhibited by the DHA (20-16% at concentrations of 0.5- 50 ⁇ M) .
• This inhibition is in concordance with SOD activity with DHA being present (Fig. 10B) .
• SOD activity is not found to be increased in basal state with DHA being present (-10/15%) , but as in Foreskin cells, loss of SOD activity inherent to the oxidative stress process is inhibited with DHA being present keeping basal SOD activity.
• retina pigmentary epithelial cells ATCC CRL-2302
• Foreskin cells undifferentiated epidermal fibroblasts
• TG 20% molar in DHA
• oil derivatives enriched with 50 or 70% molar in DHA obtained by chemical methods (CHEM) or enymatic methodes (ENZ) were used an active ingredient.
• the natural DHA (DHA20%-TG) and that incorporated into a chemically obtained triglyceride (DHA50%-TG-CHEM and DHA70%-TG-CHEM) shows an inhibitory effect in the generation of the reactive oxygen species, both 0.5 ⁇ M and 5 ⁇ M concentration, showing a lower effect at 50 ⁇ M (figure 13A) .
• This effect depends on the content of DHA, being DHA70%-TG-CHEM > DHA50%-TG-CHEM > DHA20%-TG.
• DHA glutation intracellular concentration
• ARPE-19 cells Fig. 16
• DHA induces an increase in the GSH intracellular concentration directly related to GSH de novo synthesis since the addition of BSO (specific inhibitor of GSH synthesis) eliminates the protective effect of DHA (Fig. 17) in a direct relation with a decrease in the GSH intracellular concentration (Fig. 15).
• BSO specific inhibitor of GSH synthesis
• the improvement obtained in the antioxidative activity of DHA by an enzymatic synthesis is also applicable to another omega-3 fatty acid such as ecosapentaenoic acid (EPA) .
• EPA ecosapentaenoic acid
• EPA obtained enzimatically EPA70%-TG-ENZ
• DHA70%-TG-ENZ EPA obtained enzimatically
• EPA-70%-TG-BPM EPA-70%-TG-BPM
• EPA70%-Tg-ENZ obtained enzimatically shows in Foreskin cells a remarkable antioxidative activity even higher than that for DHA (DHA70%-TG-ENZ) (Fig. 20), related to, just like for DHA, the increase of GSH intracellular concentration (Fig. 21).
• ARPE-19 cells retina pigmentary epithelial cells, ATCC CRL-2302
• ATCC CRL-2302 retina pigmentary epithelial cells
• DHA20%-TG As an active ingredient there has been used structured triglycerides derived from tuna oil (DHA20%-TG, 20% molar in DHA) or oil enriched with 70% DHA (DHA70%-TG, 70% molar in DHA), wherein through enzymatic methods the fatty acids in sn-1 and sn-3 positions have been replaced with octanoic acid.
• DHA20%-TG As an active ingredient there has been used structured triglycerides derived from tuna oil (DHA20%-TG, 20% molar in DHA) or oil enriched with 70% DHA (DHA70%-TG, 70% molar in DHA), wherein through enzymatic methods the fatty acids in sn-1 and sn-3 positions have been replaced with octanoic acid.
• the molar content of DHA is 7% in the DHA20%-TG and 22% in DHA70%-TG.
• the DHA incorporated into a normal triglyceride shows an inhibitory effect in the generation of the reactive oxygen species, both 0.5 ⁇ M and 5 ⁇ M concentration, showing a lower effect at 50 ⁇ M (figure 22) .
• This effect depends on the content of DHA, being DHA70%-TG > DHA20%-TG.
• the structured oils with a real DHA concentration 2-3 times lower, show the same activity (for 0.5 ⁇ M concentration) or higher (for 5 ⁇ M and 50 ⁇ M concentrations) in the case of DHA20%-TG.
• the efficacy of the structured triglyceride is slightly lower than optimum concentrations (0.5 ⁇ M and 5 ⁇ M) , but the behaviour at high concentrations is inverted (50 ⁇ M) showing in general a more stable and less dose-dependent behaviour.
• the cellular models used were Foreskin cells
• the CRL-2076 fibroblasts were kept growing in culture flasks in Iscove's modified Dulbecco's medium (Biological
• Enzymatically synthesized DHA-TG 70% was added at a 0,5 ⁇ M concentration, made by dissolving the oil in ethanol for the stock solution (1:100) and preparing the working solutions in a culture medium prepared with serum.
• the cells were cultured with supplemented DHA-TG medium for 3 days at 37°C.
• AAPH AAPH
• AAPH oxidises the DNA, the proteins and the lipids through the action of the formed peroxil radicals. It further acts on the endogenous defence system, since it deactivates the key enzyme, the SOD, thereby losing the protective capacity of the CAT and the GPx.
• the telomeric regions constituted by high repetitive DNA can be evaluated by in situ hibridation techniques.
• the method called flow FISH uses flow citometry in combination with the FISH technique using a pan-telomeric PNA (peptide nucleic acid) as a probe and allows to measure, using the fluorescence intensities, the average telomeric lengths at the chromosome ends in individual cells.
• the results are expressed as telomere fluorescence unit (TFU) corresponding each TFU to 1 kb of repetitive telomeres.
• TFU telomere fluorescence unit
• telomere shortening index Changes in the average length of telomeres in human fibroblasts cultured under oxidative stress conditions with or without incorporated DHA were analysed by flow-FISH (figure 23) .
• a linear regression was used to analyse the relation between the length of telomeres and the pass number of cellular populations. For all the analysed cultures, the slopes in the regressions can be understood directly as the telomere shortening index.
• the treatment with AAPH which induces an excess of intracellular free radicals, accelerate noticeably the telomere shortening index.
• the incorporation of DHA at a concentration of 0,5 ⁇ M which has been proved to increase the cell antioxidant defence, reduces said index by 50% in respect to its value without DHA.
• the incorporation of DHA is capable of reducing the telomere shortening index, even in respect to the normal control of fibroblasts.

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