Dietary supplement

Abstract

The invention relates to a composition comprising at least 50 mg / g of one or more omega-3 fatty acids, at least 10 mg / g of one or more xanthophylls, at least 1 mg / g of one or more sterols and at least 2 μg / g of one or more phycoprostanes, and to the use of said composition in particular as a food supplement for the prevention of the onset of cognitive disorders.

Description

[0001] The present invention relates to a composition, as well as a food supplement based on fatty acids and xanthophylls and their applications in particular to prevent the onset of cognitive disorders in humans or animals.

[0002] The invention is defined by the attached claims.

[0003] Cognitive processes are defined as the set of brain functions that enable us to acquire, process, memorize, and use data from the environment in order to maximize the advantages and minimize the disadvantages of external constraints. Thus, cognitive processes are at work during phases involving reasoning (from which planning, organization, and judgment arise), perception, recognition, language, emotions, memory, and learning.

[0004] Mild cognitive impairment, or cognitive frailty, is defined as alterations in cognitive function without dementia. Clinically, this impairment is associated with a score of 0.5 on a standardized assessment. via the CDR (Cognitive Drug Research Computerized Assessment System) test.

[0005] Among these cognitive disorders, age-related cognitive decline and cognitive alterations induced by prenatal stress are two phenomena that can occur during an individual's life.

[0006] Age-related cognitive decline is defined as a non-pathological decrease in cognitive functions such as information processing speed, attentional capacity, and especially working (or short-term) memory. These processes result from normal physiological changes directly correlated with age. The age at which this decline begins is still a subject of debate, but given the accelerating aging of the world's population—more than 20% of the global population is over 60, and this percentage will exceed 30% by 2050—age-related cognitive decline is one of the major challenges of the coming decades, globally, and specifically within developed countries, where it will significantly impact the economy (reduced autonomy for older people) and public policy.

[0007] In contrast to the age pyramid, cognitive disorders can affect infants and young children following prenatal stress. Indeed, for several years, the influence of stress during certain periods of pregnancy on the cognitive development of the unborn child has been studied in both humans and animals. Thus, in animals, primarily rats, it has been shown that prenatal stress in the mother induces offspring with impaired long-term memory.

[0008] It appears that intense negative stimuli, stress, can induce non-pathological alterations or reductions in the cognitive functions of young children, manifesting as hyperactivity, attention and memory deficits, language delays, more difficult temperaments, and more generally behavioral alterations such as anxious behavior, reflecting a delay from a neurodevelopmental point of view and a decline in cognitive abilities.

[0009] One of the hypothesized mechanisms by which prenatal stress translates into cognitive disorders is based on the fetus's exposure to high doses of stress hormones belonging to the corticosteroid family, such as cortisol. Cortisol crosses the placental barrier, and above a certain concentration, the fetus's protective mechanisms against corticosteroids secreted by the mother become overwhelmed, thus exposing the fetus to excessive doses of cortisol that appear to have a detrimental effect on cognitive development. Other complementary hypotheses explaining the link between prenatal stress and cognitive disorders in children have also been proposed.

[0010] The concept of stress can be defined from different perspectives, such as the biological approach: stress is then a series of metabolic reactions, following one or more exogenous factors, inducing physiological or psychological changes (fear, anxiety) within the organism. However, the concept of stress and its impacts are largely individual-dependent, and an individual's response to stress is also defined from a psychological perspective. Therefore, given that an event is only stressful when... in retrospect, Because prenatal stress is unique to each individual and can be objectively stressful, it is difficult to address its underlying causes. Furthermore, pregnancy induces hormonal and psychological changes that increase the expectant mother's sensitivity to any event that might be related to the well-being of the unborn child.

[0011] A strictly therapeutic approach to stress or anxiety, using medication, is risky in pregnant women: many psychotropic drugs used to treat psychological disorders or anxiety have teratogenic effects with direct harmful consequences for the fetus. This necessitates a case-by-case evaluation, and this approach is only used in cases of clinical psychological disorders in pregnant women, not in cases of subjective stress.

[0012] Thus, there is a significant problem concerning the discovery of solutions against the consequences of prenatal stress on cognitive disorders in children or young adults.

[0013] Several studies have demonstrated the benefits of nutritional supplementation with essential fatty acids, as well as carotenes, particularly xanthophylls, or even a combination of these fatty acids and carotenes, to prevent or at least slow the decline in cognitive function. Dietary supplements and medications have been developed, leading to encouraging results.

[0014] Thus, according to document WO2013 / 032333A1, a composition based on omega-3 fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), asthaxanthin, and glycerophospholipids, is recommended for the prevention or treatment of various disorders, including cognitive impairment. These ingredients are present in this composition as microalgae extracts; in a preferred preparation variant, they are obtained by formulating two extracts from two different algae. The natural origin of the composition's constituents is a significant advantage. However, the need for more effective compositions exists, especially considering the challenges mentioned above. Furthermore, it is important to have simple and reproducible processes for preparing such compositions.

[0015] The invention provides a solution with a composition comprising one or more omega-3 fatty acids and one or more xanthophylls, as well as one or more compounds from the sterol family and one or more phycoprostanes. It has been shown that the combination of at least one sterol and at least one phycoprostane with at least one omega-3 fatty acid and at least one xanthophyll significantly increases the effectiveness of a composition in preventing the onset of age-related cognitive decline, as well as in preventing decline associated with prenatal stress.

[0016] One composition of the invention comprises: 50 to 250 mg / g of one or more omega-3 fatty acids, 10 to 50 mg / g of one or more xanthophylls, 1 to 20 mg / g of one or more sterols, 2 to 100 µg / g of one or more phycoprostanes, and at least one oil chosen from among the medium chain triglycerides (MCTs).

[0017] In a principal indication, a composition of the invention can be used as a food supplement. The invention also relates to a food supplement comprising 50 to 250 mg / g of one or more omega-3 fatty acids, 10 to 50 mg / g of one or more xanthophylls, 20 mg / g of one or more sterols, 2 to 100 µg / g of one or more phycoprostanes, and at least one oil selected from medium-chain triglycerides (MCTs).

[0018] The invention offers a key advantage in that all of the above-mentioned constituents or ingredients can be obtained from a natural source and, in particular, can be extracted from one or more microalgae, and preferably from a single microalga. Of course, one or more of the constituents or ingredients of a composition or food supplement of the invention may be of non-natural origin and provided in the form of products manufactured by chemical synthesis.

[0019] Before describing the invention in more detail, certain terms used in this text are defined.

[0020] The term "includes" in the expression "a composition includes" or "a food supplement includes" means that the composition or supplement may incorporate any additional constituent, not expressly mentioned, in any form and from any source. It also covers a composition or supplement that contains only the listed constituents and that, consequently, the composition or supplement consists solely of those constituents.

[0021] A food supplement is defined as one or more foodstuffs whose purpose is to supplement the normal diet of a human or animal, and which constitute a concentrated source of nutrients or other substances having a nutritional or physiological effect alone or in combination; it is generally available in dose form, namely presentation forms such as capsules, lozenges, tablets, pills and other similar forms, as well as sachets of powder, ampoules of liquid, bottles fitted with a dropper and other similar forms of liquid or powder preparations intended to be taken in small measured units.

[0022] Omega-3 fatty acids are a family of unsaturated fatty acids whose hydrocarbon chain has between 4 and 36 carbon atoms, generally between 14 and 36, and whose double bond, or first double bond, counted from the terminal methyl group of the chain, is located on the third carbon-carbon bond. The unsaturates can be cis or trans, independently of each other. The most representative acids are alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), but the term "omega-3 fatty acids" is not limited to these. In addition, and particularly when the fatty acid(s) are of natural origin, they can be extracted from algae and be present in the form of free molecules but also in a derived form such as an esterified form, for example in mono-, di- or tri-esterified form, or in mixtures of these forms.

[0023] Xanthophylls are defined as molecules belonging to the carotenoid family containing one or more oxygen atoms, such as astaxanthin, cantaxanthin, vaucheriaxanthin, lutein, zeaxanthin, diadinoxanthin, neoxanthin, loroxanthin, siphonoxanthin, diatoxanthin, violaxanthin, dinoxanthin, flavoxanthin, α-cryptoxanthin, β-cryptoxanthin, and fucoxanthin. In particular, when the xanthophyll(s) are of natural origin, they can be extracted from algae and exist as free molecules, but also in a derived form such as an esterified mono- or multi-ester, or in mixtures of these forms.

[0024] Sterols are a well-known family of lipids possessing a sterane nucleus in which the carbon at position 3 bears a hydroxyl group, which may be modified, for example, by an acetyl group. They include natural sterols or phytosterols, and are grouped together in this text under the term phycosterols. Examples of phytosterols include, but are not limited to, 24-methylenecholesterol, β-sitosterol, fucosterol, isofucesterol, saringosterol, oxocholesterol acetate, crinosterol, and, more specifically, brassicasterol, stigmasterol, and campesterol.

[0025] Phycoprostanes are a family of naturally occurring, prostaglandin-like lipids resulting from non-enzymatic oxidations of fatty acids naturally present in microalgal biomass. These compounds are specifically selected from phytoprostanes, isoprostanes, and neuroprostanes, depending on the fatty acid that has undergone oxidation. Thus, these compounds can be derived from fatty acids such as α-linolenic acid (ALA), arachidonic acid (ARA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA). Phytoprostanes are primarily derived from ALA and can be selected from 9-epi-9F1t-PhytoP, ent-16-epi-16-F1t-PhytoP, 9-F1t-PhytoP, ent-16B1t-PhytoP, ent-9L1t-PhytoP, and 16(RS)-16-A1t-PhytoP. Isoprostanes are primarily derived from ARA and EPA and can be selected from 15-E2t-IsoP, 15-F2t-IsoP, 15-epi-15-F2t-IsoP, 5-F2t-IsoP, and 8(RS)-8-F3t-IsoP.Neuroprostanes are primarily derived from DHA and can be selected from 4-F3t-NeuroP, 10-F4t-NeuroP, 10-epi-10-F4t-NeuroP, 4(RS)-4-F4t-NeuroP, 14(RS)-14-F4t-NeuroP, 20(R)-20-F4t-NeuroP.

[0026] Medium-chain triglycerides (MCTs) are esters of glycerol and saturated fatty acids, with a hydrocarbon chain of approximately 6 to 12 carbon atoms. They are naturally present in coconut oil, palm kernel oil, and palm oil, but can also be obtained from other fats or oils.

[0027] The present invention is described in more detail below and its variants are set forth.

[0028] A composition or food supplement of the invention advantageously meets the following characteristics, considered alone or in one or more combinations.

[0029] It comprises 50 to 250 mg / g of one or more omega-3 fatty acids, 10 to 50 mg / g of one or more xanthophylls, 1 to 20 mg / g of one or more sterols and 2 to 100 µg / g of one or more phycoprostanes.

[0030] It comprises 50 to 200 mg / g of one or more omega-3 fatty acids, 10 to 30 mg / g of one or more xanthophylls, 1 to 8 mg / g of one or more sterols and 2 to 50 µg / g of one or more phycoprostanes.

[0031] It comprises 50 to 170 mg / g of one or more omega-3 fatty acids, 10 to 25 mg / g of one or more xanthophylls, 1 to 6 mg / g of one or more sterols and 2 to 40 µg / g of one or more phycoprostanes.

[0032] A composition or food supplement of the invention further contains, as a vehicle or support to facilitate the expression of the active ingredients, at least one oil selected from medium-chain triglycerides (MCTs). Surprisingly, it has been observed that the manufacture of a composition or food supplement is facilitated when this oil is selected from medium-chain triglycerides (MCTs). In particular, when the active ingredients are obtained from the same microalgae extract, optimal homogenization is observed in such an oil. According to one embodiment, the medium-chain triglycerides (MCTs) are of natural origin and are provided by an oil selected from coconut oil, palm kernel oil, and palm oil; they may also be obtained or derived from such an oil.

[0033] Preferred formulations of a composition or food supplement of the invention are presented below; these implementations can of course be combined: The omega-3 fatty acid or at least one of the omega-3 fatty acids is chosen from stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and mixtures thereof; the xanthophyll or at least one of the xanthophylls is fucoxanthin; the sterol or at least one of the sterols is chosen from phytosterols; the phycoprostane or at least one of the phycoprostanes is chosen from phytoprostanes, isoprostanes and neuroprostanes.

[0034] A food composition or supplement of the invention may include any additive that improves its preservation, appearance, taste, or formulation. Thus, one or more additives, such as those selected from preservatives, colorings, flavorings, disintegrating agents, lubricants, coating agents, or encapsulating agents, may be incorporated.

[0035] A major application of a composition of the invention is nutraceutical; thus, such a composition or food supplement as defined above is advantageously presented in the form of capsules, tablets, lozenges, or loose powder. It is preferably packaged in doses having a unit weight between 1 mg and 1 g. Generally, the formulation of the composition or supplement will be adapted to the individual concerned, and in particular depending on whether it is intended for a child or an adult.

[0036] A composition or dietary supplement of the invention can be used to prevent the onset of non-pathological cognitive impairment related to aging or non-pathological cognitive impairment in children or young adults who have experienced prenatal stress. For the prevention of age-related cognitive impairment, the daily intake may be between 2 and 5 mg / kg of body weight. For the prevention of cognitive impairment in children or young adults who have experienced prenatal stress, the daily intake may be between 0.05 and 0.1 mg / kg of body weight. It has been observed that an effect is seen even with very low daily doses, provided that the duration of treatment is proportionally increased.

[0037] The invention also relates to the use of a microalga to prepare a food supplement as defined above. One or more preferred microalgae are chosen from any of the following taxa Pinguiophyceae, Chrysophyceae, Bacillariophyceae, Mamiellophyceae, Prymnesiophyceae, Haptophyceae, Coccolithophyceae, Isochrysidaceae And Phaeodactylaceae. Advantageously, the microalga is the Tisochrysis lutea or the Phaeodactylum tricornutum.Such microalgae will be chosen because appropriate extraction leads to an extract whose composition meets the definition of a composition of the invention. By way of example, such an extract may comprise the following fatty acid fraction: the fatty acids, expressed as a mass percentage of the total extract, are in the form of free fatty acids between 4 and 55%, monoacylglycerol between 0.5 and 10%, diacylglycerol between 0.4 and 15%, and triacylglycerol between 2 and 55%. These fatty acids comprise 5 to 20% (w / w) of the omega-3 series and between 0.5 and 5% of the omega-6 series. More specifically, the fatty acids include ALA (alpha-linolenic acid) between 0.5 and 10%, SDA (stearidonic acid) between 0.5 and 10%, EPA (eicosapentaenoic acid) between 0.05 and 20%, and DHA (docosahexaenoic acid) between 0.1 and 10%.

[0038] As previously mentioned, one of the advantages of a dietary composition or supplement lies in its preparation process and, specifically, the natural origin of its constituents, which can all be obtained from a single microalga. Depending on the microalga used, the formulation can be obtained directly from the extract. If this is not the case, the extract will be diluted to obtain the concentrations required according to the invention. However, the invention is not limited to this implementation; thus, it is possible to have only some of the constituents of natural origin, the others being obtained through chemical synthesis, and / or that the constituents of natural origin not come from the same source, for example, not be produced from the same alga.

[0039] The measurement and adjustment of the concentrations of active ingredients in an extract, and in the composition or food supplement obtained, are carried out using analytical techniques which are part of the general knowledge of a person skilled in the art.

[0040] A process for manufacturing a composition or food supplement from a microalgae culture is described in more detail below.

[0041] According to one embodiment of the invention, said organisms are microalgae, such as those belonging to the taxa Pinguiophyceae, Chrysophyceae, Bacillariophyceae, Mamiellophyceae, Prymnesiophyceae, Haptophyceae, Coccolithophyceae, Isochrysidaceae, Phaeodactylaceae. These photosynthetic microorganisms can be strictly autotrophic, mixotrophic, or transiently heterotrophic. These organisms can be harvested from the natural environment or, preferably, cultivated.

[0042] An extract is defined as a fraction of the biomass from photosynthetic organisms obtained by a process that allows for the direct or indirect production of a composition of the invention. These extracts have a composition, expressed as a mass percentage of the total extract, of proteins between 5 and 30%, lipids between 20 and 80%, sterols between 0.1 and 2%, and chlorophyll between 0.1 and 20%.

[0043] More specifically, the lipophilic part composing the extract, expressed as a mass percentage of total extract, consists of saturated fatty acids between 15 and 45%, polyunsaturated fatty acids between 5 and 20%, xanthophylls between 1 and 20% and phycoprostane between 0.0002 and 0.007%.

[0044] For the purposes of producing the extract according to the invention, advantageously the cells are microalgae cells of the species Tisochrysis lutea of the family of Isochrysidaceae, or microalgae cells of the species Phaedactylum tricornutumof the family of Phaeodactylaceae, produced by autotrophy with respect to carbon. Microalgae production method

[0045] Microalgae are ideally cultivated in a controlled manner within suitable systems such as raceways, open ponds, or preferably closed systems like photobioreactors. The photobioreactors used can be of any existing type, such as horizontal tubular photobioreactors, vertical tubular photobioreactors (like so-called "green wall panel" systems), flat panel photobioreactors, or column photobioreactors. Preferably, biomass production will occur within a closed culture system, through autotrophy, without impacting arable land.

[0046] Biomass production is carried out according to the following cultivation methods: batch, fed-batch, continuous, semi-continuous, turbidostat or chemostat. Obtaining extracts from these microalgae

[0047] Extracts from these microorganisms are preferably obtained after concentrating the biomass by removing all or part of the water using chemical or physical processes such as centrifugation, filtration, flocculation, and sedimentation, coupled or not with drying steps such as freeze-drying, vacuum drying, drum drying, spray drying, or any other process that reduces the water content of the biomass. In addition to these steps, cell lysis processes may be implemented, such as the application of pressure, electrical currents, shear forces, the use of enzymes, or any other processes that disrupt the structure of tissues, organs, cells, or organelles.

[0048] The compounds of interest from biomass are extracted using a solid-liquid extraction process, which may involve hypercritical or subcritical fluids, and may incorporate co-treatments carried out in parallel or sequentially, such as microwaves, ultrasound, pressure, or enzymes. The solvents used, pure or in mixtures, may include acetone, hexane, ethyl acetate, methyltetrahydrofuran, heptane, methanol, natural or branched oils, ethanol, or any other solvent suitable for extracting all or part of the hydrophobic and amphiphilic compounds.

[0049] The solvent or solvent mixture is separated from the residual biomass after extraction by processes such as centrifugation and filtration, and can subsequently be concentrated, or the solvent removed, by techniques such as vacuum evaporation or any other technique allowing for the selective evaporation of the solvent in question. The extract thus obtained is lipophilic in nature while also containing amphiphilic molecules. Formulation as a food supplement

[0050] The extract is formulated using compatible matrices that allow for its dissolution to obtain a homogeneous solution of the desired extract concentration. Examples include vegetable oils such as olive oil, rapeseed oil, linseed oil, sunflower oil, grapeseed oil, palm oil, and preferably MCT oils. These matrices are composed of more than 70% by mass of a mixture of caprylic and capric acids, preferably coconut or palm oil. The extract is further enhanced with molecules that increase stability, such as synthetic or natural antioxidants. The mass proportions of matrices / additives used to obtain the supplement can reach 95% by weight relative to the weight of the dietary supplement, but are generally between 15% and 80%, preferably between 35% and 45%.

[0051] The extract, but preferably the formulated composition or the supplement obtained, may be put into the form of soft capsules, or may be formulated into powder, by any technique allowing the microencapsulation of aqueous solution involving or not a support or matrix allowing or not its homogeneous dispersibility within a polar oral solution.

[0052] The extract or supplement can be used alone or as an ingredient in a dietary supplement.

[0053] The various objects of the invention are illustrated below and their advantages highlighted in the following examples, supported by the following figures: [ Fig. 1[ ] is a representation of the effects of the complement of the invention on locomotor activity, with the diagram on the left illustrating the effects on deficits in spontaneous alternations and the diagram on the right illustrating the effects on locomotor activity. Fig. 2 ] is a representation of the effects on learning deficits induced by D-Gal according to the MWM test. Fig. 3 ] is a representation of the effects of complement and DHA on D-Galactose-induced learning deficits. Fig. 4 ] is a representation of the effects on passive avoidance deficits induced by D-Galactose in mice, with the effects on step-through latency illustrated in the left diagram and on escape latency illustrated in the right diagram, measured during the retention period. Fig. 5 ] is a representation of the effects of complement and DHA on D-Galactose-induced lipid peroxidation [ Fig. 6[ ] is a representation of the effects of complement and DHA on D-Galactose-induced expression of TNF-α in the cortex and plasma, with the effect on the cortex shown on the left diagram and the effect on the plasma shown on the right diagram. Fig. 7 ] is a representation of the effects of complement and DHA on D-Galactose-induced expression of IL-6 in the cortex (left diagram) and plasma (right diagram). Fig. 8 ] is a representation of the effect of the complement on anxiety, in the locomotion test at the center of the test space, day JPN46. Fig. 9 ] is a representation of the effect of the complement on recognition memory, in the object recognition test, day JPN47. Fig. 10 ] is a representation of the effect of the complement on recognition memory, in the novel object recognition test. Fig. 11[ ] is a representation of the effects of the complement of the invention on locomotor activity, with the diagram on the left illustrating the effects on deficits in spontaneous alternations and the diagram on the right illustrating the effects on locomotor activity. Fig. 12 ] is a representation of the effects on learning deficits induced by D-Gal according to the MWM test. Fig. 13 ] is a representation of the effects of the complement on D-Galactose-induced learning deficits. Fig. 14 ] is a representation of the effects on passive avoidance deficits induced by D-Galactose in mice, with the effects on step-through latency illustrated in the left diagram and on escape latency illustrated in the right diagram, measured during the retention period. Fig. 15 ] is a representation of the effects of complement on D-Galactose-induced lipid peroxidation [ Fig. 16[ ] is a representation of the effects of complement on D-Galactose-induced expression of TNF-α in the cortex and plasma, with the effect on the cortex shown on the left diagram and the effect on the plasma shown on the right diagram. Fig. 17 ] is a representation of the effects of complement on D-Galactose-induced expression of IL-6 in the cortex (left diagram) and plasma (right diagram). Example 1: Formulation of an extract containing the constituents of a composition of the invention.

[0054] An extract is obtained from the microalga using one of the techniques described above. Phaeodactylum tricornutum.

[0055] It is insoluble in water and is highly viscous, preventing any handling at room temperature.

[0056] The extract and palm oil are brought to room temperature (25 ± 1 °C) 24 hours before preparation. The extract is transferred to a centrifuge tube containing the oil such that the final net mass of the mixture is approximately 5 g and the mass proportion is such that the extract constitutes 25% of the total net mass of the mixture. The mixture is stirred for one minute using a vortex mixer. This stirring is repeated three times per batch. A homogeneous mixture is obtained. Example 2: Testing a natural extract of microalgae Tisochrysis lutea within the framework of the model in vivo on mitigating deficits caused by age-related cognitive decline

[0057] The dietary supplement of the invention is prepared from an extract of Tisochrysis lutea which includes in mg / g: Omega-3 fatty acids (ALA, SDA, EPA, DHA): 152.6 ± 14.4; Fucoxanthin: 20.0 ± 4.0; Sterols: 4.9 ± 0.8; Phycoprostane: 0.035 ± 0.007

[0058] The supplement is obtained by adding coconut oil at a rate of 360 mg ± 10 mg / g to said extract.

[0059] The supplement is incorporated into kibble according to 3 different formulations such that the final DHA concentrations of these batches of kibble are equal to 0.5, 1.5 and 3.0% (w:w).

[0060] A commercial oily microalgae extract comprising as a fat fraction only DHA 77% (w:w) and EPA 3% (w:w) fatty acids is also tested; it is also incorporated into a batch of kibble such that the final DHA concentration of this batch of kibble is equal to 3.1% (w:w).

[0061] An additional batch of kibble is formulated solely with coconut oil, such that the vehicle concentration is equivalent to that of the other batches, i.e. 0.01% (w:w).

[0062] The five batches of kibble thus obtained are referenced as described in Table 1 below. [Table 1] Formulated kibble Reference [DHA] in % (m:m) Coconut oil A1 0 Complement A2 0,5 Complement A3 1,5 Complement A4 3,0 Commercial oily extract A5 3,1

[0063] The model in vivo The model considered is the D-Galactose model applied to mice, which is suitable for studying age-related cognitive decline. This model effectively mimics many behavioral and molecular characteristics of brain aging in rodent models.

[0064] D-Galactose is administered subcutaneously at a dose of 150 mg / kg fresh weight of mouse per day, and the above dietary supplement is incorporated into a pellet, according to the following scheme: Between day -14 and day 51, the supplement is administered by incorporation into food pellets; Between day 01 and day 51, D-Galactose is administered subcutaneously, five days a week; Between days 43 and 51, three different behavioral tests are used to monitor the effects of the test compounds.

[0065] The effectiveness of the complement is evaluated according to the following parameters: improvement of learning deficits (spatial working memory: spontaneous alternation in the Y maze according to the Y-maze test; spatial memory by the so-called "Morris Water Maze" test and long-term contextual memory in the passive avoidance test), lipid peroxidation (LPO) levels in the hippocampus and the effect on the neuro-inflammation markers IL6 and TNFα. Improvement of learning deficits

[0066] On day 43, all animals were tested for spontaneous alternation performance in the Y-maze (YM) test, via a spatial working memory index; From day 44 to day 49, all animals were tested for spatial memory in the Morris Water Maze (MWM) test, via a spatial memory index; From day 44 to day 49, all animals were tested via the MWM test to assess spatial working memory; On days 50 and 51, the animals' long-term contextual memory was assessed using the step-by-step passive avoidance procedure (STPA), via training and retention sessions, respectively; On days 50 and 51, all animals were tested for the STPA task. Lipid peroxidation (LPO) levels in the hippocampus and their effect on the neuro-inflammation markers IL6 and TNFα

[0067] On the 51st day, after behavioral tests, the animals are euthanized.

[0068] For all animals, trunk blood is collected and centrifuged to recover the plasma, and the brain is rapidly removed. The hippocampus and cortex are dissected; the hippocampus is then used to determine lipid peroxidation levels by colorimetric method; the hemifrontal cortex and plasma are used to determine the levels of the inflammatory biomarkers interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α).

[0069] Lipid peroxidation levels (LPO) were quantified according to the modified and adapted procedure of Hermes-Lima et al. This method measures the capacity of peroxidized brain lipids to oxidize an orange ferrous oxide and xylenol complex, as demonstrated in the presence of cumer hydroperoxide (CHP). The lipid peroxidation level is determined in CHP equivalents according to: CHPE = A5801 / A5802 x [CHP (nmol)] and expressed as CHP equivalents per wet tissue weight and as a percentage compared to the data obtained for the control group (D-Galactose + vehicle).

[0070] IL6 and TNFα levels are quantified using ELISA tests with the following kits: For IL-6 quantification: ThermoScientific, EM2IL6. For TNFα quantification: ThermoScientific, EMTNFA.

[0071] For all assays, the cortex is homogenized after thawing in 50 mM Tris-150 mM NaCl buffer, pH 7.5, and sonicated for 20 s. After centrifugation (16,100 g for 15 min, 4 °C), a supernatant or plasma is used for ELISA assays according to the ELISA manufacturer's instructions. For each assay, the absorbance is read at 450 nm and the sample concentration is calculated using the standard curve. Results are expressed in pg of marker per mg of fresh tissue.

[0072] All values, except for passive avoidance latencies, are expressed as mean plus or minus the measurement standard deviation. Statistical analyses were performed separately for each compound using a one-way ANOVA (F-value), followed by Dunnett's post-hoc multiple comparison test. Passive avoidance latencies do not follow a Gaussian distribution, since the upper limit times are fixed. They were therefore analyzed using a non-parametric Kruskal-Wallis ANOVA (H-value), followed by Dunn's multiple comparison test. Values ​​with p < 0.05 were considered statistically significant.

[0073] The tests are performed on 60 male mice, divided into 6 groups of 10 mice, of which group 1 is the negative control group and groups 2-6 are the positive control groups: Group 1 is the group to which a subcutaneous saline solution is administered instead of D-Galactose and A1 kibble; Group 2 is the group to which D-Galactose and A1 kibble are administered; Group 3 is the group to which D-Galactose and A2 kibble are administered; Group 4 is the group to which D-Galactose and A3 kibble are administered; and Group 5 is the group to which D-Galactose and A4 kibble are administered; and Group 6 is the group to which D-Galactose and A5 kibble are administered.

[0074] The calculation of the human equivalent daily dose (HED) from the tested mouse daily dose is defined by the FDA (Guidance, 2005) as follows: the human HED, expressed in mg / kg body weight, is equal to the animal HED, expressed in mg / kg body weight, multiplied by the ratio of the animal safety factor (Km Animal) to the human safety factor (Km Human). The Km Human is 37 and the Km Mouse is 3. Effects on spatial memory in the spontaneous alternation of the Y-maze test:

[0075] The results are represented at the figure 1 , the first diagram (on the left) illustrating the effects of the complement of the invention on deficits of spontaneous alternations and the second diagram (on the right) illustrating the effects of the complement of the invention on locomotor activity.

[0076] On the figure 1: LOW, low dose of complement (A2); MED, medium dose of complement (A3); HI, high dose of complement (A4); N is between 9 and 10 depending on the group; * p < 0.05, *** p < 0.0001 vs. saline solution / Veh group, # p < 0.05, ## p < 0.01, ### p < 0.0001 vs. D-GAL 150 group / Veh group; Dunnett test.

[0077] It was observed that treatment with D-Galactose significantly impaired spatial working memory, compared to mice treated with saline solution.

[0078] Complement A2 showed no effect on alternating behavior. Complement A3 significantly but partially attenuated deficits induced by chronic D-galactose poisoning. Complement A4 significantly and completely attenuated deficits induced by chronic D-galactose poisoning.

[0079] Treatment with DHA alone (according to A5) very significantly but partially alleviated the deficits induced by chronic D-Gal intoxication.

[0080] Surprisingly, it appears that preventive treatment with the supplement of the invention has a superior positive effect (very significant and complete attenuation of deficiencies) compared to treatment with DHA alone (very significant and partial attenuation of deficiencies), and this for the same dose of DHA. Effects on learning deficits induced by D-Gal according to the MWM test:

[0081] The results are represented at the figure 2 .

[0082] On the Figure 2: LOW, low dose of complement (A2); MED, medium dose of complement (A3); HI, high dose of complement (A4); N is between 9 and 10 depending on the group; * p < 0.05, ** p < 0.01, *** p < 0.0001 vs. saline solution / Veh group, ## p < 0.01, ### p < 0.0001 vs. D-GAL 150 group / Veh group; Bonferroni multiple comparison test after two-way ANOVA.

[0083] Chronic D-Galactose intoxication severely impaired spatial learning, compared to the negative control group (saline solution / vehicle).

[0084] The A2 complement showed no effect on alternating behavior.

[0085] Complement A3 significantly but partially attenuated the deficits induced by chronic D-Galactose intoxication.

[0086] Complement A4 very significantly and completely attenuated the deficits induced by chronic D-Galactose intoxication.

[0087] DHA alone according to A5 very significantly but partially alleviated the deficits induced by chronic D-Galactose intoxication.

[0088] Surprisingly, it appears that preventive treatment with the supplement of the invention at dose A4 has a superior positive effect (very significant and complete attenuation of deficits) compared to treatment with DHA alone (very significant and partial attenuation of deficits), and this for the same dose of DHA. Effects of complement and DHA on D-Galactose-induced learning deficits

[0089] The results are represented at the figure 3 .

[0090] On the Figure 3 : LOW, low dose of complement (A2); MED, medium dose of complement (A3); HI, high dose of complement (A4); N is between 9 and 10 depending on the group; *** p < 0.0001 vs. saline / Veh group, / Veh; ### p < 0.0001 vs. D-GAL 150 group / Veh; Bonferroni multiple comparison test after two-way ANOVA.

[0091] Chronic D-Galactose intoxication severely impaired spatial learning, compared to the negative control group (saline solution / vehicle).

[0092] The A2 complement showed no effect on alternating behavior.

[0093] Complement A3 significantly but partially attenuated the deficits induced by chronic D-Galactose intoxication.

[0094] Complement A4 very significantly and completely alleviated the deficiencies induced by chronic D-Galactose intoxication.

[0095] Treatment with DHA alone according to A5 very significantly but partially alleviated the deficits induced by chronic D-Galactose intoxication.

[0096] Surprisingly, preventive treatment with the supplement of the invention at dose A4 appears to have a greater positive effect (highly significant and complete attenuation of deficiencies) compared to treatment with DHA alone (highly significant and partial attenuation of deficiencies), even at the same DHA dose. Furthermore, preventive treatment with the supplement at dose A3 has an identical effect (highly significant and partial attenuation of deficiencies) to treatment with DHA alone, despite the latter being twice as concentrated in DHA. Effects on D-Galactose-induced passive avoidance deficits in mice

[0097] The results are represented at the figure 4 , with the effects of the complement of the invention on the stepping latency illustrated on the diagram on the left and on the escape latency illustrated on the diagram on the right, measured during the retention period.

[0098] On the figure 4LOW, low dose of complement (A2); MED, medium dose of complement (A3); HI, high dose of complement (A4). N is between 9 and 10 depending on the group; *** p < 0.0001 vs. saline / Veh group, ### p < 0.0001 vs. D-GAL 150 group / Veh group; Dunnett's test.

[0099] Chronic D-Galactose intoxication severely impaired long-term contextual working memory, compared to the negative control group (saline / vehicle).

[0100] The A2 complement showed no effect on long-term contextual memory.

[0101] Complement A3 did not significantly mitigate the deficiencies induced by chronic D-Galactose intoxication.

[0102] Complement A4 very significantly and completely attenuated the deficits induced by chronic D-Galactose intoxication.

[0103] Treatment with DHA alone (according to A5) did not significantly attenuate the deficits induced by chronic D-Galactose intoxication.

[0104] Surprisingly, preventive treatment with the supplement of the invention at dose A4 appears to have a greater positive effect (highly significant and complete attenuation of deficiencies) compared to treatment with DHA alone (non-significant attenuation of deficiencies), even at the same DHA dose. Furthermore, preventive treatment with the supplement at dose A3 has an identical effect (non-significant attenuation of deficiencies) to treatment with DHA alone, despite the latter being twice as concentrated in DHA. Effects of complement and DHA on D-Galactose-induced lipid peroxidation

[0105] The results are shown at the figure 5 .

[0106] On the figure 5: LOW, low dose of complement (A2); MED, medium dose of complement (A3); HI, high dose of complement (A4). N is between 9 and 10 depending on the group; ** p < 0.01, *** p < 0.0001 vs. saline / Veh group, ## p < 0.01, ### p < 0.0001 vs. D-GAL 150 / Veh group; Dunnett's test.

[0107] Chronic D-Galactose intoxication greatly increased oxidative stress compared to the negative control group (saline / vehicle).

[0108] Complement A2 showed no effect on lipid peroxidation induced by chronic D-Galactose intoxication.

[0109] Complement A3 significantly but partially reduced oxidative stress induced by chronic D-Galactose toxicity.

[0110] Supplement A4 very significantly and completely reduces oxidative stress induced by chronic D-Galactose toxicity.

[0111] Treatment with DHA alone according to A5 showed no effect on oxidative stress induced by chronic D-Galactose intoxication.

[0112] Surprisingly, it appears that preventive treatment with the supplement of the invention at dose A4 has a superior positive effect (very significant and complete attenuation of oxidative stress) compared to treatment with DHA alone, and this for the same dose of DHA. Effects of complement and DHA on D-Galactose-induced TNF-α expression in cortex and plasma

[0113] The results are represented at the Figure 6 , with the effect on the cortex shown in the left diagram and the effect on the plasma shown in the right diagram.

[0114] On the Figure 6 : LOW, low dose of complement (A2); MED, medium dose of complement (A3); HI, high dose of complement (A4); N is between 9 and 10 depending on the group; *** p < 0.0001 vs. saline solution / Veh group, ### p < 0.0001 vs. D-GAL 150 group / Veh group; Dunnett test.

[0115] Chronic D-Galactose intoxication significantly increased TNF-α in the cortex and plasma, compared to the negative control group (saline / vehicle).

[0116] Complement A2 and A3 very significantly but partially reduced the increase in TNF-α induced by chronic D-Galactose intoxication in the cortex and plasma.

[0117] Complement A4 very significantly and completely reduced the level of TNF-α in the cortex and plasma.

[0118] Treatment with DHA alone according to A5 very significantly but partially reduced the increase in TNF-α induced by chronic D-Galactose intoxication in the cortex and plasma.

[0119] Surprisingly, it appears that preventive treatment with complement HI (A4) has a greater positive effect (highly significant and complete attenuation of the increase in TNF-α in the cortex and plasma) compared to treatment with DHA alone, at the same DHA dose. Furthermore, preventive treatments with complement at doses A2 and A3 have identical effects in the cortex and plasma (highly significant attenuation of the increases) to treatment with DHA alone, even though the latter is six and two times more concentrated in DHA, respectively, compared to preventive treatments with complement at doses A2 and A3. Effects of complement and DHA on D-Galactose-induced IL-6 expression in cortex and plasma

[0120] The results are represented at the Figure 7 , with the effect in the cortex shown in the left diagram and in the plasma shown in the right diagram

[0121] On the Figure 7:. LOW, low dose of complement (A2); MED, medium dose of complement (A3); HI, high dose of complement (A4); N is between 9 and 10 depending on the group; *** p < 0.0001 vs. saline solution / Veh group, ### p < 0.0001 vs. D-GAL 150 group / Veh group; Dunnett's test.

[0122] Chronic D-Galactose intoxication significantly increased IL-6 in the cortex and plasma, compared to the negative control group (saline / vehicle).

[0123] Low-dose complement (A2) showed no effect on D-Galactose intoxication-induced IL-6 concentration.

[0124] Complement at medium dose (A3) very significantly but partially reduced the increase in IL-6 levels induced by chronic D-Galactose intoxication in the cortex and plasma.

[0125] High-dose complement (A4) very significantly and completely reduced the level of IL-6 in the cortex and plasma, induced by chronic D-Galactose intoxication in the cortex and plasma.

[0126] Treatment with DHA alone according to A5 significantly, but partially, reduced the increase in IL-6 levels in the cortex and plasma induced by chronic D-Galactose intoxication.

[0127] Surprisingly, preventive treatment with complement A4 has a greater positive effect (highly significant and complete attenuation of the increase in IL-6 in the cortex and plasma) compared to treatment with DHA alone (highly significant and partial attenuation of the increase in IL-6 in the cortex and plasma), even at the same DHA dose. Furthermore, preventive treatment with complement at dose A3 has an identical effect on the cortex (non-significant attenuation of deficits) to treatment with DHA alone, despite the latter being twice as concentrated in DHA.

[0128] In conclusion: Chronic D-galactose poisoning significantly impairs spatial working memory, long-term contextual memory, and spatial learning. These behavioral alterations are also linked to biochemical changes, including increased oxidative stress and the induction of neuroinflammatory processes.

[0129] Preventive treatment with the complement of the invention is dose-dependent and, in a very significant way, completely attenuated in the case of the highest dose tested (complement A4) the deficits induced by chronic D-Galactose intoxication in terms of behavioral alteration, increased oxidative stress, and activation of neuro-inflammatory processes.

[0130] Treatment with DHA alone (A5), and with equivalent doses of DHA compared to complementary treatment with HI, very significantly, but partially reduced the deficits induced by chronic D-Galactose intoxication in terms of behavioral alteration, increased oxidative stress, and activation of neuro-inflammatory processes.

[0131] Surprisingly, preventive treatment with complement is significantly more effective at equivalent doses of DHA than preventive treatment with DHA alone in mitigating age-related cognitive decline caused by chronic D-galactose intoxication in a mouse model. Furthermore, preventive treatment with complement, at half the DHA dose, has the same positive effects as treatment with DHA alone, even with the latter being twice as concentrated in DHA, in reducing oxidative stress measured in the cortex (IL-6 and TNF-α), in plasma (TNF-α), in long-term contextual memory, and in spatial learning. And, preventive treatment with complement has identical positive effects to treatment with DHA alone, even though the latter is six times more concentrated in DHA, in the case of the reduction of oxidative stress measured in the cortex (IL-6 and TNF-α) and the plasma (TNF-α).

[0132] Thus, by applying the formula for calculating the equivalent daily dose in humans, a preventive treatment for age-related cognitive decline can be defined with a daily intake of 2 to 5 mg of supplement / kg of body weight. Example 3: Testing a microalgae extract Tisochrysis lutea in young female rats affected by prenatal stress

[0133] This example studies the resolution of cognitive deficits, anxious behavior, and impaired recognition memory induced in young female rats after prenatal ancestry stress. via the administration of a supplement based on an extract of the microalga Tisochrysis lutea corresponding to the one used in Example 2. Materials and methods

[0134] The model used in this example is a recognized model of prenatal stress induction in rats by immobilizing the pregnant female in a cylinder under intense lighting.

[0135] Pregnant female rats were randomly assigned to prenatal stress (PS) or control (CS) groups, housed individually in plastic rearing cages, and had access ad libitum to food and water except during behavioral tests. The conditions within the cages are as follows: a 12-hour light / 12-hour dark cycle photoperiod (lights turned on at 7:00 AM), in a room with a constant temperature (21°C) and humidity (50%).

[0136] The prenatal stress procedure was performed as described by Meunier et al. (2004). The restraints of the female rats were performed using a semi-random restraint procedure. The animals were placed and restrained in transparent Plexiglas cylinders (20 cm long, 7 cm in diameter) under bright light for a total of 90 minutes per day, for 4 consecutive days. To make the stress as unpredictable as possible, the 90-minute period of forced restraint was administered as follows: a single 90-minute phase, two 45-minute phases spaced 4 hours apart, two phases of 60 and 30 minutes spaced 4 hours apart, or three 30-minute phases spaced 4 and 1 hour apart, at different times of the day.

[0137] Control mothers were also manipulated, but were never placed in the restraint cylinders.

[0138] The treated female rats were allowed to naturally free themselves from the restraint of the cylinders from day 1 after birth (JPN1).

[0139] The litters were weaned at JPN21. The rats were separated from their mothers, identified according to their sex, weighed, and placed in cages of the same sex (3 rats per cage). The young rats in the same cage came from different litters to avoid any possible litter-related effects.

[0140] The conditions inside the cages are as follows: a 12-hour light / 12-hour dark cycle (lights turned on at 7:00 AM), in a room with a constant temperature (21°C) and humidity (50%), with access ad libitum to food and water, except during behavioral experiments.

[0141] In each cage, the animals received the same treatment. The animals were tested randomly and in a double-blind manner.

[0142] Forty-eight (48) female rats were used and grouped into four animal groups, constituted as follows: Group 1 consists of 12 naïve female rats, meaning their ancestry has not been subjected to prenatal stress, and they receive only 200 µL per day of vehicle solution (reference: NS / Vehicle). This group is therefore the control group. Group 2 consists of 12 naïve female rats, meaning their ancestry has not been subjected to prenatal stress, and they receive 200 µL per day of supplement (reference: NS / Supplement). Group 3 consists of 12 female rats whose ancestry has been subjected to prenatal stress, and they receive 200 µL per day of vehicle solution (reference: SP / Vehicle). Group 4 consists of 12 female rats whose ancestry has been subjected to prenatal stress, and they receive 200 µL per day of supplement (reference: SP / Supplement).

[0143] The effectiveness of the supplement was evaluated 6 weeks after birth.

[0144] The supplement (one dose) was administered by gavage once daily, 5 days a week. Administrations began after weaning, i.e., after postnatal day (PD) 25, and continued until PD 46.

[0145] The daily intake was 25.7 mg of supplement per kg of rat body weight.

[0146] The animals underwent behavioral tests between days JPN46 and JPN48, outside the treatment period with the vehicle or supplement. Therefore, the effects observed during the behavioral tests are attributable to a preventative treatment.

[0147] The behavioral tests are broken down into one anxiety assessment session and two object recognition sessions. The sessions are defined as follows: Session 1, JPN 46: Rats were individually placed in an open, square space (50 cm x 50 cm x 50 cm) made of blue plexiglass with a floor equipped with infrared LEDs. The rats were habituated to the test space during a 10-minute session, and their movements were captured by an infrared camera and analyzed using Ethovision® software (Noldus). Activity was analyzed based on the total distance traveled (m) and the percentage of time spent in the central 25 x 25 cm zone defined by the software. These data reflect the intensity of anxious behavior

[38] . Session 2, JPN 47: Two identical objects (50 mL plastic Eppendorf tubes) were placed in defined locations (on opposite edges of the central zone). Each rat was placed within the test space, and exploratory activity was recorded during a 10-minute session.Activity was analyzed in terms of the number of contacts with objects and the duration of those contacts. Session 3 JPN 48: The object from session 2 was replaced with a new object (a plastic bottle cap) whose shape, texture, and color differed from those of the familiar object. Each rat was placed back in the testing area, and exploratory activity was recorded during a 10-minute session. The activity was analyzed in a similar way to that described in session 2.

[0148] The preferential exploration index was calculated as the ratio of the number (or duration) of contacts with the object of session 2, to the total number (or duration) of contacts with both objects.

[0149] All values ​​are expressed as mean plus or minus the measurement standard deviation. Statistical analyses are performed separately for each compound using a one-way ANOVA (F-value), followed by Dunnett's post-hoc multiple comparison test.

[0150] The calculation of the human equivalent daily dose (HED) from the tested rat daily dose is defined by the FDA (Guidance, 2005) as follows: the human HED, expressed in mg / kg body weight, is equal to the animal HED, expressed in mg / kg body weight, multiplied by the ratio of the animal safety factor (Km Animal) to the human safety factor (Km Human). The Km Human is 37 and the Km Rat is 6.

[0151] The results are presented below. Locomotion in the center of the test space, day JPN46; effect of the complement on anxiety

[0152] The results are represented at the Figure 8 .

[0153] To the Figure 8 Effects of treatments on anxiety. N = 12; *** p < 0.0001 compared to the NS group / treated vehicle; #### p < 0.0001 compared to the SP group / treated vehicle; Dunnett's test

[0154] The SP / vehicle group, corresponding to individuals who have experienced prenatal stress and were treated preventively only with the vehicle, have a significantly higher percentage of movements within the peripheral zone of the open test space compared to the NS / Vehicle group (group not having experienced prenatal stress).

[0155] The SP / complement group, corresponding to individuals who had experienced prenatal stress and were treated preventively with complement, had a significantly lower percentage of displacements within the peripheral zone of the open test space compared to the SP / Vehicle group (a group that had experienced prenatal stress but was not treated with complement). Furthermore, the percentage of displacements in the SP / complement group was equivalent to that of the NS / Vehicle control group.

[0156] A higher rate of movement of individuals in the peripheral zone of the open test space than in the control modality demonstrates anxious behavior

[63] , via a protection mechanism based on identifying edges that limit the areas exposed and requiring monitoring.

[0157] Thus, prenatal stress (PS) induced a very significant anxiety behavior.

[0158] Surprisingly, it appears that the supplement very significantly and completely attenuated the anxious behavior induced by prenatal stress. Recognition test, day JPN47; effect of complement on recognition memory on object recognition

[0159] The results are represented at the Figure 9 .

[0160] During this session, the same object is presented twice to the individuals.

[0161] No statistical effect between groups was measured for this parameter.

[0162] Thus, individuals from all groups interacted equally when the identical objects were brought into contact, and their interactions, both in terms of frequency and duration, were equally distributed between the two objects (50%). Recognition test, day JPN48 (new object); effect of complement on recognition memory for the new object recognition test

[0163] The results are represented at the Figure 10 .

[0164] To the Figure 10: N = 12 ; *** p < 0.0001 compared to the NS group / treated vehicle ; ### p < 0.0001 compared to the SP group / treated vehicle ; Dunett test.

[0165] During this session, two different objects are presented once each to the individuals: one of the objects corresponds to the object presented during session 2 and the other object is a new object.

[0166] The SP / vehicle group, corresponding to individuals who experienced prenatal stress and were treated preventively only with the vehicle, had a significantly lower percentage of interactions, both in frequency and duration, with the newly presented object compared to the NS / vehicle group (the group that did not experience prenatal stress). This percentage was equal to that obtained for all groups in session 2. Thus, individuals in the SP / vehicle group had as many interactions with the old object as with the new one, and therefore, individuals in this group did not recognize the old object presented during session 2.

[0167] Conversely,The SP / Complement group, corresponding to individuals who experienced prenatal stress and were treated preventively with the complement, had a significantly higher percentage of interactions, both in frequency and duration, with the newly presented object compared to the PS / Vehicle group (negative control group). This percentage was also higher than that obtained in session 2 for all groups. Thus, individuals in the SP / Complement group had fewer interactions with the old object than with the new object, and therefore, individuals in this group recognized the old object presented during session 2. Furthermore, the percentage of interactions, both in frequency and duration, with the newly presented object in the SP / Complement group was equivalent to that of the NS / Vehicle and NS / Complement groups.

[0168] Thus, prenatal stress (PS) induced very significant recognition memory deficits in the case of the new object.

[0169] Surprisingly, it appears that the supplement made it possible to very significantly and completely mitigate the recognition memory deficits induced by prenatal stress.

[0170] In conclusion: Complement treatment has, in a very significant way, completely attenuated anxious behavior as well as recognition memory deficits induced by prenatal stress.

[0171] Prenatal stress as practiced in this experiment very significantly induces anxious behavior, and very significantly alters recognition memory in young female rats.

[0172] Thus, by applying the formula for calculating the equivalent daily dose in humans, a preventive treatment alleviating cognitive disorders caused by prenatal stress can be defined with a daily intake of 0.05 to 0.1 mg of supplement / kg of body weight. Example 4: Testing a natural extract of microalgae Phaeodactylum tricornutum within the framework of the model in vivo on mitigating deficits caused by age-related cognitive decline

[0173] The dietary supplement of the invention is prepared from an extract of Phaeodactylum tricornutum which includes in mg / g: Omega-3 fatty acids (ALA, SDA, EPA, DHA): 66.6 ± 11.5; Fucoxanthin: 20.0 ± 4.0; Sterols: 3.0 ± 0.6; Phycoprostane: 0.0025 ± 0.0005

[0174] The supplement is obtained by adding coconut oil at a rate of 410 mg ± 20 mg / g to said extract.

[0175] The supplement is incorporated into kibble according to 4 different formulations such that the quantities of supplement incorporated within the different batches of kibble correspond to human equivalent daily doses as described in Table 2, by diluting the composition described below in coconut oil to the same final mass between the formulations.

[0176] The calculation of the human equivalent daily dose (HED) from the tested mouse daily dose is defined by the FDA (Guidance, 2005) as follows: the human HED, expressed in mg / kg body weight, is equal to the animal HED, expressed in mg / kg body weight, multiplied by the ratio of the animal safety factor (Km Animal) to the human safety factor (Km Human). The Km Human is 37 and the Km Mouse is 3.

[0177] An additional batch of kibble is formulated solely with coconut oil, such that the vehicle concentration is equivalent to that of the other batches, i.e. 0.01% (w:w).

[0178] The five batches of kibble thus obtained are referenced as described in Table 2 below. [Table 2] Formulated kibble Supplement dose in human equivalent (mg of supplement / kg of body mass / day) Reference Coconut oil (vehicle) 0 Veh. Complement 1,7 D1 Complement 3,3 D2 Complement 4,2 D3 Complement 5,3 D4

[0179] The model in vivo The model considered is the D-Galactose model applied to mice, which is suitable for studying age-related cognitive decline. This model effectively mimics many behavioral and molecular characteristics of brain aging in rodent models.

[0180] D-Galactose is administered subcutaneously at a dose of 150 mg / kg fresh weight of mouse per day, and the above dietary supplement is incorporated into a pellet, according to the following scheme: Between day -28 and day 51, the supplement is administered by incorporation into food pellets; Between day 01 and day 51, D-Galactose is administered subcutaneously, five days a week; Between days 43 and 51, three different behavioral tests are used to monitor the effects of the test compounds.

[0181] The effectiveness of the complement is evaluated according to the following parameters: improvement of learning deficits (spatial working memory: spontaneous alternation in the Y maze according to the Y-maze test; spatial memory by the so-called "Morris Water Maze" test and long-term contextual memory in the passive avoidance test), lipid peroxidation (LPO) levels in the hippocampus and the effect on the neuro-inflammation markers IL6 and TNFα. Improvement of learning deficits

[0182] On day 43, all animals were tested for spontaneous alternation performance in the Y-maze (YM) test, via a spatial working memory index; From day 44 to day 49, all animals were tested for spatial memory in the Morris Water Maze (MWM) test, via a spatial memory index; From day 44 to day 49, all animals were tested via the MWM test to assess spatial working memory; On days 50 and 51, the animals' long-term contextual memory was assessed using the step-by-step passive avoidance procedure (STPA), via training and retention sessions, respectively; Lipid peroxidation (LPO) levels in the hippocampus and their effect on the neuro-inflammation markers IL6 and TNFα

[0183] On the 51st day, after behavioral tests, the animals are euthanized.

[0184] For all animals, trunk blood is collected and centrifuged to recover the plasma, and the brain is rapidly removed. The hippocampus and cortex are dissected; the hippocampus is then used to determine lipid peroxidation levels by colorimetric method; the hemifrontal cortex and plasma are used to determine the levels of the inflammatory biomarkers interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α).

[0185] Lipid peroxidation levels (LPO) were quantified according to the modified and adapted procedure of Hermes-Lima et al. This method measures the capacity of peroxidized brain lipids to oxidize an orange ferrous oxide and xylenol complex, as demonstrated in the presence of cumer hydroperoxide (CHP). The lipid peroxidation level is determined in CHP equivalents according to: CHPE = A5801 / A5802 x [CHP (nmol)] and expressed as CHP equivalents per wet tissue weight and as a percentage compared to the data obtained for the control group (D-Galactose + vehicle).

[0186] IL6 and TNFα levels are quantified using ELISA tests with the following kits: For IL-6 quantification: ThermoScientific, EM2IL6. For TNFα quantification: ThermoScientific, EMTNFA.

[0187] For all assays, the cortex is homogenized after thawing in 50 mM Tris-150 mM NaCl buffer, pH 7.5, and sonicated for 20 s. After centrifugation (16,100 g for 15 min, 4 °C), a supernatant or plasma is used for ELISA assays according to the ELISA manufacturer's instructions. For each assay, the absorbance is read at 450 nm and the sample concentration is calculated using the standard curve. Results are expressed in pg of marker per mg of fresh tissue.

[0188] All values, except for passive avoidance latencies, are expressed as mean plus or minus the measurement standard deviation. Statistical analyses were performed separately for each compound using a one-way ANOVA (F-value), followed by Dunnett's post-hoc multiple comparison test. Passive avoidance latencies do not follow a Gaussian distribution, since the upper limit times are fixed. They were therefore analyzed using a non-parametric Kruskal-Wallis ANOVA (H-value), followed by Dunn's multiple comparison test. Values ​​with p < 0.05 were considered statistically significant.

[0189] The tests are performed on 72 male mice, divided into 6 groups of 12 mice, of which group 1 is the negative control group and group 2-6 is the positive control group: Group 1 is the group to which a subcutaneous saline solution is administered instead of D-Galactose and B1 kibble; Group 2 is the group to which D-Galactose and B1 kibble are administered; Group 3 is the group to which D-Galactose and B2 kibble are administered; Group 4 is the group to which D-Galactose and B3 kibble are administered; and Group 5 is the group to which D-Galactose and B4 kibble are administered; and Group 6 is the group to which D-Galactose and B5 kibble are administered. Effects on spatial memory in the spontaneous alternation of the Y-maze test:

[0190] The results are represented at the figure 11 , the first diagram (on the left) illustrating the effects of the complement of the invention on deficits of spontaneous alternations and the second diagram (on the right) illustrating the effects of the complement of the invention on locomotor activity.

[0191] On the figure 11: Saline solution / veh corresponds to the negative control (untreated group fed with kibble formulated with the vehicle, coconut oil); DGal 150 / Veh corresponds to the positive control (D-galactose treated group fed with kibble formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is between 11 and 12 depending on the group; * p < 0.05, *** p < 0.0001 vs. the saline solution / Veh group, # p < 0.05, ## p < 0.01, ### p < 0.0001 vs. the D-GAL 150 group / Veh group; Dunnett's test.

[0192] It was observed that treatment with D-Galactose significantly impaired spatial working memory, compared to mice treated with saline solution.

[0193] Complement D1 significantly but partially attenuated the deficits induced by chronic D-galactose poisoning. Complements D2, D3, and D4 significantly and completely attenuated the deficits induced by chronic D-galactose poisoning. Effects on learning deficits induced by D-Gal according to the MWM test:

[0194] The results are represented at the figure 12 .

[0195] On the Figure 12: Saline solution / veh corresponds to the negative control (untreated group fed with kibble formulated with the vehicle, coconut oil); DGal 150 / Veh corresponds to the positive control (group treated with D-galactose and fed with kibble formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is between 11 and 12 depending on the group; * p < 0.05, ** p < 0.01, *** p < 0.0001 vs. saline solution / Veh group, ## p < 0.01, ### p < 0.0001 vs. D-GAL 150 group / Veh group; Bonferroni multiple comparison test after two-way ANOVA.

[0196] Chronic D-Galactose intoxication severely impaired spatial learning, compared to the negative control group (saline solution / vehicle).

[0197] Complement D1 very significantly but partially attenuated the deficits induced by chronic D-Galactose intoxication.

[0198] Supplements D2, D3 and D4 very significantly and completely attenuated the deficiencies induced by chronic D-Galactose intoxication. Effects of complement on D-Galactose-induced learning deficits

[0199] The results are represented at the figure 13 .

[0200] On the Figure 13: Saline solution / veh corresponds to the negative control (group not treated with D-galactose and fed with kibble formulated with the vehicle, coconut oil); DGal 150 / Veh corresponds to the positive control (group treated with D-galactose and fed with kibble formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is between 11 and 12 depending on the group; Saline solution / veh corresponds to the negative control (group not treated with D-galactose and fed with kibble formulated with the vehicle, coconut oil); DGal 150 / Veh corresponds to the positive control (group treated with D-galactose and fed with kibble formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is between 11 and 12 depending on the group; *** p < 0.0001 vs. saline / Veh group, / Veh; ### p < 0.0001 vs.group D-GAL 150 / Veh; Bonferroni multiple comparison test after two-way ANOVA. "T", time spent in the target quadrant; "O", average time spent in the other three quadrants.

[0201] Chronic D-Galactose intoxication severely impaired spatial learning, compared to the negative control group (saline solution / vehicle).

[0202] D1 and D2 supplements very significantly but partially attenuated the deficits induced by chronic D-Galactose intoxication.

[0203] Supplements D3 and D4 have very significantly and completely alleviated the deficiencies induced by chronic D-Galactose intoxication. Effects on D-Galactose-induced passive avoidance deficits in mice

[0204] The results are represented at the figure 14, with the effects of the complement of the invention on the stepping latency illustrated on the diagram on the left and on the escape latency illustrated on the diagram on the right, measured during the retention period.

[0205] On the figure 14 : Saline solution / veh corresponds to the negative control (group not treated with D-galactose and fed with kibble formulated with the vehicle, coconut oil); DGal 150 / Veh corresponds to the positive control (group treated with D-galactose and fed with kibble formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is between 11 and 12 depending on the group; *** p < 0.0001 vs. saline solution / Veh group, ### p < 0.0001 vs. D-GAL 150 group / Veh group; Dunnett's test.

[0206] Chronic D-Galactose intoxication severely impaired long-term contextual working memory, compared to the negative control group (saline / vehicle).

[0207] The D1 complement showed no effect on long-term contextual memory.

[0208] Supplements D2, D3 and D4 very significantly and completely attenuated the deficiencies induced by chronic D-Galactose intoxication. Effects of complement on D-Galactose-induced lipid peroxidation

[0209] The results are shown at the figure 15 .

[0210] On the figure 15: Saline solution / veh corresponds to the negative control (untreated group fed with kibble formulated with the vehicle, coconut oil); DGal 150 / Veh corresponds to the positive control (D-Galactose treated group fed with kibble formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is between 11 and 12 depending on the group; ** p < 0.01, *** p < 0.0001 vs. the saline solution / Veh group, ## p < 0.01, ### p < 0.0001 vs. the D-GAL 150 group / Veh group; Dunnett's test.

[0211] Chronic D-Galactose intoxication greatly increased oxidative stress compared to the negative control group (saline / vehicle).

[0212] Complement D1 significantly but partially reduced oxidative stress induced by chronic D-Galactose toxicity.

[0213] Supplements D2, D3 and D4 very significantly and completely reduced oxidative stress induced by chronic D-Galactose toxicity. Effects of complement on D-Galactose-induced TNF-α expression in cortex and plasma

[0214] The results are represented at the Figure 16 , with the effect on the cortex shown in the left diagram and the effect on the plasma shown in the right diagram.

[0215] On the Figure 16 : Saline solution / veh corresponds to the negative control (group not treated with D-galactose and fed with kibble formulated with the vehicle, coconut oil); DGal 150 / Veh corresponds to the positive control (group treated with D-galactose and fed with kibble formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is between 11 and 12 depending on the group; *** p < 0.0001 vs. the saline solution / Veh group, ### p < 0.0001 vs. the D-GAL 150 group / Veh group; Dunnett's test.

[0216] Chronic D-Galactose intoxication significantly increased TNF-α in the cortex and plasma, compared to the negative control group (saline / vehicle).

[0217] Complement D1 significantly but partially decreased the increase in TNF-α induced by chronic D-Gal intoxication in the brain and plasma.

[0218] Complement D2 significantly and completely reduced the increase in TNF-α induced by chronic D-Gal intoxication in the brain, but only partially in the plasma.

[0219] Complement D3 significantly but partially decreased the increase in TNF-α induced by chronic D-Gal intoxication in the brain, and completely in the plasma.

[0220] Complement D4 significantly and completely reduced the increase in TNF-α induced by chronic D-Gal intoxication in the brain and plasma. Effects of complement on D-Galactose-induced IL-6 expression in the cortex and plasma

[0221] The results are represented at the Figure 17 , with the effect in the cortex on the left diagram and in the plasma on the right diagram.

[0222] On the Figure 17 : Saline solution / veh corresponds to the negative control (group not treated with D-galactose and fed with kibble formulated with the vehicle, coconut oil); DGal 150 / Veh corresponds to the positive control (group treated with D-galactose and fed with kibble formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is between 11 and 12 depending on the group; *** p < 0.0001 vs. the saline solution / Veh group, ### p < 0.0001 vs. the D-GAL 150 group / Veh group; Dunnett's test.

[0223] Chronic D-Galactose intoxication significantly increased IL-6 in the cortex and plasma, compared to the negative control group (saline / vehicle).

[0224] Complements D1 and D2 significantly but partially reduced the increase in IL-6 induced by chronic D-Gal intoxication in the brain and plasma.

[0225] Supplements D3 and D4 significantly and completely reduced the increase in IL-6 induced by chronic D-Gal intoxication in the brain and plasma. In conclusion :

[0226] Chronic D-galactose intoxication significantly impairs spatial working memory, long-term contextual memory, and spatial learning. These behavioral alterations are also linked to biochemical changes, including increased oxidative stress and the induction of neuroinflammatory processes.

[0227] Preventive treatment with the supplement of the invention is dose-dependent and, in the case of the highest dose tested (complement D4), significantly and completely attenuated the deficits induced by chronic D-Galactose poisoning in terms of behavioral alterations, increased oxidative stress, and activation of neuroinflammatory processes. Furthermore, in the case of the lower and intermediate doses (complements D2 and D3), the supplement of the invention significantly and completely attenuated the deficits induced by chronic D-Galactose poisoning in terms of spatial working memory, increased oxidative stress, and activation of neuroinflammatory processes.

[0228] Thus, by applying the formula for calculating equivalent daily dose in humans, a preventive treatment for age-related cognitive decline can be defined with a daily intake of 1.7 to 5.3 mg of supplement / kg of body weight.

Claims

1. Composition comprising 50 to 250 mg / g of one or several omega-3 fatty acids, 10 to 50 mg / g of one or several xanthophylls, 1 to 20 mg / g of one or several sterols, 2 to 100 µg / g of one or several phycoprostanes, and at least one oil selected from medium-chain triglycerides (MCTs).

2. Composition according to claim 1, characterized in that it comprises 50 to 200 mg / g of one or several omega-3 fatty acids, 10 to 30 mg / g of one or several xanthophylls, 1 to 8 mg / g of one or several sterols, and 2 to 50 µg / g of one or several phycoprostanes.

3. Composition according to claim 1 or 2, characterized in that it comprises 50 to 170 mg / g of one or several omega-3 fatty acids, 10 to 25 mg / g of one or several xanthophylls, 1 to 6 mg / g of one or several sterols, and 2 to 40 µg / g of one or several phycoprostanes.

4. Composition according to any one of claims 1 to 3, characterized in that the or the at least one of the omega-3 fatty acids is selected from stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and mixtures thereof.

5. Composition according to any one of claims 1 to 4, characterized in that the or the at least one of the xanthophylls is fucoxanthin.

6. Composition according to any one of claims 1 to 5, characterized in that the or the at least one of the sterols is selected from phytosterols.

7. Composition according to any one of claims 1 to 6, characterized in that the or the at least one of the phycoprostanes is selected from phytoprostanes, isoprostanes, and neuroprostanes.

8. Composition according to any one of claims 1 to 7, characterized in that it further comprises at least one additive selected from preservative agents, colorants, flavors, disintegration agents, lubricant agents, coating or encapsulation agents,9. Composition according to any one of claims 1 to 8, characterized in that it is in the form of gel capsules, capsules, tablets, pastilles, or loose powder.

10. Composition according to any one of claims 1 to 9, characterized in that it is packaged in doses having a unit weight comprised between 10 mg and 1 g.

11. Composition according to any one of claims 1 to 10, characterized in that the medium-chain triglycerides (MCTs) are selected from coconut oil and palm oil.

12. Use of the composition according to any one of claims 1 to 11 as a food supplement.

13. Use of a microalga selected from any of the taxa Pinguiophyceae, Chrysophyceae, Bacillariophyceae, Mamiellophyceae, Prymnesiophyceae, Haptophyceae, Coccolithophyceae, Isochrysidaceae, and Phaeodactylaceae, for preparing a composition according to any one of claims 1 to 11.

14. Use according to claim 13, characterized in that said microalgae is Tisochrysis lutea or Phaeodactylum tricornutum.

15. Use of a composition according to any one of claims 1 to 11, or according to claim 12, to prevent the onset of age-related cognitive disorders, defined as a non-pathological decrease of the cognitive functions.

16. Use of a composition according to claim 15, to prevent the onset of age-related cognitive disorders, defined as a non-pathological decrease of the cognitive functions, characterized in that the daily intake is comprised between 2 to 5 mg of composition / kg of body weight.

17. Composition according to any one of claims 1 to 11, or according to claim 12, for use in preventing cognitive disorders in children or young adults having been subjected to a prenatal stress inducing hyperactivity, attention and memory deficits, language retardation, and anxious behavior.

18. Composition for use according to claim 17, characterized in that the daily intake is comprised between 0,05 to 0,1 mg of composition / kg of body weight.

Images