Food supplement

Abstract

The present 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 its use, particularly as a food supplement, for preventing the occurrence of cognitive impairment.

Description

Technical Field

[0001] The present invention relates to a composition, a food supplement based on fatty acids and xanthophylls, and its application, and in particular to preventing the occurrence of cognitive impairment in humans or animals thereby.

Background Art

[0002] The cognitive process is defined as a brain function that enables the acquisition, processing, storage, and utilization of data derived from the environment in order to maximize the advantages and minimize the disadvantages of external constraints. Thus, the cognitive process occurs during phases including inference (resulting in planning, organization, judgment), perception, recognition, language, emotion, memory, and learning.

[0003] Mild cognitive impairment or cognitive fragilities are defined as changes in cognitive function without dementia. From a clinical perspective, such impairments have a score of 0.5 in the assessment by the CDR (Cognitive Drug Research Computerized Assessment System).

[0004] Among such cognitive impairments, cognitive decline associated with aging and changes in cognitive function caused by prenatal stress are two phenomena that may be related throughout an individual's life.

[0005] Age-related cognitive decline is defined as a non-pathological decline in cognitive functions such as the speed of information processing, attention ability, and especially so-called working (or short-term) memory. These processes are due to normal physiological changes directly related to age. Although there is still room for discussion about the age at which this decline begins, considering the accelerating aging of the world population, with more than 20% of the world's population being over 60 years old and this proportion exceeding 30% by 2050, age-related cognitive decline is among the major problems in the coming decades and is considered to have a significant impact on the economy (lower autonomy in the elderly) and public policies globally, especially in developed countries.

[0006] On the opposite side of the age composition pyramid, cognitive impairment can affect infants following prenatal stress. In fact, for several years now, in humans and animals, the impact of stress during specific periods of pregnancy on fetal cognitive development has been studied. That is, in animals, mainly rats, prenatal stress on the mother has been shown to induce offspring with altered long-term memory.

[0007] Strong negative stimuli, stress, can induce non-pathological changes or decline in the cognitive functions of infants, which manifest as behavioral changes such as hyperactivity, attention and memory disorders, language delay, a more difficult temperament, and more generally anxious behavior, indicating a delay in neurodevelopment and a decline in cognitive ability.

[0008] One hypothesized mechanism by which prenatal stress manifests as cognitive impairment is that the fetus is exposed to large amounts of so-called stress hormones, such as cortisol, which belongs to the family of corticosteroids. Cortisol passes through the placental barrier and, starting from a certain concentration, saturates the fetal protective mechanism against corticosteroids secreted by the mother, thereby exposing the fetus to large amounts of cortisol, which is thought to have a negative impact on the development of cognitive functions. Other complementary hypotheses explaining the relationship between prenatal stress and the child's cognitive impairment have also been developed.

[0009] The concept of stress is defined according to different perspectives, such as a biological approach. In this case, stress is a series of metabolic reactions in an organ that occur following one or more external factors that cause physiological or psychological changes (fear, pain). However, the concept and impact of stress are highly individualized, and an individual's reaction to stress is also defined from a psychological perspective. Therefore, whether an event was stressful due to an individual's unique reaction or objectively stressful can only be said in hindsight, and it should be borne in mind that it is difficult to address the causes of prenatal stress. In addition to this, there is the fact that pregnancy induces hormonal and psychological changes, increasing the future mother's susceptibility to any event that may affect the health of the fetus.

[0010] A strict treatment approach to stress and anxiety, if it takes the form of prescribing drugs, is dangerous in the case of pregnant women. Many psychotropic drugs for treating psychological disorders or anxiety have teratogenic effects that are directly harmful to the fetus. This requires a case-by-case evaluation, and this approach is only used in the case of clinical psychological disorders in pregnant women and not in cases of so-called perceived stress.

[0011] Therefore, there are major challenges in finding solutions for cognitive impairments in children or young adults caused by prenatal stress.

[0012] Various studies have shown that supplementing nutrition with so-called essential fatty acids, as well as carotenoids, particularly xanthophyll, and furthermore combinations of said fatty acids and said carotenoids, is associated with preventing or at least limiting a decline in cognitive function. Food supplements or pharmaceuticals have been developed and positive results have been obtained.

[0013] Thus, according to document WO2013 / 032333A1, compositions based on omega-3 fatty acids, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), astaxanthin and glycerophospholipids are known, which are recommended for the prevention or treatment of various disorders, in particular cognitive disorders. SUMMARY OF THE INVENTION PROBLEM TO BE SOLVED BY THE INVENTION

[0014] Said components are present in this composition in the form of extracts of microalgae. In the preparation method of a preferred variant, they are prepared from two extracts from two different algae. The fact that the components of this composition are of natural origin is very beneficial. Nevertheless, there is still a need for a more effective composition, especially with respect to said problems. Furthermore, it is important to provide simple and reproducible means for preparing such a composition. MEANS FOR SOLVING THE PROBLEM

[0015] The present invention provides a solution of a composition comprising one or more omega-3 fatty acids, one or more xanthophylls, one or more substances of the sterol family, and one or more phycoprostanes. The combination of at least one sterol, at least one phycoprostane, at least one omega-3 fatty acid, and at least one xanthophyll has been found to significantly increase the effectiveness of the composition in preventing the onset of age-related cognitive impairment and also in preventing the onset of cognitive impairment associated with prenatal stress.

[0016] The composition of the present invention comprises 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.

[0017] In a major application, the composition of the present invention can be used as a food supplement. Further, the present invention relates to a food supplement 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 fucoprostanes.

[0018] The present invention has the major advantage that all of the above-mentioned constituents or contents are obtained from natural sources, in particular, they are obtained from one or more microalgae, preferably from a single microalgae. Of course, the constituents or contents of the composition or food supplement of the present invention may have a non-natural origin and be provided in the form of a chemically synthesized product.

[0019] Before disclosing the present invention in more detail, some terms used herein are defined.

[0020] The term "comprising" in the expressions "the composition comprises... " or "the food supplement comprises... " means that the composition or supplement can incorporate any additional constituents not specifically mentioned, in any form and from any origin. Also, the case where the composition or supplement contains only the listed constituents and, as a result, consists only of the described constituents is within the scope of this term.

[0021] A food supplement is defined as one or more foods that are intended to complement the normal diet of humans or animals and constitute a concentrated source of nutrients or other substances having nutritional or physiological effects, alone or in combination. It is generally available in per-serving forms, namely, gel capsules, lozenges, tablets, pills and other similar forms, as well as powder packs, liquid ampoules, vials with droppers, and other similar liquid or powder formulations intended to be taken in units measured in small amounts.

[0022] Omega-3 fatty acids are a family of unsaturated fatty acids, whose hydrocarbon chains have 4 to 36 carbon atoms, generally 14 to 36 carbon atoms, and whose single double bond or the first double bond is on the third carbon-carbon bond counted from the terminal methyl group of the chain. The unsaturation may be either cis- or trans-type, independently of each other. The most representative acids are alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), but the name "Omega-3 fatty acids" is not limited to these. Furthermore, especially when the fatty acids are of natural origin, they may be extracted from algae and may be in the form of free molecules, but may also be in an esterified form, such as a mono-, di- or tri-esterified ester-type derivative, or a mixture of these forms.

[0023] As xanthophylls, one or more oxygen atoms are defined in molecules belonging to carotenoids, such as astaxanthin, canthaxanthin, baucarotene xanthin, lutein, zeaxanthin, diadinoxanthin, neoxanthin, loroxanthin, siphonaxanthin, diatoxanthin, violaxanthin, dinoxanthin, flavoxanthin, alpha-cryptoxanthin, beta-cryptoxanthin and fucoxanthin.

[0024] In particular, when xanthophylls are of natural origin, they may be extracted from algae and may be in the form of free molecules, but may also be derivatives esterified in the form of single or multiple esters, or a mixture of these forms.

[0025] Sterols are a well-known family of lipids with a sterane nucleus having a hydroxyl group at the third carbon, and the hydroxyl group may be modified, for example, by an acetyl group. These include natural sterols or phytosterols, which are classified herein under the term fucosterols. By way of non-limiting example, phytosterols include 24-methylenecholesterol, β-sitosterol, fucosterol, isofucosterol, sitosterol, oxycholesterol acetate, clionosterol, and more particularly brassicasterol, stigmasterol, and campesterol.

[0026] Ficoprostanes are a family of lipids that are structurally of the prostaglandin type and are understood to be the result of the indirect enzymatic oxidation of fatty acids that are naturally present in microalgal biomass. In particular, these compounds are selected from phytoprostanes, isoprostanes, and neuroprostanes depending on the oxidized fatty acid. 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 mainly derived from ALA and are selected from 9-epi-9F1t-PhytoP, ent-16-epi-16-F1t-PhytoP, 9-F1t-PhytoP, ent-16B1t-PhytoP, ent-9L1t-PhytoP, 16(RS)-16-A1t-PhytoP. Isoprostanes are mainly derived from ARA and EPA and can be selected from 15-E2t-IsoP, 15-F2t-IsoP, 15-epi-15-F2t-IsoP, 5-F2t-IsoP, 8(RS)-8-F3t-IsoP. Neuroprostanes are mainly 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.

[0027] Medium-chain triglycerides (MCTs) should be understood as esters of glycerol having hydrocarbon chains with 6 to 12 carbon atoms and saturated fatty acids. These are naturally present in coconut palm oils such as coconut oil, palm kernel oil, and palm oil, but they can also be obtained from other greases or oils.

[0028] Hereinafter, the present invention will be described in more detail, and its modifications will be disclosed.

[0029] Advantageously, the composition or food supplement of the present invention discloses the following features, taking them alone or in any combination into consideration.

[0030] It contains one or more omega-3 fatty acids at 50 to 250 mg / g, one or more xanthophylls at 10 to 50 mg / g, one or more sterols at 1 to 20 mg / g, and one or more fucoprostanes at 2 to 100 μg / g.

[0031] It contains one or more omega-3 fatty acids at 50 to 200 mg / g, one or more xanthophylls at 10 to 30 mg / g, one or more sterols at 1 to 8 mg / g, and one or more fucoprostanes at 2 to 50 μg / g.

[0032] It contains one or more omega-3 fatty acids at 50 to 170 mg / g, one or more xanthophylls at 10 to 25 mg / g, one or more sterols at 1 to 6 mg / g, and one or more fucoprostanes at 2 to 40 μg / g.

[0033] The composition or food supplement of the present invention advantageously further contains at least one oil as a vehicle or support for promoting the expression of the active ingredient. Surprisingly, it has been observed that when this oil is selected from medium-chain triglycerides (MCTs), the production of the composition or food supplement is promoted. In particular, when the active ingredient is obtained from the same microalgae extract, optimal homogenization is observed in such oils. According to one variant, the medium-chain triglycerides (MCTs) are of natural origin and are provided by oils selected from coconut palm oil, palm kernel oil and palm oil. They may also be obtained from or derived from such oils.

[0034] The following discloses preferred formulations of the composition or food supplement of the present invention, although these may be combined, of course.

[0035] One or at least one omega-3 fatty acid is selected from stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and mixtures thereof.

[0036] One or at least one xanthophyll is fucoxanthin.

[0037] One or at least one sterol is selected from phytosterols.

[0038] One or at least one fycoprostane is selected from fycoprostane, isoprostane and neuroprostane.

[0039] The composition or food supplement of the present invention may in particular contain any additives that make it possible to improve their preservation, appearance, taste and their formulation. Thus, one or more selected from preservatives, colorants, flavorings, disintegrants, lubricants, coating agents or encapsulating agents may be incorporated.

[0040] The main use of the composition of the present invention is as a food supplement. Thus, the composition or food supplement as defined above is preferably in the form of a gel capsule, capsule, troche or loose powder. Preferably, it is packaged in a dosage having a unit weight between 1 mg and 1 g. Generally, the galenic formulation of the composition or supplement is prepared for an individual taking into account the application and depends in particular on whether it is intended for children or adults.

[0041] The composition or food supplement of the present invention can be used to prevent the occurrence of non-pathological age-related cognitive impairment or non-pathological cognitive impairment in children or young adults exposed to prenatal stress. For the prevention of age-related cognitive impairment, the daily intake can be between 2 and 5 mg per kg of body weight. For the prevention of cognitive impairment in children or young adults exposed to prenatal stress, the daily intake may be between 0.05 and 0.1 mg per kg of body weight.

[0042] Even when the daily intake is very small, it has been confirmed that there is an effect if the treatment period is extended accordingly.

[0043] The present invention relates to the use of microalgae for preparing a food supplement as defined above. The one or more desired microalgae may be selected from any of the following taxons, namely, Pinguiophyceae, Chrysophyceae, Bacillariophyceae, Mamiellophyceae, Prymnesiophyceae, Haptophyceae, Coccolithophyceae, Isochrysidaceae and Phaeodactylaceae. Desirably, the microalgae are Tisochrysis lutea or Phaeodactylum tricornutum. Such microalgae are selected because an extract that meets the definition of the composition of the present invention can be obtained by appropriate extraction. For example, such an extract contains the following fatty acid fractions. That is, the fatty acids, expressed as weight percentages relative to the total extract, are 4 to 55% in the form of free fatty acids, 0.5 to 10% in the form of monoacylglycerol, 0.4 to 15% in the form of diacylglycerol, and 2 to 55% in the form of triacylglycerol. These fatty acids are such that the omega-3 series fatty acids are in the range of 5 to 20% (m / m), and the omega-6 series fatty acids are in the range of 0.5 to 5%. More specifically, the fatty acids are 0.5 to 10% ALA (α-linolenic acid), 0.5 to 10% SDA (stearidonic acid), 0.05 to 20% EPA (eicosapentaenoic acid), and 0.1 to 10% DHA (docosahexaenoic acid).

[0044] As shown above, one interesting aspect of the composition or food supplement is its preparation method, particularly the natural origin of its components, and all of them are obtained from a single microalgae. Depending on the microalgae used, the formulation of the composition is directly obtained by the extract. Otherwise, the extract is diluted to obtain the required concentration according to the present invention. However, the present invention is not limited to this implementation. Therefore, it is also possible to consider that only a part of the components is of natural origin and the others are obtained by chemical synthesis, and / or the components of natural origin are not obtained from the same source, for example, not obtained from the same algae.

[0045] The measurement and adjustment of the concentration of the active ingredient in the extract and in the composition or the obtained food supplement are carried out using analytical techniques within the scope of the general knowledge of those skilled in the art.

[0046] The method for producing the composition or food supplement from the microalgae culture is described in more detail below.

[0047] According to a variant of the present invention, the organisms are microalgae belonging to the following taxonomic groups, namely, Pinguiophyceae, Chrysophyceae, Bacillariophyceae, Mamiellophyceae, Prymnesiophyceae, Haptophyceae, Coccolithophyceae, Isochrysidaceae, Phaeodactylaceae, etc. These photosynthetic microorganisms are strictly autotrophic, mixotrophic, or temporarily heterotrophic.

[0048] An extract refers to a fraction of biomass derived from photosynthetic organisms obtained by a method that enables the obtaining of the composition of the present invention, whether directly or indirectly. These extracts have a composition of 5 - 30% protein, 20 - 80% lipid, 0.1 - 2% sterol, and 0.1 - 20% chlorophyll, expressed as a weight percentage relative to the total extract.

[0049] More specifically, the lipophilic portion constituting the extract is composed of 15 - 45% saturated fatty acids, 5 - 20% polyunsaturated acids, 1 - 20% xanthophyll, and 0.0002 - 0.007% fucoprostane, expressed as a weight percentage relative to the total extract.

[0050] For the production of the extract according to the present invention, the cells preferably consist of microalgae cells of Isochrysis galbana, a species of the order Isochrysidales, or microalgae cells of Phaeodactylum tricornutum, a species of the genus Phaeodactylum produced by carbon autotrophy.

[0051] (Method for producing microalgae) Microalgae are ideally cultured in a controlled manner within a suitable system such as a raceway, open pond, or preferably within a closed system such as a photobioreactor. The photobioreactor used may be of an existing type. For example, it may be a horizontal tubular photobioreactor, a vertical type, so-called "green wall panel" system, a planar or columnar photobioreactor, etc. Desirably, the production of biomass is carried out in a closed cultivation system by autotrophy with zero impact on arable land.

[0052] The production of biomass is carried out according to a cultivation management method of batch, fed-batch, continuous, semi-continuous, turbidostat, or chemostat type.

[0053] (Obtaining these microalgae extracts) The extracts derived from these microorganisms are preferably obtained in a drying step by freeze-drying, vacuum drying, drum drying, spraying or any other process capable of reducing the water content in the biomass, regardless of whether they are combined or not, after concentration using chemical or physical processes such as centrifugation, filtration, aggregation, sedimentation, etc. to remove all or part of the water from the biomass.

[0054] Complementary to these processes, a cell lysis process may be carried out. For example, it is any other process capable of applying pressure, electric current, shear force, using enzymes, or destroying tissues, organs, cells or organelles.

[0055] The interesting compounds of the biomass are extracted by a solid-liquid extraction type process. This may utilize supercritical fluids or subcritical fluids, be carried out in parallel or continuously, and may include co-processing with microwaves, ultrasound, pressure, enzymes, etc. The solvent used may be pure or a mixture, and may consist of acetone, hexane, ethyl acetate, methyltetrahydrofuran, heptane, methanol, natural or branched oils, ethanol, or any other solvent capable of extracting all or part of the hydrophobic and amphiphilic compounds.

[0056] The solvent or mixture of solvents is separated from the residual biomass after extraction by a centrifugation, filtration type process and may then be concentrated. Also, the solvent may be removed by vacuum evaporation or any other technique enabling selective evaporation of the solvent under consideration. The extract thus obtained contains amphiphilic molecules while having lipophilic properties.

[0057] (Formulation as a food supplement) The preparation of the extract is carried out using a compatible base in which the extract can be dissolved in order to obtain a homogeneous solution with a desired concentration. For example, vegetable oils such as olive oil, corza oil, linseed oil, sunflower oil, grape seed oil, palm oil, and preferably MCT oil are used. It is composed of a 70% by weight mixture selected from caprylic acid and capric acid, preferably coconut oil or palm oil, and is entirely supplemented with molecules capable of improving stability such as synthetic or natural antioxidants. The compounding weight of the base / additive for obtaining the supplement can reach 95% by weight based on the weight of the food supplement. Generally, it is between 15% and 80%, preferably between 35% and 45%.

[0058] The extract, preferably the formulated composition or the obtained supplement, may be formulated in the form of soft capsules or may be formulated in powder form. For this, any technique enabling microencapsulation of an aqueous solution may or may not be used, and auxiliary agents or bases enabling the dispersion of a homogeneous extract in a polar solution that can be drunk may or may not be used.

[0059] The extract or supplement can be used alone or as a component of a food supplement.

[0060] With reference to the following drawings, different objects of the present invention are described below, and its advantages are described in the following examples.

Brief Description of the Drawings

[0061]

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Mode for Carrying Out the Invention

[0062] (Example 1: Preparation of an extract containing the components of the composition of the present invention) The extract is obtained from Phaeodactylum tricornutum by any of the techniques described above.

[0063] This is insoluble in water and highly viscous, thus hindering any operation at room temperature.

[0064] Before preparation, the extract and palm oil were kept at room temperature (25 ± 1 °C) for 24 hours.

[0065] The extract was transferred to a centrifuge tube containing oil, and the final net mass of the mixture was about 5 g, and the mass ratio of the extract to the net mass of the mixture was 25%. The mixture was stirred for 1 unit using a so-called vortex mixer. Stirring was repeated 3 times for each mixture. A uniform mixture was obtained.

[0066] (Example 2: Test of the natural extract of the microalgae Tisochrysis lutea in an in vivo model for the attenuation of deficits induced by age-related cognitive decline) The food supplement of the present invention is prepared from a Tisochrysis lutea extract, which contains the following in mg / g units: Omega-3 fatty acids (ALA, SDA, EPA, DHA): 152.6 ± 14.4; Fucoxanthin: 20.0 ± 4.0; Sterol: 4.9 ± 0.8; Ficoprostane: 0.035 ± 0.007.

[0067] The supplement is obtained by adding coconut oil to the extract at a rate of 360 ± 10 mg / g.

[0068] This supplement is incorporated into kibble according to three different formulations, and the final DHA concentration in the batch of kibble is set to 0.5, 1.5 and 3.0% (m:m).

[0069] A commercially available oily extract of microalgae, containing only 77% (m:m) DHA and 3% (m:m) EPA as the fat fraction, was also tested. For this as well, it was incorporated into kibble such that the final DHA concentration in the batch of kibble was 3.1% (m:m).

[0070] An additional batch of kibble was formulated to contain only coconut oil, with the vehicle concentration made equivalent to that of the other batches, i.e., 0.01% (m:m).

[0071] Five batches of kibble were thus obtained and are referred to as shown in Table 1 below.

[0072]

Table 1

[0073] The in vivo model investigated was the D-galactose model applied to mice, which is suitable for the study of cognitive function decline due to aging. In fact, this model mimics many behavioral and molecular characteristics of brain aging in rodent models.

[0074] D-galactose was administered subcutaneously at a daily rate of 150 mg / kg of mouse wet weight, and the above food supplement was incorporated into the pellets according to the following pattern: · Between the 14th and 51st days, the supplement was administered by incorporation into food pellets; · Between the 1st and 51st days, D-galactose was administered subcutaneously 5 days a week; · Between the 43rd and 51st days, the effects of the test compound were monitored using three different behavioral tests.

[0075] The effectiveness of the supplement was evaluated by the following parameters: improvement of learning disabilities (spatial working memory: spontaneous alternation in the Y-maze by the Y-maze test, spatial memory by the so-called "Morris water maze", and long-term contextual memory in the passive avoidance test), lipid peroxidation (LPO) rate in the hippocampus, and the effects on the neuroinflammatory markers IL-6 and TNF-α.

[0076] (Improvement of learning disabilities) · On the 43rd day, for all animals, a test of spontaneous alternation performance was conducted in the Y-maze (YM) test through the spatial working memory index. · From the 44th to the 49th day, for all animals, a test of spatial memory was conducted in the Morris water maze (MWM) test through the spatial memory index. · On the 50th and 51st days, the long-term contextual memory of the animals was evaluated. For this, a step-by-step type passive avoidance process (DTPA) was used through the movement and retention sessions. · On the 50th and 51st days, all animals were tested for STPA issues.

[0077] (Effect on lipid peroxidation (LPO) rate and neuroinflammatory markers IL-6 and TNF-α in the hippocampus) On the 51st day, after the behavioral test, the animals were euthanized.

[0078] For all animals, trunk blood was collected and centrifuged to recover plasma, and the brain was quickly recovered. The hippocampus and cortex were dissected, and the hippocampus was used to measure the peroxidation rate of lipids by colorimetry. Using the hemi-frontal cortex and plasma, the levels of the inflammatory biomarkers interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were measured.

[0079] Quantification of the lipid peroxidation (LPO) rate was performed according to the modified and adapted procedure of Hermes-Lima et al. This method measures the ability of brain peroxidized lipids to oxidize the iron(II) and xylenol orange complex in the presence of cumene hydroperoxide (HPC). The lipid peroxidation level is determined by HPC equivalents (HPCE) according to the following formula: HPCE = A5801 / A5802 × [HPC (nmol)] And it is expressed as a percentage relative to the data obtained for the control group (D-galactose + excipient) in terms of HPC equivalents per tissue wet weight.

[0080] The IL-6 and TNF-α contents were quantified by ELISA tests using the following kits: Quantification of IL-6: EM2IL6 from ThermoScientifique Quantification of TNF-α: EMTNFA from ThermoScientifique For all tests, after thawing in 50 mM Tris-buffered saline (Tris-150 mM NaCl), pH 7.5, the cortex is homogenized and sonicated for 20 seconds. After centrifugation (16100 g, 15 minutes, 4 °C), the supernatant or plasma is used for the ELISA test according to the instructions of the ELISA test manufacturer. For each test, the absorbance is read at 450 nm, and the concentration of the sample is calculated using a standard curve. The results are expressed as pg of marker per mg of tissue wet weight.

[0081] All values except passive avoidance latency are expressed as the mean value plus or minus the standard deviation of the measured values. Statistical analysis was performed separately for each compound by one-way ANOVA (F value), followed by Dunnett's post hoc multiple comparison test. Since the upper limit time is fixed, passive avoidance latency does not follow a Gaussian distribution. Therefore, it is analyzed using the Kruskal-Wallis non-parametric ANOVA (H value), followed by the Dunn multiple comparison test. A p < 0.05 value is considered statistically significant.

[0082] The test was performed using 60 male mice, which were divided into 6 groups of 10 mice each. Among them, the first group was the negative control group, and the second to sixth groups were the positive control groups: Group 1 is the group in which saline is administered subcutaneously instead of D-galactose and sham bolus A1 is given; Group 2 is the group in which D-galactose and bolus A1 are administered; Group 3 is the group in which D-galactose and bolus A2 are administered; Group 4 is the group in which D-galactose and bolus A3 are administered; Group 5 is the group in which D-galactose and bolus A4 are administered; Group 6 is the group in which D-galactose and bolus A5 are administered.

[0083] The calculation of the human daily dose based on the daily dose tested in mice is defined by the FDA (Guidance, 2005). The value of the human daily dose expressed in mg per kg of body weight (HED human) is the value of the daily dose in animals expressed in mg / kg (HED animal) multiplied by the safety factor of the animal considered (Km animal) and the safety factor of humans (Km human). Km human is equal to 37 and Km mouse is equal to 3.

[0084] (Effect on spatial memory in the Y-maze spontaneous alternation test) The results are shown in Figure 1. The first figure (left side) shows the effect of the supplement of the present invention on spontaneous alternation disorder, and the second figure (right side) shows the effect of the supplement of the present invention on spontaneous motor activity.

[0085] In Figure 1: LOW, low-dose supplement (A2); MED, medium-dose supplement (A3); HI, high-dose supplement (A4); N is composed between 9 and 10 according to the group. * p < 0.05, *** p < 0.0001 compared to the saline / vehicle group, and # p < 0.05, ## p < 0.01, p < 0.0001 compared to the D-galactose 150 / vehicle group. These are by Dunnett's test.

[0086] It was observed that treatment with D-galactose significantly changed spatial working memory compared to mice treated with saline.

[0087] Supplement A2 showed no effect on alternating behavior. Supplement A3 significantly, but partially, attenuated the disorder induced by chronic intoxication with D-galactose. Supplement A4 significantly and completely attenuated the disorder induced by chronic intoxication with D-galactose.

[0088] Treatment with DHA alone (by A5) significantly, but partially, reduced the disorder induced by chronic intoxication with D-Gal.

[0089] Surprisingly, prophylactic treatment with the supplement of the present invention has been found to have a more significant positive effect (complete attenuation of the disorder) compared to treatment with DHA alone (significant and partial attenuation of the disorder), at the same dose of DHA.

[0090] (Effect on learning disorder induced by D-Gal based on MWM test) The results are shown in Figure 2.

[0091] In Figure 2: LOW, low-dose supplement (A2); MED, medium-dose supplement (A3); HI, high-dose supplement (A4); N is composed of 9 to 10 according to the group. * p<0.05, ** p<0.01, *** p<0.0001 compared to the saline / vehicle group, # p<0.01, p<0.0001 compared to the D-galactose 150 / vehicle group. These are by Bonferroni multiple comparison test after two-way ANOVA.

[0092] Chronic intoxication with D-galactose significantly changed spatial learning compared to the negative control group (saline / vehicle).

[0093] Supplement A2 showed no effect on alternating behavior.

[0094] Supplement A3 significantly but partially attenuated the disorder induced by chronic intoxication with D-galactose.

[0095] Supplement A4 significantly and completely attenuated the disorder induced by chronic intoxication with D-galactose.

[0096] Treatment with DHA alone by A5 significantly but partially alleviated the disorder induced by chronic intoxication with D-galactose.

[0097] Surprisingly, prophylactic treatment with the supplement of the present invention at the A4 dosage has been found to have a more significant positive effect (complete and significant attenuation of the disorder) compared to treatment with DHA alone (significant and partial attenuation of the disorder). This is at the same dosage of DHA.

[0098] (Effect of supplement and DHA on learning disorders induced by D-galactose) The results are shown in Figure 3.

[0099] In Figure 3: LOW, low-dose supplement (A2); MED, medium-dose supplement (A3); HI, high-dose supplement (A4); N is composed between 9 and 10 according to the group. *** p < 0.0001 compared to the saline / vehicle group, and p < 0.0001 compared to the D-galactose 150 / vehicle group. These are by Bonferroni multiple comparison test after two-way ANOVA.

[0100] Chronic intoxication with D-galactose significantly altered spatial learning compared to the negative control group (saline / vehicle).

[0101] Supplement A2 showed no effect on alternating behavior.

[0102] Supplement A3 significantly but partially attenuated the disorder induced by chronic intoxication with D-galactose.

[0103] Supplement A4 significantly and completely attenuated the disorder induced by chronic intoxication with D-galactose.

[0104] Treatment with DHA alone by A5 significantly but partially alleviated the disorder induced by chronic intoxication with D-galactose.

[0105] Surprisingly, prophylactic treatment with the supplement of the present invention at the dosage of A4 has been found to have a more significant positive effect (complete and significant attenuation of the disorder) compared to treatment with DHA alone (significant and partial attenuation of the disorder), at the same dosage of DHA. Furthermore, prophylactic treatment at the supplement dosage of A3 has the same effect (significant and partial attenuation of the disorder) as treatment with DHA alone with a two-fold DHA concentration.

[0106] (Effect on D-galactose-induced passive avoidance impairment in mice) The results are shown in Figure 4. The effects of the supplement of the present invention are shown in the step-down latency in the left figure and the avoidance latency in the right figure, respectively, measured during the retention period.

[0107] In Figure 4, LOW, low-dose supplement (A2); MED, medium-dose supplement (A3); HI, high-dose supplement (A4); N is composed of between 9 and 10 depending on the group. *** p < 0.0001 compared to the saline / vehicle group, and p < 0.0001 compared to the D-galactose 150 / vehicle group. These are by Dunnett's test.

[0108] Chronic intoxication with D-galactose significantly altered long-term contextual working memory compared to the negative control group (saline / vehicle).

[0109] Supplement A2 showed no effect on long-term contextual memory.

[0110] Supplement A3 attenuated the disorder induced by chronic intoxication with D-galactose, although not significantly.

[0111] Supplement A4 significantly and completely attenuated the disorder induced by chronic intoxication with D-galactose.

[0112] Treatment with DHA alone (by A5) attenuated, though not significantly, the disorders induced by chronic intoxication with D-galactose.

[0113] Surprisingly, prophylactic treatment with the supplement of the present invention at the dosage of A4 has been found to have a more significant positive effect (a significant and complete attenuation of the disorder), as compared to the treatment with DHA alone (a non-significant attenuation of the disorder). Further, prophylactic treatment with the supplement at dosage A3 has the same effect (a non-significant attenuation of the disorder) as the treatment with DHA alone with a DHA concentration twice as high.

[0114] (Effect of the supplement and DHA on lipid peroxidation induced by D-galactose) The results are shown in Figure 5.

[0115] In Figure 5: LOW, low dose supplement (A2); MED, medium dose supplement (A3); HI, high dose supplement (A4); N is composed between 9 and 10 according to the group. ** p < 0.01, *** p < 0.0001 compared to the saline / vehicle group, ## p < 0.01, p < 0.0001 compared to the D-galactose 150 / vehicle group. These are by Dunnett's test.

[0116] Chronic intoxication with D-galactose significantly increased oxidative stress as compared to the negative control group (saline / vehicle).

[0117] Supplement A2 showed no effect on lipid peroxidation induced by chronic intoxication with D-galactose.

[0118] Supplement A3 significantly, though partially, decreased the oxidative stress induced by chronic intoxication with D-galactose.

[0119] Supplement A4 significantly and completely decreased the oxidative stress induced by chronic intoxication with D-galactose.

[0120] The treatment with DHA alone based on A5 showed no effect against the oxidative stress induced by chronic intoxication with D-galactose.

[0121] Surprisingly, preventive treatment with the supplement of the present invention at the dosage of A4 was found to have a significant positive effect (significant and complete attenuation of oxidative stress) compared to treatment with DHA alone at the same dosage.

[0122] (Effect of the supplement and DHA on the expression of TNF-α in the cortex and plasma induced by D-galactose) The results are shown in Figure 6. The effect on the cortex is shown in the left figure, and the effect on the plasma is shown in the right figure.

[0123] In Figure 6: LOW, low-dose supplement (A2); MED, medium-dose supplement (A3); HI, high-dose supplement (A4); N is composed of between 9 and 10 according to the group. *** p < 0.0001 compared to the saline / vehicle group, and p < 0.0001 compared to the D-galactose 150 / vehicle group. These are by Dunnett's test.

[0124] Chronic intoxication with D-galactose significantly increased TNF-α in the cortex and plasma compared to the negative control group (saline / vehicle).

[0125] Supplements A2 and A3 significantly but partially inhibited the increase in TNF-α in the cortex and plasma induced by chronic intoxication with D-galactose.

[0126] Supplement A4 significantly and completely decreased the levels of TNF-α in the cortex and plasma.

[0127] Treatment with DHA alone based on supplement A5 significantly but partially inhibited the increase in TNF-α in the cortex and plasma induced by chronic intoxication with D-galactose.

[0128] Surprisingly, prophylactic treatment with supplement HI (A4) has been found to have a significant positive effect (significant and complete attenuation of the increase in TNF-α in the cortex and plasma) compared to treatment with DHA alone containing the same dose of DHA. Furthermore, prophylactic treatment with supplements at doses A2 or A3 has an effect (attenuation of a significant increase) in the cortex and plasma, which is comparable to the effect of DHA alone having DHA concentrations 6-fold and 2-fold higher in order compared to the doses of supplements A2 and A3, respectively.

[0129] (Effect of supplements and DHA on IL-6 expression in the cortex and plasma induced by D-galactose) The results are shown in Figure 7. The effect on the cortex is shown in the left figure, and the effect on the plasma is shown in the right figure.

[0130] In Figure 7: LOW, low-dose supplement (A2); MED, medium-dose supplement (A3); HI, high-dose supplement (A4); N is composed of between 9 and 10 depending on the group. *** p < 0.0001 compared to the saline / vehicle group, and p < 0.0001 compared to the D-galactose 150 / vehicle group. These are by the Dunnett test.

[0131] The low-dose supplement (A2) showed no effect on the concentration of IL-6 induced by intoxication with D-galactose.

[0132] The medium-dose supplement (A3) has a significant but partial effect on the increase in IL-6 in the cortex and plasma induced by chronic intoxication with D-galactose.

[0133] The high-dose supplement (A4) significantly and completely suppressed the increase in IL-6 in the cortex and plasma induced by chronic intoxication with D-galactose.

[0134] Treatment with DHA alone based on A5 has a significant, but partial, effect on the increase in IL-6 in the cortex and plasma induced by chronic intoxication with D-galactose.

[0135] Surprisingly, preventive treatment with supplement HI based on A4 has a significant positive effect (significantly and completely attenuating the increase in IL-6 in the cortex and plasma) compared to treatment with DHA alone (which significantly but partially attenuates the increase in IL-6 in the cortex and plasma). This is for the same dose of DHA. Furthermore, preventive treatment with the supplement at dose A3 has an effect similar to that of treatment with DHA alone at twice the DHA concentration with respect to the cortex (not significant but attenuating the impairment).

[0136] In conclusion: Chronic intoxication with D-galactose induces extremely significant changes in spatial working memory and long-term contextual memory and impairs spatial learning. The behavioral changes are also related to biochemical changes such as an increase in oxidative stress and the induction of neuroinflammatory processes.

[0137] Preventive treatment with the supplement of the present invention is dose-dependent. In the case of the most potent test dose (preventive treatment with the supplement is A4), the impairments manifested as behavioral changes, an increase in oxidative stress, and activation of the neuroinflammatory process induced by chronic intoxication with D-galactose are extremely significantly and completely attenuated.

[0138] Treatment with an equivalent dose of DHA compared to DHA alone (A5) and supplement HI attenuates the impairments manifested as behavioral changes, an increase in oxidative stress, and activation of the neuroinflammatory process induced by chronic intoxication with D-galactose significantly but partially.

[0139] Surprisingly, preventive treatment with the supplement has a significantly higher effect at the same dosage of DHA than preventive treatment with DHA alone. This is with respect to the attenuation of age-related cognitive decline and with respect to a mouse model resulting from chronic poisoning with D-galactose. In addition, preventive treatment with the supplement has an effect equivalent to that of treatment with DHA alone at twice the DHA concentration at half the DHA dosage. This is in the case of the reduction of oxidative stress measured in the cortex (IL-6 and TNF-α) and in plasma (TNF-α), in the case of long-term contextual memory, and in the case of spatial learning. Also, preventive treatment with the supplement has the same positive effect as treatment with DHA alone, although the latter has a DHA concentration six times higher. This is in the case of the reduction of oxidative stress measured in the cortex (IL-6 and TNF-α) and in plasma (TNF-α).

[0140] Therefore, by applying the formula for calculating the human equivalent daily dosage, preventive treatment of age-related cognitive decline may be defined as taking the supplement at 2 - 5 mg per kg of body weight per day.

[0141] (Example 3: Test in young female rats subjected to prenatal stress with an extract of the microalgae Isochrysis lutea) In this example, the elimination of cognitive impairment, anxiety behavior, and changes in recognition memory in young female rats after prenatal stress of their ancestors is examined. This is done through the administration of a supplement based on the extract of the microalgae Isochrysis lutea used in Example 2.

[0142] (Apparatus and method) The model used in this example is a recognized model that induces prenatal stress in rats by fixing pregnant females in a cylinder under intense lighting.

[0143] Pregnant female rats were randomly assigned to a prenatal stress group (SP) or a control group (NS), placed individually in plastic breeding cages, and allowed free access to food and water except during the behavioral test period. The environment inside the cages was as follows: placed in a room with a photoperiod cycle of 12 hours of light / 12 hours of darkness (light on at 7:00 am), constant temperature (21°C), and constant humidity (50%).

[0144] The prenatal stress procedure was performed as described by Meunier et al. (2004). Immobilization of female rats was for the purpose of a semi-random restraint procedure. The animals were placed in a transparent ferret holder made of Plexiglas (length 20 cm, diameter 7 cm) and held for 90 minutes a day for 4 consecutive days under bright light. To make the stress as unpredictable as possible, the 90-minute forced immobilization period was applied as follows: one 90-minute phase, two 45-minute phases separated by 4 hours, two phases of 60 minutes and 30 minutes separated by 4 hours, or three 30-minute phases separated by 4 hours and 1 hour, at different times of the day.

[0145] Control mother mice were also manipulated but were never placed in the ferret holder.

[0146] The treated female rats were naturally released from the trap of the ferret holder on postnatal day 1 (PPD1).

[0147] Litters were weaned at PPD21. The rats were separated from their mothers, sexed, and weighed, and rats of the same sex were assigned to cages (3 rats per cage). Young rats in the same cage were from different litters. This was to avoid effects related to being littermates.

[0148] The environment inside the cages was as follows: placed in a room with a photoperiod cycle of 12 hours of light / 12 hours of darkness (light on at 7:00 am), constant temperature (21°C), and constant humidity (50%), and allowed free access to food and water except during the behavioral test period.

[0149] In each cage, the animals were treated the same. The animals were tested by random double-blind method.

[0150] Forty-eight female rats were used and divided into four groups of animals configured as follows: Group 1 consisted of 12 naive female rats, i.e., rats whose ancestors had not been exposed to prenatal stress, and received only 200 μL of excipient solution per day (referred to as NS / excipient). Therefore, this group is a control group.

[0151] Group 2 consisted of 12 naive female rats, i.e., rats whose ancestors had not been exposed to prenatal stress, and received 200 μL of supplement per day (referred to as NS / supplement).

[0152] Group 3 consisted of 12 female rats whose ancestors had been exposed to prenatal stress and received only 200 μL of excipient solution per day (referred to as SP / excipient).

[0153] Group 4 consisted of 12 female rats whose ancestors had been exposed to prenatal stress and received 200 μL of supplement per day (referred to as SP / supplement).

[0154] The effect of the supplement was evaluated at six weeks of age.

[0155] The supplement (one dose) was administered once a day, five days a week, by forced oral administration. The administration started after weaning, i.e., on postpartum day 25 (PPD25), and continued until PPD46.

[0156] The daily intake was 25.7 mg of supplement per kg of rat body weight.

[0157] The animals were subjected to behavioral tests on days between PPD46 and PPD48, i.e., during the period excluded from the treatment period with the excipient and supplement. Therefore, the effects observed during the behavioral tests would be essentially due to prophylactic treatment.

[0158] The behavioral tests were divided into one anxiety assessment session and two object recognition sessions. The sessions were defined as follows.

[0159] · Session 1, PPD46: The rats were placed in a square open space (50 cm × 50 cm × 50 cm × 50 cm) made of blue Plexiglas equipped with infrared light-emitting diodes on the floor. The rats were acclimated to the test space during the 10-minute session, and their movements were captured by an infrared camera and analyzed using Ethovision® (Noldus) software. Activity was analyzed based on the total distance traveled (m) and the percentage of presence in the central area of 25 cm × 25 cm defined by the software. These data have been reported for the intensity of anxiety behavior (38).

[0160] · Session 2, PPD47: Two identical objects (50-ml resin Eppendorf tubes) were placed at predetermined positions (opposite ends in the central area). Each rat was placed in the test space, and exploratory behavior was recorded during the 10-minute session. Activity was analyzed in terms of the number of interactions with the objects and the interaction time.

[0161] · Session 3, PPD48: The objects in Session 2 were replaced with new objects (resin bottle caps). Their shape, texture, and color were different from the familiar objects. Each rat was placed in the test space, and exploratory behavior was recorded during the 10-minute session. Activity was analyzed in the same way as described in Session 2.

[0162] The priority search index is calculated as the ratio of the number of interference times (or time) with an object in session 2 to the total number of interference times (or time) for each object.

[0163] All values are expressed as the mean value of the measurement plus or minus the standard deviation. For each element, one-way ANOVA (F value) followed by Dunnett's post hoc multiple comparison test is used for statistical analysis.

[0164] The calculation of the human daily dose from the daily dose tested in rats is defined by the FDA (Guidance, 2005) as follows: The value representing the human daily dose in mg per kg of body weight (HED human) is the value representing the daily dose in animals in mg / kg (HED animal) multiplied by the safety factor for the animal considered (Km animal) and the safety factor for humans (Km human). Km human is equal to 37 and Km rat is equal to 3.

[0165] The results are shown below.

[0166] (Movement in the center of the test space on day 46 of PPD; effect of supplement on anxiety) The results are shown in Figure 8.

[0167] Figure 8: Effect of treatment on anxiety: N = 12; ***p < 0.0001 for the treatment group NS / vehicle and # p < 0.0001 for the treatment group SP / vehicle. These are by Dunnett's test.

[0168] The group SP / vehicle, which was exposed to prenatal stress and treated with vehicle only, had a significantly higher percentage of movement in the peripheral area of the open test space compared to the group NS / vehicle (the group not exposed to prenatal stress). The group SP / supplement, which was exposed to prenatal stress and received preventive treatment with the supplement, had a significantly lower percentage of movement in the vicinity of the open test space compared to the group SP / excipient (the group that was exposed to prenatal stress but did not receive treatment with the supplement). Furthermore, the percentage distribution of movement in the group SP / supplement was equivalent to that of the control group NS / excipient.

[0169] The fact that the proportion of movement of individuals in the peripheral area of the open test space is higher than the control mobility is an expression of anxious behavior via a protective mechanism based on the exploration of boundaries that delimit the un-covered area (63) and is monitored.

[0170] Thus, prenatal stress (PS) triggers very serious anxious behavior.

[0171] Surprisingly, it was revealed that the supplement very significantly and completely attenuates the anxious behavior induced by prenatal stress.

[0172] (Recognition test, day 47 of PPD, effect of the supplement on recognition memory in object recognition) The results are shown in Figure 9.

[0173] During this session, the same object is presented to each individual twice.

[0174] No statistical effect regarding this parameter was measured between the groups. Therefore, all group individuals interact equally when interfering with the same object, and that interaction is equally distributed (50%) between the two objects in both frequency and duration.

[0175] (Recognition test, day 48 of PPD (new object); effect of the supplement on recognition memory for the recognition test of a new object) The results of the test are shown in Figure 10.

[0176] In Figure 10: N = 12; *** p < 0.0001 compared with the treatment group NS / vehicle, and p < 0.0001 compared with the treatment group SP / vehicle. These are by Dunnett's test.

[0177] During this session, two different objects are presented to each individual once. One of the objects corresponds to the object presented during Session 2, and the other object is a new object.

[0178] The group SP / vehicle, which was prenatally stressed and prophylactically treated with vehicle only, had a significantly lower percentage of interaction with the presented new object in both frequency and duration compared to the group NS / vehicle (the group not prenatally stressed). And this percentage is equal to that obtained for all groups in Session 2. That is, the individuals in the group SP / vehicle interact with the new object to the same extent as they do with the old object. Therefore, the individuals in this group do not recognize the old object presented in Session 2.

[0179] In contrast, the group SP / supplement, which was prenatally stressed and prophylactically treated with a supplement, had a significantly higher percentage of interaction with the new object in both frequency and duration compared to the group SP / vehicle (the negative control group). And this percentage is higher than that obtained for all groups. Therefore, the individuals in the group SP / supplement interact less with the old object compared to the new object. Thus, the individuals in this group recognize the old object presented in Session 2. Furthermore, the individuals in the group SP / supplement have a percentage of interaction with the presented new object that is equivalent to that of the individuals in the groups NS / vehicle and NS / supplement in both frequency and duration.

[0180] Therefore, prenatal stress (SP) induced a very significant impairment in recognition memory regarding novel objects.

[0181] Surprisingly, treatment with the supplement was found to significantly and completely attenuate the anxiety behavior induced by prenatal stress.

[0182] In conclusion: Treatment with the supplement significantly and completely attenuates anxiety behavior and attenuates the cognitive memory impairment induced by prenatal stress.

[0183] The prenatal stress conducted in this experiment significantly induced anxiety behavior and significantly changed the cognitive function of young female rats.

[0184] By applying the formula for calculating the human equivalent daily dose, a preventive treatment that attenuates the cognitive impairment caused by prenatal stress can be defined as a supplement with a daily dose of 0.05 - 0.1 mg per kg of body weight.

[0185] (Example 4: Test of the natural extract of microalgae Phaeodactylum tricornutum in an in vivo model regarding attenuation of disorders induced by age-related decline in cognitive function) The food supplement of the present invention is prepared from a Phaeodactylum tricornutum extract and contains the following per mg / g: Omega-3 fatty acids (ALA, SDA, EPA, DHA): 66.6 ± 11.5; Fucoxanthin: 20.0 ± 4.0; Sterol: 3.0 ± 0.6; Ficoprostane: 0.0025 ± 0.0005.

[0186] The supplement is obtained by adding coconut oil to the extract at a ratio of 410 mg ± 20 mg / g.

[0187] The supplement is incorporated into the kibble in four different formulations. The amount of supplement incorporated into different batches of kibble is made to correspond to the human equivalent daily dose described in Table 2, and the composition is diluted with coconut oil as described below to have the same mass in all formulations.

[0188] The calculation of the human daily dose from the daily dose tested in mice is defined by the FDA (Guidance, 2005) as follows: The value representing the human daily dose in mg per kg of body weight (HED human) is the value representing the daily dose in animals in mg / kg (HED animal) multiplied by the safety factor for the animal under consideration (Km animal) and the safety factor for humans (Km human). Km human is equal to 37 and Km mouse is equal to 3.

[0189] Additional batches of kibble are formulated with coconut oil only, and the excipient concentration is equal to that of the other batches, i.e., 0.01% (m:m).

[0190] The five batches of kibble thus obtained are referred to as shown in Table 2 below.

[0191]

Table 2

[0192] The in vivo model examined was the D-galactose model applied to mice, which is suitable for studying age-related decline in cognitive function. In fact, this model mimics many of the behavioral and molecular characteristics of brain aging in rodent models.

[0193] D-galactose is administered subcutaneously daily at a rate of 150 mg / kg relative to the wet weight of the mice, and the above supplement is incorporated into the pellets according to the following pattern: · Administered by incorporation into food pellets from day 28 to day 51; · D-galactose is administered subcutaneously 5 days a week from day 01 to day 51; · Between the 43rd and 51st days, the effects of the test composition were monitored using three different behavioral tests.

[0194] The effects of the supplement were evaluated based on the following parameters: improvement of learning disabilities (spatial working memory: spontaneous alternation in the Y-maze by the Y-maze test; spatial memory by the so-called "Morris water maze" and long-term contextual memory in the passive avoidance test), lipid peroxidation (LPO) rate in the hippocampus, and the effects on the neuroinflammatory markers IL-6 and TNF-α.

[0195] (Improvement of learning disabilities) · On the 43rd day, for all animals, the spontaneous alternation performance in the Y-maze (YM) test was examined via the spatial working memory index; · From the 44th to the 49th day, for all animals, the spatial memory in the Morris water maze (MWM) test was examined via the spatial memory index; · From the 44th to the 49th day, for all animals, it was examined by the MWM test to evaluate spatial working memory; · On the 50th and 51st days, through the exercise and retention sessions, the long-term contextual memory of the animals was examined using the step-through passive avoidance procedure (STPA).

[0196] (Effects on lipid peroxidation (LPO) rate in the hippocampus and neuroinflammatory markers IL-6 and TNF-α) On the 51st day, after the behavioral tests, the animals were euthanized.

[0197] For all animals, trunk blood was collected, centrifuged to recover plasma, and the brain was collected rapidly. The hippocampus and cortex were dissected, and the lipid peroxidation rate was measured using the hippocampus by colorimetry. Using the prefrontal cortex and plasma, the levels of the inflammatory biomarkers interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were measured.

[0198] Quantification of the lipid peroxidation (LPO) rate was carried out according to the modified and adapted procedure of Hermes-Lima et al. This method measures the ability of brain lipid peroxides to oxidize the ferrous iron and xylenol orange complex in the presence of cumene hydroperoxide (HPC). The lipid peroxidation level is determined by HPC equivalents according to the following formula: HPCE = A5801 / A5802 × [HPC (nmol)] And it is expressed as a percentage relative to the data obtained for the control group (D-galactose + excipient) in HPC equivalents per tissue wet weight.

[0199] IL-6 and TNF-α contents were quantified by ELISA tests using the following kits: Quantification of IL-6: EM2IL6 from ThermoScientifique Quantification of TNF-α: EMTNFA from ThermoScientifique For all tests, after thawing in 50 mM Tris-buffered saline (Tris-150 mM NaCl), pH 7.5, the cortex was homogenized and sonicated for 20 seconds. After centrifugation (16100 g, 15 minutes, 4 °C), the supernatant or plasma was used in the ELISA test according to the manufacturer's instructions. For each test, the absorbance was read at 450 nm and the concentration of the sample was calculated using a standard curve. The results are expressed as pg of marker per mg of tissue wet weight.

[0200] All values except passive avoidance latency are expressed as the mean value plus or minus the standard deviation of the measured values. Statistical analysis was performed separately for each compound by one-way ANOVA (F value), followed by Dunnett's post hoc multiple comparison test. Passive avoidance latency does not follow a Gaussian distribution because the upper time limit is fixed. Therefore, it was analyzed using the Kruskal-Wallis non-parametric ANOVA (H value), followed by Dunn's multiple comparison test. A p < 0.05 value was considered statistically significant.

[0201] The test was conducted on 72 male mice distributed into 6 groups of 12. Among them, Group 1 was the negative control group, and Groups 2 - 6 were the positive control groups.

[0202] Group 1 was the group administered with subcutaneous physiological saline instead of D - galactose and Kibble B1.

[0203] Group 2 was the group administered with D - galactose and Kibble B1.

[0204] Group 3 was the group administered with D - galactose and Kibble B2.

[0205] Group 4 was the group administered with D - galactose and Kibble B3.

[0206] Group 5 was the group administered with D - galactose and Kibble B4.

[0207] Group 6 was the group administered with D - galactose and Kibble B5.

[0208] (Effect on Spatial Memory in the Y - maze Spontaneous Alternation Test) The results are shown in Figure 11. The first figure (left side) shows the effect of the supplement of the present invention on spontaneous alternation disorder, and the second figure (right side) shows the effect of the supplement of the present invention on spontaneous locomotor activity.

[0209] In Figure 11: Saline / vehicle is the negative control group (the group fed with kibble consisting of vehicle and coconut oil without treatment with D-galactose); DGal 150 / vehicle is the positive control group (the group treated with D-galactose and fed with kibble consisting of vehicle and coconut oil); D1, D2, D3, and D4 have increasing supplement dosages; N is composed between 11 and 12 per group. *p < 0.05, ***p < 0.0001 compared to the saline / vehicle group, and #p < 0.05, ##p < 0.01, #p < 0.0001 compared to the D-galactose 150 / vehicle group. These are by Dunnett's test.

[0210] It is observed that treatment with D-galactose significantly changes spatial working memory compared to treatment with saline.

[0211] Supplement D1 significantly but partially attenuated the impairments induced by chronic intoxication with D-galactose. Supplements D2, D3, and D4 significantly and completely attenuated the impairments induced by chronic intoxication with D-galactose.

[0212] (Effect on learning impairments induced by D-Gal based on the MWM test) The results are shown in Figure 12.

[0213] In Figure 12: Saline / vehicle is the negative control group (the group fed with kibble consisting of vehicle and coconut oil without treatment with D-galactose); DGal 150 / vehicle is the positive control group (the group treated with D-galactose and fed with kibble consisting of vehicle and coconut oil); D1, D2, D3, and D4 have increasing supplement dosages; N is composed between 11 and 12 by group. * p < 0.05, ** p < 0.01, *** p < 0.0001 compared to the saline / vehicle group, and ## p < 0.01, p < 0.01 compared to the D-galactose 150 / vehicle group. These are by Bonferroni multiple comparison test after two-way ANOVA.

[0214] Chronic intoxication with D-galactose significantly changed spatial learning compared to the negative control group (saline / vehicle).

[0215] Supplement D1 significantly but partially attenuated the impairments induced by chronic intoxication with D-galactose.

[0216] Supplements D2, D3, and D4 significantly and completely attenuated the impairments induced by chronic intoxication with D-galactose.

[0217] (Effect of supplements on learning impairments induced by D-galactose) The results are shown in Figure 13.

[0218] In Figure 13: Saline / vehicle is the negative control group (the group fed with kibble consisting of vehicle and coconut oil without treatment with D-galactose); DGal 150 / vehicle is the positive control group (the group treated with D-galactose and fed with kibble consisting of vehicle and coconut oil); D1, D2, D3, and D4 have increasing supplement dosages; N is composed between 11 and 12 by group. Saline / vehicle is the negative control group (the group fed with kibble consisting of vehicle and coconut oil without treatment with D-galactose); DGal 150 / vehicle is the positive control group (the group treated with D-galactose and fed with kibble consisting of vehicle and coconut oil); D1, D2, D3, and D4 have increasing supplement dosages; N is composed between 11 and 12 by group. *** p < 0.0001 for the saline / vehicle group and p < 0.0001 for the D-galactose 150 / vehicle group. These are by Bonferroni multiple comparison test after two-way ANOVA. "T" is the time spent in the target quadrant, and "O" is the average of the time spent in the other three quadrants.

[0219] Chronic intoxication with D-galactose significantly altered spatial learning compared to the negative control group (saline / vehicle).

[0220] Supplements D1 and D2 significantly, but partially, attenuated the impairments induced by chronic intoxication with D-galactose.

[0221] Supplements D3 and D4 significantly and completely attenuated the impairments induced by chronic intoxication with D-galactose.

[0222] (Effect on D-galactose-induced passive avoidance impairment in mice) The results are shown in Figure 14. The effect of the supplement of the present invention is shown in the step-down latency in the left figure and the avoidance latency in the right figure, respectively, measured during the retention period.

[0223] In Figure 14: Saline / excipient is the negative control group (the group fed with kibble consisting of excipient and coconut oil without treatment with D-galactose); DGal 150 / excipient is the positive control group (the group treated with D-galactose and fed with kibble consisting of excipient and coconut oil); D1, D2, D3, and D4 have increasing supplement dosages; N is composed between 11 and 12 by group. It is *** p < 0.0001 for the saline / excipient group and p < 0.0001 for the D-galactose 150 / excipient group. These are by Dunnett's test.

[0224] Chronic intoxication with D-galactose significantly changed long-term contextual working memory compared to the negative control group (saline / excipient).

[0225] Supplement D1 showed no effect on long-term contextual memory.

[0226] Supplements D2, D3, and D4 significantly and completely attenuated the disorders induced by chronic intoxication with D-galactose.

[0227] (Effect of supplements on lipid peroxidation induced by D-galactose) The results are shown in Figure 15.

[0228] In Figure 15: Saline / vehicle is the negative control group (the group fed with kibble consisting of vehicle and coconut oil without treatment with D-galactose); DGal 150 / vehicle is the positive control group (the group treated with D-galactose and fed with kibble consisting of vehicle and coconut oil); D1, D2, D3, and D4 have increasing supplement dosages; N is composed between 11 and 12 per group. It is ** p<0.01, *** p<0.0001 compared to the saline / vehicle group, and ## p<0.01, p<0.0001 compared to the D-galactose 150 / vehicle group. These are by Dunnett's test.

[0229] Chronic intoxication with D-galactose significantly increases oxidative stress compared to the negative control group (saline / vehicle).

[0230] Supplement D1 significantly, but partially, decreased the oxidative stress induced by chronic intoxication with D-galactose.

[0231] Supplements D2, D3, and D4 significantly and completely decreased the oxidative stress induced by chronic intoxication with D-galactose.

[0232] (Effect of supplements on TNF-α expression in the cortex and plasma induced by D-galactose) The results are shown in Figure 16. The effect on the cortex is shown in the left figure, and the effect on the plasma is shown in the right figure.

[0233] In Figure 16: Saline / vehicle is the negative control group (the group fed with kibble consisting of vehicle and coconut oil without treatment with D-galactose); DGal 150 / vehicle is the positive control group (the group treated with D-galactose and fed with kibble consisting of vehicle and coconut oil); D1, D2, D3, and D4 have increasing supplement dosages; N is composed between 11 and 12 by group. It is *** p < 0.0001 for the saline / vehicle group and p < 0.0001 for the D-galactose 150 / vehicle group. These are by Dunnett's test.

[0234] Chronic intoxication with D-galactose significantly increased TNF-α in the brain and plasma compared with the negative control group (saline / vehicle).

[0235] Supplement D1 significantly but partially inhibited the increase in TNF-α in the brain and plasma induced by chronic intoxication with D-galactose.

[0236] Supplement D2 significantly and completely inhibited the increase in TNF-α induced by chronic intoxication with D-galactose in the brain and significantly but partially inhibited it in plasma.

[0237] Supplement D3 significantly but partially inhibited the increase in TNF-α induced by chronic intoxication with D-galactose in the brain and significantly and completely inhibited it in plasma.

[0238] Supplement D4 significantly and completely inhibited the increase in TNF-α in the brain and plasma induced by chronic intoxication with D-galactose.

[0239] (Effect of supplements on IL-6 expression in the cortex and plasma induced by D-galactose) The results are shown in Figure 17. The effect on the cortex is shown in the left figure and the effect on plasma is shown in the right figure.

[0240] In Figure 17: Saline / excipient is the negative control group (the group fed with kibble consisting of excipient and coconut oil without treatment with D-galactose); DGal 150 / excipient is the positive control group (the group treated with D-galactose and fed with kibble consisting of excipient and coconut oil); D1, D2, D3, and D4 have increasing dosages of the supplement; N is composed between 11 and 12 by group. ***p < 0.0001 compared to the saline / excipient group, and p < 0.0001 compared to the D-galactose 150 / excipient group. These are by Dunnett's test.

[0241] Chronic intoxication with D-galactose significantly increased IL-6 in the brain and plasma compared to the negative control group (saline / excipient).

[0242] Supplements D1 and D2 significantly but partially suppressed the increases in the brain and plasma induced by chronic intoxication with D-galactose.

[0243] Supplements D3 and D4 significantly and completely suppressed the increases in the brain and plasma induced by chronic intoxication with D-galactose.

[0244] (Conclusion) Chronic intoxication with D-galactose significantly induced changes in spatial working memory and long-term contextual memory and impaired spatial learning. The changes in behavior are also related to the biochemical changes that appear as increased oxidative stress and activation of neuroinflammatory processes.

[0245] The preventive treatment with the supplement of the present invention is dose-dependent. In the case of the strongest dose (Supplement 4), it significantly and completely attenuates the disorders manifested as changes in behavior, increased oxidative stress, and activation of neuroinflammatory processes induced by chronic intoxication with D-galactose. And in the case of intermediate low doses (Supplements D2 and D3), the supplement of the present invention significantly and completely attenuates the disorders manifested as spatial working memory, increased oxidative stress, and activation of neuroinflammatory processes induced by chronic intoxication with D-galactose.

[0246] Therefore, by applying the formula for calculating the human equivalent daily dose, the preventive treatment for age-related decline in cognitive function can be defined as an intake of 1.7 mg of the supplement per kg of body weight per day.

Claims

1. One or more omega-3 fatty acids of at least 50 mg / g, one or more xanthophylls containing fucoxanthin of at least 10 mg / g, phytosterols of at least 1 mg / g, and one or more fucoprostanoids selected from the group consisting of phytoprostanes, isoprostanes, and neuroprostanes of at least 2 μg / g, and Medium-chain triglycerides (MCT) that are coconut palm oil or palm oil A composition comprising The extract is obtained by extraction using one or more solvents selected from the group consisting of acetone, hexane, ethyl acetate, methyltetrahydrofuran, heptane, methanol, natural or branched oils, and ethanol, The microalgae is Phaeodactylum tricornutum, Composition.

2. In the composition of Claim 1, One or more omega-3 fatty acids of 50 to 250 mg / g, One or more xanthophylls of 10 to 50 mg / g, One or more phytosterols of 1 to 20 mg / g, and A composition characterized by containing one or more fucoprostanoids of 2 to 100 μg / g.

3. In the composition of Claim 1 or 2, One or more omega-3 fatty acids of 50 to 200 mg / g, One or more xanthophylls of 10 to 30 mg / g, One or more phytosterols of 1 to 8 mg / g, and A composition characterized by containing one or more fucoprostanoids of 2 to 50 μg / g.

4. In any one of the compositions of Claims 1 to 3, One or more omega-3 fatty acids of 50 to 170 mg / g, One or more xanthophylls of 10 to 25 mg / g, One or more phytosterols of 1 to 6 mg / g, and A composition characterized by containing one or more fucoprostanoids of 2 to 40 μg / g.

5. The composition according to any one of Claims 1 to 4, which is a food supplement.

6. The composition according to any one of Claims 1 to 4, which is a food supplement that is a liquid preparation intended to be taken in units measured in small amounts.

7. At least one of the omega-3 fatty acids is selected from stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), α-linolenic acid (ALA), and mixtures thereof, and the composition according to any one of claims 1 to 6 is characterized in that.

8. The composition according to claim 7, wherein the extract contains 0.05 to 20% eicosapentaenoic acid (EPA) in an amount indicated by weight percentage based on the total extract.

9. The composition according to any one of claims 1 to 8, further comprising at least one additive selected from preservatives, colorants, flavors, disintegrants, lubricants, coating agents, and encapsulating agents.

10. The composition according to any one of claims 1 to 5 and 7 to 8, which is in the form of a gel capsule, capsule, tablet, troche or coarse powder.

11. The composition according to any one of claims 1 to 5 and 7 to 8, which is in the form of a gel capsule or capsule.

12. The composition according to any one of claims 1 to 11, which is packaged in a dosage having a unit weight between 10 mg and 1 g.

13. For suppressing the occurrence of cognitive impairment due to aging defined as a decrease in non-pathological cognitive function, or cognitive impairment in children or young adults exposed to prenatal stress that induces non-pathological impairment, the composition according to any one of claims 1 to 12.

14. The composition according to claim 13, wherein the cognitive impairment in the child or young adult is at least one selected from the group consisting of hyperactivity, attention and memory impairment, language delay and anxiety behavior.

15. The composition according to claim 13, for suppressing cognitive impairment due to aging defined as a decrease in non-pathological cognitive function, wherein the daily dosage is in the range of 2 to 5 mg / kg body weight of the composition.

16. The composition according to claim 13, for suppressing the occurrence of cognitive impairment in children or young adults exposed to prenatal stress that induces non-pathological impairment, wherein the daily dosage is in the range of 0.05 to 0.1 mg / kg body weight of the composition. The composition according to claim 16, wherein the cognitive dysfunction in the child or young adult is at least one selected from the group consisting of hyperactivity, attention and memory disorders, language delay and anxiety behavior.

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