Food supplement

Abstract

A composition comprising at least 50 mg/g of one or several omega-3 type fatty acid(s), at least 10 mg/g of one or several xanthophyll(s), at least 1 mg/g of one or several sterol(s) and at least 2 μg/g of one or several phycoprostane(s), and its applications in particular as a food supplement in the prevention of the apparition of cognitive disorders.

Claims

1. A tablet, capsule, capsule gel, or pastille consisting essentially of coconut oil and/or palm oil; fucoxanthin; stearidonic acid, eicosapentaenoic acid and/or docosahexaenoic acid; phytosterols; and phytoprostanes, isoprostanes and/or neuroprostanes.

2. The tablet, capsule, capsule gel, or pastille according to claim 1, consisting essentially of: 50 to 250 mg/g of the stearidonic acid, eicosapentaenoic acid and/or docosahexaenoic acid; 10 to 50 mg/g of the fucoxanthin; 1 to 20 mg/g of the phytosterols; and 2 to 100 μg/g of the phytoprostanes, isoprostanes and/or neuroprostanes.

3. The tablet, capsule, capsule gel, or pastille according to claim 1, consisting essentially of: 50 to 200 mg/g of the stearidonic acid, eicosapentaenoic acid and/or docosahexaenoic acid; 10 to 30 mg/g of the fucoxanthin; 1 to 8 mg/g of the phytosterols; and 2 to 50 μg/g of the phytoprostanes, isoprostanes and/or neuroprostanes.

4. The tablet, capsule, capsule gel, or pastille according to claim 1, consisting essentially of: 50 to 170 mg/g of the stearidonic acid, eicosapentaenoic acid and/or docosahexaenoic acid; 10 to 25 mg/g of the fucoxanthin; 1 to 6 mg/g of the phytosterols; and 2 to 40 μg/g of the phytoprostanes, isoprostanes and/or neuroprostanes.

5. The tablet, capsule, capsule gel, or pastille according to claim 1, wherein the total weight of the coconut oil and/or palm oil; fucoxanthin; stearidonic acid, eicosapentaenoic acid and/or docosahexaenoic acid; phytosterols; and phytoprostanes, isoprostanes and/or neuroprostanes is between 10 mg and 1 g.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The different embodiments of the disclosure are illustrated hereinbelow and their advantages set out in the following examples, with reference to the following figures:

(2) FIG. 1 is a representation of the effects of the supplement of the disclosure on the locomotor activity, with the left-side diagram illustrating the effects on the spontaneous alternation deficits and the right-side diagram illustrating the effects on the locomotor activity.

(3) FIG. 2 is a representation of the effects on the learning deficits induced by the D-Gal according to the MWM test.

(4) FIG. 3 is a representation of the effects of the supplement and of DHA on the learning deficits induced by the D-Galactose.

(5) FIG. 4 is a representation of the effects on the passive avoidance deficits induced by the D-Galactose in mice, with the effects on the step-down latency illustrated on the left-side diagram and on the escape latency illustrated on the right-side diagram, measured during the retention period.

(6) FIG. 5 is a representation of the effects of the supplement and of DHA on the lipid peroxidation induced by the D-Galactose.

(7) FIG. 6 is a representation of the effects of the supplement and of DHA on the expression of TNF-α in the cortex and the plasma induced by the D-Galactose, with the effect on the cortex on the left-side diagram and the effect on the plasma on the right-side diagram.

(8) FIG. 7 is a representation of the effects of the supplement and of DHA on the expression of IL-6 in the cortex (left-side diagram) and the plasma (right-side diagram) induced by the D-Galactose.

(9) FIG. 8 is a representation of the effect of the supplement on anxiety, in the test of locomotion at the center of the test space, day PPD46.

(10) FIG. 9 is a representation of the effect of the supplement on the recognition memory, in the test of recognition of an object, day PPD47.

(11) FIG. 10 is a representation of the effect of the supplement on the recognition memory, in the test of recognition of a new object.

(12) FIG. 11 is a representation of the effects of the supplement of the disclosure on the locomotor activity, with the left-side diagram illustrating the effects on the spontaneous alternation deficits and the right-side diagram illustrating the effects on the locomotor activity.

(13) FIG. 12 is a representation of the effects on the learning deficits induced by the D-Gal according to the MWM test.

(14) FIG. 13 is a representation of the effects of the supplement on the learning deficits induced by the D-Galactose.

(15) FIG. 14 is a representation of the effects on the passive avoidance deficits induced by the D-Galactose in mice, with the effects on the step-down latency illustrated on the left-side diagram and on the escape latency illustrated on the right-side diagram, measured during the retention period.

(16) FIG. 15 is a representation of the effects of the supplement on the lipid peroxidation induced by the D-Galactose.

(17) FIG. 16 is a representation of the effects of the supplement on the expression of TNF-α in the cortex and the plasma induced by the D-Galactose, with the effect on the cortex on the left-side diagram and the effect on the plasma on the right-side diagram.

(18) FIG. 17 is a representation of the effects of the supplement on the expression of IL-6 in the cortex (left-side diagram) and the plasma (right-side diagram) induced by the D-Galactose.

DETAILED DESCRIPTION AND EXAMPLES

Example 1: Formulation of an Extract Containing the Constituents of a Composition of the Disclosure

(19) An extract is obtained according to any of the above-described techniques from the microalga Phaeodactylum tricornutum.

(20) It is water-insoluble and is highly viscous preventing any handling at room temperature.

(21) The extract and the palm oil are kept at room temperature (25±1° C.) for 24 h before preparation. The extract is transferred in a centrifuge tube including the oil such that the final net mass of the mixture is about 5 g and the mass proportion is such that the extract consists of 25% of the overall net mass of the mixture. The mixture is stirred for one unit using a so-called vortex mixing device. Stirring is repeated three times for each mixture. A homogeneous mixture is obtained.

Example 2: Test of a Natural Extract of the Microalga Tisochrysis lutea in the Context of the In Vivo Model on the Attenuation of the Deficits Induced by the Age-Related Cognitive Decline

(22) The food supplement of the disclosure is prepared from a Tisochrysis lutea extract which comprises in mg/g:

(23) Omega-3 type fatty acids (ALA, SDA, EPA, DHA): 152.6±14.4;

(24) Fucoxanthin: 20.0±4.0;

(25) Sterols: 4.9±0.8;

(26) Phycoprostane: 0.035±0.007.

(27) The supplement is obtained by addition of coconut oil in a proportion of 360 mg±10 mg/g to said extract.

(28) The supplement is incorporated into kibbles according to 3 different formulations such that the final concentrations of DHA in these batches of kibbles are equal to 0.5, 1.5 and 3.0% (m:m).

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

(30) An additional batch of kibbles is formulated only with the coconut oil, such that the vehicle concentration is equivalent to that of the other batches, namely 0.01% (m:m).

(31) The five batches of kibbles thus obtained are referenced as described in Table 1 hereinbelow.

(32) TABLE-US-00001 TABLE 1 Formulated kibbles Reference [DHA] in % (m:m) Coconut oil A1 0 Supplement A2 0.5 Supplement A3 1.5 Supplement A4 3.0 Commercial oily extract A5 3.1

(33) The considered in vivo model is the D-Galactose model applied to mice which is suitable for the study of the age-related cognitive decline. Indeed, this model mimics numerous behavioral and molecular characteristics of the cerebral ageing in rodents' models.

(34) The D-Galactose is administered subcutaneously in a daily proportion of 150 mg/kg of mice wet weight, and the food supplement hereinabove is incorporated into a pellet, according to the following pattern:

(35) Between day -14 and day 51, the supplement is administered by incorporation into food pellets;

(36) Between day 1 and day 51, the D-Galactose is administered subcutaneously, five days a week;

(37) Between days 43 and 51, three different behavioral tests are used to monitor the effects of the test compounds.

(38) The effectiveness of the supplement is assessed according to the following parameters:

(39) improvement of the learning deficits (spatial working memory: spontaneous alternation in the Y labyrinth according to 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 effect on the neuro-inflammation markers IL6 and TNFα.

Improvement of the Learning Deficits

(40) On day 43, all animals have been tested for the spontaneous alternation performance in the Y-maze (YM) test, via a spatial working memory index;

(41) From day 44 to day 49, all animals have been tested for the spatial memory in the Morris Water Maze (MWM) test, via a spatial memory index;

(42) From day 44 to day 49, all animals are tested via the MWM test to assess the spatial working memory;

(43) On days 50 and 51, the long-term contextual memory of the animals is assessed using the step-by-step type passive avoidance process (STPA), through exercise and retention sessions, respectively;

(44) On days 50 and 51, all animals are tested for the STPA task.

Lipid Peroxidation (LPO) Rate in the Hippocampus and Effect on the Neuro-Inflammation Markers IL6 and TNFα

(45) On the 51th day, after the behavioral tests, the animals have been euthanized.

(46) For all animals, trunk blood is collected and centrifuged to recover plasma and the brain is rapidly collected. The hippocampus and the cortex are dissected, the hippocampus is then used to determine the lipid peroxidation rates by a colorimetric method; the hemi-frontal cortex and the plasma are used to determine the level of the inflammatory biomarkers interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α)

(47) The quantification of the lipid peroxidation (LPO) rates has been carried out according to the modified and adapted procedure of Hermes-Lima et al. This method measures the capacity of the peroxidized lipids of the brain to oxidize a ferrous oxide and xylenol orange complex, set out in the presence of cumene hydroperoxide (HPC). The lipid peroxidation level is determined in HPC-equivalent according to the formula:
HPCE=A5801/A5802×[HPC (nmol)]

(48) and expressed in HPC-equivalent per wet tissue weight and in percentage with respect to the data obtained for the control group (D-Galactose+vehicle).

(49) The IL6 and TNFα contents are quantified by means of ELISA tests with the following kits:

(50) For the quantification of IL6: ThermoScientifique, EM2IL6

(51) For the quantification of TNFα: ThermoScientifique, EMTNFA

(52) For all tests, the cortex is homogenized after defrosting in a buffer of 50 mM Tris-150 mM NaCl, pH 7.5, and sonicated for 20 s. After centrifugation (16 100 g for 15 min, 4° C.), a supernatant or plasma are used for the ELISA tests in compliance with the instructions of the manufacturer of the ELISA tests. For each test, the absorbance is read at 450 nm and the concentration of the sample is calculated using the standard curve. The results are expressed in pg of marker per mg of wet tissue.

(53) All values, except the passive avoidance latencies, are expressed as an average more or less the standard deviation of the measurement. Statistical analyses are performed separately for each compound using a unidirectional ANOVA (value F), followed by a Dunnett post-hoc multiple comparison test. The passive avoidance latencies do not follow a Gaussian distribution, since the upper limit times are fixed. Hence, they are analyzed using a Kruskal-Wallis non-parametric ANOVA (value H), followed by Dunn multiple comparison test. The values with p<0.05 are considered as statistically significant.

(54) The tests are performed on 60 male mice, distributed in 6 groups of 10 mice, amongst which the group 1 is the negative control group and the groups 2-6 are the positive control groups:

(55) the group 1 is the group to which a subcutaneous saline solution is administered instead of D-Galactose and kibbles A1;

(56) the group 2 is the group to which D-Galactose and kibbles A1 are administered;

(57) the group 3 is the group to which D-Galactose and kibbles A2 are administered;

(58) the group 4 is the group to which D-Galactose and kibbles A3 are administered; and

(59) the group 5 is the group to which D-Galactose and kibbles A4 are administered; and

(60) the group 6 is the group to which D-Galactose and kibbles A5 are administered.

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

Effects on the Spatial Memory in the Y-Maze Spontaneous Alternation Test

(62) The results are represented in FIG. 1, the first diagram (to the left) illustrating the effects of the supplement of the disclosure on the spontaneous alternation deficits and the second diagram (to the right) illustrating the effects of the supplement of the disclosure on the locomotor activity.

(63) In FIG. 1: LOW, low dose of the supplement (A2); MED, medium dose of the supplement (A3); HI, high dose of the supplement (A4); N is comprised between 9 and 10 depending on the groups; * p<0.05, *** p<0.0001 vs. the saline solution/group Veh, # p<0.05, ## p<0.01, ### p<0.0001 vs. the group D-GAL 150/group Veh; Dunnett test.

(64) It is observed that the treatment with D-Galactose has significantly altered the spatial working memory, in comparison with the mice treated with the saline solution.

(65) The supplement A2 has not demonstrated any effect on the alternation behavior. The supplement A3 has quite significantly but partially attenuated the deficits induced by the chronic intoxication with D-Galactose. The supplement A4 has quite significantly and completely attenuated the deficits induced by the chronic intoxication with D-Galactose.

(66) The treatment with DHA alone (according to A5) has quite significantly but partially alleviated the deficits induced by the chronic intoxication with D-Gal.

(67) Surprisingly, it turns out that the preventive treatment with the supplement according to the disclosure has a more considerable positive effect (quite significant and complete attenuation of the deficits) in comparison with the treatment with DHA alone (quite significant and partial attenuation of the deficits), and that for the same dose of DHA.

Effects on the Learning Deficits Induced by the D-Gal According to the MWM Test

(68) The results are represented in FIG. 2.

(69) In FIG. 2: LOW, low dose of the supplement (A2); MED, medium dose of the supplement (A3); HI, high dose of the supplement (A4); N is comprised between 9 and 10 depending on the groups; * p<0.05, ** p<0.01, *** p<0.0001 vs. the saline solution/group Veh, ## p<0.01, ### p<0.0001 vs. the group D-GAL 150/group Veh; Bonferroni multiple comparison test after bidirectional ANOVA.

(70) The chronic intoxication with D-Galactose has considerably altered the spatial learning, in comparison with the negative control group (saline solution/vehicle).

(71) The supplement A2 has not demonstrated any effect on the alternation behavior.

(72) The supplement A3 has quite significantly but partially attenuated the deficits induced by the chronic intoxication with D-Galactose.

(73) The supplement A4 has quite significantly and completely attenuated the deficits induced by the chronic intoxication with D-Galactose.

(74) The DHA alone according to A5 has quite significantly but partially alleviated the deficits induced by the chronic intoxication with D-Galactose.

(75) Surprisingly, it turns out that the preventive treatment with the supplement according to the disclosure at the dose A4 has a more considerable positive effect (quite significant and complete attenuation of the deficits) in comparison with the treatment with DHA alone (quite significant and partial attenuation of the deficits), and that for the same dose of DHA.

Effects of the Supplement and of the DHA on the Learning Deficits Induced by the D-Galactose

(76) The results are represented in FIG. 3.

(77) In FIG. 3: LOW, low dose of the supplement (A2); MED, medium dose of the supplement (A3); HI, high dose of the supplement (A4); N is comprised between 9 and 10 depending on the group; *** p<0.0001 vs. the saline solution/group Veh,/Veh; ### p<0.0001 vs. the group D-GAL 150/Veh; Bonferroni multiple comparison test after bidirectional ANOVA.

(78) The chronic intoxication with D-Galactose has considerably altered the spatial learning, in comparison with the negative control group (saline solution/vehicle).

(79) The supplement A2 has not demonstrated any effect on the alternation behavior.

(80) The supplement A3 has quite significantly but partially attenuated the deficits induced by the chronic intoxication with D-Galactose.

(81) The supplement A4 has quite significantly and completely alleviated the deficits induced by the chronic intoxication with D-Galactose.

(82) The treatment with DHA alone according to A5 has quite significantly but partially alleviated the deficits induced by the chronic intoxication with D-Galactose.

(83) Surprisingly, it turns out that the preventive treatment with the supplement according to the disclosure at the dose A4 has a more considerable positive effect (quite significant and complete attenuation of the deficits) in comparison with the treatment with DHA alone (quite significant and partial attenuation of the deficits), and that for the same dose of DHA. In addition, the preventive treatment with the supplement at the dose A3 has an effect (quite significant and partial attenuation of the deficits) that is identical to the treatment with DHA alone while the latter is two times more concentrated in DHA.

Effects on the Passive Avoidance Deficits Induced by the D-Galactose in Mice

(84) The results are represented in FIG. 4, with the effects of the supplement of the disclosure on the step-down latency illustrated on the left-side diagram and on the escape latency illustrated on the right-side diagram, measured during the retention period.

(85) In FIG. 4: LOW, low dose of the supplement (A2); MED, medium dose of the supplement (A3); HI, high dose of the supplement (A4). N is comprised between 9 and 10 depending on the groups; *** p<0.0001 vs. the saline solution/group Veh, ### p<0.0001 vs. the group D-GAL 150/group Veh; Dunnett test.

(86) The chronic intoxication with D-Galactose has considerably altered the long-term contextual working memory, in comparison with the negative control group (saline solution/vehicle).

(87) The supplement A2 has not demonstrated any effect on the long-term contextual memory.

(88) The supplement A3 has allowed attenuating the deficits induced by the chronic intoxication with D-Galactose in a non-significant manner.

(89) The supplement A4 has quite significantly and completely attenuated the deficits induced by the chronic intoxication with D-Galactose.

(90) The treatment with DHA alone (according to A5) has allowed attenuating the deficits induced by the chronic intoxication with D-Galactose in a non-significant manner.

(91) Surprisingly, it turns out that the preventive treatment with the supplement of the disclosure at the dose A4 has a more considerable positive effect (quite significant and complete attenuation of the deficits) in comparison with the treatment with DHA alone (non-significant attenuation of the deficits), and that for the same dose of DHA. In addition, the preventive treatment with the supplement at the dose A3 has an effect (non-significant attenuation of the deficits) that is identical to the treatment with DHA alone while the latter is two times more concentrated in DHA.

Effects of the Supplement and of the DHA on the Lipid Peroxidation Induced by the D-Galactose

(92) The results are represented in FIG. 5.

(93) In FIG. 5: LOW, low dose of the supplement (A2); MED, medium dose of the supplement (A3); HI, high dose of the supplement (A4). N is comprised between 9 and 10 depending on the groups; ** p<0.01, *** p<0.0001 vs. the saline solution/group Veh, ## p<0.01, ### p<0.0001 vs. the group D-GAL 150/group Veh; Dunnett test.

(94) The chronic intoxication with D-Galactose has considerably increased the oxidative stress, in comparison with the negative control group (saline solution/vehicle).

(95) The supplement A2 has not demonstrated any effect on the lipid peroxidation induced by the chronic intoxication with D-Galactose.

(96) The supplement A3 has quite significantly but partially reduced the oxidative stress induced by the chronic intoxication with D-Galactose.

(97) The supplement A4 has quite significantly and completely reduced the oxidative stress induced by the chronic intoxication with D-Galactose.

(98) The treatment with DHA alone according to A5 has not demonstrated any effect on the oxidative stress induced by the chronic intoxication with D-Galactose.

(99) Surprisingly, it turns out that the preventive treatment with the supplement of the disclosure at the dose A4 has a more considerable positive effect (quite significant and complete attenuation of the oxidative stress) in comparison with the treatment with DHA alone, and that for the same dose of DHA.

Effects of the Supplement and of the DHA on the Induced Expression of TNF-α in the Cortex and the Plasma by the D-Galactose

(100) The results are represented in FIG. 6, with the effect on the cortex on the left-side diagram and the effect on the plasma on the right-side diagram.

(101) In FIG. 6: LOW, low dose of the supplement (A2); MED, medium dose of the supplement (A3); HI, high dose of the supplement (A4); N is comprised between 9 and 10 depending on the groups; *** p<0.0001 vs. the saline solution/group Veh, ### p<0.0001 vs. the group D-GAL 150/group Veh; Dunnett test.

(102) The chronic intoxication with D-Galactose has considerably increased in a significant manner the TNF-α in the cortex and the plasma, in comparison with the negative control group (saline solution/vehicle).

(103) The supplements A2 and A3 have quite significantly but partially reduced the increase of the TNF-α induced by the chronic intoxication with D-Galactose in the cortex and the plasma.

(104) The supplement A4 has quite significantly and completely reduced the level of the TNF-a in the cortex and the plasma.

(105) The treatment with DHA alone according to A5 has quite significantly but partially reduced the increase of the TNF-α induced by the chronic intoxication with D-Galactose in the cortex and the plasma.

(106) Surprisingly, it turns out that the preventive treatment with the supplement HI (A4) has a more considerable positive effect (quite significant and complete attenuation of the increase of the TNF-α in the cortex and in the plasma) in comparison with the treatment with DHA alone, and that for the same dose of DHA. In addition, the preventive treatments with the supplement at the doses A2 and A3 have effects in the case of the cortex and the plasma (quite significant attenuations of the increases) that are identical to the treatment with DHA alone while the latter is respectively six and two times more concentrated in DHA, in comparison with the preventive treatments with the supplement at the doses A2 and A3, respectively.

Effects of the Supplement and of the DHA on the Induced Expression of IL-6 in the Cortex and the Plasma by the D-Galactose

(107) The results are represented in FIG. 7, with the effect on the cortex on the left-side diagram and the effect on the plasma on the right-side diagram.

(108) In FIG. 7: LOW, low dose of the supplement (A2); MED, medium dose of the supplement (A3); HI, high dose of the supplement (A4); N is comprised between 9 and 10 depending on the groups; *** p<0.0001 vs. the saline solution/group Veh, ### p<0.0001 vs. the group D-GAL 150/group Veh; Dunnett test.

(109) The chronic intoxication with D-Galactose has considerably increased in a significant manner the IL-6 in the cortex and the plasma, in comparison with the negative control group (saline solution/vehicle).

(110) The low-dose supplement (A2) has not demonstrated any effect on the concentration of IL-6 induced by intoxication with D-Galactose.

(111) The medium-dose supplement (A3) has quite significantly but partially reduced the increase of the IL-6 content induced by the chronic intoxication with D-Galactose in the cortex and the plasma.

(112) The high-dose supplement (A4) has quite significantly and completely reduced the IL-6 content in the cortex and the plasma, induced by the chronic intoxication with D-Galactose in the cortex and the plasma.

(113) The treatment with DHA alone according to A5 has quite significantly but partially reduced the increase of the IL-6 induced by the chronic intoxication with D-Galactose in the cortex and the plasma.

(114) Surprisingly, it turns out that the preventive treatment with the supplement HI according to A4 has a more considerable positive effect (quite significant and complete attenuation of the increase of the IL-6 in the cortex and in the plasma) in comparison with the treatment with DHA alone (quite significant and partial attenuation of the increase of the IL-6 in the cortex and in the plasma), and that for the same dose of DHA. In addition, the preventive treatment with the supplement at the dose A3 has an effect in the case of the cortex (non-significant attenuation of the deficits) that is identical to the treatment with DHA alone while the latter is two times more concentrated in DHA.

(115) In conclusion:

(116) The chronic intoxication with D-Galactose has quite significantly induced an alteration of the spatial working memory, of the long-term contextual memory and has prejudiced the spatial learning. The behavioral alterations are also related to biochemical alterations manifested in an increase of oxidative stress and in an induction of neuro-inflammatory processes.

(117) The preventive treatment with the supplement of the disclosure is dose-dependent and has quite significantly and completely attenuated in the case of the strongest tested dose (the supplement D4) the deficits induced by the chronic intoxication with D-Galactose manifested in a behavioral alteration, an increase of oxidative stress and an activation of neuro-inflammatory processes.

(118) The treatment with DHA alone (A5), and at an equivalent dose of DHA in comparison with the treatment with the supplement HI, has quite significantly but partially reduced the deficits induced by the chronic intoxication with D-Galactose manifested in a behavioral alteration, an increase of oxidative stress and an activation of neuro-inflammatory processes.

(119) Surprisingly, the preventive treatment by the supplement is significantly more effective at an equivalent dose of DHA than the preventive treatment with DHA alone, with regards to the attenuation of the age-related cognitive decline on a murine model caused by a chronic intoxication with D-Galactose. In addition, the preventive treatment by the supplement, for doses of DHA that are two times lower, has positive effects which are identical to the treatment with DHA alone, while the latter is two times more concentrated in DHA, in the case of the decrease of the oxidative stress measured in the cortex (IL-6 and TNF-α), in the plasma (TNF-α), in the case of the long-term contextual memory and in the case of spatial learning. And, the preventive treatment by the supplement has positive effects which are identical to the treatment with DHA alone, while the latter is six times more concentrated in DHA, in the case of the decrease of the oxidative stress measured in the cortex (IL-6 and TNF-α) and the plasma (TNF-α).

(120) Thus, by applying the formula for calculating the human equivalent daily dose, a preventive treatment of the age-related cognitive decline may be defined with the daily intake of 2 to 5 mg of supplement/kg of body weight.

Example 3: Test of an Extract of the Microalga Tisochrysis lutea in Young Female Rats Suffering from a Prenatal Stress

(121) In this example, the resolution of the cognitive deficits, the anxious behavior and the alteration of the recognition memory, induced in young female rats after a prenatal stress of the ancestry thereof, via the administration of a supplement based on an extract of the microalga Tisochrysis lutea corresponding to that used in Example 2, is studied

Equipment and Methods

(122) The model used in this example is an acknowledged model for inducing prenatal stress in rats by immobilizing the pregnant female in a cylinder under a harsh lighting.

(123) Pregnant female rats have been randomly assigned to prenatal stress group (SP) or to control groups (NS), individually put in plastic-made breeding cages, and have had ad libitum access to food and water, except during the behavioral tests time. The conditions within the cages are as follows: photoperiod 12 h of light/12 h of darkness cycle (light turned on at 7 AM), in a room at constant temperature (21° C.) and constant humidity (50%).

(124) The prenatal stress procedure has been performed as described by Meunier et al. (2004). The immobilizations of the female rats have been the object of a semi-random constraining procedure. The animals have been place and retained in Plexiglas-made transparent ferret retainers (20 cm long, 7 cm diameter) under a bright light for a total period of 90 minutes a day, for 4 consecutive days. For the stress to be as unpredictable as possible, the 90 minutes forced immobilization period has been administered in the following manner: one single 90 min phase, two 45 min phases spaced by 4 h, two 60 and 30 min phases spaced by 4 h, or three 30 min phases spaced by 4 and 1 h, and that, at different times of the day.

(125) Control mothers have also been manipulated, but have never been placed in the ferret retainers.

(126) The treated female rats have been allowed to clear themselves naturally from the trap of the ferret retainers as of the day 1 after the birth (PPD1).

(127) The litters have been weaned on PPD21. The rats have been separated from the mothers, identified according to their gender, weighted and rats of the same gender have been distributed in cages (3 rats per cage). The young rats within the same cage originated from different litters, in order to avoid any possible litter-related effect.

(128) The conditions within the cages are as follows: photoperiod 12 h of light/12 h of darkness cycle (light turned on at 7 AM), in a room at constant temperature (21° C.) and constant humidity (50%), with ad libitum access to food and water, except during the behavioral tests time.

(129) In each cage, the animals have received the same treatment. The animals have been tested randomly and in a double-blind manner.

(130) Forty-eight (48) female rats have been used and grouped into four groups of animals, constituted in the following manner:

(131) The group 1 is composed by 12 naïve female rats, that is to say whose ancestry has not been subjected to a prenatal stress, and receiving only 200 μL a day of the vehicle solution (reference: NS/Vehicle). Hence, this group is the control group;

(132) The group 2 is composed by 12 naïve female rats, that is to say whose ancestry has not been subjected to a prenatal stress, and receiving 200 μL a day of the supplement (reference: p NS/Supplement);

(133) The group 3 is composed by 12 female rats whose ancestry has been subjected to a prenatal stress, and receiving 200 μL a day of the vehicle solution (reference: SP/Vehicle);

(134) The group 4 is composed by 12 female rats whose ancestry has been subjected to a prenatal stress, and receiving 200 μL a day of the supplement (reference: SP/Supplement);

(135) The effectiveness of the supplement has been assessed 6 weeks after birth.

(136) The supplement (one dose) has been administered by gavage once every day, 5 days a week. The administrations have started after weaning, namely after the postpartum day (PPD) 25, and has lasted until PPD46.

(137) The daily intake was 25.7 mg of supplement per kg of rat body weight.

(138) The animals have been subjected to behavioral tests during the period between the days PPD46 and PPD48, namely outside the period of treatment with the vehicle or the supplement. Hence, the effects that are therefore observed during the behavioral tests will be due to a treatment that is preventive in nature.

(139) The behavioral tests are divided into one anxiety assessment session and two objects recognition sessions. The sessions are defined as follows:

(140) Session 1, PPD 46: The rats have been individually placed in a square open space (50 cm×50 cm×50 cm×50 cm) made of blue-colored Plexiglas with a floor equipped with infrared light-emitting diodes. The rats have been accustomed to the test space during a 10 minute session and their displacements captured by an infrared camera and analyzed using the Ethovision® (Noldus) software. The activity has been analyzed according to the overall covered distance (m), and according to the percentage of presence in the 25 cm×25 cm central area defined by the software/these data report on the intensity of the anxious behavior (38).

(141) Session 2, PPD 47: Two identical objects (50 mL plastic-made Eppendorf tube) have been placed at determined locations (on two opposite edges of the central area). Each rat has been placed within the test space and the exploratory activity recorded during a 10 minute session. The activity has been analyzed in terms of number of contacts with the objects and of duration of the contacts.

(142) Session 3, PPD 48: The object of session 2 has been replaced with a new object (a plastic-made bottle cap) whose shape, texture, color differ from those of the familiar object. Each rat has been replaced in the test space and the exploratory activity has been recorded during a 10 minute session. The activity has been the object of an analysis similar to that described in session 2.

(143) The preferential exploration index has been calculated as the ratio of the number (or duration) of contacts with the object of session 2, to the overall number (or duration) of contacts with both objects.

(144) All values are expressed as an average more or less the standard deviation of the measurement. Statistical analyses are performed separately for each compound using a unidirectional ANOVA (value F), followed by a Dunnett post-hoc multiple comparison test.

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

(146) The results are presented hereinafter.

Locomotion at the Center of the Test Space, Day PPD46, Effect of the Supplement on Anxiety

(147) The results are represented in FIG. 8.

(148) In FIG. 8: Effects of the treatment on anxiety. N=12; *** p<0.0001 with respect to the treated group NS/vehicle; #### p<0.0001 with respect to the treated group SP/vehicle; Dunnett test.

(149) The group SP/Vehicle, corresponding to the individuals having been subjected to a prenatal stress and preventively treated with the vehicle alone, has a percentage of displacements that is quite significantly higher within the peripheral area of the open test space in comparison with the group NS/Vehicle (the group that has not been subjected to a prenatal stress).

(150) The group SP/supplement, corresponding to the individuals having been subjected to a prenatal stress and preventively treated with the supplement has a percentage of displacements that is quite significantly lower within the peripheral area of the open test space in comparison with the group SP/Vehicle (the group that has been subjected to a prenatal stress but not treated with the supplement). In addition, the percentage of displacements of the group SP/supplement is equivalent to that of the control group NS/vehicle.

(151) A rate of displacements of the individuals in the peripheral area of the open test space that is higher than the control modality is the demonstration of an anxious behavior [63], via a protection mechanism based on the search for boundaries limiting the uncovered areas and is to be monitored.

(152) Thus, the prenatal stress (PS) has induced a very significant anxious behavior.

(153) Surprisingly, it turns out that the supplement has quite significantly and completely attenuated the anxious behavior induced by the prenatal stress.

Recognition Test, Day PPD47; Effect of the Supplement on the Recognition Memory in the Recognition of an Object

(154) The results are represented in FIG. 9.

(155) During this session, the same object is exhibited twice to the individuals.

(156) No statistical effect between the groups has been measured for this parameter.

(157) Thus, the individuals of all groups have interacted in an equivalent manner when put in contact with identical objects and their interactions, both in terms of frequency and duration are equally distributed between the two objects (50%).

Recognition Test, Day PPD48 (new object); Effect of the Supplement on the Recognition Memory for the Test of Recognition of a New Object

(158) The results are represented in FIG. 10.

(159) In FIG. 10: N=12; *** p<0.0001 with respect to the treated group NS/vehicle; ### p<0.0001 with respect to the treated group SP/vehicle; Dunnett test.

(160) During this session, two different objects are exhibited to the individuals each once: one of the objects corresponds to the object exhibited during the session 2 and the other object is a new object.

(161) The group SP/vehicle, corresponding to the individuals having been subjected to a prenatal stress and preventively treated with the vehicle alone, has a percentage of interactions, both in terms of frequency and duration, with the exhibited new object that is quite significantly lower in comparison with the group NS/Vehicle (the group that has not been subjected to a prenatal stress). And this percentage is equal to that of the session 2 obtained for all groups. Thus, the individuals of the group SP/vehicle have as many interactions with the ancient object as with the new object and therefore the individuals of this group do not recognize the ancient object exhibited during the session 2.

(162) In contrast, the group SP/Supplement corresponding to the individuals having been subjected to a prenatal stress and preventively treated with the supplement, has a percentage of interactions, both in terms of frequency and duration, with the exhibited new object that is quite significantly higher in comparison with the group PS/Vehicle (the negative control group). And this percentage is higher than that of the session 2 obtained for all groups. Thus, the individuals of the group SP/Supplement have less interactions with the ancient object than with the new object, and therefore the individuals of this group recognize the ancient object exhibited during the session 2. In addition, the individuals of the group SP/Supplement have a percentage of interactions, both in terms of frequency and duration, with the exhibited new object that is equivalent with those of the groups NS/Vehicle and NS/Supplement.

(163) Thus, the prenatal stress (PS) has induced very considerable recognition memory deficits in the case of the new object.

(164) Surprisingly, it turns out that the supplement has allowed attenuating quite significantly and completely the recognition memory deficits induced by the prenatal stress.

(165) In conclusion:

(166) The treatment with the supplement has significantly and completely attenuated the anxious behavior as well as the recognition memory deficits induced by the prenatal stress.

(167) The prenatal stress as practiced in this experiment significantly induces an anxious behavior, and quite significantly alters the recognition memory in young female rats.

(168) Thus, by applying the formula for calculating the human equivalent daily dose, a preventive treatment attenuating the cognitive disorders caused by a prenatal stress may be defined with the daily intake of 0.05 to 0.1 mg of supplement/kg of body weight.

Example 4: Test of a Natural Extract of the Microal2a Phaeodactylum tricornutum in the Context of the In Vivo Model on the Attenuation of the Deficits Induced by the Age-Related Cognitive Decline

(169) The food supplement of the disclosure is prepared from a Phaeodactylum tricornutum extract which comprises in mg/g:

(170) Omega-3 type fatty acids (ALA, SDA, EPA, DHA): 66.6 ±11.5;

(171) Fucoxanthin: 20.0±4.0;

(172) Sterols: 3.0±0.6;

(173) Phycoprostane: 0.0025±0.0005.

(174) The supplement is obtained by addition of coconut oil in a proportion of 410 mg±20 mg/g to said extract.

(175) The supplement is incorporated into kibbles according to 4 different formulations such that the incorporated amounts of the supplement within the different batches of kibbles correspond to human equivalent daily doses as described in Table 2, and that, by dilution of the composition described hereinbelow in coconut oil with an equal final mass for all formulations.

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

(177) An additional batch of kibbles is formulated only with the coconut oil, such that the vehicle concentration is equivalent to that of the other batches, namely 0.01% (m:m).

(178) The five batches of kibbles thus obtained are referenced as described in Table 2 hereinbelow.

(179) TABLE-US-00002 TABLE 2 Human equivalent ssdupplement dose (mg of supplement/ Formulated kibbles kg of body mass/day) Reference Coconut oil (vehicle) 0 Veh. Supplement 1.7 D1 Supplement 3.3 D2 Supplement 4.2 D3 Supplement 5.3 D4

(180) The considered in vivo model is the D-Galactose model applied to mice which is suitable for the study of the age-related cognitive decline. Indeed, this model mimics numerous behavioral and molecular characteristics of the cerebral ageing in rodents' models.

(181) The D-Galactose is administered subcutaneously in a daily proportion of 150 mg/kg of mice wet weight, and the food supplement hereinabove is incorporated into a pellet, according to the following pattern:

(182) Between day -28 and day 51, the supplement is administered by incorporation into food pellets;

(183) Between day 1 and day 51, the D-Galactose is administered subcutaneously, five days a week;

(184) Between days 43 and 51, three different behavioral tests are used to monitor the effects of the test compounds.

(185) The effectiveness of the supplement is assessed according to the following parameters: improvement of the learning deficits (spatial working memory: spontaneous alternation in the Y labyrinth according to 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 effect on the neuro-inflammation markers IL6 and TNFα.

Improvement of the Learning Deficits

(186) On day 43, all animals have been tested for the spontaneous alternation performance in the Y-maze (YM) test, via a spatial working memory index;

(187) From day 44 to day 49, all animals have been tested for the spatial memory in the Morris Water Maze (MWM) test, via a spatial memory index;

(188) From day 44 to day 49, all animals are tested via the MWM test to assess the spatial working memory;

(189) On days 50 and 51, the long-term contextual memory of the animals is assessed using the step-by-step type passive avoidance process (STPA), through exercise and retention sessions, respectively.

Lipid Peroxidation (LPO) Rate in the Hippocampus and Effect on the Neuro-Inflammation Markers IL6 and TNFα

(190) On the 51th day, after the behavioral tests, the animals have been euthanized.

(191) For all animals, trunk blood is collected and centrifuged to recover plasma and the brain is rapidly collected. The hippocampus and the cortex are dissected, the hippocampus is then used to determine the lipid peroxidation rates by a colorimetric method; the hemi-frontal cortex and the plasma are used to determine the level of the inflammatory biomarkers interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α)

(192) The quantification of the lipid peroxidation (LPO) rates has been carried out according to the modified and adapted procedure of Hermes-Lima et al. This method measures the capacity of the peroxidized lipids of the brain to oxidize a ferrous oxide and xylenol orange complex, set out in the presence of cumene hydroperoxide (HPC). The lipid peroxidation level is determined in HPC-equivalent according to the formula:
HPCE=A5801/A5802×[HPC (nmol)]

(193) and expressed in HPC-equivalent per wet tissue weight and in percentage with respect to the data obtained for the control group (D-Galactose+vehicle).

(194) The IL6 and TNFα contents are quantified by means of ELISA tests with the following kits:

(195) For the quantification of IL6: ThermoScientifique, EM2IL6

(196) For the quantification of TNFα: ThermoScientifique, EMTNFA

(197) For all tests, the cortex is homogenized after defrosting in a buffer of 50 mM Tris-150 mM NaCl, pH 7.5, and sonicated for 20 s. After centrifugation (16 100 g for 15 min, 4° C.), a supernatant or plasma are used for the ELISA tests in compliance with the instructions of the manufacturer of the ELISA tests. For each test, the absorbance is read at 450 nm and the concentration of the sample is calculated using the standard curve. The results are expressed in pg of marker per mg of wet tissue.

(198) All values, except the passive avoidance latencies, are expressed as an average more or less the standard deviation of the measurement. Statistical analyses are performed separately for each compound using a unidirectional ANOVA (value F), followed by a Dunnett post-hoc multiple comparison test. The passive avoidance latencies do not follow a Gaussian distribution, since the upper limit times are fixed. Hence, they are analyzed using a Kruskal-Wallis non-parametric ANOVA (value H), followed by Dunn multiple comparison test. The values with p<0.05 are considered as statistically significant.

(199) The tests are performed on 72 male mice, distributed in 6 groups of 12 mice, amongst which the group 1 is the negative control group and the groups 2-6 are the positive control groups:

(200) the group 1 is the group to which a subcutaneous saline solution is administered instead of D-Galactose and kibbles B1;

(201) the group 2 is the group to which D-Galactose and kibbles B1 are administered;

(202) the group 3 is the group to which D-Galactose and kibbles B2 are administered;

(203) the group 4 is the group to which D-Galactose and kibbles B3 are administered; and

(204) the group 5 is the group to which D-Galactose and kibbles B4 are administered; and

(205) the group 6 is the group to which D-Galactose and kibbles B5 are administered.

Effects on the Spatial Memory in the Y-maze Spontaneous Alternation Test

(206) The results are represented in FIG. 11, the first diagram (to the left) illustrating the effects of the supplement of the disclosure on the spontaneous alternation deficits and the second diagram (to the right) illustrating the effects of the supplement of the disclosure on the locomotor activity.

(207) In FIG. 11: Sol. Saline/veh corresponds to the negative control (the group that has not been treated with D-galactose and fed with the kibbles formulated with the vehicle, coconut oil); DGal 150/Veh corresponds to the positive control (the group that has been treated with D-Galactose and fed with the kibbles formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is comprised between 11 and 12 depending on the groups; * p<0.05, *** p<0.0001 vs. the saline solution/group Veh, # p<0.05, ## p<0.01, ### p<0.0001 vs. the group D-GAL 150/group Veh; Dunnett test.

(208) It is observed that the treatment with D-Galactose has significantly altered the spatial working memory, in comparison with the mice treated with the saline solution.

(209) The supplement D1 has quite significantly but partially attenuated the deficits induced by the chronic intoxication with D-Galactose. The supplements D2, D3 and D44 have quite significantly and completely attenuated the deficits induced by the chronic intoxication with D-Galactose.

Effects on the Learning Deficits Induced by the D-Gal According to the MWM Test

(210) The results are represented in FIG. 12.

(211) In FIG. 12: Sol. Saline/veh corresponds to the negative control (the group that has not been treated with D-galactose and fed with the kibbles formulated with the vehicle, coconut oil); DGal 150/Veh corresponds to the positive control (the group that has been treated with D-Galactose and fed with the kibbles formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is comprised between 11 and 12 depending on the groups; * p<0.05, ** p<0.01, *** p<0.0001 vs. the saline solution/group Veh, ## p<0.01, ### p<0.0001 vs. the group D-GAL 150/group Veh; Bonferroni multiple comparison test after bidirectional ANOVA.

(212) The chronic intoxication with D-Galactose has considerably altered the spatial learning, in comparison with the negative control group (saline solution/vehicle).

(213) The supplement D1 has quite significantly but partially attenuated the deficits induced by the chronic intoxication with D-Galactose.

(214) The supplements D2, D3 and D4 have quite significantly and completely attenuated the deficits induced by the chronic intoxication with D-Galactose.

Effects of the Supplement on the Learning Deficits Induced by the D-Galactose

(215) The results are represented in FIG. 13.

(216) In FIG. 13: Sol. Saline/veh corresponds to the negative control (the group that has not been treated with D-galactose and fed with the kibbles formulated with the vehicle, coconut oil); DGal 150/Veh corresponds to the positive control (the group that has been treated with D-Galactose and fed with the kibbles formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is comprised between 11 and 12 depending on the groups; Sol. Saline/veh corresponds to the negative control (the group that has not been treated with D-galactose and fed with the kibbles formulated with the vehicle, coconut oil); DGal 150/Veh corresponds to the positive control (the group that has been treated with D-Galactose and fed with the kibbles formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is comprised between 11 and 12 depending on the groups; *** p<0.0001 vs. the saline solution/group Veh, Neh; ### p<0.0001 vs. the group D-GAL 150/Veh; Bonferroni multiple comparison test after bidirectional ANOVA. “T”, the time spent in the target quadrant; “O”, the average time spent in the three other quadrants.

(217) The chronic intoxication with D-Galactose has considerably altered the spatial learning, in comparison with the negative control group (saline solution/vehicle).

(218) The supplements D1 and D2 have quite significantly but partially attenuated the deficits induced by the chronic intoxication with D-Galactose.

(219) The supplements D3 and D4 have quite significantly and completely alleviated the deficits induced by the chronic intoxication with D-Galactose.

Effects on the Passive Avoidance Deficits Induced by the D-Galactose in Mice

(220) The results are represented in FIG. 14, with the effects of the supplement of the disclosure on the step-down latency illustrated on the left-side diagram and on the escape latency illustrated on the right-side diagram, measured during the retention period.

(221) In FIG. 14: Sol. Saline/veh corresponds to the negative control (the group that has not been treated with D-galactose and fed with the kibbles formulated with the vehicle, coconut oil); DGal 150/Veh corresponds to the positive control (the group that has been treated with D-Galactose and fed with the kibbles formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is comprised between 11 and 12 depending on the groups; *** p<0.0001 vs. the saline solution/group Veh, ### p<0.0001 vs. the group D-GAL 150/group Veh; Dunnett test.

(222) The chronic intoxication with D-Galactose has considerably altered the long-term contextual working memory, in comparison with the negative control group (saline solution/vehicle).

(223) The supplement D1 has not demonstrated any effect on the long-term contextual memory.

(224) The supplements D2, D3 and D4 have quite significantly and completely attenuated the deficits induced by the chronic intoxication with D-Galactose.

Effects of the Supplement on the Lipid Peroxidation Induced by the D-Galactose

(225) The results are represented in FIG. 15.

(226) In FIG. 15: Sol. Saline/veh corresponds to the negative control (the group that has not been treated with D-galactose and fed with the kibbles formulated with the vehicle, coconut oil); DGal 150/Veh corresponds to the positive control (the group that has been treated with D-Galactose and fed with the kibbles formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is comprised between 11 and 12 depending on the groups; ** p<0.01, *** p<0.0001 vs. the saline solution/group Veh, ## p<0.01, ### p<0.0001 vs. the group D-GAL 150/group Veh; Dunnett test.

(227) The chronic intoxication with D-Galactose has considerably increased the oxidative stress, in comparison with the negative control group (saline solution/vehicle).

(228) The supplement D1 has quite significantly but partially reduced the oxidative stress induced by the chronic intoxication with D-Galactose.

(229) The supplements D2, D3 and D4 have quite significantly and completely reduced the oxidative stress induced by the chronic intoxication with D-Galactose.

Effects of the Supplement on the Induced Expression of TNF-α in the Cortex and the Plasma by the D-Galactose

(230) The results are represented in FIG. 16, with the effect on the cortex on the left-side diagram and the effect on the plasma on the right-side diagram.

(231) In FIG. 16: Sol. Saline/veh corresponds to the negative control (the group that has not been treated with D-galactose and fed with the kibbles formulated with the vehicle, coconut oil); DGal 150/Veh corresponds to the positive control (the group that has been treated with D-Galactose and fed with the kibbles formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is comprised between 11 and 12 depending on the groups; *** p<0.0001 vs. the saline solution/group Veh, ### p<0.0001 vs. the group D-GAL 150/group Veh; Dunnett test.

(232) The chronic intoxication with D-Galactose has considerably increased in a significant manner the TNF-α in the cortex and the plasma, in comparison with the negative control group (saline solution/vehicle).

(233) The supplement D1 has quite significantly but partially reduced the increase of the TNF-α induced by the chronic intoxication with D-Gal in the brain and the plasma.

(234) The supplement D2 has quite significantly and completely reduced the increase of the TNF-α induced by the chronic intoxication with D-Gal in the brain, but partially in the plasmas.

(235) The supplement D3 has quite significantly but partially reduced the increase of the TNF-α induced by the chronic intoxication with D-Gal in the brain, and completely in the plasma.

(236) The supplement D4 has quite significantly and completely reduced the increase of the TNF-α induced by the chronic intoxication with D-Gal in the brain and the plasma.

Effects of the Supplement on the Induced Expression of IL-6 in the Cortex and the Plasma by the D-Galactose

(237) The results are represented in FIG. 17, with the effect on the cortex on the left-side diagram and the effect on the plasma on the right-side diagram.

(238) In FIG. 17: Sol. Saline/veh corresponds to the negative control (the group that has not been treated with D-galactose and fed with the kibbles formulated with the vehicle, coconut oil); DGal 150/Veh corresponds to the positive control (the group that has been treated with D-Galactose and fed with the kibbles formulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N is comprised between 11 and 12 depending on the groups; *** p<0.0001 vs. the saline solution/group Veh, ### p<0.0001 vs. the group D-GAL 150/group Veh; Dunnett test.

(239) The chronic intoxication with D-Galactose has considerably increased in a significant manner the IL-6 in the cortex and the plasma, in comparison with the negative control group (saline solution/vehicle).

(240) The supplements D1 and D2 have quite significantly but partially reduced the increase of the IL-6 induced by the chronic intoxication with D-Gal in the brain and the plasmas.

(241) The supplements D3 and D4 have quite significantly and completely reduced the increase of the IL-6 induced by the chronic intoxication with D-Gal in the brain and the plasma.

In Conclusion

(242) The chronic intoxication with D-Galactose has quite significantly induced an alteration of the spatial working memory, of the long-term contextual memory and has prejudiced the spatial learning. The behavioral alterations are also related to biochemical alterations manifested in an increase of oxidative stress and in an induction of neuro-inflammatory processes.

(243) The preventive treatment with the supplement of the disclosure is dose-dependent and has quite significantly and completely attenuated in the case of the strongest tested dose (the supplement D4) the deficits induced by the chronic intoxication with D-Galactose manifested in a behavioral alteration, an increase of oxidative stress and an activation of neuro-inflammatory processes. And in the cases of the intermediate lower doses (the supplements D2 and D3), the supplement of the disclosure has quite significantly and completely attenuated the deficits induced by the chronic intoxication with D-Galactose manifested in the spatial working memory, an increase of oxidative stress and an activation of neuro-inflammatory processes.

(244) Thus, by applying the formula for calculating the human equivalent daily dose, a preventive treatment of the age-related cognitive decline may be defined with the daily intake of 1.7 to 5.3 mg of supplement/kg of body weight.