Patent Application
20240016866
The present disclosure concerns a composition, as well as a food supplement based on fatty acids and xanthophylls and the applications thereof in particular to prevent the apparition of cognitive disorders in humans or animals.
The cognitive processes are defined as all cerebral functions allowing acquiring, processing, memorizing and using data originating from the environment in order to maximize the advantages and minimize the drawbacks of the external constraints. Thus, the cognitive processes are implemented during phases involving reasoning (giving rise to planning, organization, judgment), perception, recognition, language, emotions, memory and learning.
Light cognitive disorders, or cognitive fragilities, are defined as alterations of the cognitive functions without dementia. From a clinical perspective, these disorders are associated to a 0.5 score in the context of an assessment via the CDR (Cognitive Drug Research Computerized Assessment System) test.
Amongst these cognitive disorders, a cognitive decline associated with ageing and cognitive alterations induced by a prenatal stress are two phenomena that might intervene throughout the life of an individual.
The age-related cognitive decline is defined as a non-pathological decrease in the cognitive functions such as the speed of information processing, the attentional ability and especially the so-called working (or short-term) memory. These processes result from normal physiological modifications directly related to the age. The age of the start of this decline is still controversial, but given the acceleration of the ageing of the world population, more than 20% of the world population being more than 60 years, and this percentage will be more than 30% in 2050, the age-related cognitive decline is amongst the major issues of these upcoming decades, at global level, and specifically in developed countries, in which it will considerably impact the economy (less autonomy for elderly persons) and public policies.
At the opposite of the age composition pyramid, cognitive disorders may affect infants and young children following a prenatal stress. Indeed, for several years now, the influence of stress during some period of the pregnancy on the cognitive development of the unborn individual has been studied both in humans and in animals. Thus, in animals, mainly in rats, it has been demonstrated that a prenatal stress in the mother would induce a progeny having an altered long-term memory.
It turns out that intense negative stimuli, stress, may induce non-pathological alterations or decreases in the cognitive functions of young children, manifested in an hyperactivity, an attention and memory deficit, language retardation, more difficult temperaments and more generally behavioral alterations such as an anxious behavior, revealing a neurodevelopmental retardation and a decrease in the cognitive abilities.
One amongst the presumed mechanisms of the expression of the prenatal stress into cognitive disorders, is based on the contact of the fetus with large doses of so-called stress hormones, belonging to the family of corticosteroids, such as cortisol. Yet, cortisol crosses the placental barrier and, starting from a determined concentration, the protective mechanisms of the fetus against the corticoids secreted by the mother, are saturated, thereby bringing the fetus in contact with too considerable doses of cortisol which seem to have a negative effect on the cognitive development. Other complementary assumptions explain the relationship between the prenatal stress and the cognitive disorders of the child, have been developed.
The stress concept may be defined according to different perspective such as the biological approach: stress is then a series of metabolic reactions, following one or several exogenous factor(s), inducing physiological or psychological changes (fear, anguish) in the organism. However, the stress concept and its impacts is largely individual-specific, and the response of the individual to a stress is also defined from a psychological perspective. Henceforth, bearing in mind that an event is stressful only retrospectively, because of a reaction that is specific to each individual, or because it may be objectively stressful, it is difficult to act on the sources of prenatal stress. Added to this is the fact that pregnancy induces hormonal and psychological changes increasing the sensitivity of the prospective mother to every event likely to impact the well-being of the unborn infant.
The strictly therapeutic approach of stress or anxiety, in the form of drugs prescription, is dangerous in the case of a pregnant woman: numerous psychotropic drugs for the treatment of psychological disorders or anxiety have teratogenic effects with direct adverse effects on the fetus. This requires an assessment on a case-by-case basis, and this approach is used only in the case of clinical psychological disorders in the pregnant woman and not in the case of so-called subjective stresses.
Thus, there is a major problem relating to the discovery of solutions against the consequences of prenatal stress on cognitive disorders in children or young adults.
Different studies have shown the interest of a nutritional supplementation of so-called essential fatty acids, but also of carotenes and in particular xanthophylls, and even the combination of said fatty acids and said carotenes, to prevent or at least limit the decline of the cognitive functions. Food supplements or drugs have been developed leading to positive results.
Thus, there is known according to the document WO2013/032333A1, a composition based on omega-3 type fatty acids, in particular the eicosapentaenoic acid (EPA) and the docosahexaenoic acid (DHA), asthaxanthin and glycerophospholipids, which is recommended for the prevention or the treatment of different disorders and in particular cognitive disorders. Said ingredients are present in this composition in the form of extracts of microalgae; in a preferred variant of preparation, they are obtained by formulation of two extracts originating from two different algae. The natural origin of the constituents of the composition is a tremendous benefit. Nonetheless, the need for more effective compositions still exists, in particular with regards to the aforementioned issues. Furthermore, it is important to provide methods for preparing such compositions that are simple and replicable.
The disclosure brings in a solution with a composition comprising one or several omega-3 type fatty acid(s) and one or several xanthophyll(s), as well as one or several compound(s) of the family of sterols and one or several phycoprostane(s). It turned out that the combination of at least one sterol and at least one phycoprostane with at least one omega-3 type fatty acid and at least one xanthophyll significantly increases the effectiveness of a composition in the prevention of the apparition of age-related cognitive disorders but also against those associated to a prenatal stress.
A composition of the disclosure comprises:
In a primary indication, a composition of the disclosure may be used as a food supplement. Also, the disclosure concerns a food supplement which comprises at least 25 mg/g, in particular at least 50 mg/g, of one or several omega-3 type fatty acid(s), at least 5 mg/g, in particular at least 10 mg/g, of one or several xanthophyll(s), at least 0.5 mg/g, in particular at least 1 mg/g, of one or several sterol(s), and at least 1 μg/g, in particular at least 2 μg/g of one or several phycoprostane(s).
The disclosure has an essential advantage in that all of the constituents or ingredients hereinabove may be obtained from a natural source and in particular they may be extracted from one or several microalga(e), and preferably from one single microalga. Of course, the constituent(s) or ingredient(s) of a composition or of a food supplement of the disclosure may have a non-natural origin and be provided in the form of chemically synthesized products.
The different embodiments of the disclosure are illustrated hereinbelow and their advantages set out in the following examples, with reference to the following figures:
Before disclosing the disclosure in more details, some terms used in the present text are defined.
The term «comprises» in the expression «a composition comprises» or «a food supplement comprises» means that the composition or the supplement may incorporate any additional constituent or more, which is not specifically mentioned, in any form and from any origin whatsoever. It also covers a composition or a supplement that would contain only the listed constituents and consequently the composition or the supplement would consist of said constituents.
A food supplement is defined as one or several foodstuff(s) whose purpose is to complete the normal diet of a human or of an animal, and which constitute a concentrated source of nutrients or other substances having a nutritional or physiological effect alone or in combination; it is generally available in the form of doses, namely the packaging forms such as gel capsules, pastilles, tablets, pills and other similar forms, as well as powder packs, ampoules of liquid, vials fitted with a dropper and the other similar forms of liquid or powder preparations intended to be taken in units measured in a small amount.
Omega-3 type fatty acid(s) are a family of unsaturated fatty acids whose hydrocarbon chain has 4 to 36 atoms of carbon, in general from 14 to 36 atoms of carbon, and whose double bond or whose first double bond, counted starting from the terminal methyl group of the chain, is on the third carbon-carbon bond. The unsaturation(s) may be of the cis or trans type, independently from one another. The most representative acids are the alpha-linolenic acid (ALA), the eicosapentaenoic acid (EPA) and the docosahexaenoic acid (DHA), but the designation «omega-3 type fatty acids» is not restricted thereto. Furthermore, and in particular when the fatty acid(s) are from a natural origin, they may be extracted from algae and be in the form of free molecules but also in a derivative form such as an esterified form, for example in a mono-, di- or tri-esterified form, or in mixtures of these forms.
By xanthophylls, are defined the molecules belonging to the carotenoids including one or several atom(s) of oxygen such as astaxanthin, canthaxanthin, vaucheriaxanthin, lutein, zeaxanthin, diadinoxanthin, neoxanthin, loroxanthin, siphonoxanthin, diatoxanthin, violaxanthin, dinoxanthin, flavoxanthin, α-cryptoxanthin, 6-cryptoxanthin and fucoxanthin. In particular, when the xanthophyll(s) are from a natural origin, they may be extracted from algae and be in the form of free molecules but also in a derivative form such as an esterified form of mono- or multi-esters, or in mixtures of these forms.
Sterol(s) are a family of well-known lipids having a sterane core whose 3rd position carbon carries a hydroxyl group, the latter may be modified for example by an acetyl group. These include natural sterols or phytosterols, and are grouped in the present text under the term phycosterols. Without limitation, as examples of phytosterols, mention may be made to 24-methylene cholesterol, 6-sitosterol, fucosterol, isofucosterol, saringosterol, oxocholesterol acetate, crinosterol, and more particularly brassicasterol, stigmasterol and campesterol.
By phycoprostanes, it should be understood a family of lipids that are structurally of the prostaglandin type, from a natural origin, resulting from non-directly enzymatic oxidations of the fatty acids naturally present within the microalgal biomasses. In particular, these compounds are selected from phytoprostanes, isoprostanes and neuroprostanes, depending on the fatty acid that has undergone the oxidation(s). Thus, these compounds may originate from fatty acids such as the α-linolenic acid (ALA), the arachidonic acid (ARA), the eicosapentaenoic acid (EPA), or the docosahexaenoic acid (DHA). The phytoprostanes are mainly derived from ALA and may be selected from 9-epi-9F1t-PhytoP, ent-16-epi-16-F1 t-PhytoP, 9-F1t-PhytoP, ent-16B1t-PhytoP, ent-9L1t-PhytoP, 16(RS)-16-A1t-PhytoP. The isoprostanes are mainly derived from ARA and EPA and may be selected from 15-E2t-IsoP, 15-F2t-IsoP, 15-epi-15-F2t-IsoP, 5-F2t-IsoP, 8(RS)-8-F3t-IsoP. The neuroprostanes are mainly derived from DHA and may 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.
By Medium-Chain Triglycerides (MCT), it should be understood esters of glycerol and saturated fatty acids, whose hydrocarbon chain has 6 to 12 atoms of carbon. They are naturally present in coconut palm oil such as coconut oil, palm kernel oil and palm oil, but they may be obtained from other greases or oils.
The present disclosure is described hereinafter in more details and its variants are disclosed.
Advantageously, a composition or a food supplement of the invention presents the following features, considered separately or in any combination.
It comprises 25 to 250 mg/g, in particular 50 to 250 mg/g, of one or several omega-3 type fatty acid(s), 5 to 50 mg/g, in particular 10 to 50 mg/g, of one or several xanthophyll(s), 0.5 to 20 mg/g, in particular 1 to 20 mg/g, of one or several sterol(s) and 1 to 100 μg/g, in particular 2 to 100 μg/g, of one or several phycoprostane(s).
It comprises 25 to 200 mg/g, in particular 50 to 200 mg/g, of one or several omega-3 type fatty acid(s), 5 to 30 mg/g, in particular 10 to 30 mg/g, of one or several xanthophyll(s), 0.5 to 8 mg/g, in particular 1 to 8 mg/g, of one or several sterol(s) and 1 to 50 μg/g, in particular 2 to 50 μg/g, of one or several phycoprostane(s).
It comprises 25 to 170 mg/g, in particular 50 to 170 mg/g, of one or several omega-3 type fatty acid(s), 5 to 25 mg/g, in particular 10 to 25 mg/g, of one or several xanthophyll(s), 0.5 to 6 mg/g, in particular 1 to 6 mg/g, of one or several sterol(s) and 1 to 40 μg/g, in particular 2 to 40 μg/g, of one or several phycoprostane(s).
Advantageously, a composition or a food supplement of the disclosure further contains at least one oil as a vehicle or support to facilitate the expression of the active ingredients. Surprisingly, it has been observed that the production of a composition or of a food supplement is facilitated when this oil is selected from medium-chain triglycerides (MCT). In particular, when the active ingredients are obtained from the same microalga extract, an optimum homogenization is observed in such oil. According to one variant, the medium-chain triglycerides (MCT) are from a natural origin and are brought by oil selected from coconut palm oil, palm kernel oil and palm oil; they may also be obtained or derived from such oil.
Hereinafter, preferred formulations of a composition or of a food supplement of the disclosure are presented, these implementations may of course be combined: the or at least one of the omega-3 type fatty acids is selected from stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and mixtures thereof;
A composition or a food supplement of the disclosure may comprise any additive allowing improving in particular the preservation, the appearance, the taste, the formulation thereof. Thus, one or several additive(s) as those selected from preservative agents, colorants, flavors, disintegration agents, lubricant agents, coating or encapsulation agents may be incorporated therein.
A major application of a composition of the disclosure is nutraceutical, thus, such a composition or food supplement as defined hereinabove is advantageously in the form of gel capsules, capsules, tablets, pastilles or loose powder. Preferably, it is packaged in doses having a unit weight comprised between 1 mg and 1 g. In general, the galenic of the composition or of the supplement will be adapted to the considered individual and in particular depending on whether it is intended to a child or an adult.
A composition or a food supplement of the disclosure may be used to prevent the apparition of non-pathological age-related cognitive disorders or non-pathological cognitive disorders in children or young adults having been subjected to a prenatal stress. The “age-related cognitive disorders” are notably but not exclusively chosen from a deficit in spatial working memory, a deficit in attention and vigilance, a deficit in executive function, a deficit in episodic memory, and/or a deficit in perceptual and cognitive skills. The food supplement according to the disclosure has demonstrated its efficacy onto said deficits through human clinical trial(s), such as detailed in Example 5 of the present application.
In particular, the food supplement may be used towards healthy older humans aged between 55 and 75 years old. More particularly, the disclosure concerns a method for preventing the apparition of age-related cognitive disorders, defined as a non-pathological decrease of the cognitive functions, in order to improve calmness and reduce perceived stress. In the prevention of such age-related cognitive disorders, the daily intake may be comprised between 2 to 16 mg/kg, in particular 2 to 5 mg/kg of body weight. Such food supplement may be orally administrated daily during a period of between and included one month to 6 months. In particular, when the daily intake is 16 mg/kg, the food supplement may be orally administrated during 1 month or during 3 months, in particular to a healthy older humans aged between 55 and 75 years old.
In the prevention of cognitive disorders in children or young adults having been subjected to a prenatal stress, the daily intake may be comprised between 0.05 to 0.1 mg/kg of body weight. It has been noticed that an effect is observed even for very small daily intakes, provided that the duration of the treatment is proportionally extended.
The disclosure also concerns the use of a microalga to prepare a food supplement as previously defined. One or several preferred microalga(e) are selected from any one of the following taxons Pinguiophyceae, Chrysophyceae, Bacillariophyceae, Mamiellophyceae, Prymnesiophyceae, Haptophyceae, Coccolithophyceae, Isochrysidaceae and Phaeodactylaceae. Advantageously, the microalga is Tisochrysis lutea or Phaeodactylum tricornutum. Such microalgae will be selected because an appropriate extraction leads to an extract whose composition meets the definition of a composition of the disclosure. As example, such an extract may comprise the following fraction of fatty acids: the fatty acids, expressed in weight percentages with respect to the total extract, are in the form of free fatty acids between 4 and 55%, in the form of monoacyl glycerol between 0.5 and 10%, in the form of diacylglycerol between 0.4 and 15% and in the form of triacyl glycerol between 2 and 80%, in particular between 2 and 55%. These fatty acids are in the range of 2.5 to 20% (w/w), in particular in the range of 5 to 20% (w/w), of the fatty acids from the omega-3 series and between 0.5 and 5%, of the fatty acids from the omega-6 series. More specifically, in particular, the fatty acids are ALA (α-linolenic acid) between 0.5 and 10%, SDA (stearidonic acid) between 0.5 and 10%, EPA (eicosapentaenoic acid) between 0.05 and 20%, DHA (docosahexaenoic acid) between 0.1 and 10%.
As previously indicated, one of the interests of a composition or of a food supplement lies in the method of preparation thereof and specifically the natural origin of its constituents all of which may be obtained from one single microalga.
Depending on the used microalga, the formulation of the composition may be obtained directly with the extract. If such is not the case, the extract will be diluted to obtain the required concentrations according to the disclosure. Nonetheless, the disclosure is not limited to this implementation, thus, it may be considered that only one portion of the constituents is from a natural origin, the others being obtained through a chemical synthesis and/or that the constituents from a natural origin do not originate from the same source, for example, they are not produced from the same alga.
The measurement and the adjustment of the concentrations of the active ingredients in an extract, and in the composition or the obtained food supplement, are carried out using analytic techniques that fall within the general knowledges of those skilled in the art.
A method for producing a composition or a food supplement from a microalga culture is described hereinafter in more details.
According to a variant of the disclosure, said organisms are microalgae, such as those belonging to the taxons Pinguiophyceae, Chrysophyceae, Bacillariophyceae, Mamiellophyceae, Prymnesiophyceae, Haptophyceae, Coccolithophyceae, Isochrysidaceae, Phaeodactylaceae. These photosynthesis-capable microorganisms may be strictly autotrophic, mixotrophic or transiently heterotrophic. These organisms may be harvested in a natural or preferably cultivated environment.
By extract, it is referred to a fraction of the biomass derived from the photosynthesis-capable organisms obtained by a method allowing obtaining, whether directly or indirectly, a composition of the disclosure. These extracts have a composition, expressed in weight percentage with respect to the total extract, in proteins comprised between 5 and 30%, in lipids between 20 and 80%, between 0.05 and 2% of sterols, in particular between 0.1 and 2% of sterols, between 0.1 and 20% of chlorophyll.
More specifically, the lipophilic portion composing the extract, expressed in weight percentage with respect to the total extract, is constituted by saturated fatty acids between 15 and 45%, polyunsaturated acids between 5 and 20%, xanthophylls between 0.5 and 20%, in particular between 1 and 20%, and phycoprostane between 0.0001 and 0.007%, in particular between 0.0002 and 0.007%.
For the production of the extract according to the disclosure, the cells advantageously consist of microalgae cells of the species Tisochrysis lutea of the Isochrysidaceae family, or microalgae cells of the species Phaedactylum tricornutum of the Phaeodactylaceae family, produced by carbon autotrophy.
The microalgae are ideally cultivated in a controlled manner within adapted systems such as race-ways, open ponds or preferably in closed systems such as photobioreactors. The used photobioreactors may be of any existing type such as horizontal tubular photobioreactors, vertical such as so-called «green wall panel» systems, planar or column type photobioreactors. Preferably, the production of the biomass will be performed within a closed cultivation system, by zero-impact autotrophy on arable lands.
The production of the biomass is performed according to batch, fed-batch, continuous, semi-continuous, turbidostat or chemostat type culture management practices.
Obtainment of the Extracts of these Microalgae
The extracts derived from these microorganisms are preferably obtained after concentration of the biomass by elimination of all or part of water using chemical or physical processes such as centrifugation, filtration, flocculation, sedimentation, whether coupled or not, at drying steps by freeze-drying, vacuum-drying, drum-drying, atomization or any other process allowing lowering the water content of the biomass. Complementarily to these steps, cellular lysis processes may be implemented such as the applications of pressures, electric flows, shear forces, the use of enzymes, or any other processes allowing destructuring the tissues, organs, cells or organelles.
The interesting compounds of the biomass are extracted according to a solid-liquid extraction type process, which may use hypercritical fluids or subcritical fluids, which may involve co-treatments carried out in parallel or sequentially such as microwaves, ultrasounds, pressures, enzymes. The used solvents, whether pure or in a mixture, may consist of acetone, hexane, ethyl acetate, methyltetrahydrofuran, heptane, methanol, natural or branched oils, ethanol or any other solvent allowing extracting all or part of the hydrophobic and amphiphilic compounds.
The solvent or the mixture of solvents is separated from the residual biomass after extraction by centrifugation, filtration type processes and may be concentrated afterwards, or the solvent eliminated, by techniques such as vacuum evaporation or any other technique allowing for the selective evaporation of the considered solvent. The extract thus obtained has a lipophilic nature while including amphiphilic molecules.
The formulation of the extract is performed with compatible matrices, enabling the dissolution thereof in order to obtain a homogeneous solution with the desired concentration of extract such as for example, vegetable oils such that olive oil, colza oil, linseed oil, sunflower oil, grape seed oil, palm oil and preferably MCT oils, and composed to about 70 weight % of a mixture of caprylic acid and capric acid, and preferably selected from coconut oil or palm oil, the whole being supplemented with molecules allowing increasing stability such as synthetic or natural antioxidants. The weight rates of incorporation of the matrices/additives in order to obtain the supplement may reach 95 weight % with respect to the weight of the food supplement, they are generally comprised between 15 and 80%, preferably between and 45%.
The extract, but preferably the formulated composition or the obtained supplement, may be formulated in form of soft capsules, or may be formulated in form of powder, by any technique enabling the micro-encapsulation of the aqueous solution involving, or not, a support or a matrix allowing, or not, a homogeneous dispersibility thereof within a drinkable polar solution.
The extract or the supplement may be used alone or as an ingredient within a food complementation.
This disclosure includes at least the following individual embodiments. These and other embodiments of the food supplement described herein are not mutually exclusive, and are independently combinable.
In some embodiments, a food supplement comprises a water-insoluble extract of a microalga and at least one oil selected from medium-chain triglycerides (MCT), wherein the extract of microalga comprises at least 25 mg/g of one or several omega-3 type fatty acid(s), at least 5 mg/g of one or several xanthophyll(s), at least mg/g of one or several sterol(s) and at least 1 μg/g of one or several phycoprostane(s).
The extract can comprise 25 to 250 mg/g of one or several omega-3 type fatty acid(s), 5 to 50 mg/g of one or several xanthophyll(s), 0.5 to 20 mg/g of one or several sterol(s), 1 to 100 μg/g of one or several phycoprostane(s).
The extract can also comprise 25 to 200 mg/g of one or several omega-3 type fatty acid(s), 5 to 30 mg/g of one or several xanthophyll(s), 0.5 to 8 mg/g of one or several sterol(s) and 1 to 50 μg/g of one or several phycoprostane(s).
The extract can also comprise 25 to 170 mg/g of one or several omega-3 type fatty acid(s), 5 to 25 mg/g of one or several xanthophyll(s), 0.5 to 6 mg/g of one or several sterol(s) and 1 to 40 μg/g of one or several phycoprostane(s).
The medium-chain triglycerides (MCT) can be selected from coconut palm oil and palm oil.
The one or at least one of the omega-3 type fatty acids can be selected from ALA (α-linolenic acid), stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and mixtures thereof.
The at least one of the xanthophylls can be fucoxanthin.
The or at least one of the sterols is selected from phytosterols.
The or at least one of the phycoprostanes can be selected from phytoprostanes, isoprostanes and neuroprostanes.
The food supplement can further comprise at least one additive selected from preservative agents, colorants, flavors, disintegration agents, lubricant agents, coating or encapsulation agents.
The food supplement can be in the form of gel capsules, capsules, tablets, pastilles or loose powder.
The food supplement can be packaged in doses having a unit weight comprised between 10 mg and 1 g.
The microalga can be selected from any one of the taxons Pinguiophyceae, Chrysophyceae, Bacillariophyceae, Mamiellophyceae, Prymnesiophyceae, Haptophyceae, Coccolithophyceae, Isochrysidaceae and Phaeodactylaceae.
The microalga can also be Tisochrysis lutea or Phaeodactylum tricornutum.
In some embodiments, a method for preventing the apparition of age-related cognitive disorders, defined as a non-pathological decrease of the cognitive functions, or cognitive disorders in children or young adults having been subjected to a prenatal stress inducing non-pathological disorders, such as hyperactivity, attention and memory deficit, language retardation and anxious behavior, comprises the administration of an effective amount of the food supplement to a human in need thereof.
The age-related cognitive disorders can be chosen from deficit in spatial working memory, deficit in attention and vigilance, deficit in executive function, deficit in episodic memory, and deficit in perceptual and cognitive skills.
The effective amount of the food supplement can improve calmness and reduce perceived stress.
The method can comprise the administration of an effective amount of the food supplement to a healthy older human aged between 55 and 75 years old.
The effective amount can correspond to a daily intake of between 2 to 16 mg of food supplement/kg of body weight.
The food supplement can be orally administrated during between 1 month to 6 months.
The effective amount can correspond to a daily intake of between 0.05 to 0.1 mg of food supplement/kg of body weight.
An extract is obtained according to any of the above-described techniques from the microalga Phaeodactylum tricornutum. In particular its process of preparation is as follows: the P. tricornutum microalgae extract is obtained from the biomass of a microalgae culture of a strain originating in France. The biomass is first centrifuged and then filtered to remove the water. A solid-liquid extraction step is then implemented. The extract is thus obtained by extraction in a water/ethanol mixture in a proportion of 40/60 (w/w) to 1/100 (w/w), at a temperature of 20° C., i.e. at room temperature, for a period of 2 hours. It is then filtered and then dried by freeze-drying. It comes in powder form.
It is water-insoluble and is highly viscous preventing any handling at room temperature.
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.
The food supplement of the disclosure is prepared from a Tisochrysis lutea extract which comprises in mg/g:
The supplement is obtained by addition of coconut oil in a proportion of 360 mg±10 mg/g to said extract.
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% (w:w).
A commercial oily extract of microalgae comprising, as a fat fraction, only the fatty acids DHA 77% (w:w) and EPA 3% (w:w) 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% (w:w).
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% (w:w).
The five batches of kibbles thus obtained are referenced as described in Table 1 hereinbelow.
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.
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:
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
On the 51th day, after the behavioral tests, the animals have been euthanized. 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)
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)]
The IL6 and TNFα contents are quantified by means of ELISA tests with the following kits:
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.
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:
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.
The results are represented in
In
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. 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.
The treatment with DHA alone (according to A5) has quite significantly but partially alleviated the deficits induced by the chronic intoxication with D-Gal.
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.
The results are represented in
In
The chronic intoxication with D-Galactose has considerably altered the spatial learning, in comparison with the negative control group (saline solution/vehicle).
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.
The DHA alone according to A5 has quite significantly but partially alleviated the deficits induced by the chronic intoxication with D-Galactose.
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.
The results are represented in
In
The chronic intoxication with D-Galactose has considerably altered the spatial learning, in comparison with the negative control group (saline solution/vehicle).
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 alleviated the deficits induced by the chronic intoxication with D-Galactose.
The treatment with DHA alone according to A5 has quite significantly but partially alleviated the deficits induced by the chronic intoxication with D-Galactose.
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.
The results are represented in
In
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).
The supplement A2 has not demonstrated any effect on the long-term contextual memory.
The supplement A3 has allowed attenuating the deficits induced by the chronic intoxication with D-Galactose in a non-significant manner.
The supplement A4 has quite significantly and completely attenuated the deficits induced by the chronic intoxication with D-Galactose.
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.
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.
The results are represented in
In
The chronic intoxication with D-Galactose has considerably increased the oxidative stress, in comparison with the negative control group (saline solution/vehicle).
The supplement A2 has not demonstrated any effect on the lipid peroxidation induced by the chronic intoxication with D-Galactose.
The supplement A3 has quite significantly but partially reduced the oxidative stress induced by the chronic intoxication with D-Galactose.
The supplement A4 has quite significantly and completely reduced the oxidative stress induced by the chronic intoxication with D-Galactose.
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.
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.
The results are represented in
In
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).
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.
The supplement A4 has quite significantly and completely reduced the level of the TNF-α in the cortex and the plasma.
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.
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.
The results are represented in
In
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).
The low-dose supplement (A2) has not demonstrated any effect on the concentration of IL-6 induced by intoxication with D-Galactose.
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.
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.
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.
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.
In conclusion:
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.
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.
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.
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).
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.
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
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.
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%).
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 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.
Control mothers have also been manipulated, but have never been placed in the ferret retainers.
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).
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.
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.
In each cage, the animals have received the same treatment. The animals have been tested randomly and in a double-blind manner.
Forty-eight (48) female rats have been used and grouped into four groups of animals, constituted in the following manner:
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.
The daily intake was 25.7 mg of supplement per kg of rat body weight.
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.
The behavioral tests are divided into one anxiety assessment session and two objects recognition sessions. The sessions are defined as follows:
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.
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.
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.
The results are presented hereinafter.
The results are represented in
In
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).
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.
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.
Thus, the prenatal stress (PS) has induced a very significant anxious behavior.
Surprisingly, it turns out that the supplement has quite significantly and completely attenuated the anxious behavior induced by the prenatal stress.
The results are represented in
During this session, the same object is exhibited twice to the individuals.
No statistical effect between the groups has been measured for this parameter.
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%).
The results are represented in
In
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.
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.
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.
Thus, the prenatal stress (PS) has induced very considerable recognition memory deficits in the case of the new object.
Surprisingly, it turns out that the supplement has allowed attenuating quite significantly and completely the recognition memory deficits induced by the prenatal stress.
In conclusion:
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.
The prenatal stress as practiced in this experiment significantly induces an anxious behavior, and quite significantly alters the recognition memory in young female rats.
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.
The food supplement of the disclosure is prepared from a Phaeodactylum tricornutum extract which comprises in mg/g:
The supplement is obtained by addition of coconut oil in a proportion of 410 mg±20 mg/g to said extract.
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.
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.
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% (w:w).
The five batches of kibbles thus obtained are referenced as described in Table 2 hereinbelow.
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.
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:
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.
On the 51th day, after the behavioral tests, the animals have been euthanized.
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)
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)]
The IL6 and TNFα contents are quantified by means of ELISA tests with the following kits:
For the quantification of IL6: ThermoScientifique, EM2IL6
For the quantification of TNFα: ThermoScientifique, EMTNFA
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 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.
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.
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:
The results are represented in
In
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.
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.
The results are represented in
In
The chronic intoxication with D-Galactose has considerably altered the spatial learning, in comparison with the negative control group (saline solution/vehicle).
The supplement D1 has quite significantly but partially attenuated the deficits induced by the chronic intoxication with D-Galactose.
The supplements D2, D3 and D4 have quite significantly and completely attenuated the deficits induced by the chronic intoxication with D-Galactose.
The results are represented in
In
The chronic intoxication with D-Galactose has considerably altered the spatial learning, in comparison with the negative control group (saline solution/vehicle).
The supplements D1 and D2 have quite significantly but partially attenuated the deficits induced by the chronic intoxication with D-Galactose.
The supplements D3 and D4 have quite significantly and completely alleviated the deficits induced by the chronic intoxication with D-Galactose.
The results are represented in
In
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).
The supplement D1 has not demonstrated any effect on the long-term contextual memory.
The supplements D2, D3 and D4 have quite significantly and completely attenuated the deficits induced by the chronic intoxication with D-Galactose.
The results are represented in
In
The chronic intoxication with D-Galactose has considerably increased the oxidative stress, in comparison with the negative control group (saline solution/vehicle).
The supplement D1 has quite significantly but partially reduced the oxidative stress induced by the chronic intoxication with D-Galactose.
The supplements D2, D3 and D4 have quite significantly and completely reduced the oxidative stress induced by the chronic intoxication with D-Galactose.
The results are represented in
In
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).
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.
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.
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.
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.
The results are represented in
In
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).
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.
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.
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.
The preventive treatment with the supplement of the invention 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 invention 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.
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.
The food supplement of the disclosure is prepared from the water insoluble Phaeodactylum tricornutum extract obtained by solid-liquid extraction as described in Example 1 and comprises in mg/g:
Two double blind, randomized manner, human clinical trials have been conducted as detailed below.
A population of 40 free living males and females aged between 55 to 75 years with age-associated memory decline have been administrated orally 4 capsules containing 275 mg of the food supplement above or 4 capsules of a placebo (275 mg of maltodextrine) daily during a period of 12 weeks. Cognitive function battery has been realized after 4 and 12 weeks of daily supplementation. The inclusion and exclusion criteria were as below:
Be able to give written informed consent and to consume the investigational product daily for the duration of the study.
Healthy males and females aged ≥55 and ≤75 years old.
The specific cognitive functions evaluated after 4 and 12 weeks of daily supplementation were as follows:
C-Reactive Protein (CRP) is an acute-phase protein, produced exclusively in the liver. Interleukin-6 is the mediator for the synthesis by the hepatocytes of CRP. It's well admitted in scientific literature that elevated levels of blood CRP predict poorer cognitive function and increased dementia risk in cognitively healthy middle-aged and older adults. (Lewis, N. A., and Knight, J. E. (2021)). Longitudinal associations between C-reactive protein and cognitive performance in normative cognitive ageing and dementia. Age Ageing 50, 2199-2205. doi: 10.1093/ageing/afab152).
A volume of 21.5 mL of blood of the individuals of the 2 groups were collected, frozen and further, the following have been measured:
All computerised cognitive/mood assessments were identical, and were carried out via laptop computers and response boxes using the Computerised Mental Performance Assessment System (COMPASS, Northumbria University, Newcastle upon Tyne, UK). This software platform incorporates the presentation of classic and custom computerised cognitive tasks, with fully randomised parallel versions of each task delivered at each assessment for each individual. A similar selection of tasks has previously been shown to be sensitive to diverse nutritional interventions.
Nine blue squares on a black background are displayed on the screen. Some of the blue squares change to red and back to blue again in a sequence. Participants were required to remember this sequence. The task was repeated five times at each level of difficulty with the sequence span increasing from 4 upwards, until the participant could no longer correctly recall the sequences. For Corsi blocks and Corsi blocks lite, participants used the cursor to click the blocks in the exact sequence in which they were presented. For reversed Corsi blocks, they clicked on the blocks in reverse to the sequence shown.
Corsi blocks and reversed Corsi blocks were continued up to fifteen levels (i.e. up to squares in each sequence), as long as participants were making enough correct responses. As soon as they made less than 3 correct responses (out of the five in one level) the task ended. Corsi blocks lite is a shorter task with a maximum of 6 blocks in a sequence; all participants completed levels 4-6 regardless of incorrect responses. This module is particularly useful for training configurations.
The tests are described below:
Choice Reaction Time Task
Arrows pointing left and right appear on the screen at irregular intervals. The participant is required to indicate the direction of the arrow as quickly as possible, whenever an arrow is displayed.
Digit Vigilance Task
A fixed number appear on the right of the screen and a series of changing numbers appear on the left side of the screen. Participants are required to make a response when the number on the left matches the number on the right.
Stroop Task
A series of colour names (RED, YELLOW, GREEN, BLUE) are displayed on the screen. These are written in a coloured font. Participants are required to make a response based on the colour font the word is written in not the colour the word depicts. Participants are shown either congruent or incongruent stimuli, or a random mix of both.
Picture Presentation Task
A series of pictures are displayed on the screen, one at a time. The number of pictures, the rate at which they are displayed and the interstimulus interval can be modified.
Picture Recognition Task
All target pictures shown during Picture Presentation plus an equal number of decoys will be displayed on the screen one at a time. For each stimulus, participants select ‘Yes’ or ‘No’ to indicate if they have seen the picture before or not.
Word Recognition Task
All target words that were shown during Word Presentation plus an equal number of decoys will be displayed on the screen one at a time. Participants indicate if they remember seeing the word earlier or not.
Word Presentation/Word Recall (Immediate and Delayed) Task
Word Presentation
A list of words is displayed on the screen, one word at a time. The number of words, the rate at which they are displayed and the interstimulus interval can be modified.
Word recall (scored manually, see Processing Word Recall Data document for details)
Participants are given 60 seconds to write down as many of the words they were shown earlier as they can. This task can be performed immediately following presentation of the words (Immediate recall) or following a delay (Delayed recall).
Bond-Lader Mood Rating Scale
This is a series of mood scales. Each scale is a line anchored at either side by an adjective describing a mood. Participants must click (using the mouse) at a point on the scale that represents how they are feeling at that point in time. There are 16 scales in total, each scored out of 100. From these scales three composite scores are calculated describing feelings of ‘Alert’, ‘Calm’ and ‘Content’, which are also presented as a score out of 100.
Trial 2: A population of 66 healthy individuals male and female, aged between 55 and 75 years old, with age-associated memory decline have been administrated orally 2 capsules containing 275 mg of the food supplement above or 2 capsules of a placebo (275 mg of maltodextrine) daily during a total period of 24 weeks. Cognitive function battery tests have been realized after 12 and 24 weeks of daily supplementation. The inclusion and exclusion criteria were the same as for the trial described above.
The blood concentration of CRP was measured in the population after 24 weeks of daily supplementation with the food supplement, as previously described in trial 1 (ELISA technique).
The results demonstrated a significant reduction of inflammation by reduction of the concentration of hs-CRP after 24 weeks in the group who received the food supplement according to the invention versus the placebo group.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19/01820 | Feb 2019 | FR | national |
This application is a Continuation-in-Part application of U.S. Non-provisional application Ser. No. 16/798,609, filed Feb. 24, 2020, which claims the benefit of FR Application No. 19/01820, filed Feb. 22, 2019, both of which are incorporated by reference in their entirety herein.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16798609 | Feb 2020 | US |
| Child | 18374301 | US |
