WO2016083362A1

WO2016083362A1 - Composition for improving cognitive functions

Info

Publication number: WO2016083362A1
Application number: PCT/EP2015/077480
Authority: WO
Inventors: Peder Esben BILDE
Original assignee: Fonden For Helene Elsass Centeret
Priority date: 2014-11-25
Filing date: 2015-11-24
Publication date: 2016-06-02

Abstract

The present invention relates to a composition for use as a nutritional supplement or a medicament. The composition is for use in improving cognitive functions such as learning, memory and attention as well as motor skills in individuals in need thereof. Said individuals may be healthy individuals or individuals with congenital and/or developmental brain disorders. The composition may be in the form of liquid or solid dosage form.

Description

Composition for improving cognitive functions.

Field of the invention The present invention is in the field of nutritional (or pharmaceutical) supplements and more particularly relates to a composition for use in improving/enhancing cognitive and motor abilities in humans.

Background of the invention

Foods containing specific nutrients and vitamins can have a profound effect on the mental and physical fitness of the human mind and body. The relative abundance of specific nutrients could affect cognitive processes due to the fact that influences of dietary factors on neuronal function and synaptic plasticity are responsible for brain health and mental function. Certain foods are therefore frequently consumed as part of a health improving diet. Consuming adequate amount of foods with health promoting effect can be a difficult task, as a result of changing seasons, inadequate supply of the food source, shelf life of the unprocessed food as well as inconvenience in food preparation. There is therefore an increasing demand for industrially prepared compositions that specifically contain the health promoting compounds.

It is well established in the art that learning and memory require structural changes in neurons and neuronal networks in the form of down-or up- regulation of receptors, membrane channels, neurotransmitters and synaptic connections. Such structural changes depend on the availability of proteins, carbohydrates, fatty acids and other dietary supplements necessary for growth of new neural connections. Specific lipids, oils and other fatty materials are frequently consumed as part of a health improving composition. Examples of such lipids are omega-3 fatty acids and variations thereof. Omega-3 fatty are normal constituents of cell membranes and are essential for normal brain function. Omega-3 fatty acids are considered essential fatty acids, meaning that they cannot be synthesized by the human body and therefore needs to be supplied by diet or as a supplement. Omega-3 fatty acids are polyunsaturated fatty acids having the unsaturated bonds at a particular position, which contributes to the effect on human health. Omega-3 fatty acids preferably have between 18-22 carbons and 3 to 6 double bonds. These omega-3 fatty acids can be in the form of free fatty acids, (alkyl) esters thereof, glycerides thereof, phospholipid esters thereof or mixtures thereof. Such omega-3 fatty acids can be obtained e.g. from marine oils such as fish oils, krill, algae and some specific vegetable oils.

During the last decade omega-3 fatty acids such as DHA in combination with other ingredients have attracted attention as active components with an ability to reduce cognitive deficits in children affected with developmental coordination disorder (Richardson AJ, Montgomery P, Pediatrics 1 15, 1360- 1366 (2005)) and improve coordination, concentration and academic ability in "normal" children (Portwood M, Nutr. Health, 18, 233-247, (2006)).

Uridine is another micronutrient, which has been found important in mental fitness. Cells use uridine and fatty acids like DHA in the synthesis of phosphatidylcholine (PC) which is a major component of brain cell membranes. PC also provides the phosphocholine moiety needed to synthesize sphingomyelin (SM), which is the major choline containing brain phospholipid. Vitamin D is able to cross the blood brain barrier and vitamin D receptors are found in the brain suggesting that vitamin D is essential for optimal brain health. Blueberry, green tea and cocoa, are rich in a group of phytochemicals called flavonoids. Flavonoids are a class of compounds found to have a neuroprotective effect and potential to promote memory, learning and cognitive function. Flavonoids are strong antioxidants and are able to reduce the damage caused by free radicals. However, it has been suggested that the neuroprotective effect of flavonoids may be mediated by other pathways to protect vulnerable neurons, enhance existing neuronal function, stimulate neuronal regeneration and induce neurogenesis (Spenser EPJ, Genes Nutr, 243-250, (2009)). Hence it is clear from the art that numerous micronutrients independently are reported to have a positive effect on the biochemical and cellular level corresponding to cognitive functions. However, a clear understanding of how these micronutrients can work together and create a synergy corresponding to a greater effect is clearly lacking.

A clinical study on children of the effects of polyunsaturated fatty acids DHA and EPA alone or in combination with micronutrients on cognitive abilities found that DHA EPA had no effect on cognitive abilities whereas the composition of micronutrients consisting of vitamin A, B, C, Folate, Zink and Iron where found to have an effect (Osendrap SJ, 2007, NEMO study)

A similar study by Kennedy D et al. (2009) has assessed the the effect of DHA on cognitive performance in healthy children. The study assessed two doses of DHA, 400 mg or 1000 mg per day, for a period of weeks. However, the study did not find any beneficial effects of DHA on brain function. Hence the data suggest that polyunsaturated fatty acids alone is not enough to enhance cognitive function in healthy individuals. There is a clear need to find out which combinations of micronutrients will be able to improve cognitive functions in healthy individuals.

WO 2006/127627 relates to a composition of polyunsaturated fatty acids (PFUAs) with or without Uridine. The composition is used as a method of enhancing brain development and increasing intelligence by administering the composition to a subject or a pregnant or nursing mother.

A similar composition has been reported to have anti-depressant effects in a rat study (Renshaw PF, 2005).

The amount of knowledge required to function and succeed is increasing and the speed with which an individual acquires more knowledge is increasing.

Consequently, there is a need for compositions with an ability to improve cognitive skills such as learning, memory and attention in healthy individuals, individuals with congential or developmental brain disorders as well as individuals with developmental coordination disorder.

Summary of the invention

The present invention was made in view of the prior art described above, and the object of the present invention is to provide a composition with the ability to improve cognitive functions such as e.g. learning, memory, attention or motorial skills. Another object relates to the provision of a nutritional supplement for improving cognitive functions such as learning, memory, attention and motor skills administration in healthy individuals. A further object relates to the provision of a composition to improve developmental coordination disorder. To solve the problem, the present invention provides a composition comprising:

i) Uridine, such as Uridine monophosphate (UMP), or phosphates, acyl derivatives, esters or salts thereof; ii) Docosahexaenoic acid (DHA), such as DHA in triacylglyceride form, or phospholipids, esters or salts thereof;

iii) Vitamin D3, 25-Hydroxyvitamin D3, or salts thereof. That is, the inventors of the present invention in a first aspect of the invention found that the composition formulated for oral administration comprising Uridine, DHA and Vitamin D3 has an ability to improve cognitive and motor abilities in healthy individual. In some embodiments of the present invention, the composition further comprises one or more flavonoid(s) obtainable form blueberry or blueberry extract. The combination of Uridine, DHA, vitamin D3 with flavonoid(s) obtainable form blueberry is able to enhance the effect of Uridine, DHA and vitamin D on cognitive functions and motor skills.

In some embodiments the present invention may comprise a pharmaceutical composition for oral administration comprising Uridine, DHA and vitamin D3. In another embodiment of the invention the pharmaceutical composition may be used to improve cognitive and motor skills in individuals with Cerebral Palsy.

Another aspect of the present invention, concerns a method for improving cognitive functions and/or motor skills in a human subject in need thereof, comprising administering to the subject an effective amount of a composition comprising: i) Uridine, such as uridine monophosphate (UMP), or salts, phosphates, acyl derivatives or esters thereof;

ii) Docosahexaenoic acid (DHA), such as DHA in triacylglyceride form, or esters thereof;

iii) Vitamin D3, such as 25-Hydroxyvitamin D3, or salts thereof.

The composition of the present invention when consumed on a daily basis over a prolonged period of time offers the advantage of improved cognitive abilities such learning, memory, attention as well as motor abilities. The combination of the specific micronutrients of the present invention gives the recipient an improved health benefit/effect, compared to the consumption of one or more of the micronutrients separately, as the unique components work in synergy. The combination of the micronutrients in one composition makes it more convenient for individuals to get adequate intake on a daily basis compared to consuming unprocessed foods. The ability to consume this composition which constitutes a cocktail of micronutrients with a clear health benefit is a significant advantage as it would be challenging to get enough of each micronutrient through regular food consumption on a daily and continues basis. Furthermore, consuming enough of the different foods in one meal/setting would also be a significant challenge as foods rich in Uridine, DHA, Vitamin D and flavonoids such as blueberry not necessarily complement each other in a dish. In many regions of the world it would also be a significant challenge to acquire food with sufficient micronutrients through all seasons of the year. An additional advantage of the composition of the present invention is the ability to preserve micronutrients and prolong shelf life, compared to their natural form.

Brief description of the drawings Figure 1 illustrates individual dietary intake of mono- and polyunsaturated fatty acids and vitamin D. 1 -4 represents girls and 5-1 1 represents boys. Bold line represent daily minimum recommended levels of intake (A) Monounsaturated fatty acid intake (E%), recommended level: 10-15 E%. (B) Polyunsaturated fatty acid intake (E%), recommended level: 5-10 E%. (C) Vitamin D ( g) intake with (grey bars) and without (black bars) vitamin supplement, recommended level: 7.5 g. (D) correlation between dietary intake of vitamin D incl. dietary supplement and plasma 25(OH)D (vitamin D) concentration in blood (r=0.699, p<0.05, Pearson product moment correlation).

Figure 2 illustrates plasma concentrations of vitamin D (A), Docosahexaenoic acid (DHA) (B) and brain-derived neurotrophic factor (BDNF) (C) in boys (black square dots) and girls (grey triangle dots), before and after DDU- and placebo supplement. Right upper corner: Delta concentrations of vitamin D (A), DHA (B) and BDNF (C) in relation to interventions. Data are mean±SEM., ^*p < 0.05, ^**p <compared to placebo (paired t-test).

Figure 3 illustrates the performance of the subjects in two Mitii training modules; Memory (A) and Mathematics (B) during the intervention periods when children received DDU-supplement (black circles) or placebo (grey circles). The values are given as the decrease in the errors in percentage during each possible training day. The shaded areas mark the standard error for each group. Significant improvement pre compared to post intervention (p < 0.05, Two-way ANOVA RM).

Figure 4 illustrates the performance of the subjects in reading/comprehension test (A) and math test (B) in boys (black square dots) and girls (grey triangle dots) before and after DDU- and placebo supplement. Right upper corner: Performance in relation to interventions. Data are mean±SEM., ^*p<0.05, ^**p<0.01 compared to pre intervention (Two-way ANOVA RM). Figure 5 illustrates the performance of the subjects before and after DDU- and placebo supplement in CPAL: Speed of performance (A), Percentages correct answers (B). Significant interaction between pre/post and CPAL repetition (Linear mixed mode, p<0.05l), GML: Duration (C), Percentages errors (D). Significant interaction between pre/post and GML repetition (Linear mixed model, p<0.05). One Back task: Error (E), Speed of performance (F) in boys (black square dots) and girls (grey triangle dots). Right upper corner in E and F: Performance in relation to interventions. Data are mean±SEM., ^*p < 0.05 compared to pre intervention (Two-way ANOVA RM).

Figure 6 illustrates the average relative success rate for the two different groups of mice. Mice that received a supplement containing blueberry extract had a success rate of about 40%, whereas the group receiving normal supplements without blueberry extracts had a success rate of about 34%.

List of tables Table 1 : Characteristics for girls and boys together (n=14) and girls (n=6) and boys (n=8) separately.

Table 2: Composition of DDU-supplement and Placebo emulsions [wt %]. Table 3: Macronutrients given by an average of four independent days of 24 hours dietary weighing for girls and boys together (n=1 1 ) and girls (n=4) and boys (n=7) separately.

Table 4: Micronutrients given by an average of four independent days of 24 hours dietary weighing for girls and boys together (n=1 1 ) and girls (n=4) and boys (n=7) separately. Table 5: Effect of interventions on blood parameters.

Detailed description of the invention

In describing the embodiments of the invention specific terminology will be resorted to for the sake of clarity. However, the invention is not intended to be limited to the specific terms so selected, and it is understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.

As mentioned above, the invention relates to a composition with the ability to improve cognitive functions such as learning, memory, attention and motor skills. One aspect of the invention is directed to a composition comprising i) Uridine, Uridine monophosphate (UMP) or phosphates, acylderivatives, esters or salts thereof and ii) Docosahexaenoic acid (DHA) in triacylglyceride form, or as free fatty acids, as phospholipids, esters or salts thereof and iii) vitamin D (vitamin D3, 25-Hydroxyvitamin D3) or salt thereof. A preferred embodiment of the invention is a composition comprising i) Uridine monophosphate and ii) Docosahexaenoic acid in triacylglyceride form and iii) vitamin D.

Uridine is a nucleoside which has been found important in mental fitness. Uridine can be obtained from a variety of food sources such as sugarcane extract, tomatoes, beer, beetroot and broccoli. Cells use uridine and fatty acids like DHA in the synthesis of phosphatidylcholine (PC) which is a major component of brain cell membranes. PC also provides the phosphocholine moiety needed to synthesize sphingomyelin (SM), which is the major choline containing brain phospholipid. The uridine component of the composition may be in the form of uridine, uridine 5'-monophosphate, acylderivatives, esters, salts thereof or any other uridine derivative with properties similar to uridine 5'-monophosphate. Docosahexaenoic acid (DHA; 22:6) is an omega-3 polyunsaturated fatty acid (PUFA), more particular a carboxylic acid with a 22-carbon chain containing six cis double bonds. DHA is the primary structural component of the human brain, cerebral cortex, skin, sperm, testicles and retina. DHA has been linked to several health benefits e.g. anti-inflammatory, cardiovascular, brain development and anti-depressant. As mentioned above DHA is one of the components in the synthesis of phosphatidylcholine (PC). DHA can be isolated from a variety of sources. The DHA component of the composition may be in the form of a triacylglyceride, as free fatty acids, as phospholipids, esters, salts thereof or any other DHA derivative with properties similar to docosahexaenoic acid in triacylglyceride form.

Vitamin D belongs to a group of fat-soluble secosteroids with multiple functions. The vitamin D receptor (VDR) is present in many cell types including neurons and glial cells and vitamin D may regulate neurotransmission, neuroprotection and brain processes. The VDR have been located in the human cortex and hippocampus, which are key areas for cognitive function. In humans, the most important compound within the vitamin D group is vitamin D3 (also known as cholecalciferol). Vitamin D3 can be produced by the human body upon sunlight exposure. However, in periods with inadequate sunlight vitamin D needs to be ingested from dietary sources to prevent vitamin D deficiency. Vitamin D deficiency has been associated with numerous diseases such as hyperparathyroidism, osteoporosis, cardiovascular and autoimmune diseases in addition to cognitive impairment.

Vitamin D3 is metabolised in the body into its more active metabolites'. Vitamin D3 is hydroxylated in the liver to form 25-hydroxyvitamin D3 (calcidiol) followed by a second hydroxylation in the kidney to form 1 ,25- dihydroxyvitamin D3 (calcitriol). 25-hydroxyvitamin D3 is the major circulating metabolite in serum with a concentration 1000 fold higher than that of 1 ,25- dihydroxyvitamin D3, but 25-hydroxyvitamin D3 has a 500 fold lower affinity for the vitamin D receptor than 1 ,25-dihydroxyvitamin D3. The composition of the invention may in one embodiment comprise vitamin D3. In a further embodiment the composition comprises 25-hydroxyvitamin D3. In another embodiment the composition comprises 1 ,25-dihydroxyvitamin D3.

In one embodiment the DHA may be obtainable form fish, krill or alga. Cold water fish have a particularly high content of DHA. DHA can also be isolated form marine microalgae e.g. Crypthecornium cohnii and Schizochytrium. DHA isolated form krill are in the form of phospholipids (mostly phosphatidylcholine). DHA in the form of phospholipids leads to better absorption and delivery of DHA into the brain. Some microalgae produce DHA and may be a suitable source of DHA in the present invention. Microalgae belonging to a genus or class selected from Schizochytrium being the most preferred.

Blueberries are a food source rich in micronutrients such as dietary minerals and fibre, vitamins and compounds with high antioxidant capacity. In addition to blueberries, green tea and cocoa are also rich in a group of phytochemicals called flavonoids. Flavonoids are a class of compounds found to have a neuroprotective effect and to promote memory, learning and cognitive function.

In one embodiment the composition of the invention may further comprise a flavonoid obtainable from blueberry. Further, in one embodiment the flavonoid is selected from the list consisting of rutin, myricetin, quercetrin, delphinidin, cyaniding, petunidin, peonidin, malvidin anthocynins, procyanidins. In one embodiment the composition may comprise one or more flavonoids that can be isolated from blueberries. The composition of the invention may comprise flavonoid(s) in the form of blueberry or blueberry extract. Blueberries may be formed/converted into a liquid solution which can be added to the composition or from which a blueberry extract containing flavonoids is extracted. The blueberry extract may be further concentrated to increase the concentration of flavonoid(s) added to the composition.

Blueberries belong to the genus Vaccinium and are classified in the section of Cyanoccous. Flavonoid(s), blueberry or blueberry extract of the present invention may be isoladed from Vaccinium sp., Vaccinium alaskaense, Vaccinium angustifolium, Vaccinium boreale, Vaccinium caesariense, Vaccinium corymbosum, Vaccinium constablaei, Vaccinium darrowii, Vaccinium elliottii, formosum, Vaccinium fuscatum, Vaccinium hirsutum, Vaccinium myrsinites, Vaccinium myrtilloides, Vaccinium operium, Vaccinium pallidum, Vaccinium simulatum, Vaccinium tenellum, Vaccinium virgatum.

In one embodiment the blueberry, blueberry extract or flavonoid(s) is obtained from Vaccinium angustifolium Aiton. In another embodiment the blueberry, blueberry extract or flavonoid(s) is obtained from Vaccinium corymosum L.

The seed of Theobroma cacao also known as cacao tree and cocoa tree are used to make cocoa powder which is an ingredient of chocolate. The cocoa seed and cocoa powder is like blueberry a source of antioxidants e.g. flavonoids with neuroprotective effect and the ability to promote memory, learning and cognitive function. In one embodiment one or more flavonoid(s) are isolated form cocoa. In another embodiment the flavonoid is provided in the form of cocoa or cocoa extract. Cocoa may be added to the composition in the form of cocoa powder. All tea plants belong to the species Camellia sinensis. The leaves of Camellia sinensis are harvested and processed into tea. It is the production process of the leaves after harvesting that determines whether the final product will be a white tea, green tea, oolong tea or black tea. White and Green tea are the least processed and thus provides the highest level of flavonoids and other compounds with a positive impact on health. In one embodiment the flavonoid(s) is selected from the list consisting of rutin, catechins, myricetin and quercetin. In one embodiment the flavonoid(s) is provided in the form of white tea or white tea extract. In another embodiment the flavonoid(s) is provided in the form of green tea or green tea extract.

The composition of the present invention is preferably combined in one dosage form. The components of the composition are present in different concentrations.

In one embodiment the composition comprises uridine, phosphate, acyl derivatives, ester or salt thereof at a concentration of 1000 mg per dosage form. In another embodiment the dosage of uridine is within the range of 500- 5000 mg per dosage form. In another embodiment the dosage of uridine is in the range of 200-1200 mg per dosage form. In another embodiment the dosage of uridine is in the range of 500-1000 mg per dosage form. In another embodiment the dosage of uridine is in the range of 1000-2000 mg per dosage form. In another embodiment the dosage uridine is in the range of 2000-3000 mg per dosage form. In another embodiment the dosage of uridine is in the range of 3000-4000 mg per dosage form. In another embodiment the dosage of uridine is in the range of 4000-5000 mg per dosage form.

The composition further comprises docosahexaenoic acid (DHA). DHA may be in triacylglyceride form, or as free fatty acids, as phospholipids, esters or salts thereof at a concentration of 500 mg per dose form. In another embodiment the dosage of DHA is within the range of 500-5000 mg per dosage form. In another embodiment the dosage of DHA is within the range of 100-1000 mg per dosage form. In another embodiment the dosage of DHA is within the range of 1000-5000 mg per dosage form. In another embodiment the dosage of DHA is within the range of 2000-5000 mg per dosage form. In another embodiment the dosage of DHA is within the range of 3000-5000 mg per dosage form. In another embodiment the dosage of DHA is within the range of 4000-5000 mg per dosage form. The composition may comprise vitamin D in the form of vitamin D3_, 25- hydroxyvitamin D3, biologically active form or salt thereof at a concentration of 10 g per dosage form. In another embodiment the dosage of vitamin D3 is in the range of 2-20 g per dosage form. In another embodiment the dosage of vitamin D3 is in the range of 10-100 g per dosage form. In another embodiment the dosage of vitamin D3 is in the range of 10-50 g per dosage form. In another embodiment the dosage of vitamin D3 is in the range of 30-80 g per dosage form. In another embodiment the dosage of vitamin D3 is in the range of 50-100 g per dosage form. In some embodiments the composition comprises one or more flavonoid(s) obtainable from blueberry at a concentration within the range of 100-5000 mg per dosage form. In one embodiment the collective concentration of one or more flavonoid(s) is at a concentration within the range of 100-5000 mg per dosage form. In one embodiment the collective concentration of one or more flavonoid(s) is at a concentration within the range of 100-1000 mg per dosage form. In one embodiment the collective concentration of one or more flavonoid(s) is at a concentration within the range of 1000-5000 mg per dosage form. In another embodiment the concentration of one or more flavonoid(s) is within the range of 100-5000 mg per dosage form for each individual flavonoid. In another embodiment the concentration of one or more flavonoid(s) is within the range of 100-1000 mg per dosage form for each individual flavonoid. In another embodiment the concentration of one or more flavonoid(s) is within the range of 1000-5000 mg per dosage form for each individual flavonoid. In some embodiments the composition comprises blueberry extract at a concentration of 500 mg per dosage form. In another embodiment the composition comprises blueberry extract at a concentration within the range of 100-5000 mg per dosage form. In another embodiment the composition comprises blueberry extract at a concentration within the range of 100-1000 mg per dosage form dosage form. In another embodiment the composition comprises blueberry extract at a concentration within the range of 1000-5000 mg per dosage form.

In one embodiment the composition comprises per dosage form: 500-5000 mg Uridine, or salts, phosphates, acyl derivatives or esters thereof, 500-5000 mg docosahexaenoic acid (DHA) or esters thereof, 10-100 g vitamine D3 or salts thereof, and/or 100-5000 mg flavonoid obtainable from blueberry;

and/or 0,1 -10 g blueberry or blueberry extract. In one embodiment the composition comprises per dosage form: 200-1200 mg Uridine, or salts, phosphates, acyl derivatives or esters thereof, 100-1000 mg docosahexaenoic acid (DHA) or esters thereof, 2-20 g vitamin D3 or salts thereof, and/or 100-5000 mg flavonoid obtainable from blueberry;

and/or 0,1 -10 g blueberry or blueberry extract.

In a further embodiment the composition comprises per dosage form: about 290 mg Uridine, or salts, phosphates, acyl derivatives or esters thereof, about 500 mg docosahexaenoic acid (DHA) or esters thereof, about 10 g vitamin D3 or salts thereof, and/or about 145 mg flavonoid obtainable from blueberry; and/or 0,1 -10 g blueberry or blueberry extract. In one embodiment the composition is a nutritional supplement. Further the composition is formulated as a nutritional supplement contained in gelatinous capsules. The capsules may be provided in any shape suitable for oral ingestion, such as capsules having the shape of drops. In addition the composition is formulated for oral administration. In one embodiment the composition is a nutritional supplement for healthy individual. In another embodiment the composition is a nutritional supplement for subjects with congenital or developmental brain disorders. In an alternative embodiment the formulation of the composition is a solid dosage form. Further in some embodiments the composition is a tablet. In another the composition is a liquid dosage form. Further the in some embodiments the liquid composition is a capsule. In another embodiment the composition is a pharmaceutical composition.

Further the pharmaceutical composition is formulated for oral administration.

In one embodiment the pharmaceutical composition is a solid dosage form.

In another embodiment the pharmaceutical composition is a liquid dosage form. Further in a preferred embodiment the pharmaceutical composition is a capsule. In other embodiments the pharmaceutical composition is a tablet.

In some embodiments the use of the composition is for improving of cognitive functions and/or motor skills in healthy individuals. In an additional embodiment a composition of the invention is for use in improving cognitive functions and/or motor skills in individuals in need therefore. In some embodiments the composition is for use in improving cognitive functions and/or motor skills in individuals with developmental coordination disorder and congenital brain disorder. In a further embodiment the composition is for use in improving the cognitive functions and/or motor skills in healthy individuals. In another embodiment the composition is for use in improving learning in healthy individuals. In a further embodiment the composition is for improving learning in individuals with developmental coordination disorder. In another embodiment the composition is for use in improving memory in healthy individuals. In a further embodiment the composition is for improving memory in individuals with developmental coordination disorder.

In another embodiment the composition is for use in improving attention in healthy individuals. In a further embodiment the composition is for use in improving attention in individuals with developmental coordination disorder.

In another embodiment the composition is for use in improving the cognitive functions and/or motor skills in prepubescent children. In a further embodiment the composition is for use in improving cognitive functions e.g. attention, learning and/or memory in prepubescent children.

Another aspect of the invention is a method for improving cognitive functions and/or motor skills in a human subject in need thereof, comprising administering to the subject an effective amount of a composition comprising: i) Uridine, such as Uridine monophosphate, or salts, phosphates, acyl derivatives or esters thereof; ii) Docosahexaenoic acid (DHA), such as DHA in triacylglyceride form or esters thereof; iii) vitamin D3, such as 25- hydroxyvitamin D3, or salts thereof. A preferred embodiment of the invention is a method for improving cognitive functions and/or motor skills in a human subject in need thereof, comprising administering to the subject an effective amount of a composition comprising : i) uridine monophosphate and ii) docosahexaenoic acid in triacylglyceride form and iii) vitamin D3. Another embodiment of the invention is a method for improving cognitive functions and/or motor skills in a human subject in need thereof, comprising administering to the subject an effective amount of a composition which further comprises at least one flavonoid obtainable form blueberry.

An additional embodiment of the invention the composition used by the method of improving cognitive functions and/or motor skills comprises at least one flavonoid which is selected from the list consisting of rutin, myricetin, quercetrin delphinidin, cyanidin, petunidin, peonidin, malvidin, anthocynins procyanidins. In yet a further embodiment, the at least one flavonoid is provided in the form of blueberry or blueberry extract.

In another embodiment the blueberry or blueberry extract is obtained from Vaccinium spp. In another embodiment of the composition the blueberry or blueberry extract is obtained from Vaccinium angustifolium Aiton. In another embodiment of the composition the blueberry or blueberry extract is obtained from Vaccinium corymosum L.

An additional embodiment of the invention is a method of activating the transcription factor cAMP response element-binding protein (CREB) by administering a composition according to the invention.

When describing the embodiments of the present invention, the combinations and permutations of all possible embodiments have not been explicitly described. Nevertheless, the mere fact that certain measures are recited in mutually different dependent claims or described in different embodiments does not indicate that a combination of these measures cannot be used to advantage. The present invention envisages all possible combinations and permutations of the described embodiments.

The term "comprising", "comprise" and "comprises" herein are intended by the applicant to be optionally substituted with the terms "consisting of, "consist of or "consists of, respectively, in every instance. Example 1

The objective of the present study was to investigate the ability of a composition comprising Uridine, DHA, vitamin D and blueberry extract to affect cognitive and motor functions, according to the present invention.

The following example(s) are provided so as to describe to those or ordinary skill in the art how to make and use methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

SUBJECTS AND METHODS

Subjects

The present study was a randomized double-blinded placebo-controlled cross-over design. The subjects in the study were fourteen prepubescent schoolchildren (6 girls and 8 boys). The average age was 9.6±0.19 yr, body weight was 34.5±1 .5 kg and body mass index (BMI) was 16.7±0.6 kg/m2, with no difference between sex (Table 1 ). Maximal oxygen uptake (VO2 max) for the group was 1 .85±0.08 l/min with higher levels in boys (2.04±0.07 l/min) compared to girls (1 .60±0.08 l/min, p<0.01 , Table 1 ). All subjects were right- handed according to the Edinburgh Handedness Inventory (Neuropsychologia 1971 ;9:97-1 13) and none of them had any history of neurological or psychiatric disorders.

The children and their parents gave informed written and oral consent to the experiment, which was approved by the local ethics committee for the Copenhagen area. The experiments were performed in accordance with the Helsinki Declaration.

Research Design

A combination of docosahexaenoic acid (DHA), vitamin D, uridine and blueberry extract (DDU-supplement) or a placebo supplement containing medium-chain triacylglycerol was ingested in a 6 week period with a 6 week washout period between trials. The interventions were combined with a cognitive and motor training program consisting of a number of progressively challenging cognitive and motor tasks for 30 minutes a day, 3 days a week. Before the first intervention period four days of diet registration were completed. Before and after each intervention period blood sampling and testing of cognitive parameters such as attention, learning and memory was performed. Furthermore, maximal oxygen uptake (VO₂inax) was measured on a treadmill before and after the interventions to determine the physical fitness level of the children.

Dietary registration

Four days of diet registration were completed prior to the first intervention period. For data analysis of the dietary registrations the computer program: DANKOST 3000, was applied providing details about energy intake and macro- and micronutrients.

Blood sampling

Blood was drawn in the morning following a 12 hours fasting period. Blood was drawn from an antecubital vein with cuff. Plasma or serum was frozen at minus 20 or 80 degrees Celsius for further analysis.

Testing

Math test and reading/comprehension test

All children completed school tests, which included a reading/comprehension task, as well as a math test. All exercises were specifically chosen in accordance to the level of 3^rd and 4^th grades school children. Number of errors and time for completion of each task were noted. The reading/comprehension test is a standardized school test where children have a maximum of 15 minutes to complete the task. In order to note the speed of their answers the colour of the pencil was changed every fifth minute. For both the math test and the reading/comprehension test off-line counting of errors was assessed.

Psychological test

The children completed memory and attention tests by using the computer program CogState and the computer exercises were chosen to specifically measure visual attention, learning and executive functions. The program is ideal for pre and post testing design as the tasks are presented in a different way without changing the level. The total time for the test is around 30 minutes and the main outcomes from the test are given by speed and total correct moves/choices. In this study we used the following tasks: One Back Task: subjects had to recall if the card they were presented was similar to the previous card seen. Two Back Task: subjects had to recall if the card they were presented to was similar to the second previously seen card. Continuous Paired Associative Learning (CPAL) Task: subjects had to memorize the positions of up to seven abstract figures on the computer screen. Groton Maze Learning Test: subjects had to remember a specific path in a maze, which were 10 x 10 squares. This test was also presented as a recall-test after completing the other tests in the CogState program. Identification Task: subjects had to respond as fast as possible depending on what card was presented on the computer screen (red card vs. black card).

Measurement of maximal oxygen uptake (V0₂max)

All children performed a running test on a treadmill. The children were familiarized with the equipment and test procedure before the running test. During all tests the children breathed through paediatric masks adapted to their faces. Heart rate (HR) was continuously recorded (polar Electro, Finland). Respiratory gas exchanges were measured breath-by-breath using an automatic gas-analysis system (CPX MedGraphics, USA) to determine oxygen uptake (VO2) and respiratory exchange rate (RER). Calibrating of O2 and CO2 analysis systems were performed before testing using ambient air and a mix of known O₂ and CO₂ concentrations (15 % O₂ and 5.8% CO₂) and the tube flowmeter was calibrated using a 3 L syringe. For data analysis values were recorded every 5 seconds. The children performed an incremental running test to exhaustion on a treadmill. The protocol started with 1 min running at an individual maximal speed (9-1 1 .5 km/h) on slope 0% followed by stepwise 1 % incline every 1 minute until exhaustion. Criteria for reaching maximal oxygen uptake were: RER > 1 .00, a plateau in VO₂ despite increasing slope and unable to continue running despite verbal encouragement.

DDU-supplement and placebo supplement

DDU-supplement consisted of a 10 ml o/w emulsion of 500 mg docosahexaenoic acid (DHA) in triacylglyceride form, 10 g vitamin D and 1000 mg uridine (Table 2). 0.5 g blueberry extract was added. Placebo consisted of a 10 ml o/w emulsion of 2 g MCT oil (medium-chain triglycerides) and artificial colouring (Table 2). Whey protein was used as emulsifier in both types of emulsions. In addition, both emulsions contained synthetic blueberry flavour to make the flavour and odour of the two emulsions similar. An overview of the different ingredients and their concentrations in the two different emulsions is shown in Table 2.

Production: DDU-supplement and placebo emulsions were produced in 2- steps: pre-emulsification and homogenization. First, whey protein was solubilized in the water and other hydrophilic ingredients were thereafter added to the whey protein-water solution (DDU-supplement: uridine and blue berry extract, artificial flavour; Placebo: artificial colour and flavour). For pre- emulsification, the aqueous solutions were stirred with an Ultra-Turrax (Janke & Kunkel IKA-Labortechnik, Staufen, Germany) and oil mixture (DDU-supplement: DHA 500TG and vitamin D) or oil (Placebo: MCT oil) was added during first min of the 2 minutes total mixing. Pre-emulsions were then homogenized using a two valve table homogenizer at a pressure of 225 bar (GEA Niro Soavi Spa, Parma, Italy). Produced emulsions were bottled (10 ml_), purged with nitrogen to limit lipid oxidation, sealed and pasteurized (72°C). All bottles were cooled at 5°C and thereafter stored at - 20°C until the delivery day. The emulsions were taken daily in the morning together with at least 100 ml yoghurt or orange juice as part of the study subjects breakfast.

Materials: Oils used for DDU-supplement (Incromega DHA 500TG, 58% DHA) and Placebo (MCT, medium chain length C6-C12 triglycerides, whereof C8 56% and C10 43%, 99.3% triglyceride) were supplied from CRODA (East Yorkshire, England) and Sasol Germany GmbH (Witten, Germany), respectively. Vitamin D, Uridine and blueberry extract were purchased from a local dietary shop, Yamasa Corporation (Chiba, Japan) and DENK Ingerdients GmbH (Munich, Germany), respectively. Artificial flavor and whey protein were donated by A S Einar Willumsen (Broendby, Denmark) and Aria Foods (Viby J, Denmark), respectively. Artificial colours (Dr. Oetker: red, blue and green) were purchased from a local super market.

MiTii program:

During the two intervention periods the children practiced a number of progressively challenging cognitive and motor tasks for 30 minutes a day, 3 days a week. The children completed all practice sessions at home using a recent developed internet based cognitive and motor training system (Move It To Improve It: MiTii). Mitii consists of a number of modules in which the child used visual information, solved a cognitive problem (i.e. mathematical question, memory related task or similar) and responded with a motor act (i.e. bend to pick up needle and blow up balloon with the right answer). Examples of modules are Memory: the children had to memorize a specific order of images and Mathematics: the children had to solve arithmetic tasks as fast as possible. Further details are described earlier (BMC Neurol 201 1 ;1 1 :32). Each child had to perform 4 different MiTii programs each consisting of 24 modules. Program 1 was completed in the first three weeks of the first intervention period and program 2 in the last three weeks. Program 3 and 4 were used in the first and last 3 week periods of the second intervention period. All children practiced the same program at the same time. Data of the task performance were collected on a server for offline analysis.

Analysis

25(OH)D was used as measurement of plasma vitamin D concentration (J Nutr 2005;235:2739-48), and was measured by a competitive chemiluminescent immunoassay on a immunoDiagnosticSystem (iSYS). DHA (C22:6, n-3) in plasma was analyzed as earlier described (Eur J Lipid Sci Technol 2007;109:1993-009). Briefly, plasma lipids were extracted using a modification of the Folch-method, and fatty acid methyl esters (FAME) were produced using a BF₃-catalyzed method, in which hydroquinone is added as antioxidant (Hamilton, J.R. and Hamilton, S. Extraction of lipids and derivative formation, in Lipid Analysis- A practical Approach. 13-64.1992. Oxford , IRL Press.). The method has been validated on DHA, and do not induce double-bond losses. The mass-percentage contribution of DHA to the total plasma FAME-pool was analyzed using GC-FID, as earlier described (Eur J Lipid Sci Technol 2007;109:1993-009). Plasma brain- derived neurotrophic factor (BDNF) concentration was measured by a commercial available kit (Cat. No. CYT306, Chemicon international chomikine) by ELISA (Millipore, Corporation).

Blood glucose and haemoglobin concentration were measured on ABL815 (Radiometer Medical A S, Copenhagen, Denmark).

Plasma triacylglycerol (TG), total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and fatty acids (FA) were measured using enzymatic colorimetric methods (Hitachi 912 automatic analyzer). Statistics

All data was analyzed using SigmaPlot (version 1 1 .0, SYSTAT Software, San Jose, CA, USA). Data are expressed as means±SEM. Data were evaluated using two-way ANOVA with repeated measures for both intervention (DDU- supplement/placebo) and time (Pre/Post intervention). For values depended on intervention but not on time (delta values) a paired t test performed. To test for differences between boys and girls an unpaired t test was performed when variables were independent of time and intervention. A Tukey test was used as a post hoc test.

The psychological tests CPAL and GML were tested using a linear mixed model that takes into account that measurements within persons are correlated. Correlations were investigated using the Pearson product moment correlation. A significance of P<0.05 was chosen.

RESULTS

There was no difference in results whether the children completed the placebo trial first or the DDU- supplement trial first. Habitual diet

Diet registrations from three children were excluded because of inaccurate results. The diet was supplemented with vitamins and minerals on a daily basis in eight of the children. Results of the dietary registration are shown without these individual supplements when nothing else is noted.

The Nordic nutrition guidelines from 2004 and the information from the Danish Food ministry from 2013 provides the given recommendations for children in the age of 9-1 1 years.

Energy intake amounted to 208±17 kJ/kg body weight (bw) and 246±26 kJ/kg bw in girls and boys, respectively (p=0.3) (Table 3). The composition of the diet averaged 57±1 .5 percentage of total energy intake (E%) carbohydrates, 28±1 .3 E% fat and 16±0.5 E% protein. The boys consumed a higher E% carbohydrates and a tendency towards a lower E% fat (p=0.056) than the girls (Table 3).

Despite that the average intake of mono- and polyunsaturated fatty acids was within the range of recommended levels, 45% of the children were below recommended intake of both mono - and poly- unsaturated fatty acids (Figure 1A and B).

The average daily vitamin D intake from the diet was not different between sex (Table 4). In both groups vitamin D intake was lower (mean 3.89±2.66 g) than recommended (7.5 g). Eight children supplemented their daily intake with vitamins and minerals and thereby reached the recommended level of vitamin D intake (Figure 1 C).

A lower intake of calcium (55%), potassium (90%) and iodine (90%) was observed compared to recommended levels in the children (Table 4).

Blood parameters

Before the DDU-supplementation trial the average plasma concentration of vitamin D (25(OH)D) was 47.7±4.8 nmol/l. It increased to 62.4±4.5 nmol/l (p<0.001 ) after DDU-supplementation but remained unchanged in the placebo trial (pre: 52.9±4.9 nmol/l, post: 49.1 ±5.0 nmol/l). The increase in plasma concentration of vitamin D was significantly larger in DDU- supplementation trial compared to placebo (Figure 2A).

Before the DDU-supplementation trial the average plasma DHA concentration was 2.33±0.24 % of total FAME-pool and increased to 3.66±0.19 % of total FAME-pool (p<0.001 ), with no differences between sex. In the placebo trial plasma DHA concentration was unchanged (pre: 2.74±0.19, post: 2.39±0.20 % of total FAME-pool). Delta plasma concentration of DHA was significantly larger in DDU-supplementation trial compared to placebo trial (Figure 2B). Plasma brain-derived neurotrophic factor (BDNF) concentration remained unchanged following both the DDU-supplement trial and the placebo trial (Figure 2C).

There were no sex differences or differences between basal in relations to vitamin D, DHA and BDNF plasma concentrations.

There was no difference in basal plasma concentration of glucose, triacylglycerol, cholesterol, HDL-C, LDL-C, Fatty acids (FA) and haemoglobin before either intervention (Table 5). Plasma glucose and HDL-cholesterol concentrations remained unchanged during both interventions (Table 5). LDL-cholesterol concentration decreased significantly in the placebo trial and plasma FA concentration decreased significantly in response to both interventions (Table 5). There was no difference in plasma parameters between sex.

A correlation was found between dietary vitamin D intake and plasma vitamin D concentration (Figure 1 D, r=0.7, P<0.05). No correlation was found between delta DHA and delta BDNF in DDU-supplementation trial (r=-0.17, p=0.57) or placebo trial (r=-0.092, p=0.76).

Testing

MiTii practice

Motor and cognitive training resulted in significant progression of performance of cognitive and motor tasks in the program with no difference in relation to dietary supplementation. Two examples of modules are shown in Figure 3. School test

Time to complete reading/comprehension test was significantly reduced following the motor and cognitive training intervention (Figure 4A), with no decline in number of correct answers (data not shown). Interestingly, the math test was performed faster (p<0.05) following DDU-supplement than placebo (Figure 4B), with no decline in number of correct answers (data not shown). Psychological tests

We found an overall improvement of interventions in the CPAL task shown as an improvement in speed of performance (Figure 5A) and increase in percentages of correct answers (Figure 5B). The GML task was improved in response to the training intervention with a decrease in duration (Figure 5C) and errors (Figure 5D). Moreover, there was an overall decrease in percentage of error in response to both interventions in the One back Task (Figure 5E), and interestingly a significantly faster performance after DDU- supplementation compared to placebo was observed (Figure 5F).

Maximal oxygen uptake

Maximal oxygen uptake (VO2 max) remained unchanged during the intervention both in the DDU-supplementation trial (pre; 1 .85±0.09 l/min, post: 1 .96±0.09 l/min) and the placebo trial (placebo pre: 1 .86±0.07 l/min, post: 1 .92±0.08 l/min).

Conclusion

The present study investigated the effect of cognitive and motor training with and without supplementation of DHA, vitamin, uridine and blueberry extract on motor and cognitive abilities in prepubescent children. The inventors found an overall improvement of cognitive learning after 6 weeks of progressively challenging cognitive and motor tasks, 30 minutes 3 days/week independent of dietary supplementation. This was reflected in a significant progression in the cognitive and motor tasks in the MiTii program (Figure 3), a significant decrease in time to complete reading/comprehension test (Figure 4A), improvement in psychological tests (CPAL and GML tests, Figure 5A-D) and a decrease in percentage of error in the One back Task (Figure 5E). These data show improved attention and working memory after 6 weeks of cognitive training. Interestingly, after a daily supplementation of DHA (500 mg), vitamin D (10 g), uridine (1000 mg) and blueberry extract (500 mg) which resulted in a significant increase in plasma levels of DHA an vitamin D content, an even bigger improvement in math test (Figure 4B) and One Back Task (Figure 5F) was shown These observations were not apparent in the placebo trial (Figure 2). Furthermore, the physical fitness level of the children remained unchanged during the interventions. Together the result of the study show an additive effect on cognitive learning when cognitive-motor training and dietary supplementation of DHA, vitamin D uridine and blueberry are combined in children, especially when status of plasma DHA and Vitamin D concentration was low.

The participants in present study included 14 healthy, normal weight, prepubescent school children with lipid profile and maximal oxygen uptake comparable to Danish children at similar age (Prev Med 2003;37:363-7). However, the average plasma concentration of vitamin D was 47.7±4.8 nmol/l (19.12±1 .9 ng/ml) before DDU-supplementation, which is considered as vitamin D deficiency (Nimitphong H, Curr Opin Clin Nutr Metab Care 201 1 ;14:7-14). 90-95% of vitamin D formations occur as a result of exposures to sunlight. Our study was initiated in wintertime and stopped during the spring. In this period no cutaneous synthesis of vitamin D occurs in the latitude of Denmark (54-58°N), because of lack in ultraviolet B photons (290-315 nm) penetrations from the sun. Thus, vitamin D status is dependent on intake, but an adequate intake can be difficult to obtain due to limited intake of food sources containing vitamin D. This can be one of the reasons for the poor vitamin D status found in children in our study. Moreover, 4 days of habitual diet registration revealed an insufficient intake of mono- and polyunsaturated fatty acids in 45 % of the children in the present study. We observed a correlation between vitamin D intake and the plasma vitamin D concentration (Figure 1 D, r=0.7, P<0.05,) supporting that the dietary registration was accurate. The observed low intake of important fatty acids could be due to misleading recommendations from parents, school or society with advise to reduce fat in general resulting in reduced intake of important fat sources.

The length of dietary supplementation is another important factor to consider. In the NEMO study the intervention period was 12 month (Osendrap SJ, 2007) while dietary supplementation lasted only 8 weeks in the study by Kennedy et al. (2009) suggesting that cognitive changes take time to occur. In our study cognitive skills were improved after only 6 weeks, even though we did not use higher daily doses of DHA in the present study (500mg) compared to the study by Kennedy et al. (400 mg or 1000 mg). The improvement in the present study could not be attributed to changes in physical fitness level, as this remained unchanged. In the present study an internet based training program was applied allowing us to control that each session of practice was completed. The rather fast improvements obtained may be a synergistic effect conferred by the combination of nutrients, which is more effective than DHA alone. Little is known about the combination of DHA, vitamin D and uridine in relation to brain physiology in humans. In rats, the combined intake of vitamin D and DHA resulted in a synergistically protective impact in relation to cerebral ischemia (Neurol Sci 2009;30:207-12) and the administration of DHA and uridine in combination improved memory and cognitive skilled tasks in rodents (Behav Brain Res 2008;191 :1 1 -6). The mixture of important neural substrates in the present study might contribute to improve cognitive learning. However, it cannot be ruled out that the low initial status in plasma vitamin D concentrations and DHA content in the children in the present study plays a significant role for the rapid improvement in cognitive skills.

To our surprise we found no effect on the plasma concentration of BDNF (figure 2C), despite an increase in plasma DHA concentration (figure 2B). It is known that BDNF cross the blood barrier in both directions. Thus, plasma/serum drawn from an antecubital vein is believed to reflect the BDNF level in the brain. To our knowledge no one has measured exercise facilitated BDNF production in children. In adults aerobic and resistance training resulted in increased serum BDNF concentration in some studies, but not in all. Schmidt-Kassow et al. (Neuroreport 2012;23:889-93) showed in adults that acute exercise at high intensity increased the serum BDNF concentration to higher levels than low intensity exercise. In the present study maximal oxygen consumption did not change in response to 30 minutes progressively challenging cognitive and motor task program, 3 days/week, indicating that the physical activity level when practicing the cognitive and motor task program was of a too low intensity to induce changes in BDNF production. In the study by Schmidt-Kassow et al. serum BDNF concentrations returned to baseline within only 10 min of recovery, suggesting an acute clearance of the blood, which may be a reason for the lack of change in fasting levels in the present study.

In conclusion our data show that 6 weeks of cognitive and motor training improves cognitive abilities with more marked improvements when combined with daily dietary supplementation of DHA, vitamin D, uridine and blueberry extract in prepubescent children with initial poor DHA and vitamin D status. Furthermore, the data shows that daily intake of a composition of the present invention are able to optimize cognitive abilities and motor learning in children.

Example 2

The effect of a food supplement containing an extract of blue berry on the learning ability in mice (C57BL/6 mice) challenged with a motorial precision task was investigated. The contents of the supplements are seen in the table below. 30 mice (4 weeks old where mice are regarded as adult by the age of 5 weeks) was divided in 2 groups containing 15 individuals. One group was given ordinary staple food, whereas the other group was given the same staple food supplemented with a blue berry extract rich in flavonoids (ca. 200 mg/kg). The mice were given the respective foods for 2 weeks prior to being challenged and trained with the above mentioned task. The then 6 old mice were trained and challenged with the precision motorial task for 2x20 min every day for a period of 10 days. The task consisted of the mice having to stretch out its paws to grad a treat. The treats were arranged in a steps formation requiring a certain skill to be reachable. In total, 32 treats were arranged in the described manner which also presented the maximum score an individual mouse could attain. After 10 days, the mice were tested 2 times for 2 consecutive days. The results are graphically illustrated in Fig. 6.

The difference is attributed to the fact that the mice used in the study were young healthy individuals which have an inherent good capability of learning high precision motorial operations. It is expected to see a larger difference in mice having a neurological defect. The reason for studying healthy mice is that mice with a neurological effect may have a high inter individual variation which may influence the statistical variation of the results.

TABLES:

TABLE 1

Characteristics for girls and boys together (n=14) and girls (n=6) and boys (n=8) separately .

Girls + boys Girls Boys

Age (years) 9.71±0.19 9.8±0.3 9.6±0.3

Weight (kg) 34.5±1.5 32.4±2 35.4±2

Height (cm) 142±1.3 141+1.1 143±1.9

BMI (kg/m ) 16.7±0.6 16.2±0.8 17.1±0.7

V0₂max (l/min) 1.85±0.08 1.60±0.08 2.04±0.07**

V0₂max (l/min/kg) 0.55±0.02 0.49±0.02 0.59±0.04*

¹ Data are Mean ± SEM. ^*p>0.05, ^**p>0.01 vs. girls (t test).

²V0₂max: maximal oxygen uptake

TABLE 2

Composition of DDU-supplement and Placebo emulsions¹ [wt %]

Ingredients DDU-supplement Placebo

Hydrophilic ingredients

Water 62.1 58.9

Whey protein 1.01 1.01

Blueberry extract 5.00

Uridin 10.0

Artificial flavour 1.69 0.85

Red color 13.1

Blue color 1.05

Green color 4.18

Lipophilic ingredients

Incromega DHA 500TG 17.3

Vitamin D 2.90

MCT oil 20.1

¹Data are shown in wt % (weight solute/weight total solution). DHA, Docosahexaenoic acid. MCT, medium-chain triacylglycerol, ²The amount of these ingredients was adjusted according to their purity to give the desired amount of bioactive compounds (DHA: 0.5 g; Vitamin D: 10 μg) in 10 ml of the DDU emulsion. TABLE 3

Macronutrients given by an average of four independent days of 24 hours dietary weighing for girls and boys together (n=1 1 ) and girls (n=4) and boys (n=7) separately .

Girls + boys Girls Boys Rl²

Energy intake (kJ) 7797±462 6733±401 8406±586(*) Girls: 8200

Boys: 9500

Energy intake (kJ/kg bw.) 232±18 208±17 246±26

Macronutrients

Protein (E%) 16±0.5 17±0.2 15±0.6 <20

Fat (E%) 27±1.3 31 ±1.9 26±1.4(**)

Carbohydrate (E%) 57±1.5 52±2.0 59±1 .2 *

Lipid profile

Saturated fatty acid (E%) 8±0.58 9±0.7 8±0.8 <10

Monounsaturated fatty acid (E%) 12±1.42 13±3.2 1 1 ±1.4 10→15

Polyunsaturated fatty acid (E%) 7±1.23 9±2.6 7±1.3 5→10

Cholesterol (mg) 218±35.3 266±52 190±46 < 300

Added sugar (E%) 3.48±0.66 3.80±1.80 3.30±0.37 < 10

Fibers (g) 26.0±2.09 21.2±1.6 28.8±2.7 (§) >10

¹ Data mean ± SEM,^*p>0.05, (^*)p=0.079, (^**)p=0.056 (§)p=0.076 vs. girls (t test), bw = body weight. E%, percentages of total energy intake.

2 RI: Recommended intake for a child aged 10-1 1 years.

TABLE 4

Micronutrients given by an average of four independent days of 24 hours dietary weighing for girls and boys together (n=1 1 ) and girls (n=4) and boys (n=7) separately . Girls + boys Girls Boys Rl²

Micron utrients

Vitamins

Vitamin A (RE (μς)) 931 ±82.2 767±169 1026±74 600

Vitamin D ( g 4.15±0.84 4.36±1.8 4.02±0.9 7.5

Vitamin E (aTE (mg)) 14.1±3.24 12.8±5.3 14.9±4.4 7

Vitamin B1 (thiamin) (mg) 9.33±3.35 10.88±6.2 8.44±4.3 1.2

Vitamin B2 (riboflavin) (mg) 4.69±1.30 5.14±2.4 4.43±1.7 1.4

Girls:14

Vitamin B3 (niacin) (mg) 17.1±1.01 15.6±1.4 17.9±1.2

Boys: 16 Vitamin B6 (mg) 9.80±3.44 11.3±6.5 9.0±4.3 1.3

Vitamin B12 (μς) 14.2±3.70 15.5±7.0 13.5±4.7 2

Vitamin C (mg) 250±82 257±162 245±101 50

Folacin ^g) 430±73.2 296±47 507±104 200

Minerals

Calcium (mg) 873±70.63 853±68 885±108 900

Phosphor (mg) 1352±73.02 1219±29 1429±105^* 700

Girls: 2900

Potassium (mg) 2412±163 2156±166 2560±228

Boys: 3300

Magnesium (mg) 349±21.6 306±17 374±29(^*) 280

Feron (mg)g 19.9±3.0 18.1±5.0 21.0±4.0 11

Zinc (mg) 16.7±3.1 17.3±5.5 16.4±4.1 11

Copper (mg) 3.83±0.78 3.6±1.4 4.0±1.0 0.7

Iodine ^g) 119±9.1 112±14 123±12 150

Selen ^g) 59.6±7.7 48±7.0 66±11.0 40

Data mean ± SEM. ^*p>0.05, (^*)p=0.078 vs. girls (t test).

Rl: Recommended intake for a child aged 10-1 1 years.

TABLE 5

Effect of interventions on blood parameters

DDU-supplement Placebo supplement Pre Post Pre Post

TG (mmol/l) 0.75±0.04 0.82±0.05 0.68±0.05 0.94±0.14

Cholesterol (mmol/l) 4.18±0.17 4.41 ±0.15 4.53±0.16 4.21 ±0.17

HDL-C (mmol/l) 1.39±0.07 1.41 ±0.10 1.52±0.06 1.45±0.10

LDL-C(mmol/l) 2.52±0.16 2.60±0.14 2.72±0.17 2.39±0.15*

FA (μΓΤΐοΙ/Ι) 542±70 363±37n 614±77 447±69n

Glucose (mmol/l) 5.03±0.08 5.33±0.08 5.22±0.10 5.23±0.16

Hemoglobin (mmol/l) 8.20±0.38 8.66±0.17 8.24±0.28 8.51 ±0.14

¹ Data are Mean ± SEM, n=14, ^*p<0.05 compared to pre, ° p<0.05: time interference (Two-way ANOVA RM), FA, Fatty acids; HDL-C, High-density lipoprotein cholesterol; LDL-C, Low-density lipoprotein cholesterol; TG, triacylglycerol.

In the following the invention is described in non-limiting items. Item 1 . A composition comprising:

i) Uridine, such as Uridine monophosphate, 5'-uridylic

acid (UMP), or phosphates, acyl derivatives, esters or salts thereof;

ii) Docosahexaenoic acid (DHA), such as DHA in triacylglyceride form, or phospholipids, esters or salts thereof;

iii) Vitamine D3, such as 25-Hydroxyvitamin D3, or salts thereof.

Item 2. The composition according to item 1 , further comprising at least one flavonoid obtainable from blueberrry.

Item 3. The composition according to item 2, wherein said at least one flavonoid is selected from the list consisting of rutin, myricetin, quercetrin delphinidin, cyanidin, petunidin, peonidin, malvidin, anthocynins procyanidins and flavonols. Item 4. The composition according to item 2 or 3, wherein said at least one flavonoid is provided in the form of blueberry or blueberry extract,. Item 5. The composition according to item 4, wherein the blueberry or blueberry extract is from Vaccinium spp.

Item 6 The composition according to item 4, wherein the blueberry or blueberry extract is from Vaccinium angustifolium Aiton.

Item 7 The composition according to item 4, wherein the blueberry or blueberry extract is from Vaccinium corymosum L.

Item 8. The composition according to any of the preceding items, wherein said composition comprises uridine, or salts, phosphates, acyl derivatieves or esters thereof at a concentration within the range of 500 - 5000 mg per dose such as 1000 mg per dose.

Item 9. The composition according to any of the preceding items, wherein said composition comprises docosahexaenoic acid (DHA) or esters thereof at a concentration within the range of 500 - 5000 mg per dose such as 500 mg per dose.

Item 10. The composition according to any of the preceding items, wherein said composition comprises vitamin D3 or salt thereof at a concentration within the range of 10 - 100 g per does such as 10 g per dose.

Item 1 1 . The composition according to any of the preceding items, wherein said composition comprises flavonoid obtainable for blueberry at a concentration within the range of 100 - 5000 mg per dose. Item 12. The composition according to any of the preceding items, wherein said composition comprises blueberry extract at a concentration within the range of 100 - 10000 mg per does such as 500 mg per dose. Item 13. The composition according to any of the preceding items, wherein said composition comprises, per dosage form:

500-5000 mg Uridine, or salts, phosphates, acyl derivatives or esters thereof; 500-5000 mg Docosahexaenoic acid (DHA) or esters thereof;

10-100 g Vitamine D3 or salts thereof; and/or

100-5000 mg flavonoid obtainable from blueberry; and/or

0,1 -10 g blueberry or blueberry extract.

Item 14. The composition according to any of the preceding items, wherein the composition is formulated as a nutritional supplement, contained in gelatinous capsules.

Item 15. The composition according to any of the preceding items, wherein the composition is formulated for oral administration.

Item 16. The composition according to any of the preceding items, wherein the composition is a solid dosage form.

Item 17. The composition according to any of the preceding items, wherein the composition is a liquid dosage form.

Item 18. The composition according to any of the preceding items, wherein the composition is a capsule.

Item 19. The composition according to any of the preceding items, wherein the composition is a tablet. Item 20. A pharmaceutical composition comprising the composition according to any of the preceding items.

Item 21 . The pharmaceutical composition according to any of the preceding items, wherein the composition is formulated for oral administration.

Item 22. The pharmaceutical composition according to any of the preceding items, wherein the composition is a solid dosage form. Item 23. The pharmaceutical composition according to any of the preceding items, wherein the composition is a liquid dosage form.

Item 24. The pharmaceutical composition according to any of the preceding items, wherein the composition is a capsule.

Item 25. The pharmaceutical composition according to any of the preceding items, wherein the composition is a tablet.

Item 26. Use of the composition according to any of the preceding items for improving of cognitive functions and/or motor skills.

Item 27. A composition according to any of the preceding items for use in improving cognitive functions and/or motor skills. Item 28. The composition according to any of the preceding items for use in improving the cognitive functions and/or motor skills in healthy individuals.

Item 29. The composition according to any of the preceding items for use in improving learning in healthy individuals. Item 30. The composition according to any of the preceding items for use in improving memory in healthy individuals.

Item 31 . The composition according to any of the preceding items for use in improving attention in healthy individuals.

Item 32. The composition according to any of the preceding items for use in improving the cognitive functions and/or motor skills in prepubescent children.

Item 33. The composition according to any of the preceding items for use in improving cognitive functions e.g. attention, learning and/or memory in prepubescent children.

Item 34. A method for improving cognitive functions and/or motor skills in a human subject in need thereof, comprising administering to the subject an effect amount of a composition comprising:

i) Uridine, such as Uridine monophosphate (UMP), 5'-uridylic acid or salts, phosphates, acyl derivatives or esters thereof;

iii) Vitamine D3, 25-Hydroxyvitamin D3 or salts thereof.

Item 35. The method of item 34, wherein the composition further comprises at least one flavonoid obtainable from blueberry.

Item 36. The method according to item 35, wherein said at least one flavonoid is selected from the list consisting of rutin, myricetin, quercetrin delphinidin, cyanidin, petunidin, peonidin, malvidin, anthocynins procyanidins and flavonols. Item 37. The method according to item 35 or 36, wherein said at least one flavonoid is provided in the form of blueberry or blueberry extract.

Item 38. The composition according to item 37, wherein the blueberry or blueberry extract is from Vaccinium spp.

Item 39. The composition according to item 37, wherein the blueberry or blueberry extract is from Vaccinium angustifolium Aiton. Item 40. The composition according to item 37, wherein the blueberry or blueberry extract is from Vaccinium corymosum L.

Claims

1 . A composition comprising:

iv) Uridine, such as Uridine monophosphate (UMP), 5'-uridylic

acid, or phosphates, acyl derivatives, esters or salts thereof; v) Docosahexaenoic acid (DHA), such as DHA in triacylglyceride form, or phospholipids, esters or salts thereof;

vi) Vitamine D3, such as 25-Hydroxyvitamin D3, or salts thereof.

2. The composition according to claim 1 , further comprising at least one flavonoid obtainable from blueberry.

3. The composition according to claim 2, wherein said at least one flavonoid is selected from the list consisting of rutin, myricetin, quercetrin delphinidin, cyanidin, petunidin, peonidin, malvidin, anthocynins procyanidins and flavonols.

4. The composition according to claim 2 or 3, wherein said at least one flavonoid is provided in the form of blueberry or blueberry extract,.

5. The composition according to any of the preceding claims, wherein said composition comprises, per dosage form:

10-100 g Vitamin D3 or salts thereof; and/or

100-5000 mg flavonoid obtainable from blueberry; and/or

0,1 -10 g blueberry or blueberry extract.

6. The composition according to any of the preceding claims, wherein the composition is formulated as a nutritional supplement, contained in

gelatinous capsules.

7. The composition according to any of the preceding claims, wherein the composition is formulated for oral administration.

8. A pharmaceutical composition comprising the composition according to any of the preceding claims.

9. Use of the composition according to any of the preceding claims for improving of cognitive functions and/or motor skills.

10. A composition according to any of the preceding claims for use in improving cognitive functions and/or motor skills.

1 1 . The composition according to any of the preceding claims for use in improving the cognitive functions and/or motor skills in healthy individuals.

12. The composition according to any of the preceding claims for use in improving the cognitive functions and/or motor skills in prepubescent children.

13. The composition according to any of the preceding claims for use in improving cognitive functions e.g. attention, learning and/or memory in prepubescent children.

14. A method for improving cognitive functions and/or motor skills in a human subject in need thereof, comprising administering to the subject an effect amount of a composition comprising:

iv) Uridine, such as Uridine monophosphate (UMP), 5'-uridylic

acid, or salts, phosphates, acyl derivatives or esters thereof; v) Docosahexaenoic acid (DHA), such as DHA in triacylglyceride form, or esters thereof; vi) Vitamin D3, 25-Hydroxyvitamin D3 or salts thereof.

15. The method of claim 14, wherein the composition further comprises at least one flavonoid obtainable from blueberry.