Patent Application: US-201013202453-A

Abstract:
the present invention relates to the use of an hydroxylated stilbene , in particular resveratrol , in the manufacture of a neutraceutical composition for increasing the microvascular plasticity and / or microvessel density , and / or decreasing the microvessel abnormalities in the brain , in particular in the hippocampus of a mammal , in particular for the treatment of age - and condition - related decline in brain neuronal function and / or cognitive functioning in a mammal . in particular , the condition is selected from the group of alzheimer &# 39 ; s disease , dementia , depression , sleep disorders , impaired memory function , psychoses , parkinson &# 39 ; s disease , huntington &# 39 ; s chorea , epilepsy , schizophrenia , paranoia , adhd and anxiety .

Description:
within the context of the invention , the term “ nutraceutical ” as used herein , denotes the use both in the nutritional and pharmaceutical field of application . thus , the nutraceutical composition comprising a hydroxylated stilbene can find use as supplements to food and beverages , and as pharmaceutical formulations for enteral or parenteral application which may be solid formulations such as capsules or tablets , or liquid formulations , such as solutions or suspensions . as will be evident from the foregoing , the term nutraceutical composition also comprises nutritional compositions such as , for instance bars , cookies , drinks , yogurts , ice creams , beverages and the like , containing the above - specified active ingredient . the term hydroxylated stilbene as used herein , comprises compounds encompassed by the general formula i wherein a denotes a carbon - carbon double bond which may be trans or cis , and r1 , r2 , r3 , r4 , r5 and r6 , independently denote hydrogen or hydroxy , with the proviso that at least one of said r - groups denotes a hydroxy group , which hydroxy group may optionally be etherified or esterified . while the carbon - carbon double bond denoted by the symbol a may be trans or cis , formula i above is understood to also include cis / trans mixtures . however , compounds of formula i wherein a is a trans carbon - carbon bond are preferred . etherified hydroxy groups may be derived from unsubstituted or substituted , straight or branched chain alkyl groups having 1 to 26 carbon atoms , in particular 1 to 6 carbon atoms , more in particular methyl . esterified hydroxy groups may be derived from unsubstituted or substituted , straight or branched chain aliphatic carboxylic acids having 1 to 26 carbon atoms , in particular 1 to 6 carbon atoms , more in particular formic acid , and from araliphatic and aromatic carboxylic acids having 3 to 26 carbon atoms , in particular 3 to 18 carbon atoms , more in particular 6 to 12 carbon atoms . etherified hydroxy groups may further be glycoside groups and esterified hydroxy groups may further be glucuronide or sulphate groups . examples of compounds of formula i are summarized in table 1 . of primary interest for the purposes of the invention is resveratrol , in particular ( trans )- resveratrol . the nutraceutical compositions of the present invention usually contain an hydroxylated stilbene in an amount sufficient to administer to a human adult ( average weight about 70 kg ) a daily dosage from about 0 . 5 mg / day to about 2000 mg / day , preferably from about 5 mg / day to about 500 mg / day . hence , if the nutraceutical composition is a food or beverage , the amount of a hydroxylated stilbene contained therein is suitably in the range from about 0 . 5 mg to about 500 mg per serving . if the nutraceutical composition is a pharmaceutical formulation , such formulation may contain from about 0 . 5 mg to about 500 mg per solid dosage unit , e . g ., per capsule or tablet , or from about 0 . 5 mg per daily dose to about 2000 mg per daily dose of a liquid formulation . the term “ serving ” as used herein denotes an amount of food or beverage normally ingested by a human adult with a meal at a time and may range , e . g ., from about 1 g ( such as a nutritional shot ) to about 500 g . in one aspect the present invention the composition comprising an hydroxylated stilbene may be used as a nutritional supplement , e . g ., as an additive to a multivitamin preparations comprising vitamins and minerals which are essential for the maintenance of normal metabolic function but are not synthesized in the body , especially for the treatment or prevention of age - related decline in brain neuronal function and / or cognitive functioning in a mammal . in a preferred embodiment , the mammal is a human . most preferably , the human is an elderly person , except for the embodiments directed to treatment of cerebral palsy . in this respect , it is submitted that in the context of this application , an elderly person is a person of the age of 50 or more , in particular of the age of 55 or more , more in particular of the age of 60 or more , more in particular of the age of 65 or more . this rather broad definition takes into account the fact that the average age varies between different populations , on different continents , etc . most developed world countries have accepted the chronological age of 65 years as a definition of ‘ elderly ’ or older person ( associated with the age at which one may begin to receive pension benefits ), but like many westernized concepts , this does not adapt well to e . g . the situation in africa . at the moment , there is no united nations ( un ) standard numerical criterion , but the un agreed cut - off is 60 + years to refer to the older population in western world . the more traditional african definitions of an elder or ‘ elderly ’ person correlate with the chronological ages of 50 to 65 years , depending on the setting , the region and the country . according to another aspect of the invention , the compositions may be pharmaceutical compositions , preferably for enteral application , which may be solid or liquid galenical formulation . examples of solid galenical formulations are tablets , capsules ( e . g . hard or soft shell gelatine capsules ), pills , sachets , powders , granules and the like which contain the active ingredient together with conventional galenical carriers . any conventional carrier material can be utilized . the carrier material can be organic or inorganic inert carrier material suitable for oral administration . suitable carriers include water , gelatine , gum arabic , lactose , starch , magnesium stearate , talc , vegetable oils , and the like . additionally , additives such as flavouring agents , preservatives , stabilizers , emulsifying agents , buffers and the like may be added in accordance with accepted practices of pharmaceutical compounding . while the individual active ingredients are suitably administered in a single composition they may also be administered in individual dosage units . according to another aspect of the invention , the composition may be a nutritional composition , such as a food or beverage or a supplement composition for a food or beverage , comprising further ingredients , such as , for example , protein , fat , digestible carbohydrates , dietary fibres , such as indigestible carbohydrates , minerals , vitamins , organic acids , and flavouring agents . according to another aspect of the invention , the composition may be a nutrition composition specifically designed for the treatment of the aforementioned conditions , comprising several further specific active ingredients , such as the ones disclosed in wo2003 / 041701 ( n . v . nutricia ) and wo2007 / 073178 ( n . v . nutricia ). advantageously , the nutritional composition according to the invention may comprise protein , preferably intact protein . proteins enable the manufacturing of palatable products . especially elderly and ad patients benefit from the protein as it strengthens their motor skills . preferably , the nutritional composition according to the invention comprises milk protein . preferably , the nutritional composition according to the invention comprises a protein selected from the group consisting of whey protein , casein or caseinate . preferably , the nutritional composition according to the invention comprises caseinate , more preferably the nutritional composition according to the invention comprises at least 70 weight %, more preferably at least 90 weight % casein and / or caseinate , based on total protein . preferably , the proteins are included in intact ( unhydrolyzed ) form , in order to have a palatable product . such high molecular weight proteins increase the viscosity of the heat - treated liquid product , compared to the hydrolyzed forms . the present inventors were able to make an acceptable product , with good palatability and limited viscosity , by applying the measures according the invention , still avoiding precipitation . preferably , the nutritional composition according to the invention comprises between 0 . 2 and 16 gram protein per 100 ml , preferably between 0 . 2 and 10 gram protein per 100 ml , more preferably between 1 and 6 grams protein per 100 ml , more preferably between 2 and 5 grams protein per 100 ml . advantageously , the nutritional composition according to the invention may comprise fat . with regard to the type of fat , a wide choice is possible , as long as the fat is of food quality . the fat may include medium chain triglycerides ( mct , mainly 8 to 10 carbon atoms long ), long chain triglycerides ( lct ) or any combination of the two types . mcts are beneficial because they are easily absorbed and metabolized . moreover , the use of mcts will reduce the risk of nutrient malabsorption . lct sources , such as rapeseed oil , more in particular rapeseed oil low in erucic acid , sunflower oil , corn oil , palm kernel fat , coconut fat , palm oil , or mixtures thereof are preferred because they provide more energy per unit of fat . in one embodiment , the fat is a liquid fat , i . e . an oil . in one embodiment , the fat comprises 30 to 60 weight % of animal or algal fat , 40 to 70 weight % of vegetable fat and optionally 0 to 20 weight % of mcts based on total fat of the nutritional composition according to the invention . the animal fat preferably comprises a low amount of milk fat , i . e . lower than 6 weight %, especially lower than 3 weight %. in particular , a mixture of corn oil , egg oil , and / or canola oil and specific amounts of marine oil are used . egg oils , fish oils and algal oils are a preferred source of non - vegetable fats . marine oils containing dha and / or epa are preferably present in the nutritional composition according to the invention in an amount lower than 25 weight %, preferably lower than 15 weight % of the fat for obtaining a maximum health effect , such as , for instance , the prevention of cardiovascular risks . the amount of epa ranges preferably between 4 weight % and 15 weight %, more preferably between 8 weight % and 13 weight % of the fat . preferably , the nutritional composition according to the invention comprises a phospholipid , preferably 0 . 1 to 50 weight % phospholipids , based on total weight of lipids , more preferably 0 . 5 to 20 weight %, more preferably between 1 and 5 weight %, based on total weight of lipids . preferably , the nutritional composition according to the invention contains at least one selected from the group consisting of phosphatidylcholine , phosphatidylethanolamine , phosphatidylserine and phosphatidylinositol . the total amount of lipids is preferably between 10 and 30 weight % on dry matter , and / or between 2 and 6 g lipid per 100 ml for a liquid composition . inclusion of phospholipids improve the stability of the nutritional composition according to the invention . advantageously , the nutritional composition according to the invention comprises digestible carbohydrates . the digestible carbohydrates positively influence the operational skills of a subject , and add to the advantageous effect of the nutritional composition according to the invention . the nutritional composition according to the invention preferably contains between 1 and 50 gram digestible carbohydrates per 100 ml of a liquid product , more preferably between 5 and 30 grams per 100 ml , more preferably 10 to 30 grams of digestible carbohydrates per 100 ml . the total amount of digestible carbohydrates is preferably between 25 and 80 weight % on dry matter basis , preferably 40 to 80 weight %. examples of digestible carbohydrates are digestible pentoses , digestible hexoses , and digestible oligosaccharides , e . g . digestible disaccharides and digestible trisaccharides saccharides . more specifically one or more digestible carbohydrates may be chosen selected from the group of galactose , mannose , ribose sucrose , trehalose , palatinose , lactose , maltodextrose , maltose and glucose . the nutritional composition according to the invention may optionally be fortified with non - digestible carbohydrates ( dietary fibres ) such as oligosaccharides . these oligosaccharides may comprise fructo - oligosaccharides ( fos ), galacto - oligosaccharides ( gos ), trans - galacto - oligosaccharides ( tos ), xylo - oligosaccharides ( xos ), soy oligosaccharides , and the like . optionally , also higher molecular weight compounds such as inulin , resistant starch and the like may be incorporated in the composition according to the invention . in another embodiment of the present invention , the composition according to the invention may comprise a mixture of neutral and acid oligosaccharides such as disclosed in wo 2005 / 039597 ( n . v . nutricia ), the compositions therein which are incorporated herein by reference . persons suffering from neuropathies or neurological problems often experience problems with eating . their sensory capabilities and / or control of muscles has become imparted , as well as in some instances their ambition to apply proper eating habits . part of these patients may experience a general loss in appetite and a relatively large part of this patient group became malnourished . preferably , the product has an energy density of 0 . 8 to 4 . 5 kcal per gram of the composition , more preferably between 0 . 9 and 2 . 5 kcal per ml . liquid nutritional products preferably have a long shelf life . however , increasing shelf life by heat treatments often results in destabilisation of the products and / or palatability , leading to a product which is unacceptable . the nutritional composition according to the invention can be subjected to a heat treatment without major adverse effects on the palatability . hence , the nutritional composition according to the invention is preferably heat - treated , more preferably the composition is subjected to a sterilization treatment . in a preferred embodiment , the nutritional composition according to the invention is subjected to an ultra - high temperature treatment ( uht - treatment ). such uht - treatment is preferably applied in line , i . e . before the liquid final product is filled in the package of the sold unit . fig1 . effects of chronic resveratrol supplementation on locomotor behaviour in an open field . the test duration was 5 minutes and locomotion of the animals was monitored with an automated video analysis system . panel a : total distance covered during the test . panel b : time spent in the centre area of the round arena (* p = 0 . 003 ). fig2 . effects of chronic resveratrol supplementation on y - maze learning . the y - maze task consisted of 3 training days and a retention test one week later . each test day consisted of 8 trials and the performance of the animals is expressed as the number of correct choices per day (* p = 0 . 016 ). fig3 . effects of chronic resveratrol supplementation on the cholinergic system . a . cholinergic cell number in the nucleus basalis is unchanged in the resveratrol treated animals . b . cholinergic fibre density in hippocampal ca1 and c . dentate gyrus does not show an effect of resveratrol treatment . fig4 . effects of chronic resveratrol supplementation on vascular density in the brain (* p = 0 . 019 ). in the hippocampus , resveratrol treatment caused a higher vascular density . fig5 . effects of resveratrol supplementation on the occurrence of microvascular abnormalities . panel a & amp ; b : a representative photomicrograph and quantitative analysis of endothelial processes . in panel a , arrowheads are pointing at the typical endothelial processes of a capillary . panel c & amp ; d : a representative photomicrograph and quantitative analysis of endothelial vacuoles (* p & lt ; 0 . 05 ). the photomicrograph demonstrates a capillary in the hippocampus ca1 region . arrowheads are pointing at the large , empty endothelial vacuole . panel e & amp ; f : a representative photomicrograph and quantitative analysis of vascular basement membrane thickening . in panel e , the photomicrograph demonstrates an arteriole in the hippocampal vascular layer . arrowheads are pointing at healthy and thickened segments of the basement membrane . abbreviations : asterisk : microvascular lumen , art : arterioles , cap : capillaries , e : endothelia ) cell , en : endothelial cell nucleus , p : pericyte , smc : smooth muscle cell , smn : smooth muscle cell nucleus . the experiment was performed with male c57bi / 6 mice ( harlan , horst , the netherlands ). animals were individually housed under a 12 hours light / 12 hours dark cycle with lights on at 8 : 00 . food and drinking water were provided ad libitum , except for the period of behavioural testing as described below . the experiment was started with 60 mice , half of which served as control ( con ) and half of which received resveratrol in their food from the age of 1 year onwards ( res ). between the ages of 18 and 20 month , spatial learning performance of the animals was studied in a y - maze test . at the age of 24 month , brains were collected and processed for immunocytochemistry to asses cholinergic parameters and electron microscopy for several cerebrovascular analyses . the procedures concerning animal care and treatment were in accordance with the regulations of the ethical committee for the use of experimental animals of the university of groningen . resveratrol ( hope farms , woerden , the netherlands ) was provided in the food ( 150 μg resveratrol / gram ) in an amount comparable to a human dose . to rule out differences in food intake between animals on a control diet and resveratrol - supplemented diet , food intake and body weight were monitored every two weeks . to establish whether resveratrol provided in the food enters the circulation , a pilot experiment was performed in which blood was collected from mice by decapitation after 1 day and 30 days of supplementation . plasma concentrations of resveratrol were measured . after 1 day of supplementation , resveratrol levels in the plasma were 88 . 5 μg / l at the end of the dark phase or active phase , when mice consume most of their food , and 41 μg / l at the end of the light phase or resting phase ( n = 3 each ). after 1 month of supplementation , the levels were 102 . 7 and 30 . 7 μg / l at the end of active phase and rest phase , respectively ( n = 3 each ). these measurements confirm that resveratrol ingested via the food indeed entered the circulation , and the levels appeared to fluctuate over the day in a fashion that paralleled food intake . supplementation of resveratrol via food thus seemed an appropriate and simple method for further studies . to examine general explorative activity , mice were subjected to an open - field test for 5 minutes . the open field consisted of a circular arena with a diameter of 120 cm . the arena was divided in two imaginary concentric zones , a central zone ( 60 cm diameter ) and an outer zone ( 120 cm diameter ). position and locomotion of the mice was recorded and analyzed with a computerized video tracking system ( ethovision , noldus information technology , wageningen , the netherlands ). the number of visits and time spend in each of the two zones was determined . the open - field arena was thoroughly cleaned before a new animal was tested . to examine cognitive performance , the mice were subjected to a y - maze test in which they had to learn the location of a food reward in one of two accessible choice arms [ 7 ]. the test was conducted in a tubular , transparent plexiglas y maze consisting of a start arm and two test arms forming the y . all arms of the maze were 27 . 5 cm long , had a diameter of 5 cm , and were at a 120 ° angle from each other . the home cages of the mice were equipped with a small sliding door that connected to the starting arm . one of the two test arms was baited with a food reward consisting of small crumbs of the regular food . food crumbs were also placed below perforations at the end of the two test arms to prevent animals from discriminating between baited and non - baited arms by olfactory cues . small grey plastic blocks ( 1 cm high ) were placed 4 cm from the end of the arms to prevent visual inspection for food presence from a distance . a guillotine door halfway each arm could be operated manually from the experimenter &# 39 ; s position and was used to allow animals only one choice in each training trial . the experimental room contained visual cues , which served as distal spatial cues . to assure sufficient motivation for the test , the animals were food restricted to about 85 % of their original body weight . food restriction started 3 days before the test . the animals received their food daily between 16 : 00 and 18 : 00 pm . the y - maze test took place during the first half of the light phase between 10 : 00 and 14 : 00 in a room adjacent to the home cage room . the animals were carried in their home cage to the experimental room and the cage was then connected to the y - maze . the first day of the test animals were habituated to the maze by allowing them to freely explore the arms for 5 minutes . they further received two habituation trials during which they only had access to subsequently the right or left test arm , which then contained a food reward . after the habituation day , mice received a daily training session consisting of 8 trials . during the entire training phase , either the right or left arm was baited . this was constant for a given individual , but randomized between subjects and treatments . when during a trial a subject visited one of the two accessible arms , the non - visited arm was closed . after the subject retreated to the start box , the start arm connected to the start box was blocked preventing reentrance of the maze . after cleaning all arms with damped paper cloth , and re - baiting the same arm , the subject was again allowed to explore either the right or left test arm . a visit to the baited arm was recorded as a correct trial . the mice were trained in the y - maze for 3 consecutive days . ten days after the final training day , animals were subjected to another series of 8 trials to test their memory . at the age of 24 months animals were sacrificed for histochemical and electronmicroscopical analysis . mice were anaesthetized by a high dose of pentobarbital sodium salt ( nembutal , 60 mg / kg bodyweight ) and transcardially perfused with heparinised saline followed by 4 % phosphate buffered paraformaldehyde . after perfusions , brains were removed from the skull and kept in 0 . 01 m pbs overnight . a random subset of 18 brains were further processed for electron microscopy . another random subset of 22 brains was used for histological analysis . for histology , brain tissue was cryoprotected in 30 % sucrose in 0 . 1 m phosphate buffer for 48 h after which 20 μm sections were cut with a cryostat ( leica cm3050 ). to analyze possible changes in cholinergic cells and fibres , an enzymatic staining for acetylcholinesterase ( ache ) was performed . sections were postfixed in a 2 . 5 % glutardialdehyde solution in pb overnight at 4 ° c . ache histochemistry was carried out according to methods known to the skilled person . in brief , sections were incubated in an acetylthiocholine - iodide containing incubation buffer , followed by sodiumsulfide and finally intensified with a 0 . 1 % silver nitrate solution . stereological analysis was performed in every tenth section . cells positive for ache were quantified in sections containing nucleus basalis ( around bregma − 0 . 7 ) and ache fibre density measurements were performed by a computerized image analysis system quantimet ( leica ) in the dorsal hippocampus . after perfusion , 18 brains were prepared for electron microscopy using standard methods . briefly , tissue blocks were incubated in an aqueous solution of 1 % oso 4 and 5 % k 2 cr 4 o 7 ( 1 : 1 ) after thorough rinsing . subsequently , the samples were dehydrated , incubated in 1 % uranyl acetate , and embedded in glycide ether . semi - thin sections ( 0 . 3 μm ) were cut on an ultramicrotome and stained on object glasses with a 1 : 1 mixture of 1 % ethylene blue and 1 % azure ii blue . the samples were then coverslipped with dpx and analyzed under a light microscope . then , 90 nm ultra thin sections were cut of the same blocks and three non - serial sections were collected on 200 mesh copper grids . subsequently , preparations were contrasted with 5 % uranyl - acetate and reynolds lead - citrate solution . finally , the samples were analyzed and photographs were taken with a philips tm10 transmission electron microscope . for quantitative analysis , approximately 4000 μm 2 tissue surface was scanned systematically in each region , and 61 ± 10 microvascular cross sections were examined in each sample . vascular density was calculated for a standard surface area with the help of the sample grid . in addition to microvessel density , the occurrence of the following features were quantitated : luminal endothelial protrusions , large empty endothelial vacuoles and basement membrane thickening . the basement membrane was considered thickened when local exfoliations were observed in case of capillaries , or hyalinosis in case of arterioles . the number of microvessels displaying any of the noted abnormalities was counted and expressed as percentage of the total number of microvessels examined . capillaries and arterioles were analyzed separately . since arterioles were encountered at high enough number for statistical analysis only in the hippocampal vascular layer , quantitative analysis of arteriolar condition was performed for this region of the hippocampus alone . behavioural performance was statistically analyzed with a one way anova paradigm for repeated measures . immunocytochemical data and em data were statistically analyzed with an independent samples t - test ( 2 - tailed ). data are expressed as averages ± s . e . m . p & lt ; 0 . 05 was considered as significant . animals received resveratrol in their food from 1 year of age onwards . adding resveratrol to the food did not affect food intake , growth and body weight ( data not shown ). before behavioural testing started , at the age of 18 - 20 months , 4 animals died ( 2 con ; 2 res ). after behavioural testing , but before the end of the experiment at 24 months , 9 more animals died ( 4 con ; 5 res ). there were no significant differences in mortality rate and regular visual inspection did not indicate obvious differences in general health . in the open field test , con and res mice displayed the same amount of locomotion ( total distance covered ), however , the res group spent about 50 % more time in the centre of the arena than the control animals ( t - test , p = 0 . 003 ; fig1 ). also during the habituation trial of the y - maze , con and res mice displayed similar levels of activity in terms of arm visits and spontaneous alternations ( data not shown ). however , during the training phase , chronic resveratrol supplementation improved learning ( repeated measures anova for number of correct choices on day 1 to 3 : f 1 , 54 = 6 . 156 , p = 0 . 016 ; fig2 ). posthoc testing shows that res animals performed better then con animals on the second day of the trainings phase ( p & lt ; 0 . 05 ). the final level of performance on day 3 and the retention trial on day 10 were not different . analysis of cholinergic cell number in the nucleus basalis did not reveal any difference between con and res ( fig3 a ). also , cholinergic fibre density in the ca1 and dentate gyrus of the hippocampus was unchanged after res treatment ( fig3 b ). electronmicroscopical analysis of brain tissue revealed that the vascular density in the hippocampus of res mice was about 15 % higher than that in the control animals ( p = 0 . 019 ; fig4 ). the endothelial cells of cerebral microvessels exhibited two discernable abnormalities . the apical surface of the endothelial cells displayed rich , microvillus - like processes into the lumen , and empty , large vacuoles formed preferentially in the vicinity of tight junctions ( fig5 a and 5c ). the basement membrane of capillaries appeared occasionally thickened in the form of local exfoliations , while the arteriolar basement membrane , particularly between the endothelial and smooth muscle cells was affected by hyalinosis ( fig5 e ). the occurrence of basement membrane thickening and endothelial processes was not significantly affected by dietary resveratrol ( fig5 b and 5f ). however , the number of microvessels with endothelial vacuoles was significantly reduced by chronic resveratrol treatment ( fig5 d ) in the hippocampal arterioles ( p = 0 . 05 ). chronic dietary supplementation with the natural polyphenol resveratrol improved performance of aged mice in the acquisition of a y - maze task . this improvement in cognitive performance was paralleled by an increased microvascular density in the hippocampus and decreased number of vacuolar abnormalities in hippocampal microvessels . learning and memory performance in the y - maze is dependent on the hippocampus , a brain area often affected by aging in both humans and rodents . it was found that resveratrol treated animals are better able to acquire this task . although differences in general activity and anxiety are potential confounding factors in most learning task , it is unlikely that this explains the results of the y - maze test in the present study . first , during the free exploration , habituation trial before the start of training , resveratrol - supplemented and control mice showed similar levels of explorative activity in terms of arm visits and spontaneous alternations . second , during the training , all animals entered the maze with every trial and latencies were not different for treated versus untreated animals . furthermore , the data from the open field test confirm that there was no effect of resveratrol treatment on general locomotor activity , since the total distance covered by animals was the same for both groups . all together , the increased performance of the resveratrol - treated animals during the acquisition phase of the y - maze task most likely indicates an improved cognitive performance or , alternatively , a reduction in the well - established age - related decline in cognitive performance . the mechanisms behind resveratrol - induced preservation of cognitive performance during aging are most likely multi - factorial . in the present study , improved performance was paralleled by higher hippocampal microvessel density and less aberrant microvessels in the hippocampus . a higher microvascular density and increased cerebral blood flow might improve performance directly by increasing glucose and oxygen supply to relevant brain areas [ 8 ]. additionally , the microvessels potentially provide a source of trophic factors like igf - 1 and nerve growth factor ( ngf ) that support neuronal function . these factors have been shown to decrease in aging together with decreases in cortical microvascular density [ 9 ] and are also implicated in age - related cognitive decline [ 10 ]. without being bound to the following explanation , an increased microvascular density can be explained in two ways . first , resveratrol could actively stimulate angiogenesis or second , resveratrol could prevent age - related decline in angiogenesis and / or deterioration of vascular status . since resveratrol has been reported to inhibit angiogenesis in vitro [ 11 , 12 ], it seems more likely that resveratrol supplementation in the present study attenuated the age - related decline in vascular density and maintained normal levels of angiogenesis as opposed to an increase in angiogenesis and vessel density over control levels . additionally , resveratrol appeared to prevent or delay the occurrence of vascular abnormalities , another hallmark of age - related decline in vascular status . in this particular study , mice received resveratrol in a chronic fashion , doses and timing highly comparable to the experimental design . the inventors found that resveratrol produces changes associated with a longer lifespan , resulting in a physiological shift of middle - aged mice on a high - calorie diet towards that of mice on a standard diet . survival was not assessed , but focus was on neurobiological function and cerebrovascular status . it is now postulated that one of the target - systems for resveratrol to exert its beneficial effects can be found in a preserved cerebrovascular system . in contrast to the results on cerebrovascular level , no effects were found of chronic resveratrol treatment on structural parameters of the cholinergic system i . e . cholinergic cell number and fibre density , although direct effects of resveratrol on neuronal health cannot be ruled out . the lack of effects on neuronal level is actually paralleled by previous studies on aging , where no quantitative difference in the number of neurons or synapses were found . furthermore , a previous studies on dietary supplementation in tg2576 transgenic mice , a model for alzheimer &# 39 ; s disease , found a strong effect on spatial learning , but no effects on neuronal readouts , like plaque deposition [ 13 , 14 ]. in order to exhibit its direct effects on neuronal health resveratrol is required to enter the brain in sufficient levels . in this study it was shown that resveratrol is present in the blood , fluctuating according to daily food - intake . however , the bioavailability of resveratrol and relative concentrations in different tissue are still under discussion [ 15 ] and at present , it is unknown whether sufficient concentrations of resveratrol can cross the blood - brain barrier to exert its beneficial effects directly on neuronal level . this has implications on the doses , but not on the established effect . in conclusion , this is the first study showing beneficial effects of long - term dietary supplementation with the natural polyphenol resveratrol on cognitive functioning and cerebrovascular status . in this light , we propose that ingestion of resveratrol , thereby targeting the health of the cerebrovascular system , may be an effective and simple method to support successful aging . by way of example , the following liquid nutritional composition may be used as carrier for administering the hydroxylated stilbenes according to the invention , wherein the hydroxylated stilbene is resveratrol in an amount ranging from 0 . 2 mg to about 500 mg per serving . ready - to - drink oral supplement comprising per 100 ml , 100 kcal , 40 en % protein , 41 . 1 en % carbohydrate and 18 . 9 en % lipids . 10 g of protein based on whey and casein ( 2 g / 8 g ); 10 . 3 g carbohydrates based on maltodextrines and 2 . 1 g of fat based on canola oil and sunflower oil . other micronutrients according to general recommendations for medical nutrition . dong w , li n , gao d , zhen h , zhang x , li f . resveratrol attenuates ischemic brain damage in the delayed phase after stroke and induces messenger rna and protein express for angiogenic factors . j vasc surg 2008 . lu k t , chiou r y , chen l g , chen m h , tseng w t , hsieh h t , yang y l . neuroprotective effects of resveratrol on cerebral ischemia - induced neuron loss mediated by free radical scavenging and cerebral blood flow elevation . j agric food chem 2006 ; 54 ( 8 ): 3126 - 31 . virgili m , contestabile a . partial neuroprotection of in vivo excitotoxic brain damage by chronic administration of the red wine antioxidant agent , trans - 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