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L. Al Damen3, A. Stockton1, E.A.S. Al-Dujaili1,2


1. Department of Dietetics, Nutrition and Biological sciences, Queen Margaret University, Musselburgh, East Lothian EH21 6UU, UK; 2. Cardiovascular Sciences, Queens Medical Research Institute, Edinburgh University, Edinburgh, UK; 3. Faculty of Pharmacy, Middle East University, Amman, Jordan

Corresponding Author: Professor Emad Al-Dujaili, Cardiovascular Sciences, Queens Medical Research Institute, Edinburgh University, Edinburgh, UK, Email: or

J Prev Alz Dis 2018 inpress
Published online May 23, 2018,



Decline in cognitive function can be observed in normal aging, MCI, neurodegenerative disorders, cerebral hypoperfusion and post-surgery. Cognitive impairment presents with limited therapeutic options. Berries, pomegranates and grapes are polyphenol-rich fruits that have both antioxidant and anti-inflammatory effects. This review aims to explore the effects of these fruits and their biophenols on cognitive function in healthy subjects and their protective effects on cognitive deficits in normal aging, mild cognitive impairment, Alzheimer’s disease (AD), cerebral hypoperfusion using both animal models and human studies. MEDLINE® and Proquest Central data bases were searched to retrieve articles used in this general review according to specific inclusion criteria. Independently, two researchers have summarized the study characteristics and assessed quality of methodology. Meta-analysis was not possible due to the limited number of studies and marked heterogeneity, and thus results were presented as a narrative review. Biophenol-rich fruits have been shown to exert cognitive functions boosting and enhanced memory in children and healthy adults.  In patients with age related cognitive decline, MCI and AD, Biophenols-rich fruits showed a marked enhancement of memory and verbal fluency with reduced dementia risk, and in artery graft and valve surgery, pomegranate protected against memory impairment. Animal studies have shown that biophenols intake enhanced cognition in AD models presumably due to a reduction of Amyloidβ plaque deposition, inflammatory cytokines, microgliosis (glial cells hypertrophy), and brain DNA protection. In conclusion, large scale studies are urgently needed to fully evaluate the preventive and/or adjunctive therapy of biophenol-rich fruits in cognitive decline, deficit and Alzheimer disease. .

Key words: Fruits, Polyphenols, Cognitive disorders, Memory impairment, Alzheimer, Dementia

Abbreviations: AD: Alzheimer’s disease; APP: Amyloid Precursor Protein; APP/PS1: Amyloid Precursor Protein/Presenilin 1; Aβ: Amyloid β; AVLT: Auditory verbal learning task; BACE1: Beta-Secretase 1; BDNF: Brain-Derived Neurotrophic Factor; CRP: C- Reactive protein; CVLT: California Verbal Learning Test; 2CCAO:  Common carotid arteries occlusion; CGJ: Concord grape juice; Cox-2: Cycloxygenase-2; ELISA: Enzyme-linked immunosorbent assay; EGCG: Epigallocatechin gallate; fMRI: Functional Brain Activation; GSH: Glutathione; GSE: Grape seed extract; GSPE: Grape seed polyphenolic extract; GDS: Geriatric Depression Scale; hsCRP: High-sensitivity C-reactive protein; DG: Hippocampus gyrus; iNOS: Inducible nitric oxide synthase; IGF-1: Insulin-like growth factor-1; IL-1β: Interleukin 1 beta; IL 2-10: Interleukin 2 – 10; LPO: Lipid peroxidation; LPS: ipopolysaccharide; IN: Induced Neuroinflammation; LTP: Long- term potentiation; MMSE: Minin-Mental State Examination; MCI: Mild Cognitive Impairment; MFT: Modified flanker task; NFT: Neuro-Fibrillary Tangles; NO: Nitric Oxide; NF-κB: Nuclear Factor- Kappa B; NFAT: Nuclear Factor of Activated T-cell; IκB: Nuclear Factor of kappa light polypeptide gene enhancer in B-cells Inhibitor; PD: Parkinson Disease; PMT: Picture matching task; PE: Pomegranate Extract; PPE: Pomegranate Peel Extract; PGSE: Pomegranate Seed Extract; POCD: Postoperative cognitive dysfunction; PGE-2: Prostaglandin E2; PUN: Punicalagin; PGJ: Purple grape juice; RVIP: Rapid visual information processing; RT: Reaction time; ROS: Reactive Oxygen Species; RAVLT: Rey auditory verbal learning task; SOPT: Self-ordered pointing task; TNF-α: Tissue Necrosis Factor- Alpha; TEAC:Trolox-Equivalent Antioxidant Capacity; VAD:Vascular Dementia; (WBB: Wild blueberry.



The brain is known to be highly susceptible to oxidative damage because of its high metabolic load and abundance of oxidisable endogenous substances, such as the poly-unsaturated fatty acids that form the plasma membranes of neural cells (1). Oxidative stress usually results from an imbalance between free radicals and antioxidants. If the free radicals levels overwhelm the body’s ability to regulate them, then oxidative stress ensues. Free radicals can attack DNA, proteins and lipids within the cells, which contribute to several conditions and diseases, and in particular, those related to aging including neurodegenerative diseases such as Alzheimer’s disease  (2, 3), vascular dementia (VAD) and Parkinson’s disease (PD) (4). In addition to oxidative stress, inflammatory processes are also considered to be an important factor in the pathophysiology of neurodegenerative diseases (5, 6).
Since biophenols found naturally in many fruits, vegetables and herbs have both antioxidant and anti-inflammatory effects, there is an increased interest to investigate their roles in the reduction of oxidative stress and inflammatory processes involved in cognitive deficits, and thus their potential use as neuroprotective nutrients to maintain normal cognitive function and as therapeutic agents where cognitive function actually declines (7). Polyphenolics, or biophenols, represent one class of phytochemicals which includes thousands of uniquely identified organic structures that occur exclusively in foods of plant origin (e.g. fruits, vegetables, nuts, seeds, grains, tea, coffee, cocoa beans and wine).  Polyphenols are classified according to the number of phenolic rings into: phenolic acids; stilbenes; coumarins; tannins; and flavonoids.  Flavonoids found in varying concentrations in many vegetables (e.g. Broccoli, greens, onions), fruits (e.g. blueberry, blackcurrant, strawberry, cherry, grape, pomegranate, apple and orange), beverages such as green tea and red wine and in plant extracts from pine bark, Gingko biloba and Pueraria lobata . Flavonoids can be further sub-divided into the following subclasses; flavonols (e.g., quercetin rich in apple), flavones, isoflavones, flavanones (e.g., hesperidin rich in orange), anthocyanidins (e.g., cyanidin and delphinidin rich in berries) and flavan-3-ols (e.g., catechin rich in green tea and cocoa) (8).
Flavonoids have been well documented to elicit health benefits by reducing the risk factors associated with cardiovascular disease, diabetes and stroke (9). Over the last decade, interest has also grown in their ability to elicit cognitive benefits. Flavonoids particularly, anthocyanidins can cross blood-brain barrier and localize in the area of learning and memory- the hippocampus (10).  Most fruits and their pure beverages contain variable levels of flavonoids. Our choice for investigating grape, blueberry, blackcurrant, cherry and strawberry stems from the fact that they are among the richest sources of flavonoids, particularly anthocyanidins such as cyanidin and delphinine (per 100g: 158mg anthocyanidins in blackcurrants, 163.3mg anthocyanidins in blueberries, 33.4mg in cherry, 27mg in strawberry and 120mg in grape) (11). Pomegranate has also been selected in this review due to its unique multi-biophenol contents.  In addition to flavonoids such as delphinidin, cyanidin, and pelargonidin (12), pomegranate species contain a hydrolysable tannins which are known as punicalagins; these represent the largest polyphenolic antioxidants with a molecular weight of >1000 Dalton. Following ingestion, these tannins are hydrolyzed into ellagic acid and gallagic acid that are transferred to the blood circulation.  Punicalagins (alpha and beta), gallagic acid and ellagic acid account for the majority of ellagitannins found in pomegranate extract. The gut converts these ellagitannins into urolithins which are small molecules that possess antioxidant properties (13). Pomegranate juice antioxidant power was shown to be three times higher than those of red wine and green tea (14). Ellagic acid, gallic acid and punicalagins found in pomegranate were shown to inhibit lipopolysaccharide (LPS) induced nitric oxide (NO), prostaglandin E2 (PGE-2) and interleukin-6 (IL-6) production that is believed to be responsible for the anti-inflammatory response of pomegranate extract (13).
The aim of this review was to investigate the effect of fruits (berry, pomegranate, grape and others) supplementation and their biophenols on cognitive function as evidenced from animal and human studies:
•    Healthy people defined as those with no cognitive decline “human studies”.
•    Decline in cognitive function due to aging and mild cognitive impairment (MCI) “animal models and human studies”
•    Cognitive function deficits due to Alzehimer’s disease (AD)  “ animal models and human studies”
•    Cognitive function deficits due to cerebral ischemia and surgical operations “animal models and human studies”


Search strategy methods

MEDLINE® and Proquest Central data bases were searched to retrieve articles used in this general review of in vivo animal and human studies. The review preparation was completed in several steps: identification of research question, definition of inclusion criteria, literature search and selection of eligible studies. We used the following keywords: Polyphenol-rich fruits and berries and cognitive decline or memory impairment, or Alzheimer. We also searched the reference list of selected studies for more relevant research. Studies included were human randomized clinical trials or cohort studies investigating as a main outcome the link between polyphenols- rich fruits (pomegranate and grape) and berries (e.g. blueberry, strawberry and blackcurrant) on cognitive function and memory, and placebo controlled animal studies. Epidemiological and interventional studies which investigated the effect of pomegranate and grape and their polyphenols alongside berry fruits on cognitive function in healthy subjects were also considered. Independently, two researchers have summarized each study characteristics and assessed quality of methodology. Meta-analysis was not possible due to the limited number of studies and marked heterogeneity, and thus results were presented as a narrative review.


Effect of biophenol-rich fruits on cognitive Function in healthy subjects with no cognitive decline


The effect of grape supplementation on cognition in healthy individuals with no cognitive function impairment or decline has been investigated following both chronic and acute consumption. Regarding chronic consumption, Lamport et al. (15) conducted a study which indicated that biophenol-rich grape juice might be beneficial for cognition in healthy individuals with no apparent cognitive disorders. Healthy women (n= 25) aged 40-50 years, who were employed for ≥30 hours/week volunteered for the study. The women consumed daily either Concord grape juice (CGJ) (355mL containing 777mg total polyphenols of which 167 mg anthocyanins and 334 mg proanthocyanidins) or matched placebo drink for 12 week with 4 weeks washout period.  A marked improvement in spatial memory and driving skills were observed with no effects on measures of executive function, verbal memory and psychomotor skill. However, there was an evidence of sustained benefits in verbal recall and executive function after cessation of the CGJ supplementation (15).
Acute consumption was investigated in two further studies looking at the effect of an acute administration of grape juice on cognition and mood. Haskell-Ramsay et al. (16) conducted a randomized, placebo-controlled, double-blind, crossover study which examined measures of episodic memory, working memory, attention and mood following a 20 minutes absorption period of 230 ml single serving of commercially available purple grape juice (PGJ), containing 1504 mg total phenols as gallic acid equivalent and 138 mg anthocyanin, on 20 healthy young adults (average age of 21 years) (16). The second study conducted by Hendrickson and Mattes (17), enrolled a larger sample size with 35 young adult smokers (average age 26 years) in a randomized, placebo-controlled trial. The participants consumed either 600 mL of CGJ (containing around 2,100 total phenols as gallic acid equivalent and 580 mg anthocyanins) along with a standardized lunch or energy- matched placebo followed by assessments of mood and implicit memory (17). Both of these studies failed to find an acute effect of grape juice supplementation on memory. However, the first study found a significant effect of acute intake of grape juice on attention and mood as measured by enhanced overall speed on attention tasks and significantly increased calm ratings. However, it is important to notice that executive function measures weren’t evaluated thoroughly using these studies. Haskell-Ramsay et al. (16) evaluated the measures through composite score of memory reaction time (Memory reaction time (RT) = (RT of delayed word recognition + RT delayed picture recognition + RT numeric working memory)/3). In the study conducted by Hendrickson and Mattes (17), only measure for implicit memory was utilized and thus executive memory was not evaluated. No studies have been conducted to examine the acute effect of CGJ on cognition in children.

Cherry and Berry (Blueberry, Blackcurrant and Cranberry)

Blueberries have different flavonoids subclasses, but they are the richest in anthoycanins. The Effect of berries on cognition has been investigated only after acute consumption. For the aim of investigating the acute effect, a study by Dodd (18) has been conducted. The study had employed a randomized, controlled, double blind, crossover design of freeze dried blueberries (200 g fresh equivalent containing 631 mg anthocyanidins) or energy matched control, with cognition function measured at baseline, 2 hours and 5 hours post consumption. For younger adults (n = 19), an improved accuracy on a letter memory task (measuring working memory) was observed 5 hours postprandially. No effects were observed at an earlier time of 2 hours or for other measures of executive function, memory, or mood.  For a subset of participants who had consumed blueberry, blood samples taken 1 hour postprandially showed an increase in plasma levels of brain-derived neurotrophic factor (BDNF). Unfortunately, cognition was not measured after one hour and it is impossible to say whether this neurochemical change was related to any cognitive outcome. For older adults (n = 18), an improved performance on an immediate word recognition task at both 2 hours and 5 hours postprandially were observed.  No improvements in measures of executive function or mood were evidenced (18).  For evaluation of the acute effect of berry consumption in children, Whyte et al. (19, 20) conducted two studies. The first study was a small, crossover study with only 14 children (8-10 years old), using fresh whole blueberries (200 g containing 143 mg anthocyanins). The study found no effects at 2 hours for a range of executive function tasks, but did observe a marked improvement in delayed word recall using the Rey auditory verbal learning task (RAVLT).  The second study enrolled a higher number of children (n = 21) of 7-10 years old in a randomized, double-blind, cross-over study with two separate blueberry doses of 15 and 30 g freeze dried blueberry powder (containing 127 mg and 253 mg anthocyanins consequently).This study utilized four cognitive tests; AVLT which examined performance in learning, memory recall and recognition, modified flanker task (MFT) which examined response interference, Go–NoGo which examined response inhibition and picture matching task (PMT) which investigated both levels of processing and response interference. The effect on cognition was measured at baseline and at three post-intervention sessions (at 1.15, 3 and 6 hours). The 30 g dose resulted in a significant improvement in immediate word recall after 1.25 hour and the 15 g dose resulted in a trend towards significance on delayed recall. For the 15 and 30 g doses, a significant effect was seen at word recognition test 6 hours post intervention.  The 30 g dose showed an improved accuracy during MFT after 3 h, although only for cognitively demanding incongruent trials. However, there was a no significant improvement observed in Go-NoGo test for the 30 g dose and faster performance in Go-NoGo test in the placebo group compared with the blueberry group (19, 20). This study indicated a dose response relationship for anthocynanins; higher doses are linked to better cognitive function. It is also noticed that the cognitive function domains affected by blueberry supplementation varied by age. In children and older adult, the memory function was mainly enhanced while an improvement in executive function was observed in young adults.  This could be an indicative of age differences response in the capacity for particular cognitive domain improvement.
Blackcurrants are also considered a very rich source of anthocyanins. Watson et al. (21) conducted a randomized, double-blind, controlled crossover trial using 36 healthy nonsmokers’ young participants (18-35 years old) of two blackcurrant extracts; cold-pressed juice or freeze-dried powder or energy-matched control. The total polyphenols content of the two extracts were approximately 525 mg as gallic acid equivalent per 60 kg bodyweight. However, there was a slight difference in anthocyanin content between extracts; 483 mg/60 kg bodyweight for the powder and 467 mg/60 kg for the juice. Seven repetitions of the digit vigilance task, Stroop task and rapid visual information processing (RVIP) task were utilized in this study to examine acute blackcurrant effect on cognition.  A significant improvement was observed for tests of executive functions (RVIP and to some extent vigilance). Specifically, declining accuracy on a RVIP task was attenuated after taking the powdered extract. No effects were observed for the Stroop test which evaluates inhibition and attention function. Also no enhancements in measures of mood and mental fatigue were seen (21). The results of this study were consistent with the results of the executive function improvement seen in healthy young adults after blueberry consumption.
In addition to blueberry and blackcurrant, cherry contains a considerable concentration of anthocyanins. Caldwell et al. (22) conducted a pilot cross-over study assessing the acute response of cherry flavonoids consumption following administration of 300 mL cherry juice approximately containing 55 mg anthocyanins to 6 younger adults (18-35 years) and 5 older adults (≥55).  Three tests were utilized in this study; RAVLT which assessed verbal learning and memory, task-switching test measured higher executive function and pattern and letter comparison tasks assessed speed of processing at baseline and 6 hours postprandially. No significant differences were found for cognitive tasks with the exception of the task-switching test in older adults after consuming the single 300 ml serving (22). However, the results of this study couldn’t be adopted with high confidence, since the small sample size decreased its power dramatically, and there was also no energy matched control group. A second study administered the same juice in three separate 100mL aliquots each consumed at 1 hour apart. No cognitive effects were observed relative to baseline following consumption of the juice in these consecutive smaller doses. It should also be noticed that the intervention dose was lower compared to some of the earlier mentioned studies. Since the concentration of anthocyanin in cherry is far lower compared to blueburry, blackcurrant and grape; 157.7 mg/100 g in blackcurrants, 163.30 mg/100 g in blueberries and 120 mg/100g in grapes (9), a larger volumes of cherry juice should be utilized to achieve similar anthocyanin doses consumed in other similar studies.

Summary and future recommendations

A summary of human studies conducted for the evaluation of cognition effects of grape and berry in healthy individuals with no cognitive decline is shown in Table 1. There were positive effects for chronic grape supplementation on cognitive function, particularly in spatial memory. A sustained effect for executive function was also noticed after cessation of CGJ, which could mask the difference between tests groups. Future studies using chronic CGJ consumption should be conducted with longer wash out period to avoid carryover effects, or different study design. This would indicate if chronic CGJ intake produces additional benefits in executive function and verbal memory. For both berries and pomegranate, no randomized controlled studies have been performed to investigate their chronic consumption effect on healthy subjects. We would strongly justify interventional studies to be conducted very soon.
Acute blueberry and blackcurrant supplementations found to have beneficial effects on the executive function in young adults, yet no such benefits were noticed after cherry intake, and this might be due to the small sample size employed. Thus, a larger randomized control trial (RCT) using higher range of anthocyanin doses is needed to determine if cherry anthocyanins can elicit acute cognitive effects similar to those of other anthocyanin-rich fruits (24). Acute grape intake seems to show no memory benefits (episodic or implicit). Further interventional studies to evaluate the acute effect of CGJ intake on cognition that investigate measures of executive function should be conducted to explore if anthocyanin-rich supplements (blueberry, blackcurrant and grape) produce the same benefits of executive function improvement in young healthy adults. In children and older adults, the memory functions were seen to be mainly enhanced after blueberry supplementation. In addition, cherry intake was shown to enhance executive function in older adults.  It should be noted that interventional studies of blackcurrant and grape extracts in children and older adults (older than 55 years) have not been conducted. Thus, we highly recommend such studies to be performed in order to determine if these extracts can produce similar benefits in cognitive function enhancement as those seen with blueberry consumption. This would also strengthen the evidence of anthocyanins-rich fruit intake as memory improvement supplements in children. No studies using pomegranate have been published for healthy subjects to evaluate the acute or chronic effects on memory. We suggest that further interventional studies to be conducted to study the acute and chronic effects of pomegranate intake in healthy individuals.

Table 1. Summary of human studies of the effects of grape and berries (blueberry, blackcurrant and cherry) in healthy individuals with no cognitive problems

Table 1. Summary of human studies of the effects of grape and berries (blueberry, blackcurrant and cherry) in healthy individuals with no cognitive problems

*For acute intake, no duration applicable; ** Doses for total biophenols expressed as gallic acid equivalent; ***Explicit memory and executive function weren’t studied; ****Trend toward significant; ↔ Significant improvement, ↔ no significant difference, ↔ worsening



Effect of biophenols-rich fruits and extracts on Age-related decline in cognitive function or Mild Cognitive Impairment

Humans and animals cognitive functions start to decline during aging which thought to be related to increased susceptibility to long-term effects of oxidative stress and inflammation (23-25). Thus, several animal and human studies have been conducted to evaluate the effects of antioxidant and/or anti-inflammatory phytochemicals on age-related cognitive decline and the possible mechanisms of their effects.

Animal studies


The CGJ was found to have a positive impact on both cognitive function and motor function of aged rats. A study conducted by Skukitt et al. (26) showed that 10% concentrated grape fruit juice improved cognitive function in aged rats assessed through Morris water maze (Morris water maze is an age-sensitive learning paradigm that tests spatial learning and memory) and 50% concentrated grape juice produced an improvement in co-ordination motor function tests (rod walk, wire suspension, and small plank walk) (26). The cognitive benefit was explained by improvements in oxotremorine potassium evoked release of dopamine from striatal slices. Another mechanism by which grape supplementation induced such reversal of neural and behavioral aging could be explained by Balu et al. (27) work.  In this study grape seed extract (GSE) showed an inhibiting effect on the accumulation of age related oxidative DNA damage both in the spinal cord and certain brain regions such as the striatum, hippocampus and cerebral cortex of aged rats (27).


Studies that investigated berry intake in aged animals showed a significant improvement in cognitive function. In a study conducted by Baros et al. (28), long term memory has been significantly improved by administration of lyophilized extract of Vaccinium ashei berries for 30 days. The study suggested that the effect was due to the extract protective effect on DNA damage in the hippocampus and cerebral cortex. This effect could be explained by the antioxidant activity of polyphenols, including anthocyanins found in the berries (28).  In addition to oxidative stress, reduction in hippocampal neurogenesis was considered to be another contributing factor for cognitive function decline during aging. One of the key modulator of hippocampal neurogenesis is Insulin-like growth factor-1 (IGF-1), a major activator of the extracellular receptor kinase pathway that plays a vital role in learning and memory processes. A study by Casadesus et al. (31) showed that blueberry supplementation improved spatial memory in aged rats tested by radial arm water maze and also improved hippocampal neurogenesis, extracellular receptor kinase activation, and Insulin-like growth factor-1 (IGF-1) and IGF-1R levels, which led to enhancement of hippocampal neuronal plasticity (29).
There are also neuronal and behavioral changes that take place during the aging process in the absence of neurodegenerative disease. These changes may include decrements in calcium homeostasis (30), and sensitivity of several receptor systems, most notably adrenergic (31), dopaminergic (32), muscarinic (33, 34). Joseph et al. (35) examined cognitive effects of strawberry, spinach or blueberry supplementation on aged rats and showed that strawberry and berry consumption for 8 weeks (14.8 gm and 18.6 gm daily of dried aqueous extract per kilogram of diet respectively) produced a significant enhancement in several neuronal and behavioral parameters. These include: enhancement of K+-evoked release of dopamine from striatal slices, carbachol-stimulated GTPase activity and striatal Ca45 buffering in striatal synaptosomes. These have presumably led to improvement in motor behavioral performance on the rod walking and accelerod tasks, and a significant enhancement of spatial learning and memory using Morris water maze. Such findings suggest the ability of biophenol-rich supplementation to reverse age related deficits (35). Another study found that blueberry and strawberry supplementation in a model of aging rats with deteriorated motor and cognitive abilities due to exposure to whole-body irradiation, a differential effect of protection against radiation harmful effects as tested by water maze performance test (36). Blueberry supplementation was found to improve reversal learning, which relies on intact striatal functioning, whereas strawberry supplementation appeared to show better protection against spatial deficits in the maze. This study provided some evidence that blueberry and strawberry intake could offset the irradiation-induced deficits in spatial learning and memory. Blackberry was also investigated to evaluate its efficacy in reversing age-related deficits in behavioral and neuronal function. A study by Shukitt-Hale et al. (37) found that 2 % of blackberry supplementation in aged rats enhanced short-term memory as assessed by the water maze test and resulted in significant improvement in motor performance of tasks that depend on balance and co-ordination (37).

Human studies


Several studies have been conducted to investigate the effect of polyphenol-rich fruits on age-related cognition decline and MCI. Individuals with MCI are usually at increased risk of developing dementia and clinical appearance of neurodegeneration which may progress to AD (38, 39).The first study conducted by Krikorian et al. (40) enrolled 12 older adults with MCI supplemented with CGJ (range 444–621 ml/day) in a randomized, placebo-controlled study. The California verbal learning test (CVLT) was used to assess verbal memory (List acquisition performance and delayed recall), and the spatial paired associate learning test used to evaluate non-verbal memory (spatial memory). The study showed a significant improvement in verbal learning and enhancement of delayed verbal recall and spatial memory when compared to placebo (40). The second study enrolled 21 older adults with MCI used similar dosing schedule of CGJ supplementation (range 355–621 ml/day containing 742-1298 mg total polyphenols) for 16 weeks. While the study found no difference in performance using the CVLT learning tasks and recognition memory performance, patients who consumed CGJ showed less interference errors in the recognition memory task (indicative of inhibitory control in working memory). In addition, this study assessed functional magnetic resonance imaging (fMRI) changes for participants to test potential brain activation during a working memory task, and found an increased activation in the right superior parietal cortex and right middle frontal cortex following CGJ supplementation (41). These studies provided some evidence that grape intake might enhance memory in elderly patients with MCI and justify conducting larger trials on elderly patients with MCI or age related cognitive decline.


A small trial which investigated the effects of daily consumption of blueberry juice (444-621mL containing 1056-1477 mg of total polyphenols) enrolled nine elderly participants with MCI who had experienced age-related memory decline such as forgetfulness and prospective memory lapses. Following 3 months of juice consumption, patients showed significant improvement in memory function as indicated by paired associate learning and word list recall tests. There was also a reduction of associated depressive symptoms. Although the sample size was relatively small, the positive effects on memory after blueberries intake may mitigate neurodegeneration. Clearly, larger randomized trials are needed to recommend the use of blueberry supplementation as preventive intervention in cognitive function deterioration (42).
Epidemiological studies have also been conducted to examine the effect of flavonoids intake on cognitive function decline. Devore et al. (43) evaluated the effect of berries and other flavonoid-rich diet consumption on cognition decline in a large prospective cohort study. Female registered nurses (n=12,1700) aged 30–55 years were followed up for 26 years using food frequency questionnaire updated every four years. Those women who were ≥70 years old and free of stroke were invited to participate in the cognitive function tests and their data have been analyzed (n=16,010). The study calculated the intake of 31 individual flavonoids (representing six major flavonoid subclasses; anthocyanidins, flavonols, flavones, flavanones, flavan-3-ols, and polymeric flavonoids) that are commonly found in the United States diet. Blueberries and strawberries were the major foods contributing to anthocyanidin intake in this cohort, whereas tea, apples, and oranges were the major contributors to other flavonoid subclasses and total flavonoid intake.  Two measures of overall cognition (global composite score averaging all tests, and the telephone interview of cognitive status) and a verbal memory composite score; averaging four tests of episodic memory were used to evaluate the cognition status. The study concluded that higher total flavonoid intake was associated with significant slower rates of cognitive decline for all of the three stated primary outcome measures. Among flavonoids, anthocyanidins were noticed to have the highest association with cognitive benefits. However, flavonols were observed to have modest association with cognitive function. Researchers specifically found that this positive effect was due to greater consumption of blueberries and strawberries, and that berry intake appeared to delay cognitive aging by up to 2.5 years.  Interestingly, flavonoids intake originating from tea, onions, apples and oranges were not associated with delaying cognitive decline (43).


Pomegranate juice intake by older subjects with age-associated memory complaints was investigated by Bookeimer et al. (44). Participants (n=32) were randomly assigned to drink 8 ounces (236 mL) of either pomegranate juice or a flavor-matched placebo drink for 4 weeks, and then memory tests assessed by using the Buschke-Fuld selective reminding task which evaluated verbal memory. The participants were also scanned by fMRI and blood biomarkers before and after the intervention. Following 4 weeks, only the pomegranate group showed a significant improvement in the verbal memory selective test with a significant increase in plasma trolox-equivalent antioxidant capacity (TEAC) and urolithin-A glucuronide indicating an increase in free radicals scavenging. Furthermore, the pomegranate group had increased fMRI activity during verbal and visual memory tasks compared to the placebo group. These results suggested a role for pomegranate juice in augmenting memory function through task-related increase in cerebral blood flow, which in turn facilitated memory performance. Metabolic measures did also confirm the increase in polyphenols levels among the experimental group (44).

Summary and future recommendations

Table 2 summarizes the animal studies on cognitive decline related to aging following grape and berries intake. There was a significant cognitive function enhancement using Morris water maze that reflected spatial learning and memory domain improvement. Proposed mechanisms for anthocyanins rich fruits beneficial effects on age related cognitive decline included: oxidative stress reduction, enhancement of neuronal signaling in brain memory centers, enhancement of hippocampal neurogenesis, increased dopamine release from striatal slices and buffering excess striatal calcium in striatal synaptosomes.  No animal studies were conducted so far to investigate the effect of pomegranate extract on age related cognitive decline.

Table 2. Summary of animal studies of grape and berries effects on cognition decline related to aging and MCI

Table 2. Summary of animal studies of grape and berries effects on cognition decline related to aging and MCI

↑ Represents significant improvement

Human interventional studies of grape, berry and pomegranate showed a significant enhancement of cognitive functions in older subjects with age-related memory complaints and MCI, as evidenced by some of the memory and executive function measures assessed. In addition, an increased in the free radical scavenging effect was seen after pomegranate juice intake and an increase of fMRI activation in certain brain regions that suggested greater cerebral blood flow during memory tasks. Summary of human studies effects of grape, berry and pomegranate on cognition in patient with MCI or aged related cognition decline is shown Table 3.

Table 3. Summary of human studies of grape, berry and pomegranate effects on cognition in patient with MCI or aged related cognition decline

Table 3. Summary of human studies of grape, berry and pomegranate effects on cognition in patient with MCI or aged related cognition decline

*Total polyphenols expressed as gallic acid equivalent; ** Anothicyanidins were noticed to have the highest association with cognitive benefits due to greater blueberry and strawberry consumption; ↑ Significant improvement


However, all interventional studies had a small sample size which means the data were insufficiently supported. Furthermore, epidemiological studies which evaluated the association of biophenol-rich fruit with age cognition decline related to aging are really scarce.  One study showed an association of high flavonoids consumption and protection against cognitive function decline in older female nurses.  However, this study didn’t follow up patients who already diagnosed with MCI or patients with age-related memory complaints. We recommend future studies will assess MCI patients or those with age-related memory complaints to confirm the protective effect of biophenol-rich fruit consumption.
Effect of biophenol-rich fruits and extracts on Alzheimer Disease

Animal studies


Wang et al. (45) evaluated the effect of commercially available MegaNatural grape seed polyphenolic extract (GSPE) treatment for 5 months on a transgenic mouse model with Amyloid β (Aβ) accumulation. Results showed a significant decrease in oligomerization of Aβ peptides into high molecular weight Aβ species, reduced amounts of Aβ42 and Aβ40 peptides and amyloid neuritic plaque burden compared to the matched group control animals. GSPE treated mice had marked improvement of spatial memory function compared with the control group (45). A similar study using transgenic mice that have been supplemented with GSE for 9 months found similar findings of a reduced level of Aβ in the brain and serum in the treatment group mice.  In addition, GSE intake was also found to reduce microgliosis (proliferation or hypertrophy of different types of glial cells, including astrocytes, microglia, and oligodendrocytes) in the brain of Alzheimer’s mice model. GSE also decreased the levels of inflammatory cytokines: interleukin 1 beta (IL-1β), tissue necrosis factor- alpha (TNF-α) and tissue necrosis factor-gamma (IFN-γ) (46).
Tau protein phosphorylation and aggregation which lead to neurofibrillary tangles (NFT) development, may contribute to the pathophysiology of Alzheimer’s disease (AD). Several studies examined the effect of polyphenol-rich fruit on tau neuropathy. A study by Wang et al. (47) examined the effect of GSPE on a mouse model of AD, characterized by an age-dependent development of tau pathology in the brain, found that it effectively interfered with the assembly of tau peptides into neurotoxic aggregates. Moreover, oral administration of GSPE had attenuated the development of AD type tau neuropathology in the brain of the mouse model of AD through mechanisms associated with attenuation of extracellular receptor kinase signaling in the brain involved in tau hyperphosphorylation (47).


Amyloid precursor protein (APP) is a transmembrane protein best known as the precursor molecule whose sequential proteolysis generates Aβ protein. Therefore, APP processing could lead to Aβ production which is a key pathogenic feature of AD (48). Several studies evaluated the effect of biohenol-rich fruit on APP processing and subsequently, the level of Aβ production and aggregation. A study by Vepsalainen et al. (49) was carried out on transgenic mice with amyloid precursor protein/presenilin1, found that mice fed anthocyanin-rich bilberry or blackcurrant extract had lower APP C-terminal fragment levels in the cerebral cortex as compared to transgenic mice on the control diet. Both berry diets improved the spatial working memory deficit of aged transgenic mice compared to mice on the control diet, as evidenced by delayed Aβ protein processing. This would indicate a favorable effect of berry fruits on working memory enhancement. On another hand, both berry diets showed no changes in the expression or phosphorylation status of tau in mice. These data suggest that anthocyanin-rich bilberry and blackcurrant diets favorably modulate APP processing and alleviate behavioral abnormalities in a mouse model of AD (49).


The effect of dietary supplementation of pomegranate extract (PE) on memory, anxiety, and learning skills in AD mouse model possessing double Swedish APP mutation has been examined by Subash et al. (50). The study showed that the transgenic mice with APP mutation that were fed a diet containing 4% PE developed significant improvements in memory, learning, locomotors function as well as a reduction in anxiety, compared with transgenic mice fed the standard chow diet. The study suggested that pomegranates intake may slow the progression of cognitive and behavioral impairments in AD (50). The mechanism of action was investigated by Ahmed et al. (51) who used aged transgenic mice AD model to evaluate the effects of a standardized  PE on spatial function of long-term and working memory, APP and Aβ levels and other biomarkers of AD in brain tissues. The study showed that PE did not improve cognitive performance of the mice, but altered levels and ratio of the Aβ40 and Aβ42 peptides that would favor a diminution in AD pathogenesis.  This reversal could be due to the modification of γ-secretase enzyme activity (the enzyme involved in the generation of Aβ isoforms). These findings suggested an anti-amyloidogenic mechanism of PE in this aged AD animal model (51). Essa et al. (52) have also conducted a placebo-controlled study using dietary supplementation of pomegranates, figs and dates, and found that pomegranate significantly decreased Aβ40 levels in the cortex and hippocampus of the transgenic rats, decreased levels of inflammatory cytokines: IL-2, IL-3, IL-4, IL-5, IL-9 and IL-10, in the plasma following diet containing pomegranates. The authors concluded that reducing inflammatory cytokines during aging may represent one mechanism by which fruit supplements may exert their beneficial effects against neurodegenerative diseases such as AD (52).
The effects of Pomegranate Peel Extract (PPE) on spatial memory, biomarkers of neuroplasticity, oxidative stress and inflammation in mice with Aβ peptide deposition induced by chronic infusion of Aβ42 using mini-osmotic pumps for 35 days have been examined by Morzelle et al. (53) using a placebo-controlled study. Animals consumed PPE improved the escape box Barnes maze test, which reflected an enhancement in the spatial memory, a finding that was not observed in the Aβ group that was fed chow diet. Amyloid plaque density, the activity of acetylcholinesterase enzyme and level of TNF-α were also reduced, while the expression of BDNF was increased. The effect of PE on memory in APP/PS1 transgenic mice as model of AD was also studied after 3 months supplementation and found to enhance spatial memory as evidenced by decreased path length to escape the Barnes maze compared with their initial values and their control-fed counterparts. It was also found that one month of pomegranate feeding increased spontaneous alternations, which reflected working memory improvement. Brains of the pomegranate-fed mice had significantly lower TNF-α, lower nuclear factor of activated T-cell (NFAT) transcriptional activity, attenuated microgliosis and Aβ plaque deposition. This study indicated that dietary pomegranate produced brain anti-inflammatory effects in the brain that may attenuate AD progression (54)

Human Studies

Flavonoids from various sources

Few epidemiological studies linking biophenols consumption and AD were published and some of these had evaluated the association of fruit and vegetable consumption without specifically examining the importance of biophenols contents. Cohort studies by Hughes et al. (55) and Dai et al. (56) showed that moderate and large fruit and vegetable consumption at midlife ages and in elderly individuals, were associated with a decreased risk of dementia (55, 56). The relationship of flavonoids with AD risk was investigated by two large prospective epidemiologic studies. In the first study, Letenneur et al. (57) assessed the data of 1,640 participants for whom nutritional data were available at the 3-year visit and who completed at least one psychometric test at one of the visits. Cognitive function was assessed by 3 psychiatric tests at each visit. The mean flavonoid intake was 14.33 mg/day. The quartiles of flavonoid intake were divided into; 0–10.39, 10.40–13.59, 13.60–17.69, and 17.70–36.94 mg/day. The study estimated the prediction of flavonoid intake on the baseline and the annual rate of change in Minin-Mental State Examination (MMSE) score. The mean MMSE score at baseline was 27.1 and increased as flavonoid intake increased. The same pattern was observed for the other cognitive tests. Flavonoid intake was significantly associated with better cognitive performance at baseline and subjects in the two highest quartiles had a significantly better evolution than did subjects in the first quartile (57). The second study (3,777 community dwellers, free from dementia at baseline, aged 65 years or older) performed by Commenges et al. (58) that used a statistical method to impute a quantity of favonoid intake for each subject based on the questionnaires analyzed the relationship between this measurement and the risk of developing dementia in a 5-year follow-up of the cohort between 1991 and 1996. Data showed that 66 incident cases of dementia were observed in 1367 patients, and flavonoid consumption did actually decrease the risk of dementia (58).
Interestingly, a recent cross sectional study was conducted also to assess the relationship of flavonoid intake with measures of cognition in 49 participants aged 65 years and older with mild to moderate AD. The study assessed measures of mood, verbal learning and memory, working memory, semantic memory, executive function and short-term memory domains with flavonoids intake. The major source of flavonoid intake was black tea (80% contribution). Other sources included green tea, berries, red wine, apples and oranges.  Total flavonoid intake was significantly correlated with verbal fluency task which evaluates the executive function domain, although other measures of executive function, trial making task and self-ordered pointing task, were not improved significantly. Verbal fluency was also significantly correlated with the flavonoid subclasses; flavonols, flavan-3-ols and anthocyanins. This may be related to the consumption of black and green tea (the major sources of flavonols and flavan-3-ols) and berries (a major source of anthocyanins). Also, no other significant associations for other cognitive domains were identified. There was also a positive correlation found between depressive symptoms and flavonoids intake as assessed by geriatric depressive scale. On other hand, no significant association between flavonoids intake and cognitive measures was shown after controlling for depression. The reduction in association between verbal fluency and flavonoid intake to non-significance level when depression was controlled suggested that this relationship was confounded by the effect of depression on executive functioning. The authors suggested that previous epidemiological studies that have reported associations between flavonoid intake and cognitive outcomes, without controlling for depression, may have overestimated the strength of this relationship (59).
In contrast, two other epidemiological studies found no association between flavonoid intake and developing AD. Engelhart et al. (23) analyzed the data from the Rotterdam study which followed a total of 5395 participants, who were at least 55 years old for 6 years. The study examined the effect of several antioxidants, including flavonoids on the risk of developing AD. They found a non-significant decrease in the risk of Alzheimer’s disease as flavonoid intake increased (23). Another study by Kalmijn et al. (60) used 342 men and analyzed data derived from a cohort of men, aged 69-89 years, who were participants in the Zutphen Elderly Study for the effect of flavonoid intake on the risk of cognitive decline. Subjects classified in the medium or highest intake tertile, showed a non-significant decrease in risk of cognitive decline defined as a drop of more than two points in the MMSE over a 3-year period. The non-significant reduction was probably due to the small number of subjects included and the short period of follow-up for this sample (60).


We have found one study that evaluated the intake of biophenol-rich fruit in patient with AD. A 12-week, randomized, controlled trial of 49 patients with mild-to-moderate dementia AD type, aged 70 years or older where cognitive outcomes were assessed after consumption of 200 mL/day of either a cherry juice (containing 138 mg of anthocyanin) or a control juice with negligible anthocyanin content. Marked improvement was seen for category verbal fluency and tasks relating to verbal learning and memory including short term and long-term memory, as evidenced by RAVLT tasks. Other measures for semantic memory and working memory or executive functions were not significantly different between the groups. Inflammation markers such as C-reactive protein remained unaltered (61).

Summary and future recommendations

A summary of animal studies on AD-rats model using grape, berry and pomegranate is shown in Table 4. A beneficial role of grape, berry and pomegranate on cognitive and behavioral parameters in AD was observed. Most of studies targeted levels of Aβ isoforms and its disposition in the brain, modified APP processing, with the resultant decrease in the density of neuritic plaques which suggested an anti-amyloidogenic effect of the supplementation. Additionally, grape intake interfered with the assembly of tau peptides into NFTs. In fact, targeting Aβ and tau proteins showed promising results in the efforts to modify the pathological effects associated with AD.  A systematic review of several trials targeted Aβ and tau proteins provided an evidence of reducing pathological outcomes during certain drugs therapy in animal models of AD that was mostly shown as an improvement in cognition (62). Another proposed mechanism for PPE was an enhanced expression of BNDF, and presumably, the depletion of BDNF increases the progression of dementia related to AD. Thus, an increase in BDNF expression might be a potential target for the treatment of neurodegenerative diseases (63). Interestingly, pomegranate produced an anti-inflammatory effect that was indicated by changes in inflammatory cytokines levels in blood and brain of transgenic mice and attenuation of microgliosis. Inflammation can also play an important role in AD pathophysiology as evidenced by a meta-analysis conducted by Swardfager et al., (64) which concluded that AD cases seem to be accompanied by an inflammatory response and higher peripheral concentrations of cytokines (64), and this was supported also by a recent review by Gardener et al. (65). Thus, the use of biophenol-rich fruit supplementation may have a beneficial role in AD pathogenesis.

Table 4. Summary of animal studies investigating the effects of grape, berry and pomegranate on AD

Table 4. Summary of animal studies investigating the effects of grape, berry and pomegranate on AD

*Marginally significant; ↑ Significant improvement, ↔ No significant difference


The association between flavonoids intake and AD risk and cognition in AD patients along with cherry effect on cognition in AD patients is summarized in Table 5. As preventive supplements, most of studies conducted to evaluate the relationship of flavonoid with AD risk, found a positive correlation between flavonoid consumption and AD risk reduction. However, these studies didn’t specify the benefits to a particular flavonoid subclass and we can suggest that further research exploring the effects of particular class of flavonoids such as anthocyanins on AD prevention should be performed. As therapeutic and/or adjunctive role of biophenol-rich fruit on cognitive deficit seen in AD, one cross section study of flavonoids intake and one randomized trial of cherry juice intake in patients with mild-to moderate Alzheimer’s type showed a positive effect on verbal fluency and memory. However, the cherry study utilized a small dose of anthocynins compared to other studies which examined anthocynanins-rich fruit effect on cognition. We definitely recommend RCT’s of berries with varying doses to be performed that may also include further cognition measures. Also, the cross sectional study utilized a small sample size and thus the identified association between cognitive function, depression and flavonoid needs to be confirmed by a larger sample size. To our knowledge, no RCT’s were conducted to evaluate the effect of grape, pomegranate or other berry fruits such as blueberry and blackcurrant on cognitive deficit seen in AD. Performing these RCT’s would provide an additional evidence to support the use of biophenol-rich fruit supplementation as adjunctive therapy for patients with AD cognitive deficits. In addition, it is worth mentioning that dietary microbiome intervention has the potential to improve physical and emotional wellbeing in the general population, and in particular, as a treatment option for individuals with conditions as diverse as IBS, anxiety, depression and Alzheimer’s disease (76).

Table 5. Summary of human studies of the association of flavonoids and AD risk and cognition in AD patients along with the effect of cherry on cognition in AD patients

Table 5. Summary of human studies of the association of flavonoids and AD risk and cognition in AD patients along with the effect of cherry on cognition in AD patients

↑ Significant improvement, ↔ non-significant effect; *Flavonoid in general has been studies with no referral to specific subclass; **Duration is not applicable and after controlling for depression no significant relationship between  flavonoid and any cognitive measure was observed; NA = not available


Effect of biophenol-rich fruits on cognitive deficit induced by cerebral ischemia

Memory deficits could also be seen in cerebral hypoperfusion or in diffuse ischemic state. Animal and human studies have been conducted to investigate the relationship between biophenols-rich fruits and cognitive deficit induced by cerebral ischemia. Unfortunately, human studies only focused on the effect of biophenols on postoperative cognitive dysfunction (POCD) without investigating other cerebral ischaemic insults causes. Reactive oxygen species generation is regarded as one of the most important factors related to neuronal death in ischemic related areas (66-68).

Animal studies


The effect of chronic oral administration of GSE on passive avoidance memory deficit and hippocampus gyrus (DG) long-term potentiating (LTP) inhibition induced by permanent bilateral common carotid arteries occlusion (2CCAO) used as an animal model of cerebral ischemia /hypoperfsuion was examined by Sarkaki et al. (69). The study showed that oral administration of GSE for 28 days could ameliorate the passive avoidance memory deficit induced by 2CCAO manifested in longer step-down latency. Moreover, GSE increased percentage of amplitude, slope, and area under curve of LTP recorded from hippocampal DG after High-frequency stimulation (HFS) compared to placebo group (69).


The effect of pomegranate on memory deficits due to cerebral hypoperfusion has been studied in male adult rats. Hajipour et al. (70) examined the effect of two weeks oral administration of pomegranate Seed Extract (PGSE) on active avoidance memory and motor coordination activities after permanent 2CCAO in rats. Those treated with PGSE showed significant improvement in impairment of memory and motor coordination. It was stated that PGSE exhibited therapeutic potential for memory and muscular coordination, most likely due to its antioxidative and free radical scavenging actions (70). A second study by Sarkaki et al. (71) aimed to evaluate the effects of PGSE on passive and active avoidance memories due to gonadal hormone deprivation in ovariectomized rats, with and without cerebral hypoperfusion after permanent 2CCAO has concluded that hormone deprivation, such as estrogen, can alter cognitive performance. Estrogen can exert positive mnemonic effects in the inhibitory avoidance task (72) and estrogen deficits in rats (ovariectomy) impaired memory (73). This study showed that PGSE treatment has improved active and passive memories in those rats. Thus, it seemed that PGSE exhibits therapeutic potential of both active and passive avoidance memories, which is most likely related to its phytoestrogenic and antioxidative actions (71). A third study conducted on adult female rats to evaluate the effect of two weeks oral administration of PGSE on active and passive avoidance memories after permanent 2CCAO to induce permanent cerebral ischemia found consistent results with the previous two studies (74).

Human studies

Proposed mechanisms for postoperative cognitive dysfunction (POCD) following heart surgery include general hypoperfusion of the brain (global ischemia) leading to critically low levels of oxygen and glucose throughout the brain. In addition, inhaled anesthetics have been shown to induce the formation of amyloid-beta, a potentially neurotoxic protein linked to Alzheimer’s disease and acute cognitive deficits in the brain (75, 76). A pilot study was conducted by Ropacki et al. (77) to investigate the effect of biophenols in pomegranate on POCD in 10 patients undergoing elective coronary artery bypass graft and/or valve surgery to examine such relationship. The patients were given either 2 g of pomegranate extract/day or placebo one week before surgery to 6 weeks after surgery. The patients were also administered a battery of neuropsychological tests to assess memory function at 1 week before surgery (baseline), 2 and 6 weeks after surgery. The placebo group showed a significant deficit in postsurgery memory retention, while the pomegranate extract supplementation was shown to not only protected against postoperative cognitive dysfunction, but also actually had an improved memory retention performance for up to 6 weeks after surgery as compared to pre-surgery baseline performance. This study was the first to report that pomegranate extract improves POCD in humans and that it may provide long-lasting protection of heart surgery-induced memory retention deficits (77).

Summary and future recommendations

A summary of animal studies investigating the effect of grape and pomegranate on cognition deficit induced by cerebral ischemia is shown in Table 6. Animal studies showed a significant enhancement of active and passive cognitive function measures. No animal studies so far were conducted to examine such benefits using berry and thus further trials would also be needed to confirm the benefits. In relation to the effect of biophenol-rich fruit on cognition impairment following surgery in humans, only a small, pilot trial was conducted using pomegranate extract to explore its effects. We highly recommend further RCT’s with larger sample size to be conducted to support the notion if PE supplementation in pre and post operations settings could be beneficial. As far as we know, no RCT’s have been conducted to examine the effects of grape and berry on memory impairment post operatively. We suggest RCT’s to investigate grape and berry potential effect on POCD. It would also be quite important to notice that no human studies have been conducted to examine the relationship between biophenol-rich fruit supplementation and stroke induced memory dysfunction. We again recommend an RCT exploring the potential effect of grape, berry and pomegranate intake on cognitive function enhancement in stroke patients.

Table 6. Summary of animal studies of the effect of grape and pomegranate on cognition deficit induced by cerebral ischemia

Table 6. Summary of animal studies of the effect of grape and pomegranate on cognition deficit induced by cerebral ischemia



It seems that the beneficial effects of biophenols-rich fruits on cognition of healthy subjects or cognitive deficits related to aging, MCI, AD and POCD present a safe and effective approach for memory domain improvement.  It was also noticed that acute and chronic flavonoid-rich supplementations effect on cognition of healthy subjects is age, dose and subclass dependent, as we know that not all flavonoid subclasses have similar benefits on cognition. Anthocyanidins found in pomegranate, grape and berry provided such benefits. Other flavonoid subclasses investigated have shown differential effects; some (e.g. flavanones) were found to be beneficial for cognition of healthy subject. Flavanone is richest in orange juice, and studies with acute and chronic orange juice consumption indicated a marked enhancement of cognitive measures (78-80). In contrast to the positive effect of anthocyanidins and flavanones on cognition, studies of flavonols (quercetin) and flavan-3-ols (epicatechin) rich in apple showed no observed effects on measures of attention and executive functions (81). Also non-significant acute effects were reported for flavan-3-ol, epigallocatechin gallate (EGCG), rich in green tea on cognitive function measures (82).
Generally, the effect of grape, berry and pomegranate on cognitive deficits or decline could be examined for two main purposes – prevention and/or treatment. Due to the fact that there is no remedy for dementia so far, and we cannot predict when or if effective therapy will be developed, it would be important to consider the preventive role of biophenols-rich fruit on cognitive deficits associated with AD.  Dementia has shown to have a long “pre-dementia phase”, which could last for years preceding future potential AD. In this phase, patients have lower cognitive function associated with difficulties of performing instrumental activities (83). It has been proposed that interventions initiated in individuals with pre-dementia conditions such as MCI might prevent further progression of cognitive decline, and MCI may represent the final point at which intervention can be effective (84). Interventional studies with grape, berry and pomegranate for MCI cases showed an improvement in cognitive functions which suggests that supplementations could have a preventative role for AD if started at the MCI stage. However, these studies require larger sample size to substantiate the evidence. The treatment option of biophenol-rich fruit is scarcely studied and the conducted two studies (one RCT with cherry and one cross sectional study with flavonoids) in mild to moderate Alzheimer’s type dementia, showed an enhancement of specific cognitive domains which would suggest an adjunctive role for such supplementation. A recent review exploring possible nutritional treatment of AD concluded that there is a relationship between nutrition (vitamins, curcumin and mediterranean diet) and AD (85). Clearly, further studies should be conducted to justify such use. An interesting aspect of grape, berry and pomegranate intake benefit was examined to study their use to improve cognitive deficit that may occur in patients with cerebral ischemia or following surgery. These studies were also limited in number and definitely further human studies should be conducted to provide clear description of the role of these fruits in cognitive deficits of those patients.


Acknowledgements: L.A.D would like to express her thanks to Middle East University for their support to conduct this research review. Also she would like to express her greatest gratitude to her mentor, Professor Emad AL-Dujaili for his patience and support throughout the project and looking forward to work with him in future research. E.A.D has planned, supervised and edited the work. All authors have contributed in the production of the whole manuscript. All authors reviewed and approved the manuscript.

Conflict of interest: All authors confirm that the content of this review has no conflict of interests



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M.E. Clementi1, B. Sampaolese1, G. Lazzarino2, B. Giardina2


1. CNR-ICRM Institute of “Chimica del Riconoscimento Molecolare”, c/o Institute of Biochemistry and Clinical Biochemistry, Catholic University School of Medicine Largo F. Vito 1, 00168 Rome, Italy; 2. Institute of Biochemistry and Clinical Biochemistry, Catholic University School of Medicine, Largo F. Vito 1, 00168 Rome, Italy

Corresponding Author: Dr Maria Elisabetta Clementi, ICRM – Institute of Chimica del Riconoscimento Molecolare’ (CNR), c/o Institute of Biochemistry and Clinical Biochemistry, Faculty of Medicine, Catholic University, Largo F. Vito 1, 00168 Rome, Italy, Tel:++39 6 30154215, Fax:++39 6 30154309, e-mail:

J Prev Alz Dis 2015;2(1):33-37
Published online Januay 20, 2015,


Methionine sulfoxide reductase A (MsrA) has been postulated to act as a catalytic antioxidant system involved in the protection of oxidative stress-induced cell injury. MsrA has recently turned attention in coupling with the neurodegenerative disorders  and in particular with Alzheimer disease. In fact this neurodegenerative disorder depends to a deposit of beta amyloid a peptide with an oxidizable methionine in position 35 which is proved able to modulate the expression to MsrA in neuronal cells. Here, we firstly provided evidence that pretreatment with Resveratrol and Punicalagin (a potent antioxidant extracted from pomegranate), up-regulate the expression and enzymatic activity of MsrA in human neuroblastoma IMR-32 cells with beta amyloid peptides. This effect determines a lowering of oxidative potential of the cells as demonstrated by the ROS measurement and a protective effect on cellular availability. Therefore we hypothesize a possible prevent role for these molecules in Alzheimer and in other neurodegenerative diseases.

Key words: Methionine Sulfoxide Reductase, Punicalagin, Resveratrol, Alzheimer.



disease (AD) is one of the most common neurodegenerative disorders that characterized by a protein misfolding disease due to the accumulation of abnormally folded amyloid beta protein, a peptide of 1-42 aminoacid, in the brains of Alzheimer’s patients. Amyloid beta peptide is a monomeric soluble molecule which contains a single methionine residue in position 35, located in the middle of the hydrophobic C-terminal region (1, 2). Therefore the dramatic increase in polarity of the Met side chain that occurs upon oxidation has a profound effect on the hydropathy of the entire region (3). Met is highly susceptible to oxidation “in vivo”, particularly under condition of oxidative stress: at this regard the sulfoxide form has been found to comprise 10-50% of Ab in amyloid plaques of AD brains (4, 5). The oxidation of methionine to sulfoxide is a reversible reaction catalyzed in vivo by the methionine-sulfoxide reductase (MsrA) system which reduces the sulfoxide groups providing protection against oxidative stress (6, 7). To understand the relevance of this enzymatic system in AD an our recent study has evidenced elevated MsrA activity and mRNA levels in neuronal cells in response to treatment with Aβ-Met-35OX suggesting that the cells sensed the presence of sulfoxide in Aβ and upregolated Msr to provide enhanced cellular protection (8). Hence, considering that the deficit in MsrA system may participate in age-related oxidative damage (9), it is desirable to identify agents to restore and increase MsrA expression and function. In this light, one of the plausible ways to prevent cellular damage induced by oxidative stress is to augment or potentiate the cellular defense capacity through the dietary or pharmacological intake of antioxidants. Antioxidants can inhibit ROS formation, directly scavenge ROS, enzymatically detoxify accumulated ROS, and augment cellular defenses by up-regulating antioxidant gene transcription (10, 11). The accumulated epidemiological data indicate the importance of a proper dietary regimen in maintaining adequate cognitive functions and preventing or delaying AD (12).     

In this study we chosen as potential protective natural molecules Resveratrol (RSV), a natural polyphenolic compound abundantly present in grape skins and wines, and Punicalagin (2,3-S-hexahydroxydiphenoyl-4,6-(S-S)-gallagyl-D-glucose, referred to as PUN here), an ellagitannin isolated from pomegranate polyphenols, so both have been found to protect against oxidative stress-induced cell injury in many tissues (13,14), and can up-regulate the expression of endogenous catalytic antioxidant systems. In particular we investigated here the effect of resveratrol and punicalagin preconditioning on cellular vitality, oxidation potential and MsrA function, in IMR32 cells treated with beta amyloid peptides.

Materials and Methods

Aβ peptide was obtained by Peptide Speciality Laboratories GmbH (Heidelberg, Germany). Stock solution of Aβ peptide, 2.5 mM in DMSO were prepared according to the manufacturer’s instructions and stored at –20 °C. Resveratrol and Punicalagin were dissolved in ethanol as 10 mM stock solutions.  

Cell culture and treatments 

Human neuroblastoma IMR-32 cells were grown in minimum essential medium supplemented with 10% heat inactivated fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and cultured at 37 °C in an atmosphere of 5% CO2 in air. Cell differentiation was induced by 1 mM dibutyryl cAMP and 2.5 μM 5-bromodeoxyuridine, which were added to the culture medium three times for week, starting from the day after plating. After a week, the differentiated cells were plated at an appropriate density according to each experimental procedure. The cells were pretreated with resveratrol or punicalagin 20 μM, 48 h before the treatments with beta amyloid peptides which were added to each experimental set at a final concentration of  10 μM.

Direct toxicity study

For determination of vitality, IMR-32 cells were plated in 96-well plates at a density of 10,000 cells/well and incubated, after pretreatments with resveratrol or punicalagin, for 48 h in the absence (control) and in the presence of 10 μM Aβ peptides. Cell survival was evaluated by the 3-[(4,5-dimethylthiazol-2-yl)-5,3-carboxymethoxyphenyl]-2-(4-sulfophenyl)-2H tetrazolium, inner salt (MTS) reduction assay. The MTS assay is a sensitive measurement of the normal metabolic status of cells, which reflects early cellular redox changes. The intracellular soluble formazan produced by cellular reduction of the MTS was determined by recording the absorbance of each 96-well plate using the automatic microplate photometer at a wavelength of 490 nm.

Methionine Sulfoxide Reductase gene expression

Total RNA was isolated using SV total RNA isolation System (Promega) that includes the elimination of any genomic DNA by DNAse treatment. The purity and quantity of the resulting RNA were determined via the measurement of the absorbance at 280 and 260 nm respectively. The A260/A280 ratio was about 1.8. The RNA was concentrated by precipitation and re-dissolved in water RNAse free. Total RNA (1 μg) from each sample was used for first strand cDNA synthesis using the M-MLV Reverse and Oligo-dT, as random primer (SIGMA). Human GAPDH was chosen as internal control (data do not shown).

The following primer sequences were used for amplification:



MsrA sense primer: 5′-AGTACCTGAGCA AGAACCCCA-3′,

MsrA anti- sense primer:5′-TCACTCAGACCCC AGAAGACA-3′.

PCR was carried out with Red Taq Polymerase (Sigma, St. Louis, MO, USA) according to the supplier’s conditions. The PCR reaction conditions were for both genes: 94 °C for 5 min, followed by 35 cycles of 1 min denaturation at 95 °C, 1 min annealing at  60°C, 30 s polymerization at 72 °C and finally 10 min extension at 72 °C. PCR products were analyzed by electrophoresis in agarose 1.8% with ethidium bromide (1 μg/ml) in TBE 1× buffer (Tris 40 mM, EDTA 1 mM, boric acid 44 mM) for 2 h at 80 V (constant voltage) with 123 bp ladder as molecular weight marker.

Images of gels were acquired and scanned using Biorad Quantity One software. The density of the PCR bands were expressed as a ratio of the band density divided by that of the housekeeping gene, GAPDH.

Methionine Sulfoxide Reductase Activity Assay

Post treatments harvested cells were washed once in PBS buffer and lysed in 300 μl of lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1% Triton X-100, in the presence of protease inhibitor cocktail-Roche). The resulting extracts were spun down (10,0000 × g) and the soluble material was collected and analyzed for protein concentration assay. Equal amounts of protein extracts were analyzed for total MsrA activity according a new colorimetric assay (15) where MsrA activity is measured by the consumption of DTT, which can be classically detected by Ellman’s spectrophotometric assay. MsrA-catalytic methyl sulfoxides-mediated oxidation controls the concentrations of DTT in the reaction system. Thus, the decreased OD412 of DTNB, the reduced product by DTNB-DTT reaction, serve as a rapid indicator for MsrA activity. Briefly, a reaction mixture (100 μl) including MgCl2 10 mM, KCl 30 mM, Tris–HCl 25 mM, DTT 50 μM ( pH 8) and 300 μg of extracted protein was incubated for one hour at 37 °C. Due to the potential reductive activity of DTT, this reaction was performed in the air-tight and complete darkness. After reaction, 100 μL of the reaction mixture and 100 μL of dithio-bis-nitrobenzoic acid (DTNB – 4 mM) was added to a 96-well plates and incubated at 37 °C for 10 min. Absorbance at 412 nm was measured with a microplate reader. Different concentrations of recombinant MsrA were also used to create an internal control (data not shown).

Detection of ROS

The detection of ROS was performed after staining cells with DCFDA Cellular ROS Detection Assay Kit. Briefly, cells treated in different experimental conditions, were grown in 96-well microplate with 25,000 cells per well and treated successively with 2’,7’–dichlorofluorescein diacetate (DCFDA) which is initially non-fluorescent and is converted by oxidation to the fluorescent molecular DCF. DCF was then quantified using a CytoFluor Multi-well Plate Reader, with 485 nm excitation and 538 nm emission filters.


To investigate the neuroprotective effects of RSV and PUN pretreatment on neurons insulted by native and oxidized Aβ peptides we used MTS reduction assay on neuroblastoma differentiated human cells (IMR32). As shown (Fig. 1), IMR-32 cells preincubated for 48 h with 20 micromolar RSV and PUN and successively treated with Aβ (10 μM) peptides, showed an increased cell viability respect to the cells treated with amyloid beta peptides alone. In particular, the pretreatments preserve the neurons by a reduction of cellular availability of 40% and 55% determined by Aβ oxidized and reduced respectively, maintaining values approximately similar to the control.

Figure 1 Effects of Punigalagin and Resveratrol pretreatment on IMR-32 cells after 24h of incubation with oxidized and reduced beta amyloid peptides.

The cell survival is expressed as percent of cells untreated. Cells (10,000 cells/well) were cultured with substances under analysis (experimental conditions are reported materials and methods), and the availability of cells was measured by MTS assay. All values indicate means ± S.D. of seven independent experiments. Significantly different from cells untreated: *P < 0.01; Significantly different from cells treated with beta oxidized (#) and reduced ($) amyloid peptides P < 0.01.


Hence having shown the protective effect of RSV and PUN on Aβ-treated IMR-32 cells, the molecular mechanisms underlying this action was further investigated. In this light we tested the effect of RSV and PUN on MsrA expression and activity in human neuroblastoma IMR32 cells. Firstly we tested the effect of PUN and RSV on MsrA expression in human neuroblastoma IMR32. The cells were incubated with 20 micromolar of RSV and PUN for 48 h. As shown in Fig. 2 RSV and PUN pretreatment significantly increased the expression of MsrA respect to the control showing an increased antioxidant deference and the gene over-expression is maintained also after treatment with beta amyloid peptides. Moreover, as shown in Fig. 3 RSV and more PUN treatment increased also the MsrA activity to 135% and 155% respect to untreated cells. The Aβ peptides (oxidized and reduced) treatments did not modify the enzymatic activity respect to the control while the pretreatments both with RSV and PUN determine an increased activity of MsrA reaching values, in particular in cells treated with oxidized peptide, higher respect to the cells treated with protective molecules alone. These results indicate that RSV and PUN preconditioning up-regulates MsrA expression and activity in human neuroblastoma cells and these effects are amplified by the successive treatments with beta amyloid peptides. The modulated expression and activity of MsrA is evidenced also by ROS measurement reported in Figure 4. Exposure of neuroblastoma cells with beta amyloid peptides increase production of ROS whereas previous exposure with RSV and PUN defends the cells by oxidation processes. Also in this case the effect of Punicalagin is more evident respect to that of Resveratrol.

Figure 2 Panel A: Methionine Sulfoxide Reductase (MsrA) gene expression in IMR32 cells preconditioned with Resveratrol and Punicalagin before of treatments with beta amyloid peptides. GAPDH was used as internal control (data do not shown). Panel B: quantification of the intensities of MsrA bands determined by densitometric scanning of agarose gel. Results are from four independent experiments.

Significantly different from cells untreated: *P < 0.01; Significantly different from cells treated with beta oxidized (#) and reduced ($) amyloid peptides P < 0.01

From these experiments we concluded that β-amyloid treatments in neuroblastoma cells generates the production of hydrogen peroxide responsible for accelerated cellular death and that the action of natural antioxidant compounds preserves the cells through the activation of Methionin sulfoxide reductase expression and enzymatic activity.

Figure 3 Methionine Sulfoxide Reductase (MsrA) Enzymatic Activity in IMR32 cells preconditioned with Resveratrol and Punicalagin before of treatments with beta amyloid peptides.

Significantly different from cells untreated: *P < 0.01; Significantly different from cells treated with beta oxidized (#) and reduced ($) amyloid peptides P < 0.01

Figure 4 Effect of Resveratrol and Punicalagin on ROS production (expressed as Fluorescence Intensity) in IMR32 cells treated with beta amyloid peptides.

Significantly different from cells untreated: *P < 0.01; Significantly different from cells treated with beta oxidized (#) and reduced ($) amyloid peptides P < 0.01



Alzheimer disease (AD) is by far the most common cause of senile dementia. This neurodegenerative disorder of the brain is chronic and progressive and it is characterized clinically by the deterioration in the key symptoms of behavioral and cognitive abilities. Oxidative stress has been strongly implicated in the patho-physiology of this neurodegenerative disorders (5, 16-17). Central neurons are especially vulnerable to insults induced by oxidative stress, due to their higher levels of polyunsaturated fatty acids and the lower levels of brain-resident antioxidants, as well as the high oxygen consumption. ROS, produced by damaged mitochondria during oxidative stress (18), can damage proteins, nucleic acids, and membrane polyunsaturated fatty acids, causing lipid peroxidation and leading to loss of membrane integrity, and increasing permeability to Ca2+ in the plasma membrane (19). Biochemical evidence indicates that amyloid β-peptide (Aβ), a 42 amino acid neurotoxic peptide, plays a predominant role in the pathogenesis of AD associated with oxidative damage. In fact, a single Met residue in Aβ, Met35, is highly susceptible to oxidation in vivo, particularly under conditions of oxidative stress (2,20-21). The resultant sulfoxide form has been found to comprise 10–50% of Aβ in amyloid plaques of AD brain (16, 22). In addition to oxidation of Met to Met-sulfoxide (Met(O)), Met can undergo a second irreversible oxidation reaction yielding Met-sulfone (Met(O2)) that is not commonly found in vivo (6). In contrast, oxidation of Met to Met(O) is reversible and the reverse reaction is catalyzed in vivo by the methionine-sulfoxide reductase (EC, MsrA) an enzyme which reduces the sulfoxide group providing protection against oxidative stress. Previously proposed functions of this enzyme include repair of oxidatively damaged proteins, regulation of protein function and elimination of oxidants (23).

In this scenario, the use of antioxidants especially those of dietary origin have been suggested as possible useful agents for the prevention of cellular damage in neurodegenerative disorders. Based on the knowledge described above, in the present study we investigated whether Resveratrol and Punicalagin, two natural compounds extracted by grapes and pomegranate respectively, and with a well known antioxidant effect, could protect neurons against Aβ toxicity. The obtained results show that RSV and still more PUN preconditioning enhance neuron resistance to toxicity induced by beta amyloid treatments, increasing significantly the cellular availability. The molecular mechanism at the basis of this observed phenomena seems to be link to a control of oxidative cellular potential. In fact the IMR32 cells treated with RSV and PUN 48 h before the treatment with beta amyloid, maintain low levels of reactive oxygen species (ROS) unlike cells treated with Aβ peptides where the formation of ROS is considerable and plays a significant role in effecting cellular pathogenesis.

Since, previous observations individuated in oxidation of Methionine 35 of Aβ42 peptide, a key role in determining the oxidization cellular damage, we hypothesize that the lower toxicity of Aβ peptides linked to ROS formation might result from activation of methionine-sulfoxide reductase (MsrA), an important component of the cellular antioxidant system by side of RSV and PUN. In fact, the treatment with RSV and PUN increase significantly in IMR32 the MsrA expression and activity compared to control and this increase remains also after treatment with beta amyloid peptide both with oxidized and reduced methionine. To note that the protective effect exerted by PUN and RSV are been observed with a concentration easily obtainable with a portion of fruit juice considering that punicalagin reaches levels of 2 g/L of fresh pomegranate juice (24) and resveratrol is about 0,5 mg/liter in fresh grape juice (25).


 The showed data suggest an important neuroprotective role for the MsrA system in the AD brain, which may lead to development of new therapeutic approaches for AD. In this study in particular we evidenced that food-derived polyphenols such Resveratrol and Punicalagin determine a neuroprotective effect on human neuroblastoma cells insulted with beta amyloid peptides, promoting the activation of MsrA: hence a supplement of polyphenol rich foods in diet could be beneficial in prevention of AD. 

Conflict of interest: All Authors of the manuscript declare to have not any direct financial relation with the commercial identities mentioned in the present paper. 

Acknowledgements: This work was supported by grants from the Italian Ministry of Education, University, and Research (PRIN 2010-2011 20109MXHMR-006 to B.G.) and by Italian National Research Council (CNR).


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B. Vellas

UMR INSERM 1027, Gerontopole, CHU Toulouse, University Paul Sabatier, France

Corresponding Author: Bruno Vellas, M.D., PhD,

J Prev Alz Dis 2014;1(3):168-175

Published online November 25, 2014,


An evolving consensus about the need to treat AD in the presymptomatic phase has emerged following the disappointing results of several trials that enrolled subjects with mild to moderate disease, as well as accumulating research demonstrating that AD pathologic process begins decades before the appearance of symptoms. Several lessons can be learned from past prevention trials. The targeted populations were too diverse, the interventions probably not strong enough, and the time of exposure was most likely too short. We have learned from these trials that future prevention trials must be targeted, use strong interventions with known biological activity, and must be sustained with a long-term intervention. In this paper, we focus on three prevention trial approaches:

A. Targeted therapy: Preventing AD by targeting a specific population with a specific intervention. Such preventive approaches and trials must be based on biomarkers and imaging to select a study population in accordance with the mechanism of the specific intervention;

B. Multi-domain interventions targeting a larger, more diverse population over a longer time period with long-term exposure to non-specific, multi-domain intervention. The rationale for this approach stems from studies showing that several environmental factors are associated with the risk of developing dementia. These factors may include educational level, vascular and metabolic risk factors, physical activity, cognitive stimulation, and nutritional status. It may also be possible to identify healthy adults at high risk of AD and likely to benefit from intervention based on subjective memory complaint, ApoEε4 carriage, family history of AD, or the presence of frailty; and use multidomain interventions to compensate for low specificity;

C. What will be probably the future of clinical practice: A preventive approach, integrated into primary care settings that begins with longitudinal monitoring of memory function in a general population to identify decliners, followed by a specific intervention based on biomarkers and imaging discussed case by case. Finally, preventing AD will require new and improved infrastructure.

Key words: Alzheimer, primary care, drug trials, intervention trials, prevention.



In worldwide efforts to address the oncoming public health and economic crisis resulting from the rising prevalence of Alzheimer’s disease (AD), prevention has been recognized as a key goal (1, 2). Primary prevention by targeting modifiable risk factors could potentially reduce disease incidence by millions of cases by 2050 (3). Meanwhile, the field has coalesced around the idea of secondary prevention, which involves diagnosing and treating the disease before symptoms become apparent (4). An evolving consensus about the need to treat AD in the presymptomatic phase has emerged following the disappointing results of several trials that enrolled subjects with mild to moderate disease (e.g., (5-7)), as well as accumulating research demonstrating that AD pathologic process begins decades before the appearance of symptoms (e.g., (8)).

Several lessons can be learned from past prevention trials. For example, the GuidAge clinical trial — the largest preventive trial conducted in the EU — tested whether long-term use of a Ginkgo biloba extract could reduce the risk of progression to AD among subjects over age 70 who spontaneously subjective memory complaints reported to their primary-care physician (9). This randomized, placebo-controlled trial enrolled 2840 individuals and followed them for five years. At the end of the study, there were no statistically significant differences in AD incidence between the two groups. Three reasons were cited as contributing to these disappointing results: the targeted population was too diverse, the intervention with Gingko biloba was probably not strong enough, and the time of exposure was most likely too short. The population of individuals with subjective memory complaints is highly variable, with the overall incidence of dementia quite small, making it difficult to achieve statistical significance. In addition, progression to dementia is slow, so in order to demonstrate a slowing of progression, one would likely need either an intervention with a very robust effect or exposure for a very long time, taking into account that the impact of exposure may not be proportional but may increase over time. Taken together, these factors suggest that future preventive trials will need to consider novel statistical approaches with pre-defined endpoints that take into consideration the fact that the impact of intervention could depends on the time of exposure.

Phase 3 trials of solanezumab and bapineuzumab, both monoclonal antibodies directed against beta-amyloid (Aβ), provide additional lessons (6, 7). Despite the fact that both trials enrolled subjects who met criteria for mild-to-moderate AD, biomarker studies revealed that a substantial number of subjects (nearly 30% of those with mild dementia) had no amyloid in the brain (10). Moreover, amyloid-negative subjects receiving placebo showed almost no disease progression during the 18- month study period, suggesting that they did not have AD. After this trial, the sponsor Eli Lilly launched a third, targeted solanezumab trial (Expedition III), which enrolled only individuals with biomarker evidence of brain amyloid. Interestingly, the practice of conducting targeted trials in other disease areas such as oncology is credited with much of the progress achieved in developing effective drugs.

We have learned from these trials that future prevention trials must be targeted, must use strong interventions with known biological activity, and must be sustained with a long-term intervention. Here, we propose three prevention trial approaches:

  • Targeting a specific population with a specific intervention
  • Multi-domain interventions on a large, more diverse population over a longer time period.
  • What will be probably the future of clinical practice: A preventive approach, integrated in primary care setting that begins with longitudinal monitoring of memory function in a general population to identify decliners, followed by a specific intervention based on biomarkers and imaging if the disease progress


Targeted therapy: Preventing AD by targeting a specific population with a specific intervention

One approach to the development of an effective disease-slowing therapy is to select a study population in accordance with the mechanism of a specific intervention (11). Targeting therapies in this way depends on identification of biomarkers or genetic markers that provide evidence of the stage or type of disease, as hypothesized by Jack and colleagues (12) and demonstrated in subsequent studies (8, 13). For example, trials of anti-amyloid therapies would enroll subjects at early disease stages when deposition of amyloid is underway, but not so far along that neurodegeneration has ensued.

In 2011, the National Institute on Aging and the Alzheimer’s Association (NIA-AA) proposed modifications to the diagnostic criteria for AD, which included a category called “preclinical AD,” subdivided into three stages based on biomarker findings (Figure 1): stage 1 is defined by the presence of amyloid, evidenced using PET imaging or a CSF analysis; stage 2 by the presence of amyloid plus markers of neurodegeneration, indicated by hypometabolism on fluorodeoxyglucose positron emission tomography (FDG-PET), elevated CSF tau or phospho-tau, or structural MRI findings of hippocampal atrophy or cortical thinning; or stage 3, where in addition to amyloidosis and neurodegeneration, there is evidence of subtle cognitive decline (14). Subsequent refinement of the criteria added two additional preclinical stages: stage 0, where all biomarkers are normal and there is no cognitive impairment; and suspected non-Alzheimer’s pathophysiology, i.e., markers of neurodegeneration but not amyloidosis (SNAP) (15).

Figure 1. N.I.A classification (adapted from Sperling)

Vos et al. used these criteria to classify 311 cognitively normal (CDR 0) subjects living in the community. They found that 41% were classed as stage 0, 15% as stage 1, 12% as stage 2, 4% as stage 3, 23% as SNAP, and 5% remained unclassified. They also determined the 5-year progression rate to symptomatic AD (CDR ≥ 0.5). Only 2% of stage 0 subjects progressed, whereas, 11% of stage 1 subjects, 26% of stage 2 subjects, 56% of stage 3 subjects, and 5% of SNAP subjects progressed (16). These data support the temporal order of biomarkers proposed Jack et al, and its relevance for clinical progression (12, 13), in particular that amyloid accumulation begins in the preclinical stage of the disease and that this could be the appropriate time to intervene with anti-amyloid therapies.

Johnson et al used florbetapir PET imaging to assess amyloid load in healthy controls, demonstrating that the mean SUVR increases with age even among cognitively normal subjects, from 5.3% positive in those aged 50 to 59, 10.5% in those 60-69, 15.0% in those 70-79, and 33 % in those 80 years or older (17). These results suggest that it may be possible to enroll subjects based on the presence of brain amyloid (by CSF or amyloid PET), with no objective cognitive decline, possible subjective memory complaints, and preserved activities of daily living. The advantages of such a trial targeting amyloid are its specificity and the ability to treat at a very early stage before non-reversible lesions have developed. The drawbacks are the difficulty of demonstrating a slowing of cognitive decline in an already slowly-declining population and the low conversion rate to dementia. These drawbacks result in long and costly trials.

Nonetheless, a trial based on this strategy already started this year. The Anti-Amyloid Treatment in Asymptomatic Alzheimer’s (A4 trial) will be the first prevention trial in subjects determined to be at risk based on brain amyloid demonstrated with PET imaging (18). This placebo-controlled trial will use solanezumab as the treatment and a composite of well-validate neuropsychological tests known to be sensitive in the early stages of cognitive decline as the primary outcome.

The A4 trial aims to exclude older persons without cognitive impairment who, based on the absence of brain amyloid, are much less likely to develop AD. Overall, in our point of view, amyloid PET or CSF seems to be best for selecting trial participants. As tau PET imaging continues to develop, it may be useful for assessing disease-stage and perhaps response to treatment.

We must underline, however, that such trials are expensive and raise cost effectiveness issues. It will be hard to use such treatment for very long period of time, e.g., decades. There are also ethical concerns raised by treating individuals who may never develop AD with drugs that have unknown long-term safety profiles, particularly in people who may develop other chronic diseases. Moreover initiating treatment based on biomarker findings has the potential to affect the life and well-being of subjects. For example, when we detect amyloid and propose treatment in still-normal older adults, we will likely induce stress and other life-altering decisions, which must be taken into consideration. While we hope to prevent the development of AD in some older people, we must realize that not all would have gone on to develop AD and that many other diseases can also occurs at this age.

Other trials targeting the preclinical stages of AD have also begun enrolling subjects. These trials – conducted by the Alzheimer’s Prevention Initiative (API) (19) and the Dominantly Inherited Alzheimer’s Network Trials Unit (DIAN-TU) (20) are targeting individuals with autosomal dominant mutations that make them almost certain to develop early-onset AD (EOAD). A third trial, also by API, will target ApoEε4 carriers, who are at elevated risk of developing late onset AD (LOAD). All of these trials will test the efficacy of active immunotherapeutic agents.

Another possible target population for preventive trials is late MCI due to AD. Individuals at this stage have objective decline in memory, for instance evidenced by low scores in logical memory testing, and a positive amyloid signature (CSF or amyloid-PET), but generally preserved activities of daily living. The advantage of targeting this population is the fact that they have a higher likelihood of converting to AD and, because they are already symptomatic, are more likely to comply with the study protocol. However, they are difficult to screen due to the low prevalence and the high cost of screening with imaging, CSF biomarkers, or extensive cognitive testing. Moreover the cut-off at which cognitive impairment represents MCI is still unclear, and is impacted by education, sleep, general health, life events, and other factors. Finally the learning effect must be taken into consideration in this population when a repetitive test is used as a screening tool. In the GuidAge trial, for example, the learning effect with the free and cued selective reminding test (FCSRT) was observed over 2 years both in subjects with CDR 0 and CDR 0.5 (personal data). CDR-SB may be a reasonable end point in late MCI, (ADNI and MAPT personal analysis), while in those with early MCI, a composite score appears to be more appropriate. Recently a composite score composed of tests for Word Recall, Delayed Word Recall, Orientation, the CDR-SB, and the FAQ was proposed (21).

Several drugs with varying mechanisms of action have been, or plan to be, used in preclinical, MCI, and AD trials. A phase 3 trial of the gamma-secretase inhibitor semagacestat was tested in patients with mild-to- moderate AD but did not improve cognition and, in fact, was associated with a worsening of functional abilities among those receiving a high dose of the drug. There were also more adverse side effects among those receiving drug compared to placebo (5). Beta-secretase inhibitors may be more efficacious (22). Despite evidence of hepatic toxicity with some of these compounds, at least one (MK-8931) is recruiting subjects for a phase 3 study. Other approaches include monoclonal antibodies such as solanezumab and gantenerumab, which are also in phase 3 studies. Less advanced molecules targeting alpha secretase or tau protein, as well as neuroprotective compounds still in early development. Some studies have been terminated, for example a study of the microtubule stabilizer epothilone D.

Table 1 summarizes prevention trials in MCI due to AD and prodromal AD currently underway. However, questions remain about whether treating at these stages is too late. While it makes sense to treat before neurodegeneration begins, the progression of the disease is still slow in prodromal AD and MCI, suggesting that there may be some benefit to treating at these stages.

Table 1. Drug trials


Alzheimer Prevention Trials: Larger Target, Non Specific but Multi-Domain Intervention, Long-Term Exposure

An alternative approach for prevention trials is to have a larger more diverse population group with long-term exposure to non-specific, multi-domain intervention. The rationale for this approach stems from studies showing that several environmental factors are associated with the risk of developing dementia. These factors may include educational level, vascular and metabolic risk factors, physical activity, cognitive stimulation, and nutritional status. In addition, recent studies suggest a declining incidence and prevalence of AD over the last ten years, thought to be due to improvements in overall health and educational levels (23, 24). Finally, a recent autopsy study of 1599 older people compared amyloid deposition in subjects 65 yrs. and older who died between 1972 and 2006. Lower amyloid deposition was seen in the 2006 cohort and was particularly marked in the oldest age groups, providing preclinical evidence supporting recently described decreases in AD incidence (25). These accumulating data recently led the U.S. National Institute on Aging (NIA) to encourage all adults to exercise regularly, eat a healthy diet rich in fruits and vegetables, engage in social and intellectual activities, control type 2 diabetes, lower high blood pressure, lower cholesterol levels, maintain a healthy weight, stop smoking, and get treatment for depression.

Targeting the general population for interventional AD prevention trials may not be feasible or even desirable, although there is the potential to promote more informed decision-making by the general public on low-risk approaches that could improve brain health and reduce the risk of dementia (26). In addition, it may be possible to identify healthy adults at high risk of AD and likely to benefit from intervention based on subjective memory complaints (SMC, also called subjective memory impairment [SMI] or subjective cognitive impairment [SCI]), ApoEε4 carriage, family history of AD, or the presence of frailty. Multidomain interventions may compensate for low specificity in these populations.

We would like to propose two specific approaches, targeting 1) those with subjective memory complaints, and 2) physically and cognitively frail older adults.

Individuals with SMC have, by definition, no objective cognitive decline and preserved activities of daily living (ADL). The prevalence has been estimated at between 11% in 65-85 year olds (27) to over 88% in those over age 85 (28), and some studies have suggested that the presence of SMC may predict subsequent dementia (29). Progression to dementia among those with SMC is elevated in individuals with a family history of dementia, expressed concern about memory, onset over the previous 5 years, and when the concern is severe enough to motivate consultation with a primary care provider (PCP)

The advantages of targeting individuals with SMC are that there are large numbers of potential subjects who are relatively easy to identify through PCPs, and that engaging PCPs in the process may increase compliance. Disadvantages of this approach include very high heterogeneity, slow decline, and minimal conversion to MCI or AD. The endpoint for a trial in this population could be a composite score including measures of logical memory or the FCSRT and measures of executive function.

Another large population that could be targeted for non-specific multi-domain trials are older persons with physical and/or cognitive frailty. In the longitudinal Rush Memory and Aging study, frailty was shown to be associated with both cognitive decline and incident AD (30). Indeed, the definition of frailty — increased vulnerability resulting from decline across multiple physiologic systems (31) — has recently been expanded to include cognitive decline (32). In 2013, a consensus group organized by the International Academy on Nutrition and Aging (IANA) and the International Association of Gerontology and Geriatrics (IAGG) proposed a definition of cognitive frailty that includes both physical frailty and cognitive impairment (e.g., CDR 0.5) in the absence of dementia (33). Individuals meeting this definition are typically 80 yrs. or older with preserved basic ADLs, but some decline in instrumental ADL (IADLs), due mostly to physical frailty.

The advantages of targeting frail older adults for multi- domain prevention trials include the importance of intervening and potentially slowing or reversing the frailty syndrome, the large numbers of persons affected, and the ability to target these individuals through PCPs. Disadvantages include the broad heterogeneity and presence of multiple morbidities within this population and the likelihood of poor compliance to intervention. In addition the neurobiology of frailty has yet to be defined. Endpoints of a study in this population could include both physical (e.g. gait speed) and cognitive functions (memory plus executive functions)

Multi-domain prevention trials in these two populations should include both pharmacological and non-pharmacologic therapies. Possible pharmacotherapies include anti-diabetic drugs such as pioglitazone. This drug is used in the oncoming TOMMORROW trial (34), which will enroll subjects based on their APOE and TOMM40 genotype. Other agents that could be considered for prevention trials include insulin (35), selective serotonin receptor agonists (SSRIs) (36), and a variety of nutrients such as resveratrol (37), the Ginkgo biloba extract EGB 761 (38), Vitamin D (39), B vitamins, including folate (40), and omega-3 fatty acids (41).

Omega-3 fatty acids (ω-3) are poly-unsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) that are found in cold- water fish and fish oil and have been associated, in a number of epidemiologic studies, with a reduced risk of dementia (42). Based on the results of a pilot study (43), the NIA has funded a study to test the potential of ω-3 PUFAs to prevent vascular cognitive impairment. This 3- year, randomized, placebo-controlled trial of ω-3 PUFA will enroll 150 subjects age 80 and older with a CDR ≤ 0.5 (non-demented), low plasma ω-3 PUFA, and white matter hyperintensities (WMH) on MRI scans. Outcome measures include progression of WMH, progression of blood-based markers of inflammation, and cognitive decline (executive function and processing speed.

Physical exercise has been studied extensively in recent trials and found to be related to improvement of cognitive function (44), decreased MRI hippocampal brain atrophy (45), improved brain metabolism and some in amyloid deposit (ref…). Cognitive stimulation has been largely shown to improve cognition (46) and lower amyloid burden (47) in older adults.

Multi-domain intervention aims to bring together the benefits of nutritional intervention, physical exercise, cognitive stimulation, social activities, and vascular and metabolic risk control to increase the effect of each intervention, reach a threshold, and achieve clinically significant effects. The first and largest trial to have been designed is the Multi-domain Alzheimer’s Prevention Trial (MAPT) (48), a randomized, placebo-controlled study of 1680 subjects, 70 years of age or older living in the community and presenting with SMC (99% of subjects). The cohort was enriched for frail subjects with slow walking speed (4 meters test) in 11.9% of the sample and limitation in one IADL in 11.2%. Demented patients as well as those dependent for basic ADL were excluded. Subjects were randomized into four treatment rms: Omega 3 alone, Placebo alone, Omega 3 plus multi- domain intervention, and Placebo plus multi-domain intervention. The multi-domain intervention included physical and cognitive exercises, dietary counseling and weight maintenance, increased social activities, control of vascular and metabolic risk factors, and correction of vision and hearing impairments. The length of the intervention was 3 years plus 2 years of observational follow-up. Outcome measures included cognitive decline using the FCRST, cerebral and hippocampal volumes (n=500), cerebral glucose metabolism using FDG-PET (n=68) and amyloid PET scanning with florbetapir (n= 271).

The MAPT multi-domain intervention was shown be feasible with good compliance demonstrated by only 22.5% drop outs over the 3-year study. The MAPT trial is now completed and results will be released shortly.

Other multi-domain trials include the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER Study), a 2-year interventional trial targeting 1200 subjects at risk for dementia (49), the Prevention of Dementia by Intensive Vascular Care (Pre- DIVA) trial, the Vitamin D3, Omega-3, Home Exercise Healthy Ageing and Longevity Trial (DO-HEALTH), and the Healthy Aging through Internet Counseling of the Elderly (HATICE) program (50). Results presented from the FINGER study at the 2014 Alzheimer’s Association International Conference (AAIC) indicate positive effects on cognitive function (51). The Pre-Diva, MAPT and FINGER trials have been brought together under the umbrella of the European Dementia Prevention Initiative in order to share data and collaborate on new studies.

The advantages of multi-domain trials with large population targets and non-specific but multi-domain intervention delivered over a long time period are that these interventions are likely to be less expensive, easier to implement in daily clinical practice or at the population level, and safe for long-term exposure; and may act on different therapeutic targets. The disadvantages are interventions themselves are non- specific, the cohorts are highly variable and with different risks of developing dementia, and the potential for low compliance with the study protocol. For example, with regard to the variability in risk of dementia, amyloid PET scans performed in 271 subjects enrolled in the MAPT trial showed that 38.0% had significant brain amyloid (cortical SUVR > 1.17). Moreover these individuals were found to have lower cognitive function at baseline and more cognitive decline over the trial period, similar to what has been seen in observational studies (48).

In fact, these two preventive approaches: targeting a specific population with a specific intervention or targeting a larger at risk population with a multi-domain intervention are complementary and may both be appropriate at different time-points over the life time of an older adult. Indeed, this may be what is required in future clinical practice.


The future of clinical practice: A preventive approach, integrated in primary care setting that begins with longitudinal monitoring of memory function in a general population to identify decliners, followed by a specific intervention based on biomarkers and discussed case by case if the disease progress (FIG 2)

A prevention approach could start by making general recommendations to a large, diverse population (e.g., those age 50 years or older with normal cognition) on diet, physical and cognitive exercise, and risk factor control; then identify decliners through longitudinal monitoring of biomarkers or cognitive markers; and finally test interventions targeted specifically. These preventive approaches must start in primary care settings and integrate the family practitioners.

Among those with SMC and/or a family history of dementia, a tailored multi-domain intervention might be proposed, including nutrition, physical and cognitive exercise, and risk factor control, such as was used in the MAPT or FINGER trials. Ideally, these interventions could be delivered by PCPs who, at the same time, could begin longitudinal monitoring of cognition as a way to identify decliners for the next level of prevention trials. Some web resources will be probably helpful in the near future, such as the Brain Health Registry (

If subjects with early MCI, biomarkers (e.g., CSF amyloid, tau, as well as PET scans) may be considered despite the fact that they are expensive and invasive. Plasma biomarkers would greatly enhance the ability to conduct large, longitudinal progression studies. A recent study identified 10 plasma proteins that are strongly associated with structural MRI findings and appear to be able to predict conversion from MCI to AD with an accuracy of 87%, sensitivity of 85%, and specificity of 88% (52). In another study, a set of ten peripheral plasma phospholipids were identified that predicted conversion to MCI or AD over a 2–3 year timeframe with over 90% accuracy (53). However these findings have to be replicated and their clinical utility validated. At that point, it should be possible to offer multi-domain interventions to those who are biomarker-negative and oral drugs such as anti-amyloid drugs to those who are biomarker positive or those who transition to biomarker positivity during the trial. However, as mentioned earlier, there are those who believe that the MCI stage is too late to begin treatment with anti-amyloid therapy and that what is needed is better characterization of the transition from amyloid negativity to amyloid positivity. Anti-amyloid monoclonal antibodies will be also probably useful if the ongoing clinical trials of these drugs are successful.

Figure 2. A preventive approach, integrated in primary care setting that begins with longitudinal monitoring of memory function in a general population to identify decliners, followed by a specific intervention based on biomarkers and discussed case by case if the disease progress

If the disease progress to late MCI/prodromal AD stages of the disease, new therapies are needed, such as those that target tau; or it may be necessary to use combination therapies that simultaneously act on multiple therapeutic targets (e.g., amyloid plus tau). Anti- amyloid monoclonal antibodies may also be useful in these patients, depending on the outcome of ongoing studies of these agents. However these results will have to be scrutinized closely to assess the real impact of such therapies (54).

Comments and Research Directions

To achieve our goal of preventing AD, changes are needed in the way clinical trials are designed and conducted:

  • Clinical trials must target specific populations according to the intervention.
  • The interventions need to be robust and appropriate for the targeted population, e.g., a multi-domain intervention for a large heterogeneous population vs. an intervention with a specific mechanism of action for targeted populations.
  • Trials must be of sufficient length to assess long-term exposure to intervention
  • Trials will need to be implemented in clinical settings, beginning with the involvement of general practitioners and other primary care providers. These providers are in an ideal position to monitor longitudinally the cognition of elderly patients and identify decliners who can then be referred for more intensive assessment of progression using biomarkers.
  • Trial designs are needed that will enable the testing of combined approaches, including multi-domain approaches as well as combined pharmacotherapy against multiple therapeutic targets.

Moreover, preventing AD will require new and improved infrastructure. In the war against AD we need not only new weapons (drugs) but also the fleet (infrastructures, clinical research facilities) and the network (international collaborations). We must also learn from our failures to obtain clinically relevant and innovative results


Acknowledgment: Aisen P (ADCS-UCSD, San Diego CA, USA), Andrieu S (INSERM U 1027, Toulouse, France) Bain L (Elverson PA, USA), Delrieu J (Inserm U 1027, CHU, Toulouse, France), Ousset. P.J (INSERM U 1027, CHU Toulouse, France), Weiner. M (UCSF, San Francisco CA, USA). This paper was presented as a key-note lecture at AAIC 2014 in Copenhagen.

Conflicts of Interest: Scientific Board Member: Lilly, MSD, Nestlé, Roche, Sanofi. Research Grants: Abbvie, Affiris, Avid, Eisai, Envivo, Exhonit, Genentech, GSK, Lilly, MSD, Nutricia, Otsuka, Pharnext, Pfizer, Pierre-Fabre, Régénéron, Roche, Sanofi, Servier, TauRx Therapeutics, Wyeth.




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