Joanna L. Bowtell,a Zainie Aboo-Bakkar,a Myra E. Conway,b Anna-Lynne R. Adlam,c Jonathan Fulfordd

aSport and Health Sciences, University of Exeter, St Luke’s Campus, Heavitree Rd., Exeter, EX1 2LU, United Kingdom.

bDepartment of Applied Sciences, University of the West of England, Bristol, United Kingdom.

cPsychology Department, University of Exeter, EX4 4QG, United Kingdom.

dMedical School, University of Exeter, EX1 2LU, United Kingdom.

Received October 4, 2016. Accepted February 10, 2017.

Applied Physiology, Nutrition, and Metabolism, 2017, 42(7): 773-779, https://doi.org/10.1139/apnm-2016-0550

Blueberries are rich in flavonoids, which possess antioxidant and anti-inflammatory properties. High flavonoid intakes attenuate age-related cognitive decline, but data from human intervention studies are sparse. We investigated whether 12 weeks of blueberry concentrate supplementation improved brain perfusion, task-related activation, and cognitive function in healthy older adults. Participants were randomised to consume either 30 mL blueberry concentrate providing 387 mg anthocyanidins (5 female, 7 male; age 67.5 ± 3.0 y; body mass index, 25.9 ± 3.3 kg·m −2 ) or isoenergetic placebo (8 female, 6 male; age 69.0 ± 3.3 y; body mass index, 27.1 ± 4.0 kg·m −2 ). Pre- and postsupplementation, participants undertook a battery of cognitive function tests and a numerical Stroop test within a 1.5T magnetic resonance imaging scanner while functional magnetic resonance images were continuously acquired. Quantitative resting brain perfusion was determined using an arterial spin labelling technique, and blood biomarkers of inflammation and oxidative stress were measured. Significant increases in brain activity were observed in response to blueberry supplementation relative to the placebo group within Brodmann areas 4/6/10/21/40/44/45, precuneus, anterior cingulate, and insula/thalamus ( p < 0.001) as well as significant improvements in grey matter perfusion in the parietal (5.0 ± 1.8 vs –2.9 ± 2.4%, p = 0.013) and occipital (8.0 ± 2.6 vs –0.7 ± 3.2%, p = 0.031) lobes. There was also evidence suggesting improvement in working memory (2-back test) after blueberry versus placebo supplementation ( p = 0.05). Supplementation with an anthocyanin-rich blueberry concentrate improved brain perfusion and activation in brain areas associated with cognitive function in healthy older adults.

We hypothesised that cerebrovascular function will improve in response to blueberry supplementation in healthy older adults due to favourable changes in redox balance and hence NO bioavailability. To test this hypothesis, resting cerebral perfusion and task-related brain activation were measured in healthy older adults before and after 12 weeks of supplementation with placebo or blueberry concentrate providing 387 mg·d −1 anthocyanins. Cognitive function was assessed as a secondary outcome, although the study was not sufficiently powered to detect changes in cognitive performance.

Acute and chronic supplementation with fruit polyphenols has also been shown to improve peripheral vascular function. Rodriguez-Mateos and colleagues (2013) showed that a range of blueberry polyphenol doses (0.3–1.88 g polyphenols) acutely increased flow-mediated dilatation in healthy men peaking at 1 h postingestion. A number of studies have also found improvements in endothelial-dependent vasodilatation after chronic supplementation with fruit polyphenols, especially amongst study populations with impaired cardiovascular function ( Coimbra et al. 2005 ; Chaves et al. 2009 ; Poreba et al. 2009 ; Khan et al. 2014 ). Despite this evidence of improved peripheral vascular function after either acute or chronic supplementation with fruit flavonoids, the chronic effects of fruit polyphenol supplementation on cerebral perfusion have not yet been investigated.

Acute and chronic supplementation with cocoa polyphenols enhances endothelium-dependent vasodilation measured as brachial artery flow-mediated dilatation ( Hooper et al. 2012 ). Local perfusion of brain tissue is controlled by the neurovascular unit comprising neurons, astrocytes, pericytes, and endothelial cells. Therefore, endothelial cell function plays an important role in regulating cerebral blood flow through the release of vasoactive substances including nitric oxide (NO), whilst microvascular pressure is maintained through dilation of the larger upstream arteries via endothelial dependent mechanisms and vasomotor responses (for review see Cipolla 2009 ). It is plausible, therefore, that interventions that enhance peripheral vascular function may also improve cerebral perfusion and hence cognitive function. In support of this hypothesis, Sorond et al. (2013) found a strong correlation between neurovascular coupling and cognitive function in elderly participants with vascular risk factors, both of which were improved after 30 days of high flavanol cocoa consumption ( Sorond et al. 2013 ).

In a meta-analysis, flavonoid supplementation relative to a placebo control group improved cognitive performance in 9 of 15 randomised controlled trials ( Macready et al. 2009 ). Subsequent studies found that 12 weeks of wild blueberry juice supplementation (∼7 mg anthocyanins per kg −1 ·d −1 ) improved memory function (paired associative learning and word list recall) in adults with mild cognitive impairment ( Krikorian et al. 2010 ). Additionally, consumption of high flavanol cocoa (≥900 mg·d −1 ) improved cognitive function in healthy older adults after 8 weeks in comparison to a low flavanol cocoa supplement ( Mastroiacovo et al. 2015 ). Brickman et al. (2014) found that 12 weeks of high flavanol cocoa consumption enhanced task-related activation of the dentate gyrus and associated cognitive function. Similarly, 4 weeks of supplementation with pomegranate, which are rich in the polyphenols from the elligitannins family, increased task-related brain activation and cognitive function in healthy older adults ( Bookheimer et al. 2013 ). Most recently, 8 weeks of consumption of flavanone-rich orange juice improved global cognitive function in healthy older participants in a crossover randomised controlled trial ( Kean et al. 2015 ). The mechanisms most likely to be responsible for these effects include interaction with signalling cascades responsible for neurogenesis, synaptic plasticity, and neuronal repair. In addition, polyphenol supplementation may increase antioxidant capacity and hence improve redox balance and vascular function as well as reduce neuroinflammation (for review see Rendeiro et al. 2015 ).

Sample size calculations were based on change in region of interest-based fMRI signal with a population SD (<0.5%) ( Zandbelt et al. 2008 ), although accurate calculations were hampered by the limited available data on the effects of blueberry polyphenols on fMRI. A sample size of 12 participants per group was estimated based on a moderate effect size of 0.5 with 80% power and α level of 0.05. Data are reported as mean ± SEM for baseline and postsupplementation. Cognitive function, cerebral perfusion (ASL), as well as serum BDNF and CRP concentration data were analysed by 2-way mixed model ANOVA (treatment vs time) to determine whether there were any statistically significant effects of time or treatment. The changes in cognitive performance and fMRI data from pre- to postsupplementation were compared across groups using 2-tailed independent sample t tests with equal variance.

The assessment of brain activity during the cognitive task was undertaken by monitoring alterations in local blood delivery characteristics linked to task performance relative to that associated with the fixation cross by examining changes in image signal intensity. Analysis was undertaken using SPM8 software (The Wellcome Department of Cognitive Neurology, University College, London) a suite of MATLAB functions and subroutines. Preprocessing included slice time correction, spatial processing to correct for head movement and size, and warping to the Montreal Neurological Institute template (MNI305). Images were then convolved with a 3D Gaussian filter with an 8 mm full-width-at-half-maximum. The fMRI data were analyzed based on mass univariate (voxel-by-voxel) testing within the general linear model framework over the whole brain, treating each participant separately and constructing individual maps comparing the differences in response between the 2 visits. Statistical testing took place examining the differences in group responses for overall activation, size of hemodynamic response, and timing of hemodynamic activation with significant brain activation defined as arising within a region where the differences between groups in signal intensity or the time to signal peak gave rise to a p value <0.001 after no corrections had been made for multiple comparisons and the cluster size of the activated region was equal or greater than 10 voxels. For ASL analysis, ASL images were initially registered to the structural images within the FSL software package (FMRIB Software Library v5.0, Oxford University, UK) after which the structural images were registered into MNI space and the generated warping matrix applied to the ASL data so it was also present within MNI space. Difference images were obtained from the control-tag pairs followed by averaging over the 30 acquisitions at each delay time, such that a single difference image was obtained for each delay time. Quantitative perfusion calculations were then undertaken using the BASIL toolbox (Bayesian inference for arterial spin labelling MRI) ( Chappell et al. 2009 , 2010 ) within FSL. Regions of interest corresponding to the grey matter of the parietal, frontal, and occipital lobes were defined based upon the MNI structural atlas and the average perfusion values within each region determined.

All MRI was undertaken in a 1.5 T Philips Gyroscan scanner with an 8-element head coil. Within the head coil a mirror assembly was mounted such that with their head within the coil participants were able to see a screen at the end of the scanner bed onto which visual-based cognitive tasks could be projected. The assessment of brain activity during a cognitive task was undertaken using a standard single shot echo-planar dynamic imaging sequence (repetition time = 3 s, echo time = 45 ms, resolution 2.5 mm × 2.5 mm × 3.5 mm, 39 contiguous transverse-oblique slices, field of view 230 mm × 230 mm, 64 × 64 within-plane matrix, 220 dynamics) while undertaking a numerical Stroop test ( Kaufmann et al. 2008 ) (144 trials) where image presentation was alternated with the presentation of a fixation cross. This was followed by the acquisition of a high-resolution whole-brain T1-weighted anatomical scan (resolution 0.9 mm × 0.9 mm × 0.9 mm) and a pseudo-continuous arterial spin labelling (ASL) sequence to assess any modifications in brain perfusion at rest, consisted of labelling tag of 1800 ms applied to the carotid artery 20 mm inferior to a stack of 14 acquisition slices covering the whole brain. Data were recorded for 5 different delay times (400, 800, 1200, 1600, 2000 ms) with 30 pairs of control-tag images acquired for each delay time.

For protein carbonylation detection, serum proteins were denatured using 12% sodium dodecyl sulfate and derivatised using 1X 2,4-dinitrophenylhydrazine solution at room temperature for 15 min and neutralised. Carbonylated proteins were subsequently detected using Western blot analysis (antidinitrophenylhydrazine: 1/2000 and anti, glyceraldehyde 3-phosphate dehydrogenase: 1/1000, respectively) as described in Hull et al. (2015) ; 4-hydroxynonenal was detected using anti-hydroxynonenal (1/500) and secondary anti-rabbit (1/2000). Serum glutathione was assayed using a commercially available reduced glutathione/glutathione disulfide ratio detection assay kit (Abcam, Cambridge, Mass, USA). Serum samples were also analysed for brain-derived neurotrophic factor (BDNF) by commercially available enzyme-linked immunosorbent assay (Chemikine, Darmstadt, Germany) and for C-reactive protein (CRP) using a turbidometric assay.

The cognitive task undertaken while acquiring functional MRI (fMRI) data was a numerical Stroop test ( Kaufmann et al. 2008 ), chosen to challenge attention and memory. The task was selected based upon its independence on knowledge, training, practice, or educational level, particularly in terms of language or mathematical ability. In addition, the task only required the pressing of a single button of a pair of MRI-compatible response boxes (Cedrus, San Pedro, Calif., USA); one button was held in each hand to ensure ease of response within the scanner environment. The Stroop task involved the presentation of a series of single-digit number pairs shown side by side with participants requested to press the button held in the same hand as the side of the screen the larger number of the pair appeared. The definition of ‘larger’ as applied to the number pairs varied during the course of the experiment, with classification based upon either numerical value or physical size, and the experiment was split up into blocks with each block beginning with the presentation of a classification guide: either the word ‘Numerical’ or ‘Physical’ being presented indicating which parameter should be considered when deciding which number was ‘bigger’. The test was run using E-Prime version 2 software (Psychology Software Tools Inc., Sharpsburg, Penn., USA) with participant responses recorded allowing an assessment of accuracy and reaction times. In all cases participants practiced the task once they had been positioned within the scanner to ensure full familiarity with the test, the environment, and the method of responding. Responses were monitored and the test proper not begun until participants were consistently responding correctly.

The battery of tests (CogState Ltd.) selected for the present study took approximately 35 min to complete and assessed a range of cognitive domains that have previously been shown to be affected by polyphenol supplementation: psychomotor function, visual processing, executive function, verbal and spatial memory, and working memory (for review see Lamport et al. 2014 ). Specifically the tests comprised a detection task (has a card turned over, reaction time) to assess psychomotor function; the Groton maze timed chase test (chasing a visual target, moves per second) to assess speed of visual processing; the Groton maze learning test with a delayed recall component (find a hidden pathway through the maze and then recall after a delay, number of errors and duration) to assess executive function and delayed recall; identification task (is a card red, reaction time) to assess attention; international shopping list task with delayed recall (learn and recall shopping list items, number of correct responses) to assess verbal learning and delayed recall; and 1-back and 2-back memory tasks (is the card the same as 1 or 2 back, reaction time and correct responses proportion) to assess working memory. The speed and accuracy of responses were quantified.

Participants completed a battery of cognitive function tests (CogState Ltd., New Haven, Conn., USA) in a quiet room, after which a venous blood sample was taken prior to completing MRI testing. Participants were subsequently given 12 × 210 mL bottles of the appropriate liquid supplement that were identical in appearance for placebo and blueberry conditions and instructed to take a 30 mL dose diluted to 240 mL total volume with tap water every morning for the following 12 weeks. A measuring cup was provided for this purpose. Participants returned 12 weeks later to repeat all measurements, and they returned the bottles to allow assessment of compliance with the supplementation regime.

Thirty millilitres of blueberry concentrate were consumed once per day for 12 weeks and provided 387 mg anthocyanidins (34 mg malvidin, 108 mg cyanidin, 41 mg pelargonidin, 63 mg peonidin, 86 mg delphinidin, and 55 mg petunidin; analysed by high performance liquid chromatography; Chandra et al. 2001 ) and 25.5 g carbohydrate. The placebo was a synthetic blackcurrant and apple cordial (Robinsons cordial, Britvic Ltd., Hemel Hempstead, UK) with sugar added to match blueberry energy content. Participants and researchers were unaware of the condition to which they were randomized, and blinding was maintained since participants were informed that the aim of the study was simply to test the effects of fruit concentrates on brain function.

Participants were pair-matched for ACE-III score and randomised to a group, with participants and investigators blind to treatment. Measurements were taken before and after 12 weeks of supplementation with either BlueberryActive (CherryActive Ltd., Sunbury, UK) or an isoenergetic placebo. Participants were instructed to consume their habitual diet throughout.

Twenty-six participants ( Table 1 ) completed this double-blind randomized controlled trial, which was approved by the Sport and Health Sciences Ethics Committee at the University of Exeter and performed in accordance with the Declaration of Helsinki. At an initial visit, after providing their written informed consent, participants completed the Addenbrooke’s Cognitive Examination III questionnaire (ACE-III, version A), and magnetic resonance imaging (MRI) safety screening questionnaire to ensure their suitability for the trial prior to completing the cognitive function tasks to familiarize themselves with the test procedures. The ACE-III questionnaire, developed at Neuroscience Australia, assesses 5 cognitive domains: attention, memory, verbal fluency, language, and visuospatial abilities. The test took approximately 15 min to administer, and scoring was performed after the visit following the validated protocol ( Hsieh et al. 2013 ). A cut-off score of 88/100 was adopted to indicate cognitive impairment as recommended for a research context requiring high sensitivity (1.00) and lower specificity (0.96) ( Hsieh et al. 2013 ). Exclusion criteria were cognitive impairment, any contraindications to MRI, consuming more than 5 portions of fruit per day, and an age of less than 65 years. No participants were excluded on the basis of their ACE-III score.

The percentage change in performance of the 2-back tests showed weak evidence for improvement in the blueberry versus placebo groups (reaction time: placebo: 0.4 ± 0.4% vs blueberry: –1.0 ± 0.7%; p = 0.09; accuracy: placebo: –3.8 ± 2.5%; blueberry: 3.6 ± 2.7%; group by time interaction effect: p = 0.05). The change in performance for the other cognitive function tests were not significantly different between placebo or blueberry supplementation.

Performance of the Groton maze learning task (accuracy, p = 0.005), international shopping list task ( p = 0.002), and international shopping list with delayed recall ( p = 0.004, Table 2 ) improved over time, but there was no significant difference in this improvement among groups. Performance of the 1-back test tended to improve to a greater extent in the blueberry group but this was not statistically significant (speed, p = 0.094).

There was a significant decrease in serum glutathione concentration in both conditions (placebo: from 73.7 ± 3.6 to 64.4 ± 2.6; blueberry: from 70.0 ± 4.0 to 65.1 ± 3.6 μmol·L −1 ; main time effect p < 0.001), which tended to be smaller in the blueberry condition (placebo: –11.7 ± 2.8%; blueberry: –6.5 ± 2.4%; p = 0.09). However, an overall change in protein carbonylation HNE adduct or malonaldehyde formation between these groups was not evident (data not shown). There was no significant change over time in either serum hsCRP (placebo: 1.7 ± 0.6 to 1.4 ± 0.4; blueberry: 1.4 ± 0.5 to 1.4 ± 0.4 mg·L −1 ) or BDNF (placebo: 103.7 ± 10.1 to 98.8 ± 9.0; blueberry: 88.6 ± 7.3 to 97.4 ± 9.6 ng·mL −1 ) concentration, nor was there any difference between conditions.

No significant performance differences were seen between pre- versus postsupplementation visits or between groups for the number of correct responses while undertaking the numerical Stroop test. Over all cases, group accuracy percentages ranged between 98.1% and 98.8%. However, significant increases in brain activation responses were found in a number of task-associated regions following blueberry supplementation compared with placebo relative to the baseline visits (Brodman areas 4, 6, 10, 21, 40, 44, 45, precuneus, anterior cingulate, insula and thalamus, all p < 0.001, Fig. 1 ). In contrast, no significant increases in brain activity were observed following placebo compared with blueberry supplementation relative to baseline.

In this paper Top of page Introduction Materials and methods Results Discussion « References

Discussion

Chronic supplementation with blueberry concentrate providing 387 mg anthocyanins per day exerted favourable effects on cerebrovascular and cognitive function in healthy older adults. Specifically, resting-state perfusion in the gray matter of the parietal and occipital lobes and brain activation in a number of task-related different areas increased from baseline levels after 12 weeks of blueberry supplementation but not after placebo supplementation.

Improvements in task-related brain activation may reflect either increased cognitive effort due to better focus upon the numerical Stroop task or greater increases in task-related blood flow. Two other fruit polyphenol intervention studies also found increased task-specific brain activation after supplementation. Bookheimer et al. (2013) found that 4 weeks of supplementation with pomegranate juice increased verbal memory and task-related brain activation during verbal and visual memory tasks in healthy older adults. In addition, Krikorian et al. (2012) found that activation in right anterior and posterior cortical regions was increased when performing n-back tests during fMRI after 16 weeks of Concord grape juice supplementation (260 mg anthocyanins per day). In both cases, the authors attributed this response to improved vascular function, despite the absence of any direct measures of vascular function or perfusion. Similarly, 12 weeks of high flavanol cocoa consumption in a healthy older adult population improved task-related activation of the dentate gyrus, which is susceptible to aging-related functional deterioration, as well as performance of cognitive tests that rely upon the contribution of dentate gyrus (Brickman et al. 2014). In the present study, we observed increased task-related brain activation and increased resting-state cerebral perfusion in the parietal and occipital lobes after chronic blueberry supplementation. This seems to provide further support for the concept that the increased task-related brain activation observed by us and others (Bookheimer et al. 2013; Krikorian et al. 2012; Brickman et al. 2014) after chronic polyphenol supplementation can be attributed to improved cerebrovascular function.

Acute consumption of blueberry polyphenols (0.3–1.88 g) has been shown to increase endothelium-dependent vasodilatation in the brachial artery, with the response peaking 1 h after ingestion (Rodriguez-Mateos et al. 2013). In addition, a number of studies have found that chronic supplementation with fruit polyphenols improves peripheral vascular function, especially amongst study populations with impaired cardiovascular function (Coimbra et al. 2005; Poreba et al. 2009). Most recently, Khan et al. (2014) found that blackcurrant polyphenol supplementation for 6 weeks (815 mg polyphenols including 143 mg anthocyanins per day) improved flow-mediated dilatation in healthy adults consuming 2 or less portions of fruit and vegetables per day. However, the present study is the first to directly measure changes in cerebral perfusion in response to chronic fruit supplements using arterial spin labelling. The mechanism of these effects is likely to be related to improved availability of the potent vasodilator, NO, in the vasculature. There is evidence from in vitro studies that polyphenols induce activation of endothelial NO synthase via signalling through Estrogen Receptor-α via G protein, ERK and PI3K pathways (Chalopin et al. 2010). In addition, polyphenols have been shown to inhibit nicotinamide adenine dinucleotide phosphate oxidase, one of the key sources of superoxide production (Maraldi 2013), and to induce signalling through Nrf2 thus increasing endogenous antioxidant capacity (Ramirez-Sanchez et al. 2013); both of these will preserve NO bioavailability by reduced formation of peroxynitrite from the reaction of NO and superoxide. In the present study, glutathione status declined in both conditions across the 12-week study. Although this decline was slightly attenuated in the blueberry group, serum reduced glutathione status was not increased after blueberry supplementation as hypothesised given that glutathione is a downstream target of the Nrf2-ARE pathway. There was no evidence of reduced oxidative modification of proteins or lipids since serum protein carbonyls, malondialdehyde, and HNE adducts were not differentially affected by blueberry versus placebo. However, blood samples were not collected in the fasted state, and the last supplement dose was consumed at least 24 h prior to measurements, so such effects may no longer be evident at least in the extracellular compartment.

We a priori hypothesised that blueberry-induced increases in cerebrovascular perfusion would result in neurogenesis of brain areas that retain capacity for neurogenesis into adulthood such as the hippocampus. However, serum BDNF concentration, a marker of neural synaptic plasticity that has been associated with long-term memory improvements, was not affected by blueberry supplementation. Again, however, blood samples were not taken in a fasted state which may introduce significant variation and confounding, since feeding status has been shown to affect serum BDNF concentration (Karczewska-Kupczewska et al. 2012). To our knowledge, the acute or chronic effects of fruit polyphenol supplementation on human plasma BDNF have not been previously assessed and may warrant further investigation, especially given that consumption of the Mediterranean diet for 3 years has been shown to elevate BDNF in those with depression (Sanchez-Villegas et al. 2011).

Although Kolehmainen et al. (2012) found that 8 weeks of bilberry consumption, providing 1323 mg·d−1 anthocyanins (∼400 g·d−1), reduced hsCRP in middle-aged men and women with metabolic syndrome; blueberry supplementation in the present study, providing 387 mg anthocyanins per day, did not affect hsCRP concentration. The discrepancy between trials may relate to the restriction of dietary berry consumption by Kolehmainen et al. (2012), the difference in participant characteristics, and the lower dose of anthocyanins in the present study. Although the study was not powered to detect significant effects in cognitive function, consistent with previous studies (Macready et al. 2009; Mastroiacovo et al. 2015), there was some evidence of improvement in processing speed and working memory following blueberry supplementation.

In conclusion, blueberry concentrate consumed once per day (30 mL, providing 387 mg anthocyanins) for 12 weeks increased activation of brain areas associated with cognitive processes including memory and executive function, which tend to deteriorate with age. These effects of blueberry appear to be mediated by improved vascular function as suggested by the improved resting perfusion of gray matter in the parietal and occipital lobes of the brain.