Fruit Polyphenolics and Brain Aging
Nutritional Interventions Targeting Age-related
Neuronal and Behavioral Deficits
RACHEL L. GALLI,a,b BARBARA SHUKITT-HALE,a KURESH A.YOUDIM,a
AND JAMES A. JOSEPHa
aUSDA-ARS,
HNRCA at Tufts University, Boston, Massachusetts 02111, USA
bDepartment
of Psychology, Simmons College, Boston, Massachusetts 02215, USA
ABSTRACT: Nutritional interventions, in this case, increasing dietary intake of
fruits and vegetables, can retard and even reverse age-related declines in brain
function and in cognitive and motor performance in rats. Our lab has shown
that as Fischer 344 rats age their brains are increasingly vulnerable to oxidative stress. Dietary supplementation with fruit or vegetable extracts high in antioxidants (e.g., blueberry, BB, spinach, respectively) can decrease this
vulnerability to oxidative stress as assessed in vivo by examining reductions in
neuronal signaling and behavioral deficits and in vitro via H2O2-induced decrements in striatal synaptosomal calcium buffering. Examinations have also revealed that BB supplementations are effective in antagonizing other agerelated changes in brain and behavior, as well as decreasing indices of inflammation and oxidative stress in gastrocnemius and quadriceps muscles. In ongoing studies we are attempting to determine the most effective BB polyphenolic
components. To date, the anthocyanins show the most efficacy in penetrating
the cell membrane and in providing antioxidant protection. In sum, our results
indicate that increasing dietary intake of fruits and vegetables high in antioxidant activity may be an important component of a healthy living strategy designed to maximize neuronal and cognitive functioning into old age.
KEYWORDS: dietary supplementation; diet; memory; learning; phytochemicals; phytonutrients; free radicals; oxidative stress; inflammation; behavior;
cognitive behavior; blueberry; signal transduction; calcium flux; rats
As humans age they become increasingly likely to display age-related deficits in
cognitive and motor performance, even when there is no underlying disease. One
strategy to maximize the healthy life span and to retard and/or reverse the impairments associated with normal aging is through nutritional interventions. Research
has demonstrated that diets rich in fruits and vegetables are effective at reducing the
Address for correspondence: Rachel L. Galli, Ph.D., Neuroscience Laboratory, USDA-ARS,
HNRCA at Tufts University, 711 Washington St., Boston, MA 02111. Voice: 617-521-2607; fax:
617-556-3222.
rachel.galli@simmons.edu
Ann. N.Y. Acad. Sci. 959: 128–132 (2002). © 2002 New York Academy of Sciences.
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rates of certain types of cancer and cardiovascular disease.1 Our lab has examined
the effects of dietary supplementation with fruits and vegetables on the neuronal and
behavioral decrements seen with aging. To date, supplementing a rat’s diet with a
fruit or vegetable (e.g., blueberry, strawberry, spinach) has slowed and, in some cases, even reversed deficits in brain function, motor performance, and learning and
memory in old animals.2–5
The brain uses a proportionally large amount of the body’s oxygen supply, and
with age its endogenous capacity to fight oxidative stress decreases.6 The free radical theory of aging purports that as the balance between prooxidants and antioxidants shifts in favor of the prooxidants, damage to the brain results in decrements in
neuronal and behavioral functioning.7,8 We have shown that the fruits and vegetables
that are most effective at ameliorating age-related deficits (e.g., signal transduction,
motor performance, and cognitive behavior)2–4 are those highest in antioxidant capacity.9 However, cumulatively the multiple polyphenolic compounds present in
fruits and vegetables have been shown to have additional effects, including antiinflamatory, antiallergic, antiviral, and antiproliferative, which suggests that their impact on central nervous system (CNS) function and on behavioral parameters may
stem from a combination of factors.1,10–13
A number of neuronal mechanisms and behavioral measures have been shown by
us and others to be affected by age in Fischer 344 (F344) rats.5,14–20 Significant decrements occur in neuronal signal transduction, as measured by striatal dopamine release and GTPase activity as well as calcium clearance from striatal synaptosomes
and measures of neuronal membrane fluidity, which appear to be age and oxidative
stress dependent.2,3,4,21–24 Age-related changes in CNS function are also evident in
altered psychomotor and cognitive behaviors. Motor performance tests of balance,
coordination, strength, and stamina clearly illustrate age-related declines starting at
12 months and continuing through 22 months.18,19 A cognitive task assessing spatial
learning and memory, the Morris water maze, has also characterized deficits associated with normal aging,18,19 changes that parallel decrements in similar abilities in
humans.25,26 Age-sensitive neuronal and behavioral measures such as these can be
expected to be affected by nutritional interventions seeking to retard or reverse the
effects of brain aging.
Dietary supplementation with strawberry extract (STB) or spinach extract (SPC)
from 6 to 15 months of age (Male F344 rats, source: NIA colony) prevented the onset
of age-related deficits in signal transduction as measured by oxotremorine-enhanced
dopamine (OX-enhanced DA) release and carbachol-stimulated GTPase coupling/
uncoupling in isolated striatal slices, as compared to animals fed a control diet.2,3
Additionally, aged animals fed a STB-, SPC-, or blueberry extract (BB)–supplemented diet for 8 weeks (19–21 months) demonstrated similar antiaging effects. In
fact, the BB diet reversed the age-related deficits in OX-enhanced DA release. 3,4
Efficient regulation of calcium flux and homeostasis is critical for signal transduction and optimal neuronal functioning. Fruit-supplemented diets high in
polyphenolics were shown to have an antiaging effect on striatal Ca2+ uptake and recovery after depolarization in striatal synaptosomes.2,3 The BB diet was especially
effective, reversing the age-related decrement in Ca2+ recovery in the presence of hydrogen peroxide (H2O2)–induced oxidative stress, as compared to control groups. In
addition, the BB diet improved cerebellar membrane fluidity, as measured by significantly reduced anistropy, compared to aged subjects maintained on a control diet.4
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Behavioral measures showed some of the strongest antiaging effects of the fruit
polyphenolic–supplemented diet. On three psychomotor tasks, the rod walk and accelerating roto-rod, which assess balance, coordination, and resistance to fatigue,
and the inclined screen, which measures strength, stamina, and balance, the BB diet
significantly improved the performance of old animals.3,4
In addition, a measure of cognitive performance was improved by all three fruitand vegetable-supplemented diets. The Morris water maze (MWM) is a spatial
learning task that measures acquisition, working memory, reference memory, the
ability to shift set, and spatial learning strategies.18,27 The maze is a featureless circular pool of room temperature water (1 m diameter) containing a 10-cm-diameter
movable platform hidden just below the surface of the water. Rats swim well and use
distal cues to learn to navigate to the hidden platform. This age-sensitive paradigm
is a well-accepted measure of hippocampally based learning and memory. Dietary
supplementation with either STB, SPC, or BB for eight weeks antagonized the agerelated impairments in MWM performance.3,4
As might be suggested by the robust behavioral results, the physiological effects
of polyphenolic dietary supplementation is not limited to the CNS. In the periphery,
we have found that a BB diet decreases markers of inflammation and oxidative stress
in the gastrocnemius and quadriceps muscles (in preparation) and have also shown
BB supplementation to significantly increase red blood cell (RBC) membrane
fluidity.4
Ongoing studies seek to characterize which polyphenolic compounds present in
BB and other fruits and vegetables may contribute to the significant antiaging effects
described above. Initial studies have determined that anthocyanins and hydroxycinnamic acids (HCA) isolated from blueberries are able to ameliorate in vitro H2O2induced oxidative stress, with the anthocyanins significantly protecting RBCs
against reactive oxygen species even at low doses.28 Following a 24-hour fast, F344
rats were anesthetized and either anthocyanin or HCA, extracted from blueberry skin
or flesh, respectively, were administered by stomach intubation. RBC susceptibility
to H2O2-mediated oxidative stress was assessed by dichlorofluorescein assay at 0-,
1-, 6-, and 24-hours postgavage. While the total concentration of plasma anthocyanin and HCA fell off rapidly from 1 hour on, significant protection from reactive oxygen species was seen at 6 and 24 hours. Other experiments have shown that berry
anthocyanins penetrate a variety of cell types and that incorporation into the cell’s
cytosol protects against H2O2-induced oxidative stress and loss of cell function.29
Further studies will investigate the protective effects of a variety of polyphenolic
compounds in order to clarify the complete profile of phytonutrients found in fruits
and vegetables with antiaging effects.
Diets supplemented with fruits and vegetables rich in color and correspondingly
high in anthocyanins and other polyphenolic phytonutrients with antioxidant and additional bioactive properties antagonize the effects of aging in our animal model.
The amounts of BB, STB, and SPC added to the diets were matched for antioxidant
capacity2,3,9 and are roughly equivalent to a daily one cup portion for humans. In
sum, research from our lab and others suggests that increasing dietary intake of fruits
and vegetables high in antioxidant activity may be an important component of a
healthy living strategy designed to maximize neuronal and cognitive functioning
into old age.
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131
REFERENCES
1. HOLLMAN, P.C. & M.B. KATAN. 1999. Health effects and bioavailability of dietary flavonols. Free Radical Res. 31: S75–S80.
2. JOSEPH, J.A., B. SHUKITT-HALE, N.A. DENISOVA, et al. 1998. Long-term dietary strawberry, spinach, or vitamin E supplementation retards the onset of age-related neuronal signal-transduction and cognitive behavioral deficits. J. Neurosci. 18: 8047–
8055.
3. JOSEPH, J.A., B. SHUKITT-HALE, N.A. DENISOVA, et al. 1999. Reversals of age-related
declines in neuronal signal transduction, cognitive and motor behavioral deficits with
diets supplemented with blueberry, spinach or strawberry dietary supplementation. J.
Neurosci. 19: 8114–8121.
4. YOUDIM, K.A., B. SHUKITT-HALE, A. MARTIN, et al. 2000. Short-term dietary supplementation of blueberry polyphenolics: beneficial effects on aging brain performance
and peripheral tissue function. Nutr. Neurosci. 3: 383–397.
5. BICKFORD, P.C. 1993. Motor learning deficits in aged rats are correlated with loss of
cerebellar noradrenergic function. Brain Res. 620: 133–138.
6. SOKOLOFF, L., G.G. FITZGERALD & E.E. KAUFMAN. 1977. Determinants of the availability of nutrients to the brain. In Nutrition and the Brain. J.R. Wurtman & J.J. Wurtman, Eds.: 87–139. Raven Press. New York.
7. AMES, B.N., M.K. SHIGENAGA & T.M. HAGEN. 1993. Oxidants, antioxidants, and the
degenerative disease of aging. Proc. Natl. Acad. Sci. USA 90: 7915–7922.
8. HARMAN, D. 1994. Free-radical theory of aging. Increasing the functional life span.
Ann. N.Y. Acad. Sci. 717: 1–15.
9. WANG, H., G. CAO & R.L. PRIOR. 1996. Total antioxidant capacity of fruits. J. Agric.
Food Chem. 44: 701–705.
10. MIDDLETON, E., JR. 1998. Effect of plant flavonoids on immune and inflammatory cell
function. Adv. Exp. Med. Biol. 439: 175–182.
11. EASTWOOD, M.A. 1999. Interaction of dietary antioxidants in vivo: how fruit and vegetables prevent disease? Q.J.M. 92: 527–530.
12. GERRITSEN, M.E. 1998. Flavonoid inhibitors of cytokine induced gene expression. In
Flavonoids in the Living System. J. Manthey & B. Buslig, Eds.: 183–190. Plenum
Press. New York.
13. FOTSIS, T., M.S. PEPPER, E. AKTAS, et al. 1997. Flavonoids, dietary-derived inhibitors
of cell proliferation and in vitro angiogenesis. Cancer Res. 57: 2916–2921.
14. JOSEPH, J.A. R.E. BERGER, B.T. ENGEL & G.S. ROTH. 1978. Age-related changes in the
nigrostriatum: a behavioral and biochemical analysis. J. Gerontol. 33: 643–649.
15. JOSEPH, J.A., M.A. KOWATCH, T. MAKI & G.S. ROTH. 1990. Selective cross activation/inhibition of second messenger systems and the reduction of age-related deficits in the muscarinic control of dopamine release from perfused rat striata. Brain Res. 537: 40–48.
16. JOSEPH, J.A., R.T. BARTUS, D.E. CLODY, et al. 1983. Psychomotor performance in the
senescent rodent: reduction of deficits via striatal dopamine receptor up-regulation.
Neruobiol. Aging 4: 313–319.
17. INGRAM, D.K., M. JUCKER & E. SPANGLER. 1994. Behavioral manifestations of aging.
In Pathobiology of the Aging Rat. U. Mohr, D.L. Cungworth & C.C. Capen, Eds.:
149–170. ILSI. Washington.
18. SHUKITT-HALE, B., G. MOUZAKIS & J.A. JOSEPH. 1998. Psychomotor and spatial memory performance in aging male Fischer 344 rats. Exp. Gerontol. 33: 615–624.
19. SHUKITT-HALE, B. 1999. The effects of aging and oxidative stress on psychomotor and
cognitive behavior. Age 22: 9–17.
20. CANTUTI-CASTELVETRI, I., B. SHUKITT-HALE & J.A. JOSEPH. 2000. Neurobehavioral
aspects of antioxidants and aging. Int. J. Dev. Neurosci. 18: 367–381.
21. JOSEPH, J.A., J.G. STRAIN, N.D. JIMENEZ & D. FISHER. 1997. Oxidant injury in PC12
cells—a possible model of calcium deregulation in aging. I. Selectivity of protection
against oxidative stress. J. Neurochem. 69: 1252–1258.
22. DENISOVA, N.A., S.A. ERAT, J.F. KELLY & G.S. ROTH. 1998. Differential effect of
aging on cholesterol modulation of carbachol stimulated Low-Km GTPase in striatal
synaptosomes. Exp. Gerontol. 33: 249–265.
132
ANNALS NEW YORK ACADEMY OF SCIENCES
23. DENISOVA, N.A., D. FISHER, M. PROVOST & J.A. JOSEPH. 1999. The role of glutathione,
membrane sphingomyelin, and its metabolites in oxidative stress-induced calcium
‘dysregulation’ in PC12 cells. Free Radic. Biol. Med. 27: 1292–1301.
24. JOSEPH, J.A., B. SHUKITT-HALE, J. MCEWEN & B. RABIN. 1999. Magnesium activation
of GTP hydrolysis or incubation in S-Adenosyl-1-methionine reverses 56Fe-induced
decrements in oxotremorine-enhancement of K+ evoked striatal dopamine release.
Radiat. Res. 152: 637–641.
25. MUIR, J.L. 1997. Acetylcholine, aging, and Alzheimer’s disease. Pharmacol. Biochem.
Behav. 56: 687–696.
26. WEST, R.L. 1996. An application of pre-frontal cortex function theory to cognitive
aging. Psych. Bull. 120: 272–292.
27. MORRIS, R. 1984. Developments of a water maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 11: 47–60.
28. YOUDIM, K.A., B. SHUKITT-HALE, S. MACKINNON, et al. 2000. Polyphenolics enhance
red blood cell resistance to oxidative stress: in vitro and in vivo. Biochim. Biophys.
Acta 1523: 117–122.
29. YOUDIM, K.A., A. M ARTIN & J.A. JOSEPH. 2000. Incorporation of the elderberry anthocyanins by endothelial cells increases protection against oxidative stress. Free Radic.
Biol. Med. 29: 51–60.