Frozen Fruit Pulp of Euterpe oleraceae Mart. (Acai) Prevents Hydrogen Peroxide-Induced Damage in the Cerebral Cortex, Cerebellum, and Hippocampus ...
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JOURNAL OF MEDICINAL FOOD
J Med Food 12 (5) 2009, 1084–1088
Full Communication
# Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2008.0236
Frozen Fruit Pulp of Euterpe oleraceae Mart. (Acai) Prevents
Hydrogen Peroxide-Induced Damage in the Cerebral Cortex,
Cerebellum, and Hippocampus of Rats
Patricia D.S. Spada,1 Caroline Dani,1,2 Giovana V. Bortolini,1 Claudia Funchal,2
João A.P. Henriques,1,3 and Mirian Salvador1
1
Laboratório de Estresse Oxidativo e Antioxidantes, Instituto de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul;
2
Centro Universitário Metodista, Porto Alegre; and 3Universidade Luterana do Brasil, Canoas, Rio Grande do Sul, Brazil
ABSTRACT Oxidative stress is implicated in several human illnesses, including neurological disorders such as Parkinson’s
and Alzheimer’s diseases. Acai is largely consumed in Brazil and contains high levels of antioxidant compounds. This work
aims to study the antioxidant activity of acai frozen fruit pulp in the cerebral cortex, hippocampus, and cerebellum of rats
treated with the oxidizing agent hydrogen peroxide (H2O2). Pretreatment of tissue with acai decreased H2O2-induced damage
of both lipids and proteins in all tissues tested. This fruit was also able to reduce the activities of the antioxidant enzymes
superoxide dismutase and catalase to basal levels. We observed a negative correlation between the polyphenol content of acai
and the levels of lipid (r ¼ 0.689; P .05) and protein damage (r ¼ 0.569; P .05), suggesting the participation of
polyphenols in the observed antioxidant activity. These data suggest that acai has a positive contribution in the development of
age-related neurodegenerative diseases.
KEY WORDS: acai brain tissues catalase Euterpe oleraceae oxidative stress superoxide dismutase
INTRODUCTION brain consumes 20% of the total oxygen used by the body. It
is enriched with readily peroxidizable polyunsaturated fatty
S everal studies have shown that the consumption of
fruits and vegetables is associated with a reduced risk of
many diseases,1 including neurodegenerative diseases such
acids, and, although microglial cells contain glutathione in
high concentrations,15 neurons do not possess high amounts
of antioxidant defenses.16,17 The brain also has high levels
as Parkinson’s and Alzheimer’s.2–4 Fruits and vegetables
of iron, which is the key catalyst for lipid peroxidation.
synthesize a vast array of secondary chemical compounds
Additionally, many neurotransmitters are themselves auto-
that, although not involved in primary metabolism, are im-
xidize to generate reactive species.16,17 Lipid peroxidation
portant for a multitude of beneficial effects that have been
in brain tissues is associated with a progressive loss of
reported for fruits.5
membrane permeability and cellular damage, which leads to
The palm fruit of Euterpe oleraceae Mart., commonly
an increased susceptibility to various diseases.18 In order to
known as acai, is consumed in a variety of beverages and
protect the brain against oxidative damage, there exist in-
food preparations, mainly in Brazil.6 This fruit supplies
tricate and interrelated processes, which include superoxide
several antioxidant compounds such as polyphenols, carot-
dismutase (SOD) and catalase (CAT) enzymes. SOD cata-
enoids, and ascorbic acid.7–10 Acai has been demonstrated to
lyzes the dismutation of superoxide anion (O2 ) to oxygen
possess strong antioxidant capacity10,11 and potent anti-
and hydrogen peroxide (H2O2), while CAT converts H2O2
inflammatory activity in vitro and shown to inhibit lipid
to water and molecular oxygen.19 Freeze-dried acai pulp has
peroxidation in humans in vivo.9,12 The pharmacokinetics of
been reported to possess superior superoxide scavenging
acai pulp in humans has also been investigated.13
activity in vitro compared to any food tested to date.11 SOD
The brain is especially susceptible to oxidative stress.14
and CAT enzymes have an important role in maintaining
While only making up about 2% of the total body mass, the
physiological redox equilibrium, avoiding or decreasing the
oxidative stress.19
Manuscript received 12 September 2008. Revision accepted 14 January 2009. The aim of the present study was to investigate the bio-
logical effects of frozen pulp of acai in reducing the oxi-
Address correspondence to: Mirian Salvador, Laboratório de Estresse Oxidativo e An-
tioxidantes, Instituto de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul,
dative stress induced by H2O2 in the cerebellum, cerebral
RS, Brazil, E-mail: msalvado@ucs.br cortex, and hippocampus from Wistar rats.
1084ACAI PREVENTS H2O2 DAMAGE IN NERVOUS TISSUE 1085
MATERIALS AND METHODS formation at 480 nm (model UV-1700 spectrophotometer,
Shimadzu, Kyoto, Japan) in a reaction medium containing
Acai frozen fruit pulp 1 mmol=L adrenaline (pH 2.0) and 50 mmol=L glycine (pH
Acai (E. oleraceae) was obtained from the company Mais 10.2). This reaction was performed at 308C for 3 minutes.23
Fruta (Antonio Prado, RS, Brazil). The pulp was produced One unit of SOD activity is defined as the amount of enzyme
with fresh and clean fruits, free of dirt, parasites, and plant or that inhibits the rate of adrenochrome formation by 50%=g
animal debris. Only the edible portion of acai was used. of protein. The CAT-like activity assay was performed ac-
After pressing, the pulp was frozen at 208C. Immediately cording to the method described by Aebi24 by determining
prior to the assays frozen pulp was mixed with distilled the H2O2 decomposition rate at 240 nm. A total of 1 unit of
water in a blender to achieve a final concentration of 40% CAT decomposed 1 mmol of H2O2=mg of protein in 1 minute
(wt=vol). Macronutrient composition and polyphenol, ca- at pH 7.4. Protein concentration was determined by the
rotenoid, and vitamin C levels of acai are shown in Table 1. method of Bradford25 using serum bovine albumin as
No organophosphorus or carbamate pesticides were detected the standard.
in the sample, according to the assay performed as described Lipid peroxidation was monitored by the formation of
by Bastos et al.20 and Lima et al.21 thiobarbituric acid-reactive species (TBARS) during an
acid-heating reaction, which has been widely adopted as a
Animal and tissue preparation sensitive method for measurement of lipid peroxidation, as
previously described.26 The oxidative damage to proteins
Wistar rats (n ¼ 5; 10 days old) were obtained from our was assessed by the determination of carbonyl groups based
breeding colony (Centro Universitário Metodista, Porto on the reaction with dinitrophenylhydrazine, as previously
Alegre, RS, Brazil). The animals were handled under stan- described.27 All chemicals were purchased from Sigma
dard laboratory conditions of a 12-hour light=dark cycle and Chemical Co. (São Paulo, Brazil).
fixed temperature (25 28C). Food and water were avail-
able ad libitum. All experimental were conducted in ac- Statistical analyses
cordance with the Guiding Principles of the Use of Animals
in Toxicology, adopted by the Society of Toxicology in July Values were determined as parametric or nonparametric
1989 with the approval of the local ethics committee (Centro by using the Kolmogorov-Smirnoff test. Data were sub-
Universitário Metodista). Assays were performed as de- jected to analysis of variance, and means were compared
scribed by Leipnitz et al.22 In brief, animals were killed by using Tukey’s test. Relationships between variables were
decapitation without anesthesia, and the brain was rapidly assessed with Pearson’s product-moment correlation coef-
excised on a Petri dish placed on ice. The cerebral cortex, ficient. SPSS version 12.0 (SPSS, Chicago, IL) was used in
cerebellum, and hippocampus were dissected, weighed, and all statistical analyses.
kept chilled until homogenization, which was performed
using a ground glass-type Potter-Elvejhem homogenizer in RESULTS
1.5% KCl. The homogenates were centrifuged at 800 g for
10 minutes at 48C, the pellet was discarded, and the super- Tissue treatments with H2O2 induced an increase in lipid
natants were used immediately. Aliquots were treated with (TBARS) and protein (protein carbonyl groups) damage in
acai pulp (40% wt=vol) for 30 minutes, with agitation, and the cerebellum, cerebral cortex, and hippocampus of rats
1 mM H2O2 was subsequently added to the mixture. Sam- relative to the untreated control (Fig. 1). H2O2 also induced
ples were incubated for 1 hour at 308C with agitation. an increase in both SOD and CAT activities in all the as-
sayed tissues (Fig. 2). Acai treatment was neither able to
Biochemical assays change enzyme activities (Fig. 2) nor to induce oxidative
damage in lipids or proteins (Fig. 1). However, when tissues
SOD activity was determined spectrophotometrically by were pretreated with acai and then with H2O2, a significant
measuring the inhibition of self-catalytic adrenochrome decrease in lipid and protein damage and in SOD and CAT
activities was observed (Fig. 2). The reduction of lipid
Table 1. Macronutrient Composition and Total damage was around 48% in the cerebral cortex, 64% in the
Phenolic, Carotenoid, and Vitamin C Content hippocampus, and 72% in the cerebellum. Reduction of pro-
(per 100 g of Frozen Pulp) tein damage was 55% in the cerebral cortex, 36% in
the hippocampus, and 42% in the cerebellum.
Acai level
Protein (g) 0.55 0.03 DISCUSSION
Lipid (g) 0.67 0.04
Carbohydrate (g) 7.63 0.02 Brain cells are continuously threatened by the damage
Caloric value (kJ) 163.25 0.03 caused by reactive species produced during normal oxygen
Total phenolic content (mg of catechin) 1.19 0.20 metabolism or induced by exogenous sources.28 The
Total carotenoid content (mg) 1.02 0.55 mechanisms by which reactive species interfere with cel-
Vitamin C (mg) 15.70 0.97
lular function are not fully understood, but one of the most
Data are mean SD values. important events seems to be oxidative damage. The loss of1086 SPADA ET AL. FIG. 1. (A) Lipid and (B) protein damage in the cerebral cortex, hippocampus, and cerebellum of rats: untreated tissues (&), 40% (wt=vol) acai ( ), 1 mM H2O2 (&), and 40% (wt=vol) acai plus 1 mM H2O2 (&). Data are mean SD values of five independent experiments. *Different letters indicate a significant difference according to analysis of variance and Tukey’s post hoc test (P .05) for each tissue evaluated. motor function due to cell death and region-specific loss of related to oxidative stress in many epidemiological and neurons in the mammalian brain by oxidative stress is more experimental studies.10,11,34–36 prominent in the cerebral cortex, hippocampus, and cere- Brain tissue pretreatments with acai were able to prevent bellum. The cerebral cortex and hippocampus are regions oxidative damage induced by H2O2. This oxidant causes cell associated with cognition and feedback control of stress, damage through the production of hydroxyl radicals via the whereas the cerebellum is concerned with motor function.29 Haber-Weiss=Fenton reaction.19 Unlike the free radicals In vitro studies of the responses of the cerebral cortex to O2 and OH, H2O2 is very diffusible within and between vitamin E have revealed its neuroprotective role in the brain cells in vivo, causing damage in membranes, proteins, lipids, of mice.30 Green tea catechins and polyphenols have also and the cellular nucleus.19 Negative correlations between been reported for their antioxidant properties and protective total phenolic content of acai and TBARS (r ¼ 0.689; effects against oxidative stress in rat brain.11,31 P .05) and carbonyl assay (r ¼ 0.569; P .05) levels Acai, a very popular Brazilian fruit, is known to possess were observed, suggesting that the polyphenols present in antinociceptive, anti-inflammatory,32,33 and inhibitory ef- this fruit could be responsible, at least in part, for the anti- fects on nitric oxide production by the activated macrophage oxidant activity of this fruit. cell line RAW 264.7. This fruit is rich in antioxidant com- H2O2 and=or the oxidative stress produced by it are able pounds,10,11 which have been found to reduce the risks of to induce the antioxidant activity of the SOD and CAT diseases associated with chronic or acute oxidative stress enzymes,37 as observed in this work (Fig. 2). Acai treatments FIG. 2. Activities of (A) SOD and (B) CAT in the cerebral cortex, hippocampus, and cerebellum of rats: untreated tissues (&), 40% (wt=vol) acai ( ), 1 mM H2O2 (&), and 40% (wt=vol) acai plus 1 mM H2O2 (&). Data are mean SD values of five independent experiments. *Different letters indicate a significant difference according to analysis of variance and Tukey’s post hoc test (P .05) for each tissue evaluated.
ACAI PREVENTS H2O2 DAMAGE IN NERVOUS TISSUE 1087
were able to return SOD and CAT antioxidant activities to Amazonian palm berry, Euterpe oleraceae Mart. (Acai). J Agric
basal levels (Fig. 2). In fact, a negative correlation was Food Chem 2006;54:8598–8603.
observed between SOD and CAT activities and total phe- 9. Rodrigues RB, Lichtenthäler R, Zimmermann BF, Papagianno-
nolic content of acai (r ¼ 0.701 and r ¼ 0.698, P .05, poulos M, Fabricius H, Marx F, Maia JG, Almeida O: Total
respectively). Some polyphenols, such as catechin,2 have oxidant scavenging capacity of Euterpe oleracea Mart. (açaı́)
been reported to be superoxide radical, H2O2, and methyl- seeds and identification of their polyphenolic compounds. J Agric
glyoxal scavengers.38 In fact, catechin and epicatechin have Food Chem 2006;54:4162–4167.
been shown to be effective trap inhibitors.11 Acai frozen 10. Spada PD, de Souza GG, Bortolini GV, Henriques JA, Salvador
pulp has already been shown to possess SOD- and CAT-like M: Antioxidant, mutagenic, and antimutagenic activity of frozen
fruits. J Med Food 2008;11:144–151.
activities10 and the ability to scavenge superoxide radicals,11
11. Schauss AG, Wu X, Prior RL, Ou B, Huang D, Owens J, Agarval A,
which could be responsible, at least in part, for its beneficial
Jensen GS, Hart AN, Shanbro E: Antioxidant capacity and other bio-
effects against oxidative stress. activities of the freeze-dried amazonian palm berry Euterpe oler-
The data obtained in this study showed that the frozen aceae Mart. (acai). J Agric Food Chem 2006;54:8604–8610.
pulp of acai possesses antioxidant activity in the brain tissue 12. Honzel D, Carter SG, Redman KA, Schauss AG, Endres JR, Jensen
of rats. Other polyphenol-rich fruits, such as berries39 and GS: Comparison of chemical and cell-based antioxidant methods
grapes,35 have already been found to be beneficial to brain for evaluation of foods and natural products: generating multifac-
function. Thus, it is possible to assert that nutritional inter- eted data by parallel testing using erythrocytes and polymorpho-
ventions containing polyphenols, other phytochemicals, and nuclear cells. J Agric Food Chem 2008;56:8319–8325.
nutrients could inhibit, slow down the progression, or pos- 13. Mertens-Talcott SU, Rios J, Jilma-Stohlawetz P, Pacheco-
sibly prevent the development of some neurodegenerative Palencia LA, Meibohm B, Talcott ST, Derendorf H: Pharma-
diseases, such as Parkinson’s and Alzheimer’s. Chronic cokinetics of anthocyanins and antioxidant effects after the
feeding studies are warranted in light of our findings. consumption of anthocyanin-rich acai juice and pulp (Euterpe
oleraceae Mart.) in human healthy volunteers. J Agric Food
ACKNOWLEDGMENTS Chem 2008;56:7796–7802.
14. Halliwell B, Gutteridge JMC: Oxygen radicals and the nervous
We thank the Universidade de Caxias do Sul (Caxias do system. Trends Neurosci 1985;8:22–26.
Sul, RS, Brazil), Research Council of the State of Rio 15. Dringen R: Oxidative and antioxidative potential of brain mi-
Grande do Sul, and Centro Universitário Metodista for their croglial cells. Antioxid Redox Signal 2005;7:1223–1233.
help and financial support. 16. Olanow CW: An introduction to the free radical hypothesis in
Parkinson’s disease. Ann Neurol 1992;32(Suppl):S2–S9.
AUTHOR DISCLOSURE STATEMENT 17. Savory J, Rao JK, Huang Y, Letada PR, Herman MM: Age-
related hippocampal changes in Bcl-2:Bax ratio, oxidative stress,
No competing financial interests exist. redox-active iron and apoptosis associated with aluminum-
induced neurodegeneration: increased susceptibility with aging.
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