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Antioxidant Capacity and Other Bioactivities of the Freeze-Dried
Amazonian Palm Berry,
Euterpe oleraceae
Mart. (Acai)
ALEXANDER G. SCHAUSS,*,† XIANLI WU,‡,§ RONALD L. PRIOR,‡BOXIN OU,⊥
DEJIAN HUANG,|JOHN OWENS,∇AMIT AGARWAL,#GITTE S. JENSEN,X
AARON N. HART,XAND EDWARD SHANBROM∇
Natural and Medicinal Products Research, AIMBR Life Sciences, 4117 South Meridian,
Puyallup, Washington 98373, Agriculture Research Service, Arkansas Children’s Nutrition Center,
U.S. Department of Agriculture, 1120 Marshall Street, Little Rock, Arkansas 72202, Department of
Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 West Markham,
Little Rock, Arkansas 72205, Brunswick Laboratories, 6 Thatcher Lane,
Wareham, Massachusetts 02571, Food Science and Technology Program, Department of Chemistry,
National University of Singapore, Singapore 117543, Singapore, University of California,
Irvine, Building 55, 101 The City Drive South, Orange, California 92868, Natural Remedies,
19th K. M. Stone, Hosur Road, Bangalore 560100, India, and NIS Labs, 1437 Esplanade,
Klamath Falls, Oregon 97601
The fruit of
Euterpe oleraceae
, commonly known as acai, has been demonstrated to exhibit sig-
nificantly high antioxidant capacity
in vitro
, especially for superoxide and peroxyl scavenging, and,
therefore, may have possible health benefits. In this study, the antioxidant capacities of freeze-dried
acai fruit pulp/skin powder (OptiAcai) were evaluated by different assays with various free radical
sources. It was found to have exceptional activity against superoxide in the superoxide scavenging
(SOD) assay, the highest of any food reported to date against the peroxyl radical as measured by
the oxygen radical absorbance capacity assay with fluorescein as the fluorescent probe (ORACFL),
and mild activity against both the peroxynitrite and hydroxyl radical by the peroxynitrite averting capacity
(NORAC) and hydroxyl radical averting capacity (HORAC) assays, respectively. The SOD of acai
was 1614 units/g, an extremely high scavenging capacity for O2•-, by far the highest of any fruit or
vegetable tested to date. Total phenolics were also tested as comparison. In the total antioxidant
(TAO) assay, antioxidants in acai were differentiated into “slow-acting” and “fast-acting” components.
An assay measuring inhibition of reactive oxygen species (ROS) formation in freshly purified human
neutrophils showed that antioxidants in acai are able to enter human cells in a fully functional form
and to perform an oxygen quenching function at very low doses. Furthermore, other bioactivities
related to anti-inflammation and immune functions were also investigated. Acai was found to be a
potential cyclooxygenase (COX)-1 and COX-2 inhibitor. It also showed a weak effect on lipopolysac-
charide (LPS)-induced nitric oxide but no effect on either lymphocyte proliferation and phagocytic
capacity.
KEYWORDS:
Euterpe oleraceae
; acai; reactive oxygen species (ROS); antioxidant; ORACFL; NORAC;
HORAC; superoxide; SOD; TAO; cyclooxygenase (COX); macrophage phagocytosis assay; nitric oxide
assay; lymphocyte proliferation assay
INTRODUCTION
High intake of fruits and vegetables was found to positively
associate with lower chance of many diseases by epidemiologi-
cal studies and clinical trials. Antioxidant capacity was believed
to be one of the possible mechanisms, though others are also
involved. Acai, fruits of Euterpe oleraceae Martius, is consumed
in a variety of beverages and food preparations in the native
land in Brazil, Colombia, and Suriname and used medicinally
as an antidiarrheal agent (1,2). Recently, much attention has
been paid to its antioxidant capacity and possible role as a
“functional food” or food ingredient (3-6). Euterpe oleraceae
fruit pulp has been reported to quench peroxyl radicals,
peroxynitrite, and in Vitro hydroxyl radicals by the TOSC assay
(4). In another study, the antioxidant activity of acai frozen pulp
was determined on the basis of the inhibition of copper-induced
* To whom correspondence should be addressed. E-mail: alex@aibmr.com.
Phone: 253-286-2888. Fax: 253-286-2451.
†AIMBR Life Sciences.
‡U.S. Department of Agriculture.
§University of Arkansas for Medical Sciences.
⊥Brunswick Laboratories.
|National University of Singapore.
3University of California, Irvine.
#Natural Remedies.
XNIS Lab.
8604
J. Agric. Food Chem.
2006,
54,
8604
−
8610
10.1021/jf0609779 CCC: $33.50 © 2006 American Chemical Society
Published on Web 10/07/2006
peroxidation of liposome and the inhibition of the co-oxidation
of the linoleic acid and β-carotene system (5). Phytochemical
composition and nutrient analysis of acai have been presented
in our former paper (7). Here, we focus on its antioxidant
capacities evaluated by different assays with various free radical
sources to further enhance our knowledge of this fruit’s health
potential.
Free radicals are consistently formed as byproducts of aerobic
metabolism in the human body (8). They are generally reactive
oxygen or nitrogen species (ROS or RNS). The most common
ROS and RNS in ViVoare superoxide (O2•-), hydroxyl radical
(OH2•), peroxyl radical (RO2•), nitric oxide (•NO), and peroxy-
nitrite (ONOO-). These ROS have been associated with many
chronic and degenerative diseases including vascular diseases,
diabetes, cancer, and overall aging (9-11). Dietary antioxidants
are believed to be good external sources to counteract free
radicals in the body (12). A large number of methods have been
developed to evaluate total antioxidant capacity (TAC) of food
samples. Nevertheless, few of them have been used widely due
to the difficulty of measuring TAC owing to limitations
associated with methodological issues and free radical sources
(13). In this study, the TAC of acai was evaluated by a series
of oxygen radical absorbance capacity assay with fluorescein
as the fluorescent probe (ORACFL) based assays, including
hydrophilic ORACFL (H-ORACFL), lipophilic ORACFL (L-
ORACFL), peroxynitrite radical averting capacity (NORAC), and
hydroxyl radical averting capacity (HORAC). As a comparison,
total phenolics was also measured by the Folin-Ciocalteu
method. Moreover, several novel antioxidant capacity assays
including the superoxide scavenging (SOD) assay, total anti-
oxidant (TAO) assay, and inhibition of ROS formation in a
functional, cell-based assay using freshly purified human
neutrophils from healthy donors were also performed. Results
from these assays are expected to provide additional information
to help us better understand the antioxidant capacity of acai.
Bioactivities based on mechanisms other than antioxidant
activities may also contribute to the overall health benefits of
acai. In this study, we conducted several assays related to anti-
inflammation and immune functions, including the cyclooxy-
genase (COX-1 and COX-2) inhibitor assay, macrophage
phagocytosis assay, nitric oxide assay, and lymphocyte prolif-
eration assay.
MATERIALS AND METHODS
Plant Material. Freeze-dried acai (Euterpe oleracea) fruit pulp/skin
powder (OptiAcai) was obtained from K2A LLC (Provo, UT). The
berries were collected in Belem, Brazil. Within hours of harvesting,
acai berries were frozen and stored at -20 °C until transferred for freeze
drying. The freeze-dried acai powder was kept at -20 °C until analyzed.
Chemicals and Standards. ORAC-Based Assays and Total Phen-
olics. 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) was
purchased from Wako Chemicals USA (Richmond, VA). 6-Hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), fluorescein
(sodium salt) (Fl), cobalt(II) fluoride tetrahydrate, and picolinic acid
were obtained from Aldrich (Milwaukee, WI). Randomly methylated
β-cyclodextrin (Trappsol, Pharm Grade, RMCD) was obtained from
Cyclodextrin Technologies Development Inc. (High Springs, FL).
Folin-Ciocalteu’s phenol reagent, sodium carbonate, sodium hydrogen
carbonate (NaHCO3), dihydrorhodamine-123 (DHR-123), caffeic acid,
and gallic acid were all purchased from Sigma (St. Louis, MO).
Potassium phosphate dibasic (K2HPO), potassium phosphate monobasic
(KH2PO4), and hydrogen peroxide were obtained from VWR (West
Chester, PA). 3-Morpholinosydnonimine hydrochloride (SIN-1) was
provided by Toronto Research Chemicals (North York, Ontario,
Canada). Other solvents were purchased from Fisher (Fair Lawn, NJ).
SOD Assay. Hydroethidine was from Polysciences, Inc. (Warrington,
PA). Xanthine oxidase (from butter milk, catalog number X4875),
xanthine, and superoxide dismutase (from bovine erythrocytes, catalog
number S 2515) were purchased from Sigma-Aldrich (St. Louis, MO).
Manganese(III) 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride
tetrakis(methochloride) was obtained from Aldrich (Milwaukee, WI,).
TAO Assay. TAO iodine reagent was provided by Shanbrom
Technologies, LLC (Ojai, CA). A 710A+basic ion selective meter
was obtained from Thermo-Electron Corp (Waltham, MA).
Inhibition of ROS Formation in Human PMN Cells. Histopaque 1119
and 1077 are both from Sigma-Aldrich (St. Louis, MO). DCF-DA is
from Molecular Probes (Eugene, OR).
COX-1 and COX-2 Inhibitor Assay. Arachidonic acid and COX-1
and COX-2 enzymes are all purchased from Cayman Chemical (Ann
Arbor, MI).
Macrophage Phagocytosis Assay, Nitric Oxide Assay, and Lympho-
cyte Proliferation Assay. RPMI-1640 media was purchased from
Invitrogen (Carlsbad, CA) and phosphate-buffered saline (PBS) from
Hyclone (Logan, UT). Lipopolysaccharide (LPS), 3-[4,5-dimethylthi-
azol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and naphthyleth-
ylene diamine dihydrochloride (NEDD) were obtained from Sigma (St.
Louis, MO). Other reagents were of analytical grade (Bangalore, India).
Total Phenolic Analysis. The acetone/water/acetic acid (AWA)
extracts were subjected to total phenolics measurement by Folin-
Ciocalteu reagent according to the method of Wu et al. (14). The results
were expressed as milligrams of gallic acid equivalents per 100 g of
fresh weight (mg of GAE/100 g of FW).
ORACFL-Based Assays. Freeze-dried acai (0.035 g) was extracted
with 20 mL of acetone/water (50:50 v/v) for1hatroom temperature
on an orbital shaker. The extracts were centrifuged at 5900 rpm, and
the supernatant was ready for H-ORACFL, HORAC, and NORAC
analysis. An acai sample (1 g) was extracted with hexane/dichlo-
romethane two times (10 mL ×2). The supernatants were combined
for L-ORACFL analysis (15).
The H-ORACFL assay was conducted on the basis of a report by Ou
and co-workers (16), modified for the FL600 microplate fluorescence
reader (Bio-Tek Instruments, Inc., Winooski, VT). The FL600 micro-
plate fluorescence reader was used with fluorescence filters for an
excitation wavelength of 485 (20 nm and an emission wavelength of
530 (25 nm. The plate reader was controlled by software KC4 3.0.
For L-ORACFL, a sample solution was prepared according to a previous
paper (17). Then the L-ORACFL was also measured in the same plate
reader based on a published procedure (17).
The HORAC assay is based on a report by Ou and co-workers and
modified for the FL600 fluorescence microplate plate reader (Bio-TeK
Instruments, Inc., Winooski, VT) (18).
ONOO-scavenging was measured by monitoring the oxidation of
DHR-123 according to a modification of the method of Chung et al
(19). Briefly, a stock solution of DHR-123 (5 mM) in dimethylforma-
mide was purged with nitrogen and stored at -80 °C. A working
solution with DHR-123 (final concentration, fc, 5 M) diluted from the
stock solution was placed on ice in the dark immediately prior to the
study. The buffer of 90 mM sodium chloride, 50 mM sodium phosphate
(pH 7.4), and 5 mM potassium chloride with 100 M (fc) diethyl-
enetriaminepentaacetic acid (DTPA) was purged with nitrogen and
placed on ice before use. ONOO-scavenging by the oxidation of DHR-
123 was measured with a microplate fluorescence reader FL600 with
excitation and emission wavelengths of 485 and 530 nm, respectively,
at room temperature. The background and final fluorescent intensities
were measured 5 min after treatment with or without SIN-1 (fc 10 M)
or authentic ONOO-(fc 10 M) in 0.3 N sodium hydroxide. Oxidation
of DHR-123 by decomposition of SIN-1 gradually increased, whereas
authentic ONOO-rapidly oxidized DHR-123 with its final fluorescent
intensity being stable over time.
SOD Assay. An acai sample (0.02 g) was extracted with 20 mL of
an acetone/water mixture on a shaker for 1 h. The mixture was
centrifuged at 5900 rpm and 20 °C for 10 min. The supernatant was
used for SOD assay. SOD assay was carried out on the basis of an
in-house protocol (Brunswick Labs, Wareham, MA) on a Precision 2000
eight channel liquid handling system and Synergy HT microplate UV-
vis and fluorescence reader, both from Bio-Tek Inc. (Winooski, VT).
TAO Assay. Acai powder (4 g) was placed in 40 mL of 10% (w/v)
soluble polyvinyl pyrrolidone (PVP; BASF, Kollidon 17PF) and
Antioxidant Capacity and Bioactivities of Acai
J. Agric. Food Chem.,
Vol. 54, No. 22, 2006 8605
incubated for2hat37°C. The extract was centrifuged at 4000 rpm
for 10 min, and the supernatant was decanted from the tube and serially
diluted 2-fold to 1:8. Iodine reagent (0.1 mL) was mixed well with
diluted supernatant. The iodide level (µg/mL) in the sample was
determined by an Orion Iodide Sure-Flow solid-state combination
electrode (Waltham, MA) at 30 s and 30 min after addition of iodine
reagent. The TAO value is equivalent to ppm (µg/mL) of iodide formed.
Inhibition of ROS Formation in Human PMN Cells. Heparinized
blood samples were obtained from healthy volunteers upon informed
consent. The blood was immediately layered on top of a double gradient
of Histopaque 1119 and 1077 (both from Sigma-Aldrich, St. Louis,
MO), and centrifuged for 25 min at room temperature. The top layer
of mononuclear cells was removed. The second layer of cells between
the two gradients, which represents almost 100% neutrophils, was
harvested and used for the evaluation of ROS formation. Cells were
washed twice in phosphate-buffered saline without calcium or mag-
nesium.
An extract of the test product was prepared by adding acai powder
(0.5 g) to 5 mL of phosphate-buffered saline, pH 7.4. This mixture
was vortexed repeatedly and allowed to sit at room temperature for 1
h. Prior to use, insoluble particles were removed by centrifugation and
subsequent filtration using a 0.22 µm cellulose-acetate syringe filter.
This liquid was used to prepare a series of 100-fold dilutions in
phosphate-buffered saline without calcium or magnesium. Freshly
purified human neutrophils were preincubated with acai extracts over
a wide range of dilutions and then incubated at 37 °C for 90 min.
Following a wash to remove compounds within the extracts that could
interfere with the oxidation marker, cells were loaded with 0.5 µM
DCF-DA (Molecular Probes, Eugene, OR) for1hat37°C. All samples,
except for the untreated control samples, were then exposed to 167
mM H2O2for a period of 45 min to induce oxidative stress. Samples
were washed to remove the peroxide, transferred to cold RPMI, and
stored on ice in preparation for immediate acquisition by flow
cytometry, using a FACSCalibur cytometer (Becton-Dickinson, San
Jose, CA). Untreated samples were run before and after running all
other samples, to verify that spontaneous oxidation was minimal.
Intracellular levels of DFC-DA fluorescence intensity in untreated
versus challenged cells in the presence versus absence of the test product
were analyzed by flow cytometry. A standard curve of DCF-DA
fluorescence intensity as a result of treatment with known amounts of
hydrogen peroxide was used to produce an estimation of the effective-
ness of a given natural product in terms of quenched hydrogen peroxide
molecules. Data was collected in triplicate for controls and duplicate
for each sample concentration. Dose levels are reported in volumetric
parts per billion. Statistical significance was determined using Student’s
t-test.
COX Assay. Acai powder (2 g) was extracted with 50% acetone
and tested directly without further dilution. The sample was incubated
at 37 °C with Tris buffer (0.5 mL) in the reaction chamber followed
by 5 µLof100µM heme in DMSO. To the solution, 5 µL of COX-1
(or 10 µL of COX-2) enzyme solution was added (used as received
from supplier). The mixture was incubated for 1 min. A 5 µL sample
(in DMSO or ethanol) was added and incubated for 1 min. Arachidonic
acid (5 µL) was added, and the reaction rate was monitored. The oxygen
concentration was monitored in real time by an Oxytherm (Hansatech
Instrumental, Norfolk, England). The initial oxygen consumption rate
is obtained from the kinetic curve. In the presence of inhibitors, the
initial rate decreased. The IC50, the concentration at which the initial
oxygen consumption rate decreased by 50%, was used to express the
COX-1 and -2 inhibition activity.
Nitric Oxide Assay, Macrophage Phagocytosis Assay, and
Lymphocyte Proliferation Assay. Acai was diluted in media contain-
ing 5% DMSO to a concentration of 10 mg/mL. It was subjected to
sonication at 35 kHz for 10 min in a Bandelin Sonorex sonicator. The
supernatant was collected, sterilized though a 0.22 µm filter, and used
immediately for the assay.
Mouse splenocytes were isolated according to previously published
procedures with minor modifications (20).
The lymphocyte proliferation assay was performed per previously
established protocols (20,21). Splenocytes were plated in 96-well plates
onday0(0h)at5×105cells/well (200 µL per well) in RPMI-1640
media containing 10% fetal bovine serum (FBS). The plates were
incubated for2hat37°CinaCO
2incubator (5% CO2) to allow the
cells to recover. A Trypan blue dye exclusion test was performed at
the time lymphocytes were seeded in the well. Cell viability was >95%.
Acai extracts were added to the wells at the requisite concentrations,
and the plates were incubated again for 24 h at 37 °CinaCO
2incubator
(5% CO2). LPS (5 µg/mL) was also added to some wells as a positive
control for the assay. Cell proliferation was checked at 24 h using the
MTT assay.
The nitric oxide assay was performed as per previously established
protocols (22-24). Briefly, J774A.1 cells were plated in 96 well plates
onday0(0h)at5×105cells/mL (200 µL per well) in RPMI-1640
medium containing 10% FBS. The plates were incubated overnight
(for about 16 h) at 37 °CinaCO
2incubator (5% CO2). The old media
was removed from the wells after 16 h, and fresh media was added
onto the cells. LPS or herbal extract at different concentrations was
added to the wells. The cells were incubated for 48 h at 37 °Cina
CO2incubator (5% CO2). At the end of 48 h, the supernatants were
collected and used for the nitric oxide (NO) assays.
In order to perform the NO assay, 100 µL of the sample supernatant
was added in a 96-well plate. Greiss reagent (100 µL) was added to
each well, and the samples were incubated at room temperature for 10
min. After 10 min incubation, the absorbance was measured at 540
nm. A standard curve was made with different concentrations of NaNO2,
and the data was expressed in terms of micromoles of NaNO2. The
experiment was run three times with six replicates per data point.
The macrophage phagocytosis assay was performed per previously
established protocols (25). Briefly, J774A.1 cells were plated in 35
mm Petri dishes on day 0 (0 h) at 4 ×105cells/dish (2 mL per 35 mm
Petri dish) in RPMI-1640 medium containing 10% FBS. The plates
were incubated for6hat37°CinaCO
2incubator (5% CO2) to allow
the cells to adhere to the plates. The herbal extracts were added at
different concentrations and the plates were incubated overnight (for
about 16 h) at 37 °CinaCO
2incubator (5% CO2). The old media was
removed from the plates after 16 h, and fresh media was added onto
the cells. Yeast cells were added onto the plates at a 1:8 (macrophage/
yeast) ratio, and the cells were incubated again at 37 °CinaCO
2
incubator (5% CO2) for 1 h. At the end of 1 h, the supernatant was
discarded, and the cells were washed twice with phosphate-buffered
saline to remove unattached yeast cells. The cells were then fixed with
methanol, stained with Geimsa stain, and observed under the oil
immersion lens of the microscope for calculating the phagocytotic index.
Cells were counted in different fields, and a minimum of 100
macrophages were observed per sample. The phagocytotic index was
expressed in two sets of parameters: percentage infected macrophages
and average number of yeast per 100 infected macrophages. Attached
but noninternalized yeast were not counted. The experiment was run
three times with three replicates per data point.
The statistical analysis was performed using the GraphPad Prism
program. A one-way analysis of variance (ANOVA) was performed
on the data to analyze for significance, followed by a Neuman-Keuls
test to compare multiple samples. A value of P<0.05 was considered
to be significant.
RESULTS
ORACFL, HORAC, NORAC, SOD, and Total Phenolic
(TP) Content of Acai. H-ORACFL, L-ORACFL, HORAC,
NORAC, SOD, and total phenolics (TP) content of freeze-dried
acai are reported in Table 1. Total antioxidant capacity (TAC)
was calculated as sum of H-ORACFL and L-ORACFL.
Antioxidant Capacity from TAO. Antioxidant capacity of
freeze-dried acai from the TAO assay is shown in Figure 1.In
this assay, the antioxidant values of “slow-acting” (measured
at 30 min) and “fast-acting” (measured at 30 s) were differenti-
ated.
Inhibition of ROS Formation. Pretreatment of human
neutrophils with freeze-dried acai extracts prior to induction of
ROS by H2O2treatment resulted in a significant reduction in
8606
J. Agric. Food Chem.,
Vol. 54, No. 22, 2006 Schauss et al.
ROS production. The formation of ROS was significantly
inhibited, even at extremely low doses of freeze-dried acai.
(Figure 2).
COX Inhibition Effects. Inhibition of COX-1 and COX-2
by freeze-dried acai is shown in Table 2. The IC50 ratio of
COX-1 vs COX-2, which indicates the selectivity of the sample
in inhibition of COX enzymes, is also presented (Table 2).
Lymphocyte Proliferation Activity. Freeze-dried acai did
not show any effect on lymphocyte proliferation at the concen-
trations tested (5-1000 µg/mL) at a 24 h assay point. LPS (5
µg/mL), the positive control for the assay, demonstrated a 1.55-
fold increase in lymphocyte proliferation over cell controls. The
latter result is in keeping with the data usually obtained with
LPS in this assay (Figure 3).
Nitric Oxide Assay. The freeze-dried acai did not show any
effect on NO release by J774A.1 macrophages at the above
concentrations (250-2500 µg/mL) at a 48 h assay point. LPS
(5 µg/mL), the positive control for the assay, demonstrated a
15.66-fold increase in NO release over cell controls. Freeze-
dried acai at the above concentrations demonstrated a significant
dose-dependent inhibition of LPS-induced nitric oxide in this
assay (Figure 4).
Macrophage Phagocytosis Activity. Freeze-dried acai at
5-250 µg/mL increased macrophage infection by about 1.4-
1.5-fold over control values. However, this effect did not appear
to be dose dependent, and infection levels came down to control
values with 500 µg/mL acai. There was a significant increase
in the number of yeast engulfed per macrophage at 5 µg/mL
acai, but the effect was not evident at higher concentrations
(Figure 5).
DISCUSSION
An Internet search using the words “acai and antioxidant”
entered into Google.com resulted in over 200 000 hits. Other
than its antioxidant capacity, it was interesting to note how many
health benefits were reported for acai. Yet, little research has
been reported in the literature, while even less existed to support
its claimed health benefits. In this study, antioxidant capacities
of acai were investigated by different assays in an effort to fully
understand the scope of its antioxidant capacities. Moreover,
other possible bioactivities of acai related to inflammatory
processes and its effect on markers related to immune function
were also performed. Due to the complexity of the antioxidant
defense system and involvement of many different types of free
radicals in the body, a single antioxidant assay cannot provide
us a complete picture of the antioxidant capacity of a given
food in Vitro, much less in ViVo. Thus, several different
antioxidant assays were used to study the antioxidant capacity
of acai in Vitro.
Table 1.
Antioxidant Capacity from ORAC Assay with Different Free Radicals, SOD Assay, and Total Phenolics (TP) of Freeze-Dried Acai and Other
Acai Products
a
sample H-ORAC
FL
b
(
µ
mol TE/g) L-ORAC
FL
c
(
µ
mol TE/g) TAC
d
(
µ
mol TE/g) NORAC
(
µ
mol TE/g) HORAC
(
µ
mol GAE/g) SOD
(unit/g) TP
e
(mg GAE/g)
freeze-dried acai 997 30 1027 34 52 1614 13.9
a
Data was expressed as mean of duplicate measurements.
b
Hydrophilic ORAC
FL
.
c
Lipophilic ORAC
FL
.
d
Total antioxidant capacity, calculated as the sum of H-ORAC
FL
and L-ORAC
FL
.
e
Total phenolics.
Figure 1.
Total antioxidant (TAO) activity of freeze-dried acai, in which
TAO assay differentiates antioxidant into “slow-acting” (30 min) and “fast-
acting” (30 s) components.
Figure 2.
Freeze-dried acai reduced the H
2
O
2
-induced formation of
reactive oxygen species (ROS) in freshly purified human neutrophils.
Table 2.
Results from COX Assay of Freeze-Dried Acai
sample
IC
50
of
COX-1
a
(mg/mL)
IC
50
of
COX-2
a
(mg/mL)
IC
50
ratio
(COX-1 vs
COX-2)
freeze-dried acai 6.96 12.50 0.56
a
The result is reported as the IC
50
(50% enzyme activity inhibition concentration).
Figure 3.
Effect of freeze-dried acai on lymphocyte proliferation.
Antioxidant Capacity and Bioactivities of Acai
J. Agric. Food Chem.,
Vol. 54, No. 22, 2006 8607
The original ORACFL assay was designed to measure the
antioxidant capacity of foods toward peroxyl radicals, and it
can be conducted to measure both hydrophilic and lipophilic
antioxidants (16,17). From our results, the H-ORACFL of freeze-
dried acai was 996.9 µmol TE/g, which is significantly higher
than that of most dark colored berry or any fruit or vegetable
tested to date when appropriately converting fresh weight to
dry weights (14). The L-ORACFL of freeze-dried acai was 30
µmol TE/g, thereby yielding a total ORAC of 1026.9 µmol TE/
g. Contradictorily and surprisingly, the contents of anthocyanins,
proanthocyanidins, and other polyphenol compounds in this
freeze-dried product were found to be much lower than those
found in blueberry or any other berries with elevated H-ORACFL
values. To make things even more confusing, the total phenolics
in acai was found to be only 13.9 mg/g GAE. In a recent paper,
the ratio between hydrophilic ORACFL and total phenolics was
found to vary dramatically from less than 2 to more than 100
for different groups of foods (14). For most fruits and vegetables,
this ratio is about 10. However, the ratio in acai is 50, five times
greater than that found for any other fruit. This “unusual” ratio
raises questions whether acai contains much stronger antioxi-
dants than those found in other berries on an equal weight basis.
Determining which antioxidants contributed to this unusual ratio
warrants further work.
Freeze-dried acai has an oily feel when rubbed between the
fingers, suggesting that acai contains fairly large amounts of
lipophilic compounds. The L-ORACFL is 29.6 µmol TE/g, which
is higher than any berry samples tested to date (14).
HORAC and NORAC, two assays developed from ORAC,
were adopted to measure antioxidant capacity of acai toward
OH•and ONOO-, two of the major cell-killing ROS in the
human body (26). The HORAC value of freeze-dried acai was
52 µmol GAE/g, which is similar to that of grapes but lower
than that of dark colored berries (18). From our limited data
(unpublished data), the NORAC value of freeze-dried acai is
among the average of other fruits.
Superoxide (O2•-) is believed to be the cause of other ROS
formations such as hydrogen peroxide, peroxynitrite, and
hydroxyl radicals. Therefore, O2•- scavenging capacity in the
human body is the first line of defense against oxidative stress.
It has been reported that overexpression of superoxide dismutase
and catalase in transgenic flies extended life-span by as much
as one-third, perhaps, due to decreased oxidative stress reflected
by lower protein carbonyl contents (27). Superoxide scavenging
capacity in blood is considered very important in maintaining
antioxidant status. The most studied SOD from any natural
source is wheat sprout SOD, ranging from 160 to 500 units/g
for different samples (unpublished data). The SOD of acai was
1614 units/g, meaning acai has extremely high scavenging
capacity to O2•-, by far the highest of any fruit or vegetable
tested to date.
The total antioxidant (TAO) assay was developed to permit
rapid and simple determination of a sample’s antioxidant
capacity. The TAO assay is based on the iodine-iodide
oxidation-reduction (redox) reaction, with the formation of
iodide in the sample proportional to the antioxidant (or reducing)
capacity of the sample. The TAO assay also differentiates
antioxidants into a “slow-acting” component, which includes
complex organic antioxidants (e.g., phenolics) and a “fast-
acting” or “vitamin-C-like” component. The “fast-acting” an-
tioxidants were measured at 30 s, whereas the “slow-acting”
antioxidants were measured at 30 min. The combination of these
values is the total antioxidant capacity (28). The TAO assay
results for freeze-dried acai clearly showed that the antioxidant
capacity of “slow-acting” antioxidants was stronger than that
of “fast-acting” antioxidants (Figure 1).
Freeze-dried acai was also assayed for inhibition of ROS
formation in freshly purified human neutrophils. Freeze-dried
acai demonstrated a substantial inhibitory effect on the ROS
formation in human neutrophil cells (Figure 2). Freeze-dried
Figure 4.
Effects of freeze-dried acai on nitric oxide release by J774
cells (A) and on LPS-stimulated nitric oxide release by J774 cells (B).
Figure 5.
Effects of freeze-dried acai on J774 macrophage infection by
yeast cells (A) and number of yeast engulfed by J774 macrophage (B).
8608
J. Agric. Food Chem.,
Vol. 54, No. 22, 2006 Schauss et al.
acai displayed a maximum effect at a concentration of 1-10
parts per trillion (ppt, v/v). A nonmonotonic dose response was
observed, which is typical in this type of in Vitro cell-based
assay, where a complex blend of active ingredients results in
multiple factors contributing to intracellular oxidative stress. The
ROS inhibition by the freeze-dried acai extract was effective at
extremely low doses. The level of ROS formation was not
brought back to the level of the positive control at any of the
acai dilutions tested, including 0.1 ppt. This data indicates that
the active antioxidant compounds in the freeze-dried acai are
able to enter human cells in a fully functional form and perform
oxygen quenching at extremely low doses.
Other Bioactivities of Acai. Antioxidants are surely not the
only reason that we should eat fruits and vegetables. There are
hundreds and even sometimes thousands of compounds in foods.
Many of them contribute to health benefits through mechanisms
other than antioxidant activity. Proteins in acai pulps have been
found to show high antitryptic activity and considerable
inhibition activity toward human salivary R-amylase (29). The
effects of acai polyphenolics on the antiproliferation and
induction of apoptosis in HL-60 human leukemia cells have
also been investigated (30). Hence, we primarily focused on
the possible effects of freeze-dried acai on immune parameters
and some anti-inflammatory markers.
The lymphocyte proliferation assay (LPA) is a measure of
immune activation/stimulation. It measures the ability of
lymphocytes placed in short-term tissue culture to undergo
clonal proliferation when exposed to a foreign substance/
mitogen. This assay helps evaluate the immunostimulatory/
immunosuppressive activity of a mitogen. Freeze-dried acai fruit
demonstrated no significant effect on lymphocyte proliferation
across a very wide concentration range (5-1000 µg/mL) in this
assay (Figure 3)(31).
Nitric oxide (NO) is an inorganic free radical that functions
as an intracellular messenger and effector molecule. It is
produced during the conversion of arginine to citrulline and its
production is catalyzed by the enzyme nitric oxide synthase
(NOS) (32). NOS has three isoforms: NOS I, II, and III. Out
of these three isoforms, only NOS II is inducible and is produced
during macrophage activation (33). Macrophage activation is
thus accompanied by the induction of inducible nitric oxide
synthase and sustained release of NO (34). Synthesis of NO
endows macrophages with cytostatic or cytotoxic activity against
viruses, bacteria, fungi, protozoa, helminths, and tumor cells
(35,36). Freeze-dried acai fruit did not affect nitric oxide release
by J774A.1 macrophages at a concentration of 250-2500 µg/
mL (Figure 4).
Neutrophils/macrophages play a major role in phagocytosis
of microorganisms and other foreign entities that enter the body.
Compounds that increase the phagocytic capacity of these cells
are potent immunostimulators. Thus, this assay can be used to
gauge the potential immunostimulatory effect of a substance.
Freeze-dried acai was found to increase macrophage activity
slightly (1.4-1.5-fold over control values) at concentrations of
5-250 µg/mL (Figure 5). However, this effect did not appear
to be dose dependent, and activity levels came down to control
values at 500 µg/mL. There was also a significant increase in
the number of yeast engulfed per macrophage at 5 µg/mL, but
the effect was not statistically significant at higher concentra-
tions. This suggests that lower concentrations of freeze-dried
acai may be activators of macrophage phagocytosis but possibly
not at higher concentrations. Thus on the whole, it appears that
freeze-dried acai probably possesses minimal immunostimula-
tory properties at concentrations higher than 5 µg/mL. Lower
concentrations (less than 5 µg/mL) might be immunostimulatory,
but additional study for this is needed. We also observed an
inhibition in nitric oxide levels within J774A.1 macrophages
with acai treatment. Since increased nitric oxide is associated
with increased killing of microorganisms, and we have examined
only initial macrophage phagocytosis and not yeast killing at
later time points, it is probable that acai does not significantly
enhance immunity in Vitro.
Interestingly, the freeze-dried acai at 250-2500 µg/mL
demonstrated a significant dose-dependent inhibition of LPS-
induced nitric oxide (Figure 4). Since inhibition of LPS-induced
nitric oxide has been correlated with anti-inflammatory activity
(37), this result suggests that the freeze-dried acai may be used
as a potent anti-inflammatory substance and thus may find
applications in allergic and autoimmune disorders.
Only recently has the mechanism of botanicals been inves-
tigated at the molecular biology level by using COX-1 and
COX-2 inhibitory assays to measure the pain-relieving and anti-
inflammatory potential of herbal supplements (38). Freeze-dried
acai showed mild inhibition capacity in Vitro based on the
COX-1 and COX-2 assays (Table 2). The IC50 ratio of COX-1
vs COX-2 indicates the selectivity of the sample in inhibition
of COX enzymes. When the ratio is one, there is no selectivity.
If the ratio is smaller than one, the sample inhibits COX-1 better
than COX-2. If the ratio is larger than one, the sample inhibits
COX-2 better. Therefore, freeze-dried acai inhibits the COX-1
enzyme more efficiently than the COX-2 enzyme.
Conclusion. In this study, freeze-dried acai fruit pulp/skin
powder has been shown to be extremely powerful in its
antioxidant properties against superoxide (O2•-) by SOD assay.
The freeze-dried acai fruit pulp/skin powder was also shown to
be excellent against the peroxyl radical (RO2•), with the highest
reported total ORAC (1026.9 µmol TE/g) of any fruit or
vegetable, and mild against both peroxynitrite (ONOO-) and
hydroxyl radical (OH•) by ORACFL-based assays. In addition,
this freeze-dried acai was found to be a potential COX-1 and
COX-2 inhibitor. These findings may have significant value as
to this fruit’s antioxidant role in aging and disease. Although
this study proved that antioxidants in freeze-dried acai are able
to enter human cells in a fully functional form in Vitro, more
studies are warranted to determine safety and efficacy of acai
in ViVo.
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