antinflammatory

1. Journal of Ethnopharmacology 107 (2006) 291–296
Inhibitory effects of Euterpe oleracea Mart. on nitric oxide
production and iNOS expression
Maria Eline Matheusa, Sidnei Bessa de Oliveira Fernandesb, Cristiane Silva Silveirab,
Verˆonica Pinto Rodriguesb, Fabio de Sousa Menezesb, Patricia Dias Fernandesa,∗
a Departamento de Farmacologia B´asica e Cl´ınica, ICB, Caixa Postal: 68016, Universidade Federal do Rio de Janeiro, 21944-970 Rio de Janeiro, Brazil
b Departamento de Produtos Naturais e Alimentos, Faculdade de Farm´acia, Universidade Federal do Rio de Janeiro, Brazil
Received 9 August 2005; received in revised form 24 February 2006; accepted 15 March 2006
Available online 22 March 2006
Abstract
The palm Euterpe oleracea is a plant of great economic value in Brazil. Although the heart of palm extracted from its trunk is considered a
delicacy the world over, its fruits are popular only among Brazilians. In some poor regions of Brazil, there are reports on the popular use of its
juice in the treatment of several disorders, mainly those of oxidative onset as cardiovascular ones. Because of its wide utilization; because there
are very few scientific studies of this species, and to discover if its use in folk medicine for problems related with oxidation is in fact justifiable,
we decided, in this study, to evaluate the effects of Euterpe oleracea flowers, fruits and spikes fractions on: nitric oxide (NO) production, NO
scavenger capacity, and on the expression of inducible nitric oxide synthase enzyme, as well. Results showed that the fractions obtained from fruits
were the most potent in inhibiting NO production, followed by those from flowers and spikes. Only in high doses, did some fractions reduce cell
viability. Reduction on NO production was not due to NO scavenger activity. These results were accompanied by inhibition of iNOS expression.
The more pronounced effect was observed in the fractions in which the concentration of cyanidin-3-O-glucoside and cyanidin-3-O-rhamnoside
were higher. To sum up, our results indicate that fractions from Euterpe oleracea inhibits NO production by reducing the levels of inducible nitric
oxide synthase expression.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Nitric oxide; inducible nitric oxide synthase; Euterpe oleracea
1. Introduction
Nitric oxide is a water-soluble gas involved in physiolog-
ical and pathological conditions such as vasodilation, host
defense, tumor cell death and apoptosis (Walter, 1989; Rapoport
and Murad, 1993; Zhao et al., 2000; Monteiro et al., 2004).
The action of the enzyme nitric oxide synthase (NOS) in the
amino acid l-arginine leads to the production of nitric oxide
(NO). Physiological conditions involve endothelial or neuronal
NOS (eNOS or nNOS, respectively) (Bredt and Snyder, 1992;
Schmidt and Walter, 1994). In pathological conditions, over-
Abbreviations: FlT, ethanolic extracts of flowers; FrT, fruits; SpT, spikes;
FlE, ethyl acetate fraction of flowers; FrE, fruits; SpE, spikes; FlB, butanolic
fraction of flowers; FrB, fruits; SpB, spikes
∗ Corresponding author. Tel.: +55 21 2562 64 42/55 11 81111623;
fax: +55 21 2562 64 42/55 11 30917744.
E-mail address: patfern@farmaco.ufrj.br (P.D. Fernandes).
production of NO occurs after the induction and expression of
the inducible nitric oxide synthase (iNOS) in response to agents
such as interleukin 1␤, tumor necrosis factor-␣, interferon-␥
(IFN-␥) lipopolisaccharide (LPS) in different cells, including
macrophages, endothelial cells and hepatocytes (Moncada and
Higgs, 1993). The use of agents that inhibit activity and/or induc-
tion of iNOS may be a useful tool with therapeutic focus in many
inflammatory processes.
Euterpe oleracea Mart. (Arecaceae), popularly known in
Brazil as “ac¸a´ı”, is an economically important plant found
throughout the country. Although the heart of palm extracted
from its trunk is considered a delicacy the world over, its fruit
is consumed only in Brazil. There are several non-published
reports (popular relates) indicating the positive effect of the
medicinal use of a¸ca´ı juice, especially among people in the poor-
est regions of Brazil (mainly the North and the Northeast), as a
deterrent in cases of fever, pain and the flu (Menezes, personal
communication). Because its pharmacological properties and
0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2006.03.010

2. 292 M.E. Matheus et al. / Journal of Ethnopharmacology 107 (2006) 291–296
bioactive constituents have not yet been fully characterized, and
also because of the significance of its juice in folk medicine,
a study of its pharmacological effects is overdue. Moreover,
a great variety of a¸ca´ı products are now being produced and
commercialized as possessing anti-ageing properties and antiox-
idant activity (Menezes et al., 2005). They are also being used
topically for the management of inflammatory skin conditions
associated with acne, for example. However, there is no scien-
tific study that proves its antioxidant, anti-ageing qualities, or
even its anti-inflammatory activity. Therefore, as the plant is
widely used in folk medicine by the North and Northeast people
in Brazil and is also, in parts and or extracts (fractions), the basis
of several products commercially available in pharmacies and
drugstores (even with no governmental official permission), we
decided to start this study.
In this paper, we show that some fractions obtained from
different parts of Euterpe oleracea inhibited NO production by
RAW 264.7 cells stimulated with LPS and IFN-␥, and also that
some fractions developed inhibitory activity on NO production
by inhibiting iNOS enzyme expression.
2. Materials and methods
2.1. Reagents
Lipopolysaccharide (from Salmonella thyphimurium), NG-
monomethyl-l-arginine (l-NMMA), 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT), RPMI 1640
medium, fetal calf serum, 96-well microplates were purchased
from Sigma. Rutin was purchased from Merk. Nitrocellulose
membranes (250 nm) were from Bio Rad, anti-mouse iNOS anti-
body was purchased from Sigma, anti-mouse IgG antibody con-
jugated to horseradish peroxidase and enhanced chemilumines-
cence (ECL) kit were purchased from Amersham. Cyanidin-3-
O-glucoside and cyanidin-3-O-rhamnoside were acquired from
Extrasynthese (Lyon, France).
2.2. Preparations of Euterpe oleracea fractions
The plant material, separated in flowers, fruits and spikes,
was collected in the district of Imperatriz, Maranh˜ao, Brazil,
in February 2000. A herbarium sample (Voucher number 179)
has been deposited at the Atipo Ceabra Herbarium, Universi-
dadeFederaldoMaranh˜ao,Brazil.Crudeethanolicextractswere
separately obtained from the different parts by static maceration
with ethanol 70◦ (150 g/5 l; 600 g/20 l; 300 g/10 l, respectively)
for 72 h each 2.5 l of ethanol. The ethanolic extracts obtained
from fruits, flowers and spikes were, then, dried under reduced
pressure and, after total dryness and suspension in water, they
weresubmittedtoaliquid–liquidextractionprocedurewithethyl
acetate, followed by n-butanol so as to obtain fractions with
different polarities (ethyl acetate first and butanol after). Each
fraction received the following code: Total ethanolic extracts
from Flowers (FlT), Fruits (FrT), Spikes (SpT); Ethyl acetate
fraction from Flowers (FlE), Fruits (FrE), Spikes (SpE), and
Butanolic fraction from Flowers (FlB), Fruits (FrB) and Spikes
(SpB).
2.3. Chemical analysis of Euterpe oleracea fractions
A Lachrom HPLC system (Merck, Rio de Janeiro, RJ, Brazil)
equipped with a model D7000 interface, an L-7100 pump, an
L-7450A diode array detector (DAD) and an L-7612 solvent
degasser was used for the analysis of the polar fractions. For the
ethyl acetate and butanol fractions and also for the crude ethano-
lic extract, the analysis was made using a HPLC/DAD system
with a Lachrom RP-18 Column (250 mm × 4.5 mm, 4.5 ␮m par-
ticle) eluted with a binary high pressure gradient at a flow
rate of 1 ml min−1; solvent A, H2O:HCOOH, 9/1; solvent B
H2O:HCOOH:CH3CN, 4/1/5. After an initial hold of 1 min, the
percentage of solvent B was increased linearly from 12 to 30%
for 25 min; then, to 100% for an additional 9 min. The column
was then reconditioned with the initial mobile phase for about
20 min. The absorbance detection was on 518 nm (Mandello et
al., 2000). In order to calculate the standard error of the mean in
the chromatographic analysis of anthocyanins aiming to achieve
the concentration of each one there were made three injections
for each plant part extract.
2.4. Cell culture
RAW 264.7 mouse monocyte-macrophages (ATCC TIB-71)
were grown in plastic bottles in a RPMI 1640 medium sup-
plemented with 10% fetal bovine serum, penicillin (100 U/ml),
streptomycin (100 ␮g/ml), glutamine (2 mM) and HEPES
(15 mM) (from now named RPMI) in a humidified atmosphere
containing 5% CO2 and 95% air at 37 ◦C. When cultures formed
a confluent, monolayer cells were scrapped, centrifuged and put
to adhere in 96 or 12 wells plate with RPMI at a density of
2 × 106 cell/ml in final volumes of 250 ␮l or 2 ml, respectively
(Raschke et al., 1978).
2.5. Cell viability assay
The mitochondrial-dependent reduction of 3-(4,5-
dimethylthizaol-2yl)-2,5-diphenyltetrazolium bromide (MTT)
to formazan was used to measure cell respiration as an indicator
of cell viability (Denizot and Lang, 1986). Briefly, after 24 h
incubation of RAW 264.7 adherent cells with or without
fractions (1–300 ␮g/ml), supernatants were changed by 100 ␮l
of RPMI containing 500 ␮g/ml MTT and cells incubated for
1 h at 37 ◦C in a 5% CO2 atmosphere. After the medium were
aspirated, 100 ␮l of DMSO was added to the cells to dissolve
the formazan. The absorbance from each group was measured
in a Dynatech microplate reader at 570 nm. The control groups
consisted of cells with medium and was considered as 100% of
viable cells. Results are expressed as percentage of viable cells
when compared with control groups.
2.6. Nitric oxide-trapping capacity of Euterpe oleracea
fractions
To test the capacity of Euterpe oleracea fractions in trapping
nitric oxide, we used a cell-free system. SNAP (s-nitroso
n-acetyl dl-penicillamine) was used, as, when in solution,

3. M.E. Matheus et al. / Journal of Ethnopharmacology 107 (2006) 291–296 293
it liberates to the medium nitric oxide that transforms to
nitrite (Field et al., 1978). The addition of a NO scavenger
to the SNAP solution results in a decay in the supernatant
nitrite accumulation. Using this protocol, each fraction (in
doses of 100 ␮g/ml) was incubated with 1 mM of SNAP.
Positive groups were composed by rutin (at 1 mM). Cyanidin-
3-O-glucoside and cyanidin-3-O-rhamnoside were used at
200 ␮M. After 6 h of incubation, an aliquot of supernatant was
removed to quantify the nitrite accumulated by Griess reaction
(Green et al., 1982). Results are expressed as ␮M of nitrite
calculated in comparison with the sodium nitrite standard
curve.
2.7. Quantification of nitric oxide production
To evaluate NO production, nitrite concentration in the super-
natants of RAW 264.7 adherent cells was measured using the
Griess reaction (Green et al., 1982). Briefly, cells were acti-
vated with LPS (100 ng/ml) plus IFN-␥ (10 U/ml). After 24 h of
incubation with fractions (1–300 ␮g/ml), 100 ␮l of the super-
natant was collected and mixed with equal volume of Griess
Reagent (1% sulphanilamide, 0.1% naphthylethylene diamine
dihydrochloride, 10% H3PO4) for 10 min at room temperature.
The absorbance was measured at 540 nm using a Dynatech
microplate reader, and the nitrite concentration was calculated
using a standard curve of sodium nitrite.
2.8. Detection of inducible nitric oxide synthase (iNOS)
enzyme expression
After the activation of RAW 264.7 adherent cells with
LPS/IFN-␥ and addition of fractions of Euterpe oleracea, cul-
tures were incubated for 6 h. At the end of the incubation
period, the cells were washed in cold PBS and lysated in a cold
lysis buffer (10% NP40, 150 mM NaCl, 10 mM Tris HCl pH
7.6, 2 mM PMSF, 5 ␮M Leupeptin). Cell debris were removed
by centrifugation (12,000 × g, 4 ◦C, 10 min). After the protein
concentration for each aliquot were determined by the BCA
method (BCATM Protein Assay Kit, Pierce), suspensions were
boiled in an application buffer (100 mM DTT, 0.1% Bromophe-
nol Blue). For SDS-PAGE, aliquots of 25 ␮g of protein from
each sample were subjected to electrophoresis in 10% poly-
acrylamide gel. After electrophoresis, the proteins were elec-
trophoretically transferred into nitrocellulose membrane. Mem-
branes were blocked with 5% nonfat dried milk in Tris buffered
saline-Tween (TBS-T, 10 mM Tris–HCl, 150 mM NaCl, 0.1%
Tween 20) at room temperature for 2 h. After washing in TBS-T
primary antibody solution, mouse monoclonal IgG was applied
overnight at 4 ◦C against iNOS at dilution of 1:2000. Mem-
branes were washed in TBS-T and secondary antibody solution;
anti-mouse IgG antibody conjugated to horseradish peroxidase
at a dilution of 1:10,000 was, then, applied for 1 h at room
temperature. The blots were washed twice in TBS-T, incubated
in enhanced chemiluminescence reagent (ECL) and exposed to
photographic film (Kodak, Brazil). Images were collected and
bands intensity were calculated using DigDoc100 (Alpha Ease
FC software) program.
Table 1
Amount of the anthocyanins contents in the Euterpe oleracea fractions
Samples Cyanidin-3-O-glucoside
amount (%)
Cyanidin-3-O-rhamnoside
amount (%)
ST 12.0 ± 0.4 5.0 ± 0.2
FlT 31.0 ± 0.2 11.0 ± 0.3
FrT 42.0 ± 0.4 15.0 ± 0.6
SB 7.0 ± 0.4 3.0 ± 0.1
FlB 14.0 ± 0.7 8.0 ± 0.1
FrB 30.0 ± 0.6 10.0 ± 0.4
SE 9.0 ± 0.4 3.0 ± 0.1
FlE 18.0 ± 0.1 6.0 ± 0.7
FrE 25.0 ± 0.5 9.0 ± 0.1
Results are expressed as mean ± S.E.M. (n − 3) of percentage of cyanidin-3-O-
glucoside or cyanidin-3-O-rhamnoside.
2.9. Statistical analysis
The results are presented as the mean ± S.E.M. (n = 6). Sta-
tistical significance between groups was performed by the appli-
cation of analyses of variance ANOVA followed by Bonferroni’s
test. p Values less than 0.05 (p < 0.05) were used as the signifi-
cant level.
3. Results
3.1. Chemical analysis of Euterpe oleracea
Cyanidin-3-O-glucoside (C3G, retention time = 11.6 min)
and cyanidin-3-O-rhamnoside (C3R, retention time = 12.3 min)
were identified in all tested samples. The most prominent con-
centrations of both compounds were observed on the total
ethanolic extracts from fruits (FrT) with 42 and 15%, respec-
tively. When a composition of each fraction was made, it was
observed that the fruit fractions were again those with greater
concentration of C3G and C3R. The concentration of each com-
pound in each fraction is shown in Table 1.
3.2. Effects of fractions from Euterpe oleracea on NO
production and cell viability
The stable metabolite of NO nitrite, was accumulated and
measured in the supernatant medium after 24 h of incuba-
tion with LPS/IFN-␥. Nitrite concentration in the control
group (without stimulation) was 1.9 ± 0.6 ␮M, and when cells
were activated with LPS/IFN-␥, nitrite concentration was
39.5 ± 1.4 ␮M (mean ± S.E.M., six experiments in triplicate).
The inhibitor of iNOS, l-NMMA (300 ␮M) potently blocked
NO production and reduced values to 3.1 ± 0.9 ␮M. Each frac-
tion was tested in cell viability (by MTT method) and NO pro-
duction assays. Cell viability with all fractions varied between
85 and 100% to doses of 300 ␮g/ml. Even when doses were as
higher as 500/␮g ml reduction on cell viability did not overcome
42%. When fractions were evaluated on NO production, com-
parison between them showed that flowers and fruits were the
most effective in inhibiting NO been FrT the most potent with
an IC50 of 0.9 ␮g/ml. The comparison between ethyl acetate

4. 294 M.E. Matheus et al. / Journal of Ethnopharmacology 107 (2006) 291–296
Table 2
Inhibitory effects from Euterpe oleracea or pure anthocyanins on nitric oxide
production and cytotoxicity on LPS/IFN-stimulated RAW 264.7 cells, and NO
scavenger activity from SNAP
Compound IC50
NO production Cytotoxicity NO scavenger
FlT 1.2 ␮g/ml >500 ␮g/ml >500 ␮g/ml
FlE 8.3 ␮g/ml >500 ␮g/ml >500 ␮g/ml
FlB 8.5 ␮g/ml >500 ␮g/ml >500 ␮g/ml
FrT 0.9 ␮g/ml >500 ␮g/ml >500 ␮g/ml
FrE 11.2 ␮g/ml >500 ␮g/ml 73 ␮g/ml
FrB 1.3 ␮g/ml >500 ␮g/ml 47.3 ␮g/ml
ST 270 ␮g/ml >500 ␮g/ml >500 ␮g/ml
SE 30.2 ␮g/ml >400 ␮g/ml >500 ␮g/ml
SB 34.5 ␮g/ml >400 ␮g/ml >500 ␮g/ml
C3G 39.7 ␮M >400 ␮M 150 ␮M
C3R 59.3 ␮M >400 ␮M 169 ␮M
IC50 was calculated graphically and the mean value of at least six experiments
are shown.
and the butanolic fractions from the different parts of the plant
showed that those from fruits were the most potent in reducing
NO production, followed by the flower fractions. Spikes frac-
tions presented the weakest inhibitory effect. In order to test
the effects of C3G and C3R, we incubated 100 ␮M from each
one with RAW 264.7 cells activated with LPS/IFN-␥. At dose
of 400 ␮M neither C3G nor C3R reduced cell viability more
than 42%. Calculation of IC50 in NO production to both antho-
cyanins indicated values of 39.7 and 59.3 ␮M to C3G and C3R,
respectively (Table 2).
3.3. Effects of fractions from Euterpe oleracea on NO
scavenger
Previous observations in our laboratorial studies have indi-
cated some antioxidant activity for many Euterpe oleracea frac-
tions from different parts of the palm, including the ability to
scavenge the superoxide free radical (Arruda et al., 2004). In
order to investigate if inhibitory effects of fractions on NO pro-
duction was due to NO sequestration, a “cell-free” system was
used with s-nitroso n-acetyl dl-penicillamine (SNAP) as a NO
donor in the presence or absence of fractions. As a control for the
free radical scavenger substance, rutin was used. The addition of
1 mM rutin to the SNAP solution reduced, after 6 h of incubation,
the nitrite accumulated in the supernatant in 26.9%. Incuba-
tion of crescent doses of Euterpe oleracea fractions with 1 mM
SNAP lead to a reduction on the nitrite accumulated, but only in
the ethyl acetate fruit fractions (FrE) with IC50 of 73 ␮g/ml. All
otherfractionstesteddidnotreducethelevelsofNOproducedby
SNAP when compared to NO donor alone. When C3G and C3R
were tested it was observed that both significantly reduce the
nitrite accumulated in the supernatant and IC50 values obtained
was 150 and 169 ␮M, respectively (Table 2).
3.4. Effects of Euterpe oleracea fractions on induction of
iNOS protein
iNOS was detected, at 130 kDa, after 6 h of incubation of
RAW 264.7 activated cells with LPS/IFN-␥ in the presence or
absence of the fractions (100 ␮g/ml) by 10% SDS-PAGE west-
ern blotting analysis. The most potent fractions in inhibiting the
induction of iNOS were those from flowers reducing enzyme
expression in 50%. When fruits fractions were studied on iNOS
expression it could be observed that only total ethanolic extract
(FrT) was able to significantly reduce the enzyme expression
while none of spikes fractions were able to significantly reduce
iNOS enzyme expression (Fig. 1A). Incubation of C3G or C3R
with activated cells resulted in 50 and 30% reduction on iNOS
protein (Fig. 1B).
4. Discussion
Popular medicine is common practice in countries in which
the occurrence of a diversified vegetation promotes the medici-
nal use of plants. In Brazil, the fruits of “ac¸a´ı” (Euterpe oleracea)
are very popular (juice and fruit) among the native population
of the North and Northeast Brazil. There are also several non-
published reports on the popular use of its juice in the treatment
of several disorders among poor communities. However, these
indications are subjective and lack pharmacological confirma-
tion. Recently, a group described the indication of “ac¸a´ı” juice
as clinical oral contrast agent for magnetic resonance imaging
signals of the gastrointestinal tract (Cordova-Fraga et al., 2004).
Our group had demonstrated other effects of Euterpe oleracea
such as antinociceptive and anti-inflammatory (Marinho et al.,
2003; Matheus et al., 2003). As part of our continuous interest
in Brazilian native plants and our intention of confirming the
pharmacological use of “ac¸a´ı” in the treatment of inflammatory
processes in folk medicine, we studied its effects on NO pro-
duction and cell viability. Our results demonstrate that extracts
frompartsofEuterpeoleraceainhibitedLPS/IFN-␥inducedNO
production by RAW 264.7 macrophage cell line. Some fractions
also inhibited the expression of inducible nitric oxide synthase
(iNOS) without affecting cell viability.
Chemical study using the polar fractions (butanolic and ethyl
acetate) of Euterpe oleracea has lead to the identification of
anthocyanins. This class of compound has a very important
antioxidant activity (Awika et al., 2004; Del Pozo-Insfran et
al., 2004; Garcia-Alonso et al., 2004; Williams and Grayer,
2004). Antioxidant substances with important activity have also
been described in other plants (Dreikorn, 2002; Mahady, 2002;
Banerjee et al., 2003). In our study, no fraction developed NO
scavenger activity, the exception being the ethyl acetate and
n-butanolic fractions from fruits (FrE and FrB) which showed
some activity. Even when C3G and C3R were added to the SNAP
solution, no drastic reductions were observed in the nitrite levels

5. M.E. Matheus et al. / Journal of Ethnopharmacology 107 (2006) 291–296 295
Fig. 1. Effect of Euterpe oleracea fractions on iNOS expression. RAW 264.7 cells activated or not with LPS/IFN were incubated with Euterpe oleracea fractions
(100 ␮g/ml). iNOS protein was quantified as described in the method section. Results are expressed as X ± S.E.M. (n = 6) of iNOS arbitrary units. Codes used
are: M, macrophage without activation; LI, macrophages activated with LPS/IFN-␥; T, total ethanolic extract; E, ethyl acetate fraction; B, butanolic fraction; C3G,
cyanidin-3-O-ganglioside; C3R, cyanidin-3-O-rhamnoside. *p < 0.005 when compared with LI group (ANOVA followed by Mann–Whitney test).
been IC50 of 150 and 169 ␮M. The antioxidant effect observed
to anthocyanins is related to superoxide scavenger capacity and
also to the scavenger ability observed in the evaluation by DPPH
(2,2-diphenyl-1-picrylhidrazyl) method (Menezes et al., 2005),
and seems to have no correlation with effects on NO. Menezes et
al. (2005) described how the less polar fractions from fruits have
compounds from steroidal skeleton together with great amount
of fatty acids, and the more polar ones contain, in addition to
other flavonoids, glucosyl flavonoids, mainly cyanidin deriva-
tives. This observation could explain the scavenger activity of
the FrE and FrB fractions, since some radical formed from glu-
cosyl flavonoid could be trapping the NO produced.
iNOS is the enzyme responsible by NO production in
macrophages cell lines and several other cells after activation
with LPS and/or cytokines (Moncada and Higgs, 1993). Recent
studies have demonstrated that various extracts or fractions from
plants inhibited selectively the induction and/or activity of iNOS
(Matsuda et al., 2002, 2003). One of the problems in using plant
extracts and fractions is the possible cytotoxicity resulting from
the residues of the solvents used in the preparation or from other
toxic substances present in the fractions. For this reason, we
decided to test all fractions in cell viability assay. Significant
reduction on cell viability (lesser than 80% of viable cells) was
observed only with high dose (500 ␮g/ml) in almost all fractions.
This reduction might explain the effect on NO inhibition when
this dose was used. However, the reduction on NO production
induced by others doses cannot be explained by reduction on cell
viability since this parameter is higher than 90% in all groups.
In such cases the explanation may be found in the direct effect
of fraction on NO cell production.
Aiming to elucidate the mechanism by which the fractions
reduced NO production we investigated iNOS enzyme expres-
sion. The flower fractions were those which presented significant
inhibitory effect on iNOS expression. With the single exception
of total fruit extract (FrT), none of the others reduced the lev-
els of the enzyme. Comparing results from fruits, flowers and
spikes fractions on NO production by LPS/IFN-␥ activated cells,
we may conclude that the fruit fractions demonstrate the most
pronounced effect.
These reductions on iNOS levels correlate directly with the
inhibition on NO produced by LPS/IFN-␥ activated cells thus,
explaining the mechanism by which flower fractions reduced
NO production on cells. However, the absence of inhibition on
iNOS expression in fruits and spikes fractions indicate these
effects may be due to alterations on enzyme activity and not on
their synthesis. Similar results were also observed with C3G
and C3R. Both reduced the levels of nitrite accumulated on
culture supernatant and iNOS expression enzyme, suggesting

6. 296 M.E. Matheus et al. / Journal of Ethnopharmacology 107 (2006) 291–296
that the reduction on the NO produced is paralleled by enzyme
expression. It is interesting to note that apart anthocyanins con-
centration used were higher than the amount of them on each
fraction, the effects observed were not proportional. Taking into
account that the fractions developed inhibitory effect greater
than pure anthocyanins, we must remember that Euterpe oler-
acea fractions have other substances which conjoined may be
enhancing the final effect.
In conclusion, Euterpe oleracea showed potent inhibitory
effects on NO production by activated macrophage cell line
RAW 264.7. The mechanism of inhibition seems to be due to a
reduction on iNOS expression (in flower fractions) and on iNOS
activity (fruit and spike fractions).
Acknowledgments
FSM received grants from FAPERJ and FUJB and fellowship
from CNPq.
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