Antioxidant Action of an Ethanol Extract of Ptychopetalum olacoides

Abstract

Alcohol infusions of roots from Ptychopetalum olacoides Bentham (PO; Olacaceae) have been used for treating many diseases in which free radicals are likely to be implicated. Of particular interest are the uses amongst the elderly (to ameliorate cognitive functions), and by patients recovering from pathologies associated with damage to the central nervous system (such as stroke). The aim of this study was to evaluate the antioxidant properties of a PO ethanol extract (POEE) by using various in vitro systems. POEE acted as a scavenger of nitrogen oxides as well as superoxide generated by the xanthine-xanthine oxidase system. The extract also showed a high antioxidant capacity using a luminol chemiluminescence derived from a thermolabile diazocompound. We suggest that the therapeutic effects attributed to P. olacoides could be in part associated to its oxygen free radical scavenging capacity.

Full text

Phytomedicine 14 (2007) 763–769
Antioxidant activities of Ptychopetalum olacoides
(‘‘muirapuama’’) in mice brain
I.R. Siqueira
b,c
, C. Fochesatto
a
, I.L.S. Torres
b
, A.L. da Silva
b
, D.S. Nunes
d
,
E. Elisabetsky
b,c
, C.A. Netto
a,c,
a
Departamento de Bioquı
´
mica, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600, Pre
´dio Anexo, 90035-003
Porto Alegre RS, Brazil
b
Departamento de Farmacologia, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 500, sala 202, 90050-170
Porto Alegre RS, Brazil
c
PPG-Fisiologia, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 500, 90050-170 Porto Alegre RS, Brazil
d
Departamento de Quı
´
mica, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil
Abstract
Ptychopetalum olacoides (PO) roots are used by Amazonian peoples to prepare traditional remedies for treating
various central nervous system conditions in which free radicals are likely to be implicated. Following the identification
of PO ethanol extract (POEE) free-radical scavenging properties in vitro, the aim of this study was to verify the in vivo
antioxidant effect of POEE. Aging mice (14 months) were treated (i.p.) with saline, DMSO (20%) or POEE (100 mg/kg
body wt.), and the hippocampi, cerebral cortex, striata, hypothalamus and cerebellum dissected out 60 min later to
measure antioxidant enzyme activities, free-radical production and damage to macromolecules. POEE administration
reduced free-radical production in the hypothalamus, lead to significant decrease in lipid peroxidation in the cerebral
cortex, striatum and hypothalamus, as well as in the carbonyl content in cerebellum and striatum. In terms of
antioxidant enzymes, catalase activity was increased in the cortex, striatum, cerebellum and hippocampus, while
glutathione peroxidase activity was increased in the hippocampus. This study suggests that POEE contains compounds
able to improve the cellular antioxidant network efficacy in the brain, ultimately reducing the damage caused by
oxidative stress.
r2006 Elsevier GmbH. All rights reserved.
Keywords: Ptychopetalum olacoides; Marapuama; Muirapuama; Antioxidant activity; Brain; Aging
Introduction
Among the numerous hypotheses developed to under-
stand the aging process, the free-radical theory of aging
has become especially prominent (Harman, 1956). Free
radicals, such as hydrogen peroxide, superoxide anion,
hydroxyl, alkoxyl and peroxyl radicals, are continuously
produced during oxidative metabolism; in excess they
may lead to direct oxidation of critical biological
molecules (e.g., membranous lipid peroxidation, specific
protein destruction and DNA strand breaks). Accord-
ingly, cell defense mechanisms to reduce oxidative
damage include enzymatic systems: superoxide dismutase
(SOD) converts superoxide radicals into H
2
O
2
;catalase
ARTICLE IN PRESS
www.elsevier.de/phymed
0944-7113/$ - see front matter r2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2006.12.007

Corresponding author. Departamento de Bioquı
´mica, Universi-
dade Federal do Rio Grande do Sul, Rua Eurı
´pedes M. Duarte 10,
Nonoai, 305, 90830-250, Porto Alegre RS, Brazil.
Tel.: +55 51 316 5577; fax: +55 51 316 5540.
E-mail address: alex@prograd.ufrgs.br (C.A. Netto).

(CAT) is responsible for detoxification of hydrogen
peroxide (H
2
O
2
); and glutathione peroxidase (GPx)
breaks down peroxides, notably those derived from the
oxidation of membrane phospholipids. Non-enzymatic
antioxidants, such as vitamins E and C, glutathione and
carotenoids, also play important roles in antioxidant
defense mechanisms (Halliwell, 1992). Oxidative stress,
the result of an imbalance between free-radical-generat-
ing and free-radical-scavenging systems, has been asso-
ciated with various pathologies, including those affecting
the central nervous system (Halliwell, 1992).
Medicinal plants are commonly used for treating CNS
disorders in traditional medicinal systems in Brazil.
Alcoholic infusions of Ptychopetalum olacoides Bentham
(PO, Olacaceae), known as ‘‘muirapuama’’ and ‘‘mar-
apuama’’, are consumed in the Amazon for the
treatment of CNS-related conditions or during highly
stressful periods (Elisabetsky, 1987;Grenand et al.,
1987;Siqueira et al., 1998); the frequency of elders and
patients recovering from pathologies associated with
damage to the central nervous system (such as stroke)
among its users are of particular interest (Elisabetsky
and Siqueira, 1998). This plant is also widely employed
due to its alleged aphrodisiac effects. This species is
currently included in dozens of herbal drugs or multi-
vitamin dietary supplements available all around the
world that are claimed to enhance sexual, physical and
cognitive performance.
We have previously reported that a specific ethanol
extract (POEE) of PO roots possesses various central
nervous system (CNS) activities (Siqueira et al., 1998),
including mild anxiogenic effect in the hole-board test,
improvement of long-term memory retrieval in the adult
and aged mice step down paradigm (da Silva et al., 2002,
2004), and that it inhibits AChE (in vitro and ex vivo
assays, Siqueira et al., 2003), suggesting that improve-
ment in cholinergic function might be a neurochemical
correlate of the extract’s behavioral effects.
A substantial antioxidant property could be related to
some of the therapeutic properties claimed to be
associated with marapuama, as radical scavengers reverse
the loss of spatial memory and decrease damage to brain
proteins in aged gerbils and rats (Carney et al., 1991;
Socci et al., 1995). Indeed, we recently reported a marked
free-radical scavenging property of POEE in several in
vitro assays (Siqueira et al., 2002). The aim of the present
study was to evaluate the effects of POEE treatment on
the oxidative status in different brain areas of aging mice.
Materials and methods
Plant material. Roots of Ptychopetalum olacoides
Bentham (PO, Olacaceae) were collected in the State
of Para
´(Brazil) and identified by Mr. N. Rosa (voucher
MG 108036, Goeldi Museum herbarium).
Chemicals. Thiobarbituric acid and Trolox were
obtained from Merck, and ABAP from Wako Chemi-
cals Inc. (USA). 20-70-dichlorofluorescein diacetate
(DCFH-DA), 20-70-dichlorofluorescein (DCF), trichlor-
oacetic acid (TCA), 2,4-dinitrophenylhydrazine
(DNPH), guanidine hydrochloride, phenylmethylsulfo-
nyl fluoride (PMSF), 5-amino-2,3-dihydro-1,4-phtalazi-
nedione (luminol) and hydrogen peroxide stock solution
were purchased from Sigma Chemical Co.
Preparation of ethanol extract. The extractive proce-
dure has been detailed elsewhere (Siqueira et al., 1998);
briefly, milled roots were extracted (Sohxlet) with
ethanol and dried in vacuo, yielding a brown syrup
(6%). For assays, POEE was diluted in 20% DMSO.
HPLC analysis. Analytical HPLC was carried out on
a HP 1100 system equipped with a photodiode array
detector (Agilent Technologies). The extract was ana-
lyzed on a Zorbax extended C18 column (250 4.6 mm
i.d.) with the gradient: MeOH-H
2
O gradient
(10:90–100:0). The flow rate was 1 ml/min; the UV
traces were measured at 210 and 254 nm and UV spectra
(DAD) were recorded between 200 and 500 nm. Injec-
tion 20 ml (5 mg/ml). Results are presented in Fig. 1.
Animals and Treatment. Swiss albino male adult mice
(CF1 strain), 14 months of age, housed with access to
food and water ad libitum, and light-dark cycles of 12 h,
were used. DMSO 20% or POEE 100 mg/kg body wt.
were administered intraperitoneally (0.1 ml/10 g body
wt.); this dose was chosen because it proved to lessen the
cognitive deficit of aging animals in an inhibitory
avoidance task (da Silva et al., 2004).
Preparation of brain samples. Mice were decapitated
60 min after drug administration; the brain was quickly
excised. Hippocampi, cerebral cortex, striata, hypotha-
lamus and cerebellum were dissected out, and instanta-
neously placed in liquid nitrogen. Brain tissues were
homogenized in 10 vol. of ice-cold phosphate buffer
(0.1 M, pH 7.4) containing KCl 140 mM, EDTA (1 mM)
and phenylmethylsulfonyl fluoride (1 mM). The homo-
genate was centrifuged at 960 gfor 10 min; the
supernatant was used for the assays.
Free radical content. To assess the free-radical levels
we used 20-70-dichlorofluorescein diacetate (DCFH-DA)
as a probe (Sriram et al., 1997).
Lipid peroxidation assay. The formation of thiobarbi-
turic acid reactive substances (TBARS) was based on
Bromont et al. (1989).
Determination of protein carbonyl levels. Protein
carbonyl content was determined as described pre-
viously (Levine et al., 1990).
Total antioxidant reactivity (TAR) assay. The TAR
assay is based on luminol-enhanced chemiluminescence
(CL) induced by an azo initiator (Lissi et al., 1995;
Desmarchelier et al., 1997).
Superoxide dismutase (SOD) activity. SOD acti
vity was determined using a RANSOD kit (Randox
ARTICLE IN PRESS
I.R. Siqueira et al. / Phytomedicine 14 (2007) 763–769764

Laboratories, USA) which is based on the procedure
described by Delmas-Beauvieux et al. (1995).
Catalase (CAT) activity. CAT activity was deter-
mined according to Aebi (1984).
Glutathione peroxidase (GPx) activity. GPx activity
was determined according to Wendel (1981). The
activity of selenium-dependent GPx was measured
taking tert-butyl-hydroperoxide as the substrate.
Protein assay. The total protein concentration was
determined (Bradford, 1976).
Statistical analysis. Data were analyzed using ANO-
VA with post hoc analysis by Duncan’s multiple-range
test. Results are expressed as mean7standard error of
the mean (SEM).
Results
A single i.p. administration of 100 mg/kg body wt.
POEE induced changes in most of the studied indexes of
oxidative stress; different patterns were found at
different brain regions. A significant decrease (F
(2,11)
¼
3.781; po0.05) in free radical levels (DCF formation)
was observed in the hypothalamus (Table 1). For
oxidative-induced protein damage, CARB content was
decreased in the cerebellum (F
(2,15)
¼3.431; p¼
0.05), and striatum (F
(2,17)
¼5.374; p¼0.018) (Table 1).
Lipoperoxidation (TBARS levels, Fig. 2) was decreased in
the cortex (F
(2,17)
¼4.201; p¼0.033), striatum (F
(2,15)
¼
5.973; p¼0.013) and hypothalamus (F
(2,10)
¼4.241;
po0.05). No changes in TAR levels were observed in
the brain regions examined (data not shown). In terms of
antioxidant enzyme activities, there were no changes in
SOD activity (Fig. 3A), GPx activity was increased only in
the hippocampus (F
(2,10)
¼7.384; po0.01, 33%, Fig. 3B),
and CAT activity was enhanced in the striatum
(F
(2,11)
¼6.35; p¼0.015, 33%), cerebellum (F
(2,13)
¼
4.229; po0.04, 30%), and hippocampus (F
(2,11)
¼3.781;
po0.05, 47%) of POEE treated mice (Fig. 3C).
Discussion and conclusions
The working hypothesis of this study was that the
therapeutic properties traditionally claimed for PO
could be associated with its ability to increase antiox-
idant capacity, therefore attenuating free-radical gen-
eration and the consequent damage to lipids and
proteins. Because the endogenous antioxidant defenses
are not always completely effective, as is the case in
normal aging and neurodegenerative diseases, it has
been proposed that exogenous antioxidants could
effectively restrain the cumulative effects of oxidative
damage.
ARTICLE IN PRESS
min0 5 10 15 20 25 30 35
mAU
0
100
200
300
400
500
12
4
5
673
nm300 400 500
0
50
nm300 400 500
0
50
nm300 400 500
0
50
nm300 400 500
0
50
nm300 400 500
0
50
nm300 400 500
0
50
UV 254
12
75
34
nm300 400 500
mAU
0
50
100
150
200
mAUmAU mAU
mAU
mAU
mAU
6
Fig. 1. HPLC/UV analysis of ethanol extract from Ptychopetalum olacoides (POEE).
I.R. Siqueira et al. / Phytomedicine 14 (2007) 763–769 765

Confirming previously reported results of in vivo and
in vitro activities (da Silva et al., 2002;Siqueira et al.,
1998, 2003), this study shows that an acute treatment
with POEE can improve the protective defenses against
oxidative stress in specific brain areas. This is an
important step toward verifying a physiologically
relevant contribution. The data presented here show a
decrease in free-radical generation in the hypothalamus
(but not cortex, striatum or hippocampus) obtained
from POEE-treated aged mice. POEE also reduced the
levels of two markers of oxidative stress: the formation
of TBARS, an index of lipid peroxidation, was reduced
in the frontal cortex, striatum and hypothalamus, and
the carbonyl content, an index of oxidative damage to
proteins, was diminished in the cerebellum and striatum.
It is conceivable that POEE may contribute to the
maintenance of neural membrane stability by inhibiting
lipid peroxidation. Lipid peroxidation affects membrane
integrity and is accompanied by the generation of
chemically reactive aldehyde products; these are thought
to contribute significantly to the pathological effects of
lipid peroxidation by covalently modifying cellular
macromolecules, especially proteins. Carbonyl deriva-
tives are formed by the attack of highly reactive free
radicals to amino acid residues, or by reacting with
sugars and/or products of sugar oxidation, or with lipid
peroxidation products. The oxidative modification of
proteins leads to structural changes and a consequent
inactivation of many enzymes (Levine et al., 1990). The
decline in oxidized proteins observed in brain areas of
POEE-treated animals could be the direct result of
reduced lipid peroxidation; in the case of the hypotha-
lamus, such decline could also be related to an efficient
scavenging of free radicals, as expressed by DCF levels.
Despite the fact that TAR levels (indicators of
antioxidant reactivity) of brain samples were not
affected by acute treatment with POEE, DCF formation
(measure of free radical formation) was found to be
reduced only in the hypothalamus. Data indicate that
the highly active antioxidant compound(s) responsible
ARTICLE IN PRESS
Table 1. Effects of POEE (100 mg/kg body wt.) on free radical production (DCFH-DA as probe), and protein oxidative damage
(carbonyl content) in brain regions from aging mice
DCF (nmol DCF/mg protein) CARB (nmol DNPH/mg protein)
Saline DMSO POEE Saline DMSO POEE
Cerebellum 1.8870.11 1.8470.06 2.1370.11 10877115 11897253 622762
a
Frontal cortex 1.8770.39 1.4070.36 1.6370.44 15207224 18427172 17747244
Striatum 2.2170.15 2.2470.24 2.5570.23 2411776 20107299 11647337
a
Hypothalamus 2.8370.12 2.7170.18 2.3770.15
b
11437183 14777285 17047620
Hippocampus 1.6870.24 1.3670.17 1.4170.17 28567584 19267428 20757317
Data are expressed as a mean7SEM for six to eight experiments.
a
As compared to saline and DMSO groups.
b
As compared to saline group, ANOVA followed by Duncan’s test (po0.05).
0
20
40
60
80
100
120
140
hippocampus frontal cortex striatum cerebellum hypothalamus
% vs. the saline group
saline DMSO POEE
*
#
+
Fig. 2. Effects of POEE (100 mg/kg body wt.) on lipid damage (thiobarbituric acid reactive substance, TBARS) in brain regions
from aging mice. Data expressed as percentage of saline (mean7SEM, 6–8 experiments). The absolute mean TBARS values for
saline group were 0.3470.03 (cerebellum), 0.7670.07 (frontal cortex), 0.8170.06 (striatum), 0.3470.03 (hypothalamus), 0.7670.09
(hippocampus) (pmol MDA /mg protein). ANOVA followed by Duncan’s test (po0.05),
*
as compared to saline and DMSO
groups,
#
as compared to saline group,
+
as compared to DMSO group.
I.R. Siqueira et al. / Phytomedicine 14 (2007) 763–769766

for the high TAR levels observed with POEE experi-
ments in vitro (Siqueira et al., 2002) did not reach the
brain areas studied, at least in significant amounts. The
significant decrease in free-radical content found only in
the hypothalamus suggests the presence of antioxidants
unable to cross the BBB, since the structure lacks this
barrier (Weindl and Joynt, 1973). Interestingly, several
groups have suggested that the hypothalamus is a
crucial area in mediating the age-related decline of
physiological functions and alterations of biological
rhythms (Meites et al., 1987;Carlson and Sawada, 1996;
Hofman, 1997).
The diverse pattern of effects obtained in various brain
areas can be interpreted as the result of a distinct
distribution of active compound(s), as well as to
specificities in regional antioxidant organization, since it
has well recognized that the susceptibility to oxidative
stress varies among brain regions (Candelario-Jalil et al.,
2001a;Homi et al., 2002). Bickford (1993),Bickford
et al. (1992) have suggested that the motor-dependent
ARTICLE IN PRESS
BGPx
0
3
6
9
12
15
hippocampus frontal cortex striatum cerebellum hypothalamus
nmol NADPH oxidized/min/mg
protein
*
0
50
100
150
200
hippocampus frontal cortex striatum cerebellum hypothalamus
% of control
saline DMSO POEE
CCAT #
#
+
*
SOD
0
20
40
60
80
100
120
140
hippocampus frontal cortex striatum cerebellum hypothalamus
U/ mg protein
A
Fig. 3. Effects of POEE (100 mg/kg body wt.) on antioxidant enzymes (SOD 2A, CAT 2B, and GPx 2C) activities in brain regions
from aging mice. The mean CAT values from saline groups were 677759 (cerebellum), 315719.15 (frontal cortex), 3967113
(striatum), 525772 (hypothalamus), and 322721 mU/mg protein (hippocampus). Data expressed as mean7SEM, 6–8 experiments.
Anova followed by Duncan’s test (po0.05),
*
as compared to saline and DMSO groups,
#
as compared to saline group,
+
as
compared to DMSO group.
I.R. Siqueira et al. / Phytomedicine 14 (2007) 763–769 767

learning deficits observed in aged rats is strongly related
with losses in cerebellar function, which can be modified
by nutritional intervention with antioxidants (Bickford et
al., 2000). It is therefore noteworthy that the oxidative-
protective effects of POEE seem to be particularly
relevant in the cerebellum, striatum and hypothalamus.
Augmenting body defenses against free radicals seems
to be another interesting approach for treating neuro-
degenerative disorders (Nikolov and Richardson, 1998).
The ability of POEE to enhance brain CAT activity is of
importance because catalase has very low activity in the
brain (Benzi and Moretti, 1995). Additionally, it is
relevant that catalase (mitochondrial and cytosolic) has
been found to enhance respiration maintaining adequate
ATP levels (Rodriguez et al., 2000). It has been shown
that a persistent and intense decrease in hippocampal
GPx and glutathione reductase activities occurs in
experimental models of induced neuronal damage
(Candelario-Jalil et al., 2001b). Therefore, the POEE-
induced enhancement of CAT and GPx activities in
some brain structures might be an important mechanism
to avoid hydrogen peroxide accumulation.
At this point, neither the active compound(s) nor the
exact mechanism(s) by which POEE exerts its antiox-
idant activities are completely known. Nevertheless,
b-sistosterol and lupeol, both present in P. olacoides,
have been shown to possess antioxidant properties.
Beta-sistosterol has been reported to inhibit lipid
peroxidation in vitro, at low concentrations (van
Rensburg et al., 2000), while lupeol is associated with
lipid-peroxide reduction (Nagaraj et al., 2000).
Our results are consistent with previous studies
reporting neuroprotection by antioxidants, as well as
improvements in age-related neurological decline found
with other herbal drugs (Arivazhagan et al., 2001;Ward
et al., 2002). In situations of oxidative stress, especially
in the brain, it is desirable that a combination of
complementary antioxidant properties occur. Our pre-
vious studies indicate that P. olacoides possesses
compound(s) with multiple and complementary modes
of action; in this case, free-radical scavenging and
increased activity of antioxidant enzymes might be
important neuroprotective properties.
Acknowledgments
We gratefully acknowledge financial support received
from FINEP/PRONEX, FAPERGS, CAPES, CNPq
and PROPESQ-UFRGS. The authors are grateful to
Professor Kurt Hostettmann and Dr. Emerson Ferreira
Queiroz from the Laboratoire de Pharmacognosie et
Phytochimie at the Universite
´de Gene
`ve (Switzerland)
for providing the HPLC analysis. This study is
associated/protected with patent numbers PI0205432-
9/PI0307647-4 (INPI/Br).
References
Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105,
121–126.
Arivazhagan, P., Ramanathan, K., Panneerselvam, C., 2001.
Effect of DL-a-lipoic acid on status of lipid peroxidation
and antioxidants in mitochondria of aged rats. J. Nutr.
Biochem. 12, 2–6.
Benzi, G., Moretti, A., 1995. Age- and peroxidative stress-
related modifications of the cerebral enzymatic activities
linked to mitochondria and the glutathione system. Free
Radic. Biol. Med. 19, 77–101.
Bickford, P., Gould, T., Briederick, L., Chadman, K., Pollock,
A., Young, D., Shukitt-Hale, B., Joseph, J., 2000.
Antioxidant-rich diets improve cerebellar physiology and
motor learning in aged rats. Brain Res. 866, 211–217.
Bickford, P.A., 1993. Motor learning deficits in aged rats are
correlated with loss of cerebellar noradrenergic function.
Brain Res. 620, 133–138.
Bickford, P.A., Heron, C., Young, D., Gerhardt, G.A., de la
Garza, R., 1992. Impaired acquisition of novel locomotor
tasks in aged and norephinephrine-depleted F344 rats.
Neurobiol. Aging 13, 475–481.
Bradford, M.M., 1976. A rapid and sensitive method for the
quantification of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal. Biochem. 72,
248–254.
Bromont, C., Marie, C., Bralet, J., 1989. Increased lipid
peroxidation in vulnerable brain regions after transient
forebrain ischemia in rats. Stroke 20, 918–924.
Candelario-Jalil, E., Mhadu, N.H., Al-Dalain, S.M., Martinez,
G., Leo
´n, O.S., 2001a. Time course of oxidative damage in
different brain regions following transient cerebral ischemia
in gerbils. Neurosci. Res. 41, 233–241.
Candelario-Jalil, E., Al-Dalain, S.M., Castillo, R., Martinez,
G., Fernandez, O.S., 2001b. Selective vulnerability to
kainate-induced oxidative damage in different rat brain
regions. J. Appl. Toxicol. 21, 403–407.
Carlson, J.C., Sawada, M., 1996. The free radical theory of
aging and a look at the hypothalamus. Can. J. Aging 15,
44–53.
Carney, J.M., Starke-Reed, P.E., Oliver, C.N., Landum, R.W.,
Cheng, M.S., Wu, J.F., Floyd, R.A., 1991. Reversal of age-
related increase in brain protein oxidation, decrease in
enzyme activity, and loss in temporal and spatial memory
by chronic administration of the spin-trapping compound
N-tert-butyl-a-phenylnitrone. Proc. Natl. Acad. Sci. USA
88, 3633–3636.
da Silva, A.L., Bardini, S., Nunes, D.S., Elisabetsky, E., 2002.
Anxiogenic properties of Ptychopetalum olacoides Ben-
tham (Marapuama). Phytother. Res. 16, 223–226.
da Silva, A., Piato, A., Bardini, S., Netto, C., Nunes, D.,
Elisabetsky, E., 2004. Memory retrieval improvement by
Ptychopetalum olacoides in young and aging mice. J.
Ethnopharmacol. 95, 199–203.
Delmas-Beauvieux, M.C., Peuchant, E., Dumon, M.F.,
Receuver, M.C., Le Bras, M., Clerc, M., 1995. Relationship
between red cell antioxidant enzymatic system status and
lipid peroxidation during the acute phase of malaria. Clin.
Biochem. 28, 163–169.
ARTICLE IN PRESS
I.R. Siqueira et al. / Phytomedicine 14 (2007) 763–769768

Desmarchelier, C., Repetto, M., Coussio, J., Llesuy, S., Ciccia,
G., 1997. Total Reactive Antioxidant Potential (TRAP)
and Total Antioxidant Reactivity (TAR) of Medicinal
Plants used in Southwest Amazonia (Bolivia and Peru). Int.
J. Pharmacog. 35, 288–296.
Elisabetsky, E., 1987. From indigenous disease concepts to
laboratory work hypothesis: the case of ‘‘nerve tonics’’
from the Brazilian Amazon. Provisional Report Series, vol.
19. International Foundation for Science, Stockholm, p. S-
11438.
Elisabetsky, E., Siqueira, I.R., 1998. Is there a psychophar-
macological meaning for traditional tonics? In: Prender-
gast, H.D., Etkin, N., Harris, D.R., Houghton, P.J. (Eds.),
Plants for Food and Medicine. Royal Botanic Gardens,
Kew, London, pp. 373–385.
Grenand, P., Moretti, C., Jacquemin, H., 1987. Pharmacope
´es
traditionnelles en Guyane. Editions de L’Orstom, Paris,
pp. 326–328.
Halliwell, B.H., 1992. Oxygen radicals as key mediators in
neurological disease: fact or fiction? Ann. Neurol. 32,
510–515.
Harman, D., 1956. Aging: a theory based on free radical and
radiation chemistry. J. Gerontol. 11, 298–300.
Hofman, M.A., 1997. Lifespan changes in the hypothalamus.
Exp. Gerontol. 32, 559–576.
Homi, H.M., Freitas, J.J., Curi, R., Velasco, I.T., Junior, B.A.,
2002. Changes in superoxide dismutase and catalase
activities of rat brain regions during early global transient
ischemia/reperfusion. Neurosci. Lett. 333, 37–40.
Levine, R.L., Garland, D., Oliver, C.N., Amici, A., Climent,
I., Lenz, A., Ahn, B.W., Shaltiel, S., Stadman, E.R., 1990.
Determination of carbonyl content in oxidatively modified
proteins. Methods Enzymol. 186, 464–478.
Lissi, E., Salim-Hanna, M., Pascual, C., del Castillo, M.D.,
1995. Evaluation of total antioxidant potential (TRAP) and
total antioxidant reactivity from luminol-enhanced chemi-
luminescence measurements. Free Radic. Biol. Med. 18,
153–158.
Meites, J., Goya, R., Takahashi, S., 1987. Why the neuroen-
docrine system is important in the aging process. Exp.
Gerontol. 22, 1–15.
Nagaraj, M., Sunitha, S., Varalakshmi, P., 2000. Effect of
lupeol, a pentacyclic triterpene, on the lipid peroxidation
and antioxidant status in rat kidney after chronic cadmium
exposure. J. Appl. Toxicol. 20, 413–417.
Nikolov, R., Richardson, S., 1998. Antiamyloid strategies for
the treatment of Alzheimer’s Disease. Drugs Today 34,
673–689.
Rodriguez, A., Carrico, P., Mazurkiewicz, J., Melendez, J.A.,
2000. Mitochondrial or cytosolic catalase reverses MnSOD-
dependent inhibition of proliferation by enhancing respira-
tory chain activity, net ATP production, and decreasing the
steady state levels of H
2
O
2
. Free Radic. Biol. Med. 29,
801–813.
Siqueira, I.R., Lara, D.R., Gaieski, F., Silva, F.S., Nunes,
D.S., Elisabetsky, E., 1998. Psychopharmacological Prop-
erties of Ptychopetalum olacoides (Olacaceae). Pharmac.
Biol. 36, 327–334.
Siqueira, I.R., Cordova, C.S., Creczynski-Pasa, T., Elisabets-
ky, E., Nunes, D.S., Netto, C.A., 2002. Antioxidant Action
of an Ethanolic Extract of Ptychopetalum olacoides
Bentham (Olacaceae). Pharmac. Biol. 40, 374–379.
Siqueira, I.R., Fochesatto, C., da Silva, A.L., Nunes, D.S.,
Battastini, A.M., Netto, C.A., Elisabetsky, E., 2003.
Ptychopetalum olacoides, a traditional Amazonian ‘‘nerve
tonic’’, possesses anticholinesterase activity. Pharmacol.
Biochem. Behav. 75, 645–650.
Socci, D.J., Crandall, B.M., Arendash, G.W., 1995. Chronic
antioxidant treatment improves the cognitive performance
of aged rats. Brain Res. 693, 88–94.
Sriram, K., Pai, K.S., Boyd, M.R., Ravindranath, V., 1997.
Evidence for generation of oxidative stress in brain by MPTP:
in vitro and in vivo studies in mice. Brain Res. 749, 44–52.
van Rensburg, S.J., Daniels, W.M., van Zyl, J.M., Taljaard,
J.J., 2000. A comparative study of the effects of cholesterol,
beta-sitosterol, beta-sitosterol glucoside, dehydroepian-
drosterone sulphate and melatonin on in vitro lipid
peroxidation. Metab. Brain Dis. 15, 257–265.
Ward, C.P., Redd, K., Williams, B.M., Caler, R.J., Luo, Y.,
McCoy, J.G., 2002. Ginkgo biloba extract cognitive
enhancer or antistress buffer. Pharmacol. Biochem. Behav.
72, 913–922.
Weindl, A., Joynt, R.J., 1973. Barrier properties of the
subcommissural organ. Arch. Neurol. 29, 16–22.
Wendel, A., 1981. Glutathione peroxidase. Methods Enzymol.
77, 325–333.
ARTICLE IN PRESS
I.R. Siqueira et al. / Phytomedicine 14 (2007) 763–769 769