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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.
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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.
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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
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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).
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ARTICLE IN PRESS
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... We have previously reported that the ethanol extract of PO roots possesses various central nervous system activities, including reversal of reserpine-induced ptosis, mild anxiogenic effect on the holeboard , and enhancement of memory retrieval in young and aged mice (Siqueira et al., 1998; Silva et al., 2002; da Silva, 2001). Additionally, we have recently reported marked free radicals scavenging activity of PO ethanol extract (POEE) in several in vitro assays (Siqueira et al., 2002), as well as a significant inhibition of acethylcholinesterase in different brain regions (Siqueira et al., 2003). Relevant to this study, it was also found that the acute (ip) administration of POEE in mice leads to significant reduction on free radical generation, lipid peroxidation and protein-bound carbonyl content, and increased activities of the antioxidant enzymes catalase and glutathione peroxidase, pertinent indexes of oxidative status in diverse brain structures (Siqueira et al., 2004). ...
... Taken together, these results indicate a neuroprotective effect of the extract. POEE concentrations used in this study (0.2 to 0.6 Ag/ ml) were those active at free radicals scavenging in vitro assays (total antioxidant reactivity and total antioxidant potential assays, Siqueira et al., 2002). The fact that a neuroprotective effect was observed only at the highest dose used implies that the extract sample in this ex vivo assay was retained at intra and extra intracellular compartments, effective doses starting at the highest dose here used. ...
... We here observed that POEE seems to increase respiratory chain activity, which might be related to POEE-induced increase in brain catalase activity (Siqueira et al., 2004). Furthermore, the superoxide anion scavenging porperties of POEE (Siqueira et al., 2002) is of relevance, since mitochondria complexes I and II and aconitase, iron-sulfur cluster-containing enzymes, may suffer free radical inactivation (Melov et al., 1999). It is conceivable that POEE may also inhibit the generation of free radicals, along with its scavenging activity. ...
... We have previously reported that the ethanol extract of PO roots possesses various central nervous system activities, including reversal of reserpine-induced ptosis, mild anxiogenic effect on the holeboard , and enhancement of memory retrieval in young and aged mice (Siqueira et al., 1998; Silva et al., 2002; da Silva, 2001). Additionally, we have recently reported marked free radicals scavenging activity of PO ethanol extract (POEE) in several in vitro assays (Siqueira et al., 2002), as well as a significant inhibition of acethylcholinesterase in different brain regions (Siqueira et al., 2003). Relevant to this study, it was also found that the acute (ip) administration of POEE in mice leads to significant reduction on free radical generation, lipid peroxidation and protein-bound carbonyl content, and increased activities of the antioxidant enzymes catalase and glutathione peroxidase, pertinent indexes of oxidative status in diverse brain structures (Siqueira et al., 2004). ...
... Taken together, these results indicate a neuroprotective effect of the extract. POEE concentrations used in this study (0.2 to 0.6 Ag/ ml) were those active at free radicals scavenging in vitro assays (total antioxidant reactivity and total antioxidant potential assays, Siqueira et al., 2002). The fact that a neuroprotective effect was observed only at the highest dose used implies that the extract sample in this ex vivo assay was retained at intra and extra intracellular compartments, effective doses starting at the highest dose here used. ...
... We here observed that POEE seems to increase respiratory chain activity, which might be related to POEE-induced increase in brain catalase activity (Siqueira et al., 2004). Furthermore, the superoxide anion scavenging porperties of POEE (Siqueira et al., 2002) is of relevance, since mitochondria complexes I and II and aconitase, iron-sulfur cluster-containing enzymes, may suffer free radical inactivation (Melov et al., 1999). It is conceivable that POEE may also inhibit the generation of free radicals, along with its scavenging activity. ...
Article
Alcoholic infusions of Ptychopetalum olacoides Bentham (PO, Olacaceae) are used in traditional medicine by patients presenting age associated symptoms and those recovering from stroke. The aim of this study is to evaluate the neuroprotective properties of PO ethanol extract (POEE) using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD, followed by reoxygenation). Mitochondrial activity, an index of cell viability, was assessed by the MTT assay; in addition, the free radicals content was estimated by the use of dichlorofluorescein diacetate as probe. The OGD ischemic condition significantly impaired cellular viability, and increased free radicals generation. In non-OGD slices, incubation with POEE (0.6 microg/ml) increased (approximately 40%) mitochondrial activity, without affecting free radicals levels. In comparison to OGD controls, slices incubated with POEE (0.6 microg/ml) during and after OGD exposure had significantly increased cellular viability. In addition, at this same concentration, POEE prevented the increase of free radicals content induced by OGD. In view of the fact that respiratory chain inhibition and increased generation of free radicals are major consequences of the ischemic injury, this study suggests that Ptychopetalum olacoides contains useful neuroprotective compounds and, therefore, deserves further scrutiny.
... Other international publications have cited the medical use of the species (Anselmino 1932(Anselmino , 1933Steinmetz 1962;Toyota et al. 1979), contributing to the species becoming internationally known. There is a preference for root or root bark (Siqueira et al. 2002), but other parts are utilized. The preparations are varied, the most common being the tea prepared from the bark of the roots, the intake of dried and ground plant parts, and alcoholic preparations, including tinctures. ...
... The antioxidant activity of the extract also seems to contribute to its neuroprotective and pro-cognitive functions. The antioxidant potential of the ethanolic extract from root barks of P. olacoides was demonstrated against different challenges such as nitric oxide, superoxide, and peroxyl radicals (Siqueira et al. 2002). Siqueira et al. (2007) have shown that acute administration of the extract (100 mg/kg, ip) to 14-month-old mice decreased free radical production and lead to a decrease in lipid peroxidation in important cerebral areas. ...
Chapter
The Ptychopetalum olacoides Benth. (Olacaceae) is an Amazonian tree popularly known as muirapuama or marapuama, among other names, which is used for several central nervous system related problems. The roots and occasionally the bark roots are the main medicinal parts employed and are prepared as an alcoholic infusion, tinctures, and tea. Phytochemical studies revealed that the roots contain tannins, flavonoids, and several terpenoids, while the presence of alkaloids is not clear. Most studies used ethanolic or hydroalcoholic extracts prepared with the roots of the plant. These studies indicate that the species has promising potential for treating central nervous system disorders, acting as an antidepressant, an anti-stress, a neuroprotective agent, and improving cognition. Although some herbal products contain P. olacoides in their composition, clinical studies are still needed to confirm the effects observed in pre-clinical studies.
... 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. ...
... 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 PRESSTable 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 * # + for the high TAR levels observed with POEE experiments 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). ...
... 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. ...
... for the high TAR levels observed with POEE experiments 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). ...
Article
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 (100mg/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.
... Subtantial evidence from the literature indicate that P. olacoides act as a scavenger of nitrogen oxides as well as superoxide generated from the xanthine dehydrogenase/xanthine oxidase (XD/XO) system in vitro. The same authors showed that the administration of P. olacoides (100 mg/kg) significantly decreased free radical production and lipid peroxidation in different brain areas of mice in relation to the control [116,120]. P. olacoides has also been demonstrated to inhibit acethylcholinesterase activity in cognitiverelevant brain region areas of mice [121]. This evidence suggests that the plant can be used for treating neurodegenerative conditions where a hypofunctioning cholinergic neurotransmission is prominent, like Alzheimer disease and perhaps brain I/R [122]. ...
Article
Brain ischemia-reperfusion injury is a complex pathological condition that involves a cascade of events like excitotoxicity, peri-infarct depolarization, oxidative/nitrosative stress, inflammation, apoptosis, necrosis and autophagic degeneration. Since the past decades, researchers have provided significant information about the therapeutic potential and possible mechanism of action of plant extracts and derived chemicals to target multiple pathways of the ischemic cascade. Here, we summarized experimental, clinical and epidemiological therapeutic interventions of plants extracts and phytochemicals against brain ischemia-reperfusion injury. Whether oxidative stress is the cause or consequence of brain ischemia is open for debate but details about oxidative burden and inflammation following brain ischemia are also described. Furthermore, the antioxidant mechanism of these extracts/phytochemicals was reviewed. Although these plant extracts and phytochemicals showed the ability to act on the multiple steps of the ischemic cascade in in vitro and in vivo models of brain ischemia, further investigations are needed for their validation and for the development of new drugs.
... Another model of human declarative memory involves a natural preference displayed by rodents for novel objects: the object recognition tasks is considered a non-aversive, non-spatial type of memory, and have been shown to be very useful for assessing changes in neuronal function induced by drugs, aging, and cholinergic dysfunction (Deschaux et al., 1997; Puma et al., 1999; Lebrun et al., 2000). We have previously shown that an ethanol extract of Ptychopetalum olacoides Bentham (Olacoides), traditionally used by Amazonian communities as a " nerve tonic " (Elisabetsky and Siqueira, 1998), posses antioxidant properties in vitro (Siqueira et al., 2002) and in vivo (Siqueira et al., 2006 ), act as neuroprotector in hippocampal slices submitted to oxygen and glucose deprivation (Siqueira et al., 2004), and has anticholinesterase activity in vitro and in vivo at various relevant brain areas (Siqueira et al., 2003). Relevant to this study, it was shown that the extract specifically facilitates the retrieval of long-term memory (inhibitory avoidance) in mice; it is also noteworthy that the memory deficit observed in aging (14-month old) mice was reversed by orally given extract (da Silva et al., 2004). ...
... Marapuama-based remedies are employed for various conditions, including facilitating recovery from stroke, and keeping up with highly stressful psychological and/or physical circumstances [28]. A standardized ethanol extract obtained from P. olacoides (POEE) roots possesses nootropic [29], antioxidant [30,31], and neuroprotective [32] properties. The pharmacodynamic basis of POEE facilitatory effects on diverse memory types [29] includes anticholinesterase actions [33], and the involvement of dopamine D 1 and β adrenergic receptors unpublished data. ...
Article
Nootropic, antioxidant, and neuroprotective properties have been shown in a standardized ethanol extract of Ptychopetalum olacoides (POEE), a medicinal plant traditionally used by the Amazonian elderly population. It has been revealed that POEE mechanisms of action include anticholinesterase effects, and involve beta-adrenergic and dopamine D(1) receptors. The purpose of this study was to verify the role of serotonin receptors in the promnesic effects of this standardized extract. The step-down task in mice and selective serotonin antagonists were used. The study reveals that POEE promnesic effects on short-term (acquisition, consolidation and retrieval) and long-term (retrieval) declarative aversive memories are increased by 5HT(2A) (but not 5HT(1A)) serotonin antagonists (spiperone and pindolol, respectively). The observed synergism between POEE and spiperone can be interpreted as the combined effects of two subeffective doses of two 5HT antagonists, or the known synergism between an acetylcholinesterase inhibitor (POEE) and a 5HT antagonist. In conclusion it is suggested that 5HT(2A) serotonin receptors are relevant for the promnesic effects of this extract, adding to its multiple mechanisms of action.
Article
Full-text available
Antimicrobial resistance is a subject of great concern in public health and also in the designing of strategies for current therapeutic protocols all over the world. New drugs, including those necessary for a reserve armamentarium and exhibiting less side effects deserve special attention. In rural areas, particularly in Brazil, a huge number of natural products, in different artisanal preparations, mainly from plants, have been used by traditional populations to cure diseases. Despite some of these plants have been studied, many of them are awaiting to have their compounds chemically characterized and investigated their pharmacodynamics properties. Further, as well known, the environment plays a crucial role in the metabolism of these plants, yielding different and varied molecular complexes depending on the period of collection, climate conditions, kind of soil and also the plant speciation. In this report, ethanol crude extract of 10 different botanical specimens from the Amazon region of Brazil, in the Amapa State, were screened for antibacterial activity of 7 clinical resistant microorganisms utilizing as control ATCC bacterial species by the Kirby-Bauer method. Plant extracts of Geissospermum argenteum, Uncaria guianensis, Brosimum acutifolium, Copaifera reticulate, Licania macrophylla, Ptycopetalum olacoides and Dalbergia subcymosa yielded activity against Staphylococcus aureus and Pseudomonas aeruginosa, both multidrug resistant, and Staphylococcus aureus ATCC strain.
Article
Homemade remedies with Ptychopetalum olacoides (PO) roots are used by Amazonian peoples for treating various age-related conditions. We previously reported that Ptychopetalum olacoides ethanol extract significantly improved step-down inhibitory avoidance long-term memory in adult and reversed memory deficits in aging mice. Adding to previous data, this study shows that a single i.p. administration of Ptychopetalum olacoides ethanol extract (POEE 50 and 100 mg/kg) improved step-down inhibitory avoidance short-term memory (STM) 3 h after training in adult (2.5 month) mice; comparable results were obtained with POEE given p.o. at 800 mg/kg. Moreover, memory improvement was also observed in aging (14 months) mice presenting memory deficit as compared to adult mice. Furthermore, POEE (100 mg/kg) improved non-aversive memory systems in adult mice in an object recognition paradigm. Consistently with its traditional use this study add to previously reported data and reinforces that POEE facilitates memory processes. Although the acetylcholinesterase inhibitory properties described for this extract may be of relevance for improving memory processes, the molecular mechanism(s) underlying the memory improvement here reported needs further scrutiny.
Article
Full-text available
Roots of Ptychopetalum olacoides Bentham (Olacaceae), known as Marapuama, are prepared in alcoholic infusion for treating “nervous weakness” by Amazonian Caboclos. “Nervous weakness” can be described as a syndrome having several symptoms, among which the following are emphasized: lassitude, depression, sexual impotence and tremors. Based on ethnopharmacological data, we have considered the hypothesis that PO may have psychopharmacological effects, by interacting with different neurotransmitter systems: (i) the dopaminergic system, considering its use as an appetite modulator and to counteract tremors, as well as for its alleged sexual arousing properties; (ii) the noradrenergic system, again for its use against tremors and/or depression; and/or (iii) the serotonergic system, also related to depression and sexual arousal. This paper reports that P. olacoides hydroalcoholic extract potentiated yohimbine-induced lethality, rever sed reserpine-induced ptosis and prevented apomorphine-induced stereotypy. The data indicates that P. olacoides has central nervous system effects and supports the hypothesis of its interaction with dopaminergic and/or noradrenergic systems.
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The time course of oxidative damage in different brain regions was investigated in the gerbil model of transient cerebral ischemia. Animals were subjected to both common carotid arteries occlusion for 5 min. After the end of ischemia and at different reperfusion times (2, 6, 12, 24, 48, 72, 96 h and 7 days), markers of lipid peroxidation, reduced and oxidized glutathione levels, glutathione peroxidase, glutathione reductase, manganese-dependent superoxide dismutase (MnSOD) and copper/zinc containing SOD (Cu/ZnSOD) activities were measured in hippocampus, cortex and striatum. Oxidative damage in hippocampus was maximal at late stages after ischemia (48-96 h) coincident with a significant impairment in glutathione homeostasis. MnSOD increased in hippocampus at 24, 48 and 72 h after ischemia, coincident with the marked reduction in the activity of glutathione-related enzymes. The late disturbance in oxidant-antioxidant balance corresponds with the time course of delayed neuronal loss in the hippocampal CA1 sector. Cerebral cortex showed early changes in oxidative damage with no significant impairment in antioxidant capacity. Striatal lipid peroxidation significantly increased as early as 2 h after ischemia and persisted until 48 h with respect to the sham-operated group. These results contribute significant information on the timing and factors that influence free radical formation following ischemic brain injury, an essential step in determining effective antioxidant intervention.
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Seven flavonoids and three non-flavonoid antioxidants, i.e. butylated hydroxyanisole, chlorpromazine and BW 755 C, were studied as potential scavengers of oxygen free radicals. Superoxide anions were generated enzymatically in a xanthine-xanthine oxidase system and non-enzymatically in a phenazine methosulphate-NADH system, and assayed by reduction of nitro blue tetrazolium. The generation of malonaldehyde (MDA) by the ascorbate-stimulated air-oxidised boiled rat liver microsomes was considered as an index of the non-enzymatic formation of hydroxyl radicals. Flavonoids but not non-flavonoid antioxidants lowered the concentration of detectable superoxide anions in both enzymic and non-enzymic systems which generated these SOD-sensitive radicals. The most effective inhibitors of superoxide anions were quercetin, myricetin and rutin. Four out of seven investigated flavonoids seemed also to suppress the activity of xanthine oxidase as measured by a decrease in uric acid biosynthesis. All ten investigated compounds inhibited the MDA formation by rat liver microsomes. Non-flavonoid antioxidants were more potent MDA inhibitors than flavonoids. It is concluded that antioxidant properties of flavonoids are effected mainly via scavenging of superoxide anions whereas non-flavonoid antioxidants act on further links of free radical chain reactions, most likely by scavenging of hydroxyl radicals.
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A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
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Ischemic brain injury produced by stroke or cardiac arrest is a major cause of human neurological disability. Steady advances in the neurosciences have elucidated the pathophysiological mechanisms of brain ischemia and have suggested many therapeutic approaches to achieve neuroprotection of the acutely ischemic brain that are directed at specific injury mechanisms. In the second portion of this two-part review, the following potential therapeutic approaches to acute ischemic injury are considered: 1) modulation of nonglutamatergic neurotransmission, including monoaminergic systems (dopamine, norepinephrine, serotonin), γ-aminobutyric acid, and adenosine; 2) mild-to-moderate therapeutic hypothermia; 3) calcium channel antagonism; 4) an tagonism of oxygen free radicals; 5) modulation of the nitric oxide system; 6) antagonism of cytoskeletal proteolysis; 7) growth factor administration; 8) therapy directed at cellular mediators of injury; and 9) the rationale for combination pharmacotherapy. The Neuroscientist 1:164-175, 1995