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Journal of Ethnopharmacology 111 (2007) 308–314
Possible involvement of GABAA-benzodiazepine receptor in the
anxiolytic-like effect induced by Passiflora actinia extracts in mice
Luiz F. Lolli a,Cl
´
audia M. Sato a,C
´
assia V. Romanini a, Larissa De Biaggi Villas-Boasb,
Cid A. Moraes Santos b,R
´
ubia M.W. de Oliveiraa,∗
aLaboratory of Psychopharmacology, Department of Pharmacy and Pharmacology, Universidade Estadual de Maring´a,
Av. Colombo, 5790, Jardim Universit´ario, Bloco K-80, 87020-900 Maring´a, PR, Brazil
bLaboratory of Pharmacognosy, Department of Pharmacy, Universidade Federal do Paran´a, Rua Prefeito Lothario Meissner,
632, Jd. Botˆanico, 80210-170 Curitiba, PR, Brazil
Received 22 March 2006; received in revised form 18 October 2006; accepted 21 November 2006
Available online 26 November 2006
Abstract
Hydroethanol (HE) and methanol (ME) extracts obtained from the leaves of Passiflora actinia Hooker were evaluated for behavioral effects in
mice. Single-dose oral administration of HE (300 and 600 mg/kg) or ME (100 and 300 mg/kg) resulted in anxiolytic-like effects in the elevated
plus-maze. The anxiolytic-like effects were also seen after the repeated administration of the HE (100 and 300 mg/kg). Flumazenil (10mg/kg,
i.p.), a GABAA-benzodiazepine receptor antagonist, blocked the effects of ME (300mg/kg, p.o.) and HE (600 mg/kg). At higher doses, a sedative
effect produced by acute administration of HE (600 mg/kg) or ME (300 mg/kg) was indicated by the potentiation of pentobarbital-induced sleep.
With regard to memory-disrupting effects of anxiolytics, mice were evaluated by measuring the retest step-down latency 24h after foot-shock
in a passive avoidance task. In contrast to diazepam (0.5mg/kg) or piracetam (200 mg/kg), ME (30, 100 and 300 mg/kg) or HE (100, 300 and
600 mg/kg) did not influence the step-through latency in the acquisition or retention memory tasks. The present results show an anxiolytic profile
for HE and ME of Passiflora actinia. There are also indications of an involvement of GABAAsystem in this effect.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Passiflora actinia Hooker; Anxiety; Memory; Catalepsy
1. Introduction
The aerial parts of Passiflora species have been tradition-
ally used to treat anxiety, insomnia and nervousness (Dhawan
et al., 2001, 2004). Passiflora incarnata is one of the species
of Passiflora that has extensive clinical applications throughout
the world and lots of studies have been done on its chemi-
cal and pharmacological properties (Dhawan et al., 2004). In
earlier works, anxiolytic-like effects were reported with the
hydroalcoholic extract obtained from the leaves of Passiflora
incarnata at a dose of 400 mg/kg (Soulimani et al., 1997) while
the maximum effect was observed with the methanol extract
at a dose of 125 mg/kg in mice (Dhawan et al., 2001). The
harmane alkaloids and flavonoids have been suggested as the
main bioactive constituents of Passiflora incarnata (Ayoagi et
∗Corresponding author. Tel.: +55 44 32614814; fax: +55 44 32614999.
E-mail address: rmmwoliveira@uem.br (R.M.W.d. Oliveira).
al., 1974; Speroni and Minghetti, 1988; Zanoli et al., 2000; for
review see Dhawan et al., 2004). Recently, a benzoflavone moi-
ety has been isolated from the bioactive methanol extract of
Passiflora incarnata, which has exhibited significant anxiolytic
effects (Dhawan, 2003) and results in the reversal of tolerance
and dependence of several addiction-prone psychotropic drugs,
including morphine, nicotine, ethanol, diazepam and delta-9-
tetrahydrocannabinol (Dhawan, 2003; Dhawan et al., 2004).
However, up to now, because of the differences in experimental
procedures as well as in the preparation of extracts, doses, sched-
ules and routes of administration, there is no consensus about
the active constituents responsible for central effects obtained
with Passiflora extracts.
Passiflora actinia Hooker (vernacular name: ‘maracuj´
a-do-
mato’) is a native species widely distributed through Southern
Brazil. High-performance liquid chromatography (HPLC) com-
parative analysis of the active fractions of Passiflora actinia
has suggested a closer chromatographic profile with Passiflora
incarnata (Santos, 2003). Isovitexin, which is the most abundant
0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2006.11.021
L.F. Lolli et al. / Journal of Ethnopharmacology 111 (2007) 308–314 309
flavonoid in Passiflora incarnata (Menghini et al., 1993), was
present in methanol and its aqueous fractions obtained from Pas-
siflora actinia, while standard alkaloids such as harman, harmin,
harmaline, harmol or harmalol were not detected (Santos, 2003).
Interested in investigating the pharmacological properties of
Passiflora actinia extracts, Santos et al. (2003) have demon-
strated that intraperitoneal (i.p.) injections of Passiflora actinia
crude and methanol extracts cause sedative effects in mice.
These effects were observed as a pronounced decrease in the
number of entries in enclosed and open arms of the elevated
plus-maze (EPM) and a decrease in the general motor activity in
the open field test. Furthermore, a cataleptic effect has also been
detected after i.p. administration of Passiflora actinia methanol
extract (Santos et al., 2005).
Taking the above evidence into account, the aim of the present
study was to evaluate the behavioral effects of the oral admin-
istration of Passiflora actinia hydroethanol (HE) and methanol
(ME) extracts in mice. The present work also compared the two
extracts, once the first is the dosage form recommended in phar-
maceutical formulations when Passiflora is used, and the second
one, ME, is a fraction prepared in the way that Dhawan et al.
(2001) have demonstrated anxiolytic-like activity of Passiflora
incarnata leaves. Anxiolytic and sedative effects were assessed
in the EPM and pentobarbital-induced sleep, respectively. Pos-
sible effects on learning and memory functions were evaluated
through the acquisition and retention of inhibitory avoidance
step-down task.
2. Materials and methods
2.1. Animals
Male albino-Swiss mice (30–45 g), housed in groups (n=5)
with free access to food and water were used. The experiments
were carried out according to the National Institute of Health
Guide for Care and Use of Laboratory Animals, and all efforts
were made to minimize animal suffering.
2.2. Plant material and extract preparation
The leaves of Passiflora actinia Hooker were collected in
Canguiri’s Farm, Pinhais, Paran´
a, Brazil (November 2001) and
a voucher specimens were identified by Dr. Armando Cervi and
deposited at the Department of Botany Herbarium (UPCB no.
30.831), Universidade Federal do Paran´
a, PR, Brazil.
The leaves (23.85 kg) were oven-dried at 45 ◦C and ground
(7.98 kg). The dried and powdered Passiflora actinia leaves
(750 g) were defeated in a Soxhlet apparatus with petroleum
ether (37.05 g) and exhaustively and successively Soxhlet
extracted with CHCl3and MeOH. The solvents were removed
under vacuum and the residues lyophilized affording 103.86 g of
methanol extract (ME). The dosage form (hydroethanol extract
1:1, HE) was prepared according to the Brazilian Pharmacopoeia
Second Edition (Method A). The leaves (500 g) were perco-
lated with 45% ethanol in water until exhaustion and the solvent
concentrated to 500 ml.
2.3. Treatments
Diazepam (Dienpax®, Sanofi-Wintrop Laboratories, Brazil),
piracetam (Nootropil®, Rhodia, S˜
ao Paulo, Brazil), haloperidol
(Haldol®, Janssen-Cilag, S˜
ao Paulo, Brazil) and sodium pento-
barbital (Hypnol®, Crist´
alia, S˜
ao Paulo, Brazil) were solubilized
in saline (NaCl 0.9%) while flumazenil (Sigma chemicals, St.
Louis, USA) was dissolved in vehicle (salina containing Tween-
80 2%). Extracts (HE and ME) or saline were administered by
oral route (gavage) using a tuberculine seringe fitted with oral
cannula (0.1 cm ×4 cm). All drugs were administered to mice
at a volume of 10 ml/kg.
2.4. Behavioral tests
All the behavioral procedures were carried out between 8:00
and 12:00 h a.m. in a temperature controlled room (23 ±1◦C),
illuminated with a 40 W red fluorescent bulb and were video-
taped. Mice were fasted 12 h prior to drug administration and
during the experiments except for repeated treatments. Each
animal was used only once.
2.4.1. Elevated plus-maze
The method initially suggested by Handley and Mithani
(1984) was employed with minor modifications (Lister,
1987). Briefly, the apparatus comprised of two open arms
(25 cm ×10 cm) and two closed arms (25cm ×10 cm ×20 cm)
that extended from a common central platform (10 cm ×10 cm).
The entire maze was elevated to a height of 90 cm above the floor
level.
Mice received single oral administration of HE (100, 300 and
600 mg/kg), ME (30, 100 and 300 mg/kg) or saline and 30 min
later they were individually placed in the centre of the EPM for
5 min. For the repeated treatment, animals were treated twice a
day (8:00–17:00 h) for 6 days with HE (100 and 300 mg/kg), ME
(100 and 300 mg/kg) or saline; on the seventh day, 30 min after
the morning administration, mice were submitted to the EPM.
The number of open and enclosed arm entries and time spent
on open arms was registered. Subsequently, the percentage of
open arm entries (100 ×open/total entries) and the percentage of
time spent in the open arms (100 ×open/open + enclosed) were
calculated for each animal.
In another set of experiment, mice were treated with saline
or flumazenil (10 mg/kg, i.p.) 30 min before the oral adminis-
tration of the HE (600 mg/kg, p.o.) or ME extracts (300mg/kg).
Flumazenil, a specific GABAAantagonist, was used to deter-
mine the role of GABA-system in the probable action of the
extracts (Lee and Rodgers, 1991).
2.4.2. Sodium pentobarbital-induced sleeping time
Thirty minutes after HE (100, 300 and 600 mg/kg) or ME
(30, 100 and 300 mg/kg) oral administration, a dose of 50 mg/kg
of sodium pentobarbital was i.p. injected into the mice. Loss of
righting reflex was taken as sleep latency, while the time between
loss of righting reflex and the regain of right reflex was taken as
the duration of sleep.
310 L.F. Lolli et al. / Journal of Ethnopharmacology 111 (2007) 308–314
2.4.3. Step-down inhibitory avoidance
The step-down apparatus consisted of an acrylic box
(12 cm ×30 cm ×15 cm), whose floor consisted of parallel
1.0 mm diameter stainless steel bars spaced 1.0 cm apart. A
10 cm wide, 3.0 cm high, 6.0 cm long platform occupied the
center of the grid floor. In the training session (day 1), imme-
diately after stepping down placing their paws on the grid, the
animals received a 0.3 mA 15 s scrambled foot shock and were
immediately withdrawn from the cage. Twenty-four hours later,
in the text sessions (day 2), no foot shock was given and the step-
down latency was used as a measure of retention (to a ceiling of
300 s).
Drugs were administered 30 min before the training or prior
the test session, aiming thus to investigate their effect on mem-
ory acquisition or retrieval, respectively (Izquierdo et al., 1998;
Barros et al., 2001). Animals that showed a latency in the cri-
terion range (30 s) during the training session were used for the
retention test (Parle and Dhingra, 2003).
2.4.4. Open field
Thirty minutes after receiving the treatments, animals were
placed into the center of the open field in order to measure the
motor activity (Royce, 1977). The open field used to measure the
locomotion was a wooden square box, 45 cm ×45 cm with a wall
30 cm high, the floor was divided into nine smaller squares of
equal dimensions (15 cm ×15 cm). The animals could explore
the box during 5 min. Hand operated counters and stopwatches
were used to score the number of crossings (number of square
floor units entered) and rearing (number of times the animal
stood on hind legs).
2.4.5. Statistical analysis
Data are expressed as mean ±S.E.M. of the groups (n= 10).
Data were analyzed by the Student’s t-test or one-way analysis
of variance (ANOVA) followed by the Tukey’s test for multiple
comparisons.
3. Results
3.1. Elevated plus-maze
As expected for a positive control, diazepam 1mg/kg i.p.,
induced a selective anxiolytic-like effect in mice characterized
by an increase in the % of the number (t18 = 9.48, p< 0.001) and
of the time (t18 = 7.53, p< 0.001) spent in the open arms of the
EPM compared to saline. A single administration of HE (300 and
600 mg/kg) or ME (100 and 300 mg/kg) also resulted in a signif-
icant increase in the % of the number (HE F3.39 = 7.93, p< 0.05;
ME F3.39 = 11.04, p< 0.001) and the time (HE F3.39 = 7.28,
p< 0.05; ME F3.39 = 13.04, p<0.001) into the open arms of the
EPM (Fig. 1). These treatments did not result in any significant
changes in motor activity as can be observed by the number in
the enclosed arms (p> 0.05).
Although flumazenil caused no significant change in the EPM
open arms (p> 0.05 for entries or time), it completely inhibited
the anxiolytic-like effect of diazepam, HE and ME as seen by
the % of open arm entries (diazepam F3.39 = 16.58, p< 0.001;
Fig. 1. Anxiolytic-like effect of a single oral dose of Passiflora actinia
hydroethanol extract (HE; 100, 300 and 600 mg/kg) or methanol extract (ME;
30, 100 and 300 mg/kg) in mice submitted to the elevated plus-maze. Diazepam
(DZ, 1 mg/kg) or its control saline (sal), were administered intraperitoneally
(i.p.). In lower panel, the hatched and blank columns represent the percentage of
entries and of time into the open arms of the maze, respectively. Bars and vertical
lines represent the mean ±S.E.M. of each group (n= 10). **p< 0.001, compared
to control groups (ANOVA followed by Tukey’s test, except for diazepam, for
which nonpaired Student’s t-test was used).
HE F3.39 = 22.2, p< 0.001; ME, F3.39 = 10.42, p< 0.05) as well
as of the % of time (diazepam F3.39 = 21.33, p<0.001; HE
F3.39 = 43.57, p< 0.001; ME, F3.39 = 17.04, p< 0.001) spent in
the EPM (Fig. 2).
Repeated administration of HE (100 and 300 mg/kg) pro-
duced an enhanced open arm exploration as seen by an increase
in the % of entries (F2.29 = 4.86, p< 0.05) as well as % of
time (F2.29 = 10.2, p< 0.001) in the open arms of the maze
(Fig. 3). There is no significant alteration in enclosed arm entries
(p> 0.05). However, no activity was detected after repeated treat-
ment with ME tested doses (p> 0.01).
3.2. Pentobarbital-induced sleep in mice
Table 1 shows the effects of the HE (600 mg/kg) and ME
(300 mg/kg) on pentobarbital-induced sleep in mice. Animals
L.F. Lolli et al. / Journal of Ethnopharmacology 111 (2007) 308–314 311
Fig. 2. Effects of oral administration of HE (600 mg/kg) or ME (300 mg/kg)
alone or in combination with flumazenil (10 mg/kg, i.p.) on the behavior of rats
on the elevated plus-maze. *p< 0.05 and ** p< 0.001 compared to all groups
(ANOVA followed by Tukey’s test).
given sodium pentobarbital (50 mg/kg, i.p.) showed loss of
righting reflex within 3 min of administration. The two extracts
produced a prolongation of pentobarbital-induced sleeping time
in mice (HE F3.39 = 6.70, p< 0.05; ME F3.39 = 3.76, p< 0.05).
Therefore, the ME was more potent in increasing pentobarbital-
induced sleeping time in mice.
No alteration was observed considering the sleep latency
(p> 0.05).
Table 1
Sedative effect of a single-dose of Passiflora actinia HE (600 mg/kg) or ME
(300 mg/kg) on pentobarbital-induced sleep in mice
Treatment Latency (s) Sleep time (s)
Saline (10 ml/kg) 193.5 ±7.0 5102 ±847.2
HE (mg/kg)
100 208.3 ±11.06 4620 ±430.4
300 190.5 ±6.0 5588 ±619.7
600 205.5 ±21.33 8130 ±422.7*
ME (mg/kg)
30 186.8 ±5.0 6687 ±671.1
100 185.2 ±17.01 7807 ±723.9
300 167.7 ±6.0 8550 ±835.9*
Saline (10 ml/kg) 193.5 ±7.0 5102 ±847.2
HE (mg/kg)
100 208.3 ±11.06 4620 ±430.4
300 190.5 ±6.0 5588 ±619.7
600 205.5 ±21.33 8130 ±422.7*
Numbers represent the mean ±S.E.M. of the groups (n=10).
*p< 0.05 compared to saline (ANOVA followed by the Tukey’s test).
Fig. 3. Effects of repeated oral administration of Passiflora actinia HE (100,
300 and 600 mg/kg) or ME (30, 100 and 300 mg/kg) in mice submitted to the
elevated plus-maze. **p< 0.001 and *p<0.05 compared to saline group.
3.3. Step-down
In comparison to saline, diazepam (0.2 mg/kg, i.p.) admin-
istered 30 min before training, significantly reduced the latency
time (F2.29 = 8.25, p< 0.05) as compared to saline group, indi-
cating significant impairment of memory (Fig. 4). Mice orally
treated with HE (100, 300 and 600 mg/kg) or ME (30, 100 and
300 mg/kg) did not show any behavioral changes when submit-
ted to the step-down (p> 0.05).
The results of the retention test performance are demonstrated
in Fig. 4. Administration of diazepam (0.25 mg/kg, i.p.) or pirac-
etam (200 mg/kg) modified the latency time in the step-down
(F2.29 = 90.44, p< 0.001) when administered 30 min before the
test session. No significant effect was detected after HE (100,
300 and 600 mg/kg) or ME (30, 100 and 300 mg/kg).
3.4. Open field
As shown in Table 2, neither the number of crossings nor the
rearings were significantly affected by treatment with HE (100
and 600 mg/kg) or ME (30, 100 and 300 mg/kg). Only HE at
312 L.F. Lolli et al. / Journal of Ethnopharmacology 111 (2007) 308–314
Fig. 4. Effects of acute oral administration of Passiflora actinia HE (100, 300
and 600 mg/kg) or ME (30, 100 and 300 mg/kg) given 30 min before training
(upper panel) or test session (lower panel) in a step-down inhibitory avoid-
ance task. Diazepam (DZ, 0.5 mg/kg, i.p.) and piracetam (200mg/kg, i.p.) were
used as reference drugs. Bars and vertical lines represent the mean ±S.E.M. of
each group (n= 10). **p< 0.001 and *p< 0.05 compared to controls (ANOVA
followed by the Tukey’s test).
Table 2
Effects of a single-dose of Passiflora actinia HE (100, 300 and 600 mg/kg), ME
(30, 100 and 300 mg/kg) and reference drugs, diazepam (DZ, 0.2 mg/kg) and
piracetam (200 mg), upon the behavioral parameters recorded in the open field
Treatment Crossings Rearings
Saline (10 ml/kg, i.p.) 55.0 ±4.0 36.4 ±3.6
Diazepam (0.2 mg/kg, i.p.) 55.7 ±2.9 32.4 ±1.9
Piracetam (200 mg/kg, i.p.) 58.6 ±2.44 30.6 ±2.8
Numbers represent the mean ±S.E.M. of the groups (n= 10). *p< 0.05 com-
pared to saline (ANOVA followed by Tukey’s test).
300 mg/kg significantly decreased the number of rearings in the
open field (F3.39 = 2.83, p< 0.05).
4. Discussion
In the present study, we have demonstrated that both HE (300
and 600 mg/kg) and ME (100 and 300 mg/kg), following acute
oral administration, produced a dose-dependent anxiolytic-like
effect in mice as measured by an increased open arm exploration
in the EPM. Furthermore, previous administration of flumazenil
reverts the extracts-induced anxiolytic-like effects to basal lev-
els. In addition, HE (100 and 300 mg/kg) enhanced anxiolytic
efficacy following repeated oral administration when compared
to its effects following acute administration. Curiously, repeated
administration of ME, which acutely also produced anxiolytic
effects at 100 and 300 mg/kg, was devoid of any change in the
EPM parameters, suggesting tolerance to its anxiolytic effect.
The valitidy of the EPM test for evaluation of anxiolytic or
anxiogenic effects of drugs has been well documented (Lister,
1987; Pellow and File, 1986; Carobrez and Bertoglio, 2005).
As expected, in the present work, the treatment with diazepam,
a benzodiazepine anxiolytic drug, led to a significant increase
in the entries and time that mice spent in the open arms of the
EMP without changing the number of enclosed arm entries, a
well-accepted measure of motor activity (Dalvi and Rodgers,
1999). These results are in agreement with others that have
shown diazepam to produce consistent anxiolytic effect both
pre-clinically and clinically (Haefely, 1988).
Prior research in our lab (Santos et al., 2003) has reported
that acute i.p. injections of Passiflora actinia crude extract (100
and 300 mg/kg) or ME (300 and 600 mg/kg) cause sedative but
not anxiolytic effects in mice. These effects were observed as a
pronounced decrease in the number of entries in enclosed and
open arms of the EPM as well as decrease in the general motor
activity in the open field. Nevertheless, in the present study,
an oral single-dose administration of HE (300 and 600 mg/kg)
or ME (100 and 300 mg/kg) produced selective anxiolytic-like
effects in mice, as observed as an enhanced frequency of entries
and time spent on the open arms of the EPM without causing
marked change in motor activity.
In fact, both sedative and anxiolytic effects have been
described using different Passiflora extracts. For example, anx-
iolytic effects have been described for i.p. administration of
Passillora edulis and Passiflora alata hydroethanol extracts, in a
range of 50–150 mg/kg, in rats submitted to the EPM (De-Paris
et al., 2002). On the other hand, a prolongation of barbituric-
induced sleep was observed after i.p. injection of dry extract
obtained from Passiflora alata (Oga et al., 1984)orPassi-
flora incarnata (Ayoagi et al., 1974). Soulimani et al. (1997)
have observed that Passiflora incarnata aqueous extracts (400
and 800 mg/kg, i.p.) induced sedative while the hydroalcoholic
extract (400 mg/kg, i.p.) produced anxiolytic effects in mice.
Furthermore, the observed different effects were attributed to
the solvents used to prepare the extracts.
In this way, sedative effects have been related to strong doses
while anxiolytic activity with weak doses of plant extracts or ref-
erence drugs such as clorazepate (Rolland et al., 1991). Besides
the dose, utilization of different route of administration may
alter the blood concentration and thus produce different biolog-
ical activities of one same compound. Thus, it is possible that
anxiolytic effects observed with oral administration in contrast
with sedative effects obtained with i.p. administration (Santos
et al., 2003) might be due to blood concentrations achieved by
the active principle present in those extracts.
Tolerance is an undesirable side effect for therapeutic pur-
poses and may also be linked to dependence liability. Some
L.F. Lolli et al. / Journal of Ethnopharmacology 111 (2007) 308–314 313
anxiolytic drugs, such as diazepam, have been shown to produce
tolerance following repeated dosing in the conflict assay (Smith
and Barrett, 1997) and in the EPM (Fernandes et al., 1999).
Tolerance to benzodiazepines has been associated with adaptive
changes in the central nervous system, e.g. pharmacodynamic
(File, 1992; Roy-Byrne, 2005), in contrast to small changes in
pharmacokinetics (Smith and Darlington, 1994). These changes
may be attributed to the benzodiazepine binding-site or GABA
binding-site at the GABAA-receptor. Furthermore, Davis and
Gallager (1988) reported that the development of tolerance is
more likely to occur with continuous receptor occupation, indi-
cating that route and schedule of administration are also of
relevance. The current study showed HE (100 and 300mg/kg)
to be devoid of tolerance development as opposed to repeated
ME (300 mg/kg) administration. However, future investigation
are necessary to clarify the mechanisms involved in Passiflora
actinia ME tolerance.
To investigate the detailed mechanisms involved in the
anxiolytic-like effect caused by Passiflora actinia extracts, the
effect of flumazenil (10 mg/kg, i.p.), a GABAA-benzodiazepine
receptor antagonist, was evaluated. Thus, flumazenil caused
no obvious effect in mice submitted to the EPM, however it
showed a significant antagonistic effect on the anxiolytic effect
induced by diazepam as well as by the HE (600 mg/kg) and
ME (300 mg/kg). Flumazenil generally produces no marked
behavioral effects, but it is able to reverse almost all the pharma-
cological consequences of benzodiazepine anxiolytics (Lee and
Rodgers, 1991). According to our results, the anxiolytic-like
effect of Passiflora extracts was significantly reversed by the
treatment of animals with flumazenil. This suggests that HE and
ME extracts might produce anxiolytic-like effect by interaction
with central benzodiazepine GABAA-receptors.
Confirming the involvement of benzodiazepine receptor
system, ME (300 mg/kg) and HE (600 mg/kg) increased the
sleeping time induced by pentobarbital. Barbiturates have been
shown to enhance the activation of GABAAreceptors, although
its effects are more widespread and less restricted than benzodi-
azepine effects (Johnston, 1996). Some members of the family
of flavonoids have been demonstrated to have moderate binding
affinities for the benzodiazepine-site. In vivo studies revealed
that these compounds were mostly partial agonists of GABAA
receptors, and only a few flavonoids were shown to possess
antagonistic activities (Wang et al., 2005). Considering Passi-
flora species, Wolfman et al. (1994) have postulated that chrysin,
a natural monoflavonoid occurring in Passiflora incarnata,isa
ligand for central benzodiazepine receptors.
A major problem of anxiolytic compounds is that their anx-
iolytic activity cannot be easily separated from sedation (Costa
and Guidotti, 1996; Atack, 2003). At high doses, for example,
diazepam starts to reduce the activity of the rats, as hinted at by
the significantly reduced unpunished licks in the Vogel conflict
test, a parameter related to locomotor activity (Nazar et al., 1997;
Kennett et al., 1998). The anxiolytic-like effect of HE and ME
observed in the present study seems not to be associated with
any motor effects, since no significant behavior change of mice
was observed in the open field. Only ME at 30 mg/kg produced
a reduction in the number of rearings. This leads to the assump-
tion that the anxiolytic-like effect of Passiflora actinia extracts
is selective without producing benzodiazepine-like side effects
such as sedation, muscle relaxation or ataxia.
A close relationship between anxiety and memory process
has been pointed out. Some anxiolytics, such as benzodi-
azepines impair learning and memory, both in animals and
humans (McNamara and Skelton, 1991; Cole and Jones, 1995;
Olaman and McNaughton, 2001), whereas anxiogenic com-
pounds like amphetamine and -carboline improve memory
process (Venault et al., 1987). Animals treated with diazepam
prior to training of inhibitory avoidance, display amnesia for the
task during testing, suggesting that diazepam may depress mem-
ory acquisition and/or storage (Izquierdo and Ferreira, 1989).
Herein, diazepam administered before the training or the test
sessions impaired mice performance in a step-down avoidance
task. On the other hand, under the same experimental condi-
tions, piracetam significantly improved memory retention. Our
results are in agreement with others showing that some nootropic
agents, including piracetam, improve retention but not acquisi-
tion (Bhattacharya et al., 1993; Hasenohrl et al., 1998). HE and
ME at doses that produced anxiolytic-like effects did not exert
any obvious amnestic or otherwise disruptive effect in the tasks
employed in the present work.
Dissociation between anxiolytic and hypoamnestic effects
has been described for some herbal compounds. Hasenohrl et al.
(1998) have observed that combined extracts of Zingiber offici-
nale and Ginkgo biloba are active in the EPM without interfering
negatively with the performance on an inhibitory avoidance and
a water maze task, as opposed to benzodiazepines, suggesting
that the anxiolytic-like effects are dissociable from undesirable
side effects on memory processes.
In conclusion, the present study provides evidence that HE
and ME obtained from Passiflora actinia present anxiolytic-like
effects avoiding sedation when administered orally to mice. The
anxiolytic activity is likely mediated, at least partly, through
benzodiazepine receptors. Subsequent studies are, therefore,
necessary in order to verify the Passiflora extracts mechanism
of action and to elucidate the active principles involved with its
central activity.
Acknowledgments
The authors thank Dr. A. Cervi (Universidade Federal
do Paran´
a) for identification of the plant material and M.
A.Trombelli for technical support.
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