The aqueous crude extract of Montanoa frutescens produces anxiolytic-like
effects similarly to diazepam in Wistar rats: Involvement of GABAAreceptor
Miguel Carro-Jua ´reza, Juan Francisco Rodrı ´guez-Landab,n, Marı ´a de Lourdes Rodrı ´guez-Pen ˜aa,
Marı ´a de Jesu ´s Rovirosa-Herna ´ndezb, Francisco Garcı ´a-Ordun ˜ab
aLaboratorio de Comportamiento Reproductivo, Escuela de Medicina Veterinaria y Zootecnia, Universidad Auto ´noma de Tlaxcala, Tlaxcala CP. 90000, Tlaxcala, Me ´xico
bInstituto de Neuroetologı ´a, Universidad Veracruzana, Av. Dr. Luis Castelazo s/n, Col. Industrial Las A´nimas, Xalapa 91190, Veracruz, Me ´xico
a r t i c l e i n f o
Received 10 April 2012
Received in revised form
22 June 2012
Accepted 20 July 2012
Available online 31 July 2012
Open field test
a b s t r a c t
Ethnopharmacological relevance: Cihuapatli is the Nahuatl name assigned to some medicinal plants
grouped in the genus Montanoa, where Montanoa frutescens (Family: Asteraceae, Tribe: Heliantheae) is
included. The crude extract from these plants has been used for centuries in the Mexican traditional
medicine as a remedy for reproductive impairments and mood disorders. Experimental studies have
systematically corroborated the traditional use of cihuapatli on reproductive impairments and sexual
motivation, however, the effect on mood and ‘‘nervous’’ disorders, remains to be explored.
Materials and methods: The anxiolytic-like effect of aqueous crude extract of M. frutescens (25, 50 and
75 mg/kg) was investigated in male Wistar rats evaluated in the elevated plus-maze and compared
with several doses of diazepam (1, 2 and 4 mg/kg) as a reference anxiolytic drug. Picrotoxin (1 mg/kg), a
noncompetitive antagonist of the GABAAreceptor, was used in experimental procedures to evaluate if
this receptor could be involved in the anxiolytic-like effects produced by M. frutescens. To discard
hypoactivity, hyperactivity, or no changes associated with treatments, which could interfere with the
behavioral activity in the elevated plus-maze, rats were subjected to the open field test.
Results: M. frutescens at 50 mg/kg showed anxiolytic-like activity similarly to 2 mg/kg of diazepam,
without disrupts in general motor activity. The anxiolytic-like effect of M. frutescens detected in the
elevated plus-maze was blocked by picrotoxin, indicating that GABAAreceptors are involved in the
modulation of this effect.
Conclusions: The results corroborate the use of M. frutescens in folk Mexican ethnomedicine as a
potential anxiolytic agent and suggest that this effect is mediated by the GABAAreceptors. Additionally,
some sedative effects with high doses of M. frutescens were detected in the present study.
& 2012 Elsevier Ireland Ltd. All rights reserved.
Zoapatle or Cihuapatli is the Nahuatl name assigned to a group
of plants (i.e. Montanona tomentosa, Montanoa frutescens and
Montanoa grandiflora) included in the genus Montanoa. The
aqueous crude extract from these plants has been used for
centuries in the Mexican traditional medicine as a remedy for
reproductive impairments and mood disorders (Ximenez, 1615;
Levine et al., 1981; Gallegos, 1985). Early descriptions about the
use of cihuapatli aqueous crude extract as a traditional remedy
are contained in the Badianus Codex or Libellus de Medicinalibus
Indorum Herbis written in 1552 (de la Cruz-Badiano, 1552), where
botanical determinants, traditional recipes and prescriptions
were provided. Mexican traditional medicine mentions that the
prominent use of cihuapatli extract was as a contraceptive agent
during the early stages of pregnancy (de la Cruz-Badiano, 1552;
Ximenez, 1615; Hahn et al., 1981, Levine et al., 1981; Gallegos,
1983; Ponce-Monter et al., 1983) and to induce rhythmic uterine
contractions at birth. Additionally, mood and ‘‘nervous’’ disorders
(an anxiety-like behavior in the context of Mexican antique
culture) are also treated with the aqueous crude extract of
cihuapatli and early reports by Ximenez (1615) mentioned,
‘‘cihuapatli resolves the mood changes and nerves in an admirable
In order to obtain the medicinal effect, midwifes mostly
prepare the cihuapatli aqueous crude extract from M. tomentosa
as the starting material but also this decoction can be prepared
including M. frutescens or M. grandiflora (Estrada et al., 1983).
Experimental studies have confirmed that cihuapatli aqueous
crude extract obtained from M. tomentosa or its purified fractions
possesses contraceptive effects (Hahn et al., 1981), without
influencing the endocrine status (Gallegos, 1985). The effects of
Contents lists available at SciVerse ScienceDirect
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Journal of Ethnopharmacology
0378-8741/$-see front matter & 2012 Elsevier Ireland Ltd. All rights reserved.
nCorresponding author. Tel.: þ52 2288418900x13614; fax: þ52 2288418920.
E-mail address: firstname.lastname@example.org (J.F. Rodrı ´guez-Landa).
Journal of Ethnopharmacology 143 (2012) 592–598
cihuapatli aqueous crude extract prepared with M. tomentosa on
the reproductive tract obey to an oxytocic-like profile (Ortiz de
Montellano, 1975; Ponce-Monter et al., 1983) without modifying
the hematological, blood lipid, protein, electrolytic status or the
function of the liver, kidney and thyroid gland (Hahn et. al., 1981).
Recent studies have reported that the extract of cihuapatli
prepared with M. tomentosa can cross the blood-brain barrier to
exert their actions directly upon the central nervous system
(Carro-Jua ´rez et al., 2004, 2006) and in the case of copulating
male rats, administration of this extract promotes an aphrodisiac
effect by improving both sexual motivation and performance
(Carro-Jua ´rez et al., 2004). Thus, aforementioned studies have
systematically corroborated the traditional use of cihuapatli on
reproductive impairments and sexual motivation, however, the
effect on mood and ‘‘nervous’’ disorders, remains to be explored.
The oxytocic-like contraceptive effects of cihuapatli extracts
prepared with M. frutescens have shown to be more effective
and more potent than M. tomentosa and its relatives in all
evaluated experimental models (Estrada et al., 1983). Thus, we
hypothesized that the aqueous crude extract of M. frutescens (Mf)
exerts an anxiolytic-like effect similarly to diazepam, a reputed
and clinically effective anxiolytic drug.
In the present study the potential anxiolytic-like effect of
several doses of the aqueous crude extract prepared with M.
frutescens was evaluated and compared with the effect produced
by several doses of diazepam in the elevated plus-maze, a well-
accepted experimental animal model used to test the effective-
ness of clinical effective anxiolytics (Griebel et al., 1996; Varty
et al., 2002). Additionally, and by considering that the GABAA
receptor is the main target in which clinically effective anxiolytic
drugs or substances with anxiolytic potential exert their actions
(Trincavelli et al., 2012), the participation of the GABAAreceptor
in the anxiolytic-like effect of M. frutescens was explored by
means of a pre-treatment with picrotoxin, a non-competitive
antagonist of this receptor (Bormann, 2000).
2. Material and methods
Ninety-eight adult male Wistar rats, weighing 300–350 g,
were used in the experiments. The rats were housed in Plexiglas
cages (seven rats per cage; 44 cm width, 33 cm length, 20 cm
height) under a 12/12 h light/dark cycle (light on at 7:00 AM) at
an average temperature of 25 1C (72 1C) with ad libitum access
to purified water and food (Harlans, Me ´xico, S.A. de C.V.). All of
the experimental procedures were performed according to the
Guide for the care and use of laboratory animals published by
the National Institutes of Health (National Research Council,
Publication no. 85-23, revised in 1996) and the Norma Oficial
Mexicana para el Cuidado y Uso de Animales de Laboratorio
(Norma Oficial Mexicana, NOM-062-ZOO-1999). This protocol
received authorization (No. MVZ-189/12) from Ethical Internal
Committee of the Veterinary School (Universidad Auto ´noma de
2.2. Preparation of M. frutescens extract
Montanoa frutescens Cerv. (Family: Asteraceae, Tribe: Heliantheae)
was collected in its habitat in the state of Tlaxcala, Me ´xico during
September of 2011 and was authenticated by a specialist from the
Herbarium of the Universidad Auto ´noma de Tlaxcala, where
voucher specimens are preserved and the plant is cultivated
(Serial Number: MF UATX11). Doses of aqueous crude extracts
of Mf were selected on the basis of a previous pilot study and to
select the convenient doses, it was asked for the quantity of this
plant employed by traditional healer in Tlaxcala, Me ´xico. The
traditional healer recommended us to boil twenty leaves to obtain
an aqueous crude extract with ‘‘anti-nervous’’ effect in adult
subjects. Leaves of Mf were collected and prepared to be dried
during twenty days. Once dried, the material was ground into a
fine powder averaging 1 g, which was mixed with 20 ml of
distilled water. This mixture was warmed up approximately
10 min, just before boiling. The obtained infusion was filtered
and oven dried at a temperature of 55 1C and the brownish
residue of the extract yield was calculated to be 80 mg. The dried
extract of the plant was maintained at 3 1C and then used to
prepare the stock solutions of 25, 50 and 75 mg/ml. In present
investigation the solution of 75 mg was prepared initially and
after this it was diluted to obtain equivalent solutions of 50 and
25 mg. Infusion and solutions were prepared 40 min previous to
its administration to avoid modifications in the chemical proper-
ties of the extracts.
2.3. Experimental series
2.3.1. Effect of several doses of Mf on the elevate plus-maze and its
comparison with diazepam
Rats were assigned to eight independent groups (n¼7 in each
group). A first vehicle group received the vehicle (purified water,
1 ml/kg, p.o.) in which Mf extract was prepared and three
additional groups of rats received several doses (25, 50, 75 mg/
kg/ml, p.o.) of the aqueous extract of Mf. Additionally, a second
vehicle group received the vehicle in which diazepam was
dissolved (propylene glycol 40%, 1 ml/kg, i.p.) and three indepen-
dent groups of rats treated with several doses of diazepam (1, 2 or
4 mg/kg i.p.) were included to validate the experimental condi-
tions of the plus-maze apparatus and to compare the effect
produced by Mf extracts. The doses of 2 mg/kg de diazepam were
selected according previous studies describing their anxiolytic-
like effects in the elevated plus-maze (Contreras et al., 2011;
Karim et al., 2011), from this anxiolytic doses of diazepam we
include additionally one half (1 mg/kg) and the double (4 mg/kg)
of those doses to extend comparison with the different doses of
Mf. Diazepam was acquired from Laboratorios CryopharmasS.A.
de C.V., D.F., Me ´xico. The doses of Mf utilized in this study were
based in previous studies describing the ability of cihuapatli plant
to cross the blood-brain barrier in male rats (Carro-Jua ´rez et al.,
2004, 2006). All treatments were administered in a single dosage
and 30 min after the correspondent treatment; the rats were
evaluated in the elevated plus-maze (5 min) and subsequently in
the open field test (5 min).
2.3.2. Effect of the GABAAreceptor antagonism upon the anxiolytic-
like activity of Mf
To explore the participation of the GABAA receptor in the
anxiolytic-like effect of the effective dose of Mf extract in the
elevated plus-maze, four independent groups of rats were
included: the vehicle group (vehicle, n¼7) received the vehicle
of picrotoxin (saline, 0.9%) plus purified water (vehicle of Mf
extract). Another group received 1 mg/kg of picrotoxin plus
purified water (P, n¼7); a third group received saline plus
50 mg/kg of Mf extract (Mf, n¼7) and the last group received
1 mg/kg of picrotoxin plus 50 mg/kg of Mf (PþMf, n¼7). Addi-
tionally, to corroborate the participation and specificity of GABA
in this experiment, two additional groups were included: a
diazepam group (Dz, n¼7) received vehicle of picrotoxin plus
2 mg/kg of diazepam, and other group (PþDz) received 1 mg/kg
of picrotoxin plus 2 mg/kg of diazepam. Mf extract, diazepam or
their vehicle were administrated 30 min before the behavioral
M. Carro-Jua ´rez et al. / Journal of Ethnopharmacology 143 (2012) 592–598
tests and picrotoxin or its vehicle (i.p.) 30 min before the admin-
istration of Mf extract, diazepam or its vehicle. Doses schedule
was selected from previous reports in which 1 mg/kg of
picrotoxin antagonized the anxiolytic-like effects of GABAergic
compounds (Rodrı ´guez-Landa et al., 2007; 2009a). Treatments
were administered in an equivalent volume of 1 ml/kg.
2.4. Behavioral tests
2.4.1. Elevated plus-maze
The apparatus was constructed of wood and situated in a
brightly lit room. The apparatus consisted of two opposite open
and closed arms set in a plus configuration. The dimensions of the
open arms were 50 cm?10 cm (length?width) and the closed
arms were 50 cm?10 cm?40 cm (length?width?height). The
entire maze was elevated 50 cm from the floor. A digital video
camera (Sony, DCR-SR42, 40? optical zoom, Carl Zeiss lens) was
installed above of the apparatus to record the rat activity. Later,
two independent observers measured the behavioral variables
until reaching a coincidence higher than 95% in measurement.
To evaluate the effect of treatments, rats were placed at the
center of the maze, facing an open arm and the evaluated
variables were: (a) the time spent into the open arms, (b) the
number of entries into the open arms (Walf and Frye, 2007), (c)
the total number of entries (open armsþclosed arms) and (d) the
percentage of open arm entries ([open entries]/[total entries]?
100). Rats that fell to the floor were discarded from subsequent
2.4.2. Open field test
To evaluate the effect of the substances on spontaneous motor
activity, animals were individually subjected to a 5-min period to
the open field test. The open field apparatus was an opaque
plexiglass cage (44?33 cm) with walls 20-cm in height where
the floor was delineated into 12 squares (11?11 cm). A digital
video camera (Sony, DCR-SR42, 40? optical zoom, Carl Zeiss lens)
was installed above the cage to record the activity of the rat. Later,
two independent observers measured the behavioral variables. In
this study was evaluated the general motor activity of the rats to
discard hypoactivity, hyperactivity, or no changes associated with
treatments, which could interfere with the behavioral activity of
the rats in the elevated plus-maze. At the onset of the test,
animals were gently placed in one of the corners of the cage and
variables measured were: (a) the number of squares crossed by
the rat (crossing), assuming crossing when an animal passed from
one square to another with its rear legs; (b) the time spent in
rearing, assuming rearing when the rat acquired a vertical posture
with respect to the cage floor, (c) the time spent in grooming,
which according to Kalueff and Touhimaa (2004a, 2004b, 2005)
included paw licking, nose/face grooming (strokes along the
snout), head washing (semicircular movements over the top of
the head and behind the ears), body grooming/scratching (body
fur licking and scratching the body with the hind paws), leg licking,
and tail/genitals grooming (licking of the genital area and tail), and
(d) the total resting time, assuming it when rats acquired a resting
posture on floor without realize any movement.
After each test session, the elevated plus-maze apparatus and
the open field cage were carefully cleaned with a 30% ethanol
solution to remove the scent of the previously evaluated animal,
which could otherwise modify the spontaneous behavior of the
subsequent evaluated rat (Gutie ´rrez-Garcı ´a et al., 2006).
2.5. Statistical analysis
In a first statistical analysis by using the Student t test,
both vehicle groups were compared to identify any difference
produced by the vehicle (i.e. vehicle of Mf and vehicle of
diazepam) or the administration route (p.o. or i.p.). In a second
analysis the effect of treatments was analyzed using one-way
analysis of variance (ANOVA) with independent groups, when the
p values reached r0.05 the Student-Newman–Keuls post-hoc test
was applied. Results are expressed as mean7standard error.
3.1. Comparison between vehicle groups
The analysis of both vehicle groups did not reveal significant
differences in none of the evaluated variables neither in elevated
plus-maze nor in the open field test (data no shown). For this
reason, to analyze the effect of Mf and diazepam only a vehicle
group integrated by both vehicle groups was included.
3.2. Mf exhibits anxiolytic-like activity: comparison with diazepam
3.2.1. Elevated plus-maze test
[F(6,49)¼5.94, po0.001] in the time spent into the open arms.
The post-hoc test showed that the time spent into the open arms
was increased (po0.05) only in the group treated with 50 mg/kg
of Mf or 2 mg/kg of diazepam, versus the vehicle and remaining
groups (Fig. 1a).
The analysis of the number of entries into either the open or
closed armsshoweda significant
[F(6,49)¼16.56, po0.001]. The post-hoc test showed that the total
number of entries into the arms was augmented (po0.05) in the
group treated with 25 and 50 mg/kg of Mf or 1 and 2 mg/kg of
diazepam, when compared with the vehicle group (Fig. 1b).
No significant differences were detected among groups treated
with 75 mg/kg of Mf or 4 mg/kg of diazepam, when compared
with the vehicle group. Similarly, the number of open arms entries
was significantly different between treatments [F(6,49)¼23.09,
po0.001]. Compared to the vehicle group, the post-hoc test showed
that the total number of entries into the open arms was significantly
augmented (po0.05) in the group treated with 25 and 50 mg/kg of
Mf or 1 and 2 mg/kg of diazepam (Fig. 1c). No significant differences
were detected in animals treated with 75 mg/kg of Mf or 4 mg/kg of
diazepam as compared with the vehicle group. The total number of
entries into the closed arms exhibited no significant differences
among treatments [F(6,49)¼1.76, po0.127].
Consistent with these results, the analysis of the percentage of
open arm entries revealed a significant effect of treatment
[F(6,49)¼10.12, po0.001]. The 50 mg/kg of Mf or 2 mg/kg of
diazepam groups displayed a higher percentage of open arm
entries compared with the vehicle group (Fig. 1d). No significant
differences were found in the remaining groups.
3.2.2. Open field test
The effect of vehicle, Mf and diazepam on crossing, rearing,
grooming, and resting time in the open field test is showed in the
Table 1. The one-way ANOVA showed significant differences in
crossings [F(6,49)¼9.74, po0.001], among treatments. The post-hoc
test revealed that crossing was diminished only in 75 mg/kg of Mf
and 4 mg/kg of diazepam, as compared with the vehicle and
remaining groups. Similarly, significant differences [F(6,49)¼11.53,
po0.001] in time spent in rearing were found. The post-hoc test
revealed that time spent in rearing diminished only in 75 mg/kg
of Mf and 4 mg/kg of diazepam groups versus the vehicle
and remaining groups. The analysis of time spent in grooming
showed significant differences among treatments [F(6,49)¼24.97,
po0.001]. The post-hoc test revealed that grooming was lower in
M. Carro-Jua ´rez et al. / Journal of Ethnopharmacology 143 (2012) 592–598
animals treated with 75 mg/kg of Mf and 4 mg/kg of diazepam
when compared with the vehicle group; but on the other side,
compared with the vehicle group, grooming was augmented in
50 mg/kg of Mf and 2 mg/kg of diazepam groups. Finally, the
analysis of the resting time also showed significant differences
among treatments [F(6,49)¼53.02, po0.001] and it was observed
that resting time was only augmented in animals treated with
75 mg/kg of Mf and 4 mg/kg of diazepam, as compared with the
vehicle and remaining groups.
3.3. Blockade of the anxiolytic-like effect of Mf by picrotoxin
3.3.1. Elevated plus-maze test
The one-way ANOVA showed significant differences in time
spent into the open arms [F(5,36)¼5.77, po0.001]. Compared to
vehicle group, post-hoc analysis showed that this variable was
increased (po0.05) in the group treated with 50 mg/kg of Mf or
2 mg/kg of diazepam, an effect antagonized by the pretreatment
with picrotoxin (Fig. 2a).
The analysis of the number of entries into either the open or
[F(5,36)¼37.32, po0.001]. The total number of entries into the
arms augmented (po0.05) in groups treated with 50 mg/kg of Mf
or 2 mg/kg of diazepam respect to vehicle group (Fig. 2b),
picrotoxin pretreatment attenuated the effect of Mf and antag-
onized the effect of diazepam on this variable. On the other hand,
the number of open arms entries was significantly different
between treatments [F(5,36)¼44.78, po0.001]. The post-hoc
analysis showed that compared with the vehicle group, the total
number of entries into the open arms augmented (po0.05) in the
groups treated with 50 mg/kg of Mf or 2 mg/kg of diazepam;
however, pretreatment with picrotoxin, only prevented the effect
produced by diazepam but nor by Mf extract (Fig. 2c).
Finally, the analysis of the percentage of open arm entries
revealed significant effects of treatments [F(5,36)¼5.53, po0.001]
since animals treated with 50 mg/kg of Mf or 2 mg/kg of diazepam
displayed higher percentage of open arm entries compared with
the vehicle group, an effect attenuated by picrotoxin pretreatment
Fig. 1. Elevated plus-maze. (a) Time spent in open arms, (b) total entries into arms, (c) number of entries in the open arms, and (d) percentage of entries into the open arms
during 5 min of test.npo0.05 vs vehicle group (Veh), one-way ANOVA, Student-Newman–Keuls post-hoc test.
Effect of vehicle, M. frutescens aqueous extract, and diazepam on crossing, rearing, grooming and resting time in rats subjected to open field test for 5 min.
Montanoa frutescens (mg/kg)Diazepam (mg/kg)
npo0.05 vs. control group, M. frutescens (25 and 50 mg/kg), and diazepam (1 and 2 mg/kg).
þpo0.05 vs. vehicle group and remain groups in the same line, one-way ANOVA, post-hoc test Student-Newman-Keulls.
M. Carro-Jua ´rez et al. / Journal of Ethnopharmacology 143 (2012) 592–598
3.3.2. Open field test
The effect of vehicle, picrotoxin, Mf, diazepam or the combina-
tion of treatments on crossing, rearing, grooming, and resting time
in the open field test is showed in the Table 2. The one-way
ANOVA detected no significant differences in none of the evaluated
variables in this test: crossing [F(5,36)¼1.63, po0.176], rearing
[F(5,36)¼2.17, po0.079], grooming [F(5,36)¼2.06, po0.093] and
resting time [F(5,36)¼2.04, po0.095].
In the present study we explored the anxiolytic-like effects of
different doses of the aqueous crude extracts of Mf in the elevated
plus-maze and comparisons with the effects of diazepam, a clinically
effective anxiolytic, are described. Additionally, the involvement of
the GABAAreceptor in the anxiolytic-like effect of Mf extract was
also explored. The results can be summarized as follow: (i) 50 mg/kg
of Mf produced anxiolytic-like effects similarly to 2 mg/kg of
diazepam; while 25 mg/kg of Mf scarcely produced anxiolytic-like
effects comparable to 1 mg/kg diazepam; (ii) 75 mg/kg of Mf devoid
of any anxiolytic-like effects in the elevated plus-maze similarly to
4 mg/kg of diazepam, accompanied by hypoactivity in the open field
test; and, (iii) pretreatment with the GABAAantagonist, picrotoxin,
prevented the anxiolytic-like effect produced by 50 mg/kg of Mf
extract. Taken together, these findings indicate that Mf extract may
produce anxiolytic-like and sedative-like effects, reproducing a
pharmacological profile similar to diazepam, with participation of
the GABAAreceptor. Thus, present data support the notion that the
aqueous crude extract of Mf possesses anxiolytic properties as
popularly attributed by ancient Mexican cultures.
The elevated plus-maze is a well-accepted, experimental
animal model typically used to test the effectiveness of anxioly-
tics drugs (Griebel et al., 1997; Varty et al., 2002; Walf and Frye,
2007). In this test, rats or mice that display anxiety-like behavior
usually show reduction in the exploration of the open arms
evidenced by the reduction in both the number of entries into
and time spent in the open arms. Thus, animals treated with
clinically effective anxiolytic drugs like diazepam (Pellow et al.,
1985; Ferna ´ndez-Guasti et al., 2001; Wilson et al., 2004), some
neurosteroids with anxiolytic-like potency such as progesterone
or allopregnanolone (Go ´mez et al., 2002; Reddy et al., 2005;
Engin and Treit, 2007) or extracts from medicinal plants with
reputed anxiolytic properties (Molina-Herna ´ndez et al., 2004;
Grundmann et al., 2007; Dutt et al., 2010; Herrera-Ruiz et al.,
2011) increase the total time spent in the open arms, the number
of entries in the open arms, or the percentage of entries into the
open arms. Findings of this study show that animals treated with
50 mg of Mf extract reduces behavioral indicators of anxiety-like
behavior in the elevated plus-maze, which supports the notion of
Fig. 2. Elevated plus-maze. Blockade of the anxiolytic-like effect of M. frutescens (Mf) or diazepam (Dz) by picrotoxin (P): (a) time into open arms, (b) total entries into arms, (c)
entries into open arms, and (d) percentage of entries into the open arms. Veh¼vehicle.npo0.05 vs. Veh, P, PþMf, PþDz;þpo0.05 vs. Veh and P;#po0.05 vs. Veh, P and Dz;
&po0.05 vs. all groups, 1po 0.05 vs. Veh, P and PþDz; one-way ANOVA, Student-Newman–Keuls pos-hoc test.
Effect of vehicle, M. frutescens aqueous extract, picrotoxin and, treatment combination on crossing, rearing, grooming and resting time in rats subjected to open field test
for 5 min.
No significant differences among groups were found, one-way ANOVA. Picrotoxin¼1 mg/kg, M. frutescens¼50 mg/kg, Diazepam 1 mg/kg.
M. Carro-Jua ´rez et al. / Journal of Ethnopharmacology 143 (2012) 592–598
an anxiolytic effect attributed to Mf in the Mexican traditional
Animals treated with Mf extract at 75 mg/kg did not exhibit any
anxiolytic-like effect in the elevated plus-maze but exhibited
hypoactivity in the open field test, a phenomenon induced by
diazepam at 4 mg/kg, suggesting a possible sedative effect. In line
with this notion, it has been reported that 4 mg/kg of diazepam
produces sedative effects and reduces routed distance in the open
field test, an effect also detected after the administration of
extracts from plants with reputed sedative activity such as Pinelliae
ternate (Wu et al., 2011). Thus, taken together, these data obtained
from the elevated plus-maze suggest that Mf extracts possess a
similar pharmacological profile than benzodiazepines; while low
doses of the extract exert anxiolytic-like effects, high doses may
produce sedative activity similarly to diazepam. In this case,
sedative effect produced by 75 mg/kg of Mf could interfere
negatively in the behavioral performance of the rat in the elevated
plus-maze, concealing any anxiolytic-like effect.
Evidence shows that grooming behavior abnormally increase
in rats subjected to mild moderate stress (Murphy et al., 1996;
Singh et al., 1996; Jaiswal, 2002; Moyaho and Valencia, 2002) and
on the contrary, animals subjected to high-stress significantly
diminish this behavior (van Dijken et al., 1992; Perrot-Sinal et al.,
2004). Reduction of grooming behavior is prevented by anxiolytic
drugs, returning it to values of unstressed animals (Hata et al.,
1988; D’Aquila et al., 2000). This effect is considered as an
additional indicator of an anxiolytic-like effect at experimental
level and can be produced by diazepam and others substances
with well-characterized anxiolytic potency (Hata et al., 1988;
Rodrı ´guez-Landa et al., 2009b). In the present study, the vehicle
group showed the lower levels of grooming, an effect possibly
associated to the stress induced by previous session in the
elevated plus-maze, which also has been reported in other studies
using the light/dark test previous to evaluation in open field test
(Rodrı ´guez-Landa et al., 2009b). Interestingly, 50 mg/kg of Mf or
2 mg/kg of diazepam maintains grooming behavior while in
antagonism experiment show only a non-significant tendency to
increase grooming but effects of picrotoxin pretreatment on this
variable were no detected. Besides, the aforementioned profile is
associated with anxiolytic-like activity (D’Aquila et al., 2000).
Additionally, rearing was not significantly affect with the anxio-
lytic doses used in both experiments, which suggest that this
variable is not sufficiently sensible to anxiolytic agents including
diazepam, as previously reported (Rodrı ´guez-Landa et al., 2009b).
Respect to higher doses of Mf or diazepam (75 or 4 mg/kg,
respectively) exhibited a diminished grooming and rearing beha-
viors consistent with the sedative effects produced by both
treatments in this behavioral test.
Finally, the mechanism that mediates the anxiolytic-like effect
obtained with the aqueous crude extract of Mf in the elevated plus-
maze is unknown. However, it could be thought after a comparison
with the effects produced by diazepam above mentioned that a
major role of the central GABAergic system could be involved. Thus,
we observed that the pretreatment with picrotoxin, a non-compe-
titive antagonist for the GABAAchloride channel (Bormann, 2000)
prevented the anxiolytic-like effect produced by effective doses of
Mf extract in the elevated plus maze, an effect similar to that
obtained by picrotoxin pretreatment upon diazepam, suggesting the
involvement of the GABAA receptor in the anxiolytic-like effects
promoted by the compounds contained in this plant. In line with
this notion, evidence show that diazepam possesses a particular
benzodiazepine recognition site in the GABAAreceptor, which may
be selectively antagonized by bicuculline, however, picrotoxin also
may cancel the pharmacological effects of diazepam (Geller et al.,
1978; Bormann, 2000), including their anxiolytic-like effects at
experimental level (Treit et al., 1982).
The GABAAreceptor is a heteropentameric structure made up
of several subunits with recognition sites to g-aminobutyric
acid (GABA), benzodiazepines, alcohol, barbiturates, neuroster-
oids (Bormann, 2000) and natural chemical compounds (i.e. some
members of the family of terpenoids, polyacetylenic alcohols and
flavonoids) obtained from some plant extracts (Wang et al., 2005;
Nilsson and Sterner, 2011). The vast majority of GABAAreceptors
are characterized by their sensitivity to benzodiazepines and
generally their activation increase the intraneuronal concentra-
tion of chloride ion hyperpolarizing the cell, a mechanism
involved in the typical anxiolytic, hypnotic, sedative and antic-
onvulsant actions of benzodiazepines (i.e. diazepam and deriva-
tives) and other GABAergic agonists (Enna, 2007). Moreover, the
GABAAreceptor is the main target of clinically effective anxiolytic
drugs, neurosteroids (Bitran et al., 1995; Ferna ´ndez-Guasti and
Picazo, 1995; Bormann, 2000) and biologically active substances
of some medicinal plants (Zanoli et al., 2000; Avallone et al., 2000;
Grundmann et al., 2007; Costa et al., 2011). Thus, it is tempting to
suggest that the biologically active compounds contained in the
extract of Mf (i.e. kaurenoic, grandiflorenic or kauradienoic acids,
among others) target, at least partially, the central GABAergic
system to exert its actions promoting an anxiolytic-like effect.
Nonetheless, the bioactive compounds contained in the Mf
extract could also interact with other neurotransmitter systems
(i.e. serotoninergic, dopaminergic or noradrenergic) that together
could contribute to the anxiolytic-like effects of Mf extracts, a
possibility that remains to be further explored.
In conclusion, the data presented hereby reinforce the tradi-
tional use of Montanoa frutescens (‘‘resolves the mood changes and
nerves in an admirable form’’) described in the Libellus de Medici-
nalibus Indorum Herbis written in 1552 (de la Cruz-Badiano, 1552)
and in the actual Mexican traditional medicine as an anxiolytic-
like agent. Additionally, we contribute to the identification of a
partial mechanism of action subjacent to anxiolytic-like effect of
Mf extracts, which involve the GABAAreceptor.
This study was partially supported by grants to Universidad
Auto ´noma de Tlaxcala CA-UATx-006, and Universidad Veracruzana
UVE-CA-25. MLR-P received a fellowship from CONACyT (Reg.
377677) to postgraduate studies in Neuroethology (Universidad
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