1Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 29 | Number 03 | Article 349318
Anxiolytic-like activity of Aloysia virgata var. platyphylla leaves extract in mice
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Filliations
1Departamento de Farmacología,
Facultad de Ciencias Químicas,
Universidad Nacional de Asunción.
Campus UNA, 2169. San Lorenzo.
Paraguay.
*Corresponding
María L. Kennedy lukenrol@qui.una.py
Received: 06 April 2022
Accepted: 13 August 2022
Published: 18 August 2022
Anxiolytic-like activity of Aloysia virgata
var. platyphylla leaves extract in mice
Actividad ansiolítica del extracto de hojas de Aloysia virgata
var. platyphylla en ratones
Miguel A. Campuzano-Bublitz , Elena M.G. Diarte , Enrique Snead ,
Teresa Taboada , María L. Kennedy *
ABSTRACT
Background: Medicinal plants are part of traditional medicine and should be considered a
therapeutic alternative for mental diseases. Several plants belonging to the Verbenaceae
family have proved useful in treating general anxiety disorders, the most prevalent psychiatric
disorders. Objective: This research aimed to verify the extract’s safety, the effect on general
behavior, and the effect on sleeping time, as well as to evaluate the anxiolytic-like effect of
the methanol extract of Aloysia virgata var. platyphylla (Avp), in mice. Methodology: The
toxicity test was done according to the OECD guide (mice groups n=5), and general behavior
was observed during the assay. Sleeping time was assessed using the pentobarbital-induced
hypnosis method (n=8). Male Swiss albino mice (n=6) were treated with 50 to 400 mg/kg
of Avp extract and diazepam as a control. The anxiolytic-like effect was tested through
the hole board and elevated plus-maze test. Results: The Avp extract has no side effects
in tested doses, and no central nervous system depressant activity was noted. A. virgata
var. platyphylla increased exploration (number and time) in the hole board. In the elevated
plus-maze, increased number and time into open arms were evidenced compared to the
control group. Conclusion: With all these results, we concluded that the Avp extract is safe
and has a potential anxiolytic-like activity in the animal model used.
Keywords: Aloysia virgata var. platyphylla, sleeping time, hole board, elevated plus maze,
pre-clinical, anxiolytic
ORIGINAL RESEARCH
Published 18 August 2022
Doi: https://doi.org/10.17533/udea.vitae.v29n3a349318
Anxiolytic-like activity of Aloysia virgata var. platyphylla leaves extract in mice
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Filliations
1Departamento de Farmacología,
Facultad de Ciencias Químicas,
Universidad Nacional de Asunción.
Campus UNA, 2169. San Lorenzo.
Paraguay.
*Corresponding
María L. Kennedy lukenrol@qui.una.py
Received: 06 April 2022
Accepted: 13 August 2022
Published: 18 August 2022
Anxiolytic-like activity of Aloysia virgata
var. platyphylla leaves extract in mice
Actividad ansiolítica del extracto de hojas de Aloysia virgata
var. platyphylla en ratones
Miguel A. Campuzano-Bublitz , Elena M.G. Diarte , Enrique Snead ,
Teresa Taboada , María L. Kennedy *
ABSTRACT
Background: Medicinal plants are part of traditional medicine and should be considered a
therapeutic alternative for mental diseases. Several plants belonging to the Verbenaceae
family have proved useful in treating general anxiety disorders, the most prevalent psychiatric
disorders. Objective: This research aimed to verify the extract’s safety, the effect on general
behavior, and the effect on sleeping time, as well as to evaluate the anxiolytic-like effect of
the methanol extract of Aloysia virgata var. platyphylla (Avp), in mice. Methodology: The
toxicity test was done according to the OECD guide (mice groups n=5), and general behavior
was observed during the assay. Sleeping time was assessed using the pentobarbital-induced
hypnosis method (n=8). Male Swiss albino mice (n=6) were treated with 50 to 400 mg/kg
of Avp extract and diazepam as a control. The anxiolytic-like effect was tested through
the hole board and elevated plus-maze test. Results: The Avp extract has no side effects
in tested doses, and no central nervous system depressant activity was noted. A. virgata
var. platyphylla increased exploration (number and time) in the hole board. In the elevated
plus-maze, increased number and time into open arms were evidenced compared to the
control group. Conclusion: With all these results, we concluded that the Avp extract is safe
and has a potential anxiolytic-like activity in the animal model used.
Keywords: Aloysia virgata var. platyphylla, sleeping time, hole board, elevated plus maze,
pre-clinical, anxiolytic
ORIGINAL RESEARCH
Published 18 August 2022
Doi: https://doi.org/10.17533/udea.vitae.v29n3a349318
2Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 29 | Number 03 | Article 349318Miguel A. Campuzano-Bublitz, Elena M.G. Diarte, Enrique Snead, Teresa Taboada, María L. Kennedy
INTRODUCTION
Anxiety disorders are the most prevalent psychiatric
disorders. It is presumed that 3.8% of the world’s
population currently suffers from anxiety disorders
(1). Anxiety is still an underdiagnosed disease,
particularly in lower-income countries where data
about the prevalence of this disease is scarce, and
the treatment of mental illness has less importance
(2, 3). Anxiety usually occurs without obvious
symptoms but tends to manifest itself chronically,
and symptoms vary in severity over time (4). The
prevalence of anxiety has increased considerably
in recent years, attributing this to the currently
imposed lifestyle (5). Psychosocial factors include
genetic vulnerability, trauma, stress, and ultimately
end in neurobiological dysfunction and altered
neuropsychological behavior (3). Anxiety is often
related to other pathologies, such as viral diseases
(6). It is also usually considered a risk factor for
cardiovascular diseases and can cause depression,
which is a risk factor for cardiovascular diseases (7,
8). It is also related to rheumatological conditions (9).
More recently, it has been seen that the appearance
of COVID-19 increased the manifestations of anxiety
in people (10).
Anxiety disorders treatment includes drugs that
are quite effective. However, other considerations
like adverse effects, cost, safety warnings, drug
interactions, and contraindications must be
considered (3. 6). Medicinal plants have been part
of traditional medicine for centuries and still provide
alternatives for treating several diseases (11).
The use of medicinal plants as a therapeutic
alternative to treat a condition widely distributed
worldwide, such as anxiety, must be considered.
Many investigations have shown the anxiolytic
effect of medicinal plants in preclinical trials. The
roots, rhizomes, and stolons of Valeriana officinalis
L. (Valerianaceae) have sesquiterpenes and iridoids
such as valerenone, valerianol, valerenic acid, among
others, which have shown certain GABAergic activity,
reducing anxiety levels. The aerial parts of Passiflora
incarnata (Passifloraceae) contain flavonoids and
alkaloids to which sedative and anxiolytic effects
are attributed (12, 13).
In South America, several species of medicinal
plants belong to the Verbenaceae family, on which
anxiolytic activity was reported. Aloysia gratissima
var. gratissima (14), A. polystachya (15-17), A. triphylla
(18), Lippia alba, L. sidoides, and L. graveolens
(19-21) have shown their effectiveness as anxiolytic
in preclinical studies in mice, and some have also
shown anxiolytic effect in clinical trials (16). Mice
treated with these plants’ extract exhibited an
increase in number and time spent exploring in hole
board, and in the elevated plus-maze, they increased
the number of entries and time spent in the open
arms of the labyrinth.
Considering the large number of species belonging
to the Verbenaceae family that demonstrated
anxiolytic effect and the reported effect on the
central nervous system of two diterpenes isolated
from Aloysia virgata (22), in this work, the safety,
the effect on sleeping time, and the anxiolytic-like
activity of the methanol extract Aloysia virgata var.
platyphylla (Avp) in mice were evaluated.
RESUMEN
Antecedentes: Las plantas medicinales forman parte de la medicina tradicional y deben ser consideradas una alternativa
terapéutica para las enfermedades mentales. Varias plantas pertenecientes a la familia Verbenaceae han demostrado su
utilidad en el tratamiento de los trastornos de ansiedad, uno de los trastornos psiquiátricos más prevalentes. Objetivo: Esta
investigación tuvo como objetivo verificar la seguridad del extracto, el efecto sobre el comportamiento general y el efecto
sobre el tiempo de sueño, así como evaluar el efecto tipo ansiolítico del extracto metanólico de Aloysia virgata var. platyphylla
(Avp), en ratones. Metodología: La prueba de toxicidad se realizó de acuerdo con la guía de la OCDE (grupos de ratones n=5),
y se observó el comportamiento general durante el ensayo. El tiempo de sueño se evaluó mediante el método de hipnosis
inducida por pentobarbital (n=8). Se trataron ratones albinos suizos macho (n=6) con 50 a 400 mg/kg de extracto de Avp y
diazepam como control. El efecto ansiolítico se probó a través de la placa perforada y prueba del laberinto en cruz elevado.
Resultados: El extracto de Avp no tiene efectos secundarios en las dosis probadas y no se observó actividad depresora del
sistema nervioso central. A. virgata var. platyphylla aumentó la exploración (número y tiempo) en el tablero de agujeros. En
el laberinto en cruz elevado, se evidenció un mayor número y tiempo en los brazos abiertos en comparación con el grupo de
control. Conclusión: Con todos estos resultados, concluimos que el extracto de Avp es seguro y tiene una potencial actividad
ansiolítica en el modelo animal utilizado.
Palabras clave: Aloysia virgata var. platyphylla, tiempo de sueño, placa perforada, laberinto en cruz elevado, preclínico, ansiolítico
INTRODUCTION
Anxiety disorders are the most prevalent psychiatric
disorders. It is presumed that 3.8% of the world’s
population currently suffers from anxiety disorders
(1). Anxiety is still an underdiagnosed disease,
particularly in lower-income countries where data
about the prevalence of this disease is scarce, and
the treatment of mental illness has less importance
(2, 3). Anxiety usually occurs without obvious
symptoms but tends to manifest itself chronically,
and symptoms vary in severity over time (4). The
prevalence of anxiety has increased considerably
in recent years, attributing this to the currently
imposed lifestyle (5). Psychosocial factors include
genetic vulnerability, trauma, stress, and ultimately
end in neurobiological dysfunction and altered
neuropsychological behavior (3). Anxiety is often
related to other pathologies, such as viral diseases
(6). It is also usually considered a risk factor for
cardiovascular diseases and can cause depression,
which is a risk factor for cardiovascular diseases (7,
8). It is also related to rheumatological conditions (9).
More recently, it has been seen that the appearance
of COVID-19 increased the manifestations of anxiety
in people (10).
Anxiety disorders treatment includes drugs that
are quite effective. However, other considerations
like adverse effects, cost, safety warnings, drug
interactions, and contraindications must be
considered (3. 6). Medicinal plants have been part
of traditional medicine for centuries and still provide
alternatives for treating several diseases (11).
The use of medicinal plants as a therapeutic
alternative to treat a condition widely distributed
worldwide, such as anxiety, must be considered.
Many investigations have shown the anxiolytic
effect of medicinal plants in preclinical trials. The
roots, rhizomes, and stolons of Valeriana officinalis
L. (Valerianaceae) have sesquiterpenes and iridoids
such as valerenone, valerianol, valerenic acid, among
others, which have shown certain GABAergic activity,
reducing anxiety levels. The aerial parts of Passiflora
incarnata (Passifloraceae) contain flavonoids and
alkaloids to which sedative and anxiolytic effects
are attributed (12, 13).
In South America, several species of medicinal
plants belong to the Verbenaceae family, on which
anxiolytic activity was reported. Aloysia gratissima
var. gratissima (14), A. polystachya (15-17), A. triphylla
(18), Lippia alba, L. sidoides, and L. graveolens
(19-21) have shown their effectiveness as anxiolytic
in preclinical studies in mice, and some have also
shown anxiolytic effect in clinical trials (16). Mice
treated with these plants’ extract exhibited an
increase in number and time spent exploring in hole
board, and in the elevated plus-maze, they increased
the number of entries and time spent in the open
arms of the labyrinth.
Considering the large number of species belonging
to the Verbenaceae family that demonstrated
anxiolytic effect and the reported effect on the
central nervous system of two diterpenes isolated
from Aloysia virgata (22), in this work, the safety,
the effect on sleeping time, and the anxiolytic-like
activity of the methanol extract Aloysia virgata var.
platyphylla (Avp) in mice were evaluated.
RESUMEN
Antecedentes: Las plantas medicinales forman parte de la medicina tradicional y deben ser consideradas una alternativa
terapéutica para las enfermedades mentales. Varias plantas pertenecientes a la familia Verbenaceae han demostrado su
utilidad en el tratamiento de los trastornos de ansiedad, uno de los trastornos psiquiátricos más prevalentes. Objetivo: Esta
investigación tuvo como objetivo verificar la seguridad del extracto, el efecto sobre el comportamiento general y el efecto
sobre el tiempo de sueño, así como evaluar el efecto tipo ansiolítico del extracto metanólico de Aloysia virgata var. platyphylla
(Avp), en ratones. Metodología: La prueba de toxicidad se realizó de acuerdo con la guía de la OCDE (grupos de ratones n=5),
y se observó el comportamiento general durante el ensayo. El tiempo de sueño se evaluó mediante el método de hipnosis
inducida por pentobarbital (n=8). Se trataron ratones albinos suizos macho (n=6) con 50 a 400 mg/kg de extracto de Avp y
diazepam como control. El efecto ansiolítico se probó a través de la placa perforada y prueba del laberinto en cruz elevado.
Resultados: El extracto de Avp no tiene efectos secundarios en las dosis probadas y no se observó actividad depresora del
sistema nervioso central. A. virgata var. platyphylla aumentó la exploración (número y tiempo) en el tablero de agujeros. En
el laberinto en cruz elevado, se evidenció un mayor número y tiempo en los brazos abiertos en comparación con el grupo de
control. Conclusión: Con todos estos resultados, concluimos que el extracto de Avp es seguro y tiene una potencial actividad
ansiolítica en el modelo animal utilizado.
Palabras clave: Aloysia virgata var. platyphylla, tiempo de sueño, placa perforada, laberinto en cruz elevado, preclínico, ansiolítico
3Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 29 | Number 03 | Article 349318
Anxiolytic-like activity of Aloysia virgata var. platyphylla leaves extract in mice
MATERIALS AND METHODS
Plant material and extraction
The leaves of the species Aloysia virgata (Ruiz & Pav.)
Pers. var. platyphylla (Briq.) Moldenke belonging
to the Verbenaceae family were collected in
Cordillera, Paraguay. In the herbarium of Facultad
de Ciencias Químicas a voucher specimen was filed
(Universidad Nacional de Asunción, code Degen R
4.067). Dried and powdered leaves were extracted
with methanol. First, the powder was sonicated
for 30 min. at room temperature (three times) and
filtered. Subsequently, the residue was extracted
by a conventional reflux method (methanol, twice).
The material obtained after solvent evaporation was
used for biological assays.
Drugs
In our experiment diazepam (Roche) and sodium
pentobarbital (Abbott, Japan) were used. Methanol
(JT Baker) was used for plant extraction.
Experimental animals and ethical issues
Toxicity and general behavior tests were performed
in adult Swiss albino female mice. For sleeping time
and anxiolytic assays, male mice were used. All
animals came from Facultad de Ciencias Químicas
bioterium. They received daily standard animal
pellets and water ad libitum in an acclimatized room
(23-25°C, 12:12 h light-dark cycle, 50-60% humidity).
Scientifically standardized principles in compliance
with international animal welfare standards were
considered, and the Research Ethics Committee of
the Facultad de Ciencias Químicas approved the
experiment (CEI 402/18). All animals were used once
and euthanized after use by cervical dislocation.
Acute toxicity study and general behavior effect
Following the fixed doses methods described by
OECD guidelines (23), water (control group, n=5)
or methanol extracts of A. virgata var. platyphylla
(Avp) were administered orally by gavage to female
mice (n=5; doses: 5; 50; 300; 500 and 2000 mg/Kg)
and observed during 24 h. After that, mice were
followed for 14 days looking for delayed adverse
effects and finally dissected. The organs and
fundamental tissues (heart, kidney, spleen, lung,
liver, stomach, and intestine) were macroscopically
observed, and compared with those in the control
group. General effects on behavior, physiological
and neurological alterations, and neurotoxicity
symptoms were sought in treated animals and
control group (vehicle, 0.1mL/10g body weight, n=5)
5, 15, 30, 60, 120 minutes, and 24 and 48 hours after
drug administration (24).
Pentobarbital-induced hypnosis
Six groups of male mice (n=8) were treated with
vehicle (water, 0.1 mL/10 g body weight, per os,
p.o.); 50, 100, 200, and 400 mg/kg of Avp (p.o.),
or with diazepam (0.5 mg/kg, intraperitoneal, i.p.).
Each animal received sodium pentobarbital (35 mg/
kg, i.p.) 1 hour after the vehicle or the extract, and
twenty minutes after diazepam. Induction time and
sleeping time were recorded for each animal (25).
Hole-board test
The hole-board is an experimental method used to
measure anxiety and emotionality in animals. It is a
box containing 16 evenly spaced holes (10×10, 2 cm
diameter), the number and time spent in exploring
the holes (head-dipping), as well as ambulation
(peripheral and central area), rearing, grooming, and
defecation were measured (25). Six groups (male,
n=8) were treated with Avp extract (50, 100, 200,
and 400 mg/kg, p.o.), vehicle (water) or diazepam
(0,5 mg/kg, i.p.). One hour after the treatment (20
min after for diazepam-treated mice), each animal
was placed individually in the center of the hole
board and allowed to explore for 5 min freely. The
apparatus was cleaned after each trial with 10%
ethanol. The test was carried out in a light-controlled
room (red light, 15 W).
Elevated plus-maze test (EPM)
The plus maze apparatus (EPM) has been widely
validated to measure animal anxiolytic behavior.
The apparatus was made of transparent Plexiglas
and consisted of a plus-shaped maze formed by
two opposite open arms (arm length: 30.0 cm; arm
width: 5.0 cm); crossed with two arms enclosed
by walls (height: 15.0 cm). The open and enclosed
arms converge into a central platform (5.0 cm×5.0
cm). The maze is elevated at 40.0 cm from ground
level by a wood bearing, and it is placed in a room
illuminated with red light (15 W) (26-27). After
treatment (Avp extract: 50, 100, 200, and 400 mg/
kg, p.o.; vehicle (water) or diazepam, 0,5 mg/kg, i.p.),
each mouse was placed in the center on the elevated
plus-maze apparatus, with its head towards the open
arms, 60 min after the administration of vehicle and
the extract of Avp, or 20 min after diazepam. Their
behavior was observed for 5 min, and parameters
such as frequency of entrance and duration into
Anxiolytic-like activity of Aloysia virgata var. platyphylla leaves extract in mice
MATERIALS AND METHODS
Plant material and extraction
The leaves of the species Aloysia virgata (Ruiz & Pav.)
Pers. var. platyphylla (Briq.) Moldenke belonging
to the Verbenaceae family were collected in
Cordillera, Paraguay. In the herbarium of Facultad
de Ciencias Químicas a voucher specimen was filed
(Universidad Nacional de Asunción, code Degen R
4.067). Dried and powdered leaves were extracted
with methanol. First, the powder was sonicated
for 30 min. at room temperature (three times) and
filtered. Subsequently, the residue was extracted
by a conventional reflux method (methanol, twice).
The material obtained after solvent evaporation was
used for biological assays.
Drugs
In our experiment diazepam (Roche) and sodium
pentobarbital (Abbott, Japan) were used. Methanol
(JT Baker) was used for plant extraction.
Experimental animals and ethical issues
Toxicity and general behavior tests were performed
in adult Swiss albino female mice. For sleeping time
and anxiolytic assays, male mice were used. All
animals came from Facultad de Ciencias Químicas
bioterium. They received daily standard animal
pellets and water ad libitum in an acclimatized room
(23-25°C, 12:12 h light-dark cycle, 50-60% humidity).
Scientifically standardized principles in compliance
with international animal welfare standards were
considered, and the Research Ethics Committee of
the Facultad de Ciencias Químicas approved the
experiment (CEI 402/18). All animals were used once
and euthanized after use by cervical dislocation.
Acute toxicity study and general behavior effect
Following the fixed doses methods described by
OECD guidelines (23), water (control group, n=5)
or methanol extracts of A. virgata var. platyphylla
(Avp) were administered orally by gavage to female
mice (n=5; doses: 5; 50; 300; 500 and 2000 mg/Kg)
and observed during 24 h. After that, mice were
followed for 14 days looking for delayed adverse
effects and finally dissected. The organs and
fundamental tissues (heart, kidney, spleen, lung,
liver, stomach, and intestine) were macroscopically
observed, and compared with those in the control
group. General effects on behavior, physiological
and neurological alterations, and neurotoxicity
symptoms were sought in treated animals and
control group (vehicle, 0.1mL/10g body weight, n=5)
5, 15, 30, 60, 120 minutes, and 24 and 48 hours after
drug administration (24).
Pentobarbital-induced hypnosis
Six groups of male mice (n=8) were treated with
vehicle (water, 0.1 mL/10 g body weight, per os,
p.o.); 50, 100, 200, and 400 mg/kg of Avp (p.o.),
or with diazepam (0.5 mg/kg, intraperitoneal, i.p.).
Each animal received sodium pentobarbital (35 mg/
kg, i.p.) 1 hour after the vehicle or the extract, and
twenty minutes after diazepam. Induction time and
sleeping time were recorded for each animal (25).
Hole-board test
The hole-board is an experimental method used to
measure anxiety and emotionality in animals. It is a
box containing 16 evenly spaced holes (10×10, 2 cm
diameter), the number and time spent in exploring
the holes (head-dipping), as well as ambulation
(peripheral and central area), rearing, grooming, and
defecation were measured (25). Six groups (male,
n=8) were treated with Avp extract (50, 100, 200,
and 400 mg/kg, p.o.), vehicle (water) or diazepam
(0,5 mg/kg, i.p.). One hour after the treatment (20
min after for diazepam-treated mice), each animal
was placed individually in the center of the hole
board and allowed to explore for 5 min freely. The
apparatus was cleaned after each trial with 10%
ethanol. The test was carried out in a light-controlled
room (red light, 15 W).
Elevated plus-maze test (EPM)
The plus maze apparatus (EPM) has been widely
validated to measure animal anxiolytic behavior.
The apparatus was made of transparent Plexiglas
and consisted of a plus-shaped maze formed by
two opposite open arms (arm length: 30.0 cm; arm
width: 5.0 cm); crossed with two arms enclosed
by walls (height: 15.0 cm). The open and enclosed
arms converge into a central platform (5.0 cm×5.0
cm). The maze is elevated at 40.0 cm from ground
level by a wood bearing, and it is placed in a room
illuminated with red light (15 W) (26-27). After
treatment (Avp extract: 50, 100, 200, and 400 mg/
kg, p.o.; vehicle (water) or diazepam, 0,5 mg/kg, i.p.),
each mouse was placed in the center on the elevated
plus-maze apparatus, with its head towards the open
arms, 60 min after the administration of vehicle and
the extract of Avp, or 20 min after diazepam. Their
behavior was observed for 5 min, and parameters
such as frequency of entrance and duration into
4Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 29 | Number 03 | Article 349318Miguel A. Campuzano-Bublitz, Elena M.G. Diarte, Enrique Snead, Teresa Taboada, María L. Kennedy
open and closed arms were recorded. After each
test, the EPM apparatus was thoroughly cleaned
with ethanol (10%). Subsequently, the number of
both measured parameters was analyzed.
Data analysis
GraphPad Software, Inc., CA (GraphPad Prism
7.0) was used for the statistical analysis of data.
Analysis of variance (ANOVA) of one factor followed
by Dunnett’s post hoc was performed, and data
were presented as mean ± SD. When p < 0.05, the
difference was considered statistically significant.
RESULTS
Toxicity test and general behavior
The toxicity test revealed no adverse effects of
Aloysia virgata var. platyphylla, with doses of up
to 2 g/Kg of mouse body weight. Administration
of the extract did not cause the animals’ death or
toxicity symptoms during the 24-hour observation
period. Compared to the control group, all animals
presented normal behavior: postural reflex,
grooming, responses to nociceptive stimuli, and
water and food consumption (data not shown). After
macroscopical observation, no organ alteration was
evidenced with any tested doses.
Sleeping time induced by pentobarbital
In the sleep-time trial, it was observed that with the
tested doses, neither the induction time (not shown)
nor the total sleep time were affected compared
to the control group. This suggests that A. virgata
var. platyphylla has no central nervous system
depressant effect. Diazepam, used as a positive
control drug, prolonged the sleep time as expected
(Figure 1).
Figure 1: Sleep-time after barbiturate administration in mice orally
treated with A. virgata var. platyphylla extract. Veh (vehicle), Dz
(Diazepam), Avp50 (A. virgata var. platyphylla 50mg/Kg), Avp100
(100mg/Kg), Avp200 (200mg/Kg), Avp400 (400mg/Kg). Data are
expressed as mean ± SD, after one-way ANOVA, Dunnet posttest;
**p<0.01, compared with Vehicle.
Hole-board assay
The number of head dipping in the hole-board test
(Figure 2A) and the exploration time (Figure 2B)
showed a significant difference between the vehicle
group and diazepam and the groups of animals
treated with some concentrations of A. virgata var.
platyphylla extract.
A B
Figure 2. A) Number of head dipping and B) time of exploration in mice treated with A. virgata var. platyphylla in the Hole board
apparatus. Veh (vehicle), Dz (Diazepam), Avp50 (A. virgata var. platyphylla 50mg/Kg), Avp100 (100mg/Kg), Avp200 (200mg/Kg),
Avp400 (400mg/Kg). Data represent mean ± SD (n= 8); ANOVA one way, Dunnett post-test; * p<0.05, ** p<0.01, *** p<0,001, ****
p<0,0001, compared with Vehicle.
open and closed arms were recorded. After each
test, the EPM apparatus was thoroughly cleaned
with ethanol (10%). Subsequently, the number of
both measured parameters was analyzed.
Data analysis
GraphPad Software, Inc., CA (GraphPad Prism
7.0) was used for the statistical analysis of data.
Analysis of variance (ANOVA) of one factor followed
by Dunnett’s post hoc was performed, and data
were presented as mean ± SD. When p < 0.05, the
difference was considered statistically significant.
RESULTS
Toxicity test and general behavior
The toxicity test revealed no adverse effects of
Aloysia virgata var. platyphylla, with doses of up
to 2 g/Kg of mouse body weight. Administration
of the extract did not cause the animals’ death or
toxicity symptoms during the 24-hour observation
period. Compared to the control group, all animals
presented normal behavior: postural reflex,
grooming, responses to nociceptive stimuli, and
water and food consumption (data not shown). After
macroscopical observation, no organ alteration was
evidenced with any tested doses.
Sleeping time induced by pentobarbital
In the sleep-time trial, it was observed that with the
tested doses, neither the induction time (not shown)
nor the total sleep time were affected compared
to the control group. This suggests that A. virgata
var. platyphylla has no central nervous system
depressant effect. Diazepam, used as a positive
control drug, prolonged the sleep time as expected
(Figure 1).
Figure 1: Sleep-time after barbiturate administration in mice orally
treated with A. virgata var. platyphylla extract. Veh (vehicle), Dz
(Diazepam), Avp50 (A. virgata var. platyphylla 50mg/Kg), Avp100
(100mg/Kg), Avp200 (200mg/Kg), Avp400 (400mg/Kg). Data are
expressed as mean ± SD, after one-way ANOVA, Dunnet posttest;
**p<0.01, compared with Vehicle.
Hole-board assay
The number of head dipping in the hole-board test
(Figure 2A) and the exploration time (Figure 2B)
showed a significant difference between the vehicle
group and diazepam and the groups of animals
treated with some concentrations of A. virgata var.
platyphylla extract.
A B
Figure 2. A) Number of head dipping and B) time of exploration in mice treated with A. virgata var. platyphylla in the Hole board
apparatus. Veh (vehicle), Dz (Diazepam), Avp50 (A. virgata var. platyphylla 50mg/Kg), Avp100 (100mg/Kg), Avp200 (200mg/Kg),
Avp400 (400mg/Kg). Data represent mean ± SD (n= 8); ANOVA one way, Dunnett post-test; * p<0.05, ** p<0.01, *** p<0,001, ****
p<0,0001, compared with Vehicle.
5Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 29 | Number 03 | Article 349318
Anxiolytic-like activity of Aloysia virgata var. platyphylla leaves extract in mice
Elevated plus-maze test
The anxiolytic activity of A. virgata var. platyphylla
was determined in mice using the elevated plus-
maze. It was observed that the group treated
with diazepam, and those that received 50 and
200 mg/kg of A. virgata var. platyphylla, increased
the crossings in open arms compared to the group
treated with vehicle (Figure 3A). The mean time
spent in open arms was also increased in the groups
treated with diazepam (p<0.0001) and when the
animals received 50, 100, and 200 mg/kg (Figure
3B) compared to the control group.
In the number of crossings in closed arms, no
significant differences have been observed with any
of the tested doses of A. virgata var. platyphylla or
diazepam compared to the control group (figure
4A). The time spent in closed arms was significantly
reduced with diazepam and 50, 100, and 200 mg/kg
of Avp extract (figure 4B).
A B
Figure 3. A) Number of crossing and B) time spent by mice in the open arms of plus-maze after treatment with A. virgata var.
platyphylla. Veh (vehicle), Dz (Diazepam), Avp50 (A. virgata var. platyphylla 50mg/Kg), Avp100 (100mg/Kg), Avp200 (200mg/Kg),
Avp400 (400mg/Kg). Data represent mean ± SD (n= 8); ANOVA one way, Dunnett post-test; * p<0.05, ** p<0.01, *** p<0,001, ****
p<0,0001, compared with Vehicle.
A B
Figure 4. A) Number of crossing and B) time spent by mice in the closed arms of plus maze after treatment with A. virgata var.
platyphylla. Veh (vehicle), Dz (Diazepam), Avp50 (A. virgata var. platyphylla 50mg/Kg), Avp100 (100mg/Kg), Avp200 (200mg/Kg),
Avp400 (400mg/Kg). Data represent mean ± SD (n= 8); ANOVA one way, Dunnett post-test; ** p<0.01, *** p<0,001, **** p<0,0001,
compared with Vehicle.
Anxiolytic-like activity of Aloysia virgata var. platyphylla leaves extract in mice
Elevated plus-maze test
The anxiolytic activity of A. virgata var. platyphylla
was determined in mice using the elevated plus-
maze. It was observed that the group treated
with diazepam, and those that received 50 and
200 mg/kg of A. virgata var. platyphylla, increased
the crossings in open arms compared to the group
treated with vehicle (Figure 3A). The mean time
spent in open arms was also increased in the groups
treated with diazepam (p<0.0001) and when the
animals received 50, 100, and 200 mg/kg (Figure
3B) compared to the control group.
In the number of crossings in closed arms, no
significant differences have been observed with any
of the tested doses of A. virgata var. platyphylla or
diazepam compared to the control group (figure
4A). The time spent in closed arms was significantly
reduced with diazepam and 50, 100, and 200 mg/kg
of Avp extract (figure 4B).
A B
Figure 3. A) Number of crossing and B) time spent by mice in the open arms of plus-maze after treatment with A. virgata var.
platyphylla. Veh (vehicle), Dz (Diazepam), Avp50 (A. virgata var. platyphylla 50mg/Kg), Avp100 (100mg/Kg), Avp200 (200mg/Kg),
Avp400 (400mg/Kg). Data represent mean ± SD (n= 8); ANOVA one way, Dunnett post-test; * p<0.05, ** p<0.01, *** p<0,001, ****
p<0,0001, compared with Vehicle.
A B
Figure 4. A) Number of crossing and B) time spent by mice in the closed arms of plus maze after treatment with A. virgata var.
platyphylla. Veh (vehicle), Dz (Diazepam), Avp50 (A. virgata var. platyphylla 50mg/Kg), Avp100 (100mg/Kg), Avp200 (200mg/Kg),
Avp400 (400mg/Kg). Data represent mean ± SD (n= 8); ANOVA one way, Dunnett post-test; ** p<0.01, *** p<0,001, **** p<0,0001,
compared with Vehicle.
6Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 29 | Number 03 | Article 349318Miguel A. Campuzano-Bublitz, Elena M.G. Diarte, Enrique Snead, Teresa Taboada, María L. Kennedy
DISCUSION
A. virgata var. platyphylla showed no adverse
effect in the acute toxicity test. A similar result was
observed with A. gratissima var gratissima (14). The
barbiturate-induced sleep time test indicated that
this plant is devoid of depressant or stimulant effects
on the central nervous system (28), while animals
treated with diazepam prolonged sleep time as
expected for this drug used as the positive control.
Mice treated with diazepam and 50, 100, and 200 mg/kg
of the methanolic extract of A. virgata var. platyphylla
significantly increased the number and exploration
time in the hole board compared to the control
group. This result is an indication of the anxiolytic
effect. (29). Additionally, in the elevated plus maze
test, both diazepam and the Avp extract (50,
100, and 200 mg/kg) increased the entries and
permanency in the EPM open arms, which confirms
the anxiolytic-like effect of Avp. Accordingly, the
time spent in closed arms decreased in mice that
received diazepam and the same doses of Avp (50,
100, and 200 mg/kg). In this case, it was verified
that the trial is more sensitive for the time spent in
closed arms (30) since no difference was noted in the
number of entries into the closed arms. Therefore,
the hole-board and plus-maze results indicated that
A. virgata var. platyphylla has a potential anxiolytic
effect (26). In both tests with animals, the control
group was compared with diazepam (positive
control to determine anxiolytic activity), and a
statistically significant difference was observed for
each of the parameters analyzed, thus validating
each of the determinations carried out to conclude
that Avp extract has an anxiolytic-type activity.
The hole-board test offers a simple method to
measure potential anxiolytic effects. In unfamiliar
environments, mice show less mobility, and when
treated with an anxiolytic drug such as diazepam,
their mobility and the number and time of head
dipping will increase considerably. These results,
together with those obtained in the EPM where
time spent in open arms increased and time spent
in closed arms decreased in those mice treated with
an anxiolytic drug, allowed us to determine that A.
virgata var. platyphylla has an anxiolytic-like effect
(30). Several species of Verbenaceae, particularly
Aloysia, demonstrated an anxiolytic effect (12,14-
16,18, 20-22), and our results agree with that.
Although it is still pending to determine the
secondary metabolites present in this extract,
phenylethanoid compounds, such as verbascoside,
have been identified in this genus and associated
with the anxiolytic effect. The tests showed that
animals treated with verbascoside increased the
percentage of entry in EPM and the time spent
in open arms, as expected for substances with
anxiolytic effects (31). Further experiments are
required to determine the chemical composition of
the extract, as well as to elucidate the mechanism of
action involved in the anxiolytic effect demonstrated
in biological systems. Still, it is presumed that they
would act by interaction with GABA A since, in all
the tests similar response to diazepam has been
observed (32).
CONCLUSION
Conducting this research, we determined that the
extract is safe up to 2 g/Kg since there was no
evidence of toxicity, nor did it affect the general
behavior. The results indicated a possible anxiolytic
activity according to the results observed in the
hole-board and the elevated plus-maze tests.
We also observed the absence of central nervous
system depressant activity in the barbiturate-
induced sleep test.
Authors’ Contributions statement
All authors have made substantial contribution to
this work, read the final manuscript, and approved
the submission.
Acknowledgements
The authors really appreciate the collaboration of
researchers from the Department of Botany from
Facultad de Ciencias Químicas for plant collection
and identification; and Mr G López and N Kennedy
for revising the English language in the document.
Conflicts of interest
The authors declare that they have no conflicts of
interest.
Funding
This research was suppor ted by Rectorado,
Universidad Nacional de Asunción, Paraguay
(Proyecto code FCQ/04/16), and Facultad de
Ciencias Químicas, Universidad Nacional de
Asunción, Paraguay.
DISCUSION
A. virgata var. platyphylla showed no adverse
effect in the acute toxicity test. A similar result was
observed with A. gratissima var gratissima (14). The
barbiturate-induced sleep time test indicated that
this plant is devoid of depressant or stimulant effects
on the central nervous system (28), while animals
treated with diazepam prolonged sleep time as
expected for this drug used as the positive control.
Mice treated with diazepam and 50, 100, and 200 mg/kg
of the methanolic extract of A. virgata var. platyphylla
significantly increased the number and exploration
time in the hole board compared to the control
group. This result is an indication of the anxiolytic
effect. (29). Additionally, in the elevated plus maze
test, both diazepam and the Avp extract (50,
100, and 200 mg/kg) increased the entries and
permanency in the EPM open arms, which confirms
the anxiolytic-like effect of Avp. Accordingly, the
time spent in closed arms decreased in mice that
received diazepam and the same doses of Avp (50,
100, and 200 mg/kg). In this case, it was verified
that the trial is more sensitive for the time spent in
closed arms (30) since no difference was noted in the
number of entries into the closed arms. Therefore,
the hole-board and plus-maze results indicated that
A. virgata var. platyphylla has a potential anxiolytic
effect (26). In both tests with animals, the control
group was compared with diazepam (positive
control to determine anxiolytic activity), and a
statistically significant difference was observed for
each of the parameters analyzed, thus validating
each of the determinations carried out to conclude
that Avp extract has an anxiolytic-type activity.
The hole-board test offers a simple method to
measure potential anxiolytic effects. In unfamiliar
environments, mice show less mobility, and when
treated with an anxiolytic drug such as diazepam,
their mobility and the number and time of head
dipping will increase considerably. These results,
together with those obtained in the EPM where
time spent in open arms increased and time spent
in closed arms decreased in those mice treated with
an anxiolytic drug, allowed us to determine that A.
virgata var. platyphylla has an anxiolytic-like effect
(30). Several species of Verbenaceae, particularly
Aloysia, demonstrated an anxiolytic effect (12,14-
16,18, 20-22), and our results agree with that.
Although it is still pending to determine the
secondary metabolites present in this extract,
phenylethanoid compounds, such as verbascoside,
have been identified in this genus and associated
with the anxiolytic effect. The tests showed that
animals treated with verbascoside increased the
percentage of entry in EPM and the time spent
in open arms, as expected for substances with
anxiolytic effects (31). Further experiments are
required to determine the chemical composition of
the extract, as well as to elucidate the mechanism of
action involved in the anxiolytic effect demonstrated
in biological systems. Still, it is presumed that they
would act by interaction with GABA A since, in all
the tests similar response to diazepam has been
observed (32).
CONCLUSION
Conducting this research, we determined that the
extract is safe up to 2 g/Kg since there was no
evidence of toxicity, nor did it affect the general
behavior. The results indicated a possible anxiolytic
activity according to the results observed in the
hole-board and the elevated plus-maze tests.
We also observed the absence of central nervous
system depressant activity in the barbiturate-
induced sleep test.
Authors’ Contributions statement
All authors have made substantial contribution to
this work, read the final manuscript, and approved
the submission.
Acknowledgements
The authors really appreciate the collaboration of
researchers from the Department of Botany from
Facultad de Ciencias Químicas for plant collection
and identification; and Mr G López and N Kennedy
for revising the English language in the document.
Conflicts of interest
The authors declare that they have no conflicts of
interest.
Funding
This research was suppor ted by Rectorado,
Universidad Nacional de Asunción, Paraguay
(Proyecto code FCQ/04/16), and Facultad de
Ciencias Químicas, Universidad Nacional de
Asunción, Paraguay.
7Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 29 | Number 03 | Article 349318
Anxiolytic-like activity of Aloysia virgata var. platyphylla leaves extract in mice
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Anxiolytic-like activity of Aloysia virgata var. platyphylla leaves extract in mice
REFERENCES
1. Dattani S, Ritchie H, Roser M. Mental Health. Our World In Data
2021; Available at: https://ourworldindata.org/mental-health
2. Wittchen HU, Jacobi F, Rehm J, Gustavsson A, Svensson M,
Jönsson B, Olesen J, Allgulander C, Alonso J, Faravelli C,
Fratiglioni L, Jennum P, Lieb R, Maercker A, van Os J, Preisig
M, Salvador-Carulla L, Simon R, Steinhausen H-C. The size and
burden of mental disorders and other disorders of the brain in
Europe 2010. Eur Neuropsychopharmacol. 2011;21(9):655–679.
DOI: https://doi.org/10.1016/j.euroneuro.2011.07.018
3. Bandelow B, Michaelis S, Wedekind D. Treatment of anxiety
disorders. Dialogues Clin Neurosci. 2017;19(2):93-106. DOI:
https://doi.org/10.31887/DCNS.2017.19.2/bbandelow
4. Angst J, Gamma A, Baldwin DS, Ajdacic-Gross V, Rössler W. The
generalized anxiety spectrum: Prevalence, onset, course and
outcome. Eur Arch Psychiatry Clin Neurosci. 2009;259(1):37–45.
DOI: https://doi.org/10.1007/s00406-008-0832-9
5. Kidman A. Neurochemical and cognitive aspects of anxiety
disorders. Prog Neurobiol. 1989;32(5):391–402. DOI: https://doi.
org/10.1016/0301-0082(89)90029-4
6. Coughlin SS. Anxiety and depression: linkages with viral diseases.
Public Health Rev. 2012;34(2):1–17. DOI: https://doi.org/10.1007/
BF03391675
7. Gustad LT, Laugsand LE, Janszky I, Dalen H, Bjerkeset O.
Symptoms of anxiety and depression and risk of heart failure: The
HUNT Study. Eur J Heart Fail. 2014;16(8):861–870. DOI: https://
doi.org/10.1002/ejhf.133
8. Scott KM. Depression, anxiety and incident cardiometabolic
diseases. Curr Opin Psychiatry. 2014;27(4):289–293. DOI: https://
doi.org/10.1097/YCO.0000000000000067
9. Tor ta R, Pennazio F, Ieraci V. Anxiety and depression in
rheumatologic diseases: The relevance of diagnosis and
management. Reumatismo. 2014;66(1):92–97. DOI: https://doi.
org/10.4081/reumatismo.2014.769
10. Giuntella O, Hyde K, Saccardo S, Sadoff S. Lifestyle and mental
health disruptions during COVID-19. Proceedings of the National
Academy of Sciences Mar 2021, 118 (9) e2016632118. DOI: https://
doi.org/10.1073/pnas.2016632118
11. Yeboah SO, Amponsah IK, Kaba JS, Abunyewa AA. Abundance,
richness and use of medicinal plants under different land uses
in the Guinea Savanna zone of Northern Ghana, All Earth, 2022;
34:1, 202-214. DOI: 10.1080/27669645.2022.2105485
12. López-Rubalcava C, Estrada-Camarena E. Mexican medicinal
plants with anxiolytic or antidepressant activity: Focus on
preclinical research. J Ethnopharmacol. 2016;186:377–391. DOI:
https://doi.org/10.1016/j.jep.2016.03.053
13. Saki K, Bahmani M, Rafieian-Kopaei M. The effect of most
impor tant medicinal plants on two impor tant psychiatric
disorders (anxiety and depression)-a review. Asian Pac J Trop
Med. 2014;7(S1):S34–42. DOI: https://doi.org/10.1016/S1995-
7645(14)60201-7
14. Kennedy ML, Taboada T, Snead E, Diarte EMG, Coronel CM,
Arrua W, Heinechen O, Montalbetti Y, Hellion-Ibarrola MC,
Ibarrola DA, Campuzano-Bublitz MA. Evaluation of methanol
extract of Aloysia gratissima var. gratissima leaves on behavior
and anxiety in mice. Int J Pharm Sci & Res. 2021; 12(7): 3858-3865.
DOI: https://doi.org/10.13040/IJPSR.0975-8232.12(7).3858-65
15. Mora S, Díaz-Véliz G, Millán R, Lungenstrass H, Quirós S, Coto-
Morales T, Hellión-Ibarrola MC. Anxiolytic and antidepressant-
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