Key words
antidepressant - mental illness - immobility - Olax subscorpioidea - yohimbine
Introduction
Depression is an affective disorder characterized by change in mood, loss of interest
or pleasure, feelings of guilt or low self-worth, disturbed sleep or appetite, low
energy, psychomotor retardation and melancholia. It is a major health condition with
life time prevalence in the range of 10–15% [1]. World Health Organization [2] and Richelson et al., [3] estimated that 5.8% of men and 9.5% of women undergoing a depressive episode in
their life time mostly end up committing suicide. Its negative social impact and impairing
effect on daily activities and wellbeing has led to incapability and loss of productivity
[4]. Most current therapeutic interventions employed in the management of depressive
disorder are marred by their bizarre and undesirable side effects which tend to limit
their clinical usefulness. Another drawback is that available treatments remain sub-optimal,
with a delay of 3–6 weeks before their clinical effects can be achieved and lack of
efficacy is also observed in many cases [5]
[6].
In the light of the fact that plants present a large untapped source of structurally
novel compounds that might serve as lead for the development of novel drugs, the exploration
of traditional source of medicine for more promising antidepressant drugs is justified.
Studies have shown that herbs have been used as alternative therapy for depression
[7]. Olax subscorpioidea (OS) locally called ‘ifon’ is a shrub or tree that is widely distributed in Nigeria
and some parts of Africa. In Congo Republic folk medicine, stem with leaves of Olax subscorpioidea decoction, and steam bath have been used to treat rheumatism and articular pains
[8]. Ethno-botanical survey showed that OS has been indicated in the management of asthma
in South West Nigeria and as recipe for the management of cancer [9]
[10]. Its use in combination with another plant in the treatment of anxiety and mental
illness disorders has been reported. [11]. Studies have shown that stem of OS contains alkaloids, steroids, and flavonoids
together with other active ingredients in the ethanoic extract with the exception
of saponins which is present in the aqueous extract alone and demonstrates antimicrobial
activity [12]. Based on the ethnomedicinal use in mental illness and some other CNS disorders
and lack of scientific report supporting its use in these conditions, the study is
therefore, designed to evaluate antidepressant activity of Ethanol Extract of Olax subscorpioidea Leaves (EEOSL) in animal models.
Materials and Methods
Plant materials
The leaves of Olax subscorpioidea were collected in February 2012 at the Gambari Forest Reserve, Ibadan, the Oyo state
capital, Nigeria. The taxonomical identification and authentication of the plant was
done at the herbarium section of the Forestry Research Institute of Nigeria (FRIN),
Ibadan, Nigeria. A voucher specimen with identification number 109924 was deposited
and compared with the reference specimen.
Preparation of plant material and drugs
Air-dried leaves (150 g) were pulverized and soaked in 50% ethanol (1.5 L) for 48 h.
The filtrate was concentrated with a rotary evaporator to give a semisolid residue
and evaporated to dryness to form solid residue (8.7 g). It was kept in the desiccator
for further use. The dried extract was then subsequently reconstituted in distilled
water at appropriate concentrations for the various experiments. All drugs and the
extract were dissolved in distilled water and administered by intraperitoneal (i. p.)
route.
Laboratory animal
Male Swiss Albino mice (20–25 g) used in this study were obtained from the Laboratory
Animal Centre of the College of Medicine, University of Ibadan, Nigeria. The animals
were kept in well-ventilated and hygienic compartments, maintained under standard
environmental conditions and fed with standard rodent pellet (Livestock Feed PLC,
Lagos, Nigeria) and water ad libitum. The experimental procedures adopted in this
study were in accordance with the United States National Institutes of Health Guidelines
for Care and Use of Laboratory Animals in Biomedical Research (NIH, 1985).
Drugs and chemicals
Yohimbine (Sigma-Aldrich (St.Louis, MO, USA), reserpine (Pfizer Inc., New York, NY,
USA)., imipramine (Shanghai Zhongxi Pharmaceutical Co., Ltd. Shanghai, China).
Acute toxicity test
The method described by Lorke [13] was used to determine the LD50, which is the index of acute toxicity. Swiss Albino mice (20–25 g) of either sex
were used. This method involved an initial dose finding procedure, in which the animals
were divided into 3 groups of 3 animals. Doses of 10, 100 and 1 000 mg/kg were administered
intraperitonealy (i. p.). One dose for each group. The treated animals were monitored
for 24 h mortality and general behavior. From the results of the above step, 4 different
doses of (500, 800, 2 000 and 3 000 mg/kg) were chosen and administered i. p. respectively
to 4 groups of one mouse per group. The treated animals were monitored for 24 h. The
LD50 was then calculated as the geometric mean of the lowest dose showing death and the
highest dose showing no death.
Antidepressant activity
Forced swimming test (FST)
Male mice (20–24 g) were used in the forced swimming test [14]. Mice were assigned to 6 different groups (n=5 for each group). Group (1) received
distilled water (10 mL/kg), group (2–5) received OS (6.25 12.5, 25, 50 mg/kg) respectively
while group (6) received Imipramine (25 mg/kg). 30 min later, mice were dropped one
at a time into a Plexiglas cylinder (25 cm height, diameter 10 cm containing water
to a height of 10 cm at 23–25°C) and observed for 6 min. After the first 2 min of
the initial vigorous struggling, the animals were immobile. A mouse was judged immobile
if it floated in the water in an upright position and made only slight movements to
prevent sinking. The total duration of immobility was recorded during the last 4 min
of the 6 min test.
Tail suspension test
The total duration of immobility following tail suspension was measured according
to the method described for evaluating potential antidepressants [15]. Mice were assigned to 6 different groups (n=5 for each group). Group (1) received
distilled water (10 mL/kg), group (2–5) received OS (6.25 12.5, 25, 50 mg/kg) respectively
while group (6) received Imipramine (25 mg/kg). 30 min later mice were suspended on
the edge of a table, 50 cm above the floor with the help of an adhesive tape placed
approximately 1 cm from the tip of the tail. Immobility time was recorded during 6 min
period in different groups. The animal was considered to be immobile when it did not
show any movement of the body and hanged passively.
Open field Test (OFT)
In order to rule out any unspecific locomotor effect of olax subscorpioidea on antidepressant-like effect of these compounds, mice were administered with the
same regimen as in FST or TST. Their locomotor activities (crossing activity) were
evaluated in the open field paradigm. Before each test, animals were kept in the test
room at least 1 h before open-field test (OFT) for habituation. The ambulatory behaviour
was assessed in open-field described by Rodrigues et al. [16]. The main apparatus consisted of square arena (50 cm×50 cm×40 cm) high with grey
surface covering every wall. The floor of the arena was divided equally into 25 squares
(10 cm×10 cm) marked by black lines. All animals were used only once in this test.
These animals were different from those used in the FST and TST. Group (1) received
distilled water (10 mL/kg), group (2–5) received OS (6.25 12.5, 25, 50 mg/kg) respectively
while group (6) received Imipramine (25 mg/kg). 30 min after, each mouse was placed
individually at the center of the arena and allowed to explore freely. The number
of squares crossed with all paws (crossing) were observed and counted in 5 min. The
square arena was cleaned with a solution of 70% alcohol between tests and dried after
occupancy by each mouse in order to hide animal clues and to prevent each mouse from
being influenced by the odors present in the urine and feces of the previous mouse.
Yohimbine induced lethality test
To reveal whether noradrenergic system is involved in the antidepressant-like effect
of the extract, yohimbine induced lethality test was performed [17]. Mice were assigned to 6 different groups (n=5 for each group). Group (1) received
distilled water (10 mL/kg), group (2–5) received OS (6.25 12.5, 25, 50 mg/kg) respectively
while group (6) received Imipramine (25 mg/kg) 30 min prior to yohimbine administration
(35 mg/kg, intraperitoneal injection). The number of dead mice was calculated during
a 24 h period after injection of yohimbine.
Reserpine induced depression
6 groups of animal (Group 1–6) were reserpinised by administration of reserpine (2.5 mg/kg,
i. p.) 1 h after the respective drug administration. Group (1) received distilled
water (10 mL/kg), group (2–5) received OS (6.25 12.5, 25, 50 mg/kg) respectively while
group (6) received Imipramine (25 mg/kg). The acute effects of Olax subscorpioidea and Imipramine on reserpine induced diarrhea were observed. Mice were observed for
the presence of diarrhea at 1, 2, 3 & 4 h after reserpine injection.
Statistical analysis
All data are presented as mean±SEM. The results were analyzed by One way analysis
of variance (ANOVA) and post hoc tests (Student’s-Newman-Keuls) were carried out
to determine the source of significant main effect using GraphPad InStat® Biostatistics software. The level of significance for all tests was set at p<0.05.
Results
Acute toxicity test
The LD50 of Olax subscorpioidea crude extract in mice was found to be 300 mg/kg i. p. body weight.
Effect of olax subscorpioidea on immobility time of forced swimming test (FST)
OS at 6.25 mg/kg and 12.5 mg/kg significantly reduced (P<0.05) immobility time of
mice in FST as compared to the control (vehicle) while doses at 25 mg/kg and 50 mg/kg
did not reduce immobility time in mice. Clinically effective antidepressant imipramine
at 25 mg/kg produced a marked significant reduction (P<0.05) in the duration of immobility
as compared with control ([Fig. 1]).
Fig. 1 Effect of OS on immobility time of Forced Swimming Test (FST). The results are expressed
as Mean±SEM (n=5). One way ANOVA revealed that there is significant [F (5, 24)=17.22,
p<0.0001] difference between various treatment groups. *indicates significant difference
from the control P<0.05. Imp=imipramine (25 mg/kg); C=control; OS=olax susbscorpioidea
Effect of olax subscorpioidea on immobility time of tail suspension test (TST)
OS at 6.25 mg/kg and 12.5 mg/kg significantly reduced (P< 0.05) immobility time of
mice in TST as compared to the control while doses at 25 mg/kg and 50 mg/kg did not
reduce immobility time in mice. The clinically effective antidepressant imipramine
at 25 mg/kg produced a marked significant reduction (P<0.05) in the duration of immobility
as compared with control ([Fig. 2]).
Fig. 2 Effect of OS on immobility time of Tail Suspension Test (TST). The results are expressed
as mean±SEM (n=5). One way ANOVA revealed that there is significant [F (5, 24)=14.94,
p<0.0001] difference between various treatment groups. * indicates significant difference
from the control P<0.05. Imp=imipramine (25 mg/kg); C=control; OS=olax susbscorpioidea
Effect of OS on locomotor activity of OFT
Treatment with OS at 6.25 mg/kg and 12.5 mg/kg which significantly reduced duration
of immobility in FST and TST produced no significant difference in number of crossing
activity of mice in OFT. Antidepressant Imipramine at 25 mg/kg which reduced duration
of immobility of mice in FST and TST produced no significant difference in number
of crossing activity as compared to control ([Fig. 3]).
Fig. 3 Effect of OS on the locomotor activity of mice in OFT. The results are expressed
as mean±SEM (n=5). One way ANOVA revealed that there is significant [F (5, 24)=35.98,
p<0.0001] difference between various treatment groups. *Indicates significant difference
from the control P<0.05. Imp=imipramine (25 mg/kg); C=control; OS=olax susbscorpioidea.
Effect of olax subscorpioidea on yohimbine induced lethality test
Comparing with control OS (6.25–50 mg/kg) did not significantly potentiate yohimbine
toxicity in mice. Antidepressant imipramine at 25 mg/kg produced a marked significant
increase in the number of death (P<0.05) as compared with control ([Table 1]).
Table 1 Effect of Olax subscorpioidea on Yohimbine-induced lethality Test.
|
Group
|
Dose (mg/kg)
|
No of death
|
% mortality
|
|
Distilled water
|
10 mL/kg
|
2/10
|
20
|
|
Olax subscorpioidea
|
6.25
|
2/10
|
20
|
|
12.5
|
0/10
|
0
|
|
25
|
0/10
|
0
|
|
50
|
2/10
|
20
|
|
Imipramine
|
25
|
7/10
|
70*
|
The results are expressed as mean±SEM (n=5). One way ANOVA revealed that there is
significant [F (5, 24)=21.73, p<0.0001] difference between imipramine and the control
*indicates significant difference from the control P<0.05
Effect of crude extract of olax subscorpioidea in reserpine induced depression
In reserpine induced depression test, OS (6.25, 12.5, 25, 50 mg/kg) produced significant
(p<0.05) decrease in the mean feacal droppings in all the groups compared to control.
Anti-diaorrhea effect of Olax subscorpioidea was comparable to that of imipramine ([Table 2]).
Table 2 Effect of crude extract of Olax subscorpioidea in Reserpine Induced Diarrhoea.
|
Group
|
Dose mg/kg
|
60 min
|
120 min
|
180 min
|
240 min
|
|
Control
|
10 mL/kg
|
4.80±0.80
|
7.60±0.92
|
8.20±0.86
|
6.51±0.73
|
|
Olax subscorpioidea
|
6.25
|
1.60±0.50*
|
1.80± 0.66*
|
2.40±0.92*
|
2.63±1.12*
|
|
12.5
|
1.20±0.58*
|
1.20±0.58*
|
1.20±0.58*
|
2.01±0.35*
|
|
25
|
0.40±0.24*
|
0.60±0.40*
|
1.20±0.37*
|
1.78±1.09*
|
|
50
|
1.00±0.44*
|
1.20±0.37*
|
2.80±0.58*
|
1.58±0.55*
|
|
Imipramine
|
25
|
0.71±0.24*
|
0.82±0.28*
|
1.56±0.34*
|
0.92±0.35*
|
The results are expressed as mean±SEM (n=5). One way ANOVA revealed that there is
significant [F (5, 24)=10 p<0.0001; F (5, 24)=21.58 p<0.0001; F (5, 24)=17.45 p<0.0001;
F (5, 24)=19.98 p<0.0001] difference between various treatment groups
*indicates significant difference from the control P<0.05
Discussion
Behavioural studies have been shown to play an important part in the evaluation and
development of antidepressant drugs [18]. Forced swimming test (FST) and tail suspension test (TST) are 2 important behavioural
models widely and routinely used for screening new antidepressant compounds [19]. According to Porsolt [20] and Steru et al., [21] existence of significant correlation between clinical potency and potency of antidepressants
has been established. The immobility displayed by animals subjected to an unavoidable
and inescapable stress that characterizes these models has been hypothesized to reflect
behavioural despair which in turn may reflect depressive disorder in humans. This
immobile position is reduced by variety of therapeutically active antidepressants
e. g. tricyclics, monoamine oxidase-inhibitors, and newer antidepressants [22]
[23].
Forced swimming test is based on the assumption that animals forced to swim in a restricted
space will ultimately cease to apparent attempts to escape and become immobile making
only small movements necessary to keep their heads above the surface of water [24]. In this present study however, OS (6.25 and 12.5 mg/kg) produced a statistically
significant reduction in immobility time in forced swimming test, while OS (25 and
50 mg/kg) produced no effect on immobility time. Positive control antidepressant drug
imipramine produced significant reduction in immobility time. Therefore the ability
of OS to reduce immobility time in animals subjected to this stressful situation indicates
its antidepressant like activity.
Similar results were obtained in TST with OS (6.25 and 12.5 mg/kg) and imipramine
(25 mg/kg) producing significant reduction in immobility time while OS (25 and 50 mg/kg)
did not affect immobility in these animals. The test having got an advantage of being
able to detect broad spectrum of antidepressants irrespective of their mechanism of
action, is however based on the observation that rodents mostly mice after initial
escape behaviour, develop an immobile position when subjected to an inescapable stressful
situation [25]. Animals are considered as immobile when they hang passively and completely motionless.
The development of immobile posture by the animals which disengages them from active
form of coping with stressful stimuli [26] represents behavioural despair which in turn may reflect depressive disorders in
humans. Clinically effective antidepressants decrease immobility time in TST. The
ability of the extract (OS) to reduce immobility time suggests that it may possess
antidepressant activity.
While interpreting antidepressant-like effect of any test substance based on swimming
performance and exploratory behaviour of rodents in either FST or TST, it is noteworthy
that false positive results can be obtained for agents that stimulate locomotor activity
[27]. Therefore the influence of the test substance in baseline locomotion in animal
is of prime concern [28]. Agents like amphetamine, convulsants and anticholinergic which enhance locomotor
activity or cause hyperkinesias in open field test (OFT) produce false positive results
in FST and TST [27]
[29]
[30]. Hence the need of OFT as a paradigm to eliminate the bias that anti-immobility
effect could be associated with hyperkinesia [31]. As psycho-stimulant will always give false positive result in FST and TST, one
way to discern or discriminate between antidepressants and psycho-stimulants is that
antidepressants would not cause general increase in motor activity [32]. Potential antidepressant activities of selective serotonin1A agonists based on
anti-immobility activity in the forced swimming test in rats without effect on open-field
activity have been suggested [33]. The observation that OS (6.25 and 12.5 mg/kg) did not increase the number of line
crossed in open field eliminates exertion of psycho-stimulant-like action and confirms
the assumption that antidepressant-like effect of the extract in TST and FST is specific
[34]. Conversely, OS (25 and 50 mg/kg) statistically produced a significant reduction
in the number of line crossed indicating gross sedation at these doses which masks
its antidepressant effect. This probably explains why the extract at higher doses
could not exhibit anti-immobility effect seen at lower doses.
Yohimbine induced lethality test is another animal model used to further screen antidepressant
potential of olax subscorpioidea. This model has been developed not only to detect potential antidepressant properties
of new substances, but also to identify neurotransmitter systems that may be involved
in the mechanism of actions of antidepressant drugs [35]. Yohimbine, an alpha 2 adrenergic antagonist, causes increased sympathetic discharge
both in the peripheral and central nervous system. The antagonism of alpha 2 receptors
also causes an increase in the level of serotonin [36]. Antidepressant drugs by enabling more amines to reach the receptors potentiate
yohimbine induced lethality. This occurs either by their reuptake inhibition or reduced
inactivation by monoamine oxidase inhibitors. Yohimbine induced lethality test reveals
an adrenergic component of pharmacological activity of antidepressants and is sensitive
to detect MAO inhibitors, tricyclic antidepressants, noradrenaline (NA), and selective
serotonin reuptake inhibitors [37]. The present study showed that OS did not potentiate yohimbine induced lethality
at all doses thus precluding involvement of the central adrenergic mechanism in its
antidepressant action.
Studies have shown that reserpine can deplete amine stores and irreversibly inhibit
the vesicular uptake of monoamines. This reduced level of monoamines in the brain
has been implicated to be an underlining factor in the pathophysiology of depression.
In this paradigm, physiological effects such as diarrhoea, ptosis and hypothermia
are observed and these have been associated with the effect of reserpine as signs
of depression [38]. These syndromes have been inhibited or reversed by major classes of antidepressant
drugs. In this study, like imipramine significant reversal of diarrhoea by OS at all
4 dose levels used was noticed and thus indicating its antidepressant property.
In conclusion, it is obvious that administration of OS produced antidepressant-like
effect in FST and TST, which is not due to the effect of psycho-stimulant or hyperkinesia.
However, giving the fact that Forced swimming, Tail suspension, yohimbine induced
lethality and reserpine induced depression tests are not the only models of depression
by which the results obtained can be considered, interpreted and validated due to
individual differences among experimental animals and clinical studies in human, there
is therefore the need to advance the course of the study by evaluating the effect
of the extract (OS) in other animal’s behavioural model including relevant antidepressant
doses of OS on its safety and efficacy level.
