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~ 146 ~
Journal of Pharmacognosy and Phytochemistry 2014; 3(3): 146-154
E-ISSN: 2278-4136
P-ISSN: 2349-8196
JPP 2014; 3(3): 146-154
Received: 23-08-2014
Accepted: 16-09-2014
Foyet Harquin Simplice
Department of Biological Sciences,
Faculty of Science, University of
Maroua, Cameroon. P.O. Box: 814,
Maroua, Cameroon.
Abdou, Bouba Armand
Department of Agriculture, Animal
husbandry and Derived products,
High Institute of the Sahel,
University of Maroua, Cameroon.
P.O. Box: 46, Maroua.
Ngatanko Abaissou Hervé Hervé
Department of Life and Earth
Sciences, Higher Teachers’ Training
College, University of Maroua,
Cameroon. P.O. Box: 55, Maroua.
Manyi Forka Lucy
Department of Life and Earth
Sciences, Higher Teachers’ Training
College, University of Maroua, P.O.
Box: 55, Maroua, Cameroon.
Manyo Nkongho Annabel
Department of Life and Earth
Sciences, Higher Teachers’ Training
College, University of Maroua,
Cameroon.
Shu Nyenti Patence Neh
Department of Life and Earth
Sciences, Higher Teachers’ Training
College, University of Maroua,
Cameroon.
Asongalem Emmanuel Acha
Department on Biomedical Sciences,
Faculty of Health Sciences,
University of Buea, Cameroon.
Correspondence:
Foyet Harquin Simplice
Department of Biological Sciences,
Faculty of Science, University of
Maroua, Cameroon. P.O. Box: 814,
Maroua, Cameroon.
Neuroprotective and memory improvement effects of a
standardized extract of Emilia coccinea (SIMS) G. on
animal models of anxiety and depression
Foyet Harquin Simplice, Abdou, Bouba Armand, Ngatanko Abaissou
Hervé Hervé, Manyi Forka Lucy, Manyo Nkongho Annabel, Shu Nyenti
Patence Neh, Asongalem Emmanuel Acha
Abstract
The present study evaluated the putative effects of the methanolic extract of Emilia coccinea leaves
(MEC) on the central nervous system, including anxiety, depression-like behavior, and memory, in
Wistar rats. The behavioral assays included open-field, elevated plus maze, forced swimming and Y-
maze. The antioxidant activity of the extract was also measured in vitro. MEC showed a significant
antioxidant activity. It significantly increased the number of open arm entries and time spent in the open
arms of the elevated plus maze test. The rearing time as well as the time spent at the centre of the open
field was significantly increased. MEC also significantly decreased the number of lines crossed and the
climbing time on this task. In the forced swimming test, the extract was as effective as Imipramine in
inducing shortening of immobility time while after 3 days of treatment, it significantly improves spatial
memory in the Y-maze task.
Keywords: Emilia coccinea, Antioxidant, flavonoids, Anxiety, Depression, spatial memory
1. Introduction
Emilia coccinea (SIMS) G. (Asteraccae) is an annual herb commonly found throughout the
plain of the Central Africa and in dry areas up to 2000 m altitude in the eastern Africa. This
species belongs to the genus Emilia represented by about 100 species, with 50 of them found
in Africa [1]. In traditional medicine, this plant is used for the treatment of fever, convulsions
and epilepsy in children [2]. The sap is also applied to ulcers body rashes and abscesses. The
dry leaves are used for the treatment of wounds, sores and sinusitis ulcer, ringworm [3], but
also to treat jaundice, abdominal pains, and gastritis. In some tribe in the western part of
Cameroon, the infusion of the dry leaves of this plant is used as a potent sedative and
restorative. Previous phytochemical studies on E. coccinea have reported the presence of
alkaloids, tannin, saponin, steroid, terpenoid, flavonoid and cardiac glycoside [4-5]. Quantitative
estimation of the percentage of crude chemical constituents in the Nigerian E. coccinea was
0.92±0.22% of alkaloids, 0.81±0.10% of phenols, 0.96±0.10% of flavonoid, 2.30±0.20 of
saponin and 11.85±0.31 of tannin [4].
According to the WHO report, approximately 450 million people suffer from a mental or
behavioural disorder. This accounted for 12.3% of the global burden of disease, and this
percentage may rise up to 15% by 2020 from predictions. The brain is susceptible to free-
radical damage due to its comparatively high levels of oxygen metabolism and also relatively
deficient in both free-radical scavenging enzymes and antioxidant molecules as compared to
other organs. Oxidative stress by the imbalance between free radicals and the antioxidant
system is a prominent and early feature in the pathogenesis of neuronal damage [6].
Different therapeutic regimens are employed to treat anxiety and depressive disorders; but
their clinical uses are limited by their side effects such as psychomotor impairment,
potentiation of other central depressant drugs and dependence liability. In the search for new
therapeutic products for the treatment of neurological disorders, research on medicinal plant
has also contributed significantly by demonstrating pharmacological effectiveness of different
herbs in various animal models.
Various activities of the entire herb, including antibacterial, antioxidant and anti-inflammatory
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Journal of Pharmacognosy and Phytochemistry
activities have been reported in various studies [7], but no
scientific data are available for the central nervous systems
actions of the leaves of E. coccinea although this plant is
used for the treatment of some neurological disorders in the
western part of the Cameroon. The presence of flavonoids
and phenolic compounds in the leaves of E. coccinea
suggests that this plant possesses antioxidant properties and
can have neuroprotective propensity. Therefore, the aim of
this study was to examine the antidepressant-like, anxiolytic-
like and sedative actions of the methanol extract of the
methanolic extract of Emilia coccinea using animal models.
Putative anxiolytic-like and antidepressant-like properties of
E. coccinea were studied in the elevated plus-maze, open
field and forced swimming test, while the effect on short
term memory was investigated in Y-maze.
2. Methods
2.1. Plant material and extraction
Fresh leaves of E. coccinea were harvested in September
2013 at Etoug Ebe in the Centre Region of Cameroon and
authenticated at the National Herbarium-Yaoundé, where the
voucher specimen was conserved under the reference number
6297/HNC. The leaves were then washed and dried at room
temperature (24-26 °C during 10 days).
Methanolic extract was prepared as follows: after drying
fresh leaves and powdering it, 500 g of the powder were
mixed with 500 ml of the solvent at room temperature and
agitated for eight hours in a flask shaker using a magnetic
agitator. The mixture was then filtered thought a Whatman
paper. This was followed by the elimination of the solvent by
a rotavapor. The given powder yielded 8.80% of a brown
extract. The same process was done for the fresh leaves.
2.2. Experimental animals
Male Wistar albino rats (weighing 100-180 g) were obtained
from the Laboratory of Biophysics and Biochemistry of the
Department of Food Sciences and Nutrition, University of
Ngaoundéré, Cameroon. The animals were housed in
polyacrylic cages (6 animals / cage) and maintained at a
temperature and light-controlled room (25 ± 2 °C, a 12-h
cycle). The animals were acclimatized to laboratory
conditions for 7 days before the start of experiment. Prior to
and after treatment, the animals were fasted for 12 and 7 h,
respectively. However, all animals were allowed to drink
water ad libitum. Rats were treated in accordance with the
guidelines of the Cameroonian Bioethics Committee (reg
N°.FWA-IRB00001954) and in accordance with NIH- Care
and Use of Laboratory Animals manual ( 8th Edition).
2.3. Chemicals
Diazepam hydrochloride and Imipramine were purchased
from Novartis Turkey and used as reference drugs. All drugs
and extracts were freshly prepared in saline on the day of the
experiments and administered intraperitoneally (i.p.). Control
animals received 10 ml/kg body of the vehicle in the same
route of administration.
2.4. In vitro analysis
2.4.1. Determination of mineral composition
Micro and macro-elements were determined by dry ashing in
muffle furnace 500 °C. 1 g of ground sample in a porcelain
crucible was ashed in conventional resistance muffle furnace
(CMF). The ash was diluted in 5ml of diluted mixture of
HCl/HNO3 acids, following by 20 mL of hot water and
brought to 100 mL in deionised water. Ca, Mg, Na, K, Zn,
Cu, Mn, and Fe were analyzed using Atomic Absorption
Spectrometer. Phosphorous (P) was also determined as above
but analyzed using Murphy Riley reagent and read
colorimetrically [8].
2.4.2. Determination of total phenolic content
Total phenol content was determined spectrophotometrically
in the extracts by using Folin–Ciocalteu method. 0.04 mL
(0.0125 M) of the methanolic extract of E. coccinea was
added to 1.36 mL distilled water and 0.2 mL of freshly
prepared Folin–Ciocalteu reagent, followed by incubation in
the darkness for 5 min. Then, 0.4 mL of 20% sodium
carbonate solution was added. The test tubes were stirred
with the help of a vortex and the samples were incubated at
40 °C in the darkness for 30 min. The UV–vis spectra of all
the samples were recorded against the reference solution
(zero gallic acid) and the absorbance was read at 760 nm.
The measurements were done four times. For the gallic acid
standard, a calibration curve (Pearson’s correlation
coefficient: R2 = 0.999) was constructed and the total level of
phenolics for each sample was determined in terms of gallic
acid equivalents [9].
2.4.3. Determination of anti-oxidant activity
Two model systems: 2, 4-dinitrophenyl-1-picryl hydrazyl
(DPPH) radical scavenging activity and ferric Iron reduced
activity assay were used to measure the antioxidant activities
of the extract. In the two in vitro tests, ascorbic acid and
quercetin were used as standard antioxidant compounds
respectively.
2.4.3.1. Ferric reducing antioxidant power (FRAP) assay
The antioxidant capacity of the methanolic extract of E.
coccinea leaves were evaluated by determining its ability to
reduce iron (Fe3+) into Fe2+ using Oyaizu method [10]. The
methanolic extract of E. coccinea leaves (0.1 mL) were
mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and
2.5 ml of potassium hexacyanoferrate solution (K3Fe(CN)6)
at 1%. The mixture was incubated at 50 °C for 30 min. 2.5
mL of trichloroacetic acid (10%) was added and the mixture
centrifuged for 10 min. 0.5 mL was pipetted into a test tube
and mixed with 2.5 mL of distilled water and 0.5 ml of
aqueous solution of FeCl3 (0.1%). The absorbance was read
at 700 nm in a spectrophotometer. Ascorbic acid was used as
reference and the total reducing power (Ferric Iron reducing
activity) was expressed as ascorbic acid equivalent.
2.4.3.2. Free radical scavenging activity (DPPH assay
method)
The free radical scavenging activity of the methanolic extract
of E. coccinea was evaluated as described by Zhang and
Hamauzu [11]. Briefly, 2 mL of DPPH (0.1 mM prepared in
methanol) was introduced in each test tube containing 0.25
µL of the fresh extract. The mixture was stirred for 5 min and
incubated in darkness for 60 min at room temperature. For
the control tube, methanol was used in the place of the
extract while quercetin was used as reference at variable
concentration. A curve was drawn from this reference and
the absorbance read at 517 nm. Each assay was repeated four
times and the results, recorded as mean of the fourth
experiments.
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Journal of Pharmacognosy and Phytochemistry
The antioxidant activity of the extract was expressed as
grams of quercetin equivalent/100g of the extract. The
inhibition percentage was calculated from the following
equation.
PI (%) = [(DO control-DOessay) x100]/DOcontrol
2.5. Behavioral evaluation
2.5.1. Open Field Activity test (OFT)
The open field apparatus was constructed of white polywood
and measured 72 x 72 cm with 36 cm walls. Red lines were
drawn on the floor with a marker and were clearly visible
through the clear Plexiglas floor. Rats were treated (i.p) with
single administration of the methanolic extract of E. coccinea
leaves and the test were performed 30 min after the drug
administration of the extract (200 and 400 mg/kg, i.p.) or
saline (10 ml/kg). The standard drug diazepam (1 mg/kg, i.p.)
was given once 30 min before the test. The rats were placed
in the open field box for 5 min, and their behaviors were
recorded. The behaviors scored included: time spent at the
center square, number of the lines crossed on the floor of the
maze, time spent at the border of the maze, grooming
(duration of time the animal spent licking or scratching itself
while stationary), and the climbing time [12].
2.5.2. Elevated plus-maze test (EPM)
Behavior in the elevated plus-maze (EPM) is used to assess
exploratory, anxiolytic and motor activity. The possible
anxiolytic effects of the methanolic extract of E. coccinea
leaves were assessed, basically using the same method
described by Casarrubea et al. [13]. The EPM consists of four
arms, 49 cm long and 10 cm wide, arranged in such a way
that the two arms of each type were opposite to each other.
The maze was elevated 50 cm above the floor. Two arms
were enclosed by walls 30 cm high and the other two arms
were exposed. Rats were treated i.p. with single
administration of the methanolic extract of E. coccinea
leaves (200 and 400 mg/kg; i.p) or saline (10 ml/kg; i.p). The
positive control diazepam (1 mg/kg, i.p) was given once 30
min before the test. Thirty minutes after the i.p. injection of
the extract or saline, each animal was placed at the center of
the maze facing one of the enclosed arms. During a 5 min
test period, the number of open and enclosed arm entries, as
well as the time spent in open and enclosed arms, were
recorded as previously described [14]. Entry into an arm was
defined as the point when the animal places all four paws
into the arm. After the test, the maze was carefully cleaned
with wet cotton (70% ethanol solution) and allowed to dry
before the next animal.
2.5.3. Forced swimming test (FST)
The FST is the most widely used pharmacological models for
assessing antidepressant activity [15]. The development of
immobility when the rodents are placed in an inescapable
cylinder of water reflects the cessation of persistent escape-
directed behavior [16]. The possible antidepressant effects of
the methanolic extract of E. coccinea leaves were assessed,
basically using the same method described by Kawaura et al.
[17] with minor modifications. Rats were treated with single
administration of the methanolic extract of E. coccinea
leaves (200 and 400 mg/kg; i.p) or saline (10 ml/kg; i.p). The
standard drug imipramine (10 mg/kg, i.p) was given once 30
min before the test. On the first day of the experiments
(pretest session), rats were individually placed into
transparent Plexiglas cylinder (50 cm high and 20 cm wide)
filled to a 30 cm depth with water at 26 ± 1 ºC. The animals
were left to swim for 15 min before being removed, dried
and returned to their cages.
The procedure was repeated 24 h later, in a 6 min swim
session (test session) 30 min after the last dose of the
methanolic extract of E. coccinea leaves, imipramine or
saline. During the test session, the following behavioral
responses were recorded: immobility time (time spent
floating with the minimal movements to keep the head above
the water) and swimming time (time spent with active
swimming movements). Increases in active responses, such
as climbing or swimming and reduction in immobility, was
reconsidered as behavioral profiles consistent with an
antidepressant-like action [15].
3. Y-Maze test
Y-maze analysis has been shown to be a reliable,
noninvasive test to determine cognitive changes in wistar rat
through the measurement of the spontaneous alternation
behavior in the Y-maze task. The maze used in the present
study consisted of three arms (35 cm long, 25 cm high and 10
cm wide) and an equilateral triangular central area. All
animals were tested in a randomized order at the beginning
and at the end of the experimental protocol. Rats were treated
once daily with the methanolic extract of E. coccinea leaves
(200 and 400 mg/kg; i.p), diazepam (2 mg/kg, i.p.),
Diazepam (2 mg/kg, i.p.) plus E. coccinea (400 mg/kg; i.p)
or saline (10 ml/kg; i.p) during 3 consecutive days. 30 min
after the last administration of the methanolic extract of E.
coccinea leaves, diazepam or saline solution, rats were
placed at the end of one arm and allowed to move freely
through the maze for 8 min. The time limit in Y-maze test
was 8 min, and every session was stopped after 8 min. An
arm entry was counted when the hind paws of the rat were
completely within the arm. Spontaneous alternation behavior
was defined as three consecutive entries in three different
arms (i.e. A, B, C or B, C, A, etc). The percentage
alternation score was calculated using the following formula:
Total alternation number/ (Total number of entries − 2) x
100. Furthermore, total number of arm entries was used as a
measure of general activity in the animals. The maze was
wiped clean with 70% ethanol between each animal to
minimize odour cues [18-19].
3.1 Statistical analysis
Data were presented as mean ± SEM values. One-way
ANOVA followed by Tukey multicomparaison “t”-test was
performed using Graph Pad Prism version 5.00 for Windows,
Graph Pad Software, San Diego California USA,
www.graphpad.com. A probability level of 0.05 or less was
accepted as significant. Pearson’s correlation coefficient and
regression analysis were used to evaluate the connection
between the working memory errors and some parameters
like locomotion, grooming and rearing in the Y-maze test.
4. Results
4.1. Total phenolic content and in vitro antioxidant
activity of Emilia coccinea
The results of the phenolic content showed that the dry
leaves of this plant contained 863.04±5.42 mg of GAE/100 g
of dry material. This represents a very good content of total
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Journal of Pharmacognosy and Phytochemistry
phenolics compounds in the dry leaves and is four fold the
total phenolic content of the fresh leaves. The effect of
antioxidants on DPPH radical scavenging was thought to be
due to their hydrogen-donating ability. The preparations were
able to reduce the stable free radical DPPH to the yellow-
coloured 1,1-diphenyl-2-picrylhydrazyl, with 19.08±0.62 g
of quercetin equivalent/100 g of dry material, indicating a
weak activity against this radical. The total reducing power
was about 4.71±0.04 g of Vit C equivalent/100 g of dry
material (Table 1).
Mn and Zn were found to be very high (549.83±0.60 and
46.87±0.01 mg/100 g respectively) in our samples of E.
coccinea than those reported on other plants [20] (Table 2)
Table 1: Total phenolic content and in vitro antioxidant activity of Emilia coccinea
Test
Unit
Dry
Emilia coccinea
Fresh
Emilia coccinea
Water content
g/100
g
4.41± 0.03
a
86.05±0.53
b
Total phenolic content
Mg of GAE/100
g of DM
863.04
±5.42
b
204.15
±2.04
a
Total reducing powder
g of Vit C equivalent/100
g of DM
4.71
±0.0
4
b
3.24
±0.39
a
Antiradical activity (DPPH)
g of
quercetin
equivalent/100
g of DM
19.08
±0.62
b
6.40
±0.06
a
Results present as the mean ± S.E.M. of 4 experiments. Values with different letters within a line are significantly different at 0.05 level.
Table 2: Mineral composition of Emilia coccinea leaves
Elements
Mg
(g/100 g)
Mn
(mg/100 g)
Ca
(mg/100 g)
Fe
(mg/100 g )
Zn
(mg/100 g )
Cu
(mg/100 g )
E.C
0.52
±0.003
549.83
±0.60
3.26
±0.01
265.35
±0.52
46.87
±0.01
10.23
±
0.003
Results present as the mean ± S.E.M. of 4 experiments. Samples expressed in mg/100g dry weight basis (N=4).
4.2. Effects of the methanolic extract of E. coccinea leaves
in the open field test in rats
The results given in table 3 indicate that the diazepam (2.0
mg/kg) treated rats showed a significant decrease in number
of lines crossed, climbing and rearing as well as the time
spent at the border of the field. The time spent at the centre
of the field was increased significantly at the same time.
MEC treated rats (200 mg/kg) exhibited a significant
increase in the time spent at the centre of the maze while the
extract was a sedative in all of the applied doses with
decreased in the number of line crossed. The animals were
most immobile and inactive at the dose 400 mg/kg. The
number of lines crossed by the negative control group was
greater than that of the extract-treated groups, but, the
number of lines crossed by extract-treated groups was not
significantly different to that of the diazepam-treated group,
as shown in table 4.
4.3. Effects of the extract in the EPM
In the EPM, the MEC (200, 400 mg/kg, i.p.) was found to
significantly (P < 0.05) decrease the number of entries and
the time spent by the rats in the closed arms compared to the
control animals (Fig. 1 and 2). With the diazepam (2 mg/kg),
the standard drug used in this test, the number of closed arm
entries as well as the time spent in the closed arms was
significantly decreased (P < 0.05). By considering the total
number of entries in both the arms (enclosed and open arms),
as an index of locomotory activity of the animals, the
difference between the total number of lines crossed by the
saline treated animals (33±0.96) was not significantly
different from those of MEC treated animals at the doses of
200 and 400 mg/kg (25±1.30 and 28±0.84 respectively), (F
(2.86) = 0.70, P ≥ 0.05). This difference was also
nonsignificant when compared to the diazepam -treated
animals (24±1.20) (Data not shown).
Table 3: Effects of the MEC and diazepam in the open field test in rats.
Groups Dose
(mg/kg) Number of line
crossed Climbing time
(s) Rearing Time
(s) Time spent at
the center (s) Time spent at the
border (s)
Control
-
33±3.2
19.4 ±3.12
78.8±16.56
3.80±0.96
268.00±23.60
MEC
200
8.2±2.24***
5.80±2.56***
47.20±11.76
21.20±6.76*
2
67.00±23.20
MEC
400
5.8±1.76***
3.00±1.20***
24.00±9.60**
12.80±3.04
241.00±16.00
Diazepam 2 5.4±4.32*** 2.00±1.20*** 1.20±0.32*** 114.00±12.00**
#
£
166.0±44.00****
#
£
Animals were treated with single dose of the extract (200 or 400 mg/kg, i.p.) or distilled water. In the positive control, diazepam
was given only once (2 mg/kg, i.p.) 30 min prior to the test. Results present as the mean ± S.E.M. of 6 animals. Data analysis
was performed using One way ANOVA followed by Tukey multicomparaison “t” –test. *** P < 0.0001 vs. saline-treated
animals; £ P < 0.0001 vs. 200 mg/kg; # P < 0.0001 vs. group 400 mg/kg.
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Journal of Pharmacognosy and Phytochemistry
Fig 1: Effect of the methanolic extract of E. coccinea leaves on the
number of entries in open and closed arm in elevated maze test.
Experiments were performed 30 min after the administration of the
extract or diazepam (Diaz). Each column represents mean ± S.E.M.
of 6 animals. Data analysis was performed using One way ANOVA
followed by Tukey multicomparaison “t” –test. * P < 0.05 vs.
saline-treated animals; ** P < 0.01 vs. saline-treated animals.
Fig 2: Effect of the methanolic extract of E. coccinea leaves on the
time spent in open arm and closed arm in elevated maze test.
Experiments were performed 30 min after the administration of the
extract of E. coccinea or diazepam (Diaz). Each column represents
mean ± S.E.M. of 6 animals. Data analysis was performed using
One way ANOVA followed by Tukey multicomparaison “t” –test.
* P < 0.05 vs. saline-treated animals.
4.4. Effects of the extract in the FST
The figure 3 shows the effect of MEC for the duration of
immobility time in the FST model. One-way ANOVA
revealed that there were no significant differences between E.
coccinea -treatment groups (F (39.41) = 3.09, P ˃ 0.05).
Post-hoc analysis showed that the MEC (200 and 400 mg/kg)
and imipramine treated groups were significantly different
(P < 0.0001) from the vehicle treated group. MEC
significantly increased in the dose dependent manner the
duration of swimming time, indicating the antidepressant
effect of the extract. This antidepressant effect of the MEC at
the dose of 100 mg/kg was comparable to that of imipramine
(2 mg/kg).
Fig 3: Effect of the methanolic extract of E. coccinea leaves on the
immobility and swimming time in forced swimming test.
Experiments were performed 30 min after the administration of the
extract or imipramine (Imip). Each column represents mean ±
S.E.M. of 6 animals. Data analysis was performed using One way
ANOVA followed by Tukey multicomparaison “t” –test. ** P <
0.001; *** P < 0.0001 vs. saline-treated animals.
4.5. Effects of the extract in the Y-Maze task
In Y-maze task, we observed after three days administration
a significant increase of spatial memory in animal treated
with high-dose (400 mg/kg) of the methanolic extract of E.
coccinea leaves (F (3.68) = 6.47, P < 0.01) (Fig. 4a),
indicated by an increase of spontaneous alternation
percentage compared to control group, suggesting effects on
short term-memory. At the same time, the plant extract (200,
400 mg/kg) and diazepam (2 mg/kg), significantly, and at a
dose -dependent manner, decreased the total number of
arm entries of the animals when compared to control group
(F (3.09) = 8.69, p ˂0.0007) (Figure 4b). More importantly,
when linear regression was determined, no significant
positive correlation between spontaneous alternation vs.
number of entries in the maze (n=7, r2=0. 237, p=0.2678)
(Figure 5) was noted. It can also be clearly realized that
Diazepam, GABAA agonist slightly impaired short term
memory of rats, although this was not significant compared
to control animals.
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Journal of Pharmacognosy and Phytochemistry
Fig 4: Effect of the methanolic extract of E. coccinea leaves and diazepam (Diaz) on the spontaneous alternation percentage (A) and number of
entries (B) in Y-maze task. Experiments were performed 30 min after 3 days administration of the extract. Each column represents mean ±
S.E.M. of 7 animals. Data analysis was performed using One way ANOVA followed by Tukey multicomparaison “t” –test. * P < 0.05; ** P <
0.001; ** P < 0.0001 vs. control animals; £ P < 0.001 vs. 400 mg/kg treated animals.
Fig 5: Correlation between working memory errors vs. locomotor activities and rearing behavior of rats treated with the MEC at
the dose of 400 mg/kg.
5. Discussion
The present study provides behavioral evidence for the
anxiolytic and antidepressant-like activities of E. coccinea.
The EPM and OF test are regularly used to study anxiolytic
effects of plant extract while the forced swimming tests are
widely accepted behavioral models for the assessment of
antidepressant activity. E. coccinea has been used to treat
some neurological-related diseases in traditional medicine
such as convulsion and epilepsy, but its specific
neuropharmacological activities have not yet been
demonstrated. The findings of the current investigation show
for the first time that MEC, standardized in its content of
flavonoids with doses of quercetin (19.08 g /100 g of DM),
Vit C (4.71 08 g /100 g of DM), Gallic acid (863.04 mg /100
g of DM), Mn and Zn (549.83±0.60 and 46.87±0.01 mg/100
g of DM respectively) possesses a significant anxiolytic and
antidepressant properties.
The open field test is a paradigm used for evaluating the
effect of drugs on gross general behavior and is used to
measure the level of nervous excitability [21-22]. When
removed from their acclimatized home cages and placed in a
novel environment, animals express their anxiety and fear by
showing decreased ambulation and exploration,
immobilization or freezing, and modification in normal
rearing and grooming behavior. Increased micturation and
defecation due to augmented autonomic activity is also
observed. These paradigms are attenuated by classical
anxiolytics and potentiated by anxiogenic agents [23]. In the
open field behavioral task, the MEC was seen to increase
time spent at the center of the maze and decrease peripheral
square movements; the observed decrease in central square
movements could be due to the impairment of locomotory
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Journal of Pharmacognosy and Phytochemistry
activity. The decrease in locomotory (number of lines
crossed and rearing) activity in the open field test of rats
treated with the extract produces more evidence for its
central nervous depressant activity. The decrease in the
rearing activity (vertical movement), as well as grooming, an
emotional activity parameter, was also significantly affected
by treatment with the MEC. Diazepam used as a positive
control drug, also significantly reduced anxiety state in the
open-field test with some depressive side effects.
A behavioral assay extensively used for the studies of acute
behavioral stress reactivity is the elevated plus maze (EPM).
When animals were taken from their home cage and given
access to either an open maze alley or a closed maze alley,
they spent more time exploring the closed arms as a
characteristic of an approach-avoidance conflict [24]. The
EPM test has several characteristics that make it particularly
useful. It reliably detects anxiolytic and anxiogenic activity
of a variety of therapeutic and experimental drugs of
different classes. Unlike models that require extensive
conditioning, it relies on an innate conflict between
competing “drives”, the balance of which is affected by the
level of anxiety. Thus, it requires no training, deprivation,
pain or aversive stimuli. The response involves the
redirection of an ongoing activity (i.e., exploration) rather
than the suppression of behavior, which could be confounded
by sedation or ataxia. Several plants increase the exploration
of open arms in the elevated plus-maze test and are used to
diminish anxiety in folk medicine. Among them are Trichilia
catigua and Plumeria rubra [25-26].
Conventional anxiety indices in the elevated plus-maze test
comprise percent open arm entries and percent time spent in
these areas in the maze, with anxiolytics generally increasing
and anxiogenics decreasing these measures. In this regard, in
the elevated plus-maze test, the MEC (200 and 400 mg/kg,
i.p.) increased the exploration and the time spent in the open
arms in a non-dose-related way. The number of entries and
the time spent in the enclosed arms were also significantly
reduced when compared to the control group: indicating that
the MEC has an anxiolytic-like effect. As expected,
diazepam reduced the animal's natural aversion to the open
arms and promoted maze exploration. Literature reports
describe the action of benzodiazepines, such as diazepam, as
anxiolytics when used at the lowest doses, but these effects
are associated with the sedation and myorelaxant effects at
higher doses. Our results clearly indicated that the dose of
diazepam used in this study also act as sedative.
In the forced swimming test, the animals are forced to swim
in a very restricted space from which there is no way to
escape. They rapidly develop a state of despair behavior
characterized by a low motivation for escaping as shown by
the increased periods of immobility. In this experiment, the
immobility displayed by rodents when subjected to
unavoidable stress such as forced swimming is thought to
reflect a state of despair or lowered mood, which is thought
to reflect depressive disorders in humans. This behavioral
test is sensitive to serotoninergic compounds, such as the
selective serotonin reuptake inhibitor fluoxetine [12]. The
immobility time has also been shown to be reduced by
treatment with tricyclic antidepressant drugs like imipramine,
which typically increase the swimming efforts of the animal
seeking a solution to the problem and, therefore, they
decrease the duration of immobility in the forced swimming
test [27]. In this study, the single administration of the MEC
provoked significant reduction of the immobility time of rats
subjected to forced swimming when compare to the control
group. This result shows that the extract possesses
antidepressant activity on the central nervous system. It is
noteworthy that in the FST test, false positive results can be
obtained from agents that stimulate locomotory activity [28].
In the open field test, we clearly showed that the MEC
significantly reduced the locomotory activity of the animals
(number of lines crossed and rearing), this confirms the
assumption that the antidepressant-like effect of the extract in
the FST is specific [29].
Working memory allows animals to remember information
that is useful for a single session of an experiment but not for
subsequent sessions and spontaneous alternation behavior is
considered to reflect spatial working memory, which is a
form of short-term memory. The Y-maze task is a specific
and sensitive test of spatial recognition memory in rodents.
The test relies on an innate tendency of rats to explore a
novel environment [30]. The Y-maze used in this study
involves no aversive stimuli and was considered suitable for
evaluating memory and the specific part of the brain involved
in performance of this task include the hippocampus [31-33].
As shown in our results, the MEC at the dose of 100 mg/kg
did not significantly increase the number of spontaneous
alternation. However rats treated with high dose of the
methanolic extract of E. coccinea (400 mg/kg) showed a
significant improvement in spatial learning with an increase
number of spontaneous alternations and reduction of a
percentage of bias, when compared to control. This result
suggests that the plant extract (400 mg/kg) displays
improvement effect on acquisition of the short term-memory
of the rats within Y-maze task. This effect is however linked
to a marked significant decrease in exploratory behavior,
probably due to the myorelaxant effect of the extract. At this
level of our study it is not possible to suggest any possible
mechanism of action of the extract since the process for the
acquisition of short-term memory is a very complex
biological process. At the same time the implication of the
GABAA agonist in the impairment of the learning and
memory in the spontaneous alternation paradigm is clearly
evident. However, the results obtained from the linear
regression results suggest that the improvement of the
acquisition of the short term memory could not be related to
the locomotory activities of the animals treated with the
MEC. In our experiment, the rats receiving both the MEC
and diazepam did no show any sign of memory impairment.
This result suggested that the extract may counteract the
effects of diazepam, as a GABA antagonist and in that case
its anxiolytic effect will be through serotoninergic pathway.
This result may also indicate that the MEC does not act
through the GABA receptors, during the short term memory
process but through other receptors types like glutaminergic,
cholinergic or dopaminergic receptors. The implication of
these receptors in the process of learning and memory is
known well established [34].
Extracts of many plant species that contain a number of
polyphenolic compounds have been shown to present
antioxidant properties. The antioxidant activity of
polyphenolics has been attributed to their redox properties,
which allow them to act as reducing agents or hydrogen-atom
donors [35]. In the present study, a higher antioxidant activity
was observed with the MEC. A very high content of total
phenolics has been determined in the E. coccinea leaves
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Journal of Pharmacognosy and Phytochemistry
(863.04±5.42 mg of GAE/100 g of dry material, 19.08±0.62
g of quercetin equivalent/100 g of dry material), we cannot
exclude that the scavenging activity could result from their
presence, namely, on the basis of a synergistic effect with
other metabolites [36].
The presence of Cu, Zn and Mn ions in our sample, which
are metallic co-factor of anti-oxidant enzyme, give credence
on the anti-oxidant properties of E. coccinea. In this way,
antioxidant properties have been related to some of
pharmacological effects of secondary metabolites of the
plant. Weinreb et al. [37] showed that the neuroprotective
activities of the green tea are based on the antioxidant
activities of epicatechins. Thus, it is possible that both
functional and antioxidant activities of the MEC observed in
the present work are related.
In conclusion, the present study clearly demonstrated that the
methanolic extract of E. coccinea leaves treatment could
significantly prevent anxiety and depression state. The
positive effect of the treatment on memory suggests the
therapeutic potential of this extract in aging and age-related
neurodegenerative disorders where cognitive impairment is
involved. However, for other behavioral effects of MEC and
underling mechanism(s) of action, further preclinical
investigations are necessary.
6. Competing interest
The authors declare that they have no competing interests.
7. Authors' contributions
FHS, MFL, MNA and SNPN carried out the study; ABA,
and FHS, designed the experiments. FHS and NAHH wrote
the manuscript; FHS and ABA supervised the work. All
authors read and approved the final manuscript.
8. Acknowledgments
Foyet Harquin Simplice was supported by TWAS grant N°:
12-132 RG/BIO/AF/AC_G.
9. References
1. Bosch CH. Emilia coccinea (Sims) G.Don In: Grubben,
G.J.H. & Denton, O.A. (Editors). PROTA 2,
Vegetables/Légumes. PROTA, Wageningen,
Netherlands, 2004.
2. Agoha RC. Medicinal plants of Nigeria. Offsetdikker
Jifaculteit waskunden, Natnurwenten schopp; pen,
Netherlands, 1981; 22-158.
3. Burkill HM. The useful plants of West Tropical Africa
Vol 3, Families J-L. Royal Botanical Garden kew, 1984,
522.
4. Edeoga HO, Okwu DE, Mbaebie BO. Phytochemical
constituents of some nigerian medicinal plants. African
Journal of Biotechnology 2005; 4(7):685-688.
5. Teke GN, Kuiate JR, Ngouateu OB, Gatsing D.
Antidiarrhoeal and antimicrobial activities of Emilia
coccinea(Sims) G. Don extracts. Journal of
Ethnopharmacology 2007; 112:278-283
6. Uttara B, Singh VA, Zamboni P, Mahajan RT. Oxidative
Stress and Neurodegenerative Diseases: A Review of
Upstream and Downstream Antioxidant Therapeutic
Options. Current Neuropharmacology 2009; 7(1):65-74.
7. Okiei W, Ogunlesi M, Ademoye MA. An assessment of
the antimicrobial properties of extracts of various
polarities from Chasmanthera dependens, Emilia
coccinea and Cuscuta australis, herbal medications for
eye diseases. Journal of Applied Sciences 2009; 9:4076-
4080.
8. Murphy J, Riley JP. A modified single solution method
for the determination of phosphate in natural waters.
Analytica Chimica Acta 1962; 27:31-36.
9. Gao X, Bjo kL, Trajkovski V, Uggla M. Evaluation of
antioxidant activities of rosehip ethanol extracts in
different test systems. Journal of Agricultural and Food
Chemistry 2000; 80:2021-2027.
10. Oyaizu M. Studies on products of browning reactions:
antioxidative activities of products of browning reaction
prepared from glucosamine. Japanese Journal of
Nutrition 1986; 44:307-315.
11. Zhang D, Hamauzu Y. Phenolics, ascorbic acid,
carotenoids and antioxidant activity of broccoli and their
changes during conventional and microwave cooking.
Food Chemistry 2004; 88:503-509.
12. Foyet HS, Tsala ED, Bouba AA, Hritcu L. Anxiolytic
and antidepressant-like effects of the aqueous extract of
Alafia multiflora stem barks in rodents. Hindawi
Publishing Corporation. Advances in Pharmacological
Sciences, 2012.
13. Casarrubea M, Roy V, Sorbera F, Magnussonb MS,
Santangelo A, Arabo A et al. Significant divergences
between the temporal structure of the behavior in Wistar
and in the spontaneously more anxious DA/Han strain of
rats tested in elevated plus maze. Behavioral brain
research 2013; 250:166-173.
14. Adeyemi OO, Akindele AJ, Yemitan OK, Aigbe FR,
Fagbo FI. Anticonvulsant, anxiolytic and sedative
activities of the aqueous root extract of Securidaca
longepedunculata Fresen. Journal of
Ethnopharmacology 2010; 130(2):191-195.
15. Cryan JF, Markou A, Lucki I. Assessing antidepressant
activity in rodents: recent developments and future need.
Trends in Pharmacological Sciences 2002; 23(5):238-
245.
16. Ulak G, Mutlu O, Yıldız Akar F, Komsuoğlu FI, Tanyeri
P, Erden BF et al. Neuronal NOS inhibitor 1-(2-
trifluoromethylphenyl)-imidazole augment the effects of
antidepressants acting via serotonergic system in the
forced swimming test in rats. Pharmacology
Biochemistry and Behavior 2008; 90(4):563-8.
17. Kawaura K, Miki R, Urashima Y, Kawahara R, Soeda F,
Shirasaki T, Takahama K et al. Pharmacological
mechanisms of antidepressant-like effect of tipepidine in
the forced swimming test. Behavioral Brain Research
2012; 226:381-385.
18. Siedlak SL, Casadesus G, Webber KM, Pappolla MA,
Atwood CS, Smith MA et al. Chronic antioxidant
therapy reduces oxidative stress in a mouse model of
Alzheimer’s disease. Free Radical Research 2009;
43(2):156-164.
19. Hernández-Chan NG, Góngora-Alfaro JL, Álvarez-
Cervera FJ, Solís-Rodríguez FA, Heredia-López FJ,
Arankowsky-Sandoval G et al. Quinolinic acid lesions
of the pedunculopontine nucleus impair sleep
architecture, but not locomotion, exploration,
emotionality or working memory in the rat. Behavioral
Brain Research 2011; 225:482- 490.
20. Bouba AA, Njintang YN, Foyet HS, Scher J, Mbufong
CMF. Proximate composition, minerals and vitamins
~ 154 ~
Journal of Pharmacognosy and Phytochemistry
contents of some wild plants used as spaces in
Cameroun. Food Nutrition Sciences 2012; 3:423-432.
21. File SE, Fernandes C. Dizocilpine prevents the
development of tolerance to the sedative effects of
diazepam in rats. Pharmacology Biochemistry and
Behavior 1994; 47:823-6.
22. Hines TJ, Minton BR. Effects of environmental
enrichment on rat behavior in the open field test.
Proceedings of the National Conference on
undergraduate research (NCUR) 2012 Weber State
University, Ogden, 2012, 29-31.
23. Varsha J, Bharatkumar GP. Effect of hydroalcoholic
extract of Sphaeranthus indicus against experimentally
induced anxiety, depression and convulsions in rodents.
International Journal of Ayurveda Research 2010;
1(2):87-92.
24. Lapiz-Bluhm MD, Bondi CO, Doyen J, Rodriguez GA,
Bédard-Arana T, Orilak DS et al. Behavioral assays to
model cognitive and affective dimensions of depression
and anxiety in rats. Journal of Neuroendocrinology
2008; 20:1115-1137.
25. Chassot JM, Longhini R, Gazarini L, Mello JCP,
Weffort-de Oliveira RM. Preclinical evaluation of
Trichilia catigua extracts on the central nervous system
of mice. Journal of Ethnopharmacology 2011; 137:1143
-1148.
26. Chatterjee M, Verma R, Lakshmi V, Sengupta S, Verma
AK, Mahdi AA et al. Anxiolytic effects of Plumeria
rubra var. acutifolia (Poiret) L. flower extracts in the
elevated plus-maze model of anxiety in mice. Asian
Journal of Psychiatry 2013; 6(2):13-118.
27. Herrera-Ruiz M, Zamilpa A, Gonzalez-Cortazar M,
Reyes-Chilpa R, Leon E, Garcia MP, Tortoriello J,
Huerta-Reyes M. Antidepressant effect and
pharmacological evaluation of standardized extract of
flavonoids from Byrsonima crassifolia. Phytomedicine
2011, 18:1255-1261.
28. Bourin M, Fiocco AJ, Clenet F. How valuable are animal
models in defining antidepressant activity? Hum.
Psychopharmacology 2001; 16:9-21
29. Sanchez-Mateo CC, Bonkanka CX, Prado B, Rabanal
RM. Antidepressant properties of some Hypericum
canariense L and Hypericum glandulosum Ait extracts
in the forced swimming test in mice. Journal of
Ethnopharmacology 2005; 97:541-547.
30. Yusuf S, Adelaiye BA, Agunu A. Effect of Ziziphus
mauritiania (L.) seed extracts on spatial recognition
memory of rats as measured by the Y-maze test. Journal
of Natural Products 2009; 2:31-39.
31. Borght KVD, Havekes R, Bos T, Eggen BJL, Zee
EAVD. Exercise improves memory acquisition and
retrieval in theY-Maze Task: Relationship with
hippocampal neurogenesis. Behavioral Neurosciences
2007; 121(2):324-334.
32. Bagheri M, Joghataei MT, Mohseni S, Roghani M.
Genistein ameliorates learning and memory deficits in
amyloid β(1–40) rat model of Alzheimer’ s disease.
Neurobiology Learning and Memory 2011; 95:270-276.
33. Liu DS, Zhou YH, Liang ES, Li W, Lin WW, Chen FF
et al. Neuroprotective effects of the Chinese Yi-Qi-Bu-
Shen recipe extract on injury of rat hippocampal neurons
induced by hypoxia/reoxygenation. Journal of
Ethnopharmacology 2013; 145:168-174.
34. Myhrer T. Neurotransmitter systems involved in
learning and memory in the rat: a meta-analysis based on
studies of four behavioral tasks. Brain Research Reviews
2003; 41:268-287.
35. Zhang G, Hu M, He L, Fu P, Wang L, Zhou J et al.
Optimization of microwave-assisted enzymatic
extraction of polyphenols from waste peanut shells and
evaluation of its antioxidant and antibacterial activities
in vitro. Food and Bioproducts Processing 2013;
91(2):158-168.
36. Saito M, Sakagami H, Fujisawa S. Cytotoxicity and
apoptosis induction by butylated hydroxyl anisole
(BHA) and buty lated hydroxytoluene (BHT).
Anticancer Research 2003; 23:4693-4701.
37. Weinreb O, Mandel S, Bar-Am O, Yogev-Falach M,
Avramovich-Tirosh Y, Mit T et al. Multifunctional
neuroprotective derivatives of rasagiline as anti-
alzheimer's disease drugs. Neurotherapy 2009; 6:163-
174.
