Post on 23-Feb-2023
ETHIOPIAN PHARMACEUTICAL JOURNAL Ethiop. Pharm. J. 30, 21-32 (2014) http://dx.doi.org/10.4314/epj.v30i1.3
Depression is an important global public
health problem due to both its relatively high
lifetime prevalence and being a leading
cause of disability, workplace absenteeism,
decreased productivity, and high suicide
rates (WHO, 2007). A recent World Health
Organization (WHO) report predicts that de-
pression will be the leading cause of disabil-
ity and premature death in the industrial
world, and unipolar major depression to be
the second largest cause of global disease
problems in the world, only behind ischemic
heart disease by the year 2020 (Kozisek et
al., 2008). Until the late 1980s, depression
was treated with electroconvulsive therapy
and antidepressant drugs known as monoam-
ine oxidase inhibitors (MAOIs) and tricyclic
antidepressants but later on these are super-
seded by agents like selective serotonin reup-
take inhibitors (Licinio and Wong, 2005).
Although treatment with these commer-
cially available antidepressant drugs is effec-
tive, a significant number of patients do not
achieve continuous remission, despite inten-
sive management. Moreover, only 60% of
patients are responsive to currently available
antidepressants. The unpredictable clinical
response to antidepressant drugs, a need to
administer up to 6-8 weeks to see the antide-
pressant effect and high susceptibility to ad-
verse effects are major clinical problems.
Thus, new drugs are still needed to treat de-
pression-related disorders (Schloss and
Henn, 2004; Kwon et al., 2010).
Prior to the application of synthetic anti-
depressant drugs, many traditional medicines
and empirical formula were successfully
used to treat depression-like disorders. Plants
of the genus Hypericum have traditionally
been used as medicinal plants in various
INTRODUCTION
Screening of the Antidepressant-like Activity of Two Hypericum
Species Found in Ethiopia
1 Department of Pharmacology and Clinical Pharmacy, School of Pharmacy, College of Health Sciences, Addis
Ababa University, Addis Ababa, Ethiopia
The widespread use of Hypericum perforatum for the treatment of mild to moderate depression has prompted
screening of the antidepressant-like effect of other species of the genus. The present study was designed to assess
the antidepressant-like activity of the 80% methanol extract of Hypericum quartinianum and Hypericum revolutum
in behavioral despair model. Eighty percent methanol extract of H. quartinianum and H. revolutum was
investigated using learned helplessness models of depression such as tail suspension test (TST), forced swimming
tests (FST) and avoidance tests. In addition, locomotor activity was investigated with open field test (OFT). Mice
(for TST, avoidance test and OFT) and rats (for FST) were randomly assigned into different groups and treated
with distilled water (control), imipramine 64 mg/kg (standard) and extract (200 mg/kg and 400 mg/kg). At 200 and
400 mg/kg, H. revolutum was effective in reducing immobility time in the TST (43.84%, p<0.01 and 49.08%,
p<0.01, respectively) and FST (33.7%, p<0.05 and 38.4%, p<0.01, respectively). Similarly, H. quartinianum also
showed anti-immobilty effect at 200 (30.67%, p<0.01) and 400 mg/kg (41.19%, p<0.01) in TST. However, only
the larger dose produced significant anti-immobility effect in FST (35.3%, p<0.05). Moreover, both extracts at the
doses used significantly decreased the escape failure (p<0.01) and increased the intertrial crossing (p<0.05 and
p<0.01) during the resting periods in the avoidance task in a shuttle box. In OFT, the tested crude extracts did not
significantly alter locomotor activity, suggesting that it is very unlikely that the observed antidepressant effects are
false positives. These observations together provide evidence that the 80% methanolic extract of leaves of H.
quartinianum and H. revolutum display antidepressant-like actions in established models of behavioral despair
without affecting locomotion.
Keywords: Hypericum quartinianum, Hypericum revolutum, depression, antidepressant-like activity
Abebe Ejigu1 and Ephrem Engidawork1*
* Correspondence: ephrem.engidawork@aau.edu.et
ETHIOPIAN PHARMACEUTICAL JOURNAL Ethiop. Pharm. J. 30, 21-32 (2014) http://dx.doi.org/10.4314/epj.v30i1.3
A. Ejigu and E. Engidawork
Ethiop Pharm J 30, 21-32 (2014)
http://dx.doi.org/10.4314/epj.v30i1.3
22
parts of the world and Hypericum perfora-
tum occupies a special position among the
genus. It is approved for its antidepressant
effect beyond reasonable doubt (Smelcerovic
et al., 2007). Extracts from various Hypericum
species have been shown to possess anti-
microbial, anti-inflammatory and antioxidant
activities. The most commonly isolated com-
pounds from this genus include acyl-
phloroglucinols, xanthones, flavonoids, tan-
nins and, less frequently, benzopyrans (Linde
et al., 1996; Choi et al., 2005, Vázquez-
Palacios et al., 2010).
There are around eight species of Hy-
pericum in Ethiopia including H. quartinianum
and H. revolutum (Robson, 1995). H. revo-
lutum has been reported to have the highest
fidelity level in treating rheumatism and ear-
ache traditionally in Ethiopia (Hostettmann
and Marston, 1994). The pulverized fresh
leaves of H. quartinianum are also used for
the treatment of “Evil eye”, and for the treat-
ment of diarrhoea in combination with other
plants (Tadesse et al., 2005). The leaves of
the plant are also used against fungal infec-
tions in Tanzania (Moshi et al., 2007).
The wide spread use of H. perforatum for
the treatment of mild to moderate depression
has initiated screening of the antidepressant
effect of other species of the genus (Mendes
et al., 2002: Rodríguez-Landa and Contreras,
2003). Studies with other plants of the same
genus have been carried out under the stimu-
lus of the great scientific interest and eco-
nomic value acquired by other Hypericum
species (Sanchez-Mateo et al., 2002; 2005).
The present study therefore focused on
evaluating the antidepressant-like activity of
H. quartinianum and H. revolutum in different
animal models of depression.
Plant material:
The leaves of H. quartinianum and H.
revolutum were collected at the flowering
period around Menagesha (50 km midwest
of Addis Ababa) and Jimma (350 km south-
west Addis Ababa), respectively. A taxono-
mist (Melaku Wondafrash) identified the
plants and a voucher specimens (collection
numbers AE/001 and AE/002, respectively)
were deposited at the National Herbarium,
College of Natural Sciences, Addis Ababa
University.
Experimental animals: Male and female Swiss Albino mice (7-10
weeks, 22–32 g) and Wistar male rats (7-10
weeks, 180-230 g) bred at the animal house
of School of Pharmacy and purchased from
Ethiopian Health and Nutrition research In-
stitute as well as College of Natural Sciences,
Addis Ababa University were used for the
experiments. The animals were housed in
groups with free access to laboratory pellets
and tap water and maintained under standard
conditions (12/12 h light-dark cycle, and am-
bient temperature 22 ±1 °C). All animals
used in the study were cared for and treated
humanely according to the Principles of
Laboratory Animal Care (OECD, 2001) and
the protocol was approved by School of
Pharmacy Ethics Committee.
Extraction procedure:
The leaves of the Hypericum species were
shade dried and powdered by grinding with
an electric mill. About 300 g of the plant ma-
terial of each species was macerated with
80% methanol for three days at room tem-
perature. This procedure was repeated twice.
The respective extracts were filtered using
gauze and Whatman filter paper, and dried at
a temperature below 45 °C in an oven. The
yields obtained with respect to the initial dried
material were 14% for H. quartinianum and
17% for H. revolutum. The dried extracts
were reconstituted with water for oral
administration.
MATERIALS AND METHODS
ANTIDEPPRESANT-LIKE ACTIVITY OF TWO HYPERICUM SPECIES
Ethiop Pharm J 30, 21-32 (2014)
http://dx.doi.org/10.4314/epj.v30i1.3
23
Acute toxicity test:
Acute toxicity study was performed using
the limit test dose of 2000 mg/kg as de-
scribed by Organization for Economic Coop-
eration and Development guideline and In-
teragency Research Animal Committee rec-
ommendation (OECD, 2001). Six female
mice for each extract and control were fasted
for 4 h and administered with the limit dose
2000 mg/kg of the respective extract or dis-
tilled water. Animals were observed indi-
vidually for any sign of acute toxicity, mor-
bidity or mortality after dosing at least once
during the first 30 min, periodically during
the first 24 h, with special attention given for
the first 4 h and daily thereafter for a total of
14 days.
Grouping and dosing of animals:
The animals were randomly assigned into
different groups, each comprising eight ani-
mals. Group I was control and received the
vehicle used for reconstitution. Group II was
given standard drug, imipramine (64 mg/kg,
orally). Group III-V were given either the
extract of H. quartinianum or H. revolutum
at doses of 100, 200 and 400 mg/kg. The ex-
tracts and the standard drug were dissolved
in distilled water and administered orally at a
volume of 10 ml/kg.
From the acute toxicity studies as per
OECD (2001) guidelines, after having con-
sidered the safety of the plant, 1/10th of the
maximum dose (2000 mg/kg) was consid-
ered as a middle dose. Half and double of the
middle dose were considered as the low and
high dose, respectively. Dose of imipramine
(64 mg/kg) was selected based on previous
studies (Mac Sweeney et al., 1998). The test
doses were prepared freshly on the day of
the experiment. Animals were treated with
the vehicle or extract/standard 1 h before
commencement of the experiments.
Antidepressant activity test:
Forced swim test:
For forced swim test (FST), a protocol
developed by Castagné et al. (2011) was fol-
lowed with slight modification. Male rats
were forced to swim in a glass aquarium
(diameter: 20 cm, height: 40 cm) containing
15 cm of water at 25 oC in two sessions
separated by 24 h. The water was changed
after each rat’s forced swim. The first ses-
sion, lasting 15 min, was conducted prior to
drug admin-istration and without behavioral
recording. In the second session, rats were
treated according to their grouping, 24, 4,
and 1 h before the test. Swimming was de-
fined by escape behavior, i.e., diving, rigor-
ous paddling with all four legs, circling the
tank, and clambering at the walls. Immobil-
ity was judged when the rats were floating
without struggling and treading in the water
just enough to keep the nose above the water
and the total duration of immobility was re-
corded using a stopwatch. Following the test,
the animals were dried with towel and heater.
Tail suspension test:
The protocol for tail suspension test
(TST) in mice is conceptually similar to
FST, but differs in that immobility is in-
duced by suspending the animal on its tail
(Castagné et al., 2011). After 60 min of ad-
ministration of the treatment, as per the re-
spective grouping, male mice were individu-
ally hung upside down from a counter top of
75 cm of height using an adhesive tape
placed approximately1 cm from the tip of
their tails. The total duration of immobility
was recorded manually using a stopwatch
during a 6-min test session. Immobility was
defined as the absence of any limb or body
movements, except for those caused by res-
piration. A mouse was considered immobile
when it hung passively and became com-
pletely motionless.
A. Ejigu and E. Engidawork
Ethiop Pharm J 30, 21-32 (2014)
http://dx.doi.org/10.4314/epj.v30i1.3
24
Avoidance test:
Male mice were used in this paradigm and
the procedure adopted by Anisman and
Merali (2001) and Xu et al. (2004) were fol-
lowed with slight modification. On the first
day, every mouse was individually exposed
to inescapable electric foot shocks (0.3 mA
DC) on an electrified grid floor in a shuttle
box with the gate closed for 1 h in the dark
phase. The foot shocks were unpredictable
with varying duration (1–3 sec) and interval-
episodes (1–15 sec), amounting to a total
session duration of 1 h. During the shock
exposure, lights were turned off. Two con-
trol groups of mice were used in this para-
digm. Control 1 mice received the vehicle
and subjected to shock, while Control 2 ani-
mals received the vehicle without shock.
Forty-eight hours after the inescapable
shock, all mice were subjected to an avoid-
ance-escape test in a shuttle-box (Coulborn
Instruments, Germany) (19” L x 9” W x
10.75” H). The animals were placed singly
into the shuttle box and allowed to habituate
to the environment for 3 min. Following this
free exploration session, 30 stimulus-shock
trials were presented for a period of 15 min,
i.e., two trials per minute. During the first 3
sec of each trial, a light signal was presented,
followed by a 3-sec electric shock (0.3 mA)
applied via the grid floor and then by a 24-
sec resting period. Subsequently, another
trial began and the number of escape failures
were recorded. The avoidance-escape test
was repeated on the 4th and 5th days, but
without giving the adaptation time of 3 min.
In this test, treatment was carried out on 5
consecutive days on daily basis, 1 h before
the avoidance task. The number of escape
failures, referred to as a ‘noncrossing re-
sponse’ during stimuli presentation and the
number of intertrial crossings observed dur-
ing the resting period were recorded and
comparison was made with control.
Open field test:
In order to detect any association of im-
mobility in the FST and TST with changes in
motor activity, the activities of animals
treated with the extracts were tested in an
open field test (OFT) in mice (Kim et al.,
2005). The OFT apparatus consisted of a
wooden box (68 x 68 x 45 cm), with a dark
floor, subdivided into 16 equal fields. The
experimental room was a sound attenuated
dark room. The OFT, illuminated with a 40
W bulb, focusing on the field from a height
of about 100 cm, was placed in the experi-
mental room. After 60 min of treatment,
mice were placed individually in a corner
square of the OFT and the ambulation (the
number of squares crossed at periphery), to-
tal locomotion (total number of squares
crossed) and activity in the centre (number
of central squares crossed) were recorded for
5 min.
Phytochemical screening:
A preliminary phytochemical screening
was performed on the hydroalcoholic ex-
tracts to identify secondary metabolites pre-
sent using standard procedures as described
elsewhere (WHO, 1978; Sofowara, 1993).
Data analysis:
Data generated from the experiment are
expressed as mean ± S.E.M and level of sig-
nificance was set at p<0.05. The data were
analyzed by SPSS windows version 19 using
one-way analysis of variance followed by
Tukey’s post-hoc test.
Acute toxicity test
At a single dose of 2000 mg/kg, both ex-
tracts showed no signs of overt toxicity or
death in mice during the 14 days observation
period, indicating that the LD50 of both ex-
RESULTS
ANTIDEPPRESANT-LIKE ACTIVITY OF TWO HYPERICUM SPECIES
Ethiop Pharm J 30, 21-32 (2014)
http://dx.doi.org/10.4314/epj.v30i1.3
25
tracts to be above 2000 mg/kg. The general
behavior of treated animals remained unal-
tered during this period and was similar to
those of controls.
Effect of the extracts in tail suspension test
The effect of the extracts on immobility
time in TST is shown in Table 1. Whilst H.
revolutum extract at doses of 200 (44%,
p<0.01) and 400 mg/kg (49%, p<0.01) sig-
nificantly reduced immobility time, 100 mg/
kg did not show any significant change com-
pared to controls. Though no apparent
change was observed between 200 mg/kg
and 400 mg/kg, the same level of signifi-
cance in decrement as that of controls had
been observed between 400 and 200 mg/kg
compared to 100 mg/kg of the extract.
Treatment with H. quartinianum produced
a similar pattern of change in immobility
time to that of H. revolutum (Table 1). Ac-
cordingly, both 400 mg/kg (41.2%, p<0.01)
and 200 mg//kg (31%, p<0.01) significantly
reduced immobility, but no detectable
change was observed with 100 mg/kg com-
pared to controls. Once again no apparent
change was noted between the middle and
higher doses.
Comparing the two extracts, 200 mg/kg
and 400 mg/kg of H. revolutum showed sig-
nificant effect compared to 100 mg/kg of H.
quartinianum. However, no significant
change was observed with the other doses of
the two extract, although percent reduction
was tended to be better with H. revolutum.
Whilst imipiramine significantly de-
creased immobility time compared to con-
trols (p<0.01) and 100 mg/kg of both ex-
tracts (p<0.01), no statistically significant
decrement was noted when compared with
the other doses of the extract. It is of note
that maximum reduction was achieved with
impiramine.
Effect of the extracts in forced swim test
Alteration of duration of immobility in
FST by administration of the hydroalcoholic
extracts is depicted in Table 2. Lower dose
of both extracts was unable to bring any no-
table change in duration of immobility com-
pared to controls. However, a different pat-
tern emerged in FST unlike TST when the
other doses of both extracts were analyzed.
Whereas H. revolutum extract significantly
brought down immobility time at both 200
mg/kg (33.72%, p<0.05) and 400 mg/kg
(38.42%, p<0.01), only 400 mg/kg (35.3%,
p<0.05) of H. quartinianum extract did pro-
duce significant decrease when compared
against controls.
The standard drug showed a marked re-
duction in immobility time by about 50%
compared to vehicle treated group, which
was by far the highest of all. Apart from this,
Table 1. Effects of administration of the 80% methanolic extracts of Hypericum quartinianum and
Hypericum revolutum extracts on immobility time in the tail suspension test.
Treatment Dose (mg/kg) Immobility time (s) % Change
Vehicle - 160.06±6.637 -
H. revolutum 100 mg/kg 132.75±12.385b** 17.16
200 mg/kg 89.88±11.578a**,c*,d* 43.84
400 mg/kg 81.5±9.038a**,c**,d** 49.08
H. quartinianum 100 mg/kg 133.25±6.742b** 16.75
200 mg/kg 111.00±7.928a** 30.67
400 mg/kg 94.13±11.746a** 41.19
Imipramine 64 mg/kg 76.19±5.186a**,c**,d** 52.34
Values are mean ± SEM; n=6 per group; aagainst vehicle control; bagainst standard; cagainst H.
revolutum 100 mg/kg; dagainst H. quartinianum 100 mg/kg, *p<0.05; **p< 0.01.
A. Ejigu and E. Engidawork
Ethiop Pharm J 30, 21-32 (2014)
http://dx.doi.org/10.4314/epj.v30i1.3
26
the effect observed with imipramine was sig-
nificantly higher than the one produced by
100 mg/kg (p<0.05) of both extracts. How-
ever, once again no detectable changes were
noted when compared to 200 and 400 mg/kg
doses of both extracts.
Effect of the extracts on learned helplessness
paradigm
The effect of H. quartinianum and H.
revolutum extracts on the number of escape
failures in the three-day avoidance task in
the shuttle box is presented in Table 3. Con-
trol 2 animals exhibited lower number of es-
cape failures compared to control 1 animals,
which were exposed to inescapable foot
shock. The escape failure from the electric
shock during the first day of the avoidance
task was decreased (p<0.01) by about 59.3%
compared to control 1. Moreover, the escape
failure further decreased on day 2 and 3 by
about 68.4% and 65%, respectively. The in-
crement in the number of escape failures in
the learned helpless animals (control 1) as
compared to control 2 animals was indica-
tive of a behavioral deficiency caused by ex-
posure to an uncontrollable aversive situa-
tion, which is considered to be indicative of
depression.
As demonstrated in Table 3, treatment
with H. revolutum extract at a dose of 100
mg/kg did not produce significant decrease
in the number of escape failures throughout
all the three days as compared to control 1.
By contrast, 200 mg/kg (p<0.01) of the ex-
tract did show significant reduction in the
number of escape failures by 51.2%, 49.2%
and 49.4% in day 1, 2 and 3 of avoidance
task, respectively. Moreover, the extract at
400 mg/kg showed a marked decrease
(p<0.01) in the number of escape failures by
about 50.8% than control 1 animals. The es-
cape failure also decreased after the admini-
stration of H. revolutum at a dose of 400
mg/kg during the 2nd and 3rd avoidance task
(48.1%, p<0.01 and 50.54%, p<0.01, respec-
tively). However, no apparent changes were
observed among the different doses.
Similar to H. revolutum, treatment with
H. quartinianum extract at the lowest dose
(100 mg/kg) did not produce significant re-
duction in the number of escape failures in
all the three days compared to control 1 ani-
mals. However, the extract at a dose of 200
(40.2%-44%, p<0.01) and 400 mg/kg (45-
51.2%, p<0.01) exerted a remarkable de-
crease in the number of escape failures com-
pared to control 1 mice across the test days.
However, no detectable change was ob-
served across days in all doses.
No apparent change was observed when
comparison was made between doses of both
extracts (200 mg/kg and 400 mg/kg) that re-
Table 2. Antidepressant effects of the 80% methanolic extracts Hypericum revolutum and Hypericum
quartinianum in the forced swimming test in rats.
Treatment Dose (mg/kg) Immobility time (s) % Change
Vehicle - 170.50±12.132 -
H. revolutum 100 mg/kg 160.17±11.176b** 6.06
200 mg/kg 113.00±8.805a* 33.72
400 mg/kg 105.00±10.869a**,c* 38.42
H. quartinianum 100 mg/kg 154.50±13.691b** 9.38
200 mg/kg 137.50±16.051 19.35
400 mg/kg 110.33±10.70a* 35.29
Imipramine 64 mg/kg 85.67±10.330(a,c,d)** 49.85
Values are mean ± SEM; n=6 per group; aagainst vehicle control; bagainst standard; cagainst H.
revolutum 100 mg/kg; dagainst H. quartinianum 100 mg/kg, *p<0.05; **p< 0.01.
ANTIDEPPRESANT-LIKE ACTIVITY OF TWO HYPERICUM SPECIES
Ethiop Pharm J 30, 21-32 (2014)
http://dx.doi.org/10.4314/epj.v30i1.3
27
sulted in significant decrease in the number
of escape failures with control 2, although a
better percentage reduction was observed in
escape failures by control 2 than any of the
doses.
Mice treated with imipramine showed re-
duction in the number escape failures (p<
0.01) on all three days of the avoidance task
compared to control 1 animals, with the
highest percent reduction (63.8 %) of escape
failures noted on the first day. Although the
decrease in the number of escape failures
was also significant (p<0.01) relative to 100
mg/kg of both extracts, the difference was
lost when compared to increasing doses of
the extracts.
In addition to the escape failure, number
of inter-trial crossing was recorded in the
three day avoidance task during the 24 sec
resting periods and is shown in Figure 1.
There were several inter-trial crossings dur-
ing the resting periods for control 2
(7.75±1.84, 8.63±1.43 and 7.75±1.11, re-
spectively), which was significantly greater
(p<0.01) compared to control 1. The extracts
of the tested Hypericum species produced a
significantly increased (p<0.05 in all cases)
inter-trial crossings at both 200 mg/kg and
400 mg/kg doses compared to control 1 mice
across the test days. However, 100 mg/kg of
both extracts failed to significantly increase
the inter-trial crossings. Imipramine, on the
other hand, showed a marked increase
(p<0.01) on the number of inter-trial cross-
ings in all the three days compared to control
1. But, no significant difference was ob-
served amongst the doses and with
imipramine as well. Moreover, no apparent
changes were seen among treatment groups
and control 2 mice which did not take the
electric shock.
Effect of the extracts in open field test
To rule out that the changes in immobility
time observed in the TST and FST were not
Table 3. Number of escape failures observed during three days of an avoidance task in the shuttle box in
the learned helplessness test in mice.
Treatment Dose
(mg/kg)
Number of escape failures (percent reduction)
Day 1 Day2 Day 3
Vehicle control 1 - 24.88±1.43 23.38±1.647 22.00±1.524
Vehicle control 2 - 10.13±1.767(a,c)**,d*
(59.28 %)
7.38±1.792(a,c,d)**
(68.43%)
7.75±1.319(a,c,d)**
(64.77 %) 80% methanolic
extract of
Hypericum revolutum
100 20.25±2.328b**
(18.61 %)
18.88±3.270b**
(19.24 %)
18.25±2.403b**
(17.04 %)
200 12.13±1.025a**
(51.24 %)
11.88±1.008a**
(49.18 %)
11.13±1.093a*,d*
(49.41 %)
400 12.25±0.796a**
(50.76 %)
12.13±1.246a**
(48.11 %)
10.88±1.093a**,d*
(50.54 %)
80% methanolic
extract of
Hypericum quartinianum
100 18.50±3.257b*
(25.64 %)
18.75±1.731b**
(19.80 %)
19.88±3.627b**
(9.63 %)
200 14.88±3.907a**
(40.19 %)
13.88±1.747a**
(40.63 %)
12.63±1.253a*
(48.32 %) 400 12.13±5.027a**
(51.24 %)
11.63±1.626a**
(50.26 %)
12.13±1.315a**
(44.86 %)
Imipramine 64 9.00±1.626(a,c)**,d*
(63.82 %)
8.50±0.627(a,c,c)**
(63.64 %)
8.38±1.375(a,c,d)**
(61.91 %)
Values are mean ± SEM; n=8 per group; aagainst vehicle control 1; bagainst standard; cagainst H. revolutum 100
mg/kg; dagainst H. quartinianum 100 mg/kg; *p<0.05; **p< 0.01.
A. Ejigu and E. Engidawork
Ethiop Pharm J 30, 21-32 (2014)
http://dx.doi.org/10.4314/epj.v30i1.3
28
attributed to non-specific effects of the ex-
tract, the ability of the extracts to alter loco-
motion was examined using the OFT (Table
4). Doses of both extracts and imipiramine
that significantly reduced immobility time in
TST and FST failed to produce any signifi-
cant alteration in the parameters measured in
the OFT compared with controls. Even the
lowest dose (100 mg/kg) of the extracts that
was devoid of any effect on immobility time
did not significantly affect those parameters,
which is indicative of non-locomotory effect
of the extracts. It is interesting to note that
impiramine tended to decrease the number of
central square crossing compared to controls,
although the difference failed to reach statis-
tical significance.
Phytochemical screening
The results of phytochemical screening
test showed the presence of different secon-
dary metabolites in both H. quartinianum
and H. revolutum total extracts. Whilst alka-
loids, saponins, phenolic compounds, and
flavonoids were detected in both extracts,
tannins which were present in H. quartin-
ianum, were not detected in H. revolutum.
However, terpenoids, phlabotannins and
steroids were not detected in both species of
Hypeicum.
Both FST and TST are widely used to
screen new antidepressant drugs as well as to
investigate the mechanism of action of new
antidepressants. These tests are quite sensi-
tive and relatively specific to all major classes
of clinical antidepressant drugs (Kwon et al.,
2010; Castagné et al., 2011). The current
study revealed that the hydro-alcoholic ex-
tracts of H. quartinianum and H. revolutum
display antidepressant-like actions in estab-
lished models of behavioral despair, namely
FST, TST, and learned helplessness model
DISCUSSION
Figure 1. Number of inter-trial crossing observed during three days of an avoidance task in the shuttle
box in the learned helplessness test in mice (values are mean ± SEM; n=8 per groupa; against vehicle
control 1: *p<0.05; **p< 0.01).
ANTIDEPPRESANT-LIKE ACTIVITY OF TWO HYPERICUM SPECIES
Ethiop Pharm J 30, 21-32 (2014)
http://dx.doi.org/10.4314/epj.v30i1.3
29
as demonstrated by the significant reductions
in immobility and escape failure.
Doses of 200 and 400 mg/kg of both ex-
tracts significantly reduced immobility time
in TST. However, 200 mg/kg dose of H.
quartinianum was unable to replicate the ef-
fect seen in TST in FST. Moreover, 100 mg/
kg dose of both plants did not show any anti-
depressant-like activity in TST as well as
FST, suggesting that this is a sub-threshold
dose. Effect was apparent with increasing
dose pointing to the fact that a relatively
high dose of the extracts is needed to pro-
duce an antidepressant-like effect. This is in
line with reports of other Hypericum species,
where H. glandulosum and H. grandifolium
extracts were shown to exert antidepressant-
like effect at higher doses (Sanchez-Mateo et
al., 2005). It must also be considered that
utilization of a crude extract may mask the
effects of active constituents and the concen-
tration of the different biological compo-
nents might not be enough to have the re-
quired effect.
One could also note that there were incon-
sistencies in the percent reduction observed
by the same dose in the two models, namely
TST and FST. For example, reduction pro-
duced by H. revolutum at 400 mg/kg was
49.1% in TST but the corresponding value
for FST was 38.4%. Such inconsistencies
may emanate from inter-strain and interspe-
cies variations as well as difference in sensi-
tivity of the tests (El-Alfy et al., 2010).
The antidepressant activity of the extracts
were also assessed in the learned helpless-
ness model, which is one of the well-
validated animal models of depression, in
which a depressive-like state in rodents is
induced by uncontrollable and unpredictable
electrical foot-shock stress (Enkel et al.,
2010). In this test, rodents exposed to ines-
capable and unpredictable electric shock, at
one situation fail to escape even when escape
is possible. This is termed as escape failure,
and decreased by potential anti-depressant
drugs (Mac Sweeney et al., 1998).
The results of learned helplessness
showed that both H. quartinianum and H.
revolutum at 200 and 400 mg/kg reversed the
learning deficits caused by uncontrollable
and unpredictable shock, an effect similar to
that of antidepressant treatment. It can be
seen from the data produced that the higher
dose of both extracts possessed more antide-
pressant activity compared to other doses,
which is consistent with the results obtained
from the TST and FST. Although no single
animal model of depression has a generally
accepted predictive power for clinical effec-
tiveness as the magnitude of the effects
bound to show some difference, these results
clearly suggest that the extracts have a po-
tential anti-depressant activity. It is widely
accepted that chronic treatment with antide-
pressants reduces escape failure in learned
Table 4. Results of open field test in mice administered with the 80% methanolic extracts of Hypericum
quartinianum and Hypericum revolutum.
Treatment Dose (mg/kg) Number of squares crossed
Peripheral Central Total
Vehicle - 67.25±4.942 6.00±1.955 73.25±5.437
H. revolutum 100 mg/kg 89±5.822 6.38±0.498 95.38±6.050
200 mg/kg 64.38±6.514 4.13±1.342 68.50±7.136
400 mg/kg 65.14±3.508 6.71±2.190 71.86±5.068
H. quartinianum 100 mg/kg 59.75±5.243 4.38±1.164 64.13±5.184
200 mg/kg 64.00±5.060 7.25±1.645 71.25±5.706
400 mg/kg 64.13±7.311 6.75±2.320 70.88±7.786
Imipramine 64 mg/kg 59.57±8.842 2.71±0.808 62.29±8.928
Values are expressed as mean ± SEM, n=8 per group
A. Ejigu and E. Engidawork
Ethiop Pharm J 30, 21-32 (2014)
http://dx.doi.org/10.4314/epj.v30i1.3
30
helpless rats. Acute assays for antidepres-
sants have been argued to be less valid than
more chronic paradigms, which more closely
model the behavioral, endocrine and neuro-
chemical features of clinical depression, and
in which the temporal aspects of behavioral
change and drug response more closely re-
semble those of human depressive illnesses
(Semba et al., 1998). In the learned helpless-
ness model, repeated administration of ex-
tracts for 5 days was performed and shown
to induce significant reduction in the number
of escape failures, reinforcing the notion that
the extracts are endowed with a potential an-
tidepressant-like activity.
It has been reported that psychomotor
stimulant drugs such as caffeine also de-
crease immobility time, but in contrast to
antidepressants, cause marked motor stimu-
lation, indicating that the effects may be
nonspecific. Thus, the assessment of sponta-
neous locomotor activity is imperative as
part of the routine procedures for detecting
prospective antidepressant drugs. Antide-
pressants are known to reduce immobility at
doses that do not change the motor behavior
of rodents in OFTs (Kwon et al., 2010; Yu et
al., 2002). The standard OFT is commonly
used to assess locomotor, exploratory and
anxiety-like behavior in laboratory animals
(Sanchez-Mateo et al., 2002). Data obtained
from the OFT showed that crude extracts of
both H. quartinianum and H. revolutum as
well as imipramine at doses used in this
study did not significantly alter spontaneous
locomotor activity, indicating that a psy-
chostimulant effect is not responsible for the
decrease in the immobility and the observed
antidepressant effects are not false positives.
In addition, the OFT task approaches the
conflicts between the innate fear that mice
have of the central area of a novel or brightly
lit open field versus their desire to explore
new environments. In the present study, the
extracts did not significantly increase the
number of crossings to the center of the OFT
apparatus. Though further researches using
other models of anxiety are important, it can
be suggested that both extracts might not
have anxiolytic effect.
Several studies have shown that the anti-
depressant effects of H. perforatum extracts
may be due to a combination of different
biological constituents (naphtodianthrones,
flavonoids and phloroglucinols) rather than
any single compound (Bilia et al., 2002;
Vázquez-Palacios et al., 2010). Hypericin
and hyperforin are the main constituents
thought to be responsible for antidepressant
activity of the plant (Linde et al., 1996;
Vázquez-Palacios et al., 2010). Thus, it is
possible to suggest that part of the antide-
pressant activity showed in this study may at
least in part be due to the presence of poly-
phenolic substances. Alkaloids were also
detected as major compounds in both ex-
tracts, thus it seems likely that they may ac-
count for the plants’ antidepressant activity
reported here, as administration of mon-
tanine, an alkaloid isolated from Hippeas-
trum vittatum, reduced total immobility time
and enhanced struggling behavior (da Silva
et al., 2006). Total saponins extracted from
the caudexes and leaves of Panax notogin-
seng exerted antidepressant like effects in
the depression paradigms and demonstrated
that the saponins may exert these effects
through a monoamine neurotransmitter
mechanism (Xiang et al., 2011). Moreover, a
study conducted on ginseng total saponins
also showed a tendency to reduce immobility
time at different doses in FST in mice (Li et
al., 2003; Dang et al., 2009). Thus, saponins
which are also found in H. quartinianum and
H. revolutum extract might contribute to the
antidepressant-like effect of the crude extracts.
Tannins are known to cause protein dena-
turation and can coagulate and precipitate
proteins and thus lead to false negative/
positive results. The screenings for tannins
were negative for H. revolutum but are pre-
sent in methanolic extract of H. quartin-
ANTIDEPPRESANT-LIKE ACTIVITY OF TWO HYPERICUM SPECIES
Ethiop Pharm J 30, 21-32 (2014)
http://dx.doi.org/10.4314/epj.v30i1.3
31
ianum. Thus, it could be cited as one possi-
ble reason for the difference in immobility
reducing ability of the two extracts in TST
and FST. Although inconsistent findings
were obtained in the learned helplessness
paradigm across the test days, activity was
still better with H. revolutum than H. quar-
tinianum.
In conclusion, this study provides evi-
dence that the 80% methanolic extract of the
leaves of H. quartinianum and H. revolutum
displayed antidepressant-like actions in es-
tablished animal models of depression as
demonstrated by a reductions in immobility
time and escape failure. These effects were
not due to motor stimulation, indicating that
the effects were not false positive. The find-
ings in this study demonstrated the potential
use of the two medicinal plants to treat men-
tal illnesses related to depression and could
be used as an alternative therapy for depres-
sive disorders.
Acknowledgements
The financial assistance of Addis Ababa Uni-
versity is gratefully acknowledged.
CONCLUSION
Anisman H, Merali Z (2001). Rodent models of
depression: learned helplessness induced in
mice. Curr Protoc Neurosci May; Chapter 8:
Unit 8.10C (doi:10.1002/0471142301.
ns0810cs14).
Bilia AR, Gallori S, Vincieri FF (2002). St. John’s
wort and depression efficacy, safety and tol-
erability-an update. Life Sci 70: 3077-3096.
Castagné V, Moser P, Roux S, Porsolt RD
(2011). Rodent models of depression: forced
swim and tail suspension behavioral despair
tests in rats and mice. Curr Protoc Neurosci
Apr; Chapter 8:Unit 8.10A (doi: 10.1002/
0471142301.ns0810as55).
Choi WH, Hong SS, Lee SA, Han XH, Lee KS,
Lee MK, Hwang BY, Ro JS (2005). Mono-
amine oxidase inhibitory naphthoquinones
from the roots of Lithospermum
erythrorhizon. Arch Pharm Res 28: 400-404.
Dang H, Chen Y, Liu X, Wang Q, Wang L, Jia
W, Wang Y (2009). Antidepressant effects
of ginseng total saponins in the forced swim-
ming test and chronic mild stress models of
depression. Prog Neuro Psychopharmacol
Biol Psychiatry 33: 1417-1424.
da Silva AFS, Andrade JP, Bevilaqua LRM,
Souza MM, Izquierdo I, Henriques AT, Zua-
nazzi JAS (2006). Anxiolytic, antidepressant
and anticonvulsant-like effects of the alka-
loid montanine isolated from Hippeastrum
vittatum. Pharmacol Biochem Behav 85: 148
-154.
El-Alfy AT, Ivey K, Robinson K, Safwat AS,
Radwan M, Slade D, Khan I, El-Sohly M,
Ross S (2010). Antidepressant-like effect of
9-tetrahydrocannabinol and other cannabi-
noids isolated from Cannabis sativa L. Phar-
macol Biochem Behav 95: 434-442.
Enkel T, Spanagel R, Vollmay B, Schneider M
(2010). Stress triggers anhedonia in rats bred
for learned helplessness. Behav Brain Res
209: 183-186.
Hostettmann K, Marston A (1994). Search for
new antifungal compounds from higher
plants. Pure Appl Chem 66: 2231-2234.
Kim SH, Han J, Seog DH, Chung JY, Kim N,
Hong Park Y, Lee SK (2005). Antidepres-
sant effect of Chaihu-Shugan-San extract
and its constituents in rat models of depres-
sion. Life Sci 76: 1297-1306.
Kozisek ME, Middlemas D, Bylund DB (2008).
Brain-derived neurotrophic factor and its
receptor tropomyosin-related kinase B in the
mechanism of action of antidepressant thera-
pies. Pharmacol Ther 117: 30-51.
Kwon S, Lee B, Kim M, Lee H, Park HJ, Hahm
DH (2010). Antidepressant-like effect of the
methanolic extract from Bupleurum falcatum
in the tail suspension test. Prog Neuropsy-
chopharmacol Biol Psychiatry 34: 265-270.
Li YF, Gong ZH, Cao JB, Wang HL, Luo ZP, Li
J (2003). Antidepressant-like effect of ag-
matine and its possible mechanism. Eur J
Pharmacol 469: 81-88.
REFERENCES
A. Ejigu and E. Engidawork
Ethiop Pharm J 30, 21-32 (2014)
http://dx.doi.org/10.4314/epj.v30i1.3
32
Licinio J, Wong ML (2005). Depression, antide-
pressants and suicidality: a critical appraisal.
Nat Rev Drug Discov 4: 165-171.
Linde K, Ramirez G, Mulrow CD, Pauls A,
Weidenhammer W, Melchart D (1996). St
John's wort for depression-an overview and
meta-analysis of randomised clinical trials.
BMJ 313: 253-258.
Mac Sweeney CP, Lesourd M, Gandon JM
(1998). Antidepressant-like effects of alne-
spirone S 20499/ in the learned helplessness
test in rats. Eur J Pharmacol 345: 133-137.
Mendes FR, Rita MR, Car ELA (2002). Activity
of Hypericum brasiliense and Hypericum
cordatum on the central nervous system in
rodents. Fitoterapia 73: 462-471.
Moshi MJ, van den Beukel CJ, Hamza OJ,
Mbwambo ZH, Nondo RO, Masimba PJ,
Matee MI, Kapingu MC, Mikx F, Verweij
PE, van der Ven AJ (2006). Brine shrimp
toxicity evaluation of some Tanzanian plants
used traditionally for the treatment of fungal
infections. Afr J Tradit Complement Altern
Med 4: 219-225.
Organization of Economic Cooperation and De-
velopment (2001). The OECD guidelines for
testing of chemical: 420 acute oral toxicity.
France.
Robson NKB (1995). Guttiferae. In: Flora of
Ethiopia and Eritrea. Gebregziabhere T,
Hedberg O, Tadesse M, Demisew S, Friis I,
Hedberg I, Edwards S, Vol 2, Part 2, pp 135-
143.
Rodriguez-Landa JF, Contreras CM (2003). A
review of clinical and experimental observa-
tions about antidepressant actions and side
effects produced by Hypericum perforatum
extracts. Phytomedicine 10: 688-699.
Sánchez-Mateo CC, Bonkanka CX, Prado B,
Rabanal RM (2005). Antidepressant proper-
ties of some Hypericum canariense L. and
Hypericum glandulosum Ait. extracts in the
forced swimming test in mice. J Ethnophar-
macol 97: 541-547.
Sanchez-Mateo CC, Prado B, Rabanal RM
(2002). Antidepressant effects of the metha-
nol extract of several Hypericum species
from the Canary Islands. J Ethnopharmacol
79: 119-127.
Schloss P, Henn FA (2004). New insights into
the mechanisms of antidepressant therapy.
Pharmacol Ther 102: 47-60.
Semba J, Mataki C, Yamada S, Nankai M, Toru
M (1998). Antidepressant like effects of
chronic nicotine on learned helplessness
paradigm in rats. Biol Psych 43: 389–391.
Smelcerovic A, Spiteller M, Ligon AP, Smel-
cerovic Z, Raabe N (2007). Essential oil
composition of Hypericum L. species from
Southeastern Serbia and their chemotaxon-
omy. Biochem Systs Ecol 35: 99-113.
Sofowara A (1993). Medicinal Plants and Tradi-
tional Medicines in Africa. 2nd ed. Spectrum
Books, Ibadan, Nigeria.
Tadesse M, Debela H, Yehenew G (2005). Sur-
vey of medicinal plants used to treat human
diseases in Seka Chekorsa, Jimma zone,
Ethiopia. Ethiop J Health Sci 15: 89-106.
Vázquez-Palacios G, Hernandez GM, Perez
MP, Bonilla JH (2010). Nicotine and
fluoxetine induce arousing effects on sleep–
wake cycle in antidepressive doses: a possi-
ble mechanism of antidepressant-like effects
of nicotine. Pharmacol Biochem Behav 94:
503-509.
World Health Organization (1978). The promo-
tion and development of traditional medicine,
Technical Report Series, 622, Geneva.
World Health Organization (2007). Ten statisti-
cal highlights in global public health.
Xiang H, Liu Y, Zhang B, Huang J, Li Y, Yang
B, Huang Z, Xiang F, Zhang H (2011). The
antidepressant effects and mechanism of ac-
tion of total saponins from the caudexes and
leaves of Panax notoginseng in animal mod-
els of depression. Phytomedicine 18: 731-
738.
Xu C, Luo L, Tan RX (2004). Antidepressant
effect of three traditional Chinese medicines
in the learned helplessness model. J Ethno-
pharmacol 91: 345-349.
Yu ZF, Kong LD, Chen Y (2002). Antidepres-
sant activity of aqueous extracts of Curcuma
longa in mice. J Ethnopharmacol 83: 161-
165.