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

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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

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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.

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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

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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.

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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.

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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.

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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).

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Ethiop Pharm J 30, 21-32 (2014)

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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

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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)

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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

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