Effects of common daisy (Bellis perennis L.) aqueous extracts on anxiety-like behaviour and spatial memory performance in Wistar albino rats

Abstract

The effects of aqueous extract of flowers from Bellis perennis on anxiety-like behavior and memory in Wistar rats were tested. Vehicle 20 and 60 mg kg -1 B. perennis groups were performed and the animals were tested by open field and elevated plus maze tests for anxiety-like behaviour and Morris water maze test for spatial memory. In the open field, the high dose of B. perennis administrated rats spent more time at the center, showed less mobility and velocity. In the elevated plus maze, the high dose of B. perennis administrated rats spent more time in the open arms, spent less time in the closed arms, were less mobile, were slower and rotated less frequently. In the Morris water maze, the high dose of B. perennis administrated rats spent more of the time to find the platform. In conclusion, B. perennis may produce biphasic effects on both anxiety-like behaviour and learning performance of the rats.

Full text

African Journal of Pharmacy and Pharmacology Vol. 5(11), pp. 1378-1388, 22 September, 2011
Available online http://www.academicjournals.org/ajpp
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Effects of common daisy (Bellis perennis L.) aqueous
extracts on anxiety-like behaviour and spatial memory
performance in Wistar albino rats
F. Pehlivan Karakaş1*, A. Karakaş1, H. Coşkun2 and A. Uçar Turker1
1Department of Biology, Faculty of Arts and Sciences, Abant Izzet Baysal University, Bolu 14280, Turkey.
2Department of Psychology, Faculty of Arts and Sciences, Abant Izzet Baysal University, Bolu 14280, Turkey.
Accepted 17 June, 2011
The effects of aqueous extract of flowers from Bellis perennis on anxiety-like behavior and memory in
Wistar rats were tested. Vehicle 20 and 60 mg kg-1 B. perennis groups were performed and the animals
were tested by open field and elevated plus maze tests for anxiety-like behaviour and Morris water maze
test for spatial memory. In the open field, the high dose of B. perennis administrated rats spent more
time at the center, showed less mobility and velocity. In the elevated plus maze, the high dose of B.
perennis administrated rats spent more time in the open arms, spent less time in the closed arms, were
less mobile, were slower and rotated less frequently. In the Morris water maze, the high dose of B.
perennis administrated rats spent more of the time to find the platform. In conclusion, B. perennis may
produce biphasic effects on both anxiety-like behaviour and learning performance of the rats.
Key words: Bellis perennis, common daisy, anxiety, sedative, spatial memory.
INTRODUCTION
Bellis perennis L. (common daisy), a herbaceous
perennial plant of meadows, lawns and other grassy
areas, is a member of the Asteraceae family. It is native
to western, central and northern Europe, but is commonly
found as an invasive plant in North America (Tutin et al.,
1976; Panda, 2004). Common daisy has been used as a
diuretic, antispasmodic, anti-inflammatory, astringent,
expectorant, antipyretic, vulnerary, ophthalmic and
homeostatic in traditional medicine (Grieve, 1982; Bown,
1995; Baytop, 1999; Duke et al., 2002). The main
constituents are triterpenoid saponins (Hiller et al., 1988;
Schopke et al., 1990, 1991, 1992; Li et al., 2005;
Morikawa et al., 2008; Yoshikawa et al., 2008), flavonoids
(Gudej and Nazaruk, 2001) and essential oils (Avato et
al., 1997; Kavalcioglu et al., 2010). Antibacterial,
antifungal, antioxidant (Desevedavy et al., 1989; Avato et
al., 1997; Kavalcioglu et al., 2010), antihyperlipidemic
(Morikawa et al., 2008, 2010a), postpartum antihemorrhagic
(Oberbaum et al., 2005), pancreatic lipase inhibitor
*Corresponding author. E-mail: pehlivan_f@ibu.edu.tr. Tel: +90-
374- 254 1000. Fax: +90-374- 253 4642.
(Morikawa et al., 2010b) and cytotoxic activities against
HL-60 human promyelocytic leukemia cells (Li et al.,
2005) of B. perennis have also been shown. Anxiety is a
common experience in daily life which causes substantial
suffering in the general population. The data from
Western Countries report the prevalence estimates as
high as 10 to 15% of the population (Regional Health
Forum). This high percentage of the prevalence of the
anxiety disorder in general population is elevating the
importance of the results of the animal experiments
especially for the drug treatments. B. perennis may be
one of the plausible treatments for reducing anxiety. As
best as is known, there has been no reported research
that examines the plausible anxiolytic effects of B.
perennis on anxiety in the literature. Also, it would be
interesting to see whether or not its effects on anxiety
changes with gender.
For instance, juveniles may become more anxious than
adults for various reasons (for example, differences in
olfactory or visual sensations and searching behaviors for
environmental cues) and thereby are more sensitive to
the effects of either high or low doses of B. perennis than
adults. Such plausible outcome requires an experimental
research paradigm that investigates both dose treatment

(for example, high or low doses) and generation in a
single research paradigm in which has not been found in
the relevant literature so far. In addition to this, the fact
that B. perennis may have important effects on learning
performance has been disregarded in the literature.
Given the consideration mentioned earlier, if B. perennis
has an anxiolotic effect, it should enhance learning
performance. If not or if it has an anesthetic effect, then it
can decrease spatial learning performance. These
possibilities are among interesting research concerns that
need to be answered. Given the considerations
mentioned earlier, the present study was conducted to
investigate the effects of B. perennis injection on the
anxiety like behaviors via open field (Benabid et al.,
2008) and elevated plus maze (Dawson and Tricklebank,
1995) and spatial memory performance of the rats via the
Morris water maze (Hooge and De Deyn, 2001). This is
because it is still not known whether B. perennis extracts
are effective or not on anxiety like behaviors and spatial
memory.
MATERIALS AND METHODS
Plant material
Common daisy (B. perennis L.) flowers and pedicels were collected
from Abant Izzet Baysal University Campus, Bolu, Turkey in May
2009. Identification of the species was made by using “Flora of
Turkey and the East Aegean Islands” (Davis, 1975) and voucher
specimen (collection number AUT-1909) was deposited at the
Abant Izzet Baysal University (AIBU) Herbarium, Bolu, Turkey.
Preparation of the common daisy extracts
Collected common daisy flowers and pedicels were dried in oven at
40°C and then ground into a powder. For aqueous extraction, 20 g
from powdered plant sample were extracted with 200 ml water at
80°C in a water bath for 12 h and then filtered. Frozen filtrate was
lyophilized by using freeze-dryer at -65°C. Residue was dissolved
in serum physiologic (0.9% NaCl) to produce a final concentration
of 100 mg ml-1. The yield of extract (w/w) was 15%.
Animal care
Adult male W istar rats (200 to 250 g) were obtained from our
laboratory colony maintained at the Abant Izzet Baysal University
(AIBU). They were exposed from birth to 12 L (12 h of light, 12 h of
darkness, lights off at 1800 h). Animals were maintained in plastic
cages (16 × 31 × 42 cm) with pine shavings used as bedding. Food
pellets and t ap water were acc essible ad libitum. The procedures in
this study were carried out in accordance with the Animal Scientific
procedure and approved by the Institutional Animal care and Use
Committee. All lighting was provided by cool-white fluorescent
tubes controlled by automatic programmable timers. Ambient
temperatures in the animal f acilities were held constant at 22 ± 2°C
in air-ventilated rooms.
Administration of the extract
In the present study, adult (n:18) and juvenile (n:18) and t otally the
Karakaş et al. 1379
thirty six male rats were used and were randomly divided into three
groups as vehicle, 20 mg kg-1 B. perennis aqueous extract injection
(low dose) and 60mg kg-1 B. perennis aqueous extract injection
(high dose). Intraperitoneal injections were applied everyday at
13:00 h st arting from t he 1st day to t he end of the experiment. In
vehicle group, animals were injected with the same amount of the
saline to obtain the same stress conditions with the experiments
groups. Anxiety-like behaviour and spatial memory of Wistar rats
were measured at 15:00 h by a means of open field, elevated plus
maze and Morris water maze test, respectively.
Open field
Open-field test is taken place in an 80 × 80 cm arena with 40 cm
high walls. The open field has been the most widely used test in
animal psychology. In this test, an animal (usually a rodent) is
introduced into a plain and illuminated arena and its behavior is
commonly regarded as a fundamental index of general behavior. In
this experiment a video camera ( Gkb CC-28905S, Commat
LTD.ŞTĐ. Ankara/Turkey) was mounted above the arena, recording
behavior into the Ethovision videotracking system (Noldus
Ethovision, Version 6, Netherland; Commat LTD.ŞTĐ.
Ankara/Turkey) that provided a variety of behavioral measures
including distance, time in the edge, time in the center, frequency in
the edge, frequency in the center, mobility and velocity among the
different areas of the arena. All animals were then returned to the
breeding and exhibition colonies.
Elevated plus maze
The elevated plus maze consisted of the two open and two closed
10 cm wide arms in a plus-sign configuration of 55 cm off the floor.
The closed arms were enclosed by 41 cm tall black Plexiglas. All
arms were covered with contact paper to prevent the animals from
sliding off, and all surfaces were wiped with 70% alcohol between
animals. Each animal was released into one of the c losed arms and
allowed to move freely on the maze for a 5-min testing period that
was videotaped from above the maze. Animals that fell off the maze
into compartments below were placed back on the maze for the
remainder of the t esting period. An observer uninformed about
experimental conditions scored the videotapes with The Observer
Software (EthoVision XT) (Noldus Ethovision, Version 6,
Netherland; Commat LTD.ŞTĐ. Ankara/Turkey) for distance,
duration in the open arm, fr equency in the open arm, duration in the
closed arms, frequency in the closed arms, mobility and velocity.
Animals were considered to have entered an arm when all four
paws crossed onto the arm.
Morris water maze
For the s patial memory, the performances of the r ats in the Morris
water maze were evaluated. The experiments were carried out in a
circular, galvanized st eel maze (1.5 m in diameter and 60 cm in
depth), which was filled with 40 cm deep water kept at 28°C and
rendered opaque by the addition of a non-toxic, water soluble dye.
The maze was located in a large quiet test room, surrounded by
many visual cues external to the maze (for example the
experimenter, ceiling lights, r ack, pictures, etc.), which were visible
from within the pool and could be used by the rats for spatial
orientation. Locations of the cues were unchanged throughout the
period of testing. A video camera fixed to the ceiling over the center
of the maze was used for recording and monitoring movements of
the animals. There were the four equally divided quadrants in the
pool. In one of the quadrants, a platform (1.0 cm below water
surface, 10 cm in diameter) was submerged centrally and fixed in

1380 Afr. J. Pharm. Pharmacol.
position which was kept constant throughout the acquisition trials.
The rats performed the four trials per day for the f our consecutive
days (16 trials). In the swimming trials, each individual rat was
released gently into the water at a chosen quadrant except for the
one that contained the hidden platform for f acing an extra maze
cue. The rat swam and learned how to find the hidden platform.
After reaching, the rat was allowed t o stay on the platform for 15 s
and was then taken back into the cage. During the inter-trial
intervals, animals were kept in a dry home cage for 60 s.
The video camera recordings were obtained on the fifth day of
the experiment. The rat had to swim until it climbed onto the
platform submerged underneath the water. The escape platform
was kept in the s ame position relative to the distal cues. The time to
reach the platform, total distance travelled, time passed in the
correct quadrant, entrance fr equency to the correct quadrant and
immobility were measured as the indexes of the spatial memory. In
this experiment a video camera ( Gkb CC-28905S, Commat
LTD.ŞTĐ. Ankara/Turkey) was mounted above the arena, recording
behavior into the Ethovision videotracking system (Noldus
Ethovision, Version 6, Netherland; Commat LTD.ŞTĐ.
Ankara/Turkey) that provided a variety of behavioral measures
including distance, time in the edge, time in the center, frequency in
the edge, frequency in the center and immobility among the
different areas of the arena. All animals were then returned to the
breeding and exhibition colonies.
Statistical analysis of the data
Data were analyzed using SPSS (SPSS Statistical Software, SPSS
Inc., Los Angeles, CA, USA, Ver. 15.0). Data were analyzed by 2
(generation: adult and juvenile) X 3 (treatments: vehicle, low dose
and high dose of B. perennis) ANOVA analysis. Values were
considered st atistically significant at p ≤ 0.05. Data are presented
as means in the relevant paragraphs and illustrated as mean ±
SEM in Figures 1 to 3 after back transforming from ANOVA results.
The Tukey test was used to c ompare significant mean differences
in treatment condition.
RESULTS
Open field measurements
Total distance travelled (TDT)
The main effect of the treatment was significant on the
total distance travelled in the open field, (F (2, 30) =
47.89, p = 0.00001, ž2 = 0.76). Tukey test indicated that
the subjects in high doses of B. perennis (M = 1065.94)
travelled less distance than the subjects of those in
control condition (M = 2377.22) and low doses of B.
perennis (M = 2334.66), with the last two being not
significantly different from each other. The interaction
effect was significant between generation and treatment
F (2, 30) = 6.66, p = 0.004, ž2 = 0.31. This interaction
reflected the fact that the difference between the control
condition (M = 2680.64) and high dose condition (M =
1034.64) was greater in juveniles than the difference
between control condition (M = 2073.81) and high dose
condition (M = 1097.25) in adults.
Time spent at the edge of the open field (TSEO)
The main effect of the generation was significant on the
time spent at the edge of the open field, (F (1, 30) =
22.35, p = 0.0001, ž2 = 0.43). Juveniles (M = 4.91) spent
more time at the edge of the open field than adults (M =
4.75). The interaction effect between the generation and
the treatment was significant (F (2, 30) = 3.91, p = 0.03,
ž2 = 0.21). This interaction reflected the fact that adults in
control condition (M = 4.86) spent more time at the edge
of the open field than those in high dose condition (M =
4.66), but, this trend was not evident in juveniles.
Time spent at the center of the open field (TSCO)
The main effect of the generation was significant on the
time spent at the center of the open field, (F (1, 30) =
22.35, p = 0.0001, ž2 = 0.43). Adults (M = 0.25 min)
spent more time at the center of the open field than
juveniles (M = 0.09). The interaction effect between the
generation and the treatment was also significant (F (2,
30) = 3.91, p = 0.03, ž2 = 0.21). This interaction reflected
the fact that adults in control condition (M = 0.15) spent
less time at the center of the open field than those in high
dose condition (M = 0.34), but, this trend was not evident
in juveniles.
Entrance frequency to the edge of the open field
(EFEO)
The main effect of the treatment was significant on the
entrance frequency to the edge of the open field, (F (2,
30) = 8.64, p = 0.001, ž2 = 0.37). The Tukey test
indicated that the subjects in high dose condition (M =
2.50) were less frequently to enter the edge of the open
field than those in the control condition (M = 6.17) and
those in the low dose condition (M = 6.17), with the last
two being not significantly from each other. This finding
shows that high dose of B. perennis decrease the total
entrance frequency to the edge of the open field. This
also indicates that such dose decreases the anxiety level
of the rats.
Entrance frequency to the center of the open field
(EFCO)
The main effect of the treatment was significant on the
entrance frequency to the center of the open field, (F (2,
30) = 8.21, p = 0.001, ž2 = 0.35). The Tukey test
indicated that the subjects in high dose condition (M =
1.67) entered less frequently to the center of the open
field than those in the control condition (M = 5.17) and

Karakaş et al. 1381
Figure 1. (A) The total distance travelled ( TDT), (B) the time spent at the edge of the open field (TSEO), (C) the time spent at the center of the open field (TSCO), ( D) the entrance frequency to
the edge of the open field (EFEO), (E) the entrance frequency to the center of the open f ield (EFCO), (F) the mobility time and (MT), and (G) the velocity (VEL) are represented for the open
field. The black bar represents controls, the right striated bar represents the low dose of B. perennis injection and the checkered bar represents high dose of B. perennis injections for both
adults and juveniles. Data are presented as means (± S.E.M.). Different letters indicate the statistically different groups.
(A) (B) (C) (D)
(E) (F) (G)
TDT
T ot al d is ta n c e t ra v el le d (c m )
0
500
1000
1500
2000
2500
3000
3500
Control Low High Control Low High
a
b
c
b
a
c
Adult Juvenile
TSEO
T i m e s p en t a t th e e dg e o f th e o p e n f ie ld (m i n )
0
1
2
3
4
5
6
7
Control Low High Control Low High
aaab b b
Adult
Juvenile
TSCO
T im e s p en t a t t he c e n te r o f t he o p e n fie ld ( m in )
0,0
0,1
0,2
0,3
0,4
0,5
Control Low High Control Low High
a
b
c
dd
e
Adult Juvenile
EFEO
E nt r a nc e f re qu e n c y to t he e dg e o f o p en f ield
0
1
2
3
4
5
6
7
8
9
10
Control Low High Control Low High
a
b
c
a
d
e
Adult Juvenile
EFCO
E nt ra nc e fr eq u e n cy t o t h e c en te r o f o p en f ield
0
1
2
3
4
5
6
7
8
9
10
Control Low High Control Low High
a
b
c
a
d
e
Adult Juvenile
MOB
M o b ili ty (m in )
0,00
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0,08
Control Low High Control Low High
a
b
a
c
a
d
Adult
Juvenile
VEL
V e lo c i ty
0
100
200
300
400
500
600
700
Control Low High Control Low High
a
b
c
b
a
c
Adult Juvenile

1382 Afr. J. Pharm. Pharmacol.
Figure 2. (A) The total distance travelled (TDT), (B) the time spent in open arms (TSOA) (C), the entrance frequency t o the open arms (EFOA), (D) the time spent in closed arms (TSCA), (E)
the entrance fr equency to the closed arms (EFCA), (F) the mobility time (MT), (G) the rotation, and (H) the velocity (VEL) are represented for t he elevated plus maze. The black bar represents
controls, the right striated bar represents the low dose of B. perennis injection and the checkered bar represents high dose of B. perennis injections for both adults and juveniles. Data are
presented as means (± S.E.M.). Different letters indicate the statistically different groups.
(A) (B) (C) (D)
(E) (F) (G) (H)
TDT
T ot a l d is ta n c e tr a v e lle d ( cm )
0
200
400
600
800
1000
1200
1400
1600
1800
Control Low High Control Low High
aa
a a
bc
Adult Juvenile
TSOA
T im e s p en t in o p e n ar m ( m in )
0
1
2
3
4
5
Control Low High Control Low High
a
a
b
a
aa
Adult Juvenile
EFOA
E ntr an ce f req ue n cy to t he o p e n a rm
0
1
2
3
4
5
6
7
8
9
10
Control Low High Control Low High
a
a
a
a
a
a
Adult Juvenile
TSCA
T im e s p en t in c lo se d arm (m in )
0
1
2
3
4
5
6
Control Low High Control Low High
a
a
b
a
aa
Adult Juvenile
EFCA
E ntr an ce f re q u e n cy to th e c lo s e d a rm
0
1
2
3
4
5
6
7
8
9
10
Control Low High Control Low High
a
aa
a
a
a
Adult
Juvenile
MOB
M o b i li ty
0,0
0,5
1,0
1,5
2,0
2,5
3,0
Control Low High Control Low High
aa
b
a
cc
Adult Juvenile
ROT
R o t a t i o n
0
1
2
3
4
5
6
7
8
9
10
Control Low High Control Low High
a
b
c
bb
c
Adult
Juvenile
VEL
V el o ci ty ( c m / m in )
0
50
100
150
200
250
300
350
400
450
500
Control Low High Control Low High
aa
b
a
a
b
Adult
Juvenile

Karakaş et al. 1383
Figure 3. (A) The total distance travelled (TDT), (B) the time spent to find the platform (TSFP), (C) the time s pent in the
correct quadrant (TSCQ), (D) the entrance frequency to the correct quadrant (EFCQ), (E) the mobility time and (MT), and
(F) the velocity (VEL) are represented for the Morris water maze. The black bar represents controls, the right striated bar
represents the low dose of B. perennis injection and t he checkered bar represents high dose of B. perennis injections for
both adults and juveniles. Data are presented as means (± S.E.M.). Different letters indicate the statistically different
groups.
(A) (B)
TDT
Total distance travelled (cm)
0
200
400
600
800
1000
1200
Control Low High Control Low High
a
b
c
d
b
e
Adult Juvenile
TSFP
Time spent to find the platform (min)
0
10
20
30
40
50
60
70
80
Control Low High Control Low High
aa
b
c
a
d
Adult Juvenile
(C) (D)
TSCQ
Time spent in the correct quadrant (min)
0,00
0,05
0,10
0,15
0,20
0,25
Control Low High Control Low High
aa
b
c
a
d
Adult Juvenile
EFCQ
Entrance frequency to the correct quadrant
0
1
2
3
4
Control Low High Control Low High
a
b
c
c
bb
Adult Juvenile
(E) (F)
MOB
Mobility
0,00
0,02
0,04
0,06
0,08
0,10
Control Low High Control Low High
a
b
c
d
be
Adult Juvenile
VEL
Velocity
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Control Low High Control Low High
aa
bbb
c
Adult Juvenile

1384 Afr. J. Pharm. Pharmacol.
those in the low dose condition (M = 5.33), with the last
two being not significantly different from each other.
These finding shows that high dose of B. perennis
decreases the total entrance frequency to the edge of the
open field. This also indicates that such dose decreases
the anxiety level of the rats.
Mobility (MOB)
The main effect of the treatment was significant on the
mobility, F (2, 30) = 78.66, p = 0.0001, ž2 = 0.84. The
Tukey test indicated that the subjects in the control
condition (M = 0.03) were more mobile than those in low
(M = 0.007) and high doses of B. perennis (M = 0.00),
with the last two being significantly different from each
other. The main effect of the generation was also
significant on the mobility, F (1, 30) = 130.01, p =
0.00001, ž2 = 0.81. Juveniles (M = 0.03) were more
mobile than adults (M = 0.00). In addition, the interaction
effect between generation and treatment was significant,
(F (2, 30) = 79.87, p = .0001, ž2 = 0.84).
This interaction effect reflected the fact that the
difference between control condition (M = 0.06) and high
dose condition (M = 0.00) was greater in juveniles than
adults.
Velocity
The main effect of the treatment was significant on the
velocity in the open field, F (2, 30) = 48.36, p = .0001, ž2
= 0.76). The Tukey test indicated that the subject in the
high dose condition (M = 213.67) showed less velocity
than those in the control condition (M = 476.24) and
those in the low dose condition (M = 467.86), with the last
two being not significantly different from each other. The
interaction effect between generation and treatment was
also significant, (F (2, 30) = 6.67, p = 0.004, ž2 = 0.31).
This interaction reflected the fact that the difference
between the control condition (M = 536.91) and the high
dose condition (M = 207.58) was greater in juveniles than
the difference between control condition (M = 415.57)
and high dose condition (M = 219.77) in adults.
ELEVATED MAZE MEASUREMENTS
Total distance travelled (TDT)
The main effect of the generation was significant on the
total distance travelled, F (1, 30) = 18.36, p = 0.0001, ž2
= 0.38. Adults (M = 1396.96) travelled more distance than
juveniles (M = 941.05).
Time spent in open arms (TSOA)
The main effect of the treatment was significant on the
time spent in open arms, F (2, 30) = 4.24, p = 0.02, ž2 =
0.22. The Tukey test indicated that the subject in the high
dose condition (M = 2.35 min) spent more time in the
open arms than those in the low dose condition (M = 1.38
min) and those in the control condition (M = 1.59), with
the last two being not significantly different from each
other. The main effect of the generation was also
significant on the time spent in open arms, F (1, 30) =
8.84, p = 0.006, ž2 = 0.23. Adults (M = 2.22 min) spent
more time in the open arms than juveniles (M = 1.35
min). In addition the interaction effect between generation
and treatment was significant, F (2, 30) = 7.59, p = 0.002,
ž2 = 0.34. This interaction effect reflected the fact that the
difference between control condition (M = 1.35 min) and
high dose condition (M = 3.51 min) in time was greater in
adults than the difference between the control (M = 1.80
min) and the high dose condition (M = 1.20 min) in
juveniles.
Entrance frequency to open arms (EFOA)
No significant effects related to the treatment and
generation and as well as the interaction effect between
generation and treatment were found to be significant at
p >0.13.
Time spent in closed arms (TSCA)
The main effect of the treatment was significant on the
time spent in closed arms, F (2, 30) = 4.17, p = 0.03, ž2 =
0.22. The Tukey test indicated that the subject in the high
dose condition (M = 2.60 min) spent less time in the
closed arms than those in the low dose condition (M =
3.60 min) and those in the control condition (M = 3.38),
with the last two being not significantly different from
each other. The main effect of the generation was also
significant on the time spent in closed arms, F (1, 30) =
8.70, p = 0.006, ž2 = 0.23. Adults (M = 2.76 min) spent
less time in the closed arms than juveniles (M = 3.64
min). In addition, the interaction effect between
generation and treatment was significant F (2, 30) = 7.67,
p = 0.002, ž2 = 0.34. This interaction effect reflected the
fact that the difference between control condition (M =
3.62 min) and high dose condition (M = 1.44 min) in time
was greater in adults than the difference between the
control (M = 3.14 min) and the high dose condition (M =
3.78 min) in juveniles.
Entrance frequency to closed arms (EFCA)
No significant effects related to the treatment and

generation and as well as the interaction effect between
generation and treatment were found to be significant at
p >0.23.
Mobility (MOB)
The main effect of the generation was significant on the
mobility, F (1, 30) = 13.82, p = 0.001, ž2 = 0.32. Juveniles
(M = 1.42) were more mobile than adults (M = 0.65).
Velocity (VEL)
The main effect of the treatment was significant on the
velocity, F (2, 30) = 15.82, p = 0.0001, ž2 = 0.51. The
Tukey test indicated that the subjects in the high dose
condition (M = 270.47) were slower than those in the low
dose condition (M = 351.72) and those in the control
condition (M = 373.65), with the last two being not
significantly different from each other.
Rotation (ROT)
The main effect of the treatment was significant on the
rotation, F (2, 30) = 5.11, p = 0.01, ž2 = 0.25. The Tukey
test indicated that the subjects in the high dose condition
(M = 3.50) rotated less frequently than those in the low
dose condition (M = 4.83) and those in the control
condition (M = 6.17), with the last two being not
significantly different from each other.
Morris water maze measurements
Total distance travelled (TDT)
The interaction effect between generation and treatment
was significant on the total distance travelled, F (2, 28) =
3.70, p = .04, ž2 = 0.21. This interaction reflected the fact
that adults travelled more distance in high dose condition
(M = 908.33) than the control condition (M = 135.54),
whereas juveniles travelled less distance in high dose
condition (M = 516.64) than the control condition (M =
753.74).
Time spent to find the platform (TSFP)
The main effect of the treatment was significant on the
time to find the platform on the Morris water maze, F (2,
28) = 6.81, p = 0.04, ž2 = 0.33. The Tukey test indicated
that the subjects in the high dose condition (M = 44.83 s)
spent more time to find the platform than those in the low
dose condition (M = 10.37 s) and those in the control
Karakaş et al. 1385
condition (M = 17.46).
Time spent in the correct quadrant (TSCQ)
The main effect of the treatment was significant on the
time spent in the correct quadrant on the Morris Water
maze, F (2, 28) = 3.43, p = 0.05, ž2 = 0.20. The Tukey
test indicated that the subjects in the high dose condition
(M = 0.17) spent more time in the correct quadrant than
those in the low dose condition (M = 0.04 s) and those in
the control condition (M = 0.076), with the last two being
not significantly different from each other.
The entrance frequency to the correct quadrant
(EFCQ)
The interaction effect between generation and treatment
was significant on the entrance frequency to the correct
quadrant, F (2, 28) = 3.91, p = 0.03, ž2 = 0.22. This
interaction reflected the fact that adults entered more
frequently to correct quadrant in the high dose condition
(M = 3.00) than the control condition (M = 1.00), whereas
juveniles entered more frequently in the control condition
(M = 2.67) than the high dose condition (M = 1.67).
Mobility (MOB)
The interaction effect between generation and treatment
was significant on the mobility time, F (2, 28) = 8.30, p =
0.001, ž2 = 0.37. This interaction reflected the fact that
adults were more mobile in the high dose condition (M =
0.05) than the control condition (M = 0.002), whereas
juveniles were mobile in the control condition (M = 0.085)
than the high dose condition (M = 0.001).
Velocity (VEL)
The main effect of the treatment was significant on the
velocity on the Morris Water maze, F (2, 28) = 4.22, p =
0.03, ž2 = 0.23. The Tukey test indicated that the
subjects in the high dose condition (M = 1156.38) were
slower than those in the low dose condition (M = 1482.68
s) and those in the control condition (M = 1481.53), with
the last two being not significantly different from each
other. The interaction effect between generation and
treatment was also significant, F (2, 28) = 11.40, p =
0.0001, ž2 = 0.45. This interaction reflected the fact that
adults were faster in the high dose condition (M =
1619.20) than the control condition (M = 1387.17),
whereas juveniles were faster in the control condition (M
= 1575.88) than the high dose condition (M = 693.56).

1386 Afr. J. Pharm. Pharmacol.
SUMMARY AND CONCLUDING REMARKS
The results of the present study can be classified under
two main headings: Anxiety-like behaviour (in open field
apparatus and elevated plus maze) and spatial memory
performance (in Morris water maze).
Anxiety-like behaviours
Motor functions such as spontaneous activity is
measured by the open field. Open field test is also used
to measure the anxiety like behavior in rodents (Benabid
et al., 2008). The total distance traveled, the total number
of entries to the center and the edge of the open field, the
time spent in the center of the open field versus time
spent at the edge of the open field and the mobility are
frequently used parameters measured in open field test in
the literature (Pyter and Nelson, 2006). In this maze, if
the anxiety of the animal is high, the number of the
entries to the edge of the open field is increasing and the
total distance traveled is decreasing. The total number of
the entries into the center and the edges provides a built-
in control measure for general hyperactivity or sedation.
In the present study it was found that in the open field,
the subjects in the high doses of B. perennis
administration; a) travelled less distance in open field, b)
spent more time at the center of the open field, c) were
less frequently to enter both the edge and the center of
the open field, d) showed less mobility; and e) showed
less velocity than those in the control and low dose of B.
perennis administration. The elevated plus maze has
been one of popular or widely used test to measure the
anxiety like behaviours (Dawson and Tricklebank, 1995).
In this maze, if the anxiety of the animal is high, the
number of the entries to closed arms is increasing and
the total distance traveled is decreasing. The total
number of the entries into all arms provides a built-in
control measure for general hyperactivity or sedation.
Regarding elevated plus maze and open field tests, the
present study represent a difference in mobility, which
needs a further investigation. In the present study, it was
found that in the elevated plus maze, the subjects in the
high doses of B. perennis administration; a) spent more
time in the open arms, b) spent less time in the closed
arms, c) were less mobile d) were slower and e) rotated
less frequently than those in the control and low dose of
B. perennis administration.
Taken together, these results in both open field and
elevated plus maze can be explained from the two
perspectives: anxiolytic and anesthetic. With regard to
anxiolytic perspective, B. perennis may inhibit the
serotonergic activity via GABAergic system. B. perennis
may act like ‘benzodiazepines’ which are widely used in
reducing the anxiety-like behaviours. They make their
effect by binding their receptors which are found near the
GABA receptors and by making an allosteric effect. By
this way they increase the affinity of these GABA
receptors to benzodiazepines (Sinclair and Nutt, 2007).
The anxiolytic effect of saponins (Jalsrai et al., 2010),
essential oil (Bradley et al., 2007) and flavonoids (De
Almedia et al., 2009) in different plants have been
recently documented in the literature. B. perennis also
includes these major constituents. The anxiolytic
properties of the B. perennis may be attributed to these
active components. Future studies should examine the
exact mechanisms underlying the effects of B. perennis.
With regard to anesthetic perspective, it may have a
relevant effect via its anesthetic properties. In the present
study, some parameters such as less distance travelled,
less frequent entrance to the both edge and the center of
the open field, less mobility and less velocity after B.
perennis administration may suggest that it may also
show an anesthetic effect. Regarding the generation, it
was found that juveniles spent more time at the edge of
the open field than adults. Adults spent more time at the
center of the open field than juveniles. In the elevated
plus maze, adults travelled more distance than juveniles.
Adults spent more time in the open arms than juveniles.
Adults also spent less time in the closed arms than
juveniles. Taken together, these results suggest that,
juveniles were more anxious than adults in both open
field and elevated plus maze. There are the three
plausible explanations for these outcomes. First, adult
rats have more developed visual and olfactory sensations
than juvenile rats. Secondly, prepubertal rats may be
more anxious than postpubertal rats in searching for the
environmental cues. There is some evidence indicating
that anxiety impairs memory consolidation (Küçük et al.,
2008; Schwabe and W olf, 2010; Hawley et al., 2011).
Thirdly, postpubertal rats may have more experiences
than prepubertal rats in searching for environment. In the
present study, we also found that, juveniles were more
mobile than adults. This can be attributed to the fact that
energy metabolism is higher in prepubertal rats than
postpubertal rats. On the other hand, a high level of
anxiety in juvenile may suppress the appearance of this
metabolic energy and thereby decrease searching
behavior.
In addition, the findings of the present research showed
some interaction effects between treatment and
generation. Specifically, it was found that in the open
field, high dose of B. perennis (a) lead to travel less
distance in juveniles than adults, (b) eliminated the
difference between the juveniles and the adults in terms
of time spent at the edge of open field, (c) increased the
time spent at the center of the open field in adults, (d)
decreased the mobility in juveniles more than adults. This
means that the effect of B. perennis is more evident in
juveniles than adults in open field. It was also found in the
elevated plus maze, high dose of B. perennis, (a) leads to
an increase in time spent in open arms in adults, (b)
leads to a decrease in time spent in closed arms in
adults. This means that the effect of B. perennis is more

evident in adults than juveniles in elevated plus maze.
This outcome can be due to the fact that a high energy
metabolism in juveniles may sweep away the effects of B.
perennis on anxiety whereas a relatively low energy
metabolism in adults may be one of the best optimal
condition for the emergency of B. perennis effect. One
can see that, this effect was not consistent with what was
found in open field and elevated plus maze. This
difference may be due to the task difference between
open field and elevated plus maze. The open field
apparatus is testing the anxiety-like behaviour which may
be encountered in an open area. The elevated plus maze
is testing the height frightening. These aforementioned
findings also suggest that the dose-dependent effect was
stronger in adults than juveniles. Probably this indicates
that these measures relate to more complex constructs
than simple anxiety, since they are modulated differently
by the generation factor. Regarding elevated plus maze
and open field tests, the present study represent a
difference between adults and juveniles, which needs a
further investigation.
Spatial memory performance
Morris water maze is one of the most suitable tests for
the investigation of spatial memory (Hooge and De Deyn,
2001). In this test, the animal is trying to find a hidden
platform which is found below the water level by using
some cues around the experimental area. The time
passed to find the platform, the time spent in correct
quadrant, and the entrance frequencies to the correct and
the other quadrants are used as the parameters
representing the spatial memory and learning
performance of the animals. Since our focus was on
spatial memory, we used the Morris water maze test. In
the present study it was found that in the Morris water
maze, the subjects in the high doses of B. perennis
administration; a) increased the distance travelled in
adults, b) increased the time to find the platform, c)
increased the time spent in correct quadrant, d)
increased the the number of the entries to the correct
quadrant in adults, e) decrease the mobility in general but
increase the mobility in adults. Regarding the time spent
to find the platform and the distance travelled, we
assume that the high dose of B. perennis decreases the
learning performance. On the other hand, such dose
leads to an increase in time spent in the correct quadrant.
This outcome indicates the beneficial effect of B.
perennis on spatial memory. Taken together, these
outcomes can be explained via the anxiolytic and the
anesthetic properties of B. perennis. In other words, the
anxiolytic effects of B. perennis may produce facilitatory
effect on searching the platform in the correct quadrant.
Besides, high dose of B. perennis may also produce
anesthetic effect which in term may increase the time to
find the platform and the distance travelled indicating
decrement in the learning performance. The effects of
Karakaş et al. 1387
B. perennis on spatial learning performances in water
maze have also been explained from its active
ingredients. The amelioriation effect of flavonoids
(Spencer, 2009, 2010) and essential oil (Zhang et al.,
2007) on memory in different plants have been recently
documented in the literature. On the other hand, its active
ingredient namely saponins were reported to have
detrimental effect on learning in mice (Lee et al., 2010).
In the present study, the increase in distance travelled
and time spent to find the platform may be under the
effects of saponins. On the other hand, the increase in
the time spent in correct quadrant after the administration
of B. perennis may be determined by both flavonoids and
essential oil. In conclusion, B. perennis may produce
biphasic or bipolar effects (that is, both beneficial and
detrimental effects) on learning performance. Future
studies should examine the exact mechanisms
underlying the effects of B. perennis on learning and
memory.
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