




































_____________________________________________________________________________________________________ 
 
*Corresponding author: E-mail: nazmul_pharmacy@yahoo.com; 
(†)Authors contributed equally  
 
 

Asian Journal of Immunology 
 
3(1): 173-181, 2020; Article no.AJI.56428 
 

 
 

 

 

Investigation of Neuropharmacological Effects of 
Flemingia stricta (Roxb.) Leaves 

 
Md. Shahrear Biozid†1, Mohammad Nazmul Alam†1*, Md. Jainul Abeden†1,  

Md. Masudur Rahman1 and Md. Rafikul Islam1 
 

1
Department of Pharmacy, International Islamic University, Chittagong, Kumira,  

Chittagong, Bangladesh. 
 

Authors’ contributions  
 

This work was carried out in collaboration among all authors. Authors MSB, MMR, MNA and MRI 
proposed and designed the study. Authors MSB, MNA and MJA conducted all laboratory experiments. 

Authors MSB, MMR, MNA and MRI analyzed and interpreted experimental results as well as 
participated in manuscript preparations. All authors read and approved the final manuscript. 

 
Article Information 

 
Editor(s): 

(1) Dr. Darko Nozic, University of Belgrade, Serbia. 
Reviewers: 

(1) K. D. P. P. Gunathilake, Wayamba University, Sri Lanka. 
(2) Dra Poliana Guerino Marson, Federal University of Tocantins, Brazil. 

(3) Isabel C. Gomez-Betancur, University of Antioquia, Colombia. 
(4) John Ogedengbe, University of Abuja, Nigeria. 

Complete Peer review History: http://www.sdiarticle4.com/review-history/56428 

 
 
 
 

Received 02 March 2020  
Accepted 07 May 2020 

Published 18 May 2020 

 
 
ABSTRACT 
 

Background: Traditional preparation of the leaf of Flemingia stricta (Fabaceae) Roxb. , a medicinal 
plant of the Indian subcontinent, has been used for the treatment of different diseases as a herbal 
preparation. Our purpose was to analyze the neuropharmacological effects of different chemical 
extracts of Flemingia stricta Roxb. in mice. 
Methods: In present study, the anxiogenic activity of crude extracts of Flemingia stricta leaves was 
determined using standard animal behavioral models, such as hole cross and open field; Sedative 
and anxiolytic potential were evaluated by performing thiopental sodium induced sleeping times 
tests and elevated plus maze test respectively. 
Results: The crude extracts at the doses of 200 and 400mg/Kg exhibited a significant dose-
dependent suppression of movement of mice in both open field and hole cross test. In the anxiolytic 
and sedative study, extracts displayed an increased percentage of entry of mice into open arm at 
both doses which are 200 and 400mg/Kg. Plant extracts produced a significant increase in sleeping 

Original Research Article 



 
 
 
 

Biozid et al.; AJI, 3(1): 173-181, 2020; Article no.AJI.56428 
 

 

 
174 

 

duration and reduction of onset of sleep compared to control at the both doses of 200 and 400 
mg/Kg.  
Conclusion: This study clearly showed that the treated extracts have promising anxiolytic and 
sedative effect. Further studies on the prime constituent of this plant extract may provide lead 
compound in future. 
 

 
Keywords: Flemingia stricta; sedative; elevated plus maze; anxiolytic; locomotor activity; 

neuropharmacology. 
 

ABBREVIATIONS 
 

F. stricta: Flemingia stricta; EPM: Elevated plus 
maze; GABA: Gamma-aminobutyric acid; CNS: 
Central nervous System, FSM: F. stricta 
Methanol, FSH: F. stricta N-hexane, FSC:                 
F. stricta Chloroform, FSA: F. stricta Aqueous, 
FSE: F. stricta Ethanol. 
 

1. BACKGROUND 
 

From ancient times herbal drugs are called as 
green medicine for their safe and trustworthy 
health care paradigms. Furthermore, herbal 
drugs are cost effective than chemically 
manufactured drugs. Due to these benefits, 
public as well as industrial interests to the 
traditional herbal medicines are rising day by day 
[1].  People are leading a hectic life in this 21

st
 

century. Too much pressure in daily life 
increases anxiety and depression in people 
which results in psychiatric disorders around 
20% of the adult population [2-4]. This is an 
alarming situation for our future generation. 
Therefore, there is an increasing demand for the 
discovery of anxiolytic, sedative drugs.  
 

Anxiolytic and sedative drugs act on central 
nervous systems (CNS) which are extensively 
used as effective pharmacological agents to treat 
psychological disorders [5]. Benzodiazepines are 
among the most prescribed and effective anti-
anxiety drugs used worldwide [6]. Barbiturates 
and ethanol are also frequently used. Both 
barbiturates and benzodiazepines demonstrate 
their effect by binding with gamma aminobutyric 
acid receptor (GABAA receptor) [7]. 
 

Day to day use of benzodiazepines and 
barbiturates might cause psychological and 
physiological dependence which leads to drug 
tolerance. As a result, repeated uses of this type 
of drugs can decrease the effectiveness. Safety 
is the main issue of barbiturates as depressant 
because only ten times of their pharmacological 
dose might be lethal [8-12]. According to the 
statistical data, alcohol addiction in American 
society for men and women is 5% to 10% and 

3% to 5% respectively. Hence, a natural CNS 
depressant with minimum or no toxicity is 
therefore, essential [13]. 
 

Flemingia stricta (Fabaceae) Roxb. is distributed 
in Southeast Asia which includes Bangladesh, 
Bhutan, China, India, Indonesia, Laos, Myanmar, 
Philippines, Thailand and Vietnam [14,15]. It is 
an erect subshrub. It is found in hilly areas of 
Bangladesh and it has various traditional names 
given by local tribes [16]. Chakma healers 
traditionally use this plant for the treatment of 
polio. It has also several traditional benefits 
which consist of treat rheumatism followed by 
bone fracture, cough, asthma, goiter, urinary 
problems, snake bite, insect bite, leprosy, tumor 
and cancer, caries, hysteria, tuberculosis, 
insomnia and intestinal worms [17-19]. 
 

According to the literature review on this plant, it 
showed that the plant has been used as 
traditional medicine for the treatment of various 
diseases for many years. Furthermore, we also 
performed in-vitro antioxidant and phytochemical 
studies on this plant extract which paved our way 
to go for animal study [20]. Therefore, we 
undertook the study to assess the 
neuropharmacological potential of F. stricta 
leaves, by using animal models and studying the 
effect of the different chemical plant extracts on 
their exploratory behavior. 
 

2. METHODS 
 

2.1 Plant Collection and Identification 
 

Whole plants of F. stricta were collected from 
Bhatiary, Chittagong region, Bangladesh. The 
plants were identified by Dr. Shaikh Bokhtear 
Uddin, Taxonomist and Professor, Department of 
Botany, University of Chittagong, Chittagong, 
Bangladesh. 
 

2.2 Preparation and Extraction of Leaf 
Extract 

 

The collected leaves were thoroughly washed 
with distilled water and dried under the shade. 



 
 
 
 

Biozid et al.; AJI, 3(1): 173-181, 2020; Article no.AJI.56428 
 

 

 
175 

 

The dried sample was coarsely powdered (500 
g) and extracted with methanol for 3 days to 
allow the total extraction process. After that the 
plant extract was filtered with sterilized cotton 
filter and the filtrate was gathered in a beaker. 
The plant extracts then kept in a water bath at 60 
°C to evaporate the solvent from the solution. 
The container allowed to airtight for 72 h and 
filtrate thus obtained was concentrated by using 
a rotary evaporator. The extract was divided into 
two portions. One portion (2.5 g) was poured into 
glass vials to be tested as crude methanol 
extract, whereas the second portion (8 g) was 
dissolved in concentrated methanol and 
partitioned successively into four different 
extracts [21]. The fractions were then 
concentrated using a rotary evaporator.  
 

2.3 Animal 
 

Male Swiss albino mice, 3-4 weeks old, weighing 
between 20-25 g, were collected from the 
International Center for Diarrheal Disease and 
Research, Bangladesh. Animals were maintained 
under standard environmental conditions 
[temperature: (24±1)°C, relative humidity: 55%-
65% and 12 h light/12 h dark cycle] and had free 
access to feed and water ad libitum. Prior to 
experimentation, animals were familiarized in 
laboratory conditions for one week.  
 

2.4 Acute Toxicity Study 
 

Mice were divided into control and test groups (n 
= 5). The test groups received the extract per 
orally at the doses of 400, 600, 800 and 1000 
mg/Kg. Then the animals were kept in separate 
cages and were allowed to food and ad libitum. 
The animals were observed for possible 
behavioral changes, allergic reactions and 
mortality for the next 72 h [22]. 
 

2.5 Neuropharmacological Tests 
 

The study was done to find out if extracts had 
any effect on the central nervous system. Effect 
on the exploratory behavior of mice was 
evaluated by hole cross test and open field test. 
Elevated plus maze test was conducted for 
determination of anxiolytic activity whereas 
thiopental sodium induced sleeping time test was 
for sedative activity. 
 

2.6 Open Field Test 
 

The method was adopted as described by Gupta 
et al. [22]. In the open field test, the animals were 
divided into control, positive control and test 
groups containing 5 mice each. The test groups 

received extract of F. stricta at the doses of 200 
and 400 mg/Kg body weight orally whereas the 
control group received the vehicle (1% Tween 80 
in water) and standard group received Diazepam 
at the dose of 1 mg/kg (i.p). The floor of half 
square meter open field was divided into a series 
of squares each alternatively colored black and 
white. The apparatus had a 40cm height walls. 
The number of squares traveled by the animals 
was counted for 5 min at 0, 30, 60, 90, 120 min 
after oral administration of both doses of the 
extract. 
 

2.7 Hole Cross Test 
 

The apparatus was a cage of 30 cm × 20 cm × 
14 cm with a steel partition fixed in the middle, 
dividing the cage into two chambers. A hole of 
3.5 cm diameters was made at a height of 7.5 cm 
in the center of the cage. Animals were randomly 
divided into control, positive control and test 
groups containing 5 mice each. The test groups 
were treated with extract of F. stricta at the doses 
of 200 and 400 mg/Kg body weight orally 
whereas the positive control group with 
diazepam (1 mg/Kg) and control group with 
vehicle (1% Tween 80 in water). Number of 
passages of the animals through the hole from 
one chamber to the other was counted for 5 min 
at 0, 30, 60, 90 and 120 min after oral 
administration of the extract as well as diazepam 
and vehicle [23]. The apparatus was thoroughly 
cleaned after each trial. 
 

2.8 Thiopental Sodium Induced Sleeping 
Time Test 

 

For the experiment, the animals were randomly 
allocated to four groups, each with 5 mice. The 
test groups were given the leaf extract of F. 
stricta at doses of 200 and 400 mg/Kg body 
weight, while the positive control was treated with 
diazepam (1 mg/Kg) and control group with 
vehicle (1% Tween 80 in water). Thirty minutes 
later, thiopental sodium (40mg/Kg) was 
administered to each mouse to induce sleep. The 
animals were observed by placing them on 
separate chambers for the latent period (time 
between thiopental administrations to loss of 
righting reflex) and duration of sleep i.e. time 
between the loss and recovery of righting reflex. 
The onset of sleep and total sleeping time was 
recorded for control, positive control and test 
groups [24]. 
 

2.9 Elevated Plus Maze Test 
 

The method initially suggested by Handley and 
Mithani was employed with minor modifications 



 
 
 
 

Biozid et al.; AJI, 3(1): 173-181, 2020; Article no.AJI.56428 
 

 

 
176 

 

C
ontr

ol

D
ia

ze
pam

FSM
 2

00

FSM
 4

00

FSH
 2

00

FSH
 4

00

FSC
 2

00

FSC
 4

00

FSA
 2

00

FSA
 4

00

FSE
 2

00

FSE
 4

00

0

50

100

150

0 min

30 min

60 min

90 min

120 min

N
u

m
b

e
r 

o
f 

s
q

u
a
re

s
 t

ra
v
e
le

d

[25]. The apparatus consists of two open arms (5 
× 10) cm and two closed arms (5 × 10 × l5) cm 
radiating from a platform (5 × 5) cm to form a 
plus-sign figure. The apparatus was situated 
40cm above the floor. The open arms edges 
were 0.5 cm in height to keep the mice from 
falling and the closed-arms edges were 15 cm in 
height. Sixty minutes after administration of the 
test drugs, each animal was individually placed in 
the center of the EPM and was allowed 5 min for 
free exploration. Next, the number of open and 
enclosed arm entries, and time spent on open 
arms was manually registered [26]. Entry into an 
arm was defined as the point when the animal 
placed all four paws onto the arm. The 
percentage of open arm entries (100 × open/total 
entries) and the percentage of time spent in the 
open arms (100 × open/(open + enclosed)) were 
calculated for each animal. Observations made 
from an adjacent corner produced significant (p< 
0.05, p < 0.01) decreases of locomotion from its 
initial value during the period of the experiment. 
Maximum suppression of locomotor activity was 
displayed at the dose of 400 mg/Kg body weight, 
which was comparable to the reference drug 
diazepam. 
 

2.10 Statistical Analysis 
 

Statistical comparisons were performed using 
one-way ANOVA followed by post-hoc Dunnett’s 
test with the SPSS program (SPSS 20.0, USA). 
The data were calculated as the mean ± 

standard error of mean (S.E.M.) and data of this 
graphs are deposited to the authors. The values 
obtained from plant extracts were compared with 
the control group and were considered 
statistically significant when P<0.05. Graphs 
were represented by Graph Pad Prism software. 

 
3. RESULTS 
 
3.1 Neuropharmacological Tests 
 
3.1.1 Open field test 

 
The open field test of F. stricta treated groups 
represented significant and dose dependent 
decrease of number of movements of mice from 
0 minutes to 120 minutes. According to the 
study, 400 mg/Kg body weight treated groups 
showed more significant reduction of movements 
than 200 mg/Kg body weight. According to the 
chemical fractions, from 30 to 120 minutes, all 
the chemical extracts decreased the number of 
squares traveled by mice significantly at both 200 
mg/Kg and 400 mg/Kg treated groups, but 
aqueous and n-hexane extract at 200 mg/Kg 
showed the lowest effect. (Fig. 1). 
 
3.1.2 Hole cross test 

 
Hole cross test of F. stricta treated groups 
showed a decrease of movement of mice. Data 
represented in Fig. 2 suggests that the number of 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Fig. 1. Effect of extracts of F. stricta on exploratory behavior on mice (Open field test) 
(n=5); (P<0.05) Dunnett’s test as compared to control 

 



 
 
 
 

Biozid et al.; AJI, 3(1): 173-181, 2020; Article no.AJI.56428 
 

 

 
177 

 

C
ontr

ol

D
ia

ze
pam

FSM
 2

00

FSM
 4

00

FSH
 2

00

FSH
 4

00

FSC
 2

00

FSC
 4

00

FSA
 2

00

FSA
 4

00

FSE
 2

00

FSE
 4

00

0

10

20

30

0 min

30 min

60 min

90 min

120 min

N
u

m
b

e
r 

o
f 

h
o

le
s
 c

ro
s
s
e
d

C
ontr

ol

D
ia

ze
pam

FSM
 2

00

FSM
 4

00

FSH
 2

00

FSH
 4

00

FSC
 2

00

FSC
 4

00

FSA
 2

00

FSA
 4

00

FSE
 2

00

FSE
 4

00

0

50

100

150

200

Onset of sleep

Duration of sleep

T
im

e
 (

m
in

)

holes crossed from one chamber to another 
chamber by mice was decreased significantly for 
all chemical extracts of F. stricta leaves 
compared to control at doses of both 200 and 
400 mg/Kg. Maximum suppression of           
locomotor activity was found at doses of 400 
mg/Kg (Fig. 2). Among all the fractions, 
chloroform (FSC), methanol (FSM) and ethanol 
(FSE) extract showed comparatively better             
dose dependent reduction of movement of mice 
at both doses than other extracts after 60 
minutes. 
 

3.1.3 Thiopental sodium induced sleeping 
time test 

 

In the thiopental induced hypnosis test, both 200 
and 400 mg/Kg showed a significant reduction in 
the time of onset of sleep. Moreover, 200 and 
400 mg/Kg treated groups also increase the 
duration of thiopental sodium induced sleeping 
time in mice in the case of methanolic (FSM) and 
ethanolic (FSE) plant extract. Though chloroform 
(FSC) and n-hexane (FSH) demonstrated 
promising effect (Fig. 3). 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Fig. 2. Effect of extracts of F. stricta on exploratory behavior on mice. (Hole cross test) (n=5); 

(P<0.05) Dunnett’s test as compared to control 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Fig. 3. Effect of extracts of F. stricta on thiopental sodium induced sleeping time 
(n=5); (P<0.05) Dunnett’s test as compared to control 

 



 
 
 
 

Biozid et al.; AJI, 3(1): 173-181, 2020; Article no.AJI.56428 
 

 

 
178 

 

C
ontr

ol

D
ia

ze
pam

FSM
 2

00

FSM
 4

00

FSH
 2

00

FSH
 4

00

FSC
 2

00

FSC
 4

00

FSA
 2

00

FSA
 4

00

FSE
 2

00

FSE
 4

00

0

20

40

60

80

% of Entry into Open Arm

% of Time Spent in Open Arm

%
 o

f 
E

n
tr

y
 i
n

to
 o

p
e
n

 a
rm

,

%
 o

f 
T

im
e
 s

p
e
n

t 
in

 o
p

e
n

 a
rm

3.1.4 Elevated plus maze test 
 
This test is used to detect the anxiolytic activity. 
The extracts of F. stricta at the dose of 200 
mg/Kg and 400 mg/Kg significantly increased the 
percentage of entries of mice into the open arms, 
and the percentage of time spent in the open 
arms which are shown in Fig. 4. But, 400 mg/Kg 
showed more significant effect than 200 mg/Kg 
dose. Ethanol (FSE) extract showed 
comparatively more promising results than other 
extracts. But other extracts also showed 
promising effects (Fig. 4). 

 
4. DISCUSSION 
 

The result of open field and hole cross test 
demonstrated that this plant extracts reduced the 
frequency of movements of mice. Locomotor 
activity was dose dependently decreased from 
30 minutes to 120 minutes which proved its 
sedative activity. Hence, it can be stated that 
both doses of chemical extracts of F. stricta 
leaves decreased the frequency and the 
amplitude of movements. The above result also 
showed that crude extracts of F. stricta plant had 
strong sedative and hypnotic action that mainly 
mediated in the CNS by the GABAA receptor 
complex. Thus, it can be said that the plant has 
promising sedative effects. 

Thiopental sodium induced sleeping time test 
represented the depressant activity of central 
nervous system by decreasing the onset time of 
sleep as well as increasing the duration of sleep 
[27-29]. Chemical constituents of plant extracts 
might be the reason for its benzodiazepine like 
sedative activity. Previous literature review on 
this plant showed that this plant has several 
traditional uses. Furthermore, it also contains 
several chemical constituents such as tannins, 
glycosides, alkaloids, saponins, phytosterols, 
flavonoids. Elevated plus maze test is 
extensively used to determine the anxiolytic 
activity of drugs which is related to binding of 
drugs to GABAA receptor complex [30-32]. In 
this experiment, we observed that F. stricta 
increased the open arm entries along with time 
spent in open arms which precisely showed the 
anxiolytic effect of this plant extract in mice. 
 
Generally, anxiolytic and hypnotic drugs such as 
benzodiazepines and their metabolites are highly 
lipid soluble which helps these drugs to reach the 
central nervous system readily. But this 
beneficial effect might turn into nightmare due to 
its high lipid solubility which can increase the risk 
of amnesia. Amnesia is a disease related with 
memory deficit which is caused by not only brain 
damage or disease but also by the use of  
several anxiolytic and hypnotic drugs [33-35].

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Fig. 4. Effect of extracts of F. stricta on EPM test during 5 min test session 

(n=5); (P<0.05) Dunnett’s test as compared to control 
 



 
 
 
 

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179 

 

 
 

Fig. 5. Mechanism action of anxiolytic and hypnotic drugs 
 
Therefore, optimization of lipid solubility is 
essential to decrease this type of side effect. 
Thus, excessive lipid soluble property of 
anxiolytic drugs might be harmful for consume. 
Hence, we need a compound which can make a 
great balance between lipophilicity and 
hydrophilicity means not excessive lipophilic or 
hydrophilic. Polarity of water is highest among 
five chemicals. Polarity of methanol and ethanol 
is less comparably to water. On the contrary, n-
hexane and chloroform are more lipids soluble. 
According to our study, it could be said that, this 
plant might possess molecules which results in 
significant effects of F. stricta extracts made by 
different chemicals. Here, F. stricta extracts 
made by chemicals such as methanol, ethanol 
and chloroform extracts showed better effects 
than n-hexane and .aqueous extracts. However, 
further studies              are needed to investigate 
the underlying mechanisms of this plant extract. 
There is a great need of the discovery of new 
anxiolytic drugs with fewer side effects. Hence, 
our experiment might provide a good drug 
candidate for future. 
 

5. CONCLUSION 
 

All of the results were dose-dependent and 
statistically significant. According to the results of 

this experiment, it can be conclude that the crude 
extracts of F. stricta possess significant neuro-
pharmacological activity. Therefore, we can 
suggest that the extract may fulfill the therapeutic 
need for the treatment of anxiety and related 
neuropsychiatric disorders. Nevertheless, further 
exploration is required to discover the exact 
mechanisms that are responsible for this 
anxiolytic activity of this plant extract. 
 

CONSENT 
 

It is not applicable. 
 

ETHICAL APPROVAL 
 

The set of rules followed for animal experiment 
were approved by the institutional animal      
ethics committee, Department of Pharmacy, 
International Islamic University Chittagong, 
Bangladesh according to governmental 
guidelines. 
 

ACKNOWLEDGEMENT 
 

The authors wish to thank Botanist Dr. Shaikh 
Bokhtear Uddin, Professor, Department of 
Botany, University of Chittagong, Bangladesh, 
who helped to identify the plant. We would like to 
express our gratitude to the authority of the 



 
 
 
 

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180 

 

International Centre for Diarrheal Disease and 
Research, Bangladesh (ICDDRB) for providing 
the experimental mice. The authors are grateful 
to the Department of Pharmacy, International 
Islamic University Chittagong, Chittagong, 
Bangladesh, for providing research facilities. The 
authors are also grateful to their parents. 
 

COMPETING INTERESTS 
 
Authors have declared that no competing 
interests exist. 
 

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