Hindawi Publishing Corporation

Evidence-Based Complementary and Alternative Medicine

Volume 2016, Article ID 8541017, 8 pages
http://dx.doi.org/10.1155/2016/8541017

Research Article
Sedative and Anxiolytic-Like Actions of Ethanol Extract of
Leaves of Glinus oppositifolius (Linn.) Aug. DC.

Md. Moniruzzaman, Partha Sharoti Bhattacharjee, Moushumi Rahman Pretty,

and Md. Sarwar Hossain
Department of Pharmacy, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217, Bangladesh

Correspondence should be addressed to Md. Moniruzzaman; [email protected]

Received 8 March 2016; Revised 17 May 2016; Accepted 26 May 2016

Academic Editor: Ki-Wan Oh

Copyright © 2016 Md. Moniruzzaman et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Glinus oppositifolius is a small herb, widely used in the traditional medicine of Bangladesh in treatment of a variety of diseases
and disorders such as insomnia, pain, inflammation, jaundice, and fever. The present study evaluated the sedative and anxiolytic
potentials of the ethanol extract of leaves of G. oppositifolius (EEGO) in different behavioral models in mice. The sedative activity
of EEGO was investigated using hole cross, open field, rotarod, and thiopental sodium- (TS-) induced sleeping time determination
tests, where the elevated plus maze (EPM) and light-dark box (LDB) exploration tests were employed to justify the anxiolytic
potentials in mice at the doses of 50, 100, and 200 mg/kg. The results demonstrated that EEGO significantly inhibited the exploratory
behavior of the animals both in hole cross and in open field tests in a dose-dependent manner. It also decreased motor coordination
and modified TS-mediated hypnosis in mice. In addition, EEGO showed anxiolytic potential by increasing the number and time
of entries in the open arm of EPM, which is further strengthened by increase in total time spent in the light part of LDB. Therefore,
this study suggests the sedative and anxiolytic properties of the leaves of G. oppositifolius and supports the traditional use of this
plant in treatment of different psychiatric disorders including insomnia.

1. Introduction in treatment of a wide range of diseases and disorders

including pain, inflammation, jaundice, diarrhea, boils, and
Anxiety and insomnia are the most prevalent physiological malaria [4]. This herb is also useful for its diuretic, CNS
and psychological states characterized by cognitive, emo- depressant, anthelmintic, and antiviral properties in the folk
tional, and behavioral components affecting one-eighth of medicine of this country [5]. In recent years, several studies
the world population [1]. Nowadays, several sedative drugs have been conducted to identify the phytochemical constitu-
(e.g., diazepam, which is chosen as reference standard in this ents of G. oppositifolius and a number of phytochemicals
study) come with sanative potential to manage sleeping disor- including L-(−)-(N-trans-cinnamoyl)-arginine, kaempferol
ders, which also could reduce anxiety [2, 3]. However, in addi- 3-O-galactopyranoside, isorhamnetin 3-O-𝛽-D-xylopyrano-
tion to their beneficial properties, these currently available syl-(1→2)-𝛽-D-galactopyranoside, and L-phenylalanine have
sedative and anxiolytic therapies possess serious adverse and been isolated from different parts of this plant [6]. Further
side effects. Therefore, newer, more efficacious and better- studies have revealed that G. oppositifolius also contains
tolerated treatments including alternative/complementary spergulagenin derivatives [4], oppositifolone [7], and a
medicines would be a welcome addition in the therapeutic bioactive pectic polysaccharide having immunomodulatory
repertoire of insomnia and anxiety management. properties [8]. According to the aforementioned medicinal
Glinus oppositifolius (family: Molluginaceae) is a very properties and the presence of different bioactive phytochem-
common herb in Bangladesh, locally known as “gima shak.” icals, researchers tried to validate its traditional uses against
This plant is extensively used by the local people as a different diseases and disorders. Their findings revealed that
vegetable and the traditional healers as an essential ingredient G. oppositifolius is effective as an antioxidant, hypoglycemic,
2 Evidence-Based Complementary and Alternative Medicine

anti-inflammatory [4], antidiarrheal, and anthelmintic [5] 2.5. Acute Toxicity Test. Mice were divided into desired
agent. However, till now there is no scientific report revealing groups each containing 5–7 animals. EEGO was adminis-
its actions on the central nervous system. This influenced tered to the animals orally at the doses of 500, 1000, and
us to design and conduct the present study to evaluate the 2000 mg/kg. The mice were then allowed to take food and
impact of ethanol extract of leaves of G. oppositifolius in water ad libitum and observed for the next 72 h to check any
different behavioral models in mice. allergic symptoms and mortality induced by EEGO [10].

2. Materials and Methods 2.6. Sedative Activity Analysis

2.1. Plant Collection and Extraction. Glinus oppositifolius 2.6.1. Hole Cross Test. A cage having a size of 30 × 20 × 14 cm
leaves were collected from Khilgaon, Dhaka, during the with a fixed partition in the middle having a hole of 3 cm
spring of 2013. The collected samples were then identified by diameter was used in this experiment [11]. Mice were treated
Mr. Sardar Nasir Uddin, Senior Scientific Officer, Bangladesh with either vehicle or drug or EEGO and allowed to cross the
National Herbarium (Mirpur, Dhaka, Bangladesh). A hole from one chamber to another. The animals were then
voucher specimen has been deposited with a number DACB: observed for 3 min and the number of passages was recorded
38355 for further references. The powdered dried leaves before and at 30, 60, 90, and 120 min following the treatments.
(250 g) were macerated with 450 mL of ethanol (100%;
Merck, Bremen, Germany) with occasional stirring at room 2.6.2. Open Field Test. This test is a widely used model for the
temperature for three days. Then the filtrate was collected evaluation of emotional behavior of the animals, especially
and completely dried using a rotary evaporator. Finally the rodents. The method was carried out as described by
11.83 g extract (yield 4.73%) was obtained which was further Gupta et al. (1971) [12]. The open field apparatus consisted of
used in the entire set of studies. a wooden field of half square meter with a series of squares
alternatively painted in black and white. It had a wall of 50 cm
2.2. Animals. Adult Swiss albino mice (20–25 g) were pur- height and was placed in a dimly lit room. The animals were
chased from the Animal Resources Branch of the Interna- placed in the middle of the open field to explore freely and the
tional Center for Diarrheal Disease Research, Bangladesh number of squares visited by them was counted for 3 minutes
(icddr,b), and housed in standard laboratory conditions as pretreatment reading. Immediately after taking the reading
(relative humidity 55–60%; room temperature 25 ± 2∘ C; the animals were treated with vehicle, extract, or diazepam
12 h light/dark cycle) with standard diet (icddr,b formulated) and observed repeatedly at 30, 60, 90, and 120 min after the
and water ad libitum. Animals were acclimatized with the treatments.
experimental environment for a period of 14 days prior to
the experiments and then treated according to the “Ethical 2.6.3. Test for Motor Coordination (Rotarod Test). The rotarod
Principles and Guidelines for Scientific Experiments on test was performed according to the procedure described by
Animals” (1995) drafted by the Swiss Academy of Medical Dunham and Miya (1957) [13]. This test is effective for the
Sciences and the Swiss Academy of Sciences. All experi- investigation of motor impairment due to pharmacological
mental protocols employed in this study were approved by agents like muscle relaxants or CNS depressants. The appara-
the Institutional Ethics Committee of Stamford University tus consisted of a horizontal nonslippery plastic rod, rotating
Bangladesh (SUB/IAEC/14.08). at 20 rpm. The animals which can remain in the rotating
rod for more than 180 sec were selected for this study. After
2.3. Drugs and Treatments. Mice were divided into five desired treatments, each mouse was placed on the rod and
groups containing 5–7 animals each for control, standard, and the falling time of each mouse within 180 sec was recorded as
test samples, for every experiment. Standard drug diazepam an indication of muscle relaxation.
(1 mg/kg; i.p.) (Square Pharmaceuticals Ltd., Dhaka,
Bangladesh), EEGO (50, 100, and 200 mg/kg; p.o.), or vehicle 2.6.4. Thiopental Sodium-Induced Sleeping Time Determi-
(DMSO; 0.1 mL/mouse; p.o.; Merck), was administered nation. Thiopental sodium-induced sleeping time test was
to the animals, immediately after taking the pretreatment performed according to the previously described method
reading in hole cross and open field tests. For the rest of the [14]. Following desired sample or drug administration, the
models, diazepam was administered at 15 min and EEGO or animals were observed for the latent period (time to lose
vehicle at 30 min before the experiments. In sleeping time their righting reflex, immediately after thiopental sodium
determination test, the sleep inducer thiopental sodium injection) and the duration of sleep (time between the loss
(20 mg/kg) (Square) was administered 15 min posttreatment and recovery of reflex) induced by thiopental sodium.
with diazepam and 30 min of vehicle or EEGO.
2.7. Investigations for the Anxiolytic Potential
2.4. Phytochemical Screening. The crude extract of G. opposi-
tifolius leaves was qualitatively analyzed to detect the presence 2.7.1. Elevated Plus Maze Test. The elevated plus maze (EPM)
of alkaloids, glycosides, carbohydrates, saponins, flavonoids, test is a widely used model to investigate anxiolytic effects.
tannins, glucosides, and reducing sugars following standard The apparatus consists of two open arms (15 × 5 cm2 ) and
procedures [9]. two closed arms (15 × 5 × 5 cm3 ), extending from a central
Evidence-Based Complementary and Alternative Medicine 3

platform (5 × 5 cm2 ) and raised 50 cm above floor level. 25

Animals were randomly divided into each group and treated
with EEGO, vehicle, or diazepam. After the desired time, 20
each animal was placed at the center of the plus maze facing

Number of movements
its head to the closed arms and allowed free exploration for
3 min. Then the number of entries and total time spent in 15 ∗
open arms were recorded within the indicated time [15, 16]. ∗
10 ∗
∗
2.7.2. Light-Dark Box (LDB) Exploration Test. The apparatus ∗
∗
is an open-topped rectangular box (46 × 27 × 30 cm3 ), ∗
∗
divided into a small (18 × 27 cm2 ) and a large (27 × 27 cm2 ) 5
∗
∗ ∗
∗ ∗
compartment with a fixed partition containing a small hole ∗
of 3 cm diameter in the middle. The small compartment was ∗ ∗
0
closed with a lid, painted black, and illuminated with a dim Pretreatment 30 min 60 min 90 min 120 min
light. On the other hand, a 60 W electric light was used in
Control EEGO 100 mg/kg
the large compartment (painted white) to make it brightly
Diazepam EEGO 200 mg/kg
illuminated. After 30 min of successful oral gavage of vehicle, EEGO 50 mg/kg
EEGO, or diazepam, mice were placed in the middle of the
open compartment of LDB. The animals were then observed Figure 1: Effect of EEGO on hole cross test in mice. Before and after
for 3 min and the time spent in the lighted compartment and treatments with diazepam, vehicle, or EEGO the number of holes
the total number of transitions in between the compartments crossed in the hole cross box was recorded at different time points.
were registered [17]. Data were presented as Mean ± SEM (𝑛 = 5–7). ∗ 𝑝 < 0.01 compared
to control.

2.8. Statistical Analysis. The results are presented as Mean ±

SEM. The statistical analysis was performed using two-way 140
analysis of variance (ANOVA) followed by Bonferroni’s post 120
hoc test for hole cross and open field tests, where one-way
Number of movements

ANOVA followed by Dunnett’s post hoc test was employed 100

for other experiments performed in this study. All statistical
80
analyses were performed using SPSS software. Besides, the
ED50 values were calculated using GraphPad Prism and the 60 ∗
figures were drawn using SigmaPlot software. ∗ ∗
40 ∗
∗ ∗ ∗
∗
3. Results and Discussion 20
∗ ∗
∗
∗ ∗
The present study evaluated the effect of an ethanol extract 0 ∗
of G. oppositifolius on CNS using several behavioral mod-
els in mice. Our results demonstrated that, following oral Pretreatment 30 min 60 min 90 min 120 min
administration, EEGO was able to promote CNS depressant
Control EEGO 100 mg/kg
and anxiolytic effects. Moreover, EEGO at the doses of 500–
Diazepam EEGO 200 mg/kg
2000 mg/kg did not produce any allergic manifestation or EEGO 50 mg/kg
mortality of the animals during 72 h of observation period,
which is in line with the wide dietary and therapeutic uses of Figure 2: Effect of EEGO on open field test in mice. Before and after
this plant. This nontoxic profile of the extract also made us treatments with diazepam, vehicle, or EEGO the number of squares
capable of choosing the experimental doses in this study. crossed in the open field box was recorded at different time points.
The hole cross and open field tests are the most common Data were presented as Mean ± SEM (𝑛 = 5–7). ∗ 𝑝 < 0.01 compared
experimental models used to investigate the exploratory to control.
behavior of the animals. It is well established that several
drugs like benzodiazepines suppress curiosity of the animals
about a new environment resulting in a decrease in their weakness [19], decreased ambulatory activity, and sedation
locomotor activity [11, 18]. Likewise, our results demonstrated which negatively affect the rotarod performance of the
that EEGO significantly (𝑝 < 0.01) reduced locomotion of animals [20, 21]. As depicted in Figure 3, EEGO decreased the
the animals both in hole cross and in open field tests. The sup- falling latency of the animals from rotarod, significantly (𝑝 <
pressive effect was observed from 30 min and continued up to 0.01) with the doses of 100 and 200 mg/kg. The highest motor
120 min of EEGO administration (Figures 1 and 2) accounting coordination impairment and ED50 were calculated as 58%
81 and 92% of locomotor inhibition at the highest time point with 200 mg/kg and 89.66 mg/kg, respectively. In parallel,
of these tests, respectively. Besides, it is well established that diazepam at 1 mg/kg dose also produced similar pattern of
the CNS depressant drugs like benzodiazepines cause muscle effects observed with EEGO in all experiments. From these
4 Evidence-Based Complementary and Alternative Medicine

160 12

140
10
Rotarod performances (sec)

120

Sleeping latency (min)

8
100 ∗ ∗

80 6 ∗ ∗

60 ∗
4
40 ∗
2
20

0 0
Control

Diazepam

EEGO 50 mg/kg

EEGO 100 mg/kg

EEGO 200 mg/kg

Control

Diazepam

EEGO 50 mg/kg

EEGO 100 mg/kg

EEGO 200 mg/kg

Figure 3: Effect of EEGO on motor coordination of mice. Thirty min (a)
after the treatment with EEGO or vehicle and 15 min after diazepam, 200
rotarod performances by the animals were observed for 180 sec. Data ∗
were presented as Mean ± SEM (𝑛 = 5–7). ∗ 𝑝 < 0.01 compared to
∗
control. 150 ∗
Sleeping time (min)

observations, it is conceivable that, like diazepam, EEGO may 100

have the potential to act on CNS which was reflected by
its locomotor inhibitory activity as well as impaired motor
coordination effects in the animals. 50
Further evidence of the central sedative activity of EEGO
is provided by TS-induced sleep enhancing ability of the
extract. Substantial scientific reports suggested that the CNS
0
depressant barbiturates like TS bind to the barbiturate bind-
Control

Diazepam

EEGO 50 mg/kg

EEGO 100 mg/kg

EEGO 200 mg/kg

ing site of the GABAA receptor, which potentiates GABA-
mediated hyperpolarization of the neurons [22]. In our study,
the acute oral administration of EEGO significantly (𝑝 <
0.01) modulated the sleeping behavior of the animals induced
by TS. We found that EEGO decreased TS-induced onset
of sleeping and increased the sleeping duration in a dose- (b)
dependent manner (Figures 4(a) and 4(b)) with the ED50
Figure 4: Effect of EEGO on TS-induced hypnosis in mice. Thirty
value of 69.73 mg/kg. Therefore, these results strengthened
min after the treatment with EEGO or vehicle and 15 min after
the sedative and muscle relaxation potentials of the extract diazepam, TS was administered intraperitoneally. Then the latency
observed in hole cross, open field, and rotarod tests. to sleep (a) and total sleeping duration (b) induced by TS were
The elevated plus maze is a widely used behavioral observed. Data were presented as Mean ± SEM (𝑛 = 5–7). ∗ 𝑝 < 0.01
model in rodents and has been validated to investigate the compared to control.
anxiolytic potential of different pharmacological agents [23].
The open arm activities of the animals in EPM reflect a
conflict between the animal’s innate behavior to keep itself
in a protected area (e.g., closed arms) and motivation to addition, the effect of EEGO was also evaluated using LDB, a
explore in a novel environment, where the anxiolytic agents popular screening tool in research of anxiolytic or anxiogenic
induce the exploratory activities of the rodents in the open agents [25]. The present study demonstrated that the oral
arm [15, 24]. Our results demonstrated that EEGO caused administration of EEGO at 100 and 200 mg/kg doses could
a marked increase in the number of entries as well as the significantly (𝑝 < 0.01) increase the time the animals spent
time the animals spent in the open arms of EPM (Figures in the lighted area (ED50 : 82.46 mg/kg) without altering the
5(a) and 5(b)). However, the significant (𝑝 < 0.01) effect total number of transitions in between the compartments.
was observed with 100 and 200 mg/kg doses of EEGO and As expected, diazepam also exhibited similar patterns of
the ED50 values were calculated as 88.65 and 92.87 mg/kg effects of EEGO in these models (Figures 6(a) and 6(b)).
for the number of entries and time spent, respectively. In Substantial scientific reports demonstrated that, with an
Evidence-Based Complementary and Alternative Medicine 5

20 140
∗
∗
120 ∗
Number of entries in open arm

Time spent in open arm (sec)

15 ∗ ∗
100
∗
80
10
60

40
5
20

0 0
Control

Diazepam

EEGO 50 mg/kg

EEGO 100 mg/kg

EEGO 200 mg/kg

Control

Diazepam

EEGO 50 mg/kg

EEGO 100 mg/kg

EEGO 200 mg/kg

(a) (b)

Figure 5: Effect of EEGO on elevated plus maze (EPM) test in mice. Thirty min after the treatment with EEGO or vehicle and 15 min after
diazepam, animals were observed for their number of entries (a) and total time spent (b) in the open arms of EPM. Data were presented as
Mean ± SEM (𝑛 = 5–7). ∗ 𝑝 < 0.01 compared to control.

160 16
Time spent in lighted compartment (sec)

140 ∗ 14
∗
120 12
Number of transitions

100 ∗ 10

80 8

60 6

40 4

20 2

0 0
Control

Diazepam

EEGO 50 mg/kg

EEGO 100 mg/kg

EEGO 200 mg/kg

Control

Diazepam

EEGO 50 mg/kg

EEGO 100 mg/kg

EEGO 200 mg/kg

(a) (b)

Figure 6: Effect of EEGO on light-dark box (LDB) exploration test in mice. Thirty min after the treatment with EEGO or vehicle and 15 min
after diazepam, animals were observed in LDB and the time spent in the lighted part (a) and number of transitions between compartments
(b) were recorded. Data were presented as Mean ± SEM (𝑛 = 5–7). ∗ 𝑝 < 0.01 compared to control.

anxiolytic drug treatment, animals increased their transitions and consistent parameter to evaluate an anxiolytic action
in between the compartments of LDB. In contrast, there [25]. Therefore, our results and previously published reports
were no changes observed by other researchers when they suggest that EEGO may possess anxiolytic potentials along
administered standard anxiolytics to their animals. These with its sedative properties (Figure 7). Although this research
discrepancies might be due to the genetic or strain variation has reached its goals, the audience of this paper might have
of the animals used in their studies. Therefore, it has been the query regarding the effects of higher doses of EEGO on
concluded that simply the time spent in the lighted area, the above models. Therefore, it is worthy to include few more
but not the total number of transitions, is the most useful doses (higher than 200 mg/kg) in the future studies.
6 Evidence-Based Complementary and Alternative Medicine

Decreased locomotor

Sedative and anxiolytic effects of EEGO

Glinus oppositifolius activity of mice in hole
cross and open field tests

Ethanol extract Decreased motor

coordination of mice in
rotarod test
EEGO

Increased exploration in
open and lighted area of Potentiates TS-induced
elevated plus maze and hypnosis, strengthening
light-dark box, the sedative property of
respectively EEGO

Figure 7: Sedative and anxiolytic activities of EEGO.

Table 1: Groups of phytochemicals identified in EEGO.

Phytochemicals Name of the tests Expected changes Results

Mayer’s test Yellowish buff color precipitate +
Hager’s test Yellow crystalline precipitate +
Alkaloids Wagner’s test Brown or deep brown precipitate +
Dragendorff ’s test Orange or orange-brown precipitate +
Tannic acid test Buff color precipitate −
Ferric chloride test Blue-green color +
Tannins
Alkaline reagent test Yellow to red precipitate +
General test Yellow color +
Glycosides
Test for glucoside Production of brick-red precipitation +
A red or reddish violet ring is formed at the junction of two +
Molisch’s test
layers and on shaking a dark purple solution is formed
Barfoed’s test (general test for −
Carbohydrates Red precipitate
monosaccharides)
Fehling’s test A red or brick-red precipitate +
Test for reducing sugar A brick-red precipitate +
Flavonoids Hydrochloric acid reduction test Red color +
Saponins Frothing test Formation of stable foam +

Our phytochemical screening of EEGO revealed the activities. We may, therefore, conceive that the ethanol extract
presence of alkaloids, carbohydrates, flavonoids, glycosides, of G. oppositifolius contains psychoactive principles that are
saponins, and tannins (Table 1), where saponins are known sedative and anxiolytic in nature.
to show amphetamine antagonism and sedative property
and decrease spontaneous motor activity in the experimental 4. Conclusion
animal models [26]. It has also been reported that the
presence of alkaloids, glycosides, and flavonoids in plant Our preliminary pharmacological studies suggest that the
extract possess sedative and anxiolytic effect through the ethanol extract of Glinus oppositifolius leaves possesses seda-
interaction with GABAA receptors [27–29]. Considering our tive properties, reduces locomotor activity, and causes muscle
results and previously published reports, it is possible that the relaxation in experimental animals, which authorize the
abovementioned chemical components in the extract might possible anxiolytic-like actions of the extract. Therefore, these
contribute at least in part to the observed pharmacological results provide the scientific validation for the use of this
Evidence-Based Complementary and Alternative Medicine 7

plant in traditional medicine in treatment of various ailments [8] K. T. Inngjerdingen, S. C. Debes, M. Inngjerdingen et al.,
related to CNS disorders. However, further pharmacological “Bioactive pectic polysaccharides from Glinus oppositifolius
studies are required to clearly understand the sedative and (L.) Aug. DC., a Malian medicinal plant, isolation and partial
anxiolytic actions of EEGO, where our findings could stand characterization,” Journal of Ethnopharmacology, vol. 101, no. 1–
as a basis for further progress. In addition, whether these 3, pp. 204–214, 2005.
activities were generated by the actions of agonists or partial [9] A. Ghani, Medicinal Plants of Bangladesh with Chemical
agonists present in EEGO which could directly act on the Constituents and Uses, Asiatic Society of Bangladesh, Dhaka,
Bangladesh, 2nd edition, 2003.
receptor(s) responsible and/or interact with other molecular
mechanism(s) involved in the observed sedative-anxiolytic [10] C. I. B. Walker, G. Trevisan, M. F. Rossato et al., “Antinociceptive
activity of Mirabilis jalapa in mice,” Journal of Ethnopharmacol-
effects also needs to be investigated. These bioactivity-guided
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phytopharmacological works will give us the opportunity to
[11] K. Takagi, M. Watanabe, and H. Saito, “Studies of the spon-
identify pharmaceutical lead(s) with better tolerability and taneous movement of animals by the hole cross test; effect
lesser side effects in new drug development. of 2-dimethyl-aminoethanol and its acyl esters on the central
nervous system,” Japanese Journal of Pharmacology, vol. 21, no.
Disclosure 6, pp. 797–810, 1971.
[12] B. D. Gupta, P. C. Dandiya, and M. L. Gupta, “A psycho-
The present address of Md. Moniruzzaman is the Centre pharmacological analysis of behaviour in rats,” Japanese Journal
for Pain Research, Institute for Molecular Bioscience, The of Pharmacology, vol. 21, no. 3, pp. 293–298, 1971.
University of Queensland, Brisbane, QLD 4072, Australia. [13] N. W. Dunham and T. S. Miya, “A note on a simple apparatus for
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Competing Interests 209, 1957.
[14] R. A. Turner, “Anticonvulsant,” in Screening Methods in Pharma-
The authors wish to confirm that there are no known cology, Academic Press, New York, NY, USA, 1st edition, 1965.
competing interests associated with this paper and there has
[15] S. Pellow, P. Chopin, S. E. File, and M. Briley, “Validation of
been no significant financial support for this work that could open: closed arm entries in an elevated plus-maze as a measure
have influenced its outcome. of anxiety in the rat,” Journal of Neuroscience Methods, vol. 14,
no. 3, pp. 149–167, 1985.
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The authors are thankful to Md. Ripon Mia and Protik Roy laria,” Journal of Ethnopharmacology, vol. 151, no. 1, pp. 536–542,
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[17] M. Hascoët and M. Bourin, “A new approach to the light/dark
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