ORIGINAL RESEARCH

Asparagus racemosus Attenuates Anxiety-Like Behaviorin Experimental Animal Models

Debapriya Garabadu • Sairam Krishnamurthy

Received: 12 December 2013 / Accepted: 30 January 2014

� Springer Science+Business Media New York 2014

Abstract Asparagus racemosus Linn. (AR) is used

worldwide as a medicinal plant. In the present study, the

anxiolytic activity of standardized methanolic extract of

root of AR (MAR) was evaluated in open-field test (OFT),

hole-board, and elevated plus maze (EPM) tests. Rats

received oral pretreatment of MAR in the doses of 50, 100,

and 200 mg/kg daily for 7 days and then were evaluated for

the anxiolytic activity in different animal models. Both

MAR (100 and 200 mg/kg) and diazepam (1 mg/kg, p.o.)

increased the grooming behavior, number of central squares

crossed, and time spent in the central area during OFT.

Further, MAR (100 and 200 mg/kg) increased the head-dip

and head-dip/sniffing behavior, and decreased sniffing

activity in hole-board test. Furthermore, MAR (100 and

200 mg/kg) increased the percentage entries and time spent

to open arm in EPM test paradigm. The anxiolytic activity in

the experimental models was similar to that of diazepam.

MAR (100 and 200 mg/kg) enhanced the level of amygdalar

serotonin and norepinephrine. It also increased the expres-

sion of 5-HT2A receptors in the amygdala. In another set of

experiment, flumazenil attenuated the anxiolytic effect of

minimum effective dose of MAR (100 mg/kg) in OFT, hole-

board, and EPM tests, indicating GABAA-mediated mech-

anism. Moreover, the anxiolytic dose of MAR did not show

sedative-like effect in OFT and EPM tests compared to

diazepam (6 mg/kg, p.o.). Thus, the anxiolytic response of

MAR may involve GABA and serotonergic mechanisms.

These preclinical data show that AR can be a potential agent

for treatment of anxiety disorders.

Keywords Asparagus racemosus � Anxiety � Serotonin �GABA � Amygdala

Introduction

Asparagus racemosus Linn. (AR; Asparagaceae) is a

climbing plant with much branched spinus under-shrub with

tuberous, short rootstock bearing numerous fusiform, suc-

culent roots which grows in low forest areas throughout

India. In Ayurveda, it is considered as a rasayana drug. There

are about 64 formulations in ancient system of Indian med-

icine containing AR used for several disorders (Sharma et al.

2000). It is also recommended for diverse ailments such as

neuropathy, nervous disorders and certain infectious dis-

eases (Goyal et al. 2003). AR is reported to possess several

pharmacological activities including adaptogenic, anti-

stress, gastroduodenal ulcer protective, anticancer, anti-

urolithiatic, immunomodulatory and diabetic-induced anti-

nephropathic effects in experimental models (Krishnamur-

thy et al. 2013; Somania et al. 2012; Sairam et al. 2003). AR

has significant amount of steroidal saponins (I–X). The

major saponins are shatavarin I, shatavarin IV, shatavarin V,

shatavarins VI–X and immunoside (Hayes et al. 2008).

Recently, furostanol saponin and diphenyl pentendiol have

been isolated from the roots of AR (Sharma et al. 2012).

Saponins are considered to be the major active constituents.

Methanolic extract of root of AR (MAR) possesses highest

amount of saponins and exhibits maximum inhibitory effect

on acetylcholine and monoamine metabolizing enzymes

in vitro (Meena et al. 2011). MAR standardized to saponins

showed anti-depressant (Singh et al. 2009), nootropic, and

anti-amnesic effects (Ojha et al. 2010) in experimental ani-

mals. MAR intrinsically modulated the stress-related path-

ways such as hypothalamus–pituitary–adrenal cortex-axis

D. Garabadu � S. Krishnamurthy (&)

Neurotherapeutics Lab, Department of Pharmaceutics, Indian

Institute of Technology (Banaras Hindu University),

Varanasi 221005, India

e-mail: saibliss@hotmail.com; ksairam.phe@iitbhu.ac.in

123

Cell Mol Neurobiol

DOI 10.1007/s10571-014-0035-z

(HPA-axis) and brain monoaminergic systems in experi-

mentally un-manipulated animals (Krishnamurthy et al.

2013). These observations confer credibility to the wide-

spread application of AR as a drug and food supplement.

However, there is no report on the anxiolytic activity of

MAR in animal models.

Anxiety disorders comprise of different forms of abnormal

and pathological fear which are often associated with other

mental disorders (Graeff et al. 1993). These conditions are

often related to stressful life experiences, especially when

chronic and traumatic stress appears to act as a predisposing

and precipitating factor in these psychiatric conditions (Stro-

hle and Holsboer 2003). Dysregulation of neurotransmitter

systems, alteration of signal transduction pathways and re-

shaping of brain circuitry are all being explored as potential

targets for anxiolytic activity. Preclinical studies reveal that

the dysregulation of the serotonergic, noradrenergic, and

GABAergic systems are important in the pathogenesis of

anxiety disorders among several hypotheses (Graeff et al.

1993; Jiang et al. 2009; Hale et al. 2010; Spannuth et al. 2011).

It has been suggested that the neuronal circuits associated with

anxiety disorders including the amygdala form a critical

component of the neuronal network associated with regulation

of stress/emotional response (Hale et al. 2010). Serotonin-2

(5-HT2) receptors appear to be highly expressed in the

amygdala (Pompeiano et al. 1994) and thus may serve an

important modulatory role in fear and anxiety response.

Among several subtypes of 5-HT2 receptors, both 5-HT2A and

5-HT2C receptors have been shown to be highly expressed in

the amygdala (Xu and Pandey 2000). Restriction of 5-HT2A

receptors to interneurons in the amygdala suggests that

5-HT2A receptors participate in inhibitory modulation of the

amygdala circuitry. It has been reported that the 5-HT2A is the

primary receptor responsible for the serotonergic facilitation

of c-amino butyric acid (GABA) release in the amygdala

(Jiang et al. 2009). Thus, any mediator that facilitates GAB-

Aergic synaptic transmission in the amygdala such as 5-HT2A

receptor agonists can induce anxiolytic effect.

Therefore, the present study explores the anxiolytic

activity of standardized MAR in animal models. Further,

the GABAergic-mediated mechanism has been evaluated

for the minimum effective dose of MAR. Lastly, the sed-

ative effect of MAR and diazepam was evaluated in dif-

ferent animal models.

Materials and Methods

Animals

The experiments were conducted in accordance with the

Principles of laboratory animal care (NIH publication

number 85-23, revised 1985) guidelines. Male adult

Charles Foster strain albino rats (200 ± 20 g) were pur-

chased from the Central Animal House, Institute of Med-

ical Sciences, Banaras Hindu University (BHU).

Experiments on animals were approved by the Institutional

Animal Ethics Committee of BHU, Varanasi, India (Pro-

tocol No: Dean/11-12/CAEC/328). The animals were

housed in polypropylene cages under controlled environ-

mental conditions of temperature of 25 ± 1 �C and

45–55 % relative humidity and a 12:12 h light/dark cycle.

The experimental animals had free access to commercial

rat feed (Doodh dhara Pashu Ahar, India) and water

ad libitum during the experiment. All the experiments were

carried out during 08:00 h to 16:00 h. Animals were

acclimatized for at least 1 week before using them for

experiments and exposed only once to the experiment.

Chemicals and Reagents

Diazepam (DZ) was gifted by Ranbaxy, India. Antibodies

were purchased from Abcam Plc. (Cambridge, MA, USA).

All other chemicals and reagents of high-performance

liquid chromatography (HPLC) and analytical grade were

procured from Merck Pvt. Ltd. (India) and Himedia Lab-

oratories Pvt. Ltd. (India).

Plant Material

Fresh roots of AR were collected from Ayurveda garden of

Institute of Medical Sciences, Banaras Hindu University,

Varanasi, in the month of October. After washing, roots

were size reduced to 20-mesh sieve size and extracted with

methanol as solvent using soxhlet apparatus. After

extraction, methanol was evaporated to get a semisolid

mass of root extract. The yield of extract was about 10 %.

Standardization of Extract

One gram of extracted material was defatted with petro-

leum ether (60–80 �C) and successively extracted with

chloroform and ethyl acetate. Chloroform and ethyl acetate

extracts were discarded. Petroleum ether extract was con-

centrated and dissolved in methanol (15 ml), filtered and

concentrated to 5 ml. The five-milliliter concentrate was

added drop by drop with constant stirring to 25 ml of

acetone in order to precipitate the saponins. The precipitate

was filtered, collected, and dried to constant weight at

105 �C (0.622 g). Total saponin was found to be 62.2 % by

gravimetric estimation (Sairam et al. 2003).

Experimental Design

The whole study was divided into three sets of experi-

ments. In the first experiment, twenty-five male rats were

Cell Mol Neurobiol

123

equally divided into five groups; Control, DZ-1.0, MAR-

50, MAR-100, and MAR-200. The control group animals

received 0.5 % carboxy methyl cellulose (CMC) suspen-

sion orally as vehicle, while diazepam (1.0 mg/kg; p.o.;

Kumar et al. 2013) was given to DZ-1.0 group rats. The

rest three groups received standardized MAR (50, 100 and

200 mg/kg; p.o.). The doses were chosen based on results

from previous studies (Krishnamurthy et al. 2013). The

treatment was followed for seven consecutive days. All the

behavioral performances were observed and quantified

with ANY-mazeTM (Version-3.72; USA) video-tracking

system. The amygdalar tissue was collected through micro

dissection of brain by following the standard procedure

(Palkovits and Brownstein 1988). The tissues were stored

at -80 �C for neurochemical and western blot analysis.

To elucidate the GABA-mediated mechanism, the sec-

ond experiment was conducted with the minimum effective

anxiolytic dose of MAR. Twenty-five male rats were

equally divided into five groups, namely Control, DZ-1.0,

MAR-100, DZ-1.0 ? F, and MAR-100 ? F. The vehicle

was administered to control animals orally through oral

gavage, while DZ-1.0 and DZ-1.0 ? F group animals

received diazepam (1.0 mg/kg; p.o.). MAR-100 and MAR-

100 ? F received standardized MAR (100 mg/kg; p.o.).

All the treatments except flumazenil (GABAA competitive

antagonist) were followed for seven consecutive days. On

seventh day, flumazenil (10.0 mg/kg; i.p.) was adminis-

tered 30 min before the oral administration of DZ or MAR

to DZ-1.0 ? F and MAR-100 ? F groups (Foyet et al.

2012). Thereafter, all the behavioral performances were

observed and quantified with ANY-mazeTM (Version-3.72;

USA) video-tracking system.

The third set of experiment was conducted to study the

sedative effect of the anxiolytic dose of MAR. Fifteen male

rats were equally divided into three groups, namely Con-

trol, DZ-6.0, and MAR-100. The sedative dose of diazepam

(6.0 mg/kg; p.o.; You et al. 2012) was administered to DZ-

6.0 group animals. The vehicle was administered to control

animals, while MAR-100 group rats received MAR

(100 mg/kg; p.o.). The treatment was continued for seven

consecutive days. Thereafter, all the behavioral perfor-

mances were observed and quantified with ANY-mazeTM

(Version-3.72; USA) video-tracking system.

Anxiety-Like Behavioral Observations in Open-Field

Test Paradigm

An open-field apparatus, made of plywood and consisting

of a square (61 9 61 cm) with high walls (61 9 61 cm),

was used to study the locomotor activity in rats. The entire

apparatus was painted black except for 6-mm white lines

that divided the floor into 16 squares. Each animal was

placed in the periphery of the test apparatus for 5 min, and

the behaviors such as ambulation (the number of squares

crossed by the animal), rearing (number of times the ani-

mal stood on the hind limbs), grooming, and the number

and duration of central squares crossed were recorded

(Bronstein 1972).

Anxiety-Like Behavior in Hole-Board Test

The hole-board apparatus consisted of perspex box

(60 9 60 9 35 cm) with four equidistant holes 4 cm in

diameter in the floor. The floor of the box was positioned

12 cm above the ground and divided into nine

(20 9 20 cm) squares. For the hole-board experiments,

each animal was placed in the center of the hole-board and

allowed to freely explore the apparatus for 5 min. Total

number of head-dipping, sniffing, and squares crossed were

recorded. The ratio of head-dipping/sniffing was also cal-

culated as a measure of anxiety (Kong et al. 2006; Casa-

rrubea et al. 2009).

Anxiety-Like Behavior in EPM Test

The plus maze consisted of two opposite open arms,

50 9 10 cm, crossed with two opposite open arms of the

same dimensions with walls of 40 cm high. The arms were

connected with a central square (10 9 10 cm) to give the

apparatus a plus-sign appearance. The maze was kept

elevated 50 cm above the floor in a dimly lit room. The rats

were placed individually on the central square of the plus

maze facing an enclosed arm. The percentage time spent

and the numbers of entries made by the rat, during the next

5 min, on the open arms were recorded as an index of

anxiety. Further, the total arm entries were recorded as an

index of locomotor activity. An arm entry was defined

when all four limbs of the rat were on the arm (Pellow et al.

1985).

Neurochemical Estimation

The level of neurotransmitters (serotonin, norepinephrine

and dopamine) and their metabolites (serotonin and dopa-

mine) were estimated in amygdalar tissues using HPLC/

ECD (Kim et al. 1987; Garabadu et al. 2011). The protein

content was estimated using the method of Lowry et al.

(1951).

Estimation of 5-HT2A Receptor through Western Blot

Technique

For western blot analysis, the brain regions were lysed in

buffer containing complete protease inhibitor cocktail.

Protein concentrations were determined according to

Bradford (1976). A standard plot was generated using

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123

bovine serum albumin. An aliquot of each sample was

heated for 5 min at 100 �C and run on 6 % SDS-PAGE

gels (60 min, 150 V, 4 �C) for 5-HT2A proteins. The pro-

teins were electro blotted (350 mA, 1 h, and 4 �C) onto

polyvinylidene fluoride membranes and probed with spe-

cific antibodies, and the membrane was blocked for

unspecific binding with 5 % non-fat dry milk (1 h at room

temperature). The membrane was incubated overnight with

rabbit anti-5-HT2A (1:100) polyclonal primary antibodies.

After detection with the desired antibodies against the

proteins of interest, the membrane was stripped with

stripping buffer (25 mM Glycine pH 2.0, 2 % SDS) for

30 min at room temperature and re-probed overnight with

rabbit anti-b-actin polyclonal primary antibody at a dilu-

tion of 1:500 to confirm equal loading of protein. Fur-

thermore, membrane was probed with corresponding

secondary antibodies. Immunoreactive band of proteins

was detected by chemiluminescence using enhanced

chemiluminescence reagents (Amersham Bioscience,

USA). Quantification of the results was performed by

densitometric scan of films. The immunoreactive area was

determined by densitometric analysis using Biovis gel

documentation software.

Data Analysis

All data are presented as Mean ± S.E.M. The statistical

significance was determined by one-way analysis of variance

(ANOVA) followed by post-hoc Student–Newman–Keuls

test. P \ 0.05 was considered to be statistically significant.

Results

Anxiolytic Effect of MAR (50, 100, and 200 mg/kg)

in OFT Paradigm

Diazepam increased the number of grooming and central

squares crossed, and time spent in the central area in OFT

paradigm compared to control animals. MAR (100 and

200 mg/kg) increased the number of grooming and central

squares crossed, and time spent in the central area com-

pared to vehicle- and MAR (50 mg/kg)-treated rats.

However, there were no statistical significance among

groups in ambulation and rearing behaviors (Table 1).

Effect of MAR (50, 100 and 200 mg/kg) on Anxiety-

Like Behavior in Hole-Board Test

The effect of MAR (50, 100 and 200 mg/kg) on head-

dipping (A), sniffing (B), head-dipping/sniffing (C), and

number of squares crossed (D) in hole-board test para-

digm is depicted in Fig. 1. The head-dipping, sniffing, and

head-dipping/sniffing ratio were significantly higher in

diazepam-treated group rats compared with control ani-

mals. MAR (100 and 200 mg/kg) increased the number of

head-dipping and sniffing, and ratio of head-dipping/

sniffing compared with vehicle- and MAR (50 mg/kg)-

treated rats. However, there was no significant difference

among groups in number of squares crossed in hole-board

test procedure.

Anxiolytic Effect of MAR (50, 100, and 200 mg/kg)

in EPM Test Paradigm

Figure 2 illustrates the effect of MAR (50, 100, and

200 mg/kg) on percentage open-arm entries (A) and time

spent (B), and total arm entries (C) in EPM test para-

digm. Statistical analysis revealed that there was increase

in the percentage open-arm entries and time spent with

diazepam treatment compared with control animals.

MAR (100 and 200 mg/kg) increased the percentage

open-arm entries and time spent compared with vehicle-

and MAR (50 mg/kg)-treated rats. However, there were

no statistical significant differences among groups in

total arm entries.

Table 1 The effect of MAR (50, 100, and 200 mg/kg; p.o.) on the behavioral parameters in OFT

Group Ambulation (no.) Rearing (no.) Grooming (no.) Number of central

squares crossed (no.)

Time spent in

the central area (s)

Control 55.8 ± 1.83 14.0 ± 1.52 7.4 ± 0.68 5.4 ± 0.68 5.6 ± 0.28

DZ-1.0 55.8 ± 2.22 16.0 ± 1.73 14.6 ± 1.21a 13.8 ± 1.32a 13.7 ± 0.50a

MAR-50 58.6 ± 2.42 14.8 ± 1.43 6.8 ± 0.73b 5.8 ± 0.86b 5.2 ± 0.25b

MAR-100 55.6 ± 2.16 15.4 ± 1.50 16.0 ± 1.41a,c 14.4 ± 2.23a,c 13.4 ± 0.36a,c

MAR-200 57.2 ± 1.83 17.2 ± 1.43 16.2 ± 1.41a,c 15.0 ± 2.34a,c 12.9 ± 0.79a,c

All values are mean ± SEM (N = 5)a P \ 0.05 compared to Controlb P \ 0.05 compared to DZ-1.0c P \ 0.05 compared to MAR-50 [one-way ANOVA followed by Student–Newman–Keuls test]

Cell Mol Neurobiol

123

Effect of MAR (50, 100, and 200 mg/kg)

on Amygdalar Monoamines and Their Metabolites

Diazepam increased amygdalar serotonin (5-HT) and

5-hydroxyindoleaceticacid (5-HIAA) levels, and ratio of

5-HIAA/5-HT compared with Control group rats. MAR

(100 and 200 mg/kg) significantly increased the levels of

5-HT and 5-HIAA, and ratio of 5-HIAA/5-HT compared

with vehicle- and MAR (50 mg/kg)-treated animals.

Diazepam and MAR (50 mg/kg) did not change the level

of norepinephrine (NE) compared to control; however, the

level of NE was higher in MAR (100 and 200 mg/kg)-

treated rats compared with control. Moreover, there were

no significant differences among the groups in dopamine

(DA), 3, 4-dihydroxyphenylacetic acid (DOPAC), and

homovanillic acid (HVA) levels, and ratios of DOPAC/DA

and HVA/DA (Table 2).

Effect of MAR (50, 100 and 200 mg/kg) on Amygdalar

5-HT2A Receptor Expression Level

Diazepam increased level of expression of 5-HT2A recep-

tors compared with control rats. MAR (100 and 200 mg/

kg) elevated the level of expression of 5-HT2A receptors

compared with vehicle- and MAR (50 mg/kg)-adminis-

tered animals (Fig. 3).

Effect of Flumazenil Antagonism on Anxiolytic Effect

of MAR (100 mg/kg) in OFT Paradigm

The number of grooming and central squares crossed, and

time spent in the central area during OFT paradigm were

higher in both diazepam- and MAR (100 mg/kg)-treated

rats compared with Control group animals. Flumazenil

treatment reversed the effect of both diazepam and MAR

(100 mg/kg) on number of grooming and central squares

crossed, and time spent in the central area in OFT

(Table 3).

Flumazenil Antagonizes the Anxiolytic Effect of MAR

(100 mg/kg) in Hole-Board Test

The antagonistic effect of flumazenil on either diazepam or

MAR (50, 100, and 200 mg/kg)-induced head-dipping (A),

sniffing (B), head-dipping/sniffing (C), and number of

squares crossed (D) in hole-board test paradigm is depicted

in Fig. 4. The head-dipping, sniffing, and head-dipping/

sniffing were significantly higher in both diazepam and

MAR (100 mg/kg)-treated group rats compared with con-

trol animals. Flumazenil reversed the head-dipping, sniff-

ing, and head-dipping/sniffing behaviors in diazepam- and

MAR (100 mg/kg)-treated rats. However, there was no

significant difference among groups in number of squares

crossed in hole-board test procedure.

Fig. 1 The effect of MAR (50, 100, and 200 mg/kg; p.o.) on number

of head-dip (a), sniffing (b), and squares crossed (d), and ratio of

head-dip/sniffing (c) in hole-board test. All values are mean ± SEM

(N = 5). aP \ 0.05 compared to Control, bP \ 0.05 compared to DZ-

1.0, and cP \ 0.05 compared to MAR-50 [one-way ANOVA

followed by Student–Newman–Keuls test]

Cell Mol Neurobiol

123

Anxiolytic Effect of MAR (100 mg/kg) Is Reversed

with Flumazenil Antagonism in EPM Test

Figure 5 illustrates the effect of flumazenil on MAR

(100 mg/kg)-induced alterations in percentage open-arm

entries (A) and time spent (B), and total arm entries (C) in

EPM test paradigm. Diazepam and MAR (100 mg/kg)

treatment increased the percentage in open-arm entries and

time spent compared with vehicle-treated rats. Flumazenil

antagonism attenuated the effect of both diazepam and

MAR (100 mg/kg) on the percentage open-arm entries and

time spent during 5-min test paradigm. However, there

were no statistical significant differences among groups in

total arm entries.

MAR (100 mg/kg) Lacks Sedative-Like Behavior

in OFT and EPM Tests

The sedative-like behavior, in terms of ambulation in OFT

(A) and total arm entries in EPM test paradigm, of diaze-

pam (6.0 mg/kg) and MAR (100 mg/kg) is depicted in

Fig. 6. There was sedative-like effect of diazepam in both

the animal models compared to control rats. It is interesting

to note that MAR (100 mg/kg)-treated animals did not

show any sedative-like effect in both the animal models.

Discussion

The present study reveals the anxiolytic activity of MAR in

different animal models. Flumazenil, a GABAA antagonist,

attenuates the anxiolytic effect of MAR in OFT, hole-

Fig. 2 The effect of MAR on percentage of open-arm entries (a) and

time spent (b), and total arm entries (c) in EPM. All values are

mean ± SEM (N = 5). aP \ 0.05 compared to Control, bP \ 0.05

compared to DZ-1.0, and cP \ 0.05 compared to MAR-50 [one-way

ANOVA followed by Student–Newman–Keuls test]

Table 2 The effect of MAR (50, 100 and 200 mg/kg; p.o.) on the levels of monoamines and their metabolites, and their ratios in amygdala

Monoamine (ng/mg protein) Control DZ-1.0 MAR-50 MAR-100 MAR-200

5-HT 17.72 ± 1.18 25.08 ± 0.80a 19.21 ± 2.25b 25.91 ± 1.49a,c 25.33 ± 1.48a,c

5-HIAA 1.88 ± 0.13 4.12 ± 0.18a 2.01 ± 0.22b 4.63 ± 0.39a,c 4.28 ± 0.21a,c

5-HIAA/5-HT 0.10 ± 0.014 0.16 ± 0.005a 0.10 ± 0.009b 0.18 ± 0.022a,c 0.17 ± 0.012a,c

NE 7.62 ± 0.23 8.22 ± 0.73 8.78 ± 0.23 9.91 ± 0.63a 9.67 ± 0.38a

DA 11.91 ± 0.63 11.97 ± 0.29 12.73 ± 0.71 12.64 ± 0.54 12.32 ± 0.69

DOPAC 2.52 ± 0.16 2.58 ± 0.15 2.63 ± 0.21 2.80 ± 0.30 2.85 ± 0.27

DOPAC/DA 0.21 ± 0.02 0.22 ± 0.01 0.21 ± 0.03 0.23 ± 0.02 0.24 ± 0.03

HVA 4.49 ± 0.18 4.88 ± 0.23 4.95 ± 0.23 4.70 ± 0.28 4.64 ± 0.31

HVA/DA 0.38 ± 0.03 0.41 ± 0.02 0.39 ± 0.04 0.38 ± 0.03 0.38 ± 0.03

All values are mean ± SEM (N = 5)a P \ 0.05 compared to Controlb P \ 0.05 compared to DZ-1.0c P \ 0.05 compared to MAR-50 [One-way ANOVA followed by Student–Newman–Keuls test]

Cell Mol Neurobiol

123

board, and EPM tests. We, for the first time, report that

MAR exhibits anxiolytic activity through GABAA-medi-

ated mechanism. Further, the minimum effective dose of

MAR lacks the sedative-like behavioral side effect.

Most of the animal models of anxiety are based on the

principle of innate general avoidance behaviors. The OFT

is a widely used animal model for evaluating anxiolytic

drugs. In the present study, both MAR and diazepam

showed anxiolytic activity in terms of increasing the

grooming behavior, number of central squares crossed, and

time spent in the central area during OFT. It is reported that

the grooming behavior is a measure of anxiety in OFT

(Ramanathan et al. 1998). Normal aversion of a rodent to

the central area produces the anxiety and fear, which is

characterized by alteration in the behavioral parameters of

animal in OFT. Therefore, increased number of central

squares crossed and time spent in the central area during

OFT by diazepam- and MAR-treated animals indicates

anxiolytic activity. Previous reports also suggest that anx-

iolytic compounds increase the number of central squares

crossed in the open field by the rodents (Mechan et al.

2002). It has been reported that the ambulation and rearing

behavior reflects the horizontal and vertical locomotor

activity of the animals in OFT (Ramanathan et al. 1998).

Further, in the present study, diazepam did not show any

effect on both horizontal and vertical locomotion of the

animals in OFT which is similar to earlier reports (Melo

et al. 2010; Baretta et al. 2012). The horizontal and vertical

locomotor activity of the animals in OFT reflects the sed-

ative-like behavior (Baretta et al. 2012). MAR did not

exhibit any effect on both horizontal and vertical loco-

motion of the animals in OFT, indicating the absence of

Fig. 3 The effect of MAR on

the level of expression of

5-HT2A receptor in amygdala.

The blots are representative of

5-HT2A (a) in amygdala. The

results in the histogram are

expressed as ratio of relative

intensity of levels of protein

expression of 5-HT2A to b-actin.

All values are mean ± SEM of

three separate sets of

independent experiments.aP \ 0.05 compared to Control,bP \ 0.05 compared to DZ-1.0,

and cP \ 0.05 compared to

MAR-50 [one-way ANOVA

followed by Student–Newman–

Keuls test]

Table 3 The effect of flumazenil on MAR-induced changes on the behavioral parameters in OFT

Group Ambulation (no.) Rearing (no.) Grooming (no.) Number of central

squares crossed (no.)

Time spent in

the central area (s)

Control 53.6 ± 1.78 15.2 ± 1.16 7.4 ± 0.51 5.4 ± 0.68 5.36 ± 0.22

DZ-1.0 58.4 ± 1.94 17.0 ± 1.30 14.6 ± 1.36a 13.8 ± 1.32a 12.92 ± 0.47a

MAR-100 57.8 ± 2.13 15.6 ± 1.03 16.6 ± 1.08a 14.4 ± 2.23a 12.88 ± 0.44a

DZ-1.0 ? F 56.0 ± 2.68 15.4 ± 0.93 8.0 ± 0.95b,d 8.6 ± 0.68b,d 7.94 ± 0.50a,b,d

MAR-100 ? F 55.0 ± 2.39 17.6 ± 1.47 8.6 ± 0.75b,d 9.4 ± 0.93b,d 8.60 ± 0.76a,b,d

All values are mean ± SEM (N = 5)a P \ 0.05 compared to Controlb P \ 0.05 compared to DZ-1.0d P \ 0.05 compared to MAR-100 [One-way ANOVA followed by Student–Newman–Keuls test]

Cell Mol Neurobiol

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any sedative-like effect of MAR in the present experi-

mental condition. Hence, MAR exhibits anxiolytic activity

with a ceiling effect at a dose of 100 mg/kg without

altering locomotor activity in OFT paradigm.

The hole-board test is an experimental model largely

used to examine the behavior of animal placed in a board

provided with a variable number of holes and more in

general to test anxiety in rodents (File and Wardill 1975). It

has been suggested that the head-dip is considered as a

specific indicator of anxiety, and head-dip is closely related

with edge-sniff, a specific sniffing activity of animals at

hole-edge. Further, it has been reported that the edge-sniff

also reflects the level of anxiety in the animal (Casarrubea

et al. 2009). Moreover, head-dip/edge-sniffing is consid-

ered as a more sensible indicator for anxiety-related

behaviors than head-dip and edge-sniffing as it has an

important ethological meaning. Since, it represents the

behavioral expression of an underlying motivational

activity which in turn could be influenced by anxiety and

its possible pharmacological treatment (Casarrubea et al.

2009). In the present study, both diazepam and MAR

showed anxiolytic activity in terms of decrease in edge-

sniffing activity and increase in head-dip and head-dip/

sniffing behavior in hole-board test. Earlier studies report

the anxiolytic activity of diazepam in hole-board test pro-

cedure similar to our results (Casarrubea et al. 2009). It has

been suggested that the number of squares crossed in hole-

board test reflects the locomotor activity of the animals

(Ramanathan et al. 1998). Here, we did not observe any

change in the locomotor activity of either diazepam- or

MAR-treated animals similar to that of observations

obtained in OFT.

The EPM is a model based on the natural aversion of

rodents for open spaces that used the conflict between

exploration and aversion of elevated open spaces (Carobrez

and Bertoglio 2005). It is reported that the provoked

behavior profiles in the EPM include elements of neo-

phobia, exploration, and approach/avoidance conflict,

referring thus to the apparatus as an unconditioned spon-

taneous behavioral conflict test (Wall and Messier 2001).

Moreover, EPM has been widely used to understand the

biological basis of various types of anxiety disorders such

as generalized anxiety, phobia, and post-traumatic stress

disorder (Carobrez and Bertoglio 2005). In the present

study, both diazepam and MAR exhibited anxiolytic effect

in terms of increase in the percentage entries and time

spent to open arm in EPM test paradigm. Similar to OFT

and hole-board test results, herein neither diazepam nor

MAR showed any activity on locomotor activity in terms

of total arm entries in EPM test.

In the present study, the level of 5-HT, 5-HIAA, and its

ratio (5-HIAA/5-HT) were elevated in the amygdala after

EPM exposure in both diazepam- and MAR (100 and

200 mg/kg)-treated animals. Further, MAR (100 and

200 mg/kg) but not diazepam-treated rats showed increase

in the level of NE in the amygdalar tissues. Dysregulation

of neurotransmitter system such as serotonergic and nor-

adrenergic is postulated as putative factors responsible in

Fig. 4 The effect of flumazenil (F) on MAR-induced alteration in

numbers of head-dip (a), sniffing (b), and squares crossed (d), and

ratio of head-dip/sniffing (c) in hole-board test. All values are

mean ± SEM (N = 5). aP \ 0.05 compared to Control, bP \ 0.05

compared to DZ-1.0, and dP \ 0.05 compared to MAR-100 [one-way

ANOVA followed by Student–Newman–Keuls test]

Cell Mol Neurobiol

123

the genesis of anxiety, and drugs that facilitate either

serotonergic or noradrenergic or both systems exhibit

anxiolytic activity in animals (Graeff et al. 1993; Jiang

et al. 2009; Hale et al. 2010; Spannuth et al. 2011). In the

present study, diazepam exhibits anxiolytic activity prob-

ably also through enhancing serotonergic activity which is

similar to an earlier report (Bailey and Toth 2004). Based

on these observations, it can be assumed that MAR atten-

uates anxiety behavior probably through modulating both

amygdalar serotonergic and noradrenergic systems which

has to be clarified with further experiments.

It has been suggested that the amygdalar 5-HT2A

receptors inhibit anxiety-like behavior. Further, it has been

reported that the 5-HT2A is the primary receptor respon-

sible for the serotonergic facilitation of GABA release in

the amygdala (Jiang et al. 2009). Thus, it can be postulated

that any drug that activate 5-HT2A receptor-mediated

GABAergic synaptic transmission in the amygdala can

induce an anxiolytic effect. Diazepam elevated the level of

expression of 5-HT2A receptors in amygdala. It has been

documented that diazepam exhibits anxiolytic activity

through 5-HT2A-mediated mechanism (Ripoll et al. 2006).

However, we report, for the first time, that diazepam ele-

vates the level of expression of 5-HT2A receptors in

amygdala. Similarly, MAR (100 and 200 mg/kg) aug-

mented the level of expression of 5-HT2A receptors in

amygdala. As MAR (100 and 200 mg/kg) elevated the

level of 5-HT, 5-HIAA and expression of 5-HT2A in

amygdala, it can be assumed that the anxiolytic activity of

MAR may probably involve 5-HT2A-mediated serotonergic

mechanism. Serotonergic neurotransmission has long been

implicated in the pathogenesis of anxiety and several cur-

rently available anxiolytic drugs interfere with 5-HT re-

uptake or target 5-HT receptors (Millan 2003). Weisstaub

et al. (2006) report that there was pronounced anxiety-like

behaviors in 5-HT2A receptor knock-out mice. Buspirone, a

well-known anxiolytic, markedly increased 5-HT2A

receptor mRNA levels in various rat brain areas. This was

accompanied by a significant increase in the level of

5-HT2A receptor-binding sites in sub-hippocampal regions

of the rat (Chen et al. 1995). Clinically, it has been sug-

gested that there is decrease in the 5-HT2A receptor sig-

naling in the amygdalar portion of patients with anxiety

Fig. 5 The effect of flumazenil on MAR-induced alteration on

percentage of open-arm entries (a) and time spent (b), and total arm

entries (c) in EPM. All values are mean ± SEM (N = 5). aP \ 0.05

compared to Control, bP \ 0.05 compared to DZ-1.0, and dP \ 0.05

compared to MAR-100 [one-way ANOVA followed by Student–

Newman–Keuls test]

Fig. 6 The effects of DZ (6 mg/kg, p.o.) or MAR (100 mg/kg; p.o.)

on ambulation (a) and total arm entries (b) in OFT and EPM,

respectively. All values are mean ± SEM (N = 5). aP \ 0.05

compared to Control and eP \ 0.05 compared to DZ-6.0 [one-way

ANOVA followed by Student–Newman–Keuls test]

Cell Mol Neurobiol

123

(Hurlemann et al. 2009). Considering the above facts, it

can be assumed that the expression of anxiety behavior

depends upon function of amygdalar 5-HT2A receptors.

Therefore, amygdalar 5-HT2A-mediated action of MAR

may be one of the important mechanisms for its anxiolytic

activity. It is interesting to note that MAR at lower dose did

not produce any anxiolytic effect, but there was ceiling

effect at dose of 100 mg/kg. Thus, the minimum effective

dose of MAR (100 mg/kg) was considered as an optimum

dose for further mechanistic study.

The 5-HT and GABA systems are closely interlinked,

both neuroanatomically and functionally (Forchetti and

Meek 1981). Several studies revealed that there is bidi-

rectional relationship between GABA and 5-HT system-

mediated activity in brain (Forchetti and Meek 1981;

Nishikawa and Scatton 1983). Thus, evaluation of the

GABAA-mediated anxiolytic activity of MAR (100 mg/kg)

was carried out with the co-administration of flumazenil.

Flumazenil inhibits the anxiolytic activity of both MAR

(100 mg/kg) and diazepam in all the three animal models

of anxiety without altering locomotor activity. The block-

ade of anxiolytic activity by flumazenil is similar to earlier

findings (Foyet et al. 2012; You et al. 2012). These findings

indicate that the anxiolytic activity of MAR may also

involve GABAA-mediated mechanism.

The sedative effects of median dose of MAR and diaz-

epam were tested in the present study. To compare the

sedative effects of MAR (100 mg/kg), we chose the seda-

tive dose of diazepam (6 mg/kg). Diazepam significantly

reduced ambulation in OFT and total arm entries in EPM.

MAR (100 mg/kg) did not cause any change in the loco-

motor activity in either OFT or EPM test paradigms. Thus,

we found that MAR exhibited anxiolytic effect with an

absence of sedative-like behavior.

In conclusion, MAR exhibits anxiolytic activity in dif-

ferent animal models. MAR promotes the amygdalar

serotonergic and noradrenergic systems. Further, MAR

increased the level of expression of 5-HT2A receptors in

amygdala. Thus, the anxiolytic effect of MAR may be due

to the alterations in amygdalar 5-HT2A-mediated seroto-

nergic system. In addition, MAR showed GABAA-medi-

ated anxiolytic activity in different animal models. Further,

the anxiolytic dose of MAR did not show sedative-like

effect. Thus, AR can be a potential candidate in the phar-

macotherapy of anxiety.

Acknowledgments DG is thankful to Council of Scientific and

Industrial Research (CSIR), India, for student fellowship. SK is

thankful to University Grant Commission (UGC), India for the

financial support.

Conflict of interest The authors declare that they have no conflict

of interest.

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