antidepressant-like activity of beta-carotene in unstressed and chronic unpredictable mild stressed...

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Antidepressant-like activity of beta-carotene inunstressed and chronic unpredictable mild stressedmice

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* Corresponding author. Mobile: +91 9416712545.E-mail addresses: [email protected], [email protected] (D. Dhingra).

Please cite this article in press as: Dhingra, D., & Bansal, Y., Antidepressant-like activity of beta-carotene in unstressed and chronic unpmild stressed mice, Journal of Functional Foods (2014), http://dx.doi.org/10.1016/j.jff.2014.01.015

Dinesh Dhingra*, Yashika Bansal

Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar 125001, Haryana, India

A R T I C L E I N F O A B S T R A C T

Article history:

Received 15 November 2013

Received in revised form

14 January 2014

Accepted 14 January 2014

Available online xxxx

Keywords:

Antidepressant

Corticosterone

Depression

Beta-carotene

Chronic unpredictable mild stress

The antidepressant-like activity of beta-carotene in Swiss young male albino mice sub-

jected to chronic unpredictable mild stress was evaluated. Beta-carotene (50 and 100 mg/

kg, p.o.) and imipramine (15 mg/kg, p.o.) per se were administered for 21 successive days

to separate groups of unstressed and stressed mice. Higher dose (100 mg/kg) of beta-caro-

tene and imipramine significantly decreased immobility period of mice in tail suspension

test. These compounds significantly restored the reduced sucrose preference in stressed

mice. There was no significant effect on locomotor activity of mice by the drugs. Beta-car-

otene significantly reversed stress-induced increase in brain catalase, monoamine oxidase

(MAO-A), thiobarbituric acid-reactive substances (TBARS); and plasma nitrite and cortico-

sterone levels; and increased stress-induced decrease in reduced glutathione levels. Thus,

beta-carotene showed significant antidepressant-like activity in unstressed and stressed

mice probably through inhibition of MAO-A and oxidative stress. Its antidepressant-like

activity in stressed mice might also be due to decrease in plasma corticosterone levels.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Depression is an incapacitating psychiatric ailment which is

characterized by a pervasive low mood, loss of interest in

usual activities, diminished ability to experience pleasure

(anhedonia), withdrawal of interest, feelings of worthless-

ness, and suicidal tendencies (Schechter et al., 2005; Strau-

man et al., 2006). The report on Global Burden of Disease

estimates the point prevalence of depressive episodes to be

1.9% for men and 3.2% for women, and the 1-year prevalence

has been estimated to be 5.8% for men and 9.5% for women. It

is estimated that by the year 2020 if current trends for demo-

graphic and epidemiological transition continue, the burden

of depression will increase to 5.7% of the total burden of dis-

ease and it would be the second leading cause of disability-

adjusted life years (Grover, Dutt, & Avasthi, 2010). According

to the World Health Organization, more than 121 million peo-

ple worldwide suffer from depression, making depression the

fourth leading cause of disability (Gorwood, 2010). Research

over the second half of the 20th century provided extensive

evidence that abnormal monoamine neuronal function is an

important underlying pathology in depression. The mono-

amine hypothesis explains that depletion of monoamines like

serotonin, norepinephrine and dopamine in the hippocam-

pus, limbic system and frontal cortex are responsible for the

depressive symptoms (Delgado & Moreno, 1999; Tanabe &

Nomura, 2007). Thus, drugs reported to possess antidepres-

sant activity increase brain levels of norepinephrine, dopa-

mine and serotonin (Hao, Lai, Ho, & Sheen, 2013).

Monoamine oxidase (MAO) is a key enzyme that is associated

with the metabolism of these neurotransmitters. Medications

such as tricyclic antidepressants, selective serotonin reuptake

redictable

http://dx.doi.org/10.1016/j.jff.2014.01.015

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2 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 4 ) x x x – x x x

inhibitors), MAO inhibitors and specific serotonin-norepi-

nephrine reuptake inhibitors are clinically employed for drug

therapy (Anthony, Bertram, & Susan, 2010). However, these

drugs can impose a variety of side effects including sedation,

apathy, fatigue, sleep disturbance, extrapyramidal side ef-

fects, akathisia, cognitive impairment, and sexual dysfunc-

tion (Lane, 1998; Mayers & Baldwin, 2005; Segraves, 1998;

Vandel, Bonin, Leveque, Sechter, & Bizouard, 1997). Approxi-

mately two-thirds of the depressed patients respond to the

currently available treatments but the magnitude of improve-

ment is still disappointing (Mora et al., 2006).

The hypothalamic–pituitary–adrenal (HPA) axis is an

important node in the brain’s stress circuit and suggested to

play a role in several subtypes of depression (Schutter,

2012). Chronic hyperactivity of the HPA axis and resultant

excessive glucocorticoid (hypercortisolism) may be causal to

depression (Kunugi, Hori, Numakawa, & Ota, 2012). Stress is

an everyday burden, endured by most living creatures. The

failure of successful adaptation during stressful situations

will result in stress-related diseases including depression

(Maes et al., 2000; Michel et al., 2007). The chronic unpredict-

able mild stress (CUMS) model has been claimed to be one of

the more relevant animal models of depression (Willner,

Towell, Sampson, Sophokleous, & Muscat, 1987). CUMS model

is an important behavioural model that resembles human

depression (Willner, 1997). It is proposed that chronic stress

causes behavioural changes such as reduced locomotor activ-

ity, reduced food and water intake, decreased responding to

reward stimuli (Griffiths, Shanks, & Anisman, 1992) which

are reflective of clinical depression.

Initially, CUMS functions as a stimulant, increasing meta-

bolic rates and increasing the production of reactive oxygen

species (ROS). The generation of appropriate ROS would be

an effective way to induce organism’s adaptability (Parsons,

1996). However, if the concentration of the ROS exceeds the

body’s capacity to neutralize them, the superfluous ROS begin

to harm cells, tissues and organs, and result in oxidative

stress. Oxidative stress has been proposed to impair the anti-

oxidant defence system, leading to oxidative damage by

changing the balance between oxidant and antioxidant fac-

tors (Fontella et al., 2005; Yu & Chung, 2006). CUMS-induced

oxidative damage has been involved in the etiopathogenesis

of psychiatric disorders such as depression and anxiety

(Bhattacharya & Muruganandam, 2003). There is a correlation

of depressive disorders in humans with oxidative stress either

in the brain and blood (Bilici et al., 2001). Imipramine and mel-

atonin attenuated stress-induced increase in oxidative

parameters (Detanico et al., 2009). NO (nitric oxide) is an

important modulator of depression (Wang, An, & Zhang,

2008; Yildiz, Erden, Utkan, & Gacar, 2000) because NO produc-

tion is increased in depression (Suzuki, Yagi, Nakaki, Kanba, &

Asai, 2001). Nitric oxide is an important neurotransmitter in

the nervous system (Baranano, Ferris, & Snyder, 2001) and

regulates many behavioural, cognitive, and emotional pro-

cesses, including depression. Stressful conditions in rats have

also been reported to significantly increase plasma nitrite

levels, an index of nitric oxide production (Lee, Cheng, &

Sim, 2007).

There is co-existence of increased oxidative stress with

depressive symptoms in patients, as evidenced by defective

Please cite this article in press as: Dhingra, D., & Bansal, Y., Antidepressanmild stressed mice, Journal of Functional Foods (2014), http://dx.doi.org/1

plasma antioxidant defences in association with enhanced

susceptibility to lipid peroxidation (Maes et al., 2000). More-

over, preclinical studies have suggested that antioxidants in

the form of free radical scavengers may have antidepressant

properties (Zafir, Ara, & Banu, 2009). Therefore, it appears rea-

sonable to propose that exogenous antioxidants may be effec-

tive in treating depression. Thus, drugs with potential

antioxidant action could be for the treatment of depressive

disorders.

b-Carotene is a strongly coloured red–orange pigment

present abundantly in many plants. It is a known source of

vitamin A and has exceptional antioxidant and free radical

scavenging potential (Krinsky, 1989). Beta-carotene has been

reported to possess hepatoprotective, photoprotective, anti-

inflammatory and anti-tumour activities (Bai et al., 2005;

Chew, Park, Wong, & Wong, 1999; Hadad & Levy, 2012; Kat-

sumura et al., 1996; Manda & Bhatia, 2003; Stahl & Sies,

2012). Due to its antioxidant property, beta-carotene has been

reported to possess antiepileptic (Sayyah, Yousefi-Pour, &

Narenjkar, 2005), memory enhancing (Grodstein, Kang, Glynn,

Cook, & Gaziano, 2007) and anti-Alzheimer (Ono & Yamada,

2012) activities. But beta-carotene has not been studied for

its potential in the management of depression. Therefore,

the present study was designed to explore the antidepres-

sant-like effect of beta-carotene in mice subjected to chronic

unpredictable mild stress.

2. Materials and methods

2.1. Experimental animals

Swiss male albino mice (3 months old, weighing around 25–

30 g) were purchased from Disease Free Small Animal House,

Lala Lajpat Rai University of Veterinary and Animal Sciences,

Hisar (Haryana, India). Since estrogens (female sex hormones)

have been found to have antidepressant effect, so we

excluded female mice and used only male mice for the study

(Kandi & Hayslett, 2011) Animals were housed separately in

groups of 10 per cage (Polycarbonate cage size: 29 · 22

· 14 cm) under laboratory conditions with alternating light

and dark cycle of 12 h each. The animals had free access to

food and water. The animals were kept fasted 2 h before

and 2 h after drug administration. The animals were acclima-

tized for at least five days before behavioural experiments

which were carried out between 09:00 and 17:00 h. The exper-

imental protocol was approved by Institutional Animals Eth-

ics Committee (IAEC) vide letter number IAEC/136-144 of

dated 10th January, 2013. Animal care was taken as per the

guidelines of Committee for the Purpose of Control and

Supervision of Experiments on Animals (CPCSEA), Ministry

of Environment and Forests, Government of India (Registra-

tion No. 0436).

2.2. Drugs and chemicals

Imipramine hydrochloride (Sigma–Aldrich, St. Louis, MO,

USA), beta-carotene, sulphanilamide, N-(1-Naphthyl) ethy-

lenediamine dihydrochloride, meta-phosphoric acid (HiMe-

dia Laboratories Pvt. Ltd., Mumbai, India); were used in

t-like activity of beta-carotene in unstressed and chronic unpredictable0.1016/j.jff.2014.01.015


J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 4 ) x x x – x x x 3

the present study. Imipramine hydrochloride was dissolved

in normal saline (0.9% w/v sodium chloride). Beta-carotene

was dissolved in sesame oil (Sayyah et al., 2005).

2.3. Selection of doses

The doses of beta-carotene (50 and 100 mg/kg) and imipra-

mine hydrochloride (15 mg/kg) were selected on the basis of

literature (Detanico et al., 2009; Sayyah et al., 2005).

2.4. Chronic unpredictable mild stress procedure

The mice were subjected to chronic unpredictable mild stress

as described by Qing-Qiu, Siu-Po, Kam-Ming, Sam-Hip, and

Chun-Tao (2009) and Kumar, Kuhad, and Chopra (2011) with

some modifications. Animals were subjected to stress para-

digm once a day over a period of 3 weeks between 0900 and

1400 h. The order of stressors was as follows:

Pm

Weeks

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Day-1

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Day-2

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sal, Y., A4), http:/

Day-7

Week-1
I E F O T2 X T1
Week-2
I O X T2 E T1 F
Week-3
O F T1 X T2 I E
I—Immobilization for 2 h; E—Exposure to empty water bottles for

1 h; F—Exposure to foreign object for 24 h (e.g. piece of plastic); O—

overnight illumination; T2—tail pinch (60 s); X—Tilted cage at 45

degree for 7 h; T1—tail pinch (30 s).

2.5. Laboratory models employed

2.5.1. Tail suspension testThe tail suspension test (TST) is a behavioural test widely

used for evaluating antidepressant-like activity of a drug

(Steru, Chermat, Thierry, & Simon, 1985). In the test, the mice

were individually suspended 50 cm above the surface of a

floor, using an adhesive tape placed 1 cm away from the tip

of the tail. Each animal under test was both acoustically

and visually isolated from other animals during test. The

total period of immobility was recorded manually for 6 min.

Animal was considered to be immobile when it didn’t show

any body movement, hung passively and completely

motionless.

2.5.2. Sucrose preference testSucrose preference test (Willner et al., 1987) was employed

herein to determine anhedonia, one of the core symptoms

of major depression in humans. The procedure was com-

posed of training and testing courses. After 1 week of accli-

matization, mice were trained to consume 1% (w/v) sucrose

solution before the start of the CUMS protocol. In training

course, mice were deprived of food and water for 48 h and

only exposed to 1% (w/v) sucrose solution. Three days later,

after 23-h food and water deprivation, 1-h baseline test was

performed, in which mice could select between two pre-

weighed bottles, one with 1% (w/v) sucrose solution and the

other with tap water. Then, the sucrose preference was calcu-

lated according to the following formula:

ntidepressan/dx.doi.org/1

Sucrose preference ¼ sucrose solution intake ðgÞ½sucrose solution intake ðgÞ þ water intake ðgÞ� � 100

The test was again performed on the 22nd day to evaluate the

effect of stress as well as drug treatment.

2.5.3. Measurement of locomotor activityTo rule out the effects of various drug treatments on locomo-

tor activity, horizontal locomotor activities of control and test

animals were recorded for a period of 5 min using photoac-

tometer (INCO, Ambala, India) (Chhillar & Dhingra, 2012; Ku-

mar et al., 2011).

2.6. Experimental protocol

The animals were divided into 16 groups having 10 mice in

each group (see below).

2.6.1. Groups for tail suspension test (TST)Groups 1 to 4: Vehicle (Sesame oil), beta-carotene (50 and

100 mg/kg) and imipramine (15 mg/kg), respectively, were

administered orally to mice for 21 successive days, followed

by TST, 60 min after vehicle/drug administration on 22nd day.

Groups 5 to 8: Vehicle (Sesame oil), beta-carotene (50 and

100 mg/kg) and imipramine (15 mg/kg), respectively, were

administered orally 30 min before induction of stress to mice

for 21 successive days, followed by TST 60 min after vehicle/

drug administration on 22nd day.

2.6.2. Groups for sucrose preference test and locomotoractivityGroups 9 to 16: Separate mice were employed for sucrose pref-

erence test, but their treatments were same as mentioned un-

der groups 1 to 8. After subjecting animals to sucrose

preference test on 22nd day, locomotor activity scores were

measured on 23rd day.

2.7. Biochemical estimations

2.7.1. Collection of blood samplesAfter subjecting unstressed and stressed mice to TST on 22nd

day and one hour after drug administration on 23rd day, blood

(0.5–0.8 ml) was withdrawn from retro-orbital plexus of mice.

Plasma was separated using refrigerated centrifuge (Remi,

Mumbai, India) at 800g for 10 min to separate the plasma.

The plasma was used for estimation of nitrite and corticoste-

rone levels.

2.7.1.1. Estimation of plasma nitrite levels. Plasma nitrite was

measured by using the method of Green et al., 1982. A mixture

of 1% w/v sulphanilamide in 5% aqueous solution of m-phos-

phoric acid (1 part) and 0.1% (w/v) N-(1-Naphthyl) ethylenedi-

amine dihydrochloride (1 part) was prepared and kept at 0 �Cfor 60 min 0.5 ml plasma was mixed with 0.5 ml of the above

mixture and kept in dark for 10 min at room temperature. The

absorbance was read at 546 nm using UV–visible spectropho-

tometer (Varian Cary 5000 UV–VIS–NIR Spectrophotometer,

The Netherlands).

2.7.1.2. Estimation of plasma corticosterone levels. The quanti-

tative estimation of corticosterone levels in the blood plasma




was performed by the method of Bartos and Pesez (1979). To

1.0 ml of sample in ethanol, 0.50 ml of 0.10% solution of

p-nitroso-N,N-dimethylaniline in ethanol was added and the

tubes were immersed in ice water for 5 min, and then

0.50 ml of 0.10 M sodium hydroxide was added. The tubes

were plugged with cotton-wool, and were let to stand at 0 �Cfor 5 h, protected against light. To the above solution, 2.0 ml

of buffer for pH 9.8, 5.0 ml of 0.10% solution of phenol in eth-

anol and 0.50 ml of 1.0% aqueous solution of potassium ferri-

cyanide were added. The tubes were kept in a water bath at

20 ± 2 �C for 10 min. The solution was read at 650 nm using

UV–visible spectrophotometer (Varian Cary 5000 UV–VIS–NIR

Spectrophotometer, The Netherlands).

2.7.2. Biochemical estimations in brain homogenateAfter withdrawing blood samples on 23rd day, mice were sac-

rificed by decapitation and their brains were isolated. The col-

lected brain samples were washed with cold 0.25 M sucrose–

0.1 M Tris–0.02 M ethylenediaminetetraacetic acid buffer (pH

7.4) and weighed. The buffer washed brain sample was

homogenized in 9 volumes of cold 0.25 M sucrose–0.1 M

Tris–0.02 M ethylenediamine tetraacetic acid buffer (pH 7.4)

buffer and centrifuged twice at 800g for 10 min at 4 �C in cool-

ing centrifuge (Remi Instruments, Mumbai, India). The pellet

was discarded. The supernatant was then centrifuged at

14,500g for 20 min at 4 �C in cooling centrifuge. This centri-

fuged supernatant was separated into two parts:

Part I: The precipitates (mitochondrial fraction) were used

for estimation of MAO-A activity.

Part II: The remaining supernatant was used to assay lipid

peroxidation, reduced glutathione and catalase levels.

2.7.2.1. Measurement of MAO-A activity. The MAO-A activity

was assessed spectrophotometrically (Charles & McEwan,

1977; Schurr & Livne, 1976). The mitochondrial fraction of

brain was washed twice with about 100 ml of sucrose–Tris–

EDTA buffer and suspended in 9 volumes of cold sodium

phosphate buffer (10 mM, pH 7.4, containing 320 mM sucrose)

and mingled well at 4 �C for 20 min. The mixture was then

centrifuged at 23,000g for 30 min at 0 �C and the pellets were

re-suspended in cold sodium phosphate buffer. Then, 2.75 ml

sodium phosphate buffer (100 mM, pH 7.4) and 100 ll of 4 mM

5-hydroxytryptamine were mixed in a quartz cuvette which

was placed in UV–visible spectrophotometer (Varian Cary

5000 UV–VIS–NIR Spectrophotometer, The Netherlands). This

was followed by the addition of 150 ll solution of mitochon-

drial fraction to initiate the enzymatic reaction and the

change in absorbance was recorded at 280 nm for 5 min

against the blank containing sodium phosphate buffer and

5-hydroxytryptamine.

2.7.2.2. Estimation of protein concentration. Total protein con-

centration was estimated in brain homogenate by using a to-

tal protein kit (Erba, Transasia Bio-Medical Ltd., Baddi, Solan,

HP), using semi-automatic autoanalyzer (Chem 5 plus-V2

autoanalyzer; Erba Mannheim, Germany). The procedure fol-

lowed was as mentioned in the pamphlet supplied along with


the kit (Henry & Winkelman, 1974). The sample and reconsti-

tuted reagent were brought to room temperature prior to use.

The following general system parameters were used with this

kit:

Reaction type: End point

Wavelength: 546 nm (530–570 nm)

Incubation time: 20 min, RT

Sample volume: 10 ll

Reagent volume: 1.0 ml

Reagent setting with: Reagent blank

The instrument was set using above system parameters.

The following was dispensed into test tubes:

t-0

like activity of beta-carotene in.1016/j.jff.2014.01.015

Blank

unstressed an

Standard

d chronic unpredi

Test

Reconstituted reagent
1 ml 1 ml 1 ml
Sample
– 10 ll –
Test
– – 10 ll
The tubes were incubated for 20 min at room temperature.

The absorbance was read at 546 nm using the instrument as

mentioned above.

2.7.2.3. Estimation of lipid peroxidation. The thiobarbituric

acid-reactive substances (TBARS), a measure of lipid peroxi-

dation, was assayed by the method of Wills, 1965. Briefly,

0.5 ml of postmitochondrial supernatant and 0.5 ml of Tris–

HCl were incubated at 37 �C for 2 h. After incubation, 1 ml of

10% trichloroacetic acid was added and centrifuged at 300g

for 10 min. To 1 ml of supernatant, 1 ml of 0.67% thiobarbitu-

ric acid was added, and the tubes were kept in boiling water

for 10 min. After cooling, 1 ml of double distilled water was

added, and absorbance was measured at 532 nm. Thiobarbi-

turic acid-reactive substances were quantified using an

extinction coefficient of 1.56 · 105 M�1 cm�1 and expressed

as nanomole of malondialdehyde equivalents per milligram

protein.

2.7.2.4. Estimation of reduced glutathione. Reduced glutathione

was assayed by the method of Jollow, Mitchell, Zampaglione,

and Gillette (1974). Briefly, 1.0 ml of postmitochondrial super-

natant (10%) was precipitated with 1.0 ml of sulfosalicylic acid

(4%). The samples were kept at 4 �C for at least 1 h and then

subjected to centrifugation at 1200 rpm for 15 min at 4 �C.

The assay mixture contained 0.1 ml supernatant, 2.7 ml phos-

phate buffer (0.1 M, pH 7.4), and 0.2 ml 5,50-dithiobis-(2-nitro-

benzoic acid) (Ellman’s reagent, 0.1 mM, pH 8.0) in a total

volume of 3.0 ml. The yellow colour developed was read imme-

diately at 412 nm, and GSH levels were calculated using molar

extinction coefficient of 1.36 · 104 M�1 cm�1) and expressed as

micromole per milligram protein.

2.7.2.5. Estimation of catalase activity. Catalase activity was

assayed by the method of Claiborne (1985). Briefly, the assay

mixture consisted of 1.95 ml phosphate buffer (0.05 M, pH

ctable



7.0), 1.0 ml hydrogen peroxide (0.019 M), and 0.05 ml postmi-

tochondrial supernatant (10%) in a final volume of 3.0 ml.

Changes in absorbance were recorded at 240 nm. Catalase

activity was quantified using the millimolar extinction

coefficient of H2O2 (0.07 mM) and expressed as micromoles

of H2O2 decomposed per minute per milligram protein.

2.8. Statistical analysis

All the results are expressed as Mean ± S.E.M. Data were ana-

lyzed by analysis of variance (ANOVA) followed by Tukey–Kramer

multiple comparison test Graph Pad Instat (GPIS) package, ver-

sion 3.05. p < 0.05 was considered as statistically significant.

3. Results

3.1. Effect of beta-carotene and imipramine on immobilityperiods of mice in TST

Chronic unpredictable mild stress significantly increased the

immobility period as compared to vehicle treated unstressed

mice. Imipramine (15 mg/kg, p.o.) and beta-carotene (100 mg/

kg, p.o.) per se administered for 21 successive days significantly

decreased the immobility period of stressed mice (p < 0.001) and

unstressed mice (p < 0.05 and p < 0.01, respectively) as com-

pared to their respective vehicle treated controls. But lower dose

of beta-carotene (50 mg/kg, p.o.) significantly (p < 0.05) de-

creased the immobility period of stressed mice as compared

to its vehicle treated control but did not significantly decrease

the immobility period of unstressed mice (Fig. 1).

3.2. Effect of beta-carotene and imipramine on sucrosepreference

Exposure of the mice to unpredictable mild stress for 21

successive days significantly (p < 0.01) decreased sucrose

Fig. 1 – Effect of beta-carotene and imipramine on

immobility periods of mice in TST. n = 10 in each group.

Values are expressed as Mean ± S.E.M. Data were analyzed

by one-way ANOVA followed by Tukey–Kramer multiple

comparison test. F (7,57) = 20.174; p < 0.0001. a, b and

c = p < 0.001, p < 0.01 and p < 0.05, respectively, as compared

to vehicle treated unstressed mice. d and e = p < 0.001 and

p < 0.05, respectively, as compared to vehicle treated

stressed mice.


preference (%) as compared to unstressed mice. There was

no significant difference in sucrose preference (%) among all

the groups in the baseline test. Beta-carotene (50 and

100 mg/kg) and imipramine (15 mg/kg) per se administered

for 21 successive days did not show any significant change

in sucrose preference by unstressed mice. Beta-carotene (50

and 100 mg/kg) and imipramine (15 mg/kg) per se signifi-

cantly restored the reduced sucrose preference (%) in stressed

mice as compared to its vehicle treated control (Table 1).

3.3. Effect of beta-carotene and imipramine on locomotoractivity

Various treatments did not significantly affect the spontane-

ous locomotor activity in unstressed and stressed mice as

compared to their respective vehicle treated controls (data

not shown).

3.4. Effect of beta-carotene and imipramine on plasmanitrite levels

Plasma nitrite levels were significantly (p < 0.001) increased in

mice subjected to chronic unpredictable mild stress. The low-

er dose of beta-carotene (50 mg/kg) administered for 21 suc-

cessive days did not show any significant effect on plasma

nitrite levels of unstressed mice, but higher dose (100 mg/

kg) of beta-carotene and imipramine per se significantly

(p < 0.05 and p < 0.01, respectively) decreased plasma nitrite

levels in unstressed mice as compared to its control. Beta-car-

otene (50 and 100 mg/kg) and imipramine (15 mg/kg) per se

administered for 21 successive days significantly (p < 0.05,

p < 0.001 and p < 0.001, respectively) decreased plasma nitrite

levels in stressed mice as compared to its vehicle treated con-

trol (Fig. 2).

3.5. Effect of beta-carotene and imipramine on plasmacorticosterone levels

Chronic unpredictable mild stress significantly (p < 0.001) in-

creased plasma corticosterone levels as compared to vehicle

treated unstressed mice. Beta-carotene (100 mg/kg) and imip-

ramine (15 mg/kg) per se administered for 21 successive days

significantly (p < 0.01) decreased the corticosterone levels of

stressed mice as compared to their respective vehicle treated

controls, but lower dose (50 mg/kg) of beta-carotene did not

significantly decrease plasma corticosterone level in stressed

mice. Beta-carotene (50 and 100 mg/kg) and imipramine

(15 mg/kg) administered for 21 successive days did not signif-

icantly decrease plasma corticosterone levels in unstressed

mice as compared to their respective controls (Fig. 3).

3.6. Effect of beta-carotene and imipramine on brainMAO-A activity

Chronic unpredictable mild stress significantly (p < 0.001)

increased brain MAO-A activity as compared to vehicle trea-

ted unstressed mice. Beta-carotene (100 mg/kg) and imipra-

mine (15 mg/kg) per se administered for 21 successive days

significantly reduced MAO-A activity in unstressed (p < 0.05

and p < 0.01, respectively) and stressed (p < 0.001 and



Table 1 – Effect of beta-carotene and imipramine on sucrose preference (%) of unstressed and stressed mice.

S. No. Treatment for 21 days Dose (kg�1) Sucrose preference (%) – baseline test Sucrose preference (%) – after 21 days

1 Vehicle (sesame oil) 10 ml 65.13 ± 4.64 44.43 ± 6.78

2 Vehicle (sesame oil) + CUMS 10 ml 68.30 ± 6.93 19.37 ± 1.04a

3 Imipramine (U) 15 mg 61.05 ± 8.81 55.24 ± 6.95

4 Beta-carotene (U) 50 mg 65.34 ± 7.83 42.06 ± 4.26

5 Beta-carotene (U) 100 mg 47.88 ± 8.61 52.19 ± 3.95

6 Imipramine + CUMS 15 mg 54.52 ± 4.97 41.44 ± 2.56b

7 Beta-carotene + CUMS 50 mg 44.20 ± 6.79 41.85 ± 1.49b

8 Beta-carotene + CUMS 100 mg 35.51 ± 8.81 44.78 ± 5.01c

U = unstressed mice; CUMS = chronic unpredictable mild stress.

n = 10 in each group. Values are expressed as Mean ± S.E.M. The data were analyzed by one-way ANOVA followed by Tukey–Kramer multiple

comparison test.

F (7,54) = 5.416; p < 0.0001 (after 21 days).

a = p < 0.01 as compared to vehicle treated unstressed mice.

b and c = p < 0.05 and p < 0.001, respectively, as compared to vehicle treated stressed mice.


p < 0.001, respectively) mice as compared to their respective

controls. But the lower dose (50 mg/kg) of beta-carotene did

not significantly decrease MAO-A activity in unstressed mice,

but significantly (p < 0.05) decreased MAO-A activity in

stressed mice as compared to their respective controls (Fig. 4).

3.7. Effect of beta-carotene and imipramine on brainTBARS levels

TBARS levels were increased significantly (p < 0.001) in mice

subjected to stressed paradigm as compared to vehicle trea-

ted unstressed mice. Beta-carotene (50 and 100 mg/kg) and

imipramine (15 mg/kg) per se administered for 21 days signif-

icantly (p < 0.05, p < 0.001 and p < 0.001, respectively) de-

creased TBARS levels in stressed mice as compared to

vehicle treated stressed mice. Beta-carotene (100 mg/kg) and

Fig. 2 – Effect of beta-carotene and imipramine on plasma

nitrite levels. n = 10 in each group. Values are expressed as

Mean ± S.E.M. Data were analyzed by one-way ANOVA

followed by Tukey–Kramer multiple comparison test. F

(7,57) = 15.445; (p < 0.0001). a, b and c = p < 0.01, p < 0.05 and


unstressed mice. d and e = p < 0.001 and p < 0.05,

respectively, as compared to vehicle treated stressed mice.


imipramine (15 mg/kg) significantly (p < 0.05 and p < 0.05,

respectively) decreased TBARS levels in unstressed mice as

compared to its control, but lower dose of (50 mg/kg) of

beta-carotene did not significantly decrease TBARS levels in

unstressed mice (Table 2).

3.8. Effect of beta-carotene and imipramine on brainreduced glutathione levels

Reduced glutathione levels were significantly (p < 0.001) de-

creased in stressed mice as compared to vehicle treated un-

stressed mice. Beta-carotene (50 and 100 mg/kg) and

imipramine (15 mg/kg) per se administered for 21 successive

days significantly (p < 0.05, p < 0.01 and p < 0.05, respectively) in-

creased reduced glutathione levels in both stressed and un-

stressed mice as compared to their respective controls (Table 2).

Fig. 3 – Effect of beta-carotene and imipramine on plasma

corticosterone levels. n = 10 in each group. Values are

expressed as Mean ± S.E.M. Data were analyzed by one-way

ANOVA followed by Tukey–Kramer multiple comparison

test. F (7,57) = 26.514; p < 0.0001. a = p < 0.001 as compared

to vehicle treated unstressed mice. b = p < 0.01 as compared

to vehicle treated stressed mice.



Fig. 4 – Effect of beta-carotene and imipramine on brain

MAO-A activity. n = 10 in each group. Values are expressed

as Mean ± S.E.M. Data were analyzed by one-way ANOVA

followed by Tukey–Kramer multiple comparison test. F

(7,56) = 74.998; p < 0.0001. a, b and c = p < 0.001, p < 0.01 and


unstressed mice. d and e = p < 0.001 and p < 0.05,

respectively, as compared to vehicle treated stressed mice.


3.9. Effect of beta-carotene and imipramine on braincatalase activity

Catalase levels were significantly (p < 0.001) increased in

brains of stressed mice as compared to vehicle treated un-

stressed mice. Beta-carotene (50 and 100 mg/kg) and imipra-

mine (15 mg/kg) per se produced a significant (p < 0.05,

p < 0.01 and p < 0.05, respectively) decrease in the catalase lev-

els of stressed mice as compared to its control. Only higher

dose (100 mg/kg) of beta-carotene and imipramine (15 mg/

kg) per se significantly (p < 0.05) decreased catalase levels of

unstressed mice as compared to its control (Table 2).

Table 2 – Effect of beta-carotene and imipramine on brain catalaof unstressed and stressed mice.

S. No. Drug treatments Dose (kg�1) Catalase actividecomposed/m

1 Vehicle (sesame oil) (U) 10 ml 58.37 ± 0.992

2 Vehicle (sesame oil) + CUMS 10 ml 46.48 ± 0.841a

3 Imipramine (U) 15 mg 61.88 ± 0.604d

4 Beta-carotene (U) 50 mg 57.91 ± 0.751

5 Beta-carotene (U) 100 mg 62.16 ± 0.851d

6 Imipramine (CUMS) 15 mg 49.69 ± 0.254d

7 Beta-carotene (CUMS) 50 mg 49.71 ± 0.649d

8 Beta-carotene (CUMS) 100 mg 50.45 ± 0.662e

U = unstressed mice; CUMS = chronic unpredictable mild stress.

n = 10 in each group. Values are expressed as Mean ± S.E.M. The data we

comparison test.

For catalase activity; F (7,57) = 76.949; p < 0.0001.

For Reduced gutathione; F (7,57) = 86.582; p < 0.0001.

For malondialdehyde equivalents; F (7,57) = 100.1; p < 0.0001.

a, b and c = p < 0.001, p < 0.01 and p < 0.05, respectively, as compared veh

d and e = p < 0.05 and p < 0.01, respectively, as compared to vehicle treat


4. Discussion

In the present investigation, beta-carotene administered for

21 successive days showed significant antidepressant-like

activity in unstressed and chronic unpredictable mild

stressed mice. Induction of depression using unpredictable

chronic mild stress is considered as the most valid animal

model of depressive behaviour observed in humans after long

term exposure to multiple stressors (Willner, 1991, 2005).

CUMS-induced depression model can be used for evaluating

the potential antidepressants by employing behavioural tests

like TST (Steru et al., 1985) and sucrose preference test

(Willner et al., 1987). In the present study, mice that were

exposed to chronic stress exhibited greater immobility

periods in TST as compared to control animals, thus showed

depression-like behaviour. Chronic treatment with imipra-

mine (15 mg/kg, p.o.) or beta-carotene (100 mg/kg, p.o.)

produced significant decrease in immobility periods of

unstressed and stressed mice in TST, indicating significant

antidepressant-like activity. Beta-carotene did not affect the

locomotor activity of the unstressed and stressed mice as

compared to control, thus ruling out its CNS stimulant activ-

ity. Therefore, antidepressant-like activity of beta-carotene in

unstressed and stressed mice is specific and not false posi-

tive. Moreover, we employed another model, sucrose prefer-

ence test for evaluation of antidepressant-like activity of

beta-carotene in stressed mice. This test is an indicator of

anhedonia-like behavioural change, indicating loss of interest

or pleasure. Anhedonia, a main symptom of human major

depression, was modelled by inducing a decrease in

responsiveness to rewards reflected by a reduced consump-

tion and/or preference of sweetened solutions (Willner,

1997, 2005). In our study, stressed mice showed a decrease

in sucrose preference compared with unstressed mice. Su-

crose preference was significantly restored in stressed mice

by chronic administration of imipramine (15 mg/kg, p.o.)

or beta-carotene (100 mg/kg, p.o.) which suggested their

se, reduced glutathione and malondialdehyde equivalents

ty (lmol of H2O2

in/mg protein)Reduced glutathione(lmol/mg protein)

Malondialdehydeequivalents(nmol/mg protein)

0.036 ± 0.001 0.927 ± 0.0018

0.015 ± 0.002a 1.947 ± 0.0012a

0.043 ± 0.002c 0.852 ± 0.0007c

0.043 ± 0.001c 0.911 ± 0.0020

0.044 ± 0.002b 0.840 ± 0.0011c

0.021 ± 0.001d 1.473 ± 0.0022d

0.021 ± 0.001d 1.871 ± 0.0013e

0.022 ± 0.001e 1.847 ± 0.0012d

re analyzed by one-way ANOVA followed by Tukey–Kramer multiple

icle treated unstressed mice.

ed stressed mice.




antidepressant-like actions. Thus, the results obtained from

behavioural studies indicated that beta-carotene produced

significant antidepressant-like action in mice exposed to

UCMS.

Hypothalamic–pituitary–adrenal (HPA) axis is activated in

response to stress, with resultant increase in circulating

glucocorticoids such as corticosterone in rodents or cortisol

in primates. Activation of HPA axis is associated with an abnor-

mally high blood glucocorticoid levels, which may eventually

lead to depression (Pan, Zhang, Xia, & Kong, 2006). Cortisol is

known to regulate neuronal survival, neuronal excitability,

neurogenesis and memory acquisition, and high levels of cor-

tisol may thus contribute to the manifestation of depressive

symptoms by impairing these brain functions (Sousa,

Cerqueira, & Almeida, 2008). It has been reported that chronic

antidepressant treatment in rodents reduce HPA activity

(Mason & Pariante, 2006). Thus, the restoration of a normal

functional status of HPA axis may be critically involved in the

treatment of clinical depression (Pan et al., 2006). CUMS-in-

duced hyperactivity of HPA axis, causes increased serum corti-

costerone level which is supported by observations from other

studies (Swaab, Bao, & Lucassen, 2005). Beta-carotene reduced

CUMS-induced hyperactivity of HPA axis in mice, as indicated

by significant reduction of plasma corticosterone levels. There

was no significant effect on plasma corticosterone levels in un-

stressed mice, indicating that hyperactivity of HPA axis is ob-

served only in stressful conditions.

Reactive oxygen species (ROS) play a role in some neuro-

psychiatric disorders such as major depression. Activation

of immune-inflammatory process, increased monoamine

catabolism, and abnormalities in lipids may cause overpro-

duction of ROS, lipid peroxidation, and reduced antioxidant

enzyme activities, and these processes may be related to

depression (Bilici et al., 2001). In present study, 21 days of

exposure to different stressors resulted in increase of brain

TBARS, reduced glutathione and plasma nitrite levels and

decrease in brain catalase levels. This is supported by an

earlier study where CUMS impaired the antioxidant status

(increased lipid peroxidation and nitrite levels, decreased

glutathione levels and catalase activity) of brain tissue,

presumably through production of excessive reactive oxygen

species (Kumar et al., 2011).

Chronic administration of beta-carotene and imipramine

per se showed significant decrease in brain TBARS in

unstressed and stressed mice; and increase in brain reduced

glutathione in both unstressed and stressed mice; and

decrease in brain catalase levels of both unstressed and

stressed mice. Thus, beta-carotene and imipramine showed

significant antioxidant activity in mice. It has been reported

that acute and chronic treatment with imipramine improved

oxidative stress parameters in rat brain (Reus et al., 2010). The

antioxidant activity of beta-carotene is also supported by an

earlier study (Kheir-Eldin, Motawi, Gad, & Abd-ElGawad,

2001; Manda & Bhatia, 2003). Stressful situations in rats have

also been reported to significantly increase plasma nitrite lev-

els (Lee et al., 2007). Beta-carotene (100 mg/kg, p.o.) and imip-

ramine per se significantly reduced nitrosative stress as

indicated by reduction of the plasma nitrite levels of

unstressed and stressed mice as compared to their respective

control groups. Thus, beta-carotene showed a strong


protective effect against oxidative stress that plays key role

in chronic unpredictable mild stress-induced depression.

Thus, oxidative stress is probably involved in the pathophys-

iology of depression, the modulation by imipramine and beta-

carotene could be an important mechanism of action of these

drugs. Further, chronic exposure to different stressors led to

increased activity of brain MAO-A. Chronic treatment with

beta-carotene (100 mg/kg) significantly inhibited MAO-A

activity in both unstressed and stressed mice. Thus, antide-

pressant-like activity of beta-carotene may also be attributed

to inhibition of MAO-A and consequent increase in brain

monoamine levels.

In conclusion, beta-carotene showed significant antide-

pressant-like activity in unstressed and stressed mice prob-

ably through inhibition of MAO-A activity, decrease in

plasma nitrite levels and due to its antioxidant activity. Fur-

thermore, antidepressant-like activity of beta-carotene in

stressed mice might also be due to decrease in plasma cor-

ticosterone levels.

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