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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
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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Day-7
Week-1
I E F O T2 X T1Week-2
I O X T2 E T1 FWeek-3
O F T1 X T2 I EI—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
t-like activity of beta-carotene in unstressed and chronic unpredictable0.1016/j.jff.2014.01.015
4 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
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
Please cite this article in press as: Dhingra, D., & Bansal, Y., Antidepressanmild stressed mice, Journal of Functional Foods (2014), http://dx.doi.org/1
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 mlSample
– 10 ll –Test
– – 10 llThe 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
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 5
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.
Please cite this article in press as: Dhingra, D., & Bansal, Y., Antidepressanmild stressed mice, Journal of Functional Foods (2014), http://dx.doi.org/1
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
t-like activity of beta-carotene in unstressed and chronic unpredictable0.1016/j.jff.2014.01.015
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.
6 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
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
p < 0.001, 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.
Please cite this article in press as: Dhingra, D., & Bansal, Y., Antidepressanmild stressed mice, Journal of Functional Foods (2014), http://dx.doi.org/1
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.
t-like activity of beta-carotene in unstressed and chronic unpredictable0.1016/j.jff.2014.01.015
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
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.
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 7
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
Please cite this article in press as: Dhingra, D., & Bansal, Y., Antidepressanmild stressed mice, Journal of Functional Foods (2014), http://dx.doi.org/1
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.
t-like activity of beta-carotene in unstressed and chronic unpredictable0.1016/j.jff.2014.01.015
8 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
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
Please cite this article in press as: Dhingra, D., & Bansal, Y., Antidepressanmild stressed mice, Journal of Functional Foods (2014), http://dx.doi.org/1
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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