Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 3 8 – 5 0

0006-8993/$ - see frohttp://dx.doi.org/10

nCorresponding autE-mail address:

Research Report

Piperine potentiates the protective effects of curcuminagainst chronic unpredictable stress-induced cognitiveimpairment and oxidative damage in mice

Puneet Rinwa, Anil Kumarn

Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study, Panjab University,

Chandigarh PIN-160014, India

a r t i c l e i n f o

Article history:

Accepted 1 October 2012

Life event stressors are the major vulnerability factors for the development of cognitive

disorders. A vital therapeutic for stress related disorders is curcumin, derived from curry spice

Available online 23 October 2012

Keywords:

Acetlycholinestrase

Bioavailability enhancer

Chronic unpredictable stress

Cognitive impairment

Oxidative stress

nt matter & 2012 Elsevie.1016/j.brainres.2012.10.0

hor. Fax: þ91 172 2543101kumaruips@yahoo.com (

a b s t r a c t

turmeric. Dietary phytochemicals are currently used as an adjuvant therapy to accelerate their

therapeutic efficacy. Therefore, the present study was designed to investigate the effect of

curcumin and its co-administration with piperine against chronic unpredictable stress (CUS)-

induced cognitive impairment and oxidative stress in mice. Male Laca mice were subjected to

undergo a battery of stressors for a period of 28 days. Vehicle/drugs were administered daily

30 mins before CUS procedure. Chronic stress significantly impaired memory performance

(delayed latency time to reach platform in Morris water maze as well as to reach closed arm in

elevated plus maze test) and decreased locomotor activity along with sucrose consumption.

Further, there was a significant impairment in oxidative parameters (elevated malondialdehyde,

nitrite concentration and decreased reduced glutathione, catalase levels) and mitochondrial

enzyme complex activities, along with raised acetylcholinesterase and serum corticosterone

levels. Chronic treatment with curcumin (200 and 400 mg/kg, p.o.) significantly improved these

behavioral and biochemical alterations, restored mitochondrial enzyme complex activities and

attenuated increased acetylcholinesterase and serum corticosterone levels. In addition,

co-administration of piperine (20 mg/kg; p.o.) with curcumin (100 and 200 mg/kg, p.o.) signifi-

cantly elevated the protective effect as compared to their effects alone. The results clearly

suggest that piperine enhanced the bioavailability of curcumin and potentiated its protective

effects against CUS induced cognitive impairment and associated oxidative damage in mice.

& 2012 Elsevier B.V. All rights reserved.

1. Introduction

Memory impairment is a common and usual comorbidity

associated with exposure to prolonged stress (Radley et al.,

2004). Chronic stress is known to influence cognitive task in

r B.V. All rights reserved.02

.A. Kumar).

various psychiatric patients (Vanitallie, 2002). Chronic stress

increases corticosterone secretion, which causes dysregula-

tion of hypothalamic–pituitary–adrenocortical (HPA) axis

and impairment of hippocampus-dependent learning and

memory processes (Kurukulasuriya et al., 2004). Secretion of

7 41Day Day

Day 1

Day 21

Day28

40

80

120

160

200

NaïveCUS ControlC(100)

C(200)C(400)P(20)

C(100)+P(20)C(200)+P(20)

a

b,c

c,d

c,e

b,c

c,e

c,e

b,c

a

a

a

c,e

b,cc,e b,c

b

d,e

b

c,eb,c

c,dd,e

C(400) Naïve

Loco

mot

or c

ount

s (s

ec)

Fig. 1 – Effects of curcumin, piperine and their combination

on locomotor activity. Values are expressed as mean7SEM.

For statistical significance, aPo0.05 as compared to naive

group; bPo0.05 as compared to CUS control; cPo0.05 as

compared to C(100); dPo0.05 as compared to C(200); ePo0.05

as compared to P(20) (Two-way ANOVA followed by Bonfer-

roni’s post test) [(9, 44)¼15.72, 39.22 for interaction of days

and treatment]. CUS, chronic unpredictable stress; C(100),

curcumin (100 mg/kg); C(100), curcumin (200 mg/kg); C(400),

curcumin (400 mg/kg); P(20), piperine (20 mg/kg).

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 3 8 – 5 0 39

corticosterone also triggers oxidative stress and ultimately

leads to memory deficits (Sato et al., 2010). Chronic stress is

also known to develop anhedonia like condition (Willner et al.,

1992). These physiological consequences of stress depend on

the intensity and duration of the stressor and on how an

organism perceives and reacts to the noxious stimulus (Joels,

2006). Chronic stress is also referred to as unpredictable when

the subjects are unaware of the type and time of stress. Based

on these observations, chronic unpredictable stress (CUS)

experimental model has been developed to study the develop-

ment and progress of stress pathology (Willner et al., 1992) and

related neurological disorders.

Degeneration of cholinergic neurons is a main change

affecting the specific neuronal functions in the brain of

patients with Alzheimer’s disease (Selkoe, 1991). Along with

this, evidences suggest that neuronal functions are altered by

generation of reactive oxygen species which leads to oxida-

tive stress; a prominent feature in pathogenesis of cognitive

dysfunction (Massaad and Klann, 2011). Various antioxidants

have been tried for their effectiveness in reducing deleterious

effects on neurons due to oxidative stress (Jara-Prado et al.,

2003). Dietary and medicinal phyto-antioxidants these days

are used as an adjuvant therapy with each other in order to

limit their side effects and to increase their effectiveness.

Curcumin, the yellow pigment extracted from the rhizomes

of Curcuma longa, has been extensively studied for its ther-

apeutic properties, such as antioxidant (Nafisi et al., 2009),

anti-inflammatory and neuroprotective activities (Motterlini

et al., 2000). Curcumin has been reported to possess free

radical scavenging activity against neurodegeneration asso-

ciated with Alzheimer’s disease (Calabrese et al., 2008;

Mancuso et al., 2012). Studies from our laboratory have also

suggested that curcumin restored mitochondrial enzymes

complexes activities and thereby attenuated the release of

reactive oxygen species (Kumar et al., 2011). Besides, we have

also assessed improved learning and memory performance

with curcumin in different experimental models (Kumar

et al., 2009). Manganese complexes of curcumin exhibited a

great capacity to protect brain lipids against peroxidation and

enhance superoxide dismutase (SOD) activity (Vajragupta

et al., 2003). Study also showed an inhibitory effect of

Curcuminoids on acetylcholinesterase activity against

scopolamine-induced amnesia (Ahmed and Gilani, 2009).

Curcumin is also reported to reduce serum corticosterone

level in restraint stress induced memory dysfunction (Xu

et al., 2009). These reported pharmacological properties of

curcumin clearly suggest its beneficial role against stress

induced cognitive impairment.

In spite of large number of reports on the beneficial effects

of curcumin, there have been instances of toxicity reported

with high doses of curcumin (Burgos-Moron et al., 2010;

Mancuso and Barone, 2009a). There have also been clinical

reports of occurrence of side effects in patients treated with

curcumin (Mancuso and Barone, 2009b). All these reports

clearly show that a lot of research work is needed to establish

the risk–benefit profile of curcumin.

Poor bioavailability of curcumin limits its approval as a

therapeutic agent. To overcome this problem piperine, a

major alkaloid of black pepper (Piper nigrum Linn.) and long

pepper (P. longum Linn.) has been employed as a combination

therapy in the given study since piperine is known to increase

the bioavailability of many drugs (Atal et al., 1985). In light of

these reports, present study aims to investigate the protective

effect of co-administration of curcumin with piperine against

chronic unpredictable stress induced cognitive deficits and

oxidative damage in mice.

2. Results

2.1. Effect of curcumin, piperine and their combination onlocomotor activity

28 days stress paradigm significantly decreased locomotor

activity in CUS control group as compared to naıve group of

animals (Fig. 1). Chronic curcumin (200 and 400 mg/kg) treat-

ment dose dependently improved locomotor activity which

was significant as compared to control (CUS) group. Further,

curcumin (100 mg/kg) could not significantly improve loco-

motor activity all through the study period. However,

co-administration of curcumin (100 and 200 mg/kg) with piperine

(20 mg/kg) potentiated their protective effects (increased loco-

motor activity) which was significant as compared to their

effects alone (Fig. 1). [F (9, 44)¼15.72, 39.22 (po0.001)].

2.2. Effect of curcumin, piperine and their co-administration on sucrose preference test

As shown in Fig. 2, chronically stressed animals showed

significant reduction in sucrose consumption as compared

to naive group of animals. However, curcumin (200 and

400 mg/kg) treatment dose dependently and significantly

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 3 8 – 5 040

improved sucrose consumption as compared to control (CUS)

mice. Besides, curcumin (100 mg/kg) treatment did not show any

significant effect on sucrose consumption. However, co-

administration of curcumin (100 and 200 mg/kg) with piperine

(20 mg/kg) potentiated their protective effect (increased sucrose

consumption) and were significant as compared to their effects

alone (Fig. 2). The combination effect of curcumin (200 mg/kg)

and piperine (20 mg/kg) was similar to the highest dose of

curcumin (400 mg/kg) [F (9, 44)¼8.67 (po0.001)].

Naïve

CUS Contr

ol

C(100)

C(200)

C(400)

P(20)

C(100)+

P(20)

C(200)+

P(20)

C(400)

Naïve

0

20

40

60

80

100

a

b,c c,e

c,d d,e

Sucr

ose

wat

er in

take

(% o

f tot

al fl

uid

inta

ke)

Fig. 2 – Effects of curcumin, piperine and their combination

on sucrose preference test. Values are expressed as

mean7SEM. For statistical significance, aPo0.05 as com-

pared to naive group; bPo0.05 as compared to CUS control;cPo0.05 as compared to C(100); dPo0.05 as compared to

C(200); ePo0.05 as compared to P(20) (One-way ANOVA

followed by Tukey’s test) [F (9, 44)¼8.67]. CUS, chronic

unpredictable stress; C(100), curcumin (100 mg/kg); C(100),

curcumin (200 mg/kg); C(400), curcumin (400 mg/kg); P(20),

piperine (20 mg/kg).

Table 1 – Effect of curcumin, piperine and their interaction on

Treatment (mg/kg) Initial transfer latency

Day 20

Naıve 64.074.57

CUS Control 67.073.41

C(100) 66.875.03

C(200) 70.475.38

C(400) 69.374.12

P(20) 64.273.77

C(100)þP(20) 61.673.51

C(200)þP(20) 63.472.22

C(100) Naıve 65.274.12

Values are expressed as mean7SEM.

CUS, chronic unpredictable stress; C(100), curcumin (100 mg/kg); C(100),

curcumin (400 mg/kg); P(20), piperine (20 mg/kg).

For statistical significance,a Po0.05 as compared to naive group.b Po0.05 as compared to CUS control.c Po0.05 as compared to C(100).d Po0.05 as compared to C(200).e Po0.05 as compared to P(20) (Two-way ANOVA followed by Bonferroni

[F (9, 44)¼23.12, 72.30 for interaction of days and treatment].

2.3. Effect of curcumin, piperine and their interaction onlatency time in elevated plus maze (EPM) task

Initial transfer latencies (ITL) on day 20 for all animals of

different groups were relatively stable and showed no sig-

nificant variations. Following training, CUS control mice

performed poorly throughout the experiment and did not

show any change in the retention transfer latencies (RTL) on

days 21 and 28 as compared to pre-training latency on day 20,

demonstrating chronic stress-induced memory impairment.

Besides, curcumin (200 and 400 mg/kg) treated mice showed

significant and dose dependent decrease in both 1st and

2nd RTL on days 21 and 28 (Table 1). Further, curcumin

(100 mg/kg) treatment did not show any significant effect on

retention transfer latencies; however, co-administration of

curcumin (100 and 200 mg/kg) with piperine (20 mg/kg) sig-

nificantly elevated their protective effects (shortened transfer

latency) when compared to their effects alone (Table 1).

In addition, the synergistic effect of the combination was

similar to that of highest dose of curcumin (400 mg/kg).

[F (9, 44)¼23.12, 72.30 (po0.001)].

2.4. Effects of curcumin, piperine and their combinationon Morris water maze test

The change in the escape latency time to reach the hidden

platform was observed in the training/acquisition trials.

Although there was a downward trend in escape latency time

(ELT) in water-maze training session for four days, yet the mean

latency (days 24–27) was significantly prolonged in the CUS

control group as compared to the naive group, indicating a

poorer learning performance (Fig. 3). Curcumin (200 and

400 mg/kg) treatment for 28 days significantly shortened escape

latency time in dose dependent manner as compared to control

(CUS). However, curcumin (100 mg/kg) did not show any sig-

nificant improvement in memory performance as compared to

latency time in elevated plus maze paradigm.

Retention transfer latency

Day 21(1st RTL) Day 28(2nd RTL)

15.071.71 11.271.68

73.272.66a 69.473.19a

63.072.26 52.172.93

45.073.44b,c 32.372.57b,c

25.071.83c,d 17.271.36c,d

70.074.77 66.673.44

47.272.77c,e 32.272.44c,e

26.271.12d,e 18.272.12d,e

14.271.23 10.4.71.34

curcumin (200 mg/kg); C(400),

’s post test)

Day 24 Day 25 Day 26 Day 2720

40

60

80

100

NaïveCUS ControlC(100)

C(200)C(400)P(20)

C(100)+P(20)C(200)+P(20)

aa

a

c,d

b,cc,e

b,c

c,eb,cc,e

b,c

bb

C(400) Naïve

c,e

d,eMea

n es

cape

late

ncy

time

(sec

)

Fig. 3 – Effects of curcumin, piperine and their combination

on escape latency time in Morris water maze. Values are

expressed as mean7SEM. For statistical significance,aPo0.05 as compared to naive group; bPo0.05 as compared

to CUS control; cPo0.05 as compared to C(100); dPo0.05 as

compared to C(200); ePo0.05 as compared to P(20) (Two-way

ANOVA followed by Bonferroni’s post test) [F (9, 44)¼21.84,

32.44 for interaction of days and treatment]. CUS, chronic

unpredictable stress; C(100), curcumin (100 mg/kg); C(100),

curcumin (200 mg/kg); C(400), curcumin (400 mg/kg); P(20),

piperine (20 mg/kg).

Naïve

n

CUS Cotro

l

C(100)

C(200)

C(400)

P(20)

C(100)+

P(20)

C(200)+

P(20)

C(400)

Naïve

0

20

40

60

80

100

a

b,c c,e

c,d d,e

Tim

e sp

ent i

n ta

rget

qua

dran

t (se

c)

Fig. 4 – Effects of curcumin, piperine and their combination

on time spent in target quadrant in Morris water maze.

Values are expressed as mean7SEM. For statistical signifi-

cance, aPo0.05 as compared to naive group; bPo0.05 as

compared to CUS control; cPo0.05 as compared to C(100);dPo0.05 as compared to C(200); ePo0.05 as compared to

P(20) (One-way ANOVA followed by Tukey’s test) [F (9, 44)¼

4.42]. CUS, chronic unpredictable stress; C(100), curcumin

(100 mg/kg); C(100), curcumin (200 mg/kg); C(400), curcumin

(400 mg/kg); P(20), piperine (20 mg/kg).

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 3 8 – 5 0 41

control (CUS). Further, combination of curcumin (100 and

200 mg/kg) with piperine (20 mg/kg) showed significant

improvement in the learning performance as compared to their

effects alone (Fig. 3). [F (9, 44)¼21.84, 32.44 (po0.001)].

Platform was removed on day 28 to estimate the retention

of memory. CUS control group significantly failed to recollect

the location of the platform, thus spending significantly less

time in the target quadrant as compared to naive group.

However, curcumin (200 and 400 mg/kg) treatment signifi-

cantly and dose dependently increased the time spent in the

target quadrant as compared to CUS control, indicating

improvement in cognitive performance (Fig. 4). Curcumin

(100 mg/kg) treatment did not show any significant improve-

ment in retention of memory; however curcumin (100 and

200 mg/kg) and piperine (20 mg/kg) together significantly

increased the time spent in target quadrant as compared to

their effects alone (Fig. 4) [F (9, 44)¼4.42 (po0.001)]. Further,

the combination effect of curcumin (200 mg/kg) with piperine

(20 mg/kg) in both acquisition and retrieval trials were com-

parable to the highest dose of curcumin (400 mg/kg) alone.

2.5. Effect of curcumin, piperine and their co-administration on oxidative stress parameters

Stress control animals showed significant increase in oxida-

tive damage as evidence by increased MDA, nitrite concen-

tration, and depleted GSH, catalase and SOD enzyme activity

as compared to naive group (Table 2). However, curcumin (200

and 400 mg/kg) treatment dose dependently attenuated levels

of oxidative stress which was also significant as compared to

CUS control. Curcumin (100 mg/kg) treatment did not

significantly improve oxidative stress levels; however co-

administration of curcumin (100 and 200 mg/kg) with piper-

ine (20 mg/kg) significantly potentiated their protective effect

(decreased MDA [F (9, 71)¼52.14 (po0.001)], nitrite concentra-

tion [F (9, 71)¼24.22 (po0.001)], restored GSH [F (9, 71)¼62.11

(po0.001)], SOD [F (9, 71)¼35.12 (po0.001)] and catalase [F (9,

71)¼83.20 (po0.001)] levels) as compared to their effects alone

and was comparable to curcumin (400 mg/kg) (Table 2).

2.6. Effect of curcumin, piperine and their combination onbrain acetylcholine levels

Chronic unpredictable stress for 28 days significantly

increased acetlycholinestrase enzyme activity in control

(CUS) animals as compared to the naive group (Fig. 5).

Curcumin (200 and 400 mg/kg) treatment dose dependently

attenuated acetlycholinestrase activity which was significant

as compared to control (CUS) group. Curcumin (100 mg/kg)

treatment did not show significant inhibition of brain acet-

lycholinestrase activity; however co-administration of curcu-

min (100 and 200 mg/kg) with piperine (20 mg/kg) potentiated

the attenuation effect which was significant as compared to

their effects alone (Fig. 5). The highest dose combination also

proved to have similar effects with that of curcumin (400 mg/kg)

[F (9, 71)¼22.75 (po0.001)].

2.7. Effects of curcumin, piperine and their interaction onmitochondrial respiratory enzyme complex activity

Chronic stress procedure impaired mitochondrial NADH

dehydrogenase (complex I) and succinate dehydrogenase

(complex II) activity which was significant as compared to

naive group (Table 3). Further, it also significantly reduced the

number of viable cells (complex III) and levels of cytochrome

Table 2 – Effect of curcumin, piperine and their co-administration on oxidative stress parameters.

Treatment

(mg/kg)

LPO (mol

of MDA/mgpr)

GSH (lmol

of GSH/mgpr)

Nitrite (lg/ml) Catalase (lmol

of H2O2/min/mgpr)

SOD (units/mgpr)

Naıve 0.15970.006 0.07570.005 303.3713.21 0.72770.011 58.2373.51

CUS Control 0.60770.029a 0.02170.004a 777.8716.26a 0.19570.032a 11.7272.12a

C(100) 0.54670.027 0.02870.003 716711.23 0.24370.022 16.4373.43

C(200) 0.43370.022b,c 0.03770.002b,c 566.2710.22b,c 0.39670.018b,c 31.6472.61b,c

C(400) 0.32370.012c,d 0.05570.005c,d 386.6712.16c,d 0.52870.026c,d 46.2173.30c,d

P(20) 0.57970.028 0.02470.003 755.6715.79 0.18870.044 12.1671.98

C(100)þP(20) 0.44470.022c,e 0.03970.002c,e 580.8711.17c,e 0.38770.066c,e 30.3373.11c,e

C(200)þP(20) 0.34270.019d,e 0.05270.002d,e 395.2712.20d,e 0.50670.043d,e 45.2174.30d,e

C(100) Naıve 0.14770.013 0.07370.004 307.1718.62 0.73370.036 57.61.72.57

Values are expressed as mean7SEM.

MDA [F (9, 71)¼52.14], nitrite [F (9, 71)¼24.22], GSH [F (9, 71)¼62.11], SOD [F (9, 71)¼35.12] and catalase [F (9, 71)¼83.20]. CUS, chronic

unpredictable stress; C(100), curcumin (100 mg/kg); C(100), curcumin (200 mg/kg); C(400), curcumin (400 mg/kg); P(20), piperine (20 mg/kg).

For statistical significance,a Po0.05 as compared to naive group.b Po0.05 as compared to CUS control.c Po0.05 as compared to C(100).d Po0.05 as compared to C(200).e Po0.05 as compared to P(20) (One-way ANOVA followed by Tukey’s test).

Naïve

CUS Contro

l

C(100)

C(200)

C(400)

P(20)

C(100)+

P(20)

C(200)+

P(20)

C(400)

Naïve

0.00

0.02

0.04

0.06

0.08

0.10 a

b,c

c,d

c,e

d,e

µmol

of a

cety

lthio

chol

ine

iodi

de h

ydro

lyze

d/m

in/m

g pr

Fig. 5 – Effect of curcumin, piperine and their combination on

brain acetylcholinesterase activity. Values are expressed as

mean7SEM. For statistical significance, aPo0.05 as com-

pared to naive group; bPo0.05 as compared to CUS control;cPo0.05 as compared to C(100); dPo0.05 as compared to

C(200); ePo0.05 as compared to P(20) (One-way ANOVA

followed by Tukey’s test) [F (9, 71)¼22.75]. CUS, chronic

unpredictable stress; C(100), curcumin (100 mg/kg); C(100),

curcumin (200 mg/kg); C(400), curcumin (400 mg/kg); P(20),

piperine (20 mg/kg).

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 3 8 – 5 042

C oxidase enzyme (complex IV) (Table 3). Curcumin (200 and

400 mg/kg) treatment significantly and dose dependently

restored mitochondrial enzyme complex I, II activities and

levels of complex III and cytochrome C enzymes as compared

to control (CUS). Further, curcumin (100 mg/kg) treatment did

not show any significant effects on altered levels of mito-

chondrial respiratory enzymes. However, combination of

curcumin (100 and 200 mg/kg) and piperine (20 mg/kg)

showed a significant potentiation in their protective effect

i.e. restored mitochondrial enzyme complex I [F (9, 71)¼32.11

(po0.001)], II [F (9, 71)¼42.30 (po0.001)], III [F (9, 71)¼28.21

(po0.001)], IV [F (9, 71)¼66.86 (po0.001)] activities. Further,

the effect of combination of curcumin (200 mg/kg) with

piperine (20 mg/kg) on mitochondrial respiratory enzyme

complex activity was similar to the highest dose of curcumin

(400 mg/kg).

2.8. Effect of curcumin, piperine and their combination onserum corticosterone (CORT) levels

A significant elevation in the serum CORT levels in stressed

animals was noticed as compared to naive group (Fig. 6).

Treatment with curcumin (200 and 400 mg/kg) dose depen-

dently attenuated the increased levels of serum CORT which

was significant as compared to CUS control. However, curcu-

min (100 mg/kg) treatment did not show significant inhibition

of serum CORT levels. Further, co-administration curcumin

(100 and 200 mg/kg) and piperine (20 mg/kg) significantly

lowered serum CORT levels as compared to their effects alone

and was comparable to curcumin (400 mg/kg) [F (9, 71)¼

150.20 (po0.001)] (Fig. 6).

3. Discussion

Stress is an unavoidable life experience that may attribute to

oxidative stress leading to cognitive disturbances. There

seem to be a complex relationship between stressful situa-

tions, mind and body’s reaction to stress, and the onset of

cognitive disturbances (Bhutani et al., 2009). Chronic admin-

istration of various uncontrollable stresses, a procedure

known as ‘‘chronic unpredictable stress’’, is generally thought

to be the most reliable and valuable experimental model to

study stress pathology in animals (Willner et al., 1992).

Chronic unpredictable stress (CUS) have been shown to

influence different regions of brain i.e. hippocampus and

prefrontal cortex (McFadden et al., 2011), which play a critical

role in spatial navigation and memory (Churchwell et al.,

Table 3 – Effect of curcumin, piperine and their interaction on mitochondrial respiratory enzyme complex I, II, III and IVactivities.

Treatment (mg/kg) Complex I

(n mol of NADH oxidized/min/mg pr)

Complex II

(n mol/mg pr)

Complex III

(no of viable cells/well)

Complex IV

(n mol/min/mg pr)

(% of Naıve) (% of Naıve) (% of Naıve) (% of Naıve)

Naıve 10073.2 10074.10 10075.45 10074.32

CUS Control 27.3272.1a 21.4071.76a 38.4274.45a 46.473.12a

C(100) 34.573.44 27.272.55 43.572.34 50.274.25

C(200) 55.271.22b,c 51.572.63b,c 62.274.52b,c 71.3574.26b,c

C(400) 79.274.85c,d 70.4373.42c,d 85.273.58c,d 89.2475.34c,d

P(20) 30.6373.48 24.2072.88 40.6372.48 48.2073.31

C(100)þP(20) 57.2072.65c,e 50.3271.95c,e 64.2074.66c,e 73.1174.23c,e

C(200)þP(20) 75.2073.15d,e 68.2272.5d,e 84.2073.45d,e 90.1174.34d,e

C(100) Naıve 98.2074.8 100.3472.1 100.2073.12 101.4475.15

Values are expressed as mean7SEM.

Complex I [F (9, 71)¼32.11], II [F (9, 71)¼42.30], III [F (9, 71)¼28.21] and IV [F (9, 71)¼66.86]. CUS, chronic unpredictable stress; C(100), curcumin

(100 mg/kg); C(100), curcumin (200 mg/kg); C(400), curcumin (400 mg/kg); P(20), piperine (20 mg/kg).

For statistical significance,a Po0.05 as compared to naive group.b Po0.05 as compared to CUS control.c Po0.05 as compared to C(100).d Po0.05 as compared to C(200).e Po0.05 as compared to P(20) (One-way ANOVA followed by Tukey’s test).

Naïve

CUS Contro

l

C(100)

C(200)

C(400)

P(20)

C(100)+

P(20)

C(200)+

P(20)

C(400)

Naïve

0

100

200

300

400

500

a

c,d

c,eb,c

d,e

Seru

m c

ortic

oste

rone

(ng/

ml)

Fig. 6 – Effect of curcumin, piperine and their combination on

serum corticosterone (CORT) levels. Values are expressed as

mean7SEM. For statistical significance, aPo0.05 as com-

pared to naive group; bPo0.05 as compared to CUS control;cPo0.05 as compared to C(100); dPo0.05 as compared to

C(200); ePo0.05 as compared to P(20) (One-way ANOVA

followed by Tukey’s test) [F (9, 71)¼150.20]. CUS, chronic

unpredictable stress; C(100), curcumin (100 mg/kg); C(100),

curcumin (200 mg/kg); C(400), curcumin (400 mg/kg); P(20),

piperine (20 mg/kg).

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 3 8 – 5 0 43

2010). Thus in the present study, piperine has been tried as a

drug strategy with curcumin against chronic unpredictable

stress induced oxidative damage and cognitive deficits

in mice.

In the present study, memory functions were evaluated by

Morris water maze (MWM) as well as elevated plus maze

(EPM). These two tests are often used as complementary to

each other. Though elevated plus maze is primarily used for

assessment of anxiety, it has also been employed as a model

for evaluation of memory in rodents (Sharma and Kulkarni,

1992). In the study, chronic unpredictable stress resulted in

significant impairment of cognitive tasks in both Morris

water maze and elevated plus maze performance task as

compared to naıve animals. These results are consistent with

the previous finding (Hoffman et al., 2011). Curcumin treat-

ment significantly and dose dependently improved cognitive

performance in both MWM and EPM indicating its therapeu-

tic potential against chronic stress induced memory impair-

ment. These results are in line with the previous findings

from our laboratory (Kumar et al., 2009). Along with the

cognitive deficits, there was also a significant decrease in

both locomotor activity and sucrose preference in CUS con-

trol animals as compared to naıve group. The results are in

accordance with previous studies by Gronli et al. (2005) which

showed a significant decrease in locomotor activity and

sucrose consumption following chronic mild stress. Further,

curcumin in a dose dependent manner significantly restored

the decrease in locomotor activity and sucrose preference.

These results are similar to the reports from previous studies

of our laboratory (Kumar and Singh, 2008). All these beha-

vioral tests respond selectively to chronic curcumin treat-

ment thus mimicking the clinical time course of memory

restorative action.

Hippocampus is reported to play a key role in spatial

learning and memory (Bai et al., 2009). Since hippocampus

has abundant inputs from the basal forebrain cholinergic

system and thus acetylcholine (ACh) plays a crucial role in

learning and memory (Prado et al., 2006). Acetylcholine is

degraded by the enzyme acetylcholinesterase, terminating

the physiological action of the neurotransmitter. Alzheimer’s

disease affects cholinergic system resulting in decreased

activity of acetylcholinesterase (Dai et al., 2002). Stress has

been well documented to induce alterations in activity of

acetylcholinesterase enzyme (Nijholt et al., 2004). In the

present study, CUS caused a significant decrease in the

acetylcholinesterase activity leading to memory deficits, but

later was significantly restored by chronic curcumin

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 3 8 – 5 044

treatment thereby implicating retrieval and retention of

memory processes. These results are in line with the earlier

reports from our laboratory (Kumar et al., 2009). This could be

one of the mechanistic pathways for the neuroprotective

effect of curcumin in cognitive dysfunction of CUS animals.

In addition to behavioral abnormalities, chronic stress is

also involved in activation of hypothalamic–pituitary–adrenal

(HPA) axis which has also been reported in Alzheimer’s

patients (Landfield et al., 2007). A central feature of the HPA

stress response is the synthesis and secretion of glucocorti-

coids (corticosterone in mice) from the adrenal cortex. Addi-

tionally, glucocorticoids secreted during stressful events are

known to influence memory consolidation and retrieval

(Roozendaal, 2002). In the present investigation, CUS animals

showed a significant increase in serum corticosterone levels

as compared to the naive group. Furthermore, chronic admin-

istration of curcumin exhibited a slow but effective increase in

sucrose preference, resulting in the HPA axis normalization in

the CUS animals. These results are also consistent with the

previous findings showing that increased corticosterone induced

by chronic unpredictable stress can be prevented by chronic

curcumin administration (Li et al., 2009).

Corticosterone administration is also known to promote

oxidative stress and consequently causes memory deficits

(Sato et al., 2010). The role of oxygen free radicals in

neurodegeneration and cognitive decline has been well

reviewed (Serrano and Klann, 2004). A number of findings

suggest that reactive oxygen species (ROS) can accumulate

excessively in the brain and can severely attenuate the

neuronal function (Massaad and Klann, 2011). Oxidative

stress is therefore implicated as one of the causes of cognitive

impairment (Keller et al., 2005). Besides, chronic stress is said

to promote oxidative stress and demolish antioxidant

defense system of the brain (Lucca et al., 2009), which may

form the basis for impaired memory. In the present investi-

gation, CUS resulted in significant oxidative damage as

indicated by increase lipid peroxidation, nitrite concentra-

tion, and depletion of reduced glutathione levels, catalase

and superoxide dismutase activity, thus strengthening the

oxidative theory of cognitive deficits and its complications.

Curcumin being a lipophilic molecule is known to possess

strong antioxidant activity (Bengmark, 2006). Curcumin is

reported to inhibit iron-induced lipid peroxidation (Reddy

and Lokesh, 1994), iNOS expression (Bengmark, 2006) and

specifically scavenge NO-based radicals (Sreejavan and Rao,

1997). Curcumin is known to enhance the reduced glu-

tathione levels in ethanol intoxicated animals (Rajkrishnan

et al., 1999). It has been reported in literature that curcumin

increases the levels of SOD and catalase in irradiated mice

(Koiram et al., 2007). In line with the above correlates,

curcumin in the present study significantly and dose depen-

dently attenuated these oxidative stress markers.

Generation of reactive oxygen species (ROS) may also be

associated to mitochondrial dysfunction since mitochondrial

respiratory chain is the major sources of superoxide anion

(O2–) generation (Jezek and Hlavata, 2005). Since the energy

production in mitochondria is catalyzed by various mem-

brane bound protein complexes, namely NADH–ubiquinol

oxidoreductase (complex-I), succinate–ubiquinol oxidoreduc-

tase (complex-II), ubiquinol cytochrome c oxidoreductase

(complex-III) and complex IV (cytochrome C oxidase) (Jezek

and Hlavata, 2005), thus imbalance in these mitochondrial

enzymes may lead to severe oxidative damage. Further

mitochondria impairment may also result in Ca2þ dysregula-

tion and activation of NOS. NO and superoxide radical (O2–)

may react to from peroxynitrite (ONOO–) which leads to

oxidative damage in mitochondria (Clementi et al., 1998).

The results of the present study indicate that CUS caused

significant impairment in different mitochondrial enzyme

complex activities which were later restored by curcumin

treatment, suggesting a potential role for curcumin in restor-

ing ROS generation in mitochondria. Thus the results

strongly support our hypothesis that the memory deficits

observed after chronic unpredictable stress might have arisen

as a result of mitochondria dysfunction, which is the key

factor for the production of ROS generation and ultimately

causing oxidative injury to neurons, which could therefore be

prevented by antioxidant treatment.

Poor oral bioavailability of curcumin limits its therapeutic

efficacy. Studies have reported that curcumin gets reduced

through alcohol dehydrogenase, followed by conjugations

like sulfation and glucuronidation in liver and intestine

(Wahlstrom and Blennow, 1978). Thus high concentrations

of curcumin cannot be achieved and maintained in plasma

and tissues after oral ingestion. This limited therapeutic

potential of curcumin causes a major hindrance for its

clinical development. Recent clinical reports suggest that

only a small fraction of ingested curcumin reaches the

plasma level in patients thereby showing its poor oral

bioavailability (Baum et al., 2008; Mancuso et al., 2011).

Therefore, one of strategy to overcome the poor oral bioavail-

ability of curcumin involves use of bioavailability enhancers

which could potentiate the amount of oral curcumin reaching

plasma.

In the present study, piperine was co-administered with

curcumin to enhance its oral bioavailability. Piperine is a

potent inhibitor of hepatic and intestinal glucuronidation

(Atal et al., 1985), thus co-administration of piperine with

curcumin prevents intestinal and hepatic metabolism of

curcumin thereby increases free form of native curcumin,

responsible for its protective effect. However, there have been

some reports on inhibition of drug metabolizing enzymes

(CYP3A4) on combination of curcumin with piperine which

could further alter the metabolism of several drugs and

originate toxic effects (Mancuso and Barone, 2009a). Besides,

in present study no additional drug was given with curcu-

minþpiperine combination, thus care was taken to control

reported alterations in metabolism of drugs and its asso-

ciated toxic effects.

In the study we witnessed a profound increase in protective

effects of curcumin when co-administered with piperine.

However, in our study as well as previous literatures have

suggested that piperine does not exert any antioxidant

activity alone. The potentiation in the effects of combination

indicates that piperine might have increased the bioavail-

ability of curcumin possibly through inhibition of its intest-

inal glucuronidation resulting into increased absorption of

curcumin.

The present study clearly demonstrates the memory

restorative and antioxidant properties of curcumin due to

Fig. 7 – Experimental design for chronic unpredictable stress protocol. Different sets of animals were used for estimation of

locomotor activity, sucrose consumption, Morris water maze test and elevated plus maze and were studied independent of

each other. At the end of the study animals were clubbed and divided into different groups for biochemical, mitochondrial

and serum corticosterone estimations.

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 3 8 – 5 0 45

its multifactorial nature, which further shows elevated

effects on combination with a bioavailability enhancer,

piperine. Further these findings provide a scientific rationale

for the co-administration of piperine and curcumin, which

may act as a useful and potent adjuvant in the treatment of

cognitive disorders.

4. Statistical analysis

All the values were expressed as mean7SEM. The behavioral

data were analyzed by Two-way analysis of variance (ANOVA)

followed by Bonferroni’s post test to calculate the statistical

significance between various groups. All other test data were

analyzed using One way analysis of variance (ANOVA) fol-

lowed by post hoc Tukey’s test. The criterion for statistical

significance was Po0.05. All statistical procedures were

carried out using sigma stat Graph Pad Prism (Graph Pad

Software, San Diego, CA)

5. Experimental procedures

5.1. Animals

Male Laca mice (30–35 g) bred at Central Animal House (CAH)

Panjab University, Chandigarh, were used. They were housed

under standard (2572 1C, 60–70% humidity) laboratory

conditions, maintained on a 12 h natural day–night cycle,

with free access to standard food and water. Animals

were acclimatized to laboratory conditions before the test.

The experimental protocols were approved by the Institu-

tional Animal Ethical Committee (IAEC) of Panjab University

(IAEC/170–175) and conducted according to the CPCSEA

guidelines on the use and care of experimental animals.

5.2. Drugs and treatment schedule

Following drugs were used in the present study. Curcumin

and piperine were purchased from Sigma Chemicals Co.

(St. Louis, MO, USA). All other chemicals used for biochemical

and mitochondrial estimations were of analytical grade. The

animals were randomly divided into nine experimental

groups. First and second group was named as naıve and

control (CUS) group respectively. Curcumin (100, 200 and

400 mg/kg, p.o.) were treated as groups 3–5 respectively.

Piperine (20 mg/kg, p.o.) served as group 6. Co-

administration of piperine (20 mg/kg) with curcumin (100

and 200 mg/kg) was categorized as groups 7 and 8 respec-

tively. Curcumin Naıve (400 mg/kg, p.o.) (without stress pro-

cedure, treatment to naıve animals) served as group 9.

Curcumin and piperine were prepared in peanut oil and

administered orally on the basis of body weight (1 ml/100 g).

Solutions were made freshly at the beginning of each day of

the drug treatment. Drugs were administered daily 30 min

before CUS procedure for 28 days. The doses of curcumin and

piperine were selected on the basis of literature and labora-

tory reports (Mehla et al., 2010; Hlavackova et al., 2011). The

detailed experimental design for chronic unpredictable stress

protocol is shown in Fig. 7.

5.3. Chronic unpredictable stress procedures

Mice were exposed to a random pattern of mild stressors

(Murua et al., 1991) daily for 28 days. The order of stressors

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 3 8 – 5 046

used is depicted below:

C—Cold swim (8 1C, 5 min); T—Tail pinch (1 min); F—Food

and water deprivation (24 h); S—Swimming at room tempera-

ture (2472 1C, 20 min); O—Overnight illumination; N—No

stress; T1—Tail pinch (1.5 min); C1—Cold swim (10 1C,

5 min); S1—Swimming at room temperature (2472 1C,

15 min); T2—Tail pinch (2 min); C2—Cold swim (6 1C, 5 min).

5.4. Behavioral studies

5.4.1. Locomotor activityAnimal was kept in actophotometer for the first 3 min as a

habituation period before actual recording of locomotor

activity for 5 min. Each animal was placed in a square

(30 cm) closed arena equipped with infra-red light sensitive

photocells (digital actophotometer, IMCORP, India) and values

expressed as counts per 5 min. The apparatus was placed in a

darkened, light and sound attenuated and ventilated testing

room (Kumar and Garg, 2008).

5.4.2. Sucrose preference testThe sucrose preference test (SPT) was conducted on last day

of the study period. The mice were tested for sucrose

consumption as described earlier (Bhagya et al., 2011). Ani-

mals were housed individually throughout the test duration

and were presented simultaneously with two bottles in the

home cage, one containing a 1% sucrose solution, and other

containing standard drinking water during the 48 h training

session (every week before SPT). To prevent the preference to

position, the location of the two bottles (right/left) was varied

during this period. After an 18-h period of food and water

deprivation, an 8 h test session was conducted. The amount

of liquid remaining in each bottle was measured at the end of

the testing period. The sucrose preference score was

expressed as percent of total fluid intake. Sucrose preference

(SP) was calculated according to the following equation:

SP¼Sucrose intake ðgÞ

Sucrose intake ðgÞ þwater intake ðgÞ

� �� 100

5.4.3. Assessment of cognitive performance

5.4.3.1. Elevated plus maze paradigm. The elevated plus

maze (EPM) consisted of two opposite black open arms

(16�5 cm), crossed with two closed walls of the same

dimensions of 12 cm height. The arms were connected with

a central square of dimensions 5�5 cm. The entire maze was

elevated to a height of 25 cm from the floor. Acquisition and

retention of memory processes were assessed as previously

described (Sharma and Kulkarni, 1992). Acquisition of

memory was tested on day 20 of CUS procedure. Animal

was placed individually at one end of the open arm facing

away from the central square. The time taken by the animal

to move from the open arm to the closed arm was recorded

as the initial transfer latency (ITL). Animal was allowed to

explore the maze for 20 s after recording the ITL and then

returned to the home cage. If the animal could not enter

closed arm within 90 s, same was guided to the closed arm

and ITL was given as 90 s. Retention of memory was assessed

by placing the mouse again in an open arm and the retention

latency was noted on day 21 and day 28 of ITL and was

termed as the first retention transfer latency (1st RTL) and

second retention transfer latency (2nd RTL), respectively.

5.4.3.2. Morris water-maze test. Morris water-maze appara-

tus (MWM) is most commonly used model to test memory

(Morris, 1984). The MWM procedure is based on the principle

that an animal dislikes swimming and hence when placed in

a large pool of water its tendency is to escape it by searching

for a platform. MWM consisted of large circular pool (150 cm

in diameter, 45 cm in height, filled to a depth of 30 cm with

water at 2871 1C). The water was made opaque with white

colored dye. The tank was divided into four equal quadrants.

A submerged platform (10 cm�10 cm), painted white was

placed in the middle of the target quadrant of this pool, 1 cm

below surface of water. The position of platform was kept

unaltered throughout the training session. The tank was

located in a large room where there were several brightly

colored cues external to the maze; these were visible from the

pool and could be used by the mice for spatial orientation.

The position of the cues remained unchanged throughout the

study. The water maze task was carried out for four con-

secutive days from day 10th to day 13th. The mice received

four consecutive daily training trials in the following 4 days,

with each trial having a ceiling time of 120 s. For each trial,

individual mouse was gently put into the water at one of four

starting positions, the sequence of which being selected

randomly and allowed 120 s to locate submerged platform.

Then, it was allowed to stay on the platform for 20 s.

If animal failed to find the platform within 120 s, it was guided

gently onto platform and allowed to remain there for 20 s.

Acquisition trial—Each mouse was subjected to four trials on

each day. A rest period of 1 h was allowed in between each

trial. Four trials per day were repeated for four consecutive

days. Starting position on each day to conduct four acquisi-

tion trials was changed as described below and Q4 was

maintained as target quadrant in all acquisition trials.

Day1

Q1 Q2 Q3 Q4

Day2

Q2 Q3 Q4 Q1

Day3

Q3 Q4 Q1 Q2

Day4

Q4 Q1 Q2 Q3

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 3 8 – 5 0 47

Mean escape latency time (ELT) calculated for each day

during acquisition trials was used as an index of acquisition.

Retrieval trial—On fifth day (day 14th) the platform was

removed. Animal was placed in water maze and allowed to

explore the maze for 120 s. Mean time spent in all three

quadrants, i.e. Q1, Q2 and Q3 were recorded and the time

spent in the target quadrant, i.e. Q4 in search of missing

platform provided an index of retrieval. Care was taken that

relative location of water maze with respect to other objects

in the laboratory serving as prominent visual clues was not

disturbed during the total duration of study.

5.5. Dissection and homogenization

Immediately after the last behavioral test, animals were

randomized into two groups; one group was used for the

biochemical assays. For biochemical analysis, animals were

sacrificed by decapitation. Whole brain of each animal was

put on ice and weighed. A 10% (w/v) tissue homogenates

were prepared in 0.1 M phosphate buffer (pH 7.4). The

homogenates were centrifuged at 10,000�g for 15 min and

aliquots of supernatant were separated and used for bio-

chemical estimation.

5.6. Estimations of oxidative stress parameters

5.6.1. Lipid peroxidationThe extent of lipid peroxidation was determined quantita-

tively by performing the method as described by Wills (1966).

The amount of malondialdehyde (MDA) was measured by

reaction with thiobarbituric acid at 532 nm using Perkin

Elmer Lambda 20 spectrophotometer (Norwalk, CT, USA).

The values were calculated using the molar extinction co-

efficient of chromophore (1.56�10 M�1 cm�1).

5.6.2. NitriteThe accumulation of nitrite in the supernatant, an indicator

of the production of nitric oxide was determined by a

colorimetric assay with Greiss reagent (0.1% N-(1-napththyl)

ethylene diamine dihydrochloride, 1% sulfanilamide and 5%

phosphoric acid) (Green et al., 1982). Equal volumes of the

supernatant and the Greiss reagent were mixed and the

mixture was incubated for 10 min at room temperature in

the dark. The absorbance was measured at 540 nm using

Perkin Elmer Lambda 20 spectrophotometer (Norwalk, CT,

USA). The concentration of nitrite in the supernatant was

determined from sodium nitrite standard curve.

5.6.3. Reduced glutathioneReduced glutathione in the brain was estimated according to

the method of Ellman (1959). Homogenates were precipitated

with 1.0 ml of 4% sulfosalicylic acid by keeping the mixture at

4 1C for 1 h and the samples were immediately centrifuged at

1200�g for 15 min at 4 1C. The assay mixture contained

0.1 ml of supernatant, 2.7 ml of phosphate buffer of pH 8

and 0.2 ml of 0.01 M dithiobisnitrobenzoic acid (DTNB). The

yellow color developed was read immediately at 412 nm using

Perkin Elmer lambda 20 spectrophotometer (Norwalk, CT,

USA). The results were expressed as nanomoles of reduced

glutathione per milligram of protein.

5.6.4. Superoxide dismutase activitySuperoxide dismutase (SOD) activity was assayed by the

method of Kono (1978) where the reduction of nitrazobluete-

trazolium (NBT) was inhibited by the superoxide dismutase

and is measured. The assay system consists of EDTA 0.1 mM,

sodium carbonate 50 mM and 96 mM of nitro blue tetrazo-

lium (NBT). In the cuvette, 2 ml of the above mixture, 0.05 ml

of hydroxylamine and 0.05 ml of the supernatant was added

and auto-oxidation of hydroxylamine was measured for

2 min at 30 s intervals by measuring absorbance at 560 nm

using Perkin Elmer Lambda 20 spectrophotometer (Norwalk,

CT, USA).

5.6.5. CatalaseCatalase activity was determined by Luck (1971), wherein the

breakdown of hydrogen peroxide (H2O2) is measured at

240 nm. Briefly, the assay mixture consisted of 3 ml of H2O2,

phosphate buffer and 0.05 ml of supernatant of tissue homo-

genates (10%), and the change in absorbance was recorded at

240 nm using Perkin Elmer lambda 20 spectrophotometer

(Norwalk, CT, USA). The results were expressed as micro-

moles of H2O2 decomposed per milligram of protein/min.

5.6.6. ProteinThe protein content was estimated by biuret method (Gornall

et al., 1949) using bovine serum albumin as a standard.

5.7. Estimation of acetyl cholinesterase (AChE) activity

AchE is a marker of loss of cholinergic neurons in the brain

region. The AchE activity was assessed as described by

Ellman et al. (1961). The assay mixture contained 0.05 ml of

supernatant, 3 ml of sodium phosphate buffer (pH 8), 0.1 ml

of acetylthiocholine iodide and 0.1 ml of DTNB (Ellman

reagent). The change in absorbance was measured for 2 min

at 30 s intervals at 412 nm using Perkin Elmer lambda 20

spectrophotometer (Norwalk, CT, USA). Results were

expressed as micromoles of acetylthiocholine iodide hydro-

lyzed per min per mg of protein.

5.8. Mitochondrial enzyme complex estimations

Second group of animals were used for mitochondrial

enzyme complex isolation as described in the method of

Berman and Hastings (1999). The whole brain was homo-

genized in isolated buffer. Homogenates were centrifuged at

13,000 g for 5 min at 4 1C. Pellets were re-suspended in

isolation buffer with ethylene glycol tetraacetic acid (EGTA)

and spun again at 13,000 g at 4 1C for 5 min. The resulting

supernatants were transferred to new tubes and topped off

with isolation buffer with EGTA and again spun at 13,000 g at

4 1C for 10 min. Pellets containing pure mitochondria were re-

suspended in isolation buffer without EGTA

5.8.1. Complex-I (NADH dehydrogenase activity)Complex-I was measured spectrophotometrically by the

method of King and Howard (1967). The method involves

catalytic oxidation of NADH to NADþ with subsequent reduc-

tion in cytochrome C. The reaction mixture contained 0.2 M

glycyl glycine buffer pH 8.5, 6 mM NADH in 2 mM glycyl

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 3 8 – 5 048

glycine buffer and 10.5 mM cytochrome C. The reaction was

initiated by addition of requisite amount of solubilised

mitochondrial sample and followed absorbance change at

550 nm for 2 min.

5.8.2. Complex-II (succinate dehydrogenase activity)Complex-II was measured spectrophotometrically according

to King (1967). The method involves oxidation of succinate by

an artificial electron acceptor, potassium ferricyanide. The

reaction mixture contained 0.2 M phosphate buffer pH 7.8, 1%

BSA, 0.6 M succinic acid, and 0.03 M potassium ferricyanide.

The reaction was initiated by the addition of mitochondrial

sample and absorbance change was followed at 420 nm for

2 min.

5.8.3. Complex-III (MTT activity)The MTT assay is based on the reduction of 3-(4,5-dim-

ethylthiazol-2-yl)-2,5-diphenyl-H-tetrazolium bromide (MTT)

by hydrogenase activity in functionally intact mitochondria.

The MTT reduction rate was used to assess the activity of the

mitochondrial respiratory chain in isolated mitochondria by

the method of Liu et al. (1997). Briefly, 100 ml mitochondrial

samples were incubated with 10 ml MTT for 3 h at 37 1C. The

blue formazan crystals were solubilised with dimethylsulf-

oxide and measured by an ELISA reader at 580 nm filter.

5.8.4. Complex IV (cytochrome c oxidase)Cytochrome oxidase activity was assayed in brain mitochondria

according to the method of Sottocasa (Sottocasa et al., 1967).

The assay mixture contained 0.3 mM reduced cytochrome C in

75 mM phosphate buffer. The reaction was started by the

addition of solubilized mitochondrial sample and the changes

in absorbance were recorded at 550 nm for 2 min.

5.9. Serum corticosterone estimations

5.9.1. Preparation of serumAnimals were sacrificed and blood was collected immediately

thereafter between 8.00–9.00 AM. Blood collected in the test

tubes was allowed to clot at room temperature. The tubes

were then centrifuged at 2000 rpm for 10 min and the straw

colored serum was separated and stored frozen at �20 1C.

5.9.2. Corticosterone assessmentFor extraction of corticosterone the method of Silber et al.

(1958) was modified as described. 0.1–0.2 ml of serum were

treated with 0.2 ml of freshly prepared chloroform: methanol

mixture (2:1, v/v), followed by 3 ml of chloroform instead of

dichloromethane used in the procedure of Silber et al. The

step of treatment of petroleum ether was omitted. The

samples were vortexed for 30 s and centrifuged at 2000 rpm

for 10 min. The chloroform layer was carefully removed with

the help of syringe with a long 16 gauge needle attached to it

and was transferred to a fresh tube. The chloroform extract

was then treated with 0.1 N NaOH by vortexing rapidly and

NaOH layer was rapidly removed. The sample was then

treated with 3 ml of 30 N H2SO4 by vortexing vigorously. After

phase separation, chloroform layer on top was removed using

a syringe as described above and discarded. The tubes

containing H2SO4 were kept in dark for 30–60 min and

thereafter fluorescence measurements carried out in fluores-

cence spectrophotometer (make Hitachi, model F-2500) with

excitation and emission wavelength set at 472 and 523.2 nm

respectively. The standard curve depicting the fluorescence

yield versus corticosterone concentration was used for result

analysis.

Acknowledgment

Authors gratefully acknowledged the research grant of Indian

Council of Medical Research (ICMR), New Delhi for carrying

out this work.

r e f e r e n c e s

Ahmed, T., Gilani, A.H., 2009. Inhibitory effect of curcuminoids onacetylcholinesterase activity and attenuation of scopolamine-induced amnesia may explain medicinal use of turmeric inAlzheimer’s disease. Pharmacol. Biochem. Behav. 91, 554–559.

Atal, C.K., Dubey, R.K., Singh, J.J., 1985. Biochemical basis ofenhanced drug bioavailability by piperine: evidence thatpiperine is a potent inhibitor of drug metabolism. Pharmacol.Exp. Ther. 232, 258–262.

Bai, F., Zhang, Z., Watson, D.R., Yu., Shi, Y., Yuan, Y., Zang, Y., Zhu,C., Qian, Y., 2009. Abnormal functional connectivity of hippo-campus during episodic memory retrieval processing networkin amnestic mild cognitive impairment. Biol. Psychiatry 65,951–958.

Baum, L., Lam, C.W., Cheung, S.K., Kwok, T., Lui, V., Tsoh, J., Lam, L.,Leung, V., Hui, E., Ng, C., Woo, J., Chiu, H.F., Goggins, W.B., Zee,B.C., Cheng, K.F., Fong, C.Y., Wong, A., Mok, H., Chow, M.S., Ho,P.C., Ip, S.P., Ho, C.S., Yu, X.W., Lai, C.Y., Chan, M.H., Szeto, S.,Chan, I.H., Mok, V., 2008. Six-month randomized, placebo-controlled, double-blind, pilot clinical trial of curcumin inpatients with Alzheimer disease. J. Clin. Psychopharmacol. 28(1), 110–113.

Bengmark, S., 2006. Curcumin, an atoxic antioxidant and naturalNFkB, cyclooxygenase-2, lipoxygenases, and inducible nitricoxide synthase inhibitor: a shield against acute and chronicdiseases. J. Parenter. Enteral. Nutr 30, 45–51.

Berman, S.B., Hastings, T.G., 1999. Dopamine oxidation altersmitochondrial respiration and induces permeability transitionin brain mitochondria: implications for Parkinson’s disease.J. Neurochem. 73, 1127–1137.

Bhagya, V., Srikumar, B.N., Raju, T.R., Shankaranarayana, Rao.B.S,2011. Chronic escitalopram treatment restores spatial learning,monoamine levels, and hippocampal long-term potentiation inan animal model of depression. Psychopharmacology (Berl) 214,477–494.

Bhutani, M.K., Bishnoi, M., Kulkarni, S.K., 2009. Anti-depressantlike effect of curcumin and its combination with piperine inunpredictable chronic stress-induced behavioral, biochemicaland neurochemical changes. Pharmacol. Biochem. Behav. 92,39–43.

Burgos-Moron, E., Calderon-Montano, J.M., Salvador, J., Robles, A.,Lopez-Lazaro, M., 2010. The dark side of curcumin. Int.J. Cancer 126, 1771–1775.

Calabrese, V., Bates, T.E., Mancuso, C., Cornelius, C., Ventimiglia, B.,Cambria, M.T., Di Renzo, L., De Lorenzo, A., Dinkova-Kostova,A.T., 2008. Curcumin and the cellular stress response in freeradical-related diseases. Mol. Nutr. Food Res. 52, 1062–1073.

Churchwell, J.C., Morris, A.M., Musso, N.D., Kesner, R.P., 2010.Prefrontal and hippocampal contributions to encoding and

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 3 8 – 5 0 49

retrieval of spatial memory. Neurobiol. Learn. Mem. 93,

415–421.Clementi, E., Brown, G.C., Feelisch, M., Moncada, S., 1998. Persis-

tent inhibition of cell respiration by nitric oxide: crucial role of

S-nitrosylation of mitochondrial complex I and protective

action of glutathione. Proc. Natl. Acad. Sci. USA 95, 7631–7636.Dai, J., Buijs, R.M., Kamphorst, W., Swaab, D.F., 2002. Impaired axonal

transport of cortical neurons in Alzheimer’s disease is associated

with neuropathological changes. Brain Res. 948, 138–144.Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem.

Biophys. 82, 70–77.Ellman, G.L., Courtney, V., Andres, R.M., 1961. A new and rapid

colorimetric determination of acetylcholinesterase activity.

Biochem. Pharmacol. 7, 88–95.Gornall, A.G., Bardawill, C.J., David, M.M., 1949. Determination of

serum proteins by means of the biuret reaction. J. Biol. Chem.

177, 751–766.Green, L.C., Wagner, D.A., Glagowski, J., 1982. Analysis of nitrate,

nitrite and (15 N) nitrate in biological fluids. Anal. Biochem.

126, 131–138.Gronli, J., Murison, R., Fiske, E., Bjorvatn, B., Sorensen, E., Portas, C.M.,

Ursin, R., 2005. Effects of chronic mild stress on sexual behavior,

locomotor activity and consumption of sucrose and saccharine

solutions. Physiol. Behav. 84, 571–577.Hlavackova, L., Janegova, A., Ulicna, O., Janega, P., Cerna, A.,

Babal, P., 2011. Spice up the hypertension diet -curcumin and

piperine prevent remodeling of aorta in experimental L-NAME

induced hypertension. Nutr. Metab. (Lond) 17, 72.Hoffman, A.N., Krigbaum, A., Ortiz, J.B., Mika, A., Hutchinson, K.M.,

Bimonte-Nelson, H.A., Conrad, C.D., 2011. Recovery after chronic

stress within spatial reference and working memory domains:

correspondence with hippocampal morphology. Eur. J. Neurosci.

34, 1023–1030.Jara-Prado, A., Ortega-Vazquez, A., Martinez-Ruano, L., Rios, C.,

Santamaria, A., 2003. Homocysteine-induced brain lipid per-

oxidation, effects of NMDA receptor blockade, antioxidant

treatment, and nitric oxide synthase inhibition. Neurotoxicol-

ogy 5, 237–243.Jezek, P., Hlavata, L., 2005. Mitochondria in homeostasis of

reactive oxygen species in cell, tissues, and organism. Int.

J. Biochem. Cell Biol. 37, 2478–2503.Joels, M., 2006. Corticosteroid effects in the brain: U-shape it.

Trends Pharmacol. Sci. 27, 244–250.Keller, J.N., Schmitt, F.A., Scheff, S.W., Ding, Q., Chen, Q.,

Butterfield, D.A., Markesbery, W.R., 2005. Evidence of increased

oxidative damage in subjects with mild cognitive impairment.

Neurology 64, 1152–1156.King, T.E., 1967. Preparation of succinate dehydrogenase and

reconstitution of succinate oxidase. Methods Enzymol. 10,

322–331.King, T.E., Howard, R.L., 1967. Preparations and properties of

soluble NADH dehydrogenases from cardiac muscle. Methods

Enzymol. 10, 275–294.Koiram, P.R., Veerapur, V.P., Kunwar, A., Mishra, B., Barik, A.,

Priyadarshani, I.K., Mazhuvancherry, U.K., 2007. Effect of

curcumin and curcumin copper complex (1:1) on radiation-

induced changes of anti-oxidant enzymes levels in the livers

of Swiss albino mice. J. Radiat. Res. 48, 241–245.Kumar, A., Dogra, S., Prakash, A., 2009. Protective effect of

curcumin (Curcuma longa), against aluminium toxicity: possi-

ble behavioral and biochemical alterations in rats. Behav.

Brain Res 205, 384–390.Kumar, A., Garg, R., 2008. Possible nitric oxide modulation in the

protective effect of trazodone against sleep deprivation-

induced anxiety like behavior and oxidative damage in mice.

Int. J. Health Res. 1, 151–162.

Kumar, A., Prakash, A., Dogra, S., 2011. Protective effect ofcurcumin (Curcuma longa) against D-galactose-induced senes-cence in mice. J. Asian Nat. Prod. Res. 13, 42–55.

Kumar, A., Singh, A., 2008. Possible nitric oxide modulation inprotective effect of (Curcuma longa, Zingiberaceae) against sleepdeprivation-induced behavioral alterations and oxidativedamage in mice. Phytomedicine 15, 577–586.

Kono, Y., 1978. Generation of Superoxide radical during auto-oxidation of hydroxylamine and an assay for superoxidedismutase. Arch. Biochem. Biophys. 186, 189–195.

Kurukulasuriya, R., Sorensen, B.K., Link, J.T., Patel, J.R., Jae, H.S.,Winn, M.X., Rohde, J.R., Grihalde, N.D., Lin, C.W., Ogiela, C.A.,Adler, A.L., Collins, C.A., 2004. Biaryl amide glucagon receptorantagonists. Bioorg. Med. Chem. Lett. 14, 2047–2050.

Landfield, P.W., Blalock, E.M., Chen, K.C., Porter, N.M., 2007. A newglucocorticoid hypothesis of brain aging: implications forAlzheimer’s disease. Curr. Alzheimer Res. 4, 205–212.

Li, Y.C., Wang, F.M., Pan, Y., Qiang, L.Q., Cheng, G., Zhang, W.Y.,Kong, L.D., 2009. Antidepressant-like effects of curcumin onserotonergic receptor-coupled AC-cAMP pathway in chronicunpredictable mild stress of rats. Prog. Neuropsychopharma-col. Biol. Psychiatry 33, 435–449.

Liu, Y., Peterson, D.A., Kimura, H., Schubert, D., 1997. Mechanismsof cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-nium bromide (MTT) reduction. J. Neurochem. 69, 581–593.

Lucca, G., Comim, C.M., Valvassori, S.S., Reus, G.Z., Vuolo, F.,Petronilho, F., Dal-Pizzol, F., Gavioli, E.C., Quevedo, J., 2009.Effects of chronic mild stress on the oxidative parameters inthe rat brain. Neurochem. Int. 54, 358–362.

Luck, H., 1971. Catalase. In: Bergmeyer, H.U. (Ed.), Methods ofEnzymatic Analysis. Academic Press, New York, pp. 885–893.

Mancuso, C., Barone, E., 2009a. Curcumin in clinical practice:myth or reality?. Trends Pharmacol. Sci. 30, 333–334.

Mancuso, C., Barone, E., 2009b. The heme oxygenase/biliverdinreductase pathway in drug research and development. Curr.Drug Metab. 10, 579–594.

Mancuso, C., Siciliano, R., Barone, E., Preziosi, P., 2012. Naturalsubstances and Alzheimer’s disease: from preclinical studiesto evidence based medicine. Biochim. Biophys. Acta 1822,616–624.

Mancuso, C., Siciliano, R., Barone, E., 2011. Curcumin andAlzheimer disease: this marriage is not to be performed.J. Biol. Chem. 286, le3.

Massaad, C.A., Klann, E., 2011. Reactive oxygen species in theregulation of synaptic plasticity and memory. Antioxid. RedoxSignal. 14, 2013–2054.

McFadden, L.M., Paris, J.J., Mitzelfelt, M.S., McDonough, S.,Frye, C.A., Matuszewich, L., 2011. Sex-dependent effects ofchronic unpredictable stress in the water maze. Physiol.Behav. 102, 266–275.

Mehla, J., Reeta, K.H., Gupta, P., Gupta, Y.K., 2010. Protective effectof curcumin against seizures and cognitive impairment in apentylenetetrazole-kindled epileptic rat model. Life Sci. 87,596–603.

Morris., R.G.M., 1984. Developments of a water maze procedurefor studying spatial learning in the rats. J. Neurosci. Methods11, 47–60.

Motterlini, R., Foresti, R., Bassi, R., Green, C.J., 2000. Curcumin, anantioxidant andanti-inflammatory agent, induces hemeoxygenase-1 and protects endothelial cells against oxidativestress. Free Radic. Biol. Med. 28, 1303–1312.

Murua, V.S., Gomez, R.A., Andrea, M.E., Molina, V.A., 1991.Shuttle-box deficits induced by chronic variable stress: rever-sal by imipramine administration. Pharmacol. Biochem.Behav. 38, 125–130.

Nafisi, S., Adelzadeh, M., Norouzi, Z., Sarbolouki, M.N., 2009.Curcumin binding to DNA and RNA. DNA Cell Biol. 28,201–208.

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 3 8 – 5 050

Nijholt, I., Farchi, N., Kye, M., Sklan, E.H., Shoham, S., Verbeure, B.,Owen, D., Hochner, B., Spiess, J., Soreq, H., Blank, T., 2004.Stress-induced alternative splicing of acetylcholinesteraseresults in enhanced fear memory and long-term potentiation.Mol. Psychiatry 9, 174–183.

Prado, V.F., Martins-Silva, C., de Castro, B.M., Lima, R.F., Barros, D.M.,Amaral, E., Ramsey, A.J., Sotnikova, T.D., Ramirez, M.R., Kim, H.G.,Rossato, J.I., Koenen, J., Quan, H., Cota, V.R., Moraes, M.F.,Gomez, M.V., Guatimosim, C., Wetsel, W.C., Kushmerick, C.,Pereira, G.S., Gainetdinov, R.R., Izquierdo, I., Caron, M.G., Prado,M.A., 2006. Mice deficient for the vesicular acetylcholinetransporter are myasthenic and have deficits in object andsocial recognition. Neuron 51, 601–612.

Radley, J.J., Sisti, H.M., Rocher, A.B., Hao, J., McCall, T., Hof, P.R.,McEwen, B.S., Morrison, J.H., 2004. Chronic behavioral stressinduces apical dendritic reorganization in pyramidal neuronsof the medial prefrontal cortex. Neuroscience 125, 1–6.

Rajkrishnan, V., Vishwanathan, P., Rajasekharan, K.N., Menon, V.P.,1999. Neuroprotective role of curcumin from Curcuma longa onethanol-induced brain damage. Phytother. Res. 13, 571–574.

Reddy, A.C., Lokesh, B.R., 1994. Studies on the inhibitory effects ofcurcumin and eugenol on the formation of reactive oxygenspecies and the oxidation of ferrous iron. Mol. Cell. Biochem.137, 1–8.

Roozendaal, B., 2002. Stress and memory: opposing effects ofglucocorticoids on memory consolidation and memory retrie-val. Neurobiol. Learn. Mem. 78, 578–595.

Sato, H., Takahashi, T., Sumitani, K., Takatsu, H., Urano, S., 2010.Glucocorticoid generates ROS to induce oxidative injury in thehippocampus leading to impairment of cognitive function ofrats. J. Clin. Biochem. Nutr. 47, 224–232.

Selkoe, D.J., 1991. The molecular pathology of Alzheimer’s dis-ease. Neuron 6, 487–498.

Serrano, F., Klann, E., 2004. Reactive oxygen species and synapticplasticity in the aging hippocampus. Ageing Res. Rev. 3, 431–443.

Sharma, A.C., Kulkarni, S.K., 1992. Evaluation of learning andmemory mechanisms employing elevated plus-maze in ratsand mice. Prog. Neuropsychopharmacol. Biol. Psychiatry 16,117–125.

Silber, R.H., Busch, R.D., Oslapas, R., 1958. Practical procedure forestimation of corticosterone or hydrocortisone. Clin. Chem. 4,278–285.

Sottocasa, G.L., Kuylenstierna, B., Ernster, L., Berqstrand, A., 1967.An electron-transport system associated with the outer mem-brane of liver mitochondria. A biochemical and morphologicalstudy. J. Cell Biol. 32, 415–438.

Sreejavan, N., Rao, M.N.A., 1997. Nitric oxide scavenging bycurcuminoids. J. Pharm. Pharmacol. 49, 105–107.

Vajragupta, O., Boonchoong, P., Watanabe, H., Tohda, M., Kum-masud, N., Sumanont, Y., 2003. Manganese complexes ofcurcumin and its derivatives: evaluation for the radicalscavenging ability and neuroprotective activity. Free Radic.Biol. Med. 35, 44–1632.

Vanitallie, T.B., 2002. Stress: a risk factor for serious illness.Metabolism 51, 40–45.

Wahlstrom, B., Blennow, G.A., 1978. Study on the fate of curcuminin the rat. Acta Pharm. Toxicol. 43, 86–92.

Willner, P., Muscat, R., Papp, M., 1992. Chronic mild stress-induced anhedonia: a realistic animal model of depression.Neurosci. Biobehav. Rev. 16, 525–534.

Wills, E.D., 1966. Mechanism of lipid peroxide formation inanimal tissues. Biochem. J. 99, 667–676.

Xu, Y., Lin, D., Li, S., Li, G., Shyamala, S.G., Barish, P.A., Vernon,M.M., Pan, J., Ogle, W.O., 2009. Curcumin reverses impairedcognition and neuronal plasticity induced by chronic stress.Neuropharmacology 57, 463–471.