A NOOTROPIC EFFECT OF BENINCASA HISPIDA ON ACH AND CHAT ACTIVITY IN COLCHICINE INDUCED EXPERIMENTAL RAT MODEL OF ALZHEIMER'S DISEASE: POSSIBLE INVOLVEMENT OF ANTIOXIDANTS

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Roy et al. World Journal of Pharmaceutical Research
A NOOTROPIC EFFECT OF BENINCASA HISPIDA ON ACH AND
CHAT ACTIVITY IN COLCHICINE INDUCED EXPERIMENTAL RAT
MODEL OF ALZHEIMER’S DISEASE: POSSIBLE INVOLVEMENT
OF ANTIOXIDANTS
1Chandan Roy*, 2Debasis Mazumdar
1Dept. of Physiology, Berhampore Girls‟ College, University of Kalyani, Berhampore, West
Bengal, India.
2Dept. of Agricultural Statistics, Bidhan Chandra Krishi Viswavidyalaya (State Agriculture
University), Mohanpur, Nadia, West Bengal, India.
ABSTRACT
The fruit Benincasa hispida (BH) is an important ingredient of
„Kusmanda lehyam‟ (Ayurvedic medicine), which is widely used, in
nervous disorders. The present study has been designed to evaluate the
cognition facilitating effect of BH pulp extract in colchicine induced
experimental rat model of AD and to investigate the role of central
cholinergic system in the nootropic effect of BH pulp extract with the
possible involvement of antioxidant enzymes. The behavior study,
Acetylcholine concentration, cholineacetyl transferase activity,
antioxidant level such as, superoxide dismutase (SOD), catalase
(CAT), reduced glutathione (GSH) and lipid peroxidation (LPO) level
were studied in different parts of the brain such as frontal cortex (FC)
and hippocampus (HPC) in colchicine induced experimental
Alzheimer rat model before and after treatment with BH. BH (400 mg/kg p.o.) induced
statistically significant reversal of colchicine induced cognitive deficits. BH (400 mg/kg p.o.)
markedly induced frontal cortical and hippocampal concentrations of Ach and ChAt activity,
the effects being statistically significant on days 7, 14 and 21 respectively. Moreover, BH
significantly increased SOD, CAT, GSH activities and significantly decreased LPO level on
day 7, 14 and 21 respectively. The aqueous pulp extract of BH (400 mg/kg body weight)
containing vit- A, C, E results significant protection in the level of antioxidant status in
frontal cortex and hippocampas after a certain period of co administration on colchicine
World Journal of Pharmaceutical Research
SJIF Impact Factor 5.045
Volume 3, Issue 9, 1282-1296. Research Article ISSN 2277– 7105
Article Received on
25 August 2014,
Revised on 18 Sept 2014,
Accepted on 14 Oct 2014
*Correspondence for
Author
Dr. Chandan Roy
Dept. of Physiology,
Berhampore Girls‟
College, University of
Kalyani, Berhampore,
West Bengal, India.
iamchandan@rediffmail.com

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induced oxidative stress without causing any general and metabolic toxicity and possibly
thereby induced frontal cortical and hippocampal concentration of Ach and ChAT activity.
KEY WORDS: Benincasa hispida, Colchicine, Alzheimer‟s disease, Acetylcholine,
Cholineacetyltransferase, Antioxidant.
INTRODUCTION
The fruit of Benincasa hispida (Thunb) Cogn, commonly called as ash guard, belonging to
cucurbitaceae family is employed as a main ingredient in kusmanda lehyam, in Ayurvedic
system of medicine. The fruit Benincasa hispida (BH) is an important ingredient of
„Kusmanda lehyam‟ (Ayurvedic medicine), which is widely used, in nervous disorders. The
major constituents of this fruits are triterpenoids, flavanoids, glycosides, saccharides,
carotenes, vitamins, β sitosterin, and uronic acid (Nadkarni, 1976; Wollen et al., 1991;
Yashizumi et al., 1998). Some of the important isolated compounds of BH reported were
triterpenes, sterols and glycosides (Yashizumi et al., 1998) and volatile oils.
Alzheimer‟s disease (AD) is a complete neurodegenerative disorders characterized by the
loss of learning, memory and other cognitive functions. AD is characterized by degeneration
of neurons, especially pyramidal neurons in the hippocampus, entorhinal cortex, and
neocortical areas and cholinergic neurons in the median forebrain.
Colchicine, as a microtubule-disrupting agent (James & Dennis, 1981) produces marked
destruction of hippocampal granule cells, mossy fibers and septohippocampal pathways
(SHC, a cholinergic link between medial septum and vertical limb of diagonal band). In
induces neurofibrillary degeneration by binding to tubulin, the principal structural protein of
microtubule (McClure, 1972; Wilson & Fried-Kin, 1966; Walsh et al., 1986). This event is
associated with loss of cholinergic neurons and decrease in acetylcholine transferase, thereby
resulting in impairment of learning and memory (Kevin et al., 1989; Dwaine & Thomas,
1990). Oxidative stress due to increase in free radical generation of impaired endogenous
antioxidant mechanism is an important factor that has been implicated in neuronal damage
and in AD, and cognitive defects seen in elderly (Pratico & Delanty, 2000; Cantuli et al.,
2000). A number of in vitro studies have shown that antioxidants, both endogenous and
dietary, can protect nervous tissue from damage by oxidative stress. Vitamin C has been
described to be a major hydrophilic antioxidant in human plasma (Frei et al., 1989), CSF
(Spector & Lorenzo, 1973; Lonnrot et al., 1996) and the central nervous system (Rice, 2000).

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The same study showed that vitamin E prevented neuronal damage from reactive nitrogen
species (Tagami et. al., 1998). Both vitamin E and β carotene were found to protect rat
neurons against oxidative stress from exposure to ethanol (Mitchel et al., 1999). BH is rich in
vitamin C, E and beta- carotene (Roy et al., 2007).
Thus the present study was undertaken to determine the cognition facilitating effect of BH
pulp extract in colchicine induced experimental rat model of AD and to investigate the role of
central cholinergic system in the nootropic effect of BH pulp extract with the possible
involvement of antioxidant enzymes.
MATERIALS AND METHODS
Subjects
Male Holtzman strain adult albino rats approximately 120 days old and weighing 250-300gm
were used in the following studies. The animals were individually housed and maintained
under standard laboratory conditions with natural dark and light cycle (approximately 12-h
light/10-h dark cycle) and room temperature (2710C) and constant humidity (60%) in
accordance with „Institutional Ethical Committee‟ rules and regulations. Food and water were
freely available except during testing. Drinking water was supplied ad libitum. Five days
prior to behavioral training, animals were reduced to 85% of their free feeding weight by
limiting their daily ration of food. Food deprivation was maintained throughout testing except
for 3 days immediately prior to, and following surgery. Body weights of the rats were
recorded every day and maintained in the laboratory throughout the experimental period. The
behavioral procedure was carried out between 12:00 and 14:00 h.
Collection and Preparation of Water Extract from the Pulp of BH
The fruit of BH were purchased from the local market and the identity of the plant was
authenticated by the Botanical Survey of India, Howrah and kept in S.N.Pradhan Centre for
Neurosciences, University of Calcutta.The pulp of BH fruit was used throughout the
experimental study. The fruit of BH were cut into pieces, sun-dried under shady region
because sun-dry may results in loss of chemicals and ground with the help of an electrical
grinder to get a free flowing powder. This powder was subjected to extraction with water
(1:3) by simple decoction method at room temperature for 48 hours. The extract obtained was
filtered through Whatman filter paper and vaccum dried at 40o – 50o C to get a dry powder,
which was dissolved in double distilled water for final use (Roy et al., 2007).

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Treatment
The control animal was treated with artificial cerebrospinal fluid or ACSF. The BH pulp
extract was given orally through orogastric cannula at the standard dose of 400 mg/kg p.o. for
seven, fourteen and twenty one consecutive days respectively (between 10:00 and 11:00 hrs).
The dose was standardized in the laboratory.
The animals were sacrificed by cervical dislocation and the different parts of the brain like
Frontal cortex (FC), Hippocampus (HPC), Cerebral cortex (CC), Cerebellum (CB), Caudate
nucleus (CN), Pons & Medulla (PM) and Midbrain (MB), were isolated for biochemical
estimation after seven, fourteen and twenty one days respectively.
Grouping of Animal: The animals were divided into four groups:
1. Control (ACSF) rats
2. Colchicine induced Alzheimer‟s rat model
3. Control rats treated with BH pulp extract
4. Colchicine induced Alzheimer‟s rat model treated with BH pulp extract.
Behavioural Study
The apparatus used consisted of a shuttle box with two identical compartments, separated by
a hurdle. During training, each rat was placed in one compartment and after 5 sec a buzzer,
situated in the ceiling of the shuttle box, was sounded (2.8 kHz, 70 dB) (conditioned
stimulus, CS) for 3 sec, followed by electric shock (1.5 mA, 2 sec) (unconditioned stimulus,
UCS) through the grid floor. If the rat crossed to the unelectrified safe compartment during
presentation of CS, an avoidance response was recorded, otherwise UCS was applied. Each
rat was given 20 trials for 5 days, with an intertrial interval of 30 sec, before lesioning, until it
reached the criterion of 100% active avoidance response. Rats not reaching this criterion were
discarded from the study (Jaiswal & Bhattacharya, 1992). Retention of the acquired active
avoidance response, in the different treatment groups, was assessed on days 7, 14 and 21,
following lesioning with colchicine or ibotenic acid, by noting the number of trials required
to criterion of 100% active avoidance response.
Preparation of Experimental Alzheimer’s Rat Model by Colchicine
Prior to surgery, all the animals were subjected to overnight fasting though drinking water
was not withdrawn. During procedures, the animals were anaesthetized with sodium
pentobarbital (50mg/kg b.w.) and re-strained in a stereotaxic apparatus (INCO, INDIA Ltd)

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equipped with a custom-made ear bar, which prevents the damage of the tympanic
membrane. Head was fixed in such a position that lambda and bregma sutures were in the
same horizontal plane by introducing the incisor bar properly attached to the mouth. For
aseptic surgery, absolute alcohol or rectified spirit was applied. The scalp was incised and
retracted. An incision was made in the scalp and two holes were drilled in the skull for
placement of the injection cannula into the lateral cerebral ventricles. The stereotaxic
coordinates for intracerebroventricular injection were: 0.8 mm posterior to bregma, 1.8 mm
lateral to the sagittal suture and 3.6 mm below the cortical surface (Veerendrakumar &
Gupta, 2002). Subjects were infused through a 10 l Hamilton syringe with 15 gm of
colchicine (Wako chemicals) in 5 l of artificial cerebrospinal fluid (ACSF; in Mm: 147
Nacl, 2.9 Kcl, 1.6 Mgcl2, 2.2 Dextrose and 1.7 Cacl2) in lateral cerebral ventricle bilaterally.
A total volume of 10 l was delivered to the injection site and the injection cannula was left
in place for 2-3 min following infusion.
Postoperative care: After surgery, all aseptic measures and care were taken for feeding until
recovery from surgical stress. Penicillin was given post operatively to all animals for 3
consecutive days by intramuscular route. After 3 days of surgery, experiment was started and
continued routinely until sacrificed. Similar procedure was repeated thrice, each at an interval
of two days.
Biochemical Estimation
Tissue Preparation
Rats were sacrificed by cervical dislocation on day 7, 14 and 21 immediately after behavior
study. The Frontal cortex (FC) and Hippocampus (HPC) were dissected out. The tissues were
weighed and homogenized in ice-cold phosphate buffer and prepared for biochemical
estimation.
Estimation of Ach and ChAT Activity
Rats of the colchicine group were killed by decapitation at the predetermined time intervals
and the frontal cortex and hippocampus were dissected out (Glowinski & Iversen, 1966). The
tissues were homogenised in 10 volumes (w/v) of ice-cold Tris-HCl buffer (pH 7.6) and
divided into aliquots for estimation of acetylcholine (Ach) levels by a fluorimetric technique
(Speeg, 1974), choline acetyltransferase ( ChAT) activity by a radiometric method (Haba et
al., 1988).

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Estimation of SOD, CAT, GSH activity and LPO level
Catalase (CAT) activity was estimated by the method of Cohen et al., (1970); Roy et al.,
(2007), Superoxide Dismutase (SOD) was estimated by the method of Mishra & Fridovich
(1972); Roy et al., (2007), Reduced glutathione (GSH) level was measured according to the
method of Ellman (1959) and Lipid Peroxidation (LPO) was estimated by the method of
Bhattacharya et al. (2001); Roy et al., (2007).
Statistical Analysis
The data were expressed as MEAN  S.E.M. and were analyzed statistically using one way
analysis of variance (one way ANOVA) followed by multiple comparison „t‟ test. In addition
to this, two-tailed Student„t‟ test was performed to determine the level of significance
between the means. Difference below the probability level 0.05 was considered statistically
significant.
RESULTS
Results of Behavioural Parameter
Colchicine, injected i.c.v., induced marked deficits of the learned active avoidance task, as
compared to their ACSF treated counterparts, after 7, 14 and 21 days following
administration of the neurotoxins. The retention deficit was evident by day 7 and increased
progressively on days 14 and 21. BH (400 mg/kg p.o.) induced dose-related statistically
significant reversal of colchicine induced cognitive deficits, when assessed on day 7,
remained statistically non-significant (Table 1).
Results of Parameters of Cholinergic System
Colchicine, administered i.c.v., markedly reduced frontal cortical and hippocampal
concentrations of Ach and ChAT activity, as compared to the ACSF administration control
group. The effects were discernible by day 7, and increased progressively, thereafter, on days
14 and 21. BH (400 mg/kg p.o.) tended to reverse the deleterious effects of colchicine on all
these biochemical parameters, the effects being statistically significant on days 14 and 21, but
not on day 7 (Tables 2, 3 and 4).
Results of Parameters of Oxidative Stress
Colchicine, administered i.c.v., markedly reduced SOD, CAT and GSH activity and markedly
increased LPO level in different mentioned brain parts respectively, as compared to the
ACSF administration control group. The effects were discernible by day 7, and increased

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progressively, thereafter, on days 14 and 21. BH (400 mg/kg p.o) tended to reverse the
deleterious effects of colchicine on all these biochemical parameters, the effects being
statistically significant on days 7, 14 and 21 respectively (Table 5, 6. 7 and 8).
Table 1: Effect of BH on the retention of an active avoidance learning acquisition in
cognitive deficits induced by colchicine (15 μg, i.c.v.) in rats (values are Mean ± SEM)
Treatments
(mg/kg)
N
Number of trials required to achieve 100%
avoidance response
Colchicine group
Day 7
Day 14
Day 21
ACSF
6
4.9 ± 0.02
3.3±0.02
2.7±0.04
BH
6
2.9±0.03*
2.1±0.02*
1.3±0.04*
COLCHICINE
6
7.7±0.03**
8.2±0.03**
9.3±0.04**
BH+COLCHICINE
6
5.42±0.03#
3.64±0.02#
2.24±0.04#
Values are mean  SEM, n = 6; *p < 0.001, **p < 0.001 when compared with ACSF group. #p
< 0.001 when compared with colchicine treated group. Data were analyzed statistically using
one-way ANOVA Test followed by student„t‟ – test.
Table 2: Effect of BH on acetylcholine concentrations of frontal cortex and
hippocampus in colchicine (15 μg i.c.v.) administered rats (values are Mean ± SEM)
Values are mean  SEM, n = 6; *p < 0.001, **p < 0.001 when compared with ACSF group. #p
< 0.001 when compared with colchicine treated group. Data were analyzed statistically using
one-way ANOVA Test followed by student„t‟ – test.
Treatments
(mg/kg)
N
Acetylcholine concentrations (nmol/g)
Frontal cortex
Hippocampus
Day 7
Day 14
Day 21
Day 7
Day 14
Day 21
ACSF
6
24.64±0.02
26.30±0.03
23.52±0.04
30.54±0.03
28.42±0.02
31.62±0.04
BH
6
28.22±0.04*
34.66±0.06*
30.02±0.04*
33.84±0.03*
35.24±0.08*
38.22±0.04*
COLCHICINE
6
18.22±0.02**
15.44±0.04**
11.52±0.03**
23.42±0.04**
19.14±0.09**
14.44±0.06**
BH+COLCHICINE
6
23.56±0.04#
30.66±0.04#
24.86±0.05#
28.96±0.04#
33.12±0.03#
31.48±0.04#

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Table 3: Effect of BH on choline acetyltransferase activity of frontal cortex and
hippocampus in colchicine (15 μg, i.c.v.) administrated rats (Values are Mean ± SEM)
Treatments
(mg/kg)
n
Choline acetyltransferase activity (nmol/mg protein/h)
Frontal cortex
Hippocampus
Day 7
Day 14
Day 21
Day 7
Day 14
Day 21
ACSF
6
21.22±0.04
20.44±0.02
19.96±0.07
19.22±0.04
18.54±0.06
19.44±0.04
BH
6
21.44±0.02*
24.88±0.03*
22.56±0.02*
22.42±0.05*
22.54±0.04*
23.46±0.01*
COLCHICINE
6
16.44±0.04**
14.24±0.04**
11.96±0.04**
14.66±0.04**
12.22±0.02**
9.88 ±0.08**
BH+COLCHICINE
6
18.46±0.02#
21.34±0.06#
23.34±0.02#
17.62±0.02#
19.66±0.04#
16.68±0.02#
Values are mean  SEM, n = 6; *p < 0.001, **p < 0.001 when compared with ACSF group. #p
< 0.001 when compared with colchicine treated group. Data were analyzed statistically using
one-way ANOVA Test followed by student „t‟ – test.
Table 4: Effect of BH on muscarinic cholinergic receptors in frontal cortex and
hippocampus in colchicine (15 μg, i.c.v.) administered rats (Values are Mean ± SEM)
Treatments
(mg/kg)
N
(3H) QNB binding (pmoles/mg protein)
Frontal cortex
Hippocampus
Day 7
Day 14
Day 21
Day 7
Day 14
Day 21
ACSF
6
1.54±0.01
1.71±0.02
1.56±0.02
1.37±0.03
1.44±0.02
1.42±0.02
BH
6
1.56±0.02*
1.72±0.02*
1.96±0.01*
1.59±0.01*
1.66±0.01*
1.86±0.01*
COLCHICINE
6
0.83±0.02**
0.56±0.02**
0.46±0.01**
0.71±0.02**
0.53±0.02**
0.42±0.02**
BH+COLCHICINE
6
1.26±0.01#
1.33±0.01#
1.48±0.02#
1.08±0.04#
1.41±0.02#
1.58±0.01#
Values are mean  SEM, n = 6; *p < 0.001, **p < 0.001 when compared with ACSF group. #p
< 0.001 when compared with colchicine treated group. Data were analyzed statistically using
one-way ANOVA Test followed by student „t‟ – test.
Table 5: Effect of BH on SOD activity in frontal cortex and hippocampus in colchicine
(15 μg, i.c.v.) administered rats (Values are Mean ± SEM)
Treatments
(mg/kg)
N
SOD (% inhibition unit)
Frontal cortex
Hippocampus
Day 7
Day 14
Day 21
Day 7
Day 14
Day 21
ACSF
6
13.300.02
11.280.04
11.310.04
11.120.03
12.470.07
12.42±0.04
BH
6
10.310.03*
9.820.02 *
9.960.04*
9.790.05*
10.270.04*
10.81±0.02*
COLCHICINE
6
20.660.06**
19.110.05**
19.600.06**
18.580.06**
20.850.03**
21.40±0.02**
BH+COLCHICINE
6
15.380.03#
14.190.06#
14.310.07#
14.460.05#
14.430.02#
15.43±0.02#
Values are mean  SEM, n = 6; *p < 0.001, **p < 0.001 when compared with ACSF group. #p
< 0.001 when compared with colchicine treated group. Data were analyzed statistically using
one-way ANOVA Test followed by student„t‟ – test.

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Table 6: Effect of BH on CAT activity in frontal cortex and hippocampus in colchicine
(15 μg, i.c.v.) administered rats (Values are Mean ± SEM)
Treatments
(mg/kg)
N
CAT (% inhibition unit)
Frontal cortex
Hippocampus
Day 7
Day 14
Day 21
Day 7
Day 14
Day 21
ACSF
6
13.980.03
12.300.09
12.540.04
13.190.04
12.160.04
12.46±0.02
BH
6
12.27 0.04*
10.810.04*
10.660.03*
11.790.05*
10.550.03*
10.22±0.02*
COLCHICINE
6
21.650.03**
20.350.04**
19.690.05**
20.770.06**
20.130.04**
22.36±0.04**
BH+COLCHICINE
6
15.72 0.04#
14.23 0.03#
14.82 0.06#
14.40 0.06#
13.970.07#
15.72±0.06#
Values are mean  SEM, n = 6 ; *p < 0.001, **p < 0.001 when compared with ACSF group.
#p < 0.001 when compared with colchicine treated group. Data were analyzed statistically
using one-way ANOVA Test followed by student„t‟ – test.
Table 7: Effect of BH on LPO level in frontal cortex and hippocampus in colchicine (15
μg, i.c.v.) administered rats (Values are Mean ± SEM)
Treatments
(mg/kg)
N
LPO (nmol of TBARS / gm mol of tissue)
Frontal cortex
Hippocampus
Day 7
Day 14
Day 21
Day 7
Day 14
Day 21
ACSF
6
4.030.02
3.990.04
3.460.04
3.890.02
3.720.04
3.98±0.02
BH
6
2.930.01*
2.900.03*
3.190.02*
2.890.03*
3.350.02*
2.89±0.02*
COLCHICINE
6
8.520.02**
7.460.04**
7.590.03**
7.700.04**
7.190.02**
8.52±0.04**
BH+COLCHICINE
6
5.290.03#
4.630.05#
4.200.04#
4.680.03#
4.600.04#
5.02±0.02#
Values are mean  SEM, n = 6; *p < 0.001, **p < 0.001 when compared with ACSF group. #p
< 0.001 when compared with colchicine treated group. Data were analyzed statistically using
one-way ANOVA Test followed by student„t‟ – test.
Table 8: Effect of BH on GSH level in frontal cortex and hippocampus in colchicine (15
μg, i.c.v.) administered rats (Values are Mean ± SEM)
Treatments
(mg/kg)
N
Reduced glutathione (g/g of tissue)
Frontal cortex
Hippocampus
Day 7
Day 14
Day 21
Day 7
Day 14
Day 21
ACSF
6
30.540.06
29.620.11
29.610.04
23.90.06
26.650.04
29.42±0.02
BH
6
32.210.07*
31.20.08 *
28.080.08*
26.710.04*
27.680.02*
32.26±0.04*
COLCHICINE
6
19.960.05**
18.780.04**
15.260.08**
16.150.06**
17.170.04**
15.56±0.02**
BH+COLCHICINE
6
26.80.04#
26.810.03#
24.870.07#
25.330.07#
25.170.03#
24.44±0.02#
Values are mean  SEM, n = 6 ; **p < 0.001 when compared with ACSF group. #p < 0.001
when compared with colchicine treated group. Data were analyzed statistically using one-way
ANOVA Test followed by student„t‟ – test.

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DISCUSSION
The present study evaluates the nootropic effect of BH on cognition facilitating effect and
central cholinergic system in colchicine induced experimental rat model of AD with the
possible involvement of antioxidant enzymes. It is evident from the results of the present
investigation that intracerebroventricular administration of colchicine induced marked
deficits of the learned active avoidance task, as compared to their ACSF treated counterparts
after 7, 14 and 21 days in colchicines treated experimental AD group. But, treatment with
aqueous pulp extract of BH significantly decreased marked deficits of the learned active
avoidance task in BH cotreated colchicines treated experimental AD group in comparision
with only colchicines treated experimental AD group. These findings can be explained by
alterations of the parameters of oxidative stress namely lipid peroxidation level (LPO) , SOD,
CAT and GSH activity along with alterations of AchE and ChAT activity respectively. Thus,
the marked deficit in the retention of the learned active avoidance task, in rats induced by
colchicine, noted in this study, is consonant with earlier report (Emerich & Walsh, 1990).
Treatment with aqueous pulp extract of BH was able to reverse cognitive deficits induced by
colchicine, the effects being evident after 2 weeks of treatment. The reversal of cognitive
deficits, induced by colchicine, was accompanied by attenuation of its cholinotoxic effects,
indicating that the drug was capable of promoting cholinergic recovery. The findings support
clinical (Koti, 1991) and experimental (Verma & Kulkarni, 1991) observations that BH can
improve memory in states of cognitive deficits. From our present investigation, i.c.v.
administration of colchicines markedly reduced frontal cortical and hippocampal
concentrations of Ach, ChAt activity, as compared to the ACSF administration control group.
The effects were discernible by day 7, and increased progressively, thereafter, on days 14 and
21. BH (400 mg/kg p.o.) tended to reverse the deleterious effects of colchicine on all these
biochemical parameters, the effects being statistically significant on days 14 and 21, but not
on day 7. It was reported that the i.c.v. injection of colchicines significantly decreased the
number of cholinergic neurons in the medial septum /vertical limb of the diagonal band,
which project to the hippocampus and synapse on granule cells, pyramidal cells and
interneurons. Therefore, it was expected that the intracerebroventricularly administered
colchicines would preferentially act on the cholinergic neurons (Dwaine & Thomas, 1991).
Nootropic agents, like piracetam, which have been shown to facilitate central cholinergic
mechanisms (Chouinard et al., 1983; Moos et al., 1988), are known to improve memory only
in the presence of cognitive deficits.

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Intracerebroventricular infusion of colchicine causes it to bind with tubulin which is the
structural and functional protein of microtubule and thereby generates more and more
reactive oxygen species (ROS) leading to neurodegeneration and ultimately produces a
condition akin to AD or produces experimental AD model which is histopathologically
characterized by the extracellular deposition of senile plaques and the intracellular deposition
of neurofibrillary tangles. Free radicals play a crucial role in the pathogenesis of AD. Lipid
peroxidation can be used as an index for measuring the damage that occurs in membranes of
tissue as a result of free radical generation (Dianzani, 1985; Husain & Somani, 1997). In our
present study, ICV infusion of colchicine, it significantly increased the LPO level. The results
of significant elevation of LPO level in colchicine treated experimental Alzheimer‟s group is
possibly due to the generation of free radicals via auto-oxidation or through metal ion or
superoxide catalyzed oxidation process. This is possibly due to the generation of free radicals
via autooxdidation or through metal ion or superoxide catalyzed oxidation process. In the
present experiment, BH significantly decreased LPO level in a dose dependent manner
compared to other groups. So, from the result of LPO levels it may be concluded that the
protection by BH may be due to vitamin E and beta carotene which is present in BH pulp
extract.
Endogenous antioxidant status in colchicines induced experimental Alzheimer‟s rat model
was evaluated here by noting the activities of CAT, SOD and GSH as these are the important
biomarkers for scavenging free radicals (Venkateswaran & Pari, 2003). Colchicine induced
oxidative stress is further supported here by the study of antioxidant scavenger enzyme
activities.
CAT that protects the tissues from highly reactive hydroxyl radical catalyzes the reduction of
hydrogen peroxide. The primary role of CAT is to scavenge H2O2 that has been generated by
free radicals or by SOD in removal of superoxide anions and to convert it to water (Ribiere et
al., 1992). The destruction of superoxide radicals is catalyzed by SOD, is an important
defense system against oxidative damage. From our experimental results of the aforesaid
antioxidant enzyme activities in brain tissues colchicine significantly decreased SOD, CAT,
GSH activities in colchicine treated experimental Alzheimer‟s group rather than control, BH
treated group and BH pretreated colchicine treated experimental Alzheimer‟s groups. BH
containing vitamin E and beta-carotene significantly increased SOD, CAT, GSH, number of
correct choices along with significantly decreased the latency time in a dose dependent

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manner rather than other groups. Glutathione is an endogenous antioxidant, which is present
majorly in the reduced form within the cells. It prevents the hydroxyl radical generation by
interacting with free radicals. During this defensive process, reduced glutathione is converted
to oxidized form under the influence of the enzyme glutathione peroxidase (GPX). The
decreased level of reduced glutathione in colchicine treated experimental group seen in our
study indicates that there was an increased generation of free radicals and the reduced
glutathione was depleted during the process of combating oxidative stress (Reiter, 2000;
Schulz et al, 2000). This has probably been possible either from the low level of ROS
production or through a rapid dissolution of ROS that has further been strengthened from the
elevated activities of important antioxidant defense enzymes CAT and SOD, studied in this
experiment. Literature study has shown that the BH contains high level of vitamin E and
beta-carotene which protects rat neurons against oxidative stress possibly through the
presence of both vitamin E and beta-carotene. Because vitamin E (alpha tocopherol and other
tocopherol) is the most potent antioxidant that can break the propagation of free radical chain
reactions in the lipid part of biological membranes. It may be inferred from the present results
BH protects rat neurons against oxidative stress as is evidenced from our results of LPO,
CAT, SOD and GSH activities possibly by vitamin E and beta carotene which is present in
BH. So, keep in this view, i.c.v. administration of colchicines may generate oxidative stress
either by producing reactive oxygen species (ROS) or by hampering the endogenous
antioxidant enzymes leading to cholinotoxicity. The aqueous pulp extract of this plant
containing vit- A, C, E results significant protection in the level of antioxidant status in
frontal cortex and hippocampas after a certain period of co administration on colchicine
induced oxidative stress without causing any general and metabolic toxicity and possibly
thereby induced frontal cortical and hippocampal concentration of Ach and ChAT activity.
CONCLUSION
The present investigation, together with earlier reported clinical and experimental data, and
the similarly of behavioural and biochemical effects on cholinergic markers and different
antioxidant enzyme activity along with LPO level, permit the categorization of BH as a
nootropic agent. However, it may be proposed that further research on this field is essential to
find out other active ingredients present in the BH pulp extract and their specific role by
which the therapeutic importance may be evaluated and the outcome of which can be utilized
in the protection of AD.

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ACKNOELEDGMENTS
We are highly indebted to B.Sc final year students, Berhampore Girls‟ College, University of
Kalyani for their valuable co-operation. The authors have no conflicts of interest and there is
no funding agency to support of this study and its publication.
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