Clinical and Experimental Pharmacology and Physiology (2003) 30, 336–342
EFFECT OF CENTELLA ASIATICA ON COGNITION AND
OXIDATIVE STRESS IN AN INTRACEREBROVENTRICULAR
STREPTOZOTOCIN MODEL OF ALZHEIMER’S DISEASE
IN RATS
MH Veerendra Kumar and YK Gupta
Neuropharmacology Laboratory, Department of Pharmacology, All India Institute of Medical Sciences,
Ansari Nagar, New Delhi, India
SUMMARY
1. Centella asiatica, an Indian medicinal plant, has been
described as possessing central nervous system activity, such as
improving intelligence. In addition, we have demonstrated that
C. asiatica has cognitive-enhancing and anti-oxidant properties
in normal rats. Oxidative stress or an impaired endogenous
anti-oxidant mechanism is an important factor that has been
implicated in Alzheimer’s disease (AD) and cognitive deficits
seen in the elderly.
2. Intracerebroventricular (i.c.v.) streptozotocin (STZ) in
rats has been likened to sporadic AD in humans and the
cognitive impairment is associated with free radical generation
in this model. Therefore, in the present study, the effect of an
aqueous extract of C. asiatica (100, 200 and 300 mg/kg for
21 days) was evaluated in i.c.v. STZ-induced cognitive
impairment and oxidative stress in rats.
3. Male Wistar rats were injected with STZ (3 mg/kg, i.c.v.)
bilaterally on the days 1 and 3. Cognitive behaviour was
assessed using passive avoidance and elevated plus-maze
paradigms on the days 13, 14 and 21. Rats were killed on the
day 21 for estimation of oxidative stress parameters (malondi-
aldehyde (MDA), glutathione, superoxide dismutase and cata-
lase) in the whole brain upon completion of the behavioural
task.
4. Rats treated with C. asiatica showed a dose-dependent
increase in cognitive behaviour in both paradigms. A signifi-
cant decrease in MDA and an increase in glutathione and
catalase levels were observed only in rats treated with 200 and
300 mg/kg C. asiatica.
5. The present findings indicate that an aqueous extract of
C. asiatica is effective in preventing the cognitive deficits, as
well as the oxidative stress, caused by i.c.v. STZ in rats.
Key words: ageing, Alzheimer’s disease, Centella asiatica,
learning and memory, oxidative stress, streptozotocin.
Correspondence: Professor YK Gupta, Neuropharmacology Laboratory,
Department of Pharmacology, All India Institute of Medical Sciences,
Ansari Nagar, New Delhi 110029, India. Email: ykg@hotmail.com
Received 2 March 2002; revision 18 August 2002; accepted
25 November 2002.
INTRODUCTION
Ageing and age-related neurodegenerative diseases are associated
with various degrees of behavioural impairment. Important candi-
dates for the production of neuronal changes mediating these
behavioural deficits are free radicals, which are responsible for the
generation of oxidative stress.1,2 Currently, there has been an
increasing interest in the biochemical functions of natural anti-
oxidants contained in vegetables, fruits and medicinal plants,
which could be candidates for the prevention of oxidative damage
and improving memory.3
In the Indian system of medicine (Ayurveda), Centella asiatica
(Umbelliferae) has been used in various locations throughout India
for the treatment of different ailments, such as insanity, asthma,
leprosy, ulcers, eczema and wound healing.4,5 Centella asiatica is
a small herbaceous plant growing predominantly in the southern
hemisphere that is closely related to Hydrcotyle species and pro-
duces a characteristic essential oil and flavonoids.6 Some of the
triterpenes and flavonoids from C. asiatica have antitumour
activity.7,8 Centella asiatica has been reported to contain
saponins, astiaticoside and medecassocide in its leaves.9 The use
of asiaticoside in the treatment of leprosy and wound healing has
shown encouraging results.10–12 Brahmic acid, an active triter-
penoid present in the plant, has therapeutic value in the treatment
of ulcerations, extensive wounds and eczema,6,13–15 as well as an
inhibitory effect on the biosynthetic activity of fibroblast cells.16
Apart from these chemical constituents, C. asiatica also contains
asiatica and madecassic acid, which are known to possess neuro-
protective properties.17,18
The whole plant of C. asiatica has been shown to be beneficial
in improving memory19,20 and has also been reported to improve
the general mental ability of intellectually disabled children.21,22
Nalini et al.23 have shown that fresh leaf juice improves the passive
avoidance task in rats. Recently, in our laboratory, we have
demonstrated that an aqueous extract of C. asiatica has cognitive-
enhancing properties in different paradigms, such as the shuttle
box, step through, step down and elevated plus-maze, with associ-
ated decreases in brain oxidative stress parameters, in normal
rats.24
Intracerebroventricular (i.c.v.) injection of streptozotocin (STZ;
3 mg/kg, bilateral) in rats has been found to cause behavioural
impairment that resembles Alzheimer’s disease (AD).25–29 There-
fore, the aim of the present study was to evaluate the effect of
chronic treatment of an aqueous extract of C. asiatica on learning
 Centella asiatica in i.c.v. streptozotocin
and memory and oxidative stress parameters (malondialdehyde
(MDA), glutathione, superoxide dismutase (SOD) and catalase) in
i.c.v. STZ-treated rats. In the present study, it was observed that
C. asiatica significantly prevented cognitive impairment and
attenuated the oxidative stress in i.c.v. STZ-treated rats.
METHODS
Plant material and preparation of the extract
Plants were procured from the commercial market at Khari Baoli, New
Delhi. Samples were then authenticated for their correct botanical identity
by the Chief Botanist, Department of Drug Standardization, Dabur
Research Foundation, Ghaziabad, India. The whole aerial plant was dried
and ground coarsely with a grinder. For the preparation of the aqueous
extract, the coarse powder of the plant was extracted with 8 parts water, with
boiling, for 5 h and filtered through a 400 m mesh cloth to collect the
extract. The extract was concentrated and finally spray dried to yield a
greenish-brown powder. The percentage w/w yield of the aqueous extract
was 41. The loss on drying at 105C and the ash content of the powder were
5.0 and 8.0% w/w, respectively. The concentration of the known active
component asiaticoside was determined by HPLC and was found to be
3% w/w.
Animals
Studies were performed using male Wistar rats weighing 200–250 g. Rats
were obtained from the Central Animal House Facility of the All India
Institute of Medical Sciences, New Delhi, and stock bred in the depart-
mental animal house. Rats were group housed in polyacrylic cages
(38  23  10 cm) with not more than four animals per cage and were
maintained under standard laboratory conditions with a natural dark and
light cycle (approximately 14 h light/10 h dark cycle) and room temperature
25 ± 1C. Rats were allowed free access to standard dry diet (Golden Feeds,
Delhi, India) and tap water ad libitum. All behavioural procedures were
performed between 09.00 and 13.00 h. All procedures described were
reviewed and approved by the Institutional Committee for Ethical Use of
Animals.
Intracerebroventricular administration of STZ
Immediately prior to surgery, rats were anaesthetized with chloral hydrate
(240 mg/kg, i.p., in 4% solution) and positioned in a stereotaxic apparatus.
A saggital incision was made in the scalp and two holes were drilled
through the skull for placement of the injection cannula into the lateral
cerebral ventricles. Animals received either i.c.v. artificial cerebrospinal
fluid (ACSF; 10 L/site) or i.c.v. STZ (3 mg/kg bilaterally using a micro
syringe). The composition of the ACSF was (in mmol/L): NaCl 147;
KCl 2.9; MgCl2 1.6; CaCl2 1.7; dextrose 2.2. To ensure diffusion of the
administered drug, the cannula was left in place for a period of 2 min
following the injection. The stereotaxic coordinates for i.c.v. injection were
0.8 mm posterior to bregma, 1.8 mm lateral to the saggital suture and
3.6 mm beneath the cortical surface.
Experimental design
Animals were divided randomly into five groups of 10 animals each. The
first group received i.c.v. ACSF, whereas groups 2–5 were administered
i.c.v. STZ (Sigma, St Louis, MO, USA; 3 mg/kg) dissolved in ACSF
bilaterally on the days 1 and 3. Groups 3–5 were also administered an
aqueous extract of C. asiatica at concentrations of 100, 200 and 300 mg/kg,
respectively, once daily for 21 days orally through an intragastric feeding
tube. Behavioural tests were performed on days 13, 14 and 21 and markers
of oxidative stress were estimated after the last behavioural test on day 21
of the STZ injection.
337
Behavioural tests
Passive avoidance learning
Memory retention deficit was evaluated by a step-through passive avoid-
ance apparatus. The apparatus consisted of equal sized light and dark
compartments (30  20  30 cm). A 40 W lamp was fixed 30 cm above the
floor in the centre of the light compartment. The floor consisted of a metal
grid connected to a shock scrambler. The two compartments were separated
by a trapdoor that could be raised to 10 cm. On day 13 after i.c.v. STZ
injection, rats were placed in the light compartment and the time that
elapsed before each animal entered the dark compartment and had all four
paws inside the dark compartment was measured, in s, and termed ‘initial
latency’. Immediately after the rat entered the dark chamber with all the
four paws inside, the trapdoor was closed and an electric foot shock
(50 V AC) was delivered for 3 s. Five seconds later, the rat was removed
from the dark chamber and returned to its home cage. Rats that had an initial
latency of more than 60 s were excluded from further experiments. Twenty-
four hours and 8 days after the initial latency (i.e. days 14 and 21,
respectively, after STZ injection), the latency time was measured again in
the same way as in acquisition trial and termed the ‘first retention latency’
(1st RL) and ‘second retention latency’ (2nd RL); however, the foot shock
was not delivered and the latency time was recorded to a maximum of 600 s.
To improve the reliability and validity of the foot shock-avoidance test, the
grid, as well as the rats’ paws, were moistened with water before foot shock
because this is known to reduce the wide interanimal variability in paw skin
resistance.30
Elevated plus-maze
The plus-maze consists of two opposite open arms (50  10 cm), crossed
with two closed arms of the same dimensions with 40 cm high walls. The
arms are connected by a central square (10  10 cm). On day 13 after i.c.v.
STZ injection, rats were placed individually at one end of an open arm,
facing away from the central square. The time taken for the rat to move from
the open arm and enter into one of the closed arms was recorded as ‘initial
transfer latency’ (ITL). The animal was allowed to explore the maze for 30 s
after recording the ITL and returned to its home cage. Then, 24 h and 8 days
after ITL, the rat was placed similarly on the open arm and the retention
latency was noted again and termed as ‘first retention transfer latency’ (1st
RTL) and ‘second retention transfer latency’ (2nd RTL).31
Closed field activity
Spontaneous locomotor activity was assessed on days 13, 14 and 21 of i.c.v.
STZ administration. Each animal was observed over a period of 5 min in a
square closed arena (30 cm) equipped with infrared light-sensitive photo-
cells using a digital photoactometer (Techno Electronics, Lucknow, India)
and values were expressed as counts/5 min. The apparatus was placed in a
darkened, light- and sound-attenuated and ventilated testing room with
other behavioural testing apparatuses.27
The sequence of the behavioural tests conducted in the retest phase on
days 13, 14 and 21 was passive avoidance behaviour, followed by transfer
latency in the plus-maze and, finally, locomotor activity.
Biochemical tests
Following behavioural testing, animals were decapitated under ether anaes-
thesia and their brains quickly removed, cleaned with ice-cold saline and
stored at –80C.
Tissue preparation
Brain tissue samples were thawed and homogenized with 10 times (w/v)
ice-cold 0.1 mol/L phosphate buffer (pH 7.4).32 Aliquots of homogenates
from rat brain were separated and used to determine protein, lipid peroxi-
dation and glutathione. The remaining homogenates were centrifuged at
28 900 g for 60 min and the supernatant was then used for enzyme assays.
Catalase activity was determined immediately after sample preparation and
338 MH Veerendra Kumar and YK Gupta
SOD was determined within 24 h. Protein concentration was determined
according to the method of Lowry et al.33 using purified bovine serum
albumin as the standard.
Measurement of lipid peroxidation
Malondialdehyde, a measure of lipid peroxidation, was determined as
described by Jainkang et al.34 Acetic acid (1.5 mL; 20%; pH 3.5), 1.5 mL
thiobarbituric acid (0.8%) and 0.2 mL sodium dodecyl sulphate (8.1%)
were added to 0.1 mL processed tissue samples, then heated at 100C
for 60 min. The mixture was cooled with tap water and 5 mL n-
butanol : pyridine (15 : 1) and 1 mL distilled water were added. The
mixture was vortexed vigorously. After centrifugation at 2000 g for 10 min,
the organic layer was separated and absorbance was measured at 532 nm
using a spectrophotometer. The concentration of MDA is expressed as
nmol/g tissue.
Measurement of reduced glutathione
Glutathione was measured according to the method of Ellman.35 An equal
quantity of homogenate was mixed with 10% trichloroacetic acid and
centrifuged to separate the proteins. To 0.01 mL of this supernatant, 2 mL
phosphate buffer (pH 8.4), 0.5 mL 55-dithiobis(2-nitrobenzoic acid) and
0.4 mL double-distilled water were added. The mixture was vortexed and
the absorbance read at 412 nm within 15 min. The concentration of reduced
glutathione was expressed as g/g tissue.
Measurement of catalase
Catalase activity was measured according to the method of Aebi.36
Briefly 0.1 mL supernatant was added to a cuvette containing 1.9 mL of
50 mmol/L phosphate buffer (pH 7.0). The reaction was started by the
addition of 1 mL freshly prepared 30 mmol/L H2O2. The rate of decompo-
sition of H2O2 was measured spectrophotometrically from changes in
absorbance at 240 nm. The activity of catalase was expressed as units/mg
protein.
Fig. 1 Effect of aqueous extracts of Centella asiatica on step-through
latency in i.c.v. streptozotocin (STZ)-treated rats. Data are the mean±SEM.
*P < 0.05, **P < 0.01, ***P < 0.001 compared with i.c.v. STZ-treated rats.
IL, initial latency after 13 days of i.c.v. STZ administration; 1st RL, first
retention latency 24 h after IL; 2nd RL, 2nd retention latency 8 days
after initial latency. ( ), i.c.v. artificial cerebrospinal fluid; (), i.c.v.
STZ + vehicle; (), i.c.v. STZ + 100 mg/kg C. asiatica; ( ), i.c.v.
STZ + 200 mg/kg C. asiatica; ( ), i.c.v. STZ + 300 mg/kg C. asiatica.
Measurement of SOD
The SOD activity of the brain tissue was analysed according to the method
of Kakkar et al.37 The assay mixture contained 0.1 mL sample, 1.2 mL
sodium pyrophosphate buffer (pH 8.3; 0.052 mol/L), 0.1 mL phenazine
methosulphate (186 mol/L), 0.3 mL of 300 mol/L nitroblue tetrazolium
and 0.2 mL NADH (750 mol/L). The reaction was started by the addition
of NADH. After incubation at 30C for 90 s, the reaction was stopped by
the addition of 0.1 mL glacial acetic acid. The reaction mixture was stirred
vigorously and shaken with 4 mL n-butanol. The mixture was allowed to
stand for 10 min, centrifuged and the butanol layer was separated. The
colour intensity of the chromogen in the butanol was measured spectro-
photometrically at 560 nm. The concentration of SOD was expressed as
units/mg protein.
Statistical analysis
Data were processed by analysis of variance (ANOVA) followed by post-test
and individual comparisons using Student’s t-test (two tailed).
RESULTS
Effect of C. asiatica on parameters of learning and
memory in i.c.v. STZ-treated rats
Passive avoidance task
In the step-through apparatus, when tested on day 13, the mean
initial latency in different groups (i.e. i.c.v. ACSF, vehicle plus i.c.v.
STZ and the C. asiatica (100, 200 and 300 mg/kg) plus STZ rats)
was 37.4 ± 8.4, 37.0 ± 5.7, 42.7 ± 5.4, 48.5 ± 4.8 and 51.0 ± 6.9 s,
respectively. These values were not significantly different. On day
14, the mean 1st RL in the i.c.v. ACSF group was 394.2 ± 13.6 s
and there was a significant reduction in the value in the i.c.v.
Fig. 2 Effect of aqueous extracts of Centella asiatica on transfer latency
in i.c.v. streptozotocin (STZ)-treated rats. Data are the mean±SEM.
*P < 0.05, **P < 0.01 compared with i.c.v. STZ-treated rats. ITL, initial
transfer latency after 13 days of i.c.v. STZ administration; 1st RTL, 1st
retention transfer latency 24 h after ITL; 2nd RTL, 2nd retention transfer
latency 8 days after ITL. ( ), i.c.v. artificial cerebrospinal fluid; (), i.c.v.
STZ + vehicle; (), i.c.v. STZ + 100 mg/kg C. asiatica; ( ), i.c.v.
STZ + 200 mg/kg C. asiatica; ( ), i.c.v. STZ + 300 mg/kg C. asiatica.
 Centella asiatica in i.c.v. streptozotocin
STZ-treated group (166.2 ± 11.2 s). The C. asiatica-treated groups
showed significant dose-dependent recovery, the 1st RL values
being 232.6 ± 25.5, 376.4 ± 49.5 and 439.0 ± 46.5 s for 100, 200
and 300 mg/kg C. asiatica extract, respectively. On day 21, the 2nd
RL showed a similar trend of significant reversal. The 2nd RL
values in the i.c.v. ACSF, vehicle plus i.c.v. STZ and the C. asiatica
(100, 200 and 300 mg/kg) plus STZ rats were 308.4 ± 32.6,
192.7 ± 21.0, 287.4 ± 36.4, 295.7 ± 34.0 and 341.5 ± 32.7 s,
respectively. The results are summarized in Fig. 1.
Elevated plus-maze
When tested on the elevated plus-maze, the mean ITL of i.c.v.
ACSF, vehicle-treated i.c.v. STZ and C. asiatica (100, 200 and
300 mg/kg) i.c.v. STZ groups was 50.8 ± 8.4, 48.0 ± 4.9,
51.1 ± 7.5, 51.6 ± 4.8 and 47.6 ± 5.3 s, respectively. These values
did not differ significantly. On day 14, the mean 1st RTL of the
i.c.v. ACSF group was 20.2 ± 1.7 s and there was no significant
decrease in the value in the i.c.v. STZ-treated group (44.1 ± 4.9 s).
The C. asiatica-treated group showed dose-dependent decreases in
the 1st RTL values (47.1 ± 5.2, 34.1 ± 3.4 and 27.6 ± 3.1 s for 100,
200 and 300 mg/kg C. asiatica, respectively); however, only the
group administered 300 mg/kg C. asiatica showed a significant
decrease (P < 0.05). On day 21, the 2nd RTL showed a similar
Fig. 3 Effect of aqueous extracts of Centella asiatica on spontaneous
locomotor activity in i.c.v. streptozotocin (STZ)-treated rats. Data
are the mean±SEM. ( ), i.c.v. artificial cerebrospinal fluid; (), i.c.v.
STZ + vehicle; (), i.c.v. STZ + 100 mg/kg C. asiatica; ( ), i.c.v.
STZ + 200 mg/kg C. asiatica; ( ), i.c.v. STZ + 300 mg/kg C. asiatica.
Table 1 Effect of aqueous extracts of Centella asiatica on oxidative stress markers in i.c.v. streptozotocin-treated rats on day 21
Malondialdehyde
Treatment (nmol/g tissue)
i.c.v. ACSF 253.7 ± 51.000
i.c.v. STZ 438.5 ± 52.0†0
i.c.v. STZ + C. asiatica 100 mg/kg 422.3 ± 66.300
i.c.v. STZ + C. asiatica 200 mg/kg 210.1 ± 38.6**
i.c.v. STZ + C. asiatica 300 mg/kg 185.3 ± 27.8**
Data are the mean±SEM. †P < 0.001 compared with i.c.v. artificial cerebrospinal fluid (ACS)-treated rats; *P < 0.01, **P < 0.001 compared with i.c.v.
streptozotocin (STZ) vehicle-treated rats.
339
trend of significant decreases, values being 18.4 ± 3.0, 51.5 ± 8.0,
42.1 ± 4.5, 30.1 ± 1.0 and 25.6 ± 2.7 s in the i.c.v. ACSF, vehicle
plus i.c.v. STZ and the C. asiatica (100, 200 and 300 mg/kg) plus
STZ rats, respectively. Groups treated with 200 and 300 mg/kg
C. asiatica showed significant reductions in the RTL compared
with the i.c.v. ACSF group. The results are summarized in Fig. 2.
Locomotor activity
Spontaneous locomotor activity did not differ significantly between
the i.c.v. ACSF, vehicle-treated i.c.v. STZ group and the C. asiatica
(100, 200 and 300 mg kg) i.c.v. STZ groups on days 13, 14 and 21
(Fig. 3).
Effect of C. asiatica on markers of oxidative stress in
i.c.v. STZ-treated rats on day 21
Rat brain MDA levels
The MDA level in the vehicle-treated i.c.v. STZ group was
438.5 ± 52.0 nmol/g tissue, which was significantly higher than
that seen in the i.c.v. ACSF group (253.9 ± 31.6 nmol/g tissue;
P < 0.001). The C. asiatica (200 and 300 mg/kg) i.c.v. STZ
groups showed significant (P < 0.001) decreases in MDA levels
compared with the i.c.v. STZ group (210.1 ± 38.6 and
185.3 ± 27.8 nmol/g tissue, respectively; P < 0.001). However, the
100 mg/kg C. asiatica-treated i.c.v. STZ group did not show any
significant difference compared with the i.c.v. STZ group (Table 1).
Rat brain glutathione levels
Glutathione levels values of i.c.v. ACSF and vehicle-treated i.c.v.
STZ groups were 161.8 ± 7.8 and 32.0 ± 1.3 g/g tissue, respec-
tively. There was a significant (P < 0.001) decrease in the levels of
glutathione in the vehicle-treated i.c.v. STZ group compared with
the i.c.v. ACSF group. The C. asiatica (200 and 300 mg/kg) i.c.v.
STZ groups showed significant (P < 0.001) increase in glutathione
levels compared with the i.c.v. STZ group (94.2 ± 8.0 and
94.5 ± 4.5 g/g tissue, respectively; Table 1).
Rat brain SOD levels
There was no significant difference observed in SOD levels
between the i.c.v. ACSF, vehicle-treated i.c.v. STZ and C. asiatica
(100, 200 and 300 mg/kg) i.c.v. STZ groups (Table 1).
Rat brain catalase levels
There was no significant difference observed in catalase levels
between the i.c.v. ACSF and vehicle-treated i.c.v. STZ groups. In
C. asiatica (100 and 200 mg/kg) i.c.v. STZ groups, there was an
increase in catalase levels, but values not differ significantly
Glutathione Superoxide dismutase Catalase
(g/g tissue) (U/mg protein) (U/mg protein)
161.8 ± 7.800 3.2 ± 0.1 5.4 ± 1.8
32.0 ± 1.3†0 3.3 ± 0.2 6.9 ± 2.0
40.2 ± 2.700 3.2 ± 0.1 12.5 ± 2.90
94.2 ± 8.0** 3.3 ± 0.2 10.1 ± 1.50
94.5 ± 4.5** 2.9 ± 0.3 20.9 ± 3.5*
340 MH Veerendra Kumar and YK Gupta
compared with ACSF or vehicle-treated i.c.v. STZ groups. There
was a significant (P < 0.01) increase in catalase observed in
300 mg/kg C. asiatica-treated rats compared with the i.c.v. STZ
group (20.9 ± 3.57 vs 6.9 ± 2.0 units/mg protein, respectively;
Table 1).
DISCUSSION
The increase in life expectancy globally has also increased the
incidence of age-associated neurodegenerative diseases, such as
AD and Parkinson’s disease. Both ageing and age-related neuro-
degenerative disorders are associated with varying degrees of
behavioural impairment that cause significant morbidity. Among
the prime candidates responsible for producing the neuronal
changes mediating these behavioural deficits,appear to be free
radicals and the oxidative stress they generate.1 However, it is
difficult to establish whether the neuronal injury due to free radical
generation is a primary or secondary event. However, even if it is
secondary to other causes, it is still deleterious and can lead to
neuronal death.38,39 Thus, removing free radicals or preventing their
formation may be beneficial in the treatment and/or prevention of
neurodegenerative disorders. Anti-oxidants from natural products
that have been used in the ancient Indian medical system of
Ayurveda for improving learning and memory have recently drawn
much attention.
Centella asiatica has recently been shown to be effective in
reducing oxidative damage in the central nervous system (CNS),
which is attributed to its anti-oxidant properties.40 Furthermore,
C. asiatica has been shown to improve the mental capabilities/
function of rodents as well as humans.21,22 Therefore, we con-
sidered it worthwhile to investigate whether an aqueous extract of
C. asiatica, at doses that showed cognitive-enhancing and anti-
oxidant properties in normal rats,24 had a protective role against
i.c.v. STZ-induced oxidative stress and associated cognitive impair-
ment in rats.
It has been reported that there is a decrease in glucose utilization
in the brains of AD patients.41,42 This led to the hypothesis that the
cognitive dysfunction of AD is related to a reduction in central
glucose metabolism. It has been hypothesized that the cholinergic
deficit and amyloid accumulation in the brain are caused by a
significant decrease in glucose metabolism in the AD brain.43
Several studies have shown that i.c.v. administration of sub-
diabetogenic doses of STZ reduce central glucose in rats.44–46
Concomitantly, decreases in cognitive function, cholinergic and
monoaminergic neurotransmission and other related enzymes, such
as cholineacetyl transferase activity, were observed in the rat
brain.25–28,47,48 Recently, it has been shown that the generation of
free radicals following i.c.v. STZ injection is an important factor in
causing cognitive impairment in rats.49,50 Therefore, we used the
recently reported dose of i.c.v. STZ (3 mg/kg) to induce cognitive
impairment and oxidative stress in rats.49 These previous
reports25–27,49 suggest that STZ-treated rats could be regarded as
a relevant animal model of sporadic AD.
In the present study, it was observed that the vehicle-treated i.c.v.
STZ group showed significant reduction of retention latencies in
passive avoidance behaviour and no significant improvement in
RTL in the elevated plus-maze compared with the ACSF-treated
group, suggesting impairment of learning and memory in i.c.v. STZ
rats. These results are in accord with those of previous reports that
also demonstrated cognitive impairment after i.c.v. STZ in
rats.25–28,30
However, i.c.v. STZ rats that were chronically treated with
C. asiatica at 200 and 300 mg/kg, p.o., for 21 days showed
significantly increased retention latencies compared with the STZ
(vehicle)-treated animals in the passive avoidance apparatus and
significantly shorter transfer latencies on the elevated plus-maze.
The improvement in passive avoidance elevated plus-maze
behaviour indicates an improved acquisition and retention of
memory in rats treated with C. asiatica. The general stimulant or
depression activity of a CNS-active drug may affect the response
of an animal on behavioural paradigms. Therefore, the effect of
C. asiatica was also studied on spontaneous locomotor activity.
There were non-significant differences between the locomotor
activity of i.c.v. ACSF, vehicle-treated i.c.v. STZ and C. asiatica
(100, 200 and 300 mg/kg) i.c.v. STZ groups. This excludes the
possibility that any CNS depressant/stimulant activity of
C. asiatica may have contributed to changes in passive avoidance
and elevated plus-maze in vehicle-treated i.c.v. STZ rats and the
C. asiatica-treated i.c.v. STZ group.
Free radicals are normal products of cellular aerobic metabo-
lism. However, when the production of free radicals increases or
the defence mechanism of the body decreases, free radicals cause
cellular dysfunction by attacking polyunsaturated sites of bio-
logical membranes, leading to lipid peroxidation.51 In the present
study, the increase in levels of MDA, a marker of lipid peroxid-
ation, indicates increased free radical generation in vehicle
STZ-treated rats. The significantly lesser rise in MDA levels in the
brains of i.c.v. STZ rats treated with 200 and 300 mg/kg C. asiatica
compared with the vehicle-treated i.c.v. STZ rats indicates attenu-
ation of lipid peroxidation. There was a simultaneous significant
decrease in reduced glutathione levels in vehicle-treated i.c.v. STZ
rats. Glutathione is an endogenous anti-oxidant largely present in
its reduced form within cells. Glutathione reacts with the free
radicals and prevents the generation of hydroxyl radicals, the most
toxic form of free radicals. During this defence process, reduced
glutathione is converted to the oxidized form with the help of the
enzyme glutathione peroxidase. The decreased level of reduced
glutathione in vehicle-treated i.c.v. STZ seen in the present study
indicates that there was an increased generation of free radicals and
that the reduced glutathione became depleted during the process of
combating oxidative stress.52 The increase in the glutathione levels
in C. asiatica-treated i.c.v. STZ groups may be due the anti-oxidant
properties of the extract.
The levels of SOD and catalase did not differ significantly
in any of the groups (i.c.v. ACSF, vehicle-treated i.c.v. STZ rats and
C. asiatica-treated i.c.v. STZ rats). This could be because the
glutathione pathway may be playing a major role in combating
oxidative stress in the brain in i.c.v. STZ rats. The non-significant
change in the levels of catalase may be due to the fact that catalase
activity in the rat brain is very low, as reported previously.53
Therefore, there was a significant change in the level of glutathione,
but not in the levels of SOD and catalase in C. asiatica-treated i.c.v.
STZ groups compared with the vehicle-treated i.c.v. STZ group.
The increase in the levels of glutathione induced by C. asiatica
suggests that the anti-oxidant property of C. asiatica was respon-
sible for combating the oxidative stress induced by i.c.v. STZ.
Centella asiatica has recently been reported to have antilipid-
peroxidative and anti-eplileptic activity in a lithium–pilocarpine
 Centella asiatica in i.c.v. streptozotocin
model of status epilepticus.40 It has also been reported that
astiaticoside, the chief constituent of C. asiatica, possessed wound-
healing activity by increasing enzymatic (SOD, catalase, glutath-
ione peroxidase) and non-enzymatic (vitamin E and ascorbic acid)
anti-oxidant levels, thereby reducing lipid peroxide levels in
wounds.54 Lee et al.18 reported that asiatic acid and its derivatives
protect cultured neurons from glutamate-induced excitotoxicity.
In conclusion, the present study demonstrates that C. asiatica
significantly prevented cognitive impairment and attenuated the
oxidative stress induced by brain glucose metabolism impairment
in i.c.v. STZ-treated rats by its neuroprotective property. However,
the possibility of an effect of C. asiatica on neurotransmitters in
improving cognitive deficits cannot be ruled out.
ACKNOWLEDGEMENTS
We are thankful to the Indian Council of Medical Research for a
financial grant for this study and the Dabur Research Foundation
for their help in preparing extracts.
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