Neuroprotective and nootropic activity of clitorea ternatea linn.(mazreoon in unani) leaves on diabetes induced cognitive decline in experimental animals

[Downloaded free from http://www.jpbsonline.org on Saturday, January 04, 2014, IP: 117.242.184.92] || Click here to download free Android application for this journ

Original Article
Neuroprotective and nootropic activity of
Clitorea ternatea Linn.(Fabaceae) leaves
on diabetes induced cognitive decline in
experimental animals
Karuna A. Talpate, Uma A. Bhosale1, Mandar R. Zambare, Rahul S. Somani

Department of
Pharmacology, Sinhgad
College of Pharmacy,
Vadgaon, 1Department of
Pharmacology,
Smt. Kashibai Navale
Medical College and
General Hospital, Narhe,
Pune, Maharashtra, India
Address for correspondence:
Dr. Uma A. Bhosale,
E‑mail: umabhosale2000@
gmail.com

Received
: 03‑04‑12
Review completed : 13‑08‑12
Accepted
: 30‑08‑12

ABSTRACT
Purpose: Ethanol extract of Clitorea ternatea (EECT) was evaluated in diabetes‑induced cognitive decline
rat model for its nootropic and neuroprotective activity. Materials and Methods: Effect on spatial working
memory, spatial reference memory and spatial working‑reference memory was evaluated by Y maze, Morris
water maze and Radial arm maze respectively. Neuroprotective effects of EECT was studied by assaying
acetylcholinesterase, lipid peroxide, superoxide dismutase (SOD), total nitric oxide (NO), catalase (CAT) and
glutathione (GSH) levels in the brain of diabetic rats. Results: The EECT (200 and 400 mg/kg) was found to
cause significant increase in spatial working memory (P < 0.05), spatial reference memory (P < 0.001) and
spatial working‑reference (P < 0.001) in retention trials on Y maze, Morris water maze and Radial arm maze
respectively. Whereas significant decrease in acetylcholinesterase activity (P < 0.05), lipid peroxide (P < 0.001),
total NO (P < 0.001) and significant increase in SOD, CAT and GSH levels was observed in animals treated
with EECT (200 and 400 mg/kg) compared to diabetic control group. Conclusions: The present data indicates
that Clitorea ternatea tenders protection against diabetes induced cognitive decline and merits the need for
further studies to elucidate its mode of action.
KEY WORDS: Clitorea ternatea, morris water maze, neuroprotective, nootropic, radial arm maze, Y maze

D

iabetes mellitus (DM) is one of the most important and
prevalent chronic diseases that can damage any organ
in the body. Less addressed and recognized complications
of DM are cognitive dysfunction, Alzheimer’s disease and
dementia.[1,2] Like diabetes, cognitive dysfunction represents
another serious problem and is rising in prevalence worldwide,
especially among the elderly. Cognitive impairment due to
diabetes mainly occurs at two main periods; during the first
5-7 years of life when the brain system is in development;
and the period when the brain undergoes neurodegenerative
changes due to aging.[3]
Access this article online
Quick Response Code:

Website:
www.jpbsonline.org
DOI:
10.4103/0975-7406.124317

Oxidative damage to various brain regions constitutes the long
term complications, morphological abnormalities and memory
impairments.[4] Oxidative stress becomes evident by increased
lipid peroxide, nitric oxide (NO) levels and decreased glutathione
(GSH), superoxide dismutase (SOD) and catalase (CAT)levels.[5]
Neurotransmitter functions which are altered in DM include
decreased acetylcholine production, decreased serotonin turnover,
decreased dopamine activity and increased norepinephrine.[3]
Substantial experimental evidence exists for a relation between
the decline in cholinergic functions and dementia.[6]
‘Diabetes‑associated cognitive decline’ is a new term proposed
to facilitate research in Diabetic encephalopathy. Hyperglycemia,
oxidative stress and cholinergic decline play a central role
in cognitive impairment in diabetic encephalopathy. [6,7]
Anti‑diabetic and psychotropic drugs are associated with several
adverse effects and this has spurred the need for exploring
drugs without side effects especially, from traditional system
of medicine like Ayurveda, Siddha and Unani. In Ayurveda the
roots, seeds and leaves of Clitorea ternatea Linn. (Fabaceae) have
long been widely used as a brain tonic and is believed to endorse

How to cite this article: Talpate KA, Bhosale UA, Zambare MR, Somani RS. Neuroprotective and nootropic activity of Clitorea ternatea Linn.(Fabaceae)
leaves on diabetes induced cognitive decline in experimental animals. J Pharm Bioall Sci 2014;6:48-55.


48

Journal of Pharmacy and Bioallied Sciences January-March 2014 Vol 6 Issue 1

[Downloaded free from http://www.jpbsonline.org on Saturday, January 04, 2014, IP: 117.242.184.92] || Click here to download free Android application for this jour
Talpate, et al.: Clitorea ternatea: A ray of hope for diabetic dementia

memory and intelligence.[8] It is reported to have antidepressant,
anticonvulsant,[9] anti‑inflammatory, analgesic and antipyretic,[10]
local anesthetic,[11] purgative[12] and anti‑diabetic[13] activity. It is
also used for treatment of snakebite and scorpion sting in India.[14]
Since the plant is reported to be used by the traditional system
of medicine as anti‑diabetic, brain tonic and believed to promote
memory and intelligence; authors have premeditated this study to
explore the nootropic and neuroprotective potential of Ethanol
extract of Clitorea ternatea (EECT)leaves in experimental model
for DM induced cognitive decline.

Materials and Methods
Collection and identification of plant material
The leaves of Clitorea ternatea were collected in the month of
November 2009 from the local areas of Pune; Maharashtra. The
plant material was authenticated at Botanical Survey of India;
Pune and voucher specimen: BB680518 was deposited.

Preparation of extract
The leaves were dried under shade and later pulverized in a
mechanical grinder. The 100 g of powder was then extracted
with ethanol (95%) for a week. The mixture was then filtered
and the filtrate was concentrated under reduced pressure to
yield semisolid (3.70%w/w) extract. This semisolid extract
was preserved in the refrigerator in airtight container till
further use.

Selection and maintenance of animals
Adult Sprague Dawley rats of either sex, weighing between
150‑250 g were used for the study. The animals were housed
in standard polypropylene cages at room temperature
and provided with standard diet and water ad libitum.
The experimental protocol has been approved by the
Institutional Animal Ethics Committee (SCOP/IAEC/
Approval/2009‑10/10). The animals were shifted to the
laboratory one hour prior to the experiment.

Drugs and chemicals
Metformin was obtained as gift sample from Cipla
Pharmaceuticals Ltd, India, Piracetam (Nootropil, 80 mg tab),
Streptozotocin (Sigma Aldrich, USA), Diagnostic Kits (Biolab,
I n d i a ) , M DA , S O D , C AT ( S i g m a A l d r i c h , U S A ) ,
GSH (LobaChem, India) were purchased from local market.
Other chemicals of analytical grade were obtained from
Qualigens, India.

Administration of dosage
The EECT suspension was prepared in 2% acacia using
mortar and pestle and administered orally to rats at various
doses.[15] Metformin, piracetam were used as standard drugs
and administered orally at a dose of 200 mg/kg in distilled
water.

Induction of DM
Streptozotocin (STZ) dissolved in cold citrate buffer (pH 4.45, 0.1 M)
was administered intraperitoneally (55 mg/kg) to induce diabetes.
After 48 hours of STZ injection, blood samples were collected
by puncturing retro‑orbital plexus of rats under light ether
anesthesia using fine glass capillary in epindorff tubes and
serum glucose level was estimated by glucose oxidase and
peroxidaseoxidase enzymatic method. Animals with serum
glucose more than 250 mg/dl considered as diabetic and used
for the further study.[16,17]

Study Design
Phytochemical study
EECT was evaporated to residue and dilute HCl was added ‑to
it. After shaking, extract was filtered and with filtrate tests
were performed for the detection of various constituents using
conventional protocol like Mayer’s, Wagner’s, Hagner’s, and
Dragendorff’s tests for alkaloids; Baljet’s, Borntrager’s, Legal’s
tests for glycosides; foam and hemolytic tests for saponins;
Salkowski’s and Lieberman – Buchard’s tests for steroids/
triterpenoids; Gelatin and Ferric chloride tests for tannins;
Ferric chloride, Shinoda, Alkaline reagent and Lead acetate
solution test for flavonoids; Molisch’s and Benedict’s tests for
carbohydrates; Millon’s and Ninhydrin tests for proteins and
amino acids respectively.[18]

Acute toxicity study
Acute toxicity study was carried out according to the Organization
for Economic Co‑operation and Development (OECD)
Guidelines No. 423. Three animals were used for each step.
The starting dose was selected from the four fixed dose levels,
i.e., 5, 50, 300, and 2000 mg/kg body weight p.o. As per the
OECD recommendations, 300 mg/kg body weight was used
as starting dose.

Nootropic study
The EECT was tested for nootropic studies using rats as per
the method suggested by Lucian et al., Morris et al., Tuzcu and
Baydas, Carrillo et al.[19‑22] The selected animals were divided into
six groups with six animals in each. The control groups (normal
and diabetic) received 2% gum acacia 5 ml/kg/day orally. The
standard groups received metformin and piracetam in the dose
of 200 mg/kg/day, the test groups received the EECT 200 and
400 mg/kg/day orally.

Y maze test
Short‑term memory was assessed by spontaneous alternation
behavior in the Y‑maze task. The Y‑maze used in the present
study consisted of three arms (35 cm long, 25 cm high and
10 cm wide) and an equilateral triangular central area. Animal
was placed at the end of one arm and allowed to move freely
through the maze. Time limit in Y‑maze test was fixed to
49 


8 minutes hence, every session ended after 8 minutes. An
arm entry was counted when the hind paws of the rat were
completely within the arm. Spontaneous alternation behavior
was defined as entry into all three arms on consecutive choices.
The number of maximum spontaneous alternation behaviors
was then the total number of arms entered minus 2. Percent
spontaneous alternation was calculated as (actual alternations/
maximum alternations) × 100.[19] The animals were trained for
five days and % spontaneous alteration measured on 71st and
75th day [Figure 1].
Morris water maze test
On 71st day animals were tested in a spatial version of Morris
water maze. The apparatus consisted of a circular water
tank (180 cm in diameter and 60 cm high). A platform (12.5 cm
in diameter and 38 cm high) invisible to the animals, was set
inside the tank and filled with water maintained at 28 ± 2°C at
a height of 40 cm. 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
animals for spatial orientation. The position of the cues was
kept unchanged throughout the study. The water maze task
was carried out for 5 consecutive days of the post‑treatment.
The animals received daily training trials for first four of these
5 consecutive days. Each trial was of 90 seconds and with a
trial interval of approximately 30 seconds. For each trial, each
animal was put into the water at one of four starting positions,
the sequence of which being selected randomly. During test
trials, animals were placed into the tank at the same starting
point, with their heads facing the wall. The animal had to
swim until it climbs‑ the platform submerged underneath the
water. After climbing ‑the platform, the animal was allowed to
remain there for 20 seconds before the commencement of the
next trial. The escape platform was kept in the same position
relative to the distal cues. If the animal failed to reach the escape
platform within the maximum allowed time of 90 seconds, it
was gently placed on the platform and allowed to remain there
for the same time. The time to reach the platform (latency in
seconds) was measured.
A probe trial was performed wherein the extent of memory
consolidation was assessed. In the probe trial, the animal was
placed into the pool as in the training trial, except that the
hidden platform was removed from the pool. The transfer

latency (TL) to reach platform was measured on 75th day to
study spatial reference memory.[20,21]
Radial arm maze test
The radial maze is the prototype of a “multiple‑solution
problem” task. Effect of EECT on spatial working‑reference
was studied by using radial arm maze. It consisted of 8 arms,
numbered from 1 to 8 (48 × 12 cm) extending radially from a
central area (32 cm in diameter). The apparatus was placed
40 cm above the floor, and surrounded by various extra maze
cues placed at the same position during the study. At the end
of each arm there was a food cup that had a single 50 mg food
pellet. Prior to the performance of the maze task, the animals
were kept on restricted diet. Before the actual training begun,
three or four rats were simultaneously placed in the radial maze
and allowed to explore for 5 minutes to take food freely. The
food was initially made available throughout the maze, but
then ‑gradually restricted to the food cup. The animals were
shaped for 4 days to run to the end of the arms and consume
the bait. The rats were given 5 consecutive training trials to
run to the end of the arms and consume the bait. The training
trial continued until all the 5 baits were consumed or until all
5 minutes elapsed. Criterion for performance was defined as
consumption of all 5 baits or 5 min time elapse.
After adaptation all rats were trained with 1 trial per day. Briefly,
each animal was placed individually in the center of the maze
and subjected to spatial working‑reference tasks, in which
same 5 arms (no. 1, 2, 4, 5, and 7), were baited for each daily
training trial. The other 3 arms (no. 3, 6, 8) were never baited.
The training trial continued until all 5 baits were consumed or
until 5 minutes elapsed. An arm entry was counted when all
four limbs of the rat were within an arm. On 71st and 75th day;
mean spatial working‑reference was measured. Spatial working
memory was defined by the ratio; number of food rewarded
visits/number of all visits to the baited arms. This measure
represents the percentage of all visits to the baited arms that
had been reinforced with food. Spatial reference memory was
defined by the ratio; number of all visits to the baited arms/
total number of visits to all arms. This measure expresses the
number of visits to the baited arms as a percentage of the total
number of visits to all arms. The time taken to consume all 5
baits was also recorded.[19]

Neurochemical study

Figure 1: Experimental study design


50

Post‑treatment animals were sacrificed by cervical dislocation
and brain was isolated and weighed. Whole brain was rinsed
with ice cold saline (0.9% sodium chloride) and homogenized
by making 20 mg of the tissue per ml in chilled phosphate
buffer (pH 7.4). The homogenates were centrifuged at 800 g for
5 minutes at 4°C to separate the nuclear debris. The supernatant
thus obtained was centrifuged at 10500 g for 20 minutes at 4°C
to get the supernatant. Such obtained supernatant was then
used for neurochemical study.[23‑28] The protein concentration
was estimated by Lowry et al., (1951) method using bovine
serum albumin as the standard.[29]


Acetylcholinesterase assay
For estimation of acetylcholinesterase activity, Ellman’s
method named after George Ellman was used.[21] A total
of 0.4 ml supernatant was added to a cuvette containing
2.6 ml phosphate buffer (0.1 mol/L, pH 8) and 100 µL of
5,5′‑dithiobis‑(2‑nitrobenzoic acid). The contents of the cuvette
were mixed thoroughly by bubbling air and absorbance was
measured at 412 nm by a spectrophotometer. When absorbance
reaches a stable value, it was recorded as the basal reading. The
substrate of 20 µLof acetylthiocholineiodide was added and the
changes in absorbance were recorded for a period of 10 minutes
at intervals of 2 minutes. Change in the absorbance per minute
was thus determined.
The mean change in absorbance was considered for calculation
using following formula and acetylcholinesterase activity was
measured as µM/l/min/gm of tissue.[23]
R = 5.74(10−4 ) ×

∆A
Co

Where, R = Rate, in moles substrate hydrolyzed per minutes
per gm of tissue
∆A = Change in absorbance per minutes.
Co = Original concentration of tissue (20 mg/ml).
Lipid peroxide levels
Thiobarbituric acid reactive substances (TBARS) levels are measured
as an index of malondialdehyde (MDA) production which is an end
product of lipid peroxidation. MDA reacts with thiobarbituric acid
to form a red colored complex. The measurement of MDA levels
by thiobarbituric acid reactivity is the most widely used method
for assessing lipid peroxidation. For this 0.5 ml of Tris hydrochloric
acid was added in 0.5 ml of supernatant and incubated at 37°C
for 2 hours. After incubation 1 ml of 10% trichloroacetic acid was
added and centrifuged at 3000 rpm for 10 minutes, and then 1ml
of 0.67% thiobarbituric acid was added to 1 ml of supernatant. The
tubes were kept in boiling water for 10 minutes. After cooling 1 ml
double distilled water was added and absorbance was measured at
532 nm. The MDA concentrations of the samples were derived
from the standard curve prepared using known amounts of MDA
and expressed as nmol MDA/mg of protein.[24]
Total nitric oxide levels
To 100 μl of supernatant, 500 μl of Greiss reagent (1:1 solution of
1% sulphanilamide in 5% phosphoric acid and 0.1% naphthalene
diamine dihydrochloride) was added and absorbance was
measured at 546 nm. Nitrite concentration was calculated
using a standard curve for sodium nitrite and expressed as
ng/mg of protein.[25]
Superoxide dismutase levels
To 100 µl of supernatant, 1ml of sodium carbonate (1.06 g in
100 ml water), 0.4 ml of 24 mmol/L NBT (nitroblutetrazolin)

and 0.2 ml of Ethylene diamine tetra acetic acid [EDTA (37 mg
in 100 ml water)] was added, and zero minute reading was
taken at 560 nm. Reaction was initiated by addition of 0.4 ml
of 1mM hydroxylamine hydrochloride, incubated at 25°C for
5 minutes and the reduction of NBT was measured at 560 nm.
SOD level was calculated using the standard calibration curve,
and expressed in μg/mg of protein.[26]
Catalase levels
Catalase activity was assayed by the method of Claiborne (1985).
To 100 μl of supernatant, 1.9 ml of phosphate buffer (pH 7) was
added and absorbance was read at 240 nm. The reading was
taken again 1 minute after adding 1 ml of 0.019 mol/Lhydrogen
peroxide solution to the reaction mixture. One international
unit of catalase utilized is that amount that catalyzes the
decomposition of 0.019 mol/Lhydrogen peroxide/min/mg of
protein at 37°C. Catalase activity was calculated using the
standard calibration curve, and expressed as μg/mg of protein.[27]
Glutathione levels
1 ml of 10% supernatant was precipitated with 1ml of 4%
sulphosalicylic acid. The samples were kept at 4°C for at least 1
hour and then subjected to centrifugation at 1200 g for 15 minutes
at 4°C. The assay mixture contained 0.1 ml supernatant, 2.7 ml
phosphate buffer (0.1 M, pH 7.4) and 0.2 ml DTNB (5, 5, dithiobis
2‑nitro benzoic acid) Ellman’s reagent, in a total volume of 3 ml.
The yellow color developed was read immediately at 412 nm and
expressed as µg/mg of protein. The GSH concentrations of the
samples were derived from the standard curve prepared using
known amounts of GSH and expressed as ng/mg protein.[28]

Statistical analysis
The mean ± SEM values were calculated for each group. Student
t‑ test was used for statistical analysis. P < 0.05 was considered
statistically significant.

Results
Phytochemical analysis
Total extract yield was (3.70% w/w). Phytochemical screening
of the extract determined presence of alkaloids, glycosides,
steroids and flavonoids as major constituents, while tannins,
triterpenoids, saponins, carbohydrates, proteins and amino acids
were found absent [Table 1].

Acute toxicity study
The results of acute toxicity study showed no clinical signs
of toxicity and mortality in the EECT treated animals even
after administration of 2000 mg/kg dose. Hence, as per OECD
guidelines lethal dose was assigned to be more than 2000 mg/kg.
1/10th and 1/5th of this lethal dose (i.e. 200 mg/kg and 400 mg/kg)
were taken as effective doses for the study.
51 


Nootropic study
Effects of EECT on spontaneous alterations (%) in Y maze arm
Percent spontaneous alteration of diabetic controls was
significantly (P < 0.05) decreased in retention trial as
compared to normal controls, EECT (200 and 400 mg/kg),
metformin (200mg/kg) and piracetam (200 mg/kg) treated diabetic
animals showed significant (P < 0.05), increase in % spontaneous
alterations in retention trial as compared to diabetic
controls [Table 2].
Effects of EECT on spatial working and reference memory in
Radial arm maze
Spatial working‑reference memory of diabetic controls was
significantly (P < 0.01) decreased in retention trial as compared
to normal controls. Diabetic animals, treated with EECT (200
and 400 mg/kg) showed dose dependent (P < 0.001) increase
in spatial working‑reference memory in retention trial as
compared to diabetic controls. Whereas piracetam (200 mg/kg)
Table 1: Details of qualitative phytochemical tests
Chemical constituents

Test

Test for alkaloids

Mayers test
Wagners test
Hagers test
Dragendroffs test
Borntragers test
Baljet’s test
Legals test
Foam test
Haemolytic test
Salkowskitest liebermann burchard test
Salkowski test
Liebermann burchard test
Molisch’s test
Benedict’s test
Ferric chloride test
Shinoda test
Alkaline reagent test
Lead acetate test
Ferric chloride test
Gelatin test
Millons test
Ninhydrin test

Test for glycosides
Test for saponins
Test for steroids
Test for triterpenoids
Test for carbohydrates
Test for flavonoids

Test for tannins
Test for proteins
Test for amino acids

EECT
−
+
+
+
+
+
+
−
−
++
−
−
−
−
+
+
+
+
−
−
−
−

(+) Indicates present (–) Indicates absent.EECT: Ethanol extract of
Clitorea ternatea

Table 2: Effects of EECT on spontaneous alterations (%) in
Y maze arm of diabetes‑induced cognitive decline model
Treatment
(mg/kg p.o,)
Normal control (NC)
Diabetic control (DC)
DC+EECT (200)
DC+EECT (400)
DC+Metformin (200)
DC+Piracetam(200)

% Spontaneous alterations
71st Day (training trial) 75th Day (retention trial)
50.22±6.35
43.69±5.54
37.48±6.36
48.53±3.73
43.05±4.03
43.18±5.08

50.4±10.50
†34.27±2.52*
‡52.5±5.83*
‡58.98±6.77*
‡56.24±9.10*
‡61.76±1.57**

n=6, values are mean±SEM, *P<0.05, **P<0.001; †Compared
to normal control group, ‡Compared to diabetic control group.
EECT: Ethanol extract of Clitorea ternatea


52

treated diabetic animals showed significant increase in spatial
working (P < 0.001) and reference memory (P < 0.01) in
retention trial as compared to diabetic controls. Treatment with
metformin (200 mg/kg) however, produced insignificant effect
on spatial working‑reference memory [Table 3].
Effects of EECT on transfer latency in morris water maze
No significant difference observed in TL in animals
during training. In retention trial, TL was found to be
significantly (P < 0.001) increased in diabetic controls
as compared to normal controls. Two weeks repeated
t re a t m e n t w i t h E E C T ( 2 0 0 a n d 4 0 0 m g / k g ) a n d
piracetam (200 mg/kg) significantly (P < 0.001) decreased
TL in retention trial as compared to diabetic controls whereas
this effect was found insignificant in metformin (200 mg/kg)
treated animals [Table 4].

Neurochemical study
Effects of EECT on acetylcholinesterase activity
Acetylcholinesterase activity was significantly increased in
diabetic controls (P < 0.001) compared to normal controls,
suggestive of cholinergic dysfunction. While two weeks repeated
treatment with EECT (200 and 400 mg/kg) significantly decreased
acetylcholinesterase activity in diabetic animals (P < 0.05
and P < 0.001 respectively). Piracetam (200 mg/kg) treated
group showed significant (P < 0.001) decrease, whereas
metformin (200 mg/kg) showed insignificant effect in this
regard [Table 5].
Effects of EECT on lipid peroxide and total nitric oxide levels
Significant (P < 0.001) increase was observed in TBARS and
NO levels in diabetic controls compared to normal controls.
Two weeks repeated treatment with EECT (200 and 400 mg/kg)
and metformin (200 mg/kg) showed significant (P < 0.001)
reduction in TBARS and NO in diabetic animals whereas,
piracetam (200 mg/kg) though showed significant (P < 0.001)
reduction in TBARS levels; had insignificant effect on brain
NO level [Table 6].
Effects of EECT on superoxide dismutase, catalase and reduced
glutathione levels
The results showed significant (P < 0.001) decrease in
SOD, CAT and GHS levels in diabetic controls compared
to normal controls. Two weeks repeated treatment with
EECT (400 mg/kg) showed significant improvement in
SOD (P < 0.001), CAT (P < 0.01) and GSH (P < 0.001) levels
whereas, EECT (200 mg/kg) caused significant increase in
CAT (P < 0.01) and GSH (P < 0.001) levels with no significant
increase in diabetic animals. Metformin (200 mg/kg) showed
significant (P < 0.001) increase in SOD and CAT levels and
no significant effect on GSH levels. Piracetam (200 mg/
kg) showed significant increase only in SOD (P < 0.01) and
GSH (P < 0.001) levels [Table 7].


Table 3: Effects of EECT on spatial working and reference memory in radial arm maze of diabetes‑induced cognitive decline
model
Treatment (mg/kg, p.o.)

Working memory (%)

Reference memory (%)

71st Day (training trial)

71st Day (training trial)

75th Day (retention trial)

48.60±4.0
26.60±6.0
44.20±5.0
43.00±10.0
59.80±11.0
59.0±12.0

Normal control (NC)
DC+EECT (200)
DC+EECT (400)
DC+Metformin (200)
DC+Piracetam (200)

75th Day (retention trial)
82.20±1.3
23.00±2.2*
‡
75.80±5.0*
‡
89.80±11.0*
54.80±7.0
‡
98.00±1.3*

98.40±5.0
45.20±2.0
56.20±7.0
50.60±3.0
64.60±6.0
68.40±10.0

89.40±2.0
95.00±9.0**
‡
160.00±6.0***
‡
130.00±3.0***
61.60±10.0
‡
118.00±2.0**

†

†

n=6, values are mean±SEM, *P<0.05, **P<0.01, ***P<0.001; †Compared to normal control group; ‡Compared to diabetic control group.

Table 4: Effects of EECT on transfer latency in morris water
maze of diabetes‑induced cognitive decline models

Table 6: Effects of EECT on lipid peroxide and total nitrites
levels of diabetes‑induced cognitive decline models

Treatment
(mg/kg, p.o.)

Treatment
(mg/kg, p.o.)

Transfer latency (s)
71st Day (training trial) 75th Day (retention trial)

Normal control (NC)
DC+EECT (200)
DC+EECT (400)
DC+Metformin (200)
DC+Piracetam(200)

60.8±4.64
90.8±0.86
74.0±1.44
89.4±2.13
72.2±1.28
58.4±3.26

54.2±4.54
77.8±1.42**
‡
53.2±6.00*
‡
56.4±1.28**
58.8±3.83
‡
52.6±2.65**
†

to normal control group; ‡Compared to diabetic control group.

Table 5: Effects of EECT onacetylcholinesterase activity of
diabetes‑induced cognitive decline models
Treatment (mg/kg, p.o.)
Normal control (NC)
DC+EECT (200)
DC+EECT (400)
DC+Metformin (200)
DC+Piracetam(200)

Acetylcholinesterase (µM/l/min/gm of tissue)
3.67±0.47
6.36±0.30***
‡
5.45±0.25*
‡
4.07±0.42***
6.65±0.44
‡
3.15±0.20**
†

n=6, values are mean±SEM, *P<0.05, **P<0.01, ***P<0.001;
†
Compared to normal control group; ‡Compared to diabetic control
group. EECT: Ethanol extract of Clitorea ternatea

Discussion
The incidence of dementia appears to be doubled in elderly
subjects with diabetes.[30,31] The potential mechanisms for this
include direct effects of hypo or hyperglycemia and hypo or
hyperinsulinemia, and also indirect effects e.g. oxidative stress,
neurochemical changes causing cerebrovascular alterations.[32,33]
Large numbers of plants are used in treatment of cognitive
decline. Earlier studies have reported that administration of
Clitorea ternatea leaves extract significantly decrease the level
of blood glucose and glycosylated hemoglobin, increase serum
insulin to normal level in alloxan‑induced diabetic animals.[13]
The methanolic extract of Clitorea ternatea has been studied
for its effect on cognitive behavior, anxiety, depression, stress
and convulsions induced by pentylenetetrazol and maximum
electroshock. To explain these effects, the effect of Clitorea
ternatea was also studied on behavior mediated by dopamine,
noradrenaline, serotonin and acetylcholine.[9] Hence, it was

Normal Control (NC)
Diabetic Control (DC)
DC+EECT (200)
DC+EECT (400)
DC+Metformin (200)
DC+Piracetam (200)

TBARS (nmol of
MDA/mg of protein)

Total nitrites
(ng/mg of protein)

23.83±0.57
52.70±2.36*
‡
35.50±2.09*
‡
25.14±0.27*
‡
39.88±0.38*
‡
25.80±0.32*

116.4±0.36
238.3±1.07*
‡
204.1±0.98*
‡
151.5±1.15*
‡
161.3±0.40*
216.0±0.99

†

†

n=6, values are mean±SEM, *P<0.001; †Compared to
normal control group; ‡Compared to diabetic control group.

Table 7: Effects of EECT on SOD, CAT and GSH levels of
diabetes‑induced cognitive decline models
Treatment
(mg/kg, p.o.)

SOD
(µg/mg of
protein)

Normal control (NC)
108.40±0.37
Diabetic control (DC) †47.85±2.98**
DC+EECT (200)
52.35±7.48
‡
DC+EECT (400)
86.61±1.09**
DC+Metformin (200) ‡83.99±1.05**
DC+Piracetam (200) ‡61.37±0.50*

CAT
(µg/mg of
protein)

GSH
(ng/mg of
protein)

4.33±0.05
2.19±0.26**
‡
3.87±0.65*
‡
3.46±0.28*
‡
3.72±0.05**
2.64±0.34

14.97±0.73
3.30±0.01**
‡
7.27±0.09**
‡
12.42±1.4**
‡
11.84±0.54**
‡
5.37±0.55**

†

†

to normal control group; ‡Compared to diabetic control group.
EECT: Ethanol extract of Clitorea ternatea , SOD: Superoxide dismutase,
CAT: Catalase, GSH: Glutathione

thought worth to investigate the effects of Clitorea ternatea
on diabetes induced cognitive decline in experimental animals
along with its role in oxidative stress and acetylcholinesterase
activity. The cognitive decline study was focused on spatial
working and spatial reference memory based mazes.
The preliminary phytochemical screening showed presence
of alkaloids, glycosides, flavonoids, steroids in EECT leaves.
Various phytochemicals like flavonoids, anthocyanin glycosides,
pentacyclictriterpenoids, and phytosterols have been reported
from this plant.[8] The literature survey revealed cognitive
impairment in STZ‑induced diabetic animals in eight weeks.[34]
Our results are in agreement with this in diabetic animals.
Treatment of EECT was scheduled for two weeks after cognitive
decline. Results of present study showed significant improvement
in spatial working memory, spatial reference memory and spatial
working‑reference memory suggestive of nootropic activity of
EECT in diabetes induced cognitive decline models.
53 

[Downloaded free from http://www.jpbsonline.org on Saturday, January 04, 2014, IP: 117.242.184.92] || Click here to download free Android application for this jour

The increased oxidative stress in diabetes produces oxidative
damage in many regions of brain including the hippocampus[4]
this oxidative damage in the brain is increased by experimentally
induced hyperglycemia.[35] Oxidative damage to various brain
regions constitutes the long term complications, morphological
abnormalities and memory impairments. In the present study,
TBARS and NO levels were significantly increased (P < 0.001)
whereas GSH, SOD and CAT levels were markedly reduced
in the brain of diabetic controls. Treatment with EECT
significantly reduced the levels of TBARS, whereas GSH, SOD
and CAT levels were increased. Therefore, EECT might have
protective effect against diabetes induced cognitive decline
due to reduced oxidative stress. The antioxidant properties of
EECT might help to ameliorate the cognitive dysfunction in
diabetic animals.
Release of acetylcholine in the hippocampus is positively
correlated with training on a working memory task[36] and with
good performance on a hippocampus‑dependent, spontaneous
alteration task.[37] In diabetes the acetylcholinesterase levels are
found to be high; this enzyme hydrolyses acetylcholine present
in the brain and results in cognitive decline.[38] We observed
a significant rise in acetylcholinesterase activity in the brain
of diabetic rats. Two week treatment with EECT attenuated
increase in acetylcholinesterase activity in the brain of diabetic
animals. Hence, EECT treatment ameliorated cognitive decline,
cholinergic dysfunction, reduced oxidative stress, and NO in
the diabetic animals may find clinical application in treating
neuronal deficit in the diabetic patients.

Conclusion

6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.

To conclude, the results of present study showed improvement
in spatial working as well as reference memory in maze
based studies. Neurochemical study revealed decrease in
acetylcholinesterase, MDA and NO levels with increased
SOD, GSH and CAT levels in the brain. Thus, the present
data indicates that Clitorea ternatea offers protection against
diabetes induced cognitive decline and justifies the need for
further studies to elucidate its mode of action.

21.

Acknowledgment

25.

The authors are thankful to BSI Pune for identification of plant material.
The authors thank to Dr. Jain, Principal Sinhgad College of Pharmacy
Vadgaon for providing facilities to carry out of the experiments of this work.

26.

22.
23.
24.

27.

References
28.
1.
2.

3.
4.
5.


Blennow K, Leon de MJ, Zetterberg H. Alzheimer’s disease. Lancet
2006;368:387‑403.
Zhao Y, Ye W, Boye KS, Holcombe JH, Hall JA, Swindle R.
Prevalence of other diabetes‑associated complications and co
morbidities and its impact on health care charges among patients
with diabetic neuropathy. J Diabetes Complications 2010;24:9‑19.
Roriz‑Filho JS, Ticiana MS, Idiane R, Ana LC, Antonio CS, Márcia
LF, et al. (Pre) diabetes, brain aging, and cognition. Biochim Biophys
Acta2009;1792:432‑43.
Baynes JW. Role of oxidative stress in development of complications
in diabetes. Diabetes 1991;40:405‑12.
White L, Petrovitch H, Hardman J, Nelson J, Davis DG, Ross GW, et al.

54

29.
30.
31.
32.

Cerebrovascular pathology and dementia in autopsied. Honolulu‑Asia
Aging Study participants. Ann N Y Acad Sci 2002;977:9‑23.
Artola A. Diabetes, stress and ageing related changes in synaptic
plasticity in hippocampus and neocortex the same metaplastic
process? Eur J Pharmacol 2008;585:153‑62.
Bartus RT, Dean RL 3rd, Beer B, Lippa AS. The cholinergic hypothesis
of geriatric memory dysfunction.Science 1982;217:408‑17.
Mukherjee PK, Kumar V, Mal M, Houghton PJ. Acetyl cholinesterase
inhibitors from plants. Phytomedicine 2007;14:289‑300.
Jain NN, Ohal CC, Shroff SK, Bhutada RH, Somani RS, Kasture VS,
et al. Clitorea ternatea and the CNS. Pharmacol Biochem Behav
2003;75:529‑36.
Parimaladevi B, Boominathan R, Mandal SC. Evaluation of antipyretic
potential of Clitorea ternatea L. Extract in rats. Phytomedicine
2004;11:323‑6.
Kulkarni C, Pattanshetty JR, Amruthraj G. Effect of alcoholic extract
of Clitorea ternatea Linn on central nervous system in rodents. Indian
J ExpBiol 1988;26:957‑60.
Nadkarni KM. Indian Materia Medica.3rd ed. Bombay: Popular
Publication; 1976.
Daisy P Rajathi M. Hypoglycemic effects of Clitorea ternatea
,
Linn. (Fabaceae) in alloxan‑induced diabetes in rats.Trop J Pharm
Res2009;8:393‑8.
Kirtikar KR, Basu BD. Indian Medicinal Plants. Vol. 2, 2nd ed. Allahabad:
Lalit Mohan Basu; 1935.
Chauhan N, Rajvaidhya S, Dubey BK. Antiasthmatic effect of roots
of Clitorea ternatea Linn. Int J Pharm Sci Res 2012;3:1076‑9.
Trinder P Determination of glucose in blood using glucose oxidase
.
with an alternative oxygen acceptor. Ann Clin Biochem 1969;6:24‑7.
Raghavan G, Madhavan V, Rao C, Kumar V, Rawat AK, Pushpangadan P
.
Action of Asparagus racemosus against streptozotocin‑induced
oxidative stress. J Nat Prod Sci 2004;10:177‑81.
Khandelwal KR. Practical pharmacognosy.19 th ed. Pune:
Niraliprakashan; 2008.
Lucian H, Monica C, Toshitaka N. Brain serotonin depletion impairs
short‑term memory, but not Long‑term memory in rats. Physiol Behav
2007;91:652‑7.
Morris JB, Janick J, editors. Legume genetic resources with novel
value added industrial and pharmaceutical use. Perspectives on New
crops and New Uses. Alexandria, VA, USA: ASHS Press, Inc.; 1999.
p. 196‑201.
Tuzcu M, Baydas G. Effect of melatonin and vitamin E on
diabetes‑induced learning and memory impairment in rats. Eur J
Pharmacol 2006;537:106‑10.
Carrillo MP Giordano M, Santamaria A. Spatial memory: Theoretical
,
basis and comparative review on experimental methods in rodents.
Behav Brain Res 2009;203:151‑64.
Ellman GL, Courtney DK, Andres V, Featherstone RM. A new and rapid
colorimetric determination of acetylcholinesterase activity. Biochem
Pharmacol 1961;7:88‑95.
Wills ED. Mechanism of lipid peroxide formation in animals. Biochem
J 1965;99:667‑76.
Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS,
Tannenbaum SR. Analysis of nitrate, nitrite, and [15N] nitrate in
biological fluids. Anal Biochem 1982;126:131‑8.
Kono Y. Generation of superoxide radical during autoxidation of
hydroxylamine and an assay for superoxide dismutase. Arch Biochem
Biophys 1978;186:189‑95.
Claiborne A, Greenwald RA, editors. Catalase activity. Handbook of
methods for oxygen radical research. Boca Raton: CRC Press, Inc.,;
1985. p. 283‑4.
Jollow DJ, Mitchell JR, Zampaglione N, Gillette JR. Bromobenzene
induced liver necrosis: Protective role of glutathione and evidence for
3,4‑bromobenzenoxide as the hepatotoxic metabolite. Pharmacology
1974;11:151‑69.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement
with the folinphenol reagent. J Biol Chem 1951;193:265‑75.
Ott A, Stolk RP Van Harskamp F, Pols HA, Hofinan A, Breteler MM.
,
Diabetes mellitus and the risk of dementia: The Rotterdam study.
Neurology 1999;53:1937‑42.
Peila R, Rodriguez BL, Launer LJ. Type 2 diabetes, APOE gene, and
the risk for dementia and related pathologies: The Honolulu‑Asia
aging study. Diabetes 2002;51:1256‑62.
Brands AM, Biessels GJ, Haan de EH, Kappelle LJ, Kessels RP
.


The effects of type 1 diabetes on cognitive performance:
A meta‑analysis. Diabetes Care 2005;28:726‑35.
33. Lobnig BM, Krömeke O, Optenhostert‑Porst C, Wolf OT. Hippocampal
volume and cognitive performance in long‑standing type 1 diabetic
patients without macrovascular complications. Diabet Med
2005;23:32‑9.
34. Patil CS, Singh VP Kulkarni SK. Modulatory effect of sildenafil in
,
diabetes and electroconvulsive shock‑induced cognitive dysfunction
in rats. Pharmacol Rep 2006;58:373‑80.
35. Aragno MR, Mastrocola E, Brignardello M, Catalano M, Robino G,
Manti R, et al. Dehydroepiandrosterone modulates nuclear factor
kappa activation in hippocampus of diabetic rats. J Endocrinol
2002;143:3250‑8.

36. Fadda F, Mellis F, Stancampiano R. Increased hippocampal
acetylcholine release during a working memory task. Eur J Pharmacol
1996;307:R1‑2.
37. Ragozzino ME, Gold PE. Glucose injections into the medial septum
reverse the effects of intraseptal morphine infusions on hippocampal
acetylcholine output and memory. Neuroscience 1995;68:981‑8.
38. Dougherty KD, Turchin PI, Walsh TJ. Septocingulate and
septohippocampal cholinergic pathways: Involvement in working/
episodic memory. Brain Res 1998;810:59‑71.
Source of Support: Nil, Conflict of Interest: None declared.

Author Institution Mapping (AIM)

Please note that not all the institutions may get mapped due to non-availability of the requisite information in the Google Map. For AIM of other
issues, please check the Archives/Back Issues page on the journal’s website.

55 

Neuroprotective and nootropic activity of clitorea ternatea linn.(mazreoon in unani) leaves on diabetes induced cognitive decline in experimental animals

Recommended

Recommended

More Related Content

More from Younis I Munshi

More from Younis I Munshi (20)

Recently uploaded

Recently uploaded (20)

Neuroprotective and nootropic activity of clitorea ternatea linn.(mazreoon in unani) leaves on diabetes induced cognitive decline in experimental animals